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

摘要 过去二十年来，全球疟疾发病率显著下降，促使人们加大努力， 全球根除;然而，这一雄心勃勃的目标需要新颖且高效的控制工具，包括 疫苗。虽然疟原虫生命周期的生物复杂性阻碍了更快地发展 由于开发了高效疟疾疫苗，最近的技术和科学发展可以 促进进一步的进展。我们建议整合现有设施、实验模型和标准化 以及鉴定和表征间日疟原虫（Pv）红细胞前（PE）抗原的新技术， 疫苗的进一步临床开发潜力。我们的一般假设是，“免疫与 选择的PvPE抗原构建体可以在脊椎动物宿主中诱导保护性免疫应答。整体 该提案的目标是通过鉴定和表征经典和 新的候选疫苗，具有确认的保护效力，可进一步推进到临床 发展具体目的是目的1：表征优先被PvPE识别的新型PvPE抗原 子孢子接种和保护的个体;目的2：评价免疫原性和保护效力 目的3：设计自组装蛋白质纳米颗粒（SAPN） 含有抗原相关的Pv-CSP和新的PvPE选择蛋白/片段的构建体;目的4： 产生具有保护功效的抗Pv-CSP人单克隆抗体（Hu-MAB）以确定其免疫原性。 精确的表位和构象。方法：1）选择和表达早期和晚期PvPE抗原 与无菌保护相关，使用Pv蛋白微阵列。2)B、T和CTL表位的计算机预测， 和合成相应的肽序列。3)免疫学表征，即，体液（ELISA Ig/同种型）和CMI（FACS细胞因子/细胞谱）应答。4)免疫原性和保护效力 使用转基因寄生虫分析啮齿动物中的选定抗原构建体/制剂。5)猴子 免疫原性、spz对肝细胞侵袭（ISI）的离体抑制、对野生型spz的保护功效，以及 保护的持久性。6)含有Pv-CSP变体（VK 210; VK 247）的SAPN构建体的开发; 7） 在小鼠和猴子中评价纳米颗粒的免疫原性和保护功效; 8）开发 Pv-CSP Hu-MAB的免疫原性及其保护效力和精细表位特异性的分析; 9）抗原-抗体 (Ag* Ab）X射线晶体学相互作用分析。本提案的创新之处在于使用了 全面、合理和严格的Pv蛋白/表位下调选择过程，使用来自 疟疾疫苗接种保护的个人，连同蛋白质结构分析，导致合理的设计 保护性纳米颗粒制剂。该建议的意义在于提供快速的临床前 开发1-2种高效的PvPE疫苗用于未来的临床评价， 被动给药以预防疟疾。

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.