The First-in-Human Synthetic Glycan-Based Conjugate Vaccine Candidate against .

, the causative agent of shigellosis, is among the main causes of diarrheal diseases with still a high morbidity in low-income countries. Relying on chemical synthesis, we implemented a multidisciplinary strategy to design SF2a-TT15, an original glycoconjugate vaccine candidate targeting 2a (SF2a). Whereas the SF2a O-antigen features nonstoichiometric O-acetylation, SF2a-TT15 is made of a synthetic 15mer oligosaccharide, corresponding to three non-O-acetylated repeats, linked at its reducing end to tetanus toxoid by means of a thiol-maleimide spacer.

Convergent Chemoenzymatic Strategy to Deliver a Diversity of Serotype-Specific O-Antigen Segments from a Unique Lightly Protected Tetrasaccharide Core.

Progress in glycoscience is strongly dependent on the availability of broadly diverse tailor-made, well-defined, and often complex oligosaccharides. Herein, going beyond natural resources and aiming to circumvent chemical boundaries in glycochemistry, we tackle the development of an chemoenzymatic strategy holding great potential to answer the need for molecular diversity characterizing microbial cell-surface carbohydrates. The concept is exemplified in the context of , a major cause of diarrhoeal disease.

Detailed Investigation of the Immunodominant Role of O-Antigen Stoichiometric O-Acetylation as Revealed by Chemical Synthesis, Immunochemistry, Solution Conformation and STD-NMR Spectroscopy for Shigella flexneri 3a.

Shigella flexneri 3a causes bacillary dysentery. Its O-antigen has the {2)-[α-d-Glcp-(1→3)]-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-[Ac→2]-α-l-Rhap-(1→3)-[Ac→6]≈40 % -β-d-GlcpNAc-(1→} ([(E)ABAc CAc D]) repeating unit, and the non-O-acetylated equivalent defines S. flexneri X.

A Synthetic Carbohydrate Conjugate Vaccine Candidate against Shigellosis: Improved Bioconjugation and Impact of Alum on Immunogenicity.

Conjugation chemistry is among the most important parameters governing the efficacy of glycoconjugate vaccines. High robustness is required to ensure high yields and batch to batch reproducibility. Herein, we have established a robust bioconjugation protocol based on the thiol-maleimide addition.

Investigation on the Synthesis of Shigella flexneri Specific Oligosaccharides Using Disaccharides as Potential Transglucosylase Acceptor Substrates.

Chemo-enzymatic strategies hold great potential for the development of stereo- and regioselective syntheses of structurally defined bioactive oligosaccharides. Herein, we illustrate the potential of the appropriate combination of a planned chemo-enzymatic pathway and an engineered biocatalyst for the multistep synthesis of an important decasaccharide for vaccine development. We report the stepwise investigation, which led to an efficient chemical conversion of allyl α-d-glucopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→3)-2-deoxy-2-trichloroacetamido-β-d-glucopyranoside, the product of site-specific enzymatic α-d-glucosylation of a lightly protected non-natural disaccharide acceptor, into a pentasaccharide building block suitable for chain elongation at both ends.

Programmed chemo-enzymatic synthesis of the oligosaccharide component of a carbohydrate-based antibacterial vaccine candidate.

The powerful chemo-enzymatic synthesis of the pentadecasaccharide hapten involved in the first synthetic carbohydrate-based vaccine candidate against endemic shigellosis is reported. The high yielding site-selective α-D-glucosylation of a lightly protected disaccharide by an engineered transglucosylase-sucrose system gave a trisaccharide, which was chemically elongated by an efficient [5+5] process.

Linear synthesis of the branched pentasaccharide repeats of O-antigens from Shigella flexneri 1a and 1b demonstrating the major steric hindrance associated with type-specific glucosylation.

Shigella flexneri serotypes 1b and 1a are Gram-negative enteroinvasive bacteria causing shigellosis in humans. The O-antigen from S. flexneri 1b is a { → 2)-[3Ac/4Ac]-α-L-RHAP-(1 → 2)-α-L-Rhap-(1 → 3)-[2Ac]-α-L-Rhap-(1 → 3)-[α-D-Glcp-(1 → 4)]-β-D-GlcpNAc-(1 → }n branched polysaccharide ({(Ac)AB(Ac)C(E)D}n).

Non-stoichiometric O-acetylation of Shigella flexneri 2a O-specific polysaccharide: synthesis and antigenicity.

Synthetic functional mimics of the O-antigen from Shigella flexneri 2a are seen as promising vaccine components against endemic shigellosis. Herein, the influence of the polysaccharide non-stoichiometric di-O-acetylation on antigenicity is addressed for the first time. Three decasaccharides, representing relevant internal mono- and di-O-acetylation profiles of the O-antigen, were synthesized from a pivotal protected decasaccharide designed to tailor late stage site-selective O-acetylation.

CGTase-catalysed cis-glucosylation of L-rhamnosides for the preparation of Shigella flexneri 2a and 3a haptens.

