Production and characterization of antibodies neutralizing H9N2 avian influenza virus

Monoclonal antibodies (mAbs) that neutralize influenza A virus have been shown to be potent therapeutic reagents if used pre- or post – exposure to the pathogen. Whilst the majority of this research has focused on human antibody therapeutics, there is an urgent need for generation of antibodies able to neutralize avian influenza viruses (AIV). Several mAbs against H9N2 virus have previously been generated using mouse hybridomas. To facilitate development of passive immunization strategies, the variable chains of these antibodies were characterized in the context of isotype and species specific Fc fragments. Replacement of the IgG2 Fc region with an IgG1 Fc region enhanced neutralizing activity for one of the tested antibodies. Additionally, chicken chimeric antibodies were generated which showed comparable neutralization titres to those generated by the original hybridomas. To identify if antibodies could function as a single chain variable fragment antibodies (scFvs) these were produced in insect S2 cells. This confirmed that H9N2 virus can be neutralized by scFvs. However, an example with high HI titres but lost detectable neutralizing activity was found, possibly due to the loss of bivalent interaction between antigen and antibody. Antibodies showing superior neutralization activity in vitro will be subsequently tested for their potency in in vivo infection. We propose that passive immunization can reduce the impact of AIV in poultry by inducing immediate protection and bypassing immunocompromised individuals.