Cyrille Grandjean

A synthetic carbohydrate-protein conjugate vaccine candidate against Shigella flexneri 2a infection.

The protective Ag of Shigella, the Gram-negative enteroinvasive bacterium causing bacillary dysentery, or shigellosis, is its O-specific polysaccharide (O-SP) domain of the LPS, the major bacterial surface component. As an alternative to the development of detoxified LPS-based conjugate vaccines, recent effort was put into the investigation of neoglycoproteins encompassing synthetic oligosaccharides mimicking the protective Ags of the O-SP. We previously reported that when coupled to tetanus toxoid via single point attachment, a synthetic pentadecasaccharide representing three biological repeating units of the O-SP of Shigella flexneri 2a (SF2a), one of the most common Shigella serotypes, elicits a better serum anti-LPS 2a Ab response in mice than shorter synthetic O-SP sequences. In this study, we show that the pentadecasaccharide-induced anti-LPS 2a Abs protect passively administered naive mice from Shigella infection. Therefore, this three repeating units sequence, which is recognized by anti-SF2a sera from infected patients, acts as a functional mimic of the native polysaccharide Ag. Analyses of parameters influencing immunogenicity revealed that an investigational SF2a vaccine displaying a pentadecasaccharide:tetanus toxoid molar loading of 14:1 triggers a high and sustained anti-LPS Ab response, without inducing anti-linker Ab, when administered four times at a dose corresponding to 1 μg of carbohydrate. In addition, the profile of the anti-LPS Ab response, dominated by IgG1 production (Th2-type response), mimics that observed in human upon natural SF2a infection. This synthetic carbohydrate-based conjugate may be a candidate for a SF2a vaccine.

Novel Hyperbranched Glycomimetics Recognized by the Human Mannose Receptor: Quinic or Shikimic Acid Derivatives as Mannose Bioisosteres

The mannose receptor mediates the internalization of a wide range of molecules or microorganisms in a pattern recognition manner. Therefore, it represents an attractive entry for specific drug, gene, or antigen delivery to macrophages and dendritic cells. In an attempt to design novel effective synthetic mannose receptor ligands, quinic and shikimic acid were selected as putative mannose mimics on the basis of X‐ray crystallographic data from the related rat mannose‐binding lectin. As the mannose receptor preferentially binds to molecules displaying several sugar residues, fluorescein‐labeled cluster quinic and shikimic acid derivatives with valencies of two to eight were synthesized. Their mannose receptor mediated uptake was assayed on monocyte‐derived human dendritic cells by cytofluorimetric analysis. Mannose‐receptor specificity was further assessed by competitive inhibition assays with mannan, by confocal microscopy analysis, and by expression of the mannose receptor in transfected Cos‐1 cells. Constructs derived from both quinic and shikimic acid were efficiently recognized by the mannose receptor with an optimum affinity for the molecules with a valency of four. As a result, commercially available quinic and shikimic acids appear as stable mannose bioisosteres, which should prove valuable tools for specific cell delivery.

Synthesis of clustered glycoside-antigen conjugates by two one-pot, orthogonal, chemoselective ligation reactions: scope and limitations.

Major histocompatibility class II antigens have been bound to clustered glycosides for selective targeting of the dendritic cell mannose receptor. Di-, tetra-, and octavalent glycoside-antigen conjugates have been obtained after two, orthogonal, hydrazone/thioether ligations, performed by using thio derivatives of D-mannose, D-galactose, or D(-)-quinic acid, glyoxylyl (or hydrazino)-N-chloroacetylated lysinyl trees, and N-terminal hydrazino (or glyoxylyl) peptide antigens. Successful one-pot condensations have been developed to account for the nature of the antigens and the valency of the trees.

Convergent synthesis of fluorescein-labelled lysine-based cluster glycosides

Abstract The synthesis of fluorescein-labelled lysinyl trees, containing 2, 4 or 8 manno- or galactoside residues, is reported. These lysine-based cluster glycosides have been readily assembled by coupling amino-functionalized, N -chloroacetylated-L-lysinyl trees with fluorescein-isothiocyanate (FITC) and performing a thioether chemoselective ligation with fully deprotected glycoside derivatives. The reaction order is governed by the size of the lysinyl trees; the labelling/thioetherification steps can be performed in an one pot procedure, thus allowing an easy access to glycodendrimers designed to study the dendritic cellsmannose receptor.

