Protective immunogenicity of synthetic peptides selected from the amino acid sequence of Bordetella pertussis toxin subunit S1.

Five synthetic peptides identified as antigenic sites on the S1 subunit of pertussis toxin (PT) were coupled to the diphtheria toxin cross-reactive mutant protein CRM 197 or BSA. All peptide conjugates were immunogenic in animals. Two peptide-CRM-conjugates, corresponding to amino acids 1-17 and 169-186, induced especially high antibody titers against native PT in mice (Balb/c, C57/Black and outbred NMRI) as measured by ELISA. Upon in vivo PT challenge (0.5 microgram toxin) of the NMRI mice both the CRM and BSA conjugates of these two peptides fully protected the mice from PT induced leucocytosis. Some of the protected mice receiving peptide 1-17 conjugate had very low antibody titers against PT but high titers against the peptide as measured by ELISA, showing that PT-ELISA does not always reflect in vivo protection. A booster response against PT was noted upon challenge with PT in mice receiving peptide 1-17 or peptide 169-186 conjugate. Antibodies against peptide 170-186 could not be evaluated by ELISA since conjugates of this peptide (like PT itself) bind to immunoglobulins. They may also caused clustering of CHO cells. Rabbit antiserum to the peptide 1-17-CRM conjugate was highly efficient in inhibiting the ADP-ribosylating activity of PT with bovine transducin as substrate whereas the rabbit antiserum raised against the peptide 169-186-CRM conjugate neutralized the clustering effect of PT on CHO cells. Thus there is no concise correlation between the in vivo protection against PT challenge and the in vitro methods used for measuring antibody levels against PT (neutralization of the enzyme activity, the CHO cell clustering activity and titers in PT-ELISA). The CRM-conjugates of these two peptides constitute the first synthetic pertussis vaccine candidate with the ability to provide a chemically well defined, safe and efficient pertussis vaccine.

Protective immunogenicity of two synthetic peptides selected from the amino acid sequence of Bordetella pertussis toxin subunit S1.

Two peptides, corresponding to amino acids 1-17 and 169-186 of the amino acid sequence of pertussis toxin (PT) subunit S1, were synthesized and coupled to the diphtheria toxin cross-reactive mutant protein CRM 197 and evaluated for immunogenicity and protective capacity against PT challenge in vivo. The peptide-CRM conjugates induced high antibody titers against native toxin in mice (BALB/c, C57/Black, and outbred NMRI) as measured by ELISA. Upon PT challenge (0.5 microgram of toxin) of the NMRI mice, the CRM conjugates of peptides 1-17 and 169-186 fully protected the mice from PT-induced leukocytosis. Immunization with the corresponding bovine serum albumin conjugates of these two peptides also fully protected mice. Rabbit antiserum to the peptide 1-17-CRM conjugate was highly efficient in inhibiting the ADP-ribosylating activity of PT but did not neutralize the clustering effect of PT on Chinese hamster ovary cells. In contrast, the rabbit antiserum raised against the peptide 169-186-CRM conjugate neutralized the clustering effect of PT on Chinese hamster ovary cells but did not inhibit the enzymatic activity of PT. Peptide 169-186-CRM conjugates mimic the immunoglobulin binding properties of PT and also cause clustering of Chinese hamster ovary cells. The CRM conjugates of these two peptides constitute a synthetic pertussis vaccine candidate with the ability to provide a chemically well-defined, safe, and efficient pertussis vaccine.

Structural studies of the O-antigen polysaccharide of Salmonella thompson, serogroup C1 (6,7).

The structure of the O-antigen polysaccharide of Salmonella thompson, serogroup C1 (6,7) has been investigated mainly by methylation analysis, n.m.r. spectroscopy, specific degradations by a phage-associated enzyme, N-deacetylation-deamination, and f.a.b.-m.s. It is concluded that the structure involves the following repeating unit. (formula; see text) There are two populations of chains, with and without alpha-D-glucopyranosyl groups, 3-linked to an alpha-D-Manp residue, and only the latter type is hydrolysed by the phage enzyme. The alpha linkage of the third Manp residue is cleaved by the O14 phage enzyme. The structure, with or without the alpha-D-glucopyranosyl group, represents the biological repeating-unit.

Fecal colonization with pyelonephritogenic Escherichia coli in neonates as a risk factor for pyelonephritis.

The aim of the study was to investigate the properties of fecal P-fimbriated Escherichia coli strains in neonates and to relate these characteristics to the later development of acute pyelonephritis. In a 2 1/2 year prospective study of the children admitted to a particular neonatal ward, 113 children were found to be fecally colonized with a P-fimbriated Escherichia coli strain. However, only one of these children developed pyelonephritis from this strain during the first year of life. The combined results of serotyping, biochemical phenotyping and determination of outer membrane protein pattern as clonal characterization suggested that only 26 of the P-fimbriated strains belonged to a pyelonephritogenic Escherichia coli clone. It is concluded that the risk of a child colonized with an Escherichia coli strain belonging to such a clone of developing pyelonephritis, as calculated in this study, is about 4%.

Antibody responses to Escherichia coli J5 lipopolysaccharide and to Salmonella porin in patients with bacteremia.

This study was undertaken in order to evaluate whether patients with bacteremia respond with antibodies directed towards two outer membrane components of Gram-negative bacteria. The antibody responses to the core of the lipopolysaccharide molecule (LPS) of the rough E. coli J5 mutant and to a purified outer membrane protein (porin) from Salmonella were studied in bacteremic patients. Two or three serum samples were consecutively collected from 77 patients having 82 episodes of bacteremia altogether, of these 50 were caused by bacteria of the genus Enterobacteriaceae. As controls, sera from 82 age and sex matched patients and 100 healthy blood donors were analysed. The antibody titers were assessed by enzyme-linked immunosorbent assays (ELISA). None of the patients with bacteremia responded with an increase in antibody level to the E. coli J5 rough LPS. This finding indicates that the core portion of the E. coli J5 LPS contains no antigenic epitopes immunologically cross-reactive with Gram-negative bacteria causing bacteremia. By contrast, 13 patients showed significant titer increases to the porin preparation derived from Salmonella. Twelve of these patients had bacteremia caused by Gram-negative organisms belonging to Enterobacteriaceae. One patient had bacteremia with Bacteroides fragilis but also suffered from a severe peritonitis with growth of both E. coli and Klebsiella. It is suggested that the measurement of antibody response to porin may be of value for differential serological diagnosis in patients with bacteremia, to distinguish between Enterobacteriaceae and other organisms.

Structural studies of the O-antigen polysaccharides of Klebsiella O5 and Escherichia coli O8.

The O-antigen polysaccharides of Klebsiella serotype O5 and Escherichia coli serotype O8 are serologically very similar or identical. The structures of these two polysaccharides have now been re-investigated. N.m.r. spectroscopy, chromium trioxide oxidation, hydrolysis with a specific phage enzyme, and f.a.b. mass spectrometry were the principal methods used. It is concluded that the O-antigen has the following structure, in which D-Man3Me is 3-O-methyl-D-mannose and n is approximately 10. (Formula: see text) Biosynthetic studies indicate that these antigens are synthesised by addition of D-mannopyranosyl groups to the "non-reducing" end of the mannan chain, and it seems possible that addition of a 3-O-methyl-D-mannopyranosyl group involves termination.