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Comparative analysis of rubella specific antibody responses in congenitally and postnatally rubella infected… Mauracher, Christoph Andreas 1992

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(Q’\COMPARATIVE ANALYSIS OF RUBELLA SPECIFIC ANTIBODY RESPONSES INCONGENITALLY AND POSTNATALLY RUBELLA INFECTED HUMANS: A MODEL FORSELECTIVE TOLERANCEbyChristoph Andreas MauracherB.Sc., The University of Victoria, 1986A thesis submitted in partial fulfilment ofthe requirements for the degree ofDoctor of Philosophyinthe Faculty of Graduate StudiesPathology ProgramWe accept this thesis as conformingto the required standardThe University of British Columbia1992© Chris A. MauracherIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment. or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.__________________________Department of- /The University of British ColumbiaVancouver, CanadaDate ?y D76’ IDE-6 (2/88)ABSTRACTRubella virus (RV) is an enveloped, positive sense, single stranded RNA virus in the family Togaviridae. Thevirus consists of a host derived lipid bilayer membrane, two trans-membrane proteins El and E2, and the Cprotein which together with viral RNA forms the icosahedral nucleocapsid. RV causes natural infections inhumans only, where it can lead to a variety of pathological conditions. The most severe outcome, CongenitalRubella Syndrome (CRS), occurs when the fetus is infected in the first trimester of gestation. RV can beisolated from CRS patients at birth and is believed to establish a persistent infection. Although congenital RVinfection remains of clinical importance it also provides a rare opportunity for studying the effects of intrauterineviral infection on the human fetus. This thesis examines the hypothesis that intrauterine infection with RV canestablish immunological tolerance which is reflected in the serological response to RV. The study on RV-specifictolerance may help in the understanding by which mechanism self-recognition is established in the human. Theinduction of immunological tolerance may also be an important mechanism in viral persistence and play a rolein chronic inflammatory disorders such as Rubella Associated Arthritis (RAA).A comprehensive study of the antibody mediated immunity of healthy individuals responding to postnatalinfection with RV is compared to that of CRS patients. Assays were developed to measure antibody quantity,affinity and kinetics of the antibody response to whole virus as well as that to the individual RV structuralproteins, El, E2 and C. The viral proteins were purified from whole RV preparation by differentialcentrifugation followed by preparative SDS-PAGE under non-reducing conditions. Separated proteins wereanalyzed for their structural integrity by assaying their biological activity. RV protein-specific ELISAs weredeveloped and used for antibody quantitation and in IgG affinity assays using the chaotropic elution technique.Biological activity of sera was assayed by HAl assay. The specificity of antibodies to linear and topographicepitopes was investigated by using reducing and non-reducing Western blotting. Observations made during thedevelopment of protein separation protocols have led to the description of an uncoating mechanism for RV.While establishing ELJSA protocols, improved buffers were developed by including heat denatured blockingproteins and studies on a novel anti-RV IgG ELISA, which will give a more accurate assessment of protectiveimmunity than ELISA technology used to date, were initiated.Results indicate that CRS patients can produce similar amounts of IgG than control patients if measured bywhole RV ELISA. Group differences were detected at the level of the protein specific responses. CRS patientsexhibited significantly lower levels and had reduced affinities of El-specific antibodies. The most consistentfeature of this patient population was their inability to produce IgG to linear epitopes of the El protein. Thepotential role of the tolerization of anti-El Th cells due to the exposure of the immature fetal immune systemto RV is discussed. A model is proposed which can accommodate the fmdings on serological andlymphoproliferative immune responses of CRS patients and which supports the hypothesis that congenital RVinfection in the early gestational period leads to viral antigen tolerization. Tolerance to the immunodominantEl protein may lead to a sufficient depression in responder T and B cells in order to allow chronic, low gradeRV replication.IITABLE OF CONTENTSABSTRACTTABLE OF CONTENTS iiiLIST OF TABLES viiLIST OF FIGURES viiiLIST OF ABBREVIATIONS ixACKNOWLEDGEMENTS xi1. INTRODUCTION 11.1. Rubella Virus Bioloay 11.1.lClassification 11.1.2RV Morphology and Physio-Chemical Properties 21.1.3 Structural Proteins 31.1.3.1 El Glycoprotein 3l.1.3.2E2 Glycoprotein 41.1.3.3.C Protein 51.L4.Non-structural Proteins 61. 1.5.Genome Organization 61.1.6.RV Life Cycle 71.1.6.1 Entry and Uncoating 71.1.6 .2Replication and Protein Expression 81.1.6.3.Assembly 91.2. Rubella Pathology and Pathoenesis 101.2.1.History 101.2.2.Clinical and Immunological Features 10l.2.2.lRubella 101 .2.2.2.Congenital Rubella Syndrome 13a) Clinical Features 13b) Immunological Features 171.2.2.3 .Tolerance following Intrauterine Infection 181.3. Rubella Vaccine 211.3.1.History 211.3.2.Biology and Immunology 221.3.3.Side Effects of RV Vaccination 231.3.4.Vaccine Efficacy 251.4. Laboratory Diaanosis of Rubella Virus Infection 251.4.1.Virus Isolation 251.4.2.Neutralization Assay 26111l.4.3.Hemagglutination Inhibition Assay 271.4.4.Enzyme Linked linmuno-Assays (ETA) 271.4.4.1ELISA 281 .4.4.2.Western Blotting 291.4.4.Affinity Assays 291.4.5.1 .Chaotropic Elution ELISA 321.4.5.2lnhibition EUSA 331.5. Thesis Rationale and Objectives 332. MATERIALS AND METHODS 362.1. Virus Preparation. Titration and Concentration 362.l.1.BuIk Preparation of Rubella Virus (M-33 Strain) 362.l.2.Rubella Virus Titration on RK-13 Cells 362.1.3.Concentration of Rubella Virus Tissue Culture Supernatant 362.2. Preparative SDS-PAGE 372.2. 1.Electrophoresis 372.2.2.Electroelution 382.2.3.Detergent Extraction 382.2.4.Yield Determination 382.3. Metabolic Radiolabelling of Rubella Virus 392.4. Serum Separation 392.5. Gel Stainin2 402.5. 1.Colloidal Coomassie Stain 402.5.2 Silver Stain 402.6. Solid Phase linmunoassays 402.6.1.Western Blotting 402.6.2.Iinmunoprecipitation 412.6.3.ELISA 422.6.3.1.Whole RV ELISA 422.6.3.2RV Protein ELISA 432.7. Affinity Assays 432.7. 1.Chaotropic Elution ELISA 432.7.2.One Well Inhibition ELISA 442.8. Hemaalutination Inhibition Assay 452.9. Biological Function of Rubella Virus Proteins 452.9. 1.Solubility Shift of C Protein under Acidic pH 452.9.1.1 .Ultracentrifugation 462.9.1 .2Polymerase Chain Reaction for the Detection of RV RNA 462.9.2.Hemagglutination Activity of Purified El Protein 47iv2.1O.Patient Sera 472.11. Statistical Methods 483. RESULTS AN]) DISCUSSION 493.1. Section I: Virus Preparation and Use in Solid Phase Iminunoassays 493.L1.Rubella Virus Preparation 493.1.1.1.Virus Titration 493.1.2.Whole RVELISA 513.1.2.1.Coating Conditions and Antigen Treatment 513.1.2.2Jleat Denatured Sample Buffer 523. 1.3.AfflnityAssays 543.1.3.1.One Well Inhibition Assay 553.1.3.2.8M Urea Elution ELISA 573.1.3.3.Preparation of IgG Fractions from Human Sera 583.2. Section II: RV Protein Specific Immunoassays 623.2.1.Western Blottina 623.2.1. lReducing vs. Non-Reducing Conditions 623.2.1.2.Comparison of Immunoprecipitation and Western Blot 643.2.1.3.Western Blot AffinityAssay 663.2.2.Separation of RV Proteins, Their Use in Solid Phase Immunoassav 673.2.2.LFeasibility of Electroelution; Yield and Purity of RV Proteins 693.2.2.2Maximization of Protein Binding Conditions 713.2.2.3.Quantitation of Anti-El, -E2 and -C IgG by ELISA 713.2.3.Antiaenicity and Bioloaical Activity of Separated RV Structural Proteins 723.2.3.1.Function of El 743.2.3.2Function of C 753.2.4.Summarv 813.3. Section ifi: RV-Specific Humoral Immune Response in Adults FollowingRubella 823.3.1.Response to Whole Virus and Separated RV Proteins 823.3.2.Biological Activity of Sera 823.3.3.Reactivitv to Linear and Topographic Epitopes 833.3.4.Kinetics of the laG Response 843.3.5.Relative Affinity of IG Directed to RY and RV Proteins 863.3.6.Summary and Conclusion 883.3.6.1JgA Responses to RV Proteins 893.3.6.2RV Protein Specific LymphoproliferativeResponses 90V3.4. Section IV: RV-Specific Humoral Immunity in CRS Patients 923.4.1.RV and RV Protein Specific ELISA 923.4.2.Biolopical Activity of Sera in CRS Patients 943.4.3.Relative Affinity of RV Specific JaG in CRS Patients 953.4.4.Reactivity to Linear and Topoaraphic Epitopes in CRS Patients 963.4.5.Analysis of Sequential Serum Samples from CRS Patients 993.4.6.Evidence for Th Tolerance in CRS Patients 1013.4.7.Summary and Discussion 1044. SUMI’VIARY AIM]) PERSPECTIVES 1085. REFERENCES 112viLIST OF TABLESTable 1: Summary of Physical and Immunological Properties of RV Structural Proteins. 6Table 2: Clinical Features of Congenital Rubella. 15Table 3: Mechanisms of Viral Persistence. 19Table 4: Biological Reactions in Which High Affmity Antibodies are Superior to Low AffinityAntibodies. 31Table 5: Rubella IgG ELJSA Responses to Whole RV and RV Structural Proteins. 54Table 6: Relative Affmity of Anti-RV IgG in Serum or Purified IgG. 60Table 7: Percentage of Signal Remaining on Western Blot under Reducing Conditions. 84Table 8: Percentage of Signal Remaining on Western Blot under Reducing for Control and CRSPatients 97Table 9: Serological Examination of MMR Vaccinated CRS Patients. 101viiLIST OF FIGURESFigure 1: Model of Rubella Virus Structure. 2Figure 2: Model of the E1/E2 Glycoprotein Spike of RV. 4Figure 3: Strategy for the Expression of RV Structural Proteins. 8Figure 4: Model for the Topogenesis of the RV Polyprotein. 9Figure 5: Clinical Features of Rubella. 12Figure 6: Incidence of Virus Shedding in CRS Patients. 16Figure 7: Incidence of Reported Rubella and CRS Cases in the USA. 22Figure 8: Hemagglutination-Inhibition (HAl) Test. 28Figure 9: Triton X-1 14 Extraction of RV from Tissue Culture Supernatant. 46Figure 10: Micrograph of RV Infectious Focus on RK-13 Cell Monolayer. 49Figure 11: Western Blot and Total Protein Stains of RV Preparations Separated on SDS-PAGE Gels. 50Figure 12: Titration of RV Treated with Triton X-100. 52Figure 13: Determination of RV-Specific IgG by ELISA. 53Figure 14: Determination of 10.5 in Acute and Convalescent Sera. 55Figure 15: Affinity Maturation of Anti-RV IgG Measured by Inhibition ELISA. 56Figure 16: 8 M Urea Elution ELISA for Determination of Antibody Affinity. 57Figure 17: Treatment of Solid Phase Bound RV with 8M Urea. 58Figure 18: FPLC Separation of Human Serum. 59Figure 19: Western Blot and Autoradiograph of Reduced and Non-Reduced RV Preparations. 63Figure 20: Comparison of RV IgG Western Blot and Immunoprecipitation Assays. 64Figure 21: Immunoblot Analysis of RV-Specific IgG, 1gM and IgA. 65Figure 22: 8M Urea Elution of Western Blot (North-Western Blot). 66Figure 23: Yield of Radiolabelled RV Proteins after Electroelution. 68Figure 24: Western Blot of Separated RV Proteins. 69Figure 25: Comparison of Optimal Coating Concentrations for El, E2 and Cd. 70Figure 26: Western Blot and Densitometric Scan of Anti-RV Standard Serum. 72Figure 27: Effect of SDS and Temperature on the Antigenicity of Whole RV. 73Figure 28: HAl Antibody Inhibition Assay. 75Figure 29: Autoradiographs of TX-i 14 Extracted RV. 76Figure 30: Ultracentrifugation of RV Following Detergent Extraction. 78Figure 31: Proposed Model for the Entry of RV into the Host Cell. 79Figure 32: Detergent Extraction of Separated C Protein. 80Figure 33: Quantitation of IgG to Whole RV and RV Proteins. 83Figure 34: The Kinetics of the IgG Response to RV Structural Proteins. 85Figure 35: Differential IgG Affinity Maturation to the Structural Proteins of RV. 87Figure 36: Kinetics of the Appearance of RV Protein-Specific IgA. 89Figure 37: Comparison of Proliferative and IgG Responses to RV Structural Proteins. 90Figure 38: RV-, and RV Protein-Specific IgG Responses in Control and CRS Patients. 93Figure 39: Affmity Indices of IgG Directed to RV and RV Proteins in Control and CRS Patients. 95Figure 40: Western Blot under Reducing and Non-Reducing Conditions. 97Figure 41: Model for the Production of Anti-El IgG in the Absence of El-Specific Th Cells. 98Figure 42: Western Blot Analysis for Three MMR Vaccinated CRS Patients. 100viiiLIST OF ABBREVIATIONSA405 Absorbance at 405 nm wavelengthaa amino acidAU Arbitrary UnitsBCIP 5-Bromo-4-Chloro-3-Indolyl PhosphateBDV Border disease virusBis N,N’ -methylene-bis-acrylaniidebp base pairsBSA Bovine serum albuminDegrees Celsiuscb Conjugate BufferCIC Circulating Immune ComplexCRBC Chick Red Blood CellsCRS Congenital Rubella SyndromeEIA Enzyme linked inimuno assayELJSA Enzyme linked immunosorbent assayER Endoplasmic reticulumet al. et aliiHA HemagglutinationHAl Hemagglutination InhibitionIg ImmunoglobulinIU International UnitskDa kilodaltonLCMV Lymphocytic Choriomeningitis VirusM molarMBq MegabecquerelMEM Minimum Essential Mediumnil millilitre11 microlitreim micrometerMMR Mumps, Measles and Rubella vaccineMW Molecular Weightn sample numberNBT Nitro Blue TetrazoliumNGS Normal goat serumnm nanometerns non-structuralNT Neutralization titrePAGE Polyacrylamide Gel ElectrophoresisPBS Phosphate buffered salinepfu Plaque forming unitspH 1/log [Hjixp1 isoelectric pointrpm Rotation per minuteRA Rheumatoid ArthritisRAA Rubella Associated ArthritisRT Room temperatureRV Rubella virusS Svedberg unitsb Sample Buffersbb Sample Buffer (boiled)SD Standard DeviationSDS Sodium Dodecyl SulphateSFV Semliki Forest virusSI Stimulation Indexsubb Substrate BufferSV Sindbis virusTc Cytotoxic T cellTh Helper T cellTCS Tissue culture supernatantTris trishydroxymethylaminomethaneTX-100 Triton X-lOOTX-114 Triton X-114VSV Vesicular Stomatitis viruswb Wash BufferxACKNOWLEDGEMENTSI want to thank the members, past and present, of the lab: Fiona Yong, Dr. Liz Hancock, Susan Farmer,Margaret Ho, Diane Ddcarie, Ting Zhang, Dr. Beatriz Gomez, Janet Deronio and Dr. Marita LundströmHobman. Special thanks go to Bob Shukin who in most cases was a thorn in my side but whose ideas,discussions and experience helped in many crucial experiments and to Susan Farmer for the growing of muchof virus that I used.To Dr. Aubrey Tingle, my supervisor, I would like to extend my gratitude for the patience - not to mention hispatients - he had in taking me on as a graduate student and for defending me where others may have given up.If I emerge as a Doctor of Philosophy, it is his coaching which will have made it possible. My co-supervisor, Dr.Leslie Mitchell, I would like to thank for creating the stable lab environment which she created in the last yearsand especially for convincing me that all polyvinyl surfaces are not the same.Thank you to my supervisory committee, Drs. O’Kusky, Pritchard, McMaster, Chantler and Gillam, for theirinvestment of time and their wisdom to accept this thesis. I appreciated Dr. Shirley Gillam’s help and adviceon my questions regarding anything to do with Rubella virus and for the many calories which were consumedon her account.Of the agencies and individuals who funded this study I would like to thank the Research Division of theChildren’s Hospital of British Columbia, University of British Columbia and especially Mr. Roman Babicki whothrough a personal gift of fmancial support and enthusiasm enabled much of this work. It is easy for the scientistto forget that it is only through the interest and sacrifice of the community that research at university institutionsis possible. Mr. Babicki personifies this attitude which in many ways is the root for progress in science.Foremost I would like to thank my family. To my parents who endured the tortuous path of digress and successand were there with both moral and fmancial support and to my grandmother, Berta Mauracher, who was behindme since I can remember - no matter what. To my wife, Aileen, who put every thing into perspective, failures,progress and life in general. To the life she gave me, my two beautiful daughters Andrea and Marita, I dedicatethis thesis. By the time both of you chance to read these pages much time willhave elapsed, and ifI should haveforgotten, remind me of what I promised at your births.xiMuch in research is doing the wrong thing at the right time, science begins when one fmds the courage to admit it.xli1. INTRODUCTIONRubella virus (RV) causes a mild childhood illness but can cause severe birth defects if the fetus is infected inthe first trimester of gestation. The teratogenic effect of RV can cause a wide range of birth defects and lateonset sequelae collectively described as the Congenital Rubella Syndrome (CRS). RV persists in utero and canbe isolated from virtually all CRS patients at birth and is believed to cause a low grade persistent infection inmost patients over many years. The immunological dysfunction which allows the virus to persist and replicatein these individuals has not been defined. The investigations whose results are presented in this thesis examinedthe humoral responses of adults following the uncomplicated resolution of RV infection in comparison to theRV-specific humoral response of CRS patients to test the hypothesis that intrauterine exposure to a viral agentleads to virus specific immunological tolerance and that specific tolerance is reflected in the serological responseto RV. Tolerance or immunological non-responsiveness leading to viral persistence is not only of interest tothe pathology of CRS but may play an important role in the pathogenesis of other RV-associated syndromessuch as Rubella Associated Arthritis or Chronic Progressive Rubella Panencephalitis, by the chronic low gradereplication of virus in specific tissues such as the synovium or the brain.This introduction contains three sections: a summary of rubella virus biology, rubella virus induced pathologyand immunity, and a review of the use and development of diagnostic tools in the detection of RV and the RVspecific immune-responses. The rationale and objectives of this thesis will conclude the chapter.1.1 Rubella Virus Biolo2y1.1. 1.ClassificationRubella Virus (RV) is the only member of the genus Rubivirus in the family Togaviridae (Porterfield et al.,1978). The Togaviridae are defined as spherical, enveloped viruses of 60-70 mu diameter, with an icosahedralnucleocapsid. The genome is composed of a 40S (+)-sense-single stranded RNA molecule. Studies on thereplication cycle of these viruses showed significant differences between flaviviruses and the other genera of theTogaviridae, resulting in the reclassification of the flaviviruses into their own family Flaviviridae in 1985—1—(Westaway et al., 1985). The Togaviridae are now composed of the rubivirus, aiphavirus, pestivirus andarterivirus genera. The old classification of Group A and B Arboviruses is no longer officially recognized by theInternational Committee on Toxonomy and Nomenclature of Viruses.1.1.2.RV Morphology and Physiochemical PropertiesEarly electron microscopic studies of RV have shown the virus to be spherical and of 60-70 nm in diameter (vonBonsdorff and Vaheri, 1969). An electron dense core of 30 nm diameter defines the nucleocapsid. An electronlucent ring separating the capsid from the membrane is characteristic and allows this virus to be distinguishedfrom other morphologically similar togaviruses (Murphy et al., 1968). Spikes of 5-8 nm length are observed onthe virion surface which are associated with hemagglutination activity.ElE2CMembraneFigure 1. Model of Rubella Virus Structure. Capsid protein (33 kD) forms the icosahedral capsid surroundingthe viral RNA (10 kb). The nucleocapsid is enveloped by a host derived lipid-bilayer membrane in whichenvelope proteins El (58 Kd) and E2 (42-47 kD) are embedded. The envelope proteins form El-Elhomodimers and E2-El heterodimers to form surface spikes of 6-8 urn in length.-2-The virus is heat labile and can be rapidly inactivated by exposure to 56 1C. The virus remains stable forseveral days at 4#C and can be stored for indefmite periods at -70fC. Agents which extract lipids, denatureproteins or interfere with nucleic acids all lead to virus inactivation (Parkman et. al, 1964).RV is assembled from three structural proteins: El, E2 and C (Vaheri and Hovi, 1972; Payment et al., 1975).The envelope proteins El and E2 are both type 1 trans membrane proteins, acylated and highly glycosylated.In the intact virion these proteins form El-E2 heterodimers and El-El homodimers, visible by electron-microscopy. The capsid is composed of C protein and has an icosahedral symmetry of T= 3. This icosahedronsurrounds the positive sense, single strand RNA to form the nucleocapsid.1. 1.3.Structural Proteinsl.l.3.1.El GlycoproteinThe El glycoprotein is the largest of three RV structural proteins with an apparent molecular mass of 58 kDon reducing SDS-PAGE and an acidic p1 of 6.5 (Ho-Terry and Cohen, 1982). The protein is a type 1transmembrane protein, containing a 14 residue cytoplasmic tail and a 27 residue trans membrane spanningregion (Vidgren et a!., 1987). The remaining 440 residues form the bulk of this protein’s surface region. Posttranslational modifications include both fatty acid acylation in the C-terminal region (Waxham and Wolinsky,1985a) as well as N-linked glycosylation at all three potential glycosylation sites, predicted by sequence analysisof all RV strains studied (Frey et al. 1986; Clarke et al., 1987; Terry et al., 1988). The glycosylation with theseendo-H resistant glycans adds 5 kD to the molecular mass of this protein and although no direct biologicalfunction for this modification has been described, it is believed that it stabilizes El in its biologically functionaland immunologically reactive conformation (Ho-Terry et al., 1984).El contains the hemagglutinin activity as well as the majority of the defined viral neutralization domains. Thehemagglutination of day-old chick erythrocytes and pigeon erythrocytes is a function of the El monomer (Ho-Terry and Cohen, 1985) but does not correlate completely with neutralization domains (Green and Dorsett,1986). By using trypsin and S.aureus cleavage product analysis the HA activity was localized to a 13 kDfragment of El. This region, containing amino acid residues 245-285,encodes three separate epitopes conferringboth neutralizing and HAl antibody binding sites (Terry et al., 1988), showing that these two functions are-3-closely situated on the El protein. Using competitive inhibition assays with a panel of monoclonal antibodiesit was further shown that at least six non-overlapping epitopes exist on this protein (See summary in table 1).Because of the concentration of 6 of the known 7 neutralizing epitopes and the receptor-like activity of HA, itis believed that El contains the as of yet undefined viral attachment site.Figure 2. Model of the El/E2 Glycoprotein Spike of RV. N-linked sugars are indicated by (Mf located onboth El and E2. 0-linked sugars (.f.) are located on E2 only. Fatty acylation ( ) on cysteine residues arepossible on both El and E2 cytoplasmic domains.1.1 .3.2.E2 GlycoproteinE2 is the smaller of the two envelope proteins and travels as a diffuse band in electrophoresis with an apparentmolecular mass of 42-48 kD, under reducing conditions. In Therien strain and to a lesser extent in M-33 thisdiffuse band can be resolved into species termed E2a and E2b (Oker-Bloom et al., 1983). The protein has awide range of isoelectric points ranging from pH 5.0 to 8.6 (Ho-Terry and Cohen, 1982) which is believed tobe due to heterogeneous glycosylation with terminal sialic acids. The protein is highly glycosylated and if VeroEl E2-4-cells are infected with RV, in the presence of tunicamycin, a 30 kD species of E2 is isolated (Oker-Blom et a!.,1983), indicating that more than one third of this protein’s mass is derived from oligosaccharide chains.Depending on the RV strain, E2 contains 3 (M 33 and HPV 77) to 4 (Therien and RA 27/3) N-linkedglycosylation sites (Clarke et al., 1987; Vidgren et al., 1987; Frey and Marr, 1988; Nakhasi et al., 1989) whichare thought to be of the complex type, as judged by their resistance to endo-H glycosidase (Oker-Blom et al.,1983). Recently the presence of sialylated 0-linked sugars on E2 has been described (Lundstr6m et al., 1991),clarifying the existence of the 15 or more isoelectric variants of this protein.No direct biological function has been assigned to this protein although the co-expression of E2 with El isnecessary for the transport of El into the assembling virion (Hobman et al., 1990). Also one neutralizingdomain has been described for this protein (Green and Dorsett, 1986). It has to be borne in mind that E2occurs in a heterodimeric form with El and in that function might confer support or protection to the spikestructure.1.l.3.3.C ProteinCapsid is a non-glycosylated protein of apparent molecular mass of 33 ‘000 under reducing SDS-PAGE. Onthese gels, C is often observed to run as a doublet, differing in less than 1 kD in mass. This is thought to bedue to the selective usage of two translation initiation sites separated by 7 amino acid residues (Clarke et al.,1987). Under non-reducing conditions, C runs in a dimeric form with an apparent molecular mass of 66 kD(Mauracher et a!., 199 la). C has two distinct p1 forms at pH 8.8 and 9.5. This protein specifically interacts withthe 40 S viral RNA (Weiss et al., 1989) and initiates the assembly of the nucleocapsid. The exact site andmechanism of nucleocapsid assembly has remained controversial, as distinct differences in RV are observed incomparison with the well studied alphavirus assembly pathway. Alphavirus capsid contains protease activity andcleaves itself, by an autolytic event, from the El/E2/E3 polypeptide, followed by capsomere assembly in thecytoplasm (Malancon and Garoff, 1987). The capsid of RV has no inherent protease activity (Oker-Blom et al.,1984) and assembly of the capsomers has been observed to be dependent on membranes (Horzinek, 1981)suggesting that RV carries with it the signal peptide of the E2/El polyprotein (Suomalainen et a!., 1990).Four epitopes have been defmed on this protein, none of which induce neutralizing antibodies (Waxham andWolinsky., 1985a), although capsid has been implicated in the events of viral penetration (Mauracher et al.,-5-1991a).Protein MW Glycosylation Epitopes Neutralizing HA Capacity toDomains2 Activity3 Induce T Cells4El 58’OOO 3 N-linked 6 strong yes strongE2 42-48’OOO 3-4 N-linked/O-linked 2 weak no weakC 33’OOO None ‘ 4 no no mediumTable 1: Summary of Physical and Immunological Properties of RV Structural Proteins. Epitopes are defmedby competitive inhibition using monoclonal antibodies (Waxham and Wolinsky, 1985b).2Defined by monoclonalantibodies which can inhibit viral replication on Vero cells (Waxham and Wolinsky, 1985b; Green and Dorsett,1986). The ability of RV to agglutinate day old chick- or pigeon erythrocytes has been mapped to El by usingpurified protein (Ho-Terry and Cohen, 1980) or by monoclonal antibody (Green and Dorsett, 1986). Definedby studying lymphoproliferative responses of healthy adults to RV proteins El, E2 and C expressed individuallyin vaccinia virus (Chaye et al., 1992).1. 1.4.Non-Structural ProteinsLittle is known about the nature of RV non-structural (ns) proteins although their genes occupy two thirds ofthe RV genome. Most work on togavirus enzymes has been performed in SFV and SV and by analogy withthese well studied viruses and findings of RV gene arrangement and sequence, it is believed that RV encodes4 ns proteins (Schlesinger, 1987). One of these ns proteins is likely functioning as an RNA dependent RNApolymerase. The catalytic steps performed by this protein or protein complex include: initiation and elongationof full length (+) and (-) sense RNA, (+) sense sub-genomic RNA synthesis as well as capping and methylation(Schlesinger and Schlesinger, 1990). Furthermore it is believed that one ns protein acts as a virus specificprotease involved in the cleavage of the viral polyprotein. Host cell proteins may also be involved in theenzymatic activities during viral replication as prolonged inhibition of host cell RNA synthesis prior to virusinfection interferes with togavirus replication (Baric et al. 1983).1. 1.5.Genome OrganizationThe RV genome exists as an infectious, single-stranded RNA molecule with a sedimentation rate of 40S (Hoviand Vaheri, 1970). In the infected cell, an additional 24S subgenomic RNA can be isolated, similarly poly-6-adenylated and capped as the full-length genome and with an identical 3’ sequence. This mRNA gives rise toa polyprotein from which the RV structural proteins are derived (Oker-Blom et al., 1984a).The sequence for the 24S subgenomic RNA is known for wild type isolates M-33 and Therien strain (Clarkeet al., 1987; Frey et al., 1986) as well as for vaccine strains RA 27/3 and HPV 77 (Nakhasi et al., 1989; Zhenget al.,1989). The aligned sequences of the 24S RNA reveal a 95% homology, whereas little homology existsbetween the genomes of RV strains and members of the aiphavirus genus (Frey and Marr, 1988).1.1.6.RV Life CycleThe life cycle of RV is very similar to the replication strategy of the alphaviruses. In particular, the biology ofSFV and SV have been studied in much detail and much of the knowledge obtained by the study of these twoviruses has been shown to apply to RV as well, although differences in some of the details of morphology andreplication exist.1.1.6.lEntry and UncoatingTogaviruses are believed to enter the target cell by receptor mediated endocytosis, a step which can beselectively inhibited by lysosomotrophic drugs (Helenius et al., 1984). The entry mechanism of the aiphaviruseshas been described in detail (Kielian and Helenius, 1986) and there is evidence that RV also uses the endocyticpathway for cell entry (Vaananen and Kaariainen, 1980). A specific cell surface receptor has not been definedfor RV, but it has been speculated that membrane phospholipids might be involved in virus attachment(Mastromarino et al., 1990); while others interpret an infection frequency of less than 10% on a cultured cellmonolayer as an indication that the RV receptor is a cell cycle dependent protein (Hemphill et al., 1988).Nevertheless, electron microscopic evidence shows RV particles being taken up by endocytosis and deliveredto the endosome system (Dr.S.Katow, NIH, Japan, personal communication). Upon acidification of theendosome below pH 5.5 the RV El undergoes a structural change to become fusogenic (Katow and Sugiura,1988), a step which allows the limiting membranes of the RV and endosome to fuse. The low pH environmentof the endosome also causes a shift in RV C protein solubility, leading to nucleocapsid uncoating in theendosome (Mauracher et al., 1991). Upon membrane fusion the viral RNA penetrates into the cytoplasm andviral replication can be initiated.