Construction and Immunogenic Characterization of a Fusion Anti-caries DNA Vaccine against PAc and Glucosyltransferase I of Streptococcus mutans

Abstract

Glucosyltransferases (GTFs) and A cell-surface protein (PAc) are two important virulence factors of the cariogenic organism Streptococcus mutans. They may mediate sucrose-independent or sucrose-dependent attachment of Streptococcus mutans to tooth surfaces, respectively. Thus, inhibiting both virulence factors is predicted to provide better protection against caries than inhibiting a single factor. To develop a highly efficient vaccine against caries, we constructed a fusion DNA vaccine, pGLUA-P, by cloning the GLU region of GTF into a DNA vaccine, pCIA-P, which encodes two highly conservative regions of PAc. In this report, we provide evidence that fewer caries lesions were observed in rats following subcutaneous injection of pGLUA-P, compared with pCIA-P, near the submandibular gland. Our findings suggest that a multigenic DNA vaccine may be more caries-preventive than a single-gene DNA vaccine.

INTRODUCTION

S treptococcus mutans (S. mutans) has been strongly implicated as a causative organism of dental caries (Hamada and Slade, 1980; Loesche, 1986). Colonization of these micro-organisms on tooth surfaces is thought to be an important step for the initiation of dental caries. Two mechanisms, one sucrose-independent and the other sucrose-dependent, are considered to mediate this process. A 190-kDa surface protein antigen (PAc) is involved in the former mechanism, which mediates the initial adherence of S. mutans to acquired pellicles on tooth surfaces (Koga et al., 1990); while the latter mechanism is due to the synthesis of water-insoluble glucan from sucrose, a process catalyzed by glucosyltransferases (GTFs) (Kuramitsu et al., 1995).

PAc protein has two important adherent functional and immunogenic regions: an N-terminal alanine-rich region (A region) and a middle proline-rich region (P region) (Russell and Mansson-Rahemtulla, 1989). S. mutans exhibits three forms of GTF (GTF-I, GTF-SI, GTF-S), which catalyze water-soluble and water-insoluble glucan synthesis from sucrose. GTFs also have two important immunogenic and functional regions: an N-terminal catalytic (CAT) region and a C-terminal glucan-binding (GLU) region (Monchois et al., 1999). Due to the importance of GTFs and PAc in the cariogenicity of S. mutans, these proteins are rational targets for the development of an anti-caries vaccine. Antibody against a protein fusing the A region of PAc and the GLU region of GTF-I, which can synthesize water-insoluble glucan, inhibited adhesion of S. mutans to saliva-coated hydroxyapatite (S-HA) and glucan synthesis by GTFs (Yu et al., 1997). A chimeric protein consisting of the two virulence determinants has been demonstrated to enhance mucosal immune responses to the single virulence determinant alone (Zhang et al., 2002).

Since the report of Wolff et al.(1990), much attention has been paid to DNA vaccines. Compared with traditional vaccines, DNA vaccines have obvious advantages, such as: (a) long-term and stable expression of endogenously produced antigenic protein, which is similar in conformation to natural protein; (b) stronger antigenicity, with the capacity to induce both cellular and humoral immune responses; and (c) the possibility for creation of a polyvalent vaccine against several kinds of pathogens (Kowalczyk and Ertl, 1999). We have reported that an anti-caries DNA vaccine, pCIA-P, which encodes two highly conservative regions of PAc, could induce protective anti-caries immune responses (Fan et al., 2002). We have since constructed a fusion anti-caries DNA vaccine, fusing the A-P fragment of PAc to the GLU region of GTF-I, and evaluated the resulting systemic and mucosal immune responses, and anti-caries protection compared with pCIA-P in a rat model.

