RETICULOCYTE BINDING-LIKE (RBL) PROTEINS AS NEW GENERATION MALARIA VACCINES

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 这个为期一年的新项目专注于生产和分析蛋白质免疫原的初步步骤，用于未来的临床前原理验证测试疫苗试验，并通过P. knowlesi血液阶段寄生虫进行挑战。两个重要的网织红细胞结合样（RBL）重组蛋白PkNBPxa和PkNBPxb，代表这些RBL的N端粘附结构域区域。coli系统，以达到利用恒河猴动物模型在体内测试其免疫原性和功效的目的。这种动物模型将允许严格评估RBL作为疟疾疫苗候选物的潜力，用于未来的人体试验。PkNBPxa和PkNBPxb重组蛋白的表达和纯化由于其生物化学组成而极具挑战性。尽管在克隆过程中进行了密码子优化，但这些蛋白质以非常高的浓度在包涵体中表达，与培养基类型、宿主细胞或温度无关。这些候选疫苗抗原的开发需要调整程序和持久性以实现溶解性和重折叠。还花费了大量精力撰写NIH R21提案，以继续这项研究，并在恒河猴中进行计划的临床前疫苗试验。 NIH的R21提案已经过成功审查，并将获得资助，使该项目能够继续进行下一阶段的临床前疫苗试验。这个项目也是非常有价值的支持生产埃默里和疾病预防控制中心在这个问题上的合作。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This new, one-year project was focused on the preliminary steps of producing and analyzing the protein immunogens for a future pre-clinical proof-of-principle testing vaccine trial with challenge by P. knowlesi blood-stage parasites. Two important Reticulocyte Binding-Like (RBL) recombinant proteins, PkNBPxa and PkNBPxb, representing N-terminal adhesive domain regions of these RBLs were produced in the E. coli system for achieving the goal of testing their immunogenicity and efficacy in vivo using the rhesus macaque animal model. This animal model will allow the stringent evaluation of the potential of RBLs as malaria vaccine candidates for future testing in humans. The expression and purification of the PkNBPxa and PkNBPxb recombinant proteins was extremely challenging due to their biochemical composition. Despite codon optimization during the cloning process, these proteins were expressed in inclusion bodies and at very high concentrations, independent of culture media type, host cell or temperature. Development of these candidate vaccine antigens required adjustment of procedures and persistence to achieve solubility and refolding. Effort was also expended to write an NIH R21 proposal to continue this research and conduct the planned pre-clinical vaccine trial in rhesus monkeys. This NIH R21 proposal was reviewed successfully and will be funded, allowing this project to proceed to the next level of carrying out the pre-clinical vaccine trial. This project was also extremely valuable for supporting the productive Emory and CDC collaboration on this topic.