An optimized caries model of Streptococcus mutans in rats and its application for evaluating prophylactic vaccines

ABSTRACT

Dental caries is a prevalent oral disease that mainly results from Streptococcus mutans. Susceptibility to S. mutans decreased rapidly after weaning in a well-known rat model. However, owing to the lack of time to establish protective immunity ahead of challenge, the weaning rat model is suboptimal for assessing prophylactic vaccines against S. mutans infection. In this study, we found that, in adult rats, S. mutans cultured under air-restricted conditions showed dramatically increased colonization efficacy and accelerated development of dental caries compared with those cultured under air-unrestricted conditions. We propose that S. mutans cultured under air-restricted conditions can be used to develop an optimal caries model, especially for the evaluation of prophylactic efficacy against S. mutans. Therefore, we used the anti-caries vaccine, KFD2-rPAc, to reevaluate the protection against the challenge of S. mutans. In immunized rats, rPAc-specific protective antibodies were robustly elicited by KFD2-rPAc before the challenge. In addition to inhibiting the initial and long-term colonization of S. mutans in vivo, KFD2-rPAc immunization showed an 83% inhibitory efficacy against the development of caries, similar to that previously evaluated in a weaning rat model. These results demonstrate that culturing under air-restricted conditions can promote S. mutans infection in adult rats, thereby helping establish a rat infection model to evaluate the prophylactic efficacy of vaccines and anti-caries drugs.

KEYWORDS:

• Dental caries
• vaccine
• Streptococcus mutans
• prophylactic efficacy
• cariogenicity
• rat model

Introduction

Dental caries is one of the most prevalent chronic oral diseases worldwide.^1–3 Although the etiology of dental caries is described as the disruption of the healthy microbial community, Streptococcus mutans (S. mutans) is still identified as the primary microbial culprit of dental caries.⁴ Pathologically, S. mutans colonizes tooth surfaces^5,6 and synthesizes large amounts of exopolysaccharides to form cariogenic biofilms.^7,8 S. mutans metabolizes carbohydrates into lactic acid to rot teeth, resulting in dental caries.⁹

Rats are the most widely used animal for caries research^10,11 because of its convenience, ease of observation, and low cost.¹² To evaluate anti-caries efficacy,^11,13–17 most studies on anti-caries vaccines and drugs have used rat models. Similar to typical prophylactic vaccines against infectious diseases, prophylactic anti-caries vaccines should prohibit both infection by cariogenic bacteria and the development of caries. In terms of clinical usage, the best strategy to prevent cariogenic bacteria-induced caries in infants and children is to establish protection ahead of infection. To mimic this clinical scenario, the prophylactic efficacy in animal models should be evaluated after the completion of the entire vaccination schedule. Three doses of vaccination at 2-week to 4-week intervals are generally required to elicit protective immunity against caries, especially mucosal IgA responses in the saliva.^14–16

However, rats older than 28 d showed decreased susceptibility to S. mutans infection by nearly 90% compared with that of 24-day-old rats.¹² To avoid the rapid decrease in the susceptibility of rats to S. mutans infection after weaning, the challenge of S. mutans should be performed in weaning rats. A minimum time window of at least five-weeks could hardly be achieved before the rats were invulnerable to S. mutans infection. While several approaches have been attempted to enhance the colonization of S. mutans in rats, such as depletion of the native oral microbiota,¹⁸ desalivation,¹⁹ and challenging gnotobiotic rats²⁰ with S. mutans, adult rats are still seldom used to study the pathogenesis of S. mutans or evaluate the prophylactic efficacy of anti-caries vaccine candidates. In currently available weaning rat models, the vaccination process is performed almost parallel to the inoculation of bacteria for challenge.

For evaluating prophylactic anti-caries vaccines, an optimal adult rat infection model should be established firstly. We wondered whether we could increase the susceptibility of rats to S. mutans infection from the aspect of S. mutans. As a facultative anaerobe, S. mutans can grow in the presence or absence of sufficient oxygen. However, S. mutans prefers oxygen-poor²¹ conditions because of the toxicity of reactive oxygen species (ROS).^22,23 The ability of S. mutans to form biofilms can also be inhibited by a sufficient oxygen supply.²⁴ These observations imply that oxygen availability plays a prominent role in modulating the biological activity and cariogenic virulence of S. mutans.

