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
The E3 region of adenovirus codes for several membrane proteins, most of which are involved in immune evasion and prevention of host cell apoptosis. We explored the topology and targeting mechanisms of E3-6.7K, the most recently described member of this group, by using an in vitro translation system supplemented with microsomes. Here, we present evidence that E3-6.7K, one of the smallest signal-anchor proteins known, translocates across the membrane of the endoplasmic reticulum in a posttranslational, ribosome-independent, yet ATP-dependent manner, reminiscent of the translocation of tail-anchored proteins. Our analysis also demonstrated that E3-6.7K could achieve several distinct topological fates. In addition to the previously postulated type III orientation (N-luminal/C-cytoplasmic, termed NtmE3-6.7K), we detected a tail-anchored form adopting the opposite orientation (N-cytoplasmic/C-luminal, termed CtmE3-6.7K) as well as the possibility of a fully translocated form (N and C termini are both translocated, termed NCE3-6.7K). Due to the translocation of a positively charged domain, both the CtmE3-6.7K and NCE3-6.7K topologies of E3-6.7K constitute exceptions to the "positive inside" rule. The NtmE3-6.7K and NCE3-6.7K are the first examples of posttranslationally translocated proteins in higher eukaryotes that are not tail anchored. Distinct topological forms were also found in transfected cells, as both N and C termini of E3-6.7K were detected on the extracellular surface of transfected cells. The demonstration of unexpected topological forms and translocation mechanisms for E3-6.7K defies conventional thinking about membrane protein topogenesis and advises that both the mode of targeting and topology of signal-anchor proteins should be determined experimentally.