Self-assembled raccoon dog parvovirus VP2 protein confers immunity against RDPV disease in raccoon dogs: in vitro and in vivo studies

Feline kidney 81 (F81) cell line was grown in DMEM medium (Gibco, America) containing 10% fetal bovine serum (FBS) (HyClone, America), 100 mg/mL of streptomycin, 100 U/mL of penicillin in T75 flasks (Corning, America) at 37 °C in an atmosphere of 5% CO₂ and humidified air. At 60% confluency, RDPV RPSN (Chinese Academy of Agricultural Sciences) was added to the T75 flasks. After incubation for 2 h at 37 °C, the medium was changed to DMEM containing 3% FBS. The cells were then cultured continuously for about 48 h. At 80% confluency, the infected cells exhibited cytopathic effects (CPE). The T75 flasks were exposed to a freeze/thaw cycle at −20 °C to recover the virus. The virus was kept at −80 °C, and RDPV RPSN was used for all immunological tests.

The recombinant plasmid pET30a-VP2 was transferred to competent E. coli ER2566 cells by heat shock method. The positive colonies were incubated in LB medium supplemented with 50 µg/mL kanamycin, 0.2 mmol/L isopropyl β-D-thiogalactoside (IPTG). The recombinant plasmid pET30a-VP2 and Tf16 were then transformed into competent E. coli ER2566 cells (TaKaRa, China) by heat shock method. The positive colonies were incubated in LB medium supplemented with 50 µg/mL kanamycin, 20 µg/mL chloramphenicol, 2 mg/mL L-Arabinose and 0.2 mmol/L IPTG. After induction with IPTG at 25 °C for 16 h, the cells were collected and lysed by sonication system in buffer containing 50 mM Tris, 250 mM NaCl (pH 8.0) at 4 °C. The homogenate was centrifugated at 10,000g at 4 °C for 30 min. The supernatant and debris were collected and analyzed.

The collected supernatant was purified by ammonium sulfate precipitation followed by Capto Butyl ImpRes hydrophobic chromatography (GE, USA). The chromatography column was washed using a buffer containing 200 mM (NH₄)₂SO₄, 20 mM Tris, 2 mM NaCl until the UV spectra had no significant changes by NGC (Bio-Rad, America). RDPV VP2 protein was washed in buffer containing 200 mM (NH₄)₂SO₄, 20 mM Tris, 2 mM NaCl and analyzed by SDS-PAGE. Following purification with Triton X-114 (Solarbio, China) extraction, the concentration of endotoxin in the purified RDPV-VP2 protein was measured by Limulus lysate gelatin assay kit (CRL, America). Briefly, after adding a final concentration of 1% of Triton X-114 to RDPV VP2 protein, the mixture was incubated and stirred continuously on ice for 30 min. Then, the mixture was incubated and stirred continuously at 37 °C for 15 min. After centrifugation at 8000 g at 25 °C for 30 min, the RDPV VP2 protein and Triton X-114 were separated. The RDPV VP2 protein so obtained was subjected to another 2 cycles of treatment.

RDPV VP2 protein was incubated with different concentrations of buffer containing NaCl (150 mM, 250 mM, 500 mM) and at different pH (pH 7.0 and 8.0). The collected RDPV VLPs were determined by DLS, TEM, hemagglutination (HA) assay.

Twenty-five raccoon dogs were divided into 5 groups (n = 5), and were immunized by intramuscular injection. Groups A, B, C used 10 μg, 50 μg, and 100 μg RDPV VLP treated with 20 mg/ml Al(OH)₃ (Thermo, USA), respectively. In addition, group D were vaccinated with 100 μL of experimentally inactivated RDPV vaccine (HA titer 1:2¹¹). Group E was vaccinated with 100 μL PBS. Blood sample was obtained from the veins of the forelimb at 14, 28, 42, 56, 70, 84, 98 days post-inoculation (dpi). The blood samples were centrifuged at 4000 rpm/min for 15 min. The extracted serum was inactivated at 56 °C for 30 min. RDPV RPSN virus was used as antigen (HA titer 1:2⁴). The mixtures were incubated with 1.0% pig erythrocytes in a 96-well V-shaped microplates for Hemagglutination inhibition. The raccoon dogs immunized for 14 days were euthanized, the injection site was observed grossly, and evaluation of the cadavers was done to assess the safety of this vaccine.

