Enterotoxin-Based Mucosal Adjuvants Alter Antigen Trafficking and Induce Inflammatory Responses in the Nasal Tract

Abstract

The safety of nasal vaccines containing enterotoxin-based mucosal adjuvants has not been studied in detail. Previous studies have indicated that native cholera toxin (nCT) can alter antigen trafficking when applied nasally. In this study, we determined the enterotoxin-based variables that alter antigen trafficking. To measure the influence of enterotoxin-based mucosal adjuvants on antigen trafficking in the nasal tract, native and mutant enterotoxins were coadministered with radiolabeled tetanus toxoid (TT). The nCT and heat-labile enterotoxin type 1 (LTh-1) redirected TT into the olfactory neuroepithelium (ON/E). Antigen redirection occurred mainly across the nasal epithelium without subsequent transport along olfactory neurons into the olfactory bulbs (OB). Thus, no significant accumulation of the vaccine antigen TT was observed in the OB when coadministered with nCT. In contrast, neither mutant CT nor mutant LTh-1, which lack ADP-ribosyltransferase activity, redirected TT antigen into the ON/E. Thus, ADP-ribosyltransferase activity was essential for antigen trafficking across the olfactory epithelium. Accumulation of TT in the ON/E was also due to B-subunit binding to GM1 gangliosides, as was demonstrated (i) by redirection of TT by LTh-1 in a dose-dependent manner, (ii) by ganglioside inhibition of the antigen redirection by LTh-1 and nCT, and (iii) by the use of LT-IIb, a toxin that binds to gangliosides other than GM1. Redirection of TT into the ON/E coincided with elevated production of interleukin 6 (IL-6) but not IL-1␤ or tumor necrosis factor alpha in the nasal mucosa. Thus, redirection of TT is dependent on ADP-ribosyltransferase activity and GM1 binding and is associated with production of the inflammatory cytokine IL-6.Enterotoxins are powerful mucosal adjuvants; however, the mechanisms for their adjuvanticity are still being defined. Native cholera toxin (nCT) and the Escherichia coli-derived heatlabile toxin (human type 1) (nLTh-1) are both potent mucosal adjuvants for coadministered protein antigens when given by the oral, nasal, or parenteral route (4, 9-11, 33, 42, 50). Despite extensive research on these enterotoxins, mucosal adjuvants for human use remain in experimental phases, and recent studies have focused on generating nontoxic mutants of CT (mCT) and LTh-1 (mLTh-1). Detoxification of these enterotoxins was accomplished by site-directed mutagenesis of the ADP-ribosylation site located in the A subunit of these AB 5 enterotoxins (3,8,51,52,53,54). These mutants are effective mucosal adjuvants in mice and induce long-term memory for coadministered proteins given either by the nasal or parenteral route (3,51,52). In this regard, the nasal route is perhaps superior to oral delivery, since it requires much lower doses of both adjuvant and coadministered proteins/vaccines.Both nCT and nLTh-1 are part of serogroup

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

“…The use of HLT as mucosal adjuvants in human vaccines has been restricted by their intrinsic toxicity and by their propensity to traffic to the brain via the olfactory bulb (55,56). A variety of methods have been used to reduce or eliminate the toxic activities of the type II HLT in order to facilitate their use in human vaccines.…”

Prophylactic Administration of Bacterially Derived Immunomodulators Improves the Outcome of Influenza Virus Infection in a Murine Model

“…LT(R192G) has yet to be evaluated as a prophylactic immunomodulator, but another LT mutant, LT(S63K), has demonstrated some protective effects against influenza virus, respiratory syncytial virus (RSV), and Cryptococcus neoformans infections (80). Although safety concerns limit the use of native enterotoxins for intranasal or intrapulmonary use in humans (54,76), animal model studies are warranted because they enhance our understanding of the initial responses that can ultimately lead to protection of the host against infection. In addition, the use of these enterotoxins in laboratory research has the potential to be translated into clinical application by using mutated low-toxinogenic derivatives that retain their immunomodulatory properties.…”

Sublingual vaccination with influenza virus protects mice against lethal viral infection

“…or s.l., and lung, OB, and brain were removed 24 h after immunization as described in ref. 33. Tissues were weighed, and 200 l of CellLytic MT lysis buffer (Sigma) was added per 10 mg of tissue wet weight.…”