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PubMed1996

Epitope mapping of homologous and cross-reactive antigens by monoclonal antibodies to streptococcal cell membrane (mAb to SCM).

An approach to epitope mapping of a series of anti-streptococcal cell membrane (SCM) mAbs is described. Evaluations by enzyme-linked immunosorbent assay (ELISA) of one control mAb HB-35 and 13 different anti-SCM mAbs were made on homologous SCM antigen and human basement membrane antigens isolated from glomeruli (GBM) and lung (LBM). These anti-SCM mAbs were previously shown to be cross-reactive in a variety of systems with both GBM and LBM. The binding capacities were measured for all 14 mAbs on ELISA plates sensitized with SCM antigen or the cross-reactive GBM or LBM antigens, at 5 micrograms/ml or approximately 20 pM/well. From the 50% binding capacity dilution the pM of mAb bound/pM antigen-well was calculated which translated into an estimate of the ratio-number of epitopes bound. Observations with the homologous and cross-reactive antigens showed multiple reactive epitope ratios to eight mAbs whereas the other five yielded a ratio value of one or two on the tested antigens. Plates blocked with a specific dilution of one mAb evaluated the binding by a second mAb providing both binding and specificity data. One mAb (I-F-3) blocked all the other anti-SCM mAbs on all three antigen plates. An additive effect was noted by three mAbs, I-G-8, II-C-4 and II-D-8 with most of the other mAbs. The order of placement, however, made distinctive differences; II-F-4 showed an additive or enhancement effect on I-B-5 but no reciprocal effect was seen. A similar effect was made with I-G-8 and II-C-4 or I-F-7. Possible interpretations are that each mAb is binding different epitopes each fully exposed, and the order of placement of the mAbs makes no difference. Where an enhanced effect was observed it is suggested that the binding of the first mAb changed the conformation of the antigen, thereby opening and exposing additional epitope(s) to the second antibody. Or, in contrast, where the second mAb was blocked by the first, a fixing of the protein conformation is suggested thereby occluding the other epitope, as seen with I-F-3 and II-C-4. These epitope mapping procedures confirmed that all 13 anti-SCM mAbs were binding at different epitopes. The nature of basement membrane collagens and how this relates to post-streptococcal sequelae will be discussed.

Antibodies, Bacterial↗
PubMed2004

The clinical value of intracellular autoantigens B-cell epitopes in systemic rheumatic diseases.

A hallmark of autoimmune diseases is the production of autoantibodies against intracellular autoantigens. Although their pathogenetic and their etiologic relationship are not fully understood, these autoantibodies are important tools for establishing the diagnosis, classification and prognosis of autoimmune diseases. Systemic rheumatic diseases are among the most complex disorders because their clinical presentation and constellation of findings are in part reflected by the wide spectrum of autoantibodies found in the sera of patients suffering from these disorders. These autoantibodies usually target large complexes consisting of protein antigens noncovalently associated with (ribo)-nucleic acid(s), like the spliceosome or Ro/La-RNPs. In this review, we first address the main characteristics and the clinical value of several autoantibodies, with respect to their diagnostic sensitivity and specificity. Subsequently, we provide a brief overview of the antigenic determinant types that have been identified on the corresponding autoantigens. The antibody targets of autontigens include primary, secondary, tertiary and quarternary structure epitopes, as well as cryptotopes, neoepitopes and mimotopes. We next focus on antigenic structures corresponding to B-cell epitopes with high disease specificity and sensitivity for all the major autoantigens in systemic autoimmunity including the Ro/La and U1 ribonucleoprotein complexes and the Ku70/80, ribosomal P, DNA topoisomerase I, filaggrin, Jo-1 and PM/SCl-100 autoantigens. These epitopes, defined at the peptide level, can be chemically synthesized and engineered for the development of new inexpensive and easier to perform assays and the improvement of the methods for autoantibody detection. Specific examples of newly developed assays that incorporate (i) epitopes with high disease specificity and sensitivity, (ii) modified epitopes, (iii) conformational epitopes and (iv) complementary epitopes are discussed in detail. Finally, we examine the potential of combining these synthetic epitopes for future development of multiplex diagnostic tests based on miniaturized autoantigen arrays.

