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

To design a safe and effective synthetic peptide vaccine against rubell 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 951% 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. © 1993 Wiley-Liss, Inc.     

Analysis of human T-cell epitopes in the 19,000 MW antigen of Mycobacterium tuberculosis: influence of HLA-DR.

The potential number of T-cell epitopes in the 19,000 molecular weight (MW) antigen has been investigated using overlapping peptides which comprise the complete sequence. Sixteen potential epitopes could be deduced from the responses to these peptides by polyclonal T cells derived from 22 antigen-responsive donors. The majority of epitopes were not predicted by either of the major paradigms, the Rothbard motif and the amphipathic helix. A hierarchy of epitopes was indicated by the responses, which ranged from strong and frequent in the N-terminal region, to moderate or weak elsewhere. Some epitopes were restricted by single HLA-DR determinants, or families of determinants sharing structural features in common, whilst the two N-terminal peptides were recognized by donors with a diversity of DR types. The high degree of T-cell recognition of the N-terminal region may be of relevance to the design of a sub-unit vaccine capable of priming T cells against Mycobacterium tuberculosis.     

Identification of Promiscuous Epitopes from the Mycobacterial 65-Kilodalton Heat Shock Protein Recognized by Human CD4+ T Cells of the Mycobacterium leprae Memory Repertoire

By using a synthetic peptide approach, we mapped epitopes from the mycobacterial 65-kDa heat shock protein (HSP65) recognized by human T cells belonging to the Mycobacterium leprae memory repertoire. A panel of HSP65 reactive CD4(+) T-cell lines and clones were established from healthy donors 8 years after immunization with heat-killed M. leprae and then tested for proliferative reactivity against overlapping peptides comprising both the M. leprae and Mycobacterium tuberculosis HSP65 sequences. The results showed that the antigen-specific T-cell lines and clones established responded to 12 mycobacterial HSP65 peptides, of which 9 peptides represented epitopes crossreactive between the M. tuberculosis and M. leprae HSP65 (amino acids [aa] 61 to 75, 141 to 155, 151 to 165, 331 to 345, 371 to 385, 411 to 425, 431 to 445, 441 to 455, and 501 to 515) and 3 peptides (aa 343 to 355, 417 to 429, and 522 to 534) represented M. leprae HSP65-specific epitopes. Major histocompatibility complex restriction analysis showed that presentation of 9 of the 12 peptides to T cells were restricted by one of the 2 HLA-DR molecules expressed from self HLA-DRB1 genes, whereas 3 peptides with sequences completely identical between the M. leprae and M. tuberculosis HSP65 were presented to T cells by multiple HLA-DR molecules: peptide (aa 61 to 75) was presented by HLA-DR1, -DR2, and -DR7, peptide (aa 141 to 155) was presented by HLA-DR2, -DR7, and -DR53, whereas both HLA-DR2 and -DR4 (Dw4 and Dw14) were able to present peptide (aa 501 to 515) to T cells. In addition, the T-cell lines responding to these peptides in proliferation assays showed cytotoxic activity against autologous monocytes/macrophages pulsed with the same HSP65 peptides. In conclusion, we demonstrated that promiscuous peptide epitopes from the mycobacterial HSP65 antigen can serve as targets for cytotoxic CD4(+) T cells which belong to the human memory T-cell repertoire against M. leprae. The results suggest that such epitopes might be used in the peptide-based design of subunit vaccines against mycobacterial diseases.     

Epitopes recognized by CBV4 responding T cells: effect of type 1 diabetes and associated HLA-DR-DQ haplotypes

The present study aimed at characterizing the epitopes recognized by coxsackievirus B4 (CBV4)-specific T-cell lines established from 23 children with type 1 diabetes (T1D) and 29 healthy children with T1D risk-associated HLA genotypes. Responsiveness to VP1 region was dependent on the specific infection history as 55% of the T-cell lines from donors with neutralizing antibodies to CBV serotypes responded to VP1 peptides compared to none of the T-cell lines from other donors (P = 0.01). The pattern of recognized peptides was dependent of the HLA genotype. Forty-two percent of the T-cell lines from donors carrying the HLA-(DR4)-DQB1*0302 haplotype responded to VP1 peptides 71-80 compared to none of the T-cell lines from donors without this haplotype (P = 0.02). No evidence for the existence of diabetes-specific epitopes was found. Only few epitopes were exclusive recognized by T cells from diabetic children, and in each case only one or two T-cell lines were responding.     

