RESUMEN
UNLABELLED: Currently one of the most promising approaches in development of cancer virotherapy is based on the ability of oncolytic viruses to selective infection and lysis of tumor cells. AIM: The goal of the study was to identify and evaluate perspective oncolytic viruses capable of selectively destroying human glioma cells. PATIENTS AND METHODS: Original GB2m, GA14m and GB22m glioma cell cultures derived from patients were used for evaluating in vitro oncolytic activity of some typical orthopoxviruses, adenoviruses and parvoviruses. RESULTS: The oncolytic activity in the human glioma cell models was confirmed for LIVP and WR strains of vaccinia virus, Adel2 and Ad2del strains with deletions within E1B/55K gene and derived from human adenoviruses type 2 and 5, respectively. CONCLUSIONS: We consider these oncolytic viruses as promising agents for the treatment of human malignant glioma.
Asunto(s)
Glioma , Viroterapia Oncolítica/métodos , Virus Oncolíticos/fisiología , Adenoviridae/fisiología , Técnicas de Cultivo de Célula , Glioma/terapia , Glioma/virología , Humanos , Orthopoxvirus/fisiología , Parvovirus/fisiología , Células Tumorales Cultivadas/virología , Fenómenos Fisiológicos de los VirusRESUMEN
Lytic viral infection results in production of viral progeny, and lysis of the infected cells. Tumor cells are usually more sensitive to virus infection. Studies of viral oncolysis indicate that it could represent a promising alternative approach to cancer therapy. The ability of viruses to kill selectively cancer cells had been noticed for quite a long time ago. However, only in recent years, based on deeper understanding of molecular biology of viruses and the cell and due to the development of modern methods for directed modification of viruses, there emerged a real opportunity for development of virus variants with improved therapeutic potential. Adenoviruses represent one of the most studied models of oncolytic viruses. The DNA-containing viruses are very suitable for genetic manipulation and show minimal pathogenicity. The review summarizes data on directions and approaches aiming generation of highly efficient variants of oncolytic adenoviruses. The approaches include introduction of directed genetic modifications into viral genome, accelerated selection of oncolytic viral variants following treatment with mutagens, the use of adenoviruses as vectors for introduction of therapeutic gene products, optimization of viral delivery systems, minimalization of negative effects from the host immune system etc. The dynamic development of studies in the field holds promise for introduction into clinical practice of many variants of oncolytic adenoviruses in the very near future.
Asunto(s)
Infecciones por Adenoviridae/genética , Adenoviridae/fisiología , Antineoplásicos/uso terapéutico , Neoplasias/terapia , Viroterapia Oncolítica/métodos , Viroterapia Oncolítica/tendencias , Virus Oncolíticos/fisiología , Animales , Ensayos Clínicos como Asunto , Terapia Genética , Humanos , Neoplasias/genéticaRESUMEN
Thirty nine water soluble nitroxyl radicals of various classes, belonging to piperidine, pyrrolidine and imidazolidine series were synthesized. Twenty seven of them were cytotoxic in vitro with respect to the tumor cell culture A431. The CC50 of the most active nitroxyl radicals with respect to cells SW480 and A431 was within 0.16-2.5 mM at the selectivity index of 3.91-7.81 in relation to cytotoxicity of the compounds for the cells of the normal L68 phenotype and tumor cells. The tests on the antiviral activity showed that 16 out of 22 nitroxyl radicals had antiviral activity in Vero cell culture with respect to the West Nile virus and Herpes simplex virus of type II respectively. The EC50 ranged within 0.09-3.45 mM. Some of the nitroxyl radicals had only antiviral activity, but a number of the compounds had both cytotoxic properties and antiviral activity.
