Preparing Monoclonal Antibodies Against Antigenic Variation H9N2 Avian Influenza Virus And Their Application In Antigenic Variation

The first known infection of H9N2 subtype of influenza A virus (H9N2) occurred in 1966 among turkeys in Wisconsin. Since the 1990s, the virus has readily circulated among domestic poultry populations in several Asian countries, mading huge economic losses for poultry industries and having significant impacts on human health. Vaccination is used as an important strategy in the control of AIV in poultry. However, the vaccine antibody pressure makes AIV easier to alter its antigenic properties, contributing to the failure of immune and the emergence of new pandemic. In previous study, A/chicken/Shanghai/F/98 H9N2 (F) subtype avian influenza virus was selected, and was-continuously passed in SPF embryonated chicken eggs with or without homologous vaccine antibody. The antigenic variation H9N2 avian influenza virus mF50 were obtained. We identified four mutant positions, K131R, S145N, G181E and A198V in the HA gene of mF50. In this study, to investigate the molecular mechanism of antigen variation under the vaccine antibody selective pressure, sixteen monoclonal antibodies against the hemagglutitin of mF50 were generated from the BALB/C mice immunized with the antigenic variation H9N2 avian influenza virus mF50. By PCR and Site-directed mutagenesis, we constructed recombinated eukaryotic expression vector which contained different combinations of the four mutant Amino acid sites. Then we used the method of transfection and enzyme-linked immunosorbent assays (ELISAs) to investigate the relationship between the amino acid mutation and antigenic variation in HA gene. Finally, the critical amino acid position causing the antigenic variation in HA gene were analyzed.1. Development of monoclonal antibodies against the antigenic variation H9N2 avian influenza virus mF50mF50 was propagated in the allantoic cavities of 10-day-old embryonated chicken eggs and purified by differential gradient centrifugation. BALB/c mice (6 weeks) were infected with mF50 twice at an interval of 2 weeks and boosted with inactivated, purified mF50 on day 3 before the fusion. Splenocytes from immunized mice were fused with Sp2/0 cells. Hybridomas were initially screened with enzyme-linked immunosorbent assays (ELISAs) using mF50 virus-coated plates. Positive clones were further subcloned third times by limiting dilution. Ascitic fluid was generated for each hybridoma in BALB/c mice. By Western blot, sixteen monoclonal antibodies which against the hemagglutitin of H9N2 subtype of AIV were identified named 4F1,3D6,6C6,2H6,4B11,4E9,1D5,3G1,5A4,3C5,4H8,6D1,3D5,1D3, 5B9 and 2C4, respectively. The HI titer were between 26-29 and the ELISA titer were between 105-107. By HI assay, sixteen monoclonal antibodies were specifically against H9 subtype AIV, but not reacted with H5 subtype AFV, Newcastle disease virus, avian infectious bronchitis virus and avian egg drop syndrome virus. All the results confirmed that the sixteen monoclonal antibodies were specific to the hemagglutinin of mF50 virus.2. Application of monoclonal antibodies in antigenic variation of H9N2 avian influenza virusIn the previous study, we discovered that there were four amino acids substitution (K131R, S145N、G181E and A198V) in the HA gene of antigenic mutate virus mF50 compare to the parental virus F. To find out the relationship between the amino acid mutation in the HA gene and the antigenic variation, the HA genes with different mutants, which included one mutations (called rFHA131, rFHA145 rFHA181, rFHA198, respectively, two mutations (called rFHA145-181 and rFHA181-198), triple mutation (called rFHA131-181-198), quadruple mutation called rFHA131-145-181-198 and parental HA from F virus, (called rFHA), were constructed, and then inserted into the expression plasmid pCAGGS. The HA protein were expressed on 293T cells by transfection with rFHA or mutant plasmids. The HA protein expressed in 293T cells were used antigen, and the obtained monoclonal antibodies in this paper were used antibodies t to select the monoclonal antibodies against the mutant in HA gene by the cell-based ELISA test. The results showed that 3G1 monoclonal antibodies were only reacted strongly with all HA protein including mutant A198V, and 5B9 monoclonal antibodies were only reacted well with all HA protein including mutant G181E, implying that the amino acid of 181 or 198 site might contribute to the antigenic variation of H9N2 subtype avian influenza virus.