Green Tea Polyphenols Cause Apoptosis and Autophagy in HPV-16 Subgene-Immortalized Human Cervical Epithelial Cells via the Activation of the Nrf2 Pathway.

Infection with human papillomavirus (HPV) is relatively common and certain high-risk HPV strains can induce epithelial dysplasia, increasing the risk of cervical cancer. Green tea polyphenol (GTP) preparations exhibit diverse anti-inflammatory, antioxidative, and antitumor properties In Vitro and In Vivo. Topical GTP application has been recommended as a treatment for genital warts, but the effect of GTP treatment on HPV infection and HPV-associated cancer remains to be established. The present study aimed to explore the mechanism by which GTP affected HPV type 16 (HPV-16)-positive immortalized human cervical epithelial cells. Survival, apoptosis, and autophagocytosis of these cells following GTP treatment was assessed using CCK-8 assay, flow cytometry, and monodansylcadaverine (MDC) staining. These cells were further transfected with an shRNA specific for Nrf2 to generate stable Nrf2-knockdown cells. The levels of Caspase-3, Bcl-2, Bax, P53, Rb, HPV-16 E6, HPV-16 E7, P62, Beclin1 and LC3B were determined via Western blotting. These analyses revealed that GTP treatment induced autophagy and apoptosis in HPV-16-positive cells, while Nrf2 gene knockdown reversed GTP-induced autophagic and apoptotic effects. Together, these results suggested that GTP could alleviate HPV infection and HPV-associated precancerous lesions In Vitro by regulating the Nrf2 pathway, highlighting the therapeutic potential of GTP in treating HPV infection.

Optimal combination treatment regimens of vaccine and radiotherapy augment tumor-bearing host immunity.

A major obstacle to immunotherapy is insufficient infiltration of effector immune cells into the tumor microenvironment. Radiotherapy greatly reduces tumor burden but relapses often occur. Here we show that the immunosuppressive tumor microenvironment was gradually established by recruiting Tregs after radiation. Despite tumors being controlled after depletion of Tregs in the irradiated area, improvement of mice survival remained poor. A much better antitumor effect was achieved with vaccination followed by radiation than other treatments. Vaccination followed by radiation recruited more effector T cells in tumor regions, which responded to high levels of chemokines. Sequential combination of vaccination and radiotherapy could elicit distinct host immune responses. Our study demonstrated that optimal combination of irradiation and vaccination is required to achieve effective antitumor immune responses. We propose a combination regimen that could be easily translated into the clinic and offer an opportunity for rational combination therapies design in cancer treatment.

CD40 Accelerates the Antigen-Specific Stem-Like Memory CD8+ T Cells Formation and Human Papilloma Virus (HPV)-Positive Tumor Eradication.

Antigen-specific stem-like memory CD8+ T cells (Tscm) have a series of stem cell characteristics, including long-term survival, self-renewal, anti-apoptosis and persistent differentiation into cytotoxic T cells. The effective induction of tumor-specific CD8+ Tscm could persistently eradicate tumor in pro-tumor hostile microenvironment. This study was to investigate the role of CD40 in HPV16-specific CD8+ Tscm induction and its anti-tumor function. We found that CD40 activation accelerated vaccine-induced HPV16 E7-specific CD8+ Tscm formation. Comparing to other HPV-specific CD8+ T cells, CD8+ Tscm were found to be stronger and long-term anti-tumor function, in vivo and in vitro, even in the adoptive cellular transferring model. Furthermore, high frequencies of Tscm might prevent the HPV infection to move on to the development of cancer. And the CD40 effect on Tscm involved Wnt/ß-catenin activation. Our study suggest that CD40 activation supports the generation of tumor-specific CD8+ Tscm, thus providing new insight into cancer immunotherapy.

Nanoparticle Conjugation of Human Papillomavirus 16 E7-long Peptides Enhances Therapeutic Vaccine Efficacy against Solid Tumors in Mice.

