An anthrax lethal factor-neutralizing monoclonal antibody protects rats before and after challenge with anthrax toxin

Abstract

Lethal factor (LF) is a component of anthrax lethal toxin (LeTx). We generated anti-LF murine monoclonal antibodies (MAbs) that show LeTx-neutralizing activity in vitro and in vivo. Anti-LF MAbs were generated by immunization with recombinant LF, and the MAbs showing LeTx-neutralizing activity in vitro were selected. Two MAbs with the highest affinities, 5B13B1 (dissociation constant [K(d)], 2.62 nM) and 3C16C3 (K(d), 8.18 nM), were shown to recognize the same or closely overlapping epitopes on domain III of LF. The 50% inhibitory concentration of 5B13B1 (0.21 microg/ml) was approximately one-third that of 3C16C3 (0.63 microg/ml) in the in vitro LeTx-neutralization assay. The 5B13B1 antibody, which had the highest neutralizing activity, provided perfect protection against LeTx challenge in an in vivo LeTx neutralization assay using Fisher 344 rats. In addition, the antibody showed pre- and postexposure prophylactic effects in the animal experiments. This is the first report that an MAb binding to domain III of LF has neutralizing activity against LeTx. The 5B13B1 antibody may be useful in prophylaxis against anthrax poisoning.

FIG. 1.

LeTx-neutralizing activity of anti-LF MAb. MAb 5B13B1 or 3C16C3 was preincubated with LeTx (0.4 μg/ml PA plus 0.2 μg/ml LF), and the complexes were incubated with J774A.1 cells. The viability of the cells was assessed by an MTT assay. The results are expressed as percentages of viable cells. The IC₅₀s of the antibodies were deduced from the graph.

FIG. 2.

In vitro neutralization of J774A.1 cells by anti-LF MAb 5B13B1. Antibody was administered before or after challenge with LeTx (0.4 μg/ml PA plus 0.2 μg/ml LF) at different times. Anti-GST mouse MAb (0.84 μg/ml) was administered as an isotype control. After 3 h of LeTx challenge, the survival of J774A.1 cells was determined by an MTT assay. The results are expressed as percentages of viable cells.

FIG. 3.

Competitive inhibition of 5B13B1 by 3C16C3. Biotinylated 5B13B1 was competed with increasing concentrations of unlabeled 3C16C3 (•), 5B13B1 (•), anti-LF polyclonal antibody (▪), or a control antibody (anti-GST mouse MAb) (▾).

FIG. 4.

Epitope mapping. (A) Schematic representation of the construction of LF mutants. Mutants L1 (domain I), L2 (domain I, part of domain II, and domain III), L3 (domains I, II, and III), L4 (part of domain II and domains III and IV), L5 (domains I and III), and L6 (domains I, II, and IV) are shown. (B) Slot blot analysis of wild-type LF and six LF mutants using 5B13B1 (lane 1), 3C16C3 (lane 2), or AP1 (lane 3). (C) Analysis of binding of the four peptides (R2, R3, R4, and R5) of domain III to 5B13B1 or 3C16C3. (D) Competitive inhibition of 5B13B1 or 3C16C3 by the R4 peptide. The antibody was incubated with LF in the presence of increasing concentrations of the R4 or R5 peptide, and then an indirect ELISA was done.

FIG. 5.

Protection of rats from LeTx challenge by 5B13B1. LeTx (PA 80 μg plus LF 40 μg) preincubated with 5B13B1 or control antibody (anti-GST MAb) was intravenously administered to each of six Fisher 344 rats weighing 120 to 130 g. The dose of antibody was 42.4 μg/rat (twice the IC₅₀), corresponding to 1.28 molar equivalents of LF. The animals were monitored for 24 h after toxin administration.

FIG. 6.

Protection of rats from LeTx challenge by 5B13B1: postexposure prophylactic effect. LeTx (PA 80 μg plus LF 40 μg) was intravenously administered to four Fisher 344 rats weighing 120 to 130 g. After 5, 15, and 30 min, 5B13B1 or control antibody (anti-GST MAb) was administered. The dose of antibody was 42.4 μg/rat (twice the IC₅₀), corresponding to 1.28 molar equivalents of LF. The animals were monitored for 24 h after toxin administration.

FIG. 7.

Protection of rats from LeTx challenge by 5B13B1: preexposure prophylactic effect. 5B13B1 or control antibody (anti-GST MAb) was intravenously administered to four Fisher 344 rats weighing 120 to 130 g. After 0, 1, 3, or 5 days, LeTx (PA 80 μg plus LF 40 μg) was intravenously administered. The dose of antibody (Ab) was 84.8 μg/rat (four times the IC₅₀), corresponding to 2.56 molar equivalents of LF. The animals were monitored for 24 h after toxin administration.