Regulation of TNF-related apoptosis-inducing ligand-mediated death-signal pathway in human beta cells by Fas-associated death domain and nuclear factor kappaB.

Transfectants of human CM and NES2Y beta cell lines and primary islets transfected by FADD-DN (dominant-negative form of Fas-associated death domain), a mutant of FADD and/or a superrepressor of nuclear factor kappaB (NF-kappaB) (AdIkappaB(SA)2), were examined for their susceptibility to the TRAIL (TNF-related apoptosis-inducing ligand)-induced death signal pathway, compared with controls, wild-type cells, and vector transfectants in caspase fluorescence, Western blot, electrophoretic mobility shift, apoptosis, and cytotoxicity assays. FADD-DN inhibited caspase-8 activation induced by TRAIL in the transfectants of CM and NES2Y cells. TRAIL-induced apoptosis and cytotoxicity to the FADD-DN transfectants were decreased in comparison to those responses in controls (CM, p < 0.01 and p < 0.01; NES2Y, p < 0.05, and p < 0.02, respectively). When CM, NES2Y, and primary islet cells were transfected by AdIkappaB(SA)2, TRAIL-induced IkappaB degradation and nuclear translocation of NF-kappaB p50/p65 were blocked. TRAIL-induced apoptosis and cytotoxicity to AdIkappaB(SA)2 transfectants of these cells were also reduced (CM, p < 0.02 and p < 0.02; NES2Y, p < 0.01 and p < 0.01, respectively, and islet p < 0.01 for cytotoxicity). Finally, cytotoxicity induced by TRAIL in CM and NES2Y cells transfected with both FADD-DN and AdIkappaB(SA)2 was reduced, compared with that observed in these cells transfected with either FADD-DN alone or AdIkappaB(SA)2 alone, suggesting that FADD and NF-kappaB have synergistic proapoptotic regulatory effects on the susceptibility of beta cell lines and islet cells to TRAIL-induced destruction.

TNF-related apoptosis-inducing ligand death pathway-mediated human beta-cell destruction.

AIMS/HYPOTHESIS: The aim of this study is to investigate whether apoptosis in human beta cells can be related to the induction of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway. METHODS: We examined the expression of TRAIL and TRAIL receptors in two human pancreatic beta-cell lines and in human primary islet cells using RT-PCR assays and flow cytometric analyses and tested TRAIL-mediated beta-cell destruction in (51)Cr release cytotoxicity assays, Annexin-V and APO-DIREC assays. RESULTS: Most of the human beta cells express TRAIL receptors-R1, -R2, -R3, -R4 and/or TRAIL. TRAIL induced much stronger cytotoxicity and apoptosis to beta-cell lines CM and HP62 than did FasL, TNF-alpha, LTalpha1beta2, LTalpha2beta1, LIGHT, and IFN-gamma. The cytotoxicity and apoptosis induced by TRAIL to beta-cell lines CM were inhibited competitively by soluble TRAIL receptors, R1, R2, R3 or R4. Treatment of these beta cells with antibodies against TRAIL receptors was able to block the cytotoxicity of TRAIL to these cells. Beta-cell antigen-specific CTL (CD4(+) and CD8(+)) clones express TRAIL, suggesting that these cells are potential sources of TRAIL-inducing beta-cell destruction. Normal primary islet cells from most donors are resistant to the cytotoxicity mediated by TRAIL. However, treatment with an inhibitor of protein synthesis (cycloheximide) or with an enzyme (PI-PLC) that can remove TRAIL-R3 from the islet-cell membrane was able to increase the susceptibility of TRAIL-resistant primary islet cells to the TRAIL death pathway. CONCLUSION/INTERPRETATION: The TRAIL death pathway is present and can function in human islet beta cells, but unidentified inhibitors of the TRAIL death pathway are present in normal islet cells.

Cross-reactive rubella virus and glutamic acid decarboxylase (65 and 67) protein determinants recognised by T cells of patients with type I diabetes mellitus.