Treatment of patients bearing human papillomavirus (HPV)-related cancers with synthetic long-peptide (SLP) therapeutic vaccines has shown promising results in clinical trials against premalignant lesions, whereas responses against later stage carcinomas have remained elusive. We show that conjugation of a well-documented HPV-E7 SLP to ultra-small polymeric nanoparticles (NP) enhances the antitumor efficacy of therapeutic vaccination in different mouse models of HPV+ cancers. Immunization of TC-1 tumor-bearing mice with a single dose of NP-conjugated E7LP (NP-E7LP) generated a larger pool of E7-specific CD8+ T cells with increased effector functions than unconjugated free E7LP. At the tumor site, NP-E7LP prompted a robust infiltration of CD8+ T cells that was not accompanied by concomitant accumulation of regulatory T cells (Tregs), resulting in a higher CD8+ T-cell to Treg ratio. Consequently, the amplified immune response elicited by the NP-E7LP formulation led to increased regression of large, well-established tumors, resulting in a significant percentage of complete responses that were not achievable by immunizing with the non-NP-conjugated long-peptide. The partial responses were characterized by distinct phases of regression, stable disease, and relapse to progressive growth, establishing a platform to investigate adaptive resistance mechanisms. The efficacy of NP-E7LP could be further improved by therapeutic activation of the costimulatory receptor 4-1BB. This NP-E7LP formulation illustrates a "solid-phase" antigen delivery strategy that is more effective than a conventional free-peptide ("liquid") vaccine, further highlighting the potential of using such formulations for therapeutic vaccination against solid tumors. Cancer Immunol Res; 6(11); 1301-13. ©2018 AACR.

Poly(I:C) enhanced anti-cervical cancer immunities induced by dendritic cells-derived exosomes.

Dendritic cell (DC)-derived exosomes (Dexo) has been confirmed to be able to induce the specific anti-tumor immune response ex vivo and in vivo. Here, the aim of this study was to evaluate the application of the antigen-pulsed Dexo as a new vaccination platform in immunotherapy for cervical cancer. The immunogenic profile of the different Dexo was assessed by the cell proliferation, cytokines secretion and effector functions of CD8+ T cells and the splenocytes from Dexo-vaccinated mice. Furthermore, the anti-tumor immunity elicited by Dexo was further compared in cervical cancer-bearing mice. Dexo from DCs loaded with E749-57 peptide could efficiently induce the cytotoxic activity of CD8+ T cells on TC-1 tumor cells ex vivo, the proliferation and IFN-γ excretion of CD8+ T cells. Moreover, Dexo vaccine promoted the immune responses of vaccinated mice splenocytes induced by antigen E7 in vitro restimulation. Of note, poly(I:C) was significantly more potent inducer of the antigen-loaded Dexo mediated protective immunity responses for cervical cancer and further evidenced by that Dexo(E7+pIC) markedly inhibited the tumor growth and improved the survival rate of the tumor-bearing mice. We provided evidence that poly(I:C) dramatically increased the potent antitumoral immunity induced by antigen-pulsed Dexo for ameliorating cervical cancer.

Human papilloma virus-specific T cells can be generated from naïve T cells for use as an immunotherapeutic strategy for immunocompromised patients.

Human papilloma virus (HPV) is a known cause of cervical cancer, squamous cell carcinoma and laryngeal cancer. Although treatments exist for HPV-associated malignancies, patients unresponsive to these therapies have a poor prognosis. Recent findings from vaccine studies suggest that T-cell immunity is essential for disease control. Because Epstein-Barr Virus (EBV)-specific T cells have been highly successful in treating or preventing EBV-associated tumors, we hypothesized that the development of a manufacturing platform for HPV-specific T cells from healthy donors could be used in a third-party setting to treat patients with high-risk/relapsed HPV-associated cancers. Most protocols for generating virus-specific T cells require prior exposure of the donor to the targeted virus and, because the seroprevalence of high-risk HPV types varies greatly by age and ethnicity, manufacturing of donor-derived HPV-specific T cells has proven challenging. We, therefore, made systematic changes to our current Good Manufacturing Practice (GMP)-compliant protocols to improve antigen presentation, priming and expansion for the manufacture of high-efficacy HPV-specific T cells. Like others, we found that current methodologies fail to expand HPV-specific T cells from most healthy donors. By optimizing dendritic cell maturation and function with lipopolysaccharide (LPS) and interferon (IFN)γ, adding interleukin (IL)-21 during priming and depleting memory T cells, we achieved reliable expansion of T cells specific for oncoproteins E6 and E7 to clinically relevant amounts (mean, 578-fold expansion; n = 10), which were polyfunctional based on cytokine multiplex analysis. In the third-party setting, such HPV-specific T-cell products might serve as a potent salvage therapy for patients with HPV-associated diseases.

