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  • 1
    Language: English
    In: Molecular Biotechnology, Oct, 2006, Vol.32(3), p.157(8)
    Description: Byline: Sabine Nakowitsch (1), C. Kittel (1), W. Ernst (1), A. Egorov (1), R. Grabherr (1) Keywords: Influenza B virus; transduction; baculovirus; mammalian cells; RNP-complex Abstract: Recombinant baculovirus expression vectors derived from the Autographa californica nuclear polyhedrosis virus can serve as efficient gene-transfer vehicles for transient expression of recombinant proteins in a wide range of mammalian cell types and are able to produce multisubunit particles such as viruses or virus like particles. In this study, we constructed eight recombinant baculoviruses each containing one of the influenza B/Lee/40 virus genes in a bidirectional expression cassette for simultaneous mRNA and viral RNA transcription. Baculoviruses were transduced into FreeStyle.sup.TM293 in combination with the specific histone deacetylase inhibitor trichostatin A (TSA). Contransduction conditions were optimized with a set of five baculoviruses (influenza B/Lee/40 PB1, PB2, PA, and NP and the control construct NCR-NS-minus-sense orientated encoding green fluorescent protein [rGFP]), which led to GFP expression in each host cell transduced with all five constructs. Based on the optimization with five constructs, transduction with eight baculoviruses was performed at MOI 50 and 100 with high yield stocks and 1 uM TSA and led to successful rescue of infectious influenza B/Lee/40 viruses. Author Affiliation: (1) Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Vienna, Austria Article History: Registration Date: 09/04/2007
    ISSN: 1073-6085
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  • 2
    Language: English
    In: PLoS ONE, 01 January 2015, Vol.10(6), p.e0128794
    Description: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection.A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 3
    Language: English
    In: PLoS ONE, 2011, Vol.6(4), p.e18577
    Description: H5N1 influenza vaccines, including live intranasal, appear to be relatively less immunogenic compared to seasonal analogs. The main influenza virus surface glycoprotein hemagglutinin (HA) of highly pathogenic avian influenza viruses (HPAIV) was shown to be more susceptible to acidic pH treatment than that of human or low pathogenic avian influenza viruses. The acidification machinery of the human nasal passageway in response to different irritation factors starts to release protons acidifying the mucosal surface (down to pH of 5.2). We hypothesized that the sensitivity of H5 HA to the acidic environment might be the reason for the low infectivity and immunogenicity of intranasal H5N1 vaccines for mammals. ; We demonstrate that original human influenza viruses infect primary human nasal epithelial cells at acidic pH (down to 5.4), whereas H5N1 HPAIVs lose infectivity at pH≤5.6. The HA of A/Vietnam/1203/04 was modified by introducing the single substitution HA2 58K→I, decreasing the pH of the HA conformational change. The H5N1 reassortants containing the indicated mutation displayed an increased resistance to acidic pH and high temperature treatment compared to those lacking modification. The mutation ensured a higher viral uptake as shown by immunohistochemistry in the respiratory tract of mice and 25 times lower mouse infectious dose. Moreover, the reassortants keeping 58K→I mutation designed as a live attenuated vaccine candidate lacking an NS1 gene induced superior systemic and local antibody response after the intranasal immunization of mice. ; Our finding suggests that an efficient intranasal vaccination with a live attenuated H5N1 virus may require a certain level of pH and temperature stability of HA in order to achieve an optimal virus uptake by the nasal epithelial cells and induce a sufficient immune response. The pH of the activation of the H5 HA protein may play a substantial role in the infectivity of HPAIVs for mammals.
    Keywords: Research Article ; Biology ; Medicine ; Virology ; Immunology ; Infectious Diseases
    E-ISSN: 1932-6203
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  • 4
    Language: English
    In: Biotechnology journal, March 2014, Vol.9(3), pp.405-14
    Description: Egg-derived viruses are the only available seed material for influenza vaccine production. Vaccine manufacturing is done in embryonated chicken eggs, MDCK or Vero cells. In order to contribute to efficient production of influenza vaccines, we investigate whether the quality of inactivated vaccines is influenced by the propagation substrate. We demonstrate that H3N2 egg-derived seed viruses (A/Brisbane/10/07, IVR147, and A/Uruguay/716/07) triggered the hemagglutinin (HA) conformational change under less acidic conditions (0.2-0.6 pH units) than antigenically similar primary isolates. This phenotype was associated with HA1 (A138S, L194P) and HA2 (D160N) substitutions, and strongly related to decreased virus stability towards acidic pH and elevated temperature. The subsequent propagation of H3N2 and H1N1 egg-derived seed viruses in MDCK and Vero cells induced HA2 N50K (H1N1) and D160E (H3N2) mutations, improving virus growth in cell culture but further impairing virus stability. The prevention of the loss or recovery of stability was possible by cultivation at acidified conditions. Viruses carrying less stable HAs are more sensitive for HA conformational change during concentration, purification and storage. This results in decreased detectable HA antigen content - the main potency marker for inactivated influenza vaccines. Thus, virus stability can be a useful marker for predicting the manufacturing scope of seed viruses.
