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Efficacy evaluation of a bivalent subunit vaccine against classical swine fever virus and porcine circovirus type 2 – Scientific Reports

Preparation of CSFV E2 and PCV2 ORF2 recombinant proteins and vaccines

The CSFV and PCV2 vaccine antigen design was conducted based on their protein sequences with NCBI accession numbers of AAT66638 and ABV21950, respectively. Briefly, the CSFV E2 was cloned and expressed in CHO cells. The E2 recombinant protein antigens were then purified by nickel-chelating affinity chromatography. Recombinant baculovirus carrying the ORF2 gene of PCV2 (PCV2 ORF2) was used to infect High Five™ cells to express PCV2 ORF2 protein, which were purified by ion exchange chromatography. SDS-PAGE and transmission electron microscopy were used to verify protein production and VLP formation, respectively. The monovalent and bivalent vaccines were prepared by mixing antigen with an oil adjuvant, Montanide™ ISA-206 (Seppic, France), in a 1:1 ratio w/w (antigen: adjuvant).

Ethical statement

All animal experiments in this study were performed in accordance with the relevant guidelines and regulations. Studies with live CSFV were conducted in biosafety level 3 facilities, and experimental animals were kept in high containment animal biosafety level 3 facilities. The animals were sacrificed following the AVMA guidelines for the euthanasia of animals (Version 2020.0.1). The studies involving mice and pigs were reviewed and approved by the Institutional Animal Care and Use Committees (IACUC) of the National Ilan University (Committee protocol number 111–4) and the National Pingtung University of Science and Technology (NPUST-IACUC NPUST-111–01), respectively. All procedures in the current study were reported in compliance with the ARRIVE guidelines 2.0 (

Mouse immunization with the vaccine candidate

Five-week-old BALB/c mice were purchased from BioLASCO Taiwan Co., Ltd. After one week of a regular diet, the mice were randomly divided into four groups (n = 5 each group: (1) PBS control group; (2) CSFV E2 monovalent vaccine group; (3) PCV2 ORF2 monovalent vaccine group; and (4) CSFV E2 plus PCV2 ORF2 bivalent vaccine group). For immunization, the mice were intramuscularly injected with 0.1 mL of CSFV E2 (15 μg/mL), PCV2 ORF2 (50 μg/mL), or CSFV E2 (15 μg/mL) plus PCV2 ORF2 (50 μg/mL), whereas mice in the PBS control group was given the same volume of treatment vaccines. The dose was determined based on our previous PCV2 studies (unpublished) and publicly available information of the antigen contents of other CSFV E2 vaccines. Three weeks post-vaccination, the vaccinated mice were challenged with 106.3 TCID50 PCV2 (Fig. S1a). At the end of experiment, the mice were sacrificed using carbon dioxide inhalation, and their blood was collected.

Measurement of specific antibodies and viremia in mice

The blood samples were collected through the submandibular vein at weeks 4 and 6, including both day 2 and day 4 post PCV2 challenge. After centrifugation at 4 °C, the mouse sera were isolated and stored at -80 °C for future experiments.

Anti-CSFV specific antibodies were evaluated using a commercial ELISA kit, Classical Swine Fever Antibody Test Kit (SK106 CSFV E2, BioChek, The Netherlands). Measurement of anti-PCV2 specific antibodies were conducted by immunofluorescence assay (IFA)34.

To perform the neutralization test, the mouse serum samples were incubated with PCV2 along with porcine kidney 15 (PK15) cells for 3 days. After fixation with 80% acetone, PK15 cells were incubated with serum obtained from a New Zealand white rabbit immunized with purified and inactivated PCV2, followed by FITC labeled anti-rabbit antibodies (Sigma-Aldrich). The neutralizing antibody titer for PCV2 was calculated using the 50% virus neutralization test (VNT50)35.

The PCV2 viremia was detected by extraction of PCV2 nucleic acids in mouse sera with LabPrep™ Viral DNA Mini Kit. The nucleic acid samples were then analyzed using SBC Porcine Circovirus Type 2 qPCR Kit (Schweitzer Biotech Company Ltd., Taiwan).