Identification of T-cell determinants in natural immune responses to the Plasmodium falciparum apical membrane antigen (AMA-1) in an adult population exposed to malaria.

AMA-1 of Plasmodium falciparum is a promising candidate antigen in malaria vaccine development. In this study, we have mapped the immunodominant T-cell determinants in this antigen by using synthetic peptides. From the amphipathic scores, 17 putative T-cell determinants were identified. Nine of the 17 peptides complementary to the putative T-cell determinants induced proliferation of peripheral blood mononuclear cells (PBMC) from Kenyan residents who had lifelong exposure to malaria; none of these peptides induced proliferation of PBMC from donors who were not previously exposed to malaria. This indicates that AMA-1 peptides were stimulating T cells that were previously primed by prior exposure to P. falciparum. Many positive responders showed reactivity to more than one peptide, and some of the potent proliferative T epitopes were found to be localized in the highly conserved regions of AMA-1, suggesting that it may be possible to induce T-cell memory that can recognize different variant forms of the parasite. This information on the natural immune responses against the AMA-1 vaccine antigen in clinically immune adults will be helpful in the development of an AMA-1 antigen-based malaria vaccine and may also guide testing of AMA-1-based vaccine formulations.     

Conservation of Babesia bovis small heat shock protein (Hsp20) among strains and definition of T helper cell epitopes recognized by cattle with diverse major histocompatibility complex class II haplotypes

Babesia bovis small heat shock protein (Hsp20) is recognized by CD4+ T lymphocytes from cattle that have recovered from infection and are immune to challenge. This candidate vaccine antigen is related to a protective antigen of Toxoplasma gondii, Hsp30/bag1, and both are members of the alpha-crystallin family of proteins that can serve as molecular chaperones. In the present study, immunofluorescence microscopy determined that Hsp20 is expressed intracellularly in all merozoites. Importantly, Hsp20 is also expressed by tick larval stages, including sporozoites, so that natural tick-transmitted infection could boost a vaccine-induced response. The predicted amino acid sequence of Hsp20 from merozoites is completely conserved among different B. bovis strains. To define the location of CD4+ T-cell epitopes for inclusion in a multiepitope peptide or minigene vaccine construct, truncated recombinant Hsp20 proteins and overlapping peptides were tested for their ability to stimulate T cells from immune cattle. Both amino-terminal (amino acids [aa] 1 to 105) and carboxy-terminal (aa 48 to 177) regions were immunogenic for the majority of cattle in the study, stimulating strong proliferation and IFN-gamma production. T-cell lines from all individuals with distinct DRB3 haplotypes responded to aa 11 to 62 of Hsp20, which contained one or more immunodominant epitopes for each animal. One epitope, DEQTGLPIKS (aa 17 to 26), was identified by T-cell clones. The presence of strain-conserved T helper cell epitopes in aa 11 to 62 of the ubiquitously expressed Hsp20 that are presented by major histocompatibility complex class II molecules represented broadly in the Holstein breed supports the inclusion of this region in vaccine constructs to be tested in cattle.     

Characterization of conserved T- and B-cell epitopes in Plasmodium falciparum major merozoite surface protein 1

Vaccines for P. falciparum will need to contain both T- and B-cell epitopes. Conserved epitopes are the most desirable, but they are often poorly immunogenic. The major merozoite surface protein 1 (MSP-1) is currently a leading vaccine candidate antigen. In this study, six peptides from conserved or partly conserved regions of MSP-1 were evaluated for immunogenicity in B10 congenic mice. Following immunization with the peptides, murine T cells were tested for the ability to proliferate in vitro and antibody responses to MSP-1 were evaluated in vivo. The results showed that one highly conserved sequence (MSP-1#1, VTHESYQELVKKLEALEDAV; located at amino acid positions 20 to 39) and one partly conserved sequence (MSP-1#23, GLFHKEKMILNEEEITTKGA; located at positions 44 to 63) contained both T- and B-cell epitopes. Immunization of mice with these peptides resulted in T-cell proliferation and enhanced production of antibody to MSP-1 upon exposure to merozoites. MSP-1#1 stimulated T-cell responses in three of the six strains of mice evaluated, whereas MSP-1#23 was immunogenic in only one strain. Immunization with the other four peptides resulted in T-cell responses to the peptides, but none of the resulting peptide-specific T cells recognized native MSP-1. These results demonstrate that two sequences located in the N terminus of MSP-1 can induce T- and B-cell responses following immunization in a murine model. Clearly, these sequences merit further consideration for inclusion in a vaccine for malaria.     

