Preparation and protection of monoclonal antibody against H7N9 influenza virus hemagglutinin

The first human infection with novel H7N9 influenza virus was reported in March 2013 in China. To date, the H7N9 influenza viruses possess a potential pandemic threat to public health worldwide and have caused severe infection and high mortality in humans in China. The latent period of human infection of H7N9 influenza virus is generally within 7 days. Patients usually show flu like symptoms, such as runny nose, sore throat, fever, cough, headache and muscle pain. Severe patients have rapid development, characterized by severe pneumonia and dyspnea. Antibodies play a major role in protective immunity against the influenza virus infection, and passive immunization with monoclonal antibodies (MAbs) specific to viral proteins might be a potential option for humans against lethal infection of the influenza viruses. It is known that hemagglutinin (HA), is the major viral antigenic protein on the influenza virus particle and the primary target of neutralizing antibodies against influenza virus. In this study, in order to obtain the HA MAbs of influenza virus H7N9 (A/Shanghai/2/2013), study their biology characteristics, and evaluate the prophylactic and therapeutic efficacy of the HA MAbs in mouse model. The BALB/c mice were immunized with inactivated H7N9 vaccine by subcutaneous administration for three times, and boosted with inactivated H7N9 vaccine by the tail vein three days before fusion. Finally, 28 MAbs were obtained and identified by ELISA and hemagglutination inhibition test (HI). Eight IgG subtype MAbs (1A8, 2A12, 2H3, 4A11, 4C8, 4G5, 6A3, 7G2) were selected to identify their biology characteristics. The ELISA titers of ascites of the eight MAbs were 10 2 –10 6 . The eight Mabs exhibited high affinity which can reach nmol/L level and meet the requirement of affinity for monoclonal antibody drug. The immunofluorescence assay indicated that the eight MAbs had excellent specificity for HA protein expressed in 293T cell and were able to recognize the H7N9 influenza virus particles. In addition, all the eight MAbs could inhibit HA activity and the MAbs 2H3, 4A11, 4G5 and 6A3 can also present neutralization activity against the H7N9 live virus. The highest neutralizing titer was up to 1:14125. At the same time, we evaluated the prophylactic and therapeutic efficacy of these MAbs against the homologous H7N9 strain. The activity against H7N9 virus was evaluated by clinical syndromes, survival rates and lung viral titers. The results showed that in the prophylactic and therapeutic experiments, MAb 6A3 can inhibit effectively H7N9 influenza virus infection, provided 100% protection and remarkably reduced the lung viral titers in mouse model. The HA monoclonal antibodies against influenza virus H7N9 were successfully obtained. The HA MAbs not only may be as a prophylactic and therapeutic measure to prevent H7N9 influenza virus infection, but also laid the foundation for us to research the H7N9 HA antigen epitope and develop diagnostic reagents.