Adjuvant activity of GP96 C-terminal domain towards Her2/neu DNA vaccine is fusion direction-dependent

The Her2 is one of tumor-associated antigens (TAA), regarded as an ideal target of immunotherapy. DNA encoding full-length or truncated rat Her2/neu have shown protective and therapeutics potentials against Her2/neu-expressing mammary tumors. However, the efficacy of active vaccination is limited since Her2 is a self-tolerated antigen. Hence, new strategies are required to enhance both the quality and quantity of the immune response against Her2-expressing tumors. Many studies have used Her2/neu gene with cytokine or other molecules involved in regulation of immune response to enhance the potency of Her2/neu DNA vaccines. Some studies fused adjuvant gene to C-terminal domain of Her2/neu gene, while others fused the adjuvant gene N-terminally to Her2/neu gene, but no comparison on how direction of fusion could affect efficiency of DNA vaccine has ever been made. Based on previous reports demonstrating potent adjuvant activity of gp96 C-terminal domain, we chose it as adjuvant. The aim of this study was to investigate if direction of fusion could affect adjuvant activity of gp96 C-terminal domain or potency of Her2/neu DNA vaccination. To do so, we fused C-terminal domain of gp96 to downstream or C-terminal end of transmembrane and extracellular domain (TM+ECD) of rat Her2/neu and resultant immune response to DNA vaccination was evaluated. The results were compared with that of N-terminally fusion of gp96 C-terminal domain to TM+ECD of rat Her2/neu. Our results revealed that adjuvant activity of gp96 C-terminal domain is enhanced when fused N-terminally to TM+ECD of rat Her2/neu. It suggests that adjuvant activity of gp96 C-terminal domain towards Her2/neu is fusion direction-dependent.