Date of Award

1-1-2009

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (viii, 140 pages) : illustrations (some color)

Dissertation/Thesis Chair

Nicholas J Mantis

Committee Members

R. John Collier, Christina Egan, Kathleen McDonough, Kristen Bernard

Keywords

Anthrax, B-Cell Epitopes, Domain 4, Immunity, Monoclonal Antibodies, Protective Antigen, B cells, Antigenic determinants, Monoclonal antibodies, Bacterial vaccines

Subject Categories

Microbiology | Public Health

Abstract

Protective Antigen (PA) is the receptor binding subunit common to both Lethal (LT) and Edema (ET) toxins, which contribute to the mortality associated with Bacillus anthracis infection. While recombinant PA (rPA) is likely to be an important constituent of second generation anthrax vaccines, evaluating the effectiveness of candidate vaccines is currently difficult, because the specific B cell epitopes involved in toxin neutralization have not been completely defined. The only well characterized antibody, 14B7, has been shown to disrupt the association of PA with the anthrax toxin receptors (ATR) by binding to domain 4 of PA. I hypothesized that other domain 4 epitopes play a critical role in eliciting a protective immune response to anthrax. To test this hypothesis I first identified a novel rPA immunogen capable of eliciting a protective immune response in goats and mice. I next established an LT challenge mouse model to evaluate the ability of antibodies to disrupt the intoxication pathway in vivo. To identify neutralizing epitopes on domain 4 of PA, I screened a collection of murine B-cell hybridomas for monoclonal antibodies (MAbs) that reacted with the native PA protein as well as linear peptides within domain 4. Two IgG1 MAbs, 1-F1 and 2-B12, were identified that recognize distinct domain 4 linear epitopes. 1-F1 recognized residues 692-703, part of the ATR recognition region. 1-F1 blocked PA's ability to associate with ATR in an in vitro solid phase binding assay, and neutralized LT in vitro. 2-B12 recognized residues 716-727, a region not previously known to be a target of neutralizing antibodies. 2-B12 was as effective as 1-F1 in neutralizing LT in vitro, although it only partially inhibited PA binding to ATR. Mice passively administered 1-F1 or 2-B12 were protected against LT challenge. This data confirm that several epitopes on domain 4 of PA contribute to the protective immune response against anthrax intoxication. The identification of neutralizing MAbs recognizing linear peptides provides us with a powerful tool for identifying the specific epitopes involved in the protective immune response to anthrax and advances our fundamental understanding of the mechanisms by which antibodies neutralize anthrax toxin.

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