Generation and efficacy evaluation of recombinant classical swine fever virus E2 glycoprotein expressed in stable transgenic mammalian cell line

Abstract

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), which is a highly contagious swine disease that causes significant economic loses to the pig industry worldwide. The envelope E2 glycoprotein of CSFV is the most important viral antigen in inducing protective immune response against CSF. In this study, we generated a mammalian cell clone (BCSFV-E2) that could stably produce a secreted form of CSFV E2 protein (mE2). The mE2 protein was shown to be N-linked glycosylated and formed a homodimer. The vaccine efficacy of mE2 was evaluated by immunizing pigs. Twenty-five 6-week-old Landrace piglets were randomly divided into five groups. Four groups were intramuscularly immunized with mE2 emulsified in different adjuvants twice at four-week intervals. One group was used as the control group. All mE2-vaccinated pigs developed CSFV-neutralizing antibodies two weeks after the first vaccination with neutralizing antibody titers ranging from 1:40 to 1:320. Two weeks after the booster vaccination, the neutralizing antibody titers increased greatly and ranged from 1:10,240 to 1:81,920. At 28 weeks after the booster vaccine was administered, the neutralizing antibody titers ranged from 1:80 to 1:10240. At 32 weeks after the first vaccination, pigs in all the groups were challenged with a virulent CSFV strain at a dose of 1 × 10(5) TCID50. At two weeks after the challenge, all the mE2-immunized pigs survived and exhibited no obvious symptoms of CSF. The neutralizing antibody titer at this time was 20,480. Unvaccinated pigs in the control group exhibited symptoms of CSF 3-4 days after challenge and were euthanized from 7-9 days after challenge when the pigs became moribund. These results indicate that the mE2 is a good candidate for the development of a safe and effective CSFV subunit vaccine.

Figure 1. Selection and comparison of transfected cell clones expressing E2 protein using ELISA.

E2 protein expressing titers were detected and compared between 60 G418-resistant and IFA-positive cell clones (A). Among the 60 cell clones, 12 cell clones with a relative high titer of E2 were selected and further compared using ELISA (B). The no. 12 cell clone, which had the highest ELISA titer value was selected for further characterization (as shown by the arrow).

Figure 2. Immunofluorescence and flow cytometry analysis of BCSFV-E2 cell passages.

The tenth (F10), twentieth (F20), and thirtieth (F30) generations of BCSFV cells were analyzed by indirect immunofluorescence and flow cytometry. Both assays show a high percentage of cells expressing CSFV E2.

Figure 3. SDS-PAGE analysis of mE2 in culture and purified medium.

M, Molecular marker; 1, supernatant of BCSFV-E2; 2 and 3, Immunoaffinity-purified mE2; –, non-reduced; +, reduced with β-mercaptoethanol.

Figure 4. Western blot analysis of mE2 in culture supernatants.

BHK-21 cells and BCSFV-E2 cells culture supernatants were separated in the absence (+) or presence (+) of β-mercaptoethanol or treated with peptide: N-glycosidase F (PNGase F) followed by Western blot analysis with 12C4 monoclonal antibody.

Figure 5. Production of mE2 by BCSFV-E2 cells.

(A) Confluent BCSFV-E2 cells were incubated in maintenance medium for six days. ELISA was used to quantify the titers of mE2 antigen accumulated in the supernatant every day (24 h). (B) The culture medium of confluent BCSFV-E2 cells was harvested every 4–6 days and replaced with fresh medium, and the mE2 antigen titers in the harvested culture supernatants were determined by ELISA. (C) The culture supernatants of different passages of cell line were determined by ELISA.

Figure 6. ELISA antibody development after vaccination and challenge infection.

Pigs were vaccinated with mE2 antigen with four kinds of adjuvants. The pigs were given a booster vaccination four weeks after the first vaccination. The pigs were then challenged with a virulent CSFV strain at 32 weeks post-vaccination. Pigs inoculated with PBS were used as controls. The antibody titers are presented as blocking rates (A) and the reciprocal of the highest dilution of serum for which a blocking rate of ≥40% was obtained (B).

Figure 7. Rectal temperatures post-challenge.

Vaccinated and control pigs were challenged with the virulent CSFV Shimen strain. After challenge, rectal temperatures of each pig were measured daily for 14 days.