RESUMEN
Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract illness in infants, the elderly, and other high-risk individuals. Despite years of research in this field, there is no effective licensed vaccine to prevent RSV infection. We have generated candidate RSV vaccines using a recombinant vesicular stomatitis virus (rVSV) replicon in which the attachment and fusion domains of the VSV glycoprotein (G) have been deleted (rVSV-Gstem), rendering the virus propagation-defective except in the presence of complementing VSV G provided in trans. A form of this vector encoding the RSV fusion protein (F) gene expressed high levels of F in vitro and elicited durable neutralizing antibody responses as well as complete protection against RSV challenge in vivo. Mice vaccinated with rVSV-Gstem-RSV-F replicons also developed robust cellular responses characterized by both primary and memory Th1-biased CD8+ and CD4+ T cells. Furthermore, a single high dose of the Gstem-RSV-F replicon was effective against challenge with both RSV A and B subgroup viruses. Finally, addition of an RSV glycoprotein (G)-expressing Gstem vector significantly improved the incomplete protection achieved with a single low dose of Gstem-RSV-F vector alone.
Asunto(s)
Vectores Genéticos/genética , Vectores Genéticos/inmunología , Inmunidad Celular , Inmunidad Humoral , Infecciones por Virus Sincitial Respiratorio/inmunología , Virus Sincitiales Respiratorios/genética , Virus Sincitiales Respiratorios/inmunología , Vesiculovirus/genética , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Femenino , Orden Génico , Vectores Genéticos/administración & dosificación , Humanos , Inmunización , Memoria Inmunológica , Ratones , Infecciones por Virus Sincitial Respiratorio/prevención & control , Células TH1/inmunología , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/inmunologíaRESUMEN
Propagation-defective vesicular stomatitis virus (VSV) vectors that encode a truncated G protein (VSV-Gstem) or lack the G gene entirely (VSV-DeltaG) are attractive vaccine vectors because they are immunogenic, cannot replicate and spread after vaccination, and do not express many of the epitopes that elicit neutralizing anti-VSV immunity. To consider advancing non-propagating VSV vectors towards clinical assessment, scalable technology that is compliant with human vaccine manufacturing must be developed to produce clinical trial material. Accordingly, two propagation methods were developed for VSV-Gstem and VSV-DeltaG vectors encoding HIV gag that have the potential to support large-scale production. One method is based on transient expression of G protein after electroporating plasmid DNA into Vero cells and the second is based on a stable Vero cell line that contains a G gene controlled by a heat shock-inducible transcription unit. Both methods reproducibly supported production of 1 x 10(7) to 1 x 10(8) infectious units (I.U.s) of vaccine vector per milliliter. Results from these studies also showed that optimization of the G gene is necessary for abundant G protein expression from electroporated plasmid DNA or from DNA integrated in the genome of a stable cell line, and that the titers of VSV-Gstem vectors generally exceeded VSV-DeltaG.
Asunto(s)
Vectores Genéticos , Glicoproteínas de Membrana/deficiencia , Vesiculovirus/crecimiento & desarrollo , Vesiculovirus/genética , Proteínas del Envoltorio Viral/deficiencia , Animales , Chlorocebus aethiops , Glicoproteínas de Membrana/biosíntesis , Datos de Secuencia Molecular , ARN Viral/genética , Análisis de Secuencia de ADN , Células Vero , Proteínas del Envoltorio Viral/biosíntesis , Cultivo de Virus/métodos , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Recombinant vesicular stomatitis viruses (rVSVs) are being developed as potential HIV-1 vaccine candidates. To characterize the in vivo replication and dissemination of rVSV vectors in mice, high doses of a highly attenuated vector expressing HIV-1 Gag, rVSV(IN)-N4CT9-Gag1, and a prototypic reference virus, rVSV(IN)-HIVGag5, were delivered intramuscularly (IM), intranasally (IN), or intravenously (IV). We used quantitative, real-time RT-PCR (Q-PCR) and standard plaque assays to measure the temporal dissemination of these viruses to various tissues. Following IM inoculation, both viruses were detected primarily at the injection site as well as in draining lymph nodes; neither virus induced significant weight loss, pathologic signs, or evidence of neuroinvasion. In contrast, following IN inoculation, the prototypic virus was detected in all tissues tested and caused significant weight loss leading to death. IN administration of rVSV(IN)-N4CT9-Gag1 resulted in detection in numerous tissues (brain, lung, nasal turbinates, and lymph nodes) albeit in significantly reduced levels, which caused little or no weight loss nor any mortality. Following IV inoculation, both prototypic and attenuated viruses were detected by Q-PCR in all tissues tested. In contrast to the prototype, rVSV(IN)-N4CT9-Gag1 viral loads were significantly lower in all organs tested, and no infectious virus was detected in the brain following IV inoculation, despite the presence of viral RNA. These studies demonstrated significant differences in the biodistribution patterns of and the associated pathogenicity engendered by the prototypic and attenuated vectors in a highly susceptible host.