MATERIALS & METHODS

Plasmid Construction

A DNA fragment encoding the GLU region (amino acid residues 1185-1475) of the gtfB gene from S. mutans was amplified from plasmid pYNB13 (provided by Prof. H.K. Kuramitsu, State University of New York, USA) by polymerase chain-reaction [PCR: Expand High Fidelity PCR System (Boehringer, Mannheim, Germany)]. We constructed the recombinant plasmid pGLUA-P by inserting the GLU fragment into pCIA-P, which encodes the A region and P region (aa 222 to 965) of the pac gene of S. mutans, described previously (Fan et al., 2002), and we verified its viability by restriction digestion and by sequencing the completely inserted DNA (Shanghai Sangon, Shanghai, China) (Fig. 1).

Fusion Protein Expression in Cultured Cells

The expression of the recombinant fusion protein was performed in a transient transfection assay with the use of (1,3-di-oleoyloxy-2-(6-carboxy-spermyl)-propyl-amid; Roche Molecular Biochemicals, Mannheim, Germany), according to the manufacturer’s instructions. Briefly, Chinese hamster ovary cells (CHO, 3 × 10⁵ cells; purchased from the Chinese Center for Type Culture Collection, CCTCC, Wuhan, China) were incubated with DNA-liposome complexes for 5 hrs and cultured overnight with CHO medium (Hyclone, Logan, UT, USA). Recombinant fusion protein expression in CHO was evaluated with a fluorescent immunoassay with avidin-biotin-Cy3 Complex (Boster, Wuhan, China) according to the manufacture’s protocol. The slides were incubated with anti-PAc IgG antibody (provided by Prof. M.W. Russell, University at Buffalo, NY, USA) or anti-GTF antibody (provided by Prof. S.M. Michalek, University of Alabama at Birmingham, USA) at 37°C for 30 min and kept at 4°C overnight. They were then incubated with biotinylated goat anti-rabbit IgG and avidin-biotin-Cy3 complex in turn and viewed under a fluorescence microscope.

Immunization of Rats

Five groups of newborn male SD rats (5 per group) were bred and maintained in the Hubei (China) Medical Laboratory Animal Center. The animal protocols were approved by the review board of Hubei Medical Laboratory Animal Center. Rats were weaned at 18 days and raised on a cariogenic diet, Keyes 2000 (Navia, 1997). Antibiotics (ampicillin, chloramphenicol, and carbenicillin, 1.0 g/kg) were added to the diet on days 20–22, and animals were infected with S. mutans Ingbritt on days 24–26. Before and after infection, bacterial samples from the oral cavity were examined. Two days after infection, rats were immunized with 100-μL plasmid (1 μg/μL) as follows: injection of pGLUA-P into the quadriceps femoris muscle (GLUA-P/i.m.); subcutaneous injection of pGLUA-P near the submandibular gland (GLUA-P/s.c.); injection of pCIA-P into the quadriceps femoris muscle (A-P/i.m.); and subcutaneous injection of pCIA-P near the submandibular gland (A-P/s.c.). Immunizations were boosted 2 wks later. Controls were infected with 100-μL pCI vector (1 μg/μL) via injection into the quadriceps femoris muscle. On day 63, saliva samples were collected after stimulation of salivary flow by intraperitoneal injection of 1 mg pilocarpine (Sigma, St. Louis, MO, USA); blood was collected from the tail, and the mandible was removed, cleaned, and stained with murexide. The teeth were sectioned, and caries levels were scored according to the Keyes method (Keyes, 1958).

Antibody Analysis

For measurement of anti-PAc or anti-GTF IgA and IgG antibody responses in saliva and serum, each well of an ELISA plate was coated with PAc (10 μg/mL in carbonate buffer, pH 9.6, provided by Prof. M.W. Russell) (Russell et al., 1980) or rGTF (10 μg/mL in carbonate buffer, pH 9.6) (Jia et al., 2003) overnight at 4°C and then blocked with phosphate-buffered saline (PBS, pH 7.2) containing 3% bovine serum albumin (BSA). After samples were washed with PBS containing 0.1% Tween (PBST, pH 7.2), a 100-μL quantity of diluted saliva or sera was added to each well and incubated for 1.5 hrs at 37°C. Each well was washed again with PBST, and then treated with 100-μL quantities of goat anti-rat IgG or goat anti-rat IgA (1:1000; Sigma), incubated for 2 hrs at 37°C, and washed again. Next, a 100-μL quantity of alkaline-phosphatase-conjugated rabbit anti-goat IgG (1:10000; Sigma) was added to each well and incubated for 5 hrs at 37°C, followed by phosphate substrate (ρ-nitrophenylphosphate) for 30 min at 37°C. Optical density (OD) readings were taken at 405 nm. The end-point titer was defined as the highest dilution with an absorbance ≥ 0.1 above that of the sham control.

