Structural and Immunological Analysis of Anthrax Recombinant Protective Antigen Adsorbed to Aluminum Hydroxide Adjuvant

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>New anthrax vaccines currently under development are based on recombinant protective antigen (rPA) and formulated with aluminum adjuvant. Because long-term stability is a desired characteristic of these vaccines, an understanding of the effects of adsorption to aluminum adjuvants on the structure of rPA is important. Using both biophysical and immunological techniques, we compared the structure and immunogenicity of freshly prepared rPA-Alhydrogel formulations to that of formulations stored for 3 weeks at either room temperature or 37°C in order to assess the changes in rPA structure that might occur upon long-term storage on aluminum adjuvant. Intrinsic fluorescence emission spectra of tryptophan residues indicated that some tertiary structure alterations of rPA occurred during storage on Alhydrogel. Using anti-PA monoclonal antibodies to probe specific regions of the adsorbed rPA molecule, we found that two monoclonal antibodies that recognize epitopes located in domain 1 of PA exhibited greater reactivity to the stored formulations than to freshly prepared formulations. Immunogenicity of rPA-Alhydrogel formulations in mice was assessed by measuring the induction of toxin-neutralizing antibodies, as well as antibodies reactive to 12-mer peptides spanning the length of PA. Mice immunized with freshly prepared formulations developed significantly higher toxin-neutralizing antibody titers than mice immunized with the stored preparations. In contrast, sera from mice immunized with stored preparations exhibited increased reactivity to nine 12-mer peptides corresponding to sequences located throughout the rPA molecule. These results demonstrate that storage of rPA-Alhydrogel formulations can lead to structural alteration of the protein and loss of the ability to elicit toxin-neutralizing antibodies.</jats:p>

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

“…As shown in Fig. 6, the wild-type rPA-Alhydrogel formulation exhibited a significant decrease in immunogenicity over time (P Ͻ 0.05; ANOVA with Tukey posttest for trend) as has been reported previously (28). In contrast, the immunogenicity of the six-Asp mutant rPA-Alhydrogel formulation did not change significantly upon storage.…”

“…This irreversible binding interferes with methodologies that could be used to assess deamidation. Factors other than deamidation, such as structural destabilization of rPA protein upon adsorption to aluminum adjuvant (28), may have contributed to the loss of immunogenicity that we observed with the wild-type rPA formulation. Of note, however, is the observation that after 4 weeks of storage, the wild-type rPA and sixAsp mutant vaccine formulations exhibited similar immunogenicities, consistent with the idea that deamidation of susceptible Asn residues played a role in the loss of immunogenicity seen over time with the wild-type rPA formulation.…”

Aluminum Hydroxide Influences Not Only the Extent but Also the Fine Specificity and Functional Activity of Antibody Responses to Tick-Borne Encephalitis Virus in Mice

“…Indeed, in our experiments the antigen was completely adsorbed immediately after mixing with the adjuvant (data not shown). Previous studies have revealed that the interaction with aluminum hydroxide, which strongly depends on the properties of the antigen, buffer composition, pH, and physicochemical properties of the aluminum preparation used, can affect the structure of protein immunogens (15)(16)(17)(18). Flavivirus particles are probably especially prone to adsorption-related structural effects, since the organization of their E proteins in the viral membrane is subject to dynamic changes (29,30,67), can be fixed in different configurations (e.g., by the interaction with specific antibodies [67]), and can undergo extensive temperaturedependent rearrangements (29,30,67).…”

“…In the case of protein antigens, this interaction can lead to changes in the secondary or tertiary structure and can affect protein stability (15)(16)(17)(18). Since adsorption-induced effects on protein structure can potentially modulate the fine specificities and, consequently, the functional activities of antibodies elicited by immunization, such changes can affect the effectiveness of vaccination.…”

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

“…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.…”