Manufacture of aseptic, purified, cryopreserved Plasmodium vivax sporozoites (PvSPZ Challenge) for controlled human malaria infection (CHMI)

 描述(申请人提供)：间日疟原虫(PV)是第二大人类疟疾寄生虫，每年导致超过8000万例病例，包括严重的致命疾病。预防和控制受到新出现的耐药性和休眠肝期寄生虫(称为催眠虫)复发的挑战。唯一一种治疗复发的方法是伯喹，它会在G6PD缺乏症患者中导致危及生命的急性溶血性贫血，G6PD缺乏症是最常见的人类遗传疾病，在疟疾流行国家影响8%的人。这种治疗障碍导致PV反复发作，加剧了控制问题。Sanaria的基于子孢子(SPZ)的疫苗对肺吸虫病的高水平(100%)、长期(至少12个月)保护效力的展示是一个重要的里程碑，表明这种疫苗将构成遏制和消除肺吸虫病的可行方法。我们相信，同样的疫苗方法也可以用于PV。在PF疫苗的开发中，受控人类疟疾感染(CHMI)一直是进步的引擎，加快了候选疫苗的测试。最近，Sanaria的PfSPZ Challenges的开发使PF CHMI发生了革命性的变化，无菌、纯化、冷冻保存、完全感染的PfSPZ来自于PF配子细胞的体外培养，使志愿者能够通过皮内、肌肉和静脉注射在非洲3个国家和欧洲2个以前从未进行过CHMI的国家成功感染。相比之下，基于光伏SPZ的产品的开发受到了缺乏体外培养光伏寄生虫的可用技术的影响，以至于产生感染了CHMI的蚊子需要用膜喂养来自光伏患者的新鲜的受光伏感染的血液。我们现在通过使用PV配子体感染的非人类灵长类动物(NHP)来产生PvSPZ，从而克服了这一主要限制。事实上，我们是唯一一家拥有由NHP感染的血液制成的瓶装PvSPZ的实验室，在一天内从2000只蚊子身上生产了多达8000万瓶PvSPZ。这些低温保存的PvSPZ在体外以传统的单层形式感染肝细胞系3-6天，并在微模式共培养的原代人肝细胞12-21天感染，以及2)体内感染NHP。我们现在建议通过使用允许的博利维链霉菌的特定无菌菌落作为PV感染血液的来源来生产无菌、纯化、冷冻保存的传染性PvSPZ(PvSPZ挑战)。这条新的管道将产生符合cGMP的受控批次的PvSPZ，包括从人类分离的各种原代和克隆性PV株。Sanaria的这一创新将提供一致的、质量可控的冷冻保存PvSPZ库存，以促进包括CHMI在内的PV体外和体内研究的良好控制和重复性。这项使能技术将支持在世界各地的CHMI中开发和测试抗PV药物和疫苗，就像PfSPZ挑战赛为PF CHMI所做的那样。当接种抗疟疾化学预防药物PvSPZ化学预防疫苗(PvSPZ-CVac)时，它也将构成预防PV疟疾的一种有效疫苗方法的基础。

 DESCRIPTION (provided by applicant): Plasmodium vivax (Pv), the second most important human malaria parasite, causes more than 80 million cases annually including severe, fatal disease. Prevention and control are challenged by emerging drug resistance and relapses from dormant liver stage parasites called hypnozoites. The only therapy against relapse, primaquine, causes life threatening acute hemolytic anemia in patients with G6PD deficiency, the most prevalent human genetic disorder, affecting 8% of people in malaria-endemic nations. This barrier to treatment results in repeated Pv attacks, aggravating the problem of control. The demonstration of high level (100%), long-lasting (at least 12 months) protective efficacy of Sanaria's sporozoite (SPZ)-based vaccines against Pf malaria is a significant milestone, and indicates that such vaccines will constitute a viable approach to containing and eliminating Pf. We believe that the same vaccine approach could work for Pv. In the development of the Pf vaccines, controlled human malaria infection (CHMI) has been an engine of progress, accelerating the testing of vaccine candidates. Pf CHMI has recently been revolutionized by the development of Sanaria's PfSPZ Challenge, aseptic, purified, cryopreserved, fully infectious PfSPZ derived from in vitro cultures of Pf gametocytes, enabling the successful infection of volunteers by intradermal, intramuscular, and intravenous injection in 3 countries in Africa and 2 countries in Europe that had never conducted CHMI before. In contrast, development of Pv SPZ-based products has suffered from lack of available technology to culture Pv parasites in vitro, such that generating infected mosquitoes for CHMI required membrane feeding on fresh, Pv-infected blood from Pv patients. We have now overcome this major limitation by using Pv gametocyte- infected Saimiri boliviensis non-human primates (NHPs) to produce PvSPZ. In fact we are the only laboratory with an inventory of vialed PvSPZ made from NHP-infected blood, having produced as much as 80 million PvSPZ vialed in 1 day from 2,000 mosquitoes. These cryopreserved PvSPZ are 1) infectious to hepatocyte cell lines in vitro in traditional monolayer formats over 3-6 days and in micro-patterned co-cultured primary human hepatocytes over 12-21 days, and 2) infectious to NHPs in vivo. We now propose to produce aseptic, purified, cryopreserved, infectious PvSPZ (PvSPZ Challenge) by using a specific germ free colony of the permissive S. boliviensis as the source for Pv-infected blood. This novel pipeline will generate cGMP-compliant, controlled batches of PvSPZ including a wide variety of primary and clonal Pv lines isolated from humans. This innovation by Sanaria will offer a consistent, quality-controlled stock of cryopreserved PvSPZ to promote well-controlled, reproducible in vitro and in vivo studies in Pv including CHMI. This enabling technology will support the development and testing of anti-Pv drugs and vaccines in CHMIs world-wide, just as PfSPZ Challenge has done for Pf CHMIs. It will also form the basis of a powerful vaccine approach to preventing Pv malaria when administered with anti-malarial chemoprophylaxis, the PvSPZ chemoprophylaxis vaccine (PvSPZ-CVac).