L de Haan

Musosal immunoadjuvant activity of recombinant Escherichia coli heat-labile enterotoxin and its B subunit: Induction of systemic IgG and secretory IgA responses in mice by intranasal immunization with influenza virus surface antigen

The Escherichia coli heat-labile enterotoxin (LT) is a very potent mucosal immunogen. LT also has strong adjuvant activity towards coadministered uni elated antigens and is therefore of potential inter-est for development of mucosal vaccines. However; despite the great demand for such mucosal vaccines, the use of LT holotoxin us an adjuvant is essentially precluded by its toxicity. LT is composed of an A subunit, carrying the toxic ADP-ribosylation activity and a pentamer of identical B subunits, which mediates binding to ganglioside G(M1), the cellular receptor for the toxin. In this paper; we demonstrate that recombinant enzymatically inactive variants of LT, including the LTB pentamer by itself; retain the immunoadjuvant activity of LT holotoxin in a murine influenza model. Mice were immunized intranasally (i.n.) with influenza virus subunit antigen, consisting mostly of the isolated surface glycoprotein hemagglutinin (HA), supplemented with either recombinant LTB (rLTB), a nontoxic LT mutant (E112K, with a Glu112-->Lys substitution in the A subunit), or LT holotoxin, and the induction of systemic IgG and local S-IgA responses was evaluated by direct enzyme-linked immunosorbent assay (ELISA). immunization with subunit antigen alone resulted in a poor systemic IgG response and no detectable S-IgA. However; supplementation of the antigen with E112K or rLTB resulted in a substantial stimulation of the serum IgG level and in induction of a strong S-IgA response in the nasal cavity. The adjuvant activity of E112K or rLTB under these conditions was essentially the same as that of the LT holotoxin. The present results demonstrate that nontoxic variants of LT rLTB in particular; represent promising immunoadjuvants for potential application in an i.n. influenza virus subunit vaccine. Nontoxic LT variants may also be used in i.n. vaccine formulations directed against other mucosal pathogens. In this respect, it is of interest that LT(B)-stimulated antibody responses after i.n. immunization were also observed at distant mucosal sites, including the urogenital system. This, in principle, opens the possibility to develop i.n. vaccines against sexually transmitted infectious diseases

Role of GM1 binding in the mucosal immunogenicity and adjuvant activity of the Escherichia coli heat‐labile enterotoxin and its B subunit

Escherichia coli (E. coliu200a) heat‐labile toxin (LT) is a potent mucosal immunogen and immunoadjuvant towards co‐administered antigens. LT is composed of one copy of the A subunit, which has ADP‐ribosylation activity, and a homopentamer of B subunits, which has affinity for the toxin receptor, the ganglioside GM1. Both the ADP‐ribosylation activity of LTA and GM1 binding of LTB have been proposed to be involved in immune stimulation. We investigated the roles of these activities in the immunogenicity of recombinant LT or LTB upon intranasal immunization of mice using LT/LTB mutants, lacking either ADP‐ribosylation activity, GM1‐binding affinity, or both. Likewise, the adjuvant properties of these LT/LTB variants towards influenza virus subunit antigen were investigated. With respect to the immunogenicity of LT and LTB, we found that GM1‐binding activity is essential for effective induction of anti‐LTB antibodies. On the other hand, an LT mutant lacking ADP‐ribosylation activity retained the immunogenic properties of the native toxin, indicating that ADP ribosylation is not critically involved. Whereas adjuvanticity of LTB was found to be directly related to GM1‐binding activity, adjuvanticity of LT was found to be independent of GM1‐binding affinity. Moreover, a mutant lacking both GM1‐binding and ADP‐ribosylation activity, also retained adjuvanticity. These results demonstrate that neither ADP‐ribosylation activity nor GM1 binding are essential for adjuvanticity of LT, and suggest an ADP‐ribosylation‐independent adjuvant effect of the A subunit.

The role of ADP-ribosylation and G M1 -binding activity in the mucosal immunogenicity and adjuvanticity of the Escherichia coli heat-labile enterotoxin and Vibrio cholerae cholera toxin

The mucosal route of vaccination has attracted a great deal of attention recently. Not only is mucosal application of vaccines, for example, orally or intranasally, particularly convenient, it also offers the possibility to induce locally produced and secreted S‐IgA antibodies in addition to systemic IgG antibodies. These IgA antibodies are known to play a key role in protection against pathogens that invade the host through mucosal surfaces. Induction of such responses is not readily achieved by currently used vaccination strategies, which generally involve intramuscular or subcutaneous injection with inactivated pathogens or antigens thereof. For the induction of a mucosal immune response, the vaccine needs to be applied locally. However, local vaccination with non‐replicating antigens is usually ineffective and may result in tolerance unless a mucosal immunoadjuvant is included. The most potent mucosal immunoadjuvants known to date are probably cholera toxin (CT) and the closely related Escherichia coli heat‐labile enterotoxin (LT). Although CT and LT have become standard adjuvants for experimental mucosal vaccines, the intrinsic toxicity has thus far precluded their use as adjuvants for human vaccine formulations. In the present review, the mucosal immunogenic and adjuvant properties of LT and CT are described, with special emphasis on the functional role of the individual subunits on their immune‐stimulatory properties.

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

The 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, GM1‐binding properties of the LTB‐pentamer have been suggested to be involved. In recent studies we found that GM1‐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 immunoglobulin‐isotype 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 LTB‐specific, 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.