AIMS/HYPOTHESIS: To examine the cross-reaction between viral and beta-cell protein determinants and to further understand the potential role of this mechanism in Type I (insuline-dependent) diabetes mellitus. METHODS: Immune responses to a panel of 28 viral and beta-cell protein peptides representing selected sequences of rubella virus (RV), Coxsackie virus, human 38 KDa31G and glutamic acid decarboxylase (GAD 65 and 67) proteins in proliferation or cytotoxicity assays have been studied using uncloned and cloned T-cell cohorts from a group of 60 Type I diabetic patients. RESULTS: Peptide GAD65(252-266) induced the responses of patients with recent onset diabetes in proliferation assays at the highest frequency (77%), whereas GAD67(212-226) stimulated the cellular responses at the highest rate (61%) in patients with late-onset diabetes. RVE1(157-176) was recognised by all groups of patients at the highest frequency and the largest amplitude among the viral peptides tested. T-cell clones specific to GAD65(252-266), GAD65(274-286) or GAD67(212-226) were tested in cytotoxicity assays for their responses to rubella virus peptides. Each of these T-cell clones cross-reacted with two to four rubella virus peptides, including RVE1(157-176) and RVE2(87-107). Analysis of the sequences of cross-reactive viral and glutamic acid decarboxylase antigens showed that these epitopes shared similar peptide binding motifs to HLA DR3/DR4. There is a statistically significant correlation between the response amplitude of patient's peripheral blood mononuclear cells to RVE1(157-176), RVE2(87-107) and GAD65(274-286) in patients with recent onset diabetes, and to RVE1(157-176) and GAD67(212-226) in patients with late onset diabetes. CONCLUSION/INTERPRETATION: Cross-reactive glutamic acid decarboxylase and rubella virus determinants identified by T-cell clones were also recognised at high frequencies by general T-cell populations of Type I diabetic patients.

Rubella virus vaccine associated arthropathy in postpartum immunized women: influence of preimmunization serologic status on development of joint manifestations.

OBJECTIVE: To measure preimmunization rubella virus (RV)-specific IgG levels and to relate these to the development of acute and chronic (persistent or recurrent) joint manifestations following rubella vaccination. METHODS: Specific IgG was determined by whole RV enzyme immunoassays (EIA) (Abbott Rubazyme and M33, an in-house method), immunoblot, neutralization domain peptide (BCH-178c) EIA, and neutralization bioassay in prevaccine samples of 268 RV seronegative women (Abbott absorbance < 0.999 units) who had received monovalent live attenuated RA27/3 strain RV vaccine in a clinical trial that recorded joint manifestations. RESULTS: Of rubella vaccinated women tested for prevaccine antibodies, 21.7% were actually positive (> or = 10 IU/ml) by M33 EIA, 33.2% had Abbott values > or = 0.250 units, and 47.6% had RV protein-specific antibody (immunoblot), while only 17.6% were positive (> or = 10 IU/ml) by neutralization domain peptide EIA and 12.7% had neutralization titers > or = 1:8. Seropositivity by the various methods was compared to recorded occurrence of acute and chronic arthropathy (arthralgia and/or arthritis) after RV vaccination. Relative to women who had no joint manifestations, prevaccine seropositivity rates for subjects with acute arthropathy were significantly (p < 0.05) lower in the Abbott test (< 0.250 units), BCH-178c peptide EIA, and neutralization bioassay, while those who also developed chronic arthropathy had significantly lower prevaccine seropositivity rates for the Abbott (< 0.250 units) and M33 EIA and neutralization bioassay. CONCLUSION: Results suggest that risk for arthropathy following RA27/3 rubella vaccination may be higher in women who have very low prevaccine levels of antibody, particularly in assays measuring functional (neutralizing) antibodies.

CD4+ and CD8+ T-cell clones from congenital rubella syndrome patients with IDDM recognize overlapping GAD65 protein epitopes. Implications for HLA class I and II allelic linkage to disease susceptibility.