Investigation towards bivalent chemically defined glycoconjugate immunogens prepared from acid-detoxified lipopolysaccharide of Vibrio cholerae O1, serotype Inaba

A free amino group present on the acid-detoxified lipopolysaccharide (pmLPS) of V. cholerae O1 serotype Inaba was investigated for site-specific conjugation. Chemoselective pmLPS biotinylation afforded the corresponding mono-functionalized derivative, which retained antigenicity. Thus, pmLPS was bound to carrier proteins using thioether conjugation chemistry. Induction of an anti-LPS antibody (Ab) response in BALB/c mice was observed for all conjugates. Interestingly, the sera had vibriocidal activity against both Ogawa and Inaba strains opening the way to a possible bivalent vaccine. However, the level of this Ab response was strongly affected by both the nature of the linker and of the carrier. Furthermore, no switch from IgM to IgG, i.e. from a T cell-independent to a T cell-dependent immune response was detected, a result tentatively explained by the possible presence of free polysaccharide in the formulation. Taken together, these results encourage further investigation towards the development of potent pmLPS-based neoglycoconjugate immunogens, fully aware of the challenge faced in the development of a cholera vaccine that will provide efficient serogroup coverage.

Grafting of synthetic mannose receptor-ligands onto onion vectors for human dendritic cells targetingElectronic supplementary information (ESI) available: full experimental details. See http://www.rsc.org/suppdata/cc/b2/b206980f/

A practical preparation of onion vesicles targeted to dendritic cells involves the grafting of mannose-mimetic clusters, bearing a hydrazino group, onto the surface of onion vesicles containing an aldehyde functionalized lipid.

Synthesis and mannose receptor-mediated uptake of clustered glycomimetics by human dendritic cells: Effect of charge

Effect of charge and shape of multivalent lysine-based cluster glycomimetics on their mannose receptor-mediated uptake by human dendritic cells has been evaluated: The capture is strongly affected by the shape of the ligands. The effect of charge is less pronounced although positive charges on the ligands seem to favor non-specific endocytosis capture.

Access to Antigens Related to Anthrose Using Pivotal Cyclic Sulfite/Sulfate Intermediates

Anthrose is the upstream terminal unit of the tetrasaccharide side chain from a major glycoprotein of Bacillus anthracis exosporium and is part of important antigenic determinants. A novel entry to anthrose-containing antigens and precursors is described. The synthetic route, starting from D(+)-fucose, makes use of intermediates featuring a cyclic sulfite or sulfate function which serves successively as a protecting and a leaving group.

Convergent synthesis of D-(–)-quinic and shikimic acid-containing dendrimers as potential C-lectin ligands by sulfide ligation of unprotected fragments

The preparation of D-(–)-quinic and (–)-shikimic acid-derived dendrimers with valencies of 4, 8 and 16, respectively, as potential C-lectin ligands is reported. D-(–)-Quinic and shikimic acids were branched to an (S-tert-butylthio-L-cysteine)-containing tripeptide on solid phase to furnish compounds 1 and 3. These intermediates were reduced upon treatment with tri-n-butylphosphine and linked to N-chloroacetylated L-lysinyl cores via a nucleophilic substitution performed in aqueous DMF.

A new tartaric acid-based linker for the synthesis of C-terminal peptide α-oxo-aldehydes

There is a need for simple methods allowing the synthesis of peptides bearing an aldehyde moiety at the C-terminus which can be used in chemical ligation studies [1], With the synthesis of chemical libraries in mind, we needed: (1) a new solid-phase methodology permitting the formation of a C-terminal aldehyde function during the separation of the product from the solid support. We excluded multistep procedures such as generation of soluble 1,2-diols or 1,2-amino alcohols followed by a periodic oxidation in solution; (2) the aldehyde moiety must not be an aldehyde function to avoid stability and racemization problems; (3) a cleavage procedure giving directly fully deprotected peptide aldehydes in high yield and compatible with aqueous or partial aqueous conditions; (4) a mild cleavage step compatible with all the amino acids; (5) cleavage conditions which could be easily automated and permitting the solubilization of diverse structures; and (6) a procedure using only cheap starting materials and minimal or no chemical steps in solution for the elaboration of the linker. The strategy described in Scheme 1 fulfills all these criteria. An 2,3-O-isopropylidene-D-tartrate (IPT) based linker is the precursor of the 1,2diol moiety leading to a C-terminal oxo-aldehyde moiety following a periodic oxidation. This solid-phase periodic oxidation is performed on a fully deprotected peptide and leads to the formation of the -oxo-aldehyde moiety and to the cleavage of the product from the solid support. To this end, we used resins well solvated in aqueous media such as PEGA or PEG-PS solid supports. The acetonide protecting group is stable during standard Fmoc/tert-butyl solid-phase peptide synthesis. It is simultaneously and easily deprotected during the removal of the side chain protecting groups.