A NATURAL-BASED NANOADJUVANT ENHANCES HUMORAL AND CELLULAR IMMUNE RESPONSE INCORPORATED INTO AN INACTIVATED ENTEROTOXIGENIC Escherichia coli (ETEC) VACCINE.

Congreso; LXXI REUNIÓN SAI; 2023

In a previous study, we have demonstrated the adjuvant potential of a Minthostachys verticillata essential oil-based nanoemulsion (NEO), an Argentinian plant, into an inactivated enterotoxigenic Escherichia coli (ETEC) vaccine administered subcutaneously in mice. We observed anti-ETEC IgG levels similar to incomplete Freund's adjuvant (IFA), without a granuloma induction in the area of inoculation. In addition, the binding between NEO and ETEC was demonstrated by SEM. The aim of this study was to evaluate the IgG subclasses, the functionality of antibodies and the cytokines profile induced by NEO incorporated in an experimental inactivated vaccine against ETEC. The NEO was synthetized with 20% v/v of EO, 1% v/v of a surfactant mixture (0.75% Tween 80 and 0.25% Span 60) and 79% v/v of distillated water. Experimental vaccines were prepared as follow: a) formaldehyde-inactivated ETEC at 1x109 UFC/mL and saline solution, b) ETEC with NEO (0.5 mg/mL), c) ETEC with incomplete Freund's adjuvant (IFA) (50%), d) ETEC with EO (0,5 mg/mL) and e) ETEC with surfactant mixture (Tween80/Span60 0,5 mg/mL). Mice were divided into six groups of four animals each and vaccines were administrated subcutaneously on days 1, 14, 28 and 42. One group received only saline solution as a negative control. Animals were kept under controlled temperature, provided ad libitum access to food and water and were sacrificed 7 days after the last inoculation. For each group, a pool of sera was obtained and anti-ETEC IgG titers as wells as the IgG1 and IgG2a subclasses were determined through the indirect ELISA method. As in previous study, results showed that IgG titers of the group that received the vaccines formulated with EO as adjuvant were significantly increased compared to group that received ETEC (p˂0.0001), reaching similar values of those in IFA group. All groups vaccinated showed IgG1 levels higher than control group, being IFA the adjuvant that achieved the highest levels (p<0.001). The IgG2a levels in all groups were also higher than control group; however, IFA, ETEC and surfactant mixture-groups induced significant IgG2a levels compared with other groups (p<0.01). The functionality of the synthesized antibodies was evaluated by the opsonophagocytic assay performing serum dilutions (1/100, 1/200, 1/500, 1/750 and 1/1000). NEO induced antibodies with opsonophagocytic ability against ETEC similar to IFA up to 1/750 serum dilution. IL-10 and IFN-γ levels were measured in splenocyte culture supernatants from immunized mice. Only the group immunized with NEO as adjuvant showed a significant increase in IFN-γ compared to the other groups (p<0.001) and the group immunized with IFA showed a significant increase in IL-10 compared to the other groups (p<0.001). In conclusion, NEO as adjuvant was able to activate humoral and cellular immune response in mice with functional antibodies against ETEC and will continue to be studied for its incorporation into vaccines.