CPV strain JX624771 and CDV strain WL01 were isolated from giant panda in Sichuan Province of China and cultured in Vero cells in our laboratory. Madin–Derby canine kidney (MDCK) cell line was cultured at 37°C in 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, United States). Vero cells expressing canine signaling lymphocyte activation molecules (SLAM) were cultured in DMEM (Gibco, United States) supplemented with 10% fetal calf serum (Gibco, United States) containing 0.1 mg/ml Zeocin (Invitrogen, United States).

DNA template of VP2 gene was extracted from MDCK cell pellets infected with CPV using MiniBEST Viral DNA/RNA Extraction Kit Ver.5.0 (Takara, Japan). A pair of primers with Xho I and Hind III was designed according to DNA sequences on Genbank (DQ354068.1) and used to amplify the whole sequence of VP2 gene (Table 1). PCR was performed using Taq Plus DNA Polymerase (Vazyme, Nanjing, China) or PCR Hero™ (Foregene, Chengdu, China) in a Bio-Rad cycler (United States). The PCR procedure was as follows: denaturation at 95°C for 30 s; denaturation at 95°C for 15 s, annealing at 65°C for 15 s; and polymerization at 72°C for 130 s with 30 cycles; extension at 72°C for 10 min. PCR products were detected in 1% agarose gel and visualized under UV light. The VP2 gene was amplified and cloned into pMD19-T plasmid vector (TaKaRa, Japan) to generate a recombinant plasmid of pMD19-T VP2.

The CDV epitopes identified in previous studies were applied for construction of CPV VP2 chimeras, including a B cell epitope and a CTL epitope of CDV N protein, a Th epitope, a T cell epitope and a B cell epitope of CDV F protein (Beauverger et al., 1993; Obeid et al., 1995; Ghosh et al., 2001; Yi et al., 2016; Table 2).

The chimeric protein included two combinations of VP2N and VP2L. VP2N was constructed with a N-terminus containing additional 78 amino acids ligated into the VP2 backbone. In brief, the epitope of CTL-B of CDV-N protein and the TH-T-B epitope of CDV-F protein were inserted into N terminal region of CPV-VP2 (Figure 1; Casal et al., 1995). As for VP2L, 53 additional amino acids including TH-T-B epitopes of CDV F protein were ligated into N terminus of VP2, and 22 amino acids containing CTL-B epitope of CDV N protein were inserted into the site utilizing the deletion of 218–233 AA at the Loop2 region of CPV VP2 (Figure 1; Rueda et al., 1999). In order to keep each epitope relatively independent, the epitopes were separated from one another by the linker with amino acid sequences GGGGS or GG.

The DNA sequences of each CDV epitope were optimized for expression in Escherichia coli with in silico analysis¹, and were chemically synthesized and cloned into pMD19-T vector. Subsequently, the epitopes DNA sequences were conjugated into CPV VP2 gene using Splicing by Overlapping Extension (SOE) PCR. At last, VP2N/VP2L genes were cloned into pMD19-T vector with Xho I and Hind III site, respectively.

The plasmids of pMD19T-VP2/VP2N/VP2L were transformed into E. coli DH5α competent cells, and the E. coli containing plasmid of pMD19T-VP2/VP2N/VP2L were cultured in 5 ml LB broth containing 100 μg/ml of ampicillin at 37°C overnight. The recombinant plasmids were extracted with E.Z.N.A Plasmid Mini Kit (Omega, United States) and confirmed by Xho I and Hind III digestion. The inserts in the wild type and chimeric constructs were confirmed by Sanger DNA sequencing.

The full-length VP2/VP2N/VP2L genes were subcloned in frame into expression vector of pCold-TF (TaKaRa, Japan) at Xho I and Hind III sites, and the recombinant expression vectors pCold-TF-VP2/VP2N/VP2L were transformed into E. coli BL21 (DE3), respectively. After induction with 0.2 mmol/l IPTG, the cultures were collected and crushed by ultrasonic to confirm the presence and the distribution of the target recombinant protein by SDS-PAGE. Subsequently, the recombinant VP2/VP2N/VP2L protein was purified with Ni-NTA affinity chromatography (Qiagen, Germany). Since the purified recombinant protein had a large soluble Tag TF-Tag (46 kDa), 1a0 U Thrombin protease (Solarbio, China) was added to each 1 mg of purified recombinant protein for digestion at 4°C overnight. Lastly, the label and target proteins were separated by affinity chromatography.

