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Many studies have investigated the Mincle-mediated agonist activity of α,α'-trehalose-6,6́-glycolipids, however, none have considered how the position, or absence, of the ester moiety influences Mincle-mediated agonist activity. We prepared a variety of 6-C-linked α,α'-trehalose glycolipids containing inverted esters, ketone, carboxy or no carbonyl moieties. Modelling studies indicated that these derivatives bind to the CRD of Mincle in a manner similar to that of the prototypical Mincle agonist, trehalose dibehenate (TDB), with NFAT-GFP reporter cell assays confirming that all compounds, apart from derivatives with short alkyl chains, led to robust Mincle signalling. It was also observed that a carbonyl moiety was needed for good Mincle-mediated signalling. The ability of the compounds to induce mIL-1 β and mIL-6 production by bone marrow-derived macrophages (BMDMs) further demonstrated the agonist activity of the compounds, with the presence of a carbonyl moiety and longer lipid chains augmenting cytokine production. Notably, a C20 inverted ester led to levels of mIL-1β that were significantly greater than those induced by TDB. The same C20 inverted ester also led to a significant increase in hIL-1β and hIL-6 by human monocytes, and exhibited no toxicity, as demonstrated using BMDMs in an in vitro Sytox Green assay. The ability of the inverted ester to enhance antigen-mediated immune responses was then determined. In these studies, the inverted ester was found to augment the OVA-specific Th1/Th7 immune response in vitro, and exhibit adjuvanticity that was better than that of TDB in vivo, as evidenced by a significant increase in IgG antibodies for the inverted ester but not TDB when using OVA as a model antigen., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Mincle, a C-type lectin receptor (CLR), activates the innate immune system by recognizing certain complex lipid compounds. In this study, we designed and synthesized trehalose disteate (TDS) and dibehenate (TDB), containing a polar-functional group in the middle of fatty acid moieties, based on a model of the Mincle-glycolipids interaction. The modified fatty acids were prepared using hydroxy fatty acids as common intermediates, and conjugated with an appropriate trehalose moiety to synthesize the desired trehalose diesters. TDE derivatives containing the modified fatty acid have different Mincle-mediated signaling activities depending on the position of the functional group and the length of the lipids. The newly developed TDE derivatives exhibit signaling activity comparable or superior to that of TDS or TDB, and the results suggest that Mincle tolerates polar functional groups at a certain position of the lipid chain of TDE. The introduction of the polar functional groups into the lipid moiety of the glycolipids also resulted in improved solubility in polar solvents, which would be advantageous for various analyses and applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

The covalent fusion of immunostimulatory adjuvants to immunogenic antigens is a promising strategy for the development of effective synthetic vaccines for infectious diseases. Herein, we describe the conjugation of a mycobacterial peptide antigen from the 6 kDa early secretory antigenic target (ESAT6) to a suitably functionalised trehalose dibehenate (TDB), a potent glycolipid adjuvant targeting macrophage inducible C-type lectin (Mincle).

The cell wall of Mycobacterium tuberculosis is composed of diverse glycolipids which potentially interact with the human immune system. To overcome difficulties in obtaining pure compounds from bacterial extracts, we recently synthesized three forms of mycobacterial diacyltrehalose (DAT) that differ in their fatty acid composition, DAT 1 , DAT 2 , and DAT 3 . To study the potential recognition of DATs by human T cells, we treated the lipid-binding antigen presenting molecule CD1b with synthetic DATs and looked for T cells that bound the complex. DAT 1 - and DAT 2 -treated CD1b tetramers were recognized by T cells, but DAT 3 -treated CD1b tetramers were not. A T cell line derived using CD1b-DAT 2 tetramers showed that there is no cross-reactivity between DATs in an IFN-γ release assay, suggesting that the chemical structure of the fatty acid at the 3-position determines recognition by T cells. In contrast with the lack of recognition of DAT 3 by human T cells, DAT 3, but not DAT 1 or DAT 2 , activates Mincle. Thus, we show that the mycobacterial lipid DAT can be both an antigen for T cells and an agonist for the innate Mincle receptor, and that small chemical differences determine recognition by different parts of the immune system.

The first asymmetric total synthesis of three structures proposed for mycobacterial diacyl trehaloses, DAT 1 , DAT 2 , and DAT 3 is reported. The presence of two of these glycolipids, DAT 1 and DAT 3 , within different strains of pathogenic M. tuberculosis was confirmed, and it was shown that their abundance varies significantly. In mass spectrometry, synthetic DAT 2 possessed almost identical fragmentation patterns to presumptive DAT 2 from Mycobacterium tuberculosis H37Rv, but did not coelute by HPLC, raising questions as the precise relationship of the synthetic and natural materials. The synthetic DATs were examined as agonists for signaling by the C-type lectin, Mincle. The small differences in the chemical structure of the lipidic parts of DAT 1 , DAT 2 , and DAT 3 led to drastic differences of Mincle binding and activation, with DAT 3 showing similar potency as the known Mincle agonist trehalose dimycolate (TDM). In the future, DAT 3 could serve as basis for the design of vaccine adjuvants with simplified chemical structure.

