The natural diversity of the elt operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Enterotoxigenic Escherichia coli (ETEC)-associated diarrhea represents a major cause of mortality and morbidity among children and travelers, respectively, in most developing countries in Latin America, Africa, and Asia (3, 33). ETEC secretory diarrhea involves a rather straightforward pathogenesis plan, requiring first colonization of small intestine epithelial cells by means of filamentous adhesins collectively known as colonization factors (CFs) and, at a second stage, production of at least one out of two enterotoxin types, the heat-stable toxin (ST) and/or the heat-labile toxin (LT) (28, 36). One of the most complex aspects of ETEC pathogenesis is the remarkable antigen heterogeneity. At least 150 O:H serotypes have been found among ETEC strains isolated from humans, although a more restricted number of serotype combinations is detected among strains isolated from patients requiring medical intervention, also characterized, in some cases, by a conserved set of virulence-associated factors and a common clonal origin (29,30,46). Moreover, the ETEC phenotypic heterogeneity is also well illustrated by the encoded virulence-associated factors, including more than 20 known CFs and production of LT, ST, or both enterotoxins (10,33,46). Two types of ST, STa and STb (also known as ST-I and ST-II), have been differentiated based on biological and chemical features (7,11). Similarly, LTs produced by ETEC strains are also a heterogeneous group of toxins. Two major LT families have been identified, LT-I and LT-II. LT-II is rarely found among human-derived ETEC strains, but two natural variants have been reported, LT-IIa and LT-IIb, based on differences in the subunit sequences (14, 16). LT-I shows a rather high similarity with cholera toxin (CT) (over 80% amino acid identity), and both have been intensively studied as virulence factors and modulators of immune responses in mammalian species, including humans (18,28).The known natural variability of LT-I toxins expressed by ETEC strains has been mainly restricted to the differences detected between LTs produced by human (LTh)-and pig (LTp)-derived strains. Initial evidence based on the antigenicities and electrophoretic mobilities of LTh and LTp indicated that the toxins differ in their primary amino acid sequences (19,42). Sequencing of the elt operons encoding LTh and LTp revealed differences in the primary sequences of the toxins, which share over 95% identity along the complete amino acid sequence (45). Altogether, six amino acid replacements were detected between the A subunits (K4R, K213E, and N238D) and the B subunits (S4T, A46E, and E102K) of LTh and LTp derived from th...

Production and release of heat-labile toxin (LT) by wild-type enterotoxigenic Escherichia coli (ETEC) strains, isolated from diarrheic and asymptomatic Brazilian children, was studied under in vitro and in vivo conditions. Based on a set of 26 genetically diverse LT(+) enterotoxigenic E. coli strains, cell-bound LT concentrations varied from 49.8 to 2415 ng mL(-1). The amounts of toxin released in culture supernatants ranged from 0% to 50% of the total synthesized toxin. The amount of LT associated with secreted membrane vesicles represented <5% of the total toxin detected in culture supernatants. ETEC strains secreting higher amounts of LT, but not those producing high intracellular levels of cell-bound toxin, elicited enhanced fluid accumulation in tied rabbit ileal loops, suggesting that the strain-specific differences in production and secretion of LT correlates with symptoms induced in vivo. However, no clear correlation was established between the ability to produce and secrete LT and the clinical symptoms of the infected individuals. The present results indicate that production and release of LT by wild-type human-derived ETEC strains are heterogeneous traits under both in vitro and in vivo growth conditions and may impact the clinical outcomes of infected individuals.

Heat-labile toxins (LT) encompass at least 16 natural polymorphic toxin variants expressed by wild-type enterotoxigenic Escherichia coli (ETEC) strains isolated from human beings, but only one specific form, produced by the reference ETEC H10407 strain (LT1), has been intensively studied either as a virulence-associated factor or as a mucosal/transcutaneous adjuvant. In the present study, we carried out a biological/immunological characterization of a natural LT variant (LT2) with four polymorphic sites at the A subunit (S190L, G196D, K213E, and S224T) and one at the B subunit (T75A). The results indicated that purified LT2, in comparison with LT1, displayed similar in vitro toxic activities (adenosine 3',5'-cyclic monophosphate accumulation) on mammalian cells and in vivo immunogenicity following delivery via the oral route. Nonetheless, the LT2 variant showed increased adjuvant action to ovalbumin when delivered to mice via the transcutaneous route while antibodies raised in mice immunized with LT2 displayed enhanced affinity and neutralization activity to LT1 and LT2. Taken together, the results indicate that the two most frequent LT polymorphic forms expressed by wild ETEC strains share similar biological features, but differ with regard to their immunological properties.

