Dubey Sheri, Clair James, Fu Tong-Ming, Guan Liming, Long Romnie, Mogg Robin, Anderson Kiersten, Collins Kelly B, Gaunt Christine, Fernandez V Rose, Zhu Lan, Kierstead Lisa, Thaler Scott, Gupta Swati B, Straus Walter, Mehrotra Devan, Tobery Timothy W, Casimiro Danilo R, Shiver John W
Department of Vaccine and Biologics Research, Merck Research Laboratories, West Point, PA 19486, USA.
J Acquir Immune Defic Syndr. 2007 May 1;45(1):20-7. doi: 10.1097/QAI.0b013e3180377b5b.
An effective vaccine for HIV is likely to require induction of T-cell-mediated immune responses, and the interferon-gamma (IFNgamma) enzyme-linked immunospot (ELISPOT) assay has become the most commonly used assay for measuring these responses in vaccine trials. We optimized and validated the HIV ELISPOT assay using an empirical method to establish positivity criteria that results in a < or =1% false-positive rate. Using this assay, we detected a broad range of HIV-specific ELISPOT responses to peptide pools of overlapping 20mers, 15mers, or 9mers in study volunteers receiving DNA- or adenovirus vector-based HIV vaccines and in HIV-seropositive donors. We found that 15mers generally had higher response magnitudes than 20mers and lower false-positive rates than 9mers. These studies show that our validated ELISPOT assay using 15mer peptide pools and the positivity criteria of > or =55 spots per 10(6) cells and > or =4-fold over mock (negative control) is a sensitive and specific assay for the detection of HIV vaccine-induced cell-mediated immunity.
一种有效的HIV疫苗可能需要诱导T细胞介导的免疫反应，而干扰素-γ（IFNγ）酶联免疫斑点（ELISPOT）检测已成为疫苗试验中测量这些反应最常用的检测方法。我们采用经验方法优化并验证了HIV ELISPOT检测，以建立阳性标准，使假阳性率≤1%。使用该检测方法，我们在接受基于DNA或腺病毒载体的HIV疫苗的研究志愿者以及HIV血清阳性供体中，检测到了对重叠20肽、15肽或9肽肽库的广泛HIV特异性ELISPOT反应。我们发现，15肽通常比20肽具有更高的反应强度，且比9肽具有更低的假阳性率。这些研究表明，我们使用15肽肽库并采用每10^6个细胞≥55个斑点且比模拟（阴性对照）高≥4倍的阳性标准进行验证的ELISPOT检测，是一种检测HIV疫苗诱导的细胞介导免疫的灵敏且特异的检测方法。
