Role of G_M1 binding in the mucosal immunogenicity and adjuvant activity of the Escherichia coli heat‐labile enterotoxin and its B subunit

Abstract

SUMMARYEscherichia coli (E. coli ) 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 G M1 . Both the ADP-ribosylation activity of LTA and G M1 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, G M1 -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 G M1 -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 G M1 -binding activity, adjuvanticity of LT was found to be independent of G M1 -binding affinity. Moreover, a mutant lacking both G M1 -binding and ADPribosylation activity, also retained adjuvanticity. These results demonstrate that neither ADPribosylation activity nor G M1 binding are essential for adjuvanticity of LT, and suggest an ADP-ribosylation-independent adjuvant effect of the A subunit. INTRODUCTIONinvestigators have shown that the enzymatic activity of LTA does not play a major role in the immunogenicity of LT.8-11 The Escherichia coli (E. coli ) heat-labile toxin (LT ) and Vibrio On the other hand, recombinant LTB alone is clearly less cholerae cholera toxin (CT ) are exceptionally potent mucosal immunogenic than LT mutants which lack ADP-ribosylation immunogens and immunoadjuvants.1-5 In addition, LT and activity,12 suggesting that the presence of LTA, but not CT have been found to abrogate tolerance towards necessarily its enzymatic activity, does contribute to the co-administered antigens.1,3 Both are heterohexameric proteins antitoxin antibody response. The role of the A subunit in composed of one copy of the A subunit, which has ADPadjuvanticity is even less clear. Several studies have shown ribosylation activity, and five copies of the B subunit. The that recombinant LTB and CTB lack the capacity to stimulate B-pentamer has high affinity for the toxin receptor, ganglioside systemic and mucosal antibody responses towards G M1 . Delivery of the A subunit to the cytosol of the target cell co-administered antigens.13-18 Furthermore, using an LT results in persistent synthesis of cAMP.6,7 mutant lacking ADP-ribosylation activity, Lycke et al.16 Even though the role of the individual subunits of LT showed that the adjuvant effect of LT and CT is directly and CT in the toxic mechanism are well defined, their role in linked to the enz...

Suppression of inhibitor formation against FVIII in a murine model of hemophilia A by oral delivery of antigens bioencapsulated in plant cells

“…[38][39][40] To evaluate the receptor-binding ability of CTB-HC and CTB-C2 fusion proteins produced in tobacco chloroplasts, GM1-binding ELISA was performed. As observed in Figure 2E, CTB-HC and CTB-C2 fusion protein extracts along with purified CTB protein showed strong binding affinity to GM1.…”

Adjuvants modulating mucosal immune responses or directing systemic responses towards the mucosa

“…However, the results obtained with LT-B and CT-B alone as mucosal adjuvants are highly inconsistent. Studies have shown that neither LT-B nor CT-B enhance immune responses to mucosal co-administered protein antigens when given orally [165,231], whereas some other reports have suggested that LT-B and CT-B could display mucosal adjuvant activity when intranasally administered (large doses) in combination with proteins (large doses) [46,47,52,165]. Antibody responses can be observed when LT-B or CT-B are directly conjugated to the antigen itself [165] and are given either orally or intranasally (Tab.…”

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).…”