Autoantigens↗
PubMed2005

Interactions between helper T-cell epitopes of hepatitis C virus.

The premise of this work is that within a given hepatitis C virus (HCV) protein there exists an array of Th1 and Th2 epitopes, each of which can provide synergistic (positive or negative) effects upon other epitopes by intramolecular, cytokine-mediated immunoregulation of helper T-cell responses. To address this question, we constructed minigene plasmids pHCVTh1, pHCVTh1X3 and pHCVThR, and HCV NS3 full-length plasmid pHCVNS3. 293T cells were transfected with these plasmids and cell lysates from the transfected cells were used to stimulate PBMC from a patient with chronic HCV infection. IL-2 and IFN-gamma in the supernatant of the cultured PBMC were tested and proliferation of the PBMC was measured. The results demonstrate that interactions exist among helper T-cell epitopes; the synergistic effects of suppressive Th2 epitopes upon Th1 epitopes will inhibit the responses induced by Th1 epitopes, which may contribute to chronic infection by HCV; synergistic effects among Th1 epitopes induce higher levels of IFN-gamma, which may suggest a new strategy for HCV vaccine development. Further, stimulation of an HCV NS3 specific clone with cell lysates from 293T cells transfected with different constructs shows that the HCV NS3 clone could respond to all suggesting that the epitope-specific suppression may be due to an imbalance of Type 1 and Type 2 cytokines or regulatory T-cells.

Amino Acid Sequence↗
PubMed1998

Enhancement of the immunogenicity of a synthetic peptide bearing a VP3 epitope of hepatitis A virus.

The immune responses elicited in mice by different forms of the VP3(110-121) B-epitope of the hepatitis A virus (HAV) were studied. Different forms of incorporation in liposomes were tested, encapsulation, rather than surface exposure, being the best antigenic preparation. Three larger peptides of the VP3 epitope, two of them containing a hepatitis B virus T-epitope, and a third containing a putative T-epitope of HAV (VP3(102-121)) were assayed. While this latter T-epitope induced an enhancement of the response against the VP3 B-epitope, the artificially coupled T-epitopes failed to induce a significant increase. The administration of two multiple antigenic peptide (MAP) constructs, the first containing the VP3(110-121) and VP1(11-25) HAV sequences and the second only the VP1(11-25) sequence, also suggested the presence of a T-epitope, since the response against the VP1 peptide was increased in the first construct.

Animals↗
PubMed2004

Phage display for epitope determination: a paradigm for identifying receptor-ligand interactions.

Antibodies that react with many different molecular species of protein and non-protein nature are widely studied in biology and have particular utilities, but the precise epitopes recognized are seldom well defined. The definition of epitopes by X-ray crystallography of the antigen-antibody complex, the gold standard procedure, has shown that most antibody epitopes are conformational and specified by interactions with topographic determinants on the surface of the antigenic molecule. Techniques available for the definition of such epitopes are limited. Phage display using either gene-specific libraries, or random peptide libraries, provides a powerful technique for an approach to epitope identification. The technique can identify amino acids on protein antigens that are critical for antibody binding and, further, the isolation of peptide motifs that are both structural and functional mimotopes of both protein and non-protein antigens. This review discusses techniques used to isolate such mimotopes, to confirm their specificity, and to characterize peptide epitopes. Moreover there are direct practical applications to deriving epitopes or mimotopes by sequence, notably the development of new diagnostic reagents, or therapeutic agonist or antagonist molecules. The techniques developed for mapping of antibody epitopes are applicable to probing the origins of autoimmune diseases and certain cancers by identifying "immunofootprints" of unknown initiating agents, as we discuss herein, and are directly applicable to examination of a wider range of receptor-ligand interactions.

Binding Sites↗
PubMed2003

Advances in B-cell epitope analysis of autoantigens in connective tissue diseases.

