RESUMEN
BACKGROUND: Oncolytic virotherapy is an attractive drug platform of cancer gene therapy, but efficacy and specificity are important prerequisites for success of such strategies. Previous studies determined that Apoptin is a p53 independent, bcl-2 insensitive apoptotic protein with the ability to specifically induce apoptosis in tumor cells. Here, we generated a conditional replication-competent adenovirus (CRCA), designated Ad-hTERT-E1a-Apoptin, and investigated the effectiveness of the CRCA a gene therapy agent for further clinical trials. RESULTS: The observation that infection with Ad-hTERT-E1a-Apoptin significantly inhibited growth of the melanoma cells, protecting normal human epidermal melanocytes from growth inhibition confirmed cancer cell selective adenoviral replication, growth inhibition, and apoptosis induction of this therapeutic approach. The in vivo assays performed by using C57BL/6 mice containing established primary or metastatic tumors expanded the in vitro studies. When treated with Ad-hTERT-E1a-Apoptin, the subcutaneous primary tumor volume reduction was not only observed in intratumoral injection group but in systemic delivery mice. In the lung metastasis model, Ad-hTERT-E1a-Apoptin effectively suppressed pulmonary metastatic lesions. Furthermore, treatment of primary and metastatic models with Ad-hTERT-E1a-Apoptin increased mice survival. CONCLUSIONS: These data further reinforce the previously research showing that an adenovirus expressing Apoptin is more effective and advocate the potential applications of Ad-hTERT-E1a-Apoptin in the treatment of neoplastic diseases in future clinical trials.
Asunto(s)
Adenoviridae/genética , Adenoviridae/patogenicidad , Antineoplásicos/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Viroterapia Oncolítica , Proteínas E1A de Adenovirus/metabolismo , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular , ADN Recombinante/genética , Expresión Génica , Humanos , Neoplasias Pulmonares/secundario , Melanoma/patología , Ratones , Especificidad de Órganos , Tejido Subcutáneo/patología , Análisis de Supervivencia , Telomerasa/metabolismo , Transgenes/genéticaRESUMEN
Two recombinant fowlpox viruses (rFPV-P1 and rFPV-IL18-2AP12A) containing foot-and-mouth disease virus (FMDV) capsid polypeptide, 3C coding regions of O/NY00 were evaluated to determine their abilities to induce humoral and cellular responses in the presence or absence of swine IL-18 as genetic adjuvant. The ability to protect swine against homologous virus challenge was examined. All swine were given booster vaccinations at 21 days after the initial inoculation and were challenged 10 days after the booster vaccination. Control groups were inoculated with wild-type fowlpox virus (wtFPV). All animals vaccinated with rFPV-P12A and rFPV-IL18-P12A developed specific anti-FMDV ELISA antibody and neutralizing antibody and T-lymphocyte proliferation was observed. Cellular immune function was evaluated via examination of IFN-gamma production in swine peripheral blood serum. The results demonstrate the potential viability of a fowlpox virus-based recombinant vaccine in the control and prevention of FMDV infections.
