Adjuvant formulation structure and composition are critical for the development of an effective vaccine against tuberculosis

Abstract

One third of the world is infected with Mycobacterium tuberculosis (Mtb) with eight million new cases of active tuberculosis (TB) each year. Development of a new vaccine to augment or replace the only approved TB vaccine, BCG, is needed to control this disease. Mtb infection is primarily controlled by TH1 cells through the production of IFN-γ and TNF which activate infected macrophages to kill the bacterium. Here we examine an array of adjuvant formulations containing the TLR4 agonist GLA to identify candidate adjuvants to pair with ID93, a lead TB vaccine antigen, to elicit protective TH1 responses. We evaluate a variety of adjuvant formulations including alum, liposomes, and oil-in-water emulsions to determine how changes in formulation composition alter adjuvant activity. We find that alum and an aqueous nanosuspension of GLA synergize to enhance generation of ID93-specific TH1 responses, whereas neither on their own are effective adjuvants for generation of ID93-specific TH1 responses. For GLA containing oil-in-water emulsions, the selection of the oil component is critical for adjuvant activity, whereas a variety of lipid components may be used in liposomal formulations of GLA. The composition of the liposome formulation of ID93/GLA does alter the magnitude of the TH1 response. These results demonstrate that there are multiple solutions for an effective formulation of a novel TB vaccine candidate that enhances both TH1 generation and protective efficacy.

Keywords: Alum; BCG; Bacillus Calmette–Guérin; GLA; Liposomes; Mtb; Mycobacterium tuberculosis; Oil-in-water emulsions; SE; TB; Vaccine formulation; glucopyranosyl lipid adjuvant; stable emulsion; tuberculosis.

Fig. 1

Alum and GLA synergize to promote protective TH1 responses to ID93. One month after the third immunization splenocytes from immunized mice were stimulated with ID93 and analyzed for (A) IL-5 production or (B and C) cytokine production by CD4 T cells. (D and E) Mtb burdens in the lung and spleen were determined four weeks after challenge with a low dose of aerosolized Mtb. Data are representative at least two experiments with three to four mice per group for immunogenicity and seven mice per group for Mtb challenge. * indicates P < 0.05 compared to the unadjuvanted group.

Fig. 2

Squalene is required for an oil-in-water emulsion formulation of GLA to augment protective TH1 responses to ID93. One month after the third immunization splenocytes from immunized mice were stimulated with ID93 and analyzed for (A) IL-5 production or (B and C) cytokine production by CD4 T cells. (D and E) Mtb burdens in the lung and spleen were determined three weeks after challenge with a low dose of aerosolized Mtb. Data are representative at least two experiments with three to four mice per group for immunogenicity and seven mice per group for Mtb challenge. * and ***indicate P < 0.05 and P < 0.001 compared to the unadjuvanted group, respectively.

Fig. 3

Liposomal formulations of GLA enhance protective TH1 responses to ID93. One month after the third immunization splenocytes from immunized mice were stimulated with ID93 and analyzed for (A) IL-5 production or (B and C) cytokine production by CD4 T cells. (D and E) Mtb burdens in the lung and spleen were determined three weeks after challenge with a low dose of aerosolized Mtb. Data are representative at least two experiments with three to four mice per group for immunogenicity and seven mice per group for Mtb challenge. * indicates P < 0.05 compared to the unadjuvanted group.

Fig. 4

The SE formulation induces more terminally differentiated ID93 specific TH1 responses than the liposome formulation of ID93/GLA. (A) Splenocytes and (B) lung from immunized mice were analyzed for ID93 specific CD4 T cell responses one week after the final immunization. (C) Splenocytes were also analyzed for the frequency of ID93 specific CD4 T cells by tetramer staining. Representative samples for ID93/GLA-SE and ID93/GLA-liposome immunizations are shown on the top and bottom rows respectively. (D) The frequency of tetramer positive cells was determined for each group. (E) The frequencies of MPEC and SLEC were determined based on expression of either: CD27 and CD62L, PD1 and KLRG1, or Ly6C and T-bet. Data are representative of at least two experiments with three to four mice per group.

Fig. 5

ID93/GLA-SE is more immunogenic and protective than ID93/GLA-anionic. One month after the third immunization (A) splenocytes and (B) lung lymphocytes from immunized mice were stimulated with ID93 and analyzed for cytokine production by CD4 T cells. (C and D) Mtb burdens in the lung and spleen were determined four weeks after challenge with a low dose of aerosolized Mtb. Data are representative of at least two experiments with three to four mice per group for immunogenicity and seven mice per group for Mtb challenge. *, **, ***, and **** indicate P < 0.05, 0.01, 0.001 and 0.0001 compared to the saline immunized group, respectively.