Supercharged green fluorescent protein delivers HPV16E7 DNA and protein into mammalian cells in vitro and in vivo.

Macromolecules including DNA and proteins serve as important human therapeutics but are limited by their general inability to cross cell membranes. Supercharged proteins have been known as potent tools for delivery of macromolecules into mammalian cells. Thus, the use of these delivery systems is important to reduce the human papillomavirus (HPV)-associated malignancies through improvement of vaccine modalities. In this study, we used a supercharged green fluorescent protein (+36 GFP) for delivery of the full-length HPV16 E7 DNA and protein into mammalian cells and evaluated immune responses, and protective/therapeutic effects of different formulations in C57BL/6 tumor mice model. Our results showed that the complexes of E7 DNA/+36 GFP and also E7 protein/+36 GFP form stable nanoparticles through non-covalent binding with an average size of ∼ 200-300 nm. The efficient delivery of E7 DNA or protein by +36 GFP was detected in HEK-293T cell line for 4 h and 24 h post-transfection. Mice immunization with E7 protein/+36 GFP nanoparticles elicited a higher Th1 cellular immune response with the predominant IgG2a and IFN-γ levels than those induced by E7 protein, E7 DNA, E7 DNA/+36 GFP and control groups (p < .05). Moreover, the E7 DNA/+36 GFP and E7 protein/+36 GFP nanoparticles similarly protected mice against TC-1 tumor challenge (∼67%) as compared to E7 DNA and E7 protein (∼33%), respectively. These data suggest that +36 GFP may provide a promising platform to improve protein and DNA delivery in vitro and in vivo.

Fungal mannosylation enhances human papillomavirus 16 E7 therapeutic immunity against TC-1 tumors.

Cervical cancer, resulting from infection with human papillomavirus (HPV)16, remains the fourth most common cancer in women worldwide. Recently, three prophylactic HPV vaccines targeting high-risk HPVs (particularly HPV16 and HPV18) have been implemented to protect younger women. However, individuals with pre-existing infections have no benefit from prophylactic vaccines. Thus, there is an urgent need to develop therapeutic vaccines. HPV16 E7 has been widely utilized as a target for immune therapy of HPV16-associated lesions or cancers, reflecting the sustained existence of this virus in cancerous cells. We developed mannosylated HPV16 E7 (mE7) expressed from Pichia pastoris as a therapeutic vaccine against HPV16-associated cancer. Unmannosylated E7 (E7) was also generated from Pichia pastoris as a control. Mannosylation enhanced the uptake of mE7 by mannose receptors of bone marrow-derived dendritic cells (BMDCs), while the uptake of E7 was unaffected. mE7-uptake BMDCs in vitro induced more IFN-γ secretion by splenocytes of immunized mice than E7. Vaccination of C57BL/6 mice with mE7 combined with adjuvant monophosphoryl lipid A (MPL) elicited stronger Th1 (type 1 T helper cell) responses and E7-specific T cell responses than E7. The mE7 vaccine induced the increased production of IFN-γ, IL-2 and TNF-α, elicited more E7-specific IFN-γ-secreting CD8+ T cells in spleen and peripheral blood mononuclear cells (PMBCs) and promoted stronger E7-specific cytotoxic CD8+ T cell responses compared with E7. Furthermore, TC-1 tumor challenged mice were used to confirm the antitumor activity of the vaccines. As a result, mE7 generated complete antitumor activity against TC-1 tumors, while E7 only provided partial antitumor activity. Taken together, mE7 can be a promising immunotherapy for treating cervical cancer.

