Scalable Synthesis of Versatile Rare Deoxyamino Sugar Building Blocks from d-Glucosamine.

We report the syntheses of 1,3,4-tri-O-acetyl-2-amino-2,6-dideoxy-ß-d-glucopyranose and allyl 2-amino-2,6-dideoxy-ß-d-glucopyranoside from d-glucosamine hydrochloride. The potential of these two versatile scaffolds as key intermediates to a diversity of orthogonally protected rare deoxyamino hexopyranosides is exemplified in the context of fucosamine, quinovosamine, and bacillosamine. The critical C-6 deoxygenation step to 2,6-dideoxy aminosugars is performed at an early stage on a precursor featuring an imine moiety or a trifluoroacetamide moiety in place of the 2-amino group, respectively. Robustness and scalability are demonstrated for a combination of protecting groups and incremental chemical modifications that sheds light on the promise of the yet unreported allyl 2,6-dideoxy-2-N-trifluoroacetyl-ß-d-glucopyranoside when addressing the feasibility of synthetic zwitterionic oligosaccharides. In particular, allyl 3-O-acetyl-4-azido-2,4,6-trideoxy-2-trifluoroacetamido-ß-d-galactopyranoside, an advanced 2-acetamido-4-amino-2,4,6-trideoxy-d-galactopyranose building block, was achieved on the 30 g scale from 1,3,4,6-tetra-O-acetyl-ß-d-glucosamine hydrochloride in 50% yield and nine steps, albeit only two chromatography purifications.

Combination of High-Resolution Multistage Ion Mobility and Tandem MS with High Energy of Activation to Resolve the Structure of Complex Chemoenzymatically Synthesized Glycans.

Carbohydrates, in particular microbial glycans, are highly structurally diverse biomolecules, the recognition of which governs numerous biological processes. Of special interest, glycans of known monosaccharide composition feature multiple possible isomers, differentiated by the anomerism and position of their glycosidic linkages. Robust analytical tools able to circumvent this extreme structural complexity are increasing in demand to ensure not only the correct determination of naturally occurring glycans but also to support the rapid development of enzymatic and chemoenzymatic glycan synthesis. In support to the later, we report the use of complementary strategies based on mass spectrometry (MS) to evaluate the ability of 14 engineered mutants of sucrose-utilizing α-transglucosylases to produce type/group-specific Shigella flexneri pentasaccharide bricks from a single lightly protected non-natural tetrasaccharide acceptor substrate. A first analysis of the reaction media by UHPLC coupled to high-accuracy MS led to detect six reaction products of enzymatic glucosylation out of the eight possible ones. A seventh structure was evidenced by an additional step of ion mobility at a resolving power (Rp) of approximately 100. Finally, a Rp of about 250 in ion mobility made it possible to detect the eighth and last of the expected structures. Complementary to these measurements, tandem MS with high activation energy charge transfer dissociation (CTD) allowed us to unambiguously characterize seven regioisomers out of the eight possible products of enzymatic glucosylation. This work illustrates the potential of the recently described powerful IMS and CTD-MS methods for the precise structural characterization of complex glycans.

Exploratory N-Protecting Group Manipulation for the Total Synthesis of Zwitterionic Shigella sonnei Oligosaccharides.

Shigella sonnei surface polysaccharides are well-established protective antigens against this major cause of diarrhoeal disease. They also qualify as unique zwitterionic polysaccharides (ZPSs) featuring a disaccharide repeating unit made of two 1,2-trans linked rare aminodeoxy sugars, a 2-acetamido-2-deoxy-l-altruronic acid (l-AltpNAcA) and a 2-acetamido-4-amino-2,4,6-trideoxy-d-galactopyranose (AAT). Herein, the stereoselective synthesis of S. sonnei oligosaccharides comprising two, three and four repeating units is reported for the first time. Several sets of up to seven protecting groups were explored, shedding light on the singular conformational behavior of protected altrosamine and altruronic residues. A disaccharide building block equipped with three distinct N-protecting groups and featuring the uronate moiety already in place was designed to accomplish the iterative high yielding glycosylation at the axial 4-OH of the altruronate component and achieve the challenging full deprotection step. Key to the successful route was the use of a diacetyl strategy whereby the N-acetamido group of the l-AltpNAcA is masked in the form of an imide.

