Classical swine fever (CSF), caused by the RNA-containing Pestivirus C, is one of the most dangerous viral diseases of the Suidae family and is accompanied by hemorrhagic syndrome [1][2]. Classical swine fever virus (CSFV) primarily targets endothelial cells and macrophages. Concurrently, it triggers T-cell apoptosis, leading to significant immunosuppression. In most cases, the infection follows an acute course, culminating in one of three outcomes: fatality, recovery with the development of virus-neutralizing antibodies, or progression to a chronic infection [3][4]. In countries where CSF is endemic, effective live attenuated vaccines are commonly used. However, the ability of vaccine strains to replicate within the host complicates differentiating vaccinated animals from infected ones (the DIVA strategy). This challenge underlies the restrictive vaccination policies adopted in non-endemic regions. Thus, within the Russian Federation, there is a clear need to develop effective and safe marker vaccines. Such vaccines are essential for controlling potential CSF outbreaks and are a prerequisite for eventually achieving CSF-free zone status, officially recognized by the World Organisation for Animal Health [5][6].

In the context of developing experimental subunit vaccines, various expression systems have been successfully employed for the biosynthesis of the major glycoprotein E2. We previously generated a prokaryotic equivalent of glycoprotein E2 [7]. Its synthesis predominantly resulted in inclusion body formation, necessitating extensive chromatographic purification and refolding steps. Consequently, the yield of purified, bioactive product was relatively low. As an alternative to Escherichia coli expression, gene expression in mammalian cells can be considered. Its primary advantages are post-translational modifications closely resembling the native ones, along with recombinant protein solubility and folding compatible with high-level overexpression [8]. To facilitate future scale-up, mammalian cell expression must be cost-effective and high yielding. This necessitated development of an optimized genetic construct, selection of a suitable production cell line as a potential producer of the recombinant vaccine antigen, and adaptation of established transfection protocols to meet specific requirements of this bioprocess.

The objective of this study was to optimize transient transfection conditions in mammalian production cell lines for CSFV E2 antigen expression. The following tasks were completed to achieve this objective:

a) assess suitability of CHO-K1, PK-15, and BHK-21/13 cell lines for transient expression of a gene encoding a fragment of glycoprotein E2;

b) compare efficiency of widely available non-viral DNA delivery methods (calcium-phosphate and cationic transfection);

c) validate developed methods for monitoring expression efficiency: immunofluorescence assay (IFA) on cell culture, quantitative assessment of messenger RNA (mRNA), and antigen-capture enzyme-linked immunosorbent assay (ELISA).

Generation of recombinant DNA. A nucleotide sequence encoding a truncated E2 protein Shimen strain of CSFV (GenBank ID AF092448.2) was cloned into pVAX1 vector (Evrogen, Russia) using BamHI and EcoRI restriction sites. Sanger sequencing confirmed the identity of the generated construct. The recombinant plasmid was transformed into E. coli strain DH5α (Novagen, Germany), amplified, and isolated using a Midiprep system (Evrogen, Russia). Plasmid concentration was measured using a Nano-500 spectrophotometer (Allsheng, China). Sample purity was assessed by the absorbance ratio at 260 and 280 nm; a ratio between 1.8 and 2.0 indicates an absence of polypeptide or free-nucleotide contamination.

Cell culture. Monolayers of Chinese hamster ovary (CHO-K1, ATCC® CCL-61), Syrian hamster kidney (BHK-21/13, ATCC® CCL-10), and porcine kidney (PK-15, ATCC® CCL-33) cells were cultivated in 75 mL flasks and 100 mm diameter plates (Biologix Group Ltd., China). The growth medium consisted of DMEM-F12 (PanEco company, Russia) supplemented with 10% fetal bovine serum (HyClone, Australia), 100 IU/mL penicillin and streptomycin (PanEco company, Russia), and 20 mM L-glutamine (Sigma-Aldrich, USA). Cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2, starting at an initial concentration of 10 × 106 cells/mL. Routine contamination screening was performed. No contamination by bacteria, mycoplasma, or bovine viral diarrhea virus was detected.

