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PubMed2026

Proteome-wide curation of experimentally validated HPV T-cell epitopes identifies key gaps in our understanding of cellular immunity to HPV and informs vaccine design.

BACKGROUND: Human papillomavirus (HPV) drives both malignant and benign tumours. Current prophylactic vaccines are type-restricted, not optimised for T-cell induction, and lack therapeutic efficacy. Although T-cells are critical for both preventing and clearing HPV infection, experimentally validated HPV T-cell epitopes remain fragmented across the literature, limiting systematic evaluation of cellular immune targets. METHODS: We curated experimentally validated HPV T-cell epitopes from the Immune Epitope Database (IEDB). Epitopes were mapped across HPV proteins and genotypes, and analysed for response rate, sequence conservation across 454 representative HPV genomes, and HLA restriction patterns. RESULTS: 485 unique experimentally validated HPV epitopes have been described (133 studies; 1,494 functional assays). Consistent with research focus and viral biology, E6 and E7 proteins account for >60% of known HPV epitopes despite accounting for ~10% of the viral proteome. High-risk HPV types, especially HPV16 and HPV18, were the most studied (p <.001) and were enriched for CD8+ epitopes (p <.001). We identified major knowledge gaps, including: underrepresentation of structural proteins such as L2; limited epitope coverage for low-prevalence HPV genotypes; a bias towards common HLA alleles. In silico analysis indicated greater conservation of epitopes in L1/L2 and across high-risk HPV types. Conserved, commonly detected, and HLA-promiscuous epitopes were highlighted and we provide panels of candidate epitopes for consideration in immune monitoring, broad-spectrum prophylactic vaccines, and high-risk targeted therapeutic vaccines. CONCLUSION: This study provides the first comprehensive atlas of experimentally validated HPV T-cell epitopes and ranked epitope candidates for translational application. We demonstrate that our understanding of HPV T-cell immunity is constrained by biases in antigen, genotype and HLA focus and by incomplete epitope mapping. Addressing these gaps will be essential for a comprehensive assessment of cellular immunity and for utilising T-cells in next-generation vaccines.

Epitopes, T-Lymphocyte↗

PubMed1996

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 coimmunization 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.

Animals↗

PubMed2005

Inefficient cross-presentation limits the CD8+ T cell response to a subdominant tumor antigen epitope.

CD8(+) T lymphocytes (T(CD8)) responding to subdominant epitopes provide alternate targets for the immunotherapy of cancer, particularly when self-tolerance limits the response to immunodominant epitopes. However, the mechanisms that promote T(CD8) subdominance to tumor Ags remain obscure. We investigated the basis for the lack of priming against a subdominant tumor epitope following immunization of C57BL/6 (B6) mice with SV40 large tumor Ag (T Ag)-transformed cells. Immunization of B6 mice with wild-type T Ag-transformed cells primes T(CD8) specific for three immunodominant T Ag epitopes (epitopes I, II/III, and IV) but fails to induce T(CD8) specific for the subdominant T Ag epitope V. Using adoptively transferred T(CD8) from epitope V-specific TCR transgenic mice and immunization with T Ag-transformed cells, we demonstrate that the subdominant epitope V is weakly cross-presented relative to immunodominant epitopes derived from the same protein Ag. Priming of naive epitope V-specific TCR transgenic T(CD8) in B6 mice required cross-presentation by host APC. However, robust expansion of these T(CD8) required additional direct presentation of the subdominant epitope by T Ag-transformed cells and was only significant following immunization with T Ag-expressing cells lacking the immunodominant epitopes. These results indicate that limited cross-presentation coupled with competition by immunodominant epitope-specific T(CD8) contributes to the subdominant nature of a tumor-specific epitope. This finding has implications for vaccination strategies targeting T(CD8) responses to cancer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Characterization of intertype specific epitopes on adenovirus hexons.