The characterization of autoantibody specificities in rheumatic diseases is important in both diagnostic and basic research areas. Identification of the epitopes recognized by autoantibodies and their clinical and biological significance is not a trivial task. Epitopes may range in complexity from simple linear sequences of amino acids to complex quaternary structures. In addition to this structural complexity the frequency with which an autoantigen and its epitopes are recognized in a patient population may be useful in diagnosis, defining disease subgroups, and may offer information on disease prognosis. In this review recent advances in the epitope mapping of autoantigens in connective tissue diseases are discussed, with particular emphasis placed on the methodologies used to identify epitopes and the classification of the structural features of epitopes. To illustrate the identification of epitope structure, clinically relevant autoantigens, including CENP-A, PM/Scl-100, fibrillarin, filaggrin, Ro-52, and dsDNA, are discussed as examples of each type of epitope.

Amino Acid Sequence↗
PubMed2003

Targeted destruction of the polymerized human serum albumin binding site within the preS2 region of the HBV surface antigen while retaining full immunogenicity for this epitope.

The 55-amino acid (a.a.) preS2 region of the hepatitis B virus (HBV) envelope protein is highly immunogenic, and antibodies against this epitope confer seroprotection against HBV infections. Accordingly, various experimental and clinical studies for developing and evaluating HBV vaccines that include this particular epitope have been reported. However, a pitfall in using preS2 epitopes as part of a vaccinating antigen is that polymerized human serum albumin (pHSA), which is a normal constituent of the human serum, binds to and makes complexes with this particular region. Consequently, it is most likely that the antigen epitope is masked by serum pHSA and subsequently not detected by the immune system. To overcome these limitations, a novel single a.a substitute of the preS2 region was designed that corresponds to a tyrosine to serine exchange at position 140 of preS2. Competitive enzyme-linked immunosorbent assay showed that this substitution completely abolishes pHSA-binding activities in the mutated preS2 peptide, and CD spectra analysis revealed that this property might have been induced by slight conformational changes in its secondary structure. Nevertheless, the original B-cell epitope was still preserved in the mutated preS2 as determined by experimental immunization in mice. In this regard, the preS2(120-145/Y140S) sequence may be an HBV vaccine where epitopes, with intrinsic properties have been deleted without affecting the immunogenicity of the epitope itself. It is expected that the inclusion of this point mutated preS2 epitope will improve the efficacy of conventional preS2-containing HBV vaccines.

Antibody Specificity↗
PubMed1997

Antigenicity and immunogenicity of multiple antigen peptides (MAP) containing P. vivax CS epitopes in Aotus monkeys.

Using linear synthetic peptides corresponding to the Plasmodium vivax circumsporozoite (CS) protein of the common type, we have identified several T and B-cell epitopes recognized by human individuals. Three T-cell epitopes studied (p6) from the amino, (p11) from the central and (p25) from the carboxyl regions, were widely recognized by lymphocytes of immune donors. A series of six peptides, in addition to p11, representing the central repeat domain of the CS(p11-p17) protein were used in ELISA assays to map the B-cell epitopes of this region. P11 was the peptide most frequently recognized by sera containing antibodies to the homologous CS protein as determined by IFAT. The sequences corresponding to peptides p6, p11 and P25 as well as that representing a universal T-cell epitope derived from the tetanus toxin were used to assemble eight different Multiple Antigen Peptides (MAP). The immunogenicity of these MAP was analysed in Aotus monkeys. Groups of two animals were immunized with each MAP and both antibody response, T-lymphocyte proliferation and in vitro gamma-IFN production were evaluated. Two MAPs containing the same B-cell epitope and either a promiscuous CS-protein derived T-cell epitope (p25) or the tetanus toxin epitope (p-tt30) proved to be the most immunogenic and induced high levels of anti-peptide antibodies that recognized the native protein. Except for animals immunized with MAP VII, there was no correlation between antibody levels, lymphocyte proliferation or gamma-IFN production in vitro. The broad recognition of these epitopes by individuals which had been exposed to malaria, the capacity of these MAPs to induce antibodies, recognize the cognate protein, and in vitro gamma-IFN production encourages further analyses of the potential of these proteins as malaria vaccine candidates for human use.

