Introduction
Raccoon dog parvovirus (RDPV) is responsible for causing an acute, highly contagious, infectious viral disease, which may lead to fatal hemorrhagic enteritis in raccoon dogs of all ages [
24,
30]. Inactivated virus vaccines have been the mainstay of the classical vaccine strategy for RDPV, which despite their higher efficacy suffer from the limitation of incomplete inactivation, thus posing a challenge in vaccine development [
1]. The amount of formaldehyde used in the production process of inactivated vaccines often exceeds the safety standards causing concerns during use. This is further complicated by the fact that at present there is no parvovirus enteritis vaccine dedicated to raccoon dogs in the market. Therefore, it is imperative to develop a safe alternative vaccine dedicated to this disease [
7].
RDPV is an icosahedral non-enveloped virus belonging to the Parvoviridae family. It consists of a single strand of 20–25 nm DNA, two non-capsid proteins NS1, NS2, and three capsid proteins VP1, VP2 and VP3 [
27]. The VP2 protein is found amongst all of the immunogenic epitopes and is capable of generating specific antibody response [
12].
Virus-like particles (VLPs) spontaneously assembled by VP2 protein can mimic the three-dimensional structure of natural viruses. VLPs determine the type of antigen and host range, and can stimulate the immune response mediated by B cells. NS gene is known to be essential for replication, and VP gene encodes various forms of the structural protein. As the main capsid protein of the virus, VP2 can self-assemble into VLPs. [
5,
9,
15].
The trigger factor (Tf) is a protein with a size of about 50 ku, which is an important member of the
Escherichia coli molecular chaperones. Tf transiently attaches to a point on the ribosome, forming a protective area and restricting the access of proteases and other downstream factors to the nascent polypeptide chain. This helps in preventing the newly synthesized polypeptide chain from aggregating during folding [
10]. Previous studies have shown that several VP2 proteins could be co-expressed in soluble form with Tf using the Prokaryotic expression system [
15,
26,
28].
In the present work, we studied the co-expression of VP2 protein and Tf16 using recombinant plasmid pET30 in E. coli. A novel attempt was made to purify the RDPV VP2 protein and its self-assembly into VLPs was also studied. Further, the immune efficacy of RDPV VLPs vaccine was evaluated in vivo.
Materials and methods
Virus and cells
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.
Plasmid construction
For optimization of expression of RDPV VP2 protein, the sequence of RDPV VP2 was optimized based on E. coli. The complete RDPV VP2 gene was restriction digested by HindIII, NdeI (TaKaRa, China), and cloned into pET30a (pET30a-VP2) by T4 DNA ligase (TaKaRa, China).
RDPV VP2 protein expression and co-expression
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.
Purification of RDPV VP2 protein
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 VLPs self-assembly and characterization
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.
Raccoon dog immunization with RDPV VLPs
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.
Changes in the physiological parameters of immunized raccoon dogs upon virus challenge
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 evaluation of raccoon dogs post-inoculation
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.
Ethics approval and consent to participate
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.
Discussion
At present, the inactivated vaccine is the only strategy available to fight RDPV disease. However, the major drawback of this approach is the use of formaldehyde as an inactivating agent. It is found to be toxic to raccoon dogs and causes local inflammation at the site of inoculation, which in some cases may lead to suppuration, and ulceration [
6,
7]. A recent approach has been the development of VLP vaccines which has received widespread attention. VLPs mimic virus particles and present considerable epitopes and are, therefore, capable of stimulating multiple immune responses [
17]. Previous studies have shown that a variety of VLPs protein could be expressed in various expression systems, such as mammalian, insects, yeast, and
E. coli [
4,
12,
14,
16,
20,
29]. To the best of our knowledge, there are no reports on the expression of RDPV VP2 till date. Alignment studies of RDPV VP2 genes and CPV VP2 genes have shown 99.9% identity at the nucleotide level, which indicated a very high degree of sequence homology [
30]. Previous researchers have successfully reported the co-expression of CPV VP2 with Tf16 in
E. coli [
26]. Working on similar lines, an attempt was made to co-express RDPV VLPs and Tf16 in
E. coli. The soluble RDPV VP2 expressed was found to induce a strong immune response in raccoon dogs. This is one of the pioneer reports studying the expression of RDPV VP2, and also evaluating the protective effect of drugs through HE staining and IHC.
Escherichia coli has significant advantages in expressing recombinant proteins, such as high expression and low cost. However, due to the highly toxic effects of
E. coli endotoxin, contamination of the vaccination with it may cause body temperature to rise, disturb metabolic function, trigger the coagulation cascade and induce shock. Therefore, complete removal of endotoxin is a crucial step in the manufacturing process [
23,
25]. Preliminary studies had suggested several methods for endotoxin removal such as the use of an organic solvent (1-octanol), CsCl density gradient centrifugation, anion exchange chromatography, detergent extraction (Triton X-100, Triton X-114), nickel affinity column chromatography, and use of Polymyxin B-immobilized cartridge [
3,
8,
22,
31]. Considering the need for large-scale production of RDPV VP2 protein in the future, we attempted to standardize a new, efficient and cost-effective method for endotoxin removal. Following a single-cycle Triton X-114 extraction, the concentration of endotoxin in RDPV VP2 protein was reduced by more than 92%. It is being suggested that subsequent phase extraction cycles would further reduce the contamination with endotoxin (Table
1). These data are consistent with previous reports which have shown the efficacy of Triton X-114 extraction to reduce endotoxin concentration in expressed VP2 protein [
28].
