Toward suicidal automation of porphyric Leishmania for photodynamic vaccination

描述（由申请人提供）：利什曼原虫被认为是疫苗递送的通用载体，因为它们不仅进化出了感染抗原呈递细胞（APC）的先天能力，而且还只存在于巨噬细胞的抗原处理吞噬溶酶体中。此外，一些物种相对无害，引起人类单纯性皮肤利什曼病，已知可自愈，导致终身免疫。对感染活利什曼原虫的人群进行免疫接种（利什曼化）确实取得了成功，但它仍然会产生皮肤疾病，这可能使人衰弱，因此是不可取的。有可能通过分子遗传方法来降低利什曼原虫的致病性或构建自杀性突变体，从而提高利什曼原虫活疫苗接种的安全边际。这些活疫苗的开发尚未超出实验阶段，因为它们产生不完全保护和/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吞噬溶酶体的能力，但随后可诱导积累尿卟啉以在其中进行选择性光解。及时诱导它们的自我毁灭，从而将疫苗释放到所需的部位。光动力接种这些自杀突变体的仓鼠确实被发现模拟“利什曼化”，但没有皮肤疾病，对抗内脏利什曼病。此外，免疫是在没有明显的寄生虫持久性和长期记忆的情况下产生的，正如其在幼稚动物身上的适应性转移所表明的那样。然而，发现BALB/c小鼠的类似光动力疫苗对皮肤利什曼病的保护不完全。在该模型中看到的较不可靠的结果是由于突变体从外部给予的利什曼特异性尿卟啉症和/或光解诱导剂中逃逸。我们建议通过纠正突变体缺陷来改善自杀设计：[1]转染卟啉突变体，使其在吞噬酶体阶段特异性表达一种额外的酶，以及时产生α -氨基乙酰酸（ALA），从而导致尿卟啉症的自我诱导；和[2]对突变体进行进一步的基因工程，表达荧光素酶，用于吞噬溶酶体特定阶段的发光，以增强它们在尿卟啉发生后的细胞溶解。当突变体到达吞噬溶酶体进行分化时，相关的转基因将在已知的3'-UTR序列的调控下进行阶段特异性表达。利用ALA合酶和荧光素酶构建[1]和[2]组合突变体，试图实现其自诱导尿卟啉发生和荧光素诱导发光。因此，这种自动化设计有望使突变体的光解作用系统和及时，而不依赖于接种部位的外部诱导剂。这种自杀性突变体的应用将增加免疫原性和安全边际，从而增强其作为光动力疫苗接种通用疫苗平台的潜力。公共卫生相关性：本申请中提出的工作是开发，改进和评估寄生原生动物利什曼原虫的诱导自杀突变体，这些突变体具有进入和生活在我们的免疫细胞中的天然能力，这些免疫细胞专门接受疫苗，使其有效。这项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传染病的疫苗。

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.