Asunto(s)
Cisteína Endopeptidasas/inmunología , Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/prevención & control , Interleucina-18/inmunología , Enfermedades de los Porcinos/prevención & control , Porcinos/inmunología , Proteínas Virales/inmunología , Vacunas Virales/inmunología , Proteasas Virales 3C , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/farmacología , Animales , Anticuerpos Antivirales/sangre , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Cisteína Endopeptidasas/genética , Ensayo de Inmunoadsorción Enzimática/veterinaria , Fiebre Aftosa/inmunología , Virus de la Fiebre Aftosa/genética , Virus de la Viruela de las Aves de Corral/genética , Virus de la Viruela de las Aves de Corral/inmunología , Interferón gamma/sangre , Interleucina-18/genética , Pruebas de Neutralización/veterinaria , Plásmidos/genética , Plásmidos/inmunología , Distribución Aleatoria , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/virología , Vacunación/métodos , Vacunación/veterinaria , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/farmacología , Proteínas Virales/genética , Vacunas Virales/farmacologíaRESUMEN
We designed and constructed a fuse expression gene OAAT and staphylococcal enterotoxin A (SEA) on the basis of the OAAT designed and constructed which consists of the structural protein VP1 genes from serotypes A and O FMDV, 5 major VP1 immunodominant epitopes from two genotypes of Asia1 serotype, and 3 Th2 epitopes originating from the non-structural protein, 3ABC gene and structural protein VP4 gene. The recombinant plasmids pEA was constructed using SEA as a genetic adjuvant. Expressions of target gene from the pEA in Hela cell were verified by IFA and Western blotting. The experiment of BALB/c mice immunized with the DNA vaccines showed that pA and pEA could induce simultaneously specific antibodies against serotypes A, Asia1, and O FMDV, and the highest antibody titres were found in the pEA and inactivated vaccine groups compared to pA vaccinating mice. Compared with the control, the levels of IL-2, IFN-gamma, IL-4, and IL-10 expression by splenic lymphocytes from mice immunized with pA and pEA were significantly increased. In addition, we found that the levels of IL-2, IFN-gamma and IL-4 from the mice immunized with pEA was higher than mice immunized with pA did. The results of viral challenge in guinea pigs showed the pA, pEA and inactivated vaccine provided full protection in 2/4, 2/4, 3/4, 3/4 and 4/4, 4/4 guinea pigs from challenge with FMDV O/NY00 and Asial/YNBS/58, respectively. The results demonstrated fuse protein OAAT and SEA may be potential immunoge against FMDV, furthermore, SEA may be an effective genetic adjuvant for DNA vaccine.
Asunto(s)
Antígenos Virales/inmunología , Epítopos/inmunología , Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/prevención & control , Vacunas de ADN/inmunología , Vacunas Virales/inmunología , Adyuvantes Inmunológicos/genética , Animales , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Enterotoxinas/genética , Enterotoxinas/inmunología , Fiebre Aftosa/inmunología , Cobayas , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/inmunologíaRESUMEN
An outbreak of fever and dyspnea with high incidence rate and case fatality rate occurred among pigs in a pigfarm in Jilin province, China, in 2000. The paramyxovirus-like particles could be observed in lungs, spleens and kidneys of the dead pigs under the transmission electron microscope. A Newcastle disease virus (NDV) isolate designated as JL01 was determined as the causal agent for the disease outbreak. The purified virus was reinoculated into pigs, and then the pigs infected with the virus showed similar symptoms, and the HI antibody of NDV could be detected from the reinoculated pigs. The MDT, ICPI and EID50 of the JL01 isolate was 55.2h, 1.60 and 10(-7.5)/0.1 mL respectively. The F gene of JL01 was cloned and sequenced, and the results showed that the identities of F gene shared by JL01 and avirulent NDV reference strains were from 91.5% to 98.5%, and could be ascribed to NDV genotype I. Thus, the swine NDV JL01 strain should be an avirulent strain with gene variation, but the virulence of JL01 was just the same as velogenic strains.
Asunto(s)
Enfermedad de Newcastle/virología , Virus de la Enfermedad de Newcastle/aislamiento & purificación , Filogenia , Enfermedades de los Porcinos/virología , Animales , Pollos , Perros , Caballos , Ratones , Microscopía Electrónica , Virus de la Enfermedad de Newcastle/clasificación , Virus de la Enfermedad de Newcastle/genética , Virus de la Enfermedad de Newcastle/ultraestructura , Reacción en Cadena de la Polimerasa , Conejos , Análisis de Secuencia de ADN , Porcinos , Proteínas Virales/genéticaRESUMEN
DNA-based vaccination is an attractive alternative for overcoming the disadvantages of inactivated virus vaccines; however, DNA vaccines alone often generate only weak immune responses. In this study, the efficacy of LMS as a chemical adjuvant on a DNA vaccine (pVIR-P12A-IL18-3C) encoding the P1-2A and 3C genes of the FMDV and swine IL-18, which provides protection against FMDV challenge, was tested. All test pigs were administered booster vaccinations 28 days after the initial inoculation, and were challenged with 1000 ID50 FMDV O/NY00 20 days after the booster vaccination. Positive and negative control groups were inoculated with inactivated virus vaccine and PBS respectively. The DNA vaccine plus LMS induced greater humoral and cell-mediated responses than the DNA vaccine alone, as evidenced by higher concentrations of neutralizing and specific anti-FMDV antibodies, and by higher concentrations of T-lymphocyte proliferation and IFN-y production, respectively. FMDV challenge revealed that the DNA vaccine plus LMS provided higher protection than the DNA vaccine alone. This study demonstrates that LMS may be useful as an adjuvant for improving the protective efficiency of DNA vaccination against FMDV in pigs.
