Novel B Cell Reagents

记忆B细胞是体液免疫的指示和维持的关键贡献者。表征 记忆B细胞对SIV的应答在引发中和抗体的疫苗策略中将是重要的 反应在这项研究应用中，我们建议开发试剂，专门识别猕猴 记忆B细胞群。在原理研究的证明中，我们已经产生了单克隆七鳃鳗VLR 对人类记忆B细胞具有特异性的抗体。我们建议开发新的单克隆VLR 猕猴记忆B细胞的抗体。与哺乳动物的抗体不同， 七鳃鳗利用富含亮氨酸的重复序列作为基本结构单元。完全不同的蛋白质结构 再加上七鳃鳗进化距离很远， 识别哺乳动物免疫球蛋白基抗体检测不到的抗原结构， 结构或耐受性原因。为了产生单克隆VLR抗体，我们将免疫海七鳃鳗， 具有纯化的原代B细胞或B细胞系的幼虫。将通过流式细胞术筛选VLR抗体表达文库。 与记忆B细胞或亚群特异性相互作用的单克隆VLR抗体 将其用作亲和试剂以鉴定由VLR抗体识别的抗原。的VLR 来自这一核心的抗体将提供给研究小组，以分析记忆B细胞对 疫苗接种或SIV攻毒。在一个补充的方法，我们将产生传统的哺乳动物 针对FCRL 4的单克隆抗体，FCRL 4是一种免疫受体，在人体中由以下细胞亚群表达： 粘膜相关的记忆B细胞，其具有对HIV gp120蛋白的抗体特异性富集。一 由融合至IgG的Fc结构域的FCRL 4的细胞外结构域组成的融合蛋白将用作 免疫原和特异性单克隆抗体将通过流式细胞术鉴定。长期 这些研究的目标是产生新的研究试剂，以促进记忆B细胞的分析 对SIV攻击或疫苗接种作出反应。

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.