Enzyme-linked immunosorbent assays for virulence properties of Campylobacter jejuni clinical isolates.

To evaluate the capacity of enzyme-linked immunosorbent assays (ELISAs) to identify pathogenic strains among clinical fecal isolates of Campylobacter jejuni, 40 consecutively obtained strains from 39 sick patients and 1 asymptomatic person were tested by respective ELISAs for enterotoxin production in culture filtrates and for the invasive virulence antigen of bacterial cells. Of the 40 strains, 14 produced the enterotoxin; 15 strains, two of which were also enterotoxigenic, were invasive; and 11 strains had no detectable virulence property. The presence or absence of these virulence properties was confirmed by the demonstration that viable cells of all 12 randomly selected enterotoxigenic or invasive strains tested, but none of 9 nonpathogenic strains tested, caused fluid secretion in rat ligated ileal loops.

Immunisation of volunteers with a synthetic peptide vaccine for enterotoxigenic Escherichia coli.

A completely synthetically produced peptide vaccine, consisting of the 18-aminoacid Escherichia coli heat-stable toxin and the 26-aminoacid epitope of the heat-labile toxin B subunit, was given orally to thirteen volunteers. It raised antitoxin titres to both toxin components four-fold in serum samples and seven-fold in jejunal aspirates over preimmunisation control titres. Jejunal aspirates taken after immunisation from vaccinees, but not controls, neutralised the secretory activity of both toxins in appropriate biological assays.

Pathogenic properties of Campylobacter jejuni: assay and correlation with clinical manifestations.

The pathogenic properties of 20 strains of Campylobacter jejuni isolated from persons with clearly defined clinical manifestations were determined. Cell-free broth filtrates were examined for (i) enterotoxin production by Chinese hamster tissue culture assay and an enzyme-linked immunosorbent assay (ELISA) employing GM1 ganglioside and affinity-purified antiserum to Escherichia coli heat-labile toxin, (ii) cytotoxin production by Vero and HeLa cell tissue culture lines, and (iii) their ability to cause fluid secretion in rat ligated ileal loops. Viable bacteria were examined for invasive properties by an ELISA with the immunoglobulin fraction of antiserum to Formalin-killed bacteria of an invasive strain, and by their effect on fluid secretion and morphology in rat ligated ileal loops.

Mucosal antitoxin response in volunteers to immunization with a synthetic peptide of Escherichia coli heat-stable enterotoxin.

Peroral immunization of volunteers on four weekly occasions with 750 micrograms of a conjugate containing 3,000 antigen units of a synthetically produced peptide of hyperantigenic Escherichia coli heat-stable (ST) toxin, conjugated with the heat-labile toxin B subunit as a carrier, raised serum immunoglobulin G antitoxin titers to ST by fourfold and intestinal immunoglobulin A antitoxin titers to ST by sevenfold over control values at five weeks postimmunization. The ability of jejunal aspirates from the immunized volunteers to neutralize ST in the suckling mouse assay correlated with the intestinal immunoglobulin A ST antitoxin response determined by enzyme-linked immunosorbent assay.

Immunological relationship of the B subunits of Campylobacter jejuni and Escherichia coli heat-labile enterotoxins.

The application of dissociation techniques, involving gel filtration in the presence of guanidine, to a semipurified preparation of Campylobacter jejuni heat-labile enterotoxin yielded a material whose functional and immunological properties resemble those of the B subunits of cholera toxin and Escherichia coli heat-labile toxin (LT). The C. jejuni toxin B subunit reacted with GM1 ganglioside in an enzyme-linked immunosorbent assay, but lacked the holotoxin's cytotonic activity in the Chinese hamster ovary tissue culture assay and its ability to cause fluid secretion in rat ileal ligated loops.

A completely synthetic toxoid vaccine containing Escherichia coli heat-stable toxin and antigenic determinants of the heat-labile toxin B subunit.

The immunodeterminant regions of the Escherichia coli heat-labile toxin B subunit were identified by determining the antigenicity, by using enzyme-linked immunosorbent assays, of synthetically produced peptides corresponding to various segments of its 124-amino-acid sequence. The addition of the 18-amino-acid sequence of heat-stable toxin (ST) to some of these peptides enhanced their B subunit antigenicity. Peptide residues containing the 26 amino acids of B subunit sequence 58 to 83 joined to the 18-amino-acid sequence of ST yielded a 44-amino-acid peptide whose antigenicity was 50% that of both native B subunit and ST.

