Sialidase inhibitors related to zanamivir: synthesis and biological evaluation of 4H-pyran 6-ether and ketone

Synthesis of 5R-Acetamido-4S-amino-4H-pyran-6R-O-( -ethyl)propyl and 6R-(1-oxo-2-ethyl)butyl 2-carboxylic acids (4 and 5) and their evaluation as inhibitors of influenza virus sialidase is described. Both compounds showed good inhibitory activity with marked selectivity for influenza A sialidase.     

Human CD8+ and CD4+ T lymphocyte memory to influenza A viruses of swine and avian species.

Recently, an avian influenza A virus (A/Hong Kong/156/97, H5N1) was isolated from a young child who had a fatal influenza illness. All eight RNA segments were of avian origin. The H5 hemagglutinin is not recognized by neutralizing Abs present in humans as a result of infection with the human H1, H2, or H3 subtypes of influenza A viruses. Subsequently, five other deaths and several more human infections in Hong Kong were associated with this avian-derived virus. We investigated whether influenza A-specific human CD8+ and CD4+ T lymphocytes would recognize epitopes on influenza A virus strains derived from swine or avian species, including the 1997 H5N1 Hong Kong virus strains. Our results demonstrate that adults living in an urban area of the U.S. possess influenza A cross-serotype reactive CD8+ and CD4+ CTL that recognize multiple epitopes on influenza A viruses of other species. Bulk culture cytotoxicity was demonstrated against avian and human influenza A viruses. Enzyme-linked immunospot assays detected precursor CTL specific for both human CTL epitopes and the corresponding A/HK/97 viral sequences. We hypothesize that these cross-reactive CTL might provide partial protection to humans against novel influenza A virus strains introduced into humans from other species.     

Evidence of infection with H4 and H11 avian influenza viruses among Lebanese chicken growers

Human infections with H5, H7, and H9 avian influenza viruses are well documented. Exposure to poultry is the most important risk factor for humans becoming infected with these viruses. Data on human infection with other low pathogenicity avian influenza viruses is sparse but suggests that such infections may occur. Lebanon is a Mediterranean country lying under two major migratory birds flyways and is home to many wild and domestic bird species. Previous reports from this country demonstrated that low pathogenicity avian influenza viruses are in circulation but highly pathogenic H5N1 viruses were not reported. In order to study the extent of human infection with avian influenza viruses in Lebanon, we carried out a seroprevalence cross-sectional study into which 200 poultry-exposed individuals and 50 non-exposed controls were enrolled. We obtained their sera and tested it for the presence of antibodies against avian influenza viruses types H4 through H16 and used a questionnaire to collect exposure data. Our microneutralization assay results suggested that backyard poultry growers may have been previously infected with H4 and H11 avian influenza viruses. We confirmed these results by using a horse red blood cells hemagglutination inhibition assay. Our data also showed that farmers with antibodies against each virus type clustered in a small geographic area suggesting that unrecognized outbreaks among birds may have led to these human infections. In conclusion, this study suggests that occupational exposure to chicken is a risk factor for infection with avian influenza especially among backyard growers and that H4 and H11 influenza viruses may possess the ability to cross the species barrier to infect humans.     

The hemagglutinin-esterase of mouse hepatitis virus strain S is a sialate-4-O-acetylesterase

By comparative analysis of the hemagglutinin-esterase (HE) protein of mouse hepatitis virus strain S (MHV-S) and the HE protein of influenza C virus, we found major differences in substrate specificities. In striking contrast to the influenza C virus enzyme, the MHV-S esterase was unable to release acetate from bovine submandibulary gland mucin. Furthermore, MHV-S could not remove influenza C virus receptors from erythrocytes. Analysis with free sialic acid derivatives revealed that the MHV-S HE protein specifically de-O-acetylates 5-N-acetyl-4-O-acetyl sialic acid (Neu4, 5Ac2) but not 5-N-acetyl-9-O-acetyl sialic acid (Neu5,9Ac2), which is the major substrate for esterases of influenza C virus and bovine coronaviruses. In addition, the MHV-S esterase converted glycosidically bound Neu4,5Ac2 of guinea pig serum glycoproteins to Neu5Ac. By expression of the MHV esterase with recombinant vaccinia virus and incubation with guinea pig serum, we demonstrated that the viral HE possesses sialate-4-O-acetylesterase activity. In addition to observed enzymatic activity, MHV-S exhibited affinity to guinea pig and horse serum glycoproteins. Binding required sialate-4-O-acetyl groups and was abolished by chemical de-O-acetylation. Since Neu4,5Ac2 has not been identified in mice, the nature of potential substrates and/or secondary receptors for MHV-S in the natural host remains to be determined. The esterase of MHV-S is the first example of a viral enzyme with high specificity and affinity toward 4-O-acetylated sialic acids.     

