Tripalmitoyl-S-glyceryl-cysteine-dependent OspA vaccination of toll-like receptor 2-deficient mice results in effective protection from Borrelia burgdorferi challenge

Abstract

Toll-like receptor 2 (TLR2) is a transmembrane signal transducer for tripalmitoyl-S-glyceryl-cysteine (Pam(3)Cys)-modified lipoproteins, including OspA from the Lyme disease spirochete Borrelia burgdorferi. The Pam(3)Cys modification provides adjuvant activity for inducing humoral responses, suggesting that TLR2 could function as the adjuvant receptor for the OspA vaccine. The importance of TLR2 in the humoral response to OspA was confirmed, because overall levels of immunoglobulin G (IgG) were reduced in TLR2-deficient mice, when compared with those in wild-type mice. However, the levels of production of IgG1 were similar in both mouse strains, and the levels of induction of protective immunity were comparable. Unlipidated OspA was not immunogenic in wild-type or TLR2-deficient mice, indicating the lipid modification was active in the absence of TLR2. These findings indicate that the Pam(3)Cys modification of bacterial lipoprotein has adjuvant properties independent of TLR2 signaling.

FIG. 1.

The OspA vaccine fails to stimulate NO production in macrophages from TLR2^−/− C3H/HeJ mice. Bone marrow macrophages from wild-type (WT) and TLR2^−/− C3H/HeJ mice were incubated with 100 ng of LPS per ml or the indicated concentrations of OspA, detergent-free OspA, unlipidated OspA, or sonicated B. burgdorferi. Supernatants were collected at 24 h and assayed for nitrate production by the Greiss reaction. Values are means ± standard deviations for triplicate samples.

FIG. 2.

The OspA vaccine fails to induce proliferation of splenocytes from TLR2^−/− C3H/HeJ mice. Single-cell suspensions of splenocytes taken from wild-type and TLR2^−/− C3H/HeJ mice were incubated with 100 ng of LPS per ml or the indicated concentrations of unlipidated OspA, lipidated OspA (OspA), sonicated B. burgdorferi, or Pansorbin (heat-treated Staphylococcus aureus) for 72 h. Splenocyte proliferation was determined by [³H]thymidine uptake during the final 20 h of culture. Values are means ± standard deviations for triplicate samples.

FIG. 3.

Effect of TLR2 deficiency on anti-OspA IgG production in OspA-vaccinated C3H/HeJ mice. Anti-OspA titers of sera collected from wild-type (WT) and TLR2^−/− C3H/HeJ mice 1 week following the secondary immunization with OspA, unlipidated OspA, or PBS controls were determined by ELISA with OspA-coated plates.

FIG. 4.

Effect of TLR2 deficiency on isotype distribution in OspA-vaccinated C3H/HeJ mice. Sera collected from OspA-vaccinated wild-type (WT) and TLR2^−/− C3H/HeJ mice 1 week following secondary immunization were assessed for OspA-specific IgG1 (A), IgG2a (B), IgG2b (C), IgG3 (D), IgM (E), and IgE (F) by ELISA on OspA-coated plates.