Salmonella species are among the world's most prevalent pathogens. Because the cell wall interfaces with the host, we designed a lipidomics approach to reveal pathogen-specific cell wall compounds. Among the molecules differentially expressed between Salmonella Paratyphi and S. Typhi, we focused on lipids that are enriched in S. Typhi, because it causes typhoid fever. We discovered a previously unknown family of trehalose phospholipids, 6,6'-diphosphatidyltrehalose (diPT) and 6-phosphatidyltrehalose (PT). Cardiolipin synthase B (ClsB) is essential for PT and diPT but not for cardiolipin biosynthesis. Chemotyping outperformed clsB homology analysis in evaluating synthesis of diPT. DiPT is restricted to a subset of Gram-negative bacteria: large amounts are produced by S. Typhi, lower amounts by other pathogens, and variable amounts by Escherichia coli strains. DiPT activates Mincle, a macrophage activating receptor that also recognizes mycobacterial cord factor (6,6'-trehalose dimycolate). Thus, Gram-negative bacteria show convergent function with mycobacteria. Overall, we discovered a previously unknown immunostimulant that is selectively expressed among medically important bacterial species., (© 2019 Reinink et al.)

Trehalose glycolipids (TGLs) are promising vaccine adjuvants, however effects of glycolipid presentation in the in vitro evaluation, and ultimate selection, of lead vaccine adjuvants are often overlooked. To this end, we synthesised a variety of TGLs and determined how the physicochemical presentation of these lipids influenced the cytokine response by bone marrow derived macrophages (BMMs). The TGLs were presented to wild-type and Mincle -/- BMMs as micellar solutions, coated on plates, coated on beads or surfactant solubilised. Medium to long-chain TGLs, either coated on plates or surfactant solubilised, resulted in the highest BMM activation. Stimulation of BMMs with TGLs coated on beads led to a decreased cytokine response, as compared to TGLs alone. All the TGL responses were Mincle dependent, however the mode of presentation did not have the same effect for each individual TGL. This was most apparent for the C22 trehalose monoester, which showed reduced activity compared to its diester counterpart when presented on a plate, but similar activity to the diester when presented as micelles or on beads. Taken together, our findings support the use of several in vitro assays for selecting lead vaccine adjuvants, particularly if structural differences between the adjuvants are pronounced. Graphical abstract The mode of glycolipid presentation, such as micellar solutions, coated on plates, coated on beads or surfactant solubilised, influences the immune response to trehalose glycolipids.

The macrophage inducible C-type lectin (Mincle) is a pattern recognition receptor that recognizes trehalose dimycolate (TDM), and trehalose dibehenate (TDB) and related trehalose diesters, and thus represents a promising target for the development of vaccine adjuvants based on the trehalose glycolipid scaffold. To this end, we report on the synthesis of a series of long-chain α-branched, β-modified trehalose monoesters and diesters to explore how glycolipid structure affects signaling through Mincle. Key steps in our synthetic strategy include a Fráter-Seebach α-alkylation to install the C 20 aliphatic lipid on a malic acid derivative, and the formation of a β,γ-epoxide as an intermediate from which modifications to the β-position of the lipid can be made. Biological evaluation of the derivatives using nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cell lines expressing mMincle or hMincle revealed that the hMincle agonist activity of all diesters was superior to that of the current lead trehalose glycolipid adjuvant TDB, while the activity of several monoesters was similar to that of their diester counterparts for mMincle, but all showed reduced hMincle agonist activity. Taken together, diesters 2d-g are thus potent Mincle agonists and promising vaccine adjuvants.

Effective Th1-stimulating vaccine adjuvants typically activate antigen presenting cells (APCs) through pattern recognition receptors (PRRs). Macrophage inducible C-type lectin (Mincle) is a PRR expressed on APCs and has been identified as a target for Th1-stimulating adjuvants. Herein, we report on the synthesis and adjuvanticity of rationally designed brartemicin analogues containing long-chain lipids and demonstrate that they are potent Mincle agonists that activate APCs to produce inflammatory cytokines in a Mincle-dependent fashion. Mincle binding, however, does not directly correlate to a functional immune response. Mutation studies indicated that the aromatic residue of lead compound 9a has an important interaction with Mincle Arg183. In vivo assessment of 9a highlighted the capability of this analogue to augment the Th1 response to a model vaccine antigen. Taken together, our results show that lipophilic brartemicin analogues are potent Mincle agonists and that 9a has superior in vivo adjuvant activity compared to TDB.

