RESUMEN
BACKGROUND: In vivo targeting of human papillomavirus (HPV) derived antigens to dendritic cells might constitute an efficient immunotherapeutic strategy against cervical cancer. In previous works, we have shown that the extra domain A from murine fibronectin (mEDA) can be used to target antigens to toll-like receptor 4 (TLR4) expressing dendritic cells and induce strong antigen-specific immune responses. In the present study, we have produced a bivalent therapeutic vaccine candidate consisting of the human EDA (hEDA) fused to E7 proteins from HPV16 and HPV18 (hEDA-HPVE7-16/18) and evaluate its potential as a therapeutic vaccine against cervical cancer. MATERIALS AND METHODS: Recombinant fusion proteins containing HPV E7 proteins from HPV16 and HPV18 virus subtypes fused to hEDA were produced and tested in vitro on their capacity to bind TLR4 and induce the production of tumor necrosis factor-α or interleukin (IL)-12 by human monocytes and dendritic cells. The immunogenicity and potential therapeutic activity of the vaccine in combination with cisplatin or with the TLR3 agonist molecules polyinosinic-polycytidylic acid (Poly IC) or Poly ICLC was evaluated in mice bearing subcutaneous or genital orthotopic HPV16 TC-1 tumors. RESULTS: hEDA-HPVE7-16/18 prototype vaccine binds human TLR4 and stimulate TLR4-dependent signaling pathways and IL-12 production by human monocyte-derived dendritic cell. Vaccination with hEDA-HPVE7-16/18 induced strong HPVE7-specific Cytotoxic T lymphocyte (CTL) responses and eliminated established tumors in the TC-1-based tumor model. The antitumor efficacy was significantly improved by combining the fusion protein with cisplatin or with the TLR-3 ligand Poly IC and especially with the stabilized analog Poly ICLC. Moreover, hEDA-HPVE7-16/18+Poly ICLC induced full tumor regression in 100% of mice bearing orthotopic genital HPV tumors. CONCLUSION: Our results suggest that this therapeutic vaccine formulation may be an effective treatment for cervical tumors that do not respond to current therapies.
Asunto(s)
Vacunas contra el Cáncer/administración & dosificación , Proteínas de Unión al ADN/inmunología , Fibronectinas/inmunología , Neoplasias Experimentales/prevención & control , Proteínas Oncogénicas Virales/inmunología , Proteínas E7 de Papillomavirus/inmunología , Infecciones por Papillomavirus/prevención & control , Animales , Vacunas contra el Cáncer/inmunología , Células Dendríticas/inmunología , Femenino , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/inmunología , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/inmunología , Humanos , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/virología , Infecciones por Papillomavirus/inmunología , Infecciones por Papillomavirus/virología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
The development of tools for efficient targeting of antigens to antigen presenting cells is of great importance for vaccine development. We have previously shown that fusion proteins containing antigens fused to the extra domain A from fibronectin (EDA), an endogenous TLR4 ligand, which targets antigens to TLR4-expressing dendritic cells (DC), are highly immunogenic. To facilitate the procedure of joining EDA to any antigen of choice, we have prepared the fusion protein EDAvidin by linking EDA to the N terminus of streptavidin, allowing its conjugation with biotinylated antigens. We found that EDAvidin, as streptavidin, forms tetramers and binds biotin or biotinylated proteins with a Kd ~ 2.6 × 10(-14) mol/L. EDAvidin favours the uptake of biotinylated green fluorescent protein by DC. Moreover, EDAvidin retains the proinflammatory properties of EDA, inducing NF- κß by TLR4-expressing cells, as well as the production of TNF- α by the human monocyte cell line THP1 and IL-12 by DC. More importantly, immunization of mice with EDAvidin conjugated with the biotinylated nonstructural NS3 protein from hepatitis C virus induces a strong anti-NS3 T cell immune response. These results open a new way to use the EDA-based delivery tool to target any antigen of choice to DC for vaccination against infectious diseases and cancer.