A cross protective multivalent vaccine for Shigella and ETEC

项目摘要：尽管志贺氏菌和产肠毒素大肠杆菌(ETEC)是最常见的两种 发展中国家和国际上儿童中重度腹泻的重要细菌病因 对于旅行者来说，目前还没有针对这些感染的许可疫苗。此应用程序的目标是 开发一种可注射的联合疫苗来预防这两种细菌的肠道感染。 为了实现这一点，我们开发了一种ETEC候选疫苗(MecVax)，它使用表位和结构- 基于疫苗学平台的MEFA(多表位融合抗原)可产生多价融合蛋白 在一个蛋白质上有多个表位。MecVax包括两种蛋白质，一种是刺激对 ETEC的定植因子抗原(CFAs)和另一种能刺激免疫的两种肠毒素。第一 有七种最常见的CFA的表位，而第二种有不耐热的肠毒素的表位 (Lt)和耐热肠毒素(Sta)。用MecVax免疫的动物产生功能性抗体 CFA和两种毒素，在受到ETEC挑战时受到保护。开发一种安全的免疫原性 StA的抗原是创新的，因为这种小蛋白(19AA)不是天然的免疫原性。 为了扩大对志贺氏菌病的保护，我们开发了志贺氏菌的MEFA，它包括不同志贺氏菌的表位。 在所有志贺氏菌和侵袭性大肠杆菌中常见的毒力蛋白。为了进一步扩展 除了保护，志贺氏菌Mefa还包括志贺毒素的表位(包括产生大肠杆菌的志贺毒素- STEC)。因为这种疫苗是基于所有毒株共同的毒力蛋白，所以这种MEFA是 预期对所有志贺氏菌物种和血清型具有保护作用，而不受其他血清型的限制 完全基于内毒素抗原的疫苗。 目前还没有单一的动物模型可以有效地评估对甲氧西林的保护性免疫反应。 因此，我们使用多种检测方法来确定对MEFA疫苗的免疫反应。 小鼠的抗体反应量化了对MEFA融合中特定表位的反应，而我们使用 功能抗体检测以确定抗体是否阻断黏附(对ETEC)、侵袭(对志贺氏菌)和 中和毒素(针对LT、STA和志贺毒素)。然后，我们用一只兔子在动物模型中确定保护作用 定植模型(ETEC和志贺氏菌)和预防疾病的猪模型(ETEC)和 小鼠致死性肺攻击模型(志贺氏菌)。结合使用这些化验方法和动物模型， 我们将建立ETEC-志贺氏菌联合疫苗的临床前证据。 中心假设是，与目前的口服疫苗候选者不同，可注射的志贺氏菌-ETEC Mefa 疫苗将刺激更高的血清抗体效价，并在受试者最脆弱的时候提供保护 自然暴露会提高当地的肠道免疫力。一种有效的志贺氏菌-ETEC疫苗将防止数百人 每年减少数百万腹泻临床病例，挽救20万人的生命。

Project Summary: Even though Shigella and enterotoxigenic Escherichia coli (ETEC) are the two most important bacterial causes of moderate-to-severe diarrhea in children in developing countries and in international travelers, there are currently no licensed vaccines for these infections. The goal of this application is the development of an injectable combination vaccine to protect from these two bacterial enteric infections. To accomplish this, we developed an ETEC vaccine candidate (MecVax) using the epitope- and structure- based vaccinology platform, MEFA (Multi-Epitope Fusion Antigen) which results in a polyvalent fusion protein with multiple epitopes on a single protein. MecVax includes two proteins, one to stimulate immunity to the colonization factor antigens (CFAs) of ETEC and another to stimulate immunity to the two enterotoxins. The first has epitopes for the seven most common CFAs while the second has epitopes for the heat-labile enterotoxin (LT) and the heat stable enterotoxin (STa). Animals immunized with MecVax develop functional antibodies to the CFAs and the two toxins and are protected when challenged with ETEC. Developing a safe and immunogenic antigen for STa is innovative since this small protein (19AA) is not naturally immunogenic. To extend protection to shigellosis, we developed a MEFA for Shigella which includes epitopes for the different virulence proteins that are common among all strains of Shigella and invasive E. coli. To further extend protection, the Shigella MEFA also includes epitopes for shiga toxins (including shiga toxin producing E coli - STEC). Because this vaccine is based on the virulence proteins which are common to all strains, this MEFA is expected to protect against all Shigella species and serotypes and not be limited by serotype as are the other vaccines which are based solely on the LPS antigen. There is no single animal model which can effectively evaluate the protective immune responses to the MEFA vaccines; thus, we use a combination of assays to determine the immune responses to the MEFA vaccines. Antibody responses in mice quantitate the responses to the specific epitopes in MEFA fusion while we use functional antibody assays to determine if the antibodies block adhesion (for ETEC), invasion (for Shigella) and neutralize toxins (for LT, STa and Shiga toxins). We then determine protection in animal models using a rabbit colonization model (both ETEC and Shigella) and protection against disease in a pig model (for ETEC) and a mouse lethal pulmonary challenge model (for Shigella). Using a combination of these assays and animal models, we will build the preclinical evidence for the combined ETEC-Shigella combination vaccine. The central hypothesis is that unlike current oral vaccine candidates, an injectable Shigella-ETEC MEFA vaccine will stimulate higher serum antibody titers and protect when the subject is most vulnerable and that natural exposure will boost local intestinal immunity. An effective Shigella-ETEC vaccine will prevent hundreds of millions diarrhea clinical cases and save > 200,000 lives annually.