Novel B Cell Reagents

记忆B细胞是指示和维持体液免疫的关键因素。表征 记忆B细胞对SIV的反应在疫苗策略中将是重要的，以诱导中和抗体 回应。在这项研究应用中，我们建议开发专门识别猕猴的试剂 记忆B细胞群。在原理研究的证明中，我们已经制造了单克隆性七鳃鳗VLR 针对人类记忆B细胞的抗体。我们建议开发新的单克隆性VLR 猕猴记忆B细胞的抗体。与哺乳动物抗体不同，无颌海的VLR抗体 七鳃鳗利用富含亮氨酸的重复序列作为基本结构单位。截然不同的蛋白质结构 结合海鳗进化的巨大距离，可以制造出VLR抗体 识别未被哺乳动物免疫球蛋白抗体检测的抗原结构 结构性或耐受性原因。为了产生单抗VLR，我们将免疫海鳗 具有纯化的原代B细胞或B细胞系的幼虫。VLR抗体表达文库将通过Flow进行筛选 与记忆B细胞或亚群特异相互作用的细胞术和VLR单抗 其将被用作亲和试剂来鉴定VLR抗体识别的抗原。VLR 来自这个核心的抗体将被提供给研究小组，以分析记忆B细胞对 接种疫苗或SIV挑战。在一个互补的方法中，我们将产生传统的哺乳动物 抗FCRL4的单抗是一种免疫受体，在人类中由以下亚群表达 粘膜相关记忆B细胞，其对HIV gp120蛋白具有丰富的抗体特异性。一个 由FCRL4胞外区与免疫球蛋白Fc区融合而成的融合蛋白将作为 免疫原和特异性单抗将通过流式细胞仪进行鉴定。从长远来看 这些研究的目标是产生新的研究试剂，以促进对记忆B细胞的分析 应对SIV挑战或接种疫苗。

Memory B cells are key contributors to the indiction and maintenance of humoral immunity. Characterization of the memory B cells response to SIV will be important in an vaccine strategy to elicit a neutralizing antibody response. In this research application we propose to develop reagents to specifically identify macaque memory B cell populations. In proof of principle studies we have produced monoclonal lamprey VLR antibodies that are specific for memory B cells in humans. We propose to develop novel monoclonal VLR antibodies to macaque memory B cells. Unlike mammalian antibodies, the VLR antibodies of the jawless sea lampreys utilize leucine-rich repeats as basic structural units. The radically different protein architecture combined with the large evolutionary distance ofthe sea lamprey allows VLR antibodies to be made that recognize antigenic structures which are not detetcted by mammalian immunoglobulin-based antibodies for structural or tolerogenic reasons. To generate monoclonal VLR antibodies, we will immunize sea lamprey larvae with purified primary B cells or B cell lines. VLR antibody expression libraries will be screened by flow cytometry and monoclonal VLR antibodies that specifically interact with memory B cells or subpopulations thereof will be used as affinity reagents to identify the antigen recognized by the VLR antibody. The VLR antibodies from this core will be provided to research groups to analyze the memory B cell responses to vaccination or SIV challenge. In a complementary approach, we will generate conventional mammalian monoclonal antibodies to FCRL4, an immunoreceptor that In humans is expressed by a subpopulation of mucosa associated memory B cells with enrichment in antibody specificity for the HIV gp120 protein. A fusion protein consisting ofthe extracellular domain of FCRL4 fused to the Fc-domain of IgG will serve as the immunogen and the specific monoclonal antibodies will be identified by flow cytometry. The long term goal of these studies is to generate novel reseach reagents that will facilitate the analysis of memory B cells responding to SIV challenge or vaccination.