DNA vaccine encoding human papillomavirus antigens flanked by a signal peptide and a KDEL sequence induces a potent therapeutic antitumor effect

Abstract

Cellular immune responses play a critical role in the eradication of intracellular infections and malignant cells through the recognition and subsequent removal of the infection or malignant cells. Effective antigen presentation is crucial for stimulating the immune system against malignant cells. Calreticulin (CRT) has been used to improve antigen presentation. However, CRT overexpression has been previously associated with the development of pancreatic and breast cancer. The import and retention signals of CRT in the endoplasmic reticulum (ER) can be used to overcome CRT overexpression. The present study describes the potent antitumor effect of a DNA vaccine encoding human papillomavirus type 16 E6 and E7 antigens flanked by ER import and retention signals (SP-E6E7m-KDEL). The effect of this vaccine was compared with that of E6 and E7 antigens fused to human full-length CRT (hCRT-E6E7m). In the present study, the effectiveness of SP-E6E7m-KDEL for inducing an interferon-γ antigen-specific, response and its therapeutic effect against tumors was demonstrated, which was as effective as immunization against those antigens fused to CRT. This simplified strategy, using ER import and retention signal peptides to direct antigens to this organelle, provides an efficient alternative to traditional vaccines and, more importantly, a safe and potent system to induce a therapeutic antitumor response.

Keywords: DNA vaccination; E6; E7; HPV16; KDEL sequence; antigen targeting; calreticulin; endoplasmic reticulum; signal peptide.

Figure 1.

Construction of DNA vaccines. These gene constructs involve the fusion of the human papillomavirus 16 E6 and E7 proteins to different signal sequences. rCRT, rabbit calreticulin; hCRT, human calreticulin, KDEL, lysine-aspartic acid-glutamic acid-leucine peptide sequence; SP, signal peptide.

Figure 2.

Detection of the recombinant proteins through western blotting. HEK-293 cells transfected with constructs expressing rCRT-E7, hCRT-E6E7m, SP-E6E7m-KDEL or the empty vector. After 24 h, the cell protein extracts were subjected to SDS-PAGE and incubated with specific antibodies. Lane 1, rCRT-E7; Lane 2, hCRT-E6E7m; Lane 3, SP-E6E7m-KDEL; Lane 4, empty vector. A molecular-weight size marker was loaded on lane M. rCRT, rabbit calreticulin; hCRT, human calreticulin, KDEL, lysine-aspartic acid-glutamic acid-leucine peptide sequence; SP, signal peptide.

Figure 3.

Subcellular localization of recombinant proteins. HEK-293 cells were transfected with constructs expressing rCRT-E7, hCRT-E6E7m, SP-E6E7m-KDEL or the empty vector. After 24 h, the cells were fixed and incubated with specific primary antibodies and secondary antibodies conjugated with fluorochromes. The samples were examined using a confocal fluorescence microscope. Original magnification, ×630. rCRT, rabbit calreticulin; hCRT, human calreticulin, KDEL, lysine-aspartic acid-glutamic acid-leucine peptide sequence; SP, signal peptide; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 4.

Enzyme linked immunosorbent assay detection of IFN-γ production by human papillomavirus E7/E6-specific splenocytes. Groups of three mice (6–8 weeks old) were immunized in the abdominal area with 1 µg of DNA on days 0 and 7 using a gene gun system. After 7 days, the splenocytes were harvested and stimulated in vitro with E7 or E6 peptide for 48 h. The culture supernatants were collected, and IFN-γ expression levels were determined. *P<0.05. rCRT, rabbit calreticulin; hCRT, human calreticulin, KDEL, lysine-aspartic acid-glutamic acid-leucine peptide sequence; IFN-γ, interferon-γ; SD, standard deviation.

Figure 5.

Antitumor effects of therapeutic DNA vaccination. Groups of 5 mice were injected with 1×10⁴ TC-1 cells in the tail vein. The mice were immunized with 1 µg of the corresponding DNA constructs using a gene gun system on the third and tenth days following tumor implantation and were subsequently sacrificed on day 25. The lungs were fixed with 4% paraformaldehyde, and tumor foci present on the epithelium were counted. The values and bars represent the means and standard deviations of tumor foci, respectively. *P<0.05 (rCRT-E7, hCRT-E6E7m and SP-E6E7m-KDEL vs. ‘empty’ control). rCRT, rabbit calreticulin; hCRT, human calreticulin, KDEL, lysine-aspartic acid-glutamic acid-leucine peptide sequence; IFN-γ, interferon-γ; SD, standard deviation.

Figure 6.

Histological changes in the tumor foci following therapeutic DNA vaccination. The lungs collected from the antitumor therapeutic assay were embedded in paraffin and processed for H&E staining. These images represent general structural changes observed in the organization of tumor cells in the foci between each treatment. Magnification, ×100. rCRT, rabbit calreticulin; hCRT, human calreticulin, KDEL, lysine-aspartic acid-glutamic acid-leucine peptide sequence; SP, signal peptide.