A DNA vaccine based on a shuffled E7 oncogene of the human papillomavirus type 16 (HPV 16) induces E7-specific cytotoxic T cells but lacks transforming activity

ABSTRACT Recombinant adenovirus or DNA vaccines encoding herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) genetically fused to human papillomavirus type 16 (HPV-16) oncoproteins (E5, E6, and E7) induce antigen-specific CD8+ T-cell responses and confer preventive resistance to transplantable murine tumor cells (TC-1 cells). In the present report, we characterized some previously uncovered aspects concerning the induction of CD8+ T-cell responses and the therapeutic anticancer effects achieved in C57BL/6 mice immunized with pgD-E7E6E5 previously challenged with TC-1 cells. Concerning the characterization of the immune responses elicited in mice vaccinated with pgD-E7E6E5, we determined the effect of the CD4+ T-cell requirement, longevity, and dose-dependent activation on the E7-specific CD8+ T-cell responses. In addition, we determined the priming/boosting properties of pgD-E7E6E5 when used in combination with a recombinant serotype 68 adenovirus (AdC68) vector encoding the same chimeric antigen. Mice challenged with TC-1 cells and then immunized with three doses of pgD-E7E6E5 elicited CD8+ T-cell responses, measured by intracellular gamma interferon (IFN-γ) and CD107a accumulation, to the three HPV-16 oncoproteins and displayed in vivo antigen-specific cytolytic activity, as demonstrated with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled target cells pulsed with oligopeptides corresponding to the H-2Db -restricted immunodominant epitopes of the E7, E6, or E5 oncoprotein. Up to 70% of the mice challenged with 5 × 105 TC-1 cells and immunized with pgD-E7E6E5 controlled tumor development even after 3 days of tumor cell challenge. In addition, coadministration of pgD-E7E6E5 with DNA vectors encoding pGM-CSF or interleukin-12 (IL-12) enhanced the therapeutic antitumor effects for all mice challenged with TC-1 cells. In conclusion, the present results expand our previous knowledge on the immune modulation properties of the pgD-E7E6E5 vector and demonstrate, for the first time, the strong antitumor effects of the DNA vaccine, raising promising perspectives regarding the development of immunotherapeutic reagents for the control of HPV-16-associated tumors.

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Regression of Established Human Papillomavirus Type 16 (HPV-16) Immortalized Tumors In Vivo by Vaccinia Viruses Expressing Different Forms of HPV-16 E7 Correlates with Enhanced CD8+ T-Cell Responses That Home to the Tumor Site

Journal of Virology ◽

10.1128/jvi.75.20.9654-9664.2001 ◽

2001 ◽

Vol 75 (20) ◽

pp. 9654-9664 ◽

Cited By ~ 68

Author(s):

Abigail Lamikanra ◽

Zhen-Kun Pan ◽

Stuart N. Isaacs ◽

Tzyy-Choou Wu ◽

Yvonne Paterson

Keyword(s):

