RESUMEN
The frequency that multiple different subtypes of hepatitis C virus (HCV) simultaneously infect a given individual is controversial. To address this question, heteroduplex mobility analysis (HMA) of portions of the HCV core and envelope 1 region was optimized for sensitive and specific detection of mixtures of HCV genomes of different genotype or subtype. Using the standard HCV genotyping approach of 5'-untranslated region (UTR) analysis, 28 of 374 (7.5%) chronic hepatitis C research subjects were classified as having either multiple-subtype HCV infections (n = 21) or switching HCV subtypes over time (n = 7), the latter pattern implying viral superinfection. Upon retesting of specimens by HMA, 25 of 28 multiple-subtype results could not be reproduced. All three patients with positive results were injection drug users with potential multiple HCV exposures. To address the hypothesis of tissue sequestration of multiple-subtype HCV infections, liver (n = 22), peripheral blood mononuclear cell (n = 13), perihepatic lymph node (n = 16), and serum (n = 19) specimens from 23 subjects with end-stage hepatitis C were collected and analyzed by the HMA technique. Whereas 5'-UTR results implicated mixed-subtype HCV infections in 2 subjects, HMA testing revealed no evidence of a second HCV subtype in any tissue compartment (0 of 70 compartments [0%]) or within any given subject (0 of 23 subjects [0%]). In summary, a large proportion of mixed-genotype and switching-genotype patterns generated by 5'-UTR analysis were not reproducible using the HMA approach, emphasizing the need for additional study.
Asunto(s)
Hepacivirus/clasificación , Hepacivirus/genética , Hepatitis C Crónica/virología , Análisis Heterodúplex/métodos , ARN Viral/genética , Regiones no Traducidas 5'/genética , Secuencia de Bases , Dermatoglifia del ADN , Genotipo , Hepacivirus/aislamiento & purificación , Humanos , Leucocitos Mononucleares/virología , Hígado/virología , Ganglios Linfáticos/virología , Datos de Secuencia Molecular , Filogenia , Polimorfismo de Longitud del Fragmento de Restricción , Suero/virología , Proteínas del Núcleo Viral/genética , Proteínas del Envoltorio Viral/genéticaRESUMEN
We have generated replication-competent (VSV-C/E1/E2) and nonpropagating (VSVDeltaG-C/E1/E2) vesicular stomatitis virus (VSV) contiguously expressing the structural proteins of hepatitis C virus (HCV; core [C] and glycoproteins E1 and E2) and report on their immunogenicity in murine models. VSV-C/E1/E2 and VSVDeltaG-C/E1/E2 expressed high levels of HCV C, E1, and E2, which were authentically posttranslationally processed. Both VSV-expressed HCV E1-E2 glycoproteins were found to form noncovalently linked heterodimers and appeared to be correctly folded, as confirmed by coimmunoprecipitation analysis using conformationally sensitive anti-HCV-E2 monoclonal antibodies (MAbs). Intravenous or intraperitoneal immunization of BALB/c mice with VSV-C/E1/E2 or VSVDeltaG-C/E1/E2 resulted in significant and surprisingly comparable HCV core or E2 antibody responses compared to those of control mice. In addition, both virus types generated HCV C-, E1-, or E2-specific gamma interferon (IFN-gamma)-producing CD8(+) T cells, as determined by enzyme-linked immunospot (ELISPOT) analysis. Mice immunized with VSVDeltaG-C/E1/E2 were also protected against the formation of tumors expressing HCV E2 (CT26-hghE2t) and exhibited CT26-hghE2t-specific IFN-gamma-producing and E2-specific CD8(+) T-cell activity. Finally, recombinant vaccinia virus (vvHCV.S) expressing the HCV structural proteins replicated at significantly lower levels when inoculated into mice immunized with VSV-C/E1/E2 or VSVDeltaG-C/E1/E2, but not with control viruses. Our data therefore illustrate that potentially safer replication-defective VSV can be successfully engineered to express high levels of antigenically authentic HCV glycoproteins. In addition, this strategy may therefore serve in effective vaccine and immunotherapy-based approaches to the treatment of HCV-related disease.
Asunto(s)
Vacunas contra Hepatitis B/inmunología , Hepatitis C/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Proteínas del Envoltorio Viral/inmunología , Replicación Viral , Animales , Formación de Anticuerpos/inmunología , Linfocitos T CD8-positivos/inmunología , Cricetinae , Modelos Animales de Enfermedad , Estudios de Evaluación como Asunto , Femenino , Vectores Genéticos , Células HeLa , Vacunas contra Hepatitis B/genética , Vacunas contra Hepatitis B/farmacología , Hepatitis C/prevención & control , Anticuerpos contra la Hepatitis C/inmunología , Humanos , Inmunización , Interferón gamma/inmunología , Ratones , Ratones Endogámicos BALB C , Conformación Proteica , Procesamiento Proteico-Postraduccional/genética , Procesamiento Proteico-Postraduccional/inmunología , Virus Vaccinia/genética , Virus Vaccinia/inmunología , Virus de la Estomatitis Vesicular Indiana/genéticaRESUMEN
Serological antibody assays used in hepatitis C virus diagnosis have improved in sensitivity and specificity. However, detection of active viremia or monitoring levels of virus during or after patient treatment is most commonly undertaken using nucleic acid-based technologies. Advancements in diagnostic technologies and implications for managing patients with hepatitis C in various clinical settings are discussed.