Identifying the targets of protective immunity to severe falciparum malaria

摘要 本R 01应用的目标是发现自然获得的保护，以防止严重的 恶性疟原虫疟疾，并开发它们作为新的血液阶段候选疫苗。在~100 目前正在研究的疟疾候选疫苗中，60%以上是基于四种寄生虫 抗原和最先进的疫苗，RTS，S，只产生适度的保护1，2。 在以往的研究中，我们开发了一种高度创新的全蛋白质组差异筛选策略， 鉴定由抗性个体表达的抗体识别的寄生虫抗原的子集 而不是易感个体。使用这种策略，我们发现了裂殖体出口抗原-1（PfSEA-1）， 244-kDa寄生虫抗原，其是抗体的靶标，所述抗体在鞭毛期捕获寄生虫， 在n=785名2岁儿童的队列中，与严重疟疾疾病的显著保护相关。 这是第一次证明特异性阻断出口的抗体可以预防严重的疟疾 人类（科学4） 在平行研究中，我们还鉴定了PfGARP，这是一种以前未被识别的候选疫苗， 在滋养体感染的红细胞中，红细胞膜的外表面。抗高度不变的 PfGARP（PfGARP-A，aa 411-673）的羧基末端在体外抑制寄生虫生长达99%， 对照组（P < 0.001）。大量的机理分析表明， 抗PfGARP与感染的RBC表面的结合诱导寄生虫程序性细胞死亡， 核固缩形态学、半胱天冬酶激活、线粒体去极化、DNA 断裂和细胞内钙的释放。此外，非人灵长类动物接种 PfGARP作为脂质包封的mRNA配制导致对恶性疟原虫攻击的显著保护 与对照组相比（Nature 5）。 这些初步结果是基于使用来自抗性和敏感性的血清进行的差异筛选。 个体的抗性定义仅基于寄生虫血症。以前的工作表明， 儿童仅在一两次发作后就对严重疟疾产生了抵抗力，这种保护作用不同于 简单控制寄生虫血症的反应6.在本提案中，我们将利用这一观察结果， 鉴定作为抗体应答的靶标的寄生虫抗原，所述抗体应答是在免疫应答期间获得和表达的。 在严重疟疾发作的恢复阶段，并防止今后发生严重疟疾。 我们将：1）在肯尼亚西部的一个完全流行地区进行病例对照研究， 确定患有严重疟疾的婴儿和儿童以及匹配的对照组。2)进行全蛋白质组差异分析 使用来自该病例对照研究的血清进行筛选，以及3）向下选择用于后续疫苗研究的候选者 使用一套测定（生长抑制、人类免疫流行病学研究和鼠疫苗试验）。

ABSTRACT The goal of this R01 application is to discover the targets of naturally acquired protection against severe Plasmodium falciparum malaria and to develop them as novel blood-stage vaccine candidates. Of the ~100 malaria vaccine candidates currently under investigation, more than 60% are based on only four parasite antigens and the most advanced vaccine, RTS,S, generates only modest protection 1, 2. In previous studies, we developed a highly innovative whole proteome differential screening strategy which identifies the subset of parasite antigens that are recognized by antibodies expressed by resistant individuals but not susceptible individuals. Using this strategy, we discovered Schizont Egress Antigen-1 (PfSEA-1), a 244-kDa parasite antigen that is the target of antibodies which arrest parasites at the schizont stage and are associated with significant protection from severe malarial disease in a cohort of n=785 two yr old children. This is the first demonstration that antibodies that specifically block egress can protect against severe malaria in humans (Science 4). In parallel studies, we also identified PfGARP, a previously unrecognized vaccine candidate which localized to the exofacial surface of the RBC membrane in trophozoite infected RBC. Antibodies to the highly invariant carboxyl terminal of PfGARP (PfGARP-A, aa 411-673) inhibit parasite growth in vitro by 99% compared to controls (P < 0.001) by killing trophozoite stage parasites. Numerous mechanistic assays demonstrated that the binding of anti-PfGARP to the surface of the infected RBC induces parasite programed cell death as evidenced by pyknotic nuclear morphology, caspase activation, mitochondrial depolarization, DNA fragmentation, and release of intracellular calcium. In addition, vaccination of non-human primates with PfGARP formulated as a lipid encapsulated mRNA results in significant protection from P. falciparum challenge compared to controls (Nature 5). These preliminary results were based on differential screening using sera from resistant and susceptible individuals with the definition of resistance based solely on parasitemia. Previous work has demonstrated that children develop resistance to severe malaria after only one or two episodes, and this protection is distinct from responses that simply control parasitemia 6. In the current proposal, we will capitalize on this observation to identify parasite antigens that are targets of antibody responses which are acquired and expressed during the convalescent phase of an episode of severe malaria and protect against future episodes of severe malaria. We will: 1) conduct a case-control study at our field site in a holoendemic region of western Kenya to identify infants and children with severe malaria and matched controls. 2) perform whole proteome differential screening using sera from this case-control study, and 3) down select candidates for follow-on vaccine studies using a suite of assays (growth inhibition, human immunoepidemiologic studies, and murine vaccine trials).