INFLUENCE OF HOST CELL DEFENCE DURING INFLUENZA VACCINE PRODUCTION IN MDCK CELLS

For cell culture-based influenza vaccine production virus yield optimisation is of crucial importance. In particular, with the recent threat of the new H1N1 pandemic, not only seasonal vaccines but also pre-/pandemic vaccines have to be supplied in large quantities. In vivo influenza replication is limited by the immune system, but for production cell lines the impact of cellular defence mechanisms on virus yield is unknown. In influenza-infected adherent Madin-Darby canine kidney (MDCK) cells the interferon (IFN) response and subsequent induction of the antiviral state was monitored. Virus yield and host cell signalling intensity were strain-dependent. By over-expression of viral antagonists IFN-signalling could be reduced up to 90%. However, maximum virus titre determined by real-time PCR and HA-assay was not altered significantly. Stimulation of the antiviral state by conditioned medium led to enhanced IFN-signalling, which initially slowed down virus replication but had only minor effects on final virus titres. Interestingly, minireplicon assays revealed that canine Mx proteins are lacking the antiviral activity against influenza of their human or mouse counterparts. In summary, for MDCK cell culture-based influenza virus production host cell defence mechanisms seem to play only a minor role for final virus yields. Antiviral mechanisms of these epithelial cells may slow down influenza replication, which in vivo gains time for the immune system to be activated, but do not reduce maximum virus titres obtained in the bioprocess

Influenza A virus propagation in MDCK: Intracellular virus replication, virus release and cell-cycle preferential infection analysis

Cell culture-based processes for vaccine manufacturing offer advantages over egg-based processes in terms of product uniformity and sterility, production time and scaling up capacity1,2. Regarding influenza vaccines, MDCK cells are one of the host cell lines currently used to manufacture licensed products; however, virus titers remain lower compared to those obtained in eggs and further increase of specific and volumetric yields is required. To identify bottlenecks in influenza A virus (IAV) production, we thoroughly studied IAV replication in MDCK cells. For this, we analyzed different features of the infection process such as viral RNA replication, intracellular localization of viral components, virus release and morphology of the particles, and the preferential infection in different cell-cycle phases. Using synchronous infections, we found that production of infectious particles dropped much earlier than the production of total particles. Furthermore, we found that the maximum virus release rate was reached when all viral RNA species attained their maximum intracellular concentration. Using qPCR we determined that the vRNA maximum concentration per cell was 10‑fold higher than the specific viral titers obtained, indicating that vRNA concentration does not limit IAV particle assembly. When we evaluated the morphology of particles released using electron microscopy, we observed that a higher fraction of the viral particles produced at late times possess an abnormal morphology, concurring with the increased production of non-infectious viruses. Using imaging flow cytometry, we determined that the export of influenza viral genome segments (ribonucleoprotein complexes, vRNPs) from the nucleus to the cytoplasm strongly correlated with the onset of virus release. However, our results also suggest that the induction of apoptosis caused that virus assembly became deficient producing more non-infectious particles at late infection times. Lastly, using low MOI infections and imaging flow cytometry, we found that -in contrast to previous publications- IAV did not preferentially infect a specific cell cycle phase and no cell cycle arrest induction was observed during the time frame of the experiment (9 hpi). In summary, the data presented here offers a comprehensive overview of the dynamics of IAV infection in MDCK and might contribute to the development of molecular or cell culture-based strategies to improve IAV production in MDCK cells. 1 Gallo-Ramírez et al, 2015. Exp. Rev. Vaccines 14 (9). 2 Pardue et al. 2011. Exp. Rev. Vaccines 10 (8)

Multiscale Modeling of Influenza A Virus Infection Supports the Development of Direct-Acting Antivirals

