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Elucidation of antigenic epitopes on the rubella virus E1 glycoprotein Chaye, Helena H. 1993

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We accept this thesis as conformingto the required stELUCIDATION OF ANTIGENIC EPITOPES ON THE RUBELLA VIRUS El GLYCOPROTEINbyHelena Hojung ChayeB.Sc., The University of British Columbia, 1986A thesis submitted in partial fulfilment ofthe requirement for the degree ofDoctor of PhilosophyinThe Faculty of Graduate StudiesGenetics ProgramThe University of British Columbia1993© Helena H. ChayeIn 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.(Signature)( FIV-bDepartment of  TA-1-tvi__06, The University of British ColumbiaVancouver, CanadaDate ^L4C_7.0 2 6/5 \DE-6 (2/88)ABSTRACTRubella virus (RV), a positive-stranded RNA virus, is the only member of the genus Rubivirus withinthe Togaviridae family. The virus consists of a host derived lipid bilayer membrane, membrane glycoproteinspikes, and the C protein which together with viral RNA forms the icosahedral nucleocapsid. Althoughclinical rubella is a relatively mild disease, RV remains an important human pathogen because of itsteratogenic effects in utero which can result in new born infants with congenital rubella syndrome (CRS).Complications such as polyarticular arthralgia and arthritis following vaccination or infection are commonand rare cases of progressive panencephalitis have been reported.To obtain a better understanding of the immunopathology of RV infection, the degree of antigenicityof the structural proteins in humans were examined for both cellular and humoral immune responses. Itwas found that of the structural proteins, El glycoprotein was the dominant antigen recognized by the studypopulation sampled from normal individuals with no history of rubella associated conditions. It was alsofound that the CRS patients had immune responses distinct from that of the normal population. In CRSpatients E2 was the dominant antigen to which both cellular and humoral immune responses were elicited.The implications of these observation with respect to proposed mechanisms of persistent infection arediscussed.Twenty three synthetic peptides spanning the entire El sequence were screened with humanperipheral blood lymphocytes and the corresponding sera to examine the distribution of antigenic domains.El glycoprotein was also the target of viral neutralizing (VN) and hemagglutinin (HA) epitope mappingstudies. Deletion mutants of El were constructed from El cDNAs and expressed in in vitro, in COS cellsand in E.coli. The mutants were screened with monoclonal antibodies with VN and HA inhibiting activities.The mutants containing the functional epitopes were further studied by using synthetic peptides. HAepitope was mapped to amino acid residues E1 214 to E1 2„ while two VN epitopes mapped to amino acidresidues E1 214 to E1 2, and E1 219 to E1 232. The potential . use of the defined epitopes in the developmentof subunit vaccine is discussed.TABLE OF CONTENTSABSTRACTTABLE OF CONTENTSLIST OF TABLES^ viLIST OF FIGURES viiLIST OF ABBREVIATIONS^ viiiACKNOWLEDGEMENTS xi1.^INTRODUCTION^ 11.1 Rubella Virus 11.1.1. Classification^ 11.1.2. Morphology 21.1.3. Nucleic Acid and Genome Organization and Replication^ 21.1.4. Non-structural proteins^ 41.1.5. Structural proteins Capsid protein^ E2 Glycoprotein El Glycoprotein^ 81.1.6. RV Entry^ 111.2 Rubella Pathogenesis and Pathology^111.2.1. Clinical Features^ 111.2.2. Congenital Rubella Syndrome^ 141.2.3. Rubella Associated Arthritis 171.2.4. Rubella Vaccine^ 191.2.5. Host Responses to Viral Infections^ 201.2.6. Immune Responses to RV^ 231.3 Epitope Mapping^ 241.3.1. Epitope mapping using expressed proteins from E.coli^ 241.3.2. Epitope mapping using expressed proteins frommammalian cells^ 241.3.3. Epitope mapping using synthetic peptides^ 271.3.4. Functional eptiopes of Rubella virus structural proteins^ 291.4 Project Rationale and Thesis Objectives^322. MATERIALS and METHODS2.1 Materials^332.2 Methods2.2.1. Propagation of bacterial strains^ 332.2.2. Preparation of competent cells and transformants^ 342.2.3. Growth of transformants and preparation of plasmid DNA^ 362.2.3.1. Small scale plasmid preparation^ 362.2.3.2. Large scale plasmid preparation 372.2.3.3. Isolation of single-stranded DNA 382.2.4. Expression vectors^ 382.2.5. Construction of deletion mutants^ 422.2.6. Polymerase chain reaction 432.2.7. Restriction endonuclease digestion and DNA modification^ 442.2.7. Purification of oligonucleotides^ 452.2.8. Identification of El recombinants 452.2.8.1. Colony hybridization 452.2.8.2. Dideoxy sequencing of DNA^ 462.2.9. Separation of nucleotides (DNA sequencing gel)^ 472.2.10. Expression of El recombinants^ 482.2.10.1. In vitro transcription 482.2.10.2. In vitro translation 482.2.10.3. Transfection of COS cells^ 492.2.11. Detection of El recombinants 502.2.11.1. Monoclonal antibodies 502.2.11.2. Immunoprecipitation^ 512.2.11.3. Immunoblotting/dot blotting 522.2.12. Electorphoresis^ 522.2.12.1. Separation of DNA fragment^ 522.2.12.2. Separation of protein^ 532.2.12.3. Coomassie blue staining 532.2.12.4. Electroelution^ 542.2.12.5. Enzyme linked immunoadsorbant assay^ 542.2.13. T-cell proliferation assay 552.2.14. Antigen preparation for T-cell proliferation assays^ 552.2.14.1. Vaccinia recombinants^ 552.2.14.2. El peptides^ 562.2.15. Statistical methods 562.2.16. Study group^ 563. RESULTS and DISCUSSION3.1. Section I: Mapping the hemagglutinin and viral neutralizing epitopes of the RV El dlycoprotein 3.1.1. In vitro transcription and translation of the El deletion mutants^ 59iv3.1.2. Expression in COS cells^ 633.1.3. Expression in E.coli strain BL21(DE3)/pLysS^ 653.1.4. Synthetic peptide ELISA^ 693.1.5. Summary of HI and VN epitope mapping^ 693.1.6. Discussion of Section I^ 733.2. Section II: Cellular responses to RV structural proteins^783.2.1. Proliferative responses to RV structural proteins^ 783.2.2. Antibody responses to RV structural prOteins 793.2.3. Discussion of Section II^ 853.3. Section III: Human T- and B-cell epitopes of El dlycoprotein^893.3.1. Lymphocyte proliferative response to El peptides^ 893.3.2. Antibody response to El peptides^ 943.3.3. Discussion of Section III^ 954. SUMMARY and PERSPECTIVES 995. REFERENCES^ 104LIST of TABLESTable I.^Congenital features of rubella^ 14Table II.^Methods used to localize epitopes in virus^ • 31Table III.^Summary of T-cell epitopes of the RV structural proteins^ 31Table IV.^List of oligonucleotides used in the construction of the El deletion mutants^35Table V.^Synthetic peptides screened with monoclonal antibodies^ 35Table VI.^Summary of properties of monoclonal antibodies directed against El^51Table VII.^El synthetic peptides^ 58Table VIII.^ELISA immunoreactivity of individual structural proteins and whole virus.^84Table IX.^Amphipathic scores of El peptides^ 92Table X.^Lymphocyte proliferative responses to El synthetic peptides^ 93Table Xl.^B-cell response to El peptides^ 93viLIST of FIGURESFigure 1. Schematic representation of the synthesis and processing of RV structural proteins. 4Figure 2. Comparative diagram of the genomes of RV and Sindbis Virus.^ 5Figure 3. Model of the El/E2 glycoprotein spike of RV.^ 10Figure 4. Representation of the pathogenesis of RV infection:^ 12Figure 5. The relationship between the time of maternal RV infection and fetal development.^13Figure 6. Physical map of the expression vectors pSPT18/19 and pCMV5.^ 40Figure 7. Physical map of the expression vector pET.^ 41Figure 8. Schematic diagram of the cDNA fragments used for the construction of El mutants. 61Figure 9. Immunoprecipitation of in vitro translated El mutants.^ 62Figure 10. Immunoblot of El mutants expressed in COS cells. 65Figure 11. Immunoblot of El mutants expressed in E.coli.^68Figure 12. Coomasie brilliant blue stained SDS-PAGE of mutants expressed in E.coli.^70Figure 13. Position of El peptides relative to the mutant m7.^ 70Figure 14. ELISA of the peptides EP11 to EP15 and EP25 with MAbs 21B9H and.3D9F.^71Figure 15. ELISA of the peptides EP24 to EP26 with MAbs 21B9H and 16A10E.^71Figure 16. Summary of the VN and HA epitope mapping studies.^ 72Figure 17. Proliferative response to RV antigens expressed from vaccinia recombinants.^80Figure 18. Proliferative response to RV structural proteins in the study group.^81Figure 19. Comparison of proliferative and IgG responses to the RV structural proteins.^83Figure 20. Predicted structure of El protein by conventional structural analysis algorithm.^91Figure 21. Immunoblot analysis of human sera against M33 RV antigens.^ 91Figure 22. Peptide stimulated T-cell proliferative responses.^ 92viLIST of ABBREVIATIONSas^ amino acidATP adenosine triphosphateAU^ Arbitrary UnitsBCIP 5-Bromo-4-Chloro-3-Iridyol PhosphateBSA^ bovine serum albumin°C Degrees CelsiusCB^ conjugate bufferCIC circulating immune complexCRS^ Congenital Rubella SyndromeCTP cytidine triphosphateDNase^ deoxyribonucleaseddNTPs dideoxynucleoside triphosphatesdNTPs^ deoxynucleoside triphophatesEDTA ethylene diaminetetraacetic acidELISA^ enzyme linked immunosorbent assayendo H endo-f3-N-acetylglucosaminidase HER^ endoplasmic reticulumFMDV Foot and Mouth Disease VirusGTP^ guanosine triphosphateHA hemagglutininHAI^ hemagglutination inhibitingHBV Hepatitis B VirusIg^ immunoglobulinIU international unitskDa^ kilodaltonLCMV^ Lymphocytic Choriomeningitis VirusmicrogrammicrolitreM^ molarMEM Minimum Essential MediumMHC^ Major Histocompatibility complexmg milligramml^ millilitremM millimolarmoi^ multiplicity of infectionMMR mumps, measles, rubella vaccineMW^ molecular weightNBT Nitro-Blue Tetrazoliumns^ non-structuralnt nucleotidePAGE^ polyacrylamide gel electrophoresisPBL peripheral blood lymphocytesPBS^ phosphate-buffered salinePMSF phenylmethyisulfonyl fluorideRAA^ Rubella Associated ArthritisRNase ribonucleaseRSV^ Respiratory Syncytial VirusRV Rubella VirusS^ Svedberg UnitSB sample bufferSD^ standard deviationSDS sodium dodecyl sulfateSFV^ Semliki Forest VirusSI stimulation indexSV^ Sindbis VirusTBS tris-buffered salineTTP^ thymidine triphosphateUTP uridine triphosphatexACKNOWLEDGEMENTSI would like to extend by heartfelt gratitude to my supervisor Dr. Gillam for giving me the opportunity toundertake this project and for her endless patience throughout, especially during those years in which Isuffered severe growing pains, and for her understanding and encouragement to the very end. I would alsolike to thank my supervisory committee, Drs. McMaster, Tufaro, Beatty for their critical comments andsuggestions throughout this project.I would like to thank Dr. Tingle for the patient samples and for taking the time to provide criticisms of theproliferation data.I want to thank the members of Shirley's lab, Helen, David, Nina and Qui for their help. To my friendsMarita, Tom, Chris and Bob, with whom I've shared a mid-night beer or two, for helpful discussions andcriticisms.Lastly but most importantly, I want to thank my family. To my parents for their support and unconditionalfaith inspite of my past failures, I am forever indebted.This thesis is dedicated to my family ... Mom, Dad, John and Mikey.1. INTRODUCTIONRubella virus (RV) causes a relatively mild German measles disease most commonin children. When contracted in utero, RV can cause a wide variety of birth defects,collectively described as congenital rubella syndrome (CRS) (Oxford and Obery, 1985).Moreover, there is an increasing evidence that RV is a significant human pathogeninvolved in panencephalitis (Townsend et al., 1975) and polyarticular arthritis (Chantleret al., 1981; 1982; 1985).Presently available rubella vaccines were developed with limited informationregarding the genetics of the virus and the molecular basis for its virulence.Complications such as polyarticular arthralgia and arthritis following vaccination arecommon (Chantler et al., 1982). Rare cases of progressive panencephalitis have alsobeen attributed to adverse complications resulting from vaccination (Marvin, 1975;Townsend et al., 1975). In addition to these problems, RV grows to relatively low titreand its structural proteins are difficult to purify. Thus, in light of these problems, the needfor a new approach to designing a safer and effective rubella vaccine is evident. Thisthesis will discuss the methodology with which immunologically functional epitopes wereelucidated and the potential role - of these epitopes in the RV vaccine development.1.1 RUBELLA VIRUS 1.1.1. ClassificationRV is the only member of the genus Rubivirus in the Togaviridae family (Porterfieldet al., 1978) which also include Alphavirus, Pestivirus and Arterivirus. Togaviruses are1enveloped RNA viruses whose genome consists of a single molecule of single-strandedpositive polarity RNA complexed with a single species protein to form an icosahedralnucleocapsid. Surrounding the nucleocapsid is a host cell-derived lipid bilayer in whichglycoprotein spike complexes are embedded.1.1.2. MorphologyElectron microscopy studies of RV have shown that the virus is spherical andapproximately 60-70 nm in diameter with a 30 nm dense core enveloped by a lipid bilayer(Murphy et al., 1968; Von Bonsdorff and Vaheri, 1969). Spikes of 5-8 nm in length onthe surface of the virion are associated with hemagglutinin activity (Holmes et al., 1969).The site of budding seems to vary with the host cell type. Maturation in BHK-21cells occur primarily in cytoplasmic vesicles, Golgi and vacuoles, and to a lesser extentat the plasma membrane. In Vero cells, however, virions bud exclusively from the plasmamembrane (Bardeletti et al., 1979).1.1.3. Nucleic Acid and Genome Organization and ReplicationThe RV genome is an infectious single-stranded RNA molecule with asedimentation rate of 40S (Vaheri and Hovi, 1972). Infected cells contain a 40S genomicand a 24S subgenomic mRNA both of which are capped and polyadenylated (Oker-Blomet al., 1984; Kalkinnen et al., 1984). The subgenomic 24S mRNA corresponds to the 3'one-third of the 40S RNA and encodes the structural proteins E1, E2 and C (Oker-Blomet al., 1984). The sequence for the 24S subgenomic mRNA is known for wild type M332(Clarke et al., 1987, 1988) and Therein strain (Frey et al., 1986; Frey and Marr, 1988) aswell as for vaccine strains RA27/3 and HPV77 (Nakhasi et al., 1989a; Zheng et al., 1989).In comparison to M33 strain, 31 amino acid changes in RA27/3 and 5 amino acidchanges in HPV77 were found. Whether these changes are important for attenuation isyet to be determined.The RV genome is 9757 nucleotides in length and has a G/C content of 69.5%(Dominguez et al., 1990). It contains two long open reading frames (ORF's): a 5'proximal ORF of 6656 nucleotides and a 3' proximal ORF of 3189 nucleotides. Thegenome organization is similar to that of alphaviruses. Sequences homologous to threehighly conserved regions among alphaviruses were found on the RV genomic RNA: astem-loop structure at the 5' end of the genome, a 51 nucleotide sequence near the 5'end of the genome and a 20 nucleotide sequence at the subgenomic RNA start site(Dominguez et al., 1990).The 24S subgenomic mRNA is 3346 nucleotides in length and contains a 3189nucleotide of open reading frame encoding NH 2-C- E2-E1-COOH (Frey and Marr, 1988).The nucleotide sequences of 24S mRNA from various RV strains indicate 95% homology,although little homology was found with the alphavirus subgenomic mRNA (Frey andMarr, 1988).The first event leading to the expression of the structural proteins is the synthesisof 24S subgenomic mRNA from the negative sense template intermediate (Oker-Blom,1984). The 24S mRNA encodes a 110,000 dalton polypeptide (p110) that isproteolytically processed to yield three structural proteins C, E2, and El (Oker-Blom et3al., 1983) (Fig. 2). The capsid protein C, a non-glycosylated protein of 33 kD, is rich inbasic amino acids and proline (Clarke et al., 1987). El (58 kD) and E2 (42-47 kD) areboth type I membrane glycoproteins comprise the viral spikes located on the virionsurface (Oker-Blom et al., 1983). C protein remains in the cytoplasm whereas the E2 andEl proteins are co-translationally translocated into the endoplasmic lumen.GENOM1C 40S RNA5'4^ I-(A)N 3'24S RNA(A)n 3'POLYPROTEIN (110kD)NH2^ COOHC (33kD)^E2 (30kD) El (52kD)11111111111,11115111MEMIE2 (42-47kD) El (58-62kD)Fig. 1. Schematic representation of the synthesis and processing of the structuralproteins of RV c = non-glycosylated El and E2, ma= glycosylated E2;—= glycosylatedEl (from Wolinsky, 1990).1.1.4. Non-structural ProteinsThe non-structural proteins are encoded by the 5' two-thirds of the 40S genome(Dominguez et al., 1990). Amino acid comparisons between the non-structural proteinsof RV and alphaviruses revealed only one short (122 amino acid) region of significanthomology indicating that these viruses are distantly related. This region of homology islocated at the N-terminus of nsP3 in the alphavirus genome (Fig. 1). RV non-structural46K El 1■Poly ASG RNA■Poly,A• C E3 E2ElSG RNASIN ■RUB ■0 51 nucleotide conserved regionprotein ORF contains two global amino acid motifs (helicase and replicase motifs) whichare conserved in a large number of positive polarity RNA viruses (Dominguez et al.,1990).The order of the helicase motif and the nsP3 homology region in the RV and alphavirusesis reversed with respect to each other indicating a genetic rearrangement during theevolution of the viruses. To date there is no data pertaining to the RV non-structuralproteins beyond the genetic organization. The RV non-structural proteins are yet to bedefined and analyzed but are thought to be similar to those of alphaviruses.0^1.0^2.0^3.0^4.0^5.0^6:0^7.0^8.0^9.0^10.0^11.0^12.01 ^nsP1^nsP2^nsP3^nsP4Helicas• motifRegion of homology between SIN and RUBReplicas, motif• SG RNA start site conserved regionFig. 