Cervical Cancer-Associated Human Papillomavirus 16 E7 Oncoprotein Inhibits Induction of Anti-Cancer Immunity by a CD4+ T Cell Dependent Mechanism (original) (raw)
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Gene Therapy, 2000
Infection of genital epithelial cells with human papillomavirus (HPV) types 16 and 18 is closely associated with the development of cervical carcinoma. The transforming potential of these high-risk HPV depends on the expression of the E6 and E7 early viral gene products. Since the expression of E6 and E7 is selectively maintained in premalignant and malignant cervical lesions these proteins are attractive candidates for immunotherapeutic and prophylactic strategies. This report describes the construction, characterization and the in vivo immunotherapeutic potential of recombinant Semliki Forest virus (SFV) expressing the HPV16 E6 and E7 proteins (SFVE6E7). Western blot analysis and immunofluorescence staining demonstrated expression of E6 and E7 in BHK cells infected with SFVE6E7. Immunization of mice with SFVE6E7 resulted in an efficient in vivo priming of HPV-specific CTL activity. The induced CTL lysed murine tumor cells transformed with the HPV16 genome and EL-4 cells loaded with an immunodominant class I-binding HPV E7 peptide. CTL could reproducibly be induced by immunization with three injections of as few as 10 5 infectious units of SFVE6E7. Protection from tumor challenge was studied using the tumor cell line TC-1. Immunization with 5x10 6 SFVE6E7 particles protected 40% of the mice from tumor challenge. These results indicate that E6E7 expression by the efficient and safe recombinant SFV system represents a promising strategy for immunotherapy or immunoprophylaxis of cervical carcinoma.
CD4+ T Cell-Mediated Antigen-Specific Immunotherapy in a Mouse Model of Cervical Cancer
Cancer Research, 2005
A major agenda for tumor immunology is the generation of specific immune responses leading to the destruction of incipient and frank neoplasia. In this report, we show that a novel HPV16 E7 fusion protein can produce objective therapeutic responses against incipient cervical cancer in genetically engineered mice that express in the cervix the HPV16 early region genes implicated as causative agents in human cervical cancer. Although nonresponsive toward the HPV16 E7 oncoprotein in the CD8+ T-cell compartment by virtue of MHC haplotype, the mice were capable of mounting an induced CD4+ T-cell response against E7, and in addition developed spontaneous anti-E7 antibodies. HPV16/CD4−/− mice showed increased tumor burden indicative of CD4-mediated immune surveillance. Seeking to enhance the CD4 response, we immunized mice bearing incipient cervical cancer with a recombinant protein fusing E7 with a mycobacterial heat shock protein. The incidences of cervical carcinoma and of high-grade dy...
Gene Therapy, 2005
Despite promising preclinical results of various therapeutic anticancer immunization strategies, these approaches may not be effective enough to eradicate tumors in cancer patients. While most animal models are based on fastgrowing transplantable tumors, malignancies in, for example, cervical cancer patients in general develop much more slowly, which may lead to immune suppression and/or immune tolerance. As a consequence, the immunomodulating signal of any therapeutic immunization regimen should be sufficiently potent to overcome this immunocompromised condition. In previous studies, we demonstrated that an experimental vaccine against human papillomavirus (HPV)-induced cervical cancer, based on Semliki Forest virus (SFV), induces robust HPV-specific cellular immune responses in mice. Now we studied whether this strategy is potent enough to also prime a cellular immune response in immune-tolerant HPV transgenic mice, in which CTL activity cannot be induced using protein or DNA vaccines. We demonstrate that, depending on the route of immunization, SFV-expressing HPV16 E6 and E7 indeed has the capacity to induce HPV16 E7-specific cytotoxic T cells in HPVtransgenic mice.
