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.

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.

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).

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.

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]

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 the O-specific polysaccharide of Citrobacter freundii O3a,3b,1c.

The following structure of the O-specific polysaccharide of Citrobacter freundii O3a,3b,1c containing D-mannose and D-rhamnose was established using sugar analysis and NMR spectroscopy, including computer-assisted analysis of the 13C NMR spectrum, 2D COSY, H,H-relayed COSY, heteronuclear 13C, 1H correlation (HETCOR), and rotating-frame NOE spectroscopy (ROESY):-->4)-alpha-D-Manp-(1-->3)-beta-D-Rhap-(1-->4) -beta-D-Rhap-(1-->.

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.

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.

[Antigenic polysaccharides of bacteria. 22. Structure of the O-specific polysaccharide chain of Proteus hauseri lipopolysaccharide]. / Antigennye polisakharidy bakterii. 22. Struktura O-spetsificheskoi polisakharidnoi tsepi lipopolisakharida Proteus hauseri.

The following structure of the repeating unit of the Proteus hauseri O-specific polysaccharide was established on the basis of monosaccharide composition and 13C NMR data of the polysaccharide and products of its Smith degradation and partial cleavage with hydrogen fluoride: (Formula: see text).

[Intrageneric O-antigenic relations of Providencia bacteria]. / Vnutrirodovye O-antigennye sviazi bakterii Providencia

The authors present literature and personal data on the intragenera O-antigenic relations of the strains of a collection of bacteria belonging to Providencia genus, including standard strains of 53 serological O-groups whose antigenic interrelations were studied serologically in the tests of agglutination and adsorption of agglutinins. Experimental results confirmed the presence of unilateral relations by the O-antigen between the serological groups 01-021, 03-021 and bilateral relations between the serological groups 01-03; there were also revealed relations by the O-antigens between the serological groups 09-036, 014-023, 018-035, 039-053, and 039-027 of a unilateral character. The data obtained added information to the antigenic structure of Providencia and were of significance for the preparation of diagnostic O-sera of Providencia.

[Experimental study of the protective activity of vaccines made from 7 Pseudomonas aeruginosa immunotypes]. / Eksperimental'noe izuchenie protektivnoi aktivnosti vaktsin iz semi immunotipov Pseudomonas aeruginosa.

Monovaccines were prepared from seven P. aeruginosa immunotypes (according to Fisher's classification) and three P. aeruginosa production strains belonging to immunotypes 2, 3, and 7. The immunogenic potency of these monovaccines was studied in direct and cross experiments on the active protection of mice. The study revealed that the monovaccines prepared from P. aeruginosa of seven immunotypes possessed both specific and cross protective activity. Protective cross activity was revealed also in the vaccines prepared from the production strains. When tested in mice challenged with the corresponding homologous strains, the monovaccines prepared from immunotypes 2, 3, 4 and 7 proved to possess higher immunogenic potency than monovaccines prepared from immunotypes 1, 5 and 6.

[An antigenic analysis and the protective properties of the R-form lipopolysaccharides of gram-negative bacteria]. / Antigennyi analiz i protektivnye svoistva lipopolisakharidov R-form nekotorykh gramotritsatel'nykh bakterii.

The antigenic and immunogenic properties of the R-form lipopolysaccharides (R-LPS) of Pseudomonas aeruginosa, Salmonella minnesota, Escherichia coli and Shigella were studied. The results of the study revealed the existence of antigenic relationship between P.aeruginosa R-LPS and R-LPS Escherichia and Shigella. In serological tests no antigenic relationship between P. aeruginosa R-LPS and Salmonella R-LPS was revealed, but as shown in earlier experiments of the protection of mice, Re-glycolipid stimulated protective immunity against Pseudomonas infection in the animals. On the basis of R-LPS obtained from selected P. aeruginosa and Salmonella strains a vaccine was prepared which proved to be effective against infection caused by P. aeruginosa S-strain in experiments on mice. The vaccine induced protection in 40-100% of immunized mice, depending on the scheme of immunization. The vaccine may probably be effective against infections caused by other gram-negative bacteria.

[Serotyping of bacteria of the genus Providencia using the indirect hemagglutination test and the indirect hemagglutination inhibition test]. / Serotiporovanie bakterii roda Providencia pri pomoshchi reaktsii nepriamoi gemaggliutinatsii i reaktsii tormozheniia nepriamoi gemaggliutinatsii.

The experiments with the collection of strains and diagnostic sera available in the USSR confirmed the data contained in the literature on the good prospects of using the indirect hemagglutination test for the serotyping of bacteria belonging to the genus Providencia. Certain differences were observed in cross reactions, revealed by the indirect hemagglutination test and by agglutination: the interrelations of serogroups O14 and O25, O23 and O25, O23 and O29, O29 and O45, O37 and O49, determined by means of the indirect hemagglutination test, could not be detected in the same sera by the slide and tube agglutination tests. For the first time the usefulness of aqueous-saline extracts of agar cultures in the indirect hemagglutination inhibition test to specify more precisely the interrelations between individual bacterial serogroups of the genus Providencia has been shown.