Antitumor HPV E7-specific CTL activity elicited by in vivo engineered exosomes produced through DNA inoculation.

We recently proved that exosomes engineered in vitro to deliver high amounts of HPV E7 upon fusion with the Nefmut exosome-anchoring protein elicit an efficient anti-E7 cytotoxic T lymphocyte immune response. However, in view of a potential clinic application of this finding, our exosome-based immunization strategy was faced with possible technical difficulties including industrial manufacturing, cost of production, and storage. To overcome these hurdles, we designed an as yet unproven exosome-based immunization strategy relying on delivery by intramuscular inoculation of a DNA vector expressing Nefmut fused with HPV E7. In this way, we predicted that the expression of the Nefmut/E7 vector in muscle cells would result in a continuous source of endogenous (ie, produced by the inoculated host) engineered exosomes able to induce an E7-specific immune response. To assess this hypothesis, we first demonstrated that the injection of a Nefmut/green fluorescent protein-expressing vector led to the release of fluorescent exosomes, as detected in plasma of inoculated mice. Then, we observed that mice inoculated intramuscularly with a vector expressing Nefmut/E7 developed a CD8+ T-cell immune response against both Nef and E7. Conversely, no CD8+ T-cell responses were detected upon injection of vectors expressing either the wild-type Nef isoform of E7 alone, most likely a consequence of their inefficient exosome incorporation. The production of immunogenic exosomes in the DNA-injected mice was formally demonstrated by the E7-specific CD8+ T-cell immune response we detected in mice inoculated with exosomes isolated from plasma of mice inoculated with the Nefmut/E7 vector. Finally, we provide evidence that the injection of Nefmut/E7 DNA led to the generation of effective antigen-specific cytotoxic T lymphocytes whose activity was likely part of the potent, therapeutic antitumor effect we observed in mice implanted with TC-1 tumor cells. In summary, we established a novel method to generate immunogenic exosomes in vivo by the intramuscular inoculation of DNA vectors expressing the exosome-anchoring protein Nefmut and its derivatives.

Antitumor effect of therapeutic HPV DNA vaccines with chitosan-based nanodelivery systems.

BACKGROUND: Cervical cancer is the second-most-common cause of malignancies in women worldwide, and the oncogenic activity of the human papilloma virus types (HPV) E7 protein has a crucial role in anogenital tumors. In this study, we have designed a therapeutic vaccine based on chitosan nanodelivery systems to deliver HPV-16 E7 DNA vaccine, considered as a tumor specific antigen for immunotherapy of HPV-associated cervical cancer. We have developed a Nano-chitosan (NCS) as a carrier system for intramuscular administration using a recombinant DNA vaccine expressing HPV-16 E7 (NCS-DNA E7 vaccine). NCS were characterized in vitro for their gene transfection ability. RESULTS: The transfection of CS-pEGFP NPs was efficient in CHO cells and the expression of green fluorescent proteins was well observed. In addition, NCS-DNA E7 vaccine induced the strongest E7-specific CD8+ T cell and interferon γ responses in C57BL/6 mice. Mice vaccinated with NCS-DNA E7 vaccine were able to generate potent protective and therapeutic antitumor effects against challenge with E7-expressing tumor cell line, TC-1. CONCLUSIONS: The strong therapeutic effect induced by the Chitosan-based nanodelivery suggest that nanoparticles may be an efficient carrier to improve the immunogenicity of DNA vaccination upon intramuscular administration and the platform could be further exploited as a potential cancer vaccine candidate in humans.

The effect of helper epitopes and cellular localization of an antigen on the outcome of gene gun DNA immunization.

