Anti-Metalloprotease P-I Single-Domain Antibodies: Tools for Next-Generation Snakebite Antivenoms

Abstract

In order to address the global antivenom crisis, novel antivenoms need to present high therapeutic efficacy, broad neutralization ability against systemic and local damage, sufficient safety, and cost-effectiveness. Due to biological characteristics of camelid single-domain antibodies (VHH) such as high affinity, their ability to penetrate dense tissues, and facility for genetic manipulation, their application in antivenoms has expanded considerably. VHHs that are active against the metalloprotease BjussuMP-II from the snake Bothrops jararacussu were selected. After isolation of BjussuMP-II, a camelid was immunized with the purified toxin in order to construct the recombinant phage library. Following a round of biopanning, 52% of the selected clones were able to recognize BjussuMP-II in an ELISA assay. After sequencing, seven sequence profiles were identified. One selected clone (VHH61) showed cross-reactivity to B. brazili venom, but did not recognize the Crotalus and Lachesis genera, indicating specificity for the Bothrops genus. Through in vitro tests, the capacity to neutralize the toxicity triggered by BjussuMP-II was observed. Circular dichroism spectroscopy indicated a robust secondary structure for VHH61, and the calculated melting temperature ( T M ) for the clone was 56.4°C. In silico analysis, through molecular docking of anti-BjussuMP-II VHHs with metalloprotease, revealed their potential interaction with amino acids present in regions critical for the toxin’s conformation and stability. The findings suggest that anti-BjussuMP-II VHHs may be beneficial in the development of next-generation antivenoms.

Revolutionizing snakebite care with novel antivenoms: Breakthroughs and barriers

“…This technology capitalizes on the connection between the genetic blueprint (genotype) of antibodies and their observable characteristics (phenotype). This connection is established by integrating antibody genes, specifically single-chain variable fragments (scFvs) or single-domain antibody fragments (VHHs or Nanobodies) [ 15 , 67 ], into the DNA of bacteriophages. These modified bacteriophages display the antibody peptide sequence on their outer coat, facilitating the study and analysis of antibodies within a phage context [ 35 ].…”

“…The potential of larger biomolecular therapies, including monoclonal human single chain variable fragments (scFvs) and complete monoclonal human IgGs show promise for inhibiting snake venom components in the future [ 15 , [87] , [88] , [89] ]. However, their size can impede tissue penetration, affecting efficacy against viper bites.…”

“…The escalating rates of snakebite-related mortality and amputations in low- and middle-income countries can be attributed to the inadequate availability of antivenoms, substandard healthcare services, variations in antivenom efficacy, and limited ability to mitigate local damage caused by venom variation intra- and interspecifically. Additionally, adverse reactions to antivenom further complicate this issue [ [12] , [13] , [14] , [15] , [16] ].…”