Figure 4

Antibody binding to H9N2 infected MDCK cells. (A) Representative dot plots of uninfected MDCK cells (left column), H9N2 Influenza infected MDCK (middle column) and overlays of specific antibody responses (right panels) are depicted. Top panels shown FSC-SSC dot plots, second line of panels indicate 7AAD live/dead discrimination in FL3 channel. Third row shows staining of live uninfected (left) and H9N2 infected (middle) MDCK cells with rabbit-anti-M2 antibody and the respective overlay (right panel). Fourth row shows staining of live uninfected (left) and H9N2 infected (middle) MDCK cells with rabbit-anti-HA antibody and the respective overlay (right panel). Fifth row shows staining of live uninfected (left) and H9N2 infected (middle) MDCK cells with naïve rabbit control antibody and the respective overlay (right panel). (B) Representative dot plots specific antibody responses of one of five vaccinated chickens to uninfected MDCK cells (left column), H9N2 Influenza infected MDCK (middle column) and overlays of (right panels) are depicted for each indicated treatment group (top to bottom: his-M2 protein, M2e peptide, M2e tetramer and HSP70 tetramer), followed by negative and H9N2 vaccinated positive chicken reference serum. (C) The average value of the mean fluorescence intensity (MFI) of the H9N2 infected MDCK cells minus the MFI of the uninfected MDCK cells (as depicted in (B)) +/− SEM from five animals in each treatment group (indicated on the X-axis as M2 protein, M2e Peptide, M2e Tetramer and HSP70 Tetramer respectively) as well as triplicates +/− SEM of a negative and positive reference sample from naive and H9N2 vaccinated chickens (GD Deventer, The Netherlands) is shown as a Delta MFI. No significant differences between treatment groups were observed.