-7-1.1 .6.2Replication and Protein ExpressionNaked RV RNA is infectious (Maes et al. 1966). Hence, the first event in replication is the translation of thenon-structural proteins needed to produce full length 40S (+)-sense RNA and 24S (+)-sense subgenomic RNA,via a (-)-sense RNA intermediate (Oker-Blom, 1984b). The 24S subgenomic RNA is required as a templatefor translation of the structural proteins. This message is capped, polyadenylated and from the 5’ end encodesfor: C, E2 and El (Figure 3). The molecular mechanism which controls the production of 24S mRNA forstructural protein translation, or full length 40S RNA for nucleocapsid packaging remains unclear.40 S (—11.000 b)1AA(A)—24 S (—3500 b)5l 1AA(A)3Ii translationp110 ‘NH2;- : COOH. I I—capsidC33KJ, processingenvelope—II IIE2 El30K 53K,, glycosylation ,E2a (47K) 58KE2b (42K)Figure 3. Strategy for the expression and processing of RV structural proteins from a 24S + sense RNA subgenomic fragment. (From: Oker-Blom, 1984b)C protein remains in the cytoplasm whereas the E2 and El proteins are transported to the endoplasmic lumenand processed as transmembrane proteins. Therefore it is believed that translation, proteolytic cleavage andpost-translational modifications occur simultaneously at the different sites of this polyprotein. A translocationsignal sequence at the COO - terminal of the C protein allows the entry of the polyprotein into the lumen of theER where a series of trans-membrane and signal sequences allow for the correct insertion of El and E2(illustrated in detail in figure 4).-8-A C E2 ElI I I INH** **Figure 4. Model for the Topogenesis of the Rubella Virus Polyprotein. A) The 110 kD polyprotein is shownwith the location of signal peptide sequences (white boxes) at the COOH termini of C and E2 and thetransmembrane anchor sequences of E2 and El (black boxes). The asterisks (**) indicate published N-terminussequences of El and E2 (Kalkkinen et a!., 1984). The polyprotein is not drawn to scale. B) Two cleavages bysignal peptidase (white arrow head) release the capsid and separate the E2 and El proteins. A third cleavagesite (black arrow head) has been proposed to occur in an arginine-rich region in the cytoplasmic tail of £2(Vidgren et al., 1987). (Figure B adapted from: McDonald, 1991).The membrane proteins are extensively post-translationally modified. Both El and E2 are acylated and Nglycosylated (Waxham and Wolinsky, 1985a; Hobman and Gillam, 1989). E2 is additionally glycosylated with0-linked saccharides (Lundstr 6m et al., 1991). Although El and E2 are co-translationally inserted into themembrane, their fates in intracellular transporting are quite different. E2 can be fully processed alone whereasEl is dependent on E2 for complete processing and for transport to the cell surface (Hobman, 1989).1. l.6.3.AssemblyDepending on the host cell, the RV envelope proteins may either congregate in the plasma membrane or in theGolgi apparatus membrane to await assembled nucleocapsid for the budding process (Bardeletti et al., 1979,Hobman, 1989). Capsid protein of RV differs from the C of aiphaviruses in that it does not contain auto-B capsidNcytoplasmER lumenE2-9-proteolytic capacity and it is therefore believed that the E2 signal peptide remains with the RV C protein(Suomalainen et al., 1990; McDonald et al., 1991). Although this explains the microscopic observations thatcapsid and assembling capsomere units are found in association with cellular membrane systems (Horzinek,1981), the C protein and the assembled nucleocapsid have complete hydrophobic properties under physiologicalpH conditions (Mauracher et al., 1991). In SV, the C protein specifically interacts with viral RNA sequences(Weiss et al., 1989) and the encapsidation process is thought to be catalyzed by such an RNA-protein bindingevent in RV also. The assembled nucleocapsids can be readily detected in the cytoplasm and seen to bud intoeither the Golgi or from the plasma membrane (Oshiro et al., 1969) thereby completing the virus life cycle.Capsid protein has recently been shown to also bind to ribosome subunits (Singh et al., 1991) and such bindingactivity could possibly be involved in the control of the transition between protein synthesis and virion assembly.1.2. Rubella Pathoaenesis and Patholov1.2.1.HistoryThe first description of a disease induced by RV infection was described as “R6theln” by a German physicianin the 18th century (in: Forbes, 1969), likely leading to the common name of “German Measles”. In 1866 theEnglish-speaking medical establishment (maybe because of the annoyance of having to pronounce an umlaut),fmally followed the lead of the Scottish physician H. Veale in renaming this exanthematous rash and febriledisease to “Rubella” (Veale, 1866). To complicate matters, this disease obtained a third English identifier,following the attempt to classify acute exanthematous diseases into “First” through “Sixth” disease; Rubella,henceforth was known also as “Third disease” (Marcy and Kibrick, 1972). The viral etiology of rubella wasconfirmed in 1938 by human to human transmission studies (Hiro and Tasaka, 1938), thereby fulfilling Koch’spostulates.1.2.2.Clinical and Immunological Features1.2.2. LRubellaa) Clinical FeaturesThe clinical features of this disease can range from a completely inapparent infection to the characteristicchildhood presentation of low grade fever, adenopathy, malaise and exanthem. The exanthematous rash evolves-10-rapidly and in most cases spreads from the head to the trunk, where by the second day it may appear as a pinpoint or reticular pattern. The rash usually disappears by day four causing no scarring. Other epithelial surfacessuch as the conjunctivae or the oro-pharyngeal region may also show signs of inflammation. In more severecases, the infection results in encephalitis and in rare cases (< 0.05%) severe encephalitis can have a fataloutcome (Center for Disease Control, 1975). In cases of post-pubertal infection, the disease generally has amore severe outcome. Especially in young women the disease is associated with articular complications rangingfrom acute arthralgia to chronic arthritis (Smith et al., 1987). This outcome will be discussed in more detail insection 1.3.4.. The common symptoms of Rubella can be readily confused with similar illnesses presenting witha maculopapular rash, malaise and adenopathy. In one particular study of patients having a rubelliform rash,adenopathy, generalized malaise and joint pain only 36% were confirmed by laboratory tests to have had rubella.The remaining patients were infected with parvovirus (7%) or had an unlcnown etiology (57%) (Shirley et al.,1987). Defmite diagnosis can therefore only be made following laboratory studies involving either virus isolationor serological analysis.Rubella is highly communicable with spread, most probably, occurring by droplet infection or direct mucosalcontact. Mucosal epithelial cells and submucosal lymphoid tissue serve as the initial infection site from wherethe virus spreads to regional lymph nodes. Rapid viral replication in affected lymph nodes leads to enlargementand tenderness which may start 5-10 days prior to the outbreak of a rash (Green et al., 1965). An incubationperiod of 7-9 days precedes the onset of viremia and shedding of the virus from nasopharyngeal secretions andstool (Heggie and Robbins, 1969). The exanthem commences at 16-2 1 days following initial exposure andcoincides with the appearance of IgG in the blood stream. As the rash onset coincides with the generation ofanti-RV IgG it has been hypothesised that the exanthem is caused by immune complex mediated vessel injury(Heggie and Robbins, 1969). The viremic phase quickly subsides with the mounting of the specific immuneresponse and in general virus can not be isolated from biological fluids one week after initiation of rash (Davieset a!., 1971). In cases of experimental RV infection or following vaccination these symptoms may occur up toa week earlier than natural infections (Anderson, 1949).b) Immunological FeaturesThe initial antibody response to RV infection or RV vaccination is the 1gM response, which in most cases is—11—102464-.-—.. -0rm,14 1 2 1 2 3 10I LJ IMonths YearsFigure 5. Clinical Features of Rubella. Patterns and kinetics of viral excretion, rash (shaded area) and antibodyresponses during primary Rubella. (HAl) Hemagglutination lithibiting antibody, (CF) Complement Fixingantibody, (rIgM) 1gM treated with reducing agent. (From: Cooper and Buimovici-Klein, 1985)specific to the El protein (Partanen et al., 1985). This response is transient, lasting on average for one month(see Fig. 5) but cases have been reported where 1gM responses are detected for periods of months or yearsfollowing initial Rubella infection (Al-Nakib et al., 1975). 1gM persistence is not necessarily correlated withspecific pathology but the occurrence of persisting 1gM can make it difficult to confidently time initial RVinfection by serological means. All remaining immunoglobulin classes are stimulated within weeks of infectionincluding IgG, IgD and IgE (Salonen et al., 1985). The IgA response is felt to be transient, restricted to IgAland restricted to C protein (Partanen et al., 1985; Stokes et al., 1986). However more recent studies have shownthat the serum IgA response may be long lasting and directed to all three proteins of RV (Chapter 3; Zhanget al., 1992). The IgG response is by far the dominant serological response and is directed to all three structuralproteins of RV. When analyzed by immunoprecipitation techniques it is observed that the majority of RVspecific IgG is directed to El with significantly lower levels being directed to E2 and C (Katow and Sugiura,1985, de Mazancourt et al., 1986). This holds true also if sera are analyzed by immunoblot or protein specificELISA (Zhang et al., 1992,Chaye et al., 1992). The RV specific IgG response is predominantly made up by theIgG 1 subclass with small amounts of IgG3 and IgG4 (Sarnesto et al., 1985).The levels of IgG and its subclasses correlate well with HAl titres (Stokes et al., 1986) and with neutralizing100RAELISA,4%..HAI.\76543211234 6 8 10 12Days Before Days After Onset of Rash-12-antibody titres (Stewart et a!. ,1967) and it has been assumed that these responses play a major role in viralclearance. The affinity of RV-specific IgG increases rapidly in the months following infection or vaccination(Hedman and Rousseau, 1989) and these biologically active antibodies are maintained indefmitely if infectionoccurs in childhood and contributes to the long term resistance to RV reinfection (Wolinsky, 1990). Circulatingimmune complexes (CIC) containing RV-specific antibodies and antigen are frequently detected followinginfection (Ziola et al., 1983).The cellular response to RV is vigorous and in healthy responders is maintained at low levels over anindefinite period (Buimovici-Klein et al., 1977). RV-specific cellular responses are demonstrable by lymphoproliferative responses, lymphocyte-mediated cytotoxicity and are MI{C restricted (Ilonen et al., 1986). Recentwork has shown that El shows the highest reactivity in RV protein specific lymphoproliferative responses inadult humans, followed by C protein with weakest reactivities measured to E2 (Chaye et al., 1992).The predominance of both anti-El B- and T-cell responses in RV specific immunity have led to the conclusionthat immunity to El is central to protection from viral reinfection. This is further supported by the almostexclusive distribution of neutralizing domains on the El protein.1.2.2. 2.Congenital Rubella Syndromea) Clinical FeaturesThe characteristically uneventful outcome of childhood rubella stands in marked contrast to the devastatingoutcome of in utero RV infection. The connection between matemal rubella in the first trimester of pregnancyand the high incidence of congenital malformations, especially cataract formation, was first recognized in 1941by an Australian ophthalmologist in the months following a Rubella epidemic (Gregg, 1941). Following thisinitial description, the teratogenic effects of this virus were quickly established and instigated the global effortfor vaccine development and production.Maternal RV viremia prior to conception is not associated with an infection risk to the fetus (Enders et al.,1988), however once gestation commences the risk of in utero infection becomes high, with few fetuses escapinginfection if maternal Rubella occurs in the first month of gestation (South and Sever, 1988). The rate of fetalinfection diminishes to an estimated 25% -30% following the first trimester (Miller et al., 1982). The severity-13-of birth defects and late onset sequelae strongly correlate with the gestational age of the fetus at infection. Ingeneral it is believed that the closer infection occurs to conception the more severe the outcome will be.Congenital malformations are expected if infection occurs prior to 16 weeks. Birth defects are rarely describedif infection occurs later than 17 weeks; late onset sequelae such as neurological deficits or endocrine disorders(hypothyroidism or diabetes mellitus) are however commonly described in congenitally infected patients and maybe attributed to RV exposure (Munro et al. 1987).RV readily infects placental tissue and virus can usually be isolated from this site (Alford et al., 1964). Duringearly gestation, the placenta is observed to contain scattered foci of necrotic trophoblast tissue as well as vascularendothelium (Tondury and Smith, 1966). The invasion of the virus into the feto-matemal circulation allows foran embolic mode of transmission. This can explain the disseminated infection of the fetus affecting almost everyorgan system (Bellanti et al., 1965). The mechanism by which the virus interferes with organogenesis is not clearbut several theories have been proposed to explain the observations of organ dysfunction and lowered cellnumbers in organs which otherwise appear anatomically normal. It has been suggested that virus can directlydamage cells or can persistently infect fetal cells and thereby interfere with the ontogeny of organs (Rawls andMelnick, 1966). A more indirect mechanism has been proposed to be mediated by soluble factors, secreted byRV infected cells, inhibiting regular mitotic division (Plotkin et al., 1967). More recently it has been describedthat cells, persistently infected with RV, do not react well to growth factors and produce little collagen incomparison to uninfected cells (Yoneda et al., 1986). The general observations of damaged larger vessels bothin the placenta and the CR5 fetus can explain lowered cranial size and birth weight as well as ischemic necrosisobserved in neural tissues of some CRS patients. Immune complex mediated injury may also contribute muchto the morbidity and mortality observed in perinatal and late onset sequelae. Inflammatory damage, rangingfrom acute neutrophil infiltration to chronic inflammatory lesions, is commonly described in lung or the brainat autopsy in CRS patients (Desmond et al., 1967). Increased loads of CIC, containing anti-RV IgG, have alsobeen described in CRS patients with active clinical complications and have been interpreted as being indicativefor the systemic, low grade replication of RV in these patients (Coyle et al., 1982). Table 2 lists some of thecommon clinical features of CRS.Viral persistence is one of the hallmarks of the CRS. Virus may be isolated from most organs at birth andRV is actively secreted in urine, stool and naso-pharyngeal secretions in more than 80% of CRS patients in their-14-Feature IncidenceaIntrauterine growth retardation (low birthweight) TCThrombocytopenic purpura TCHepatosplenomegaly TCMeningoencephalitis TCBone lesions (radiographic) TCLarge anterior tontanelle TCAdenopathy, generalized TUHepatitis TUCloudy cornea TUHemolytic anemia TUPneumoraa due to rubella TtJMyocarditis due to rubella TUDeafness. sensorineural PC (DU)Central language disorders PDCMental retardation PDCBehavioral disorders PDCSpastic diplegia PCPatent ductus arteriosus PCPulmonic Stenosis PC (?DU)Cataract (and microphthalmia) PCRetinopathy PCGlaucoma PU (DU)Severe myopia PDUInguinal hernia PUCryptorchidism PUa Transient (T): permarwnt (P); developmental (D); common(C); or uncommon (U).Table 2. Clinical Features of Congenital Rubella (From: Cooper and Buimovici-Klein, 1985).first month of life (See Fig. 6). Thereafter, the rate of viral excretion diminishes and is only rarely detected oncethe patient has reached the age of 2 years. However it is felt that RV persists in organs of most CRS patientsas virus has been isolated from patients 20 years and older (Menser et al., 1967; Weil et al., 1975). With theadvent of PCR technology a more accurate estimate of the incidence and preferred organ distribution ofpersisting virus in this patient group may be forthcoming.Persistence of RV in these infants has remained a paradox, as this virus readily resists clearance despite thepresence of maternally acquired immunity as well as the autologous anti-RV 1gM response. As CRS patientsdo not exhibit generalized immunosuppression after infancy and are capable of responding to the routinepediatric vaccines it has been proposed that a form of RV-specific tolerance exists. In several animal modelsit has been shown that in utero infection lead to immunological tolerance resulting in the persistence of the virus.-15-I009071/85’84%80705OI8I62%6050‘z 4026/8033%30wC-,20II/s8lrh100/20’O%C0-I -4 5-8 9-12 13-20 /MONTHS YEARSAGE Of INrANTS AND CHILDRENFigure 6: Incidence of virus shedding by age in CRS patients (Cooper and Krugman, 1967).Examples such as LCMV in mice (Traub, 1938), Borna agent in rats (Hirano et al., 1983) and Border diseasevirus (BDV) in sheep (Barlow, 1983), have described that infection during the early ontogeny of the immune-system can lead to inmunologic non-responsiveness of the fetus leading to a lifelong, low grade persistentinfection of the individual. Immunological tolerance to these agents is rarely complete but rather a state of splittolerance is established. In the case of LCMV or the Borna agent, cytotoxic T cells (Tc) and delayed typehypersensitivity (DTH) reactions to viral antigens are not detectable. However helper T cell (Th) cell responsesare believed to remain intact as IgG specific to the three LCMV structural proteins is produced; this hypothesisis supported by the observation that adult athymic nude mice, infected with LCMV, only respond with a transient1gM response (Buchmeier et al., 1980). However the IgG antibody in the tolerant animals is of low affinity andshows weak or no in vitro neutralization activity. The similarities between the immunity of LCMV tolerized miceand the CRS patients will be further discussed in section 1 .2.2.2.c.Other explanations for viral persistence mayinclude cryptic viral infections in immunologically preferred sites such as lens, salivary glands, cartilage or neuraltissue. Virus might re-emerge from these sites following the waning of passively acquired IgG immunity. Amore plausible explanation is that the presence of in vitro neutralizing antibody, transferred from mother tofetus, may be of no consequence for the replication of virus in vivo. This suggests that the cytotoxic responses-16-are most important in protective immunity and as the cellular response is not transferred from mother to fetus,the viral infection can not be cleared until an autologous cellular responses is mounted. Recent evidence hasshown (Chaye et al., 1992) that viral-specific T cell responses to the El protein are significantly reduced in CRSpatients and that this perturbation in the viral specific T cell repertoire may lead to deficiency in viral clearance.b) Immunological FeaturesThe congenitally infected fetus actively produces RV-specific 1gM at 15 to 20 weeks of gestation (Daffos et al.,1984). A positive RV 1gM titre in cord blood is therefore interpreted as serologic evidence for intrauterineinfection with RV. However this does not correlate with the development of birth defects nor does the absenceof virus-specific 1gM preclude this syndrome (Enders and Jonatha, 1985). The newborn carries passivelytransmitted maternal IgG, which in the first year will be removed at an average half-life rate of 21 days(Hamilton, 1987). IgG of all subclasses can cross the placenta (McNabb et al., 1986) and CRS patients or theircord blood normally test positive for HAl and neutralizing IgG, reflecting the passively acquired maternal IgG(Sato et al., 1979). Nevertheless neither the maternal IgG nor colostrum/breast milk derived IgA is capable ofeliminating viral replication. Over the first year, the infants mount their own RV-specific antibody response,switching from the endogenous 1gM response to IgG and IgA. Because of the reported long term persistenceof RV, it has long been speculated that a form of immunological tolerance must exist to allow viral persistence.This RV-specific tolerance can not be complete as CRS patients produce a RV-specific IgG to both envelopeand capsid proteins and by inference must therefore also possess Th cell responses. Much controversy existsin the literature on the quantitative levels and protein-specific responses of both the humoral and cellular RVresponses in the CRS patients. Initially it was observed that CRS patients quickly lost HAl antibodies in theirfirst year of life , indicating a specific loss of anti-El antibodies (Cooper et al., 1971; Ueda et al., 1987). It wasthen proposed that tolerance in RV patients was indicated by their inability to produce IgG to RV El, whilemaintaining reactivity to E2 and C. A 1985 study comparing the protein-specific antibody reactivity of CRS- andcontrol patients using immunoprecipitation technique showed that CRS patients had significantly lower levelsof El- and elevated levels of E2-specific IgG (Katow and Sugiura, 1985), although these fmdings were notconfirmed by others (de Mezancourt et al., 1986). The affinity of antibody directed to RV has been found tobe low in CRS children, in comparison to age-matched RV sero-positive controls (Fitzgerald et al., 1988). Both-17-lowered affinity and non-responsiveness to key proteins or epitopes may explain viral persistence by deficientantibody mediated viral clearance mechanisms.The study of RV-specific T-cell responses, measured by3H-thymidine uptake or interferon release, has beendescribed to be selectively reduced in CRS patients (Buimovici-Klein and Cooper, 1985). Moreover, theseauthors report that the degree of tolerization is dependent on the gestational age at infection, with infection inthe first 16 weeks leading to the most pronounced reduction in T cell responses to whole RV. Others havereported that CRS patients have reduced ratios of CD4 + /CD8 + cells, as well as an overall reduction ofactivated Th cells as indicated by significantly lower Ia positive cells than age-matched control patients(Rabinowe et al., 1986). Although suppression of RV-specific responses are suggested in CRS patients, arudimentary Th cell response must exist to support anti-RV IgG production to the viral antigens. Recentevidence from studies using recombinant RV proteins or RV peptides in lymphoproliferative assays, suggeststhat only responses to the El protein are selectively suppressed, whereas responses to the E2 and C protein arenormal or elevated in CRS patient groups (Chaye et al., 1992; Ou et al. 1992). This thesis will propose a modelwhich explains the selective loss of anti-El T cells in the presence of IgG specific to all three RV proteins forthe CRS patient group.1.2.3 .Tolerance following Intrauterine Virus InfectionBoth RNA and DNA viruses have developed a variety of means of producing persistent infections in their hosts(Table 3). Indeed it can be speculated that the earliest viral pathogens of the emerging human species had tobe capable of inducing persistent infections as the human hosts were distributed in small pockets of populationwhich rarely intermingled. This is supported by epideniiological studies on isolated populations in the Amazon,where it has been shown that the dominant viral infections are caused by persistently infecting viral species(Black et al., 1970). Only with the onset of organized agriculture did human populations reach basic communitysizes which were large enough to support viruses which do not establish persistence in the host (ie: influenza,smallpox) (Fenner et a!, 1974). Immunological tolerance to a virus (defined as the virus-specific refractorinessin the immune response following prior exposure to this virus) may be one mechanism which viruses haveevolved to establish persistence in the vertebrate host. Animal models such as the neonatally LCMV infected-18-Mechanisms of Viral PersistenceAvoiding Immunological Surveillance1) Lowering or avoiding the presentation of antigen on MHC I and II moleculesa) Down-regulation by direct virus infection [Adenovirus (Tanaka et al., 1985)].b) Infection of cells devoid of IvIHC-I, eg: neural cells [HSV I (Fitzpatrick et al., 1991)].2) Infection of cellular constituents of the immune system [HIV (Hirsch and Curran, 1990)].3) Generalized Immunosuppression [Measles virus (McChesney and Oldstone, 1987), FUV (Hirsch andCurran, 1990)].4) Induction of Tolerance [Lymphocytic Choriomeningitis virus (LCMV) in mice (Buchmeier et al., 1980);Rubella virus (Coyle et al., 1982; Mauracher et al., 1992c)].5) Infection of an immunologically privileged site [Papilloma viruses (Carson et al., 1986)1.Alter Replication and Transcription of Virus1) Production of Defective Interfering Particles [Adenovirus, Rhabdovirus (Huang, 1988)].2) Generation of immunological variants [liv (Ruscher et al., 1988)].3) Loss of viral protein from the surface of the infected cell.a) Capping and antigen modulation by anti-viral IgG [Measles (Fujinanii and Oldstone, 1984)].b) Budding of virion into Golgi or ER [HIV (Narayan et al., 1988); Rubella (Hobman, 1989)].4) Insertion of genomic information, with transient expression (latency) [Herpes viridae (Fitzpatrick andBielefeldt, 1991)].Other Mechanisms1) Poor antigenicity [Scrapie and other agents causing spongiform encephalopathies (Chesebro, 1985)].2) Continuous infection of host or population by vector mediated infection from exogenous pool or zoonoticinfections [Dengue virus (Burke et al., 1987)].Table 3. Mechanisms of Viral Persistence. For each mechanism, an example of a human or other vertebrateviris is given. Potential mechanisms for RV persistence are listed, however this virus may use other mechanismsin its persistence.mouse, which leads to a life-long infection (Traub, 1938), have led to the concept of immunological tolerance(Bumet and Fenner, 1949) and the formulation of Burnet’s clonal selection theory. These have also inspiredextensive studies of tolerance induction by LCMV infection in the mouse model as well as in other virus/animal-19-models (Nash, 1985). The persistently LCMV-infected mouse was initially reported to be completely tolerantto the virus, but it was later reported that these animals were capable of producing antibody to all three LCMVproteins. These however were found as deposited IC in spleen and kidney (Oldstone and Dixon, 1969) wherethey could induce IC-mediated immunopathology. These virus-specific IgGs were found to be non-neutralizingai-id of low functional affinity (Buchmeier et a!., 1980). The Tc and DTH responses are absent in these animals.Although studies on in utero infected animal models has clarified many concepts in immunological tolerance itwas the introduction of the transgenic mouse model which allowed a more detailed interpretation of toleranceinduction in both B and T cells. With the controlled expression of transgenic proteins in specific organs the roleof both periferal and central tolerance could be investigated, as well as the time point in lymphocyte ontogenywhen tolerization occurred. Once it was recognized that the Th cell was an essential regulator of B cellprogression, the issue was raised whether B cell tolerance was essential for self-education or if tolerance of theT cell responses was sufficient in the regulation of antibody responses. Mice which have been made doubletransgenic for hen egg lysozyme (HEL) and for high affinity anti-HEL antibody have provided evidence for Bcell anergy (Goodnow et al., 1990), independent of T cell control. The model using MHC transgenic mice hasshown that exposure of the pre-B cell to high levels of high density toleragen can lead to clonal B-cell deletionin the bone marrow (Nemazee and BiYrki, 1989). On the other hand studies using a peripherally expressed,soluble transgene product (human insulin) have shown that B cell anergy does not occur but that antibody non-responsiveness is solely due to peripheral T cell tolerance (Whiteley and Kapp, 1989). Recent work using atransgenic G membrane protein of Vesicular Stomatitis virus (VSV) has also provided supportive evidence thatB cell tolerance to a monovalent, periferally expressed protein is regulated by Th cells (Zinkernagel et al., 1990).In these transgenic mice B cell tolerance remained complete when animals were challenged with recombinantG protein but tolerance was broken if whole VSV was used as an immunogen. Presumably the anergic anti-Gprotein B cells could be stimulated into IgG production by T cells specific to other viral proteins. Toleragenconcentration, tissue distribution and multivalency clearly play a key role in B cell tolerance, with exposure ofthe immature B cell to multivalent antigens (ie: MHC) leading to clonal deletion. In encountering solublemonomeric proteins or membrane proteins of low copy number clonal anergy may occur but it now seemsevident that the fate of the B cell is controlled by the underlying Th cells repertoire (Nossal, 1991). Suppressor-20-T cells in B cell tolerance (Theopold and Köhler, 1990) and the anti-idiotype network theory (Jeme, 1974) mayalso play a role in the regulation of self-recognition but are beyond the scope of this introduction.Many similarities exist between the tolerant state of mice following neonatal or intrauterine LCMV exposureand humans infected with RV in utero during the first trimester of gestation. In both cases there is an IgGresponse which is of low affinity and which lack in vitro neutralizing activity. IC formation and depositionfollowed by IC-mediated inflammation has been described in both models. Whether CRS patients show acomplete lack of virus-specific Tc and DTH reactivity, as is the case in the murine model, remains speculative.Recent data suggests that lymphoproliferative responses to the E2 and C protein of RV remain intact in CRSpatients and it is likely that Te responses to the these minor T cell antigens are present (Chaye et a!., 1992).It is proposed that tolerance is induced in CRS patients to the immunodominant El protein, rather than to allviral proteins as has been reported in the LCMV model.1.3 Rubella Vaccine1.3.1.HistoryFollowing the descriptions of the devastating outcome of congenital rubella (Gregg, 1941), a worldwide effortwas undertalcen to develop measures to prevent fetal RV infection. Initially, gamma globulin was administeredto pregnant women recently exposed to rubella (described in Chase, 1984) but it was not until 1962 when theetiological agent of rubella was finally isolated by two independent groups (Weller and Neva, 1962; Parkmanet al., 1962) that vaccine development could be initiated. Between 1965 and 1967, several live attenuated RVstrains were developed and tested as vaccines. Of these HPV 77/DE 5 and RA 27/3 have been licensed inEurope and North America. Currently the RA 27/3 strain of RV which is passaged on human diploidfibroblasts (Plotkin et al., 1969) is the commonly used vaccine strain in Canada and the USA. This vaccine wasnot licensed for use in North America until 1979 but has been used in many European countries from 1969onward. With the development of less virulent vaccine strains the public health offices of North Americachanged their recommended vaccine scheduling. The USA health policy in 1969 recommended universalvaccination of children at age one year and a one time vaccination effort of children up to age 12 (Ilimnan etal., 1983). This vaccine policy was in effect until the late 1970s when it became clear that the incidence of CRS-21-30 Vaccwe Licensed — Rubella, lolalr—5. —— Rubella. aIS year olds28 N 14Congenilal rubella+26 \24 \ 1222 \20 100ag1812 6c) (I,a a10 s_I0a-8 /‘“ 4/6 —.. •_.