Structure of the O-specific polysaccharide of Citrobacter braakii O7a,3b,1c.

The following structure of the O-specific polysaccharide of Citrobacter braakii O7a,3b,1c was established using sugar and methylation analyses and NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and 1H, 13C heteronuclear single-quantum coherence (HSQC) experiments: (struture: see text). The main D-mannan chain of the polysaccharide studied has the same structure as the O-specific polysaccharide of Escherichia coli O9, Klebsiella pneumoniae O3, and Hafnia alvei PCM 1223.

Structure of a neutral O-specific polysaccharide of the bacterium Providencia alcalifaciens O5.

A neutral polysaccharide containing D-galactose, 2-acetamido-2-deoxy-D-glucose, and 3-acetamido-3,6-dideoxy-D-glucose (Qui3NAc) in the ratios 2:1:1 was obtained by mild acid degradation of lipopolysaccharide of the bacterium Providencia alcalifaciens O5 followed by gel chromatography and ion-exchange chromatography or treatment with anhydrous hydrogen fluoride. On the basis of full acid hydrolysis, methylation, and 1H- and 13C-NMR spectroscopy, including two-dimensional correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), H-detected heteronuclear 1H,13C single-quantum coherence (HSQC), and nuclear Overhauser effect spectroscopy (NOESY), the following structure of the linear tetrasaccharide repeating unit of the polysaccharide was established:

R-form lipopolysaccharides (LPS) of Gram-negative bacteria as possible vaccine antigens.

The antigenic and immunogenic properties of R-form lipopolysaccharides (LPS) of Pseudomonas aeruginosa, Salmonella spp., Escherichia coli and Shigella spp. were studied. The results showed the presence of antigenic relationships among P. aeruginosa R mutants with different structures of the LPS core lipid A region and also among E. coli, Shigella and P. aeruginosa R-LPS, but not with S. minnesota Re-LPS. Vaccines prepared with R-LPS proved to be effective preparations for the active immunization of mice against P. aeruginosa infection. The vaccine stimulated 40-100% protection in mice depending upon the scheme of immunization.

Structure of the O-specific polysaccharide of the bacterium Providencia alcalifaciens O23 containing a novel component: an amide of D-glucuronic acid with N(epsilon)-(1-carboxyethyl)lysine.

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of the bacterium Providencia alcalifaciens O23 and found to contain D-glucose, D-galactose, 2-acetamido-2-deoxy-D-galactose, and N epsilon-(1-carboxyethyl)-N alpha-(D-glucuronoyl)lysine. On the basis of full and partial acid hydrolyses, selective solvolysis with anhydrous hydrogen fluoride, and 1H- and 13C-NMR spectroscopy, including two-dimensional correlation spectroscopy (COSY), H-detected heteronuclear 1H, 13C multi-quantum coherence (HMQC), and rotating-frame nuclear Overhauser effect spectroscopy (ROESY), the following structure of the liner tetrasaccharide repeating unit of the polysaccharide was established [structure: see text]

Pseudomonas aeruginosa antigens as potential vaccines.

