Divisions of Parasitology, CSIR-Central Drug Research Institute, Lucknow, India.
Molecular and Structural Biology, CSIR-Central Drug Research Institute, Lucknow, India.
PLoS One. 2014 Jan 24;9(1):e86073. doi: 10.1371/journal.pone.0086073. eCollection 2014.
Th1 immune responses play an important role in controlling Visceral Leishmaniasis (VL) hence, Leishmania proteins stimulating T-cell responses in host, are thought to be good vaccine targets. Search of such antigens eliciting cellular responses in Peripheral blood mononuclear cells (PBMCs) from cured/exposed/Leishmania patients and hamsters led to the identification of two enzymes of glycolytic pathway in the soluble lysate of a clinical isolate of Leishmania donovani--Enolase (LdEno) and aldolase (LdAld) as potential Th1 stimulatory proteins. The present study deals with the molecular and immunological characterizations of LdEno and LdAld. The successfully cloned and purified recombinant proteins displayed strong ability to proliferate lymphocytes of cured hamsters' along with significant nitric-oxide production and generation of Th1-type cytokines (IFN-γ and IL-12) from stimulated PBMCs of cured/endemic VL patients. Assessment of their prophylactic potentials revealed ∼ 90% decrease in parasitic burden in rLdEno vaccinated hamsters against Leishmania challenge, strongly supported by an increase in mRNA expression levels of iNOS, IFN-γ, TNF-α and IL-12 transcripts along with extreme down-regulation of TGF-β, IL-4 and IL-10. However, animals vaccinated with rLdAld showed comparatively lesser prophylactic efficacy (∼ 65%) with inferior immunological response. Further, with a possible implication in vaccine design against VL, identification of potential T-cell epitopes of both the proteins was done using computational approach. Additionally, in-silico 3-D modelling of the proteins was done in order to explore the possibility of exploiting them as potential drug targets. The comparative molecular and immunological characterizations strongly suggest rLdEno as potential vaccine candidate against VL and supports the notion of its being effective T-cell stimulatory protein.
Th1 免疫反应在控制内脏利什曼病（VL）中发挥重要作用，因此，刺激宿主 T 细胞反应的利什曼抗原被认为是良好的疫苗靶点。在治愈/暴露/利什曼病患者和仓鼠的外周血单个核细胞（PBMCs）中寻找刺激细胞反应的抗原，导致在利什曼原虫 Donovan 临床分离株的可溶性裂解物中鉴定出两种糖酵解途径的酶-烯醇酶（LdEno）和醛缩酶（LdAld）作为潜在的 Th1 刺激蛋白。本研究对 LdEno 和 LdAld 的分子和免疫学特性进行了研究。成功克隆和纯化的重组蛋白显示出强烈的增殖能力，可以刺激治愈的仓鼠淋巴细胞，同时显著增加一氧化氮的产生和 Th1 型细胞因子（IFN-γ 和 IL-12）的产生，从治愈/地方性 VL 患者的刺激 PBMCs 中产生。评估它们的预防潜力表明，在 rLdEno 疫苗接种的仓鼠中，寄生虫负荷减少了约 90%，对利什曼挑战的强烈支持，伴随着 iNOS、IFN-γ、TNF-α 和 IL-12 转录物的 mRNA 表达水平的增加，以及 TGF-β、IL-4 和 IL-10 的极端下调。然而，用 rLdAld 疫苗接种的动物显示出相对较低的预防功效（约 65%），免疫反应也较差。此外，由于可能对 VL 疫苗设计有影响，使用计算方法鉴定了两种蛋白的潜在 T 细胞表位。此外，还对蛋白质进行了计算机模拟 3-D 建模，以探索将其用作潜在药物靶点的可能性。比较分子和免疫学特性强烈表明 rLdEno 是一种有潜力的 VL 疫苗候选物，并支持其作为有效的 T 细胞刺激蛋白的观点。
