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PubMed1996

Characterization of epitopes involved in the neutralization of Pasteurella haemolytica serotype A1 leukotoxin.

Defined segments of the leukotoxin A gene (lktA) from an A1 serotype of Pasteurella haemolytica were cloned into a plasmid vector and expressed as LacZ alpha fusion proteins. These fusion proteins were electrophoresed in SDS-PAGE gels and their immunoblotting reactivities with several monoclonal antibodies characterized. The epitope recognized by a strongly neutralizing monoclonal antibody was localized to a 32 amino acid region near the C terminus of the leukotoxin A (LktA) molecule. The epitope recognized by a non-neutralizing antibody was localized to a 33 amino acid region immediately adjacent. Smaller recombinant peptides containing these epitopes were not antigenic, but a polypeptide encompassing 229 amino acids at the C terminus evoked neutralizing antibodies when used to immunize specific-pathogen-free lambs. The distributions of linear epitopes recognized by this antiserum and by antisera raised to full-length recombinant LktA and to native LktA produced by P. haemolytica serotype A1 were determined by their reactivities with a set of overlapping 10 amino acid synthetic peptides. This revealed a complex distribution of linear epitopes at the C-terminal end of LktA. Toxin-neutralizing antibodies in convalescent sheep serum were shown to be directed against conformational epitopes by selective absorption of antibodies directed against linear epitopes.

Amino Acid Sequence↗
PubMed1998

Conservation of the amino-terminal epitope of elongation factor Tu in eubacteria and Archaea.

An epitope of elongation factor Tu (EF-Tu), which is found in organisms in both the bacterial and archaeal domains, was recently defined by mAb 900. To localize the conserved epitope within the EF-Tu molecule and to determine its sequence, SPOTScan analysis of synthetic peptides, Western blot analysis of purified EF-Tu domains and site-directed mutagenesis studies were used. Analysis of mAb 900 binding to overlapping 15-mer peptides encompassing the complete sequence of EF-Tu of Escherichia coli was inconclusive, suggesting three distinct regions may be epitopes. Western blot analysis of EF-Tu domains 1-3 of Thermus thermophilus suggested that the epitope was located at the N terminus. This was confirmed by site-directed mutagenesis of EF-Tu domain 1 of Mycoplasma hominis. By C-terminal truncation of the N-terminal 15-mer peptide the epitope was mapped to EF-Tu residues 1-6. Replacement of each of the residues in the epitope peptide demonstrated that only positions 5 and 6 were indispensable for antibody binding. These data provide evidence that the highly conserved epitope recognized by mAb 900 in the bacterial and archaeal domains is located at the very end of the N terminus of the EF-Tu molecule.

Amino Acid Sequence↗
PubMed1997

Immune response to a Murray Valley encephalitis virus epitope expressed in the flagellin of an attenuated strain of Salmonella.

Recent developments in vaccine construction include the use of attenuated, avirulent strains of Salmonella as carriers of foreign antigens. These recombinant strains can elicit a heterologous immune response when injected into animals, demonstrating potential for their use in the construction of many vaccines. In the present study, a B-cell epitope of Murray Valley encephalitis virus (MVE) was identified and expressed in a Salmonella strain to evaluate its potential to induce a specific immune response to MVE. A synthetic oligonucleotide encoding the B-cell epitope (residues E201-224) of the envelope protein of MVE was inserted into the cloned flagellin gene of the Salmonella strain. The construct was sequenced to ensure correct orientation of the epitope. Expression of the epitope was demonstrated by Western blot analysis and immunogold electron microscopy with monoclonal antibody specific to the epitope. Electron microscopy analysis revealed multiple copies of the epitope along the flagella. The recombinant Salmonella carrying the hybrid flagellin gene elicited an immune response to the MVE epitope in a mouse model. The MVE-specific antibodies partially neutralised the virus in vitro. The significance of this system for engineering vaccines for other medically important flaviviruses is discussed.

