Design of a B cell tetramer to track PfMSP2-specific B cells

项目摘要 原生动物寄生虫疟原虫是疟疾的病原体， 当今世界面临的重大公共卫生挑战。抗体在预防严重的 由血液感染引起的疾病。但是，需要在较长时间内反复感染 以诱导保护性体液免疫。目前还不清楚保护性抗体是否来自于短寿命的 浆母细胞通过滤泡外反应或长寿浆细胞通过生发中心。诱导 后者是有效疫苗的主要目标。虽然疟原虫特异性抗体可在 尽管这些抗体反应的细胞生物学基础仍然存在于临床疟疾个体的血清中， 未知在这里，我们建议开发一种疟原虫特异性B细胞四聚体来追踪抗原特异性B细胞 这些反应填补了我们对疟原虫体液反应理解的空白。利用血液阶段 抗原，裂殖子表面蛋白2（MSP 2），一种用于血液阶段疟疾疫苗的候选抗原，我们将 产生两个B细胞四聚体，一个含有全长重组恶性疟原虫MSP 2蛋白， 另一种包含嵌合PfMSP 2/8蛋白。前者能在体外形成原纤维，而后者则不能 没有因此，我们预测嵌合蛋白质将提供诸如随时间增加的稳定性和免疫原性等益处。 鉴定出更多数量的MSP 2-特异性B细胞，其结合更广泛数量的表位， PfMSP 2的四聚体比仅由rPfMSP 2组成的四聚体更好。在子目标1中，我们将监测MSP 2原纤维形成， 在小鼠免疫后测定每种B细胞四聚体离体识别抗原特异性B细胞的能力 使用rPfMSP 2或rPfMSP 2/8。然后在子目标2中，我们将确认B细胞的结合特异性 通过ELISA通过基于MSP 2的B细胞受体序列产生单克隆抗体， 四聚体阳性B细胞。在使用前必须确认B细胞四聚体的结合特异性 鉴定来自疟疾流行地区的个体或个体的血液中的MSP 2特异性B细胞 参与疫苗研究。

Project Summary The protozoan parasite Plasmodium is the causative agent of malaria, which remains one of the most prominent public health challenges in the world today. Antibodies play a primary role in protection from severe disease caused by blood-stage infection. However, repeated infections over an extended period are required to induce protective humoral immunity. It is unclear if protective antibodies are derived from short-lived plasmablasts through an extrafollicular response or long-lived plasma cells via the germinal center. Induction of the latter is the primary goal of effective vaccines. While Plasmodium-specific antibodies are detectable in the serum of individuals with clinical malaria, the cell biology underlying these antibody responses remains unknown. Here we propose developing a Plasmodium-specific B cell tetramer to track antigen-specific B cell responses to close gaps in our understanding of the humoral response to Plasmodium. Using the blood-stage antigen, merozoite surface protein 2 (MSP2), a candidate antigen for a blood-stage malaria vaccine, we will generate two B cell tetramers, one containing a full-length recombinant P. falciparum MSP2 protein and another encompassing a chimeric PfMSP2/8 protein. The former can form fibrils in vitro, while the latter does not. Hence, we predict the chimeric protein will offer such benefits as increased stability over time and the identification of a greater number of MSP2-specific B cells that bind a broader number of epitopes across PfMSP2 than a tetramer composed solely of rPfMSP2. In sub-aim 1, we will monitor MSP2 fibril formation and determine each B cell tetramer's ability to recognize antigen-specific B cells ex vivo after immunization of mice with either rPfMSP2 or rPfMSP2/8. Then in sub-aim 2, we will confirm the specificity of the B cells for binding MSP2 by ELISA through the generation of monoclonal antibodies based on the B-cell receptor sequences of tetramer positive B cells. Confirmation of the B cell tetramers' binding specificity is essential before using them to identify MSP2-specific B cells in the blood of individuals from malaria endemic areas or individuals participating in vaccine studies.