To fully characterize human glutamic acid decarboxylase (GAD)65 protein T-cell epitopes associated with insulin-dependent diabetes mellitus (IDDM), CTL clones specific to GAD65 protein antigens were isolated from two congenital rubella syndrome (CRS)-associated IDDM patients. Overlapping nonamer T-cell epitopes recognized by both CD4+ or CD8+ CTL clones within peptides GAD65(252-266) and GAD65(274-286) were identified as sequences bounded by GAD65(255-266) with 6/9 overlapping residues, and GAD65(276-285) with 8/9 overlapping residues, respectively, using two panels of overlapping peptide analogs in cytotoxicity assays. HLA restrictive elements of the T-cell clones were also identified using a panel of B cell lines with different HLA phenotypes as targets in cytotoxicity assays. The antigenic GAD65 peptides elicited cytotoxic responses of peptide-specific CD4+ T-cell clones in the context of HLA DRB1*0404. The CD8+ T-cell clone specific to GAD65(255-263) was found to be restricted by HLA A3 and A11. Similarly, the CD8+ T-cell clone specific to GAD65(277-285) killed peptide-sensitized target cells expressing HLA B35 and B15. The observed HLA restriction of these overlapping epitopes implies that a tandem of [DRB1*0404-A11(3)] and/or a tandem of [DRB1*0404-B35(15)] might predispose CRS patients to development of IDDM.

Identification of rubella virus T-cell epitopes recognized in anamnestic response to RA27/3 vaccine: associations with boost in neutralizing antibody titer.

Rubella virus (RV)-specific cell-mediated immunity (CMI) and antibodies were measured in healthy adolescents reimmunized with measles-mumps-rubella (MMR) vaccine. Lymphocyte proliferation to RV synthetic peptides was determined before and at 2, 4 and 10 weeks after, MMR. After MMR, increased CMI was observed with 16 peptides, including six containing antibody neutralization (NT) domains. Positive CMI (stimulation index > or =2.0) to E1(254-285) and C(1-29) before vaccination was significantly associated with a boost in NT titers, while positive CMI at weeks 2 or 4 to E1(254-285), E1(301-314), E1(389-408), E1(462-481), E2(134-150), E2(140-156), E2(168-179), C(1-29) and C(88-111) showed the same association.

Serologic responses to measles, mumps, and rubella (MMR) vaccine in healthy infants: failure to respond to measles and mumps components may influence decisions on timing of the second dose of MMR.

Measles, mumps, and rubella-specific IgG antibodies were evaluated in 134 healthy infants routinely immunized with trivalent live attenuated measles-mumps-rubella (MMR) vaccine at one year of age. Blood samples were collected just before, and at 1, 3, and 12 months after MMR. Specific IgG was measured by commercial enzyme immunoassays. Before vaccination, 98.5%, 99.2%, and 98.5% of the infants tested were seronegative for measles, mumps, and rubella, respectively. One year after MMR, 16.4% and 22.4% of vaccinees lacked demonstrable antibody to measles and mumps while none were found to be seronegative for rubella. Response profile analysis revealed primary failure rates of 12.1% (measles) and 8.6% (mumps) while 4% (measles) and 13.8% (mumps) of the infants responded initially but became seronegative within one year. These observations suggest that earlier administration (at age 18 months) of the second dose of MMR may be more desirable than revaccination at school entry.

A new categorization of HLA DR alleles on a functional basis.

In this analysis, we introduce a new categorization of HLA DR alleles which are important members of HLA class II genes encoding cell surface glycoproteins that function to present antigenic peptides to T cells. We have grouped all HLA DR molecules into seven different functional categories on the basis of their ability to bind and present antigenic peptides to T cells and their association with susceptibility or resistance to disease. This novel categorization of DR alleles on the basis of function allows for the prediction of seven similar subregion structures (supertypes or supermotifs) within pocket 4 of HLA DR peptide binding groove as the molecular basis for grouping these alleles. The physicochemical characteristics of HLA DR supertype residues, charge in particular, may influence the selectivity for binding peptide, dominate promiscuous T-cell recognition of antigenic peptides, and affect HLA DR disease associations. To rationalize the functional categories of DR alleles, we have further combined the seven DR supertype patterns into three groups based on the charges of residues within the supertypes. Grouping HLA DR alleles into functional categories may assist in understanding the mechanistic basis of autoimmunity, resolving current paradoxes in HLA disease associations, and developing new immunotherapy strategies.