In the present study, we found that culturing S. mutans under air-restricted conditions, simply using a screw-cap-sealed tube filled with brain heart infusion (BHI) broth, could upregulate the expression of some virulence genes and cause exacerbated dental caries in challenged adult rats. By using S. mutans cultured under the air-restricted condition for challenging rats, we reevaluated the immune responses elicited by the vaccine candidate, KFD2-rPAc, and the prophylactic efficacy against S. mutans infection and caries.

Materials and methods

Bacteria culture and measurements

The S. mutans strain Ingbritt was stored in our laboratory and propagated in brain heart infusion (BHI) broth (Oxoid). S. mutans was seeded 1:100 into BHI broth and statically cultured for the indicated time at 37°C. Under air-restricted culture, S. mutans was added to BHI-filled 15 mL tubes, and the tubes were tightly sealed with a screw cap. For air-unrestricted culture, S. mutans was added to 15 mL BHI broth in a 150 mL conical flask with an air-permeable membrane. For sampling bacteria, after completely resuspending all bacteria, 200 μL of suspension was collected and pipetted into 96-well plates. The optical density at 600 nm (OD₆₀₀) was recorded by a spectrophotometer (BioTek). The total bacteria were serially diluted with PBS, and the bacteria were cultured on solid MSB medium for 48 hours in anaerobic conditions at 37°C (Becton Dickinson, CA, USA). The number of colony forming units (CFUs) was quantified. To measure dissolved oxygen levels, a dissolved oxygen meter (INESA Scientific Instrument, Shanghai) was inserted into the middle of the broth without agitation, and the dissolved oxygen value was recorded.

RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT‒PCR)

After culturing at 37°C for 12 hours, S. mutans bacteria were harvested by centrifugation at 12,000 × g at 4°C for 1 minute and immediately washed in cold diethyl pyrocarbonate (DEPC)-treated water 2 times. The bacteria were transferred to 2.0 mL screw cap microcentrifuge tubes containing 0.5 g glass beads (0.1 mm diameter, APPLYGEN) and lysed in a beater homogenizer at 4°C for a total of 120 s (homogenized four times for 30 s with 1 min intervals). The total RNA of the samples was then isolated with the FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme, Nanjing, China). The purity and concentration of the RNA were determined by gel electrophoresis and a Nanodrop 2000 instrument (Thermo Scientific) (A260/A280 ratio ≥ 2.0). Amplification of RNA and synthesis of cDNA were performed with a HiScript II One Step qRT‒PCR SYBR Green Kit (Vazyme, Nanjing, China) and a real-time PCR equipment (Bio-Rad). All primers are provided in Supplementary Table S1. The relative gene expression was normalized to double reference genes (16S rRNA and gyrA) as previously described.²⁵

Rats

Specific pathogen-free female Wistar rats were purchased from the Wuhan Institute of Biological Products and bred in the Animal Center of Wuhan Institute of Virology (WIV). After arriving, all rats were labeled with random number generated by Microsoft Excel. According to the number order, rats were randomly assigned into groups. All groups of rats were raised in individually ventilated cages (IVCs) under specific pathogen-free conditions. Animal experiments were reviewed and approved by the Institutional Review Board of WIV and performed according to Relations for the Administration of Affairs Concerning Experimental Animals in China (1988). The investigation complied with ARRIVE 2.0 guidelines for preclinical animal studies.

