Virus Production

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Udo Reichl - One of the best experts on this subject based on the ideXlab platform.

  • continuous influenza Virus Production in a tubular bioreactor system provides stable titers and avoids the von magnus effect
    PLOS ONE, 2019
    Co-Authors: Felipe Tapia, Daniel Wohlfarth, Volker Sandig, Ingo Jordan, Yvonne Genzel, Udo Reichl
    Abstract:

    Continuous cell culture-based influenza vaccine Production could significantly reduce footprint and manufacturing costs compared to current batch processing. However, yields of influenza Virus in continuous mode can be affected by oscillations in Virus titers caused by periodic accumulation of defective interfering particles. The generation of such particles has also been observed previously in cascades of continuous stirred tank reactors (CSTRs) and is known as the "von Magnus effect". To improve Virus yields and to avoid these oscillations, we have developed a novel continuous tubular bioreactor system for influenza A Virus Production. It was built using a 500 mL CSTR for cell growth linked to a 105 m long tubular plug-flow bioreactor (PFBR). Virus propagation took place only in the PFBR with a nominal residence time of 20 h and a Production capacity of 0.2 mL/min. The bioreactor was first tested with suspension MDCK cells at different multiplicities of infection (MOI), and then with suspension avian AGE1.CR.pIX cells at a fixed nominal MOI of 0.02. Maximum hemagglutinin (HA) titers of 2.4 and 1.6 log10(HA units/100 μL) for suspension MDCK cells and AGE1.CR.pIX cells, respectively, were obtained. Flow cytometric analysis demonstrated that 100% infected cells with batch-like HA titers can be obtained at a MOI of at least 0.1. Stable HA and TCID50 titers over 18 days of Production were confirmed using the AGE1.CR.pIX cell line, and PCR analysis demonstrated stable Production of full-length genome. The contamination level of segments with deletions (potentially defective interfering particles), already present in the Virus seed, was low and did not increase. Control experiments using batch and semi-continuous cultures confirmed these findings. A comparison showed that influenza Virus Production can be achieved with the tubular bioreactor system in about half the time with a space-time-yield up to two times higher than for typical batch cultures. In summary, a novel continuous tubular bioreactor system for cell culture-based influenza Virus Production was developed. One main advantage, an essentially single-passage amplification of Viruses, should enable efficient Production of vaccines as well as vectors for gene and cancer therapy.

  • Efficient influenza A Virus Production in high cell density using the novel porcine suspension cell line PBG.PK2.1.
    Vaccine, 2019
    Co-Authors: Gwendal Gränicher, Volker Sandig, Yvonne Genzel, Erdmann Rapp, Juliana Coronel, Alexander Pralow, Pavel Marichal-gallardo, Michael W. Wolff, Alexander Karlas, Udo Reichl
    Abstract:

    Abstract Seasonal and pandemic influenza respiratory infections are still a major public health issue. Vaccination is the most efficient way to prevent influenza infection. One option to produce influenza vaccines is cell-culture based Virus propagation. Different host cell lines, such as MDCK, Vero, AGE1.CR or PER.C6 cells have been shown to be a good substrate for influenza Virus Production. With respect to the ease of scale-up, suspension cells should be preferred over adherent cells. Ideally, they should replicate different influenza Virus strains with high cell-specific yields. Evaluation of new cell lines and further development of processes is of considerable interest, as this increases the number of options regarding the design of manufacturing processes, flexibility of vaccine Production and efficiency. Here, PBG.PK2.1, a new mammalian cell line that was developed by ProBioGen AG (Germany) for Virus Production is presented. The cells derived from immortal porcine kidney cells were previously adapted to growth in suspension in a chemically-defined medium. Influenza Virus Production was improved after Virus adaptation to PBG.PK2.1 cells and optimization of infection conditions, namely multiplicity of infection and trypsin concentration. Hemagglutinin titers up to 3.24 log10(HA units/100 µL) were obtained in fed-batch mode in bioreactors (700 mL working volume). Evaluation of Virus propagation in high cell density culture using a hollow-fiber based system (ATF2) demonstrated promising performance: Cell concentrations of up to 50 × 106 cells/mL with viabilities exceeding 95%, and a maximum HA titer of 3.93 log10(HA units/100 µL). Analysis of glycosylation of the viral HA antigen expressed showed clear differences compared to HA produced in MDCK or Vero cell lines. With an average cell-specific productivity of 5000 virions/cell, we believe that PBG.PK2.1 cells are a very promising candidate to be considered for next-generation influenza Virus vaccine Production.