—‘ S. \5/4.5’,.2-.-—2 S.-0 01966 67 ‘68 •69 70 ‘7 172’7374 75767778 79 ‘80YearFigure 7: Incidence of reported rubella and CRS cases in the USA, 1966-8 1 (Hinman et a!., 1983).and reported rubella cases in females of child bearing age had not dropped to levels that were initially expected(Figure 7). In 1978-1979 it then became common practice to immunize females of child bearing age and toadvise post partum immunization of RV seronegative mothers (Preblud, 1986). This change in vaccine policyled to a significant drop in reported cases of CRS and has remained vaccine policy in both the USA and Canada.1.3.2.Biology and ImmunologyThe mechanism of RV attenuation has remained unclear although both wild type strains and attenuated vaccinestrains have been sequenced (Zheng et al., 1989). Attenuated strains vary minimally in protein sequence, withonly 5 amino acid substitutions occurring in the structural polyprotein of the HPV 77 RV vaccine straincompared to wild type isolates. The low virulence and slow replication rates of vaccine strains are thought tobe due to a deficit in cell attachment and entry (Nakhasi et al., 1989), although other mechanisms of attenuation(eg: host protease dependence) can not be ruled out.Immunity to vaccine strain virus is believed to be life long (Plotkin et al., 1973). After MMR vaccination at12 months or older, 95% of children seroconvert by HAl technique and this immunity has shown to beprotective for at least 15 years in 90% of vaccinees (Robinson et al., 1982).-22-1.3.3.Side Effects of RV VaccinationControversy over the safety of rubella vaccine has only recently become of public concern. In the early seventies,neuropathies (Kilroy et al., 1970) and transient arthritis (Spruance and Smith, 1971) were describe in childrenreceiving the HPV 77/DE 5 strain and led to the withdrawal of this strain and the licensing of the RA 27/3strain in the USA and Canada (in Howson et al., 1992). Although the RA 27/3 strain has been shown to causeminimal side effects in children (Polk et al., 1982) it is still associated with significant adverse effects inadolescent and adult female vaccinees. Acute arthritis is estimated to occur in 13% -15% of adult RA 27/3vaccine recipients (Reviewed in: Howson and Fineberg, 1992). Case reports of severe and chronic arthritis havealso been reported following the administration of RA 27/3 in adult females (Tingle et al., 1986). In the prelicensure trials of the RA 27/3 vaccine this strain was determined to be safe for use in children. Subsequentlythese results were interpreted to hold true for adults as well, leading to the extension of vaccine administrationto women of child bearing age without further evaluation of potential side effects (Tingle, 1990). Ongoingdouble blinded, randomized and placebo-controlled clinical trials of rubella vaccine in adult seronegative womenat the BC’s Children’s Hospital Vaccine Evaluation Centre will provide a more definitive estimate of theincidence of acute and chronic forms of arthritis following RV vaccination.The pathogenesis of Rubella Associated Arthritis (RAA) has remained unresolved (Ford et al., 1986).Rubella can be isolated from synovial fluid and in peripheral lymphocytes in individuals with RAA, in the yearsfollowing RV infection or immunization (Ogra et al., 1975; Chantler et al., 1985). Such fmdings have led to theproposal that RV can persist in the cells of the synovial fluid or the synovial membrane. RAA is mostcommonly associated with peripheral joints (Sauters and Utsinger, 1978) which are at temperatures of 5 #- 6 #Cbelow the body’s core temperature. This is of significance in light of reports that RV readily establishespersistent infections of synovial cell cultures at 32#C but not at 36 #C (Cunningham and Fraser, 1985). Recentlyothers have shown that wild type and vaccine strains of RV replicate to different extents in human synovial celllines or in human synovial membrane organ cultures (Mild and Chantler, 1992). They showed that strains whichhave been reported to have a higher incidence of RAA are also characterised by a high degree ofsynoviotropism. Although it is likely that RV may persist in local tissues it remains unclear if this non-lytic virusis sufficiently virulent to cause direct damage in the joint or whether persistent RV antigen is connected withthe initiation of immunopathology. It thus remains an enigma why some individuals mount a response which-23-leads to rapid viral clearance and uncomplicated outcomes in Rubella while others, especially adolescent andadult females, mount immune responses which may not clear RV in localized organs and lead to thedevelopment of acute or even chronic arthritis.The pathogenesis of infectious arthritis has been summarized as “..a chronic T-lymphocyte - and macrophagedependent response to foreign or autoantigens present in the synovial tissue. “(Decker et a!., 1984), however thequestion remains what initially attracts these cells to the synovium. A role of CIC and their deposition in thesynovial space has maintained itself as an attractive hypothesis for involvement in the pathogenesis of RAA aswell as in the manifestations of other forms of infectious arthritis (Inman et a!., 1987). IC are involved ininflammatory responses of CRS patients (Tardieu et al., 1980) and are most likely responsible for the vesseldamage which presents as the rubelliform rash in acute Rubella (Heggie and Robbins, 1969). It is feasible thatRV antibody and low grade locally persisting viral antigen may result in IC deposition and lead to the initiationof an inflammatory response. Studies regarding the presence of immune complexes have shown that levels ofcic in RAA patients are not significantly increased in comparison to healthy Rubella patients (Singh et a!.,1986), however others have shown that cic containing anti-RV IgG are increased following RV vaccination(Coyle et al., 1982) but this study did not compare anti-RV containing CIC in healthy and RAA patients. Astudy investigating the levels of CIC containing anti-RV IgG or RV antigen as well as IC in the joint space mayclaril’ the role of IC-mediate injury in RAA.Reactivity of the virus-specific immunity with self-antigens has been proposed (Lund and Chantler, 1991),describing cross-reactivity of rubella specific epitopes with an unidentified protein derived from synovialepithelium. The hypothesis of molecular mimicry remains of interest and studies showing cross-reactivity of viralspecific T - and B-cells may shed further light on the pathogenesis of RAA.A recent model for the pathogenesis of RA has involved the role of undefmed infectious agents assuperantigens (Paliard et a!., 1991), and the role of RV in this context is being currently investigated.Interestingly the RV capsid molecule shares structural similarities with the well defmed S. aureus enterotoxinsuperantigen (Marrack and Kappler, 1990), in that both contain an 8 membered b-pleated barrel as their corestructure.Evaluation of the proposed immunopathological mechanisms in RAA requires a good understanding of the-24-“normal” adult immune response in order to be able to define differences in the immune responses of RAApatients which may be involved in the induction of the inflammatory processes. Furthermore similarities mayexist in the immune repertoire of CRS- and RAA patients as both patients group may develop inefficientimmune responses which lead to viral persistence.1.3.4.Viral Vaccine EfficacyThe recent controversy in the public media and medical literature (reviewed in: Howson et al., 1992) over RVvaccine and its induction of infrequent debilitating side-effects, has obscured the benefit that this vaccineprogram has brought to the public. The incidence of CRS in the USA has dropped from 106 cases in 1979 to20 cases in 1985 with a continuing downward trend, so that CRS is hoped to be on the verge of elimination inNorth-America (Cochi et al., 1989). In the case of seronegative females, it should be remembered that theincidence ofjoint-manifestations following RA 27/3 administration is significantly lower than following wild RVinfection, to which these vaccinees would have been susceptible (Tingle, personal communication).1.4. Laboratory Diaanosis of Rubella Virus InfectionIn order to initiate the comparative study on the humoral immune responses to RV and RV proteins incongenitally- and post natally infected individuals, new assays had to be developed and established assays hadto be adapted for application to the individual RV antigens. This section will review the most commonly usedassays for the detection of RV or RV-specific immunity.1.4. 1.Virus IsolationThe defmitive diagnosis of RV infection is achieved by the isolation of this virus by tissue culture techniques.The virus will infect a broad range of cell lines. Virus samples can be isolated from pharyngeal swabs ornasopharyngeal secretions at the time of onset of the exanthem. In the congenitally infected infant, virus isreadily isolated from stool, urine, nasopharynx, blood and cerebrospinal fluid. Virus can also be isolated fromblood, synovial and breast milk lymphocytes (Chantler and Tingle, 1982), but these preparations do not yieldvirus in all cases and are not used for routine clinical diagnosis. Excretions or cell preparations are mostcommonly taken up in solutions of physiological salt solutions containing 1% BSA or 5-10% fetal calf serum and-25-transferred immediately onto the cell line of choice for viral propagation. Two commonly used cell lines, AfricanGreen Monkey kidney cells (Vero) and Baby Hamster kidney cells (BHK-12) are used for growing high yieldvirus. The virus causes little cytopathic effect in these cells and virions can be readily isolated from tissue culturesupernatants following the second day of infection. Other cell lines such as primary African Green Monkeykidney cells have been used to detect RV by interference with echovirus 11 replication and Rabbit kidney cells(RK-13) are extensively used for RV titre determination as this cell line exhibits a detectable cytopathic effect(Nawa, 1979). With the advent of sensitive molecular probing assays, direct detection of RV genome mayreplace viral isolation by tissue culture. Reports of RNA hybridization for the detection of RV RNA inchorionic villus samples (ITo-Terry et al., 1986) and the use of PCR technology in direct RV detection(Eggerding et al., 1991) indicates the direction of future virus detection.Serological testing for antigen specific antibody responses, remains the primary diagnostic means for RVinfection. Serological techniques are independent of the narrow time interval when virus can be isolated andthereforee can be used retrospectively to confirm and time RV infection. The following paragraphs willintroduce the serological techniques which have found application in RV serology and some of the problemsassociated with their application and interpretation.1.4.2.Neutralization AssayThe neutralization test (NT) is one of the oldest assays in viral diagnosis and serology, dating back to the earlywork on vaccinia virus (Stemberg, 1898 reviewed in: Lennette et al., 1989). Stemberg observed that certain serawere capable of rendering vaccinia virus non-infective to a susceptible host, and termed this activity“neutralization”. This serologic test has been the “goldstandard” against which other serological techniques havebeen measured. In rubella serology this assay has been in use since the isolation of this agent and directlymeasures the individuals capacity to neutralize RV infection in vitro. However because NTs are labour intensivethey have not found much application in routine diagnosis and are mainly employed in research laboratories forthe standardization of new serodiagnostic tools (Steward et al., 1967) or in the characterization of RV specificmonoclonal antibodies (Waxham and Wolinsky, 1985b; Brush, 1989). In the case of RV neutralization it wassoon realized that serum components other than immunoglobulins had the capacity of neutralizing the virus.-26-These have been determined to be complement, b-Iipoproteins (Clarke and Cassals, 1958) and phospholipids(Mastromarino et al., 1990). Such non-specific inhibitors have to be removed by pretreatment of sera with 57°Cincubation followed by kaolin treatment or by precipitation with manganese chioride/heparin (Kawano andMinamishima, 1987) prior to the determination of neutralization titres.1.4.3.Hemagglutination Inhibition (HAl) AssayMany viruses from varied families are capable of agglutinating erythrocytes from a variety of species. Thishemagglutination (HA) phenomenon forms the basis for the serodiagnostic test of antibodies which bind to thevirus and thereby inhibit HA (HAl). RV was observed to bind to day-old chick erythrocytes as well as whitegoose erythrocytes and this interaction can be selectively inhibited by antibodies as well as the non-specificinhibitors described above. The HAl assay had been extensively tested and compared for sensitivity andspecificity to the NT (Lennette et a!., 1967; Field et al., 1967) and has been found to show a good correlationwith NT in human test populations. The standard protocol (U.S. Dept. of Health, Education and Welfare, 1970)recommended pretreatment of sera with manganese chioride/heparin and adult chicken erythrocytes for theremoval of non-immunoglobulin inhibitors of HA and has set a HAl titre of 1:8 as the cut-off for protectiveimmunity. The HA! assay will provide results in one day and demands a fraction of the costs of the NT;therefore HAT was quickly adapted by most laboratories as the mainstay of rubella serology from the mid 1960sto 1980s, and provided the reference standard to which novel immunoassays had to be compared (Herrmann,1985). The molecular mechanism of the HA interaction is not completely understood in that the receptor onthe erythrocyte is not defined. The HA activity on the virion on the other hand has been studied extensively.HA activity resides on the El protein of RV and is dependent on intact conformation as well as completeglycosylation (Qui and Gillam, 1992). Binding sites for HAl antibodies have been mapped to a 40 amino acidregion on El. These are in close proximity to the defined neutralizing epitopes but do not correlate in all cases(Terry et a!., 1988; Waxham and Wolinsky l985a).1.4.4.Enzyme-Linkedlinmunoassays (EIA)Enzyme-linked immunoassays have found much application in serodiagnosis since the large scale availability ofenzyme-conjugated monoclonal and polyclonal antibodies directed to immunoglobulins first described in 1969-27-Figure 8. Hemagglutination-Inhibition (HAl) Test. Erythrocyte sedimentation [ A] correlates to the presenceof antibodies which have bound to El protein and inhibited the cross-linking of cells by the virion.Hemagglutination [ ©] means the absence or the diluting out of antibodies, as the virus exerts its agglutinationproperties. Each row represents a two-fold serum dilution from 1:8 to 1:512. Top row illustrates a negativeserum, the remaining three are positive with titres of 1:64,32 and 16 respectively (From: Lennette et al., 1989).(Avrameas, 1969; Engvall and Perlmann, 1971). Conjugation procedures and substrate systems have improveddramatically in the last 15 years and have led to the large scale acceptance of this technique. Althoughfluorescent- and radiolabelled immunoassays find application in specialized fields, the enzyme conjugates, mainlyalkaline phosphatase and horseradish peroxidase, have emerged as the most versatile and applicable toimmunodetection and diagnosis (Lennette et al., 1989). For RV serology in most clinical and researchlaboratories, EIAs have replaced the older HA! and NT assays because of the increased sensitivity and economyof the EIA (Steece et al., 1984).The two following paragraphs will briefly review two applications of RV specific EIA which are central to thisthesis: the enzyme-linked immunosorbent assay (ELISA) and Western blot.1.4.4.1 Enzyme-Linked linmunosorbent Assay (ELISA)The use of ELISA in RV serodiagnosis was initially described in 1977 (Bidwell et al., 1977). By the early 1980’sit had become the main laboratory diagnostic test for rubella. As this test allows the determination of levelsof all immunoglobulin classes, it lends itself to automation and can be readily scaled up to screen largepopulations (Leinikki et al, 1978; Buimovici-Klein, 1980). A world-wide serum reference system has allowedfor the uniform and standardized interpretation of ELISA results (WHO, 1971). A good correlation wasobserved between ELISA seropositivity and positive HAl titres (Shekarchi et al., l981;Herrmann. 1985: Stokes-28-et al., 1986) in normal patient populations while others have found this correlation to be poor in CRS patients(Hancock et al., 1986). The commercially available whole RV EUSAs measure antibodies directed mainly tosurface RV epitopes of the envelope proteins and do not necessarily measure antibodies which are functionallysignificant for in vitro neutralization or which provide the host with protective immunity. This stands in contrastto functional antibody tests, such as HAl and NT, which measure antibody to a narrow range of epitopes on theEl protein.1.4.4. 2.Westem BlottingThe electrophoretic transfer of proteins onto nitrocellulose from SDS-PAGE gels and the detection of proteinby enzyme-conjugated antibody was first described in 1979 (Towbin et al., 1979). Although Western blottingmost commonly employes enzyme-conjugated detection antibodies (immunoblot) the use of flourescingantibodies have come into use over the last years. In the case of RV serology, Western blotting allows thefurther dissection of the humoral immune response by determining the protein specificity of immunoglobulins.However disadvantages inherent to this technique (ie: protein denaturation, long processing time) have givenit few applications in large scale serology, with the exception of AIDS serology (Steckelberg and Cockerill, 1988),so that this technique has remained a tool largely used in research laboratories.Serological techniques for the analysis of Rubella immunity have evolved as a consequence of the demand forassays capable of rapidly, repetitively and cost-effectively handling large numbers of samples. Today, ELJSAshave filled this demand and provide a sensitive and specific test for the presence of anti-RV imniunoglobulins.These assays are therefore of use in the diagnosis of previous virus exposure - but do not provide a final answeras to whether the detected immune response will protect an individual from reinfection. This problem isillustrated well by CRS patients who test strongly positive on RV EUSA but remain susceptible to reinfection(Cooper et al., 1971).1.4.5.Affinity AssaysThe above discussed RV serology assays have been developed to measure RV- or RV protein specificity of-29-immunoglobulin classes, their quantitation and to some extent their biological function but provide limitedinformation about the affmity of these iminunoglobulins. The high affmity of the immunoglobulin molecule toits antigen is an important functional parameter and is ultimately involved with antigen-specificity as well as thebiological activity of the molecule (Table 4). As the terms affinity and avidity will be used extensively in the textand have often been used interchangeably in the literature, the brief defmitions below will help in theclarification of these terms. The term “Affmity”describes the quantitative interaction of hapten and antibody.The Law of Mass Action can be applied to this interaction and the stability of the antibody:hapten complex canbe expressed and experimentally defined as the association equilibrium constant, K. For an accurateexperimental determination of the K, both antigen and antibody should be present in pure and homogeneousform. The determination of K is therefore most precise in a system involving monoclonal antibodies interactingwith monovalent or haptenic antigens (Steward and Steensgaard, 1983). The term avidity or “functional affinity”is used to qualitatively describe the strength of antigen: antibody interaction. Although directly dependent onthe affinity of the antigen binding site with the antigen, it is strongly influenced by antigen valence andimmunoglobulin class. This can be best explained by a set of hypothetical IgG and 1gM molecules with identicalantigen binding sites for a multivalent antigen. Both molecules have identical affinities however the 1gMmolecule will bind more avidly as it can combine with more sites on the antigen.It has been realized for many years that the affinity of antibodies progressively increases followingimmunization and this phenomenon has been termed affinity maturation. The mechanism by which such anincrease of the average affinity occurs in response to immunologic challenge has received much attention andseveral underlying molecular mechanisms have found acceptance: somatic mutations (Milstein, 1987), antigenicselection (Allen et al., 1987) and a unifring hypothesis, combining the latter two and proposing hypermutationof the IgG V region on a DNA level during the G0 phase prior to B-cell progression (Manser, 1990).The affinity maturation of anti-RV IgG responses following primary rubella infection occurs over a three tofour month period (Hedman and Rousseau, 1989), with antibody affmity increasing by an estimated 100 fold.This range of increase in affmity is supported by studies in rabbits using DNP-lysine antigen, indicating that IgGfrom the acute response rose in affinity from 5x10 to 108 lM’ in the months following antigenic challenge(Eisen and Siskind, 1964). The well defined increase in the affinity of anti-RV IgG has been recently beenexploited to determine the time point when RV infection occurred (Hedman et al., 1989). This is of significant-30-Reaction ReferencePassive hemagglutination (Levine and Levytska, 1967)Complement fixation (Fauci et al., 1970)Hemolysis (Warner and Ovary, 1970)Immune elimination of antigen (Alpers et al., 1972)Virus neutralization (Blank et al., 1972)Damage to DNP-sensitized liposomes (Six et a!., 1973)Protection against bacterial infections (Ahlstedt et al., 1974)Blocking antibodies (Adkinson et al., 1979)Hemolysis typing (Hedman et al., 1989)Table 4: Biological Reactions in which High Affmity Antibodies are Superior to Low Affinity Antibodies.diagnostic importance in determining intrauterine exposure if recent RV infection during early pregnancy issuspected as the determination of affinity will provide a good estimate if Rubella occurred before or afterconception.Antibody affmity considerations are also of importance in the study of immunological tolerance. The LCMVmouse model of tolerance was initially described to show complete immunological tolerance following neonatalvirus inoculation (Traub, 1938), but was later shown to be a form of split tolerance. Cytotoxic responses werelacking in these animals (explaining persistent virus replication) but IgG responses to all three viral structuralproteins were present. In most animals LCMV-specific antibodies were found as CIC or IC deposited in thereticuloendothelial system or in the kidneys. These antibodies were of low affinity in comparison to those foundin adult responding mice and had no in vitro neutralizing activity. It remains unclear if the decrease in antibodyaffinity during immunological tolerance is due to preferential deletion of high affinity clones or if the lack of Thcell results in inefficient stimulation of B-cell clones in the germinal centres. However recent evidence indicates-31-that high affinity responders to self or toleragen are selectively purged (Nossal, 1991). Predominance of lowaffinity responses to a replicating antigen is therefore an indication that split immunological tolerance hasoccurred.A wide range of methods are available for the determination of antibody affinity. Equilibrium dialysis andanimonium sulphate precipitation have found much application in studies using monoclonal antibodies but areof little use for the determination of functional affinities of sera to complex mixtures of antigens such as theRubella virion. Two methods will be introduced here which will be extensively described in the body of thethesis.1.4.5.1 .Chaotropic Elution AssaysAntigen-antibody interactions can be disrupted by exposure to pH extremes, chaotropic agents and detergents.Studies on immune complexes described that low affmity antibodies could be readily dissociated by chaotropicagents, whereas high affmity interactions were resistant to these conditions and could only be dissociated byconditions leading to the denaturation of both antigen and antibodies (Hogben et al., 1986). Protein denaturantscould therefore be used to selectively disrupt the binding of low affmity antibodies by choosing the reagentconcentration and the exposure time of the immune complex to the denaturant. Urea acts as a denaturant byinterfering with hydrophobic interactions of polypeptide chains (Kamoun, 1988) and exposure of immunecomplexes for short periods to 8M urea, or other denaturants such as 4M isothiocyanate, leads to selectiveelution of low affinity antibodies from solid phase bound immune complexes. This can provide a measurementof the average affinity of serum antibodies by calculating the amount of total immunoglobulin which haveresisted the chaotropic elution (elution ratio [ER(%)]. This ratio, also referred to as the affinity index, isbelieved to be related to the average affmities of the circulating immunoglobulins directed to the antigen system,as these techniques have been successful in demonstrating affmity maturation in response to a variety ofantigens: RV and RV vaccine (Thomas and Morgan-Capner, 1988; Mauracher et al., 1992a), Toxoplasmosis(}{edman et al., 1989), Respiratory syncytial virus (Meurman et al., 1992) and Varicella zoster virus (Tilley andJunker, 1992).It is still argued whether the chaotropic elution ELISA indirectly measures affinity or the avidity ofimmunoglobulins. In the titles of papers and abstracts which use this method both terms are used, at times-32-interchangeably. As chaotropic agents most likely directly interfere with the epitope-antibody binding site itwould seem reasonable to suggest that this method measures antibody affinity. To resolve this issue theinteraction of low and high affinity anti-dinitrophenol (DNP) monoclonal antibodies with variably substitutedDNP-albumin could be investigated by chaotropic elution ELJSA. If this assay can selectively differentiatebetween high and low affmity antibodies, independent of DNP concentration on the albumin molecules, thenit could become possible to conclude that the chaotropic elution assay indirectly allows for the assessment ofantibody affinity.1.4.5.2lnhibition ELISAIn the ELISA technique, affinity of a serum can be estimated by the slope of the titration curve, especially ifnon-repeating antigens are used (Steward and Lew, 1985). Low affinity monoclonal antibodies need to bepresent in far higher concentration to produce similar absorbance values as high affmity antibody. Similarobservations have also been made in inhibition ELISA, where low affinity monoclonal antibodies require higherconcentrations of liquid phase inhibitor to decrease solid phase binding (Rath et al., 1988). This principle wasadapted in a One-Well Inhibition ELISA where a constant amount of antibody was added to wells with constantamounts of coated antigen. The amount of liquid phase inhibitor was variable and the amount of inhibitorrequired to bring about a 50% decrease in absorbance (I) was found to correlate well with the average affinityof sera (Steward et al., 1991). These authors used polyclonal sera directed to an 18 an peptide representing theneutralizing domain of the foot-and-mouth disease virus and were able to verify the affinity values obtained bythe inhibition ELISA with those obtained by the ammonium sulphate precipitation technique.1.5. Thesis Rationale and Thesis ObiectivesRubella virus is a pathogen which provides the rare opportunity to study the influence of intrauterine viralinfections on human immunity. The study of CRS patients as a model for the induction of immunologicaltolerance is of advantage, as RV biology, pathology and molecular genetics are well established and clinicalspecimens of both pre- and postnatally infected individuals were readily available in our laboratory. This thesistested the hypothesis that intrauterine infection of the human fetus in the first trimester of gestation withRubella virus leads to development of immunological tolerance. A study of the tolerization of the human-33-immune system with RV will help in the understanding which mechanisms for immunological self/non-selfrecognition are operational in humans. Furthermore, the induction of immunological tolerance may be animportant mechanism for viral persistence. Persistence of RV in CRS patients has been well documented.However, RV persistence may also play a role in the pathogenesis of chronic inflammatory disorders such asRAA. The identification of immune responses which may be inefficient in clearing viral infections may thereforebe of relevance in both the congenitally-infected patient and patients developing chronic rubella associatedarthritis.The objectives of the thesis are discussed in point form below and also reflect the titles under which theindividual sections of the results and discussion chapter are presented. The development of reagents andtechniques, needed for the comprehensive evaluation of RV-specific humoral responses, were initiallyestablished. Once these assays had been established it was possible to first defme the normal adult responseto RV infection followed by the analysis of antibody responses to RV in CRS patients.I) Preparations and Characterization of RV for Use in Solid Phase Immimoassays. In order to comparethe antibody responses of CRS and control patients, new virus specific immunoassays had to be developed andstandardized. It was therefore necessary to produce a large and uniform preparation of RV which would providea large enough stock to be used for all further experiments. This virus stock was defined for its RV titre,antigenicity and purity. Whole RV ELISA methods were optimalized using this preparation and this RVpreparation was also used in the evaluation of suitable methodologies of anti-RV IgG affinity assays.II) RV Protein Specific Immunoassays. The objective of the methodological development was to havetechniques available which could measure the following parameters of the RV-specific antibody response: 1)Quantitation of IgG to whole RV and RV proteins El, E2 and C, 2) Measurement of relative affinities of IgGdirected to whole RV and RV proteins El, P2 and C, 3) Measurement of biological activity of sera by HAlassay and 4) Determination of specificity of antibodies to linear and topographic epitopes of the viral proteins.The development of new techniques was necessary in order to initiate the comprehensive study of patientsinfected with RV in the pre- and postnatal period. These included methods for evaluating the specificity andaffinity of antibodies to individual RV proteins. Immunoprecipitation, Western blotting and ELISA using-34-separated RV proteins were evaluated as techniques suitable for the detection of RV protein-specificimmunoglobulins and needed to be capable of measuring both antibody quantity and affinity. It had beenreported that both El and E2 proteins contained mainly conformationally dependent epitopes and it wasnecessary to show that purified RV antigens retained their native structure, before using these proteins incomparative immunoassays.III) RV-SpecifIc Antibody Responses in Postnatally Infected Patients. In order to determine if a state of RVspecific tolerance existed in the CRS patients it was first necessary to characterize the humoral immune responseof healthy patients to RV. Although antibody responses to RV in adults have been evaluated for more than fourdecades a comprehensive study of biological activity (HAl), whole virus and virus protein specificity, antibodyaffmity and linear/topographic epitope specificity of RV-specific antibody responses has never been undertaken.Once the RV-specific antibody response had been defined in healthy responders it was possible to analyze theresponses of CRS patients. Differences in the responses of these patient groups may reveal defects in theimmune repertoire which would suggest a state of immunological tolerance in CRS patients.IV) RV-SpecificAntibodyResponses in Congenitally Infected Patients. To test the hypothesis that intrauterineexposure to RV had established immunological tolerance, the RV-specific antibody responses of CRS patientshad to be analyzed and compared to those of the control population. If differences in the humoral responsewere detected, a model of tolerance could be proposed which could accommodate serological findings andcurrent data on cellular responses in this patient group.