Synthetic mycobacterial diacyl trehaloses reveal differential recognition by human T cell receptors and the 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, DAT1, DAT2, and DAT3. 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. DAT1- and DAT2-treated CD1b tetramers were recognized by T cells, but DAT3-treated CD1b tetramers were not. A T cell line derived using CD1b-DAT2 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 DAT3 by human T cells, DAT3, but not DAT1 or DAT2, 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.

Formation of Lung Inducible Bronchus Associated Lymphoid Tissue Is Regulated by Mycobacterium tuberculosis Expressed Determinants.

Mycobacterium tuberculosis (Mtb) is the causative agent of the infectious disease tuberculosis (TB), which is a leading cause of death worldwide. Approximately one fourth of the world's population is infected with Mtb. A major unresolved question is delineating the inducers of protective long-lasting immune response without inducing overt, lung inflammation. Previous studies have shown that the presence of inducible Bronchus-Associated Lymphoid Tissue (iBALT) correlate with protection from Mtb infection. In this study, we hypothesized that specific Mtb factors could influence the formation of iBALT, thus skewing the outcome of TB disease. We infected non-human primates (NHPs) with a transposon mutant library of Mtb, and identified specific Mtb mutants that were over-represented within iBALT-containing granulomas. A major pathway reflected in these mutants was Mtb cell wall lipid transport and metabolism. We mechanistically addressed the function of one such Mtb mutant lacking mycobacteria membrane protein large 7 (MmpL7), which transports phthiocerol dimycocerosate (PDIM) to the mycobacterial outer membrane (MOM). Accordingly, murine aerosol infection with the Mtb mutant Δmmpl7 correlated with increased iBALT-containing granulomas. Our studies showed that the Δmmpl7 mutant lacking PDIMs on the surface overexpressed diacyl trehaloses (DATs) in the cell wall, which altered the cytokine/chemokine production of epithelial and myeloid cells, thus leading to a dampened inflammatory response. Thus, this study describes an Mtb specific factor that participates in the induction of iBALT formation during TB by directly modulating cytokine and chemokine production in host cells.

Species-Specific Structural Requirements of Alpha-Branched Trehalose Diester Mincle Agonists.

Despite the ever present need for an effective Mycobacterium tuberculosis (Mtb) vaccine, efforts for development have been largely unsuccessful. Correlates of immune protection against Mtb are not wholly defined, but Th1 and likely Th17 adaptive immune responses have been demonstrated to be necessary for vaccine-mediated protection. Unfortunately, no approved adjuvants are able to drive a Th17 response, though recent clinical trials with CAF01 have demonstrated proof of concept. Herein we present the discovery and characterization of a new class of potential Th17-inducing vaccine adjuvants, alpha-branched trehalose diester molecules (αTDE). Based off the Mtb immunostimulatory component trehalose dimycolate (TDM), we synthesized and evaluated the immunostimulatory capacity of a library of structural derivatives. We evaluated the structure activity relationship of the compounds in relation to chain length and engagement of the Mincle receptor, production of innate cytokines from human and murine cells, and a pro-Th17 cytokine profile from primary human peripheral blood mononuclear cells. Murine cells displayed more structural tolerance, engaging and responding to a wide array of compound chain lengths. Interestingly, human cells displayed a unique specificity for ester chains between 5 and 14 carbons for maximal immune stimulating activity. Evaluation of two distinct αTDEs, B16 and B42, in concert with a recombinant Mtb antigen demonstrated their ability to augment a Th17 immune response against a Mtb antigen in vivo. Collectively this data describes the species-specific structural requirements for maximal human activity of alpha-branched trehalose diester compounds and demonstrates their capacity to serve as potent Th17-inducing adjuvants.

Influence of trehalose 6,6'-diester (TDX) chain length on the physicochemical and immunopotentiating properties of DDA/TDX liposomes.