Complete Genome Sequence of an Avian-Like H4N8 Swine Influenza Virus Discovered in Southern China

We report here the complete genomic sequence of an avian-like H4N8 swine influenza virus containing an H5N1 avian influenza virus segment from swine in southern China. Phylogenetic analyses of the sequences of all eight viral RNA segments demonstrated that these are wholly avian influenza viruses of the Asia lineage. To our knowledge, this is the first report of interspecies transmission of an avian H4N8 influenza virus to domestic pigs under natural conditions.     

The TLR4-TRIF pathway protects against H5N1 influenza virus infection

Prestimulation of the TLR4 pathway with lipopolysaccharide (LPS) protects mice from lethal infection with H5N1 influenza virus. Here, we reveal that the TLR4-TRIF pathway is required for this protective effect by using mice whose TLR4-related molecules were knocked out. Microarray analysis of primary mouse lung culture cells that were LPS pretreated and infected with an H5N1 virus indicated that TLR3 mRNA was upregulated. Primary lung culture cells of TLR3 knockout mice showed no response to LPS pretreatment against H5N1 virus infection, suggesting that TLR3 is also involved in the preventive effect of LPS. Our data suggest that the TLR4-TRIF axis has an important role in stimulating protective innate immunity against H5N1 influenza A virus infection and that TLR3 signaling is involved in this pathway.     

Single and coexpression of CXCR4 and CXCR5 identifies CD4 T helper cells in distinct lymph node niches during influenza virus infection

Influenza virus infection results in strong, mainly T-dependent, extrafollicular and germinal center B cell responses, which provide lifelong humoral immunity against the homotypic virus strain. Follicular T helper cells (T(FH)) are key regulators of humoral immunity. Questions remain regarding the presence, identity, and function of T(FH) subsets regulating early extrafollicular and later germinal center B cell responses. This study demonstrates that ICOS but not CXCR5 marks T cells with B helper activity induced by influenza virus infection and identifies germinal center T cells (T(GC)) as lymph node-resident CD4(+) ICOS(+) CXCR4(+) CXCR5(+) PSGL-1(lo) PD-1(hi) cells. The CXCR4 expression intensity further distinguished their germinal center light and dark zone locations. This population emerged strongly in regional lymph nodes and with kinetics similar to those of germinal center B cells and were the only T(FH) subsets missing in influenza virus-infected, germinal center-deficient SAP(-/-) mice, mice which were shown previously to lack protective memory responses after a secondary influenza virus challenge, thus indicting the nonredundant functions of CXCR4- and CXCR5-coexpressing CD4 helper cells in antiviral B cell immunity. CXCR4-single-positive T cells, present in B cell-mediated autoimmunity and regarded as "extrafollicular" helper T cells, were rare throughout the response, despite prominent extrafollicular B cell responses, revealing fundamental differences in autoimmune- and infection-induced T-dependent B cell responses. While all ICOS(+) subsets induced similar antibody levels in vitro, CXCR5-single-positive T cells were superior in inducing B cell proliferation. The regulation of T cell localization, marked by the single and coexpression of CXCR4 and CXCR5, might be an important determinant of T(FH) function.     