Chemical synthesis of an octadecapeptide with the biological and immunological properties of human heat-stable Escherichia coli enterotoxin.

An eighteen-amino-acid peptide having the linear amino acid sequence of human heat-stable enterotoxin (ST) has been synthesized by solid phase peptide synthesis. The purified peptide could be obtained in yields approaching 25% after purification by size, charge, and high-performance ligand chromatography. This material was pure and identical to native ST by analytical high-performance ligand chromatography, amino acid analysis, paper electrophoresis and thin-layer chromatography.

Properties of crude Campylobacter jejuni heat-labile enterotoxin.

The amount of crude Campylobacter jejuni enterotoxin present in culture products was quantitated by comparing the response of these preparations with that of pure Escherichia coli heat-labile toxin (LT) in the Chinese hamster ovary assay and in enzyme-linked immunosorbent assays that used GM ganglioside or antisera to LT or both. Maximum C. jejuni enterotoxin production was achieved by growth at 42 degrees C for 24 h under agitation in supplemented GC medium.

Enzyme-linked immunosorbent assay for Escherichia coli heat-stable enterotoxin.

The sensitivity of an enzyme-linked immunosorbent assay (ELISA) to detect pure native Escherichia coli heat-stable toxin (ST) and to identify ST-producing strains among clinical isolates was determined. Two synthetically produced ST preparations were used to raise hyperimmune antisera in rabbits and goats: ST(S), which has the same antigenicity as native ST; and ST(C), which is 15-fold more immunogenic. These antisera were used in the double-sandwich technique as either crude double-species antisera or pure single-species antibody.

Properties of cross-linked toxoid vaccines made with hyperantigenic forms of synthetic Escherichia coli heat-stable toxin.

The ability of hyperantigenic preparations of synthetically produced Escherichia coli heat-stable toxin (ST) to provide an immunogenically more potent vaccine when cross-linked by the glutaraldehyde reaction to the heat-labile toxin B subunit was assessed. Three synthetic ST preparations were evaluated: ST(S) had the same antigenicity and toxicity (secretory potency in the suckling mouse assay) as native ST, ST 1056 had 3.5-fold more antigenicity and 1% toxicity, and ST(C) had 15-fold greater antigenicity and 31% toxicity.

Differences in cross-protection in rats immunized with the B subunits of cholera toxin and Escherichia coli heat-labile toxin.

Although cholera toxin (CT), Escherichia coli heat-labile toxin (LT), and their B subunits are known to be immunologically related, the ability of each to raise an antitoxin response that provides equally strong cross-protection against active challenge with pure heterologous toxin has not been examined previously. We immunized rats with pure preparations of the B subunits of human LT, porcine LT, and CT. Immunization with either of the LT B subunits raised greater than or equal to fourfold increases in specific mucosal immunoglobulin A antitoxin titers to homologous and heterologous LT and CT B subunits, thereby providing strong protection against active challenge in ligated ileal loops with all three respective holotoxins and with a viable LT-producing E.

Immunological properties of purified Klebsiella pneumoniae heat-stable enterotoxin.

Klebsiella pneumoniae heat-stable enterotoxin was purified to apparent homogenicity by the same techniques used to purify Escherichia coli heat-stable enterotoxin. The two toxins had the same potency in the suckling mouse assay and showed immunological cross-reactivity in enzyme-linked immunosorbent assay, neutralization of secretory activity by specific hyperimmune antisera, and protection against active challenge in rats immunized with a vaccine containing synthetically produced E. coli heat-stable enterotoxin.

Protection in rabbits immunized with a vaccine of Escherichia coli heat-stable toxin cross-linked to the heat-labile toxin B subunit.

Rabbits and rats were immunized with a vaccine consisting of synthetically produced Escherichia coli heat-stable toxin cross-linked by the carbodiimide reaction to the B subunit of biologically produced porcine heat-labile toxin. The vaccine contained 50% of each toxin component by weight and antigenicity; the toxicity of the heat-stable enterotoxin component was reduced by greater than 600-fold. Two or three peroral immunizations with vaccine containing 1,000 antigen units of each component raised greater-than-threefold increases in specific mucosal immunoglobulin A antitoxin titers to each component in all animal groups.

Protection against human and porcine enterotoxigenic strains of Escherichia coli in rats immunized with a cross-linked toxoid vaccine.