2. Comparative diagram of the genomes of RV (RUB) and Sindbis virus (SIN).The location within the non-structural protein ORF of regions of nucleotide homology (51nucleotide conserved region and SG (structural genes) RNA start site conserved regionsencoding homologous amino acid sequence (helicase motif, SIN and RUB homology, andreplicase motif) are shown. ORFs are denoted by boxes and untranslated regions by lines(from Dominguez et al., 1990). *5Nucleotide sequence analysis of Semliki Forest Virus (SFV) and Sindbis Virus (SV)genomic RNAs revealed that four genes encode the non-structural proteins (Strauss etal., 1984; Takkinen, 1986). Translation begins at an AUG codon, 60-80 nucleotides 5'to the cap site (Ou et al., 1983). The non-structural proteins are synthesized as twopolyprotein precursors which are then cleaved to produce the four non-structural proteins(Schlessinger and Schlessinger, 1990). One or more of these non-structural proteinsmust possess an RNA dependent RNA polymerase activity whose functions includeinitiation and elongation of the full length positive and negative sense RNA and positivesense 26S subgenomic RNA (Lemm and Rice, 1993), as well as capping and methylation(Schlessinger and Schlessinger 1990).Bowden and Westaway (1985) found other polypeptides of 150,000 and 87,000daltons in RV infected cells. They also identified several other minor RV specific proteinswith sizes ranging from 17,000 to 111,000 daltons. Much work is required to elucidatethe identity and functions of these proteins before the molecular mechanism for replicationcan be defined.1.1.5. Structural Proteins1.1.5.1. Capsid ProteinThe capsid (C) protein is associated with the 40S genomic RNA to formnucleocapsids (Vaheri and Hovi, 1972). Sequence analysis of RV cDNA indicate that Cprotein is rich in arginine and proline (Clarke et al., 1987). C is often detected as adoublet on SDS-PAGE under reducing conditions, differing in less than 1 kD. This6phenomenon may occur as a result of two closely spaced translation initiation sitesseparated by 7 amino acid residues (Clarke et al., 1987). Under non-reducing conditions,C runs in a dimeric form with an apparent molecular weight of 66 kD suggesting disulfidebond linkages in native proteins (Mauracher et al., 1991).Unlike alphaviruses, RV capsid has no inherent autoprotease activity (Clarke et al.,1987; McDonald et al.,1991). Studies with monoclonal antibodies have mapped four non-overlapping epitopes, the significance of which is not yet clear (Waxham and Wolinsky,1985a, b). E2 GlycoproteinRV E2 glycoprotein forms the spike complex on the virion surface and migrates asa diffuse band (42-47 kD) on a reducing SDS-PAGE. E2 synthesized in the presence oftunicamycin has a molecular weight of 30 kD indicating that it is heavily glycosylated(Oker-Blom et al., 1983; Clarke et al., 1988). The number of potential N-linkedglycosylation sites vary in different strains of RV as determined by nucleic acidsequences. There are three N-linked glycosylation sites in M33 and HPV77, whereasboth Therein and RA27/3 contain four glycosylation sites (Clarke et al., 1987; Vidgren etal., 1987; Frey and Marr, 1988; Nakhasi et al., 1989). The carbohydrate moieties on RVglycoproteins are of the complex endo-H resistant type (Oker-Blom et al., 1983), andsialiated 0-linked sugars are also present on E2 (Lundstrom et al., 1991).Biological functions of E2 are not clearly defined. E2 is necessary for transport ofEl to the Golgi and cell surface (Hobman et al., 1990). In addition, strain specific7epitopes and a neutralizing epitope have been found on E2 (Dorsett et al., 1985; Greenand Dorsett, 1986).Due to the poor immunogenicity of E2, few monoclonal antibodies have beenisolated (Green and Dorsett, 1986; Waxham and Wolinsky, 1985a, b). Digestion of intactvirions with mixed glycosidases indicates that the carbohydrate moieties on E2 are lessaccessible than those on El (Ho-Terry and Cohen, 1984), suggesting that E2 may beburied under El in the spike complex. El GlycoproteinEl (58 kD) is the most well studied RV structural protein. Post translationalmodifications of El include fatty acid acylation in the C-terminal region (Waxham andWolinsky, 1985a; Hobman et al., 1990) as well as the N-linked glycans (Frey et al., 1986;Clarke et. al.,1987; Terry et. al.,1988; Hobman et al., 1991). The glycosylation of El isthought to stabilize the conformation of El in its biologically and immunologicallyfunctional state (Ho-Terry et. a1.,1984).El contains the hemagglutinin activity as well as a number of neutralizationdomains (Waxham and Wolinsky 1983, 1985a; Green and Dorsett, 1986). Using trypsinand staphylococcus V8 protease digestion, three non-overlapping hemagglutinin domainswere mapped between residues 245-285 of El (Ho-Terry and Cohen, 1 . 985; Terry .,1988). Competitive inhibition assays with a panel of monoclonal antibodies defined sixnon-overlapping epitopes on El with viral neutralizing and/or hemagglutinin activities(Waxham and Wolinsky, 1985a). There are three N-linked glycosylation sites on El8(Clarke et al., 1987). The role of carbohydrate in the presentation of antigenic andimmunogenic epitopes on El were examined by Qiu et al. (1992). They have constructeda panel of vaccinia recombinants expressing glycosylation mutants of E1, and haveshown that the single glycosylation mutants (G1, G2 and G3) but not the double mutants(G23) or the triple mutant (G123) were capable of inducing antibodies with viralneutralizing activities. Among the single glycosylation mutants, only G2 and G3 wereactive in producing antibodies with hemagglutination inhibiting activities. They alsoobserved that all the El monoclonal antibodies to El used in the study recognized all theglycosylation mutants. This study indicates that although carbohydrate on El may notbe directly involved in the antigenic structures of E1, it is important in maintaining a stableconformation for expression of immunogenic epitopes.From the predicted topological structure of the glycoprotein spike complex (Fig. 3),and the localization of hemagglutination and viral neutralization activities on E1, it isthought that El mediates binding to the viral receptors on the host cell surface. Inaddition, immunological studies suggest that antibodies to El play a major role inprotective immunity to RV (Katow and Suguira, 1985).9Fig 3. Model of the El/E2 glycoprotein spike of RV. N-linked sugars are indicatedby ( 0) located on both El and E2 whereas 0-linked sugars ( ■ ) are located on E2only (from Ph.D thesis of C. Mauracher, 1992).101.1.6. RV EntryEntry of togaviruses into the host cells is thought to occur via receptor mediatedendocytosis (Helenius et. al.,1982). Bound virions accumulate in coated pits which arethen endocytosed to form coated vesicles. The vesicles fuse with endosomes resultingin acidification of the vesicles. This causes a conformational change in the viralglycoprotein complex resulting in fusion of the viral envelope with the endosomalmembrane (Tycko and Maxfield, 1982).Although the entry and uncoating of RV have not been studied as extensively asalphaviruses, sufficient evidence suggest that RV also uses the endocytic pathway for cellentry (Vaananen and Kaariainen, 1980). pH of <5.0 causes structural changes in RV Elallowing RV membrane to fuse with endosomes (Katow and Suguira, 1988). The acidicenvironment of the endosomes is also thought to cause the RV C protein to becomehydrophobic resulting in nucleocapsid uncoating in the endosome and releasing the viralRNA into the cytoplasm for replication initiation (Mauracher et al., 1991). To date aspecific membrane receptor has not been identified for RV. Regardless of the multiplicityof infection, it has not been possible to infect 100% of a cultured cell monolayer with RVand has led to the speculation that RV receptor is expressed in a cell cycle manner(Hemphill et al.,1988).1.2. Rubella Pathogenesis and Pathology 1.2.1. Clinical FeaturesThe clinical symptoms of RV infection can range from subclinical to the11Days Before7 6 5 4 3 2 1 1 2 3 4^6^8^10 12 14^1 2^1 2 3^10JDays After Onset of Rash^Months^Years1024EL1SAI^Virus in Throat.^A^•^S^•HNEUTRALIZINGti1 00 6432.4.*\CF^XI^I^1^ir""I" 7^7^7^-I^IalgM \A" T00HAI^""k--.characteristic features of adenopathy, general malaise, low grade fever and exanthem(Cooper and Buimovici-Klein, 1985). In more severe cases, the RV infection associatedwith progressive panencephalitis which manifests 10 to 20 years after the infection(Townsend et al., 1985). The general representation of the RV pathogenesis is shownin Fig. 4. The clinical infection spans only a short portion of the time scale, lasting fromshortly before the onset of the rash to shortly after the recession of the characteristicrash. Furthermore, many cases of RV infection pass without clinical symptoms includinga discernible rash (Wolinsky, 1990).Fig. 4. Rerpresentation of the pathogenesis of RV infection from the time ofinfection. HAl=hemagglutinin inhibiting IgG; CF=complex fixing anti-RV IgG (from Cooperand Buimovici-Klein, 1985).1280I^I CR INFECTIONUNCONFIRMED CRSNM CONFIRMED CRS6040200RV infection becomes a more serious medical concern when contracted during thefirst trimester of pregnancy leading to fetal infection. The virus is highly teratogenic andin utero infection often results in a variety of birth defects collectively called congenitalrubella syndrome (CRS) (Fig. 5). Cataracts, mental retardation, deafness, congenitalheart disease are some of the more common defects (Table I) (Cooper and Krugman,1969).2^4^6^8^10^12^14^16^18^20 >20BIWEEKLY INTERVAL OF GESTATIONFig. 5. The relationship between the apparent time of maternal rubella infectionand the consequence of the infection for fetal development. The data are based on 422infants registered in the National Congenital Rubella (from Wolinsky, 1990).13Table I. Congenital feature of Rubella (from Oxford and Obery, 1985).Feature^ Incidence'Intrauterine growth retardation (low birthweight)^ TCThrombocytopenic purpura^ TCHepatosplenomegaly^ TCfvteningoencephafitis TCBone lesions (radiographic)^ TCLarge anterior fontanelle TCAdenopathy. generalized TUHepatitis^ TUCloudy cornea^ TUHemotytic anemia T11Pneumonia due to rubella^ TUMyocarditis 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 microphthatmia)^ PCRetinopathy^ PCGlaucoma PU (DU)Severe myopia PDUtnguinaf hernia^ PUCryptocchidism PUTransient (r); permanent (P); developmental (D): common(C); or uncommon (U).1.2.2. Congenital Rubella SyndromeRubella was first described in 1814, but did not attract serious attention until 1942when Dr. Norman Gregg recognized the teratogenicity of the virus (reviewed in Cooperand Buimovici-Klein, 1985). Following Gregg's initial observation, world wide studiesestablished that a syndrome of defects resulted from intra-uterine infection. Congenitalrubella, due to the multitude of symptoms, is clinically complex (Cooper and Buimovici-Klein, 1985).14Prospective studies show that infection during the first four weeks of pregnancyresults in congenital defects up to 90% of the infants (Fig. 5). The incidence of congenitaldefects resulting from intrauterine infection in the first four months of pregnancy has beenestimated at between 15% to 50%. Also, intrauterine infections during the first 8 weeksmay result in spontaneous abortions at a rate of 20% (Oxford and Obery, 1985). Therate of fetal infection diminishes to an estimated 25-30% following the first trimester (Milleret al., 1982). The severity of birth defects and late onset sequelae correlate strongly withthe gestational age of the fetus at infection. The rate of infection and the severity of thedefects are independent of the severity of the rubella in the adult (Oxford and Obery,1985).The mechanism by which the virus interferes with fetal development is not clear.It has been suggested that RV infection interferes with regular mitotic division and thatinfected cells secrete factors that inhibit mitosis of cell in culture (Plotkin and Vaheri,1967). Increased numbers of chromosomal breaks have also been observed in RVinfected cells, but it is not clear whether this is a direct consequence of abnormal mitosis.Yoneda et al (1986) observed that cells persistently infected with RV responded poorlyto growth factors and produced little collagen in comparison to uninfected cells. Thisfinding is consistent with general observation of organ dysfunction and retarded growthin congenital rubella syndrome (CRS) patients.The mechanisms proposed are predicated on the hypothesis that RV hasestablished a persistent infection in CRS patients. Virus can be isolated from mostorgans at birth and is actively secreted in urine, stool and nasopharyngeal secretions in15more than 80% of CRS patients in the first month of life (Cooper and Krugman, 1967).The rate of viral excretion decreases as patients get older, however, viruses have beenisolated from CRS patients 20 years and older (Menser et al., 1967; Well et al., 1975).Proposed mechanisms for viral persistence are as varied as those for theteratogenicity of the virus. First, Abernathy et al (1990) maintained a RV persistent Verocell line in the presence of anti-RV antibodies for 45 weeks. The investigators suggestthat since RV buds both at the cytoplasmic membrane and into the intracellular vacuoles,the passage of virus to daughter cells occurs during cell division. In this proposed model,the virus avoids exposure to antibodies and is thereby protected from antibody mediatedclearance. The same study reported that the persistent virus population treated withantibody was less cytopathic. Accordingly, they proposed that in the presence of anti-serum, the only repository for virus was the infected cells. Those infected with cytopathicvirus were killed, resulting in survival of less cytopathic virus population. These resultsmay explain persistent infection of RV in some individuals regardless of the presence ofvigorous humoral response.Contrary to the above, second plausible mechanism for viral persistence arisesfrom the observations of reduced immunity. This reduced immunity coined asimmunological tolerance has also been observed in other viral infections. In utero infections leading to viral persistence have been documented with LymphocyticChoriomeningitis Virus (LCMV) in mice (Traub, 1983), Borna agent in rats (Hirano et al.,1983) and Border Disease virus in sheep (Barlow, 1983). In case of LCMV or Bornaagent, viral- specific cytotoxic T cell response is absent. RV specific T cell response16studies have shown reduced levels in CRS patients (Buimovici-Klein and Cooper, 1985).The degree of impairment was greater in children infected during the first trimester ofgestation. It is possible that the lack of a cytotoxic T cell response allows the virus topersist in vivo. Another study reported that CRS patients have reduced ratios ofCD4+/CD8+ in addition to overall reduced levels of RV specific activated T-helper cells(Rabinow et al., 1986). Antigen specific proliferation assay revealed that CRS patientsexhibited higher lymphocytic response to E2 than the normal population (Chaye et al.,1992b). The poor immunogenicity of E2 in the normal population may have evolved asa protective mechanism against the observed adverse effects arising from E2 immunityin the CRS patients. This is further supported by the E2 induced autoimmunelymphocytic hypophysitis-like syndrome in hamsters that could be prevented by neonatalthymectomy (Yoon et al., 1991). The inability of CRS patients to produce high affinityanti-rubella IgG (Fitzgerald et al., 1988), the reduced levels of El reactive IgG (Katow andSuguira, 1985) and deletion on HAI IgG (Cooper et al., 1971) have all been proposed aspossible mechanisms of decreased antibody mediated viral clearance of RV resulting inviral persistence.1.2.3. Rubella Associated ArthritisThe pathogenesis of rubella associated arthritis (RAA) either from wild typeinfection or from vaccination remains a matter of speculation (Ford et al., 1986). RV hasbeen isolated from synovial fluid and in peripheral lymphocytes in individuals with RAAyears after infection or immunization (Ogra et al., 1975; Chantler et al., 1985). Suchfindings have led to the proposal that RV can persist in the cells of synovial fluid or the17synovial membrane. RAA is most commonly associated with peripheral joints (Sauter andUtsinger, 1978). The lower temperature of the peripheral joints may be critical with theestablishment of persistent infection since it has been reported that RV readily establishespersistent infections of synovial cell cultures at 32°C but not at 36°C (Cunningham andFraser, 1985). More recently, Miki and Chantler (1992) have shown that wild type andvaccine strains of RV replicate at different rates in human synovial cell lines and in humansynovial membrane organ cultures. They also reported that strains which have beenreported to have a higher incidence of RAA exhibited a high degree of synoviotropism.Although persistence of RV seems to be associated with RAA, the pathology remainsunclear. RAA may either be a direct consequence of non-lytic strain of RV or an indirectconsequence of persistent infection triggered immunopathogenesis.Circulating immune complexes (CIC) and their deposition in the synovial space isone hypothesis that is favoured in describing the pathogenesis of RAA (Inman et al.,1987). CIC are involved in inflammatory responses of CRS patients (Tardieu et al., 1980)and are most likely responsible for the vessel damage which presents as a rubelliformrash in acute Rubella. Information regarding CIC involvement in RAA is incomplete. Nosignificant increase of CIC was observed in RAA patients compared to healthy rubellavaccinees (Singh et al., 1986). Conversely, others have shown increase of CICcontaining anti-RV IgG following RV vaccination (Coyle et al., 1982). However, since nocomparative study was done between the healthy and RAA patients, this study is notconclusive.Reactivity of the virus-specific immunity with self-antigen has been proposed18(personal communication with Dr. J.K. Chantler) describing cross-reactivity of rubellaspecific epitopes with an unidentified protein derived from synovial epithelium. Anotherstudy reported cross-reactivity between pituitary cell proteins and RV glycoproteins Eland E2 (Yoon et al., 1991). They found that hamsters injected with either El or E2vaccinia recombinants developed autoimmune lymphocytic hypophysitis evidenced by theinduction of autoantibodies against pituitary cells and by lymphocytic infiltration of thepituitary. These studies suggest that molecular mimicry may be significant in themechanism of RAA pathogenesis.A recent model for the pathogenesis of RAA has involved the role of undefinedinfectious agents as superantigens (Paliard et al., 1991), and the