Rapid Deamidation of Recombinant Protective Antigen when Adsorbed on Aluminum Hydroxide Gel Correlates with Reduced Potency of Vaccine

D'Souza, Ajit Joseph M.; Mar, Kevin D.; Huang, Joanne; Majumdar, Sumit; Ford, Brandi M.; Dyas, Beverly; Ulrich, Robert G.; Sullivan, Vincent J.

doi:10.1002/jps.23422

Cited by 36 publications

(33 citation statements)

References 26 publications

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“…While a standard ensemble of release and stability tests applies to rPA for insuring consistency in purity, strength, and quality, cumulative evidence has indicated that deamidation is the principle stability issue confronting rPA as API and formulated DP (Verma et al 2013;D'Souza et al 2013;Powell et al 2007;Ribot et al 2006;Zomber et al 2005). Figure 6.27 illustrates correlation between the progression of charged isoforms, loss of biological activity, and increased deamidation at specific sites ).…”

Section: Stabilitymentioning

confidence: 95%

“…While anion exchange chromatography (IEX) has been used to isolate isoforms for structural and functional study (Ribot et al 2006), isoelectric focusing (IEF) in gel and capillary format (cIEF) have been useful in monitoring rPA charge heterogeneity (Ribot et al 2006;Powell et al 2007;Zomber et al 2005). Peptide mapping and amino acid sequencing by mass spectrometry (LC/MS and LC-MS/MS) enable the direct connection between charge heterogeneity and deamidation, as noted above (Verma et al 2013;D'Souza et al 2013;Powell et al 2007;Zomber et al 2005). Figure 6.2 shows a ribbon structure depiction of rPA and the most labile asparagines ).…”

Section: 0mentioning

confidence: 99%

See 1 more Smart Citation

Analytical Control Strategy of Bacterial Subunit Vaccines

Lee¹,

Turula

Powell³

et al. 2014

Vaccine Analysis: Strategies, Principles, and Control

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Section: Stabilitymentioning

confidence: 95%

Section: 0mentioning

confidence: 99%

Analytical Control Strategy of Bacterial Subunit Vaccines

Lee¹,

Turula

Powell³

et al. 2014

Vaccine Analysis: Strategies, Principles, and Control

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“…In that previous study, we demonstrated that six-Asp mutant rPA possesses lower immunogenicity than wild-type rPA. Recently, D'Souza et al (9) confirmed that rPA adsorbed to Alhydrogel rapidly deamidates and further confirmed that this deamidation resulted in the loss of immunogenicity. Taken together, these results provide substantial support for the idea that the loss of stability of rPA vaccines is due, at least in part, to deamidation.…”

mentioning

confidence: 87%

“…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).…”

mentioning

confidence: 99%

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

Verma

Ngundi

Burns

2016

Clin Vaccine Immunol

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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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“…For example, as is evident in phase 1 clinical trials, rPA vaccines do not address the need for an improved dosing schedule over the current vaccine, AVA, and induce equivalent levels, at best, of toxin neutralizing antibodies. In addition, rPA adsorbed to aluminum hydroxide adjuvant has been shown to lose its ability to induce toxin neutralizing antibodies as it is stored 47,48 . For obvious reasons, long-term stability is an important characteristic of a stock-piled vaccine.…”

Section: Experimental Anthrax Vaccinesmentioning

confidence: 99%

Design and Testing of Novel Anthrax Vaccines Utilizing a Tobacco Mosaic Virus Expression System

McComb¹

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Rapid Deamidation of Recombinant Protective Antigen when Adsorbed on Aluminum Hydroxide Gel Correlates with Reduced Potency of Vaccine

Cited by 36 publications

References 26 publications

Analytical Control Strategy of Bacterial Subunit Vaccines

Analytical Control Strategy of Bacterial Subunit Vaccines

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

Design and Testing of Novel Anthrax Vaccines Utilizing a Tobacco Mosaic Virus Expression System

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