Clinical and Vaccine …, 2010
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 antigenspecific 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-␥) 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-2D b-restricted immunodominant epitopes of the E7, E6, or E5 oncoprotein. Up to 70% of the mice challenged with 5 ؋ 10 5 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. Cancers pose unique challenges to therapeutic vaccines. Tumor-associated antigens are often self-antigens to which the patient is tolerant. In the case of virus-associated tumors, the viral oncoproteins commonly lack high-avidity T-cell epitopes and thus can evade immune surveillance. Cancer patients frequently show immunological abnormalities, such as T-cell anergy, peripheral and central tolerance, regulatory T cell (Treg)-mediated immunosuppression, and functional T-cell exhaustion (9, 31, 36). Therapeutic cancer vaccines, unlike prophylactic vaccines, thus need to be formulated not only to induce T-cell responses but also to overcome immunological unresponsiveness to tumor antigens. Cervical cancer is the second most common cause of cancer death in women, claiming approximately 400,000 to 500,000 lives each year worldwide (32). Cervical cancer affects ϳ1% of all women and is the most common cause of cancer death in women under the age of 50. Virtually all cases of cervical cancers are associated with human papillomavirus (HPV) infections (2, 37). Prevalence of sexually transmitted infections
Journal of immunotherapy (Hagerstown, Md. : 1997)
We have previously shown that a novel DNA vaccine technology of codon optimization and the addition of ubiquitin sequences enhanced immunogenicity of a herpes simplex virus 2 polynucleotide vaccine in mice, and induced cell-mediated immunity when administered in humans at relatively low doses of naked DNA. We here show that a new polynucleotide vaccine using the same technology and encoding a fusion protein of the E6 and E7 oncogenes of high-risk human papillomavirus type 16 (HPV16) is immunogenic in mice. This vaccine induces long-lasting humoral and cell-mediated immunity and protects mice from establishment of HPV16-E7-expressing tumors. In addition, it suppresses growth of readily established tumors and shows enhanced efficacy when combined with immune checkpoint blockade targeted at PD-L1. This vaccine also facilitates rejection of HPV16-E7-expressing skin grafts that demonstrate epidermal hyperplasia with characteristics of cervical and vulvar intraepithelial neoplasia. Clinic...
European Journal of Immunology, 2009
The role of thymic versus peripheral epithelium in the regulation of the antigen-specific CD8 T-cell repertoire is still largely unresolved. We generated TCR-b chain transgenic mice in which an increased frequency of peripheral CD8 T cells recognizes an epitope from a viral oncoprotein (HPV16E7) in the context of H-2D b MHC class I. When T cells from these mice developed through the thymus of mice expressing functional E7 protein from a keratin 14 promoter, no major perturbation to transgenic T-cell development in the thymus was observed in these double-transgenic mice. In contrast, peripheral CD8 T-cell responses in the single-transgenic, K14E7 mice, including those unrelated to E7 antigen, are reduced whereas CD4 T-cell responses and antibody production are unchanged in these mice. Peripheral non-responsiveness among CD8 T cells was mediated largely by CD4 1 CD25 1 T cells. This suggested that epithelium expressing HPV16E7 protein induces Treg that specifically down-regulate CD8 T-cell responses in the periphery. This may have important consequences for the treatment of cervical pre-cancers and provides a model for understanding differential suppression of T and B lymphocyte subsets by Treg.
Iranian journal of cancer prevention
Cervical cancer has been shown to be highly associated with human papillomavirus (HPV) infection. The viral oncogenes E6 and E7 are constantly expressed by the tumor cells and are therefore potent targets for therapeutic genetic vaccination. In the present study, it was investigated the potential effect of HPV-16 E6, E7 and L1 co-administration to activate specific cytotoxic T lymphocytes in tumor mice models. The HPV-16 E6, E7 and L1 genes from Iranian isolate were separately inserted into the mammalian expression vector, pcDNA3, to construct the DNA vaccine candidates. Tumor-bearing Animals (C57BL/6 mice) were immunized with the vaccine candidate; then, Lymphocyte Proliferation Assay (LPA) and relative tumor volume measurements were carried out in order to examine the immunological effects of the vaccine. Obtained results showed that co-administration of the HPV-16 E6, E7 and L1 DNA induced HPV-16 specific cellular immune responses and also protected against TC-1-induced tumor in ...
