Pre-existing antibodies directed against a tetramerizing domain enhance the immune response against artificially stabilized soluble tetrameric influenza neuraminidase.

The neuraminidase (NA) is an abundant antigen at the surface of influenza virions. Recent studies have highlighted the immune-protective potential of NA against influenza and defined anti-NA antibodies as an independent correlate of protection. Even though NA head domain changes at a slightly slower pace than hemagglutinin (HA), NA is still subject to antigenic drift, and therefore an NA-based influenza vaccine antigen may have to be updated regularly and thus repeatedly administered. NA is a tetrameric type II membrane protein, which readily dissociates into dimers and monomers when expressed in a soluble form. By using a tetramerizing zipper, such as the tetrabrachion (TB) from Staphylothermus marinus, it is possible to stabilize soluble NA in its active tetrameric conformation, an imperative for the optimal induction of protective NA inhibitory antibodies. The impact of repetitive immunizations with TB-stabilized antigens on the immunogenicity of soluble TB-stabilized NA is unknown. We demonstrate that TB is immunogenic in mice. Interestingly, preexisting anti-TB antibodies enhance the anti-NA antibody response induced by immunization with TB-stabilized NA. This immune-enhancing effect was transferable by serum and operated independently of activating Fcγ receptors. We also demonstrate that priming with TB-stabilized NA antigens, enhances the NA inhibitory antibody responses against a heterosubtypic TB-stabilized NA. These findings have implications for the clinical development of oligomeric vaccine antigens that are stabilized by a heterologous oligomerizing domain.

Antibodies directed towards neuraminidase restrict influenza virus replication in primary human bronchial epithelial cells.

Influenza neuraminidase (NA) is implicated in various aspects of the virus replication cycle and therefore is an attractive target for vaccination and antiviral strategies. Here we investigated the potential for NA-specific antibodies to interfere with A(H1N1)pdm09 replication in primary human airway epithelial (HAE) cells. Mouse polyclonal anti-NA sera and a monoclonal antibody could block initial viral entry into HAE cells as well as egress from the cell surface. NA-specific polyclonal serum also reduced virus replication across multiple rounds of infection. Restriction of virus entry correlated with the ability of the serum or monoclonal antibody to mediate neuraminidase inhibition (NI). Finally, human sera with NI activity against the N1 of A(H1N1)pdm09 could decrease H6N1 virus infection of HAE cells, highlighting the potential contribution of anti-NA antibodies in the control of influenza virus infection in humans.

In Vivo Therapy with M2e-Specific IgG Selects for an Influenza A Virus Mutant with Delayed Matrix Protein 2 Expression.

The ectodomain of matrix protein 2 (M2e) of influenza A viruses is a universal influenza A vaccine candidate. Here, we report potential evasion strategies of influenza A viruses under in vivo passive anti-M2e IgG immune selection pressure in severe combined immune-deficient (SCID) mice. A/Puerto Rico/8/34-infected SCID mice were treated with the M2e-specific mouse IgG monoclonal antibodies (MAbs) MAb 65 (IgG2a) or MAb 37 (IgG1), which recognize amino acids 5 to 15 in M2e, or with MAb 148 (IgG1), which binds to the invariant N terminus of M2e. Treatment of challenged SCID mice with any of these MAbs significantly prolonged survival compared to isotype control IgG treatment. Furthermore, M2e-specific IgG2a protected significantly better than IgG1, and even resulted in virus clearance in some of the SCID mice. Deep sequencing analysis of viral RNA isolated at different time points after treatment revealed that the sequence variation in M2e was limited to P10H/L and/or I11T in anti-M2e MAb-treated mice. Remarkably, in half of the samples isolated from moribund MAb 37-treated mice and in all MAb 148-treated mice, virus was isolated with a wild-type M2 sequence but with nonsynonymous mutations in the polymerases and/or the hemagglutinin genes. Some of these mutations were associated with delayed M2 and other viral gene expression and with increased resistance to anti-M2e MAb treatment of SCID mice. Treatment with M2e-specific MAbs thus selects for viruses with limited variation in M2e. Importantly, influenza A viruses may also undergo an alternative escape route by acquiring mutations that result in delayed wild-type M2 expression. IMPORTANCE Broadly protective influenza vaccine candidates may have a higher barrier to immune evasion compared to conventional influenza vaccines. We used Illumina MiSeq deep sequence analysis to study the mutational patterns in A/Puerto Rico/8/34 viruses that evolve in chronically infected SCID mice that were treated with different M2e-specific MAbs. We show that under these circumstances, viruses emerged in vivo with mutations in M2e that were limited to positions 10 and 11. Moreover, we discovered an alternative route for anti-M2e antibody immune escape, in which a virus is selected with wild-type M2e but with mutations in other gene segments that result in delayed M2 and other viral protein expression. Delayed expression of the viral antigen that is targeted by a protective antibody thus represents an influenza virus immune escape mechanism that does not involve epitope alterations.

