Viral proteins as serological antigens Development and clinical applications Linn Persson Berg Department of Infectious Diseases Institute of Biomedicine Sahlgrenska Academy, University of Gothenburg Gothenburg 2022 Cover illustration: Viruses and antibodies by Linn Persson Berg. Viral proteins as serological antigens © Linn Persson Berg 2022 Linn.persson.berg@gu.se ISBN 978-91-8009-809-0 (PRINT) ISBN 978-91-8009-810-6 (PDF) To my family with all my love AN ENMÄRK V E Printed in Borås, Sweden 2022 Lycka är att få vara med er! Printed by Stema Specialtryck AB Trycksak3041 0234 S T Cover illustration: Viruses and antibodies by Linn Persson Berg. Viral proteins as serological antigens © Linn Persson Berg 2022 Linn.persson.berg@gu.se ISBN 978-91-8009-809-0 (PRINT) ISBN 978-91-8009-810-6 (PDF) To my family with all my love Printed in Borås, Sweden 2022 Lycka är att få vara med er! Printed by Stema Specialtryck AB Viral proteins as serological antigens Development and clinical applications Linn Persson Berg Department of Infectious Diseases, Institute of Biomedicine Sahlgrenska Academy, University of Gothenburg Gothenburg, Sweden ABSTRACT Serological methods are based on the detection of antibodies and antigens in mainly serum but also in other body fluids such as cerebrospinal fluid (CSF). Conventional whole virus antigens are widely used in viral serological assays. These antigens usually contain a mixture of proteins from the virus of interest together with residual cell components from antigen production, which can cause diagnostic problems with cross-reactive antibodies between closely related viruses and antibodies that bind to cellular components. The methods can become more specific by using antigens based on recombinant single viral proteins that differ between closely related viruses but to which the immune system reacts strongly (immunodominant proteins). The aim of the research has been to develop specific serological assays to detect antibodies to varicella-zoster virus (VZV), Epstein-Barr virus (EBV) and measles virus (MeV). This has been accomplished by recombinantly producing single, specific, immunodominant viral proteins, VZV glycoprotein E (gE), EBV glycoprotein 350 (gp350) and the core part of the MeV nucleocapsid protein (NCORE), for use as serological antigens in enzyme-linked immunosorbent assay (ELISA). In Paper I, we show that VZVgE functions well as ELISA antigen to detect anti-VZVgE IgG antibodies. The antigen has thereafter been used in the routine diagnostics at the Department of Clinical Microbiology, Sahlgrenska University Hospital. In Paper II, we demonstrate that EBVgp350 performs well as serological antigen in ELISA for the detection of anti-EBVgp350 IgG. In Paper III, we found that patients with multiple sclerosis (MS) and their clinically healthy siblings with similar MS findings in CSF, i.e. a suspect hyperimmune phenotype, still show an increased IgG response to MeV in both serum and CSF compared with healthy controls when the previously used complex MeV whole virus antigen was replaced with MeV NCORE. Our results Viral proteins as serological antigens Development and clinical applications Linn Persson Berg Department of Infectious Diseases, Institute of Biomedicine Sahlgrenska Academy, University of Gothenburg Gothenburg, Sweden ABSTRACT Serological methods are based on the detection of antibodies and antigens in mainly serum but also in other body fluids such as cerebrospinal fluid (CSF). Conventional whole virus antigens are widely used in viral serological assays. These antigens usually contain a mixture of proteins from the virus of interest together with residual cell components from antigen production, which can cause diagnostic problems with cross-reactive antibodies between closely related viruses and antibodies that bind to cellular components. The methods can become more specific by using antigens based on recombinant single viral proteins that differ between closely related viruses but to which the immune system reacts strongly (immunodominant proteins). The aim of the research has been to develop specific serological assays to detect antibodies to varicella-zoster virus (VZV), Epstein-Barr virus (EBV) and measles virus (MeV). This has been accomplished by recombinantly producing single, specific, immunodominant viral proteins, VZV glycoprotein E (gE), EBV glycoprotein 350 (gp350) and the core part of the MeV nucleocapsid protein (NCORE), for use as serological antigens in enzyme-linked immunosorbent assay (ELISA). In Paper I, we show that VZVgE functions well as ELISA antigen to detect anti-VZVgE IgG antibodies. The antigen has thereafter been used in the routine diagnostics at the Department of Clinical Microbiology, Sahlgrenska University Hospital. In Paper II, we demonstrate that EBVgp350 performs well as serological antigen in ELISA for the detection of anti-EBVgp350 IgG. In Paper III, we found that patients with multiple sclerosis (MS) and their clinically healthy siblings with similar MS findings in CSF, i.e. a suspect hyperimmune phenotype, still show an increased IgG response to MeV in both serum and CSF compared with healthy controls when the previously used complex MeV whole virus antigen was replaced with MeV NCORE. Our results indicate that the reactivity is indeed specific and not caused by cross-reacting SAMMANFATTNING PÅ SVENSKA autoantibodies to cellular proteins. In Paper IV, patients with MS show higher IgG levels in both serum and CSF to MeV and EBVgp350 compared with healthy controls. In addition, we observed that patients with serologically Serologiska metoder baseras på påvisning av antikroppar och antigen i främst serum verified acute infectious mononucleosis have higher serum IgG levels to men även i cerebrospinalvätska (CSV). Många virusserologiska metoder använder EBVgp350 at follow-up after 10 years compared with healthy controls, helvirusantigen som består av flera olika proteiner från viruset samt kvarvarande suggesting that EBV-induced mononucleosis affects the immune system in a cellulära komponenter från antigenproduktionen. Helvirusantigener kan ge upphov till powerful and long-lasting way. diagnostiska problem med korsreaktiva antikroppar mellan närbesläktade virus och antikroppar som binder till cellulära komponenter. Metoderna kan bli mer specifika In Paper V, patients with MS treated with interferon beta (IFNβ) had higher genom att använda antigen baserade på enstaka virala proteiner som skiljer sig från anti-EBVgp350 and anti-MeV NCORE IgG levels in serum compared with närbesläktade virus men som immunförsvaret reagerar starkt emot (immundominanta). healthy blood donors. Following initiation of treatment with the monoclonal Syftet med vår forskning har varit att utveckla specifika serologiska analyser som kan antibody natalizumab, patients' serum IgG levels decreased against both användas för att påvisa antikroppar mot varicella-zoster virus (VZV), Epstein-Barr antigens, whereas levels were relatively stable during previous IFNβ treatment. virus (EBV) och mässlingsvirus (MeV). Sådan förbättrad antigenproduktion har Another finding was that all 728 patients with MS in the study were EBV IgG utförts genom att rekombinant framställa enstaka, specifika, immundominanta seropositive while 10 of the 144 blood donors in the control group were EBV virusproteiner, VZV glykoprotein E (gE), EBV glykoprotein 350 (gp350) och MeV IgG seronegative. This finding further strengthens the potential role of EBV in nukleokapsid protein (NCORE), som serologiska antigen i enzymbunden the pathogenesis of MS. immunosorbentanalys (ELISA). Delarbete I visar att VZVgE fungerar bra som ELISA antigen. VZVgE har därefter kunnat användas i rutindiagnostiken på Mikrobiologen, The developed ELISA methods can, through increased specificity, offer new Sahlgrenska universitetssjukhuset. I Delarbete II så talar resultaten för att EBVgp350 diagnostic possibilities for detecting antibodies to EBV, VZV and MeV in viral fungerar bra som serologiskt antigen i ELISA. Delarbete III fastslår att patienter med infections, for control of immunity after infection/vaccination, in multipel skleros (MS) och deras kliniskt friska syskon med MS-liknande fynd i CSV, epidemiological investigations and in autoimmune diseases such as MS. dvs en misstänkt hyperimmun fenotyp, har ett ökat IgG-svar i både serum och CSV mot MeV jämfört med friska kontroller även när det tidigare använda komplexa MeV Keywords: Serology, ELISA, IgG, varicella-zoster virus, Epstein-Barr virus, helvirusantigenet bytts ut mot MeV NCORE. Resultaten indikerar att reaktiviteten är measles virus, viral glycoproteins, multiple sclerosis specifik och inte kan förklaras av korsreagerande autoantikroppar. I Delarbete IV uppvisar patienter med MS högre IgG-nivåer i serum och CSV mot MeV och ISBN 978-91-629-809-0 (PRINT) EBVgp350 jämfört med friska kontroller. Resultaten visar även att patienter som haft ISBN 978-91-629-810-6 (PDF) mononukleos (körtelfeber) har högre IgG-nivåer mot EBVgp350 i serum vid uppföljning efter tio år jämfört med friska kontroller, vilket indikerar att EBV orsakad mononukleos påverkar immunförsvaret på ett kraftfullt och långvarigt sätt. I Delarbete V uppvisar patienter med MS under interferon beta (IFNβ) behandling högre IgG- nivåer i serum mot EBVgp350 och MeV NCORE jämfört med friska blodgivare. Efter behandlingsstart med den monoklonala antikroppen natalizumab sjönk patienternas IgG-nivåer mot bägge dessa antigen, medan nivåerna var relativt stabila under den föregående IFNβ-behandlingen. Alla 728 patienter med MS i studien var EBV IgG- seropositiva medan 10 av de 144 blodgivarna i kontrollgruppen var EBV IgG- seronegativa. Detta fynd stärker ytterligare EBV:s potentiella roll i patogenesen bakom MS. De här utvecklade ELISA-metoderna kan genom ökad specificitet erbjuda nya diagnostiska möjligheter för att påvisa antikroppar mot EBV, VZV och MeV vid virusinfektioner, vid kontroll av immunitet efter infektion/vaccination, vid epidemiologiska undersökningar och även vid autoimmuna sjukdomar såsom MS. indicate that the reactivity is indeed specific and not caused by cross-reacting SAMMANFATTNING PÅ SVENSKA autoantibodies to cellular proteins. In Paper IV, patients with MS show higher IgG levels in both serum and CSF to MeV and EBVgp350 compared with healthy controls. In addition, we observed that patients with serologically Serologiska metoder baseras på påvisning av antikroppar och antigen i främst serum verified acute infectious mononucleosis have higher serum IgG levels to men även i cerebrospinalvätska (CSV). Många virusserologiska metoder använder EBVgp350 at follow-up after 10 years compared with healthy controls, helvirusantigen som består av flera olika proteiner från viruset samt kvarvarande suggesting that EBV-induced mononucleosis affects the immune system in a cellulära komponenter från antigenproduktionen. Helvirusantigener kan ge upphov till powerful and long-lasting way. diagnostiska problem med korsreaktiva antikroppar mellan närbesläktade virus och antikroppar som binder till cellulära komponenter. Metoderna kan bli mer specifika In Paper V, patients with MS treated with interferon beta (IFNβ) had higher genom att använda antigen baserade på enstaka virala proteiner som skiljer sig från anti-EBVgp350 and anti-MeV NCORE IgG levels in serum compared with närbesläktade virus men som immunförsvaret reagerar starkt emot (immundominanta). healthy blood donors. Following initiation of treatment with the monoclonal Syftet med vår forskning har varit att utveckla specifika serologiska analyser som kan antibody natalizumab, patients' serum IgG levels decreased against both användas för att påvisa antikroppar mot varicella-zoster virus (VZV), Epstein-Barr antigens, whereas levels were relatively stable during previous IFNβ treatment. virus (EBV) och mässlingsvirus (MeV). Sådan förbättrad antigenproduktion har Another finding was that all 728 patients with MS in the study were EBV IgG utförts genom att rekombinant framställa enstaka, specifika, immundominanta seropositive while 10 of the 144 blood donors in the control group were EBV virusproteiner, VZV glykoprotein E (gE), EBV glykoprotein 350 (gp350) och MeV IgG seronegative. This finding further strengthens the potential role of EBV in nukleokapsid protein (NCORE), som serologiska antigen i enzymbunden the pathogenesis of MS. immunosorbentanalys (ELISA). Delarbete I visar att VZVgE fungerar bra som ELISA antigen. VZVgE har därefter kunnat användas i rutindiagnostiken på Mikrobiologen, The developed ELISA methods can, through increased specificity, offer new Sahlgrenska universitetssjukhuset. I Delarbete II så talar resultaten för att EBVgp350 diagnostic possibilities for detecting antibodies to EBV, VZV and MeV in viral fungerar bra som serologiskt antigen i ELISA. Delarbete III fastslår att patienter med infections, for control of immunity after infection/vaccination, in multipel skleros (MS) och deras kliniskt friska syskon med MS-liknande fynd i CSV, epidemiological investigations and in autoimmune diseases such as MS. dvs en misstänkt hyperimmun fenotyp, har ett ökat IgG-svar i både serum och CSV mot MeV jämfört med friska kontroller även när det tidigare använda komplexa MeV Keywords: Serology, ELISA, IgG, varicella-zoster virus, Epstein-Barr virus, helvirusantigenet bytts ut mot MeV NCORE. Resultaten indikerar att reaktiviteten är measles virus, viral glycoproteins, multiple sclerosis specifik och inte kan förklaras av korsreagerande autoantikroppar. I Delarbete IV uppvisar patienter med MS högre IgG-nivåer i serum och CSV mot MeV och ISBN 978-91-629-809-0 (PRINT) EBVgp350 jämfört med friska kontroller. Resultaten visar även att patienter som haft ISBN 978-91-629-810-6 (PDF) mononukleos (körtelfeber) har högre IgG-nivåer mot EBVgp350 i serum vid uppföljning efter tio år jämfört med friska kontroller, vilket indikerar att EBV orsakad mononukleos påverkar immunförsvaret på ett kraftfullt och långvarigt sätt. I Delarbete V uppvisar patienter med MS under interferon beta (IFNβ) behandling högre IgG- nivåer i serum mot EBVgp350 och MeV NCORE jämfört med friska blodgivare. Efter behandlingsstart med den monoklonala antikroppen natalizumab sjönk patienternas IgG-nivåer mot bägge dessa antigen, medan nivåerna var relativt stabila under den föregående IFNβ-behandlingen. Alla 728 patienter med MS i studien var EBV IgG- seropositiva medan 10 av de 144 blodgivarna i kontrollgruppen var EBV IgG- seronegativa. Detta fynd stärker ytterligare EBV:s potentiella roll i patogenesen bakom MS. De här utvecklade ELISA-metoderna kan genom ökad specificitet erbjuda nya diagnostiska möjligheter för att påvisa antikroppar mot EBV, VZV och MeV vid virusinfektioner, vid kontroll av immunitet efter infektion/vaccination, vid epidemiologiska undersökningar och även vid autoimmuna sjukdomar såsom MS. LIST OF PAPERS Scientific papers not included in the thesis: This thesis is based on the following studies, referred to in the text by their Roman numerals. i. Lind L, Studahl M, Persson Berg L, Eriksson K. CXCL11 production in cerebrospinal fluid distinguishes I. Thomsson E, Persson L, Grahn A, Snäll J, Ekblad M, herpes simplex meningitis from herpes simplex Brunhage E, Svensson F, Jern C, Hansson G.C, Bäckström encephalitis. Journal of Neuroinflammation. M, Bergström T. Recombinant glycoprotein E produced in 2017;14(1):134 mammalian cells in large-scale as an antigen for varicella- ii. Widgren K, Persson Berg L, Mörner A, Lindquist L, zoster-virus serology. Journal of Virological Methods. Tegnell A, Giesecke J, Studahl M. Severe chickenpox 2011;175(1):53-9. disease and seroprevalence in Sweden - implications for II. Persson Berg L, Thomsson E, Hasi G, Bäckström M, general vaccination. International Journal of Infectious Bergström T. Recombinant Epstein-Barr virus glycoprotein Diseases. 2021;111:92 350 as a serological antigen. Journal of Virological Methods. 2020;284:113927. III. Persson L, Longhi S, Enarsson J, Andersen O, Haghigi S, Nilsson S, Lagging M, Johansson M, Bergström T. Elevated antibody reactivity to measles virus NCORE protein among patients with multiple sclerosis and their healthy siblings with intrathecal oligoclonal immunoglobulin G production. Journal of Clinical Virology. 2014;61(1):107-12. IV. Jons D, Persson Berg L, Sundström P, Haghighi S, Axelsson M, Thulin M, Bergström T, Andersen O. Follow- up after infectious mononucleosis in search of serological similarities with presymptomatic multiple sclerosis. Multiple Sclerosis and Related Disorders. 2021;56:103288. V. Persson Berg L, Eriksson M, Longhi S, Kockum I, Warnke C, Thomsson E, Bäckström M, Olsson T, Fogdell-Hahn A, Bergström T. Serum IgG levels to Epstein-Barr and measles viruses in patients with multiple sclerosis during natalizumab and interferon beta treatment. Submitted manuscript. LIST OF PAPERS Scientific papers not included in the thesis: This thesis is based on the following studies, referred to in the text by their Roman numerals. i. Lind L, Studahl M, Persson Berg L, Eriksson K. CXCL11 production in cerebrospinal fluid distinguishes I. Thomsson E, Persson L, Grahn A, Snäll J, Ekblad M, herpes simplex meningitis from herpes simplex Brunhage E, Svensson F, Jern C, Hansson G.C, Bäckström encephalitis. Journal of Neuroinflammation. M, Bergström T. Recombinant glycoprotein E produced in 2017;14(1):134 mammalian cells in large-scale as an antigen for varicella- ii. Widgren K, Persson Berg L, Mörner A, Lindquist L, zoster-virus serology. Journal of Virological Methods. Tegnell A, Giesecke J, Studahl M. Severe chickenpox 2011;175(1):53-9. disease and seroprevalence in Sweden - implications for II. Persson Berg L, Thomsson E, Hasi G, Bäckström M, general vaccination. International Journal of Infectious Bergström T. Recombinant Epstein-Barr virus glycoprotein Diseases. 2021;111:92 350 as a serological antigen. Journal of Virological Methods. 2020;284:113927. III. Persson L, Longhi S, Enarsson J, Andersen O, Haghigi S, Nilsson S, Lagging M, Johansson M, Bergström T. Elevated antibody reactivity to measles virus NCORE protein among patients with multiple sclerosis and their healthy siblings with intrathecal oligoclonal immunoglobulin G production. Journal of Clinical Virology. 2014;61(1):107-12. IV. Jons D, Persson Berg L, Sundström P, Haghighi S, Axelsson M, Thulin M, Bergström T, Andersen O. Follow- up after infectious mononucleosis in search of serological similarities with presymptomatic multiple sclerosis. Multiple Sclerosis and Related Disorders. 2021;56:103288. V. Persson Berg L, Eriksson M, Longhi S, Kockum I, Warnke C, Thomsson E, Bäckström M, Olsson T, Fogdell-Hahn A, Bergström T. Serum IgG levels to Epstein-Barr and measles viruses in patients with multiple sclerosis during natalizumab and interferon beta treatment. Submitted manuscript. CONTENT ABBREVIATIONS ............................................................................................. III DEFINITIONS IN SHORT ................................................................................... VI 1 INTRODUCTION ........................................................................................... 1 1.1 Human immune system ......................................................................... 2 1.1.1 Barrier protection .......................................................................... 2 1.1.2 Innate immune system ................................................................... 3 1.1.3 Adaptive immune system .............................................................. 3 1.2 Immunoglobulins .................................................................................. 5 1.2.1 Production ..................................................................................... 5 1.2.2 Basic structure ............................................................................... 7 1.2.3 Cleavage with proteases ................................................................ 8 1.2.4 Isotypes .......................................................................................... 9 1.2.5 Effector mechanisms ................................................................... 11 1.2.6 Antibody-antigen interaction ....................................................... 13 1.2.7 Antibody diversity ....................................................................... 16 1.2.8 Antibody response to viral infections .......................................... 17 1.3 Viruses ................................................................................................ 20 1.3.1 Herpesviridae .............................................................................. 22 1.3.2 Alphaherpesvirinae ..................................................................... 24 1.3.3 Varicella-zoster virus .................................................................. 24 1.3.4 Betaherpesvirinae ........................................................................ 30 1.3.5 Gammaherpesvirinae .................................................................. 32 1.3.6 Epstein-Barr virus ........................................................................ 32 1.3.7 Measles virus ............................................................................... 36 1.4 Viral serology ...................................................................................... 40 1.4.1 Serological methods in virology .................................................. 43 1.4.2 Advantages of serological methods ............................................. 52 1.4.3 Limitations of serological methods ............................................. 53 i CONTENT ABBREVIATIONS ............................................................................................. III DEFINITIONS IN SHORT ................................................................................... VI 1 INTRODUCTION ........................................................................................... 1 1.1 Human immune system ......................................................................... 2 1.1.1 Barrier protection .......................................................................... 2 1.1.2 Innate immune system ................................................................... 3 1.1.3 Adaptive immune system .............................................................. 3 1.2 Immunoglobulins .................................................................................. 5 1.2.1 Production ..................................................................................... 5 1.2.2 Basic structure ............................................................................... 7 1.2.3 Cleavage with proteases ................................................................ 8 1.2.4 Isotypes .......................................................................................... 9 1.2.5 Effector mechanisms ................................................................... 11 1.2.6 Antibody-antigen interaction ....................................................... 13 1.2.7 Antibody diversity ....................................................................... 16 1.2.8 Antibody response to viral infections .......................................... 17 1.3 Viruses ................................................................................................ 20 1.3.1 Herpesviridae .............................................................................. 22 1.3.2 Alphaherpesvirinae ..................................................................... 24 1.3.3 Varicella-zoster virus .................................................................. 24 1.3.4 Betaherpesvirinae ........................................................................ 30 1.3.5 Gammaherpesvirinae .................................................................. 32 1.3.6 Epstein-Barr virus ........................................................................ 32 1.3.7 Measles virus ............................................................................... 36 1.4 Viral serology ...................................................................................... 40 1.4.1 Serological methods in virology .................................................. 43 1.4.2 Advantages of serological methods ............................................. 52 1.4.3 Limitations of serological methods ............................................. 53 i 1.5 Multiple sclerosis ................................................................................ 58 ABBREVIATIONS 2 AIM ........................................................................................................... 63 3 PATIENTS AND METHODS ......................................................................... 64 a.a. Amino acid 3.1 Patients ................................................................................................ 64 APC Antigen-presenting cell 3.1.1 Paper 1 ......................................................................................... 64 BBB Blood-brain barrier 3.1.2 Paper II ........................................................................................ 65 B cell B lymphocyte 3.1.3 Paper III ....................................................................................... 66 bp Base pair 3.1.4 Paper IV....................................................................................... 67 CHO Chinese hamster ovary 3.1.5 Paper V ........................................................................................ 68 CH Constant heavy chain 3.2 Methods ............................................................................................... 70 CL Constant light chain 3.2.1 Antigen production ...................................................................... 70 CLIA 3.2.2 Varicella-zoster virus glycoprotein E .......................................... 70 Chemiluminescence immunoassay 3.2.3 Epstein-Barr virus glycoprotein 350 ........................................... 71 CMIA Chemiluminescence microparticle immunoassay 3.2.4 Measles virus nucleocapsid antigen ............................................ 72 CMV Cytomegalovirus 3.2.5 Western blot ................................................................................ 72 CNS Central nervous system 3.2.6 Immunofluorescence ................................................................... 73 CSF Cerebrospinal fluid 3.2.7 ELISA ......................................................................................... 74 CSR Class swish recombination 3.3 Statistical methods .............................................................................. 75 DAMP Damage-associated molecular pattern 3.4 Ethics ................................................................................................... 76 DMT Disease modifying therapy 4 RESULTS AND DISCUSSION ....................................................................... 77 DNA Deoxyribonucleic acid 4.1 Paper I ................................................................................................. 77 ds Double-stranded 4.2 Paper II ................................................................................................ 83 4.3 Paper III .............................................................................................. 87 EBNA1/2 Epstein-Barr virus nuclear antigen 1/2 4.4 Paper IV .............................................................................................. 90 EBNA1+/- EBNA1 seropositive/seronegative 4.5 Paper V ................................................................................................ 94 EBV Epstein-Barr virus 5 CONCLUSION ............................................................................................ 99 EBVgp350 EBV glycoprotein 350 6 FUTURE PERSPECTIVES ........................................................................... 101 EIA Enzyme immunoassay ACKNOWLEDGEMENT .................................................................................. 103 ELISA Enzyme-linked immunosorbent assay REFERENCES ................................................................................................ 106 Fab Fragment antigen binding ii iii 1.5 Multiple sclerosis ................................................................................ 58 ABBREVIATIONS 2 AIM ........................................................................................................... 63 3 PATIENTS AND METHODS ......................................................................... 64 a.a. Amino acid 3.1 Patients ................................................................................................ 64 APC Antigen-presenting cell 3.1.1 Paper 1 ......................................................................................... 64 BBB Blood-brain barrier 3.1.2 Paper II ........................................................................................ 65 B cell B lymphocyte 3.1.3 Paper III ....................................................................................... 66 bp Base pair 3.1.4 Paper IV....................................................................................... 67 CHO Chinese hamster ovary 3.1.5 Paper V ........................................................................................ 68 CH Constant heavy chain 3.2 Methods ............................................................................................... 70 CL Constant light chain 3.2.1 Antigen production ...................................................................... 70 CLIA 3.2.2 Varicella-zoster virus glycoprotein E .......................................... 70 Chemiluminescence immunoassay 3.2.3 Epstein-Barr virus glycoprotein 350 ........................................... 71 CMIA Chemiluminescence microparticle immunoassay 3.2.4 Measles virus nucleocapsid antigen ............................................ 72 CMV Cytomegalovirus 3.2.5 Western blot ................................................................................ 72 CNS Central nervous system 3.2.6 Immunofluorescence ................................................................... 73 CSF Cerebrospinal fluid 3.2.7 ELISA ......................................................................................... 74 CSR Class swish recombination 3.3 Statistical methods .............................................................................. 75 DAMP Damage-associated molecular pattern 3.4 Ethics ................................................................................................... 76 DMT Disease modifying therapy 4 RESULTS AND DISCUSSION ....................................................................... 77 DNA Deoxyribonucleic acid 4.1 Paper I ................................................................................................. 77 ds Double-stranded 4.2 Paper II ................................................................................................ 83 4.3 Paper III .............................................................................................. 87 EBNA1/2 Epstein-Barr virus nuclear antigen 1/2 4.4 Paper IV .............................................................................................. 90 EBNA1+/- EBNA1 seropositive/seronegative 4.5 Paper V ................................................................................................ 94 EBV Epstein-Barr virus 5 CONCLUSION ............................................................................................ 99 EBVgp350 EBV glycoprotein 350 6 FUTURE PERSPECTIVES ........................................................................... 101 EIA Enzyme immunoassay ACKNOWLEDGEMENT .................................................................................. 103 ELISA Enzyme-linked immunosorbent assay REFERENCES ................................................................................................ 106 Fab Fragment antigen binding ii iii FAMA Fluorescent antibody to membrane antigen PML Progressive multifocal leukoencephalopathy Fc Fragment crystallizable PPMS Primary-progressive multiple sclerosis GC Germinal center PRR Pattern recognition receptor Gp Glycoprotein RBC Red blood cell HHV6A/6B/7/8 Human herpesvirus 6A/6B/7/8 RIA Radioimmunoassay HIV Human immunodeficiency virus RNA Ribonucleic acid HSV 1/2 Herpes simplex virus type 1/2 ROC Receiver operating characteristic Ig Immunoglobulin RRMS Relapsing-remitting multiple sclerosis IM Infectious mononucleosis RV Rubella virus IFN Interferon SHM Somatic hypermutation IFNβ Interferon beta SPMS Secondary progressive multiple sclerosis IQR Interquartile range ss Single-stranded JCV JC polyomavirus TBE Tick-borne encephalitis NCORE The core fragment (a.a. 1–392) of the measles virus T cell T lymphocyte nucleocapsid protein TFF Tangential flow filtration MeSH Medical Subject Headings VCA Viral capsid antigen MeV Measles virus VCA+/- VCA seropositive/seronegative MHC-I/II Major histocompatibility complex class I and class II VCAM-1 Vascular cell adhesion molecule 1 MRI Magnetic resonance imaging VH Variable heavy chain MS Multiple sclerosis VL Variable light chain NAT Natalizumab VZV Varicella-zoster virus NCBI National Center for Biotechnology Information VZVgB VZV glycoprotein B NK cells Natural killer cells VZVgE VZV glycoprotein E OCB Oligoclonal bands VZVgE-ag VZVgE antigen OD Optical density VZVwhole-ag VZV whole virus antigen PAMP Pathogen-associated molecular pattern WHO World Health Organization PBS Phosphate-buffered saline PCR Polymerase chain reaction iv v FAMA Fluorescent antibody to membrane antigen PML Progressive multifocal leukoencephalopathy Fc Fragment crystallizable PPMS Primary-progressive multiple sclerosis GC Germinal center PRR Pattern recognition receptor Gp Glycoprotein RBC Red blood cell HHV6A/6B/7/8 Human herpesvirus 6A/6B/7/8 RIA Radioimmunoassay HIV Human immunodeficiency virus RNA Ribonucleic acid HSV 1/2 Herpes simplex virus type 1/2 ROC Receiver operating characteristic Ig Immunoglobulin RRMS Relapsing-remitting multiple sclerosis IM Infectious mononucleosis RV Rubella virus IFN Interferon SHM Somatic hypermutation IFNβ Interferon beta SPMS Secondary progressive multiple sclerosis IQR Interquartile range ss Single-stranded JCV JC polyomavirus TBE Tick-borne encephalitis NCORE The core fragment (a.a. 1–392) of the measles virus T cell T lymphocyte nucleocapsid protein TFF Tangential flow filtration MeSH Medical Subject Headings VCA Viral capsid antigen MeV Measles virus VCA+/- VCA seropositive/seronegative MHC-I/II Major histocompatibility complex class I and class II VCAM-1 Vascular cell adhesion molecule 1 MRI Magnetic resonance imaging VH Variable heavy chain MS Multiple sclerosis VL Variable light chain NAT Natalizumab VZV Varicella-zoster virus NCBI National Center for Biotechnology Information VZVgB VZV glycoprotein B NK cells Natural killer cells VZVgE VZV glycoprotein E OCB Oligoclonal bands VZVgE-ag VZVgE antigen OD Optical density VZVwhole-ag VZV whole virus antigen PAMP Pathogen-associated molecular pattern WHO World Health Organization PBS Phosphate-buffered saline PCR Polymerase chain reaction iv v DEFINITIONS IN SHORT Immunodominant Antigens that are easily recognized by the immune system and thus are of most Antibodies Immunoglobulin molecules having a specific importance for the specificity of the induced amino acid sequence by virtue of which they antibody response. interact only with the antigen (or a very Intrathecal antibody Antibodies produced in the central nervous similar shape) that induced their synthesis in production system and secreted to the cerebrospinal cells of the lymphoid series, especially fluid. plasma cells (Medical Subject Headings MeSH, National Center for Biotechnology Paratope Local surface sites on antibodies which react Information NCBI). with antigen determinant sites on antigens (epitopes) (MeSH, NCBI). Antigens Substances that are recognized by the immune system and induce an immune Serology Diagnostic identification of antibodies and reaction (MeSH, NCBI). antigens in serum and other body fluids including cerebrospinal fluid. Antigen-antibody reactions The processes triggered by interactions of antibodies with their antigens (MeSH, NCBI). Antibody affinity A measure of the binding strength between antibody and a simple hapten or antigen determinant. It depends on the closeness of stereochemical fit between antibody combining sites and antigen determinants, on the size of the area of contact between them, and on the distribution of charged and hydrophobic groups. It includes the concept of "avidity," which refers to the strength of the antigen-antibody bond after formation of reversible complexes (MeSH, NCBI). Epitope Site on an antigen that interact with specific antibodies (MeSH, NCBI). MS trait A suspect hyperimmune phenotype in clinically healthy siblings of patients with MS. vi vii DEFINITIONS IN SHORT Immunodominant Antigens that are easily recognized by the immune system and thus are of most Antibodies Immunoglobulin molecules having a specific importance for the specificity of the induced amino acid sequence by virtue of which they antibody response. interact only with the antigen (or a very Intrathecal antibody Antibodies produced in the central nervous similar shape) that induced their synthesis in production system and secreted to the cerebrospinal cells of the lymphoid series, especially fluid. plasma cells (Medical Subject Headings MeSH, National Center for Biotechnology Paratope Local surface sites on antibodies which react Information NCBI). with antigen determinant sites on antigens (epitopes) (MeSH, NCBI). Antigens Substances that are recognized by the immune system and induce an immune Serology Diagnostic identification of antibodies and reaction (MeSH, NCBI). antigens in serum and other body fluids including cerebrospinal fluid. Antigen-antibody reactions The processes triggered by interactions of antibodies with their antigens (MeSH, NCBI). Antibody affinity A measure of the binding strength between antibody and a simple hapten or antigen determinant. It depends on the closeness of stereochemical fit between antibody combining sites and antigen determinants, on the size of the area of contact between them, and on the distribution of charged and hydrophobic groups. It includes the concept of "avidity," which refers to the strength of the antigen-antibody bond after formation of reversible complexes (MeSH, NCBI). Epitope Site on an antigen that interact with specific antibodies (MeSH, NCBI). MS trait A suspect hyperimmune phenotype in clinically healthy siblings of patients with MS. vi vii 1 INTRODUCTION The term serology comes from studies of blood serum, where immunological reactions are investigated, particularly in vitro, with a focus on antigen- antibody reactions. Immunoglobulins (Ig), also called antibodies, are produced by B lymphocytes and are the humoral component in the adaptive immune system. Antibodies can be produced in response to various antigens. Medical Subject Headings (MeSH) by National Center for Biotechnology Information (NCBI) defines antibodies as: “Immunoglobulin molecules having a specific amino acid sequence by virtue of which they interact only with the antigen (or a very similar shape) that induced their synthesis in cells of the lymphoid series (especially plasma cells)”. MeSH defines antigens as: “Substances that are recognized by the immune system and induce an immune reaction”. Antigens can be part of microorganisms, other foreign substances, or, in the case of autoimmune diseases, the body's own molecules. In medical terminology, the term serology often refers to the diagnostic identification of antibodies and antigens in both serum and other body fluids including cerebrospinal fluid (CSF). Serological methods are important for diagnosing infections and autoimmune diseases, to control immunity after infection/vaccination, and in many other situations, such as for epidemiological purposes to determine the prevalence of a particular infection. Virological diagnostic research has recently focused strongly on molecular biological methods, but serological methods remain important. It is therefore warranted to continue to improve these methods. 1 1 INTRODUCTION The term serology comes from studies of blood serum, where immunological reactions are investigated, particularly in vitro, with a focus on antigen- antibody reactions. Immunoglobulins (Ig), also called antibodies, are produced by B lymphocytes and are the humoral component in the adaptive immune system. Antibodies can be produced in response to various antigens. Medical Subject Headings (MeSH) by National Center for Biotechnology Information (NCBI) defines antibodies as: “Immunoglobulin molecules having a specific amino acid sequence by virtue of which they interact only with the antigen (or a very similar shape) that induced their synthesis in cells of the lymphoid series (especially plasma cells)”. MeSH defines antigens as: “Substances that are recognized by the immune system and induce an immune reaction”. Antigens can be part of microorganisms, other foreign substances, or, in the case of autoimmune diseases, the body's own molecules. In medical terminology, the term serology often refers to the diagnostic identification of antibodies and antigens in both serum and other body fluids including cerebrospinal fluid (CSF). Serological methods are important for diagnosing infections and autoimmune diseases, to control immunity after infection/vaccination, and in many other situations, such as for epidemiological purposes to determine the prevalence of a particular infection. Virological diagnostic research has recently focused strongly on molecular biological methods, but serological methods remain important. It is therefore warranted to continue to improve these methods. 1 1.1 HUMAN IMMUNE SYSTEM 1.1.2 INNATE IMMUNE SYSTEM Pathogenic microorganisms such as bacteria, fungi and viruses are constantly The development of the innate immune system occurred earlier in evolution present in our environment, but the human body has ways to protect us against compared with the adaptive immune system, and it is found in both disease-causing pathogens. An individual with a well-functioning immune invertebrates and vertebrates. Innate immunity is important for the survival of system is termed immunocompetent. Some individuals are organisms and the basic mechanisms are thus conserved among animals (4). immunocompromised i.e. have a weakened immune system due to either The immune cells that are part of the innate response are neutrophils, primary immune deficiencies or secondary/acquired immune deficiencies due monocytes, macrophages, dendritic cells, eosinophils, basophils, mast cells to acquired conditions e.g. immunosuppressive medications or malnutrition. In and natural killer cells (NK cells). The complement system, cytokines and the classical partition of the human body's defenses against infections, the acute phase proteins are also important parts of the system. Some cytokines defense has been divided into three different levels (1): such as interferons (IFN) have a direct antiviral effect. IFN can directly interfere with viral replication and stimulate cell-mediated immunity to • Barrier protection respond to viral infection (5). The immune cells and molecules are present on • Innate immune system the surfaces as well as inside the body's organs and tissues and are immediately • Adaptive immune system ready to act to fight invasive pathogens. The response occurs after recognition of pathogen-derived structures and endogenous danger signals. The pattern recognition receptors (PRRs) on the 1.1.1 BARRIER PROTECTION immune cells i.e. C-type lectin receptors, Toll-like receptors, NOD-like receptors, and RIG-I-like receptors recognize pathogen-associated molecular The human body's first defenses against disease-causing pathogens are patterns (PAMPs) on pathogens and damage-associated molecular patterns physical, chemical, and biological barriers. The skin, which display dense (DAMPs) (6). The innate immune response has been considered to lack connections of epithelial cells, provides mechanical protection against immunological memory (1), but this view has been questioned in recent years pathogens entering the body. We are also protected from unwanted invaders and there are now arguments that the concept of immunological memory must by our mucous membranes that line body cavities and canals in contact with be expanded, as also the innate immune system can mount resistance to the outside world, such as the gastrointestinal, respiratory, and urogenital reinfection (6-9). tracts. The tough structure of mucous membranes can prevent pathogens from invading. The structures vary, but they all have a surface layer of tightly 1.1.3 ADAPTIVE IMMUNE SYSTEM connected epithelial cells and a deeper layer of connective tissue. The mucus membranes also protect us by secreting mucus, a viscous fluid with inhibitory Only vertebrates have an adaptive immune system with special molecular substances including bactericidal and antiviral substances, where pathogens characteristics, including B and T lymphocytes (B and T cells) and lymphoid may be entrapped and killed. There are also dynamic forces that can transport organs such as the spleen and thymus (10). This branch of the immune system away intruders, e.g. intestinal motility and cilia in the airways. The normal evolved in jawed fish about 500 million years ago (10). The adaptive immune microbial flora does not normally cause disease and can prevent other more response is highly specific, and B and T cells are activated only when they pathogenic microbes from gaining a foothold by competing for encounter their cognate antigen. The adaptive immune system has an microenvironments more effectively. The role of bacteria in the normal flora immunological memory which means that reinfection with a previously is more established than the role of viruses (human virome) (2, 3). encountered pathogen induces a more vigorous and rapid response (1, 4, 10). The innate and adaptive immune systems cooperate to fight invasive pathogens. For example, antigen-presenting cells (APCs) from the innate immune system can capture antigens and present peptides from these antigens 2 3 1.1 HUMAN IMMUNE SYSTEM 1.1.2 INNATE IMMUNE SYSTEM Pathogenic microorganisms such as bacteria, fungi and viruses are constantly The development of the innate immune system occurred earlier in evolution present in our environment, but the human body has ways to protect us against compared with the adaptive immune system, and it is found in both disease-causing pathogens. An individual with a well-functioning immune invertebrates and vertebrates. Innate immunity is important for the survival of system is termed immunocompetent. Some individuals are organisms and the basic mechanisms are thus conserved among animals (4). immunocompromised i.e. have a weakened immune system due to either The immune cells that are part of the innate response are neutrophils, primary immune deficiencies or secondary/acquired immune deficiencies due monocytes, macrophages, dendritic cells, eosinophils, basophils, mast cells to acquired conditions e.g. immunosuppressive medications or malnutrition. In and natural killer cells (NK cells). The complement system, cytokines and the classical partition of the human body's defenses against infections, the acute phase proteins are also important parts of the system. Some cytokines defense has been divided into three different levels (1): such as interferons (IFN) have a direct antiviral effect. IFN can directly interfere with viral replication and stimulate cell-mediated immunity to • Barrier protection respond to viral infection (5). The immune cells and molecules are present on • Innate immune system the surfaces as well as inside the body's organs and tissues and are immediately • Adaptive immune system ready to act to fight invasive pathogens. The response occurs after recognition of pathogen-derived structures and endogenous danger signals. The pattern recognition receptors (PRRs) on the 1.1.1 BARRIER PROTECTION immune cells i.e. C-type lectin receptors, Toll-like receptors, NOD-like receptors, and RIG-I-like receptors recognize pathogen-associated molecular The human body's first defenses against disease-causing pathogens are patterns (PAMPs) on pathogens and damage-associated molecular patterns physical, chemical, and biological barriers. The skin, which display dense (DAMPs) (6). The innate immune response has been considered to lack connections of epithelial cells, provides mechanical protection against immunological memory (1), but this view has been questioned in recent years pathogens entering the body. We are also protected from unwanted invaders and there are now arguments that the concept of immunological memory must by our mucous membranes that line body cavities and canals in contact with be expanded, as also the innate immune system can mount resistance to the outside world, such as the gastrointestinal, respiratory, and urogenital reinfection (6-9). tracts. The tough structure of mucous membranes can prevent pathogens from invading. The structures vary, but they all have a surface layer of tightly 1.1.3 ADAPTIVE IMMUNE SYSTEM connected epithelial cells and a deeper layer of connective tissue. The mucus membranes also protect us by secreting mucus, a viscous fluid with inhibitory Only vertebrates have an adaptive immune system with special molecular substances including bactericidal and antiviral substances, where pathogens characteristics, including B and T lymphocytes (B and T cells) and lymphoid may be entrapped and killed. There are also dynamic forces that can transport organs such as the spleen and thymus (10). This branch of the immune system away intruders, e.g. intestinal motility and cilia in the airways. The normal evolved in jawed fish about 500 million years ago (10). The adaptive immune microbial flora does not normally cause disease and can prevent other more response is highly specific, and B and T cells are activated only when they pathogenic microbes from gaining a foothold by competing for encounter their cognate antigen. The adaptive immune system has an microenvironments more effectively. The role of bacteria in the normal flora immunological memory which means that reinfection with a previously is more established than the role of viruses (human virome) (2, 3). encountered pathogen induces a more vigorous and rapid response (1, 4, 10). The innate and adaptive immune systems cooperate to fight invasive pathogens. For example, antigen-presenting cells (APCs) from the innate immune system can capture antigens and present peptides from these antigens 2 3 to T cells to activate them. The adaptive immune response can in turn both 1.2 IMMUNOGLOBULINS regulate and stimulate the innate immune system. Cytotoxic T cells that express CD8 on the cell surface identify and kill cells that synthesize aberrant peptides that are presented on major histocompatibility 1.2.1 PRODUCTION complex class I (MHC-I) molecules, which occur in virus-infected cells. As a Hematopoietic stem cells in the bone marrow can develop into B cells in an token of the importance of this defense mechanism, certain viruses can prevent antigen-independent process. After leaving the bone marrow, B cells will MHC-I molecules from reaching the surface of virus-infected cells and thus colonize secondary lymphoid organs and there, in an antigen-dependent hide from the cytotoxic T cells (i.e. immune evasion). However, when NK cells process, they can differentiate into memory B cells and antibody-producing detect cells with fewer MHC-I molecules than normal, they will kill the cell. plasma cells. The process is initiated after a mature B cell encounters and This is an example of how the innate and adaptive immune response interacts recognizes its cognate antigen. There are T cell-independent antigens, often to eliminate pathogens. The main function of CD4+ T helper cells is to polysaccharide antigens, that directly activate B cells, but most antigens are T stimulate and regulate other immune cells. T helper cells can for instance cell-dependent, which means that after antigen recognition, the B cell requires increase the functional capacity of phagocytic cells and stimulate B cells to cooperation with a T helper cell that recognizes the same antigen for final become more efficient at producing immunoglobulins (antibodies). activation (13, 18). After B cell activation, cell division and differentiation into memory B cells and plasma cells can occur. The humoral immunity is one of the main components of the adaptive immunity and immunoglobulins are important for providing immunity after B cells can produce both soluble and membrane-bound immunoglobulins infection/vaccination. Secreted immunoglobulins can neutralize pathogens and simultaneously. This is done by splicing at the mRNA level, so that the same act as effector molecules to stimulate other parts of the immune system to fight main structure can be combined with a hydrophilic short tail that provides a pathogens. The human body can produce antibodies with almost infinite soluble molecule, or with a partially hydrophobic longer tail that gives a specificity. membrane-bound molecule. B cells have mainly membrane-bound immunoglobulins (the main part of the B cell receptor) while plasma cells are After antigen recognition and activation of various B cells in the body, these specialized in producing and secreting immunoglobulins (19). Plasma cells are cells will produce antibodies that can recognize and bind to different antigens. larger than B cells and the morphology is characteristic with a small, The antibody response in the human body is thus polyclonal because different asymmetrically placed nucleus, large cytoplasm with abundant rough antibodies recognize different antigens. Monoclonal antibodies are generated endoplasmic reticulum and Golgi apparatus. The morphology of the plasma by a single B-cell clone, and they all recognize the same antigen. Antibody- cell reflects its main function of producing and secreting Ig molecules (19). secreting cells have been utilized to produce highly specific monoclonal antibodies that can be used for both therapeutic and diagnostic purposes (11). Plasma cells can be divided into two main groups, plasmablasts and long-lived (memory) plasma cells. Plasmablasts are short-lived (20) while long-lived Antibody-secreting cells do have a downside, which includes involvement in plasma cells can have a lifespan similar to long-lived memory B cells (21). In the pathogenesis of certain autoimmune diseases, including antinuclear/anti- this context, it has been previously demonstrated that long-lived plasma cells DNA antibodies in systemic lupus erythematous and anti-citrullinated protein can maintain antibody titers for long periods and in some instances throughout (i.e. proteins carrying the rare amino acid citrulline) antibodies in rheumatoid life (22). These long-lived plasma cells are found in the bone marrow but also arthritis. Patients with severe rheumatoid arthritis who have insufficient effect in the human intestine (23-25). on other disease-modifying antirheumatic drugs may be treated with anti- CD20 monoclonal antibodies that depletes B cells, such as rituximab (Mabthera®) (12). Antibody-secreting cells are also involved in multiple myeloma (13) and IgG4-related disease (14-17). 4 5 to T cells to activate them. The adaptive immune response can in turn both 1.2 IMMUNOGLOBULINS regulate and stimulate the innate immune system. Cytotoxic T cells that express CD8 on the cell surface identify and kill cells that synthesize aberrant peptides that are presented on major histocompatibility 1.2.1 PRODUCTION complex class I (MHC-I) molecules, which occur in virus-infected cells. As a Hematopoietic stem cells in the bone marrow can develop into B cells in an token of the importance of this defense mechanism, certain viruses can prevent antigen-independent process. After leaving the bone marrow, B cells will MHC-I molecules from reaching the surface of virus-infected cells and thus colonize secondary lymphoid organs and there, in an antigen-dependent hide from the cytotoxic T cells (i.e. immune evasion). However, when NK cells process, they can differentiate into memory B cells and antibody-producing detect cells with fewer MHC-I molecules than normal, they will kill the cell. plasma cells. The process is initiated after a mature B cell encounters and This is an example of how the innate and adaptive immune response interacts recognizes its cognate antigen. There are T cell-independent antigens, often to eliminate pathogens. The main function of CD4+ T helper cells is to polysaccharide antigens, that directly activate B cells, but most antigens are T stimulate and regulate other immune cells. T helper cells can for instance cell-dependent, which means that after antigen recognition, the B cell requires increase the functional capacity of phagocytic cells and stimulate B cells to cooperation with a T helper cell that recognizes the same antigen for final become more efficient at producing immunoglobulins (antibodies). activation (13, 18). After B cell activation, cell division and differentiation into memory B cells and plasma cells can occur. The humoral immunity is one of the main components of the adaptive immunity and immunoglobulins are important for providing immunity after B cells can produce both soluble and membrane-bound immunoglobulins infection/vaccination. Secreted immunoglobulins can neutralize pathogens and simultaneously. This is done by splicing at the mRNA level, so that the same act as effector molecules to stimulate other parts of the immune system to fight main structure can be combined with a hydrophilic short tail that provides a pathogens. The human body can produce antibodies with almost infinite soluble molecule, or with a partially hydrophobic longer tail that gives a specificity. membrane-bound molecule. B cells have mainly membrane-bound immunoglobulins (the main part of the B cell receptor) while plasma cells are After antigen recognition and activation of various B cells in the body, these specialized in producing and secreting immunoglobulins (19). Plasma cells are cells will produce antibodies that can recognize and bind to different antigens. larger than B cells and the morphology is characteristic with a small, The antibody response in the human body is thus polyclonal because different asymmetrically placed nucleus, large cytoplasm with abundant rough antibodies recognize different antigens. Monoclonal antibodies are generated endoplasmic reticulum and Golgi apparatus. The morphology of the plasma by a single B-cell clone, and they all recognize the same antigen. Antibody- cell reflects its main function of producing and secreting Ig molecules (19). secreting cells have been utilized to produce highly specific monoclonal antibodies that can be used for both therapeutic and diagnostic purposes (11). Plasma cells can be divided into two main groups, plasmablasts and long-lived (memory) plasma cells. Plasmablasts are short-lived (20) while long-lived Antibody-secreting cells do have a downside, which includes involvement in plasma cells can have a lifespan similar to long-lived memory B cells (21). In the pathogenesis of certain autoimmune diseases, including antinuclear/anti- this context, it has been previously demonstrated that long-lived plasma cells DNA antibodies in systemic lupus erythematous and anti-citrullinated protein can maintain antibody titers for long periods and in some instances throughout (i.e. proteins carrying the rare amino acid citrulline) antibodies in rheumatoid life (22). These long-lived plasma cells are found in the bone marrow but also arthritis. Patients with severe rheumatoid arthritis who have insufficient effect in the human intestine (23-25). on other disease-modifying antirheumatic drugs may be treated with anti- CD20 monoclonal antibodies that depletes B cells, such as rituximab (Mabthera®) (12). Antibody-secreting cells are also involved in multiple myeloma (13) and IgG4-related disease (14-17). 