The purified recombinant proteins were transferred onto nitrocellulose filter membrane (NC membrane) after SDS-PAGE. Then NC membrane was blocked by 1% BSA for 2 h. After wash with Western washing buffer (TBS-T) for 3 times, the membranes were incubated overnight at 4°C with 1:5000 diluted mouse anti-CPV or anti-CDV monoclonal antibody (Jilin Wuxing Animal Health Care Co., Ltd., China). The membranes were washed by TBS-T again for 5 times and incubated with Horseradish Peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (Sangon Biotech Co. Ltd., Shanghai) for 2 h. After another wash for 5 times with 5 min each time, electrochemiluminescence chemiluminescence (ECL) coloring solution (Sangon Biotech Co. Ltd., China) was added for color development under dark condition. The results were photographed with gel imaging system (Gel Doc™ XR⁺, Bio-Rad).

After purification with Ni-NTA affinity chromatography, the recombinant protein was concentrated by ultrafiltration to remove the imidazole. The concentration of the recombinant protein was determined by the BCA Protein Assay Kit (Boster Biological Technology, United States). Subsequently, purified recombinant proteins of VP2/VP2L/VP2N were placed in a 14 kD dialysis bag with sufficient amount of dialysis buffer (50 mM NaH₂PO₄, 250 mM NaCl, 0.5%Triton X-100, 2 mM Dithiothreitol, pH8.0). The whole buffer system was placed on a magnetic agitator for slight stirring, and dialysis was conducted at 4°C for 16 h, during which the liquid was changed for 3 times. At last, 20 μl of the assembled sample was dropped on the copper net and placed for 2 min. The phosphotungstic acid solution was added for negative staining for 2–3 min, and the morphology of VLPs were observed by transmission electron microscope (TEM). Moreover, the hemagglutination test (HA) was used to detect the hemagglutination activity of the recombinant protein. The highest dilution concentration that could agglutinate 50% of porcine acetaldehyded erythrocytes (Guangzhou Hongquan Bio-tel Co. Ltd., China) was taken as the reading end point.

Fifty female BALB/c mice aged 6 weeks were randomly divided into 5 groups (n = 10). Group A, group B and group C were subcutaneously immunized with 50 μg of VP2, VP2L and VP2N recombinant protein, respectively. Group D was immunized with 100 μl CPV/CDV bivalent live-attenuated vaccine (Nobivac® Puppy DP, Intervet) as a positive control, and Group E injected with 100 μl PBS as negative control. Each group was boosted with the same dosage at 14-and 28-days post immunization (dpi). All animal experiments were conducted in compliance with protocols approved by Sichuan Provincial Laboratory Animal Management Committee (Permit Number: XYXK (Sichuan) 2019–187). The protocols for this experiment were performed according to the guidelines of the Ethics and Animal Welfare Committee (EAWC) of Sichuan Agricultural University. The mice were euthanized via exposure to carbon dioxide until complete cessation of breathing is observed for a minimum of 2 min by a trained technician during the experimental period as approved by EAWC.

At 0, 14, 28, 42, 56 days after first immunization, peripheral blood was obtained and serum was used to determine the antibody levels against CPV or CDV using an indirect ELISA method. Briefly, 96-well microtiter plates were coated with CPV JX624771 or CDV WL01 in 0.1 mol/l carbonate/bicarbonate buffer (pH 9.6) and incubated at 4°C overnight. After three washes in PBS-T, the plates were blocked with 250 μl PBS-T containing 1% BSA at 37°C for 2 h. After three washes in PBS-T, 100 μl of diluted mice serum (1:100) in PBS-T was added and incubated at 37°C for 1 h. Then mouse serum (1:100) with PBS-T containing 1% BSA was added, and plates were again incubated for at 37°C 1 h. After three washes in PBS-T, 100 μl diluted goat anti-mouse IgG peroxidase conjugate (Sangon Biotech Co. Ltd., China) in PBS-T containing 1% BSA at a 1:5, 000 dilution was then added for at 37°C for 1 h. The plates were washed three times, and the colorimetric reaction was developed using 100 μl tetramethylbenzidine substrate solution (Sangon Biotech Co. Ltd., China) at 37°C for 15 min. Color development was stopped with 50 μl of 2 N H₂SO₄, and OD was read at 450 nm.

