In vivo neutralization of dendrotoxin-mediated neurotoxicity of black mamba venom by oligoclonal human IgG antibodies

Abstract

The black mamba (Dendroaspis polylepis) is one of the most feared snake species of the African savanna. It has a potent, fast-acting neurotoxic venom comprised of dendrotoxins and α-neurotoxins associated with high fatality in untreated victims. Current antivenoms are both scarce on the African continent and present a number of drawbacks as they are derived from the plasma of hyper-immunized large mammals. Here, we describe the development of an experimental recombinant antivenom by a combined toxicovenomics and phage display approach. The recombinant antivenom is based on a cocktail of fully human immunoglobulin G (IgG) monoclonal antibodies capable of neutralizing dendrotoxin-mediated neurotoxicity of black mamba whole venom in a rodent model. Our results show the potential use of fully human monoclonal IgGs against animal toxins and the first use of oligoclonal human IgG mixtures against experimental snakebite envenoming.

Fig. 1

Polyclonal phage ELISA signals for the output phages for each selection. A significant increase in binding signal is observed from round 2 to 3 for all selections, and for selections against Dp5, Dp6, and Dp7, a high degree of cross-reactive binding exists for the phages against all three antigens (Dp5, Dp6, and Dp7). Negative control antigens: Cbtx α-cobratoxin, b-Gal β-galactosidase, Strep Streptavidin. All experiments were performed in triplicates on distinct samples. Error bars represent standard deviations

Fig. 2

Schematic overview of selected results from the employed discovery process. Here, demonstrated for human IgGs against the antigen, Dp8. a Polyclonal phage ELISA signals against different venom fractions and negative control antigens (Cbtx α-cobratoxin, b-Gal β-galactosidase, Strep Streptavidin). b Monoclonal scFv ELISA signals against Dp8. c Summary of DNA sequencing results. Sequences are defined as unique based on V_H and V_L CDR3 sequences. d Monoclonal IgG ELISA signals for converted clones demonstrating retained binding for the majority of the clones upon conversion from the scFv format

Fig. 3

Example of MALDI-TOF MS analysis of antigen-antibody complex pull-down experiments. a IgG 361_01_F07 pull-down from venom fraction Dp5. b IgG 361_01_F07 pull-down from whole venom. c IgG 363_01_F07 pull-down from venom fraction Dp6. d IgG 363_01_F07 pull-down from whole venom. Dtx Dendrotoxin, 3FTx Three-finger toxin

Fig. 4

Kaplan-Meier survival curves for antibody cocktails. Here, shown for a Cocktail 1 and b Cocktail 2 at different molar ratios against black mamba whole venom administered i.c.v., demonstrating full protection at an IgG:toxin molar ratio of 4:1 for Cocktail 1 and 3:1 for Cocktail 2. Cocktail 1 contains the IgGs: 361_01_F07 (anti-Dp5), 363_01_F07 (anti-Dp6), 365_01_G06 (anti-Dp7), 367_01_H01 (anti-Dp8). Cocktail 2 contains the IgGs: 363_01_F07 (anti-Dp6), 365_01_G06 (anti-Dp7), 367_01_H01 (anti-Dp8). Each individual curve represents survival of a cohort of 4 animals

Fig. 5

Kaplan–Meier survival curves for antibody cocktails. Here, shown for a Cocktail 3, b Cocktail 4, and c clone 367_01_H01 against black mamba whole venom administered i.c.v. at an IgG:toxin molar ratio of 3:1. Cocktail 3 contains the IgGs: 363_01_F07 (anti-Dp6) and 367_01_H01 (anti-Dp8). Cocktail 4 contains the IgGs: 365_01_G06 (anti-Dp7) and 367_01_H01 (anti-Dp8). Each individual curve represents survival of a cohort of 4 animals

Fig. 6

Schematic representation of the genetic elements present in the pINT3-hg1 vector