-35-2. MATERIALS AN]) METHODS2.1. Virus Preparation. Titration and Concentration2.1. 1.Bulk Preparation of Rubella Virus (M-33 Strain)Rubella Virus, M-33 strain (VR-3 15, ATCC, Rockville MA, USA) (Parkman et al. 1962) was grown by infecting80% confluent monolayers of Vero Cells (CRL 1586, ATCC) as follows. Vero cells were grown in completeMEM containing 1% Glutamine (30 mg/nil), 100 units/mI and 0.1 mg/nil of Penicillin and Streptomycinrespectively to prevent bacterial contamination (all from Gibco, Grand Island NY, USA), 20mM Hepes (Sigma,St. Louis MO, USA) and 2% fetal calf serum (1-lyClone Laboratories, Logan UT, USA) using T-175 Falcontissue culture flasks (Becton-Dickinson, Lincoln Park NJ, USA) at 36°C in 5% CO2. Supernatant was aspiratedand cells were washed once with 10 ml PBS at 36°C. RV (at an m.o.i. of 10) was then added in 30 nil ofmedium without fetal calf serum. Virus was left to absorb at 36#C for 2 hours. 120 ml of complete mediumwas then added and cells were returned to the incubator for 3 days. The primary virus harvest at three daysfollowing infection was used as the starting material for further viral purification. The technical help and themany innovations which Ms. S.Farmer introduced to the growing of RV is acknowleged and greatly appreciated.2.1.2.Rubella Virus Titration on RK-13 CellsRK-13 cells (CCL 37, ATCC) were seeded into 96-well Falcon plates, at 0.2 ml per well, at a concentration of20,000cells/ml, using M199 Medium, 1% penicillinlstreptomycin and 2% fetal calf serum (suppliers as above).At 48 hours, medium was removed and the wells were washed with warm PBS. Tissue culture supematantscontaining RV were diluted in 10 fold dilution series and 50 11 loaded in triplicate onto the confluent RK-13cells followed by a one hour incubation 36°C. Following virus absorbtion, 150 11 of complete medium was addedto each well and infectous foci were scored after 3-4 days.2.1.3.Concentration of Rubella Virus Tissue Culture SupernatantTissue culture supernatant was clarified by centrifugation on a Silencer H-103 N centrifuge at 3000 rpm(G av= 1000) for 15 minutes. Pooled preparations were then brought to 0.5 M NaC1 and 10% w/v polyethylene-36-glycol 20,000 (BDH, Poole, England) while stirring slowly at 4°C for 2 hours. The suspension was thenprecipitated by centrifugation in a Sorvall GSA rotor in a RC-5B Sorvall Centrifuge (DuPont & Co, NewtonCT, USA) at 8000 rpm (G av 7000) for thirty minutes. The virus pellet was resuspended in TE buffer (10 mMTris, 5 mM EDTA, pH 7.4)2.2. Preparative SDS-PAGE2.2. 1.ElectrophoresisRV proteins were separated on 1.5mm thick slab gels using the Protean II mini-gel system (BioRad, RichmondCA, USA). The discontinuous buffer system according to Laemmli (L.aemmli, 1970) was used. Thepolyacrylamide separating gels (10 %I0.25 % Acrylamide/Bis, 0.375M Tris-HC1, 0.05% Ammonium Persulphate,0.05% Temed, pH 8.8) were overlaid with stacking gels (4 %I0. 1% Acrylamide/Bis, 0.125 M Tris-HC1, .05%Ammonium Persulphate, .05% Temed, pH 6.8) and a custom-made preparative teflon comb used for wellformation. In order to counteract “smiling” of the dye front, the comb was cut with the reference wellsexceeding the preparative well by 1.5mm in length. In all cases, gels were poured and left to polymerize overnight in the dark at 4°C.Sample buffer for preparative PAGE was devoid of reducing agents, contained less SDS than commonly usedand was prepared monthly as double strength stock (0.25 M Tris-HC1, pH 6.8,0.5% SDS, 20% Glycerol, .02%Bromophenol Blue). 50 11 of RV preparation was diluted in 300 11 dH2O followed by the addition of 350 11of Sample buffer. Tubes were incubated in a 60C waterbath for 3 minutes and loaded into the preparativewells. The reference wells received 50 11 of pre-stained molecular markers (Sigma, St. Louis MO, USA).Electrophoresis was performed at 40 niAmps per gel, constant current, until the dye-front reached the bottomof the slab. The location of RV protein bands in the slab gel was detected by excising a thin lane of gelcontaining half the lane of pre-stained MW markers and the edge of the preparative lane containing RV,followed by transfer to nitrocellulose and immunoblotting. The nitrocellulose strips were developed using ahuman anti-RV serum containing IgG to El, E2 and C (See immunoblotting section for methods). These bandswere then used as a guide in the excision of gel strips containing antigen.-37-2.2.2.ElectroelutionGel strips were cut into 1 cm sections and electroeluted using a Model 422 electroelution attachment of theBioRad Protean II system. Elution buffer (25 mM Tris base, 192 mlvi Glycine, 0.1 % SDS) was filtered througha 0.22 im filter. Electroeluates of each protein were collected, frozen at -70°C until enough of each protein wasprepared to provide sufficient material for completing the anticipated patient study. Each preparation wasassayed by immunoblot and the preparations of each viral protein, showing no contamination with other RVstructural proteins, were pooled. The C antigen pool showed residual contamination with El and was furtherpurified using Triton X-l 14 extraction (see below).2.2.3.Detergent ExtractionCapsid preparations were treated with KCI for SDS removal as follows. 100 11 of 0.4 M KC1 were added to 1ml of C preparation at room temperature. The preparation of the C antigen pool was then put onto ice andprecipitation of the potassium lauryl sulphate salt allowed to equilibrate for 30 minutes. Tubes were thencentrifuged for 1 minute at 10,000 rpm on a bench-top centrifuge and the SDS-free C preparation transferredto new tubes and left on ice. Triton X-114 (Sigma, St. Louis MO, USA) was added to a final concentration of0.5% and the tubes rotated at 4 #C for 1 hour and then brought to room temperature. Triton X-l 14 reachesits clouding point at 17°C, above which it can be removed from an aqueous solution by centrifugation. Thedetergent free C preparation was then aliquoted and stored at -70 °C.2.2.4.Yield Determination of ElectroelutionL-[355]methionine labelled RV (see below) was loaded into two wells of reducing 9% SDS-PAGE slab gels witheach well containing antigen being flanked by pre-stained MW markers. Gels were run until the BPB dye frontreached the bottom of the gel. The region of the lanes containing labelled virus located between the MW the58- and 26 kD markers was excised, fragmented and added to 2 ml scintillant (ScintiVerse E, Fisher Scientific,Fair Lawn, NJ) with the addition of0.4 nil of a 1% SDS solution. The other lane was electroeluted as describedabove and the total volume of 0.4 ml of electroeluate was added to an additional scintillation vial. Vials werekept ovemight, in the dark, and counted the following day on a Beckman LS6800 Scintillation Counter. Yieldswere determined by comparing the counts obtained from the electroeluate and the fragmented gel strip.-38-2.3. Metabolic RadioIabellin of Rubella VirusVero cells were grown in T- 175 flasks to a confluence of 80% and infected with RV (M 33 strain) as describedabove. After adsorption of virus 30 ml of medium was aspirated and 150 ml of fresh complete MEM (as definedabove was added. After 24 hours the medium was changed with complete MEM lacking L-methionine.Following a 2 hour incubation this medium was aspirated and 150 of fresh medium added (1:1 of completeMEM and MEM lacking L-methionine with the addition of 18.5 MBq ofL-[35S]methionine) (Amersham,Arlington Heights IL, USA). After a further 24 hour incubation, virus was harvested and concentrated byultracentrifugation. Tissue culture supernatant was centrifuged at 27,000 rpm (G=9O,OOO) for 2 hours, inpolyallomer tubes, each holding 38.5 ml TCS. Pellets were washed once with 10 ml ice cold PBS and spun for30 minutes at 27,000rpm. The supernatant was discarded and pellets taken up in 385 11 TBS.2.4. Serum SeparationSeparation of IgG, IgA and 1gM from human serum was performed using anion exchange chromatography. Serawere centrifuged on a bench-top centrifuge for 10 minutes at 10’OOO rpm to remove particulate material andchylomicrons. Samples were then diluted 1:2 in running buffer and filtered through 0.22 im Millipore filters.Solutions used in chromatography were as follows:Running buffer (Buffer A): 20 mM Tris-HC1, pH 8.3,5 mM NaN3High salt buffer (Buffer B): 20 mM Tris-HC1, pH 7.1,500 mM NaC1, 5 mM NaN3Column rinse buffer 1 200 mM NaCI, 1% Triton X-100Column rinse buffer 2 1 M Acetic AcidDialysis buffer 50 mM Ammonium acetate, 10 mlvi NaCl, pH 7.2All buffers were filtered through 0.22 im Millex-GV filters (Millipore, Bedford MA, USA) and stored in thecold room. As chromatography was performed at room temperature, buffers were equilibrated to abienttemperature and degassed before use. Separation was performed on a Mono Q HR5/5 (Pharmacia, Uppsala,Sweden) column attached to a LKB 215OHPLC system (LKB, Bromma, Sweden). Sample (200 1L) was loadedonto the column and proteins were eluted by a step-gradient illustrated in Figure 18. Following each increaseof Buffer B, eluted proteins were pooled. The three pools collected, contained separated serum immunoglobulinclasses in the following order: IgG in pool 1, IgA in pool 2 and 1gM in pool 3. Each pool was dialysed over-39-night at 4 #C and lyophilized. Ammonium sulphate was used as salt in the dialysis buffer in order to allow theremoval of this volatile salt during lyophilization. Samples were taken up in 200 11 of dH2O before analysis.2.5. Gel Stamina2.5.1.Colloidal Coomassie StainCoomassie Brilliant Blue G-250 (Sigma, St.Louis, MO) was prepared according to Neuhoff et a!., (1988) asfollows: 4 grams of Coomassie Brilliant Blue G-250 was dissolved in 250 ml of hot 7.5% acetic acid. Ammoniumsulphate (44 grams) was added slowly and the solution cooled to 4 #C. The recrystallized dye was washed withcold 7.5% acetic acid, 18% ammonium sulphate solution and dried. The staining solution was obtained bydissolving 0.5 g of the recrystallized G-250 in 10 mIs of water which is added to 500 ml of a 0.45 M ammoniumsulphate, 2% phosphoric acid solution. Befor staining, gels were fixed in 10% acetic acid, 1% trichioroaceticacid and then agitated over night in the colloidal stain working solution. Gels were briefly rinsed in 20%methanol, 10% acetic acid and dried.2.5.2.Silver StainSilver staining was performed with a commercially available kit (Bio-Rad, Richmond, CA). All glass plates werecleaned with Chromerge prior to gel casting and deionized water was used in all reagents.2.6. Solid Phase Immunoassays2.6.1.Western BlottingFor gel electrophoresis separation of RV proteins for immunoblotting either reducing or non-reducing samplebuffer was used. Buffers used were as follows:Sample buffer 2X (Reducing): 0.1 M Tris-HC1, pH 6.8,20% Glycerol, 0.5% SDS, 3% Mercaptoethanol,0.01% BPBSample buffer 2X (Non-Reducing): 0.1 M Tris-HC1, pH 6.8,20% Glycerol, 0.5% SDS, 0.01% BPBStacking Gel: 4% Acrylamide, 0.1% Bis, 0. 125M Tris-HC1, pH 6.8,0.05% Temed andAPSSeparating Gel: 9% Acrylamide, 0.2% Bis, 0.375M Tris-HC1, pH 8.8,0.05% Temed and-40-APSRunning buffer: 200 mM Glycine, 25 mM Tris, 0.1 % SDSTransfer buffer: 200 mM Glycine, 25 mM Tris, 0.1 % SDS, 20 % MethanolImmunoblot Sample Buffer: PBS, pH 7.3,3% skim milk powder, 0.02% Tween 20Immunoblot Conjugate Buffer: Same as sample buffer but without Tween 20Substrate buffer: 50 mlvi Tris-HC1, pH 9.6,140mM NaCl, 5 mlvi MgC12,0.3mg/mi BCIP,0.3 mg/mi NBTSDS-PAGE was performed on Mini-Protean II apparatuses gels using a preparative 0.75 mm comb to formwells. RV preparation (20 11) was diluted with 300 11 using either non-reducing or reducing sample buffer,heated at 60°C for 3 minutes and loaded. Separation was performed at 30 mAmps constant current run untilthe BPB dye front reached the bottom of the gel. Proteins were then eloctrophoretically transferred tonitrocellulose for 30 minutes at 30 mAmps constant current using Transfer buffer. Nitrocellulose sheets werethen blocked in immunoblot sample buffer for one hour. Sheets were then cut into 4mm wide strips and addedto test tubes containing 300 11 of the appropriate serum sample dilution in immunoblot sample buffer. Stripswere incubated over night at 4°C, washed in three changes of PBS-Tween with a last wash in Conjugate buffer.Strips were added to test tubes containing 300 11 conjugate buffer with 1 ig/mi alkaline phosphatase-conjugatedGoat IgG anti-Human IgG c-chain. Following a 2 hour incubation and a further wash, strips were developedin Substrate buffer. Quantitative analysis of immunoblot strips was performed by densitometry using a Model620 Video Densitometer (BioRad, Richmond CA, USA). Using the reflectance mode, the apparatus integratedthe intensity of each band and quantitated signal strength as OD x mm.2.6.2.InimunoprecipitationThe conventional protein A-sepharose immunoprecipitation technique was used, with modifications describedby others (Loo et al., 1986). Protein A-sepharose suspension (Pharmacia, Uppsala, Sweden) was washed inSample buffer (20 mM Tris-HCI, 150 mM NaCl, 1 % TX-100, pH 7.3) and resuspended in a minimal volumeof Sample buffer. Patient serum (30 11) was diluted in 200 11 of Sample buffer and added to 50 11 washed beadslurry, the resulting suspension was incubated at 4 C for 4 hours on a rotator. Beads were washed 3 times and35S-methionine-labelled RV was added, in a volume of 200 Ii Lysis buffer (20 mM Tris-HCI, 150 NaC1, 1%-41-sodium dioxycholate, 1% TX—100, 0.1% SDS, 5 mM EGTA, 1 mM EDTA) and the mixture incubated over nightat 4 #C. Beads were washed 4 times and then boiled for 4 minutes in reducing SDS-PAGE Sample buffer torelease antibody and bound RV antigen, spun at 10,000rpm for 1 minute and loaded onto 10% SDS-PAGE gelsand separated as described above. Following electrophoresis, gels were soaked in Amplify (Amersham,Arlington Heights, IL) for 5 minutes, dried and autoradiographed using X-OMAR, AR diagnostic film (EastmanKodak, Rochester, NY).2.6.3.Enzyme Linked Immunosorbent Assays (ELISA)ELISA was used to quantitate immunoglobulins directed to whole RV or separated RV structural proteins. AllELISAs were performed in 96-well, flat-bottom, microtitre plates using either: Immulon-2 (Dynatech, Chantilly,VA) or Falcon 3915 (Becton Dickinson Labware, Lincoln Park, NJ). ELISA buffers are listed below:Coating buffers a) Carbonate (pH 9.6): 15 mM Na2CO3,35 mM NaHCO3,0.02% Azide, filtered.b) PBS (pH 7.3): 40mM NaC1, 16mMNa2HPO4,1.5mM KH2PO4,3mM KC1,0.02%AzideBlocking buffer (bb): PBS, 2% Normal Goat serum, 0.02% Tween-20, filtered (0.22 im).Sample buffer (Sb): Same as Blocking buffer.Heat Denatured Sample buffer (sbb): Boil 25 ml of SB for 5 minutes, then immediately mix with 25 ml ofice cold SB and transfer onto ice (Mauracher et al., 1991b).Wash buffer (wb): PBS, 0.1% Normal Goat serum, 0.02% Tween-20.Conjugate buffer (cb): PBS, 2% Normal Goat SerumSubstrate buffer (subb): 1 M diethanolamine, 1 mM MgCl, pH 9.6,2 mg/mi p-nitrophenylphosphate disodium2.6.3.1.Whole Virus ELISAPurified RV was coated at a dilution of 1:3000 in PBS Coating buffer onto Immulon-2 plates by overnightincubation at 4 #C overnight using 100 11/well. Plates were warmed to RT for 1 hour, flicked and blocked withsb for one hour at RT with 200 11 buffer per well. Serum samples were diluted in sbb and serial two-folddilutions were added in triplicate at 100 il/well. Following incubation at 36 #C for 2 hours the plated were-42-washed 3 times in wb then alkaline phosphatase-conjugated goat anti-human IgG( c-chain) antibody (Kirkgaardand Perry Laboratories, Gaithersburg, MD, USA), diluted to a fmal concentration of 1 ig/nil in cb, was addedat 100 Ji/well. Following a further 2 hour incubation at 36 lC, the plates were washed 4 times and developedby adding 170 11/well subb, followed by incubation at 36C. Each plate contained five duplicate dilutions ofa standard serum, whose concentration of anti-RV IgG was previously determined to be 943 lU/mi, by titrationversus the WHO reference serum (WHO, 1971). Standart serum was diluted serially in two-fold dilution startingat 1:1000. The end-point of development was reached when the 1:1000 dilution of the standard curve reacheda net absorbance of 1.0 AU at 405nm. Plates were analysed on a microplate reader (Model 3550, Bio-Rad)using a wavelength of 405 nm. Using the five point standard curve immunoglobulins in the samples werequantified and assigned JJJ/ml of anti-RV IgG using quadratic regression (Microplate Manager , software, BioRad).2.6.3.2RV Protein EUSAElectrophoretically purified RV structural proteins, El, E2 and C, were coated onto Immulon-2 plates using PBSbuffer at 44C overnight. Stock preparations of each protein were prepared, divided into aliquots and stored at-70 #C. Optimal coating concentrations varied for each viral protein preparation: El was coated at 1:60, E2 at1:80 and C at 1:40 using PBS Coating buffer. ELISA was performed as described above. The standard serum(943 lU/nil) was analyzed on non-reducing immunoblot and the relative amounts of IgG directed to each viralprotein were determined by densitometry. The percentages of protein specific IgGs were expressed as arbitraryunits (AU) in realation to the 943 TU/tril of whole virus specific antibody. Accordingly this standard contained:424 AU/mi anti-El, 405 AU/mi anti-E2 and 114 AU/nil anti-C.2.7. Affinity Assays2.7.1.Chaotropic Elution ELISAEUSA buffers and general protocol were followed as described above and has been described previously forboth whole RV as well as for separated RV protein (Mauracher et al., 1992a). Serum samples were diluted insbb and incubated in duplicate dilution series in RV antigen coated microtitre plates: one series to be exposedto Elution buffer (eb) (8 M urea, 150 mM NaC1, 20 mM Tris-HC1, 0.02% Tween-20, pH 9.6) and the control-43-series to be exposed to wb. After a 2 hour sample incubation step at 364C, plates were washed twice with wb,then one dilution series was exposed for 4 minutes to 200 il/well of eb while the control dilution series wastreated with the an equivalent amount of wb. Following elution, the plates were washed twice and routineELISA protocol was resumed. The end-point of the assay was reached when the least dilution of the standardcurve reached 1 absorbance unit. Elution ratios [ER(%)] were determined for each serum sample by selectingthe dilutions which fell between absorbance units of 0.7 and 1.5 and were calculated according to the followingformula:ER(%) = [(A405 Urea eluted - A405 Background) % (A405 Control - A405 Background)] x 1002.7.2.One Well Inhibition ELISAThis assay was adapted from the whole RV ELISA described above and was only employed using whole viralantigen preparations. A similar assay was previously described for use with peptide antigens (Rath et al., 1988).Microtitre plates were coated with a constant concentration of whole RV and blocked. A dilution series ofliquid phase RV antigen was then added to the plates followed by the addition of a constant dilution of serum.Constant serum concentrations were determined by choosing the concentration of serum which fell into middleof the linear region of the titration curve on whole RV IgG ELISA. The wells which received the highestconcentration of liquid phase antigen will result in the lowest absorbance reading, following the developmentof the plates, as antibody binding to the solid phase will have been prevented by the competing liquid phaseantigen. The concentration of liquid phase antigen needed to bring about 50% inhibition of antibody binding(‘.s was related to antibody affinity, with low affmity antibodies requiring more liquid phase antigen to bringabout 10.5 (Andersson, 1970). Whole RV was coated onto Falcon plates in carbonate coating buffer at a dilutionof 1:3000 and sera titred out from dilutions of 1:10 to 1 :50,000in sb. A dilution which yielded 0.5 absorbanceunits after one hour incubation was chosen as the serum concentration to be used in the inhibition assay. Incase of FPLC purified IgG fractions, the lyophilizate was taken up in 100 11 sb and then further diluted as serumsamples. RV preparation was diluted from 1:5 through 1:5000 in S nil test tubes using ELISA sb, the finaldilution contained no virus and was taken as the infmitely diluted virus. 50 11 of each dilution was added to eachwell in triplicate, using Falcon plates which had been coated overnight with whole RV (1:3000) and blocked with-44-sb for 1 hour at RT. A double strength dilution of the serum sample was then added to each well at 50 11/welland incubated for 2 hours at 36 41C. Routine ELISA protocol was employed in all the following steps. Thedilution of liquid phase inhibitor to bring about a reduction of 50% net absorbance was determined by fittinga best fit line through the linear portion of the inhibition curve.2.8. Hema1utination Inhibition AssayRV Gilchrist strain (Whittaker MA Bioproducts, Walkersville, MD, USA) was used as hemagglutinin. HA titresof this preparation were determined for each virus lot by serially diluting the HA antigen in two fold dilutionstarting at 1:4. The last dilution yielding complete agglutination of at total of 100 11 of a 0.125% solution of 1day old chick eiythrocytes (PML Microbiologics, Tualatin, OR) was assigned the concentration of 1HA unit.Following HA determination the viral antigen was aliquoted and stored at -70#C. All serum samples (200 11)were pre-treated for 15 minutes with 200 11 of MnCI2/heparin solution (0.5 M MnC12,2500 lU/mi Porcineheparin) at 4 lC. Next, 200 11 of a 50% chicken erythrocyte (PML Microbiologics, Tualatin, OR) solution inHSAG buffer (25 mM HEPES, 140mM NaC1, 1 mM CaCl2, 1% BSA, 0.25%o Gelatin, pH 6.5) was added andincubated at 4 #C for 1 hour. An additional 600 11 of HSAG buffer was then added and the tubes centrifugedfor 10 minutes at 1000 x G and the supernatant (now at a serum dilution of 1:8) was collected. Pre-treatedserum (50 11) was serially diluted two fold in “U”-shaped polyvinylserocluster plates (Costar, Cambridge, MA)and 25 11 RV hemagglutinin, containing 4 HA units, was added to each well. After 1 hour of incubation at 4C,50 11 of a 0.25% solution of 1 day old chick erythrocytes was added and the plates interpreted for HAl titrefollowing 3 hours and again after an overnight incubation at 4C.2.9. Bioloaical Function of RV Proteins2.9.1.Solubility Shift of C Protein at Acidic pHTissue culture supernatant (TCS) containing L-[35S]-methionine labelled RV was extracted in 1% TX-i 14(Bordier, 1981) (Calbiochem, SanDiego, CA), replacing Tris-HC1 buffers withMcllvanes citrate-phosphate buffer(Mcllvane, 1962) in order to accommodate detergent extraction over pH range 4.0 to 9.0 (Figure 9). Extractionwas performed with cold solutions by rotating tubes end-over-end at 4 4C for 1 hour. This solution was thenoverlaid onto a sucrose cushion (6% sucrose, 0.02% TX-i 14, with pH adjusted accordingly) and the tube was-45-allowed to warm to 36 #C. Tubes were then centrifuged for 1 minute on a benchtop centrifuge, and the aqueousphase removed. Detergent pellets were washed once in Mcllvane’s buffer and then resuspended in water to theoriginal volumes.Add TX-114 Rotate Transfer onto Sucrose Centrifuge4°C 220CFigure 9. Triton X-1 14 extraction of RV from Tissue Culture Supernatant. Ice cold TCS is made to 1% TX-114, and extraction performed by end-over-end rotation at 44C. TCS is then overlaid onto a 6% sucrose cushion(II) and the tube warmed to RT. The TCS turns opaque as TX-i 14 reaches clouding point Followingcentrifugation TX-i 14 is concentrated as a pellet (I below the sucrose cushion and the TCS is devoid ofdetergent. Hydrophobic proteins are in the pellet, hydrophilic proteins remain in the TCS.2.9.1.1 .UltracentifugationTCS containing high titres of RV was treated at either pH 7.0 or 5.0 with or without 1% TX-i 14 and with 1%SDS. The aqueous supernatants from the TX-i 14 extractions and the SDS and non-detergent treated TCS werethen placed on 22% sucrose cushions and centrifuged for 3 hours at 39,000rpm in a SW41 Ti rotor (Beckman,Palo Alto, CA). The pellet was resusupended in dH2O and analyzed by Western blot or by PCR.2.9.1.2Polymerase Chain Reaction for Detection of RV RNAAn aliquot of 50 11 from the resuspended pellets from ultracentifuged TCS was solubilized in 4 M guanidinium-46-isothiocyanite, phenol/chiorophorm extracted and RNA precipitated in the presence of tRNA (Maniatis et a!.,1982). Complementary DNA was synthesized using M-MLV reverse transcriptase (BRL, Gaithersburg, MD).One tenth of the volume was added to the PCR mix, containing 10 c4vl of each forward and reverse primershown below and 1 U Taq polymerase (Bio/Can Scientific, Mississauga, ONT) (Saiki et al., 1988). The primersused were both 24-mers which amplified a 287 bp fragment from the El gene of RV: 5’-TrGAACTT’CAGCC-CCAAGGGGCCC-3’ and 5’-TCCCCGG1TfGCCAACGCCACTCC-3’. Thermal cycles of 95#C for 45 sec,6841C for 15 sec and 72#C for 30 sec, were performed on a Perkin-Elmer-Cetus thermal cycler (Mauracher etal., 1991a). The work of Mr. R.Shukin in the development and design of this protocol is acknowledged.2.9.2.Hemagglutination Activity of Purified El ProteinAn aliquot of El and C protein preparation was treated with KC1 to remove SDS, as described above, andemployed to competitively bind HAl antibodies (C protein was used as the negative control). Pooled RVseropositive human serum with an HAl titre of 1:256 was used as the source of IgG. Five rows of theserocluster plates received 5 wells of 25 11 each of serum dilutions of 1:40, 80, 160 and 320. To each row ofserum dilutions, a dilution series of El (or C) protein was added, starting with 25 11 of El preparation, 12.5,6.25,3.1 and 0 11 of El. Each well now contained serum with El or C protein and incubated at 4 tIC for 1 hour.Each well then received 25 11 of RV containing 4 HA units and received 25 11 of a 0.5% CRBC solution aftera further 1 hour incubation at 4tIC. Negative controls included cells incubated with either El or C protein butno serum, CRBC with serum and no HA antigen and CRBC in buffer only.2.10. Patient SeraBlood samples were collected by venipuncture and sera were stored at -70 tIC until needed. CRS patients werediagnosed clinically and were shown to be positive for cord blood anti-RV 1gM and/or by RV isolation duringthe neonatal period. Time of intrauterine infection was established for all CRS patients on the basis of maternalhistory and by serological confirmation. The age of patients at the time of serum collection was 1 to 34 years.One CRS patient in the study was also diagnosed with chronic progressive rubella panencephalitis. Controlpatients were adults and were either mothers of CRS patients used in the study or individuals who were infectedduring the 1984/85 rubella epidemic in British Columbia.