Rational design of adjuvants targeting the C-type lectin Mincle

Abstract

The advances in subunit vaccines development have intensified the search for potent adjuvants, particularly adjuvants inducing cellmediated immune responses. Identification of the C-type lectin Mincle as one of the receptors underlying the remarkable immunogenicity of the mycobacterial cell wall, via recognition of trehalose-6,6′-dimycolate (TDM), has opened avenues for the rational design of such molecules. Using a combination of chemical synthesis, biological evaluation, molecular dynamics simulations, and protein mutagenesis, we gained insight into the molecular bases of glycolipid recognition by Mincle. Unexpectedly, the fine structure of the fatty acids was found to play a key role in the binding of a glycolipid to the carbohydrate recognition domain of the lectin. Glucose and mannose esterified at O-6 by a synthetic α-ramified 32-carbon fatty acid showed agonist activity similar to that of TDM, despite their much simpler structure. Moreover, they were seen to stimulate proinflammatory cytokine production in primary human and murine cells in a Mincle-dependent fashion. Finally, they were found to induce strong Th1 and Th17 immune responses in vivo in immunization experiments in mice and conferred protection in a murine model of Mycobacterium tuberculosis infection. Here we describe the rational development of new molecules with powerful adjuvant properties. mycobacteria | glycolipid | innate immunity

The Mincle ligand trehalose dibehenate differentially modulates M1‐like and M2‐like macrophage phenotype and function via Syk signaling

“…Thus, the ability to deplete tumor-associated macrophages (TAMs) or convert them from an M2-like phenotype to a more tumor-suppressive phenotype represents a promising new approach for anti-cancer therapies [32]. As TDB and related trehalose glycolipids exhibit anti-cancer activities and have been found to be effective adjuvants [9][10][11][12][13][14][15], we compared the effect of TDB on M1and M2-like macrophages.…”

Synthetic mycobacterial molecular patterns partially complete Freund’s adjuvant

“…Recently, a rationally determined minimal chemical structure of TDM for Mincle recognition was produced (called GlcC14C18) (fig. 4A and B) which was reported to retain activity as a Mincle agonist without the ‘toxicity’ associated with TDM 23 . We compared TDM and GlcC14C18 at a 10 µg dose in Mincle +/+ and Mincle -/-mice to confirm if GlcC14C18 produced less pathological inflammation and whether inflammation was Mincle -dependent in our mouse model.…”

“…While in vitro assays showed clear synergy between TDM and MDP, in vivo experiments were limited by overt immunopathology from TDM. That TDM was able to synergize with MDP drove us to test GlcC14C18, which had been branded as a similarly efficacious, but non-toxic Mincle agonist 23 . GlcC14C18 together with N -glycolyl MDP was able to partly recapitulate CFA in the OVA model.…”

“…TDM is thought to be the main Mincle ligand in mycobaria; however, there are other ligands. Purified from H37Rv, trehalose-6,6’-monomycolate, glucose monomycolate, diacyl-trehalose and triacyl-trehalose were shown to stimulate mouse and human Mincle (plus glycerol monomycolate stimulates human Mincle only) 23 . It is possible that the phenotype of reduced immunity was because the sensing of these other MAMPs was decreased.…”

“…Emulsification was accomplished using all-plastic syringes and repeated passage through an 18-G blunt-end needle. Where IFA was complemented with mycobacterial components: MDP (Invivogen) was diluted in the PBS fraction of the adjuvant before emulsification; TDM (Sigma) or GlcC14C18 23 were dissolved in the IFA fraction before emulsification as described previously 17 ; heat-killed mycobacteria in saline were diluted in PBS and added to the PBS fraction of the adjuvant before emulsification for mice to receive an equivalent of 10 8 CFU. To make heat-killed mycobacteria, cultures were grown to equivalent 600-nm absorbance at mid-log phase, were pelleted and washed three times with saline, then heat-killed at 100°C for 30 minutes and frozen at −80°C until use.…”