Convergent Chemoenzymatic Strategy to Deliver a Diversity of Shigella flexneri 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 in vitro 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 Shigella flexneri, a major cause of diarrhoeal disease. Aiming at a broad serotype coverage S. flexneri glycoconjugate vaccine, a non-natural lightly protected tetrasaccharide was designed for compatibility with (i) serotype-specific glucosylations and O-acetylations defining S. flexneri O-antigens, (ii) recognition by suitable α-transglucosylases, and (iii) programmed oligomerization following enzymatic α-d-glucosylation. The tetrasaccharide core was chemically synthesized from two crystalline monosaccharide precursors. Six α-transglucosylases found in the glycoside hydrolase family 70 were shown to transfer glucosyl residues on the non-natural acceptor. The successful proof of concept is achieved for a pentasaccharide featuring the glucosylation pattern from the S. flexneri type IV O-antigen. It demonstrates the potential of appropriately planned chemoenzymatic pathways involving non-natural acceptors and low-cost donor/transglucosylase systems to achieve the demanding regioselective α-d-glucosylation of large substrates, paving the way to microbial oligosaccharides of vaccinal interest.

ADP-heptose is a newly identified pathogen-associated molecular pattern of Shigella flexneri.

During an infection, the detection of pathogens is mediated through the interactions between pathogen-associated molecular patterns (PAMPs) and pathogen recognition receptors. ß-Heptose 1,7-bisphosphate (ßHBP), an intermediate of the lipopolysaccharide (LPS) biosynthesis pathway, was recently identified as a bacterial PAMP. It was reported that ßHBP sensing leads to oligomerization of TIFA proteins, a mechanism controlling NF-κB activation and pro-inflammatory gene expression. Here, we compare the ability of chemically synthesized ßHBP and Shigella flexneri lysate to induce TIFA oligomerization in epithelial cells. We find that, unlike bacterial lysate, ßHBP fails to initiate rapid TIFA oligomerization. It only induces delayed signaling, suggesting that ßHBP must be processed intracellularly to trigger inflammation. Gene deletion and complementation analysis of the LPS biosynthesis pathway revealed that ADP-heptose is the bacterial metabolite responsible for rapid TIFA oligomerization. ADP-heptose sensing occurs down to 10-10 M. During S. flexneri infection, it results in cytokine production, a process dependent on the kinase ALPK1. Altogether, our results rule out a major role of ßHBP in S. flexneri infection and identify ADP-heptose as a new bacterial PAMP.

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. Major variables were determined and acceptable margins were investigated for a synthetic pentadecasaccharide-tetanus toxoid conjugate, which is a promising vaccine candidate against Shigella flexneri serotype 2a infection. The optimized process is applicable to any thiol-equipped hapten and provides an efficient control of the hapten:carrier ratio. Moreover, comparison of four S. flexneri 2a glycoconjugates only differing by their pentadecasaccharide:tetanus toxoid ratio confirmed preliminary findings indicating that hapten loading is critical for immunogenicity with an optimal ratio here in the range of 17 ± 5. In addition, the powerful influence of alum on the immunogenicity of a Shigella synthetic carbohydrate-based conjugate vaccine candidate is demonstrated for the first time, with a strong anti-S. flexneri 2a antibody response sustained for more than one year.