Vaccination and challenge of pigs

Twenty cesarean-derived, colostrum-deprived, 4-week-old SPF pigs purchased from Agricultural Technology Research Institute (ATRI), Taiwan were randomly assigned into 4 groups (n = 5 each group: (1) Vaccine + CSFV (VC) group; (2) Non-vaccine + CSFV (NC) control group; (3) Vaccine + PCV2 (VP) group; (4) Non-vaccine + PCV2 (NP) control group). After one-week observation, each pig in the treatment groups was intramuscularly immunized with 2 mL of the CSFV/PCV2 bivalent vaccine, containing 50 μg of CSFV E2 and 50 μg of PCV2 ORF2 antigens, twice with an interval of 2 weeks, whereas pigs in the control groups were given the same volume of normal saline (0.9% NaCl) (Fig. 3a). To monitor the safety of the CSFV/PCV2 bivalent vaccine in pigs, the rectal temperatures, BWG, clinical signs, and CSFV/PCV2 nucleic acids of each pig were measured before sacrifice at week 7 (CSFV challenge) or week 12 (PCV2 challenge). The monitoring periods after viral challenges were not the same because the disease progression of the two viruses were different. A mean rectal temperature over 40.5℃ was considered as fever36.

One week before challenge, pigs in VC and NC groups were transferred to Animal Health Research Institute for CSFV challenge; VP and NP groups were transferred to National Pingtung University of Science and Technology in Taiwan for PCV2 challenge. Previously established challenge procedures were used. For CSFV challenge, four weeks after the 1st vaccination, pigs in VC and NC groups were intramuscularly challenged with 2 mL of CSFV ALD strain (1 × 105.41 FAID50). For PCV2 challenge, pigs in VP and NP groups were administered with 2 mL of PCV2d THF0601-7 strain (1 × 106 TCID50 /mL) via intranasal and intramuscular routes on the first day of challenge, followed by intranasal inoculation of 1 mL of viral suspension using a syringe on 2 consecutive days. The pigs were euthanized through exsanguination under a surgical plane of anesthesia induced by intramuscular administration of Stresnil® (Azaperone), followed by intravenous Zoletil® (tiletamine-zolazepam mixture) injection. Tissues were harvested and fixed for future examination.

Pathological and clinical examination

For histopathological examination, tissue sections were fixed in 10% neutral-buffered formalin. The scoring system of 0 (normal) to 4 (most severe) was used for the evaluation of the severity of lymphoid depletion, interstitial pneumonia, infarcts and hemorrhages in the spleen. In addition, clinical signs of CSFV were evaluated by a scoring system of 0 (normal) to 3 (most severe)37. The histopathological changes and clinical syndromes mentioned above were estimated by two veterinary pathologists blinded to treatment allocation.

Serological examination

All pig serum samples were isolated and measured for the presence of anti-CSFV or anti-PCV2 antibodies. For the detection of CSFV-specific antibodies, a commercial ELISA kit, IDEXX CSFV Ab Test Kit (IDEXX Laboratories, Inc., USA), was used to perform the tests. The CSFV ELISA antibody levels were expressed as the blocking % which 30 and 40 were interpreted as negative and positive, respectively. A blocking % between 30 and 40 was considered as suspected. The neutralization antibody titer against CSFV ALD strain was determined in duplicate wells and expressed as the log2 of the reciprocal of the highest serum dilution that 50% of the wells were protected from infection, calculated using the Reed-Muench Method. A Porcine Circovirus type 2 Antibody Test Kit (SK105, BioChek), which is an ELISA kit, was used to detect PCV2-specific antibodies according to the manufacturer’s recommended procedures.

Quantitative RT-PCR for viral quantification

CSFV RNA was extracted from serum and tissue samples using MagNA Pure 24 Total NA Isolation Kit (Roche Molecular Systems, Inc., USA). The quantitative reverse transcription PCR (qRT-PCR) was performed with the LightCycler Multiplex RNA Virus Master Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. A CSFV-containing blood sample of known concentration was ten-fold serially diluted from 108 to 101 TCID50/mL and used to establish a standard curve.

PCV2 DNA was extracted using AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit (Axygen®; Corning, USA). The qPCR was performed using PowerUp SYBR Green Master Mix (Applied Biosystems™, Thermo Fisher Scientific Inc., USA) on QuantStudio™ 3 Real-Time PCR Systems (Applied Biosystems™, Thermo Fisher Scientific Inc., USA). Viral DNA concentrations were expressed as Log10 PCV2 genomic copies/mL in serum and tissue.

The specific primer and/or probe sets used for the detection of CSFV and PCV2 are summarized in Table S1.

Statistical analysis

Data analyses were performed using the generalized linear models (GLM) procedure of SAS® (version 9.4, SAS Institute Inc., Cary, NC, USA). A two-sided p value of < 0.05 was considered statistically significant.