A chimeric protein that functions as both an anthrax dual-target antitoxin and a trivalent vaccine

Abstract

Effective measures for the prophylaxis and treatment of anthrax are still required for counteracting the threat posed by inhalation anthrax. In this study, we first demonstrated that the chimeric protein LFn-PA, created by fusing the protective antigen (PA)-binding domain of lethal factor (LFn) to PA, retained the functions of the respective molecules. On the basis of this observation, we attempted to develop an antitoxin that targets the binding of lethal factor (LF) and/or edema factor (EF) to PA and the transportation of LF/EF. Therefore, we replaced PA in LFn-PA with a dominant-negative inhibitory PA (DPA), i.e., PA(F427D). In in vitro models of anthrax intoxication, the LFn-DPA chimera showed 3-fold and 2-fold higher potencies than DPA in protecting sensitive cells against anthrax lethal toxin (LeTx) and edema toxin (EdTx), respectively. In animal models, LFn-DPA exhibited strong potency in rescuing mice from lethal challenge with LeTx. We also evaluated the immunogenicity and immunoprotective efficacy of LFn-DPA as an anthrax vaccine candidate. In comparison with recombinant PA, LFn-DPA induced significantly higher levels of the anti-PA immune response. Moreover, LFn-DPA elicited an anti-LF antibody response that could cross-react with EF. Mice immunized with LFn-DPA tolerated a LeTx challenge that was 5 times its 50% lethal dose. Thus, LFn-DPA represents a highly effective trivalent vaccine candidate for both preexposure and postexposure vaccination. Overall, we have developed a novel and dually functional reagent for the prophylaxis and treatment of anthrax.

FIG. 1.

Construction, expression, purification, and identification of the chimeras. (a) Schematic representation of each chimera constructed in this study. The furin cleavage site in the PA moiety and the mutation sites are indicated by arrows. (b) SDS-PAGE analysis of chimeric protein expression and purification. The expression host E. coli BL21-CodonPlus (DE3)-RIL carrying pGEX-LFn-PA was induced with 0.2 mM IPTG at 18°C for 6 h, and the cells were collected and disrupted. The insoluble debris (lane 4) and clarified supernatant (lane 5) were examined by SDS-PAGE, and the target protein bands are indicated with a triangle. The host cell containing pGEX-6p-1 was used as the control (lane 2, debris of the cell lysate; lane 3, supernatant of the cell lysate). The chimeric proteins LFn-PA (lane 6), LFn-DPA (lane 7), and mLFn-PA (lane 8) were purified by the methods described in Materials and Methods and analyzed by SDS-PAGE. A molecular weight standard (lane 1) was used as the reference for the target proteins. (c) Western blot analysis of the chimeras LFn-PA (lane 3) and LFn-DPA (lane 4) using the anti-PA polyclonal antibody. LF (lane 1) and PA (lane 2) were used as the negative and positive controls, respectively. (d) Western blot analysis of the chimeras LFn-PA (lane 3) and LFn-DPA (lane 4) using the anti-LF polyclonal antibody. PA (lane 1) and LF (lane 2) were used as the negative and positive controls, respectively.

FIG. 2.

Cytotoxicity assay of the chimeras. (a) RAW 264.7 cells were treated with a mixture containing a fixed concentration of LF (50 nM) and serially diluted chimeras (ranging in concentration from 1.25 nM to 320 nM in 2-fold increments). (b) RAW 264.7 cells were treated with a mixture containing a fixed concentration of chimeras (10 nM) and various concentrations of LF (ranging from 2.5 nM to 640 nM in 2-fold increments). In all the assays, an equal amount of PA was used for comparison. Data points represent the mean ± SEM values for triplicate assays.

FIG. 3.

Inhibition of anthrax toxin activity in vitro. (a) Inhibition of LeTx. RAW 264.7 cells were treated with a fixed concentration of LeTx (20 nM PA plus 5 nM LF) in the presence of various concentrations of DPA or LFn-DPA, and cell viability was determined by the alamarBlue method. Cells treated with a fixed concentration of LeTx alone were taken as the positive control. (b) Inhibition of EdTx. CHO-K1 cells were treated with a fixed concentration of EdTx (20 nM PA plus 5 nM EF) in the presence of various concentrations of DPA or LFn-DPA. After incubation for 2 h, the concentration of intracellular cAMP was determined. The untreated cells or cells treated with a fixed concentration of EdTx were used as the negative and positive controls, respectively. The results are represented as the means ± SEMs of at least three assays. The IC₅₀s were calculated by nonlinear regression analysis using GraphPad Prism (version 5) software.

FIG. 4.

Serum antibody responses in mice. Mice (8 mice per group) were immunized intraperitoneally with three 0.24-nmol doses of PA or LFn-DPA at 2-week intervals. Sera were collected 1 week after each injection and tested by ELISA for PA-specific (a), LF-specific (b), and EF-specific (c) IgG Abs. The control group injected with PBS alone with alum adjuvant was used as the control. The isotypes of PA-specific (d), LF-specific (e), and EF-specific (f) IgG from the group immunized with LFn-DPA were determined. The values are presented as the mean log₁₀ titers ± SEMs. The statistical difference in the PA-specific IgG levels between the LFn-DPA and PA groups after each immunization was determined by the two-tailed Student's t test, and the P values are shown.