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 provide protection through postexposure 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 serum levels of in vitro toxin neutralization capacity. Using solid-phase epitope mapping and confirmatory assays, we identified several neutralization-associated humoral epitopes and demonstrated that select antipeptide 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.

Figure 1. AVA vaccination results in high titers of antibodies to PA

The endpoint titer or last dilution at which a sample was positive for antibodies to PA (a). Each symbol represents one participant and the line shows the mean titer (1:100) of all individuals. The demographics of the participants at the indicated endpoint titer for anti-protective antigen antibodies are listed (b).

Figure 2. Over 50% of AVA vaccinated individuals fail to generate antibodies with detectable in vitro toxin neutralization activity

The percent viability of macrophages in response to lethal toxin (LT) neutralization by sera from all vaccinated and non-vaccinated individuals is shown (a). Each symbol represents the average viability from three independent experiments using sera from each individual and the horizontal line indicates the overall median viability. The number and percent of individuals who were considered high, medium, low, or very low responders are shown (b). The percent viability of macrophages in response to LT neutralization is shown for each individual and is grouped based upon the number of years post vaccination for each participant (c). Each symbol represents the average viability from three independent experiments for each individual and the horizontal lines indicate the overall median viability of macrophages for each group.

Figure 3. In vitro LT neutralization correlates with anti-PA titer and the number of years post vaccination

Antibody neutralization of the toxin was correlated with PA titer (p<0.0001; a), number of vaccinations (p<0.0001; b), and number of years post last vaccination (p<0.0001; c), but not with age (p = 0.22; d) as determined by using a Spearman' correlation coefficient. Each symbol represents one individual.

Figure 4. Thirteen common antigenic regions, spanning all four domains of PA, were identified using solid phase epitope mapping

Sera were used in a standard solid-phase peptide assay and compared to sera from non-vaccinated controls. Shown are serum antibody responses from individuals with high neutralization (n=6; a), moderate neutralization (n=12; b), and low neutralization (n=12; c). The locations of the indicated antigenic regions mapped onto the crystal structure of PA are shown (d). The table shows the amino acid number, sequence, and functional domain for each epitope.

Figure 5. Peptide-specific antibodies mediate protection against in vitro and in vivo LT challenge

Plasma samples from AVA-vaccinated participants with high titers of peptide-specific antibodies were passaged and eluted six times over the corresponding peptide-specific column. Following column-enrichment and –depletion, the samples were used in an in vitro neutralization assay to determine the neutralization activity of the specific peptides. Mean neutralization (±SD) values are shown for 4 individuals in each group (a). Six-week-old A/J mice (n= 5 mice/group) were injected intraperitoneally with 30 μg of peptide-specific antibodies and then were challenged three hours later with 3 × LD₅₀ of LT. Control mice received either saline or IgG purified from the serum of a non-vaccinated control. Shown is the combined percent survival of the mice from two independent experiments (b).