Individuals with metabolic dysregulation of cellular glycosylation often experience severe influenza disease, with a poor immune response to the virus and low vaccine efficacy. Here, we investigate the consequences of aberrant cellular glycosylation for the glycome and the biology of influenza virus. We transiently induced aberrant N-linked glycosylation in cultured cells with an oligosaccharyltransferase inhibitor, NGI-1. Cells treated with NGI-1 produced morphologically unaltered viable influenza virus with sequence-neutral glycosylation changes (primarily reduced site occupancy) in the hemagglutinin and neuraminidase proteins. Hemagglutinin with reduced glycan occupancy required a higher concentration of surfactant protein D (an important innate immunity respiratory tract collectin) for inhibition compared to that with normal glycan occupancy. Immunization of mice with NGI-1-treated virus significantly reduced antihemagglutinin and antineuraminidase titers of total serum antibody and reduced hemagglutinin protective antibody responses. Our data suggest that aberrant cellular glycosylation may increase the risk of severe influenza as a result of the increased ability of glycome-modified influenza viruses to evade the immune response. IMPORTANCE People with disorders such as cancer, autoimmune disease, diabetes, or obesity often have metabolic dysregulation of cellular glycosylation and also have more severe influenza disease, a reduced immune response to the virus, and reduced vaccine efficacy. Since influenza viruses that infect such people do not show consistent genomic variations, it is generally assumed that the altered biology is mainly related to host factors. However, since host cells are responsible for glycosylation of influenza virus hemagglutinin and neuraminidase, and glycosylation is important for interactions of these proteins with the immune system, the viruses may have functional differences that are not reflected by their genomic sequence. Here, we show that imbalanced cellular glycosylation can modify the viral glycome without genomic changes, leading to reduced innate and adaptive host immune responses to infection. Our findings link metabolic dysregulation of host glycosylation to increased risk of severe influenza and reduced influenza virus vaccine efficacy.

Background: Although ferret antisera used in influenza surveillance did not detect antigenic drift of A(H1N1)pdm09 viruses during 2015-16 season, low vaccine effectiveness was reported in adults. We investigated the immune basis of low responses to circulating A(H1N1)pdm09 viruses following vaccination. Methods: Over 300 paired adult (18-49 yrs) pre and post-vaccination sera collected in 6 seasons (2010-11 to 2015-16) were analyzed by hemagglutination inhibition assays to evaluate the antibody responses to thirteen A(H1N1) viruses circulated from 1977 to 2016. Microneutralization and serum adsorption assays were used to verify 163K- and 223R-specificity of antibodies. Results: Individual antibody profiles to A(H1N1) viruses revealed three priming patterns: USSR/77-, TW/86- or NC/99-priming. Over 20% of adults had reduced titers to cell-propagated circulating 6B.1 and 6B.2 A(H1N1)pdm09 viruses compared to the A/California/07/2009 vaccine virus X-179A. Significantly reduced antibody reactivity to circulating viruses bearing K163Q was only observed in USSR/77-primed cohort, whereas significantly lower reactivity caused by egg-adapted Q223R change was detected across all 3 cohorts. Conclusion: Both 163K-specificity driven by immune priming and 223R-specificity from egg-adapted changes in the vaccine contributed to low responses to circulating A(H1N1)pdm09 viruses following vaccination. Our study highlights the need to incorporate human serology in influenza surveillance and vaccine strain selection.

Specific residues of influenza A virus (IAV) PB1-F2 proteins may enhance inflammation or cytotoxicity. In a series of studies, we evaluated the function of these virulence-associated residues in the context of different IAV subtypes in mice. Here, we demonstrate that, as with the previously assessed pandemic 1968 (H3N2) IAV, PB1-F2 inflammatory residues increase the virulence of H1N1 IAV, suggesting that this effect might be a universal feature. Combining both inflammatory and cytotoxic residues in PB1-F2 enhanced virulence further, compared to either motif alone. Residues from these virulent motifs have been present in natural isolates from human seasonal IAV of all subtypes, but there has been a trend toward a gradual reduction in the number of virulent residues over time. However, human IAV of swine and avian origin tend to have more virulent residues than do the human-adapted seasonal strains, raising the possibility that donation of PB1 segments from these zoonotic viruses may increase the severity of some seasonal human strains. Our data suggest the value of surveillance of virulent residues in both human and animal IAV to predict the severity of influenza season.

PB1-F2 is an accessory protein of most human, avian, swine, equine, and canine influenza A viruses (IAVs). Although it is dispensable for virus replication and growth, it plays significant roles in pathogenesis by interfering with the host innate immune response, inducing death in immune and epithelial cells, altering inflammatory responses, and promoting secondary bacterial pneumonia. The effects of PB1-F2 differ between virus strains and host species. This can at least partially be explained by the presence of multiple PB1-F2 sequence variants, including premature stop codons that lead to the expression of truncated PB1-F2 proteins of different lengths and specific virulence-associated residues that enhance susceptibility to bacterial superinfection. Although there has been a tendency for human seasonal IAV to gradually reduce the number of virulence-associated residues, zoonotic IAVs contain a reservoir of PB1-F2 proteins with full length, virulence-associated sequences. Here, we review the molecular mechanisms by which PB1-F2 may affect influenza virulence, and factors associated with the evolution and selection of this protein.

