Monoclonal Antibodies against the Fusion Peptide of Hemagglutinin Protect Mice from Lethal Influenza A Virus H5N1 Infection

ABSTRACT The HA2 glycopolypeptide (gp) is highly conserved in all influenza A virus strains, and it is known to play a major role in the fusion of the virus with the endosomal membrane in host cells during the course of viral infection. Vaccines and therapeutics targeting this HA2 gp could induce efficient broad-spectrum immunity against influenza A virus infections. So far, there have been no studies on the possible therapeutic effects of monoclonal antibodies (MAbs), specifically against the fusion peptide of hemagglutinin (HA), upon lethal infections with highly pathogenic avian influenza (HPAI) H5N1 virus. We have identified MAb 1C9, which binds to GLFGAIAGF, a part of the fusion peptide of the HA2 gp. We evaluated the efficacy of MAb 1C9 as a therapy for influenza A virus infections. This MAb, which inhibited cell fusion in vitro when administered passively, protected 100% of mice from challenge with five 50% mouse lethal doses of HPAI H5N1 influenza A viruses from two different clades. Furthermore, it caused earlier clearance of the virus from the lung. The influenza virus load was assessed in lung samples from mice challenged after pretreatment with MAb 1C9 (24 h prior to challenge) and from mice receiving early treatment (24 h after challenge). The study shows that MAb 1C9, which is specific to the antigenically conserved fusion peptide of HA2, can contribute to the cross-clade protection of mice infected with H5N1 virus and mediate more effective recovery from infection.

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Characterization of a Human H5N1 Influenza A Virus Isolated in 2003

Journal of Virology ◽

10.1128/jvi.79.15.9926-9932.2005 ◽

2005 ◽

Vol 79 (15) ◽

pp. 9926-9932 ◽

Cited By ~ 70

Author(s):

Kyoko Shinya ◽

Masato Hatta ◽

Shinya Yamada ◽

Ayato Takada ◽

Shinji Watanabe ◽

...

Keyword(s):

Hong Kong ◽

Avian Influenza ◽

Influenza A Virus ◽

Influenza A ◽

Mainland China ◽

Virus Infections ◽

Influenza A Viruses ◽

H5n1 Avian Influenza Virus ◽

H5n1 Influenza ◽

Avian Influenza A Virus

ABSTRACT In 2003, H5N1 avian influenza virus infections were diagnosed in two Hong Kong residents who had visited the Fujian province in mainland China, affording us the opportunity to characterize one of the viral isolates, A/Hong Kong/213/03 (HK213; H5N1). In contrast to H5N1 viruses isolated from humans during the 1997 outbreak in Hong Kong, HK213 retained several features of aquatic bird viruses, including the lack of a deletion in the neuraminidase stalk and the absence of additional oligosaccharide chains at the globular head of the hemagglutinin molecule. It demonstrated weak pathogenicity in mice and ferrets but caused lethal infection in chickens. The original isolate failed to produce disease in ducks but became more pathogenic after five passages. Taken together, these findings portray the HK213 isolate as an aquatic avian influenza A virus without the molecular changes associated with the replication of H5N1 avian viruses in land-based poultry such as chickens. This case challenges the view that adaptation to land-based poultry is a prerequisite for the replication of aquatic avian influenza A viruses in humans.

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Viral Factors Important for Efficient Replication of Influenza A Viruses in Cells of the Central Nervous System

Journal of Virology ◽

10.1128/jvi.02273-18 ◽

2019 ◽

Vol 93 (11) ◽

Cited By ~ 6

Author(s):

Jurre Y. Siegers ◽

Marco W. G. van de Bildt ◽

Zhanmin Lin ◽

Lonneke M. Leijten ◽

Rémon A. M. Lavrijssen ◽

...

