Induction of immune responses to functional determinants of a cell surface streptococcal antigen.

Antibodies directed against the cell surface adhesin, termed streptococcal antigen I/II of Streptococcus mutans can protect against dental caries. Streptococcal antigen I/II (SA I/II) interacts with salivary glycoproteins and promotes adhesion to the tooth surface. Topical application of monoclonal antibodies which recognize a domain within residues 816-1213 (fragment 3) prevents colonization by S. mutans in primates. In this study the immunogenicity and antigenicity of fragment 3 was investigated in five strains of mice. Fragment 3 induced an immune response following immunization with whole cells of S. mutans in all strains of mice. Immunization with recombinant fragment 3 also induced T-cell proliferative and antibody responses both to fragment 3 and to the SA I/II. Antibody responses to the previously defined adhesion determinants (residues 1005-1044) were weak or undetectable. Immunization of three representative strains of mice with a recombinant polypeptide (residues 975-1044) comprising this adhesion epitope and an adjacent T-cell epitope (residues 975-1004) elicited both T- and B-cell responses to the polypeptide and to native SA I/II. The B-cell epitopes overlapped with the adhesion determinant. These findings provide a means of directing immune responses to functional determinants of SA I/II.     

A protein fragment of streptococcal cell surface antigen I/II which prevents adhesion of Streptococcus mutans.

Attachment of Streptococcus mutans to the tooth surface involves a cell surface protein with an M(r) of 185,000, termed streptococcal antigen (SA) I/II. Four overlapping fragments of the gene encoding SA I/II were amplified by polymerase chain reaction, cloned, and expressed in Escherichia coli. The recombinant polypeptides were assayed for adhesion-binding activity to salivary receptors and for recognition by a panel of monoclonal antibodies (MAbs) raised against SA I/II. Two of the MAbs which are known to prevent colonization of S. mutans in vivo bound the recombinant polypeptide comprising residues 816 to 1161. In vitro adhesion of S. mutans to saliva-coated hydroxyapatite beads was also inhibited specifically by a polypeptide (residues 816 to 1213) encompassing the same region. The evidence from the MAbs preventing colonization of S. mutans and the adherence inhibition assay suggests that an adhesion-binding activity resides within the portion of SA I/II comprising residues 816 to 1213, which is highly conserved among oral streptococcal species.     

The reactivity of naturally sensitized human CD4 cells and IgG antibodies to synthetic peptides derived from the amino terminal sequences of a 3800 MW Streptococcus mutans antigen.

Natural immunity to synthetic peptides (SP) derived from the sequences of a 3800 MW streptococcal antigen (SA) was found in human subjects. Significant serum IgG antibodies were detected both to the native SA and to peptides consisting of residues 3-13, 1-15 and 1-20. Inhibition studies confirmed cross-reactivity between the native SA and SP. A series of short peptides with deletions at the amino and carboxy termini were then tested to determine the sequence of B-cell epitopes. Residues 8-13 and 1-6 bound significant serum IgG antibodies, but residues 8-13 were more effective and consistent in inhibiting human antibodies than residues 1-6. These results suggest that residues 8-13 constitute a major B-cell epitope but that residues 1-6 may represent a minor B-cell epitope. The human CD4 subset of T cells was then examined by stimulating the cells with SA or SP and measuring the uptake of [3H]thymidine [( 3H]TdR). The cells were found to be sensitized in vivo to both the native SA and the SP and cross-reactivity between the SA and SP was shown by enrichment and depletion experiments on antigen-coated monocytes. As with the B-cell epitope, the series of short peptides was used to stimulate CD4 cells, in order to determine the T-cell epitope. Residues 6-15 were the shortest SP which stimulated significant [3H]TdR uptake and this peptide was designated as a T-cell epitope. The results suggest that natural oral immunization with Streptococcus mutans induces serum antibodies and T-cell sensitization to a peptide in which a T-cell epitope (residues 6-15) overlaps with a B-cell epitope (residues 8-13). Furthermore, a comparison between linear and cycled peptides suggests that unlike immunogenicity which is commonly enhanced by the more rigid cyclized peptides, antigenicity is favoured by linear peptides. This was evident not only for antibodies but also for T-cell proliferative responses.     

