Induction of H7N9-Cross-Reactive Antibody-Dependent Cellular Cytotoxicity Antibodies by Human Seasonal Influenza A Viruses that are Directed Toward the Nucleoprotein

Abstract

Antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC) against avian influenza virus subtypes, including H7N9 and H5N1, have been detected in human sera. Using NK cell activation and NK cytotoxicity assays, we compared ADCC-mediating antibodies (ADCC-Abs) in sera collected from healthy infants, children and adults against H7N9 virus-infected cells and recombinant hemagglutinin (HA), neuraminidase (NA), and nucleoprotein (NP) proteins. High titers of ADCC-Abs against H7N9 virus-infected cells were detected in sera from adults and children but not infants. ADCC-Abs titers directed against H7N9 HA or NA proteins. Further analysis showed that ADCC-Abs titers were significantly higher toward H7N9 NP, as compared with H7N9 HA or NA proteins, and correlated strongly with ADCC-Abs titers against H7N9 virus-infected cells. Indeed, ADCC-Abs to NPs of seasonal H1N1 and H3N2 viruses correlated strongly with ADCC-Abs to H7N9 NP, suggesting that seasonal influenza infections and vaccinations may induce these cross-reactive antibodies. Targeting ADCC-Abs to internal proteins may be a potential mechanism of universal vaccine design.

Keywords: ADCC; antibody; antibody-dependent cellular cytotoxicity; influenza virus; nucleoprotein.

Figure 1.

Antibody-dependent cellular cytotoxicity–mediating antibodies (ADCC-Abs) that recognize A(H7N9) virus–infected cells in sera of healthy subjects do not directly target H7N9 hemagglutinin (HA) or neuraminidase (NA) proteins. ADCC-Ab titers were measured against A/Anhui/01/2013 H7N9 virus–infected A549 cells (A), A/Anhui/01/2013 rHA protein (B), or A/Anhui/01/2013 rNA protein (C) in sera from infants (n = 10), children (n = 52), and adults (n = 16). Bars indicate median titers. Titers were compared using separate Mann–Whitney U tests (**P < .05; ns = not statistically significant).

Figure 2.

H7N9-specific antibody-dependent cellular cytotoxicity–mediating antibodies (ADCC-Abs) are generated toward the H7N9 nucleoprotein. ADCC-Ab titers against recombinant A/Anhui/01/2013 (H7N9) nucleoprotein (NP) in sera from infants (n = 10), children (n = 52), and adults (n = 16) (A). Linear correlation analysis of ADCC-Ab titers against H7N9 NP and H7N9 virus-infected cells measured by NK cell activation assay (B) or cytotoxicity assay (cytotoxicity assay results have been published elsewhere [14]) (C). Bars indicate median titers. Titers were compared using separate Mann–Whitney U tests (**P < .05; ns = not statistically significant). In (B) and (C) some dots represent >2 samples.

Figure 3.

Antibody-dependent cellular cytotoxicity–mediating antibodies (ADCC-Abs) to H7N9 nucleoprotein (NP) correlate strongly with ADCC-Abs to seasonal influenza NP. ADCC-Ab titer against recombinant NP from A/Puerto Rico/8/1934 (H1N1), A/California/07/2009 (H1N1pdm09), or A/Hong Kong/1/1968 (H3N2) in sera from infants (I, n = 10), children (C, n = 52), and adults (A, n = 16) (A). Linear correlation analysis of H7N9 NP ADCC-Ab titers versus titers to NPs of A/Puerto Rico/8/1934 (H1N1, B), A/California/07/2009 (H1N1pdm09, C), or A/Hong Kong/1/1968 (H3N2, D). Bars indicate median titers. Titers were compared using separate Mann–Whitney U tests (**P < .05; ns = not statistically significant). In B–D, some dots represent >2 samples.