Influenza A viruses are respiratory pathogens that cause seasonal epidemics with up to 500,000 deaths each year. Yet there are currently only two classes of antivirals licensed for treatment and drug-resistant strains are on the rise. A major challenge for the discovery of new anti-influenza agents is the identification of drug targets that efficiently interfere with viral replication. To support this step, we developed a multiscale model of influenza A virus infection which comprises both the intracellular level where the virus synthesizes its proteins, replicates its genome, and assembles new virions and the extracellular level where it spreads to new host cells. This integrated modeling approach recapitulates a wide range of experimental data across both scales including the time course of all three viral RNA species inside an infected cell and the infection dynamics in a cell population. It also allowed us to systematically study how interfering with specific steps of the viral life cycle affects virus production. We find that inhibitors of viral transcription, replication, protein synthesis, nuclear export, and assembly/release are most effective in decreasing virus titers whereas targeting virus entry primarily delays infection. In addition, our results suggest that for some antivirals therapy success strongly depends on the lifespan of infected cells and, thus, on the dynamics of virus-induced apoptosis or the host's immune response. Hence, the proposed model provides a systems-level understanding of influenza A virus infection and therapy as well as an ideal platform to include further levels of complexity toward a comprehensive description of infectious diseases

Modeling the intracellular dynamics of influenza virus replication to understand the control of viral RNA synthesis

Influenza viruses transcribe and replicate their negative-sense RNA genome inside the nucleus of host cells via three viral RNA species. In the course of an infection, these RNAs show distinct dynamics, suggesting that differential regulation takes place. To investigate this regulation in a systematic way, we developed a mathematical model of influenza virus infection at the level of a single mammalian cell. It accounts for key steps of the viral life cycle, from virus entry to progeny virion release, while focusing in particular on the molecular mechanisms that control viral transcription and replication. We therefore explicitly consider the nuclear export of viral genome copies (vRNPs) and a recent hypothesis proposing that replicative intermediates (cRNA) are stabilized by the viral polymerase complex and the nucleoprotein (NP). Together, both mechanisms allow the model to capture a variety of published data sets at an unprecedented level of detail. Our findings provide theoretical support for an early regulation of replication by cRNA stabilization. However, they also suggest that the matrix protein 1 (M1) controls viral RNA levels in the late phase of infection as part of its role during the nuclear export of viral genome copies. Moreover, simulations show an accumulation of viral proteins and RNA toward the end of infection, indicating that transport processes or budding limits virion release. Thus, our mathematical model provides an ideal platform for a systematic and quantitative evaluation of influenza virus replication and its complex regulation. Copyright © 2012 by the American Society for Microbiology. [accessed November 2nd 2012

Herstellung moderner Grippeimpfstoffe : Zellkultur statt Hühnerei

Today, about 95 % of the available influenza vaccine doses are still produced in embryonated chicken eggs. However, since the 2009 H1N1 influenza pandemic many people have realized that in case of a real threat, it might be difficult to guarantee the timely supply of a sufficient amount of vaccines. Therefore, it is of utmost importance to improve existing processes and to develop alternative manufacturing methods. The cell culture-based processes discussed in this manuscript represent a promising option to establish scalable and robust production processes. Probably these processes have not been pushed up to their limits yet. Further optimization concerning the design of bioprocesses and the optimization of biological systems involved could contribute significantly to increase yields and product quality. Copyright © 1999–2012 John Wiley & Sons, Inc. All Rights Reserved. [accessed November 29th 2012

Elucidating bottlenecks in influenza virus replication to optimize vaccine production

Influenza viruses cause seasonal epidemics with up to 500,000 deaths each year and have the potential of becoming pandemic. The most effective way to prevent a severe infection is vaccination. However, ensuring an adequate and timely supply with vaccines poses a serious challenge as manufacturing capacities are limited and yields heavily depend on the host system's ability to produce large amounts of virus particles. To optimize influenza vaccine production in mammalian cell culture, we developed a quantitative model of the viral life cycle. It comprises key steps of intracellular replication from virus entry to the release of progeny virus particles. By integrating a variety of published data sets on different aspects of infection, we were able to capture the time course of viral protein and genome synthesis. Further analyses showed that the virus can regulate these dynamics by controlling the stability and the transport of its genomic RNAs. Interestingly, simulations also predict an accumulation of viral genomes and proteins toward the end of infection indicating that virus assembly and budding are potential bottlenecks and that in principle cells could release more virions. Hence, viral control mechanisms and the steps involved in virus release may provide promising targets for the optimization of cell culture-based vaccine production. Modeling can contribute to this optimization by facilitating a systems-levels understanding of the influenza virus life cycle