role of RV in this contextis being currently investigated. RV capsid molecule shares structural similarities with theS. aureus enterotoxin superantigen defined by Marrack and Kappler (1990), in that bothcontain a 8 membered f3-pleated barrel as their core structure.1.2.4. Rubella VaccineBetween 1965 and 1967, several live attenuated RV strains were developed andtested as vaccines. Of these, HPV77/DE5 and RA27/3 have been licensed in Europe andNorth America. Currently RA27/3 strain is commonly used as vaccine in Canada and theUSA. The mechanism of RV attenuation remains unclear. Sequences of the structuralproteins of both the wild type and the vaccine strains revealed only limited differences(Nakhasi et al., 1989a, 1989b). The low virulence and slow replication rates of vaccinestrains are thought to be due to a deficit in cell attachment and entry (Nakhasi et al.,191989).RA27/3 has served well in immunization of children since its licensure. However,it has been associated with significant adverse effects in a significant proportion ofadolescent and adult female vaccinees (Polk et al., 1982). Acute arthritis is estimated tooccur in 13-15% of adult RA27/3 vaccine recipients (Howson and Fineberg, 1992), andcases of severe, chronic arthritis have been reported following the administration ofRA27/3 in adult females (Tingle et al., 1986).1.2.5. Host Response to Viral InfectionsHost response to viral infection occurs in two phases (for review see Stites et al.,1984). Initially the response is largely non-antigen specific and involves stimulating theproduction of interferons and the activation of natural killer (NK) cells. Virus-infected cellsare more susceptible than normal cells to NK-lysis. Once virus starts replicating, antigenspecific immune responses are triggered. This involves T-cells and B-cells, both of whichare derived from bone marrow stem cells. In this section, only the antigen specificimmune response to virus infection will be discussed.B-cells undergo differentiation to plasma cells which secrete the various classesof immunoglobulins. This usually occurs under the influence of activated T-cells. T-cellsundergo thymic differentiation and education, during which they mature into differentsubclasses identifiable by cell surface antigens and by function. The two major T-cellfunctional subclasses are cytotoxic T lymphocytes (CTLs) and helper T lymphocyte(ThCs). CTLs are usually CD8+ and lyse virus infected cells. ThCs, which are usually20CD4+, are divided further into two subsets T H 1 and TH2. TH 1 cells produce gamma-interferon and IL-2 and promote cell-mediated effector responses; whereas TH2 cellsproduce IL-4, IL-5, IL-6 and IL-10, cytokines which produce B-cell development and canaugment humoral responses (Scott, 1993).The immune response to virus infection is generally characterized by the inductionof a response from both CTLs and T hCs (Whitton and Oldstone, 1990). Classicallydescribed antiviral CTLs are restricted by class I major histocompatibility (MHC) antigensin their recognition of viral antigens, while T hCs are restricted by class II MHC antigens.Expression of MHC class II molecules is restricted to specialized antigen-presenting cells(macrophages, dendritic cells, B-cells), whereas MHC class I molecules are expressedon most cells.It is widely accepted that T-cell receptors recognize degraded form of viral antigensbound to class I or class II MHC molecules (Williams and Smith, 1990; Teyton et al.,1990; Parham 1990). The processing pathways utilized by endogenously derived andexogenously derived antigens are distinct (Germain, 1988). Briefly, exogenous proteinantigens and polypeptides present in a virus (if entry is by endocytosis), are degraded inan acidic environment of the endosome and the resulting processed fragments associatewith class II molecules in the trans-Golgi or an endosomal compartment. Endogenouslyderived viral antigens are synthesized and degraded into fragments in the cytoplasm andsubsequently transported into the endoplasmic reticulum for association with class I MHCmolecules.B-cell receptor for antigen is immunoglobulin (Ig), initially expressed as a21membrane protein. During their development, B-cells express IgM on their surface,followed by expression of different subclasses IgG, IgA, IgE upon activation. Once B-cellhas switched its subclass from IgM, it becomes committed to secrete the. switchedsubclass of lg. When B-cell is activated by association with an antigen and bylymphokines secreted by T-cells, it differentiates into mature plasma cells which secreteone class of Igs. Memory cells are also generated.Antibodies play an important role in the control of virus infection (Whitton andOldstone, 1990). IgM and IgG can be effective in neutralizing viral infectivity. This mayresult from the antibody preventing virus attachment to specific cellular receptors byassociating with viral antigens that recognize cell receptors. Complexing of viruses withIgG antibody will also facilitate their phagocytosis by macrophages via Fc receptors onthese cells.Both humoral and cellular immune responses function together to clear virusesonce infected and to protect the host from future infection. However, it is thought thatcytotoxic T-cells are essential in viral clearance. For example, in persistently infectedmice by lymphocytic choriomeningitis virus, CTLs were not readily detectable (Jamiesonand Ahmed, 1985). This persistence, however, was terminated by adoptive transfer ofsyngeneic virus-specific MHC-restricted CTLs.221.2.6. Immune Response to RVContact with RV by infection or vaccination initially elicits an IgM response, mostlyto El (Salonen et al., 1985; Zhang et al., 1991). The IgM response is transient usuallylasting for approximately a month and is followed by production of other immunoglobulinclasses - IgG, IgE and IgA (Salonen et al., 1985; Zhang et al., 1991). IgG production isthe dominant serological response to all three structural proteins. Analysis by bothimmunoprecipitation and immunoblotting revealed that majority of RV IgG is directed toEl with lower levels specific for E2 and C (Katow and Suguira, 1985; deMazancourt etal., 1986; Zhang et al., 1991).IgGs to RV may have hemagglutination inhibiting and viral neutralizing properties(Green and Dorsett, 1986; Waxham and Wolinsky, 1985). The levels of IgG to RVcorrelate well with hemagglutination inhibiting titres and with neutralizing antibody titres(Stokes et al., 1969) and it is assumed that these responses play a positive role in viralclearance and protection (Waxham and Wolinsky, 1985a). Circulating immune complexescontaining RV specific antibody and antigen are frequently found after infection (Ziola etal., 1983) but, in most cases, their presence has not been associated with any of thecomplications following RV infection or vaccination (Singh et al., 1986).Much less is known about the importance of cellular responses to RV infection.RV-specific cellular responses have been demonstrated using lymphocyte proliferationassays and lymphocyte mediated cytotoxicity assays (Buimovici-Klein and Cooper, 1985;Vesikari and Buimovici-Klein, 1974; Ilonen and Salmi, 1986). Cell-mediated cytotoxicityhas been implicated in pathogenicity of RV infection (Martin et al., 1989). Ilonen and23Salmi (1986) noted that the RV-specific cellular responses are MHC restricted. Similarly,Ou et al. (1992a,b,c) isolated T-cell clones against the E2 glycoprotein and the C proteinfrom RV seropositive individuals and found that HLA restrictions are associated with HLADR7 and HLA DR4 for E2 and C epitopes respectively.Early cellular studies have been limited to the responses to whole RV in immuneand susceptible individuals (Buimovici-Klein et al., 1979; Vesikari and Buimovici-Klein,1974; Kauffman et al., 1974). The results of these studies demonstrated developmentof cellular immune response over time. Only recently RV protein specific lymphocyteproliferation assays have become possible. Ou et al (1992a,b) have mapped T-cellspecific epitopes on E2 and C using synthetic peptides. In addition to E2 and C,McCarthy et al (1993) also identified T-cell epitopes on the structural protein E1.1.3. Epitope Mapping 1.3.1. Epitope mapping using expressed proteins from E.coli Antigenic and/or immunogenic epitopes have been localized by expressing partsof the viral proteins using prokaryotic expression systems and determining the antigenicreactivity of the expressed products. The inserted viral genes are expressed in E. coli either as fusion proteins or on their own. The products are usually analyzed either bywestern blot techniques and/or radio-immunoprecipitations (Rosenberg et al., 1987; Terryet al., 1989; Studier et al., 1990; Wolinsky et al., 1991). A number of functionalcontinuous epitopes on viral proteins have been identified by means of prokaryoticexpression vectors such as RV (Terry et al., 1989; Wolinsky et al., 1991; Chaye et al.,241992a), feline leukemia virus (Nunberg et al., 1984), hepatitis B virus (Offensperger et al.,1985; Milich, 1988), infectious bronchitis virus (Lenstra et al., 1990), and humancytomegalovirus (Kniess et al., 1991).Prokaryotic expression system may not be the appropriate choice for epitopeanalysis of all viral proteins. First, because expression levels depend on the growth ofthe host bacteria, any interference by the viral proteins may result in low levels ofexpression. For example, inhibition of  E.coli growth have been observed upon expressionof the vesicular stomatitis virus G protein and hepatitis B virus S protein which result inlow expression levels (Rose and Shafferman, 1981). Second, the expressed productsmay lose their biological activity after purification (Kleid et al., 1981). Purification ofrecombinant proteins require denaturation in a chaotropic agents followed by renaturation.Furthermore, viral proteins expressed in E.coli may lack the appropriate post-translationalmodifications which may influence the tertiary structure of the protein. Thus the lack ofthese modifications may result in conformation that is no longer functional. Theprokaryotic system is therefore most useful for antigens where activity does not dependon conformation or post-translational modifications such as glycosylation or formation ofdisulfide bonds. However, for antigens which are conformation independent, prokaryoticexpression systems can be valuable tools for expression. For example, hepatitis B virusHBcAg produced in E.coli is immunogenically active and confers protection against HBVchallenge in immunized animals (Milich, 1988).251.3.2. Epitope mapping using expressed eukaryotic systemsExpression of viral proteins in eukaryotic cell lines is a commonly used system forthe analysis of the viral protein, post-translation modifications and the viral assembly(reviewed in Rutgers, 1990). It is also important in the analysis of viral epitopes whichare conformation dependent and are influenced by the post-translational modifications.However, for the purposes of subunit vaccine design, expression in eukaryotic cells hashad limited success. First, the viral protein domains containing the immunologicallyfunctional epitopes should be produced in large quantities. Second, they should beproduced in systems that will permit relatively simple purification. Third, the epitopesshould be conformation independent such that once purified from the rest of the proteins,they will retain their biological functions.A number of viral polypeptides have been expressed by using recombinant DNAtechniques in a variety of eukaryotic cell systems (Cane and Gould,1988; Emini et al.,1988; Luckow and Summers, 1989; Putnak et al., 1988). Genetically engineered vacciniaviruses have been used to express epitopes of hepatitis B virus, herpes simplex virus andinfluenza hemagglutinin (reviewed in Mackett, 1990). Recombinant baculovirusescontaining genes for animal virus proteins have also been shown to induce protectiveimmunity in rats and rabbits (Luckow and Summers, 1988). Manipulations of S.cerevisiae genome resulted in expressions of the hepatitis B virus core protein and theHIV gag p55 (reviewed in Rutgers et al., 1990).The expression efficacy for a particular viral protein appear to depend on thecompatibility of the foreign protein to be expressed and the system chosen as well as the26purpose for which the expressed protein will be used. RV structural proteins have beenexpressed both in the eukaryotic and in the prokaryotic expression systems. The proteinsexpressed in the mammalian cells were used to study processing and transport of thestructural proteins (Clarke et al., 1988; Hobman et al., 1989, 1990). Vacciniarecombinants were used to study the peripheral blood lymphocyte proliferative responseto the individual structural proteins (Chaye et al., 1992b). Prokaryotic systems werechosen for the expression of antigens for epitope mapping (Terry et al., 1989; Wolinskyet al., 1991; Chaye et al., 1992a). Each system chosen were particularly useful for thepurposes of that study.1.3.3. Epitope mapping using synthetic peptidesSince the advent of automated systems for rapid synthesis of peptides with highyields, a plethora of antigenic and immunogenic studies of viral proteins have beenreported using synthetic peptides as antigens and as immunogens (Heber-Katz andDietzschold, 1986; Milich, 1988; Roehrig et al., 1989). Common methods used to localizeepitopes in proteins have included measuring the cross-reactivity of antibodies raisedagainst intact protein to synthetic peptides and measuring the cross-reactivity ofmonoclonal antibodies to the peptides. These experiments are usually carried out inenzyme linked immunoassay (ELISA) where peptides are either directly bound to theplate or are used to competitively inhibit binding of antibodies to proteins already boundto the plates.Various approaches in the use of peptides for mapping antigenic sites are used.One of the methods incorporate results of predictive algorithms that provide amino acid27sequences that will likely form secondary structures such as a-helices or 13-turns(Jameson and Wolf, 1988). The most commonly used algorithm is the hydrophilicity plot(Hopp and Woods, 1982). While many of the known antigenic sites have structures thatthese algorithms predict, not all sites with these secondary structures are antigenic.Another approach is the measurement of the affinity of antibodies elicited to the nativeantigen for synthetic peptides (Steward and Howard, 1987). The higher the affinity, thecloser the resemblance of the peptide to the determinant expressed on the native protein.It is thought that the degree of affinity reflect the complementarity of the antibody to thepeptide. A series of overlapping peptides spanning a region of interest have also beenused to map antigenic epitopes. It was found that although B-cell epitopes only consistof 5-7 amino acids, longer peptides provided better binding capacity. Therefore, thisapproach allows for fine mapping without compromising the binding capacity of thepeptides to the antibodies.Synthetic peptides have also proven to be instrumental in the T-cell epitopemapping (Brett et al., 1991; Fayolle et al., 1991; Ou et al., 1992; Wallace et al., 1991).Generally, a series of overlapping peptides are screened with T-cell clones, T-cell linesor with peripheral blood lymphocytes, in the latter to determine immunodominant epitopes.As with the studies using antibodies, algorithms for T-cell epitopes have been developed(Margalit et al., 1987; Rothbard and Taylor, 1988). Again as with B-cell epitopes, thesealgorithms are predictive tools and thus the predicted epitopes are not necessarilyantigenic, however, they can provide a measure of confirmation once epitopes have beenlocalized.281.3.4. Functional epitopes of Rubella virus structural proteinsIn 1985, Waxham and Wolinsky (1985) mapped hemagglutination and viralneutralization activities to the E1 glycoprotein. Since this study, much effort has beenfocused on delineating functional epitopes on E1 (Terry et al., 1988, 1989; Lozzi et al.,1990; Wolinsky et al., 1991). There are two prevailing reasons for these efforts. First,at present, serological techniques with whole RV as a target antigen for detection ofantibodies to RV are most commonly used for laboratory diagnosis of acute andcongenital rubella infections and for determination of rubella immunity. These serologicaltechniques lack defined specificity against antigenic determinants such as hemagglutininand virus neutralizing epitopes of RV. For example, women, seronegative as measuredby HI assay (<1:8), were shown to have moderate levels of RV specific antibodies,measurable by ELISAs using whole RV (Tingle et al., 1983). Sera from CRS patientshave higher levels of antibodies directed against E2 but with low or no reactivity to E1(Chaye et al., 1992b). Since ELISAs employing whole RV fails to distinguish between thevarious antibody specificities, it is necessary to define the functional epitopes of RVstructural proteins for diagnostic assays to assess the immunity against RV.Second, functional epitopes are essential in the development of a non-infectiousrubella vaccine. Subunit vaccines containing only those epitopes which will elicit protectiveimmunity without the adverse side effects can only be achieved by accurately definingimportant epitopes. Immunosuppressive epitopes and/or autoreactive epitopes can bedeleted in the construction of vaccines to limit the potential adverse effects of vaccination.Numerous methods of viral epitope localization have been developed (Table II)29(Van Regenmortel, 1990). Epitope analyses of RV El have utilized recombinant DNAtechnology (Terry et al., 1989; deMazancourt and Perricaudet, 1989; Wolinsky et al.,1991), peptide analyses (Lozzie et al., 1990; Terry et al., 1988; Mitchell et al., 1992) andcompetitive binding assays with monoclonal antibodies (Waxham and Wolinsky, 1985a).Six independent epitopes have been identified which are thought to be important for viralinfectivity and hemagglutination (Green and Dorsett, 1986; Waxham and Wolinsky,1985a). Epitopes that react with monoclonal antibodies that have hemagglutination andviral neutralization activities have been localized to El residues El 245 to El 285 (Terry etal., 1988) and residues E1 202 to E1283 (Wolinsky et al., 1991). The latter