RBL Binding Domain Malaria Candidate Vaccines

描述（由申请人提供）：本探索性R21提案侧重于评估两种网织红细胞结合样（RBL）蛋白候选疫苗在具有血期挑战的猕猴体内的有效性，首次为这类merozoite疫苗抗原提供原理证明，并为未来的间日疟原虫RBL抗原准备临床前试验。除非用抗疟疾药物治疗，否则恒河猴的诺氏疟原虫血期感染在接种后1-2周就会致命。我们建议测试诺氏疟原虫RBL结合域作为免疫原的潜力，以减弱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RBLs，则可以提高在临床试验中推进同源恶性疟原虫和间日疟原虫靶点作为疫苗的信心。该项目建立在该小组发现间日疟原虫网状细胞结合蛋白（pvrbp）的基础上，随后在其他物种（恶性疟原虫、cynomolgi疟原虫、P. coatneyi疟原虫、P. reichenowi疟原虫和P. knowlesi疟原虫）中鉴定了这些蛋白，并鉴定和制备了两种P. knowlesi RBL结合域的免疫原。RBL蛋白（也被称为网状红细胞结合同源（RH）蛋白超家族）位于分裂子的顶极，与红细胞结合，被认为是指导分裂子侵入的工具。每种疟原虫中都存在多个rbl基因（诺氏疟原虫中只有2个，而约氏疟原虫中多达14个），数据表明，每种疟原虫都保持了使用一种或另一种编码配体或可能的组合作为粘附分子的能力，以确保进入红细胞。至关重要的是，每个物种似乎都至少有两个rbl，这是分裂子侵入所必需的。在Aim 1中，我们使用恒河猴模型来验证RBL红细胞结合区域免疫可以限制致命感染的假设，并且多个RBL结合区域的组合将提供更大的保护。在Aim 2中，我们将验证间日疟原虫RBL蛋白同源结合区域的抗血清将在体外抑制merozoites侵袭的假设，作为可能在NHPs中使用这些蛋白进行临床前疫苗试验的前奏曲。我们将从表达的间日疟原虫RBL蛋白中优化同源结合域的表达和纯化，表征功能（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。无论疫苗测试的结果如何，我们都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干预生物学靶点。

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.