The specification and differentiation of eighteen intertype specific (IT) epitopes of adenovirus hexons defined by monoclonal antibodies are given by two groups of hexon types: on which they are and on which they are not present. The close specificity relationship among some groups of epitopes determined by pairwise analysis according to their presence on the hexon types indicate that they could be continuous (sequential), overlapping or discontinuous (topographic) epitopes. Based on the identification of the hydrophilic regions and the localization of beta-turns sixteen IT epitope sites were predicted on human adenovirus (HAdV) type 2 and nineteen on HAdV-41 hexon's amino acid (aa) sequences beside the type and genus specific ones. The 16 predicted IT epitopes on HAdV-2 hexon show good coincidency with the 14 IT epitopes demonstrated with monoclonal antibodies (MAbs) on this hexon type. The predicted number of common epitopes between HAdV types 2 and 41 also corresponds well with the eight IT epitopes determined by MAbs, but the 17 predicted non-common epitopes indicate the possibility of the existence of more epitopes on these hexon types. The location of the predicted epitopes of HAdV-2 were determined by alignment of their sequence numbers on the three dimensional ribbon representation of the hexon subunit. Most of the predicted IT epitopes were found between the type and genus specific epitopes i.e. in the "upper" regions of pedestal domains P1 and P2 orientated toward the virion surface and in the "lower" part of loop 1 region orientated inside the virion. Two peptides representing potential IT epitopes were synthesized corresponding to residues 309-320 from the "lower" part of loop 1 and 399-408 from the "upper" part of P1 region of HAdV-2 hexon. Antibodies raised against the peptide-carrier conjugates recognized different purified native hexon types in ELISA.

Adenoviruses, Human↗

PubMed2000

Multiepitope vaccines intensively increased levels of antibodies recognizing three neutralizing epitopes on human immunodeficiency virus-1 envelope protein.

A few neutralizing antibodies against human immunodeficiency virus-1 (HIV-1) envelope proteins have been shown to be highly effective at neutralizing different strains in vitro, and exist at very low levels in the sera of HIV-1-infected individuals. Based on our hypothesis that epitope vaccination may be a novel strategy for inducing high levels of antibodies against HIV-1, we prepared multiepitope vaccines using three neutralizing epitopes (GPGRAFY, ELDKWA and RILAVERYLKD) on HIV-1 envelope proteins. The PI [C-G-(ELDKWA-GPGRAFY)2-K] and PII (CG-GPGRAFY-G-ELDKWA-G-RILAVERYLKD) peptides were synthesized and conjugated to a carrier protein, bovine serum albumin (BSA). After vaccination, both the PI-BSA and PII-BSA multiepitope vaccines induced high levels of epitope-specific antibodies to the three neutralizing epitopes (antibody titre: 1 : 12,800-102,400). The recombinant glycoprotein 160 (rgp160) subunit vaccine induced strong antibody responses to rgp160, but only very weak epitope-specific antibody responses to the three epitopes. The epitope-specific antibodies were isolated from rabbit sera by single epitope-peptide-conjugated sepharose columns. A yield of 51 microg of epitope-specific antibodies/ml of serum (mean value) was obtained and identified to recognize these epitopes, while 0.35 microg of protein was isolated from 1 ml of pooled preserum by C-(ELDKWAG)4- or C-(RILAVERYLKD-G)2-K- and C-(GPGRAFY)4-sepharose columns. The levels of these epitope-specific antibodies induced in rabbits were much greater than 1 microg/ml, a level that is considered to confer long-term protection against some viruses. Moreover, these antibodies recognized the neutralizing epitopes on peptides and rgp41. Based on the fact that a very low level of ELDKWA epitope-specific antibodies exist in HIV-1-infected individuals, these results suggesting that synthetic epitope vaccines could induce high levels of multiepitope-specific neutralizing antibodies indicate a new strategy for developing an effective neutralizing antibody-based epitope/peptide vaccine against HIV-1.

AIDS Vaccines↗

PubMed1992

p185 HER2/neu epitope mapping with murine monoclonal antibodies.

