title: Temperature-mediated recombinant anthrax protective antigen aggregate development: Implications for toxin formation and immunogenicity.
abstract: Anthrax vaccines containing recombinant PA (rPA) as the only antigen face a stability issue: rPA forms aggregates in solution after exposure to temperatures ⩾40°C, thus losing its ability to form lethal toxin (LeTx) with Lethal Factor. To study rPA aggregation's impact on immune response, we subjected rPA to several time and temperature combinations. rPA treated at 50°C for 30min formed high mass aggregates when analyzed by gel electrophoresis and failed to form LeTx as measured by a macrophage lysis assay (MLA). Aggregated rPA-formed LeTx was about 30 times less active than LeTx containing native rPA. Mice immunized with heat-treated rPA combined with Al(OH)3 developed antibody titers about 49 times lower than mice immunized with native rPA, as measured by a Toxicity Neutralization Assay (TNA). Enzyme Linked Immunosorbent Assay (ELISA) of the same immune sera showed anti-rPA titers only 2-7 times lower than titers elicited by native rPA. Thus, rPA's ability to form LeTx correlates with its production of neutralizing antibodies, and aggregation significantly impairs the protein's antibody response. However, while these findings suggest MLA has some value as an in-process quality test for rPA in new anthrax vaccines, they also confirm the superiority of TNA for use in vaccine potency.
authors.affiliation: Center for Biologics Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, MD 20993, United States. Electronic address: jc.amador@itesm.mx.
authors.forename: Juan C
authors.initials: JC
authors.lastname: Amador-Molina
authors.affiliation: Center for Biologics Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, MD 20993, United States.
authors.forename: Esther D
authors.initials: ED
authors.lastname: Valerdi-Madrigal
authors.affiliation: Center for Biologics Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, MD 20993, United States.
authors.forename: Rocío I
authors.initials: RI
authors.lastname: Domínguez-Castillo
authors.affiliation: Center for Biologics Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, MD 20993, United States.
authors.forename: Lev A
authors.initials: LA
authors.lastname: Sirota
authors.affiliation: Center for Biologics Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, MD 20993, United States.
authors.forename: Juan L
authors.initials: JL
authors.lastname: Arciniega
mesh_terms: D000818:Animals; D022122:Anthrax Vaccines / Q000737:chemistry; D000907:Antibodies, Bacterial / Q000097:blood; D057134:Antibodies, Neutralizing / Q000097:blood; D000942:Antigens, Bacterial / Q000737:chemistry*; D000992:Antitoxins / Q000097:blood; D001427:Bacterial Toxins / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006358:Hot Temperature; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D009500:Neutralization Tests; D066329:Protein Aggregates; D055550:Protein Stability; D000067996:RAW 264.7 Cells; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D022122:Anthrax Vaccines; D000907:Antibodies, Bacterial; D057134:Antibodies, Neutralizing; D000942:Antigens, Bacterial; D000992:Antitoxins; D001427:Bacterial Toxins; D066329:Protein Aggregates; D011994:Recombinant Proteins; C030325:anthrax toxin
keywords: Aggregation; Anthrax; Protective antigen; Stability; Vaccine