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Summary: Type I interferon (IFNα/β) pathways are fine-tuned to elicit antiviral protection while minimizing immunopathology; however, the initiating stimuli, target tissues, and underlying mechanisms are unclear. Using models of physiological and dysregulated IFNα/β receptor (IFNAR1) surface expression, we show here that IFNAR1-dependent signals set the steady-state IFN signature in both hematopoietic and stromal cells. Increased IFNAR1 levels promote a lung environment refractory to early influenza virus replication by elevating the baseline interferon signature. Commensal microbiota drive the IFN signature specifically in lung stroma, as shown by antibiotic treatment and fecal transplantation. Bone marrow chimera experiments identify lung stromal cells as crucially important for early antiviral immunity and stroma-immune cell interaction for late antiviral resistance. We propose that the microbiota-driven interferon signature in lung epithelia impedes early virus replication and that IFNAR1 surface levels fine-tune this signature. Our findings highlight the interplay between bacterial and viral exposure, with important implications for antibiotic use. : Bradley, Finsterbusch, et al. identify lung stroma as the target of microbiota-driven signals that set the interferon signature in these cells. Antibiotic treatment reduces gut microbiota and the lung stromal interferon signature and facilitates early influenza virus replication in lung epithelia, effects that can be reversed by fecal transplantation. Keywords: type I interferon (IFN), tonic IFN signaling, lung, microbiota, influenza, anti-viral protection, gut, antibiotics

The highly pathogenic avian influenza (HPAI) H5N1 influenza virus has been a public health concern for more than a decade because of its frequent zoonoses and the high case fatality rate associated with human infections. Severe disease following H5N1 influenza infection is often associated with dysregulated host innate immune response also known as cytokine storm but the virological and cellular basis of these responses has not been clearly described. We rescued a series of 6:2 reassortant viruses that combined a PR8 HA/NA pairing with the internal gene segments from human adapted H1N1, H3N2, or avian H5N1 viruses and found that mice infected with the virus with H5N1 internal genes suffered severe weight loss associated with increased lung cytokines but not high viral load. This phenotype did not map to the NS gene segment, and NS1 protein of H5N1 virus functioned as a type I IFN antagonist as efficient as NS1 of H1N1 or H3N2 viruses. Instead we discovered that the internal genes of H5N1 virus supported a much higher level of replication of viral RNAs in myeloid cells in vitro, but not in epithelial cells and that this was associated with high induction of type I IFN in myeloid cells. We also found that in vivo during H5N1 recombinant virus infection cells of haematopoetic origin were infected and produced type I IFN and proinflammatory cytokines. Taken together our data infer that human and avian influenza viruses are differently controlled by host factors in alternative cell types; internal gene segments of avian H5N1 virus uniquely drove high viral replication in myeloid cells, which triggered an excessive cytokine production, resulting in severe immunopathology., Author summary Some avian influenza viruses, including highly pathogenic H5N1 virus, cause severe disease in humans and in experimental animal models associated with excessive cytokine production. We aimed to understand the virological mechanism behind the cytokine storm, and particularly the contribution of internal gene segments that encode the viral polymerase and the non-structural proteins, since these might be retained in a pandemic virus. We found that the internal genes from an H5N1 avian influenza virus allowed virus to replicate to strikingly higher levels in myeloid cells compared to internal genes of human adapted strains. The higher viral RNA levels did not lead to higher viral load but drove excessive cytokine production and more severe outcome in infected mice. The remarkable difference in viral replication in myeloid cells was not observed in lung epithelial cells, suggesting that cell type specific differences in host factors were responsible. Understanding the molecular basis of excessive viral replication in myeloid cells may guide future therapeutic options for viruses that have recently crossed into humans from birds.

To examine the range of selective processes that potentially operate when poorly binding influenza viruses adapt to replicate more efficiently in alternative environments, we passaged a virus containing an attenuating mutation in the hemagglutinin (HA) receptor binding site in mice and characterized the resulting mutants with respect to the structural locations of mutations selected, the replication phenotypes of the viruses, and their binding properties on glycan microarrays. The initial attenuated virus had a tyrosine-to-phenylalanine mutation at HA1 position 98 (Y98F), located in the receptor binding pocket, but viruses that were selected contained second-site pseudoreversion mutations in various structural locations that revealed a range of molecular mechanisms for modulating receptor binding that go beyond the scope that is generally mapped using receptor specificity mutants. A comparison of virus titers in the mouse respiratory tract versus MDCK cells in culture showed that the mutants displayed distinctive replication properties depending on the system, but all were less attenuated in mice than the Y98F virus. An analysis of receptor binding properties confirmed that the initial Y98F virus bound poorly to several different species of erythrocytes, while all mutants reacquired various degrees of hemagglutination activity. Interestingly, both the Y98F virus and pseudoreversion mutants were shown to bind very inefficiently to standard glycan microarrays containing an abundance of binding substrates for most influenza viruses that have been characterized to date, provided by the Consortium for Functional Glycomics. The viruses were also examined on a recently developed microarray containing glycans terminating in sialic acid derivatives, and limited binding to a potentially interesting subset of glycans was revealed. The results are discussed with respect to mechanisms for HA-mediated receptor binding, as well as regarding the species of molecules that may act as receptors for influenza virus on host cell surfaces.

