Department of Veterinary Medicine, College of Veterinary Medicine & Institute of Veterinary Science, Kangwon National University, Chuncheon, Republic of Korea
Correspondence: Tae-Wook Hahn, College of Veterinary Medicine & Institute of Veterinary Science, Kangwon National University, 192–1 Hyoja-dong, Chuncheon, Gangwon-do, Republic of Korea, 200–701. Tel.: + 82 33 2508671;
The surface adhesin P97 mediates the adherence of Mycoplasma hyopneumoniae to swine cilia. Two reiterated repeats R1 and R2 are located at the C-terminus of P97. The purpose of this study was to evaluate the immunogenicity of Montanide adjuvant IMS 1113 plus soluble subunit proteins rR1, rR1R2 and their chimeric forms coupled with B subunit of the heat-labile enterotoxin of Escherichia coli (LTB). Each recombinant protein in this study was capable of eliciting anti-R1 specific humoral antibodies (IgG), mucosal antibodies (IgG and IgA) and IFN-γ production. The chimeric protein rLTBR1R2 elicited the quickest humoral antibody response among the recombinant proteins. Serum and bronchoalveolar lavage analysis revealed that each recombinant protein was capable of inducing both Th1 and Th2 responses. Importantly, all of the proteins induced an anti-R1-specific Th2-biased response in both humoral and mucosal compartments, similar to the response observed in a natural infection or vaccination process. These observations indicate that rR1, rR1R2, rLTBR1 and rLTBR1R2 with IMS 1113 might represent a promising subunit vaccine strategy against porcine enzootic pneumonia in pigs.
Mycoplasma hyopneumoniae (Mhp) causes the respiratory disease porcine enzootic pneumonia (PEP) in swine. A prerequisite for the colonization of respiratory tract by Mhp is the attachment of bacteria to the respiratory cilia. The adhesion of Mhp results in the disruption of the respiratory mucociliary apparatus and acute inflammation of airways (Thacker, 2006). P97 is a well characterized immunodominant cell surface adhesin of Mhp (Klinkert et al., 1985; Zhang et al., 1995; Minion et al., 2000). The C-terminal portion of P97 contains two repeat regions (R1 and R2), and the cilium binding site is located in the R1 region (AAKPV/E) (Minion et al., 2000). In the last two decades, different strategies have been used to develop vaccines using P97 (King et al., 1997; Shimoji et al., 2003; Chen et al., 2006, 2008; Conceicao et al., 2006; Ogawa et al., 2009; Okamba et al., 2010). Nevertheless, subunit vaccine production using Escherichia coli has several advantages over other methods. It is safer, simpler and less costly than other methods, such as live delivery systems, DNA vaccines and RNA vaccines (Hansson et al., 2000).
Previous investigations have reported several issues in developing vaccine with P97, for instance, low level expression, insolubility and type of immune response (Chen et al., 2001, 2006, 2008; Shimoji et al., 2003; Conceicao et al., 2006; Okamba et al., 2007, 2010). To overcome these problems, we evaluated the conformational advantage of the R1 repeat surrounding region and/or LTB (B subunit of the heat-labile enterotoxin of E. coli) protein fusion on the expression pattern and type of anti-R1 response. We hypothesized that including the R1 repeat surrounding region and/or LTB fusion would help soluble expression of proteins. Also, this might affect the conformation of final proteins and the type of induced immunity.
Adjuvants are frequently combined with subunit vaccines to enhance the specific immune response against the co-administered antigen. Aluminum salts (alum) and oil-based emulsions are among the most commonly used adjuvants in animal vaccines (Freund et al., 1948; Gupta et al., 1995; Bowersock & Martin, 1999). In the past few years, a number of new oil-based adjuvants with better efficiency and innocuity have been registered under the name Montanide® IMS (Aucouturier et al., 2000). In particular, IMS 1113 has shown significant efficiency and safety for use in pig and mouse models (Aucouturier et al., 2000; To et al., 2010). However, until now, Montanide® adjuvants have not been tested with Mhp subunit vaccines. The purpose of the present study was to evaluate the immunogenicity of IMS 1113 combined with soluble subunit and chimeric proteins containing Mhp P97 C-terminal repeat regions.