European Journal of Immunology, 1994
We have used a mouse model that utilizes the exclusively epithelial nature of human papillomavirus (HPV) infections to investigate the in vivo immune response to the E7 protein of human papillomavirus type-16. A keratinocyte cell line expressing E7 protein has been established and grafted onto syngeneic mice using a transplantation technique that permits the reformation of a differentiated epithelium on a granulation tissue bed. In this way viral antigens may be presented to the immune system in a way comparable to natural infection. A delayed-type hypersensitivity (DTH) response was studied post grafting by intradermal challenge with recombinant E7 protein. A significant response to E7 has been demonstrated in this way; however, priming with a low amount of HPV-16 E7 antigen induces immunological unresponsiveness, as measured by a loss of DTH reactivity to the protein, and persistence of keratinocytes expressing E7. Lymphocytes from mice exhibiting DTH reactivity have been shown to proliferate when stimulated with purified recombinant E7 protein in vitro, while immunoperoxidase staining of tissue from the sites of immunologically-induced inflammation has defined the cell infiltrate to be phenotypically characteristic of DTH. The observations reported here have important implications for vaccine strategy.
Clinical Cancer Research, 2008
The study aims to evaluate the effect of a human papillomavirus type 16 (HPV16) E6 and E7 synthetic long peptides vaccine on the antigen-specificT-cell response in cervical cancer patients. Experimental Design: Patients with resected HPV16-positive cervical cancer were vaccinated with an overlapping set of long peptides comprising the sequences of the HPV16 E6 and E7 oncoproteins emulsified in Montanide ISA-51. HPV16-specificT-cell immune responses were analyzed by evaluating the magnitude, breadth, type, and polarization by proliferation assays, IFNg-ELISPOT, and cytokine production and phenotyped by theT-cell markers CD4, CD8, CD25, and Foxp3. Results: Vaccine-induced T-cell responses against HPV16 E6 and E7 were detected in six of six and five of six patients, respectively.These responses were broad, involved both CD4 + and CD8 + T cells, and could be detected up to 12 months after the last vaccination. The vaccine-induced responses were dominated by effector type CD4 + CD25 + Foxp3type 1cytokine IFNg-producing Tcells but also included the expansion of Tcells with a CD4 + CD25 + Foxp3 + phenotype. Conclusions: The HPV16 E6 and E7 synthetic long peptides vaccine is highly immunogenic, in that it increases the number and activity of HPV16-specific CD4 + and CD8 + Tcells to a broad array of epitopes in all patients. The expansion of CD4 + and CD8 + tumor-specific T cells, both considered to be important in the antitumor response, indicates the immunotherapeutic potential of this vaccine. Notably, part of the vaccine-inducedTcells display a CD4 + CD25 + Foxp3 + phenotype that is frequently associated with regulatory T-cell function, suggesting that strategies to disarm this subset of Tcells should be considered as components of immunotherapeutic modalities against HPV-induced cancers. Cervical cancer is the second most common cancer in women worldwide (1), and it is the first cancer recognized by the WHO to be 100% attributable to the infection with a high-risk type of human papillomavirus (HPV; ref. 2). HPV type 16 (HPV16) is the most common carcinogenic type and is found in around 50% of invasive cervical tumors worldwide (3, 4). The occurrence of HPV-induced cancer is strongly associated with failure to mount a strong HPV-specific type 1 T-helper and CTL response (5-7), the lack of CD8 + T cells migrating into the tumor, the induction of HPV16-specific regulatory T cells, and the influx of regulatory T cells into the tumor (8, 9). Currently, a preventive vaccine, providing protection against HPV16 and HPV18, has been registered. This vaccine is fully protective against persistent infection and the associated development of high-grade genital lesions (reviewed in ref. 10). Notably, the prophylactic vaccine showed no benefit in women who were already infected with the HPV types covered by this vaccine (reviewed in ref. 10). Therefore, there is still a need for a so-called therapeutic vaccine that is able to protect those who are already infected. Not only because the lack of resources among the nations and individuals that have the greatest need will limit the benefits of prophylactic vaccination (reviewed in ref. 10), but also because an estimated 5 million cervical cancer deaths will occur in the next 20 years due to existing HPV infections (11). All HPV16-induced cervical cancer cells expresses the HPV16 E6 and E7 oncoproteins (3), which makes these proteins excellent target antigens for T cell-mediated immunotherapeutic strategies (12). We have developed a long E6 and E7