Linking physicochemical characterization to functional properties is crucial for defining critical quality attributes during development of subunit vaccines toward optimal safety and efficacy profiles. We investigated how the trehalose 6,6'-diester (TDX) chain length influenced the physicochemical and immunopotentiating properties of the clinically tested liposomal adjuvant composed of dimethyldioctadecylammonium (DDA) bromide and analogues of trehalose-6,6'-dibehenate (TDB). TDB analogues with symmetrically shortened acyl chains [denoted X: arachidate (A), stearate (S), palmitate (P), myristate (Myr) and laurate (L)] were incorporated into DDA liposomes and characterized with respect to size, polydispersity index, charge, thermotropic phase behavior and lipid-lipid interactions. Incorporation of 11 mol% TDX into DDA liposomes significantly decreased the polydispersity index when TDA, TDS, TDP and TDMyr were incorporated, whereas both the initial size and the charge of the liposomes were unaffected. The long-term colloidal stability was only decreased when including TDL in DDA liposomes. The fatty acid length of TDX affected the phase transition of the liposomes, and for the DDA/TDP and DDA/TDS liposomes a homogeneous distribution of the lipids in the bilayer was indicated. The membrane packing was studied further by using the Langmuir monolayer technique. Incorporation of TDS improved the packing of the lipid monolayer, as compared to the other analogues, suggesting the most favorable stability. Finally, immunization of mice with the recombinant tuberculosis fusion antigen Ag85B-ESAT-6-Rv2660c (H56) and the physicochemically most optimal formulations (DDA/TDB, DDA/TDS and DDA/TDP) induced comparable T-cell responses. In conclusion, of the investigated TDB analogues, incorporation of 11 mol% TDS or TDP into DDA liposomes resulted in an adjuvant system with the most favorable physicochemical properties and an immunological profile comparable to that of DDA/TDB.

Long-chain lipids are required for the innate immune recognition of trehalose diesters by macrophages.

Going to any length? Trehalose diesters of various chain lengths have been synthesised in order to determine the effect of lipid length on innate immune recognition, as determined by NO and cytokine production by macrophages. In this work, we show that longer lipids (C(20) -C(26)) are required for macrophage activation, with C(22) giving optimal activity.

Naked plasmid DNA formulation: effect of different disaccharides on stability after lyophilisation.

Since plasmid DNA (pDNA) is unstable in solution, lyophilisation can be used to increase product shelf life. To prevent stress on pDNA molecules during lyophilisation, cryo- and lyoprotectants have to be added to the formulation. This study assessed the effect of disaccharides on naked pDNA stability after lyophilisation using accelerated stability studies. Naked pDNA was lyophilised with sucrose, trehalose, maltose or lactose in an excipient/DNA w/w ratio of 20. To one part of the vials extra residual moisture was introduced by placing the vials half opened in a 25 degrees C/60% RH climate chamber, before placing all vials in climate chambers (25 degrees C/60% RH and 40 degrees C/75% RH) for stability studies. An ex vivo human skin model was used to assess the effect of disaccharides on transfection efficiency. Lyophilisation resulted in amorphous cakes for all disaccharides with a residual water content of 0.8% w/w. Storage at 40 degrees C/75% RH resulted in decreasing supercoiled (SC) purity levels (sucrose and trehalose maintained approximately 80% SC purity), but not in physical collapse. The addition of residual moisture (values between 7.5% and 10% w/w) resulted in rapid collapse except for trehalose and decreasing SC purity for all formulations. In a separate experiment disaccharide formulation solutions show a slight but significant reduction (<3% with sucrose and maltose) in transfection efficiency when compared to pDNA dissolved in water. We demonstrate that disaccharides, like sucrose and trehalose, are effective lyoprotectants for naked pDNA.

Diacyltrehalose of Mycobacterium tuberculosis inhibits lipopolysaccharide- and mycobacteria-induced proinflammatory cytokine production in human monocytic cells.

The lipids located in the outer layer of Mycobacterium tuberculosis, which include sulfolipid, phthiocerol dimycocerosate (PDIM), diacyltrehalose, and polyacyltrehalose, may play a role in host-pathogen interactions. These lipids were purified using thin-layer chromatography, and their ability to induce proinflammatory cytokines in human monocytes and in a human acute monocytic leukemia cell line (THP-1) was examined. None of the lipids tested induced significant interleukin (IL)-12p40 or tumor necrosis factor (TNF)-alpha production in monocytic cells. Diacyltrehalose significantly inhibited lipopolysaccharide- and M. tuberculosis-induced IL-12p40, TNF-alpha, and IL-6 productions in human monocytes, whereas other lipids had no effect. However, diacyltrehalose was unable to inhibit peptidoglycan-induced IL-12p40 production. These results suggest that diacyltrehalose is a mycobacterial factor capable of modulating host immune responses.

Serodiagnosis of tuberculosis: comparison of immunoglobulin A (IgA) response to sulfolipid I with IgG and IgM responses to 2,3-diacyltrehalose, 2,3,6-triacyltrehalose, and cord factor antigens.

