RESUMEN
Measles remains a principal cause of worldwide mortality, in part because young infants cannot be immunized effectively. Development of new vaccines has been hindered by previous experience with a formalin-inactivated vaccine that predisposed to a severe form of disease (atypical measles). Here we have developed and tested potential DNA vaccines for immunogenicity, efficacy and safety in a rhesus macaque model of measles. DNA protected from challenge with wild-type measles virus. Protection correlated with levels of neutralizing antibody and not with cytotoxic T lymphocyte activity. There was no evidence in any group, including those receiving hemagglutinin-encoding DNA alone, of 'priming' for atypical measles.
Asunto(s)
Hemaglutininas Virales/uso terapéutico , Vacuna Antisarampión/uso terapéutico , Sarampión/prevención & control , Vacunación , Vacunas de ADN/uso terapéutico , Proteínas Virales de Fusión/uso terapéutico , Animales , Anticuerpos Antivirales/sangre , Vías de Administración de Medicamentos , Exantema , Hemaglutininas Virales/genética , Inmunización Secundaria , Macaca mulatta , Pruebas de Neutralización , Neumonía , Piel/patología , Vacunas Atenuadas/uso terapéutico , Proteínas Virales de Fusión/genéticaRESUMEN
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
We report here the generation and characterization of EGF-IL-18 fusion protein as an anti-tumor reagent. The epidermal growth factor (EGF) and interleukin-18 (IL-18) fusion protein was shown to induce interferon-gamma (IFNgamma) expression and secretion in KG-1 cells, and to promote PBMNC proliferation. It also stimulated activation of CD4+ T cells, and increased other immune responses. Moreover, EGF-IL-18 could induce significant tumor regression in SMMC-7721-xenografted Balb/c nude mice when administered together with peritumoral injection of X-ray-irradiated NK-92 cells, and this regression is associated with arresting of the tumor cells in G1 phase and induction of apoptosis.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Sistema Inmunológico/efectos de los fármacos , Interleucina-18/farmacología , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Péptidos/farmacología , Proteínas Virales de Fusión/farmacología , Animales , Antineoplásicos/uso terapéutico , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/enzimología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Receptores ErbB/efectos de los fármacos , Receptores ErbB/metabolismo , Femenino , Fase G1 , Humanos , Sistema Inmunológico/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Interferón gamma/metabolismo , Interleucina-18/uso terapéutico , Células K562 , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/efectos de la radiación , Leucocitos Mononucleares/efectos de los fármacos , Neoplasias Hepáticas Experimentales/inmunología , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Péptidos/uso terapéutico , Factores de Tiempo , Proteínas Virales de Fusión/uso terapéutico , Rayos X , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cancer is a devastating disease that affects millions of patients every year, and causes an enormous economic burden on the health care system and emotional burden on affected families. The first line of defense against solid tumors is usually extraction of the tumor, when possible, by surgical methods. In cases where solid tumors can not be safely removed, chemotherapy is often the first line of treatment. As metastatic cancers often become vigorously resistant to treatments, the development of novel, more potent and selective anti-cancer strategies is of great importance. Adenovirus (Ad) is the most commonly used virus in cancer clinical trials, however, regardless of the nature of the Ad-based therapeutic, complete responses to treatment remain rare. A number of pre-clinical studies have shown that, for all vector systems, viral spread throughout the tumor mass can be a major limiting factor for complete tumor elimination. By expressing exogenous cell-fusion proteins, many groups have shown improved spread of Ad-based vectors. This review summarizes the research done to examine the potency of Ad vectors expressing fusogenic proteins as anti-cancer therapeutics.
