A Multivalent Vaccine Containing Actinobacillus pleuropneumoniae and Mycoplasma hyopneumoniae Antigens Elicits Strong Immune Responses and Promising Protection in Pigs

Actinobacillus pleuropneumoniae (App) and Mycoplasma hyopneumoniae (Mhp) cause porcine pleuropneumonia and mycoplasmal pneumonia, respectively, and have serious impacts on the swine industry because they retard the growth of pigs. To protect pigs against these diseases, we have developed a multivalent vaccine consisting of App bacterins, APP RTX toxins (Apx toxins), and Mhp bacterin and adhesin protein. This vaccine induced the production of higher levels of antibodies against App and Mhp than the commercial vaccine (Nisseiken Swine APM Inactivated Vaccine). Furthermore, the vaccine efficiently protected pigs against virulent App challenge, showing promise as an efficient vaccine for the prevention of two important respiratory diseases, porcine pleuropneumonia and mycoplasmal pneumonia.

huge economic impacts in the swine industry 14 . Vaccination can effectively reduce its clinical signs and the severity of lung lesions, and is therefore widely used to prevent Mhp infection 13,15 . The current commercial vaccines against Mhp include bacterin vaccines, subunit vaccines, and combination vaccines 13,14 . Although these vaccines can reduce the severity of Mhp infection, vaccines that can confer better protective efficacy and reduce transmission are still required.
P97 protein is a surface adhesin that is highly conserved among strains of Mhp 16,17 . P97 is well characterized and its fragments have been expressed in Escherichia coli (E. coli) and evaluated as candidate vaccine components 13 . Recombinant repeat regions of P97 conferred good protection and represented a promising candidate for developing Mhp vaccines [18][19][20] .
Coinfection with App and Mhp exacerbates the severity of these respiratory diseases in pigs 21,22 , and results in more severe lung lesions and poorer growth. Furthermore, coinfection or sequential infection with App and Mhp is commonly detected in the field. Therefore, the control of App and Mhp coinfection is important for reducing economic losses.
I n t h i s s t u d y, w e p r o d u c e d a multicomponent vaccine containing App serotype 1, 2, 5 bacterins, Apx toxoids, inactivated Mhp, and recombinant P97 (rP97) from Mhp with the aim of reducing injection-induced stress in pigs. The immunogenicity and protective efficacy of the vaccine against virulent App challenge were evaluated and compared with those of a commercial vaccine. The results suggest that this vaccine is a promising candidate to protect pigs against App and Mhp.

Strains, growth conditions, and primers
App serotypes 1, 2, and 5 were isolated from the lungs of infected pigs in Korea and are available in the Laboratory of Infectious Diseases, Kangwon National University. App strains were grown in mycoplasma broth base (PPLO; Oxoid Ltd., UK) supplemented with 10 µg/mL of nicotinamide adenine dinucleotide (NAD; Merck, Germany) at 37°C and 5% CO 2 atmosphere. The primers used to identify App are listed in Table 1.
Mhp was isolated from a sample from an infected pig and was cultured in modified Friis medium 23 at 5% CO 2 and 37°C.

Preparation of inactivated App
Whole-cell, formalin-inactivated bacterins were prepared from App serotypes 1, 2, and 5 as described previously 24 . Briefly, bacteria were grown to reach the mid-exponential phase in PPLO containing 10 µg/mL NAD at 37°C. The bacteria were harvested by centrifugation (8000 x g for 20 min at 4°C) and the pellets were washed twice with phosphate-buffered saline (PBS). Formalin was added at 2% of the total volume and the mixtures were incubated for 4 h at 37°C with shaking at 180 rpm. After three washes with PBS, the inactivated bacteria were resuspended in PBS, subcultured to verify successful inactivation, and then frozen at -70°C until use.

Production of Apx toxoids
Apx I, Apx II, and ApxIII were produced, optimized, and purified from App serotypes 1, 2, and 5 as described in our previous study 25 . The purified Apx toxins were inactivated by 0.4% formalin 26 overnight at 4°C and the formalin was removed by ultrafiltration using Amicon Ultra-15 centrifugal filters with a 10-kDa molecular weight cutoff (Merck Millipore Ltd., Ireland). The collected toxoids were stored at -70°C until use. The purity and the concentration of the toxoids were estimated using SDS-PAGE as previously described 25 .