Point mutation of a rubella virus E1 protein T-cell epitope by substitution of single amino acid reversed the restrictive HLA-DR polymorphism: a possible mechanism maintaining HLA polymorphism.

The influence of single amino acid substitutions within a rubella E1 protein T-cell epitope, E1(273-284) on T-cell recognition was studied. Substitutions of an uncharged amino acid A for an E or for a T and substitution of a T for S were found to not significantly reduce the T-cell responses. However, substitution of a charged residue such as E for hydrophobic residues (I, V, or W); D for Q; or a relatively larger size amino acid for polar residues completely abolished the cytotoxicities mediated by E1(273-284)-specific T-cell clone. A set of single amino acid-substituted peptide analogs of E1(273-284) not eliciting cytotoxicity of the T-cell clone was used to test the influence of point mutation of the epitope on HLA DR restrictions. A panel of B-cell lines with different DR4 subtypes was used as targets in cytotoxicity assays to determine the restrictive HLA molecules. Results showed that modification of the T-cell epitope by point mutation could reverse the HLA DR restriction from one allele to other alleles. A model based on these results has been proposed to explain the mechanism balancing major histocompatibility complex (MHC) polymorphism in outbred populations.

Promiscuous T-cell recognition of a rubella capsid protein epitope restricted by DRB1*0403 and DRB1*0901 molecules sharing an HLA DR supertype.

Two T cell clones derived from different donors with HLA-DRB1*0403 or DRB1*0901 phenotype recognize a rubella capsid peptide, C(265-273) in the context of several different HLA-DR molecules in addition to DRB1*0403 and DRB1*0901. All DR molecules restricting the T-cell clones have in common residues, R or Q at position beta 70, R at position beta 71, and E at position beta 74 in pocket '4' of the DR peptide binding groove, suggesting that a DR subregion structure or supertype, "Q/RRE" underlies the promiscuous T-cell recognition of this peptide. Single amino acid substituted analogs of peptide C(263-275) at anchor position 4 for natural residue R were tested for their ability to induce clonal T-cell cytotoxic responses. The results indicated that a positively charged residue, R or K, was required for T-cell recognition, suggesting a possible mechanism of electrostatic interactions between the negatively charged residue E at position beta 74 of these DR molecules and the positively charged residue at anchor position 4 of the peptide in T-cell recognition.

HLA-DR class II associations with rubella vaccine-induced joint manifestations.

HLA class II (HLA-DR) frequencies were examined in relation to incidence of acute arthralgia or arthritis in 283 white women who had received RA27/3 rubella vaccine (n = 146) or placebo (n = 137) postpartum. Leukocyte DNA was molecularly typed for HLA-DRB1 gene expression. Univariate analysis revealed higher frequencies of DR2 (odds ratio [OR], 4.8; 95% confidence interval [CI], 1.2-18.8) and DR5 (OR, 7.5; 95% CI, 1.5-37.5) but lower frequencies of DR4 (OR, 2.3; 95% CI, 1.1-4.9) and DR6 (OR, 2.8; 95% CI, 1.4-5.8), in rubella vaccinees compared with placebo recipients with arthropathy. Logistic regression modelling of DR, treatment, age, time postpartum, and arthropathy revealed that the odds of developing arthropathy was 1.9 times greater (95% CI, 1.07-3.44) after rubella vaccine than placebo. Risk for arthropathy (regardless of rubella vaccination) was also influenced by DR interactions: odds were 8 times greater in individuals with both DR1 and DR4 (95% CI, 1.45-44.02) and 7.1 times greater with both DR4 and DR6 present (95% CI, 1.85-27.52), suggesting that coexpression of these specificities may predispose to postpartum arthropathy.

