Functional characterization of CTL against gp100 altered peptide ligands

In this study, four modified gp100 peptides were designed by combining amino acids from the melanoma peptide antigen gp100((209-217)) with preferred primary and auxiliary HLA-A *0201 anchor residues previously identified from combinatorial peptide library screening with recombinant HLA-A*0201. These modified peptides demonstrated stronger binding affinity for the HLA-A*0201 molecule compared to wild-type gp100 peptide. Nine CTL lines generated from patients immunized with the g209-2 M peptide and one CTL line from a non-immunized patient were tested for the ability to respond to these modified gp100 peptides. Stimulation of CTL by two of four modified peptides induced higher levels of IFN-gamma secretion than the wild-type gp100 peptide, demonstrating that higher peptide binding affinity for HLA molecules does not necessarily equate to functional activity of CTL. Two major and one minor CTL recognition pattern were observed, irrespective of previous peptide immunization, suggesting that multiple, rationally designed modified tumor peptides for the same epitope stimulate a broad CTL response by activating multiple CTL capable of cross-reacting with the natural antigenic peptide.     

Random screening of proteins for HLA-A*0201-binding nine-amino acid peptides is not sufficient for identifying CD8 T cell epitopes recognized in the context of HLA-A*0201

HLA-A2 is the most frequent HLA molecule in Caucasians with HLA-A*0201 representing the most frequent allele; it was also the first human HLA allele for which peptide binding prediction was developed. The Bioinformatics and Molecular Analysis Section of the National Institutes of Health (BIMAS) and the University of Tübingen (Syfpeithi) provide the most popular prediction algorithms of peptide/MHC interaction on the World Wide Web. To test these predictions, HLA-A*0201-binding nine-amino acid peptides were searched by both algorithms in 19 structural CMV proteins. According to Syfpeithi, the top 2% of predicted peptides should contain the naturally presented epitopes in 80% of predictions (www.syfpeithi.de). Because of the high number of predicted peptides, the analysis was limited to 10 randomly chosen proteins. The top 2% of peptides predicted by both algorithms were synthesized corresponding to 261 peptides in total. PBMC from 10 HLA-A*0201-positive and CMV-seropositive healthy blood donors were tested by ex vivo stimulation with all 261 peptides using crossover peptide pools. IFN-gamma production in T cells measured by CFC was used as readout. However, only one peptide was found to be stimulating in one single donor. As a result of this work, we report a potential new T cell target protein, one previously unknown CD8-T cell-stimulating peptide, and an extensive list of CMV-derived potentially strong HLA-A*0201-binding peptides that are not recognized by T cells of HLA-A*0201-positive CMV-seropositive donors. We conclude that MHC/peptide binding predictions are helpful for locating epitopes in known target proteins but not necessarily for screening epitopes in proteins not known to be T cell targets.     

Coupling in silico and in vitro analysis of peptide-MHC binding: a bioinformatic approach enabling prediction of superbinding peptides and anchorless epitopes

The ability to define and manipulate the interaction of peptides with MHC molecules has immense immunological utility, with applications in epitope identification, vaccine design, and immunomodulation. However, the methods currently available for prediction of peptide-MHC binding are far from ideal. We recently described the application of a bioinformatic prediction method based on quantitative structure-affinity relationship methods to peptide-MHC binding. In this study we demonstrate the predictivity and utility of this approach. We determined the binding affinities of a set of 90 nonamer peptides for the MHC class I allele HLA-A*0201 using an in-house, FACS-based, MHC stabilization assay, and from these data we derived an additive quantitative structure-affinity relationship model for peptide interaction with the HLA-A*0201 molecule. Using this model we then designed a series of high affinity HLA-A2-binding peptides. Experimental analysis revealed that all these peptides showed high binding affinities to the HLA-A*0201 molecule, significantly higher than the highest previously recorded. In addition, by the use of systematic substitution at principal anchor positions 2 and 9, we showed that high binding peptides are tolerant to a wide range of nonpreferred amino acids. Our results support a model in which the affinity of peptide binding to MHC is determined by the interactions of amino acids at multiple positions with the MHC molecule and may be enhanced by enthalpic cooperativity between these component interactions.     

