High-throughput screening of novel malaria vaccine candidates using human immune sera

疟疾仍然是热带国家居民发病和死亡的主要原因之一。恶性疟原虫寄生虫对化疗药物的耐药性增加的证据突出了对有效疫苗的迫切需要。疟疾基因组数据库建立后，我们现在可以自由访问基因组数据，以寻找新的候选疫苗。然而，传统方法表达重组蛋白的困难是疟疾疫苗研究的瓶颈之一。我们将基于麦胚无细胞系统的高通量蛋白生产方法应用于疟疾疫苗的研究。为了筛选新型疟疾疫苗候选基因，我们选择并克隆了180多个在裂殖子期表达的基因，然后通过PCR高通量制备转录模板，再通过麦胚无细胞系统合成蛋白质。利用这种方法，我们成功地获得了149个重组裂殖子蛋白。与此同时，我们成功地从生活在泰缅边境沿着的无症状村民中获得了约30份人类免疫血清，并从泰国曼谷的医院获得了约30份有症状的疟疾患者血清。本研究中使用的所有人血清样本均由泰国公共卫生部和日本爱媛大学的机构伦理委员会审查和批准。最后，我们用5例疟疾患者的免疫血清，通过ELISA方法筛选这些重组蛋白，用于鉴定新型疟疾疫苗候选物。作为初步筛选的结果，我们确定了10个裂殖子蛋白作为抗原。因此，这种利用麦胚无细胞系统的策略将是新型疟疾候选疫苗发现研究的重大突破。

Malaria remains one of the leading causes of both morbidity and mortality in humans residing in the tropical countries. The evidence of increasing resistance of the Plasmodium falciparum parasite to chemotherapeutic agents highlights the critical need for an effective vaccine. After the establishment of malaria genome database, we have now free access to the genome data to search for novel vaccine candidates. However, one of the bottlenecks for the malaria vaccine research is the difficulty of the recombinant protein expression using conventional methods. We applied a high-throughput protein production method based on the wheat germ cell-free system to the malaria vaccine research. To identify novel malaria vaccine candidates, we selected and cloned more than 180 genes which are expected to be expressed in merozoite stage, then prepared transcription templates through PCR-based high throughput procedures, followed by protein synthesis by wheat germ cell-free system. Using this approach, we succeeded in obtaining 149 recombinant merozoite proteins. At the same time, we successfully obtained approximately 30 human immune sera from asymptomatic villagers living along the Thai-Myanmar border, and approximately 30 symptomatic malaria patient sera from hospital in Bangkok, Thailand. All human serum samples used in this study were reviewed and approved by the Institutional Ethics Committee of the Thai Ministry of Public Health and of the Ehime University, Japan. Finally, we tried to screen these recombinant proteins for identifying novel malaria vaccine candidates with five cases of the malaria immune sera by ELISA. As a result of this preliminary screening, we identified ten merozoite proteins as antigens. Accordingly, this strategy using wheat germ cell-free system will be a major breakthrough in the novel malaria vaccine candidate discovery research.