Development of neutralizing monoclonal antibodies against the pandemic H1N1 virus (2009) using plasmid DNA immunogen

Abstract

Reported here is the development and characterization of eighteen mouse monoclonal antibodies (MAbs) against the 2009 pandemic H1N1 influenza virus (A/Mexico/InDRE4487/2009). To our knowledge, this is the first report on pandemic (pH1) H1N1 MAbs developed using plasmid DNA encoding the viral surface glycoprotein, hemagglutinin (HA). All eighteen MAbs were specific for A/Mexico/InDRE4487/2009 HA. Ten MAbs were found to cross-react with A/Swine/Indiana/81 using a dot blot assay. However, there was no cross-reactivity detected against any other strains of influenza A viruses despite screening against all 16 hemagglutinin subtypes. Examination of these MAbs identified individual antibodies suitable for use in several practical applications including ELISA, immunoblot and immunofluorescence assays. Analysis of the kinetics of each MAb revealed significant binding affinities (K(D)<10(-8) M) confirming the antibodies are highly specific for A/Mexico/InDRE4487/2009 HA. Functional analysis demonstrated the panel of MAbs included antibodies with HA inhibition and virus neutralization activities. Not all MAbs inhibited hemagglutination or neutralized the virus. Furthermore, the panel of MAbs was not found to be cross-reactive against additional strains tested in hemagglutination inhibition assays. Finally, the MAbs were tested in competitive ELISA (cELISA) using reference serum antibodies developed against different clusters of H1 (pH1, α, β, γ, and δ). The developed MAbs outcompeted serum antibodies of pH1 in 16/18, 15/18 (γ), 3/18 (α), 2/18 (δ1) and 1/18 (β) samples. Overall, this panel of MAbs proved specific and highly sensitive for A/Mexico/InDRE4487/2009 HA and could potentially serve as immunodiagnostic tools for the rapid detection of this specific strain of influenza virus.

Keywords: H1N1; HA; HI; IFA; Influenza virus; MAb; MDCK; Madin-Darby Canine Kidney; NA; Neutralizing monoclonal antibody; Pandemic; hemagglutination inhibition; hemagglutinin; immunofluorescence assay; monoclonal antibody; neuraminidase.