In DNA vaccination, CD4(+) T-cell help can be enhanced by fusion of a gene encoding an immunization protein with a foreign gene or its part providing T(h) epitopes. To study the effect of helper epitope localization in a protein molecule, the influence of the vicinity of the helper epitope, and the impact of chimeric protein cellular localization, we fused the helper epitope p30 from tetanus toxin (TT, aa 947-967) with the N- or C-terminus of the mutated E7 oncoprotein (E7GGG) of human papillomavirus type 16, enlarged the p30 epitope with the flanking residues containing potential protease-sensitive sites and altered the cellular localization of the fusion constructs by signal sequences. The p30 epitope enhanced the E7-specific response, but only in constructs without added signal sequences. After localization of the fusion proteins into the endoplasmic reticulum and endo/lysosomal compartment, the TT-specific T(h)2 response was increased. The synthetic Pan DR epitope (PADRE) induced a stronger E7-specific response than the p30 epitope and its stimulatory effect was not limited to nuclear/cytoplasmic localization of the E7 antigen. These results suggest that in the optimization of immune responses by adding helper epitopes to DNA vaccines delivered by the gene gun, the cellular localization of the antigen needs to be taken into account.

Immunomodulatory effects of IP-10 chemokine along with PEI600-Tat delivery system in DNA vaccination against HPV infections.

Although DNA vaccines represent an attractive approach for generating antigen-specific immunity, improvement of their potency is highly demanded. In the present study, three strategies including linkage to immunostimulatory molecules (N-terminal of gp96), co-administration of chemokines (IP-10 or RANTES) and PEI600-Tat as non-viral gene delivery system have been applied to enhance DNA vaccine efficacy against HPV infections. We found that C57BL/6 immunization with E7-NT-gp96 fusion gene led to increased level of IFN-γ compared to E7 alone. The fused genes showed considerable protective potency in tumor mice model. In addition, E7-NT-gp96 delivered with PEI600-Tat was more protective against E7-expressing tumors comparing with E7-NT-gp96 alone. Our results showed that co-administration of IP-10 with E7-NT-gp96 delivered by PEI600-Tat elicits significant IFN-γ production and consequently a strong preventive response against TC-1 tumor cells in contrast to increased tumor growth by RANTES co-delivery. Also in therapeutic experiment, our data showed that co-immunization of IP-10 at the same inoculation site of TC-1 along with E7-NT-gp96 delivery by PEI600-Tat is able to significantly suppress TC-1 tumor growth. The successful treatment by this immunization protocol was associated with the elevated levels of IFN-γ and IL-2 production in the lymph nodes. These data indicated that fusion of NT-gp96 to E7 in combination with IP-10 co-administration and PEI600-Tat delivery system can synergistically enhance the potency of HPV DNA vaccines. Therefore, this approach suggests a combinational therapeutic strategy against cervical and other HPV-related cancers.

Potential antitumor applications of a monoclonal antibody specifically targeting human papilloma virus 16 E7 49-57 peptide.

Our study aims to evaluate whether the approach of TCRm mAb has therapeutic potential against HPV-induced tumors. In the present study, we generated a murine IgG2a mAb 6C10 specifically recognizing HPV-16-E7(49-57) epitope (RAHYNIVTF) in the polypeptides and in complex with a MHC class I molecule. Analysis of the primary structure shows that the 6C10 Ab displays a novel sequence in the CDR of the heavy chain, compared to the sequences in the Kabat database, which suggests the Ab has completed its affinity maturation. The 6C10 Ab can specifically recognize E7 and Trx-E7(30-67) protein in ELISA, and can also specifically bind to T2 cell carrying HPV-16-E7(49-57) peptide. In the TC-1 cell tumor-bearing mouse model, 6C10 exhibits tumor suppression activity when compared to the isotype control Ab. 6C10 Ab has showed tumor-inhibition potency in a mouse model and this Ab may have the prospect of cancer therapy.

Purified herpes simplex type 1 glycoprotein D (gD) genetically fused with the type 16 human papillomavirus E7 oncoprotein enhances antigen-specific CD8+ T cell responses and confers protective antitumor immunity.

