Rapid Generation of Human-Like Neutralizing Monoclonal Antibodies in Urgent Preparedness for Influenza Pandemics and Virulent Infectious Diseases

BackgroundThe outbreaks of emerging infectious diseases caused by pathogens such as SARS coronavirus, H5N1, H1N1, and recently H7N9 influenza viruses, have been associated with significant mortality and morbidity in humans. Neutralizing antibodies from individuals who have recovered from an infection confer therapeutic protection to others infected with the same pathogen. However, survivors may not always be available for providing plasma or for the cloning of monoclonal antibodies (mAbs). Methodology/Principal FindingsThe genome and the immunoglobulin genes in rhesus macaques and humans are highly homologous; therefore, we investigated whether neutralizing mAbs that are highly homologous to those of humans (human-like) could be generated. Using the H5N1 influenza virus as a model, we first immunized rhesus macaques with recombinant adenoviruses carrying a synthetic gene encoding hemagglutinin (HA). Following screening an antibody phage display library derived from the B cells of immunized monkeys, we cloned selected macaque immunoglobulin heavy chain and light chain variable regions into the human IgG constant region, which generated human-macaque chimeric mAbs exhibiting over 97% homology to human antibodies. Selected mAbs demonstrated potent neutralizing activities against three clades (0, 1, 2) of the H5N1 influenza viruses. The in vivo protection experiments demonstrated that the mAbs effectively protected the mice even when administered up to 3 days after infection with H5N1 influenza virus. In particular, mAb 4E6 demonstrated sub-picomolar binding affinity to HA and superior in vivo protection efficacy without the loss of body weight and obvious lung damage. The analysis of the 4E6 escape mutants demonstrated that the 4E6 antibody bound to a conserved epitope region containing two amino acids on the globular head of HA. Conclusions/SignificanceOur study demonstrated the generation of neutralizing mAbs for potential application in humans in urgent preparedness against outbreaks of new influenza infections or other virulent infectious diseases.研究背景：由严重急性呼吸综合征冠状病毒（SARS coronavirus）、H5N1、H1N1以及近期的H7N9流感病毒等病原体引发的新发传染病暴发，已造成人类出现大量死亡与发病病例。感染康复者体内的中和抗体可对其他感染同一病原体的个体提供治疗性保护。然而，康复者往往难以提供血浆或用于单克隆抗体（monoclonal antibodies, mAbs）的克隆制备。 研究方法与主要结果：恒河猴与人类的基因组及免疫球蛋白基因具有高度同源性，因此本研究探究是否可制备出与人类抗体高度同源的中和性单克隆抗体（即类人中和单抗）。本研究以H5N1流感病毒为模型，首先通过携带编码血凝素（hemagglutinin, HA）的合成基因的重组腺病毒免疫恒河猴。随后，从免疫猴的B细胞构建抗体噬菌体展示文库并进行筛选，将筛选得到的恒河猴免疫球蛋白重链与轻链可变区克隆至人类IgG恒定区，由此获得与人抗体同源性超过97%的人-恒河猴嵌合单克隆抗体。所筛选得到的单克隆抗体可有效中和H5N1流感病毒的3个进化分支（0、1、2型）。体内保护实验结果显示，即便在感染H5N1流感病毒后3天内给药，该类单克隆抗体仍可有效保护小鼠。尤为关键的是，单抗4E6对HA的结合亲和力达到亚皮摩尔级，且体内保护效果优异，不会导致小鼠体重下降及明显的肺组织损伤。对4E6逃逸突变株的分析表明，4E6抗体结合于HA球状头部上包含两个氨基酸残基的保守表位区域。 研究结论与意义：本研究成功制备出中和性单克隆抗体，有望用于人类应对新型流感暴发或其他烈性传染性疾病的紧急防控准备。