Novel malaria transmission-blocking vaccine development using cell-free protein synthesis system

尽管不能为接种者提供直接保护，但靶向寄生虫性阶段表面表达的抗原的疫苗被认为是疟疾控制的一种有希望的策略，并且这些疟疾传播阻断疫苗（TBV）在过去十年中受到越来越多的关注。这阻断了蚊子中肠内的寄生虫发育周期，从而减少或防止寄生虫传播到其他个体。TBV还有望防止在抗疟疾药物治疗或针对寄生虫无性阶段的其他预防性疫苗过程中出现的逃逸突变体的传播。迄今为止，仅研究了这种类型疫苗的四种候选分子诱导保护性免疫的潜力。自从2002年10月完成恶性疟原虫基因组测序以来，我们现在可以免费获得基因组数据来寻找新的候选疫苗。然而，恶性疟原虫基因外显子中AT含量高，用常规方法表达重组蛋白会受到很大的抑制，这是恶性疟原虫疫苗研制的瓶颈之一。我们的策略是在不使用合成基因的情况下，基于恶性疟原虫的基因组序列表达重组蛋白以鉴定每种蛋白。为了鉴定新的TBV候选基因，我们选择了190个预期仅在恶性疟原虫配子体阶段表达的基因。将这些基因克隆到质粒中，并通过基于PCR的程序制备转录模板，然后通过麦胚无细胞系统高通量合成重组蛋白。利用这种方法，我们成功地获得了120个重组蛋白。筛选这些重组蛋白，以确定新的TBV候选人与传播阻断的人血清，我们已经确定了15个新的配子母细胞抗原作为TBV候选人。

Although not providing direct protection for vaccinees, vaccines targeting antigens expressed on the surface of the sexual stages of the parasites are considered one promising strategy for malaria control, and these malaria transmission-blocking vaccines (TBV) have received increased attention over the past decade. This blocks parasite developmental cycles within the mosquito midgut, consequently reducing or preventing parasite transmission to other individuals. TBVs are also expected to prevent the spread of escape mutants emerged during the course of anti-malaria drug treatment or other prophylactic vaccines targeting asexual stages of the parasites. Only four candidate molecules of this type of vaccine are studied for their potential to induce protective immunity, to date. Since the genomic sequence of P falciparum was completed in October 2002, we have now free access to the genome data to search for novel vaccine candidates. However, one of the bottlenecks for the vaccine development is the high AT content in exons of P. falciparum, which will considerably inhibit the recombinant protein expression using conventional methods. Our strategy is to express recombinant proteins for the characterization of each protein based on the genome sequences of P. falciparum without using synthetic gene. In order to identify the novel TBV candidates, we selected 190 genes, which are expected to be expressed only in gametocyte stage of P falciparum. These genes were cloned into plasmids and templates were prepared for transcription through PCR-based procedures, followed by high throughput recombinant protein synthesis by wheat germ cell-free system. Using this approach, we succeeded in obtaining 120 recombinant proteins. After the screening of these recombinant proteins to identify novel TBV candidates with transmission-blocking human sera, we have identified 15 novel gametocyte antigens as TBV candidates.