  • CAP, a new human suspension cell line for influenza Virus Production
    Applied microbiology and biotechnology, 2012
    Co-Authors: Yvonne Genzel, Ilona Behrendt, J. Rödig, Erdmann Rapp, Claudia Kueppers, Stefan Kochanek, Gudrun Schiedner, Udo Reichl
    Abstract:

    Forced by major drawbacks of egg-based influenza Virus Production, several studies focused on the establishment and optimization of cell-based Production systems. Among numerous possible host cell lines from duck, monkey, canine, chicken, mouse, and human origin, only a few will meet regulatory requirements, accomplish industrial standards, and result in high Virus titers. From primary Virus isolation up to large-scale manufacturing of human vaccines, however, the most logical choice seems to be the use of human cell lines. For this reason, we evaluated the recently established CAP cell line derived from human amniocytes for its potential in influenza Virus Production in suspension culture in small scale shaker flask and stirred tank bioreactor experiments. Different human and animal influenza Viruses could be adapted to produce hemagglutination (HA) titers of at least 2.0 log10 HA units/100 μL without further process optimization. Adjusting trypsin activity as well as infection conditions (multiplicity of infection, infection medium) resulted in HA titers of up to 3.2 log10 HA units/100 μL and maximum cell-specific Virus productivities of 6,400 virions/cell (for human influenza A/PR/8/34 as a reference). Surface membrane expression of sialyloligosaccharides as well as HA N-glycosylation patterns were characterized. Overall, experimental results clearly demonstrate the potential of CAP cells for achieving high Virus yields for different influenza strains and the option to introduce a highly attractive fully characterized human cell line compliant with regulatory and industrial requirements as an alternative for influenza Virus vaccine Production.

Georg A Funk - One of the best experts on this subject based on the ideXlab platform.

  • hiv replication elicits little cytopathic effects in vivo analysis of surrogate markers for Virus Production cytotoxic t cell response and infected cell death
    Journal of Medical Virology, 2006
    Co-Authors: Georg A Funk, Annette Oxenius, Marek Fischer, Milos Opravil, Beda Joos, Markus Flepp, Rainer Weber, Huldrych F Gunthard
    Abstract:

    Several potential mechanisms for viral destruction of HIV-infected cells have been described. The hypothesis was examined that if HIV were cytopathic, a positive relation between the in vivo Virus Production or CTL activity and infected cell death should be observed. In a regression analysis no significant relation was found between surrogate markers for in vivo Virus Production or the Virus-specific CTL response and death rates of productively infected cells. In a subgroup of patients the hypothesis is rejected that HIV replication elicits a large (R2 > 0.25) cytopathic effect (P < 0.05, N = 36). It is concluded that HIV replication elicits little cytopathic effect in productively infected cells and that CD4+ T lymphocytes are eroded by other mechanisms. J. Med. Virol. 78:1141–1146, 2006. © 2006 Wiley-Liss, Inc.

  • HIV replication elicits little cytopathic effects in vivo: analysis of surrogate markers for Virus Production, cytotoxic T cell response and infected cell death.
    Journal of medical virology, 2006
    Co-Authors: Georg A Funk, Annette Oxenius, Marek Fischer, Milos Opravil, Beda Joos, Markus Flepp, Rainer Weber, Huldrych F Gunthard, Sebastian Bonhoeffer
    Abstract:

    Several potential mechanisms for viral destruction of HIV-infected cells have been described. The hypothesis was examined that if HIV were cytopathic, a positive relation between the in vivo Virus Production or CTL activity and infected cell death should be observed. In a regression analysis no significant relation was found between surrogate markers for in vivo Virus Production or the Virus-specific CTL response and death rates of productively infected cells. In a subgroup of patients the hypothesis is rejected that HIV replication elicits a large (R2 > 0.25) cytopathic effect (P 

Yvonne Genzel - One of the best experts on this subject based on the ideXlab platform.