In order to obtain further information on the biological role of the HER2/neu oncoprotein, 7 new monoclonal antibodies (MAbs) were produced against the p185HER2 extracellular domain. These MAbs, together with two others previously produced, were used to investigate the p185HER2 expression in breast carcinomas and compare the recognized antigenic determinants. The 7 reagents (MGR4,5,6,7,8,9 and 10), were shown to define five distinct epitopes. Three of these MAbs (MGR5,7,10), as well as one previously produced (MGR2), recognize the same epitope (Epitope-1) which seems, therefore, to be highly immunogenic for the murine immune system. Epitope-2 recognized by the MGR4 MAb, appears to be closely related to epitope-1 due to a cross inhibition between MGR4 and MGR10, but not MGR2. Epitope-2 is the only one of the 5 also present on the product of the neu oncogene, the rat analogue of the human HER2/neu gene. None of the reagents against epitope-1 and epitope-2 were found to mediate receptor internalization, whereas MGR6 as well as a previously produced MAb (MGR3), both of which define epitope-3 and MGR8 which defines epitope-4, were found to do so. Epitope-4 like the neu-specific peptide recognized by the reference c-neu Ab3 MAb, was detectable on all p185HER2 positive breast cancer, independently from the quantitative content of the oncoprotein, at variance with the other 4 epitopes whose availability on p185HER2 for the relevant MAbs varied with the degree of overexpression. Epitope-5, recognized by the MGR9 MAb, on the contrary to the other epitopes, was prevalently localized at the basal membrane level of the tumor nodule.

Animals↗

PubMed1997

Biochemical characterization of Candida albicans epitopes that can elicit protective and nonprotective antibodies.

We previously reported that the immunoglobulin M (IgM) monoclonal antibody (MAb) B6.1 protects mice against disseminated candidiasis, whereas the IgM MAb B6 does not. Both MAbs are specific for an adhesin fraction isolated from the cell surface of Candida albicans, but their epitope specificities differ. In the present study, we examined the surface locations of both epitopes and obtained structural information regarding the B6.1 epitope. Immunofluorescence confocal microscopic analysis of C. albicans yeast forms showed that epitope B6.1 is displayed rather homogeneously over the entire cell surface, whereas epitope B6 appears to have a patchy distribution. Both antibodies were essentially nonreactive with the surfaces of mycelial forms of the fungus, indicating that neither epitope is expressed on the surfaces of these forms. For isolation of the B6.1 epitope, the adhesin fraction consisting of cell surface phosphomannan was subjected to mildly acidic (10 mM HCl) hydrolysis and was fractionated into acid-labile and acid-stable portions by size exclusion chromatography. Antibody blocking experiments showed that the B6.1 epitope is an acid-labile moiety of the phosphomannan and that the B6 epitope is located in the acid-stable fraction. The B6 epitope appeared to be mannan because it was stable to heat (boiling) and protease treatments but was destroyed by alpha-mannosidase digestion. The B6.1 epitope eluted from the size exclusion column in two fractions. Mass spectroscopic analyses showed that one fraction contained material with the size of a mannotriose and that the other was a mixture of mannotriose- and mannotetraose-size substances. Dose response inhibition tests of the fractions indicated that the B6.1 epitope is associated with the mannotriose. Nuclear magnetic resonance (NMR) spectroscopic analysis of the epitope yielded data consistent with a beta-(1-->2)-linked mannotriose. The fine structure of the B6 epitope is under investigation. Information derived from these investigations will be useful both in understanding protective versus nonprotective antibody responses to C. albicans and in improving anti-Candida vaccine formulations.

Agglutination Tests↗

PubMed2006

CED: a conformational epitope database.

