Protective Effect of Immunization with Heat-Labile Enterotoxin in Gnotobiotic Rats Monocontaminated with Enterotoxigenic Escherichia coli

The protective effect of active immunization with a purified preparation of the polymyxin-release form of Escherichia coli heat-labile enterotoxin (LT), administered using a parenteral prime and peroral boosts given after ablation of gastric secretion by means of cimetidine, was assessed in gnotobiotic rats which were challenged by monocontamination with enterotoxigenic strains of E. coli. Water transport was evaluated by the in vivo marker perfusion technique at weekly intervals over a 3-week period after contamination. Water transport in unimmunized control rats was consistently in absorption in those contaminated by a nontoxigenic strain, in secretion during only week 2 in those contaminated by an LT⁺/− strain, in secretion during weeks 2 and 3 in those contaminated by an LT⁺/ST⁺ (heat-stable enterotoxin) strain, and consistently in absorption in those contaminated by an −/ST⁺ strain. Rats immunized with a booster dosage of 250 μg had a significant increase (P < 0.001) in net water absorption as compared to unimmunized rats, with values in the borderline range of absorption, when challenged with either the LT⁺/− or LT⁺/ST⁺ strains. Rats immunized with a 10-fold-higher boosting dosage had a significant increase (P < 0.001) in net water absorption as compared to those boosted at the lower dosage; water absorption was within the normal range. There was no difference between the ileal bacterial counts of unimmunized and immunized rats challenged by the various strains. These observations indicate that this immunization program provides complete protection in an animal model against challenge by intestinal contamination with enterotoxigenic strains of E. coli which produce LT, either alone or in combination with ST.     

Protective Effect of Immunization of Rats with Holotoxin or B Subunit of Escherichia coli Heat-Labile Enterotoxin

The relative immunogenicities of three forms of the Escherichia coli heatlabile enterotoxin (LT), the holotoxin, its B subunit, and the polymyxin-release form (PM LT) were compared by immunizing rats with various dosages of each given exclusively by the parenteral (IP/IP) or peroral (PO/PO) routes or by a combination of the two (IP/PO). The degree of protection was evaluated by challenge in ligated ileal loops, and the serum antitoxin response was determined by an enzyme-linked immunosorbent assay with homologous antigens. When given by the PO/PO route, each LT antigen provided only weak protection against the toxin and virtually none against viable LT-producing strains; serum antitoxin titers were not significantly increased. When the toxins were given after a parental primary immunization by either the IP/IP or the IP/PO routes, each LT antigen provided a dose-related increase in serum antitoxin titers and in the degree of protection against the toxin as well as against viable strains which produce LT alone (LT⁺/ST⁻) or in combination with the heat-stable toxin (LT⁺/ST⁺). The degree of protection against viable bacteria, particularly the LT⁺/ST⁺ strain, was stronger in animals which received booster immunizations by the PO route. When expressed on the basis of molar equivalents, holotoxin provided significant protection (a protection index of >5 against toxin challenge and >50% reduced secretion with bacterial challenge) with 4 to 15 times fewer moles than PM LT and up to 50 times fewer moles than the B subunit. These observations indicate that, on the basis of molar equivalents, the holotoxin (which contains one A plus five or six B subunits) is a more potent immunogen than either PM LT (which contains one A and probably one B subunit) or the B subunit.     

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.     

Immunization with Escherichia coli Enterotoxin Protects Against Homologous Enterotoxin Challenge

Immunization of rabbits with the culture filtrate of an enterotoxigenic strain of Escherichia coli 078: H12 resulted in the appearance of anti-enterotoxin activity in rabbit sera and protection of rabbit small intestine against homologous enterotoxin challenge.     