Adult↗
PubMed1998

Mapping the T cell epitopes of the Babesia bovis antigen 12D3: implications for vaccine design.

The Babesia bovis antigen 12D3 was analysed to identify potential T-cell epitopes. Two predictive algorithms identified 13 possible sites but there was minimal agreement between the different predictive methods. Experimental determination of the T-cell epitopes recognized by nine cattle was achieved using a panel of overlapping peptides which identified seven different epitopes, five of which were clustered together around residues 210-320 of the molecule. No T cell epitopes were located within the tightly disulphide bonded core of 12D3. Using a series of truncated peptides, the location of two of the epitopes was mapped to residues 35-43 and 266-275. The sequences of these two epitopes was compared with a database of previously described binding motifs for MHC II alleles and each epitope was found to contain three sequence motifs recognized by HLA-DR alleles. The BoLA-DRB3 alleles occurring in these cattle were determined by a sequence specific oligonucleotide hybridization assay. Within those cattle whose T cells proliferated in response to 12D3, there was a consistent pattern of epitope recognition and presence of particular DRB3 alleles. The implications for effective vaccine design are discussed.

Alleles↗
PubMed1995

Monoclonal antibody 9EG7 defines a novel beta 1 integrin epitope induced by soluble ligand and manganese, but inhibited by calcium.

The monoclonal antibody 9EG7 has been previously found to recognize an epitope induced by manganese on the integrin beta 1 chain (Lenter, M., Uhlig, H., Hamann, A., Jeno, P., Imhof, B., and Vestweber, D. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 9051-9055). Here we show that treatment of beta 1 integrins with manganese or soluble integrin ligands (e.g. fibronectin and RGD peptide) induced the 9EG7 epitope. This epitope was also induced upon EGTA treatment to remove calcium, and the addition of calcium inhibited 9EG7 epitope induction by manganese or by ligand. Further emphasizing the importance of the 9EG7 epitope, the 9EG7 antibody itself stimulated adhesion mediated by multiple beta 1 integrins, and conversely, ligands for alpha 2 beta 1, alpha 3 beta 1, alpha 4 beta 1, and alpha 5 beta 1 all stimulated 9EG7 expression. Together these results support a model whereby (i) calcium inhibits beta 1 integrin function because it prevents the appearance of a conformation favorable to ligand binding and (ii) manganese enhances beta 1 integrin function because it induces the same favorable conformation that is induced by adding ligand, or removing calcium. Notably, other beta 1-stimulating agents (magnesium and mAb TS2/16) did not induce 9EG7 expression unless ligand was also present. Thus, although 9EG7 may reliable detect the ligand-bound conformation of beta 1 integrins, its expression does not always correlate with integrin "activation". Finally, mouse/chicken beta 1 chimeric molecules were used to map the 9EG7 epitope to beta 1 residues 495-602 within the cysteine-rich region, and antibody cross-blocking studies showed that the 9EG7 epitope is distinct from all previously defined human beta 1 epitopes.

Amino Acid Sequence↗
PubMed2006

Autoantibodies to intracellular autoantigens and their B-cell epitopes: molecular probes to study the autoimmune response.

A common laboratory finding in systemic autoimmune diseases is the presence of autoantibodies against intracellular autoantigens. Although their pathogenesis is not fully understood, autoantibodies are important tools for establishing diagnosis, classification, and prognosis of autoimmune diseases. Autoantibodies mainly target multicomponent complexes containing both protein antigens and (ribo)-nucleic acid(s), such as the spliceosome or Ro/La RNPs. In this review, we address the main characteristics and the clinical value of the main autoantibody types with respect to their disease association, and we describe the corresponding autoantigens, their biologic function, and their B-cell antigenic determinants (epitopes). The structural characteristics and clinical associations of these epitopes, and their utility as tools to investigate the autoimmune response, are discussed in detail. New insights into the pathogenetic role of epitopes in systemic autoimmunity are also examined. In this regard, using the defined structures of the B-cell antigenic epitopes, complementary epitopes can be designed according to the "molecular recognition" theory. These complementary epitopes can be used as probes to study pathogenetic and immunoregulatory aspects of the anti-idiotypic response. The origin of humoral autoimmunity and the spreading of the epitopes in systemic lupus erythematosus are also discussed. Finally, the ability of post-translational modifications to induce autoreactive immune attack via the generation of neo-epitopes is summarized.

