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首页> 外文期刊>Vaccine >Development of an interferon-gamma ELISPOT assay to detect human T cell responses to HSV-2
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Development of an interferon-gamma ELISPOT assay to detect human T cell responses to HSV-2

机译：开发用于检测人T细胞对HSV-2应答的干扰素-γELISPOT测定法

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Background: The need for an HSV-2 vaccine is great considering the increasing prevalence of HSV-2 despite the widespread use of antiviral drugs. Human clinical trials of HSV-2 vaccines that elicit neutralizing antibodies have proven to be only partially effective suggesting that induction of effective T cell responses to HSV-2 is also a critical component to an efficacious vaccine. A sensitive and specific assay to measure HSV-specific T cell responses is a necessary part of vaccine development and thus we undertook the development of an interferon-gamma (IFN-gamma) ELISPOT assay to measure T cell responses to HSV-2.Methods: PBMC from HSV-seronegative (HSVneg) (n = 35), HSV-1-seropositive (HSV-1+/2-) (n = 20) and HSV-2-seropositive (HSV-2+) subjects (n = 26) were screened by IFN-gamma ELISPOT for T cell responses using 34 peptide pools representing 16 HSV-2 proteins including mostly virion and immediate-early (IE) proteins.Results: Overall, 85% of HSV-2+ subjects had a positive response to the HSV-2 peptide pools and on average, HSV-2+ subjects responded to 3 peptide pools (range 1-10). The most frequent responses were to gD-2, UL39, UL46, ICP0, UL49, gB-2, and ICP4. In contrast, only 2 of 35 (6%) HSVneg subjects had detectable T cell responses and in both cases, responses were of low magnitude relative to responses in HSV-2+ subjects and were directed at a single peptide pool. The response rate to the HSV-2 peptide pools in HSV-1+/2- subjects was 40% suggesting that the HSV-2 peptide pools contain a significant number of type-common T cell epitopes. The IFN-gamma ELISPOT assay detected CD4 and CD8 T cells directed at HSV-2 peptides as confirmed by intracellular cytokine staining and flow cytometry.Conclusion: We have developed a quantitative IFN-gamma ELISPOT assay that detects both CD4 and CD8 T cells to HSV-2 peptides. This assay does not require large quantities of PBMC to generate dendritic cells for T cell stimulation, making it an ideal assay for monitoring the immunogenicity of candidate HSV-2 vaccines designed to elicit T cell responses to HSV-2 specific epitopes
机译：背景：尽管抗病毒药物的广泛使用，但考虑到HSV-2的患病率上升，对HSV-2疫苗的需求很大。引发中和抗体的HSV-2疫苗的人类临床试验仅被证明部分有效，这表明诱导对HSV-2的有效T细胞应答也是有效疫苗的关键组成部分。敏感而特异的测定方法来测量HSV特异性T细胞反应是疫苗开发的必要部分，因此我们进行了干扰素-γ（IFN-γ）ELISPOT测定法的开发，以测定对HSV-2的T细胞反应。来自HSV血清阴性（HSVneg）（n = 35），HSV-1-血清阳性（HSV-1 + / 2-）（n = 20）和HSV-2-血清阳性（HSV-2 +）受试者的PBMC（n = 26通过IFN-γELISPOT筛选T细胞反应，使用34个代表16种HSV-2蛋白的肽库（包括病毒颗粒和即刻早期（IE）蛋白）对T细胞反应进行了筛选。结果：总体而言，HSV-2 +受试者中有85％的阳性反应平均而言，HSV-2 +受试者对3个肽库有反应（范围1-10）。最常见的响应是对gD-2，UL39，UL46，ICP0，UL49，gB-2和ICP4的响应。相比之下，在35名（6％）HSVneg受试者中，只有2名具有可检测的T细胞应答，并且在两种情况下，应答均相对于HSV-2 +受试者的应答为低幅度，并且针对单个肽库。在HSV-1 + / 2-受试者中，对HSV-2肽库的响应率为40％，这表明HSV-2肽库包含大量的典型T细胞表位。经细胞内细胞因子染色和流式细胞术证实，IFN-γELISPOT检测可检测到针对HSV-2肽的CD4和CD8 T细胞。结论：我们开发了定量IFN-γELISPOT检测方法，可检测CD4和CD8 T细胞对HSV的表达-2肽。该测定不需要大量的PBMC即可产生树突状细胞来刺激T细胞，使其成为监测候选HSV-2疫苗免疫原性的理想测定方法，该疫苗旨在引起T细胞对HSV-2特异性表位的反应

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