IMPROVING MANUFACTURING AND POTENCY OF CRYOPRESERVED MALARIA SPOROZOITE VACCINE

描述（由申请人提供）：Sanaria的无菌、纯化、冷冻保存的恶性疟原虫（Pf）子孢子（SPZ）产品的开发在过去一年中获得了国际认可，主要是由于四项发现：1）PfSPZ攻毒（感染性PfSPZ）诱导志愿者疟疾，2）PfSPZ疫苗（辐射减毒PfSPZ）诱导前所未有水平的PfSPZ特异性IFN-？-γ。在非人灵长类动物（NHP）的肝脏中产生CD 8 + T细胞，3）纯化的、冷冻保存的约氏疟原虫（Py）SPZ在小鼠中诱导71-100%的保护，以及4）我们在荷兰的同事报道，PfSPZ感染服用氯喹的志愿者诱导100%的保护，持续至少28个月。目前计划在NIAID、NIH疫苗研究中心（Bethesda，USA）和坦桑尼亚的Ifakara健康研究所（IHI）开展PfSPZ疫苗静脉注射（IV）试验，计划在坦桑尼亚、英国、美国、荷兰、德国和瑞士开展PfSPZ攻毒试验，并计划在荷兰和美国开展PfSPZ Cvac（PfSPZ攻毒，使用氯喹）试验。PySPZ在小鼠中的研究和PfSPZ和PySPZ的体外试验数据表明，冷冻保存的SPZ的效力比新鲜SPZ的效力低2.7至6倍。Sanaria的所有产品都依赖于无菌纯化PfSPZ的稳定和冷冻保存。对稳定化/冷冻保存的改进至关重要，当实施时，将使PfSPZ的感染性和效力加倍，通过增加批次大小降低PfSPZ产品批次的生产和质量控制放行测定的成本，并通过延长解冻后可使用一小瓶PfSPZ产品的时间来减少临床中的疫苗损失/浪费。我们的目标是将商品成本（COGS）整体降低至少75%。五个具体目标包括实现这一目标的工作：目标1。将达到100%感染和80%体内保护效力所需的PySPZ数量减少50%：体外测定和体内感染性（ID 50）以及针对攻击的保护是读数。目标2.将冷冻保存PfSPZ的感染性和效力增加50%：将使用体外测定的组合将目的1开发的方法应用于PfSPZ以进行读数。目标3.在NHP中确定，与使用我们当前方法生产的PfSPZ相比，通过目标1和2中优化的方法冷冻保存的PfSPZ诱导INF-？-频率显著增加。当用于体外刺激来自免疫的NHP的PBMC时和当用于体内免疫NHP时，分泌CD 8 + T细胞。目标4。开发在多个规定时间间隔生产PfSPZ在制品批次的能力，这些批次可合并为PfSPZ散装产品，然后合并为大批量PfSPZ成品。目标是将工艺保持时间延长至6天，并将批量增加至180，000剂。目标5。延长PfSPZ在解冻后和配制后的临床存活：将使用体内和体外试验检测旨在延长临床稳定性的冷冻保存、解冻的PySPZ和PfSPZ稀释剂制剂，目标是将临床稳定性增加至至少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.