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...

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) produces both heat-labile (LT) and heat-stable (ST) enterotoxins and is a major cause of diarrhea in infants in developing countries and in travelers to those regions. In addition to inducing fluid secretion, LT is a powerful mucosal adjuvant capable of promoting immune responses to coadministered antigens. In this study, we examined purified A subunit to further understand the toxicity and adjuvanticity of LT. Purified A subunit was enzymatically active but sensitive to proteolytic degradation and unable to bind gangliosides, and even in the presence of admixed B subunit, it displayed low cyclic AMP (cAMP) induction and no enterotoxicity. Thus, the AB5 structure plays a key role in protecting the A subunit from proteolytic degradation and in delivering the enzymatic signals required for secretion. In contrast, the A subunit alone was capable of activating dendritic cells and enhanced immune responses to multiple antigens following intranasal immunization; therefore, unlike toxicity, LT adjuvanticity is not dependent on the AB5 holotoxin structure or the presence of the B subunit. However, immune responses were maximal when signals were received from both subunits either in an AB5 structure or with A and B admixed. Furthermore, the quality of the immune response (i.e., IgG1/IgG2 balance and mucosal IgA and IL-17 secretion) was determined by the presence of an A subunit, revealing for the first time induction of Th17 responses with the A subunit alone. These results have important implications for understanding ETEC pathogenesis, unraveling immunologic responses induced by LT-based adjuvants, and developing new mucosal vaccines.

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Using capillary microscopy, internal coalescence between the internal aqueous compartments in a single water-in-oil-in-water (W1/O/W2) double-emulsion globule was investigated. Globules were prepared using either sodium dodecyl sulfate (SDS) (ionic) or Tween 80 (nonionic) as the water-soluble surfactants and Span 80 as the oil-soluble surfactant. Concentrations of both the hydrophobic and hydrophilic surfactants were varied until coalescence between the internal aqueous droplets was observed. Internal coalescence was observed when the concentration of SDS or Tween 80 in the internal aqueous phase was 1-3 CMC (critical micelle concentration) or 50-100 CMC, respectively, and occurred preferentially when the concentration in the internal phase was significantly greater than in the external phase. Internal coalescence always occurred in conjunction with coalescence between the internal W 1 and external W2 aqueous phases (external coalescence). When W1 and W2 were not too dissimilar in their overall surfactant concentrations, only external coalescence occurred. These phenomena were shown to also depend on the concentration of the Span 80 in the oil phase, the stabilizing ability of which was confirmed. Spontaneous emulsification was observed at low concentrations (<25 CMC Tween 80, <0.5 CMC SDS) of the hydrophilic surfactants in the internal aqueous phase. Deformation of W 1 occurred at high concentrations (>100 CMC Tween 80, >3 CMC SDS).

One option for achieving global polio eradication is to replace the oral poliovirus vaccine (OPV), which has the risk of reversion to wild-type virulence, with the inactivated poliovirus vaccine (IPV) vaccine. Adjuvants and alternate routes of immunization are promising options that may reduce antigen dose in IPV vaccinations, potentially allowing dose sparing and cost savings. Use of adjuvants and alternate routes of immunization could also help promote mucosal immunity, potentially mimicking the protection against intestinal virus shedding seen with OPV. In the current study, we examined the impact of combining the novel adjuvant dmLT with trivalent IPV for dose sparing, induction of mucosal immunity and increasing longevity of anti-poliovirus (PV) responses in a mouse model following either intradermal (ID) or intramuscular (IM) delivery. We found that non-adjuvanted ID delivery was not superior to IM delivery for fractional dose sparing, but was associated with development of mucosal immunity. Vaccination with IPV+dmLT promoted serum anti-PV neutralizing antibodies with fractional IPV doses by either IM or ID delivery, achieving at least five-fold dose sparing above non-adjuvanted fractional doses. These responses were most noticeable with the PV1 component of the trivalent vaccine. dmLT also promoted germinal center formation and longevity of serum anti-PV neutralizing titers. Lastly, dmLT enhanced mucosal immunity, as defined by fecal and intestinal anti-PV IgA secretion, when included in IPV immunization by ID or IM delivery. These studies demonstrate that dmLT is an effective adjuvant for either IM or ID delivery of IPV. Inclusion of dmLT in IPV immunizations allows antigen dose sparing and enhances mucosal immunity and longevity of anti-PV responses.