Monocional Antibody and Protein Core

在HIVRAD应用程序中用于开发HIV候选疫苗的方法基于 一种利用广泛有效的人源单抗的反向疫苗策略 识别病毒包膜(Env)上的同源表位并将这些结构植入 用作免疫原的支架蛋白。这些表位-支架免疫原然后在体内用于 诱导具有与亲本单抗相似的抗病毒活性的单抗。 核心B的拟议工作分为两个具体目标： 目标1.在项目1和2中产生的人单抗的生产。在这个目标中，我们将扩大规模 根据对V2或V2/V3的特异性，生产项目1和2中产生的人单抗 第四代中和表位(QNE)，以及它们介导各种抗病毒功能的能力。我们会 产生足够数量的单抗以进行广泛的免疫化学、功能和结晶学 根据项目1和2工作范围的要求进行研究。这些单抗将由293个细胞产生 用含有所选单抗的重链基因和相应轻链基因的载体 将从抗原特异的B细胞中扩增出来。 目标2.项目1、2和核心C的蛋白质生产。在这个目标中，我们将生产：a)生物素标记 用于单抗选择的抗原特异性B细胞染色的重组表位-支架蛋白 项目1和2中的分子方法开发，以及b)表位-支架蛋白用作 项目1和2中的抗原和动物研究核心(核心C)中的免疫原。这些表位-支架 蛋白质将主要在293个人类胚胎肾脏细胞中产生，或者在其他哺乳动物中产生 或者昆虫细胞。在真核细胞中的生产将支持糖基化，这是保存 V2和V2/V3 QNEs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。 我们在人源性抗HIV-1单抗研制和重组生产方面的丰富经验 支架蛋白确保了这些试剂的高质量，这些试剂对成功是必不可少的 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.