T Cells ◽

Human Papillomavirus ◽

Vaccinia Virus ◽

Antigen Expression ◽

Listeriolysin O ◽

Necrosis Factor Alpha ◽

Specific Lysis ◽

Hpv 16 ◽

Human Papillomavirus Type 16

ABSTRACT Using vaccinia virus as a live vector, we show that the expression of human papillomavirus type 16 (HPV-16) E7 fused to a nonhemolytic portion of the Listeria monocytogenes virulence factor, listeriolysin O (LLO), induces an immune response that causes the regression of established HPV-16 immortalized tumors in C57BL/6 mice. The vaccinia virus construct expressing LLO fused to E7 (VacLLOE7) was compared with two previously described vaccinia virus constructs: one that expresses unmodified E7 (VacE7) and another that expresses E7 in a form designed to direct it to intracellular lysosomal compartments and improve major histocompatibility complex class II-restricted responses (VacSigE7LAMP-1). C57BL/6 mice bearing established HPV-16 immortalized tumors of 5 or 8 mm were treated with each of these vaccines. Fifty percent of the mice treated with VacLLOE7 remained tumor free 2 months after tumor inoculation, whereas 12 to 25% of the mice were tumor free after treatment with VacSigE7LAMP-1 (depending on the size of the tumor). No mice were tumor free in the group given VacE7. Compared to VacE7, VacSigE7LAMP-1 and VacLLOE7 resulted in increased numbers of H2-Db-specific tetramer-positive CD8+ T cells in mouse spleens that produced gamma interferon and tumor necrosis factor alpha upon stimulation with RAHYNIVTF peptide. In addition, the highest frequency of tetramer-positive T cells was seen in the tumor sites of mice treated with VacLLOE7. An increased efficiency of E7-specific lysis by splenocytes from mice immunized with VacLLOE7 was also observed. These results indicate that the fusion of E7 with LLO not only enhances antitumor therapy by improving the tumoricidal function of E7-specific CD8+ T cells but may also increase the number of antigen-specific CD8+ T cells in the tumor, the principle site of antigen expression.

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Identification and functional analysis of sequence rearrangements in the long control region of human papillomavirus type 16 Af-1 variants isolated from Ugandan penile carcinomas

Journal of General Virology ◽

10.1099/0022-1317-81-12-2969 ◽

2000 ◽

Vol 81 (12) ◽

pp. 2969-2982 ◽

Cited By ~ 26

Author(s):

Maria Lina Tornesello ◽

Franco M. Buonaguro ◽

Luigi Buonaguro ◽

Immacolata Salatiello ◽

Elke Beth-Giraldo ◽

...

Keyword(s):

Human Papillomavirus ◽

Control Region ◽

Expression Vectors ◽

Long Control Region ◽

Hpv 16 ◽

Expression Levels ◽

Human Papillomavirus Type 16 ◽

Transforming Activity ◽

E6 And E7 ◽

Cat Expression

Human papillomavirus type 16 (HPV-16) is the predominant HPV isolate found in malignancies of male and female lower genital tracts. However, only a small percentage of individuals infected with high-risk HPVs develop a genital neoplasia, suggesting that additional events at both the cellular and the virus level are necessary for the progression to cancer, including genetic mutations/rearrangements of viral sequences involved in the oncogenic process. In this study, the genetic stability of the long control region (LCR) (nt 7289–114), which regulates expression levels of oncoproteins E6 and E7, was analysed in HPV-16 isolates from penile carcinoma (PC) biopsies of patients recruited from Uganda, one of the countries with the highest incidence of genital cancers in both men and women. Nucleotide changes within the LCR region typical of the African-1 (Af-1) lineage were observed in all HPV-16 isolates. Two out of five samples showed further rearrangements of the enhancer region. The functional activity of LCR with Af-1 mutations and/or rearrangements was evaluated by cloning each LCR into CAT expression vectors, followed by transfection in several epithelial and non-epithelial cell lines. CAT expression levels driven by a rearranged LCR were significantly higher than those driven by Af-1 or European prototype LCRs. Furthermore, in the NIH3T3 focus formation assay, the transforming activity of E6 and E7 genes, driven by a mutated or rearranged LCR, was 1·4- to 3·0-fold higher, respectively. These results indicate that rearrangements within the LCR of HPV-16 isolated from African PCs are frequently found (2 out of 5, 40%). It is also shown that increased HPV LCR activity is associated with an increased E6/E7-mediated in vitro transforming activity, suggesting that natural variants can play a major role in the pathogenesis of genital carcinomas.

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Induction of CD8 T Cells by Vaccination with Recombinant Adenovirus Expressing Human Papillomavirus Type 16 E5 Gene Reduces Tumor Growth

Journal of Virology ◽

10.1128/jvi.74.19.9083-9089.2000 ◽

2000 ◽

Vol 74 (19) ◽

pp. 9083-9089 ◽

Cited By ~ 43

Author(s):

Dai-Wei Liu ◽

Yeou-Ping Tsao ◽

Chang-Hsun Hsieh ◽

Jer-Tsong Hsieh ◽

John T. Kung ◽

...