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 % -ß-d-GlcpNAc-(1→} ([(E)ABAc CAc D]) repeating unit, and the non-O-acetylated equivalent defines S. flexneri X. Propyl hepta-, octa-, and decasaccharides sharing the (E')A'BAc CD(E)A sequence, and their non-O-acetylated analogues were synthesized from a fully protected BAc CD(E)A allyl glycoside. The stepwise introduction of orthogonally protected mono- and disaccharide imidate donors was followed by a two-step deprotection process. Monoclonal antibody binding to twenty-six S. flexneri types 3a and X di- to decasaccharides was studied by an inhibition enzyme-linked immunosorbent assay (ELISA) and STD-NMR spectroscopy. Epitope mapping revealed that the 2C -acetate dominated the recognition by monoclonal IgG and IgM antibodies and that the BAc CD segment was essential for binding. The glucosyl side chain contributed to a lesser extent, albeit increasingly with the chain length. Moreover, tr-NOESY analysis also showed interaction but did not reveal any meaningful conformational change upon antibody binding.

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-ß-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. Successful differentiation between hydroxyl groups features the selective acylation of primary alcohols and acetalation of a cis-vicinal diol, followed by a controlled per-O-benzylation step. Moreover, we describe the successful use of the pentasaccharide intermediate in the [5 + 5] synthesis of an aminoethyl aglycon-equipped decasaccharide, corresponding to a dimer of the basic repeating unit from the O-specific polysaccharide of Shigella flexneri 2a, a major cause of bacillary dysentery. Four analogues of the disaccharide acceptor were synthesized and evaluated to reach a larger repertoire of O-glucosylation patterns encountered among S. flexneri type-specific polysaccharides. New insights on the potential and limitations of planned chemo-enzymatic pathways in oligosaccharide synthesis are provided.

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 2.1.4.19) can efficiently couple an α-L-rhamnosyl acceptor to a maltodextrin molecule with an α-(1→4) linkage, albeit in mixture with the α-(1→3) regioisomer, thus giving two glucosylated acceptors in a single reaction. Optimisation of the CGTase coupling reaction with ß-cyclodextrin as the donor substrate and methyl or allyl α-L-rhamnopyranoside as acceptors resulted in good conversion yields (42-70%) with adjustable glycosylation regioselectivity. Moreover, the efficient chemical conversion of the products of CGTase-mediated cis-glucosylation into protected building blocks (previously used in the synthesis of O-antigen fragments of several Shigella flexneri serotypes) was substantiated. These novel chemoenzymatic strategies towards useful, convenient intermediates in the synthesis of S. flexneri serotypes 2a and 3a oligosaccharides might find applications in developments towards synthetic carbohydrate-based vaccine candidates against bacillary dysentery.

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)]-ß-D-GlcpNAc-(1 → }n branched polysaccharide ({(Ac)AB(Ac)C(E)D}n). It is identical to that from S. flexneri 1a, except for the 2C-acetate. A concise synthesis of the disaccharide ED, trisaccharides (Ac)C(E)D and C(E)D, tetrasaccharides B(Ac)C(E)D and BC(E)D, and pentasaccharides AB(Ac)C(E)D and ABC(E)D is described starting from a 2-N-acetyl-D-glucosaminide acceptor and using the imidate glycosylation chemistry. The E residue was efficiently introduced via a potent stereoselective [E + D] coupling. In contrast, harsh conditions and appropriate tuning of the donor were required for a high yielding [C + ED] glycosylation. Irrespective of the level of steric bulk at residue C, glycosylation at O-3D of the ED acceptor generated a major change of conformation of the D residue within the obtained C(E)D trisaccharide, as attested by NMR data. Proper manipulation of the constrained C(E)D trisaccharide was necessary to proceed with the stepwise chain elongation at O-3C of an acceptor having the 2C-O-acetyl already in place. The protected intermediates went through a one- to three-step deprotection sequence to give the propyl glycoside targets, as portions of the O-antigens from both S. flexneri 1a and 1b. Protecting group removal was clearly associated with conformational relief, yielding oligosaccharides, for which NMR data were consistent with a (4)C1 conformation for the 3,4-di-O-glycosylated residue D, as in the native bacterial polymers.

