Transgenic mouse for modifying antiviral mAb specificity

描述（由申请方提供）：本项目的目的是富集对SIV天然包膜刺突具有亲和力的鼠单克隆抗体的诱导和分离。这些实验将利用免疫耐受现象，将免疫小鼠的免疫应答“集中”在SIVmac 239和HIV-1的天然包膜刺突的特定区域或结构上。该项目的第一个具体目标是通过单体包膜蛋白的转基因表达来创建对典型的非中和决定簇具有免疫耐受性的小鼠品系。将产生在诱导型启动子控制下表达SIV包膜序列的转基因系。在包含第二个特定目的的实验中，对病毒包膜蛋白免疫耐受的转基因动物将用于将抗体应答“集中”在接种物中存在的表位上，但不在转基因蛋白中。为了做到这一点，将用化学灭活的病毒粒子颗粒免疫动物，以使包膜以其天然构型呈现（已经充分确定，针对HIV和SIV的广泛中和抗体需要对天然复合物具有亲和力）。第三个具体目标是使用全病毒体结合和中和测定作为主要筛选来分离中和mAb。待使用的灭活病毒粒子免疫原是1）在内源序列的背景下携带异源表位的病毒粒子和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表位被呈递在天然SIV包膜复合物上;因为转基因宿主将对SIV表位耐受，所以抗体应答将“集中”于异源HIV决定簇。在第二种情况下，要测试的假设是，天然包膜复合物含有构象诱导的表位，不存在于单体包膜蛋白。

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.