Identification of a novel HLA-A2-restricted cytotoxic T lymphocyte epitope from cancer-testis antigen PLAC1 in breast cancer

Identification of cytotoxic T lymphocyte (CTL) epitopes from tumor antigens is essential for the development of peptide vaccines against tumor immunotherapy. Among all the tumor antigens, the caner-testis (CT) antigens are the most widely studied and promising targets. PLAC1 (placenta-specific 1, CT92) was considered as a novel member of caner-testis antigen, which expressed in a wide range of human malignancies, most frequently in breast cancer. In this study, three native peptides and their analogues derived from PLAC1 were predicted by T cell epitope prediction programs including SYFPEITHI, BIMAS and NetCTL 1.2. Binding affinity and stability assays in T2 cells showed that two native peptides, p28 and p31, and their analogues (p28-1Y9?V, p31-1Y2L) had more potent binding activity towards HLA-A*0201 molecule. In ELISPOT assay, the CTLs induced by these four peptides could release IFN-γ. The CTLs induced by these four peptides from the peripheral blood mononuclear cells (PBMCs) of HLA-A*02⁺ healthy donor could lyse MCF-7 breast cancer cells (HLA-A*0201⁺, PLAC1⁺) in vitro. When immunized in HLA-A2.1/K^b transgenic mice, the peptide p28 could induce the most potent peptide-specific CTLs among these peptides. Therefore, our results indicated that the peptide p28 (VLCSIDWFM) could serve as a novel candidate epitope for the development of peptide vaccines against PLAC1-positive breast cancer.     

Identification of a new HLA-A*0201-restricted cytotoxic T lymphocyte epitope from CML28

Identification of cytotoxic T lymphocyte (CTL) epitopes from additional tumor antigens is essential for the development of specific immunotherapy of malignant tumors. CML28, a recently discovered cancer-testis (CT) antigen from chronic myelogenous leukemia, is considered to be a promising target of tumor-specific immunotherapy. Because HLA-A*0201 is one of the most common histocompatibility molecule in Chinese, we aim at identifying CML28 peptides presented by HLA-A*0201. A panel of CML28-derived antigenic peptides was predicted using a computer-based program. Four peptides with highest predicted score were synthesized and tested for their binding affinities to HLA-A*0201 molecule. Then these peptides were assessed for their immunogenicity to elicit specific immune responses mediated by CTLs both in vitro, from PBMCs sourced from four healthy HLA-A*0201⁺ donors, and in vivo, in HLA-A*0201 transgenic mice. One of the tested peptides, CML28_(173–181), induced peptide-specific CTLs in vitro as well as in vivo, which could specifically secrete IFN-γ and lyse major histocompatibility complex (MHC)-matched tumor cell lines endogenously expressing CML28 antigen and CML28_(173–181) pulsed Jurkat-A2/Kb cells, respectively. These results demonstrate that CML28_(173–181) is a naturally processed and presented CTL epitope with HLA-A*0201 motif and has a promising immunogenicity both in vitro and in vivo. As CML28 is expressed in a large variety of histological tumors besides chronic myelogenous leukemia, we propose that the newly identified epitope, CML28_(173–181), would be of potential use in peptide-based, cancer-specific immunotherapy against a broad spectrum of tumors.     

