Epitopes and Mechanism of Action of the Clostridium difficile Toxin A-Neutralizing Antibody Actoxumab

Abstract

The exotoxins toxin A (TcdA) and toxin B (TcdB) are produced by the bacterial pathogen Clostridium difficile and are responsible for the pathology associated with C. difficile infection (CDI). The antitoxin antibodies actoxumab and bezlotoxumab bind to and neutralize TcdA and TcdB, respectively. Bezlotoxumab was recently approved by the FDA for reducing the recurrence of CDI. We have previously shown that a single molecule of bezlotoxumab binds to two distinct epitopes within the TcdB combined repetitive oligopeptide (CROP) domain, preventing toxin binding to host cells. In this study, we characterize the binding of actoxumab to TcdA and examine its mechanism of toxin neutralization. Using a combination of approaches including a number of biophysical techniques, we show that there are two distinct actoxumab binding sites within the CROP domain of TcdA centered on identical amino acid sequences at residues 2162-2189 and 2410-2437. Actoxumab binding caused the aggregation of TcdA especially at higher antibody:toxin concentration ratios. Actoxumab prevented the association of TcdA with target cells demonstrating that actoxumab neutralizes toxin activity by inhibiting the first step of the intoxication cascade. This mechanism of neutralization is similar to that observed with bezlotoxumab and TcdB. Comparisons of the putative TcdA epitope sequences across several C. difficile ribotypes and homologous repeat sequences within TcdA suggest a structural basis for observed differences in actoxumab binding and/or neutralization potency. These data provide a mechanistic basis for the protective effects of the antibody in vitro and in vivo, including in various preclinical models of CDI.

Keywords: Clostridium difficile infection; TcdA; epitope mapping; monoclonal antibody; toxin neutralization.

Fig. 1.

Actoxumab prevents the binding of TcdA to HT29 and Vero cells. (a) Flow cytometry analysis of HT29 cells preincubated with increasing concentrations of TcdA-Atto488 at 4 °C in the presence or absence of vehicle, actoxumab (200 μg/ml), or actoxumab-Fab (200 μg/ml). Following incubation, MFI was measured with excitation and emission wavelengths of 488 nm and 530 nm, respectively. A representative experiment is shown. (b) Flow cytometry analysis of HT29 cells preincubated with 800 ng/ml TcdA-Atto488 at 4 °C in the presence or absence of vehicle, actoxumab, or bezlotoxumab. MFIs were calculated as per panel (a). Values are means ± standard deviation of two independent experiments. acto = actoxumab; bezlo = bezlotoxumab. *p < 0.05 compared to TcdA alone by paired two-tailed t-test. (c) Western blot of membranes isolated from Vero cells following incubation with 1 μg/ml TcdA in the presence of vehicle, actoxumab, or bezlotoxumab (200 μg/ml). The top panel shows TcdA and the bottom panel shows cadherin (loading control).

Fig. 2.

TcdA constructs used in this study and binding of actoxumab by Western blotting. (a) Domain organization of TcdA showing the CROP domain with LRs and SRs highlighted. The CROP fragment constructs (A0, A1, A2, A3, A4, and A5) used in this study are also shown. (b) Western blot of TcdA and TcdB holotoxins and of constructs A0, A2, A3, A4, B2, and B3.

Fig. 3.

SEC-MALLS analysis of actoxumab/TcdA immunocomplexes. Light scattering and molecular mass distributions of immunocomplexes formed at various antibody:antigen molar ratios as a function of elution time. (a) Chromatograms of TcdA (green), actoxumab (red), and of actoxumab/TcdA immunocomplexes formed at 1:1 actoxumab:TcdA molar ratio (blue). (b) Chromatograms of actoxumab/A1 immunocomplexes formed at 1:5 (red), 1:1 (blue), 2:1 (pink), and 5:1 (green) actoxumab:A1 molar ratios. (c) Chromatograms of actoxumab-Fab alone (red) and of actoxumab-Fab/TcdA immunocomplexes formed at 1:5 (green), 1:1 (pink), 5:1 (dark red), and 10:1 (blue) Fab:TcdA molar ratios.

Fig. 4.

Negative-stain EM images of actoxumab-Fab bound to TcdA. Representative class averages of actoxumab-Fab bound to TcdA holotoxin (6996 total particles, in dataset). (a) Individual classes for a single-site Fab (204 particles) or (b) two-site Fabs (40 particles) bound to the TcdA CROP domain at Fab:toxin ratio of 3:1. Side length of each panel is 52.7 nm (scale bar represents 10 nm). (c) Schematic representation of TcdA with arrows showing regions where Fab fragments bind (model based on EM data from Ref. [33]). (d) Gallery of selected particles from the two-site class (top), with schematic (below) outlining individual components (TcdA white, Fab red).

Fig.5.

Identification of actoxumab epitopes by HDX-MS analysis. (a) Summary of HDX-MS analysis showing regions of TcdA CROP domain (construct A1) that have significantly lower deuterium incorporation in the presence of actoxumab, with % difference in deuteration level in the presence versus the absence of actoxumab indicated. (b) Peptide map showing regions of protein construct A1 containing lower (in blue) or higher (in red) deuterium incorporation following preincubation with actoxumab. Each gray box represents a distinct peptide identified by MS and is subdivided by incubation time point (10, 60, 600, 6000, and 10,000 s), as indicated. Sequences underlined in red are identical to each other and correspond to the putative epitopes of actoxumab. The numbering used is that of full-length TcdA. (c) Model of the TcdA CROP domain showing the putative actoxumab epitopes centered on LR3 and LR5. LRs are shown in dark gray, SRs in light gray, and regions protected from deuteration (putative actoxumab epitopes) in blue.

Fig. 6.

Alignment of actoxumab epitope sequences in TcdA. Residues within the putative actoxumab epitopes at LR3 and LR5 of ribotype 087 (VPI 10463) TcdA (as identified by HDX-MS) are shown by position (1–28) and compared to the homologous repeat sequences (LR1, LR2, LR4, LR6, and LR7) in the TcdA CROP domain and to the corresponding LR3 and LR5 epitopes within TcdA of ribotypes 027, 078, and 012. Residues that are different from those found at the same position in the actoxumab epitopes (LR 3 and 5) of ribotype 087 TcdA are marked in red. The ability of actoxumab to bind to individual homologous regions (as determined in this report) orto neutralize TcdA from various ribotypes (as demonstrated in Ref. [35]) is indicated (-, no binding; +, moderate neutralization; ++, high binding/neutralization).

Fig. 7.

Proposed model of actoxumab epitope 2 at LR 5. Epitope positions 4–7 (KGPN) and 28 (L) are highlighted to show conformation.