RESUMO
The immunogenicity of adenovirus serotype 5 (Ad5) vectors has been shown to be suppressed by neutralizing antibodies (NAbs) directed primarily against the hexon hypervariable regions (HVRs). However, the role of NAbs directed against other capsid components, particularly the adenovirus fiber, remains unclear. Here we show that Ad5 NAbs target both hexon and fiber following vaccination and natural infection. Utilizing neutralization assays with capsid chimeric vectors, we observed that NAb responses to hexon appeared dominant and NAb responses against fiber were subdominant in sera from vaccinated mice, vaccinated humans, and naturally exposed humans. A novel chimeric Ad5 vector in which both the hexon HVRs and the fiber knob were exchanged nearly completely evaded Ad5-specific NAbs both in vitro and in vivo.
Assuntos
Infecções por Adenovirus Humanos/imunologia , Adenovírus Humanos/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/genética , Adenovírus Humanos/fisiologia , Animais , Proteínas do Capsídeo/genética , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Vetores Genéticos/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , VacinaçãoRESUMO
The immunogenicity of adenovirus serotype 5 (Ad5) vectors has been shown to be suppressed by neutralizing antibodies (NAbs) directed primarily against the hexon hypervariable regions (HVRs). We previously reported that replacing all seven HVRs with those from the rare serotype virus Ad48 resulted in a chimeric Ad5HVR48(1-7) vector that largely evaded preexisting Ad5 immunity in mice and rhesus monkeys. In this study, we evaluated the extent to which Ad5-specific NAbs are directed against various HVRs. We constructed partial HVR-chimeric Ad5 vectors with only a subset of HVRs exchanged, and we utilized these vectors in both NAb assays and murine immunogenicity studies with and without baseline Ad5 immunity. Our results demonstrate that Ad5-specific NAbs target multiple HVRs, suggesting that replacing all HVRs is required to optimize evasion of anti-Ad5 immunity. These data have important implications for the development of novel vectors for both vaccines and gene therapy.
Assuntos
Infecções por Adenovirus Humanos/imunologia , Adenovírus Humanos/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/genética , Animais , Proteínas do Capsídeo/genética , Terapia Genética/instrumentação , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Testes de NeutralizaçãoRESUMO
Targeting dendritic cell (DC) functions such as migration is a pivotal mechanism used by HIV-1 to disseminate within the host. The HIV-1 envelope protein is the most important of the virally encoded proteins that exploits the migratory capacity of DCs. In the present study, we elucidated the signaling machinery involved in migration of immature DCs (iDCs) in response to HIV-1 envelope protein. We observed that M-tropic HIV-1 glycoprotein 120 (gp120) induces phosphorylation of the nonreceptor tyrosine kinase, Pyk2. Inhibition of Pyk2 activity using a pharmacologic inhibitor, kinase-inactive Pyk2 mutant, and Pyk2-specific small interfering RNA blocked gp120-induced chemotaxis, confirming the role of Pyk2 in iDC migration. In addition, we also illustrated the importance of Pyk2 in iDC migration induced by virion-associated envelope protein, using aldithriol-2-inactivated M-tropic HIV-1 virus. Further analysis of the downstream signaling mechanisms involved in gp120-induced migration revealed that Pyk2 activates p38 mitogen-activated protein kinase, which in turn activates the F-actin-binding protein, leukocyte-specific protein 1, and enhances its association with actin. Taken together, our studies provide an insight into a novel gp120-mediated pathway that regulates DC chemotaxis and contributes to the dissemination of HIV-1 within an infected person.
Assuntos
Movimento Celular , Células Dendríticas/metabolismo , Quinase 2 de Adesão Focal/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Western Blotting , Quimiotaxia , Citometria de Fluxo , Quinase 2 de Adesão Focal/antagonistas & inibidores , Quinase 2 de Adesão Focal/genética , Proteína gp120 do Envelope de HIV/genética , Humanos , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas dos Microfilamentos/genética , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Receptores CCR5/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tirfostinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
The K1L protein of vaccinia virus is required for its growth in certain cell lines (RK-13 and human). The cowpox host-range protein CP77 has been shown to complement K1L function in RK-13 cells, despite a lack of homology between the two proteins except for ankyrin repeats. We investigated the role of ankyrin repeats of K1L protein in RK-13 cells. The growth of a recombinant vaccinia virus, with K1L gene mutated in the most conserved ankyrin repeat, was severely impaired. Infection with the mutant virus caused shutdown of cellular and viral protein synthesis early in infection. We also investigated the interaction of K1L protein with cellular proteins and found that K1L interacts with the rabbit homologue of human ACAP2, a GTPase-activating protein with ankyrin repeats. Our result suggests the importance of ankyrin repeat for host-range function of K1L in RK-13 cells and identifies ACAP2 as a cellular protein, which may be interacting with K1L.