Amino Acid Sequence↗
PubMed1997

The relationship between colonization and haemagglutination inhibiting and B cell epitopes of Porphyromonas gingivalis.

Passive immunization with the monoclonal antibody 61BG1.3 selectively prevents colonization by Porphyromonas gingivalis in humans (Booth V, Ashley FP, Lehner T. Infect Immun 1996; 64:422-7). The protective MoAb recognizes the beta component of the RI protease of P. gingivalis which is formed by proteolytic processing of a polyprotein precursor termed PrpR1. This subunit is both a haemagglutinin and an antigen which is recognized by sera from patients with periodontitis. In this study the relationship was investigated between a colonization epitope which is recognized by the MoAb 61BG1.3, a haemagglutinating and B cell epitope which are recognized by sera from patients with periodontitis. B cell epitopes were mapped by Western blotting with a series of truncated recombinant polypeptides spanning the adhesion domain within residues 784-1130 of PrpR1 and by ELISA using a panel of synthetic peptides spanning the same sequence. The epitope which is recognized by the protective MoAb was mapped within residues 907-931 of PrpR1, while serum responses of patients were directed predominantly to the adjacent carboxy-terminal sequence within residues 934-1042. The haemagglutinating epitope was mapped to residues 1073-1112. In view of our previous findings that the MoAb 61BG1.3 prevents colonization of P. gingivalis in vivo and inhibits haemagglutination, these two epitopes may be in proximity in the native protein. Active or passive immunization strategies which target the protective or haemagglutinating epitopes of the adhesion domain of PrpR1 may provide a means of preventing infection with P. gingivalis.

Adult↗
PubMed1997

Epitopic characterization of the human wild-type and mutant ras proteins using membrane-bound peptides.

Overlapping octapeptides encompassing the entire sequences of the human oncogene products Ha-ras, K-ras and N-ras protein were synthesized as spots on polypropylene membrane sheets. The binding of anti-ras protein monoclonal antibodies (mAbs) to the membrane-bound peptides was assessed using an enzyme-linked immunosorbent assay. Epitopes of 10 of 18 mAbs to the human ras proteins were mapped and identified by this procedure. The epitopes of nine of the mAbs are within residues 28-39 in the constant domain common to the three ras proteins, whereas the epitope of the tenth (mAb 21) spans residues 136-144 in Ha-ras. The minimal lengths of epitopes of all ten of the mAbs were further precisely mapped using peptides of varying length, and the tolerance for mAb binding of mutated epitopes was determined by systematically replacing each residue in the epitope with each of the 20 common amino acids. The results show that most of these mAbs have essentially the same binding specificity, namely for the sequence YDPT (residues 32-35) or for slightly longer sequences containing these residues. This site is in the switch 1 region (residues 32-38) in the ras effector loop, indicating that some of the same residues important for the interaction of ras with other proteins (GTPase-activating protein, neurofibromin or raf) are highly antigenic. In addition, we investigated epitopes and specificity of five mAbs against the activated human ras proteins by the same procedure. The information gained from this study should be useful both for study of the complicated functions of ras proteins and clinical detection of ras oncogenes in human tumor cells.

Amino Acid Sequence↗
PubMed1995

Anti-La monoclonal antibodies recognizing epitopes within the RNA-binding domain of the La protein show differential capacities to immunoprecipitate RNA-associated La protein.

