Broad spectrum Shigella subunit vaccine based on conserved proteins

摘要 志贺氏菌属是全球腹泻和痢疾的主要原因。中低龄2-5岁儿童 收入国家受影响最大。在年轻人腹泻疾病中，死亡率仅次于轮状病毒 儿童和反复发作的疾病会导致终生残疾。在工业化国家，志贺氏菌暴发 发生在日托中心和医疗机构。志贺氏菌属快速获取基因元素， 赋予抗菌素抗药性。一种安全、有效和负担得起的疫苗可以使一个重大的公共卫生 影响，但目前没有一项获得批准。基于志贺氏菌O抗原的候选药物正在临床开发中。 然而，这种方法不切实际且成本高昂，需要多种疫苗来预防由 不同O抗原的志贺氏菌株。该建议旨在发展一种安全、实用、有效的广泛- 基于高度保守的志贺氏菌III型分泌系统(TTSS)蛋白的频谱志贺氏菌疫苗， 侵袭质粒抗原(IPA)B、IPAH和毒力抗原病毒(ICSA)。我们的实验室是第一个 报道了志贺菌IPAB在小鼠体内的高免疫原性和广泛的保护性，以及与IPAB的相关性。 以及病毒-血清免疫球蛋白(IgG1)水平对人类志贺氏菌病的临床保护作用。本报告中的初步数据 应用表明，病毒还能诱导有效的保护性免疫。另外，我们发现， 居住在流行地区的成年人对志贺氏菌具有自然免疫力，其IPAB-、病毒-和 IPAH-血清抗体。针对这些蛋白质的母体免疫球蛋白通过以下途径更有效地传递给婴儿 将胎盘与抗O-多糖的免疫球蛋白进行比较。高水平的抗体也存在于 母乳。这种强大的母源免疫力与6个月内感染的低发生率是一致的。 生活的一部分。总之，我们的发现为广谱亚基(IPAB，IPAH， 和/或病毒)志贺氏菌疫苗。这种非肠道注射的疫苗预计耐受性良好，并能产生强效。 幼儿是免疫的主要目标群体。 在目标1中，我们将使用一种创新的无细胞蛋白来纯化和鉴定志贺氏菌IPAB、IpaH和Virg 综合系统。这一技术突破使高质量疫苗抗原的生产成为可能。 并且可以很容易地放大以用于制造。在目标2中，我们将评估IPAB、IPAH和 目的是在小鼠和豚鼠体内诱导保护性免疫并提供广谱保护。这些蛋白质 将单独或联合非肠道给药，并以明矾为佐剂--这一策略很容易实施 转化成人类的。在目标3中，我们将研究IPAB、IPAH和病毒抗体通过 小鼠和豚鼠感染模型的被动转移实验及靶向感染的具体步骤 每种抗体均采用体外细胞培养。我们拥有独特的专业知识、工具和新颖的技术来生产 预防多重耐药志贺氏菌的简单有效的新型亚单位疫苗。如果成功，这个概念 可以很容易地在人类第一阶段研究中进行测试。

ABSTRACT Shigella spp. are a major global cause of diarrhea and dysentery. Children 2-5 years of age in low- and middle- income countries are the most affected. Mortality is second only to rotavirus among diarrheal diseases in young children, and repeated bouts of disease cause lifelong disability. In industrialized nations, Shigella outbreaks have occurred in day care centers and medical institutions. Shigella spp. rapidly acquire genetic elements that confer antimicrobial resistance. A safe, effective, and affordable vaccine could make a major public health impact, yet none is currently approved. Candidates based on the Shigella-O-antigen are in clinical development. However, that approach is impractical and costly, requiring multiple vaccines to prevent disease caused by Shigella strains with different O antigens. This proposal seeks to develop a safe, practical, and effective broad- spectrum Shigella vaccine based on highly conserved Shigella Type III secretion system (TTSS) proteins, invasion plasmid antigen (Ipa) B, IpaH, and the virulence antigen VirG (IcsA). Our laboratory was the first to report the high immunogenic and broad protective capacity of Shigella IpaB in mice, and the association of IpaB- and VirG- serum IgG (IgG1) levels with clinical protection against shigellosis in humans. Preliminary data in this application demonstrate that VirG also elicits potent and protective immunity. In addition, we have found that adults living in endemic regions who acquire natural immunity to Shigella have high levels of IpaB-, VirG-, and IpaH- serum antibodies. Maternal IgG against these proteins are more efficiently transferred to infants through the placenta as compared to IgG against the O-polysaccharide. High levels of antibodies are also present in breast milk. This robust maternally-derived immunity is consistent with the low incidence of infection at <6 months of life. Together, our findings provide a strong premise for the success of a broad-spectrum subunit (IpaB, IpaH, and/or VirG) Shigella vaccine. Given parenterally, this vaccine is expected to be well-tolerated and to elicit robust immunity in young children, the main target group. In Aim 1, we will purify and characterize Shigella IpaB, IpaH, and VirG using an innovative cell-free protein synthesis system. This technological breakthrough allows the production of high-quality vaccine antigens at large yield and can be easily scaled-up for manufacturing. In Aim 2, we will evaluate the capacity of IpaB, IpaH, and VirG to elicit protective immunity and to confer broad-spectrum protection in mice and guinea pigs. The proteins will be administered parenterally, alone or combined, with alum as adjuvant ‒ a strategy that can be readily translated to humans. In Aim 3, we will investigate the protective role of IpaB, IpaH, and VirG antibodies through passive transfer experiments in mouse and guinea pig infection models and specific steps of infection targeted by each antibody using in vitro cell culture. We have unique expertise, tools, and novel technology to produce a simple and efficacious new subunit vaccine to prevent multidrug-resistant Shigella. If successful, this concept could be easily tested in Phase I studies in humans.