Elsevier

Vaccine

Volume 27, Issue 32, 9 July 2009, Pages 4302-4308
Vaccine

In vivo and in vitro adjuvant activities of the B subunit of Type IIb heat-labile enterotoxin (LT-IIb-B5) from Escherichia coli

https://doi.org/10.1016/j.vaccine.2009.05.027Get rights and content

Abstract

The pentameric B subunit of the Escherichia coli LT-IIb enterotoxin (LT-IIb-B5) activates TLR2 signaling in macrophages. Herein we demonstrate that LT-IIb-B5, in contrast to a TLR2-nonbinding point mutant, induces functional activation of bone marrow-derived dendritic cells and stimulates CD4+ T cell proliferation, activities which suggested that LT-IIb-B5 might function as an adjuvant in vivo. Indeed, in an intranasal mouse immunization model, LT-IIb-B5 augmented specific mucosal and serum antibody responses to a co-administered immunogen, at levels which were almost comparable to those induced by intact LT-IIb holotoxin, a potent but toxic adjuvant. Therefore, LT-IIb-B5 displays useful adjuvant properties which, combined with lack of enterotoxicity and relative stability against degradation, may find application in mucosal vaccines.

Introduction

The heat-labile enterotoxins of Escherichia coli and Vibrio cholerae are structurally related and can be classified into two major types based on genetic, biochemical, and immunological properties [1]. The Type I subfamily includes cholera toxin and E. coli LT-I, whereas the Type II subfamily comprises the antigenically cross-reactive E. coli LT-IIa and LT-IIb [1], [2]. Both Type I and Type II enterotoxins display a similar AB5 oligomeric structure, wherein an enzymatically active and toxic A subunit is noncovalently inserted into the pore of the doughnut-shaped B pentameric subunit [3], [4]. The B pentamer in itself is nontoxic but mediates intracellular delivery of the A subunit following high-affinity binding to membrane gangliosides. The internalized A subunit subsequently catalyzes ADP-ribosylation of the Gsα component of adenylate cyclase, leading to dramatic and unregulated elevation of intracellular cAMP [1]. In intoxicated gut epithelial cells, cAMP elevation results in massive secretion of electrolytes and water into the gut lumen, clinically manifested as diarrhea [1].

The heat-labile enterotoxins have attracted considerable attention due to their exceptional mucosal adjuvant properties [5], although their intrinsic enterotoxicity precludes their use as adjuvants for human vaccines. It therefore became imperative to identify immunoenhancing activities that can be separated from the enzymatic/toxic activity of the A subunit, and this has been the subject of intensive investigation [6], [7], [8]. Our own efforts have focused on the LT-II toxins, which possess immunostimulatory properties that are quite distinct from those of cholera toxin and LT-I (reviewed in Refs. [2], [7]).

In a study examining innate immune interactions of LT-II toxins and their B pentamers, we found that the latter activate nuclear factor (NF)-κB, whereas the intact molecules do not [9]. In subsequent studies, the ability of the LT-II B pentamers to activate NF-κB (and induce production of NF-κB-dependent cytokines) was attributed to stimulation of Toll-like receptor 2 (TLR2) [10], [11]. Intriguingly, the NF-κB-inducing activity of the B pentamer of LT-IIb (designated LT-IIb-B5) is strongly antagonized by the LT-IIb holotoxin, although not by catalytically defective point mutants [12]. This implied that the antagonistic mechanism is cAMP-dependent, which was confirmed in control experiments using a permeable cAMP analog or a cAMP synthesis inhibitor [12]. It is thus conceivable that the demonstrated mucosal adjuvanticity of the LT-IIb holotoxin [13] may be exerted under relatively non-inflammatory conditions, as previously suggested for cholera toxin [14], [15]. Also implicit in the findings on NF-κB activation by LT-IIb-B5[10], [12] was the notion that this B pentamer may display NF-κB-dependent adjuvant activities, such as induction of costimulatory molecules and immunoenhancing cytokines in antigen-presenting cells [16], [17].

In this study, we examined whether LT-IIb-B5 can induce maturation and activation of bone marrow-derived dendritic cells (BMDC) in a way that could provide functional costimulation to CD4+ T cells. Moreover, using an established mouse mucosal immunization model, we investigated whether LT-IIb-B5 can promote specific antibody responses to a co-administered protein immunogen, namely the AgI/II adhesin from Streptococcus mutans[18], [19]. Our findings indicate that LT-IIb-B5 displays useful adjuvant properties which, combined with lack of enterotoxicity and relative stability against degradation [1], [2], [7], suggest its potential for use in mucosal vaccines.

Section snippets

Enterotoxins and other reagents

The construction of recombinant plasmids encoding His-tagged versions of wild-type LT-IIb or LT-IIb-B5 has been previously described [9]. A single-point substitution mutation (S74D) in the LT-IIb-B5 was engineered by means of site-directed mutagenesis (QuikChange® kit, Stratagene, La Jolla, CA). LT-IIb-B5 and derivatives were expressed in E. coli DH5αF’Kan (Life Technologies, Gaithersburg, MD) transformed with the appropriate plasmids, and the proteins were extracted from the periplasmic space

LT-IIb-B5-induced gene expression profile in BMDC

TLR-mediated activation of dendritic cells represents an important mechanism of adjuvant action [16], [25]. Since LT-IIb-B5 can bind and activate the TLR2/TLR1 heterodimer [10], [11], we performed a TLR pathway-focused real-time PCR array in LT-IIb-B5-stimulated BMDC to get an insight on how LT-IIb-B5 may modulate these cells for adjuvant function. For gene expression analysis, stimulated BMDC from BALB/c mice were compared with control unstimulated cells (Table 1). The threshold line for

Discussion

Induced production of immunostimulatory cytokines and upregulation of costimulatory molecules in dendritic cells are required for effective activation of T cells and prevention of tolerance [32], [33]. Although TLR2 can function as an adjuvant receptor [34], [35], engagement of dendritic cell TLR2 by microbial ligands does not necessarily result in immunostimulation. Indeed, TLR2 signaling may also lead to the generation of regulatory dendritic cells and induction of T cell tolerance, which

Acknowledgements

This work was supported by U.S. Public Health Service Grants DE13833 (to TDC), DE06746 (to MWR), and DE015254 and DE017138 (to GH) from the National Institutes of Health.

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