Characterization of an overlapping CD8+ and CD4+ T-cell epitope on rubella capsid protein.

A synthetic peptide corresponding to rubella virus capsid protein residues 263 to 275 which contains an epitope recognized by a cloned CD4+ cytotoxic T-lymphocyte (CTL) line was used to induce CD8+ T-cell lines specific to this peptide. A peptide-specific CD8+ CTL clone was derived and characterized. This peptide-specific CD8+ CTL clone exhibited cytotoxicity against target cells infected by a vaccinia recombinant virus expressing rubella virus capsid protein, but not by target cells infected by vaccinia recombinant virus expressing rubella virus E1 or E2 envelope proteins. Analysis of HLA class I restriction of the CD8+ CTL clone revealed that A11 and A3 were restrictive elements. Fine mapping with truncated and overlapping peptide analogs revealed a nonamer sequence, C(264-272), as the T-cell epitope eliciting stronger cytotoxicity. Two anchor residues for binding to HLA A11 and A3 were identified at position 2 (isoleucine) and at position 9 (histidine) or at position 8 (arginine) of the epitope sequence. The identification of overlapping CD4+ and CD8+ T-cell epitopes within the capsid protein sequence C(263-275) implicates a strategy for using such epitopes in a candidate peptide-based rubella vaccine.

Randomised double-blind placebo-controlled study on adverse effects of rubella immunisation in seronegative women.

BACKGROUND: The objective of our study was to investigate the association of adverse clinical musculoskeletal and neurological events in healthy postpartum women with live attenuated (RA27/3 strain) rubella-virus vaccine, and to assess the frequency of acute and recurrent arthralgia and arthritis and associations with acute and recurrent muscle pain (myalgia) and neurological manifestations (paraesthesias). METHODS: We used a randomised placebo-controlled, double-blind design in a community setting. 636 women were enrolled and, after 90 women dropped out, 546 healthy women aged 18-41 years, who were rubella seronegative on routine screening were immunised parenterally with either monovalent live attenuated (RA27/3 strain) rubella vaccine (n = 270) or saline placebo (n = 276) in the postpartum period. Outcome measures were the occurrence of acute and persistent or recurrent joint manifestations (arthralgia or arthritis) at 1, 3, 6, 9, and 12 months after immunisation. Occurrence of muscle pain (myalgia), and neurological symptoms (paraesthesia) was also assessed at the same times. FINDINGS: 543 women completed 1-month follow-up. 456 women completed the 12-month assessment. There were no differences at the time of immunisation between rubella vaccine and placebo groups in distribution of age, ethnic origin, parity, time between delivery and immunisation, breastfeeding history, or histories of earlier rubella vaccination or joint complaints. Results indicated a significantly higher incidence (p = 0.006; odds ratio = 1.73 [95% CI = 1.17-2.57]) of acute joint manifestations in rubella-vaccine recipients (30%) than in placebo recipients (20%). Frequency of chronic (recurrent) arthralgia or arthritis was only marginally significant (p = 0.042; 1.58 [1.01-2.45]). INTERPRETATION: RA27/3 rubella vaccine given to seronegative women during the postpartum period was significantly associated with development of acute arthralgia or arthritis. Although the numbers of women assessed and length of follow-up revealed only marginally significant differences in persistent or recurrent joint manifestations between rubella vaccine and placebo recipients, it is possible that susceptible women who are given rubella vaccination may experience this outcome.

Recognition of contiguous allele-specific peptide elements in the rubella virus E1 envelope protein.

Peptides which bind to human HLA-DRB1 class II molecules in an allele-specific fashion were derived from the immunodominant E1 envelope protein of rubella virus. Two nonoverlapping E1 peptide epitopes were recognized by rubella virus-specific T cells in the context of independent HLA alleles when presented either separately or as a contiguous polypeptide containing both epitopes. Direct binding analysis of potential peptide epitopes to distinct HLA molecules provides a direct approach for selecting antigenic peptides useful for epitope-based vaccine targeted to multiple HLA types.

