Alf A. Lindberg

Structural studies on the O-specific side-chains of the cell-wall lipopolysaccharide from Salmonella typhimurium 395 ms

The structure of the O-specific side-chains of the cell-wall lipopolysaccharide of Salmonella typhimurium 395 MS has been investigated. Methylation analyses of the original lipopolysaccharide, of the material obtained on mild hydrolysis of the lipopolysaccharide with acid, and of a product obtained by acetalation of all of the, free hydroxyl groups in the lipopolysaccharide, followed by alkaline deacetylation, have provided the essential information in this study. The mixtures of sugars obtained on acid hydrolysis of the different methylated products were analysed, as alditol acetates, by g.l.c.-mass spectrometry. As a result of these studies, a detailed structure of the repeating unit of these side-chains is presented.

Safety, infectivity, immunogenicity, and in vivo stability of two attenuated auxotrophic mutant strains of Salmonella typhi, 541Ty and 543Ty, as live oral vaccines in humans.

Two Salmonella typhi mutants, 541Ty (Vi+) and 543Ty (Vi-), auxotrophic for p-aminobenzoate and adenine, were evaluated as live oral vaccines. 33 volunteers ingested single doses of 10(8), 10(9), or 10(10) vaccine organisms, while four others received two 2 X 10(9) organism doses 4 d apart. No adverse reactions were observed. Vaccine was recovered from coprocultures of 29 of 37 vaccinees (78%) and from duodenal string cultures of two; repeated blood cultures were negative. The humoral antibody response to S. typhi O, H, Vi, and lysate antigens in serum and intestinal fluid was meager. In contrast, all vaccinees manifested cell-mediated immune responses. After vaccination, 69% of vaccinees overall and 89% of recipients of doses greater than or equal to 10(9) responded to S. typhi particulate or purified O polysaccharide antigens in lymphocyte replication studies but not to antigens of other Salmonella or Escherichia coli. All individuals, postvaccination, demonstrated a significant plasma-dependent mononuclear cell inhibition of wild S. typhi.

Rapid and sensitive detection of Salmonella (O:6,7) by immunomagnetic monoclonal antibody-based assays.

An immunomagnetic technique to detect and identify Salmonella serogroup C1 has been developed. Monoclonal antibodies (MAbs) specific for the O antigen 6,7 of Salmonella lipopolysaccharide (LPS) coupled to magnetic beads were used to isolate the salmonellae. Captured bacteria were easily identified by acridine orange staining and measured by enzyme immunoassays with a conjugate anti-LPS MAb as the detector probe. The whole detection process required 2-3 h and the sensitivity was 10(3)-10(4) bacteria/ml. The presence of blood (10%, v/v) or stool (1%, w/v) components did not interfere with the immunomagnetic assay performance.

Glycoprotein conjugate vaccines.

The polysaccharide capsule which surrounds bacterial species like Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis, Salmonella typhi, is a potent virulence factor. It protects the bacterium from phagocytosis, but capsule specific antibodies plus complement binding to the capsule opsonise the organism for phagocytosis and elimination. Purified capsules elicit T-independent antibody responses without a memory function, and are often poorly immunogenic in infants where much of invasive H. influenzae type b (Hib) and pneumococcal infection is seen. Covalent linkage of the polysaccharide, or fractions thereof, to immunogenic carrier proteins creates glycoconjugates which are T-dependent antigens and which prime for boosting either with the glycoconjugate or the capsular polysaccharide; During the 1990s, four Hib glycoconjugate vaccines have been introduced and in countries that have vaccinated the majority of children, the success has been stunning. In countries with very high immunization coverage the disease has been virtually eliminated and, to a decline of over 95% in countries with slightly lower vaccine rates. Worldwide use of Hib glycoconjugate vaccines offers the possibility of elimination of invasive Hib disease. Pneumococcal (11 serotypes with coverage of approximately 85% of invasive disease) and meningococcal (A, C, W 135, Y but not B) glycoconjugates are in pre-registration phases and offer the prospect of being as successful as the Hib glycoconjugates.

Structural studies of the saccharide part of the cell envelope lipopolysaccharide from Haemophilus influenzae strain AH1-3 (lic3 +)☆

The structure of the saccharide part of the lipopolysaccharide from Haemophilus influenzae strain AH1-3 (lic3+) has been investigated. The saccharide was obtained from the lipopolysaccharide by mild acid hydrolysis followed by high-performance anion-exchange chromatography, and isolated fractions were studied by methylation analysis, NMR spectroscopy, and FAB mass spectrometry. The major saccharide is a heptasaccharide with the following structure, [formula: see text] in which Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid and PEA is 2-aminoethyl phosphate. Hep is identified as L-glycero-D-manno-heptose. The absolute configuration of the phosphorylated heptose is tentative only.

Molecular characterization of the O‐acetyl transferase gene of converting bacteriophage SF6 that adds group antigen 6 to Shigella flexneri

Bacteriophage SF6 antigenically converts Shigella flexneri serotype Y strains (‐;3,4) to type 3b carrying group antigen 6,3,4 by means of an O‐acetylation of the O‐antigenic polysaccharide chain. The gene for O‐acetyl transferase of bacteriophage SF6 has been cloned, identified and sequenced. The predicted O‐acetyl transferase protein encoded by this gene was found to consist of 333 amino acids, (37 185 daltons) and to have some similarity with the galactose‐1‐phosphate uridylyltransferase protein of Escherichia coli. The gene has been shown to function in a live vaccine strain of S. flexneri Y type (ΔaroD), making it a 3b type. The converted type 3b strain, SFL1104, was found to elicit significant protection against challenge by both wild‐type serotypes 3b and Y in a guinea‐pig keratoconjunctivitis model.