The HPIV2 V protein inhibits type I interferon (IFN) induction and signaling. To manipulate the V protein, whose coding sequence overlaps that of the polymerase-associated phosphoprotein (P), without altering the P protein, we generated an HPIV2 virus in which P and V are expressed from separate genes (rHPIV2-P+V). rHPIV2-P+V replicated like HPIV2-WT in vitro and in non-human primates. HPIV2-P+V was modified by introducing two separate mutations into the V protein to create rHPIV2-L101E/L102E and rHPIV2-Delta122-127. In contrast to HPIV2-WT, both mutant viruses were unable to degrade STAT2, leaving virus-infected cells susceptible to IFN. Neither mutant, nor HPIV2-WT, induced significant amounts of IFN-beta in infected cells. Surprisingly, neither rHPIV2-L101E/L102E nor rHPIV2-Delta122-127 was attenuated in two species of non-human primates. This indicates that loss of HPIV2's ability to inhibit IFN signaling is insufficient to attenuate virus replication in vivo as long as IFN induction is still inhibited.

The use of viral vectors as vaccine candidates has shown promise against a number of pathogens. However, preexisting immunity to these vectors is a concern that must be addressed when deciding which viruses are suitable for use. A number of properties, including the existence of antigenically distinct subtypes, make influenza viruses attractive candidates for use as viral vectors. Here, we evaluate the ability of influenza viral vectors containing inserts of foreign pathogens to elicit antibody and CD8+ T cell responses against these foreign antigens in the presence of preexisting immunity to influenza virus in mice. Specifically, responses to an H3N1-based vector expressing a 90 amino acid polypeptide derived from the protective antigen (PA) of Bacillus anthracis or an H1N1-based vector containing a CD8+ T cell epitope from the glycoprotein (GP) of lymphocytic choriomeningitis virus were evaluated following infections with either homosubtypic or heterosubtypic influenza viruses. We found that mice previously infected with influenza viruses, even those expressing HA and NA proteins of completely different subtypes, were severely compromised in their ability to mount an immune response against the inserted epitopes. This inhibition was demonstrated to be mediated by CD8+ T cells, which recognize multiple strains of influenza viruses. These CD8+ T cells were further shown to protect mice from a lethal challenge by a heterologous influenza subtype. The implication of these data for the use of influenza virus vectors and influenza vaccination in general are discussed.

The replicative properties of influenza virus hemagglutinin (HA) mutants with altered receptor binding characteristics were analyzed following intranasal inoculation of mice. Among the mutants examined was a virus containing a Y98F substitution at a conserved position in the receptor binding site that leads to a 20-fold reduction in binding. This mutant can replicate as well as wild-type (WT) virus in MDCK cells and in embryonated chicken eggs but is highly attenuated in mice, exhibiting titers in lungs more than 1,000-fold lower than those of the WT. The capacity of the Y98F mutant to induce antibody responses and the structural locations of HA reversion mutations are examined.

Previously, we identified several attenuating mutations in the L polymerase protein of human parainfluenza virus type 2 (HPIV2) and genetically stabilized those mutations using reverse genetics [Nolan SM, Surman S, Amaro-Carambot E, Collins PL, Murphy BR, Skiadopoulos MH. Live-attenuated intranasal parainfluenza virus type 2 vaccine candidates developed by reverse genetics containing L polymerase protein mutations imported from heterologous paramyxoviruses. Vaccine 2005;39(23):4765-74]. Here we describe the discovery of an attenuating mutation at nucleotide 15 (15(T-->C)) in the 3' genomic promoter that was also present in the previously characterized mutants. We evaluated the properties of this promoter mutation alone and in various combinations with the L polymerase mutations. Amino acid substitutions at L protein positions 460 (460A or 460P) or 948 (948L), or deletion of amino acids 1724 and 1725 (Delta1724), each conferred a temperature sensitivity (ts) phenotype whereas the 15(T-->C) mutation did not. The 460A and 948L mutations each contributed to restricted replication in the lower respiratory tract of African green monkeys, but the Delta1724 mutation increased attenuation only in certain combinations with other mutations. We constructed two highly attenuated viruses, rV94(15C)/460A/948L and rV94(15C)/948L/Delta1724, that were immunogenic and protective against challenge with wild-type HPIV2 in African green monkeys and, therefore, appear to be suitable for evaluation in humans.