Vaccinations are extremely effective at combating infectious diseases. Many conserved antigen (Ag) targets, however, are poorly immunogenic. Protein subunit vaccines frequently elicit only humoral immune responses and fail to confer protection against serious intracellular pathogens. These barriers to vaccine development are often overcome by the use of appropriate adjuvants. Heat-labile enterotoxins (HLT) produced by enterotoxigenic strains of Escherichia coli are potent adjuvants when administered by mucosal or systemic routes. The efficacy of the type II HLT, however, has not been well-defined when administered by the intradermal (ID) route. Using a murine ID immunization model, the adjuvant properties of LT-IIb and LT-IIc, two type II HLTs, were compared with those of LT-I, a prototypical type I HLT. While all three HLT adjuvants enhanced Ag-specific humoral responses to similar levels, LT-IIb and LT-IIc, in contrast to LT-I, induced a more vigorous Ag-specific CD8+ T cell response and proffered faster clearance of Listeria monocytogenes in a challenge model. Additionally, LT-IIb and LT-IIc induced distinct differences in the profiles of the Ag-specific CD8+ T cell responses. While LT-IIc stimulated a robust and rapid primary CD8+ T cell response, LT-IIb exhibited slower CD8+ T cell expansion and contraction kinetics with the formation of higher percentages of effector memory cells. In comparison to LT-I and LT-IIc, LT-IIb evoked better long-term protection after immunization. Furthermore, LT-IIb and LT-IIc enhanced the total number of dendritic cells (DC) in the draining lymph node (DLN) and expression of costimulatory molecules CD80, CD86, and CD40 on DCs. In contrast to LT-I, LT-IIb and LT-IIc induced less edema, cellular infiltrates, and general inflammation at the site of ID injection. Thus, LT-IIb and LT-IIc are attractive comprehensive ID adjuvants with unique characteristic that enhance humoral and cellular immunity to a co-administered protein Ag.

Heat-labile toxins (LTs) have ADP-ribosylation activity and induce the secretory diarrhea caused by enterotoxigenic Escherichia coli (ETEC) strains in different mammalian hosts. LTs also act as adjuvants following delivery via mucosal, parenteral, or transcutaneous routes. Previously we have shown that LT produced by human-derived ETEC strains encompass a group of 16 polymorphic variants, including the reference toxin (LT1 or hLT) produced by the H10407 strain and one variant that is found mainly among bacterial strains isolated from pigs (LT4 or pLT). Herein, we show that LT4 (with six polymorphic sites in the A (K4R, K213E, and N238D) and B (S4T, A46E, and E102K) subunits) displays differential in vitro toxicity and in vivo adjuvant activities compared with LT1. One in vitro generated LT mutant (LTK4R), in which the lysine at position 4 of the A subunit was replaced by arginine, showed most of the LT4 features with an ϳ10-fold reduction of the cytotonic effects, ADP-ribosylation activity, and accumulation of intracellular cAMP in Y1 cells. Molecular dynamic studies of the A subunit showed that the K4R replacement reduces the N-terminal region flexibility and decreases the catalytic site crevice. Noticeably, LT4 showed a stronger Th1-biased adjuvant activity with regard to LT1, particularly concerning activation of cytotoxic CD8 ؉ T lymphocytes when delivered via the intranasal route. Our results further emphasize the relevance of LT polymorphism among human-derived ETEC strains that may impact both the pathogenicity of the bacterial strain and the use of these toxins as potential vaccine adjuvants.Enterotoxigenic Escherichia coli (ETEC) 3 is a major etiological agent of diarrhea, afflicting both young children and travelers in developing countries, with high morbidity and mortality rates. ETEC also causes diarrheal disease in livestock, especially in piglets, representing an economically relevant problem (1, 2). ETEC pathogenicity is directly linked to the production of fimbrial or afimbrial colonization factor antigens and heat-stable and/or heat-labile toxin (LT) (1). LT belongs to the family of AB 5 toxins consisting of an enzymatically active A subunit, proteolytically processed into the larger and enzymatic active A1 domain and the shorter A2 domain and five B subunits that mediate binding to glycolipid and glycoprotein receptors of host cells. The B monomer (11.5 kDa) pentamer interacts with the A subunit (28 kDa) via noncovalent binding to the A2 domain. The A1 domain is responsible for ADP-ribosylation of stimulatory G protein, leading to uncontrolled elevation of the intracellular cAMP concentration. Consequently, ion permeases open, and chloride anions and water molecules are released, a hallmark of the watery diarrhea caused by ETEC infection (3).The structure of LT is closely related to the biological functions of the toxin. The A subunit has an overall globular structure and is linked to the compact cylinder-like structure formed by the five B monomers, which expose the residues involved with bind...

