Treatment of influenza A (H1N1) virus infections in mice and ferrets with cyanovirin-N

Cyanovirin-N (CV-N), a protein derived from Nostoc ellipsosporum, neutralizes influenza virus infectivity by binding to specific high-mannose oligosaccharides (oligomannose-8 and -9) at glycosylation sites on the viral hemagglutinin HA1 subunit. Mouse-adapted viruses lose sensitivity to CV-N due to HA1 mutations that eliminate these glycosylation sites. Recently we created a hybrid (reassortant) influenza A/WSN/33 (H1N1) virus containing the HA gene of A/New Caledonia/20/99 (H1N1) with an Asp225Gly mutation in the HA1, that was lethal to mice yet retained sensitivity to CV-N. We then utilized this model system to test the efficacy of CV-N against influenza. CV-N efficacy was dose-responsive from 0.0625 to 1 mg/kg/day when administered intranasally (i.n.) twice daily for 4 days starting 4 h prior to virus exposure. In a second study, survival benefit was seen with CV-N treatments (0.5 mg/kg/day for 4 days) beginning at −4 or +6 h, but was significantly reduced at +12 h. The early treatment resulted in up to 100% survival and 1000-fold reduction in lung virus titer on day 3 of the infection. In contrast, ribavirin (a positive control—75 mg/kg/day) treatment resulted in 30% survival and 30-fold decrease in lung virus titers. Lung consolidation scores and lung weights were significantly reduced by CV-N and ribavirin treatment on day 6 of the infection. Ferrets infected with a non-animal adapted influenza A/Charlottesville/31/95 (H1N1) virus were treated intranasally with CV-N (50 μg twice daily for 5 days starting 24 h before virus challenge). They exhibited 100-fold lower viral titers in nasal washes than placebos 1 day after treatment, but virus titers were equivalent on days 2–7. CV-N has the potential for prophylaxis and early initiation of treatment of influenza virus infections.     

Synthesis and biological activity of water-soluble polymer complexes of arbidol

We have synthesized water-soluble complexes between the antiviral drug arbidol and polymer compounds with molecular masses of 19–31 kDa representing copolymers of acrylamide (AA) and 2-acrylamido-2-methylpropanesulfonic acid (AAMPS). The complexes are less toxic than arbidol and retain the high level of antiviral activity of this drug. The content of arbidol in the obtained complexes is within 26.4–32.1 mass%. The antiviral activity of the synthesized polymeric complexes against all studied viruses, including human epidemic influenza virus A (H3N2), bird highly pathogenic influenza virus A (H5N1), herpes type 1 virus (HSV-1), and adenovirus type III (AV-III) is comparable to the antiviral effect of nonmodified arbidol. The in vitro toxicity of the obtained complexes is about one order of magnitude lower than that of nonmodified arbidol; the pharmacological index, four times that of the initial low-molecular-weight drug. The synthesized water-soluble polymer complexes of arbidol can be useful in pharmacology since they can serve as the basis for new effective and safe parent antiviral substances and related formulations.     

Treatment of influenza A (H1N1) virus infections in mice and ferrets with cyanovirin-N

Cyanovirin-N (CV-N), a protein derived from Nostoc ellipsosporum, neutralizes influenza virus infectivity by binding to specific high-mannose oligosaccharides (oligomannose-8 and -9) at glycosylation sites on the viral hemagglutinin HA1 subunit. Mouse-adapted viruses lose sensitivity to CV-N due to HA1 mutations that eliminate these glycosylation sites. Recently we created a hybrid (reassortant) influenza A/WSN/33 (H1N1) virus containing the HA gene of A/New Caledonia/20/99 (H1N1) with an Asp225Gly mutation in the HA1, that was lethal to mice yet retained sensitivity to CV-N. We then utilized this model system to test the efficacy of CV-N against influenza. CV-N efficacy was dose-responsive from 0.0625 to 1 mg/kg/day when administered intranasally (i.n.) twice daily for 4 days starting 4 h prior to virus exposure. In a second study, survival benefit was seen with CV-N treatments (0.5 mg/kg/day for 4 days) beginning at −4 or +6 h, but was significantly reduced at +12 h. The early treatment resulted in up to 100% survival and 1000-fold reduction in lung virus titer on day 3 of the infection. In contrast, ribavirin (a positive control—75 mg/kg/day) treatment resulted in 30% survival and 30-fold decrease in lung virus titers. Lung consolidation scores and lung weights were significantly reduced by CV-N and ribavirin treatment on day 6 of the infection. Ferrets infected with a non-animal adapted influenza A/Charlottesville/31/95 (H1N1) virus were treated intranasally with CV-N (50 μg twice daily for 5 days starting 24 h before virus challenge). They exhibited 100-fold lower viral titers in nasal washes than placebos 1 day after treatment, but virus titers were equivalent on days 2–7. CV-N has the potential for prophylaxis and early initiation of treatment of influenza virus infections.     