Materials and methods
Production of recombinant proteins
Genomic DNA from Mhp 7–25 (a Korean field isolate) was extracted using the Genomic DNA Extraction Mini kit (RBC Biosciences). Genomic DNA from enterotoxigenic E. coli was obtained by boiling method. The E. coli heat-labile enterotoxin subunit B gene (eltb), the C-terminal repeat regions of the p97 gene (r1 and r1r2) and the LTB fusion products (ltbr1 and ltbr1r2) were amplified using PCR. Table 1 shows the PCR conditions used to amplify these genes. The 5′ ends of the LTBF and R1F primers contained a 4-base pair sequence (CACC) that facilitates the directional cloning of PCR products into the pENTR/SD vector (Gateway system, Invitrogen). The LTBR and R1F primers had a BamHI restriction site that allowed the in-frame fusion between ltb and r1. The cloning of genes (r1, r1r2, ltbr1 and ltbr1r2) into the pENTR/SD and pETDEST42™ vectors (Gateway system, Invitrogen) was performed according to the manufacturer's instructions. For protein expression, E. coli BL21-competent cells were transformed by electroporation with the expression plasmids and grown in 1 L of Luria–Bertani medium containing 100 μg mL−1 ampicillin. Protein expression was induced with 1 mM of isopropyl-β-d-1-thiogalactopyranoside (IPTG) for 3 h. The expressed proteins were purified using HisPur Ni-NTA affinity chromatography (Thermo Scientific) according to the manufacturer's instructions.
Table 1. Primer sequences and PCR conditions used in this study
Primer designed in this study. Other primer sequences were obtained from previous study (Conceicao et al., 2006).
Escherichia coli heat-labile enterotoxin subunit B gene (eltb)
Discontinuous sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli (1970). The recombinant proteins were electrophoresed in 14% separating gel, and the Mhp whole proteins were electrophoresed in 10% separating gel. Gels were then either stained with Coomassie blue or set up for Western blotting. In Western blotting, the recombinant proteins were probed with anti-His antibodies (Santa Cruz Biotechnology) and anti-cholera toxin antibodies (Sigma-Aldrich) (cholera toxin is homologous to LTB). The membranes were blocked with 5% non-fat dry milk and were then incubated with 1 : 100 rabbit His probe antibody, 1 : 3000 rabbit IgG anti-cholera toxin, or 1 : 100 mouse sera against recombinant protein for 1 h at room temperature. After three washes with TBS-T (Tris-buffered saline with 0.05% Tween 20), the membranes were incubated for 1 h at room temperature with 1 : 2000 goat anti-rabbit IgG-horseradish peroxidase (HRP) or 1 : 2000 goat anti-mouse IgG-HRP (Santa Cruz Biotechnology). The reactions were developed with 3,3’-diaminobenzidinetetrahydrochloride (Sigma-Aldrich). To characterize possible polymerization of rLTBR1, non-heated samples of purified fusion proteins were analyzed by SDS-PAGE as described above.
Immunization of mice
Female BALB/c mice aged 7–8 weeks (day 0) were obtained from Charles River Technology (Orient Bio Inc.) and maintained in SPF IVC cages (Threeshine Inc.). Mice (n =5 per group) were immunized with recombinant proteins (four groups), with Respisure® (Zoetis Inc.), a commercial vaccine against PEP, or phosphate-buffered saline (PBS) (control group). Recombinant proteins containing molar equivalent amounts of rR1 (rR1, 12 μg; rR1R2, 25.2 μg; rLTBR1, 21.7 μg; rLTBR1R2, 34.9 μg) were administered intramuscularly with the adjuvant IMS 1113 (Seppic) at a 1 : 1 ratio on days 0, 15 and 30. Respisure® was administered intramuscularly at 1/20 of the swine dose at days 0 and 15. PBS 30 μL was inoculated at days 0, 15 and 30. Blood samples were collected from the retro-orbital sinus at 0, 15, 30, 45 and 60 days post inoculation (DPI). The sera were processed and stored at –20 °C. At day 60, the mice were sacrificed, and bronchoalveolar lavages (BALs) were performed with 1.5 mL cold sterile PBS (de Haan et al., 1995). The animal experiments were conducted according to the Institutional Animal Care and Use Committee guidelines at Kangwon National University.