4 5 1.2.2 BASIC STRUCTURE Immunoglobulins (Ig) are large molecules with an approximate molecular weight of 150kD. These molecules have a basic Y-shaped structure consisting of two heavy and two light polypeptide chains. There are two types of light chains, lambda (λ) and kappa (κ), based on small polypeptide differences. An Ig molecule has identical light chains, meaning that each individual antibody has either lambda or kappa light chains (26). The heavy chains of an individual Ig molecule are also identical, and the chains are joined by two disulfide bonds. Each of the light chains is joined to one of the heavy chains by a single disulfide bond. Each light and heavy chain can be divided into a constant and a variable part. The variable heavy chain is often abbreviated VH, where V stands for variable and H for heavy chain. Correspondingly, the abbreviation for the variable part of the light chain is VL where L stands for light. The constant part of the heavy and light chains is abbreviated CH and CL respectively, where C stands for constant. The heavy and light chains are composed of a special type of protein domains that have a similar globular structure termed Ig fold or Ig domain (27). The structure is the result of a special folding of the protein in the tertiary structure. These Ig domains exist in several other molecules, the Ig superfamily, suggesting that such motifs have been useful protein modules during evolution (28). The basic structure of immunoglobulins is similar, but there is a large variation in the composition of antigen-binding sites, also termed paratopes, in order to enable binding to the large numbers of different antigens that the body encounters (26). Each B cell can only express a certain type of immunoglobulin and these identical immunoglobulins all have the same specificity for binding antigen. All daughter cells of the original B cell will inherit the same antibody specificity, which means that all plasma cells from the original B cell will secrete antibodies that target the inducing antigen. The paratope consists of the outer part of the variable heavy and light chains (VH + VL). Each individual Ig molecule has two identical paratopes because the two arms are composed of identical chains (26). The two arms of the Y- shaped Ig molecule are joined to the trunk by a flexible polypeptide chain termed the hinge region. The flexible hinge region allows the two antigen- binding arms to form different angles relative to each other, allowing binding of two different antigens if they are not too far apart. The maximum angle of the antibody arms must be able to cover the distance between the two antigens. Figure 1. B cell activation and differentiation into antibody-producing plasma cells. 6 7 1.2.2 BASIC STRUCTURE Immunoglobulins (Ig) are large molecules with an approximate molecular weight of 150kD. These molecules have a basic Y-shaped structure consisting of two heavy and two light polypeptide chains. There are two types of light chains, lambda (λ) and kappa (κ), based on small polypeptide differences. An Ig molecule has identical light chains, meaning that each individual antibody has either lambda or kappa light chains (26). The heavy chains of an individual Ig molecule are also identical, and the chains are joined by two disulfide bonds. Each of the light chains is joined to one of the heavy chains by a single disulfide bond. Each light and heavy chain can be divided into a constant and a variable part. The variable heavy chain is often abbreviated VH, where V stands for variable and H for heavy chain. Correspondingly, the abbreviation for the variable part of the light chain is VL where L stands for light. The constant part of the heavy and light chains is abbreviated CH and CL respectively, where C stands for constant. The heavy and light chains are composed of a special type of protein domains that have a similar globular structure termed Ig fold or Ig domain (27). The structure is the result of a special folding of the protein in the tertiary structure. These Ig domains exist in several other molecules, the Ig superfamily, suggesting that such motifs have been useful protein modules during evolution (28). The basic structure of immunoglobulins is similar, but there is a large variation in the composition of antigen-binding sites, also termed paratopes, in order to enable binding to the large numbers of different antigens that the body encounters (26). Each B cell can only express a certain type of immunoglobulin and these identical immunoglobulins all have the same specificity for binding antigen. All daughter cells of the original B cell will inherit the same antibody specificity, which means that all plasma cells from the original B cell will secrete antibodies that target the inducing antigen. The paratope consists of the outer part of the variable heavy and light chains (VH + VL). Each individual Ig molecule has two identical paratopes because the two arms are composed of identical chains (26). The two arms of the Y- shaped Ig molecule are joined to the trunk by a flexible polypeptide chain termed the hinge region. The flexible hinge region allows the two antigen- binding arms to form different angles relative to each other, allowing binding of two different antigens if they are not too far apart. The maximum angle of the antibody arms must be able to cover the distance between the two antigens. Figure 1. B cell activation and differentiation into antibody-producing plasma cells. 6 7 Figure 2. Immunoglobulins are composed of two light (L) chains and two heavy (H) chains. The variable parts of the light and heavy chains are abbreviated V and the constant parts C. The paratopes (i.e. antigen- binding sites) are binding to specific epitopes on antigens (i.e. antigenic determinants). Figure 3. Cleavage of an immunoglobulin molecule with papain or pepsin protease. Fragment crystallizable (Fc). Fragment antigen binding (Fab). 1.2.4 ISOTYPES Immunoglobulins are divided into different isotypes (antibody classes) based on differences in the constant region of the heavy chain (CH). There are five basic structures of CH named alpha (α), delta (δ), epsilon (ε), mu (µ) and gamma (γ). These five different CH correspond to the five antibody classes IgA (α), IgD (δ), IgE (ε), IgM (µ) and IgG (γ) (26). The variation between isotypes include differences in the amino acid sequence of the Ig domains and the 1.2.3 CLEAVAGE WITH PROTEASES number of Ig domains in the CH. IgA and IgG can be further divided into Immunoglobulins can be cleaved by proteolytic enzymes (proteases) into subclasses based on differences in the properties of the alpha and gamma functionally distinct fragments. This process has been used to determine which chains (29, 30). Alpha is further divided into the two subclasses IgA1 and part of the Ig molecule that is responsible for different functions. Cleavage of IgA2, while gamma contains the four subclasses, IgG1, IgG2, IgG3 and IgG4 Ig with papain results in three fragments of the molecule. Papain cleaves the (30). The different structures of the antibody classes reflect their diverse molecule above the disulfide bonds that bind the two arms that contain the biological functions with various effector mechanisms, antigenic determinants, paratopes. The two separated identical arms are named Fab fragments, which and biological half-life (26). The different antibody classes bind to their stands for fragment antigen binding. These fragments have monovalent respective Fc receptor on immune cells. antigen binding. The trunk of the Y-structured molecule has no antigen binding activity but is in intact Ig molecules important for interactions with cells and effector molecules. This part of the molecule is called Fc fragment, which stands for fragment crystallizable, because it has been observed that it easily forms crystalline structures. Pepsin cleaves the Ig molecule below the disulfide bonds that hold the two arms of the Y-shaped Ig molecule together. The two antigen-binding arms will thus be linked in a fragment and can bind antigens with both arms (divalent antigen binding). This fragment is called F(ab’)2 fragment and has the same antigen-binding properties as the original antibody, but since it does not Figure 4. The five antibody classes are based on differences in the constant region of the contain the Fc fragment, it cannot interact with cells and/or effector molecules. heavy chain (CH). The five basic structures of CH are named alpha (α), delta (δ), epsilon The Fc fragment will be cleaved in several smaller fragments by pepsin. (ε), mu (µ) and gamma (γ) and these five different CHs correspond to the five antibody classes IgA (α), IgD (δ), IgE (ε), IgM (µ) and IgG (γ). 8 9 Figure 2. Immunoglobulins are composed of two light (L) chains and two heavy (H) chains. The variable parts of the light and heavy chains are abbreviated V and the constant parts C. The paratopes (i.e. antigen- binding sites) are binding to specific epitopes on antigens (i.e. antigenic determinants). Figure 3. Cleavage of an immunoglobulin molecule with papain or pepsin protease. Fragment crystallizable (Fc). Fragment antigen binding (Fab). 1.2.4 ISOTYPES Immunoglobulins are divided into different isotypes (antibody classes) based on differences in the constant region of the heavy chain (CH). There are five basic structures of CH named alpha (α), delta (δ), epsilon (ε), mu (µ) and gamma (γ). These five different CH correspond to the five antibody classes IgA (α), IgD (δ), IgE (ε), IgM (µ) and IgG (γ) (26). The variation between isotypes include differences in the amino acid sequence of the Ig domains and the 1.2.3 CLEAVAGE WITH PROTEASES number of Ig domains in the CH. IgA and IgG can be further divided into Immunoglobulins can be cleaved by proteolytic enzymes (proteases) into subclasses based on differences in the properties of the alpha and gamma functionally distinct fragments. This process has been used to determine which chains (29, 30). Alpha is further divided into the two subclasses IgA1 and part of the Ig molecule that is responsible for different functions. Cleavage of IgA2, while gamma contains the four subclasses, IgG1, IgG2, IgG3 and IgG4 Ig with papain results in three fragments of the molecule. Papain cleaves the (30). The different structures of the antibody classes reflect their diverse molecule above the disulfide bonds that bind the two arms that contain the biological functions with various effector mechanisms, antigenic determinants, paratopes. The two separated identical arms are named Fab fragments, which and biological half-life (26). The different antibody classes bind to their stands for fragment antigen binding. These fragments have monovalent respective Fc receptor on immune cells. antigen binding. The trunk of the Y-structured molecule has no antigen binding activity but is in intact Ig molecules important for interactions with cells and effector molecules. This part of the molecule is called Fc fragment, which stands for fragment crystallizable, because it has been observed that it easily forms crystalline structures. Pepsin cleaves the Ig molecule below the disulfide bonds that hold the two arms of the Y-shaped Ig molecule together. The two antigen-binding arms will thus be linked in a fragment and can bind antigens with both arms (divalent antigen binding). This fragment is called F(ab’)2 fragment and has the same antigen-binding properties as the original antibody, but since it does not Figure 4. The five antibody classes are based on differences in the constant region of the contain the Fc fragment, it cannot interact with cells and/or effector molecules. heavy chain (CH). The five basic structures of CH are named alpha (α), delta (δ), epsilon The Fc fragment will be cleaved in several smaller fragments by pepsin. (ε), mu (µ) and gamma (γ) and these five different CHs correspond to the five antibody classes IgA (α), IgD (δ), IgE (ε), IgM (µ) and IgG (γ). 8 9 IgA IgM IgA is the predominant antibody class on mucous membranes, the second most Secreted IgM is pentameric or hexameric common antibody class in serum and has the highest production of all isotypes (36). The pentameric form consists of five in humans (31). IgA is the most important isotype for our mucosal defense and Y-shaped basic Ig structures and the can neutralize pathogens and microbial toxins in the gastrointestinal tract (32). hexameric form of six (36-38). IgM is In serum, IgA appears predominately in monomeric form but occurs on expressed early during the maturation of B mucosal surfaces mainly as a dimer (26, 31, 33). Secretory IgA in dimeric form cells and early in an immunological is composed of a secretory component and two Y-shaped basic IgA response to a pathogen. IgM can activate the immunoglobulin structures that are linked by a joint chain (31). IgA is complement cascade through the classical transmitted via breast milk and can therefore contribute to the defense against pathway and thus help to clear infections by infections in breastfeeding infants. antibody-dependent cellular phagocytosis and antibody-dependent cell cytotoxicity (38). Figure 6. A secreted hexameric IgM molecule. IgG Secreted IgG is monomeric and the major antibody class in serum followed by IgA and IgM (26). Many cells in the immune system have Fc-gamma receptors Figure 5. A secretory IgA molecule. on their surfaces and IgG is the predominant antibody class in secondary IgD immune responses (26). IgG has many effector mechanisms including neutralization, opsonization and activation of the complement system. IgG can As with IgM, IgD is expressed early on mature B cells as a major part of the B cross the placenta to the fetus and can thereby give the newborn a passively cell receptor (34). IgD is mainly found in membrane-bound form, although transferred immunity from the mother. The half-life of most IgG subtypes is small amounts of IgD can be detected in serum. The secreted IgD molecule has approximately 3 weeks (39, 40). The neonatal Fc receptor can prolong the half- a monomeric form. IgD can activate basophils and thereby induce the life of IgG molecules (35). production of antimicrobial peptides, inflammatory cytokines and B-cell activating factors (34). IgE 1.2.5 EFFECTOR MECHANISMS Antibodies have effector mechanisms to protect the body from being harmed Secreted IgE has a monomeric form. Through its Fc moiety, IgE can bind to by foreign substances such as microorganisms (41). The main effector Fc receptors on mast cells and basophilic granulocytes with very high affinity. mechanisms of antibodies are: This high affinity causes IgE molecules to be bound to Fc receptors of basophilic granulocytes even before any antigen is recognized by the cells. Neutralization When an antigen then binds to IgE, the cell can immediately secrete various mediators. IgE is often associated with allergies and hypersensitivity, but IgE Direct antibody neutralization is the process by which antibodies bind to is also involved in the immune protection against parasitic helminths (35). antigens, thereby reducing or inhibiting the biological activity of pathogens/microbial components (41). Antibody neutralization can thus prevent harmful effects of pathogens/microbes in the body. As an example, 10 11 IgA IgM IgA is the predominant antibody class on mucous membranes, the second most Secreted IgM is pentameric or hexameric common antibody class in serum and has the highest production of all isotypes (36). The pentameric form consists of five in humans (31). IgA is the most important isotype for our mucosal defense and Y-shaped basic Ig structures and the can neutralize pathogens and microbial toxins in the gastrointestinal tract (32). hexameric form of six (36-38). IgM is In serum, IgA appears predominately in monomeric form but occurs on expressed early during the maturation of B mucosal surfaces mainly as a dimer (26, 31, 33). Secretory IgA in dimeric form cells and early in an immunological is composed of a secretory component and two Y-shaped basic IgA response to a pathogen. IgM can activate the immunoglobulin structures that are linked by a joint chain (31). IgA is complement cascade through the classical transmitted via breast milk and can therefore contribute to the defense against pathway and thus help to clear infections by infections in breastfeeding infants. antibody-dependent cellular phagocytosis and antibody-dependent cell cytotoxicity (38). Figure 6. A secreted hexameric IgM molecule. IgG Secreted IgG is monomeric and the major antibody class in serum followed by IgA and IgM (26). Many cells in the immune system have Fc-gamma receptors Figure 5. A secretory IgA molecule. on their surfaces and IgG is the predominant antibody class in secondary IgD immune responses (26). IgG has many effector mechanisms including neutralization, opsonization and activation of the complement system. IgG can As with IgM, IgD is expressed early on mature B cells as a major part of the B cross the placenta to the fetus and can thereby give the newborn a passively cell receptor (34). IgD is mainly found in membrane-bound form, although transferred immunity from the mother. The half-life of most IgG subtypes is small amounts of IgD can be detected in serum. The secreted IgD molecule has approximately 3 weeks (39, 40). The neonatal Fc receptor can prolong the half- a monomeric form. IgD can activate basophils and thereby induce the life of IgG molecules (35). production of antimicrobial peptides, inflammatory cytokines and B-cell activating factors (34). IgE 1.2.5 EFFECTOR MECHANISMS Antibodies have effector mechanisms to protect the body from being harmed Secreted IgE has a monomeric form. Through its Fc moiety, IgE can bind to by foreign substances such as microorganisms (41). The main effector Fc receptors on mast cells and basophilic granulocytes with very high affinity. mechanisms of antibodies are: This high affinity causes IgE molecules to be bound to Fc receptors of basophilic granulocytes even before any antigen is recognized by the cells. Neutralization When an antigen then binds to IgE, the cell can immediately secrete various mediators. IgE is often associated with allergies and hypersensitivity, but IgE Direct antibody neutralization is the process by which antibodies bind to is also involved in the immune protection against parasitic helminths (35). antigens, thereby reducing or inhibiting the biological activity of pathogens/microbial components (41). Antibody neutralization can thus prevent harmful effects of pathogens/microbes in the body. As an example, 10 11 antibodies can bind to envelope proteins on viruses, thereby preventing viral Antibody-dependent enhancement binding and penetration into host cells. The antibodies will thus inhibit viral replication, further dissemination and progression of the viral disease (41). There are microorganisms that can exploit the Fc receptor and complement Antibodies can also neutralize toxins by binding to them, thereby inhibiting pathways to enhance the disease through antibody-mediated responses (45, the toxins from binding to cellular receptors. Many vaccines on the market 46). This is termed antibody-dependent enhancement (41, 45, 46). An example stimulate the production of neutralizing antibodies and thus protect against of this phenomenon is when antibody opsonization of dengue viruses enhances disease (42). viral infection. This process can occur in patients who are reinfected with another serotype of dengue virus than the one responsible for the previous Opsonization infection (47). The antibodies to the dengue serotype from the previous infection may enhance the entry of the current dengue virus into Opsonization is the process by which immune cells can recognize and target monocytes/macrophages, which can lead to increased viral replication, an foreign substances by binding to opsonins (e.g. IgM, IgG and complement increased inflammatory response and aggravation of the disease (41, 45, 47). component C1) that cover the substances, for antibody-dependent cell cytotoxicity and/or phagocytosis. The problem of antibody-dependent enhancement is also reflected in the difficulty of producing an effective and safe vaccine against dengue virus (48). Antibody-dependent cellular phagocytosis is the process by which pathogens The first licensed dengue vaccine, CYD-TDV or Dengvaxia® was in 2016 are coated with antibodies to promote phagocytosis. Fc receptors on recommended by the World Health Organization (WHO) for considered use in phagocytic cells can recognize and attach to antibody coated pathogens, highly endemic regions (49). enabling phagocytosis of the pathogen. The vaccine was later found to have different effects depending on the dengue Antibody-dependent cell cytotoxicity is the immune process in which certain serostatus of the vaccinated individual (50). For individuals with a previous immune cells with Fc receptors can recognize and kill cells that have dengue infection (seropositive individuals), the vaccine is safe and effective pathogenic antigens on their surface and are coated with antibodies. Antibody- against symptomatic dengue disease (50, 51). In contrast, seronegative dependent cellular phagocytosis and antibody-dependent cell cytotoxicity are individuals without a previous dengue infection, three years after the first effective mechanisms for the clearance of encapsulated bacteria, viruses, and vaccination and onwards, have an increased risk of developing severe dengue virus-infected cells (43). fever if they become infected with the dengue virus (50). These findings have led to the suspension of the CYD-TDV vaccination program in the Philippines, Complement activation and the WHO stated in its 2018 recommendation that: “Countries should consider introduction of the dengue vaccine CYD-TDV only if the IgM and IgG can both initiate a cascade of enzymatic reactions to activate the minimization of risk among seronegative individuals can be assured” (52). complement system (antibody-mediated complement activation) (38, 44) where the ultimate consequences are: • Lysis of pathogenic microorganisms. Complement factors 1.2.6 ANTIBODY-ANTIGEN INTERACTION can create a pore in the membrane of the microorganism that disturbs the osmotic balance, leading to the death of the The antigen-binding parts of the Ig molecule consist of amino acids (a.a.) in microorganism. groups, termed complementarity-determining regions (35). These a.a. are not • Coating of pathogens by complement factors for in line in the primary sequence of the polypeptide chain but are joined together opsonization. by the folding of the protein in the tertiary structure. The paratopes (antigen- • Stimulation of inflammation by affecting the permeability of binding sites), are located in the outermost part of the variable Ig domains. blood vessels. This leads to a greater influx of inflammatory Paratopes differ in their three-dimensional structure, charge, and hydrophobic cells and various immune defense molecules. properties. Which means that different antibodies can bind various structures on antigens. 12 13 antibodies can bind to envelope proteins on viruses, thereby preventing viral Antibody-dependent enhancement binding and penetration into host cells. The antibodies will thus inhibit viral replication, further dissemination and progression of the viral disease (41). There are microorganisms that can exploit the Fc receptor and complement Antibodies can also neutralize toxins by binding to them, thereby inhibiting pathways to enhance the disease through antibody-mediated responses (45, the toxins from binding to cellular receptors. Many vaccines on the market 46). This is termed antibody-dependent enhancement (41, 45, 46). An example stimulate the production of neutralizing antibodies and thus protect against of this phenomenon is when antibody opsonization of dengue viruses enhances disease (42). viral infection. This process can occur in patients who are reinfected with another serotype of dengue virus than the one responsible for the previous Opsonization infection (47). The antibodies to the dengue serotype from the previous infection may enhance the entry of the current dengue virus into Opsonization is the process by which immune cells can recognize and target monocytes/macrophages, which can lead to increased viral replication, an foreign substances by binding to opsonins (e.g. IgM, IgG and complement increased inflammatory response and aggravation of the disease (41, 45, 47). component C1) that cover the substances, for antibody-dependent cell cytotoxicity and/or phagocytosis. The problem of antibody-dependent enhancement is also reflected in the difficulty of producing an effective and safe vaccine against dengue virus (48). Antibody-dependent cellular phagocytosis is the process by which pathogens The first licensed dengue vaccine, CYD-TDV or Dengvaxia® was in 2016 are coated with antibodies to promote phagocytosis. Fc receptors on recommended by the World Health Organization (WHO) for considered use in phagocytic cells can recognize and attach to antibody coated pathogens, highly endemic regions (49). enabling phagocytosis of the pathogen. The vaccine was later found to have different effects depending on the dengue Antibody-dependent cell cytotoxicity is the immune process in which certain serostatus of the vaccinated individual (50). For individuals with a previous immune cells with Fc receptors can recognize and kill cells that have dengue infection (seropositive individuals), the vaccine is safe and effective pathogenic antigens on their surface and are coated with antibodies. Antibody- against symptomatic dengue disease (50, 51). In contrast, seronegative dependent cellular phagocytosis and antibody-dependent cell cytotoxicity are individuals without a previous dengue infection, three years after the first effective mechanisms for the clearance of encapsulated bacteria, viruses, and vaccination and onwards, have an increased risk of developing severe dengue virus-infected cells (43). fever if they become infected with the dengue virus (50). These findings have led to the suspension of the CYD-TDV vaccination program in the Philippines, Complement activation and the WHO stated in its 2018 recommendation that: “Countries should consider introduction of the dengue vaccine CYD-TDV only if the IgM and IgG can both initiate a cascade of enzymatic reactions to activate the minimization of risk among seronegative individuals can be assured” (52). complement system (antibody-mediated complement activation) (38, 44) where the ultimate consequences are: • Lysis of pathogenic microorganisms. Complement factors 1.2.6 ANTIBODY-ANTIGEN INTERACTION can create a pore in the membrane of the microorganism that disturbs the osmotic balance, leading to the death of the The antigen-binding parts of the Ig molecule consist of amino acids (a.a.) in microorganism. groups, termed complementarity-determining regions (35). These a.a. are not • Coating of pathogens by complement factors for in line in the primary sequence of the polypeptide chain but are joined together opsonization. by the folding of the protein in the tertiary structure. The paratopes (antigen- • Stimulation of inflammation by affecting the permeability of binding sites), are located in the outermost part of the variable Ig domains. blood vessels. This leads to a greater influx of inflammatory Paratopes differ in their three-dimensional structure, charge, and hydrophobic cells and various immune defense molecules. properties. Which means that different antibodies can bind various structures on antigens. 12 13 Antigens can consist of many different types of molecules, such as Epitopes can either be located on the surface of the protein, facing the liquid polysaccharides, lipids, nucleic acids, small molecules (e.g. trinitrophenol) phase, or be located inside the protein in hydrophobic parts. In the latter case, although proteins are probably the most biologically important antigens. The the epitope is termed cryptic because it is not exposed unless the protein is part of the antigen that the paratope binds to is termed antigenic determinant denatured. Cryptotopic epitopes in viruses are hidden from the immune or epitope. A polypeptide chain can contain various linear epitopes, and, after response until the virion dissociates. Viral cryptotopes can be relatively folding of the protein, several new non-linear or discontinuous epitopes conserved in genetically related viruses because such antigenic structures are (further explained below). part of the virus' internal structures. Consequently, such domains may share essential functions such as viral assembly and thus tend to vary less compared The bonds between antibodies and antigens are non-covalent and reversible, with proteins on the surface of viruses. formed by a combination of hydrogen bonds, hydrophobic interactions, electrostatic and van der Waals forces. That the antibody-antigen bonds are Affinity reversible means that they can alternate between binding and detaching. Antibodies recognizes epitopes in their native form. Epitopes can be classified The individual binding strength between a paratope and an epitope is termed into two main groups, continuous epitopes (often called linear epitopes), and affinity. The binding strength depends on the proximity of stereochemical fit discontinuous epitopes (often referred to as conformational epitopes). between the paratope and epitope, the size of the contact surface between them and on the distribution of charged and hydrophobic groups. The affinity Continuous epitopes consist of any sequential (linear) a.a. residues that can constant Ka can be affected by temperature, pH, and solvent. Ka can range from bind to a paratope. In contrast, discontinuous epitopes are composed of a.a. below 105 mol−1 to above 1012 mol−1 (41). The equilibrium dissociation residues which are separated in the primary sequence, but which come in constant (Kd) and the affinity are inversely related, which means the higher the proximity in the three-dimensional folded shape. Discontinuous epitopes are affinity, the lower the Kd. more common than continuous epitopes, and it is estimated that more than 90% of antibodies recognize such targets (53). It is possible to determine the affinity of monoclonal antibodies since these antibodies are homogeneous and only bind to a single epitope. In contrast, polyclonal antibodies are heterogeneous and will thus contain a mixture of antibodies that will bind to various epitopes with different affinity. Thus, it is only possible to measure an average affinity for polyclonal antibodies. Avidity Avidity is the measure of the total binding strength of the antibody-antigen binding after formation of reversible complexes. The avidity depends on the number of interacting paratopes and epitopes, the affinity of each paratope- epitope binding and the structural arrangement of the interacting parts. IgM molecules that are pentamers have ten paratopes and hexameric IgM has twelve paratopes (36, 37). This gives IgM relatively high avidity to bind antigens even though the affinity between each paratope and epitope is not so high. Figure 7. Figure showing a continuous (linear) epitope consisting of sequential amino acid residues in the primary sequence and a discontinuous (conformational) epitope where the amino acid residues are apart in the primary sequence but comes in proximity in the three-dimensional form. 14 15 Antigens can consist of many different types of molecules, such as Epitopes can either be located on the surface of the protein, facing the liquid polysaccharides, lipids, nucleic acids, small molecules (e.g. trinitrophenol) phase, or be located inside the protein in hydrophobic parts. In the latter case, although proteins are probably the most biologically important antigens. The the epitope is termed cryptic because it is not exposed unless the protein is part of the antigen that the paratope binds to is termed antigenic determinant denatured. Cryptotopic epitopes in viruses are hidden from the immune or epitope. A polypeptide chain can contain various linear epitopes, and, after response until the virion dissociates. Viral cryptotopes can be relatively folding of the protein, several new non-linear or discontinuous epitopes conserved in genetically related viruses because such antigenic structures are (further explained below). part of the virus' internal structures. Consequently, such domains may share essential functions such as viral assembly and thus tend to vary less compared The bonds between antibodies and antigens are non-covalent and reversible, with proteins on the surface of viruses. formed by a combination of hydrogen bonds, hydrophobic interactions, electrostatic and van der Waals forces. That the antibody-antigen bonds are Affinity reversible means that they can alternate between binding and detaching. Antibodies recognizes epitopes in their native form. Epitopes can be classified The individual binding strength between a paratope and an epitope is termed into two main groups, continuous epitopes (often called linear epitopes), and affinity. The binding strength depends on the proximity of stereochemical fit discontinuous epitopes (often referred to as conformational epitopes). between the paratope and epitope, the size of the contact surface between them and on the distribution of charged and hydrophobic groups. The affinity Continuous epitopes consist of any sequential (linear) a.a. residues that can constant Ka can be affected by temperature, pH, and solvent. Ka can range from bind to a paratope. In contrast, discontinuous epitopes are composed of a.a. below 105 mol−1 to above 1012 mol−1 (41). The equilibrium dissociation residues which are separated in the primary sequence, but which come in constant (Kd) and the affinity are inversely related, which means the higher the proximity in the three-dimensional folded shape. Discontinuous epitopes are affinity, the lower the Kd. more common than continuous epitopes, and it is estimated that more than 90% of antibodies recognize such targets (53). It is possible to determine the affinity of monoclonal antibodies since these antibodies are homogeneous and only bind to a single epitope. In contrast, polyclonal antibodies are heterogeneous and will thus contain a mixture of antibodies that will bind to various epitopes with different affinity. Thus, it is only possible to measure an average affinity for polyclonal antibodies. Avidity Avidity is the measure of the total binding strength of the antibody-antigen binding after formation of reversible complexes. The avidity depends on the number of interacting paratopes and epitopes, the affinity of each paratope- epitope binding and the structural arrangement of the interacting parts. IgM molecules that are pentamers have ten paratopes and hexameric IgM has twelve paratopes (36, 37). This gives IgM relatively high avidity to bind antigens even though the affinity between each paratope and epitope is not so high. Figure 7. Figure showing a continuous (linear) epitope consisting of sequential amino acid residues in the primary sequence and a discontinuous (conformational) epitope where the amino acid residues are apart in the primary sequence but comes in proximity in the three-dimensional form. 14 15 response are stimulated by special follicular dendritic cells (which present intact antigens in immune complexes) and T follicular helper cells to generate memory B cells and plasma cells (57-61). B cells will within the GCs proliferate extensively. Mutations will occur in the variable genes that will lead to diversified B-cells receptors. This process is termed somatic hypermutation (SHM) (13). B cells with receptors that have higher affinity for the antigen will be selected for survival and further proliferation. This process of generating B cells and plasma cells that produce high affinity antibodies is termed affinity maturation (13, 62). During proliferation, some of the daughter cells can undergo a class swish Figure 8. Hexameric IgM molecules have twelve paratopes compared with IgG molecules recombination (CSR). It is the process by which B cells rearrange their DNA that have two paratopes. This gives IgM molecules relatively high avidity to bind antigens in the constant part of the heavy chain (CH) to change it to another CH, thereby even though the affinity of each paratope-epitope binding generally is lower compared producing another antibody isotype (e.g. from IgM to IgA, IgE or IgG) with with IgG molecules. retained antigen specificity but with other effector functions (13, 63, 64). 1.2.7 ANTIBODY DIVERSITY 1.2.8 ANTIBODY RESPONSE TO VIRAL INFECTIONS It is possible to produce antibodies against virtually all foreign substances Many viruses can induce a potent and long-lasting antibody response (65, 66). provided that the substance is at least as large as required to form an epitope. To generate long-lived plasma cells that produce high affinity antibodies, B The number of different antibody specificities appears almost infinite. During cells require cooperation with their cognate T helper cells. Activation of T the development of B cells in the bone marrow, the genes encoding the variable helper cells is therefore essential for the development of a long-lasting and parts of the Ig molecule are built up randomly by somatic recombination from effective antibody response. The procedure in which professional antigen- hereditary DNA segments (54-56). presenting cells (APCs) take up antigen, process antigen into peptides and The three gene segments that encode the variable part of the heavy chain (V ) present these antigenic peptides on MHC class II molecules to T helper cells H are named variable (V), diverse (D) and joining (J). The variable part of the together with co-stimulatory signals to activate the helper T cells is therefore light chain is constructed by two gene segments V and J. The final V and V important. H L genes are constructed in a process termed the V(D)J recombination, which The surface of many viruses consists of one or a few proteins and is highly greatly contributes to Ig diversity (54-56). Each B cell produces only a specific organized with a repetitive structure. Such structures are unusual in the human type of Ig molecule. A certain B cell will survive if it recognizes a foreign body and thus both the innate and the adaptive immune system have evolved substance, while B cells that bind too strongly to the body’s own molecules or to recognize these structures as antigenic “danger signals” (67). These do not recognize foreign substances at all, will die. structures can thus be considered as pathogen-associated molecular patterns A mature B cell expresses IgM or IgD as its membrane-bound B cell receptor. (PAMPs) (67). These repetitive motifs and other danger signals, including viral B cells in secondary lymphoid tissue are activated by encountering their nucleic acids such as dsRNA, U-rich ssRNA and hypomethylated DNA can be cognate antigen, but most require stimuli from an activated T helper cell that detected by pattern-recognition receptors (PRRs) on cells in the innate immune recognizes the same antigen to begin proliferating in a process termed clonal system. This will lead to the induction of immune signals in the form of expansion. When B cells in B-cell follicles begin to proliferate, the follicle can inflammatory cytokines (including interferons), the recruitment of be transformed into a germinal center (GC). These GCs are distinct neutrophiles and the activation of professional APCs, in particular dendritic microanatomical parts in B-cell follicles where B cells during an immune cells in viral infections (65). The uptake and transport of viruses by dendritic 16 17 response are stimulated by special follicular dendritic cells (which present intact antigens in immune complexes) and T follicular helper cells to generate memory B cells and plasma cells (57-61). B cells will within the GCs proliferate extensively. Mutations will occur in the variable genes that will lead to diversified B-cells receptors. This process is termed somatic hypermutation (SHM) (13). B cells with receptors that have higher affinity for the antigen will be selected for survival and further proliferation. This process of generating B cells and plasma cells that produce high affinity antibodies is termed affinity maturation (13, 62). During proliferation, some of the daughter cells can undergo a class swish Figure 8. Hexameric IgM molecules have twelve paratopes compared with IgG molecules recombination (CSR). It is the process by which B cells rearrange their DNA that have two paratopes. This gives IgM molecules relatively high avidity to bind antigens in the constant part of the heavy chain (CH) to change it to another CH, thereby even though the affinity of each paratope-epitope binding generally is lower compared producing another antibody isotype (e.g. from IgM to IgA, IgE or IgG) with with IgG molecules. retained antigen specificity but with other effector functions (13, 63, 64). 1.2.7 ANTIBODY DIVERSITY 1.2.8 ANTIBODY RESPONSE TO VIRAL INFECTIONS It is possible to produce antibodies against virtually all foreign substances Many viruses can induce a potent and long-lasting antibody response (65, 66). provided that the substance is at least as large as required to form an epitope. To generate long-lived plasma cells that produce high affinity antibodies, B The number of different antibody specificities appears almost infinite. During cells require cooperation with their cognate T helper cells. Activation of T the development of B cells in the bone marrow, the genes encoding the variable helper cells is therefore essential for the development of a long-lasting and parts of the Ig molecule are built up randomly by somatic recombination from effective antibody response. The procedure in which professional antigen- hereditary DNA segments (54-56). presenting cells (APCs) take up antigen, process antigen into peptides and The three gene segments that encode the variable part of the heavy chain (V ) present these antigenic peptides on MHC class II molecules to T helper cells H are named variable (V), diverse (D) and joining (J). The variable part of the together with co-stimulatory signals to activate the helper T cells is therefore light chain is constructed by two gene segments V and J. The final V and V important. H L genes are constructed in a process termed the V(D)J recombination, which The surface of many viruses consists of one or a few proteins and is highly greatly contributes to Ig diversity (54-56). Each B cell produces only a specific organized with a repetitive structure. Such structures are unusual in the human type of Ig molecule. A certain B cell will survive if it recognizes a foreign body and thus both the innate and the adaptive immune system have evolved substance, while B cells that bind too strongly to the body’s own molecules or to recognize these structures as antigenic “danger signals” (67). These do not recognize foreign substances at all, will die. structures can thus be considered as pathogen-associated molecular patterns A mature B cell expresses IgM or IgD as its membrane-bound B cell receptor. (PAMPs) (67). These repetitive motifs and other danger signals, including viral B cells in secondary lymphoid tissue are activated by encountering their nucleic acids such as dsRNA, U-rich ssRNA and hypomethylated DNA can be cognate antigen, but most require stimuli from an activated T helper cell that detected by pattern-recognition receptors (PRRs) on cells in the innate immune recognizes the same antigen to begin proliferating in a process termed clonal system. This will lead to the induction of immune signals in the form of expansion. When B cells in B-cell follicles begin to proliferate, the follicle can inflammatory cytokines (including interferons), the recruitment of be transformed into a germinal center (GC). These GCs are distinct neutrophiles and the activation of professional APCs, in particular dendritic microanatomical parts in B-cell follicles where B cells during an immune cells in viral infections (65). The uptake and transport of viruses by dendritic 16 17 cells to lymph nodes are important for the activation of the adaptive immune In primary viral infections, IgM is often generated first and can sometimes system. already be detected within one to two weeks after the onset of symptoms (69, 70). The maximum IgM response occurs approximately three to six weeks after Dendritic cells are potent inducers of T cell activation where naïve T cells can the onset of symptoms and a decline is then seen over a period of a couple of differentiate into cytotoxic T cells and T helper cells. Effective priming of T months. Sometimes, however, the IgM response may persist longer. cells is further facilitated by the prolonged stimulation of virus-derived antigens, which often occurs during viral infections due to viral replication in IgG production usually begins a little later than IgM production. The IgG cells of the infected host (65, 66). Most B cells require the help of cognate T response is more long-lasting compared with the IgM response and generally helper cells for final activation. The B cell will present antigenic peptides on reaches its peak within four to twelve weeks after the onset of symptoms and MHC class II molecules for activated helper T cells. In response, the T helper persist for months or years (70). Some viruses such as herpesviruses, which cell will synthesize effector molecules, both cell-bound (mainly CD40L which remain latent in the body but also measles virus, can induce a lifelong IgG will bind to CD40 on the B-cell) and secreted molecules to activate the B-cell. response (22). Other viruses e.g. SARS-CoV-2 has a much faster decline in IgG levels after infection (71). Previous research suggests that many human viruses have a suitable size for passive transport through the lymphatic system without the need for cell- In connection with a reinfection/reactivation with the same virus, memory B mediated transport to reach lymph nodes where the virus in its native form can cells and long-lived plasma cells will be activated, leading to an early secretion interact with B cells (65). The highly repetitive surfaces of many viruses of high IgG levels. Upon reinfection/reactivation, IgM will in some cases be facilitate the cross-linking of B-cell receptors, which is a strong B-cell produced at detectable levels and in other cases not. Reactivation of VZV often activation signal. Cross-linking of B-cell receptors can also stimulate a T-cell- elicits a detectable anti-VZV IgM response (72, 73). The antibody response independent antigenic response with IgM production (65, 66). IgM can activate described above is a generalized description and it will differ depending on the the complement cascade through the classical pathway. pathogen and the individual's immunocompetence. Other effector molecules (pentraxins) produced in response to the viral infection can also activate the complement system. Complement factor C2 can bind to B-cell receptors and facilitate the stimulation of these receptors. IgM antibodies, together with complement, can, by binding to viruses, increase the uptake of these immune complexes by APCs, which is an important step in the priming process of T helper cells and facilitates the capture of virus particles in lymphoid organs (68). The increased uptake of these immune complexes by APCs can also lead to optimal display of viral antigens by specialized follicular dendritic cells in lymph nodes, which is important for the optimal development of the antibody response. Prior to infection/vaccination with a particular virus, there are no detectable Figure 9. A generalized depiction of the IgM and IgG antibody response during a primary antibodies against that virus. An exception is newborns who through the and a secondary infection. placenta may have received a passive transmission of the mother's IgG antibodies without being virus-infected themselves. An individual who lacks antibodies to a particular virus is called seronegative while an individual who has antibodies is called seropositive. The term seroconversion is used when a seronegative individual begins to produce detectable antibodies to a certain virus due to infection/vaccination and thus becomes seropositive. 18 19 cells to lymph nodes are important for the activation of the adaptive immune In primary viral infections, IgM is often generated first and can sometimes system. already be detected within one to two weeks after the onset of symptoms (69, 70). The maximum IgM response occurs approximately three to six weeks after Dendritic cells are potent inducers of T cell activation where naïve T cells can the onset of symptoms and a decline is then seen over a period of a couple of differentiate into cytotoxic T cells and T helper cells. Effective priming of T months. Sometimes, however, the IgM response may persist longer. cells is further facilitated by the prolonged stimulation of virus-derived antigens, which often occurs during viral infections due to viral replication in IgG production usually begins a little later than IgM production. The IgG cells of the infected host (65, 66). Most B cells require the help of cognate T response is more long-lasting compared with the IgM response and generally helper cells for final activation. The B cell will present antigenic peptides on reaches its peak within four to twelve weeks after the onset of symptoms and MHC class II molecules for activated helper T cells. In response, the T helper persist for months or years (70). Some viruses such as herpesviruses, which cell will synthesize effector molecules, both cell-bound (mainly CD40L which remain latent in the body but also measles virus, can induce a lifelong IgG will bind to CD40 on the B-cell) and secreted molecules to activate the B-cell. response (22). Other viruses e.g. SARS-CoV-2 has a much faster decline in IgG levels after infection (71). Previous research suggests that many human viruses have a suitable size for passive transport through the lymphatic system without the need for cell- In connection with a reinfection/reactivation with the same virus, memory B mediated transport to reach lymph nodes where the virus in its native form can cells and long-lived plasma cells will be activated, leading to an early secretion interact with B cells (65). The highly repetitive surfaces of many viruses of high IgG levels. Upon reinfection/reactivation, IgM will in some cases be facilitate the cross-linking of B-cell receptors, which is a strong B-cell produced at detectable levels and in other cases not. Reactivation of VZV often activation signal. Cross-linking of B-cell receptors can also stimulate a T-cell- elicits a detectable anti-VZV IgM response (72, 73). The antibody response independent antigenic response with IgM production (65, 66). IgM can activate described above is a generalized description and it will differ depending on the the complement cascade through the classical pathway. pathogen and the individual's immunocompetence. Other effector molecules (pentraxins) produced in response to the viral infection can also activate the complement system. Complement factor C2 can bind to B-cell receptors and facilitate the stimulation of these receptors. IgM antibodies, together with complement, can, by binding to viruses, increase the uptake of these immune complexes by APCs, which is an important step in the priming process of T helper cells and facilitates the capture of virus particles in lymphoid organs (68). The increased uptake of these immune complexes by APCs can also lead to optimal display of viral antigens by specialized follicular dendritic cells in lymph nodes, which is important for the optimal development of the antibody response. Prior to infection/vaccination with a particular virus, there are no detectable Figure 9. A generalized depiction of the IgM and IgG antibody response during a primary antibodies against that virus. An exception is newborns who through the and a secondary infection. placenta may have received a passive transmission of the mother's IgG antibodies without being virus-infected themselves. An individual who lacks antibodies to a particular virus is called seronegative while an individual who has antibodies is called seropositive. The term seroconversion is used when a seronegative individual begins to produce detectable antibodies to a certain virus due to infection/vaccination and thus becomes seropositive. 18 19 1.3 VIRUSES its entire length. The icosahedral symmetry consists of 20 identical triangular structures, which form an almost spherical structure. Complex capsids are Viruses exist everywhere where there is biological life, and viruses, especially capsids with structures other than helical or icosahedral, as seen for some bacteriophages that infect bacteria, are the most abundant biological entities on viruses, e.g. poxviruses. the planet (74). Viruses can infect all types of living organisms, e.g. plants, algae, fungi, archaea, bacteria, and all animals including humans. Viruses require living cells to produce new viruses and they lack a complete machinery for energy production and protein synthesis. Viruses must therefore use cellular systems to produce their components and for energy production. The whole virus particle, called virion, can thus be seen as a transport container for transferring the viral genome to host cells where the virus can induce changes in the cell so that the cell will start to produce new viruses. Viral genome Viruses can have DNA or RNA as genetic material. The genome can be double stranded (ds) or single stranded (ss). The viral nucleic acid encodes all virus- specific proteins, both structural proteins, enzymes, regulatory elements, and factors necessary for propagation. There are some general differences between DNA and RNA viruses. DNA viruses usually have longer genomes because Figure 10. The viral genome is surrounded by a protective protein shell called capsid. these viruses are genetically more stable compared with RNA viruses. RNA This figure shows the two common capsid symmetries in viruses, icosahedral and helical. viruses are more prone to mutate, giving them a more genetically unstable Viral envelope genome compared with DNA viruses. Most DNA viruses replicate their genome in the cell nucleus where the virus can use the cell machinery. In Some viruses also have a lipid envelope surrounding the capsid. The contrast, most RNA viruses replicate in the cytoplasm. substructure with the core and capsid in these viruses is termed nucleocapsid. The viral genome does not encode for the lipids in the envelope, instead the In the human genome, some viral genetic material remains from previous virus will acquire them from host cell membranes. The viral envelope will thus retroviruses (a group of viruses that can copy their RNA to DNA using a viral consist of a bilayer of phospholipids just like our cell membranes, but the reverse transcriptase) that during evolution have managed to integrate their integrated proteins are primarily encoded by the viral genome. Viruses will genetic material into our genome. Viruses' ability to introduce new genetic acquire envelopes from different host cell membranes, but it is virus-specific information into cells has made them useful tools in genetic engineering. exactly from where the acquisition will take place. Two examples of where the Viral capsid envelope is retrieved are the Golgi apparatus and the outer cell membrane. The viral genome is surrounded by a protective protein shell termed capsid. The viral envelope is protective and blocks water, chemicals, and enzymes The capsid has a rigid and durable structure, which protects the nucleic acid from entering the virus, but most envelopes are quite sensitive to detergents, from external influences such as changes in pH, temperature, and chemical drying and heat. An intact envelope is important for virus-cell interactions, e.g. composition. Inside the capsid, there is room for the viral genome and some for viral attachment and entry into host cells. Enveloped viruses are thus viral proteins. The capsid consists of many copies of a small number of viral generally easier to inactivate compared with naked viruses, which lack proteins, in a few cases only one type of protein. The structural forms of viral envelope. Few enveloped viruses can cope with the environment in the capsids are termed helical, icosahedral, and complex. The helical symmetry gastrointestinal tract. The viral capsid is generally quite resistant to variations consists of repeating protein subunits that coat the nucleic acid in a helix along in pH, temperature, and chemical composition in the environment. 20 21 1.3 VIRUSES its entire length. The icosahedral symmetry consists of 20 identical triangular structures, which form an almost spherical structure. Complex capsids are Viruses exist everywhere where there is biological life, and viruses, especially capsids with structures other than helical or icosahedral, as seen for some bacteriophages that infect bacteria, are the most abundant biological entities on viruses, e.g. poxviruses. the planet (74). Viruses can infect all types of living organisms, e.g. plants, algae, fungi, archaea, bacteria, and all animals including humans. Viruses require living cells to produce new viruses and they lack a complete machinery for energy production and protein synthesis. Viruses must therefore use cellular systems to produce their components and for energy production. The whole virus particle, called virion, can thus be seen as a transport container for transferring the viral genome to host cells where the virus can induce changes in the cell so that the cell will start to produce new viruses. Viral genome Viruses can have DNA or RNA as genetic material. The genome can be double stranded (ds) or single stranded (ss). The viral nucleic acid encodes all virus- specific proteins, both structural proteins, enzymes, regulatory elements, and factors necessary for propagation. There are some general differences between DNA and RNA viruses. DNA viruses usually have longer genomes because Figure 10. The viral genome is surrounded by a protective protein shell called capsid. these viruses are genetically more stable compared with RNA viruses. RNA This figure shows the two common capsid symmetries in viruses, icosahedral and helical. viruses are more prone to mutate, giving them a more genetically unstable Viral envelope genome compared with DNA viruses. Most DNA viruses replicate their genome in the cell nucleus where the virus can use the cell machinery. In Some viruses also have a lipid envelope surrounding the capsid. The contrast, most RNA viruses replicate in the cytoplasm. substructure with the core and capsid in these viruses is termed nucleocapsid. The viral genome does not encode for the lipids in the envelope, instead the In the human genome, some viral genetic material remains from previous virus will acquire them from host cell membranes. The viral envelope will thus retroviruses (a group of viruses that can copy their RNA to DNA using a viral consist of a bilayer of phospholipids just like our cell membranes, but the reverse transcriptase) that during evolution have managed to integrate their integrated proteins are primarily encoded by the viral genome. Viruses will genetic material into our genome. Viruses' ability to introduce new genetic acquire envelopes from different host cell membranes, but it is virus-specific information into cells has made them useful tools in genetic engineering. exactly from where the acquisition will take place. Two examples of where the Viral capsid envelope is retrieved are the Golgi apparatus and the outer cell membrane. The viral genome is surrounded by a protective protein shell termed capsid. The viral envelope is protective and blocks water, chemicals, and enzymes The capsid has a rigid and durable structure, which protects the nucleic acid from entering the virus, but most envelopes are quite sensitive to detergents, from external influences such as changes in pH, temperature, and chemical drying and heat. An intact envelope is important for virus-cell interactions, e.g. composition. Inside the capsid, there is room for the viral genome and some for viral attachment and entry into host cells. Enveloped viruses are thus viral proteins. The capsid consists of many copies of a small number of viral generally easier to inactivate compared with naked viruses, which lack proteins, in a few cases only one type of protein. The structural forms of viral envelope. Few enveloped viruses can cope with the environment in the capsids are termed helical, icosahedral, and complex. The helical symmetry gastrointestinal tract. The viral capsid is generally quite resistant to variations consists of repeating protein subunits that coat the nucleic acid in a helix along in pH, temperature, and chemical composition in the environment. 