Transfection process. The conditions for calcium-phosphate (CaP) and cationic (PEI) transfection of the production cell lines are detailed in the Table.

Cell viability was assessed 24 hours post-transfection in 96-well culture plates (Corning Incorporated, USA) using a colorimetric methyl thiazole tetrazolium (MTT) assay. To each well containing 100 µL of fresh culture medium, 10 µL of MTT reagent (Wuhan Servicebio Technology Co., Ltd., China) were added. The plates were then incubated for 3.5 hours under standard conditions. The formazan crystals that formed were dissolved by adding an equal volume of dimethyl sulfoxide. The absorbance in each well was then measured at a wavelength of 570 nm. The percentage of viable cells was calculated from the ratio of wells containing transfected cells to the wells with the intact cells.

Assessment of messenger RNA expression levels. The transcriptional activity of the E2 gene fragment was assessed by quantitatively determining its product – specific mRNAs. Nucleic acids were extracted from transfected cells using the RIBO-sorb reagent kit (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia). The isolated samples were treated with DNase (Syntol, Russia) at a ratio of 1 unit per 10 ng of DNA and incubated for 1 hour at 37 °C, followed by enzyme inactivation at 80 °C for 5 minutes. For amplification, the following reaction mixture was prepared (per one 20 µL sample): 4 µL of prepared PCR 5× qPCRmix-HS SYBR premix, 5 pM each of forward (5’-CGTCAACCAAT GAGATAGGGCTGT-3’) and reverse (5’-GCACAGCCCGAATC CGAAGT-3’) primers, 100 units of MMLV reverse transcriptase (Evrogen, Russia), 15 ng of RNA template, and ddH2O to a final volume of 20 µL. A ten-fold serial dilution of the recombinant plasmid pVAX1-trE2 was used to generate a calibration curve. Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) was performed using a C1000 thermocycler with a CFX96 optical module (Bio-Rad Laboratories, Inc., USA) according to the following program: 1 – reverse transcription at 37 °C for 5 minutes; 2 – DNA denaturation at 95 °C for 5 minutes; 3 – 35 cycles of: 94 °C for 30 seconds, 56 °C for 30 seconds, 72 °C for 25 seconds; 4 – final elongation at 72 °C for 10 minutes. Melting curve analysis of the amplicons was performed using the CFX Manager software (Bio-Rad Laboratories, Inc., USA). The number of mRNA copies in each sample was normalized and is presented per 1 µg of the total RNA.

Immunofluorescence assay on cell culture. Transfected cells were stained with an FITC-conjugated polyclonal swine antiserum to CSFV (Federal Center for Toxicological, Radiation and Biological Safety) at a working dilution of 1:500, as previously described by the authors [9].

Isolation of the target protein. 48–72 hours after transfection, cells were mechanically removed from the surface of the culture vials using scrapers. Cells from the suspension were pelleted by centrifugation at 3,000 g for 15 minutes. The pellet was resuspended in 1 mL of chilled RIPA buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1 mM ethylenediaminetetraacetic acid; 1% Triton X-100; 0.1% sodium deoxycholate; 0.1% sodium dodecyl sulfate; 0.1 mM phenylmethylsulfonyl fluoride) and incubated on ice for 30 minutes. Subsequently, the cells were pelleted again by centrifugation. Protein presence in the supernatant was confirmed by analytical electrophoresis on a 15% polyacrylamide gel under denaturing conditions. Reactivity with specific antisera was assessed in ELISA using a previously developed test system designed to monitor the specificity of the recombinant CSFV antigen [10].

Statistical analysis was performed using GraphPad Prism 10.4 software (GraphPad Software, USA).

