Fig. 3: Anti-DNP antibodies recruiting, complement dependent cytotoxicity (CDC), and antibody-dependent cellular cytotoxicity (ADCC) of neuraminidase expressing cells induced by zan-DNP.

a and c Confocal micrographs of binding of biotinylated anti-DNP antibodies (visualized by streptavidin-PE) to the surface of A/Puerto Rico/8/1934 (H1N1) (a) and A/Aichi/2/1968 (H3N2) (c) virus-infected MDCK cells mediated by zan-DNP (n = 3). Nuclear staining is shown in blue, and streptavidin-PE staining is shown in red. b, d Binding of anti-DNP antibodies to influenza A(H1N1) (b) and A(H3N2) (d) virus-infected MDCK cells determined by quantitating the fluorescence of cell-bound streptavidin-PE in the presence of anti-DNP antibodies as a function of the concentration of zan-DNP (n = 3). All the virus-infected cells were used 24 h post-infection for the above studies. e Flowcytometry analyses of recruitment of anti-DNP antibodies to virus-infected cells mediated by zan-DNP in vivo (n = 3). A/California/07/2009 (H1N1)pdm09 virus-infected mice (3 mice/group) were first given zan-DNP and anti-DNP antibodies (3 days post-infection). 12 h later, the cells digested from the lung of virus-infected/uninfected mice were stained first with anti-hemagglutinin antibodies and then labeled with dye conjugated secondary antibodies against anti-hemagglutinin antibodies and anti-DNP antibodies. Left: selected flow cytometry plots; Right: summary graph. Statistical difference of antibody stained positive cells (%) between indicated two groups was analyzed by two-sided t test (*P < 0.05, **P < 0.01, ***P < 0.001, see exact P values in Supplementary Table 1). f CDC mediated killing of N1 neuraminidase transduced and nontransduced HEK 293 cells mediated by zan-DNP in the presence of anti-DNP antibodies and complement-preserved rabbit serum (n = 3). g Antibody-dependent cellular cytotoxicity (ADCC) of N1 neuraminidase transduced and nontransduced HEK 293 cells mediated by zan-DNP in the presence of anti-DNP antibodies and human FcγRIIIa expressing effector cells (n = 3). ADCC activity is proportional to the amount of firefly luciferase produced by effector cells (detected as the luminescence intensity). The coding sequence of N1 neuraminidase for the transduction of HEK 293 cells is from strain A/Puerto Rico/8/1934 (H1N1); Data are presented as mean values ± SD (n = 3).