Type 1 herpes virus (HSV-1) glycoprotein D (gD) enhances antigen-specific immune responses, particularly CD8(+) T cell responses, in mice immunized with DNA vaccines encoding hybrid proteins genetically fused with the target antigen at a site near the C-terminal end. These effects are attributed to the interaction of gD with the herpes virus entry mediator (HVEM) and the concomitant blockade of a coinhibitory mechanism mediated by the B- and T-lymphocyte attenuator (BTLA). However, questions concerning the requirement for endogenous synthesis of the antigen or the adjuvant/antigen fusion itself have not been addressed so far. In the present study, we investigated these points using purified recombinant gDs, genetically fused or not with type 16 papilloma virus (HPV-16) E7 oncoprotein. Soluble recombinant gDs, but not denatured forms, retained the ability to bind surface-exposed cellular receptors of HVEM-expressing U937 cells. In addition, in vivo administration of the recombinant proteins, particularly gD genetically fused with E7 (gDE7), promoted the activation of dendritic cells (DC) and antigen-specific cytotoxic CD8(+) T cells. More relevantly, mice immunized with the gDE7 protein developed complete preventive and partial therapeutic antitumor protection, as measured in mice following the implantation of TC-1 cells expressing HPV-16 oncoproteins. Collectively, these results demonstrate that the T cell adjuvant effects of the HSV-1 gD protein did not require endogenous synthesis and could be demonstrated in mice immunized with purified recombinant proteins.

Dendritic cells treated with HPV16mE7 in a three-dimensional model promote the secretion of IL-12p70 and IFN-γ.

Although the human papillomavirus (HPV) DNA therapeutic vaccine represents a promising approach to the prevention and treatment of cervical cancer, the mechanism of the HPV DNA vaccine is poorly understood. Moreover, current strategies have met with only limited success in preclinical and dendritic cell-based (DC-based) clinical research. In addition, two-dimensional (2-D) DC monolayers poorly mimic the physiology function in vivo. We used a three-dimensional (3-D) DC culture model in vitro to explore the immune mechanism of the HPV DNA vaccine. DCs were generated from peripheral blood monocytes with interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cells, growing in 3-D collagen gel, were treated with pcDNA3.1-HPV16mE7 in vitro for 48 h. Compared to DCs treated with E7 in a 2-D culture model, the expression of co-stimulatory molecules CD80 and CD40 were significantly increased in the 3-D model (p<0.05), and a remarkable increase of IL-12 p70 was observed. However, we did not detect any obvious change in IL-10 in 3-D culture. In addition, we found that IFN-γ expression increased when HPV16mE7-DC cells were co-cultured with T-cells for 96 h in the 3-D model, and HPV16mE7-DCs stimulated the proliferation of T lymphocytes more efficiently in the 3-D model than in the 2-D model (p<0.05). These results suggest that DCs in 3-D culture model have a notable effect on the enhancement of the HPV16 DNA vaccine's immune reaction and indicate that the DC-based 3-D model is a novel approach to study the HPV vaccine.

Human papillomavirus 16 E7 protein inhibits interferon-γ-mediated enhancement of keratinocyte antigen processing and T-cell lysis.

Infection of epithelium with human papillomavirus (HPV) 16 is generally prolonged, suggesting an ineffective virus-specific immune response, and prolonged infection promotes anogenital cancer. To determine whether poor antigen presentation by HPV-infected keratinocytes (KCs) contributes to prolonged HPV infection, KCs and KCs expressing HPV 16 E7 protein (E7-KCs) were compared for susceptibility to T-cell-mediated lysis directed to ovalbumin (OVA) processed for presentation by the KCs. Interferon (IFN)-γ efficiently enhanced susceptibility to lysis of KCs presenting OVA, but not of E7-KCs similarly presenting OVA. E7-KCs also exhibited impaired IFN-γ-induced upregulation of transcription of major histocompatibility complex class I antigen processing and presentation-associated genes, and of membrane SIINFEKL-H-2K(b) complexes. Thus, expression of HPV 16 E7 protein in KCs may inhibit enhancement by IFN-γ of KC sensitivity to T-cell lysis, by impairing antigen presentation.

E6 and E7 fusion immunoglobulin from human papilloma virus 16 induces dendritic cell maturation and antigen specific activation of T helper 1 response.

