Neutralizing Activity of Vaccine-Induced Antibodies to TwoBacillus anthracisToxin Components, Lethal Factor and Edema Factor

Abstract

Anthrax vaccine adsorbed (AVA; BioThrax), the current FDA-licensed human anthrax vaccine, contains various amounts of the three anthrax toxin components, protective antigen (PA), lethal factor (LF), and edema factor (EF). While antibody to PA is sufficient to mediate protection against anthrax in animal models, it is not known if antibodies to LF or EF contribute to protection in humans. Toxin-neutralizing activity was evaluated in sera from AVA-vaccinated volunteers, all of whom had antibody responses to LF and EF, as well as PA. The contribution of antibodies to LF and EF was assessed using mouse macrophage J774A.1 cells by examining neutralization of LF-induced lysis using alamarBlue reduction and neutralization of EF-induced cyclic AMP increases by enzyme-linked immunosorbent assay. Antibody responses to LF and EF were low compared to those to PA, and the amount of LF or EF in the assay could exceed the amount of antibodies to LF or EF. Higher titers were seen for most individuals when the LF or EF concentration was limiting compared to when LF or EF was in excess, initially suggesting that antibody to LF or EF augmented protection. However, depletion of LF and EF antibodies in sera did not result in a significant decrease in toxin neutralization. Overall, this study suggests that AVA-induced LF and EF antibodies do not significantly contribute to anthrax toxin neutralization in humans and that antibodies to PA are sufficient to neutralize toxin activity.

Neutralizing Monoclonal Antibody to Edema Toxin and Its Effect on Murine Anthrax

“…This, in turn, suggests that vaccines that also elicit neutralizing Abs to EF may be more effective than those focused only on eliciting Abs to PA. Unfortunately, our results showing that most of the MAbs generated were not neutralizing, combined with similar findings in a prior report (19) and the observation that depletion of EF-binding Abs from human immune sera does not significantly affect neutralizing activity (40), suggest that neutralizing Abs to EF may be relatively uncommon. However, epitope mapping of protective and nonprotective epitope domains in EF using MAbs could result in the identification of antigenic sites that elicit useful Abs and the incorporation of such domains in future vaccines.…”

The Major Neutralizing Antibody Responses to Recombinant Anthrax Lethal and Edema Factors Are Directed to Non-Cross-Reactive Epitopes

“…RAW 264.7 mouse macrophages were grown in Dulbecco's modified Eagle's medium containing 4 mM L-glutamine (ATCC) supplemented with 10% fetal calf serum at 37°C in a 5% CO 2 -95% air atmosphere. To determine toxin neutralization, we utilized a technique adapted from Taft and Weiss (33). RAW 264.7 cells were harvested to 2 ϫ 10 6 cells/ml, and 100 l was added to a 96-well flat-bottomed tissue culture plate for 2 h. Serum dilutions were then prepared similarly to those for the in vitro LeTx neutralization assay, starting at 1:50 and ending at 1:1,600, and incubated with EdTx (composed of a 3:1 PA-to-EF ratio [75 ng/well PA and 25 ng/well EF; final well volume ϭ 100 l]) for 1 h at RT.…”

A Three-Dose Intramuscular Injection Schedule of Anthrax Vaccine Adsorbed Generates Sustained Humoral and Cellular Immune Responses to Protective Antigen and Provides Long-Term Protection against Inhalation Anthrax in Rhesus Macaques

“…The primary immunogen in AVA is anthrax toxin protective antigen (PA). Anti-PA IgG antibodies are considered to protect against anthrax by neutralizing the toxin, inhibiting spore germination, and enhancing phagocytosis and killing of spores by macrophages (1,11,12,16,39,42,49,50,55,56).…”