Visceral leishmaniasis (VL) represents one of the most challenging infectious diseases worldwide. The reason that once infected, patient develops immunity against Leishmania parasite has paved way to develop prophylactic vaccines against disease, but only some of these have moved ahead for clinical trials. Herein, the study to explore novel and potential vaccine candidates was extended to pathogenic form of parasite, that is, amastigote form which is less explored due to complexity of its purification process. Methods and results. Classical protocol of purification of splenic amastigotes was modified to obtain highly pure amastigotes which was confirmed by Western blotting in support with proteomics studies. Fractionation and sub‐fractionation of purified splenic amastigotes revealed four sub‐fractions, belonging to 97 to 68 kDa and 68 to 43 kDa ranges, which showed long‐lasting protection with remarkable Th1‐type cellular responses in hamsters vaccinated with these sub‐fractions (LTT, NO, QRT‐PCR). Further proteomics analysis, to identify and understand the precise nature and function of these protective protein sub‐fractions, identified a total of 47 proteins including twenty‐five hypothetical proteins/unknowns. Amastigote stage has potential Th1‐stimulatory vaccine candidates, notably, among identified proteins, major were uncharacterized proteins/hypothetical proteins, which once characterized may serve as novel and potential vaccine candidates/drug targets.

中文翻译：

---

纯化的利什曼原虫的脾吻合动物-免疫免疫方法研究Th1刺激性多蛋白。

内脏利什曼病（VL）是全球最具挑战性的传染病之一。病人一旦感染，就会产生针对利什曼原虫的免疫力的原因寄生虫为开发预防疾病的疫苗铺平了道路，但只有其中一些已向前推进临床试验。在此，探索新型和潜在候选疫苗的研究扩展到了寄生虫的致病性形式，即，由于其纯化过程的复杂性而较少进行研究的鞭毛体形式。方法和结果。修改了纯化脾脏吻合蛋白的经典方法以获得高纯度的吻合蛋白，这在蛋白质组学研究的支持下通过蛋白质印迹法得到了证实。纯化的脾吻张虫的分馏和亚分馏显示出四个亚馏分，分别属于97至68 kDa和68至43 kDa的范围，在接种了这些亚馏分的仓鼠中显示出持久的保护作用，并具有明显的Th1型细胞反应（ LTT，NO，QRT-PCR）。为了进一步鉴定和理解这些保护性蛋白质亚组分的精确性质和功能，进一步进行了蛋白质组学分析，共鉴定出47种蛋白质，其中包括25种假设的蛋白质/未知蛋白质。鞭毛阶段具有潜在的Th1刺激性疫苗候选物，尤其是在已鉴定的蛋白质中，主要是未表征的蛋白质/假设蛋白，它们一旦被表征，就可以作为新型和潜在的候选疫苗/药物靶标。