    Keywords: Hemagglutinin Antigen Content ; Inactivated Vaccines ; Influenza Virus ; Temperature Stability ; Ph Stability ; Hemagglutinins -- Genetics ; Influenza Vaccines -- Immunology ; Influenza, Human -- Immunology ; Vaccines, Inactivated -- Immunology
    ISSN: 18606768
    E-ISSN: 1860-7314
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  • 5
    Language: English
    In: Biotechnology Journal, January 2010, Vol.5(1), pp.17-23
    Description: Recent outbreaks of influenza A highlight the importance of rapid and sufficient supply for pandemic and inter‐pandemic vaccines. Classical manufacturing methods for influenza vaccines fail to satisfy this demand. Alternatively, cell culture‐based production systems and virus‐like particle (VLP)‐based technologies have been established. We developed swine‐origin pandemic H1N1 influenza VLPs consisting of hemagglutinin (A/California/04/2009) and matrix protein. Hemagglutinin and matrix protein were co‐expressed in insect cells by the baculovirus expression system. VLPs were harvested from infection supernatants, purified and used for intraperitoneal immunization of BALB/c mice. Immunization induced high serum antibody titers against A/California/04/2009 as well as hemagglutination inhibiting antibodies. Additionally, we compared VLP production in two different insect cell lines, Sf9 and BTI‐TN5B1‐4 (High Five™). Taken together VLPs represent a potential strategy for the fight against new pandemic influenza viruses.
    Keywords: H1n1 ; Influenza Vaccine ; Insect Cells ; Pandemic Influenza ; Virus‐Like Particle
    ISSN: 1860-6768
    E-ISSN: 1860-7314
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  • 6
    Language: English
    In: Vaccine, 27 April 2011, Vol.29(19), pp.3517-3524
    Description: The isolation and cultivation of human influenza viruses in embryonated hen eggs or cell lines often leads to amino acid substitutions in the haemagglutinin (HA) molecule. We found that the propagation of influenza A H3N2 viruses on Vero cells may trigger the appearance of HA destabilising mutations, affecting viral resistance to low pH or high temperature treatment. Two ΔNS1 reassortants, containing the HA sequences identical to the original human H3N2 influenza virus isolates were constructed. Passages of these viruses on Vero cells led to the appearance of single mutations in the HA L194P or HA G75R subunits that impaired virus stability. The original HA sequences and the stable phenotypes of the primary isolates were preserved if reassortants were passaged by infection at pH 5.6 and cultivation in medium at pH 6.5. Corresponding ΔNS1 reassortants were compared for their immunogenicity in ferrets upon intranasal immunisation. Vaccine candidates containing HA mutations demonstrated significantly lower immunogenicity compared to those without mutations. Thus, the retaining of the original HA sequences of human viruses during vaccine production might be crucial for the efficacy of live attenuated influenza vaccines.
    Keywords: Influenza A Virus ; Ns1 Deletion ; Vero Cells ; Mutation in Ha ; Ha Stability ; Immunogenicity ; Medicine ; Biology ; Veterinary Medicine ; Pharmacy, Therapeutics, & Pharmacology
    ISSN: 0264-410X
    E-ISSN: 1873-2518
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  • 7
    Language: English
    In: Journal of Virology, 2011, Vol. 85(21), p.11139
    Description: In general, antibiotics are not rated as substances that inhibit or support influenza virus replication. We describe here the enhancing effect of the polyene antibiotic amphotericin B (AmB) on influenza virus growth in Vero cells. We show that isolation rates of influenza A and B viruses from clinical samples can be dramatically enhanced by adding AmB to the culture medium. We demonstrate that AmB promotes the viral uptake and endocytic processing of the virus particles. This effect is specific for Vero and human nasal epithelial cells and was not observed in Madin-Darby canine kidney cells. The effect of AmB was subtype specific and more prominent for human seasonal influenza strains but absent for H5N1 human viruses. The AmB-enhancing effect seemed to be solely due to the viral hemagglutinin function. Our results indicate that the use of AmB may facilitate influenza virus isolation and production in Vero cells.
