Aryl Trehalose Derivatives as Vaccine Adjuvants for Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis (Mtb) continues to be a major health threat worldwide, and the development of Mtb vaccines could play a pivotal role in the prevention and control of this devastating epidemic. Th17-mediated immunity has been implicated in disease protection correlates of immune protection against Mtb. Currently, there are no approved adjuvants capable of driving a Th17 response in a vaccine setting. Recent clinical trial results using trehalose dibehenate have demonstrated a formulation-dependant proof of concept adjuvant system CAF01 capable of inducing long-lived protection. We have discovered a new class of Th17-inducing vaccine adjuvants based on the natural product Brartemicin. We synthesized and evaluated the capacity of a library of aryl trehalose derivatives to drive immunostimulatory reresponses and evaluated the structure-activity relationships in terms of the ability to engage the Mincle receptor and induce production of innate cytokines from human and murine cells. We elaborated on the structure-activity relationship of the new scaffold and demonstrated the ability of the lead entity to induce a pro-Th17 cytokine profile from primary human peripheral blood mononuclear cells and demonstrated efficacy in generating antibodies in combination with tuberculosis antigen M72 in a mouse model.

Figure 1.

Representative Mincle ligands TDM, sTDCM, TDB, Vizantin, Brartemicin and C18MeBrar.

Figure 2.

Bovine Mincle (Protein data bank 4KZV) with trehalose (light blue) CRD.

Figure 3.

TNFα cytokine production in response to synthetic aryl trehalose compounds and TDB as reference in murine assays. A) The indicated compound was dissolved in IPA, serially diluted in IPA and then dried to the bottom of a tissue culture plate. B) The indicated compound was dissolved in DMSO, serially diluted in cell culture media. RAW cells were added to both conditions and incubated at 37 °C 18–24 hr followed by assessment of TNFα production by ELISA. C) The indicated compound was dissolved in isopropanol, serially diluted in vehicle and then dried to the bottom of a tissue culture plate. D) The indicated compound was dissolved in DMSO and serially diluted in cell culture media. Freshly prepared human PBMCs were added to both conditions and incubated at 37 °C. TNFα production was measured by ELISA, n=3 experimental replicates for A, B displayed as the average. Data presented in C, D are from an individual human donor and is representative of 3 donors evaluated.

Figure 4.

Cytokine production from primary human mononuclear cells in response to stimulation with synthetic trehalose diester compounds. The indicated compound was dissolved in 50% isopropanol/isooctane, serially diluted in vehicle and then dried to the bottom of a tissue culture plate. Cryopreserved PBMCs were thawed and applied to the compound-coated plates and incubated at 37°C; supernatant was harvested 24 h later and analyzed for TNFα, IL-1β, IL-23, and IL-6 via a multiplex cytokine array (Luminex). Data presented are from an individual representative donor and are shown as percent maximal cytokine induced at each dose level.

Figure 5.

Cytokine production of UM1046–48, UM1068 and UM1087, Brartemicin, TDB and UM1024. A) The indicated compound was dissolved in ethanol, and dried to the bottom of a tissue culture plate. RAW cells were added and incubated at 37 °C for 18–24 hours. TNFα secretion was measured by ELISA. B) The indicated compound was dissolved in DMSO and serially diluted in media. RAW cells were added to culture wells and incubated at 37 °C for 18–24 hours. TNFα secretion was measured by ELISA. C) The indicated compound was dissolved in ethanol, and dried to the bottom of a tissue culture plate. hPBMCs were added and incubated at 37 °C for 18–24 hours. IL-6 secretion was measured by ELISA. D) The indicated compound was dissolved in DMSO and serially diluted in media and added to fresh hPBMCs. Cells were incubated at 37 °C for 18–24 hours after which IL-6 secretion was measured by ELISA. n=3 for all experiments and displayed as the average.

Figure 6.

Activation of mouse and human Mincle in response TDM, TDB and UM1024. The indicated compounds were plate coated and HEK cells transfected with human or mouse Mincle and an NF-κB-driven SEAP reporter were incubated with the compounds for 24 h followed by assessment of the supernatants for SEAP levels. Data are represented as fold change in OD650 over vehicle treated cells. Graphs are mean values from three independent experiments ± SEM.

Figure 7.

High-ranking docking pose of UM1024 (purple) in bovine Mincle (Protein data bank 4KZV) overlays with trehalose (light blue) and maintains similar interactions with bound calcium (yellow).

Figure 8.

Serum antibody levels from mice in response to vaccination with recombinant M72 antigen and various Mincle stimulating adjuvants. BALB/c mice, 10 per group, were vaccinated twice, IM, with 1 μg antigen plus 10 nmol of the indicated adjuvant or volume-matched blank liposomes. Serum was analyzed 14 days post-secondary vaccination for presence of anti-M72 total IgG, IgG1, and IgG2a antibodies via ELISA.

Figure 9.

Induction of IL-17 producing CD4+ T cells from UM1024 adjuvanted M72 vaccine. BALB/c mice, 10 per group, were immunized two times i.m. with 0.1 μg antigen plus 2, 10 or 50 nmol of the indicated adjuvant. Spleens were harvested from 3 mice per group at 5 days post-secondary vaccination. Splenocytes were restimulated with 1 μg whole antigen and transport inhibitors followed by surface staining for CD3, CD4 and CD8 and intracellular cytokine staining for IL-17A. Data represent percentage of live, CD3⁺/CD4⁺ cells that are also positive for IL-17A upon antigen restimulation.

Scheme 1.

Synthesis of aryl trehalose library Reagents and conditions: (a) Aryl acid, N,N’-Dicyclohexylcarbodiimide, DPTS, methylene chloride; (b) Dowex 50W8X, methylene chloride, methanol.

Scheme 2.

Coupling of 2-hydroxy benzoic ads to trehalose Reagents and conditions: (a) Aryl acid, potassium trimethylsilanolate; (b) i, trimethylsilyltriflate, methylene chloride, pyridine; ii, 2, toluene, 18-crown-6, 80 °C; (c) Dowex 50W8X, methylene chloride, methanol.

Scheme 3.

Preparation of trehalose caffeic acid derivative UM1021 Reagents and conditions: (a) Aryl acid, DCC, DPTS, CH₂Cl₂; (b) DBU (c) Dowex 50W8X, CH₂Cl₂, MeOH

Scheme 4.

Expanded SAR of lead Mincle agonist UM1024 Reagents and conditions: (a) Aryl acid, potassium trimethylsilanolate; (b) i, trimethylsilyltriflate, methylene chloride, pyridine; ii, 2, toluene, 18-crown-6, 80 °C; (c) Dowex 50W8X, methylene chloride, methanol.