Development of an Oncolytic Influenza A Virus Vector Expressing Human CXCL10 for Enhanced T-Cell Responses

Influenza A virus (IAV) vectors with truncated NS1 proteins combine strong innate adjuvanticity with genetic flexibility and are attractive platforms for immune modulation. We engineered an NS1-truncated A/Puerto Rico/8/34 (H1N1) virus, PR8/NS124_SS_CXCL10, to express human CXCL10 from the NS segment and compared its biological and immunological properties with the parental NS124 vector in mice. The CXCL10-expressing virus replicated efficiently in embryonated chicken eggs and MDCK cells and secreted high levels of CXCL10, but showed profoundly reduced replication in mouse lungs and peritoneal cavity, indicating a strongly attenuated in vivo phenotype. After intraperitoneal immunization, both vectors induced rapid local cytokine and innate-cell recruitment, although early inflammatory responses and viral RNA loads were lower with PR8/NS124_SS_CXCL10 than with NS124. Despite this attenuation, PR8/NS124_SS_CXCL10 elicited significantly higher frequencies of systemic antigen-specific CD8⁺ and CD4⁺ effector-memory T cells producing IFN-γ, TNF-α, and IL-2, and promoted robust recall CD8⁺ and CD4⁺ T-cell responses in the lungs following low-dose homologous challenge. In a stringent heterologous challenge model with A/Aichi/2/68 (H3N2), however, mice primed intranasally with the CXCL10 vector experienced greater weight loss than NS124-primed animals, consistent with enhanced T-cell–driven immunopathology in the context of insufficient early viral control. These data show that CXCL10 expression in an NS1-attenuated IAV backbone simultaneously enforces replication restriction and amplifies T-cell immunogenicity, supporting its potential as a chemokine-armed platform for immune modulation and oncolytic virotherapy while underscoring the need to carefully balance mucosal priming and recall in chemokine-expressing influenza vaccines.