Kanoi Bernard N, Takashima Eizo, Morita Masayuki, White Michael T, Palacpac Nirianne M Q, Ntege Edward H, Balikagala Betty, Yeka Adoke, Egwang Thomas G, Horii Toshihiro, Tsuboi Takafumi
Division of Malaria Research, Proteo-Science Center, Ehime University, Matsuyama, Ehime 790-8577, Japan.
Division of Malaria Research, Proteo-Science Center, Ehime University, Matsuyama, Ehime 790-8577, Japan.
Vaccine. 2017 Feb 7;35(6):873-881. doi: 10.1016/j.vaccine.2017.01.001. Epub 2017 Jan 12.
The key targets of protective antibodies against Plasmodium falciparum remain largely unknown. In this study, we determined immunoreactivity to 1827 recombinant proteins derived from 1565 genes representing ∼30% of the entire P. falciparum genome, for identification of novel malaria vaccine candidates. The recombinant proteins were expressed by wheat germ cell-free system, a platform that can synthesize quality plasmodial proteins that elicit biologically active antibodies in animals. Sera were obtained from indigenous residents of a malaria endemic region in Northern Uganda who were enrolled at the start of a rainy season and prospectively monitored for symptomatic malaria episodes for a year. Immunoreactivity to sera was determined by AlphaScreen; a homogeneous high-throughput system that detects protein interactions. Our analysis revealed antibody responses to 128 proteins that significantly associated with protection from symptomatic malaria. From 128 proteins, 53 were down-selected as the most plausible targets of host protective immune response by virtue of having a predicted signal peptide and/or transmembrane domain(s), or confirmed localization on the parasite surface. The 53 proteins comprised of not only previously characterized vaccine candidates but also uncharacterized proteins. Proteins involved in erythrocyte invasion; RON4, RON2 and CLAG3.1 and pre-erythrocytic proteins; SIAP-2, TRAP and CelTOS, were recommended for prioritization for further evaluation as vaccine candidates. The findings clearly demonstrate that generation of the protein library using the wheat germ cell-free system coupled with high throughput immunoscreening with AlphaScreen offers new options for rational discovery and selection of potential malaria vaccine candidates.
针对恶性疟原虫的保护性抗体的关键靶点在很大程度上仍不清楚。在本研究中，我们测定了对来自1565个基因的1827种重组蛋白的免疫反应性，这些基因约占恶性疟原虫整个基因组的30%，以鉴定新型疟疾疫苗候选物。重组蛋白由小麦胚无细胞系统表达，该平台可以合成能在动物体内引发具有生物活性抗体的优质疟原虫蛋白。血清取自乌干达北部疟疾流行地区的当地居民，这些居民在雨季开始时登记入组，并对有症状的疟疾发作进行了为期一年的前瞻性监测。通过AlphaScreen测定血清的免疫反应性；AlphaScreen是一种检测蛋白质相互作用的均相高通量系统。我们的分析揭示了对128种蛋白质的抗体反应，这些反应与预防有症状疟疾显著相关。在这128种蛋白质中，53种因其具有预测的信号肽和/或跨膜结构域，或已证实定位于寄生虫表面，而被选为宿主保护性免疫反应最合理的靶点。这53种蛋白质不仅包括先前已鉴定的疫苗候选物，还包括未鉴定的蛋白质。参与红细胞入侵的蛋白质；RON4、RON2和CLAG3.1以及前红细胞期蛋白质；SIAP-2、TRAP和CelTOS，被推荐优先作为疫苗候选物进行进一步评估。这些发现清楚地表明，利用小麦胚无细胞系统生成蛋白质文库并结合AlphaScreen进行高通量免疫筛选，为合理发现和选择潜在的疟疾疫苗候选物提供了新的选择。
