Background
Foot-and-mouth disease virus (FMDV) is a highly transmissible and extremely contagious RNA virus, infecting domestic as well as wild cloven-hooved animals [
1]. Vaccination campaigns are the way of choice to eradicate FMDV in endemic countries and in case of an outbreak in an FMDV-free country, vaccination is a useful strategy to limit spread [
2].
Vaccines produced to combat FMDV have a long history, going back to first attempts in the early 1900s and Waldmann’s first inactivated vaccine, developed in 1937 [
3]. The most common production cell line is the mammalian baby hamster kidney cell (BHK21, clone 13), adapted to grow in suspension by Capstick et al. [
4], and processed for large-scale fermenters by Telling and Elsworth [
5]. To achieve adaption of the virus to the production cell line, the virus is first passaged in BHK21 adherent cells until a rapid cytopathogenic effect develops, and then further propagated and expanded in stationary or roller systems [
6]. In industry practice, viruses are passaged in suspension cell culture to expand the virus to large scale. Preparation of master and working seed stocks for the vaccine production process are the last steps of a successful virus adaption in both cell culture systems [
7]. In the vaccine production process, the raw materials, including serum-containing cell culture media, need special attention. Serum as well as other components such as animal tissue hydrolysates are poorly defined, resulting in significant lot-to-lot variation of the product [
8,
9]. On top of their substantial costs, animal-derived products can contain viruses, mycoplasmal bacteria or prions, and therefore require special risk assessments by the supplier and the user [
9,
10]. Because of this, attempts to find alternatives to serum in vaccine production have been of major importance for many years [
11].
Today, cell culture media can be divided in different types based on their content of animal-derived products. Serum-free media (SFM) do not require the addition of serum for optimal cell growth but may contain other additives derived from animals such as lactalbumin, casein, insulin, lipids or sterols [
12]. Animal-component-free media (ACFM) are media in which none of the components are animal-derived [
11]. Protein-free media (PFM) are free of supplemental polypeptide factors but may contain hydrolyzed peptide fragments from animal or plant sources. Finally, chemically defined media (CDM) comprise well-characterized constituents of low molecular weight and are, in most cases, free of proteins [
11,
12]. BHK21 cells have already been adapted to grow in serum-free or animal-component-free cell media for rabies vaccine production [
13,
14]. With adaption to serum-free conditions, BHK21 cells switch from anchorage-dependent to suspension growth [
13,
14] and fundamental changes in cell structure take place [
15,
16]. On the other hand, selective pressures during the adaption of viral strains to BHK21 cells, whether as adherent or as suspension cells, can lead to capsid alterations that influence the antigenicity and stability of the virus particle.
The first part of the study examines the adaption of the virus to an adherent and a suspension cell culture system, the viral sequence changes that take place during subsequent passaging as and their possible impact on receptor tropism, particle stability and antigenicity. The second part of the study compares virus production in an animal-component-free medium and virus production in serum-supplemented growth medium and the possible differences in quality and quantity of the viral harvest.
Methods
Cells
The adherent BHK21C13 cell line (CCLV-RIE 179 in the Collection of Cell Lines in Veterinary Medicine, Friedrich-Loeffler-Institut [FLI], Greifswald, Germany; originally derived from the American Type Culture Collection (ATCC) specimen CCL-10™; short: BHK179) and the adherent BHK21 “clone Tübingen” cell line (CCLV-RIE 164, short: BHK164) were cultured in Minimum Essential Medium Eagle (MEM), supplemented with Hanks’ and Earle’s salts (Sigma, St. Louis, USA) with 10% fetal bovine serum (FBS) during maintenance and passaging, and with 5% FBS during infection experiments. Cells were incubated in flasks with sealed caps at 37 °C.