Nonpeptidic antigens from the Mycobacterium tuberculosis cell wall are the focus of extensive studies to determine their potential role as protective antigens or serological markers of tuberculous disease. Regarding this latter role and using an enzyme-linked immunosorbent assay, we have made a comparative study of the immunoglobulin G (IgG), IgM, and IgA antibody responses to four trehalose-containing glycolipids purified from M. tuberculosis: diacyltrehaloses, triacyltrehaloses, cord factor, and sulfolipid I (SL-I). Sera from 92 tuberculosis patients (taken before starting antituberculosis treatment) and a wide group of control individuals (84 sera from healthy donors, including purified protein derivative-negative, -positive, healed, and vaccinated individuals, and 52 sera from nontuberculous pneumonia patients), all from Spain, were studied. The results indicated a significantly elevated IgG and IgA antibody response in tuberculosis patients, compared with controls, with all the antigens used. SL-I was the best antigen studied, showing test sensitivities and specificities for IgG of 81 and 77.6%, respectively, and of 66 and 87.5% for IgA. Using this antigen and combining IgA and IgG antibody detection, high test specificity was achieved (93.7%) with a sensitivity of 67.5%. Currently, it is widely accepted that it is not possible to achieve sensitivities above 80% in tuberculosis serodiagnosis when using one antigen alone. Thus, we conclude that SL-I, in combination with other antigenic molecules, could be a useful antigen for tuberculosis serodiagnosis.

Occurrence of an antigenic triacyl trehalose in clinical isolates and reference strains of Mycobacterium tuberculosis.

A careful re-examination of the glycolipid content of clinical isolates and reference strains of the tubercle bacillus, Mycobacterium tuberculosis, led to the identification of a glycoconjugate that passed unnoticed in earlier studies. Nuclear magnetic resonance spectroscopy, gas chromatography-mass spectrometry and chemical degradations were used to identify the glycolipid as a 2,3,6-triacyl trehalose. The glycolipid contains a phthienoic acyl substituent, a family of multimethyl-branched, alpha,beta-unsaturated fatty acids specific for virulent strains of the tubercle bacillus. It reacted with sera from tuberculosis patients with a specificity and sensitivity of 96.2% and 76%, respectively. Comparable data were obtained with the 2,3-diacyl trehaloses of M. tuberculosis and M. fortuitum and with the triacyl trehaloses of M. fortuitum, suggesting that the antigens from the latter species may be used for the serodiagnosis of tuberculosis.

Mycobacterium fortuitum glycolipids for the serodiagnosis of pulmonary tuberculosis.

Glycolipids belonging to the family of acylated trehaloses were isolated from Mycobacterium fortuitum, a rapidly growing mycobacterial species, and tested in the serologic diagnosis of human pulmonary tuberculosis by enzyme-linked immunosorbent assay. Di- and tri-O-acylated trehaloses from M. fortuitum reacted with serum antibodies of patients with pulmonary tuberculosis at higher titers than did with sera from healthy donors. With both glycolipids, the sensitivity of the test was above 0.80 at a chosen specificity of 0.98. Individuals with treated tuberculosis showed lower antibody titers compared with their initial reactivities. These data show that M. fortuitum could be used as a surrogate source of antigens for tuberculosis serodiagnosis.

Comparison of two 2,3-diacyl trehalose antigens from Mycobacterium tuberculosis and Mycobacterium fortuitum for serology in tuberculosis patients.

Immunoglobulin G antibodies against two 2,3-diacyl trehalose (DAT) antigens from Mycobacterium tuberculosis (DATT) and Mycobacterium fortuitum (DATF) were studied by enzyme-linked immunosorbent assay of 356 serum samples. The sera were obtained from non-tuberculosis-infected individuals (282 serum samples) and tuberculosis patients (74 serum samples). Non-tuberculosis-infected individuals were healthy people (120 serum samples; positive purified-protein-derivative skin test, 60 patients; negative purified-protein-derivative skin test, 60 patients) patients with nontuberculosis lung disease (59 serum samples), contacts of sputum-smear-positive tuberculosis patients (57 serum samples), and human immunodeficiency virus-infected patients with nontuberculosis lung disease (46 serum samples). Of the 74 patients with tuberculosis, 14 were human immunodeficiency virus infected. The sensitivity of the method using DATT was 44.5%, and that with DATF was 48.6%. The specificities with both antigens were 99.1%. There were no significant differences between the mean values for both antigens (P = 0.2815). We therefore concluded that both antigens were interchangeable. As M. fortuitum, a fast-growing mycobacterium, could be a good source of antigen DAT, these results deserve consideration in the serology of tuberculosis.

