The study of heterosubtypic neutralising antibody responses against H5N1 influenza viruses in human subjects using a comparative serology approach

Background: The continuous rapid genetic and antigenic evolution of H5 subtype influenza viruses has major implications for the sensitivity of serologic assays and can limit the efficacy of prepandemic human vaccines and the ability to undertake effective serosurveillance in susceptible populations. Materials and Methods: A panel of serum samples collected from the Italian population between 1992 and 2007 and previously found to be positive for antibodies against H5N1 as determined by serial radial hemolysis (SRH) were evaluated using pseudotype-based neutralisation assays (PPN) in combination with haemagglutination-inhibition (HI) assays using a clade 1 and a clade 2 H5N1 serologic antigen. Results: Sixty-eight human sera were evaluated for their ability to neutralize A/Vietnam/1194/04 H5 and A/chicken/Italy/13474/99 H7 pseudotypes. IC50 neutralizing antibody titres against the Group 1 A/Viet Nam/1194/04 H5 pseudotypes ranged from < 40 (cut-off) to 1:5120- 10240. Seven of 68 (10.3%) sera were found to be negative against A/Vietnam/1194/04 pseudotypes. IC50 neutralizing antibody titres against the Group 2 A/chicken/Italy/13474/99 H7 pseudotype ranged from < 40 (cut-off) to 1:160-320. Forty-seven of 68 (69.1%) sera were found to be negative against A/chicken/Italy/13474/99 H7. Conclusions: From the results obtained it can be concluded that the pseudotype assay can efficiently measure cross-reactive antibody responses that are not detected by the HI assay. It is postulated that these responses are directed against epitopes on the HA2 stalk. All three serologic assays (PPN, SRH, HI) measure antibodies with different (functional) overlapping specificities, contributing to a comprehensive analysis of humoral immunity to influenza viruses