Prediction of HLA class I-restricted T-cell epitopes of islet autoantigen combined with binding and dissociation assays

Identification of cognate peptides recognized by human leucocyte antigen (HLA)/T cell receptor (TCR) complex provides insight into the pathogenic process of type 1 diabetes (T1D). We hypothesize that HLA-binding assays alone are inadequate metrics for the affinity of peptides. Zinc transporter-8 (ZnT8) has emerged in recent years as a novel, major, human autoantigen. Therefore, we aim to identify the HLA-A2-restricted ZnT8 epitopes using both binding and dissociation assays. HLA class I peptide affinity algorithms were used to predict candidate ZnT8 peptides that bind to HLA-A2. We analyzed 15 reported epitopes of seven β-cell candidate autoantigens and eight predicted candidate ZnT8 peptides using binding and dissociation assays. Using IFN-γ ELISpot assay, we tested peripheral blood mononuclear cells (PBMCs) from recent-onset T1D patients and healthy controls for reactivity to seven reported epitopes and eight candidate ZnT8 peptides directly ex vivo. We found five of seven recently reported epitopes in Chinese T1D patients. Of the eight predicted ZnT8 peptides, ZnT8(153-161) had a strong binding affinity and the lowest dissociation rate to HLA-A*0201. We identified it as a novel HLA-A*0201-restricted T-cell epitope in three of eight T1D patients. We conclude that ZnT8(153-161) is a novel HLA-A*0201-restricted T-cell epitope. We did not observe a significant correlation (P = 0.3, R = - 0.5) between cytotoxic T cell (CTL) response and peptide/HLA*0201 complex stability. However, selection of peptides based on affinity and their dissociation rate may be helpful for the identification of candidate CTL epitopes. Thus, we can minimize the number of experiments for the identification of T-cell epitopes from interesting antigens.     

Identification and HLA restriction of naturally derived Th1-cell epitopes from the secreted Mycobacterium tuberculosis antigen 85B recognized by antigen-specific human CD4(+) T-cell lines

Antigen 85B (Ag85B/MPT59) is a major secreted protein from Mycobacterium tuberculosis which is a promising candidate antigen for inclusion in novel subunit vaccines against tuberculosis (TB). The present study was undertaken to map naturally derived T-cell epitopes from M. tuberculosis Ag85B in relation to major histocompatibility complex (MHC) class II restriction. Antigen-specific CD4(+) T-cell lines were established from HLA-typed TB patients and Mycobacterium bovis BCG vaccinees by stimulation of peripheral blood mononuclear cells with purified Ag85B in vitro. The established T-cell lines were then tested for proliferation and gamma interferon (IFN-gamma) secretion in response to 31 overlapping synthetic peptides (18-mers) covering the entire sequence of the mature protein. The results showed that the epitopes recognized by T-cell lines from TB patients were scattered throughout the Ag85B sequence whereas the epitopes recognized by T-cell lines from BCG vaccinees were located toward the N-terminal part of the antigen. The T-cell epitopes represented by peptides p2 (amino acids [aa] 10 to 27), p3 (aa 19 to 36), and p11 (aa 91 to 108) were frequently recognized by antigen-specific T-cell lines from BCG vaccinees in both proliferation and IFN-gamma assays. MHC restriction analysis demonstrated that individual T-cell lines specifically recognized the complete Ag85B either in association with one of the self HLA-DRB1, DRB3, or DRB4 gene products or nonspecifically in a promiscuous manner. At the epitope level, panel studies showed that peptides p2, p3, and p11 were presented to T cells by HLA-DR-matched as well as mismatched allogeneic antigen-presenting cells, thus representing promiscuous epitopes. The identification of naturally derived peptide epitopes from the M. tuberculosis Ag85B presented to Th1 cells in the context of multiple HLA-DR molecules strongly supports the relevance of this antigen to subunit vaccine design.     

