Characterization of a Mutant Escherichia coli Heat-Labile Toxin, LT(R192G/L211A), as a Safe and Effective Oral Adjuvant

Abstract

Despite the fact that the adjuvant properties of the heat-labile enterotoxins of Escherichia coli (LT) and Vibrio cholerae (CT) have been known for more than 20 years, there are no available oral vaccines containing these molecules as adjuvants, primarily because they are both very potent enterotoxins. A number of attempts with various degrees of success have been made to reduce or eliminate the enterotoxicity of LT and CT so they can safely be used as oral adjuvants or immunogens. In this report we characterize the structural, enzymatic, enterotoxic, and adjuvant properties of a novel mutant of LT, designated LT(R192G/L211A), or dmLT. dmLT was not sensitive to trypsin activation, had reduced enzymatic activity for induction of cyclic AMP in Caco-2 cells, and exhibited no enterotoxicity in the patent mouse assay. Importantly, dmLT retained the ability to function as an oral adjuvant for a coadministered antigen (tetanus toxoid) and to elicit anti-LT antibodies. In vitro and in vivo data suggest that the reduced enterotoxicity of this molecule compared to native LT or the single mutant, LT(R192G), is a consequence of increased sensitivity to proteolysis and rapid intracellular degradation in mammalian cells. In conclusion, dmLT is a safe and powerful detoxified enterotoxin with the potential to function as a mucosal adjuvant for coadministered antigens and to elicit anti-LT antibodies without undesirable side effects.Bacterially derived enterotoxins, such as the heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli (ETEC) and the closely related cholera enterotoxin (CT) produced by Vibrio cholerae, promote secretory diarrhea during microbial infection. Each enterotoxin is composed of an enzymatically active A-subunit noncovalently associated with a pentameric B-subunit. Induction of intestinal fluid secretion occurs after a series of events involving both changes to toxin structure and activation of intracellular signaling pathways (reviewed in reference 14). The B-subunit mediates binding and internalization of the toxin to cells; subsequent proteolytic cleavage and disulfide bond reduction separate the A-subunit into two domains, the enzymatically active A1 subunit and a smaller A2 peptide. Transport of A1 into the cytoplasm results in ADP-ribosylation of Gs␣, followed by irreversible activation of adenylate cyclase and increases in intracellular levels of cyclic AMP (cAMP). In intestinal epithelial cells, this induction of cAMP causes a disregulation of cAMP-sensitive ion transport mechanisms, inhibiting intracellular salt absorption, increasing electrolyte transport into the gut lumen, and creating an osmotic gradient favoring intestinal water secretion (12).An enzymatically active A-subunit is required for intestinal fluid secretion but also enables LT to act as an oral adjuvant, boosting the immune response to coadministered antigens and inducing both humoral and cellular immune responses in both the systemic and mucosal compartments. However, even low doses of LT (and CT) induce side ef...

Comparison of Memory B Cell, Antibody-Secreting Cell, and Plasma Antibody Responses in Young Children, Older Children, and Adults with Infection Caused by Vibrio cholerae O1 El Tor Ogawa in Bangladesh

“…We hypothesize that these reduced responses following vaccination compared to infection may reflect the absence of the immunoadjuvantative properties of CT in current oral cholera vaccines. Experimental studies in animal models evaluating oral adjuvanticity of the toxin have demonstrated that CT stimulates a greater mucosal immune response than CtxB alone (4,6); unfortunately, the use of CT as a vaccine component is limited due to its toxicity, although mutant toxoids are being explored for their use in vaccines (28,40). The precise differences in immune stimulation between wild-type disease and current vaccines remain to be elucidated, and further studies are warranted to account for the agespecific differences in long-term vaccine efficacy seen with current vaccine formulations.…”

A Double Mutant Heat-Labile Toxin from Escherichia coli, LT(R192G/L211A), Is an Effective Mucosal Adjuvant for Vaccination against Helicobacter pylori Infection

“…In order to further reduce the enterotoxicity of mLT, an additional mutation was introduced (L211A) to create a double mutant, LT(R192G/L211A) (dmLT). This molecule is essentially nontoxic compared to native LT in a patent mouse enterotoxicity assay which measures the increase in intestinal weight resulting from toxin-induced fluid secretion (11). Furthermore, dmLT has been found to strongly potentiate immune responses to various parenterally and mucosally administered vaccines, e.g., tetanus toxoid and experimental whole-cell vaccines against enterotoxigenic Escherichia coli (J. Holmgren et al, unpublished data), Streptococcus pneumoniae, and H. pylori, making it a promising adjuvant to include in future vaccines (11)(12)(13).…”

Transcutaneous Immunization with a Band-Aid Prevents Experimental Otitis Media in a Polymicrobial Model

“…IHF (integration host factor), is a member of the DNABII family of DNA-binding proteins and was purified from E. coli (15). The systemic and mucosal adjuvant LT(R192G/L211A), or dmLT, is a double mutant of E. coli heat-labile enterotoxin (21). Formulations consisted of the following: (i) 10 g of rsPilA admixed with 10 g dmLT, (ii) 10 g of IHF admixed with 10 g of dmLT, (iii) 5 g of IHF plus 5 g of rsPilA (for 10 g of antigen in total) admixed with 10 g of dmLT, (iv) 10 g of chimV4 admixed with 10 g of dmLT, and (v) 10 g of dmLT alone.…”

“…To do so, our lead candidates, chimV4, rsPilA, and IHF, were administered by band-aid either singly or in combination. Immunogens were admixed with the potent adjuvant LT(R192G/L211A) or dmLT, a double mutant of Escherichia coli heat-labile enterotoxin (21), to potentiate the immune response induced by the incorporated antigens, and efficacy was assessed in a polymicrobial model that more closely mimics the natural progression of disease in children in whom an upper respiratory tract viral infection predisposes to development of bacterial OM (22,23). These data confirm the utility of TCI via simple band-aid delivery to induce an immune response that prevents the development of NTHIinduced OM and further reveal the kinetics of inhibition that were unique to each immunogen tested.…”