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，主要是因为四个发现：1)PfSPZ挑战(传染性PfSPZ)在志愿者中诱导疟疾；2)PfSPZ疫苗(辐射减毒PfSPZ)在非人类灵长类动物(NHP)的肝脏中诱导前所未有的PfSPZ特异性、产生干扰素的CD8+T细胞；3)纯化、冷冻保存的约氏疟原虫(Py)SPZ在小鼠中诱导71-100%的保护4)我们在荷兰的同事报告说，服用氯喹的志愿者感染PfSPZ可产生100%的保护作用，至少持续28个月。现在计划在NIAID、美国贝塞斯达的NIH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(IV)的试验，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，在荷兰和美国进行PfSPZ CVac(用氯喹注射的PfSPZ挑战)试验。PfSPZ和PySPZ的体外检测数据表明，冷冻保存的SPZ的效力比新鲜的SPZ低2.7~6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。对稳定性/冷冻保存的改进至关重要，实施后将使PfSPZ的传染性和效力翻一番，通过增加批次来降低大量PfSPZ产品的制造成本和质量控制释放分析，并通过延长PfSPZ产品可以使用的解冻后时间来减少临床上的疫苗损失/浪费。我们的目标是将商品成本(COGS)总体降低至少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%体内保护效果所需的吡咯烷酮的数量减少50%：体外检测和体内传染性(ID50)以及对挑战的保护是读数。目的2.提高冷冻保存的PfSPZ的感染力和效价50%：将开发的Aim 1方法应用于PfSPZ，结合体外检测读数。目的3.在NHP中建立经AIMS 1和AIMS 2中优化的方法冷冻保存的PfSPZ，与用我们现有方法制备的PfSPZ相比，在体外刺激免疫NHP的PBMC和用于体内免疫NHP时，PfSPZ诱导分泌干扰素-βCD8+T细胞的频率显著增加。目标4.发展能力，在多个确定的时间间隔内生产多批正在加工的PfSPZ材料，这些材料可以组合成PfSPZ大宗产品，然后再大量生产PfSPZ最终产品。目标是将过程保持时间延长到6天，并将批量增加到180,000剂。目的5.延长PfSPZ在临床解冻后的存活时间和处方：为延长临床稳定性而设计的稀释剂配方中的冷冻保存、解冻后的PfSPZ和PfSPZ将通过体内和体外试验进行测试，目标是增加到至少4小时，在临床上稳定。 公共卫生意义：为了保护个人免受疟疾的侵袭并帮助根除这种疾病，需要一种高效的疫苗，Sanaria已经开发了一种平台技术，由活的恶性疟原虫子孢子(PfSPZ)制造，以制造这种疫苗。两种产品正在临床开发中：一种是名为PfSPZ疫苗的减毒活整体疫苗，另一种是使用抗疟疾药物的传染病活疫苗，名为PfSPZ-CVac的疫苗。此外，Sanaria还开发了一种活的、有传染性的PfSPZ产品，PfSPZ Challenges，用于测试疟疾药物和疫苗。这三种产品都需要低温保存。这项建议旨在提高冷冻保存的PfSPZ的效力，这将提高生产效率，并减少疫苗剂量所需的PfSPZ的数量。该项目的成功将大大降低疫苗制造成本，从而使旅行者和发展中国家人口能够更便宜地获得疫苗。

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. PUBLIC HEALTH RELEVANCE: To protect individuals against malaria and to aid in eradicating this disease, a highly effective vaccine is needed, and Sanaria has developed a platform technology, manufactured from live Plasmodium falciparum sporozoites (PfSPZ), to make such a vaccine. Two products are in clinical development: a live attenuated whole organism vaccine called the PfSPZ Vaccine, and a live infectious vaccine administered with an antimalarial drug, a vaccine called PfSPZ-CVac. In addition Sanaria has developed a live, infectious PfSPZ product, PfSPZ Challenge, for use in testing malaria drugs and vaccines. All three products require cryopreservation for storage at low temperature. This proposal aims to increase the potency of cryopreserved PfSPZ, which will increase the efficiency of manufacturing and reduce the numbers of PfSPZ required in vaccine doses. Success in this project will greatly reduce cost of vaccine manufacture, thereby making the vaccine available more cheaply for travelers and developing world populations.