Bet Anne, Maze Emmanuel Atangana, Bansal Anju, Sterrett Sarah, Gross Antoine, Graff-Dubois Stéphanie, Samri Assia, Guihot Amélie, Katlama Christine, Theodorou Ioannis, Mesnard Jean-Michel, Moris Arnaud, Goepfert Paul A, Cardinaud Sylvain
Retrovirology. 2015 Feb 10;12:15. doi: 10.1186/s12977-015-0135-y.
CD8+ T cells recognize HIV-1 epitopes translated from a gene's primary reading frame (F1) and any one of its five alternative reading frames (ARFs) in the forward (F2, F3) or reverse (R1-3) directions. The 3' end of HIV-1's proviral coding strand contains a conserved sequence that is directly overlapping but antiparallel to the env gene (ARF R2) and encodes for a putative antisense HIV-1 protein called ASP. ASP expression has been demonstrated in vitro using HIV-transfected cell lines or infected cells. Although antibodies to ASP were previously detected in patient sera, T cell recognition of ASP-derived epitopes has not been evaluated. We therefore investigated the ex vivo and in vitro induction of ASP-specific T cell responses as a measure of immune recognition and protein expression during HIV-1 infection.
A panel of overlapping peptides was initially designed from the full-length ASP sequence to perform a global assessment of T cell responses. Recognition of ASP-derived antigens was evaluated in an IFN-γELISpot assay using PBMCs from HIV-1 seropositive and seronegative individuals. Eight of 25 patients had positive responses to ASP antigens and none of the seronegative donors responded. As a complimentary approach, a second set of antigens was designed using HLA-I binding motifs and affinities. Two ASP-derived peptides with high predicted binding affinities for HLA-A02 (ASP-YL9) and HLA-B07 (ASP-TL10) were tested using PBMCs from HIV-1 seropositive and seronegative individuals who expressed the matching HLA-I-restricting allele. We found that HLA-I-restricted ASP peptides were only recognized by CD8+ T cells from patients with the relevant HLA-I and did not induce responses in any of the seronegative donors or patients who do not express the restrictive HLA alleles. Further, ASP-YL9-specific CD8+ T cells had functional profiles that were similar to a previously described HLA-A02-restricted epitope (Gag-SL9). Specific recognition of ASP-YL9 by CD8+ T cells was also demonstrated by tetramer staining using cells from an HLA-A02 HIV-infected patient.
Our results provide the first description of CD8+ T cell-mediated immune responses to ASP in HIV-1-infected patients, demonstrating that ASP is expressed during infection. Our identification of epitopes within ASP has implications for designing HIV vaccines.
CD8 + T细胞可识别从基因的主要阅读框（F1）以及其五个可变阅读框（ARF）中任何一个在正向（F2、F3）或反向（R1 - 3）方向上翻译的HIV-1表位。HIV-1前病毒编码链的3'端包含一个保守序列，该序列与env基因（ARF R2）直接重叠但方向相反，并编码一种假定的反义HIV-1蛋白，称为ASP。已使用HIV转染的细胞系或感染细胞在体外证实了ASP的表达。尽管先前在患者血清中检测到了针对ASP的抗体，但尚未评估T细胞对ASP衍生表位的识别。因此，我们研究了ASP特异性T细胞反应的体外和体内诱导情况，以此作为HIV-1感染期间免疫识别和蛋白表达的一种衡量指标。
最初从全长ASP序列设计了一组重叠肽，以对T细胞反应进行全面评估。使用来自HIV-1血清阳性和血清阴性个体的外周血单核细胞（PBMC），通过IFN-γ酶联免疫斑点试验（ELISpot）评估对ASP衍生抗原的识别。25名患者中有8名对ASP抗原呈阳性反应，而血清阴性供者均无反应。作为一种补充方法，利用HLA-I结合基序和亲和力设计了第二组抗原。使用来自表达匹配的HLA-I限制性等位基因的HIV-1血清阳性和血清阴性个体的PBMC，测试了两种对HLA-A02（ASP-YL9）和HLA-B07（ASP-TL10）具有高预测结合亲和力的ASP衍生肽。我们发现，HLA-I限制性ASP肽仅被具有相关HLA-I的患者的CD8 + T细胞识别，在任何血清阴性供者或不表达限制性HLA等位基因的患者中均未诱导出反应。此外，ASP-YL9特异性CD8 + T细胞具有与先前描述的HLA-A02限制性表位（Gag-SL9）相似的功能特征。使用来自一名HLA-A02 HIV感染患者的细胞进行四聚体染色，也证实了CD8 + T细胞对ASP-YL9的特异性识别。
我们的结果首次描述了HIV-1感染患者中CD8 + T细胞介导的对ASP的免疫反应，表明ASP在感染期间表达。我们对ASP内表位的鉴定对设计HIV疫苗具有重要意义。
