Tan Yunrui, Ng Qingyong, Jia Qiang, Kwang Jimmy, He Fang
Animal Health Biotechnology, Temasek Life Sciences Laboratory, Singapore, Singapore.
Animal Health Biotechnology, Temasek Life Sciences Laboratory, Singapore, Singapore Department of Microbiology Faculty of Medicine, National University of Singapore, Singapore, Singapore
J Virol. 2015 Apr;89(7):3712-22. doi: 10.1128/JVI.03014-14. Epub 2015 Jan 21.
Highly pathogenic avian influenza virus subtype H5N1 continues to be a severe threat to public health, as well as the poultry industry, because of its high lethality and antigenic drift rate. Neutralizing monoclonal antibodies (MAbs) can serve as a useful tool for preventing, treating, and detecting H5N1. In the present study, humanized H5 antibody 8A8 was developed from a murine H5 MAb. Both the humanized and mouse MAbs presented positive activity in hemagglutination inhibition (HI), virus neutralization, and immunofluorescence assays against a wide range of H5N1 strains. Interestingly, both human and murine 8A8 antibodies were able to detect H5 in Western blot assays under reducing conditions. Further, by sequencing of escape mutants, the conformational epitope of 8A8 was found to be located within the receptor binding domain (RBD) of H5. The linear epitope of 8A8 was identified by Western blotting of overlapping fragments and substitution mutant forms of HA1. Reverse genetic H5N1 strains with individual mutations in either the conformational or the linear epitope were generated and characterized in a series of assays, including HI, postattachment, and cell-cell fusion inhibition assays. The results indicate that for 8A8, virus neutralization mediated by RBD blocking relies on the conformational epitope while binding to the linear epitope contributes to the neutralization by inhibiting membrane fusion. Taken together, the results of this study show that a novel humanized H5 MAb binds to two types of epitopes on HA, leading to virus neutralization via two mechanisms.
Recurrence of the highly pathogenic avian influenza virus subtype H5N1 in humans and poultry continues to be a serious public health concern. Preventive and therapeutic measures against influenza A viruses have received much interest in the context of global efforts to combat the current and future pandemics. Passive immune therapy is considered to be the most effective and economically prudent preventive strategy against influenza virus besides vaccination. It is important to develop a humanized neutralizing monoclonal antibody (MAb) against all of the clades of H5N1. For the first time, we report in this study that a novel humanized H5 MAb binds to two types of epitopes on HA, leading to virus neutralization via two mechanisms. These findings further deepen our understanding of influenza virus neutralization.
高致病性禽流感病毒H5N1亚型因其高致死率和抗原漂移率，仍然对公共卫生以及家禽业构成严重威胁。中和性单克隆抗体（MAb）可作为预防、治疗和检测H5N1的有用工具。在本研究中，人源化H5抗体8A8由鼠源H5单克隆抗体制备而来。人源化单克隆抗体和鼠源单克隆抗体在针对多种H5N1毒株的血凝抑制（HI）、病毒中和及免疫荧光试验中均表现出阳性活性。有趣的是，人源和鼠源8A8抗体在还原条件下的蛋白质印迹试验中均能检测到H5。此外，通过对逃逸突变体进行测序，发现8A8的构象表位位于H5的受体结合域（RBD）内。通过对HA1的重叠片段和替代突变体形式进行蛋白质印迹分析，鉴定出8A8的线性表位。构建了在构象表位或线性表位上具有单个突变的反向遗传H5N1毒株，并在一系列试验中进行了表征，包括HI、附着后及细胞-细胞融合抑制试验。结果表明，对于8A8而言，由RBD阻断介导的病毒中和依赖于构象表位，而与线性表位的结合则通过抑制膜融合促进中和作用。综上所述，本研究结果表明，一种新型人源化H5单克隆抗体可结合HA上的两种表位，通过两种机制导致病毒中和。
高致病性禽流感病毒H5N1亚型在人类和家禽中的再次出现仍然是一个严重的公共卫生问题。在全球应对当前和未来大流行的努力背景下，针对甲型流感病毒的预防和治疗措施备受关注。除疫苗接种外，被动免疫疗法被认为是对抗流感病毒最有效且经济上最审慎的预防策略。开发一种针对所有H5N1进化枝的人源化中和单克隆抗体非常重要。我们在本研究中首次报告，一种新型人源化H5单克隆抗体可结合HA上的两种表位，通过两种机制导致病毒中和。这些发现进一步加深了我们对流感病毒中和作用的理解。
