Alum-absorbed subunit vaccine to prevent intestinal amebiasis

描述（由申请方提供）：我们建议生产溶组织内阿米巴亚单位疫苗，并在小鼠中测试其免疫原性和对肠道阿米巴病的保护作用。溶组织内阿米巴由于其低感染剂量和食物和水-骨传播而被NIH列为B类生物防御病原体，并且是非洲、亚洲和拉丁美洲儿童腹泻的重要原因。候选疫苗抗原是E.溶组织Gal/GalNAc凝集素，介导寄生虫粘附和接触依赖性细胞毒性。LecA含有天然Gal/GalNAc凝集素的所有毒力中和抗体表位，事实上，儿童免疫力的替代标志物是针对LecA的肠道伊加。LecA在许多研究者的实验室中作为阿米巴病啮齿动物模型的疫苗的有效性强调了我们方法的可行性。 拟议研究的创新方面包括生产第一种针对肠道寄生虫的疫苗，使用抗原（LecA），其中已绘制了毒力中和表位，并利用新型阿米巴结肠炎小鼠模型进行疗效研究。 我们的方法将是进行明矾配制的LecA疫苗的临床前开发。我们小组发表的初步研究已经证明了这种原型疫苗在小鼠模型中的有效性。提出了两个具体目标来进行这项工作：在具体目标1中，我们将表达和纯化从E。大肠杆菌中的Gal/GalNAc凝集素的LecA片段，使用可扩展到cGMP的标准操作程序。我们将对纯化的蛋白质进行内毒素、DNA和污染宿主细胞蛋白质的质量控制，将其吸收到明矾中并测试其稳定性和批间变异性。TechLab将完全负责目标1。在具体目标2中，我们将比较不同的给药间隔和剂量，以检查明矾吸收的LecA胃肠外疫苗的免疫原性（细胞和体液）：（a）抗原特异性攻毒前IFN-g+、CD 4 + T细胞对LecA的应答以及粘膜和血清抗体应答的幅度，以及（B）疫苗有效性和持久性。UVA将进行所有免疫接种，细胞介导的免疫反应测试，体液反应和疫苗试验。TechLab将提供LecA明矾吸附疫苗和体液反应ELISA分析试剂。 这项研究建立在研究人员在阿米巴病方面15年的合作基础上，TechLab的Lyerly博士和UVa的Petri博士（Gal/GalNAc凝集素的发现者）。靠近TechLab和UVa（在I-81上开车2小时），为这种协作环境提供了便利。 这些研究的成功完成将为LecA疫苗的cGMP生产和毒性研究（未来I期人体临床试验所需）做好准备，最大限度地提高其免疫原性和有效性，从而为人类阿米巴病疫苗的I期临床试验奠定基础。 公共卫生相关性：溶组织内阿米巴是人体肠道、肝脏和其他器官的一种致病寄生虫，可用作通过供水传播的生物战剂。每年，它感染全世界5000万人，估计造成40，000至100，000人死于腹泻、痢疾和肝脓肿。该项目详细介绍了溶组织内阿米巴的近GMP重组疫苗的生产，并测试了疫苗的免疫原性和对小鼠肠道疾病的保护。

DESCRIPTION (provided by applicant): We propose to produce a subunit vaccine for Entamoeba histolytica and test it in mice for immunogenicity and for protection from intestinal amebiasis. Entamoeba histolytica is a Category B NIH Biodefense pathogen due to its low infectious dose and food and water-bone transmission, and an important cause of diarrhea in children in Africa, Asia and Latin America. The candidate vaccine antigen is the LecA domain of the E. histolytica Gal/GalNAc lectin that mediates parasite adherence and contact-dependent cytotoxicity. LecA contains all of the virulence neutralizing antibody epitopes of the native Gal/GalNAc lectin, and in fact a surrogate marker of immunity in children is intestinal IgA against LecA. Feasibility of our approach is underscored by the effectiveness of LecA in many investigators' laboratories as a vaccine in rodent models of amebiasis. Innovative aspects of the proposed research include production of the first vaccine against an enteric parasite, the use of an antigen (LecA) where virulence-neutralizing epitopes have been mapped, and utilization of the novel mouse model of amebic colitis for efficacy studies. Our approach will be to conduct preclinical development of the LecA vaccine formulated in alum. Preliminary studies published from our group have demonstrated the effectiveness of this prototype vaccine in the murine model. Two specific aims are proposed to conduct this work: In Specific Aim 1, we will express and purify from E. coli the LecA fragment of the Gal/GalNAc lectin using standard operating procedures that are scalable to cGMP. We will quality control the purified protein for endotoxin, DNA, and contaminating host cell proteins, absorb it to alum and test its stability and lot-to-lot variability. TechLab will be entirely responsible for Aim 1. In Specific Aim 2, we will compare different dosing intervals and amounts to examine the immunogenicity, both cellular and humoral, of the alum-absorbed LecA parenteral vaccine for (a) the magnitude of an antigen-specific pre-challenge IFN-g+, CD4+ T cell response to LecA, and mucosal and serum antibody responses, and (b) vaccine efficacy and durability. UVA will perform all immunizations, cell mediated immune response tests, humoral responses, and vaccine trials. TechLab will provide the LecA alum-absorbed vaccine and reagents for ELISA analysis of humoral responses. The research proposed builds on the 15 year collaboration in amebiasis of the investigators, Dr. Lyerly of TechLab and Dr. Petri (the discoverer of the Gal/GalNAc lectin) from UVa. The proximity of TechLab and UVa (a 2 hour drive on I-81) facilitates this collaborative environment. Successful completion of these studies will ready the LecA vaccine for cGMP manufacture and toxicity studies (required for future phase I human clinical trials), maximize its immunogenicity and efficacy, and thereby set the stage for phase I clinical trials of an amebiasis vaccine for humans. PUBLIC HEALTH RELEVANCE: Entamoeba histolytica, a pathogenic parasite of the human intestine, liver and other organs, could be utilized as a bio-warfare agent spread through water supplies. Annually, it infects 50 million people worldwide and causes an estimated 40,000 to 100,000 deaths due to diarrhea, dysentery, and liver abscess. This project details the production of a near-GMP recombinant vaccine for Entamoeba histolytica and tests the vaccine for immunogenicity and for protection from intestinal disease in mice.