Factors Affecting Release of Heat-Labile Enterotoxin by Enterotoxigenic Escherichia coli

Various conditions affecting the release of heat-labile enterotoxin (LT) by enterotoxigenic Escherichia coli have been examined. The pH of a defined medium containing three amino acids, M-9 salts, and 0.5% glucose decreased to less than 7.0 in early log phase of growth, and no extracellular LT was detected. Adjustment of the pH at 8 h from 6.0 to 8.0 resulted in a concomitant increase in LT activity in culture supernatants. The release of cell-associated LT was significantly reduced by preincubation with protease inhibitors and increased by preincubation with trypsin. Cell-associated LT was not released by pH adjustment of cells grown at 21 degrees C; however, polymyxin B treatment released a toxin species active in only the pigeon erythrocyte lysate (PEL) assay system. As the growth temperature was increased, polymyxin B released toxin species which exhibited both PEL and Y-1 adrenal tumor cell activity. Polymyxin B extracts of enterotoxigenic E. coli in early log phase grown at 37 degrees C possessed only PEL activity, whereas extracts from cells in late-log and stationary phases had biological activity in both assay systems. Also, LT released by pH adjustment from mid-log to stationary phase was active in both PEL and Y-1 adrenal tumor cell assays. Gel electrophoresis of polymyxin B extracts revealed at least three molecular weight species active in either the PEL (22,000 daltons and 30,000 daltons) or both the PEL and the Y-1 adrenal tumor cell assay (72,000 daltons), depending on the growth temperature. These observations may help to explain the chemical and biological heterogeneity of most LT preparations and facilitate purification of LT by increasing the yield of enterotoxin.     

Influence of Route of Administration on Immediate and Extended Protection in Rats Immunized with Escherichia coli Heat-Labile Enterotoxin

The effect of route of administration, dosage, and number of boosts employed during immunization with the polymyxin-release form of Escherichia coli heat-labile (LT) enterotoxin on the degree and duration of protection afforded was evaluated in rats which were challenged by the ligated loop technique. Increasing the boosting dosage by fivefold from 50 to 250 μg resulted in a marked increase in protection against challenge with toxin in rats immunized either just by the parenteral route (i.p./i.p.) or by a parenteral prime, followed by peroral boosts (i.p./p.o.) in rats pretreated with cimetidine to ablate gastric secretions; such was not the case, however, even with a 50-fold increase in dosage in rats immunized just by the peroral route (p.o./p.o.). Four weekly peroral boosts were required to achieve the strongest degree of protection. Increasing the boosting dosage also increased the degree of protection against challenge with viable LT⁺/ST⁻ and LT⁺/ST⁺ strains (ST indicates heat-stable enterotoxin) in rats immunized by the i.p./p.o., but not by the i.p./i.p., route; no protection was evident against an LT⁻/ST⁺ strain. Protection was lost within 3 weeks after immunization in rats immunized by the i.p./i.p. route. In contrast, protection was extended over the 3-month observation period in those immunized by the i.p./p.o. route; the degree of protection was enhanced in rats which received an additional boost at 2 months. These observations establish the fact that immunization with LT is similar to that with cholera toxin in that arousal of the local immune intestinal response by means of peroral immunization provides maximal extended protection.     

Polymyxin B-Induced Release of Low-Molecular-Weight, Heat-Labile Enterotoxin from Escherichia coli

Polymyxin B-induced release of enterotoxin from Escherichia coli strain H-10407 was demonstrated. Incubation of E. coli cells derived from 6-h cultures with polymyxin caused the rapid release of enterotoxin with a molecular weight of approximately 20,000, as estimated by the gel filtration technique. The rapidity of the release of enterotoxin indicates that it probably resides in the periplasmic space of the cell. The low-molecular-weight enterotoxin possessed vascular permeability factor and diarrheagenic activities, both of which were found to be heat-labile. The permeability factor activity of this enterotoxin was neutralized by antisera prepared against crude E. coli enterotoxin, Vibrio cholerae enterotoxin (choleragen), and V. cholerae toxoid (choleragenoid), respectively. Supernatant fluids of 6-h E. coli cultures did not contain this molecular form of enterotoxin but did contain very high-molecular-weight, heat-labile enterotoxin. Incubation of cells derived from older (18 h) cultures with polymyxin caused the release of both low- (20,000) and high-molecular-weight forms of enterotoxin. We concluded that either the 20,000-dalton form of heat-labile enterotoxin is not released by E. coli under in vitro growth conditions or that enterotoxin released in this form is rapidly destroyed or inactivated.     

