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

性状（由申请方提供）：利什曼原虫属天生具有作为潜在的通用疫苗载体开发的特别有利的属性。利用转基因技术可以使真核原生动物表达外源蛋白用于疫苗接种。此外，利什曼原虫对疫苗接种的佐剂作用从观察到的人单纯皮肤利什曼病自发治愈后总是随之产生终身持久的免疫力中得到证实。此外，在自然感染中，利什曼原虫不仅能抵抗体液溶解因子，而且只寄生于抗原呈递细胞（APC），e.巨噬细胞/树突状细胞，但也生活在它们的吞噬溶酶体中-疫苗递送的理想位点。我们的长期目标是安全地利用利什曼原虫的属性将疫苗导向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，以促进其装载光敏剂（PS），即。e.尿卟啉（URO）和酞菁（Pc），从而使它们具有光敏性以产生ROS用于细胞溶解，从而在APC的ROS抗性吞噬溶酶体中释放疫苗。正如我们最近的研究所示，利什曼原虫可以有效地装载PS，以至于它们在暴露于非常昏暗的光线下时容易发生光解，例如。G.荧光素酶/荧光素介导的发光（半自动发光）。利什曼原虫阶段特异性URO积累和发光发射目前正在通过转基因方法进行研究，用于在APC中自动吞噬利什曼原虫的光解（自动光）。值得注意的是，用尿卟啉利什曼原虫接种动物，然后原位照射（光照后）， 证据表明其对实验性利什曼病具有免疫预防活性。随后，显示负载Pc的利什曼原虫的光灭活（预光）成功地递送替代疫苗，即。e.将卵清蛋白（OVA）呈递给APC，APC在体外和体内有效地呈递该抗原以激活OVA特异性T细胞。最近，我们获得的初步证据表明，利什曼原虫光解完成时，更一致的敏化URO和PC比每个单独。我们现在提出了两个具体的目标，详细研究这种双重光敏化（DP）的利什曼原虫在前，后和自动光照条件下，如下：[1]通过评估其在体外APC和小鼠体内的存活来评估DP光灭活利什曼原虫的安全性;[2]评价DP光灭活利什曼原虫将OVA作为替代疫苗递送至APC以在体外和体内引发T细胞免疫应答的功效。DP后利什曼原虫的完全光解预计不仅可以确定它们作为疫苗载体的安全使用的消除，而且还可以有效地递送和释放疫苗，以提高其免疫效力。因此，预计完成所提出的研究将产生这些载体的最终版本，其不仅对利什曼病而且对其他传染性/非传染性疾病的光动力学免疫治疗和预防是最佳的。 公共卫生相关性：本申请中提出的工作是评价遗传和化学相结合的寄生虫光灭活方法的安全性和有效性。这些研究的结果将有助于我们开发针对感染性和非感染性疾病的预防和治疗性光医学。

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.