Alum-absorbed subunit vaccine to prevent intestinal amebiasis

描述（由申请人提供）：我们建议生产一种溶组织内阿米巴亚单位疫苗，并在小鼠身上测试其免疫原性和对肠道阿米巴病的保护作用。溶组织内阿米巴是一类B类NIH生物防御病原体，因其感染剂量低，可通过食物和水骨传播，是非洲、亚洲和拉丁美洲儿童腹泻的重要原因。候选疫苗抗原是溶组织乳杆菌Gal/GalNAc凝集素的LecA结构域，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，儿童免疫的替代标记是针对LecA的肠道IgA。在许多研究人员的实验室中，LecA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，使用已绘制出毒力中和表位的抗原（LecA），以及利用新型阿米巴性结肠炎小鼠模型进行疗效研究。我们的方法是对明矾配制的LecA疫苗进行临床前开发。我们小组发表的初步研究已经在小鼠模型中证明了这种原型疫苗的有效性。在具体目标1中，我们将使用可扩展到cGMP的标准操作程序从大肠杆菌中表达和纯化Gal/GalNAc凝集素的LecA片段。我们将对纯化蛋白进行质量控制，以检测内毒素、DNA和污染宿主细胞蛋白，将其吸收到明矾中，并测试其稳定性和批次间变异性。TechLab将完全负责Aim 1。在特异性目标2中，我们将比较不同的给药间隔和剂量，以检查铝吸收的LecA肠外疫苗的细胞和体液免疫原性(a)抗原特异性攻击前IFN-g+、CD4+ T细胞对LecA的反应以及粘膜和血清抗体反应的大小，以及(b)疫苗的功效和持久性。UVA将执行所有免疫、细胞介导免疫反应测试、体液反应和疫苗试验。TechLab将提供LecA铝吸附疫苗和用于体液反应ELISA分析的试剂。这项提议的研究建立在TechLab的Lyerly博士和UVa的Petri博士（Gal/GalNAc凝集素的发现者）在阿米巴病方面长达15年的合作基础上。靠近TechLab和UVa（在I-81上开车2小时）促进了这种协作环境。这些研究的成功完成将使LecA疫苗为cGMP生产和毒性研究（未来一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一期临床试验奠定基础。

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.