group hassubsequently narrowed the epitope to El m to E1 239 using a set of nested syntheticpeptides (Wolinsky et al., 1993).Protective immunity to viral infection requires activation of helper T cells specificfor viral antigens. A T-helper function is required for the production of neutralizingantibodies and the activation of cytotoxic precursors into cytotoxic effector cells (reviewedin Milich, 1989). Ou et al (1992a,b,c, 1993) have identified T-cell epitopes by screeningoverlapping synthetic peptides with peripheral blood lymphocytes (PBL) from immunedonors and subsequently with T-cell lines/clones derived from the PBL of immune donors.They have identified E1 358-E1 377 , E254-E274 and C255-C280 as the immunodominant T-cellepitopes. Furthermore, McCarthy et al (1993) applying essentially the same methodologyhave identified T-cell epitopes located on all of the structural proteins. List of all T-cellepitopes defined in these studies are summarized in Table Ill. These studies will providethe basis for future construction of an effective subunit vaccine for RV.30Table II. Methods used to localize epitopes in virus (from Van Regenmortel, 1990)Method^Type ofcpitopc recognizedI X-ray crystallography ofantigen-Fab complexes2 Study of cross-reactive bindingof natural or synthetic peptidefragments with viral antibodies3 Study of- cross-reactive bindingof fusion proteins with viralantibodies4 Study of- cross-reactive bindingof virus with anti-peptideantibodies5 Analysis of viral mutants withmonoclonal antibodies6 Competitive binding assayswith pairs of monoclonalantibodiesDiscontinuous cpitopc reactingwith homologous antibodyContinuous epitopc cross-reactingwith heterologotrs antibodyCross-reactive continuous CptlOpCCross-reactive Continuous CpitOpCNeutrafization,epitopcs anddiscontinuous epitopesOnly relative position of epitopesis definedTable ill. Summary of T-cell epitopes of the RV structural proteins.El^202-207El^226-239El^240-247El^248-261El^272-291El^307-326El^358-377E2^31-55E2 56-75E2^81-105E2 54-74C^9-18C^64-97C^119-152C^205-233C^255-280DLVEYYIHGPDWASPVCQPDCSRLVGATPERPRLRLVDADGEVWVTPVIGSQARKCGLHII HAHTTSDPWH PPGPLGLKFVEGLAPGGGNCHLTVNGEDVQLPFLGHDGHHGGTLRVGQHYRNASDVKOGHWKQGGWGCYNLSDWVCHTKHMDFWCVEHDRPPPATPTPLMEDLQKALEAGNRGRGQRRDWSRAPPPPEERQETRSQTPAPKPSPELGPPTNPFQAAVARGLRPPLHDPDTEAPTEACVRAYNQPAGCVRGVWGKGERTYAEQDFRVPLPPHTTERIETRSARHPWRIRFGAP311.4. Project Rationale and Thesis Objectives Although the history of Rubella dates back to 1941, little is known about thehumoral and cellular immune responses to RV. Due to lack of this information togetherwith the limited understanding of the immunopathology of RV infection, the developmentof an effective vaccine has been difficult.The objective of this thesis is to define functional epitopes on the El glycoproteinwhich may be used in the construction of subunit vaccines. Site specific deletion mutantsof the El glycoprotein expressed both in the eukaryotic and the prokaryotic expressionsystems were screened with monoclonal antibodies with hemagglutination inhibiting andviral neutralizing activities. The deletion mutants containing HA and VN domains werefurther divided into synthetic peptides and screened on ELISAs. The human B- and T-cellepitopes on El glycoprotein were analyzed and the results were discussed in relation tothe HA and the VN epitopes defined with the monoclonal antibodies. The potential useof these epitopes in the construction of subunit vaccines will be discussed.322. Materials and Methods2.1. Materials DNA modifying enzymes and restriction endonucleases were purchased fromBethesda Research Laboratories, Promega Biotec, New England Biolabs, BoehringerMannheim, Pharmacia and United States Biochemical Corporation. All enzymes wereused as specified by the manufacturer unless indicated otherwise. The oligonucleotidesHC-1, HC-2, HC-3, HC-4 and HC-5 were synthesized on an Applied Biosystemsoligonucleotide synthesizer by T. Atkinson (Biotechnology Laboratory, UBC) (Table IV).Human anti-rubella serum was a gift from Dr. Aubrey Tingle (Department of Pediatrics,University of British Columbia). Mouse monoclonal antibodies to El were producedpreviously in this lab. Tissue culture agents were from Gibco and Sigma. Synthetic Elpeptides were provided by Dr. P. Chong at Connaught Laboratories (Table V). Humanperipheral blood lymphocytes representative of asymptomatic immune study group weregenerously donated by volunteers and the patients samples were provided by Dr. AubreyTingle. COS cells were obtained from Dr. David Russell (Department of MolecularGenetics, University of Texas, Dallas). E.coli strains DH5a and DH5a F' cells werepurchased from BRL and BL21(DE3)/pLysS was given by Dr. William F. Studier (BiologyDepartment, Brookhaven National Laboratory, Upton, New York).2.2. Methods 2.2.1. Propagation of bacterial strainsE.coli strains DH5a and DH5aF' from Bethesda Research Laboratories were used33for the propagation of recombinant clones. DH5a cells containing recombinant plasmidswere grown in LB medium (1% tryptone; 0.5% yeast extract; 1% NaCI) containing 100ug/mi ampicillin (AP) for selection of antibiotic resistance. DH5aF' cells were propagatedin 2xYT (1.6% Tryptone, 1% Yeast Extract, 0.5% NaCI). E.coli strain BL21(DE3)/pLysScells were grown in medium with 100 pg/ml ampicillin plus 25 µg/ml chloramphenicol tomaintain the plasmid pLysS. For long term storage the bacterial strains were stored in15% glycerol at -70°C.2.2.2. Preparation of competent cells and transformationCompetent cells were prepared using a method described in Promega Biotectechnical bulletin 018. Briefly, E.coli cells were grown in 20 ml of LB broth until theabsorbance at 600 nm reached 0.15-0.3. Cells were centrifuged at 5000 rpm in a SorvallSS34 rotor at 4°C for five minutes, and the supernatant was discarded. The bacterialpellet was resuspended in 10 ml of cold solution A (10 mM 3-[N-morpholino]propanesulfonic acid (MOPS) (pH 7.0); 10 mM RbCI), and centrifuged as above. Cellswere then resuspended in 10 ml of cold solution B (10 mM MOPS (pH 6.5); 10 mM RbCI;50 mM CaCl 2) and incubated in ice for 30 minutes. After pelleting the cells as above, cellpellets were resuspended in 1 ml of solution B plus 15% glycerol, and quick frozen in 0.2ml aliquots in dry ice-ethanol and stored at -70°C.For plasmid transformation, 0.2 ml of competent cells were incubated on ice with10-50 ng of plasmid DNA for 30 minutes. After a two minute heat shock at 37°C, 1 mlof LB broth was added to the transformation mixture and the cells were allowed torecover at 37°C for one hour before plating onto selective media. For M1334Table IV. OligonucleotidesHC-1 CCATGGGGCATGGCCCCGATTGGGGCHC-2 CCATGGGGAACCAACAGTCCCGGTHC-3 CCATGGGGCATGGCCCCGATTGGCHC-4 CCATGGATGACAATTCGGGCTCCHC-5 CCATGGGGGACGCTCTGGCGTTable V. Synthetic peptides screened with MAbsEP11^GQLEVQVPPDPGDLVEYIMNEP12^IMNYTGNQQSRWGLGSPNCHEP13^NCHGPDWASPVCQRHSPDCSEP14^PDCSRLVGATPERPRLRLVDEP15^RLVDADDPLLRTAPGPGEVWEP24^VPPDPGDLVEYIMNYTGNEP25^DPGDLVEYIMNYTGNQQSRWGLGSPNCHGPDWASPEP26^GLGSPNCHGPDWASPtransformations, the cells were plated with 50 ul of 5-bromo-4-chloro-3-indoyl-13-D-galactoside (Xgal) (2%),10 isopropylthio-13-D-galactoside (IPTG) (100 mM), 50 ul freshexponential DH5aF', and 3 ml soft agarose (0.7% in 2YT at 55 °C) on YT withoutantibiotic.2.2.3. Growth of transformants and preparation of plasmid DNA2.2.3.1. Small scale plasmid preparationColonies containing plasmids were picked into 2 ml of 100 lag LB-ampicillin (AP)per ml or LB with 100 fag/m1 AP and 25 µg/m1 chloramphenicol (Cam) and the bacteriawere grown to saturation. M13 transformants were grown in 5 ml of YT containing 50 ulof an overnight culture of DH5aF' for 6 hours at 37 °C. Plasmid and M13 RF DNA wereisolated by the alkaline lysis method. Briefly, bacterial cells from 1.5 ml culture werepelleted for one minute in a microfuge. The pellet was resuspended in 100 of 50 mMglucose; 10 mM EDTA; 25 mM Tris-HCI (pH8.0) and lysed by the addition of 200 µl of 0.2N NaOH/1 °/0 SDS for five minutes at 0 °C. Chromosomal DNA and proteins wereprecipitated by incubating the lysis mixture with 150 RI of cold potassium acetate (3MK+;5M CH 3C00 - (pH 4.8)) at 0 °C for five minutes, followed by centrifuging in a microfuge forfive minutes at 4°C. The supernatant was extracted with an equal volume ofphenol:chloroform (1:1), and the DNA was precipitated with two volumes of ethanol atroom temperature for five minutes. Plasmid DNA was recovered by centrifugation in amicrofuge for five minutes at room temperature, washed in 70% ethanol, dried in a SpeedVac Concentrator, and resuspended in 50 pi of TE containing 20 µg/ml RNase A.36Aliquots were used for restriction analysis or subcloning. Large scale plasmid DNA preparationsThe protocol is a procedure obtained from Promega Biotec technical bulletin 009(developed by Dr. P. Krieg and Dr. D. Melton of Harvard University) with modifications.Cells grown in selective media overnight in 250 ml cultures were pelleted by centrifugationat 5000 rpm in a Sorvall GSA rotor at 4 °C for five minutes. The supernatant wasdiscarded and each pellet was resuspended in five nil of 50 mM Glucose; 10 mM EDTA;25 mM Tris-HCI (pH 8.0) containing 20 mg lysozyme followed by 20 minute incubationon ice. Cells were lysed by addition of 12 ml of 0.2 N NaOH, 1% SDS and incubation onice for 10 minutes. Chromosomal DNA and proteins were precipitated with eight ml ofcold potassium acetate solution (see mini-prep procedure) on ice for 20 minutes, followedby centrifugation at 10,000 rpm in a SS34 rotor at 4 °C for 15 minutes. RNAse A (100 ug)was added to the cleared lysate followed by incubation at 37°C for 30-45 minutes. Thelysate was extracted twice with equal volumes of phenol:chloroform, and the nucleic acidswere recovered in siliconized Corex tubes by centrifuging at 10,000 rpm for 10 minutesat RT in a SS34 rotor. The pellet was dried, and dissolved in 1.60 ml of sterile water.DNA was selectively precipitated by addition of 0.4 ml of 4 M NaCI and 2.0 ml 13%polyethylene glycol (PEG mw 8,000), mixing and incubation on ice for 60 minutes. Theplasmid DNA was pelleted in siliconized Corex tubes at 10,000 rpm for 15 minutes at 4°Cin a SS34 rotor, washed with 70% ethanol, dried and dissolved in TE.372.2.3.3. Isolation of single-stranded DNAFive ml of YT media containing 50 ul of overnight culture of DH5aF' cells wasinoculated with cored plaques of recombinant M13 phage and was grown for 6 hours at37°C. 1.5 ml cultures of DH5aF' containing M13mp18 or M13mp19 recombinants werepelleted by centrifugation in a microfuge for 5 minutes at room temperature. The pelletwas subjected to the plasmid mini-prep procedure for isolation of RF DNA. Phage wereprecipitated from the supernatant by addition of 300 41 of 20% PEG 80012.5 M NaCI,vortexing, and incubating at room temperature for 15 minutes. Phage were pelleted bycentrifugation for 15 minutes in the microfuge. The supernatant was discarded and thepellet was resuspended in 0.1 ml TE and extracted once with 50 of phenol, once with50 j.11 phenol:chloroform (1:1) and finally once with 100 p1 of chloroform. The aqueouslayer was ethanol precipitated with one tenth volume of 3 M sodium acetate (pH 5.5).The DNA was spun down in a microfuge, washed with 70% ethanol, dried, andresuspended in 30 gl TE.2.2.4. Expression vectorsThe multiple cloning sites of pSPT18 and pSPT19 (Pharmacia) are flanked byoppositely oriented T7 and SP6 RNA polymerase promoters which allow transcription ofeither strand of inserted DNA (Fig. 6). Synthetic mRNAs from cDNAs cloned into thesevectors were used to direct translation in a rabbit reticulocyte lysate system.For transient expression of RV cDNA in COS cells, pCMV5 (D. Russell, Texas)was used (Fig. 6). This vector directs transcription by the human cytomegalovirus major38immediate early gene promoter. pCMV5 contains the SV40 origin of replication allowingreplication in COS cells as well as a prokaryotic origin of replication and ampicillinresistance gene for growth and selection in E.coli.E.coli expression vectors pET8c, pET3a,b,c and pET3xa,xb,xc were supplied byDr. F.W. Studier (New York) (Fig. 7). All pET translation vectors place the cloned cDNAunder the control of T7 promoter and an efficient translation initiation signal for the gene10 protein of T7 phage. The letters 'a', 'b' and 'c' with the pET3 and pET3x vector denotethe three reading frames relative to the gene 10 initiation codon. Vectors pET3a,b,c carrya fragment that codes for the first 11 amino acids of the gene 10 resulting in a fusionprotein. Translation products from vectors pET3xa, xb, xc are hybrid protein with 261amino acid fusion domain from the amino-terminus of the gene 10. Vector pET8c allowsdirect joining of the coding sequence to the gene 10 initiation codon at the Ncol site.Vaccinia recombinants used in the T-cell proliferative studies have beenconstructed in the laboratory.391,CS1OS, T1(AC0 co^—us co a. x cnmRNAflonpCMV54.i- kbAmpsFig. 6. Physical map of th expression vectors pSPt18/19 (Pharmacia)and pCMV5(Andersson et al., 1989).40A,c-4515to \t\X\ 65't‘• "4 \O 99pET-3 pET-3a,b orcFig. 7. Physical map of the expression vector pET and its derivative (Studier etal., 1990). Vectors ET3a,b,c carry fragment that codes for the first amino acids of thegene 10 and vectors pETxa, xb, xc (not shown in the figure) carry a fragment that codesfor the first 261 amino acids of the gene 10.412.2.5. Construction of deletion mutantsA series of in-frame deletions and truncations were generated by restrictionendonuclease subfragment excisions using the natural sites available within the-El codingsequence in p3'E2/E1 plasmid (Hobman et al., 1988). For expression in COS cells andin E.coli, the appropriate fragments were excised from p3'E2/E1 and subcloned intovectors pCMV5 and pET, respectively. Small fragments of El were amplified bypolymerase chain reactions prior to subcloning. The constructions of mutant forms of Elare as follows (Fig. 8):1. m1: the Xhol fragment (450 nt) was excised from plasmid p3'E2/E1 andproduct religated.2. m2: the fragment (560 nt) from BamHI to Hindlll sites was removed fromp3'E2/E1 and the product religated.3. m3: the fragment (670 nt) from Smal to Hindlll was removed, the ends filledby repair and religated.4. m4: the fragment (1057 nt) from Ncol to Smal deletion, the ends filled byrepair, and religated.5. m5: the fragment (1147 nt) from Ncol site to BamHl was removed, endsfilled by repair, and religated in the presence of BamHl linker(pGGGATCCC) to introduce the correct reading frame.6. m6: the fragment (670 nt) from Smal-Hindlll sites was excised from m1, theends filled by repair, and religated.427. m7: 787 nucleotide Ncol-Xhol fragment removed from m6.8. FP1-FP5: cDNA fragments were amplified by the polymerase chain reaction(Erlich, 1989) using synthetic oligonucleotides (Table IV) as shown below,subcloned into pET3xb vector (Fig 7), and sequenced to check formutations which may have accumulated during the amplification. Thesynthetic oligonucleotides used in the amplifications were:FP 1 (CCATGGGGAACCAACAGTCCCGGT^andCCATGGGGGACGCTCTGGCGT),FP 2 (CCATGGGGGAGGTCCAGGTCCCG^andCCATGGATGACAATTCGGGCTCC),FP 3 (CCATGGGGCATGGCCCCGATTGGGC^andCCATGGGGGACGCTCTGGCGT),FP 4 (CCATGGGGAACCAACAAGTCCCGGT^andGCCAACGCCACTCCCCTGACT),FP5 (CCATGGGGAACCAACAGTCCCGGT^andCCATGGATGACAATTCGGGCTCC).2.2.6. Polymerase chain reaction (PCR):El sequences of FP1 to FP5 (Fig. 8) were amplified by PCR with the DNA thermalcycler (Perkin-Elmer Cetus) (Erlich, 1989). PCR mixtures contained 50 mM KCI; 10 mMTris-HCI (pH 8.3); 15 mM MgCl 2 , 200 11M each of the four dNTP's; 0.01% gelatin; 0.1%Triton-X-100; 2 units of Taq-Pol; and 1 of p3'E2/E1 plasmid; 1 pl of 10 mM primers per4350 .1.1 reaction mix. Thermal cycle parameters were 95 °C for two minutes, 60 °C for 30seconds, 72°C for one minute and total of 30 cycles. PCR amplified products were gelpurified, T7 DNA polymerase treated and ligated to pET3xb vector restricted with BamHland blunt ends filled by repaired.2.2.7. Restriction endonuclease digestions and DNA modifications:All restriction digestion reactions were performed according to assay conditionsspecified by the suppliers.DNA fragments were ligated using T4 DNA ligase in 50 mM Tris-HCI (pH7.6); 10mM MgCl 2 ; 1 mM ATP; 1 mM DTT; 5% (w/v) polyethylene glycol for 2 hours at roomtemperature. Reactions were diluted five-fold with TE prior to transformation.DNA fragments with 5' overhangs were blunt ended with E.coli DNA polymeraseI Klenow enzyme in 50 mM Tris-HCI (pH 7.2); 10 mM MgSO4 ; 10 mM OTT; 50 mM BSA;80 11M dNTP's for 30 minutes at room temperature. The enzyme was inactivated byheating at 70 °C for 5 minutes.Removal of terminal 5' phosphates from DNA fragments with 5' overhangs wasdone using calf intestinal alkaline phosphatase (CIP) in 50 mM Tris-HCI (pH 9.0); 1 mMMgCl2 ; 0.1 mM ZnCl2 ; 1 mM spermidine for two successive 30 minute incubation periodsof 15 minute at 37 °C and 15 minutes at 56°C. CIP reactions were terminated by additionof 0.3% SDS and phenol:chloroform extraction followed by ethanol precipitation.Purification of DNA fragments from agarose gels or enzyme reaction mixtures wasroutinely done using GENECLEANTM . Desired fragments were excised from ethidium44bromide stained TAE agarose gels and the gel matrix was solubilized in 2-3 volumes ofsaturated sodium iodide at 55°C. DNA was removed from the agarose solutions byvortexing the mixture with a suspension of glassmilk TM , and a brief spin in a microfuge.Contaminants were washed away from the glass bound DNA by three successive washeswith cold NaCl/ethanol/water (NEW) solution. The DNA was eluted from the glass beadswith TE or water by incubating at 55°C for 3 minutes.2.2.7. Purification of oligonucleotides:Deoxyribo-oligonucleotides were synthesized by T.Atkinson (UBC) (Table III). Thecrude oligonucleotides were purified by electrophoresis through a 20% acrylamide gelcontaining 7M urea and 50 mM TBE buffer. The gel slice containing the oligonucleotidewas incubated overnight at 37°C in 0.5 M ammonium acetate; 10 mM Mg(OAc) 2 . Themixture was centrifuged to remove the gel slices and the supernatant was concentratedto 2 M ammonium acetate followed by ethanol precipitation. Oligonucleotides wereresuspended in TE to use in polymerase chain reactions and as primers