Rational design of a consensus bothropic metalloproteinase as an immunogen for broadly protective antivenom...

Antivenoms remain the only specific therapy for snakebite envenoming. In Bothrops venoms, Snake Venom Metalloproteinases (SVMPs) are abundant and highly diverse toxins responsible for the major local and hemotoxic effects induced by envenoming. However, currently available bothropic antivenoms fail to fully neutralize these effects, frequently resulting in permanent and disabling sequelae in affected patients. Here, we describe a rational approach to improve the SVMP neutralization potential of bothropic antivenoms using a consensus toxin. Forty SVMP sequences belonging to PI, P-II, and P-III classes from snakes of the Bothrops genus were analyzed, and a consensus amino acid sequence corresponding to the M12 metalloproteinase domain was determined through multiple sequence alignment. Based on the structural organization of SVMPs, we designed a recombinant construct containing an N-terminal hexahistidine tag, followed by a metalloproteinase pro-domain from Bothrops and a Tobacco Etch Virus (TEV) protease recognition site inserted between the pro-domain and the catalytic domain, enabling the expression of a consensus PI SVMP zymogen (pro_crSVMP-I) in the heterologous system Komagataella phaffii. Cleavage of the pro-domain and histidine tag by TEV protease yielded the mature form of the recombinant consensus PI SVMP (crSVMP-I). The consensus protein exhibited high immunogenicity in Swiss mice, whose anti-crSVMP-I immunoglobulins recognized in vitro, PI, P-II, and P-III SVMPs from 24 distinct Bothrops species. In addition, these antibodies were able to prevent or attenuate cutaneous hemorrhage induced by a bothropic venom mixture (B. jararaca, B. jararacussu, B. alternatus, B. neuwiedi, and B. moojeni) as well as by isolated venoms from B. atrox, B. asper, and B. leucurus in an in vivo experimental model. Together, these findings support the use of crSVMP-I as a rational immunogen with the potential to expand the efficacy and coverage of bothropic antivenoms.