Asunto(s)
Adenoviridae/genética , Vectores Genéticos/genética , Neoplasias/terapia , Proteínas Virales de Fusión/uso terapéutico , Adenoviridae/metabolismo , Animales , Fusión Celular , Vectores Genéticos/metabolismo , Humanos , Neoplasias/fisiopatología , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismoAsunto(s)
Hemaglutininas Virales/uso terapéutico , Vacuna Antisarampión/uso terapéutico , Sarampión/prevención & control , Vacunas de ADN/uso terapéutico , Proteínas Virales de Fusión/uso terapéutico , Animales , Salud Global , Hemaglutininas Virales/genética , Macaca mulatta , Vacuna Antisarampión/efectos adversos , ARN Viral/genética , Proteínas Virales de Fusión/genéticaRESUMEN
Malignant gliomas are the most common primary brain tumors in adults and, with few exceptions, have a dismal prognosis despite the therapeutic use of surgery, radiation therapy, and chemotherapy. Because CNS gliomas rarely metastasize, they represent an attractive target for gene therapy through local gene delivery. Here we report on the use of two different fusogenic membrane glycoproteins (FMGs), the measles virus proteins F and H (MV-F and MV-H) and a mutated form of the retroviral envelope protein of the gibbon ape leukemia virus (GALV.fus), as a novel class of therapeutic transgenes in gliomas. Transfection of U87 and U118 cells with MV-F and MV-H cDNA or GALV.fus cDNA led in 48 hr to massive syncytial formation followed by cell death. FMG-mediated cytotoxicity in the U87 and U118 cell lines was superior to the cytotoxicity caused by transfection with HSV-tk cDNA followed by ganciclovir (GCV) treatment at all time points. At high-density cell seeding, addition of tumor cells transfected with MV-F and H killed at least 1 log more cells than by HSV-tk + GCV treatment, indicating higher bystander effect. Similar results were obtained with GALV.fus. The mechanism of syncytial death in cultured glioma cell lines was predominantly apoptotic. Transfection of U87 cells with F + H or GALV.fus expression constructs completely suppressed their tumorigenicity. Treatment of established U87 xenografts in nude mice with a combination of F and H adenoviruses at 1:1 ratio led to complete tumor regression, significantly higher antitumor effect, and prolongation of survival as compared with control animals treated with a GFP adenovirus. In summary, the viral fusogenic membrane glycoproteins (GALV and the MV-F + MV-H combination) are potent therapeutic transgenes with potential utility in the gene therapy of gliomas.
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Terapia Genética/métodos , Glioma/genética , Glioma/terapia , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/uso terapéutico , Animales , Apoptosis , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Fusión Celular , Ganciclovir/farmacología , Vectores Genéticos/genética , Células Gigantes/metabolismo , Células Gigantes/patología , Glioma/metabolismo , Glioma/patología , Hemaglutininas Virales/efectos adversos , Hemaglutininas Virales/genética , Hemaglutininas Virales/metabolismo , Hemaglutininas Virales/uso terapéutico , Humanos , Lentivirus/genética , Virus de la Leucemia del Gibón/genética , Virus del Sarampión/genética , Glicoproteínas de Membrana/efectos adversos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mutación/genética , Trasplante de Neoplasias , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Transgenes/genética , Células Tumorales Cultivadas , Proteínas Virales de Fusión/efectos adversos , Proteínas Virales de Fusión/metabolismoRESUMEN
Newcastle disease virus (NDV) of paramyxovirus and Marek's disease virus (MDV) of herpesvirus, two of the most serious threats to the poultry industry, can give rise to complex co-infections that hinder diagnosis and prevention. In the current study, two different peptides, derived from the MDV gH (gHH2L) and gB (gBH3), respectively, exhibit antiviral activity against NDV in vitro. The potent inhibitory effect of heptad repeat 2 from fusion glycoprotein of the NDV on MDV infection also has been demonstrated. Plaque formation and embryo infectivity assays confirmed these antiviral results. Furthermore, each tandem peptide consisting of two motifs from different viruses exhibits more potent antiviral activity than the constituent peptides. The current work provides a new strategy for developing novel peptides and vaccines against virus infection and co-infections.
