Combination of the cationic surfactant dimethyl dioctadecyl ammonium bromide and synthetic mycobacterial cord factor as an efficient adjuvant for tuberculosis subunit vaccines

Abstract

Recombinant, immunodominant antigens derived from Mycobacterium tuberculosis can be used to effectively vaccinate against subsequent infection. However, the efficacy of these recombinant proteins is dependent on the adjuvant used for their delivery. This problem affects many potential vaccines, not just those for tuberculosis, so the discovery of adjuvants that can promote the development of cell-mediated immunity is of great interest. We have previously shown that the combination of the cationic surfactant dimethyl dioctadecyl ammonium bromide and the immunomodulator modified lipid A synergistically potentiates Th1 T-cell responses. Here we report a screening program for other adjuvants with reported Th1-promoting activity and identify a second novel adjuvant formulation that drives the development of Th1 responses with an extremely high efficacy. The combination of dimethyl dioctadecyl ammonium bromide and the synthetic cord factor trehalose dibehenate promotes strong protective immune responses, without overt toxicity, against M. tuberculosis infection in a vaccination model and thus appears to be a very promising candidate for the development of human vaccines.

FIG. 1.

Effective induction of Th1 responses with DDA-based adjuvant formulations is dependent on efficient immunomodulators. IFN-γ (in nanograms per milliliter) from restimulated PBMCs was measured as an indicator of systemic immune responses. Mice (four animals per group) were bled 1 week after the final immunization, and PBMCs were pooled within groups and restimulated with the vaccine antigen in triplicate wells. The figure shows representative data from three separate experiments. Values are the means of triplicate assays ± standard errors of the means. The level of response observed with the DDA-ESAT-6 combination is indicated by the dotted line for ease of comparison. **, values significantly above this level (P < 0.01).

FIG. 2.

Kinetics of the IFN-γ response in C57BL/6 mice immunized with subunit vaccines. Mice were immunized with either BCG or ESAT in a variety of adjuvants, and IFN-γ from the restimulation of PBMCs (in nanograms per milliliter) was measured as an indicator of systemic immune responses. Mice (four animals per group) were used for tissue samples either the day before the first vaccination (day −1), 1 week after the final vaccination (day 7), 3 weeks after the final vaccination (day 21), or 8 weeks after the final vaccination (day 56). Cells from blood or spleen were pooled within groups and restimulated in triplicate wells. Kinetic studies were performed with spleen and blood cells for all adjuvants assessed, using at least two time points after vaccination, with comparable results.

FIG. 3.

Efficacy of MPL and TDB as immunomodulators in an ESAT-6 subunit vaccine. Testing of MPL and TDB in combination revealed a strong additive potential as assessed by IFN-γ production, but this was not reflected in a correspondingly high level of protection. (A) Systemic immune responses determined by IFN-γ production after in vitro restimulation with ESAT-6 of PBMCs harvested from mice (four per group) 1 week after the final immunization (blood was pooled within groups). Results are presented as means of triplicate wells ± standard deviations. (B) Protective efficacy of the vaccines, expressed as the log₁₀ reduction in bacterial load in the lung, compared to the unimmunized group (naïve). Results are from four mice per group, assayed individually. N.A., not applicable. * and **, values significantly different from the naïve control (P < 0.05 and P < 0.01, respectively).