The suspension cell line BHK21C13-2P (originally derived from the European Collection of Authenticated Cell Cultures specimen 84,111,301; short: BHK-2P) was either maintained in Glasgow MEM (Thermo Fisher Scientific), supplemented with tryptose phosphate (Sigma-Aldrich) and sodium hydrogen carbonate (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) with 5% FBS or was adapted to grow in the animal-component-free medium Cellvento™ BHK-200 (Merck KGaA, Darmstadt, Germany) in TubeSpin® bioreactors (TPP Techno Plastic Products AG, Trasadingen, Switzerland). The cells were maintained in a shaker incubator with 320 rpm (rpm) at 37 °C, 5% CO2 and 80% relative humidity.
The Chinese hamster ovary (CHO) cell lines CHO-K1 (ATCC CCL-61, held as CCLV-RIE 134), lacking the known FMDV integrin receptors [
17], and the heparan sulfate (HS)-deficient CHO677 [
18] (CRL 2244, held as CCLV-RIE 1524) were maintained in Ham’s MEM mixed 1:2 with Iscove’s Modified Dulbecco’s Medium (Thermo Fisher Scientific) and with 10% FBS at 37 °C in sealed flasks.
Viruses and virus titrations
The FMDV isolates A
24 Cruzeiro and O
1 Manisa were selected from archival stocks at the FLI. Their passage history and origin can be found in Additional file
1: Table S1.
Viral titers were estimated by endpoint titration with the Spearman-Kärber method [
19,
20] and expressed as 50% tissue culture infectious dose (TCID
50) per milliliter. Titrations for virus grown on all cell lines were performed on the adherent BHK164 to avoid biasing the results by titrating BHK179-passaged virus on BHK179 cells.
Virus adaption and passaging
Both virus strains were serially passaged on BHK179 monolayers for 20 passages. In suspension cells, the viruses were passaged until stable adaption to the suspension cell line was achieved. Adaption was defined as a decrease in cell viability to values under 10% within less than 24 h post infection (hpi). Adaption of the virus to growth in BHK-2P as well as passaging the virus on BHK179 was done two times independently. FMDV strain A24 Cruzeiro was fully adapted to BHK-2P after 19 passages (16 in the second experiment) and O1 Manisa after 22 (19) passages. The adapted viruses will be referred to as A24–179, A24-2P, O1–179 and O1-2P, respectively.
RNA extraction, RT-PCR and sequencing
FMDV RNA of the original stocks of A
24 Cruzeiro and O
1 Manisa, of the virus passage 20 in BHK179 and of the final passages in BHK-2P of both adaption experiments was extracted using TRIzol® LS Reagent (Invitrogen, Carlsbad, CA, USA) and the RNeasy® Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturers’ instructions. A previously described method was used for RT-PCR and sequencing of the nearly complete open reading frame [
21].
The nucleotide sequences were assembled and mapped with Geneious (Biomatters Limited) against the complete published sequence for A24 Cruzeiro (GenBank accession no. AY593768) and O1 Manisa (AY593823) followed by an alignment of original and passaged virus sequences.
To find the passage in which each mutation was fixed in the suspension system, the passages in which a rapid drop in cell viability was observed for the first time were chosen for additional sequencing. For the adherent cell system no such indicator existed and therefore the passages were sequenced in arbitrary intervals.
Structure analysis
Amino acid sequences of the original virus and the final passages in BHK179 and BHK-2P were used to model virus capsid protomers using the Geno3D algorithm [
22]. The X-ray crystal structures of A
24 Cruzeiro [
23] (Protein Data Bank accession 1ZBE) and O
1 Manisa [
24] (1FOD) served as templates. In total, ten possible structures were generated and the best-fitting model was further analyzed with the UCSF Chimera package [
25]. Chimera was developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311). VIPERdb [
26] was used to extract contact information for specific residues.
In-silico analysis
The complete genomes of FMDV strains representing possible vaccine strains [
27] as well as representative strains for different topotypes within the seven serotypes were downloaded from GenBank. Multiple sequence alignments for all serotypes were performed using the MUSCLE algorithm as implemented in Geneious and the amino acids at the positions of interest were tabulated.
