PDF(Pubmed)
Abstract：
Centrin1 gene deleted Leishmania donovani parasite (LdCen1-/-) was developed and extensively tested experimentally as an intracellular stage-specific attenuated and immunoprotective live parasite vaccine candidate ex vivo using human PBMCs and in vivo in animals. Here we report manufacturing and pre-clinical evaluation of current Good-Laboratory Practice (cGLP) grade LdCen1-/- parasites, as a prerequisite before proceeding with clinical trials. We screened three batches of LdCen1-/- parasites manufactured in bioreactors under cGLP conditions, for their consistency in genetic stability, attenuation, and safety. One such batch was preclinically tested using human PBMCs and animals (hamsters and dogs) for its safety and protective immunogenicity. The immunogenicity of the CGLP grade LdCen1-/- parasites was similar to one grown under laboratory conditions. The cGLP grade LdCen1-/- parasites were found to be safe and non-toxic in hamsters and dogs even at 3 times the anticipated vaccine dose. When PBMCs from healed visceral leishmaniasis (VL) cases were infected with cGLP LdCen1-/-, there was a significant increase in the stimulation of cytokines that contribute to protective responses against VL. This effect, measured by multiplex ELISA, was greater than that observed in PBMCs from healthy individuals. These results suggest that cGLP grade LdCen1-/- manufactured under cGMP complaint conditions can be suitable for future clinical trials.
摘要：
开发了Centrin1基因缺失的多尼利什曼原虫寄生虫（LdCen1-/-），并作为使用人PBMC和动物体内的细胞内阶段特异性减毒和免疫保护性活寄生虫疫苗候选物进行了广泛的实验测试。在这里，我们报告了当前良好实验室规范（cGLP）等级LdCen1-/-寄生虫的制造和临床前评估，作为进行临床试验之前的先决条件。我们筛选了在cGLP条件下在生物反应器中制造的三批LdCen1-/-寄生虫，因为它们在遗传稳定性方面的一致性，衰减，和安全。使用人PBMC和动物(仓鼠和狗)临床前测试一个这样的批次的安全性和保护性免疫原性。CGLP级LdCen1-/-寄生虫的免疫原性与在实验室条件下生长的类似。发现cGLP级LdCen1-/-寄生虫在仓鼠和狗中是安全且无毒的,即使是预期疫苗剂量的3倍。当来自已治愈的内脏利什曼病（VL）病例的PBMC感染cGLPLdCen1-/-时，促进针对VL的保护性反应的细胞因子的刺激显着增加。这个效果,通过多重ELISA测量，高于在健康个体的PBMC中观察到的。这些结果表明，在cGMP投诉条件下生产的cGLP等级LdCen1-/-可适用于未来的临床试验。
