HA Protein Of Influenza Virus Research Articles

In Silico Prediction and Evaluation of E. coli Expressed Recombinant HA Protein of Avian Influenza Virus

In Silico Prediction and Evaluation of E. coli Expressed Recombinant HA Protein of Avian Influenza Virus

Vigna radiata (L.) R. Wilczek Extract Inhibits Influenza A Virus by Targeting Viral Attachment, Penetration, Assembly, and Release.

Vigna radiata (L.) R. Wilczek (mung bean) is a Chinese functional food with antioxidant, antimicrobial and anti-inflammatory activities. However, little is known about its antiviral activity. We aimed to investigate the antiviral activity and mechanisms of action of Vigna radiata extract (VRE) against influenza virus. HPLC was conducted to analyze the components of the VRE. The anti-influenza viral activity of VRE in Mardin-Darby canine kidney (MDCK) cells was evaluated by virus titration assays, hemagglutination assays, quantitative RT-PCR assays, cellular α-glucosidase activity assays and neuraminidase activity assays. Chromatographic profiling analysis identified two major flavonoids, vitexin and isovitexin, in the ethanol extract of Vigna radiata. Through in vitro studies, we showed that VRE, at concentrations up to 2,000 μg/ml, exhibited no cytotoxicity in MDCK cells. VRE protected cells from influenza virus-induced cytopathic effects and significantly inhibited viral replication in a concentration-dependent manner. A detailed time-of-addition assay revealed that VRE may act on both the early and late stages of the viral life cycle. We demonstrated that 1) VRE inhibits virus entry by directly blocking the HA protein of influenza virus; 2) VRE inhibits virus entry by directly binding to cellular receptors; 3) VRE inhibits virus penetration; 4) VRE inhibits virus assembly by blocking cellular α-glucosidase activity, thus reducing HA protein trafficking to the cell surface; and 5) VRE inhibits virus release by inhibiting viral neuraminidase activity. In summary, Vigna radiata extract potently interferes with two different subtypes of influenza viruses at multiple steps during the infectious cycle, demonstrating its broad-spectrum potential as an anti-influenza preventive and therapeutic agent. Continued development of Vigna radiata-derived products into antiviral therapeutics is warranted.

Baculovirus surface display of the HA protein of H5N2 avian influenza virus and its immunogenicity against a lethal challenge with H5N1 virus in chickens

In the present study, we have generated several H5N2 HA recombinant baculoviruses for production of a HA subunit vaccine against the lethal H5N2 avian influenza virus (AIV). The effective display of functional HA on the cell membrane and baculoviral envelope was examined. Our results reveal that chickens immunized with the chimeric AIV HA protein fused with the baculovirus gp64 cytoplasmic domain (CTD) induced higher HI titer. To further increase the expression level of the H5N2 AIV HA protein, the HA gene of H5N2 AIV was amplified and cloned into three novel baculovirus surface display vectors BacDual DisplayEGFP-2HA, BacDual DisplayEGFP-3HA, BacDual DisplayEGFP-4HA which contains multiple expression cassettes for higher level display of HA proteins on the cell membrane and baculovirus envelope. To determine the optimum conditions for producing HA protein, various MOI, infection times, and shaker times for virus transfection were tested. Our results reveal that the conditions of an MOI of 5, 3 day post infection, and 15 min of shaker time have higher efficiency for HA protein production. Our results reveal that the baculovirus surface display vector pBacDual DisplayEGFP-4HA increases significantly the expression level of the H5N2 AIV HA protein. Chickens that received two doses of BacDual DisplayEGFP-4HA cell lysates formulated with Montanide ISA70 adjuvant elicited efficient immunogenicity and had an average HI titer of 7 log2 at 2 weeks post-vaccination. Challenge studies revealed that vaccinated chickens with HI titers 5 log2 were completely protected against the lethal H5N1 AIV challenge. Furthermore, HI titers could be maintained at 5 log2 for 20 weeks for laying hens. This study suggests that the HA protein expression from the baculovirus surface display system could be a safe and efficacious subunit vaccine for chickens.

