CpG-ODN/Coa-ASC16 nanostructured adjuvant platform as a safe and effective alternative in Bothrops diporus antivenom production

Abstract

In the field of vaccines, adjuvants have been optimized to enhance immune responses while reducing adverse effects.Transferring these advances to antivenom production could improve antibody quality and animal welfare. Here, we evaluateda novel adjuvant platform, CpG-ODN/Coa-ASC16, which combines immunostimulatory CpG-ODN oligodeoxynucleotideswith the biodegradable nanostructure Coa-ASC16, co-formulated with a hemorrhagic antigenic model basedon Bothrops diporus venom (B.dV). Immunization with B.dV/CpG-ODN/Coa-ASC16 elicited IgG titers equivalent tothose induced by Freund’s adjuvant, with significantly higher IgG1 levels. Antibodies generated with both formulationsexhibited progressively higher avidity, reflecting effective affinity maturation. Sera from B.dV/CpG-ODN/Coa-ASC16-immunized mice recognized major venom proteins and neutralized key toxic activities—proteolytic, coagulant, and indirecthemolytic—at levels comparable to Freund’s-induced sera. Functionally, sera from B.dV/CpG-ODN/Coa-ASC16-immunized mice conferred protection against a lethal venom challenge, resulting in survival of a fraction of animals andprolonged time to death in non-survivors. Importantly, histopathological analyses revealed minimal tissue alterations in themice treated with CpG-ODN/Coa-ASC16, in sharp contrast to the severe abscesses and granulomas caused by Freund’sadjuvant. Overall, this study provides new evidence that CpG-ODN/Coa-ASC16 can be effectively combined with hemorrhagicBothrops venom to generate robust, high-affinity antibodies that efficiently neutralize the major venom toxins whilemarkedly reducing local tissue damage. These findings position CpG-ODN/Coa-ASC16 as a safer and ethical alternativeplatform for antivenom production.