# Immunogenicity of two representative American consensus scorpion neurotoxins from the genera Tityus and Centruroides

## TL;DR

This study shows that a designed toxin from the Centruroides genus is more effective at generating antibodies that neutralize scorpion venom than one from the Tityus genus.

## Contribution

The novel contribution is the rational design and evaluation of two consensus scorpion toxins for their immunogenicity and antivenom potential.

## Key findings

- rNATx elicited a stronger immune response and neutralized more venom than rSATx.
- Anti-rNATx antibodies neutralized up to 3LD50 of Centruroides venoms with 1-1.5 mg.
- rNATx may improve antivenom efficacy against scorpion envenomation.

## Abstract

Two consensus scorpion toxins derived from venoms of the genera Centruroides (NATx, North America), and Tityus (SATx, South America) were rationally designed and recombinantly expressed in Escherichia coli Origami. Both recombinant proteins were produced as inclusion bodies and subsequently purified and refolded in vitro to obtain biologically active isoforms. The expression yields were approximately 1 mg/L for rNATx and 0.5 mg/L for rSATx. Structural characterization by circular dichroism spectroscopy revealed that rNATx and rSATx exhibited folding patterns typical of scorpion β-toxins. The purified recombinant toxins were used as immunogens to raise polyclonal antibodies in New Zealand rabbits. The resulting antisera were evaluated for their capacity to neutralize isolated toxins and whole scorpion venoms. rNATx elicited a stronger immune response and showed superior immunogenicity compared to rSATx. Notably, 1-1.5 mg of anti-rNATx antibodies were sufficient to neutralize up to 3LD50 of venoms from the most medically relevant Centruroides species in México. On the other hand, approximately 20 mg of anti-rSATx antibodies were required to neutralize only 2LD50 of Tityus venoms. Additionally, T-cell subsets quantified by flow cytometry showed that rNATx is more immunogenic and probably confers improved antivenom efficacy, highlighting its potential application in the development of broad-spectrum antivenoms against scorpion envenomation.

Scorpion stings are considered a neglected health problem in tropical areas, with incidences reaching several hundred or thousands each year. Scorpion sting envenomation causes various symptoms, including pain, dizziness, nausea, vomiting, and in some cases, death. In the American continent, two genera of buthid scorpions are of medical importance: Centruroides and Tityus. Treating scorpion envenomations in American tropical countries involves the use of specific antivenoms; however, their efficacy may vary among scorpion species. In this work, we demonstrate that the de novo design of two scorpion toxins, here named rNATx and rSATx, which are representative of the genera Centruroides and Tiyus, respectively, was able to elicit an immune response in rabbits, whose antibodies were able to neutralize scorpion venoms and native toxins isolated from American buthid scorpions.