The influenza B virus encodes a unique protein, NB, a membrane protein whose function in the replication cycle is not, as yet, understood. We engineered a recombinant influenza B virus lacking NB expression, with no concomitant difference in expression or activity of viral neuraminidase (NA) protein, an important caveat since NA is encoded on the same segment and initiated from a start codon just 4 nt downstream of NB. Replication of the virus lacking NB was not different to wild-type virus with full-length NB in clonal immortalized or complex primary cell cultures. In the mouse model, virus lacking NB induced slightly lower IFN-α levels in infected lungs, but this did not affect virus titres or weight loss. In ferrets infected with a mixture of viruses that did or did not express NB, there was no fitness advantage for the virus that retained NB. Moreover, virus lacking NB protein was transmitted following respiratory droplet exposure of sentinel animals. These data suggest no role for NB in supporting replication or transmission in vivo in this animal model. The role of NB and the nature of selection to retain it in all natural influenza B viruses remain unclear.

Influenza viruses bind to host cell surface glycans containing terminal sialic acids, but as studies on influenza binding become more sophisticated, it is becoming evident that although sialic acid may be necessary, it is not sufficient for productive binding. To better define endogenous glycans that serve as viral receptors, we have explored glycan recognition in the pig lung, because influenza is broadly disseminated in swine, and swine have been postulated as an intermediary host for the emergence of pandemic strains. For these studies, we used the technology of “shotgun glycomics” to identify natural receptor glycans. The total released N- and O-glycans from pig lung glycoproteins and glycolipid-derived glycans were fluorescently tagged and separated by multidimensional HPLC, and individual glycans were covalently printed to generate pig lung shotgun glycan microarrays. All viruses tested interacted with one or more sialylated N-glycans but not O-glycans or glycolipid-derived glycans, and each virus demonstrated novel and unexpected differences in endogenous N-glycan recognition. The results illustrate the repertoire of specific, endogenous N-glycans of pig lung glycoproteins for virus recognition and offer a new direction for studying endogenous glycan functions in viral pathogenesis.

Viral vectors based on influenza virus, rabies virus (RV), and vaccinia virus (VV) were used to express large polypeptide segments derived from the Bacillus anthracis protective antigen (PA). For the infectious influenza virus vector and recombinant VV constructs, the receptor binding domain (RBD or domain 4) or the lethal and edema factor binding domain (LEF or domain 1′) were engineered into functional chimeric hemagglutinin (HA) glycoproteins. In the case of the RV vector, the viral glycoprotein (G) was used as a carrier for RBD in an inactivated form of the vector. These constructs were examined by using multiple homologous and heterologous prime/boost immunization regimens in order to optimize the induction of α-PA antibody responses. Several immunization combinations were shown to induce high titers of antibody recognizing the anthrax RBD and LEF domains, as well as the full-length PA protein in mice. The heterologous prime/boost immunization regimens that involved an initial intranasal administration of a live influenza virus vector, followed by an intramuscular boost with either the killed RV vector or the VV vector, were particularly effective, inducing antigen-specific antibodies at levels severalfold higher than homologous or alternative heterologous protocols. Furthermore, sera from several groups of the immunized mice demonstrated neutralization activity in an in vitro anthrax toxin neutralization assay. In some cases, such toxin-neutralizing activity was notably high, indicating that the mechanisms by which immunity is primed by live influenza virus vectors may have beneficial properties.

Avian influenza viruses (AIV) are an important emerging threat to public health. It is thought that sialic acid (sia) receptors are barriers in cross-species transmission where the binding preferences of AIV and human influenza viruses are sias α2,3 versus α2,6, respectively. In this study, we show that a normal fully differentiated, primary human bronchial epithelial cell model is readily infected by low pathogenic H5N1, H5N2 and H5N3 AIV, which primarily bind to sia α2,3 moieties, and replicate in these cells independent of specific sias on the cell surface. NHBE cells treated with neuraminidase prior to infection are infected by AIV despite removal of sia α2,3 moieties. Following AIV infection, higher levels of IP-10 and RANTES are secreted compared to human influenza virus infection, indicating differential chemokine expression patterns, a feature that may contribute to differences in disease pathogenesis between avian and human influenza virus infections in humans.