Intravesical Bovis bacillus Calmette-Guérin (BCG) therapy is the most effective immunotherapy for bladder cancer, but it sometime causes serious side effects because of its inclusion of live bacteria. It is necessary to develop a more active but less toxic immunotherapeutic agent. Trehalose 6,6′-dimycolate (TDM), the most abundant hydrophobic glycolipid of the BCG cell wall, has been reported to show various immunostimulatory activities such as granulomagenesis and adjuvant activity. Here, we developed cationic liposomes incorporating TDM purified from Mycobacterium bovis BCG Connaught, and we investigated the antitumor effect of the cationic liposome TDM (Lip-TDM). Lip-TDM exerted an antitumor effect in bladder cancer, colon cancer, and melanoma-bearing mouse models that was comparable or even superior to that of BCG, with no body weight loss or granuloma formation. The antitumor effect of Lip-TDM disappeared in two types of mice: those with depletion of CD8+ T cells, and those with knockout of macrophage-inducible C-type lectin (Mincle) which recognize TDM. Lip-TDM treatment enhanced the maturation and migration of dendritic cells in the tumor microenvironment in a Mincle-dependent manner. Our results elucidate mechanisms that underlie Lip-TDM treatment and suggest that Lip-TDM has potential as a safe and effective treatment for various cancers. [ABSTRACT FROM AUTHOR]

The innate immune receptor Mincle senses lipid-based molecules derived from pathogens, commensals and altered self. Based on emerging structure–activity relationships we design simple alkyl 6-O-acyl-β- D -glucosides that are effective agonists of Mincle and signal with potency on par with the prototypical ligand trehalose dimycolate. [ABSTRACT FROM AUTHOR]

An enantioselective synthesis of (+)-corynomycolic acid, and its elaboration to esters of trehalose, glucose and glycerol, is described. Trehalose dicorynomycolate and trehalose monocorynomycolate activate human and mouse Mincle as effectively as trehalose dicorynomycolate (cord factor). Glucose monomycolate is revealed to be a potent activator of both mouse and human Mincle. Glycerol monocorynomycolate signals through human Mincle, with the activity predominantly residing in the 2′S-isomer. [ABSTRACT FROM AUTHOR]

Summary: Cord factor, also called trehalose-6,6′-dimycolate (TDM), is a potent mycobacterial adjuvant. We herein report that the C-type lectin MCL (also called Clec4d) is a TDM receptor that is likely to arise from gene duplication of Mincle (also called Clec4e). Mincle is known to be an inducible receptor recognizing TDM, whereas MCL was constitutively expressed in myeloid cells. To examine the contribution of MCL in response to TDM adjuvant, we generated MCL-deficient mice. TDM promoted innate immune responses, such as granuloma formation, which was severely impaired in MCL-deficient mice. TDM-induced acquired immune responses, such as experimental autoimmune encephalomyelitis (EAE), was almost completely dependent on MCL, but not Mincle. Furthermore, by generating Clec4e gfp reporter mice, we found that MCL was also crucial for driving Mincle induction upon TDM stimulation. These results suggest that MCL is an FcRγ-coupled activating receptor that mediates the adjuvanticity of TDM. [Copyright &y& Elsevier]

[Display omitted] • Trehalose diamide glycolipids were prepared in excellent yield. • Trehalose diamides signal through the macrophage inducible C-type lectin. • Trehalose diamides are effective vaccine adjuvants. Many studies have investigated how trehalose glycolipid structures can be modified to improve their Macrophage inducible C-type lectin (Mincle)-mediated adjuvanticity. However, in all instances, the ester-linkage of α,ά-trehalose to the lipid of choice remained. We investigated how changing this ester-linkage to an amide influences Mincle signalling and agonist activity and demonstrated that Mincle tolerates this functional group change. In in vivo vaccination studies in murine and ovine model systems, using OVA or Mannheimia haemolytica and Mycoplasma ovipneumoniae as vaccine antigens, respectively, it was demonstrated that a representative trehalose diamide glycolipid was able to enhance antibody-specific immune responses. Notably, IgG titres against M. ovipneumoniae were significantly greater when using trehalose dibehenamide (A-TDB) compared to trehalose dibehenate (TDB). This is particularly important as infection with M. ovipneumoniae predisposes sheep to pneumonia. [ABSTRACT FROM AUTHOR]