DISCOVERY AND PRECLINICAL DEVELOPMENT OF EFFICACIOUS P. vivax PRE-ERYTHROCYTIC STAGE MALARIA VACCINES

摘要 过去20年全球疟疾发病率的显著下降刺激了更大的努力，以实现其 全球根除；然而，这一雄心勃勃的目标需要新颖和高效的控制工具，包括 疫苗。尽管疟原虫生命周期的生物学复杂性阻碍了更快的进展 高效疟疾疫苗的开发，最近的技术和科学发展可能 促进进一步进步。我们建议将现有的设施、实验模型和标准化 以及识别和表征间日疟原虫(PV)红细胞前(PE)抗原的新技术 疫苗具有进一步临床开发的潜力。我们的一般假设是“免疫与 选定的PvPE抗原构建体可以在脊椎动物宿主中诱导保护性免疫反应。整体而言 这项提案的目标是通过识别和表征经典和 具有已证实保护效果的新候选疫苗，可进一步推进到临床 发展。具体目标是目标1：优先识别的新型PvPE抗原的特性 接种和保护子孢子的个体.目标2：免疫原性和保护效果的评价 在动物模型中构建新的PvPE蛋白结构；目标3：自组装蛋白纳米颗粒(SAPN)的设计 含有抗原性相关的PV-CSP和新的PvPE选择的蛋白质/片段的构建物；目的4： 抗PV-CSP人源单抗的制备及其保护性研究 精确的表位和构象。方法有：1)早期和晚期PVPE抗原的筛选和表达 与无菌保护相关，使用PV蛋白质微阵列。2)在B、T和CTL表位的电子预测中， 和相应的多肽序列的合成。3)免疫学特征，即体液(酶联免疫吸附试验 Ig/异型)和CMI(FACS细胞因子/细胞图谱)反应。4)免疫原性和保护效果 使用转基因寄生虫对啮齿动物体内选定的抗原结构/配方进行分析。5)猴子 免疫原性，体外抑制SPZ对肝细胞的侵袭，对野生型SPZ的保护作用，以及 保护的耐久性。6)开发含有PV-CSP变体的SAPN构建体(VK210；VK247)；7) 评价纳米粒在小鼠和猴子体内的免疫原性和保护效果；8)开发 PV-CSP Hu-MAbs及其保护效果和良好表位特异性分析9)抗原抗体 (Ag：：AB)相互作用的X射线结晶学分析这一建议的创新之处在于使用了 使用独特的血清/细胞进行全面、合理和严格的PV蛋白/表位下调选择过程 疟疾疫苗接种受保护个体，以及导致合理设计的蛋白质结构分析 保护性纳米颗粒配方。这项提议的意义在于提供快速的临床前 开发1-2种用于未来临床评估的高效PVPE疫苗和可能用于临床评估的PV-HuMAB 用于预防疟疾的被动给药。

Abstract A significant global malaria incidence reduction in the last two decades has stimulated greater efforts toward its global eradication; however, this ambitious goal demands novel and highly efficacious control tools, including vaccines. Although the biological complexity of the Plasmodium life cycle has prevented faster progress towards the development of a highly efficacious malaria vaccine, recent technological and scientific developments could facilitate further progress. We propose to integrate established facilities, experimental models, and standardized and novel techniques to identify and characterize P. vivax (Pv) pre-erythrocytic (PE) antigens showing vaccine potential for further clinical development. Our general hypothesis is that “immunization with selected PvPE antigen constructs can induce protective immune responses in the vertebrate host.” The overall goal of this proposal is to accelerate Pv vaccine development by identifying and characterizing classical and novel vaccine candidates with confirmed protective efficacy that could be further advanced to clinical development. Specific aims are Aim 1: Characterization of novel PvPE antigens recognized preferentially by sporozoite-vaccinated and protected individuals; Aim 2: Evaluation of the immunogenicity and protective efficacy of novel PvPE protein constructs in animal models; Aim 3: Design of self-assembled protein nanoparticle (SAPN) constructs containing antigenically relevant Pv-CSP and novel PvPE selected proteins/fragments; Aim 4: Generation of anti-Pv-CSP human monoclonal antibodies (Hu-MABs) with protective efficacy to define their precise epitopes and conformations. Methods are 1) selection and expression of early and late PvPE antigens associated with sterile protection, using Pv protein microarrays. 2) In silico prediction of B, T and CTL epitopes, and synthesis of the corresponding peptide sequences. 3) Immunological characterization, i.e., humoral (ELISA Igs/isotypes) and CMI (FACS cytokines/cell profiles) responses. 4) Immunogenicity and protective efficacy analyses of selected antigen constructs/formulations in rodents, using transgenic parasites. 5) Monkey immunogenicity, ex-vivo inhibition of spz invasion (ISI) to liver cells, protective efficacy to wild-type spz, and durability of protection. 6) Development of SAPN constructs containing the Pv-CSP variants (VK210; VK247); 7) Evaluation of the nanoparticles´ immunogenicity and protective efficacy in mice and monkeys; 8) Development of Pv-CSP Hu-MABs and analyses of their protective efficacy and fine epitope specificity; 9) Antigen-antibody (Ag::Ab) interactions analyses by X-ray crystallography. The innovation of this proposal is the use of a comprehensive, rational, and rigorous Pv protein/epitope down-selection process using unique sera/cells from malaria vaccinated protected individuals, together with protein structural analyses leading to the rational design of protective nanoparticle formulations. The significance of this proposal is to provide rapid preclinical development of 1-2 highly efficacious PvPE vaccines for future clinical evaluation and Pv-HuMABs that could be administered passively for malaria prophylaxis.