Genome-wide screening of human T-cell epitopes in influenza A virus reveals a broad spectrum of CD4 T-cell responses to internal proteins, hemagglutinins, and neuraminidases

We performed a genome-wide screening for T-cell epitopes using synthetic peptides that encompass all of the influenza A viral proteins, including subtype variants for hemagglutinin (HA; H1, H3, and H5) and neuraminidase (NA; human and avian N1 and N2) proteins, based on the sequence information of recently circulating strains. We identified a total of 83 peptides, 54 of them novel, to which specific T cells were detectable in interferon-γ (IFN-γ) enzyme-linked immunosorbent spot assays using peripheral blood mononuclear cells from four healthy adult donors. The surface glycoproteins, HA and NA, major components of vaccines, expressed many T-cell epitopes. HA and matrix protein 1 expressed more T-cell epitopes than other viral proteins, most of which were recognized by CD4⁺ T cells. We established several cytotoxic CD4⁺ T-cell lines from these donors. We also analyzed H1 and H3 HA-specific T-cell responses using the peripheral blood mononuclear cells of 30 hospital workers. Fifty-three percent of donors gave a positive response to H3 HA peptides, whereas 17% gave a positive response to H1 HA peptides. Our genome-wide screening is useful in identifying T-cell epitopes and is complementary to the approach based on the predicted binding peptides to well-studied HLA-A, -B, and -DR alleles.     

Human T cells recognize multiple epitopes of an immediate early/tegument protein (IE62) and glycoprotein I of varicella zoster virus.

Infection with varicella zoster virus (VZV) elicits persistent cell-mediated immunity directed against the immediate early (IE62) protein and the glycoprotein I (gp I) in most healthy subjects. In these experiments, synthetic peptides corresponding to residues of the IE62 protein and gp I were used to identify linear amino acid sequences of these immunogenic VZV proteins that were recognized by peripheral blood T lymphocytes from VZV-immune individuals of known major histocompatibility complex (MHC) type. All of 12 VZV-immune donors had T-cell proliferative responses, defined as a stimulation index (SI) greater than or equal to 2.0, to at least two of ten synthetic IE62 peptides; the mean number of IE62 peptides recognized by T cells from VZV-immune donors was seven. Five of the ten IE62 peptides stimulated T cells from 75% to 83% of the VZV-immune donors; the other five IE62 peptides were recognized by T cells from 42% to 67% of the subjects. All VZV-immune donors also had T proliferation responses to at least two of ten synthetic gp I peptides; the mean number of peptides recognized was six. Six of the ten gp I peptides were recognized by T cells from 67% to 92% of the VZV-immune donors; the frequency of donors responding to the other gp I peptides ranged from 42% to 58%. None of five nonimmune donors demonstrated T-cell proliferation to any of the IE62 or gp I peptides. A combination of two IE62 peptides provided epitopes that could be recognized by T cells from all twelve VZV-immune donors, regardless of DR type. Similarly, one gp I peptide in combination with either of two other gp I peptides induced proliferation of T cells from all immune subjects. Memory T cells with specificity for multiple short amino acid sequences of the IE62 protein and gp I were detected in subjects who had had primary VZV infection more than 20 years earlier. These observations indicate that natural VZV infection elicits a diverse cell-mediated immune response to viral proteins that is not restricted to only one or two immunodominant regions. Although the usefulness of peptide vaccines remains to be established, multiple epitopes of the IE62 protein and gp I were identified that could be presented by antigen-presenting cells (APC) and recognized by T cells from most subjects in an "outbred" human population.     

Primary human T-cell responses to the major outer membrane protein of Chlamydia trachomatis.