Autoantibodies↗
PubMed2005

Novel simian immunodeficiency virus CTL epitopes restricted by MHC class I molecule Mamu-B*01 are highly conserved for long term in DNA/MVA-vaccinated, SHIV-challenged rhesus macaques.

Simian immunodeficiency virus (SIV) infection of rhesus macaques provides an excellent model for investigating the basis of protective immunity against human immunodeficiency virus (HIV). One limitation of this model, however, has been the availability of a small number of known MHC class I-restricted CTL epitopes for investigating virus-specific immune responses. We assessed CTL responses against SIV Gag in a cohort of DNA/modified vaccinia virus Ankara (MVA)-vaccinated/simian-human immunodeficiency virus (SHIV)-challenged rhesus macaques. Here, we report the identification of five novel SIV CTL epitopes in Gag for the first time (Gag(39-46) NELDRFGL, Gag(169-177) EVVPGFQAL, Gag(198-206) AAMQIIRDI, Gag(257-265) IPVGNIYRR and Gag(296-305) SYVDRFYKSL) that are restricted by the common MHC class I molecule Mamu-B*01. CTL responses to these epitopes were readily detected in cryopreserved PBMC in multiple animals up to 62 weeks post-infection, both by IFN-gamma enzyme-linked immunospot assay and intracellular IFN-gamma staining. Importantly, viral sequencing results revealed that these epitopes are highly conserved in the SIV-challenged macaques over a long period of time, indicating functional constraints in these regions. Moreover, the presence of CTL responses targeting these epitopes has been confirmed in two independent cohorts of rhesus macaques that have been challenged by SHIV or SIV. Our findings provide valuable candidates for poly-epitope vaccines and for long-term quantitative monitoring of epitope-specific CD8(+) responses in the context of this common Mamu class I allele. It may thus help increase the supply of rhesus macaques in which epitope-specific immunity can be studied in the context of SIV vaccine design.

Animals↗
PubMed2000

Delineation of a conserved B cell epitope on bonnet monkey (Macaca radiata) and human zona pellucida glycoprotein-B by monoclonal antibodies demonstrating inhibition of sperm-egg binding.

To circumvent autoimmune oophoritis after immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing B cell epitope(s) and devoid of oophoritogenic T cell epitopes as immunogens have been proposed. In this study, bonnet monkey (Macaca radiata) ZP glycoprotein-B (bmZPB) was expressed as polyhistidine fusion protein in Escherichia coli. Rabbit polyclonal antibodies against recombinant bmZPB (r-bmZPB) significantly inhibited human sperm-oocyte binding. To map B cell epitopes on ZPB, a panel of 7 murine monoclonal antibodies (mAbs) was generated against r-bmZPB. All 7 mAbs, when tested in an indirect immunofluorescence assay, reacted with bonnet monkey ZP, and only 6 recognized human zonae. Monoclonal antibodies MA-809, -811, -813, and -825 showed significant inhibition in the binding of human spermatozoa to human ZP in a hemizona assay. Epitope-mapping studies using multipin peptide synthesis strategy revealed that these 4 mAbs recognized a common epitope corresponding to amino acids (aa) 136-147 (DAPDTDWCDSIP). Competitive binding studies revealed that the synthetic peptide corresponding to the identified epitope (aa 136-147) inhibited the binding of MA-809, -811, -813, and -825 to r-bmZPB in an ELISA and to bonnet monkey ZP in an indirect immunofluorescence assay. The epitopic domain corresponding to aa 136-147 of bmZPB was completely conserved in human ZPB. These studies will further help in designing ZP-based synthetic peptide immunogens incorporating relevant B cell epitope for fertility regulation in humans.

