Recombinant Antigen-Enterotoxin A2/B Chimeric Mucosal Immunogens Differentially Enhance Antibody Responses and B7-Dependent Costimulation of CD4⁺T Cells

Abstract

Cholera toxin (CT) produced by Vibrio cholerae and the labile toxins (LT) from Escherichia coli are structurally related heat-labile enterotoxins (HLE) that have been employed as adjuvants to augment both mucosal and systemic immune responses to coadministered antigens (Ag) (3, 12). These oligomeric toxins consist of an A subunit noncovalently coupled to five B polypeptides (42). After proteolytic cleavage and reduction of an intrachain disulfide bond, the A subunit is cleaved into a toxic A1 and a linking A2 polypeptide. Initial studies using HLE as mucosal adjuvants in animal models led to the conclusion that their adjuvanticity was due to the toxic ADPribosyltransferase activity of the A1 subunit (31). ADP ribosylation of the Gs␣ subunit of adenylate cyclase results in abnormally high levels of intracellular cyclic AMP (cAMP) (24, 42) and subsequent chloride ion efflux into the lumen of the gut, which is ultimately responsible for the characteristic watery diarrhea.Due to the toxic nature of the holotoxins, many investigators have tried to dissociate the toxicity associated with the A1-subunit from the adjuvanticity of the AB 5 complex and have attempted to address the immunostimulatory effects of B subunit receptor-mediated interactions. Earlier studies using commercial CTB preparations contaminated with intact CT made it impossible to distinguish between the adjuvanticity associated with ADP-ribosyltransferase activity and the binding properties of the AB 5 complex. This issue was further complicated by the synergistic effect of holotoxin on the adjuvanticity of the B subunit (45, 48). However, with the aid of recombinant techniques, mutant constructs of CT and LT-I, which lacked ADP-ribosyltransferase activity, were shown to retain many of the adjuvant properties of the native toxin (11,15,52,53). Studies comparing recombinantly produced wild type and a LT-I B subunit (LT-I B) mutant that lacks GM1 binding further demonstrated that both immunogenicity and adjuvanticity were dependent upon GM1 binding (35). Additional experiments have demonstrated that the up-regulation of various costimulatory molecules on Ag-presenting cells (APCs) by LT-I B or nontoxic derivatives of CT was abrogated when GM1 binding was blocked (34,52). These studies demonstrate that the GM1 binding properties of the type I HLE appear to be necessary for their adjuvant properties.Two types of HLE have been distinguished on the basis of distinct immunoreactivity (22, 37): type I HLE are represented by CT and LT-I (25, 37); type II HLE include E. coli 23). Although type I and type II HLE are structurally homologous and catalyze similar enzymatic reactions, comparison of the predicted amino acid sequences reveals considerable variability between type I and type II enterotoxin B subunits (22,(37)(38)(39). This extensive diversity imparts different ganglioside-binding properties to the respec-* Corresponding author. Mailing address:

Mutants of Type II Heat-Labile Enterotoxin LT-IIa with Altered Ganglioside-Binding Activities and Diminished Toxicity Are Potent Mucosal Adjuvants

“…Full activation of antigen-specific T cells requires interaction between the T-cell receptor and MHC-II, as well as interaction between CD28 and B7 and/or between CD40 and CD40L on the surfaces of T cells and antigen-presenting cells (APC) (30). Since it was previously reported that CT, LT-I, LT-IIa, and LT-IIb modulate the expression of MHC-II and costimulatory molecules (CD40, CD80, and CD86) on the surface of APC (2,33,36,41), the effects of the mutant enterotoxins LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) on the levels of expression of CD40, CD80 (B7-1), CD86 (B7-2), and MHC-II on the surfaces of resting B cells (B220 ϩ ) and dendritic cells (CD11c ϩ ) isolated from the spleens of naïve mice were examined.…”

Characterization of aSalmonellaTyphimurium ghost carrying an adjuvant protein as a vaccine candidate for the protection of chickens against virulent challenge

“…The current study demonstrated that the oligomeric complex of recombinant LTB in the outer membrane of the ghost appeared to be assembled in its native pentameric form with the ability to bind to GM1 ganglioside receptors, as indicated by data from the GM1 binding assays (Figure 2). As the LTB protein is genetically coupled with the ghost cells, it might be associated with improved presentation of the coupled Salmonella Typhimurium antigens by antigen-presenting cells, thereby enhancing the induction of antigen-specific mucosal and systemic immune responses (Martin et al, 2001;Ekong et al, 2009). The role of the LTB protein as a mucosal adjuvant has been widely studied (Freytag & Clements 2005); however, because LTB binds to the GM1 receptors of all dividing eukaryotic cells, it can act as an adjuvant protein via parenteral delivery (Yamamoto et al, 1997;Agren et al, 1999;Weltzin et al, 2000;Conceição et al, 2006).…”

Gonococcal transferrin binding protein chimeras induce bactericidal and growth inhibitory antibodies in mice

“…The subunit chimeras complex with Ctb pentamers and result in the production of robust and protective immune responses to A2-fused antigens [23][24][25][26][27][28][29]. We focused our efforts on two Tbp-specific domains, the NB domain from TbpB and the L2 domain from TbpA based largely on surface exposure and strain-to-strain sequence conservation.…”

“…The non-toxic A2 domain of CtA passes through the central pore of the B subunit which allows for the tethering of the activity domain to Ctb [21]. Previous investigators have demonstrated that the replacement of the toxic A1 moiety of CtA with heterologous proteins genetically fused with the A2 subunit, allowed for production of holotoxin-like chimeras [23][24][25][26][27][28][29]. Employing this approach for TbpA, we focused on the surface-exposed loop 2 (L2).…”