Oral delivery of Escherichia coli persistently infected with M2e-displaying bacteriophages partially protects against influenza A virus.

We describe a novel live oral vaccine type. Conceptually, this vaccine is based on a non-lytic, recombinant filamentous bacteriophage that displays an antigen of interest. To provide proof of concept we used the amino-terminal part of a conserved influenza A virus epitope, i.e. matrix protein 2 ectodomain (M2e) residues 2 to 16, as the antigen of interest. Rather than using the phages as purified virus-like particles as a vaccine, these phages were delivered to intestinal Peyer's patches as a live bacterium-phage combination that comprises Escherichia coli cells that conditionally express invasin derived from Yersinia pseudotuberculosis. Invasin-expressing E. coli cells were internalized by mammalian Hep-2 cells in vitro and adhered to mouse intestinal microfold (M) cells ex vivo. Invasin-expressing E. coli cells were permissive for recombinant filamentous bacteriophage f88 that displays M2e and became persistently infected. Oral administration of the live engineered E. coli-invasin-phage combination to mice induced M2e-specific serum IgG antibodies. Mice that had been immunized with invasin-expressing E. coli cells that carried M2e2-16 displaying fd phages seroconverted to M2e and showed partial protection against challenge with influenza A virus. Oral delivery of a live vaccine comprising a bacterial host that is targeted to Peyer's patches and is persistently infected with an antigen-displaying phage, can thus be exploited as an oral vaccine.

Neutralizing inhibitors in the airways of naïve ferrets do not play a major role in modulating the virulence of H3 subtype influenza A viruses.

Many insights regarding the pathogenesis of human influenza A virus (IAV) infections have come from studies in mice and ferrets. Surfactant protein (SP)-D is the major neutralizing inhibitor of IAV in mouse airway fluids and SP-D-resistant IAV mutants show enhanced virus replication and virulence in mice. Herein, we demonstrate that sialylated glycoproteins, rather than SP-D, represent the major neutralizing inhibitors against H3 subtype viruses in airway fluids from naïve ferrets. Moreover, while resistance to neutralizing inhibitors is a critical factor in modulating virus replication and disease in the mouse model, it does not appear to be so in the ferret model, as H3 mutants resistant to either SP-D or sialylated glycoproteins in ferret airway fluids did not show enhanced virulence in ferrets. These data have important implications for our understanding of pathogenesis and immunity to human IAV infections in these two widely used animal models of infection.

Endogenous Murine BST-2/Tetherin Is Not a Major Restriction Factor of Influenza A Virus Infection.

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.

A GFP expressing influenza A virus to report in vivo tropism and protection by a matrix protein 2 ectodomain-specific monoclonal antibody.

The severity of influenza-related illness is mediated by many factors, including in vivo cell tropism, timing and magnitude of the immune response, and presence of pre-existing immunity. A direct way to study cell tropism and virus spread in vivo is with an influenza virus expressing a reporter gene. However, reporter gene-expressing influenza viruses are often attenuated in vivo and may be genetically unstable. Here, we describe the generation of an influenza A virus expressing GFP from a tri-cistronic NS segment. To reduce the size of this engineered gene segment, we used a truncated NS1 protein of 73 amino acids combined with a heterologous dimerization domain to increase protein stability. GFP and nuclear export protein coding information were fused in frame with the truncated NS1 open reading frame and separated from each other by 2A self-processing sites. The resulting PR8-NS1(1-73)GFP virus was successfully rescued and replicated as efficiently as the parental PR8 virus in vitro and was slightly attenuated in vivo. Flow cytometry-based monitoring of cells isolated from PR8-NS1(1-73)GFP virus infected BALB/c mice revealed that GFP expression peaked on day two in all cell types tested. In particular respiratory epithelial cells and myeloid cells known to be involved in antigen presentation, including dendritic cells (CD11c+) and inflammatory monocytes (CD11b+ GR1+), became GFP positive following infection. Prophylactic treatment with anti-M2e monoclonal antibody or oseltamivir reduced GFP expression in all cell types studied, demonstrating the usefulness of this reporter virus to analyze the efficacy of antiviral treatments in vivo. Finally, deep sequencing analysis, serial in vitro passages and ex vivo analysis of PR8-NS1(1-73)GFP virus, indicate that this virus is genetically and phenotypically stable.

