Transgenic porphyric Leishmania as suicidal live vaccines against leishmaniasis

描述（由申请人提供）：我们制备了转基因利什曼原虫，其可能用于针对利什曼病和其他疾病的光动力学疫苗接种。这些锥虫原生动物是天然缺乏血红素生物合成，从而使它们依赖于外源性供应的四吡咯，使功能性呼吸复合物。我们利用利什曼原虫的这些特殊缺陷来制造自杀性突变体。由于这些细胞内寄生虫天然感染树突状细胞并驻留在巨噬细胞的吞噬溶酶体中，因此当它们被发出信号以进行溶酶体内自杀性细胞溶解时，这些突变体可以作为有效的载体来递送前药或疫苗以用于它们的活化或呈递。为了工程化这样的突变体，利什曼原虫属（Leishmania spp.）用编码血红素生物合成途径中的第2和第3种酶的哺乳动物基因转染（Sah等人，2002. J. Biol. Chem 277，14902-9）。因此，当暴露于外部信号，即该途径中第一种酶的δ-氨基乙酰丙酸（ALA）产物时，这些转染子呈现卟啉状。当巨噬细胞被这些转染子感染并暴露于ALA时，卟啉病在宿主细胞和它们的细胞内利什曼原虫中都发生了。然而，在巨噬细胞中形成的卟啉被迅速代谢到背景水平，而利什曼原虫中的卟啉由于缺乏血红素代谢途径而积累并持续存在。这些巨噬细胞内突变体的卟啉病的发展导致其选择性细胞溶解，这可以通过光照来调节和增强。这种细胞内突变体的选择性破坏改变了体外感染的巨噬细胞的全局表达谱，表明增强的免疫原性和寄生虫消除，如通过微阵列分析所确定的。这得到了初步体内数据的支持，表明在叙利亚金黄仓鼠模型中通过该接种方案对实验性黑热病具有保护作用。在本申请中，我们提出通过承担以下具体目标来进一步探索该活疫苗模型的功效：[1]通过特别注意不存在利什曼原虫持续性和残留致病性来在仓鼠模型中复制针对黑热病的初步实验中的阳性结果;以及[2]将发现扩展到其他动物模型，例如：G. BALB/c小鼠，通过用皮肤种属（即亚马逊利什曼原虫）攻毒对皮肤利什曼病易感。这些探索性研究的结果有助于评估自杀性突变体不仅用作抗利什曼病活疫苗，而且用作抗其他传染性和非传染性疾病的疫苗供应者的潜力。

DESCRIPTION (provided by applicant): We prepared transgenic Leishmania, which are potentially useful for photodynamic vaccination against leishmaniasis and other diseases. These trypanosomatid protozoa are naturally deficient in heme biosynthesis, thereby rendering them dependent on the exogenous supply of tetrapyrroles for making functional respiratory complexes. We exploit these peculiar defects in Leishmania to produce suicidal mutants. Since these intracellular parasites naturally infect dendritic cells and reside in the phagolysosomes of macrophages, such mutants may serve as effective carriers to deliver pro-drugs or vaccines for their activation or presentation when they are signaled to commit intralysosomal suicidal cytolysis. To engineer such mutants, Leishmania spp. were transfected with mammalian genes encoding the 2nd and 3rd enzymes in heme biosynthesis pathway (Sah et al. 2002. J. Biol. Chem 277, 14902-9). These transfectants were thus rendered porphyric when exposed to an external signal, i.e. delta-aminolevulinate (ALA) - products of the 1st enzyme in this pathway. When macrophages were infected with these transfectants and exposed to ALA, porphyria developed both in the host cells and in their intracellular Leishmania. However, porphyrins formed in macrophages are metabolized rapidly to the background level, while those in Leishmania accumulate and persist due to the absence of heme metabolic pathway. The development of porphyria of these intra- macrophage mutants resulted in their selective cytolysis, which can be regulated and enhanced by light illumination. This selective destruction of intracellular mutants altered the global expression profiles of in vitro infected macrophages, suggestive of enhanced immunogenicity and parasite elimination, as determined by microarray analyses. This is supported by preliminary in vivo data, indicative of protection via this scheme of vaccination against experimental kala-azar in the Syrian Golden hamster model. We propose in this application to further explore the efficacy of this live vaccine model by undertaking the following specific aims: [1] To replicate the positive outcome in the preliminary experiments against kala-azar in the hamster model by paying special attention to the absence of Leishmania persistence and residual pathogenicity; and [2] To extend the findings to additional animal models, e. g. BALB/c mouse, susceptible to cutaneous leishmaniasis by challenging them with the cutaneous species, i.e. Leishmania amazonensis. The results of these exploratory studies help evaluate the potential of the suicidal mutants for use as live vaccines not only against leishmaniasis but also as vaccine purveyors against other infectious and non-infectious diseases.