Plasmodium vivax recombinant CS protein vaccine for malaria

描述(申请人提供)：间日疟原虫重组CS蛋白疫苗间日疟原虫(PV)每年导致数亿疟疾病例，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。大洋洲、南亚和南美洲最近报告了由PV引起的严重疟疾死亡。在过去的十年中，出现了抗药性光伏病毒。在发达国家的旅行者和军人中，以及在出现地方性PV的国家的人群中，PV疫苗有着巨大的潜在市场。光伏疫苗的研究一直被忽视。在全世界大多数疟疾流行地区，肺结核病和小儿麻痹症共存，这一事实对部署仅对肺鼠疫有效的疫苗构成了技术和伦理上的限制。如果不消灭小儿麻痹症，就无法根除疟疾。已被重复证明可在人类中预防PF疟疾的唯一亚单位疟疾疫苗是基于PF环子孢子蛋白(PfCSP)的。这些保护性疫苗诱导出针对该分子中心重复区域的抗体，该区域在所有PF分离株中都是保守的，并针对C末端侧翼区域的T细胞表位。目前还不可能成功地生产出包含N-末端侧翼区的PfCSP重组蛋白疫苗。PvCSP的两个主要等位基因(210和247)基于中心重复序列的变化，这使得PvCSP rec蛋白疫苗的开发变得复杂。在第一阶段，我们构建了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-端(蛋白1)、C-端(蛋白2)或N-端和C-端(蛋白3)结合的PvCSP重组蛋白。所有基因的表达水平都足以产生GMP。蛋白2聚集，使纯化和鉴定变得困难。均可在小鼠体内诱导识别表达PvCSP 210或247的PvCSP和PV子孢子的抗体，并抑制PV子孢子对人肝细胞的侵袭和发育。蛋白2不能诱导针对Pv247重复序列的抗体，也不能在T细胞研究中进行评估。蛋白3比蛋白1诱导更好的免疫反应，由于蛋白3基本上包括具有所有B和T细胞表位的全长蛋白，我们选择了蛋白3作为第二阶段。在第二阶段，我们将：1)优化蛋白3的表达并产生主克隆和主细胞库，2)定义cGMPs下的规模化发酵过程，3)用4种不同的佐剂配制候选蛋白，4)通过比较小鼠和猴子的免疫原性来选择最佳的蛋白质/佐剂/制剂，5)确定工艺规范，生成用于cGMP制造的技术转移记录和文件，6)制造疫苗，进行释放试验，释放原料药并进行稳定性研究。到第二阶段结束时，PvCSP蛋白3疫苗/佐剂将可用于临床前毒理学研究和第一/2a阶段临床试验。公共卫生相关性：间日疟原虫(PV)每年造成数亿疟疾病例，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。大洋洲、南亚和南美洲最近报告了由PV引起的严重疟疾死亡。在过去的十年中，出现了抗药性光伏病毒。在发达国家的旅行者和军人中，以及在出现地方性PV的国家的人群中，PV疫苗有着巨大的潜在市场。该项目将开发和制造这样一种疫苗。

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.