A long peptide from MELOE-1 contains multiple HLA class II T cell epitopes in addition to the HLA-A*0201 epitope: an attractive candidate for melanoma vaccination

CD4⁺ T cells contribute importantly to the antitumor T cell response, and thus, long peptides comprising CD4 and CD8 epitopes may be efficient cancer vaccines. We have previously identified an overexpressed antigen in melanoma, MELOE-1, presenting a CD8⁺ T cell epitope, MELOE-1_36–44, in the HLA-A*0201 context. A T cell repertoire against this epitope is present in HLA-A*0201+ healthy subjects and melanoma patients and the adjuvant injection of TIL containing MELOE-1 specific CD8⁺ T cells to melanoma patients was shown to be beneficial. In this study, we looked for CD4⁺ T cell epitopes in the vicinity of the HLA-A*0201 epitope. Stimulation of PBMC from healthy subjects with MELOE-1_26–46 revealed CD4 responses in multiple HLA contexts and by cloning responsive CD4⁺ T cells, we identified one HLA-DRβ1*1101-restricted and one HLA-DQβ1*0603-restricted epitope. We showed that the two epitopes could be efficiently presented to CD4⁺ T cells by MELOE-1-loaded dendritic cells but not by MELOE-1+ melanoma cell-lines. Finally, we showed that the long peptide MELOE-1_22–46, containing the two optimal class II epitopes and the HLA-A*0201 epitope, was efficiently processed by DC to stimulate CD4⁺ and CD8⁺ T cell responses in vitro, making it a potential candidate for melanoma vaccination.     

CTL recognition of a novel HLA-A*0201-binding peptide derived from glioblastoma multiforme tumor cells

Genetic instability of tumor cells can result in translation of proteins that are out of frame, resulting in expression of neopeptides. These neopeptides are not self-proteins and therefore should be immunogenic. By eluting peptides from human glioblastoma multiforme (GBM) tumor cell surfaces and subjecting them to tandem mass spectrometry, we identified a novel peptide (KLWGLTPKVTPS) corresponding to a frameshift in the 3′ beta-hydroxysteroid dehydrogenase type 7 (HSD3B7) gene. HLA-binding algorithms predicted that a 9-amino acid sequence embedded in this peptide would bind to HLA-A*0201. We confirmed this prediction using an HLA-A*0201 refolding assay followed by live cell relative affinity assays, but also showed that the 12-mer binds to HLA-A*0201. Based on the 9-mer sequence, optimized peptide ligands (OPL) were designed and tested for their affinities to HLA-A*0201 and their abilities to elicit anti-peptide and CTL capable of killing GBM in vitro. Wild-type peptides as well as OPL induced anti-peptide CTL as measured by IFN-γ ELISPOTS. These CTL also killed GBM tumor cells in chromium-51 release assays. This study reports a new CTL target in GBM and further substantiates the concept that rational design and testing of multiple peptides for the same T-cell epitope elicits a broader response among different individuals than single peptide immunization.     

Identification of peptide epitopes of MAGE-1, -2, -3 that demonstrate HLA-A3-specific binding

 The MAGE gene family of tumour antigens are expressed in a wide variety of human cancers. We have identified 43 nonamer peptide sequences, from MAGE-1, -2 and -3 proteins that contain binding motifs for HLA-A3 MHC class I molecules. The T2 cell line, transfected with the cDNA for the HLA-A3 gene, was used in a MHC class I stabilisation assay performed at 37°C and 26°C. At 37°C, 2 peptides were identified that stabilised HLA-A3 with high affinity (fluorescence ratio, FR >1.5), 4 peptides with low affinity (FR 1.11 – 1.49) and 31 peptides that did not stabilise this HLA haplotype (FR <1.1). At 26°C, 12 peptides were identified that stabilised HLA-A3 with high affinity, 8 peptides with low affinity and 17 peptides that did not stabilise this HLA haplotype. Two peptides stabilised HLA-A3 at both temperatures. Small changes in one to three amino acids at positions distinct from the anchor residues altered peptide affinity. Data were compared to a similar study in which a peptide competition assay was used to investigate MAGE-1 peptide binding to several HLA haplotypes. This study demonstrates that anchor residues do not accurately predict peptide binding to specific HLA haplotypes, changes in one to three amino acids at positions distinct from anchor residues influence peptide binding and alternative methods of determining peptide binding yield different results. We are currently investigating the ability of these peptides to induce antitumour cytotoxic T lymphocyte activity as they may be of potential therapeutic value. Received: 4 January 1996 / Accepted: 20 March 1996     

