Toward suicidal automation of porphyric Leishmania for photodynamic vaccination

性状（由申请方提供）：利什曼原虫属被有利地认为是疫苗递送的通用载体，因为它们不仅进化出感染抗原呈递细胞（APC）的先天能力，而且还专门驻留在巨噬细胞的抗原加工吞噬溶酶体中。此外，有些种类相对无害，可引起人类单纯性皮肤利什曼病，已知该疾病可自愈，导致终身免疫。用活的利什曼原虫对人类群体进行免疫接种（利什曼化）确实是成功的，但它仍然产生皮肤病，这是潜在的使人衰弱的，因此是不希望的。通过分子遗传学手段减弱利什曼原虫的致病性或构建自杀性突变体，有可能提高活利什曼原虫疫苗接种的安全范围。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，它保留了其自然的能力，归巢的吞噬溶酶体的APC，但随后可诱导积累尿卟啉选择性光解其中。因此，及时诱导它们在其中的自我破坏将疫苗释放到期望的位点。用这些自杀突变体对仓鼠进行光动力学疫苗接种确实被发现模拟“利什曼化”，但没有皮肤病，对抗内脏利什曼病。此外，免疫引起的寄生虫没有明显的持久性和长期记忆，如其过继转移到幼稚动物。然而，发现BALB/c小鼠的类似光动力疫苗接种对皮肤利什曼病的保护不完全。在该模型中观察到的不太稳健的结果可归因于突变体从外部施用的利什曼特异性尿卟啉症诱导剂和/或光解中逃逸。我们建议通过纠正突变体的缺陷来改进自杀设计，如下：[1]转染卟啉原突变体，使吞噬溶酶体阶段特异性表达额外的酶，以及时产生δ-氨基乙酰丙酸（ALA），从而导致尿卟啉症的自我诱导;和[2]突变体的进一步遗传工程化以表达用于吞噬溶酶体阶段的荧光素酶-特异性产生光以增强它们在尿卟啉生成后的细胞溶解。相关转基因将置于已知3 '-UTR序列的调控下，以在突变体到达吞噬溶酶体进行分化时进行阶段特异性表达。将构建使用ALA合酶加荧光素酶的[1]和[2]的组合突变体，以试图实现它们的尿卟啉生成的自诱导以及荧光素诱导的光发射。因此，这种自动化设计预计将使突变体的光解系统和及时，独立的外部诱导剂施加到接种部位。这种自杀突变体的应用将增加免疫原性和安全裕度，从而增强它们作为用于光动力接种的通用疫苗平台的潜在用途。公共卫生相关性：本申请中提出的工作是开发，改进和评估寄生原虫利什曼原虫的诱导性自杀突变体，这些突变体具有进入并生活在我们专门接受疫苗的免疫细胞中的天然能力，以使其有效。这项研究的结果将帮助我们生产一种通用载体，用于预防和治疗传染性和非传染性疾病。

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.