A Single VHH-Based Toxin-Neutralizing Agent and an Effector Antibody Protect Mice against Challenge with Shiga Toxins 1 and 2

ABSTRACTShiga toxin-producingEscherichia coli(STEC) is a major cause of severe food-borne disease worldwide, and two Shiga toxins, Stx1 and Stx2, are primarily responsible for the serious disease consequence, hemolytic-uremic syndrome (HUS). Here we report identification of a panel of heavy-chain-only antibody (Ab) VH(VHH) domains that neutralize Stx1 and/or Stx2 in cell-based assays. VHH heterodimer toxin-neutralizing agents containing two linked Stx1-neutralizing VHHs or two Stx2-neutralizing VHHs were generally much more potent at Stx neutralization than a pool of the two-component monomers tested in cell-based assays andin vivomouse models. We recently reported that clearance of toxins can be promoted by coadministering a VHH-based toxin-neutralizing agent with an antitag monoclonal antibody (MAb), called the “effector Ab,” that indirectly decorates each toxin molecule with four Ab molecules. Decoration occurs because the Ab binds to a common epitopic tag present at two sites on each of the two VHH heterodimer molecules that bind to each toxin molecule. Here we show that coadministration of effector Ab substantially improved the efficacy of Stx toxin-neutralizing agents to prevent death or kidney damage in mice following challenge with Stx1 or Stx2. A single toxin-neutralizing agent consisting of a double-tagged VHH heterotrimer—one Stx1-specific VHH, one Stx2-specific VHH, and one Stx1/Stx2 cross-specific VHH—was effective in preventing all symptoms of intoxication from Stx1 and Stx2 when coadministered with effector Ab. Overall, the availability of simple, defined, recombinant proteins that provide cost-effective protection against HUS opens up new therapeutic approaches to managing disease.

Use of Pertussis Toxin Encoded by ptxGenes from Bordetella bronchiseptica To Model the Effects of Antigenic Drift of Pertussis Toxin on Antibody Neutralization

ABSTRACT Recently, concern has been voiced about the potential effect that antigenic divergence of circulating strains of Bordetella pertussis might have on the efficacy of pertussis vaccines. In order to model antigenic drift of pertussis toxin, a critical component of many pertussis vaccines, and to examine the effects of such drift on antibody neutralization, we engineered a strain of B. pertussis to produce a variant pertussis toxin molecule that contains many of the amino acid changes found in the toxin encoded byBordetella bronchiseptica ptx genes. This altered form of the toxin, which is efficiently secreted by B. pertussisand which displays significant biological activity, was found to be neutralized by antibodies induced by vaccination as readily as toxin produced by wild-type B. pertussis. These findings suggest that significant amino acid changes in the pertussis toxin sequence can occur without drastically altering the ability of antibodies to recognize and neutralize the toxin molecule.

Expression and Immunogenicity of a Mutant Diphtheria Toxin Molecule, CRM197, and Its Fragments inSalmonella typhi Vaccine Strain CVD 908-htrA

ABSTRACT Mutant diphtheria toxin molecule CRM197 and fragments thereof were expressed in attenuated Salmonella typhi CVD 908-htrA, and the constructs were tested for their ability to induce serum antitoxin. Initially, expressed proteins were insoluble, and the constructs failed to induce neutralizing antitoxin. Soluble CRM197 was expressed at low levels by utilizing the hemolysin A secretion system from Escherichia coli.

Identification of the Helicobacter pylori VacA Toxin Domain Active in the Cell Cytosol

ABSTRACT Cells exposed to Helicobacter pylori toxin VacA develop large vacuoles which originate from massive swelling of membranous compartments at late stages of the endocytic pathway. When expressed in the cytosol, VacA induces vacuolization as it does when added from outside. This and other evidence indicate that VacA is a toxin capable of entering the cell cytosol, where it displays its activity. In this study, we have used cytosolic expression to identify the portion of the toxin molecule responsible for the vacuolating activity. VacA mutants with deletions at the C and N termini were generated, and their activity was analyzed upon expression in HeLa cells. We found that the vacuolating activity of VacA resides in the amino-terminal region, the whole of which is required for its intracellular activity.