-47-2.11. Statistical MethodsStatistical analysis using the unpaired T-test was used for most population comparisons and distributions testedfor normality using the Martinez and Iglewicz test. Level of confidence was set at a=O.05 for all analyses.Populations showing non-normal distribution were analyzed using the Mann-Whitney U test.Analysis of variance (ANOVA) was used when multiple comparisons of populations were performed. One-Way AJ’TOVA was used with level of confidence set at a=O.05. Means were separately analyzed with theFisher’s LSD post-hoc test.Means and standard deviations (SD) were determined using descriptive statistic on the software package. Allerror bars in the figures of this thesis are — 1 SD.All statistical calculations were performed on NCSS 5.01 software (J.L.Hintze, 1987). The help of Mrs.R.Milner and Mr. M.McKinnon from the Statistical Research Support Unit, Research Centre, in the statisticaldesign is greatly appreciated.-48-3. RESULTS AND DISCUSSION3.1. Section I: Vfrus Preparation and Use in Solid Phase Immunity and Affiiilty Assays.At the start of the project preparations of RV had to be established which were uniform, well characterized andwould be of sufficient quantity to provide antigen for both phases of the experimental part of the thesis:methodology development and patient studies. This was important to eliminate RV antigen preparation as avariable in the comparative studies of the immune responses of pre- and postnatally RY infected patients.3.1. 1.Rubella Virus PreparationThe RV stock, which was used for all direct antigen assays and for the preparation of purified RV proteins El,E2 and C was obtained from a pooi of 24 liters of tissue culture supernatant (TCS), and purified as one batch.The virus was obtained from ATCC (M-33 strain, ATCC VR-553) and further experiments verified the stockto contain infectious RV. This wild type RV isolate was used as antigen in all serological assays, as responsesto wild type infection (both in post- and prenatally infected individuals) were analyzed.3.1.1.1.Virus TitrationThe original TCS contained RV at a concentration of 2.7 x 106 pfu/ml, as determined by virus titration on RK13 cells (Nawa, 1979). The morphology of the infectious foci (Fig. 11) on RK-13 cells were characteristic forthe cytopathic effect caused by RV in this cell line (Nawa, 1979).-5—-fFigure 10. Micrograph of RV Infectious Focus on RK-13 cell Monolayers. Phase contrast microscopy (500 x)of a RK 13 cell monolayer 3 days after in vitro infection.-49-Figure 1 1A shows whole protein stain and Western blots of the RV concentrate (lane 1), the starting TCS (lane2) and TCS obtained from mock-infected Veto cells. The final concentrated viral preparation contained 2 x 1010pflilml and had a 150 fold increase in antigenic activity per gram protein. Protein bands for El, E2 and C wereElEl -E1E2CdElAElE2— 97 E2 E2-68 ElE2-45CFigure 11. A) Western Blot and Colloidal Coomassie Blue Stain of RV Preparations. 0.5 11 RV concentrate(1), 10 11 starting TCS (2) or mock-infected Vero cell TCS (3). Prestained MW markers (4). MW markers areindicated on the right side of the gel. Western blots were developed with anti-RV reference standard humanserum, anti-human IgG (c-chain) alkaline phosphatase-conjugate and BCIP/NBT stain. Relative positions of RVproteins are indicated on the left side. El, E2 and C refer to monomeric forms of RV structural proteins, Cddimeric capsid. Dimeric forms of the envelope proteins are indicated as El :El (homodimer) and El :E2(heterodimer).B) Silver Stain and Autoradiograph of concentrated RV Separated by Reducing SDS-PAGE . Lanes 1 through4 contain a BSA titration (20,5, 1 and 0.2ng/band). RV preparations, contained 1/10 volume of35S-methioninelabelled RV, were loaded in doubling dilutions in lane 7,6 and 5 (10,5 and 2.5 11 of RV concentrate respectively).Lane 8 contains MW markers. The corresponding autoradiograph of lane 7 is shown to the right with relativepositions of RV proteins indicated. Arrow heads in the gel indicate possible El and C bands.12 3 MW 1 2 3E2Non-Reducing1 2 34-*BSAMW——97-45—27— —27B1 2 3 45 6 7 8Reducing.z44 ElE2797684527-50-not clearly identifiable by either colloidal Coomassie or silver staining. The detection limit for protein in thesilver stained gel in figure 1 lB lies between 1- .2 ng BSA per band. If the faint bands () in lane 5 areinterpreted to represent the El and C protein, then the final protein concentration of RV antigen can beestimated to lie in the order of 1 ig/mi. The relative molecular masses of the RV structural proteins, asdetermined by reducing SDS-PAGE were: El (58 kD), E2 (47-42 kD) and C (33 kD). Under non-reducingconditions, the capsid protein was detectable in its dimeric form (Cd) at 68 kD and El travelled at a lowerapparent molecular mass of 55 kD, in comparison to its position on gels run under reducing conditions whereits apparent mass was estimated to be at 58 kD (Baron et al., 1991, Mauracher et al., 1992a).3.1.2Whole Rubella Virus ELISAAlthough basic conditions for whole RV ELTSA had been established (Bidwell et al., 1977; Tingle et al., 1989)and numerous commercial suppliers have marketed reliable anti-RV IgG ELISAs (Dimech et al., 1992), a seriesof modifications to the whole RV ELISA protocol were developed. These improved both sensitivity andspecificity of the RV ELISA.3.1.2.1.Coating Conditions and Antigen TreatmentWhole RV was coated onto 96-well ELISA plates (Falcon 3915 or Immulon-2) diluted at 1:100 - 1:25 ,000inPBS (pH 7.3) followed by blocking in NGS-containing Sample buffer. The addition of Triton X-l00 to the wholeRV preparation improved signal strength (Fig. 12). Whole RV was made to a fmal concentration of either 2%,0.5% or 0.2% TX-100 and compared to RV, without any detergent added, in a titration of antigen using constantconcentration of detection antibody. Whole RV, in the absence of detergent, showed a shallow titration curvewith a saturated region at dilutions stronger that 1:500. A marked increase in signal was observed betweendilutions of 1:200 and 1:6400 for viral preparations which had been treated with detergent. A strong prozonewas observed in the preparations containing 0.5% and 2% TX-100 suggesting that detergent may compete forhydrophobic binding sites on the polystyrene plastic surface. The increase of the signal in the detergent-treatedvirus may result from disaggregation of the virus or to the disruption of the viral envelope leading to increasedexposure of the capsid as well as E2 protein.-51-2.0-1.5-1.0-a1.0C’)0.5-0.0— I I I I I —100 200 400 800 1600 3200 6400 12800 25600Reciprocal Dilution of Whole Rubella VirusFigure 12. Titration of RV preparations Treated with Triton X-100. Virus was brought to a final concentrationof 0.2% (1), 0.5% () or 2% () TX-100 using a 20% stock solution. As a control an equivalent amount of PBSwas added to the preparation which received no detergent (0).3.1.2.2Jleat Denatured Sample BufferDuring the course of these investigations it was frequently observed that sera from certain patients had highbackgrounds on RV IgG ELISA. Such high background could result in false positive RV serology. Increasedbackgrounds were especially common in infants (Dr. L.A.Mitchell, Dept. of Pathology, UBC, personalcommunication), who had RV vaccine as well as several other vaccinations during the same period. It wasobserved that much of the non-specific background in such patients could be removed by pre-adsorption ofserum on NGS blocked microtitre plates (Fig. 13). This additional step was time consuming and costly and it—0-0 No TX—100n—s 0.2% TX—laOh--a 0.5% TX—laOa--. 2% TX—100///e-L/A/-52-was found that it could be replaced by including heat-denatured protein in the Sample buffer (Fig. 13)(Mauracher et al., 1991b). Briefly, SB was boiled for 5 mm. and then mixed with an equal volume of ice coldSB. The resulting buffer, termed “SBb. for boiled sample buffer, contained heat denatured and aggregatedproteins which remained in suspension. Table 5 shows the quantitation of serum IgG directed to whole RV aswell as the individual structural proteins: El, E2 and C. Two vaccinees, 2 and 3 showed no difference in theirquantitative level of anti-RV IgG, expressed in TU/ml when either SB or SBb was used as a diluent. Theseobservations indicate that sample buffer containing the heat denatured protein did not reduce background levelsby masking RV-specific signals. A value of 20 lU/mi of anti-RV IgG was determined in the pre-immunizationserum of patient 1 using SB. While this value was in the range of positive immunity (ie., 15 lU/nil), anequivalent dilution prepared in SBb reduced RV-specific IgG levels to 0 lU/mi. RV seronegativity was alsoconfirmed by determination of a HAl titre of < 1:8. These observations suggest that SBb can aid in thereduction of non-specific binding, without interfering with RV antigen-specific binding.0.3-ECIC)° 02-0C)C0-DL.0Cl)-Q0.0- I0 100 200 300 400Reciprocal Serum Dilution Reciprocal Serum DilutionFigure 13. Determination of RV-Specific IgG by ELISA. A single patient’s serum sample, taken prior to RA27/3 immunization, was serially diluted in regular sample buffer (0) or in SBb (1). An equivalent dilution seriesin regular sample buffer was preadsorbed on NGS-blocked micropates (s). Negative control (o).-53-G—E) Regular SBn-n BoHed SB0—4 Neg. Serum--- Preabsorbed—e Neg. Serum160 260 360 400 0Proteins may be structurally altered by binding to plastic surfaces (Van Regemnortel, 1990). Also, it has beenshown that immunoglobulins which do not bind to liquid phase antigen will recognize new epitopes (cryptotopes)revealed by structural deformation of protein occurring during solid phase binding (Friguet et al., 1984).Similarly, new epitopes could potentially be created by the juxtapositioning of monomeric proteins onto solidphase (neotopes). It is possible that such cryptotopes and neotopes are exposed or created during heatdenaturation and aggregation respectively. The decrease of non-specific binding, described above could beattributed to the competitive removal of antibodies reactive to denatured NGS constituents in the liquid phaseof the diluent. Immunoglobulins reactive to such common blocking agents as BSA and normal animal sera havebeen previously reported (Levinson and Goldman, 1988) and are thought to have arisen as a result of activationof the immune system with dietary antigens or by vaccine contaminants.Vaccinee Weeks lgG rubella ELISA (lU/mI)post- Regular sample buffer Boiled sample buffervaccineEl E2 C Whole El E2 C WholeRV RV1 0 4 5 8 20 0 0 0 06 21 64 28 62 7 21 10 2454 51 49 16 156 20 20 4 522 1 0 0 1 0 0 0 0 06 10 8 4 28 12 8 6 3064 103 70 10 306 108 75 12 3213 0 0 0 1 0 0 0 1 06 19 26 4 60 20 22 3 6454 58 46 6 180 60 42 7 198Table 5. Rubella IgG ELISA Responses to Whole RV and RV Structural Proteins. The level of IgG directedto viral antigen was determined using the two sample buffers SB and SBb in three individuals at sequential timeintervals following immunization with RA 27/3 Rubella vaccine. (From: Mauracher et al., 1991b)3. 1.3.Affinity AssaysFor the measurement of affinity, the ideal experimental situation requires that both antibody and antigen bepresent in pure form (Steward and Steensgaard, 1983), such as monoclonal antibody reacting with haptenicantigen. Well established techniques, such as equilibrium dialysis (Eisen and Karush, 1949) and ammoniumsulphate precipitation (Steward and Petty, 1972) have found application in such antibody-antigen studies.However if the affinity of IgG responses from sera are to be estimated in an antigen system which containsmultiple proteins, then the traditional techniques for affinity measurement are not applicable (Hedman et al.,-54-1988). Two techniques which had been previously reported for their ability to measure average affinities of IgGfrom serum (Steward et al., 1991; Inouye et al., 1984) were investigated for their applicability in the RV antigensystem. Experiments were performed to establish the ability of these techniques to demonstrate affinitymaturation of anti-RV serum IgG following primary RV infection in humans.3.1.3.1.One-Well Inhibition ELISAThis ability of this technique to measure relative affinities of sera to RV was evaluated using patient seracollected at sequential time intervals after onset of natural Rubella infection to determine if the affinitymaturation of the IgG response in the first 3 months following infection could be demonstrated. Prior to theseexperiments it was established that the concentration of liquid phase antigen required to bring about a 50%reduction of the signal obtained with no competitor antigen (l) was independent of the constant antibodya)0CQL.0cn10Figure 14: Determination of‘05 in Acute and Convalescent Sera. Serum samples were obtained at 12 days(acute) and at 8 months (convalescent) following diagnosis of Rubella rash. The specific absorbance of theserum in the absence of competing antigen is shown on the left side of the y-axis (0.49 and 0.67 for theconvalescent and acute sample respectively), with the 50% absorbance values indicated on the right hand sideof the axis. The‘0.5 values were extrapolated from the best fit line through the linear region of the titrationcurve and are indicated as underlined values on top of the x-axis.-55-.—. 12 Days Post Symptom•.- 8 Months Post Symptom0 10’OOO 1000 100Inhibitor Dilution (1/x)concentration chosen, as long these antibody concentrations were on the linear region of the RV ELISAtitration curve. For all sera tested the 10.5 were identical at serum dilutions of 1:250 to 1:1000. It was howeverrequired to plot RV EUSA titration curves for each serum in order to establish the linear range dilutions.Figure 14 illustrates the determination of the Io for an acute (12 days) and convalescent (8 months) serumsample following the onset of a rubelliform rash in an adult patient.The‘0.5 values were determined in three separate experiments for paired (acute and convalescent) serumsamples from 3 patients. The results shown in Figure 15 reveal that this technique could demonstrate affinitymaturation of anti-RV IgG in the months following Rubella, although patient numbers were not of sufficientsize to determine statistical significance.1000-1-00 -10812 18 6 8Days MonthsTime foflowing Onset of Rubella RashFigure 15. Affinity Maturation of Anti-RV IgG Measured by Inhibition EUSA. TheI05values were measuredin three individual patients (0, , 1) at 8, 12 and 18 days or 6 and 8 months following the onset of a rubelliformrash. Each patient sample was tested three times. Error bars indicate SD.-56-3.1.3.2.8M Urea Elution ELISALow affinity antibodies are easily eluted from their antigen by briefly exposing the antibody-antigen complex todenaturants such as 8 M urea, whereas high affmity antibodies are resistant to such treatment. This differentialsensitivity of low and high affmity antibodies to chaotropic agents provides the basis for the 8 M urea elutionEUSA. It was initially established that a 4 minute exposure of antibodies complexed to solid phase-bound RVto 8 M urea, provided the optimal conditions for differentiating the low affinity anti-RV IgG in acute sera fromhigh affinity anti-RV IgG in convalescent sera (Fig. 16). The antigenicity of solid phase bound RV was notadversely affected by exposure to 8 M urea, nor was antigen eluted from the microtitre plate by such treatment.This was established by exposing RV-coated and blocked niicrotitre plates to a 4 minute wash of 8 M urea priorto the addition of a 1:1000 dilution of anti-RV standard serum (Fig. 17). This simple technique allowed therapid determination of relative affinities of anti-RV IgG in human sera and was capable of demonstrating theaffinity maturation of these antibodies in the months following Rubella infection.16 065.5%°—° 6 months post rash (PBS)10- --o 6 months post rash (Urea)- 2 weeks post rash (PBS)o-.-- 2 weeks post rash (Urea)0.8-0.6-0-< 0.4-0.2-- - - -i.— —‘s.-.400 800 1600 6400Dilution FactorFigure 16. 8M Urea Elution ELISA for Determination of Antibody Affinity. Paired serum samples from apatient taken at 2 weeks () and 6 months (I following the onset of a Rubella rash. The dilution series washedin WB only are indicated by solid lines, the 8 M urea washed dilution series is indicated as a dashed line. Aboveeach point the calculated elution ratio [ER( %)] is given, showing that ER( %) remains relatively constant overa wide absorbance range.57.9 %4.8 %a.• 52.9 X5.1 %54.0 %7.6 Z3200-57-1•0000C0.5-0.0-1000 2000 4000 8000 16000 32000Dilution of Whole RV Preparation (1/x)Figure 17. Treatment of Solid Phase Bound Rubella Virus with 8M Urea. Whole RV was coated in two-folddilutions onto microtitre plates and then either washed for 4 minutes with 8 M urea wb (o) or with wb (Iv, inorder to determine whether 8M urea affected the antigenicity of RV. Standard ELISA protocol was thenfollowed using a 1:1000 constant dilution of anti-RV standard serum to detect RV antigen.3.1.3.3.Preparation of IgG Fractions from Human SeraBoth techniques described above were capable of measuring low affinity anti-RV responses in the first weeksfollowing Rubella. Although the primary response is initially characterized by a low affinity IgG response, it isalso typical to detect high levels of antigen-specific 1gM in the first weeks following infection. Hence, it wasimportant to determine if the presence of 1gM in RV antisera influenced affinity measurements of anti-RV IgG.For that purpose, lgG fractions were prepared by separating this immunoglobulin class from IgA and 1gM usingFPLC anion-exchange chromatography. The FPLC system allowed rapid and repetitive runs of 100 11 serum.The elution profile of33%, 48% and 70% buffer B, allowed the best separation of three immunoglobulin classesG, A and M (Fig. 18A). The overall yield of IgG was 60%, with the yield of IgG in the purified IgG pooi being50%. Losses of protein may have occurred during filtration of sera, losses of protein in peak trails which werenot included in the pooled fractions or during dialysis. The dialysed Ig fractions were lyophilized and stored at -70#C. IgG fractions and sera were compared for three patients, each containing an acute and a convalescentsample, using the One-Well Elution ELISA and the 8 M Urea Elution EUSA. It was shown that the presenceof RV specific 1gM could adversely influence the measurement of IgG affinity when using the One-Well-58-e—o PBS WASH0-, 8M UREAAECI1)C”Cci)C-)C0-c0(I,-oC04-,-4-,Cci)C)C0(-)CD-D00’0CDEEFigure 18. A: Elution Profile of Serum Separation. Each fraction contained 1 ml and a step gradient was runas indicated by the solid line. Eluted protein is indicated by the dashed line. Three pools were collected, eachcontaining 5 mIs. B: Quantitation of immunoglobulins in each of the pooled fractions (G, A or M) and serum(SER).1001 .0.50ci)4-9-DFraction NumberBEZZI g GZI g ALEn Ig M1400-1200-1000-800-400200G A M SERPooled Fractions-59-Inhibition ELISA (Table 6). It is likely that RV-specific 1gM binds liquid phase antigen resulting in theincreased requirement of liquid phase antigen to compete out IgG binding. This results in a shift to the rightof the inhibition titration curve and in an overestimation of the In contrast, the affinity measurements usingER(%) 150Serum IgG Fraction Serum IgG FractionPatient 1 acute 8 5 7 50convalescent 53 48 390 400Patient 2 acute 1 2 20 63convalescent 52 47 398 410Patient 3 acute 12 18 50 60convalescent 70 71 450 450Table 6. Relative Affinity of Anti-RV IgG in Serum or Purified IgG. Serum samples from three patients werecollected during the acute and convalescent period following Rubella. IgG fraction, prepared by ion-exchangechromatography, and whole sera were compared for their relative anti-RV IgG affinity measured by using both8M Urea Elution ELISA (ER(%)) and the One-Well Inhibition EUSAthe Elution EUSA were not affected by the presence of RV-specific 1gM. The overall amount of RV-specificIgG binding to the solid surface may well be decreased by competing 1gM, this however should not affect theratio of low and high affinity antibodies, as measurements of ER(%) were performed in antigen excess (ie: inthe linear region of the titration curve).Comparison of the One-Well-Inhibition ELISA and Elution EUSAThe inhibition EUSA showed two main disadvantages over the Elution EUSA:1) The presence of anti-RV 1gM interfered with the determination of affinity values when using the inhibitionassay. The valence of 1gM may give this molecule an advantage over IgG for binding antigen in the fluidphase. The reason why this interference of 1gM with affinity measurement of IgG is observed remainssomewhat of a puzzle. Other groups using a similar assay with peptide antigen, have not observed any-60-difficulty with 1gM in acute sera (Dr. M.W.Steward. personal communication).2) The amount of antigen required for the liquid phase inhibitor exceeds the requirement of antigen by theElution ELISA by 100 to 1000 fold. This would have provided a significant obstacle for the use of purifiedRV proteins.Based on the above describe observations, it was decided to proceed with the Elution ELISA as the assay ofchoice, which would be employed to study the relative affinities of sera to whole virus as well as to the separatedRV structural proteins El, E2 and C.-61-3.2. Section II: Protein Specific Immunity: Antibody Quantitation and Affinity toSeparated RV El, E2 and C.Rubella virus contains three structural proteins which are immunogenic in humans. However it is the El-specificantibodies which have hemagglutination inhibition activity and the majority of the in vitro neutralization activities.In mouse monoclonal antibody studies 4 El-specific epitopes have been defined which have both in vitroneutralizing (in absence of complement)- and hemagglutination inhibition activity. A further 2 epitopes havebeen described to have hemagglutination inhibition activity only. On the other hand only 1 epitope has beendefmed on E2 which can neutralize RV replication in vitro (Green and Dorsett, 1986). It is therefore believedthat the response to the envelope protein El is paramount in protective humoral immunity. As whole RVassays, such as those described above, do not differentiate between protein specific responses, it is necessary todevelop protein specific immunoassays to quantitate the antibody responses directed to individual viral proteins.Assays such as immunoprecipitation (Katow and Sugiura, 1985), purified RV protein ELISA (Mauracher et a!.,1992a) and Western blotting (Zhang et a!., 1992) have allowed the investigation of protein-specific immuneresponses following Rubella infections. This section will discuss the development and some applications of thelatter two techniques referenced above.3.2.1.Western Blotting3.2.1.lReducing versus Non-Reducing ConditionsFigure 19 illustrates a Western blot and autoradiograph of gel separating viral proteins under reducing ( + 2-ME) in the left lane and non-reducing conditions (- 2-ME) in the right lane. This SDS-PAGE separation wasunorthodox in that both wells were positioned side by side to each other and the diffusing of reducing agentduring the stacking run into the right lane, which contains non-reducing sample buffer, illustrates the decreaseof both El and E2 antigenicity in the presence of reducing agents. Capsid, under non-reducing conditionstravels as a dimer (Cd). Its antigenicity is not stronger in the absence of reducing agents, and Cd is poorly visiblein the Western blot in Figure 19. The autoradiograph of the Western blot shows that the increase in signalstrength observed in the immunoblots is due solely to antigenicity and not to quantity differences. The apparentmolecular mass of El was observed to increase by about 4 kD upon reduction. This suggests that native El isheld in a compact tertiary conformation. El contains an average of 5 mole % cysteine residues (Clarke et a!.,-62-1987) which likely contribute to the highly folded conformation of this protein. Although E2 contains a similardistribution of cysteines, its apparent MW is not affected by reducing conditions. The C protein travelled as adimer under non-reducing conditions, and the transition from the 66 kD dimer to the 33 kD monomer can seenin the autoradiograph in Figure 20A. As the addition of reducing agents selectively affected the antigenicityFigure 19. Western Blot and Autoradiograph of Reduced and Non-Reduced Preparations of RV. A: Westernblot developed with polyclonal human anti-RV serum as primary antibody. Both lanes contain equal amountsofL-[35S]methionine labelled RV, run in the presence (+ 2-ME) or absence (- 2-ME) of 2-mercaptoethanol.Reduced ( ) and non-reduced ( ) viral structural proteins are indicated at the margin: (D) homodimeric andheterodimeric forms of El and E2, (Cd) capsid dimer, as well as the monomeric forms of El, E2 and C. B:Autoradiograph of the Western blot illustrated in A. (Mauracher et al., 1992a)of El and to a lesser extent that of E2. Western blot protocols were put into practice which excluded the useof reducing agents. It became apparent that non-reducing blots were about between 1 - 2 log units moresensitive than those performed under reducing conditions and evidence by others indicates that the non-reducingblots may shows equal sensitivity as RV IgG ELISA (Dr.L.A.Mitchell and Ms.M.Ho, Dept. of Pathology andPediatrics, UBC, personal communication). Before the development of the immunoblotting technique for thedetection of RV specific IgG, immunoprecipitation of radiolabelled RV was the assay of choice for the detection-63-A BWestern Blot2-ME+ —D$El =-E2I<Cd<ElAutoradiograph2-ME+DE1E1E2i <E2ct.of protein-specific IgG, and had been used in two studies to determine the protein specific responses in rubellaresponders (Katow and Sugiura, 1985; de Mazancourt et al, 1986). If the immunoblot technique was to replacethe accepted methodology for the measurement of protein specific responses it had to be demonstrated thatinmiunoblot provided similar response profiles as observed with immunoprecipitation.3.2. 1.2.Comparison of Immunoprecipitation and Western Blot under Non-Reducing ConditionsSix patient sera were compared for their RV protein-specific IgG responses using immunoprecipitation andimniunoblot assays performed under non-reducing conditions (Fig. 20). The results show that immunoprecipitation overestimates the amount of anti-E2 IgG present in serum. Lane 2 contains a serum with no E2 specificantibody as determined by the immunoblot technique. This may be due to two factors. IfL-[35S]-methionineis used as a metabolic label then differential distributions of this amino acid in the RV proteins has to be takeninto account. El contains only 3 methionine residues whereas E2 and C contain 7 and 6 respectively (ClarkeImmunoblot Immunoprecipitation1 2 3 4 5 6 1 2 3 4 5 .6CdElE2 • E2cFigure 20. Comparison of Anti-RV IgG Western Blot and Immunoprecipitation Assays. Six serum samples fromone negative control and 5 RV serology positive patients were subjected to comparative analysis by Immunoblotor Immunoprecipitation. Lane 1: negative control; lanes 2-4 responders to wild RV infection; lanes 5 and 6:CRS patients.et al., 1987). This problem could be overcome by replacing methionine with tritiated amino acids in order toensure equal labelling of RV proteins. The problem of co-precipitation of El and E2 was more difficult tosolve. Monoclonal mouse IgGs directed against El often precipitate E2 protein (Brush, 1989) as dimerized-64-forms of the envelope protein are resilient to dissociation even in the presence of ionic detergents. Thediscrepancy between immunoprecipitation and Western blot techniques with respect to detection of E2 signalsmay therefore be due to co-precipitation in the immunoprecipitation assay. The two techniques correlate wellin respect to El- and C-specific signals.-- PatIent 1 Patlent2-. Patient 3 -MW1 23456789 10111284J — DIgG4E2L—- -1 23456789101112— r— i E -IgAL’_ :__ _E1 2 3 4 5 6 789 10 1112rEFrL084 I1gM 58 IFigure 21. Immunoblot Analysis of RV-Specific IgG, 1gM and IgA antibodies at intervals following wild RubellaInfection. Western blots of RV proteins separated by non-reducing SDS-PAGE were developed withrepresentative sera obtained at the following time iptervals after the diagnosis of a rubelliform rash: lanes 1, 5and 9: 0-6 days (early acute phase); lanes 2,6 and 10:7-30 days (acute phase); lanes 3,7 and 11: 1-11 months(convalescent phase); lanes 4, 8 and 12: 1-3 years (late convalescent).Western blotting provided a good alternative to immunoprecipitation and showed considerable advantages:a) independence from radiolabelled reagents; b) technical difficulties such as unequal protein labelling and coprecipitation are avoided; c) rapid results ; d) more amenable to the processing of large samples and e) allowingthe determination of RV protein-specific 1gM and IgA antibodies in addition to IgG.The immunoblot technique was sensitive enough to detect IgA and 1gM directed to the individual RV proteins(Fig. 21). An analysis of three patients, recovering from wild type RV infection shows the rapid increase of RVspecific 1gM in the first months of RV infection, followed by its decline within a year following infection. Both-65-D.H. V.S.Figure 22. 