The heat-labile toxins (LT) produced by enterotoxigenic Escherichia coli display adjuvant effects to coadministered antigens, leading to enhanced production of serum antibodies. Despite extensive knowledge of the adjuvant properties of LT derivatives, including in vitro-generated non-toxic mutant forms, little is known about the capacity of these adjuvants to modulate the epitope specificity of antibodies directed against antigens. This study characterizes the role of LT and its non-toxic B subunit (LTB) in the modulation of antibody responses to a coadministered antigen, the dengue virus (DENV) envelope glycoprotein domain III (EDIII), which binds to surface receptors and mediates virus entry into host cells. In contrast to non-adjuvanted or alum-adjuvanted formulations, antibodies induced in mice immunized with LT or LTB showed enhanced virus-neutralization effects that were not ascribed to a subclass shift or antigen affinity. Nonetheless, immunosignature analyses revealed that purified LT-adjuvanted EDIII-specific antibodies display distinct epitope-binding patterns with regard to antibodies raised in mice immunized with EDIII or the alum-adjuvanted vaccine. Notably, the analyses led to the identification of a specific EDIII epitope located in the EF to FG loop, which is involved in the entry of DENV into eukaryotic cells. The present results demonstrate that LT and LTB modulate the epitope specificity of antibodies generated after immunization with coadministered antigens that, in the case of EDIII, was associated with the induction of neutralizing antibody responses. These results open perspectives for the more rational development of vaccines with enhanced protective effects against DENV infections.

Cholera toxin (CT) and heat-labile enterotoxins (LTs) produced by Vibrio cholerae and by some enterotoxigenic Escherichia coli (ETEC) strains, respectively, belong to the family of AB (A 1 B 5 )-type bacterial enterotoxins. The A 1 B 5 enterotoxins have an enzymatically active A subunit (28 kDa) that is noncovalently associated to a pentamer composed of five B polypeptides (11.5 kDa each). While the A polypeptide confers the catalytic activity, the B pentamer is responsible for binding to gangliosides and/or galactose-containing surface receptors found on the surface of eukaryotic cells (39). In addition to their roles as virulence determinants directly involved with the watery diarrhea caused by these bacterial pathogens, both CT and LT have received considerable interest due to their remarkable immunomodulatory effects in different mammalian species when they are delivered via parenteral, mucosal, or transcutaneous routes (3,15,21,28,31,38,43).Based on genetic, biochemical, and immunological features, two major groups of heat-labile enterotoxins have been described (17, 20). Type I enterotoxins produced by ETEC strains share high similarity with V. cholerae CT and include the reference LT produced by the H10407 strain (here termed LT-I) while type II enterotoxins (LT-IIa, LT-IIb, and LT-IIc) are usually isolated from ETEC strains derived from nonhuman hosts (16,33,37). Although the A peptides of type I and type II enterotoxins are highly homologous, the two toxin groups differ largely in the B subunit amino acid sequences (Ͻ14% sequence identity). The different B subunit sequences reflect, at least in part, differential receptor specificity and some distinct immunological features of type I and type II enterotoxins (1,5,20). Indeed, previous studies showed that LT-I binds with high affinity to the GM1 ganglioside and, with lower affinity, to a few other galactose-containing surface components such as GM2, asialo-GM1, GD2, GD1b, and paraglobosides while LT-IIa binds most avidly to the GD1b ganglioside and less strongly to other ganglioside receptors (GT1b, GD2, GD1a, GM1, and GM2) (13,40). Interestingly, the B pentamer of LT-IIa (LT-IIaB 5 ), but not the holotoxin, interacts with Toll-like receptor 2 (TLR2) to induce secretion of proinflammatory cytokines (19).Several reports demonstrated that the adjuvant and immunomodulatory properties of LT-IIa correlate with the presence of a functional B subunit (1,32,34). Coadministration of LT-IIa and purified antigens via the nasal route promotes enhanced antigen-specific mucosal (IgA) and systemic (IgG) antibody responses and in vitro activation of T lymphocytes (31). In contrast, a receptor-binding-deficient mutant of LT-IIa lost most of the immunomodulatory properties (34). Nonetheless, while a plethora of studies have characterized the adjuvant properties of LT-I when it is delivered via parenteral

In the present study, we evaluated the immunological responses induced by dengue vaccines under experimental conditions after delivery via a transcutaneous (TC) route. Vaccines against type 2 Dengue virus particles (DENV2 New Guinea C (NGC) strain) combined with enterotoxigenic Escherichia coli (ETEC) heat-labile toxin (LT) were administered to BALB/c mice in a three-dose immunization regimen via the TC route. As a control for the parenteral administration route, other mouse groups were immunized with the same vaccine formulation via the intradermic (ID) route. Our results showed that mice vaccinated either via the TC or ID routes developed similar protective immunity, as measured after lethal challenges with the DENV2 NGC strain. Notably, the vaccine delivered through the TC route induced lower serum antibody (IgG) responses with regard to ID-immunized mice, particularly after the third dose. The protective immunity elicited in TC-immunized mice was attributed to different antigen-specific antibody properties, such as epitope specificity and IgG subclass responses, and cellular immune responses, as determined by cytokine secretion profiles. Altogether, the results of the present study demonstrate the immunogenicity and protective properties of a dengue vaccine delivered through the TC route and offer perspectives for future clinical applications.