Infection and caries generation in the adult rat

The S. mutans Ingbritt infection procedure was performed as previously described,¹⁴ except for the age of rats and the culturing condition of S. mutans. Briefly, 12- to 13-week-old rats were randomly divided into two groups (18 rats per group). After acclimatization for 3 d, rats were raised on cariogenic diet Keyes 2000¹⁸ and fed with antibiotics (ampicillin, chloramphenicol, carbenicillin,1.0 g/kg diet and 1.0 g/L water) for 5 consecutive days to deplete the native oral flora. Twelve hours before the inoculation of S. mutans Ingbritt, three rats in each challenge group were sacrificed and set as the caries baseline. S. mutans bacteria were proliferated under either air-restricted conditions or air-unrestricted conditions for 12 hours. The teeth of fifteen rats in each challenge group were inoculated three times (once daily) with 1.7 × 10⁹ CFUs of the freshly proliferated S. mutans Ingbritt. The day that the inoculation was completed was set as 0 day post complete inoculation (dpi). On 1 dpi, the numbers of S. mutans Ingbritt on all 30 rats’ teeth were quantified on MSB medium. On 30 dpi, 58 dpi, and 86 dpi, the number of S. mutans Ingbritt on teeth was also quantified and the rats were sacrificed to determine the caries score.

Oral sample collection and analysis

The collection and analysis of oral samples were conducted as previously described with slight modifications.¹⁵ Briefly, swabs presoaked in PBS were used to collect bacteria on the occlusal surfaces of molar teeth and then transferred to 500 μL physiological saline solution. Next, these samples were dissociated from the swabs by vortexing for 30 s, and the numbers of S. mutans were subsequently quantified on solid MSB medium.

Tooth treatment and caries score analysis

To determine caries scores, the rats were sacrificed and then the molar teeth were collected, cleaned, and stained with murexide (0.4% in 70% ethanol) as previously described.^14–16 Next, the molar teeth were washed, labeled with random number, hemisectioned, photographed by a stereomicroscope (Phenix Optics Co., Ltd.). The caries scores were determined by blind method following Keyes scoring protocol²⁶ as previously described.¹⁶

Protein expression and purification

The protein expression and purification were performed as previously described.¹³ In brief, flagellin-rPAc fusion proteins (KFD2-rPAc) were expressed in E. coli BL21 (DE3) and purified by affinity chromatography on a Ni-NTA column (Qiagen, Hilden, Germany). The purified protein was dialyzed with PBS at 4°C, eliminated the residual LPS, and stored at −80°C. Then the concentration and purity of the purified protein were determined and verified that the residual endotoxin content in purified protein for immunization was less than 0.005 EU/μg.

Rat immunization and challenge

Seventeen 18-day-old weaning female rats were divided into three groups (6 rats for the two immunized groups and 5 rats for the uninfected control group). After acclimatization for 3 d, two groups of rats were intranasally immunized three times with 7 μg KFD2-rPAc in 10 μL PBS or PBS alone at 28 d intervals. Serum and saliva were collected on day 14 post the last immunization and analyzed for antibody responses by ELISA as previously described.¹⁶ On day 15 post the last immunization, native flora was depleted by antibiotics, and then the rats were challenged by 1.7 × 10⁹ CFU of S. mutans grown under air-restricted conditions for 3 d (once daily), as described above in the infection experiments section. On 1 dpi and 86 dpi, the numbers of S. mutans Ingbritt CFUs on tooth occlusal surface of rats were quantified. Rats were sacrificed at 86 dpi and the caries score was determined. The inhibitory ratio of caries score was calculated by the formula described previously.¹⁴

Biofilm inhibition assay

After being statically cultured under air-restricted conditions or air-unrestricted conditions at 37°C for 12 hours, the S. mutans Ingbritt bacteria was diluted at 1:100 by Tryptic Soy Broth (TSB) supplied with 1% (w/v) sucrose. Then 100 μL of diluted bacteria with rat serum or saliva were seeded into wells of a sterile 96-well flat-bottomed plastic plate with a lid. The plates were anaerobically cultured at 37°C for 12 hours to form biofilms. Next, the biofilms were gently washed three times with double distilled H₂O (ddH₂O), stained by crystal violet (Beyotime Biotechnology, Shanghai) for 10 min, and washed three times again with ddH₂O. At last, the attached crystal violet was extracted with 200 µL of 30% acetic acid from each well and was evaluated at 570 nm by spectrophotometer (BioTek).