Asunto(s)
Antivirales/farmacología , Glicoproteínas/farmacología , Mardivirus/efectos de los fármacos , Virus de la Enfermedad de Newcastle/efectos de los fármacos , Péptidos/farmacología , Proteínas Virales de Fusión/farmacología , Animales , Antivirales/uso terapéutico , Línea Celular , Embrión de Pollo , Glicoproteínas/genética , Glicoproteínas/uso terapéutico , Mardivirus/genética , Enfermedad de Marek/prevención & control , Pruebas de Sensibilidad Microbiana , Enfermedad de Newcastle/prevención & control , Virus de la Enfermedad de Newcastle/genética , Péptidos/genética , Péptidos/uso terapéutico , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/uso terapéutico , Ensayo de Placa ViralAsunto(s)
Membrana Celular/metabolismo , Sistemas de Liberación de Medicamentos , Oligonucleótidos/uso terapéutico , Proteínas/metabolismo , Proteínas Recombinantes de Fusión/uso terapéutico , Transporte Biológico , Técnicas de Transferencia de Gen , Terapia Genética/tendencias , Humanos , Proteínas de Fusión Oncogénica/uso terapéutico , Proteínas Virales de Fusión/uso terapéuticoAsunto(s)
Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Fármacos Anti-VIH/uso terapéutico , Proteína gp41 de Envoltorio del VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Fragmentos de Péptidos/uso terapéutico , Proteínas Virales de Fusión/uso terapéutico , Secuencia de Aminoácidos , Enfuvirtida , Humanos , Datos de Secuencia MolecularRESUMEN
Vaccinia virus recombinants encoding the hemagglutinin or fusion protein of measles virus have been constructed. Infection of cell cultures with the recombinants led to the synthesis of authentic measles proteins as judged by their electrophoretic mobility, recognition by antibodies, glycosylation, proteolytic cleavage, and presentation on the cell surface. Mice vaccinated with a single dose of the recombinant encoding the hemagglutinin protein developed antibodies capable of both inhibiting hemagglutination activity and neutralizing measles virus, whereas animals vaccinated with the recombinant encoding the fusion protein developed measles neutralizing antibodies. Mice vaccinated with either of the recombinants resisted a normally lethal intracerebral inoculation of a cell-associated measles virus subacute sclerosing panencephalitis strain.
Asunto(s)
Antígenos/uso terapéutico , Encefalitis/prevención & control , Hemaglutininas Virales/uso terapéutico , Vacuna Antisarampión/uso terapéutico , Virus del Sarampión/inmunología , Sarampión/complicaciones , Proteínas Recombinantes de Fusión/uso terapéutico , Proteínas Recombinantes/uso terapéutico , Vacunas Sintéticas/uso terapéutico , Proteínas Virales de Fusión/uso terapéutico , Animales , Anticuerpos Antivirales/biosíntesis , Células Cultivadas , ADN/genética , Encefalitis/etiología , Hemaglutininas Virales/inmunología , Virus del Sarampión/genética , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/inmunología , Virus Vaccinia/genética , Proteínas Virales de Fusión/inmunología , Proteínas Virales/biosíntesisRESUMEN
Polymorphic epithelial mucin, encoded by the MUC1 gene, is present at the apical surface of glandular epithelial cells. It is over-expressed and aberrantly glycosylated in most breast tumors, resulting in an antigenically distinct molecule and a potential target for immunotherapy. This transmembrane protein, when produced by tumor cells, is often cleaved into the circulation, where it is detectable as a tumor marker (CA 15.3) by various antibodies, allowing for early detection of recurrences and evaluation of treatment efficacy. The objective of the current study was to examine the clinical and environmental safety and immunogenicity of a live recombinant vaccinia virus expressing the human MUC1 and IL2 genes (VV TG5058), referred to here as TG1031. The study was an open-label phase 1 and 2 trial in nine patients with advanced inoperable breast cancer recurrences to the chest wall. The patients were vaccinated intramuscularly with a single dose of TG1031; three patients were treated at each of three progressive dose levels ranging from 5x10(5) to 5x10(7) plaque-forming units. A boost injection at their original dose level was administered in patients responding immunologically, clinically, or both. Vaccination resulted in no significant clinical adverse effects, and there was no environmental contamination by live TG1031. All patients had been vaccinated as children, and patients treated at the highest dose level mounted a significant anti-vaccinia antibody response. None of the nine patients had a significant increase in MUC1-specific antibody titers after one single injection, whereas five patients had a detectable increase in vaccinia virus antibody titers. Peripheral blood mononuclear cells of one patient at the intermediate dose level showed a proliferative response to in vitro culture with vaccinia virus, with a stimulation index of 6. A second patient treated at the intermediate dose level had a stimulation index of 7 to MUC1 peptide and of 14 after a boost injection. This patient had a concomitant decrease in carcinoembryonic antigen serum levels and remained clinically stable for 10 weeks. Evidence of MUC1-specific cytotoxic T lymphocytes was detected in two patients. Immunohistochemical analysis revealed an increase in T memory cells (CD45RO) in tumor biopsies after vaccination. The absence of serious adverse events, together with the documentation of immune stimulations in vivo, warrant the further use of TG1031 in immunotherapy trials of breast cancer.