Recombinant Plasodium falciparum CelTOS vaccine

描述(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，并造成相当于国内生产总值1%的损失，并引起旅行者和军人的严重关切。Protein Potential的目标是单独开发或为以下两个主要市场的90%保护性亚单位重组疫苗的开发和商业化做出贡献：1)来自发达国家的旅行者；2)发展中国家的婴幼儿。这种疫苗的黄金标准是通过受辐射感染的蚊子的叮咬用整个减毒的恶性疟原虫(PF)子孢子(SPZ)免疫，这可以保护90%以上的实验挑战志愿者至少10个月。这种保护作用主要由CD8+T细胞介导，其次是CD4+T细胞，以对抗SPZ和感染肝细胞中表达的多种寄生虫蛋白，其次是抗多种SPZ蛋白的抗体。到目前为止，只有针对PF环子孢子蛋白(PfCSP)的免疫反应才能重复性地预防志愿者的PF感染，但这种保护作用在2-3周内只有40%左右。因此，人们一直在加紧努力识别其他SPZ或肝脏(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靶标。PfCelTOS，又称PfAg2，是通过对Pf基因组的分析而发现的。伯氏疟原虫CelTOS参与了恶性疟原虫对肝脏的侵袭，恶性疟原虫CelTOS可被8/8人受试者的T细胞识别，在约氏疟原虫鼠疟模型系统中接种疫苗时，约氏疟原虫CelTOS可保护%的小鼠免受感染。这些发现使PfCelTOS成为一种独立的红细胞期前PF疫苗的首选候选者，或者是旨在诱导保护性T细胞和抗体反应的多蛋白PF疫苗的一部分。因此，我们已经在巴斯德毕赤酵母中生产了全长的重组PfCelTOS蛋白，并表明用该蛋白免疫的杂交小鼠产生了识别PF SPZ的抗体(间接荧光抗体试验中的滴度为12,800)，并在体外阻断了PF SPZ在肝细胞中的侵袭和发展(血清稀释度为1：20时抑制率为68%)。在此，我们建议对该蛋白的生产和纯化进行优化。许多疫苗专家认为，重组病毒和重组(Rec)蛋白的顺序免疫是目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PF CelTOS rec蛋白单独与佐剂的免疫原性，以及在PRIME Boost(序贯免疫)策略中表达PfCelTOS的rec病毒的免疫原性，为猕猴第二阶段研究和这些免疫原的首次临床试验提供数据支持。此外，为了加强将PfCelTOS rec蛋白转移到第二阶段的论点，我们将生产一种PyCelTOS rec蛋白，并进行研究，以实现对用PyCelTOS免疫原免疫的小鼠的显著保护性免疫。与公共卫生相关：疟疾每年导致5亿临床病例和100-300万人死亡，每年造成非洲国内生产总值1%的损失，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和商业化90%保护性疟疾疫苗，年收入可能达到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。该项目的成功将大大降低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.