Shigellosis, a major cause of diarrhea worldwide, exhibits high morbidity and mortality in children. Specificity of Shigella immunity is determined by the structure of the main protective O-antigen polysaccharide component incorporated into the lipopolysaccharide (LPS) molecule. Endotoxicity, however, precludes LPS clinical use. Thus, there is still no vaccine against the most prevalent shigellosis species (serotype S. flexneri 2a), despite ongoing efforts focused on inducing serotype-specific immunity. As LPS is highly heterogenous, we hypothesized that more homogenous pools of LPS might be less toxic. We developed a method to generate a homogenous S. flexneri 2a LPS subfraction, Ac-3-S-LPS, containing long chain O-specific polysaccharide (S-LPS) and mainly tri-acylated lipid A, with no penta- and hexa-acylated, and rare tetra-acylated lipid A. Ac-3-S-LPS had dramatically reduced pyrogenicity and protected guinea pigs from shigellosis. In volunteers, 50 mu g of injected Ac-3-S-LPS vaccine was safe, with low pyrogenicity, no severe and few minor adverse events, and did not induce pro-inflammatory cytokines. In spite of the profound lipid A modification, the vaccine induced a prevalence of IgG and IgA antibodies. Thus, we have developed the first safe immunogenic LPS-based vaccine candidate for human administration. Homogenous underacetylated LPSs may also be useful for treating other LPS-driven human diseases. (C) 2019 The Authors. Published by Elsevier Ltd.

Solvolysis of polysaccharides with trifluoroacetic acid is efficient for selective cleavage of certain glycosidic linkages. This method was successfully used for structure elucidation of polysaccharides and obtaining oligosaccharides from polysaccharides for construction of glycoconjugate vaccines in cases of O-specific polysaccharides of bacteria Enterobacter cloacae and Shigella flexneri.

Muropeptides are fragments of peptidoglycan that trigger innate immune responses by activating nucleotide-binding oligomerization domain (NOD) 1 and NOD2. Muropeptides from Gram-negative bacteria contain a meso-diaminopimelic acid (meso-DAP) residue in either a terminal or a non-terminal position. While the former ones are known to be recognized by NOD1, much less is known about recognition of muropeptides with non-terminal meso-DAP, which are most abundant moieties of Gram-negative peptidoglycans. Here, we developed a novel system to assess biological activity of muropeptides, based on CRISPR/Cas9-mediated knockout (KO) of NOD1 and NOD2 genes in modified HEK293T cells. Using NOD1/NOD2 knockout and overexpression systems, as well as human monocytes and macrophages, we refine the current view of muropeptide recognition. We show that NOD2 can recognize different natural muropeptides containing a meso-DAP residue (preferably in a non-terminal position), provided they are present at micromolar concentrations. NOD2 accepts muropeptides with long and branched peptide chains and requires an intact N-acetylmuramyl residue. Muropeptides with non-terminal meso-DAP can activate NOD1 as well, but, in this case, probably require peptidase pre-processing to expose the meso-DAP residue. Depending on NOD1/NOD2 ratio in specific cell types, meso-DAP-containing muropeptides can be recognized either primarily via NOD2 (in monocytes) or via NOD1 (in monocyte-derived macrophages and HEK293T-derived cells). The dual NOD1/NOD2 agonism of meso-DAP-containing muropeptides should be taken into account when assessing cellular responses to muropeptides and designing muropeptide immunostimulants and vaccine adjuvants.

O-Polysaccharide (O-antigen) accompanied by a minor mannan was isolated from the lipopolysaccharide of Escherichia coli O39 and studied by component analyses, methylation, Smith degradation, mass spectrometry, and 1D and 2D NMR spectroscopy. In addition, a new approach, solvolysis with anhydrous trifluoroacetic acid, was applied to cleave selectively the rhamnosidic linkage. The following structure of the O-polysaccharide was established: α--D-Galpl-->3-->3)-β-D-Quip4N(R3Hb)-(1-->2)-α-D-Manp-(l-->4)-α-L-Rhap-(1-->3)-α-D-GlcpNAc-(1--> where D-Qui4N(R3Hb) indicates 4,6-dideoxy-4-[(R)-3-hydroxybutanoylamino]-d-glucose. The O-antigen gene cluster of E. coli O39 has been sequenced. The gene functions were tentatively assigned by a comparison with sequences in the available databases and found to be in agreement with the O-polysaccharide structure.

Muramyl peptides are potent activators of innate immune cells but, for an unknown reason, are weak inducers of pro-inflammatory cytokines in dendritic cells (DCs). Here, we show that in DCs stimulated by glucosaminyl muramyl tripeptide (GMtri) isolated from S. typhi, elevation of mRNA coding for tumor necrosis factor (TNF) and several other pro-inflammatory cytokines and chemokines is more short-lived than in lipopolysaccharide (LPS)-stimulated DCs and in GMtrior LPS-stimulated macrophages (Mphi). In addition, TNF and CCL4 mRNAs in GMtri-stimulated DCs are translated less efficiently than in LPS-stimulated DCs or in GMtrior LPS-stimulated Mphi. We found that Mphi, but not DCs, secrete interleukin-1beta (IL-1β) within first 1-2 h of GMtri stimulation. Addition of recombinant IL-1β results in an increase of TNF production by GMtri-stimulated DCs up to the levels observed in LPS-stimulated DCs and in GMtrior LPS-stimulated Mphi. Thus, the weak production of TNF by GMtri-stimulated DCs is likely caused by their inability to secrete IL-1β, which is an important autocrine regulator of cellular responses to muramyl peptides.

