Transgenic porphyric Leishmania as suicidal live vaccines against leishmaniasis

转基因卟啉利什曼原虫作为针对利什曼病的自杀性活疫苗

• 批准号: 7078078
• 负责人: Kwang Poo Chang
• 金额: $ 22.8万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7078078
• 关键词: Leishmania; active immunization; cytolysis; hamsters; intracellular; leishmaniasis; live vaccine; macrophage; model; mutant; porphyrias

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.

描述（由申请人提供）：我们制备了转基因利什曼原虫，其可用于针对利什曼病和其他疾病的光动力疫苗接种。这些锥虫原生动物天然缺乏血红素生物合成，从而使它们依赖于外源四吡咯的供应来制造功能性呼吸复合物。我们利用利什曼原虫的这些特殊缺陷来产生自杀突变体。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，因此当它们被告知要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使其激活或呈递。为了设计这样的突变体，利什曼原虫属。用编码血红素生物合成途径中第二和第三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δ-氨基乙酰丙酸（ALA）——该途径中第一种酶的产物时，这些转染子就会呈现卟啉状。当巨噬细胞被这些转染子感染并暴露于 ALA 时，宿主细胞及其细胞内利什曼原虫中都会出现卟啉症。然而，巨噬细胞中形成的卟啉会迅速代谢至背景水平，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症的发展导致它们的选择性细胞溶解，这可以通过光照射来调节和增强。细胞内突变体的这种选择性破坏改变了体外感染巨噬细胞的整体表达谱，表明免疫原性增强和寄生虫消除，正如微阵列分析所确定的。初步体内数据支持了这一点，表明通过叙利亚金仓鼠模型中的实验性黑热病疫苗接种方案可提供保护。我们建议在本申请中通过实现以下具体目标来进一步探索这种活疫苗模型的功效：[1]通过特别注意在仓鼠模型中复制黑热病初步实验的积极结果，特别注意缺乏利什曼原虫的持久性和残留致病性； [2] 将研究结果扩展到其他动物模型，例如。 g。 BALB/c 小鼠，通过用皮肤物种（即亚马逊利什曼原虫）攻击它们，易患皮肤利什曼病。这些探索性研究的结果有助于评估自杀突变体不仅用作针对利什曼病的活疫苗，而且还用作针对其他传染性和非传染性疾病的疫苗供应商的潜力。