Anti-caries DNA vaccine-induced secretory immunoglobulin A antibodies inhibit formation of Streptococcus mutans biofilms in vitro

Abstract

Aim: To investigate the effects of anti-caries DNA vaccine-induced salivary secretory immunoglobulin A (S-IgA) antibodies on Streptococcus mutans (S. mutans) adherence and biofilms formation in vitro.

Methods: Adult female Wistar rats were intranasally immunized with the anti-caries DNA vaccine pGJA-P/VAX. Their saliva samples were collected at different times after the immunization, and S-IgA antibody level in the saliva and its inhibition on S. mutans adherence were examined. The effects of S-IgA in the saliva with the strongest inhibitory effects were examined at 3 different stages, ie acquired pellicles, biofilm formation and production of mature biofilms. The number of viable bacteria and depth of the biofilm at 16 h in each stage were determined using counting colony forming units and using a confocal laser scanning microscopy (CLSM). The participation of S-IgA in acquired pellicles and its aggregation with S. mutans were also observed under CLSM.

Results: The S-IgA titer in saliva reached its peak and exhibited the strongest inhibition on S. mutans adhesion at 10 weeks after the immunization. The colonies and depth of the biofilm in the saliva-pretreated group were 41.79% and 41.02%, respectively, less than the control group. The colonies and depth of the biofilm in the co-culture group were 27.4% and 22.81% less than the control group. The assembly of S. mutans and S-IgA was observed under CLSM after co-cultivation. In the mature-stage biofilm, no differences were observed between the different groups.

Conclusion: These results demonstrate that the anti-caries DNA vaccine induces the production of specific S-IgA antibodies that may prevent dental caries by inhibiting the initial adherence of S. mutans onto tooth surfaces, thereby reducing the accumulation of S. mutans on the acquired pellicles.

Figure 1

The specific secretory anti-Pac and anti-Glu S-IgA antibody levels detected in individual rats after immunization with pGJA-P/VAX or pVAX1. Mean±SD. n=3. ^bp<0.05, ^cP<0.01 vs 0 week.

Figure 2

Adherence inhibition analysis of salivary sample. (A) The adherence of S. mutans at different time points was significantly lower in saliva of immunized rats than that in the control group, but there were no significant differences between the 4th, 10th, and 16th week. (B) Different dilutions of saliva could inhibit the adherence of S. mutans, and the lowest dilution with effect was 1:16. Mean±SD. n=3. ^bp<0.05, ^cP<0.01 vs control.

Figure 3

Scanning electron microscopy (SEM) investigation. Surface of hydroxyapatite disks at 2000× (A) and 5000× (B) magnification (HA disk was a standard hexagonal structure). S. mutans 16 h biofilm at 8000× (C) and 20000× (D) magnification (S. mutans biofilm uniformly covered the surface of the HA disks). The surface of the hydroxyapatite disks at 2000× (E) and 5000× (F) magnification after biofilm resuspension (few remaining bacteria were observed: arrows).

Figure 4

Effect of S-IgA pretreated HA disks on biofilm formation. Acquired pellicle formed by saliva from groups A and B. The pellicle formed by saliva from group A could be observed to have more S-IgA (green) on the HA disks than the pellicle formed by saliva from group B.

Figure 5

Confocal microscopic image showing distribution of S-IgA and S. mutans in depositon after co-cultivating saliva containing S-IgA and S. mutans onto acquied pellicles. Two fluorescence images were taken from the same microscopic field. 1: Fluorescence imaging of S. mutans (red); 2: Fluorescence imaging of S-IgA (green); 3: Merged fluorescence imaging of 1 and 2. (A) The green fluorescence generally presents around the red fluorescence, indicating S-IgA could assemble S. mutans together. (B) The green fluorescence could be seen superposed with the red fluorescence, indicating S-IgA could cover the surface of S. mutans. (C) The green fluorescence is around or superposed with the red fluorescence, demonstrating S-IgA distributes around and among S. mutans, indicating S-IgA may have the effect by both covering and assembling the S. mutans.