Mechanistic Analysis of the Effect of Deamidation on the Immunogenicity of Anthrax Protective Antigen

Abstract

The spontaneous modification of proteins, such as deamidation of asparagine residues, can significantly affect the immunogenicity of protein-based vaccines. Using a "genetically deamidated" form of recombinant protective antigen (rPA), we have previously shown that deamidation can decrease the immunogenicity of rPA, the primary component of new-generation anthrax vaccines. In this study, we investigated the biochemical and immunological mechanisms by which deamidation of rPA might decrease the immunogenicity of the protein. We found that loss of the immunogenicity of rPA vaccine was independent of the presence of adjuvant. We assessed the effect of deamidation on the immunodominant neutralizing B-cell epitopes of rPA and found that these epitopes were not significantly affected by deamidation. In order to assess the effect of deamidation on T-cell help for antibody production elicited by rPA vaccine, we examined the ability of the wild-type and genetically deamidated forms of rPA to serve as hapten carriers. We found that when wild-type and genetically deamidated rPA were modified to similar extents with 2,4-dinitrophenyl hapten (DNP) and then used to immunize mice, higher levels of anti-DNP antibodies were elicited by wild-type DNP-rPA than those elicited by the genetically deamidated DNP-rPA, indicating that wild-type rPA elicits more T-cell help than the genetically deamidated form of the protein. These results suggest that a decrease in the ability of deamidated rPA to elicit T-cell help for antibody production is a possible contributor to its lower immunogenicity. Inhalation anthrax is a serious, often fatal, disease caused by Bacillus anthracis. Because of the fatal nature of the disease and the ease of dispersion of B. anthracis spores, B. anthracis is one of the most feared of all bioterror weapons. Due to the low incidence of natural anthrax disease in humans, anthrax vaccines are not given routinely. However, the potential for the use of B. anthracis as a bioterror agent has prompted stockpiling of anthrax vaccines by national governments. Long-term stability is a highly desired characteristic of a stockpiled vaccine since a long shelf life significantly decreases the cost of the stockpile.Many anthrax vaccines are based on protective antigen (PA), which is a nontoxic component of anthrax toxin. Anthrax toxin is a critical virulence factor of B. anthracis and is essential for disease symptomatology and progression (1, 2). The toxin is composed of PA, lethal factor (LF), and edema factor (EF). PA binds to cell receptors, heptamerizes, and then binds LF or EF to form lethal toxin (LT) and edema toxin (ET), respectively (3). Internalization of LT and ET leads to introduction of the enzymatically active effector proteins LF and EF into the cell cytosol where they exert their cytotoxicity (2). Vaccines based on PA or a recombinant form of PA (rPA) elicit antibodies, in particular functional toxinneutralizing antibodies (TNAs), that have been correlated with protection against the disease (4-6).Unfortunately...

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Binding of the von Willebrand Factor A Domain of Capillary Morphogenesis Protein 2 to Anthrax Protective Antigen Vaccine Reduces Immunogenicity in Mice

“…These results may also suggest a role for receptors on APCs in the presentation of intact surface-localized PA to B cells. With regard to stability, deamidation of asparagine residues in PA was associated with decreased potency, reduced levels of TNAs, and impaired binding of PA to cells (43).…”

Structural Characterization and Physicochemical Stability Profile of a Double Mutant Heat Labile Toxin Protein Based Adjuvant

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