Amino Acid Sequence↗
PubMed1998

Effective lysis of HIV-1-infected primary CD4+ T cells by a cytotoxic T-lymphocyte clone directed against a novel A2-restricted reverse-transcriptase epitope.

Most HIV-specific cytotoxic T-lymphocyte (CTL) epitopes have been identified using peptide-pulsed and recombinant vaccinia virus-infected targets. These systems may not accurately reflect the ability of epitopes to be presented by HIV-infected T cells. Recent studies suggest, in fact, that some CTL epitopes are poorly presented on HIV-infected cells. In this study, we have identified a novel A2.1-restricted HIV reverse-transcriptase (RT) epitope and investigated the presentation of this epitope by HIV-infected primary CD4+ T cells and T-cell lines. A CD8+ CTL clone, isolated from a seropositive subject that recognized a novel A2-restricted epitope KYTAFTIPSI (aa 293-302) in RT, was used for these studies. Primary CD4+ T cells and the CD4+ T-cell line T1 were infected with virus from T1-nPLAP, a cell line stably transfected with HXB-nPLAP, a molecular construct of HIV linked to a placental alkaline phosphatase (PLAP) marker gene. A uniformly infected cell population, obtained by immunomagnetic selection for PLAP expression, was used as targets in CTL assays. HIV-infected T cells were lysed by CTL recognizing this RT epitope as effectively as peptide-pulsed targets. This suggests that some RT epitopes are good targets for CTL recognition.

Amino Acid Sequence↗
PubMed2001

Characterization of a new H-2D(k)-restricted epitope prominent in primary influenza A virus infection.

Influenza A virus infection of mice has been used extensively as a model to investigate the mechanisms of antigen presentation to cytotoxic T lymphocytes (CTL) and the phenomenon of immunodominance in antiviral CTL responses. The different virus-encoded epitopes that are recognized in H-2(b) and H-2(d) mice have been characterized and their relative immunodominance has been well-studied. In H-2(k) mice, four different K(k)-restricted influenza virus epitopes have been described, but the dominance hierarchy of these epitopes is unknown and there is also an uncharacterized D(k)-restricted response against the virus. In this study, a D(k)-restricted epitope derived from the influenza virus A/PR/8/34 polymerase protein PB1, corresponding to amino acid residues 349-357 (ARLGKGYMF), was identified. This peptide is the major epitope within the PB1 polymerase and is at least as dominant as any of the four K(k)-restricted epitopes that are recognized in CBA mice following primary influenza virus infection. The PB1 epitope is only the fourth D(k)-presented peptide to be reported and the sequence of this epitope confirms a D(k)-restricted peptide motif, consisting of arginine at position two, arginine or lysine at position five and a hydrophobic residue at the carboxy terminus.

Animals↗
PubMed1995

Clustering of B and T epitopes within short sequence regions of the nicotinic acetylcholine receptor.

The epitope repertoire of B cells, due to their selective ability to process their specific antigen and the potential bias imposed on the resulting peptides by the surface immunoglobulins bound to the antigen, may influence the T-helper repertoire. Immunization of C57B1/6 mice with Torpedo acetylcholine receptor (TAChR) causes experimental autoimmune myasthenia gravis (EAMG). Anti-TAChR CD4+ cells recognize epitopes within three sequence regions of the TAChR alpha subunit ('dominant epitopes'). Immunization of mice with denatured or synthetic TAChR antigens sensitizes CD4+ cells to other TAChR sequence regions ('cryptic epitopes'). We investigated here whether clustering of B and T epitopes within the same short sequence segments occurs during the anti-TAChR response, as previously described for the response to hexogenous antigens unrelated to homologous self proteins. Twelve 19-20 residue synthetic sequences of the TAChR alpha, gamma and delta subunits, containing dominant or cryptic CD4+ epitopes for C57B1/6 mice, were tested for ability to induce anti-peptide antibody production. C57B1/6 mice were immunized with the individual peptides. Ten peptides stimulated antibody production. Therefore > 80% of these short TAChR sequences also contain B epitopes. Therefore also in the anti-TAChR response leading to EAMG T and B cell epitopes frequently reside within the same short sequence segment.