in thesequencing reactions. Some of the oligonucleotides were kinased with T4 polynucleotidekinase in 50 mM Tris-HCI (pH 7.5); 10 mM DTT; 10 mM MgCl 2 containing wither 30 1.1,Ci(32P]-ATP for screening colonies for recombinants.2.2.8. Identification of El recombinants2.2.8.1. Colony hybridizationFive recombinants FP1 to FP5 were screened using the colony hybridizationprotocol described in Maniatis (1982). Using sterile toothpick, colonies were transferred45onto agar plate with Hybond-N TM (Amersham) filter and onto master plate. The plateswere inverted and incubated overnight at 37°C. The bacteria colonies were lysed bytreating the filter with 10% SDS for 3 minutes. The filter paper was then transferred toWhatman 3MM saturated with denaturing solution (0.5 M NaOH, 1.5 M NaCI) for 5minutes followed by 5 minute incubation with the neutralization solution (1.5 M NaCI, 0.5M Tris-HCI (pH 8.0)). The filter was air dried colony side up on a sheet of 3 MM paper.The dried filter was placed onto UV transilluminator for 3 minutes to chelate the DNA tothe filter paper.The filters were soaked in 6xSSC for 5 minutes and then transferred to prewashingsolution (50 mM Tris-HCI (pH 8.0), 1 M NaCI, 1 mM EDTA, 10% SDS) for 1 hour at 42°C.Following 1 hour prehybridization at 60 °C, filters were hybridized with P32-labelledoligonucleotide probes overnight in hybridization solution at 60°C. After three 10 minutewashes in 6xSSC at 65 °C, the filters were wrapped in saran wrap and autoradiographed.SSC: 0.15M NaCI, 0.015 M NaCitrate (pH 7.0); 10xDenhardt's solution: 0.1% each officoll, polyvinylpyrolidone, BSA; Prehybridization solution: 50% formamide; 6xSSC; 5xDenhardt's solution; 0.1% SDS; 1004g/m1 denatured, salmon sperm DNA; Hybridizationsolution: prehybridization solution plus [32 P]-labelled probe. Dideoxy sequencing of DNASequenaseTM (modified T7 DNA polymerase) was used for sequence determinationof the cDNA constructs. The DNA template was annealed to 1 ul of primer in 2 ul of 5X sequencing buffer (5 X buffer=200 mM Tris-HCI (pH 7.5); 50 mM MgCl 2 ; 250 mM46NaCI). The tube was warmed to 65°C for 2 minutes and allowed to cool to roomtemperature slowly. Two gl of labelling mix (1,51.0 dGTP, 1.5 .t.N.4 dCTP, 1.5 gM dTTP),1 gl 0.1 M DTT, 0.5 gl [a- 35S]dATP or [a-32 P]clATP (10 gei/u1), and 2 gl Sequenase n"(diluted 1:8 in TE) was added to the template/primer mixture and incubated for 5 minutesat room temperature. The template/primer mixture was distributed (3.5 gl to each tube)to 4 pre-warmed tubes containing 2.5 gl of each of the following:G: 80 .1,1\A of each dATP, dTTP, dCTP, dGTP, 8 gM ddGTPA: 80 gM of each dATP, dTTP, dCTP, dGTP, 8 gM ddATPT: 80 gM of each dATP, dTTP, dCTP, dGTP, 8 gM ddTTPC: 80 gM of each dATP, dTTP, dCTP, dGTP, 8 gM ddCTPThe tubes were incubated for 5 minutes at 37°C and the reactions were terminated by theaddition of 4µl of stop buffer (95% formamide, 20 mM EDTA, 0.05% bromophenol blue,0.05% xylene cyanol FF). The samples were heated to 72 °C for 2 minutes immediatelyprior to loading onto a gel.2.2.9. Separation of nucleotides (DNA sequencing gel)Three ul of DNA sequencing reactions were loaded onto 6% and 8%polyacrylamide gels (19:1 acrylamide:bis-methylacrylamide, 8 M urea, 0.06% ammoniumpersulfate, 20 gl TEMED, 50 mM TBE). Electrophoresis was performed at 1600V, 37watts. The gels were dried onto Whatman 3 MM paper using vacuum gel drier at 80°Cand exposed to X-ray film at room temperature overnight for [a- 35S] and for [a-32 P],exposure was done at -70°C.472.2.10. Expression of El recombinants2.2.10.1. In vitro transcriptionPlasmid constructs p3'E2/E1, m4, m5 were linearized with Hindlll, m2 and m3 werelinearized with BamHl and Smal, respectively. Linearized plasmid DNAs were purifiedusing GENECLEAN and templates were added to 50 ill of transcription reaction mixturecontaining 40 mM Tris-HCI (pH 7.5), 6 mM MgCl 2 , 2 mM spermidine, 10 mM NaCI, 10mM dithiothreitol, 500 U of RNasin (Promega Biotec) per ml, 100 ptg of nuclease-freebovine serum albumin per ml, 0.5 mM each ATP, CTP, UTP and 0.05 mM GTP. After60 minute incubation at 40°C, DNA template was digested with 15 units of DNase I at37°C for 15 minutes. The newly transcribed RNA templates were extracted withphenol:chloroform, precipitated with ethanol, and stored in diethyl pyrocarbonate (DEPC)-treated water at -70°C. In vitro translationSP6-derived transcripts were translated in a nuclease-treated rabbit reticulocytelysate system (Promega) containing 0.02 mM amino acid mixture minus methionine orminus cysteine; [35S]-methionine or [35S]-cysteine at 1200 1.1Ci/m1; RNAsin at 1600units/ml; and RNA at 40 µg/ml in a 25 pi volume. After incubation at 30°C for 1 hour, thetranslation products were immunoprecipitated with anti-RV human sera or monoclonalantibodies to El.482.2.10.3. Transfection of COS cellsCOS cells were transfected with plasmid DNA containing deletion mutants of El(m1, m2, and m3) using method described by Adam and Rose (1985). Subconfluentmonolayer of cell grown in Dulbecco modified Eagle medium (DMEM) plus 5% fetal calfserum were washed twice with Tris-saline (25 mM Tris-HCI (pH 7.4), 140 mM NaCI, 3 mMKCl2 , 1 mM CaCl 2 , 0.5 mM MgCl2 , 0.9 mM Na2HPO4). Cells were incubated with DEAE-dextran (Mr=5 X 105 ; 500 lag/m1) and plasmid DNA (4 µg/ml) in Tris-saline at 37 °C for 30minutes. The DNA solution was then removed and replaced with DMEM plus 40 uMchloraquin for 3 hours at 37 °C. After removal of chloraquin solution, the cells wereshocked with 10% dimethylsulfoxide/DMEM for 3 minutes at room temperature. Finally,the monolayer was washed three times with Tris-saline and incubated at 37°C for 40hours in DMEM plus 5% calf serum. The monolayer was scraped and lysed in lysatebuffer (25 mM Tris-HCI (pH 7.4); 100 mM NaCI; 1 mM EDTA; 1% Nonidet P-40)containing 1 mM PMSF prior to immunoblotting. Transformation of E.coli strain BL21(DE3)/pLysSExpression of the truncated and deleted El constructs was directed by inducibleT7 RNA polymerase engineered in the E.coli strain BL21(DE3)/pLysS. This straincontains a copy of T7 RNA polymerase gene located in the chromosome under thecontrol of the inducible lacUV5 promoter, and a plasmid which encodes for constitutiveexpression of lysozyme and abrogates the need for sonication. Cultures were grown at37°C in L-broth containing ampicillin (100 pig/m1) and chloramphenicol (25 pig/m1) forselection of plasmid pLysS. T7 RNA polymerase was induced by addition of49isopropyithiogalactoside (IPTG) (0.04 mM) when the culture reached optical density of0.8-0.99 at 600 nm. Induced cultures were allowed to grow for additional 2 hours at 37°Cand were subsequently harvested by centrifugation. The pellets were resuspended in1/50 volume of DNase I buffer (50 mM Tris-HCI (pH 7.5), 5 mM EDTA, 10 mM MgSO 4),freeze/thawed twice to Iyse the cells and then treated with DNasel (1mg/m1) for 15minutes at room temperature. 5-10 samples were analyzed by electrophoresis on 12%SDS-PAGE. Expressed proteins were detected by immunoblotting. Expression ofrecombinants from pET3x vectors were sufficiently high and can be visualized bycoomasie brilliant blue staining. Bands corresponding to recombinant proteins were cutout and electroeluted for 3 hours at 10mA using Bio-Rad electroelution apparatus(Electroeluter 422, Bio-Rad, Richmond). The eluates were lyophilized and solubilized in8M urea (50 mM Tris-HCI (pH 7.5), 5 mM EDTA) by incubating with rocking motion for1 hour at room temperature. Supernatant was collected following 30 minute centrifugationand analyzed on immunoblots.2.2.11. Detection of El recombinants2.2.11.1. Monoclonal antibodiesEl monoclonal antibodies were generated and characterized previously in thislaboratory. Properties of the monoclonal antibodies used are summarized in Table VI.3D9F, 3D9D, and 12B2D were characterized to have hemagglutinin inhibiting (HI) activity1:16384, 1:8192, and 1:4096, respectively. 21 B9H, 12B2D, and 16A10E were found tohave viral neutralizing (VN) activity. 21 B9H neutralizes both M33 and RA27/3 strains withthe addition of complement whereas, 16A10E neutralizes M33 only.50Table VI. Summary of properties of monoclonal antibodies directed against El2.2.11.2. ImmunoprecipitationHuman polyclonal anti-rubella serum was preincubated with Protein A-Sepharose(Pharmacia) for at least four hours at 4°C in binding buffer (100 mM Tris-HCI (pH 7.4);400 mM NaCI) with constant mixing. The serum-coated beads were washed twice withbinding buffer, and once in lysate buffer (25 mM Tris-HCI (pH 7.4); 100 mM NaCI; 1 mMEDTA; 1% Nonidet P-40). [35S]-labelled antigen from in vitro translation was mixedovernight at 4°C with the coated beads in lysate buffer. Beads were washed once withlysate buffer, twice with wash buffer (25mM triethanolamine; 172 mM NaCI; 1% SDS; 1mM EDTA), three times with 10 mM Tris-HCI (pH 7.4), and once with water. Antigen-antibody complexes were dissociated from the Protein A-Sepharose by boiling in 1 X SDSdissociation buffer for 5 minutes, vortexing and pelleting the beads by centrifugation.Supernatants were collected and separated on SDS-PAGE, and then fluorographed.512.2.11.3. Immunoblotting/dot blottingCOS cell lysates and E.coli bacterial lysates were separated by SDS-PAGE andtransferred to nitrocellulose filters using a Bio-Rad Trans-Blot apparatus for 60 minutesat 65 volts in 25 mM Tris-HCI; 192 mM Glycine (pH 8.3); 20% methanol. The filters wereblocked for 60 minutes to overnight in TBS containing 4% powdered skimmed milk.Membranes were then incubated with human anti-RV serum or monoclonal antibodies for2 hours, washed with TBS/0.3% Tween-20 and treated with goat anti-human or goat anti-mouse IgG conjugated to alkaline phosphatase (BRL) for two hours. Blots were washedas above and developed with NBT (nitro blue tetrazolium)/BCIP (5-bromo-4-chloro-3-indoyl phosphate). All incubations were done at room temperature. For dot blot analysisone pl of purified recombinants expressed in E.coli (m7, FP1-FP5) were dotted ontonitrocellulose filters presoaked in TBS. Following overnight air dry, the filter was handledas an immunoblot as described above.2.2.12. Electrophoresis2.2.12.1. Separation of DNA fragmentThe buffers used in agarose gel electrophoresis were 1XTAE (40 mM Tris-acetate;1 mM EDTA (pH 8.0) and 1XTBE (89 mM Tris-borate (pH 8.0); 89 mM boric acid; 2 mMEDTA) for separation of small fragments. The gel concentration varied from 1% to 2%agarose with 1 jig/ml ethidium bromide for visualization. DNA samples were diluted to8% sucrose; 20 mM EDTA (pH 8.0); 0.05% bromophenol blue; 0.05% xylene cyanol andseparated by electrophoresis on 10 cm submarine horizontal agarose gels.522.2.12.2. Separation of proteinProteins were separated using a discontinuous gel system described by Laemmli(1970). Samples were adjusted to 62.5 mM Tris-HCI (pH6.8); 10% glycerol; 2% SDS; 2%2-mercaptoethanol and denatured at 95°C for 3 minutes. Stacking gels consisted of 4%polyacrylamide, and separating gels contained either 10% or 12% polyacrylamide. Gelswere run at constant voltage of 100 volts until the markers have run to the desiredposition. The stacking gel was trimmed away, and the proteins were either fixed in 10%acetic acid for 15 minutes for fluorograph or transferred to nitrocellulose membrane forimmunoblot analysis. Fixed gels were immersed in the fluorographic agent Amplify(Amersham) for 15 minutes, dried under vacuum and exposed to X-ray film at -70°C.Solutions used for electrophoresis:5X Stacking gel buffer: 0.625 M Tris-HCI (pH 6.8), 0.5% SDS5X Separating gel buffer: 1.875 M Tris-HCI (pH 8.8). 0.5%SDS5X Gel running buffer: 0.125 M Tris-HCI; 0.96M glycine, 0.5% SDS (pH 8.3)Polyacrylamide Stock: 30% acrylamide, 0.8% N'N'-bis methylene acrylamideGels were polymerized by adding ammonium persulfate to 0.05% and TEMED(N'N'N'N'-Tetramethylenediamine) to 0.1%. Coomassie blue stainingEl recombinants expressed in E.coli were separated by SDS-polyacrylamide gelsand simultaneously fixed with methanol:glacial acetic acid and stained with CoomassieBrilliant Blue R250 (0.25 g of Coomassie Brilliant Blue R250 is dissolved in 90 ml of53methanol:H20 (1:1 v/v) and 10 ml of glacial acetic acid). Gel was immersed in stainingsolution and place on a shaker for 30 minutes. Gel was then destained (destainingsolution: 90 ml of methanol:H 20 and 10 ml of glacial acetic acid) by changing thedestaining solution 3 to 4 times. ElectroelutionPolyacrylamide strips of recombinants expressed in E.coli were cut into 1 cmsections and electroeluted using a model 422 electroelution attachment of the BioRadProtean II system. Elution buffer (25 mM Tris base, 192 mM Glycine, 0.1% SDS) wasfiltered through a 0.22 pm filter. Electroeluates were lyophilized and resuspended in lxsample buffer. Each preparation was assayed by immunoblot for antigenicity. Enzyme linked immunoadsorbant assay (ELISA)El peptides were coated onto lmmulon-2 plates (Dynatech, Chantilly VA, USA) incarbonate buffer (15 mM Na 2CO3 , 35 mM NaHCO3 (pH 9.5)). Following one hourblocking in 0.5% milk-PBS, the plates were incubated with monoclonal antibodies dilutedin 0.5% skim milk-PBS. The two hour incubation was followed by the addition of alkalinephosphatase-conjugated goat anti-mouse IgG antibody (BRL) diluted 1:3000. The plateswere developed in substrate buffer (1M Diethanolamine, 5 mM MgCI, 2 mg/ml p-nitro-phenylphosphate (pH 9.6)) and read at 405 nm on a Bio-Rad mibroplate reader (Bio-Rad,Richmond CA, USA).542.2.13. T-cell proliferation assayPeripheral blood lymphocytes (PBL) were isolated on Ficoll-Hypaque densitygradient (Boyum, 1968) to give 5x10 5 cells/ml in RPM' 1640 supplemented with 10%autologous plasma. For antigen-specific response, lx10 5 cells per well were incubatedin 96-well flat bottom plates with varying concentrations of expressed antigens in triplicate.Following 7 day incubation at 37°C with antigen, the cells were pulse labelled with [ 3 H]-thymidine (1 [ICl/well) for 6 hours, harvested and washed onto glass-fibre filters withdistilled water. After the filters were air dried overnight, 3 ml of ACS II (Amersham)scintillation fluid was added to determine the incorporation of [ 3H]-thymidine.2.2.14. Antigen preparations for T-cell proliferation assays2.2.14.1. Vaccinia recombinantsCV-1 cells were infected with vaccinia virus recombinants (E1, E2 and C, preparedin this laboratory) at 5 PFU/cell (Chaye et al., 1992b). At 40 hours post infection, themonolayers were washed with phosphate-buffered saline (PBS), scraped and suspendedin MEM (modified Eagle's medium) media. The isolated cellular extracts (1x108 PFU/ml)were irradiated with a germicidal lamp at a distance of 3 cm for 5 minutes to inactivatethe virus. Inactivated wild-type vaccinia virus harvested in the same manner as therecombinants were used to monitor the proliferative responses to the vaccinia virus.Media alone was used as the control to monitor the spontaneous proliferations. Noresidual vaccinia virus infectivity was observed in the inactivated cellular extracts byplaque assay.552.2.14.2. El peptidesEl peptides EP1 to EP23 (Table VII) were synthesized and provided by Dr. P.Chong at Connaught, Canada. Each peptide was prepared by dissolving in ethanol andair drying in sterile tissue culture hood overnight and resuspended in sterile PBS (2mg/ml). Peptides were diluted in RPM' 1640 before adding to the proliferation assays.2.2.15. Statistical methodsThe T-cell proliferation data were analyzed using a mixed effects analysis ofvariance model. Proliferative responses to each of the structural proteins E1, E2, and C,were compared within each group and in between the two groups.2.2.16 Study groupa) T-cell epitope mapping:Eleven (5 females; 7 males) healthy individuals with no known conditionsassociated with rubella virus infection.b) Antigen specific lymphocyte proliferation study:Group A: fourteen adults from the hospital staff (seven males; seven females) whoexhibited no rubella associated symptoms. Their ages ranged from 24 to 48 years, withthree individuals having previously received rubella vaccines. All donors had documentedhistories of having received smallpox vaccinations.Group B: four CRS patients, with ages.of 2, 5, 24, and 25 years. All had a CRSdiagnosis confirmed by clinical and serological criteria, and for each, RV exposure in56utero had occurred prior to 16 weeks of gestation. One patient had a late onsetsequelae, presenting with progressive senso-neural deafness at age 16. None of thepatients had received smallpox vaccinations; the oldest of the two individuals hadreceived measles-rubella vaccines.57Table VII. El synthetic peptides.EP1^EEAFTYLCTAPGCATQTPVPVREP2 VPVRLAGVGFESKIVDGGCFEP3^FAPWDLEATGACICEIPTDVEP4 PTDVSCEGLGAWVPTAPCARIEP5^CARIWNGTQRACTFWAVNAYSEP6 GSYYKQYHPTACEVEPAFGHEP7^AFGHSDAACWGFPTDTVMSVEP8 SVFALASYVQHPHKTVRVKFEP9^VKFHTETRTVWQLSVAGVSCEP10 VSCNVTTEHPFCNTPHGQLEEP11^GQLEVQVPPDPGDVLEYIMNEP12 IMNYTGNQQSRWGLGSPNCHEP13^NCHGPDWASPVCQRHSPDCSEP14 PDCSRLVGATPERPRLRLVDEP15^RLVDADDPLLRTAPGPGEVWEP16 GEEWVTPVIGSQARKCGLHIEP17^LHIRAGPPYGHATVEMPEWIHEP18 IHAHTTSDPWHPPGPLGLKFEP19^LKFKTVRPVALPRALAPPRNEP20 PRNVRVTGCYQCGTPALVEGEP21^VEGLAPGGGNACHLTVNGEDVEP22 GEDVGAFPPGKFVTAALEP23^LNTPPPYQVSCGGESDRASAGH3. RESULTS and DISCUSSION3.1. Section I: Mapping the hemagglutinin and viral neutralizing epitopes of the RV El glvcoprotein Epitopes important for viral infectivity and hemagglutination have been mapped tothe El glycoprotein (Green and Dorsett, 1986; Ho-Terry et al., 1985; Waxham andWolinsky, 1985a). In this study, a panel of El deletion mutants and subset of E1-specificmonoclonal antibodies were used for analysis of hemagglutination and viral neutralizationepitopes of the El protein. The deletion mutants were expressed in the cell-free rabbitreticulocyte system, in COS cells and in E.coli and were subsequently screened with themonoclonal antibodies. Finally, synthetic peptides derived from the predicted El aminoacid sequence were also screened.3.1.1. In vitro transcription and translation of the El deletion mutantsAll RV cDNA constructs for in vitro expression were derived from p3'E2/E1described in Hobman et al (1988). In addition to containing the entire El gene, the wild-type El construct p3'E2/E1 also contains the capsid protein translation start site as wellas nucleotides specifying the first eight amino acids of C and 69 