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.

Enterotoxigenic Escherichia coli (ETEC) represents one of the most important etiological agents of diarrhea in developing countries and characteristically produces at least one of two enterotoxins: heat-labile toxin (LT) and heat-stable toxin (ST). It has been previously shown that the production and release of LT by human-derived ETEC strains are variable. Although the natural genetic polymorphisms of regulatory sequences of LT-encoding (eltAB) genes may explain the variable production of LT, the knowledge of the transcriptional and posttranscriptional aspects affecting LT expression among ETEC strains is not clear. To further understand the factors affecting LT expression, we evaluated the impact of the natural polymorphism in noncoding regulatory sequences of eltAB among clinically derived ETEC strains. Sequence analyses of seven clinically derived strains and the reference strain H10407 revealed polymorphic sites at both the promoter and upstream regions of the eltAB operon. Operon fusion assays with GFP revealed that specific nucleotide changes in the Pribnow box reduce eltAB transcription. Nonetheless, the total amounts of LT produced by the tested ETEC strains did not strictly correspond to the detected LTspecific mRNA levels. Indeed, the stability of LT varied according to the tested strain, indicating the presence of posttranscriptional mechanisms affecting LT expression. Taken together, our results indicate that the production of LT is a strain-specific process and involves transcriptional and posttranscriptional mechanisms that regulate the final amount of toxin produced and released by specific strains.

Introduction: Heat-labile toxins (LT), produced by enterotoxigenic Escherichia coli (ETEC) strains, exert potent adjuvant effects when admixed or linked to antigens delivered via mucosal, transcutaneous or parenteral routes. There is limited information regarding the impact of preexisting immunity on the immunomodulatory properties of LT, which is frequently observed among people infected with ETEC. Aims: In the present study, we evaluated the effect of anti-LT antibodies on the adjuvant and inflammatory activities triggered by LT admixed with a specific vaccine antigen following subcutaneous administration to mice. Material and Methods/Results: Animals were immunized with dengue virus nonstructural protein (NS1), as model antigen, in combination with native LT in the presence of LT-specific antibodies. Exposure to anti-LT antibodies did not impair the humoral adjuvanticity of LT regarding to the magnitude of the serum anti-NS1 IgG titers. In addition, anti-toxin antibodies did not reduce neutrophil migration nor edema formation after s.c. administration of LT. Nonetheless, administration of LT admixed with anti-LT antibodies changed the local cytokine production profile and modulated the NS1specific T cell responses to a Th1-type pattern. Conclusion: These results indicate that preexisting immunity does not affect the humoral adjuvant activities but may modulate different aspects of both innate and adaptive immune responses induced by parenterally administered LT.

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