Bioinformatic analysis identified a 188-amino acid fragment (residues 690–878) characterized by the highest density of CSFV-specific B-cell epitopes. According to pBLAST analysis results, the sequence identity of this fragment among representatives of CSFV genotypes 1 and 2 ranges from 91.19 to 98.74%. Furthermore, it exhibits no significant homology with the corresponding region of the bovine viral diarrhea virus proteome, which is a critical factor in selecting a diagnostic antigen sequence. The final amino acid sequence, flanked by a hexahistidine tag, was reverse-translated. The synthesized nucleotide sequence, containing a Kozak consensus sequence upstream of the start codon, was then cloned into the target pVAX1 vector. The construct scheme is given in Figure 1.

Cell viability was assessed 24 hours after transfection in the CHO-K1, PK-15, and BHK-21/13 cell lines (Fig. 2).

The chart shows that the percentage of viable cells following both transfection methods, under the specified incubation periods with the transfection agents, was at least 87%. Furthermore, cell viability exceeded 93.6% when using cationic transfection with incubation period of 6–12 hours, as well as with CaP transfection when the primary incubation period was limited to no more than 6 hours. Lower viability rates (87.2–92.8%) were recorded for CaP transfection when the primary incubation was extended up to 12 hours. This is likely attributable to the cytotoxic effect exerted by calcium precipitates during extended incubation periods.

The next phase of the study involved a comparative assessment of various methods for monitoring transfection efficiency, specifically direct IFA on transfected cells, quantitative mRNA analysis in real-time RT-PCR, and antigen-capture ELISA.

According to IFA performed on cell cultures, transfection efficiency at 48 hours post-transfection varied from 60 to 90%. The highest efficiency was recorded in monolayers of the CHO-K1 line: 87% for CaP transfection and 90% for PEI transfection. A slightly lower proportion of fluorescent cells was observed for the PK-15 line: 82% for CaP and 85% for PEI transfection. The lowest efficiency was observed for the BHK-21/13 line: 80 and 65% for CaP and PEI transfection, respectively (Fig. 3).

In addition to IFA, which serves as a fundamental method for assessing transfection efficiency, an analysis of mRNA expression was conducted. It is established that measurements based on protein and mRNA levels are complementary and essential for a comprehensive assessment of gene expression. The real-time RT-PCR protocol was initially validated using a ten-fold serial dilution of the control plasmid pVAX1-trE2 (starting concentration 50 ng/µL). It was determined that the linearity of the reaction (R²) was 0.994, the slope (Ct threshold between two DNA dilutions) was –3.748, the theoretical limit of detection (y-intercept) was 29.288, and the reaction efficiency (E) was 94.8%. The results of specific mRNA level quantification, determined by real-time RT-PCR, are given in Figure 4.

It is known that within a cell, the ratio of protein to mRNA is governed by translation and protein degradation processes, which are regulated at the gene-specific level [11]. The overall correlation between mRNA and protein concentrations in multicellular eukaryotes is considered significant but moderate compared to that in bacteria and yeasts: the number of protein molecules per transcript can vary substantially [12][13]. Therefore, a more objective assessment of the expression system’s functionality, particularly in a production setting, is the quantitative protein yield coupled with the determination of its specificity using an accessible serological method (Fig. 5).

According to the calculated data, the rE2 protein, with a predicted molecular mass of 17.3 kDa, is stable and soluble. These parameters did not differ among the protein variants expressed by all three producer lines, which may indicate similar patterns of post-translational modifications. This is supported by the comparable activity of all protein variants in ELISA, with a positiveness degree ranging from 5.1 to 6.2. The highest recombinant protein yield was achieved using the CHO-K1 line (35.6–47.4 mg/L), while the lowest yield was observed with BHK-21/13 (19.3–24.1 mg/L). This finding aligns with the established influence of cell origin on transfection efficiency under comparable conditions [14]. Although cationic transfection demonstrated greater efficiency in terms of transcript levels, the CaP transfection method yielded an average of (8.7 ± 1.4)% more functional product.