The major outer membrane protein (MOMP) of Chlamydia trachomatis is the main candidate antigen for a synthetic vaccine against chlamydial infection. Antibodies to surface-exposed epitopes on MOMP neutralize chlamydial infectivity but little is known about T-cell recognition of the molecule. We have measured primary human T-cell responses to recombinant fragments of MOMP as well as to the whole organism and synthetic MOMP peptides. Using antigen-pulsed low density cells (LDC) we were able to stimulate proliferative responses with T cells from most naive individuals. This response was antigen dose dependent and displayed an absolute requirement for dendritic cells in the antigen-presenting cell (APC) population. Several T-cell epitopes were identified in MOMP and one which stimulated T cells from 80% of donors was resolved as a 12 amino acid synthetic peptide. Dual cell surface labelling and cell cycle analysis by FACS revealed that both CD4+ and CD8+ T cells were stimulated in these cultures. The fact that we were able to obtain proliferative responses and interferon-gamma (IFN-gamma) production to MOMP using cells from cord bloods confirmed that these are genuine primary responses. These experiments have identified a region on MOMP, to which T cells from most humans make a primary response, which may be useful in a chlamydial vaccine. The approach is useful for vaccine development in general.     

Human Th1 cell lines recognize the Mycobacterium tuberculosis ESAT-6 antigen and its peptides in association with frequently expressed HLA class II molecules

We have used a synthetic-peptide approach to map epitope regions of the Mycobacterium tuberculosis ESAT-6 antigen recognized by human T cells in relation to major histocompatibility complex (MHC) restriction. ESAT-6-specific CD4+ T-cell lines were established by stimulating peripheral blood mononuclear cells from 25 HLA-DR-typed tuberculosis patients with complete antigen in vitro. The established T-cell lines were then screened for proliferation and interferon-gamma (IFN-gamma) secretion in response to eight overlapping 20-mer peptides covering the ESAT-6 sequence. The response of the T-cell lines to ESAT-6 and peptides from a human leucocyte antigen (HLA)-heterogeneous group of donors suggested the presence of multiple epitopes and promiscuous recognition of the antigen. Analysis of antigen and peptide recognition in the presence of anti-HLA class I and class II antibodies suggested that the T-cell lines recognized ESAT-6 in association with HLA-DR and -DQ molecules. Furthermore, testing of selected T-cell lines with ESAT-6 and the peptides in the presence of autologous and allogeneic HLA-DR- and -DQ-typed antigen-presenting cells identified HLA-DR2, -DR52 and -DQ2 amongst the HLA molecules involved in the presentation of ESAT-6 and its peptides to human Th1 cells. In addition, the T-cell lines were cytotoxic for monocytes and macrophages pulsed with ESAT-6 and peptides. In conclusion, the recognition of ESAT-6 by IFN-gamma-secreting and cytotoxic CD4+ T cells in association with frequently expressed HLA class II molecules supports the application of this antigen to either specific diagnosis or subunit vaccine design.     

Prediction of HLA class I-restricted T-cell epitopes of islet autoantigen combined with binding and dissociation assays

Identification of cognate peptides recognized by human leucocyte antigen (HLA)/T cell receptor (TCR) complex provides insight into the pathogenic process of type 1 diabetes (T1D). We hypothesize that HLA-binding assays alone are inadequate metrics for the affinity of peptides. Zinc transporter-8 (ZnT8) has emerged in recent years as a novel, major, human autoantigen. Therefore, we aim to identify the HLA-A2-restricted ZnT8 epitopes using both binding and dissociation assays. HLA class I peptide affinity algorithms were used to predict candidate ZnT8 peptides that bind to HLA-A2. We analyzed 15 reported epitopes of seven β-cell candidate autoantigens and eight predicted candidate ZnT8 peptides using binding and dissociation assays. Using IFN-γ ELISpot assay, we tested peripheral blood mononuclear cells (PBMCs) from recent-onset T1D patients and healthy controls for reactivity to seven reported epitopes and eight candidate ZnT8 peptides directly ex vivo. We found five of seven recently reported epitopes in Chinese T1D patients. Of the eight predicted ZnT8 peptides, ZnT8_153–161 had a strong binding affinity and the lowest dissociation rate to HLA-A*0201. We identified it as a novel HLA-A*0201-restricted T-cell epitope in three of eight T1D patients. We conclude that ZnT8_153–161 is a novel HLA-A*0201-restricted T-cell epitope. We did not observe a significant correlation (P = 0.3, R = ? 0.5) between cytotoxic T cell (CTL) response and peptide/HLA*0201 complex stability. However, selection of peptides based on affinity and their dissociation rate may be helpful for the identification of candidate CTL epitopes. Thus, we can minimize the number of experiments for the identification of T-cell epitopes from interesting antigens.     