20 21 Viral glycoproteins much is still unknown when it comes to how various herpesviruses cause latent infection in host cells, as the area has not been sufficiently studied. Integrated in the envelope are viral proteins. Many of these envelope proteins are glycoproteins, which means that the proteins have carbohydrate side chains. These viral glycoproteins are sometimes called spike proteins. The protein moiety is encoded by the virus but the glycans reflect the host cell machinery of the infected cell. Viral glycoproteins are synthesized in the same way as cellular glycoproteins. The glycoproteins are transmembrane, and the outer part (the ectodomain) is often involved in attachment and viral entry into host cells. The internal domain is often important for viral assembly. These types of viral glycoproteins are important targets for antibodies because they are often present in relatively large amounts on the surface of the virus and often also on virus-infected cells. In naked viruses, the capsid proteins have similar functions to the envelope proteins for attachment and viral entry into host cells. Some naked viruses may have protruding proteins that originate from the capsid. 1.3.1 HERPESVIRIDAE The Herpesviridae family consists of more than 100 identified viruses that are known to infect a diverse range of species, e.g. birds, reptiles, and mammals including humans (75). Herpesviruses have a linear dsDNA genome of 125– 241 kilobase pairs (75). The genome contains 70–170 genes that encode viral proteins (76). The genome is genetically stable compared with the genome of Figure 11. Schematic illustration of a herpesvirus. many RNA viruses. The genome is densely packed within an icosahedral capsid. Surrounding the nucleocapsid is the tegument, which is an amorphous Herpesviruses have evolved to adapt to their respective hosts. Herpesvirus layer containing viral proteins. Herpesviruses are enveloped viruses with viral infections are common in all animal species investigated and the viruses are glycoproteins in the lipid bilayer. They have a spherical shape and measure spread around the globe. Many of the human herpesviruses can cause around 150–200 nm. asymptomatic primary infections in early childhood, while VZV is the human herpesvirus that most often causes symptoms in primary infection, and then in After primary infection, herpesviruses will establish a lifelong latent/persistent the form of chickenpox (varicella). Severe disease due to human herpesviruses infection with a subdued gene expression. Herpesviruses can later reactive and is seen mainly in immunocompromised individuals, fetuses, and newborns. start the production of new viruses, which can enable the spread to new hosts. It has been shown that certain herpesviruses such as herpes simplex virus Members of the Herpesviridae family are classified into three subfamilies (HSV) and varicella-zoster virus (VZV) can retain their latent genomes as based on biological characteristics: Alphaherpesvirinae (α), Betaherpesvirinae closed circular molecules (episomes) in the host cell nucleus (77). Studies have (β) and Gammaherpesvirinae (γ) (75, 80). All three subfamilies are represented demonstrated that human herpesviruses 6A and 6B (HHV-6A and HHV-6B) among the nine known herpesviruses that infect humans. and certain other herpesviruses, including Marek’s disease virus, can integrate their viral genome into the genome of the infected host cell (78, 79). However, 22 23 Viral glycoproteins much is still unknown when it comes to how various herpesviruses cause latent infection in host cells, as the area has not been sufficiently studied. Integrated in the envelope are viral proteins. Many of these envelope proteins are glycoproteins, which means that the proteins have carbohydrate side chains. These viral glycoproteins are sometimes called spike proteins. The protein moiety is encoded by the virus but the glycans reflect the host cell machinery of the infected cell. Viral glycoproteins are synthesized in the same way as cellular glycoproteins. The glycoproteins are transmembrane, and the outer part (the ectodomain) is often involved in attachment and viral entry into host cells. The internal domain is often important for viral assembly. These types of viral glycoproteins are important targets for antibodies because they are often present in relatively large amounts on the surface of the virus and often also on virus-infected cells. In naked viruses, the capsid proteins have similar functions to the envelope proteins for attachment and viral entry into host cells. Some naked viruses may have protruding proteins that originate from the capsid. 1.3.1 HERPESVIRIDAE The Herpesviridae family consists of more than 100 identified viruses that are known to infect a diverse range of species, e.g. birds, reptiles, and mammals including humans (75). Herpesviruses have a linear dsDNA genome of 125– 241 kilobase pairs (75). The genome contains 70–170 genes that encode viral proteins (76). The genome is genetically stable compared with the genome of Figure 11. Schematic illustration of a herpesvirus. many RNA viruses. The genome is densely packed within an icosahedral capsid. Surrounding the nucleocapsid is the tegument, which is an amorphous Herpesviruses have evolved to adapt to their respective hosts. Herpesvirus layer containing viral proteins. Herpesviruses are enveloped viruses with viral infections are common in all animal species investigated and the viruses are glycoproteins in the lipid bilayer. They have a spherical shape and measure spread around the globe. Many of the human herpesviruses can cause around 150–200 nm. asymptomatic primary infections in early childhood, while VZV is the human herpesvirus that most often causes symptoms in primary infection, and then in After primary infection, herpesviruses will establish a lifelong latent/persistent the form of chickenpox (varicella). Severe disease due to human herpesviruses infection with a subdued gene expression. Herpesviruses can later reactive and is seen mainly in immunocompromised individuals, fetuses, and newborns. start the production of new viruses, which can enable the spread to new hosts. It has been shown that certain herpesviruses such as herpes simplex virus Members of the Herpesviridae family are classified into three subfamilies (HSV) and varicella-zoster virus (VZV) can retain their latent genomes as based on biological characteristics: Alphaherpesvirinae (α), Betaherpesvirinae closed circular molecules (episomes) in the host cell nucleus (77). Studies have (β) and Gammaherpesvirinae (γ) (75, 80). All three subfamilies are represented demonstrated that human herpesviruses 6A and 6B (HHV-6A and HHV-6B) among the nine known herpesviruses that infect humans. and certain other herpesviruses, including Marek’s disease virus, can integrate their viral genome into the genome of the infected host cell (78, 79). However, 22 23 1.3.2 ALPHAHERPESVIRINAE VZV has the smallest genome of the known human herpesviruses (88). The • Herpes simplex virus type 1 (HSV-1, HHV-1) linear dsDNA genome has approximately 125,000 base pairs (bp) and consists • Herpes simplex virus type 2 (HSV-2, HHV-2) of at least 70 genes, all but 6 of which have homologues in HSV (88, 89). The • Varicella-zoster virus (VZV, HHV-3) genome has a unique long coding region and a unique short coding region (88, 89). Herpes simplex virus type 1 and 2 The genome is encased in an icosahedral capsid. The nucleocapsid is in turn HSV-1 and HSV-2 are ubiquitous around the globe but with geographic surrounded by tegument. Outside the tegument is the lipid envelope, which variation in seroprevalence (81). The estimated global prevalence of HSV-1 is contains glycoproteins encoded by the viral genome as well as cellular two-thirds of all people aged 0–49 years (81). In a study regarding HSV-1 in membrane proteins. Swedish adults, the seroprevalence was 79.4% for people who were 35–95 years old (82). The estimated global prevalence of HSV-2 is 13% for people Glycoproteins aged 15–49 years, with the highest seroprevalence in Africa (81). In Sweden, the HSV-2 seroprevalence in adults aged 35–95 years was measured at 12.9% The VZV genome encodes the glycoproteins gB, gC, gE, gH, gK, gI, gL, gM, (82). gN (89-91). The glycoproteins incorporated into the viral envelope are important for attachment and virus entry into host cells (90, 91). These Both viruses infect mucoepithelial cells as their primary target and establish structural components also have other important functions for the pathogenesis latency in sensory neurons. They both cause herpetic blisters; HSV-1 can and replication of the virus, such as viral assembly and egress (89-93). induce both oral and genital lesions while HSV-2 mainly causes genital lesions. Both viruses can be reactivated after latency and cause new The glycoproteins gB, gH , gL, gM, and gN are core proteins that are conserved mucoepithelial lesions. Complications of the primary or reactivated infections among the three subfamilies of the herpesviruses (94). Based on the homology include herpesvirus infections of the central nervous system (CNS), keratitis, with HSVgB, it is likely that VZVgB has similar functions and is critical for hepatitis, pancreatitis, and pneumonitis. viral entry into host cells (89, 95). VZVgE is essential for VZV replication and infectivity in T cells and skin (93, 96-99). VZVgE (open-reading frame 68) is the most produced glycoprotein in VZV- 1.3.3 VARICELLA-ZOSTER VIRUS infected cells and the most common viral protein integrated into the viral envelope (100). Both VZVgE and VZVgB are targets for cytotoxic T cells and Varicella-zoster virus (VZV) is, as described above, a member of the can induce the production of neutralizing antibodies (101, 102). VZVgE is Herpesviridae family and belongs to the Alfaherpesvirinae subfamily. The probably the most immunogenic VZV glycoprotein (100, 102-105). genus of the virus is Varicellovirus and the species is Human alphaherpesvirus 3. VZV is described in more detail because the virus is a central topic in the Epidemiology thesis. VZV infections occur only naturally in humans and the virus has no animal The virus reservoir. VZV infections are widespread around the world, especially in temperate climates where children become infected at a young age. In the There is one serotype of VZV, seven established clades (1–6 and 9) and two absence of vaccination programs, it is estimated that over 90% of adolescents additional putative clades awaiting confirmation (83-87). VZV causes in temperate countries are infected with the virus. A recent Swedish study chickenpox at primary infection, which primarily affects young children. VZV showed a seroprevalence of 66.7% in 5-year-old children and 91.5% in 12- can later reactivate from latency to cause herpes zoster (shingles). VZV mainly year-olds (106). The seroprevalence in Swedish adults aged 35–95 years was infects epithelial cells, T cells and neurons where the virus establishes latency. measured at 97.9% (82). 24 25 1.3.2 ALPHAHERPESVIRINAE VZV has the smallest genome of the known human herpesviruses (88). The • Herpes simplex virus type 1 (HSV-1, HHV-1) linear dsDNA genome has approximately 125,000 base pairs (bp) and consists • Herpes simplex virus type 2 (HSV-2, HHV-2) of at least 70 genes, all but 6 of which have homologues in HSV (88, 89). The • Varicella-zoster virus (VZV, HHV-3) genome has a unique long coding region and a unique short coding region (88, 89). Herpes simplex virus type 1 and 2 The genome is encased in an icosahedral capsid. The nucleocapsid is in turn HSV-1 and HSV-2 are ubiquitous around the globe but with geographic surrounded by tegument. Outside the tegument is the lipid envelope, which variation in seroprevalence (81). The estimated global prevalence of HSV-1 is contains glycoproteins encoded by the viral genome as well as cellular two-thirds of all people aged 0–49 years (81). In a study regarding HSV-1 in membrane proteins. Swedish adults, the seroprevalence was 79.4% for people who were 35–95 years old (82). The estimated global prevalence of HSV-2 is 13% for people Glycoproteins aged 15–49 years, with the highest seroprevalence in Africa (81). In Sweden, the HSV-2 seroprevalence in adults aged 35–95 years was measured at 12.9% The VZV genome encodes the glycoproteins gB, gC, gE, gH, gK, gI, gL, gM, (82). gN (89-91). The glycoproteins incorporated into the viral envelope are important for attachment and virus entry into host cells (90, 91). These Both viruses infect mucoepithelial cells as their primary target and establish structural components also have other important functions for the pathogenesis latency in sensory neurons. They both cause herpetic blisters; HSV-1 can and replication of the virus, such as viral assembly and egress (89-93). induce both oral and genital lesions while HSV-2 mainly causes genital lesions. Both viruses can be reactivated after latency and cause new The glycoproteins gB, gH , gL, gM, and gN are core proteins that are conserved mucoepithelial lesions. Complications of the primary or reactivated infections among the three subfamilies of the herpesviruses (94). Based on the homology include herpesvirus infections of the central nervous system (CNS), keratitis, with HSVgB, it is likely that VZVgB has similar functions and is critical for hepatitis, pancreatitis, and pneumonitis. viral entry into host cells (89, 95). VZVgE is essential for VZV replication and infectivity in T cells and skin (93, 96-99). VZVgE (open-reading frame 68) is the most produced glycoprotein in VZV- 1.3.3 VARICELLA-ZOSTER VIRUS infected cells and the most common viral protein integrated into the viral envelope (100). Both VZVgE and VZVgB are targets for cytotoxic T cells and Varicella-zoster virus (VZV) is, as described above, a member of the can induce the production of neutralizing antibodies (101, 102). VZVgE is Herpesviridae family and belongs to the Alfaherpesvirinae subfamily. The probably the most immunogenic VZV glycoprotein (100, 102-105). genus of the virus is Varicellovirus and the species is Human alphaherpesvirus 3. VZV is described in more detail because the virus is a central topic in the Epidemiology thesis. VZV infections occur only naturally in humans and the virus has no animal The virus reservoir. VZV infections are widespread around the world, especially in temperate climates where children become infected at a young age. In the There is one serotype of VZV, seven established clades (1–6 and 9) and two absence of vaccination programs, it is estimated that over 90% of adolescents additional putative clades awaiting confirmation (83-87). VZV causes in temperate countries are infected with the virus. A recent Swedish study chickenpox at primary infection, which primarily affects young children. VZV showed a seroprevalence of 66.7% in 5-year-old children and 91.5% in 12- can later reactivate from latency to cause herpes zoster (shingles). VZV mainly year-olds (106). The seroprevalence in Swedish adults aged 35–95 years was infects epithelial cells, T cells and neurons where the virus establishes latency. measured at 97.9% (82). 24 25 Transmission (112-115). During latency, there is limited gene expression and replication (107). The infection is mainly transmitted by aerosols from vesicular fluid from the rash and is therefore highly contagious (107). The virus can also be spread Immunity from direct contact with the rash and possibly via infected respiratory tract secretions (107). The infection is transmitted mainly from individuals with Primary VZV infection/vaccination will lead to the production of VZV- chickenpox rash and to a lesser extent from individuals with zoster rash. specific antibodies and cell-mediated immune responses that will induce Varicella is thought to be contagious from one to two days before the onset of immunity to chickenpox. However, both clinical and subclinical reinfection the rash, but little conclusive evidence is available in the literature (108). The can occur after natural infection/vaccination (83, 86, 116, 117). The actual estimated incubation period is 14–16 days with an interval of 10–21 days. The frequency of reinfections is unknown, but several studies suggest that disease is contagious until all the lesions have developed into crusts, which reinfections and establishment of latency by reinfecting strains are more normally takes about 5–7 days. Immunocompromised individuals can be common than previously thought and that recombination occurs (83, 86, 116). contagious for an extended period. If the exposure is sufficient, active herpes zoster can be transmitted to a VZV-susceptible individual and cause Reactivation chickenpox. The main component of the defense against VZV is the cell-dependent Primary VZV infection immunity and when this immunity to VZV wanes, the virus can reactive and cause herpes zoster, but reactivation can also be subclinical (118). Receiving The virus infects via the oral cavity and upper respiratory tract where the virus immunosuppressive drugs as well as being immunocompromised for other will replicate in the mucosal epithelium and spread to regional lymph nodes reasons are risk factors for VZV reactivation, along with old age as cell- where T cells can be infected. This is followed by a low-grade T-cell- mediated immunity decreases with age (119). associated viremia with the spread of the virus to the skin and possibly other organs where further replication will occur. Common symptoms in infected Herpes zoster causes a rash in the dermatome that is innervated by the ganglion individuals are fever, malaise, and the characteristic, generalized rash with from which the virus is reactivated. Reactivation of VZV will in many cases small itchy blisters over the body, especially over the upper part of the body be complicated by neurological pain. Some individuals will experience and in the facial region (107). Chickenpox is in most cases a self-limiting persistent pain, termed postherpetic neuralgia, for months or even years after disease, but several complications have been described (107). These reactivation (120). Other complications that can occur are encephalitis, manifestations include bacterial superinfection of skin, lungs, bones and blood, meningitis, VZV facial palsy (Ramsay Hunt Syndrome), myelitis, VZV involvement of CNS such as cerebellar ataxia, meningitis, myelitis and vasculopathy, ocular disorders (keratitis and retinopathy), gastrointestinal encephalitis, and hemorrhagic/ischemic conditions e.g. stroke and even death disorders e.g. ulcers, hepatitis and pancreatitis and secondary bacterial (107, 109). infections of the skin, pneumonia and sepsis (107, 121-125). For adult patients suffering from CNS involvement following herpes zoster, sequelae with High-risk groups that are vulnerable for the development of severe disease and neurological complications are common (121, 126). Reactivations with complications include immunocompromised individuals, pregnant women, complications can occur without vesicles; this is termed zoster sine herpete and infants under one year of age (107, 110). Adults are also at greater risk of (127). developing more severe illness compared with children (106, 107, 111). Although individuals from these high-risk groups are more likely to develop Treatment severe complications, VZV infections are so common that the incidence of these manifestations will also be substantial among non-risk groups (106). Antiviral drugs that inhibit VZV replication can be used to treat VZV-infected After primary infection, the virus establish latency in neurons in peripheral individuals with severe disease and individuals who are considered at risk of ganglia, i.e. dorsal root ganglia, cranial nerve ganglia and autonomic ganglia such development. The nucleoside analogue acyclovir and its prodrug valaciclovir can be used. Another option is famiciclovir, which is a prodrug 26 27 Transmission (112-115). During latency, there is limited gene expression and replication (107). The infection is mainly transmitted by aerosols from vesicular fluid from the rash and is therefore highly contagious (107). The virus can also be spread Immunity from direct contact with the rash and possibly via infected respiratory tract secretions (107). The infection is transmitted mainly from individuals with Primary VZV infection/vaccination will lead to the production of VZV- chickenpox rash and to a lesser extent from individuals with zoster rash. specific antibodies and cell-mediated immune responses that will induce Varicella is thought to be contagious from one to two days before the onset of immunity to chickenpox. However, both clinical and subclinical reinfection the rash, but little conclusive evidence is available in the literature (108). The can occur after natural infection/vaccination (83, 86, 116, 117). The actual estimated incubation period is 14–16 days with an interval of 10–21 days. The frequency of reinfections is unknown, but several studies suggest that disease is contagious until all the lesions have developed into crusts, which reinfections and establishment of latency by reinfecting strains are more normally takes about 5–7 days. Immunocompromised individuals can be common than previously thought and that recombination occurs (83, 86, 116). contagious for an extended period. If the exposure is sufficient, active herpes zoster can be transmitted to a VZV-susceptible individual and cause Reactivation chickenpox. The main component of the defense against VZV is the cell-dependent Primary VZV infection immunity and when this immunity to VZV wanes, the virus can reactive and cause herpes zoster, but reactivation can also be subclinical (118). Receiving The virus infects via the oral cavity and upper respiratory tract where the virus immunosuppressive drugs as well as being immunocompromised for other will replicate in the mucosal epithelium and spread to regional lymph nodes reasons are risk factors for VZV reactivation, along with old age as cell- where T cells can be infected. This is followed by a low-grade T-cell- mediated immunity decreases with age (119). associated viremia with the spread of the virus to the skin and possibly other organs where further replication will occur. Common symptoms in infected Herpes zoster causes a rash in the dermatome that is innervated by the ganglion individuals are fever, malaise, and the characteristic, generalized rash with from which the virus is reactivated. Reactivation of VZV will in many cases small itchy blisters over the body, especially over the upper part of the body be complicated by neurological pain. Some individuals will experience and in the facial region (107). Chickenpox is in most cases a self-limiting persistent pain, termed postherpetic neuralgia, for months or even years after disease, but several complications have been described (107). These reactivation (120). Other complications that can occur are encephalitis, manifestations include bacterial superinfection of skin, lungs, bones and blood, meningitis, VZV facial palsy (Ramsay Hunt Syndrome), myelitis, VZV involvement of CNS such as cerebellar ataxia, meningitis, myelitis and vasculopathy, ocular disorders (keratitis and retinopathy), gastrointestinal encephalitis, and hemorrhagic/ischemic conditions e.g. stroke and even death disorders e.g. ulcers, hepatitis and pancreatitis and secondary bacterial (107, 109). infections of the skin, pneumonia and sepsis (107, 121-125). For adult patients suffering from CNS involvement following herpes zoster, sequelae with High-risk groups that are vulnerable for the development of severe disease and neurological complications are common (121, 126). Reactivations with complications include immunocompromised individuals, pregnant women, complications can occur without vesicles; this is termed zoster sine herpete and infants under one year of age (107, 110). Adults are also at greater risk of (127). developing more severe illness compared with children (106, 107, 111). Although individuals from these high-risk groups are more likely to develop Treatment severe complications, VZV infections are so common that the incidence of these manifestations will also be substantial among non-risk groups (106). Antiviral drugs that inhibit VZV replication can be used to treat VZV-infected After primary infection, the virus establish latency in neurons in peripheral individuals with severe disease and individuals who are considered at risk of ganglia, i.e. dorsal root ganglia, cranial nerve ganglia and autonomic ganglia such development. The nucleoside analogue acyclovir and its prodrug valaciclovir can be used. Another option is famiciclovir, which is a prodrug 26 27 that will be metabolized by the liver to penciclovir which has antiviral effects DNA cannot be detected. Serological methods for the detection of anti-VZV (128). Treatment should be started early after the first symptoms to be IgM and IgG in CSF and in acute and convalescence serum samples can be effective, but treatment is always recommended in Sweden for patients with used as a complement to PCR diagnostics (152-154). Serological methods are severe VZV disease. Treatment can reduce virus excretion, shorten the course also important for controlling immunity after infection/vaccination. of the disease, and reduce the risk of complications. VZV serology Vaccines In one study, both serum IgM and IgG against VZV were detected within seven There are effective and safe attenuated varicella vaccines, all of which are days after disease onset (69). A study of the antibody response in seronegative based on the VZV Oka strain (129-132). Varicella vaccines can be given in children vaccinated against VZV showed that 40% had seroconverted two single or double dose schedules and are part of the child immunization weeks after vaccination and that 97% demonstrated detectable anti-VZV programs of several countries, where the vaccine has significantly reduced the antibodies six weeks after vaccination (155). Another study showed that burden of disease (133-135). VZV vaccine is not yet part of the child antibodies to VZV in CSF could be detected in 37% of patients with a VZV immunization program in Sweden but can be obtained on the Swedish market. infection in the CNS after an interval of seven days from onset of symptoms (151). The attenuated Oka strain has also been used to produce live herpes zoster vaccine (Zostavax®) but with a much higher concentration of virus compared VZV, HSV-1 and HSV-2 are, as previously described, genetically related with the chickenpox vaccines (131). The Zostavax® vaccine is given in a viruses which all belong to the subfamily of alphaherpesviruses. They have single dose and has been on the market in Sweden since 2013. Recently, on the homologous proteins and previous studies indicate that the viruses share market since 2020, there is an adjuvanted recombinant vaccine based on the common epitopes on gB (156, 157). These cross-reactive epitopes may give highly immunogenic VZVgE (Shingrix®) (136-139). In contrast to the herpes rise to cross-reactive antibodies (157-163). zoster vaccine based on the attenuated Oka strain, this vaccine can be administered to immunocompromised individuals and induces a more robust The fluorescent antibody to membrane antigen (FAMA) method is considered immune response with a longer protection period, even in elderly individuals one of the most sensitive methods for detecting anti-VZV antibodies, but the (140-142). method is laborious, cannot be automated, requires experience in handling VZV, and interpretation is subjective (164, 165). The method is thus not Viral diagnosis suitable for testing large amounts of sera in routine analysis. Time-resolved fluorescence immunoassay is a sensitive method that uses purified whole cell Chickenpox and herpes zoster can often be diagnosed clinically except in the antigen extract to detect anti-VZV antibodies, but there are enzyme case of unusual disease presentations such as infection in the absence of rash immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA) methods and atypical rash. For example, CNS manifestations often occur in the absence that are more user-friendly and robust (166, 167). of rash (122, 143-145). Laboratory tests are often necessary to diagnose atypical VZV cases and to link complications to VZV infections. Polymerase VZV serological assays mainly use VZV-infected cell lysates as antigens to chain reaction (PCR) is the preferred diagnostic method and analyses can be detect VZV-specific antibodies, but there are some methods based on performed on sample materials such as secretion from the vesicles, serum, glycoproteins (166, 168-171). However, whether the serological assays use CSF, and saliva (121, 143, 145-150). VZV-infected cells, glycoproteins, or a mixture of viral proteins from VZV- infected cell lysates, most of them contain VZVgB, which may lead to cross- PCR can be used to analyze and quantify VZV DNA during the acute phase reactivity that can cause diagnostic problems. but the viral load in CSF will gradually decrease during the disease. Detection of VZV DNA in CSF has in studies been mainly possible during the first seven days of disease (122, 151). Thus, the time window for detecting VZV DNA by PCR is limited. Diagnostic difficulties can arise in atypical cases when VZV 28 29 that will be metabolized by the liver to penciclovir which has antiviral effects DNA cannot be detected. Serological methods for the detection of anti-VZV (128). Treatment should be started early after the first symptoms to be IgM and IgG in CSF and in acute and convalescence serum samples can be effective, but treatment is always recommended in Sweden for patients with used as a complement to PCR diagnostics (152-154). Serological methods are severe VZV disease. Treatment can reduce virus excretion, shorten the course also important for controlling immunity after infection/vaccination. of the disease, and reduce the risk of complications. VZV serology Vaccines In one study, both serum IgM and IgG against VZV were detected within seven There are effective and safe attenuated varicella vaccines, all of which are days after disease onset (69). A study of the antibody response in seronegative based on the VZV Oka strain (129-132). Varicella vaccines can be given in children vaccinated against VZV showed that 40% had seroconverted two single or double dose schedules and are part of the child immunization weeks after vaccination and that 97% demonstrated detectable anti-VZV programs of several countries, where the vaccine has significantly reduced the antibodies six weeks after vaccination (155). Another study showed that burden of disease (133-135). VZV vaccine is not yet part of the child antibodies to VZV in CSF could be detected in 37% of patients with a VZV immunization program in Sweden but can be obtained on the Swedish market. infection in the CNS after an interval of seven days from onset of symptoms (151). The attenuated Oka strain has also been used to produce live herpes zoster vaccine (Zostavax®) but with a much higher concentration of virus compared VZV, HSV-1 and HSV-2 are, as previously described, genetically related with the chickenpox vaccines (131). The Zostavax® vaccine is given in a viruses which all belong to the subfamily of alphaherpesviruses. They have single dose and has been on the market in Sweden since 2013. Recently, on the homologous proteins and previous studies indicate that the viruses share market since 2020, there is an adjuvanted recombinant vaccine based on the common epitopes on gB (156, 157). These cross-reactive epitopes may give highly immunogenic VZVgE (Shingrix®) (136-139). In contrast to the herpes rise to cross-reactive antibodies (157-163). zoster vaccine based on the attenuated Oka strain, this vaccine can be administered to immunocompromised individuals and induces a more robust The fluorescent antibody to membrane antigen (FAMA) method is considered immune response with a longer protection period, even in elderly individuals one of the most sensitive methods for detecting anti-VZV antibodies, but the (140-142). method is laborious, cannot be automated, requires experience in handling VZV, and interpretation is subjective (164, 165). The method is thus not Viral diagnosis suitable for testing large amounts of sera in routine analysis. Time-resolved fluorescence immunoassay is a sensitive method that uses purified whole cell Chickenpox and herpes zoster can often be diagnosed clinically except in the antigen extract to detect anti-VZV antibodies, but there are enzyme case of unusual disease presentations such as infection in the absence of rash immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA) methods and atypical rash. For example, CNS manifestations often occur in the absence that are more user-friendly and robust (166, 167). of rash (122, 143-145). Laboratory tests are often necessary to diagnose atypical VZV cases and to link complications to VZV infections. Polymerase VZV serological assays mainly use VZV-infected cell lysates as antigens to chain reaction (PCR) is the preferred diagnostic method and analyses can be detect VZV-specific antibodies, but there are some methods based on performed on sample materials such as secretion from the vesicles, serum, glycoproteins (166, 168-171). However, whether the serological assays use CSF, and saliva (121, 143, 145-150). VZV-infected cells, glycoproteins, or a mixture of viral proteins from VZV- infected cell lysates, most of them contain VZVgB, which may lead to cross- PCR can be used to analyze and quantify VZV DNA during the acute phase reactivity that can cause diagnostic problems. but the viral load in CSF will gradually decrease during the disease. Detection of VZV DNA in CSF has in studies been mainly possible during the first seven days of disease (122, 151). Thus, the time window for detecting VZV DNA by PCR is limited. Diagnostic difficulties can arise in atypical cases when VZV 28 29 1.3.4 BETAHERPESVIRINAE HHV-6A, HHV-6B and HHV-7 • Cytomegalovirus (CMV, HHV-5) Human herpesvirus 6A (HHV-6A) HHV-6A and HHV-6B where previously considered to be the same virus • Human herpesvirus 6B (HHV-6B) species (184, 185). Although closely related, HHV-6A and HHV-6B have • Human herpesvirus 7 (HHV-7) genetic, biological, and immunological characteristics that are so different that • they were recently classified as two separate species by the International Cytomegalovirus Committee on Taxonomy of Viruses (186, 187). The herpesviruses in the subfamily Betaherpesvirinae infects white blood The viral genome of HHV-6 can be integrated into the genome of a small cells, but the prototype member of this family, CMV, can also infect several proportion of somatic cells and, in rare instances, into germ cell chromosomes, other cells in the body. CMV establishes lifelong latency in which may result in the offspring carrying a copy of the viral genome in all monocytes/macrophages but may be reactivated periodically. It is also possible nucleated cells in the body (78). Inherited chromosomally integrated HHV-6 to become reinfected with another strain of the virus. CMV is spread through is uncommon and the frequency has been estimated at 0.2–2.9% depending on body fluids such as urine, saliva, blood, semen, and breast milk, but also the region and population (78). through organ transplants. Less is known about HHV-6A and its role in human disease compared with The seroprevalence among women of reproductive age show geographic HHV-6B. HHV-6A and B are closely related to HHV-7 (184). All three belong variation, from 45% to approaching 100% in different parts of the world with to the genus Roseolovirus, are T-lymphotropic viruses and, like other the highest numbers in Asia, Africa and South America and lowest in the USA herpesviruses, establish lifelong latent infection (185). Both HHV-6B and and Western Europe (172). The CMV seroprevalence in Sweden is higher HHV-7 are ubiquitous viruses with high seroprevalence in most countries (82, compared with the general figures for Western Europe (172). In Swedish adults 185). aged 35–95 years the seroprevalence was measured at 83.2% (82). Primary infection with HHV-6B usually occur early in life after the loss of Primary CMV infection in immunocompetent young individuals is often protective maternal antibodies. The infection with HHV-7 usually occurs a asymptomatic. CMV reactivations are also often asymptomatic in little later. The viruses are primarily transmitted through saliva (185). Both the immunocompetent individuals. CMV infections can in immunocompetent primary infection and reactivations can be asymptomatic or symptomatic adults occasionally cause symptoms of protracted fever, malaise, sweating, (185). myalgia, fatigue and headache and increased transaminases (173-175). HHV-6B and HHV-7 (but less frequently) can cause Roseola Infantum, also CMV mainly causes severe disease, including hepatitis, pneumonitis, known as exanthem subitum, a common childhood illness with symptoms myocarditis, pancreatitis, and retinitis, in immunocompromised individuals including fever and skin rash (188, 189). Known complications of the infection where the immune system is unable to control the infection (176, 177). are febrile seizures and status epilepticus (190). Immunocompromised Neonatal CMV infection in premature infants and immunocompromised individuals, especially transplant recipients, have an increased risk of severe newborns can be aggravated by the immaturity of the immune system at this disease in both primary infection and reactivation with the viruses (191, 192). age and may include sepsis-like conditions, hepatitis, and thrombocytopenia (178-180). CMV infections in these individuals can be treated with antiviral drugs such as valganciclovir/ganciklovir and preventive measures may be indicated in individuals with high risk of severe disease (177, 178). CMV can have teratogenic effect and congenital CMV infection can cause permanent disabilities of the CNS and internal organs (181, 182). There is currently no vaccine against CMV, but research is ongoing (183). 30 31 1.3.4 BETAHERPESVIRINAE HHV-6A, HHV-6B and HHV-7 • Cytomegalovirus (CMV, HHV-5) Human herpesvirus 6A (HHV-6A) HHV-6A and HHV-6B where previously considered to be the same virus • Human herpesvirus 6B (HHV-6B) species (184, 185). Although closely related, HHV-6A and HHV-6B have • Human herpesvirus 7 (HHV-7) genetic, biological, and immunological characteristics that are so different that • they were recently classified as two separate species by the International Cytomegalovirus Committee on Taxonomy of Viruses (186, 187). The herpesviruses in the subfamily Betaherpesvirinae infects white blood The viral genome of HHV-6 can be integrated into the genome of a small cells, but the prototype member of this family, CMV, can also infect several proportion of somatic cells and, in rare instances, into germ cell chromosomes, other cells in the body. CMV establishes lifelong latency in which may result in the offspring carrying a copy of the viral genome in all monocytes/macrophages but may be reactivated periodically. It is also possible nucleated cells in the body (78). Inherited chromosomally integrated HHV-6 to become reinfected with another strain of the virus. CMV is spread through is uncommon and the frequency has been estimated at 0.2–2.9% depending on body fluids such as urine, saliva, blood, semen, and breast milk, but also the region and population (78). through organ transplants. Less is known about HHV-6A and its role in human disease compared with The seroprevalence among women of reproductive age show geographic HHV-6B. HHV-6A and B are closely related to HHV-7 (184). All three belong variation, from 45% to approaching 100% in different parts of the world with to the genus Roseolovirus, are T-lymphotropic viruses and, like other the highest numbers in Asia, Africa and South America and lowest in the USA herpesviruses, establish lifelong latent infection (185). Both HHV-6B and and Western Europe (172). The CMV seroprevalence in Sweden is higher HHV-7 are ubiquitous viruses with high seroprevalence in most countries (82, compared with the general figures for Western Europe (172). In Swedish adults 185). aged 35–95 years the seroprevalence was measured at 83.2% (82). Primary infection with HHV-6B usually occur early in life after the loss of Primary CMV infection in immunocompetent young individuals is often protective maternal antibodies. The infection with HHV-7 usually occurs a asymptomatic. CMV reactivations are also often asymptomatic in little later. The viruses are primarily transmitted through saliva (185). Both the immunocompetent individuals. CMV infections can in immunocompetent primary infection and reactivations can be asymptomatic or symptomatic adults occasionally cause symptoms of protracted fever, malaise, sweating, (185). myalgia, fatigue and headache and increased transaminases (173-175). HHV-6B and HHV-7 (but less frequently) can cause Roseola Infantum, also CMV mainly causes severe disease, including hepatitis, pneumonitis, known as exanthem subitum, a common childhood illness with symptoms myocarditis, pancreatitis, and retinitis, in immunocompromised individuals including fever and skin rash (188, 189). Known complications of the infection where the immune system is unable to control the infection (176, 177). are febrile seizures and status epilepticus (190). Immunocompromised Neonatal CMV infection in premature infants and immunocompromised individuals, especially transplant recipients, have an increased risk of severe newborns can be aggravated by the immaturity of the immune system at this disease in both primary infection and reactivation with the viruses (191, 192). age and may include sepsis-like conditions, hepatitis, and thrombocytopenia (178-180). CMV infections in these individuals can be treated with antiviral drugs such as valganciclovir/ganciklovir and preventive measures may be indicated in individuals with high risk of severe disease (177, 178). CMV can have teratogenic effect and congenital CMV infection can cause permanent disabilities of the CNS and internal organs (181, 182). There is currently no vaccine against CMV, but research is ongoing (183). 30 31 1.3.5 GAMMAHERPESVIRINAE Glycoproteins • Human herpesvirus 8 (HHV-8) or Kaposi’s sarcoma- associated herpesvirus (KSHV) The glycoproteins encoded by the EBV genome include gB (gp110), gH Epstein-Barr virus (EBV, HHV-4) (gp85), gL (gp25), gM, gN, gp42, gp78, gp150, gp220, gp350 and BMRF2 • (208). The viral glycoproteins in the envelope are essential for infecting host Human herpesvirus 8 (HHV-8) cells. The two glycoproteins gp220 and gp330 are encoded by the same gene (BLLF1) but gp220 will be shorter due to internal splicing of mRNA (209, The prevalence of HHV-8 varies around the world. Studies have shown 210). EBVgp350 consist of 907 a.a., with an extracellular N-terminal segment, seroprevalence below 10% in adult populations in North America and northern a transmembrane segment and a small portion located on the inside of the viral Europe but significantly higher proportion, 20–80%, in endemic areas in the membrane (211). The protein has extensive glycosylation with N- and O- Mediterranean region, the Xinjiang region of China and sub-Saharan Africa linked oligosaccharide chains (209, 212). EBVgp350 is the major viral (193-196). The virus can be transmitted through several routes, including envelope protein and can be found on the plasma membrane of virus- saliva, sexual, and parenteral transmission (195, 197). HHV-8, like all replicating cells (208, 210, 213). The initial attachment between the virus and members of the herpesvirus family, can establish and maintain lifelong latent the B cells is between EBVgp350 and the CD21 receptor (211, 214-219). The infection with potential later reactivation. In immunocompromised primary target of EBV-neutralizing antibodies is EBVgp350 (220-222). individuals, HHV-8 may cause Kaposi’s sarcoma, multicentric Castelman’s disease and pleural effusion lymphomas (152, 194, 198-200). Seroprevalence and Transmission EBV is one of the most widespread human viruses and the seroprevalence in adults is usually over 90% (204, 205). EBV is easily transmitted from one 1.3.6 EPSTEIN-BARR VIRUS individual to another, primarily through saliva (204, 223, 224). Excretion of EBV has also been observed in saliva from asymptomatic individuals (223). In Epstein-Barr virus (EBV) is a member of the Herpesviridae family and belongs addition, the virus can be transmitted through blood, solid organ transplants to the Gammaherpesvirinae subfamily. Lymphocryptovirus is the virus genus, and hematopoietic cell transplants (204). and the species is Human gammaherpesvirus 4. EBV is described more extensively because the virus is a central topic in the thesis. EBV infection The virus It is common to be infected with the virus early in life. When the primary infection occurs in young, immunocompetent children it is often asymptomatic EBV was discovered in 1964 by the two researchers Epstein and Barr (201). or causes symptoms that are indistinguishable from other mild childhood The virus was identified from Burkitt's lymphoma tumor cells and was the first infections (225, 226). Infectious mononucleosis (IM) caused by EBV occurs virus to be associated with human cancer (201, 202). EBV soon proved to be mainly when adolescents and young adults undergo a primary infection (204, a ubiquitous virus spread throughout the world (203-205). EBV has a linear 227). Symptoms of IM includes sore throat, cervical lymphadenopathy, fever, dsDNA genome with approximately 172,000 bp (206), which encodes more upper respiratory symptoms, fatigue, headache, myalgia, subclinical hepatitis than 85 genes (207). with increased levels of alanine aminotransferase, enlarged liver and/or spleen and skin rash (204, 227). For some individuals, fatigue will continue for several EBV is currently divided into type 1 and type 2 based on the differences in the weeks to months (204). EBV mainly infects and transforms B cells but can gene for Epstein-Barr virus nuclear antigen 2 (EBNA-2) (207). The dsDNA infect other cells e.g. epithelial cells, T cells and NK cells (228, 229). The virus genome is encased in an icosahedral capsid. The nucleocapsid is in turn has a latent and a productive (lytic) cycle and it establishes a lifelong persistent surrounded by tegument. Outside the tegument is the lipid envelope, which infection in B cells after primary infection (230). EBV can be reactivated contains glycoproteins encoded by the viral genome. intermittently and can then be spread to other hosts (231). 32 33 1.3.5 GAMMAHERPESVIRINAE Glycoproteins • Human herpesvirus 8 (HHV-8) or Kaposi’s sarcoma- associated herpesvirus (KSHV) The glycoproteins encoded by the EBV genome include gB (gp110), gH Epstein-Barr virus (EBV, HHV-4) (gp85), gL (gp25), gM, gN, gp42, gp78, gp150, gp220, gp350 and BMRF2 • (208). The viral glycoproteins in the envelope are essential for infecting host Human herpesvirus 8 (HHV-8) cells. The two glycoproteins gp220 and gp330 are encoded by the same gene (BLLF1) but gp220 will be shorter due to internal splicing of mRNA (209, The prevalence of HHV-8 varies around the world. Studies have shown 210). EBVgp350 consist of 907 a.a., with an extracellular N-terminal segment, seroprevalence below 10% in adult populations in North America and northern a transmembrane segment and a small portion located on the inside of the viral Europe but significantly higher proportion, 20–80%, in endemic areas in the membrane (211). The protein has extensive glycosylation with N- and O- Mediterranean region, the Xinjiang region of China and sub-Saharan Africa linked oligosaccharide chains (209, 212). EBVgp350 is the major viral (193-196). The virus can be transmitted through several routes, including envelope protein and can be found on the plasma membrane of virus- saliva, sexual, and parenteral transmission (195, 197). HHV-8, like all replicating cells (208, 210, 213). The initial attachment between the virus and members of the herpesvirus family, can establish and maintain lifelong latent the B cells is between EBVgp350 and the CD21 receptor (211, 214-219). The infection with potential later reactivation. In immunocompromised primary target of EBV-neutralizing antibodies is EBVgp350 (220-222). individuals, HHV-8 may cause Kaposi’s sarcoma, multicentric Castelman’s disease and pleural effusion lymphomas (152, 194, 198-200). Seroprevalence and Transmission EBV is one of the most widespread human viruses and the seroprevalence in adults is usually over 90% (204, 205). EBV is easily transmitted from one 1.3.6 EPSTEIN-BARR VIRUS individual to another, primarily through saliva (204, 223, 224). Excretion of EBV has also been observed in saliva from asymptomatic individuals (223). In Epstein-Barr virus (EBV) is a member of the Herpesviridae family and belongs addition, the virus can be transmitted through blood, solid organ transplants to the Gammaherpesvirinae subfamily. Lymphocryptovirus is the virus genus, and hematopoietic cell transplants (204). and the species is Human gammaherpesvirus 4. EBV is described more extensively because the virus is a central topic in the thesis. EBV infection The virus It is common to be infected with the virus early in life. When the primary infection occurs in young, immunocompetent children it is often asymptomatic EBV was discovered in 1964 by the two researchers Epstein and Barr (201). or causes symptoms that are indistinguishable from other mild childhood The virus was identified from Burkitt's lymphoma tumor cells and was the first infections (225, 226). Infectious mononucleosis (IM) caused by EBV occurs virus to be associated with human cancer (201, 202). EBV soon proved to be mainly when adolescents and young adults undergo a primary infection (204, a ubiquitous virus spread throughout the world (203-205). EBV has a linear 227). Symptoms of IM includes sore throat, cervical lymphadenopathy, fever, dsDNA genome with approximately 172,000 bp (206), which encodes more upper respiratory symptoms, fatigue, headache, myalgia, subclinical hepatitis than 85 genes (207). with increased levels of alanine aminotransferase, enlarged liver and/or spleen and skin rash (204, 227). For some individuals, fatigue will continue for several EBV is currently divided into type 1 and type 2 based on the differences in the weeks to months (204). EBV mainly infects and transforms B cells but can gene for Epstein-Barr virus nuclear antigen 2 (EBNA-2) (207). The dsDNA infect other cells e.g. epithelial cells, T cells and NK cells (228, 229). The virus genome is encased in an icosahedral capsid. The nucleocapsid is in turn has a latent and a productive (lytic) cycle and it establishes a lifelong persistent surrounded by tegument. Outside the tegument is the lipid envelope, which infection in B cells after primary infection (230). EBV can be reactivated contains glycoproteins encoded by the viral genome. intermittently and can then be spread to other hosts (231). 32 33 EBV complications Viral diagnosis EBV infections are primarily kept under control by the cell-mediated immune Detection of heterophilic antibodies may contribute to the diagnosis of system and are therefore particular a problem in individuals with this type of individuals with compatible clinical manifestations of EBV (263, 264). The immune deficiency (232-234). Immunocompromised individuals are at risk of sensitivity of these tests is lower in young children due to lack of production developing smooth muscle sarcoma and EBV-driven B lymphoproliferative of heterophilic antibodies (264). False positive tests are also seen in several diseases, which, in immunosuppressed patients who have undergone solid other conditions including acute infections, cancer and autoimmune diseases organ transplantation or hematopoietic stem cell transplantation, are referred (204). Serological methods are frequently used to determine the EBV infection to as posttransplant lymphoproliferative disease (233, 235-237). EBV- status of patients. In immunocompromised individuals where the serological associated malignancies such as Burkitt’s lymphoma, diffuse large B cell response may take unusual courses, PCR is the preferred method for lymphoma, Hodgkin’s lymphoma, nasopharyngeal carcinoma, T/NK cell diagnosing current infection and for monitoring changes in viral load (265- lymphoma and gastric carcinoma may develop in seemingly 267). immunocompetent individuals (228, 232, 238, 239). EBV serology Chronic active EBV disease is very uncommon in Europe, but such cases are slightly more common in South America and Asia (240). Another serious but The two antigens primarily used to detect EBV-specific antibodies are viral rare complication associated with EBV is infection-associated capsid antigen (VCA) and Epstein-Barr virus nuclear antigen 1 (EBNA1) (268- hemophagocytic lymphohistiocytosis (241-244). Infection with EBV, 272). Anti-VCA IgM antibodies can often be detected when symptoms of EBV especially a primary infection in the form of IM, has been associated with an disease occur (204, 273, 274). Anti-VCA IgG can be detected as early as the increased risk of developing multiple sclerosis (MS) (245-247). This will be onset of symptoms and is often detected during the first month of illness (204, described in more detail under 1.5 Multiple sclerosis. 273, 274). Anti-VCA IgM usually begins to decline after about two to three months but may persist longer, while anti-VCA IgG will persist throughout life Treatment and vaccine (22, 263, 273, 274). There is currently no clinically effective antiviral treatment for EBV (248- The generation of antibodies to EBNA1 takes longer compared with the 250). In patients with EBV-associated diseases who are on immunosuppressive production of anti-VCA antibodies. Anti-EBNA1 IgG can be detected no medications, a dose reduction may be performed (251). Other options for earlier than four weeks after the onset of symptoms, but it often takes around patients with EBV-associated B lymphoproliferative diseases are treatment three months before these antibodies can be detected (204, 272, 275). Thus, with anti-CD20 monoclonal antibodies, which depletes B cells, such as detection of anti-EBNA1 IgG during an acute illness can be used as a marker rituximab (Mabthera®) or ocrelizumab (Ocrevus®) and cytotoxic to rule out primary EBV infection. Overall, however, there is a large variation chemotherapy (251-257). New possibilities for the treatment of EBV- in the serological EBV response between different individuals (204, 272-274). associated cancers are immune cell therapies such as adoptive T-cell therapy When anti-EBV antibodies can be detected also depends on the type of assay (258). platform and the specific type of antigen used (204, 272). There is currently no available EBV vaccine to prevent infection, disease, and The combination of anti-EBNA1 IgG, anti-VCA IgM and anti-VCA IgG associated diseases with the virus, but research is ongoing (258-260). The EBV detection can be used to determine the stage of EBV infection (276). Detection glycoprotein 350/220 has been attempted as a prophylactic vaccine candidate of anti-VCA IgM with or without anti-VCA IgG but without detection of anti- based on its immunological properties (258, 259, 261, 262). An EBVgp350- EBNA1 IgG indicates acute infection while detection of anti-VCA IgG and based vaccine has in a phase two trial shown possible protection against IM anti-EBNA1 IgG without anti-VCA IgM is typical of previous infection. but not against infection (262). 34 35 EBV complications Viral diagnosis EBV infections are primarily kept under control by the cell-mediated immune Detection of heterophilic antibodies may contribute to the diagnosis of system and are therefore particular a problem in individuals with this type of individuals with compatible clinical manifestations of EBV (263, 264). The immune deficiency (232-234). Immunocompromised individuals are at risk of sensitivity of these tests is lower in young children due to lack of production developing smooth muscle sarcoma and EBV-driven B lymphoproliferative of heterophilic antibodies (264). False positive tests are also seen in several diseases, which, in immunosuppressed patients who have undergone solid other conditions including acute infections, cancer and autoimmune diseases organ transplantation or hematopoietic stem cell transplantation, are referred (204). Serological methods are frequently used to determine the EBV infection to as posttransplant lymphoproliferative disease (233, 235-237). EBV- status of patients. In immunocompromised individuals where the serological associated malignancies such as Burkitt’s lymphoma, diffuse large B cell response may take unusual courses, PCR is the preferred method for lymphoma, Hodgkin’s lymphoma, nasopharyngeal carcinoma, T/NK cell diagnosing current infection and for monitoring changes in viral load (265- lymphoma and gastric carcinoma may develop in seemingly 267). immunocompetent individuals (228, 232, 238, 239). EBV serology Chronic active EBV disease is very uncommon in Europe, but such cases are slightly more common in South America and Asia (240). Another serious but The two antigens primarily used to detect EBV-specific antibodies are viral rare complication associated with EBV is infection-associated capsid antigen (VCA) and Epstein-Barr virus nuclear antigen 1 (EBNA1) (268- hemophagocytic lymphohistiocytosis (241-244). Infection with EBV, 272). Anti-VCA IgM antibodies can often be detected when symptoms of EBV especially a primary infection in the form of IM, has been associated with an disease occur (204, 273, 274). Anti-VCA IgG can be detected as early as the increased risk of developing multiple sclerosis (MS) (245-247). This will be onset of symptoms and is often detected during the first month of illness (204, described in more detail under 1.5 Multiple sclerosis. 273, 274). Anti-VCA IgM usually begins to decline after about two to three months but may persist longer, while anti-VCA IgG will persist throughout life Treatment and vaccine (22, 263, 273, 274). There is currently no clinically effective antiviral treatment for EBV (248- The generation of antibodies to EBNA1 takes longer compared with the 250). In patients with EBV-associated diseases who are on immunosuppressive production of anti-VCA antibodies. Anti-EBNA1 IgG can be detected no medications, a dose reduction may be performed (251). Other options for earlier than four weeks after the onset of symptoms, but it often takes around patients with EBV-associated B lymphoproliferative diseases are treatment three months before these antibodies can be detected (204, 272, 275). Thus, with anti-CD20 monoclonal antibodies, which depletes B cells, such as detection of anti-EBNA1 IgG during an acute illness can be used as a marker rituximab (Mabthera®) or ocrelizumab (Ocrevus®) and cytotoxic to rule out primary EBV infection. Overall, however, there is a large variation chemotherapy (251-257). New possibilities for the treatment of EBV- in the serological EBV response between different individuals (204, 272-274). associated cancers are immune cell therapies such as adoptive T-cell therapy When anti-EBV antibodies can be detected also depends on the type of assay (258). platform and the specific type of antigen used (204, 272). There is currently no available EBV vaccine to prevent infection, disease, and The combination of anti-EBNA1 IgG, anti-VCA IgM and anti-VCA IgG associated diseases with the virus, but research is ongoing (258-260). The EBV detection can be used to determine the stage of EBV infection (276). Detection glycoprotein 350/220 has been attempted as a prophylactic vaccine candidate of anti-VCA IgM with or without anti-VCA IgG but without detection of anti- based on its immunological properties (258, 259, 261, 262). An EBVgp350- EBNA1 IgG indicates acute infection while detection of anti-VCA IgG and based vaccine has in a phase two trial shown possible protection against IM anti-EBNA1 IgG without anti-VCA IgM is typical of previous infection. but not against infection (262). 