Design and Synthesis of Benzenesulfonamide Derivatives as Potent Anti-Influenza Hemagglutinin Inhibitors

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Immune responses of chickens inoculated with a recombinant fowlpox vaccine coexpressing HA of H9N2 avain influenza virus and chicken IL-18

Control of the circulation of H9N2 avian influenza virus (AIV) is a major concern for both animal and public health, and H9N2 AIV poses a major threat to the chicken industry worldwide. Here, we developed a recombinant fowlpox virus (rFPV-HA) expressing the haemagglutinin (HA) gene of the A/CH/JY/1/05 (H9N2) influenza virus and a recombinant fowlpox virus (rFPV-HA/IL18) expressing the HA gene and chicken interleukin-18 (IL-18) gene. Recombinant plasmid pSY-HA/IL18 was constructed by cloning chicken IL-18 expression cassette into recombinant plasmid pSY-HA containing the HA gene. Two rFPVs were generated by transfecting two recombinant plasmids into the chicken embryo fibroblast cells pre-infected with S-FPV-017, and assessed for their immunological efficacy on one-day-old White Leghorn specific-pathogen-free chickens challenged with the A/CH/JY/1/05 (H9N2) strain. There was a significant difference in HI antibody levels (P < 0.05) elicited by either rFPV-HA or rFPV-HA/IL18. The level of splenocyte proliferation response in the rFPV-HA/IL18-vaccinated group was significantly higher (P < 0.05) than that in the rFPV-HA group. After challenge with 10^6.5 ELD₅₀ H9N2 AIV 43 days after immunization, rFPVs vaccinated groups could prevent virus shedding and replication in multiple organs in response to H9N2 AIV infection, and rFPV-HA/IL18 vaccinated group had better inhibition of viruses than rFPV-HA vaccinated group. Our results show that the protective efficacy of the rFPV-HA vaccine could be enhanced significantly by simultaneous expression of IL-18.     

Characterization of a fully human monoclonal antibody against extracellular domain of matrix protein 2 of influenza A virus

The extra-cellular domain of the influenza virus matrix protein 2 (M2e) is highly conserved between influenza A virus strains compared to hemagglutinin and neuraminidase, and has long been viewed as a potential and universal vaccine target. M2e induces no or only weak and transient immune responses following infection, making it difficult to detect M2e-specific antibodies producing B-cells in human peripheral blood lymphocytes. Recently, using a single-cell manipulation method, immunospot array assay on a chip (ISAAC), we obtained an M2e-specific human antibody (Ab1-10) from the peripheral blood of a healthy volunteer. In this report, we have demonstrate that Ab1-10 reacted not only to seasonal influenza A viruses, but also to pandemic (H1N1) 2009 virus (2009 H1N1) and highly pathogenic avian influenza A virus, and that the antibody-bound M2e of 2009 H1N1 inactivated the virus with high affinity (∼10⁻¹⁰ M). More importantly, it inhibited 2009 H1N1 viral propagation in vitro. These results suggest that Ab1-10 might be a potential candidate for antibody therapeutics for a wide range of influenza A viruses.     

