DNA vaccine encoding HPV-16 E7 with mutation in L-Y-C-Y-E pRb-binding motif induces potent anti-tumor responses in mice
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.
References (37)
- et al.
Transforming growth factor-beta in the gastrointestinal and hepatic tumor microenvironment
Gastroenterology
(2011) - et al.
Antitumor efficacy of Venezuelan equine encephalitis virus replicon particles encoding mutated HPV16 E6 and E7 genes
Vaccine
(2004) Diseases associated with human papillomavirus infection
Virology
(2013)- et al.
Specific N-methylations of HPV-16 E7 peptides alter binding to the retinoblastoma suppressor protein
J. Biol. Chem.
(1992) - et al.
Identification of HPV-16 E7 peptides that are potent antagonists of E7 binding to the retinoblastoma suppressor protein
J. Biol. Chem.
(1990) - et al.
Modified HPV16 E7/HSP70 DNA vaccine with high safety and enhanced cellular immunity represses murine lung metastatic tumors with downregulated expression of MHC class I molecules
Gynecol. Oncol.
(2007) - et al.
The future of human DNA vaccines
J. Biotechnol.
(2012) - et al.
The role of regulatory T lymphocytes in the induced immune response mediated by biological vaccines
Immunobiology
(2006) - et al.
An improved rearranged Human Papillomavirus Type 16 E7 DNA vaccine candidate (HPV-16 E7SH) induces an E7 wildtype-specific T cell response
Vaccine
(2006) - et al.
A prime/boost strategy by DNA/fowlpox recombinants expressing a mutant E7 protein for the immunotherapy of HPV-associated cancers
Virus Res.
(2012)
Immune responses against human papillomavirus (HPV) infection and evasion of host defense in cervical cancer
J. Infect. Chemother.
Molecular determinants for the complex formation between the retinoblastoma protein and LXCXE sequences
J. Biol. Chem.
Modified HPV16 E7 genes as DNA vaccine against E7-containing oncogenic cells
Virology
The region of the HPV E7 oncoprotein homologous to adenovirus E1a and Sv40 large T antigen contains separate domains for Rb binding and casein kinase II phosphorylation
EMBO J.
De-oncogenic HPV E6/E7 vaccine gets enhanced antigenicity and promotes tumoricidal synergy with cisplatin
Acta Biochim. Biophys. Sin.
Identification of T- and B-cell epitopes of the E7 protein of human papillomavirus type 16
J. Virol.
Cervical cancer vaccines: recent advances in HPV research
Viral Immunol.
Current cervical cancer prevention strategies including cervical screening and prophylactic human papillomavirus vaccination: a review
Curr. Opin. Oncol.
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