Cell line and cell culture media
The cell line HEK293-E2-CD154 (Suárez et al. 2017): Human kidney embryonic cells (ATCC CRL-1573) adapted to stably transformed suspension growth that produce the E2-CD154 protein in the culture supernatant was used in all experiments. Three times a week, cell passaging was routinely performed in 125 mL plastic erlenmeyer flasks (Corning Inc., USA), seeding 15 mL of culture media with 0.3 × 106 cells/mL. Flasks were shaken at 110 rpm on an orbital shaker (IKA, Germany) in an incubator (ASSAB Box 1715-17225, Sweden) with a temperature set at 37 °C in a humidified atmosphere with 5% of CO2. At each passaging, a sample of culture supernatant was taken to check the E2-CD154 protein expression. Cultures were grown up to 1 × 106 cells/mL and then a new passaging was performed. ProS293CDM (Invitrogen, USA), CDM4HEK293 (HyClone, USA), SFM4HEK293 (HyClone, USA) and CPCHO (CIM, Cuba) culture media were used to growth cells.
Cell growth assessment
HEK293 E2-CD154 cells were seeded in four replicates at 0.3 × 106 cells/mL in a final volume of 30 mL using ProS293CDM, CDM4HEK293, SFM4HEK293 and CPCHO culture media. Cultures were carried out in 125 mL erlenmeyer flasks under the same culture conditions that those in maintenance passagings. Cell number from each culture was determined in duplicate every 24 h using a Neubauer emocytometer and a phase contrast microscope (ZEISS, Germany). Cell viability in the cultures was assessed using the Trypan blue dye exclusion method. Supernatant samples of each culture were taken every 24 h for quantification of E2-CD154 protein. The growth parameters of the cells in each medium were calculated using the following formulas:
$${\text{Cellular}}\;{\text{density:\;D}}\left( {\frac{{{\text{cells}}}}{{{\text{mL}}}}} \right) = \frac{{{\text{number}}\;{\text{of}}\;{\text{cells}}}}{4} \times {\text{dilution}}\;{\text{factor}} \times 10^{4}$$
$${\text{Cellular viability: V }}\left( \% \right) = \frac{\text{number of viable cells }}{{{\text{number of viable cells}} + {\text{number of dead cells}}}} \times 100$$
$${\text{Specific growth rate: }}\mu \left( {{\text{h}}^{ - 1} } \right) = \frac{{logN - logN_{0} }}{0.301} \times \left( {t - t_{0} } \right)$$
where N number of cells at time t, No: number of cells at time to, t: final time, to: initial time, 0.301: log2.
$${\text{Duplication time: td }}\left( {\text{h}} \right) = \frac{1}{\mu }$$
$${\text{Productivity: P }}\left( {{\text{E}}2 - {\text{CD}}154\,{\text{pg}}/{\text{cell}}} \right) = \frac{{{\text{E}}2 - {\text{CD}}154 {\text{pg in the supernatant}}}}{\text{number of viable cells}}.$$
E2-CD154 production process in fermenter
Two fermentation process using the culture medium CDM4HEK293 and SFM4HEK293 (Batch 1 and Batch 2, respectively) were performed. These processes were carried out in a fermenter (BIOSTAT B Plus, Spain) with 8 L of culture effective volume in a 10 L glass reactor and a rotary filter (Sartorius, Germany) operated on a continuous infusion regimen at 37 °C of temperature, stirring at 150 rpm, pO2 = 20%, pH = 7,3 and an inoculum of 0.3 × 106 cells/mL. The culture supernatants obtained in these conditions were concentrated using tangential ultrafiltration technology (100 kDa PESU cassette) until 150 mg of E2-CD154 protein/L. After that, the proteins were dialyzed against three volumes of 50 mmol/L of phosphate buffer and 0.3 mol/L sodium chloride, pH 7.2 ± 0.2. These materials were filtered by 0.2 µm to sterilize them.
SDS-PAGE and Western blotting assays
E2-CD154 protein samples were separated by electrophoresis on SDS-PAGE gels at 10% as previously described (Sambrook et al. 1989), under reducing conditions (5% β-mercaptoethanol, 1% glycerol, 0.4% SDS and 12.5 mM Tris–HCl pH 6.6). In all cases, 10 μL of the sample was applied directly from the culture supernatant or 1 mL of precipitated sample. The specific E2CD154 protein concentration was not taken into account to apply the samples in the SDS-PAGE assays. Samples separated by SDS-PAGE were transferred to a Hybond-C nitrocellulose membrane using a semi-dry Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, USA). Western blot technique was performed using a monoclonal antibody against E2 protein conjugated to horseradish peroxidase diluted 1: 5000 (MAb-CBSSE2.3-HRP, CIGB Sancti Spíritus, Cuba).
