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.

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.

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.

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.

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.

Influence of surfactant concentration on the antitumor activity of emulsified components of mycobacteria.

The influence of Tween 80 content on the antitumor activity of emulsified mycobacterial components administered intralesionally was studied in mice. The number of treated animals in which there was complete regression of tumor depended on the concentration of Tween in each of the emulsions. An additional variable was the size distribution of the mineral oil droplets, which depended on whether the emulsions were prepared by ultrasonication or by mechanical grinding. Emulsions of mycobacterial components (cell walls of Bacillus Calmette-Guérin and trehalose-6,6'-dimycolate) prepared by ultrasonication contained smaller oil droplets than did those prepared by grinding. Ground emulsions retained antitumor activity over a wider range of Tween concentrations than did ultrasonically prepared emulsions. The latter required Tween at concentrations in an optimal range above which the tumor-regressive potency was diminished. Emulsions of trehalose-6,6'-dimycolate containing the lowest concentration of Tween needed to produce a relatively stable preparation contained larger oil droplets and were immunotherapeutically less active than were those prepared with an optimal concentration of Tween. Emulsions of B. Calmette-Guérin cell walls retained antitumor activity even in the absence of added Tween.

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.

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.

Evaluation of a novel 2,3-diacyl-trehalose-2'-sulphate (SL-IV) antigen for case finding and diagnosis of leprosy and tuberculosis.

Serum IgG and IgM antibodies against a 2,3-diacyl-trehalose-2'-sulphate (SL-IV) antigen using ELISA were determined in controls (n = 288) and in leprosy (n = 210) and tuberculosis (n = 99) patients. In all assays, the amount of antigen per well was 100.0 ng and sera were diluted 1/250. In the case of leprosy, anti-SL-IV IgG and IgM antibody titres increased from the tuberculoid towards the lepromatous pole of the spectrum. In the tested population, the sensitivity of the assay was 93.2% in multibacillary leprosy and 33.3% in paucibacillary leprosy (specificity of 88.7%). Multibacillary patients with erythema nodosum leprosum (ENL) had lower titres than non-ENL. ELISA results were similar to those obtained using the Mycobacterium leprae phenolic glycolipid-I (PGL-I) antigen. In the case of tuberculosis (pulmonary and extrapulmonary), significant titres of anti SL-IV IgG and IgM antibodies were detected in about 75% of the patients using a cutoff point of 0.150, and in 51.6% using a cutoff of 0.300 (specificities were, respectively, 88% and 100%). We concluded that the determination of IgG and IgM antibodies against SL-IV was useful in leprosy and tuberculosis case finding program using a cutoff point of 0.150, and for serodiagnosis using a cutoff of 0.300.

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.

Immunotherapy with nonviable microbial components.

Structural components of microorganisms have been studied for immunopotentiating effect with the aid of transplantable (line 10) tumors in syngeneic guinea pigs. Microbial components were associated with oil droplets, suspended in Tween-saline, and injected intralesionally. BCG cell walls, given in this way, produced regression and cure of 50-60% of established tumors, as did viable BCG. Lipid extraction markedly reduced the tumor-regressing potency of cell walls, but P3, a trehalose mycolate present in the extract, restored full activity to the cell wall residue. P3 alone was nonsensitizing and had no antitumor activity, but it enhanced the latter property of various other microbial products. For example, the cure rates produced by cell walls of M. tuberculosis, M. bovis, M. phlei, or M. smegmatis were enhanced from 20-60% to as much as 90% by addition of P3. P3 also conferred antitumor activity on products from unrelated microbes, such as cell walls of E. coli, and in combination with endotoxins from rough Re mutant salmonellae, it produced cure rates of up to 93%. These results suggest that P3 is essential to the immunopotentiating activity of mycobacteria and that it may be broadly applicable in immunotherapy of cancer with microbial agents.

Revised structure of a trehalose-containing immunoreactive glycolipid of Mycobacterium tuberculosis.

Nuclear magnetic resonance spectroscopy, fast-atom bombardment mass spectrometry as well as various chemical degradations and chromatographic techniques were used to re-examine the structure of a highly immunoreactive glycolipid previously described in Mycobacterium tuberculosis (strain Canetti) as a 2,3-diacyl trehalose 2'-sulfate (labelled SL-IV). Ion exchange chromatography allowed the recognition of a neutral and an acidic glycolipid, indistinguishable on conventional silica gel. The neutral glycolipid was shown to be serologically identical to SL-IV and its structure was established as 2,3-diacyl trehalose. It corresponded to the non-chemically defined highly observed immunoreactive lipid previously recognized by others in M. tuberculosis (H37Rv).

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.

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.

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.

[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.

[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.