TY - JOUR
T1 - Temperature-mediated recombinant anthrax protective antigen aggregate development
T2 - Implications for toxin formation and immunogenicity
AU - Amador-Molina, Juan C.
AU - Valerdi-Madrigal, Esther D.
AU - Domínguez-Castillo, Rocío I.
AU - Sirota, Lev A.
AU - Arciniega, Juan L.
N1 - Funding Information:
This project was supported in part by the Biomedical Advanced Research and Development Authority and by an appointment to the Research Participation Program at the Center for Biologics Evaluation and Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration.
Publisher Copyright:
© 2016 Elsevier Ltd
PY - 2016/7/29
Y1 - 2016/7/29
N2 - 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 30 min 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.
AB - 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 30 min 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.
KW - Aggregation
KW - Anthrax
KW - Protective antigen
KW - Stability
KW - Vaccine
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U2 - 10.1016/j.vaccine.2016.06.057
DO - 10.1016/j.vaccine.2016.06.057
M3 - Article
C2 - 27364097
AN - SCOPUS:84977558805
SN - 0264-410X
VL - 34
SP - 4188
EP - 4195
JO - Vaccine
JF - Vaccine
IS - 35
ER -