Broad spectrum Shigella subunit vaccine based on conserved proteins

摘要 志贺菌是全球腹泻和痢疾的主要原因。2-5岁的儿童在低-和中等- 收入国家受影响最大。死亡率在年轻人的呼吸道疾病中仅次于轮状病毒 反复发作的疾病会导致终身残疾。在工业化国家，志贺氏菌的爆发 发生在日托中心和医疗机构。志贺菌快速获得遗传元素， 赋予抗微生物剂抗性。一种安全、有效和负担得起的疫苗可以使一个主要的公共卫生 影响，但目前没有批准。基于志贺氏菌-O-抗原的候选物正在临床开发中。 然而，这种方法是不切实际和昂贵的，需要多种疫苗来预防疾病引起的 O抗原不同的志贺菌菌株。该提案旨在开发一种安全、实用、有效的广泛的- 基于高度保守的志贺氏菌III型分泌系统（TTSS）蛋白的谱志贺氏菌疫苗， 侵袭质粒抗原（Ipa）B、IpaH和毒力抗原VirG（IcsA）。我们的实验室是第一个 报道了志贺氏菌IpaB在小鼠中的高免疫原性和广泛的保护能力，以及IpaB- VirG-血清IgG（IgG 1）水平，具有临床保护作用，可预防人类志贺氏菌病。初步数据显示， 应用表明，VirG还具有较强的保护性免疫力。此外，我们发现， 生活在流行地区的成年人获得对志贺氏菌的天然免疫力， IpaH-血清抗体。母体抗这些蛋白质的IgG通过以下途径更有效地转移给婴儿： 胎盘与抗O-多糖的IgG相比。高水平的抗体也存在于 母乳。这种强有力的母源性免疫与<6个月时感染的低发生率一致 生命总之，我们的发现为广谱亚基（IpaB，IpaH， 和/或VirG）志贺氏菌疫苗。胃肠外给药，预期该疫苗耐受性良好， 免疫力的主要目标群体是幼儿。 在目标1中，我们将使用创新的无细胞蛋白纯化和表征志贺氏菌IpaB，IpaH和VirG 合成系统这一技术突破使得大规模生产高质量的疫苗抗原成为可能 并且可以容易地按比例放大用于制造。在目标2中，我们将评估IpaB、IpaH和 VirG在小鼠和豚鼠中引发保护性免疫并赋予广谱保护。的蛋白质 将单独或与明矾作为佐剂联合胃肠外给药， 翻译给人类。在目标3中，我们将通过以下途径研究IpaB、IpaH和VirG抗体的保护作用： 在小鼠和豚鼠感染模型中的被动转移实验和靶向感染的具体步骤 通过使用体外细胞培养的每种抗体。我们拥有独特的专业知识、工具和新颖的技术， 预防多重耐药志贺菌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的I期研究中进行测试。

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.