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 cell-mediated 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.

Keywords: glycolipid; innate immunity; mycobacteria.

Fig. 1.

Identification of natural and synthetic ligands of Mincle. (A) Chemical structures of TDM, TDB, GlcC14C18, ManC14C18, GlcC16, and GlcC4C12. (B–E) EC₅₀ values determined from the activation curves of HEK-hMincle (B and D) and HEK-mMincle (C and E) after incubation with mycobacterial natural compounds (B and C) or synthetic analogs (D and E). EC₅₀ data are presented as the mean ± SEM calculated from at least three independent experiments (SI Appendix, Fig. S2). All of the compound structures are described in SI Appendix, Fig. S1.

Fig. 2.

Comparison of conformational rearrangements observed from MD simulations in regions D142–Q148 and N170–D177 of Mincle interacting with GlcMCM or Glc3OHC18. The X-ray crystallographic structure of Mincle (Protein Data Bank ID code 4KZV) is shown in blue for reference. The hydrophobic groove is represented as a molecular surface to show how the C18 alkyl chain of GlcMCM (green) binds into the cleft on conformational rearrangement of loop N170–D177, whereas Glc3OHC18 (pink) cannot be properly inserted in the groove.

Fig. 3.

Newly identified Mincle ligands induce proinflammatory cytokines by human and mouse primary cells. BMDCs (A), BMDMs (B), and moMФs (C) were stimulated with 1 µg of plate-bound lipid for 18 h, and TNF-α release in the culture supernatant was determined by ELISA. Mincle and TLR2 dependence were investigated by preincubating cells for 30 min at 37 °C with 5 µg/mL anti-mMincle, anti-hMincle, anti-TLR2, or isotype control antibodies before stimulation with lipid. The Pam₃CSK₄ lipopeptide served as a control TLR2 ligand at a concentration of 100 ng/mL. Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

GlcC14C18 and ManC14C18 have adjuvant activity in vivo. Mice (n = 5) were immunized intradermally three times with 10 µg of the M. tuberculosis antigen Ag85A in DDA, DDA/TDB, DDA/GlcC14C18, or DDA/ManC14C18. The immune response was monitored 3 wk after the last immunization. IL-2 (A), IFN-γ (B), and IL-17 (C) release in the culture supernatant of splenocytes after restimulation with Ag85A, along with serum anti-Ag85A IgG2b titers (D), were determined by ELISA. Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.