The La (SS-B) autoimmune antigen is an RNA-binding protein that is present in both the nucleus and cytoplasm of eukaryotic cells, where it is found associated with RNA polymerase III transcripts. We have investigated the capacity of anti-La monoclonal antibodies SW1, SW3, and SW5 to immunoprecipitate human La ribonucleoprotein particles. Distinct differences were observed for SW3 in comparison with SW1 and SW5. While SW1 and SW5 precipitated ribonucleoproteins containing pre-tRNA, pre-5S rRNA, hY RNAs, pre-U6 snRNA or the viral EBER1 and VA RNAs, SW3 precipitated only ribonucleoproteins containing VA RNAs or (the precursor of) 7-2 RNA. Mapping of the epitopes recognized by SW1, SW3, and SW5 revealed that all three monoclonal antibodies recognize an epitope within the domain of the protein formed by the ribonucleoprotein motif. Cross-competition studies suggested that the epitope recognized by SW1 and SW5 are identical but distinct from the epitope recognized by SW3. Further analyses of the recognition of La from other species by these monoclonal antibodies revealed that they all reacted with bovine La and were not reactive with La from rodents and Xenopus laevis. Replacement of a single amino acid in the human protein by its murine counterpart abolished recognition by SW1 and SW5, but had no effect on recognition by SW3. Taken together, our results indicate that SW1 and SW5 recognize the same epitope and that SW3 recognizes a distinct epitope, both of which are located in the RNA-binding domain of La, and that the accessibility of these epitopes is differentially influenced by the association of La with various RNA polymerase III transcripts.

Amino Acid Sequence↗
PubMed1997

Mapping and mutational analysis of the IgE-binding epitopes on Ara h 1, a legume vicilin protein and a major allergen in peanut hypersensitivity.

Peanut allergy is a significant health problem because of the prevelance and potential severity of the allergic reaction. Serum IgE from patients with documented peanut hypersensitivity reactions and overlapping peptides were used to identify the IgE-binding epitopes on the major peanut allergen, Ara h 1. At least twenty-three different linear IgE-binding epitopes, located throughout the length of the Ara h 1 protein, were identified. All of the epitopes were 6-10 amino acids in length, but there was no obvious sequence motif shared by all peptides. Four of the peptides appeared to be immunodominant IgE-binding epitopes in that they were recognized by serum from more than 80% of the patients tested and bound more IgE than any of the other Ara h 1 epitopes. Mutational analysis of the immunodominant epitopes revealed that single amino acid changes within these peptides had dramatic effects on IgE-binding characteristics. The identification and determination of the IgE-binding capabilities of core amino acids in epitopes on the Ara h 1 protein will make it possible to address the pathophysiologic and immunologic mechanisms regarding peanut hypersensitivity reactions specifically and food hypersensitivity in general.

Adult↗
PubMed2004

Epitope structure of the Bordetella pertussis protein P.69 pertactin, a major vaccine component and protective antigen.

Bordetella pertussis is reemerging in several countries with a traditionally high vaccine uptake. An analysis of clinical isolates revealed antigenic divergence between vaccine strains and circulating strains with respect to P.69 pertactin. Polymorphisms in P.69 pertactin are mainly limited to regions comprised of amino acid repeats, designated region 1 and region 2. Region 1 flanks the RGD motif, which is involved in adherence. Although antibodies against P.69 pertactin are implicated in protective immunity, little is known about the structure and location of its epitopes. Here we describe the identification by pepscan analysis of the locations of mainly linear epitopes recognized by human sera and mouse monoclonal antibodies (MAbs). A total of 24 epitopes were identified, and of these only 2 were recognized by both MAbs and human antibodies in serum. A number of immunodominant epitopes were identified which were recognized by 78 to 93% of the human sera tested. Blocking experiments indicated the presence of high-avidity human antibodies against conformational epitopes. Human antibodies against linear epitopes had much lower avidities, as they were unable to block MAbs. Pepscan analyses revealed several MAbs which bound to both region 1 and region 2. The two regions are separated by 289 amino acids in the primary structure, and we discuss the possibility that they form a single conformational epitope. Thus, both repeat regions may serve to deflect the immune response targeted to the functional domain of P.69 pertactin. This may explain why the variation in P.69 pertactin is so effective, despite the fact that it is limited to only two small segments of the molecule.

Amino Acid Sequence↗
PubMed1999

Acute-phase-specific heptapeptide epitope for diagnosis of parvovirus B19 infection.