HLA-DR restrictive supertypes dominate promiscuous T cell recognition: association of multiple HLA-DR molecules with susceptibility to autoimmune diseases.

The association of individual autoimmune diseases with multiple HLA molecules has remained an enigma. That T cells can recognize the same peptide presented by several different HLA-DR alleles (i.e., promiscuous recognition) has been well documented. To explain this, we propose the "DR restrictive supertype pattern (DR RSP)" hypothesis. We focus on the known amino acid polymorphisms at positions beta 70, beta 71, and beta 74 located within pocket 4 of the DR molecule and their potential influence on promiscuous T cell recognition. We have shown that HLA-DR alleles may be grouped, on the basis of their polymorphisms at these positions, into at least 6 sets of DR alleles, 4 of which share, respectively, one of the RSP: A (Q/RR/KA), D (DE/R[K]A/L), E (Q/RRE), or R (QKR/Q) and 2 of which share the restrictive patterns Q (DRQ) or a (QAA). Most of the RSP have been shown to be associated with promiscuous T cell recognition of antigenic peptides. We also provide a rationale on how different DR alleles, exhibiting a particular RSP, might be capable of binding an antigenic peptide and presenting it, in a promiscuous fashion, to peptide-specific T cells. By identifying these RSP represented by DR alleles that have been clinically associated with certain autoimmune disease, we also extend the DR RSP hypothesis to account for the association of certain autoimmune diseases with multiple HLA-DR alleles.

Lymphocytes from site of disease indicate probable microbiological etiology of "infective-immune" diseases such as rheumatoid arthritis.

Synovial lymphocytes, from the site of disease, by their response to microbiological antigen stimulation as measured by the [3H]thymidine uptake method, indicate the microbiological causes of reactive arthritis and also oligoarthritis unassociated with enteric or genital symptoms. In the study of the etiology and pathogenesis of rheumatoid arthritis, the application of the same procedures gives an indication that the disease is an immune response to a variety of common infective agents, both viral and bacterial. The demonstration of antigens or nucleic acid of an infective agent at the site of disease, in association with a specific local immune response suggests the pathogenetic importance of the agent. Recent studies of relationships between epitopes of infective agents and MHC gene products suggest several ways in which infective agents can directly cause a disease such as rheumatoid arthritis without any requirement for autoimmune contributions. Because the infective agent may be the primary determining factor and the one most amenable to correction or eradication, the term "infective-immune" is suggested in preference to "autoimmune" for these immune-mediated diseases.

Characterization of the HLA-restrictive elements of a rubella virus-specific cytotoxic T cell clone: influence of HLA-DR4 beta chain residue 74 polymorphism on antigenic peptide-T cell interaction.

The influence of glutamic acid (E)-alanine (A) dimorphism at position 74 of the DR4 beta chain on cytotoxic T cell recognition of an antigenic rubella virus peptide, E1(273-284), was studied using a panel of B cell lines and B cell transfectants expressing different HLA-DRB1 alleles as antigen-presenting cells and targets in 51Cr-release assays. Only B cell lines expressing the DRB1*0403, DRB1*0406 or DRB1*0407 subtypes which shared a residue, E, at position 74 in the DR4 beta chain when sensitized with E1(273-284) elicited strong cytotoxic T lymphocyte responses. However, in direct binding and antibody inhibition assays, it was shown that biotinylated E1(272-285) could bind to DR molecules with residues other than E at position 74, including DRB1*0401, DRB1*0404 and DRB1*1101 expressed on transfectants. E1(272-285) bound with similar affinity to the transfectant with DRB1*0403, which has E at position 74, as well as the transfectant with DRB1*0404, which does not. When T-B cell engagement rates were compared in cell conjugate assays, the percentage of T-B conjugates was higher when peptide-pulsed transfectants with DRB1*0403 were used than with transfectants expressing DRB1*0404. Hence, the HLA DR beta 1 polymorphism at position 74, while not critical for the binding affinity of E1(272-285) to the HLA molecule, appears to be a primary determinant of restricted recognition and subsequent activation of the peptide-specific T cells.