Acid sensitivity
The protocol of Martín-Acebes et al. [
28] was used with modifications. Equal amounts of virus (A
24 Cruzeiro and O
1 Manisa, original isolates as well as adapted to BHK179 and BHK-2P) were mixed at a final dilution of 1:100 with phosphate-buffered saline (PBS) solutions of different pH within the range of pH values commonly seen in the suspension cell system (7.5, 7.0, 6.8, 6.5). An additional solution with a pH of 5.5 was used as a positive control for FMDV inactivation. The mixtures were incubated for 30 min at room temperature and then neutralized with 1 M Tris-HCl (pH 8.0). The remaining infectivity in each sample was determined by titration on BHK164 cells as described above. Experiments were performed three times independently.
Infectivity testing on CHO cells
A procedure described by Jackson et al. [
29] was used to quantify the capacity of the virus strains to infect the FMDV receptor-deficient cell lines CHO-K1 and CHO677. As a modification of the original protocol, the CHO cell preparations were titrated on BHK164. The test was conducted in duplicates and performed three times independently.
Virus neutralization test (VNT)
The VNT was performed on BHK164 cells with A
24 Cruzeiro, A
24–179, A
24-2P, O
1 Manisa, O
1–179, and O
1-2P as described by the World Organization for Animal Health (OIE) [
30]. A bovine serum, collected 21 days after infection with an earlier passage of the A
24Cruzeiro stock virus, was used to neutralize the serotype A virus isolates. Another bovine serum, from an animal infected with an earlier passage of the O
1 Manisa stock virus, also collected at 21 dpi, was used to examine the serotype O virus isolates. The highest dilution in which 50% of the wells did not show any CPE defined the neutralization titer. Titers are expressed as the log
10 of the reciprocal of that dilution. To determine the relationship between the original and adapted virus isolates, the r
1 value was calculated by dividing the neutralization titer against the adapted isolate by the neutralization titer against the original virus isolate [
30]. All experiments were performed independently in duplicates for a total of three times.
Virus infection kinetics
BHK-2P cells were seeded at a density of 1 × 106 cells/mL and infected with the adapted A24-2P or O1-2P at an MOI of 0.1. BHK179 cells were cultured in T25 culture flasks until confluency and infected with A24–179 or O1–179 under the same conditions as the BHK-2P cells. Samples to determine the viral titer were taken after 0 and 4 h and then every 2 h until a total incubation time of 24 h.
Because the determination of cell death and viability is different between adherent and suspension cells, cytopathic effect (CPE, in %) was documented for BHK179 cells, while cell number and cell viability were assessed for BHK-2P cells. Cell death in suspension cell culture cannot be visually evaluated under a microscope and therefore determination of cell viability is necessary. Additionally, the cell density of an infected culture is compared to an equally seeded negative culture to account for the rapid growth of a healthy suspension culture. Cell numbers and cell viability have been determined by trypan blue staining with an automated cell counter (TC20™, Bio-Rad).
Determination of viral yield
Adherent and suspension cells (cell count 3.7 × 10
7) were infected at a multiplicity of infection (MOI) of 0.1 and incubated for 20 h. The supernatant was clarified of cell debris by centrifugation for 10 min at 3200×g at 4 °C, followed by purification through a 30% (wt/vol) sucrose cushion in 40 mM sodium phosphate buffer (pH 7.6) with 100 mM NaCl (buffer P as in [
31]), centrifuged at 125,755×g in a SW32Ti rotor (Beckman Coulter, Optima LE-70) for 2 h 50 min at 10 °C. Pellets were resuspended in 400 μL buffer P and loaded onto 15% to 45% (wt/vol) sucrose gradients in buffer P. Ultracentrifugation was performed in a SW32Ti rotor at 96,281×g for 3 h at 10 °C. Gradients were fractionated from the bottom of the gradient into one milliliter fractions. All fractions were heated to 70 °C for 30 min before analysis. Absorption at 260 nm was measured twice with a spectrophotometer (NanoDrop™ 2000, Thermo Fisher Scientific). FMDV protein was detected in duplicate by a standard serotype-specific double-antibody sandwich ELISA [
30]. The experiment was performed three times.