Amino Acid Sequence↗
PubMed1997

Identification of epitopes of monoclonal antibodies to porcine zona pellucida 3 beta glycoprotein, a homologue of the mouse/human sperm receptor.

PROBLEM: Immunization with zona pellucida (ZP) glycoproteins leads to a block in fertility with a variable degree of ovarian dysfunction. To avoid autoimmune oophoritis, synthetic peptides corresponding to B cell epitope(s) and devoid of oophoritogenic T cell epitopes as immunogens have been proposed. The main objective of the present study is to define the epitopes recognized by monoclonal antibodies (mAbs) generated against porcine ZP3 beta, a homologue of the designated primary sperm receptor in mice and humans. METHODS: A multipin synthetic peptides approach has been used to map the epitopes recognized by mAbs. Dodecapeptides with an overlap of 6 amino acids corresponding to a precursor pZP3 beta-deduced amino acid sequence (excluding the signal sequence) were synthesized on polypropylene pins and were tested for their reactivity with mAbs by enzyme-linked immunoadsorbent assay (ELISA). The ability of synthetic peptides corresponding to the identified epitopes to inhibit the binding of mAbs to pZP3 beta in a competitive inhibition ELISA was investigated to confirm the above findings. RESULTS: Reactivity of the mAbs with the pin-bound peptides in ELISA-identified epitopes for mAb-451 to EEKLVF (166-171) and mAb-462/470 to FKAPRP (250-255) amino acid residues. mAb-30 recognized QPVWQDEGQRLR (23-34) and VICRCC (316-321) amino acid residues. Competitive inhibition with synthetic peptides encompassing the motifs corresponding to 23-34 and 316-321 for binding of mAb-30 to pZP3 beta revealed the epitopic domain to be 23-34 amino acids. Synthesis of overlapping octapeptides further identified WQDE as the minimum motif for binding of mAb-30, and the replacement of one amino acid at a time with glycine revealed tryptophan as the critical residue. CONCLUSIONS: Collectively, these results describe peptide epitopes that will help in the design of an immunocontraceptive vaccine based on synthetic peptides corresponding to pZP3 beta or its homologues in other species.

Amino Acid Sequence↗
PubMed1994

Mapping of multiple HLA class II-restricted T-cell epitopes of the mycobacterial 70-kilodalton heat shock protein.

By combining a DNA subclone and synthetic-peptide approach, we mapped epitopes of the immunogenic mycobacterial 70-kDa heat shock protein (HSP70) recognized by human CD4+ T-cell clones and lines. In addition, we identified the respective HLA-DR molecules used in antigen presentation. The donor groups used were healthy persons immunized with killed Mycobacterium leprae and tuberculoid leprosy patients. The results show that the N-terminal part of the HSP70 molecule contains three different T-cell epitopes, of which two were presented by DR7 (amino acids [aa] 66 to 82 and 210 to 226) and one was presented by DR3 (aa 262 to 274). The C-terminal part contains one epitope (aa 413 to 424) presented by HLA-DR2. The C-terminal epitope shows extensive homology to the corresponding region of the human HSP70 sequence. All of the T-cell epitopes identified were presented by only one particular HLA-DR molecule. We also found that HLA-DR5 and DRw53 can present HSP70 to T cells, demonstrating the presence of additional epitopes not yet defined at the peptide level. On the basis of the donors used in this study, recognition of HSP70 at the epitope level seems to be ruled by the restriction elements expressed by the donor rather than by any difference in reactivity between healthy individuals and patients. In conclusion, mycobacterial HSP70 is relevant to subunit vaccine design since it contains a variety of T-cell epitopes presented in the context of multiple HLA-DR molecules.

Amino Acid Sequence↗