RESUMO
The magnitude and durability of immune responses induced by replication-defective adenovirus serotype 5 (ADV5) vector-based vaccines were evaluated in the simian-human immunodeficiency virus/rhesus monkey model. A single inoculation of recombinant ADV5 vector constructs induced cellular and humoral immunity, but the rapid generation of neutralizing anti-Ad5 antibodies limited the immunity induced by repeated vector administration. The magnitude and durability of the immune responses elicited by these vaccines were greater when they were delivered as boosting immunogens in plasmid DNA-primed monkeys than when they were used as single-modality immunogens. Therefore, administration of ADV5-based vectors in DNA-primed subjects may be a preferred use of this vaccine modality for generating long-term immune protection.
Assuntos
Adenovírus Humanos/imunologia , Anticorpos Antivirais/sangue , Vetores Genéticos/imunologia , Infecções por HIV/imunologia , Imunização Secundária , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Linfócitos T/imunologia , Vacinação , Vacinas Virais/imunologia , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Proteínas E1 de Adenovirus/genética , Proteínas E3 de Adenovirus/genética , Adenovírus Humanos/genética , Animais , Avaliação Pré-Clínica de Medicamentos , Deleção de Genes , Vetores Genéticos/genética , Anticorpos Anti-HIV/sangue , HIV-1/genética , HIV-1/imunologia , Injeções Intramusculares , Macaca mulatta , Testes de Neutralização , Plasmídeos/genética , Síndrome de Imunodeficiência Adquirida dos Símios/sangue , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Vacinas Virais/administração & dosagemRESUMO
Recombinant adenovirus (Ad) gene transfer vectors are effective at transferring exogenous genes to a variety of cells and tissue types both in vitro and in vivo. However, in the process of gene transfer, the Ad vectors induce the expression of target cell genes, some of which may modify the function of the target cell and/or alter the local milieu. To develop a broader understanding of Ad vector-mediated induction of endogenous gene expression, genes induced by first-generation E1(-) E4(+) Ad vectors in primary human umbilical vein endothelial cells were identified by cDNA subtraction cloning. The identified cDNAs included signaling molecules (lymphoid blast crisis [LBC], guanine nucleotide binding protein alpha type S [Galpha-S], and mitogen kinase [MEK5]), calcium-regulated/cytoskeletal proteins (calpactin p11 and p36 subunits, vinculin, and spinocerebellar ataxia [SCA1]), growth factors (insulin-like growth factor binding protein 4 and transforming growth factor beta2), glyceraldehyde-6-phosphate dehydrogenase, an expressed sequence tag, and a novel cDNA showing homology to a LIM domain sequence. Two- to sevenfold induction of the endogenous gene expression was observed at 24 h postinfection, and induction continued up to 72 h, although the timing of gene expression varied among the identified genes. In contrast to that observed in endothelial cells, the Ad vector-mediated induction of gene expression was not found following Ad vector infection of primary human dermal fibroblasts or human alveolar macrophages. Empty Ad capsids did not induce endogenous gene expression in endothelial cells. Interestingly, additional deletion of the E4 gene obviated the upregulation of genes in endothelial cells by the E1(-) E3(-) Ad vector, suggesting that genes carried by the E4 region play a central role in modifying target cell gene expression. These findings are consistent with the notion that efficient transfer of exogenous genes to endothelial cells by first-generation Ad vectors comes with the price that these vectors also induce the expression of a variety of cellular genes.
Assuntos
Proteínas E1 de Adenovirus/fisiologia , Proteínas E4 de Adenovirus/fisiologia , Adenovírus Humanos/fisiologia , Regulação Viral da Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos/fisiologia , Proteínas E1 de Adenovirus/genética , Proteínas E4 de Adenovirus/genética , Adenovírus Humanos/genética , Capsídeo/metabolismo , Células Cultivadas , DNA Complementar , Endotélio Vascular/citologia , Deleção de Genes , Vetores Genéticos/genética , Humanos , CinéticaRESUMO
OBJECTIVE: To assess the safety of adenovirus-mediated transfer of the RA538 (Ad-RA538) for the treatment of cancer and to furthermore in preparation for a clinical trial of Ad-RA538. METHODS: RT-PCR was used to detect the transcription of Ad-RA538 in HeLa cells infected with extracts from HeLa cells previously infected with Ad5-RA538. Cell counting was made to observe the effects of Ad-RA538 on the growth of the normal human fetal lung cell line 2BS. The virus was intraperitoneally injected into 2 groups of BalB/C mice at a dosage of 10(7) pfu and 10(9) pfu. Blood samples were taken from the mice to test the liver and renal function. PCR were used to screen the vital organs for the presence of adenovirus DNA. Microscopic examination of the vital organs was performed to observe the pathogenicity of Ad-RA538. RESULTS: Ad-RA538 was a replication-defective virus. It could infect 2BS cells effectively, but could not inhibit 2BS cell growth. No mouse died and no signs of general toxicity were seen following intraperitoneal injection of Ad-RA538. The adenoviral vector was present in the liver, spleen, kidney and stomach of mice injected with 10(9) pfu Ad-RA538. Six and 12 days after injection, mild inflammation was observed in the liver of mice received 10(9) pfu Ad-RA538. CONCLUSION: Ad-RA538 is safe both in vivo and in vitro, and clinical trials of Ad-RA538 can be performed.