Photo-inactivation of Leishmania for safe and effective delivery of surrogate vac

描述（由申请人提供）：利什曼原虫天生具有特别有利的属性，可作为潜在的通用疫苗载体加以开发。转基因生物技术的发展使这些真核原生动物能够表达外源蛋白用于疫苗接种。此外，观察到人类单纯性皮肤利什曼病自发治愈后，终身持久的免疫总是随之而来，利什曼原虫对疫苗接种的辅助作用是显而易见的。此外，在自然感染中，利什曼原虫不仅抵抗体液溶解因子并只寄生于抗原呈递细胞（APC），即巨噬细胞/树突状细胞，而且还生活在它们的吞噬溶酶体中，这是一个理想的疫苗递送位点。我们的长期目标是安全地利用利什曼原虫的特性将疫苗归巢到APC，以提高其免疫效力。为此，我们对利什曼原虫进行了遗传和化学修饰，以促进它们与光敏剂（PS），即uroporphyrin （URO）和酞青素（Pc）的负载，从而使它们具有光敏性，产生用于细胞溶解的ROS，从而在APC的抗ROS吞噬溶酶体中释放疫苗。正如我们最近的研究表明，利什曼原虫可以有效地装载ps，以至于它们在暴露于非常微弱的光线下容易光解，例如荧光素酶/荧光素介导的发光（半自动光）。目前正在研究利什曼原虫阶段特异性URO积累和发光发射的转基因方法，以实现利什曼原虫在apc（自动发光）的吞噬体内光解的自动化。值得注意的是，接种带有尿卟啉利什曼原虫的动物疫苗，然后就地照明（光照后）提供了初步的效果

DESCRIPTION (provided by applicant): Leishmania spp. is innately endowed with exceptionally favorable attributes for exploitation as a potential universal vaccine carrier. Transgenic biotechnology is well-developed for these eukaryotic protozoa to express foreign proteins for vaccination. In addition, adjuvanticity of Leishmania on vaccination is evident from the observation that life-long lasting immunity ensue invariably after spontaneous cure of human simple cutaneous leishmaniasis. Moreover, in natural infection, not only do Leishmania resist humoral lytic factors and parasitize exclusively the antigen-presenting cells (APC), i. e. macrophages/dendritic cells, but also live in their phagolysosomes - a desirable site for vaccine delivery. It has been our long-term objective to safely harness the attributes of Leishmania for homing vaccines to APC to enhance their immune efficacy. Toward that end, we have genetically and chemically modified Leishmania to facilitate their loading with photosensitizers (PS), i. e. uroporphyrin (URO) and phthalocyanines (Pc), thereby rendering them photosensitive to produce ROS for cytolysis to release vaccines in the ROS-resistant phagolysosomes of APC. As shown by our recent study, Leishmania can be PS-loaded so effectively that they are susceptible to photolysis on exposure to very dim light, e. g. luciferase/luciferin-mediated luminescence (semi-auto-light). Leishmania stage-specific URO accumulation and luminescence emission are currently under study by transgenic approach for automation of Leishmania photolysis intraphagolysosomally in APCs (auto-light). Significantly, vaccination of animals with uroporphyric Leishmania followed by illumination in situ (post-light) provided initial evidence for its immuno-prophylactic activity against experimental leishmaniasis. Subsequently, photo-inactivation of Pc-loaded Leishmania (Pre-light) was shown to successfully deliver a surrogate vaccine, i. e. ovalbumin (OVA) to APC, which presented this antigen effectively to activate OVA-specific T cells in vitro and in vivo. Most recently, we obtained preliminary evidence, showing that Leishmania were photolysed to completion far more consistently when sensitized with both URO and Pc than with each individually. We now propose 2 specific aims to study this double photosensitization (DP) of Leishmania in detail under pre-, post- and auto-light conditions as follows: [1] To evaluate the safety of DP-photo-inactivated Leishmania by assessing their survival in vitro in APCs and in vivo in mice; [2] To evaluate the efficacy of DP-photo-inactivated Leishmania to deliver OVA as a surrogate vaccine to APCs for presentation to elicit T cell immune responses in vitro and in vivo. Complete photolysis of Leishmania after DP is expected not only to ascertain their elimination for safe use as a vaccine carrier but also to deliver and release vaccines effectively to increase their immune efficacy. Completion of the proposed studies is thus expected to produce a final version of these carriers optimal for photodynamic immuno-therapy & -prophylaxis not only against leishmaniasis but also other infectious/non-infectious diseases. PUBLIC HEALTH RELEVANCE: The work proposed in this application is to evaluate the safety and efficacy of a combined genetic and chemical approach to photo-inactivation of parasites. The outcome of the proposed studies will help us develop prophylactic and therapeutic photomedicines against infectious and non-infectious diseases.