Asunto(s)
Antivirales/farmacología , Citotoxinas/farmacología , Radicales Libres/farmacología , Herpes Genital/tratamiento farmacológico , Herpesvirus Humano 2/metabolismo , Óxidos de Nitrógeno/farmacología , Fiebre del Nilo Occidental/tratamiento farmacológico , Virus del Nilo Occidental/metabolismo , Animales , Antioxidantes/farmacología , Línea Celular Tumoral , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Humanos , Células VeroRESUMEN
Complete nucleotide sequence of genomic RNA of hepatitis A virus (HAV) rapidly replicating strain MB-7 was determined. Comparison of nucleotide and deduced amino-acid sequences demonstrated the highest level of identity of MB-7 with strain HAS-15 (above 99%) and high homology with other HAV strains (HM 175/7, CR326, and GBM/HFS) used in production of anti-hepatitis A vaccines. MB-7 was classified as subgenotype IA. Phylogenetic analysis showed that MB-7 is most closely related to the strain HAS-15 and the HAV variants circulating in Russia. Comparative analysis of genomic differences between MB-7 and HAS-15 with other HAV strains revealed among changes characteristic of MB-7 those typical of the described earlier rapidly replicating HAV strains (nt. 149-162 in 5'-untranslated region and changes in the VP3 and 2C genes). These results suggest the functional importance of changes in above-mentioned regions of HAV genome for the increased replication level of MB-7 in vitro.
Asunto(s)
Virus de la Hepatitis A Humana/genética , Hepatitis A/virología , ARN Viral/genética , Replicación Viral , Secuencia de Bases , Virus de la Hepatitis A Humana/clasificación , Humanos , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ARN , Homología de Secuencia de Ácido Nucleico , Proteínas Virales/genéticaRESUMEN
Blood serum samples collected from patients with acute hepatitis symptoms admitted to Infectious Disease Hospitals of Novosibirsk, Barnaul, and Irkutsk were studied. The serum samples were tested for the IgM and IgG antibodies to HEV using ELISA. Seropositive samples were tested using RT-PCR for HEV RNA. Two HEV strains were isolated, and thus HEV infection was identified for West Siberia. One of this strains is classified as HEV genotype I; the other, as genotype III. Cell culturing of these strains in green monkey kidney (4647) cells showed an ability of HEV genotype I strain to cause persistent infection.
Asunto(s)
Virus de la Hepatitis E/metabolismo , Hepatitis E/metabolismo , Replicación Viral/fisiología , Animales , Línea Celular , Chlorocebus aethiops , Genotipo , Anticuerpos Antihepatitis/sangre , Hepatitis B/sangre , Hepatitis B/complicaciones , Hepatitis B/genética , Hepatitis B/metabolismo , Hepatitis E/sangre , Hepatitis E/complicaciones , Hepatitis E/genética , Virus de la Hepatitis E/genética , Virus de la Hepatitis E/aislamiento & purificación , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Filogenia , ARN Viral/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Siberia , Cultivo de VirusRESUMEN
The anticancer drug Cancerolysin has been developed, by using the mutant Adel2 variant of human adenovirus serotype 5 designed at the State Research Center of Virology and Biotechnology. Cancerolysin possesses a high degree of replication activity for complementary cells 293 and p53-deficient tumor cells and, at the same time, has significant replication limitations in normal human cells. Preclinical studies of the drug on laboratory animals (mice, rabbits, guinea pigs) have demonstrated its harmlessness and safety. When stored at -40 and -70 degrees C, the drug showed no significant activity throughout the control observational period (1 year).
Asunto(s)
Adenoviridae , Antineoplásicos , Efecto Citopatogénico Viral , Viroterapia Oncolítica , Adenoviridae/genética , Anafilaxia , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/inmunología , Antineoplásicos/toxicidad , Línea Celular Transformada , Línea Celular Tumoral , Cobayas , Humanos , Terapia de Inmunosupresión , Inyecciones Intraperitoneales , Inyecciones Subcutáneas , Macrófagos Peritoneales/inmunología , Ratones , Ratones Endogámicos BALB C , Fagocitosis , Conejos , Replicación ViralRESUMEN
Site-directed mutagenesis was used to construct human adenovirus serotype 5 (Ad5) variants defective in E1A or E1B. Mutant Adel3 with deletion from E1A was markedly attenuated in permissive cell cultures regardless of the p53 status, and replicated efficiently only in cells of the complementing 293 line. Mutant Adel2 with deletion from E1B55K infected the 293 line cells and p53-deficient human tumor cells (A431, SW480, HEp2) with efficiencies similar to those of Ad5, whereas its replication in normal p53-positive cells was substantially limited. Thus, Adel2 proved to be capable of selective infection and lysis of p53-deficient human tumor cells in vitro. On intratumor injection, Adel2 dramatically suppressed the growth of human epidermoid carcinoma (A431) in nude mice. Adel2 is thus a promising model for designing therapeutic agents against p53-deficient human tumors.
