SINGLE CELL ANALYSIS OF A MULTI-TIER AIDS VACCINE

开发有效的艾滋病毒疫苗可能是生物医学研究界目前面临的最大挑战。一种共识正在形成，即有效的疫苗必须激活免疫反应的体液和细胞两个方面，此外，还必须考虑到艾滋病毒分离株之间的巨大变异性。这种疫苗的开发和改进关键取决于模型系统，其中可以精确定量特异性B和T细胞应答。我们的初步研究表明，当首先作为DNA，然后作为重组牛痘病毒，最后作为纯化蛋白（即，D-V-P疫苗接种方案）。该项目的总体目标是使用小鼠模型来全面评估D-V-P作为以高免疫原性形式递送多个HIV包膜的载体。我们将应用我们实验室中成熟的方法来测量单细胞水平上的乳腺癌特异性B细胞和CD 4+和CD 9 + T细胞应答。最初，我们将使用D-V-P方案用单个HIV包膜免疫小鼠期间的特异性细胞应答表征。将使用来自HIV-1毒株IIIB和MN的两种不同的、充分表征的包膜。该项目的第二部分将测试D-V-P方案在同时呈递多个包膜以引起更大多样性的B和T细胞应答方面的有效性。在该系列的第一个实验中，IIIB和MN将被包括在D-V-P方案的每个步骤中。然后，我们将讨论仅在一个或两个疫苗接种步骤中施用IIIB或MN的免疫学后果。最后，我们将评估小鼠对基于D-V-P方案的拟议多包膜临床疫苗的应答。这些研究将为合理评价和完善多步骤、多载体疫苗接种策略提供必要的信息。

The development of an effective HIV vaccine is perhaps the greatest challenge currently facing the biomedical research community. A consensus is emerging that an effective vaccine must activate both the humoral and cellular arms of the immune response and, in addition, take into account the tremendous variability among HIV isolates. The development and refinement of such a vaccine is crucially dependent on model systems in which specific B and T cell responses can be precisely quantified. Our preliminary studies show that a single HIV envelope elicits strong antibody responses in mice when administered first as DNA, then as recombinant vaccinia virus, and finally as purified protein (i.e., a D-V-P vaccination regimen). The overall aim of this project is to use a mouse model to comprehensively evaluate the D-V-P as a vehicle for the delivery of multiple HIV envelopes in a highly immunogenic form. We will apply methodologies that are well-established in our laboratory to measure envelope-specific B cell and CD4+ and CD9+ T cell responses at the single cell level. Initially, we will characterize specific cellular responses during the immunization of mice with single HIV envelopes using the D-V-P regimen. Two distinct, well characterized envelopes from the HIV-1 strains IIIB and MN will be used. The second part of this project will test the effectiveness of the D-V-P regimen in presenting multiple envelopes simultaneously to elicit a greater diversity of B and T cell responses. In the first experiments in this series, both IIIB and MN will be included in each step of the D-V-P regimen. We will then address the immunological consequences of administering IIIB or MN at only one or two of the vaccination steps. Finally, we will evaluate the response of mice to a proposed multi-envelope clinical vaccine based on the D-V-P regimen. These studies will provide information that is essential for the rational evaluation and refinement of multi-step, multi-vector vaccination strategies.