Propagation of Brazilian Zika virus strains in static, microcarrier-based and suspension cultures using BHK and Vero cells
June 12-17, 2016
The spread of Zika virus (ZIKV) in the Americas results in an urgent need for the development of a ZIKV vaccine. Current strategies for ZIKV propagation in animal cells rely mainly on adherent Vero and C6/36 cells. This work focused on understanding ZIKV replication in animal cell culture to develop an inactivated or live-attenuated ZIKV vaccine in microcarrier culture or, preferably, in suspension cells, so that low cell-specific yields can be overcome by the establishment of high-cell density processes.
First, adherent cells (Vero and BHK-21) were infected with different Brazilian ZIKV isolates. Comparing both cell lines, maximum infectious titers and cell-specific yields (1–48 PFU/cell) of respective virus strains were similar, whereas process yields across different strains strongly varied by two log-scales.
Scale-up of Vero cells in bioreactors using 6 g/L Cytodex 1 resulted in maximum cell concentrations of 5.3 × 106 cells/mL. However, low cell-specific yields of 0.0002 PFU/cell indicated poor virus replication. Using suspension-adapted BHK-21 cells grown in a chemically-defined medium, higher virus titers were achieved when infections were initiated at the mid/late exponential growth phase at MOI 0.001. Nevertheless, cell-specific yields did not exceed 0.0002 PFU/cell. Subsequent RT-qPCR data indicated a poor virus release as intracellular viral RNA levels were 20-fold higher than extracellular levels.
At small-scale, centrifugal spinoculation was evaluated to enhance ZIKV infection in suspension BHK-21 cells, with no significant improvements. In a further investigation with these cells in a perfusion bioreactor using an ATF-2 filtration system, a maximum cell concentration of 14 × 106 cells/mL was achieved with a final titer of 4.6 × 106 PFU/mL and an increased cell-specific yield of 0.09 PFU/cell.
Overall, the present results demonstrate that ZIKV propagation in microcarrier- and suspension-based systems is challenging regarding virus yields. Future investigations will focus on improving cell-specific yields by adapting Zika virus isolates to suspension cell lines, and on increasing maximum titers by process intensification.
Alexander Nikolay, Leda Castilho, Amilcar Tanuri, Udo Reichl, and Yvonne Genzel, "Propagation of Brazilian Zika virus strains in static, microcarrier-based and suspension cultures using BHK and Vero cells" in "Vaccine Technology VI", Laura Palomares, UNAM, Mexico Manon Cox, Protein Sciences Corporation, USA Tarit Mukhopadhyay, University College London, UK Nathalie Garçon, BIOASTER Technology Research Institute, FR Eds, ECI Symposium Series, (2016). http://dc.engconfintl.org/vaccine_vi/82