A novel method to incorporate bioactive cytokines as adjuvants on the surface of virus particles.

Cytokines have been used extensively as adjuvants in vaccines. However, practical considerations limit their use; diffusion from antigen, short half-lives and additional production costs. To address these problems we have developed a technology that efficiently produces inactivated, whole-virus influenza vaccine bearing membrane-bound cytokines. To provide "proof of principle," we chose chicken interleukin-2 (IL-2) and chicken granulocyte-macrophage colony-stimulating factor. Fusion constructs were generated in which their coding regions were linked to the influenza virus transmembrane encoding domains of the neuraminidase and hemagglutinin genes, respectively. These fusion constructs were used to establish stable Madin-Darby Canine Kidney cell lines, constitutively expressing membrane-bound cytokine. Cell surface expression was verified by immunofluorescence and cytokine-specific bioassays. Influenza virus harvested from infected cytokine-bearing cells was purified, inactivated, and confirmed to include membrane-bound cytokine by immunofluorescence, Western blotting and bioassay. Cytokine bioactivity was preserved using several standard virus inactivation protocols. Both cytokine-bearing influenza vaccines are now being tested for immunogenicity in vivo. Initial experiments indicate that chickens injected with IL-2-bearing influenza have elevated antiviral antibody levels, compared to chickens given conventional vaccine. In conclusion, this technology offers a novel method to utilize cytokines and other immunostimulatory molecules as adjuvants for viral vaccines.