The association of heat-stable enterotoxin (STa) produced by enterotoxigenic Escherichia coli 431 with isolated rat intestinal epithelial cells and brush border membranes was characterized. Specific binding of strain 431 I25-STa to a single class of specific high-affinity receptors was saturable and temperature dependent and reached a maximum between 5 and 10 min. A 1,000-fold excess of unlabeled 431 STa competitively displaced 90 to 95% of radiolabeled enterotoxin bound to brush border membranes. In contrast, specific binding of 431 125I-STa to intestinal cells ranged from 40 to 65%. The number of STaspecific receptors on rat intestinal cells determined by Scatchard analysis was 47,520 + 14,352 (mean ± standard error of the mean) per cell, with affinity constants (KaS) of 2.55 x 1011 and 4.32 x 10i liters/mol determined for intestinal cells and brush border membranes, respectively. Villus intestinal cells appeared to possess about twice as many STa receptors as did crypt cells. Dissociation of specifically bound 431 125I_STa from intestinal cells and brush border membranes was minimal (2 to 5%). In addition, neither the rate nor the extent of dissociation was increased by a 1,000-fold excess of unlabeled homologous 431 STa. Binding experiments with 431 125I-STa and brush border membranes showed that purified unlabeled STaS from enterotoxigenic E. coli strains 667 (class 1 porcine enteropathogen), B-41 (bovine enteropathogen), and human strains 213C2 (Mexico) and 153961-2 (Dacca, Bangledesh) exhibited patterns of competitive inhibition similar to those of homologous unlabeled 431 STa (class 2 enteropathogen). A lipid extract which contained gangliosides and glycolipids exhibited dose-dependent competitive inhibition of heat-labile enterotoxin binding to brush border membranes but did not inhibit binding of 431 125I-STa. Purified heatlabile enterotoxin from strain 286C2 did not inhibit binding of 431 STa to brush border membranes. Pronase treatment of brush border membranes reduced binding of 431 125I-STa by about 30%, suggesting that the STa receptor was a protein or a glycoprotein. The putative STa receptor was radiolabeled with 431 1251_STa and solubilized with sodium deoxycholate. One major radioactive band was detected by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, followed by radioautography. These data suggested that STas bind essentially irreversibly to a specific receptor on the cell surface of intestinal cells before activation of guanylate cyclase.The watery diarrhea associated with enterotoxigenic Escherichia coli strains is caused by one or more enterotoxins (38). The heat-labile enterotoxin (LT) consists of two kinds of subunits similar to cholera toxin (17, 31), the B protomer of five polypeptides which binds to GM1 ganglioside (18,30) and the A subunit which catalyzes ADP ribosylation of a regulatory subunit of adenylate cyclase (10,16,30). Inactivation of the regulatory subunit leads to activation of adenylate cyclase and, subsequently, increased levels of intracellular cyclic AMP. T...

Five heat-stable enterotoxins (STs) produced by enterotoxigenic Escherichia coli strains of porcine, bovine, and human host origins have been purified to apparent homogeneity. The STs with biological activity in suckling mice and piglets (STas) contained 18 amino acid residues, 10 amino acids with a high proportion of acidic amino acids, and 6 half-cystines. The carboxy-terminal and amino-terminal residues of all STaS were tyrosine and asparagine, respectively. All five STa preparations were homogeneous by several criteria: (i) a single symmetrical peak on gel filtration, (ii) a single fluorescent band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (iii) single carboxyl-terminal and amino-terminal residues, and (iv) amino acid analysis data that indicated a stoichiometric relationship between the component amino acids. The isoelectric

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Antiserum was raised against the purified heat-stable enterotoxin (ST) produced by enterotoxigenic Escherichia coli strain 431, a class II porcine enteropathogen. The antiserum was used to examine the antigenic determinants of STs produced by enterotoxigenic strains of different host origins and develop a sensitive radioimmunoassay specific for ST having biological activity in suckling mice and piglets (STA). The antiserum neutralized one effective dose of toxin at a dilution of 1:5,000 and neutralized approximately 40 microgram of toxin per ml of serum. In the radioimmunoassay, protein A-bearing staphylococci was used as the primary solid-phase adsorbent. The purified STs produced by a class I enteropathogen (strain 667) and by a bovine enterotoxigenic strain (B-41) exhibited patterns of competitive inhibition identical to those of homologous unlabeled strain 431 ST in the radioimmunoassay when specific antibody to strain 431 ST was used. The levels of ST in culture supernatants determined by the suckling mouse assay correlated with the concentrations of toxin measured by the radioimmunoassay. The antiserum was specific for STA produced by enterotoxigenic E. coli of porcine, bovine, and human origins and did not react with heat-labile enterotoxin or with ST that had biological activity in piglets but not in suckling mice (STB). These results suggest that STA molecules having different host origins share at least one antigenic determinant.

The response to recombinant vaccines for Lyme disease was studied to determine serum antibody levels effective in protecting against tick-transmitted infection. Data presented here demonstrate a significant correlation between antibody to an epitope on outer surface protein A (OspA) and protection against infection with Borrelia burgdorferi in canines and mice. A competitive enzyme-linked immunosorbent assay was developed to measure antibody to a site on OspA, defined by monoclonal antibody LA-2. Comparison of LA-2 titers against infection of canines and mice following vaccination and challenge established a predicted value for LA-2 titers. The statistical relationship between serum antibody levels and protection was calculated by logistic regression analysis. The statistical model predicted that an LA-2 titer of 0.32 g equivalents (eq) per ml correlated to an 80% predicted probability of protection for both mice and dogs. This value was used to classify mice and dogs as to their protected status at the time of tick exposure. The LA-2 cutoff titer (0.32 g eq/ml) correctly classified all dogs (n ‫؍‬ 13) and mice (n ‫؍‬ 44) that failed to become infected. By contrast, 20 of 22 dogs and 28 of 31 mice with titers of less than 0.32 g eq/ml became infected. On the basis of these results, we conclude that an LA-2 titer is a reliable indicator of immune status for estimating immune protection following use of OspA-based vaccines for B. burgdorferi sensu stricto. MATERIALS AND METHODS Vaccines. Mice were vaccinated with four different OspA formulations as described previously (13). Briefly, the gene for OspA was isolated from B. burgdorferi sensu stricto ZS7, recombined, and expressed in Escherichia coli as either a whole lipidated protein or fused protein with NS-1 of influenza virus. The fusion protein is not lipidated. The codons for the N-terminal cysteine of OspA reside directly adjacent to the terminal codons of NS-1. Consequently, a primary amino group on the OspA moiety of the fusion protein is not available. The OspA gene of B. afzellii ACA-1 was also isolated and expressed, fused with NS-1, in E. coli. All but one of the experimental formulations for vaccination of mice were adsorbed to aluminum hydroxide gel. The ratio for adsorption was 1 mg of protein per 100 mg of aluminum hydroxide gel (13). Vaccination protocol. Outbred BALB/c and C3H/HeJ mice received three vaccinations at 2-week intervals. Serum samples were collected 1 week after the last vaccination and 1 week before challenge with infected Ixodes scapularis ticks. Details regarding the numbers of ticks used to challenge mice were reported previously (11). In an attempt to determine the effectiveness of the Lyme disease vaccine formulation in the normal regimen of canine vaccination, we incorporated the OspA vaccine into the normal, recommended canine schedule. Dogs were randomly assigned to one of four groups. At 8 to 9 weeks of age, all dogs received a dose of canine distemper-measles virus vaccine, modified live virus (MLV), and a dose of canine par...