Healthy human subjects have CD4+ T cells directed against H5N1 influenza virus

It is commonly perceived that the human immune system is naive to the newly emerged H5N1 virus. In contrast, most adults have been exposed to influenza A H1N1 and H3N2 viruses through vaccination or infection. Adults born before 1968 have likely been exposed to H2N2 viruses. We hypothesized that CD4(+) T cells generated in response to H1N1, H3N2, and H2N2 influenza A viruses also recognize H5N1 epitopes. Tetramer-guided epitope mapping and Ag-specific class II tetramers were used to identify H5N1-specific T cell epitopes and detect H5N1-specific T cell responses. Fifteen of 15 healthy subjects tested had robust CD4(+) T cell responses against matrix protein, nucleoprotein, and neuraminidase of the influenza A/Viet Nam/1203/2004 (H5N1) virus. These results are not surprising, because the matrix protein and nucleoprotein of influenza A viruses are conserved while the neuraminidase of the H5N1 virus is of the same subtype as that of the circulating H1N1 influenza strain. However, H5N1 hemagglutinin-reactive CD4(+) T cells were also detected in 14 of 14 subjects examined despite the fact that hemagglutinin is less conserved. Most were cross-reactive to H1, H2, or H3 hemagglutinin epitopes. H5N1-reactive T cells were also detected ex vivo, exhibited a memory phenotype, and were capable of secreting IFN-gamma, TNF-alpha, IL-5, and IL-13. These data demonstrate the presence of H5N1 cross-reactive T cells in healthy Caucasian subjects, implying that exposure to influenza A H1N1, H3N2, or H2N2 viruses through either vaccination or infection may provide partial immunity to the H5N1 virus.     

Pre-clinical evaluation of a replication-competent recombinant adenovirus serotype 4 vaccine expressing influenza H5 hemagglutinin

Background

Influenza virus remains a significant health and social concern in part because of newly emerging strains, such as avian H5N1 virus. We have developed a prototype H5N1 vaccine using a recombinant, replication-competent Adenovirus serotype 4 (Ad4) vector, derived from the U.S. military Ad4 vaccine strain, to express the hemagglutinin (HA) gene from A/Vietnam/1194/2004 influenza virus (Ad4-H5-Vtn). Our hypothesis is that a mucosally-delivered replicating Ad4-H5-Vtn recombinant vector will be safe and induce protective immunity against H5N1 influenza virus infection and disease pathogenesis.

Methodology/Principal Findings

The Ad4-H5-Vtn vaccine was designed with a partial deletion of the E3 region of Ad4 to accommodate the influenza HA gene. Replication and growth kinetics of the vaccine virus in multiple human cell lines indicated that the vaccine virus is attenuated relative to the wild type virus. Expression of the HA transgene in infected cells was documented by flow cytometry, western blot analysis and induction of HA-specific antibody and cellular immune responses in mice. Of particular note, mice immunized intranasally with the Ad4-H5-Vtn vaccine were protected against lethal H5N1 reassortant viral challenge even in the presence of pre-existing immunity to the Ad4 wild type virus.

Conclusions/Significance

Several non-clinical attributes of this vaccine including safety, induction of HA-specific humoral and cellular immunity, and efficacy were demonstrated using an animal model to support Phase 1 clinical trial evaluation of this new vaccine.     

Direct ex vivo analyses of HLA-DR1 transgenic mice reveal an exceptionally broad pattern of immunodominance in the primary HLA-DR1-restricted CD4 T-cell response to influenza virus hemagglutinin

The recent threat of an avian influenza pandemic has generated significant interest in enhancing our understanding of the events that dictate protective immunity to influenza and in generating vaccines that can induce heterosubtypic immunity. Although antigen-specific CD4 T cells are known to play a key role in protective immunity to influenza through the provision of help to B cells and CD8 T cells, little is known about the specificity and diversity of CD4 T cells elicited after infection, particularly those elicited in humans. In this study, we used HLA-DR transgenic mice to directly and comprehensively identify the specificities of hemagglutinin (HA)-specific CD4 T cells restricted to a human class II molecule that were elicited following intranasal infection with a strain of influenza virus that has been endemic in U.S. human populations for the last decade. Our results reveal a surprising degree of diversity among influenza virus-specific CD4 T cells. As many as 30 different peptides, spanning the entire HA protein, were recognized by CD4 T cells, including epitopes genetically conserved among H1, H2, and H5 influenza A viruses. We also compared three widely used major histocompatibility class II algorithms to predict HLA-DR binding peptides and found these as yet inadequate for identifying influenza virus-derived epitopes. The results of these studies offer key insights into the spectrum of peptides recognized by HLA-DR-restricted CD4 T cells that may be the focus of immune responses to infection or to experimental or clinical vaccines in humans.     

