VanBuskirk Kelley M, O'Neill Matthew T, De La Vega Patricia, Maier Alexander G, Krzych Urszula, Williams Jack, Dowler Megan G, Sacci John B, Kangwanrangsan Niwat, Tsuboi Takafumi, Kneteman Norman M, Heppner Donald G, Murdock Brant A, Mikolajczak Sebastian A, Aly Ahmed S I, Cowman Alan F, Kappe Stefan H I
Seattle Biomedical Research Institute, Seattle, WA 98109, USA.
Proc Natl Acad Sci U S A. 2009 Aug 4;106(31):13004-9. doi: 10.1073/pnas.0906387106. Epub 2009 Jul 22.
Falciparum malaria is initiated when Anopheles mosquitoes transmit the Plasmodium sporozoite stage during a blood meal. Irradiated sporozoites confer sterile protection against subsequent malaria infection in animal models and humans. This level of protection is unmatched by current recombinant malaria vaccines. However, the live-attenuated vaccine approach faces formidable obstacles, including development of accurate, reproducible attenuation techniques. We tested whether Plasmodium falciparum could be attenuated at the early liver stage by genetic engineering. The P. falciparum genetically attenuated parasites (GAPs) harbor individual deletions or simultaneous deletions of the sporozoite-expressed genes P52 and P36. Gene deletions were done by double-cross-over recombination to avoid genetic reversion of the knockout parasites. The gene deletions did not affect parasite replication throughout the erythrocytic cycle, gametocyte production, mosquito infections, and sporozoite production rates. However, the deletions caused parasite developmental arrest during hepatocyte infection. The double-gene deletion line exhibited a more severe intrahepatocytic growth defect compared with the single-gene deletion lines, and it did not persist. This defect was assessed in an in vitro liver-stage growth assay and in a chimeric mouse model harboring human hepatocytes. The strong phenotype of the double knockout GAP justifies its human testing as a whole-organism vaccine candidate using the established sporozoite challenge model. GAPs might provide a safe and reproducible platform to develop an efficacious whole-cell malaria vaccine that prevents infection at the preerythrocytic stage.
恶性疟原虫疟疾是在按蚊通过吸血传播疟原虫子孢子阶段时引发的。经辐射的子孢子在动物模型和人类中可提供针对后续疟疾感染的无菌保护。这种保护水平是目前的重组疟疾疫苗所无法比拟的。然而，减毒活疫苗方法面临着巨大障碍，包括开发准确、可重复的减毒技术。我们测试了是否可以通过基因工程在早期肝脏阶段使恶性疟原虫减毒。恶性疟原虫基因减毒寄生虫（GAPs）具有子孢子表达基因P52和P36的单个缺失或同时缺失。基因缺失通过双交换重组完成，以避免基因敲除寄生虫的基因回复。基因缺失在整个红细胞周期、配子体产生、蚊虫感染和子孢子产生率方面均不影响寄生虫复制。然而，这些缺失导致寄生虫在肝细胞感染期间发育停滞。与单基因缺失系相比，双基因缺失系表现出更严重的肝内生长缺陷，且不能持续存在。这种缺陷在体外肝脏阶段生长试验和携带人肝细胞的嵌合小鼠模型中进行了评估。双敲除GAPs的强表型证明了其作为全生物体疫苗候选物在既定的子孢子攻击模型中进行人体试验的合理性。GAPs可能为开发一种有效的全细胞疟疾疫苗提供一个安全且可重复的平台，该疫苗可在红细胞前期预防感染。
