RESUMEN
Currently, adenovirus (Ad) is being considered as a vector for the treatment of cystic fibrosis as well as other diseases. However, the cytotoxic T lymphocyte (CTL) response to Ad could limit the effectiveness of such approaches. Since the CTL response to virus infection is often focused on one or a few immunodominant epitopes, one approach to circumvent this response is to create vectors that lack these immunodominant epitopes. The effectiveness of this approach was tested by immunizing mice with human group C adenoviruses. Three mouse strains (C57BL/10SnJ [H-2b], C3HeB/FeJ [H-2k], and BALB/cByJ [H-2d]) were immunized with wild-type Ad or Ad vectors lacking the immunodominant antigen(s), and the CTL responses were measured. In C57BL/10 (B10) mice, a single inoculation intraperitoneally (i.p.) led to the recognition of an immunodominant antigen in E1A. When B10 mice were inoculated multiple times either i.p. or intranasally with wild-type Ad or an Ad vector lacking most of the E1 region, subdominant epitopes outside this region were recognized. In contrast, C3H mice inoculated with wild-type Ad recognized an epitope mapping within E1B. When inoculated twice with Ad vectors lacking both E1A and E1B, no immunorecessive epitopes were recognized. The immune response to Ad in BALB/c mice was more complex. CTLs from BALB/c mice inoculated i.p. with wild-type Ad recognized E1B in the context of the major histocompatibility complex (MHC) class I Dd allele and a region outside E1 associated with the Kd allele. When BALB/c mice were inoculated with E1-deleted Ad vectors, only the immunodominant Kd-restricted epitope was recognized, and Dd-restricted CTLs did not develop. This report indicates that the emergence of CTLs against immunorecessive epitopes following multiple administrations of Ad vectors lacking immunodominant antigens is dependent on haplotype and could present an obstacle to gene therapy in an MHC-diverse human population.
Asunto(s)
Adenovirus Humanos/inmunología , Antígenos Virales/inmunología , Epítopos de Linfocito T/inmunología , Vectores Genéticos/inmunología , Antígenos H-2/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/inmunología , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/inmunología , Proteínas E3 de Adenovirus/inmunología , Adenovirus Humanos/genética , Administración Intranasal , Animales , Línea Celular Transformada , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/inmunología , Vectores Genéticos/genética , Haplotipos , Antígeno de Histocompatibilidad H-2D , Humanos , Inmunización , Epítopos Inmunodominantes/inmunología , Inyecciones Intraperitoneales , Interferón gamma/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Células Tumorales CultivadasRESUMEN
Adenovirus (Ad) vectors for gene therapy are made replication defective by deletion of E1 region genes. For isolation, propagation, and large-scale production of such vectors, E1 functions are supplied in trans from a stable cell line. Virtually all Ad vectors used for clinical studies are produced in the 293 cell, a human embryonic kidney cell line expressing E1 functions from an integrated segment of the left end of the Ad type 5 (Ad5) genome. Replication-competent vector variants that have regained E1 sequences have been observed within populations of Ad vectors grown on 293 cells. These replication-competent variants presumably result from recombination between vector and 293 cell Ad5 sequences. We have developed Ad2-based vectors and have characterized at the molecular level examples of replication-competent variants. All such variants analyzed are Ad2-Ad5 chimeras in which the 293 cell Ad5 E1 sequences have become incorporated into the viral genome by legitimate recombination events. A map of Ad5 sequences within the 293 cell genome developed in parallel is consistent with the proposed recombination events. To provide a convenient vector production system that circumvents the generation of replication-competent variants, we have modified the Ad2 vector backbone by deleting or rearranging the protein IX coding region normally present downstream from the E1 region such that the frequency of recombination between vector and 293 cell Ad5 sequences is greatly reduced. Twelve serial passages of an Ad2 vector lacking the protein IX gene were carried out without generating replication-competent variants. In the course of producing and testing more than 30 large-scale preparations of vectors lacking the protein IX gene or having a rearranged protein IX gene, only three examples of replication-competent variants were observed. Use of these genome modifications allows use of conventional 293 cells for production of large-scale preparations of Ad-based vectors lacking replication-competent variants.
