A CXCL10-Expressing Influenza Vector Induces Robust Adaptive Immunity Despite Strong Attenuation

Background/Objectives: Although influenza A viruses with partially truncated NS1 proteins are substantially attenuated and immunogenic due to enhanced innate immune activation; residual NS1-mediated antagonism of antiviral innate responses may support viral replication in the lower respiratory tract and constrain optimal immune responses. Strategies to further improve their immunogenicity and protective efficacy by incorporating immunomodulatory cytokines, such as IL-2, have been successfully explored. Methods: Here, we extended this approach to chemokine expression by engineering an NS1-truncated PR8-based virus (PR8/NS124) to express the immunomodulatory chemokine CXCL10 from the NS segment and compared it with the parental vector. Results: The recombinant NS124_SS_CXCL10 virus replicated to high titers in embryonated chicken eggs and MDCK cells. In vivo, however, CXCL10 expression reduced viral replication in mouse lungs by ~10⁴-fold, resulting in a near-non-replicating phenotype. In contrast to the parental virus, the vector did not induce weight loss and exhibited a strongly attenuated phenotype. This effect was associated with altered innate immune signaling, including increased IRF7 expression and early induction of IFN-α responses in the lungs, together with modulation of TLR-dependent sensing pathways in the upper respiratory tract. Despite severely impaired replication, intranasal immunization induced antigen-specific T-cell responses comparable to those elicited by the parental vector. Following intraperitoneal immunization, when replication of both vectors was minimal, the CXCL10-expressing vector induced significantly higher frequencies of antigen-specific CD8⁺ and CD4⁺ effector-memory T cells. This was accompanied by enhanced antigen-specific T-cell recall responses in the lungs following intranasal challenge. Importantly, the CXCL10-expressing vector demonstrated protective efficacy comparable to that of the parental NS124 vector against heterologous H3N2 challenge while exhibiting an improved safety profile. Conclusions: These findings support the incorporation of CXCL10 as a strategy to improve the safety and T-cell immunogenicity of NS1-truncated influenza vectors.