Statistical analysis and data presentation
Linear mixed-effects models using R (
http://www.r-project.org) and lme4 [
32] were used to evaluate the differences between treatment groups, with replicates as random effects. The packages car and phia were applied to calculate Wald chi-square tests for fixed effects and their interactions.
P-values < 0.001 were taken as significant.
Discussion
BHK21 cells have been shown to change during passaging and even lose their susceptibility for FMDV [
33]. Additionally, the change from adherent to suspension cell culture comes along with profound changes to the cells that the virus needs to adapt to [
34]. The first part of this study focused on the sequence changes in the viral genome that take place when adapting to either an adherent or a suspension BHK cell culture system and their influence on virus receptor tropism, antigenicity and particle stability.
The passaging of FMDV O
1 Manisa in either cell culture system resulted in three substitutions in the VP1 capsid protein (K41 N, E83K, K210E). Earlier studies by Gullberg and colleagues found that the switch from K to E at position 210 at the VP1/2A junction in a serotype O FMDV results in the formation of virus particles containing the uncleaved VP1-2A product [
31] and is also linked to the E83K substitution within VP1 [
35].This substitution is responsible for the inhibition of the cleavage of the VP1/2A junction [
35] and provides a selective advantage in the BHK cell culture system, but did not enable the virus to successfully infect CHO cells [
36,
37].
The third substitution K41 N is located close to the fivefold symmetry axis of the virus particle at the interface between two VP1 molecules and results in a reduction of positive charge at the interface similar to K210E. Mutations in this particular region have been implicated in the ability to infect cells independently of receptors such as integrin, HS, chondroitin sulfate or sialic acid [
38‐
40]. While it is not clear what selective advantage the inhibition of the VP1/2A junction might have, in sum these mutations seem to allow the use of a receptor on BHK21 cells that is neither integrin nor HS. However, experiments with receptor-deficient CHO cell lines revealed no extended tropism of O
1-2P and O
1–179 in comparison to the original O
1 Manisa isolate.
For A
24 Cruzeiro, the acquired substitutions E194K in VP1 and C56R in VP3 in A
24–179 reflect an adaption for utilization of HS as receptor. According to Fry et al., the HS binding pocket consists of three sites: VP3 residues 55–60 form one of the walls, while residues 84–88 shape the base. The other two walls are composed of residue 133–138 of VP2 and the C-terminus of VP1 (residues 195–197) [
23]. The amino acid change from histidine to arginine at position 56 of VP3 has been described as a characteristic feature for HS attachment of serotype O viruses [
41]. Together with the second substitution (E194K in VP1), the HS-binding pocket of the capsid acquires a clearly more positive charge. While type A FMDV might show lower affinity to HS than type O [
42], the beta-B “knob” regions between residues 55 to 62 of VP3 are structurally very similar between the serotypes [
23]. A previous study examining mutations in serotype A capsid proteins after cell culture adaptation also found a switch of C56 to R in a BHK21 culture system, while the E194K mutation was only fixed in strains passaged on IB-RS-2 cells and in a small minority of BHK21 derived isolates [
40].
The sequence changes in A
24-2P were not as distinct. The switch from a positively charged amino acid to a neutral glutamine (H85Q) at the base of the HS-binding pocket does not support the acquisition of HS as receptor during the course of cell culture adaption. Similar to O
1 Manisa, the A
24-2P isolate obtained a positively charged amino acid (E95K) close to the fivefold symmetry axis in VP1. As already discussed for O
1 Manisa, these substitutions suggest that A
24-2P uses a yet unknown “third” receptor on BHK-2P suspension cells. This assumption is supported through studies using an A
24 Cruzeiro mutant (A-SIR #42) that harbors the same E95K change in VP1 [
17]. Further studies revealed this amino acid change to be responsible for utilizing Jumonji C-domain containing protein 6 to infect cells in an integrin- and HS-independent way [
43]. Indeed, in the present study, A
24-2P was the only mutant capable of infecting CHO677 cells, which do neither have surface integrins nor HS.
