The venom of the scorpion Centruroides limpidus, which causes the highest number of stings in Mexico, is neutralized by two recombinant antibody fragments
Introduction
In Mexico, 281 species of scorpions have been described (Santibáñez-López et al., 2015), positioning it as one of the countries with the greatest diversity in the world. Annually, about 300,000 stings are reported. Scorpion toxins affect the normal functioning of excitable cell ion channels, and in particular those that affect voltage-gated sodium channels (Nav+) have been considered of medical importance because they cause typical envenoming symptoms (Dehesa-Dávila and Possani, 1994; Escalona and Possani, 2013; Gordon et al., 1998) that could end in a fatal situation for humans (Gordon et al., 1998; Possani et al., 1999; Rodríguez De La Vega and Possani, 2005).
One of the states in the country with the highest accident rates is the State of Morelos, where the cases reported annually exceed 27 thousand (Riaño-Umbarila et al., 2017; Secretaria de Salud, 2019). In this region, all cases of scorpion sting envenoming are caused by the species Centruroides limpidus. Originally, the name of this scorpion was Centruroides limpidus limpidus and the acronyms for its toxins were Cll followed by a number like Cll1 and Cll2. The current name now is Centruroides limpidus, so the new toxin names are Cl followed by a number like Cl13. The venom of this species, has been extensively characterized (Alagón et al., 1988; Cid-Uribe et al., 2019; Dehesa-Dávila et al., 1996; Martin et al., 1994; Olamendi-Portugal et al., 2017; Ramírz et al., 1994), with an LD50 of 15 μg/20 g of body weight determined in CD1 mice which were injected intraperitoneally (Riaño-Umbarila et al., 2017). Three main toxins named Cll1, Cll2 and Cl13 have been identified in the venom of C. limpidus, corresponding to 1.5 %, 3.5 % and 2.1 % of the venom respectively. These toxins show different characteristics in terms of specificity for sodium channels, as well as stability and toxicity. LD50 shown by toxins Cll1, Cll2 and Cl13 were 1.7 μg, 1.5 μg and 0.5 μg, respectively (Olamendi-Portugal et al., 2017; Riaño-Umbarila et al., 2017). While Cll1 and Cll2 showed greater sequence similarity and toxic activity, Cl13 toxin is more diverse at the sequence level and exhibits greater activity on voltage-gated sodium channels (Olamendi-Portugal et al., 2017; Schiavon et al., 2006).
In the last two decades, the generation of recombinant antibodies potentially neutralizing of scorpion toxins and venoms has yielded excellent results. In many cases, the original source of these antibodies has been the product of animal immunization (Aubrey et al., 2004; Ben Abderrazek et al., 2011; Hmila et al., 2008; Juárez-González et al., 2005; Mousli et al., 1999; Selisko et al., 2004). Previous work by our group made it possible to generate single chain variable fragments (scFvs) capable of neutralizing some toxins and whole venoms from medically relevant Mexican scorpions (Riaño-Umbarila et al., 2019, 2013, 2011, 2005; Rodríguez-Rodríguez et al., 2016, 2012). Recently described scFv 10FG2, showed the ability to recognize and neutralize a significant number of toxins, including Cll1 and Cll2 (Riaño-Umbarila et al., 2019). This scFv was not able to recognize Cl13 toxin in ELISA tests which was confirmed by surface plasmon resonance (SPR) assays. These results explain why it had not been possible to neutralize 100 % the effect of C. limpidus venom (Riaño-Umbarila et al., 2019). Additionally, they allowed to understand why mice that survived when treated with scFv 10FG2, showed envenoming signs related to the presence of the third toxic component (Cl13). We know that neutralization of scorpion venom is only achieved when the lethal effect of the most abundant toxic components is inhibited (Riaño-Umbarila et al., 2016, 2011). It is important to note that in some venoms only 1 or 2 components are lethal, while in this case there are 3 of them.
Widening the cross-reactivity of antibodies is a useful strategy when targets show a high level of sequence identity as is the case of scorpion toxins. However, when comparing the primary structure of Cl13 toxin with other toxin sequences from Mexican scorpion venoms, important differences are observed in the regions that we know make contact with the previously generated scFvs, thereby explaining the lack of recognition of this toxin (López-Giraldo et al., 2020). These observations have also allowed to understand why efforts to broaden cross-reactivity against Cl13 were not successful. For this reason, hybridoma technology was used as an alternative source of monoclonal antibodies and specific scFvs derived from them. The subsequent processes of directed evolution and phage display allowed the generation of a scFv (11F) capable of neutralizing Cl13 toxin. This scFv, administered in conjunction with the scFv 10FG2, resulted in the full neutralization of the whole venom of Centruroides limpidus scorpion.
Section snippets
Purification of Cl13 toxin
Cl13 toxin was purified from the whole venom of the scorpion C. limpidus following the protocol reported in (Olamendi-Portugal et al., 2017).
Immunization of mice with Cl13 toxin
Mice used in this process were maintained under constant surveillance and supplied with water and food ad libitum following the standards established by the Bioethics Committee of the Institute of Biotechnology from UNAM applied to the project IBT-UNAM-290PDCPN 2014−01-246824. Once a week, during four months, six female Balb/c mice were immunized
Isolation of monoclonal antibody B7 (mAb B7)
The sera of 6 Balb/c mice (M1-M6) that were subjected to the immunization scheme described above (section 2.2), were evaluated by ELISA assays. In all cases, the presence of polyclonal antibodies that recognize Cl13 toxin was confirmed (Fig. S1). The spleen cells of these mice were used for the generation of hybridomas through a cell fusion process with SP2/0 myeloma cells. The characterization of 500 hybridomas in ELISA tests, allowed the identification of a hybridoma that showed a very good
Discussion
In Mexico, the commercial antivenom against scorpion stings (Alacramyn®), is produced through hyper-immunization of horses, using a mash of the venomous glands of four species toxic to humans. Antibodies present in equine serum are processed to obtain F-(ab')2 type antibody fragments (Dehesa-Dávila and Possani, 1994). This antivenom is made up of a collection of antibody fragments of polyclonal and polyvalent character. One of the drawbacks of this process is that the antibodies generated are
Conclusions
The objective of this work was to achieve the neutralization of the whole venom of Centruroides limpidus by means of using scFvs. For this purpose, the hybridoma methodology was used to obtain monoclonal antibodies and the production of the corresponding scFvs. In this way, it was possible to generate the monoclonal antibody B7, which showed good recognition toward Cl13 toxin, translating into an advance in the neutralization of the venom. The corresponding single-chain variable fragment was
CRediT authorship contribution statement
Guillermo Fernández-Taboada: Experiments, Data collection and Formal analysis, Preparation and Visualization of the manuscript. Ilse V. Gómez-Ramírez: Experiments and Data collection. Lidia Riaño-Umbarila: Review and editing of the manuscript, General supervision. Alejandro Olvera-Rodríguez: Supervision during generation of monoclonal antibodies and Formal analysis. Luis Fernando Losoya-Uribe: Acquisition data. Baltazar Becerril Lujan: Writing, review and editing of the manuscript, Project
Acknowledgements
Guillermo Fernández Taboada and Ilse Gómez Ramirez are doctoral students from Programa de Doctorado en Ciencias Bioquimicas and Biomédicas, respectively in Universidad Nacional Autónoma de México (UNAM) and have received a fellowship No. 278647 and 296771, respectively, from the Consejo Nacional de Ciencia y Tecnología (CONACYT). This study was supported by grants from Programa de Apoyo a Proyectos de Investigación e Inovación Tecnológica (PAPIIT IN) [grant number 201918] to BB and
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