Evaluation of antibody and cellular immune response
Purified rR1 was used to coat the wells of 96-well Maxi-Sorp microtiter plates (NUNC, Thermo Scientific) at 0.5 μg per well. After overnight incubation at 4 °C, the wells were washed three times with PBS-T (phosphate-buffered saline with 0.05% Tween 20). The plates were blocked with PBS containing 5% (w/v) bovine serum albumin (BSA). Subsequently, triplicate samples (50 μL) of 1 : 20 diluted serum or BALs were added and incubated at 37 °C for 1 h. After three PBS-T washes, 1 : 2000 goat anti-mouse IgG HRP or 1 : 1000 goat anti-mouse IgA HRP (Santa Cruz Biotechnology) was added and incubated at 37 °C for 1 h. The reactions were developed with 3,3’,5,5’-tetramethylbenzidine (TMB) substrate (BioLegend Inc.) according to the manufacturer's instructions. To determine the isotypes of IgG antibodies in serum and BAL, peroxidase-labeled goat anti-mouse IgG1 and goat anti-mouse IgG2a (Fitzgerald Industries International) were used.
At day 60, the spleens from immunized mice were aseptically removed, and splenocytes were isolated using the procedure of Bastos et al. (2002). The splenocytes were counted, plated at 107 cells mL−1 in RPMI-1640 (GenDepot) supplemented with 10% fetal bovine serum (GenDepot) and incubated for 72 h with either 15 μg mL−1 of rR1 protein or PBS. The production of interferon (IFN)-γ was determined using a commercial ELISA kit following the manufacturer's instructions (BioLegend Inc.). All tests were performed in duplicate for five mice.
Statistical analysis was performed using graphpad prism 5 software (GraphPad Software Inc.). Two-way anova combined with the Bonferroni post-test was used to compare the serum IgG levels of different experimental groups. A paired t-test was used to evaluate the differences between IgG1 and IgG2a levels. One-way analysis of variance (anova) combined with the Bonferroni post-test was used to evaluate the differences in all other statistical analyses. Values of P <0.05 were considered significant.
Production and characterization of recombinant proteins
All proteins were expressed in the soluble fraction and purified using Ni-NTA affinity chromatography. Protein rR1 was expressed as three proteins with masses of 17, 18 and 23 kDa; rR1R2 was expressed as two proteins with masses of 20 and 38 kDa; rLTBR1 was expressed as two proteins with masses of 27 and 33 kDa; and rLTBR1R2 was expressed as two proteins with masses of 41 and 52 kDa (Fig. 1a). The estimated molecular weights of rR1, rR1R2, rLTBR1 and rLTBR1R2 are 14.1, 29.6, 25.5 and 41.4 kDa, respectively (dnastar Lasergene Version 7.0). The slower migration and higher apparent molecular weights of the recombinant proteins are due to the abundance of residues with positive charge and the conformation of the P97 C-terminal repeat region (Conceicao et al., 2006). Western blotting was used to characterize the recombinant proteins. All of the recombinant proteins were recognized by anti-His antibodies (Fig. 1b). Anti-cholera toxin antibodies recognized only the chimeric proteins that contained the LTB fusion (Fig. 1c). The ability of the LTB fusion proteins to form oligomers was verified by electrophoresis of non-heated samples of rLTBR1 and rLTBR1R2 (Fig. 1d and e). Oligomerization is associated with the adjuvant activity of LTB (Sixma et al., 1991; de Haan et al., 1996, 1998).
Systemic antibody response against recombinant proteins
To evaluate the immunogenicity of the recombinant proteins, sera from the immunized mice were analyzed using an indirect ELISA (Fig. 2a). The total IgG levels in rLTBR1- and rLTBR1R2-immunized mice rose and reached a peak at 45 DPI before declining at 60 DPI. The antibody levels in rR1- and rR1R2-immunized mice continued to rise even after 45 DPI, and showed an increasing trend even at 60 DPI. At 45 DPI, the rLTBR1-, rLTBR1R2- and rR1R2-immunized groups had significantly higher antibody levels than the other groups (P <0.001). However, no significant difference was observed among these three groups at 45 DPI. Notably, total IgG rose more rapidly (D15; P <0.001) in the rLTBR1R2-immunized group than in the other groups. At 45 DPI, the rLTBR1, rLTBR1R2 and rR1R2 mouse groups had approximately 2.7-fold higher antibody levels than the rR1 group. The proteins rLTBR1 (D30, D45 and D60; P <0.001) and rLTBR1R2 (D15 and D30; P <0.001) elicited higher antibody levels than the corresponding rR1 and rR1R2 proteins. No antibody response was detected in the control group.