[Effect of plasmids on the virulence of Pseudomonas aeruginosa strains in experiments on mice]. / Vliianie plazmid na virulentnost' shtammov Pseudomonas aeruginosa v éksperimentakh na myshakh.

A comparative study of virulence of P. aeruginosa strains PAO containing and not containing plasmids has been made. A number of plasmids which are present in strains PAO decrease their virulence for mice 3-7 times. The virulence-affecting plasmids considerably differ in their biological properties. Bacterial mutations rpm, selected as mutations stabilizing RP4 plasmid in PAO cells, have also been found to affect virulence of bacteria, decreasing its level several times. The introduction of plasmids into PAO cells carrying mutations rpm is not accompanied by decrease of virulence.

[Etiologic role of bacteria of the genus Providencia in acute intestinal diseases]. / Ob étiologicheskoi roli bakterii roda Providencia pri ostrykh kishechnykh zabolevaniiakh.

A study was made of morphological, cultural, biochemical properties and antigenic structure (by O-antigen) of 59 strains isolated in group acute intestinal affection of children at the pioneer camp. By the combination of biochemical properties (31 tests) all the cultures isolated were referred to Providencia alcalifaciens of biotype 7. Serological typing with the aid of experimental diagnostic agglutinating O-sera showed these strains to be referred to serological group O2. Identity of the O-antigen of the isolated cultures to the standard O2 strain was confirmed by the results of cross reactions of agglutinin absorption. The results of bacteriological and clinico-epidemiological studies offered a possibility of regarding the isolated Providencia alcalifaciens of serological group O2 as the causative agents of the group intestinal disease in children.

[Structure of the O-specific polysaccharides and the protective activity of the lipopolysaccharides in Pseudomonas aeruginosa of 7 immunotypes (after Fisher)]. / Struktura O-spetsificheskikh polisakharidov i protektivnaia aktivnost' lipopolisakharidov Pseudomonas aeruginosa semi immunotipov (po Fisheru).

The structure of O-specific polysaccharides and the protective activity of lipopolysaccharides (LPS) obtained from seven P. aeruginosa immunotypes (according to Fisher's classification) have been studied. The structure of O-specific polysaccharides, immunotypes 2, 3, 4, 5 and 6, is identical to that of polysaccharides of serotypes 011; 0(2a), 2c; 01; 010a, 10b; 07a, 7d respectively. No structural analogs of O-polysaccharide characteristic of immunotypes 1 and 7 have been detected among serotypes classified according to the scheme of Lányi-Bergan-Akatova-Smirnova. The specific character of O-polysaccharides is confirmed by the results of the passive hemagglutination inhibition test, but the data of the passive hemagglutination test indicate that LPS of different immunotypes are antigenically related. As revealed in experiments on the active immunization of mice, LPS of seven immunotypes possess more or less pronounced cross protective properties. The causes of the cross protective activity observed in experiments with these LPS are discussed.

[Experimental vaccinal prophylaxis of Pseudomonas aeruginosa burn sepsis]. / Vaktsinoprofilaktika sinegnoinogo ozhogovogo sepsisa v éksperimente.

After the injection of P. aeruginosa live culture under the burned skin of mice sepsis develops within the first 24 hours, finally leading to the death of the animals. The microorganisms can be isolated from the blood, liver, kidneys and mesenterial lymph nodes till day 3 and from the spleen till day 5. After the intraperitoneal injection of P. aeruginosa live culture into mice, sepsis also develops within 24 hours, and the culture can be isolated from the blood and parenchymatous organs till day 3. The LD50 of the culture is equal to 5.1 X 10(6) microbial cells when introduced intraperitoneally and to 30 microbial cells in experimental burn sepsis. Experimental burn sepsis clearly demonstrates the effectiveness of Pseudomonas acellular protein vaccine: its index of effectiveness exceeds 3,000.

[The serological characteristics of the O antigens of Pseudomonas aeruginosa isolated from patients with a Pseudomonas infection during immunotherapy]. / Serologicheskaia kharakteristika O-antigenov Pseudomonas aeruginosa, vydelennykh ot bol'nykh sinegnoinoi infektsiei v protsesse immunoterapii.

Serologic characteristics of P. aeruginosa O-antigens isolated from patients with P. aeruginosa infection were studied over the course of treatment with anti-P. aeruginosa sheep immunoglobulin. The preparation was used in 54 patients with nongeneralized forms of P. aeruginosa infection (infected wounds, pleural empyema) externally or intraperitoneally. From the clinical material collected from the patients a total of 54 P. aeruginosa strains were isolated. Serologic typing of the isolated strains with factor or group diagnostic agglutinating sera has revealed the O-group composition of the isolated strains; 66% of them were classified with O-groups 2,3, and 6. Serologic variants of the strains isolated from patients proved to be stable over the course of the disease immunotherapy. Analysis of the results of bacteriologic control of immunotherapy. efficacy and the clinical data has demonstrated the efficacy of immunotherapy in 61.1% of cases and its partial effect in 20.4% of cases of P. aeruginosa infection.