Natural symptomatic but not experimental influenza infection

Natural symptomatic but not experimental influenza infection increased Ab-mediated NK cell activation to the internal protein M1 and to a lesser extent NP. Donor infection history, strain/subtype of infecting virus, route of infection and infectious dose may affect the capacity of a given influenza infection to boost Ab-dependent NK cell activation. During natural influenza infection Ab-dependent NK cell activation was increased to the HA of the infecting virus and M1 following seroconversion. Surprisingly, when the NK cell activating Abs to HA and M1 were serially diluted it was revealed that in some cases the endpoint titres were identical in pre- and post-seroconversion samples. These results suggest that the total levels of NK cell activating Abs were not increased post-seroconversion but that the Abs induced or expanded after seroconversion are more potent activators of NK cells. This could be due to a number of factors including enhanced Ab avidity for its Ag or altered glycosylation of the IgG Fc region, which can change the binding affinity for Fc receptors (Ferrara et al., 2006; Ferrara et al., 2011; Shinkawa et al., 2003) and lead to altered immune effector functions like ADCC. Differential Fc glycosylation or increased Ab avidity post-seroconversion may explain the observation that NK cell activation at a single IgG concentration is variable in samples that demonstrate similar quantities of NK cell activating Abs by titration. The role of NK cell activating Abs to internal influenza proteins in the control of influenza infection is controversial. Animal models of influenza infection suggest that Abs to conserved Ags like M1 and NP are important for heterosubtypic immunity (LaMere et al., 2011b). Likewise, passive transfer of immune serum from mice vaccinated with NP into naïve or B-cell deficient mice decreased influenza viral load (Carragher et al., 2008). Since Abs to internal influenza Ags cannot prevent viral entry by neutralisation (Gerhard et al., 1997; Mozdzanowska et al., 1999), anti-NP IgG activity is likely mediated through complex interactions with effector nobiletin (LaMere et al., 2011b). In B cell deficient mice the antiviral action of NP reactive Abs is Fc receptor dependent and significantly reduced in Fc receptor γ-common chain knockout (FcRγ−/−) mice (LaMere et al., 2011b). The nature of these antiviral effects and their relevance to human influenza infection is unclear. Specifically it is unknown if M1 and NP Abs activate effector cells that directly kill infected targets (ADCC) or act indirectly by cytokine-mediated stimulation of other antiviral cell types. Indeed, this study found NK cell activating Abs to highly conserved influenza Ags, but we were not able to detect Ab-mediated killing of target cells infected with rVVs expressing either NP or M1. Internal influenza proteins are present in the extracellular environment following their release from dead or infected cells (LaMere et al., 2011b) and NP is present on the surface of influenza-infected cells in vitro as early as one hour post-infection (Bodewes et al., 2013; Virelizier et al., 1977; Yewdell et al., 1981). In spite of a robust rVV infection system as evidenced by consistently high levels of ADCC against rVV-HA infected cells, we found that only a small percentage (up to ~25%) of target CEM cells infected with rVV-NP had NP on the cell surface, which may be insufficient for detection of Ab-mediated killing in the assays employed. Influenza and vaccinia viruses differ in genome composition, replication cycle and tissue tropism therefore it is not known if the rVV expression system accurately reflects an in vivo influenza infection. Influenza-infected cells have been shown to express relatively low levels of surface NP compared to surface glycoproteins HA and NA, which were approximately 10-fold more abundant (Yewdell et al., 1981). Future studies using influenza-infected cells as targets in Ab-mediated killing assays would be informative, but such assays are problematic since influenza-infected cells are susceptible to HA and NA-specific ADCC in polyclonal serum. To carry out Ab-mediated killing assays specifically targeting surface NP and M1 on influenza-infected cells would require monoclonal Abs (mAbs) with known ADCC activity. However most currently available mAbs are not isolated with their native Fc regions and glycosylation patterns, which can significantly impact ADCC functionality (Arnold et al., 2007; Mahan et al., 2016; Vidarsson et al., 2014).