Pseudomonas aeruginosa is one of the most important opportunistic bacterial pathogens in humans and animals. This organism is ubiquitous and has high intrinsic resistance to antibiotics due to the low permeability of the outer membrane and the presence of numerous multiple drug efflux pumps. Various cell-associated and secreted antigens of P. aeruginosa have been the subject of vaccine development. Among pseudomonas antigens, the mucoid substance, which is an extracellular slime consisting predominantly of alginate, was found to be heterogenous in terms of size and immunogenicity. High molecular mass alginate components (30-300 kDa) appear to contain conserved epitopes while lower molecular mass alginate components (10-30 kDa) possess conserved epitopes in addition to unique epitopes. Surface-exposed antigens including O-antigens (O-specific polysaccharide of LPS) or H-antigens (flagellar antigens) have been used for serotyping due to their highly immunogenic nature. Chemical structures of repeating units of O-specific polysaccharides have been elucidated and these data allowed the identification of 31 chemotypes of P. aeruginosa. Conserved epitopes among all serotypes of P. aeruginosa are located in the core oligosaccharide and the lipid A region of LPS and immunogens containing these epitopes induce cross-protective immunity in mice against different P. aeruginosa immunotypes. To examine the protective properties of OM proteins, a vaccine containing P. aeruginosa OM proteins of molecular masses ranging from 20 to 100 kDa has been used in pre-clinical and clinical trials. This vaccine was efficacious in animal models against P. aeruginosa challenge and induced high levels of specific antibodies in human volunteers. Plasma from human volunteers containing anti-P. aeruginosa antibodies provided passive protection and helped the recovery of 87% of patients with severe forms of P. aeruginosa infection. Vaccines prepared from P. aeruginosa ribosomes induced protective immunity in mice, but the efficacy of ribosomal vaccines in humans is not yet known. A number of recent studies indicated the potential of some P. aeruginosa antigens that deserve attention as new vaccine candidates. The outer core of LPS was implicated to be a ligand for binding of P. aeruginosa to airway and ocular epithelial cells of animals. However, heterogeneity exists in this outer core region among different serotypes. Epitopes in the inner core are highly conserved and it has been demonstrated to be surface-accessible, and not masked by O-specific polysaccharide. The use of an in vivo selection/expression technology (IVET) by a group of researchers identified a number of P. aeruginosa proteins that are expressed in vivo and essential for virulence. Two of these in vivo-expressed proteins are FptA (ferripyochelin receptor protein) and a homologue of an LPS biosynthetic enzyme. Our laboratory has identified a highly conserved protein, WbpM, and P. aeruginosa with a deficiency in this protein produces only rough LPS and became serum sensitive. Results from these studies have provided the foundation for a variety of vaccine formulations.

Structural and serological studies of lipopolysaccharides of Citrobacter O35 and O38 antigenically related to Salmonella.

Structural analysis using 13C NMR spectroscopy and methylation showed that lipopolysaccharides (LPSs) of Citrobacter freundii O35 and Salmonella arizonae O59 have structurally identical O-specific polysaccharide chains, and those of C. freundii O38 and Salmonella kentucky differ only in the presence of O-acetyl groups in the former. Serological relationships between the structurally similar LPSs were demonstrated using inhibition of ELISA, rocket immunoelectrophoresis, double gel diffusion, and immunoblotting. The O-acetyl groups present in C. freundii O38 LPS are of little importance for its serological specificity. A cross-reaction was observed in immunoblotting between O-antisera to C. freundii O35 and S. arizonae O59 and a structurally related LPS of Pseudomonas aeruginosa O11a, 11b (Lányi-Bergan classification).

Mouse protection induced by Pseudomonas aeruginosa PAC1R and its defective mutants, Salmonella minnesota Re-mutant and Escherichia coli O14.

Pseudomonas aeruginosa PAC1R and its defective mutants (acetone-killed bacteria), Salmonella minnesota Re mutant (acetone-killed bacteria and Re-LPS) and Escherichia coli O14 (acetone-killed bacteria and enterobacterial common antigen, ECA) were studied in a mouse active protection test. Immunized mice were challenged with wild-type P. aeruginosa strains. It was established that P. aeruginosa LPS-defective mutants induced cross-immunity against different Fisher immunotypes of P. aeruginosa. S. minnesota Re-LPS and ECA gave mice protection against P. aeruginosa.

The structure of the O-specific polysaccharide of Salmonella arizonae O21 (Arizona 22) containing N-acetylneuraminic acid.

The O-specific polysaccharide of S. arizonae O21 was found to contain 2-acetamido-2-deoxy-D-glucose, 2-acetamidino-2,6-dideoxy-L-galactose, N-acetylneuraminic acid, and O-acetyl groups. On the basis of 1H and 13C NMR studies of the intact and O-deacetylated polysaccharide and oligosaccharide fragments obtained by solvolysis with anhydrous hydrogen fluoride, partial methanolysis and partial hydrolysis, it was concluded that the O-specific polysaccharide has the following structure: [formula: see text]

The structure of the O-specific polysaccharide of Citrobacter O16 containing glycerol phosphate.

