FLOW CYTOMETRY STUDY OF T CELL RESPONSES TO HIV VACCINES

对HIV-1特异性人类CD4+和CD8+T细胞免疫进行更好的检测将有助于对HIV-1疫苗的评估。这样的分析方法应该是可靠的、可重复性的，并且能够对转运的临床标本进行高通量分析。我们开发和优化了一种基于流式细胞术的检测方法，用于检测HIV-1感染患者队列中人类T细胞对巨细胞病毒(CMV)的特异性CD4+和CD8+反应。这种“细胞因子流式细胞术”(CFC)分析表明，针对CMV的CD4+T细胞反应的存在与CMV相关终末器官疾病的消失或缓解相关。最近，这项检测的结果被证明与使用携带CMV特定表位的主要组织相容性复合体(MHC)I类四聚体获得的结果相关。由于CFC法似乎能够可靠和特异地检测这种抗原特异性反应，我们还开发了相关的检测方法，能够检测人类CD4+和CD8+T细胞对其他艾滋病相关机会性感染的特异性反应，包括由结核分枝杆菌、禽型分枝杆菌复合体、隐球菌和人乳头瘤病毒引起的感染。我们现在建议设计一种类似的氟氯化碳检测方法，用于检测和定量针对HIV-1表位的CD4+和CD8+人类T细胞反应。初步实验表明，有可能检测到针对全病毒疫苗制剂和针对HIV-1 Gag的特定表位的此类反应。为了开发、优化和标准化这种氯氟化碳检测方法，并将其与使用MHC Class I/HIV-1多肽四聚体(特定目标1)获得的结果相关联，我们与领先的流式细胞仪试剂和设备制造商和分销商Becton Dickinson Biosciences建立了合作关系。为了进一步评估这种氯氟化碳分析方法，我们计划分析接触过HIV-1但没有感染HIV-1的患者、处于HIV-1疾病进展的不同阶段的患者或HIV-1感染的无进展患者(特定目标2)中的HIV-1特异性免疫反应。最后，为了确定这一检测结果是否有助于HIV-1疫苗的设计和开发，我们与几个正在测试HIV-1疫苗的研究小组(包括免疫反应公司、CHIRON、VaxGen和宾夕法尼亚大学David Weiner博士的实验室)建立了合作关系，并计划分析HIV-1血清阳性和血清阴性疫苗受试者中的HIV-1特异性T细胞反应(特定目标3)。

The evaluation of HIV-1 vaccines will be facilitated by the availability of better assays for HIV-1-specific human CD4+ and CD8+ T cell immunity. Such assays should be robust, reproducible, and amenable to high throughput analysis of trans-shipped clinical specimens. We have developed and optimized a flow cytometry-based assay to detect specific CD4+ and CD8+ human T cell responses to cytomegalovirus (CMV) in cohorts of HIV-1-infected patients. This "cytokine flow cytometry" (CFC) assay has shown that the presence of CD4+ T cell responses against CMV is correlated with the absence or resolution of CMV-associated end organ disease. More recently, results from this assay have been shown to correlate with results obtained using major histocompatibility complex (MHC) Class I tetramers bearing specific epitopes of CMV. Since the CFC assay appears to reliably and specifically detect such antigen-specific responses, we have also developed related assays capable of detecting specific human CD4+ and CD8+ T cell responses to other AIDS-related opportunistic infections, including those caused by Mycobacterium tuberculosis, the Mycobacterium avium complex, cryptococcus, and human papilloma virus. We now propose to devise a similar CFC assay for the detection and quantitation of CD4+ and CD8+ human T cell responses against epitopes of HIV-1. Preliminary experiments indicate that it is possible to detect such responses against a whole virus vaccine preparation and against defined epitopes of HIV-1 Gag. To develop, optimize, and standardize this CFC assay, and to correlate it with results obtained using MHC Class I/HIV-1 peptide tetramers (Specific Aim 1), we have formed a collaboration with Becton Dickinson Biosciences, a leading flow cytometry reagent and equipment manufacturer and distributor. To further evaluate this CFC assay, we have made plans to analyze HIV-1-specific immune responses in patients who have been exposed to but not infected by HIV-1, who are in varying stages of HIV-1 disease progression, or who are HIV-1-infected nonprogressors (Specific Aim 2). Finally, to determine whether the results from this assay may facilitate the design and development of HIV-1 vaccines, we have established collaborations with several research groups that are testing HIV-1 vaccines (including The Immune Response Corporation, Chiron, VaxGen, and the laboratory of Dr. David Weiner at University of Pennsylvania) and we have made plans to analyze HIV-1-specific T cell responses in both HIV- 1-seropositive and -seronegative vaccinees (Specific Aim 3).