Role of Toll-like receptors in host responses to a virulence antigen of Streptococcus mutans expressed by a recombinant, attenuated Salmonella vector vaccine

Abstract

In the present study, we investigated the role of Toll-like receptors (TLRs) in host responses to the saliva-binding region (SBR) of Streptococcus mutans expressed by a recombinant, attenuated Salmonella vaccine. C57BL/6 wild type (wt), TLR2-/-, TLR4-/- and MyD88-/- mice were immunized by the intranasal route on days 0, 18 and boosted on day 98 with Salmonella typhimurium BRD 509 containing a plasmid encoding SBR. Serum and saliva samples were collected throughout the experiment and assessed for antibody activity by ELISA. Evidence is provided that the induction of a serum IgG2a (Th1-type) anti-SBR antibody response involved TLR2 signaling, whereas the anti-Salmonella response involved signaling through TLR4. The adaptor molecule MyD88 was not essential for the induction of a primary Th1-type response to SBR or Salmonella, but was necessary for a secondary response to SBR. Furthermore, the absence of TLR2, TLR4 or MyD88 resulted in enhanced Th2-type serum IgG1 anti-SBR and anti-Salmonella responses. Mucosal IgA responses to SBR were TLR2-, TLR4- and MyD88-dependent, while IgA responses to Salmonella were TLR4- and MyD88-dependent.

Fig. 1

Time course of the serum IgG anti-SBR (A, C, E) and anti-Salmonella (B, D, F) antibody response in mice following i.n. immunization with a Salmonella vaccine clone expressing SBR under the control of the T7 promoter. Both groups of mice were given a single dose of 10⁹ cfu of the Salmonella clone on days 0, 18 and 98. The levels of IgG (A–B), IgG1 (C–D) and IgG2a (E–F) antibody activity to SBR and Salmonella were determined by ELISA. The results are expressed as the mean ×/÷ SD. ^ Indicates time of immunization. Values were significantly different from those of wt mice at P < 0.05 (*) and P < 0.005 (**).

Fig. 2

Time course of the serum IgG anti-SBR (A, C, E) and anti-Salmonella (B, D, F) antibody response in C57BL/6, TLR2^−/− and TLR4^−/− mice following i.n. immunization with a Salmonella vaccine clone expressing SBR under the control of the T7 promoter. Immunization procedure is as described in legend to Figure 1. The results are expressed as the mean ×/÷ SD. ^ Indicates time of immunization. Values were significantly different from those of wt mice at P < 0.05 (*) and values were significantly different between TLR2^−/− and TLR4^−/− at P <0.05 (‡).

Fig. 3

Ratio of serum IgG2a/IgG1 antibodies responses to the cloned antigen SBR (A) and to the Salmonella (B). The results are shown as the mean ×/÷ SD of the IgG2a/IgG1 ratios from pooled serum samples. Values were significantly different from those of wt mice at P <0.05 (*) and P <0.005 (**).

Fig. 4

% specific IgA antibody/total IgA antibody level to SBR (A) and to Salmonella (B). Immunization procedure is as described in legend to Figure 1. The results are expressed as the means of the % specific IgA antibody/total IgA antibody ×/÷ SD. Values were significantly different from those of wt mice at P < 0.05 (*) and P < 0.005 (**). ^ Indicates time of immunization.

Fig. 5

T-cell proliferative responses of SBR (A) or Salmonella (B)-specific CD4⁺ T cells isolated 2 wks after the last immunization of mice. Immunization procedure is as described in legend to Figure 1. CD4⁺ T cells (2 × 10⁶/ ml) were isolated from the spleens and were co-cultured with irradiated, T cell-depleted splenic feeder cells (5 × 10⁶/ml) in the presence or absence of SBR (2 µg/ml) or of killed Salmonella (10⁷ equivalent cfu/ml). Proliferation of antigen-specific CD4⁺ T cells was determined following the addition of 0.5 microCi/well of [³H]TdR for the last 18 h of a 96-h culture. The results are expressed as the mean ×/÷ SD. Values were significantly different from those of wt mice at P < 0.05 (*) and P < 0.005 (**).