Mutating the anchor residues associated with MHC binding inhibits and deviates CD8+ T cell mediated protective immunity against malaria

We investigated whether immune responses induced by immunization with plasmid DNA are restricted predominantly to immunodominant CD8+ T cell epitopes, or are raised against a breadth of epitopes including subdominant CD8+ and CD4+ T cell epitopes. Site-directed mutagenesis was used to change one or more primary anchor residues of the immunodominant CD8+ T cell epitope on the Plasmodium yoelii circumsporozoite protein, and in vivo protective efficacy and immune responses against defined PyCSP CD8+ and/or CD4+ epitopes were determined. Mutation of the P2 but not P9 or P10 anchor residues decreased protection and completely abrogated the antigen-specific CD8+ CTL activity and CD8+ dependent IFN-gamma responses to the immunodominant CD8+ epitope and overlapping CD8+/CD4+ epitope. Moreover, mutation deviated the immune response towards a CD4+ T cell IFN-gamma dependent profile, with enhanced lymphoproliferative responses to the immunodominant and subdominant CD4+ epitopes and enhanced antibody responses. Responses to the subdominant CD8+ epitope were not induced. Our data demonstrate that protective immunity induced by PyCSP DNA vaccination is directed predominantly against the single immunodominant CD8+ epitope, and that although responses can be induced against other epitopes, these are mediated by CD4+ T cells and are not capable of conferring optimal protection against challenge.     

Natural antibody responses against the non-repeat-sequence-based B-cell epitopes of the Plasmodium falciparum circumsporozoite protein.

Synthetic peptides and human serum or plasma samples from regions of Brazil, Papua New Guinea, and Kenya in which malaria is endemic were used to identify B-cell epitopes localized outside the repeat region of the circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum. In agreement with recent observations, our results confirm the presence of two non-repeat-region-based B-cell epitopes of the CS protein. Of these two epitopes, only the region I epitope (KPKHKKLKQPGDGNP) was previously shown to be recognized by human sera. In this study, we show that human immune sera from malarious regions recognize another B-cell epitope, ENANANNAV, that resides carboxyl to the repeat region. The present study reveals that (i) the repeat-sequence (NANP)-based B-cell epitope of the CS protein is an immunogenic but not immunodominant epitope; (ii) the natural expression of antibody responses to the two non-repeat-region-based B-cell epitopes of the CS protein varies in different populations in which malaria is endemic; (iii) although the host immune responses to the non-repeat-region-based B-cell epitopes increase as a function of host age, this increase is not statistically significant for the region I epitope but is significant for the other epitope; and (iv) the Th1R T-cell site but not the Th2R or Th3R T-cell site induces an antibody response in the human host. This study confirms the immunogenic potential of non-repeat-region-based B-cell epitopes and suggests that antibody pressures may also contribute to the maintenance of the antigenic diversity of the CS protein.     

Plasmodium vivax promiscuous T-helper epitopes defined and evaluated as linear peptide chimera immunogens

Clinical trials of malaria vaccines have confirmed that parasite-derived T-cell epitopes are required to elicit consistent and long-lasting immune responses. We report here the identification and functional characterization of six T-cell epitopes that are present in the merozoite surface protein-1 of Plasmodium vivax (PvMSP-1) and bind promiscuously to four different HLA-DRB1* alleles. Each of these peptides induced lymphoproliferative responses in cells from individuals with previous P. vivax infections. Furthermore, linear-peptide chimeras containing the promiscuous PvMSP-1 T-cell epitopes, synthesized in tandem with the Plasmodium falciparum immunodominant circumsporozoite protein (CSP) B-cell epitope, induced high specific antibody titers, cytokine production, long-lasting immune responses, and immunoglobulin G isotype class switching in BALB/c mice. A linear-peptide chimera containing an allele-restricted P. falciparum T-cell epitope with the CSP B-cell epitope was not effective. Two out of the six promiscuous T-cell epitopes exhibiting the highest anti-peptide response also contain B-cell epitopes. Antisera generated against these B-cell epitopes recognize P. vivax merozoites in immunofluorescence assays. Importantly, the anti-peptide antibodies generated to the CSP B-cell epitope inhibited the invasion of P. falciparum sporozoites into human hepatocytes. These data and the simplicity of design of the chimeric constructs highlight the potential of multimeric, multistage, and multispecies linear-peptide chimeras containing parasite promiscuous T-cell epitopes for malaria vaccine development.     

