SINGLE CELL ANALYSIS OF A MULTI-TIER AIDS VACCINE

开发一种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，即一种有效的疫苗必须同时激活免疫反应的体液和细胞手臂，此外，还必须考虑到艾滋病毒分离株之间的巨大差异。这种疫苗的开发和改进关键依赖于模型系统，在模型系统中可以精确地量化特定的B和T细胞反应。我们的初步研究表明，单个HIV包膜首先作为DNA，然后作为重组痘苗病毒，最后作为纯化蛋白(即D-V-P疫苗接种方案)在小鼠中引起强烈的抗体反应。该项目的总体目标是使用小鼠模型来综合评价D-V-P作为以高度免疫原性形式递送多个艾滋病毒信封的载体的作用。我们将应用我们实验室成熟的方法，在单细胞水平上测量包膜特异性B细胞以及CD4+和CD9+T细胞的反应。首先，我们将使用D-V-P方案对单一HIV包膜的小鼠免疫期间的特定细胞反应进行表征。将使用来自HIV-1毒株IIIB和MN的两个截然不同、特征良好的信封。这个项目的第二部分将测试D-V-P方案的有效性，即同时呈现多个信封，以引起更大多样性的B和T细胞反应。在本系列的第一个实验中，IIIB和MN将被包括在D-V-P方案的每一步中。然后，我们将讨论在接种疫苗的一个或两个步骤中仅注射IIIB或MN的免疫学后果。最后，我们将评估小鼠对基于D-V-P方案的多包膜临床疫苗的反应。这些研究将提供对合理评估和改进多步骤、多媒介疫苗接种策略至关重要的信息。

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.