Keyword(s):

Central Nervous System ◽

Influenza A ◽

H5n1 Virus ◽

Influenza Viruses ◽

Virus Infections ◽

Influenza A Viruses ◽

Cns Disease ◽

Efficient Replication ◽

Hpai H5n1 ◽

In Cells

ABSTRACTCentral nervous system (CNS) disease is one of the most common extrarespiratory tract complications of influenza A virus infections. Remarkably, zoonotic H5N1 virus infections are more frequently associated with CNS disease than seasonal or pandemic influenza viruses. Little is known about the interaction between influenza A viruses and cells of the CNS; therefore, it is currently unknown which viral factors are important for efficient replication. Here, we determined the replication kinetics of a seasonal, pandemic, zoonotic, and lab-adapted influenza A virus in human neuron-like (SK-N-SH) and astrocyte-like (U87-MG) cells and primary mouse cortex neurons. In general, highly pathogenic avian influenza (HPAI) H5N1 virus replicated most efficiently in all cells, which was associated with efficient attachment and infection. Seasonal H3N2 and to a lesser extent pandemic H1N1 virus replicated in a trypsin-dependent manner in SK-N-SH but not in U87-MG cells. In the absence of trypsin, only HPAI H5N1 and WSN viruses replicated. Removal of the multibasic cleavage site (MBCS) from HPAI H5N1 virus attenuated, but did not abrogate, replication. Taken together, our results showed that the MBCS and, to a lesser extent, the ability to attach are important determinants for efficient replication of HPAI H5N1 virus in cells of the CNS. This suggests that both an alternative hemagglutinin (HA) cleavage mechanism and preference for α-2,3-linked sialic acids allowing efficient attachment contribute to the ability of influenza A viruses to replicate efficiently in cells of the CNS. This study further improves our knowledge on potential viral factors important for the neurotropic potential of influenza A viruses.IMPORTANCECentral nervous system (CNS) disease is one of the most common extrarespiratory tract complications of influenza A virus infections, and the frequency and severity differ between seasonal, pandemic, and zoonotic influenza viruses. However, little is known about the interaction of these viruses with cells of the CNS. Differences among seasonal, pandemic, and zoonotic influenza viruses in replication efficacy in CNS cells,in vitro, suggest that the presence of an alternative HA cleavage mechanism and ability to attach are important viral factors. Identifying these viral factors and detailed knowledge of the interaction between influenza virus and CNS cells are important to prevent and treat this potentially lethal CNS disease.

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A Single Immunization with CoVaccine HT-Adjuvanted H5N1 Influenza Virus Vaccine Induces Protective Cellular and Humoral Immune Responses in Ferrets

Journal of Virology ◽

10.1128/jvi.00549-10 ◽

2010 ◽

Vol 84 (16) ◽

pp. 7943-7952 ◽

Cited By ~ 25

Author(s):

R. Bodewes ◽

J. H. C. M. Kreijtz ◽

G. van Amerongen ◽

M. M. Geelhoed-Mieras ◽

R. J. Verburgh ◽

...

Keyword(s):

Cell Culture ◽

Influenza Virus ◽

Immune Responses ◽

Influenza A Virus ◽

Virus Vaccine ◽

Influenza A ◽

H5n1 Virus ◽

The Novel ◽

Highly Pathogenic ◽

H5n1 Influenza

ABSTRACT Highly pathogenic avian influenza A viruses of the H5N1 subtype continue to circulate in poultry, and zoonotic transmissions are reported frequently. Since a pandemic caused by these highly pathogenic viruses is still feared, there is interest in the development of influenza A/H5N1 virus vaccines that can protect humans against infection, preferably after a single vaccination with a low dose of antigen. Here we describe the induction of humoral and cellular immune responses in ferrets after vaccination with a cell culture-derived whole inactivated influenza A virus vaccine in combination with the novel adjuvant CoVaccine HT. The addition of CoVaccine HT to the influenza A virus vaccine increased antibody responses to homologous and heterologous influenza A/H5N1 viruses and increased virus-specific cell-mediated immune responses. Ferrets vaccinated once with a whole-virus equivalent of 3.8 μg hemagglutinin (HA) and CoVaccine HT were protected against homologous challenge infection with influenza virus A/VN/1194/04. Furthermore, ferrets vaccinated once with the same vaccine/adjuvant combination were partially protected against infection with a heterologous virus derived from clade 2.1 of H5N1 influenza viruses. Thus, the use of the novel adjuvant CoVaccine HT with cell culture-derived inactivated influenza A/H5N1 virus antigen is a promising and dose-sparing vaccine approach warranting further clinical evaluation.