Hierarchic T-Cell Help to Non-Linked B-Cell Epitopes

The induction of antibodies against peptides requires the presence of a T helper cell epitope. In the absence of an added T-cell epitope only 10% of the mice, or less depending on the strain, gave an antibody response to a series of peptides of the measles virus (MV) fusion (F) protein. After co-immunization with a non-covalently coupled T-cell epitope more than 60% of the peptides became immunogenic. Considerable differences became apparent when BALB/c mice were immunized with peptides in the presence of different T-cell epitopes. An immunodominant T-cell epitope of the MV-F protein was more efficient than a subdominant or a cryptic T-cell epitope in providing help to a non-linked B-cell epitope. There is both a ranking order of the amount of help which B-cell epitopes require and a ranking order for the help T-cell epitopes are able to provide. The capability of a T-cell epitope to provide help to a B-cell epitope correlated with its own immunogenicity, i.e. the intensity of the antibody response to the peptide representing the T-cell epitope. The data suggest that for each MHC class II allele there is an optimal T-cell epitope which can provide help to a maximal number of B-cell epitopes and that such a peptide can be identified by its ability to induce antibodies against itself. By using this strategy, the authors were able to induce antibodies which cross-reacted with the MV.     

Epitope maps of the Escherichia coli heat-labile toxin B subunit for development of a synthetic oral vaccine.

Linear B- and T-cell epitopes spanning all 103 amino acids of the Escherichia coli heat-labile toxin B subunit (LT-B) were assessed in mice orally immunized with native LT or with recombinant Salmonella enteritidis expressing LT-B. Oral administration of native LT induced mucosal immunoglobulin A (IgA) antibodies reactive with an epitope at residues 85 to 91, while IgA induced by recombinant Salmonella LT-B reacted with an epitope at residues 36 to 44. Serum IgG anti-LT-B antibodies from mice orally immunized with either LT or with recombinant Salmonella LT-B were directed to both epitopes. A single T-cell epitope spanning residues 34 to 42 was identified by T-cell proliferative and cytokine responses. When a 20-mer peptide (residues 26 to 45) with B- and T-cell epitopes was given orally to BALB/c (H-2(d)) and B10 congenic (I-A(d), I-A(b), and I-A(k)) mice, significant fecal IgA and serum IgG anti-LT-B antibodies were induced. The peptide also induced LT-B-specific T-cell proliferative responses in these mice. Orally administered LT-B peptide (residues 26 to 45) induced a cytokine profile indicative of both T helper 1- and 2-type cells. The remarkable immunogenicity of this 20-mer peptide makes it a candidate for a vaccine to protect against enterotoxigenic E. coli.     

Characterization of the conformational epitope of Guy's 13, a monoclonal antibody that prevents Streptococcus mutans colonization in humans

Guy's 13 is a mouse monoclonal antibody which recognizes streptococcal antigen I/II (SA I/II), a major cell surface glycoprotein of Streptococcus mutans. In a number of clinical trials, this antibody has been shown to prevent colonization in the human oral cavity. The aim of this study was to identify the SA I/II epitope recognized by Guy's 13. The data suggest that the epitope is conformational, delimited by two noncontiguous regions of the antigen: residues 45 to 457, within the N-terminal half of SA I/II, and residues 816 to 983, within the C-terminal half. In fluid-phase immunoassays a strict requirement for the simultaneous presence of both regions was demonstrated for antibody binding. Furthermore, these two regions of SA I/II were shown to have the ability to interact with each other in the absence of Guy's 13 antibody, suggesting that the normal conformation of SA I/II might be determined by the interaction of these two regions.     

Cloning and expression of the gene for the Avi-3 antigen of Mycobacterium avium and mapping of its epitopes.

