Abstract
Anthrax vaccine adsorbed (AVA) containing protective antigen (PA) is the only FDA-approved anthrax vaccine in the United States. Characterization of the binding of AVA-induced anti-PA human antibodies against the PA antigen after vaccination is crucial to understanding mechanisms of the AVA-elicited humoral immune response. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is often coupled with a short liquid chromatography gradient (e.g., 5–10 min) for the characterization of protein interactions. We recently developed a long-gradient (e.g., 90 min), sub-zero temperature, ultra-high performance liquid chromatography HDX-MS (UPLC-HDX-MS) platform that has significantly increased separation power and limited back-exchange for the analysis of protein samples with high complexity. In this study, we demonstrated the utility of this platform for mapping antibody–antigen epitopes by examining four fully human monoclonal antibodies to anthrax PA. Antibody p1C03, with limited neutralizing activity in vivo, bound to a region on domain 1A of PA. p6C04 and p1A06, with no neutralizing activities, bound to the same helix on domain 3 to prevent oligomerization of PA. We found p6C01 strongly bound to domain 3 on a different helix region. We also identified a secondary epitope for p6C01, which likely leads to the blocking of furin cleavage of PA after p6C01 binding. This novel binding of p6C01 results in highly neutralizing activity. This is the first report of this distinct binding mechanism for a highly neutralizing fully human antibody to anthrax protective antigen. Studying such epitopes can facilitate the development of novel therapeutics against anthrax.
Highlights
IntroductionBacillus anthracis has long been recognized as a potential bioterrorist threat due to the transmissibility of its spores and the high mortality rate from inhalational infection
Unlike the short liquid chromatography gradient times (e.g., 5–10 min) typically used in the routine Hydrogen deuterium exchange mass spectrometry (HDX-MS) studies [13,14,15,16,17], we have recently developed a subzero-temperature, long gradient, ultrahigh pressure liquid chromatography system located in a low-cost refrigerator with significantly improved separation power while limiting back-exchange for HDX-MS to separate deuterated protein fragments with high complexity [18]
Only a few of the antibodies were able to efficiently neutralize lethal toxin, measured in both a standard in vitro lethal toxin neutralization assay and in vivo mouse model, where A/J mice were challenged with lethal toxin after administration of antibody
Summary
Bacillus anthracis has long been recognized as a potential bioterrorist threat due to the transmissibility of its spores and the high mortality rate from inhalational infection. Such fears were realized in 2001, when spores circulated by a bioterrorist through the. Lethal toxin and edema toxin, via a tripartite system of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). Both LF and EF require PA to gain entry into the cell before intoxication; PA is the target of immunization and passive immunotherapies for anthrax infection [2]
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