Transgenic mouse for modifying antiviral mAb specificity

描述(申请人提供)：本项目的目标是为诱导和分离具有对SIV天然包膜尖峰的亲和力的小鼠单克隆抗体而丰富。这些实验将利用免疫耐受现象，将免疫小鼠的免疫反应集中在SIVmac239和HIV-1天然包膜尖峰的特定区域或结构上。该项目的第一个具体目标是通过转基因表达单体包膜蛋白来创造对典型的非中和决定簇具有免疫耐受性的小鼠品系。在可诱导启动子的控制下，将建立表达SIV包膜序列的转基因株系。在包含第二个特定目的的实验中，对病毒包膜蛋白具有免疫耐受性的转基因动物将被用来将抗体反应集中在疫苗中存在的表位上，而不是转基因蛋白中。为此，将用化学灭活的病毒粒子免疫动物，以便以其天然的形式呈现包膜(众所周知，针对HIV和SIV的广泛中和抗体需要与天然的复合体有亲和力)。第三个具体目的是以全病毒粒子结合和中和试验为主要筛选方法分离中和单抗。要使用的灭活病毒粒子免疫原是1)在内源序列的上下文中携带异源表位的病毒粒子，以及2)具有与内源表达的包膜相同的包膜的病毒粒子。在第一种情况下，HIV表位呈现在天然的SIV包膜复合体上；因为转基因宿主对SIV表位具有耐受性，抗体反应将“集中”在异源的HIV决定簇上。在第二种情况下，要检验的假设是天然的包膜复合体包含单体包膜蛋白中不存在的构象诱导的表位。

DESCRIPTION (provided by applicant): The goal of this project is to enrich for the induction and isolation of murine monoclonal antibodies with affinity for native envelope spikes of SIV. These experiments will take advantage of the phenomenon of immune tolerance to "focus" the immune response of immunized mice on particular regions or structures of the native envelope spike of SIVmac239 and HIV-1. The first specific aim of this project is to create strains of mice that are immunologically tolerant to typical, non-neutralizing determinants through transgenic expression of monomeric envelope protein. Transgenic lines will be created expressing SIV envelope sequences under the control of an inducible promoter. In experiments comprising the second specific aim, transgenic animals that are immunologically tolerant to viral envelope proteins will be used to "focus" the antibody response on epitopes present in the inoculum but not in the transgenic protein. To do this, animals will be immunized with chemically inactivated virion particles, in order to present the envelope in its native configuration (it is well established that broadly neutralizing antibodies against HIV and SIV need to have affinity for the native complex). The third specific aim is to isolate neutralizing mAbs using whole virion binding and neutralization assays as the primary screens. Inactivated virion immunogens to be used are 1) virions bearing heterologous epitopes in the context of the endogenous sequence and 2) virions with envelope identical to the endogenously expressed envelope. In the first case, HIV epitopes are presented on native SIV envelope complexes; because the transgenic host will be tolerant to SIV epitopes, the antibody response will be "focused" on the heterologous, HIV determinant. In the second case, the hypothesis to be tested is that native envelope complexes contain conformationally induced epitopes that are not present in the monomeric envelope protein.