Toward suicidal automation of porphyric Leishmania for photodynamic vaccination

描述(申请人提供)：利什曼原虫。它们被认为是疫苗传递的通用载体，因为它们不仅进化出一种固有的感染抗原提呈细胞(APC)的能力，而且专门驻留在巨噬细胞的抗原处理吞噬小体中。此外，一些物种相对无害，会导致人类单纯性皮肤利什曼病，这种疾病已知会自动愈合，导致终身免疫。活体利什曼原虫(利什曼化)对人类人群的免疫确实取得了成功，但它仍然会产生皮肤病，这可能会使人虚弱，因此是不受欢迎的。通过分子遗传学方法降低活体利什曼原虫的致病性或构建自杀性突变体，有可能提高活体利什曼原虫接种的安全边际。这些活疫苗的开发还没有超过实验阶段，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，它保留了它们天然的归巢到APC吞噬溶酶体的能力，但随后可以诱导积累尿卟啉在其中进行选择性光解。及时诱导它们在其中自毁，从而将疫苗释放到理想的位置。用这些有自杀倾向的突变体对仓鼠进行光动力免疫，确实可以模拟利什曼病，但没有皮肤病，对抗内脏利什曼病。此外，这种免疫力是在寄生虫没有明显的持久性和长期记忆的情况下激发的，通过将其收养转移到幼小动物身上证明了这一点。然而，对BALB/c小鼠进行类似的光动力疫苗接种后，发现它们对皮肤利什曼病的保护作用较差。在这个模型中看到的不那么强劲的结果是由于突变株逃脱了外部给药诱导的利什曼原虫特异性尿卟啉和/或光解。我们建议通过纠正突变体的缺陷来改进自杀设计，如下：[1)转染吞噬体期特异性表达的卟啉突变体，适时地产生一种额外的酶，以产生β-氨基酮戊酸(ALA)，从而导致尿失禁症的自我诱导；以及[2]进一步对突变体进行基因工程，以表达荧光素酶，用于吞噬体期特异性光的产生，以增强其在尿卟啉形成后的细胞溶解。当突变体到达吞噬小体进行分化时，相关的转基因将被置于已知3‘-UTR序列的调控下，进行阶段特异性表达。将构建使用丙氨酸合成酶和荧光素酶的[1]和[2]的组合突变体，以尝试实现它们对尿卟啉发生的自我诱导以及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光解变得系统和及时，而不依赖于接种部位施加的外部诱导剂。这种自杀性突变体的应用将提高免疫原性和安全性，从而增强它们作为光动力疫苗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、改进和评估寄生原虫利什曼原虫的可诱导自杀突变，这种突变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，使其有效。这项研究的结果将有助于我们生产一种通用的载体来提供预防和治疗传染病和非传染性疾病的疫苗。

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.