  • continuous influenza Virus Production in a tubular bioreactor system provides stable titers and avoids the von magnus effect
    PLOS ONE, 2019
    Co-Authors: Felipe Tapia, Daniel Wohlfarth, Volker Sandig, Ingo Jordan, Yvonne Genzel, Udo Reichl
    Abstract:

    Continuous cell culture-based influenza vaccine Production could significantly reduce footprint and manufacturing costs compared to current batch processing. However, yields of influenza Virus in continuous mode can be affected by oscillations in Virus titers caused by periodic accumulation of defective interfering particles. The generation of such particles has also been observed previously in cascades of continuous stirred tank reactors (CSTRs) and is known as the "von Magnus effect". To improve Virus yields and to avoid these oscillations, we have developed a novel continuous tubular bioreactor system for influenza A Virus Production. It was built using a 500 mL CSTR for cell growth linked to a 105 m long tubular plug-flow bioreactor (PFBR). Virus propagation took place only in the PFBR with a nominal residence time of 20 h and a Production capacity of 0.2 mL/min. The bioreactor was first tested with suspension MDCK cells at different multiplicities of infection (MOI), and then with suspension avian AGE1.CR.pIX cells at a fixed nominal MOI of 0.02. Maximum hemagglutinin (HA) titers of 2.4 and 1.6 log10(HA units/100 μL) for suspension MDCK cells and AGE1.CR.pIX cells, respectively, were obtained. Flow cytometric analysis demonstrated that 100% infected cells with batch-like HA titers can be obtained at a MOI of at least 0.1. Stable HA and TCID50 titers over 18 days of Production were confirmed using the AGE1.CR.pIX cell line, and PCR analysis demonstrated stable Production of full-length genome. The contamination level of segments with deletions (potentially defective interfering particles), already present in the Virus seed, was low and did not increase. Control experiments using batch and semi-continuous cultures confirmed these findings. A comparison showed that influenza Virus Production can be achieved with the tubular bioreactor system in about half the time with a space-time-yield up to two times higher than for typical batch cultures. In summary, a novel continuous tubular bioreactor system for cell culture-based influenza Virus Production was developed. One main advantage, an essentially single-passage amplification of Viruses, should enable efficient Production of vaccines as well as vectors for gene and cancer therapy.

  • Efficient influenza A Virus Production in high cell density using the novel porcine suspension cell line PBG.PK2.1.
    Vaccine, 2019
    Co-Authors: Gwendal Gränicher, Volker Sandig, Yvonne Genzel, Erdmann Rapp, Juliana Coronel, Alexander Pralow, Pavel Marichal-gallardo, Michael W. Wolff, Alexander Karlas, Udo Reichl
    Abstract:

    Abstract Seasonal and pandemic influenza respiratory infections are still a major public health issue. Vaccination is the most efficient way to prevent influenza infection. One option to produce influenza vaccines is cell-culture based Virus propagation. Different host cell lines, such as MDCK, Vero, AGE1.CR or PER.C6 cells have been shown to be a good substrate for influenza Virus Production. With respect to the ease of scale-up, suspension cells should be preferred over adherent cells. Ideally, they should replicate different influenza Virus strains with high cell-specific yields. Evaluation of new cell lines and further development of processes is of considerable interest, as this increases the number of options regarding the design of manufacturing processes, flexibility of vaccine Production and efficiency. Here, PBG.PK2.1, a new mammalian cell line that was developed by ProBioGen AG (Germany) for Virus Production is presented. The cells derived from immortal porcine kidney cells were previously adapted to growth in suspension in a chemically-defined medium. Influenza Virus Production was improved after Virus adaptation to PBG.PK2.1 cells and optimization of infection conditions, namely multiplicity of infection and trypsin concentration. Hemagglutinin titers up to 3.24 log10(HA units/100 µL) were obtained in fed-batch mode in bioreactors (700 mL working volume). Evaluation of Virus propagation in high cell density culture using a hollow-fiber based system (ATF2) demonstrated promising performance: Cell concentrations of up to 50 × 106 cells/mL with viabilities exceeding 95%, and a maximum HA titer of 3.93 log10(HA units/100 µL). Analysis of glycosylation of the viral HA antigen expressed showed clear differences compared to HA produced in MDCK or Vero cell lines. With an average cell-specific productivity of 5000 virions/cell, we believe that PBG.PK2.1 cells are a very promising candidate to be considered for next-generation influenza Virus vaccine Production.

