Augmented immunogenicity of the HPV16 DNA vaccine via dual adjuvant approach: integration of CpG ODN into plasmid backbone and co-administration with IL-28B gene adjuvant

Abstract

Therapeutic human papillomavirus (HPV) DNA vaccine is an attractive option to control existed HPV infection and related lesions. The two early viral oncoproteins, E6 and E7, are continuously expressed in most HPV-related pre- and cancerous cells, and are ideal targets for therapeutic vaccines. We have previously developed an HPV 16 DNA vaccine encoding a modified E7/HSP70 (mE7/HSP70) fusion protein, which demonstrated significant antitumor effects in murine models. In this study, we employed multifaceted approach to enhance the potency of the HPV16 DNA vaccine. Strategies including inserting CpG oligodeoxynucleotide (CpG ODNs) into the vaccine vector backbone, selecting cytokine gene adjuvants, combining plasmids encoding mE6/HSP70 and mE7/HSP70, and utilizing electroporation for vaccination. Our findings revealed that mice immunized with CpG-modified vaccines, coupled with an IL-28B gene adjuvant exhibited heightened antigen-specific CD8⁺ T cell responses. Additionally, the combination of mE6/HSP70 and mE7/HSP70 plasmids synergistically enhanced the specific CD8⁺ T cell response. Furthermore, vaccination with CpG-modified mE7/HSP70 and mE6/HSP70 plasmids, alongside the Interleukin-28B (IL-28B) gene adjuvant, generated substantial preventive and therapeutic antitumor effects against HPV E6- and E7-expressing tumors in C57BL/6 mice. These results suggested that integrating these multiple strategies into an HPV DNA vaccine holds promise for effectively controlling HPV infection and related diseases.

Keywords: CpG oligodeoxynucleotide; E6; E7; HPV DNA vaccine; HSP70; IL-28B gene adjuvant.

Fig. 1

Map for CpG-modified vector. The optimized CpG ODN sequence (5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’) was synthesized by Sangon (Shanghai, China). 20 tandem copies of this sequence were inserted into the backbone of pVAX1EI via the PamC I restriction site, resulting in the CpG-modified immunization vector pmVAX1EI

Fig. 2

Western blot was performed to assess the expression of mE6/HSP70, mE7/HSP70, IL-28B and IL-15 in lysates from 293T cells transfected with either pVAX1EI (A) or pmVAX1EI (B) vectors, 48 h post-transfection. Control samples were prepared from cells transfected with empty pVAX1EI or pmVAX1EI vectors

Fig. 3

Effects of cytokine gene adjuvants on the quantity of IFN-γ-specific CD8⁺ T cells and mRNA levels of granzyme B in isolated splenocytes. (A) The quantity of IFN-γ-secreting E7-specific CD8⁺ T cells was assessed using ELISPOT. The spot counts were presented as the mean ± SE for 4 mouse samples per vaccinated group, normalized to 5 × 10⁵ splenocytes. (B) Representative ELISPOT images from each vaccinated group were shown. (C) The mRNA levels of specific granzyme B were analyzed via real-time PCR and normalized relative to GAPDH. *:P<0.05, ***:P<0.001, ****:P<0.0001

Fig. 4

Effects of CpG ODN modified plasmids on the quantity of IFN-γ -specific CD8⁺ T cells and mRNA levels of granzyme B in isolated splenocytes. (A) The quantity of IFN-γ-secreting E7-specific CD8⁺ T cells was assessed using ELISPOT. The spot counts were presented as the mean ± SE for 4 mouse samples per vaccinated group, normalized to 5 × 10⁵ splenocytes. (B) Representative ELISPOT images from each vaccinated group are shown. (C) The mRNA levels of specific granzyme B were analyzed via real-time PCR and normalized relative to GAPDH. *:P<0.05, **:P<0.01, ***:P<0.001, ****:P<0.0001

Fig. 5

Effects of CpG-modified plasmids combining mE7/HSP70 and mE6/HSP70 on the quantity of IFN-γ-specific CD8⁺ T cells and mRNA levels of granzyme B in isolated splenocytes. (A) The quantity of IFN-γ-secreting E7-specific CD8⁺ T cells was assessed using ELISPOT. The spot counts were presented as the mean ± SE for 4 mouse samples per vaccinated group, normalized to 5 × 10⁵ splenocytes. (B) Representative ELISPOT images from each vaccinated group are shown. (C) The mRNA levels of specific granzyme B were analyzed via real-time PCR and normalized relative to GAPDH. *:P<0.05, **:P<0.01, ***:P<0.001, ****:P<0.0001

Fig. 6

The analysis of prophylactic immune activity in vivo. C57BL/6 mice were immunized with 15 µg of plasmid via electroporation, administered twice at a 10-day interval. 7 days following the final vaccination, each mouse was challenged subcutaneously with 7.5 × 10⁴ TC-1 tumor cells, and the growth of tumors was monitored for a period of 60 days

Fig. 7

The analysis of therapeutic immune activity in vivo. Each C57BL/6 mouse was subcutaneously inoculated with 7.5 × 10⁴ TC-1 tumor cells. Three days post-inoculation, the mice were immunized with 15 µg of plasmid via electroporation, administered twice at a 10-day interval. Subsequently, tumor growth was monitored for a period of 60 days