Twenty-five raccoon dogs were divided into 5 groups (n = 5). Groups A, B, C were immunized with either 10 μg, 50 μg, or 100 μg RDPV VLP treated with 20 mg/mL Al(OH)₃ gel. Additionally, group D was vaccinated with 100 μL of experimentally inactivated RDPV vaccine (HA titer 1:2¹¹), while group E was vaccinated with 100 μL of PBS. Raccoon dogs described above were struck with RDPV RPSN virus (HA titer 1:2¹¹) containing 10 mL oral MEM medium (Gibco, USA) at 21 dpi. The diet, mental health and the stool samples of raccoon dogs were evaluated. Stool samples were collected and tested with canine parvovirus detection kit (Sinohp, China). Additionally, 1 g stool samples were mixed evenly with 1 mL PBS, and the mixture was centrifuged at 10,000g/min for 5 min. The HA titer was estimated from the collected supernatant. Blood samples were collected and analyzed with a Blood Chemistry Analyzer (ABAXIS, USA). The extracted serum was used to detect HI titer. Stool and blood samples from every group were collected and labelled at 8 o'clock every morning.

Pathological examination was carried out for the raccoon dogs at 11 dpi. Various organs i.e. heart, liver, spleen, lungs, kidneys and intestinal tract were collected for examination. The specified organs were removed and stored in 10% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). Immunohistochemical (IHC) examination was performed using rabbit anti-RPSN polyclonal antibody (anti-RPSN pAb) to evaluate paraffin-embedded sections. Target area (×200) of the tissue was selected by Eclipse Ci-L microscope camera. The positive cumulative optical density value of each field of view was marked as A, while the area of the corresponding tissue pixel was measured and recorded as B. The areal density (C) was calculated as: A/B.

All animal experiments were approved by Animal Experiment Committee of Specialty Products Research Institute of the Academy of Agricultural Sciences. The protocols were performed in accordance with the guidelines for the Welfare and Ethics of Laboratory Animals of China.

Results showed that a 65 kDa VP2 protein was expressed within the inclusion bodies of induced pET30a-VP2 cells (Fig. 1a). The pET30a-VP2 and Tf16 were transferred to E. coli ER2566, where the soluble VP2 protein and protein Tf16 (56 kDa), were co-expressed (Fig. 1b). Western blot analysis of RDPV VP2 expression confirmed that the mouse anti-CPV antibody (Sinohp, China) could bind the target RDPV VP2 protein (Fig. 1c).

Upon purification by ammonium sulfate precipitation and hydrophobic chromatography, the Tf16 protein (56 kDa) could be removed, and the RDPV VP2 protein (65 kDa) had a purity of 90% (Fig. 2b). This was followed by 3 cycles of Triton X-114 extraction, which showed the concentration of endotoxin in the purified RDPV VP2 protein to be less than 125 EU/mL as estimated by limulus lysate gelatin test kit (Table 1; Fig. 2c) (Charles River, USA). The recovery of RDPV VP2 protein was approximately 72%. Through the BCA protein determination kit (Solarbio, China), the total yield of purified RDPV VP2 protein was estimated to be about 6 g per liter of bacteria, and the recovered protein was 18 mg, which is higher than the previously described protocol by Ji et al. [15].

DLS results indicated that the size of RDPV VLPs was about 19.10 nm at pH 7.0 and 23.85 nm at pH 8.0 (Fig. 3a). This implied that pH seemed to play a decisive role in the assembly of virus-like particles. The optimal concentration of NaCl was also analyzed at pH 8.0. Results indicated that the size of RDPV VLPs did not change significantly with the increasing concentration of NaCl (Fig. 3b). TEM result showed that purified RDPV VP2 protein could self-assemble into the particles at pH 8.0 and 250 mM NaCl, with a dimension of approximately 24 nm (Fig. 3c). HA assay showed a high HA titer of RDPV VLPs (1:2¹⁸).

