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BST-2 (tetherin, CD317, HM1.24) restricts virus growth by tethering enveloped viruses to the cell surface. The role of BST-2 during influenza A virus infection (IAV) is controversial. Here, we assessed the capacity of endogenous BST-2 to restrict IAV in primary murine cells. IAV infection increased BST-2 surface expression by primary macrophages, but not alveolar epithelial cells (AEC). BST-2-deficient AEC and macrophages displayed no difference in susceptibility to IAV infection relative to wild type cells. Furthermore, BST-2 played little role in infectious IAV release from either AEC or macrophages. To examine BST-2 during IAV infection in vivo, we infected BST-2-deficient mice. No difference in weight loss or in viral loads in the lungs and/or nasal tissues were detected between BST-2-deficient and wild type animals. This study rules out a major role for endogenous BST-2 in modulating IAV in the mouse model of infection.

The long pentraxin, pentraxin 3 (PTX3), can play beneficial or detrimental roles during infection and disease by modulating various aspects of the immune system. There is growing evidence to suggest that PTX3 can mediate antiviral activity in vitro and in vivo. Previous studies demonstrated that PTX3 and the short pentraxin serum amyloid P express sialic acids that are recognized by the hemagglutinin (HA) glycoprotein of certain influenza A viruses (IAV), resulting in virus neutralization and anti-IAV activity. In this study, we demonstrate that specificity of both HA and the viral neuraminidase for particular sialic acid linkages determines the susceptibility of H1N1, H3N2, and H7N9 strains to the antiviral activities of PTX3 and serum amyloid P. Selection of H3N2 virus mutants resistant to PTX3 allowed for identification of amino acid residues in the vicinity of the receptor-binding pocket of HA that are critical determinants of sensitivity to PTX3; this was supported by sequence analysis of a range of H3N2 strains that were sensitive or resistant to PTX3. In a mouse model of infection, the enhanced virulence of PTX3-resistant mutants was associated with increased virus replication and elevated levels of proinflammatory cytokines in the airways, leading to pulmonary inflammation and lung injury. Together, these studies identify determinants in the viral HA that can be associated with sensitivity to the antiviral activities of PTX3 and highlight its importance in the control of IAV infection.

Seasonal influenza A viruses (IAV) originate from pandemic IAV and have undergone changes in antigenic structure, including addition of glycans to the hemagglutinin (HA) glycoprotein. The viral HA is the major target recognized by neutralizing antibodies and glycans have been proposed to shield antigenic sites on HA, thereby promoting virus survival in the face of widespread vaccination and/or infection. However, addition of glycans can also interfere with the receptor binding properties of HA and this must be compensated for by additional mutations, creating a fitness barrier to accumulation of glycosylation sites. In addition, glycans on HA are also recognized by phylogenetically ancient lectins of the innate immune system and the benefit provided by evasion of humoral immunity is balanced by attenuation of infection. Therefore, a fine balance must exist regarding the optimal pattern of HA glycosylation to offset competing pressures associated with recognition by innate defenses, evasion of humoral immunity and maintenance of virus fitness. In this review, we examine HA glycosylation patterns of IAV associated with pandemic and seasonal influenza and discuss recent advancements in our understanding of interactions between IAV glycans and components of innate and adaptive immunity.

Otitis media (OM) (a middle ear infection) is a common childhood illness that can leave some children with permanent hearing loss. OM can arise following infection with a variety of different pathogens, including a coinfection with influenza A virus (IAV) and Streptococcus pneumoniae (the pneumococcus). We and others have demonstrated that coinfection with IAV facilitates the replication of pneumococci in the middle ear. Specifically, we used a mouse model of OM to show that IAV facilitates the outgrowth of S. pneumoniae in the middle ear by inducing middle ear inflammation. Here, we seek to understand how the host inflammatory response facilitates bacterial outgrowth in the middle ear. Using B cell-deficient infant mice, we show that antibodies play a crucial role in facilitating pneumococcal replication. We subsequently show that this is due to antibody-dependent neutrophil extracellular trap (NET) formation in the middle ear, which, instead of clearing the infection, allows the bacteria to replicate. We further demonstrate the importance of these NETs as a potential therapeutic target through the transtympanic administration of a DNase, which effectively reduces the bacterial load in the middle ear. Taken together, these data provide novel insight into how pneumococci are able to replicate in the middle ear cavity and induce disease.

