Exceptionally potent human monoclonal antibodies are effective for prophylaxis and treatment of tetanus in mice
Marco Pirazzini, Alessandro Grinzato, Davide Corti, Sonia Barbieri, Oneda Leka, Francesca Vallese, Marika Tonellato, Chiara Silacci-Fregni, Luca Piccoli, Eaazhisai Kandiah, Giampietro Schiavo, Giuseppe Zanotti, Antonio Lanzavecchia, Cesare Montecucco
Marco Pirazzini, Alessandro Grinzato, Davide Corti, Sonia Barbieri, Oneda Leka, Francesca Vallese, Marika Tonellato, Chiara Silacci-Fregni, Luca Piccoli, Eaazhisai Kandiah, Giampietro Schiavo, Giuseppe Zanotti, Antonio Lanzavecchia, Cesare Montecucco
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Research Article Neuroscience

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.

Authors

Marco Pirazzini, Alessandro Grinzato, Davide Corti, Sonia Barbieri, Oneda Leka, Francesca Vallese, Marika Tonellato, Chiara Silacci-Fregni, Luca Piccoli, Eaazhisai Kandiah, Giampietro Schiavo, Giuseppe Zanotti, Antonio Lanzavecchia, Cesare Montecucco

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Figure 5

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

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TT104-Fab prevents TeNT toxicity by interfering with toxin binding to PS...
(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.