It has already been shown that adherent BHK cells offer a limited range of surface molecules such as integrins that can be utilized as receptors by FMDV, and BHK cells in suspension culture often have none at all [
34]. For this reason, there is a selective pressure in favor of alternative entry mechanisms for the virus. The observed differences between the serotypes may indicate that FMDV type A is more malleable or has a higher mutation rate, resulting in a more variable adaption to different BHK cell lines. Conversely, FMDV serotype O may have a preference for certain mutations that result in a more universal outcome of adaption. This hypothesis is supported by a recently published study by Anil and colleagues, which also showed different mutations occurring in an FMDV serotype A strain depending on passaging in suspension or monolayer BHK cells [
44].
Nevertheless, important vaccine quality aspects such as viral antigenicity and particle stability appear to be unaffected by the acquired amino acid substitutions. The r
1-value, which determines the serological relationship between the original virus and the passaged mutant [
30], was higher than 1 for all isolates, indicating that the neutralizing epitopes on the capsid surface are unchanged and immunization with the passaged viruses confers protection like the original isolate does. As for particle stability, one of the main reasons for instability is an increased sensitivity towards low pH. Viral genome release inside the cell is induced through endosomal acidification [
28] and there are known sequence mutations that lead to a more labile or a more stable virus capsid. For the O
1 Manisa viruses in the study, no differences in acid sensitivity were observed. In contrast, the virus variant A
24–179 showed a significantly stronger decrease in viral titer at pH 6.5 than the original virus or A
24-2P. Several studies indicate that virion stability is influenced through amino acid replacements preferentially located at the N terminus of VP1 or the pentameric interface [
27,
45,
46]. However, none of the previously described mutations or amino acid substitutions were detected in any of the virus variants generated during passaging in this study.
The switch from media that contain serum and other animal-derived components to a serum-free or even completely animal-component-free system is a major step forward in the production of vaccines. It can bring many advantages such as lower cost, reduced risk of contamination and a cleaner product recovery [
9,
12]. The second part of the study examined virus production in an animal-component-free medium compared to virus production in serum-supplemented growth medium and their influence on quality and quantity of the viral harvest.
In the production process of an FMDV vaccine, the virus is harvested as soon as the majority of the cells are dead [
8]. Therefore, viral infection kinetics were recorded to find the best time point to harvest the virus. In a monolayer cell culture system, the total cell count is limited due to the available surface area, but growth in a suspension cell system is rapid and unlimited as long as sufficient nutrients and oxygen are available [
47]. The maximum viral titers of the kinetic experiments were similar between both cell culture systems. These results are consistent with other studies comparing roller and suspension systems [
45]. Furthermore, the maximum viral titers were not influenced by the type of cell culture medium. Kinetic experiments even revealed a quicker virus release when using ACFM. This might be because ACFM contain fewer inhibitory ingredients [
46]. On top of reduced biological risks and lower costs through the use of ACFM, the shorter process time also could be an important factors to be taken into account for vaccine producers. The decrease in post-peak titers over time was independent of serum content in the media.
Sucrose density gradient profiles revealed no significant differences in the content of 146S particles between BHK-2P in ACFM and BHK179 in serum-containing media for serotype O preparations. For serotype A preparations, there were no significant differences in contents of 146S particles between the preparation in ACFM and serum-containing growth medium, but the viral yield from the adherent cell culture system exhibited a significantly higher content of 146S particles than the suspension cell preparations. It is also striking that A24-2P yielded 146S and 75S particles in nearly equal amounts, independent of the cell culture media. In addition, all preparations contained high amounts of free RNA. This might lead to the assumption that the packaging of viral RNA into the particle is impaired, which leads to increased free RNA and empty capsid formation. However, not all of the free RNA is of viral origin. The process of purifying the virus from the cell culture supernatant does not completely remove free cellular RNA, which then accumulates in the top fraction of the sucrose gradient.