34 35 1.3.7 MEASLES VIRUS Transmission Measles virus (MeV) belongs to the Paramyxoviridae family within the Orthoparamyxovirinae subfamily. The viral genus is Morbillivirus and the MeV is highly contagious and infects cells in the respiratory tract. When species Measles morbillivirus. infected individuals cough or sneeze, the virus is transmitted via airway droplets and airborne transmission (284, 285). The virus Measles infection MeV is closely related to rinderpest virus, which was a pathogen in cattle before it was eradicated by vaccination in 2011 (277, 278) and it is possible Humans are the natural host of MeV and there is no animal reservoir although that MeV developed as a zoonotic infection when humans and cattle started to non-human primates can become infected with the virus (286-288). The live close together (279). MeV has a single negative-stranded RNA genome. incubation period is often about 10–14 days but may be as long as 23 days The size of the genome is relatively conserved to 15,894 nucleotides, but small (289). Symptoms usually begin with fever, cough, coryza and conjunctivitis, discrepancies have been reported (280, 281). The genome encodes six which after around three to four days are followed by the characteristic structural proteins, nucleocapsid protein, matrix protein, phosphoprotein, large erythematous, maculopapular rash. The small white papules on the oral protein, hemagglutinin protein and fusion protein (282). mucosa, termed Koplik spots, appear a day or two before the rash and can be used clinically to diagnose the measles disease before the rash occurs. MeV is divided into eight clades, A to H, and 24 genotypes based on the diversity of the 450 nucleotides in the carboxyterminal of the nucleocapsid Complications can affect many organs in the body and include pneumonia, protein (283). There is only one serotype of MeV. The genome is encased otitis media, laryngotracheobronchitis, stomatitis, diarrhea, and neurological within a helical capsid. Surrounding the capsid is the envelope where complications. Risk groups for complications are children under 5 years of age, hemagglutinin and fusion glycoproteins are incorporated. adults over 20 years of age, pregnant women, and immunocompromised individuals (287). Pneumonia causes the highest MeV-related morbidity and mortality. The neurological complications are rare but serious. MeV can cause acute encephalitis (290) and may trigger the autoimmune disease acute disseminated encephalomyelitis, which can occur during or shortly after the measles episode (291). Severely immunocompromised individuals who are unable to eliminate MeV infection due to impaired cellular immunity may develop measles inclusion body encephalitis within months after the acute infection, with progressive MeV infection in the brain causing neurological deterioration that often leads to death (291, 292). Neurological complications can also occur several years after the acute disease as in subacute sclerosing panencephalitis (293-295). The disease occurs mainly in children who are infected with MeV before the age of two. In these cases, the immune system in response to the progressive MeV infection in the brain will cause extensive brain damage that will lead to progressive loss of motor and cognitive functions, seizures, and death (293, 294). The pathogenesis behind the disease has not been fully established (295). Laboratory support for the diagnosis if often dependent on intrathecal anti-MeV IgG detection because MeV RNA is rarely detected in CSF, although MeV has been discovered in brain tissues in patients with the Figure 12. Schematic illustration of a measles virus. disease (293, 294, 296). 36 37 1.3.7 MEASLES VIRUS Transmission Measles virus (MeV) belongs to the Paramyxoviridae family within the Orthoparamyxovirinae subfamily. The viral genus is Morbillivirus and the MeV is highly contagious and infects cells in the respiratory tract. When species Measles morbillivirus. infected individuals cough or sneeze, the virus is transmitted via airway droplets and airborne transmission (284, 285). The virus Measles infection MeV is closely related to rinderpest virus, which was a pathogen in cattle before it was eradicated by vaccination in 2011 (277, 278) and it is possible Humans are the natural host of MeV and there is no animal reservoir although that MeV developed as a zoonotic infection when humans and cattle started to non-human primates can become infected with the virus (286-288). The live close together (279). MeV has a single negative-stranded RNA genome. incubation period is often about 10–14 days but may be as long as 23 days The size of the genome is relatively conserved to 15,894 nucleotides, but small (289). Symptoms usually begin with fever, cough, coryza and conjunctivitis, discrepancies have been reported (280, 281). The genome encodes six which after around three to four days are followed by the characteristic structural proteins, nucleocapsid protein, matrix protein, phosphoprotein, large erythematous, maculopapular rash. The small white papules on the oral protein, hemagglutinin protein and fusion protein (282). mucosa, termed Koplik spots, appear a day or two before the rash and can be used clinically to diagnose the measles disease before the rash occurs. MeV is divided into eight clades, A to H, and 24 genotypes based on the diversity of the 450 nucleotides in the carboxyterminal of the nucleocapsid Complications can affect many organs in the body and include pneumonia, protein (283). There is only one serotype of MeV. The genome is encased otitis media, laryngotracheobronchitis, stomatitis, diarrhea, and neurological within a helical capsid. Surrounding the capsid is the envelope where complications. Risk groups for complications are children under 5 years of age, hemagglutinin and fusion glycoproteins are incorporated. adults over 20 years of age, pregnant women, and immunocompromised individuals (287). Pneumonia causes the highest MeV-related morbidity and mortality. The neurological complications are rare but serious. MeV can cause acute encephalitis (290) and may trigger the autoimmune disease acute disseminated encephalomyelitis, which can occur during or shortly after the measles episode (291). Severely immunocompromised individuals who are unable to eliminate MeV infection due to impaired cellular immunity may develop measles inclusion body encephalitis within months after the acute infection, with progressive MeV infection in the brain causing neurological deterioration that often leads to death (291, 292). Neurological complications can also occur several years after the acute disease as in subacute sclerosing panencephalitis (293-295). The disease occurs mainly in children who are infected with MeV before the age of two. In these cases, the immune system in response to the progressive MeV infection in the brain will cause extensive brain damage that will lead to progressive loss of motor and cognitive functions, seizures, and death (293, 294). The pathogenesis behind the disease has not been fully established (295). Laboratory support for the diagnosis if often dependent on intrathecal anti-MeV IgG detection because MeV RNA is rarely detected in CSF, although MeV has been discovered in brain tissues in patients with the Figure 12. Schematic illustration of a measles virus. disease (293, 294, 296). 36 37 Immunity It is also preferable to analyze different sample materials to increase the chance of detecting MeV RNA (299, 303). Natural infection generally provides lifelong immunity with a stable and long- lasting anti-MeV IgG response (22). Immunity after vaccination is also long Measles serology but the anti-MeV IgG response decreases with time (297) and breakthrough cases occur, especially after only one vaccine dose (286, 298, 299). Vaccine- Although PCR is the preferred method for diagnosing acute measles (300), a modified measles usually comes with milder symptoms and may therefore be measles diagnosis can also be established by serological detection of anti-MeV more difficult to diagnose clinically (298-300). IgM antibodies or by a significant increase in anti-MeV IgG levels between paired sera taken in the acute and convalescent phase. Treatment Negative measles serology in a patient with true measles infection may be due There is no specific antiviral treatment for measles. The management of to early sampling before the patient has seroconverted. Samples taken within patients with MeV infection is therefore focused on supportive therapy with three days after the onset of the rash have a higher risk of becoming anti-MeV possible vitamin A supplementation, rehydration in severe diarrhea and IgM-negative and sampling within 10 days have a higher risk of becoming antibiotic treatment in secondary bacterial infections. IgG-negative (287, 299). Vaccination Cases of vaccine-modified measles, i.e. breakthrough infection, also have a higher risk of becoming anti-MeV IgM-negative (287, 299). It is well known MeV has been a leading cause of morbidity and mortality in the world before that false positive anti-IgM MeV responses occur and may be due to vaccines were developed against the disease. The control of MeV is largely nonspecific reactions such as other antibodies or rheumatoid factor (287). It due to vaccination with safe and effective attenuated live vaccines. Vaccines can be difficult to obtain paired serum samples collected during the acute and have been available since the 1960s and the fact that MeV has only one convalescence phase and the diagnosis in these cases comes retrospectively, serotype has facilitated the use of the same type of vaccine during decades. which is often not so useful. IgG avidity assays can be useful in identifying WHO recommends two doses of MeV vaccine in all countries' childhood measles reinfections (305). immunization programs and that countries striving to eliminate MeV need to achieve 95% coverage across the country (301). Serological analyzes are important to monitor the MeV seroprevalence in populations to gain knowledge about population immunity. Many different Diagnosis commercial assays are used to determine MeV seroprevalence including hemagglutination inhibition, neutralization assays, microtiter plate assays and A clinical diagnosis can be established based on typical clinical symptoms, but automated immunoassays (306). The MeV nucleocapsid protein is known to it can be more difficult early in the disease when the typical rash is not present induce a long-lasting antibody response and is therefore used in many and in vaccine-modified measles (298, 299). Koplik spots have been serological assays (307, 308). considered a typical marker for measles, but a study demonstrated that the spots can also occur in other viral infections such as rubella (302). PCR MeV RNA can be detected by PCR in materials from nasopharynx and pharynx swabs, oral fluid, and urine. To increase the likelihood of detecting MeV RNA, samples should preferably be collected within seven days after the onset of the rash, but it may be possible to detect MeV RNA for months in some individuals after both natural infection and vaccination (287, 303, 304). 38 39 Immunity It is also preferable to analyze different sample materials to increase the chance of detecting MeV RNA (299, 303). Natural infection generally provides lifelong immunity with a stable and long- lasting anti-MeV IgG response (22). Immunity after vaccination is also long Measles serology but the anti-MeV IgG response decreases with time (297) and breakthrough cases occur, especially after only one vaccine dose (286, 298, 299). Vaccine- Although PCR is the preferred method for diagnosing acute measles (300), a modified measles usually comes with milder symptoms and may therefore be measles diagnosis can also be established by serological detection of anti-MeV more difficult to diagnose clinically (298-300). IgM antibodies or by a significant increase in anti-MeV IgG levels between paired sera taken in the acute and convalescent phase. Treatment Negative measles serology in a patient with true measles infection may be due There is no specific antiviral treatment for measles. The management of to early sampling before the patient has seroconverted. Samples taken within patients with MeV infection is therefore focused on supportive therapy with three days after the onset of the rash have a higher risk of becoming anti-MeV possible vitamin A supplementation, rehydration in severe diarrhea and IgM-negative and sampling within 10 days have a higher risk of becoming antibiotic treatment in secondary bacterial infections. IgG-negative (287, 299). Vaccination Cases of vaccine-modified measles, i.e. breakthrough infection, also have a higher risk of becoming anti-MeV IgM-negative (287, 299). It is well known MeV has been a leading cause of morbidity and mortality in the world before that false positive anti-IgM MeV responses occur and may be due to vaccines were developed against the disease. The control of MeV is largely nonspecific reactions such as other antibodies or rheumatoid factor (287). It due to vaccination with safe and effective attenuated live vaccines. Vaccines can be difficult to obtain paired serum samples collected during the acute and have been available since the 1960s and the fact that MeV has only one convalescence phase and the diagnosis in these cases comes retrospectively, serotype has facilitated the use of the same type of vaccine during decades. which is often not so useful. IgG avidity assays can be useful in identifying WHO recommends two doses of MeV vaccine in all countries' childhood measles reinfections (305). immunization programs and that countries striving to eliminate MeV need to achieve 95% coverage across the country (301). Serological analyzes are important to monitor the MeV seroprevalence in populations to gain knowledge about population immunity. Many different Diagnosis commercial assays are used to determine MeV seroprevalence including hemagglutination inhibition, neutralization assays, microtiter plate assays and A clinical diagnosis can be established based on typical clinical symptoms, but automated immunoassays (306). The MeV nucleocapsid protein is known to it can be more difficult early in the disease when the typical rash is not present induce a long-lasting antibody response and is therefore used in many and in vaccine-modified measles (298, 299). Koplik spots have been serological assays (307, 308). considered a typical marker for measles, but a study demonstrated that the spots can also occur in other viral infections such as rubella (302). PCR MeV RNA can be detected by PCR in materials from nasopharynx and pharynx swabs, oral fluid, and urine. To increase the likelihood of detecting MeV RNA, samples should preferably be collected within seven days after the onset of the rash, but it may be possible to detect MeV RNA for months in some individuals after both natural infection and vaccination (287, 303, 304). 38 39 1.4 VIRAL SEROLOGY Seroprevalence Serological methods are important in the field of virology and are frequently Serological methods are important for epidemiological investigations. used. The assays are used for controlling immunity after infection/vaccination, Seroprevalence is the number of individuals who have antibodies, i.e. are to screen individuals for potential infections and to diagnose individuals with seropositive, against the virus of interest in the studied population. viral disease. The methods should be sensitive, specific, reproducible, rapid, and not too expensive. Viral serological assays are based on antigen-antibody Sensitivity and specificity reactions. The presence of antibodies to a particular virus and/or viral antigen indicates whether the individual from whom the sample was taken has been The reliability of diagnostic assays is often described by the specificity and infected with the specific virus. Most viral serological assays that detect sensitivity of the test. Sensitivity is a measure of the analysis' ability to detect antibodies measure the IgM and IgG responses. all true positive cases of a particular disease. If the sensitivity is low, there will be individuals with the disease who will not be detected, these are called false Diagnostics negatives. An infected individual's antibody isotype pattern may indicate whether the Specificity is a measure of the analysis' ability to distinguish between individual is undergoing a primary infection or a reinfection/reactivation. The individuals with disease and individuals without disease. The analysis should detection of IgM antibodies to a particular virus indicates that it is a become negative when the sample is derived from a healthy uninfected current/recent infection. If only IgM is detected but not IgG, it is likely to be a individual. Samples from healthy individuals who become positive are termed primary infection. To give an example, if anti-VZV IgM but not anti-VZV IgG false positives. If many individuals are false positive, the assay has low is detected, it indicates that it is a primary VZV infection. If both anti-VZV specificity. IgM and anti-VZV IgG antibodies are detected, it may be a primary infection, but it may also be due to reactivation because anti-VZV IgM antibodies are Antibody levels often produced upon VZV reactivation (72, 73). The antibody level is the amount of antibodies produced against a particular Antibody detection against a certain virus is performed in some cases on two antigen. Antibody levels are often described as antibody titers. The word titer occasions, first during the initial disease (acute phase) and then during the comes from titrate which means dilute. Classically, samples have been diluted convalescent phase, after about 2–4 weeks. It is then possible to decide whether in dilution series, e.g. 1/2, 1/4, 1/8 and so on. The antibody titer is the highest an individual seroconverts. Seroconversion means that an individual who does dilution that still gives a positive result in the analysis, and it is expressed as not have detectable antibodies to the examined virus, i.e. is seronegative, has the inverse of that dilution level. Titers are thus not an absolute measure but a detectable antibodies in the follow-up sample and thus becomes seropositive. relative concept. Seroconversion thus indicates that the individual has a primary infection. It is also possible to examine whether an individual has undergone a The word titer is often used synonymously with antibody level regardless of reactivation/reinfection by comparing antibody levels in samples taken during whether the level of antibodies is determined using dilution series or not. the acute and convalescence phase, where a significantly increased level Newer methods often do not use dilution series to determine antibody levels. between the samples indicates a current/recent reactivation or reinfection with In ELISA, the color intensity can be measured in a spectrophotometer as the virus. optical density (OD). It provides a relative measurement of the antibody level in the sample. There are also methods that provide even more quantifiable If only IgG antibodies can be detected in the samples and not IgM, this results where there are international standards to follow. indicates that the person has been infected or immunized with the virus before. Whether IgM is detectable or not in reactivations/reinfections varies between different viral infections and is also affected by the host's immune response. 40 41 1.4 VIRAL SEROLOGY Seroprevalence Serological methods are important in the field of virology and are frequently Serological methods are important for epidemiological investigations. used. The assays are used for controlling immunity after infection/vaccination, Seroprevalence is the number of individuals who have antibodies, i.e. are to screen individuals for potential infections and to diagnose individuals with seropositive, against the virus of interest in the studied population. viral disease. The methods should be sensitive, specific, reproducible, rapid, and not too expensive. Viral serological assays are based on antigen-antibody Sensitivity and specificity reactions. The presence of antibodies to a particular virus and/or viral antigen indicates whether the individual from whom the sample was taken has been The reliability of diagnostic assays is often described by the specificity and infected with the specific virus. Most viral serological assays that detect sensitivity of the test. Sensitivity is a measure of the analysis' ability to detect antibodies measure the IgM and IgG responses. all true positive cases of a particular disease. If the sensitivity is low, there will be individuals with the disease who will not be detected, these are called false Diagnostics negatives. An infected individual's antibody isotype pattern may indicate whether the Specificity is a measure of the analysis' ability to distinguish between individual is undergoing a primary infection or a reinfection/reactivation. The individuals with disease and individuals without disease. The analysis should detection of IgM antibodies to a particular virus indicates that it is a become negative when the sample is derived from a healthy uninfected current/recent infection. If only IgM is detected but not IgG, it is likely to be a individual. Samples from healthy individuals who become positive are termed primary infection. To give an example, if anti-VZV IgM but not anti-VZV IgG false positives. If many individuals are false positive, the assay has low is detected, it indicates that it is a primary VZV infection. If both anti-VZV specificity. IgM and anti-VZV IgG antibodies are detected, it may be a primary infection, but it may also be due to reactivation because anti-VZV IgM antibodies are Antibody levels often produced upon VZV reactivation (72, 73). The antibody level is the amount of antibodies produced against a particular Antibody detection against a certain virus is performed in some cases on two antigen. Antibody levels are often described as antibody titers. The word titer occasions, first during the initial disease (acute phase) and then during the comes from titrate which means dilute. Classically, samples have been diluted convalescent phase, after about 2–4 weeks. It is then possible to decide whether in dilution series, e.g. 1/2, 1/4, 1/8 and so on. The antibody titer is the highest an individual seroconverts. Seroconversion means that an individual who does dilution that still gives a positive result in the analysis, and it is expressed as not have detectable antibodies to the examined virus, i.e. is seronegative, has the inverse of that dilution level. Titers are thus not an absolute measure but a detectable antibodies in the follow-up sample and thus becomes seropositive. relative concept. Seroconversion thus indicates that the individual has a primary infection. It is also possible to examine whether an individual has undergone a The word titer is often used synonymously with antibody level regardless of reactivation/reinfection by comparing antibody levels in samples taken during whether the level of antibodies is determined using dilution series or not. the acute and convalescence phase, where a significantly increased level Newer methods often do not use dilution series to determine antibody levels. between the samples indicates a current/recent reactivation or reinfection with In ELISA, the color intensity can be measured in a spectrophotometer as the virus. optical density (OD). It provides a relative measurement of the antibody level in the sample. There are also methods that provide even more quantifiable If only IgG antibodies can be detected in the samples and not IgM, this results where there are international standards to follow. indicates that the person has been infected or immunized with the virus before. Whether IgM is detectable or not in reactivations/reinfections varies between different viral infections and is also affected by the host's immune response. 40 41 Intrathecal antibody production space, lumbar blockage by a local stenosis/tumor, impaired drainage by arachnoid villi, or reduced CSF production (315). CNS infections such as The CNS is protected by the blood-brain barrier (BBB), which consists of the meningitis can lead to decreased CSF flow due to increased CSF viscosity, endothelial barrier between the CNS and cerebral capillaries, as well as the meningeal adhesions, and deposition of cells and protein complexes in the blood-CSF barrier, where CSF is produced as an ultrafiltrate of the blood arachnoid villi that will cause flow obstruction (315). mainly by the choroid plexus. (309, 310). Most proteins in CSF are blood- derived but some proteins are produced in the CNS, which is called intrathecal To investigate whether an increased levels of antibodies in CSF are due to production. Some proteins such as albumin are derived only from the blood, intrathecal production or to an increased amount of blood-derived antibodies, while other proteins such as neuron-specific enolase are produced intrathecally both the antibody levels and a reference protein must be measured and (311, 312). Antibodies in CSF are both derived from blood and can be compared in CSF and blood. Albumin is often used as a reference protein produced intrathecally. In healthy individuals, CSF antibody levels are low because it is only produced by the liver and is thus a useful indicator of the compared with blood levels. Proteins including our antibodies are transferred proportion in CSF that are derived from blood (320, 321). Other choices may from blood to CSF primarily through diffusion across the blood-CSF barrier be to measure a reference antibody response to another common virus or to (309). measure the total IgG levels in CSF and blood (322, 323). Many formulae have been used to distinguish between an intrathecal antibody response and blood- The transport rate depends mainly on the molecular radius of the proteins and derived antibodies including Ig synthesis rate, Ig index, Igloc, CSF/serum the protein concentration in the blood (309, 313). Smaller proteins have a lower quotient diagrams (Reibergram) and isoelectric focusing for detection of concentration gradient between blood and CSF compared with larger proteins. oligoclonal bands (OCB) (314, 321, 324-327). When analyzing intrathecal This is reflected by the concentration gradient for different subclasses of antibody production, any erythrocytes in the CSF must be considered as it may antibodies where IgG has a lower gradient compared with IgM, which is larger be a sign of barrier damage that can occur due to traumatic lumbar puncture, in size compared with IgG. The concentration gradient between CSF and neurosurgical surgery, or other causes. serum for IgG and IgM is estimated to be 1:429 and 1:3300, respectively (314). The concentration of blood-derived proteins in CSF is also dependent on the CSF flow rate, which varies between individuals and can be affected by factors 1.4.1 SEROLOGICAL METHODS IN VIROLOGY such as age and various diseases (312, 315, 316). One theory is that decreased CSF flow causes a reduction in CSF exchange which leads to an increased There are several different serological methods for detecting viral antigens and blood-derived protein concentration in CSF (312, 314, 315). In diseases such antibodies against viruses. Many are heterogeneous assays, also called as Guillain-Barré syndrome, brain tumors and chronic inflammatory separation assays, because these assays must separate the product to be demyelinating polyneuropathy, there is an increase of blood-derived proteins measured from unreacted materials before detection. Although there are many in CSF, which is probably due to a decrease in CSF outflow leading to reduced different methods, many are based on the same principles. The principles CSF turnover (314-316). include a capture system (antigen or capture antibody), addition of the analyte (the substance i.e. antigen/antibody, that the assay is designed to measure) and In a viral infection of the CNS, antibodies can be produced against the virus a detection system. Immunoassays are based on the specific binding that occurs by antibody-secreting cells. A significant antibody response in CSF indicates between an antibody and the specific antigen that it recognizes. Solid-phase an intrathecal antibody production, but the response in CSF must be compared immunoassays are methods that use a solid phase in their capture system. with serum antibody levels because, as previously explained, antibodies in Heterogeneous assays have separation steps where a common separation CSF can be derived from blood and the antibody levels in CSF are thus strategy is to wash away unbound material. The basic principles of partially dependent on the protein level in the blood. Various diseases may heterogeneous solid-phase immunoassays for the detection of increase the concentration of antibodies in the CNS, which may be due to antibodies/antigens including the enzyme-linked immunosorbent assay decreased CSF flow and/or alterations of the barriers (309, 314-319). The (ELISA) are described below (328-330). Examples of other viral serological decrease in CSF flow could be due to decreased circulation in the subarachnoid assays will also be presented. 42 43 Intrathecal antibody production space, lumbar blockage by a local stenosis/tumor, impaired drainage by arachnoid villi, or reduced CSF production (315). CNS infections such as The CNS is protected by the blood-brain barrier (BBB), which consists of the meningitis can lead to decreased CSF flow due to increased CSF viscosity, endothelial barrier between the CNS and cerebral capillaries, as well as the meningeal adhesions, and deposition of cells and protein complexes in the blood-CSF barrier, where CSF is produced as an ultrafiltrate of the blood arachnoid villi that will cause flow obstruction (315). mainly by the choroid plexus. (309, 310). Most proteins in CSF are blood- derived but some proteins are produced in the CNS, which is called intrathecal To investigate whether an increased levels of antibodies in CSF are due to production. Some proteins such as albumin are derived only from the blood, intrathecal production or to an increased amount of blood-derived antibodies, while other proteins such as neuron-specific enolase are produced intrathecally both the antibody levels and a reference protein must be measured and (311, 312). Antibodies in CSF are both derived from blood and can be compared in CSF and blood. Albumin is often used as a reference protein produced intrathecally. In healthy individuals, CSF antibody levels are low because it is only produced by the liver and is thus a useful indicator of the compared with blood levels. Proteins including our antibodies are transferred proportion in CSF that are derived from blood (320, 321). Other choices may from blood to CSF primarily through diffusion across the blood-CSF barrier be to measure a reference antibody response to another common virus or to (309). measure the total IgG levels in CSF and blood (322, 323). Many formulae have been used to distinguish between an intrathecal antibody response and blood- The transport rate depends mainly on the molecular radius of the proteins and derived antibodies including Ig synthesis rate, Ig index, Igloc, CSF/serum the protein concentration in the blood (309, 313). Smaller proteins have a lower quotient diagrams (Reibergram) and isoelectric focusing for detection of concentration gradient between blood and CSF compared with larger proteins. oligoclonal bands (OCB) (314, 321, 324-327). When analyzing intrathecal This is reflected by the concentration gradient for different subclasses of antibody production, any erythrocytes in the CSF must be considered as it may antibodies where IgG has a lower gradient compared with IgM, which is larger be a sign of barrier damage that can occur due to traumatic lumbar puncture, in size compared with IgG. The concentration gradient between CSF and neurosurgical surgery, or other causes. serum for IgG and IgM is estimated to be 1:429 and 1:3300, respectively (314). The concentration of blood-derived proteins in CSF is also dependent on the CSF flow rate, which varies between individuals and can be affected by factors 1.4.1 SEROLOGICAL METHODS IN VIROLOGY such as age and various diseases (312, 315, 316). One theory is that decreased CSF flow causes a reduction in CSF exchange which leads to an increased There are several different serological methods for detecting viral antigens and blood-derived protein concentration in CSF (312, 314, 315). In diseases such antibodies against viruses. Many are heterogeneous assays, also called as Guillain-Barré syndrome, brain tumors and chronic inflammatory separation assays, because these assays must separate the product to be demyelinating polyneuropathy, there is an increase of blood-derived proteins measured from unreacted materials before detection. Although there are many in CSF, which is probably due to a decrease in CSF outflow leading to reduced different methods, many are based on the same principles. The principles CSF turnover (314-316). include a capture system (antigen or capture antibody), addition of the analyte (the substance i.e. antigen/antibody, that the assay is designed to measure) and In a viral infection of the CNS, antibodies can be produced against the virus a detection system. Immunoassays are based on the specific binding that occurs by antibody-secreting cells. A significant antibody response in CSF indicates between an antibody and the specific antigen that it recognizes. Solid-phase an intrathecal antibody production, but the response in CSF must be compared immunoassays are methods that use a solid phase in their capture system. with serum antibody levels because, as previously explained, antibodies in Heterogeneous assays have separation steps where a common separation CSF can be derived from blood and the antibody levels in CSF are thus strategy is to wash away unbound material. The basic principles of partially dependent on the protein level in the blood. Various diseases may heterogeneous solid-phase immunoassays for the detection of increase the concentration of antibodies in the CNS, which may be due to antibodies/antigens including the enzyme-linked immunosorbent assay decreased CSF flow and/or alterations of the barriers (309, 314-319). The (ELISA) are described below (328-330). Examples of other viral serological decrease in CSF flow could be due to decreased circulation in the subarachnoid assays will also be presented. 42 43 Indirect detection will therefore bind to several different epitopes on the primary antibody. This allows amplification of the signal because several antibodies can bind to the Indirect detection is a useful principle for detecting human antibodies in same primary antibody. This can amplify the signal of the assay, but it can also various sample materials including serum and CSF. The antigen is coated on a lead to a higher background and reduce the overall signal due to the increased solid phase, which may be a glass slide, polystyrene microtiter plate, risk of non-specific binding. nitrocellulose membrane, nylon membrane, polyvinylidene difluoride membrane, polystyrene beads, or magnetic beads. During the incubation Direct detection period, the antigen will adhere to the solid phase by passive adsorption (329, 331, 332). Unbound antigen is then washed away. The remaining areas of the The sample with the antigen of interest is coated on a solid surface e.g. a glass solid phase where the antigen has not been bound can be blocked to avoid slide or a microtiter plate. After incubation, unbound material from the sample residual binding capacity and thus to avoid non-specific binding. This can be is washed away. Blocking can be performed to avoid non-specific binding. A performed by using blocking agents e.g. bovine serum albumin or non-fat dry complementary reporter-labeled antibody is then added to the solid-bound milk. The sample is then added and if there are complementary antibodies to antigen. After another round of incubation, unbound reporter-labeled the antigen, these so-called primary antibodies will bind to the antigen. In antibodies are washed away. The next step is to add a substrate that will react serum and CSF samples, there are polyclonal antibodies that will bind to with the reporter. Depending on the reporter and substrate, the detection may different epitopes on the antigen. be based on fluorescence, color, or luminescence. A direct detection method is faster than the other methods because it involves fewer steps. One limitation with this method is that the complementary antibody must be conjugated to a reporter, which can be quite costly and time consuming. There is also a risk that the immune reactivity of the antibody is negatively affected by the labeling with the reporter. The risk of cross- reactivity and non-specific binding is reduced in this method by not using a secondary antibody, but this also removes the possibility of amplifying the assay signal. Complex samples with many proteins can be a problem because it is possible that many of the proteins are not of interest but that they take up space on the solid surface and thereby reduce the sensitivity of the assay. Figure 13. Basic principle of indirect detection of antibodies in a sample. After incubation, all unbound antibodies and other unbound components in the sample will be washed away. A conjugate will then be added. The conjugate consists of a reporter-labeled, secondary antibody from another animal species that targets the Fc portion of the primary antibody. These secondary antibodies can be directed against different antibody isotypes, e.g. anti-IgM, anti-IgG, or against more than one isotype, depending on which isotype the assay is to detect. These secondary antibodies will bind if there are primary antibodies to bind to. After incubation, the unbound secondary antibodies will be washed away. For detection, a substrate is added which, together with the reporter, gives rise to fluorescence, color, or luminescence to measure the analytes in Figure 14. Basic principle for direct antigen detection. the sample. Many reporter-labeled secondary antibodies are polyclonal and 44 45 Indirect detection will therefore bind to several different epitopes on the primary antibody. This allows amplification of the signal because several antibodies can bind to the Indirect detection is a useful principle for detecting human antibodies in same primary antibody. This can amplify the signal of the assay, but it can also various sample materials including serum and CSF. The antigen is coated on a lead to a higher background and reduce the overall signal due to the increased solid phase, which may be a glass slide, polystyrene microtiter plate, risk of non-specific binding. nitrocellulose membrane, nylon membrane, polyvinylidene difluoride membrane, polystyrene beads, or magnetic beads. During the incubation Direct detection period, the antigen will adhere to the solid phase by passive adsorption (329, 331, 332). Unbound antigen is then washed away. The remaining areas of the The sample with the antigen of interest is coated on a solid surface e.g. a glass solid phase where the antigen has not been bound can be blocked to avoid slide or a microtiter plate. After incubation, unbound material from the sample residual binding capacity and thus to avoid non-specific binding. This can be is washed away. Blocking can be performed to avoid non-specific binding. A performed by using blocking agents e.g. bovine serum albumin or non-fat dry complementary reporter-labeled antibody is then added to the solid-bound milk. The sample is then added and if there are complementary antibodies to antigen. After another round of incubation, unbound reporter-labeled the antigen, these so-called primary antibodies will bind to the antigen. In antibodies are washed away. The next step is to add a substrate that will react serum and CSF samples, there are polyclonal antibodies that will bind to with the reporter. Depending on the reporter and substrate, the detection may different epitopes on the antigen. be based on fluorescence, color, or luminescence. A direct detection method is faster than the other methods because it involves fewer steps. One limitation with this method is that the complementary antibody must be conjugated to a reporter, which can be quite costly and time consuming. There is also a risk that the immune reactivity of the antibody is negatively affected by the labeling with the reporter. The risk of cross- reactivity and non-specific binding is reduced in this method by not using a secondary antibody, but this also removes the possibility of amplifying the assay signal. Complex samples with many proteins can be a problem because it is possible that many of the proteins are not of interest but that they take up space on the solid surface and thereby reduce the sensitivity of the assay. Figure 13. Basic principle of indirect detection of antibodies in a sample. After incubation, all unbound antibodies and other unbound components in the sample will be washed away. A conjugate will then be added. The conjugate consists of a reporter-labeled, secondary antibody from another animal species that targets the Fc portion of the primary antibody. These secondary antibodies can be directed against different antibody isotypes, e.g. anti-IgM, anti-IgG, or against more than one isotype, depending on which isotype the assay is to detect. These secondary antibodies will bind if there are primary antibodies to bind to. After incubation, the unbound secondary antibodies will be washed away. For detection, a substrate is added which, together with the reporter, gives rise to fluorescence, color, or luminescence to measure the analytes in Figure 14. Basic principle for direct antigen detection. the sample. Many reporter-labeled secondary antibodies are polyclonal and 44 45 Sandwich/capture assays less reporter-labeled standard analyte will bind to the capture antibodies/antigen. Unbound reporter-labeled standard analyte will be washed In a sandwich assay, also called capture assay, a monoclonal antibody (capture away in following steps, and this will weaken the signal in the assay when the antibody) is immobilized on the solid phase to capture soluble antigen. After substrate is added. In a competitive assay, the generated signal is thus inversely incubation, washing and blocking, the patient sample is added. If the proportional to the amount of bound antigens/antibodies in the patient´s complementary antigen is present in the patient’s sample, it will bind to the sample. capture antibody. The same incubation, washing, conjugate and substrate steps as described above for direct or indirect detection can then be performed. For direct detection in the sandwich assay, a secondary reporter-labeled antibody directed against the antigen will be added. For indirect detection in the sandwich assay, the secondary antibody that attaches to the antigen will be unlabeled. This secondary antibody will in turn be detected by a reporter- labeled antibody from another animal species. It is crucial that the reporter- labeled antibody does not bind to the capture antibody. It is also important that the capture antibody and the secondary antibody bind to different epitopes that are located far enough apart on the antigen. Figure 16. Basic principles for competitive/inhibition assays for antibody or antigen detection. Structural arrangement Solid-phase immunoassays have immobilized antigen/capture antibodies on a solid surface that provide a high concentration of epitopes located to a specific area. The advantage is that this gives a higher chance of forming antibody- antigen complexes over a certain time frame compared with if the antigen/capture antibodies were free in solution. When a paratope from an IgG molecule has bound to an epitope, the other paratope is more likely to bind an identical epitope if the epitope is close, which is the case when the antigen is immobilized on a solid surface compared to if it is free in solution. Figure 15. Basic principle for a direct and indirect sandwich assay. Competitive/inhibition assays The basic principles described above with direct and indirect detection, and sandwich assays can all be adapted to competitive/competition assays, also called inhibition assays. The distinguishing feature of these assays is that the sample with an unknown amount of antigen/antibodies of interest will compete for binding with a reporter-labeled standard analyte for attachment to a limited number of binding sites on capture antibodies/antigen. The higher the Figure 17. A higher quantity of antibody-antigen complexes is formed during a certain time frame if the antigen is immobilized on a solid phase compared to complexes formed concentration of the antibodies/antigens of interest in the patient’s sample, the when the antigen is free in solution. 46 47 Sandwich/capture assays less reporter-labeled standard analyte will bind to the capture antibodies/antigen. Unbound reporter-labeled standard analyte will be washed In a sandwich assay, also called capture assay, a monoclonal antibody (capture away in following steps, and this will weaken the signal in the assay when the antibody) is immobilized on the solid phase to capture soluble antigen. After substrate is added. In a competitive assay, the generated signal is thus inversely incubation, washing and blocking, the patient sample is added. If the proportional to the amount of bound antigens/antibodies in the patient´s complementary antigen is present in the patient’s sample, it will bind to the sample. capture antibody. The same incubation, washing, conjugate and substrate steps as described above for direct or indirect detection can then be performed. For direct detection in the sandwich assay, a secondary reporter-labeled antibody directed against the antigen will be added. For indirect detection in the sandwich assay, the secondary antibody that attaches to the antigen will be unlabeled. This secondary antibody will in turn be detected by a reporter- labeled antibody from another animal species. It is crucial that the reporter- labeled antibody does not bind to the capture antibody. It is also important that the capture antibody and the secondary antibody bind to different epitopes that are located far enough apart on the antigen. Figure 16. Basic principles for competitive/inhibition assays for antibody or antigen detection. Structural arrangement Solid-phase immunoassays have immobilized antigen/capture antibodies on a solid surface that provide a high concentration of epitopes located to a specific area. The advantage is that this gives a higher chance of forming antibody- antigen complexes over a certain time frame compared with if the antigen/capture antibodies were free in solution. When a paratope from an IgG molecule has bound to an epitope, the other paratope is more likely to bind an identical epitope if the epitope is close, which is the case when the antigen is immobilized on a solid surface compared to if it is free in solution. Figure 15. Basic principle for a direct and indirect sandwich assay. Competitive/inhibition assays The basic principles described above with direct and indirect detection, and sandwich assays can all be adapted to competitive/competition assays, also called inhibition assays. The distinguishing feature of these assays is that the sample with an unknown amount of antigen/antibodies of interest will compete for binding with a reporter-labeled standard analyte for attachment to a limited number of binding sites on capture antibodies/antigen. The higher the Figure 17. A higher quantity of antibody-antigen complexes is formed during a certain time frame if the antigen is immobilized on a solid phase compared to complexes formed concentration of the antibodies/antigens of interest in the patient’s sample, the when the antigen is free in solution. 46 47 However, it is important to consider how the three-dimensional structure of the reporter in these assays such as acridinium, together with the substrate, can antigen can change when used in serological assays. Antigens and antibodies create a chemiluminescent reaction, which emits light and is measured as can be conformationally altered by immobilization to solid phases (329). Some relative light units. The signal is directly proportional to the concentration of assays, including Western blot, use denatured proteins, which limit the three- antigen/antibodies in the sample. Assays using magnetic microparticles as dimensional structure of the proteins and the presentation of conformational solid phase are often termed chemiluminescence microparticle immunoassays epitopes. Other assays, such as immunofluorescence with virus-infected cells, (CMIA). CLIA methods have been widely used in virology laboratories due to will to a greater extent retain the natural forms of the proteins. the ability to analyze many samples in a relatively short time, the high degree of automation and most importantly, the fact that the methods are sensitive and Enzyme-linked immunosorbent assay specific (271, 339-341). Enzyme-linked immunosorbent assay (ELISA) and enzyme immunoassay Line immunoassay (EIA) were developed at the same time by different research groups in Sweden and the Netherlands (333, 334). The methods are based on the same principles The principle of line immunoassay is that antigens to be tested are adsorbed in for detection of antibodies/antigens as described above (328, 335). The separate bands on a membrane strip of e.g. nylon. The patient sample is added methods can detect small quantities of antigens and antibodies in fluid samples and if it contains complementary antibodies to any of the antigens, these such as serum and CSF. In both ELISA and EIA, an enzyme is used as the antibodies will bind to the respective antigen during the incubation period. reporter to detect biological molecules. The most used enzymes are probably Unbound antibodies are then washed away. To detect the bound antibodies, a alkaline phosphatase and horseradish peroxidase. The enzyme signal will secondary antibody conjugated to an enzyme is added. After incubation, increase over time by continuing to turnover more substrate unless a stop washing is performed to remove unbound secondary antibodies. The substrate solution is added to the assay. Microtiter plates are often used as a solid phase is then added. Positive reactions are seen as bands on the strip. Each test strip in these assays. ELISA/EIA methods are widespread throughout the world with also contains control bands to determine that sufficient sample material has many adaptations (336). been added to the strip. Radioimmunoassay Western blot The radioimmunoassay (RIA) was first described in 1960 by Rosalyn Yalow Western blot is an assay technique where sample identification occurs in part and Solomon Berson for measuring endogenous plasma insulin (337). The through molecular weight and specific antibody binding. After denaturation of development of the RIA was awarded the Nobel Prize in 1977, but only Yalow the proteins in the antigen, they are separated by electrophoresis, usually in was able to receive the prize since Berson passed away in 1972 (337). The polyacrylamide gel. The proteins migrate in the gel and the final position basic principles of solid-phase immunoassays apply to RIA. Radioisotopes are depends on the size, conformation, and charge of the protein. Small proteins used as reporters for the reaction. Although the methods are sensitive and travel longer compared with large proteins. The separated proteins can then be specific, the problems with radioisotopes when it comes to laboratory safety stained directly on the gel with e.g. Coomassie blue or transferred with and the aspect of disposal of radioactive waste have prompted the development electrical voltage to a membrane, e.g. nitrocellulose membrane. If the proteins of other techniques that use other reporters for the reactions. are transferred to a membrane, the same overarching principles as previously presented can be used for detection of antigen/antibodies. Chemiluminescence immunoassays Direct detection to determine if the protein of interest is present can be done Chemiluminescence immunoassays (CLIA) determine the concentration of by adding reporter-labeled antibodies and substrate. Indirect detection can be antigen/antibodies by the intensity of the luminescence that is emitted from the performed by adding a patient sample or a monoclonal antibody along with a chemical reaction in the assay (338). The basic principles of solid-phase secondary reporter-labeled antibody (conjugate). The reporter can for example immunoassays also apply to CLIA. These methods can have direct or indirect be an enzyme which, together with the substrate, creates a color change on the detection, be sandwich assays and be competitive or non-competitive. The membrane. The thickness of the band gives an indication of the amount of 48 49 However, it is important to consider how the three-dimensional structure of the reporter in these assays such as acridinium, together with the substrate, can antigen can change when used in serological assays. Antigens and antibodies create a chemiluminescent reaction, which emits light and is measured as can be conformationally altered by immobilization to solid phases (329). Some relative light units. The signal is directly proportional to the concentration of assays, including Western blot, use denatured proteins, which limit the three- antigen/antibodies in the sample. Assays using magnetic microparticles as dimensional structure of the proteins and the presentation of conformational solid phase are often termed chemiluminescence microparticle immunoassays epitopes. Other assays, such as immunofluorescence with virus-infected cells, (CMIA). CLIA methods have been widely used in virology laboratories due to will to a greater extent retain the natural forms of the proteins. the ability to analyze many samples in a relatively short time, the high degree of automation and most importantly, the fact that the methods are sensitive and Enzyme-linked immunosorbent assay specific (271, 339-341). Enzyme-linked immunosorbent assay (ELISA) and enzyme immunoassay Line immunoassay (EIA) were developed at the same time by different research groups in Sweden and the Netherlands (333, 334). The methods are based on the same principles The principle of line immunoassay is that antigens to be tested are adsorbed in for detection of antibodies/antigens as described above (328, 335). The separate bands on a membrane strip of e.g. nylon. The patient sample is added methods can detect small quantities of antigens and antibodies in fluid samples and if it contains complementary antibodies to any of the antigens, these such as serum and CSF. In both ELISA and EIA, an enzyme is used as the antibodies will bind to the respective antigen during the incubation period. reporter to detect biological molecules. The most used enzymes are probably Unbound antibodies are then washed away. To detect the bound antibodies, a alkaline phosphatase and horseradish peroxidase. The enzyme signal will secondary antibody conjugated to an enzyme is added. After incubation, increase over time by continuing to turnover more substrate unless a stop washing is performed to remove unbound secondary antibodies. The substrate solution is added to the assay. Microtiter plates are often used as a solid phase is then added. Positive reactions are seen as bands on the strip. Each test strip in these assays. ELISA/EIA methods are widespread throughout the world with also contains control bands to determine that sufficient sample material has many adaptations (336). been added to the strip. Radioimmunoassay Western blot The radioimmunoassay (RIA) was first described in 1960 by Rosalyn Yalow Western blot is an assay technique where sample identification occurs in part and Solomon Berson for measuring endogenous plasma insulin (337). The through molecular weight and specific antibody binding. After denaturation of development of the RIA was awarded the Nobel Prize in 1977, but only Yalow the proteins in the antigen, they are separated by electrophoresis, usually in was able to receive the prize since Berson passed away in 1972 (337). The polyacrylamide gel. The proteins migrate in the gel and the final position basic principles of solid-phase immunoassays apply to RIA. Radioisotopes are depends on the size, conformation, and charge of the protein. Small proteins used as reporters for the reaction. Although the methods are sensitive and travel longer compared with large proteins. The separated proteins can then be specific, the problems with radioisotopes when it comes to laboratory safety stained directly on the gel with e.g. Coomassie blue or transferred with and the aspect of disposal of radioactive waste have prompted the development electrical voltage to a membrane, e.g. nitrocellulose membrane. If the proteins of other techniques that use other reporters for the reactions. are transferred to a membrane, the same overarching principles as previously presented can be used for detection of antigen/antibodies. Chemiluminescence immunoassays Direct detection to determine if the protein of interest is present can be done Chemiluminescence immunoassays (CLIA) determine the concentration of by adding reporter-labeled antibodies and substrate. Indirect detection can be antigen/antibodies by the intensity of the luminescence that is emitted from the performed by adding a patient sample or a monoclonal antibody along with a chemical reaction in the assay (338). The basic principles of solid-phase secondary reporter-labeled antibody (conjugate). The reporter can for example immunoassays also apply to CLIA. These methods can have direct or indirect be an enzyme which, together with the substrate, creates a color change on the detection, be sandwich assays and be competitive or non-competitive. The membrane. The thickness of the band gives an indication of the amount of 48 49 antigen. Western blot provides good control of the specificity of antibody Hemagglutination inhibition assay binding and is often used as a confirmatory test. The hemagglutination inhibition assay was among the first serological assays Immunofluorescence in diagnostic virology. The method is based on the hemagglutination process where viral hemagglutinin glycoproteins on the surface of certain viruses e.g. Immunofluorescence methods have been widely used in virology laboratories. influenza virus, binds to the sialic acid receptors on the surface of erythrocytes The methods can detect antigen/antibodies by using a fluorochrome as a and cause the red blood cells (RBCs) to agglutinate together in complexes. The reporter which together with added substrate can emit fluorescent color. presence of specific antibodies will inhibit this process and thus the viruses Common fluorochromes are fluorescein which gives a yellow-green color and will not agglutinate the erythrocytes. The method is limited for viruses with rhodamine which gives a red color (70). A fluorescence microscope is used to hemagglutination glycoproteins e.g. influenza virus, MeV, rubella virus (RV) observe the reaction. Virus-infected cells that attach to glass slides are often and dengue virus (342-344). used in these assays. In direct immunofluorescence, the primary antibody labeled with a fluorochrome together with the substrate can directly detect the Latex agglutination test viral protein. This can be used to detect virus-infected cells. In a latex agglutination test, latex beads are coated with the virus of interest, In indirect immunofluorescence, antibodies to a particular virus can be e.g. RV. These beads are added to the patient’s serum sample. If the serum detected. The patient's sample is added to virus-infected cells. If the sample sample contains antibodies to the virus, the antibodies will agglutinate with the contains antibodies to the virus, the antibodies will attach to the virus-infected beads. The agglutination titer is the highest serum dilution where there are cells. These antibodies can then be detected using a secondary antibody labeled enough antibodies for agglutination to occur (345). Most patients, with a with a fluorochrome. Immunofluorescence is a rather labor-intensive method, symptomatic primary EBV infection, will develop heterophilic antibodies i.e. which cannot be automated, and which takes a long time to master. An IgM antibodies produced due to the polyclonal response of EBV-infected B advantage of the method is that it is possible to locate where on the virus- cells. These antibodies can agglutinate mammalian erythrocytes. Detection of infected cell the antibodies bind. heterophilic antibodies is a rapid, simple, and widely used method for diagnosing EBV-induced mononucleosis (264). Monospot, a commonly used test, is a latex agglutination assay that uses equine erythrocytes, and if the patient's sample contains heterophilic antibodies, the antibodies will agglutinate the erythrocytes (204, 264). Complement fixation test The complement fixation test is another example of an early serological method. The serum sample from the patient is heated so that the complement proteins but not the antibodies in the sample are destroyed. Subsequently, a standardized amount of complement proteins and the antigen of interest are added to the sample. If the serum sample contains antibodies to the added antigen, the formation of antibody-antigen complexes will occur. If complexes are formed, the complement proteins will bind to the complexes. In the next step, animal RBCs carrying a complement-binding antibody are added. If large amounts of antigen-antibody complexes have been formed, there will be no Figure 18. An immunofluorescence photograph showing fluorescent stained anti- free complement proteins in the solution as they will have bound to the varicella-zoster virus glycoprotein E (VZVgE) monoclonal IgG antibodies that have complexes, but if there are not enough antigen-antibody complexes to bind all attached to VZV-infected cells. the complement proteins, the remaining ones will bind and lyse the RBCs (70). 