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Fibronectinas/inmunología , Proteínas Recombinantes de Fusión/inmunología , Receptor Toll-Like 4/inmunología , Animales , Células Presentadoras de Antígenos/metabolismo , Línea Celular , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Fibronectinas/química , Fibronectinas/genética , Hepacivirus/inmunología , Humanos , Inmunoterapia Activa , Ligandos , Ratones , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Estreptavidina/química , Linfocitos T/inmunología , Linfocitos T/patología , Receptor Toll-Like 4/metabolismoRESUMEN
The Extradomain A from fibronectin (EDA) has an immunomodulatory role as fusion protein with viral and tumor antigens, but its effect when administered with bacteria has not been assessed. Here, we investigated the adjuvant effect of EDA in mice immunizations against Salmonella enterica subspecies enterica serovar Enteritidis (Salmonella Enteritidis). Since lipopolysaccharide (LPS) is a major virulence factor and the LPS O-polysaccharide (O-PS) is the immunodominant antigen in serological diagnostic tests, Salmonella mutants lacking O-PS (rough mutants) represent an interesting approach for developing new vaccines and diagnostic tests to differentiate infected and vaccinated animals (DIVA tests). Here, antigenic preparations (hot-saline extracts and formalin-inactivated bacterins) from two Salmonella Enteritidis rough mutants, carrying either intact (SEΔwaaL) or deep-defective (SEΔgal) LPS-Core, were used in combination with EDA. Biotinylated bacterins, in particular SEΔwaaL bacterin, decorated with EDAvidin (EDA and streptavidin fusion protein) improved the protection conferred by hot-saline or bacterins alone and prevented significantly the virulent infection at least to the levels of live attenuated rough mutants. These findings demonstrate the adjuvant effect of EDAvidin when administered with biotinylated bacterins from Salmonella Enteritidis lacking O-PS and the usefulness of BEDA-SEΔwaaL as non-live vaccine in the mouse model.
Asunto(s)
Vacunas Bacterianas/inmunología , Fibronectinas/inmunología , Lipopolisacáridos/inmunología , Salmonella enteritidis/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/genética , Femenino , Fibronectinas/química , Lipopolisacáridos/genética , Ratones , Ratones Endogámicos BALB C , Estructura Terciaria de Proteína , Salmonella enteritidis/genética , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunologíaRESUMEN
The complement system and Toll-like receptors (TLR) are key innate defense systems which might interact synergistically on dendritic cells (DC) to reinforce adaptive immunity. In a previous work, we found that the extra domain A from fibronectin EDA (an endogenous ligand for TLR4) can favour antigen delivery to DC and induce their maturation. Given the potential of anaphylatoxins to cause inflammation and activation of myeloid cells, we hypothesized that a fusion protein between EDA, and anaphylatoxins C3a, C4a or C5a together with an antigen might improve the immunogenicity of the antigen. Naked DNA immunization with a construct expressing the fusion protein between C5a, EDA and the cytotoxic T cell epitope SIINFEKL from ovalbumin, induced strong antigen specific T cell responses. The purified recombinant fusion protein EDA-SIINFEKL-C5a induced activation of dendritic cells, the production of proinflammatory cytokines/chemokines and stimulated antigen presenting cell migration and NK cell activation. As compared to EDA-SIINFEKL, the fusion protein EDA-SIINFEKL-C5a did not induce the production of the immunosuppressive molecules IL-10, CCL17, CCL1, CXCL12 or XCL1 by DC. Moreover, EDA-SIINFEKL-C5a induced strong specific T cell responses in vivo and protected mice against E.G7-OVA tumor growth more efficiently than EDA-SIINFEKL or SIINFEKL-C5a recombinant proteins. Our results suggest that fusion proteins containing EDA, the anaphylatoxin C5a and the antigen may serve as a suitable strategy for the development of anti-tumor or anti-viral vaccines.