Bacterial peptidoglycan and its muropeptide derivatives potently activate mammalian innate immune system and are promising immunomodulators and vaccine adjuvants. However, their effects on human antigen-presenting cells, such as dendritic cells (DCs) and Mphi, are not fully understood. Lysozyme treatment of PG from Salmonella typhi yielded three muropeptides, GlcNAc–MurNAc–L-Ala–D-isoGlu–meso-DAP (GM-3P), GlcNAc–MurNAc–L-Ala–D-isoGlu–meso-DAP–D-Ala (GM-4P), and a dimer (GM-4P)2, in which two GM-4P monomers are linked through their peptidic moieties. All three muropeptides induced TNF-α and IL-6 production by Mphi (GM-3P>GM-4P>>(GM-4P)2), but failed to trigger TNF-α, IL-6 and IL-12p70 production by immature DCs. At the same time, muropeptide-stimulated DCs abundantly produced inflammatory chemokines IL-8, MIP-1α and MIP-1β, as well as displayed signs of phenotypic and functional maturation. Thus, muropeptide-dependent pro-inflammatory cytokine production is repressed in DCs. While this defect may be partly compensated in vivo by muropeptide-activated Mphi, neither Mphi nor DCs produce Th1- or Th17-polarizing cytokines upon muropeptide stimulation, which may contribute to the preferential induction of Th2 responses by muropeptides and should be taken into account when designing muropeptide-based immunomodulators and adjuvants.

Shigella flexneri type 2a is the first, and type 1b is the second, most prevalent isolates from patients with shigellosis in Russia. The O-specific polysaccharides (OPSs, O-antigens) of S. flexneri types 1–5 possess a common →2)-α-l-RhapIII-(1→2)-α-l-RhapII-(1→3)-α-l-RhapI-(1→3)-β-d-GlcpNAc-(1→ backbone and differ from each other in its glucosylation or/and O-acetylation at various positions, the modifications being responsible for various O-factors. It was suggested that O-factor 6 expressed by type 1b is associated with O-acetylation of RhaI at position 2 but more than one O-acetyl group has been detected in the type 1b OPS [Kenne, L. et al. Eur. J. Biochem.1978, 91, 279–284]. In this work, O-acetylation of RhapI in the type 1b OPS was confirmed by NMR spectroscopy and location of an additional O-acetyl group at position either 3 (major) or 4 (minor) of RhapIII was determined. Type 1a differs from type 1b in the lack of O-acetylation of RhapI only. In type 2a, in addition to two reported major O-acetyl groups at position 6 of GlcNAc and position 3 of RhapIII [Kubler-Kielb, J. et al. Carbohydr. Res.2007, 342, 643–647], a minor O-acetyl group was found at position 4 of RhaIII. Therefore, RhapIII is O-acetylated in the same manner in all three S. flexneri serotypes studied.

The O-specific polysaccharide (O-antigen) structure of a Shigella flexneri type 4a strain from the Dysentery Reference Laboratory (London, UK) was elucidated in 1978 and its characteristic feature was found to be alpha-D-glucosylation of GlcNAc at position 6, which defines O-factor IV. Our NMR spectroscopic studies of the O-specific polysaccharides of two other strains belonging to S. flexneri type 4a (G1668 from Adelaide, Australia, and 1359 from Moscow, Russia) confirmed the carbohydrate backbone structure but revealed in both strains an additional component, ethanolamine phosphate (EtnP), attached at position 3 of one of the rhamnose residues: [structure: see text]. Phosphorylation has not been hitherto reported in any S. flexneri O-antigen. Reinvestigation of the O-specific polysaccharide of S. flexneri type 4b showed that it is not phosphorylated and confirmed its structure established earlier.

On the basis of chemical and NMR data the partial structure of lipid A from lipooligosaccharide (LOS) of Neisseria meningitidis group B, strain BC5S No 125 was established. Lipid A consisted of disaccharide 2-deoxy-6-O-[2-deoxy-2-(3-hydroxytetradecanoylamino)- beta-gluco-pyranosyl]-2-(3-hydroxytetradecanoylamino)- alpha-glucopyranose carrying the beta-(2-aminoethyl)pyrophosphate residue at 0-4' and the pyrophosphate or phosphate residue at 0-1. On hydrolysis of the acidic form of LOS with 1% acetic acid the substituent at 0-1 was practically completely removed whereas that at 0-4' was stable. The analogous hydrolysis of the Mg-salt of LOS was accompanied by splitting off the pyrophosphate linkage in the substituent at 0-4'. Hydrolysis of LOS at pH 4.5 in the presence of SDS led mainly to a lipid A preparation retaining both pyrophosphate residues.