Asunto(s)
Proteínas E1 de Adenovirus/genética , Adenovirus Humanos/genética , Proteínas de la Cápside , Cápside/genética , Vectores Genéticos , Recombinación Genética , Adenovirus Humanos/fisiología , Secuencia de Bases , Línea Celular Transformada , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Replicación del ADN , ADN Viral , Variación Genética , Genoma Viral , Células HeLa , Humanos , Datos de Secuencia Molecular , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Replicación ViralRESUMEN
The human melanoma tumor Ags, MART1 and gp100, are specifically recognized by HLA-A2-restricted CD8+ CTLs derived from melanoma patients and appear to be involved in tumor regression. In order to develop immunizing vectors for the treatment of patients with metastatic melanoma, replication-defective recombinant adenoviruses, Ad2CMV-MART1 and Ad2CMV-gp100, which encode these tumor Ags, have been generated. Infection of non-Ag expressing HLA-A2+ cell lines A375 and MDA-231 with the vectors resulted in recognition by Ag-specific CTLs as demonstrated by specific target cell lysis and release of cytokines, including IFN-gamma, TNF-alpha, and granulocyte-macrophage-CSF. Sodium butyrate and TNF-alpha can further augment adenovirus-mediated transgene expression and increase recognition by specific CTLs. Although adenovirus-infected cells expressed the E3/19K protein at detectable levels, significant reduction of surface MHC class I expression was observed in only 3 of 10 tumor cell lines infected with either Ad2CMV-MART1 or Ad2CMV-gp100. Because of the suspected homology between the human MART1 and gp100 genes and their murine counterparts, we immunized C57BL/6 mice with these recombinant adenoviruses and demonstrated that immunization with Ad2CMV-gp100 could protect mice from murine melanoma B16 challenge administered intradermally. Depletion of CD8+ but not CD4+ T cells in vivo from Ad2CMV-gp100-vaccinated mice eliminated the protective effect. The anti-gp100 T cells induced by Ad2CMV-gp100 vaccinated appeared to be responsible for the protection. Thus, these recombinant adenoviruses encoding tumor Ags may be useful as vaccines to induce specific T cell immunity for cancer therapy.
Asunto(s)
Adenoviridae/genética , Virus Defectuosos/genética , Vectores Genéticos/genética , Inmunoterapia Activa , Melanoma Experimental/prevención & control , Melanoma/terapia , Glicoproteínas de Membrana/inmunología , Proteínas de Neoplasias/inmunología , Proteínas Recombinantes de Fusión/inmunología , Linfocitos T Citotóxicos/inmunología , Vacunas Sintéticas , Proteínas E3 de Adenovirus/fisiología , Animales , Antígenos de Neoplasias , Butiratos/farmacología , Ácido Butírico , Línea Celular , ADN Recombinante , Regulación Viral de la Expresión Génica/efectos de los fármacos , Antígeno HLA-A2/inmunología , Humanos , Epítopos Inmunodominantes/genética , Epítopos Inmunodominantes/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Antígeno MART-1 , Melanoma/inmunología , Melanoma Experimental/inmunología , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Proteínas Recombinantes de Fusión/genética , Factor de Necrosis Tumoral alfa/farmacología , Vacunación/métodos , Vacunas Sintéticas/uso terapéutico , Antígeno gp100 del MelanomaRESUMEN
A new adenovirus-based vector (Ad2/CFTR-1) has been constructed in which the cDNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis (CF) gene product, replaces the early region 1 coding sequences, E1a and E1b. The virus retains the E3 region. Ad2/CFTR-1 and a related construct encoding beta-galactosidase replicate in human 293 cells which provide E1 gene functions in trans. Replication of these recombinant viruses was not detected in a variety of other cells, although very limited viral DNA synthesis and transcription from the E4 and L5 regions could be measured. These E1-deletion vectors were also deficient in cellular transformation, shut-off of host cell protein synthesis, and production of cytopathic effects, even at high multiplicities of infection. Ad2/CFTR-1 produced CFTR protein in a variety of cells including airway epithelia from CF patients. Expression of functional CFTR protein in a CF airway epithelial monolayer was detected by correction of the Cl- transport defect characteristic of CF. Surprisingly low multiplicities of infection (0.1 moi) were sufficient to generate CFTR Cl- current across a CF epithelial monolayer in vitro. These data, together with the lack of obvious toxicity, suggest that Ad2/CFTR-1 should be suitable for CF gene therapy.
