TY - JOUR
T1 - Use of site-directed mutagenesis to model the effects of spontaneous deamidation on the immunogenicity of Bacillus anthracis protective antigen
AU - Verma, Anita
AU - McNichol, Beth
AU - Domínguez-Castillo, Rocío I.
AU - Amador-Molina, Juan C.
AU - Arciniega, Juan L.
AU - Reiter, Karine
AU - Meade, Bruce D.
AU - Ngundi, Miriam M.
AU - Stibitz, Scott
AU - Burns, Drusilla L.
PY - 2013/1
Y1 - 2013/1
N2 - Long-term stability is a desired characteristic of vaccines, especially anthrax vaccines, which must be stockpiled forlarge-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 mutantrPA, 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.
AB - Long-term stability is a desired characteristic of vaccines, especially anthrax vaccines, which must be stockpiled forlarge-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 mutantrPA, 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.
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U2 - 10.1128/IAI.00863-12
DO - 10.1128/IAI.00863-12
M3 - Article
C2 - 23115046
AN - SCOPUS:84871865748
SN - 0019-9567
VL - 81
SP - 278
EP - 284
JO - Infection and Immunity
JF - Infection and Immunity
IS - 1
ER -