The Avi-3 antigen, which is found only in Mycobacterium avium culture sonic extracts, is species specific and results in strong skin test activity in guinea pigs sensitized with heat-killed M. avium. Its gene was cloned by using a previously developed single-probe method and was sequenced. The gene encoded a 194-amino-acid polypeptide with a molecular weight of 21,500. A recombinant Avi-3 antigen expressed in Escherichia coli reacted with monoclonal and polyclonal antibodies raised against the native Avi-3 antigen. To identify epitopes on this protein for immunodiagnostic purposes, various parts of the Avi-3 antigen were expressed as beta-galactosidase fusion proteins, using pUR and pURS expression vectors. The clones screened by both antibody reactivity and T-cell proliferative activity defined fragments with coexisting B- and T-cell epitopes. A B-cell epitope (Asn-176 to Ala-186) and two T-cell epitopes (Glu-75 to Ile-86 and Arg-155 to Leu-164) were thus defined. The synthetic polymerized peptides of the T-cell epitopes were proven to elicit a delayed cutaneous hypersensitivity reaction in guinea pigs. This mapping method would be useful in the development of a subunit vaccine consisting of an immunodominant B-cell epitope linked to a T-cell epitope in the vicinity.     

Mapping major and minor T-cell epitopes in vitro and their immunogenic or tolerogenic effect in vivo in non-human primates.

The immunogenicity of synthetic peptides of in vitro mapped T- and B-cell epitopes from a Streptococcus mutans cell-surface antigen were investigated in non-human primates. Peptide (1-15) contains T-cell (7-15) and B-cell (8-13) epitopes, but is only immunogenic if dimerized (1-15)2 or linked to the carrier tetanus toxoid (1-15)TT. Monomers and dimers of T- and B-cell epitopes were prepared and used to immunize macaques. Immunogenicity was assayed in lymphocytes by the uptake of [3H]thymidine and serum antibodies by a solid-phase radioimmunoassay. Macaques immunized with the dimerized (1-15)2 or carrier-linked peptide (1-15)TT exhibited in vitro T-cell proliferative responses to peptides (1-15) and (7-15). T cells from animals immunized with peptides (1-15), (7-15) or (7-15)2 failed to elicit an immune response. In order to establish if these non-immunogenic peptides might induce tolerance, the same macaques were challenged with the immunogenic peptide (1-15)TT. The results suggest that T-cell responses to peptide (1-15) were reestablished, but instead of responding to peptide (7-15) they were stimulated by a hitherto silent epitope (1-7). Tolerance to the major T-cell epitope (7-15) and the expression of a minor (silent) T-cell epitope (1-7) was associated with B-cell tolerance, suggesting that T-cell help for antibodies resides in the major T-cell epitope (7-15). However, short-term T-cell lines revealed T-cell responses to peptides (1-7) and (7-15) in both tolerized and immunized macaques, but the relative frequency of the minor epitope (1-7)-reactive lines was significantly higher in tolerized animals, whilst that for the major epitope (7-15) was higher in immunized animals. These findings suggest that the silent epitope (1-7) is really cryptic, in that it can be detected if the cell lines are first expanded in vitro with the whole peptide (1-15) and then stimulated with the truncated peptides (1-7) or (7-15). The results are consistent with the concept of a hierarchy of major and minor T-cell epitopes, now demonstrated in non-human primates, in which tolerance to the major T-cell epitope is associated with tolerance to antibody formation and the emergence of a minor T-cell epitope.     

Anaplasma marginale major surface protein 2 CD4+-T-cell epitopes are evenly distributed in conserved and hypervariable regions (HVR), whereas linear B-cell epitopes are predominantly located in the HVR

Organisms in the genus Anaplasma express an immunodominant major surface protein 2 (MSP2), composed of a central hypervariable region (HVR) flanked by highly conserved regions. Throughout Anaplasma marginale infection, recombination results in the sequential appearance of novel MSP2 variants and subsequent control of rickettsemia by the immune response, leading to persistent infection. To determine whether immune evasion and selection for variant organisms is associated with a predominant response against HVR epitopes, T-cell and linear B-cell epitopes were localized by measuring peripheral blood gamma interferon-secreting cells, proliferation, and antibody binding to 27 overlapping peptides spanning MSP2 in 16 cattle. Similar numbers of MSP2-specific CD4(+) T-cell epitopes eliciting responses of similar magnitude were found in conserved and hypervariable regions. T-cell epitope clusters recognized by the majority of animals were identified in the HVR (amino acids [aa] 171 to 229) and conserved regions (aa 101 to 170 and 272 to 361). In contrast, linear B-cell epitopes were concentrated in the HVR, residing within hydrophilic sequences. The pattern of recognition of epitope clusters by T cells and of HVR epitopes by B cells is consistent with the influence of protein structure on epitope recognition.     

