In-Vitro Culture of Plasmodium falciparum Sporozoites for Malaria Vaccine

 描述（由申请方提供）：Sanaria的平台技术是生产无菌、纯化、冷冻保存的恶性疟原虫（Pf）子孢子（SPZ）。该技术已经生产了三种通过针头和注射器给药的产品，所有这些产品都在临床试验中- PfSPZ疫苗（辐射减毒PfSPZ），PfSPZ挑战（用于控制人类疟疾感染的传染性PfSPZ [CHMI]）和PfSPZ-CVac（PfSPZ挑战，作为疫苗给予抗疟疾药物）。PfSPZ疫苗在NIH的临床试验中保护100%（6/6）志愿者免受CHMI。NIH研究结果的重现性、保护的持久性、对异源Pf寄生虫的保护以及改变疫苗方案后的剂量减少正在美国7个地点的6项临床试验中进行评估，欧洲和非洲。到2014年3月，我们的马里合作者已经通过直接静脉接种（DVI）以4周的间隔对100多名志愿者进行了两次注射;注射进行得非常顺利，耐受性非常好。在7个国家进行了7项PfSPZ攻毒临床试验;其中5项试验实现了100%感染。PfSPZ-CVac方法的试验于2014年5月在德国开始。我们为CHMI提供疟疾疫苗和产品的整个PfSPZ方法得到了巨大的国际支持。Sanaria的产品依赖于无菌蚊子中PfSPZ的生产。我们的第一阶段SBIR项目提出开发一种在培养物（体外）中生产PfSPZ的方法，该方法将消除对蚊子的需要。我们估计这将使疫苗生产成本降低约80%。第一阶段SBIR是成功的。我们开发了体外生产Pf卵囊的方法，其效率比我们在蚊子中生产卵囊时高39倍。我们在体外反复产生PfSPZ，其以与从蚊子新鲜解剖的PfSPZ相同的效率侵入并发育至成熟的表达Pf裂殖子表面蛋白1的6天肝期宿主。这是疟疾研究史上的“第一次”。虽然我们将在II期中显著提高PfSPZ体外生产的效率和质量，但我们现在实际上可以在体外生产足够的PfSPZ以支持PfSPZ体外释放的CHMI临床试验。在这个II期项目中，我们将优化体外生产临床级PfSPZ的方法。我们将利用3D细胞培养技术，充分优化PfSPZ的体外生产方法，然后证明这些PfSPZ可以在体外和体内可重复地在人肝细胞中侵入和发育，并完成Pf生命周期。这将使用输注人血的人肝嵌合小鼠在体内完成。我们将建立纯化PfSPZ的方法和定量纯度的测定。我们的生产、质量和监管团队将确保所有试剂和工艺均符合cGMP，并足以进行生产、进行工程运行，并向FDA提交IND前包装，用于PfSPZ体外接种的CHMI临床试验，以证明对人类的感染性，从而为我们的疫苗从蚊子生产转向体外生产PfSPZ奠定基础。

 DESCRIPTION (provided by applicant): Sanaria's platform technology is production of aseptic, purified, cryopreserved Plasmodium falciparum (Pf) sporozoites (SPZ). This technology has produced three products administered by needle and syringe, all of which are in clinical trials - PfSPZ Vaccine (radiation attenuated PfSPZ), PfSPZ Challenge (infectious PfSPZ for controlled human malaria infection [CHMI]) and PfSPZ-CVac (PfSPZ Challenge given with antimalarial drugs as a vaccine). PfSPZ Vaccine protected 100% (6/6) volunteers against CHMI in a clinical trial at the NIH. The reproducibility of the NIH findings, durability of protection, protection against heterologous Pf parasites, and reduced numbers of doses with altered vaccine regimens are being assessed in 6 clinical trials at 7 sites in the U.S., Europe, and Africa. By March 2014, our Malian collaborators had injected >100 volunteers twice at a 4 week interval by direct venous inoculation (DVI); the injections have gone perfectly and been extremely well tolerated. 7 clinical trials of PfSPZ Challenge have been conducted in 7 countries; 100% infection was achieved in five of the trials. A trial of the PfSPZ-CVac approach begins in Germany in May 2014. There is tremendous international support for our whole PfSPZ approach for malaria vaccines and products for CHMI. Sanaria's products rely on production of PfSPZ in aseptic mosquitoes. Our Phase I SBIR project proposed developing a method for producing PfSPZ in culture (in vitro) that would eliminate the need for mosquitoes. We estimated this would reduce cost of producing vaccine by ~80%. The Phase I SBIR was successful. We developed methods for producing Pf oocysts in vitro with an efficiency 39 times greater than when we produce oocysts in mosquitoes. We repeatedly produced PfSPZ in vitro that invaded and developed to mature 6 day liver stage schizonts expressing Pf merozoite surface protein 1 with the same efficiency as PfSPZ freshly dissected from mosquitoes. This is a "first" in the history of malaria research. Although we will significantly improve efficiency and quality of in vitro production of PfSPZ in Phase II, we could now actually manufacture enough PfSPZ in vitro to support a CHMI clinical trial of PfSPZ Challenge-in vitro. In this Phase II projet we will optimize methods for manufacture of clinical grade PfSPZ in vitro. We will use 3D cell culture technologies to fully optimize methods for production of PfSPZ in vitro, and then demonstrate these PfSPZ can reproducibly invade and develop in human hepatocytes in vitro and in vivo, and complete the Pf life cycle. This will be done in vivo using human liver chimeric mice transfused with human blood. We will establish a method for purifying PfSPZ and an assay for quantifying purity. Our manufacturing, quality, and regulatory teams will ensure all reagents and processes are compliant with cGMPs and adequate for manufacturing, conduct engineering runs, and submit a pre-IND package to FDA for a CHMI clinical trial of PfSPZ Challenge-in vitro to demonstrate infectivity to humans, thereby establishing a rationale for moving from mosquito- to in vitro-produced PfSPZ for our vaccines.