Haddad Diana, Bilcikova Erika, Witney Adam A, Carlton Jane M, White Charles E, Blair Peter L, Chattopadhyay Rana, Russell Joshua, Abot Esteban, Charoenvit Yupin, Aguiar Joao C, Carucci Daniel J, Weiss Walter R
Naval Medical Research Center, Walter Reed Army Institute of Research, Silver Spring, MD, USA.
Infect Immun. 2004 Mar;72(3):1594-602. doi: 10.1128/IAI.72.3.1594-1602.2004.
We describe a novel approach for identifying target antigens for preerythrocytic malaria vaccines. Our strategy is to rapidly test hundreds of DNA vaccines encoding exons from the Plasmodium yoelii yoelii genomic sequence. In this antigen identification method, we measure reduction in parasite burden in the liver after sporozoite challenge in mice. Orthologs of protective P. y. yoelii genes can then be identified in the genomic databases of Plasmodium falciparum and Plasmodium vivax and investigated as candidate antigens for a human vaccine. A pilot study to develop the antigen identification method approach used 192 P. y. yoelii exons from genes expressed during the sporozoite stage of the life cycle. A total of 182 (94%) exons were successfully cloned into a DNA immunization vector with the Gateway cloning technology. To assess immunization strategies, mice were vaccinated with 19 of the new DNA plasmids in addition to the well-characterized protective plasmid encoding P. y. yoelii circumsporozoite protein. Single plasmid immunization by gene gun identified a novel vaccine target antigen which decreased liver parasite burden by 95% and which has orthologs in P. vivax and P. knowlesi but not P. falciparum. Intramuscular injection of DNA plasmids produced a different pattern of protective responses from those seen with gene gun immunization. Intramuscular immunization with plasmid pools could reduce liver parasite burden in mice despite the fact that none of the plasmids was protective when given individually. We conclude that high-throughput cloning of exons into DNA vaccines and their screening is feasible and can rapidly identify new malaria vaccine candidate antigens.
我们描述了一种用于鉴定疟原虫红细胞前期疫苗靶抗原的新方法。我们的策略是快速测试数百种编码约氏疟原虫基因组序列外显子的DNA疫苗。在这种抗原鉴定方法中，我们测量小鼠经子孢子攻击后肝脏中寄生虫负荷的降低情况。然后可以在恶性疟原虫和间日疟原虫的基因组数据库中鉴定约氏疟原虫保护性基因的直系同源物，并将其作为人类疫苗的候选抗原进行研究。一项开发该抗原鉴定方法的初步研究使用了192个来自生命周期子孢子阶段表达基因的约氏疟原虫外显子。总共182个（94%）外显子通过Gateway克隆技术成功克隆到DNA免疫载体中。为了评估免疫策略，除了编码约氏疟原虫环子孢子蛋白的特征明确的保护性质粒外，还用19种新的DNA质粒对小鼠进行了疫苗接种。通过基因枪进行的单质粒免疫鉴定出一种新型疫苗靶抗原，该抗原可使肝脏寄生虫负荷降低95%，并且在间日疟原虫和诺氏疟原虫中有直系同源物，但在恶性疟原虫中没有。肌肉注射DNA质粒产生的保护性反应模式与基因枪免疫所见不同。尽管单个质粒注射时没有一种具有保护作用，但用质粒库进行肌肉免疫可降低小鼠肝脏中的寄生虫负荷。我们得出结论，将外显子高通量克隆到DNA疫苗中并进行筛选是可行的，并且可以快速鉴定新的疟疾疫苗候选抗原。