  • CAP, a new human suspension cell line for influenza Virus Production
    Applied microbiology and biotechnology, 2012
    Co-Authors: Yvonne Genzel, Ilona Behrendt, J. Rödig, Erdmann Rapp, Claudia Kueppers, Stefan Kochanek, Gudrun Schiedner, Udo Reichl
    Abstract:

    Forced by major drawbacks of egg-based influenza Virus Production, several studies focused on the establishment and optimization of cell-based Production systems. Among numerous possible host cell lines from duck, monkey, canine, chicken, mouse, and human origin, only a few will meet regulatory requirements, accomplish industrial standards, and result in high Virus titers. From primary Virus isolation up to large-scale manufacturing of human vaccines, however, the most logical choice seems to be the use of human cell lines. For this reason, we evaluated the recently established CAP cell line derived from human amniocytes for its potential in influenza Virus Production in suspension culture in small scale shaker flask and stirred tank bioreactor experiments. Different human and animal influenza Viruses could be adapted to produce hemagglutination (HA) titers of at least 2.0 log10 HA units/100 μL without further process optimization. Adjusting trypsin activity as well as infection conditions (multiplicity of infection, infection medium) resulted in HA titers of up to 3.2 log10 HA units/100 μL and maximum cell-specific Virus productivities of 6,400 virions/cell (for human influenza A/PR/8/34 as a reference). Surface membrane expression of sialyloligosaccharides as well as HA N-glycosylation patterns were characterized. Overall, experimental results clearly demonstrate the potential of CAP cells for achieving high Virus yields for different influenza strains and the option to introduce a highly attractive fully characterized human cell line compliant with regulatory and industrial requirements as an alternative for influenza Virus vaccine Production.

Huldrych F Gunthard - One of the best experts on this subject based on the ideXlab platform.

  • hiv replication elicits little cytopathic effects in vivo analysis of surrogate markers for Virus Production cytotoxic t cell response and infected cell death
    Journal of Medical Virology, 2006
    Co-Authors: Georg A Funk, Annette Oxenius, Marek Fischer, Milos Opravil, Beda Joos, Markus Flepp, Rainer Weber, Huldrych F Gunthard
    Abstract:

    Several potential mechanisms for viral destruction of HIV-infected cells have been described. The hypothesis was examined that if HIV were cytopathic, a positive relation between the in vivo Virus Production or CTL activity and infected cell death should be observed. In a regression analysis no significant relation was found between surrogate markers for in vivo Virus Production or the Virus-specific CTL response and death rates of productively infected cells. In a subgroup of patients the hypothesis is rejected that HIV replication elicits a large (R2 > 0.25) cytopathic effect (P < 0.05, N = 36). It is concluded that HIV replication elicits little cytopathic effect in productively infected cells and that CD4+ T lymphocytes are eroded by other mechanisms. J. Med. Virol. 78:1141–1146, 2006. © 2006 Wiley-Liss, Inc.

  • HIV replication elicits little cytopathic effects in vivo: analysis of surrogate markers for Virus Production, cytotoxic T cell response and infected cell death.
    Journal of medical virology, 2006
    Co-Authors: Georg A Funk, Annette Oxenius, Marek Fischer, Milos Opravil, Beda Joos, Markus Flepp, Rainer Weber, Huldrych F Gunthard, Sebastian Bonhoeffer
    Abstract:

    Several potential mechanisms for viral destruction of HIV-infected cells have been described. The hypothesis was examined that if HIV were cytopathic, a positive relation between the in vivo Virus Production or CTL activity and infected cell death should be observed. In a regression analysis no significant relation was found between surrogate markers for in vivo Virus Production or the Virus-specific CTL response and death rates of productively infected cells. In a subgroup of patients the hypothesis is rejected that HIV replication elicits a large (R2 > 0.25) cytopathic effect (P 

Stanley M. Lemon - One of the best experts on this subject based on the ideXlab platform.

