Influence of cell type and cell culture media on the propagation of foot-and-mouth disease virus with regard to vaccine quality

Abstract

Background: Suspension culture of BHK cells allows large-scale virus propagation and cost-efficient vaccine production, while the shift to animal-component-free cell culture media without serum is beneficial for the quality and downstream processing of the product. Foot-and-mouth disease virus is still endemic in many parts of the world and high-quality vaccines are essential for the eradication of this highly contagious and economically devastating disease.

Methods: Changes to the viral genome sequence during passaging in an adherent and a suspension cell culture system were compared and the impact of amino acid substitutions on receptor tropism, antigenicity and particle stability was examined. Virus production in suspension cells in animal-component-free media and in serum-containing media as well as in adherent cells in serum-containing media was compared. Infection kinetics were determined and the yield of intact viral particles was estimated in all systems using sucrose density gradient centrifugation.

Results: Capsid protein sequence alterations were serotype-specific, but varied between cell lines. But The A₂₄-2P virus variant had expanded its receptor tropism, but virus neutralization tests found no changes in the antigenic profile in comparison to the original viruses. There were no differences in viral titer between a suspension and an adherent cell culture system, independent of the type of media used. Also, the usage of a serum-free suspension culture system promoted viral growth and allowed an earlier harvest. For serotype O isolates, no differences were seen in the yield of 146S particles. Serotype A preparations revealed a decreased yield of 146S particles in suspension cells independent of the culture media.

Conclusion: The selective pressure of the available surface receptors in different cell culture systems may be responsible for alterations in the capsid coding sequence of culture-grown virus. Important vaccine potency characteristics such as viral titer and the neutralization profile were unaffected, but the 146S particle yield differed for one of the tested serotypes.

Keywords: Animal-component-free media; BHK21; Foot-and-mouth disease virus; Serum-free media; Suspension cells.

Fig. 1

3D structure with mutations acquired during adaption of FMDV strains A₂₄ Cruzeiro and O₁ Manisa. Panels (a), (b), and (c) present detailed structural models of the virus capsid pentamers. The X-ray crystal structure of 1FOD served as template for the 3D models. The structure of a single protomer is highlighted in panel (a) (blue: VP1, green: VP2, red: VP3). While O₁ Manisa (panel a) acquired the same three mutations in the VP1 region of the capsid (K210E: yellow dots, E83K: orange dots, K41 N: red dots) independent of the culture system, A₂₄ Cruzeiro developed different mutations in VP1 (blue dots) and VP3 (red dots) in adherent cell culture (panel c) and suspension cell culture (panel b). However, these mutations lie in similar locations on the particle

Fig. 2

Sensitivity of O₁ Manisa and A₂₄ Cruzeiro and their derivatives to acidic pH Equal amounts of virus of the different passages and culture systems were incubated in buffers of different pH for 30 min, then neutralized and titrated. Titers are expressed relative to the value obtained using PBS at pH 7.5. Experiments were performed three times independently. Significance code: “***” p ≤ 0.001

Fig. 3

Virus infection kinetics on monolayer and suspension BHK cells. Virus infection kinetics were performed using the virus variants A₂₄-2P to infect the suspension BHK-2P cell line, maintained in BHK200 (red line) or GMEM + 5% FCS (blue line) and A₂₄–179 to infect the monolayer BHK179 cell line (green line) at an MOI = 0.1 (panel a). The virus variant O₁-2P was used to infect the suspension BHK-2P cell line, maintained in BHK200 (red line), and the virus variant O₁–179 was used to infect the monolayer BHK179 cell line (green line) under the same conditions (panel b) as described for serotype A. All preparations were sampled for the first time after 4 h and every 2 h thereafter. CPE and cell viability are given in percent (%). Titers are shown in log₁₀ TCID₅₀ relative to time 0

Fig. 4

Sucrose gradient profiles of passaged A₂₄ Cruzeiro and O₁ Manisa. Identical numbers of BHK179 and BHK-2P cells, maintained in GMEM + 5% FCS or ACFM were infected with an MOI = 0.1 and virus was harvested after 20 hpi. The harvested virus was concentrated by ultracentrifugation and sedimented through a 15–45% sucrose density gradient. The peaks corresponding to 146S (fraction 9) and empty 75S particles (fraction 13) are indicated. FMDV protein content is shown as absorbance at 492 nm obtained with antibodies specific for O₁ (panel a, − 179: solid, grey line, -2P: dotted, black line) and A₂₄ (panel c, − 179: solid, grey line, -2P: dotted, black line for ACFM; dotted, grey line for serum-containing GMEM) in a standard FMDV antigen ELISA. RNA content corresponds to absorbance at 260 nm as measured with a spectrophotometer (panel b: O₁ isolates, panel d: A₂₄isolates, − 179: solid, grey line, -2P: dotted, black line for ACFM; dotted, grey line for serum-containing GMEM). For both assays, absorbances were normalized by subtracting the absorbance reading of fraction 2