Asunto(s)
Anafilatoxinas/inmunología , Complemento C5a/inmunología , Ectodisplasinas/inmunología , Linfocitos T Citotóxicos/inmunología , Receptor Toll-Like 4/agonistas , Anafilatoxinas/genética , Animales , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Complemento C5a/genética , Citocinas/metabolismo , Ectodisplasinas/genética , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Femenino , Ratones , Ratones Endogámicos C57BL , Neoplasias/inmunología , Neoplasias/prevención & control , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunologíaRESUMEN
The development of effective vaccines against HIV-1 infection constitutes one of the major challenges in viral immunology. One of the protein candidates in vaccination against this virus is p24, since it is a conserved HIV antigen that has cytotoxic and helper T cell epitopes as well as B cell epitopes that may jointly confer enhanced protection against infection when used in immunization-challenge approaches. In this context, the adjuvant effect of EDA (used as EDAp24 fusion protein) and poly(I:C), as agonists of TLR4 and TLR3, respectively, was assessed in p24 immunizations using a recombinant Listeria monocytogenes HIV-1 Gag proteins (Lm-Gag, where p24 is the major antigen) for challenge in mice. Immunization with EDAp24 fusion protein together with poly(I:C) adjuvant induced a specific p24 IFN-γ production (Th1 profile) as well as protection against a Lm-Gag challenge, suggesting an additive or synergistic effect between both adjuvants. The combination of EDA (as a fusion protein with the antigen, which may favor antigen targeting to dendritic cells through TLR4) and poly(I:C) could thus be a good adjuvant candidate to enhance the immune response against HIV-1 proteins and its use may open new ways in vaccine investigations on this virus.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Fibronectinas/farmacología , Productos del Gen gag/inmunología , Proteína p24 del Núcleo del VIH/inmunología , Poli I-C/farmacología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/química , Animales , Antígenos Virales/inmunología , Femenino , Fibronectinas/administración & dosificación , Fibronectinas/química , Proteína p24 del Núcleo del VIH/administración & dosificación , Proteína p24 del Núcleo del VIH/química , Inmunidad Celular/inmunología , Listeria monocytogenes/inmunología , Listeriosis/prevención & control , Ratones , Ratones Endogámicos BALB C , Poli I-C/administración & dosificación , Poli I-C/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Cervical carcinoma is one of the most common cancers in women worldwide. It is well established that chronic infection of the genital tract by various mucosatropic human papillomavirus (HPV) types causes cervical cancer. Cellular immunity to E7 protein from HPV (HPVE7) has been associated with clinical and cytologic resolution of HPV-induced lesions. Thus, we decided to test if targeting of HPVE7 to dendritic cells using a fusion protein containing the extra domain A (EDA) from fibronectin, a natural ligand for TLR4, and HPVE7 (EDA-HPVE7) might be an efficient vaccine for the treatment of cervical carcinoma. We found that EDA-HPVE7 fusion protein was efficiently captured by bone marrow derived dendritic cells in vitro and induced their maturation, with the upregulation of maturation markers and the production of IL-12. Immunization of mice with EDA-HPVE7 fusion protein induced antitumor CD8(+) T cell responses in the absence of additional adjuvants. Repeated intratumoral administration of EDA-HPVE7 in saline was able to cure established TC-1 tumors of 5-7 mm in diameter. More importantly, intravenous injection with EDA-HPVE7 in combination with the TLR ligand polyinosinic-polycytidylic acid (pIC), or with low doses of cyclophosphamide and the TLR9 ligand CpG-B complexed in cationic lipids, were able to eradicate large established TC-1 tumors (1.2 cm in diameter). Thus, therapeutic vaccination with EDA-HPVE7 fusion protein may be effective in the treatment of human cervical carcinoma.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Fibronectinas/inmunología , Proteínas E7 de Papillomavirus/inmunología , Vacunas contra Papillomavirus/inmunología , Neoplasias del Cuello Uterino/inmunología , Neoplasias del Cuello Uterino/terapia , Adyuvantes Inmunológicos , Animales , Células de la Médula Ósea/inmunología , Linfocitos T CD8-positivos/inmunología , Ciclofosfamida/farmacología , Células Dendríticas/inmunología , Femenino , Fibronectinas/metabolismo , Células HEK293 , Humanos , Interleucina-12/biosíntesis , Ratones , Ratones Endogámicos C57BL , Oligodesoxirribonucleótidos/farmacología , Papillomaviridae/inmunología , Proteínas E7 de Papillomavirus/metabolismo , Infecciones por Papillomavirus/inmunología , Poli I-C/farmacología , Proteínas Recombinantes de Fusión/inmunología , Neoplasias del Cuello Uterino/virología , Vacunas Sintéticas/inmunologíaRESUMEN