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Amino Acid Residues in the Fusion Peptide Pocket Regulate the pH of Activation of the H5N1 Influenza Virus Hemagglutinin Protein

Journal of Virology ◽

10.1128/jvi.02238-08 ◽

2009 ◽

Vol 83 (8) ◽

pp. 3568-3580 ◽

Cited By ~ 74

Author(s):

Mark L. Reed ◽

Hui-Ling Yen ◽

Rebecca M. DuBois ◽

Olga A. Bridges ◽

Rachelle Salomon ◽

...

Keyword(s):

Amino Acid ◽

Influenza A Virus ◽

Influenza A ◽

Coiled Coil ◽

Fusion Peptide ◽

Influenza Viruses ◽

Amino Acid Residues ◽

H5n1 Influenza ◽

Coiled Coil Domain ◽

Ha Protein

ABSTRACT The receptor specificity and cleavability of the hemagglutinin (HA) protein have been shown to regulate influenza A virus transmissibility and pathogenicity, but little is known about how its pH of activation contributes to these important biological properties. To identify amino acid residues that regulate the acid stability of the HA protein of H5N1 influenza viruses, we performed a mutational analysis of the HA protein of the moderately pathogenic A/chicken/Vietnam/C58/04 (H5N1) virus. Nineteen HA proteins containing point mutations in the HA2 coiled-coil domain or in an HA1 histidine or basic patch were generated. Wild-type and mutant HA plasmids were transiently transfected in cell culture and analyzed for total protein expression, surface expression, cleavage efficiency, pH of fusion, and pH of conformational change. Four mutations to residues in the fusion peptide pocket, Y23H and H24Q in the HA1 subunit and E105K and N114K in the HA2 subunit, and a K58I mutation in the HA2 coiled-coil domain significantly altered the pH of activation of the H5 HA protein. In some cases, the magnitude and direction of changes of individual mutations in the H5 HA protein differed considerably from similar mutations in other influenza A virus HA subtypes. Introduction of Y23H, H24Q, K58I, and N114K mutations into recombinant viruses resulted in virus-expressed HA proteins with similar shifts in the pH of fusion. Overall, the data show that residues comprising the fusion peptide pocket are important in triggering pH-dependent activation of the H5 HA protein.

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Efficacy of Orally Administered T-705 on Lethal Avian Influenza A (H5N1) Virus Infections in Mice

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01051-06 ◽

2006 ◽

Vol 51 (3) ◽

pp. 845-851 ◽

Cited By ~ 105

Author(s):

Robert W. Sidwell ◽

Dale L. Barnard ◽

Craig W. Day ◽

Donald F. Smee ◽

Kevin W. Bailey ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Influenza A ◽