To compare their relative immunogenicities, we used synthetically produced Escherichia coli heat-stable toxin coupled to a protein carrier and the B subunit of porcine heat-labile toxin separately in graded dosages to immunize rats. Equivalent antigen unit dosages of each toxin raised approximately the same level of mucosal immunoglobulin A (IgA) antitoxin response and degree of protection against a challenge with respective heat-stable- or heat-labile-toxin-producing viable bacteria. Conjugation conditions were identified, therefore, which yielded a vaccine of these toxins, cross-linked by the carbodiimide reaction, that consisted of equal antigenic proportions of each toxin component as determined by enzyme-linked immunosorbent assay and expressed in antigen units.

Cloning and molecular characterization of the B subunit of Escherichia coli heat-labile enterotoxin.

We have constructed a plasmid containing the gene for production of the B subunit of the heat-labile enterotoxin (LT-B) from a human isolate of Escherichia coli, strain H10407. The 0.8-kilobase gene fragment encoding synthesis of LT-B was cloned onto plasmid pBR322 after sequential digestion of the enterotoxin plasmid of strain H10407 with restriction endonucleases PstI and HindIII.

Peroral immunization of rats with Escherichia coli heat-labile enterotoxin delivered by microspheres.

The antigenicity of the Escherichia coli heat-labile enterotoxin was not protected against the adverse effect of gastric acidity when the toxin was given together with bicarbonate for peroral immunization to rats, but immunization with the heat-labile enterotoxin encapsulated in pH-dependent microspheres aroused the same strong degree of serum and mucosal antitoxin responses and of protection against challenge as was achieved by peroral immunization after ablation of gastric secretions by pretreatment with cimetidine.

Vaccine for enterotoxigenic Escherichia coli based on synthetic heat-stable toxin crossed-linked to the B subunit of heat-labile toxin.

Synthetically produced Escherichia coli heat-stable toxin (ST) was conjugated to the nontoxic B subunit of the heat-labile toxin (LT) by the carbodiimide reaction. Modifying the molar ratio of toxins mixed and the ratio of carbodiimide added to the toxins permitted synthesis of conjugates with any desired degree of proportional antigenicity for each toxin component. Immunization of rats by the parenteral/peroral routes with cross-linked vaccine containing 39% ST and 61% B subunit antigenicity, with 0.

Properties of synthetically produced Escherichia coli heat-stable enterotoxin.

The properties of a synthetically produced peptide composed of the same primary structure of 18 amino acids described for human Escherichia coli heat-stable enterotoxin were compared with those of purified heat-stable toxin obtained by bacterial growth. The dosage required to evoke fluid secretion in the suckling mouse and rat ligated ileal loop assays was the same for both toxins. The antigenicity of the two toxins was similar when assayed by enzyme-linked immunosorbent assay with hyperimmune antiserum to either toxin.

Immunological and physicochemical characterization of heat-labile enterotoxins isolated from two strains of Escherichia coli.

Heat-labile enterotoxins from Escherichia coli strains of human and porcine origins had identical subunit composition, arrangement, and size in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunological differences recently described by others were shown to reside exclusively in the B subunits.

Arousal of mucosal secretory immunoglobulin A antitoxin in rats immunized with Escherichia coli heat-labile enterotoxin.

Specific serum and mucosal antitoxin levels were determined by enzyme-linked immunosorbent assays in rats immunized with Escherichia coli heat-labile enterotoxin (LT). Immunization by means of a parenteral prime followed by peroral boosts was the only approach that aroused titers of both serum immunoglobulin G (IgG) antitoxin and mucosal secretory IgA antitoxin that were increased fourfold or more over control values. Primary parenteral immunization was effective when given either intraperitoneally or subcutaneously with either Freund complete adjuvant or alum as the adjuvant.

Development of a vaccine of cross-linked heat-stable and heat-labile enterotoxins that protects against Escherichia coli producing either enterotoxin.

A vaccine of cross-linked heat-stable (ST) and heat-labile (LT) toxins that protects against heterologous serotypes of strains of Escherichia coli which produce either the LT or ST enterotoxin was developed by conjugating ST to LT by the carbodiimide reaction. Three interrelated factors were found to affect the composition and properties of the final conjugate: (i) the amount of carbodiimide added to the toxins, (ii) the initial ratio of ST to LT, and (iii) the duration of the conjugation reaction. Optimal conjugation conditions were identified as a carbodiimide-to-toxin ratio of 10:1 by weight, an initial molar ratio of ST to LT of 100:1, and a conjugation reaction time of 96 h.