Playing hide and seek: how glycosylation of the influenza virus hemagglutinin can modulate the immune response to 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.

A single amino acid substitution in the hemagglutinin of H3N2 subtype influenza A viruses is associated with resistance to the long pentraxin PTX3 and enhanced virulence in mice.

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.

Antibodies mediate formation of neutrophil extracellular traps in the middle ear and facilitate secondary pneumococcal otitis media.

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.

Serum amyloid P is a sialylated glycoprotein inhibitor of influenza A viruses.

Members of the pentraxin family, including PTX3 and serum amyloid P component (SAP), have been reported to play a role in innate host defence against a range of microbial pathogens, yet little is known regarding their antiviral activities. In this study, we demonstrate that human SAP binds to human influenza A virus (IAV) strains and mediates a range of antiviral activities, including inhibition of IAV-induced hemagglutination (HA), neutralization of virus infectivity and inhibition of the enzymatic activity of the viral neuraminidase (NA). Characterization of the anti-IAV activity of SAP after periodate or bacterial sialidase treatment demonstrated that α(2,6)-linked sialic acid residues on the glycosidic moiety of SAP are critical for recognition by the HA of susceptible IAV strains. Other proteins of the innate immune system, namely human surfactant protein A and porcine surfactant protein D, have been reported to express sialylated glycans which facilitate inhibition of particular IAV strains, yet the specific viral determinants for recognition of these inhibitors have not been defined. Herein, we have selected virus mutants in the presence of human SAP and identified specific residues in the receptor-binding pocket of the viral HA which are critical for recognition and therefore susceptibility to the antiviral activities of SAP. Given the widespread expression of α(2,6)-linked sialic acid in the human respiratory tract, we propose that SAP may act as an effective receptor mimic to limit IAV infection of airway epithelial cells.

Addition of glycosylation to influenza A virus hemagglutinin modulates antibody-mediated recognition of H1N1 2009 pandemic viruses.

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.

Influenza-induced inflammation drives pneumococcal otitis media.

Influenza A virus (IAV) predisposes individuals to secondary infections with the bacterium Streptococcus pneumoniae (the pneumococcus). Infections may manifest as pneumonia, sepsis, meningitis, or otitis media (OM). It remains controversial as to whether secondary pneumococcal disease is due to the induction of an aberrant immune response or IAV-induced immunosuppression. Moreover, as the majority of studies have been performed in the context of pneumococcal pneumonia, it remains unclear how far these findings can be extrapolated to other pneumococcal disease phenotypes such as OM. Here, we used an infant mouse model, human middle ear epithelial cells, and a series of reverse-engineered influenza viruses to investigate how IAV promotes bacterial OM. Our data suggest that the influenza virus HA facilitates disease by inducing a proinflammatory response in the middle ear cavity in a replication-dependent manner. Importantly, our findings suggest that it is the inflammatory response to IAV infection that mediates pneumococcal replication. This study thus provides the first evidence that inflammation drives pneumococcal replication in the middle ear cavity, which may have important implications for the treatment of pneumococcal OM.

Cross-reactive influenza-specific antibody-dependent cellular cytotoxicity antibodies in the absence of neutralizing antibodies.

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.

Glycosylation of the hemagglutinin modulates the sensitivity of H3N2 influenza viruses to innate proteins in airway secretions and virulence in mice.

Collectins in airway fluids and membrane-associated lectins such as the macrophage mannose receptor (MMR) recognize mannose-rich glycans on the envelope glycoproteins of influenza A viruses. In this study, we used a reverse genetic approach to examine the role of particular N-linked glycosylation sites on the hemagglutinin (HA) of A/Beijing/353/89 (Beij/89, H3N2) in determining sensitivity to lectin-mediated immune defenses and virulence in mice. We generated 7:1 reassortant viruses on an A/PR/8/34 'backbone' with Beij/89 HA or HA lacking one or more glycosylation sites. Asn(165) was an important determinant of sensitivity to mouse collectins and virulence but did not alter susceptibility of airway macrophages to infection. Removal of both Asn(165) and Asn(246) led to a further increase in virulence, characterized by enhanced virus replication, pulmonary inflammation and vascular leak. These studies define the importance of particular glycans on H3 HA in determining sensitivity to airway collectins and virulence in mice.

