Garg Ravendra, Gupta Shraddha K, Tripathi Parul, Hajela K, Sundar S, Naik S, Dube Anuradha
Division of Parasitology, Central Drug Research Institute, Lucknow, India.
Vaccine. 2006 Apr 5;24(15):2900-9. doi: 10.1016/j.vaccine.2005.12.053. Epub 2006 Jan 17.
Most of the studies for the identification of prophylactic antigens that elicit T cell responses were concentrated on membrane proteins of Leishmania donovani. This study was taken up to assess L. donovani soluble promastigote antigens for their ability to stimulate proliferation of peripheral blood mononuclear cells (PBMCs) from cured visceral leishmaniasis (VL) patients, endemic and non-endemic controls and lymphocytes/peritoneal macrophages of cured hamsters. The soluble protein was subjected to sequential precipitation with saturated ammonium sulphate (20%, 40%, 60% and 80%), of which largely 80% fractioned protein showed significant cellular responses in cured patients and hamsters. This fraction was further fractionated into five sub fractions by preparative SDS-PAGE and subjected to re-evaluation for their ability to induce cellular responses. Out of these, only F2 sub fraction belonging to the MW of 97.4-68 kDa stimulated remarkable lymphoproliferative and IFN-gamma responses in cured VL patients and in endemic controls. Similarly, significant lymphoproliferative responses and nitric oxide production were also noticed in cured Leishmania infected animals indicating an element of uniformity in responses between hamster and human. F2 sub fraction, when evaluated for its prophylactic efficacy with BCG against L. donovani challenge in hamster exhibited significant parasite inhibition in spleen (71.1%; p<0.001) and liver (68.2%; p<0.001) as compared to their unvaccinated counterpart. The vaccinated animals showed significant lymphoproliferative response and nitric oxide production but leishmania specific IgG level were suppressed. The results indicate the presence of immunostimulatory and protective molecules in F2 sub fraction which may further be exploited for the development of a vaccine against VL, hitherto an unrealized goal.
大多数旨在鉴定能引发T细胞反应的预防性抗原的研究都集中在杜氏利什曼原虫的膜蛋白上。本研究旨在评估杜氏利什曼原虫可溶性前鞭毛体抗原刺激治愈的内脏利什曼病（VL）患者、流行区和非流行区对照以及治愈仓鼠的淋巴细胞/腹腔巨噬细胞外周血单核细胞（PBMC）增殖的能力。将可溶性蛋白用饱和硫酸铵（20%、40%、60%和80%）进行连续沉淀，其中大部分80%分级的蛋白在治愈的患者和仓鼠中显示出显著的细胞反应。该级分通过制备性SDS-PAGE进一步分级为五个亚级分，并对其诱导细胞反应的能力进行重新评估。其中，只有分子量为97.4 - 68 kDa 的F2亚级分在治愈的VL患者和流行区对照中刺激了显著的淋巴细胞增殖和IFN-γ反应。同样，在治愈利什曼原虫感染的动物中也观察到显著的淋巴细胞增殖反应和一氧化氮产生，表明仓鼠和人类之间的反应具有一定的一致性。当用F2亚级分与卡介苗联合评估其对仓鼠杜氏利什曼原虫攻击的预防效果时，与未接种的对照组相比，在脾脏（71.1%；p<0.001）和肝脏（68.2%；p<0.001）中显示出显著的寄生虫抑制作用。接种疫苗的动物表现出显著的淋巴细胞增殖反应和一氧化氮产生，但利什曼原虫特异性IgG水平受到抑制。结果表明F2亚级分中存在免疫刺激和保护分子，这可能进一步用于开发抗VL疫苗，这是一个迄今尚未实现的目标。
