Development of a live attenuated vaccine for visceral leishmaniasis

摘要 由原生动物寄生虫利什曼原虫引起的感染包括皮肤（CL）、粘膜（ML）和内脏（ML）感染。 利什曼病（VL）。世界卫生组织将利什曼病归类为一种被忽视的热带疾病， 目前全球感染，每年约有200万新病例。从以下疾病中康复的患者 利什曼病发展保护性免疫力，防止再感染，这一切表明，疫苗， 可行在过去，利什曼化，一个过程中，故意感染低剂量的利什曼原虫 人畜共患皮肤利什曼病（ZCL）的主要病原体引起可控制的皮肤损害， > 90%的预防再感染，是ZCL流行地区的常见做法。根据现行 由于可能出现包括不愈合在内的并发症， 病变然而，这些研究表明，不引起疾病的减毒活寄生虫可能会被感染。 一种治疗利什曼病的有效疫苗遗传减毒L. infantum和L. donovani包括LdCen-/- 在动物模型中显示出作为疫苗的前景。然而，使用减毒活L. Donovani作为疫苗 由于这种利什曼原虫物种的内脏化潜力，在人类中可能引起安全性问题。减毒 交叉保护VL的皮肤营养性利什曼原虫可能是一种更安全的利什曼原虫疫苗 因为不良事件（例如，疫苗接种部位病变的发展）将易于监测， 使用批准的非药物干预治疗，如局部热疗。多种临床和 临床前动物研究表明，感染皮肤营养型利什曼原虫如利什曼原虫。重大而 L. tropica对L. donovani或L.婴儿使用CRISPR-Cas 技术，我们已经产生了无抗生素选择标记的中心蛋白基因缺陷型L.主要（LmCen-/-）。 LmCen-/-多次通过小鼠的全基因组测序证实了LmCen-/-的稳定缺失 在寄生虫基因组中没有其他突变的中心蛋白基因。我们的初步研究结果表明，LmCen-/-是 高度减毒的并且在免疫受损的动物中不引起疾病。我们还发现 用LmCen-/-寄生虫免疫诱导仓鼠中的疾病保护性Th 1应答并保护它们 防治白蛉传播的L. donovani。类似地，LmCen-/-免疫还保护免受 CL由L.少校这些发现表明LmCen-/-是一种有前途的利什曼病疫苗。我们 行业合作伙伴Gennova Biumma已经在cGMP条件下建立了LmCen-/-生产， 美国食品药品管理局批准的设施在这个项目中，我们建议（目标1）进行表型，遗传和 GMP-LmCen-/-寄生虫的生物化学表征和（目的2）确定GMP-LmCen-/-是否诱导 来自活动性VL患者的外周血单核细胞（PBMC）中的保护生物标志物， 无症状者和治愈VL的人。这个项目的科学前景，如果成功， 为推进LmCen-/-寄生虫作为抗人类利什曼病的疫苗提供基础。

Abstract Infections caused by the protozoan parasite Leishmania include cutaneous (CL), mucosal (ML), and visceral leishmaniasis (VL). The WHO classifies leishmaniasis as a neglected tropical disease with over 12 million current infections globally, and approximately 2 million new cases annually. Patients who recover from leishmaniasis develop protective immunity against reinfection, which altogether indicates that a vaccine is feasible. In the past, leishmanization, a process in which deliberate infections with a low dose of Leishmania major, etiologic agent of zoonotic cutaneous leishmaniasis (ZCL) causes a controlled skin lesion and provides > 90% protection against reinfection, was a common practice in ZCL-endemic regions. Under the current regulatory environment such practice is not acceptable due to possibility of complications including non-healing lesions. However, these studies suggest that live-attenuated parasites which don not cause a disease could be an effective vaccine for leishmaniasis. Genetically attenuated L. infantum and L. donovani including LdCen-/- have shown promise as a vaccine in animal models. However, using live-attenuated L. donovani as a vaccine in humans could raise safety concerns due to visceralizing potential of this Leishmania species. Attenuated dermatotrophic Leishmania that cross-protects against VL could be a safer vaccine against Leishmania because adverse events (e.g. development of a lesion as vaccination site) will be easy to monitor and can be treated using approved non-pharmacological interventions such as topical thermotherapy. Several clinical and preclinical animal studies have shown that an infection with dermatotrophic Leishmania such as L. major and L. tropica confers cross-protection against VL caused by L. donovani or L. infantum. Using CRISPR-Cas technology, we have generated antibiotic selection marker free centrin gene deficient L. major (LmCen-/-). Whole genome sequencing of LmCen-/- passed through mice multiple times has confirmed stable deletion of centrin gene without other mutations in the parasite genome. Our preliminary findings show that LmCen-/- are highly attenuated and fail to cause disease in immunocompromised animals. We have also found that immunization with LmCen-/- parasites induces disease protective Th1 response in hamsters and protects them against sand fly transmitted VL caused by L. donovani. Similarly, LmCen-/- immunization also protects against CL caused by L. major. These findings indicate that LmCen-/- is a promising vaccine for leishmaniasis. Our industry partner Gennova Biopharma has already established LmCen-/- production under cGMP conditions at their US-FDA approved facility. In this project, we propose to (Aim 1) perform phenotypic, genetic and biochemical characterization of GMP-LmCen-/-parasites and (Aim 2) determine whether GMP-LmCen-/- induce biomarkers of protection in peripheral blood mononuclear cells (PBMCs) from the patients with active VL, asymptomatics and those who have cured VL. The scientific promise of this project, if successful, could provide foundation for advancing LmCen-/- parasites as a vaccine against leishmaniasis in humans.