The CPV JX624771 strain was grown in MDCK cells and cell-free virus fluids were harvested. After inactivation by 0.025% β-propiolactone at 4°C for 24 h, CPV JX624771 was incubated at 37°C for 2 h and used as hemagglutination inhibition (HI) antigen. The HI test was performed at 14, 28, 42, 56 dpi as described previously (Senda et al., 1986). Briefly, the hemagglutination (HA) test was conducted in 96-well V-shaped microplates to determine the HA units of CPV JX624771. Twenty-five μL of serial 2-fold dilutions of inactivated CPV JX624771 with PBS was mixed with 25 μl of porcine erythrocyte suspension (Guangzhou Hongquan Bio-tel Co. Ltd., China) in each well and incubated at 4°C for 2 h. HA titers were determined and 4 HA units of inactivated CPV JX624771 were prepared. Serial 2-fold dilutions of heat inactivated serum were made in 25 μl of PBS in 96-well microplates and mixed with 25 μl CPV JX624771 containing 4 HA units. The mixture was incubated at 37°C for 30 min and 50 μl of porcine erythrocyte suspension was added for another incubation at 4°C for 1 h. The HI titer of serum was determined according to the reciprocal of the highest serum dilution which was a complete absence of HA.

The sera neutralizing antibody titers obtained from mice were determined by serum neutralization test (SNT) with a monolayer of Vero-SLAM cells at 56 dpi. Briefly, 50 μl of heat inactivated (56°C for 30 min) test sera were diluted serially 2-fold and mixed with equal volume of CDV WL01 strain (100 TCID₅₀) in 96-well tissue culture plates. The plates were incubated at 37°C in 5% CO₂ for 1 h. 100 μl of Vero-SLAM cells suspension (1 × 10⁶ cells/mL) was added and incubated at 37°C for 7 days for assessment of cytopathic effect (CPE). In addition, 100 TCID₅₀ CDV virus diluent were set as positive control, and negative serums were added as negative controls. The highest dilution of serum showing complete inhibition of CPE was taken as the endpoint. The serum neutralization titer was expressed as the reciprocal of the final dilution of serum in the serum/virus mixture, which neutralized an estimated 100 TCID₅₀ of the virus at 50% endpoint estimated according to the method of Kärber.

Mouse spleens were removed under sterile conditions and ground through a sterile cuprous mesh (200 meshes) at 56 dpi. The spleen cells were immersed in RPMI 1640 medium (Gibco, United States) with 10% fetal calf serum, added to the lymphocyte separation medium (Sangon Biotech Co. Ltd., China), homogenized, and centrifuged at 1, 000 r/min for 10 min. Pellets were discarded; buoyant cells were washed thrice in RPMI 1640 medium with 10% fetal calf serum. The T lymphocytes in 96-well plates (1 × 10⁵ cells/well) were cocultured with 100 TCID₅₀ CPV JX624771 or CDV WL01 in RPMI 1640 supplemented with 10% fetal calf serum, and maintained at 37°C in a humidified 5% CO₂ atmosphere for 68 h. Meanwhile, negative control group was added with PBS. Each sample had 3 replicates. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (5 mg/ml) was added to each well and incubated for 4 h at 37°C in 5% CO₂. After culture for 4 h, the medium was gently removed, and 150 μl DMSO was added to each well. The plates were gently shaken to dissolve the crystals. The absorbance of each well was determined at 490 nm using a microplate reader, and the stimulatory index (SI) was calculated (SI = OD₄₉₀ value of the stimulus group/OD₄₉₀ value of the negative control group).

Analysis of variance (ANOVA) and other statistical analysis were performed using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, United States). Data was shown as the mean ± SEM, with the significant difference set at 0.05 (p < 0.05).

To obtain the soluble protein expressed in E. coli, pCold TF DNA was used as expression plasmid in this study. Trigger factor (TF) is a prokaryotic ribosome-associated chaperone protein of 48 kDa which facilitates co-translational folding of nascent polypeptides, and tag removal from the expressed fusion protein. As a result, the recombinant protein TF-VP2 was successfully expressed in the supernatant (Figure 2A).

The supernatant after ultrasonic crushing was collected, and the recombinant protein in the supernatant was further purified by Ni-NTA affinity chromatography. The purified TF-VP2 was ultrafiltered to remove imidazole, and the appropriate amount of Thrombin was added to remove TF label overnight at 4°C by enzyme digestion. After overnight digestion, the solubilizing label of the recombinant protein TF-VP2 was removed by protease, and there was a distinct TF label at 56 kDa and a VP2 protein at 68 kDa (Lane 3; Figure 2B). Due to 6 × His tag on the removed TF tag, the protein solution after enzyme digestion was passed through Ni column again by Ni-NTA affinity chromatography to collect the flow fluid and soluble VP2 protein was obtained with high purity (Lane 2; Figure 2B). Furthermore, the recombinant proteins of both TF-VP2L and TF-VP2N were soluble, indicating that the soluble expression of the recombinant protein was not affected by CDV antigenic epitopes in the appropriate region of VP2 (Figure 2C). After purification by Ni-NTA affinity chromatography and Thrombin digestion, the pure recombinant protein without promoter label was obtained (Figure 2D).