carboxyl-terminalresidues of E2, including the putative El signal sequence (Hobman et al., 1988). The in-frame deletions and truncations were generated by using the available restriction siteswithin the El coding sequence (Fig. 8). The mutants m2, m3, m4 and m5 wereconstructed as described in the Material and Methods (page 42). The constructs werelinearized with HindlIl for wild-type, E1, m4, m5, with Smal for m2 and BamHI for m3 (Fig.8). The linearized constructs were transcribed with SP6 RNA polymerase and59subsequently translated in a rabbit reticulocyte lysate system without microsomes asdescribed in Hobman et al. (1988). Fluorographs from translation productsimmunoprecipitated with monoclonal antibodies 21B9H and 3D9F and human anti-RVserum are shown in Fig. 9. Mutants m2 and m3 were immunoprecipitated by bothhemagglutination inhibiting (3D9F) and viral neutralizing (21 B9H) monoclonal antibodies(Fig. 9(i)). These results suggest that the binding capacity of both MAbs is independentof the presence of carbohydrate moieties on El fragments. Cell-free translation of RNAsfrom p3'E2/E1, m2, and m3 produced proteins with apparent molecular mass of 61, 40,and 35 kDa, respectively. The higher molecular weights observed in the translationproducts than predicted from amino acid sequences for m2 (33 kDa) and m3 (28 kDa)are due to the presence of eight amino acids of C protein and 69 carboxy-terminalresidues of E2 in m2 and m3 which were not cleaved in the in vitro translation system.In contrast to m2 and m3, N-terminal deletion mutants m4 and m5 were not precipitatedby either of the two monoclonal antibodies but were precipitated by the human serum(Fig. 9(ii)). These results suggest that the epitopes recognized by hemagglutinationinhibiting and viral neutralizing monoclonal antibodies are present within the N-terminalEl peptide domain upstream from the Smal site at position. The results from theseexperiments indicate that the epitopes for 21 B9H and 3D9F are contained within the N-terminal half of E1, m3 (Fig. 8), from amino acid residues 1 to 253.60El Hx^•£450560£670£1037£1147&450 £670£787 4,670a26933 aa40 as26 as26 aa13 aaENElml ^m2 ^M3 ^m4m5m6 ^m7 ^aa193m7 ^FP2 ^FP1FP3FP4FP5Fig. 8. Schematic representation of the cDNA fragments used for construction ofEl mutants. The deletions are denoted in terms of translated amino acid residues formutants ml to m7. The sizes of PCR products (FP1 to FP5) are also given in amino acidresidues. E=EcoRl; X=Xhol; S=Smal; B=BamHl; H=Hindlll; N=Ncol. N-linkedglycosylations are indicated by Y. The top line shows the cDNA fragment encoding RVEl.61Figure 9. Translation of SP6-derived El mRNA and deletion mutant mRNAs inrabbit reticulocyte lysates. mRNAs were translated in presence of 35S-methionine, andthe translated products were separated on 12% Laemmli gels and fluographed. (i)Immunoprecipitation of El and the mutants m2, m3 with MAbs 21 B9H (A) or 3D9F (B).Neg denotes transcription/translation of a construct with no RV El sequence. (ii)Immunoprecipitation of mutants m4 and m5 with MAb 21B9H (A), 3D9F (B) or humananti-RV serum (C). Protein molecular weight standards are indicated (kDa).623.1.2. Expression in COS cellsThe cDNA inserts from p3'E2/E1, ml , m2, and m3 were subcloned into theeukaryotic expression vector pCMV5, downstream from the human cytomegalovirusimmediate early gene promoter (Andersson et al., 1989). COS cells were transfected withrecombinant plasmids and cell lysates were isolated at 48 hours post-transfection forimmunoblot analysis. Wild-type El and mutants (m1, m2, and m3) all reacted withmonoclonal antibodies that exhibit viral neutralizing (Fig. 10A) and hemagglutinationinhibiting activities (Fig. 10B). Mutant 1 lacks the 450 amino acid Xhol fragment (E1 45 toE1 481 ) but was still recognized by both monoclonal antibodies suggesting that therespective epitopes are not contained within this fragment. Similarly, both viralneutralizing and hemagglutination inhibiting monoclonal antibodies recognized the mutantm2 with the 560 amino acid deletion at the carboxy-terminus and the mutant m3 with 670amino acid deletion also at the carboxy-terminus. Thus, the recognition of mutants m1,m2 and m3 by both monoclonal antibodies suggests that the corresponding epitopes arenot contained within the Xhol fragment (E1 45 to E1 481 ) or the fragment between Smal andHindlIl (El m to E 1481 ) (Fig. 8).El protein contains three functional N-linked glycosylation sites (Hobman et al.,1991). In mutant m1, one glycosylation site is retained, while in mutants m2 and m3, allthree glycosylation sites are retained. The observed apparent molecular weights of ml(37 kDa), m2 (42 kDa), and m3 (38 kDa) suggest that they were translocated andglycosylated, as the estimated molecular weights based on the predicted amino acidsequence for m1, m2 and m3 are 36, 33 and 28 kDa, respectively. The presence of63oligosaccharides on the mutants did not appear to affect the recognition of epitopes by21 B9H and 3D9F suggesting that the binding capacity of both MAbs is independent ofcarbohydrate moieties on El fragments.64Fig. 10. Immunoblot analysis of El mutants expressed in COS cells. COS cellswere transfected as described in Hobman et al., 1988. After 48 hour transfection, thetransfected COS cells were scraped off the plates and analyzed by immunoblotting. Elantigens were detected using MAb 21 B9H (A) and 3D9F (B). The relative mobilities ofprotein standards (kDa) are indicated. VEC = COS cells transfected with pCMV5 vector.3.1.3. Expression in E.coli strain BL21(DE3)/pLysSSince the expression of the smaller mutants were not detected in transfected COScells, E. coli pET vectors were used to express these mutants. Mutant cDNAs (m4 andm6) were inserted into the Ncol site of the pET8c vector (Rosenberg et al., 1987) andexpressed in E.coli as non-fusion proteins. The cell lysates from induced E.coli cultures65were separated on a 0.1% SDS-12% PAGE and RV El-specific polypeptides weredetected by immunoblotting using monoclonal antibodies (Fig. 11). Lack of recognitionof m4 but recognition of m6 (band at 20 KDa) by hemagglutination inhibiting and viralneutralizing monoclonal antibodies confirm that hemagglutination and viral neutralizingepitopes are located within these two regions (El l to E1 44 and E1 193 to E1 269). Todetermine which contains these epitopes, mutant m7 was constructed (Fig. 8) andexpressed in E.coli as a fusion protein using vector pET3xa (Studier et al., 1990).Expressed fusion protein from m7 was recognized by both hemagglutination inhibiting andviral neutralizing monoclonal antibodies (Fig. 11), suggesting that hemagglutinin and viralneutralizing epitopes are located within the region E1 193 and E1269 and not in the regionEl i to E1 44 . The region E1 193 to E1269 was further divided into two smaller fragments (FP-1 and FP-2) using the polymerase chain reactions with synthetic oligonucleotides (TableIV). Mutants FP-1 (E1 214 to E1 264) (33.4 KDa) and FP-2 (E1 193 to E1 226) (32.6 KDa) wereexpressed as fusion proteins in E.coli using pET3xa vector (Studier et al., 1990) (Fig. 12).The combined apparent molecular mass of the fusion partner and the El fragments are33.4 KDa for FP-1 and 32.6 KDa for FP-2. Both hemagglutination inhibiting and viralneutralizing monoclonal antibodies reacted with mutant FP-1 but not with mutant FP-2(Fig. 11) suggesting that the corresponding epitopes are contained within the regionsE1 214 to E1 254. Mutant FP-1 was further subdivided into three small constructs: FP-3(E1 226 to E 1 254) (31.7 KDa), FP-4 (E1 214 to El m) (31.9 KDa) and FP-5 (E1 214 to El 226)(30.3 KDa). The denoted molecular masses are of the fusion partner and the Elfragment. Binding of viral neutralizing and hemagglutination inhibiting monoclonal66antibodies to the expressed fusion proteins is shown in Fig. 11A and 11 B, respectively.Both hemagglutination inhibiting and viral neutralizing monoclonal antibodies recognizedmutant FP-4, but not mutants FP-3 and FP-5. The failure of FP-3 and FP-5 to.react withthe monoclonal antibodies is not due to the low levels of expression, as abundantexpressed proteins were observed in SDS-PAGE stained with Coomassie brilliant blue(Fig. 12). It is possible that the hemagglutination and viral neutralizing epitopes may beinterrupted by the break at the amino acids around residues E1226, or the epitopes on FP-5 and FP-3 may be buried under the large fusion partners and inaccessible to the MAbs.Thus it is concluded from these results that the epitopes defined by monoclonalantibodies 3D9F (HI) and 21B9H (VN) map to a domain of 27 amino acids (E1 214 toE 1 240)•67Fig. 11. Immunoblot analysis of El fusion proteins expressed in E.coli.Expression of El mutants was induced with the addition ofisopropylthiogalactoside when the culture reached an optical density of 0.8-0.9 at 600 nm. Induced cultures were harvested 2-4 hours after induction.Expressed proteins were separated on 12% Laemmli gels and detected byimmunoblotting. Blots were detected with MAbs 21 B9H (A) and 3D9F (B).RV = RV antigen preparation; UNI = uninduced E.coli culture.683.1.4. Synthetic peptide ELISATo define the epitopes further, six overlapping synthetic peptides (EP11 to EP15and EP25) spanning the m7 region (Fig. 13) were synthesized and coated onto ELISAplates and probed with MAbs. Rabbit anti-peptide sera were used as positive controlsto ensure that the peptides were sufficiently bound to the plates. Peptide-specific ELISAresults were observed only with the 35 amino acid peptide EP25 by VN MAb 21 B9H (Fig.14). EP25 was then divided into two smaller peptides, EP24 (17 aa) and EP26 (15 aa)(Fig. 13). Although, MAb 21B9H reacted strongly with EP25, it failed to recognize EP24or EP26 in a peptide specific ELISA (Fig. 15). However, another viral neutralizing MAb(16A10E) recognized both EP25 and EP26 (Fig. 15), suggesting that there are twodistinct viral neutralizing epitopes on E1. Three HI MAbs (3D9F, 3D5D and 12B2D) failedto recognize any of the synthetic peptides tested.3.1.5. Summary of HI and VN epitope mappingCombining the data obtained from the studies of the truncated forms of El and thepeptide analysis, the results are summarized as follows (Fig. 16):1) The viral neutralizing epitope defined by MAb 21B9H mapped toamino acid residues 214 to 233 (QQSRWLGLGSPNCHGPDWASP).2) The viral neutralizing epitope defined by MAb 16A10E mapped toamino acid residues 219 to 233 (GLGSPNCHGPDWASP).3)^The hemagglutinin epitope defined by MAb 3D9F mapped to aminoacid residues 214 to 240 (QQSRWGLGSPNCHGPDWASPVCQRHSP).69aa193 as 269Fig. 12. Coomassie brilliant blue stained SDS-PAGE of El fusion proteins expressed in E.coli. 2-4 hours afterinduction, cultures were centrifuged and resuspended in 1/10 the volume of lysate buffer. 5111 of the concentratedcell lysate loaded per well. uni=uninduced; VEC=induced fusion partner without El sequences; M=rnolecular weightmarker (kDa).EP11GOLE VO VPPOPGOLVEYI MNEP12IMNYTONOOSPI WGLGSPNCHEP13HONG POWASPVCQRHSPOCSEP14PO CS RLV GATP ER PRLRLVOEP15RLVDADOPLLRTA PG PGEVEP24DPG LVEYIMNYTGNEP26GLGsPNCHGPDwAsPEP25DPG LV EY IMN Y TO NGO S R WG LOS PNCHGPD WASPFig. 13. Position of peptides relative to the mutant m7. The numbersindicate the positions of each peptide in El protein.70mawit' 7- 1.4$;, EP24^ EP25^ EP26Peptide concentration (100 ng/ul)Absorbance at 405 nm0.^EPI5^EP25Peptide concentration (100 ug/ul)IN Negative sera mAb 21 B9H EmAb 3D9FFig. 14. Recognition of El peptides EP11 to EP15 and EP25 by viralneutralizing MAb 21 B9H and hemagglutinin inhibiting MAb 3D9F. 100 ng/ulof synthetic peptides were bound to immulon-2 plates and probed withmonoclonal antibodies at 1:2000 dilutions. The negative sera are normalBalb/C mouse sera not exposed to RV.Absorbance at 405 nm3.532.521.510.50Kg Negative sera mAb 2169H 1^1 mAb 16A10EFig. 15. Recognition of El peptides EP24, EP25 and EP26 by VNmonoclonal antibodies 21B9H and 16A10E at 1:2000 dilutions. Thenegative sera are normal Balb/C mouse sera not exposed to RV_7188193.^213^218^ 233^239^ a a 26 9EP11 118 ea]EP12 (208a1EP13 120aa1EP14 120 ea!EP15EP24 (17aaEP26 U15881EP26 (358813388P2P1P3'P4P5 40 aa26 aa26 as13 aamAbVN^HAI+Fig. 16. Summary of the results using peptides (EP11 to EP14, EP24 toEP26) and PCR products expressed as fusion proteins (FP-1 to FP-5). VN= viral neutralizing monoclonal antibody 21B9H, HAI = hemagglutinininhibiting monoclonal antibody 3D9F.723.1.6. Discussion of Section IUsing in vitro and in vivo expression systems, twelve El mutants were constructedand expressed in order to identify the location of epitopes recognized by E1-specificmonoclonal antibodies. Due to the nature of the experiments used in this study, theepitopes that have been mapped are linear in structure and conformational-independent.Any epitopes that are dependent on native conformation may not have been located.There appears to be no general rule whether neutralizing epitopes are linear orconformational (Alexander and Elder, 1984; Long et al., 1986; Wright et al., 1989). Forconstruction of synthetic peptide vaccines, it is necessary to define functional epitopeswhich can be mimicked by linear polypeptides fragments (Dietzschold et al., 1990).In general, the oligosaccharide side chains of viral glycoproteins do not act asepitopes per se, but only modulate the expression of neighboring epitopes constituted byresidues of the underlying polypeptide backbone. The presence of carbohydratespreserves the conformational integrity of some epitopes that lose antigenicity upondeglycosylation (van Regenmortel, 1990). In addition, attachment of additionaloligosaccharide may prevent monoclonal antibody from binding to its underlying epitope.However, the majority of neutralizing antibodies are not dependent on the presence ofcarbohydrates. Deglycosylated virus adsorbs neutralizing antibody from sera as efficientlyas glycosylated virus (van Regenmortel, 1990). The likelihood of epitopes to be eitherhidden under carbohydrate moiety or by the folding induced by carbohydrate moiety isless for those epitopes which are conformation independent. This characteristic isimportant in the design of subunit vaccines. The epitopes contained in the vaccines73should be accessible to circulating immunoglobulins to elicit protective immunoglobulinproduction. The monoclonal antibodies in this study recognize epitopes regardless of thepresence or absence of carbohydrates on El and its mutants. This suggests thatvaccines containing epitopes defined in this study will likely be accessible by the host'simmunoglobulins.Fig. 16 summarizes all the data obtained in this section. Epitopes for viralneutralizing monoclonal antibodies 21B9H and 16A10E mapped to amino acid residues214 to 233 and 219 to 233, respectively. The hemagglutination epitope defined by 3D9Fmapped to amino acids 214 to 240. The inability of the monoclonal antibody 21 B9H torecognize peptides EP12, EP13 and EP26 as well as the expressed mutant proteins FP2,FP3 and FP5 suggests that residues 214 to 219 and 226 to 233 are critical for antibody-peptide interaction. Alternatively, the epitope, upon binding to the ELISA plate, may havebeen altered such that the monoclonal antibody no longer recognized its epitope (Tanget al., 1988). The structural data suggest that epitopes on native proteins consist of 15-20 residues with a smaller subset of 5-6 of these residues contributing most of the bindingenergy (Laver et al., 1990). Since EP26 is only 15 amino acids in length, it is possiblethat the critical 5-6 amino acid residues are not available for binding to the solid support.EP25 (35-mer) is recognized by the monoclonal antibody 21B9H suggesting that theepitope on this larger peptide is in the appropriate form. The surrounding extra aminoacid residues may be required for appropriate recognition of the epitope by monoclonalantibody 21B9H.In contrast to monoclonal antibody 21 B9H, EP26 reacted positively with74monoclonal antibody 16A10E. This result suggests that there are two distinct viralneutralizing epitopes close together or overlapping on a linear peptide. However,monoclonal antibody 16A10E failed to recognize peptides EP12 or EP13 and the mutantproteins FP2, FP3, and FPS. This result implies that the epitope for monoclonal antibody16A10E overlaps the break regions of the above mentioned peptide (Fig. 13) and thedeletion products and is, hence, mapped to residues E1 216 to El 233.Using various fusion protein constructs the hemagglutination epitope, as definedby monoclonal antibody 3D9F, mapped to FP4 (E1 214 to E1 240). Since monoclonalantibody 3D9F failed to recognize any synthetic peptides that included peptide EP25(El m to E1 233), this implies that the epitope recognized by monoclonal antibody 3D9Frequires additional residues at the C-terminus of peptide EP25. Positive recognition ofFP1 (E1 214 to E1 264) and FP4 (E1 214 to E1 240) by monoclonal antibodies further supportsthis conclusion. However, the absence of positive identification of EP13 (E1 224 to E1 243)and FP3 (E1 226 to E1 264) by the monoclonal antibody makes conclusion difficult. As withthe epitopes for the viral neutralizing monoclonal antibodies used in this study, thehemagglutination epitope may have been altered during the binding of the smallerpeptides to the plates, resulting in negative data for EP13. On mutant FP3, the epitopemay not have been retained due to the fusion partner. On the other hand, properrecognition of the monoclonal antibody 3D9F epitope may require additional residues atthe N-terminus of EP13 and FP3. Though the antibody binding residues may only bewithin 5-6 amino acids of FP4, the surrounding residues may be required to maintain thestability of the antibody-antigen complex (Laver et al., 1990).75Terry et al. (1988) have identified three epitopes(EP 1 ,EP2 , EP 3) within the Elregion E1245 to E1 285. Monoclonal antibodies recognizing epitopes EP, and EP 2 show bothhemagglutination inhibiting and viral neutralizing activity, while monoclonal antibodyrecognizing EP 3 epitope show only viral neutralizing activity. It is not unexpected toobserve that the epitopes mapped in this study do. not overlap the epitopes mapped byTerry et al. (1988), but are adjacent to the EP 2 epitope. It is possible that this study andthe study of Terry et al. (1988) have independently mapped three