The O-specific polysaccharide, obtained by mild acid degradation of Citrobacter O16 lipopolysaccharide, consists of D-glucose, D-galactose, 2-acetamido-2-deoxy-D-galactose, glycerol and phosphate in the ratios 2:2:2:1:1. Selective cleavage of the polysaccharide was carried out by Smith degradation, N-deacetylation-deamination and dephosphorylation with 48% hydrofluoric acid, which was accompanied by unexpected splitting of one of the glycosidic linkages. The structures of the oligosaccharides thus obtained were established using 1H- and 13C-NMR spectroscopy, including one-dimensional NOE, two-dimensional rotating-frame NOE, homonuclear and heteronuclear 13C, 1H correlation spectroscopy, and, for the Smith degradation product, positive- and negative-ion-mode fast-atom-bombardment MS and MS/MS with collision-induced dissociation. On the basis of these data and the results of methylation analysis, it was concluded that the O-specific polysaccharide has the following repeating unit structure: [formula: see text]

Immunological studies of an artificial antigen with specificity of a common polysaccharide antigen of Pseudomonas aeruginosa.

Synthetic D-rhamnan, with the structure of Pseudomonas aeruginosa common polysaccharide antigen (CPA), was conjugated with BSA. The artificial antigen obtained, and the natural antigens, lipopolysaccharides (LPS) of P. aeruginosa and Pseudomonas cerasi with rhamnan chains of the same structure, were studied by ELISA with rabbit antibodies to the D-rhamnan-BSA conjugate and to the P. cerasi O-antigen. Immunological relations between the LPS of P. aeruginosa and P. cerasi determined by CPA as well as between these LPS and D-rhamnan-BSA were revealed by ELISA. O-antiserum to P. cerasi possesses protective activity in the mouse passive protection test when mice are challenged with some P. aeruginosa strains; the antiserum to the D-rhamnan-BSA does not possess protective activity in mice.

Structure of the O-specific polysaccharide of Salmonella arizonae O45.

The O-specific polysaccharide of Salmonella arizonae O45 (Arizona 11) is acidic and has a branched hexasaccharide repeating unit containing two residues of L-fucose, one residue each of D-galactose, D-ribose, D-glucuronic acid, and 2-acetamido-2-deoxy-D-glucose, and an O-acetyl group. It was studied with the help of 1H and 13C NMR spectroscopy, including 1D selective spin-decoupling and homonuclear Hartmann-Hahn spectroscopy, 2D homonuclear and 13C-1H heteronuclear shift-correlated (COSY) and NOE (ROESY) spectroscopy, as well as by methylation analysis, and selective cleavages with anhydrous HF (or dilute HCl) and lithium in ethylenediamine to yield two different tetrasaccharide fragments. As a result, the following structure of the polysaccharide was established: [formula: see text] Anomalous 13C chemical shifts were observed in the spectrum of the trisaccharide fragment alpha-L-Fucp-(1-->2)-beta-D-Gal p-(1-->3)-beta-D-Glc pNAc, structurally related to the Le(d) blood-group determinant, and rationalised by inter-residue proton-proton interactions.

Specific and non-specific mouse protection induced by different chemotypes of the Pseudomonas aeruginosa lipopolysaccharides.

Lipopolysaccharides (LPS) of Pseudomonas aeruginosa were studied by the mouse active, cross-protection test. The primary structure of O-specific polysaccharides (O-repeating units) of different chemotypes was determined and their cross-protective activity demonstrated. Low doses of LPS (0.1-1 micrograms) stimulated chemotype-specific protection against P. aeruginosa in mice. This immunity was associated with the primary structure of the LPS and it lasted for 14 days after the first or second immunization. High doses of LPS (10-100 micrograms) induced cross-protection against P. aeruginosa in mice. The cross-protective capacity was caused evidently by the secondary structure or conformation of LPS molecule, i.e. by the common conformational protective determinant. This cross-protection lasted for only 5 days after the first or second immunization.

The structure of the O-specific polysaccharide chain of the lipopolysaccharide of Salmonella arizonae O61.