Characterization of a trypsin-dependent avian influenza H5N1-pseudotyped HIV vector system for high throughput screening of inhibitory molecules

In this study, we have generated and characterized an avian influenza H5N1 hemagglutinin (HA), neuraminidase (NA) and M2 ion channel pseudotyped HIV-based vector system (HaNaM-pseudotyped HIV vector). The cleavage site of the HA protein was modified to necessitate trypsin-dependent maturation of the glycoprotein. HA, NA and M2 were efficiently incorporated in HIV vector particles which could transduce different cell lines in a trypsin-dependent manner. Results also showed that the presence of avian influenza M2 and NA proteins maximized both vector production and transduction and that transduction was highly sensitive to the specific NA inhibitor oseltamivir (Tamiflu). H5N1 HaNaM-pseudotyped HIV vector system was also adapted for cell-based high throughput screening of drug candidates against influenza virus infection, and its high sensitivity to the specific oseltamivir validates its potential utility in the identification of new influenza inhibitors. Overall, the trypsin-dependent H5N1-pseudotyped HIV vector can mimic avian influenza virus infection processes with sufficient precision to allow for the identification of new antivirals and to study avian influenza virus biology in a lower biosafety level laboratory environment.     

PP2A mediates diosmin p53 activation to block HA22T cell proliferation and tumor growth in xenografted nude mice through PI3K–Akt–MDM2 signaling suppression

Hepatocellular carcinoma is a common type of cancer with poor prognosis. This study examines the in vitro and in vivo mechanisms of diosmin on human hepato-cellular carcinoma HA22T cell proliferation inhibition. HA22T cells were treated with different diosmin concentrations and analyzed with Western blot analysis, MTT assay, wound healing, flow cytometry, siRNA transfection assays and co-immuno-precipitation assay. The HA22T-implanted xeno-graft nude mice model was applied to confirm the cellular effects. Diosmin showed strong HA22T cell viability inhibition in a dose dependent manner and significantly reduced the cell proliferative proteins as well as inducing cell cycle arrest in the G2/M phase through p53 activation and PI3K–Akt–MDM2 signaling pathway inhibition. However, protein phosphatase 2A (PP2A) siRNA or PP2A inhibitor totally reversed the diosmin effects. The HA22T-implanted nude mice model further confirmed that diosmin inhibited HA22T tumor cell growth and down regulated the PI3K–Akt–MDM2 signaling and cell cycle regulating proteins, as well as activating PP2A and p53 proteins. Our findings indicate that HA22T cell proliferation inhibition and tumor growth suppression by diosmin are mediated through PP2A activation.     

CL-385319 inhibits H5N1 avian influenza A virus infection by blocking viral entry

CL-385319, an N-substituted piperidine, is effective in inhibiting infection of H1-, H2-, and to a lesser extent, H3-typed influenza A viruses by interfering with the fusogenic function of the viral hemagglutinin. Here we show that CL-385319 is effective in inhibiting infection of highly pathogenic H5N1 influenza A virus in Madin-Darby Canine Kidney (MDCK) cells with an IC50 of 27.03±2.54 μM. This compound with low cytotoxicity (CC50=1.48±0.01 mM) could also inhibit entry of pseudoviruses carrying hemagglutinins from H5N1 strains that were isolated from different places at different times, while it had no inhibitory activity on the entry of VSV-G pseudotyped particles. CL385319 could not inhibit N1-typed neuraminidase activity and the adsorption of H5-typed HA to chicken erythrocytes at the concentration as high as 1 mg/ml (2.8 mM). Computer-aid molecular docking analysis suggested that CL-385319 might bind to the cavity of HA2 stem region which was known to undergo significant rearrangement during membrane fusion. Pseudoviruses with M24A mutation in HA1 or F110S mutation in HA2 were resistant to CL-385319, indicating that these two residues in the cavity region may be critical for CL-385319 bindings. These findings suggest that CL-385319 can serve as a lead for development of novel virus entry inhibitors for preventing and treating H5N1 influenza A virus infection.     

Type II transmembrane serine proteases as potential target for anti-influenza drug discovery

Introduction: The outbreak of an influenza pandemic as well as the continued circulation of seasonal influenza highlights the need for effective antiviral therapies. The emergence of drug-resistant strains further necessitates the development of novel antivirals that target the host factors crucial for viral replication.

Area covered: This review summarizes the current understanding of the structural and functional properties of type II transmembrane serine proteases (TTSPs) as a proteolytic activator of influenza virus infection and discusses their potential as antiviral targets. It also explores the experimental evidence accumulated for inhibitors of TTSPs as novel, broad-spectrum antivirals against various influenza virus subtypes. The review also provides an overview of the properties of small molecules, proteins, and peptides that efficiently inhibit the proteolytic activation of the influenza virus.