Statistical Analysis

Differences in anti-PAc and anti-GTF specific antibodies and caries protection among the test groups and control group were evaluated by analysis of variance. Antibody responses were compared by the 10-log-rank statistic. p < 0.05 was regarded as significant.

RESULTS

Expression of Recombinant Fusion Protein in vitro

Many cells positively stained with a fluorescent red were found in the cytoplasm of pGLUA-P-transfected CHO cells incubated with both anti-PAc IgG and anti-GTF antibody and in pCIA-P-transfected CHO cells incubated with anti-PAc IgG antibody, whereas no such specific products could be found in cells transfected with pCI vector. Analysis of the data indicates that the plasmid pGLUA-P has the ability to express both PAc and GTF protein in eukaryotic cells (Fig. 2).

Antibody Responses to PAc

We evaluated the ability of pGLUA-P to induce PAc-specific antibody responses in both serum and saliva by immunizing rats through different routes (Fig. 3A ). The anti-PAc IgG ELISA end-point titers in the serum of rats immunized by anti-caries DNA vaccines were significantly higher than those of control rats (p < 0.01). Mean serum PAc-specific IgG antibody titers in the GLUA-P/i.m. and A-P/i.m groups were significantly higher than those in the GLUA-P/s.c. and A-P/s.c. groups (p < 0.05). Salivary anti-PAc IgA antibody responses in the GLUA-P/s.c. and A-P/s.c. groups were significantly higher than those in the control, GLUA-P/i.m., or A-P/i.m. group (p < 0.01). There was no notable difference in serum anti-PAc IgG antibody responses between the GLUA-P/i.m. group and the A-P/i.m. group (p > 0.05). There was no notable difference in serum and saliva anti-PAc antibody responses between the GLUA-P/s.c. group and the A-P/s.c. group (p > 0.05).

Antibody Responses to GTF

We evaluated the ability of pGLUA-P to induce GTF-specific antibody responses in both serum and saliva by immunizing rats through different routes (Fig. 3B ). No GTF-specific antibody response was detected in serum or saliva from rats immunized with pCIA-P. Anti-GTF IgG ELISA end-point titers in serum of rats immunized with pGLUA-P were significantly higher than those of control rats (p < 0.01). Mean serum GTF-specific IgG antibody titer in the GLUA-P/i.m. group was significantly higher than that in the GLUA-P/s.c. group (p < 0.05). Salivary anti-GTF IgA levels in the GLUA-P/s.c. group were significantly higher than those in either the GLUA-P/i.m. group or the control group (p < 0.01).

Caries Protection

Groups of rats immunized with anti-caries DNA vaccines had significantly less caries experience than was observed in the control group (p < 0.01) (Fig. 3C ). Subcutaneous administration of DNA vaccines near the mandibular gland afforded better protection against caries than did intramuscular injection (p < 0.01). Group GLUA-P/s.c. rats displayed the fewest enamel lesions (p < 0.01), while slight dentinal lesions (p < 0.05) were seen in all groups.

