FLOW CYTOMETRY STUDY OF T CELL RESPONSES TO HIV VACCINES

对HIV-1特异性人类CD4+和CD8+ T细胞免疫的更好测定将促进对HIV-1疫苗的评价。这样的分析应该是可靠的，可重复的，并适用于转运临床标本的高通量分析。我们开发并优化了一种基于流式细胞术的检测方法，用于检测hiv -1感染患者群体中CD4+和CD8+人类T细胞对巨细胞病毒（CMV）的特异性反应。这种“细胞因子流式细胞术”（CFC）检测显示，CD4+ T细胞对巨细胞病毒的反应与巨细胞病毒相关的终末器官疾病的不存在或消退相关。最近，该试验的结果已被证明与使用携带CMV特异性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检测似乎可靠且特异性地检测这种抗原特异性反应，我们也开发了相关的检测方法，能够检测特异性的人类CD4+和CD8+ T细胞对其他艾滋病相关的机会性感染的反应，包括由结核分枝杆菌、禽分枝杆菌复合体、隐球菌和人乳头瘤病毒引起的感染。我们现在建议设计一个类似的CFC检测和定量CD4+和CD8+人类T细胞对HIV-1表位的反应。初步实验表明，有可能检测到针对整个病毒疫苗制剂和针对HIV-1 Gag的确定表位的这种反应。为了开发、优化和标准化CFC检测，并将其与MHC I类/HIV-1肽四聚体（Specific Aim 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CFC检测，我们计划分析暴露于但未感染HIV-1的患者、处于HIV-1疾病进展的不同阶段的患者或HIV-1感染的非进展患者的HIV-1特异性免疫反应（特异性目标2）。最后，为了确定该试验的结果是否有助于HIV-1疫苗的设计和开发，我们已经与几个正在测试HIV-1疫苗的研究小组（包括the Immune Response Corporation、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，我们已经制定了计划，分析HIV-1血清阳性和-血清阴性疫苗接种者的HIV-1特异性T细胞反应（Specific Aim 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).