Expert opinion: TTSPs activate a wide range of influenza virus subtypes including avian influenza viruses, both in vitro and in vivo, via proteolytic cleavage of influenza hemagglutinin (HA) into infection-competent fusogenic conformation. Other viruses such as SARS-, MERS-coronaviruses and human metapneumoviruses may use the same host cell proteases for activation, implying that TTSP inhibition might be a novel strategy for developing broad-spectrum antiviral agents for respiratory viral infections.     

A peptide targeted against phosphoprotein and leader RNA interaction inhibits growth of Chandipura virus – An emerging rhabdovirus

The fatal illness caused by Chandipura virus (CHPV), an emerging pathogen, presently lacks any therapeutic option. Previous research suggested that interaction between the virally encoded phosphoprotein (P) and the positive sense leader RNA (le-RNA) may play an important role in the viral lifecycle. In this report, we have identified a β-sheet/loop motif in the C-terminal domain of the CHPV P protein as essential for this interaction. A synthetic peptide encompassing this motif and spanning a continuous stretch of 36 amino acids (Pep_208–243) was found to bind the le-RNA in vitro and inhibit CHPV growth in infected cells. Furthermore, a stretch of three amino acid residues at position 217–219 was identified as essential for this interaction, both in vitro and in infected cells. siRNA knockdown-rescue experiments demonstrated that these three amino acid residues are crucial for the leader RNA binding function of P protein in the CHPV life cycle. Mutations of these three amino acid residues render the peptide completely ineffective against CHPV. Effect of inhibition of phosphoprotein–leader RNA interaction on viral replication was assayed. Peptide Pep_208–243 tagged with a cell penetrating peptide was found to inhibit CHPV replication as ascertained by real time RT-PCR. The specific inhibition of viral growth observed using this peptide suggests a new possibility for designing of anti-viral agents against Mononegavirale group of human viruses.     

Polymer-bound 6′ sialyl-N-acetyllactosamine protects mice infected by influenza virus

To develop a mouse model for testing receptor attachment inhibitors of human influenza viruses, the human clinical virus isolate in MDCK cells A/NIB/23/89M (H1N1) was adapted to mice by serial passaging through mouse lungs. The adaptation enhanced the viral pathogenicity for mice, but preserved the virus receptor binding phenotype, preferential binding to 2–6-linked sialic acid receptors and low affinity for 2–3-linked receptors. Sequencing of the HA gene of the mouse-adapted virus A/NIB/23/89-MA revealed a loss of the glycosylation sites in positions 94 and 163 of HA1 and substitutions 275Asp → Gly in HA1 and 145Asn → Asp in HA2. The four mouse strains tested differed significantly in their sensitivity to A/NIB/23/89-MA with the sensitivity increasing in the order of BALB/cJCitMoise, C57BL/6LacSto, CBA/CaLacSto and A/SnJCitMoise strains. Testing of protective efficacy of the polyacrylamide conjugate bearing Neu5Acα2-6Galβ1-4GlcNAc trisaccharide under conditions of lethal or sublethal virus infection demonstrated a strong protective effect of this preparation. In particular, aerosol treatment of mice with the polymeric attachment inhibitor on 24–110 h after infection completely prevented mortality in sensitive animals and lessened disease symptoms in more resistant mouse strains.     

Serum amyloid P component inhibits influenza A virus infections: in vitro and in vivo studies

Serum amyloid P component (SAP) binds in vitro Ca(2+)-dependently to several ligands including oligosaccharides with terminal mannose and galactose. We have earlier reported that SAP binds to human influenza A virus strains, inhibiting hemagglutinin (HA) activity and virus infectivity in vitro. These studies were extended to comprise five mouse-adapted influenza A strains, two swine influenza A strains, a mink influenza A virus, a ferret influenza A reassortant virus, a influenza B virus and a parainfluenza 3 virus. The HA activity of all these viruses was inhibited by SAP. Western blotting showed that SAP bound to HA trimers, monomers and HA1 and HA2 subunits of influenza A virus. Binding studies indicated that galactose, mannose and fucose moieties contributed to the SAP reacting site(s). Intranasal administration of human SAP to mice induced no demonstrable toxic reactions, and circulating antibodies against SAP were not detected. Preincubation of virus (A/Japan/57) with SAP prevented primary infection of mice and development of antiviral antibodies. After a single intranasal administration of SAP (40 microg) 1 h before primary infection with virus (2LD(50)), nine out of 10 mice survived on day 10 and these mice approached normal body weight, whereas control mice (one out of five surviving on day 10) died. The data provide evidence of the potential of intranasally administered SAP for prophylactic treatment of influenza A virus infections in humans.     

