RESUMO
The generation of successful anticancer vaccines relies on the ability to induce efficient and long-lasting immune responses to tumor antigens. In this scenario, dendritic cells (DCs) are essential cellular components in the generation of antitumor immune responses. Thus, delivery of tumor antigens to specific DC populations represents a promising approach to enhance the efficiency of antitumor immunotherapies. In the present study, we employed antibody-antigen conjugates targeting a specific DC C-type lectin receptor. For that purpose, we genetically fused the anti-DEC205 monoclonal antibody to the type 16 human papillomavirus (HPV-16) E7 oncoprotein to create a therapeutic vaccine to treat HPV-associated tumors in syngeneic mouse tumor models. The therapeutic efficacy of the αDEC205-E7 mAb was investigated in three distinct anatomical tumor models (subcutaneous, lingual and intravaginal). The immunization regimen comprised two doses of the αDEC205-E7 mAb coadministered with a DC maturation stimulus (Polyinosinic:polycytidylic acid, poly (I:C)) as an adjuvant. The combined immunotherapy produced robust antitumor effects on both the subcutaneous and orthotopic tumor models, stimulating rapid tumor regression and long-term survival. These outcomes were related to the activation of tumor antigen-specific CD8+ T cells in both systemic compartments and lymphoid tissues. The αDEC205-E7 antibody plus poly (I:C) administration induced long-lasting immunity and controlled tumor relapses. Our results highlight that the delivery of HPV tumor antigens to DCs, particularly via the DEC205 surface receptor, is a promising therapeutic approach, providing new opportunities for the development of alternative immunotherapies for patients with HPV-associated tumors at different anatomical sites.
Assuntos
Antígenos CD/imunologia , Vacinas Anticâncer/administração & dosagem , Células Dendríticas/imunologia , Lectinas Tipo C/imunologia , Antígenos de Histocompatibilidade Menor/imunologia , Neoplasias Experimentais/prevenção & controle , Proteínas E7 de Papillomavirus/imunologia , Infecções por Papillomavirus/prevenção & controle , Receptores de Superfície Celular/imunologia , Adjuvantes Imunológicos , Animais , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Feminino , Humanos , Memória Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/virologia , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/virologia , Poli I-C/administração & dosagemRESUMO
The presence of IL-10, produced either by tumor cells or immunosuppressive cells, is frequently associated with a poor prognosis for cancer progression. It may also negatively impact anticancer treatments, such as immunotherapies, that otherwise would promote the activation of cytotoxic T cells capable of detecting and destroying malignant cells. In the present study, we evaluated a new adjuvant approach for anticancer immunotherapy using a plasmid vector encoding a soluble form of the IL-10 receptor (pIL-10R). pIL-10R was coadministered to mice with a DNA vaccine encoding the type 16 human papillomavirus (HPV-16) E7 oncoprotein genetically fused with glycoprotein D of herpes simplex virus (HSV) (pgDE7h). Immunization regimens based on the coadministration of pIL-10R and pgDE7h enhanced the antitumor immunity elicited in mice injected with TC-1 cells, which express HPV-16 oncoproteins. The administration of the DNA vaccines by in vivo electroporation further enhanced the anticancer effects of the vaccines, leading to the activation of tumor-infiltrating polyfunctional E7-specific cytotoxic CD8+ T cells and control of the expansion of immunosuppressive cells. In addition, the combination of immunotherapy and pIL-10R allowed the control of tumors in more advanced growth stages that otherwise would not be treatable by the pgDE7h vaccine. In conclusion, the proposed treatment involving the expression of IL-10R enhanced the antitumor protective immunity induced by pgDE7h administration and may contribute to the development of more efficient clinical interventions against HPV-induced tumors.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Células Epiteliais/fisiologia , Papillomavirus Humano 16/fisiologia , Imunoterapia/métodos , Neoplasias Experimentais/imunologia , Infecções por Papillomavirus/imunologia , Vacinas contra Papillomavirus/imunologia , Receptores de Interleucina-10/imunologia , Animais , Tolerância Imunológica , Interleucina-10/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Proteínas E7 de Papillomavirus/genética , Vacinas contra Papillomavirus/genética , Receptores de Interleucina-10/genética , Vacinas de DNA , Proteínas do Envelope Viral/genéticaRESUMO
In vivo electroporation (EP) has reignited the clinical interest on DNA vaccines as immunotherapeutic approaches to control different types of cancer. EP has been associated with increased immune response potency, but its capacity in influencing immunomodulation remains unclear. Here we evaluated the impact of in vivo EP on the induction of cellular immune responses and therapeutic effects of a DNA vaccine targeting human papillomavirus-induced tumors. Our results demonstrate that association of EP with the conventional intramuscular administration route promoted a more efficient activation of multifunctional and effector memory CD8+ T cells with enhanced cytotoxic activity. Furthermore, EP increased tumor infiltration of CD8+ T cells and avoided tumor recurrences. Finally, our results demonstrated that EP promotes local migration of antigen presenting cells that enhances with vaccine co-delivery. Altogether the present evidences shed further light on the in vivo electroporation action and its impact on the immunogenicity of DNA vaccines.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Eletroporação/métodos , Memória Imunológica , Neoplasias/terapia , Vacinas contra Papillomavirus/administração & dosagem , Vacinas de DNA/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Movimento Celular , Citotoxicidade Imunológica , Humanos , Imunidade Celular , Imunogenicidade da Vacina , Injeções Intramusculares , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Neoplasias/imunologia , Neoplasias/virologia , Papillomaviridae/imunologia , Papillomaviridae/isolamento & purificação , Papillomaviridae/fisiologia , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/imunologia , Vacinas contra Papillomavirus/uso terapêutico , Recidiva , Vacinação/métodos , Vacinas de DNA/administração & dosagemRESUMO
