Plasmodium vivax recombinant CS protein vaccine for malaria

描述（由申请人提供）：间日疟原虫重组CS蛋白疫苗间日疟原虫（Pv）每年造成数亿例疟疾病例，旅行者中的疟疾病例与恶性疟原虫（Pf）一样多，并造成巨大的经济负担。最近在大洋洲、南亚和南美洲报告了由Pv导致死亡的严重疟疾。在过去的十年中，出现了耐药PV。在来自发达国家的旅行者和军人中，以及在有地方性Pv的国家的人群中，Pv疫苗有巨大的潜在市场。Pv疫苗的研究一直被忽视。Pf和Pv在全世界大多数疟疾流行地区共存的事实对部署仅对Pf有效的疫苗提出了技术和伦理限制。不消除Pv就不能根除疟疾。唯一的亚单位疟疾疫苗已被证明可重复地预防人类Pf疟疾是基于Pf环子孢子蛋白（PfCSP）。这些保护性疫苗引发针对分子的中心重复区的抗体，其在Pf的所有分离株中是保守的，并且针对C-末端侧翼区中的T细胞表位。还不可能成功地生产包括N-末端侧翼区的PfCSP重组（rec）蛋白疫苗。PvCSP rec蛋白疫苗的开发由于以下事实而变得复杂：基于中心重复序列的变异，存在PvCSP的2个主要等位基因（210和247）。在阶段I中，我们构建了将3个拷贝的PvCSP 210重复序列和3个拷贝的PvCSP 247重复序列与PvCSP的N-末端（蛋白1）、C-末端（蛋白2）或N-和C-末端（蛋白3）组合的PvCSP rec蛋白。所有这些均以足以用于GMP生产的水平表达。蛋白质2聚集使得纯化和表征困难。所有这些都在小鼠中诱导了识别PvCSP和表达PvCSP 210或247的Pv子孢子的抗体，并抑制了Pv子孢子在人肝细胞中的侵袭和发育。蛋白2不诱导针对Pv 247重复序列的抗体，并且不能在T细胞研究中进行评估。蛋白质3比蛋白质1诱导更好的免疫应答，并且由于蛋白质3基本上包括具有所有B和T细胞表位的全长蛋白质，因此我们选择蛋白质3用于II期。在第二阶段，我们将：1）优化蛋白3表达并产生主克隆和主细胞库，2）定义cGMP下规模发酵程序的规范，3）用4种不同佐剂配制候选蛋白，4）通过小鼠和猴中的免疫原性比较向下选择最佳蛋白/佐剂/制剂，5）定义工艺规范，产生cGMP生产的技术转移记录和文件，6）制造疫苗，进行释放测定，释放散装药物物质并进行稳定性研究。到II期结束时，PvCSP蛋白3疫苗/佐剂将可用于临床前毒理学研究和I/2a期临床试验。公共卫生相关性：间日疟原虫（Pv）每年造成数亿例疟疾病例，与恶性疟原虫（Pf）一样多的旅行者疟疾病例，以及巨大的经济负担。最近在大洋洲、南亚和南美洲报告了由Pv导致死亡的严重疟疾。在过去的十年中，出现了耐药PV。在来自发达国家的旅行者和军人中，以及在有地方性Pv的国家的人群中，Pv疫苗有巨大的潜在市场。该项目将开发和生产这种疫苗。

DESCRIPTION (provided by applicant): Plasmodium vivax recombinant CS protein vaccine Plasmodium vivax (Pv) is responsible for hundreds of millions of malaria cases annually, as many cases of malaria in travelers as P. falciparum (Pf), and a substantial economic burden. Severe malaria with mortality due to Pv has recently been reported from Oceania, south Asia, and South America. During the last decade, drug resistant Pv has emerged. There is a huge potential market for a Pv vaccine in travelers and military from the developed world, and among populations in countries with endemic Pv. Pv vaccine research has been neglected. The fact that Pf and Pv co-exist in most malaria endemic areas worldwide presents technical and ethical constraints for deployment of a vaccine effective only against Pf. Malaria cannot be eradicated without eliminating Pv. The only subunit malaria vaccines that have been reproducibly shown to prevent Pf malaria in humans are based on the Pf circumsporozoite protein (PfCSP). These protective vaccines elicit antibodies against the central repeat region of the molecule, which is conserved in all isolates of Pf, and against T cell epitopes in the C-terminal flanking region. It has not been possible to produce successfully a PfCSP recombinant (rec) protein vaccine that includes the N-terminal flanking region. Development of a PvCSP rec protein vaccine has been complicated by the fact that there are 2 major alleles of the PvCSP (210 and 247) based on variation in sequence of the central repeats. In Phase I we constructed PvCSP rec proteins that combined 3 copies of PvCSP 210 repeats and 3 copies of PvCSP 247 repeats with N-terminus (Protein 1), C- terminus (Protein 2) or N- and C-termini (Protein 3) of the PvCSP. All were expressed at levels adequate for GMP production. Protein 2 aggregated making purification and characterization difficult. All induced in mice antibodies that recognized PvCSP and Pv sporozoites expressing PvCSP 210 or 247, and inhibited Pv sporozoite invasion and development in human hepatocytes. Protein 2 did not induce antibodies against the Pv247 repeats, and could not be evaluated in T cell studies. Protein 3 induced better immune responses than Protein 1, and since Protein 3 includes essentially the full-length protein with all B and T cell epitopes, we have selected Protein 3 for Phase II. In Phase II we will: 1) Optimize protein 3 expression and generate a master clone and master cell bank, 2) Define specifications for fermentation procedure at scale under cGMPs, 3) Formulate candidate protein with 4 different adjuvants, 4) Down select optimal protein/adjuvant/formulation by comparative immunogenicity in mice and monkeys, 5) Define specifications for process, generate technical transfer records and documentation for cGMP manufacture, 6) Manufacture vaccine, conduct release assays, release bulk drug substance and conduct stability studies. By end of Phase II PvCSP Protein 3 vaccine/adjuvant will be available for pre-clinical toxicology studies and Phase 1/2a clinical trials. PUBLIC HEALTH RELEVANCE: Plasmodium vivax (Pv) is responsible for hundreds of millions of malaria cases annually, as many cases of malaria in travelers as P. falciparum (Pf), and a substantial economic burden. Severe malaria with mortality due to Pv has recently been reported from Oceania, south Asia, and South America. During the last decade, drug resistant Pv has emerged. There is a huge potential market for a Pv vaccine in travelers and military from the developed world, and among populations in countries with endemic Pv. This project will develop and manufacture such a vaccine.