Development of an auxotrophic oral live Shigella flexneri vaccine

An oral live attenuated Shigella flexneri vaccine candidate strain was constructed by making it auxotrophic and dependent on aromatic metabolites not available in mammalian tissues. An aroD gene of Escherichia coli K12 strain NK 5131, inactivated by insertion in it of the Tn 10 transposon, was transduced using phage P1 into a virulent S. flexneri serotype Y strain (Sfl 1) isolated from a patient with bacillary dysentery. One of the transductant strains Sfl 114 was found to invade HeLa cells in vitro, to cause plaque formation in HeLa monolayers (i.e. maintain intracellular multiplication in vitro), but to be unable to cause keratoconjunctivitis in guinea-pig eyes. When the strain was fed to Macacca fascicularis monkeys it was well tolerated, excreted for 1-4 days, and found to elicit a local intestinal sIgA and serum IgA, IgM and IgG responses. Monkeys challenged with 100 ID50 dose (1 X 10(11) bacteria) of the virulent parent Sfl 1 strain were completely protected from development of diarrhoea. Coloscopy of the monkeys and the sampling of intestinal biopsies showed that the vaccine protected against the surface epithelial erosions and ulcerations seen in unimmunized monkeys. Killing of invading virulent shigellae apparently took place intracellularly in the mucosa suggesting that cellular immune mechanisms played a role in the elicited host defence. The constructed S. flexneri Sfl 114 strain has the properties of a promising shigella vaccine and will next be the subject of studies with human volunteers.

Antigenic similarities in lipopolysaccharides of Haemophilus and Neisseria and expression of a digalactoside structure also present on human cells

Monoclonal antibodies raised against Haemophilus influenzae and Neisseria gonorrhoeae were used to investigate similarities or differences in the lipopolysaccharide antigens of pathogenic and commensal strains of several Gram-negative bacteria indigenous to mucosal surfaces of humans. In immunoblotting experiments, 20 of 36 monoclonal antibodies showed cross-reactions between species of Neisseria and Haemophilus. The common epitopes were present on N. gonorrhoeae, N. meningitidis, N. lactamica, H. influenzae including biogroup aegyptius, and occasionally H. parainfluenzae. No other commensal Neisseria or Gram-negative organisms tested reacted with the monoclonal antibodies with one exception; a single strain of pathogenic Escherichia coli was recognised by a N. gonorrhoeae-specific monoclonal antibody. One monoclonal antibody, raised against N. gonorrhoeae lipopolysaccharide, reacted with N. gonorrhoeae (32 of 59 strains), N. meningitidis (9 of 26 strains), H. influenzae (6 of 16 strains). An epitope expressed by H. influenzae and implicated in its virulence was also present on 14 of 59 strains of N. gonorrhoeae and was shown to comprise a digalactoside structure, alpha-galactosyl-1,4-beta-galactose (Gal alpha 1,4Gal beta), also found on human cells.

Chemical structure of the lipopolysaccharide of Haemophilus influenzae strain I-69 Rd−/b+

The chemical structure of the lipopolysaccharide of a deep-rough mutant (strain I-69 Rd−/b+) of Haemophilus influenzae was investigated. The hydrophilic backbone of lipid A was shown to consist of a β-(1′,6)-linked D-glucosamine disaccharide with phosphate groups at C-1 of the reducing D-glucosamine and at C-4′ of the nonreducing one. Four molecules of (R)-3-hydroxytetradecanoic acid were found directly linked to the lipid A backbone, two by amide and two by ester linkage (positions 2, 2′ and 3, 3′, respectively). Laser-desorption mass spectrometry showed that both 3-hydroxytetradecanoic acids linked to the non-reducing glucosamine carry tetradecanoic acid at their 3-hydroxyl group, so that altogether six molecules of fatty acid are present in lipid A. The lipopolysaccharide was the first described to contain only one sugar unit linked to lipid A. This, sugar in accordance with a previous report [Zamze et al. (1987) Biochem. J. 245, 583–587], was shown to be a dOclA phosphate. The phosphate group was found at position 4, but the analytical procedures employed (permethylation and methanolysis followed by gas-liquid chromatography/mass spectrometry) also revealed dOclA 5-phosphate. Since a cyclic 4,5-phosphate could be ruled out by 31P-NMR, we conclude that, in this lipopolysaccharide, a mixture of dOclA 4- and 5-phosphate is present. By methylation analysis of the dephosphorylated, deacylated and reduced lipopolysaccharide the attachment site of the dOclA was assigned to position C-6′ of the nonreducing glucosamine of lipid A. The anomeric linkages present in the lipopolysaccharide were assessed by 1H-NMR and 13C-NMR of deacylated lipopolysaccharide. The saccharide backbone of this Haemophilus influenzae lipopolysaccharide possesses the following structure:

Chemical Composition of Cell-wall Polysaccharide of Rough Mutants of Salmonella typhimurium

SUMMARY: Quantitative analyses of monosaccharide constituents of the cell-wall polysaccharide of the smooth form and rough mutants of Salmonella typhimurium were made by gas-liquid chromatography of glycitol acetates produced from acid hydrolysates of phenol+water extracts of cell-wall preparations. The presence, in varying amounts, of sugars characteristic of the S-specific repeating unit was detected in all rough mutants investigated. Definite conclusions about the core structure of the different rough mutants could not be drawn by using only the basis of the monosaccharide composition of the cell-wall polysaccharide. Ribose was found in the cell-wall polysaccharide of the smooth form in small amounts.