Keyword(s):

T Cells ◽

Human Papillomavirus ◽

Cd8 T Cells ◽

Recombinant Adenovirus ◽

Tumor Vaccine ◽

Tumor Rejection ◽

Hpv 16 ◽

Human Papillomavirus Type 16 ◽

E5 Protein ◽

Tumor Protection

ABSTRACT The potential of the E5 protein as a tumor vaccine candidate has not been explored yet. In this study, we evaluate the human papillomavirus type 16 (HPV-16) E5 protein delivered by an adenovirus vector as a tumor vaccine for cervical lesions. The results demonstrate that a single intramuscular injection of a recombinant adenovirus carrying the HPV-16 E5 gene into syngeneic animals can reduce the growth of tumors which contain E5 gene expression. Moreover, the E5 vaccine-induced tumor protection occurs through CD8 T cells but not through CD4 T cells in in vitro assays. In addition, our studies using knockout mice with distinct T-cell deficiencies confirm that cytotoxic T-lymphocyte-induced tumor protection is CD8 dependent but CD4 independent. Hence, HPV-16 E5 can be regarded as a tumor rejection antigen.

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Single-Dose, Therapeutic Vaccination of Mice with Vesicular Stomatitis Virus Expressing Human Papillomavirus Type 16 E7 Protein

Clinical and Vaccine Immunology ◽

10.1128/cvi.00343-07 ◽

2008 ◽

Vol 15 (5) ◽

pp. 817-824 ◽

Cited By ~ 17

Author(s):

John B. Liao ◽

Jean Publicover ◽

John K. Rose ◽

Daniel DiMaio

Keyword(s):

T Cells ◽

Single Dose ◽

Human Papillomavirus ◽

Tumor Cells ◽

Vesicular Stomatitis Virus ◽

Cytotoxic T Cells ◽

Therapeutic Vaccination ◽

Human Papillomavirus Type 16 ◽

Vesicular Stomatitis ◽

Syngeneic Tumors

ABSTRACT We are developing recombinant attenuated vesicular stomatitis virus (VSV) as a vaccine vector to generate humoral and cell-mediated immune responses. Here, we explore the use of VSV vaccines for cancer immunotherapy. Immunotherapy targeting high-risk human papillomavirus (HPV) lesions has the potential to benefit HPV-infected individuals and cervical cancer patients by generating cytotoxic T cells that kill tumor cells that express viral antigens. A single dose of VSV expressing the HPV type 16 (HPV16) E7 oncogene was used for therapeutic vaccination of mice bearing TC-1 syngeneic tumors, which express HPV16 E7. HPV16 E7-specific T cells were generated and displayed cytotoxic activity against the tumor cells. By 14 days postvaccination, average tumor volumes were 10-fold less in the vaccinated group than in mice that received the empty-vector VSV, and regression of preexisting tumors occurred in some cases. This antitumor effect was CD8 T-cell dependent. Our results demonstrate antitumor responses to HPV16 E7 and suggest that recombinant-VSV-based vaccination should be explored as a therapeutic strategy for cervical carcinoma and other HPV-associated cancers.

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Nasal Immunization of Mice with Human Papillomavirus Type 16 (HPV-16) Virus-Like Particles or with the HPV-16 L1 Gene Elicits Specific Cytotoxic T Lymphocytes in Vaginal Draining Lymph Nodes

Journal of Virology ◽

10.1128/jvi.73.11.9063-9071.1999 ◽

1999 ◽

Vol 73 (11) ◽

pp. 9063-9071 ◽

Cited By ~ 76

Author(s):

Catherine Dupuy ◽

Dominique Buzoni-Gatel ◽

Antoine Touzé ◽

Daniel Bout ◽

Pierre Coursaget

Keyword(s):

