Use of Site-Directed Mutagenesis To Model the Effects of Spontaneous Deamidation on the Immunogenicity of Bacillus anthracis Protective Antigen

Abstract

c Long-term stability is a desired characteristic of vaccines, especially anthrax vaccines, which must be stockpiled for large-scale use in an emergency situation; however, spontaneous deamidation of purified vaccine antigens has the potential to adversely affect vaccine immunogenicity over time. In order to explore whether spontaneous deamidation of recombinant protective antigen (rPA)-the major component of new-generation anthrax vaccines-affects vaccine immunogenicity, we created a "genetically deamidated" form of rPA using site-directed mutagenesis to replace six deamidation-prone asparagine residues, at positions 408, 466, 537, 601, 713, and 719, with either aspartate, glutamine, or alanine residues. We found that the structure of the six-Asp mutant rPA was not significantly altered relative to that of the wild-type protein as assessed by circular dichroism (CD) spectroscopy and biological activity. In contrast, immunogenicity of aluminum-adjuvanted six-Asp mutant rPA, as measured by induction of toxin-neutralizing antibodies, was significantly lower than that of the corresponding wild-type rPA vaccine formulation. The six-Gln and six-Ala mutants also exhibited lower immunogenicity than the wild type. While the wild-type rPA vaccine formulation exhibited a high level of immunogenicity initially, its immunogenicity declined significantly upon storage at 25°C for 4 weeks. In contrast, the immunogenicity of the six-Asp mutant rPA vaccine formulation was low initially but did not change significantly upon storage. Taken together, results from this study suggest that spontaneous deamidation of asparagine residues predicted to occur during storage of rPA vaccines would adversely affect vaccine immunogenicity and therefore the storage life of vaccines.

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

“…The purified rPA proteins obtained using this procedure were approximately 95 to 99% pure as determined by SDS-PAGE. We have previously shown that wild-type rPA exhibits only minor amounts of deamidation (8).…”

“…The residues that were changed were Asn 408 , Asn 466 , Asn 537 , Asn 601 , Asn 713 , and Asn 719 . We have used this "genetically deamidated" form of the protein as a model for the naturally deamidated form of the protein that would be expected to result upon prolonged vaccine storage (8). In that previous study, we demonstrated that six-Asp mutant rPA possesses lower immunogenicity than wild-type rPA.…”

“…Deamidation of Asn occurs in vivo on a time scale ranging from a few seconds to centuries, depending on the local environment (10). In theory, deamidation might adversely affect the interaction of rPA with the aluminum adjuvant of the vaccine, might alter the epitope structure of the antigen, and/or might affect the ability of the protein to elicit T-cell help for antibody production which, in turn, might affect the vaccine immunogenicity (11)(12)(13)(14)(15).Earlier studies have demonstrated that certain Asn residues of rPA deamidate during the purification process and upon storage (8,16,17). Previously, we generated a mutant form of rPA in which the six Asn residues that are the most prone to deamidation were changed to Asp residues (six-Asp mutant rPA).…”

“…Earlier studies have demonstrated that certain Asn residues of rPA deamidate during the purification process and upon storage (8,16,17). Previously, we generated a mutant form of rPA in which the six Asn residues that are the most prone to deamidation were changed to Asp residues (six-Asp mutant rPA).…”

“…Unfortunately, progress to develop new-generation rPA-based anthrax vaccines has been hampered by vaccine instability (7), which is believed to be due, at least in part, to spontaneous deamidation of the protein upon long-term storage (8,9). Deamidation results in posttranslational conversion of asparagine (Asn) to aspartate (Asp) or isoaspartic acid (isoAsp).…”