Epitope analysis of HLA-DR-restricted helper T-cell responses to Der p II, a major allergen molecule of Dermatophagoides pteronyssinus

T-cell epitopes of Der p II, a major allergen of Dermatophagoides pteronyssinus , were analyzed by using human T-cell clones. We tested 38 cloned T cells from two Japanese patients with allergic rhinitis, and identified at least two peptides (K33-T47 and 158-C73) as helper T-cell epitopes. The former epitope was shown to be restricted by HLA-DRB1* 1502, and the latter by HLA-DRB1* 0405, both of which are typical Japanese HLA-DR alleles, suggesting that those T-cell epitopes might be important for the onset of house-dust mite allergy in the Japanese population. We prepared 15 analog peptides of the HLA-DRB1* 1502-restricted 15-mer peptide. Of those 15 residues, five (F35, L37, A39, F41, and E42) were critical for the epitope activity, and three residues (F35, A39, and E42) seemed to be included in anchor motifs for HLA-DRB1* 1502. The epitope peptide was also recognized by HLA-DRB1* 1502-positive healthy donors; however, only allergic T cells showed Th2 functions. Antigen-presenting cells of nonallergic donors were able to activate allergic T cells to express Th2 function. This seemed to suggest that antigen recognition of T cells, as well as additional unknown factors which promote Th2, rather than Th1, responses, might be important for the onset of house-dust mite allergy.     

弓形虫ROP2基因的克隆及在大肠杆菌中的表达

目的 :构建弓形虫棒状体蛋白 (ROP2 )基因重组质粒并在大肠杆菌中表达 ,用于筛选弓形虫新的诊断抗原和疫苗分子。方法 :根据ROP2基因已知序列 ,设计合成一对引物 ,用PCR方法从弓形虫RH株基因组DNA中扩增出ROP2基因片段 ,再克隆到pGEX 4T 1质粒 ,重组质粒经酶切及PCR鉴定 ,而后进行测序 ;重组质粒在大肠杆菌BL2 1 (DE3)中进行ITPG诱导表达 ,表达产物经SDS PAGE及WesternBlot分析。结果 :ROP2基因PCR产物大小与预期相符 ,约 1 0 4 3bp ,重组质粒经酶切及PCR鉴定表明获得正确重组子 ,测序结果与已知序列基本吻合 ;SDS PAGE及免疫印迹显示ROP2融合蛋白表观分子量约为 5 5kDa ,表达产物约占菌体总蛋白1 7% ,具有一定的免疫反应性。结论 :克隆并表达了弓形虫ROP2基因 ,为下一步弓形虫诊断及疫苗研究奠定了基础     

Immunoprevalence of the CD4+ T-cell response to HIV Tat and Vpr proteins is provided by clustered and disperse epitopes, respectively

Recent studies have suggested including nonstructural proteins as Tat and Vpr in HIV vaccines. However, little is known about the CD4+ T-cell response that these small proteins induce in humans. We have therefore evaluated these responses by in vitro priming experiments of CD4+ T lymphocytes harvested in healthy donors. In the Tat protein, only one peptide primed CD4+ T cells of eight HLA unrelated healthy donors. T cells induced by this peptide recognized immature DC loaded with the native Tat protein and are restricted by multiple HLA-DR molecules, in agreement with its binding capacity. This peptide was therefore processed in an appropriate manner and was highly immunoprevalent. CD4+ T-cell response to Vpr peptides was more disperse and involved six different peptides depending on the HLA-DR molecules of the donors. Two overlapping peptides were T-cell stimulating in at least half of the donors. T-cell response to Vpr in multiple donors is the result of a combination of several CD4+ T-cell epitopes with good to moderate immunoprevalence. Altogether, our results show that the frequency of responders to HIV Tat or Vpr proteins relies on one or multiple CD4+ T-cell epitopes, respectively.     