In Vitro Effects of a High-Molecular-Weight Heat-Labile Enterotoxin from Enteroaggregative Escherichia coli

The pathogenic mechanisms of enteroaggregative Escherichia coli (EAggEC) infection are not fully elucidated. In this work we show that an ammonium sulfate precipitate of culture supernatant of EAggEC strain 049766 increased the potential difference (PD) and the short-circuit current (Isc) in rat jejunal preparations mounted in Ussing chambers. The precipitate contained two major proteins of 108 and 116 kDa, which were partially copurified by chromatography in DEAE-cellulose. This chromatographic fraction (peak I) increased jejunal PD and Isc in a dose-dependent manner, accompanied by a decrease in tissue electrical resistance. These effects were inhibited by incubation of peak I at 75°C for 15 min or for 1 h with proteinase K at 37°C. Rabbit polyclonal antibodies against peak I containing both the 108- and 116-kDa proteins inhibited the enterotoxic effect. Specific polyclonal antibodies raised against the 108-kDa but not against the 116-kDa protein inhibited the enterotoxic effect, suggesting that the 108-kDa protein is the active toxic species. Moreover, another EAggEC strain (065126) producing the 116-kDa protein but not the 108-kDa protein had no effect on rat jejunal mucosa in the Ussing chamber. The >100-kDa fraction derived from prototype EAggEC strain 042, which also expressed both 108- and 116-kDa proteins, also produced an enterotoxic effect on rat jejunal preparations in Ussing chambers; however, the same strain cured of its 65-MDa adherence plasmid did not. A subclone derived from the 65-MDa plasmid expressing the 108-kDa toxin (and not the 116-kDa protein) elicited rises in Isc. Tissue exposed to any preparation containing the 108-kDa toxin exhibited similar histopathologic changes, characterized by increased mucus release, exfoliation of cells, and development of crypt abscesses. Our data suggest that some EAggEC strains produce a ca. 108-kDa enterotoxin/cytotoxin which is encoded on the large virulence plasmid.     

Nutritional Requirements for Synthesis of Heat-Labile Enterotoxin by Enterotoxigenic Strains of Escherichia coli

Optimal growth conditions have been established for production of heat-labile enterotoxin (LT) by both porcine and human strains of enterotoxigenic (ENT⁺) Escherichia coli. There were no unusual growth factor requirements, and some strains produced fairly high levels of LT in a basal salts medium containing 0.5% glucose if the pH was carefully controlled. Several amino acids markedly stimulated LT synthesis when added to the basal salts-glucose medium. Methionine and lysine were the most stimulatory for both human and porcine strains. Either aspartic acid or glutamic acid further enhanced LT synthesis in the presence of methionine and lysine, with aspartic acid being more stimulatory for porcine strains and glutamic acid more stimulatory for human strains. There were no apparent vitamin requirements and no unusual cations needed for toxin synthesis except that Fe³⁺ was slightly stimulatory for porcine strains. The stimulation by Fe³⁺ was observed only in the presence of the three amino acids, suggesting that the effect was indirect rather than on toxin synthesis. The carbon source also influenced the yield of LT. Glucose supported maximal synthesis, but other carbon sources which exhibit a high degree of catabolite repression also supported high levels of synthesis. Little or no LT was released below pH 7.0; therefore, because the pH drops during growth from 7.5 to 6.8, even in highly buffered media, it was necessary to adjust the pH to 8.0 to effect complete release of cell-associated toxin. The defined medium containing three amino acids reduced the amount of UV-absorbing material in culture supernatants about fivefold and increased LT activity for various strains from two- to fivefold over a complex Casamino Acids-yeast extract medium. Conditions found to be optimal for synthesis of LT were inhibitory for the heat-stable enterotoxin.     

Purification and Chemical Characterization of the Heat-Labile Enterotoxin Produced by Enterotoxigenic Escherichia coli

