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

光灭活利什曼原虫以安全有效地输送替代疫苗

• 批准号: 8386400
• 负责人: Kwang Poo Chang
• 金额: $ 23.18万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-25 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8386400
• 关键词: Acquired Immunodeficiency Syndrome; Adjuvanticity; Animals; Antigen-Presenting Cells; Antigens; Automation; Biological Assay; Biotechnology; CD8B1 gene; Cell Count; Cell Survival; Cells; Cellular Immunity; Communicable Diseases; Complementary DNA; Cutaneous Leishmaniasis; Cytolysis; Data; Dendritic Cells; Disease; Effectiveness; Endotoxins; Engineering; Enzymes; Epitopes; Eukaryotic Cell; Evaluation; Exposure to; Figs - dietary; Growth; Homing; Human; Immune; Immune response; Immunity; In Situ; In Vitro; Infection; Injection of therapeutic agent; Investigation; Leishmania; Leishmaniasis; Lesion; Life; Light; Lighting; Luciferases; Lytic; Malaria; Measurement; Mediating; Methods; Microscopic; Monoclonal Antibodies; Mus; Organ; Outcome; Ovalbumin; Parasites; Particulate; Phagolysosome; Pharmaceutical Preparations; Phase; Photosensitization; Photosensitizing Agents; Post-Translational Protein Processing; Process; Production; Property; Prophylactic treatment; Proteins; Protocols documentation; Protozoa; Reagent; Resistance; Safety; Singlet Oxygen; Site; Skin; Specificity; Staging; T cell response; T-Cell Activation; T-Lymphocyte; Therapeutic; Time; Transfection; Transgenes; Transgenic Organisms; Uroporphyrins; Vaccination; Vaccines; Viral; Work; chemical genetics; cost; in vivo; luciferin; luminescence; macromolecule; macrophage; novel vaccines; photolysis; phthalocyanine; prophylactic; recombinant peptide; residence; scale up; tumor; vaccine delivery

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.

描述（由申请人提供）：利什曼原虫属。天生具有作为潜在的通用疫苗载体开发的异常有利的属性。转基因生物技术已经很成熟，可以让这些真核原生动物表达外源蛋白用于疫苗接种。此外，从观察到人类单纯性皮肤利什曼病自然治愈后必然会产生终生持久的免疫力，可以明显看出利什曼原虫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并专门寄生抗原呈递细胞（APC），即。 e.巨噬细胞/树突状细胞，但也生活在其吞噬溶酶体中 - 疫苗输送的理想场所。我们的长期目标是安全地利用利什曼原虫的特性，将疫苗导向 APC，以增强其免疫功效。为此，我们对利什曼原虫进行了基因和化学改造，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，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，利什曼原虫可以如此有效地装载 PS，以至于它们在暴露于非常暗的光线（例如光线）时很容易发生光解。 g。荧光素酶/荧光素介导的发光（半自动发光）。目前正在通过转基因方法研究利什曼原虫阶段特异性 URO 积累和发光发射，以实现利什曼原虫在 APC 中吞噬溶酶体内光解的自动化（自动光）。值得注意的是，对动物进行尿卟啉利什曼原虫疫苗接种，然后进行原位照明（光照后）提供了初步的结果。 其针对实验性利什曼病的免疫预防活性的证据。随后，负载 PC 的利什曼原虫（Pre-light）的光灭活被证明可以成功提供替代疫苗，即。 e.卵清蛋白 (OVA) 呈递给 APC，后者将这种抗原有效地在体外和体内激活 OVA 特异性 T 细胞。最近，我们获得了初步证据，表明当同时使用 URO 和 Pc 致敏时，利什曼原虫光解完成的一致性比单独使用时要一致得多。我们现在提出两个具体目标，详细研究利什曼原虫在光照前、光照后和自动光照条件下的双重光敏化 (DP)，如下所示： [1] 通过评估 DP 光灭活利什曼原虫的存活率来评估其安全性APC 体外实验和小鼠体内实验； [2] 评估 DP 光灭活利什曼原虫将 OVA 作为替代疫苗递送至 APC 的功效，以在体外和体内引发 T 细胞免疫反应。 DP后利什曼原虫的完全光解不仅可以确定其被消除以安全地用作疫苗载体，而且可以有效地递送和释放疫苗以提高其免疫功效。因此，拟议研究的完成预计将产生这些载体的最终版本，该最终版本不仅适用于利什曼病，而且适用于其他传染性/非传染性疾病的光动力免疫治疗和预防。 公共健康相关性：本申请中提出的工作是评估遗传和化学相结合的光灭活寄生虫方法的安全性和有效性。拟议研究的结果将帮助我们开发针对传染性和非传染性疾病的预防性和治疗性光药物。