Cervical cancer is a major public health problem and one of the leading causes of cancer deaths in women. Virtually all cases of cervical cancer, as well as a growing share of anal and head/neck tumors, are associated with human papillomavirus (HPV) infection. Despite the effectiveness, the available prophylactic vaccines do not benefit women with cervical lesions or cancer. Therefore, the search of new immunotherapeutic approaches to treat HPV-induced tumors is still a priority. The present study characterizes a therapeutic antitumor vaccine based on the genetic fusion of the Herpes simplex virus-1 (HSV-1) glycoprotein D (gD) with the E7 oncoprotein from HPV-16 (gDE7). Two subcutaneous doses of gDE7, admixed with poly (I:C), conferred complete and long-lasting therapeutic antitumor protection on mice previously challenged with tumor cells expressing the HPV-16 oncoproteins. The vaccine induced multifunctional E7-specific CD8+ T cells with cytotoxic activity and effector memory phenotype (CD44+ CD62Llow). In addition, gDE7 admixed with poly (I:C) vaccination controlled the expansion of tumor-induced regulatory T cells and myeloid-derived suppressor cells. More importantly, gDE7 activated mouse CD11c+ CD8α+ and human BDCA3+ dendritic cells (DC), specialized in antigen cross-presentation to CD8+ T cells, under in vitro conditions. These results indicated that the activation of a specific DC population, mediated by gD, improved the antigen-specific immune responses and the therapeutic performance induced by antitumor vaccines. These results open perspectives for the clinical testing of gDE7-based vaccines under the concept of active immunization as a tool for the therapeutic control of cancer. Mol Cancer Ther; 16(9); 1922-33. ©2017 AACR.
Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Neoplasias/etiologia , Neoplasias/patologia , Papillomaviridae/imunologia , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Apresentação Cruzada/imunologia , Células Dendríticas/metabolismo , Feminino , Humanos , Imunização , Memória Imunológica , Camundongos , Camundongos Knockout , Neoplasias/terapia , Proteínas E7 de Papillomavirus/imunologia , Poli I-C , Especificidade do Receptor de Antígeno de Linfócitos T , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Active anticancer immunotherapeutic approaches have been shown to induce cellular or humoral immune responses in patients, but, thus far, the observed outcomes did not ensure their recommendation for clinical use. The induction of tumor-specific CD8(+) T cells, although required for the clearance of most solid tumors, was shown to be insufficient for the development of a successful immunotherapeutic approach. The suppressive immune environment triggered by tumors, including the expansion of myeloid-derived suppressor cells (MDSC), is detrimental to the development of antitumor immune responses and precludes the generation of more promising clinical outcomes. In this work, we characterized the CD8(+) T-cell population specifically involved in the control of tumor growth and the role of MDSCs after administration of an antitumor therapeutic DNA vaccine targeting human papillomavirus type 16 (HPV-16)-associated tumors. Activation of cytotoxic high-avidity CD8(+) T cells with an effector memory phenotype was found in mice grafted with tumor cells expressing the HPV-16 oncoproteins. In addition, MDSC antibody depletion further enhanced the immunotherapeutic effects of the vaccine, resulting in the complete eradication of tumor cells. Collectively, the current results indicate that the simultaneous control of MDSCs and activation of high-avidity tumor-specific effector memory CD8(+) T cells are key features for tumor protection by immunotherapeutic approaches and deserve further testing under clinical conditions. Mol Cancer Ther; 15(8); 1920-30. ©2016 AACR.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Papillomavirus Humano 16/imunologia , Memória Imunológica , Ativação Linfocitária/imunologia , Células Supressoras Mieloides/imunologia , Infecções por Papillomavirus/imunologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Imunização , Interferon gama/biossíntese , Camundongos , Células Supressoras Mieloides/metabolismo , Neoplasias/etiologia , Neoplasias/mortalidade , Neoplasias/patologia , Neoplasias/terapia , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/virologia , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Carga Tumoral/imunologia , Vacinas de DNA/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Bacillus subtilis spores represent a suitable platform for the adsorption of proteins, enzymes and viral particles at physiological conditions. In the present work, we demonstrate that purified spores can also adsorb DNA on their surface after treatment with cationic molecules. In addition, we demonstrate that DNA-coated B. subtilis spores can be used as particulate carriers and act as an alternative to gold microparticles for the biolistic (gene gun) administration of plasmid DNA in mice. Gene gun delivery of spores pre-treated with DODAB (dioctadecyldimethylammonium bromide) allowed efficient plasmid DNA absorption and induced protein expression levels similar to those obtained with gold microparticles. More importantly, we demonstrated that a DNA vaccine adsorbed on spores can be loaded into biolistic cartridges and efficiently delivered into mice, which induced specific cellular and antibody responses. Altogether, these data indicate that B. subtilis spores represent a simple and low cost alternative for the in vivo delivery of DNA vaccines by the gene gun technology.