BACKGROUND: Antigen epitopes provide valuable information useful for disease prevention, diagnosis, and treatment. Recently, more and more databases focusing on different types of epitopes have become available. Conformational epitopes are an important form of epitope formed by residues that are sequentially discontinuous but close together in three-dimensional space. These epitopes have implicit structural information, making them attractive for both theoretical and applied biomedical research. However, most existing databases focus on linear rather than conformational epitopes. DESCRIPTION: We describe CED, a special database of well defined conformational epitopes. CED provides a collection of conformational epitopes and related information including the residue make up and location of the epitope, the immunological property of the epitope, the source antigen and corresponding antibody of the epitope. All entries in this database are manually curated from articles published in peer review journals. The database can be browsed or searched through a user-friendly web interface. Most epitopes in CED can also be viewed interactively in the context of their 3D structures. In addition, the entries are also hyperlinked to various databases such as Swiss-Prot, PDB, KEGG and PubMed, providing wide background information. CONCLUSION: A conformational epitope database called CED has been developed as an information resource for investigators involved in both theoretical and applied immunology research. It complements other existing specialised epitope databases. The database is freely available at http://web.kuicr.kyoto-u.ac.jp/~ced.

Animals↗

PubMed1996

Dimorphic sequences of rap-1 genes encode B and CD4+ T helper lymphocyte epitopes in the Babesia bigemina rhoptry associated protein-1.

The rhoptry-associated protein-1 (RAP-1) of Babesia bigemina induces protective immune responses in cattle and contains neutralization-sensitive B cell epitopes. RAP-1 variants containing blocks of sequence dimorphism in the amino and carboxy terminal ends are encoded by four nonallelic genes in B. bigemina. Epitopes recognized by RAP-1 specific monoclonal antibodies (MAbs) and bovine CD4+ T cell clones were mapped to determine whether these epitopes are localized in the amino and carboxy terminal dimorphic regions. Four B cell epitopes, including a neutralization-sensitive epitope, required both the amino terminal variant type 1 (NT-1) and non-dimorphic sequences for conformation. Intrachain disulfide bonds were required for at least one of these epitopes, since reduction and alkylation of cysteine residues abolished MAb binding. A fifth B cell epitope was mapped to the carboxy terminal variant type 1 (CT-1). As expected, the neutralizing MAb and two other MAbs requiring NT-1 for epitope binding recognized only the two RAP-1 variants with the NT-1 sequence, while the MAb binding an epitope in CT-1 did not bind RAP-1 variants with CT-2. In contrast, the fourth MAb requiring NT-1 for binding recognized all rap-1 gene products, indicating that dimorphic residues are not part of the epitope recognized by this MAb. Bovine CD4+ T cell clones characterized previously as responding in a strain dependent fashion recognized at least one epitope in CT-1, and did not cross-react with CT-2. A second group of bovine CD4+ T cell clones that responded to multiple parasite strains recognized an epitope in a non-dimorphic region of RAP-1. These data indicate that dimorphic regions of RAP-1 encode unique B and T helper lymphocyte epitopes and may be required for enhanced protective immune responses in cattle.

Animals↗

PubMed2002

Definition of permissible and immunogenic HLA antigens based on epitope analysis of the HLA specific antibodies produced in sensitized patients.

