N-terminally fusion of Her2/neu to HSP70 decreases efficiency of Her2/neu DNA vaccine

Abstract

DNA vaccines consisted of tumor-associated antigen (TAA) are well suited for immunotherapy against tumor. The construct can contain TAA fused to an appropriate molecule (biologic adjuvant) to improve the efficacy of anti-tumor immune response. Heat shock protein 70 (HSP70) has been shown to be an excellent candidate, capable of cross-priming TAA by antigen presenting cells leading to a robust T-cell response. However, the relationship between strong T-cell responses and tumor rejection is not always mutually exclusive, for which TAA loss or activation of suppressive mechanisms may occur. HSP70 fused to downstream of Her2/neu as DNA vaccine has been shown to be efficient against Her2-expressing tumors. In this study, we examined if N-terminally fusion of Her2/neu to HSP70 could also improve efficiency of Her2/neu DNA vaccine. Therefore, mice with an established Her2/neu expressing tumor were immunized with DNA vaccine consisting of extracellular and trans-membrane domain (EC+TM) of rat Her2/neu alone or N-terminally fused to HSP70 and immune response was evaluated. Administration of rat Her2/neu led to partial control of tumor progression. Surprisingly, fusion of HSP70 to N-terminal of rat Her2/neu led to tumor progression. Our result proposes that fusion direction of biologic adjuvant is an important consideration when Her2/neu is used.

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

IFN-γ and IL-4 level 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 pHSP70/Her2 led to a significant increase in IFN-γ level (black columns). In addition, IL-4 level (white columns) was significantly different between pHer2- or pHSP70/Her2-vaccinated animals. Results are represented as the mean ± SD

Fig. 5

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⁺. Percentage of Tregs (a) and Treg/T effector ratio were determined (b). Tumor MNCs were labeled with FITC-conjugated anti-CD4 or -CD8, freshly. CD4⁺ cell percentage was observed as the marginal increase in the pHSP70/Her2- and pHer2-vaccinated animals (c)