T cells from multiple sclerosis patients recognize multiple epitopes on Self-IgG

The highly diversified variable regions of immunoglobulin (Ig) molecules contain immunogenic determinants denoted idiotopes. We have previously reported that T cells from multiple sclerosis (MS) patients recognize IgG from autologous cerebrospinal fluid (CSF), and mapped a T-cell epitope to an IgG idiotope. To test the ability of CSF IgG molecules to elicit a broad polyclonal T-cell response in MS, we have analysed T-cell responses in the blood and CSF against idiotope peptides spanning complementarity determining region (CDR) 3 and somatic mutations within the variable regions of monoclonal CSF IgG. Consistent with a diversified idiotope-specific T-cell repertoire, CD4(+) T cells from both patients recognized several idiotope peptides presented by HLA-DR molecules. Mutations were critical for T-cell recognition, as T cells specific for a mutated CDR1 peptide did not recognize corresponding germline-encoded peptides. One T-cell clone recognized both an idiotope peptide and the B-cell clone expressing this idiotope, compatible with endogenous processing and presentation of this idiotope by B cells. These results suggest that mutated CSF IgG from MS patients carry several T-cell epitopes, which could mediate intrathecal IgG production and inflammation in MS through idiotope-driven T-B-cell collaboration.     

Identification and characterization of host-protective T-cell epitopes of a major surface glycoprotein (gp63) from Leishmania major

By using a series of overlapping synthetic peptides that cover more than 75% of the amino acid sequence of the major surface glycoprotein (gp63) from Leishmania major, 11 T-cell epitopes in CBA and BALB/c mice have been identified. Six of the peptides were recognized by T cells of CBA mice recovered from L. major infection, while one was recognized by the T cells from BALB/c mice recovered from the infection following sublethal doses of gamma-irradiation. Lymph node cells from mice immunized with the peptides also responded to a number of the same peptides (seven in CBA and one in BALB/c). Peptide p10-28 induced proliferative T-cell responses in both CBA and BALB/c mice. Five of the peptides (p10-28, p22-40, p289-309, p459-471 and p467-482) induced vigorous T-cell response in CBA mice but were not recognized by T cells from recovered mice. Four other peptides (p321-336, p364-476, p372-385 and p378-396) were recognized by T cells from recovered CBA mice but could not induce a T-cell response in normal CBA mice. Three peptides (p146-171, p289-309 and p395-414) were both able to induce a T-cell response and were recognized by T cells from recovered mice. However, only two peptides (p146-171 and p467-482) were able to activate T cells, which also recognized epitopes expressed by antigen-presenting cells infected with promastigotes. T cells induced by p146-171 and p467-171 or a mixture of these two peptides were mainly CD4+ and produced interleukin (IL-2) and interferon-gamma (IFN-gamma) but not IL-4 upon antigen stimulation in vitro. These two peptides also induced a classical delayed type hypersensitivity (DTH) response in CBA mice. Furthermore, CBA mice immunized with a mixture of the two peptides in Coryne parvum or entrapped in liposomes induced significant resistance against L. major infection. The implications of these results in terms of a synthetic vaccine against leishmaniasis and the mechanism of the induction of Th1 and Th2 cells are discussed.     

Development of a Malaria T-Cell Vaccine for Blood Stage Immunity

We have defined a strategy for the development of a T-cell vaccine for blood stage immunity, taking into consideration the central role of T cells and MHC restriction in malaria immune responses. We have used the AMPHI computer algorithm lo identify putative T-cell epitopes from conserved regions of 11 Plasmodium falciparum asexual stage proteins. Ten of the eleven proteins arc currently candidates for vaccine development. Using this algorithm we selected 22 putative T-cell epitope peptides und 8 control peptides. These peptides were used to test the T-cell responses of three defined populations of Caucasians who have (1) recovered from P. falciparum malaria. (2) been exposed, but never clinically infected, (3) never been exposed or infected. Preliminary analysis of our data shows population differences in T-tell responses to putative T-cell epitope peptides. Ultimately, these studies will help to identify those T epitopes that can be incorporated into a T-cell vaccine for protective immunity.