Mucosal immunogenicity and adjuvant activity of the recombinant A subunit of the Escherichia coli heat‐labile enterotoxin

Abstract

SUMMARYThe Escherichia coli heat-labile enterotoxin (LT ) is an exceptionally effective mucosal immunogen and mucosal immunoadjuvant towards coadministered antigens. Although, in general, the molecular basis of these properties is poorly understood, both the toxic ADP-ribosylation activity of the LTA subunit and the cellular toxin receptor, ganglioside, G M1 -binding properties of the LTB-pentamer have been suggested to be involved. In recent studies we found that G M1 -binding is not essential for the adjuvanticity of LT, suggesting an important role for the LTA subunit in immune stimulation. We now describe the immunomodulatory properties of recombinant LTA molecules with or without ADP-ribosylation activity, LTA(His) 10 and LTA-E112K(His) 10 , respectively. These molecules were expressed as fusion proteins with an N-terminal His-tag to allow simple purification on nickel-chelate columns. Their immunogenic and immunoadjuvant properties were assessed upon intranasal administration to mice, and antigen-specific serum immunoglobulinisotype and -subtype responses and mucosal secretory immunoglobulin A (IgA) responses were monitored using enzyme-linked immunosorbent assay. With respect to immunogenicity, both LTA(His) 10 and LTA-E112K(His) 10 failed to induce antibody responses. On the other hand, immunization with both LT and the non-toxic LT-E112K mutant not only induced brisk LTBspecific, but also LTA-specific serum and mucosal antibody responses. Therefore, we conclude that linkage of LTA to the LTB pentamer is essential for the induction of LTA-specific responses. With respect to adjuvanticity, both LTA(His) 10 and LTA-E112K(His) 10 were found to stimulate serum and mucosal antibody responses towards coadministered influenza subunit antigen. Remarkably, responses obtained with LTA(His) 10 were comparable in both magnitude and serum immunoglobulin isotype and subtype distributions to those observed after coimmunization with LT, LT-E112K, or recombinant LTB. We conclude that LTA, by itself, can act as a potent adjuvant for intranasally administered antigens in a fashion independent of ADP-ribosylation activity and association with the LTB pentamer. INTRODUCTIONB subunits. The A subunit carries the toxic ADP-ribosylation activity of the toxin, while the B-pentamer has high affinity for The Escherichia coli heat-labile enterotoxin (LT ) and its close the cellular toxin receptor, ganglioside G M1 . Intoxication of homologue from Vibrio cholerae, cholera toxin (CT ), are wellintestinal epithelial cells with LT or CT results in persistent known powerful mucosal immunogens and adjuvants. Both synthesis of cAMP and concomitant excessive electrolyte and toxins have proven to be valuable tools for unravelling mechanfluid secretion to the intestinal lumen (for reviews see refs. 4,5). isms of induction of mucosal immune responses, and have Over the last 10 years, several strategies have been develbecome standard adjuvants for experimental mucosal vacoped in order to investigate whether the toxic properties of cines.1-3 How...

Oral Administration of Recombinant Cholera Toxin Subunit B Inhibits IL-12-Mediated Murine Experimental (Trinitrobenzene Sulfonic Acid) Colitis

“…Previous studies of CT and related bacterial toxins (such as E. coli heat-labile toxin) have emphasized the fact that the adjuvant and toxic effects of the toxin resides primarily in its A subunit, the part of the molecule that has ADP ribosyltransferase activity and accounts for its ability to activate adenylate cyclase and to generate cAMP (5). This is supported by the observation that recombinant LT-A subunit or CT-A bound to protein A (an alternative binding molecule that targets the B cells) both retain potent adjuvant activity (6,7). Of interest, it appears that the A subunit can function as an adjuvant even in the absence of ribosyltransferase activity (7).…”

“…The adjuvant effect of CT-holotoxin had been attributed to CT-A (4,5) and indeed it has recently been shown that CT-A linked to protein A, a molecule that binds to Ig on the surface of B cells or that recombinant A subunit of Escherichia coli heatlabile enterotoxin (rLT-A), a toxin molecule functionally related to CT-A, also exerts powerful adjuvant effects (6,7). Whether or not adjuvant effects are also inherent in the activity of CT-B has until recently been obscured by the fact that purified CT-B preparations used in earlier studies were probably contaminated by small amounts of CT-A (8,9).…”

Distribution of intranasal instillations in mice: effects of volume, time, body position, and anesthesia

“…In published studies, volumes of substances intranasally instilled into mice range from 5 l (22) to 100 l (3), with little justification for the chosen volumes. Intranasal delivery is often carried out after intraperitoneal (9, 19) or inhalation (5, 14, 15) anesthesia but has also been performed with fully awake mice (1,6,17). The position of the mouse during intranasal delivery has also varied between studies, with horizontal (13) and head-down supine (12) positions having been used.…”

GM1 Binding-Deficient Exotoxin Is a Potent Noninflammatory Broad Spectrum Intradermal Immunoadjuvant

“…Application to the nasal mucosal environment in mice resulted in specific translocation of exotoxin (or exotoxin subunits) and coadministered Ags to the CNS, raising concerns about undesirable neurotoxicity (18,19). To reduce toxicity, modified exotoxins are described in which the A subunit is mutated or deleted to reduce or eliminate ribosylation activity (20,21). However, these formulations may still retain toxicity due to residual ribosylation activity, or there is an undesirable loss of adjuvanticity (15,22).…”