Lu Jiahai, Guo Zhongmin, Pan Xinghua, Wang Guoling, Zhang Dingmei, Li Yanbin, Tan Bingyan, Ouyang Liping, Yu Xinbing
Sun Yat-sen University, Guangzhou 510080, China.
Respir Res. 2006 Mar 23;7(1):43. doi: 10.1186/1465-9921-7-43.
Avian influenza virus H5N1 has demonstrated considerable pandemic potential. Currently, no effective vaccines for H5N1 infection are available, so passive immunotherapy may be an alternative strategy. To investigate the possible therapeutic effect of antibody against highly pathogenic H5N1 virus on a mammal host, we prepared specific equine anti-H5N1 IgGs from horses vaccinated with inactivated H5N1 virus, and then obtained the F(ab')2 fragments by pepsin digestion of IgGs.
The horses were vaccinated with inactivated H5N1 vaccine to prepare anti-H5N1 IgGs. The F(ab')2 fragments were purified from anti-H5N1 hyperimmune sera by a protocol for 'enhanced pepsin digestion'. The protective effect of the F(ab')2 fragments against H5N1 virus infection was determined in cultured MDCK cells by cytopathic effect (CPE) assay and in a BALB/c mouse model by survival rate assay.
By the protocol for 'enhanced pepsin digestion', total 16 g F(ab')2 fragments were finally obtained from one liter equine antisera with the purity of over 90%. The H5N1-specific F(ab')2 fragments had a HI titer of 1:1024, and the neutralization titre of F(ab')2 reached 1: 2048. The in vivo assay showed that 100 microg of the F(ab')2 fragments could protect BALB/c mice infected with a lethal dose of influenza H5N1 virus.
The availability of highly purified H5N1-specific F(ab')2 fragments may be promising for treatment of influenza H5N1 infection. Our work has provided experimental support for the application of the therapeutic equine immunoglobulin in future large primate or human trials.
H5N1禽流感病毒已显示出相当大的大流行潜力。目前，尚无针对H5N1感染的有效疫苗，因此被动免疫疗法可能是一种替代策略。为了研究抗高致病性H5N1病毒抗体对哺乳动物宿主的可能治疗效果，我们用灭活的H5N1病毒对马进行免疫接种，制备了特异性马抗H5N1 IgG，然后通过胃蛋白酶消化IgG获得F(ab')2片段。
用灭活的H5N1疫苗对马进行免疫接种以制备抗H5N1 IgG。通过“增强胃蛋白酶消化”方案从抗H5N1超免疫血清中纯化F(ab')2片段。通过细胞病变效应（CPE）试验在培养的MDCK细胞中以及通过存活率试验在BALB/c小鼠模型中确定F(ab')2片段对H5N1病毒感染的保护作用。
通过“增强胃蛋白酶消化”方案，最终从一升马抗血清中获得了总计16 g纯度超过90%的F(ab')2片段。H5N1特异性F(ab')2片段的血凝抑制（HI）效价为1:1024，F(ab')2的中和效价达到1:2048。体内试验表明，100 μg的F(ab')2片段可以保护感染致死剂量H5N1流感病毒的BALB/c小鼠。
高纯度的H5N1特异性F(ab')2片段的可得性可能为治疗H5N1流感感染带来希望。我们的工作为治疗性马免疫球蛋白在未来大型灵长类动物或人体试验中的应用提供了实验支持。