The specificity of the antibody response against P97 was analyzed by Western blotting against Mhp whole cell proteins as antigens. P97 from Mhp was specifically detected by 60 DPI rR1, rR1R2, rLTBR1 and rLTBR1R2 antisera. Figure 2b shows the detection of P97 and its proteolytic products (Djordjevic et al., 2004) by 60 DPI rLTBR1 and rLTBR1R2 immune sera. P97-specific antibodies were not detected in day 0 serum or 60 DPI PBS-immunized serum (control sera).
Mucosal antibody response against recombinant proteins
Mucosal immunity (IgA and IgG) was examined by indirect ELISA using BALs collected at 60 DPI. Mice immunized with recombinant proteins produced significantly higher IgG levels compared with PBS-immunized mice (P <0.0001) or Respisure®-immunized mice (P <0.001) (Fig. 2c). The highest IgG levels were detected in rLTBR1 and rLTBR1R2 BALs. However, no significant difference was observed between the IgG levels of these two groups. BALs from the control group did not show IgG, whereas a low antibody response was observed in the Respisure®-immunized group.
The IgA antibody levels in BALs are shown in Fig. 2d. All of the mouse groups immunized with recombinant proteins produced significantly more IgA than the PBS- (P <0.0001) and Respisure®-inoculated groups (P <0.001). Similar to the IgG levels, the highest IgA levels were observed in the rLTBR1 and rLTBR1R2 groups. However, the difference between these two groups was not significant. Additionally, a low-level IgA response was observed in 60 DPI BALs of the Respisure®-immunized group.
IgG isotypes elicited by recombinant proteins
In mice, IgG1 is associated with a Th2-biased response (humoral immunity), whereas IgG2a is indicative of a Th1-like response (cell-mediated immunity) (Stevens et al., 1988). Mice immunized with recombinant proteins produced IgG1 and IgG2a in both sera and BALs. Regardless of whether systemic or mucosal samples were analyzed, the ratio of IgG1/IgG2a was always high in the immunized animals (Fig. 3a and b).
Cell-mediated immune response against the recombinant proteins
The mean IFN-γ concentration in splenocyte supernatants from different mouse groups upon in vitro stimulation with rR1 is shown in Fig. 4. IFN-γ production was observed in all immunized groups. IFN-γ levels were highest in splenocyte supernatants from rR1-immunized mice. IFN-γ production in splenocytes from rR1-, rLTBR1- and rLTBR1R2-inoculated mice was significantly greater (P <0.001, P <0.001 and P <0.05, respectively) than production in the control mouse group. Among the immunized groups, a significant difference was only observed between the rR1- and rR1R2-inoculated mice.
In the present study, the immunogenicity of four recombinant proteins (rR1, rR1R2, rLTBR1 and rLTBR1R2) with adjuvant IMS 1113, was assessed in mice. In protein antigens, there are two types of epitopes, namely conformational epitopes and linear or sequential epitopes. Conformational epitopes are composed of amino acids that are located far apart in their linear sequence but become juxtaposed when the protein is folded in its native shape. Linear epitopes are composed of a particular stretch of consecutive amino acids in the primary structure of a protein (Yamashita et al., 2011). Protein tags have been observed to improve expression and solubility of the fused antigen (Sorensen & Mortensen, 2005). The B subunit of cholera toxin or heat-labile enterotoxin (LT) can also be utilized as a protein tag (Harakuni et al., 2005). Also, it is desirable to include a large portion of the full length immunogens to retain the conformation and epitopes involved in immunogenicity. Here we report the effect of R1-repeat surrounding region and/or LTB fusion on anti-R1 immunity.
To identify optimal expression conditions, cultures of different bacterial expression hosts (BL21 (DE3), Rosetta (DE3) and BL21 (DE3) pLysS) harboring the expression plasmids were induced at various temperatures, durations and IPTG concentrations. Among these expression hosts, BL21 (DE3) provided high-level soluble expression of all four constructs used in this study. All of the recombinant proteins were expressed as two or three protein bands. A similar observation was reported for the expression of constructs containing the P97 C-terminal R1 repeat (Conceicao et al., 2006). The R1 region of the constructs used in this study contains 11 repeating units of AAKPV/E. Furthermore, each protein used in this study contains the motif and sequence previously shown to be involved in binding heparin and mucin (Jenkins et al., 2006). The ability of Mhp to interact with heparin and mucin is considered important in the pathogenesis and infection process of Mhp (Jenkins et al., 2006).