8M Urea Elution of Western Blot. Paired serum samples from Patient A and B, taken during theacute (18 or 12 days) and convalescent phase (8 months) following natural Rubella infection. Strips labelled“a” represent nitrocellulose strips washed in PBS buffer following serum incubation. Strips “b” show blotsincubated with the same sera as in lanes “a” ,but were washed for 3 minutes in 8M urea elution buffer. Positionof El and C is indicated on the right side. Anti-C IgG was readily eluted by urea, both in acute andconvalescent samples whereas anti-El IgG resisted urea wash in the convalescent samples, indicating adifferentially higher affinity of anti-El IgG in the convalescent sample.PAGE, one strip was washed in PBS, the other to be washed for 3 minutes in elution buffer, as described abovefor affmity ELISA. As was observed with whole RV ELISA, signal strength was not effected when blots werewashed with urea prior to the addition of antibody. The paired, acute and convalescent, serum samples obtainedfrom patients following Rubella showed antibodies reactivity to El and C, but not to E2. The El-specific IgGmatured in its affinity and became resistant to elution in the 8 month samples. In contrast the anti-C IgG wasnot resistant to urea elution in either the 18, 12 day or 8 month samples, suggesting that these antibodies didIgG and IgA responses are maintained in the first three years following primary rubella. The kinetics of protein-specific responses of IgG and IgA are discussed further in section ifi.3.2.1.3.Western Blot AffinityAssayThe principle of chaotropic elution of low affinity antibodies was applied to Western blots (Fig. 22). Patient serawere applied in duplicate to nitrocellulose strips containing blotted RV proteins, separated by reducing SDS12d 8ma b a bElC8ma b—18da b-66-not undergo affinity maturation.These results indicated that the affinities of antibodies directed to the individual viral proteins may not beequally distributed. Differential affinities of antibodies to proteins of a viral pathogen had not been reportedpreviously and the possibility that one protein was immunodominant and was giving rise to both higher levelsas well as higher affinity antibodies was investigated further (Section ifi).Most investigators which have analyzed the specificity of anti-RV antibodies have employed eitherimmunoprecipitation (Katow and Sugiura, 1985; de Mazancourt et al., 1986) or immunoblotting under reducingconditions (Partanen et al., 1985; Cusi et a!., 1989). With the new observations reported here, concerningsensitivity and applicability of imniunoblotting performed under non-reducing conditions to the detection of Igof all classes (Mauracher et al., 1992; Zhang et al., 1992) this technique may find application in serology duringthe early post-conception period to verify RV infection. The ability to determine specificity of IgA for individualRV proteins may aid in the understanding of the role of both serum and mucosal IgA in the protection againstviral reinfection and provide a technique to further our understanding of the role of persisting serum IgA as anindicator of persistent RV infection (Morris et a!., 1985).Some disadvantages remain inherent to the Western blot technique. The technique is more time consumingand costlier than ELJSA and the quantitation of peaks by densitometry can introduce bias in the interpretationof protein specific signals. It became desirable to obtain RV proteins El, E2 and C in pure form and to utilizethese proteins in the EUSA protocol.3.2.2.Seyaration of RV Proteins. Their Use in Solid Phase linmunoassaysMany strategies for the separation of viral proteins have been described. The properties of El, E2 and C, suchas size, charge or solubility, can be used to purify these proteins. The starting material for the separation ofthe RV proteins was the highly concentrated virus stock described earlier in section I, which was highly antigenicand also immunogenic in animals. The goal of the protein purification strategy was not necessarily to arrive atpreparations which showed high levels of purity for each protein, but to arrive at preparations which were purein respect to other RV proteins. Hence the term “RV protein separation” will be used rather than “proteinpurification”.Although the envelope proteins El and E2 can be readily separated from the capsid protein using detergent-67-extraction (Mauracher et al., 1991a) (Fig. 29), the separation of El from E2 was more difficult. Both envelopeproteins have similar solubilities and have overlapping p1 ranges due to glycosylation with complex sialylated N-linked sugars (Lundstr dm et al., 1990). More importantly they form heterodimers which are resistant to all butthe most severe denaturing conditions. As described above, non-reducing immunoblots showed similar immunoreactivities as immunoprecipitations and supported the concept that SDS-PAGE under non-reducing conditionsand low heat may yield separated RV structural proteins with antigenic properties similar to that of nativeBAFigure 23. Yield of Radiolabelled RV Proteins after Electroelution. A: Bio-Rad model 422 electro-eluterassembly. The vertical glass tube is filled with elution buffer. The negative electrode is at the top, the positiveelectrode is below the membrane cap. RV macromolecules are carried by the electrical current outof the gel slice, through the frit and into the membrane cap (400 11). The molecules are retained by adialysis membrane (MW cutoff 12-15 kD) which is moulded into the cap. (From: Bio-Rad, manual 87-01330387,1987) B: Yield of radiolabelled RV proteins by electroelution. Counts per minute (cpm) were determined ingel slices before and after electroelution (PRE and POST), “Top 1 nil” indicated a 1 ml sample of elution buffertaken from above the frit.Top 1 mlGel SiloElectro EluateHg. 1. Side view of Model 422 Electro-Eluter Assembly. The vertical glasstube is filled with elution buffer. The negative electrode is at the top. Thepositive electrode is below the membrane cap. Macromolecules are carriedby the electrical current out of the gel stice. through the tnt arid into themembrane cap. The molecules are retained by a dialysis membrane whichis molded into the cap.Viral Protein PRE Electro Eluate Top 1 ml POST Yield %Gel Slice Gel SliceC 1737 1960 120 76 112El 939 923 65 62 98E2 2067 2025 108 72 98-68-protein. Using preparative SDS-PAGE, followed by electroelution provided a feasible strategy for RV proteinseparation (Mauracher et al., 1 992a).3.2.2.1..Feasibility of Electroelution; Yield and Purity of RV Proteins.The yield of this separation technique was initially established as follows: L-[35S]methionine labelled RV wasseparated on 10% acrylamide slab gels and the relative positions of the viral proteins were determined by anovernight autoradiograph of one gel lane. The RV proteins El, E2 and C were excised from the gel, with one-half being electroeluted and the other half stored for the determination of radioactivity prior to elution. Samplesof electroeluted proteins in the membrane cap, the electroeluted gel slices above the frit and elution buffer inthe glass tube were solubilized in the scintillant and counted for radioactivity and was compared to the countsin the gel slices which were not electroeluted. Gel slices and electroeluate were added to I ml of scintillant andleft in the dark over night. Counts per minute from pre- and post-eluted gel fragments, electroeluate and elutionbuffer as well as yield determinations are shown in Figure 23. The yield of protein was 90 - 100%. This was1 2 3 4DCdElE2Figure 24. Western Blot of Separated RV proteins. Preparations of capsid dimer (Cd), El and E2 proteins wererun on non-reducing SDS-PAGE in lanes 1, 2 and 3 respectively. Purified whole RV was loaded in lane 4, allviral structural proteins were visualized including the dimerized species of El and E2 (D). (From Mauracheret al., 1992a)-69-also confirmed by electroeluting known amounts of BSA and determining protein concentrations followingelectroelution (data not shown).The monomeric species of the RV envelope proteins, El and E2, and the C dimer differ in mass by about10 kD each. This difference in MW is sufficient to separate these proteins on 10% SDS-PAGE slab gels. Gelswere run until the 27 kD pre-stained MW marker reached the gel front, then the proteins were excised usingthe pre-stained MW markers as guides and electroeluted. Each electroeluate was checked for purity byWestern blot and the preparations shown to be free of the other two respective viral proteins were pooled.Figure 24 illustrates a Western blot of the stock preparations of separated RV El, E2 and Cd, each antigenpreparation is pure in respect to viral protein contamination.EC11.0ECU,UCa0E10 20 40 SO 160 320R.ciprocal DIutIon of MtIgen PreparationFigure 25. Comparison of Optimal Coating Concentration for El, E2 and Cd. Anti-RV protein IgG EUSAs wereperformed with a constant concentration of anti-RV reference serum on microtitre plates coated with serialdilutions ofEl, E2 or Cd containing 0.1% SDS (•ff•) and followingremoval of SDS by KCI precipitation (I-(From: Mauracher et al., 1992a)C-70-3.2.2.2Maximization of Protein BindingPolystyrene, in the format of 96-well microtitre plates, was chosen as the solid phase for the adsorption of thepurified RV proteins. Different plates supplied by Nunc, Costar, Falcon and Dynatech were tested for theirabilities to bind RV proteins under various coating conditions: PBS, phosphate buffer pH 7.4, carbonatebuffered saline pH 9.4, carbonate buffer pH 9.4, distilled water and drying protein onto solid phase in distilledwater. Immulon-2 plates in combination with PBS as the coating buffer were found to be optimal for thecoating of each RV protein. Each protein preparation had an optimal coating concentration (Fig. 25) and thedilution yielding peak absorbances were established to be: 1:40, 1:80 and 1:20 for El, E2 and Cd respectively.The titration of the El and E2 preparation exhibited decreased absorbance values at concentrations of SDS of0.01% and 0.005% ,whereas Cd was not affected by the presence of SDS. When SDS was removed from theantigen preparations by KC1 precipitation prior to coating, the marked prozone seen at dilutions of 1: lOand 1:20was not observed. However, a loss of up to 50% of the protein was realized during KC1 precipitation. Thedecrease of El and E2 antigen signals at low dilutions may be due to the competition of SDS with protein forhydrophobic binding sites on the plate or actual electrostatic repulsion of SDS-protein complexes from thecharged polystyrene surface.3.2.2.3.Quantitation of anti-El, -E2 and -c IgG by ELISAIgG directed to the individual RV proteins was determined in a similar manner to that directed to whole RV.The laboratory standard serum containing 943 lU/mi of anti-RV IgG was analyzed on non-reducing blots andthe ratios of IgG directed to each protein were determined. The 943 lU/mi of anti-RV activity were thenassigned to anti-El, -E2 and -C IgGs according to the percentage determined by Western blot. Figure 26 showsa Western blot of the standard serum with its densitometric trace. The dimeric forms of the envelope proteins,El dimer (El d) and El-E2 heterodimer make up about 35% of the total signal on the blot, and were includedin the calculations. The slowest travelling dimer band was assumed to be Eld. A second dimer band, belowthe El d’ often stains more weakly, and was presumed to be the El -E2 heterodimer. The signal from this bandis attributed to both anti-El and -E2 IgG and was divided according to the ratio of El/E2 monomer specificIgG. E2 homodimers has thus far not been described. It is not known if distinct epitopes exist for the dimericEl or E2 forms, but all the described El- and E2-specific mouse monoclonal antibodies show reactivities to both-71-monomeric and dimeric forms of El or E2 (J.Wolinsky, University of Texas, personal communication).The standard serum was blotted and scanned in four separate experiments and the serum was assigned thefollowing concentrations of RV protein specific antibodies, expressed in arbitrary units per millilitre of serum(AU/ml): El (456 AU/mI), E2 (390 AU/nil) and C (97 AU/nil). Five dilutions of the standard serum wereincluded on all plates coated with either detergent solubilized RV or electrophoretically separated El, E2 orCd. The concentration of protein-specific IgG could then be determined by performing quadratic regressionon the absorbance values obtained for the dilutions used for the construction the standard curve.V (i1.41.41.00.80.0.40.20.c I I I I it itEld El-E2 Cd El E2Figure 26. Western Blot and Densitometric Scan of Anti-RV Standard Serum. 48.4% of total signal strengthwas attributed to anti-El IgG (13.9% from Eld, 9.6% from El-E2 and 24.9% from El); 41.4% due to anti-E2IgG (11.5% from El-E2 and 29.9% from E2) and 10.2% due to anti-C IgG (10.2 from Cd). The El-E2heterodimer can be seen as a doublet on the blot, but is poorly resolved, as a peak with a shoulder, on thedesitometric scan.3.2.3.Antipenicity and Bioloical Activity of Separated RV Structural ProteinsAs described above it was established that the antigenicity of the El and E2 proteins were sensitive todenaturation by reducing agents. It was important to determine the effects of SDS and heat, the maindenaturants involved in non-reducing SDS-PAGE, on the antigenicity of the separated RV proteins. Antibodyquantitation and affinity measurements were dependent on intact proteins, which had retained their epitopesduring the purification protocol.Initial results indicated that denaturation by low concentrations of SDS (0.25%) was reversible but that boiling-72-of RV in non-reducing sample buffer lead to irreversible denaturation leading to a loss of antigenicity asmeasured by EUSA (Fig. 27). Whole RV preparations were diluted 1:10 in a 0.25% solution of SDS and theneither boiled for 3 minutes or Left at RT. As a control, a preparation was diluted in PBS only. All preparationswere then serially diluted from 1:10 to 1:1280. The control titration curve is non-linear at dilutions of 1:10 and1: l6Oand becomes linear beyond that. The virus, treated with SDS without heating, showed an equal end-pointtitre to the control preparation, indicating that denaturation by SDS was reversible. In the saturated region ofn—c No SOS2.0 u—n .25 % SDS-c .25 % SDS,E boiledC10 20 40 80 160 320 640 1280Viral Antigen Dilution (1/x)Figure 27. Effect of SDS and Temperature on the Antigenicity of Whole RV. RV preparation were either nottreated with SDS (0) or treated with SDS (0.25%) and subsequently held at either 60#C (1) or boiled () for3 minutes. Each RV treatment group was coated in serial dilution onto microtitre plates and its antigenicitywas tested by RV IgG-specific ELISA, using a constant dilution of human anti-RV reference serum (From:Mauracher et al., 1992a).the titration curve a noted depression of absorbance was observed indicating that SDS at concentrations above.001% interfered with binding of protein to the solid phase. Heat denaturation in presence of SDS led toirreversible loss of antigenicity indicated by a lower end-point titre of this preparation. This treatment reachesthe same plateau as the unboiled SDS-treated preparation in the saturated region of the titration curve, againsuggesting that the loss of absorbance is due to binding inhibition rather than denaturation. Although the above-73-experiment indicated that denaturation of RV proteins due to the employed SDS-PAGE conditions (0.25% SDS,60C, non-reducing conditions) were minimal, the effect of separation on the biological activities of El and Cprotein were further investigated to determine if the purified RV proteins had retained their antigenic integrity.Biological activity of a protein is generally dependent on its structure. It was therefore reasoned that ifseparated viral proteins had maintained their biological functions, then their structure and also their structurallydependent epitopes had remained intact throughout the purification procedure. Both El and C proteinpreparations were analyzed for some of their biological activities.3.2.3.1J?unction of ElThe El protein of RV contains the majority of the currently identified neutralization domains (Waxham andWolinsky, 1985b) as well as the region involved in the agglutination of chick and goose erythrocytes. Both theseactivities are sensitive to denaturation (Ho-Terry et al., 1985; Qui and Gillam, 1992). This protein is not onlythought to be involved in attachment of the virus to the target cell, but is also involved in low pH induced virusendosome membrane fusion, through low pH (<5.5) activation of fusogenic properties (Katow and Sugiura,1988). Separated El protein preparations were analyzed for their HA- and HAl antibody binding activity. Theseparated El protein by itself was not capable of hemagglutinating chick red blood cells (CRBC) (Dr. S.Gillam,Dept. of Pathology, UBC, personal communication). This was not surprising as CRBC agglutination requireswhole virion or cross-linked El. The HA activity of the electrophoretically separated El was measuredindirectly by its capability to compete for HAl antibodies with native Rubella virions (Fig. 28). The Elpreparation was capable of competing for HAl antibodies in human serum. El preparations at a volume of 25and 12.5 11, were capable of binding sufficient antibody in the 1:160 diluted antiserum to allow virus to cross-link CRBC and form a mat. At volumes of 6.25 11 or less of competing El, sufficient HAl IgG was left toinhibit CRBC crosslinking. The equivalent series using C preparations showed that the competition for HAlantibodies was exclusively due to El. As HAl antibodies recognize structural domains on El (Ho-Terry et al.,1985), it was possible to conclude that the separated El had maintained its native structure during purificationprocedures.An alternate approach for directly measuring intact HA activity of El preparations would have been to letthe KC1 treated El preparation interact with CREC followed by the exposure of such sensitized erythrocytes-74-Volume of lnhibftor25 12.5 6.25 3.1 00000000000000000000000000000000OG0000025 12.5 6.25 3.1 000:of0*0000)oooooFigure 28. HAT Antibody Competition Assay. El and C preparations were used to compete for HAT antibodieswith whole rubella virions. Four dilutions of the anti-RV standard serum (1:40, 1:80, l:160,and 1:320) with aHAT titre of 1:160 were added to 5 wells per row. To each row, containing constant serum dilutions, varyingamounts of either El or C preparations were added (25, 12.5,6.2,3.1 or 0 11). Each well was then brought toa fmal volume of 50 11. After a 1 hour incubation at 44C each well received 25 11 of 4 HA units of RV.Following 30 minutes incubation each well was loaded with 25 11 of a 0.02% solution of CRBC. Negativecontrols included the use of RV negative serum at l:40with both El and C antigen. Serum controls tested bothnegative and positive serum for endogenous HA activity. Cell control tested for button formation in the absenceof both serum and HA antigen.to HAl positive antisera in the presence of complement. Lysis of the cells would have indicated binding ofElonto the erythrocytes, thereby proving biologic activity of the HA domain of El.3.2.3.2.Function of C proteinThe biological activities of the RV C and E2 proteins are less well defmed. Although E2 protein was recentlydescribed to contain both N- and 0-liniced oligosaccharides (Lundstr6m et al., 1991) and is proposed to be anabsolute requirement for the transport of El to the cell surface, followed by virion assembly (Hobman, 1989),1 :40E 1:801:160>1:3200.E 1:400C.2 1:8001:1601:320ElCNeg. HAl Serum, 1:8Neg. HAl Serum, 1:8Serum ControlCell Control-75-a clear biological function of E2 in the assembled virion has not been established. Hence, it was not possibleto analyze the biological function of purified E2 preparation. Similarly the capsid protein of RV has nodescribed biological function in the assembled virion, other than the described morphological feature of thenucleocapsid. By inference, from the work on the related aiphaviruses, SFV and SV, it is proposed that the Cprotein initially binds to the 5’ end of the full length + sense RNA (Weiss et a!., 1989) to initiate nucleocapsidassembly, leading to the budding of the assembled nucleocapsid from the plasma or trans-Golgi membrane(Hobman, 1989).During experimentation on the detergent extraction of RV envelope proteins, using TX-i 14, it was noted thatthe capsid protein, which under physiological pH conditions is not soluble in detergent, became detergent-solubleat pH 4.5 (Fig. 29A). This change in solubility properties was examined further using detergent extractions ofA Sucrose Cushion RuV Triton X-114 PelletpH 45 7.3 9.6 45 7.3 9.6EDTA— + — + — + — + — + — + BpH 4 5 55 6, ,,a. mA —ElE2 Elc E2CFigure 29. Autoradiographs of TX-i 14 Extracted RV. L-[35S]methionine-labelled RV in TCS was detergentextracted and separated by reducing SDS-PAGE. A: Extraction performed at pH 4.5,7.3 and 9.6 ,in the presenceand absence of 10 mM EDTA, showing the detergent pellets and the corresponding 6% sucrose cushion. Thelane marked RuV contains untreated rubella virus. B: Detergent pellets from TX-i 14 extraction performed atp1-I 4-9 are shown in the first seven lanes from the left. Lane V contains untreated rubella virus. (From:Mauracher et al., 1991a)TCS containing radiolabelled RV performed at narrow pH intervals (Fig. 29B). C protein underwent a solubilityshift from being hydrophilic to hydrophobic below pH 5.5. RV proteins are not acid precipitated at pH 4.5,nordoes Triton X-114 change its properties within the pH range used in these experiments. The appearance of the6.5 7 8 V-76-capsid band in the detergent phase below pH 5.5 can be explained by a p11-induced structural change of thisprotein, either causing an intrinsic change in its solubility properties or allowing it to interact more strongly andco-solubilize with the lipid soluble El. To rule out the latter, TX-i 14 extractions were performed on cell lysatesof insect cells expressing the cloned RV capsid gene, independent of El, E2 or the RV RNA (baculovirusexpressed C protein was generously supplied by Dr. S.GiIlam, Dept. of Pathology, UBC). This recombinantcapsid protein also underwent the solubility shift described for the wild-type capsid, indicating that the proposedsolubility shift was an intrinsic feature of the C protein (Mauracher et a!., 1991a). To establish that the observedcapsid solubility shift also represented a property of the intact nucleocapsid, the effect of TX-i 14 extraction andSDS treatment at pH 5 and 7 on the sedimentation of the virion and nucleocapsid through 22% sucrose wasdetermined (Fig. 30A). The relative concentration of virion pelleting through the sucrose cushion followingtreatment at pH 5 or 7 in the absence of detergent was compared to the equivalent volume of TCS loadeddirectly onto the gel. The pronounced “smile” and lowered apparent MW is caused by the displacement of Elby large amounts of BSA present in the TCS. TCS treated with 1% SDS at both neutral and acidic pH resultedin the disassembly of both the virion and the capsid into individual structural proteins, which remain buoyantin 22% sucrose, resulting in the absence of viral proteins in the pellet. Intact capsid can be differentiated fromdisassembled capsid by its ability to pellet through the 22% sucrose cushion. Extraction of TCS with 1% TX- 114at neutral pH did not interfere with the capsid structure as C protein was detected in the pellet of theultracentrifuged aqueous supernatant of the pH 7 extraction. If the TCS was acidified following the additionof TX-i 14, capsid was removed by its solubility in the detergent phase, resulting in the absence of a detectableC protein in the pellet upon ultracentrifugation. Viral RNA was detected in the pellets of all treatment groups(Fig 30B). The naked 40S genome of RV pellets through 22% sucrose (Oker-Blom et al., 1984). The presenceof RV genome in the pellet following TX-i 14 extraction at low pH indicates that RNA had been released intothe aqueous supernatant following the solubilization of the proteinaceous structure of the nucleocapsid into thedetergent phase.Recent observations made on the cytopathic vacuoles of SFV infected cells (Froshauer et a!., 1988) haveshown that the virus-encoded RNA polymerase remains associated with the limiting membrane of these vacuolesand that ribosome-RNA-capsid complexes form upon fusion of the viral envelope. This would be consistent witha proposed acid-induced intra-endosomal uncoating strategy, where the solubility shift of the capsid protein at-77-A MW SDS TX-tM None TCS Vp7 5 7 5 7 5 pH.- —84D-58D- ElE226 CB M None TX-114 SOS Neg.5 75 757Figure 30. Ultracentrifugation of RV Following Detergent Extraction A: Western blot analysis of resuspendedpellets following ultracentrifugation. TCS containing i0 pfulml of RV was treated with I % SDS at pH 5 or7 p1-I 5 or 7) or with no detergent at both pH 5 or 7 (None, pH 5 or 7). , overlaid onto 22% sucrose andcentrifuged at 39’OOO rpm. Alternatively TCS was brought to a final concentration of 1% TX-i 14 and thenacidified or left at neutral pH. Following extraction , the aqueous supernatant was overlaid onto sucrose cushionand centrifuged (TX-i 14, pH 5 or 7). The lane labelled Vp contains 0.1 11 purified RV, lane TCS contains theequivalent amount of RV that was loaded onto the gel for each of the above described treatment groups.Molecular weights (x 1000) are indicated on the left margin, and the identity of the RV proteins on the right.B: Visualization of RV-specific PCR products. cDNA synthesized from the resuspended pellets of the sixtreatment groups described above was amplified by PCR using El-specific primers delineating a 287-hpfragment. Lane labelled M contains HaeIJI digest of pUC 19. Lane labelled Neg. contains a negative controlfor contaminating DNA sequences. (From: Mauracher et al., 1991a)pH 5.5 leads to the disassembly of the capsid within the endosome and the release of the viral RNA (Fig. 31).This concomitant shift in structure and properties of both El and C below pH 5.5 suggests that the solubilityshift of the capsid plays a role in capsid uncoating. It has been questioned whether the acidification of theendosome may necessarily lead to the exposure of the nucleocapsid to an acidic environment (Kielian andHelenius, 1986). These authors reasoned that the viral membrane remains impermeable to H + ions and thatthe endosomal milieu would, therefore, not affect nucLeocapsid integrity. Experiments using ion fluxmeasurements across artificial bilayers upon SFV membrane fusion have shown that ion permeability of the viralmembrane occurs only after injurious events such as sonication, freeze-thawing or storage of the virus at RT(Young et al., 1983). These observations support the hypothesis that the intact viral membrane of freshly-78-Figure 31. Proposed Model for the Entry of RV into the Host Cell. Following attachment to the cell surfacereceptor (1) the virus is taken up via the receptor and internalized into a coated vesicle (2). The low pH in theendosome induces the El protein to become fusogenic (Katow and Sugiura, 1988) and facilitates thefusion between virus and lysosome membranes. In parallel, the low pH induces a solubility shift of thecapsid, allowing it to associate with membrane, causing intra-endosomal uncoating (3). Upon membranefusion, viral RNA is released into the cytoplasm. (From: Mauracher et al., I 991a)harvested virions remains impermeable to small ions. However this group concludes that endocytosis in itselfcan compromise membrane integrity. Recent evidence suggests that the spike proteins in SFV act as an ionchannel upon activation by the low pH environment of the endosome (Kempf et al., 1988). The currenthypothesis on the entry of togaviridae suggests that within the endosome a low p11-induced conformationalchange of the viral spike proteins takes place resulting in a) a proton influx into the virion, b) the low pH-induced solubility shift of the nucleocapsid leading to the uncoating of the viral RNA and c) the fusion of viraland endosomal membrane (Mauracher et al., 1991a; Schlegel and Kempf, 1992).The investigation of the solubility properties has led to the formulation of a novel biological property for thecapsid and has lead to a more complete understanding of the events occurring during early events in viralreplication. The property of togavirus capsid proteins to undergo a structural shift leading to solubility changesand the unique b-barrel structure common to many RNA virus capsid proteins (Fuller and Argos, 1987) makesthis event in viral replication a good target for drug intervention. Rhinovirus (picornaviridae) employs a similar-79-entry strategy to the togaviridae, in that the low pH of the endosome leads to the uncoating of the nucleocapsid(McKinlay et a!., 1986). This event can be specifically inhibited by WIN 51711,a drug which tightly associateswith the capsid and causes the protein shell to become less flexible, thereby preventing uncoating of RNA(Badger et al., 1988). It would be of interest to screen similar compounds for their ability to interfere with thesolubility shift of the RV C protein in an effort to identify a chemotherapeutic route to interfere with thereplication of RV and other togaviruses.MW pH5 pH7TXAq TXAq58— El•E237-26— CFigure 32. Detergent Extraction of Separated C protein. Preparations of separated C protein were treated with50 mM KC1 to remove SDS and then adjusted to pH 7 and 5 with 1 N HCI. Preparations were then extractedin 1% TX-i 14, and both the detergent phase (TX) and the aqueous phase (Aq) separated on 10% SDS-PAGEunder reducing conditions. Lane RV contains whole RV, all other lanes contain pre-stained MW markers,indicated on the left margin.The description of a function of C protein, that is structurally dependent, allowed the analysis of theelectroeluted C protein. The C preparation was extracted in TX-i 14 in both acidic and neutral pH, followingthe removal of SDS by KC1 precipitation (Fig. 32). C protein retained its characteristic biological function and-80-became detergent soluble under acidic pH, suggesting that its structure was not irreversibly altered during non-reducing SDS-PAGE.3.2.4.SummaryBoth Western blotting under non-reducing conditions and RV protein ELISA can be used to study the responseof all immunoglobulin classes to the viral proteins. ELISA has the advantage of being faster, requiring fewerreagents and also providing easier interpretation of quantitative antibody measurement. For the quantitationand the assignment of units of protein-specific antibodies, care has to be taken to use both serum samples andthe standard serum at dilutions in the linear region of the titration curve. Software programs which are capableof performing quadratic regressions and can transform absorbance data are of great value (ie: BioRad’sMicroplate Manager , especially if they can analyze data directly from the microplate reader.Much of the work in the development of protein-specific ELISA was concerned with the determination of theoptimal conditions for binding proteins to polystyrene microtitre plates. Although coating buffers were ofimportance in maximizing signal to background ratios , the crucial variable was found to be the type ofmicrotitre plate used in the ELISA. The presence of non-ionic detergent (ie: Tween-20), at concentrations lowerthan 0.05% ,in the sample buffer was also critical in obtaining good signals, particularly when using El and E2protein preparations. It is possible that the non-ionic detergent replaces the protein bound SDS and allows there-naturation of the protein on the plate surface.Of interest was the development of what initially appeared to be an insignificant observation, namely the pHinduced solubility shift of RV C protein. One of the values of the academic environment is that it allows anindividual to pursue an interesting phenomena on an intuitive basis. In the case of C protein the work lead toan additional study which culminated in the proposal of a new uncoating mechanism for RNA virus. Ultimatelythese extra studies became an important part in this thesis, by establishing that RV C maintained at least oneof its biochemical properties following purification-81-3.3. Section ifi: RV-Specific Humoral Immune Responses in Adults Following RubellaWith the establishment of RV-specific antibody techniques it became possible to perform comparative analysisof the immune responses of patient populations following infection with RV. The first subject populationstudied was composed of adults who had a confirmed clinical and serological diagnosis of Rubella. Thesesubjects will frequently be referred to as the “normal” or “control” study group. Subjects were randomly chosenfrom the study sample inventory list and were not selected on the basis of their clinical histories or the outcomeof RV infection.It is necessary to define the normal RV-specific antibody response in an adult population in order to be ableto compare and determine differences in groups developing pathological conditions followingRV infection, suchas CRS or RAA. If such group differences in the immune response are detected, it may become possible todetermine correlations with the pathogenesis or the etiology of the disorder. It is also important to establishthe range in normal immune responses to RV as defmed in adults using current, as well as newly developed,laboratory techniques.3.3.1.Response to Whole Virus and Separated RV ProteinsThe levels of IgG in a group of 15 adults who had confirmed clinical rubella at least six months prior toobtaining the serum sample, were determined using EUSA employing whole RV or individual RV proteins.The average value of IgG directed to whole RV was 202 JJJ/ml — 65 (X — SD), which was in accordance withpreviously reported anti-RV IgG levels (Dimechi et al., 1992). Currently RV-specific IgG levels A15 ITJ/ml areconsidered protective (WHO, 1971). Hence all individuals tested in this group are considered immune to RVre-infection. The response to El was dominant (208 AU/mi .