IMPROVING MANUFACTURING AND POTENCY OF CRYOPRESERVED MALARIA SPOROZOITE VACCINE

描述（由申请人提供）：Sanaria无菌，纯化，冷冻保存的恶性疟原虫（Pf）孢子子（SPZ）产品的开发在过去一年中获得了国际认可，主要是由于四个发现：1)PfSPZ挑战（感染性PfSPZ）在志愿者中诱导疟疾，2)PfSPZ疫苗（辐射减毒PfSPZ）诱导前所未有水平的PfSPZ特异性IFN-？3)纯化的，冷冻保存的P. yoelii (Py) SPZ在小鼠中诱导了71-100%的保护作用，4)我们荷兰的同事报告说，服用氯喹的志愿者感染PfSPZ诱导了100%的保护作用，持续至少28个月。目前计划在美国贝塞斯达的NIAID、美国国立卫生研究院疫苗研究中心和坦桑尼亚的Ifakara卫生研究所进行PfSPZ疫苗静脉注射试验，计划在坦桑尼亚、英国、美国、荷兰、德国和瑞士进行PfSPZ挑战试验，并在荷兰和美国进行PfSPZ Cvac（氯喹给药的PfSPZ挑战）试验。在小鼠中进行的PySPZ研究以及PfSPZ和PySPZ的体外实验数据表明，冷冻保存的SPZ效力比新鲜的SPZ低2.7至6倍。Sanaria的所有产品都依赖于无菌、纯化的PfSPZ的稳定和冷冻保存。对稳定/冷冻保存的改进是至关重要的，一旦实施，将使PfSPZ的传染性和效力加倍，通过增加批次的大小，降低制造成本和质量控制释放分析的成本，并通过延长一瓶PfSPZ产品解冻后的使用时间，减少临床疫苗的损失/浪费。我们的目标是将整体的商品成本（COGS）至少降低75%。五个具体目标包括实现这一目标的工作：在体内达到100%感染和80%保护效果所需的PySPZ数量减少50%：体外测定和体内感染（ID50）和抗攻击保护是读数。目标2。将冷冻保存的PfSPZ的传染性和效力提高50%:Aim 1开发的方法将应用于PfSPZ，使用体外检测的组合进行读数。目标3。在NHPs中证实，与使用我们现有方法制造的PfSPZ相比，用Aims 1和2中优化的方法冷冻保存的PfSPZ诱导INF-？-在体外刺激免疫NHPs的pbmc和体内免疫NHPs时分泌CD8+ T细胞。目标4。培养以多个确定的时间间隔批量生产PfSPZ材料的能力，这些材料可以组合成PfSPZ散装产品，然后是大量的PfSPZ最终产品。目标是将工艺保持时间延长至6天，并将批量增加到18万剂。目标5。延长PfSPZ解冻后在临床中的存活时间和配方：冷冻保存、解冻的PySPZ和PfSPZ稀释剂配方旨在延长临床稳定性，将使用体内和体外试验进行测试，目标是将临床稳定性增加到至少4小时。

DESCRIPTION (provided by applicant): The development of Sanaria's aseptic, purified, cryopreserved Plasmodium falciparum (Pf) sporozoite (SPZ)-based products has received international recognition during the past year, due principally to four findings: 1) PfSPZ Challenge (infectious PfSPZ) induced malaria in volunteers, 2) PfSPZ Vaccine (radiation attenuated PfSPZ) induced unprecedented levels of PfSPZ-specific, IFN-?-producing CD8+ T cells in livers of non-human primates (NHPs), 3) purified, cryopreserved P. yoelii (Py) SPZ induced 71-100% protection in mice, and 4) our colleagues in the Netherlands' report that PfSPZ infection of volunteers taking chloroquine induced 100% protection that lasted for at least 28 months. Trials of PfSPZ Vaccine administered intravenously (IV) are now planned for the NIAID, NIH Vaccine Research Center, Bethesda, USA, and the Ifakara Health Institute (IHI) in Tanzania, and PfSPZ Challenge trials are planned for Tanzania, UK, USA, the Netherlands, Germany, and Switzerland, and PfSPZ Cvac (PfSPZ Challenge administered with chloroquine) in the Netherlands and USA. PySPZ studies in mice and data from in vitro assays with PfSPZ and PySPZ indicate potency of cryopreserved SPZ is 2.7 to 6-fold less than that of fresh SPZ. All of Sanaria's products rely on stabilization and cryopreservation of aseptic, purified PfSPZ. Improvements to stabilization/cryopreservation are critically important and when implemented will double the infectivity and potency of PfSPZ, reducing the cost of manufacturing and quality control release assays for lots of PfSPZ products by increasing the size of lots, and reducing vaccine loss/wastage in the clinic by prolonging the time after thawing that a vial of PfSPZ product can be used. Our goal is to reduce the cost of goods (COGS) overall by at least 75%. Five Specific Aims encompass work to accomplish this goal: Aim 1. Reduce by 50% the numbers of PySPZ required to achieve 100% infection and 80% protective efficacy in vivo: In vitro assays and the in vivo infectivity (ID50) and protection against challenge are the readouts. Aim 2. Increase by 50% the infectivity and potency of cryopreserved PfSPZ: Methods developed Aim 1 will be applied to PfSPZ using a combination of in vitro assays for readouts. Aim 3. Establish in NHPs that PfSPZ, cryopreserved by methods optimized in Aims 1 and 2 as compared to PfSPZ manufactured using our current methods induce a significant increase in the frequency of INF-?-secreting CD8+ T cells when used to stimulate PBMCs from immunized NHPs in vitro and when used to immunize NHPs in vivo. Aim 4. Develop the capacity to create batches of in process PfSPZ material at multiple, defined intervals that can be combined into PfSPZ bulk product and then large lots of PfSPZ final product. The goal is to extend the process hold-time up to 6 days and to increase lot size to 180,000 doses. Aim 5. Extend PfSPZ survival in the clinic post-thaw and formulation: Cryopreserved, thawed PySPZ and PfSPZ in diluent formulations designed to extend in-clinic stability will be tested using in vivo and in vitro assay with the goal of increasing to a minimum of four hours, stability in the clinic.