Chimeric proteins from this study were fused with LTB. LTB has been identified as a potent immunoadjuvant and has been demonstrated to enhance mucosal as well as systemic antibody responses against the co-administered or fused antigens. It has been suggested that the stimulatory activity of LTB depends on its ability to form functional oligomers that bind to the cell receptor, GM1 ganglioside (Sixma et al., 1991; de Haan et al., 1996, 1998). Western blot results from this study show that the chimeric proteins were able to form oligomeric structures. For mice immunization, the subunit and chimeric proteins were co-administered with Montanide adjuvant IMS 1113. In the past, this adjuvant has proved to be a very efficient adjuvant enhancing the specific immune response, without inducing local reactions (Aucouturier et al., 2000; To et al., 2010).
In the past, expression of the R1 region by an attenuated strain of Salmonella enterica serovar Typhimurium aroA (Chen et al., 2006) or Erysipelothrix rhusiopathiae YS-19 (Shimoji et al., 2003) failed to induce the antibody response against the P97 adhesin. It is important to note that the P97 adhesin can induce an early antibody response following Mhp infection in pigs (Klinkert et al., 1985; Zhang et al., 1995). In addition, both vaccination and Mhp infection in pigs have been reported to induce humoral and mucosal antibodies against the P97 C-terminal R1 repeat (Okamba et al., 2010). In the present study, all four recombinant proteins induced an anti-P97R1 specific immune response in both systemic and mucosal compartments. In this study, recombinant proteins containing equivalent amounts of rR1 were used to immunize the BALB/c mice. However, the anti-R1 antibody level rose much more quickly in rLTBR1R2-immunized mice than in rLTBR1-immunized mice. Additionally, rR1R2-immunized mice showed a stronger and quicker anti-R1 antibody response. This result suggests that the conformation of the R1 repeat in rLTBR1R2 and rR1R2 is more effective for inducing an antibody-mediated response.
Protection by Mhp vaccines has been shown to occur through the induction of mucosal, humoral and cellular immune responses (Thacker et al., 2000; Okamba et al., 2010). Vaccination induces humoral antibodies, mucosal IgG, mucosal IgA and IFN-γ-secretory lymphocytes (Thacker et al., 2000; Okamba et al., 2010). Each recombinant protein in this study was capable of inducing specific anti-P97 humoral antibodies (IgG), mucosal antibodies (IgG and IgA) and IFN-γ production. This result is interesting because mucosal IgG has not been reported after immunization with recombinant P97 (King et al., 1997; Chen et al., 2001; Conceicao et al., 2006). We also observed a low-level humoral and mucosal anti-P97 response in mice immunized with Respisure® vaccine.
ELISA results from this study revealed that each recombinant protein is capable of inducing both Th1 and Th2 responses. Interestingly, we found that IgG1 predominated in all of the immunized groups, suggesting a bias towards a Th2-type response in both the humoral and mucosal compartments. Vaccination or Mhp inoculation also elicits Th2-biased humoral and mucosal immunity against P97 (Okamba et al., 2010). Mice immunized with Respisure® vaccine in this study also showed a Th2-biased response. However, previous investigations were unable to generate this type of immune response against P97 (Conceicao et al., 2006; Okamba et al., 2007, 2010). It is interesting to note that we observed a similar immune response trends in the group of mice immunized with rLTBR1 without IMS 1113 (Fig. 5).
In summary, the data presented here suggest that P97 C-terminal repeats produced in soluble form have a conformational advantage and elicit an anti-P97 immune response similar to that observed in natural infection or vaccination processes. The recombinant proteins combined with IMS 1113 induced not only an antibody-mediated response but also a cell-mediated immune response against P97. Particularly interesting is the induction of mucosal IgG and IgA by this immunization. Thus, the results showing the immunogenicity of subunit and chimeric P97 C-terminal repeats with IMS 1113 adjuvant appear promising and warrant further testing in pigs.
The authors have no conflict of interest to declare. This research was supported by the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries and was supported technically by the Institute of Veterinary Science, Kangwon National University, Republic of Korea. This study was also supported by 2013 Research Grant from Kangwon National University (No. 120131835), Republic of Korea.