Statistics

Statistical analysis was performed with GraphPad Prism 9.5. To analyze the differences between groups, one-way ANOVA or unpaired two-tailed Student’s t test was used for normally distributed data with homogeneous variance, and the Mann – Whitney U test was used for nonnormally distributed data. Pearson’s correlation coefficient and spearman’s correlation coefficient were calculated for normally and nonnormally distributed data respectively.

Results

Expression of virulence genes of S. mutans is upregulated under air-restricted culture conditions

To test whether the different culturing conditions would affect S. mutans proliferation, virulence, or cariogenicity, we attempted to culture S. mutans under air-restricted conditions simply by using a screw-cap-sealed tube filled with BHI broth (Figure 1(a) red, air-restricted), in contrast to S. mutans cultured in a traditional 150-mL conical flask with a large air-filled space and air-permeable membrane cover (Figure 1(a) blue, air-unrestricted).¹⁴ For air-unrestricted culture, accompanied by the rapid proliferation of bacteria within the first 8 hours post-inoculation (hpi) (Figure 1(b), blue line), the dissolved oxygen level in the broth rapidly decreased (Figure 1(c), blue line). Once the proliferation of S. mutans slowed down, the dissolved oxygen level in the broth gradually recovered (Figure 1(c), blue line).

Figure 1. Bacteria proliferation and the expression of virulence genes of S. mutans under air-restricted and air-unrestricted culturing conditions.

(a) In the air-unrestricted group, S. mutans was seeded into a 150-mL conical flask containing 15 mL BHI broth with an air-permeable membrane. In the air-restricted group, S. mutans was seeded into a 15 mL polystyrene centrifuge tube containing 15 mL BHI broth. Then the S. mutans was cultured statically at 37°C for indicated time. (b) The concentration of total bacteria measured by OD₆₀₀ (n = 6 samples per group). (c) The dissolved oxygen in BHI broth (n = 3 samples per group). (d) The concentration of alive S. mutans at 24 hours after inoculation measured on MSB plates (n = 3 samples per group). (e) S. mutans bacteria were collected by centrifugation at 12 hours after inoculation. Total RNA was extracted, and then transcription levels of the virulence genes were qualified by qRT‒PCR (n = 6 samples per group). Changes in virulence gene expression levels were shown after normalization to double reference genes (16S rRNA and gyrA). All data represent the mean ± SEM. Significance was determined by Student’s t test. *, p < .05; **, p < .01; ***, p < .001; ****, p < .0001; ns, nonsignificant.

In contrast, S. mutans Ingbritt cells cultured under air-restricted conditions (Figure 1(a) red) proliferated more rapidly than that cultured under air-unrestricted conditions at 4 and 6 hpi (Figure 1(b) red). Accordingly, the dissolved oxygen levels in the broth decreased rapidly in the first 6 hpi and remained at a very low level thereafter (Figure 1(c), red line). At 12 hpi, bacteria reached the plateau phase (Figure 1(b)) with similar total bacterial density and live bacterial density when cultured under either air-unrestricted or air-restricted conditions (Figure 1(b,d)). These results suggest that air restriction affects the initial proliferation rate of S. mutans but has little influence on the number of proliferated S. mutans at 12 hpi and thereafter. Meanwhile, the transcriptional levels of the colonization-associated gene spaP ²⁷ and biofilm formation-associated genes gtfB, gtfC, and gbpB ²⁸ were all upregulated under air-restricted conditions (Figure 1(e)). The upregulation of these well-known virulence genes suggests that air-restricted cultures facilitate the cariogenicity of S. mutans.

S. mutans cultured under air-restricted conditions causes higher colonization efficacy and exacerbated caries lesions in adult rats

Therefore, we investigated whether air-restricted culture conditions could facilitate the cariogenicity of S. mutans and develop a caries model in adult rats. Instead of the commonly used 18-day-old weaning rats, adult female rats aged 12- to 13-week-old were selected to test the cariogenicity of S. mutans in vivo. As previously described,¹⁶ the rats were first depleted of native flora. Two groups of rats were inoculated with S. mutans proliferating under air-unrestricted (air-unrestricted group) or air-restricted conditions (air-restricted group). Before inoculation of S. mutans, three rats in each group were sacrificed to evaluate the baseline caries lesions (−2 dpi). On day 1 post inoculation (1 dpi), S. mutans adhering to the occlusal tooth surface was sampled and quantified (Figure 2(a)). At 30, 58, and 86 dpi, five rats from each group were sacrificed after oral sampling to evaluate the development of caries (Figure 2(a)).