The major capsid protein VP2 of human parvovirus B19, when studied in a denatured form exhibiting linear epitopes, is recognized exclusively by immunoglobulin G (IgG) antibodies of patients with acute or recent B19 infection. By contrast, conformational epitopes of VP2 are recognized both by IgG of the acute phase and by IgG of past immunity. In order to localize the VP2 linear epitope(s) specific for acute-phase IgG, the entire B19 capsid protein sequence was mapped by peptide scanning using well-characterized acute-phase and control sera. A unique heptapeptide epitope showing strong and selective reactivity with the acute-phase IgG was detected and characterized. By using this linear epitope (VP2 amino acids 344 to 350) and virus-like particles exhibiting conformational VP2 epitopes, an innovative approach, second-generation epitope-typing enzyme immunoassay, was set up for improved diagnosis of primary infections by human parvovirus B19.

Acute Disease↗
PubMed1997

A novel antibody-dependent cellular cytotoxicity epitope in gp120 is identified by two monoclonal antibodies isolated from a long-term survivor of human immunodeficiency virus type 1 infection.

Two monoclonal antibodies (MAbs), 42F and 43F, were isolated some 14 months apart from a single long-term survivor of human immunodeficiency virus type 1 (HIV-1) infection. These MAbs were found to be indistinguishable in terms of their isotypes, specificities, affinities, and biological activities. Both 42F and 43F directed substantial antibody-dependent cellular cytotoxicity (ADCC) against cells infected with four divergent lab-adapted strains of HIV-1, but no neutralizing activity against these strains was detectable. The ability of MAbs 42F and 43F, as well as that of MAbs against two other gp120 epitopes, to direct ADCC against uninfected CD4+ cells to which recombinant gp120SF2 had been adsorbed (i.e., "innocent bystanders") was demonstrated to be less efficient by at least an order of magnitude than their ability to direct ADCC against HIV-1-infected cells. Flow cytometry analyses showed that 42F and 43F also bind to native primary isolate Envs from clades B and E expressed on cell surfaces. By direct binding and competition assays, it was demonstrated that the 42F/43F epitope lies in a domain of gp120 outside the previously described CD4-binding site and V3 loop ADCC epitope clusters. Immunoblot analysis revealed that the 42F/43F epitope is not dependent on disulfide bonds or N-linked glycans in gp120. Epitope mapping of 42F and 43F by binding to linear peptides demonstrated specificity of these MAbs for a sequence of 10 amino acids in the C5 domain comprising residues 491 to 500 (Los Alamos National Laboratory numbering for the HXB2 strain). Thus, 42F and 43F define a new ADCC epitope in gp120. Because of the relative conservation of this epitope and the fact that it appears to have been significantly immunogenic in the individual from which these MAbs were derived, it may prove to be a useful component of HIV vaccines. Furthermore, these MAbs may be used as tools to probe the potential importance of ADCC as an antiviral activity in HIV-1 infection.

Amino Acid Sequence↗
PubMed1997

Generation of cytotoxic T lymphocytes against immunorecessive epitopes after multiple immunizations with adenovirus vectors is dependent on haplotype.