distinct epitopes on E1.The mechanisms of viral neutralization by these monoclonal antibodies are not yet clear.Neutralization by monoclonal antibodies may prevent infection directly or indirectly bybinding to the glycoprotein and preventing receptor recognition or by binding to a site inproximity to the receptor binding site, causing steric hinderance or a conformationalchange that the receptor-binding domain is masked or altered (loro, 1988).Waxham and Wolinsky (1985a) have defined six non-overlapping antigenicepitopes on El using monoclonal antibodies in competitive inhibition studies. They furthermapped the hemagglutination and viral neutralizing epitopes within the 82 amino aciddomain of El (E1 202 to E1 283) using viral neutralizing monoclonal antibodies E1-18 and El -20 (Wolinsky et al., 1991). Subsequent to this study, the same group screened a seriesof five overlapping synthetic peptides from this region (Wolinsky et al., 1993). Theepitope was mapped to E1 208 to E1239 (peptide SP15) with the minimal amino-terminalrequirements of E1 221 and E1223 for monoclonal antibodies E1-18 and E1-20, respectively.The peptide SP15 induced viral neutralizing antibodies in mice and rabbits whichrecognized SP15, peptides overlapping SP15 and rubella virus. The findings in this study76are consistent with the epitopes defined by the monoclonal antibodies 21 B9H and16A10E, and further support the hypothesis that these epitopes may be critical forprotective host humoral immune response.The significance of the epitopes within the region E1 214 to E1 240 defined in thisstudy is further noted by Mitchell et al. (1992). A synthetic peptide corresponding toresidues 213 to 239 was used as a target antigen in ELISA to assess the antibodyresponses of patients during acute and convalescent phases of wild rubella infection. Itwas found that the El peptide-reactive antibodies closely paralleled the RV-specificantibodies measured by RV ELISA, hemagglutination inhibiting and viral neutralizingassays (Mitchell et al., 1992). This result suggests that the epitopes defined may behemagglutination and viral neutralizing epitopes for human antibodies of RV El and mayprove useful in determining effective RV immunity in diagnostic assay for rubella inaddition to their usefulness in the construction of subunit vaccines.773.2. Section II: Cellular response to RV structural proteins 3.2.1. Proliferative responses to RV structural proteinsThis study was designed to identify an RV structural protein that may play a pivotalrole in eliciting a T-cell immune response and to determine the difference, if any in theproliferative profile between the normal individuals with no rubella-associated symptomsand those suffering from CRS. Peripheral blood lymphocytes were stimulated in vitro withrecombinant vaccinia virus expressing E1, E2 or C protein. Cellular extracts from wild-type vaccinia virus were used as the control to monitor the proliferative responses specificto the vaccinia virus. As shown in Fig. 17, depending on the background response towild-type vaccinia virus, the optimal concentration of each antigen inducing the greatestresponse varied with each subject in the study. The counts per minute value used in thestatistical analysis were those obtained at the optimal concentration of each antigen. Theproliferative responses were expressed as stimulation index (SI). The results indicatedthat each individual in both study groups exhibited differential response to E1, E2 and C(Fig. 18). El was the predominant antigen to which a majority of the subjects elicitedlymphocyte proliferative responses. Relatively large proportions of individuals hadproliferative responses to C which is an internal protein. Proliferative responses to E2was observed mostly for the individuals with CRS (Fig. 18).The response levels to each structural protein in the two subject groups werecompared (Fig. 19). Statistical analyses by using nonparametric statistics was undertakento determine the significant levels. For group A (control), it was found that thelymphoproliferative response to El was dominant, followed by the response to C, with the78weakest response being directed to E2. The response to El was significantly higher thanthe response to E2 (p=0.002), whereas statistical differences between the El and Cresponses were weak (p=0.1). For group B (CRS), no significant differences wereobserved in the responses to the individual viral proteins; however, it was observed thatthe proliferative response to E2 was the dominant response of the three proteins in theseindividuals. For CRS patients, a decrease in the E1-specific response and an increasein the E2-specific response were observed contrary to the statistically significant differentresponses observed for El and E2 in the control population.3.2.2. Antibody response to RV structural proteinsSeparated RV structural proteins were used in an ELISA system to quantitate IgGresponses directed to whole RV as well as to E1, E2 and C. The results of the antigenicspecific ELISA (Table VIII) showed that circulating anti-RV IgG in the control populationwas predominantly directed to E1, at levels significantly higher than those of anti-E2 (p< 0.002) and anti-C IgG (p < 0.0001). CRS patients had significantly elevated levels ofanti-E2 IgG (p < 0.05) in comparison to those of the control, whereas no significantdifference was detected in their anti-El responses (p < 0.3). The increase in the E2/E1ratio, which had been previously reported for CRS patients (Katow and Suguira, 1985),seems to be due primarily to an increase in the serological response to E2 in this patientpopulation.79-41010-1^102^ 103-1041000E1010-1 -310-210ADilutions DilutionsFig. 17. Proliferative response to recombinant vaccinia virus expressing RVantigens E1, E2 or C. Peripheral blood lymphocytes were stimulated with inactivatedvaccinia recombinant expressing RV antigen. Stimulated lymphocytes were harvestedat day 7 following a 6 hour pulse with [3F1]-thymidine. Representative proliferationresponse as shown by incorporation of [3H]-thymidine into DNA at different antigendilutions when background response to wild-type vaccinia virus was either high (A) orlow (B). o= El recombinant; ■= E2 recombinant; o= C recombinant;t = wild type.800ceECOControl^CRS2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4Patient NumberFig. 18. Proliferative response to RV structural proteins in the study groups.Preceding the calculation of stimulation index (SI), the raw values were corrected for thespontaneous background response, which ranged from 100 to 500 cpm. Sl=cpm(vacciniarecombinant - spontaneous) - cpm(wild type - spontaneous), where spontaneous refersto spontaneous background response. High background response to wild type vacciniavirus was observed for six individuals (no. 3, 4, 5, 11, 12, and 13) in group A. The optimalconcentration of antigen used in the statistical analysis was a 10 -2 dilution for highbackground response and a 10 -1 to 10 -2 dilution for low background response. Control =individuals without rubella associated symptoms; CRS = patients with congenital rubellasyndrome.=E1 emos —E2 ^ =C81Comparison of the B-cell and lymphoproliferative responses in the two patientpopulations showed similarities in their protein specificities. The control populationshowed the immunodominance of El in both the cellular and humoral responses overboth E2 and C (Fig. 19, top panel). The increase of the serological response to E2observed for the CRS population was also reflected by an increase in E2-specificproliferative responses (Fig. 19, lower panel). However, in the analysis of thelymphoproliferative and B-cell responses of individual CRS patients or control individuals,a poor correlation between circulating IgG levels and stimulation indices to the individualproteins was observed, as demonstrated by the results for patients 2 and 14 (Fig. 18;Table VIII).82El E2 CEl E2 CCRS: Proliferative Response20-1.0-0.0control: Proliferative Response -6.0Control: Serological Response2.00.0El^E2^CCRS: Serological Response El^E2^C-6.00.0Fig. 19. Comparison of proliferative and IgG responses to E1, E2 and C in thecontrol and CRS study group. Values of SI and arbitrary unite per minute are based ona natural logarithm scale. AU = arbitrary units of IgG to each structural protein; •means; error bars indicate the standard errors of the means. The dots indicatemeasuring point averages for each patient.83Table VIII. ELISA immunoreactivity of individual structural proteins and wholevirus by using sera from subjects in group A (control) and group B (CRS patients) (fromChaye et al., 1992b).GroupandsubjectIgG response(IU/ml) towhole RVIgG response (AU/ml) to:El E2Group A1 555 1,010 65 402 145 250 50 403 273 280 50 1504 1 60 10 105 120 200 50 256 290 290 67 507 0 18 80 08 0 40 0 09 160 290 40 1510 172 250 170 3011 945 150 0 012 125 210 40 2013 360 420 90 7014 46 90 50 0Group B1 265 2,960 87 112 35 0 35 03 147 49 93 134 257 41 108 11843.2.3. Discussion of Section IIStudies of immune response to RV infection in humans have indicated that of thestructural proteins studied, envelope glycoproteins, in particular E1, are the primarytargets for induction of humoral immune responses (deMazancourt et al., 1986; Katowand Sugiura, 1985; Zhang et al., 1991). Immune responses to non-structural proteinsmay be important in RV immunity; however, such proteins are not available forimmunological studies. Thus, this study has been limited to the analyses of the structuralproteins. The results of early studies with rubella subunit vaccine composed of only viralenvelope proteins showed that the vaccine was capable of stimulating both humoral andcellular responses in rabbits (Cappel and DeCuyper, 1976). The present study showedthat the El glycoprotein is the most antigenic of the three structural proteins in elicitingboth cellular and humoral responses. This parallel pattern in the cellular and humoralresponses is not unexpected. T-cell and B-cell cooperation for antibody production is notrandom in that the B-cell selects the T-cell with which to cooperate (Celada and Sercarz,1988). This selection is postulated to take place at the level of antigen processing andpresentation by the B-cells. The region of the antigen bound by the paratope of theimmunoglobulin may be protected from enzymatic degradation during processing and thuswill be presented intact. Therefore, B-cell epitope and T-cell epitopes may be found onthe same antigen (French et al., 1989; Nicholas et al., 1988). This hypothesis is furthersupported by the pattern observed for the individuals with CRS, in whom both cellular andhumoral responses to E2 were relatively elevated when compared to the responses ofnormal individuals. The relatively reduced levels of immune response, both humoral and85cellular, against E2 glycoprotein in the control individuals may be a result of antigeniccompetition between the glycoproteins El and E2. In the natural infection by influenzavirus, suppression of the anti-neuraminidase response has been observed to -be due toantigenic competition (Johansson et al., 1987). This competition is affected by therelative amounts of competing immunogens, resulting in an immune response that ispredominantly directed against hemagglutinin. The low level of immune response to E2in RV infection may be due to the inaccessibility of the polypeptide chain to the host'simmune system because of masking by glycosylation (Ho-Terry and Cohen, 1984).Katow and Sugiura proposed that the elevated level of immune response to E2 is a directconsequence of CRS (Katow and Sugiura, 1985). This is further supported by workreported by Williams et al. (1992). This group found that patients with the retinaldegeneration of retinitis pigmentosa had elevated levels of E2 antibodies than the normalindividuals.Persistent RV infection have been reported by a number of investigators (Chantleret al., 1982; Cunningham and Fraser, 1985; Oxford and Potter, 1971), though themechanism for establishing persistence is still not clear. Recently, Williams et al. (1993)observed defective phagocytosis by cultured human retinal pigment epithelial cells withpersistent rubella virus infection, which may be a possible mechanism of establishingpersistence. Extrathymic tolerance resulting in the absence of neutralizing antibodies tothe virus may interfere with viral clearance and contribute to persistence (Nossal, 1991).Persistent infection may cause slow release of the viral antigens, leading to continualimmunostimulation and increased immune response,to antigens that are not seen by the86immune system for rapidly cleared viral infection (Oldstone, 1989). RV infections havebeen implicated in a number of disorders, such as arthritis, diabetes, and encephalitis(Ginsberg-Fellner et al., 1985; Martin et al., 1989; Marvin, 1975). Williams et al. (1993)Nath and Wolinsky (1990) found that irrespective of the antibody titre to whole RV, therelative proportion of the IgG response to El was diminished and that to E2 was elevatedin MS (multiple sclerosis) patients when compared to the control population. Nodifference in C was observed. Perhaps in susceptible individuals, vigorous immuneresponses directed against E2 may result in autoimmunity as an indirect consequenceof persistent infection and may result in autoimmune conditions.In addition to the cellular responses, humoral responses were also studied. A lackof correlation between the proliferative responses to RV structural proteins and thepresence of RV-specific IgG in sera was observed (Table VIII). This discrepancy inprotein recognition between T- and B-cell repertoires has also been detected in bovineherpesvirus infection (Hutchings et al., 1990) and in RV infection (Williams et al., 1992).The presence of lymphocyte responsiveness in the absence of circulating antibody is ofclinical importance because it is a direct evidence of previous exposure to antigen. Theabsence of detectable antibody titre does not always correlate well with susceptibility toclinical infection (Brody, 1966; Horstman et al., 1970). Consequently, it is possible tohave no circulating antibody levels and be protected against clinical reinfection. Whetherthe individuals who showed conflicting T- and B-cell assay results are susceptible toclinical infection was not determined here in this study. But what this study does indicateis that serology studies alone are not accurate indicators of whether an individual is87susceptible or not. Lymphocyte proliferation assays, in addition to immunoblots and/orELISAs may, provide more accurate information on whether an individual is immune thanimmunoblot analysis and/or ELISAs alone. At present, laboratory diagnosis for RVinfection employs techniques such as virus neutralization assays, hemagglutinin assaysand ELISAs which rely on the presence of anti-RV antibodies. The results of theseassays are used to determine whether an individual should be immunized. These resultsare especially important to women planning to become pregnant since the risks of fetaldamage associated with re-exposure to RV are not fully understood. It was alsoobserved that immunization of those who are sero-negative but have been previouslyexposed to the RV may suffer symptoms similar to RAA (personal communication withDr. A. Tingle). No experimentally substantiated explanations have been forwarded toexplain these observations. Thus, serology studies alone may not be sufficient todetermine whether an individual is protected from future infection. It would also beinformative to investigate the changes with time in the T-cell proliferative profiles towardseach of the structural proteins in parallel with humoral analysis. These studies wouldchart the development of immunity of RV infection or vaccination in greater detail andallow a better understanding of the clinical development of RV infection and immunity.883.3. Section III: Human T- and B-cell epitopes of El qlycoprotein 3.3.1. Lymphocyte proliferative response to El peptides23 overlapping peptides (EP1 to EP23) covering approximately 90% of the Elprotein sequence have been synthesized and provided by Dr. P. Chong at ConnaughtLaboratories (Fig. 20, Table VII). The lengths of the peptides were chosen based on thehigh index of hydrophillic (3-turns and a-helix as judged by secondary structure predictionanalysis according to Hopp and Woods (1981). These regions are likely to be exposedand antigenic. Long peptides (17 to 22 residues) were synthesized to mimic epitopes onnative El protein because they have more chance of having a conformation similar to thatof the corresponding portions of the native protein than short peptides (Rothbard andTaylor, 1988).Sera and peripheral blood lymphocytes were isolated from 10 seropositive healthydonors with no history of clinical rubella. Nine of the ten donors were found to react toRV structural proteins in immunoblot analysis using non-reducing conditions (Fig. 21).This immunoblot suggests that 9 of the 10 donors have RV-specific circulating antibodies.Lymphocyte proliferative responses of human PBL were determined in vitro by directstimulation with RV or with El peptides. All individuals responded to RV, although thelevels of response varied considerably between different donors (Table X). Eachindividual showed an unique response profile to the El peptides (Table X). PeptidesEP12 (residues 207-226), EP17 (residues 289-308), EP19 (residues 324-343), and EP21(residues 358-377) were recognized by six or more individuals, while EP8 (residues 140-159) and EP9 (residues 157-176) were not recognized by any of the 10 individuals in the89study group (Table X and Fig. 22). Since the regions of overlap between adjacentpeptides are generally one to three amino acids, it is possible that epitopes presented inthe overlap regions were not detected in this study.T-cell antigenic sites are postulated to be amphipathic helices, with one facepredominantly polar and the opposite face predominantly apolar (Margalit et al., 1987).Amphipathic scores of domains present in peptides EP8, EP9, EP12, EP17, and EP19are listed in Table IX. Amphipathic score is a method of predicting T-cell epitope and ahigh amphipathic score does not necessarily indicate the presence of a T-cell epitope, asit is true in converse, where a low score does not exclude a particular domain as a T-cellepitope. The high amphipathic score and lack of response observed for peptides EP8and EP9 are not unexpected. Since the algorithm used to ascertain the amphipathicscore does not take into account of MHC restrictions (Margalit et al., 1987). MHCrestriction is an important factor in T-cell epitope selection in vivo and thus this type ofpredictive methods has inherent limitations.90160^16120^40^60^ero^100^120^110Flaraluo Number200^221^240^250^260 •^3,7,7^321^340^250^Zeo^• 0RosIduo Number1.