Pulmonary fibrosis encompasses several respiratory diseases characterized by epithelial cell injury, inflammation and fibrosis. Transforming growth factor (TGF)-ß1 is one of the main profibrogenic cytokines involved in the pathogenesis of lung fibrosis. It induces fibroblast differentiation into myofibroblasts, which produce high levels of collagen and concomitantly loss of lung elasticity and reduction of the respiratory function. In the present study, we have investigated the effects of P17 (a TGF-ß inhibitor peptide) on IMR-90 lung fibroblast differentiation in vitro, as well as on the inhibition of the development of bleomycin-induced pulmonary fibrosis in mice. It was found that in IMR-90 cells, P17 inhibited TGF-ß1-induced expression of connective tissue growth factor and α-smooth muscle actin. In vivo, treatment of mice with P17 2days after bleomycin administration decreased lung fibrosis, areas of myofibroblast-like cells and lymphocyte infiltrate. P17 also reduced mRNA expression of collagen type I, fibronectin and the fibronectin splice isoform EDA in the lung, and increased the expression of IFN-γ mRNA. Finally, therapeutic treatment with P17 in mice with already established fibrosis was able to significantly attenuate the progression of lung fibrosis. These results suggest that P17 may be useful in the treatment of pulmonary fibrosis.
Asunto(s)
Fibroblastos/efectos de los fármacos , Péptidos/farmacología , Fibrosis Pulmonar/prevención & control , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Actinas/genética , Actinas/metabolismo , Animales , Bleomicina , Western Blotting , Línea Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Músculo Liso/química , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
Immunosuppressive activity of regulatory T cells (Treg) may contribute to the progression of cancer or infectious diseases by preventing the induction of specific immune responses. Using a phage-displayed random peptide library, we identified a 15-mer synthetic peptide, P60, able to bind to forkhead/winged helix transcription factor 3 (FOXP3), a factor required for development and function of Treg. P60 enters the cells, inhibits FOXP3 nuclear translocation, and reduces its ability to suppress the transcription factors NF-κB and NFAT. In vitro, P60 inhibited murine and human-derived Treg and improved effector T cell stimulation. P60 administration to newborn mice induced a lymphoproliferative autoimmune syndrome resembling the reported pathology in scurfy mice lacking functional Foxp3. However, P60 did not cause toxic effects in adult mice and, when given to BALB/c mice immunized with the cytotoxic T cell epitope AH1 from CT26 tumor cells, it induced protection against tumor implantation. Similarly, P60 improved the antiviral efficacy of a recombinant adenovirus expressing NS3 protein from hepatitis C virus. Functional inhibition of Treg by the FOXP3-inhibitory peptide P60 constitutes a strategy to enhance antitumor and antiviral immunotherapies.
Asunto(s)
Factores de Transcripción Forkhead/antagonistas & inhibidores , Péptidos/farmacología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Vacunas/inmunología , Animales , Femenino , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos ICR , Microscopía Confocal , Biblioteca de Péptidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T Reguladores/metabolismo , TransfecciónRESUMEN
BACKGROUND/AIMS: Vaccination strategies able to induce strong T-cell responses might contribute to eradicate hepatitis C virus (HCV) infection. We previously demonstrated that fusion of an antigen to the extra domain A from fibronectin (EDA) targets the antigen to TLR4-expressing dendritic cells (DC) and improves its immunogenicity. Here, we studied if fusion of EDA with the non-structural HCV protein NS3 might constitute an effective immunogen against HCV. METHODS: Recombinant NS3 and the fusion protein EDA-NS3 were produced and purified from E. coli, and tested in vitro for their capacity to activate maturation of DC and to favour antigen presentation. HHD transgenic mice expressing the human HLA-A2 molecule were immunized with recombinant proteins in the absence or presence of poly(I:C) and anti-CD40 agonistic antibodies and responses elicited by vaccination were tested in vitro, and in vivo, by their capacity to downregulate intrahepatic expression of HCV-NS3 RNA. RESULTS: EDA-NS3, but not NS3 alone, upregulated the expression of maturation markers, as well as Delta-like 1 and Delta-like 4 Notch ligands in DC and induced the production of IL-12. Mice immunized with EDA-NS3 had strong and long lasting NS3-specific CD4+ and CD8+ T-cell responses and, in combination with poly(I:C) and anti-CD40, downregulated intrahepatic expression of HCV-NS3 RNA. CONCLUSIONS: Recombinant EDA-NS3 may be considered for the development of vaccines against HCV infection.