Gr-1+ cells, but not neutrophils, limit virus replication and lesion development following flank infection of mice with herpes simplex virus type-1.

Neutrophils are prominent in epidermal and dermal layers of human herpetic lesions and are rapidly recruited into the skin follow epidermal abrasion and infection of mice with herpes simplex virus type-1 (HSV-1). Herein, we demonstrate that early production of neutrophil-attracting chemokines KC/MIP-2 is associated with transient recruitment of neutrophils into the skin of HSV-1-infected mice in temporal association with the development of herpetic lesions. Treatment of HSV-1-infected mice with a Ly6G-specific mAb induced systemic neutropenia, but surprisingly did not alter virus replication or lesion development. In contrast, depletion of Gr-1(+) cells with mAb RB6-8C5 led to enhanced virus growth and lesion severity. Thus, while neutrophils are prominent in zosteriform lesions of HSV-1-infected mice, they do not appear to play a major role in controlling virus replication or lesion development and/or healing. In contrast, Gr-1(+) cells limit both virus replication and lesion development in the zosteriform model.

Pandemic H1N1 influenza A viruses are resistant to the antiviral activities of innate immune proteins of the collectin and pentraxin superfamilies.

Acquired immune responses elicited to recent strains of seasonal H1N1 influenza viruses provide limited protection against emerging A(H1N1) pandemic viruses. Accordingly, pre-existing or rapidly induced innate immune defenses are of critical importance in limiting early infection. Respiratory secretions contain proteins of the innate immune system, including members of the collectin and pentraxin superfamilies. These mediate potent antiviral activity and act as an initial barrier to influenza infection. In this study, we have examined the sensitivity of H1N1 viruses, including pandemic virus strains, for their sensitivity to collectins (surfactant protein [SP]-D and mannose-binding lectin [MBL]) and to the pentraxin PTX3. Human SP-D and MBL inhibited virus-induced hemagglutinating activity, blocked the enzymatic activity of the viral neuraminidase, and neutralized the ability of H1N1 viruses to infect human respiratory epithelial cells in a manner that correlated with the degree of glycosylation in the globular head of the hemagglutinin. Recent seasonal H1N1 viruses expressed three to four N-glycosylation sequons on the head of hemagglutinin and were very sensitive to inhibition by SP-D or MBL, whereas A(H1N1) pandemic viruses expressed a single N-glycosylation sequon and were resistant to either collectin. Of interest, both seasonal and pandemic H1N1 viruses were resistant to PTX3. Thus, unlike recent seasonal H1N1 strains of influenza virus, A(H1N1) pandemic viruses are resistant to the antiviral activities of innate immune proteins of the collectin superfamily.

Loss of a single N-linked glycan from the hemagglutinin of influenza virus is associated with resistance to collectins and increased virulence in mice.

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.

Antiviral activity of the long chain pentraxin PTX3 against influenza viruses.

Proteins of the innate immune system can act as natural inhibitors of influenza virus, limiting growth and spread of the virus in the early stages of infection before the induction of adaptive immune responses. In this study, we identify the long pentraxin PTX3 as a potent innate inhibitor of influenza viruses both in vitro and in vivo. Human and murine PTX3 bound to influenza virus and mediated a range of antiviral activities, including inhibition of hemagglutination, neutralization of virus infectivity and inhibition of viral neuraminidase. Antiviral activity was associated with binding of the viral hemagglutinin glycoprotein to sialylated ligands present on PTX3. Using a mouse model we found PTX3 to be rapidly induced following influenza infection and that PTX3-/- mice were more susceptible than wild-type mice to infection by PTX3-sensitive virus strains. Therapeutic treatment of mice with human PTX3 promoted survival and reduced viral load in the lungs following infection with PTX3-sensitive, but not PTX3-resistant, influenza viruses. Together, these studies describe a novel antiviral role for PTX3 in early host defense against influenza infections both in vitro and in vivo and describe the therapeutic potential of PTX3 in ameliorating disease during influenza infection.