在1型人类免疫缺陷病毒（Human immunodeficiency virus type 1，HIV-1）疫苗研发中大部分的策略均以失败告终，而一项以猕猴巨细胞病毒68-1亚型（68-1 Rhesus cytomegalovirus，68-1 RhCMV）为载体的猴免疫缺陷病毒（Simian immunodeficiency virus，SIV）疫苗设计，却为恒河猴应对SIV感染提供了强大的预防保护。对该疫苗保护机制的研究显示MHC-E介导的非经典CD8+ T细胞反应发挥了关键作用；此外，对HIV-1感染者中HLA-E介导的CD8+ T细胞反应的研究再次验证了HLA-E在人体中的重要免疫功能。与此同时靶向pMHC的类T细胞受体（T cell receptor, TCR）抗体研究显示，这类抗体能够识别MHC呈递的胞内抗原，在肿瘤治疗中展现出强大的治疗效果。目前类TCR抗体在HIV-1研究中多靶向呈递HIV-1抗原肽的经典HLA-Ia分子，靶向HLA-E限制的HIV-1 T细胞表位的类TCR抗体研究仍是一片空白。本研究挑选HLA-E*0103分子，展示在SIV疫苗研究中显示出保护作用的表位肽SIV RL9，以及其在HIV-1 gag基因上同源的抗原肽HIV RL9，采用原核细胞表达单链融合蛋白（single chain trimer, SCT）形式的肽-MHC复合物。基于结构信息对SCT形式进行理性设计，成功构建稳定且具有功能的SCT-HIV RL9-E1及SCT-SIV RL9-E1融合蛋白作为免疫原，进行小鼠免疫接种。随后我们建立SCT-pHLA稳转细胞系，并以此为基础搭建小鼠单克隆抗体高通量流式筛选平台，成功获得4类结合pHLA-E复合物的抗体，其中11株抗体特异性识别呈递HIV RL9抗原肽的HLA-E复合物，展现出类TCR抗体的性质。设计实验对抗体表位进行初步探索，并基于人工智能进行结构预测，发现以3F3为代表的类TCR抗体，对于HIV RL9抗原肽序列及HLA-E分子均有相互作用；而抗体1D3则在相互作用位点预测中显示并不和HIV RL9抗原肽相互作用，提示呈递不同抗原肽的HLA-E分子结构上的差异，以及识别这类结构差异的特殊类TCR抗体的存在。基于靶向HLA-E限制的HIV-1 T细胞表位的类TCR抗体，开发工程化改造T细胞，在体外实验中展现出对HIV-1感染细胞的杀伤能力，为基于这类抗体研发新型的抗体药物及细胞疗法，用于HIV-1感染的防控提供了重要理论和技术支撑。
Most vaccine strategies failed to provide effective protection against HIV-1 infection. Recently, one SIV vaccine based on RhCMV 68-1 strain, provided strong and unusual protection against SIV infection in rhesus macaques, wherein the vaccine induced quite an unconventional CD8+ T cell response restricted by MHC-E. This unconventional activation is believed to confer the vaccine its unique protective effect. Similary, HLA-E restricted HIV-1–specific CD8+ T cell response also confers protection against HIV infection in humans. TCR-like antibodies, a type of antibody that targets pMHC has shown high efficacy in the treatment of cancer. Likewise, TCR-like antibodies have been used to target peptide- HLA-Ia complex as a promising prospect for the treatment for HIV. However, there has yet to be research into the application of TCR-like antibodies in HIV targeting HLA-E presenting HIV peptides.However, there has yet to be research into the application of TCR-like antibodies were mainly designed to target the complexes of peptide and classical HLA-Ia molecule, none of them focused on HLA-E-restricted HIV-1 CD8+ T cell epitopes.In this study, the HLA-E*0103 molecule was selected to display peptide SIV RL9, to which all rhesus macaques immunized with the RhCMV 68-1 vaccine mounted responses, and its close homolog, HIV RL9 from HIV-1 gag p24 protein. We chose prokaryotic expression system to produce inclusion bodies and refold to harvest soluble pHLA complex in the form of single chain trimer (SCT). Finally, the stable and functional SCT-HIV RL9-E1 and SCT-SIV RL9-E1 fusion proteins were successfully optimized and expressed, then used in mice immunization. Then SCT-pHLA stably transfected cell lines were developed, and a high-throughput flow cytometry technology for mouse monoclonal antibody screening was established based on the cell lines. We obtained 4 classes of antibodies that bind to the pHLA-E complex, of which 11 antibodies specifically recognize the HLA-E presenting HIV RL9 antigen peptide, exhibiting the properties of TCR-like antibodies. In the preliminary exploration of their binding sites and the structure prediction model based on artificial intelligence, we found that the antibody 3F3 interacted with both HIV RL9 antigen peptide sequence and HLA-E molecule, consistent with its specificity. The interaction sites of 1D3, another TCR-like antibody against HLA-E restricted HIV RL9 peptide, oddly did not include the HIV RL9 antigen peptide region, suggesting its recognization of structure differences of HLA-E when presenting different antigen peptides. Based on these TCR-like antibodies targeting HLA-E-restricted HIV-1 T cell epitopes, we have made simple exploration of its detection function to HIV-1 infected cells, and its application in HIV-1 cell therapy, suggesting its brilliant prospect to develop as new antibody drugs and cell therapy to fight with HIV-1 infection.
