Design, synthesis, and validation of recombinant antigens for hyperimmune serum production

Introduction: Hyperimmune sera, developed over a century ago, rely on specific immune responses induced by target antigens.Their production involves immunizing large animals, followed by serum collection and processing to obtain neutralizing antibodies.The Ezequiel Dias Foundation (FUNED) is a key producer of these sera in Brazil, supplying eight types to the Ministry of Health.These therapies are effective for treating envenomations, bacterial toxin-related conditions (e.g., tetanus and diphtheria), and rabies.However, the production process has seen little innovation, particularly in antigen sourcing, which often depends on live organisms, posing biosafety risks, batch variability, and supply limitations.Advances in biotechnology offer a solution, enabling the industrial-scale production of recombinant antigens with improved quality control.The Molecular Toxinology Service at FUNED is exploring this approach to address antigen-related challenges.Objectives: To modernize the production of hyperimmune sera by replacing natural antigens with recombinant antigens.This study presents proof-of-concept strategies for laquetic, bothropic, and tetanic sera.Methodology: Recombinant antigens were designed based on natural antigen sequences.For laquetic serum, major toxins from Lachesis muta venom were identified, and key epitopes were arranged in tandem.The corresponding gene was cloned into pET-24b and expressed in E. coli.For bothropic serum, a consensus sequence representing snake venom metalloproteinases (SVMPs) from Bothrops venoms was designed to elicit broadly specific antibodies targeting hemorrhagic toxins.This sequence was expressed in Komagataella phaffii.For tetanic serum, a validated fragment of the tetanus toxin (fragment C) was expressed in E. coli to enable safer production.All recombinant antigens were purified, characterized, and used to immunize animals with Montanide ISA50 adjuvant.The resulting sera were evaluated for crossreactivity with natural antigens by ELISA.Results: All recombinant antigens were successfully expressed and purified.Immunization with these antigens elicited strong, specific immune responses.The resulting sera demonstrated significant crossreactivity with natural antigens. Conclusion:The recombinant antigens developed by FUNED showed promising results for hyperimmune serum production.Further in vivo neutralization tests are required to confirm their efficacy.