The goal of this study was to develop an accurate protocol whereby detection of acceptable HLA-A and -B mismatches is based on epitope analysis of HLA class I specific antibodies detected in the serum of highly sensitized patients awaiting a kidney retransplant. A total of 400 serum samples from 44 highly sensitized patients with panel reactive antibodies (PRA) of > or = 60% were collected during a 3-year follow-up period. All patients had been sensitized from a previous graft. In order to define the specificities of the HLA class I specific antibodies, two techniques were used in parallel: the antihuman globulin augmented complement-dependent cytotoxicity (CDC) technique and an enzyme-linked immunoabsorbent assay (ELISA) technique. Epitope identification was based on class I HLA antigen sequencing, where the unique epitope configuration on one HLA antigen represented the private epitope of the specific HLA antigen, and epitopes shared by more than one HLA antigen represented public determinants. The epitope prediction for the immunogenic HLA epitopes was based on an MHC database. For each highly sensitized patient, antibody specificities against actual and 'at risk' epitopes were defined. Following epitope analysis, all HLA antigens that did not express the actual and/or 'at risk' immunogenic epitopes were considered as acceptable mismatches of epitope analysis. The cytotoxicity of highly sensitized patients was determined using two different panels of selected, separated T lymphocytes. HLA class I specific IgG antibodies against 69 actual and 86 'at risk' epitopes were detected. In all patients, a large number of acceptable mismatches were defined. These included a large number of HLA antigens, corresponding to both HLA-A and -B loci. Our study introduces an accurate protocol for the detection of acceptable mismatches in highly sensitized patients. According to this protocol, the detailed description of immunogenic HLA specific epitope targets, against which HLA class I specific antibodies are directed, is a useful tool for the detection of acceptable mismatches in highly sensitized patients. This may lead to reduced production of HLA class I specific antibodies and, consequently, improved graft survival.

Adolescent↗

PubMed1997

Sequences of antigenic epitopes of streptokinase identified via random peptide libraries displayed on phage.

Though streptokinase (SK) is widely used to treat humans with thrombotic disease, it is antigenic and anti-SK antibody causes allergic reactions and neutralizes SK's therapeutic effects. To pinpoint the fine structure of two immunodominant, continuous epitopes in SK, we used unconstrained 15 and 6-mer random peptide libraries displayed on phage (theoretical complexity of 3.2 x 10(19) and 0.64 x 10(8) unique sequences). The first epitope, recognized by both human Ab and murine monoclonal (m)Abs, was previously localized to the amino terminus of SK. Repeated panning and selection experiments against a 15-mer peptide phage library, using a representative mAb (A2.5) to this epitope, identified a dominant structural motif (GP[R/L]WL) corresponding to amino acids 3 to 7 of native SK, which was consistent with previous epitope mapping. These findings were further confirmed by: (1) the fact that a synthetic peptide spanning the epitope of A2.5 (AGPEWLL) specifically inhibited the binding of A2.5 to SK and (2) the finding that mAb 9D10, which competes with mAb A2.5 for binding to SK, independently selected, from a different random hexamer library, an epitope sequence spanning residues 4 to 9 that overlaps the A2.5 epitope. Similar studies of the second epitope in SK, which is immunodominant for murine but not human antibodies, identified a consensus sequence KS(K/L)P(F/Y) corresponding to amino acids 59 to 63 of SK; this was confirmed by epitope peptide binding experiments. This epitope is cleaved and destroyed when SK reacts with human but not murine plasminogen. Thus, pinpointing the sequences of antigenic epitopes of SK: (1) provides a potential explanation for species differences in SK's antigenicity, (2) demonstrates the overlapping fine structure of epitopes recognized by competitive mAbs, (3) confirms previous epitope mapping studies and (4) has the potential to identify antigenic sequences that lead to allergic reactions in patients treated with SK.

Amino Acid Sequence↗

PubMed1989

Neutralizing epitopes of lymphocytic choriomeningitis virus are conformational and require both glycosylation and disulfide bonds for expression.

Lymphocytic choriomeningitis virus (Armstrong strain) bears two overlapping epitopes, GP-1A (A) and GP-1D (D), recognized by neutralizing antibodies on the major surface glycoprotein GP-1. Both are discontinuous conformational epitopes that require prior formation of disulfide bridges and addition of N-linked oligosaccharides. Using monoclonal antibodies specific for each of these epitopes, as well as for conformation-independent epitopes, we have investigated the requirements for biosynthesis and folding of the epitopes. The carbohydrate residues themselves do not appear to comprise critical informational components of these epitopes, but are required for proper folding of the nascent glycopeptide chain within the rough endoplasmic reticulum. These epitopes differ in their resistance to denaturation; epitope D is retained when denatured with SDS under nonreducing conditions, whereas epitope A is lost. Monoclonal antibodies to epitope A cross-react with several strains of LCMV. However, epitope D is detected in only a subset of isolates derived from the Armstrong strain of LCMV. By RNA sequence analysis, we have mapped a single amino acid change distinguishing those virions containing epitope D. Acquisition of binding activity of the epitope D-specific monoclonal correlates with a Thr----Ala or Thr----Lys mutation at amino acid 173 of the GP-1 molecule and concomitant disruption of a consensus N-linked glycosylation site.