Heat-labile enterotoxin (LT) produced by a human strain of enterotoxigenic Escherichia coli (286C(2)) was purified to homogeneity from pH extracts of fermentor-grown cells by ultrafiltration, (NH(4))(2)SO(4) fractionation, hydrophobic chromatography on norleucine-Sepharose 4B, hydroxylapatite chromatography, and Bio-Gel P-150 filtration. Purified LT preparations exhibited biological activity comparable to that of cholera toxin in four bioassays specific for the two enterotoxins (Y-1 adrenal tumor cells, Chinese hamster ovary cells, pigeon erythrocyte lysates, and skin permeability test). The overall yield of LT protein was 20%, which represented a 500-fold purification over pH extracts. A native molecular weight of 73,000 was determined by gel electrophoresis. The toxin dissociated upon treatment with sodium dodecyl sulfate, pH 7.0, into two components with molecular weights of 44,000 and 30,000. Purified LT preparations were remarkably stable over a wide range of storage conditions, temperatures, and pH's. The biological activity was increased by incubation with trypsin and completely destroyed by pronase and proteinase K, whereas deoxyribonuclease I, ribonuclease, and phospholipase D had no effect. The amino acid composition of purified LT was quite different from that of cholera toxin. Neither carbohydrate nor lipopolysaccharide was present in purified preparations. The purification scheme appeared applicable to LT produced by other human and porcine enterotoxigenic strains, but reflected the amount of LT produced by each strain. These data show that LT and cholera toxin share many common chemical and physical properties, but must be purified by different techniques.     

Immunological Cross-Reactivity Between a Heat-Labile Enterotoxin(s) of Escherichia coli and Subunits of Vibrio cholerae Enterotoxin

Heat-labile enterotoxin from Escherichia coli is immunologically related to both subunits, A and B, of cholera enterotoxin as demonstrated by neutralization and immunodiffusion.     

Inhibition of Class II Major Histocompatibility Complex Antigen Processing by Escherichia coli Heat-Labile Enterotoxin Requires an Enzymatically Active A Subunit

Escherichia coli heat-labile enterotoxin (LT) and cholera toxin (CT) were found to inhibit intracellular antigen processing. Processing was not inhibited by mutant LT with attenuated ADP-ribosyltransferase activity, CT B or LT B subunit, which enhanced presentation of preexisting cell surface peptide-class II major histocompatibility complex complexes. Inhibition of antigen processing correlated with A subunit ADP-ribosyltransferase activity.Escherichia coli heat-labile enterotoxin (LT) and cholera toxin (CT) are related ADP-ribosylating toxins with five identical B subunits that bind to cell surface ganglioside receptors and an enzymatically active A subunit that enters the cell and catalyzes the ADP-ribosylation of guanine nucleotide binding proteins of the adenylate cyclase complex, causing constitutive activation of adenylate cyclase and increased intracellular cyclic AMP (cAMP).LT and CT are potent mucosal adjuvants (7, 8, 12, 20, 22, 23, 29, 31–33). Some degree of A subunit enzymatic activity is required for oral adjuvant function (20, 23, 32, 33). While ADP-ribosyltransferase activity enhances adjuvanticity, it also confers toxicity. For an optimal adjuvant, reduced toxicity would be desirable, and mutant LT (6, 9–11, 15, 17, 21, 26, 34) and CT (5, 35) molecules have been constructed with altered A subunits, reduced ADP ribosylation activity, and reduced toxicity, yet with maintained adjuvant function (9–11, 13, 25, 26, 35). Mutation studies with LT revealed that residues at positions 7, 110, and 112 of LT A subunit (LTA) are important for ADP-ribosyltransferase activity (6, 21, 28), with Glu-112 providing a catalytic role. A conservative mutation (Asp to Glu) at position 112 produced a mutant toxin, rLT-E112D, with substantially reduced (<2% of wild type) but detectable ADP-ribosyltransferase activity (6).CT and LT affect many components of immune responses, including antigen presentation (3, 4, 18), with inhibitory as well as enhancing effects. We previously showed that CT enhances macrophage presentation of cell surface peptide-class II major histocompatibility complex (MHC-II) complexes to T cells but inhibits intracellular antigen processing (24). However, the effects of LT have not been similarly investigated. Furthermore, mutant LT molecules provide tools to determine the role of A subunit enzymatic activity in immunomodulation and toxicity.The present study was designed to investigate the effects of LT and mutant LT molecules on antigen processing and presentation by macrophages. In particular, we examined the effects of LT on the processing and presentation of a model antigen expressed in bacteria (a system to which LT has natural relevance) by using Escherichia coli strain HB101 expressing the Crl-HEL fusion protein (HB101.Crl-HEL) (27), which contains the HEL(48-61) epitope. LT, the mutant toxin rLT-E112D, and recombinant LTB (rLTB) (Table ​(Table1)1) were prepared as described previously (6, 15). rLTB was produced by using a vector encoding LTB and the A2 fragment of LT (LTA2), but subsequent chromatographic purification produced isolated rLTB, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Trypsin-cleaved LT was produced as described previously (15) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Highly purified CT was purchased from List Biologicals (Campbell, Calif.). Recombinant CTB (rCTB) was a gift from Jan Holmgren (University of Gøteborg, Gøteborg, Sweden) and was prepared as described previously (30).