Literature describes production of the recombinant glycoprotein E2 of CSFV in mammalian cells. For instance, in the work of R.-H. Hua et al. [15], the generation of a stable BHK-21 cell line using the pCAG vector is reported, achieving a productivity of up to 45 mg/L. The recombinant protein provided 100% protection to pigs upon lethal challenge. Tian H. et al. [16] successfully created a truncated E2 protein containing CSFV-specific domains, which induced seroconversion in rabbits, i.e. in PK-15 cells transfected with the retroviral vector pBABE puro. The use of the CHO cell line also enabled significant yields of recombinant E2: L. Feng et al. [17] generated stable rCHO transgenic cells with a dynamic Txnip promoter, forming the basis for an inducible expression strategy. Thus, mammalian cell expression is an effective tool for the large-scale biosynthesis of complex glycoproteins and is applicable in the development of candidate subunit vaccines [18][19][20].

This study investigated the application of standard transfection methods on several mammalian production cell lines for the generation of the recombinant CSFV E2 antigen, along with methods for monitoring expression efficiency. It was established that the CHO-K1, PK-15, and BHK-21/13 cell lines are successfully transfected under standard conditions with an efficiency of up to 90%. Among these, the CHO-K1 line demonstrated the highest transfection efficiency as well as the highest overall yield of the target protein. Given that all cell lines considered are characterized by high proliferation rates, ease of cultivation (including in suspension), and adaptation to serum-free media, they possess significant potential for generating clonal producer lines tailored to specific bioprocess requirements. It was also established that both transfection methods examined – CaP and PEI – provided sufficiently high transfection efficiency and comparable yields of the target protein. Their advantages include cost-effectiveness and reproducibility.

A comparative assessment of transfection efficiency monitoring methods – IFA, quantitative PCR, and indirect ELISA – demonstrated that their combined application is advisable during the expression system design phase. While conducting IFA with FITC-labeled polyclonal antibodies provides valuable information during the initial stages of protein synthesis, it necessitates continuous screening of cell lines and fetal bovine sera for contamination with bovine viral diarrhea virus, which can lead to false-positive results. Quantitative mRNA detection does not always correlate with target product yield; therefore, the use of this method is justified primarily for an initial assessment of gene transcriptional activity. In a production setting, the most reliable indicator is the total yield of the target protein. This is conveniently monitored using quantitative or semi-quantitative ELISA, particularly when working with stable expression cell lines.

Although this study utilized a nucleotide sequence encoding a truncated antigen intended for diagnostic purposes – specifically, a fragment of the E2 glycoprotein with the highest density of CSFV-specific domains – the described approaches are applicable to the development of a recombinant subunit vaccine against CSFV. This application is viable provided there is a well-designed construct encoding the full-length glycoprotein.

Contribution of the authors: Galeeva A. G. – development of the study design, literature analysis, conducting experiments, preparation of the paper text; Kuznetsova Yu. A. – conducting experiments, data collection; Akhunova A. R. – conducting experiments, preparation of the paper text; Khammadov N. I. – bioinformatics analysis, literature analysis; Khaertynov K. S. – conducting experiments, data interpretation; Mukhammadiev Rin. S. – conducting experiments, data interpretation; Efimova M. A. – administering, study result analysis and generalization.

Вклад авторов: Галеева А. Г. – концепция исследования, работа с литературой, проведение экспериментов, подготовка текста; Кузнецова Ю. А. – проведение экспериментов, сбор материала; Ахунова А. Р. – проведение экспериментов, подготовка текста; Хаммадов Н. И. – биоинформатический анализ, работа с литературой; Хаертынов К. С. – проведение экспериментов, интерпретация данных; Мухаммадиев Рин. С. – проведение экспериментов, интерпретация данных; Ефимова М. А. – администрирование, анализ и обобщение результатов исследования.