Immunological characterization of rationally-designed vaccines against plague in mice and non-human primate models

鼠疫耶尔森氏菌（Yersinia pestis，Yp）是腺鼠疫和肺鼠疫的病原体。瘟疫的数量越来越多 全球病例（2010-2017年），包括美国，约18%的病死率可能反映了气候变化， 啮齿动物携带者的射程变化2017年马达加斯加爆发疫情，约2400例（>75%为肺炎）和约9% 导致世卫组织（2018年4月）加强了开发新一代亚单位和活细胞的必要性， 减毒鼠疫疫苗Yp能够在死亡的宿主中存活，然后在多年的沉默后复活， 令人担忧。此外，抗真菌的Yp菌株天然存在或已被有意开发，并且目前 没有FDA批准的鼠疫疫苗可用。最后，由荚膜组成的双组分亚单位疫苗 抗原F1和T3 SS组分和效应物LcrV（低钙应答V抗原），其仅产生 体液免疫应答，在非洲绿色猴（AGM）中提供可变保护，并产生不良T 细胞介导的人体免疫反应。由于细胞免疫对保护也至关重要，我们将重点放在 首先鉴定Yp的新毒力基因，然后组合删除这些基因以开发新的 减毒活疫苗株。我们的数据表明，两种这样的候选疫苗是100%减毒的， 在小鼠/大鼠中诱导腺鼠疫/肺鼠疫，并产生长期稳健的体液和细胞免疫 为啮齿类动物提供100%的保护，以防止肺鼠疫的发展。无临床症状 的疾病或组织病理学病变，在免疫期间或当接种疫苗的动物 随后在肺鼠疫模型中暴露于野生型Yp CO 92。我们假设， 这些突变体的免疫学特征及其在高等动物如食蟹猴中的测试 猕猴（CM）和AGM，将为未来的临床前和临床研究提供依据。是有先例 使用减毒活疫苗对抗鼠疫，因为EV 76毒株在世界某些地区用于人类 瘟疫流行的地方然而，疫苗是反应原性的，代表自发突变，并引起 铁负荷过高的患者。提出了三个目标：目标1，证明疗效， Yp CO 92（东方生物变种）产生的两种候选疫苗对其他Yp的免疫应答 biovars（Antiqua和Medievalis），以及CO 92的F1-负突变体在腺和肺炎小鼠模型中的作用。 我们对突变体的数据表明IL-17（一种Th 17细胞因子）、Th 1-IFN-γ和抗体在 保护在目的2中，我们将通过使用ROR突变体来研究这种保护的机制基础（一种选择的突变体）。 /-小鼠，其缺乏Th 17细胞，以及IFN-γ和伊加k/o小鼠，并辨别它们与嗜中性粒细胞的联系 招募和粘膜免疫，以对抗淋巴腺/肺鼠疫模型中的Yp感染。在目标3中，CM 和AGM将与一种突变体一起使用，以证明其在引起淋巴腺和/或 肺鼠疫以及反应原性。保护性免疫的相关性将被建立。 这些创新的机制/翻译方法将导致有效的新一代鼠疫疫苗。

Yersinia pestis (Yp) is the causative agent of bubonic and pneumonic plague. The increasing number of plague cases globally (2010-2017), including the U.S., with a ~18% case fatality rate may reflect climate changes and a rodent carrier range shift. An outbreak of 2017 in Madagascar with ~2400 cases (>75% pneumonic) and ~9% causalities has led WHO (April 2018) to intensify the need for developing new generation subunit and live- attenuated plague vaccines. Yp’s ability to persist in dead hosts, and then resurge after years of silence, is alarming. Also, antibiotic-resistant Yp strains occur naturally or have been intentionally developed and currently no FDA-approved plague vaccine is available. Finally, two-component subunit vaccines composed of capsular antigen F1 and a T3SS component and effector LcrV (low calcium response V antigen), which only generate a humoral immune response, provide variable protection in African green monkeys (AGM) and generate poor T cell-mediate immune responses in humans. Since the cellular immunity is also critical for protection, we focused first on identifying new virulence genes of Yp and then to delete such genes in combination to develop novel live-attenuated vaccine strains. Our data indicated that two such vaccine candidates were 100% attenuated in inducing bubonic/pneumonic plague in mice/rats and generated long-term robust humoral and cellular immune responses to provide 100% protection to rodents against developing pneumonic plague. No clinical symptoms of the disease or histopathological lesions were noted either during immunization or when the vaccinated animals were subsequently exposed to wild-type Yp CO92 in a pneumonic plague model. We hypothesize that further immunological characterization of these mutants and their testing in higher animals, such as cynomolgus macaques (CM) and AGM, will provide a rationale for future preclinical and clinical studies. There is a precedent for using a live-attenuated vaccine against plague, as EV76 strain is used in humans in some parts of the world where plague is endemic. However, the vaccine is reactogenic, represents a spontaneous mutant, and causes disease in patients with over iron load. Three Aims have been proposed: Aim 1, to demonstrate efficacy and immune responses of two vaccine candidates generated from Yp CO92 (biovar Orientalis) against other Yp biovars (Antiqua and Medievalis), and the F1-minus mutant of CO92 in bubonic and pneumonic mouse models. Our data with the mutants indicated a potential role of IL-17 (a Th17 cytokine), Th1-IFN-γ, and antibodies in protection. In Aim 2, we will study the mechanistic basis of this protection (one chosen mutant) by using RORt- /- mice, which lack Th17 cells, as well as IFN-γ, and IgA k/o mice and to discern their links to neutrophil recruitment and mucosal immunity, to combat Yp infection in bubonic/pneumonic plague models. In Aim 3, CM and AGM will be used with one mutant to demonstrate its short- and long-term efficacy in causing bubonic and/or pneumonic plague as well as reactogenicity. The correlates of protective immunity will then be established. These innovative mechanistic/translational approaches will result in effective new generation plague vaccines.