TABLE 1

Toxin composition and enzymatic activity

Solid-Phase Microtiter Radioimmunoassay Blocking Test for Detection of Antibodies to Escherichia coli Heat-Labile Enterotoxin

The development of a solid-phase microtiter radioimmunoassay blocking test to detect serum antibody to Escherichia coli heat-labile enterotoxin is described. The assay is easy to perform and quantitate, and it is sensitive and specific.     

Effect of Purified Escherichia coli Heat-Stable Enterotoxin on Intestinal Cyclic Nucleotide Metabolism and Fluid Secretion

Enterotoxigenic Escherichia coli cause diarrhea by elaborating two enterotoxins. The large-molecular-weight, heat-labile toxin causes intestinal secretion by stimulating cyclic adenosine 5'-monophosphate production. The mechanism by which the small-molecular-weight, heat-stable enterotoxin induces secretion is unclear. The present study tested the hypothesis that heat-stable enterotoxin induces secretion by altering intestinal cyclic nucleotide concentrations. This was studied in suckling mice by using highly purified E. coli heat-stable enterotoxin obtained from a strain pathogenic for humans. At 3 min after administration of this toxin, intestinal cyclic guanosine 5'-monophosphate (GMP) levels were increased 10-fold. Cyclic GMP levels decreased thereafter, but still were greater than control levels at 120 min. Cyclic adenosine 5'-monophosphate levels fell to one-half of control levels at 3 min and remained below control levels for 120 min. When the time course of enterotoxin-induced secretion was compared with changes in cyclic GMP levels, fluid secretion was not evident until 15 to 30 min after enterotoxin administration. Thus, the increase in intestinal cyclic GMP concentration preceded measurable fluid secretion. And finally, administration of the 8-bromo analog of cyclic GMP evoked fluid secretion, the time course of which was similar to that induced by enterotoxin. These, and other data, strongly suggest that E. coli heat-stable enterotoxin induces intestinal secretion by increasing intestinal cyclic GMP levels.     

Salmonella enterica Serovar Enteritidis Ghosts Carrying the Escherichia coli Heat-Labile Enterotoxin B Subunit Are Capable of Inducing Enhanced Protective Immune Responses

The Escherichia coli heat-labile enterotoxin B subunit (LTB) is a potent vaccine adjuvant. Salmonella enterica serovar Enteritidis ghosts carrying LTB (S. Enteritidis-LTB ghosts) were genetically constructed using a novel plasmid, pJHL187-LTB, designed for the coexpression of the LTB and E lysis proteins. S. Enteritidis-LTB ghosts were characterized using scanning electron microscopy to visualize their transmembrane tunnel structures. The expression of LTB in S. Enteritidis-LTB ghost preparations was confirmed by immunoblot and enzyme-linked immunosorbent assays. The parenteral adjuvant activity of LTB was demonstrated by immunizing chickens with either S. Enteritidis-LTB ghosts or S. Enteritidis ghosts. Chickens were intramuscularly primed at 5 weeks of age and subsequently boosted at 8 weeks of age. In total, 60 chickens were equally divided into three groups (n = 20 for each): group A, nonvaccinated control; group B, immunized with S. Enteritidis-LTB ghosts; and group C, immunized with S. Enteritidis ghosts. Compared with the nonimmunized chickens (group A), the immunized chickens (groups B and C) exhibited increased titers of plasma IgG and intestinal secretory IgA antibodies. The CD3⁺ CD4⁺ subpopulation of T cells was also significantly increased in both immunized groups. Among the immunized chickens, those in group B exhibited significantly increased titers of specific plasma IgG and intestinal secretory IgA (sIgA) antibodies compared with those in group C, indicating the immunomodulatory effects of the LTB adjuvant. Furthermore, both immunized groups exhibited decreased bacterial loads in their feces and internal organs. These results indicate that parenteral immunization with S. Enteritidis-LTB ghosts can stimulate superior induction of systemic and mucosal immune responses compared to immunization with S. Enteritidis ghosts alone, thus conferring efficient protection against salmonellosis.     