Analysis of the immunological humoral response to Mycobacterium tuberculosis glycolipid antigens (DAT, PGLTb1) for diagnosis of tuberculosis in HIV-seropositive and -seronegative patients.

Using an enzyme immunoassay (EIA) test, the concentrations of IgG antibodies against 2,3 diacyl trehalose (DAT) and phenolic glycolipid Tb1 (PGLTb1) were measured in the sera of 153 patients with active tuberculosis, 50 of whom were coinfected with HIV, and in the sera of 152 healthy blood donors, 149 asymptomatic HIV-seropositive patients, 12 HIV-seronegative patients with conditions simulating tuberculosis, 23 HIV-seropositive patients with disseminated infection caused by mycobacteria other than tuberculosis and 24 HIV-seropositive patients with pulmonary disease from whom mycobacteria was not isolated in culture. A slightly lower percentage (74%) of the HIV-seropositive than the HIV-seronegative (77%) tuberculosis patients were positive for anti-DAT and anti-PGLTb1 IgG antibodies, with a specificity ranging from 91 to 95%. There was no significant difference between EIA sensitivity in smear-positive and smear-negative patients with pulmonary tuberculosis for all HIV immune statuses and sites of disease (pulmonary vs. extrapulmonary). In HIV-seropositive patients, however, sensitivity was always lower for disseminated tuberculosis than for localized tuberculosis. Combining data for both the smear test and the EIA maximized sensitivity. The main value of the EIA test could be to provide early complementary information by antibody detection in patients with tuberculosis, particularly those with a negative smear test.

Development of an enzyme-linked immunosorbent assay (ELISA) combined with a streptavidin-biotin and enzyme amplification method to detect anti-2,3-di-o-acyltrehalose (DAT) antibodies in patients with tuberculosis.

IgG antibodies against the 2,3-di-o-acyltrehalose glycolipid of Mycobacterium tuberculosis were determined in a set of 49 sera from patients with pulmonary tuberculosis and 65 negative control subjects. We compared a conventional ELISA method using a beta-galactosidase anti-human IgG conjugate developed with ONPG, with an amplification ELISA system constituted of an anti-human IgG biotinylated conjugate, a streptavidin-alkaline phosphatase complex, and NADP as a substrate. The resulting NAD was measured by using a redox enzymatic recycling system of alcohol dehydrogenase, diaphorase and iodonitrotetrazolium as chromogen. With specificity set at 92.31% in both methods, we obtained a sensitivity of 42.86% in the conventional method and a sensitivity of 61.22% in the amplified method. We conclude that by using a more sensitive method we can detect cases that otherwise could be identified as false negatives.

Synthesis of 4,6:2',3':4',6'-tri-O-cyclohexylidene-alpha,alpha'-trehalose 2-palmitate: an intermediate for the synthesis of mycobacterial 2,3-di-O-acyl-alpha,alpha'-trehalose antigens.

The aim was to 'triprotect' trehalose by placing various acetals, or related protecting groups, across the 4,6-, 2',3'-, and 4',6'-positions, leaving the 2,3-positions free for subsequent acylation. Isopropylidene and ethylidene acetals were studied, with the formation of a small amount of 4,6:2',3':4',6'-tri-O-isopropylidene-alpha,alpha'-trehalose. 4,6:4',6'-Di-O-benzylidene-2',3'-O-(tetraphenyldisiloxane-1,3-d iyl)-alpha, alpha'-trehalose 2,3-diacetate was prepared in low yield. 1,1-Dimethoxycyclohexane reacted with methyl alpha-D-glucopyranoside to afford the 4,6-O-cyclohexylidene derivative, isolated as the diacetate; mild acid cleavage of the acetal gave the 2,3-diacetate. 4,6:2',3':4',6'-Tri-O-cyclohexylidene-alpha,alpha'-trehalose is the major product of the reaction between alpha,alpha'-trehalose and 1,1-dimethoxycyclohexane. 2,3:4,6:2',3':4',6'-Tetra-O-cyclohexylidene-, 4,6:4',6'-di-O-cyclohexylidene-, and 4,6-O-cyclohexylidene-alpha,alpha'-trehaloses were also isolated in lower yields, all acetals being characterised as their peracetates. The proportions of the different trehalose acetals were dependent upon the molar ratio of 1,1-dimethoxycyclohexane and particularly on the reaction temperature. The triprotected trehalose acetal was acylated with palmitic acid, with excellent regioselectivity, affording the 2-O-palmitoyl ester. This 2-monoacylated, triprotected trehalose is a key intermediate for the synthesis of 2,3-di-O-acyl-alpha,alpha'-trehalose glycolipid antigens, isolated from Mycobacterium fortuitum and Mycobacterium tuberculosis.

