Single-Chain Variable Fragments: Targeting Snake Venom Phospholipase A₂ and Serine Protease

Area of Science:

• * Immunology and Toxinology
• * Molecular Biology and Recombinant Antibody Engineering

Background:

• * Snakebite envenomation is a significant global health concern, necessitating the development of effective antivenoms.
• * Snake venom phospholipase A2s (PLA2s) and snake venom serine proteases (SVSPs) are key toxins responsible for severe envenomation.
• * Current antivenom limitations drive the need for innovative therapeutic strategies.

Purpose of the Study:

• * To design and evaluate immunogenic peptides targeting conserved epitopes of Crotalus atrox PLA2s and SVSPs.
• * To generate single-chain variable fragment (scFv) libraries from immune responses to these peptides.
• * To establish a foundation for developing recombinant monoclonal antibody-based antivenoms.

Main Methods:

• * Epitope-directed design of degenerate 15-mer peptides targeting PLA2s and SVSPs.
• * Immunization of mice with peptides, followed by extraction of IgG mRNA from lymphocytes.
• * Construction and sequencing of scFv libraries using PCR and cDNA.
• * ELISA to assess peptide immunogenicity.

Main Results:

• * Over 80% of sequenced plasmids contained diverse scFv sequences, with significant variation in complementarity-determining regions (CDRs), especially CDR3.
• * The snake venom serine protease (SP) peptide demonstrated broader immunogenicity in mice compared to the Asp49 peptide.
• * Generated scFv libraries show potential for developing targeted antivenom components.

Conclusions:

• * Peptide-based immunization successfully generated diverse scFv libraries targeting snake venom toxins.
• * The SP peptide exhibits strong immunogenic properties, suggesting its utility in antivenom development.
• * These scFvs are a promising basis for creating recombinant human monoclonal antibodies for snakebite treatment.