Permissive recognition of a mycobacterial T-cell epitope: localization of overlapping epitope core sequences recognized in association with multiple major histocompatibility complex class II I-A molecules.

Most T-cell epitopes are recognized in the context of a single or limited number of major histocompatibility complex (MHC) class II molecules. We have shown previously, however, that the immunodominant p61-80 epitope from the Mycobacterium tuberculosis 19,000 MW protein is recognized in a genetically permissive manner. In this study, permissive recognition of p61-80 was analysed in three murine MHC haplotypes (H-2b,d and k) with respect to: (i) T-cell-epitope core structure; (ii) I-A/I-E class II MHC restriction; and (iii) the identification of critical amino acid residues within the core region. Overlapping epitope core sequences composed of 6 to 8 amino acids were identified for each of the three H-2 haplotypes by T-cell epitope scanning (PEPSCAN) using peptide-specific T-cell lines. The epitope core sequences recognized by peptide and 19,000 MW protein-specific T cells were similar. In all three haplotypes, responses to p61-80 were restricted by class II MHC I-A molecules. To identify residues within the epitope core critically required for recognition, single substitution (alanine or leucine) analogue peptides were tested for their capacity to stimulate p61-80-specific T-cell hybridomas. A heterogeneous pattern of reactivity was observed, even among individual hybridomas derived from the same H-2 haplotype. Although every core residue could be defined as critical for at least one hybridoma, only one critical substitution (74Val-->Ala) was common to all hybridomas. The identification and structural analysis of genetically permissive epitopes of mycobacteria may be a useful strategy for the rational design of peptide-based vaccines for tuberculosis.     

Induction of Multiepitopic and Long-Lasting Immune Responses Against Tumour Antigens by Immunization with Peptides, DNA and Recombinant Adenoviruses Expressing Minigenes

The development of immunization strategies to induce strong and multiepitopic T-cell responses against tumour antigens is needed for anti-tumour immunotherapy. However, a common finding after immunization with complex antigens is the preferential induction of immune responses against immunodominant epitopes. In this study, with the aim of inducing multiepitopic responses against several common tumour antigens, we have designed a minigene construct encoding four human leucocyte antigen (HLA)-A2-restricted epitopes belonging to tumour antigens CEA (CEA-691 and CEA-571), MAGE2 (MAGE2-157) and MAGE3 (MAGE3-112), as well as the universal PADRE epitope recognized by T helper lymphocytes. To optimize the activation of immune responses against these epitopes, we have used different antigen formats (short peptides encompassing individual epitopes and DNA plasmids or adenoviral constructs expressing the minigene) in single or combined immunization schedules. A single immunization with either DNA plasmid or recombinant adenovirus induced a monospecific immune response against the immunodominant epitope CEA-571, whereas immunization with the peptide pool induced responses against all epitopes. Combination of peptide priming followed by a boost with the plasmid and the recombinant adenovirus expressing the minigene induced stronger, multi-specific and long-lasting immune responses, overcoming the immunodominance imposed by the main T-cell epitope. Moreover, these combined immunization strategies were able to induce responses that were able to recognize Mel624 HLA-A2⁺ tumour cells expressing MAGE2. These results suggest that heterologous immunization strategies combining peptides and DNA or recombinant adenoviruses can be useful to broaden the specificity and enhance the efficacy of subunit vaccines.     

Epitope specificity and isoforms of the mycobacterial 19-kilodalton antigen.

