Department of Immunology, Graduate Program in Immunology, Mahidol University, Bangkok 10700, Thailand.
Virol J. 2013 May 14;10:148. doi: 10.1186/1743-422X-10-148.
Novel effective anti-influenza agent that tolerates influenza virus antigenic variation is needed. Highly conserved influenza virus M2 protein has multiple pivotal functions including ion channel activity for vRNP uncoating, anti-autophagy and virus assembly, morphogenesis and release. Thus, M2 is an attractive target of anti-influenza agents including small molecular drugs and specific antibodies.
Fully human monoclonal single chain antibodies (HuScFv) specific to recombinant and native M2 proteins of A/H5N1 virus were produced from huscfv-phagemid transformed E. coli clones selected from a HuScFv phage display library using recombinant M2 of clade 1 A/H5N1 as panning antigen. The HuScFv were tested for their ability to inhibit replication of A/H5N1 of both homologous and heterologous clades. M2 domains bound by HuScFv of individual E. coli clones were identified by phage mimotope searching and computerized molecular docking.
HuScFv derived from four huscfv-phagemid transformed E. coli clones (no. 2, 19, 23 and 27) showed different amino acid sequences particularly at the CDRs. Cells infected with A/H5N1 influenza viruses (both adamantane sensitive and resistant) that had been exposed to the HuScFv had reduced virus release and intracellular virus. Phage peptide mimotope search and multiple alignments revealed that conformational epitopes of HuScFv2 located at the residues important for ion channel activity, anti-autophagy and M1 binding; epitopic residues of HuScFv19 located at the M2 amphipathic helix and cytoplasmic tail important for anti-autophagy, virus assembly, morphogenesis and release; epitope of HuScFv23 involved residues important for the M2 activities similar to HuScFv2 and also amphipathic helix residues for viral budding and release while HuScFv27 epitope spanned ectodomain, ion channel and anti-autophagy residues. Results of computerized homology modelling and molecular docking conformed to the epitope identification by phages.
HuScFv that bound to highly conserved epitopes across influenza A subtypes and human pathogenic H5N1clades located on different functional domains of M2 were produced. The HuScFv reduced viral release and intracellular virus of infected cells. While the molecular mechanisms of the HuScFv await experimental validation, the small human antibody fragments have high potential for developing further as a safe, novel and mutation tolerable anti-influenza agent especially against drug resistant variants.
需要一种新型有效的抗流感药物，该药物能够耐受流感病毒的抗原变异。高度保守的流感病毒 M2 蛋白具有多种关键功能，包括 vRNP 脱壳的离子通道活性、抗自噬和病毒组装、形态发生和释放。因此，M2 是抗流感药物（包括小分子药物和特异性抗体）的一个有吸引力的靶点。
从使用重组 1 型 A/H5N1 M2 作为淘选抗原的重组 M2 噬菌体展示文库中选择的 Huscfv 噬菌体转化的大肠杆菌克隆中，产生了针对 A/H5N1 病毒的重组和天然 M2 蛋白的全人源单链抗体（HuScFv）。测试了 HuScFv 抑制同源和异源 1 型 A/H5N1 病毒复制的能力。通过噬菌体模拟肽搜索和计算机分子对接鉴定与单个大肠杆菌克隆的 HuScFv 结合的 M2 结构域。
从四个 Huscfv 噬菌体转化的大肠杆菌克隆（2 号、19 号、23 号和 27 号）中获得的 HuScFv 显示出不同的氨基酸序列，特别是在 CDRs 中。与 HuScFv 接触的感染 A/H5N1 流感病毒（包括金刚烷胺敏感和耐药）的细胞病毒释放和细胞内病毒减少。噬菌体肽模拟搜索和多重比对显示，HuScFv2 的构象表位位于对离子通道活性、抗自噬和 M1 结合重要的残基处；HuScFv19 的表位残基位于对自噬、病毒组装、形态发生和释放重要的 M2 两性螺旋和胞质尾；HuScFv23 的表位涉及与 HuScFv2 相似的对 M2 活性重要的残基，以及对病毒出芽和释放重要的两性螺旋残基，而 HuScFv27 的表位跨越了外显子、离子通道和抗自噬残基。计算机同源建模和分子对接的结果与噬菌体鉴定的表位一致。
产生了与流感 A 亚型和人致病性 H5N1 分支上不同功能域上的高度保守表位结合的 HuScFv。HuScFv 减少了感染细胞的病毒释放和细胞内病毒。虽然 HuScFv 的分子机制有待实验验证，但这些小的人源抗体片段具有很高的潜力，可以进一步开发成为一种安全、新型且耐受突变的抗流感药物，特别是针对耐药变体。