Real-time measurement of in vitro peptide binding to soluble HLA-A*0201 by fluorescence polarization

Measuring the interaction of class I human leukocyte antigens (HLA) and their peptide epitopes acts as a guide for the development of vaccines, diagnostics, and immune-based therapies. Here, we report the development of a sensitive biochemical assay that relies upon fluorescence polarization to indicate peptide interactions with recombinant soluble HLA proteins. It is a cell- and radioisotope-free assay that has the advantage of allowing the direct, real-time measurement of the ratio between free and bound peptide ligand in solution without separation steps. Peptide/HLA assay parameters were established using several HLA A*0201-specific fluorescein isothiocyanate-labeled peptides. Optimal loading of synthetic peptides into fully assembled soluble HLA-A*0201 complexes was enabled by thermal destabilization at 53 degrees C for 15 min, demonstrating that efficient peptide exchange does not require the removal of endogenous peptides from the reaction environment. An optimal ratio of three beta-2 microglobulin molecules per single HLA heavy chain was determined to maximize peptide binding. Kinetic binding studies indicate that soluble HLA-A*0201/peptide interactions are characterized by a range of moderate k(on) values (1 x 10(4) to 8.7 x 10(4) M(-1) s(-1)) and slow k(off) values (1.9 x 10(-4) to 4.3 x 10(-4) s(-1)), consistent with parameters for native HLA molecules. Testing of the A*0201-specific peptides with 48 additional class I molecules demonstrates that the unique peptide binding behavior of individual HLA molecules is maintained in the assay. This assay therefore represents a versatile tool for characterizing the binding of peptide epitopes during the development of class I HLA-based vaccines and immune therapies.     

Identification of an HLA-A*0201-restricted CD8+ T-cell epitope encoded within Leptospiral immunoglobulin-like protein A

Leptospirosis is an important zoonosis in humans. Immunity against leptospiral infection was thought to be primarily humoral, and limited studies have addressed the role of CD8⁺ T cells. Leptospiral immunoglobulin-like protein A (LigA) is an important protective antigen of Leptospira and a potential target for Leptospira-specific cell-mediated immunity. In this study, twenty LigA-derived peptides were tested their binding affinity and stability for the HLA-A*0201 molecule. Peptides with high binding affinity and stability for HLA-A*0201 were then assessed their capacity to elicit specific cytotoxic T-lymphocyte (CTL) responses using cytotoxicity, ELISPOT assays for IFN-γ and HLA-A*0201-peptide tetramer assays. We identified a HLA-A*0201-restricted epitope, LigA_305–313 KLIVTPAAL in Leptospira LigA. CTLs specific for LigA_305–313 were elicited both in HLA-A2.1/K^b transgenic mice and in patients with a clinical and/or laboratory diagnosis of leptospirosis. Staining of the HLA-A*0201–LigA_305–313 tetramer revealed the presence of LigA_305–313-specific CTLs in peripheral blood mononuclear cells (PBMCs) sourced from five patients infected with three different serovars of Leptospira. In conclusion, we report the existence of specific cytotoxic CD8⁺ T cells in patients with leptospirosis and we suggest that the newly identified epitope, LigA_305–313, will be helpful in enhancing the understanding of the mechanism of immunity to leptospirosis.     

Synthesis and ex vivo profiling of chemically modified cytomegalovirus CMVpp65 epitopes.