Amino Acid Sequence↗

PubMed2005

Epitope mapping and functional analysis of sigma A and sigma NS proteins of avian reovirus.

We have previously shown that avian reovirus (ARV) sigmaA and sigmaNS proteins possess dsRNA and ssRNA binding activity and suggested that there are two epitopes on sigmaA (I and II) and three epitopes (A, B, and C) on sigmaNS. To further define the location of epitopes on sigmaA and sigmaNS proteins and to further elucidate the biological functions of these epitopes by using monoclonal antibodies (MAbs) 62, 1F9, H1E1, and 4A123 against the ARV S1133 strain, the full-length and deletion fragments of S2 and S4 genes of ARV generated by polymerase chain reaction (PCR) were cloned into pET32 expression vectors and the fusion proteins were overexpressed in Escherichia coli BL21 strain. Epitope mapping using MAbs and E. coli-expressed deletion fragments of sigmaA and sigmaNS of the ARV S1133 strain, synthetic peptides, and the cross reactivity of MAbs to heterologous ARV strains demonstrated that epitope II on sigmaA was located at amino acid residues 340QWVMAGLVSAA350 and epitope B on sigmaNS at amino acid residues 180MLDMVDGRP188. The MAbs (62, 1F9, and H1E1) directed against epitopes II and B did not require the native conformation of sigmaA and sigmaNS, suggesting that their binding activities were conformation-independent. On the other hand, MAb 4A123 only reacted with complete sigmaNS but not with truncated sigmaNS fusion proteins in Western blot, suggesting that the binding activity of MAb to epitope A on sigmaNS was conformation-dependent. Amino acid sequence analysis and the binding assays of MAb 62 to heterologous ARV strains suggested that epitope II on sigmaA was highly conserved among ARV strains and that this epitope is suitable as a serological marker for the detection of ARV antibodies following natural infection in chickens. On the contrary, an amino acid substitution at position 183 (M to V) in epitope B of ARV could hinder the reactivity of the sigmaNS with MAb 1F9. The sigmaNS of ARV with ssRNA-binding activity could be blocked by monoclonal antibody 1F9. The epitope B on sigmaNS is required for ssRNA binding because its deletion fully abolished the ssRNA binding activity of sigmaNS.

Amino Acid Sequence↗

PubMed2002

Stimulatory and inhibitory epitopes in the T cell responses of mice to Der p 1.

BACKGROUND: The responses of mice to the mite allergen Der p 1 have been used to study the mechanisms of allergic sensitization and the development of new types of immunotherapy. Many of the studies require a knowledge of the T cell epitopes, and because Der p 1 is polymorphic, the effect of natural amino acid substitution in the allergen. The intranasal administration of peptides containing T cell epitopes can induce a mucosal tolerance but it is not known if the major activity is limited to stimulatory peptides and if, as found for autoimmunity, some epitopes are not inhibitory. OBJECTIVE: To determine and compare the sequences of Der p 1 which contain stimulatory epitopes for the high responding H-2(b) and H-2(q) mice and the sequences which induce tolerance by intranasal administration of peptides. METHODS: T cell responses of mice immunized with Der p 1 were measured by in vitro T cell stimulation assays so an extensive study of epitope recognition and intranasal tolerance could be made. Synthetic peptides were used to examine the stimulatory and inhibitory ability of all Der p 1 sequences and to map the major H-2(b) epitope in detail. This included the effect of the common polymorphic amino acid 124 substitution found within this epitope. RESULTS: Three and two regions, respectively, were found to contain stimulatory T cell epitopes for H-2(b) and H-2(q) mice. The peptides in these regions were also the most active at inducing intranasal tolerance for the responding haplotype. The correspondence between inhibitory and stimulatory peptides was maintained for the fine mapping of the major H-2(b) epitope. This was found about a core region of 118-126 which was overlapping but separate to a consensus sequence for the binding of endogeneous peptides. Peptides with alanine at the naturally polymorphic residue 124 stimulated and inhibited responses to Der p 1 more effectively, while peptides with the valine 124 variant were immunogenic but poorly cross-reactive. CONCLUSIONS: The intranasal administration of peptides representing each of five epitopes recognized by two strains of mice were able to induce mucosal tolerance and the major tolerizing activity was limited to these epitopes. The position of the core major epitope for C57 mice, which differs from a previously predicted epitope, and its specificity for the natural alanine 124 variant is described.