Design, synthesis and biological evaluation of amino acids-oleanolic acid conjugates as influenza virus inhibitors

Viral entry inhibitors are of great importance in current efforts to develop a new generation of anti-influenza drugs. Inspired by the discovery of a series of pentacyclic triterpene derivatives as entry inhibitors targeting the HA protein of influenza virus, we designed and synthesized 32 oleanolic acid (OA) analogues in this study by conjugating different amino acids to the 28-COOH of OA. The antiviral activity of these compounds was evaluated in vitro. Some of these compounds revealed impressive anti-influenza potencies against influenza A/WSN/33 (H1N1) virus. Among them, compound 15a exhibited robust potency and broad antiviral spectrum with IC50 values at the low-micromolar level against four different influenza strains. Hemagglutination inhibition (HI) assay and docking experiment indicated that these OA analogues may act in the same way as their parent compound by interrupting the interaction between HA protein of influenza virus and the host cell sialic acid receptor via binding to HA, thus blocking viral entry.

Abstract #4367 Blood endotoxin levels may contribute to gender differences in the link between depressive symptoms and ex vivo inflammatory responses

Viral entry inhibitors are of great importance in current efforts to develop a new generation of anti-influenza drugs. Inspired by the discovery of a series of pentacyclic triterpene derivatives as entry inhibitors targeting the HA protein of influenza virus, we designed and synthesized 32 oleanolic acid (OA) analogues in this study by conjugating different amino acids to the 28-COOH of OA. The antiviral activity of these compounds was evaluated in vitro. Some of these compounds revealed impressive anti-influenza potencies against influenza A/WSN/33 (H1N1) virus. Among them, compound 15a exhibited robust potency and broad antiviral spectrum with IC50 values at the low-micromolar level against four different influenza strains. Hemagglutination inhibition (HI) assay and docking experiment indicated that these OA analogues may act in the same way as their parent compound by interrupting the interaction between HA protein of influenza virus and the host cell sialic acid receptor via binding to HA, thus blocking viral entry.

Plant molecular farming of virus‐like nanoparticles as vaccines and reagents

The use of plants for the production of virus-like nanoparticles (VNPs) dates back to separating natural empty capsids of plant viruses from whole virions nearly 70 years ago, through to the present use of transgenic plants or recombinant Agrobacterium tumefaciens and/or plant virus-derived vectors for the transient expression of engineered viral or other structural proteins in plants-a production system also known as molecular farming. Plant production of heterologous proteins has major advantages in terms of convenience-whole plants are generally used, and processes do not need to be sterile-and cost, as bulk biomass production is significantly cheaper than by any other method. Plant-made VNPs in current use for nanotechnology include whole virions and naturally occurring empty capsids of plant viruses, and particles made by reassembly of coat protein (CP) purified from virions or by recombinant expression. Engineered VNP-forming animal or human virus CPs expressed in plants include L1 protein from human papillomaviruses, human norovirus CP, hepatitis B surface and core antigens, influenza virus HA protein and HIV Gag polyprotein forming large enveloped particles by budding, orbi- and rotavirus particles that require assembly of four co-expressed proteins, and polio- and foot and mouth disease viruses which require proteolytic processing of a polyprotein precursor to form 4-component VNPs. Both plant and animal virus-derived plant-made VNPs can be used for surface and internal display of heterologous peptides or even whole proteins. A significant recent development has been the production of pseudovirions in plants, comprising plant or animal virus CPs and RNA or DNA pseudogenomes that can be used to deliver nucleic acid payloads into cultured cells or specific tissues or tumors in whole animals. This article is characterized under: Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Therapeutic Approaches and Drug Discovery > Emerging Technologies Diagnostic Tools > in vivo Nanodiagnostics and Imaging.

Effect of annexin II-mediated conversion of plasmin from plasminogen on airborne transmission of H9N2 avian influenza virus

Airborne transmission plays an important role in dissemination of H9N2 subtype avian influenza virus. Annexin II (A2)-mediated activation of plasminogen (PLG) promotes cleavage of the influenza virus HA protein and viral replication, resulting in enhanced pathogenesis. In this study, airborne transmission competent and defective strains of H9N2 influenza virus, SH7 and SH14, respectively, were used to investigate the effect of A2 on airborne spread. The results showed that A2 protein was increased in SH7 virions compared with SH14 particles, the binding ability of the SH7-infected MDCK cells to PLG was significantly higher than the SH14-infected cells, and influence efficiency of the PLG on replicated ability of SH7 virus was significantly stronger than that of SH14 virus, who spread without airborne route, indicating that the annexin 2 (A2) can bind PLG and contributes to SH7 with high replication ability. Furthermore, the copies of SH7 in the airborne infected chickens under inhibited by 6-AHA were significantly decreased, suggesting that the release of H9N2 avian influenza virus were reduced by inhibiting the conversion of PLG to PL, ultimately resulting in reduced airborne transmission of H9N2 avian influenza virus. In summary, A2-mediated conversion of PLG to PL plays a role in the airborne transmission capacity of H9N2 avian influenza viruses, and this interaction may represent potential targets for prevention and treatment of influenza virus infection.