Avian influenza viruses of the H7 hemagglutinin (HA) subtype present a significant public health threat, as evidenced by the ongoing outbreak of human A(H7N9) infections in China. When evaluated by hemagglutinin inhibition (HI) and micro-neutralization (MN) assays, H7 viruses and vaccines induce lower level of neutralizing antibodies (nAb) than do their seasonal counterparts, making it difficult to develop and evaluate pre-pandemic vaccines. We have previously shown that purified recombinant H7 hemagglutinin (HA) appear to be poorly immunogenic in that they induce low levels of HI and MN antibodies. Here, we immunized mice with whole inactivated reverse genetics reassortant (RG) viruses expressing HA and NA from 3 different H7 viruses [A/Shanghai/2/2013 (H7N9), A/Netherlands/219/2003 (H7N7) and A/New York/107/2003 (H7N2)], or with human A(H1N1)pdm09 [A/California/07/2009-like] or A(H3N2) [A/Perth16/2009] viruses. Mice produced equivalent titers of antibodies to all viruses as measured by ELISA. However, the antibody titers induced by H7 viruses were significantly lower when measured by HI and MN assays. Despite inducing very low levels of nAb, H7 vaccines conferred complete protection against homologous virus challenge in mice, and the serum antibodies directed against the HA head region were capable of mediating protection. The apparently low immunogenicity associated with H7 viruses and vaccines may be at least partly related to measuring antibody titers with the traditional HI and MN assays, which may not provide a true measure of protective immunity associated with H7 immunization. This study underscores the need for development of additional correlates of protection for pre-pandemic vaccines.IMPORTANCE H7 avian influenza viruses present a serious risk to human health. Preparedness efforts include development of pre-pandemic vaccines. For seasonal influenza viruses, protection is correlated with antibody titers measured by hemagglutination inhibition (HI) and virus microneutralization (MN) assays. Since H7 vaccines typically induce low HI and MN titers, they have been considered to be poorly immunogenic. We show that in mice H7 whole inactivated virus (WIV) vaccines were as immunogenic as seasonal WIVs, as they induced similar levels of overall serum antibodies. However, a larger fraction of the antibodies induced by H7 WIV was non-neutralizing in vitro. Nevertheless, the H7 WIV completely protected mice against homologous viral challenge, and antibodies directed against the HA-head were the major contributor toward immune protection. Vaccines against H7 avian influenza viruses may be more effective than HI and virus neutralization assays suggest, and such vaccines may need other methods for evaluation.

Zoonotic A(H7N9) avian influenza viruses emerged in China in 2013 and continue to be a threat to human public health, having infected over 800 individuals with a mortality rate approaching 40%. Treatment options for people infected with A(H7N9) include the use of neuraminidase (NA) inhibitors. However, like other influenza viruses, A(H7N9) can become resistant to these drugs. The use of monoclonal antibodies is a rapidly developing strategy for controlling influenza virus infection. Here we generated a murine monoclonal antibody (3c10-3) directed against the NA of A(H7N9) and show that prophylactic systemic administration of 3c10-3 fully protected mice from lethal challenge with wild-type A/Anhui/1/2013 (H7N9). Further, post-infection treatment with a single systemic dose of 3c10-3 at either 24, 48 or 72 h post A(H7N9) challenge resulted in both dose- and time-dependent protection of up to 100% of mice, demonstrating therapeutic potential for 3c10-3. Epitope mapping revealed that 3c10-3 binds near the enzyme active site of NA, and functional characterization showed that 3c10-3 inhibits the enzyme activity of NA and restricts the cell-to-cell spread of the virus in cultured cells. Affinity analysis also revealed that 3c10-3 binds equally well to recombinant NA of wild-type A/Anhui/1/2013 and to a variant NA carrying a R289K mutation known to infer NAI resistance. These results suggest that 3c10-3 has the potential to be used as a therapeutic to treat A(H7N9) infections either as an alternative to, or in combination with, current NA antiviral inhibitors.

Avian H7N9 influenza virus infection with fatal outcomes continues to pose a pandemic threat and highly immunogenic vaccines are urgently needed. In this report we show that baculovirus-derived recombinant H7 hemagglutinin protein, when delivered with RIG-I ligand, induced enhanced antibody and T cell responses and conferred protection against lethal challenge with a homologous H7N9 virus. These findings indicate the potential utility of RIG-I ligands as vaccine adjuvants to increase the immunogenicity of recombinant H7 hemagglutinin.