Administration, Intranasal↗

PubMed2007

Ab and T cell epitopes of influenza A virus, knowledge and opportunities.

The Immune Epitope Database and Analysis Resources (IEDB) (www.immuneepitope.org) was recently developed to capture epitope related data. IEDB also hosts various bioinformatics tools that can be used to identify novel epitopes as well as to analyze and visualize existing epitope data. Herein, a comprehensive analysis was undertaken (i) to compile and inventory existing knowledge regarding influenza A epitopes and (ii) to determine possible cross-reactivities of identified epitopes among avian H5N1 and human influenza strains. At present, IEDB contains >600 different epitopes derived from 58 different strains and 10 influenza A proteins. By using the IEDB analysis resources, conservancy analyses were performed, and several conserved and possibly cross-reactive epitopes were identified. Significant gaps in the current knowledge were also revealed, including paucity of Ab epitopes in comparison with T cell epitopes, limited number of epitopes reported for avian influenza strains/subtypes, and limited number of epitopes reported from proteins other than hemagglutinin and nucleoprotein. This analysis provides a resource for researchers to access existing influenza epitope data. At the same time, the analysis illustrates gaps in our collective knowledge that should inspire directions for further study of immunity against the influenza A virus.

Animals↗

PubMed1993

Autoreactive epitopes defined by diabetes-associated human monoclonal antibodies are localized in the middle and C-terminal domains of the smaller form of glutamate decarboxylase.

The gamma-aminobutyrate-synthesizing enzyme glutamate decarboxylase (GAD; L-glutamate 1-carboxy-lyase, EC 4.1.1.15) is a major target of autoantibodies associated with both early and late stages of pancreatic beta-cell destruction and development of type 1 diabetes. We have used five monoclonal anti-islet-cell antibodies (MICAs 1,2,3,4, and 6) derived from a newly diagnosed diabetic patient to probe the autoimmune epitopes in the enzyme. All the MICAs specifically recognized the smaller GAD protein, GAD65, and did not recognize the nonallelic GAD67 protein. A series of N-terminal, C-terminal, and internal deletion mutants, as well as protein footprinting, were used to identify the target regions in GAD65. Immunoprecipitation revealed two major native epitope areas in the GAD65 molecule. The first, defined by MICAs 1 and 3, is destroyed by deleting 41 amino acids at the C terminus but is also dependent on intact amino acids 244-295. This epitope (or epitopes) may span both middle and C-terminal domains of the protein. The second conformational epitope region, defined by MICAs 4 and 6, is dependent on intact amino acids 245-295 but is not affected by deletion of 110 amino acids at the C terminus and is therefore confined to domain(s) in the middle of the molecule. MICA 2 recognizes a linear epitope close to the C terminus. Thus, the N-terminal domain of GAD65, which differs most significantly from GAD67, does not harbor the MICA epitopes. Rather subtle amino acid differences in the middle and C-terminal domains define the GAD65-specific autoimmune epitopes. Analysis of sera from 10 type 1 diabetic patients suggests that MICAs 1, 3, 4, and 6 represent a common epitope recognition in this disease, whereas the MICA 2 epitope is rare. Furthermore, autoantibodies in some sera are restricted to the MICA 1/3 epitope, suggesting that this epitope may represent a single dominant epitope in the early phases of beta-cell autoimmunity.