A better understanding of immunity to influenza virus is needed to generate cross-protective vaccines. Engagement of Ab-dependent cellular cytotoxicity (ADCC) Abs by NK cells leads to killing of virus-infected cells and secretion of antiviral cytokines and chemokines. ADCC Abs may target more conserved influenza virus Ags compared with neutralizing Abs. There has been minimal interest in influenza-specific ADCC in recent decades. In this study, we developed novel assays to assess the specificity and function of influenza-specific ADCC Abs. We found that healthy influenza-seropositive young adults without detectable neutralizing Abs to the hemagglutinin of the 1968 H3N2 influenza strain (A/Aichi/2/1968) almost always had ADCC Abs that triggered NK cell activation and in vitro elimination of influenza-infected human blood and respiratory epithelial cells. Furthermore, we detected ADCC in the absence of neutralization to both the recent H1N1 pandemic strain (A/California/04/2009) as well as the avian H5N1 influenza hemagglutinin (A/Anhui/01/2005). We conclude that there is a remarkable degree of cross-reactivity of influenza-specific ADCC Abs in seropositive humans. Targeting cross-reactive influenza-specific ADCC epitopes by vaccination could lead to improved influenza vaccines.

Seasonal influenza A viruses (IAV) originate from pandemic IAV and have undergone changes in antigenic structure, including addition of glycans to the viral hemagglutinin (HA). Glycans on the head of HA promote virus survival by shielding antigenic sites, but highly glycosylated seasonal IAV are inactivated by soluble lectins of the innate immune system. In 2009, human strains of pandemic H1N1 [A(H1N1)pdm] expressed a single glycosylation site (Asn(104)) on the head of HA. Since then, variants with additional glycosylation sites have been detected, and the location of these sites has been distinct to those of recent seasonal H1N1 strains. We have compared wild-type and reverse-engineered A(H1N1)pdm IAV with differing potential glycosylation sites on HA for sensitivity to collectins and to neutralizing Abs. Addition of a glycan (Asn(136)) to A(H1N1)pdm HA was associated with resistance to neutralizing Abs but did not increase sensitivity to collectins. Moreover, variants expressing Asn(136) showed enhanced growth in A(H1N1)pdm-vaccinated mice, consistent with evasion of Ab-mediated immunity in vivo. Thus, a fine balance exists regarding the optimal pattern of HA glycosylation to facilitate evasion of Ab-mediated immunity while maintaining resistance to lectin-mediated defenses of the innate immune system.

BACKGROUND: Glycosylation on the globular head of the hemagglutinin (HA) protein of influenza virus acts as an important target for recognition and destruction of virus by innate immune proteins of the collectin family. This, in turn, modulates the virulence of different viruses for mice. The role of particular oligosaccharide attachments on the HA in determining sensitivity to collectins has yet to be fully elucidated. METHODS: When comparing the virulence of H3N2 subtype viruses for mice we found that viruses isolated after 1980 were highly glycosylated and induced mild disease in mice. During these studies, we were surprised to find a small plaque variant of strain A/Beijing/353/89 (Beij/89) emerged following infection of mice and grew to high titres in mouse lung. In the current study we have characterized the properties of this small plaque mutant both in vitro and in vivo. RESULTS: Small plaque mutants were recovered following plaquing of lung homogenates from mice infected with influenza virus seed Beij/89. Compared to wild-type virus, small plaque mutants showed increased virulence in mice yet did not differ in their ability to infect or replicate in airway epithelial cells in vitro. Instead, small plaque variants were markedly resistant to neutralization by murine collectins, a property that correlated with the acquisition of an amino acid substitution at residue 246 on the viral HA. We present evidence that this substitution was associated with the loss of an oligosaccharide glycan from the globular head of HA. CONCLUSION: A point mutation in the gene encoding the HA of Beij/89 was shown to ablate a glycan attachment site. This was associated with resistance to collectins and increased virulence in mice.