Select Human Anthrax Protective Antigen Epitope‐Specific Antibodies Provide Protection from Lethal Toxin Challenge

Abstract

Bacillus anthracis remains a serious bioterrorism concern, and the currently licensed vaccine remains an incomplete solution for population protection from inhalation anthrax and has been associated with concerns regarding efficacy and safety. Thus, understanding how to generate long lasting protective immunity with reduced immunizations or providing protection through post exposure immunotherapeutics are long sought goals. Through evaluation of a large military cohort, we characterized the levels of antibodies against protective antigen and found that over half of anthrax vaccinees had low levels of in vitro toxin neutralization capacity in their sera. Using solid phase epitope mapping and confirmatory assays, we identified several neutralization-associated humoral epitopes and demonstrated that select anti-peptide responses mediated protection in vitro. Finally, passively transferred antibodies specific for select epitopes provided protection in an in vivo lethal toxin mouse model. Identification of these antigenic regions has important implications for vaccine design and the development of directed immunotherapeutics.

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

“…Cysteine substitutions at any of these 4 amino acids resulted in the inhibition of PA activity by at least 100-fold 64 . Additionally, antibodies directed against the epitope P232 to V247, which contain two of these residues, were found in 4 of 6 serum samples from AVA vaccinated human donors with high neutralizing serum titers 78 . Affinity purified antibodies directed against P232 to V247 (designated as PA6 in this study) were tested against LT and shown to provide 50% protection of RAW 264.7 cells.…”

“…78 . This shows that antibodies directed against this region contribute to neutralization, but it is unknown whether they are sufficient on their own for providing protection against anthrax toxin.…”

“…Since the mice were not challenged with toxin, however, it is impossible to know if the antibodies induced by vaccination were sufficient for in vivo protection. In another study, human antibodies directed against the sequence between N162 and N180 of PA (designated as site PA4 in this study) were affinity purified from AVA vaccinated donors and tested for their ability to neutralize LT in vitro and in vivo 78 . This study showed that furin cleavage site directed antibodies protected 70% of RAW 264.7 macrophages after treatment with LT but only 1 of 10 A/J mice survived challenged with 3 x LD50 LT after treatment with 30 μg of affinity purified PA4 specific antibodies 78 .…”

“…In another study, human antibodies directed against the sequence between N162 and N180 of PA (designated as site PA4 in this study) were affinity purified from AVA vaccinated donors and tested for their ability to neutralize LT in vitro and in vivo 78 . This study showed that furin cleavage site directed antibodies protected 70% of RAW 264.7 macrophages after treatment with LT but only 1 of 10 A/J mice survived challenged with 3 x LD50 LT after treatment with 30 μg of affinity purified PA4 specific antibodies 78 . It appears that, although furin cleavage is an attractive target for focused therapies from a functional standpoint, there are limited levels of protection that can be achieved with antibodies directed against this epitope.…”

“…Neutralizing titers of 1:800 were observed with from serum of vaccinated animals in a cellular LT cytotoxicity assay but no in vivo survival data was reported. Finally, Crowe and colleagues report that sera from AVA vaccinated human donors reacted with the peptide Y192 to P205 in a solid-phase epitope mapping experiment, but no neutralization assays or toxin survival experiments in animals were performed 78 . These studies show that antibodies targeting the LF/EF binding site of PA are likely critical for toxin neutralization and support including this as a potential vaccine target.…”