We report the enzymatic synthesis of α-D-glucopyranosyl-(1→4)-α-L-rhamnopyranoside and α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranoside by using a wild-type transglucosidase in combination with glucoamylase and glucose oxidase. It was shown that Bacillus circulans 251 cyclodextrin glucanotransferase (CGTase, EC 2.1.

Dynamic aspects of antibody:oligosaccharide complexes characterized by molecular dynamics simulations and saturation transfer difference nuclear magnetic resonance.

Carbohydrates are likely to maintain significant conformational flexibility in antibody (Ab):carbohydrate complexes. As demonstrated herein for the protective monoclonal Ab (mAb) F22-4 recognizing the Shigella flexneri 2a O-antigen (O-Ag) and numerous synthetic oligosaccharide fragments thereof, the combination of molecular dynamics simulations and nuclear magnetic resonance saturation transfer difference experiments, supported by physicochemical analysis, allows us to determine the binding epitope and its various contributions to affinity without using any modified oligosaccharides. Moreover, the methods used provide insights into ligand flexibility in the complex, thus enabling a better understanding of the Ab affinities observed for a representative set of synthetic O-Ag fragments.

Effects of backbone substitutions on the conformational behavior of Shigella flexneri O-antigens: implications for vaccine strategy.

The O-antigen (O-Ag), the polysaccharide part of the lipopolysaccharide, is the major target of the serotype-specific protective humoral response elicited upon host infection by Shigella flexneri, the main causal agent of the endemic form of bacillary dysentery. The O-Ag repeat units (RUs) of 12 S. flexneri serotypes share the tetrasaccharide backbone →2)-α-l-Rhap-(1 → 2)-α-l-Rhap-(1 → 3)-α-l-Rhap-(1 → 3)-β-d-GlcpNAc-(1→, with site-selective glucosylation(s) and/or O-acetylation defining the serotypes.

Rational design and immunogenicity of liposome-based diepitope constructs: application to synthetic oligosaccharides mimicking the Shigella flexneri 2a O-antigen.

We have designed chemically defined diepitope constructs consisting of liposomes displaying at their surface synthetic oligosaccharides mimicking the O-antigen of the Shigella flexneri 2a lipopolysaccharide (B-cell epitope) and influenza hemagglutinin peptide HA 307-319 (Th epitope). Using well controlled and high-yielding covalent bioconjugation reactions, the two structurally independent epitopes were coupled to the lipopeptide Pam(3)CAG, i.e.

Efficient synthesis of six tri- to hexasaccharide fragments of Shigella flexneri serotypes 3a and/or X O-antigen, including a study on acceptors containing N-trichloroacetylglucosamine versus N-acetylglucosamine.

Six tri- to hexasaccharide fragments of the {2)-[alpha-D-Glcp-(1-->3)]-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-[Ac-->2]-alpha-L-Rhap-(1-->3)-beta-D-GlcpNAc-(1-->}(n) polymer ([(E)AB(Ac)CD](n)) were synthesized as their propyl glycosides. All targets share the (E)AB sequence. Following a thorough investigation on the use of N-trichloroacetylglucosamine- versus N-acetylglucosamine-containing tri- and tetrasaccharide acceptors, the successful strategy was based on an efficient combination of the trichloroacetimidate chemistry, a trichloroacetyl used as permanent N-protection, and an allyl aglycon as temporary and/or permanent anomeric protection of selected building blocks.

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.

Characterization of functional oligosaccharide mimics of the Shigella flexneri serotype 2a O-antigen: implications for the development of a chemically defined glycoconjugate vaccine.

Protection against reinfection with noncapsulated Gram-negative bacteria, such as Shigella, an enteroinvasive bacterium responsible for bacillary dysentery, is mainly achieved by Abs specific for the O-Ag, the polysaccharide part of the LPS, the major bacterial surface Ag. The use of chemically defined glycoconjugates encompassing oligosaccharides mimicking the protective determinants carried by the O-Ag, thus expected to induce an efficient anti-LPS Ab response, has been considered an alternative to detoxified LPS-protein conjugate vaccines. The aim of this study was to identify such functional oligosaccharide mimics of the S.

Borano-nucleotides: new analogues to circumvent HIV-1 RT-mediated nucleoside drug-resistance.

Alpha-boranophosphates suppress RT-mediated resistance when the catalytic rate of incorporation (kpol) of the analogue 5'-triphosphate is responsable for drug resistance, such as in the case of K65R mutant and ddNTPs, and Q151M toward AZTTP and ddNTPs. This suppression is also observed with BH3-d4T and BH3-3TC toward their clinically relevant mutants Q151M and M184V. Moreover, the presence of the borano (BH3-) group renders the incorporation of the analogue independent from amino-acid substitutions in RT.

On the preparation of carbohydrate-protein conjugates using the traceless Staudinger ligation.

[reaction: see text] The nature of a linker used for preparing glycoconjugate vaccines is of utmost importance as it may lead to immunogenic biomolecules. We report the conjugation of carbohydrate haptens to protein carriers leading to potential vaccines using the traceless Staudinger ligation. The ligation relies on the selective transfer of a phosphane substituent to an azide to form a native amide bond in the final product upon release of an oxidized phosphane byproduct.

