Jacques Prandi

Diacylated Sulfoglycolipids Are Novel Mycobacterial Antigens Stimulating CD1-restricted T Cells during Infection with Mycobacterium tuberculosis

Mycobacterial lipids comprise a heterogeneous group of molecules capable of inducing T cell responses in humans. To identify novel antigenic lipids and increase our understanding of lipid-mediated immune responses, we established a panel of T cell clones with different lipid specificities. Using this approach we characterized a novel lipid antigen belonging to the group of diacylated sulfoglycolipids purified from Mycobacterium tuberculosis. The structure of this sulfoglycolipid was identified as 2-palmitoyl or 2-stearoyl-3-hydroxyphthioceranoyl-2′-sulfate-α-α′-d-trehalose (Ac2SGL). Its immunogenicity is dependent on the presence of the sulfate group and of the two fatty acids. Ac2SGL is mainly presented by CD1b molecules after internalization in a cellular compartment with low pH. Ac2SGL-specific T cells release interferon γ, efficiently recognize M. tuberculosis–infected cells, and kill intracellular bacteria. The presence of Ac2SGL-responsive T cells in vivo is strictly dependent on previous contact with M. tuberculosis, but independent from the development of clinically overt disease. These properties identify Ac2SGL as a promising candidate to be tested in novel vaccines against tuberculosis.

Mycolic acids constitute a scaffold for mycobacterial lipid antigens stimulating CD1-restricted T cells.

CD1-restricted lipid-specific T lymphocytes are primed during infection with Mycobacterium tuberculosis, the causative agent of tuberculosis. Here we describe the antigenicity of glycerol monomycolate (GroMM), which stimulates CD1b-restricted CD4(+) T cell clones. Chemical characterization of this antigen showed that it exists as two stereoisomers, one synthetic isomer being more stimulatory than the other. The hydroxyl groups of glycerol and the mycolic acid length are critical for triggering the T cell responses. GroMM was presented by M. tuberculosis-infected dendritic cells, demonstrating that the antigen is available for presentation during natural infection. Ex vivo experiments showed that GroMM stimulated T cells from vaccinated or latently infected healthy donors but not cells from patients with active tuberculosis, suggesting that GroMM-specific T cells are primed during infection and their detection correlates with lack of clinical active disease.

Fatty Acyl Structures of Mycobacterium tuberculosis Sulfoglycolipid Govern T Cell Response

CD1b-restricted T lymphocytes recognize a large diversity of mycobacterial lipids, which differ in their hydrophilic heads and the structure of their acyl appendages. Both moieties participate in the antigenicity of lipid Ags, but the structural constraints governing binding to CD1b and generation of antigenic CD1b:lipid Ag complexes are still poorly understood. Here, we investigated the structural requirements conferring antigenicity to Mycobacterium tuberculosis sulfoglycolipid Ags using a combination of CD1b:lipid binding and T cell activation assays with both living dendritic cells and plate-bound recombinant soluble CD1b. Comparison of the antigenicity of a panel of synthetic analogs, sharing the same trehalose-sulfate polar head, but differing in the structure of their acyl tails, shows that the number of C-methyl substituents on the fatty acid, the configuration of the chiral centers, and the respective localization of the two different acyl chains on the sugar moiety govern TCR recognition and T lymphocyte activation. These studies have major implications for the design of sulfoglycolipid analogs with potential use as tuberculosis subunit vaccines.

Fine tuning by human CD1e of lipid-specific immune responses

CD1e is a member of the CD1 family that participates in lipid antigen presentation without interacting with the T-cell receptor. It binds lipids in lysosomes and facilitates processing of complex glycolipids, thus promoting editing of lipid antigens. We find that CD1e may positively or negatively affect lipid presentation by CD1b, CD1c, and CD1d. This effect is caused by the capacity of CD1e to facilitate rapid formation of CD1–lipid complexes, as shown for CD1d, and also to accelerate their turnover. Similar results were obtained with antigen-presenting cells from CD1e transgenic mice in which lipid complexes are assembled more efficiently and show faster turnover than in WT antigen-presenting cells. These effects maximize and temporally narrow CD1-restricted responses, as shown by reactivity to Sphingomonas paucimobilis-derived lipid antigens. CD1e is therefore an important modulator of both group 1 and group 2 CD1-restricted responses influencing the lipid antigen availability as well as the generation and persistence of CD1–lipid complexes.

