Elsevier

Journal of Virological Methods

Volume 206, 15 September 2014, Pages 12-18
Journal of Virological Methods

DNA vaccine encoding HPV-16 E7 with mutation in L-Y-C-Y-E pRb-binding motif induces potent anti-tumor responses in mice

https://doi.org/10.1016/j.jviromet.2014.05.013Get rights and content

Highlights

  • Design and construction of a novel modified E7 gene with mutation in LYCYE pRb-binding motif.

  • Modified E7 gene with mutations in aa 23-25 leads to the increased presentation of E7, stronger CD8+ T cell immune responses as well as potent therapeutic antitumor effects.

  • Therapeutic DNA vaccine encoding mutant E7 antigen is able to significantly reduce tumor volume against TC-1.

  • Mutant E7 DNA vaccine might be a promising novel therapeutic approach to control HPV-16-associated tumor formation.

Abstract

Cervical cancer is the second most common cancer among women worldwide and remains a clinical problem despite improvements in early detection and therapy. The human papillomavirus (HPV) type 16 (HPV16) E7 oncoprotein expressed in cervical carcinoma cells are considered as attractive tumor-specific antigen targets for immunotherapy. Since the transformation potential of the oncogenes, vaccination based of these oncogenes is not safe. In present study, DNA vaccine expressing the modified variant with mutation in pRb-binding motif of the HPV-16 E7 oncoprotein was generated.

A novel modified E7 gene with mutation in LYCYE motif was designed and constructed and the immunogenicity and antitumor effect of therapeutic DNA vaccines encoding the mutant and wild type of E7 gene were investigated. The L-Y-C-Y-E pRb-binding motif of E7 proteins has been involved in the immortalization and transformation of the host cell.

The results showed that the mutant and wild type HPV-16 E7 vectors expressed the desired protein. Furthermore, the immunological mechanism behind mutant E7 DNA vaccine can be attributed at least partially to increased cytotoxic T lymphocyte, accompanied by the up-regulation of Th1-cytokine IFN-γ and TNF-β and down-regulation of Th3-cytokine TGF-β. Immunized mice with mutant plasmid demonstrated significantly stronger cell immune responses and higher levels of tumor protection than wild-type E7 DNA vaccine.

The results exhibit that modified E7 DNA vaccine may be a promising candidate for development of therapeutic vaccine against HPV-16 cancers.

Introduction

Cervical cancer is the second most common cause of women deaths in all over the world. It would result in death of approximately 250,000–290,000 women each year globally, particularly in developing countries. Clinical, molecular and epidemiological investigations have identified human papilloma virus (HPV) as the major cause of cervical cancer (Trottier and Burchell, 2009).

Virtually all cervical cancers (about 99%) contain the genes of high-risk HPVs, most commonly types 16, 18, 31, and 45. In addition, HPV may play a role in certain carcinomas of the head and neck region and perhaps other cancers (Cubie, 2013). Therefore, it is necessary to develop therapeutic vaccines to reduce infection or HPV-related cancers especially cervical cancer (Elfstrom et al., 2014, Tran et al., 2014).

As the late proteins L1 and L2 are not detected in cervical cancer or infected basal cells, most therapeutic vaccines target the HPV early proteins such as E6 and E7. These oncogenic proteins are critical to the induction and maintenance of cellular transformation and are co-expressed in the majority of HPV-containing carcinomas (Morrow et al., 2013).

When a cell is infected with HPV, the E7 abrogate retinoblastoma (Rb) protein function, preventing it from interacting with E2F. Because E2F is now free, it promotes further rounds of cell division (Ghittoni et al., 2010). E7 also alter cytokine expression pattern, resulting in immune evasion (Sasagawa et al., 2012).

DNA vaccines targeting the E7 antigen offer a potentially effective procedure in HPV therapeutic vaccine development against E7-expressing tumors. DNA vaccines represent a promising strategy for generating antigen-specific immunotherapy because of their simplicity, stability, safety, and capacity for repeated administration (Li et al., 2012).

Although some experts believe that DNA vaccines are safer than live recombinant vaccines, others have raised concerns that the injected DNA might become integrated into the host genome, potentially inactivating tumor suppressor genes or activating oncogenes (Peng et al., 2006). DNA vaccines encoding E7 oncoprotein can either stably integrate into the genome or are maintained in an episomal form allowing for extended expression of HPV antigens (Eiben et al., 2003). In order to prevent vaccination-induced cellular transformation, modification in the pRb binding sites is necessary to eliminate the potential for oncogenic transformation while preserving critical epitopes (Ohlschlager et al., 2006).

The HPV16 E7 protein represents a zinc finger-binding phosphoprotein with two Cys-X-X-Cys domains composed of 98 amino acids. HPV16 E7 protein binds Rb through an L-Y-C-Y-E (conserved region 1; aa 21–26) motif (Cassetti et al., 2004, Munger et al., 2001). It has been shown that the transformation potential of the E7 oncoprotein is mainly localized in its pRb binding site (Smahel et al., 2001). As this interaction is probably required for carcinogenic progression in human patients, then therapeutic blockade of this activity could provide new treatment strategies in cervical carcinoma (Pang et al., 2013).

Previous study have demonstrated that mutation affecting only Cys of the repeats, which are conserved between different HPV E7 proteins, severely reduced the transforming activity but did not totally destroy it (Cassetti et al., 2004, Shi et al., 1999, Smahel et al., 2001). In order to design an E7 DNA vaccine with reduced transformation capacity and increased stability, three point mutations were introduced into the L-Y-C-Y-E pRb-binding motif (23 Tyr to Gly, 24 Cys to Gly, 25 Tyr to Gly) and mutated E7 gene was designed as DNA vaccine and administrated in tumor cell expressing HPV16 antigens. The immunogenicity and antitumor effect of the mutated vaccine was compared with wild E7 DNA vaccine.

Section snippets

Mice

6 ± 8-week-old female C57BL/6 mice were purchased from the Pasteur Institute (Karaj, Iran) and kept in the laboratory animal facility of Golestan University of medical science. All animals were fed with enough food and water to pass adaptation period, and treated with 6.00–18.00-h light/dark cycle. Approved protocols were applied to all animal experiments with consideration of recommendations for the accurate use and care of laboratory animals by the ethical commission of Golestan University.

Cell lines

Construction of mutant HPV-16 E7 DNA vaccine

The accuracy of inserted E7 fragment was confirmed by restriction enzyme analysis and sequencing. Western blots were performed for protein expression analysis. At 48 h post transfection, cell lysates were analyzed by western blot using anti-E7 antibody. As shown in Fig. 1, a very strong band of approximately 13 kDa, corresponding to E7, was observed in cells transfected with pcDNA3-mutant E7 (lane 4). This E7 band was also visible in cells transfected with pcDNA3-wild type E7 (lane 3). Cell

Discussion

The human HPV E7 protein is expressed selectively in cervical cancer cells and thus is considered the prime target for cell mediated immunotherapy (Hung et al., 2007). Therefore, the goal of immunization against HPV-induced malignancies is to boost cellular immune system that eliminates cancerous cells. Several E7-specific therapeutic vaccines are being developed currently (Morrow et al., 2013, Tomson et al., 2004). Among them, DNA vaccination has emerged as an attractive strategy for

Conflict of interest

All the authors have no conflicting interests.

Acknowledgments

The authors appreciate the financial support of the Research Deputy at Golestan Medical University Gorgan, Iran Grant No: 9011250224. This project was extracted from an MSc thesis.

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