人源中和性抗高致病性禽流感病毒H5N1基因工程全抗体的研制

运用噬菌体表面呈现技术,从禽流感病人恢复期血中获得淋巴细胞,通过基因工程手段,构建了人源抗H5NI禽流感病毒基因工程抗体文库.用纯化的人源H5N1禽流感病毒颗粒(A/Anhui/1/2005)及重组血凝素蛋白HA(A/Viet Nam/1203/2004)对Fab噬菌体抗体库进行富集筛选,成功地获得了抗禽流感病毒H5N1血凝素蛋白HA的人源单抗Fab段基因,并在大肠杆菌中获得有效表达.通过序列测定确定抗体轻重链型别,然后将阳性克隆的轻链和重链Fd段基因分别克隆入全抗体表达载体pAC-L-Fc后转染昆虫Sf9细胞,利用杆状病毒/昆虫细胞系统实现全抗体的分泌型表达.用ELISA、IFA和流式细胞术对所获人源单抗的功能特性进行鉴定.结果表明,我们获得了2株特异性针对H5N1禽流感病毒血凝素蛋白HA而与甲1型和甲3型人流感病毒无交叉反应的人源单抗(AVFlulgG01、AVFlulgG03).微量中和试验结果表明,除A/Guangdong/1/2006外,AVFlu-IgG01能够广泛地中和HA基因进化上属于Clade 2的中国南方、北方及中部地区的H5N1禽流感病毒分离株,同时还对属于Clade Ⅰ的越南H5N1分离株A/Viet Nam/1203/2004具有中和活性;AVFluIgG03虽然不能中和A/Viet Nam/1203/2004,但是对属于Clade 2的所有中国H5N1分离株均具有中和作用.人源中和性抗禽流感病毒H5N1基因工程全抗体的获得不仅为高致病性禽流感病毒H5N1的预防和治疗带来了希望,同时也为其疫苗研制提供了新的思路.

Generation of Neutralizing Recombinant Human Antibodies for Targeting Highly Pathogenic Avian Influenza A(H5N1) Virus

Two human Fab antibodies against avian influenza A (H5N1) virus were obtained by panning a H5N1 patient-derived antibody phage library using purified virions of the H5N1 patient isolate A/Anhui/1/2005 and HA protein of the H5N1 reference viruse A/Viet Nam/1203/2004. After testing the binding properties and antiviral function to H5N1 virus, the selected Fab antibodies were converted to full human IgG antibodies with recombinant baculovirus/insect cell system. Both mAbs, AVFluIgG01 and AVFluIgG03, bound to HA in immunofluorescence assay (IFA) without cross-reaction with the other substypes of influenza A viruses (H1N1, H3N2). The cross-reactivity of the two antibodies for different strains of H5N1 was tested in vitro by micro-neutralization assays. In vitro, mAb AVFluIgG01 potently neutralized not only the selected well-characterized Clade 2 H5N1 viruses isolated from mainland of China except A/Guangdong/1/2006, but also the Clade 1 representative isolate A/Viet Nam/1203/2004; and AVFluIgG03 neutralized all the selected Clade 2 H5N1 viruses isolated from mainland of China, but had no neutralizing activity with the Clade 1 H5N1 virus A/Viet Nam/1203/2004. The results bring new prospect for the prophylaxis or treatment of H5N1 virus infection and may provide a clue for novel vaccine development.