Assuntos
Biolística/métodos , Portadores de Fármacos/química , Esporos Bacterianos/química , Vacinas de DNA/química , Adsorção , Animais , Bacillus subtilis/química , Portadores de Fármacos/administração & dosagem , Ouro/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Compostos de Amônio Quaternário/química , Esporos Bacterianos/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologiaRESUMO
Recently, Bacillus subtilis spores were shown to be endowed with strong adjuvant capacity when co-administered with purified antigenic proteins. In the present study we assessed whether spores possess adjuvant properties when combined with DNA vaccines. We showed that B. subtilis spores promoted the activation of dendritic cells in vitro and induced migration of pro-inflammatory cells after parenteral administration to mice. Likewise, co-administration of spores with a DNA vaccine encoding the human papillomavirus type 16 (HPV-16) E7 protein enhanced the activation of antigen-specific CD8(+) T cell responses in vivo. Mice immunized with the DNA vaccine admixed with spores presented a protective immunity increase to previously implanted tumor cells, capable of expressing HPV-16 oncoproteins. Finally, we observed that the adjuvant effect can vary accordingly to the number of co-administered spores which may be ascribed with the ability to induce. Collectively, the present results demonstrate for the first time that B. subtilis spores can also confer adjuvant effects to DNA vaccines.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Bacillus subtilis/imunologia , Esporos Bacterianos/imunologia , Vacinas de DNA/imunologia , Animais , Bacillus subtilis/fisiologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Citocinas/imunologia , Células Dendríticas/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Interferon gama/imunologia , Masculino , Camundongos Endogâmicos C57BL , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/imunologia , Vacinas de DNA/administração & dosagemRESUMO
Millions of people worldwide are currently infected with human papillomavirus (HPV), herpes simplex virus (HSV) or human immunodeficiency virus (HIV). For this enormous contingent of people, the search for preventive and therapeutic immunological approaches represents a hope for the eradication of latent infection and/or virus-associated cancer. To date, attempts to develop vaccines against these viruses have been mainly based on a monovalent concept, in which one or more antigens of a virus are incorporated into a vaccine formulation. In the present report, we designed and tested an immunization strategy based on DNA vaccines that simultaneously encode antigens for HIV, HSV and HPV. With this purpose in mind, we tested two bicistronic DNA vaccines (pIRES I and pIRES II) that encode the HPV-16 oncoprotein E7 and the HIV protein p24 both genetically fused to the HSV-1 gD envelope protein. Mice i.m. immunized with the DNA vaccines mounted antigen-specific CD8⺠T cell responses, including in vivo cytotoxic responses, against the three antigens. Under experimental conditions, the vaccines conferred protective immunity against challenges with a vaccinia virus expressing the HIV-derived protein Gag, an HSV-1 virus strain and implantation of tumor cells expressing the HPV-16 oncoproteins. Altogether, our results show that the concept of a trivalent HIV, HSV, and HPV vaccine capable to induce CD8⺠T cell-dependent responses is feasible and may aid in the development of preventive and/or therapeutic approaches for the control of diseases associated with these viruses.
Assuntos
Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/prevenção & controle , Herpes Simples/prevenção & controle , Infecções por Papillomavirus/prevenção & controle , Vacinas de DNA/imunologia , Vacinas de DNA/uso terapêutico , Vacinas contra a AIDS/imunologia , Vacinas contra a AIDS/uso terapêutico , Alphapapillomavirus/genética , Alphapapillomavirus/imunologia , Animais , Antígenos Virais/genética , Feminino , HIV/genética , HIV/imunologia , Infecções por HIV/imunologia , Herpes Simples/imunologia , Vacinas contra o Vírus do Herpes Simples/imunologia , Vacinas contra o Vírus do Herpes Simples/uso terapêutico , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infecções por Papillomavirus/imunologia , Vacinas contra Papillomavirus/imunologia , Vacinas contra Papillomavirus/uso terapêutico , Simplexvirus/genética , Simplexvirus/imunologia , Vacinas de DNA/genéticaRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Herpesvirus Humano 1 , Papillomavirus Humano 16 , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/farmacologia , Linfócitos T/efeitos dos fármacos , Vacinas de DNA/imunologia , Proteínas do Envelope Viral/genética , Animais , Antígenos CD8/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas E7 de Papillomavirus/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Linfócitos T/imunologia , Proteínas do Envelope Viral/farmacologiaRESUMO
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 × 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.