Exceptionally potent human monoclonal antibodies are effective for prophylaxis and treatment of tetanus in mice

Abstract

We used human monoclonal antibodies (humAbs) to study the mechanism of neuron intoxication by tetanus neurotoxin and to evaluate these antibodies as a safe preventive and therapeutic substitute for hyperimmune sera to treat tetanus in mice. By screening memory B cells from immune donors, we selected 2 tetanus neurotoxin-specific mAbs with exceptionally high neutralizing activities and extensively characterized them both structurally and functionally. We found that these antibodies interfered with the binding and translocation of the neurotoxin into neurons by interacting with 2 epitopes, whose identification pinpoints crucial events in the cellular pathogenesis of tetanus. Our observations explain the neutralization ability of these antibodies, which we found to be exceptionally potent in preventing experimental tetanus when injected into mice long before the toxin. Moreover, their Fab derivatives neutralized tetanus neurotoxin in post-exposure experiments, suggesting their potential for therapeutic use via intrathecal injection. As such, we believe these humAbs, as well as their Fab derivatives, meet the requirements to be considered for prophylactic and therapeutic use in human tetanus and are ready for clinical trials.

Keywords: Immunoglobulins; Neuroscience; Therapeutics; Toxins/drugs/xenobiotics.

Figure 1. TeNT polypeptide chains and domain-specific recognition by TT humAbs.

(A) Schematic structure of TeNT (top panel) and TeNT-HC (middle panel). Bottom panel shows the L chain (red) and HC subdomains HC-N (violet) and HC-C (green) and as they appear in Western blotting (WB) using TIG as the primary antibody. Whole TeNT has a molecular weight of 150 kDa, corresponding to the L chain plus the H chain (left line). Reduction with DTT generated 2 bands corresponding to HC (HN plus HC, 100 kDa) and L (50 kDa). Recombinant HC has a molecular weight of approximately 50 kDa. The L chain has a weak signal, possibly due to a low immunoreactivity of L-specific IgGs in TIG. Single asterisk indicates a redox isomer of TeNT; double-asterisk indicates single-chain TeNT; plus sign indicates degradation fragments. (B) Schematic structure of HC (top), HC-N (middle), and HC-C (bottom) and how they appear in Western blotting stained for TIG. (C and D) Summary tables of recognition of TeNT domains and subdomains by TT humAbs as detected by Western blotting. The specificity of TT-humAbs was determined by at least 3 independent trials per antibody.

Figure 2. Preliminary screening for TeNT neutralization by TT humAbs assayed in vitro and in vivo.

(A) TeNT (50 pM) was diluted in complete culture medium alone (positive control) or supplemented with a 100X molar excess of the indicated humAb. The mixture was then added to CGNs for 12 hours, and TeNT activity was evaluated by monitoring the cleavage of VAMP-2 with an antibody recognizing only the intact form. SNAP-25 was used as a loading control. (B) Effect of different TeNT/humAbs ratios for the humAbs displaying toxin-neutralizing activity on CGNs. (C) Immunofluorescence analysis performed with an antibody specific for intact VAMP-2 (green) to assay for the TeNT-neutralizing activity of TT39, TT104, TT109, and TT110 preincubated with TeNT (100:1 molar ratio) and added to the primary culture of CGNs. Control CGNs (NC) are labeled in green, whereas neurons treated with TeNT alone (PC) do not display this signal because of the complete cleavage of VAMP-2. CGNs treated with the indicated humAbs displayed intermediate signals depending on the neutralization activity of the humAbs. Images are representative of 3 independent experiments. Scale bars: 25 μm. (D) Mice were injected i.p. with TeNT (4 ng/kg, black trace) alone or preincubated with the indicated molar ratios of humAb/TeNT, and survival is plotted as a function of time after toxin injection. P values are shown in the panels and were determined by Mantel-Cox test. The number of mice in each group is indicated in the panels. NC, negative control; PC, positive control.

Figure 3. Cryo-EM structure of the [TeNT-HC]-[TT104-Fab]-[TT110-Fab] ternary complex.

(A) Structure of HN (yellow) and L (red) domains in complex with TT110-Fab (light and dark blue for the variable L and H chains, respectively). (B) Structure of the HC-C (green) and HC-N (purple) domains in complex with TT104-Fab (light and dark blue for the variable L and H chains, respectively). (C and D) Overall structure of the TeNT-Fabs complex colored as in A and B.

Figure 4. Model of [TeNT-HC]-[TT104-Fab] interaction in complex with GT1b.

(A) The HC-N (purple) and HC-C (green) domains of TeNT complexed with TT104-Fab (light and dark blue), and the oligosaccharide portion of GT1b (yellow; bound as in pdb:1FV3). The nidogen binding region is shown in red. (B) View of the model after 180° horizontal rotation.

Figure 5. TT104-Fab prevents TeNT toxicity by interfering with toxin binding to PSGs and nidogen.

