Comprehensive screening and identification of the novel malaria transmission-blocking vaccine candidate antigens

疟疾传播阻断疫苗（TBVs）通过诱导针对在性阶段寄生虫上特异性表达的抗原的抗体来预防疟疾的传播。由于具有良好特征的TBV候选者只有四种，因此有必要为成功的TBV开发确定新的TBV候选者。为了鉴定新的TBV候选基因，我们从基因组和转录组数据库中检索了192个基因，这些基因预计只在恶性疟原虫配子细胞阶段表达。将这些基因克隆到质粒中，制备了170个模板cDNA克隆，通过pcr方法进行转录，然后在小麦无生殖细胞系统中高通量合成重组蛋白。利用这种方法，我们成功地获得了148个重组蛋白。在对这些重组蛋白进行筛选后，我们发现了27种新的TBV候选抗原，这些抗原与含有寄生虫传播阻断抗体的疟疾患者血清相结合。将这些候选基因亚克隆到pEU质粒中，利用小麦无生殖细胞系统进行大规模表达，共表达和亲和纯化了22个目标基因。为了获得抗血清，我们将这些蛋白免疫到小鼠体内。在确认这些抗血清对寄生虫的免疫反应性后，我们检查了血清的传播阻断功效。最后，我们鉴定了两种新的寄生虫抗原，它们在小鼠体内诱导了传播阻断抗体。因此，该方法将有助于发现新的传播阻断候选疫苗。

Malaria transmission-blocking vaccines (TBVs) prevent the transmission of malaria by inducing antibodies against antigens specifically expressed on the sexual stage parasites. Since well-characterized TBV candidates are only four, it would be necessary to identify novel TBV candidates for a successful TBV development. In order to identify the novel TBV candidates, we searched a combined dataset from genome and transcriptome databases and selected 192 genes, which are expected to be expressed only in gametocyte stage of P. falciparum. These genes were cloned into plasmids and 170 of the template cDNA clones 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 148 recombinant proteins. After the screening of these recombinant proteins to identify novel TBV candidates with malaria patient sera harboring parasite transmission-blocking antibodies, we have identified 27 novel antigens. After subclone these candidate genes into pEU plasmid for the large-scale expression using the wheat germ cell-free system, we have expressed and affinity purified 22 targets. In order to obtain antisera, we immunized these proteins into mice. After the confirmation of the immunoreactivity of these antisera against parasites, we examined the transmission-blocking efficacy of the sera. Finally we identified the two novel parasite antigens, which induced transmission-blocking antibodies in mice. Accordingly, this approach will be useful for the novel transmission-blocking vaccine candidate discovery.