Protective biomarkers for the development of vaccines against malaria

描述(申请人提供)：对于第一阶段的SBIR拨款，我们构建了一个恶性疟原虫(PF)3D7蛋白质微阵列，包含代表1,200个已知和假设蛋白质的2,320个多肽，或整个PF蛋白质组的~23%。我们发现，打印在这些阵列上的单个蛋白可以捕获存在于感染者血清中的抗体，并且可以使用荧光二次抗体来定量捕获抗体的量。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图谱。我们用居住在马里的人的血清对该阵列进行了探测，并确定了一组潜在的疫苗候选抗原，与来自同一村庄的年龄匹配的易感儿童相比，受保护儿童对这些候选疫苗的抗体显著升高。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，与同样接种但未受保护的受试者相比，受保护志愿者的血清中优先识别这些生物标记物和潜在的疫苗抗原，这些受试者由受辐射的PF子孢子感染蚊子的叮咬免疫。这些结果验证了在第一阶段资助中开发的PF蛋白质组微阵列用于识别与免疫保护相关的抗体的实用性。在这个第二阶段的应用中，我们建议应用第一阶段开发的蛋白质组微阵列的扩展版本(包含4,253个Pf蛋白)来确定在几个减毒的子孢子疫苗临床试验中登记的个体的抗体反应。通过比较疫苗保护者和未保护者的抗体谱，我们的目的是识别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.正在领导一项重大努力，开发纯化的代谢活性、无菌、瓶装和冷冻保存的肺炎子孢子(PfSPZ)疫苗，以防止感染和传播，并通过针头和注射器注射给药。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，拉德布大学奈门根医学中心(RUNMC)的研究小组发表了一篇关于通过在服用氯喹化学预防措施防止寄生虫血症的同时，通过携带活的(非辐射的)PfSPZ的蚊子叮咬来免疫志愿者来实现对PF的完全保护。这一保护现已被证明可持续两年。Sanaria和RUNMC共同证明，他们可以通过针头和注射器注射活的PfSPZ感染志愿者，用纯化的活的PfSPZ免疫氯喹治疗的志愿者的研究正在进行中，以确定他们是否可以复制蚊子叮咬后的保护。来自这些试验的大约1500个血清样本将用于这里提出的生物标记物发现项目。其目的将是识别和验证这些保护的替代生物标志物，并开发一种经过验证的测试，旨在用作FDA要求的关键测试，以支持子孢子疫苗的许可。 公共卫生相关性：一种有效的疟疾疫苗将极大地惠及全世界疟疾流行地区数百万婴儿、儿童、孕妇和其他面临疟疾疾病和死亡风险的人，但迄今为止，尽管付出了巨大努力，但开发重组蛋白和病毒载体疫苗的努力令人失望。这一建议利用了最近的发现，这些发现表明，几种减毒活的全身疫苗可以提供强大的保护，使其免受寄生虫的感染。这项研究旨在探索包含4253个PF蛋白的微阵列和数百个子孢子疫苗临床试验样本，以识别和验证保护的替代抗体生物标记物，并开发一种验证测试，旨在用作FDA要求的关键测试，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. PUBLIC HEALTH RELEVANCE: An effective malaria vaccine would greatly benefit millions of infants, children, pregnant women and others at risk of malaria disease and death throughout the malaria-endemic world, but thus far efforts to develop recombinant protein and viral vector vaccines have been disappointing despite intense efforts. This proposal takes advantage of recent discoveries showing that several live attenuated whole organism vaccines can provide robust protection from infection by the parasite. The study proposed here aims to probe a microarray containing 4,253 Pf proteins with hundreds of sporozoite vaccine clinical trial specimens to identify and validate surrogate antibody 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.