Effects of Site-Directed Mutagenesis of Escherichia coli Heat-Labile Enterotoxin on ADP-Ribosyltransferase Activity and Interaction with ADP-Ribosylation Factors

Escherichia coli heat-labile enterotoxin (LT), an oligomeric protein with one A subunit (LTA) and five B subunits, exerts its effects via the ADP-ribosylation of G_sα, a guanine nucleotide-binding (G) protein that activates adenylyl cyclase. LTA also ADP-ribosylates simple guanidino compounds (e.g., arginine) and catalyzes its own auto-ADP-ribosylation. All LTA-catalyzed reactions are enhanced by ADP-ribosylation factors (ARFs), 20-kDa guanine nucleotide-binding proteins. Replacement of arginine-7 (R7K), valine-53 (V53D), serine-63 (S63K), valine 97 (V97K), or tyrosine-104 (Y104K) in LTA resulted in fully assembled but nontoxic proteins. S63K, V53D, and R7K are catalytic-site mutations, whereas V97K and Y104K are amino acid replacements adjacent to and outside of the catalytic site, respectively. The effects of mutagenesis were quantified by measuring ADP-ribosyltransferase activity (i.e., auto-ADP-ribosylation and ADP-ribosylagmatine synthesis) and interaction with ARF (i.e., inhibition of ARF-stimulated cholera toxin ADP-ribosyltransferase activity and effects of ARF on mutant auto-ADP-ribosylation). All mutants were inactive in the ADP-ribosyltransferase assay; however, auto-ADP-ribosylation in the presence of recombinant human ARF6 was detected, albeit much less than that of native LT (Y104K > V53D > V97K > R7K, S63K). Based on the lack of inhibition by free ADP-ribose, the observed auto-ADP-ribosylation activity was enzymatic and not due to the nonenzymatic addition of free ADP-ribose. V53D, S63K, and R7K were more effective than Y104K or V97K in blocking ARF stimulation of cholera toxin ADP-ribosyltransferase. Based on these data, it appears that ARF-binding and catalytic sites are not identical and that a region outside the NAD cleft may participate in the LTA-ARF interaction.     

Levels of Expression and Immunogenicity of Attenuated Salmonella enterica Serovar Typhimurium Strains Expressing Escherichia coli Mutant Heat-Labile Enterotoxin

The effects of heterologous gene dosage as well as Salmonella typhimurium strain variability on immune response toward both the heterologous antigen, the nontoxic mutant of the Escherichia coli heat-labile enterotoxin LTK63, and the carrier Salmonella strain have been analyzed. Effects of a single integration into the host DNA and different-copy-number episomal vectors were compared in S. typhimurium Δcya Δcrp Δasd strains of two different serotypes, UK-1 and SR-11. Expression of the enterotoxin in the different Salmonella isolates in vitro was found to vary considerably and, for the episomal vectors, to correlate with the plasmid copy number. LTK63-specific serum immunoglobulin G (IgG) and mucosal immunoglobulin A (IgA) antibodies were highest in mice immunized with the high-level-expression strain. High anti-LTK63 IgG and IgA titers were found to correspond to higher anti-Salmonella immunity, suggesting that LTK63 exerts an adjuvant effect on response to the carrier. Statistically significant differences in anti-LTK63 immune response were observed between groups of mice immunized with the attenuated Δcya Δcrp UK-1 and SR-11 derivatives producing the antigen at the same rate. These data indicate that the same attenuation in S. typhimurium strains of different genetic backgrounds can influence significantly the immune response toward the heterologous antigen. Moreover, delivery of the LTK63 enterotoxin to the immune system by attenuated S. typhimurium strains is effective only when synthesis of the antigen is very high during the initial phase of invasion, while persistence of the S. typhimurium strain in deep tissues has only marginal influence.