Currently, adenovirus (Ad) is being considered as a vector for the treatment of cystic fibrosis as well as other diseases. However, the cytotoxic T lymphocyte (CTL) response to Ad could limit the effectiveness of such approaches. Since the CTL response to virus infection is often focused on one or a few immunodominant epitopes, one approach to circumvent this response is to create vectors that lack these immunodominant epitopes. The effectiveness of this approach was tested by immunizing mice with human group C adenoviruses. Three mouse strains (C57BL/10SnJ [H-2b], C3HeB/FeJ [H-2k], and BALB/cByJ [H-2d]) were immunized with wild-type Ad or Ad vectors lacking the immunodominant antigen(s), and the CTL responses were measured. In C57BL/10 (B10) mice, a single inoculation intraperitoneally (i.p.) led to the recognition of an immunodominant antigen in E1A. When B10 mice were inoculated multiple times either i.p. or intranasally with wild-type Ad or an Ad vector lacking most of the E1 region, subdominant epitopes outside this region were recognized. In contrast, C3H mice inoculated with wild-type Ad recognized an epitope mapping within E1B. When inoculated twice with Ad vectors lacking both E1A and E1B, no immunorecessive epitopes were recognized. The immune response to Ad in BALB/c mice was more complex. CTLs from BALB/c mice inoculated i.p. with wild-type Ad recognized E1B in the context of the major histocompatibility complex (MHC) class I Dd allele and a region outside E1 associated with the Kd allele. When BALB/c mice were inoculated with E1-deleted Ad vectors, only the immunodominant Kd-restricted epitope was recognized, and Dd-restricted CTLs did not develop. This report indicates that the emergence of CTLs against immunorecessive epitopes following multiple administrations of Ad vectors lacking immunodominant antigens is dependent on haplotype and could present an obstacle to gene therapy in an MHC-diverse human population.

Adenovirus E1A Proteins↗
PubMed1998

Gag protein epitopes recognized by ELA-A-restricted cytotoxic T lymphocytes from horses with long-term equine infectious anemia virus infection.

Most equine infectious anemia virus (EIAV)-infected horses have acute clinical disease, but they eventually control the disease and become lifelong carriers. Cytotoxic T lymphocytes (CTL) are considered an important immune component in the control of infections with lentiviruses including EIAV, but definitive evidence for CTL in the control of disease in carrier horses is lacking. By using retroviral vector-transduced target cells expressing different Gag proteins and overlapping synthetic peptides of 16 to 25 amino acids, peptides containing at least 12 Gag CTL epitopes recognized by virus-stimulated PBMC from six long-term EIAV-infected horses were identified. All identified peptides were located within Gag matrix (p15) and capsid (p26) proteins, as no killing of target cells expressing p11 and p9 occurred. Each of the six horses had CTL recognizing at least one Gag epitope, while CTL from one horse recognized at least eight different Gag epitopes. None of the identified peptides were recognized by CTL from all six horses. Two nonamer peptide epitopes were defined from Gag p26; one (18a) was likely restricted by class I equine leukocyte alloantigen A5.1 (ELA-A5.1) molecules, and the other (28b-1) was likely restricted by ELA-A9 molecules. Sensitization of equine kidney target cells for CTLm killing required 10 nM peptide 18a and 1 nM 28b-1. The results demonstrated that diverse CTL responses against Gag epitopes were generated in long-term EIAV-infected horses and indicated that ELA-A class I molecules were responsible for the diversity of CTL epitopes recognized. This information indicates that multiple epitopes or whole proteins will be needed to induce CTL in horses with different ELA-A alleles in order to evaluate their role in controlling EIAV.

Amino Acid Sequence↗
PubMed1998

'Epitope fingerprinting' using overlapping 20-mer peptides of the MUC1 tandem repeat sequence.

The ISOBM TD-4 Workshop antibodies 122-177 were tested for reactivity with 20 overlapping MUC1 tandem repeat 20-mer peptides by an ELISA, in order to determine the complete amino acid sequences of the epitopes. Of the 56 antibodies studied, 30 showed specific binding and thus the epitopes were characterized. The epitopes appear to be 'broader' when compared to those deduced from studies using smaller peptides. Interassay variation is remarkably small, allowing for precise grouping of clusters with very similar epitope patterns. Five groups of antibodies show remarkable similarity: BC3 and VU-4-H5; BC4W154, C595 and Mc5; MF06 and B27.29; VU-11-D1 and VU-11-E2; Ma552, VU-3-C6, 7540MR and BC4E549. We have used the term 'epitope fingerprinting' to refer to the 'fine structure' of the epitope with all its essential and flanking amino acids. We believe this method is more precise than the usual epitope mapping with short peptides.