The O-specific polysaccharide was obtained by mild degradation of the Salmonella arizonae O61 lipopolysaccharide with acid. It contained 2-acetamido-2-deoxy-D-glucose, 2-acetamidino-2,6-dideoxy-L-galactose (FucAm), and 7-acetamido-3,5,7,9-tetradeoxy-5-[(R)-3-hydroxybutyramido]-D- glycero-L-galacto-nonulosonic acid (Sug). On the basis of partial acid hydrolysis with 0.1 M HCl, solvolysis with anhydrous HF in methanol, and 1H- and 13C-NMR analysis (including 1H/13C inversely correlated spectroscopy for localisation of N-acyl substituents), it was concluded that the O-specific polysaccharide had the following structure. ----3)-alpha-L-FucAm-(1----3)-alpha-D-GlcNAc-(1----8)-beta-Sug+ ++-(2---- The O-antigen of S. arizonae O61 is structurally related to that of Pseudomonas aeruginosa O12, thus explaining the known serological cross-reactivity between these micro-organisms.

Antibodies against the common polysaccharide and protein protective antigens of Pseudomonas aeruginosa in normal blood donors.

Screening of normal plasma obtained from 172 blood donors from the Helsinki area and from 46 blood donors from the Moscow area was performed in order to reveal 'natural' antibodies to the common polysaccharide (rhamnan) and protein antigens of P. aeruginosa. Antibodies were detected by ELISA. Among blood donors from the Helsinki area high titres of antibodies to the protein antigens were detected in 42 active blood donors (24.4%) and very high titres in nine (5.3%) highly-active blood donors, whereas in the Moscow area in 15 (34.9%) and in one case (2.3%), respectively. Antibodies to the common polysaccharide antigen were determined in the Helsinki area in 23 active blood donors (13.4%) and in one (0.5%) highly active blood donor, whereas in the Moscow area in four active blood donors (8.6%). The plasma contained both polysaccharide and anti-protein antibodies. The level of antibodies to the polysaccharide antigen was lower than the level of antibodies to the protein antigens. There was no statistically significant difference between the corresponding values of blood donor groups from the Helsinki and Moscow areas.

Clinico-immunological trials of Pseudomonas aeruginosa vaccine.

Pseudomonas aeruginosa vaccine (PV) containing predominantly cell-wall protein protective antigens was tested for safety and immunogenicity by immunization of 119 volunteers. The criteria for safety and immunogenicity were the absence of serious post-vaccinal reactions or complications either during immunization or 12 months later. There were mild (19 donors or 15.9%) and moderate (three donors or 2.5%) febrile reactions after immunization and in two volunteers the body temperature increased up to 38 degrees C, however it decreased to normal values within 24 h. We observed in 43 (36.1%) of volunteers mild and in five (4.2%) moderate local reactions which disappeared within 24 h. Using the ELISA and passive mouse protection test it was shown that PV induces the formation of specific antibodies. A high level of specific antibodies persisted for the 5-month period of observation. The antibody titres increased in 94-97% of volunteers and moreover in 45.6% the antibody titres (the number of ELISA units) increased 2.5-3-fold and more. Anti-P. aeruginosa plasma was used for the treatment of 46 patients with severe forms of P. aeruginosa infection (40 adults and six infants aged up to 2 years) and 87% of the patients recovered.

Detection of common polysaccharide antigen of Pseudomonas aeruginosa with O-antiserum to Pseudomonas cerasi.

The identity of the structures of common polysaccharide antigen (CPA) of Pseudomonas aeruginosa and O-antigen of Pseudomonas cerasi was used for immunochemical study of polysaccharide antigens of seven immunotypes (IT) of P. aeruginosa. ELISA performed with O-antiserum to P. cerasi showed that CPA is present in all seven ITs in different amounts. In SDS-PAGE this antigen behaves as a lipopolysaccharide (LPS) and is detected by immunoblotting technique in five of seven ITs.

Experimental studies on the protective efficacy of a Pseudomonas aeruginosa vaccine.

Pseudomonas aeruginosa vaccine (PV) containing cell proteins with molecular weight (Mr) 20,000-100,000 and up to 0.08% (w/v) admixture of lipopolysaccharide was obtained by water-salt extraction and subsequent ultrafiltration. PV protects mice against experimental P. aeruginosa infection, stimulates production of specific protective antibodies in rabbit and does not provoke obvious toxicity in laboratory animals.