全人源抗体组合对H5N1型高致病性禽流感病毒感染的阻断作用及机制

Highly pathogenic avian influenza virus (HPAIV) of the subtype H5N1 is a highly contagious disease of birds caused by A influenza viruses. The circulation in humans by the highly pathogenic H5N1 avian flu in the past few years, with either high mortality or morbidity, have caused most pandemics and have heightened fear that the next influenza pandemic is due. One of the possible effective countermeasure against influenza is a resurgence of human antibody-based therapy..In previous study, we isolated and characterized human antibody scFv 4F5 with broad heterosubtypic neutralizing activity against diverse clades of H5N1 influenza A virus. They bound to a conserved peptide in the HA1 domain, and showed satisfactory antiviral effects when tested in influenza challenge experiments using embryonated chicken eggs. The purpose of the present study was to test the protection of antibody scFv4F5 in influenza virus-infected mice. In order to increase the survival of the infected mice, the mixture of extracellular and intracellular antibodies were used to block HPAIV proliferation and infection. The viral replication, CD8+ T cell responses and apoptosis were examed before and after antibodies treatment. On the other way, the mRNA expression and protein level of IFN-α/β，IFN-γ，IL-6，IL-10 were detected by ELISA, FACS or real time-PCR techniques. Pathological injury of the lung and macrophage infiltration were detected by immunohistochemistry. All of the study was to explore the mechanism of immunoprotective effects, and provide the theorotic basis for its clinical application.

人感染H5N1型高致病性禽流感病毒(HPAIV)后呈高发病率和高死亡率的特征，我国每年都有散发病例的出现。目前，人源治疗性抗体成为国内外禽流感特效治疗药物的研究热点。申请者运用噬菌体抗体库技术获得了具有广谱中和活性的全人源小分子抗体scFv4F5，在以鸡胚为模型的试验中显示较好的保护效果。本研究拟构建HPAIV感染Balb/c小鼠模型，scFv4F5以细胞内和细胞外抗体组合形式进行病毒感染后阻断，以期待通过同时抑制病毒的增殖和病毒的感染来提高小鼠的生存率，筛选最佳组合方案。在此基础上，利用ELISA，FACS，real time-PCR及免疫组化检测抗体阻断前后小鼠体内病毒复制动力学，CD8+ T细胞的增殖活化及凋亡水平，IFN-α/β，IFN-γ，IL-6等细胞因子转录水平的变化，肺组织损伤病理变化及巨噬细胞等分布特征，探讨抗体组合对感染小鼠的免疫保护效应机制，为临床试验提供理论基础。

人感染H5N1 型高致病性禽流感病毒(HPAIV)后呈高发病率和高死亡率的特征，我国每年都有散发病例的出现。目前，人源治疗性抗体成为国内外禽流感特效治疗药物的研究热点。本研究在前期研究基础上进一步构建细胞内重组质粒pCAG-scFv 4F5-GFP和细胞外全分子IgG1重组质粒，其中pCAG-scFv 4F5-GFP体外转染细胞后有较高的荧光强度，并能正常表达重组抗体。IgG1转染细胞株经过2-3轮亚克隆后获得稳定分泌抗体的细胞株，表达量可高达20mg/100ml，亲和层析技术纯化后纯度可达95%。.构建HPAIV感染Balb/c小鼠模型，分别从小鼠四肢和腹腔注射pCAG –scFv 4F5-GFP质粒和全分子IgG1抗体，当IgG1用量为15mg/kg，重组质粒cFv4F5为30μg时，该抗体组合对500 TCID50 A/Jiangsu/1/2007(H5N1)感染的小鼠达到66.67%的存活率，具有较好的保护率。进一步探讨抗体组合对感染小鼠的免疫保护效应机制，从病毒复制动力学，CD8+ T细胞的增殖活化及凋亡水平， IFN-α/β，IFN-γ，IL-6，IL-10等细胞因子转录及表达水平的变化等方面进行研究。细胞感染后抗体组合主要通过抑制IFN-α/β，IFN-γ，IL-6 ，IL-10等因子的分泌表达从而缓解对机体细胞的损伤。病毒复制动力学表明病毒滴度在第4天达到最高，第8天降低至感染前水平。CD8+T细胞在感染给药后第8天达到最高值，12天后降低至感染前水平，肺组织切片显示存活小鼠中H5N1病毒表达量较死亡小鼠中多。本抗体组合为抗禽流感病毒人源抗体运用中的一个创新思路，为临床试验提供理论基础。