In vivo neutralization of coral snake venoms with an oligoclonal nanobody mixture in a murine challenge model

Abstract

Oligoclonal mixtures of broadly-neutralizing antibodies can neutralize complex compositions of similar and dissimilar antigens, making them versatile tools for the treatment of e.g., infectious diseases and animal envenomations. However, these biotherapeutics are complicated to develop due to their complex nature. In this work, we describe the application of various strategies for the discovery of cross-neutralizing nanobodies against key toxins in coral snake venoms using phage display technology. We prepare two oligoclonal mixtures of nanobodies and demonstrate their ability to neutralize the lethality induced by two North American coral snake venoms in mice, while individual nanobodies fail to do so. We thus show that an oligoclonal mixture of nanobodies can neutralize the lethality of venoms where the clinical syndrome is caused by more than one toxin family in a murine challenge model. The approaches described may find utility for the development of advanced biotherapeutics against snakebite envenomation and other pathologies where multi-epitope targeting is beneficial.

Fig. 1. Antibody responses of two immunized camelids over time.

Antibody binding signals observed for serum samples collected at different time points from two camelids immunized with a mixture of 18 elapid venoms. A Response of llama 0406 to the 18 venoms included in the immunization mixture. B Response of alpaca 0541 to the 18 venoms included in the immunization mixture. Values correspond to the means of two replicates (n = 2). The signal at day 0 represents the response of the preimmune sera. Source data are provided as a Source Data file.

Fig. 2. Binding kinetics of V_HHs to purified toxins.

Biotinylated toxins captured on streptavidin biosensors were dipped in decreasing concentrations of each of the V_HHs, followed by dissociation in kinetics buffer. Binding data were fitted using a 1:1 model. The colors represent the different V_HH concentrations: black is 200 nM, pink is 67 nM, green is 22 nM, dark purple is 7.4 nM, light purple is 2.5 nM, and cyan is 0.8 nM. A–C Anti-PLA₂ V_HHs binding PLA₂N. D–F Anti-αNTx V_HHs binding αNTx DH. Source data are provided as a Source Data file.

Fig. 3. In vitro neutralization of toxin activity by V_HHs.

A Inhibition of PLA₂ enzymatic activity by V_HHs. The maximal enzyme activity observed with toxin alone was set to 100%. The normalized enzymatic activity of PLA₂s from various elapid genera (PLA₂N, Nn19, and Hh3) preincubated for 30 min at RT with anti-PLA₂ V_HHs at a 1:20 toxin to V_HH molar ratio. Bars represent the mean of two replicates. B Neutralization of αNTx-mediated blocking of muscle-type nAChR current. Dose-response curves from patch clamp experiments with increasing concentrations of V_HHs to prevent the blocking of nAChR by 15 nM αNTx DH or 5 nM scNTx. Error bars represent standard deviation of independent cells performed in a 384-well plate. n = 16. Source data are provided as a Source Data file.

Fig. 4. Kaplan-Meier survival curves for mice challenged with PLA₂N or αNTx DH with or without adding V_HHs.

A, C 3 LD₅₀s of PLA₂N (A) or αNTx DH (C) were preincubated with either PBS or one of the V_HHs and injected in mice using the i.v. route. n = 3. B, D The mice were injected with 3 LD₅₀s of PLA₂N (B) or αNTx DH (D) using the s.c. route followed by immediate i.v. injection with PBS or one of the V_HHs. Toxin to V_HH molar ratio is presented in parentheses. n = 3. * Indicates a significant difference to PBS control (P < 0.05) in a Mantel-Cox log-rank test. Source data are provided as a Source Data file.

Fig. 5. Kaplan-Meier survival curves for mice challenged with αNTx DH followed by injection of different V_HH constructs.

The mice were injected with 3 LD₅₀s of αNTx DH using the s.c. route followed by immediate i.v. injection with PBS, or the V_HH TPL0629_01_D11 as a monovalent V_HH construct, and as bivalent constructs (bivalent V_HH, or V_HH-Fc). Toxin to antibody construct molar ratios are presented in parentheses. Note that a molar ratio of 1:1.25 of the bivalent constructs is equivalent to a 1:2.5 molar ratio between toxin and binding sites. n = 3. * Indicates a significant difference to PBS control (P < 0.05) in a Mantel-Cox log-rank test. Source data are provided as a Source Data file.

Fig. 6. Kaplan-Meier survival curves for mice challenged with whole venoms preincubated with an oligoclonal mixture of V_HHs, individual V_HHs, or the commercial antivenom, Coralmyn.

3 LD₅₀s of venom from (A). M. fulvius or (B). M. diastema, were preincubated with either PBS, the individual V_HHs, the relevant V_HH oligoclonal mixture prepared for the specific venom, or the polyclonal F(ab’)₂-based antivenom, Coralmyn. Approximate toxin to V_HH or F(ab’)₂ molar ratios are shown in parentheses. Calculations are based on total protein content in the Coralmyn and oligoclonal mixtures. n = 3. * Indicates a significant difference to PBS control (P < 0.05). ^# Indicates a significant difference to Coralmyn (P < 0.05) in a Mantel-Cox log-rank test. Source data are provided as a Source Data file.