(A) Western blot analysis of CGNs treated with 50 pM TeNT alone (PC) or preincubated with the indicated TT104-Fab/TeNT molar ratios. After 12 hours, TeNT activity was evaluated by monitoring the cleavage of VAMP-2, as in Figure 2. Data are representative of 3 independent experiments. (B) Survival of mice injected i.p. with either TeNT alone (4 ng/kg) or premixed with a 5:1 molar ratio TT104-Fab/TeNT. The number of mice in each group is shown in the panel. (C) Fluorescence in CGNs treated with a mixture of 50 nM A555-TeNT-HC (red) or 50 nM CpV-BoNT/A-HC (BoNT/A-HC, green) preincubated with either culture medium or a 2:1 molar ratio of TT104-Fab/TeNT-HC for 2 hours and observed with a confocal microscope. Images are representative of 3 independent experiments. Scale bars: 30 μm. (D) Immunofluorescence staining of the LAL muscle injected in vivo with A555-TeNT-HC (1 μg) or CpV-BoNT/A-HC (1 μg) preincubated with vehicle or a 2:1 molar ratio of TT104-Fab/TeNT and observed 2 hours later with a confocal microscope. Images are representative of 3 independent experiments. Scale bars: 50 μm; 10 μm (enlarged insets). (E) Purified GT1b (0.5 g/well, left panel), recombinant nidogen-1/-2 (250 ng/well, middle left and middle right panels), or their combination (right panel) were adsorbed by overnight incubation on ELISA plates, and the binding of the indicated concentrations of either TeNT alone (gray bars) or TeNT preincubated with TT110-Fab (white bars) was tested as described previously (12). Data are reported as the percentage of the highest value in the graph and were averaged from at least 3 independent experiments (each dot represent a single well). Data represent the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed t test.

Figure 6. TT110-Fab neutralizes TeNT toxicity by preventing the translocation of the L chain into the nerve terminal cytosol.

(A) Western blotting of CGNs treated with 50 pM TeNT alone (PC) or preincubated with the indicated TT104-Fab/TeNT molar ratio. After 12 hours, CGNs were lysed and immunoblotted for VAMP-2 and SNAP-25 as in Figure 2. Blots are representative of 3 independent experiments. (B) Survival curve for mice injected i.p. with either TeNT alone (4 ng/kg) or premixed with 5 times the molar excess of TT104-Fab. The number of mice in each group is indicated in the panel. (C) Scheme illustrating entry of the TeNT L chain into the neuronal cytosol via either canonical receptor–mediated cell uptake and translocation across the membrane of SVs triggered by the acidification of their lumen induced by proton pump activity of the V-ATPase (light blue), or low-pH translocation across the plasma membrane in the presence of bafilomycin A1 (Baf-A1). (D) Western blot analysis showing the inhibition of TeNT L chain membrane translocation by TT110-Fab. CGNs were incubated at 4°C for 15 minutes with either TeNT (10 nM) or TeNT preincubated with TT110-Fab. The culture medium was then replaced with a 37°C buffer for 10 minutes at pH 7.4 or pH 5.0. Samples were then incubated for 12 hours with normal medium (PC) or normal medium supplemented with Baf-A1 (100 nM). Membrane translocation was assessed according to VAMP-2 cleavage. SNAP-25 served as a loading control. Results are representative of 3 independent experiments. (E) ANS fluorescence binding experiment showing the pH-induced conformational change of TeNT blocked by TT110-Fab. TeNT (0.35 μM) or TeNT preincubated with TT110-Fab was incubated at pH 7.0 in the presence of 50 μM ANS and liposomes. The conformational change was triggered by lowering the pH with sequential addition of specific volumes of HCl and evaluated following the ANS fluorescence intensity at 470 nm. Results are representative of 2 independent experiments.

Figure 7. TT104 and TT110 humAbs allow long-lasting prophylactic protection against TeNT, and their Fab derivatives prevent tetanus development after toxin challenge.

(A) Time course for testing the prophylactic activity of humAbs. Mice were i.p. preinjected with either TT104 (400 ng/kg) or TT110 (400 ng/kg) or their combination (200 ng/kg plus 200 ng/kg), or with TIG (3.5 IU/kg roughly corresponding to 250 IU/70 kg) for 15 or 7 days. TeNT (4 ng/kg) was then inoculated i.p., and the animals were observed for 200 hours for tetanus symptoms. (B and C) Prophylactic profiles for TT104 (B) and TT110 (C) injected alone. (D) Survival curves for mice treated with TT104 plus TT110 in combination compared with TIG. (E) Time course for testing TeNT neutralization by Fabs in a post-exposure challenge. TeNT (4 ng/kg) was delivered via i.p. injection. At the indicated time points, the combination of TT104 and TT110 Fab derivatives (1.2 μg/kg) or TIG (7 IU/kg) was injected i.p., and the animals were observed for 200 hours. (F) Survival plot for mice injected with TeNT and treated with either TT104 plus TT110 Fab derivatives (1.2 μg/kg, orange traces) or TIG (7 IU/kg, cyan traces) after 6 or 12 hours. Statistical significance was calculated with a Mantel-Cox test. The number of mice in each group is indicated in the panel. B, C, D, and F display the same lethality curve for the saline group, as this curve was derived from the data for all the mice treated with TeNT alone, plotted together.