Development of an Effective Single-Dose PCV2/CSFV Bivalent Subunit Vaccine Against Classical Swine Fever Virus and Porcine Circovirus Type 2

Abstract

Background/Objectives: Porcine Circovirus Type 2 (PCV2) impairs pigs' immune systems and increases susceptibility to co-infections, including Classical Swine Fever (CSF), a highly contagious disease listed by the World Organisation for Animal Health (WOAH) as notifiable. Therefore, swine operations in CSF-endemic regions are encouraged to immunize piglets with both PCV2 and CSFV vaccinations. Currently, there is no commercially available bivalent vaccine for PCV2/CSFV. Methods: In this study, a total of twenty 4-week-old SPF pigs were administered our formulated PCV2/CSFV bivalent subunit vaccine, containing soluble CSFV-E2 (50 µg) and PCV2-ORF2 (100 µg) antigens with a porcine-specific CpG adjuvant. After 4 weeks of vaccination, all pigs were evaluated for efficacy against PCV2 and CSFV. Results: Pigs were only immunized once and showed significantly increased neutralizing or ELISA antibody titers against both viruses four weeks post-vaccination. After viral challenges, vaccinated pigs displayed no clinical signs or lesions and had markedly reduced CSFV and PCV2 viral loads in the serum and tissues compared to controls. Conclusions: These results demonstrate that a single dose of the PCV2/CSFV bivalent subunit vaccine is safe and effective in young pigs, induces strong antibody responses, and suppresses viral replication, making it a promising tool for swine disease control and cost-effective vaccination strategies.

Keywords: Classical swine fever; Porcine circovirus type 2; bivalent subunit vaccine; single dose.

Figure 1

The schematic representation of the animal experimental design. The bleeding times were represented by three symbols: ●, vaccination trial: vaccinated pigs (Groups A and C) and non-vaccinated pigs (Groups C and D) were immunized with the PCV2/CSFV bivalent vaccine or saline, respectively, at week 0 (1st vaccination); ▲, CSFV challenge trial: pigs in Groups A and B were challenged with the CSFV ALD strain (10^5.44 FAID₅₀/total dose per pig) at the beginning of week 5. Blood samples were collected at days 0, 4, 7, 10, and 14 post-challenge, and pigs were sacrificed on day 14 post-challenge (week 7); ■, PCV2 challenge trial: pigs in Groups C and D were challenged with the PCV2d THF0601-7 strain (10^6.90 TCID₅₀/total dose per pig) at the beginning of week 6. Blood samples were collected at weeks 0, 2, 3, 4, 5, 6, and 7 post-challenge, and pigs were sacrificed at week 7 post-challenge (week 13).

Figure 2

The expression of CSFV-E2 and PCV2-ORF2 proteins from ExpiCHO^TM stable cell line. CSFV-E2 (A) and PCV2-ORF2 (B) proteins were expressed by Western blot. CSFV-E2 protein reduced with or without reagent β-ME (Lane 1 and Lane 2, respectively). Monoclonal antibody (mAb) WH303 and PCV2 capsid polyclonal antibody were used for the Western blot, respectively. Lane M indicates protein markers.

Figure 3

The effect of bivalent vaccine after vaccination in response to CSV or PCV2 sera antibody titers. Sera samples from all pigs were collected and evaluated for CSFV E2-specific Ab (A) and PCV2 ELISA Ab (B) responses, respectively. Results are values ≥ 40% and ≥ 0.5 that are considered positive by the IDEXX CSFV Ab test (blocking%) and BioChek PCV2 Ab test (S/P ratio), respectively. Data with different letters (^a,b) indicate that the groups are significantly (p < 0.05) different from each other at the same time point.

Figure 4

The evaluation of the clinical symptoms of pigs before (day 0) and after the CSFV challenge. Rectal temperatures (A) and clinical scores (B) were determined in pigs. All pigs were challenged with CSFV (10^5.44 FAID₅₀/total dose per pig) four weeks after vaccination and sacrificed on day 14 post-challenge. Data were expressed as mean ± SEM of rectal temperatures. In addition, the non-parametric Mann–Whitney U test was used to analyze clinical scores, and data were expressed as the median and 95% confidence intervals. Data with different letters (^a,b) indicate that the groups are significantly (p < 0.05) different from each other at the same time point.

Figure 5

Serological responses in pigs challenged with CSFV or PCV2. Vaccinated (Groups A and C) and non-vaccinated (Groups B and D) pigs received a CSF/PCV bivalent vaccine or saline on day −28 and were challenged on day 0. Sera were analyzed for CSFV E2-specific (A), PCV2 ELISA (B,C), and CSFV-neutralizing (D) antibodies. Results are values ≥ 40% and ≥ 0.5 that are considered positive by the IDEXX CSFV Ab test (blocking%) and BioChek PCV2 Ab test (S/P ratio), respectively. Data are presented as mean ± SEM. Different letters (^a,b) indicate significant differences (p < 0.05) at the same time point.

Figure 5

Serological responses in pigs challenged with CSFV or PCV2. Vaccinated (Groups A and C) and non-vaccinated (Groups B and D) pigs received a CSF/PCV bivalent vaccine or saline on day −28 and were challenged on day 0. Sera were analyzed for CSFV E2-specific (A), PCV2 ELISA (B,C), and CSFV-neutralizing (D) antibodies. Results are values ≥ 40% and ≥ 0.5 that are considered positive by the IDEXX CSFV Ab test (blocking%) and BioChek PCV2 Ab test (S/P ratio), respectively. Data are presented as mean ± SEM. Different letters (^a,b) indicate significant differences (p < 0.05) at the same time point.

Figure 6

Viral loads in tissue were determined after two challenge trials. Tissues in pigs were collected and examined for viral loads of CSFV (A) or PCV2 (B) after euthanasia. Data in CSFV challenge trial were expressed as mean ± SEM of log₁₀ TCID₅₀/g, and in PCV2 trial as mean ± SEM of log₁₀ copies/g. Data with different letters (^a,b) indicate that the groups are significantly (p < 0.05) different from each other at the same time point.