Asunto(s)
Fibronectinas/uso terapéutico , Hepacivirus/inmunología , Hepatitis C/prevención & control , Proteínas Recombinantes/uso terapéutico , Proteínas Virales de Fusión/uso terapéutico , Proteínas no Estructurales Virales/uso terapéutico , Vacunas Virales/uso terapéutico , Animales , Antivirales/uso terapéutico , Línea Celular , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Quimioterapia Combinada , Escherichia coli/metabolismo , Femenino , Fibronectinas/biosíntesis , Antígeno HLA-A2/genética , Antígeno HLA-A2/inmunología , Antígeno HLA-A2/metabolismo , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatitis C/inmunología , Hepatitis C/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Poli I-C/uso terapéutico , Estructura Terciaria de Proteína , ARN Viral/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Virales de Fusión/biosíntesis , Proteínas no Estructurales Virales/biosíntesis , Proteínas no Estructurales Virales/genética , Vacunas Virales/biosíntesisRESUMEN
Chronic infection by hepatitis C virus (HCV) is characterized by the absence of efficient antiviral T-cell responses. Thus, vaccination strategies to induce strong anti-HCV T-cell responses are of paramount importance for prophylactic and therapeutic purposes. Dendritic cells (DCs) are the most potent antigen presenting cells; therefore, immunization with these cells loaded with viral antigens offers a new approach for induction of antiviral immunity. Here we show that immunization with DCs transfected with an adenovirus encoding non-structural 3 protein, from HCV (AdNS3), induced multiepitopic CD4 T helper cell 1 (Th1) and CD8 T-cell responses in different mouse strains. These responses prevented the growth of a tumorexpressing HCV proteins, in short- and long-term experiments. Moreover, immunization with AdNS3-transfected DCs did not induce anti-adenoviral antibodies, as compared to direct immunization with AdNS3, but elicited T-cell responses even in the presence of pre-existing anti-adenoviral antibodies. Finally, responses induced by this protocol down-regulated the expression of HCV RNA in the liver. In conclusion, DCs transfected with AdNS3 may prove to be an efficient anti-HCV vaccine.
Asunto(s)
Adenoviridae , Células Dendríticas/metabolismo , Células Dendríticas/trasplante , Vectores Genéticos , Hepacivirus/inmunología , Transducción Genética , Vacunas contra Hepatitis Viral/administración & dosificación , Proteínas no Estructurales Virales/genética , Adenoviridae/inmunología , Animales , Línea Celular , Línea Celular Tumoral , Células Dendríticas/inmunología , Femenino , Vectores Genéticos/inmunología , Hepatitis C/inmunología , Hepatitis C/prevención & control , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Linfocitos T/inmunología , Vacunas contra Hepatitis Viral/genética , Vacunas contra Hepatitis Viral/inmunología , Proteínas no Estructurales Virales/biosíntesis , Proteínas no Estructurales Virales/inmunologíaRESUMEN
Development of vaccination strategies against hepatitis C virus (HCV) is of paramount importance. With this aim, we tested the ability of dendritic cell-activating reagents polyinosinic-polycytidylic acid (poly(I:C)) and anti-CD40, as adjuvants to induce T-cell responses against HCV. Immunization of mice with these adjuvants induced dendritic cell maturation in vivo. Also, joint administration of poly(I:C) and anti-CD40 plus HCV antigens had a synergistic effect on the induction of anti-HCV T-cell responses. CD4 responses displayed a Th1 cytokine profile, and CD8 responses could be induced by immunization with a minimal CD8 epitope. Addition of a low amount of NS3 protein (as a source of Th epitopes) to the immunization mixture enhanced CD8 responses, whereas immunization with higher doses of NS3 induced both CD4 and CD8 responses. Surprisingly, immunization with NS3 protein but not with CD8 epitopes was able to induce CD8 responses and able to recognize cells expressing HCV antigens endogenously. Moreover, immunization with these adjuvants activated NK cells, which in turn helped to induce Th1 responses. Finally, this combined immunization protocol afforded long-lasting T-cell responses, suggesting that this strategy may prove to be useful in vaccination and/or treatment of HCV infection.