Elucidating bottlenecks in influenza virus replication to optimize vaccine production

Influenza viruses cause seasonal epidemics with up to 500,000 deaths each year and have the potential of becoming pandemic. The most effective way to prevent a severe infection is vaccination. However, ensuring an adequate and timely supply with vaccines poses a serious challenge as manufacturing capacities are limited and yields heavily depend on the host system's ability to produce large amounts of virus particles. To optimize influenza vaccine production in mammalian cell culture, we developed a quantitative model of the viral life cycle. It comprises key steps of intracellular replication from virus entry to the release of progeny virus particles. By integrating a variety of published data sets on different aspects of infection, we were able to capture the time course of viral protein and genome synthesis. Further analyses showed that the virus can regulate these dynamics by controlling the stability and the transport of its genomic RNAs. Interestingly, simulations also predict an accumulation of viral genomes and proteins toward the end of infection indicating that virus assembly and budding are potential bottlenecks and that in principle cells could release more virions. Hence, viral control mechanisms and the steps involved in virus release may provide promising targets for the optimization of cell culture-based vaccine production. Modeling can contribute to this optimization by facilitating a systems-levels understanding of the influenza virus life cycle

Elucidating the dynamics of influenza virus replication by mathematical modeling

Influenza viruses transcribe and replicate their negative-sense RNA genome inside the nucleus of host cells via three viral RNA species. These RNAs are regulated in a quantitative and temporal manner such that each species shows distinct dynamics during infection. The molecular mechanisms behind this regulation have received much attention in the past. However, so far the wealth of available data could not be integrated into a consistent description of the viral life cycle. We developed a mathematical model of influenza virus infection on the single cell level to gain a quantitative understanding of virus replication. It encompasses key steps from virus entry to the release of progeny virions and focuses in particular on the regulation of viral RNA synthesis. We find that two control mechanisms are essential and sufficient for the model to capture a variety of published experimental data: (I) the early control of virus replication by a recently proposed mechanism in which viral proteins stabilize replicative intermediates (cRNA); (II) the nuclear export of genome copies (vRNA) at later stages which may directly cause the previously described shutdown of positive-strand RNA synthesis. Simulations also suggest that the transport of viral precursors or budding may limit virion release as viral proteins and genomes accumulate in the cytoplasm toward the end of infection. Thus, modeling provides a valuable tool to gain a systems-level understanding of influenza virus replication

The role of the type I interferon response in mammalian cell culture based influenza vaccine production

For cell culture based Influenza vaccine production processes virus yield optimization is of crucial importance. A well characterized factor limiting Influenza replication in vivo is the type I interferon (IFN) response. Therefore, we want to analyse whether induction of type I IFN and subsequent expression of antiviral genes is also of importance in a Madin-Darby canine kidney (MDCK) cell culture based Influenza production process. qRT-PCR and luciferase reporter assays were used to determine the expression of IFN-beta and Mx1 in MDCK cells infected with different Influenza viruses. All strains tested showed significant induction of IFN-beta, but levels, as well as kinetics of induction and the amount of resulting Mx1 expression varied strain dependently. As a first approach to examine the influence of IFN-beta expression on virus yield and virus replication dynamics, we transiently transfected MDCK cells with a plasmid containing the sequence of A/PR/8/34 NS1, which acts as a type I IFN antagonists. Infection of these cells with different Influenza strains showed at least 5 fold reduced levels of IFN-beta induction. For A/PR/8/34 delNS1 this inhibition of IFN induction resulted in faster virus production and slightly higher maximal virus yield compared to control cells. However, for virus strains with functional NS1 no significant effect of NS1-overexpression on virus yield or replication kinetics was found. A possible explanation could be that in cell culture with MDCK cells and MDCK-adapted Influenza viruses, expression of IFN and IFN-stimulated genes is not a yield limiting factor. This is also supported by experiments showing that pre-treatment of MDCK cells with IFN-containing medium does not affect virus yield in our system