DISCUSSION

Streptococcus mutans has two important virulence factors: PAc and GTFs. PAc participates in the hydrophobic interaction between S. mutans and acquired pellicles on tooth surfaces (Hamada et al., 1989; Koga et al., 1990). Immunization with PAc protected monkeys from dental caries (Russell, 1992, 1994). Although anti-PAc antibodies markedly suppressed the adhesion of S. mutans to S-HA in the absence of sucrose, they did not inhibit sucrose-dependent adhesion of the organism. GTFs produce water-soluble and/or water-insoluble glucans, which play important roles in dental plaque formation of S. mutans by facilitating the accumulation of the organism on tooth surfaces. Antibodies against synthesized peptides derived from the CAT and GLU region of GTFs could inhibit glucan synthesis by S. mutans (Chia et al., 1993; Smith et al., 1994). In contrast, Cope and Mooser (1993) reported that rabbit antibodies against peptides originating from the CAT region could not inhibit glucan synthesis by GTFs. Antibodies against 22 peptides originating from the GLU region could recognize S. sobrinus glucan-binding protein, which can promote colonization by enabling the organism to adhere to glucans (Smith et al., 1993). In addition, Yu et al.(1997) reported that antibodies against a recombinant protein fusing the A region of PAc with the GLU region of GTF-I could markedly inhibit glucan synthesis by GTF-I as well as in vitro colonization by S. mutans in the absence and presence of sucrose; however, antibodies against recombinant protein (PAcA-SB), fusing the A region with the CAT region of GTF-I, could not inhibit glucan synthesis by GTF-I or S. mutans colonization to SH beads in the presence of sucrose (Yu et al., 1997). Zhang et al.(2002) constructed a genetic chimeric protein SBR-GLU, consisting of the two virulence determinants, the saliva-binding region (SBR) of PAc and the glucan-binding region (GLU) of GTF of Streptococcus mutans. The chimeric protein SBR-GLU significantly enhanced mucosal immune responses to SBR and GLU and systemic immune responses to SBR in mice and effectively protected against the colonization of S. mutans. These vaccines against these two virulence factors might provide better protection against dental caries than vaccines against only one factor. We selected the GLU region as our target antigen fragment, which was connected to the A-P fragment, and cloned it into eukaryotic expression plasmid pCI. The recombinant GLUA-P fusion protein could be expressed correctly in vitro in CHO cells. In this study, rats immunized with pGLUA-P via s.c. displayed significantly fewer enamel and Ds lesions than those immunized with pCIA-P. To our knowledge, this is the first time a DNA vaccine encoding gene fragments of both virulence factors has been found to result in better protection against dental caries than a vaccine encoding one factor alone. These findings are consistent with other anti-caries fusion protein studies in rats. Mitoma et al.(2002) reported that rats receiving immune milk containing antibodies to PAcA-GB, which involves fusion of the saliva-binding alanine-rich region (PAcA) of PAc and the glucan-binding (GB) domain of GTF-I, had significantly less caries development than controls.

A conventional DNA vaccination plasmid might encode only a single antigen; however, this may not offer ideal protection against pathogens possessing several virulence factors. A DNA vaccination plasmid has obvious advantages: Several kinds of gene fragments can be easily joined and expressed together in vivo compared with the complex connection between two different proteins in vitro. Galvin et al.(2000) reported that a HIV-1 gag/env multigenic DNA vaccine could strongly express HIV-1 Gag and Env under the regulation of a cytomegalovirus (CMV) immediate-early (IE) promoter/enhancer and induce dramatic immune responses to HIV-1 in Macaca mulatta. Our fusion anti-caries DNA vaccine pGLUA-P also has a CMV promoter/enhancer and could express fusion GLUA-P protein in CHO cells. After rats were immunized with pGLUA-P via s.c., specific antibodies could be detected in serum and saliva.