Suppression of multidrug-resistant HIV-1 reverse transcriptase primer unblocking activity by alpha-phosphate-modified thymidine analogues.

A dipeptide insertion between codons 69 and 70 together with the amino acid substitution T215Y in the reverse transcriptase (RT)-coding region of human immunodeficiency virus type 1 (HIV-1) strains are known to confer phenotypic resistance to zidovudine (AZT) and stavudine (d4T). Phenotypic resistance correlates with an increased ATP-dependent phosphorolytic activity. Nucleoside alpha-boranophosphate diastereoisomers derived from AZT and d4T were tested as substrates of a multidrug-resistant HIV-1 RT (designated as SS RT) bearing a Ser-Ser insertion at codons 69-70 and other drug resistance-related mutations, in DNA polymerization assays and ATP-mediated excision reactions.

Synthesis of two linear PADRE conjugates bearing a deca- or pentadecasaccharide B epitope as potential synthetic vaccines against Shigella flexneri serotype 2a infection.

The blockwise synthesis of the 2-aminoethyl glycosides of a deca- and a pentadecasaccharide made of two and three repeating units, respectively, of the Shigella flexneri serotype 2a specific polysaccharide is reported. The strategy relies on trifluoromethanesulfonic acid mediated glycosylation of a pentasaccharide building block acting as a glycosyl donor and a potential glycoside acceptor. Both targets were made available in amounts large enough for their subsequent conversion into glycoconjugates.

Mechanistic insights into the suppression of drug resistance by human immunodeficiency virus type 1 reverse transcriptase using alpha-boranophosphate nucleoside analogs.

A class of amino acid substitutions in drug-resistant HIV-1 reverse transcriptase (RT) is responsible for the selectively impaired incorporation of the nucleotide analog inhibitor into DNA. We have shown previously that alpha-boranophosphate nucleoside analogs suppress RT-mediated resistance when the catalytic rate is responsible for drug resistance such as in the case of K65R and dideoxy (dd)NTPs, and Q151M toward AZTTP and ddNTPs. Here, we extend this property to BH3-d4TTP and BH3-3TCTP toward their clinically relevant mutants Q151M and M184V, respectively.

A structural basis for the inhibition of the NS5 dengue virus mRNA 2'-O-methyltransferase domain by ribavirin 5'-triphosphate.

Ribavirin is one of the few nucleoside analogues currently used in the clinic to treat RNA virus infections, but its mechanism of action remains poorly understood at the molecular level. Here, we show that ribavirin 5'-triphosphate inhibits the activity of the dengue virus 2'-O-methyltransferase NS5 domain (NS5MTase(DV)). Along with several other guanosine 5'-triphosphate analogues such as acyclovir, 5-ethynyl-1-beta-d-ribofuranosylimidazole-4-carboxamide (EICAR), and a series of ribose-modified ribavirin analogues, ribavirin 5'-triphosphate competes with GTP to bind to NS5MTase(DV).

Preparation of synthetic glycoconjugates as potential vaccines against Shigella flexneri serotype 2a disease.

The synthesis of three neoglycopeptides incorporating carbohydrate haptens, differing in length, covalently linked to a non natural universal T helper peptide is disclosed. They were synthesized according to a blockwise strategy based on the condensation of appropriate di-, tri-, and tetrasaccharide trichloroacetimidate donors onto an azidoethyl 2-acetamido-2-deoxybeta-D-glucopyranoside acceptor. Use of thiol-maleimide coupling chemistry allowed site-selective efficient conjugation.

The Y181C substitution in 3'-azido-3'-deoxythymidine-resistant human immunodeficiency virus, type 1, reverse transcriptase suppresses the ATP-mediated repair of the 3'-azido-3'-deoxythymidine 5'-monophosphate-terminated primer.

Resistance to zidovudine (3'-azido-3'-deoxythymidine, AZT) by the human immunodeficiency virus, type 1, requires multiple amino acid substitutions such as D67N/K70R/T215F/K219Q in the viral reverse transcriptase (RT). In this background of AZT resistance, additional "suppressive" substitutions such as Y181C restore sensitivity to AZT. In order to characterize the mechanism of this AZT resistance suppression, the Y181C substitution was introduced into both wild-type and AZT-resistant reverse transcriptase.

Structural analysis of the activation of ribavirin analogs by NDP kinase: comparison with other ribavirin targets.

Ribavirin used in therapies against hepatitis C virus (HCV) is potentially efficient against other viruses but presents a high cytotoxicity. Several ribavirin triphosphate analogs modified on the ribose moiety were synthesized and tested in vitro on the RNA polymerases of HCV, phage T7, and HIV-1 reverse transcriptase. Modified nucleotides with 2'-deoxy, 3'-deoxy, 2',3'-dideoxy, 2',3'-dideoxy-2',3'-dehydro, and 2',3'-epoxy-ribose inhibited the HCV enzyme but not the other two polymerases.