The effect of substituting unnatural hydrophobic amino acids into the critical MHC binding residues of an HLA-A*0201-restricted cytomegalovirus CMVpp65 epitope, NLVPMVATV, has been investigated. A new set of peptides containing the amino acids tert-butyl glycine (Tgl), cyclohexyl glycine (Chg), neo-pentyl glycine (Npg), cyclohexyl alanine (Cha) and cyclo leucine (Cyl), at either position 2, to mimic Leu, or position 9, to mimic Val, have been synthesised. Immunological profiling using class I MHC stabilisation assays to assess MHC binding affinity, and enzyme-linked immunospot (ELISPOT) assays to assess the ability of the modified peptides to re-stimulate a specific cytotoxic T-lymphocyte (CTL) response, compared to the native epitope, have been performed. It was found that the majority of the unnatural substitutions resulted in a decrease in either HLA-A*0201 binding affinity or cytotoxic T-cell activity. However, the HLA-A*0201 binding affinity was unrelated to the ability to re-stimulate a T-cell response. Minimisation and molecular dynamics studies proved helpful in dissecting the ELISPOT responses. Two principal peptide binding modes were found by minimisation, designated kinked and straight. Peptides that bound in a kinked conformation were poor at re-stimulating a T-cell response. Of the peptides that bound in a straight conformation, molecular dynamics (MD) simulations revealed that those capable of re-stimulating the strongest responses had the greatest degree of flexibility (as determined by RMSD values across the MD simulation) around the P6 residue, one of the residues important for T-cell receptor recognition.     

An HLA-A3-binding prostate acid phosphatase-derived peptide can induce CTLs restricted to HLA-A2 and -A24 alleles

We previously reported peptide vaccine candidates for HLA-A3 supertype (-A3, -A11, -A31, -A33)-positive cancer patients. In the present study, we examined whether those peptides can also induce cytotoxic T lymphocyte (CTL) activity restricted to HLA-A2, HLA-A24, and HLA-A26 alleles. Fourteen peptides were screened for their binding activity to HLA-A*0201, -A*0206, -A*0207, -A*2402, and -A*2601 molecules and then tested for their ability to induce CTL activity in peripheral blood mononuclear cells (PBMCs) from prostate cancer patients. Among these peptides, one from the prostate acid phosphatase protein exhibited binding activity to HLA-A*0201, -A*0206, and -A*2402 molecules. In addition, PBMCs stimulated with this peptide showed that HLA-A2 or HLA-A24 restricted CTL activity. Their cytotoxicity toward cancer cells was ascribed to peptide-specific and CD8⁺ T cells. These results suggest that this peptide could be widely applicable as a peptide vaccine for HLA-A3 supertype-, HLA-A2-, and -A24-positive cancer patients.     

Effect of secondary anchor amino acid substitutions on the immunogenic properties of an HLA-A*0201-restricted T cell epitope derived from the Trypanosoma cruzi KMP-11 protein

The TcTLE peptide (TLEEFSAKL) is a CD8⁺ T cell HLA-A*0201-restricted epitope derived from the Trypanosoma cruzi KMP-11 protein that is efficiently processed, presented and recognized by CD8⁺ T cells from chagasic patients. Since the immunogenic properties of wild-type epitopes may be enhanced by suitable substitutions in secondary anchor residues, we have studied the effect of introducing specific mutations at position 3, 6 and 7 of the TcTLE peptide. Mutations (E3L, S6V and A7F) were chosen on the basis of in silico predictions and in vitro assays were performed to determine the TcTLE-modified peptide binding capacity to the HLA-A*0201 molecule. In addition, the functional activity of peptide-specific CD8⁺ T cells in HLA-A2⁺ chagasic patients was also interrogated. In contrast to bioinformatics predictions, the TcTLE-modified peptide was found to have lower binding affinity and stability than the original peptide. Nevertheless, CD8⁺ T cells from chronic chagasic patients recognized the TcTLE-modified peptide producing TNF-α and INF-γ and expressing CD107a/b, though in less extension than the response triggered by the original peptide. Overall, although the amino acids at positions 3, 6 and 7 of TcTLE are critical for the peptide affinity, they have a limited effect on the immunogenic properties of the TcTLE epitope.     

