Characterization of a suspension Vero cell line for viral vaccine production

Abstract

Vero cells, as approved by the World Health Organization, have been the most commonly used continuous cell line for viral vaccine production over the last 25 years, but their adherent phenotype continues to limit productivity. Adapting to a suspension culture would overcome this restriction and reduce production costs. First, a Vero suspension isolate was obtained and metabolically characterized. Second, RNA sequencing analysis was used to identify differentially expressed genes between adherent and suspension cells, which revealed complete downregulation of adhesion and matrix-associated genes. Additionally, signaling pathways involving Wnt and other tyrosine kinase receptors were identified as potential leads for growth optimization. In particular, supplementation with fibroblast growth factor 2 allowed for a 20% increase in cell density. Finally, a comparative viral productivity assay revealed a 30% increase in poliovirus production in suspension Vero cells compared to adherent cells depending on the serotype, as well as a 140% increase in respiratory syncytial virus production and a 150% increase in yellow fever virus production. This work establishes the potential of the suspension Vero cell line as a new cell platform for viral vaccine production.

Fig. 1. Growth kinetics and metabolite concentrations of suspension cells () versus adherent cells () over 9 days.

A Cell concentration, B concentrations of glucose () and lactate measured in the supernatant (), C concentrations of glutamine () and ammonium ions () measured in the supernatant. The error bars represent the standard deviation for N = 3 distinct cultures for each condition.

Fig. 2. A principal component analysis of the top 250 differentially expressed genes.

It was realized with RNA-Seq DRaMA using EdgeR.

Fig. 3. Key upregulated or downregulated genes in suspension Vero cells over 20 passages compared to adherent cells.

Examples of upregulated genes associated with (A) cytokine activity or (B) intrinsic apoptotic signaling pathways in response to endoplasmic reticulum stress, and (C) examples of downregulated genes associated with extracellular matrix or adhesion.

Fig. 4. A global comparison of gene expression in suspension and adherent cells.

A A volcano plot of differentially expressed genes (DEGs) between adherent and suspension cells on Passage 25. The red dots represent significant DEGs (log2 fold change greater than 2). A positive log fold change indicates that the gene is upregulated in suspension cells compared to adherent cells, while a negative log fold change indicates that the gene is downregulated in suspension cells. B Venn diagram depicting the differences in expression between adherent and suspension Vero cell lines on Passage 25. Downregulated genes in adherent versus suspension Vero are indicated.

Fig. 5. Downregulation of metabolic pathways and genes during adaptation to suspension.

A Representation of cell-cell junction organization metabolic pathway (Reactome), key downregulated genes in suspension cells associated with (B) cell-cell junction organization metabolic pathway and gene ontology (GO) bicellular tight junction, C GO transmembrane receptor protein tyrosine kinase activity, D Wnt signaling pathway and/or GO Wnt-protein binding.

Fig. 6. An increase in cell density (%) 3 days after passage, depending on the concentration of basic fibroblast growth factor (bFGF) in the medium.

The error bars represent the standard deviation between different cultures with N = 2 for 1.25, 2.5, and 20 ng/mL, N = 3 for 5 ng/mL; and N = 4 for 10 ng/mL.

Fig. 7. A comparison of viral titers obtained from suspension and adherent cells.

The figure shows Antigen D maximal titers with (A) poliovirus Serotype 1 (PV1) and (B) poliovirus Serotype 3 (PV3), as well as the maximal infectious titer of suspension versus adherent Vero cells infected with (C) yellow fever virus or (D) respiratory syncytial virus (RSV). The error bars represent the standard deviation for N = 2 distinct cultures for each condition of PV1, PV3, and RSV infections and N = 3 distinct cultures for each condition of YFV infection. For YFV, mean titers were compared. The data were checked for normality using the Shapiro–Wilk test. The means were compared using the two-tailed and unpaired t-test (p value = 0.0028, df = 6).