Long-lasting immunoprotective and therapeutic effects of a hyperstable E7 oligomer based vaccine in a murine human papillomavirus tumor model Marı ´a L. Cerutti 1 , Leonardo G. Alonso 1 , Silvio Tatti 2 and Gonzalo de Prat-Gay 3 1 XBio SA, Concepcio ´n Arenal 4220, Ciudad de Buenos Aires, Argentina 2 Hospital de Clı ´nicas, Universidad de Buenos Aires, Buenos Aires, Argentina 3 Fundacio ´n Instituto Leloir and Instituto de Investigaciones Bioquı ´micas Buenos Aires, CONICET, Av. Patricias Argentinas 435, Buenos Aires, Argentina Cervical cancer and many other anogenital and oropharyngeal carcinomas are strongly associated with high-risk human papillomavirus (HPV) persistent infections. HPV E7 oncoprotein is the major viral transforming factor, emerging as a natural candidate for immunotherapy, since it is constitutively expressed in HPV-induced cancer cells. We have previously shown that E7 can self-assemble into soluble and homogeneous spherical oligomers, named E7 soluble oligomers (E7SOs). These are highly resistant to thermal denaturation, providing an additional advantage given the demand for highly stable vaccine formulations. Here, we present a new chemically stabilized form of the E7SOs (E7SOx) and analyzed its effect in a murine HPV-tumor model. Vaccination of female mice with low doses of E7SOx combined with a CpG-rich oligonucleotide (ODN) as adjuvant elicits a strong long-lasting protection against E7-expressing tumor cells, preventing tumor outgrowth after rechallenge 90-days later. Therapeutic experiments showed that E7SOx/ODN vaccination significantly delays tumor growth and extends the time of survival of the treated mice in a dose-dependent manner. These proof-of-principle preclinical experiments denote the potential applicability of our E7SOx-based vaccine to the treatment of cervical cancer and other mucosal HPV-related neoplastic lesions. In addition to thermal, chemical and proteolysis stability, the combined recombinant and chemical modification nature of the E7SOx vaccine candidate, results in low-cost, of particular interest in developing countries, where most of the cervical cancer cases occur and the most affected population is at reproductive age. Persistent infection by high-risk human papillomavirus (HPV) genotypes is the necessary factor for the development of cervi- cal cancer, the second cause of female gynecologic cancer mor- tality worldwide. The incidence of cervical cancer on women is quadrupled in developing countries, where 80% of the cases occur. In addition to cancer of the uterine cervix, these same high-risk HPVs are also associated with other anogenital tumors and, in less proportion, to oropharyngeal cancers. 1 Cervical cancer results from a spectrum of precursor lesions that are histologically very well defined, namely, the cervical intraepithelial neoplasias (CIN). These types of lesions are routinely detected in the course of cytological screening programs (Paps) and are usually removed surgically or eliminated via laser therapy or cryotherapy. Thus, drug or immunological therapies are valuable alternative or comple- mentary options to treat both, benign warts and precancerous lesions. Two virus-like particle-based vaccines recently devel- oped are highly efficacious for preventing the infection. How- ever, despite their approximately 100% efficacy at preventing HPV infection, these vaccines are unlikely to have a thera- peutic effect on preexisting neoplastic processes, and thus no immediate impact on the incidence of cervical cancer. 2,3 This means that effective treatment for the female world popula- tion between 15 and 75 years old is still required, where can- cer deaths over the next 20 years due to persistent high risk HPV infections is estimated to be 5 millions. 4 Human papillomaviruses are nonenveloped viruses con- taining an 8 Kb double stranded DNA genome. This genome encodes eight proteins, two of them, E6 and E7 cooperate for transformation by HPV. 5,6 The primary event of transforma- tion by E6 is promoting the proteasomal degradation of p53. 7 E7 was early described to bind the retinoblastoma tumor suppressor (pRb), 8 and target it for degradation via the ubiq- uitin proteasome pathway. 9 In any case, E7 is the major transforming protein in HPV, since its interaction with pRb disrupts growth-suppressive pRb-E2F complexes that regulate the G1/S phase transition. 7,9 Along with their oncogenic properties, E6 and E7 were also shown to participate in immune evasion mechanisms (reviewed in 10 ). Both proteins, mostly from high-risk types, have been shown to down-regu- late the expression of several IFN-inducible genes and down- regulate the expression of components of the antigen process- ing and presentation Pathways. Key words: HPV, vaccine, therapy, E7, cancer DOI: 10.1002/ijc.26294 History: Received 26 Apr 2011; Accepted 28 Jun 2011; Online 21 Jul 2011 Correspondence to: Gonzalo de Prat-Gay, Fundacio ´n Instituto Leloir. Av. Patricias Argentinas 435, Buenos Aires (C1405BWE), Argentina, Tel.: þ54-11-5238-7500, Fax: þ54-11-5238-7501, E-mail: gpg@leloir.org.ar Infectious Causes of Cancer Int. J. Cancer: 000, 000–000 (2011) V C 2011 UICC International Journal of Cancer IJC