A natural oil-based nanoadjuvant enhances the immunogenicity of enterotoxigenic Escherichia coli (ETEC) in an experimental vaccine

Enterotoxigenic Escherichia coli (ETEC) is the leading cause of post-weaning diarrhea in piglets. The development of novel adjuvanted vaccines that can be administered directly to piglets remains a priority for the swine industry. Minthostachys verticillata essential oil (EO) has shown adjuvant effects, but its poor stability and solubility limit its use, that could be solved by emulsification. This study aimed to evaluate the effect of an EO-based nanoadjuvant to enhance the immunogenicity of ETEC in an experimental vaccine using mice as a preliminary model. A nanoemulsion (NEO) was formulated with EO (20 % v/v), Tween 80 (0.75 % v/v), and Span 60 (0.25 % w/v) using a high-energy method. The interaction between NEO and ETEC was analyzed by a scanning electron microscope. Experimental vaccines were prepared with inactivated ETEC strain combined with NEO (0.5, 0.75, and 1 mg/mL of EO) or EO (1 mg/mL) as adjuvants. Controls included Incomplete Freund's Adjuvant (IFA), Tween 80/Span 60 as a vehicle control, saline, and non-adjuvanted formulations. Balb/c mice were subcutaneously injected with the experimental vaccines, with four doses administered every 14 days. Antigen-specific antibody titers (IgG, IgG1, IgG2a), opsonizing capacity, and CD4⁺/CD69⁺ and CD8⁺/CD69⁺ T cells activation were evaluated. Splenic mononuclear cell proliferation and cytokine production (IFN-γ and IL-10) were also measured. Hepatic enzyme levels and malondialdehyde (MDA) concentrations were assessed to evaluate toxicity. NEO induced anti-ETEC IgG with significant opsonizing potential, increase in the percentage of CD4⁺/CD69⁺ and CD8⁺/CD69⁺ T cells, and production of IFN-γ. It caused no local reactogenicity, did not alter hepatic enzyme levels, and did not increase MDA concentrations. In conclusion, NEO demonstrated adjuvant potential, activating both humoral and cellular immune responses against ETEC without evidence of toxicity.