Preparation of inactivated Mhp
The Mhp vaccine strain HID3140 was cultured in modified Friis medium 23 for 10 days at 37°C and 5% CO 2 until it reached 5 x 10 8 colorchanging units (CCU). Mhp was inactivated as described by Matthijs et al. 23 . Briefly, binary ethyleneimine (BEI) was added to the liquid culture and incubated at 37°C for 24 h with shaking set to 100 rpm. Then, BEI was neutralized by adding sodium thiosulfate (Sigma Aldrich, USA) and cultures were incubated for 24 h with shaking (100 rpm). Subcultures of the inactivated Mhp were incubated for one week to confirm the inactivation.

Construction and expression of rP97 in E. coli
The plasmid pET-DEST42 containing the fusion gene LTB and R1R2 of P97 was previously constructed in our lab 18 and transformed into E. coli BL21. The rP97 was expressed and purified from E. coli lysate using HisPur Ni-NTA affinity chromatography (Thermo Fisher Scientific, USA) 18 . The concentration of the harvested protein P97 was measured using the Pierce BCA protein assay kit.
Eighteen 3-week-old pigs of both sexes were used in this study. All pigs were negative for App antibodies (IDEXX APP-ApxIVAb Test, IDEXX Laboratories Inc., USA), and Mhp antibodies (Mycoplasma hyopneumoniae Antibody Test Kit, IDEXX Laboratories Inc., USA). The 18 pigs were randomly separated into three groups, as shown in Table 2. Pigs in group 1 were immunized intramuscularly with the test vaccine twice over a 3-week interval. Pigs in group 2 were vaccinated intramuscularly twice with a commercial vaccine as per the manufacturer's instructions. The commercial vaccine used in this study (Nisseiken Swine APM Inactivated Vaccine; Nisseiken Co. Ltd., Japan) contains 1 x 10 8 CFU inactivated Mhp, 5 x 10 9 CFU for each inactivated App serotype 1, 2, and 5, and recombinant proteins ApxI, II, and III (80-120 µg). Pigs in group 3 were injected with PBS formulated with adjuvant as a negative control group.
The experimental design is shown in Fig. 1. Blood samples were taken at 0, 1, 3, 5, and 6 weeks after the first immunization for examination of antibodies. At 2 weeks after the second vaccination, two pigs from each group were sacrificed and their lungs were subjected to examination for histopathology. The remaining pigs were challenged intratracheally as previously described 2,11 with 1 x 10 5 CFU of each wild-type App serotypes 1, 2, and 5. All pigs were sacrificed at 7 days postchallenge, and their lungs were collected for isolation of the challenge Apps and determination of their lung lesion score, as described by Inzana et al. 27 . Briefly, a score of 0 indicates healthy lung; 1, < 25% lung involvement with localized congestion only; 2, 25-50% lung Journal of Pure and Applied Microbiology involvement with consolidation or congestion only and no necrosis or pleural lesions; 3, 51-75% lung involvement with some consolidation, congestion, mild pleural lesions, and congestion; 4, 76-100% lung involvement with pneumonia and consolidation, extensive pleural adhesions, and necrotic lesions.

Isolation of App from challenged pigs
To determine the presence of the challenge Apps, the cranial lobe and the portions of lung that exhibited lesions were seared superficially, incised, sampled with a sterile cotton swab, and then streaked on a BHI agar plate containing 10 µg/mL NAD. After 24 h of incubation at 37°C and 5% CO 2 , the colonies were tested for NAD dependence and satellitism. The identity of suspected colonies was confirmed by nested polymerase chain reaction (PCR) 28 . Briefly, 5 µL of the suspended colony was mixed with the PCR premix containing 2 x Master Mix (Enzynomics, Korea), and 10 pmol of the primers listed in Table 1. The first PCR was carried out using primers P1, P2, and the nested PCR was carried out using primers P3, P4. Thermal cycling conditions included initial denaturation at 95°C for 5 min; followed by 35 cycles of 95°C for 1 min, 52°C for 1 min, 72°C for 1 min; and a final extension at 72°C for 10 min 28 .