Asunto(s)
Anticuerpos/inmunología , Antígenos Virales/inmunología , Linfocitos T CD4-Positivos/inmunología , Antígenos CD40/inmunología , Linfocitos T CD8-positivos/inmunología , Hepacivirus/inmunología , Hepatitis C/inmunología , Inmunización , Péptidos/inmunología , Poli I-C/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos/administración & dosificación , Especificidad de Anticuerpos , Antígenos Virales/administración & dosificación , Diferenciación Celular , Línea Celular Tumoral , Citocinas/biosíntesis , Células Dendríticas/citología , Sinergismo Farmacológico , Epítopos de Linfocito T/inmunología , Humanos , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Péptidos/administración & dosificación , Péptidos/síntesis química , Poli I-C/administración & dosificación , Factores de Tiempo , Proteínas no Estructurales Virales/inmunologíaRESUMEN
Vaccination strategies based on the in vivo targeting of Ags to dendritic cells (DCs) are needed to improve the induction of specific T cell immunity against tumors and infectious agents. In this study, we have used a recombinant protein encompassing the extra domain A from fibronectin (EDA), an endogenous ligand for TLR4, to deliver Ags to TLR4-expressing DC. The purified EDA protein was shown to bind to TLR4-expressing HEK293 cells and to activate the TLR4 signaling pathway. EDA also stimulated the production by DC of proinflammatory cytokines such as IL-12 or TNF-alpha and induced their maturation in vitro and in vivo. A fusion protein between EDA and a cytotoxic T cell epitope from OVA efficiently presented this epitope to specific T cells and induced the in vivo activation of a strong and specific CTL response. Moreover, a fusion protein containing EDA and the full OVA also improved OVA presentation by DC and induced CTL responses in vivo. These EDA recombinant proteins protected mice from a challenge with tumor cells expressing OVA. These results strongly suggest that the fibronectin extra domain A may serve as a suitable Ag carrier for the development of antiviral or antitumoral vaccines.
Asunto(s)
Fibronectinas/metabolismo , Linfocitos T Citotóxicos/metabolismo , Receptor Toll-Like 4/metabolismo , Secuencias de Aminoácidos , Animales , Médula Ósea/metabolismo , Diferenciación Celular , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/metabolismo , Femenino , Fibronectinas/química , Fibronectinas/genética , Humanos , Interleucina-12/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/patología , Ovalbúmina/inmunología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Linfocitos T Citotóxicos/inmunología , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genética , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
The induction of protective or therapeutic cellular immunity against hepatitis C virus (HCV) is a difficult goal. In a previous work we showed that immunization with a recombinant adenovirus encoding HCV-NS3 (RAdNS3) could partially protect mice from challenge with a vaccinia virus encoding HCV antigens. We sought to investigate whether systemic administration of an immunostimulatory monoclonal antibody directed against the lymphocyte surface molecule CD137 could enhance the immunity elicited by RAdNS3. It was found that treatment with anti-CD137 mAb after the administration of a suboptimal dose of RAdNS3 enhanced cytotoxic and T helper cell responses against HCV NS3. Importantly, the ability of RAdNS3 to induce protective immunity against challenge with a recombinant vaccinia virus expressing HCV proteins was markedly augmented. Thus, combination of immunostimulatory anti-CD137 mAb with recombinant adenoviruses expressing HCV proteins might be useful in strategies of immunization against HCV.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Hepacivirus/inmunología , Receptores de Factor de Crecimiento Nervioso/inmunología , Receptores del Factor de Necrosis Tumoral/inmunología , Vacunas contra Hepatitis Viral/inmunología , Adenoviridae/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Antígenos CD , Células Cultivadas , Femenino , Hepacivirus/efectos de los fármacos , Hepatitis C/inmunología , Hepatitis C/prevención & control , Hepatitis C/virología , Humanos , Ratones , Ratones Endogámicos BALB C , Receptores de Factor de Crecimiento Nervioso/administración & dosificación , Receptores del Factor de Necrosis Tumoral/administración & dosificación , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Vacunas contra Hepatitis Viral/administración & dosificación , Carga Viral/métodosRESUMEN
