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

描述(申请人提供)：利什曼原虫。作为一种潜在的通用疫苗载体，与生俱来就具有非常有利的开发属性。转基因生物技术为这些真核原生动物表达外源蛋白用于疫苗接种而发展得很好。此外，从观察到人类单纯性皮肤利什曼病自然治愈后总是会产生终身免疫的观察，可以明显看出利什曼对疫苗接种的佐剂作用。此外，在自然感染中，利什曼原虫不仅抵抗体液溶解因子，仅寄生抗原提呈细胞(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--疫苗传递的理想场所。我们的长期目标是安全地利用利什曼原虫的属性，将疫苗归巢到APC，以提高它们的免疫效力。为此，我们对利什曼原虫进行了基因和化学修饰，以便于它们装载光敏剂(PS)，即尿卟啉(Uro)和酞菁(Pc)，从而使它们对光敏感，产生ROS用于细胞溶解，从而在APC的抗ROS吞噬溶酶体中释放疫苗。我们最近的研究表明，利什曼原虫可以非常有效地负载PS，以至于它们在非常暗的光下容易光解，例如荧光素酶/荧光素介导的发光(半自动光)。利什曼原虫特定阶段的尿素积累和发光发射目前正在通过转基因方法进行研究，以实现利什曼原虫在APC(自动发光)体内的光解自动化。值得注意的是，对动物接种尿卟啉性利什曼原虫疫苗后进行原位光照(光照后)提供了初步的结果。 其对实验性利什曼病具有免疫预防活性的证据。随后，携带Pc的利什曼原虫(Pc-LIGH)的光灭活被证明成功地将一种替代疫苗，即卵白蛋白(OVA)递送到APC，该疫苗在体外和体内都能有效地呈递这种抗原来激活OVA特异性T细胞。最近，我们获得了初步证据，表明利什曼原虫在同时用尿素和Pc敏化时，光解完成的一致性远远高于单独用Uro和Pc敏化。我们现在提出两个具体目标来详细研究利什曼原虫在前、后和自动光照条件下的这种双重光敏化作用：[1]通过评估DP光灭活利什曼原虫在APC和小鼠体内的存活率来评价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作为替代疫苗递送到APC以在体外和体内诱导T细胞免疫反应的有效性。DP后利什曼原虫的完全光解不仅有望确保它们作为疫苗载体的安全使用而消除，而且还能有效地输送和释放疫苗，以提高其免疫效力。因此，拟议研究的完成有望产生这些载体的最终版本，最适合光动力免疫疗法--不仅预防利什曼病，而且预防其他传染性/非传染性疾病。

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.