A GM-CSF-Flt3L Fused Adjuvant Enhances Immune Responses and Protective Efficacy of Influenza DNA Vaccine in Mice

Background: DNA vaccines are widely used due to their low production cost, ease of large-scale manufacturing, and rapid responsiveness to emerging epidemics. However, their relatively modest immunogenicity in larger species often requires potent adjuvants to elicit robust protective responses. GM-CSF and Flt3L are two cytokine adjuvants targeting dendritic cells (DCs). Although their combination has been reported to augment immune responses, the mechanism underlying the complementary effects of a genetically fused GM-CSF-Flt3L adjuvant plasmid remains incompletely understood. Methods: We constructed a GM-CSF-Flt3L fusion adjuvant plasmid and compared it with single-adjuvant plasmids for their effects on innate, humoral, and cellular immunity, as well as protective efficacy against lethal homologous virus challenge. Results: Compared with single-adjuvant plasmids, GM-CSF-Flt3L synergistically promoted the maturation of bone marrow-derived dendritic cells (BMDCs) and activated draining lymph node DCs, differentially expanded DC subsets, and enhanced the recruitment of migratory DCs. This adjuvant significantly elevated hemagglutinin (HA)-specific serum IgG titers, hemagglutination inhibition (HI) titers, and the responses of T follicular helper (TFH) cells and germinal center B (GCB) cells, while also enhancing the capacity of HA-specific CD4+ and CD8+ T cells to secrete IFN-γ and TNF-α. Following lethal homologous virus challenge, the GM-CSF-Flt3L group exhibited markedly reduced lung viral loads and no significant pathological damage in lung tissues. Conclusions: These findings demonstrate that the GM-CSF-Flt3L fusion adjuvant complementarily enhances humoral and cellular immune responses induced by the influenza DNA vaccine and improves protective efficacy, highlighting its potential as an effective DNA vaccine adjuvant.