50 51 antigen. Western blot provides good control of the specificity of antibody Hemagglutination inhibition assay binding and is often used as a confirmatory test. The hemagglutination inhibition assay was among the first serological assays Immunofluorescence in diagnostic virology. The method is based on the hemagglutination process where viral hemagglutinin glycoproteins on the surface of certain viruses e.g. Immunofluorescence methods have been widely used in virology laboratories. influenza virus, binds to the sialic acid receptors on the surface of erythrocytes The methods can detect antigen/antibodies by using a fluorochrome as a and cause the red blood cells (RBCs) to agglutinate together in complexes. The reporter which together with added substrate can emit fluorescent color. presence of specific antibodies will inhibit this process and thus the viruses Common fluorochromes are fluorescein which gives a yellow-green color and will not agglutinate the erythrocytes. The method is limited for viruses with rhodamine which gives a red color (70). A fluorescence microscope is used to hemagglutination glycoproteins e.g. influenza virus, MeV, rubella virus (RV) observe the reaction. Virus-infected cells that attach to glass slides are often and dengue virus (342-344). used in these assays. In direct immunofluorescence, the primary antibody labeled with a fluorochrome together with the substrate can directly detect the Latex agglutination test viral protein. This can be used to detect virus-infected cells. In a latex agglutination test, latex beads are coated with the virus of interest, In indirect immunofluorescence, antibodies to a particular virus can be e.g. RV. These beads are added to the patient’s serum sample. If the serum detected. The patient's sample is added to virus-infected cells. If the sample sample contains antibodies to the virus, the antibodies will agglutinate with the contains antibodies to the virus, the antibodies will attach to the virus-infected beads. The agglutination titer is the highest serum dilution where there are cells. These antibodies can then be detected using a secondary antibody labeled enough antibodies for agglutination to occur (345). Most patients, with a with a fluorochrome. Immunofluorescence is a rather labor-intensive method, symptomatic primary EBV infection, will develop heterophilic antibodies i.e. which cannot be automated, and which takes a long time to master. An IgM antibodies produced due to the polyclonal response of EBV-infected B advantage of the method is that it is possible to locate where on the virus- cells. These antibodies can agglutinate mammalian erythrocytes. Detection of infected cell the antibodies bind. heterophilic antibodies is a rapid, simple, and widely used method for diagnosing EBV-induced mononucleosis (264). Monospot, a commonly used test, is a latex agglutination assay that uses equine erythrocytes, and if the patient's sample contains heterophilic antibodies, the antibodies will agglutinate the erythrocytes (204, 264). Complement fixation test The complement fixation test is another example of an early serological method. The serum sample from the patient is heated so that the complement proteins but not the antibodies in the sample are destroyed. Subsequently, a standardized amount of complement proteins and the antigen of interest are added to the sample. If the serum sample contains antibodies to the added antigen, the formation of antibody-antigen complexes will occur. If complexes are formed, the complement proteins will bind to the complexes. In the next step, animal RBCs carrying a complement-binding antibody are added. If large amounts of antigen-antibody complexes have been formed, there will be no Figure 18. An immunofluorescence photograph showing fluorescent stained anti- free complement proteins in the solution as they will have bound to the varicella-zoster virus glycoprotein E (VZVgE) monoclonal IgG antibodies that have complexes, but if there are not enough antigen-antibody complexes to bind all attached to VZV-infected cells. the complement proteins, the remaining ones will bind and lyse the RBCs (70). 50 51 Neutralization assay has begun to experience neurological symptoms, which is when most patients seek medical attention. Serological assays do not require active parts of the Neutralization assay is a clinically relevant method for identifying the presence virus for analysis, which may facilitate sampling. and magnitude of functional antibodies that can prevent viral infection (346). The patient's serum in different dilutions and a defined amount of the infectious virus will be added to cell cultures. The cells will be infected by the virus if there are not enough neutralizing antibodies that will bind to the surface of the 1.4.3 LIMITATIONS OF SEROLOGICAL METHODS virions and prevent them from entering the cells. Whether the virus has It is not feasible to perform serological analyzes without having an aim for the infected the cell culture can be determined by the presence or absence of investigation. It is thus important to decide on the basis of the patient's history cytopathic cell effects or other evidence of ongoing infection using and symptoms which antigens/antibodies are to be analyzed. immunoreactive techniques. The neutralization titer is the highest serum dilution that prevents infection of the cells. Neutralization assays are labor Serological interpretation intensive but may be warranted to use in some cases to examine immunity including after vaccination. The interpretation of serological results can be complicated. The serological response is affected by the type of virus, whether it is a primary infection, a chronic infection, a reactivation or a reinfection, the length of time that has elapsed since the time of infection and the immune system of the infected 1.4.2 ADVANTAGES OF SEROLOGICAL METHODS individual. The test result can also be affected by the method used to detect Many of the serological methods currently in use are sensitive, specific, antigen/antibodies. Viruses have different abilities to induce antibody reproducible, fast, and easy to perform. Some of the methods can be automated, production and individuals have different abilities to generate antibodies. The which minimizes the risk of human error and increases the ability to analyze normal level of antibodies in healthy people also varies depending on, among many samples in a short time. Serological analyzes are often inexpensive, other things, the patient's age and immunocompetence. Serological results making them cost-effective. Some serological assays are suitable as screening should be interpreted in a clinical context where the patient's disease panorama methods for infectious diseases in blood donors, during pregnancy, before and disease course are considered. It is also important to consider whether it is transplantations and before surgeries. In addition, serological methods are a common or rare viral disease and whether the individual has received blood important for viral epidemiological studies in the population and to increase products as this may result in a passive transmission of antibodies that can be our knowledge of various viral diseases. detected in assays and cause confusion. Serological assays can help determine different phases of an infection, for Antibody dynamics instance to define different stages of hepatitis B. Serological tests for hepatitis B include analyzes of several hepatitis B-specific antibodies and antigens. The A serological analysis provides a snapshot of a dynamic situation and repeated different combinations of serological responses to these antibodies and sampling may therefore be necessary for conclusions to be drawn. When antigens can be used to determine if a patient has acute or chronic hepatitis B. antibodies cannot be detected against a particular virus, it may be because the Hepatitis B serology can also demonstrate if an individual is immune to individual is uninfected by the virus, but it may also be because antibodies have hepatitis B due to previous infection/vaccination or is susceptible to the not yet been produced. For some viral infections it can take quite some time disease. from the time of infection until seroconversion occurs e.g. in infections with hepatitis C virus. The patient may thus have symptoms and be contagious, but Serology can be used in later stages of a disease and after a disease outbreak the diagnosis cannot be based on antibody detection, this situation is called the when it is no longer possible to detect viral RNA/DNA by PCR. To give an serological window phase of infection (347-349). This is an important reason example, serological methods are the primary diagnostic method for why many western countries use PCR for screening blood donors (350). The diagnosing tick-borne encephalitis (TBE) because there is little chance of window phase for serological diagnosis of some infections e.g. human detecting viral RNA during the second phase of the disease when the patient 52 53 Neutralization assay has begun to experience neurological symptoms, which is when most patients seek medical attention. Serological assays do not require active parts of the Neutralization assay is a clinically relevant method for identifying the presence virus for analysis, which may facilitate sampling. and magnitude of functional antibodies that can prevent viral infection (346). The patient's serum in different dilutions and a defined amount of the infectious virus will be added to cell cultures. The cells will be infected by the virus if there are not enough neutralizing antibodies that will bind to the surface of the 1.4.3 LIMITATIONS OF SEROLOGICAL METHODS virions and prevent them from entering the cells. Whether the virus has It is not feasible to perform serological analyzes without having an aim for the infected the cell culture can be determined by the presence or absence of investigation. It is thus important to decide on the basis of the patient's history cytopathic cell effects or other evidence of ongoing infection using and symptoms which antigens/antibodies are to be analyzed. immunoreactive techniques. The neutralization titer is the highest serum dilution that prevents infection of the cells. Neutralization assays are labor Serological interpretation intensive but may be warranted to use in some cases to examine immunity including after vaccination. The interpretation of serological results can be complicated. The serological response is affected by the type of virus, whether it is a primary infection, a chronic infection, a reactivation or a reinfection, the length of time that has elapsed since the time of infection and the immune system of the infected 1.4.2 ADVANTAGES OF SEROLOGICAL METHODS individual. The test result can also be affected by the method used to detect Many of the serological methods currently in use are sensitive, specific, antigen/antibodies. Viruses have different abilities to induce antibody reproducible, fast, and easy to perform. Some of the methods can be automated, production and individuals have different abilities to generate antibodies. The which minimizes the risk of human error and increases the ability to analyze normal level of antibodies in healthy people also varies depending on, among many samples in a short time. Serological analyzes are often inexpensive, other things, the patient's age and immunocompetence. Serological results making them cost-effective. Some serological assays are suitable as screening should be interpreted in a clinical context where the patient's disease panorama methods for infectious diseases in blood donors, during pregnancy, before and disease course are considered. It is also important to consider whether it is transplantations and before surgeries. In addition, serological methods are a common or rare viral disease and whether the individual has received blood important for viral epidemiological studies in the population and to increase products as this may result in a passive transmission of antibodies that can be our knowledge of various viral diseases. detected in assays and cause confusion. Serological assays can help determine different phases of an infection, for Antibody dynamics instance to define different stages of hepatitis B. Serological tests for hepatitis B include analyzes of several hepatitis B-specific antibodies and antigens. The A serological analysis provides a snapshot of a dynamic situation and repeated different combinations of serological responses to these antibodies and sampling may therefore be necessary for conclusions to be drawn. When antigens can be used to determine if a patient has acute or chronic hepatitis B. antibodies cannot be detected against a particular virus, it may be because the Hepatitis B serology can also demonstrate if an individual is immune to individual is uninfected by the virus, but it may also be because antibodies have hepatitis B due to previous infection/vaccination or is susceptible to the not yet been produced. For some viral infections it can take quite some time disease. from the time of infection until seroconversion occurs e.g. in infections with hepatitis C virus. The patient may thus have symptoms and be contagious, but Serology can be used in later stages of a disease and after a disease outbreak the diagnosis cannot be based on antibody detection, this situation is called the when it is no longer possible to detect viral RNA/DNA by PCR. To give an serological window phase of infection (347-349). This is an important reason example, serological methods are the primary diagnostic method for why many western countries use PCR for screening blood donors (350). The diagnosing tick-borne encephalitis (TBE) because there is little chance of window phase for serological diagnosis of some infections e.g. human detecting viral RNA during the second phase of the disease when the patient 52 53 immunodeficiency virus (HIV) has been shortened by introducing combination have such low levels of antibodies that they fall below the established detection tests that detect both HIV antigen and anti-HIV antibodies (351, 352). limit for the assay. Some individuals produce such low levels of antibodies that they may never Depending on how the analysis is to be used, it may be more important to reach the established detection limit and these individuals may therefore be prioritize sensitivity or specificity. When screening for transmissible infections seronegative in the serological analysis even though they have been such as HIV and hepatitis C, it is important to use very sensitive assays so as infected/vaccinated. This may be the case for some immunocompromised not to miss any true positive cases. The lack of specificity can be supplemented individuals who are unable to generate an adequate antibody response to by other more specific methods. However, in rare cases, even with further infections (353). There are also rare cases of individuals with hepatitis C virus analyzes, it can be difficult to determine with certainty whether the reactivity infection who will not generate antibodies to the virus (354). Another example is due to a true antibody response or if it is non-specific (357). The is that approximately 5–10% of healthy individuals do not generate anti- disadvantage of the lack of specificity includes not being able to use donated EBNA1 IgG after an EBV infection (204, 272). Antibodies are also not blood products due to false positive or indeterminate test results and possible generated at an exact time after a certain infection because there are differences negative effects of notifying individuals of deviating test results (358). in the kinetics of individuals' antibody production. For instance, it has been shown that most individuals generate anti-EBVgp350 IgG before the onset of Predictive values clinical symptoms (274, 355) but this is not true for everyone (274, 356). The variations between different individuals' antibody responses can be a challenge The positive predictive value is the proportion of patients with a positive test when developing and interpreting serological analyzes. result who truly have the disease, while the negative predictive value is the proportion of patients with a negative test who truly do not have the disease. If the serological diagnosis needs to be determined by taking acute and These predictive values depend on the sensitivity and specificity of the assay convalescence samples, the long time between samples will relatively often but also on the prevalence of the disease in the population. When implementing allow the patient to recover before the diagnosis is determined. As an example, a diagnostic test for a particular disease in a clinical laboratory, it is therefore a primary VZV infection or reactivation can be correctly diagnosed by important to consider how the prevalence of that disease will affect the analyzing acute and convalescence samples, to detect seroconversion or performance of the assay. significant titer increase, but it will probably not be so useful as it takes a long time for the antibodies to be generated and PCR can provide a faster diagnosis Cross-reactivity in atypical cases (107). The sensitivity and specificity of all viral serological methods depend on the Sensitivity and specificity binding between antibodies and antigen. Whole virus antigens are often used as serological antigens. These antigens are produced by infecting cell cultures In the perfect serological assay, all individuals with the viral antigens or with the virus of interest, allowing virus replication to take place, and finally antibodies of interest would be positive and all individuals lacking the extracting the virus proteins. Whole virus antigens contain various proteins antigens/antibodies would be negative. Unfortunately, it is impossible to from the virus but also residues from the infected cells. Thus, antibodies with develop these perfect assays. Serological analyzes have different established different specificity may attach to many different epitopes on the viral proteins, limits for what counts as positive or negative results. The test may also have a which may increase the sensitivity of the assay, but there is a risk that the gray area where it is uncertain whether the detected reactivity is due to true remaining cell components give rise to unwanted responses. antibody/antigen reactivity or if it is false/non-specific. The limits are established after analysis of many samples that have been determined to be true The disadvantage of whole virus antigens is also that closely related viruses positive or negative. For serological analyzes, it is practically impossible to set may have similar epitopes and if many proteins are included in the antigen, the limits that provide both 100% sensitivity and specificity. Some true negative risk increases that these similar epitopes, called cross-reactive epitopes, are samples may show non-specific reactivity and some true positive samples may included in the antigen. Cross-reactive antibodies that target an epitope on a particular virus can then instead bind to a cross-reactive epitope on a closely 54 55 immunodeficiency virus (HIV) has been shortened by introducing combination have such low levels of antibodies that they fall below the established detection tests that detect both HIV antigen and anti-HIV antibodies (351, 352). limit for the assay. Some individuals produce such low levels of antibodies that they may never Depending on how the analysis is to be used, it may be more important to reach the established detection limit and these individuals may therefore be prioritize sensitivity or specificity. When screening for transmissible infections seronegative in the serological analysis even though they have been such as HIV and hepatitis C, it is important to use very sensitive assays so as infected/vaccinated. This may be the case for some immunocompromised not to miss any true positive cases. The lack of specificity can be supplemented individuals who are unable to generate an adequate antibody response to by other more specific methods. However, in rare cases, even with further infections (353). There are also rare cases of individuals with hepatitis C virus analyzes, it can be difficult to determine with certainty whether the reactivity infection who will not generate antibodies to the virus (354). Another example is due to a true antibody response or if it is non-specific (357). The is that approximately 5–10% of healthy individuals do not generate anti- disadvantage of the lack of specificity includes not being able to use donated EBNA1 IgG after an EBV infection (204, 272). Antibodies are also not blood products due to false positive or indeterminate test results and possible generated at an exact time after a certain infection because there are differences negative effects of notifying individuals of deviating test results (358). in the kinetics of individuals' antibody production. For instance, it has been shown that most individuals generate anti-EBVgp350 IgG before the onset of Predictive values clinical symptoms (274, 355) but this is not true for everyone (274, 356). The variations between different individuals' antibody responses can be a challenge The positive predictive value is the proportion of patients with a positive test when developing and interpreting serological analyzes. result who truly have the disease, while the negative predictive value is the proportion of patients with a negative test who truly do not have the disease. If the serological diagnosis needs to be determined by taking acute and These predictive values depend on the sensitivity and specificity of the assay convalescence samples, the long time between samples will relatively often but also on the prevalence of the disease in the population. When implementing allow the patient to recover before the diagnosis is determined. As an example, a diagnostic test for a particular disease in a clinical laboratory, it is therefore a primary VZV infection or reactivation can be correctly diagnosed by important to consider how the prevalence of that disease will affect the analyzing acute and convalescence samples, to detect seroconversion or performance of the assay. significant titer increase, but it will probably not be so useful as it takes a long time for the antibodies to be generated and PCR can provide a faster diagnosis Cross-reactivity in atypical cases (107). The sensitivity and specificity of all viral serological methods depend on the Sensitivity and specificity binding between antibodies and antigen. Whole virus antigens are often used as serological antigens. These antigens are produced by infecting cell cultures In the perfect serological assay, all individuals with the viral antigens or with the virus of interest, allowing virus replication to take place, and finally antibodies of interest would be positive and all individuals lacking the extracting the virus proteins. Whole virus antigens contain various proteins antigens/antibodies would be negative. Unfortunately, it is impossible to from the virus but also residues from the infected cells. Thus, antibodies with develop these perfect assays. Serological analyzes have different established different specificity may attach to many different epitopes on the viral proteins, limits for what counts as positive or negative results. The test may also have a which may increase the sensitivity of the assay, but there is a risk that the gray area where it is uncertain whether the detected reactivity is due to true remaining cell components give rise to unwanted responses. antibody/antigen reactivity or if it is false/non-specific. The limits are established after analysis of many samples that have been determined to be true The disadvantage of whole virus antigens is also that closely related viruses positive or negative. For serological analyzes, it is practically impossible to set may have similar epitopes and if many proteins are included in the antigen, the limits that provide both 100% sensitivity and specificity. Some true negative risk increases that these similar epitopes, called cross-reactive epitopes, are samples may show non-specific reactivity and some true positive samples may included in the antigen. Cross-reactive antibodies that target an epitope on a particular virus can then instead bind to a cross-reactive epitope on a closely 54 55 related virus. This type of cross-reactivity can, for instance, cause diagnostic in these cases is not a marker for acute/recent infection. Diagnosing problems in analyzes of antibodies against flaviviruses (359-361) and breakthrough infections of TBE virus and MeV in vaccinated individuals can alphaherpesviruses (157-159, 162, 323). Narrowing down the number of viral be complicated by a later or absent IgM response (287, 371). proteins in the antigen to a single protein or to peptides can lead to higher specificity but also loss of sensitivity. This is especially important to keep in Immune complexes mind when using peptide antigens as they only consist of short stretches of amino acids with few epitopes and probably no conformational epitopes. In some viral infections such as hepatitis B, immune complexes can cause diagnostic problems. Antibodies to hepatitis B bound to viral antigen in Non-specific binding complexes can prevent antibody detection in the serological assays and the patient becomes false negative (372). It is important to consider the risk of non-specific binding in the serological assays and how this may interfere with the performance of the analyzes. A Technical difficulties disadvantage of the passive adhesion of antigen to a solid phase is the risk of attachment of unwanted proteins remaining from antigen production. The As with all diagnostic methods, technical problems can arise when performing antigen may have cell residues or other components left over from production serological analyzes. Pipetting errors can occur both when the assays are that may adhere to the solid phase and cause unwanted binding to antibodies performed manually and mechanically, even if the risk of manual errors is in the patient's sample. Antibodies in human samples can also directly attach higher. Self-thawing freezers should not be used to store reagents as the freeze- to parts of the solid phase where antigen has not bound and create false positive thaw cycles can destroy protein activity, causing the proteins to be denatured reactions. The extent to which this occurs depends on the components of the or inactivated. The buffer system used must be compatible with other reagents. individual sample and how well the blockage of the wells prevents this non- Buffers containing phosphate can interfere with the commonly used enzyme specific binding. False positive reactions can also occur due to nonspecific alkaline phosphatase and reduce enzyme activity. Samples with endogenous binding of antibodies to antigens through protein-to-protein interactions. phosphates can also interfere with the enzyme. When the plates are washed, Nonspecific reactions due to unwanted binding to the secondary antibody are too high a vacuum can dry out the coated protein and too low a vacuum can also well known (362). Another established factor for interference in leave residual liquid in the wells. It is also important not to let the wells dry serological analyzes is the rheumatoid factor (287, 363-365). In addition, both out between different steps as drying can mean loss of protein activity. false negative and false positive reactions can occur due to unwanted interactions between reactants and buffer components (362, 366). A well-known problem with ELISA is the "edge effect" which is a phenomenon where the optical density (OD) values of the peripheral wells IgM assays compared with the central wells of the microtiter plate have higher or lower values than expected. This is often due to differences in temperature or lighting Diagnostic problems with cross-reactivity and non-specific binding that cause between the central and peripheral wells. The microplates are often incubated false positive results are usually a larger problem in IgM assays compared with at 37 °C instead of room temperature to shorten the incubation time. The IgG assays and it has therefore generally been more difficult to develop problem may be that the peripheral wells reach higher temperatures faster specific IgM assays (163, 365, 367-369). This may be because IgM molecules compared with the central wells. If the substrate is light-sensitive and the plates generally have lower affinity compared with IgG molecules, but still have are developed in a room with strong light, the wells closest to the light source relatively high avidity due to the many paratopes present on an IgM molecule. may have a higher development effect compared with the other wells. Other diagnostic problems with IgM assays includes polyclonal antibody responses. In response to certain viral infections, such as a primary infection with EBV, B cells can produce IgM against several different viruses, which is called polyclonal B-cell activation (370). In some infections, the IgM antibody response remains for a long time even if the infection is no longer in an active phase. It can then be difficult to determine the stage of infection because IgM 56 57 related virus. This type of cross-reactivity can, for instance, cause diagnostic in these cases is not a marker for acute/recent infection. Diagnosing problems in analyzes of antibodies against flaviviruses (359-361) and breakthrough infections of TBE virus and MeV in vaccinated individuals can alphaherpesviruses (157-159, 162, 323). Narrowing down the number of viral be complicated by a later or absent IgM response (287, 371). proteins in the antigen to a single protein or to peptides can lead to higher specificity but also loss of sensitivity. This is especially important to keep in Immune complexes mind when using peptide antigens as they only consist of short stretches of amino acids with few epitopes and probably no conformational epitopes. In some viral infections such as hepatitis B, immune complexes can cause diagnostic problems. Antibodies to hepatitis B bound to viral antigen in Non-specific binding complexes can prevent antibody detection in the serological assays and the patient becomes false negative (372). It is important to consider the risk of non-specific binding in the serological assays and how this may interfere with the performance of the analyzes. A Technical difficulties disadvantage of the passive adhesion of antigen to a solid phase is the risk of attachment of unwanted proteins remaining from antigen production. The As with all diagnostic methods, technical problems can arise when performing antigen may have cell residues or other components left over from production serological analyzes. Pipetting errors can occur both when the assays are that may adhere to the solid phase and cause unwanted binding to antibodies performed manually and mechanically, even if the risk of manual errors is in the patient's sample. Antibodies in human samples can also directly attach higher. Self-thawing freezers should not be used to store reagents as the freeze- to parts of the solid phase where antigen has not bound and create false positive thaw cycles can destroy protein activity, causing the proteins to be denatured reactions. The extent to which this occurs depends on the components of the or inactivated. The buffer system used must be compatible with other reagents. individual sample and how well the blockage of the wells prevents this non- Buffers containing phosphate can interfere with the commonly used enzyme specific binding. False positive reactions can also occur due to nonspecific alkaline phosphatase and reduce enzyme activity. Samples with endogenous binding of antibodies to antigens through protein-to-protein interactions. phosphates can also interfere with the enzyme. When the plates are washed, Nonspecific reactions due to unwanted binding to the secondary antibody are too high a vacuum can dry out the coated protein and too low a vacuum can also well known (362). Another established factor for interference in leave residual liquid in the wells. It is also important not to let the wells dry serological analyzes is the rheumatoid factor (287, 363-365). In addition, both out between different steps as drying can mean loss of protein activity. false negative and false positive reactions can occur due to unwanted interactions between reactants and buffer components (362, 366). A well-known problem with ELISA is the "edge effect" which is a phenomenon where the optical density (OD) values of the peripheral wells IgM assays compared with the central wells of the microtiter plate have higher or lower values than expected. This is often due to differences in temperature or lighting Diagnostic problems with cross-reactivity and non-specific binding that cause between the central and peripheral wells. The microplates are often incubated false positive results are usually a larger problem in IgM assays compared with at 37 °C instead of room temperature to shorten the incubation time. The IgG assays and it has therefore generally been more difficult to develop problem may be that the peripheral wells reach higher temperatures faster specific IgM assays (163, 365, 367-369). This may be because IgM molecules compared with the central wells. If the substrate is light-sensitive and the plates generally have lower affinity compared with IgG molecules, but still have are developed in a room with strong light, the wells closest to the light source relatively high avidity due to the many paratopes present on an IgM molecule. may have a higher development effect compared with the other wells. Other diagnostic problems with IgM assays includes polyclonal antibody responses. In response to certain viral infections, such as a primary infection with EBV, B cells can produce IgM against several different viruses, which is called polyclonal B-cell activation (370). In some infections, the IgM antibody response remains for a long time even if the infection is no longer in an active phase. It can then be difficult to determine the stage of infection because IgM 56 57 1.5 MULTIPLE SCLEROSIS autopsy studies showing active inflammation and demyelination in the CNS also during end stages of MS and obvious degeneration of both white and grey Multiple sclerosis (MS) is a chronic inflammatory disease in the CNS. The matter of the brain in early stages of MS (378). A new theory of the course of etiology and pathogenesis are not established, but most hypotheses suggest an the disease is that there is a diffuse smouldering pathological process were the interaction between genes, especially those that control the immune system, disease affects the entire CNS (376). The theory behind smouldering MS is and environmental risk factors such as low vitamin D levels, low sun exposure that MS is a single-stage disease and that even early in the disease, not only and smoking (373, 374). These risk factors are believed to trigger autoimmune relapses occur, which cause acute focal damage with axonal damage but also reactions to myelin, which involves both T and B cells. slower processes such as demyelination and energy deficits that eventually lead to neurological deterioration with atrophy of the brain and the spinal cord The damage mechanisms involve both demyelinating and axonal damage, the (376). It is unclear to what extent these delayed processes are dependent on the latter of which leads to irreversible disability. Demyelination and axonal focal inflammation. Later, post-neurodegenerative processes occur and with damage reduce or inhibit the impulse transmission along the axons. The name increasing age, age-related neurodegenerative processes also contribute to multiple sclerosis comes from multiple scarring that occurs due to the patients losing neurological function (376). The finding that accumulation of inflammation-causing lesions in the CNS. They are visible as hyperintense T2- disability in patients with RRMS is not associated with relapses supports this weighted lesions on magnetic resonance imaging (MRI) and active lesions may theory of smouldering MS (379). be detected as contrast-enhancing lesions on T1-weighted MRI scans, indicating damage of the blood-brain barrier (BBB). Epidemiology Phenotypes of MS The onset of MS occurs mainly in young adults, between 20–40 years of age, mean at 32 years (380). In Europe, the disease is a major cause of non- Based on clinical features, MS is traditionally divided into three different traumatic neurological impairment (381). Twice as many women as men suffer phenotypes, relapsing-remitting MS (RRMS), primary-progressive MS from the disease (380). The total prevalence of MS is estimated at 2.8 million (PPMS) and secondary progressive MS (SPMS). Approximately 90% of patients: 35.9 per 100,000 population (380). Sweden is one of the countries patients initially follow the RRMS course with acute exacerbations (also with the highest incidence and prevalence of MS in the world with almost 200 termed relapses or attacks) from which they fully or partially recover (375). per 100,000 persons with MS and 900–1000 new cases annually (382, 383). Although patients with RRMS may experience clinical stability between The prevalence of MS is uneven worldwide with higher prevalence in relapses, MRI reveals that lesion formation occurs mostly without new or temperate climates in both the southern and northern hemispheres, but the aggravated symptoms. increasing incidence of MS with latitude gradient is not as evident in more recent studies in the northern hemisphere (384, 385). The overall prevalence The neurological symptoms vary depending on where the lesion (s) occur in of the disease is increasing globally and especially in females (380, 384-386). the CNS. Common symptoms associated with onset of MS are regional motor and/or sensory impairment and impaired vision due to optic neuritis. After 20– Diagnosis 30 years of RRMS course, the disease will subsequently convert into SPMS. During this stage, there is a slow progression of disability with or without Diagnosing MS is performed by weighing the patient's history, clinical superimposed relapses. Patients with PPMS have a similar progressive decline symptoms, disease course, detection of CSF-specific oligoclonal IgG bands in their neurological function from the onset of the disease. (OCB), findings on MRI and fulfilling the criteria of dissemination in time and space according to the revised McDonald's criteria from 2017 (387). The Immunopathology of MS improved criteria enable earlier diagnosis and thereby earlier treatment of the disease, which can improve the course of the disease. When an MS diagnosis Traditionally, MS has been conceived as a two-stage disease, where the first cannot be established after the first exacerbation, the patient is diagnosed with stage is dominated by recurrent inflammation and the second stage is clinically isolated syndrome. dominated by a neurodegenerative phase (376, 377). Contrasting this view are 58 59 1.5 MULTIPLE SCLEROSIS autopsy studies showing active inflammation and demyelination in the CNS also during end stages of MS and obvious degeneration of both white and grey Multiple sclerosis (MS) is a chronic inflammatory disease in the CNS. The matter of the brain in early stages of MS (378). A new theory of the course of etiology and pathogenesis are not established, but most hypotheses suggest an the disease is that there is a diffuse smouldering pathological process were the interaction between genes, especially those that control the immune system, disease affects the entire CNS (376). The theory behind smouldering MS is and environmental risk factors such as low vitamin D levels, low sun exposure that MS is a single-stage disease and that even early in the disease, not only and smoking (373, 374). These risk factors are believed to trigger autoimmune relapses occur, which cause acute focal damage with axonal damage but also reactions to myelin, which involves both T and B cells. slower processes such as demyelination and energy deficits that eventually lead to neurological deterioration with atrophy of the brain and the spinal cord The damage mechanisms involve both demyelinating and axonal damage, the (376). It is unclear to what extent these delayed processes are dependent on the latter of which leads to irreversible disability. Demyelination and axonal focal inflammation. Later, post-neurodegenerative processes occur and with damage reduce or inhibit the impulse transmission along the axons. The name increasing age, age-related neurodegenerative processes also contribute to multiple sclerosis comes from multiple scarring that occurs due to the patients losing neurological function (376). The finding that accumulation of inflammation-causing lesions in the CNS. They are visible as hyperintense T2- disability in patients with RRMS is not associated with relapses supports this weighted lesions on magnetic resonance imaging (MRI) and active lesions may theory of smouldering MS (379). be detected as contrast-enhancing lesions on T1-weighted MRI scans, indicating damage of the blood-brain barrier (BBB). Epidemiology Phenotypes of MS The onset of MS occurs mainly in young adults, between 20–40 years of age, mean at 32 years (380). In Europe, the disease is a major cause of non- Based on clinical features, MS is traditionally divided into three different traumatic neurological impairment (381). Twice as many women as men suffer phenotypes, relapsing-remitting MS (RRMS), primary-progressive MS from the disease (380). The total prevalence of MS is estimated at 2.8 million (PPMS) and secondary progressive MS (SPMS). Approximately 90% of patients: 35.9 per 100,000 population (380). Sweden is one of the countries patients initially follow the RRMS course with acute exacerbations (also with the highest incidence and prevalence of MS in the world with almost 200 termed relapses or attacks) from which they fully or partially recover (375). per 100,000 persons with MS and 900–1000 new cases annually (382, 383). Although patients with RRMS may experience clinical stability between The prevalence of MS is uneven worldwide with higher prevalence in relapses, MRI reveals that lesion formation occurs mostly without new or temperate climates in both the southern and northern hemispheres, but the aggravated symptoms. increasing incidence of MS with latitude gradient is not as evident in more recent studies in the northern hemisphere (384, 385). The overall prevalence The neurological symptoms vary depending on where the lesion (s) occur in of the disease is increasing globally and especially in females (380, 384-386). the CNS. Common symptoms associated with onset of MS are regional motor and/or sensory impairment and impaired vision due to optic neuritis. After 20– Diagnosis 30 years of RRMS course, the disease will subsequently convert into SPMS. During this stage, there is a slow progression of disability with or without Diagnosing MS is performed by weighing the patient's history, clinical superimposed relapses. Patients with PPMS have a similar progressive decline symptoms, disease course, detection of CSF-specific oligoclonal IgG bands in their neurological function from the onset of the disease. (OCB), findings on MRI and fulfilling the criteria of dissemination in time and space according to the revised McDonald's criteria from 2017 (387). The Immunopathology of MS improved criteria enable earlier diagnosis and thereby earlier treatment of the disease, which can improve the course of the disease. When an MS diagnosis Traditionally, MS has been conceived as a two-stage disease, where the first cannot be established after the first exacerbation, the patient is diagnosed with stage is dominated by recurrent inflammation and the second stage is clinically isolated syndrome. dominated by a neurodegenerative phase (376, 377). Contrasting this view are 58 59 Treatment The patients in Paper V were treated with NAT and the mechanisms of action of that therapy will therefore be explained in more detail. NAT, is a There is currently no curative treatment for the disease, but for patients with recombinant, humanized monoclonal IgG4 antibody that binds to the α4 RRMS, there are several disease-modifying treatments that reduce the relapse subunit of α4β1 integrins (also known as very late antigen-4) and α4β7 rate, number of lesion formation and disability development. There is also integrins (392, 393). The α4β1 integrin, which is an adhesion molecule found evidence that the degeneration is mitigated with lower rate of brain and cervical at high levels on the surface of all leukocytes except neutrophils, binds to cord atrophy development. Treatment should be started in close proximity to vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells (394-396). the onset of the disease. The treatment is usually highly effective early in the The interaction between VCAM-1 and α4β1 integrins is important for course of the disease and may reduce the disease's impact on the long-term leukocyte adhesion and transmigration of cells across the BBB to the CNS health-related quality of life. The goal of treatment is to reduce the (395-398). inflammatory activity and thereby improve prognosis. The binding of NAT to α4β1 integrins therefore inhibits leukocyte migration Interferon beta (IFNß) was the first disease modifying therapy (DMT) into brain tissue, which reduces inflammation and prevents the formation of approved for RRMS and has been used since the mid-1990s (375). IFNß are lesions (392, 396, 399). The treatment effect of NAT is high, but the treatment antiviral cytokines that have a moderate effect on inflammation in MS. The may give rise to serious side effects such as progressive multifocal side effects are usually quite mild and the most common are skin irritation at leukoencephalopathy (PML) (393, 400). PML is caused by infection of the the injection site and influenza-like symptoms. Other treatments for MS CNS by JC polyomavirus (JCV). Treatment with NAT has also been associated include glatiramer acetate, dimethyl fumarate, diroximel fumarate, with herpesvirus infections of the CNS and primary central nervous system teriflunomide, fingolimod, ponesimod, ozanimod and the monoclonal lymphoma (401-403). antibodies natalizumab (NAT) (Tysabri®), alemtuzumab (Lemtrada®) and the anti-CD20-directed monoclonal antibodies rituximab, ocrelizumab and Etiology ofatumumab that depletes circulating B-cells. All of these medications reduce the inflammatory component of the disease through various mechanisms of The etiology behind MS is not yet fully understood but is thought to be due to action. The anti-CD20 B-cell depleting monoclonal antibody rituximab has complex interactions between genes and environmental factors. EBV, vitamin shown a high treatment effect and the off-label use of this drug has increased D, and smoking are among the most well-established environmental risk in Sweden (388-390). factors for developing the disease (245, 373, 374, 404-409). IM, which is caused by a delayed primary EBV infection, has been shown to particularly The choice of therapy is personalized and based on clinical factors such as increase the risk of MS development (246, 247, 410-412). Whether EBV is a number and severity of exacerbations, early disability development, the lesion prerequisite for developing MS is still unknown, and some researchers load and rate of new lesions on MRI, the course of the disease, age, question whether there are any true EBV seronegative patients with MS (413- comorbidities, and several other factors that include tolerability and safety. 415). A recent study suggesting EBV as a cause of MS showed that the risk of Treatment with the monoclonal antibodies NAT, alemtuzumab and those developing MS increased 32-fold after EBV infection and that increased serum targeting anti-CD20 may be more effective but there is a higher risk of severe levels of neurofilament light chain (a marker of neuroaxonal degeneration) side effects. MS exacerbations can be treated with high doses of corticosteroids occurred only after EBV seroconversion (245). or by plasmapheresis when steroids seem insufficient to halt relapse progression. Unfortunately, it has been more difficult to develop effective Antibody response therapies for progressive multiple sclerosis. The explanation may be that the pathogenic mechanisms are not as well understood, which causes problems in Most patients with MS show an intrathecal antibody production that can be developing efficient medications (391). However, ocrelizumab has recently detected as OCB by electrophoresis of CSF (416, 417). OCB have previously been approved for inflammatory active PPMS and siponimod for inflammatory been considered stable and represent a signature pattern for individual patients, active SPMS. but it has now been shown that these bands can change over a longer period of time but that the change in most cases probably is not affected by disease 60 61 Treatment The patients in Paper V were treated with NAT and the mechanisms of action of that therapy will therefore be explained in more detail. NAT, is a There is currently no curative treatment for the disease, but for patients with recombinant, humanized monoclonal IgG4 antibody that binds to the α4 RRMS, there are several disease-modifying treatments that reduce the relapse subunit of α4β1 integrins (also known as very late antigen-4) and α4β7 rate, number of lesion formation and disability development. There is also integrins (392, 393). The α4β1 integrin, which is an adhesion molecule found evidence that the degeneration is mitigated with lower rate of brain and cervical at high levels on the surface of all leukocytes except neutrophils, binds to cord atrophy development. Treatment should be started in close proximity to vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells (394-396). the onset of the disease. The treatment is usually highly effective early in the The interaction between VCAM-1 and α4β1 integrins is important for course of the disease and may reduce the disease's impact on the long-term leukocyte adhesion and transmigration of cells across the BBB to the CNS health-related quality of life. The goal of treatment is to reduce the (395-398). inflammatory activity and thereby improve prognosis. The binding of NAT to α4β1 integrins therefore inhibits leukocyte migration Interferon beta (IFNß) was the first disease modifying therapy (DMT) into brain tissue, which reduces inflammation and prevents the formation of approved for RRMS and has been used since the mid-1990s (375). IFNß are lesions (392, 396, 399). The treatment effect of NAT is high, but the treatment antiviral cytokines that have a moderate effect on inflammation in MS. The may give rise to serious side effects such as progressive multifocal side effects are usually quite mild and the most common are skin irritation at leukoencephalopathy (PML) (393, 400). PML is caused by infection of the the injection site and influenza-like symptoms. Other treatments for MS CNS by JC polyomavirus (JCV). Treatment with NAT has also been associated include glatiramer acetate, dimethyl fumarate, diroximel fumarate, with herpesvirus infections of the CNS and primary central nervous system teriflunomide, fingolimod, ponesimod, ozanimod and the monoclonal lymphoma (401-403). antibodies natalizumab (NAT) (Tysabri®), alemtuzumab (Lemtrada®) and the anti-CD20-directed monoclonal antibodies rituximab, ocrelizumab and Etiology ofatumumab that depletes circulating B-cells. All of these medications reduce the inflammatory component of the disease through various mechanisms of The etiology behind MS is not yet fully understood but is thought to be due to action. The anti-CD20 B-cell depleting monoclonal antibody rituximab has complex interactions between genes and environmental factors. EBV, vitamin shown a high treatment effect and the off-label use of this drug has increased D, and smoking are among the most well-established environmental risk in Sweden (388-390). factors for developing the disease (245, 373, 374, 404-409). IM, which is caused by a delayed primary EBV infection, has been shown to particularly The choice of therapy is personalized and based on clinical factors such as increase the risk of MS development (246, 247, 410-412). Whether EBV is a number and severity of exacerbations, early disability development, the lesion prerequisite for developing MS is still unknown, and some researchers load and rate of new lesions on MRI, the course of the disease, age, question whether there are any true EBV seronegative patients with MS (413- comorbidities, and several other factors that include tolerability and safety. 