Dual-antigen AdV5 vaccines against Crimean-Congo hemorrhagic fever virus induce superior humoral and Th1-biased cellular immunity

Platforms based on adenoviral vectors have proven their efficiency in developing vaccines and therefore can be considered potential candidates for immunization strategies against CCHFV. In this study, four replication-deficient recombinant human adenovirus serotype 5 (AdV5) vectors were engineered to express CCHFV antigens: a dual-antigen NP-P2A-Gc construct and monocistronic constructs for nucleoprotein (NP), glycoprotein complex precursor (GPC), and mature glycoprotein (Gc). Following GMP-compatible production and purification, vaccine candidates were evaluated in BALB/c mice ( n = 4 per group) for immunogenicity and in IFNAR −/− mice ( n = 4 per group) for protective efficacy using antigenically homologous or heterologous prime-boost regimens. Animals received priming immunization on day 0 and booster on day 28 followed by lethal CCHFV challenge (1000 TCID 50 ) on day 42. Immune responses were characterized via ELISA, virus neutralization tests, cytokine profiling, ELISpot, and flow cytometry. Post-challenge survival, viral loads in target organs, and safety profiles were assessed. All AdV5 constructs successfully expressed the target antigens and induced rapid seroconversion, with 75–100% of animals exhibiting antibody responses after priming with a single dose. The dual-antigen formulations elicited the strongest Th1-polarized responses particularly nucleoprotein + Gc combinations with interferon-γ production more than 30-fold above baseline and preferential expansion of CD4 + T helper cells (60–70% of CD3 + populations). Neutralizing antibody titers varied across groups (80–160 arbitrary units in high responders) but did not directly correlate with survival outcomes. After lethal challenge, multiple vaccine formulations achieved 100% survival throughout the 10-day observation period, while only control groups showed no protection. All vaccinated animals demonstrated significant viral load reduction of two to four logs in liver and spleen tissues, with some achieving complete clearance of the virus. Furthermore, no off-target vector dissemination was detected in examined tissues confirming safety profile of the constructs. Immunization with adenovirus serotype 5 (AdV5) vectors expressing CCHFV antigens elicited potent humoral and cellular immune responses, conferring 100% protection against lethal viral challenge across the majority of evaluated formulations. Sterilizing immunity was observed in some animals, along with evidence that protection operates through multiple immune mechanisms including both neutralizing and non-neutralizing antibody-mediated pathways. These results strongly support further clinical development of AdV5-based CCHFV vaccines, with multiple formulation strategies showing high efficacy and providing flexibility for clinical advancement. GMP-compatible production methods have been successfully implemented for these candidates, supporting their advancement toward non-human primate studies and Phase I clinical trials.