Figure 2. Caries development in adult rats challenged by S. mutans cultured under air-restricted and air-unrestricted conditions.

(a) Diagrammatic scheme of the S. mutans challenge experiment in adult rats. Briefly, 12- to 13-week-old female rats were fed Keyes 2000 diet with multiple antibiotics for five consecutive days. At 12 hours before the S. mutans challenge, the water and food containing antibiotics were removed. Then, the rats were orally infected with 1.7 × 10⁹ CFU freshly cultured S. mutans for three consecutive days (once a day). (b and c) Oral samples of the tooth occlusal surface were collected from all challenged rats on 1 dpi (n = 15 rats in each group), and on 30 dpi, 58 dpi, 86 dpi (n = 5 rats per group), before rats were sacrificed. The CFUs of S. mutans in the oral samples were quantified on MSB plates. (d) Representative photographs of caries lesions on the indicated days post-challenge. (e and f) Caries scores of rats in two challenge groups before challenge (‒2 dpi) (n = 3 rats per group) and at 30 dpi, 58 dpi, and 86 dpi (n = 5 rats per group). Total score = score of enamel lesions (E) + score of slight dentinal lesions (Ds) + moderate dentinal lesions (Dm) + extensive dentinal lesions (Dx). No Dx observed through the whole experiment. All data represent the mean ± SEM. Significance was determined by Student’s t test. *, p < .05; **, p < .01; ***,p< . 001; ns, nonsignificant.

At 1 dpi, the number of S. mutans on rat teeth in the air-restricted group was 3.8 times higher than that in the air-unrestricted group (Figure 2(b)). Between the two groups, a significant difference in the number of S. mutans was detected at 30 dpi (p = .0154), 58 dpi (p = .0202), and 86 dpi (p = .0159) (Figure 2(c)). These results suggested that S. mutans cultured under air-restricted conditions has a much higher capacity to colonize the tooth surfaces of adult rats.

As for dental caries, there was no difference either in caries lesions (Figure 2(d)) or in caries score (Figure 2(e)) between the air-restricted and air-unrestricted groups before S. mutans inoculation (−2 dpi). However, caries developed more rapidly and showed more severe carious lesions and caries scores in the air-restricted group than in the air-unrestricted group at 30, 58, and 86 dpi (Figure 2(d–f). At 30 dpi, caries rarely developed in the air-unrestricted group, whereas slight dentinal lesions (Ds) and moderate dentinal lesions (Dm) developed in the air-restricted group (Figure 2(f)). At 58 dpi, caries development in the air-unrestricted group was limited only to the enamel, while carious lesions were readily observed in the dentin of the air-restricted group (Figure 2(f)). At 86 dpi, Dm appeared in all rats in the air-restricted group; however, only a slight level of Ds was observed in the air-unrestricted group (Figure 2(f)). The positive correlation between the number of colonized S. mutans on teeth and the total caries score (Supplementary Figure S1) implied a possible association between the colonization efficacy of S. mutans and subsequent caries outcome in adult rats. Taken together, these observations indicate that S. mutans cultured under air-restricted conditions has a higher colonization ability on the tooth surface and causes significantly exacerbated carious lesions in adult rats. This may help establish an S. mutans-induced caries model in adult rats to evaluate the prophylactic effects of the vaccine.