Enterotoxigenic Escherichia coli strains produce a plasmid-encoded heat-labile enterotoxin (LT) (15, 34) related to cholera toxin (CT) (9, 35). LT is composed of two subunits, A and B, which are exported to the periplasmic space, where they assemble into an AB₅ multimeric complex (16). Several mutants of LT-A have been constructed, and in particular, a nontoxic mutant which contains a substitution of serine 63 with lysine (LTK63) has been shown to maintain the structural and immunogenic properties of wild-type LT (21, 27, 28). LTK63 has also been found to display the strong mucosal adjuvant activity pertaining to wild-type LT. Efficient induction of mucosal immune response, specifically in the mouse vagina, has been achieved via the intranasal route of immunization (10). For the development of oral vaccines, however, it would be desirable to exploit the properties of LTK63 for enhancing antigen-specific immune response in the intestinal mucosa by means of oral delivery of the potent mucosal adjuvant.Oral delivery of antigens by live vaccines is known to lead to a more effective production of antigen-specific antibodies in mucosal secretions than oral administration of the soluble antigen (36, 39). Several antigen delivery systems which use as carriers mutant intracellular pathogens that have lost the ability to persist and produce the disease while retaining limited growth in vivo have been developed. In particular, attenuated Salmonella mutants are suitable immunological carriers for virulence determinants from other enteric bacteria in that they can induce humoral immune response selectively at the site of colonization, the gut mucosa. Vaccine strains of Salmonella have been successfully attenuated by introducing different types of mutations (5, 8, 23, 26). Notably, Salmonella strains with a galactose epimerase (galE) mutation (18) or deletions in genes for the biosynthesis of aromatic compounds (aro mutants) (11, 12, 17, 19) or in the adenylate cyclase (cya) and cyclic AMP receptor protein (crp) genes (6) are the most extensively characterized.Delivery of the B subunit of the E. coli enterotoxin (LT-B) by a galE mutant of Salmonella typhimurium has been shown to elicit low levels of anti-LT-B serum and mucosal antibodies. Since the vector used for expression of LT-B was rapidly lost in vivo, i.e., in the absence of the antibiotic required for selection of the plasmid, the level of immune response could be correlated only with the amount of antigen expressed during the initial phase of invasion (3).Recently, direct comparison between the aroA aroD/pnirB and the Δcya Δcrp Δasd/asd⁺ delivery systems for the ability to induce humoral and cellular immunity after a single immunization showed that the former vaccine strain had greater potential as a carrier for antigen delivery (20). However, the balanced lethal asd system for in vivo selection of plasmids expressing heterologous antigens in the attenuated Δcya Δcrp Δasd strains is still very attractive in that asd⁺ plasmids do not require antibiotic resistance markers for selection while stably maintained in vivo (24). In addition, the Δcya Δcrp Δasd/asd⁺ delivery system has been reported to induce protective immunity against several pathogens (25, 29, 40). Most of these studies have restricted analysis of the immune response to antigens expressed from the same asd⁺ plasmid carried by Δcya Δcrp Δasd mutants usually of the same S. typhimurium serotype. In this work, we have analyzed the influence of heterologous gene dosage, and thus level of expression, as well as S. typhimurium strain variability on immune response toward both the heterologous antigen, a nontoxic mutant of E. coli LT, and the carrier Salmonella strain. Effects of a single integration into the host DNA and episomal vectors at different copy numbers were compared in S. typhimurium strains of two different Δcya Δcrp Δasd serotypes, UK-1 and SR-11.     

Protection against Lethal Leptospirosis after Vaccination with LipL32 Coupled or Coadministered with the B Subunit of Escherichia coli Heat-Labile Enterotoxin

Leptospirosis, a worldwide zoonosis, lacks an effective, safe, and cross-protective vaccine. LipL32, the most abundant, immunogenic, and conserved surface lipoprotein present in all pathogenic species of Leptospira, is a promising antigen candidate for a recombinant vaccine. However, several studies have reported a lack of protection when this protein is used as a subunit vaccine. In an attempt to enhance the immune response, we used LipL32 coupled to or coadministered with the B subunit of the Escherichia coli heat-labile enterotoxin (LTB) in a hamster model of leptospirosis. After homologous challenge with 5× the 50% lethal dose (LD₅₀) of Leptospira interrogans, animals vaccinated with LipL32 coadministered with LTB and LTB::LipL32 had significantly higher survival rates (P < 0.05) than animals from the control group. This is the first report of a protective immune response afforded by a subunit vaccine using LipL32 and represents an important contribution toward the development of improved leptospirosis vaccines.     