CD25(+) regulatory T (T reg) cells suppress the activation/proliferation of other CD4(+) or CD8(+) T cells in vitro. Also, down-regulation of CD25(+) T reg cells enhance antitumor immune responses. In this study, we show that depletion of CD25(+) T reg cells allows the host to induce both CD4(+) and CD8(+) antitumoral responses following tumor challenge. Simultaneous depletion of CD25(+) and CD8(+) cells, as well as adoptive transfer experiments, revealed that tumor-specific CD4(+) T cells, which emerged in the absence of CD25(+) T reg cells, were able to reject CT26 colon cancer cells, a MHC class II-negative tumor. The antitumoral effect mediated by CD4(+) T cells was dependent on IFN-gamma production, which exerted a potent antiangiogenic activity. The capacity of the host to mount this antitumor response is lost once the number of CD25(+) T reg cells is restored over time. However, CD25(+) T reg cell depletion before immunization with AH1 (a cytotoxic T cell determinant from CT26 tumor cells) permits the induction of a long-lasting antitumoral immune response, not observed if immunization is conducted in the presence of regulatory cells. A study of the effect of different levels of depletion of CD25(+) T reg cells before immunization with the peptide AH1 alone, or in combination with a Th determinant, unraveled that Th cells play an important role in overcoming the suppressive effect of CD25(+) T reg on the induction of long-lasting cellular immune responses.
Asunto(s)
Inhibidores de la Angiogénesis/fisiología , Linfocitos T CD4-Positivos/inmunología , Neoplasias del Colon/inmunología , Regulación hacia Abajo/inmunología , Interferón gamma/fisiología , Activación de Linfocitos/inmunología , Péptidos/inmunología , Receptores de Interleucina-2/biosíntesis , Subgrupos de Linfocitos T/inmunología , Traslado Adoptivo , Inhibidores de la Angiogénesis/biosíntesis , Inhibidores de la Angiogénesis/metabolismo , Animales , Antígenos de Neoplasias/administración & dosificación , Antígenos de Neoplasias/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/trasplante , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/prevención & control , Epítopos de Linfocito T/administración & dosificación , Epítopos de Linfocito T/inmunología , Femenino , Memoria Inmunológica/inmunología , Interferón gamma/biosíntesis , Interferón gamma/metabolismo , Depleción Linfocítica , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Trasplante de Neoplasias/inmunología , Péptidos/administración & dosificación , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/trasplanteRESUMEN
Hepatitis C virus (HCV) chronic infection is characterized by low or undetectable cellular immune responses against HCV antigens. Some studies have suggested that HCV proteins manipulate the immune system by suppressing the specific antiviral T-cell immunity. We have previously reported that the expression of HCV core and E1 proteins (CE1) in dendritic cells (DC) impairs their ability to prime T cells in vitro. We show here that immunization of mice with immature DC transduced with an adenovirus encoding HCV core and E1 antigens (AdCE1) induced lower CD4(+)- and CD8(+)-T-cell responses than immunization with DC transduced with an adenovirus encoding NS3 (AdNS3). However, no differences in the strength of the immune response were detected when animals were immunized with mature DC subsequently transduced with AdCE1 or AdNS3. According to these findings, we observed that the expression of CE1 in DC inhibited the maturation caused by tumor necrosis factor alpha or CD40L but not that induced by lipopolysaccharide. Blockade of DC maturation by CE1 was manifested by a lower expression of maturation surface markers and was associated with a reduced ability of AdCE1-transduced DC to activate CD4(+)- and CD8(+)-T-cell responses in vivo. Our results suggest that HCV CE1 proteins modulate T-cell responses by decreasing the stimulatory ability of DC in vivo via inhibition of their physiological maturation pathways. These findings are relevant for the design of therapeutic vaccination strategies in HCV-infected patients.