415). A recent study suggesting EBV as a cause of MS showed that the risk of Treatment with the monoclonal antibodies NAT, alemtuzumab and those developing MS increased 32-fold after EBV infection and that increased serum targeting anti-CD20 may be more effective but there is a higher risk of severe levels of neurofilament light chain (a marker of neuroaxonal degeneration) side effects. MS exacerbations can be treated with high doses of corticosteroids occurred only after EBV seroconversion (245). or by plasmapheresis when steroids seem insufficient to halt relapse progression. Unfortunately, it has been more difficult to develop effective Antibody response therapies for progressive multiple sclerosis. The explanation may be that the pathogenic mechanisms are not as well understood, which causes problems in Most patients with MS show an intrathecal antibody production that can be developing efficient medications (391). However, ocrelizumab has recently detected as OCB by electrophoresis of CSF (416, 417). OCB have previously been approved for inflammatory active PPMS and siponimod for inflammatory been considered stable and represent a signature pattern for individual patients, active SPMS. but it has now been shown that these bands can change over a longer period of time but that the change in most cases probably is not affected by disease 60 61 progression or immunosuppressive therapy (418). Patients with other 2 AIM autoimmune or infectious diseases may also present OCB in CSF, why it cannot be regarded as a specific marker for MS (419, 420). A small proportion of the IgG antibodies in OCB are directed against viral antigens and myelin The overall aim was to develop specific serological assays to detect IgG proteins, but the vast proportion of these proteins have unknown targets (421). antibodies to varicella-zoster virus (VZV), Epstein-Barr virus (EBV) and measles virus (MeV) and to use these assays for clinical applications. Patients with MS demonstrate higher EBV seroprevalence (245, 405, 413, 414, 422, 423) and increased serum levels of anti-EBV antibodies (404, 423-426) The specific aims were: compared with healthy controls, but the intrathecal antibody response to EBV • To produce and evaluate recombinant, single is relatively low compared with the intrathecal response to certain other immunodominant viral proteins, VZV glycoprotein E, EBV neurotropic viruses such as MeV, RV and VZV (427-431). The intrathecal glycoprotein 350 and the core part of the MeV nucleocapsid production of anti-MeV, anti-RV and anti-VZV IgG, termed the MRZ reaction, protein as serological antigens in enzyme linked is a common finding in patients with MS and can be used as a complement to immunosorbent assay. other diagnostics (429-431). A few studies have also shown an increase in serum anti-MeV IgG levels in patients with MS compared with healthy • To use the newly developed serological assays to examine controls (432-434). Not all neurotropic viruses are positively associated with antibody responses to VZV, EBV and MeV in patients with MS; for example, not CMV, and individuals who are CMV seropositive have multiple sclerosis. shown a reduced risk of developing MS (245, 435-437). Why patients with MS have this increased IgG antibody response to certain • To study the role of antibody response to EBV as a potential neurotropic viruses compared with healthy controls is unknown and therefore part of the pathogenesis of multiple sclerosis. requires further research. Due to the association between MS and EBV, the idea has arisen to use the antibody response to EBV and/or the EBV viral load as surrogate markers for disease activity and treatment effect in MS (438-443). Some studies support that disease activity is correlated with the antibody response to EBV (440, 441) while other studies do not (442-444). Further studies on the association are therefore warranted. Most serological studies on the antibody response to EBV in patients with MS have used EBNA (especially EBNA1) and VCA as antigens (404, 407, 408, 414, 424, 425, 428, 440-444). Analysis of antibodies to EBVgp350 may therefore, as a sensitive and specific antigen, provide new information on the humoral immune response to EBV in patients with MS. Some healthy siblings of patients with MS demonstrate a suspect hyperimmune phenotype with OCB in CSF and increased IgG antibody levels against certain neurotropic viruses in both CSF and serum compared with healthy controls (445-447). These siblings have been termed siblings with MS trait (446, 447). In this thesis, improved serological methods are used to examine antibody responses to certain viral antigens in patients with MS. 62 63 progression or immunosuppressive therapy (418). Patients with other 2 AIM autoimmune or infectious diseases may also present OCB in CSF, why it cannot be regarded as a specific marker for MS (419, 420). A small proportion of the IgG antibodies in OCB are directed against viral antigens and myelin The overall aim was to develop specific serological assays to detect IgG proteins, but the vast proportion of these proteins have unknown targets (421). antibodies to varicella-zoster virus (VZV), Epstein-Barr virus (EBV) and measles virus (MeV) and to use these assays for clinical applications. Patients with MS demonstrate higher EBV seroprevalence (245, 405, 413, 414, 422, 423) and increased serum levels of anti-EBV antibodies (404, 423-426) The specific aims were: compared with healthy controls, but the intrathecal antibody response to EBV • To produce and evaluate recombinant, single is relatively low compared with the intrathecal response to certain other immunodominant viral proteins, VZV glycoprotein E, EBV neurotropic viruses such as MeV, RV and VZV (427-431). The intrathecal glycoprotein 350 and the core part of the MeV nucleocapsid production of anti-MeV, anti-RV and anti-VZV IgG, termed the MRZ reaction, protein as serological antigens in enzyme linked is a common finding in patients with MS and can be used as a complement to immunosorbent assay. other diagnostics (429-431). A few studies have also shown an increase in serum anti-MeV IgG levels in patients with MS compared with healthy • To use the newly developed serological assays to examine controls (432-434). Not all neurotropic viruses are positively associated with antibody responses to VZV, EBV and MeV in patients with MS; for example, not CMV, and individuals who are CMV seropositive have multiple sclerosis. shown a reduced risk of developing MS (245, 435-437). Why patients with MS have this increased IgG antibody response to certain • To study the role of antibody response to EBV as a potential neurotropic viruses compared with healthy controls is unknown and therefore part of the pathogenesis of multiple sclerosis. requires further research. Due to the association between MS and EBV, the idea has arisen to use the antibody response to EBV and/or the EBV viral load as surrogate markers for disease activity and treatment effect in MS (438-443). Some studies support that disease activity is correlated with the antibody response to EBV (440, 441) while other studies do not (442-444). Further studies on the association are therefore warranted. Most serological studies on the antibody response to EBV in patients with MS have used EBNA (especially EBNA1) and VCA as antigens (404, 407, 408, 414, 424, 425, 428, 440-444). Analysis of antibodies to EBVgp350 may therefore, as a sensitive and specific antigen, provide new information on the humoral immune response to EBV in patients with MS. Some healthy siblings of patients with MS demonstrate a suspect hyperimmune phenotype with OCB in CSF and increased IgG antibody levels against certain neurotropic viruses in both CSF and serum compared with healthy controls (445-447). These siblings have been termed siblings with MS trait (446, 447). In this thesis, improved serological methods are used to examine antibody responses to certain viral antigens in patients with MS. 62 63 3 PATIENTS AND METHODS 3.1.2 PAPER II 3.1 PATIENTS 3.1.1 PAPER 1 Figure 20. A schematic figure of the analyzed serum material in Paper II. A total of 360 serum samples were analyzed for detection of IgG antibodies to Epstein-Barr virus glycoprotein 350. Abbreviations: Viral capsid antigen (VCA), Epstein-Barr virus nuclear antigen 1 (EBNA1), seropositive (+), seronegative (-). Figure 19. Figure depicting the analyzed serum samples in Paper I. Serum samples from five different groups were analyzed in Paper I. A total of 454 samples from patients with ischemic stroke were analyzed from four stroke The analyzed serum samples in Paper II were sent to the Department of units in western Sweden together with 100 population-based controls matched Clinical Microbiology, Sahlgrenska University Hospital in Gothenburg, for age and sex. The other three groups consisted of blood donors (n = 100), Sweden in 2014 and 2015 for clinical analysis of anti-EBNA1 and anti-VCA students (n = 100) and serum samples with low anti-VZV IgG titers (n = 100). antibodies. The samples were analyzed using ARCHITECT i4000SR A total of 854 samples were analyzed in the study. All samples were coded immunoassay analyzer (Abbott). before analysis. A schematic illustration of the patient material can be seen in Figure 19 and the characteristic of the five groups are presented in Table 1. The individuals agreed to store the samples in Biobank Väst after analysis for possible later use. For the study, a total of 360 serum samples from Biobank Table 1. Age and sex of the participants in the five different groups from Väst were collected and anonymized for analysis of anti-EBVgp350 IgG whom serum samples were analyzed in Paper I. Abbreviation: Not antibodies. Of these 360 samples, 120 were VCA and EBNA1 IgG seropositive determined (n.d.). (VCA+EBNA1+), 120 were VCA and EBNA1 IgG seronegative (VCA- EBNA-), and 120 were VCA IgG seropositive but EBNA1 IgG seronegative Mean age (VCA+ EBNA1-). The patient material is illustrated in Figure 20. Total N (years) Female N (%) Patients with stroke 454 57.4 159 (35%) Controls 100 56.4 36 (36%) Blood donors 100 n.d. n.d. Students 100 26.9 51 (51%) Low titer 100 37.9 n.d. 64 65 3 PATIENTS AND METHODS 3.1.2 PAPER II 3.1 PATIENTS 3.1.1 PAPER 1 Figure 20. A schematic figure of the analyzed serum material in Paper II. A total of 360 serum samples were analyzed for detection of IgG antibodies to Epstein-Barr virus glycoprotein 350. Abbreviations: Viral capsid antigen (VCA), Epstein-Barr virus nuclear antigen 1 (EBNA1), seropositive (+), seronegative (-). Figure 19. Figure depicting the analyzed serum samples in Paper I. Serum samples from five different groups were analyzed in Paper I. A total of 454 samples from patients with ischemic stroke were analyzed from four stroke The analyzed serum samples in Paper II were sent to the Department of units in western Sweden together with 100 population-based controls matched Clinical Microbiology, Sahlgrenska University Hospital in Gothenburg, for age and sex. The other three groups consisted of blood donors (n = 100), Sweden in 2014 and 2015 for clinical analysis of anti-EBNA1 and anti-VCA students (n = 100) and serum samples with low anti-VZV IgG titers (n = 100). antibodies. The samples were analyzed using ARCHITECT i4000SR A total of 854 samples were analyzed in the study. All samples were coded immunoassay analyzer (Abbott). before analysis. A schematic illustration of the patient material can be seen in Figure 19 and the characteristic of the five groups are presented in Table 1. The individuals agreed to store the samples in Biobank Väst after analysis for possible later use. For the study, a total of 360 serum samples from Biobank Table 1. Age and sex of the participants in the five different groups from Väst were collected and anonymized for analysis of anti-EBVgp350 IgG whom serum samples were analyzed in Paper I. Abbreviation: Not antibodies. Of these 360 samples, 120 were VCA and EBNA1 IgG seropositive determined (n.d.). (VCA+EBNA1+), 120 were VCA and EBNA1 IgG seronegative (VCA- EBNA-), and 120 were VCA IgG seropositive but EBNA1 IgG seronegative Mean age (VCA+ EBNA1-). The patient material is illustrated in Figure 20. Total N (years) Female N (%) Patients with stroke 454 57.4 159 (35%) Controls 100 56.4 36 (36%) Blood donors 100 n.d. n.d. Students 100 26.9 51 (51%) Low titer 100 37.9 n.d. 64 65 3.1.3 PAPER III 3.1.4 PAPER IV Figure 21. A schematic figure of the analyzed patient material in Paper III. Figure 22. The patient material in Paper IV. Abbreviations: Infectious mononucleosis Abbreviations: Multiple sclerosis (MS). A suspect hyperimmune phenotype seen in (IM). Cerebrospinal fluid (CSF). clinically healthy siblings of patients with MS (MS trait). The patient material in Paper IV consisted of serum samples collected from 42 The patient material was the same as in a previous study except for one male individuals with serologically verified acute IM sometime between 2003 and sibling without MS trait where sample material was lacking (445). There were 2007. Serum samples from the same individuals (post-IM patients) were nine clinically healthy siblings of patients with MS who in the previous study obtained at follow-up approximately 10 years after their IM episode. A serum presented two or more OCB in CSF and several showed increased anti-MeV sample was obtained from all 42 individuals and 21 also contributed with CSF. IgG levels in CSF and/or serum compared with healthy controls (445). These As a control group, serum and CSF samples were obtained from 17 healthy siblings were categorized as having MS trait (a suspect hyperimmune controls that denied a history of IM. Of these healthy controls, 15 were EBV phenotype) (445). In total, the patient material consisted of paired serum and seropositive. In addition to these healthy controls, serum samples from 24 EBV CSF samples from 47 patients with MS, 9 siblings with MS trait, 37 siblings seropositive blood donors were included. In total, the sample material in the without MS trait and 50 healthy controls. Of the patients with MS, 22 had EBV seropositive control group consisted of 39 serum samples and 15 CSF RRMS, 22 SPMS and 3 PPMS. The control group had fewer women and a samples. The patients with MS included in the study were sampled between lower median age compared with the groups with patients and siblings. The 1996 and 1997 and both serum and CSF samples from these individuals were patient material is displayed in Figure 21. The age and sex distribution between analyzed. Patients with MS were age and sex matched with the post-IM the groups is displayed in Table 2. patients at follow-up. Of the patients with MS, 13 had RRMS and 9 had SPMS. The patient material is presented in Figure 22 and Table 3. Table 2. Age and sex of participants in Paper III. Table 3. Age, sex and analyzed sample material from the participants in Median age Paper IV. Of the 17 healthy controls that contributed with cerebrospinal Total N (range years) Female N (%) fluid (CSF), 15 were Epstein-Barr virus seropositive. Patients with MS 47 46 (22–63) 31 (66%) Total N Median age Siblings with MS trait 9 43 (34–58) 5 (56%) (range years) Female N (%) Infectious mononucleosis (serum) 42 18 (11–34) 24 (57%) Siblings without MS trait 37 46 (22–66) 24 (65%) Follow-up (serum) 42 28 (22–43) 24 (57%) Healthy controls 50 32 (18–57) 15 (30%) Follow-up (CSF) 21 27 (22–40) 13 (62%) Healthy controls (serum) 39 23 (18–34) 21 (54%) Healthy controls (CSF) 17 25 (20–46) 11 (65%) Multiple sclerosis (serum and CSF) 22 35 (18–45) 16 (73%) 66 67 3.1.3 PAPER III 3.1.4 PAPER IV Figure 21. A schematic figure of the analyzed patient material in Paper III. Figure 22. The patient material in Paper IV. Abbreviations: Infectious mononucleosis Abbreviations: Multiple sclerosis (MS). A suspect hyperimmune phenotype seen in (IM). Cerebrospinal fluid (CSF). clinically healthy siblings of patients with MS (MS trait). The patient material in Paper IV consisted of serum samples collected from 42 The patient material was the same as in a previous study except for one male individuals with serologically verified acute IM sometime between 2003 and sibling without MS trait where sample material was lacking (445). There were 2007. Serum samples from the same individuals (post-IM patients) were nine clinically healthy siblings of patients with MS who in the previous study obtained at follow-up approximately 10 years after their IM episode. A serum presented two or more OCB in CSF and several showed increased anti-MeV sample was obtained from all 42 individuals and 21 also contributed with CSF. IgG levels in CSF and/or serum compared with healthy controls (445). These As a control group, serum and CSF samples were obtained from 17 healthy siblings were categorized as having MS trait (a suspect hyperimmune controls that denied a history of IM. Of these healthy controls, 15 were EBV phenotype) (445). In total, the patient material consisted of paired serum and seropositive. In addition to these healthy controls, serum samples from 24 EBV CSF samples from 47 patients with MS, 9 siblings with MS trait, 37 siblings seropositive blood donors were included. In total, the sample material in the without MS trait and 50 healthy controls. Of the patients with MS, 22 had EBV seropositive control group consisted of 39 serum samples and 15 CSF RRMS, 22 SPMS and 3 PPMS. The control group had fewer women and a samples. The patients with MS included in the study were sampled between lower median age compared with the groups with patients and siblings. The 1996 and 1997 and both serum and CSF samples from these individuals were patient material is displayed in Figure 21. The age and sex distribution between analyzed. Patients with MS were age and sex matched with the post-IM the groups is displayed in Table 2. patients at follow-up. Of the patients with MS, 13 had RRMS and 9 had SPMS. The patient material is presented in Figure 22 and Table 3. Table 2. Age and sex of participants in Paper III. Table 3. Age, sex and analyzed sample material from the participants in Median age Paper IV. Of the 17 healthy controls that contributed with cerebrospinal Total N (range years) Female N (%) fluid (CSF), 15 were Epstein-Barr virus seropositive. Patients with MS 47 46 (22–63) 31 (66%) Total N Median age Siblings with MS trait 9 43 (34–58) 5 (56%) (range years) Female N (%) Infectious mononucleosis (serum) 42 18 (11–34) 24 (57%) Siblings without MS trait 37 46 (22–66) 24 (65%) Follow-up (serum) 42 28 (22–43) 24 (57%) Healthy controls 50 32 (18–57) 15 (30%) Follow-up (CSF) 21 27 (22–40) 13 (62%) Healthy controls (serum) 39 23 (18–34) 21 (54%) Healthy controls (CSF) 17 25 (20–46) 11 (65%) Multiple sclerosis (serum and CSF) 22 35 (18–45) 16 (73%) 66 67 3.1.5 PAPER V material was lacking from 130 patients. The paired serum samples from the patients in the NAT group were obtained by sampling immediately prior to the first infusion of NAT, at time point 3 (t3) and the last available sample during NAT therapy, at time point 4 (t4). The median time between the samples was 12 months with an interquartile range (IQR) of 7–24 months. For 170 patients in the initial NAT group, serum samples had been taken during previous IFNβ treatment at t1 and t2 (before the start of NAT therapy). The median time between samples was 13 months with an IQR of 7–25 months. In the current study, 14 of the 170 patients in the IFN subgroup lacked material for the samples taken before (t3) and during (t4) NAT treatment why only 156 patients had paired samples taken both during IFN treatment and before and during NAT treatment. A total of 728 individual patients with MS were included in the present study. A sex- and age-matched control group with 144 blood donors was also included in the study. The characteristics of these three groups are presented in Table 4 and a schematic illustration of the patient material is presented in Figure 23. Table 4. Characteristics of the participants in the natalizumab (NAT) group, the interferon (IFN) subgroup and the healthy controls. Δt is duration of time in months between samples taken at t1–t2 and t3–t4, with interquartile range (IQR). Figure 23. Schematic figure showing the patient material in Paper V. Serum samples were collected on two occasions from 170 patients with multiple sclerosis during Median age Median ∆t treatment with interferon beta (IFNβ) at time point 1 (t1) and t2. The 170 patients in the Total N (range) Female months (IQR) IFN subgroup were then treated with natalizumab (NAT). The paired serum samples from the patients in the NAT group were obtained immediately prior to the first infusion of NAT, at time point 3 (t3) and the last available sample during NAT therapy, at t4. The IFN subgroup t1–t2 170 37 (16–58) 106 (62%) 13 (7–25) initial NAT group consisted of 1157 patients. Of these, 313 were excluded in a previous study due to insufficient quantification of antibodies to JC polyomavirus or previous NAT group t3–t4 714 37 (12–63) 500 (70%) 12 (7–24) treatment with intravenous immunoglobulin. Serum material was also lacking from 130 patients for the antiviral IgG assays in the current study. The NAT group analyzed in this Blood donors 144 35 (18–63) 100 (69%) study consisted of 714 patients, of whom 156 were also part of the IFN subgroup. The patient material in Paper V was obtained from patients with MS enrolled in the Swedish pharmacovigilance study for NAT (IMSE), where post marketing surveillance of NAT was performed (448, 449). All 1157 patients in the initial NAT group were treated with NAT before March 2010 (448-450). In a previous study, 313 patients were excluded due to insufficient quantification of anti-JCV antibodies or previous treatment with intravenous immunoglobulins, while samples from the remaining 844 patients were analyzed (450). The antiviral analyzes in the present study could be performed for 714 patients who had enough serum left for the analyzes, while serum 68 69 3.1.5 PAPER V material was lacking from 130 patients. The paired serum samples from the patients in the NAT group were obtained by sampling immediately prior to the first infusion of NAT, at time point 3 (t3) and the last available sample during NAT therapy, at time point 4 (t4). The median time between the samples was 12 months with an interquartile range (IQR) of 7–24 months. For 170 patients in the initial NAT group, serum samples had been taken during previous IFNβ treatment at t1 and t2 (before the start of NAT therapy). The median time between samples was 13 months with an IQR of 7–25 months. In the current study, 14 of the 170 patients in the IFN subgroup lacked material for the samples taken before (t3) and during (t4) NAT treatment why only 156 patients had paired samples taken both during IFN treatment and before and during NAT treatment. A total of 728 individual patients with MS were included in the present study. A sex- and age-matched control group with 144 blood donors was also included in the study. The characteristics of these three groups are presented in Table 4 and a schematic illustration of the patient material is presented in Figure 23. Table 4. Characteristics of the participants in the natalizumab (NAT) group, the interferon (IFN) subgroup and the healthy controls. Δt is duration of time in months between samples taken at t1–t2 and t3–t4, with interquartile range (IQR). Figure 23. Schematic figure showing the patient material in Paper V. Serum samples were collected on two occasions from 170 patients with multiple sclerosis during Median age Median ∆t treatment with interferon beta (IFNβ) at time point 1 (t1) and t2. The 170 patients in the Total N (range) Female months (IQR) IFN subgroup were then treated with natalizumab (NAT). The paired serum samples from the patients in the NAT group were obtained immediately prior to the first infusion of NAT, at time point 3 (t3) and the last available sample during NAT therapy, at t4. The IFN subgroup t1–t2 170 37 (16–58) 106 (62%) 13 (7–25) initial NAT group consisted of 1157 patients. Of these, 313 were excluded in a previous study due to insufficient quantification of antibodies to JC polyomavirus or previous NAT group t3–t4 714 37 (12–63) 500 (70%) 12 (7–24) treatment with intravenous immunoglobulin. Serum material was also lacking from 130 patients for the antiviral IgG assays in the current study. The NAT group analyzed in this Blood donors 144 35 (18–63) 100 (69%) study consisted of 714 patients, of whom 156 were also part of the IFN subgroup. The patient material in Paper V was obtained from patients with MS enrolled in the Swedish pharmacovigilance study for NAT (IMSE), where post marketing surveillance of NAT was performed (448, 449). All 1157 patients in the initial NAT group were treated with NAT before March 2010 (448-450). In a previous study, 313 patients were excluded due to insufficient quantification of anti-JCV antibodies or previous treatment with intravenous immunoglobulins, while samples from the remaining 844 patients were analyzed (450). The antiviral analyzes in the present study could be performed for 714 patients who had enough serum left for the analyzes, while serum 68 69 3.2 METHODS cultured in a perfusion bioreactor where VZVgE was secreted into the medium. This was done to yield larger quantities of VZVgE. The perfusion culture was 3.2.1 ANTIGEN PRODUCTION set up in a 3 L Biobundle bioreactor and 12.5 L of cell-free harvest could later be collected. The harvest was centrifuged, pre-filtered and then concentrated The quality of viral serological methods has improved over time, but further by tangential flow filtration (TFF). The protein was purified from the improvements are warranted. We decided to use single, immunodominant viral concentrate by using 1 mL HiTrap chelating columns loaded with Co2+. proteins to develop new antigens with both high specificity and sensitivity. The Imidazole was used to elute the bound protein. Western blot was used to expression system for antigen production is important to consider as residues analyze the fractions. from the antigen production can cause non-specific reactions. We have focused on expression systems without human or primate components to avoid the risk of autoantibodies to these components. In 3.2.3 EPSTEIN-BARR VIRUS GLYCOPROTEIN 350 addition, it is important to consider which cells should be used to recombinantly generate proteins, since post-translational modifications, such The EBV transmembrane envelope protein EBVgp350 consists of 907 a.a. as glycosylation of viral proteins, depend on the host cell (451, 452). We have with an 860 a.a. long extracellular N-terminal segment. EBV DNA constructs used Chinese hamster ovary (CHO) cells to express the two viral glycoproteins encoding different parts of EBVgp350, a.a. 1–506, 751–860 and 502–860, (VZVgE and EBVgp350) used as serological antigens in our studies because were derived from EBV strain B95-8 (GenBank accession number M10593). these cells are well established, including their pattern of glycosylation, for The three EBV DNA constructs were synthesized and ligated into mammalian recombinant protein production (453). pcDNA6myc-His vectors and adapted for expression in CHO cells. The His6 tag from the vector was left on the C-terminal end of the protein for expression. The VZVgE and EBVgp350 antigens were developed in collaboration with the core facility Mammalian Protein Expression at the University of Gothenburg, The two DNA constructs a.a. 1–506 and 502–860 were combined to generate Sweden. The MeV nucleocapsid antigen was produced by our collaborators at a construct encoding the whole extracellular domain a.a. 1–860 of EBVgp350. Centre National de la Recherche Scientifique, Université Aix-Marseille, The cloning steps were performed using Escherichia coli XL-1 Blue. The Marseille, France. recombinant EBVgp350 constructs were produced by transient transfection of FreeStyle™ CHO-suspension cells. The NovaCHOice® Transfection kit was used according to the manufacturer's instructions to perform the transfections. Small-scale 5 ml transfections were performed in 50 ml TubeSpin™ tubes and 3.2.2 VARICELLA-ZOSTER VIRUS GLYCOPROTEIN E large-scale transfections with EBVgp350 a.a. 1–506 and 1–860 were performed in 3 L Biobundle bioreactors. The candidate protein for developing a VZV specific antigen was the immunodominant gE. A BLAST analysis did not show extensive homologies The supernatants from the cell cultures were harvested, centrifuged, and pre- between VZVgE and HSVgE (data not shown). An expression vector filtered followed by TFF concentration. Protein was purified from the containing the gene segment encoding a.a. 1–539, corresponding to the signal concentrate using a 1 mL Hitrap™ Chelating HP column loaded with 0.1 M sequence a.a. 1–24 and the extracellular domain a.a. 25–539 of VZVgE was CoCl2, at a flow rate of 1 mL/min. Imidazole was used to elute bound protein. generated. This was done by PCR amplification of VZV DNA encoding a.a. The collected fractions were analyzed by Western blot. Then, the 20 mM 1–539 from VZV strain Dumas. The amplified PCR fragment was cut with imidazole fractions were pooled and dialyzed against 3 × 3 L phosphate- restriction enzymes and cloned into pcDNA6/myc-His A vectors. buffered saline (PBS) using Spectra/Por® 4 dialysis membrane tubing. The final protein concentrations were measured with Thermo Scientific™ The vector was transfected into CHO K1 cells. The cells were cultured and NanoDrop 2000 to 0.7 mg/ml for EBVgp350 a.a. 1–860 and 0.6 mg/ml for a.a. cloned in several cycles to generate a viable production of VZVgE. Several 1–506. stable clones that secreted high levels of VZVgE were produced and one clone was adapted to growth in serum-free suspension culture. The cells were then 70 71 3.2 METHODS cultured in a perfusion bioreactor where VZVgE was secreted into the medium. This was done to yield larger quantities of VZVgE. The perfusion culture was 3.2.1 ANTIGEN PRODUCTION set up in a 3 L Biobundle bioreactor and 12.5 L of cell-free harvest could later be collected. The harvest was centrifuged, pre-filtered and then concentrated The quality of viral serological methods has improved over time, but further by tangential flow filtration (TFF). The protein was purified from the improvements are warranted. We decided to use single, immunodominant viral concentrate by using 1 mL HiTrap chelating columns loaded with Co2+. proteins to develop new antigens with both high specificity and sensitivity. The Imidazole was used to elute the bound protein. Western blot was used to expression system for antigen production is important to consider as residues analyze the fractions. from the antigen production can cause non-specific reactions. We have focused on expression systems without human or primate components to avoid the risk of autoantibodies to these components. In 3.2.3 EPSTEIN-BARR VIRUS GLYCOPROTEIN 350 addition, it is important to consider which cells should be used to recombinantly generate proteins, since post-translational modifications, such The EBV transmembrane envelope protein EBVgp350 consists of 907 a.a. as glycosylation of viral proteins, depend on the host cell (451, 452). We have with an 860 a.a. long extracellular N-terminal segment. EBV DNA constructs used Chinese hamster ovary (CHO) cells to express the two viral glycoproteins encoding different parts of EBVgp350, a.a. 1–506, 751–860 and 502–860, (VZVgE and EBVgp350) used as serological antigens in our studies because were derived from EBV strain B95-8 (GenBank accession number M10593). these cells are well established, including their pattern of glycosylation, for The three EBV DNA constructs were synthesized and ligated into mammalian recombinant protein production (453). pcDNA6myc-His vectors and adapted for expression in CHO cells. The His6 tag from the vector was left on the C-terminal end of the protein for expression. The VZVgE and EBVgp350 antigens were developed in collaboration with the core facility Mammalian Protein Expression at the University of Gothenburg, The two DNA constructs a.a. 1–506 and 502–860 were combined to generate Sweden. The MeV nucleocapsid antigen was produced by our collaborators at a construct encoding the whole extracellular domain a.a. 1–860 of EBVgp350. Centre National de la Recherche Scientifique, Université Aix-Marseille, The cloning steps were performed using Escherichia coli XL-1 Blue. The Marseille, France. recombinant EBVgp350 constructs were produced by transient transfection of FreeStyle™ CHO-suspension cells. The NovaCHOice® Transfection kit was used according to the manufacturer's instructions to perform the transfections. Small-scale 5 ml transfections were performed in 50 ml TubeSpin™ tubes and 3.2.2 VARICELLA-ZOSTER VIRUS GLYCOPROTEIN E large-scale transfections with EBVgp350 a.a. 1–506 and 1–860 were performed in 3 L Biobundle bioreactors. The candidate protein for developing a VZV specific antigen was the immunodominant gE. A BLAST analysis did not show extensive homologies The supernatants from the cell cultures were harvested, centrifuged, and pre- between VZVgE and HSVgE (data not shown). An expression vector filtered followed by TFF concentration. Protein was purified from the containing the gene segment encoding a.a. 1–539, corresponding to the signal concentrate using a 1 mL Hitrap™ Chelating HP column loaded with 0.1 M sequence a.a. 1–24 and the extracellular domain a.a. 25–539 of VZVgE was CoCl2, at a flow rate of 1 mL/min. Imidazole was used to elute bound protein. generated. This was done by PCR amplification of VZV DNA encoding a.a. The collected fractions were analyzed by Western blot. Then, the 20 mM 1–539 from VZV strain Dumas. The amplified PCR fragment was cut with imidazole fractions were pooled and dialyzed against 3 × 3 L phosphate- restriction enzymes and cloned into pcDNA6/myc-His A vectors. buffered saline (PBS) using Spectra/Por® 4 dialysis membrane tubing. The final protein concentrations were measured with Thermo Scientific™ The vector was transfected into CHO K1 cells. The cells were cultured and NanoDrop 2000 to 0.7 mg/ml for EBVgp350 a.a. 1–860 and 0.6 mg/ml for a.a. cloned in several cycles to generate a viable production of VZVgE. Several 1–506. stable clones that secreted high levels of VZVgE were produced and one clone was adapted to growth in serum-free suspension culture. The cells were then 70 71 3.2.4 MEASLES VIRUS NUCLEOCAPSID ANTIGEN was identified by using Penta-His™ Antibody as the primary antibody and To produce the MeV antigen the immunodominant MeV nucleocapsid protein alkaline phosphatase goat anti-mouse IgG as secondary antibody. Detection was used. The plasmid pet21a/NFlag-H6, which encodes MeV nucleocapsid was performed by using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue protein (strain Edmonston B) with an N-terminal flag sequence (454) and a C- tetrazolium as substrate. MeV NCORE was identified by anti-measles IgG terminal hexahistidine tag (455) was inserted in Escherichia coli, strain Rosetta antibodies in human serum or mouse monoclonal antibody 5.227 against the [DE3] pLysS (Novagen) for antigen expression. nucleocapsid protein. The conjugates used were either horseradish peroxidase polyclonal rabbit anti-human IgG or anti-mouse IgG. The substrate used was The MeV nucleocapsid protein, was detected in the soluble fraction of the 4-chloro-1-naphtol. bacterial lysate and the protein was purified from the bacterial lysate by immobilized metal affinity chromatography using Chelating Sepharose Fast Flow Resin preloaded with Ni2+ ions (Amersham Pharmacia Biotech) as Figure 24. Western blot using previously described (455, 456). The NCORE fragment (a.a. 1–392) was varicella-zoster virus glycoprotein obtained from the nucleocapsid protein by limited proteolysis using trypsin E (VZVgE) as antigen and a (455). The core part of the nucleocapsid protein was chosen as serological human serum sample containing antigen because it is conserved and structurally ordered while the diversity of anti-VZVgE IgG antibodies. the carboxyterminal is the basis for the division of MeV into clades and Molecular marker was Mark 12 Unstained standard. genotypes (283). The NCORE antigen was purified to homogeneity (>95%) in two steps: immobilized metal affinity chromatography and gel filtration. 3.2.6 IMMUNOFLUORESCENCE 3.2.5 WESTERN BLOT Indirect immunofluorescence was used in Paper I to analyze the samples that showed discordant results between the VZV ELISA methods. Green monkey After antigen production, gel staining and Western blot were performed in kidney cells AH1 cell line (SBL/83) were infected with VZV according to Paper I, II and III to identify VZVgE, EBVgp350 and MeV NCORE. Western internal diagnostic routine at the Department of Clinical Microbiology, blot was also used in Paper I to analyze serum samples that showed discordant Sahlgrenska University Hospital in Gothenburg, Sweden. The cells were fixed results between the two VZV ELISA methods used in the study. The antigens with acetone to glass slips and the serum samples were then added. If the VZVgE, EBVgp350 and MeV NCORE were denatured and then separated by gel sample contained anti-VZV IgG, the antibodies attached to the virus-infected electrophoresis. In Paper I, VZVgE was stained directly on the gel with silver, cells. Fluorescein-labeled goat anti-human IgG was used as conjugate. or the Novex Colloidal Blue Staining Kit. In Paper III, MeV NCORE was stained directly on the gel with Coomassie blue. VZVgE, EBVgp350 and MeV NCORE Figure 25. An were also transferred with electrical voltage to nitrocellulose membranes or immunofluorescence Immobilon-P membranes. photograph showing fluorescent stained To detect VZVgE, either the mouse monoclonal antibody VZVgE sc-56994 or anti-varicella-zoster virus (VZV) IgG serum samples from patients were used. For the mouse monoclonal, the used antibodies that have conjugates were alkaline phosphatase goat anti-mouse Ig or horseradish attached to VZV- peroxidase goat anti-mouse Ig. For the human antibodies, the used conjugate infected cells. was horseradish peroxidase polyclonal rabbit anti-human IgG. The substrates used to detect the mouse monoclonal were BCIP/NBT developing solution or chemiluminescent substrate ImmobilonTM Western. To detect human anti- VZV IgG antibodies, 4-chloro-1-naphtol was used as the substrate. EBVgp350 72 73 3.2.4 MEASLES VIRUS NUCLEOCAPSID ANTIGEN was identified by using Penta-His™ Antibody as the primary antibody and To produce the MeV antigen the immunodominant MeV nucleocapsid protein alkaline phosphatase goat anti-mouse IgG as secondary antibody. Detection was used. The plasmid pet21a/NFlag-H6, which encodes MeV nucleocapsid was performed by using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue protein (strain Edmonston B) with an N-terminal flag sequence (454) and a C- tetrazolium as substrate. MeV NCORE was identified by anti-measles IgG terminal hexahistidine tag (455) was inserted in Escherichia coli, strain Rosetta antibodies in human serum or mouse monoclonal antibody 5.227 against the [DE3] pLysS (Novagen) for antigen expression. nucleocapsid protein. The conjugates used were either horseradish peroxidase polyclonal rabbit anti-human IgG or anti-mouse IgG. The substrate used was The MeV nucleocapsid protein, was detected in the soluble fraction of the 4-chloro-1-naphtol. bacterial lysate and the protein was purified from the bacterial lysate by immobilized metal affinity chromatography using Chelating Sepharose Fast Flow Resin preloaded with Ni2+ ions (Amersham Pharmacia Biotech) as Figure 24. Western blot using previously described (455, 456). The NCORE fragment (a.a. 1–392) was varicella-zoster virus glycoprotein obtained from the nucleocapsid protein by limited proteolysis using trypsin E (VZVgE) as antigen and a (455). The core part of the nucleocapsid protein was chosen as serological human serum sample containing antigen because it is conserved and structurally ordered while the diversity of anti-VZVgE IgG antibodies. the carboxyterminal is the basis for the division of MeV into clades and Molecular marker was Mark 12 Unstained standard. genotypes (283). The NCORE antigen was purified to homogeneity (>95%) in two steps: immobilized metal affinity chromatography and gel filtration. 3.2.6 IMMUNOFLUORESCENCE 3.2.5 WESTERN BLOT Indirect immunofluorescence was used in Paper I to analyze the samples that showed discordant results between the VZV ELISA methods. Green monkey After antigen production, gel staining and Western blot were performed in kidney cells AH1 cell line (SBL/83) were infected with VZV according to Paper I, II and III to identify VZVgE, EBVgp350 and MeV NCORE. Western internal diagnostic routine at the Department of Clinical Microbiology, blot was also used in Paper I to analyze serum samples that showed discordant Sahlgrenska University Hospital in Gothenburg, Sweden. The cells were fixed results between the two VZV ELISA methods used in the study. The antigens with acetone to glass slips and the serum samples were then added. If the VZVgE, EBVgp350 and MeV NCORE were denatured and then separated by gel sample contained anti-VZV IgG, the antibodies attached to the virus-infected electrophoresis. In Paper I, VZVgE was stained directly on the gel with silver, cells. Fluorescein-labeled goat anti-human IgG was used as conjugate. or the Novex Colloidal Blue Staining Kit. In Paper III, MeV NCORE was stained directly on the gel with Coomassie blue. VZVgE, EBVgp350 and MeV NCORE Figure 25. An were also transferred with electrical voltage to nitrocellulose membranes or immunofluorescence Immobilon-P membranes. photograph showing fluorescent stained To detect VZVgE, either the mouse monoclonal antibody VZVgE sc-56994 or anti-varicella-zoster virus (VZV) IgG serum samples from patients were used. For the mouse monoclonal, the used antibodies that have conjugates were alkaline phosphatase goat anti-mouse Ig or horseradish attached to VZV- peroxidase goat anti-mouse Ig. For the human antibodies, the used conjugate infected cells. was horseradish peroxidase polyclonal rabbit anti-human IgG. The substrates used to detect the mouse monoclonal were BCIP/NBT developing solution or chemiluminescent substrate ImmobilonTM Western. To detect human anti- VZV IgG antibodies, 4-chloro-1-naphtol was used as the substrate. EBVgp350 72 73 3.2.7 ELISA 3.3 STATISTICAL METHODS Indirect ELISA methods were used in the studies to analyze IgG antibodies to In Paper II, a receiver operating characteristic (ROC) curve was performed in VZVgE, EBVgp350 and MeV NCORE. The following were analyzed: GraphPad Prism 7.3 to evaluate the diagnostic accuracy of the indirect ELISA method for detecting anti-EBVgp350 IgG. A two-sided statistical test was Paper I: Anti-VZVgE IgG in serum samples. performed for the ROC curve in which a p-value smaller than 0.05 was Paper II: Anti-EBVgp350 IgG in serum samples. considered significant. Paper III: Anti-MeV NCORE IgG in serum and CSF samples. Paper IV: Anti-VZVgE, anti-EBVgp350 and anti-MeV NCORE IgG in serum and CSF samples. In Paper III, the independent groups were compared using the Mann-Whitney Paper V: Anti-EBVgp350 and anti-MeV NCORE IgG in serum samples. U test. Patients with MS and their siblings were compared using Wilcoxon signed-rank test. IBM SPSS statistics 20 was used for the statistical analyzes. The statistical tests were two-sided and p-values smaller than 0.05 were considered significant. In Paper IV, the Mann-Whitney U test was used to compare patients with IM with the control group. Where applicable, non-parametric ANOVA was used for comparisons. Wilcoxon signed-rank test was used for pairwise comparison between patients with IM and the same patients later at follow-up. To model age-adjusted group differences, quantile regression was used (457). P-values smaller than 0.05 were considered significant. In Paper V, the statistical analyzes were performed using SPSS Statistics 27. Figure 26. The figure illustrates the indirect ELISA method and an example of an ELISA The Mann-Whitney U test was used to compare anti-EBVgp350 and anti-MeV plate ready for measurement in a spectrophotometer. In the example, the plate is coated NCORE IgG levels in patients with MS during IFNβ treatment at t1 and blood with measles virus NCORE antigen. The serum samples are in two-fold dilutions from 1/200 donors. Wilcoxon signed-rank test was used to compare the anti-EBVgp350 to 1/25600. The negative control is in row 11 and the positive control is in row 12. and anti-MeV N IgG levels, between the samples collected during IFNβ Samples from patients with MS, their siblings or healthy controls are in row 1–10. COREtreatment at t1 and t2 and before and during NAT treatment at t3 and t4. The The antigens were diluted with carbonate buffer and then coated on Nunc statistical tests were two-sided and p-values <0.008 were considered MaxiSorp™ 96-well high protein binding ELISA plates. After incubation and significant due to Bonferroni correction for multiple tests. washing, the plates were blocked with non-fat dry milk diluted in PBS to avoid nonspecific binding. After blocking, patients' serum and CSF samples were added to the plates. If the samples contained complementary IgG to the viral antigens on the plates, the antibodies attached to the antigen. After incubation, the plates were washed to remove unbound antibodies and other components. A diluted conjugate, Alkaline Phosphatase AffiniPure F(ab')₂ Fragment Goat Anti-Human IgG, was then added to all wells. After incubation, all unbound conjugate was washed away. Substrate solution, phosphatase substrate dissolved and diluted in diethanolamine buffer, was added to the wells and if secondary antibodies conjugated to enzyme were present, the enzyme induced a color change of the substrate to yellow. Positive and negative control samples were added to all assays in order to control the quality of each analyzed ELISA plate. The optical density (OD) values were measured in a spectrophotometer. 74 75 3.2.7 ELISA 3.3 STATISTICAL METHODS Indirect ELISA methods were used in the studies to analyze IgG antibodies to In Paper II, a receiver operating characteristic (ROC) curve was performed in VZVgE, EBVgp350 and MeV NCORE. The following were analyzed: GraphPad Prism 7.3 to evaluate the diagnostic accuracy of the indirect ELISA method for detecting anti-EBVgp350 IgG. A two-sided statistical test was Paper I: Anti-VZVgE IgG in serum samples. performed for the ROC curve in which a p-value smaller than 0.05 was Paper II: Anti-EBVgp350 IgG in serum samples. considered significant. Paper III: Anti-MeV NCORE IgG in serum and CSF samples. Paper IV: Anti-VZVgE, anti-EBVgp350 and anti-MeV NCORE IgG in serum and CSF samples. In Paper III, the independent groups were compared using the Mann-Whitney Paper V: Anti-EBVgp350 and anti-MeV NCORE IgG in serum samples. U test. Patients with MS and their siblings were compared using Wilcoxon signed-rank test. IBM SPSS statistics 20 was used for the statistical analyzes. The statistical tests were two-sided and p-values smaller than 0.05 were considered significant. In Paper IV, the Mann-Whitney U test was used to compare patients with IM with the control group. Where applicable, non-parametric ANOVA was used for comparisons. Wilcoxon signed-rank test was used for pairwise comparison between patients with IM and the same patients later at follow-up. To model age-adjusted group differences, quantile regression was used (457). P-values smaller than 0.05 were considered significant. In Paper V, the statistical analyzes were performed using SPSS Statistics 27. Figure 26. The figure illustrates the indirect ELISA method and an example of an ELISA The Mann-Whitney U test was used to compare anti-EBVgp350 and anti-MeV plate ready for measurement in a spectrophotometer. In the example, the plate is coated NCORE IgG levels in patients with MS during IFNβ treatment at t1 and blood with measles virus NCORE antigen. The serum samples are in two-fold dilutions from 1/200 donors. Wilcoxon signed-rank test was used to compare the anti-EBVgp350 to 1/25600. The negative control is in row 11 and the positive control is in row 12. and anti-MeV N IgG levels, between the samples collected during IFNβ Samples from patients with MS, their siblings or healthy controls are in row 1–10. COREtreatment at t1 and t2 and before and during NAT treatment at t3 and t4. The The antigens were diluted with carbonate buffer and then coated on Nunc statistical tests were two-sided and p-values <0.008 were considered MaxiSorp™ 96-well high protein binding ELISA plates. After incubation and significant due to Bonferroni correction for multiple tests. washing, the plates were blocked with non-fat dry milk diluted in PBS to avoid nonspecific binding. After blocking, patients' serum and CSF samples were added to the plates. If the samples contained complementary IgG to the viral antigens on the plates, the antibodies attached to the antigen. After incubation, the plates were washed to remove unbound antibodies and other components. A diluted conjugate, Alkaline Phosphatase AffiniPure F(ab')₂ Fragment Goat Anti-Human IgG, was then added to all wells. After incubation, all unbound conjugate was washed away. Substrate solution, phosphatase substrate dissolved and diluted in diethanolamine buffer, was added to the wells and if secondary antibodies conjugated to enzyme were present, the enzyme induced a color change of the substrate to yellow. Positive and negative control samples were added to all assays in order to control the quality of each analyzed ELISA plate. The optical density (OD) values were measured in a spectrophotometer. 74 75 3.4 ETHICS 4 RESULTS AND DISCUSSION The samples from patients and controls were approved for use in Paper I by the Research Ethics Committee at the University of Gothenburg with ref. no. 4.1 PAPER I Ö 469-99. There is a need for rapid, simple, and reliable serological assays for the The samples in Paper II were collected for clinical purposes and sent to the determination of anti-VZV IgG responses, both for the diagnosis of VZV Department of Clinical Microbiology, Sahlgrenska University Hospital, infections and for the analysis of immunity to the virus. In Paper I, Gothenburg, Sweden for analysis of anti-EBV antibodies. The serum samples immunodominant VZVgE was recombinantly expressed in CHO cells and used in the study were deidentified prior to analysis of anti-EBVgp350 IgG used as a serological antigen in indirect ELISA to develop a sensitive and antibodies and the results cannot be traced back to individuals. specific assay for the detection of anti-VZV IgG antibodies. Ethical approval to use the samples in Paper III was given by the Research VZVgE production Ethics Committee in Gothenburg, ref. no. 361-96. VZVwhole-ag contains, in addition to many of the proteins in VZV, also The material from post-IM patients was approved for use in Paper IV by the cellular components. When narrowing down the number of proteins used in an Research Ethics Committee in Umeå, ref. no. 2017-484-32M, which is an antigen, the sensitivity may decrease and if only a single protein is to be used, updated version of the prior application ref. no. 2011-198-31M with previous it must be immunodominant for the serological analysis to achieve sufficient update ref. no. 2013-226-32M. The material from patients with MS was sensitivity. Glycoproteins are major components of the virus' outer surfaces approved for use in the study by the Research Ethics Committee in where they are exposed on the viral envelope. Thus, these glycoproteins are Gothenburg, ref. no. 361-96 with updates S8-97 and R584-98. accessible to the body's immune system and are common targets for human antibodies. VZVgE has been found to be the most immunogenic glycoprotein In Paper V, ethical approval was obtained from the Stockholm Regional on VZV (100, 102-105) and was also an appropriate candidate antigen in the Ethical Committee and the Swedish Ethical Review Authority ref. no. development of a new, more specific serological VZV assay due to the lack of 2006/845-31/1, ref. no. 2005/535-31/1, ref. no. 2009/1977-32 (updated 2010- extensive homologies with HSVgE. 08-06) and ref. no. 2019-04420. Large-scale production and purification of recombinant VZVgE expressed in Some sample materials included in the studies, such as the serum samples in CHO K1 cells was performed successfully. CHO cells were used because they Paper II and serum samples from blood donors in Paper I and V, are not are well established for recombinant mammalian protein production (453). covered by the Swedish act (2003:460) on ethical review of research involving CHO cells are also known to produce proteins with complex N-glycans and humans. The samples were not collected for use in the studies and the samples short core O-glycans. This is important to consider because VZVgE contain were deidentified before analysis so the results cannot be traced back to carbohydrates with N- or O-linked glycosylation and the glycosylation pattern individuals. Ethical permission is therefore not required in this context. depends on the host cell. Recombinant VZVgE was analyzed by liquid chromatography electrospray mass spectrometry and two sialylated O-linked glycans were confirmed. N-glycans were not analyzed but are likely to be present because CHO cells, as previously mentioned, are known to generate complex N-glycans. VZVgE had previously been produced in CHO cells but only on a small scale (100). In Paper I, VZVgE was generated under well- monitored conditions and therefore protein production should be reproducible. Recombinant VZVgE was generated in CHO cells adapted to growth in suspension and the protein production could be performed on a large scale in perfusion bioreactor culture. 76 77 3.4 ETHICS 4 RESULTS AND DISCUSSION The samples from patients and controls were approved for use in Paper I by the Research Ethics Committee at the University of Gothenburg with ref. no. 4.1 PAPER I Ö 469-99. There is a need for rapid, simple, and reliable serological assays for the The samples in Paper II were collected for clinical purposes and sent to the determination of anti-VZV IgG responses, both for the diagnosis of VZV Department of Clinical Microbiology, Sahlgrenska University Hospital, infections and for the analysis of immunity to the virus. In Paper I, Gothenburg, Sweden for analysis of anti-EBV antibodies. The serum samples immunodominant VZVgE was recombinantly expressed in CHO cells and used in the study were deidentified prior to analysis of anti-EBVgp350 IgG used as a serological antigen in indirect ELISA to develop a sensitive and antibodies and the results cannot be traced back to individuals. specific assay for the detection of anti-VZV IgG antibodies. Ethical approval to use the samples in Paper III was given by the Research VZVgE production Ethics Committee in Gothenburg, ref. no. 361-96. VZVwhole-ag contains, in addition to many of the proteins in VZV, also The material from post-IM patients was approved for use in Paper IV by the cellular components. When narrowing down the number of proteins used in an Research Ethics Committee in Umeå, ref. no. 2017-484-32M, which is an antigen, the sensitivity may decrease and if only a single protein is to be used, updated version of the prior application ref. no. 2011-198-31M with previous it must be immunodominant for the serological analysis to achieve sufficient update ref. no. 2013-226-32M. The material from patients with MS was sensitivity. Glycoproteins are major components of the virus' outer surfaces approved for use in the study by the Research Ethics Committee in where they are exposed on the viral envelope. Thus, these glycoproteins are Gothenburg, ref. no. 361-96 with updates S8-97 and R584-98. accessible to the body's immune system and are common targets for human antibodies. VZVgE has been found to be the most immunogenic glycoprotein In Paper V, ethical approval was obtained from the Stockholm Regional on VZV (100, 102-105) and was also an appropriate candidate antigen in the Ethical Committee and the Swedish Ethical Review Authority ref. no. development of a new, more specific serological VZV assay due to the lack of 2006/845-31/1, ref. no. 2005/535-31/1, ref. no. 2009/1977-32 (updated 2010- extensive homologies with HSVgE. 08-06) and ref. no. 2019-04420. Large-scale production and purification of recombinant VZVgE expressed in Some sample materials included in the studies, such as the serum samples in CHO K1 cells was performed successfully. CHO cells were used because they Paper II and serum samples from blood donors in Paper I and V, are not are well established for recombinant mammalian protein production (453). covered by the Swedish act (2003:460) on ethical review of research involving CHO cells are also known to produce proteins with complex N-glycans and humans. The samples were not collected for use in the studies and the samples short core O-glycans. This is important to consider because VZVgE contain were deidentified before analysis so the results cannot be traced back to carbohydrates with N- or O-linked glycosylation and the glycosylation pattern individuals. Ethical permission is therefore not required in this context. depends on the host cell. Recombinant VZVgE was analyzed by liquid chromatography electrospray mass spectrometry and two sialylated O-linked glycans were confirmed. N-glycans were not analyzed but are likely to be present because CHO cells, as previously mentioned, are known to generate complex N-glycans. VZVgE had previously been produced in CHO cells but only on a small scale (100). In Paper I, VZVgE was generated under well- monitored conditions and therefore protein production should be reproducible. Recombinant VZVgE was generated in CHO cells adapted to growth in suspension and the protein production could be performed on a large scale in perfusion bioreactor culture. 76 77 Comparison between VZVgE and VZVwhole-ag The ELISA analyses using VZVwhole-ag generally yielded higher anti-VZV IgG levels in serum compared with VZVgE. This was not as pronounced for The new serological VZVgE antigen (VZVgE-ag) was compared with a patients with stroke and the age-matched elderly controls. The older subjects conventional whole virus antigen (VZVwhole-ag) in indirect ELISA. Of the generally showed higher anti-VZVgE IgG levels compared with the younger 854 serum samples analyzed, 846 (99.1%) showed consistent results. A total participants i.e. blood donors and students (Figure 28). Overall, the results of 830 samples were VZV IgG seropositive and 16 were seronegative with showed that VZVgE had high specificity and sensitivity as a serological both methods. Only eight samples showed discrepant results between the antigen in ELISA. analyzes. These samples were all VZV IgG seropositive with VZVwhole-ag but seronegative with VZVgE-ag. The discordant samples were further Figure 28. Tukey box plot analyzed by Western blot using VZVgE as antigen and by indirect displaying the optical density immunofluorescence using VZV-infected cells. From these further analyzes, it values for the 854 serum samples diluted 1/800 and was determined that one sample was VZV IgG seropositive, two seronegative analyzed by indirect ELISA and five remained indeterminate. A flow chart of these results is presented in using varicella-zoster virus Figure 27. (VZV) whole virus antigen (VZVwhole-ag) and VZVgE antigen (VZVgE-ag). The figure also shows the results for the combined group with older participants (n = 554), i.e. stroke patients (n = 454) and age-matched elderly controls (n = 100) and the group with younger participants (n = 200), i.e. blood donors (n = 100) and students (n = 100). Importance of VZV serology methods VZV serology methods are not normally of great value in diagnosing acute VZV infections, i.e. chickenpox and herpes zoster. These diseases can usually be diagnosed clinically, but in case of uncertainty, PCR is the preferred laboratory analysis (72). However, VZV is one of the most common viruses that causes infections in the CNS and the infections give rise to a wide range of CNS manifestations, often without rash (122, 126, 143-145). Serological assays of anti-VZV antibodies are in these cases an important complement to PCR analyzes (126, 458). The time window for detecting VZV DNA is limited and when VZV DNA cannot be found, detection of intrathecally produced anti- Figure 27. Flowchart showing the results of the 854 serum samples analyzed by indirect ELISA using varicella-zoster virus (VZV) whole antigen (VZVwhole-ag) and VZVgE VZV antibodies can lead to the diagnosis (122, 126, 151). antigen (VZVgE-ag). 78 79 Comparison between VZVgE and VZVwhole-ag The ELISA analyses using VZVwhole-ag generally yielded higher anti-VZV IgG levels in serum compared with VZVgE. This was not as pronounced for The new serological VZVgE antigen (VZVgE-ag) was compared with a patients with stroke and the age-matched elderly controls. The older subjects conventional whole virus antigen (VZVwhole-ag) in indirect ELISA. Of the generally showed higher anti-VZVgE IgG levels compared with the younger 854 serum samples analyzed, 846 (99.1%) showed consistent results. A total participants i.e. blood donors and students (Figure 28). Overall, the results of 830 samples were VZV IgG seropositive and 16 were seronegative with showed that VZVgE had high specificity and sensitivity as a serological both methods. Only eight samples showed discrepant results between the antigen in ELISA. analyzes. These samples were all VZV IgG seropositive with VZVwhole-ag but seronegative with VZVgE-ag. The discordant samples were further Figure 28. Tukey box plot analyzed by Western blot using VZVgE as antigen and by indirect displaying the optical density immunofluorescence using VZV-infected cells. From these further analyzes, it values for the 854 serum samples diluted 1/800 and was determined that one sample was VZV IgG seropositive, two seronegative analyzed by indirect ELISA and five remained indeterminate. A flow chart of these results is presented in using varicella-zoster virus Figure 27. (VZV) whole virus antigen (VZVwhole-ag) and VZVgE antigen (VZVgE-ag). The figure also shows the results for the combined group with older participants (n = 554), i.e. stroke patients (n = 454) and age-matched elderly controls (n = 100) and the group with younger participants (n = 200), i.e. blood donors (n = 100) and students (n = 100). Importance of VZV serology methods VZV serology methods are not normally of great value in diagnosing acute VZV infections, i.e. chickenpox and herpes zoster. These diseases can usually be diagnosed clinically, but in case of uncertainty, PCR is the preferred laboratory analysis (72). However, VZV is one of the most common viruses that causes infections in the CNS and the infections give rise to a wide range of CNS manifestations, often without rash (122, 126, 143-145). Serological assays of anti-VZV antibodies are in these cases an important complement to PCR analyzes (126, 458). The time window for detecting VZV DNA is limited and when VZV DNA cannot be found, detection of intrathecally produced anti- Figure 27. Flowchart showing the results of the 854 serum samples analyzed by indirect ELISA using varicella-zoster virus (VZV) whole antigen (VZVwhole-ag) and VZVgE VZV antibodies can lead to the diagnosis (122, 126, 151). antigen (VZVgE-ag). 78 79 There may exist several VZV CNS infections that remain undiagnosed due to that they are at risk of detecting cross-reactive HSV antibodies because they the lack of clinical awareness in the absence of the characteristic rash. Once contain VZVgB, which is likely to have cross-reactive epitopes with HSVgB the diagnostic question has emerged, the time window for detection of VZV (156, 157). Detection of cross-reactive antibodies can cause false positive DNA may already be closed. This may well be the case in some patients with results that lead to misdiagnosis or incorrect assumptions about immunity stroke, even though the link between stroke and VZV is now established (123, (157-161). 