新型甲型H1N1流感病毒神经氨酸酶抑制剂细胞水平评价体系的建立

本文旨在建立以2009新型甲型H1N1流感病毒神经氨酸酶 (neuraminidase, NA) 为靶点的神经氨酸酶抑制剂 (neuraminidase inhibitors, NAIs) 细胞水平评价体系。NA有促进流感病毒释放的作用, 本研究应用重组病毒技术, 通过将表达NA [A/California/04/2009 (H1N1)] 的质粒、表达流感病毒血凝素蛋白HA (hemagglutinin) 的质粒以及表达敲除外壳基因并带有荧光素酶报告基因的HIV-1基因组共转染至病毒生成细胞, 产生以HA、NA为外壳蛋白包裹HIV-1核心的重组病毒。在病毒释放前加入不同浓度的化合物, 收集病毒上清液感染细胞, 通过测定感染率来反映化合物对重组病毒NA的抑制作用。经用阳性对照药物奥司他韦及其羧酸盐证实本模型能够反映化合物对病毒NA的抑制作用; 在此基础上, 本研究还建立了奥司他韦耐药株评价模型。本研究所建立的体系可用于针对新型甲型H1N1流感病毒及其临床耐药株神经氨酸酶抑制剂的寻找和评价。     

Primary structure of influenza virus genome regions coding for polypeptides from the major antigenic sites of H3 hemagglutinin

Nucleotide sequences for some regions of the hemagglutinin (HA) gene of influenza virus A/Leningrad/385/80 (H3N2) were analyzed. A double-stranded complementary DNA was synthesized on the influenza genome RNA in the presence of synthetic oligodeoxyribonucleotides A GCAAAAGCAGG and A GTAGAAACAAG and inserted into the Pst I-site of the pBR322 plasmid through G-C-tailing. Nucleotide sequences were determined by a solid phase modification of the Maxam and Gilbert procedure. A comparison of our data with those for influenza virus A/Bangkok/1/79, which is the nearest sequenced prototype of the strain investigated, revealed only two base changes in the variable region of the HA gene. One of them was accompanied by a change in the coded amino acid (Asp53----Tyr) located in the antigenic site E of the HA glycoprotein. There also were some point mutations in the constant region of the gene. The results obtained are discussed in terms of the evolution of "Hong Kong" influenza viruses during their circulation.     

Aprotinin and similar protease inhibitors as drugs against influenza

Efforts to develop new antiviral chemotherapeutic approaches are focusing on compounds that target either influenza virus replication itself or host factor(s) that are critical to influenza replication. Host protease mediated influenza hemagglutinin (HA) cleavage is critical for activation of virus infectivity and as such is a chemotherapeutic target. Influenza pathogenesis involves a “vicious cycle” in which host proteases activate progeny virus which in turn amplifies replication and stimulates further protease activities which may be detrimental to the infected host. Aprotinin, a 58 amino acid polypeptide purified from bovine lung that is one of a family of host-targeted antivirals that inhibit serine proteases responsible for influenza virus activation. This drug and similar agents, such as leupeptin and camostat, suppress virus HA cleavage and limit reproduction of human and avian influenza viruses with a single arginine in the HA cleavage site. Site-directed structural modifications of aprotinin are possible to increase its intracellular targeting of cleavage of highly virulent H5 and H7 hemagglutinins possessing multi-arginine/lysine cleavage site. An additional mechanism of action for serine protease inhibitors is to target a number of host mediators of inflammation and down regulate their levels in virus-infected hosts. Aprotinin is a generic drug approved for intravenous use in humans to treat pancreatitis and limit post-operative bleeding. As an antiinfluenzal compound, aprotinin might be delivered by two routes: (i) a small-particle aerosol has been approved in Russia for local respiratory application in mild-to-moderate influenza and (ii) a proposed intravenous administration for severe influenza to provide both an antiviral effect and a decrease in systemic pathology and inflammation.     