H5n1 Virus ◽

Arterial Oxygen Saturation ◽

Arterial Oxygen ◽

Yield Reduction ◽

Virus Infections ◽

H5n1 Influenza ◽

Virus Exposure

ABSTRACT T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) was inhibitory to four strains of avian H5N1 influenza virus in MDCK cells, with the 90% effective concentrations ranging from 1.3 to 7.7 μM, as determined by a virus yield reduction assay. The efficacy was less than that exerted by oseltamivir carboxylate or zanamivir but was greater than that exerted by ribavirin. Experiments with mice lethally infected with influenza A/Duck/MN/1525/81 (H5N1) virus showed that T-705 administered per os once, twice, or four times daily for 5 days beginning 1 h after virus exposure was highly inhibitory to the infection. Dosages from 30 to 300 mg/kg of body weight/day were well tolerated; each prevented death, lessened the decline of arterial oxygen saturation (SaO2), and inhibited lung consolidation and lung virus titers. Dosages from 30 to 300 mg/kg/day administered once or twice daily also significantly prevented the death of the mice. Oseltamivir (20 mg/kg/day), administered per os twice daily for 5 days, was tested in parallel in two experiments; it was only weakly effective against the infection. The four-times-daily T-705 treatments at 300 mg/kg/day could be delayed until 96 h after virus exposure and still significantly inhibit the infection. Single T-705 treatments administered up to 60 h after virus exposure also prevented death and the decline of SaO2. Characterization of the pathogenesis of the duck influenza H5N1 virus used in these studies was undertaken; although the virus was highly pathogenic to mice, it was less neurotropic than has been described for clinical isolates of the H5N1 virus. These data indicate that T-705 may be useful for the treatment of avian influenza virus infections.

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Comparison of polyclonal antiserum versus monoclonal antibodies for the rapid diagnosis of influenza A virus infections by immunofluorescence in clinical specimens.

Journal of Clinical Microbiology ◽

10.1128/jcm.22.5.877-879.1985 ◽

1985 ◽

Vol 22 (5) ◽

pp. 877-879 ◽

Cited By ~ 16

Author(s):

I Shalit ◽

P A McKee ◽

H Beauchamp ◽

J L Waner

Keyword(s):

Monoclonal Antibodies ◽

Influenza A Virus ◽

Influenza A ◽

Rapid Diagnosis ◽

Polyclonal Antiserum ◽

Virus Infections ◽

Clinical Specimens

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In Vitro and In Vivo Antiviral Activity of Gingerenone A on Influenza A Virus Is Mediated by Targeting Janus Kinase 2

Viruses ◽

10.3390/v12101141 ◽

2020 ◽

Vol 12 (10) ◽

pp. 1141

Author(s):

Jiongjiong Wang ◽

Richard A. Prinz ◽

Xiufan Liu ◽

Xiulong Xu

Keyword(s):

Antiviral Activity ◽

Influenza A Virus ◽

Influenza A ◽

H5n1 Virus ◽

Immune Cell ◽

Janus Kinase ◽

Virus Infections ◽

Dual Inhibitor ◽

Cell Functions

Janus kinase (JAK) inhibitors have been developed as novel immunomodulatory drugs and primarily used for treating rheumatoid arthritis and other inflammatory diseases. Recent studies have suggested that this category of anti-inflammatory drugs could be potentially useful for the control of inflammation “storms” in respiratory virus infections. In addition to their role in regulating immune cell functions, JAK1 and JAK2 have been recently identified as crucial cellular factors involved in influenza A virus (IAV) replication and could be potentially targeted for antiviral therapy. Gingerenone A (Gin A) is a compound derived from ginger roots and a dual inhibitor of JAK2 and p70 S6 kinase (S6K1). Our present study aimed to determine the antiviral activity of Gin A on influenza A virus (IAV) and to understand its mechanisms of action. Here, we reported that Gin A suppressed the replication of three IAV subtypes (H1N1, H5N1, H9N2) in four cell lines. IAV replication was also inhibited by Ruxolitinib (Rux), a JAK inhibitor, but not by PF-4708671, an S6K1 inhibitor. JAK2 overexpression enhanced H5N1 virus replication and attenuated Gin A-mediated antiviral activity. In vivo experiments revealed that Gin A treatment suppressed IAV replication in the lungs of H5N1 virus-infected mice, alleviated their body weight loss, and prolonged their survival. Our study suggests that Gin A restricts IAV replication by inhibiting JAK2 activity; Gin A could be potentially useful for the control of influenza virus infections.

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