Asunto(s)
Adenoviridae/genética , Eliminación de Gen , Genes Inmediatos-Precoces , Genes p53 , Replicación Viral/genética , Adenoviridae/fisiología , Proteínas E1A de Adenovirus/genética , Proteínas E1B de Adenovirus/genética , Animales , Secuencia de Bases , Cartilla de ADN , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Células Tumorales CultivadasRESUMEN
The results of polymerase chain reaction and of DNA sequencing of the Adel2 mutant variant of adenovirus serotype 5, passaged 10 times and capable of selectively infecting and lysing the p53-deficient human tumor cells, are indicative of a high stability of its genotype and of the phenotypic properties acquired by it in successive passage on 293 cells. The absence of admixtures of wild-type adenovirus was clearly shown in the cultivation and passage processes. It was revealed in an experimental analysis of virus-productive properties of the studied continuous cell culture 293 by using the method of multilayer cultivation, that the maximal Adel2 yield is obtained at the 50% cytopathic effect. Virus doses, that are effective for cell-culture contamination, are within a range of 100-10 TCPE50 per cell. In order to spare the viral material, the infecting dose of 10 TCPE50 per cell was chosen to infect a cell monolayer.
Asunto(s)
Adenoviridae/crecimiento & desarrollo , Mutación , Adenoviridae/genética , Secuencia de Bases , Línea Celular , Cartilla de ADN , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
Immunogenicity of recombinant vaccinia virus strain (VR26) expressing Venezuelan equine encephalomyelitis (VEE) virus structural protein genes was studied by oral immunization. Sera of animals immunized with VR26 contained antibodies specific to VEE virus, among which antibodies with virus-neutralizing activity were present. Evaluation of the protective efficiency of oral immunization with VR26 demonstrated a high level of animal protection from lethal doses of VEE virus. Rabbits immunized orally were highly resistant (protection index 142.9) to intranasal infection, which is of priority importance for antiVEE vaccine. Comparative analysis of the results of scarification and oral immunization with VR26 indicates that the type of immune response depends on the method of immunization. These results demonstrate good prospects of oral vaccination with recombinant VR26 strain for immunoprophylaxis of VEE.
Asunto(s)
Virus de la Encefalitis Equina Venezolana/genética , Genes Virales , Virus Vaccinia/inmunología , Proteínas Estructurales Virales/genética , Vacunas Virales/inmunología , Administración Oral , Animales , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , Encefalomielitis Equina Venezolana/prevención & control , Pruebas de Neutralización , Conejos , Recombinación Genética , Virus Vaccinia/genética , Vacunas Virales/administración & dosificaciónRESUMEN
Venezuelan equine encephalomyelitis (VEE) virus-based vectors for expression of heterologous genes have been constructed using full-length cDNA copy of the interstrain recombinant virus (Trinidad Donkey and 230 strain). Gene cassettes carrying the subgenomic mRNA promoter of VEE virus and the preS2-S gene of hepatitis B virus (HBV) were inserted before or after the genes of structural viral proteins. Live virus stocks were obtained by transfection of chick embryo fibroblasts with in vitro transcribed full-length RNA. Insertions of gene cassettes before the structural region resulted in expression of HBsAg (VEHB-25 and VEHB-361 viruses), whereas insertions in the 3' region did not. Recombinant virus VEHB-25 expressed HBsAg during 5 passages in Vero cells. VEHB-25 stimulated immune response to HBsAg and was less virulent than the parental virus.
Asunto(s)
Encefalomielitis Equina Venezolana/genética , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Precursores de Proteínas/genética , Proteínas del Envoltorio Viral/genética , Animales , Línea Celular , Embrión de Pollo , Chlorocebus aethiops , Clonación Molecular , Encefalomielitis Equina Venezolana/inmunología , Antígenos de Superficie de la Hepatitis B/ultraestructura , Microscopía Electrónica , Plásmidos , Regiones Promotoras Genéticas , Precursores de Proteínas/ultraestructura , ARN Mensajero/genética , ARN Viral/genética , Conejos , Recombinación Genética , Pase Seriado , Células VeroRESUMEN
A recombinant strain of vaccinia virus (VR26) containing a DNA-copy of the subgenomic 26S RNA of Venezuelan equine encephalomyelitis virus (VEE) inserted into the coding region of thymidine kinase (TK) gene was produced. This subgenomic RNA contained the genes for all structural proteins of the VEE virus, the strain Trinidad donkey (TRD). VR26 effectively expressed VEE virus glycoproteins on the membranes of the infected cells. Blood sera of VR26-immunized animals were found to contain VEE virus-specific antibodies. VR26-immunized mice and rabbits showed a high level of resistance to subcutaneous inoculation with the pathogenic TRD strain of VEE virus. VR26 also provided a high level of protection in animals against aerogenic infection. The absence of virus-neutralizing antibodies in most VR26-immunized animals resistant to inoculation with high doses of VEE suggests the dominant role of the cell component in the immune response. The immune response induced by the recombinant VR26 strain was stable as demonstrated by the resistance of the animals to a challenge with VEE virus 7 months after immunization. The experimental results suggest that this recombinant strain may be considered as a candidate for vaccine preparation.
