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

项目摘要/摘要 尽管疟疾疫苗对全球公共卫生的重要性很大，但发展缓慢，只有一种 少数候选人正在开发中，最先进的候选人表现出适度的效力。当前的用法 用于基因组/蛋白质组筛选的生物信息学、动物模型和更高效的制造设施 重组/合成抗原可以极大地加速新型疫苗的开发。我们建议 利用这些进展来识别和开发针对恶性疟原虫和P。 间日疟原虫，占全球疟疾病例的99%。α-螺旋螺旋卷曲模体，蛋白质 已知的在疟原虫蛋白质组中广泛分布的结构在水溶液中自发折叠， 能够在体外诱导出抑制寄生虫生长的抗体。我们之前在电子计算机上发现了220种蛋白质。 在这两个寄生虫蛋白质组中，合成并检测了170个蛋白质组。与人源抗原性高的人血清 流行区和小鼠的免疫原性导致了145个片段的选择。他们中的几个展示了 显著的恶性疟原虫/间日疟原虫交叉反应，以及重要的体外寄生虫抑制活性，这 与疟疾临床免疫力相关。此外，恶性疟原虫多表位免疫小鼠 构建物(PF-181)保持了单个表位的特异性和寄生虫抑制活性。我们的将军 假说是“含有交叉反应α-螺旋螺旋线圈基序的疟原虫多表位构建物 诱导猴对同源和异源寄生虫攻击的保护。总体目标是 恶性疟原虫和间日疟原虫红细胞合成多表位构建物疫苗潜能的测定 包含α螺旋螺旋线圈图案。这一目标将通过以下具体目标实现：1) 含α-螺旋的恶性疟原虫和间日疟原虫多表位构建物的设计、合成与鉴定 2)选择恶性疟原虫和恶性疟原虫的免疫原性和保护效果进行评价。 间日疟原虫多表位构建及其在Aotus猴体内对抗同源和异源寄生虫的研究 α-螺旋卷曲片段/构建体的体外鉴定及动物免疫应答 免疫接种。建议的方法是：a)多表位构建的F-moc多肽的合成，然后是高效液相色谱 纯化、质谱仪(MS)和圆二色谱(CD)分析； 多表位选择构建及其对同源和异源保护效果的评价 挑战使用恶性疟原虫(Santa Lucia)和间日疟原虫(SAL-I)寄生虫株；c)血清的酶联免疫吸附试验和IFAT试验 来自免疫动物的；d)体外抗体功能检测(GIA、ADRB、OPA)。本研究的创新之处 是高效、高性价比的方法，使用了全面合理和严格的表位向下- 从疟原虫蛋白质组电子表位鉴定中筛选出数百个蛋白质片段用于多聚体检测 表位设计及其在猴体内保护效果研究的评价。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.