Newly developed model can be adapted for evaluating prophylactic effect of vaccine against infection and dental caries

To validate the infection model for evaluating prophylactic vaccines against caries, the subunit vaccine, KFD2-rPAc, which was reported in our previous study,¹³ was used as a representative anti-caries vaccine. As illustrated in Figure 3(a), two groups of 18-day-old female weaned rats were intranasally immunized with either PBS or KFD2-rPAc. On day 70 after the initiation of immunization (D70), rPAc-specific serum IgG, serum IgA, and salivary IgA levels were 10⁵, 10⁴, and 10² respectively (Figure 3(b,c)). In addition, the in vitro biofilm inhibition assay²⁹ showed that serum and saliva from the KFD2-rPAc-immunized group exhibited significant inhibitory effects on S. mutans biofilm formation (Figure 3(d,e)). These results indicate that intranasal immunization with KFD2-rPAc can induce effective systemic and local mucosal immune responses as reported previously,¹³ which are functional in the inhibition of S. mutans biofilm formation in vitro.

Figure 3. The immune responses induced by intranasal immunization of KFD2-rPAc in rats ahead of S. mutans challenge.

(a) Scheme of immunization on rats. 18-day-old weaning female rats were intranasally immunized with PBS alone or 7 μg of KFD2-rPAc in 10-μL aliquot three times with 28 d intervals (n = 6 rats per group). The serum and the saliva were collected after the 3rd immunization (D70). (b and c) The rPAc-specific serum IgG, serum IgA, and salivary IgA responses in rats on day 70 were tested by ELISA. (d and e) The inhibitory effect of 100-fold diluted rat serum and 20-fold diluted rat saliva on biofilm formation. All data represent the mean ± SEM. Significance was determined by Student’s t test. *, p < .05; ****, p < .0001.

To assay the prophylactic effect of the vaccine, an air-restricted cultured S. mutans challenge experiment was performed on these 12- to 13-week-old rats which finished three doses intranasal immunization (Figure 4(a)). At 1 dpi, the number of S. mutans cells in the KFD2-rPAc-immunized group was approximately 72% lower than that in the PBS group (Figure 4(b)). Correlation analysis revealed that the number of S. mutans at 1 dpi negatively correlated with both rPAc-specific serum IgG and rPAc-specific salivary IgA levels before inoculation (Figure 4(c)).

Figure 4. The prophylactic effect of KFD2-rPAc against S. mutans infection in adult rats post-immunization.

(a) The challenge, and sampling schedule on rats after the third immunization. On 15 d after the third immunization (−7 dpi), two groups of immunized rats were fed a Keyes 2000 diet with multiple antibiotics for 5 consecutive days. At 12 hours before S. mutans challenge, the water and food containing antibiotics were removed. Then, the rats were orally infected with 1.7 × 10⁹ CFU fresh S. mutans cultured under air-restricted conditions for three consecutive days (once a day). Oral samples of the tooth occlusal surface were collected from all challenged rats on 1 dpi and 86 dpi before rats were sacrificed. The uninfected control group of rats which had not experienced immunization and challenge throughout the experiment were also sacrificed on 86 dpi. (b) The CFUs of S. mutans from the oral samples collected on 1 dpi (n = 6 samples per group). (c) Spearman’s correlation of CFUs of S. mutans on 1 dpi with rPAc-specific serum IgG titer (left) and rPAc-specific salivary IgA (right) on –8 dpi. (d) The CFUs of S. mutans from the oral samples collected on 86 dpi (n = 6 samples per group). (e) Spearman’s correlation of CFUs of S. mutans on 86 dpi with rPAc-specific serum IgG titer (left) and rPAc-specific salivary IgA (right) on –8 dpi. For (B) and (D), all data represent the mean ± SEM. Significance was determined by Student’s t test. For (C) and (E), dotted lines represent 95% confidence intervals. The correlation coefficients (r) and p values are also shown. *, p < .05; **, p < .01; ***, p < .001.

On 86 dpi, rPAc-specific antibodies in the KFD2-rPAc immunized group were still sustained at levels similar as those on −8 dpi (Supplementary Figure S2). The number of S. mutans cells in the KFD2-rPAc-immunized group was approximately 85% lower than that in the PBS group (Figure 4(d)). Compared to 1 dpi, the difference in the number of S. mutans between the KFD2-rPAc-immunized group and the PBS group increased by approximately 3.5 times on 86 dpi (Supplementary Figure S3). Between the number of S. mutans on the tooth surface and rPAc-specific antibody responses, a significant negative correlation was maintained at 86 dpi (Figure 4(e) and Supplementary Figure S4). These results suggested that intranasal immunization-induced rPAc-specific antibody responses inhibited S. mutans colonization in vivo.