E2-CD154 quantification by sandwich ELISA
The E2-CD154 protein expression levels of the HEK293-E2-CD154 cell line were determined by a sandwich ELISA using the specific anti-E2-CSFV monoclonal antibodies 1G6 and CBSSE2.3-HRP (horseradish peroxidase-conjugated) (CIGB Sancti Spíritus, Cuba), as capture and detector antibodies, respectively. An E2-CD154 protein (Suárez et al. 2017) with a purity degree higher than 95% was used as standard.
Molecular weight estimation of the E2-CD154 protein and prediction of its potential N-glycosylation sites
Masslynl and NetNGlyc programs were used to estimate the molecular weight and N-glycosylation sites of the E2-CD154 protein, respectively. The NetNGlyc program was also used to predict which of these potential sites are effectively N-glycosylated.
N-Glycosylation profile from E2-CD154 proteins produced by recombinant HEK293 cells
The E2-CD154 proteins obtained from both fermenter processes were denatured at 70 °C for 10 min in 0.1% SDS, 5% β-mercaptoethanol and after cooled to room temperature. Nonidet P-40 (NP-40) was added to a final concentration of 1% before the PNGase F addition. Digestions were carried out using 5U of PNGase F per µg of glycoprotein at 37 °C during 2 h. The digestion results were visualized in a 10% SDS-PAGE gel under reducing conditions. N-Glycans derived from the PNGase F deglycosylation reactions were derivatized with 2-aminobenzamide (2AB) by a reductive amination reaction. The 2AB derivatives obtained were then analyzed by high-resolution liquid chromatography in normal phase (NP-HPLC) (Montesino et al. 2008).
Oligosaccharides identification using the lectin specific binding
The ‘‘DIG Glycans Differentiation Kit’’ (Roche, Germany) and the Concanavalin A were used to identify specific E2-CD154-attached carbohydrate structures following the manufacturer’s instructions. Lectins specificities included in the assay are the followings: Concanavalin A recognizes terminal glucosamines and mannoses, Galanthusnivalis agglutinin (GNA) recognizes terminal mannoses and α(1-3), α(1-6) or α(1-2) linked to mannose, Sambucusnigra agglutinin (SNA) recognizes sialic acid linked α(2-6) to galactose and Phaseolus vulgaris fitohemaglutinina (PHA) recognizes β(1-4) linked galactoses.
Immunogenicity assay in mice
The immunogenicity induced by the chimeric proteins E2-CD154.1 and E2-CD154.2 (Batch 1 and 2, respectively) was evaluated using Balb/C female mice eight to 10 weeks old and weighing 18–20 g obtained from the Center for the production of Laboratory Animals (CENPALAB, Havana, Cuba). The trial was conducted in the Animal House at CIGB. Mice were maintained in cages (× for each one) under 12:12 h light/dark regimen and fed with a pellet diet (produced by CENPALAB, Havana, Cuba) and water ad libitum. The sampling exercise and all procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals. Mice were randomly assigned to 3 experimental groups with 10 mice each one. Groups 1 and 2 were immunized with 12.5 μg/mL of the E2-CD154.1 and E2-CD154.2 proteins, respectively. Both E2-CD154 proteins were experimentally formulated in Montanide ISA50TM V2 (SEPPIC) using a 60/40 proportion of aqueous/oil phase. “Water in oil” emulsions were obtained using an Ultra-Turrax T25 basic homogenizer (IKA Works Inc.). Group 3 was immunized with PBS1X in the same “Water in oil” emulsion. In all cases, 100 µL of final preparations was administered intraperitoneally in each mouse. Mice were immunized at days 0 and 21 and serum samples were taken on days 0 and 28 to measure the neutralizing antibody responses (NAb).
Neutralizing antibody detection
Serum samples were screened for their capacity to neutralize the cell culture adapted Margarita strain of the CSFV from the National Center for Animal and Plant Health (CENSA, Mayabeque, Cuba) using the Neutralizing Peroxidase Linked Assay, NPLA (Terpstra et al. 1984). The assay was revealed with theanti E2 Mab CBSSE2.3 (CIGB-SS, Cuba) conjugated to horseradish peroxidase followed by DAB substrate. The presence of viral replication was determined by visual inspection at the optical microscope. The last serum dilution without any signal of virus replication was considered as the neutralizing titer.
Statistics
Cell density and viability of cultures are expressed as the average of two independent counts for each sample from at least two biological replicates of each different culture condition. The error bars represent the standard deviations of the biological replicates. All data were compared by Ordinary one-way ANOVA and Tukey’s multiple comparisons Test using the statistical software Graph Pad Prismv.6.0 (GraphPad, USA).