In Silico Analysis of Six Known Leishmania major Antigens and In Vitro Evaluation of Specific Epitopes Eliciting HLA-A2 Restricted CD8 T Cell Response

Abstract

BackgroundAs a potent CD8+ T cell activator, peptide vaccine has found its way in vaccine development against intracellular infections and cancer, but not against leishmaniasis. The first step toward a peptide vaccine is epitope mapping of different proteins according to the most frequent HLA types in a population.Methods and FindingsSix Leishmania (L.) major-related candidate antigens (CPB,CPC,LmsTI-1,TSA,LeIF and LPG-3) were screened for potential CD8+ T cell activating 9-mer epitopes presented by HLA-A*0201 (the most frequent HLA-A allele). Online software including SYFPEITHI, BIMAS, EpiJen, Rankpep, nHLApred, NetCTL and Multipred were used. Peptides were selected only if predicted by almost all programs, according to their predictive scores. Pan-A2 presentation of selected peptides was confirmed by NetMHCPan1.1. Selected peptides were pooled in four peptide groups and the immunogenicity was evaluated by in vitro stimulation and intracellular cytokine assay of PBMCs from HLA-A2+ individuals recovered from L. major. HLA-A2− individuals recovered from L. major and HLA-A2+ healthy donors were included as control groups. Individual response of HLA-A2+ recovered volunteers as percent of CD8+/IFN-γ+ T cells after in vitro stimulation against peptide pools II and IV was notably higher than that of HLA-A2− recovered individuals. Based on cutoff scores calculated from the response of HLA-A2− recovered individuals, 31.6% and 13.3% of HLA-A2+ recovered persons responded above cutoff in pools II and IV, respectively. ELISpot and ELISA results confirmed flow cytometry analysis. The response of HLA-A2− recovered individuals against peptide pools I and III was detected similar and even higher than HLA-A2+ recovered individuals.ConclusionUsing in silico prediction we demonstrated specific response to LmsTI-1 (pool II) and LPG-3- (pool IV) related peptides specifically presented in HLA-A*0201 context. This is among the very few reports mapping L. major epitopes for human HLA types. Studies like this will speed up polytope vaccine idea towards leishmaniasis.

Assessment of Protection Induced by DNA and Live Vaccine Encoding Leishmania MHC Class I Restricted Epitopes against L. major Challenge in Balb/c Mice Model

“…CD4 + and CD8 + T-cells are activated by sensing short peptides presented within MHC class II and class I context respectively. Researchers now harness Immunoinformatics tools to predict potential CD4 + /CD8 + T-cell epitopes in silico and then select in vitro/in vivo evaluated immunogenic ones to arrange them together in a polytope construct [10,11]. Polytopes or T-cell vaccines as novel subunit vaccines are highly preferred since they carry minimal immunogenic part of the protein where whole protein might have gift by Prof. Genevieve Milon, Pasteur Institute of Paris) were cultured for weeks to concentrate secreted antibody in about 1500 ml medium.…”

“…Here we studied the protective effect of a previously designed and evaluated polytope construct composed of few peptides in tandem [10,17]. Briefly the polytope included HLA-A2 restricted epitopes from 4 known Leishmania vaccine candidates (CPB and CPC, 5 peptides, LmSTI-1, 4 peptides and LPG-3, 4 peptides) and 4 recently reported H-2Kd restricted epitopes from non-candidates.…”

“…Herein standard plate based protocol with some modifications was used [10,25]. Briefly, pre-stimulated splenocytes were harvested from 6 well plates.…”

Identification of Conserved Peptides Comprising Multiple T Cell Epitopes of Matrix 1 Protein in H1N1 Influenza Virus

“…Use of a single computational algorithm for T cell epitope prediction may lead to a substantial number of false positives and false negatives. Taking this into consideration, the consensus approach (i.e., combining and comparing the different prediction algorithms to identify epitopes) has been applied in various studies, and its immunogenic potential has been verified in vitro and in vivo (11,41). The immunogenicity of peptides depends on multiple factors mediating peptide immune response, such as HLA-peptide binding, TAP transport efficiency, C-terminal cleavage, and binding stability of the peptide-HLA complex.…”

Identification and Validation of Targets for Cancer Immunotherapy: From the Bench-to-Bedside

“…Other online software which can be used to identify epitopes includes EpiJen, Rankpep, nHLApred, NetCTL and Multipred [65].…”