Mapping T‐cell epitopes of rubella virus structural proteins E1, E2, and C recognized by T‐cell lines and clones derived from infected and immunized populations

Abstract

To design a safe and effective synthetic peptide vaccine against rubella virus (RV) infection, it is necessary to identify immunodominant T-cell epitopes of RV structural proteins. To define such epitopes, 49 overlapping synthetic peptides (17-34 residues in length) corresponding to more than 95% of the amino acid sequence of RV virion proteins E1 (23 peptides) and C (11 peptides) and all of E2 (15 peptides) were synthesized and tested for their capacities to induce proliferative responses of rubella-specific T-cell lines and T-cell clones derived from 4 study groups (5 women infected with RV in pregnancy, 5 patients with congenital rubella syndrome, 5 seropositive healthy donors, and 5 RV vaccine recipients). The most frequently recognized epitopes were E1-21 (residues 358-377) with 11/20 responders, E2-4 (residues 54-74) with 6/20 responders, and C11 (residues 255-280) with 11/20 responders, respectively. E1-10 (residues 174-193), E1-16 (residues 272-291) and E1-18 (residues 307-326) were responded to strongly by corresponding T-cell clones, and were recognized by 4 or 5 T-cell lines. T-cell lines derived from three congenital rubella syndrome patients did not respond to any of the synthetic peptides. The results showed that more T-cell epitopes were present in E1 (19/23) and C (10/11) than in E2 (8/15). The identification of T cell sites recognized frequently by RV-infected or -immunized populations could provide the basis for selecting candidate T-cell epitopes for the development of an effective synthetic vaccine against rubella.

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

“…Peptides V1, V4, V5, V8, V11, V13, V14, V15 are antigenic peptides containing epitopes identified by T-cells from RV seropositive healthy donors or RV vaccinees or both in our previous studies [32,33,43,44]. RV peptides V2, V3, V6, V7, V9, V10, V12 were shown earlier to be recognised by peripheral blood T-cells of CRS patients [31] and included five (V3, V6, V7, V9, V12) which stimulated cellular responses of a CRS patient who also had Type I diabetes. Positive cellular responses to whole RV virus were shown in 77 % of patients with recent onset diabetes and 84 % of patients with late onset Type I diabetes and all 3 patients with CRS-associated Type I diabetes.…”

“…All four GAD peptide-specific T-cell clones also responded with two or more rubella peptides suggesting that cross-reaction of viral antigen with GAD antigens could play a part in the pathogenesis of Type I diabetes triggered by RV infection. Peptide V3, RVE1(157±176) was first found to induce cellular responses in a patient with CRS-associated Type I diabetes and only 10 % RV seropositive healthy people and vaccinees in our previous study [31] and hence was included in the RV peptide panel for this study. Cross-reactive RV determinants such as V3, RVE1(157±176) were found to induce a higher frequency of cellular response in the cohort of patients with Type I diabetes than two CXV peptides which shared 80 % homology to GAD65 peptides, suggesting that these RV determinants could be more important in the pathogenesis of Type I diabetes than those of CXV.…”

“…A panel of peptides of human beta-cell proteins of GAD65, GAD67 and 38 KDa 31G (positions shown in Table 1) and a panel of peptides of viral proteins [rubella virus E1, E2 and C and Coxsackie B4 virus P2-C (positions shown in Table 2 and Table 3)] were synthesised in an automated ABI 430 A peptide synthesiser (Syndrgy, Foster City, Calif., USA) using standard solid-phase methods [31]. The identity and the purity of the peptides had been analysed by mass spectrometry.…”

“…For the proliferation assays using peripheral blood mononuclear cells (PBMC) as responder cells, 1 10 5 cells per well were incubated with peptide at a concentration of 10 mmol/l, or with heat-inactivated whole RV virus at 1 10 6 pfu/ml or with phytohaemagglutinin (PHA) at a final concentration of 10 mg/ml as stimulation (positive) controls in complete Opti-MEM I medium (Gibco BRL, Missisauga, Ont., Canada) containing 2 mmol/l l-glutamine, 1 mmol/l sodium pyruvate, 25 mmol/l HEPES, 50 mmol/l penicillin, 50 mmol/l streptomycin and 5 10 ±5 mol/l 2-mercaptoethanol supplemented with 10 % autologous plasma for 7 days as described previously [31,32]. Negative control (background) wells received only complete medium supplemented with autologous plasma.…”

“…Negative control (background) wells received only complete medium supplemented with autologous plasma. For the assays using cloned T-cells as responder cells, a total of 1 10 4 cells per well were incubated with autologous, gamma-irradiated (2500 rad) PBMC (5 10 4 cells per well) in complete RPMI 1640 medium containing 2 mmol/l l-glutamine, 25 mmol/l HEPES, 50 mmol/l penicillin, 50 mmol/l streptomycin, and 5 10 ±5 mol/l 2-mercaptoethanol, 10 % FCS and peptide at a concentration of 10 mmol/l in 96-well flat-bottom plates for 3 days as described previously [31,32]. The proliferation assays with the beta-cell peptides, viral antigens and PHA as stimulators including negative controls were carried out on the same assay plate for comparison.…”