Amino Acid Sequence↗
PubMed2001

CD8(+) lymphocytes respond to different HIV epitopes in seronegative and infected subjects.

HIV-1-specific cytotoxic T-lymphocyte (CTL) responses have been detected at a low frequency in many HIV-1-exposed, persistently seronegative (HEPS) subjects. However, it is unclear how CTLs could protect against HIV acquisition in HEPS subjects, when high levels of circulating CTL fail to prevent disease progression in most seropositive subjects. To address this issue we studied CD8(+) lymphocyte responses to a panel of HIV-1 CTL epitopes in 91 HEPS and 87 HIV-1-infected Nairobi sex workers. HIV-specific responses in seropositive women focused strongly on epitopes rarely or never recognized in HEPS subjects, who targeted epitopes that were subdominant or unrecognized in infected women. These differences in epitope specificity were restricted by only those HLA class I alleles that are associated with a reduced risk of HIV-1 infection in this cohort. Late seroconversion in HEPS donors was associated with a switch in epitope specificity and/or immunodominance to those epitopes preferentially recognized by HIV-1-infected women. The likelihood of detecting HIV-1-specific responses in HEPS women increased with the duration of viral exposure, suggesting that HIV-1-specific CD8(+) responses are acquired over time. The association between differential recognition of distinct CTL epitopes and protection from HIV-1 infection may have significant implications for vaccine design.

CD8-Positive T-Lymphocytes↗
PubMed2002

Identification of immunodominant regions among promiscuous HLA-DR-restricted CD4+ T-cell epitopes on the tumor antigen MAGE-3.

The molecular characterization of the CD4(+) T-cell epitope repertoire on human tumor antigens would contribute to both clinical investigation and cancer immunotherapy. In particular, the identification of promiscuous epitopes would be beneficial to a large number of patients with neoplastic diseases regardless of their HLA-DR type. MAGE-3 is a tumor-specific antigen widely expressed in solid and hematologic malignancies; therefore, is an excellent candidate antigen. We used a major histocompatability complex (MHC) class II epitope prediction algorithm, the TEPITOPE software, to predict 11 sequence segments of MAGE-3 that could form promiscuous CD4(+) T-cell epitopes. In binding assays, the synthetic peptides corresponding to the 11 predicted sequences bound at least 3 different HLA-DR alleles. Nine of the 11 peptides induced proliferation of CD4(+) T cells from both healthy subjects and melanoma patients. Four immunodominant regions (residues 111-125, 146-160, 191-205, and 281-295), containing naturally processed epitopes, were recognized by most of the donors, in association with 3 to 4 different HLA-DR alleles, thus covering up to 94% of the alleles expressed in whites. On the contrary, the other promiscuous regions (residues 161-175 and 171-185) contained epitopes not naturally processed in vitro. The immunodominant epitopes identified will be useful in the design of peptide-based cancer vaccines and in the study of the functional state of tumor-specific CD4(+) T cells in patients bearing tumors expressing MAGE-3.

Algorithms↗
PubMed2006

Predicting population coverage of T-cell epitope-based diagnostics and vaccines.

