The Promotion of Peony Seed Proteolysis Product to the Immune Efficacy Induced by ptfA Gene DNA Vaccine of Avian Pasteurella multocida

Fowl cholera, caused by avian Pasteurella multocida is a contagious disease of poultry and fowl, particularly layer and parent breeders because of its economic impact on the breeding industry around the world (Jonathan et al., 2012; Yu et al., 2013; Pushpa et al., 2017). Currently available vaccine to prevent this disease is mainly attenuated vaccine. However, the antigenic structure of avian P. multocida is complicated and prone to mutation. Therefore, the protective efficacy of live attenuated vaccine is not very high. The immunoprotection of attenuated vaccine is not long enough, the protective efficacy is not high enough and has the possibility of excretion of virion (Mariana and Hirst, 2000). Therefore, it is necessary to develop novel and more effective vaccines for controlling this infectious disease.
       
Compared to traditional vaccines, genetically engineered vaccines represent a new generation of vaccines that include DNA vaccines, recombinant subunit vaccines, etc. Among these, DNA vaccines have become a bellwether in the field of vaccine research because of their advantages. There are several reports of DNA vaccines developed against P. multocida (Ahmad et al., 2014). However, the effectiveness of most DNA vaccines are not necessarily superior to traditional vaccines. In previous study, we prepared a chitosan nanoparticle DNA vaccine based on the ptfA gene of P. multocida and determined its protective efficacy. Although the DNA vaccine could provide a certain level of protection in experimental animals, it did not exceed the level of protection conferred by live attenuated vaccine (Gong et al., 2018). Therefore, it is necessary to select new adjuvant to improve the immune efficacy of DNA vaccines against avian P. multocida.
       
Peony is a perennial woody plant belonging to Paeonia family. It is one of the traditional famous flowers in China, and has the reputation of “king of flowers” (Shi et al., 2015; Guo et al., 2017). It not only has a high ornamental value, but also has certain medicinal and edible value. For example, the peony flower contains astragalin and flavonoids, the peony leaf contains polysaccharides and peony anthocyanin and the peony root bark contains paeonol and other medical and health care ingredients (Fan et al., 2012; Guo et al., 2017). The peony seed is rich in protein, monoterpene glycosides, linoleic acid and other unsaturated fatty acids, so it has high utilization potential (Liu et al., 2017; Liu et al., 2018a; Deng et al., 2018; Liu et al., 2018b). In this study, the immune response and protective efficacy of the ptfA DNA vaccine of avian P. multocida with digested protein extracted from penoy seed as adjuvant was evaluated in vaccinated chickens. The goal of this baseline work is to perform further advanced research for development of safe and effective vaccine against avian pasteurellosis and novel vaccine adjuvant.

Bacterial strains, vaccine and chickens
 
Avian P. multocida (CVCC474, serotype A:1) was purchased from Chinese Institute of Veterinary Drug Control (IVDC). Attenuated live vaccine was purchased from Qilu Animal Health Products Co., Ltd. Peony seed meal were conserved in the Laboratory of HeNan University of Science and Technology in China. The recombinant protein PtfA (rPtfA) protein was prepared according to the recommended method (Gong et al., 2016). Healthy day old broilers were purchased from the Animal Center Laboratory of College of Medical Technology and Engineering of Henan University of Science and Technology, China and were maintained and handled using procedures consistent with regulations for experimental animals in China. The study protocol was approved by the Animal Monitoring Committee of Henan University of Science and Technology (Permit Number 2019-0006; 20 February 2019). The research was carried out from 2019-02 to 2019-09 in Henan University of Science and Technology, China.
 
Extraction of peony seed protein
 
After crushing, peony seed meal was added with distilled water at the ratio of 1:25. Sodium hydroxide (NaOH) solution was used to adjust the pH value at 9.25 and heated in a water bath at 53^oC for 68 minutes. Then the supernatant was absorbed after high speed centrifugation and the pH was adjusted to 3.5 with HCL. The precipitate was obtained after high-speed centrifugation. Then the precipitate was freeze-dried to obtain a peony seed protein powder, Protein content was determined by the Coomassie Brilliant Dye binding method of Bradford and stored frozen for use.
 
Preparation of peony seed proteolysis product
 
The peony seed protein powder was dissolved in deionized water and preheated in a water bath at 80^oC for 20 min, then adjusted the pH to at 6.5. Subsequently, the bromelain was added and the mixture was placed in a 48^oC water bath for 4 h. After the reaction, the mixture was inactivated in boiling water for 10 min, then rapidly cooled in an ice bath and centrifuged at 10,000 r/min for 10 min. The supernatant was concentrated in vacuum and then lyophilized to obtain peony seed proteolysis product, which was stored at -20^oC for reserve.

