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 利什曼病(VL)。世界卫生组织将利什曼病列为一种被忽视的热带疾病，有超过1200万人 全球目前的感染情况，每年约有200万新病例。康复的病人 利什曼病产生针对再次感染的保护性免疫，这总体上表明疫苗是 可行。在过去，利什曼病，一种故意感染低剂量利什曼原虫的过程 人畜共患皮肤利什曼病(ZCL)的主要病原体可引起受控皮肤损害，并提供 &gt；90%的保护率，在ZCL流行地区是一种常见的做法。在海流下 监管环境这种做法是不可接受的，因为可能会出现并发症，包括无法愈合 损伤。然而，这些研究表明，不会导致疾病的减毒寄生虫可能是 一种有效的利什曼病疫苗。包括LdCen-/-在内的遗传减毒L.infantum和L.donovani 已经在动物模型中显示出作为疫苗的前景。然而，使用减毒活的杜氏乳杆菌作为疫苗 由于这种利什曼原虫的内脏潜在性，可能会引起安全问题。衰减 对VL具有交叉保护作用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，作为疫苗接种部位的病变的发展)将很容易监测，并且可以 使用经批准的非药物干预措施进行治疗，如局部热疗。几个临床和 临床前动物研究表明，皮肤营养性利什曼原虫感染，如梅杰利什曼原虫和利什曼原虫 热带乳杆菌对由杜氏乳杆菌或婴儿乳杆菌引起的VL具有交叉保护作用。使用CRISPR-CAS 技术，我们已经产生了抗生素选择标记无中心素基因缺陷型LmCen-/-。 多次通过小鼠的LmCen-/-的全基因组测序证实稳定的缺失 寄生虫基因组中没有其他突变的中心素基因。我们的初步发现表明，LmCen-/-是 高度减毒，不会导致免疫功能低下的动物患病。我们还发现， LmCen-/-寄生虫免疫仓鼠诱导疾病保护性Th1反应并保护它们 对杜氏沙蝇传播的VL有明显的抑制作用。同样，LmCen-/-免疫也可以预防 L。梅杰引起的CL。这些发现表明LmCen-/-是一种很有前途的利什曼病疫苗。我们的 行业合作伙伴Gennova Biophma已经在cGMP条件下建立了LmCen/-生产 他们的美国FDA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-/-寄生虫的生化特征及(Aim 2)确定GMP-LmCen-/-是否诱导 活动期VL患者外周血单个核细胞(PBMC)保护的生物标志物 无症状患者和已治愈的VL患者。这个项目的科学前景，如果成功，可能会 为发展LmCen-/-寄生虫作为人类利什曼病疫苗奠定了基础。

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.