[Evaluation of two glycolipids from M. tuberculosis in the serodiagnosis of tuberculosis].

Sera from 112 healthy controls and 120 pulmonary tuberculous patients (61 untreated patients and 59 active patients) were assayed, by ELISA, to test the activity of IgG and IgM antibodies against antigen of 2, 3-diacyl-trehalose-2'-sulphate (SL IV) a phenolglycolipid antigen (PGLTb1) and purified protein derivative (PPD) from M. tuberculosis. Respectively, for SL IV-IgG-ELISA, SL IV-IgM-ELISA, PGLTb1-IgG-ELISA, PGLTb1-IgM-ELISA, PPD-IgG-ELISA, the specificities were of 96.43, 96.43, 96.43, 96.43 and 95.53%; the sensitivities were of 51.67, 32.50, 14.17, 18.33 and 33.33%; the efficiencies were of 73.28, 63.36, 53.88, 56.03 and 62.93%; the predictive values for a positive result were of 96.87, 86.67, 80.95, 84.62 and 88.64%. Among the three antigens tested, SL IV was found to be better than PGLTb1 and PPD.

Screening of synthetic trehalose 6,6'-diesters and trehalose 6-monoesters as potential immunoreactants for the serodiagnosis of tuberculosis.

The absence of serological cross-reactivity between trehalose 2,3-diester (DAT, formerly SL-IV) and synthetic trehalose 6,6'-diesters and trehalose 6-monoesters was established by ELISA testing using polyclonal immune sera raised in rabbits sensitized with "DAT". From the screening of fifteen synthetic trehalose 6,6'- and 6-esters, "mirror" pseudo cord factor no. 1, "mirror" amides no. 5 and 6, cord factor analogues 7 and 8 and trehalose 6-monoesters 10 and 11 were selected for future, more extensive serological analysis. Paired comparisons of analogues among these fifteen substances showed that serodiagnostic discrimination power was more a function of the carbon chain length of their substituent groups--as well as of their position--than of the "mirror" constitution of the molecules. More exhaustive testing of these seven compounds is needed to select the synthetic product most efficient in the ELISA serodiagnosis of tuberculosis.

[Detection of antibodies to sulpholipid IV from mycobacterium tuberculosis in pulmonary tuberculosis patients].

Serum IgG and IgM antibodies against a novel 2,3-diacyl-trehalose-2'-sulphate (SLIV) antigen using ELISA were determined in control (n = 112) and in pulmonary tuberculosis (n = 120) patients. The specificity and sensitivity of SLIV-IgG-ELISA were 96% and 52%, respectively. Those of SLIV-IgM-ELISA were 95% and 33%, respectively. Compared with PPD-IgG-ELISA, SLIV-IgG-ELISA showed that the specificity was about the same but the sensitivity was higher.

Synthesis of alpha,alpha-trehalose 2,3- and 2,3'-diesters with palmitic and stearic acid: potential immunoreactants for the serodiagnosis of tuberculosis.

Regioselective monoacylation, by the stannylation method, of 4,6:4',6'-di-O-benzylidene-alpha,alpha-trehalose with palmitoyl or stearoyl chloride afforded the 2-palmitate and 2-stearate of the diacetal, whereas partial diacylation led to the corresponding 2,3'-dipalmitate and 2,3'-distearate. Protection of the monoesters in the 2',3' positions by cyclizing silylation with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane, followed by acylation of the silyl ethers, gave the fully protected 2,3-dipalmitate, 2,3-distearate, and 2-palmitate-3-stearate. Small proportions of other isomers and triesters were also produced in these reactions. Desilylation and debenzylidenation of the diesters finally furnished 2,3- and 2,3'-di-O-palmitoyl-2,3- and 2,3'-di-O-stearoyl-, and 2-O-palmitoyl-3-O-stearoyl-alpha,alpha-trehalose.