Monocional Antibody and Protein Core

本HIVRAD应用程序中用于开发HIV候选疫苗的方法基于 反向疫苗学策略，其使用具有广泛和有效的人单克隆抗体（mAb）， 抗病毒活性，以鉴定病毒包膜（Env）上的同源表位，并将这些结构移植到 支架蛋白用作免疫原。然后将这些表位-支架免疫原在体内用于 引发具有与亲本mAb所显示的抗病毒活性相似的抗病毒活性的Ab。 核心B的拟议工作分为两个具体目标： 目标1.项目1和2中产生的人mAb的生产。为此，我们将扩大 基于对V2或V2/V3的特异性，生产项目1和2中生成的人mAb 第四中和表位（QNE），以及它们介导多种抗病毒功能的能力。我们将 产生足够数量的mAb，以进行广泛的免疫化学、功能和晶体学研究， 根据项目1和2的工作范围要求进行研究。这些mAb将由293个细胞产生 用含有所选mAb的重链和相应轻链基因的质粒转染， 将从抗原特异性B细胞扩增。 目标2.项目1、2和核心C的蛋白质生产。为此，我们将生产：a）生物素标记的 用于染色待选择用于mAb的抗原特异性B细胞的重组表位-支架蛋白 在项目1和2中通过分子方法的开发，和B）用作 在项目1和2中作为抗原，在动物研究核心（核心C）中作为免疫原。这些表位支架 蛋白质将主要在293个人胚胎肾细胞中产生，或者在其它哺乳动物细胞中产生。 或昆虫细胞。在真核细胞中的生产将支持糖基化，这是保存细胞所需的。 V2和V2/V3 QNE的构象表位，而在大肠杆菌中的生产可能有助于研究 线性表位 我们在开发人源抗HIV-1单克隆抗体和生产重组 支架蛋白质确保了这些试剂的高质量，这对于成功的 完成拟议的HIVRAD工作范围。

The approach used in this HIVRAD application for the development of HIV candidate vaccines is based on a reverse vaccinology strategy which uses human monoclonal antibodies (mAbs) with broad and potent anti-viral activity to identify cognate epitopes on the virus envelope (Env) and engraft these structures into scaffold proteins to be used as immunogens. These epitope-scaffold immunogens are then used in vivo to elicit Abs with anti-viral activities similar to those displayed by the parental mAbs. The proposed work for Core B is divided into two specific aims: Aim 1. Production of human mAbs generated in Projects 1 and 2. In this aim, we will scale up production of human mAbs generated in Projects 1 and 2 on the basis of their specificity for V2 or for V2/V3 quaternary neutralizing epitopes (QNEs), and their ability to mediate a variety of anti-viral functions. We will produce sufficient quantities of mAbs to perform extensive immunochemical, functional, and crystallographic studies as required by the work scopes of Projects 1 and 2. These mAbs will be produced by 293 cells transfected with plasmids containing the heavy and corresponding light chain genes of selected mAbs which will have been amplified from antigen-specific B cells. Aim 2. Protein production for Project 1, 2 and Core C. In this aim, we will produce: a) biotin-tagged recombinant epitope-scaffold proteins for staining antigen-specific B cells to be selected for mAb development by molecular methods in Projects 1 and 2, and b) epitope-scaffold proteins to be used as antigens in Projects 1 and 2 and as immunogens in Animal Studies Core (Core C). These epitope-scaffold proteins will be produced primarily in 293 human embryonic kidney cells, or alternatively in other mammalian or insect cells. Production in eukaryotic cells will support glycosylation which is needed to preserve the conformational epitopes of V2 and the V2/V3 QNEs, while production in ¿ coli may be useful for the study of linear epitopes. Our extensive experience in the development of human anti-HIV-1 mAbs and production of recombinant scaffolded proteins ensures the high quality of these reagents which are essential for the successful completion ofthe proposed HIVRAD work scope.