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.