Characterization of Receptor-Mediated Signal Transduction by Escherichia coli Type IIa Heat-Labile Enterotoxin in the Polarized Human Intestinal Cell Line T84

Escherichia coli type IIa heat-labile enterotoxin (LTIIa) binds in vitro with highest affinity to ganglioside GD1b. It also binds in vitro with lower affinity to several other oligosialogangliosides and to ganglioside GM1, the functional receptor for cholera toxin (CT). In the present study, we characterized receptor-mediated signal transduction by LTIIa in the cultured T84 cell model of human intestinal epithelium. Wild-type LTIIa bound tightly to the apical surface of polarized T84 cell monolayers and elicited a Cl(-) secretory response. LTIIa activity, unlike CT activity, was not blocked by the B subunit of CT. Furthermore, an LTIIa variant with a T14I substitution in its B subunit, which binds in vitro to ganglioside GM1 but not to ganglioside GD1b, was unable to bind to intact T84 cells and did not elicit a Cl(-) secretory response. These findings show that ganglioside GM1 on T84 cells is not a functional receptor for LTIIa. The LTIIa receptor on T84 cells was inactivated by treatment with neuraminidase. Furthermore, LTIIa binding was blocked by tetanus toxin C fragment, which binds to gangliosides GD1b and GT1b. These findings support the hypothesis that ganglioside GD1b, or possibly a glycoconjugate with a GD1b-like oligosaccharide, is the functional receptor for LTIIa on T84 cells. The LTIIa-receptor complexes from T84 cells were associated with detergent-insoluble membrane microdomains (lipid rafts), extending the correlation between toxin binding to lipid rafts and toxin function that was previously established for CT. However, the extent of association with lipid rafts and the magnitude of the Cl(-) secretory response in T84 cells were less for LTIIa than for CT. These properties of LTIIa and the previous finding that enterotoxin LTIIb binds to T84 cells but does not associate with lipid rafts or elicit a Cl(-) secretory response may explain the low pathogenicity for humans of type II enterotoxin-producing isolates of E. coli.     

Intranasal Immunization with Pneumococcal Polysaccharide Conjugate Vaccines with Nontoxic Mutants of Escherichia coli Heat-Labile Enterotoxins as Adjuvants Protects Mice against Invasive Pneumococcal Infections

Host defenses against Streptococcus pneumoniae depend largely on phagocytosis following opsonization by polysaccharide-specific immunoglobulin G (IgG) antibodies and complement. Since colonization of the respiratory mucosa is the first step in pneumococcal pathogenesis, mucosal immune responses may play a significant role. In addition to inducing systemic immune responses, mucosal vaccination with an effective adjuvant has the advantage of inducing mucosal IgA antibodies. The heat-labile enterotoxin (LT) of Escherichia coli is a well-studied mucosal adjuvant, and adjuvant activity of nontoxic LT mutants has been demonstrated for several protein antigens. We investigated the immunogenicity of pneumococcal polysaccharide conjugate vaccines (PNC) of serotypes 1 and 3 in mice after intranasal (i.n.) immunization by using as an adjuvant the nontoxic LT mutant LT-K63 or LT-R72, which has minimal residual toxicity. Pneumococcal serotype-specific antibodies were measured in serum (IgM, IgG, and IgA) and saliva (IgA), and vaccine-induced protection was evaluated by i.n. challenge with virulent pneumococci of the homologous serotype. When administered with LT mutants, i.n. immunization with both conjugates induced systemic and mucosal immune responses, and serum IgG antibody levels were significantly higher than after subcutaneous immunization. All mice immunized i.n. with PNC-1 and LT mutants were protected against bacteremia and cleared the pneumococci from the lung 24 h after i.n. challenge; pneumococcal density correlated significantly with serum IgG antibody levels. Similarly, the survival of mice immunized i.n. with PNC-3 and LT mutants was significantly prolonged. These results demonstrate that i.n. vaccination with PNC and potent adjuvants can protect mice against invasive and lethal pneumococcal infections, indicating that mucosal vaccination with PNC may be an alternative vaccination strategy for humans.