Rubella reimmunization: comparative analysis of the immunoglobulin G response to rubella virus vaccine in previously seronegative and seropositive individuals.

Rubella virus (RV)-specific immunoglobulin G (IgG) antibodies were studied in military recruits undergoing unselected immunization with live attenuated measles, mumps, and rubella virus (MMR) vaccine. Three different whole-RV enzyme immunoassays (EIAs) and an epitope-specific EIA with a synthetic peptide (BCH-178c) representing a heutralization domain on the RV E1 envelope protein were used. Before vaccination, 84.2, 87.7, and 84.5% of the subjects tested (n = 399) were found to be seropositive (> 10 IU/ml or assay equivalent) by the three whole-RV EIAs, respectively, while only 82.5% were seropositive by the BCH-178c EIA. Although prevaccination seropositivity rates were similar for the whole-RV EIAs (sensitivity, 94 to 100%), many sera considered seropositive by the whole-RV EIAs had E1 peptide EIA antibody levels of < 10 IU/ml (sensitivity, 77.4 to 80.7%). One month after vaccination, 97.8, 97.2, and 93.5% of the subjects who were followed (n = 356) were seropositive by the three whole-RV EIAs, respectively, while 89% had BCH-178c peptide-specific IgG titers of > 10 IU/ml. After vaccination, depending on the assay used, up to 20.6% of initially seropositive individuals exhibited a greater than fourfold increase in RV-specific IgG, while up to 47.3% showed a greater than twofold increase. Increased antibody titers after vaccination (seroboosting) were most frequently associated with low levels of BCH-178c peptide-specific IgG before vaccination. RV protein-specific IgG was also studied by immunoblot assays in a subset (n = 56) of individuals receiving the MMR vaccine. Of these, 89.4 and 91.1% exhibited RV protein (E1, E2, and C protein)-specific IgG before and after vaccination, respectively. Seroboosting (two- to fourfold increase in EIA titers of individuals seropositive by the whole-RV EIA before vaccination) was usually accompanied by a shift in the IgG immunoblot pattern from a single (E1) to multiple (E1-E1, E1-C, or E1-E2-C) specificities, suggesting exposure of new epitopes as a result of viral replication.

Recognition of altered self major histocompatibility complex molecules modulated by specific peptide interactions.

Antigen-specific and major histocompatibility complex (MHC)-restricted recognition by the T cell receptor involves multiple structural contacts over a large molecular surface area. Using a human T cell clone specific for a rubella viral peptide restricted by subsets of HLA DR4 molecules, we identified structurally diverse combinations of peptide-MHC complexes which were functionally equivalent to T cell recognition. Presentation of the rubella-derived peptide on DR4 molecules with an E-74 polymorphism triggered T cell recognition, as did presentation of a single amino acid-substituted peptide in the context of DR4 molecule which lacked the E-74 site. Peptide binding and molecular modeling analysis indicates the structural and functional complementarity of T cell recognition for a specific amino acid side chain, whether contributed by the peptide or by the MHC molecule.

Use of synthetic peptides to map regions of rubella virus capsid protein recognized by human T lymphocytes.

Synthetic peptides (SPs), 18-29 amino acids long, representing selected sequences of rubella virus (RV) capsid (C) protein were used in lymphocyte proliferation assays to identify antigenic regions recognized by T lymphocytes from healthy RV-reactive adults. Four SPs, C(1-29), C(90-114), C(108-134) and C(255-300), stimulated proliferation of peripheral blood mononuclear cells and RV-specific T-cell lines from the same donors. C(1-29V), an SP analogue containing an RA27/3 RV vaccine strain sequence, stimulated higher levels of proliferation in T cells obtained from RV-vaccinated subjects than did the comparable wild-type (M33 strain) RV sequence.