Toward suicidal automation of porphyric Leishmania for photodynamic vaccination

通过光动力疫苗接种实现卟啉利什曼原虫的自杀自动化

基本信息

批准号：
7914385
负责人：
Kwang Poo Chang
金额：
$ 19.25万
依托单位：
ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-13 至 2012-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7914385
关键词：
5-Aminolevulinate synthase Address Adoptive Transfer Aminolevulinate Animal Model Animals Antigen-Presenting Cells Attenuated Attenuated Vaccines Automation Bacterial Luciferases Cells Communicable Diseases Cutaneous Leishmaniasis Cytolysis Development Enzymes Escape Mutant Esters Exposure to Firefly Luciferases Generations Genes Genetic Engineering Goals Hamsters Healed Homing Human Immune Immunity Immunization Inbred BALB C Mice Language Leishmania Life Light Lighting Luciferases Modeling Molecular Genetics Mus Outcome Outcome Study Parasites Pathogenicity Phagolysosome Photobacterium Population Porphyrins Production Protozoa Public Health Regulation Renilla Research Safety Simulate Singlet Oxygen Site Staging Testing Toxic effect Transfection Transgenes Transgenic Organisms Untranslated Regions Uroporphyrins Vaccination Vaccines Visceral Leishmaniasis Work antigen processing clinical practice design healing heme biosynthesis immunogenic immunogenicity improved light emission long term memory luciferin macrophage mutant new technology novel photolysis prevent public health relevance residence skin disorder suicidal vaccine delivery

项目摘要

DESCRIPTION (provided by applicant): Leishmania spp. are favorably considered as a universal carrier for vaccine delivery, since they have evolved not only an innate ability to infect antigen-presenting cells (APC) but also to reside exclusively in the antigen-processing phagolysosomes of macrophages. In addition, some species are relatively innocuous, causing human simple cutaneous leishmaniasis, which is known to heal spontaneously, resulting in life-long immunity. Immunization of human populations with live Leishmania (Leishmanization) has been indeed successful, but it still produces the skin disease, which is potentially debilitating and thus undesirable. It is possible to increase the safety margin of live Leishmania for vaccination by molecular genetic approaches to attenuate their pathogenicity or to construct suicidal mutants. These live vaccines have not been developed beyond the experimental stage, since they produced incomplete protection and/or persisted in animal models. We have recently developed a different type of transgenic Leishmania, which retains their natural ability of homing to the phagolysosomes of APC, but is inducible subsequently to accumulate uroporphyrin for selective photolysis therein. Timely induction of their self-destruction therein thus releases vaccines to the desirable site. Photodynamic vaccination of hamsters with these suicidal mutants was indeed found to simulate "Leishmanization", but without the skin disease, against visceral leishmaniasis. Moreover, the immunity was elicited without apparent persistence of the parasites and with long-term memory, as shown by its adoptive transfer to naive animals. Similar photodynamic vaccination of BALB/c mice was however found to protect them less completely against cutaneous leishmaniasis. The less robust outcome seen in this model is attributable to the escape of the mutants from externally administered inducers of Leishmania-specific uroporphyria and/or photolysis. We propose to improve the suicidal designs by rectifying the mutant deficiencies as follows: [1] Transfection of the porphyrinogenic mutants for phagolysosomal stage-specific expression of an additional enzyme to timely produce delta-aminolevulinate (ALA), thereby resulting in the self-induction of uroporphyria; and [2] Further genetic engineering of the mutants to express luciferase for phagolysosomal stage-specific generation of light to enhance their cytolysis after uroporphyrinogenesis. The relevant transgenes will be placed under the regulation of known 3'-UTR sequences for stage-specific expression as the mutants reach phagolysosomes for differentiation. Combination mutants of [1] and [2] using an ALA synthase plus a luciferase will be constructed in attempt to achieve their self-induction of uroporphyrinogenesis as well as luciferin-inducible emission of light. This design of automation is thus expected to render photolysis of the mutants systemic and timely, independent of the external inducers applied to the site of inoculation. Application of such suicidal mutants will increase both immunogenicity and safety margin, thereby enhancing their potential use as a universal vaccine platform for photodynamic vaccination. PUBLIC HEALTH RELEVANCE: The work proposed in this application is to develop, improve and evaluate inducible suicidal mutants of parasitic protozoa, Leishmania, which have the natural ability to enter and live in our immune cells specialized in accepting vaccines to make them effective. The outcome of the study will help us produce a universal carrier to deliver vaccines for preventing and treating infectious and non-infectious diseases.

描述（由申请人提供）：利什曼尼亚属。被认为是疫苗输送的通用载体，因为它们不仅进化出了先天感染抗原呈递细胞（APC）的能力，而且还可以专门驻留在巨噬细胞的抗原处理的吞噬体中。此外，某些物种是相对无害的，导致人类简单的皮肤利什曼病，该皮肤是自发治愈的，导致了终身的免疫力。现场利什曼原虫（Leishmanization）对人类人群的免疫接种确实是成功的，但它仍然会产生皮肤疾病，这可能会使人衰弱，因此不受欢迎。通过分子遗传方法，可以增加利什曼原虫的活利什曼原虫的安全边缘，以减轻其致病性或构建自杀突变体。这些活疫苗尚未在实验阶段之外开发，因为它们产生了不完全的保护和/或在动物模型中持续存在。我们最近开发了一种不同类型的转基因利什曼原虫，它保留了其自然归巢到APC的吞噬体的能力，但随后可以诱导地积累尿中肾蛋白以进行选择性光解。及时诱导其自我毁灭，因此将疫苗释放到理想的部位。确实发现，使用这些自杀突变体对仓鼠的光动力疫苗接种可以模拟“利什曼化”，但没有皮肤病，针对内脏利什曼病。此外，这种免疫力是在没有明显持续存在的寄生虫和长期记忆的情况下引起的，如它的继承转移到幼稚的动物所示。然而，发现BALB/C小鼠的类似光动力疫苗接种，可以使它们完全不受皮肤利什曼病的保护。在此模型中看到的不太强大的结果归因于突变体从利什曼尼亚特异性泌尿肌畸形和/或光解的外部施用的诱导剂中逃脱。我们建议通过纠正突变体缺陷来改善自杀设计，如下所示：[1]转染卟啉症突变体的转染，用于吞噬溶糖体阶段特异性表达以及时产生delta-氨基苯甲酸酯（ALA）的特异性表达，从而导致Uropphyria的自我诱导; [2]突变体的进一步基因工程表达荧光素酶，以用于吞噬性阶段特异性的光的发光，以增强泌尿核化发生后的细胞解析。随着突变体到达吞噬物体进行分化，相关转基因将被置于已知的3'-UTR序列的调节下。使用ALA合酶和荧光素酶的[1]和[2]的组合突变体将被构建，以尝试实现其对尿素发生的自我诱导以及光的荧光素诱导的光发射。因此，这种自动化的设计有望使突变体的全身性和及时的光解，与应用于接种部位的外部诱导剂无关。这种自杀突变体的应用将增加免疫原性和安全边缘，从而增强其作为光动力疫苗接种的通用疫苗平台的潜在用途。公共卫生相关性：本应用中提出的工作是开发，改善和评估寄生虫原生动物利什曼尼亚的自杀诱导的自杀突变体，这些突变体具有自然的能力，可以自然地进入和生活在我们的免疫细胞中，专门接受疫苗以使其有效。该研究的结果将有助于我们生产一种通用载体，以提供预防和治疗传染病和非感染疾病的疫苗。