T CELL RECEPTOR REPERTOIRE CHANGES DURING VACCINATION

描述(摘自申请者的摘要)：细胞免疫反应 由候选HIV-1疫苗诱导的病毒以难以检测和 量化。这很可能是由于有限的细胞免疫 体外试验的反应和高度定性的性质 量一量。因为看起来强烈的、多样化的和 成功的HIV-1需要持久的细胞免疫反应 疫苗接种，开发一种更敏感和更定量的方法来 直接测量体内的细胞反应将有助于比较 可选择的疫苗接种策略。为了测量细胞免疫 在体内的反应，研究人员计划对CD4+和CD8+T细胞进行量化 金丝雀痘载体和质粒后受体(TCR)谱系的变化 注射。使用一种新的能够检测TCRβ链转录本的方法 小到单个Vβ亚家族的1%的扩张， 研究人员将立即追踪新的T细胞克隆的出现 在接种疫苗后。CD_4和CD_8的数量、频率和半衰期 因此，接种疫苗后体内诱导的克隆性扩张将是 准确地量化。这项用于细胞分析的新技术 免疫反应是基于TCRβ链转录本的检测 具有新的CDR3(VDJ)区域，并能够量化低 T细胞谱系的频率变化以其他方式无法检测 目前可用的体内替代方法(Vβ亚家族特有 半定量聚合酶链式反应、Vβ特异性单抗流式细胞术 信使核糖核酸分型，或MHC四聚体分析)。看到的T细胞克隆 接种后体内扩张的疫苗将使用组合进行纯化 使用Vβ亚家族特异性单抗进行细胞分选 TCR CDR3序列分析，并将其抗原特异性 由经典的体外试验测定。因此，调查人员将 使用一种新的高分辨率T细胞谱系分析方法来量化 不同接种方式诱导的细胞免疫反应。这个 金丝雀痘载体和DNA诱导T细胞谱系改变的程度 人体内的注射将与仅由 迄今为止有效的慢病毒疫苗接种策略，感染前 减弱的SIV。这一方法的发展可能有助于 未来候选者诱导的细胞免疫反应的评价 疫苗。

DESCRIPTION (Adapted from applicant's abstract): Cellular immune responses induced by candidate HIV-1 vaccines are notoriously difficult to detect and quantitate. This is likely the result of both limited cellular immune responses and the highly qualitative nature of the in vitro assays used to measure them. Since it appears that the induction of strong, diverse, and long lasting cellular immune responses will be required for successful HIV-1 vaccination, development of a more sensitive and quantitative method to directly measure cellular responses in vivo will assist in comparing alternative vaccination strategies. In order to measure cellular immune responses in vivo, the investigators plan to quantitate CD4+ and CD8+ T-cell receptor (TCR) repertoire changes following canarypox vector and plasmid injection. Using a new assay capable of detecting TCR beta chain transcript expansion as small as 1 percent of a single V beta subfamily, the investigators will track the emergence of novel T-cell clones immediately following vaccination. The number, frequency, and half-life of CD4 and CD8 clonal expansions induced in vivo following vaccination therefore will be accurately quantified. This new technique for the analysis of cellular immune responses is based on the detection of TCR beta chain transcripts bearing novel CDR3 (VDJ) regions, and is capable of quantifying low frequency T-cell repertoire changes otherwise undetectable by the alternative in vivo methods presently available (V beta subfamily-specific semi-quantitative PCR, FACS using V beta-specific monoclonal antibodies, TCR mRNA spectratyping, or MHC tetramer analyses). The T-cell clones seen expanding in vivo following vaccination will be purified using a combination of cell sorting using V beta subfamily-specific monoclonal antibodies and TCR CDR3 sequence analysis, and their antigenic specificity will be determined by classical in vitro assays. The investigators therefore will use a new, high resolution, method of T-cell repertoire analysis to quantify cellular immune responses induced by different modes of vaccinations. The extent of T-cell repertoire changes induced by canarypox vectors and DNA injections in humans will be compared with that induced by the only effective lentiviral vaccination strategy to date, pre-infection with attenuated SIV. The development of this methodology may assist in the evaluation of cellular immune responses induced by future candidate vaccines.