Mucosal immunity is the first line of defense against pathogens to prevent systemic or local infection. An important factor for protective mucosal immunity is the induction of specific secretory IgA (SIgA) antibodies. Jespersgaard et al.(1999) showed that immunizing mice intranasally with recombinant peptides including the CAT or GLU region of GTF-I could induce specific IgA antibody activity in serum, saliva, vaginal washes, and fecal samples. Saito et al.(2001) reported that immunizing mice nasally with PAc protein and a non-toxic A subunit mutant of cholera toxin (mCT) E112K elicited significant PAc-specific SIgA in saliva and in nasal secretions. When we immunized SD rats with pGLUA-P via s.c., a significantly higher specific salivary SIgA antibody was induced compared with immunization via i.m., and notable serum anti-PAc IgG antibody responses were also induced, suggesting that this immunization route could elicit a systemic and mucosal immune reaction. Our previous research gave the same results (Fan et al., 2002). Thus, subcutaneous injection near the submandibular gland immunization with plasmid might be a promising genetic immunization strategy against oral infectious diseases.

In conclusion, we have constructed a fusion anti-caries DNA vaccine pGLUA-P that expressed the GLU region of GTF-I and both A and P regions of PAc protein and demonstrated that pGLUA-P could express recombinant GLUA-P protein in eukaryotic cells and induce specific immune responses in vivo. Immunization with the polyvalent DNA vaccine pGLUA-P provided better protection than the single gene DNA vaccine pCIA-P.

Figure 1.

Construction and verification of plasmid pGLUA-P. (A) The GLU region of GTF-I of S. mutans was cloned into the eukaryotic expression plasmid pCIA-P to obtain the recombinant plasmid pGLUA-P. (B) Restriction enzyme digestion of pGLUA-P and pCI. pGLUA-P, A-P fragment, GLU fragment, and pCI were measured as 7.1 kb, 2.2 kb, 0.9 kb, and 4.0 kb, respectively. Lane 1: GeneRuler 1 kb DNA ladder. Lane 2: pGLUA-P digested by SmaI. Lane 3: pCI digested by SalI. Lane 4: pGLUA-P digested by SalI and XhoI. Lane 5: pGLUA-P digested by SalI. Lane 6: γDNA/HindIII marker.

Figure 2.

Recombinant fusion protein could be expressed in eukaryotic cells. (A) Recombinant fusion protein detected by anti-GTF antibody in the cytoplasm of CHO cells transfected with pGLUA-P. (B) Recombinant fusion protein detected by anti-PAc antibody in the cytoplasm of CHO cells transfected with pGLUA-P. (C) CHO cells transfected with pCIA-P, detected by anti-PAc antibody. (D) CHO cells transfected with pCI vector. Bar, 5 μm.

Figure 3

Antibody levels and caries scores of rats immunized with various treatments. Data are expressed as means ± standard deviation. Groups of rats were immunized with plasmids as follows: intramuscular injection of pGLUA-P (GLUA-P/i.m.); subcutaneous injection near the submandibular gland of pGLUA-P (GLUA-P/s.c.); intramuscular injection of pCIA-P (A-P/i.m.); subcutaneous injection of pCIA-P (A-P/s.c.) near the submandibular gland; and intramuscular injection of pCI (control). *Significantly different from the control group at p < 0.01; **significantly different from the control and pCIA-P groups at p < 0.01; #significantly different from DNA-vaccine-immunized group via i.m. at p < 0.01; ^significantly different from DNA-vaccine-immunized group via s.c. at p < 0.05; and +significantly different from pCIA-P immunized group via s.c. at p < 0.05. (A) Saliva- and serum-specific anti-PAc antibody levels (five rats per group). ▪ Serum anti-PAc IgG; ♦ salivary anti-PAc IgA. (B) Saliva- and serum-specific anti-GTF antibody levels (five rats per group). • Serum anti-GTF IgG; ▴ salivary anti-GTF IgA. (C) Keyes caries scores (five rats per group). • Enamel lesion; ▪ slight dentinal lesion; ♦ moderate dentinal lesion.

Footnotes

Notes

†

authors contributing equally to the work;

Acknowledgements

The authors thank H.K. Kuramitsu for providing the pYNB13 plasmid carrying the gtfB gene of S. mutans GS-5, Prof. M.W. Russell for providing PAc protein and anti-PAc antibody, and S.M. Michalek for providing anti-GTF antibody. This study was supported by grant No. 39770799 from the National Science Foundation of China.

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