Vaccination protocol
 
The ptfA-DNA vaccine was constructed and prepared in a large scale production, as previously described (Gong et al., 2018). Prior to vaccination, healthy 1-day old chickens (n = 150) were reared in a purpose-built animal house where natural light, temperature and humidity were controlled. After adaptation to the new feeding environment, chickens were assigned to six groups (n= 25 chickens/group) namely DNA vaccine group, peony seed proteolysis product adjuvant-DNA vaccine group (abbreviated as adjuvant-DNA vaccine group), peony seed proteolysis product gavage group (abbreviated as gavage group), live attenuated vaccine group, pcDNA3.1 (+) group and normal saline control group. Gavage was performed at 4 weeks of age. Before the gavage, peony seed proteolysis product was dissolved in normal saline at the concentration of 5 mg/mL. Chickens in the gavage group was gavaged with above solution at the dose of 0.2 mL/chicken per day and continued for two weeks. During the gavage, chickens in other groups were given normal feed. Vaccination was performed at 6 weeks of age by IM injection. Chickens in the DNA vaccine group and the gavage group were injected with 200 µL of recombinant plasmid pcP suspension, containing 200µg of DNA. Chickens in the adjuvant-DNA vaccine group were vaccinated with 200 µL of DNA suspension, which contained 200µg of plasmid pcP and 25% of peony seed proteolysis product. Chickens in the vector pcDNA3.1 (+) group and normal saline control group were given 200 µL (1 µg/µL) empty vector pcDNA3.1(+) suspension and 200 µL of sterile normal saline respectively. Chickens in the attenuated live vaccine group were vaccinated with 0.5 mL P. multocida attenuated live vaccine. All chickens were vaccinated three times at 2-week intervals (dose?). Following gavage and each round of vaccination, chickens were closely observed for any adverse reactions. Any chicken that presented signs of depression, lack of appetite other clinical signs of illness, were isolated to a quiet feeding environment and fed more palatable feed until they recovered.
 
Detection of serum antibody
 
Following the first vaccination, blood samples were collected from the wing veins weekly for 6 weeks prior to challenge. Then, serum antibodies were detected using an indirect enzyme-linked immunosorbent assay (ELISA), with the rPtfA protein of avian P. multocida and 108cfu/mL of avian P. multocida suspensionas a coating antigen and rabbit anti-chicken IgG-horseradish peroxidase as a second antibody, according to the previously published method (Gong et al., 2018).
 
Peripheral bood lymphocyte proliferation assay and cytokine secretion test
 
Two weeks after each vaccination, blood samples were collected from vaccinated chickens. Lymphocytes were then separated from the peripheral blood and the concentration was adjusted to 2×10⁷ cells/mL. A volume of 50 µL of cell suspension was seeded into a 96-well culture plate. Each well was pulsed with 50 µL of 20 μg/mL P. multocida rPtfA protein (experimental well) or a 50 µL culture medium (negative control). The plates were incubated at 37oC under 5% CO₂ for 72 h, followed by the addition of 10 µL 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cultured for 3 h. After centrifugation for 10 min, the supernatant was discarded and 150 µL of dimethyl sulfoxide (DMSO) added. The plate was then incubated for 10 min until all crystals had dissolved. The A value of each well was measured at 570 nm. Stimulation index (SI) values were estimated using the following equation: SI = A (experimental well)/A (negative control well). At the same time, peripheral blood lymphocytes, activated by the rPtfA protein of P.  multocida, were prepared as described above. The cells were cultured at 37oC under 5% CO₂ for 72 h and the supernatants harvested.Then the IFN-γ, IL-2, IL-4 and IL-6 concentrations secreted by the PBL was measured.
 
Challenge experiment
 
Two weeks after the third vaccination, all chickens were challenged with the virulent avian P. multocida strain CVCC474 (5 LD₅₀/chicken) by intramuscular injection. Following challenge, chickens were reared for further 14 days. Chickens were closely observed for clinical signs of illness every day post-challenge. Chickens showing signs of depression and/or inappetance were isolated and kept under further observation. Chickens that were anorexic, or dyspneic and those with hemorrhagic diarrhea or other abnormal gastro-intestinal signs, were withdrawn from the experiment and euthanized by intravenous injection of pentobarbital sodium. Survival time and survival number of each group were calculated.

Serum antibody concentrations
 
The humoral immune response is an important factor in the protection against avian P. multocida infection. In this study, we detect the levels of antibodies were detected in vaccinated chickens. The results showed serum antibodies in the DNA vaccine group, adjuvant-DNA vaccine group, gavage group and attenuated live vaccine group increased following vaccination (Fig 1). From 3 weeks after the first vaccination, antibodies detected in the DNA vaccine group, adjuvant-DNA vaccine group and gavage group were higher than those detected in the attenuated live vaccine group (P<0.05) when the rPtfA protein was adopted as coating antigen (Fig 1A). On the contrary, from 2 weeks after the first vaccination, antibodies detected in the attenuated live vaccine group were higher than those detected in the DNA vaccine group, adjuvant-DNA vaccine group and gavage group (P<0.05) when the coating antigen was avian P. multocida suspension (Fig 1B). No significant differences in antibody levels among the DNA vaccine group, adjuvant-DNA vaccine group and gavage group were observed, regardless of whether the coating antigen was the rPtfA protein or the avian P. multocida suspension. After vaccination with the DNA vaccines, chickens mainly produced antibodies against the PtfA protein of avian P. multocida because there was only one foreign gene in the ptfA gene DNA vaccine. However, chickens vaccinated with the attenuated live vaccine produced antibodies against a variety of P. multocida antigens. This may be the reason why the antibody concentrations detected in the DNA vaccine group, adjuvant-DNA vaccine group and gavage group were higher than those in the attenuated live vaccine group when the rPtfA protein was used as a coating antigen, and vice versa. These results indicated that the antibodies induced by the ptfA gene DNA vaccine could not be enhanced when peony seed proteolysis product was used as adjuvant or was gavaged to chickens before vaccination.
 

