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dc.contributor.authorAbboud, Nareen
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 70-04, Section: B, page: 2185.;Advisors: Arturo Casadevall.
dc.description.abstractBacillus anthracis is the causative agent of the disease anthrax, which is a powerful agent for bioterrorism and biological warfare. There is a need for a safer and more effective vaccine against Bacillus anthracis that could generate stable and prolonged immunity in humans than the currently available US licensed anthrax vaccine absorbed (AVA) vaccine. AVA is poorly immunogenic, has questionable efficacy in some hosts, and is associated with significant side effects. Protective antigen (PA) is the major antigenic component in this vaccine and experimental vaccines for protection against anthrax.;In this work, we dissected the PA molecule to identify protein fragments that are both immunogenic and elicit protective antibodies. Our goal was to understand the relative immunogenicity of the four domains of PA with the eventual goal of generating the necessary information to make a more effective vaccine against anthrax. To study this, we generated rPA proteins suitable for immunization studies to correlate the functional efficacy of discrete domains of PA with their ability to elicit immune responses in four genetically diverse strains of mice. Dependent on genetic background, significant antibody responses were made to domain 1 of PA and full length PA (all four domains). Surprisingly, immunization with domain 4 of PA was significantly less effective in eliciting high antibody responses, in contrast to the observation that many neutralizing monoclonal antibodies (mAbs) bind to this domain. Further analysis of the antisera from all immunized mouse strains indicated that the predominant antibody type produced as a result of these immunizations is of the IgG1 subclass, which is the subclass of antibodies associated with protection against anthrax toxin. We were able to demonstrate a sharp dose response of antibody amount and PA neutralizing capacity in certain strains of mice.;Given the ability to stimulate protective immunity in a range of animal models including primates, there is considerable interest in the development of passive antibody therapy directed against PA. For B. anthracis , the vast majority of B-cell epitopes reported to date are derived from PA. The epitopes described thus far tend to cluster to three discrete regions within the PA molecule. Overall, no B-cell epitopes had been described in domain 1. However, we have shown that both monoclonal- and polyclonal-murine antibodies to domain 1 are neutralizing. Therefore, we sought to identify further dominant antigenic epitopes to understand the minimal immunogenic region of PA that will allow for precise direction of potent immune responses. By use of overlapping synthetic peptides spanning the entire length of PA, we mapped epitopes recognized by five mAbs to PA. Of the three peptidic B-cell epitopes described, two are targets of neutralizing mAbs, which are able to efficiently protect animals from anthrax toxin challenge in vivo. These two epitopes map to regions on PA that are in close proximity to the furin cleavage site. Preventing furin cleavage precludes PA heptamerization, and in turn edema factor and lethal factor binding to PA. We demonstrated that the activity of both anti-domain 1 mAbs occurs via slowing down the proteolytic digestion of PA by furin, in agreement with the function of this domain and PA-mAb docking models. We further demonstrate that peptides containing these epitopes were immunogenic and elicited antibody responses that bound and neutralized PA.;The heavy chain isotype of antibody directed against PA is in general dominated by the IgG1 isotype. Since IgG2a is effective in viral infections, this phenomenon was examined more rigorously by generating IgG2a and IgG2b switch variants from the protective IgG1 mAb 19D9 and comparing isotype advantages in toxin neutralization. We explored the role of Fc receptors (FcRs) in toxin protection by infecting macrophages deleted for the common gamma chain of FcRs. The IgG2a isotype-switch variant showed isotype differences in the protective efficacy of the mAbs, but no measurable differences were revealed in fine specificity. Both IgG1 and IgG2a mAbs protected wild-type macrophages from lethal toxin mediated cytotoxicity at various levels, but failed to protect FcRgamma-/- macrophages. This observation clearly demonstrates that antibody protection against anthrax toxin is dependent on the isotype of the antibody and therefore is modulated by Fc-dependent functions.;Taken together, our results are relevant to the development of an anthrax vaccine and anti-PA passive antibody therapies. The finding that the epitope specificity did not determine the nature of the FcR interaction and outcome on toxin activity suggests a novel approach for the treatment of anthrax. (Abstract shortened by UMI.).
dc.publisherProQuest Dissertations & Theses
dc.titleAnthrax vaccine development by epitope targeting and antibody isotype

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