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

Leishmaniasis is currently a serious health as well as economic problem in underdeveloped and developing countries in Africa, Asia, the Near and Middle East, Central and South America and the Mediterranean region. Cutaneous leishmaniasis is highly endemic in Iran, remarkably in Isfahan, Fars, Khorasan, Khozestan and Kerman provinces. Since effective prevention is not available and current curative therapy is expensive, often poorly tolerated and not always effective, alternative therapies including vaccination against leishmaniasis are of priority to overcome the problem. Although Th1 dominant response is so far considered as a pre-requisite for the immune system to overcome the infection, CD8+ T cell response could also be considered as a potent arm of immune system fighting against intracellular Leishmania. Polytope vaccine strategy may open up a new way in vaccine design against leishmaniasis, since they act as a potent tool to stimulate multi-CD8 T cell responses. Clearly there is a substantial need to evaluate the promising epitopes from different proteins of Leishmania parasite species. Some new immunoinformatic tools are now available to speed up this process, and we have shown here that in silico prediction can effectively evaluate HLA class I-restricted epitopes out of Leishmania proteins

The response of individual volunteers to <i>L. major</i> lysate.

<p>Freezed/thawed antigens of <i>L. major</i> were used to stimulate the PBMCs in culture as previous disease indicator. The responses of <i>L. major</i> recovered individuals were potentially detected at CD4 level. Each point represents response of each individual. Horizontal bars represent the median value of CD4⁺/IFN γ⁺ T cells in the related group. Statistical analysis shows a significant difference between R.A2⁺ (HLA-A2⁺ recovered individuals) and R.A2⁻ (HLA-A2⁻ recovered individuals) groups with H.A2⁺ individuals (HLA-A2⁺ healthy donors) with <i>p</i> value<0.05.</p

Status of CD8⁺/IFN-γ⁺ T cell response of recovered HLA-A2⁺ individuals compared to HLA-A2⁻ individuals.

<p>6 out of 19 (31.6%) and 2 out of 15 (13.3%) HLA-A2⁺ recovered individuals responded above cutoff value (horizontal bar in each plot defined as mean + 2SD CD8⁺/IFN-γ⁺ response in HLA-A2⁻ controls) against peptide pools II and IV, respectively. Fischer's exact probability test showed that the response in peptide pool II is statistically significant.</p

HLA-A2 super-type binding possibility of selected peptides predicted by NetMHCPan1.1.

<p>*strong binder (Strong binders have an IC₅₀ less than 50 nM).</p><p>**weak binder (Weak binders have an IC₅₀ more than 50 nM and less than 500 nM.</p

<i>L. major</i> specific proteins used as candidate antigens for 9-mer epitope screening.

a<p>The full sequences of the proteins were extracted from GeneDB data of <i>L. major</i> strain MHOM/IL/80/Fredlin.</p

HLA-A2 screening by one step PCR.

<p>One step PCR-SSP method was used to screen for HLA-A2 positives among all samples included (recovered individuals and healthy donors). Lane 1 shows 100 bp DNA ladder marker, lane 4 shows the PCR reaction of T2 cells as positive control, lane 3, 5 and 6 is related to negative samples and lane 2 is related to a positive sample.</p

Sequence Specific Primers used in PCR reactions for typing HLA-A2 and sub typing HLA-A*0201 allele.

<p>Sequence Specific Primers used in PCR reactions for typing HLA-A2 and sub typing HLA-A*0201 allele.</p

Characteristics of <i>in silico</i> predicted <i>L. major</i> specific CD8⁺ T cell 9-mer peptides restricted to HLA-A*0201 allele.

a<p>Amino acid position as in the protein sequence.</p>b<p>Threshold set on 5% (percent of whole protein peptides that should be tested).</p>c<p>Specific binding threshold set on 2% (percent of whole protein peptides that should be tested).</p>d<p>Proteasomal cleavage.</p>e<p>Cut off score set on 0.5 (Threshold of binding).</p>f<p>Threshold for epitope selection set on >0.75 (Threshold of binding).</p>g<p>Promiscuous epitope predicted as moderate or high binder.</p

Detection of secreted IFN-γ from PBMCs stimulated against peptide pool II and IV by ELISA in culture supernatants.

<p>IFN-γ production was measured in culture supernatants of HLA-A2⁺ recovered individuals (R.A2⁺), HLA-A2⁻ recovered individuals (R.A2⁻), and HLA-A2⁺ healthy donors (H.A2⁺) stimulated against peptide pools II and IV. Each point represents the net result of individual experiments. Horizontal bars represent the median value of the IFN-γ concentration in the related group. The response of R.A2⁺ individuals was detected higher than that of R.A2⁻ ones in peptide pools II and IV.</p