^4 (54 '74 )^6006-125)^8440-159)^101,7' %I)^12(202225)^1412,0 2591^16(272-291(^18 (371-3201^20(341 - 360)^22 074-30011.11221^3636-57)^5(7101)^7022.41)^9457-170)^11840.2091^13 (224243)^15 (256- ^17(2055310)^19024-3443/^21 (356.3101 23(391.412)■..41 1 1\iStl itkiP,ti 11.0Fig. 20. Predicted structure of RV El protein by conventional structural analysisalgorithms. (A) Secondary structure analysis of local average a-helix and 13-turnspotentials according to Chou and Fasman (1978); (B) Hydrophilicity plots predicted by themethod of Hopp and Woods (1981). The values are derived from the average ofheptapeptide windows and are plotted at the midpoint of each segment.2 3 4 5 6 7 8 9 0Fig. 21. Immunoblot analysis of participants' sera against M33 RV antigens undernon-reducing conditions. Sera 1 to 10 were used to at a 1:80 dilution, M is the mixtureof negative control serum form four individuals. E1, E2 and C refer to the structuralproteins of RV. Relative mobilities of protein standards (kDa) are indicated at the left.91Table IX. Amphipathic scores of El peptidesPeptidenumber Peptide sequence ASEP8 (140-159) SVFALASYVQHPHKTVRVKF 24.3EP9 (157-176) VKFHTETRTVVVQLSVAGVSC 24.3EP12 (207-226) IMNYTGNQQS RWGLGSPNCH 27.9EP17 (289-308) LHIRAGPYGHATVEMPEWIH 16.7EP19 (324-343) LKFKTVRPVALPRALAPPRN 69.8EP21 (358-377) VEGLAPGGGNCHLTVNGEDV 69.8Number of Samples with SI > 2.0108642 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23PeptidesFig. 22. Peptide stimulated T-cell proliferative responses. Each bar indicates thenumber of peripheral blood lymphocyte samples which had a stimulation index of greaterthan 2 for each peptide. Peptides are numbered 1 to 23 spanning the entire Elsequence.92Table X. Lymphocyte proliferative responses to El synthetic peptides aCell proliferation indexPeptides 1 2 3 4 5 6 7 S 9 10EP1 1-22 6.0 0.9 1.7 1.9 1.1 0.8 1.4 1.8 0.7 1.0EP2 19-38 3.7 1.3 0.9 2.9 0.2 0.4 1.3 0.5 0.2 1.0EP3 38-57 3.0 1.0 0.6 2.5 3.5 1.4 7_1 1.8 0.6 1.9EP4 54-74 2.9 0_7 0.7 1.9 5.1 0.9 6.4 8.3 0:9 1.4EP5 71-91 4.2 0.7 0.9 0.8 2.5 1.3 7.1 1.1 1.6 0.8EP6 106-125 0.9 0.S 1.5 1.7 2.9 0.8 3.8 0.6 0.7 5.5EP7 122-141 2.4 0.5 0.9 0.8 1.2 0.5 2.5 1.3 0.7 1.9EPS 140-159 0.9 0.7 0.3 1.0 1.3 0.3 0.7 1.3 0.2 0.6EP9 157-176 1.3 1.3 0.9 0.5 12 0.5 1.2 0.7 1.0 0.4EPIO 174-193 3.0 0_9 1.5 4.5 1.0 0.7 4.3 1.6 0.5 1.5EP1I 190-209 1.9 1.0 1.5 2.3 3.5 1.4 1.2 1.4 1.3 8.8EP12 207-226 2.4 0.7 0.9 2-5 3.2 1.0 4.8 3.6 L2 5.1EPI3 224-243 1.1 0.5 0.6 0.8 L4 1.0 3.0 1.3 1.1 2.6EPI4 240-259 1.8 0.7 .08 1.8 1.5 1.3 2.5 1_4 OA 1.0EP15 256-275 1.6 0.6 0.8 1.8 1.3 0.7 4.3 1.1 0.6 0.9EP16 272-291 1.6 0.4 0.8 2.3 1.6 1.7 1.3 1.7 2.0 1.6EP17 289-308 2.8 1.2 2.2 0.7 1.3 3.8 4..3 2.2 1.4 2.0EP18 307-326 0.9 0.7 0.9 1.9 2.0 0.6 1.0 3_3 0.8 1.1EP19 324-343 33 1.1 2.1 3.1 6.1 1.9 8.7 2.6 0.6 5.3EP20 341-360 1.1 0.6 1.2 2.2 6.2 0.3 53 0.8 0.6 6.9EP21 358-377 1.4 2.0 2.0 3.4 3.5 0.8 5.5 0.2 0.6 3.5EP22 374-390 1.4 0.7 0.9 1.2 1.5 0.7 6.1 0.9 0.7 1.3EP23 391-412 0.5 1.1 0.9 0.8 1.7 0.6 4.3 1.0 .0.6 1.5RV 5.0 3.7 5_1 7.8 3.4 2.8 24 33 2.6 20°Cell proliferation indices were calculated as described under Materials and Methods. Boldface numbers represent proliferative indicesgreater than 2.Table Xl. B-cell response to El peptidesaELISAPeptide 1 2 3 4 5 6 9 10EP1 ++EP2 + + + + +EP3 +EP4 + + + + +EP5 ++EP6EP7EP8EP9 + + + + + + + +EPIOEP1I +EP12 +EP13 ++ +EPI4 +EP1S +EP16 +EP17 + +EP18 .EPI9 + + +EP20 + + + + + +EP21EP22 ++ ++ + + ++ ++EP23 +"Responses of sera from 10 individuals who displayed positive reactivity indicative of antibody binding to peptide are expressed in termsof relative reactivity as described under Materials and Methods. IgG antibody reactions were tested at a serum dilution of 1:64.933.3.2. Antibody response to El peptidesB-cell epitopes may be linear as well as conformational dependent. Usingsynthetic peptides, only the linear B-cell epitopes are detected. The purpose of this partof the study was to ascertain a crude picture and to see whether a correlation existsbetween human B-cell epitopes and the epitopes mapped in the Part I of the study. Thefrequently recognized B-cell epitopes were EP2 (residues 19-38), EP4 (residues 54-74),EP9 (residues 157-176), EP20 (residues 341-360), and EP22 (residues 374-390) (TableXI). No reactivity was observed with peptides EP6, EP7, EP8, EP10, EP18, and EP21(Table X). Since individual synthetic peptides are not covalently bound to the plates, thepeptide ELISA may not work as well as for all peptides. To verify the assay, individualpeptides were coated onto microtitre plates and probed with peptide-specific rabbitantisera raised against individual El peptides. All peptides were recognized by theirrespective antipeptide antisera at the reactivity titre >1/1600. These results establish thatall peptides are adsorbed to the microtitre plates and their antigenic determinants areaccessible to antibodies and synthetic peptides. To check the possibility of non-specificbinding between synthetic peptides and antibodies, synthetic peptides were tested byusing normal IgG's from rabbits. . No non-specific binding was found. Detection of RV-specific IgM antibodies is an important criterion for diagnosis of acute RV infection. Alldonors in this study are seropositive healthy adults, having no history of acute RVinfection within the last 5 years. Since no RV-specific IgM response in the sera wasdetected in the immunoblot analysis, peptide-specific IgM responses were not determined.943.3.3. Discussion of Section IIIIdeal synthetic vaccines should have the following characteristics: (i) containepitopes important for protective antibody production; (ii) contain T-cell recognition sitesthat can induce antibody production, cellular immunity, and prime memory T-cells to thepathogen; (iii) be silent for cross reactive domains to self antigen/and or forimmunosuppressive domains; and (iv) provide long lasting immunity and not requirefrequent boosters. Synthetic peptides representing only B-cell epitopes are generally poorimmunogens and need to be coupled to carrier proteins. However, such complexes arelimited in application due to carrier-induced suppression and failure to prime T-cellmemory response to the pathogen. Suitable carrier proteins for human use are not yetknown. Studies of hepatitis B virus and foot and mouth disease virus showed thatcomposite peptides containing both T- and B-cell sites yields a more efficient immunogenthan B-cell epitopes alone (Milich, 1988; Milich et al., 1988)At the time the synthetic peptides were being designed the minimum length of T-cell epitopes was thought to be 8-12 amino acids and 6-10 residues for a B-cell epitope(Dyson et al., 1988; Rothbard and Taylor, 1988). Accordingly, peptides of 17 or moreamino acids were synthesized.. Using 23 overlapping synthetic peptides, severalimmunodominant T- and B-cell epitopes of El were identified via in vitro proliferationassay and peptide specific ELISA. The frequently recognized T-cell and the common B-cell epitopes were EP2, EP4, EP9, EP21 and EP22 (Table XI). Comparing T- and B-cellresponses to El peptides in each individual, a poor correlation between the reactivity ofthe antibody and the stimulation index of peptides was observed (Tables X and XI). This95is not surprising since overlapping T- and B-cell sites are generally unusual (Milich, 1988)though viral-specific peptides containing overlapping domains have been documented(Cohen et al., 1984; Milich, 1988). The identified viral neutralizing epitopes on El arelocated within the peptides EP12, EP13, EP14, EP15 and EP16 (Chaye et al., 1992;Wolinsky et al., 1992, 1993). However none of these epitopes was found to be a sitefrequently recognized by B cells. The studies of Rothbard and Taylor (1988), and morerecent studies of Rammensee et al. (1993) and Germain (1993) found that peptide lengthfor Class I antigen is 8 or 9 residues (reviewed in Rammensee et al., 1993) and for ClassII antigen is 12 to 20 residues (reviewed in Germain, 1993). Since, the design of theassay preferentially selects for Class II restricted CD4+ (Ou et al., 1992a)there may havebeen epitopes that were missed by this panel of peptides.A hierarchy of immune responsiveness to the individual peptides was expectedin the population depending on MHC restriction (Milich, 1988b). This was observed in thisstudy with the 23 overlapping peptides. Individuals 1, 4, 5, and 7 responded to 50% ormore of the peptides. The order of response to peptides differed between individuals.Peptides EP12, EP17, EP19 and EP21 induced proliferation responses in the majority ofthe individuals in the study and indicated substantial T-cell stimulating activities of thesepeptides. Unfortunately the phenotypes of the donors' MHC antigens have not beendetermined. Whether the T-cell responses of these peptides are restricted by a commonclass II MHC antigen or whether they are compatible with more than one antigen type arenot known. The role of MHC class I or class II restricted RV-specific T cells in RVinfection and protection has not been reported. A low RV antigen-specific response is96shown to be associated with HLA-DW2 (Ilonen and Salmi, 1986). Recently, Ou et al.(1992b, 1992c) have isolated human CD4+ cytotoxic T-cell clones reactive to E2 and Cproteins of RV. Recognition of capsid epitopes by T-cell clones is associated with HLA-DR4 or DRw9 (Ou et al., 1992c), whereas the E2 epitope is associated with HLA-DR7(Ou et al., 1992b).Defining immunodominant epitopes on an antigen is critical for determiningdomains to be included in subunit vaccines. Undesirable determinants such as thoseresponsible for eliciting autoimmunity should be eliminated in constructing subunitvaccine. RV infection is known to result in complications of autoimmunity in nature. Ifdeterminants which induce responses which cross react with self-antigen in context withMHC can be eliminated and such complications would be limited.Another advantage of elucidating T-helper sites is that they can be attached toantibody binding sites of other RV viral proteins, E2 and C. Viral neutralizing epitopeshave been mapped to E2 as well as to El (Waxham and Wolinsky, 1985). In hepatitisB virus and influenza virus, cytotoxic T cell sites that are crucial for viral clearance havebeen mapped to non-envelope viral proteins. Recently identified immunodominantepitopes on C protein of RV can be incorporated in the peptide vaccine (Ou et al., 1992c).Composite vaccine containing all potential immunologically functional domains couldprovide protective immunity and aid in priming a subset of lymphocytes important for viralclearance. Subunit vaccine for hepatitis B virus containing intramolecularfinterstructuralT-cell epitopes and B-cell epitopes was found to be efficient in producing protectiveantibodies (Milich et al., 1988). By selecting immunodominant epitopes for both T and97B cells, potentially harmful or immunosuppressive domains can be eliminated. Rubellavirus has been associated with autoimmune conditions (Chantler et al., 1982; Ginsberg-Feltner et al., 1985). If domains that induce such responses can be excluded, thedangers of vaccination can be reduced.984. SUMMARY AND PERSPECTIVESPeripheral blood lymphocytes from volunteers with no history of rubella associatedconditions and patients with congenital rubella syndrome were studied for recognition ofRV E1, E2 and C structural proteins. The corresponding sera were also tested usingantigen specific ELISAs. Two interesting phenomena were observed from these studies.In normal individuals, of the RV structural proteins, El was the dominant antigen for bothhumoral and cellular immune responses while in CRS patients, immune responses to E2were dominant. These studies confirmed the hypothesis that El is the dominantimmunogen for protective immunity against RV infection.The implication of these results with respect to differential immunity to El and E2in CRS patients is less clear than the role of El immunity in the normal population.Studies in the past have shown reduced levels of cell mediated immune response in CRSpatients than in the normal population (Buimovici-Klein and Cooper, 1985). Others haveshown the reduced El specific IgG (Katow and Suguira, 1985), inability to produce high-affinity IgG (Fitzgerald et al., 1988) and lack of HAI IgG (Cooper et al., 1971) in CRSpatients. The results of our study contribute to the overall understanding of the immuneresponses observed in CRS patients. Different profiles of immune responses observedin CRS patients than that of normal individuals may contribute to the manifestation of thesymptoms suffered by CRS patients.The remainder of the thesis focused on the El glycoprotein to define theimmunogenically functional epitopes. First, . El cDNAs with various deletions wereexpressed in vitro using cell-free rabbit reticulocyte expression system, in COS cells and99in E.coli. Appropriate vectors were manipulated to express the desired El cDNAsequences. The expressed proteins were screened with monoclonal antibodies with viralneutralizing and hemagglutination inhibiting activities. These studies showed that theepitopes recognized by these monoclonal antibodies were independent of carbohydratemoieties on the protein; ie, the epitopes were not affected by the changes in theconformation arising from glycosylation and/or no glycosylation. It was thus concludedthat the epitopes were linear on the El glycoprotein. Synthetic peptides correspondingto the domain localized by using fusion proteins expressed in E.coli were synthesized andscreened in ELISAs. The results defined the viral neutralizing epitopes to residues 214to 233 and to residues 219 to 233. The epitope recognized by hemagglutination inhibitingmonoclonal antibody was mapped to residues 214 to 240.In addition to the epitopes defined by the monoclonal antibodies, the epitopesrecognized by human T-cells and human antisera were also determined. Twenty-threeoverlapping synthetic peptides spanning the entire sequence of El were synthesized.These peptides were used as antigens in the lymphocyte proliferation assays usinghuman peripheral lymphocytes. They were also used as antigens in antigen specificELISA using the corresponding human antisera. Peptides EP12 (residues 207 to 226),EP17 (residues 289 to 308), EP19 (residues 324 to 343) and EP21 (residues 358 to 377)were recognized by greater than 50% of the individuals tested. The amphipathicityanalysis showed that EP12, EP17, EP19 and EP21 have relatively high amphipathicscores (>15). ELISA experiments revealed that EP9 (residues 157 to 176), EP22(374 to 390), EP2 (residues 19 to 38), EP4 (54 to 74) and EP20 (residues 341 to 360)100were frequently recognized by the sera tested. This study provides the basis for whicha subunit vaccine may be designed. It has been shown that subunit vaccine comprisingof both T and B cell epitopes is more effective in eliciting protective immunity. It alsoeliminates the need for carrier molecules, of which a suitable molecule has not beendeveloped for humans.In summary this project has confirmed the hypothesis that the El glycoprotein isthe dominant antigen for which protective immunity is elicited. We have definedimmunogenically functional epitopes using mouse monoclonal antibodies as well as theepitopes most frequently recognized by human T and B cells in the human populationsample selected.The results of this study have provided useful starting points for the future studiesin the field of RV immunopathology. It is suggested by this study that distinct immunityprofile exists with respect to the different structural proteins in the normal and CRSpopulations. Studies of larger samples of populations separately consisting of CRS,patients with rubella associated arthritis (RAA), and vaccine recipients would be pertinentto the greater understanding of the functions of each structural proteins in theimmunopathology of rubella associated conditions. Furthermore, HLA studies of thesevarious populations may provide information to link susceptibility of these rubellaassociated conditions to a MHC genotype. By comparative analysis, it is possible todefine antigens which elicit the hostile immune responses in susceptible individuals.These epitopes then can be deleted from subunit vaccines. Both cellular and humoralimmune responses should be studied.101In addition to defining immunosuppressive and autoreactive epitopes, it is alsoimportant to study the cellular mechanisms of these disorders; ie, whether the pathologyof these disorders are driven by the cellular mechanism or humoral mechanism. Someautoimmune disorders are results of autoreactive immunoglobulins while others are dueto autoreactive T-cells.The epitopes defined using mouse monoclonal antibodies should be used todetermine their immunogenicity. Antigenicity of a protein does not automatically translateinto immunogenicity in vivo. It will be also important to the design of subunit vaccine todetermine the optimum distance between the functional epitopes which will elicit thegreatest protective immunity.Although synthetic peptides are valuable in fine epitope mapping, their usefulnessis inherently limited by the very same characteristic for which they are so valuable, theirsmall size. Epitopes defined using synthetic peptides are linear and usually conformationindependent. However, not all functional epitopes are linear and independent of theprotein's tertiary structure. We may have missed immunogenically significant epitopeswhich are conformation dependent. As it was suggested in the epitope mapping studies,the proper folding may be required for expression of epitopes important forhemagglutination. Moreover, glycosylation mutant studies (Qiu et al., 1992) showed thatonly single mutants induced viral neutralization antibodies. 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