A Novel PLGA-based nano-adjuvant augments mucosal and systemic immunity in chicks through intestinal targeting and IgA production

The ongoing dissemination of the H9N2 avian influenza virus presents a significant challenge to the effective control and prevention of avian diseases. Consequently, this study developed a multipleimmune-regulatory adjuvant, PEI-LSP-RA-PLGA, to enhance the immune efficacy of H9N2 vaccines. The adjuvant was prepared by a double-layer nanoparticle preparation technology, with Lagenaria siceraria (Molina) Standl. Polysaccharide (LSP) and retinoic acid (RA) encapsulated into PLGA nanoparticles modified by polyethylenimine (PEI), forming PEI-LSP-RA-PLGA. The nanoparticle size is 200 nm, and the zeta potential is 13 mV. It has good stability and an antigen-sustained-release effect lasting 21 days. PEI-LSP-RA-PLGA was used as an inactivated H9N2 vaccine to immunize chicks. The results showed that PEI-LSP-RA-PLGA nanoparticles could promote the secretion of serum IgG antibodies, It increased by 132.83 % compared with the control group. Intestinal IgA antibodies, it increased by 115.12 % compared with the control group, and various cytokines, enhance the function of immune organs, promote the differentiation of spleen T lymphocytes, and improve the structural morphology of the small intestine. The in vivo imaging findings indicated that the PEI-LSP-RA-PLGA nanoadjuvant exhibited an excellent sustained-release effect at the injection site and demonstrated long-term intestinal targeting. This led to an increase in the number of intestinal IgA+ cells, thus boosting the intestinal immune function. The sequencing and confirmatory test results showed that PEI-LSP-RA-PLGA produced an effective intestinal targeting effect through the CCR9 and CCR6 signaling pathways under the action of the chemokines CCL20 and CCL25. After targeting the intestinal tract, an effective mucosal immune response was generated through the Toll-like receptor pathway, the NOD-like receptor pathway, and the immune network of IgA production. To sum up, PEI-LSP-RA-PLGA is a multi-targeted vaccine adjuvant that can simultaneously induce long-term systemic immunity and intestinal mucosal immunity, showing great application potential in improving the effectiveness of vaccines.