Influenza A viruses (IAV) express the PB1-F2 protein from an alternate reading frame within the PB1 gene segment. The roles of PB1-F2 are not well understood, but appear to involve modulation of host cell responses. As shown in previous studies, we find that PB1-F2 of mammalian IAV frequently have premature stop codons that are expected to cause truncations of the protein, whereas avian IAV usually express a full-length 90 amino acid PB1-F2. However, in contrast to other avian IAV, recent isolates of highly pathogenic H5N1 influenza viruses had a high proportion of PB1-F2 truncations (15% since 2010; 61% of isolates in 2013) due to several independent mutations that have persisted and expanded in circulating viruses. One natural H5N1 IAV containing a mutated PB1-F2 start codon (i.e., lacking ATG) was 1000-fold more virulent for BALB/c mice than a closely-related H5N1 containing intact PB1-F2. In vitro, we detected expression of an in-frame protein (C-terminal PB1-F2) from downstream ATGs in PB1-F2 plasmids lacking the well-conserved ATG start codon. Transient expression of full-length, truncated (25 amino acids), and PB1-F2 lacking the initiating ATG in mammalian and avian cells had no effect on cell apoptosis or interferon expression in human lung epithelial cells. Full length and C-terminal PB1-F2 mutants co-localized with mitochondria in A549 cells. Close monitoring of alterations of PB1-F2 and their frequency in contemporary avian H5N1 viruses should continue, as such changes may be markers for mammalian virulence. IMPORTANCE: Although most avian influenza viruses are harmless for humans, some (such as highly pathogenic H5N1 avian influenza viruses) are capable of infecting humans and causing severe disease with a high mortality rate. A number of risk factors potentially associated with adaptation to mammalian infection have been noted. Here we demonstrate that the protein PB1-F2 is frequently truncated in recent isolates of highly pathogenic H5N1 viruses. Truncation of PB1-F2 has been proposed to act as an adaptation to mammalian infection. We show that some forms of truncation of PB1-F2 may be associated with increased virulence in mammals. Our data support the assessment of PB1-F2 truncations for genomic surveillance of influenza viruses.

BACKGROUND: Vaccines against avian influenza viruses often require high hemagglutinin (HA) doses or adjuvants to achieve serological titers associated with protection against disease. In particular, viruses of the H7 subtype frequently do not induce strong antibody responses following immunization. OBJECTIVES: To evaluate whether poor immunogenicity of H7 viruses is an intrinsic property of the H7 hemagglutinin. METHODS: We compared the immunogenicity, in naive mice, of purified recombinant HA from two H7 viruses [A/Netherlands/219/2003(H7N7) and A/New York/107/2003(H7N2)] to that of HA from human pandemic [A/California/07/2009(H1N1pdm09)] and seasonal [A/Perth16/2009(H3N2)] viruses. RESULTS: After two intramuscular injections with purified hemagglutinin, mice produced antibodies to all HAs, but the response to the human virus HAs was greater than to H7 HAs. The difference was relatively minor when measured by ELISA, greater when measured by hemagglutination inhibition assays, and more marked still by microneutralization assays. H7 HAs induced little or no neutralizing antibody response in mice at either dose tested. Antibodies induced by H7 were of significantly lower avidity than for H3 or H1N1pdm09. CONCLUSIONS: We conclude that H7 HAs may be intrinsically less immunogenic than HA from seasonal human influenza viruses.

Mice are widely used for studying influenza virus pathogenesis and immunology because of their low cost, the wide availability of mouse-specific reagents, and the large number of mouse strains available, including knockout and transgenic strains. However, mice do not fully recapitulate the signs of influenza infection of humans: transmission of influenza between mice is much less efficient than in humans, and influenza viruses often require adaptation before they are able to efficiently replicate in mice. In the process of mouse adaptation, influenza viruses acquire mutations that enhance their ability to attach to mouse cells, replicate within the cells, and suppress immunity, among other functions. Many such mouse-adaptive mutations have been identified, covering all 8 genomic segments of the virus. Identification and analysis of these mutations have provided insight into the molecular determinants of influenza virulence and pathogenesis, not only in mice but also in humans and other species. In particular, several mouse-adaptive mutations of avian influenza viruses have proved to be general mammalian-adaptive changes that are potential markers of pre-pandemic viruses. As well as evaluating influenza pathogenesis, mice have also been used as models for evaluation of novel vaccines and anti-viral therapies. Mice can be a useful animal model for studying influenza biology as long as differences between human and mice infections are taken into account.

Ferrets are a useful animal model for human influenza virus infections, since they closely mimic the pathogenesis of influenza viruses observed in humans. However, a lack of reagents, especially for flow cytometry of immune cell subsets, has limited research in this model. Here we use a panel of primarily species cross-reactive antibodies to identify ferret T cells, cytotoxic T lymphocytes (CTL), B cells, and granulocytes in peripheral blood. Following infection with seasonal H3N2 or H1N1pdm09 influenza viruses, these cell types showed rapid and dramatic changes in frequency, even though clinically the infections were mild. The loss of B cells and CD4 and CD8 T cells, and the increase in neutrophils, were especially marked 1-2 days after infection, when about 90% of CD8+ T cells disappeared from the peripheral blood. The different virus strains led to different kinetics of leukocyte subset alterations. Vaccination with homologous vaccine reduced clinical symptoms slightly, but led to a much more rapid return to normal leukocyte parameters. Assessment of clinical symptoms may underestimate the effectiveness of influenza vaccine in restoring homeostasis.