Tick-borne encephalitis virus (TBEV) is a tick-borne flavivirus that induces severe central nervous system disorders. It has recently raised concerns due to an expanding geographical range and increasing infection rates. Existing vaccines, though effective, face low coverage rates in numerous TBEV endemic regions. Our previous work demonstrated the immunogenicity and full protection afforded by a TBEV vaccine based on virus-like particles (VLPs) produced in Leishmania tarentolae cells in immunization studies in a mouse model. In the present study, we explored the impact of adjuvants (AddaS03TM, Alhydrogel®+MPLA) and administration routes (subcutaneous, intramuscular) on the immune response. Adjuvanted groups exhibited significantly enhanced antibody responses, higher avidity, and more balanced Th1/Th2 response. IFN-γ responses depended on the adjuvant type, while antibody levels were influenced by both adjuvant and administration routes. The combination of Leishmania-derived TBEV VLPs with Alhydrogel® and MPLA via intramuscular administration emerged as a highly promising prophylactic vaccine candidate, eliciting a robust, balanced immune response with substantial neutralization potential.

Department of Experimental Biology Faculty of Science Masaryk University Kamenice 735 5 CZ 62500 Brno Czech Republic

Department of Recombinant Vaccines Intercollegiate Faculty of Biotechnology University of Gdansk and Medical University of Gdansk Abrahama 58 80 307 Gdansk Poland

Institute of Parasitology Biology Centre of the Czech Academy of Sciences Branisovska 31 CZ 37005 Ceske Budejovice Czech Republic

Laboratory of Emerging Viral Infections Veterinary Research Institute Hudcova 70 CZ 62100 Brno Czech Republic

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