The topography and specificity of B- and T-cell stimulatory epitopes from the 19-kDa protein of Mycobacterium tuberculosis were investigated by using overlapping synthetic peptides. Murine antisera identified two cryptic epitopes (residues 11 to 30 and 61 to 80) and one species-specific immunodominant epitope (residues 140 to 159). Immunoglobulins G1 and G2a antibody isotypes varied for the respective peptide immunogens but without relationship to the T-cell cytokine profiles which were characterized by high gamma interferon and low interleukin 5 levels. Antisera to recombinant M. tuberculosis 19-kDa protein (rGST-19) cross-reacted with homologous proteins of similar size from organisms of the Mycobacterium avium-intracellulare complex. Two-dimensional gel electrophoresis revealed differences in the number, relative mobility, and charge of isoforms of the 19-kDa protein, possibly reflecting posttranslational modifications. The immunodominant T-cell epitope from the M. tuberculosis 19-kDa protein (residues 61 to 80) and the corresponding peptide sequence from Mycobacterium avium subsp. intracellulare (residues 64 to 83), differing at five residues, were both recognized in a genetically permissive manner. Peptides 61-80 and 64-83 stimulated cross-reactive responses in BALB/c (H-2d) mice, while in the C57BL/10 (H-2b) strain, responses to peptide 61-80 were species specific. In purified protein derivative-positive healthy individuals, the M. avium subsp. intracellulare peptide stimulated stronger responses than did the M. tuberculosis peptide, whereas patients with active tuberculosis had enhanced in vitro T-cell responses to both peptides.     

Human T- and B-cell epitopes of E1 glycoprotein of rubella virus

The identification of T- and B-cell sites recognized frequently by human populations could provide the basis for selecting the candidate T- and B-cell epitopes for the development of an effective synthetic vaccine against rubella. Rubella virus E1 glycoprotein has been shown to be the predominant antigen to which the majority of human populations develop lymphocyte proliferative and antibody responses. To define the T- and B-cell epitopes of E1 glycoprotein of rubella virus, 23 overlapping synthetic peptides corresponding to more than 90% of the amino acid sequence of E1 were synthesized and tested for their capacities to induce proliferative and antibody responses of 10 seropositive individuals. The most frequently recognized T-cell epitopes were EP19 (residues 324–343), with 7 of 10 responders, and both EP12 (residues 207–226) and EP17 (residues 289–308), with 6 of 10 responders, respectively. Two immunodominant linear B-cell epitopes were mapped to residues 157 to 176 (EP9, 8/10) and 374 to 390 (EP22, 6/10) by using peptide-specific enzyme linked immunosorbent assay.     

Antigen structure influences helper T-cell epitope dominance in the human immune response to HIV envelope glycoprotein gp120

The development of an effective vaccine against HIV/AIDS has been hampered, in part, by a poor understanding of the rules governing helper T-cell epitope immunodominance. Studies in mice have shown that antigen structure modulates epitope immunodominance by affecting the processing and subsequent presentation of helper T-cell epitopes. Previous epitope mapping studies showed that the immunodominant helper T-cell epitopes in mice immunized with gp120 were found flanking flexible loops of the protein. In this report, we show that helper T-cell epitopes against gp120 in humans infected with HIV are also found flanking flexible loops. Immunodominant epitopes were found to be located primarily in the outer domain, an average of 12 residues C-terminal to flexible loops. In the less immunogenic inner domain, epitopes were found an average of five residues N-terminal to conserved regions of the protein, once again placing the epitopes C-terminal to flexible loops. These results show that antigen structure plays a significant role in the shaping of the helper T-cell response against HIV gp120 in humans. This relationship between antigen structure and helper T-cell epitope immunodominance may prove to be useful in the development of rationally designed vaccines against pathogens such as HIV.     

Contribution of HLA class I allele expression to CD8+ T-cell responses against Epstein-Barr virus

Most immune responses to viral infections involve CD8+ T cells recognizing viral peptides of typically 9-10 amino acids in the groove of major histocompatibility complex (MHC) class I. Importantly, CD8+ T-cell responses appear to focus on few viral epitopes, a phenomenon termed immunodominance. While the understanding of this phenomenon has been based largely on experimental mice models, it is imperative to evaluate its contribution in humans, as the design of peptide-based vaccines may be influenced by immunodomination. Here, we present evidence that immunodominance can be detected among Epstein-Barr virus (EBV) epitopes associated with two of the most frequent class I alleles in Western Europe, human leucocyte antigen (HLA)-A2 and HLA-B7. CD8+ T-cell responses to HLA-A2-associated EBV epitopes were significantly reduced in individuals coexpressing HLA-B7. The impairment of HLA-A2-associated responses correlated with a dominant response to an HLA-B7 epitope. The data demonstrate a hierarchy in the human cellular immune response to immunodominant EBV epitopes presented by separate HLA class I alleles. This may have implications for EBV vaccine development as well as for the interpretation of isolated analysis of immunodominant responses to EBV.     

