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

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 这个为期一年的新项目的重点是为未来的临床前原理验证测试疫苗试验生产和分析蛋白质免疫原的初步步骤，该试验将挑战诺氏疟原虫的血液期寄生虫。在大肠杆菌系统中表达了两种重要的网织红细胞结合样(RBL)重组蛋白PkNBPxa和PkNBPxb，PkNBPxa和PkNBPxb代表了这些RBL的N-末端粘附区，以达到利用恒河猴动物模型在体内检测其免疫原性和有效性的目的。这一动物模型将允许严格评估RBL作为疟疾疫苗候选者的潜力，以供未来在人类身上进行测试。PkNBPxa和PkNBPxb重组蛋白的表达和纯化因其生化组成而极具挑战性。尽管在克隆过程中对密码子进行了优化，但这些蛋白以包涵体的形式表达，并以非常高的浓度表达，与培养介质类型、宿主细胞或温度无关。开发这些候选疫苗抗原需要调整程序和持久性，以实现溶解和复性。还努力撰写了一份NIH R21提案，以继续这项研究，并在恒河猴身上进行计划的临床前疫苗试验。这项NIH R21提案已被成功审查，并将获得资金，使该项目能够继续进行下一阶段的临床前疫苗试验。该项目对于支持埃默里和疾病预防控制中心在这一主题上富有成效的合作也是极其有价值的。

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.