Discovery and preclinical evaluation of Plasmodium falciparum and P. vivax coiled coil antigens for malaria vaccine development

项目总结/摘要 尽管全球公共卫生非常重要，但疟疾疫苗的开发一直很缓慢， 少数正在开发的候选药物和最先进的药物显示出适度的功效。目前使用 生物信息学的基因组/蛋白质组筛选，动物模型，更有效的生产设施， 重组/合成抗原可显著加速新型疫苗的开发。我们建议 利用这些进展来鉴定和开发针对恶性疟原虫和疟原虫的候选疫苗。 间日疟原虫，占全球疟疾病例的99%以上。α-螺旋卷曲螺旋基序，蛋白质 广泛分布在疟原虫蛋白质组中的已知在水溶液中自发折叠的结构， 能够在体外诱导抑制寄生虫生长的抗体。我们之前在计算机上鉴定了220种蛋白质 在这两种寄生虫的蛋白质组中，合成并测试了其中的170种。人血清的高抗原性， 流行区，和小鼠中的免疫原性导致145个片段的选择。其中一些显示 显著的恶性疟原虫/间日疟原虫交叉反应性，重要的是体外寄生虫抑制活性， 与疟疾临床免疫相关。此外，用恶性疟原虫多表位免疫小鼠， 构建体（Pf-181）保持了单个表位特异性和寄生虫抑制活性。我们的一般 假设是“含有交叉反应α-螺旋卷曲螺旋基序的疟原虫多表位构建体 在猴中诱导保护以抵抗同源和异源寄生虫攻击。总体目标是 确定恶性疟原虫和间日疟原虫红细胞合成多表位构建体的疫苗潜力 含有α-螺旋卷曲螺旋基序。将通过以下具体目标实现这一目标： 恶性疟原虫和间日疟原虫α-螺旋多表位构建体的设计、合成和鉴定 卷曲螺旋基序; 2）评价选择的恶性疟原虫和恶性疟原虫的免疫原性和保护效力。 针对Aotus猴中同源和异源寄生虫攻击的间日疟原虫多表位构建体; 3） α-螺旋卷曲螺旋片段/构建体的体外表征和动物诱导的免疫应答 次免疫所提出的方法是：a）多表位构建体的F-moc肽合成，随后进行HPLC 纯化、质谱法（MS）和圆二色性（CD）分析; B）猴免疫 选择的多表位构建体和对同源和异源的保护效力的评估 使用恶性疟原虫（圣露西亚）和间日疟原虫（Sal-I）寄生虫株的攻击; c）血清的ELISA和IFAT测试 d）抗体功能体外测定（GIA、ADRB、OPA）。 是一种高效、经济的方法，采用全面合理、严谨的表位下调- 从疟原虫蛋白质组中的计算机表位鉴定中选择数百个蛋白质片段用于多聚- 表位设计及其在猴中的保护效力研究的评价。它将加速发展 人类使用的疟疾疫苗

PROJECT SUMMARY/ABSTRACT Despite the great global public health importance, malaria vaccine development has been slow with only a handful of candidates under development and the most advanced displaying modest efficacy. The current use of bioinformatics for genome/proteome screening, animal models, and more efficient manufacturing facilities for recombinant/synthetic antigens could significantly accelerate the development of novel vaccines. We propose to use these advances to identify and develop vaccine candidates targeting both, Plasmodium falciparum and P. vivax parasites, responsible for >99% of the malaria cases worldwide. Alpha-helical coiled-coil motifs, protein structures widely distributed in the Plasmodium proteomes known to spontaneously fold in aqueous solution, are able to induce antibodies that inhibit parasite growth in vitro. We previously in silico identified 220 proteins in both parasite proteomes, 170 of which were synthesized and tested. High antigenicity with human sera from endemic areas, and immunogenicity in mice led to selection of 145 fragments. Several of them displayed significant P. falciparum/P. vivax cross reactivity, and importantly in vitro parasite inhibition activity, which correlated with malaria clinical immunity. Furthermore, mice immunization with a P. falciparum poly-epitope construct (Pf-181) maintained individual epitopes specificity and parasite inhibitory activity. Our general hypothesis is that “Plasmodium poly-epitope constructs containing cross-reacting α-helical coiled-coil motifs induce protection in monkey against homologous and heterologous parasite challenges. The overall goal is to determine the vaccine potential of P. falciparum and P. vivax erythrocytic synthetic poly-epitope constructs containing α-helical coiled-coil motifs. This goal will be approached through the following specific aims: 1) Design, synthesis and characterization of P. falciparum and P. vivax poly-epitope constructs containing α-helical coiled-coil motifs; 2) Evaluation of the immunogenicity and protective efficacy of selected P. falciparum and P. vivax poly-epitope constructs against homologous and heterologous parasite challenges in Aotus monkeys; 3) In vitro characterization of α-helical coiled-coil fragments/constructs and immune responses elicited by animal immunization. Methods proposed are: a) F-moc peptide synthesis of poly-epitope constructs, followed by HPLC purification, mass spectrometry (MS) and circular dichroism (CD) analyses; b) Monkey immunization with selected poly-epitope constructs and evaluation of protective efficacy to homologous and heterologous challenges using P. falciparum (Santa Lucia) and P. vivax (Sal-I) parasite strains; c) ELISA and IFAT test of sera from immunized animals; d) antibody functional in vitro assays (GIA, ADRB,OPA).The innovation of this study is the highly efficient and cost-effective approach, using a comprehensive rational and rigorous epitope down- selection of hundreds of protein fragments from in silico epitope identification in Plasmodium proteomes for poly- epitope design and evaluation of their protective efficacy studies in monkeys. It will accelerate the development of malaria vaccines for human use.