Protective biomarkers for the development of vaccines against malaria

描述（由申请人提供）：对于I期SBIR资助，我们构建了恶性疟原虫（Pf）3D7蛋白质微阵列，其中含有2，320个单独的多肽，代表1，200种已知和假设的蛋白质，或整个Pf蛋白质组的约23%。我们表明，打印在这些阵列上的单个蛋白质捕获了感染个体血清中存在的抗体，并且捕获的抗体的量可以使用荧光二抗进行定量。通过这种方式，可以确定自然或实验暴露或免疫后产生的抗体的完整谱。我们用来自马里居民的血清探测了阵列，并确定了一组潜在的疫苗抗原候选物，与来自同一村庄的年龄匹配的易感儿童相比，受保护儿童的抗体显着升高。在另一项研究中，我们描述了一组生物标志物和潜在的疫苗抗原，与类似的接种疫苗但未受保护的受试者相比，这些抗原在受保护的志愿者的血清中优先识别，所述志愿者通过被照射的Pf子孢子感染的蚊子叮咬而免疫。这些结果验证了Pf蛋白质组微阵列在I期资助中开发的用于鉴定与免疫保护相关的抗体的实用性。在这个II期申请中，我们建议应用I期开发的蛋白质组微阵列的扩展版本（包含4，253个Pf蛋白）来确定参加几个减毒子孢子疫苗临床试验的个体的抗体应答。通过比较受保护的疫苗接种者与未受保护的疫苗接种者的抗体谱，我们的目标是鉴定与子孢子疫苗介导的保护相关的替代抗体生物标志物。 现在我们的合作者Sanaria Inc.开发纯化的代谢活性、无菌、瓶装和冷冻保存的Pf子孢子（PfSPZ）疫苗，其预防感染和传播，并通过针头和注射器注射施用。第一个这样的PfSPZ疫苗已经通过辐射减毒，被称为PfSPZ疫苗。最近，Radboud大学Nijmengen医学中心（RUNMC）的研究小组发表了关于通过携带活性（非辐照）PfSPZ的蚊子叮咬免疫志愿者，同时服用氯喹化学预防以预防寄生虫血症，从而实现对Pf的完全保护。这种保护已经持续了两年。Sanaria和RUNMC一起表明，它们可以通过针头和注射器给予活PfSPZ感染志愿者，并且用纯化的活PfSPZ免疫氯喹治疗的志愿者以确定它们是否可以复制蚊子叮咬后的保护作用的研究正在进行中。来自这些试验的大约1，500份血清标本将用于此处提出的生物标志物发现项目。目的是鉴定和验证这些保护性替代生物标志物，并开发一种经验证的检测方法，用作FDA要求的关键检测方法，以支持子孢子疫苗的许可。

DESCRIPTION (provided by applicant): For the Phase I SBIR grant we constructed a Plasmodium falciparum (Pf) 3D7 protein microarray containing 2,320 individual polypeptides representing 1,200 known and hypothetical proteins, or ~23 % of the entire Pf proteome. We showed that the individual proteins printed on those arrays captured antibodies present in sera from infected individuals and the amount of captured antibody could be quantified using fluorescent secondary antibody. In this way, the complete profile of antibodies that results after natural or experimental exposure or immunization can be determined. We probed the array with serum from people residing in Mali and identified a panel of potential vaccine antigen candidates against which antibodies were significantly elevated in protected children compared to age-matched susceptible children from the same village. In another study, we described a panel of biomarkers and potential vaccine antigens that were preferentially recognized in sera of protected volunteers immunized by the bite of irradiated Pf sporozoites infected mosquitoes as compared to similarly vaccinated but unprotected subjects. These results validate the utility of the Pf proteome microarray developed in the Phase I grant to identify antibodies associated with immune protection. In this Phase II application, we propose to apply an expanded version of the proteome microarray developed in Phase I (containing 4,253 Pf proteins) to determine antibody response in individuals enrolled in several attenuated sporozoite vaccine clinical trials. By comparing the antibody profiles from vaccinees which are protected with those who are not, we aim to identify surrogate antibody biomarkers associated with sporozoite vaccine mediated protection. There is now a major effort led by our collaborator, Sanaria Inc., to develop purified metabolically active, aseptic, vialed, and cryopreserved Pf sporozoite (PfSPZ) vaccines that prevent infection and transmission and are administered by needle and syringe injection. The first such PfSPZ vaccine has been attenuated by irradiation and is called the PfSPZ Vaccine. Recently, the group at Radboud University Nijmengen Medical Center (RUNMC) published on achieving complete protection against Pf by immunizing volunteers by the bite of mosquitoes carrying viable (non-irradiated) PfSPZ while taking chloroquine chemoprophylaxis to prevent parasitemia. This protection has now been shown to be sustained for 2 years. Together, Sanaria and RUNMC have shown that they can infect volunteers by needle and syringe administration of viable PfSPZ, and studies to immunize chloroquine treated volunteers with purified viable PfSPZ to determine if they can duplicate the protection seen after administration by mosquito bite are pending. Approximately 1,500 serum specimens from these trials will be used for the biomarker discovery project proposed here. The aim will be to identify and validate these surrogate biomarkers of protection and to develop a validated test intended to be used as a pivotal FDA-required assay to support licensure of the sporozoite vaccine.