Comparative analysis of DR1 binding with H1N1 Hemagglutinin derived peptides

Abstract Binding of antigenic peptides to class II MHC molecules (MHCII) and the activity of the “editing” molecule HLA-DM (DM) on the resulting peptide-MHCII complexes are factors critical to the antigen presentation pathway. Different algorithms are available to predict peptide-MHC binding and identify CD4+ T-cell epitopes within pathogenic proteins. Fewer algorithms are focused on MHCII as compared to MHCI., and their reliability is also less solid. Possibly one reason for such difference lies in the greater complexity of peptide binding to MHCII, of which open-ended binding site features a larger flexibility and allows interaction with longer sequences. To validate algorithm predictions, we have compared the experimental IC50 of a library of overlapping 18-mers offset by 5 amino acids spanning the sequence of H1N1 influenza virus HA protein while interacting with HLA-DR1 with the one indicated by different prediction platforms online. The IC50 values for each peptide in the presence and absence of DM were measured using competition binding assay. The ex-vivo CD4+ T cell response to these sequences has already been published, on which basis they have been categorized into immunodominant, subdominant, weak and negative. Comparative analysis shows that measured IC50 is different from predictive IC50, with measured IC50 with DM showing immunodominant peptide as a top hit. Our data also shows that presence of DM reduced the affinity of all the peptides triggering the smaller CD4+ T cell response or negative, whereas the effect on the other peptides was less consistent. Thus, currently available algorithms are still inadequate to predict epitope for MHCII and DM is one important factor that should be considered when designing algorithms for MHCII.

Influenza virus differentially activates mTORC1 and mTORC2 signaling to maximize late stage replication.

Influenza A virus usurps host signaling factors to regulate its replication. One example is mTOR, a cellular regulator of protein synthesis, growth and motility. While the role of mTORC1 in viral infection has been studied, the mechanisms that induce mTORC1 activation and the substrates regulated by mTORC1 during influenza virus infection have not been established. In addition, the role of mTORC2 during influenza virus infection remains unknown. Here we show that mTORC2 and PDPK1 differentially phosphorylate AKT upon influenza virus infection. PDPK1-mediated phoshorylation of AKT at a distinct site is required for mTORC1 activation by influenza virus. On the other hand, the viral NS1 protein promotes phosphorylation of AKT at a different site via mTORC2, which is an activity dispensable for mTORC1 stimulation but known to regulate apoptosis. Influenza virus HA protein and down-regulation of the mTORC1 inhibitor REDD1 by the virus M2 protein promote mTORC1 activity. Systematic phosphoproteomics analysis performed in cells lacking the mTORC2 component Rictor in the absence or presence of Torin, an inhibitor of both mTORC1 and mTORC2, revealed mTORC1-dependent substrates regulated during infection. Members of pathways that regulate mTORC1 or are regulated by mTORC1 were identified, including constituents of the translation machinery that once activated can promote translation. mTORC1 activation supports viral protein expression and replication. As mTORC1 activation is optimal midway through the virus life cycle, the observed effects on viral protein expression likely support the late stages of influenza virus replication when infected cells undergo significant stress.

Anti-influenza virus activity of high-mannose binding lectins derived from genus Pseudomonas

Lectin PFL binding high-mannose glycan derived from Pseudomonas fluorescens and other homologous lectins: PML derived from Pseudomonas mandelii and PTL derived from Pseudomonas taiwanensis were examined for antiviral activity. The cDNA of these lectin genes were synthesized, cloned, expressed in Escherichia coli. The expressed lectins were purified by gel filtrations, and supplied to cultures infected with several strains of influenza virus. These three lectins have inhibited propagation of influenza viruses with a similar extent, 50% of inhibition-dose was around ten nanomolar concentration. An immunofluorescent microscopy, a microarray analysis, and several infection experiments with different time periods of lectin addition or using the competitor substrates indicated that binding of these lectins with high-mannose glycan on HA protein of influenza virus could block the virus entry into the host cells, thereby resulting in inhibition of the virus propagation. These Pseudomonas-derived lectins would be protential and attractive antiviral agents targeting glycoproteins of enveloped viruses including influenza virus.