To investigate whether the immune responses could mirror anti-caries effect and assess the protective efficacy of the vaccine, caries lesions were determined on 86 dpi. In the rats of KFD2-PAc immunized and challenged groups, only enamel lesions (E) and slight dentinal lesions (Ds) were observed (Figure 5(a)), similar to the rats without challenge (uninfected). The caries score in the KFD2-rPAc group was not significantly different from that of the uninfected control group (Figure 5(b,c)). The E and Ds caries scores in the KFD2-rPAc group were significantly lower than those in the PBS group (Figure 5(b)). The KFD2-rPAc vaccination group showed an 83% inhibitory efficacy in preventing caries development (Figure 5(c)). Additionally, similar to previous studies,¹³ a positive correlation was observed between the number of S. mutans cells (1 or 86 dpi) and the total caries score (Figure 5(d)). Moreover, negative correlations were observed between total caries score and rPAc-specific serum IgG and salivary IgA levels (Figure 5(e)). Therefore, the newly developed model challenged with air-restricted cultured S. mutans can be used to assess the anti-caries efficacy of prophylactic vaccines.

Figure 5. The prophylactic efficacy of KFD2-rPAc against caries development and the correlation analysis for protective factors.

(a) Representative photographs of caries lesion on 86 dpi. (b) Caries scores of enamel lesions (E), slight dentinal lesions (Ds), moderate dentinal lesions (Dm), and extensive dentinal lesions (Dx) of rats (n = 5 to 6 rats per group). (c) Total score = score of enamel lesions (E) + score of slight dentinal lesions (Ds) + moderate dentinal lesions (Dm) + extensive dentinal lesions (Dx). The inhibitory rate of vaccine-immunized group labeled above the error bar. (d) Pearson’s correlation of total caries score with CFUs of S. mutans in oral samples on 1 dpi (left) and on 86 dpi (right). (e) Spearman’s correlation of total caries score with rPAc-specific serum IgG (left) and rPAc-specific salivary IgA (right) on −8 dpi. For (B) and (C), all data represent the mean ± SEM. Significance was determined by one-way ANOVA. For (D) and (E), dotted lines represent 95% confidence intervals. The correlation coefficients (r) and p values are also shown. **, p < .01; ***, p < .001; ****, p < .0001; ns, nonsignificant.

Discussion

The rapid decrease in the susceptibility of rats to S. mutans infection after weaning makes it difficult to develop a rat model of S. mutans infection after completion of the vaccination schedule. Therefore, to evaluate prophylactic efficacy of caries vaccines, an optimal rat infection model should be established.

In this study, we found that the simple air-restricted culture method caused a subtle change in the proliferation cycles of cultured S. mutans (Figure 1). Surprisingly, the transcriptional levels of the four well-known virulence factors significantly increased under air-restricted conditions. In adult rats, S. mutans under the air-restricted culturing method could colonize the rat tooth surface more readily and cause prominent carious lesions than under air-unrestricted culturing (Figure 2). Compared to the conventional 18- to 21-day-old weaning rat model, this newly established caries model in 12- to 13-week-old adult rats substantially extended the period between vaccine immunization and S. mutans challenge, offering at least 10-weeks for the generation of protective immunity upon vaccination. Therefore, this adult rat caries model offers the opportunity to fully evaluate the prophylactic efficacy of vaccines against S. mutans infection and caries development.