Enzyme-linked immunosorbent assay (ELISA)
A Mhp antibody test kit (IDEXX Laboratories Inc., Westbrook, USA) was used to evaluate antibody production against Mhp in sera from the pigs. In addition, purified rP97 was coated on wells at 10 ng per well to detect specific antibodies against rP97.  To evaluate the levels of antibodies against App, the 42-kDa outer membrane protein (OMP) was extracted 29 and 100 ng of the purified OMP was coated onto each well for ELISA. Plates coated with a mixture of 10 ng of the purified Apx toxins from each App serotypes, were used to detect Apx-specific antibodies.
The antigens were incubated in 100 µL coating buffer overnight at 4°C using ELISA plates (Nunc, Denmark). After washing three times with 200 µL of PBS containing 0.05% Tween 20 (PBST; Merck, USA) and blocking for 2 h with 1% bovine serum albumin (Merck, USA) in PBST, 1/1,000 dilutions of individual sera were added and incubated for 2 h at 37°C. This was followed by three washes, then the horseradish peroxidase-conjugated secondary antibodies (1/5,000) (Goat anti-pig IgG H&L; Abcam, UK) were incubated for 1 h. Then the plates were washed with PBST, and incubated with 3,3′,5,5′-tetramethylbenzidine substrate (Abcam, UK) for 3 min at room temperature. After adding 2N H 2 SO 4 , the absorbance was measured at 450 nm using an EPOCH reader (BioTek Instruments, Inc., USA).

Statistical analysis
Statistical analysis and preparation of graphs were performed using GraphPad Prism v6 software (GraphPad Software, USA). An analysis of variance (ANOVA) was used to evaluate the differences between experimental groups.

Induction of antibodies against Mhp
To evaluate the immunogenicity of the test vaccine against Mhp, the levels of antibodies against Mhp or P97 were determined using the commercial ELISA kit or an in-house P97-based indirect ELISA.
Analysis of the anti-Mhp IgG levels in the sera demonstrated that the pigs immunized with the test vaccine generated significantly higher antibody levels than the negative control group at 3 weeks (p= 0.014) and 5 weeks (p < 0.001) after the first vaccination ( Fig. 2.A). Furthermore, the antibody titer against Mhp in the test vaccine group was 2.6 times higher than that in the commercial vaccine group, and this difference was significant (p < 0.001) (Fig. 2.A).
Significant levels of P97-specific antibodies (p < 0.001) were observed in the serum samples from the test vaccine group, while they were not detectable in the sera of pigs immunized with the commercial vaccine or in pigs in the negative control group at 3 and 5 weeks after the first vaccination (Fig. 2.B). These results illustrate that the test vaccine showed good ability to induce production of antibodies against Mhp.

Induction of antibodies against App
The 42 kDa OMP is predominant and is conserved among App serotypes 30,31 . Therefore, App OMP was used as a coating antigen in ELISA to evaluate the antibody levels against App as a previous report 32 and in the commercial kits (VDProAPP2 AB ELISA and VDProAPP5 AB ELISA; Median Diagnostics Inc., Korea). The levels of OMPspecific antibodies were increased dramatically in the sera of pigs vaccinated with the test vaccine and the commercial vaccine at 2 weeks after the second vaccination and remained at a high level at one week post challenge (Fig. 3.A). In addition, significantly higher levels of IgG against OMP were observed in the test vaccine group than in the commercial vaccine group at these time points (p< 0.001 and p= 0.018, respectively) (Fig. 3.A).
The same pattern was seen for the levels of IgG against Apx toxins in the test vaccine group (Fig. 3.B), which were significantly higher than those in the commercial vaccine and negative control groups (p < 0.001). In contrast, there was no significant difference between the antibody levels in the commercial vaccine and negative control groups. These results demonstrated that the test vaccine could induce better production of antibodies against App and Apx toxins than the commercial vaccine.

