RBL Binding Domain Malaria Candidate Vaccines

描述(由申请人提供)：这项探索性的R21提案侧重于评估两种候选网织红细胞结合(RBL)蛋白疫苗在有血液期挑战的恒河猴中的有效性，首次为这类裂殖子疫苗抗原提供原理证明，并为未来的临床前试验准备间日疟原虫RBL抗原。恒河猴诺氏疟原虫血液期感染在接种后1-2周内变得致命，除非用抗疟疾药物治疗。我们建议测试诺氏疟原虫RBL结合域作为免疫原发挥作用的潜力，以减轻这种寄生虫通常的致命性。如果能够在猴子身上实现对保守的关键生物学靶点的保护，如RBL，就可以增强人们对推进同源恶性疟原虫和间日疟原虫靶标(S)作为疫苗(S)在临床试验中应用的信心。这个项目建立在这个小组发现间日疟原虫网织红细胞结合蛋白(PvRBPs)的基础上，随后在其他物种(恶性疟原虫、食蟹猴疟原虫、Coatneyi、莱氏疟原虫和诺氏疟原虫)中鉴定了这些蛋白，并将两个诺氏疟原虫RBL结合区作为免疫原进行了鉴定和制备。RBL蛋白(又称网织红细胞结合同源(RH)蛋白超家族)位于裂殖子顶端，与红细胞结合，被认为在裂殖子入侵过程中起重要作用。每种疟原虫中都存在多个RBL基因(诺氏疟原虫只有2个，约氏疟原虫多达14个)，数据表明，每个物种都保持了将一种或另一种编码的配体或可能的组合用作黏附分子的能力，以确保进入红细胞。关键的是，每个物种似乎至少有两个对裂殖子入侵至关重要的RBL。在目标1中，我们建议使用恒河猴模型来检验以下假设：用RBL红细胞结合域免疫可以限制致死性感染，并且多个RBL结合域区域的组合将提供更好的保护。在目标2中，我们将检验间日疟原虫RBL蛋白同源结合结构域的抗血清在体外可以抑制裂殖子入侵的假设，作为可能在NHP中使用这些蛋白进行临床前疫苗试验的前奏。我们将致力于从表达的间日疟原虫RBL蛋白中优化同源结合结构域的表达和纯化，鉴定功能(即结合)，开发和测试这些RBL或特异性抗血清在体外抑制入侵的能力。不管疫苗测试的结果如何，我们将获得知识和额外的工具来研究RBL作为红细胞入侵的关键配体和可能的生物干预目标。 与公共卫生相关：疟疾疫苗将是帮助控制疟疾的极其宝贵的工具。需要新的方法和新的模式，以加快测试，并为理论上合理的疟疾疫苗候选提供某种形式的验证。这一提议直截了当，同时也具有创新性、风险性和探索性。我们将了解基于RBL的疟疾疫苗是否能够提供对红细胞攻击的保护，并开发有价值的工具来推进与裂殖子入侵红细胞相关的研究。这项研究还将获得关于RBL免疫应答的有价值的信息，并为基于间日疟原虫RBL免疫原的潜在临床前疟疾疫苗试验铺平道路。

DESCRIPTION (provided by applicant): This exploratory R21 proposal focuses on evaluating two Reticulocyte Binding-Like (RBL) protein vaccine candidates in rhesus monkeys with a blood-stage challenge to test for efficacy, providing for the first-time a proof of principle for this category of merozoite vaccine antigens, and also preparing P. vivax RBL antigens for future pre-clinical trials. Plasmodium knowlesi blood-stage infections in rhesus macaques become lethal 1-2 weeks after inoculation unless treated with anti-malarial drugs. We are proposing to test the potential of P. knowlesi RBL binding domains to function as immunogens that will attenuate the usual lethality of this parasite. If protection can be achieved against a conserved key biological target in monkeys, like the RBLs, there can be heightened confidence for advancing the homologous P. falciparum and P. vivax target(s) as a vaccine(s) in clinical trials. This project builds upon this group's discovery of the P. vivax Reticulocyte Binding Proteins (PvRBPs), the subsequent identification of these proteins in other species, (P. falciparum, P. cynomolgi, P. coatneyi, P. reichenowi, and P. knowlesi), and the identification and preparation as immunogens of two P. knowlesi RBL binding domains. The RBL proteins (also known as the reticulocyte-binding homologue (RH) protein superfamily) are located at the apical pole of merozoites, bind erythrocytes and are believed to be instrumental in directing merozoite invasion. Multiple rbl genes exist in each Plasmodium species (with only 2 in P. knowlesi, yet as many as 14 in P. yoelii), and data suggests that each species has maintained the ability to use one or another of the encoded ligands, or possibly combinations, as adhesion molecules to ensure entry into red blood cells. Critically, each species appears to have at least two RBLs that are essential for merozoite invasion. In Aim 1 using a rhesus monkey model we propose to test the hypothesis that immunization with an RBL red blood cell binding domain can limit a lethal infection, and that the combination of multiple RBL binding domain regions will provide greater protection. In Aim 2, we will test the hypothesis that antisera to homologous binding domain regions from P. vivax RBL proteins will inhibit invasion of merozoites in vitro, as a prelude to possible pre-clinical vaccine trials using these proteins in NHPs. We will work to optimize the expression and purification of homologous binding domain regions from expressed P. vivax RBL proteins, characterize functionality (i.e. binding), develop and test the ability of these RBLs or specific antisera to inhibit invasion in vitro. Regardless of the outcome from vaccine testing, we will have gained knowledge and additional tools to study the RBLs as critical ligands for red blood cell invasion and as possible biological targets of intervention. PUBLIC HEALTH RELEVANCE: A malaria vaccine would be extremely valuable tool to help control malaria. Novel approaches and new paradigms are needed that can expedite the testing and provide some form of validation for theoretically sound malaria vaccine candidates. This proposal is straightforward, while also being innovative, risky and exploratory. We will learn whether RBL-based malaria vaccines can provide protection against erythrocytic challenge and also develop valuable tools for advancing investigations relating to merozoite invasion of red blood cells. This research will also gain valuable information on the immune response to the RBLs and pave the way for potential pre- clinical malaria vaccine trials based on P. vivax RBL immunogens.