Immunogenicity in mice of anthrax recombinant protective antigen in the presence of aluminum adjuvants.

The only US-licensed anthrax vaccine for human use, as well as several experimental vaccines containing solely purified recombinant protective antigen (rPA), are formulated using aluminum hydroxide (Al(OH)3) as an adjuvant. It has been suggested that effective adjuvanticity of aluminum salts for protein antigens depends, at least partially, on the degree of adsorption of the antigen to the adjuvant. On the other hand, the ease of antigen desorption from the adjuvant in a quantitative fashion may facilitate the assessment of vaccine characteristics in the laboratory. In this regard, aluminum phosphate (AlPO4), although deemed a "weaker" adjuvant than Al(OH)3, appears superior to the latter. To investigate the possibility of formulating rPA vaccines with AlPO4, as well as the significance of the adsorption of this antigen to the aluminum salt for adjuvanticity, we studied the effect of AlPO4 and Al(OH)3 on the induction of anti-rPA antibodies in mice. In a first immunization experiment the adjuvanticity of AlPO4 combined with rPA was examined. Antibodies against rPA were measured using an ELISA. Results indicated that AlPO4 is able to significantly increase the antibody response to rPA, irrespective of its degree of adsorption to the adjuvant. Based on these results, in a second experiment mice were immunized twice, with different formulations of rPA containing either AlPO4 or Al(OH)3, and rPA-antibodies were measured using ELISA and an in vitro toxin neutralization assay. Comparable immune responses to rPA were obtained with both aluminum salts. Additionally, results with AlPO4 as adjuvant confirmed that, in this mouse model, binding of the protein to the adjuvant is not essential for adjuvanticity, whereas the amount of adjuvant has an influence on the antibody response induced.

Validation of an anti-PA-ELISA for the potency testing of anthrax vaccine in mice.

The potency test for the anthrax vaccine currently licensed for human use in the United States (Anthrax Vaccine Adsorbed) involves the protection of actively immunized guinea pigs from a lethal challenge with a virulent strain of Bacillus anthracis. Lethal challenge tests entail the use of specialized containment facilities for the safe and secure handling of the challenge strain. This potential difficulty, plus humane considerations, have prompted us to investigate non-lethal, alternative immunogenicity assays that could be considered as potency tests not only for the current vaccine, but also for vaccines under development. Immunogenicity tests will require suitable measurement of an antibody response to relevant antigens, by methods such as enzyme linked immunosorbent assay (ELISA) or a toxin neutralization assay. Any assay chosen for this purpose should be adequately validated and reproducible by other laboratories. Validation of an analytical procedure requires the demonstration that the assay is suitable for its intended purpose. The objective of this work was to study the performance of an anti-PA-ELISA designed to assess the antibody response to anthrax vaccines in mice. Validation studies were performed according to the guidelines of the International Conference of Harmonization (ICH), and we have established the working range of the assay (37-1159 EU/mL) on the bases of the following parameters: linearity (20-1159 EU/mL; r2=0.99; p-value=0.21), accuracy (91-118% recovery), precision (< or =20%CV, repeatability; < or =9 and < or =21%CV, intermediate precision per day and per analyst, respectively), detection limit (5 EU/mL), and quantification limit (37 EU/mL). We believe that assay specificity and the above characteristics are adequate to allow this ELISA to be considered for use in a mouse immunogenicity (potency) test of anthrax vaccines, and for the standardization of reagents.