流感病毒裂解疫苗及单价病毒液的血凝素稳定性研究

目的 通过对连续三年生产的流行性感冒(流感)病毒裂解疫苗成品和单价病毒液(MVP)血凝素(HA)的定期检测,了解成品和MVP的HA含量的变化情况,以便更准确、有效地配制半成品.  方法 2003-2005年,每年选择一定数量的成品和MVP,按一定时间间隔抽样,采用单向免疫扩散法检测成品和MVP的HA含量.观察期至少为1年.  结果 成品存放至半年,HA含量都在控制范围之内.但存放至1年,个别毒株HA含量降至低于控制范围.MVP的HA稳定性因毒株的不同而异.   结论 一般情况下,流感疫苗在成品检定合格后7个月内完成使用,因此,在此期间,成品的HA含量在合格范围之内.不同毒株的MVP,HA降幅不同,可能是由于编码HA蛋白的RNA4节段的核苷酸序列在不同亚型,甚至在同一亚型不同变异株间的差异所造成的.     

Mechanism of a Decrease in Potency for the Recombinant Influenza A Virus Hemagglutinin H3 Antigen During Storage

The recombinant hemagglutinin (rHA)-based influenza vaccine Flublok® has recently been approved in the United States as an alternative to the traditional egg-derived flu vaccines. Flublok is a purified vaccine with a hemagglutinin content that is threefold higher than standard inactivated influenza vaccines. When rHA derived from an H3N2 influenza virus was expressed, purified, and stored for 1 month, a rapid loss of in vitro potency (~50%) was observed as measured by the single radial immunodiffusion (SRID) assay. A comprehensive characterization of the rHA protein antigen was pursued to identify the potential causes and mechanisms of this potency loss. In addition, the biophysical and chemical stability of the rHA in different formulations and storage conditions was evaluated over time. Results demonstrate that the potency loss over time did not correlate with trends in changes to the higher order structure or hydrodynamic size of the rHA. The most likely mechanism for the early loss of potency was disulfide-mediated cross-linking of rHA, as the formation of non-native disulfide-linked multimers over time correlated well with the observed potency loss. Furthermore, a loss of free thiol content, particularly in specific cysteine residues in the antigen's C-terminus, was correlated with potency loss measured by SRID.     

Novel N‐(1‐thia‐4‐azaspiro[4.5]decan‐4‐yl)carboxamide derivatives as potent and selective influenza virus fusion inhibitors

Hemagglutinin is the surface protein of the influenza virus that mediates both binding and penetration of the virus into host cells. We here report on the synthesis and structure–activity relationship of some novel N‐(1‐thia‐4‐azaspiro[4.5]decan‐4‐yl)‐carboxamide compounds carrying the 5‐chloro‐2‐methoxybenzamide structure, designed as influenza virus fusion inhibitors. The carboxamides ( 1a–h, 2a–h ) have a similar backbone structure as the fusion inhibitors that we reported on previously. Compounds 2b and 2d displayed inhibitory activity against influenza A/H3N2 virus replication (average antiviral EC₅₀: 2.1 µM for 2b and 3.4 µM for 2d ). Data obtained in the hemolysis inhibition assay supported that these compounds act as inhibitors of the influenza virus hemagglutinin‐mediated fusion process.     

Sialobiology of influenza: molecular mechanism of host range variation of influenza viruses

The gene pool of influenza A viruses in aquatic birds provides all of the genetic diversity required for human and lower animals. Host range selection of the receptor binding specificity of the influenza virus hemagglutinin occurs during maintenance of the virus in different host cells that express different receptor sialo-sugar chains. In this paper, functional roles of the hemagglutinin and neuraminidase spikes of influenza viruses are described in the relation to 1) host range of influenza viruses, 2) receptor binding specificity of human and other animal influenza viruses, 3) recognition of sialyl sugar chains by Spanish influenza virus hemagglutinin, 4) highly pathogenic and potentially pandemic H5N1, H9N2, and H7N7 avian influenza viruses and molecular mechanism of host range variation of influenza viruses, 5) role of the neuraminidase spike for the host range of influenza viruses, and 6) Development of anti-influenza drugs.