Structural reorganization of the antigen-binding groove of human CD1b for presentation of mycobacterial sulfoglycolipids

The mechanisms permitting nonpolymorphic CD1 molecules to present lipid antigens that differ considerably in polar head and aliphatic tails remain elusive. It is also unclear why hydrophobic motifs in the aliphatic tails of some antigens, which presumably embed inside CD1 pockets, contribute to determinants for T-cell recognition. The 1.9-Å crystal structure of an active complex of CD1b and a mycobacterial diacylsulfoglycolipid presented here provides some clues. Upon antigen binding, endogenous spacers of CD1b, which consist of a mixture of diradylglycerols, moved considerably within the lipid-binding groove. Spacer displacement was accompanied by F’ pocket closure and an extensive rearrangement of residues exposed to T-cell receptors. Such structural reorganization resulted in reduction of the A’ pocket capacity and led to incomplete embedding of the methyl-ramified portion of the phthioceranoyl chain of the antigen, explaining why such hydrophobic motifs are critical for T-cell receptor recognition. Mutagenesis experiments supported the functional importance of the observed structural alterations for T-cell stimulation. Overall, our data delineate a complex molecular mechanism combining spacer repositioning and ligand-induced conformational changes that, together with pocket intricacy, endows CD1b with the required molecular plasticity to present a broad range of structurally diverse antigens.

Synthesis of Diacylated Trehalose Sulfates: Candidates for a Tuberculosis Vaccine

Tuberculosis remains a major world-wide health problem and results in the loss of almost 2 million lives annually, with the majority of deaths occurring in the developing world. Despite the discovery of active antibiotics in the 1960s and the production of the Bacillus Calmette–Gu rin (BCG) vaccine in the early 20th century, tuberculosis is still not under control. The increasing incidence of human immunodeficiency virus (HIV) epidemics and the emergence of multidrug-resistant strains of Mycobacterium tuberculosis, the causative agent of tuberculosis, impair the eradication of the disease. New therapeutic approaches and the development of new vaccines to fight tuberculosis are thus urgently needed. Recently, a diacylated sulfoglycolipid, acyl2SGL (1), [2] was characterized and identified as a new mycobacterial antigen able to stimulate populations of CD1b-restricted human T lymphocytes during infection with M. tuberculosis. Furthermore, these acyl2SGL-specific activated T cells were shown to: 1) release interferon-g (IFN-g), 2) recognize M. tuberculosis infected antigen-presenting cells, and 3) kill intracellular mycobacteria in vitro. In light of these properties, sulfoglycolipid 1 seemed to be a promising candidate for the development of a new tuberculosis vaccine. Compound 1 is specific to the M. tuberculosis species and is not present in M. bovis BCG, the species used in the current vaccine. The structure of acyl2SGL encompasses an a,a-d-trehalose core, which is esterified at the 2-position with a palmitic (or stearic) acid, esterified at the 3-position with a hydroxyphthioceranoic acid, and O-sulfated at the 2’-position. Hydroxyphthioceranoic acids are a family of complex dextrorotatory fatty acids specific to the Mycobacterium genus, which contain a hydroxy group and methyl groups arranged in a 2,4,6 pattern. All methyl-substituted stereocenters are of the l series, whereas the configuration of the hydroxy-substituted carbon atom has not been assigned. Compound 1 was isolated in tiny amounts (about 1 mgl ) from cultures of M. tuberculosis. Its low availability limits its further development as a potential tuberculosis vaccine. We therefore devised a synthetic route to this class of diacylated sulfated trehalose compounds and hypothesized that the hydroxyphthioceranoic acid, which is not readily available from natural sources, might be replaced by simpler fatty acids. Herein we report the preparation of various sulfoglycolipid (SGL) analogues of the natural compound acyl2SGL (1) in which the hydroxyphthioceranoic acid has been replaced by less complex acids. Some of these analogues were able, like the natural product, to activate the acyl2SGLspecific T-cell clone with the production of (IFN-g). Of the utmost importance, it was found that small modifications to the structure of the hydroxyphthioceranoic acid substituent can modulate the immunogenicity of the analogues. Recent interest in the synthesis of mycobacterial sulfoglycolipids has led to the preparation of a tetraacylated trehalose sulfate. The elaboration of the trisubstituted a,a-d-trehalose core of the sulfoglycolipids on the basis of the pioneering synthetic studies of Goren and co-workers, and Baer and Wu was straightforward. 7] The known compound 4,6,4’,6’-dibenzylidene a,a-d-trehalose (2) was acylated selectively at the 2position (or 2’-position) by using palmitoyl chloride (or another acyl chloride) in pyridine to give a trehalose derivative 3 in 45% yield (Scheme 1). This direct acylation reaction avoided the dibutylstannylene procedure, which involves toxic tin derivatives and which was found to be difficult to carry out on a larger scale. The use of the bifunctional reagent 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPSCl2) enabled the selective protection of the 2’and 3’-positions of 3 in one step and gave alcohol 4 in good yield (60 %). Owing to the steric crowding around the alcohol functionality of 4, the nucleophilicity of the oxygen atom was low, and the esterification of compound 4 with fatty-acid derivatives proved to be difficult. The best yields of 5 (up to [*] J. Guiard, Dr. M. Gilleron, Dr. J. Prandi, Dr. G. Puzo D partement des M canismes Mol culaires des Infections Mycobact riennes, Institut de Pharmacologie et de Biologie Structurale du CNRS, UMR 5089, Universit de Toulouse III 205 route de Narbonne, 31077 Toulouse Cedex (France) Fax: (+ 33)5-61-17-59-94 E-mail: Jacques.Prandi@ipbs.fr