Infection with seasonal influenza virus elicits CD4 T cells specific for genetically conserved epitopes that can be rapidly mobilized for protective immunity to pandemic H1N1 influenza virus

In recent years, influenza viruses with pandemic potential have been a major concern worldwide. One unresolved issue is how infection or vaccination with seasonal influenza virus strains influences the ability to mount a protective immune response to novel pandemic strains. In this study, we developed a mouse model of primary and secondary influenza infection by using a widely circulating seasonal H1N1 virus and the pandemic strain of H1N1 that emerged in Mexico in 2009, and we evaluated several key issues. First, using overlapping peptide libraries encompassing the entire translated sequences of 5 major influenza virus proteins, we assessed the specificity of CD4 T cell reactivity toward epitopes conserved among H1N1 viruses or unique to the seasonal or pandemic strain by enzyme-linked immunospot (ELISpot) assays. Our data show that CD4 T cells reactive to both virus-specific and genetically conserved epitopes are elicited, allowing separate tracking of these responses. Populations of cross-reactive CD4 T cells generated from seasonal influenza infection were found to expand earlier after secondary infection with the pandemic H1N1 virus than CD4 T cell populations specific for new epitopes. Coincident with this rapid CD4 T cell response was a potentiated neutralizing-antibody response to the pandemic strain and protection from the pathological effects of infection with the pandemic virus. This protection was not dependent on CD8 T cells. Together, our results indicate that exposure to seasonal vaccines and infection elicits CD4 T cells that promote the ability of the mammalian host to mount a protective immune response to pandemic strains of influenza virus.     

Synthesis of a polymeric 4-N-linked sialoside which inhibits influenza virus hemagglutinin

A multiple sialic acid-bearing polymer 7 has been made in which a novel 4-N-substituted sialoside 5 has been coupled to polyacrylamide. The conjugate 7 has been found to inhibit the agglutination of influenza virus to red blood cells with HAI inhibition constants of around 10(-6) M, based on the sialic acid concentration.     

A Human CD4+ T Cell Epitope in the Influenza Hemagglutinin Is Cross-Reactive to Influenza A Virus Subtypes and to Influenza B Virus

The hemagglutinin protein (HA) of the influenza virus family is a major antigen for protective immunity. Thus, it is a relevant target for developing vaccines. Here, we describe a human CD4(+) T cell epitope in the influenza virus HA that lies in the fusion peptide of the HA. This epitope is well conserved in all 16 subtypes of the HA protein of influenza A virus and the HA protein of influenza B virus. By stimulating peripheral blood mononuclear cells (PBMCs) from a healthy adult donor with peptides covering the entire HA protein based on the sequence of A/Japan/305/1957 (H2N2), we generated a T cell line specific to this epitope. This CD4(+) T cell line recognizes target cells infected with influenza A virus seasonal H1N1 and H3N2 strains, a reassortant H2N1 strain, the 2009 pandemic H1N1 strain, and influenza B virus in cytotoxicity assays and intracellular-cytokine-staining assays. It also lysed target cells infected with avian H5N1 virus. We screened healthy adult PBMCs for T cell responses specific to this epitope and found individuals who had ex vivo gamma interferon (IFN-γ) responses to the peptide epitope in enzyme-linked immunospot (ELISPOT) assays. Almost all donors who responded to the epitope had the HLA-DRB1*09 allele, a relatively common HLA allele. Although natural infection or standard vaccination may not induce strong T and B cell responses to this highly conserved epitope in the fusion peptide, it may be possible to develop a vaccination strategy to induce these CD4(+) T cells, which are cross-reactive to both influenza A and B viruses.     