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/fisiología , Linfocitos T Citotóxicos/inmunología , Proteínas del Núcleo Viral/fisiología , Proteínas del Envoltorio Viral/fisiología , Animales , Ligando de CD40/farmacología , Tolerancia Inmunológica , Inmunización , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Factor de Necrosis Tumoral alfa/farmacología , Proteínas del Envoltorio Viral/inmunología , Proteínas no Estructurales Virales/inmunologíaRESUMEN
Hepatitis C virus (HCV) infection has a strong tendency to evolve to chronicity despite up-regulation of proapoptotic cytokines in the inflamed liver. The mechanisms responsible for persistent viral replication in this inflammatory environment are obscure. It is conceivable that viral replication would be facilitated if the infected hepatocytes are rendered resistant to cytokine-induced cytotoxicity. In this study, we investigated if an adenovirus encoding HCV core and E1 (RAdCE1) could reduce liver cell injury in different in vivo models of cytokine-mediated hepatotoxicity in mice. We show that RAdCE1 markedly attenuates hepatocellular apoptosis and the increase in serum transaminase levels after concanavalin A (con A) challenge. This protective effect is accompanied by an inhibition of nuclear translocation of nuclear factor kappaB (NF-kappaB); reduced expression of inducible nitric oxide synthase (iNOS); decreased hepatic messenger RNA levels of chemokines macrophage inflammatory protein 2 (MIP-2), monocyte chemoattractant protein 1 (MCP-1), and interferon-inducible protein 10 (IP-10); and abrogation of liver leukocyte infiltration. RAdCE1 also causes a reduction in serum transaminase levels and inhibits hepatocellular apoptosis in mice given tumor necrosis factor (TNF)-alpha plus D-galactosamine. In conclusion, HCV structural antigens can protect liver cells against the proapoptotic effects of proinflammatory cytokines. The antiapoptotic status of infected liver cells may represent a mechanism favoring viral persistence. Our findings also suggest that, in chronic hepatitis C, the burden of hepatocellular damage mainly affects noninfected liver cells.
Asunto(s)
Adenoviridae/genética , Citocinas/fisiología , Expresión Génica/fisiología , Hepacivirus/genética , Hepatopatías/etiología , Hepatopatías/prevención & control , Proteínas del Núcleo Viral/genética , Proteínas del Envoltorio Viral/genética , Enfermedad Aguda , Animales , Núcleo Celular/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Concanavalina A , Citocinas/antagonistas & inhibidores , Combinación de Medicamentos , Femenino , Galactosamina , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Recombinación Genética , Transducción Genética , Factor de Necrosis Tumoral alfaRESUMEN
Cellular immune response plays an important role in the clearance of hepatitis C virus (HCV). Thus, development of efficient ways to induce anti-viral cellular immune responses is an important step toward prevention and/or treatment of HCV infection. With this aim, we have constructed a replication-deficient recombinant adenovirus expressing HCV NS3 protein (RAdNS3). The efficacy of RAdNS3 was tested in vivo by measuring the protection against infection with a recombinant vaccinia virus expressing HCV-polyprotein (vHCV1-3011). Immunisation with 10(9)pfu of RAdNS3 induced anti-NS3 humoral, T helper and T cytotoxic responses. We identified eight epitopes recognised by IFN-gamma producing cells, five of them exhibiting lytic activity. Moreover, we show that RAdNS3 immunised mice were protected against challenge with vHCV1-3011 and that this protection was mediated by CD8(+) cells. In conclusion, our results suggest that adenoviral vectors encoding NS3 might be useful for the induction of prophylactic and/or therapeutic anti-HCV immunity.