    Keywords: Amphotericin B -- Metabolism; Antifungal Agents -- Metabolism; Influenza A Virus -- Drug Effects; Influenza B Virus -- Growth & Development; Virus Replication -- Drug Effects; Virus Replication -- Growth & Development; Virus Replication -- Drug Effects;
    ISSN: 1098-5514
    ISSN: 10985514
    ISSN: 0022538X
    Source: American Society of Microbiology
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  • 8
    Language: English
    In: Biotechnology Journal, March 2014, Vol.9(3), pp.405-414
    Description: Egg‐derived viruses are the only available seed material for influenza vaccine production. Vaccine manufacturing is done in embryonated chicken eggs, MDCK or Vero cells. In order to contribute to efficient production of influenza vaccines, we investigate whether the quality of inactivated vaccines is influenced by the propagation substrate. We demonstrate that H3N2 egg‐derived seed viruses (A/Brisbane/10/07, IVR147, and A/Uruguay/716/07) triggered the hemagglutinin (HA) conformational change under less acidic conditions (0.2–0.6 pH units) than antigenically similar primary isolates. This phenotype was associated with HA (A138S, L194P) and HA (D160N) substitutions, and strongly related to decreased virus stability towards acidic pH and elevated temperature. The subsequent propagation of H3N2 and H1N1 egg‐derived seed viruses in MDCK and Vero cells induced HA N50K (H1N1) and D160E (H3N2) mutations, improving virus growth in cell culture but further impairing virus stability. The prevention of the loss or recovery of stability was possible by cultivation at acidified conditions. Viruses carrying less stable HAs are more sensitive for HA conformational change during concentration, purification and storage. This results in decreased detectable HA antigen content – the main potency marker for inactivated influenza vaccines. Thus, virus stability can be a useful marker for predicting the manufacturing scope of seed viruses. To date, egg‐derived viruses are the only available seed material for influenza vaccine production. Further manufacturing in embryonated chicken eggs, MDCK or Vero cells is associated with hemagglutinin (HA) substitutions, which are strongly related to decreased virus stability. Viruses carrying less stable HAs are more likely to undergo conformational change during concentration, purification and storage and result in preparations with decreased detectable HA antigen content. The preservation or recovery of stability is possible by cultivation of virus under acidified conditions.
    Keywords: Hemagglutinin Antigen Content ; Inactivated Vaccines ; Influenza Virus ; Ph Stability ; Temperature Stability
    ISSN: 1860-6768
    E-ISSN: 1860-7314
    Source: John Wiley & Sons, Inc.
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  • 9
    Language: English
    In: Journal of virology, November 2011, Vol.85(21), pp.11139-45
    Description: In general, antibiotics are not rated as substances that inhibit or support influenza virus replication. We describe here the enhancing effect of the polyene antibiotic amphotericin B (AmB) on influenza virus growth in Vero cells. We show that isolation rates of influenza A and B viruses from clinical samples can be dramatically enhanced by adding AmB to the culture medium. We demonstrate that AmB promotes the viral uptake and endocytic processing of the virus particles. This effect is specific for Vero and human nasal epithelial cells and was not observed in Madin-Darby canine kidney cells. The effect of AmB was subtype specific and more prominent for human seasonal influenza strains but absent for H5N1 human viruses. The AmB-enhancing effect seemed to be solely due to the viral hemagglutinin function. Our results indicate that the use of AmB may facilitate influenza virus isolation and production in Vero cells.
    Keywords: Amphotericin B -- Metabolism ; Antifungal Agents -- Metabolism ; Influenza A Virus -- Drug Effects ; Influenza B Virus -- Drug Effects ; Virus Replication -- Drug Effects
    E-ISSN: 1098-5514
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  • 10
    In: AIDS, 2005, Vol.19(17), pp.1957-1966
    Description: OBJECTIVE:: The human monoclonal antibodies (mAb) 2F5, 2G12, and 4E10 are three of the most broadly neutralizing antibodies against HIV-1. Although they have been shown to prevent de novo infection in vivo, their potential for treatment of chronic infection is less clear. One major obstacle may be the emergence of resistant viruses during mAb treatment. DESIGN:: To assess whether escape mutants can be generated in vitro which are resistant to all three mAbs, two neutralization-sensitive T-cell line-adapted viruses and two primary isolates were passaged in the presence of increasing concentrations of 2F5, 2G12, 4E10, and a 1: 1: 1 mixture. To get insight into viral escape in vivo, viruses were isolated from eight patients treated with repeated infusions of 2F5/2G12/4E10. RESULTS:: In vitro, viruses resistant to a single mAb emerged after 3–22 weeks. Generation of viruses resistant to the triple-combination was a slower process characterized by recurrent loss of virus replication. Some generated triple-resistant viruses seemed to be impaired in their replicative fitness. Neutralization resistance to 2F5 and partly 4E10 could be attributed to amino acid mutations in the mAb epitopes, but not for 2G12. In vivo, none of the patients developed detectable viruses that escaped neutralization by all three mAbs within the 77-day observation period. Virus escape occurred only to 2G12 in three patients. CONCLUSIONS:: In summary, the findings of the in vivo study and the difficulty in generating multi-resistance in vitro together with the fact that some generated viruses seemed to have impaired replication fitness indicate that 2F5, 2G12, and 4E10 may be useful for therapy in HIV-1 infection.
    Keywords: Fitness ; Amino Acids ; Replication ; Monoclonal Antibodies ; Chronic Infection ; Lymphocytes T ; Mutation ; Epitopes ; Human Immunodeficiency Virus 1 ; AIDS: Clinical Aspects ; Virus ; HIV-1;
    ISSN: 0269-9370
    E-ISSN: 14735571
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