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. The latter was obtained via a convergent route involving the imidate glycosylation chemistry. Binding studies to five protective mIgGs showed that none of the acetates adds significantly to broad antibody recognition. Yet, one of the five antibodies had a unique pattern of binding. With IC50 in the micromolar to submicromolar range mIgG F22-4 exemplifies a remarkable tight binding antibody against diversely O-acetylated and non-O-acetylated fragments of a neutral polysaccharide of medical importance.

Expedient synthesis of l-heptose derived septacidin building blocks from l-glucose.

Bacterial natural products containing heptosides such as septacidin represent interesting scaffolds for the development of drugs to combat antimicrobial resistance. However, very few synthetic strategies have been reported to grant access to these derivatives. Here, we have devised a synthetic pathway to l-glycero-l-glucoheptoside, a key building block en route to septacidin, directly from l-glucose. Importantly, we show that carbon homologation at C6, encompassing oxidation of the C6-OH followed by methylenation, is significantly influenced by the nature of the C4-moiety. In order to observe the effect of various patterns, namely azide (N3), p-methoxybenzyloxy (OPMB), and benzyloxy (OBn), a thorough analysis was conducted on the corresponding l-glucosides. The results unveiled a distinct trend where the efficiency of methylenation followed the trend OBn > OPMB > N3. Finally, the C6-alkene was dihydroxylated in the presence of osmium tetroxide to yield the expected l/d-glycero-l-glucoheptosides. The lead building block, which features a C-4 azide, was delivered as a phenyl thioglycoside. Added to the suitable masking of the 6,7-diol, this combination enables further functionalization to achieve versatile compounds of biological interest. The study insights into the interplay between substitution at C-4 and carbon homologation at C-6 provide valuable guidance for future endeavors in the synthesis of these carbohydrate molecules.

A Shigella flexneri 2a synthetic glycan-based vaccine induces a long-lasting immune response in adults.

Shigella is a leading cause of moderate to severe diarrhea worldwide and of diarrhea-associated deaths in children under 5 years of age in low-and middle-income countries. A vaccine against shigellosis is in high demand. SF2a-TT15, a synthetic carbohydrate-based conjugate vaccine candidate against Shigella flexneri 2a (SF2a) was found safe and strongly immunogenic in adult volunteers. Here, SF2a-TT15 at 10 µg oligosaccharide (OS) vaccine dose is shown to induce a sustained immune response in magnitude and functionality in the majority of volunteers followed up 2 and 3 years post-vaccination. High levels of either one of the humoral parameters as well as the number of specific-IgG memory B-cells determined 3 months after vaccination were good predictors of the durability of the immune response. This study is the first to examine the long-term durability of antibody functionality and memory B-cell response induced by a Shigella vaccine candidate.

Further characterization of Shigella-specific (memory) B cells induced in healthy volunteer recipients of SF2a-TT15, a Shigella flexneri 2a synthetic glycan-based vaccine candidate.

