Live attenuated influenza viruses produced in a suspension process with avian AGE1.CR.pIX cells

Background: Current influenza vaccines are trivalent or quadrivalent inactivated split or subunit vaccines administered intramuscularly, or live attenuated influenza vaccines (LAIV) adapted to replicate at temperatures below body temperature and administered intranasally. Both vaccines are considered safe and efficient, but due to differences in specific properties may complement each other to ensure reliable vaccine coverage. By now, licensed LAIV are produced in embryonated chicken eggs. In the near future influenza vaccines for human use will also be available from adherent MDCK or Vero cell cultures, but a scalable suspension process may facilitate production and supply with vaccines. Results: We evaluated the production of cold-adapted human influenza virus strains in the duck suspension cell line AGE1.CR.pIX using a chemically-defined medium. One cold-adapted A (H1N1) and one cold-adapted B virus strain was tested, as well as the reference strain A/PR/8/34 (H1N1). It is shown that a medium exchange is not required for infection and that maximum virus titers are obtained for 1x10-6 trypsin units per cell. 1 L bioreactor cultivations showed that 4x106 cells/mL can be infected without a cell density effect achieving titers of 1x108 virions/mL after 24 h. Conclusions: Overall, this study demonstrates that AGE1.CR.pIX cells support replication of LAIV strains in a chemically-defined medium using a simple process without medium exchanges. Moreover, the process is fast with peak titers obtained 24 h post infection and easily scalable to industrial volumes as neither microcarriers nor medium replacements are required. © 2012 Lohr et al.; licensee BioMed Central Ltd. [accessed 2013 November 18th

Process optimization for semi-continuous virus production at high cell densities

Background. Unlike production of recombinant proteins, continuous production of viral vaccines at high cell densities (HCD) is still constrained by host cell lysis during virus propagation and limited virus recovery from culture broth. Nevertheless, advanced fed-batch [1] and perfusion strategies can be applied to achieve a high-yield virus production processes. In this study, the development of a high-yield semi-continuous process for the production and purification of the modified vaccinia Ankara virus isolate MVA-CR19 and influenza A/PR/8 in HCD cultivations of the suspension cell line AGE1.CR.pIX (ProBioGen AG, Berlin) is presented. Methods. Depending on the required scale, high cell concentrations (~ 50×106 cell mL-1) were achieved either through medium renewal by periodic centrifugation (semi-perfusion) in 50 mL cultivations or using an alternating tangential flow (ATF) perfusion system for 1 L bioreactors. Process development and optimization comprised three phases: 1) assessment of different fed-batch and medium exchange strategies for the propagation of MVA-CR19 or influenza A/PR/8 viruses in 50 mL cultivations; 2) scale-up and process optimization of the high-yield process strategy to a 1 L bioreactor with the ATF system, and 3) integration of a purification process step using magnetic sulfated cellulose particles (MSCP). For both viruses, conventional batch cultivation (no addition/medium exchange after infection) was compared with processes applying fed-batch, periodic medium exchange and the combination of both during virus propagation. Results. Perfusion and semi-perfusion at a feeding rate of 0.05 nL/cell×d was suitable to propagate AGE1.CR.pIX cells above 60×106 cells/mL with neither limitation nor overload of nutrients. For infections in 50 mL, the application of a combined strategy comprising an initial fed-batch phase followed by a periodic virus harvest phase resulted in the highest product yield with a more than 10-fold increase, compared to the conventional batch processes at 4 to 8×106 cell/mL [2]. Additionally, a 3-fold increase in both cell-specific yield (virus/cell) and volumetric productivity (virus/L×d) could be obtained. Although product harvesting was suboptimal when up-scaling to a 1 L bioreactor with ATF-system, comparable increases in virus yields and productivity with respect to the conventional batch process were observed. In all cases, cell-specific yields and volumetric productivities reached their peak values at the peak virus concentrations, indicating that the process should be stopped at that time point. Eventually, selection of the optimal pore size of the membrane of the ATF-system allowed semi-continuous harvesting of the produced viruses and its purification with MSCPs with a recovery of about 50%. Conclusion. Compared to conventional batch processes, the developed HCD process offers significantly higher productivities including the option to integrate a purification step in a semi-continuous mode. Overall, the results show that there is a great potential for semi-continuous HCD processes for the production of viral vaccines in larger scales, which could intensify the discussion towards the establishment of true continuous production process

A Regenerative Endodontic Approach in Mature Ferret Teeth Using Rodent Preameloblast-conditioned Medium

Background: This study evaluated the effectiveness of a regenerative endodontic approach to regenerate the pulp tissue in mature teeth of ferret. The presence of odontoblast-like cells in the newly-formed tissue of teeth treated with or without preameloblast-conditioned medium was evaluated based on morphological criteria. Materials and methods: Twenty-four canines from six ferrets were treated. The pulp was removed, and the apical foramen was enlarged. After inducing the formation of a blood clot, a collagen sponge with or without preameloblast-conditioned medium was placed underneath the cementoenamel junction. The samples were analyzed at the eighth week of follow-up. Results: Vascularized connective tissue was observed in 50% of teeth, without differences between groups. The tissue occupied the apical third of the root canals. Odontoblast-like cells were not observed in any group. Conclusion: Revitalization of mature teeth is possible, at least in the apical third of the root canal. Further experimental research is needed to produce more reliable outcomes

Establishment of Fruit Bat Cells (Rousettus aegyptiacus) as a Model System for the Investigation of Filoviral Infection

Marburg virus and several species of Ebola virus are endemic in central Africa and cause sporadic outbreaks in this region with mortality rates of up to 90%. So far, there is no vaccination or therapy available to protect people at risk in these regions. Recently, different fruit bats have been identified as potential reservoirs. One of them is Rousettus aegyptiacus. It seems that within huge bat populations only relatively small numbers are positive for filovirus-specific antibodies or filoviral RNA, a phenomenon that is currently not understood. As a first step towards understanding the biology of filoviruses in bats, we sought to establish a model system to investigate filovirus replication in cells derived from their natural reservoir. Here, we provide the first insights into this topic by monitoring filovirus infection of a Rousettus aegyptiacus derived cell line, R06E. We were able to show that filoviruses propagate well in R06E cells, which can, therefore, be used to investigate replication and transcription of filovirus RNA and to very efficiently perform rescue of recombinant Marburg virus using reverse genetics. These results emphasize the suitability of the newly established bat cell line for filovirus research