Agallou Maria, Athanasiou Evita, Koutsoni Olga, Dotsika Eleni, Karagouni Evdokia
Laboratory of Cellular Immunology, Department of Microbiology, Hellenic Pasteur Institute , Athens , Greece.
Front Immunol. 2014 Jun 10;5:268. doi: 10.3389/fimmu.2014.00268. eCollection 2014.
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.
利什曼病是一个严重的全球性健康问题，目前尚无疫苗。CD4(+)和CD8(+) T细胞的激活对于产生针对寄生虫的保护性免疫至关重要。疫苗设计的最新趋势已转向基于表位的疫苗，这类疫苗更具特异性、安全性且易于生产。在本研究中，使用在线可用算法分析了四种已知的婴儿利什曼原虫抗原蛋白，即半胱氨酸蛋白酶A（CPA）、组蛋白H1、KMP-11和利什曼原虫真核起始因子（LeIF），以预测其与H2(d) MHC I类和II类分子的结合表位。基于计算机模拟分析，合成了包括高分MHC I类和II类限制性表位在内的八种肽。用每种在完全弗氏佐剂/不完全弗氏佐剂中乳化的合成肽免疫MHC相容的BALB/c小鼠，验证了肽的免疫原性。CPA_p2、CPA_p3、H1_p1和LeIF_p6在体外肽再刺激后诱导了强烈的脾细胞增殖。此外，除LeIF_p1和KMP-11_p1外，大多数肽诱导了IFN-γ分泌，而KMP-11_p1对IL-10的产生显示出抑制作用。CPA_p2、CPA_p3、LeIF_p3和LeIF_p6诱导产生IFN-γ的CD4(+) T细胞，表明是TH1型反应。此外，CPA_p2、CPA_p3和H1_p1还诱导了CD8(+) T细胞的产生。免疫小鼠中肽特异性IgG的诱导也表明肽序列中存在B细胞表位。结合免疫信息学工具和实验验证，我们证明CPA_p2、CPA_p3、H1_p1、H1_p3、CPA_p2、LeIF_p3和LeIF_p6可能包含诱导保护性细胞毒性和/或TH1型免疫反应的潜在表位，支持了基于肽的利什曼病疫苗开发的可行性。
