Introduction

Recombinant vectored vaccines produced in cell culture are receiving increased attention in the fight against infectious diseases. More and more vaccines are available that are based on this technology and research efforts to develop new vaccines or to improve current manufacturing processes have intensified over the last years (Ura et al., 2020). One such system is based on the recombinant vesicular stomatitis virus (rVSV). In addition to its use as a vaccine vector, VSV has been used extensively in many areas of research, for example as an oncolytic virus or as a gene delivery tool (Lichty et al., 2004)(Munis et al., 2020).
VSV is a replication-competent virus with a single-stranded, negative-sense RNA genome. The native glycoprotein, VSV-G, is responsible for viral entry into the cell. When genetically engineered to express the glycoprotein of another virus, rVSV can be used as a vaccine vector by delivering foreign antigens (Munis et al., 2020). The advantage of such a vectored vaccine is the increased safety during manufacturing, since the production of live-attenuated or inactivated vaccines of highly pathogenic viruses (e.g. HIV, Ebola) would require stringent biosafety standards. The recent success story of the EMA and FDA-approved Ebola vaccine rVSV-ZEBOV showcases the potential of the rVSV platform (Henao-Restrepo et al., 2017). rVSV-ZEBOV is a replication competent virus in which VSV-G was replaced by a Zaire Ebolavirus glycoprotein (ZEBOV), which is the main antigen of the Ebolavirus. Several rVSV-based vaccines are in development, for example against measles, Lassa fever and Middle East respiratory syndrome (MERS) (Henao-Restrepo et al., 2017; Kiesslich and Kamen, 2020; Munis et al., 2020).
In the light of the progress achieved with rVSV-ZEBOV, three novel rVSV constructs have been described recently, which carry different glycoproteins of the Human Immunodeficiency Virus (HIV) (Mangion et al., 2020). These HIV-vaccine candidates were produced in adherent Vero cells in tissue culture plates and it was demonstrated that they induced an HIV gp140-specific antibody response when administered to mice. The rVSV-B6-A74Env(PN6)/SIVtm construct was selected for further studies in non-human primates.
In the current race for a COVID-19 vaccine, recombinant vectored vaccines produced in cell culture are amongst the most promising (Ura et al., 2020). For example, ChAdOx1 nCoV-19, developed by the University of Oxford, is based on a chimpanzee adenovirus-vectored vaccine expressing the SARS-CoV-2 spike protein (Folegatti et al.,) and its safety, efficacy, and immunogenicity is being assessed in a phase III clinical trial (NCT04516746). Besides, several rVSV-based COVID-19 vaccine candidates expressing the SARS-CoV-2 spike protein are being evaluated in preclinical trials (University of Manitoba, Canada; University of Western Ontario, Canada; Aurobindo Pharma, India; Israel Institute for Biological Research/Weizmann Institute of Science, Israel; FBRI SRC VB VECTOR, Russia) and a phase I clinical trial (Merck Sharp & Dohme/IAVI; NCT04569786) (World Health Organization, 2020). The COVID-19 vaccine candidate rVSVInd-msp -SF-Gtc , is a temperature-sensitive construct. It is based on a recombinant VSVInd(GML) mutant, and shows avirulent in vivoreduced cytopathic effect in vitro at 37 °C, but replicates well at 31 °C (Kim et al., 2015). This attenuation was used as a strategy to further increase the safety of rVSV for its use as a human vaccine. rVSVInd-msp -SF-Gtc is expressing the SARS-CoV-2 spike protein gene, the honeybee melittin signal peptide gene and the VSV-G protein transmembrane domain gene.
Currently, the rVSV-ZEBOV vaccine is manufactured under serum-free conditions in adherent Vero cells using the roller bottle technology (Monath et al., 2019). To reduce manufacturing costs, more scalable bioprocesses involving microcarrier bioreactors and fixed-bed bioreactors have been studied recently (Kiesslich et al., 2020). However, these adherent cell processes still have scale-up limitations, for example the cell expansion steps during the seed train operation require cell detachment from and re-attachment to surfaces, usually involving enzymatic solutions such as trypsin. Suspension cell systems are considered superior with regards to process scale-up, since the transfer of cells to successively larger bioreactor vessels is straightforward.
Adherently growing Vero cells are the most used continuous cell line in viral vaccine manufacturing. For example, vaccines against Ebola, influenza, Japanese encephalitis, polio, rabies, rotavirus and smallpox are available on the market and vaccines against other infectious diseases are under development, using this cell line. The many advantages of this cell line are its broad susceptibility to many viruses, the long-term experience in cell culture and the regulatory portfolio associated with vaccine manufacturing organization and health authorities worldwide (Kiesslich and Kamen, 2020).
Adaptation of the Vero cell line to grow in suspension culture to significantly improve this cell culture manufacturing platform has been of interest for many years (Litwin, 1992; Paillet et al., 2009). Lately, studies have reported the successful adaptation using proprietary media (Rourou et al., 2019; Shen et al., 2019). Shen et al. showed that Vero cells can grow in suspension culture in serum-free batch and perfusion bioreactors, and successfully applied their system to the production of rVSV-GFP, which uses the native glycoprotein VSV-G for viral entry into the cell.
In this work, we further explore the Vero suspension system described previously (Shen et al., 2019), and demonstrate its applicability to relevant rVSV-based vaccine candidates. Using rVSV-ZEBOV as a model for rVSV, we focus on small scale experiments to optimize the multiplicity of infection (MOI) and investigate effects of different cell densities. Next, we compare the production of rVSV-ZEBOV in this system to the production in Vero cells that were adapted to grow in suspension culture in a commercially available medium. In addition, we show the production of newly developed candidate vaccines against HIV (rVSV-HIV) and COVID-19 (rVSVInd-msp -SF-Gtc ). Based on these results, we demonstrate production in batch bioreactor for all three rVSV variants.