重组腺相关病毒载体介导双特异性广谱中和抗体抗人类免疫缺陷病毒活性研究

Prevention and treatment of acquired immune deficiency syndrome (AIDS) has always been the hotspots and difficulties concerned by China and all over the world. Recently, the most significant progress in the field was found that broad-neutralizing antibodies (BrNAb) isolated from those HIV infected individuals naturally could be applied to reduce the copies of virus in AIDS patients significantly. Recombinant adeno-associated virus (rAAV) is an ideal carrier with good safety and low immunogenicity. Furthermore, rAAV carrying exogenous gene can gives a continuous expression of gene for a long duration. In our previous study, BrNAb genes were constructed into the reverse terminal repeat sequences of rAAV, and the neutralization activities of the expressed antibodies were carried out in vitro. The results indicated that broad spectrum neutralization and continuous expression could be observed. According to the high mutation rates and immune escape mechanism of HIV, the structures of BrNAbs will be optimized by computational simulation in this project. The major goals are (i) constructing rAAVs with carrying bispecific antibody gene which own the abilities for broad spectrum neutralizating and T-cell activation, so that the expressed bispecific antibodies can both recognize HIV protein and CD3 receptor; (ii) investigating binding abilities and broad spectrum neutralizating activities of the expressed antibodies in vitro against HIV-1 pseudovirions; (iii) assessing the abilities for continuous expression and T-cell activation in Balb/c mice. This project might also provide theoretical and experimental basis for clinical application of bispecific antibodies.

艾滋病（AIDS）的预防与治疗一直是我国及全世界所关注的热点和难点。近期该领域的一个重大进展是利用从自然感染者体内分离出的HIV-1广谱中和抗体（BrNAb）对HIV进行治疗，该方法能够显著减少HIV患者体内病毒拷贝数。我们前期研究利用重组腺相关病毒（rAAV）安全性好、免疫原性低、在体内表达时间长等特点，将BrNAb构建于rAAV反向末端重复序列中，体外中和结果表明rAAV所表达抗体具有广谱中和活性及持续性。基于HIV高突变性及其免疫逃逸相关机制，本项目拟对已知BrNAbs进行结构优化，在rAAVs载体上构建既具广谱中和活性又可激活辅助T细胞的双特异性抗体基因，以使表达抗体能够分别识别HIV蛋白和CD3受体；评价抗体对HIV-1假病毒的结合及广谱中和活性；Balb/c小鼠体内评价抗体的持续表达能力及激活T细胞活性。该研究为双特异性BrNAbs在预防与治疗HIV的临床应用提供理论实验依据。

艾滋病（AIDS）的预防与治疗一直是我国及全世界所关注的热点和难点。近期该领域的一个重大进展是利用从自然感染者体内分离出的HIV-1广谱中和抗体（bNAbs）对HIV-1进行治疗，该方法能够显著减少HIV-1患者体内病毒拷贝数。我们前期研究利用重组腺相关病毒8型（rAAV8）安全性好、免疫原性低、在体内表达时间长等特点，将bNAb构建于rAAV8反向末端重复序列中，体外中和结果表明rAAV8所表达抗体具有广谱中和活性及持续性。为了提高 bNAbs 的广谱性，我们将 bNAbs（PG9、PGT123或NIH45-46）的单链可变抗体片段（scFv）与全长ibalizumab（iMab）以scFv-IgG形式串联构建双特异性bNAb（BibNAb）。此外，我们描述了由rAAV8介导的BibNAb基因递送的可行性，仅通过单次注射即可实现BibNAb的长期表达，无需持续的被动注射。结果表明，BibNAb具有针对20个HIV-1假病毒的中和活性。单次注射rAAV8载体后，BibNAb在血清中的表达和中和活性持续至少24周。目前，关于使用rAAV8载体对抗HIV-1的BibNAb基因递送的研究很少。使用rAAV8载体介导BibNAb的基因递送可能有望应用于HIV-1的被动免疫。该研究为BibNAbs在预防与治疗HIV-1的临床应用提供理论实验依据。