Healthy Raccoon dogs were randomly divided into 5 groups and immunized with either different doses of RDPV VLPs (10 μg, 50 μg, 100 μg) treated with Al(OH)3 or 100 μL inactivated RDPV vaccine or PBS to examine whether RDPV VLPs could induce specific humoral immune responses. There were no sarcomas in the injection site after necropsy, which indicated that the absorption of the RDPV VLPs was good (Fig. 4a). There was a strong induction of High-titer HI antibodies which peaked (1:512) at 42 dpi, in the group receiving 100 μg RDPV VLPs as well as the vaccine group. In addition, a rise was seen in the humoral immune response. This was evident as a direct relation between an increase in HI antibody expression level in VLPs group with different doses of VLPs. In comparison with 100 μg group, the antibodies in the 10 μg and 50 μg groups did not increase significantly after day 14 post-inoculation. During the three-month immunization, however, the HI antibody titer remained above 1:80, which may be considered as conferring immune protection.

In order to determine whether the site of injection showed any pathological effects, the raccoon dogs were euthanized on day 14 after immunization. The site of injection was dissected and visually examined for the presence of any visible pathological signs such as the formation of nodules.

On day 21 after receiving a single dose of VLPs vaccine, the raccoon dogs were challenged orally with RDPV virus and the animals were observed for the development of any symptoms, which were recorded (Fig. 5). The animals in groups receiving 50 μg or 100 μg of Al(OH)₃ treated RDVP VLPs or the vaccine group showed normal physical condition. However, animals in the group receiving groups 10 μg RDPV VLPs or receiving PBS showed the appearance of clinical symptoms related to RDPV approximately 4–5 dpi. There was 100% morbidity in the group receiving PBS. The clinical symptoms manifested were depression, diarrhea, and loss of appetite. These symptoms were most obvious in 7–8 days and began to gradually decrease after 9–10 days. Observation of the nasal cavity of sick raccoon dogs showed dryness along with general dehydration, dry skin and decreased elasticity. In addition, three of the five raccoon dogs of group PBS died (Fig. 5d). Almost all of the raccoon dogs began excreting the virus on the 4th day as detected using canine parvovirus detection kit. HA assay showed that the rates of viral excretion rose sharply on the 4th day (Fig. 5e). HI assay showed the antibodies in the serum of group 10 μg, 50 μg, 100 μg, vaccine began to rise sharply on the fourth day, while the antibody in the serum of group PBS only began to rise significantly on the 5th day (Fig. 5f). These results indicate that RDPV VLPs vaccine can protect raccoon dogs against RDPV RPSN infection, and 10 μg of RDPV VLPs is the lowest threshold for immune effectiveness. Changes in the number of white blood cells and lymphocytes also confirmed the effectiveness of the RDPV VLPs vaccine (Fig. 5g, h).

The dead raccoon dogs in group receiving PBS were dissected, and their organs were removed for further study. Animals in of group receiving 100 μg RDPV VLPs were euthanized for subsequent necropsy on day 11. Comparison of these two groups, changes in the physiological parameters of immunized raccoon dogs. Changes in the physiological parameters of immunized raccoon dogs upon virus challenge showed severe lesions except for the heart and kidneys (Fig. 6a, e). The results showed that RDPV RPSN may cause bleeding and necrosis in the liver and spleen, and at the same time, could also lead to enteritis (Fig. 6b, c, f). Additionally, the lobes of lungs were reddened and swollen, which may be due to hemorrhage in the surrounding lung (Fig. 6d). The H&E analysis of kidneys showed no microscopic lesions (Fig. 6o, p). However, other organs and tissues show several degrees of microscopic lesions. Of those, serious lesions were found throughout the intestinal tract. IHC evaluation was done for various organs (heart, liver, spleen, lung, kidney and intestinal tract) to check for the presence of virus in the tissues. The area density results from IHC showed proliferation of virus in tissues of heart, liver, spleen, lung, kidney, and intestinal tract in the group receiving PBS, while the tissues of the group receiving 100 μg VLPs showed almost no infection. The results also confirmed that IHC was able to identify parvovirus infection in tissues. These results also indicated that immunized animals were protected against tissue damage induced by RDPV RPSN.