T Cells ◽

Human Papillomavirus ◽

Immune Responses ◽

Self Assembly ◽

Genital Tract ◽

Hpv 16 ◽

Virus Like Particles ◽

Mammalian Expression ◽

Human Papillomavirus Type 16 ◽

L1 Protein

ABSTRACT Human papillomavirus type 16 (HPV-16) infects the genital tract and is closely associated with the development of cervical cancer. HPV-16 initiates infection at the genital mucosal surface; thus, mucosal immune responses are likely to contribute to defense against HPV-16 infection. However, little information is available regarding the induction of immune responses in the genital tract mucosa. In this study, we evaluated the potential of intranasally administered papillomavirus vaccines to elicit both systemic and vaginal immune responses. HPV-16 virus-like particles (VLPs) produced by self-assembly of L1 protein and the HPV-16 L1 gene cloned into a mammalian expression vector were used as vaccines. Intranasally administered VLPs induced serum immunoglobulin G (IgG) and vaginal IgA secretory antibodies. Very weak serum IgG and vaginal IgA responses were found after DNA immunization. Both splenic and vaginal lymphocytes could be activated by intranasal immunization with VLPs and the HPV-16 L1 gene. Activated CD4+ Th1-like T cells were shown to synthesize gamma interferon, and activated CD8+ T cells were demonstrated to be cytotoxic.

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An improved rearranged Human Papillomavirus Type 16 E7 DNA vaccine candidate (HPV-16 E7SH) induces an E7 wildtype-specific T cell response

Vaccine ◽

10.1016/j.vaccine.2005.12.061 ◽

2006 ◽

Vol 24 (15) ◽

pp. 2880-2893 ◽

Cited By ~ 41

Author(s):

Peter Öhlschläger ◽

Michaela Pes ◽

Wolfram Osen ◽

Matthias Dürst ◽

Achim Schneider ◽

...

Keyword(s):

Human Papillomavirus ◽

T Cell ◽

Dna Vaccine ◽

Cell Response ◽

Vaccine Candidate ◽

T Cell Response ◽

Hpv 16 ◽

Human Papillomavirus Type 16

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Multiple ASF/SF2 Sites in the Human Papillomavirus Type 16 (HPV-16) E4-Coding Region Promote Splicing to the Most Commonly Used 3′-Splice Site on the HPV-16 Genome

Journal of Virology ◽

10.1128/jvi.00462-10 ◽

2010 ◽

Vol 84 (16) ◽

pp. 8219-8230 ◽

Cited By ~ 33

Author(s):

Monika Somberg ◽

Stefan Schwartz

Keyword(s):

Cervical Cancer ◽

Human Papillomavirus ◽

Splice Site ◽

Binding Sites ◽

Mrna Levels ◽

Coding Region ◽

Cervical Lesions ◽

Hpv 16 ◽

Human Papillomavirus Type 16 ◽

E6 And E7

ABSTRACT Our results presented here demonstrate that the most abundant human papillomavirus type 16 (HPV-16) mRNAs expressing the viral oncogenes E6 and E7 are regulated by cellular ASF/SF2, itself defined as a proto-oncogene and overexpressed in cervical cancer cells. We show that the most frequently used 3′-splice site on the HPV-16 genome, site SA3358, which is used to produce primarily E4, E6, and E7 mRNAs, is regulated by ASF/SF2. Splice site SA3358 is immediately followed by 15 potential binding sites for the splicing factor ASF/SF2. Recombinant ASF/SF2 binds to the cluster of ASF/SF2 sites. Mutational inactivation of all 15 sites abolished splicing to SA3358 and redirected splicing to the downstream-located, late 3′-splice site SA5639. Overexpression of a mutant ASF/SF2 protein that lacks the RS domain, also totally inhibited the usage of SA3358 and redirected splicing to the late 3′-splice site SA5639. The 15 ASF/SF2 binding sites could be replaced by an ASF/SF2-dependent, HIV-1-derived splicing enhancer named GAR. This enhancer was also inhibited by the mutant ASF/SF2 protein that lacks the RS domain. Finally, silencer RNA (siRNA)-mediated knockdown of ASF/SF2 caused a reduction in spliced HPV-16 mRNA levels. Taken together, our results demonstrate that the major HPV-16 3′-splice site SA3358 is dependent on ASF/SF2. SA3358 is used by the most abundantly expressed HPV-16 mRNAs, including those encoding E6 and E7. High levels of ASF/SF2 may therefore be a requirement for progression to cervical cancer. This is supported by our earlier findings that ASF/SF2 is overexpressed in high-grade cervical lesions and cervical cancer.

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