The suitability of the adult rat caries model for studying prophylactic vaccines was confirmed by applying the previously reported subunit mucosal vaccine, KFD2-rPAc (Figures 3–5).¹³ Regarding rPAc-specific antibody responses and the functional inhibition of S. mutans biofilm formation (Figure 3), KFD2-rPAc intranasal immunization before challenge exhibited similar efficacy in inducing protective antibody responses as the previous weaning rat challenge model, in which KFD2-rPAc was intranasally immunized in parallel with infection.¹³

At 1 dpi, the reduced number of S. mutans in the KFD2-rPAc-immunized group compared to that in the PBS group and the negative correlation between the number of S. mutans and the protective rPAc-specific antibody (Figure 4) demonstrated that KFD2-rPAc vaccination prohibited the initial infection of S. mutans. Therefore, this suggests that the infection model is suitable for evaluating the prophylactic effect of the vaccine against S. mutans infection. From 1 to 86 dpi, the difference in the number of S. mutans between the KFD2-rPAc-immunized group and the PBS group increased by approximately 3.5 times (Supplementary Figure S3). This is consistent with previous reports that protective antibodies can also reduce S. mutans colonization after infection.^15,30

Compared to a previous study in which KFD2-rPAc vaccination and S. mutans infection were performed simultaneously in weaning rats,¹³ a full schedule of KFD2-rPAc vaccination could be completed before the challenge; therefore, the prophylactic efficacy of KFD2-rPAc against caries (84% vs. 83% of the inhibitory effect) was confirmed in a new rat model. We propose that the KFD2-rPAc vaccination induces a prophylactic effect on inhibiting the initial colonization of S. mutans on teeth and exhibits a persistent effect on reducing the colonization of S. mutans, both of which could contribute to the prophylactic effect against the development of caries. In contrast, the current study demonstrates the advantages of the optimized rat caries model in terms of feasibility, convenience, and conformity. This optimized adult rat caries model is a better tool for infection studies and the evaluation of prophylactic vaccines against both S. mutans infection and dental caries.

Our study revealed once again that the air/oxygen supply is a critical factor for the proliferation ability before reaching the plateau phase (Figure 1) and for the cariogenicity of this facultative anaerobe, S. mutans (Figures 1 and 2). The results of our study are consistent with previous observations that oxygen could affect the virulence of S. mutans,^31,32 such as biofilm formation.²⁴ One study reported that virulence genes involved in biofilm formation, such as gtfB but not gtfC, were upregulated under anaerobic conditions compared with those under aerobic conditions.³¹ However, under air-restricted culture conditions, not only the gtfB, but also the gtfC were upregulated. In addition, the expression of two other virulence genes, spaP and gbpB, was upregulated. A panel of genes regulated under air-restricted culture conditions warrants further study.

In conclusion, we found that S. mutans cultured under air-restricted conditions can exacerbate dental caries in adult rats. In consistent with the previous weaning rat model, in which challenge was performed accompanied by immunization, the newly established model performed a challenge after completion of the full vaccination schedule. To the best of our knowledge, this is the first report of an effective adult rat caries model, and the first prophylactic vaccine model against the initial infection of S. mutans. In particular, the adult rat caries model is more suitable for evaluating the immune responses elicited by immunization and the correlated prophylactic protection of vaccine candidates against the initial infection of S. mutans and the development of caries. We also believe that this model can be adopted with high compliance in studies on many aspects of S. mutans.

Author contributions

Liu, Bowen: Contributed to conception, design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript.

Li, Min: Contributed to data analysis, drafted manuscript, and critically revised the manuscript.

Li Xian: Contributed to data acquisition, drafted manuscript.

Yang, Jingyi: Contributed to conception, design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript.

Yan, Huimin: Contributed to conception, design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript.

All authors gave final approval and agrees to be accountable for all aspects of work ensuring integrity and accuracy.

Acknowledgments

We especially thank Professor Yanyi Wang of the Wuhan Institute of Virology, CAS for her kind support and help on the project. We thank Xuefang An, Fan Zhang, and Li Li in the core facility of Wuhan Institute of Virology, CAS for their technical support and kind help in animal experiments.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website at https://doi.org/10.1080/21645515.2024.2345943.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the National Key Research & Development Program of China [grant number: 2021YFC2302602], the National Natural Science Foundation of China [31970878], and the Guangzhou Science and Technology Project [201803010042].