After blotting, CPV or CDV monoclonal antibody was used as primary antibody for incubation and binding. Goat anti-mouse antibody labeled with HRP was used as secondary antibody. After ECL coloring, the results showed that specific bands at expected size of about 70 kDa were found on the NC membrane indicating that the purified recombinant proteins could be recognized by specific CPV antibody (Figure 3A). In addition, after incubation and binding with CDV monoclonal antibody as primary antibody, specific bands were also found on the NC membrane for the recombinant chimeric proteins, which indicated that chimeric epitopes constructs were successful and could react with specific CDV antibody (Figure 3B). The results above suggested that recombinant proteins of VP2/VP2N/VP2L displayed a single band with a molecular weight of 68 kDa/76 kDa/71.5 kDa, indicating that these proteins retained their specific immunoreactivity.

Electron microscopy revealed the presence of spherical particles of diameter in the range of 22 nm to 26 nm, which exhibited the same dimensions as natural CPV virions. The results indicated that the insertion of foreign epitopes into the appropriate position of VP2 protein does not affect the formation of VLPs, and the wild or chimeric VP2 proteins were capable of self-assembly into VLPs with diameters similar to CPV (Figure 4A).

HA test was conducted with 1% porcine erythrocyte suspension to detect the HA titer of the wild and chimeric VLPs in each group. Consequently, the HA titer of VP2 VLPs was 1:256, VP2L VLPs and VP2N VLPs were 1:128, indicating that the prepared VLPs had similar HA activity as wild CPV virions (Figure 4B).

Each mouse was bled to determine the titers of specific antibodies at 0, 14, 28, 42 and 56 dpi, respectively. The changes of CPV or CDV specific antibody levels in serum were detected by indirect ELISA, which showed that CPV or CDV specific antibodies were efficiently induced by immunizing with wild or chimeric VLPs. In contrast, no specific antibody was detected in negative control group injected with PBS (Figure 5). The results revealed that specific antibody levels in each immunization group increased continuously, reaching the peak at 42 dpi, and then maintained at a high level. At 42 dpi, there was no significant difference in antibody levels among VLPs immunization groups (p > 0.05), indicating that CPV specific antibodies could be produced in mice after VLPs immunization in each group, and the addition of CDV antigenic epitopes did not affect the production of specific antibodies to CPV (Figure 5A). Moreover, CDV specific antibodies in the two chimeric VLPs immunization groups also increased rapidly at 28 dpi, and the antibody level reached the peak at 42 dpi and slightly decreased at 56 dpi. In contrast, no CDV specific antibody was detected in PBS group and wild VP2 VLPs group. These results indicated that chimeric VLPs could effectively display the major epitopes of CDV, which could simultaneously induce CDV specific antibodies in mice (Figure 5B).

Specific HI antibody of CPV in mice serum was detected, and the change of HI antibody level was in harmony with that detected by indirect ELISA, which reached the peak at 42 dpi and tended to be stable after the third immunization (Figure 6A). Compared with the live attenuated vaccine group, there was significant difference (p < 0.05), and there was no significant difference among the VLPs immunization groups (p > 0.05).

SNT was applied to detect the titer of CDV neutralizing antibody in mice serum at 56 dpi in order to evaluate whether the chimeric proteins could effectively induce neutralizing antibody against CDV in mice. The results indicated that CDV specific neutralizing antibodies was produced in both the VLPs immunization group and the live attenuated vaccine group at 56 dpi (Figure 6A). CDV neutralizing antibody was not detected in VP2 VLPs group and PBS group. Compared with two chimeric VLPs, the average neutralizing antibody titer of VP2N VLPs group was 1:100.4, which was slightly higher than that of VP2L VLPS group (1:95.7), and there was no significant difference after analysis (p > 0.05). Moreover, the neutralizing antibody titer of live attenuated vaccine group was 1:74.0. There was significant difference between VP2N and VP2L groups at 56 dpi (p < 0.05; Figure 6B).

CPV JX624771 or CDV WL01 virus were used to stimulate the splenic lymphocytes at 56 dpi, and PBS was used as the negative control. Splenic lymphocytes from mice in all immunization groups could proliferate under the stimulation of CPV, and the SI of VP2N VLPs group was significantly higher than live attenuated vaccine group (p < 0.01). However, there was no significant difference among three VLPs immunization groups (p > 0.05). With the stimulation of CDV, spleen lymphocytes in the chimeric VLPs group could respond to the stimulation and proliferate significantly. The SI of VP2N VLPs group was 2.18, which was significantly different from that of VP2L VLPs group (p < 0.05; Figure 7).