Lethal factor antibodies contribute to lethal toxin neutralization in recipients of anthrax vaccine precipitated

Abstract

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.

Keywords: Anthrax; Anthrax vaccine adsorbed; Anthrax vaccine precipitated; Bacillus anthracis; Lethal factor; Lethal toxin; Neutralization; Protective antigen.

Fig. 1. AVP vaccinees produce robust IgG responses to Lethal Factor and have higher levels of Lethal Toxin neutralization compared to matched AVA vaccinees

A) End-point titers of serum IgG to recombinant Lethal Factor (LF) from AVP (n=39) compared to matched AVA (n=78) vaccinees. Two AVA data points are below the axis and not shown. B) Levels of serum IgG to recombinant Protective Antigen (PA) in the same samples shown in A) and C). C) 50% effective dilution (ED₅₀) values for serum neutralization of Lethal Toxin determined in a J774A.1 macrophage-based Lethal Toxin neutralization assay. Samples are the same as those shown in A) and B). Red lines show mean ± SEM for all panels. p-values determined by unmatched t-tests.

Fig. 2. Correlation of Lethal Factor (LF) and Protective Antigen (PA) IgG levels with Lethal Toxin (LT) neutralization values in AVA and AVP vaccines

A) Linear regression showing correlation between serum LF titer and LT neutralization ED₅₀ values in AVP but not matched AVA samples. B) Linear regression showing correlation between serum PA IgG levels and LT neutralization ED₅₀ values in both AVP and matched AVA sample groups.

Fig. 3. Purified Lethal Factor (LF) antibodies from AVP serum samples neutralize Lethal Toxin (LT)

A) End-point IgG titers of reactivity to Protective Antigen (PA) or LF in samples of purified LF antibodies from four independent AVP vaccinees before (pre) and after (post) purification. Post-purification samples were brought to the starting pre-purification volumes. B) Example of results showing the capacity of affinity purified LF antibodies from AVP Sample 4 to neutralize LT-mediated killing of the J774a macrophage cell line (blue bars). A neutralizing PA- specific human monoclonal antibody is included as a positive control (gray bars). Black and red bars are from the control conditions indicated. C) Post-purification LT ED₅₀, LF titer and PA titer of all four affinity purified LF antibody samples from four independent AVP vaccinees. Two samples with LF titers of at least 1:320 neutralized LT in vitro.

Fig. 4. Purified Lethal Factor (LF) antibodies with Lethal Toxin (LT)-neutralizing activity bind to LF domain 4

A) LF domain preparations separated by SDS polyacrylamide gel electrophoresis and stained with Coomassie blue. B) Average binding of serum IgG from 39 AVP and 39 unvaccinated individuals to each LF domain by ELISA. Error bars are SEM. C) Reactivity to the LF domains before and after LF purification for AVP samples 1 and 4 by ELISA. Optical density thresholds for positivity to each domain as determined by the mean + 2 SD above the mean of values from 54 unvaccinated controls for LF1, LF2a/3, LF 2b and LF4 were 0.228, 0.150, 0.534 and 0.299, respectively.