Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 2 de 2
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Virol Methods ; 213: 26-37, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25486083

RESUMO

Advancement of new vaccines based on live viral vectors requires sensitive assays to analyze in vivo replication, gene expression and genetic stability. In this study, attenuated canine distemper virus (CDV) was used as a vaccine delivery vector and duplex 2-step quantitative real-time RT-PCR (RT-qPCR) assays specific for genomic RNA (gRNA) or mRNA have been developed that concurrently quantify coding sequences for the CDV nucleocapsid protein (N) and a foreign vaccine antigen (SIV Gag). These amplicons, which had detection limits of about 10 copies per PCR reaction, were used to show that abdominal cavity lymphoid tissues were a primary site of CDV vector replication in infected ferrets, and importantly, CDV gRNA or mRNA was undetectable in brain tissue. In addition, the gRNA duplex assay was adapted for monitoring foreign gene insert genetic stability during in vivo replication by analyzing the ratio of CDV N and SIV gag genomic RNA copies over the course of vector infection. This measurement was found to be a sensitive probe for assessing the in vivo genetic stability of the foreign gene insert.


Assuntos
Vírus da Cinomose Canina/fisiologia , Portadores de Fármacos , Expressão Gênica , Produtos do Gene gag/biossíntese , Vetores Genéticos , Instabilidade Genômica , Replicação Viral , Abdome/virologia , Animais , Encéfalo/virologia , Vírus da Cinomose Canina/genética , Furões , Produtos do Gene gag/genética , Tecido Linfoide/virologia , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Vacinas contra a SAIDS/administração & dosagem , Vacinas contra a SAIDS/genética , Vírus da Imunodeficiência Símia/genética , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética
2.
PLoS One ; 9(9): e106597, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25215861

RESUMO

Though vaccination with live-attenuated SIV provides the greatest protection from progressive disease caused by SIV challenge in rhesus macaques, attenuated HIV presents safety concerns as a vaccine; therefore, live viral vectors carrying HIV immunogens must be considered. We have designed a replication-competent vesicular stomatitis virus (VSV) displaying immunogenic HIV-1 Env trimers and attenuating quantities of the native surface glycoprotein (G). The clade B Env immunogen is an Env-VSV G hybrid (EnvG) in which the transmembrane and cytoplasmic tail regions are derived from G. Relocation of the G gene to the 5'terminus of the genome and insertion of EnvG into the natural G position induced a ∼1 log reduction in surface G, significant growth attenuation compared to wild-type, and incorporation of abundant EnvG. Western blot analysis indicated that ∼75% of incorporated EnvG was a mature proteolytically processed form. Flow cytometry showed that surface EnvG bound various conformationally- and trimer-specific antibodies (Abs), and in-vitro growth assays on CD4+CCR5+ cells demonstrated EnvG functionality. Neither intranasal (IN) or intramuscular (IM) administration in mice induced any observable pathology and all regimens tested generated potent Env-specific ELISA titers of 10(4)-10(5), with an IM VSV prime/IN VSV boost regimen eliciting the highest binding and neutralizing Ab titers. Significant quantities of Env-specific CD4+ T cells were also detected, which were augmented as much as 70-fold by priming with IM electroporated plasmids encoding EnvG and IL-12. These data suggest that our novel vector can achieve balanced safety and immunogenicity and should be considered as an HIV vaccine platform.


Assuntos
Vetores Genéticos/metabolismo , HIV-1/metabolismo , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Vacinas Atenuadas/imunologia , Vírus da Estomatite Vesicular Indiana/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Formação de Anticorpos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular , Feminino , Imunização , Pulmão/imunologia , Contagem de Linfócitos , Camundongos Endogâmicos BALB C , Conformação Proteica , Multimerização Proteica , Baço/imunologia , Replicação Viral , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...