Experimental Validation of Multi-Epitope Peptides Including Promising MHC Class I- and II-Restricted Epitopes of Four Known Leishmania infantum Proteins

Abstract

Leishmaniasis is a significant worldwide health problem for which no vaccine exists. Activation of CD4(+) and CD8(+) T cells is crucial for the generation of protective immunity against parasite. Recent trend in vaccine design has been shifted to epitope-based vaccines that are more specific, safe, and easy to produce. In the present study, four known antigenic Leishmania infantum proteins, cysteine peptidase A (CPA), histone H1, KMP-11, and Leishmania eukaryotic initiation factor (LeIF) were analyzed for the prediction of binding epitopes to H2(d) MHC class I and II molecules, using online available algorithms. Based on in silico analysis, eight peptides including highly scored MHC class I- and II-restricted epitopes were synthesized. Peptide immunogenicity was validated in MHC compatible BALB/c mice immunized with each synthetic peptide emulsified in complete Freund's adjuvant/incomplete Freund's adjuvant. CPA_p2, CPA_p3, H1_p1, and LeIF_p6 induced strong spleen cell proliferation upon in vitro peptide re-stimulation. In addition, the majority of the peptides, except of LeIF_p1 and KMP-11_p1, induced IFN-γ secretion, while KMP-11_p1 indicated a suppressive effect on IL-10 production. CPA_p2, CPA_p3, LeIF_p3, and LeIF_p6 induced IFN-γ-producing CD4(+) T cells indicating a TH1-type response. In addition, CPA_p2, CPA_p3, and H1_p1 induced also the induction of CD8(+) T cells. The induction of peptide-specific IgG in immunized mice designated also the existence of B cell epitopes in peptide sequences. Combining immunoinformatic tools and experimental validation, we demonstrated that CPA_p2, CPA_p3, H1_p1, H1_p3, CPA_p2, LeIF_p3, and LeIF_p6 are likely to include potential epitopes for the induction of protective cytotoxic and/or TH1-type immune responses supporting the feasibility of peptide-based vaccine development for leishmaniasis.

Keywords: CD4+IFN-γ+ T cells; CD8+IFN-γ+ T cells; Leishmania eukaryotic initiation factor; cysteine peptidase A; histone H1; in silico analysis; kinetoplastid membrane protein 11; lymphocyte proliferation.

Figure 1

Multi-epitope peptide-specific proliferative responses and cytokine secretion. (A) Proliferative responses. Spleen cells from BALB/c mice (n = 3/group) immunized either with individual peptide emulsified in CFA/IFA or PBS alone, were re-stimulated in vitro with the respective peptide (10 μg/ml) for 72 h. Cultures were pulsed for the final 18 h with 1 μCi of [³H]-TdR and results are depicted as Δcpm ± SD as described in Section “Materials and Methods.” Spleen cells derived from mice immunized with PBS alone, stimulated in vitro with ConA (Δcpm: 39743 ± 843) were used for comparison purposes. (B) IFN-γ and (C) IL-10 secretion. Cytokines were detected in culture supernatants of spleen cells from immunized BALB/c mice (n = 5/group), re-stimulated in vitro with the respective peptide (10 μg/ml) for 72 h, by ELISA. The results are expressed as pg/ml ± SD. Significant differences between groups of mice immunized with each synthetic peptide emulsified in CFA/IFA and the group of mice immunized with CFA/IFA alone are indicated by * (P < 0.05).

Figure 2

Multi-epitope peptide-specific cytokine production by CD4⁺ and CD8⁺ T cells. Spleen cells from BALB/c mice (n = 5/group) immunized either with individual peptide emulsified in CFA/IFA or PBS alone, were re-stimulated in vitro with the respective peptide (10 μg/ml) for 48 h, and analyzed for CD4⁺ and CD8⁺ T cells producing IL-4 and IFN-γ. (A–C) Representative FACS plots of intracellular staining used to define IL-4- and IFN-γ-expressing CD4⁺ and CD8⁺ T cells in spleens derived from immunized mice. Values represent the percentages of (A) IL-4⁺ cells among CD4⁺ T cells and (B,C) IFN-γ⁺ cells among CD4⁺ and CD8⁺ T cell populations. (D) Quantification of IL-4 and IFN-γ producing T cells in immunized mice. All data were normalized to CFA/IFA control group. Each bar represents the mean proportion of IL-4- and IFN-γ-producing CD4⁺ and CD8⁺ T cells induced by the respective peptide.

Figure 3

Multi-epitope peptide-specific antibody production. BALB/c mice (n = 9/group) immunized either with individual peptide emulsified in CFA/IFA or PBS alone, were bled 15 days post third immunization and sera were separated. (A,B) total IgG Abs, and (C) IgG1 and IgG2a Abs against each peptide were assessed by ELISA. The results are expressed as OD₄₅₀ ± SD. Significant differences between groups of mice immunized with each synthetic peptide emulsified in CFA/IFA and the group of mice immunized with CFA/IFA alone are indicated by * (P < 0.05).

Figure 4

Synthetic multi-epitope peptides docking. Ribbon diagram of 3D structural analysis of interactions between (A) HLA-A2 molecule (PDB-code: 1HHG) and the synthetic peptides CPA_p2, CPA_p3, H1_p3, and LeIF_p3, and (B) HLA-DRB1 molecule (PDB-code: 2SEB) and the synthetic peptides CPA_p2 and LeIF_p3. Candidate peptides were predicted to locate onto the peptide-binding cleft of the HLA molecules by using ClusPro program. The side and top view are shown, the α strands were shown in green, the β strands in blue, and the multi-epitope peptides in magenta.