The Bioinformatics Analyses Reveal Novel Antigen Epitopes in Major Outer Membrane Protein of Chlamydia trachomatis

Purpose: The aim of this study was to predict the T-cell and B-cell epitopes in major outer membrane protein (MOMP) of Chlamydia trachomatis (CT) by using online software and also to analyse the secondary structure of MOMP through bioinformatics tools. Materials and Methods: The predictions of secondary structure of MOMP protein were carried out using SOPMA software, and the prediction of B-cell epitopes in MOMP protein was carried out using IEDB and LEPS software, while the T-cell epitopes were predicted by the software of IEBD and SYFPEITHI. The predictions from the software were combined with MOMP protein characteristics, including surface features, hydrophilicity, flexibility, accessibility and plasticity, to analyse the common epitope areas’ response by T-cells and B-cells. Results: In the secondary structure of CT MOMP, the alpha-helices accounted for 41.62% of total amino acid, while the beta sheets and random coil accounted for 19.80% and 32.49%, respectively. Predictions combined with MOMP protein surface features, hydrophilicity, flexibility, accessibility and plasticity were further characterised, and three high-score B-cell epitope areas were found as located in 24–31, 307–311 and 318–327 amino acids of MOMP protein, respectively; in the meanwhile, three high-score T-cell epitope areas were found in 234–236, 323–329 and 338–343 amino acids of MOMP using major histocompatibility complex (MHC) class I HLA-A 0201 restrictive T-cell epitope analyser. Conclusion: We established the methods by using the biological information network technologies for looking the T-cell antigen epitopes and B-cell antigen epitopes in MOMP of CT, and three novel T-cell epitopes as well as three novel B-cell epitopes were identified in the current study. It provides important information for further studying the antigenicity of CT MOMP protein and also provides useful information for developing highly efficient subunit vaccines for CT.     

B-Cell epitope mapping of DNA topoisomerase I defines epitopes strongly associated with pulmonary fibrosis in systemic sclerosis

We hypothesized that B-cell epitope mapping of DNA Topoisomerase I (type-I topoisomerase, or Topo I) may define epitopes strongly associated with pulmonary interstitial fibrosis (PIF) in systemic sclerosis (SSc). B-cell epitope mapping of Topo I was performed using 63 20-mer peptides overlapping by eight residues and spanning the entire length of the Topo I sequence. These peptides, coupled to polystyrene pins, were tested for antibody binding by enzyme-linked immunosorbent assays (ELISAs) using immunoglobulin G fractions from anti-Topo I, anticentromere, anti-U3RNP-positive, and normal sera. Four major epitopes were recognized by anti-Topo I sera, but not from the control sera: WWEEERYPEGIKWKFLEHKG (205-224, epitope I), RIANFKIEPPGLFRGRGNHP (349-368, epitope II), PGHKWKEVRHDNKVTWLVSW (397-416, epitope III), and ELDGQEYVVEFDFLGKDSIR (517-536, epitope IV). Peptide-epitopes were then synthesized in their soluble forms and ELISA systems were developed. Epitopes II to IV are localized at highly exposed sites of the Topo I tertiary structure, whereas epitope I is localized at a less accessible site. In a cohort of 81 patients with SSc with clinical data on the evolution of their disease, patients with antibodies in their sera recognizing at least three of the four epitopes had 3.1 times (P = 0.02) the hazard of developing PIF compared with patients whose sera recognized no epitopes or only one or two of the four epitopes. The discrimination was much stronger than that achieved by the simple determination of Topo I antibodies by counterimmunoelectrophoresis and immunoblot (hazard ratio 1.7, P = 0.30) in the same patients. B-cell epitope mapping of the anti-Topo I response has identified four major epitopes which cumulatively show a strong association with the development of PIF in SSc.