Shigellosis is common worldwide, and it causes significant morbidity and mortality mainly in young children in low- and middle- income countries. To date, there are not broadly available licensed Shigella vaccines. A novel type of conjugate vaccine candidate, SF2a-TT15, was developed against S. flexneri serotype 2a (SF2a). SF2a-TT15 is composed of a synthetic 15mer oligosaccharide, designed to act as a functional mimic of the SF2a O-antigen and covalently linked to tetanus toxoid (TT). SF2a-TT15 was recently shown to be safe and immunogenic in a Phase 1 clinical trial, inducing specific memory B cells and sustained antibody response up to three years after the last injection. In this manuscript, we advance the study of B cell responses to parenteral administration of SF2a-TT15 to identify SF2a LPS-specific B cells (SF2a+ B cells) using fluorescently labeled bacteria. SF2a+ B cells were identified mainly within class-switched B cells (SwB cells) in volunteers vaccinated with SF2a-TT15 adjuvanted or not with aluminium hydroxide (alum), but not in placebo recipients. These cells expressed high levels of CXCR3 and low levels of CD21 suggesting an activated phenotype likely to represent the recently described effector memory B cells. IgG SF2a+ SwB cells were more abundant than IgA SF2a + SwB cells. SF2a+ B cells were also identified in polyclonally stimulated B cells (antibody secreting cells (ASC)-transformed). SF2a+ ASC-SwB cells largely maintained the activated phenotype (CXCR3 high, CD21 low). They expressed high levels of CD71 and integrin α4ß7, suggesting a high proliferation rate and ability to migrate to gut associated lymphoid tissues. Finally, ELISpot analysis showed that ASC produced anti-SF2a LPS IgG and IgA antibodies. In summary, this methodology confirms the ability of SF2a-TT15 to induce long-lived memory B cells, initially identified by ELISpots, which remain identifiable in blood up to 140 days following vaccination. Our findings expand and complement the memory B cell data previously reported in the Phase 1 trial and provide detailed information on the immunophenotypic characteristics of these cells. Moreover, this methodology opens the door to future studies at the single-cell level to better characterize the development of B cell immunity to Shigella.

Applying pairwise combinations of amino acid mutations for sorting out highly efficient glucosylation tools for chemo-enzymatic synthesis of bacterial oligosaccharides.

Iterative saturation mutagenesis and combinatorial active site saturation focused on vicinal amino acids were used to alter the acceptor specificity of amylosucrase from Neisseria polysaccharea , a sucrose-utilizing α-transglucosidase, and sort out improved variants. From the screening of three semirational sublibraries accounting in total for 20,000 variants, we report here the isolation of three double mutants of N. polysaccharea amylosucrase displaying a spectacular specificity enhancement toward both sucrose, the donor substrate, and the allyl 2-acetamido-2-deoxy-α-D-glucopyranoside acceptor as compared to the wild-type enzyme. Such levels of activity improvement have never been reported before for this class of carbohydrate-active enzymes. X-ray structure of the best performing enzymes supported by molecular dynamics simulations showed local rigidity of the -1 subsite as well as flexibility of loops involved in active site topology, which both account for the enhanced catalytic performances of the mutants. The study well illustrates the importance of taking into account the local conformation of catalytic residues as well as protein dynamics during the catalytic process, when designing enzyme libraries.

Toward a Multivalent Synthetic Oligosaccharide-Based Conjugate Vaccine against Shigella: State-of-the-Art for a Monovalent Prototype and Challenges.

This review focuses on the molecular glycovaccine concept, a promising option to develop a Shigella glycoconjugate vaccine. Subsequent to original developments involving, as main vaccine component, the detoxified Shigella lipopolysaccharide randomly conjugated at multiple sites to a carrier protein, novelty stems from the use of rationally designed, well-defined chemically synthesized oligosaccharide haptens conceived as functional surrogates of the main surface antigen, linked via single-point attachment onto a carrier. The concept and design of such a fine-tuned Shigella glycovaccine are presented by way of SF2a-TT15, a neoglycoprotein featuring a synthetic 15-mer oligosaccharide, which constitutes an original vaccine prototype targeting Shigella flexneri 2a, one of the predominant circulating strains in endemic settings. The clinical testing of SF2a-TT15 is summarized with the first-in-human phase I trial in young healthy adults showing a good safety profile and tolerability, while inducing bactericidal antibodies towards S. flexneri 2a bacteria. The proof-of-concept of this novel approach being established, an ongoing phase IIa clinical study in the nine-month-old infant target population in endemic area was launched, which is also outlined. Lastly, some challenges to move forward this original approach toward a multivalent cost-effective Shigella synthetic glycan conjugate vaccine are introduced.

Protecting group principles suited to late stage functionalization and global deprotection in oligosaccharide synthesis.