Assessment of promoter elements of the germ cell-specific proacrosin gene

Presence of the simian virus 40 (SV40) has recently been demonstrated in a relatively high percentage of human mesotheliomas and it is associated with the development of these malignancies in pleural cavities. Therefore, we have initiated a study to identify candidate peptides presented by the human HLA-A*0201 molecule for vaccination approaches against SV40 and monitoring of SV40 directed human immune responses. Initial screening of SV40 large T (Tag) domains required for transformation of cells for HLA-A*0201 binding motifs revealed ten possible binding peptides. Screening of these candidate peptides showed that seven of the ten peptides could bind and stabilize HLA-A*0201 molecules. In an in vitro immunization assay the two peptides with the highest binding affinity for HLA-A*0201, Tag aa 396-405 and aa 577-585, were tested for their ability to induce peptide specific cytotoxic T cells in two healthy donors. One donor developed cytotoxic T cells against Tag aa 396-405 and in T cell cultures of both donors Tag aa 577-585 specific T cells were initiated. The T cells against Tag aa 577-585 not only recognized and killed peptide pulsed cells, but, most importantly, SV40 transformed human mesothelial cells. This is the first demonstration of the induction of SV40 specific human cytotoxic T lymphocytes that recognize endogenously processed peptides from SV40. This peptide identification study opens the possibility to investigate immune responses against SV40 in mesothelioma patients and in individuals exposed to SV40.     

Proteasomal cleavage does not determine immunogenicity of gp100-derived peptides gp100<Subscript>209-217</Subscript> and gp100<Subscript>209-217T210M</Subscript>

Immune responses against tumor-associated antigens rely on efficient epitope presentation. The melanoma-associated antigen (Ag) gp100 contains HLA-A*0201 ligands that are characterized by low to medium binding affinity, among which gp100_209-217 is the most prominent (Kawakami et al., J Immunol 154:3961–3968, 1995). While this epitope is a natural T-cell target, it primes with low-efficiency T-cell responses during immunization. A modified gp100 epitope, gp100_209-217T210M, that contains a Thr to Met substitution at position 2 of the antigenic nonamer is characterized by high binding affinity for HLA-A*0201 and elicits strong and clinically effective T-cell responses. This higher affinity is believed to represent the sole reason for enhanced immunogenicity. Contrasting with this observation is the unpredictable relationship between affinity and immunogenicity observed in other antigen systems. In addition, we noted a striking difference between the capability of endogenously processed gp100_209-217 and gp100_209-217T210M to induce T-cell responses in an in vitro model. Therefore, we questioned whether factors other than HLA-affinity might play a role in determining the immunogenicity of these epitopes. In the present study, we evaluated the in vitro proteasomal cleavages of 23meric precursor peptides encompassing the native sequence (gp100_201-223) or the modified sequence (gp100_201-223T210M). Here we show that the standard proteasome liberates the C-termini of both antigenic peptides but not the N-termini. Quantitative analysis of the digestion products revealed that more of the fragments displaying the final C-termini were produced from the wild-type precursor. However, a stronger TCR engagement was observed when fractions of digested gp100_201-223T210M were used to activate an HLA-A*0201–expressing target T-cell clone. This difference was also found using separately produced, synthetic nonamers. In conclusion, the high binding affinity of gp100_209-217T210M seems to compensate for possible differences in proteasomal cleavage at the biological level. Since the final antigenic nonamer is not directly produced by the proteasome, additional further factors may influence the antigenic peptide availability, such as post-proteasomal processing and intracellular peptide transport.Abbreviations CTL Cytotoxic T lymphocyte - MFI Mean fluorescence intensity - MS Mass spectrometry - TAA Tumor-associated antigen - TCR T-cell receptor     

Neural models for predicting viral vaccine targets

We applied artificial neural networks (ANN) for the prediction of targets of immune responses that are useful for study of vaccine formulations against viral infections. Using a novel data representation, we developed a system termed MULTIPRED that can predict peptide binding to multiple related human leukocyte antigens (HLA). This implementation showed high accuracy in the prediction of the promiscuous peptides that bind to five HLA-A2 allelic variants. MULTIPRED is useful for the identification of peptides that bind multiple HLA-A2 variants as a group. By implementing ANN as a classification engine, we enabled both the prediction of peptides binding to multiple individual HLA-A2 molecules and the prediction of promiscuous binders using a single model. The ANN MULTIPRED predicts peptide binding to HLA-A*0205 with excellent accuracy (area under the receiver operating characteristic curve--AROC>0.90), and to HLA-A*0201, HLA-A*0204 and HLA-A*0206 with high accuracy (AROC>0.85). Antigenic regions with high density of binders ("antigenic hot-spots") represent best targets for vaccine design. MULTIPRED not only predicts individual 9-mer binders but also predicts antigenic hot spots. Two HLA-A2 hot-spots in Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) membrane protein were predicted by using MULTIPRED.     