459). One study showed that in patients with VZV vasculopathy, analysis of intrathecal anti-VZV antibodies was a more sensitive method compared with The indirect ELISA method developed in Paper I with VZVgE as antigen was detection of VZV DNA to diagnose the disease (154). shown in a later study to reduce the risk of detecting cross-reactive anti-HSV- 1 IgG antibodies in patients with PCR confirmed CNS infections with HSV or Patients' history of VZV infection can in rare cases be unknown and/or VZV, compared with the use of VZVwhole-ag (323). The increased specificity unreliable as several other viruses can cause viral rashes with reddish or pink of the indirect VZVgE ELISA method is an improvement in VZV serology spots, which for individuals without medical training can lead to misdiagnosis diagnostics to prevent false positive results due to cross-reactive antibodies. of the disease. Laboratory determination of VZV immunity is therefore VZVgE has been used as serological antigen in indirect ELISA for the necessary in some cases. The following are examples of when determination detection of anti-VZV IgG in the Department of Clinical Microbiology, of VZV immunity may need to be performed (i) immunocompromised Sahlgrenska University Hospital. The laboratory is the national reference individuals exposed to VZV (ii) before the introduction of immunosuppressive laboratory for herpesviruses. medications in patients to determine the need for VZV vaccination and after vaccination to control the immune response (iii) healthcare professionals with The VZVgE ELISA method for the analysis of anti-VZV IgG antibodies was unknown VZV serostatus exposed to VZV or considering vaccination (iiii) compared with the routine clinical assays, VZVwhole-ag ELISA and indirect pregnant women exposed to VZV and with uncertain history of previous immunofluorescence using VZV-infected cells, to evaluate the performance of infection (iiiii) women with unknown serostatus considering vaccination the VZVgE ELISA. The results showed that the VZVgE ELISA had high before attempting pregnancy and also other adults with unknown serostatus sensitivity and higher specificity compared with the routinely used methods considering vaccination. and these serological assays were then removed from the routine diagnostics (data not shown). Moreover, the serological VZVgE antigen developed by us Although immunity to VZV is mainly due to the cell-dependent immune has been successfully used in studies in Finland for the detection of anti-VZV response and this immune response is the best indicator of immunity, it is a IgG antibodies (461, 462). In addition, a recent study has successfully reported more labor-intensive and complicated procedure to analyze this cell-dependent the use of another VZVgE antigen in a diagnostic CLIA method (463). response. Thus, for routine laboratory analysis of VZV immunity, the detection of anti-VZV IgG is the primary method (460). Sensitive and specific VZVgE vaccine serological methods for anti-VZV IgG detection are also important for evaluating VZV seroprevalence in the population. One limitation when VZV The Shingrix® vaccine, which is based on VZVgE, has proved to induce a seroprevalence in Sweden was estimated in a recent study was the risk of false- strong immune response (136-139) and even give rise to a more robust positive results due to cross-reactive anti-HSV IgG antibodies (106). immunological memory with a longer protection period compared with the Zostavax® vaccine, which is based on a higher dose of the attenuated Oka Cross-reactivity strain (140-142). The higher protective effect of the Shingrix® vaccine may be due in part to different glycosylation patterns caused by the generation of the VZV serology methods are widely used and of high value, but it is important vaccines in different cell types (464). Recombinant VZVgE in the Shingrix® that they are specific in the detection of anti-VZV IgG as the results are used vaccine is expressed in CHO cells while the attenuated Oka strain in the to diagnose VZV CNS infections and to guide vaccination decisions and Zostavax® vaccine is expressed in human fibroblasts. Thus, VZVgE glycan therapeutic interventions for VZV-exposed pregnant women and occupancy and glycan structures may show differences between the two immunocompromised individuals. There are many different VZV serology vaccines, which may affect the induction of the immune response (464). The methods used in laboratories around the world. Common to most methods is strong immune response to VZVgE expressed in CHO cells in the Shingrix® 80 81 There may exist several VZV CNS infections that remain undiagnosed due to that they are at risk of detecting cross-reactive HSV antibodies because they the lack of clinical awareness in the absence of the characteristic rash. Once contain VZVgB, which is likely to have cross-reactive epitopes with HSVgB the diagnostic question has emerged, the time window for detection of VZV (156, 157). Detection of cross-reactive antibodies can cause false positive DNA may already be closed. This may well be the case in some patients with results that lead to misdiagnosis or incorrect assumptions about immunity stroke, even though the link between stroke and VZV is now established (123, (157-161). 459). One study showed that in patients with VZV vasculopathy, analysis of intrathecal anti-VZV antibodies was a more sensitive method compared with The indirect ELISA method developed in Paper I with VZVgE as antigen was detection of VZV DNA to diagnose the disease (154). shown in a later study to reduce the risk of detecting cross-reactive anti-HSV- 1 IgG antibodies in patients with PCR confirmed CNS infections with HSV or Patients' history of VZV infection can in rare cases be unknown and/or VZV, compared with the use of VZVwhole-ag (323). The increased specificity unreliable as several other viruses can cause viral rashes with reddish or pink of the indirect VZVgE ELISA method is an improvement in VZV serology spots, which for individuals without medical training can lead to misdiagnosis diagnostics to prevent false positive results due to cross-reactive antibodies. of the disease. Laboratory determination of VZV immunity is therefore VZVgE has been used as serological antigen in indirect ELISA for the necessary in some cases. The following are examples of when determination detection of anti-VZV IgG in the Department of Clinical Microbiology, of VZV immunity may need to be performed (i) immunocompromised Sahlgrenska University Hospital. The laboratory is the national reference individuals exposed to VZV (ii) before the introduction of immunosuppressive laboratory for herpesviruses. medications in patients to determine the need for VZV vaccination and after vaccination to control the immune response (iii) healthcare professionals with The VZVgE ELISA method for the analysis of anti-VZV IgG antibodies was unknown VZV serostatus exposed to VZV or considering vaccination (iiii) compared with the routine clinical assays, VZVwhole-ag ELISA and indirect pregnant women exposed to VZV and with uncertain history of previous immunofluorescence using VZV-infected cells, to evaluate the performance of infection (iiiii) women with unknown serostatus considering vaccination the VZVgE ELISA. The results showed that the VZVgE ELISA had high before attempting pregnancy and also other adults with unknown serostatus sensitivity and higher specificity compared with the routinely used methods considering vaccination. and these serological assays were then removed from the routine diagnostics (data not shown). Moreover, the serological VZVgE antigen developed by us Although immunity to VZV is mainly due to the cell-dependent immune has been successfully used in studies in Finland for the detection of anti-VZV response and this immune response is the best indicator of immunity, it is a IgG antibodies (461, 462). In addition, a recent study has successfully reported more labor-intensive and complicated procedure to analyze this cell-dependent the use of another VZVgE antigen in a diagnostic CLIA method (463). response. Thus, for routine laboratory analysis of VZV immunity, the detection of anti-VZV IgG is the primary method (460). Sensitive and specific VZVgE vaccine serological methods for anti-VZV IgG detection are also important for evaluating VZV seroprevalence in the population. One limitation when VZV The Shingrix® vaccine, which is based on VZVgE, has proved to induce a seroprevalence in Sweden was estimated in a recent study was the risk of false- strong immune response (136-139) and even give rise to a more robust positive results due to cross-reactive anti-HSV IgG antibodies (106). immunological memory with a longer protection period compared with the Zostavax® vaccine, which is based on a higher dose of the attenuated Oka Cross-reactivity strain (140-142). The higher protective effect of the Shingrix® vaccine may be due in part to different glycosylation patterns caused by the generation of the VZV serology methods are widely used and of high value, but it is important vaccines in different cell types (464). Recombinant VZVgE in the Shingrix® that they are specific in the detection of anti-VZV IgG as the results are used vaccine is expressed in CHO cells while the attenuated Oka strain in the to diagnose VZV CNS infections and to guide vaccination decisions and Zostavax® vaccine is expressed in human fibroblasts. Thus, VZVgE glycan therapeutic interventions for VZV-exposed pregnant women and occupancy and glycan structures may show differences between the two immunocompromised individuals. There are many different VZV serology vaccines, which may affect the induction of the immune response (464). The methods used in laboratories around the world. Common to most methods is strong immune response to VZVgE expressed in CHO cells in the Shingrix® 80 81 vaccine strengthens the argument for also expressing the serological VZVgE 4.2 PAPER II antigen in CHO cells. In Paper II, a sensitive and specific serological assay was developed using The anti-VZV IgG response was higher in serum samples from older EBVgp350 as antigen. Serum samples with previously known serostatus individuals, i.e. ischemic stroke patients and their age-matched controls against VCA and EBNA1 were analyzed with the new EBVgp350 ELISA compared with younger individuals, i.e. blood donors and students. This method for the detection of anti-EBVgp350 IgG antibodies. suggests that reactivity to this protein may increase with age and possible reactivation. The high antibody levels against VZVgE suggest that the Comparison between longer and shorter EBVgp350 constructs glycoprotein is a strong inducer of the immune response even in older individuals, which may be a contributing factor to the Shingrix vaccine's EBVgp350 is extensively glycosylated (209, 218) and glycosylation is success in protecting against herpes zoster. important for the antigenicity of the protein (451, 466). As with VZVgE, the glycosylation of EBVgp350 depends on the host cell used to express the Anti-VZV IgG antibody levels induced after vaccination are lower compared protein (451). CHO cells, which are mammalian cells, are known to generate to the levels obtained after natural infection, which is why anti-VZV IgG levels less numerous but still quite similar glycosylation to that expressed in virus- after vaccination are more difficult to detect in serological assays (465). It may infected human host cells (467). CHO cells have also been successfully used therefore be beneficial to have a serological assay based on the same in the past to express EBVgp350 (468, 469) and these cells were therefore glycoprotein as that used in the Shingrix® vaccine. A chickenpox vaccine selected to generate the recombinant EBVgp350. In paper II, two protein using VZVgE is not currently on the market, but it would be beneficial as it is constructs consisting of a.a. 1–860 or a.a. 1–506 were successfully produced likely that immunocompromised individuals who cannot be vaccinated with in CHO cells. the live attenuated chickenpox vaccine can tolerate a subunit vaccine. The two protein constructs were then used as serological antigens in indirect ELISA. A total of 21 VCA and EBNA1 IgG seropositive (VCA+EBNA1+) samples and 21 VCA and EBNA1 seronegative (VCA-EBNA1-) samples were analyzed by indirect ELISA using the two antigens. The seroreactivity against the longer protein construct, i.e. a.a. 1–860, was found to be higher compared with that against the shorter protein segment, i.e. a.a. 1–506. The antibody responses to different parts of EBVgp350 show great variation between individuals (466). This may partly explain why the analyzed serum samples in Paper II show higher anti-EBV IgG reactivity against the longer protein, i.e. the a.a. 1–860 construct, which contains more epitopes compared with the 1–506 protein. The higher IgG reactivity detected against the longer protein may be due to reactive sequences located between a.a. 741 and a.a. 860 (466) which thus only the longer construct contains. Previous studies analyzing anti-EBVgp350 antibody responses have either used a long protein such as a.a. 1–860, or a short construct, i.e. a.a. 4–450, why it has not previously been possible to compare the reactivity of antigens at different protein lengths of EBVgp350 (274, 356). 82 83 vaccine strengthens the argument for also expressing the serological VZVgE 4.2 PAPER II antigen in CHO cells. In Paper II, a sensitive and specific serological assay was developed using The anti-VZV IgG response was higher in serum samples from older EBVgp350 as antigen. Serum samples with previously known serostatus individuals, i.e. ischemic stroke patients and their age-matched controls against VCA and EBNA1 were analyzed with the new EBVgp350 ELISA compared with younger individuals, i.e. blood donors and students. This method for the detection of anti-EBVgp350 IgG antibodies. suggests that reactivity to this protein may increase with age and possible reactivation. The high antibody levels against VZVgE suggest that the Comparison between longer and shorter EBVgp350 constructs glycoprotein is a strong inducer of the immune response even in older individuals, which may be a contributing factor to the Shingrix vaccine's EBVgp350 is extensively glycosylated (209, 218) and glycosylation is success in protecting against herpes zoster. important for the antigenicity of the protein (451, 466). As with VZVgE, the glycosylation of EBVgp350 depends on the host cell used to express the Anti-VZV IgG antibody levels induced after vaccination are lower compared protein (451). CHO cells, which are mammalian cells, are known to generate to the levels obtained after natural infection, which is why anti-VZV IgG levels less numerous but still quite similar glycosylation to that expressed in virus- after vaccination are more difficult to detect in serological assays (465). It may infected human host cells (467). CHO cells have also been successfully used therefore be beneficial to have a serological assay based on the same in the past to express EBVgp350 (468, 469) and these cells were therefore glycoprotein as that used in the Shingrix® vaccine. A chickenpox vaccine selected to generate the recombinant EBVgp350. In paper II, two protein using VZVgE is not currently on the market, but it would be beneficial as it is constructs consisting of a.a. 1–860 or a.a. 1–506 were successfully produced likely that immunocompromised individuals who cannot be vaccinated with in CHO cells. the live attenuated chickenpox vaccine can tolerate a subunit vaccine. The two protein constructs were then used as serological antigens in indirect ELISA. A total of 21 VCA and EBNA1 IgG seropositive (VCA+EBNA1+) samples and 21 VCA and EBNA1 seronegative (VCA-EBNA1-) samples were analyzed by indirect ELISA using the two antigens. The seroreactivity against the longer protein construct, i.e. a.a. 1–860, was found to be higher compared with that against the shorter protein segment, i.e. a.a. 1–506. The antibody responses to different parts of EBVgp350 show great variation between individuals (466). This may partly explain why the analyzed serum samples in Paper II show higher anti-EBV IgG reactivity against the longer protein, i.e. the a.a. 1–860 construct, which contains more epitopes compared with the 1–506 protein. The higher IgG reactivity detected against the longer protein may be due to reactive sequences located between a.a. 741 and a.a. 860 (466) which thus only the longer construct contains. Previous studies analyzing anti-EBVgp350 antibody responses have either used a long protein such as a.a. 1–860, or a short construct, i.e. a.a. 4–450, why it has not previously been possible to compare the reactivity of antigens at different protein lengths of EBVgp350 (274, 356). 82 83 Analysis of samples of the VCA-EBNA1- IgG seronegative samples with the addition of two standard deviations. According to the ROC curve, the cut-off point had a The longer protein construct with higher reactivity was then used for indirect sensitivity of 90% and a specificity of 98%. ELISA analysis of the entire clinical material with 360 serum samples diluted 1/400 and with known IgG serostatus against VCA and EBNA1. The samples Anti-EBVgp350 IgG was detected in 108/120 (90%) of the VCA+EBNA1+ that were VCA+EBNA1+ and many of the VCA+ EBNA1- samples showed serum samples. Of the VCA IgG seropositive but EBNA1 IgG seronegative reactivity against EBVgp350. The samples that were both VCA and EBNA1 (VCA+ EBNA1-) samples, 79/120 (66%) were EBVgp350 IgG seropositive. IgG seronegative generally showed no anti-EBVgp350 IgG reactivity. The Only 2/120 (2%) of the VCA-EBNA1- IgG seronegative samples had results are presented in Figure 29. detectable anti-EBVgp350 IgG. Thus, a total of 118/120 (98%) of these samples were anti-EBVgp350 IgG seronegative. The results are illustrated in Figure 30. Figure 30. Schematic figure showing how many of the serum samples (n = 360) with known serostatus against viral capsid antigen (VCA) and Epstein-Barr virus nuclear Figure 29. Serum samples (n = 360) with known serostatus to viral capsid antigen (VCA) antigen 1 (EBNA1) were Epstein-Barr virus gp350 (EBVgp350) IgG seropositive (+) and and Epstein-Barr virus nuclear antigen 1 (EBNA1) were analyzed by indirect ELISA in seronegative (-). dilution 1/400 for detection of IgG antibodies to Epstein-Barr virus glycoprotein 350 (EBVgp350). In total there were 120 VCA and EBNA1 seropositive samples (VCA+EBNA1+), 120 VCA seropositive but EBNA1 IgG seronegative samples Analysis of anti-EBNA1 IgG and anti-VCA IgM/IgG can often identify the (VCA+EBNA1-) and 120 VCA and EBNA1 IgG seronegative samples (VCA-EBNA1-). stage of an EBV infection, but in some special situations, it can be difficult. In The Tukey box plot show the anti-EBVgp350 IgG reactivity in these samples measured as these cases, it may be beneficial to perform an additional EBV serological optical density. analysis to increase the chance of correctly determining the patient's history of EBV infection. The antibody response to EBV varies between individuals. It A receiver operating characteristic (ROC) curve was generated to analyze the is known that some individuals do not produce detectable anti-EBNA1 IgG diagnostic performance of the EBVgp350 ELISA method. The levels even though it has been long enough since they became infected with VCA+EBNA1+ samples (n = 120) were defined as true positive samples and the virus (204, 272). The kinetics of generating anti-EBV antibodies to various the VCA-EBNA1- samples (n = 120) were defined as true negative samples in EBV antigens also differ between individuals (204, 272-274, 355, 356). This the analysis. The ROC curve showed that the EBVgp350 ELISA method had may partly explain why some VCA+EBNA1+ IgG seropositive serum samples high sensitivity and specificity. The OD value for cut-off between EBVgp350 did not show detectable anti-EBVgp350 IgG and why two VCA-EBNA1- IgG seropositive and seronegative samples was set at 0.162, based on the mean negative samples were EBVgp350 IgG seropositive. 84 85 Analysis of samples of the VCA-EBNA1- IgG seronegative samples with the addition of two standard deviations. According to the ROC curve, the cut-off point had a The longer protein construct with higher reactivity was then used for indirect sensitivity of 90% and a specificity of 98%. ELISA analysis of the entire clinical material with 360 serum samples diluted 1/400 and with known IgG serostatus against VCA and EBNA1. The samples Anti-EBVgp350 IgG was detected in 108/120 (90%) of the VCA+EBNA1+ that were VCA+EBNA1+ and many of the VCA+ EBNA1- samples showed serum samples. Of the VCA IgG seropositive but EBNA1 IgG seronegative reactivity against EBVgp350. The samples that were both VCA and EBNA1 (VCA+ EBNA1-) samples, 79/120 (66%) were EBVgp350 IgG seropositive. IgG seronegative generally showed no anti-EBVgp350 IgG reactivity. The Only 2/120 (2%) of the VCA-EBNA1- IgG seronegative samples had results are presented in Figure 29. detectable anti-EBVgp350 IgG. Thus, a total of 118/120 (98%) of these samples were anti-EBVgp350 IgG seronegative. The results are illustrated in Figure 30. Figure 30. Schematic figure showing how many of the serum samples (n = 360) with known serostatus against viral capsid antigen (VCA) and Epstein-Barr virus nuclear Figure 29. Serum samples (n = 360) with known serostatus to viral capsid antigen (VCA) antigen 1 (EBNA1) were Epstein-Barr virus gp350 (EBVgp350) IgG seropositive (+) and and Epstein-Barr virus nuclear antigen 1 (EBNA1) were analyzed by indirect ELISA in seronegative (-). dilution 1/400 for detection of IgG antibodies to Epstein-Barr virus glycoprotein 350 (EBVgp350). In total there were 120 VCA and EBNA1 seropositive samples (VCA+EBNA1+), 120 VCA seropositive but EBNA1 IgG seronegative samples Analysis of anti-EBNA1 IgG and anti-VCA IgM/IgG can often identify the (VCA+EBNA1-) and 120 VCA and EBNA1 IgG seronegative samples (VCA-EBNA1-). stage of an EBV infection, but in some special situations, it can be difficult. In The Tukey box plot show the anti-EBVgp350 IgG reactivity in these samples measured as these cases, it may be beneficial to perform an additional EBV serological optical density. analysis to increase the chance of correctly determining the patient's history of EBV infection. The antibody response to EBV varies between individuals. It A receiver operating characteristic (ROC) curve was generated to analyze the is known that some individuals do not produce detectable anti-EBNA1 IgG diagnostic performance of the EBVgp350 ELISA method. The levels even though it has been long enough since they became infected with VCA+EBNA1+ samples (n = 120) were defined as true positive samples and the virus (204, 272). The kinetics of generating anti-EBV antibodies to various the VCA-EBNA1- samples (n = 120) were defined as true negative samples in EBV antigens also differ between individuals (204, 272-274, 355, 356). This the analysis. The ROC curve showed that the EBVgp350 ELISA method had may partly explain why some VCA+EBNA1+ IgG seropositive serum samples high sensitivity and specificity. The OD value for cut-off between EBVgp350 did not show detectable anti-EBVgp350 IgG and why two VCA-EBNA1- IgG seropositive and seronegative samples was set at 0.162, based on the mean negative samples were EBVgp350 IgG seropositive. 84 85 Anti-EBVgp350 IgG has been studied as a potential reverse marker for the 4.3 PAPER III severity of EBV-induced disease (274, 355). The results from a study showed that patients with higher anti-EBVgp350 IgG levels had milder IM disease Patients with MS show increased IgG antibody reactivity to certain neurotropic compared with patients with lower IgG levels (274). Another study found no viruses including MeV compared with healthy controls. The mechanisms correlation between peak levels of various EBV antibodies, including anti- behind the increased response to these viruses are unknown and therefore EBVgp350, and the severity of IM symptoms (355). A third study on the require further investigation. The MeV reactivity detected in serological assays subject demonstrated that the generation of EBVgp350-neutralizing antibodies may be specific to MeV antigens or nonspecific against other components of was associated with the EBV viral load in blood during IM (356). the viral antigen. In Paper III, we examined the anti-MeV IgG specificity in patients with MS, their clinically healthy siblings and healthy controls using a Furthermore, our study presented in Paper IV, showed that IM induces a strong more specific serological assay with the conserved core portion of the MeV antibody response to EBV and that individuals with IM even after 10 years nucleocapsid protein (MeV NCORE) as the serological antigen. have increased anti-EBVgp350 IgG levels compared with healthy controls (470). Further research with sensitive and specific EBVgp350 serological Patients with MS assays may clarify whether anti-EBVgp350 IgG is a marker for milder EBV disease, which may strengthen the logic for the development of an EBVgp350 Serum samples diluted 1/400 and CSF samples diluted 1/40 from the subjects vaccine. were analyzed by indirect ELISA using MeV NCORE as antigen. Patients with MS showed higher levels of anti-MeV NCORE IgG measured as OD in both EBVgp350 vaccine serum (p < 0.001) and CSF (p < 0.001) compared with healthy controls. Moreover, patients with MS had higher IgG levels in CSF (p < 0.001) but not There is currently no EBV vaccine available on the market, but research is in serum (p = 0.2) compared with the whole group of 46 healthy siblings. These ongoing (258-260). A vaccine based on EBVgp350 produced in CHO cells results are presented in Figure 31. showed in a phase two trial, a possible effect to prevent IM but not against asymptomatic EBV infection (262). EBVgp350 is still an important candidate protein for a subunit vaccine against EBV and serological methods that analyze the IgG response to this protein may therefore be needed in the future to measure antibody responses after vaccination (258-260, 471). Figure 31. IgG antibody reactivity to measles virus nucleocapsid antigen (MeV NCORE) in serum samples diluted 1/400 and cerebrospinal fluid (CSF) samples diluted 1/40, from patients with MS (n = 47), siblings with MS trait (n = 9), siblings without MS trait (n = 37) and healthy controls (n = 50). Reactivity is measured as optical density and median values with interquartile range are illustrated in the figure. 86 87 Anti-EBVgp350 IgG has been studied as a potential reverse marker for the 4.3 PAPER III severity of EBV-induced disease (274, 355). The results from a study showed that patients with higher anti-EBVgp350 IgG levels had milder IM disease Patients with MS show increased IgG antibody reactivity to certain neurotropic compared with patients with lower IgG levels (274). Another study found no viruses including MeV compared with healthy controls. The mechanisms correlation between peak levels of various EBV antibodies, including anti- behind the increased response to these viruses are unknown and therefore EBVgp350, and the severity of IM symptoms (355). A third study on the require further investigation. The MeV reactivity detected in serological assays subject demonstrated that the generation of EBVgp350-neutralizing antibodies may be specific to MeV antigens or nonspecific against other components of was associated with the EBV viral load in blood during IM (356). the viral antigen. In Paper III, we examined the anti-MeV IgG specificity in patients with MS, their clinically healthy siblings and healthy controls using a Furthermore, our study presented in Paper IV, showed that IM induces a strong more specific serological assay with the conserved core portion of the MeV antibody response to EBV and that individuals with IM even after 10 years nucleocapsid protein (MeV NCORE) as the serological antigen. have increased anti-EBVgp350 IgG levels compared with healthy controls (470). Further research with sensitive and specific EBVgp350 serological Patients with MS assays may clarify whether anti-EBVgp350 IgG is a marker for milder EBV disease, which may strengthen the logic for the development of an EBVgp350 Serum samples diluted 1/400 and CSF samples diluted 1/40 from the subjects vaccine. were analyzed by indirect ELISA using MeV NCORE as antigen. Patients with MS showed higher levels of anti-MeV NCORE IgG measured as OD in both EBVgp350 vaccine serum (p < 0.001) and CSF (p < 0.001) compared with healthy controls. Moreover, patients with MS had higher IgG levels in CSF (p < 0.001) but not There is currently no EBV vaccine available on the market, but research is in serum (p = 0.2) compared with the whole group of 46 healthy siblings. These ongoing (258-260). A vaccine based on EBVgp350 produced in CHO cells results are presented in Figure 31. showed in a phase two trial, a possible effect to prevent IM but not against asymptomatic EBV infection (262). EBVgp350 is still an important candidate protein for a subunit vaccine against EBV and serological methods that analyze the IgG response to this protein may therefore be needed in the future to measure antibody responses after vaccination (258-260, 471). Figure 31. IgG antibody reactivity to measles virus nucleocapsid antigen (MeV NCORE) in serum samples diluted 1/400 and cerebrospinal fluid (CSF) samples diluted 1/40, from patients with MS (n = 47), siblings with MS trait (n = 9), siblings without MS trait (n = 37) and healthy controls (n = 50). Reactivity is measured as optical density and median values with interquartile range are illustrated in the figure. 86 87 The ODCSF/ODserum values were calculated for each patient. When comparing Siblings with MS trait the OD ratios between different groups, the patients with MS had a higher ratio compared with both healthy controls (p < 0.001) and clinically healthy siblings Siblings with MS trait showed higher anti-MeV NCORE IgG levels compared (p < 0.001). with healthy controls in serum (p = 0.01) and CSF (p = 0.002). In addition, the siblings with MS trait showed higher anti-MeV NCORE IgG levels in CSF (p = The previous study that analyzed the identical patient material (except for a 0.04) but not in serum (p = 0.08) compared with siblings without MS trait. The male sibling without trait) showed similar results but used MeV whole virus results are presented in Figure 31. Siblings with MS trait demonstrated higher antigen generated in human fibroblasts in indirect ELISA (445). The antibody ODCSF/ODserum values compared with healthy controls (p = 0.008), while response to MeV is part of the common and characteristic MRZ reaction seen siblings without trait did not (p = 0.5). in patients with MS where intrathecally produced antibodies to MeV, RV and VZV are detected (431, 472-475). The antibody response in serum to MeV is Approximately 16 years after the first sampling, a follow-up was performed not as closely investigated but a few studies, including the precursor study to regarding the siblings with MS trait, where it was found that none of these the present one, have shown that serum anti-MeV IgG reactivity is also siblings had developed MS. The underlying pathophysiology to why some increased in patients with MS compared with healthy controls (432, 433, 445). clinically healthy siblings of patients with MS demonstrate this immunological reaction is unknown. One hypothesis is that these siblings are genetically The new and advantageous in the present study is the specific MeV NCORE susceptible, but that additional environmental triggers are required to develop antigen that has been used in the serological assay. MS is considered an the disease which may not occur (446, 447). autoimmune disease and MeV whole virus antigens commonly used in serological assays may increase the risk of autoantibody detection as there may be residual human/primate cellular components in the antigen remaining after antigen production. The risk of autoantibody detection decreases when the recombinantly produced MeV NCORE antigen lacking human/primate components is used. Thus, the diagnostic specificity for detecting anti-MeV- specific IgG increases when this ELISA method is used. Although we cannot completely rule out the risk of non-specific binding or cross-reactive antibodies, it is more likely that the increased anti-MeV NCORE IgG levels detected in serum and CSF samples from patients with MS in Paper III are due to MeV-specific antibodies. Intrathecal antibody production in the form of OCB in CSF is not a specific marker for MS but may in some clinical contexts support an MS diagnosis (320, 387, 445, 476). This has been considered in the 2017 revision of the McDonald's criteria for diagnosing MS (387). MS can sometimes be difficult to diagnose and the MRZ response can in these situations be used to support the MS diagnosis (430, 477-480). Even in patients who do not present OCB in CSF, there may be a positive MRZ reaction that can support an MS diagnosis (474, 480, 481). In addition, the MRZ reaction may be a useful marker for distinguishing MS from other autoimmune diseases with similar symptoms (477-480). 88 89 The ODCSF/ODserum values were calculated for each patient. When comparing Siblings with MS trait the OD ratios between different groups, the patients with MS had a higher ratio compared with both healthy controls (p < 0.001) and clinically healthy siblings Siblings with MS trait showed higher anti-MeV NCORE IgG levels compared (p < 0.001). with healthy controls in serum (p = 0.01) and CSF (p = 0.002). In addition, the siblings with MS trait showed higher anti-MeV NCORE IgG levels in CSF (p = The previous study that analyzed the identical patient material (except for a 0.04) but not in serum (p = 0.08) compared with siblings without MS trait. The male sibling without trait) showed similar results but used MeV whole virus results are presented in Figure 31. Siblings with MS trait demonstrated higher antigen generated in human fibroblasts in indirect ELISA (445). The antibody ODCSF/ODserum values compared with healthy controls (p = 0.008), while response to MeV is part of the common and characteristic MRZ reaction seen siblings without trait did not (p = 0.5). in patients with MS where intrathecally produced antibodies to MeV, RV and VZV are detected (431, 472-475). The antibody response in serum to MeV is Approximately 16 years after the first sampling, a follow-up was performed not as closely investigated but a few studies, including the precursor study to regarding the siblings with MS trait, where it was found that none of these the present one, have shown that serum anti-MeV IgG reactivity is also siblings had developed MS. The underlying pathophysiology to why some increased in patients with MS compared with healthy controls (432, 433, 445). clinically healthy siblings of patients with MS demonstrate this immunological reaction is unknown. One hypothesis is that these siblings are genetically The new and advantageous in the present study is the specific MeV NCORE susceptible, but that additional environmental triggers are required to develop antigen that has been used in the serological assay. MS is considered an the disease which may not occur (446, 447). autoimmune disease and MeV whole virus antigens commonly used in serological assays may increase the risk of autoantibody detection as there may be residual human/primate cellular components in the antigen remaining after antigen production. The risk of autoantibody detection decreases when the recombinantly produced MeV NCORE antigen lacking human/primate components is used. Thus, the diagnostic specificity for detecting anti-MeV- specific IgG increases when this ELISA method is used. Although we cannot completely rule out the risk of non-specific binding or cross-reactive antibodies, it is more likely that the increased anti-MeV NCORE IgG levels detected in serum and CSF samples from patients with MS in Paper III are due to MeV-specific antibodies. Intrathecal antibody production in the form of OCB in CSF is not a specific marker for MS but may in some clinical contexts support an MS diagnosis (320, 387, 445, 476). This has been considered in the 2017 revision of the McDonald's criteria for diagnosing MS (387). MS can sometimes be difficult to diagnose and the MRZ response can in these situations be used to support the MS diagnosis (430, 477-480). Even in patients who do not present OCB in CSF, there may be a positive MRZ reaction that can support an MS diagnosis (474, 480, 481). In addition, the MRZ reaction may be a useful marker for distinguishing MS from other autoimmune diseases with similar symptoms (477-480). 88 89 4.4 PAPER IV with an asymptomatic/mild infection and that this is the reason behind the increased anti-EBVgp350 IgG levels that are detected at 10-year follow-up in Several studies have shown that the risk of developing MS increases after IM post-IM patients. It has previously been shown that the amount of EBV virus (246, 247, 373). However, this relationship has not been prospectively in blood and the amount of CD8+ T cells correlate with the severity of the investigated from the time of IM but mainly retrospectively from when the MS disease (482) and that the amount of EBV virus in blood is associated with the disease was diagnosed. In Paper IV, the antibody response to EBVgp350, production of EBVgp350 neutralizing antibodies (356). VZVgE and MeV NCORE was examined in sera from patients with acute IM and 10 years later at follow-up. The antibody responses in the patients with acute The increased serum levels of anti-EBVgp350 in post-IM patients compared IM was compared with the seroreactivity to these viruses, both in healthy with healthy controls were not reflected in increased levels of anti-EBNA1 controls and in patients with MS. and/or anti-VCA IgG antibodies. There are biological differences in the antibody response to various viral antigens from the same virus both in terms Serum antibodies to EBV in patients with IM of kinetics and general antibody production. It is known that not all individuals infected with EBV show a detectable IgG response to EBNA1 (272). The levels of anti-EBNA1 and anti-EBVgp350 IgG in the serum samples diluted 1/1000, from the 42 subjects with acute IM, were generally low and EBVgp350 is an immunodominant envelope glycoprotein and the primary significantly lower compared with the 39 EBV seropositive healthy controls. target for EBV-neutralizing antibodies (220-222). Therefore, it is likely that Most patients with acute IM had lower levels of anti-VCA IgG than the healthy acute IM may induce a higher production of anti-EBVgp350 IgG compared controls, but many were, unlike the healthy controls, anti-VCA IgM with asymptomatic/mild EBV infections. Another reason for the higher anti- seropositive. EBVgp350 IgG levels in post-IM patients compared with healthy controls may be more prevalent recurrent EBV reactivations, but this question has not been At follow-up, anti-VCA, anti-EBNA1 and anti-EBVgp350 IgG levels had studied. The exact composition of the complex VCA antigen is unknown to the increased in patients with previous IM. The post-IM patients’ anti-EBVgp350 general research community, but it is likely that EBVgp350 is part of the IgG levels were higher at follow-up compared with the 39 healthy controls (p content. It is possible that our immunodominant EBVgp350 antigen may detect = 0.007). However, there was no significant difference in anti-EBNA1 or anti- higher levels of EBVgp350-specific IgG antibodies compared with the VCA VCA IgG levels between patients at follow-up and controls, although the post- antigen which also consists of many other components to which the IM patients showed a tendency of having higher levels against the latter antigen EBVgp350-specific antibodies are less likely to attach. (Table 5). Serum antibodies to VZV and MeV in patients with IM The finding that individuals with a history of IM showed higher levels of anti- EBVgp350 IgG compared with healthy controls at follow-up after 10 years Serum levels of both anti-MeV NCORE and anti-VZVgE IgG were higher during suggests that a primary EBV infection in the form of IM affects the immune acute IM compared with the levels in the same subjects at follow-up (Table 5). system in a different and more powerful way compared with an asymptomatic The increased antibody reactivity to both VZV and MeV in the patients with primary EBV infection. EBV establishes latency after the primary infection to acute IM may be due to EBV-induced polyclonal B-cell activation. later reactivate. The virus will remain in the host for life and the virus will induce a lifelong antibody response. Anti-EBV IgG antibody levels have been There was no significant difference in serum anti-VZVgE IgG levels in post- shown to be stable with no apparent decrease over time (22). Thus, even if the IM patients at follow-up compared with healthy controls. IgG antibody levels EBV seropositive healthy controls acquired their EBV infection earlier to VZV normally show a moderate decrease over time (22). Post-IM patients compared with the participants with IM, the levels of anti-EBV IgG antibodies at follow-up and healthy controls showed similar anti-MeV NCORE IgG levels are not likely to be lower due to the difference in the duration of the EBV in serum. In an age-adjusted analysis, the post-IM patients showed a moderate infection. One hypothesis is that primary EBV infection in the form of IM, increase compared with healthy controls (p = 0.014). IgG antibody levels to with a higher viral set-point, induces a stronger response from the immune MeV are generally stable over time after natural infection (22) but declines system that give rise to higher levels of anti-EBVgp350 antibodies compared with time after vaccination (297). 90 91 4.4 PAPER IV with an asymptomatic/mild infection and that this is the reason behind the increased anti-EBVgp350 IgG levels that are detected at 10-year follow-up in Several studies have shown that the risk of developing MS increases after IM post-IM patients. It has previously been shown that the amount of EBV virus (246, 247, 373). However, this relationship has not been prospectively in blood and the amount of CD8+ T cells correlate with the severity of the investigated from the time of IM but mainly retrospectively from when the MS disease (482) and that the amount of EBV virus in blood is associated with the disease was diagnosed. In Paper IV, the antibody response to EBVgp350, production of EBVgp350 neutralizing antibodies (356). VZVgE and MeV NCORE was examined in sera from patients with acute IM and 10 years later at follow-up. The antibody responses in the patients with acute The increased serum levels of anti-EBVgp350 in post-IM patients compared IM was compared with the seroreactivity to these viruses, both in healthy with healthy controls were not reflected in increased levels of anti-EBNA1 controls and in patients with MS. and/or anti-VCA IgG antibodies. There are biological differences in the antibody response to various viral antigens from the same virus both in terms Serum antibodies to EBV in patients with IM of kinetics and general antibody production. It is known that not all individuals infected with EBV show a detectable IgG response to EBNA1 (272). The levels of anti-EBNA1 and anti-EBVgp350 IgG in the serum samples diluted 1/1000, from the 42 subjects with acute IM, were generally low and EBVgp350 is an immunodominant envelope glycoprotein and the primary significantly lower compared with the 39 EBV seropositive healthy controls. target for EBV-neutralizing antibodies (220-222). Therefore, it is likely that Most patients with acute IM had lower levels of anti-VCA IgG than the healthy acute IM may induce a higher production of anti-EBVgp350 IgG compared controls, but many were, unlike the healthy controls, anti-VCA IgM with asymptomatic/mild EBV infections. Another reason for the higher anti- seropositive. EBVgp350 IgG levels in post-IM patients compared with healthy controls may be more prevalent recurrent EBV reactivations, but this question has not been At follow-up, anti-VCA, anti-EBNA1 and anti-EBVgp350 IgG levels had studied. The exact composition of the complex VCA antigen is unknown to the increased in patients with previous IM. The post-IM patients’ anti-EBVgp350 general research community, but it is likely that EBVgp350 is part of the IgG levels were higher at follow-up compared with the 39 healthy controls (p content. It is possible that our immunodominant EBVgp350 antigen may detect = 0.007). However, there was no significant difference in anti-EBNA1 or anti- higher levels of EBVgp350-specific IgG antibodies compared with the VCA VCA IgG levels between patients at follow-up and controls, although the post- antigen which also consists of many other components to which the IM patients showed a tendency of having higher levels against the latter antigen EBVgp350-specific antibodies are less likely to attach. (Table 5). Serum antibodies to VZV and MeV in patients with IM The finding that individuals with a history of IM showed higher levels of anti- EBVgp350 IgG compared with healthy controls at follow-up after 10 years Serum levels of both anti-MeV NCORE and anti-VZVgE IgG were higher during suggests that a primary EBV infection in the form of IM affects the immune acute IM compared with the levels in the same subjects at follow-up (Table 5). system in a different and more powerful way compared with an asymptomatic The increased antibody reactivity to both VZV and MeV in the patients with primary EBV infection. EBV establishes latency after the primary infection to acute IM may be due to EBV-induced polyclonal B-cell activation. later reactivate. The virus will remain in the host for life and the virus will induce a lifelong antibody response. Anti-EBV IgG antibody levels have been There was no significant difference in serum anti-VZVgE IgG levels in post- shown to be stable with no apparent decrease over time (22). Thus, even if the IM patients at follow-up compared with healthy controls. IgG antibody levels EBV seropositive healthy controls acquired their EBV infection earlier to VZV normally show a moderate decrease over time (22). Post-IM patients compared with the participants with IM, the levels of anti-EBV IgG antibodies at follow-up and healthy controls showed similar anti-MeV NCORE IgG levels are not likely to be lower due to the difference in the duration of the EBV in serum. In an age-adjusted analysis, the post-IM patients showed a moderate infection. One hypothesis is that primary EBV infection in the form of IM, increase compared with healthy controls (p = 0.014). IgG antibody levels to with a higher viral set-point, induces a stronger response from the immune MeV are generally stable over time after natural infection (22) but declines system that give rise to higher levels of anti-EBVgp350 antibodies compared with time after vaccination (297). 90 91 Table 5. IgG antibody reactivity to Epstein-Barr virus gp350 Analysis of serum and CSF samples from patients with MS (EBVgp350), Epstein-Barr virus nuclear antigen 1 (EBNA1), viral capsid antigen (VCA), measles virus nucleocapsid antigen (MeV NCORE) and The 22 patients with MS showed higher serum levels of anti-EBVgp350 IgG varicella-zoster virus glycoprotein E (VZVgE). The analyzed serum (p = 0.006) and anti-MeV NCORE IgG (p < 0.0001) compared with the 39 samples were diluted 1/1000 and the cerebrospinal fluid (CSF) samples healthy controls. However, serum levels of anti-VZVgE IgG were not higher diluted 1/10. The sample material was collected from patients with acute infectious mononucleosis (IM), these patients at follow-up (FU), healthy in patients with MS compared with controls. In CSF, patients with MS had controls (HC) and patients with MS (MS). The reactivity is presented as slightly higher IgG antibody levels against both EBVgp350 (p = 0.048) and optical density (OD) or sample to cut-off (S/CO). Median values with VZVgE (p = 0.01) compared with controls. More pronounced were the interquartile range (IQR) are presented. EBV IgG seropositive (EBV +). increased CSF levels of anti-MeV NCORE IgG in the patients compared with the controls (p < 0.0001) (Table 5). The CSF/serum ratio for MeV NCORE and VZVgE but not for EBVgp350 was higher in patients with MS compared with Median (IQR) EBVgp350 EBNA1 VCA VCA MeV NCORE VZVgE healthy controls. IgG OD IgG S/CO IgG S/CO IgM S/CO IgG OD IgG OD Serum samples It is well known that patients with MS show increased IgG responses to all IM (n=42) 0.1 (0.062) 0.38 (0.27) 7.0 (8.04) 10.3 (13.3) 0.86 (0.89) 1.19 (1.02) three viruses, EBV, VZV and MeV, compared with healthy controls, but that FU (n=42) 1.16 (1.85) 15.4 (13.5) 56.1 (30.4) 0.24 (0.35) 0.53 (0.59) 0.59 (0.53) the increased antibody response to VZV and MeV have mainly been reported HC EBV+ (n=39) 0.68 (0.84) 17.0 (14.0) 41.8 (34.9) 0.13 (0.56) 0.48 (0.56) 0.53 (0.40) in CSF while the increased antibody response to EBV has mainly been seen in MS (n=22) 1.98 (1.24) 2.5 (1.2) 0.75 (1.0) sera (428, 472-475, 483). Previous studies of antibody responses to different viruses in patients with MS show no direct correlation between serum and CSF CSF samples IgG levels (431, 484). In this study, patients with MS showed a moderate FU (n=21) 0.31 (0.42) 0.21 (0.17) 0.15 (0.05) increase in anti-EBVgp350 IgG levels in CSF compared with healthy controls. HC (n=17) 0.18 (0.24) 0.17 (0.17) 0.15 (0.13) In previous studies, anti-EBV IgG levels in CSF have often been relatively low HC EBV+ (n=15) 0.18 (0.22) 0.17 (0.17) 0.15 (0.13) compared with serum levels (428, 483). The antibody response to EBVgp350 MS (n=22) 0.31 (0.41) 1.78 (1.41) 0.46 (0.81) in patients with MS has not been extensively investigated in contrast to the carefully studied antibody responses to EBNA1 and VCA. Further studies are therefore needed to conclude whether patients with MS have increased levels CSF antibodies to EBV, VZV and MeV in patients with IM of anti-EBVgp350 IgG in CSF compared with healthy controls. The total IgG levels in CSF were within the normal range in the healthy An established risk factor for developing MS is primary EBV infection in the controls and in the post-IM patients at follow-up. None of these individuals form of IM (246, 247, 373) and a recent study provides serological support that showed OCB in CSF. The CSF samples were diluted 1/10 in the serological late EBV infections increases the risk of developing MS (485). A feature in analysis. Anti-EBVgp350 IgG levels were significantly higher in post-IM individuals with presymtomatic MS is higher antibody reactivity to EBV patients at follow-up compared with the whole group with 17 healthy controls compared with healthy controls, especially to EBNA1 (424, 425, 486, 487). (p = 0.01) but not compared with only the 15 EBV IgG seropositive controls However, the antibody reactivity to EBVgp350 has not been studied in this (p = 0.08). The CSF/serum ratio was determined based on the results of paired context. In conclusion, our results show that patients with acute IM display samples with CSF (diluted 1/10) and serum (diluted 1/1000) giving the increased levels of anti-EBVgp350 IgG in serum at follow-up after 10 years. CSF/serum dilution ratio of 1/100. The CSF/serum ratio for EBVgp350 IgG One hypothesis, that may relate to this finding, is that primary EBV infection was lower among post-IM patients at follow-up compared with the healthy in the form of IM can affect the immune system in a powerful and unusual controls while no differences between the groups were seen for MeV NCORE way, which can be a triggering factor for continued biological events that after and VZVgE. That the CSF/serum ratio for EBVgp350 was lower for the post- many years can lead to autoimmune diseases such as MS. Our present results IM patients at follow-up compared with healthy controls may be due to the support the argument for continuing to investigate the role of EBV in the increased serum levels of anti-EBVgp350 IgG in the patients. pathogenesis of MS. 92 93 Table 5. IgG antibody reactivity to Epstein-Barr virus gp350 Analysis of serum and CSF samples from patients with MS (EBVgp350), Epstein-Barr virus nuclear antigen 1 (EBNA1), viral capsid antigen (VCA), measles virus nucleocapsid antigen (MeV NCORE) and The 22 patients with MS showed higher serum levels of anti-EBVgp350 IgG varicella-zoster virus glycoprotein E (VZVgE). The analyzed serum (p = 0.006) and anti-MeV NCORE IgG (p < 0.0001) compared with the 39 samples were diluted 1/1000 and the cerebrospinal fluid (CSF) samples healthy controls. However, serum levels of anti-VZVgE IgG were not higher diluted 1/10. The sample material was collected from patients with acute infectious mononucleosis (IM), these patients at follow-up (FU), healthy in patients with MS compared with controls. In CSF, patients with MS had controls (HC) and patients with MS (MS). The reactivity is presented as slightly higher IgG antibody levels against both EBVgp350 (p = 0.048) and optical density (OD) or sample to cut-off (S/CO). Median values with VZVgE (p = 0.01) compared with controls. More pronounced were the interquartile range (IQR) are presented. EBV IgG seropositive (EBV +). increased CSF levels of anti-MeV NCORE IgG in the patients compared with the controls (p < 0.0001) (Table 5). The CSF/serum ratio for MeV NCORE and VZVgE but not for EBVgp350 was higher in patients with MS compared with Median (IQR) EBVgp350 EBNA1 VCA VCA MeV NCORE VZVgE healthy controls. IgG OD IgG S/CO IgG S/CO IgM S/CO IgG OD IgG OD Serum samples It is well known that patients with MS show increased IgG responses to all IM (n=42) 0.1 (0.062) 0.38 (0.27) 7.0 (8.04) 10.3 (13.3) 0.86 (0.89) 1.19 (1.02) three viruses, EBV, VZV and MeV, compared with healthy controls, but that FU (n=42) 1.16 (1.85) 15.4 (13.5) 56.1 (30.4) 0.24 (0.35) 0.53 (0.59) 0.59 (0.53) the increased antibody response to VZV and MeV have mainly been reported HC EBV+ (n=39) 0.68 (0.84) 17.0 (14.0) 41.8 (34.9) 0.13 (0.56) 0.48 (0.56) 0.53 (0.40) in CSF while the increased antibody response to EBV has mainly been seen in MS (n=22) 1.98 (1.24) 2.5 (1.2) 0.75 (1.0) sera (428, 472-475, 483). Previous studies of antibody responses to different viruses in patients with MS show no direct correlation between serum and CSF CSF samples IgG levels (431, 484). In this study, patients with MS showed a moderate FU (n=21) 0.31 (0.42) 0.21 (0.17) 0.15 (0.05) increase in anti-EBVgp350 IgG levels in CSF compared with healthy controls. HC (n=17) 0.18 (0.24) 0.17 (0.17) 0.15 (0.13) In previous studies, anti-EBV IgG levels in CSF have often been relatively low HC EBV+ (n=15) 0.18 (0.22) 0.17 (0.17) 0.15 (0.13) compared with serum levels (428, 483). The antibody response to EBVgp350 MS (n=22) 0.31 (0.41) 1.78 (1.41) 0.46 (0.81) in patients with MS has not been extensively investigated in contrast to the carefully studied antibody responses to EBNA1 and VCA. Further studies are therefore needed to conclude whether patients with MS have increased levels CSF antibodies to EBV, VZV and MeV in patients with IM of anti-EBVgp350 IgG in CSF compared with healthy controls. The total IgG levels in CSF were within the normal range in the healthy An established risk factor for developing MS is primary EBV infection in the controls and in the post-IM patients at follow-up. None of these individuals form of IM (246, 247, 373) and a recent study provides serological support that showed OCB in CSF. The CSF samples were diluted 1/10 in the serological late EBV infections increases the risk of developing MS (485). A feature in analysis. Anti-EBVgp350 IgG levels were significantly higher in post-IM individuals with presymtomatic MS is higher antibody reactivity to EBV patients at follow-up compared with the whole group with 17 healthy controls compared with healthy controls, especially to EBNA1 (424, 425, 486, 487). (p = 0.01) but not compared with only the 15 EBV IgG seropositive controls However, the antibody reactivity to EBVgp350 has not been studied in this (p = 0.08). The CSF/serum ratio was determined based on the results of paired context. In conclusion, our results show that patients with acute IM display samples with CSF (diluted 1/10) and serum (diluted 1/1000) giving the increased levels of anti-EBVgp350 IgG in serum at follow-up after 10 years. CSF/serum dilution ratio of 1/100. The CSF/serum ratio for EBVgp350 IgG One hypothesis, that may relate to this finding, is that primary EBV infection was lower among post-IM patients at follow-up compared with the healthy in the form of IM can affect the immune system in a powerful and unusual controls while no differences between the groups were seen for MeV NCORE way, which can be a triggering factor for continued biological events that after and VZVgE. That the CSF/serum ratio for EBVgp350 was lower for the post- many years can lead to autoimmune diseases such as MS. Our present results IM patients at follow-up compared with healthy controls may be due to the support the argument for continuing to investigate the role of EBV in the increased serum levels of anti-EBVgp350 IgG in the patients. pathogenesis of MS. 92 93 4.5 PAPER V IgG levels against EBVgp350 decreased during NAT treatment in 509 of 714 patients (71%). In Paper V, we wanted to identify whether the IgG reactivity to EBV and/or MeV is altered in patients with MS during interferon beta (IFNβ) treatment There was a slight decrease in IgG levels to MeV NCORE between the samples and/or after initiation of Natalizumab (NAT) treatment. A secondary goal was collected at t1 and t2 from the subgroup of patients with MS during IFNβ to identify if any patient with MS was EBV IgG seronegative. therapy (p = 0.006), but only 95 of 170 patients (56%) demonstrated an antibody decline, which makes this observation of reduction somewhat less Patients with MS vs healthy controls convincing (Figure 32). The patients in the NAT group showed a more pronounced decline in IgG antibodies to MeV NCORE between the samples When comparing serum IgG levels between patients with MS and the 144 obtained at t3 and t4 (p < 0.0001). In total, 538 of 714 patients (75%) healthy blood donors, samples taken at t1 from the subgroup of 170 patients demonstrated a decline in anti-MeV NCORE IgG levels after initiating NAT who had samples collected during previous IFNβ treatment were used (see therapy. Figure 23 for definition of the different time points). The reason why these samples were used in the comparison was that the patients were most treatment naïve at this time before starting treatment with NAT. Patients with MS had higher levels of both anti-EBVgp350 (p = 0.0006), and anti-MeV NCORE IgG (p < 0.0001) compared with the healthy blood donors. Why patients with MS have increased levels of anti-EBV and anti-MeV IgG in sera compared with healthy controls is unknown. By using our recombinantly produced antigens EBVgp350 and MeV NCORE which are based on single, immunodominant viral proteins lacking human/primate cellular residues, we reduced the risk of detecting autoantibodies to cellular components and/or cross-reactive antibodies. We have thus enhanced the specificity for detecting EBV- and MeV-specific antibodies. Our results suggest that patients with MS truly have an increased reactivity to these viruses. The increased anti-EBV and anti-MeV IgG levels in patients with MS have led to suggestions for the use of these antibody responses, primarily EBV, as surrogate markers for MS disease activity and/or treatment efficacy (440- 444, 488, 489). Changes in antibody levels during treatment The changes in IgG levels between the 170 paired samples collected during Figure 32. Delta (∆) optical density (OD) values are shown as dots in the figure to IFNβ treatment at t1 and t2 and the 714 paired samples collected before at t3 illustrate the difference between the IgG antibody levels for anti-EBVgp350 and anti-MeV and during NAT treatment at t4 are shown in Figure 32. The IgG levels against NCORE IgG, respectively, for each paired serum sample obtained during IFNβ treatment (n EBVgp350 did not change in the IFN subgroup between t1 and t2 and only 93 = 170) at t1 and t2 and before and during NAT treatment (n = 714) at t3 and t4. ∆OD of 170 patients (55%) had lower IgG levels in the t2 follow-up sample values were obtained by subtracting the OD value of the first sample taken at t1 or t3 compared with the first sample obtained at t1. For the 714 patients who started from the OD value of the second sample taken at t2 or t4 (i.e. t2 minus t1; t4 minus t3). ∆OD values above zero indicate an increased antibody level and values below zero NAT treatment, IgG levels against EBVgp350 decreased between the samples indicate a decreased antibody level. collected before starting treatment at t3 and during treatment at t4 (p < 0.0001). 94 95 4.5 PAPER V IgG levels against EBVgp350 decreased during NAT treatment in 509 of 714 patients (71%). In Paper V, we wanted to identify whether the IgG reactivity to EBV and/or MeV is altered in patients with MS during interferon beta (IFNβ) treatment There was a slight decrease in IgG levels to MeV NCORE between the samples and/or after initiation of Natalizumab (NAT) treatment. A secondary goal was collected at t1 and t2 from the subgroup of patients with MS during IFNβ to identify if any patient with MS was EBV IgG seronegative. therapy (p = 0.006), but only 95 of 170 patients (56%) demonstrated an antibody decline, which makes this observation of reduction somewhat less Patients with MS vs healthy controls convincing (Figure 32). The patients in the NAT group showed a more pronounced decline in IgG antibodies to MeV NCORE between the samples When comparing serum IgG levels between patients with MS and the 144 obtained at t3 and t4 (p < 0.0001). In total, 538 of 714 patients (75%) healthy blood donors, samples taken at t1 from the subgroup of 170 patients demonstrated a decline in anti-MeV NCORE IgG levels after initiating NAT who had samples collected during previous IFNβ treatment were used (see therapy. Figure 23 for definition of the different time points). The reason why these samples were used in the comparison was that the patients were most treatment naïve at this time before starting treatment with NAT. Patients with MS had higher levels of both anti-EBVgp350 (p = 0.0006), and anti-MeV NCORE IgG (p < 0.0001) compared with the healthy blood donors. Why patients with MS have increased levels of anti-EBV and anti-MeV IgG in sera compared with healthy controls is unknown. By using our recombinantly produced antigens EBVgp350 and MeV NCORE which are based on single, immunodominant viral proteins lacking human/primate cellular residues, we reduced the risk of detecting autoantibodies to cellular components and/or cross-reactive antibodies. We have thus enhanced the specificity for detecting EBV- and MeV-specific antibodies. Our results suggest that patients with MS truly have an increased reactivity to these viruses. The increased anti-EBV and anti-MeV IgG levels in patients with MS have led to suggestions for the use of these antibody responses, primarily EBV, as surrogate markers for MS disease activity and/or treatment efficacy (440- 444, 488, 489). Changes in antibody levels during treatment The changes in IgG levels between the 170 paired samples collected during Figure 32. Delta (∆) optical density (OD) values are shown as dots in the figure to IFNβ treatment at t1 and t2 and the 714 paired samples collected before at t3 illustrate the difference between the IgG antibody levels for anti-EBVgp350 and anti-MeV and during NAT treatment at t4 are shown in Figure 32. The IgG levels against NCORE IgG, respectively, for each paired serum sample obtained during IFNβ treatment (n EBVgp350 did not change in the IFN subgroup between t1 and t2 and only 93 = 170) at t1 and t2 and before and during NAT treatment (n = 714) at t3 and t4. ∆OD of 170 patients (55%) had lower IgG levels in the t2 follow-up sample values were obtained by subtracting the OD value of the first sample taken at t1 or t3 compared with the first sample obtained at t1. For the 714 patients who started from the OD value of the second sample taken at t2 or t4 (i.e. t2 minus t1; t4 minus t3). ∆OD values above zero indicate an increased antibody level and values below zero NAT treatment, IgG levels against EBVgp350 decreased between the samples indicate a decreased antibody level. collected before starting treatment at t3 and during treatment at t4 (p < 0.0001). 