Modification of eukaryotic initiation factor 4F during infection by influenza virus.

Influenza virus infection of cells is accompanied by a striking shutoff of cellular protein synthesis, resulting in the exclusive translation of viral mRNAs. The mechanism for control of cellular protein synthesis by influenza virus is poorly understood, but several translation properties of influenza virus mRNAs which are potentially involved have been described. Influenza virus mRNAs possess the surprising ability to translate in the presence of inhibitory levels of inactive (phosphorylated) eukaryotic initiation factor 2 (eIF-2). In addition, influenza virus mRNAs were shown to be capable of translating in cells during the late phase of adenovirus infection but not in cells infected by poliovirus. Since both adenovirus and poliovirus facilitate virus-specific translation by impairing the activity of initiation factor eIF-4F (cap-binding protein complex) but through different mechanisms, we investigated the translation properties of influenza virus mRNAs in more detail. We show that influenza virus infection is associated with the significant dephosphorylation and inactivation of eIF-4E (cap-binding protein), a component of eIF-4F, and accordingly that influenza virus mRNAs possess a moderate ability to translate by using low levels of eIF-4F. We also confirm the ability of influenza virus mRNAs to translate in the presence of high levels of inactive (phosphorylated) eIF-2 but to a more limited extent than reported previously. We suggest a potential mechanism for the regulation of protein synthesis by influenza virus involving a decreased requirement for large pools of active eIF-4F and eIF-2.     

Multifunctional CD4 cells expressing gamma interferon and perforin mediate protection against lethal influenza virus infection

CD4 effectors generated in vitro can promote survival against a highly pathogenic influenza virus via an antibody-independent mechanism involving class II-restricted, perforin-mediated cytotoxicity. However, it is not known whether CD4 cells activated during influenza virus infection can acquire cytolytic activity that contributes to protection against lethal challenge. CD4 cells isolated from the lungs of infected mice were able to confer protection against a lethal dose of H1N1 influenza virus A/Puerto Rico 8/34 (PR8). Infection of BALB/c mice with PR8 induced a multifunctional CD4 population with proliferative capacity and ability to secrete interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-α) in the draining lymph node (DLN) and gamma interferon (IFN-γ) and IL-10 in the lung. IFN-γ-deficient CD4 cells produced larger amounts of IL-17 and similar levels of TNF-α, IL-10, and IL-2 compared to wild-type (WT) CD4 cells. Both WT and IFN-γ(-/-) CD4 cells exhibit influenza virus-specific cytotoxicity; however, IFN-γ-deficient CD4 cells did not promote recovery after lethal infection as effectively as WT CD4 cells. PR8 infection induced a population of cytolytic CD4 effectors that resided in the lung but not the DLN. These cells expressed granzyme B (GrB) and required perforin to lyse peptide-pulsed targets. Lethally infected mice given influenza virus-specific CD4 cells deficient in perforin showed greater weight loss and a slower time to recovery than mice given WT influenza virus-specific CD4 cells. Taken together, these data strengthen the concept that CD4 T cell effectors are broadly multifunctional with direct roles in promoting protection against lethal influenza virus infection.     

Control of pathogenic CD4 T cells and lethal immunopathology by signaling immunoadaptor DAP12 during influenza infection

Immunopathology is a major cause of influenza-associated morbidity and mortality worldwide. However, the role and regulatory mechanisms of CD4 T cells in severe lung immunopathology following acute influenza infection are poorly understood. In this paper, we report that the emergence of immunopathogenic CD4 T cells is under the control of a transmembrane immunoadaptor DAP12 pathway during influenza infection. We find that the mice lacking DAP12 have unaltered viral clearance but easily succumb to influenza infection as a result of uncontrolled immunopathology. Such immunopathology is associated with markedly increased CD4 T cells displaying markedly increased cytotoxicity and Fas ligand expression. Furthermore, the immunopathogenic property of these CD4 T cells is transferrable. Thus, depletion of CD4 T cells or abrogation of Fas/Fas ligand signaling pathway improves survival and immunopathology. We further find that DAP12 expressed by dendritic cells plays an important role in controlling the immunopathogenic CD4 T cells during influenza infection. Our findings identify a novel pathway that controls the level of immune-pathogenic CD4 T cells during acute influenza infection.     