- 115) and significantly (p=0.005) higher than themeasured responses to E2 (56 AU/mi — 16) or C (33 AU/mi — 18) (Fig. 33).3.3.2.Bioloical Activity of SeraPatient sera shown to be RV positive by the above described EUSAs were also analyzed by HAl assay. Themedian titre for this population was 1:32, with values of <1:8 being interpreted as negative (U. S. Dept. of-82-Health, Education and Welfare, 1970). The comparison of serological data obtained by traditional HAlControl Rubella PaUentsa500E 400.300-E -r- TD I +-200- + I’0 H_.1__ •_100. I0Whole RV El E2 CFigure 33: Quantitation of IgG to Whole RV and RV Proteins. Levels of IgG measured by whole RV EUSAare expressed in lU/mI, whereas levels of IgG to separated RV proteins are expressed in AU/mi. Thehorizontal bars indicate the mean, error bars indicate - 1SD. Values for individual patients are indicated bydots.techniques and by solid phase irnmunoassay were in agreement. Similar experiments, performed on a largerscale (Herrmann, 1985) showed a good correlation between HAl and ELISA titres, leading ultimately to thesubstitution of HAT by ETA. Although these two approaches for the detection of RV-specific antibodies givesimilar results in this patient population, the data collected during this study show that the presence of highlevels of anti-RV or anti-El immunoglobulins does not necessarily correlate with the presence of biologicallyactive antibodies detected by HAl (see Section IV).3.3.3.Reactivity to Linear and Topographic EpitopesAs described above, El and E2 were sensitive to denaturation by reducing agents. By performing Western blotsit was possible to determine the distribution of reactivities to continuous and discontinuous (topographic)-83-epitopes among anti-El, -E2 and -C IgG (Table 7).El l0%±4%E2 46%±41%C 125% ± 83%Table 7: Percentage of Signal Remaining on Western Blot under Reducing Conditions. 14 convalescent patientsera were sampled on reducing and non-reducing blot, and analyzed by densitometry. The ODmm for eachprotein was detern-thied and ratios of protein specific signal strength determined for reducing/non-reducingconditions. The mean ratio — one standard deviation for these 14 patients is shown.Conclusions from the data shown in Table 7 were that in normal adults responding to RV infection, the Elspecific response was mainly directed to topographic epitopes with only 10% of the antibodies recognizing linearepitopes. A similar predominance of El specific antibodies to topographic epitopes has been reported in miceinjected with whole RV (Wolinsky et al., 1991). Antibodies directed to E2 were equally distributed betweenspecificities for continuous and discontinuous epitopes, however individual variability was observed. Antibodiesdirected to the C protein appeared to recognize both reduced and non-reduced protein. In many casesimmunoblot bands were stronger under reducing-conditions, suggesting that linear epitopes may remaininaccessible to antibodies when capsid is in its dimeric form.3.3.4.Kinetics of the IG ResponseUsing the Western blotting techniques, the kinetics of IgG responses directed to RV structural proteins wasstudied over the first years followingRV infection (Fig. 34). The response to El was observed to be rapid andcould be detected within the first several weeks following the clinical diagnosis of Rubella. The response to thisprotein reached maximal levels in the months following infection and remains relatively constant in the yearsfollowing infection, with a slight drop-off observed in the second year following infection. The responses to Cdwere observed to be weaker and transient, with levels being maximal at three months. Responses to the E2protein differed from El- and C-specific responses, in that they exhibited a slow onset, with weak responses firstobserved at three months- and plateauing occurring by 12 months post Rubella.-84-Why the response to the RV E2 protein exhibits a slow onset remains speculative. The virus is cleared fromthe blood stream within days of the onset of the rubelliform rash and is not detectable in biological fluidsE*D0CVCILE*a0CVLCV03 Months 1 Year 2 YearsTime Post infectionFigure 34. The Kinetics of the IgG Response to RV Structural Proteins. 9 subjects were followed throughoutthe first two years following Rubella. Serum samples at consecutive time intervals were analyzed by non-reducing Western blot. Each time point represents the average density of the protein-specific bands for 9patients — SD. The top figure shows the mean value for each protein at the given time interval, with a fittedcurve drawn through each point. The bottom figure shows the SD for each point, representing measurementson nine patients.o Elu E20.6- A C0.4-0.2-0.00 Days 10 Days-85-shortly after the onset of the IgG response; it therefore is difficult to account for a continued B-cell responseto E2, long after the virus has been cleared. It is possible that this viral protein may be retained in lymph nodes,as described for other protein antigens (Szakal et al., 1989), or that replicating virions persist. This howeverdoes not explain why only the E2 response is specifically stimulated in the months following infection. Moreintriguing is the hypothesis that continued stimulation of E2 clones is driven by cross-reactivity to selfdeterminants. Reactivities of El- and E2-specific T cells have been reported to be involved in autoimmunedisease in animal models (Yoon et al., 1991) and amino acid sequence homologies have been reported for E2and the human proteolipid protein of myelin (Wolinsky, 1990). Direct evidence for crossreactivity of E2 specificantibodies to self constituents has remained elusive. Recent evidence has demonstrated that RV specific antiseracrossreact with components of human synovial cells (Lund and Chantler, 1991). Therefore it would be ofinterest to investigate if the slow increase of anti-E2 IgG is associated with synovial tissue cross- reactivity. Theanalysis of clonal distribution of E2-specific B cells may support this hypothesis, as it would be expected thatfewer clones would be detected in the late response if self-constituents were indeed responsible for the ongoingE2-specific antibody response.3.3.5.Relative Affinity of IG directed to RV and RV ProteinsSequential serum samples from seven individuals undergoing serologically confirmed primary RV infection werecollected at time periods of 10-20 days, 60-90 days, 360 days and 720 days following the onset of a rubelliformrash. Relative affinities of these sera were determined at each time point for IgG directed to whole RV andthe individual RV structural proteins. Mean IgG ER(%) values (— 1 SD) are shown in Figure 35. Measurementof IgG anti-El affmity in acute phase sera showed initial low ER( %) values of 25% (— 4%) with subsequentdevelopment of intermediate affmity values of 52% (-‘ 11 %) at 3 months post-rash and final maturation to highaffinity levels of 85% (— 7%) and 87% (— 3%) at 1 and 2 year intervals, respectively. In contrast, IgG anti-E2and anti-C responses exhibited minimal increases over the two year post-infection follow-up period. In contrastrelative affmity of anti-E2 and anti-C did not increase significantly (p “0.3) over the two year period investigated,with ER( %) values measured at 20 (— 8%) in the acute phase and 31% (— 8%) after 2 years post infection forE2 and 21 (— 10%) to 36% (-‘8%) for C respectively. Measurements of IgG affinity to whole RV correspondedto the results obtained with purified El protein, with ER(%) values increasing from 23% (— 4%) in the initial-86-10 days post-infection, to 52% (— 11%) at 3 months, to significantly (p \0.000l) elevated levels of 85% (— 7%)and 87% (—3%) at 1 and 2 years following Rubella. Striking differences were noted in the maturation of IgGaffinity responses to individual RV structural proteins. Whereas IgG affinity to the El envelope proteinparalleled results obtained with whole RV, IgG responses to the E2 and C protein underwent minimal affinitymaturation, remaining at low levels throughout the 2 year post-infection period. The affinity response detectedin the anti-El lgG is supportive evidence that this protein is important in eliciting both neutralizing antibodyresponses (Waxham and Wolinsky, 1985a) and may represent the main target for the cell-mediated immunityalso (Chaye et al., 1992). The basis for the immunodominance of El remains unclear but may reflect the abilityof antigen-processing cells to present the individual viral proteins to the human immune system (Johansson et—S0-4-,0C0-4-,DU110—20 60—90 360Days Post Onset of Rubefla RashFigure 35. Differential IgG Affinity Maturation to the Structural Proteins of RV. Elution ratios of IgG directedto each of the viral antigens El, E2 and C, as well as whole RV, were determined at sequential time intervalsfollowing diagnosis of clinical rubella in seven subjects. Each colunm represents the average ER(%)determination — 1 SD for the seven patients. (From: Mauracher et al., 1992a)720-87-al., 1987). The high level of glycosylation of E2 with N- and 0-linked sugars, both containing sialic acid(Lundstr 6m et al., 1991), may be associated with increased resistance to proteolytic cleavage and subsequentreduced binding to the MHC class II complex (Sharon and Lis, 1982). In addition, both E2 and C may be lessphysically accessible within the intact virion. Capsid is an internal viral protein, while E2 may be masked bycarbohydrate shielding (Klenk, 1990) and by virtue of its physical relationship with the El protein in theformation of surface spikes (Ho-Terry and Cohen, 1984). As it has been reported that B-cells specific forinternal or shielded viral proteins receive less effective help from virus-specific Th cells (Scherle and Gerhard,1989), it is possible that a lowered affmity of the E2 and C protein is due to decreased antigen-specificstimulation and Th support.Alternatively, the low affinity response to E2 and C could be explained by protein denaturation andsubsequent loss of antigenicity during purification procedures or during binding to the poiyvinyl solid phase(Friguet et al., 1984). This possibility seems unlikely as it would be expected that El, being most susceptibleto denaturants, would most easily be affected by alterations of high affmity binding sites. Furthermore it wasshown in a previous section that by a variety of techniques denaturation of the separated El and C proteins wasminimal.3.3.6Summary and ConclusionsThe findings on the humoral responses to RV in adults are summarized below:L El-Specific Response: the antibody response to this protein is immunodominant and significantly (p = 0.005)higher than antibody responses to E2 or C. The kinetics of the anti-El response is characterized by a rapidonset in the first several weeks following infection and is maintained in the years following Rubella. Theaffmity of these antibodies is high. Anti-El IgG reacts mainly to topographic epitopes (90%), with only 10%being reactive to linear epitopes. Both IgA and 1gM are reactive to this protein.L E2-Specific Response: the antibody response to E2 is significantly lower than the El-specific responses.Kinetic studies showed that the onset of these antibodies is slow, reaching maximal levels in the first yearfollowing Rubella. The IgG affmity remains low, anti-E2 IgG is reactive to both topographic and linearepitopes.L C-Specific Response: IgG responses against this protein are weakest and are transient. Peak reactivity occurs-88-in the months following Rubella and falls of in the years following infection. Similar to E2-specific responsesthe affinity of IgG directed to C remains low. Epitopes on this protein are not sensitive to denaturation andit appears that the majority of anti-C IgG is reactive to linear epitopes.3.3.6.1JA Responses to RV ProteinsThe Western blotting technique had as its primary advantage an increased specificity over immunoprecipitationand the ability to analyze the IgG-,IgA- and 1gM-specific responses to RV structural proteins (see Fig. 22). Inthe investigation of the kinetics of the IgA responses of 33 male and 67 female adults over the years followingRV infection it was observed that only females produced IgA antibodies specific to RV E2 (Fig. 36) (Mitchellet al., 1992). No significant differences between male and female were detected in the protein-specific responsesof either IgG or 1gM. The clinical implications of differences in the recognition of E2 by serum IgA betweenmales and females remains unclear. It was speculated above that E2 may have possible cross-reactive epitopeswith self-determinants and this taken together with the well-known increased incidence of RAA in females maysuggests an involvement of the serological responses of E2-specific IgA in the initiation of joint inflammationfollowing Rubella or RV immunization.0-6 Days 1-4 WeeksE2ICIC‘CICCOto1-11 Months 1-3 YearsFigure 36. Kinetics of the Appearance of RV Protein-Specific IgA as Determined by Western Blot. The kineticsof appearance of El-, E2- or C-specific IgA were determined in males (checkers) and females (hatched)patients. Sera were collected sequentially from each patient during the early acute (0-6 days post onset of rash),acute (1-4 weeks), early convalescent (1-11 months) and late convalescent (1-3 years) phase of rubella infection.Specificities were determined by class-specific immunoblot assays. Results shown are the relative percentagesof female or male patients at each interval. (From: Mitchell et al., 1992a)El C C7/ ,•//Ii 1011— .. t:_El E2 C El E2 C-89-3.3.6.2RV Protein Specific LymphoproliferativeResponsesFollowing the characterization of antibody responses to the individual proteins of RV it became possible tocompare these to lymphoproliferative responses. The proteins obtained by preparative SDS-PAGE were ofinsufficient concentration when used by others in stimulation assays with human lymphocytes (Dr. D.K.Ford,Arthritis Society, Vancouver; Dr.D.Ou, Dept. of Pathology,UBC). A collaborative study was therefore initiated(Ms.H.H.Chaye and Dr.S.Gillam, Dept. of Pathology, UBC) to compare both B and T cell responses in 14randomly chosen adults, without previous knowledge of their immune status to RV. IgG responses wereanalyzed by protein-specific EUSA and T cell responses were determined by using RV El, E2, and C obtainedfrom a vaccinia virus expression system in lymphoproliferative assays (Chaye et al., 1992). In the latter assaystimulation indices (SI) greater than three were considered sigiiflcant. The comparison of the B- and T cellresponses in these patients is illustrated in Figure 37.Proliferative Response Serological Response• E2: 4<C• I -0- 1.129I I -- I I0El E2 C El E2 CFigure 37: Comparison of Proliferative and IgG Responses to RV Structural Proteins. Values of stimulationindices (SI) for the proliferative responses and A.U./ml for the protein specific IgG responses are on a naturallog scale. Error bars indicate - the standard error. Proteins used in the serological study were obtained bypreperative SDS-PAGE from whole virus, whereas proteins used in the lymphoproliferative assays was derivedfrom cloning RV protein genes into vaccinia virus expression vectors. (From: Chaye et al., submitted)-90-The comparison of the RV-specific serological and lymphoproliferative responses in this population showedsimilarities in their protein specificities. The response to El was immunodominant in both responses over thereactivity to E2- and C-specific responses, again suggesting that the response to the El protein plays aparamount role in the induction of protective immunity.These data suggest that the El protein may be the best candidate for developing subunit vaccines for RV, asit gives rise to both a vigorous antibody and lymphoproliferative response. Investigation of the precise regionsof El that are involved with neutralization and hemagglutination have been reported to lie between an residues244 and 300 of the El protein (Terry et al., 1988). Synthetic peptides constructed from this and other region’sprimary sequence are currently being investigated as potential B- and T cell recognition sites and may lead tothe assignment of dominant T-cell epitopes as well as the sites of continuous B-cell epitopes on El. Thisapproach may ultimately identify small regions from the El protein which can be used to induce a protectiveimmune response to RV, thereby avoiding RV epitopes which may be involved with adverse vaccine side effects.Observations concerning the sensitivity of the El protein to denaturation by both detergents and reducingagents have revealed that, by far, the majority of the El and E2 epitopes are topographic and structurally-dependent. The use of native antigen will therefore be an important consideration in the development ofsensitive immunoassays using bio-engineered RV proteins. The development of expression systems allowing thelarge scale production of pure, correctly folded and glycosylated RV El is currently under investigation at manycentres. The use of baculovirus as an expression vector for the production of cloned RV El and E2 inSpodoptera frugiperda cells has been reported (Oker-Blom et al., 1989; Seto et al., 1991) and is proposed toprovide a more economical alternative to the use of whole RV virus in immunodiagnostic assays (Sepp dñen eta!., 1991).-91-3.4. Section IV: RV-Specific Humoral Immune Responses in CRS PatientsCongenital Rubella Syndrome (CRS) patients and their immune status have been investigated since the initialdescription of this syndrome in 1941 (Gregg, 1941). Although this manifestation of RV infection has beendramatically reduced in the developed world by the implementation of a successful vaccination programme, andhas therefore lost much of its clinical urgency, the disease remains of interest as a model for viral persistenceand tolerance induction. The infection of the human fetus with RV early in gestation, clinically presenting asCRS, leads to long term persistence of the virus in these individuals. Virus persistence is thought to result fromRV-specific tolerance although little direct evidence has been presented to support such a mechanism forcontinued viral replication. It has remained a paradox why CRS patients mount a humoral and cellular responseyet remain incapable of clearing the virus. To explain viral persistence, the affinity of anti-RV IgG (Fitzgeraldet a!., 1988), HAl activity (Cooper et a!., 1971) or humoral and cellular responses to the individual RV structuralproteins have been investigated (Katow and Sugiura, 1985; de Mazancourt et al., 1986; Chaye et al., 1992), inan effort to defme a partial tolerant state to RV arising from either selective anergy of neutralizing IgG, clonaldeletion of T-effector cells, lowered concentrations of El specific antibodies or the inability to produce a highaffmity antibody response. RV-specific antibody responses of CRS patients were compared to those of normaladult control patients, described in Section ifi. The observed differences in how CRS and Control patientsresponded to RV support the hypothesis that intrauterine RV exposure has led to virus-specific tolerance in CRSpatients.3.4.1.RV and RV-protein Specific ELISACRS patients (n= 15) and adult Rubella control patients (n= 15) were compared for their quantitative IgGresponses to whole RV and the separated RV structural proteins (Fig. 38). Whole RV-specific IgG antibodylevels as measured by whole RV ELISA were 202-65 lU/mi (X -SD)for the control group, whereas the CRSgroup demonstrated lower levels at 124—55 lU/mi. However, these differences were not statistically significantat the p \0.05 level. No differences in the responses to the E2 and C protein were determined between the CRSand control group but significant differences were observed in the El-specific responses. In the control group,-92-the majority of RV-specific IgG antibody was observed in the anti-El response (208-115 AU/mi) which wasgreater than either the E2- or C-specific IgG response. In the CRS group, the anti-El IgG levels wereconsiderably lower (47 -25 AU/mi) and significantly lower (p =0.004) than those measured in the control group.Control Rubella Patients CRS Patients500 500— 300T 300D + D‘ 200‘‘ 20001100. I • 1000• I j0 IiWhole RV El E2 C Whole RV El E2 CFigure 38: IgG Responses to RV and RV Proteins in Control and CRS Patients. Results are expressed inlU/mi for whole RV-specific IgG and AU/mi for RV protein specific IgG. Error bars indicate -. 1 Standarddeviation around the mean. Values for individual patients are also shown (L). (From Mauracher et al., 1992c)The RV-specific immune response of CRS patients in this group would normally be considered to be protective,as it was higher than 15 lU/mi of anti-RV IgG, the suggested cutoff value for the definition of protectiveimmunity (WHO, 1971). Yet RV is known to persist in CRS patient populations and RV reinfections have beenreported in school aged CRS patients (Hardy et al., 1970), which has been taken to be indicative of anunderlying defect in the RV-specific response in these patients. The selective loss of El antibodies has beenpreviously described in a small CRS population using immunoprecipitation techniques (Katow and Sugiura,1985). A subsequent study by others (de Mazancourt et al., 1986) did not report such a decrease in El specific-93-IgG. However, as the CRS patients studied were considerably younger (\1 year), maternal IgG may haveinterfered with determination of autologous RV-specific IgG. Furthermore, no steps were taken to correct forthe unequal percent distribution of methionine in the RV structural proteins when 35S methionine labelledpreparations were used in their irnmunoprecipitations. The above study confirms Katow’s and Sugiura’s fmdingsand suggests that CRS patients have significantly reduced levels of El specific antibodies. Although anti-Ellevels were reduced, the majority of CRS patient did have circulating anti-El IgG. Therefore the biologicalactivity and affinity of these antibodies was investigated further.3.4.2.Biological Activity of Sera in CRS PatientsHAT antibody levels were determined in sera from 11 CRS patients ranging in age from 1 to 27 years of ageand were compared to those of control patients described in section ifi. The median HAT titre of the controlgroup was 1:64 and compared to a median titre of (1:8 in the CRS group. HAT values in the CRS patientgroup did not exhibit a normal distribution and the two populations were therefore compared using non-parametric statistical analysis (Mann-Whitney U test) for the 1/log2. The HAl titre showed significantly lowertitres in the CRS group as compared to the control group (p=O.003). A lack of biological activity of the anti-RVIgG response in CRS patients, as measured by HAl, has been previously described (Cooper et al., 1971; Uedaet al., 1975). Whether this correlated with a concomitant lack of in vitro neutralizing antibodies was notdetermined. In the few CRS patient samples for which both NT and HAl tests were performed in thelaboratory, the two tests gave coinciding results, with negative HAl titres correlating to negative NT tests.However it has been demonstrated by the use of monoclonal antibodies, that HA and neutralizing domains donot necessarily represent the same epitopes (Brush, 1988; Waxham and Wolinsky, 1988). A possible mechanismfor the lack of HAl activity of the El specific IgG in CRS patients is a lack of high affinity antibody. In manyCRS patients it was observed that partial HAT was occurring, similar to the soft radial hemolysis patterndescribed for low affinity anti-RV IgG by the Radial Hemolysis typing test (Hedman et al., 1989). HAT titredeterminations have been criticized because of inaccuracy in the two-fold dilution steps, and it has thereforeoften been suggested that any given HAT titre should be interpreted as a range. plus/minus one dilution fromthe determined HAT titre. Statistical analysis of HAT titres are nevertheless valid as the variance observed in-94-any given population reflects both random- and measurement error.3.4.3.Relative Affinity of-RV Specific IpG in CRS PatientsAffinity indices were determined for IgG directed to whole RV as well as to the separated structural proteinsEl, E2 and C in both CRS- and a control group patients (Fig. 39). Serum from adult control group (n= 5)showed high affinity IgG directed to whole virus and El, but had significantly lower affinity of IgG directed toE2 and C (p \O.OOl),correlating with the data presented earlier in Figure 35. The CRS patient population wassubdivided according to age into a child (n=8, age range 1-4 years) and adult (n=7, age range 17-34) groups.x0CCtuuWhole RV806040/20 ///0—-Child AdultControl CRS Control CRSChild Adultx0C>‘C9-Figure 39. Affinity Indices of IgG directed to RV and RV Structural Proteins in CRS and Control Patients. 15adult control patients in the convalescent phase of Rubella (white bars) were assayed on affinity ELISA. Resultsare expressed as the affinity index on the y-axis. A CRS population of 15 patients was divided into childhoodgroup (hatched bars, n=8) and adult groups (cross-hatched, n=7). Error bars are — SD.-95-Child Adult Child AdultControl CRS Control CRSThe latter group was age-matched to the control group. The affinity of IgG to whole RV was observed to besignificantly lower in both CRS groups (p=0.00l) as determined by one-way ANOVA. The affinity of anti-Elantibodies also was significantly reduced (p =0.0001) in both CRS groups if compared by ANOVA. The FisherLSD test also showed significant differences in the means of both the child and adult IgG anti-El affmities whencompared to the control group. No statistically significant differences were observed in the low affmity responseto E2 in any study group (p =0.36). ANOVA analysis of the C-specific IgG affinity showed significant differencesin the populations (p=0.0001). By using post-hoc analysis it was shown that the mean affmity of anti-C IgG inthe adult CRS patients was significantly higher than the affinities of either the child CRS- or the control patient’santi-C IgG. No differences were detected in the affinities of anti-C IgG of child CRS- and control patients.Antibody affinity is an important functional parameter of the humoral response and the inability to switchfrom low to high affmity antigen-specific responses has been an observed feature of immunological tolerancein animal models (Steward and Steensgaard, 1983). The inability of young CRS patients to produce high affinityIgG to El was marked. Age may be a factor in the affinity maturation but it has been recently shown thatinfants at age one can produce high affmity IgG (O.Meurman and K.Hedman, manuscript in preparation). Theadult CRS patients of this study group appear to have partially regained the ability to produce higher affinityIgG to El and interestingly were observed to produce high affinity IgG to C protein. It is conceivable thatcontinued antigen stimulation by persistent RV may have facilitated the maturation of the IgG responses in thispatient group. The observed decrease in both quantity and affinity of the El-specific IgG in the CRS populationmay well be responsible for deficient antibody-mediated viral clearance mechanisms.3.4.4.Reactivity to Linear and Topographic Epitopes in CRS PatientsThe presence of 2-mercaptoethanol reduces much of the antigenicity of the envelope proteins El and E2 butnot that of C, by the destruction of epitopes dependent on tertiary protein structure (Mauracher et al., 1992a).In the analysis of CRS patient sera by reducing and non-reducing Western blots it was observed that thesesubjects were deficient in their ability to produce IgG reactive with reduced El. In contrast no group differenceswere observed in the reactivities of E2- and C-specific antibodies in these two populations (Table 8). Thisobservation provided the most consistent feature which differentiated the CRS population from controlresponders. In respect to the other antibody parameters measured (HAl, affinity, anti-El levels) some CRSselective non-responsiveness to linear epitopes in CRS patients in CRS patients has been further investigated-96-Control Patients CRS Patients p-valueEl 10% 4% 0.5 — 1.1 \0.00lE2 46% —41% 31 —40 0.1C 125% —83% 96 —54 0.5Table 8. Percentage of Signal Remaining on Western Blot under Reducing Conditions for Control and CRSPatients. Control patients (n= 14) were compared to CRS patients (n= 24) for the fraction of their RV proteinspecific IgG reactive to linear epitopes.patients were always found to lie within the range of control responders; in respect to antibodies to linearizedEl (El red)’ all CRS patients showed significantly reduced responses to linear epitopes in comparison to normalcontrol patients. Immunoblot results of two representative patients are illustrated in the Figure 40. ThisNormal Rubella CRSNon-ReducingReducing.,. 