BACKGROUND: T cells recognize a complex between a specific major histocompatibility complex (MHC) molecule and a particular pathogen-derived epitope. A given epitope will elicit a response only in individuals that express an MHC molecule capable of binding that particular epitope. MHC molecules are extremely polymorphic and over a thousand different human MHC (HLA) alleles are known. A disproportionate amount of MHC polymorphism occurs in positions constituting the peptide-binding region, and as a result, MHC molecules exhibit a widely varying binding specificity. In the design of peptide-based vaccines and diagnostics, the issue of population coverage in relation to MHC polymorphism is further complicated by the fact that different HLA types are expressed at dramatically different frequencies in different ethnicities. Thus, without careful consideration, a vaccine or diagnostic with ethnically biased population coverage could result. RESULTS: To address this issue, an algorithm was developed to calculate, on the basis of HLA genotypic frequencies, the fraction of individuals expected to respond to a given epitope set, diagnostic or vaccine. The population coverage estimates are based on MHC binding and/or T cell restriction data, although the tool can be utilized in a more general fashion. The algorithm was implemented as a web-application available at http://epitope.liai.org:8080/tools/population. CONCLUSION: We have developed a web-based tool to predict population coverage of T-cell epitope-based diagnostics and vaccines based on MHC binding and/or T cell restriction data. Accordingly, epitope-based vaccines or diagnostics can be designed to maximize population coverage, while minimizing complexity (that is, the number of different epitopes included in the diagnostic or vaccine), and also minimizing the variability of coverage obtained or projected in different ethnic groups.

Algorithms↗
PubMed1997

A linear region of a monoclonal antibody conformational epitope mapped on p185HER2 oncoprotein.

Analysis of epitopes recognized by therapeutic monoclonal antibodies (mAb) is critical in clinical applications and in structure/function studies of target antigen. mAb MGr6 recognizes the extracellular domain of the p185HER2 oncoprotein and is a promising candidate for cancer immunodiagnosis and immunotherapy. Thus, epitope location and structure on p185HER2 need to be investigated. The use of MGr6-selected phage-displayed peptides for epitope analysis served to dissect the MGr6 epitope into at least two subregions, mimicked by CHSDC- and (L)P-(L)K(L) phage displayed peptides, respectively. Comparison of amino acid sequences of CHSDC peptides with the p185HER2 protein sequence and analysis of MGr6 reactivity with p185HER2 deletion mutants identified the linear subregion CCHEQCAAG of the MGr6 epitope, corresponding to amino acids 235-243 of the p185HER2 protein. This continuous subregion is part of a larger conformational epitope, and other amino acids, including a proline, a lysine and leucine residues contained in (L)P-(L)K(L) phage-displayed peptides appear to contribute to the formation of the MGr6 epitope surface.

3T3 Cells↗
PubMed2001

Epitope mapping of the indirect T cell response to allogeneic class I MHC: sequences shared by donor and recipient MHC may prime T cells that provide help for alloantibody production.

Indirect allorecognition occurs when T cells recognize donor MHC presented as peptide epitopes by recipient APC, but the precise nature of the epitopes involved remains unclear. Rejection of rat MHC class I-disparate PVG.R8 (RT1.A(a)) grafts by PVG.RT1(u) (RT1.A(u)) recipients is mediated by indirectly restricted CD4 T cells that provide help for the generation of alloantibody. In this study, epitope mapping was performed using a functionally relevant readout (alloantibody production) to identify key peptides that prime an indirect alloimmune response, leading to graft rejection. PVG.RT1(u) rats were immunized with a series of overlapping 15-mer peptides (peptides 1-18) that spanned the alpha1 and alpha2 domains of the RT1.A(a) molecule. Several peptides were able to accelerate both the alloantibody response to the intact RT1.A(a) Ag and PVG.R8 heart graft rejection. An immunodominant epitope was identified within the hypervariable region of the alpha1 domain. Fine mapping of this region with a second series of peptides overlapping by single amino acids confirmed the presence of an eight-amino acid core determinant. Additional "subdominant" epitopes were identified, two of which were located within regions of amino acid homology between the RT1.A(a) and RT1.A(u) molecules and not, as had been expected, within other hypervariable regions. The contribution of self-epitopes to indirect allorecognition was emphasized by the demonstration that i.v. administration of a 15-mer peptide encompassing one of the subdominant self-determinants diminished the recipient's ability to mount an alloantibody response on challenge with intact A(a) alloantigen. Our findings suggest that cryptic self-epitopes recognized by autoreactive T cells may contribute to allograft rejection and should be considered when designing novel strategies for inducing tolerance to alloantigen.

Amino Acid Sequence↗