94 95 The time span was short and similar, approximately one year, between the production. It is also possible that NAT has more direct effects on B-cell serum samples collected during IFN treatment at t1 – t2 and before (t3) and function (492, 494). during (t4) NAT treatment. The results showing that anti-EBVgp350 and anti- MeV NCORE IgG decline after initiating NAT therapy but are relatively stable Patients with MS have an increased risk of developing certain infectious during IFNβ treatment suggest that the decrease is not a general decrease due diseases in comparison with the general population (495, 496). The use of to time and increased age in the sampled individuals. disease-modifying treatments may increase this risk. It has been established that NAT therapy increases the risk of PML and that the decrease in antibodies The anti-EBV IgG antibody response usually lasts for life without any to JCV may be linked to this increased risk (400, 450, 497). Primary central significant reduction (22). Anti-MeV IgG antibody levels are generally stable nervous system lymphoma and herpesvirus infections of the CNS are and persist throughout life after natural infection in healthy individuals (22) associated with NAT therapy but less is known about these associations and but decline with age after vaccination (297). In contrast, the anti-MeV IgG whether lower antibody levels to certain herpesviruses may increase the risk response in patients with MS tends to increase over time both after natural of developing the diseases (401-403). infection and vaccination (433). Our results thus suggest that the decrease in anti-EBVgp350 and anti-MeV NCORE IgG is associated to the initiation of NAT Seroprevalence therapy. An antibody response to EBVgp350 could be detected in 718 patients with MS. There are studies that show a mild decrease in total serum IgG levels during Only 10 patients did not show detectable antibody levels. All samples where NAT treatment (490-492). One of these studies could only demonstrate a mild an antibody response to EBVgp350 could not be detected were also analyzed IgG decline in the longitudinal part of the study (491). In another study, no using EBNA1 and VCA as antigens. The 10 EBVgp350 seronegative MS decrease could be observed (493). In the study prior to the current one, on the patients showed detectable levels of anti-EBNA1 and/or anti-VCA IgG, same material, it was shown that the IgG antibody response to JCV and two indicating that all patients with MS were EBV IgG seropositive. Among the herpesviruses, CMV and VZV, differed in patients with MS after starting NAT control group with blood donors, 14 of 144 (9.5%) were EBVgp350 IgG therapy. Anti-JCV and anti-VZV IgG declined but, in contrast, anti-CMV IgG seronegative. Further analysis of these EBVgp350 seronegative patients increased slightly (450). (Our VZVgE antigen was used for anti-VZV IgG showed that four blood donors had detectable antibodies to EBNA1 and/or detection.) This shows that NAT does not suppress IgG reactivity against all VCA but 10 (6.9%) were both anti-EBNA1 and anti-VCA IgG seronegative. viruses, which may suggest that the reduction of anti-EBVgp350 and anti-MeV NCORE IgG is not part of a general IgG decline. In the IFNβ subgroup, 15 of the 170 samples (8.8%) collected at t1 had undetectable IgG antibody levels against MeV NCORE and at t2, 18 of 170 Previous studies have not found any change in anti-EBNA1 IgG levels during samples (10.5%) were seronegative. In the NAT group, 80 of 714 samples NAT treatment (443, 444, 489). One study found an increase in anti-VCA IgG (11.2%) were seronegative at t3 and 108 of 714 samples (15.1%) were during NAT treatment (444), while another was unable to detect a change seronegative at t4. In total, 41 of 144 blood donors (28.5%) were IgG MeV (489). That these antibody responses did not decrease after initiating NAT NCORE seronegative. therapy as we show with decreased anti-EBVgp350 IgG levels may be due to different biological purposes with the antibodies, different sensitivity of the Patients with MS have a high EBV seroprevalence and it is unclear if there are viral antigens and/or the small samples sizes in some of these studies. No any patients with MS who are not infected with the virus (413, 414, 424). The decrease in serum anti-MeV IgG levels during NAT treatment has been few EBVgp350 IgG seronegative samples in this study were also analyzed with observed in previous studies with relatively small sample sizes (491, 493). the antigens EBNA1 and VCA and the results showed that all patients with MS were EBV IgG seropositive. The different assays for the detection of anti-EBV It is possible that NAT treatment causes changes in patients’ immune responses IgG have different sensitivities and specificities and it is unlikely that any assay that affect the production of anti-EBV and anti-MeV IgG antibodies. Affinity has both 100% sensitivity and specificity (276). So, it is more robust to use maturation of B cells requires support from T helper cells and insufficient help several different assays to determine EBV seroprevalence, but a secure can lead to impaired plasma cell production which can affect the antibody definition of seronegativity remains however elusive. There are few EBV 96 97 The time span was short and similar, approximately one year, between the production. It is also possible that NAT has more direct effects on B-cell serum samples collected during IFN treatment at t1 – t2 and before (t3) and function (492, 494). during (t4) NAT treatment. The results showing that anti-EBVgp350 and anti- MeV NCORE IgG decline after initiating NAT therapy but are relatively stable Patients with MS have an increased risk of developing certain infectious during IFNβ treatment suggest that the decrease is not a general decrease due diseases in comparison with the general population (495, 496). The use of to time and increased age in the sampled individuals. disease-modifying treatments may increase this risk. It has been established that NAT therapy increases the risk of PML and that the decrease in antibodies The anti-EBV IgG antibody response usually lasts for life without any to JCV may be linked to this increased risk (400, 450, 497). Primary central significant reduction (22). Anti-MeV IgG antibody levels are generally stable nervous system lymphoma and herpesvirus infections of the CNS are and persist throughout life after natural infection in healthy individuals (22) associated with NAT therapy but less is known about these associations and but decline with age after vaccination (297). In contrast, the anti-MeV IgG whether lower antibody levels to certain herpesviruses may increase the risk response in patients with MS tends to increase over time both after natural of developing the diseases (401-403). infection and vaccination (433). Our results thus suggest that the decrease in anti-EBVgp350 and anti-MeV NCORE IgG is associated to the initiation of NAT Seroprevalence therapy. An antibody response to EBVgp350 could be detected in 718 patients with MS. There are studies that show a mild decrease in total serum IgG levels during Only 10 patients did not show detectable antibody levels. All samples where NAT treatment (490-492). One of these studies could only demonstrate a mild an antibody response to EBVgp350 could not be detected were also analyzed IgG decline in the longitudinal part of the study (491). In another study, no using EBNA1 and VCA as antigens. The 10 EBVgp350 seronegative MS decrease could be observed (493). In the study prior to the current one, on the patients showed detectable levels of anti-EBNA1 and/or anti-VCA IgG, same material, it was shown that the IgG antibody response to JCV and two indicating that all patients with MS were EBV IgG seropositive. Among the herpesviruses, CMV and VZV, differed in patients with MS after starting NAT control group with blood donors, 14 of 144 (9.5%) were EBVgp350 IgG therapy. Anti-JCV and anti-VZV IgG declined but, in contrast, anti-CMV IgG seronegative. Further analysis of these EBVgp350 seronegative patients increased slightly (450). (Our VZVgE antigen was used for anti-VZV IgG showed that four blood donors had detectable antibodies to EBNA1 and/or detection.) This shows that NAT does not suppress IgG reactivity against all VCA but 10 (6.9%) were both anti-EBNA1 and anti-VCA IgG seronegative. viruses, which may suggest that the reduction of anti-EBVgp350 and anti-MeV NCORE IgG is not part of a general IgG decline. In the IFNβ subgroup, 15 of the 170 samples (8.8%) collected at t1 had undetectable IgG antibody levels against MeV NCORE and at t2, 18 of 170 Previous studies have not found any change in anti-EBNA1 IgG levels during samples (10.5%) were seronegative. In the NAT group, 80 of 714 samples NAT treatment (443, 444, 489). One study found an increase in anti-VCA IgG (11.2%) were seronegative at t3 and 108 of 714 samples (15.1%) were during NAT treatment (444), while another was unable to detect a change seronegative at t4. In total, 41 of 144 blood donors (28.5%) were IgG MeV (489). That these antibody responses did not decrease after initiating NAT NCORE seronegative. therapy as we show with decreased anti-EBVgp350 IgG levels may be due to different biological purposes with the antibodies, different sensitivity of the Patients with MS have a high EBV seroprevalence and it is unclear if there are viral antigens and/or the small samples sizes in some of these studies. No any patients with MS who are not infected with the virus (413, 414, 424). The decrease in serum anti-MeV IgG levels during NAT treatment has been few EBVgp350 IgG seronegative samples in this study were also analyzed with observed in previous studies with relatively small sample sizes (491, 493). the antigens EBNA1 and VCA and the results showed that all patients with MS were EBV IgG seropositive. The different assays for the detection of anti-EBV It is possible that NAT treatment causes changes in patients’ immune responses IgG have different sensitivities and specificities and it is unlikely that any assay that affect the production of anti-EBV and anti-MeV IgG antibodies. Affinity has both 100% sensitivity and specificity (276). So, it is more robust to use maturation of B cells requires support from T helper cells and insufficient help several different assays to determine EBV seroprevalence, but a secure can lead to impaired plasma cell production which can affect the antibody definition of seronegativity remains however elusive. There are few EBV 96 97 seronegative patients with suspected clinically isolated syndrome or MS but in 5 CONCLUSION these cases, EBV seronegativity may be a useful biomarker to identify patients where a different diagnosis is more likely and further investigations are warranted. In Paper I, VZVgE was shown to work well as serological antigen in indirect ELISA. The new method had high sensitivity and specificity compared with Table 6. Epstein-Barr virus glycoprotein 350 (EBVgp350) IgG an ELISA method using VZVwhole-ag. The use of VZVgE reduces the risk of seronegative samples from patients with multiple sclerosis during detecting cross-reactive anti-HSV IgG antibodies compared with using VZV treatment with interferon (IFN) (3/170) and before and during treatment antigens that contain many VZV proteins including VZVgB, as is the case in with natalizumab (NAT) at time point 3 (t3) (11/714) and t4 (14/714). In most commercial VZV assays. At the Department of Clinical Microbiology, addition, the EBVgp350 IgG seronegative samples were assayed with the Sahlgrenska University Hospital, the VZVgE antigen has been successfully EBNA1 and VCA antigens. Abbreviations: seronegative (-), seropositive used as an ELISA antigen in the routine diagnostics for detection of anti-VZV (+), gray zone (GRZ). IgG antibodies for approximately a decade. A sensitive and specific ELISA method using EBVgp350 as serological Patient EBVgp350 EBNA1 VCA antigen was developed in Paper II for the detection of anti-EBVgp350 IgG 1 – + + antibodies. Two protein constructs of different lengths were produced and 2 – GRZ + tested as antigens in indirect ELISA. The longer protein construct containing 3 – + + a.a. 1–860 gave higher reactivity compared with the shorter construct 4 – + + containing a.a. 1–560 thus suggesting that longer EBVgp350 constructs should 5 – + + be used as serological antigens and in possible vaccine development. 6 – + + 7 – + + In Paper III, Paper IV, and Paper V, the recombinant, immunodominant 8 – – + MeV NCORE antigen devoid of human/primate components was used as 9 t3+ t4– + + serological antigen in indirect ELISA for detection of anti-MeV IgG 10 t3+ t4– + + antibodies. Our results indicate that the increased anti-MeV IgG antibody 11 t3+ t4– + + levels detected in serum and CSF in patients with MS and their siblings with 12 IFN+ NAT– + + MS trait compared with healthy controls are indeed due to MeV-specific 13 IFN– NAT– + + antibodies and not caused by cross-reacting autoantibodies. 14 IFN– NAT– + + 15 IFN– NAT+ + + Previously, mainly the antibody responses to EBV VCA and EBNA1 have been analyzed in patients with MS. We now add knowledge by analyzing anti- EBVgp350 IgG, which can provide new pieces of the puzzle to understand the possible role of EBV in the pathogenesis of MS. Our finding in Paper IV, that patients with mononucleosis have increased levels of anti-EBVgp350 IgG at follow-up after 10 years, supports the hypothesis that a primary EBV infection in the form of mononucleosis can affect the immune system in a powerful and unusual way that can be a trigger for continued biological events that after many years may possibly result in MS disease. In Paper V, we observed that natalizumab treatment of patients with MS was associated with a moderate decrease in serum anti-EBVgp350 and anti-MeV NCORE IgG antibodies. In contrast, in a previous study based on the same 98 99 seronegative patients with suspected clinically isolated syndrome or MS but in 5 CONCLUSION these cases, EBV seronegativity may be a useful biomarker to identify patients where a different diagnosis is more likely and further investigations are warranted. In Paper I, VZVgE was shown to work well as serological antigen in indirect ELISA. The new method had high sensitivity and specificity compared with Table 6. Epstein-Barr virus glycoprotein 350 (EBVgp350) IgG an ELISA method using VZVwhole-ag. The use of VZVgE reduces the risk of seronegative samples from patients with multiple sclerosis during detecting cross-reactive anti-HSV IgG antibodies compared with using VZV treatment with interferon (IFN) (3/170) and before and during treatment antigens that contain many VZV proteins including VZVgB, as is the case in with natalizumab (NAT) at time point 3 (t3) (11/714) and t4 (14/714). In most commercial VZV assays. At the Department of Clinical Microbiology, addition, the EBVgp350 IgG seronegative samples were assayed with the Sahlgrenska University Hospital, the VZVgE antigen has been successfully EBNA1 and VCA antigens. Abbreviations: seronegative (-), seropositive used as an ELISA antigen in the routine diagnostics for detection of anti-VZV (+), gray zone (GRZ). IgG antibodies for approximately a decade. A sensitive and specific ELISA method using EBVgp350 as serological Patient EBVgp350 EBNA1 VCA antigen was developed in Paper II for the detection of anti-EBVgp350 IgG 1 – + + antibodies. Two protein constructs of different lengths were produced and 2 – GRZ + tested as antigens in indirect ELISA. The longer protein construct containing 3 – + + a.a. 1–860 gave higher reactivity compared with the shorter construct 4 – + + containing a.a. 1–560 thus suggesting that longer EBVgp350 constructs should 5 – + + be used as serological antigens and in possible vaccine development. 6 – + + 7 – + + In Paper III, Paper IV, and Paper V, the recombinant, immunodominant 8 – – + MeV NCORE antigen devoid of human/primate components was used as 9 t3+ t4– + + serological antigen in indirect ELISA for detection of anti-MeV IgG 10 t3+ t4– + + antibodies. Our results indicate that the increased anti-MeV IgG antibody 11 t3+ t4– + + levels detected in serum and CSF in patients with MS and their siblings with 12 IFN+ NAT– + + MS trait compared with healthy controls are indeed due to MeV-specific 13 IFN– NAT– + + antibodies and not caused by cross-reacting autoantibodies. 14 IFN– NAT– + + 15 IFN– NAT+ + + Previously, mainly the antibody responses to EBV VCA and EBNA1 have been analyzed in patients with MS. We now add knowledge by analyzing anti- EBVgp350 IgG, which can provide new pieces of the puzzle to understand the possible role of EBV in the pathogenesis of MS. Our finding in Paper IV, that patients with mononucleosis have increased levels of anti-EBVgp350 IgG at follow-up after 10 years, supports the hypothesis that a primary EBV infection in the form of mononucleosis can affect the immune system in a powerful and unusual way that can be a trigger for continued biological events that after many years may possibly result in MS disease. In Paper V, we observed that natalizumab treatment of patients with MS was associated with a moderate decrease in serum anti-EBVgp350 and anti-MeV NCORE IgG antibodies. In contrast, in a previous study based on the same 98 99 sample material, anti-CMV IgG levels were slightly increased by this 6 FUTURE PERSPECTIVES treatment, which argues against a general IgG decline as an explanation (450). We can also conclude that all 728 patients with MS in the study were EBV IgG seropositive while 10 of the 144 blood donors in the control group were This thesis aims at improving serodiagnosis of viruses, including EBV, VZV seronegative. This finding further strengthens the potential role of EBV in the and MeV, through methodological development. Below are some clinical pathogenesis of MS. applications of such refined serology. Our specific ELISA methods using VZVgE, EBVgp350 and MeV N as VZVgE-based serological methods including our VZVgE ELISA may be CORE serological antigens can, through increased specificity offer new diagnostic important in immunization control after vaccination with VZVgE subunit possibilities for detecting antibodies to these antigens in viral infections, in vaccines. Specific and sensitive VZV serological assays such as our VZVgE controlling immunity after infection/vaccination, in epidemiological ELISA can also be used to diagnose VZV infections of the CNS when the time investigations and in autoimmune diseases such as MS. window for detection of VZV DNA in CSF is closed. VZV infections such as VZV vasculopathy can be difficult to diagnose in the absence of the characteristic rash and further research in this area may establish the incidence of VZV infections in the CNS. For example, future studies may identify the incidence of VZV-induced stroke by analyzing anti-VZV antibodies in acute and convalescent serum samples from stroke patients and/or by detecting intrathecally produced anti-VZV antibodies in CSF from these patients one to two weeks after the stroke episode. Moreover, specific serological assays can in some clinical contexts be used to support an MS diagnosis. The intrathecal MRZ reaction can be employed as a diagnostic complement to other laboratory investigations and EBV seronegativity may be used as a marker for patients who warrant further investigation. EBV infection increases the risk of developing MS, but it has not been determined whether this virus is a prerequisite for developing MS. Additional large-scale seroprevalence studies of patients with accurately diagnosed MS can identify whether there are EBV seronegative patients with MS or whether all patients are seropositive. In these seroprevalence studies, it is important to use several specific assays in order not to miss any true EBV seropositive cases. If all patients with MS are EBV seropositive, it would further strengthen the role of EBV in the pathogenesis of MS. In addition, our EBVgp350 ELISA may be used to further investigate the serological relationship between EBV and MS. Our next step will be to analyze the anti-EBVgp350 IgG reactivity in CSF samples from more patients with MS, including the reactivity in CSF samples collected before and after patients start treatment with natalizumab. Immunosuppressive treatments are becoming more common, but a major disadvantage is severe side effects such as the higher risk of developing severe 100 101 sample material, anti-CMV IgG levels were slightly increased by this 6 FUTURE PERSPECTIVES treatment, which argues against a general IgG decline as an explanation (450). We can also conclude that all 728 patients with MS in the study were EBV IgG seropositive while 10 of the 144 blood donors in the control group were This thesis aims at improving serodiagnosis of viruses, including EBV, VZV seronegative. This finding further strengthens the potential role of EBV in the and MeV, through methodological development. Below are some clinical pathogenesis of MS. applications of such refined serology. Our specific ELISA methods using VZVgE, EBVgp350 and MeV N as VZVgE-based serological methods including our VZVgE ELISA may be CORE serological antigens can, through increased specificity offer new diagnostic important in immunization control after vaccination with VZVgE subunit possibilities for detecting antibodies to these antigens in viral infections, in vaccines. Specific and sensitive VZV serological assays such as our VZVgE controlling immunity after infection/vaccination, in epidemiological ELISA can also be used to diagnose VZV infections of the CNS when the time investigations and in autoimmune diseases such as MS. window for detection of VZV DNA in CSF is closed. VZV infections such as VZV vasculopathy can be difficult to diagnose in the absence of the characteristic rash and further research in this area may establish the incidence of VZV infections in the CNS. For example, future studies may identify the incidence of VZV-induced stroke by analyzing anti-VZV antibodies in acute and convalescent serum samples from stroke patients and/or by detecting intrathecally produced anti-VZV antibodies in CSF from these patients one to two weeks after the stroke episode. Moreover, specific serological assays can in some clinical contexts be used to support an MS diagnosis. The intrathecal MRZ reaction can be employed as a diagnostic complement to other laboratory investigations and EBV seronegativity may be used as a marker for patients who warrant further investigation. EBV infection increases the risk of developing MS, but it has not been determined whether this virus is a prerequisite for developing MS. Additional large-scale seroprevalence studies of patients with accurately diagnosed MS can identify whether there are EBV seronegative patients with MS or whether all patients are seropositive. In these seroprevalence studies, it is important to use several specific assays in order not to miss any true EBV seropositive cases. If all patients with MS are EBV seropositive, it would further strengthen the role of EBV in the pathogenesis of MS. In addition, our EBVgp350 ELISA may be used to further investigate the serological relationship between EBV and MS. Our next step will be to analyze the anti-EBVgp350 IgG reactivity in CSF samples from more patients with MS, including the reactivity in CSF samples collected before and after patients start treatment with natalizumab. Immunosuppressive treatments are becoming more common, but a major disadvantage is severe side effects such as the higher risk of developing severe 100 101 infections. As an example, rituximab, which depletes B cells, can greatly ACKNOWLEDGEMENT reduce the antibody response to pathogens, thereby increasing the risk of infection, including a propensity for viral infections in the CNS. Further research on the monitoring of antibody responses to certain viruses could show Ett stort tack till alla de patienter och kontroller som har bidragit till whether there are critically low antibody levels where the risk of active forskningen. infections increases sharply. This can lead to preventive measures such as Tack alla ni som har hjälpt och stöttat mig genom åren med detta vaccination, antiviral therapy, and/or reduction of immunosuppressive therapy. avhandlingsarbete. Det har tagit sin tid men nu går jag äntligen i mål. EBVgp350 has been one of the top EBV vaccine candidates and if such a Tomas – Ett stort tack för att du har gett mig möjligheten att få dyka ner i vaccine were to come, our novel EBVgp350 ELISA may be used to analyze virologins värld. Doktorandtiden och min ST inom klinisk mikrobiologi är inte the antibody response after vaccination. The ELISA method may also be used bara ett arbete för mig utan även ett stort intresse. Tack för alla år med både to investigate whether there is an association between antibody reactivity and gladare och mörkare tider som du har handlett mig i mitt doktorandarbete. Din the severity and/or treatment effect for various EBV disease manifestations vänlighet och positiva inställning till både arbete och det övriga livet har betytt such as IM and EBV-induced tumors. mycket för mig. Marie Studahl och Jan Lycke – Jag är tacksam över att ni har ställt upp och varit mina bihandledare under dessa år. Jag hoppas att vårt samarbete kommer att ge fortsatt utdelning framöver. Medförfattare – Jag vill tacka alla mina medförfattare för samarbetet kring artiklarna i denna avhandling. Maria Johansson – Från mina första stapplande experiment har du funnits där för mig. Jag hade varit vilse på virologen utan dig. Du är en av de snällaste och mest hjälpsamma personer jag känner. Du är också en av de personer som arrangerar så många av de roliga aktiviteterna på virologen som julbastu och skidresor till Hemsedal. Anette and Carolina – Tack för att ni alltid tar er tid att svara på en förvirrad doktorands frågor och hjälper till med experiment. Alla nuvarande och tidigare doktorander, post docs, examensstudenter med flera på 3:e våningen – Tack för alla trevliga fikastunder, diskussioner och allmänt häng. Det har gjort det roligt att komma till jobbet. Ett extra tack till Charlotta som alltid fanns där när jag behövde prata av mig, vilket verkligen behövdes ibland. Du är en toppenbra konferens- och kurskompis. Tack för alla fina minnen! Rickard – Alltid lika roligt att prata och umgås med dig. Jag har många roliga minnen från Smögenmöten, julbastu och fester. 102 103 infections. As an example, rituximab, which depletes B cells, can greatly ACKNOWLEDGEMENT reduce the antibody response to pathogens, thereby increasing the risk of infection, including a propensity for viral infections in the CNS. Further research on the monitoring of antibody responses to certain viruses could show Ett stort tack till alla de patienter och kontroller som har bidragit till whether there are critically low antibody levels where the risk of active forskningen. infections increases sharply. This can lead to preventive measures such as Tack alla ni som har hjälpt och stöttat mig genom åren med detta vaccination, antiviral therapy, and/or reduction of immunosuppressive therapy. avhandlingsarbete. Det har tagit sin tid men nu går jag äntligen i mål. EBVgp350 has been one of the top EBV vaccine candidates and if such a Tomas – Ett stort tack för att du har gett mig möjligheten att få dyka ner i vaccine were to come, our novel EBVgp350 ELISA may be used to analyze virologins värld. Doktorandtiden och min ST inom klinisk mikrobiologi är inte the antibody response after vaccination. The ELISA method may also be used bara ett arbete för mig utan även ett stort intresse. Tack för alla år med både to investigate whether there is an association between antibody reactivity and gladare och mörkare tider som du har handlett mig i mitt doktorandarbete. Din the severity and/or treatment effect for various EBV disease manifestations vänlighet och positiva inställning till både arbete och det övriga livet har betytt such as IM and EBV-induced tumors. mycket för mig. Marie Studahl och Jan Lycke – Jag är tacksam över att ni har ställt upp och varit mina bihandledare under dessa år. Jag hoppas att vårt samarbete kommer att ge fortsatt utdelning framöver. Medförfattare – Jag vill tacka alla mina medförfattare för samarbetet kring artiklarna i denna avhandling. Maria Johansson – Från mina första stapplande experiment har du funnits där för mig. Jag hade varit vilse på virologen utan dig. Du är en av de snällaste och mest hjälpsamma personer jag känner. Du är också en av de personer som arrangerar så många av de roliga aktiviteterna på virologen som julbastu och skidresor till Hemsedal. Anette and Carolina – Tack för att ni alltid tar er tid att svara på en förvirrad doktorands frågor och hjälper till med experiment. Alla nuvarande och tidigare doktorander, post docs, examensstudenter med flera på 3:e våningen – Tack för alla trevliga fikastunder, diskussioner och allmänt häng. Det har gjort det roligt att komma till jobbet. Ett extra tack till Charlotta som alltid fanns där när jag behövde prata av mig, vilket verkligen behövdes ibland. Du är en toppenbra konferens- och kurskompis. Tack för alla fina minnen! Rickard – Alltid lika roligt att prata och umgås med dig. Jag har många roliga minnen från Smögenmöten, julbastu och fester. 102 103 Nina – Tack för din hjälp med dataprogram och inköp av datorer och för att till er boende i Göteborg för barnpassning. Barnen blir alltid glada av att få du är så trevlig att umgås med. Jag blir alltid glad när jag hamnar bredvid dig vara med er. på fester och middagar. Persson släkten på pappas sida – Det är fint att vi håller kontakten och ses Läkarkollegor på klinisk mikrobiologi – Tack för att ni gör mikrobiologen även nu när pappa inte är med oss. Ett extra tack till Märta som ställer upp och till en rolig arbetsplats. Ett extra tack till mina corpsarvänner Anders och passar de små. Gustaf samt till Diana som har följt mig genom min ST även då vi randade oss på infektionskliniken. Familjen Berg – Claes-Henriks systrar Cecilia och Christina med familj samt hans mor Ingalena, mormor Elsie och hans avlidne far Peter. Tack för trevliga Personal och chefer på klinisk mikrobiologi och vårdhygien – Tack alla ni resor, gemensamma sommarsemestrar och barnpassning. som har hjälpt och stöttat mig med olika saker genom åren både i min roll som doktorand och som ST-läkare. Det är ni som skapar en trivsam och bra Mamma Gerd och pappa Torsten – Tack för att ni har varit så bra föräldrar. arbetsplats. Jag önskar att ni hade fått vara med längre och att vi hade fått möjlighet att dela fler fina stunder i livet. Examensarbetare – Tack till Marcus och Sandra som jag har handlett under deras examensarbeten. Susanne – Min syster och goda vän. Det är fint att få umgås med dig så mycket nu för tiden. Jag upplever att vi under de senaste åren efter våra föräldrars Sigvard Olofsson – Tack för arrangerade av de fantastiska bortgång har kommit närmare varandra vilket jag verkligen uppskattar. Smögenkonferenserna och för att du vid något tillfälle har sagt att jag är framtiden inom virologin. Daniella och Juliana – Mina underbara barn som jag älskar över allt annat. Jag är så glad och tacksam att få vara er mamma. Ni är mina solstrålar och när Professorer på virologen – Jag är tacksam för att ni skapar en bra ni skrattar så känns det som om hela världen ler. forskningsmiljö där vi i nästa generation uppmanas att ta för oss. Claes-Henrik – Linn + CH = sant Maria, Sofia, Daniel, Mari – Gamla vänner från uppväxten i Sundsvall som Du är min man, livskamrat och bästa vän. Jag älskar dig verkligen! När du är jag har gjort mycket roligt tillsammans med. Det är fint att vi fortfarande kan borta känns det så tomt och jag saknar dig även om du bara går jour på hålla kontakten fast vi bor i olika städer. sjukhuset. Livet är så mycket bättre med dig vid min sida. De tuffa tiderna är lättare med ditt stöd och de glada tiderna blir lyckligare när de delas med dig. Banken – Tack för all vänskap och allt roligt som vi gjort genom åren med Jag hoppas att vi ska få bli gamla och grå tillsammans. middagar, firanden och resor. Det betyder mycket att ha goda vänner som er! Hanna – Så mycket roligt vi har gjort tillsammans från det att vi lärde känna My time as a PhD student has largely been funded by the Sahlgrenska varandra i Tyskland. Vilken tur att vi sedan började studera i samma stad i Academy, for which I am very grateful. Partial financial support for the Sverige! research carried out in this thesis comes from grants from the Swedish state according to the ALF agreement. I would also like to thank the Gothenburg Uffe och Gabbe – Jag är verkligen glad att Claes-Henrik har så fina vänner Medical Society and Edit Jacobson Donation Fund in Gothenburg for financial och att ni direkt inkluderade mig som en del av gruppen. Jag har så många bra support for the research. minnen från middagar, engagerade diskussioner, sunkrundor och resor. Gunnel, Glenn, Linda, Jessica – Ni är den närmsta släkten på mammas sida och jag uppskattar att vi fortsätter att fira högtider tillsammans. Ett extra tack 104 105 Nina – Tack för din hjälp med dataprogram och inköp av datorer och för att till er boende i Göteborg för barnpassning. Barnen blir alltid glada av att få du är så trevlig att umgås med. Jag blir alltid glad när jag hamnar bredvid dig vara med er. på fester och middagar. Persson släkten på pappas sida – Det är fint att vi håller kontakten och ses Läkarkollegor på klinisk mikrobiologi – Tack för att ni gör mikrobiologen även nu när pappa inte är med oss. Ett extra tack till Märta som ställer upp och till en rolig arbetsplats. Ett extra tack till mina corpsarvänner Anders och passar de små. Gustaf samt till Diana som har följt mig genom min ST även då vi randade oss på infektionskliniken. Familjen Berg – Claes-Henriks systrar Cecilia och Christina med familj samt hans mor Ingalena, mormor Elsie och hans avlidne far Peter. Tack för trevliga Personal och chefer på klinisk mikrobiologi och vårdhygien – Tack alla ni resor, gemensamma sommarsemestrar och barnpassning. som har hjälpt och stöttat mig med olika saker genom åren både i min roll som doktorand och som ST-läkare. Det är ni som skapar en trivsam och bra Mamma Gerd och pappa Torsten – Tack för att ni har varit så bra föräldrar. arbetsplats. Jag önskar att ni hade fått vara med längre och att vi hade fått möjlighet att dela fler fina stunder i livet. Examensarbetare – Tack till Marcus och Sandra som jag har handlett under deras examensarbeten. Susanne – Min syster och goda vän. Det är fint att få umgås med dig så mycket nu för tiden. Jag upplever att vi under de senaste åren efter våra föräldrars Sigvard Olofsson – Tack för arrangerade av de fantastiska bortgång har kommit närmare varandra vilket jag verkligen uppskattar. Smögenkonferenserna och för att du vid något tillfälle har sagt att jag är framtiden inom virologin. Daniella och Juliana – Mina underbara barn som jag älskar över allt annat. Jag är så glad och tacksam att få vara er mamma. Ni är mina solstrålar och när Professorer på virologen – Jag är tacksam för att ni skapar en bra ni skrattar så känns det som om hela världen ler. forskningsmiljö där vi i nästa generation uppmanas att ta för oss. Claes-Henrik – Linn + CH = sant Maria, Sofia, Daniel, Mari – Gamla vänner från uppväxten i Sundsvall som Du är min man, livskamrat och bästa vän. Jag älskar dig verkligen! När du är jag har gjort mycket roligt tillsammans med. Det är fint att vi fortfarande kan borta känns det så tomt och jag saknar dig även om du bara går jour på hålla kontakten fast vi bor i olika städer. sjukhuset. Livet är så mycket bättre med dig vid min sida. De tuffa tiderna är lättare med ditt stöd och de glada tiderna blir lyckligare när de delas med dig. Banken – Tack för all vänskap och allt roligt som vi gjort genom åren med Jag hoppas att vi ska få bli gamla och grå tillsammans. middagar, firanden och resor. Det betyder mycket att ha goda vänner som er! Hanna – Så mycket roligt vi har gjort tillsammans från det att vi lärde känna My time as a PhD student has largely been funded by the Sahlgrenska varandra i Tyskland. Vilken tur att vi sedan började studera i samma stad i Academy, for which I am very grateful. Partial financial support for the Sverige! research carried out in this thesis comes from grants from the Swedish state according to the ALF agreement. I would also like to thank the Gothenburg Uffe och Gabbe – Jag är verkligen glad att Claes-Henrik har så fina vänner Medical Society and Edit Jacobson Donation Fund in Gothenburg for financial och att ni direkt inkluderade mig som en del av gruppen. Jag har så många bra support for the research. minnen från middagar, engagerade diskussioner, sunkrundor och resor. 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Johannsen E, Luftig M, Chase MR, Weicksel S, Cahir-McFarland E, Illanes D, et al. Proteins of purified 229. Kim WY, Montes-Mojarro IA, Fend F, Quintanilla-Martinez L. Epstein-Barr Virus-Associated T and Epstein-Barr virus. Proc Natl Acad Sci U S A. 2004;101(46):16286-91. NK-Cell Lymphoproliferative Diseases. Front Pediatr. 2019;7:71. 209. Beisel C, Tanner J, Matsuo T, Thorley-Lawson D, Kezdy F, Kieff E. Two major outer envelope 230. Tsurumi T, Fujita M, Kudoh A. Latent and lytic Epstein-Barr virus replication strategies. Rev Med glycoproteins of Epstein-Barr virus are encoded by the same gene. J Virol. 1985;54(3):665-74. Virol. 2005;15(1):3-15. 210. Hummel M, Thorley-Lawson D, Kieff E. An Epstein-Barr virus DNA fragment encodes messages for 231. Tamoto N, Nagata K, Hara S, Nakayama Y, Kuwamoto S, Matsushita M, et al. Subclinical Epstein- the two major envelope glycoproteins (gp350/300 and gp220/200). J Virol. 1984;49(2):413-7. Barr Virus Primary Infection and Lytic Reactivation Induce Thyrotropin Receptor Autoantibodies. Viral 211. Tanner J, Whang Y, Sample J, Sears A, Kieff E. Soluble gp350/220 and deletion mutant glycoproteins Immunol. 2019;32(9):362-9. block Epstein-Barr virus adsorption to lymphocytes. J Virol. 1988;62(12):4452-64. 232. Taylor GS, Long HM, Brooks JM, Rickinson AB, Hislop AD. The immunology of Epstein-Barr virus- 212. Serafini-Cessi F, Malagolini N, Nanni M, Dall'Olio F, Campadelli-Fiume G, Tanner J, et al. induced disease. Annu Rev Immunol. 2015;33:787-821. Characterization of N- and O-linked oligosaccharides of glycoprotein 350 from Epstein-Barr virus. 233. Latour S, Winter S. Inherited Immunodeficiencies With High Predisposition to Epstein-Barr Virus- Virology. 1989;170(1):1-10. Driven Lymphoproliferative Diseases. Front Immunol. 2018;9:1103. 213. Gong M, Kieff E. Intracellular trafficking of two major Epstein-Barr virus glycoproteins, gp350/220 234. Rickinson AB, Long HM, Palendira U, Münz C, Hislop AD. Cellular immune controls over Epstein- and gp110. J Virol. 1990;64(4):1507-16. Barr virus infection: new lessons from the clinic and the laboratory. Trends Immunol. 2014;35(4):159-69. 214. Fingeroth JD, Weis JJ, Tedder TF, Strominger JL, Biro PA, Fearon DT. Epstein-Barr virus receptor of 235. Parvaneh N, Filipovich AH, Borkhardt A. Primary immunodeficiencies predisposed to Epstein-Barr human B lymphocytes is the C3d receptor CR2. Proc Natl Acad Sci U S A. 1984;81(14):4510-4. virus-driven haematological diseases. Br J Haematol. 2013;162(5):573-86. 215. Frade R, Barel M, Ehlin-Henriksson B, Klein G. gp140, the C3d receptor of human B lymphocytes, is 236. Shabani M, Nichols KE, Rezaei N. Primary immunodeficiencies associated with EBV-Induced also the Epstein-Barr virus receptor. Proc Natl Acad Sci U S A. 1985;82(5):1490-3. lymphoproliferative disorders. Crit Rev Oncol Hematol. 2016;108:109-27. 216. Nemerow GR, Wolfert R, McNaughton ME, Cooper NR. Identification and characterization of the 237. Prockop SE, Vatsayan A. Epstein-Barr virus lymphoproliferative disease after solid organ Epstein-Barr virus receptor on human B lymphocytes and its relationship to the C3d complement receptor transplantation. Cytotherapy. 2017;19(11):1270-83. (CR2). J Virol. 1985;55(2):347-51. 238. Shah KM, Young LS. Epstein-Barr virus and carcinogenesis: beyond Burkitt's lymphoma. Clin 217. Shannon-Lowe C, Rowe M. Epstein Barr virus entry; kissing and conjugation. Curr Opin Virol. Microbiol Infect. 2009;15(11):982-8. 2014;4:78-84. 239. Takada K. Epstein-Barr virus and gastric carcinoma. Mol Pathol. 2000;53(5):255-61. 218. Szakonyi G, Klein MG, Hannan JP, Young KA, Ma RZ, Asokan R, et al. Structure of the Epstein-Barr 240. Cohen JI. Optimal treatment for chronic active Epstein-Barr virus disease. Pediatr Transplant. virus major envelope glycoprotein. Nat Struct Mol Biol. 2006;13(11):996-1001. 2009;13(4):393-6. 219. Tanner J, Weis J, Fearon D, Whang Y, Kieff E. Epstein-Barr virus gp350/220 binding to the B 241. Chellapandian D, Das R, Zelley K, Wiener SJ, Zhao H, Teachey DT, et al. Treatment of Epstein Barr lymphocyte C3d receptor mediates adsorption, capping, and endocytosis. Cell. 1987;50(2):203-13. virus-induced haemophagocytic lymphohistiocytosis with rituximab-containing chemo-immunotherapeutic 220. Hoffman GJ, Lazarowitz SG, Hayward SD. Monoclonal antibody against a 250,000-dalton regimens. Br J Haematol. 2013;162(3):376-82. glycoprotein of Epstein-Barr virus identifies a membrane antigen and a neutralizing antigen. Proc Natl Acad 242. Kelesidis T, Humphries R, Terashita D, Eshaghian S, Territo MC, Said J, et al. Epstein-Barr virus- Sci U S A. 1980;77(5):2979-83. associated hemophagocytic lymphohistiocytosis in Los Angeles County. J Med Virol. 2012;84(5):777-85. 221. Thorley-Lawson DA, Geilinger K. Monoclonal antibodies against the major glycoprotein (gp350/220) 243. Rouphael NG, Talati NJ, Vaughan C, Cunningham K, Moreira R, Gould C. Infections associated with of Epstein-Barr virus neutralize infectivity. Proc Natl Acad Sci U S A. 1980;77(9):5307-11. haemophagocytic syndrome. Lancet Infect Dis. 2007;7(12):814-22. 222. Thorley-Lawson DA, Poodry CA. Identification and isolation of the main component (gp350-gp220) 244. Morimoto A, Nakazawa Y, Ishii E. Hemophagocytic lymphohistiocytosis: Pathogenesis, diagnosis, and of Epstein-Barr virus responsible for generating neutralizing antibodies in vivo. J Virol. 1982;43(2):730-6. management. Pediatr Int. 2016;58(9):817-25. 116 117 203. Henle W, Hummeler K, Henle G. Antibody coating and agglutination of virus particles separated from 223. Niederman JC, Miller G, Pearson HA, Pagano JS, Dowaliby JM. Infectious mononucleosis. Epstein- the EB3 line of Burkitt lymphoma cells. J Bacteriol. 1966;92(1):269-71. Barr-virus shedding in saliva and the oropharynx. The New England journal of medicine. 204. Dunmire SK, Hogquist KA, Balfour HH. Infectious Mononucleosis. Curr Top Microbiol Immunol. 1976;294(25):1355-9. 2015;390(Pt 1):211-40. 224. Niederman JC. Infectious mononucleosis: observations on transmission. Yale J Biol Med. 1982;55(3- 205. de-Thé G, Day NE, Geser A, Lavoué MF, Ho JH, Simons MJ, et al. Sero-epidemiology of the Epstein- 4):259-64. Barr virus: preliminary analysis of an international study - a review. IARC Sci Publ. 1975(11 Pt 2):3-16. 225. Sumaya CV. Primary Epstein-Barr virus infections in children. Pediatrics. 1977;59(1):16-21. 206. Baer R, Bankier AT, Biggin MD, Deininger PL, Farrell PJ, Gibson TJ, et al. DNA sequence and 226. Tamir D, Benderly A, Levy J, Ben-Porath E, Vonsover A. Infectious mononucleosis and Epstein-Barr expression of the B95-8 Epstein-Barr virus genome. Nature. 1984;310(5974):207-11. virus in childhood. Pediatrics. 1974;53(3):330-5. 207. Smatti MK, Al-Sadeq DW, Ali NH, Pintus G, Abou-Saleh H, Nasrallah GK. Epstein-Barr Virus 227. Luzuriaga K, Sullivan JL. Infectious mononucleosis. The New England journal of medicine. Epidemiology, Serology, and Genetic Variability of LMP-1 Oncogene Among Healthy Population: An 2010;362(21):1993-2000. Update. Front Oncol. 2018;8:211. 228. Young LS, Rickinson AB. Epstein-Barr virus: 40 years on. Nat Rev Cancer. 2004;4(10):757-68. 208. Johannsen E, Luftig M, Chase MR, Weicksel S, Cahir-McFarland E, Illanes D, et al. Proteins of purified 229. Kim WY, Montes-Mojarro IA, Fend F, Quintanilla-Martinez L. Epstein-Barr Virus-Associated T and Epstein-Barr virus. Proc Natl Acad Sci U S A. 2004;101(46):16286-91. NK-Cell Lymphoproliferative Diseases. Front Pediatr. 2019;7:71. 209. Beisel C, Tanner J, Matsuo T, Thorley-Lawson D, Kezdy F, Kieff E. Two major outer envelope 230. Tsurumi T, Fujita M, Kudoh A. Latent and lytic Epstein-Barr virus replication strategies. Rev Med glycoproteins of Epstein-Barr virus are encoded by the same gene. J Virol. 1985;54(3):665-74. Virol. 2005;15(1):3-15. 210. Hummel M, Thorley-Lawson D, Kieff E. An Epstein-Barr virus DNA fragment encodes messages for 231. Tamoto N, Nagata K, Hara S, Nakayama Y, Kuwamoto S, Matsushita M, et al. Subclinical Epstein- the two major envelope glycoproteins (gp350/300 and gp220/200). J Virol. 1984;49(2):413-7. Barr Virus Primary Infection and Lytic Reactivation Induce Thyrotropin Receptor Autoantibodies. Viral 211. Tanner J, Whang Y, Sample J, Sears A, Kieff E. Soluble gp350/220 and deletion mutant glycoproteins Immunol. 2019;32(9):362-9. block Epstein-Barr virus adsorption to lymphocytes. J Virol. 1988;62(12):4452-64. 232. Taylor GS, Long HM, Brooks JM, Rickinson AB, Hislop AD. The immunology of Epstein-Barr virus- 212. Serafini-Cessi F, Malagolini N, Nanni M, Dall'Olio F, Campadelli-Fiume G, Tanner J, et al. induced disease. Annu Rev Immunol. 2015;33:787-821. Characterization of N- and O-linked oligosaccharides of glycoprotein 350 from Epstein-Barr virus. 233. Latour S, Winter S. Inherited Immunodeficiencies With High Predisposition to Epstein-Barr Virus- Virology. 1989;170(1):1-10. Driven Lymphoproliferative Diseases. Front Immunol. 2018;9:1103. 213. Gong M, Kieff E. Intracellular trafficking of two major Epstein-Barr virus glycoproteins, gp350/220 234. Rickinson AB, Long HM, Palendira U, Münz C, Hislop AD. Cellular immune controls over Epstein- and gp110. J Virol. 1990;64(4):1507-16. Barr virus infection: new lessons from the clinic and the laboratory. Trends Immunol. 2014;35(4):159-69. 214. Fingeroth JD, Weis JJ, Tedder TF, Strominger JL, Biro PA, Fearon DT. Epstein-Barr virus receptor of 235. Parvaneh N, Filipovich AH, Borkhardt A. Primary immunodeficiencies predisposed to Epstein-Barr human B lymphocytes is the C3d receptor CR2. Proc Natl Acad Sci U S A. 1984;81(14):4510-4. virus-driven haematological diseases. Br J Haematol. 2013;162(5):573-86. 215. Frade R, Barel M, Ehlin-Henriksson B, Klein G. gp140, the C3d receptor of human B lymphocytes, is 236. Shabani M, Nichols KE, Rezaei N. Primary immunodeficiencies associated with EBV-Induced also the Epstein-Barr virus receptor. Proc Natl Acad Sci U S A. 1985;82(5):1490-3. lymphoproliferative disorders. Crit Rev Oncol Hematol. 2016;108:109-27. 216. Nemerow GR, Wolfert R, McNaughton ME, Cooper NR. Identification and characterization of the 237. Prockop SE, Vatsayan A. Epstein-Barr virus lymphoproliferative disease after solid organ Epstein-Barr virus receptor on human B lymphocytes and its relationship to the C3d complement receptor transplantation. Cytotherapy. 2017;19(11):1270-83. (CR2). J Virol. 1985;55(2):347-51. 238. Shah KM, Young LS. Epstein-Barr virus and carcinogenesis: beyond Burkitt's lymphoma. Clin 217. Shannon-Lowe C, Rowe M. Epstein Barr virus entry; kissing and conjugation. Curr Opin Virol. Microbiol Infect. 2009;15(11):982-8. 2014;4:78-84. 239. Takada K. Epstein-Barr virus and gastric carcinoma. Mol Pathol. 2000;53(5):255-61. 218. Szakonyi G, Klein MG, Hannan JP, Young KA, Ma RZ, Asokan R, et al. Structure of the Epstein-Barr 240. Cohen JI. Optimal treatment for chronic active Epstein-Barr virus disease. Pediatr Transplant. virus major envelope glycoprotein. Nat Struct Mol Biol. 2006;13(11):996-1001. 2009;13(4):393-6. 219. Tanner J, Weis J, Fearon D, Whang Y, Kieff E. Epstein-Barr virus gp350/220 binding to the B 241. Chellapandian D, Das R, Zelley K, Wiener SJ, Zhao H, Teachey DT, et al. Treatment of Epstein Barr lymphocyte C3d receptor mediates adsorption, capping, and endocytosis. Cell. 1987;50(2):203-13. virus-induced haemophagocytic lymphohistiocytosis with rituximab-containing chemo-immunotherapeutic 220. Hoffman GJ, Lazarowitz SG, Hayward SD. Monoclonal antibody against a 250,000-dalton regimens. Br J Haematol. 2013;162(3):376-82. glycoprotein of Epstein-Barr virus identifies a membrane antigen and a neutralizing antigen. Proc Natl Acad 242. Kelesidis T, Humphries R, Terashita D, Eshaghian S, Territo MC, Said J, et al. Epstein-Barr virus- Sci U S A. 1980;77(5):2979-83. associated hemophagocytic lymphohistiocytosis in Los Angeles County. J Med Virol. 2012;84(5):777-85. 221. Thorley-Lawson DA, Geilinger K. Monoclonal antibodies against the major glycoprotein (gp350/220) 243. Rouphael NG, Talati NJ, Vaughan C, Cunningham K, Moreira R, Gould C. Infections associated with of Epstein-Barr virus neutralize infectivity. Proc Natl Acad Sci U S A. 1980;77(9):5307-11. haemophagocytic syndrome. Lancet Infect Dis. 2007;7(12):814-22. 222. Thorley-Lawson DA, Poodry CA. Identification and isolation of the main component (gp350-gp220) 244. Morimoto A, Nakazawa Y, Ishii E. 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Raffel J, Dobson R, Gafson A, Mattoscio M, Muraro P, Giovannoni G. Multiple sclerosis therapy and test. Journal of virological methods. 2004;119(1):25-30. Epstein-Barr virus antibody titres. Mult Scler Relat Disord. 2014;3(3):372-4. 128 129 428. Castellazzi M, Tamborino C, Cani A, Negri E, Baldi E, Seraceni S, et al. Epstein-Barr virus-specific 444. Castellazzi M, Delbue S, Elia F, Gastaldi M, Franciotta D, Rizzo R, et al. Epstein-Barr Virus Specific antibody response in cerebrospinal fluid and serum of patients with multiple sclerosis. Multiple sclerosis Antibody Response in Multiple Sclerosis Patients during 21 Months of Natalizumab Treatment. Dis (Houndmills, Basingstoke, England). 2010;16(7):883-7. Markers. 2015;2015:901312. 429. Brettschneider J, Tumani H, Kiechle U, Muche R, Richards G, Lehmensiek V, et al. IgG antibodies 445. Haghighi S, Andersen O, Rosengren L, Bergström T, Wahlström J, Nilsson S. Incidence of CSF against measles, rubella, and varicella zoster virus predict conversion to multiple sclerosis in clinically abnormalities in siblings of multiple sclerosis patients and unrelated controls. Journal of neurology. isolated syndrome. PloS one. 2009;4(11):e7638. 2000;247(8):616-22. 430. Jarius S, Eichhorn P, Franciotta D, Petereit HF, Akman-Demir G, Wick M, et al. The MRZ reaction as 446. Poser CM. Multiple sclerosis trait: the premorbid stage of multiple sclerosis. A hypothesis. Acta Neurol a highly specific marker of multiple sclerosis: re-evaluation and structured review of the literature. Journal Scand. 2004;109(4):239-43. of neurology. 2017;264(3):453-66. 447. Poser CM. The multiple sclerosis trait and the development of multiple sclerosis: genetic vulnerability 431. Reiber H, Ungefehr S, Jacobi C. The intrathecal, polyspecific and oligoclonal immune response in and environmental effect. Clin Neurol Neurosurg. 2006;108(3):227-33. multiple sclerosis. Multiple sclerosis (Houndmills, Basingstoke, England). 1998;4(3):111-7. 448. Holmén C, Piehl F, Hillert J, Fogdell-Hahn A, Lundkvist M, Karlberg E, et al. A Swedish national 432. Adams JM, Imagawa DT. Measles antibodies in multiple sclerosis. Proceedings of the Society for post-marketing surveillance study of natalizumab treatment in multiple sclerosis. Multiple sclerosis Experimental Biology and Medicine Society for Experimental Biology and Medicine (New York, NY). (Houndmills, Basingstoke, England). 2011;17(6):708-19. 1962;111:562-6. 449. Piehl F, Holmén C, Hillert J, Olsson T. Swedish natalizumab (Tysabri) multiple sclerosis surveillance 433. Ahlgren C, Odén A, Bergström T, Lycke J. Serum and CSF measles antibody levels increase over time study. Neurol Sci. 2011;31 Suppl 3:289-93. in patients with multiple sclerosis or clinically isolated syndrome. Journal of neuroimmunology. 450. Warnke C, Ramanujam R, Plavina T, Bergström T, Goelz S, Subramanyam M, et al. 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PloS one. 2014;9(4):e94497. 457. Koenker R, Hallock KF. Quantile regression. Journal of economic perspectives. 2001;15(4):143-56. 440. Kvistad S, Myhr KM, Holmøy T, Bakke S, Beiske AG, Bjerve KS, et al. Antibodies to Epstein-Barr 458. Koskiniemi M, Rantalaiho T, Piiparinen H, von Bonsdorff CH, Färkkilä M, Järvinen A, et al. Infections virus and MRI disease activity in multiple sclerosis. Multiple sclerosis (Houndmills, Basingstoke, England). of the central nervous system of suspected viral origin: a collaborative study from Finland. J Neurovirol. 2014;20(14):1833-40. 2001;7(5):400-8. 441. Farrell RA, Antony D, Wall GR, Clark DA, Fisniku L, Swanton J, et al. Humoral immune response to 459. Nagel MA, Gilden D. The relationship between herpes zoster and stroke. Curr Neurol Neurosci Rep. EBV in multiple sclerosis is associated with disease activity on MRI. Neurology. 2009;73(1):32-8. 2015;15(4):16. 442. Ingram G, Bugert JJ, Loveless S, Robertson NP. 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Jääskeläinen AJ, Moilanen K, Bühler S, Lappalainen M, Vapalahti O, Vaheri A, et al. Serological 478. Hottenrott T, Dersch R, Berger B, Endres D, Huzly D, Thiel J, et al. The MRZ reaction helps to microarray for detection of HSV-1, HSV-2, VZV, and CMV antibodies. Journal of virological methods. distinguish rheumatologic disorders with central nervous involvement from multiple sclerosis. BMC Neurol. 2009;160(1-2):167-71. 2018;18(1):14. 463. Kombe Kombe AJ, Xie J, Zahid A, Ma H, Xu G, Deng Y, et al. Detection of Circulating VZV- 479. Venhoff N, Thiel J, Rizzi M, Venhoff A, Rauer S, Endres D, et al. The MRZ-Reaction and Specific Glycoprotein E-Specific Antibodies by Chemiluminescent Immunoassay (CLIA) for Varicella-Zoster Autoantibody Detection for Differentiation of ANA-Positive Multiple Sclerosis From Rheumatic Diseases Diagnosis. Pathogens. 2022;11(1). With Cerebral Involvement. Front Immunol. 2019;10:514. 464. Nordén R, Nilsson J, Samuelsson E, Risinger C, Sihlbom C, Blixt O, et al. Recombinant Glycoprotein 480. Feki S, Gargouri S, Mejdoub S, Dammak M, Hachicha H, Hadiji O, et al. The intrathecal polyspecific E of Varicella Zoster Virus Contains Glycan-Peptide Motifs That Modulate B Cell Epitopes into Discrete antiviral immune response (MRZ reaction): A potential cerebrospinal fluid marker for multiple sclerosis Immunological Signatures. Int J Mol Sci. 2019;20(4). diagnosis. Journal of neuroimmunology. 2018;321:66-71. 465. Ndumbe PM, Cradock-Watson J, Levinsky RJ. Natural and artificial immunity to varicella zoster virus. 481. Stich O, Kluge J, Speck J, Rauer S. Oligoclonal restriction of antiviral immunoreaction in oligoclonal J Med Virol. 1988;25(2):171-8. band-negative MS patients. Acta Neurol Scand. 2015;131(6):381-8. 466. D'Arrigo I, Cló E, Bergström T, Olofsson S, Blixt O. Diverse IgG serum response to novel glycopeptide 482. Balfour HH, Jr., Odumade OA, Schmeling DO, Mullan BD, Ed JA, Knight JA, et al. Behavioral, epitopes detected within immunodominant stretches of Epstein-Barr virus glycoprotein 350/220: diagnostic virologic, and immunologic factors associated with acquisition and severity of primary Epstein-Barr virus potential of O-glycopeptide microarrays. Glycoconj J. 2013;30(7):633-40. infection in university students. J Infect Dis. 2013;207(1):80-8. 467. Jenkins N, Parekh RB, James DC. Getting the glycosylation right: implications for the biotechnology 483. Ruprecht K, Wildemann B, Jarius S. Low intrathecal antibody production despite high seroprevalence industry. Nat Biotechnol. 1996;14(8):975-81. of Epstein-Barr virus in multiple sclerosis: a review of the literature. Journal of neurology. 2018;265(2):239- 468. Servat E, Ro BW, Cayatte C, Gemmell L, Barton C, Rao E, et al. Identification of the critical attribute(s) 52. of EBV gp350 antigen required for elicitation of a neutralizing antibody response in vivo. Vaccine. 484. Jacobi C, Lange P, Reiber H. Quantitation of intrathecal antibodies in cerebrospinal fluid of subacute 2015;33(48):6771-7. sclerosing panencephalitis, herpes simplex encephalitis and multiple sclerosis: discrimination between 469. Jackman WT, Mann KA, Hoffmann HJ, Spaete RR. Expression of Epstein-Barr virus gp350 as a single microorganism-driven and polyspecific immune response. Journal of neuroimmunology. 2007;187(1- chain glycoprotein for an EBV subunit vaccine. Vaccine. 1999;17(7-8):660-8. 2):139-46. 470. Jons D, Persson Berg L, Sundström P, Haghighi S, Axelsson M, Thulin M, et al. Follow-up after 485. Biström M, Jons D, Engdahl E, Gustafsson R, Huang J, Brenner N, et al. Epstein-Barr virus infection infectious mononucleosis in search of serological similarities with presymptomatic multiple sclerosis. Mult after adolescence and human herpesvirus 6A as risk factors for multiple sclerosis. European journal of Scler Relat Disord. 2021;56:103288. neurology. 2021;28(2):579-86. 471. Liu H, Gemmell L, Lin R, Zuo F, Balfour HH, Jr., Woo JC, et al. Development of an Improved Epstein- 486. Ascherio A, Munger KL, Lennette ET, Spiegelman D, Hernán MA, Olek MJ, et al. Epstein-Barr virus Barr Virus (EBV) Neutralizing Antibody Assay to Facilitate Development of a Prophylactic gp350-Subunit antibodies and risk of multiple sclerosis: a prospective study. Jama. 2001;286(24):3083-8. EBV Vaccine. Mediterr J Hematol Infect Dis. 2020;12(1):e2020016. 487. Sundström P, Juto P, Wadell G, Hallmans G, Svenningsson A, Nyström L, et al. An altered immune 472. Jarius S, Eichhorn P, Jacobi C, Wildemann B, Wick M, Voltz R. The intrathecal, polyspecific antiviral response to Epstein-Barr virus in multiple sclerosis: a prospective study. Neurology. 2004;62(12):2277-82. immune response: Specific for MS or a general marker of CNS autoimmunity? J Neurol Sci. 2009;280(1- 488. Rosche B, Laurent S, Conradi S, Hofmann J, Ruprecht K, Harms L. Measles IgG antibody index 2):98-100. correlates with T2 lesion load on MRI in patients with early multiple sclerosis. PloS one. 2012;7(1):e28094. 473. Huss A, Buttmann M, Brecht I, Weishaupt A, Otto M, Tumani H. Validation of a multiplexing 489. Dominguez-Mozo MI, Perez-Perez S, Villar LM, Oliver-Martos B, Villarrubia N, Matesanz F, et al. technique to determine the intrathecal, polyspecific antiviral immune response in multiple sclerosis. Expert Predictive factors and early biomarkers of response in multiple sclerosis patients treated with natalizumab. Rev Mol Diagn. 2016;16(12):1353-6. Sci Rep. 2020;10(1):14244. 474. Brecht I, Weissbrich B, Braun J, Toyka KV, Weishaupt A, Buttmann M. Intrathecal, polyspecific 490. Warnke C, Stettner M, Lehmensiek V, Dehmel T, Mausberg AK, von Geldern G, et al. Natalizumab antiviral immune response in oligoclonal band negative multiple sclerosis. 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