Protective efficacy of vaccine against App challenge
To evaluate the protection against virulent Apps provided by the test vaccine, pigs in all three groups were challenged with virulent App serotypes 1, 2, and 5. The lung lesion scores of the challenged pigs are shown in Fig. 4 and 5. Nonchallenged pigs in all groups showed no lung lesions. The test vaccine group showed lower lung lesion scores than the commercial vaccine and negative control groups with mean scores of 0.6 ± 0.5, 1.5 ± 0.7, and 2.4 ± 0.5, respectively. A significant difference in lung lesion scores was observed between the test vaccine group and the negative control group (p= 0.002). No significant difference in lung lesion scores was detected between the commercial vaccine and negative control groups (p= 0.196).
The protection provided by the vaccines was also assessed by the recovery of App from challenged pigs as shown in Table 2. The recovery rates from the lungs of pigs in the test vaccine, the commercial vaccine, and the negative control groups challenged with Apps were 20% (1/5), 100% (2/2), and 100% (5/5), respectively.

DisCUssiON
Coinfection with Mhp and App is very frequent in swine farms 13 . These diseases cause more severe lesions in pigs than single infections and lead to major economic losses in the swine industry 13 . Therefore, there is a strong demand for the development of multivalent vaccines against Mhp and App, because these reduce the number of vaccinations and hence reduce the high labor costs of the vaccinations and the level of injectioninduced stress in pigs. To address this demand, in this study, we developed a promising multidisease vaccine and compared the immune responses and the protective efficacy it induced against Mhp and App infections in pigs with those induced by a commercial vaccine. To our knowledge, this is the first reported development of a multivalent vaccine comprising a new formulation of Mhp bacterin, P97, App serotypes 1, 2, 5, and ApxI, II, and III toxoids.
A previous report has demonstrated that several of the OMPs from App, which are present in App bacterins, are conserved across all serotypes 33 . A vaccine containing a combination of these proteins and Apx toxoids can protect challenged pigs against lung lesion formation The test vaccine developed in the present study effectively stimulated the production of antibodies against 42 kDa OMP and Apx toxins. The antibody levels were significantly higher than those induced by the commercial vaccine and in the negative control group at 5 weeks after the first vaccination and at one week post challenge (Fig. 3.A and B). This may be because in the test vaccine, the Apx toxoids were extracted directly from cultures of App, while the Apx proteins in the commercial vaccine were expressed in and purified from E. coli. The immune responses and protective efficacy induced by the recombinant Apx toxins and the inactivated native Apx toxins were also studied. Mei et al. (2006) reported that higher antibody levels were induced by native ApxI toxoids than by recombinant full-length ApxIA 3 . Yan et al. (2007) also reported higher antibody titers and a higher protection rate in mice injected with native ApxII compared with recombinant ApxIIA 35 . In our study, we observed significantly higher levels of antibodies against Apx toxins in pigs immunized with the test vaccine than in those immunized with the commercial vaccine. Our results are consistent with those of previous studies in which native Apx toxoids induced a better immune response and protection than recombinant Apx toxins.
The formulation, induction of immune responses, and protective efficacy of the Mhp vaccine were evaluated and reported in our previous work 36 . The Mhp vaccine completely protected vaccinated pigs against Mhp challenge. Thus, a challenge experiment with Mhp was not conducted in this study. In this study, the antibody levels against Mhp and rP97 produced in the test vaccine pigs were significantly higher than those in the other groups at 3 and 5 weeks after the first vaccination ( Fig. 2.A and B). These results suggest that the test vaccine could effectively induce the production of antibodies against both App and Mhp.
The protective efficacy of the test vaccine was evaluated by challenge with virulent App. The test vaccine provided better protection for challenged pigs as assessed by lung lesion scores ( Fig. 4 and 5) and the rate of recovery of the challenge App from the lung tissues of pigs in each group ( Table 2). The higher specific antibody levels in the test vaccine-immunized pigs may have contributed to the lower lung lesion scores in this group. The highest rates of recovery of the challenge App were recorded in the negative control group and the commercial vaccine group. This result implied that the test vaccine could more effectively protect pigs and eliminate the challenge App bacteria than the commercial vaccine. Further studies are required to assess the protective efficacy of this vaccine against challenge with highly virulent Mhp.
In conclusion, this multicomponent vaccine induced relatively high levels of antibodies against both App and Mhp, and could effectively protect pigs against App challenge, indicating that it could be a candidate vaccine for prevention of these two economically important respiratory diseases.