Asunto(s)
Adyuvantes Farmacéuticos/administración & dosificación , Levamisol , Vacunas de ADN , Vacunas Virales , Animales , Anticuerpos Antivirales/sangre , Fiebre Aftosa/inmunología , Fiebre Aftosa/prevención & control , Fiebre Aftosa/virología , Virus de la Fiebre Aftosa/inmunología , Virus de la Fiebre Aftosa/patogenicidad , Interferón gamma/metabolismo , Interleucina-18/administración & dosificación , Interleucina-18/inmunología , Levamisol/administración & dosificación , Levamisol/inmunología , Activación de Linfocitos/inmunología , Pruebas de Neutralización , Porcinos , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/prevención & control , Enfermedades de los Porcinos/virología , Linfocitos T/inmunología , Resultado del Tratamiento , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/genética , Vacunas Virales/inmunologíaRESUMEN
Two recombinant fowlpox viruses (rFPV-ORF5-ORF3 and rFPV-IL-18-ORF5-ORF3) containing the ORF5/ORF3 cDNAs of PRRSV (strain Chang Chun) and IL-18 of swine were constructed and evaluated for theirs abilities to induce humoral and cellular responses in piglets. In addition, their abilities to protect piglets against homologous virus challenge were examined. All piglets were given booster vaccinations at 21 days after the initial inoculation, and all piglets were challenged at 60 after the initial inoculation. Control groups were inoculated with wild-type fowlpox virus (wtFPV). All animals vaccinated with rFPV-ORF5-ORF3 and rFPV-IL-18-ORF5-ORF3 developed specific anti-PRRSV ELISA antibody and neutralizing antibody, as well as T-lymphocyte proliferation response. To evaluate the cellular immune function, IFN-gamma production in pigs serum and T-lymphocytes (CD4 and CD8 T cells) in peripheral blood were examined. Following challenge with a pathogenic strain of PRRSV (strain Chang Chun), piglets inoculated with recombinant fowlpox virus (rFPV) showed lower (P<0.05) temperature, viremia and virus load in bronchial lymph nodes than control animals, suggesting the establishment of partial protection against PRRSV infection. The results demonstrated the potential use of a fowlpox virus-based recombinant vaccine in the control and prevention of PRRSV infections.
Asunto(s)
Virus de la Viruela de las Aves de Corral/inmunología , Interleucina-18/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/inmunología , Adyuvantes Inmunológicos/genética , Animales , Anticuerpos Antivirales/sangre , Temperatura Corporal , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Virus de la Viruela de las Aves de Corral/genética , Inmunización Secundaria , Interferón gamma/biosíntesis , Interferón gamma/sangre , Interleucina-18/genética , Ganglios Linfáticos/virología , Pruebas de Neutralización , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Porcinos , Linfocitos T/inmunología , Vacunas Sintéticas/genética , Proteínas del Envoltorio Viral/genética , Vacunas Virales/genética , ViremiaRESUMEN
The expression of the hemagglutinins of Avian influenza virus H5 H7and H9 subtypes was studied in this article by Pichia pastoris, one of the eukaryotis expression systems. Three reconstructed expression plasmids and engineering strains, named pPIC9K-H5HA, pPIC9K-H7HA, pPIC9K-H9HA and GS115/pPIC9K-H5HA, GS115/pPIC9K-H7HA, GS115/pPIC9K-H9HA repectively, were obtained. The reconstructed yeast engineering strains were identified by MD and MM plate selecting and PCR. The induced interests proteins were examined by SDS-PAGE and Western-bloting,the results showed that the interest genes were expressed exactly. And this will be helpful in the future study of antigen detection and antibody detection kit, as well in the subunit vaccines developing.