A monoclonal antibody to Bacillus anthracis protective antigen defines a neutralizing epitope in domain 1

Abstract

Antibody (Ab) responses to Bacillus anthracis toxins are protective, but relatively few protective monoclonal antibodies (MAbs) have been reported. Protective antigen (PA) is essential for the action of B. anthracis lethal toxin (LeTx) and edema toxin. In this study, we generated two MAbs to PA, MAbs 7.5G and 10F4. These MAbs did not compete for binding to PA, consistent with specificities for different epitopes. The MAbs were tested for their ability to protect a monolayer of cultured macrophages against toxin-mediated cytotoxicity. MAb 7.5G, the most-neutralizing MAb, bound to domain 1 of PA and reduced LeTx toxicity in BALB/c mice. Remarkably, MAb 7.5G provided protection without blocking the binding of PA or lethal factor or the formation of the PA heptamer complex. However, MAb 7.5G slowed the proteolytic digestion of PA by furin in vitro, suggesting a potential mechanism for Ab-mediated protection. These observations indicate that some Abs to domain 1 can contribute to host protection.

FIG. 1.

(A) Inverse Ab titers of BALB/c mice immunized with PA₈₃ protein. Mice were immunized with 2 μg (C mice) or 10 μg (D mice) of PA₈₃ protein. Mouse D3 was chosen for hybridoma generation using standard hybridoma technology. (Naïve mice, no responses.) (B) Analysis of cellular toxicity in the presence of PA₈₃ MAbs by the MTT assay. MAbs 7.5G and 10F4 can protect MSH alveolar and J774 macrophage monolayers against LeTx-mediated toxicity as compared to medium alone and LeTx alone. Bars denote the average results for four wells, and error bars (too small to be visible) denote standard deviations. (C) Lung function as measured by WBP in mice treated with MAb before administration of LeTx. MAbs 7.5G and PBS were administered intraperitoneally 24 h prior to LeTx injection. Bars denote the average results for three mice, and error bars indicate standard deviations. TV, tidal volume; Ti, inspiratory time; Te, expiratory time; PIF, peak inspiratory flow; PEF, peak expiratory flow; *, P <0.05.

FIG. 2.

(A) Analysis of Ab binding to PA₆₃ and PA₈₃ by ELISA. MOPC21 IgG1 was used as a negative control for the ELISA. Insert, schematic of ELISA. GAM-AP, alkaline phosphatase-labeled goat anti-mouse Ab reagent. (B) Analysis of Ab binding to expressed PA domains (12.05 μM). MAb 7.5G (upper panel) bound to all expressed domains: (i) domains 1 and 2 (1-2); (ii) domains 1, 2, and 3 (1-3); (iii) domains 1, 2, 3, and 4 (1-4); and (iv) domain 1. MAb 10F4 (lower panel) bound primarily to domains 1 to 4 with minimal binding to domains 1and 2 and 1 to 3. ELISAs were done twice with similar results. PA₈₃ was used as a positive control. OD₄₀₅, optical density at 405 nm. (C) SDS-PAGE analysis of MAb 7.5G epitope mapping. MW, molecular weight markers (in kDa); lane 1, PA₈₃; lane 2, PA₈₃ digested with trypsin; lane 3, PA₈₃ digested with furin; lane 4, PA₈₃ digested with trypsin plus MAb 7.5G; lane 5, PA₈₃ digested with furin plus MAb 7.5G; lane 6, PA₈₃ digested with trypsin plus MOPC21; lane 7, PA₈₃ digested with furin plus MOPC21.

FIG. 3.

(A) Binding of ¹⁸⁸Re-PA₈₃ to J774 macrophages at 4°C in the absence and presence of MAb 7.5G. Molar ratios of PA₈₃: MAb were 1:1 and 1:50. (B) Binding of ¹⁸⁸Re-PA₈₃ and LF to macrophages in the absence and presence of MAb 7.5G. (C) Binding of ¹⁸⁸Re-PA₈₃ to J774 macrophages with blocked Fc receptors. These experiments were done for 15 and 60 min at 4°C with similar results. (D) PA₆₃ oligomer formation in the presence and absence of MAb 7.5G. Lane 1, PA₆₃; lane 2, PA₆₃ with MAb 7.5G.

FIG. 4.

(A) Cleavage of PA₈₃ with furin in the absence and presence of MAb 7.5G over several time intervals. MW, molecular weight markers (in kilodaltons); lanes 1, 3, 5, 7, 9, and 11, PA₈₃ with furin; lanes 2, 4, 6, 8, 10, and 12, PA₈₃ with furin plus MAb; lane 13, undigested PA₈₃. Aliquots of PA₈₃ were removed at 30-s and 1-, 2-, 3-, 4-, and 5-min time intervals for SDS-PAGE analysis. (B) ImageQuant analysis of bands. Key in 30-s graph applies to all panels.