Animals↗

PubMed2006

Effective induction of type 1 cytotoxic T cell responses in mice with DNA vaccine encoding two hepatitis C virus cytotoxic T lymphocyte epitopes.

The aims of this study were to explain whether a multiple cytotoxic T lymphocyte (CTL) epitope-based anti-hepatitis C virus (HCV) DNA vaccine can induce specific CTL responses to each HCV CTL epitope independently and long-term CD8(+) T cell memory responses, and to determine the cytokine secretion pattern and subtype of epitope-specific cytotoxic T cells. A multi-CTL epitope gene, which consists of two epitopes of HCV (H-2(d)-restricted HCV core(133142) and E1(315322)), was cloned into the eukaryotic expression vector pcDNA3.1. BALB/c mice (H-2(d) restricted) were vaccinated intramuscularly with this multi-CTL epitope-based DNA vaccine. The epitope-specific CTLs against target cells (P815,H-2(d) restricted) pulsed with various CTL epitope peptides were detected by lactate dehydrogenase release assay, and the precursor frequency of epitope-specific CTLs was determined by limiting dilution analysis. Cytokines (interleukin [IL]-2, IL-4, and interferon-) in culture supernatants were determined by enzyme-linked immunosorbent assay. The multi-CTL epitope-based DNA vaccine directed against two HCV CTL epitopes could induce specific CTL responses to each of the two CTL epitopes independently and long-term CD8(+) T cell memory responses. The epitope-specific cytotoxic T cells produced helper T cell type 1 cytokines. This work demonstrated that multiepitope DNA vaccination is a potential strategy to control HCV infection.

Animals↗

PubMed1995

All rabbits immunized with type A influenza virions have a serum haemagglutination-inhibition antibody response biased to a single epitope in antigenic site B.

Nine rabbits were immunized with type A influenza virions and the epitope specificities of the secondary serum haemagglutination-inhibition (HI) antibody response were analysed with a panel of neutralizing monoclonal (MAb) antibody double escape mutants. Each of the latter was made by sequential selection using a MAb directed to an epitope of a discrete antigenic site, site A, site B or site D, of the haemagglutinin (HA). Thus the epitope reactivity of the escape mutants was represented as A+ B- D-, A- B+ D- and A- B- D+. The HI antibody response of all antisera was biased to the site B epitope. In 9/12 antisera, obtained from seven rabbits immunized with whole virions, the site B epitope was predominant, representing 65-82% of the total HI antibody. The restriction of HI antibody was unaffected by strain of rabbit, route of inoculation (intravenous or subcutaneous), use of Freund's adjuvant, and up to four immunizing injections. In 3/7 rabbits immunized with whole virus, there was a HI antibody response to the HC2 (site A) or HC10 (site D) epitope, but not both, of equal magnitude to the site B epitope. The HI antibody response in one of the rabbits (#40) became more biased to the site B epitope between the third and fourth immunizing doses. Two further rabbits were immunized with virions which had been partially digested with bromelain and then purified from free HA. Both of these made equal HI antibody responses to the site B epitope and the site D epitope, possibly because their remaining HA spikes were better exposed. Overall, these data demonstrate an unexpected degree of restriction in the production of biologically relevant antibody, such that some rabbits (e.g. #45) mount an HI antibody response which is essentially epitope-specific. Implications for epitope specificity of HI antibody stimulated by human influenza vaccines, and also for the generation of antigenic drift variants are discussed. The reason for the non-responsiveness of the immune system to the many other HI epitopes of the HA is not known.

Animals↗