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，疟疾仍然是最常见的 当今世界突出的公共卫生挑战。抗体在预防传染性非典型肺炎中起主要作用 由血液期感染引起的疾病。然而，需要在较长时间内反复感染。 诱导保护性体液免疫。目前尚不清楚保护性抗体是否来自短暂的 通过毛囊外反应的浆母细胞，或通过生发中心的长寿命浆细胞。诱导 后者是有效疫苗的首要目标。虽然疟原虫特异性抗体在 临床疟疾患者的血清，这些抗体反应背后的细胞生物学仍然存在 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 弥补我们对疟原虫体液反应理解上的差距。使用血液台 裂殖子表面蛋白2(MSP2)是血液期疟疾疫苗的候选抗原，我们将 产生两个B细胞四聚体，其中一个含有全长重组恶性疟原虫MSP2蛋白和 另一种包括嵌合的PfMSP2/8蛋白。前者可以在体外形成纤维，而后者可以。 不。因此，我们预测嵌合蛋白将提供这样的好处，如随着时间的推移，稳定性增加和 更多数量的MSP2特异性B细胞与更广泛数量的表位结合 PfMSP2比单独由rPfMSP2组成的四聚体要好。在次级目标1中，我们将监测MSP2原纤维形成和 免疫小鼠后测定各B细胞四聚体对抗原特异性B细胞的体外识别能力 与rPfMSP2或rPfMSP2/8结合。然后在子目标2中，我们将确认B细胞结合的特异性 MSP2通过酶联免疫吸附试验产生基于B细胞受体序列的单抗 四聚体阳性B细胞。在使用B细胞四聚体之前，确认它们的结合特异性是至关重要的 鉴定疟疾流行区个体或个体血液中MSP2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.