Neutralizing Nanobodies against Venoms from Naja haje Species Captured in North Africa

Abstract

Snakebite envenoming (SBE) remains a severely neglected public health issue, particularly affecting tropical and subtropical regions, with Africa experiencing an estimated 435,000 to 580,000 snakebites annually, leading to high morbidity and mortality rates, especially across Africa and Asia. Recognized as a Neglected Tropical Disease, SBE management is further complicated by the inadequate efficacy of current antivenom treatments. Of particular concern are cobras (Naja sp.), whose neurotoxins can induce rapid fatal respiratory paralysis. In this study, we investigate the potential of nanobodies as a promising next-generation of immunotherapeutics against cobra venoms. Through a dual strategy of the characterization of venom toxic fractions from cobras captured for the first time in Algeria and Tunisia biotopes, coupled with in vitro assays to evaluate their interactions with acetylcholine receptors, and subsequent immunization of dromedaries to produce specific nanobodies, we identified two lethal fractions, F5 and F6, from each venom, and selected five nanobodies with significant binding and neutralizing of 3DL50 (0.74 mg/kg). The combination of these nanobodies demonstrated a synergistic effect, reaching 100% neutralizing efficacy of 2DL50 lethal venom fraction (0.88 mg/kg) doses in mice. Additionally, our findings highlighted the complex mechanism of cobra venom action through the lethal synergism among its major toxins.

Keywords: LD50; Naja haje; cobra venom; nanobody; neutralizing capacity.

Figure 1

Size exclusion chromatography (SEC) profiles of Naja haje cobra venom extracts and estimation of main protein fractions. (a) Size exclusion chromatography of Nht venom with glacial acetic acid elution; (b) size exclusion chromatography of Nha venom with ammonium acetate elution.

Figure 1

Size exclusion chromatography (SEC) profiles of Naja haje cobra venom extracts and estimation of main protein fractions. (a) Size exclusion chromatography of Nht venom with glacial acetic acid elution; (b) size exclusion chromatography of Nha venom with ammonium acetate elution.

Figure 2

Analysis of Nht venom fractions via capillary electrophoresis, using an Agilent 2100 Bioanalyzer with a Protein 80 Kit: (a) Electropherogram of the Nht crude venom; (b) Electropherogram of NhtF5; (c) Electropherogram of NhtF6. The separation of proteins is shown within the molecular weight range of 1.6 to 95 kDa.

Figure 3

Binding capacity of the 18 selected nanobodies towards NhtF5/NhtF6 toxins determined by ELISA, in standardized conditions.

Figure 4

Specificity of NhtF5 and NhtF6 toxic fractions of cobra venom towards nAChRs subtypes. (a) Specificity of NhtF5 (100 nM) and NhtF6 (100 nM) towards (α1)₂β1δε-nAChRs (muscle-type); (b) Specificity of NhtF5 (100 nM) and NhtF6 (100 nM) towards α7-nAChRs ECD (extracellular domain); (c) Dose–Response curve (IC50) of NhtF5 and NhtF6 towards (α1)₂β1δε-nAChRs using I¹²⁵α-Bungarotoxin (α-Bgtx). Statistical significance is denoted as ***: p < 0.001, ns: p > 0.05.

Figure 5

(a) Nanobody–(α1)₂β1δε-nAChRs receptors’ binding affinity; (b) Nanobody-α7-nAChRs ECD receptors’ binding affinity; (c) ELISA evaluation of nanobody binding affinity to α-Bungarotoxin. ELISA plates were coated with α-bungarotoxin at a concentration of 1 µg/mL. Six nanobodies, initially at 10 µg/mL, were serially diluted and applied to assess binding affinity. Optical density was measured at 492 nm, to quantify the interaction between each nanobody and α-bungarotoxin. Statistical significance is denoted as ***: p < 0.001 and ****: p < 0.0001.

Figure 6

Map of the region from where Nh was collected. (a) Mareth, Tunisia; (b) Ghardaia, Algeria.