A replication-defective tick-borne encephalitis virus generated by NS1 trans-complementation confers robust protective efficacy

Tick-borne encephalitis virus (TBEV) poses a severe threat to public health, causing neurological disorders with high morbidity and mortality in endemic regions. Therefore, developing safe and effective strategies for TBEV vaccines has long been a focus of attention. Herein, leveraging our well-established NS1 trans -complementation platform, we successfully constructed a high-titer replication-defective TBEV (TBEV-ΔNS1) using the BHK-21 cell line stably expressing Omsk hemorrhagic fever virus (OHFV) NS1 (designated BHK NS1 ). Retention of the NS1 deletion in TBEV-ΔNS1 was confirmed by continuous passaging in BHK NS1 cells, as no replicative virus was detected in naive BHK-21 cells. The safety profile of TBEV-ΔNS1 was further validated, as administration of a high dose of TBEV-ΔNS1 to ICR mice did not induce any clinical symptoms. Notably, TBEV-ΔNS1 conferred complete protection against lethal wild-type (WT) TBEV challenge in ICR mice, whereas non-immunized control mice exhibited 100% mortality, significant weight loss, and viremia. This protective efficacy is tightly correlated with robust humoral and cellular immune responses, as demonstrated by the induction of high titers of TBEV-specific IgG, neutralizing antibodies, and IFN-γ-secreting CD8 + T cells following a single immunization. Collectively, our findings provide a proof-of-concept for the NS1 trans -complementation platform in TBEV vaccine development.