A genome-wide CRISPR/Cas9 gene knockout screen identifies immunoglobulin superfamily DCC subclass member 4 as a key host factor that promotes influenza virus endocytosis

Influenza virus infection is dependent on host cellular factors, and identification of these factors and their underlying mechanisms can provide important information for the development of strategies to inhibit viral infection. Here, we used a highly pathogenic H5N1 influenza virus to perform a genome-wide CRISPR/Cas9 gene knockout screen in human lung epithelial cells (A549 cells), and found that knockout of transmembrane protein immunoglobulin superfamily DCC subclass member 4 (IGDCC4) significantly reduced the replication of the virus in A549 cells. Further studies showed that IGDCC4 interacted with the viral hemagglutinin protein and facilitated virus internalization into host cells. Animal infection studies showed that replication of H5N1 virus in the nasal turbinates, lungs, and kidneys of IGDCC4-knockout mice was significantly lower than that in the corresponding organs of wild-type mice. Half of the IGDCC4-knockout mice survived a lethal H5N1 virus challenge, whereas all of the wild-type mice died within 11 days of infection. Our study identifies a novel host factor that promotes influenza virus infection by facilitating internalization and provides insights that will support the development of antiviral therapies.     

Age-associated change in the frequency of memory CD4+ T cells impairs long term CD4+ T cell responses to influenza vaccine

We investigated the relationship of memory CD4+ T cells with the evolution of influenza virus-specific CD4+ T cell responses in healthy young and elderly people. Elderly individuals had a similar frequency of CD69+CD4+ T cells producing IFN-gamma and TNF-alpha at 1 wk, but a lower frequency of these CD4+ T cells at 3 mo after influenza vaccination. Although the elderly had a higher frequency of central memory (CM; CCR7+CD45RA-) CD4+ T cells, they had a significantly lower frequency of effector memory (EM; CCR7-CD45RA-) CD4+ T cells, and the frequency of the latter memory CD4+ T cells positively correlated with the frequency of influenza virus-specific CD69+CD4+ T cells producing IFN-gamma at 3 mo. These findings indicate that the elderly have an altered balance of memory CD4+ T cells, which potentially affects long term CD4+ T cell responses to the influenza vaccine. Compared with the young, the elderly had decreased serum IL-7 levels that positively correlated with the frequency of EM cells, which suggests a relation between IL-7 and decreased EM cells. Thus, although the healthy elderly mount a level of CD4+ T cell responses after vaccination comparable to that observed in younger individuals, they fail to maintain or expand these responses. This failure probably stems from the alteration in the frequency of CM and EM CD4+ T cells in the elderly that is related to alteration in IL-7 levels. These findings raise an important clinical question about whether the vaccination strategy in the elderly should be modified to improve cellular immune responses.     

Preexisting influenza-specific CD4+ T cells correlate with disease protection against influenza challenge in humans

Protective immunity against influenza virus infection is mediated by neutralizing antibodies, but the precise role of T cells in human influenza immunity is uncertain. We conducted influenza infection studies in healthy volunteers with no detectable antibodies to the challenge viruses H3N2 or H1N1. We mapped T cell responses to influenza before and during infection. We found a large increase in influenza-specific T cell responses by day 7, when virus was completely cleared from nasal samples and serum antibodies were still undetectable. Preexisting CD4+, but not CD8+, T cells responding to influenza internal proteins were associated with lower virus shedding and less severe illness. These CD4+ cells also responded to pandemic H1N1 (A/CA/07/2009) peptides and showed evidence of cytotoxic activity. These cells are an important statistical correlate of homotypic and heterotypic response and may limit severity of influenza infection by new strains in the absence of specific antibody responses. Our results provide information that may aid the design of future vaccines against emerging influenza strains.