HIV-1交叉抗体10E8/iMab中和病毒的作用机制

While the search for an efficacious HIV-1 vaccine remains elusive, the emergence of a new generation of virus-neutralizing monoclonal antibodies (mAbs) has re-ignited the field of passive immunization as an alternative strategy for HIV-1 prevention. However, the plasticity of HIV-1 demands additional improvements to these mAbs in order to better ensure their clinical utility. We will explore characterization and exquisite neutralizing mechanism of HIV-1 crossmab 10E8/iMab. We believe that focusing on neutralizing crossmab will provide better understanding for novel mechanism of action of bispecific antibody. In our previous study, 10E8/iMab possesses exquisite potency and breadth against HIV-1 while retaining normal antibody structure that neutralized a majority of a large, multi-clade panel of pseudoviruses( N=118) with mean IC50 of 0.001 μg/mL, as well as neutralization breadth (as assessed by >50% neutralization) of 100%. These antibodies also potently neutralized 99% of the viruses in a second panel of 200 HIV-1 isolates belonging to clade C, the dominant subtype accounting for ~50% of new infections worldwide. Here, we sought to understand 10E8/iMab antiviral activity, especially first-pass clearance, activity against productive infection, activity against virus/ binding virus and cell to cell transmission by different neutralizing assay. Moreover, we would figure out the relationships of antiviral activity and antibody affinity, paratope and epitope and inter-dependency action, antibody configuration. Finally, we would carry out the efficacy test in rhesus macaques by passive admonition prior to SHIVs challenge. Our results of bispecific antibodies could help us to better understand the novel inter-dependency of dual mechanism of action enables 10E8/iMab to potently inhibit HIV-1 entry. These results could generate potential useful additions to the armamentarium for HIV-1/AIDS therapy and prevention.

目前HIV-1疫苗研究仍在深入研究，被动免疫中和抗体作为替代成为了研究热点，鉴于HIV-1的高度适应性，仍需优化现有抗体才能满足临床应用。双特异性抗体作为新型抗体的代表，具有极强抗病毒活性，故对双特异性交叉抗体的理解可为揭示新型抗体的抗病毒机制提供理论基础。我们前期研究发现，具有天然构象的交叉抗体10E8/iMab能以约0.002μg/mL的IC50中和HIV-1特征病毒库所有毒株（N =118），广谱度达到100%，还能中和全球感染率50%以上、属C亚型的200个分离株。本研究从抗体中和病毒特征入手，利用不同类型中和实验、亲和力检测、构象分析和动物攻毒保护，探索①抗体首轮清除和抑制生产性感染的效率，抑制不同阶段病毒和不同病毒传播方式的效力；②靶向表位对效力的影响，亲和力及差异和构象与效力的关系；③抗体对SHIVs攻毒的恒河猴的保护性。研究成果将为新的抗病毒制剂的设计提供新的手段。

尽管中和抗体效力强大，但其对已建立的HIV-1感染效力有限。接受单抗治疗时，由于体内存在/出现病毒逃逸型突变株，几乎所有患者体内都会出现病毒载量的反弹。以双特异性抗体为代表的高效广谱新型中和抗体可能通过多重靶位、效力叠加和多重抗病毒效应等新颖的设计在理论上克服的局限。.我们前期发现，10E8/iMab交叉抗体能以约0.002μg/ML的IC50中和目前公认的HIV-1 特征病毒库达99%。本研究中，我们围绕10E8/iMab的中和模式和作用机制开展深入研究，首先明确了10E8/iMab的活性增强来自于10E8功能区，iMab功能区靶向CD4分子以非接触性中和干扰病毒感染细胞，锚定10E8功能区在病毒进入细胞的路径上；其次，该抗体在针对GFP毒株的首轮清除率检测中体现极高的有效性；同时，其可以对病毒的胞间传播发生完全的抑制效应（N=5），MPI几乎达到了100%，且能以约0.06μg /ML的IC50进行吸附后中和的病毒抑制被检测的TF毒株（N=6）。在以天然构象为基本形态的两个功能区靶向不同作用分子：CD4和MPER的亲和力检测中，我们发现单个抗体分子左右功能区的亲和力的差异，并未影响其抗病毒的效应，提示交叉抗体的稳定构象能够克服这一差异，从而发挥抗病毒作用。该抗体以30mg/Kg进行被动免疫时候，可发挥对SHIV攻击恒河猴的保护作用，体现了其体内外效力的一致性，提示其可作为单抗免疫防治策略的候选抗体，并为后续新型抗体的设计提供重要的研究思路。.课题针对APRIL分子在HIV-1感染和抗病毒治疗关联效应分析和机制研，发现APRIL的血浆水平与HIV-1疾病进展标志物和免疫活性为负相关，与与功能性CD8+T细胞和NK细胞计数呈百分率正相关，提示APRIL可能会介导一种保护性反应，为后续抗体的抗病毒免疫治疗，提供了一个有效的诊疗指标和分子标记，也为新型治疗手段的途径和策略提供了潜在的参考。

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