Chemical synthesis is a powerful tool to access homogeneous complex glycans, which relies on protecting group (PG) chemistry. However, the overall efficiency of chemical glycan assembly is still low when compared to oligonucleotide or oligopeptide synthesis. There have been many contributions giving rise to collective improvement in carbohydrate synthesis that includes PG manipulation and stereoselective glycoside formation and some of this chemistry has been transferred to the solid phase or adapted for programmable one pot synthesis approaches. However, after all glycoside bond formation reactions are completed, the global deprotection (GD) required to give the desired target OS can be challenging. Difficulties observed in the removal of permanent PGs to release the desired glycans can be due to the number and diversity of PGs present in the protected OSs, nature and structural complexity of glycans, etc. Here, we have reviewed the difficulties associated with the removal of PGs from densely protected OSs to obtain their free glycans. In particularly, this review focuses on the challenges associated with hydrogenolysis of benzyl groups, saponification of esters and functional group interconversion such as oxidation/reduction that are commonly performed in GD stage. More generally, problems observed in the removal of permanent PGs is reviewed herein, including benzyl, acyl (levulinoyl, acetyl), N-trichloroacetyl, N-2,2,2-trichloroethoxycarbonyl, N-phthaloyl etc. from a number of fully protected OSs to release the free sugar, that have been previously reported in the literature.

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

Shigella, 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 Shigella flexneri 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. We report on the scale-up feasibility under GMP conditions of a high yielding bioconjugation process established to ensure a reproducible and controllable glycan/protein ratio. Preclinical and clinical batches complying with specifications from ICH guidelines, WHO recommendations for polysaccharide conjugate vaccines, and (non)compendial tests were produced. The obtained SF2a-TT15 vaccine candidate passed all toxicity-related criteria, was immunogenic in rabbits, and elicited bactericidal antibodies in mice. Remarkably, the induced IgG antibodies recognized a large panel of SF2a circulating strains. These preclinical data have paved the way forward to the first-in-human study for SF2a-TT15, demonstrating safety and immunogenicity. This contribution discloses the yet unreported feasibility of the GMP synthesis of conjugate vaccines featuring a unique homogeneous synthetic glycan hapten fine-tuned to protect against an infectious disease.

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 â†’ 2)-α-l-Rhap-(1 â†’ 3)-α-l-Rhap-(1 â†’ 3)-ß-d-GlcpNAc-(1→, with site-selective glucosylation(s) and/or O-acetylation defining the serotypes. To investigate the conformational basis of serotype specificity, we sampled conformational behaviors during 60 ns of molecular dynamic simulations for oligosaccharides representing three RUs of each one of the O-Ags corresponding to serotypes 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, X and Y, respectively. The calculated trajectories were checked by nuclear magnetic resonance (NMR) for 1a, 2a, 3a and 5a O-Ags. The simulations predict that in all O-Ags, but 1a and 1b, serotype-specific substitutions of the backbone do not induce any new backbone conformations compared with the linear type O-Ag Y, although they restrain locally the accessible conformational space. Moreover, the influence of any given substituent on the backbone is independent of the eventual presence of other substituents. Finally, only slight differences in conformational behavior between terminal and inner RUs were observed. These results suggest that the reported serotype-specificity of the protective immune response is not due to recognition of distinct backbone conformations, but to binding of the serotype-defining substituents in the O-Ag context. The gained knowledge is discussed in terms of impact on the development of a broad-serotype coverage vaccine.

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. Additionally, these complementary pieces of information give evidence to the ability of the studied mAb to recognize internal as well as terminal epitopes of its cognate polysaccharide antigen. Hence, we show that an appropriate combination of computational and experimental methods provides a basis to explore carbohydrate functional mimicry and receptor binding. The strategy may facilitate the design of either ligands or carbohydrate recognition domains, according to needed improvements of the natural carbohydrate:receptor properties.