Assembly and immunological properties of a bivalent virus-like particle (VLP) for avian influenza and Newcastle disease

Avian influenza virus (AIV) and Newcastle disease virus (NDV) are both important pathogens in poultry worldwide. The protection of poultry from avian influenza and Newcastle disease can be achieved through vaccination. We embarked on the development of a bivalent vaccine that would allow for a single immunization against both avian influenza and Newcastle disease. We constructed a chimeric virus-like particle (VLP) that is composed of the M1 protein and HA protein of avian influenza virus and a chimeric protein containing the cytoplasmic and transmembrane domains of AIV neuraminidase protein (NA) and the ectodomain of the NDV hemagglutinin-neuraminidase (HN) protein (NA/HN). The single immunization of chickens with the chimeric VLP vaccine induced both AIV H5- and NDV-specific antibodies. The HI titers and specific antibodies elicited by the chimeric VLPs were statistically similar to those elicited in animals vaccinated with the corresponding commercial monovalent vaccines. Chickens vaccinated with chimeric VLP vaccine and then challenged with the Newcastle disease F48E9 virus displayed complete protection. Overall, the chimeric VLP vaccine elicits strong immunity and can protect against Newcastle disease virus challenge.

Structure modeling and spatial epitope analysis for HA protein of the novel H1N1 influenza virus

In recent months, a novel influenza virus H1N1 broke out around the world. With bioinformatics technology, the 3D structure of HA protein was obtained, and the epitope residues were predicted with the method developed in our group for this novel flu virus. 58 amino acids were identified as potential epitope residues, the majority of which clustered at the surface of the globular head of HA protein. Although it is located at the similar position, the epitope of HA protein for the novel H1N1 flu virus has obvious differences in the electrostatic potential compared to that of HA proteins from previous flu viruses.

Safety and Immunogenicity of H5N1 Influenza Vaccine Based on Baculovirus Surface Display System of Bombyx mori

Avian influenza virus (H5N1) has caused serious infections in human beings. This virus has the potential to emerge as a pandemic threat in humans. Effective vaccines against H5N1 virus are needed. A recombinant Bombyx mori baculovirus, Bmg64HA, was constructed for the expression of HA protein of H5N1 influenza virus displaying on the viral envelope surface. The HA protein accounted for approximately 3% of the total viral proteins in silkworm pupae infected with the recombinant virus. Using a series of separation and purification methods, pure Bmgp64HA virus was isolated from these silkworm pupae bioreactors. Aluminum hydroxide adjuvant was used for an H5N1 influenza vaccine. Immunization with this vaccine at doses of 2 mg/kg and 0.67 mg/kg was carried out to induce the production of neutralizing antibodies, which protected monkeys against influenza virus infection. At these doses, the vaccine induced 1:40 antibody titers in 50% and 67% of the monkeys, respectively. The results of safety evaluation indicated that the vaccine did not cause any toxicity at the dosage as large as 3.2 mg/kg in cynomolgus monkeys and 1.6 mg/kg in mice. The results of dose safety evaluation of vaccine indicated that the safe dose of the vaccine were higher than 0.375 mg/kg in rats and 3.2 mg/kg in cynomolgus monkeys. Our work showed the vaccine may be a candidate for a highly effective, cheap, and safe influenza vaccine for use in humans.

Cellular virology

Cellular virology

Enhancement of mucosal immune responses to the influenza virus HA protein by alternative approaches to DNA immunization

DNA immunization provides many advantages as an approach to prevent infectious diseases. However, although previous studies using this approach have demonstrated immune responses in serum, they were not successful in inducing significant levels of antibodies in secretions. In this study, plasmid DNAs expressing the influenza virus hemagglutinin glycoprotein have been evaluated for their ability to induce antibody responses in serum and saliva when used alone or along with either liposomes or bioadhesive polymers as mucosal delivery vehicles. Significant levels of virus-specific Ig in serum as well as secretory IgA in saliva were detected in mice following mucosal DNA immunization. These antibodies were found to block the infectivity of the virus using a plaque reduction assay. Our findings thus indicate that mucosal DNA immunization with specific delivery systems can elicit virus-specific antibody responses in serum as well as IgA responses at mucosal surfaces.