 
 
Lymphocyte proliferation assay
 
DNA vaccines can induce effective cellular immune responses.The lymphocyte proliferation and cytokine secretion levels are common indexes that were used to evaluate cellular immune function (Stone et al., 2009; Shebannavar et al., 2010). To investigate the cellular immune response induced by the DNA vaccines, an MTT assay was performed to assess the proliferation of peripheral blood lymphocytes two weeks after the each vaccination, and the results are shown in Fig 2. After the first vaccination, there were no difference among the four vaccine groups. Following the second vaccination, the SI value in the attenuated live vaccine group was significantly higher than those in the DNA vaccine group, adjuvant-DNA vaccine group and gavage group (P<0.05) and no difference among the latter three groups was detected. After the third vaccination, the SI value in the attenuated live vaccine group was significantly higher than those in the adjuvant-DNA vaccine group and gavage group (P<0.05). Again, the SI values in the adjuvant-DNA vaccine group and gavage group were higher than those in the DNA vaccine group after the third vaccination. The results indicated when peony seed proteolysis product was used as adjuvant or was gavaged before vaccination, the proliferation of peripheral blood lymphocytes induced by DNA vaccine could be promoted to a certain extent.
 

 
Cytokine assay results
 
In this study, the concentrations of Th1 cytokines IFN-γ, IL-2 and the concentrations of Th2 cytokine IL-4 and IL-6 secreted by lymphocyte of the vaccinated chickens were detected and the results were shown as Fig 3. After the first vaccination, no significant differences were detected in the concentrations of the four cytokines among the four vaccine groups. After the second and third vaccination, the concentrations of the four cytokines detected in the attenuated live vaccine group were higher than other three vaccine groups (P<0.05). Additional, the concentrations of IFN-γ and IL-2 in the adjuvant-DNA vaccine group and gavage group were higher than those in the DNA vaccine group after the second and third vaccination (P<0.05) (Fig 3A, 3B). No significant differences in the concentrations of IL-4 among the adjuvant-DNA vaccine group, gavage group and DNA vaccine group were observed throughout the period of study (Fig 3C). Following the third vaccination, the concentrations of IL-6 in the adjuvant-DNA vaccine group and gavage group were higher than those in the DNA vaccine group (P<0.05), but no significant differences were found among them after the second vaccination (Fig 3D). Thus, it can be seen that the attenuated live vaccine could induce good Th1 and Th2 responses. The peony seed proteolysis product could promote Th1 response induced by the ptfA gene DNA vaccine to some degree, accompanied by weaker effect on the Th2 response.
 

 
Challenge study
 
Challenge experiments are one of the important indices used to evaluate the protective efficacy of vaccines. In the present study, the experimental groups of chickens were challenged with the live virulent avian P. multocida culture (strain CVCC474) two weeks following the last vaccination. The number of surviving chickens was counted every day till day 14 post-challenge. Chickens in the pcDNA3.1(+) group and normal saline group began to die from the first day onward after the challenge. None of the chickens in the two control groups survived more than 5 days after the challenge. Chickens in the attenuated live vaccine group began to die from the 3^rd day and the number of surviving chickens remained unchanged from the 4^th day onward. Chickens died from the 2^nd day in the DNA vaccine group, adjuvant-DNA vaccine group and gavage group till 14 days after the challenge. The survival numbers in these three groups were 13, 18 and 15, respectively. The protective efficacy of attenuated live vaccine, DNA vaccine, adjuvant-DNA vaccine and gavage-DNA vaccine were 92%, 52%, 72% and 60%. respectively (Fig 4). The results of challenge experiment demonstrated that peony seed proteolysis product could enhance the immune response induced by a ptfA-DNA vaccine in chickens, especially when it was used as DNA vaccine adjuvant. However, the protective efficiency provided by adjuvant-DNA vaccine still inferior to that provided by the attenuated live vaccine. Therefore, further measures should be taken to improve the immune efficacy of peony seed proteolysis product adjuvant DNA vaccine such as optimizing the dosage of seed proteolysis product adjuvant, optimizing the inoculation method.
 

Peony seed proteolysis product was obtained from peony seed protein digested with bromelain and was added to the ptfA-DNA vaccine as adjuvant or was gavaged to chickens before DNA vaccination. The peony seed proteolysis product could not enhance the humoral immune response induced by the ptfA gene DNA vaccine. However, it could promote Th1 response to some degree, accompanied by weaker effect on the Th2 response. In addition, peony seed proteolysis product could improve the protective efficiency of ptfA gene DNA vaccine, especially when it was adopted as DNA vaccine adjuvant.