Asunto(s)
ADN Complementario/genética , ADN Viral/genética , Virus de la Encefalitis Equina Venezolana/genética , Regulación Viral de la Expresión Génica/inmunología , ARN Mensajero/genética , ARN Ribosómico/genética , ARN Viral/genética , Recombinación Genética/inmunología , Virus Vaccinia/inmunología , Animales , Encefalomielitis Equina Venezolana/inmunología , Encefalomielitis Equina Venezolana/prevención & control , Regulación Viral de la Expresión Génica/genética , Sueros Inmunes/inmunología , Inmunización/métodos , Ratones , Conejos , Recombinación Genética/genética , Factores de Tiempo , Vaccinia/inmunología , Vaccinia/prevención & control , Virus Vaccinia/genéticaRESUMEN
Nine peptides were synthesized for detailed mapping of VEE virus E2-2 and E2-6 sites responsible for the formation of protective antibodies that neutralize the virus and block hemagglutination. The sequence of the peptides over-lapped the regions of amino acid residues 30-75 and 202-250 of VEE virus E2 protein in which antigenic mutations caused by monoclonal antibodies to E2-2 and E2-6 sites had been mapped. None of the synthesized peptides reacted in the enzyme immunoassay with a panel of 17 Mabs to VEE virus E2 protein. However, eight peptides reacted with polyclonal antiviral serum and two of them elicited antiviral antibody production. The E2-2 site might be associated with amino acid residues 30-45, and the region of E2 residues 57-62 in which antigenic mutations are observed is not a linear type antigenic determinant, but participates in the formation of antigenic determinants of the conformational type. The mapping of residues 202-250 demonstrated that all the peptides in this region were well recognized by polyclonal antiviral serum. The residues 235-240 were shown to form a linear epitope which provided a crossover between VEE and EEE viruses and was not recognized by 19 types of monoclonal antibodies cross-reacting with VEE and EEE viruses.
Asunto(s)
Sitios de Unión de Anticuerpos/inmunología , Virus de la Encefalitis Equina Venezolana/química , Mapeo Peptídico/métodos , Proteínas del Envoltorio Viral/análisis , Proteínas Virales de Fusión/análisis , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Antígenos Virales/análisis , Antígenos Virales/inmunología , Virus de la Encefalitis Equina Venezolana/inmunología , Epítopos/análisis , Epítopos/inmunología , Hibridomas/inmunología , Inmunización , Técnicas Inmunológicas , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutación/inmunología , Péptidos/síntesis química , Ratas , Proteínas del Envoltorio Viral/inmunología , Proteínas Virales de Fusión/inmunologíaRESUMEN
Thirty-three monoclonal antibodies (Mabs) interacting with the structural proteins of Eastern equine encephalomyelitis (EEE) virus were prepared. The mutual arrangement of the antigenic sites on the E1 and E2 glycoproteins was studied by competitive radioimmunoassay. At least four nonoverlapping sites were found on the E1. The E2 glycoprotein contained at least seven partially overlapping antigenic sites. Mabs to the sites E2-2 and E2-3 neutralized viral infectivity and blocked hemagglutination. Mabs to the site E2-1 blocked hemagglutination. Mabs to sites E2-2, 3, and 7 protected mice against lethal infection although the protective Mabs to sites E2-2b and E2-7 did not neutralize the virus. The antibodies to the other three sites of E2 and to all sites of E1 did not have any biological activity. The experimental results indicate the dominant role of E2 in antiviral immunity, over 98% of the observed protective effect being associated with the E2-2 site.