A Conserved E7-derived Cytotoxic T Lymphocyte Epitope Expressed on Human Papillomavirus 16-transformed HLA-A2+ Epithelial Cancers

Human Papillomavirus 16 (HPV-16) has been identified as the causative agent of 50% of cervical cancers and many other HPV-associated tumors. The transforming potential/tumor maintenance capacity of this high risk HPV is mediated by two viral oncoproteins, E6 and E7, making them attractive targets for therapeutic vaccines. Of 21 E6 and E7 peptides computed to bind HLA-A*0201, 10 were confirmed through TAP-deficient T2 cell HLA stabilization assay. Those scoring positive were investigated to ascertain which were naturally processed and presented by surface HLA molecules for CTL recognition. Because IFNγ ELISpot frequencies from healthy HPV-exposed blood donors against HLA-A*0201-binding peptides were unable to identify specificities for tumor targeting, their physical presence among peptides eluted from HPV-16-transformed epithelial tumor HLA-A*0201 immunoprecipitates was analyzed by MS³ Poisson detection mass spectrometry. Only one epitope (E7_11–19) highly conserved among HPV-16 strains was detected. This 9-mer serves to direct cytolysis by T cell lines, whereas a related 10-mer (E7_11–20), previously used as a vaccine candidate, was neither detected by MS³ on HPV-transformed tumor cells nor effectively recognized by 9-mer specific CTL. These data underscore the importance of precisely defining CTL epitopes on tumor cells and offer a paradigm for T cell-based vaccine design.     

MHC binding peptides for designing of vaccines against Japanese encephalitis virus: A computational approach

Japanese encephalitis (JE), a viral disease has seen a drastic and fatal enlargement in the northern states of India in the current decade. The better and exact cure for the disease is still in waiting. For the cause an in silico strategy in the development of the peptide vaccine has been taken here for the study. A computational approach to find out the Major Histocompatibility Complex (MHC) binding peptide has been implemented. The prediction analysis identified MHC class I (using propred I) and MHC class II (using propred) binding peptides at an expectable percent predicted IC (50) threshold values. These predicted Human leukocyte antigen [HLA] allele binding peptides were further analyzed for potential conserved region using an Immune Epitope Database and Analysis Resource (IEDB). This analysis shows that HLA-DRB1*0101, HLA-DRB3*0101, HLA-DRB1*0401, HLA-DRB1*0102 and HLA-DRB1*07:01% of class II (in genotype 2) and HLA-A*0101, HLA-A*02, HLA-A*0301, HLA-A*2402, HLA-B*0702 and HLA-B*4402% of HLA I (in genotype 3) bound peptides are conserved. The predicted peptides MHC class I are ILDSNGDIIGLY, FVMDEAHFTDPA, KTRKILPQIIK, RLMSPNRVPNYNLF, APTRVVAAEMAEAL, YENVFHTLW and MHC class II molecule are TTGVYRIMARGILGT, NYNLFVMDEAHFTDP, AAAIFMTATPPGTTD, GDTTTGVYRIMARGI and FGEVGAVSL found to be top ranking with potential super antigenic property by binding to all HLA. Out of these the predicted peptide FVMDEAHFTDPA for allele HLA-A*02:01 in MHC class I and NYNLFVMDEAHFTDP for allele HLA-DRB3*01:01 in MHC class II was observed to be most potent and can be further proposed as a significant vaccine in the process. The reported results revealed that the immune-informatics techniques implemented in the development of small size peptide is useful in the development of vaccines against the Japanese encephalitis virus (JEV).