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

Abstract

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.

Fig. 1

Protocol of DNA vaccination. Thirteen days after tumor implantation, the animals were injected with DNA vaccine intramuscularly three times at weekly intervals. Two weeks after the last vaccination, immunological evaluation was performed

Fig. 2

Rate of tumor growth registered from 1 day before beginning of vaccination until 2 weeks after the last vaccination. Tumor masses were measured every other day with calipers in the two perpendicular diameters

Fig. 3

Proliferative response. Spleen cells were re-stimulated with the TUBO cell-raised tumor extract in the presence of BrdU. Results are shown as SI and represent the mean ± SD

Fig. 4

CTL assay. Each group of mice (n = 5) were vaccinated three times with saline (filled diamonds), empty vector (open squares), pHer2 (open triangles), or pHer2/CT (ex symbols). Spleen cells were collected 2 weeks after the last vaccination, co-cultured with target TUBO cells at 25:1, 50:1, and 100:1 effector/target ratios, and incubated for 4 h at 37°C with 5% CO₂. Cytotoxicity was measured by LDH release assay

Fig. 5

IFN-γ and IL-4 responses in DNA-vaccinated mice. The level of responses was determined by a DuoSet R&D ELISA system after 2 weeks of the last immunization. Spleen cells were stimulated with TUBO cell-raised tumor extract in RPMI 1640 10% FBS for 90 h. Vaccination with pHer2/CT or pCT/Her2 led to a significant increase in IFN-γ level (black columns) and IL-4 secretion (white columns)

Fig. 6

Freshly isolated spleen cells and tumor MNCs were stained with anti-CD4⁺ FITC, anti-CD25 PE, and anti-foxp3 PECy5. After gating on CD4⁺CD25⁺ cells, Tregs were defined as CD25^high and foxp3⁺ (a). Tumor MNCs were labeled with FITC conjugated anti-CD4⁺ or -CD8⁺, freshly (b). We found a reverse correlation between Tregs and CD4⁺ percentage. As Tregs decrease, CD4⁺ cells increase at the tumor site (c)