A major difference between two currently licensed anthrax vaccines is presence (United Kingdom Anthrax Vaccine Precipitated, AVP) or absence (United States Anthrax Vaccine Adsorbed, AVA) of quantifiable amounts of the Lethal Toxin (LT) component Lethal Factor (LF). The primary immunogen in both vaccine formulations is Protective Antigen (PA), and LT-neutralizing antibodies directed to PA are an accepted correlate of vaccine efficacy; however, vaccination studies in animal models have demonstrated that LF antibodies can be protective. In this report we compared humoral immune responses in cohorts of AVP (n=39) and AVA recipients (n=78) matched 1:2 for number of vaccinations and time post-vaccination, and evaluated whether the LF response contributes to LT neutralization in human recipients of AVP. PA response rates (≥95%) and PA IgG concentrations were similar in both groups; however, AVP recipients exhibited higher LT neutralization ED50 values (AVP: 1464.0 ± 214.7, AVA: 544.9 ± 83.2, p<0.0001) and had higher rates of LF IgG positivity (95%) compared to matched AVA vaccinees (1%). Multiple regression analysis revealed that LF IgG makes an independent and additive contribution to the LT neutralization response in the AVP group. Affinity purified LF antibodies from two independent AVP recipients neutralized LT and bound to LF Domain 1, confirming contribution of LF antibodies to LT neutralization. This study documents the benefit of including an LF component to PA-based anthrax vaccines.

Understanding immune responses to native antigens in response to natural infections can lead to improved approaches to vaccination. This study sought to characterize the humoral immune response to anthrax toxin components, capsule and spore antigens in individuals (n = 46) from the Kayseri and Malatya regions of Turkey who had recovered from mild or severe forms of cutaneous anthrax infection, compared to regional healthy controls (n = 20). IgG antibodies to each toxin component, the poly-γ-D-glutamic acid capsule, the Bacillus collagen-like protein of anthracis (BclA) spore antigen, and the spore carbohydrate anthrose, were detected in the cases, with anthrax toxin neutralization and responses to Protective Antigen (PA) and Lethal Factor (LF) being higher following severe forms of the disease. Significant correlative relationships among responses to PA, LF, Edema Factor (EF) and capsule were observed among the cases. Though some regional control sera exhibited binding to a subset of the tested antigens, these samples did not neutralize anthrax toxins and lacked correlative relationships among antigen binding specificities observed in the cases. Comparison of serum binding to overlapping decapeptides covering the entire length of PA, LF and EF proteins in 26 cases compared to 8 regional controls revealed that anthrax toxin-neutralizing antibody responses elicited following natural cutaneous anthrax infection are directed to conformational epitopes. These studies support the concept of vaccination approaches that preserve conformational epitopes.

Edema toxin (ET), composed of edema factor (EF) and protective antigen (PA), is a virulence factor of Bacillus anthracis that alters host immune cell function and contributes to anthrax disease. Anthrax vaccine precipitated (AVP) contains low but detectable levels of EF and can elicit EF-specific antibodies in human recipients of AVP. Active and passive vaccination of mice with EF can contribute to protection from challenge with Bacillus anthracis spores or ET. This study compared humoral responses to ET in recipients of AVP (n ϭ 33) versus anthrax vaccine adsorbed (AVA; n ϭ 66), matched for number of vaccinations and time postvaccination, and further determined whether EF antibodies elicited by AVP contribute to ET neutralization. AVP induced higher incidence (77.8%) and titer (229.8 Ϯ 58.6) of EF antibodies than AVA (4.2% and 7.8 Ϯ 8.3, respectively), reflecting the reported low but detectable presence of EF in AVP. In contrast, PA IgG levels and ET neutralization measured using a luciferase-based cyclic AMP reporter assay were robust and did not differ between the two vaccine groups. Multiple regression analysis failed to detect an independent contribution of EF antibodies to ET neutralization in AVP recipients; however, EF antibodies purified from AVP sera neutralized ET. Serum samples from at least half of EF IgG-positive AVP recipients bound to nine decapeptides located in EF domains II and III. Although PA antibodies are primarily responsible for ET neutralization in recipients of AVP, increased amounts of an EF component should be investigated for the capacity to enhance next-generation, PA-based vaccines.