Recombinant Plasodium falciparum CelTOS vaccine

描述（由申请人提供）：疟疾每年在非洲造成约5亿临床病例，多达270万人死亡，并使非洲国内生产总值损失100亿美元，对旅行者和军队构成严重关切。Protein Potential的目标是单独开发或参与开发和商业化一种具有90%保护性的亚基重组疫苗，用于两个主要市场，年收入可能达到10亿美元：1)发达国家的旅行者和2)发展中国家的婴幼儿。这种疫苗的黄金标准是通过受辐照感染的蚊子叮咬，用整个减毒恶性疟原虫（Pf）孢子子（spz）进行免疫接种，这可以保护90%以上的实验挑战志愿者至少10个月。这种保护被认为主要是由CD8+ T细胞介导的，CD4+ T细胞在较小程度上抵抗spz和受感染肝细胞中表达的多种寄生虫蛋白，其次是针对多种spz蛋白的抗体。到目前为止，只有针对Pf环孢子子蛋白（PfCSP）的免疫反应被证明可以在志愿者中重复地预防Pf感染，但这种保护在2-3周内仅为40%左右。因此，人们一直在努力确定其他spz或肝（红细胞前）期蛋白，这些蛋白是辐照spz疫苗产生的高度保护性免疫的目标。PfCelTOS，也被称为PfAg2，是通过对Pf基因组的分析发现的。柏氏疟原虫CelTOS已被证明与spz侵袭肝脏有关，恶性疟原虫CelTOS被8/8接种了辐照恶性疟原虫spz的人类志愿者的T细胞识别，而约利疟原虫CelTOS作为疫苗在约利疟原虫啮齿动物疟疾模型系统中保护了64%的小鼠免受感染。这些发现已经确定PfCelTOS作为独立的红细胞前期Pf疫苗的主要候选者，或设计用于引发保护性T细胞和抗体反应的多蛋白Pf疫苗的一部分。因此，我们在毕赤酵母中制备了全长重组PfCelTOS蛋白，并表明用该蛋白免疫的远亲小鼠产生识别Pf spz的抗体（间接荧光抗体测试滴度为12,800），并在体外阻断Pf spz在肝细胞中的侵袭和发展（在血清稀释为1:20时抑制68%）。在此，我们提出优化该蛋白的生产和纯化。许多疫苗学家认为重组病毒和重组蛋白序贯免疫是当前优化同一受者抗体和T细胞反应的最佳方法。因此，我们将评估PfCelTOS rec蛋白单独与佐剂的免疫原性，以及表达PfCelTOS的rec病毒的主要增强（顺序免疫）策略，以提供数据支持恒河猴II期研究设计和这些免疫原的首次临床试验。此外，为了加强将PfCelTOS rec蛋白转移到II期的论证，我们将生产PyCelTOS rec蛋白，并在使用PyCelTOS免疫原免疫的小鼠中进行研究，以获得显著的保护性免疫。公共卫生相关性：疟疾每年造成5亿临床病例和1至300万人死亡，每年使非洲国内生产总值损失100亿美元，是旅行者和军事人员严重关切的问题。Protein Potential的目标是为初级市场开发并商业化一种保护性高达90%的疟疾疫苗，年收入有望达到10亿美元；1)来自发达国家的旅行者，2)发展中国家的婴儿、幼儿和少女。这一项目的成功将大大减少这种疟疾疫苗的开发成本和上市时间。

DESCRIPTION (provided by applicant): Malaria causes an estimated 500 million clinical cases, up to 2.7 million deaths, and a loss of >1% of GDP in Africa annually, and poses a serious concern for travelers and military. Protein Potential's goal is to develop alone or contribute to the development and commercialization of a >90% protective subunit recombinant vaccine for 2 primary markets with a potential for >$1 billion in annual revenues: 1) Travelers from the developed world and 2) Infants and young children in the developing world. The gold standard for such a vaccine is immunization with whole attenuated Plasmodium falciparum (Pf) sporozoites (spz) by the bite of irradiated infected mosquitoes, which protects greater than 90% of experimentally challenged volunteers for at least 10 months. This protection is thought to be primarily mediated by CD8+ T cells, and to a lesser extent CD4+ T cells, against multiple parasite proteins expressed in spz and infected hepatocytes, and secondarily by antibodies against multiple spz proteins. Thus far only immune responses directed against the Pf circumsporozoite protein (PfCSP) have been shown to reproducibly prevent Pf infection in volunteers, but this protection is only about 40% for 2-3 weeks. Thus, there have been intense efforts to identify other spz or liver (pre-erythrocytic) stage proteins that are targets of the highly protective immunity engendered by the irradiated spz vaccine. PfCelTOS, also know as PfAg2, was discovered through analysis of the Pf genome. P. berghei CelTOS has been shown to be involved in spz invasion of the liver, P. falciparum CelTOS was recognized by T cells from 8/8 human volunteers immunized with irradiated P. falciparum spz, and P. yoelii CelTOS protected 64% of mice against infection in the P. yoelii rodent malaria model system when administered as a vaccine. These findings have established PfCelTOS as a prime candidate for being a stand-alone pre-erythrocytic stage Pf vaccine, or part of a multi-protein Pf vaccine designed to elicit protective T cell and antibody responses. Accordingly, we have produced a full length, recombinant PfCelTOS protein in Pichia pastoris, and shown that outbred mice immunized with this protein produce antibodies that recognize Pf spz (titer in indirect fluorescent antibody test, 12,800) and block invasion and development of Pf spz in hepatocytes in vitro (68% inhibition at serum dilution of 1:20). Herein, we propose to optimize production and purification of this protein. Many vaccinologists consider sequential immunization with recombinant virus and recombinant (rec) proteins as the best current approach to optimize antibody and T cell responses in the same recipients. Therefore, we will assess the immunogenicity of the Pf CelTOS rec protein alone with adjuvant, and in a prime boost (sequential immunization) strategy with rec virus expressing PfCelTOS to provide data to support design of Phase II studies in rhesus monkeys and the first clinical trials of these immunogens. In addition, to strengthen the argument for moving the PfCelTOS rec protein to Phase II, we will produce a PyCelTOS rec protein, and conduct studies to achieve significant protective immunity in mice immunized with PyCelTOS immunogens. PUBLIC HEALTH RELEVANCE: Malaria causes 500 million clinical cases and 1-3 million deaths annually, is responsible for >1% loss of GDP in Africa annually and is a serious concern for travelers and military personnel. Protein Potential's goal is to develop and commercialize a >90% protective malaria vaccine for primary markets with a potential for >$1 billion annual revenues; 1) travelers from the developed world, and 2) infants, young children, and adolescent girls in the developing world. Success in this project will significantly decrease the cost of development and time to market for this malaria vaccine.