1•Cd ElElE2E2 CA Ai•i 11 -Cd El E2‘IE2 CFigure 40. Western Blot under Reducing and Non-Reducing Conditions of Representative Control- and CRSPatients. Two blots, and their corresponding densitometric scan, are shown for a adult control patient and oneCRS patient, Serum concentrations were equal for the reducing and non-reducing Western blots.-97-AElE2 . ()Degradation byor DCA h/Figure 41. Model for the Production of IgG to Topographic Epitopes in the Absence of El-Specific Th Cells.A hypothesis is proposed which explains the production the IgG response to topographic epitopes (A) and thelack of IgG responses to linear El epitopes (B) in the CRS population by a lack of El -specific Th cells. Th cellsto El are proposed to have been tolerized in this population (crossed-out anti-El Th) due to exposure of thefetal immune system to RV.Scenario A depicts a particular clone reactive to a topographic epitope of El. The IgG product of this clone willbe detected on whole RV/El protein ELISA or non-reducing Western blot. As the IgG recognizes the intactEl, it will be capable of binding the intact virion encountered in the initial viremic phase. By phagocytosis andpresentation of processed antigen in context of class II molecules, this clone will present peptides of all threestructural proteins on its surface, and will in turn be able to receive the second activation signal by Th cells(Noelle and Snow, 1990) specific to the three viral proteins. The lack of anti-El Th cells does therefore notprevent clonal activation.On the other hand, the scenario illustrated in situation B, depicts a clone producing IgG to a linear epitope onEl (vvv, partially or completely buried within the highly folded El protein. The most likely circumstance inwhich this IgG molecule will recognize its antigen is once the virus and its proteins have been degraded, as anoutcome of local inflammatory responses or by exo-peptidases of dendritic cells in the germinal centres of thelymph node (Steinman, 1991, Liu et al., 1992). This clone will therefore bind partially degraded species of theEl protein, and only present El peptides on its class II molecule. However, as Th cells to El are lacking, thisclone will not receive any further stimulation and will remain in anergy. IgG directed to peptides or linearizedEl will therefore not be produced in patients lacking El specific Th cells. This model accommodates theserological findings in CRS patients and lends support to the hypothesis that interuterine exposure has led toimmunological tolerance. (From Mauracher et al., 1992c)Ig GB-CellIgG directed toClass IItopographic El epitopeTCRB-CellIgG directed tolinear El epitopeClonal Activation Clonal Anergy-98-by others using solid phase bound peptides representing the a sequence adjacent to the putative neutralizingand hemagglutination regions of RV El (Terry et al., 1988), between amino acid residues 213-239 of the RVEl. It was demonstrated that Rubella patients as well as RV vaccinated individuals showed reactivity to thispeptide in the weeks following virus exposure (Mitchell et al., l992b). The CR5 patients on the other hand didnot show reactivity to this peptide which again suggests the inability of this population to produce IgG to linearEl epitopes (Mauracher et al., 1992b, Mitchell et al., 1992b). Is the non responsiveness of CR5 patients tolinearized El protein as well as El peptide based on an underlying deficiency of El specific Th cell responsesand does this represent a specific tolerization of the El specific cellular response. If so, the ability of CR5patients to make IgG to conformationally dependent epitopes has to be explained. It is unlikely that theseepitopes are T cell independent as El does not contain either multivalent epitopes nor are the carbohydratemoieties on this protein thought to be antigenic. A model to explain production of IgO directed to topographicepitopes of El in the absence of El-specific Th cells is proposed in figure 41.3.4.5.Analysis of Sequential Serum Samples from CRS PatientsThree CRS patients were retrospectively studied pre- and post MMR vaccination over a 2 to 6 year period.Results obtained following IgG in-miunoblot analysis of sequential sera from these patients are shown in Figure42 and serological work-up is summarized in Table 9. The cord blood from patients A and B exhibited strongEl responses in presence of both E2- and C-specific IgG as well as IgG specific to the reduced proteins; no cordblood was available for patient C. Cord blood contains maternal IgG at equal or higher titres than the maternalserum, and it was therefore not surprising that this sample contained reduced El reactive IgG. Following thefirst year of age, these patients lose the reduced El-specific IgG and HAl antibody responses which becomeundetectable in all three patients. Patients A and B did not respond to vaccine, with antibody levels remainingsimilar pre- and post-MMR vaccine. Patient C was unique in that no RV-specific antibodies were detected inthe pre-MMR vaccine sample and the post-vaccine samples contained El-specific IgG only. However theseantibodies were not reactive to linearized El nor did they show any HAl activity. Cord blood showed matemallyderived HAl activity and high affinity El-specific responses which were then replaced by endogenously producedIgG of lowered affmity with no HAl activity.-99-* I * *A Cord ly 4, ly2m ly6m 3y Age12m) 16m1 12yJ post MMRi +I IICdElb.ElE2WE2C -.1:* I *B Cord 6m 4, ly5m 2Y4m(imi (lyl+ + +C 1y7m 2y4m 3y8m 8y(8m1 12y1 (6y)— + — + — + — +naFigure 42. Western Blot Analysis under Reducing and Non-Reducing Conditions for Three MMR VaccinatedCRS Patients. Patients A, B and C were prospectively studied following MMR vaccine (arrow). A cord samplewas available for patient A and B. Patient age is indicated on the top of each series, and time following MMRvaccination is indicated within square brackets [J.-100-Table 9. Serological Examination of MMR Vaccinated CRS Patients. Patient samples in this table are indicatedby an asterix on the Western blots in Figure 42.3.4.6.Evidence for Th Tolerance in CRS PatientsAn immunological mechanism for the prolonged persistence of RV in the CRS infant remains speculative. Thevirus is not cleared from fetal or infant tissues although maternally transferred IgG, as well as in some casesautologous 1gM, can neutralize the virus in vitro. It is therefore likely that immunoglobulins are of little use inbringing a well established viral infection under control and that the cellular response is of primazy importance.The cellular response plays a key role in preventing primary viral infections in the human, as demonstrated byclinical frndings in X-linked agammaglobulinemia (XLA) and in congenital thymic aplasia (CTA) patients. XLApatients are deficient in IgG production but produce normal levels of T cells. These patients are usuallydiagnosed once their maternally derived IgG has been catabolized, late in the first year of life, resulting mostoften in the patient’s susceptibility to chronic bacterial infections. These patient also have difficulties in clearingPatient AAgePost MMR:Avidity index Who/c RVElE2CigG (iUJmi) Whole RVElE2CHAlSfltHElredBirth ly 2m ly Sm2m Sm77,9 51 4881 19 2935 37 3234 32 37145 163 15082 17 1724 58 4117 19 201:32 1:8 1:8yes no noPatient BBirth 2y 4mly77 6078 4436 5533 26112 7653 1725 5010 51:32 < 1:8yes noPatient Cly 7m 2y 4m8m/ 16/ 32/ // /1 693 503 31 1<1:8 <1:8/ no-101-enteric viral infections (ie: polio virus) but have little difficulty in terminating systemic viral infections (Saulsburyet a!., 1980). On the other hand patients with complete CTA, incapable of producing T cell responses, oftensuccumb to overwhelming viral or mycobacterial infections (Cooper and Butler, 1989).If cellular responses are of prime importance then the long term persistence of RV in fetal tissues can bereadily explained, as maternal T cells do not cross the placenta and fetal T cells responses are largely suppressedin utero and for the first months after birth (Papadogiannakis et al., 1990). The gradual decline in the sheddingof RV in CRS patients over the first two years of life may be due to the initiation of the endogenous cellularresponse to RV. Although CRS patients have been shown to have a specific deficiency in their lymphoproliferative responses to whole virus (Fuccillo et a!., 1973) others have reported that a non responsiveness to RV isstrongly dependent on the time period of intrauterine exposure, with CRS patients who were exposed after 12weeks of gestation showing near normal responses (Buimovici-Klein and Cooper, 1985). Non responsivenessof lymphocytes measured by whole RV proliferative assays remains unresolved, and the investigation oflymphoproliferative responses in CRS- and control patients using synthetic peptides representing RV proteinsmay help in a more definitive analysis of the T cell immunity.In the comparison of the B- and T-cel! responses of CRS and control patients to individual RV proteins, itwas demonstrated that CRS patients showed strong lymphoproliferative responses to E2 and C, but wereselectively deficient in their responses to El (Chaye et a!., 1992). As these experiments were performed withvaccinia virus expressed proteins, the background response to vaccinia in some of these patient may have causedfalse positive responses, in spite of steps taken to correct for vaccinia-specific signals. Preliminary results oflymphoproliferative assays using 30 an peptides representing the complete El, E2 and C regions have shown that3 out of 4 CRS patients are completely non-responsive to El peptides, whereas all CRS patients react to E2 andC peptides. Of the control responders, 12 out of 12 adult control responders reacted to peptides from all threeRV proteins (Dr. Dawei Ou, Dept. of Pathology, UBC, personal communication). These observation supportthe above hypothesis that the selective antibody non responsiveness to linearized El or El peptides representsan underlying immunological tolerance of the T cell response to El.Antigenic sites recognized by antibody may be regulated by Th cells and has been described in detail for themurine response to staphylococcal nuclease or sperm whale myoglobin (Berzofsky, 1985). Cognate interactions-102-between B- and T cells are thought to regulate T cell-dependent antibody responses, with the activated Th cellproviding the secondary signal to the B cell, via class II-CD4 interactions (Noelle and Snow, 1990). The restingB cell can bind antigen by surface IgG which is thought to provide the first signal for cell activation. Antigenis then phagocytosed, degraded and re-expressed on the cell surface in context of class II molecules (Tony andParker, 1985). Th cells, recognizing peptides originating from the phagocytosed antigen, can then bind to theB cell and provide the secondary stimulus to induce G0 to G1 transition of the B cell with fmal progression ofthe B cell to antibody secretion being promoted by IL-4 and IL-5. The existence of this pathway of B cellactivation is supported by observations of a hierarchy of help for B cells responding to internal or externalantigens of influenza virus in adoptive Th cell transfer experiments using athymic mice (Scherle and Gerhard,1988). In this model B cells expressing antibody to surface antigens (HA or NA) internalize whole virion andcan be activated by Th cells specific to either internal or external proteins (M, NP, HA or NA). In contrast,B cells producing IgG to internal proteins (ie: NP) can only be activated by NP-specific Th cells. It is believedthat the B cell will encounter NP only when virions have been degraded, the NA-specific cell will therefore onlybind and phagocytose NP antigen. A more recent experiment has described responses of mice which have beenmade transgenic for the G protein of Vesicular Stomatitis virus (VSV) (Zinkernagel et al., 1990). If these miceare immunized with cloned and expressed G protein no antibody responses are mounted. However if wholeVSV virus is used as the inoculum IgG responses to all VSV proteins, including G protein, are made. Thisexperiment was designed to further support the hypothesis that B cell tolerance is regulated at the Th cell level,but also demonstrates that B cells, producing antibody to viral surface structures, can be activated by Th cellswith activities to other viral proteins.A similar scenario is proposed to exist in CRS patients where the individual, a primo to the stimulation of theRV-specific IgG response, has lost El-specific T cells as a result of intrauterine tolerization of the immunesystem. The model takes the observations made by Scherle and Gerhard one step further by differentiating notonly between internal and external proteins of a virus but also between internal and external epitopes of theimmune dominant viral protein (See Fig. 41).-103-3.4.7.Summary and ConclusionThe responses of the humoral immune response in CR5 patients was found to differ from the response of adultcontrol patients in the following manner:L No significant differences in the IgG responses between CR5 and control patients was detected when usingwhole RV ELISA.L When these two populations were analyzed by RV protein-specific ELJSA, a significantly lower (p = 0.004) IgGresponse to El was demonstrated. No significant difference was shown in the E2- and C-specific response.L The affinity of IgG directed to whole RV or El was significantly lower in the CR5 patients as compared toadult control patients.L The CR5 patient population was characterized by their inability to produce IgO to linear El epitopes. IgGwas produced to linear E2 and C epitopes. This is also reflected by the significantly reduced responses to Elpeptide (an 213-239), as demonstrated by others.L The CRS patients showed significantly lowered HAl titres. It appears that the El-specific antibodies presentin this group are not biologically active, in the sense that lack HAl activity.L A comparative study of B and T cells responses revealed that both serological and lymphoproliferativeresponses to El are significantly reduced in this population, as shown by Chaye et al..The observations on the RV-speciflc humoral and cellular responses of CR5 patients identilS’ this populationto be selectively tolerant to the El protein of RV. As this protein appears to be immunodominant and providesthe main target for both serological and lymphoproliferative responses it is hypothesised that viral persistencein CRS patients is the outcome of the lack of a major part of the T cell response. Similar dominance of oneprotein acting as the main T cell antigen has also been reported in other viruses {ie: influenza A (Wraith, 1987)and BHV I (Hutchings et al., 1990)]. The reactivity of cytotoxic T cells in Rubella and CRS patients has notbeen investigated but it is hypothesised that protein specificities of the Tc cells are similar to that of the Th cellsmeasured by lymphoproliferative assays (Thomson and Marker, 1989; Ou et al., submitted). Why the E2- andC- specific T cells may still be present in the tolerized patient population remains speculative, but the sameproperties which make these proteins poor T cell antigens may also make them poor toleragens. Cross-reactivityof T cell epitopes of RV capsid or the RV encoded nonstructural (ns) protein with other common viral capsid-104-or ns proteins may also be possible. The capsid structure has been highly conserved in many RNA viruses andit may be possible that peptide sequences of RV C protein are shared with other capsid proteins or RNAbinding proteins. However amino acid homologies between RV C protein and those of the related SFV andSV are between 10 and 20% only (Frey and Marr, 1988). These authors report that significant homologies existin the nonstructural protein coding region for RV and that of many other RNA viruses. The ns proteins of RVhave not been considered as T cell antigens but may potentially play a role in T cell immunity as is the case forDNA viruses (ie: EBNA in Eppstein-Barr virus). An amino acid sequence homology search for C protein ofRV in GeneBank has not identified any sequence homologies with other viral capsid proteins but has identifiedshort stretches of homology with bacterial and viral RNAIDNA binding proteins. The possibility of anti-RVB-cell activation by cross-reactive Th cells may therefore be valid.The fmding of predominantly low affmity anti-RV IgG in the CRS group may also be taken as indirectevidence for T cell tolerance. In the absence of a strong Th cell response it may be conceivable that B cells donot obtain sufficient stimulation to undergo multiple replication cycles during which affmity maturation canoccur. This may explain not only low affmity antibody but also the observed fall in anti-El IgG titres in the CRSpatients.Similarities exist in the description of tolerance in CRS patients and the perinatally LCMV infected mousemodel. Murine LCMV membrane proteins contain conformationally dependent neutralizing domains (Wrightet a!., 1989), yet the anti-LCMV IgG in these perinatally infected mice was not capable of clearing the viralinfection. In this model, ineffective virus clearance correlated with low Tc cell responses and low delayed typehypersensitivity, but is unrelated to the levels of in vitroneutralizing antibody or NK cells (Thomson and Marker,1989). Although IgG does play an auxiliary role in the rapid clearance of virus in healthy animals (Cerny et al.,1988), the persistence of LCMV in susceptible mice is thought to be solely due to absent LCMV-specific Tc cell(Oldstone, 1989). Nevertheless, virus specific Th cells must be present as IgG production to viral proteinsrequires Th support - LCMV infected athymic mice only produce 1gM specific to LCMV. Further it has beenwell established that in the neonatally infected mice IgG directed to LCMV proteins are of low affinity and arecommonly present as dC or as deposited as IC in the kidneys or other organs (Buchmeier et al., 1980). Thisresponse patterns shows striking similarities to the observations made on low affinity anti-RV IgG responses-105-(Fig. 39) and high levels of anti-RV IgG dC (Coyle et a!., 1982) in CRS patients. The protein specificity ofthe cellular response or IgG reactivity to linearized proteins have not been analyzed in LCMV tolerant mice andcould reveal a similar scenario as in the CRS patients, where protective immunity may be dependent on thecellular response to a single protein.The selective non responsiveness of CRS patients to linearized El in their serologic responses may haverelevance to the interpretation and future development of immunodiagnostic assays for Rubella and othersystemic viral infections. CRS patients are characterized by a persisting RV infection, yet are seropositive withcommercially available solid phase immunoassays and therefore are interpreted to have “protective immunity’.It is well established that neither the endogenously produced IgG nor the maternally derived IgG, having in vitroneutralizing activity, clears RV in the CRS infants. Therefore, it has to be questioned whether anti-RV IgG hasa function in the clearance or the prevention of Rubella in vivo. It can be speculated that the only reason whythe presence of IgG correlates with protective immunity in the healthy responders is because it reflects thepresence of a functional T cell response. Serological assays for RV should therefore be designed to detect IgGwhich will correlate to the presence of Th cells specific to the El protein. As suggested by the model shownin Figure 41, responses to topographic and structurally dependent El epitopes may arise in the absence of ElTh cells whereas IgG to linearized El are dependent on El specific Th cells. Therefore it might be moreappropriate to use denatured El protein as the solid-phase antigen. Such an immunoassay would be capableof differentiating between the responses of control patients and the non-protective responses of CRS patients.Much effort is currently being invested to produce expressed El in native form, for example by the use ofbaculovirus expression systems (Oker-Bloom, 1989), as it is believed that IgG specific to the intact virion or itssurface proteins are most relevant to protective immunity. Interactions of antibodies with the structurallydependent neutralizing domains of the virus leading to neutralization in the tissue culture well should not bedirectly extrapolated to be representative to protective immunity in the organism. This concept was alsounderlying the design of the experiments described in this thesis, where the biological function of purifiedproteins was assessed in order to prove that these proteins had maintained their native structure.On the basis of the presented data, it would be reasonable to speculate that more specific serological RVassay, correlating better with protective immunity, would entail the use of recombinant El, followed by-106-deglycosylation and cystine cleavage and blockage. A positive signal in such an assay will likely correlate wellwith functional T cell immunity and protection. Bacterially expressed RV proteins have been described andproduced by independent groups (Londesborough et al., 1992; Dr. S.Gillam and H. Chaye, UBC, personalcommunication) and El has been expressed at high concentration in CHO cells (Dr.T.Hobman, University ofCalifornia (San Diego), LaJolla, CA) and could potentially be employed in ELISA test using linearized RV Elprotein. The use of insect cell derived El, used in native conformation for anti-RV IgG ELISA, may be anexpensive way of measuring irrelevant IgG and the use of synthetic peptides, cyclized or not, may be a costlyand elaborate alternative for arriving at a product providing similar result as the linearized El ELISA.-107-SUMMARY AN]) PERSPECTIVESThe hypothesis that intrauterine RV exposure leads to the development of RV-specific tolerance was tested bya comparative analysis of the RV protein-specific antibody responses in CRS and control patients. In order toexamine the antibody responses of these patient groups a series of techniques had to be developed oroptimalized. ELJSA techniques using whole RV were improved by including non-ionic detergent, resulting indisrupted virions and leading to increased sensitivity of the assay. The introduction of buffers, containing heatdenatured blocking proteins, further improved the performance of RV EUSA as well as ELJSAs using otherantigen systems (Mauracher et al., 1991b). For the determination of RV protein-specific antibody responses,both Western blot and ELJSA using separated proteins were developed. Western blotting techniques weredeveloped using non-reducing conditions (Mauracher et al., 1992a; Zhang et al., 1992). This approach provideda better alternative than immunoprecipitation and allowed the detection of IgG, IgA and 1gM to the individualRV structural proteins. Furthermore it showed that SDS-PAGE under low temperature and non-reducingconditions did not selectively destroy the antigenicity of RV antigens. Preparative SDS-PAGE followed byelectroelution was then employed to produce separated preparations of the individual RV structural proteinsEl, E2 and Cd. That proteins retained their antigenicity and much of their structural integrity was shown asboth separated El and C were demonstrated to have maintained their biological functions. Optimal conditionsfor coating these proteins onto ELJSA microtitre plates were determined and pooled preparations of eachprotein were used to examine RV protein-specific IgG quantities and affmities.IgG affinity assays were initially evaluated using whole virus preparations. Two techniques, the chaotropicelution ELTSA and the one-well inhibition ELISA were examined in detail for the determination of relativeaverage affinities of anti-RV IgG in human sera. The chaotropic elution EUSA using 8 M urea as a denaturantgave reproducible results and demonstrated affmity maturation of the anti-RV response following RV infectionin adults. It was more applicable than the inhibition affinity assay as it was independent of RV-specific 1gM andrequired less antigen, which was an important consideration once affinity assays were used in separated RVprotein EUSAs. Biological activity of antibodies were determined by HAT, an assay which has long beenconsidered the standard to which other RV serology assays are compared.The separated RV proteins were analyzed for their structural integrity. This was felt to be important as the-108-majority of El and E2 epitopes had been shown to be structurally dependent (Mauracher et al., 1992b). Asbiological activity of proteins is generally dependent on structure, it was inferred that intact RV protein functionwould indicate structural integrity. The function of El protein is well defined and includes HA activity whichhas been localized to a defined region on the protein and is known to be sensitive to denaturation (Terry et al.,1988; Qui et al., 1992). The ability of separated El to compete for HAl IgG with native virions showed thatthe structurally dependent HA domain had remained intact in the El preparations. During the investigationsof alternative methodologies for RV protein purification it was discovered that the C protein became detergent-soluble at pH below pH 5.5. This led to the further investigation of the solubility shift of this protein andresulted in the proposal of the intralysosomal uncoating mechanism of RV nucleocapsid (Mauracher et al.,1991a). This event is believed to be dependent on structural changes in the protein and has recently been shownto be applicable in SFV also, which has lead to the more broadly applicable proposal of intralysosomal capsiduncoating in the Togaviridae family (Schiegel and Kempf, 1992). Purified C protein was shown to have retainedits biochemical property to undergo the low pH induced solubility change, indicating that at least one of thisprotein’s biological functions had remained intact throughout the separation protocol. It may be of interest toinvestigate compounds which may interfere with this property of the C protein, such as the WIN compounds(Badger et a!., 1988) which have been shown to inhibit the uncoating of picorna viruses. If low pH-induceduncoating is shown to be a common mechanism for RNA viruses which enter the cell by endocytosis such asRetroviridae or Flaviviridae, then such compounds may prove to become a viable alternative to anti-viral drugsbased on nucleotide analogs.Once the immunological techniques had been established, it became possible to investigate the RV-specificantibody responses of adults with uncomplicated outcomes of Rubella in comparison with those of the CRSpatient group. Healthy RV responders predominantly produced antibodies directed to the El protein whichwere of high affinity and appeared rapidly following infection. Of these, on average 90% were directed toconformationally-dependent epitopes with 10% being directed to linear epitopes. The E2-speciflc response wasinteresting in that it ethibited slow onset with IgG increasing in quantity as late as one year following infection.It remains unclear if this indicated that RV antigen may persist in the lymph or if crossreactivity between RVE2 and other antigens or self-constituents exists. The affinity of both anti-E2 and C IgG was observed to remainlow throughout the first several years following Rubella infection, whereas El-specific IgG rapidly matured to-109-high affinity in the months following Rubella. This demonstrated that differential affinity to individual proteinsof a pathogen existed (Mauracher et a!., 1991) and indicated the importance of investigating the protein-specificimmunity in the response to pathogens. The immunodominant role of El in the antibody response was alsodemonstrated in the lymphoproliferative response (Chaye et al., 1992), indicating that the El protein may bethe most suitable candidate for the development of subunit vaccines.The comparative analysis of humoral responses to RV between CRS and normal control patients showed thatthe CRS patient group had significantly lowered IgG responses to the El protein, as measured on RV proteinELISA. These results supported fmdings by others (Katow and Sugiura, 1986). The most prominent featurewhich differentiated the IgG responses of CRS patients from those in control patients was the selective inabilityof the CRS group to produce IgG to linearized El. A model has been proposed which hypothesises that CRSpatients have been selectively tolerized to the El protein of RV. This is reflected in serological responses inthat IgG directed to linear epitopes is absent and the affmity of IgG to whole RV as well as El is low. Studieson the serological and cellular responses of CRS- and control patients to RV structural proteins suggest thatthe Th cell responses to El are significantly reduced in CRS patients (Chaye et al., 1992) and ongoing studiesusing peptides representing sequences of the RV structural proteins have provided further evidence thatlymphoproliferative responses to El peptides are absent in most CRS patients whereas responses to E2 and Cremain intact (Ou et al., 1992).This thesis has provided evidence to support the hypothesis that intrauterine RV exposure can lead toimmunological tolerance. Tolerance is proposed to be specific to the El protein and the absence of normalcellular and serological responses may explain viral persistence in the CRS patient. Viral persistence may alsobe involved in the pathogenesis of RAA and it now is possible to determine if a similar deficit exists in thecellular responses of patients developing arthritis following RV infection.Models for tolerance induction in the human are rare and it is proposed that further studies on well defmedCRS patients will contribute to the understanding of the development of the human immune system. With theavailability of peptides from all viral proteins, the fine specificity of both the B and T cell response can bestudied. 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