The immunogenicity of DNA vaccines can be improved by designing plasmids that encode secreted vaccine fusion proteins that (i) target antigen-presenting cells and (ii) display bivalent antigen. We extended this principle to a combinatorial and simultaneous delivery of 2 influenza virus antigens, hemagglutinin and neuraminidase. Using distinct dimerization motifs prevented mixing of the two antigens in the endoplasmic reticulum of transfected cells, maintaining antigen-presenting cell targeting and bivalency of each. Such a combinatorial DNA vaccine induced B and T cell responses against hemagglutinin and neuraminidase in mice at levels comparable to single-antigen vaccines, and increased protection against homologous influenza virus. In addition, vaccinated mice showed enhanced resistance to challenge with heterologous H1N1 and H5N1 viruses compared with both single-antigen and, importantly, conventional inactivated virus vaccines. This combinatorial DNA vaccine technology can be expanded to more influenza antigens as well as other pathogens, and therefore aid in the development of more potent and broadly reactive DNA vaccines.
Keywords: APC-targeted; cross-reactive protection; hemagglutinin; influenza DNA vaccine; neuraminidase.
© The Author(s) 2026. Published by Oxford University Press on behalf of The American Association of Immunologists.