Immune Responses and Therapeutic Antitumor Effects of an Experimental DNA Vaccine Encoding Human Papillomavirus Type 16 Oncoproteins Genetically Fused to Herpesvirus Glycoprotein D

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-2D^b-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.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 ∼1% 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 with oncogenic genotypes of HPV varies from 20 to 80% of sexually active adults depending on the study population, with HPV type 16 (HPV-16) representing the most epidemiological relevant oncogenic virus type (2, 37). Two vaccines that express the major capsid protein-inducing serotype-specific HPV neutralizing antibodies have recently become available for preventive vaccination (14). While these vaccines can prevent virus infections with the corresponding HPV genotypes, they cannot affect viral clearance in already infected women or inhibit the development of HPV-associated malignancies. In contrast, therapeutic vaccines targeting HPV oncoproteins, mainly E6 and E7, which are expressed by all transformed epithelial cells, can activate antigen-specific cytotoxic CD8⁺ T-cell responses and eradicate infected cells before or after the malignant transformation event (18, 21, 27). In contrast to the conventional vaccines, such oncoprotein-based anti-HPV vaccine formulations may be used under either preventive or therapeutic conditions regarding the establishment and uncontrolled growth of the cancer cells under both experimental or clinical conditions (18).Although DNA vaccines have been intensively studied as a promising immunization strategy for the control of HPV-associated tumors, vectors expressing HPV-16 E7 or E6 oncoprotein alone have shown low antigen-specific CD8⁺ T-cell activation and lack of protective antitumor effects in mice (5, 21, 30). Improved activation of antigen-specific CD8⁺ T-cell responses by anti-HPV DNA vaccines were achieved after genetic fusion of the E7 or E6 oncoprotein with different carrier proteins carrying cell targeting signals or mediators of immune responses (8-19). The focus of our DNA vaccines targeting HPV-induced cancers has been the augmentation of adaptive immune responses through the blockade of an immune inhibitory pathway based on the expression of hybrid proteins genetically fused with glycoprotein D (gD) of herpes simplex virus (HSV) (24,25). HSV gD binds the herpes virus entry mediator (HVEM) and competes for the same binding site as the B- and T-lymphocyte attenuator (BTLA). BTLA provides inhibitory signals to T and B cells upon binding to HVEM (7, 38). Blockade of the HVEM-BTLA pathway during activation of an adaptive immune response has been associated with increased immune responses, particularly E7-specific CD8⁺ T-cell responses, to the antigen encoded by the DNA vaccine (25).We reported previously that expression of HPV-16 E5, E6, and E7 oncoproteins within HSV type 1 (HSV-1) gD, either by an adenovirus vector or a DNA vaccine, induces a potent CD8⁺ T-cell response that confers preventive protection to mice challenged with transplantable E6- and E7-expressing TC-1 cells (25). Herein we characterized previously unknown aspects of the antigen-specific immune responses elicited in mice immunized with the DNA vaccine and report, for the first time, the therapeutic antitumor effects of the pgD-E7E6E5 vector in mice. The results indicate that the DNA vaccine encoding chimeric oncoproteins genetically fused to the HSV-1 gD protein represents a promising approach for the therapeutic control of HPV-associated tumors.