  • Intracellular Proton Conductance of the Hepatitis C Virus p7 Protein and Its Contribution to Infectious Virus Production
    PLoS pathogens, 2010
    Co-Authors: Ann L. Wozniak, Stephen Griffin, David J. Rowlands, Mark Harris, Stanley M. Lemon, Steven A. Weinman
    Abstract:

    The hepatitis C Virus (HCV) p7 protein is critical for Virus Production and an attractive antiviral target. p7 is an ion channel when reconstituted in artificial lipid bilayers, but channel function has not been demonstrated in vivo and it is unknown whether p7 channel activity plays a critical role in Virus Production. To evaluate the contribution of p7 to organelle pH regulation and Virus Production, we incorporated a fluorescent pH sensor within native, intracellular vesicles in the presence or absence of p7 expression. p7 increased proton (H+) conductance in vesicles and was able to rapidly equilibrate H+ gradients. This conductance was blocked by the viroporin inhibitors amantadine, rimantadine and hexamethylene amiloride. Fluorescence microscopy using pH indicators in live cells showed that both HCV infection and expression of p7 from replicon RNAs reduced the number of highly acidic (pH

  • intracellular proton conductance of the hepatitis c Virus p7 protein and its contribution to infectious Virus Production
    PLOS Pathogens, 2010
    Co-Authors: Ann L. Wozniak, Stephen Griffin, David J. Rowlands, Mark Harris, Stanley M. Lemon, Steven A. Weinman
    Abstract:

    The hepatitis C Virus (HCV) p7 protein is critical for Virus Production and an attractive antiviral target. p7 is an ion channel when reconstituted in artificial lipid bilayers, but channel function has not been demonstrated in vivo and it is unknown whether p7 channel activity plays a critical role in Virus Production. To evaluate the contribution of p7 to organelle pH regulation and Virus Production, we incorporated a fluorescent pH sensor within native, intracellular vesicles in the presence or absence of p7 expression. p7 increased proton (H+) conductance in vesicles and was able to rapidly equilibrate H+ gradients. This conductance was blocked by the viroporin inhibitors amantadine, rimantadine and hexamethylene amiloride. Fluorescence microscopy using pH indicators in live cells showed that both HCV infection and expression of p7 from replicon RNAs reduced the number of highly acidic (pH<5) vesicles and increased lysosomal pH from 4.5 to 6.0. These effects were not present in uninfected cells, sub-genomic replicon cells not expressing p7, or cells electroporated with viral RNA containing a channel-inactive p7 point mutation. The acidification inhibitor, bafilomycin A1, partially restored Virus Production to cells electroporated with viral RNA containing the channel inactive mutation, yet did not in cells containing p7-deleted RNA. Expression of influenza M2 protein also complemented the p7 mutant, confirming a requirement for H+ channel activity in Virus Production. Accordingly, exposure to acid pH rendered intracellular HCV particles non-infectious, whereas the infectivity of extracellular virions was acid stable and unaffected by incubation at low pH, further demonstrating a key requirement for p7-induced loss of acidification. We conclude that p7 functions as a H+ permeation pathway, acting to prevent acidification in otherwise acidic intracellular compartments. This loss of acidification is required for productive HCV infection, possibly through protecting nascent Virus particles during an as yet uncharacterized maturation process.

  • regulation of hepatitis c Virus translation and infectious Virus Production by the microrna mir 122
    Journal of Virology, 2010
    Co-Authors: Rohit K Jangra, Stanley M. Lemon
    Abstract:

    miR-122 is a liver-specific microRNA that positively regulates hepatitis C Virus (HCV) RNA abundance and is essential for Production of infectious HCV. Using a genetic approach, we show that its ability to enhance yields of infectious Virus is dependent upon two miR-122-binding sites near the 5′ end of the HCV genome, S1 and S2. Viral RNA with base substitutions in both S1 and S2 failed to produce infectious Virus in transfected cells, while Virus Production was rescued to near-wild-type levels in cells supplemented with a complementary miR-122 mutant. A comparison of mutants with substitutions in only one site revealed S1 to be dominant, as an S2 but not S1 mutant produced high Virus yields in cells supplemented with wild-type miR-122. Translation of HCV RNA was reduced over 50% by mutations in either S1 or S2 and was partially rescued by transfection of the complementary miR-122 mutant. Unlike the case for Virus replication, however, both sites function equally in regulating translation. We conclude that miR-122 promotes replication by binding directly to both sites in the genomic RNA and, at least in part, by stimulating internal ribosome entry site (IRES)-mediated translation. However, a comparison of the replication capacities of the double-binding-site mutant and an IRES mutant with a quantitatively equivalent defect in translation suggests that the decrement in translation associated with loss of miR-122 binding is insufficient to explain the profound defect in Virus Production by the double mutant. miR-122 is thus likely to act at an additional step in the Virus life cycle.