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Domain specificity of the human antibody response to Bacillus anthracis protective antigen.

ABSTRACT: Protective antigen (PA) is the cell surface recognition moiety of the Bacillus anthracis A-B toxin system, and the active immunogenic component in the currently licensed human anthrax vaccine (BioThrax, or AVA). The serum antibody response to the PA protein is polyclonal and complex both in terms of the antibody combining sites utilized to bind PA and the PA-associated epitopes recognized. We have cloned, sequenced, and expressed a large panel of PA-specific human monoclonal antibodies from seven AVA-immunized donors. Dot blots, Western blots, and radiolabeled antigen capture assays employing both proteolytic fragments of PA and engineered PA sub-domain fusion proteins were used to determine the region (domain) of the PA monomer to which each of the cloned human antibodies bound. The domain specificity of the isolated monoclonals was highly biased towards the amino-terminal 20kDa fragment of PA (PA(20)), with the majority (62%) of independently arising antibody clones reacting with determinants located on this PA fragment. A similar bias in domain specificity was also demonstrated in the serum response of AVA-vaccinated donors. Since PA(20) is cleaved from the remainder of the monomer rapidly following cell surface binding and has no known role in the intoxication process, the immunodominance of PA(20)-associated epitopes may directly affect the efficacy of PA-based anthrax vaccines.

SUBMITTER: Reason DC

PROVIDER: S-EPMC2536639 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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Domain specificity of the human antibody response to Bacillus anthracis protective antigen.

Reason Donald C DC Ullal Anuska A Liberato Justine J Sun Jinying J Keitel Wendy W Zhou Jianhui J

Vaccine 20080602 32

Protective antigen (PA) is the cell surface recognition moiety of the Bacillus anthracis A-B toxin system, and the active immunogenic component in the currently licensed human anthrax vaccine (BioThrax, or AVA). The serum antibody response to the PA protein is polyclonal and complex both in terms of the antibody combining sites utilized to bind PA and the PA-associated epitopes recognized. We have cloned, sequenced, and expressed a large panel of PA-specific human monoclonal antibodies from seve ...[more]

PMID: 18565627

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Project description:BackgroundAnthrax is caused by Bacillus anthracis that produce two exotoxins, lethal toxin and edema toxin. The lethal toxin is composed of the lethal factor (LF) complexed with the cell binding protective antigen (PA83, 83 kDa). Likewise, the edema factor (EF) binds to the PA83 to form the edema toxin. Once PA83 is bound to the host cell surface, a furin-like protease cleaves the full-length, inactive protein into 63 kDa and 20 kDa antigens (PA63 and PA20). PA63 forms a heptamer and is internalized via receptor mediated endocytosis forming a protease-stable pore, which allows EF and LF to enter the cell and exert their toxic effects.Both proteolytically cleaved protective antigens (PA63 and PA20 fragments) are found in the blood of infected animals. The 63 kDa protective antigen PA63 fragment has been thoroughly studied while little is known about the PA20.MethodsIn this study we examined the role of PA20 using high throughput gene expression analysis of human peripheral blood mononuclear cells (PBMC) exposed to the PA20. We constructed a PA mutant in which a Factor Xa proteolytic recognition site was genetically engineered into the protective antigen PA83 to obtain PA20 using limited digestion of this recombinant PA83 with trypsin.ResultsGlobal gene expression response studies indicated modulation of various immune functions and showed gene patterns indicative of apoptosis via the Fas pathway in a subset of the lymphoid cells. This finding was extended to include observations of increased Caspase-3 enzymatic activity and the identification of increases in the population of apoptotic, but not necrotic cells, based on differential staining methods. We identified a list of approximately 40 inflammatory mediators and heat-shock proteins that were altered similarly upon exposure of PBMC to either rPA20 or B. anthracis spores/vegetative cells.ConclusionThis study shows that the PA20 has an effect on human peripheral blood leukocytes and can induce apoptosis in the absence of other PA components.

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Domain specificity of the human antibody response to Bacillus anthracis protective antigen.

Publications

Domain specificity of the human antibody response to Bacillus anthracis protective antigen.

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