Per the previous reports that state that VP2 protein could be purified by density gradient ultracentrifugation [
13,
19], a similar approach has been taken to purify the VLPs in our study. Hydrophobic chromatography used to purify VLPs has shown better recovery
vis density gradient ultracentrifugation. Salt concentration and pH are known factors that affect the formation and stability of VLP [
21]. Our experiments also indicated that RDPV VP2 protein could self-assemble to VLPs at pH 8.0 and 250 mM NaCl, which had a similar size, shape and a high HA titer (1:2
18) to RDPV.
To assess the immunogenicity of RDPV VLPs, these were inoculated intramuscularly to the raccoon dogs. In contrast to other studies which have reported high titers of HI antibodies after two doses of immunization [
15,
26], we observed the presence of high titers of HI antibody (1:4096) against RDPV after a single injection of 100 μg VLP. An immune response was observed in all the groups in groups receiving either 10 μg, 50 μg, 100 μg RDPV VLP or inactivated vaccine. No side-effects were observed in any of the groups as compared to controls, indicating the safety of RDPV VLP vaccine and implying that the above preparation process may be used for large-scale preparation of RDPV VP2. Further, the animals were challenged with the virus to evaluate the immune protection offered by the VLP vaccine.
The mechanism of VLP vaccine in inducing proliferation of CD4 + T cells and CD8 + T cells in mice has been well documented [
9,
29]. VLP vaccines are recognized and engulfed by antigen-presenting cells in a manner similar to that of a viral infection. The vaccine can also directly stimulate dendritic cells, promote dendritic cells to produce pro-inflammatory factors, and induce cellular immunity [
11]. Studies have also extensively documented that VLPs mainly stimulate T-helper type 1 (Th1) response in guinea pigs [
15,
26]. Owing to these consistencies, similar analyses were not attempted in the present work.
We have found that the incubation period of RDPV is approximately 4 days, the clinical symptoms are most obvious in 7–8 days, and the symptoms begin to weaken gradually in 9–10 days. Listlessness, loss of appetite, loose stools are the common symptoms of the struck raccoon dogs. HA test reached detectable titers in 10 μg, PBS after 4 days. CPV test strips indicated that raccoon dogs could excrete virus through the stools after 4 days. A complete blood count showed a significant reduction in the numbers of both white blood cells and lymphocytes in the raccoon dog. This is in accordance with the recent study which has reported the decrease in WBC count upon challenge with RDPV [
7]. We found a rapid increase in the HI antibody titers upon challenge test, which may indicate the involvement of memory B cells. Interestingly, the raccoon dogs in the group with 10 μg RDPV not only showed clinical symptoms and white blood cell changes, but also better survival as compared to control. Cumulatively, these results indicated that 10 μg of RDPV VLPs may be the lowest threshold for immune effectiveness.
Study shows that CPV causes hemorrhagic gastroenteritis in adult dogs and myocarditis in puppies [
2]. We found the presence of lesions in various tissues including heart, lungs, liver, spleen and intestinal tract as examined by H&E. RDPV RPSN mainly targeting the intestinal tract. IHC results confirmed this as RDPV RPSN could be detected in 6 types of tissue, suggesting that RDPV RPSN was not only present but also able to replicate in these tissues. This suggests that RDPV was a pantropic virus, which could replicate in heart, liver, spleen, lung, kidney, intestinal tract. Since H&E staining can detect histological lesions, but may not detect parvovirus infection, thus, a combination of H&E staining and IHC can more clearly identify a parvovirus infection. These results are also consistent with findings in another study [
18]. Sick raccoon dogs having obvious clinical symptoms show damage to multiple organs in a short period of time, and the mortality rate is extremely high. Therefore, the study needs to be replicated with a similar design, focusing on the identification of parvovirus infection.
In a nutshell, we have attempted to develop an effective method for preparing RDPV VP2 protein, which includes co-expression with Tf16 in E. coli, followed by purification, endotoxin removal and self-assembly. RDPV VP2 can assemble into a VLP similar to natural RDPV. Animal experiments have shown that VLP can stimulate the long-term immune response of raccoon dogs to RDPV, and no obvious side effects have been observed in the safety test of raccoon dogs. In vivo experiments also showed that immunization with RDPV VLP significantly lowered blood viral load upon subsequent challenge by the virus. The RDPV VLPs vaccine caused a higher specific humoral immune response against RDPV, and 10 g RDPV VLPs would be the lowest threshold of immune effect. In addition, this research test also shows that H&E staining and IHC can more clearly recognize parvovirus infection. This study provides new insights into the pathogenesis and clinical characterization of RDPV. RDPV VLPs vaccine has the potential to be exploited as a new commercial vaccine candidate against RDPV infection. This study also verified that the RDPV VLPs expressed by E. coli system may become a safe, effective, and economic candidate vaccine.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.