Exploring the venom of the forest cobra snake: Toxicovenomics and antivenom profiling of Naja melanoleuca

Abstract

A toxicovenomic analysis of the venom of the forest cobra, also known as black cobra, Naja melanoleuca, was performed. Envenomings by this elapid species are characterized by a progressive descending paralysis which starts with palpebral ptosis and, in severe cases, ends up with respiratory arrest and death. A total of 52 different proteins were identified in this venom. The most abundant protein family was the three-finger toxin (3FTx) family, which comprises almost 57.1wt% of the venom, followed by phospholipases A (PLA) (12.9wt%). In addition, several other protein families were identified in a much lower percentage in the venom. A toxicity screening of the fractions, using the mouse lethality assay, identified four peaks as those having toxicity higher than that of the crude venom. These fractions predominantly contain α-neurotoxins of the 3FTx family. This toxicovenomic characterization agrees with the clinical and experimental manifestations of envenomings by this species, in which a strong neurotoxic effect predominates. Therefore, our findings suggest that immunotherapy against envenomings by N. melanoleuca should be directed towards the neutralization of 3FTxs; this has implications for the improvement of current antivenoms and for the development of novel antivenoms based on biotechnological approaches. A screening of the immunoreactivity of three antivenoms being distributed in sub-Saharan Africa revealed that they immunoreact with the fractions containing α-neurotoxins, although with different antibody titers.

How the Cobra Got Its Flesh-Eating Venom: Cytotoxicity as a Defensive Innovation and Its Co-Evolution with Hooding, Aposematic Marking, and Spitting

“…Thus there is convergence between the two groups in upright hooding displays being associated with defensive cytotoxic function, and as they have evolved the function independently the underlying chemical mechanisms are not homologous. Hemachatus + Naja venoms have a high concentration of the cytotoxic 3FTx unique to this clade [36,37,38,39,40,41,42,43,44,45]. Similarly O. hannah venoms have the highest concentration of L-amino acid oxidase of any snake venom and also the most derived forms of venom L-amino acid oxidase [23,46,47,48,49,50,51].…”

Third Generation Antivenomics: Pushing the Limits of the In Vitro Preclinical Assessment of Antivenoms

“…This figure corresponded to 19.6 mg of N. melanoleuca venom proteins per gram of IgG molecules. Assuming that the degree of toxin immunorecognition by an antivenom represents a measure of its capability to neutralize the venom’s lethal activity, a 10 mL vial of EchiTAb-Plus-ICP ® (40 mg IgG/mL; calculated ED 50 for N. melanoleuca (Uganda) = 0.66 mg/kg (95% confidence interval 0.49–0.92 mg/kg for 18–20 g mice) [33]), could theoretically neutralize 8–16 LD 50 s. Native to the central and western parts of the African continent, the forest cobra represents the largest true cobra ( Naja ) species [34]. It yields 0.5–1.1 g venom proteins per milking [35].…”

“…However, the antivenomics data highlight the poor immunorecognition of venom 3FTxs, the most abundant (57% of the venom proteome) and lethal components of forest cobra venom. Most relevant, α-neurotoxins eluting between 8–14 min comprise 24% of N. melanoleuca venom toxins, exhibit LD 50 s <0.2 µg/g mouse [33] and show no affinity for EchiTAb-Plus-ICP ® antibodies. This means that a bite injecting the whole venom load can inoculate as much as 120–240 mg of 3FTxs toxins, quantities capable of killing 4–8 75 kg humans, and against which the antivenom is not effective.…”

Strategies in ‘snake venomics’ aiming at an integrative view of compositional, functional, and immunological characteristics of venoms

“…Protein peaks resolved by the RP-HPLC step of the venomics protocol are collected, normalized for concentration, and coated onto microwell plates. Then, the presence of antibodies toward each chromatographic fraction, in a given antivenom, can be determined by ELISA [73–79]. Although this combined HPLC/ELISA immunoprofiling approach provides a general view of the immunorecognition/immunogenicity of the different venom components along its full chromatographic elution profile, it is also not exempt from limitations.…”

“…This concept was developed with the purpose of identifying which venom components should be targeted by novel neutralizing agents under development, such as recombinant human antibodies or synthetic peptide inhibitors [82]. Several investigations on elapid snake venoms have succeeded in pinpointing the main targets to be inhibited by using this experimental ‘toxicovenomics’ approach [73, 74, 78, 79]. …”