Crystal structure of human CD1e reveals a groove suited for lipid-exchange processes

CD1e is the only human CD1 protein existing in soluble form in the late endosomes of dendritic cells, where it facilitates the processing of glycolipid antigens that are ultimately recognized by CD1b-restricted T cells. The precise function of CD1e remains undefined, thus impeding efforts to predict the participation of this protein in the presentation of other antigens. To gain insight into its function, we determined the crystal structure of recombinant CD1e expressed in human cells at 2.90-Å resolution. The structure revealed a groove less intricate than in other CD1 proteins, with a significantly wider portal characterized by a 2 Å-larger spacing between the α1 and α2 helices. No electron density corresponding to endogenous ligands was detected within the groove, despite the presence of ligands unequivocally established by native mass spectrometry in recombinant CD1e. Our structural data indicate that the water-exposed CD1e groove could ensure the establishment of loose contacts with lipids. In agreement with this possibility, lipid association and dissociation processes were found to be considerably faster with CD1e than with CD1b. Moreover, CD1e was found to mediate in vitro the transfer of lipids to CD1b and the displacement of lipids from stable CD1b–antigen complexes. Altogether, these data support that CD1e could have evolved to mediate lipid-exchange/editing processes with CD1b and point to a pathway whereby the repertoire of lipid antigens presented by human dendritic cells might be expanded.

Simplified Deoxypropionate Acyl Chains for Mycobacterium tuberculosis Sulfoglycolipid Analogues: Chain Length is Essential for High Antigenicity

The longer, the better: Increasing the lengths of the 1,3-methyl-branched fatty acyl chain units in mycobacterial diacylated sulfoglycolipid (Acyl2 SGL) analogues led to dramatic improvements in their antigenic properties and gave products more potent than the natural antigen Acyl2 SGLs.

A convenient synthesis of glucose monomycolate.

An efficient synthesis of glucose monomycolate, an important lipidic antigen from Mycobacterium tuberculosis is described.

Protective efficacy of a lipid antigen vaccine in a guinea pig model of tuberculosis

The bacillus Calmette Guérin (BCG) vaccine, the only licensed vaccine against TB, displays partial and variable efficacy, thus making the exploitation of novel vaccination strategies a major priority. Most of the current vaccines in pre-clinical or clinical development are based on the induction of T cells recognizing protein antigens. However, a large number of T cells specific for mycobacterial lipids are induced during infection, suggesting that lipid-based vaccines might represent an important component of novel sub-unit vaccines. Here, we investigated whether immunization with defined mycobacterial lipid antigens induces protection in guinea pigs challenged with M. tuberculosis. Two purified mycobacterial lipid antigens, the diacylated sulfoglycolipids (Ac2SGL) and the phosphatidyl-myo-inositol dimannosides (PIM2) were formulated in biophysically characterized liposomes made of dimethyl-dioctadecyl-ammonium (DDA) and synthetic trehalose 6,6-dibehenate (TDB). In three protection trials, a reduction of bacterial load in the spleen of inoculated animals was consistently observed compared to the unvaccinated group. Moreover, a reduction in the number of lesions and severity of pathology was detected in the lungs and spleen of the lipid vaccine group compared to unvaccinated controls. As the degree of protection achieved is similar to that observed using protein antigens in the same guinea pig model, these promising results pave the way to future investigations of lipid antigens as subunit vaccines.