Extracellular vesicle-inspired liposomes boost immune response against pathogen antigens

Developing effective vaccines against some diseases remains a global health priority. However, targeting immunity against a specific disease-related antigen requires efficient adjuvant systems that adequately promote vaccination efficacy. Extracellular vesicles have been identified as a potential adjuvant platform technology but are still limited in their reproducibility and safety. Here, we present a synthetic extracellular vesicle mimetic (EVM) platform that enhances antigen immunogenicity by incorporating the large extracellular loop (LEL) of the tetraspanin CD9, a protein abundant on natural extracellular vesicles. Liposomes with defined lipid composition were functionalized with recombinant CD9-LEL and pathogen antigens-KMP-11 from Leishmania and the SARS-CoV-2 Spike (S) protein. BALB/c mice were immunized intraperitoneally with nanovesicles carrying CD9-LEL, the pathogen antigens alone, or both. Antigen-specific IgG responses were measured by ELISA, and vesicle uptake by immune cells was assessed via flow cytometry. CD9-LEL alone did not elicit an immune response, but its co-presentation with antigens significantly enhanced specific anti-pathogen IgG titers. Notably, KMP-11 alone failed to induce detectable antibodies, while nanovesicles carrying both CD9-LEL and KMP-11 triggered strong IgG1 and IgG2b responses. Similarly, nCD9 + S induced higher IgG titers than S alone. CD9-LEL promoted uptake of nanovesicles by peritoneal macrophages, suggesting improved antigen presentation. Combining EVMs with classical adjuvants further modulated the immune profile: alum favored IgG1 (Th2 bias), while CpG ODNs enhanced IgG2a and IgG2b (Th1 bias), crucial for intracellular pathogen clearance. The platform demonstrated safety and adaptability. In conclusion, CD9-LEL-decorated EVMs represent a promising, scalable vaccine strategy capable of boosting antigen immunogenicity and directing immune polarization, offering a versatile tool for combating challenging or emergent infectious diseases.