Immune response induced by new adjuvant strategy to produce crotalic anti-PLA2

Abstract

Snake envenomation is a serious medical problem and antivenoms are the main treatment. Antisera are produced by immunization of horses with snake venom using complete Freund's adjuvant and incomplete (booster) but it causes severe local reactions. A new adjuvant strategy is here proposed to increase efficiency in antisera production under much less morbilty to immunized animals. Previous works showed that CpG-ODN formulated with a 6-O-ascorbyl palmitate nanostructure (Coa-ASC16) was more efficient as adjuvant than CpG-ODN alone using ovalbumin (OVA) as an antigen model. Here, we evaluated the immune response induced by this adjuvant strategy using crotalic PLA2 enzyme as antigen. Balb/c mice were subcutaneously immunizated on days 0, 15 and 30 with PLA2/CpG-ODN/Coa-ASC16 or PLA2/Freund's Adyuvant (complete first and incomplete-booster) (dose/mice: crotalic PLA2: 10-15μg, CpG-ODN: 30 μg). On day 50, mice were sacrificed. In both immunized group mice, the antibody titers in plasma were high (dilution1/24800), with a similar IgG1/IgG2a ratio. The IgG antibodies were then purified by affinity Sepharose-proteinG column. Indirect hemolytic activity neutralization of the PLA2(2.5 μg) with specific antibodies (1.7-4.5 μg range; IgG anti-PLA2) were made by radial hemolisis. The evaluation did not show difference neutralizing capacity of the antibodies produced by CpG-ODN/Coa-ASC16 or Freund's Adyuvant. Macroscopic and microscopic analysis at the site of injection of mice inoculated with Freund's adjuvant showed local damage (with non-infectious abscesses) and hypertrophy of inguinal lymph nodes, whereas mice injected with CpG-ODN/Coa-ASC16 did not. Our results show that CpG-ODN/Coa-ASC16 produces a humoral response as strong and specific as Freund´sadjuvant, with minor or null local deleterious effect, So, this complex emerge as a new adjuvant, a very attractive alternative for anticrotalic sera production.