Synthetic antigen-binding fragments (Fabs) against S. mutans and S. sobrinus inhibit caries formation

Abstract

Streptococcus mutans and Streptococcus sobrinus are the main causative agents of human dental caries. Current strategies for treating caries are costly and do not completely eradicate them completely. Passive immunization using nonhuman antibodies against Streptococcal surface antigens has shown success in human trials, however they often invoke immune reactions. We used phage display to generate human antigen-binding fragments (Fabs) against S. mutans and S. sobrinus. These Fabs were readily expressed in E. coli and bound to the surface S. mutans and S. sobrinus. Fabs inhibited sucrose-induced S. mutans and S. sobrinus biofilm formation in vitro and a combination of S. mutans and S. sobrinus Fabs prevented dental caries formation in a rat caries model. These results demonstrated that S. mutans and S. sobrinus Fabs could be used in passive immunization strategies to prevent dental caries. In the future, this strategy may be applied towards a caries therapy, whereby Fabs are topically applied to the tooth surface.

Figure 1

Phage display selection of Fabs against S. mutans and S. sobrinus. (a) Phage display selection scheme for isolating Fabs against S. mutans and S. sobrinus. Five rounds of phage display were performed against S. mutans or S. sobrinus. During the fourth and fifth rounds of selection, subtractive panning steps were included. For the S. sobrinus and S. mutans selections, members of the Fab-phage library were removed that bound E. coli and S. mutans and S. sobrinus, respectively. (b) The frequency of Fab-phage eluted during different rounds of selection against S. mutans and S. sobrinus. (c) Frequency of the top CDRL3 and CDRH3 sequences of Fabs against S. mutans and S. sobrinus.

Figure 2

Binding Analyses of anti-S. mutans and ant-S. sobrinus Fabs. (a) Fab ELISA of Fab SM-10, SM-12, SM-C-1, SM-C-4, SS-2, and SS-C-1 against S. mutans and S. sobrinus. E. coli stain DH10B and bovine serum albumin (BSA) were used as negative controls. Binding of Fabs to bacteria- and BSA-coated microtiter plates was detected using HRP-conjugated anti-His antibody. Error bars represent the standard deviation from three independent experiments. (b) Immunofluorescence of selected Fabs against S. mutans and S. sobrinus. Non-specific Fab against the human cell surface receptor EphB6 was used as a negative control. Binding of Fabs to bacteria-coated microtiter plates were detected via FITC-conjugated anti-FLAG antibody.

Figure 3

Binding analysis of Fabs to the cell surface of S. mutans, S. sobrinus, and E. coli using flow cytometry. Fabs binding to S. mutans (a). S. sobrinus (b) and E. coli (c) A nonspecific Fab (NS Fab) against human cell surface receptor EphB6 was used a control.

Figure 4

Inhibition of in vitro biofilm formation. In vitro inhibition of biofilm formation by anti-S. mutans or anti-S. sobrinus Fabs. S. mutans and S. sobrinus were cultured in brain-heart infusion (BHI) with 1% sucrose to induce biofilm formation in presence of Fabs. Positive control: S. mutans or S. sobrinus on BHI with 1% sugar. Negative control: S. mutans or S. sobrinus on BHI. Error bars indicate standard deviation from three independent experiments.

Figure 5

Inhibition of dental caries in rat model. (a) Micro-CT analysis of interproximal caries in S. mutans- and S. sobrinus-infected rats. For in vivo experiments, 15 infection-free rats were randomly divided equally into three groups (group 1, 2 and 3). All groups were infected with S. mutans and S. sobrinus. Group 1 did not receive Fabs. Group 2 only received 2 weeks of Fab treatment. Group 3 received 4 weeks of Fab treatment. At the end of the fourth week, rats were sacrificed, mandibles and maxilla were collected and fixed with 70% ethanol for 24 hours and analyzed by micro-CT. (b) Number of rats in each group showing caries. Caries were detected and counted by micro-CT. (c). Percentage of molars showing caries. Each rat has 12 molars. We checked for caries using micro-CT in a total of 60 molar teeth in each group (5 rats in each group). This experiment was repeated 2 times. Error bars indicate standard deviation. * represents P-values < 0.0001.