Identification and characterization of B-cell epitopes of 3FTx and PLA2 toxins from Micrurus corallinus snake venom

Abstract

The main goal of this work was to develop a strategy to identify B-cell epitopes on four different three finger toxins (3FTX) and one phospholipase A2 (PLA2) from Micrurus corallinus snake venom. 3FTx and PLA2 are highly abundant components in Elapidic venoms and are the major responsibles for the toxicity observed in envenomation by coral snakes. Overlapping peptides from the sequence of each toxin were prepared by SPOT method and three different anti-elapidic sera were used to map the epitopes. After immunogenicity analysis of the spot-reactive peptides by EPITOPIA, a computational method, nine sequences from the five toxins were chemically synthesized and antigenically and immunogenically characterized. All the peptides were used together as immunogens in rabbits, delivered with Freund's adjuvant for a first cycle of immunization and Montanide in the second. A good antibody response against individual synthetic peptides and M. corallinus venom was achieved. Anti-peptide IgGs were also cross-reactive against Micrurus frontalis and Micrurus lemniscatus crude venoms. In addition, anti-peptide IgGs inhibits the lethal and phospholipasic activities of M. corallinus crude venom. Our results provide a rational basis to the identification of neutralizing epitopes on coral snake toxins and show that their corresponding synthetic peptides could improve the generation of immuno-therapeutics. The use of synthetic peptide for immunization is a reasonable approach, since it enables poly-specificity, low risk of toxic effects and large scale production.

High-throughput immuno-profiling of mamba (Dendroaspis) venom toxin epitopes using high-density peptide microarrays

“…1), supported by the presence of clear baselines in the binding profiles (Fig. 2 and Supplementary Figs S1–7), and it is in agreement with the findings in a previous study with antivenom and members of the three-finger toxin protein family12. As a conservative estimate, the lower 70-percentile of median signals was used to determine the mean and standard deviations of the background populations, and a peptide was classified as target for antivenom antibodies if the median signal intensity was more than 10 standard deviations above the mean (see method section for details).…”

“…The results obtained clearly demonstrate the power of this high-throughput approach compared to similar methods based on SPOT synthesis6712 and mutation analysis11. Furthermore, toxin epitopes recognised by antivenom antibodies were unveiled for all but two of the manually curated toxins from every mamba and four cobra species endemic to sub-Saharan Africa.…”

“…Recently, studies involving immunoassay quantification of antivenom binding to immobilised synthetic peptides (SPOT synthesis), corresponding to individual segments of the amino acid sequence of a given toxin, have added valuable molecular insight by elucidating which sequences contain linear elements of epitopes recognised by given antivenoms67121314. These kind of meticulous epitope mapping experiments have also been performed on toxins from Tityus scorpions151617 and Loxosceles spiders18.…”

Biological characterization of the Amazon coral Micrurus spixii snake venom: Isolation of a new neurotoxic phospholipase A2

“…Q2,3 In Micrurus venom there is a considerable quantity of different toxins such as C-type lectins, natriuretic peptides precursors (NPP), L-amino acid oxidase (LAAO), metalloproteinases, Kunitz-type inhibitors, serine proteases, lysosomal acid lipase, hyaluronidase and alkaline phosphomonesterases (Corrêa-Netto et al, 2011;Leão et al, 2009). The main toxins presents in Micrurus venoms are aneurotoxins 3FTx (three-finger toxins) and b-neurotoxins or phospholipase A 2 (PLA 2 ) with variable proportions in the proteome of poisons of various species being primarily responsible for toxicity (Castro et al, 2015). The PLA 2 s are considered to be important toxins in snake venoms, especially in the Elapidae family.…”

“…Those toxins are responsible for neurotoxic, myotoxic, and edematogenic activities (Castro et al, 2015) and in some species of Micrurus, act on blood coagulation, play an important role in homeostatic changes triggered by the venoms (Cecchini et al, 2015). Therefore, the aim of this study is to promote the biochemical and functional characterization of crude venom from M. spixii, to purify and characterize one neurotoxic PLA 2 , in addition to demonstrating the microbicidal potential of this venom species.…”