Human papilloma virus (HPV) 16 causes cervical cancer. Induction of oncogenesis by HPV 16 is primarily dependent on the function of E6 and E7 proteins, which inactivate the function of p53 and pRB, respectively. Thus, blocking the activity of the E6 and E7 proteins from HPV 16 is critical to inhibiting oncogenesis during infection. We have expressed and purified soluble HPV 16 E6 and E7 fusion immunoglobulin (Ig), which were combined with the constant region of an Ig heavy chain, in a mammalian system. To assess whether soluble E6 and E7 fusion Igs induce effective cellular immune responses, immature dendritic cells (DCs) were treated with these fusion proteins. Soluble E6 and E7 fusion Igs effectively induced maturation of DCs. Furthermore, immunization with soluble E6 and E7 fusion Igs in mice resulted in antigen-specific activation of T helper 1 (Th1) cells. This is the first comprehensive study to show the molecular basis of how soluble HPV 16 E6 or E7 fusion Igs induces Th1 responses through the maturation of DCs. In addition, we show that DC therapy using soluble HPV E6 and E7 fusion Igs may be a valuable tool for controlling the progress of cervical cancer.

E7 properties of mucosal human papillomavirus types 26, 53 and 66 correlate with their intermediate risk for cervical cancer development.

Epidemiological studies have demonstrated that 15 different mucosal human papillomavirus (HPV) types of the genus alpha of the HPV phylogetic tree are classified as high risk for cervical cancer development. Three additional HPV types of the same genus, HPV26, 53 and 66, are classified as probable high-risk types. In this study, we have characterized the biological properties of the E7 oncoproteins from these three HPV types. All of the corresponding E7 proteins were able to associate with retinoblastoma protein (pRb) and up-regulated the expression of several positive cell cycle regulators, i.e. CDK2, cyclin A and cylin E. However, HPV26 E7 appears to be more efficient than HPV53 and 66 E7 in up-regulating the transcription of cyclin A. Unlike E7 from the high-risk type HPV16 protein, HPV26, 53 and 66 did not efficiently promote pRb degradation. In addition, E7 from these viruses was able to promote proliferation of primary human keratinocytes and circumvent G1 arrest imposed by overexpression of p16(INK4a), but with less efficiency than the high-risk HPV16 E7. Together, our data show that in vitro properties of these E7 proteins correlate with the epidemiological classification of HPV26, 53 and 66 as HPV types with an intermediate risk for cervical cancer development.

Skin hyperproliferation and susceptibility to chemical carcinogenesis in transgenic mice expressing E6 and E7 of human papillomavirus type 38.

The oncoproteins E6 and E7 of human papillomavirus type 38 (HPV38) display several transforming activities in vitro, including immortalization of primary human keratinocytes. To evaluate the oncogenic activities of the viral proteins in an in vivo model, we generated transgenic mice expressing HPV38 E6 and E7 under the control of the bovine homologue of the human keratin 10 (K10) promoter. Two distinct lines of HPV38 E6/E7-expressing transgenic mice that express the viral genes at different levels were obtained. In both lines, HPV38 E6 and E7 induced cellular proliferation, hyperplasia, and dysplasia in the epidermis. The rate of occurrence of these events was proportional to the levels of HPV38 E6 and E7 expression in the two transgenic lines. Exposure of the epidermis of nontransgenic mice to UV led to p21WAF1 accumulation and cell cycle arrest. In contrast, keratinocytes from transgenic mice continued to proliferate and were not positive for p21WAF1, indicating that cell cycle checkpoints are altered in keratinocytes expressing the viral genes. Although the HPV38 E6/E7-expressing transgenic mice did not develop spontaneous tumors during their life span, two-stage carcinogen treatment led to a high incidence of papillomas, keratoacanthomas, and squamous-cell carcinomas in HPV38 mice compared with nontransgenic animals. Together, these data show that HPV38 E6 and E7 display transforming properties in vivo, providing further support for the role of HPV38 in carcinogenesis.