Complementation between avirulent Newcastle disease virus and a fusion protein gene expressed from a retrovirus vector: requirements for membrane fusion

The cDNA derived from the fusion gene of the virulent AV strain of Newcastle disease virus (NDV) was expressed in chicken embryo cells by using a retrovirus vector. The fusion protein expressed in this system was transported to the cell surface and was efficiently cleaved into the disulfide-linked F1-F2 form found in infectious virions. The cells expressing the fusion gene grew normally and could be passaged many times. Monolayers of these cells would plaque, in the absence of trypsin, avirulent NDV strains (strains which encode a fusion protein which is not cleaved in tissue culture). Fusion protein-expressing cells would not fuse if mixed with uninfected cells or uninfected cells expressing the hemagglutinin-neuraminidase (HN) protein. However, the fusion protein-expressing cells, if infected with avirulent strains of NDV, would fuse with uninfected cells, suggesting that fusion requires both the fusion protein and another viral protein expressed in the same cell. Fusion was also seen after transfection of the HN protein gene into fusion protein-expressing cells. Thus, the expressed fusion protein gene is capable of complementing the virus infection, providing an active cleaved fusion protein required for the spread of infection. However, the fusion protein does not mediate cell fusion unless the cell also expresses the HN protein. Fusion protein-expressing cells would not plaque influenza virus in the absence of trypsin, nor would influenza virus-infected fusion protein-expressing cells fuse with uninfected cells. Thus, the influenza virus HA protein will not substitute for the NDV HN protein in cell-to-cell fusion.

Avian cells expressing the newcastle disease virus hemagglutinin-neuraminidase protein are resistant to newcastle disease virus infection

The cDNA derived from the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) gene was inserted into a replication-competent Schmidt-Ruppin Rous sarcoma virus-derived vector. Chick embryo cells transfected with this vector expressed HN-sized protein which could be precipitated with anti-HN antibody. These cells adsorbed avian red blood cells and the cell surfaces exhibited neuraminidase activity while cells transfected with an antisense version of the gene were negative for hemadsorption and neuraminidase. The cells transfected with the retroviral vector containing the HN gene were resistant to infection by NDV and influenza virus, viruses which bind to sialic acid containing receptors, but sensitive to vesicular stomatitis virus (VSV). Cells transfected with the antisense version of the HN gene were sensitive to NDV, influenza virus, and VSV infection. Thus the HN protein-expressing cells are likely resistant to NDV and influenza virus due to the destruction of the cellular receptors by the neuraminidase of the HN protein. The expression of the influenza virus HA protein using the same retrovirus vector has been reported previously (L. A. Hunt, D. W. Brown, H. L. Robinson, C. W. Naeve, and R. G. Webster, 1988, J. Virol. 62, 3014–3019) . Cells infected with this vector were sensitive to infection with influenza virus, NDV, and VSV. Thus expression of a viral surface protein does not necessarily confer resistance of the cell to the homologous virus.

Capability of multiplication of viruses in host tissues

The ability of viruses to replicate depends on a variety of cellular functions. Approaches to identifying specific determinants of host range or tissue tropism require closely related viruses with different host ranges, and cells that differ in their ability to support replication. With several viruses, such approaches have identified structures and processes that determine tropism and the susceptibility of cells to infection. With some viruses, e.g. picornaviruses and retroviruses, susceptibility to infection may be determined by the presence of structures on the cell surface that serve as receptors in attachment and penetration. With other viruses, the ubiquitous presence of receptors on cell surfaces precludes determination of susceptibility at this level. With myxoviruses and paramyxoviruses, the ability to multiply in a tissue depends on a host cell protease that is required to activate the virus protein responsible for penetration, i.e. the F protein of parainfluenza viruses and the HA protein of influenza virus, and the presence of a suitable host protease is a determinant of host range, tissue tropism, and virulence of these viruses.