Modified adenovirus vectors for use in vaccines and gene therapy
A technology of viral vector, adenovirus, used in immunization, medical field
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Embodiment 1
[0046] Example 1. Protection of mature dendritic cells from CTL-mediated lysis
[0047] First, experiments were performed to replicate the findings of others: immature dendritic cells are sensitive to CTL-mediated killing whereas mature dendritic cells are protected. For this, blood was isolated from a healthy adult individual and subsequently used to isolate peripheral blood mononuclear cells by the ficoll procedure. Monocytes were isolated from the cell population using the Variomacs technique and CD14-specific antibodies (Miltenyi Biotec GmbH) known to those skilled in the art. These monocytes were cultured for 6 days in the presence of 100 ng / ml IL-4 (Bioscource International, Inc.) and 800 IU / ml GM-CSF (Leucomax; Novartis) to obtain immature dendritic cells. Correct differentiation of monocytes to immature dendritic cells was monitored by FACScalibur measuring cell surface expression with antibodies (from BD Pharmingen). As known to those of ordinary skill in the art of...
Embodiment 2
[0052] Example 2. Adenovirus-mediated induction of sensitized mature dendritic cells
[0053] As described above, the procedures for obtaining mature and immature dendritic cells from human peripheral blood mononuclear cells, loading the cells with europium, and loading the gp100 peptide have been roughly described. Dendritic cells were incubated with the replication-deficient adenoviral vector for 48 hours (1000 of the virus particles per cell (vp / cell)). Control groups were not incubated with adenoviral vector. The adenovirus vector used was based on adenovirus serotype 5 (Ad5), but deleted the entire adenovirus El region. In addition, the vector used was genetically engineered to carry a fiber molecule from adenovirus serotype 35, giving it a better tropism for dendritic cells. WO 00 / 03029 and WO 02 / 24730 describe in detail how to generate Ad5 viruses carrying fibers from various (other) serotypes. The vector contains a eukaryotic expression cassette in the E1 region con...
Embodiment 3
[0056] Example 3. Regulation of sensitization of mature dendritic cells by recombinant adenovirus
[0057] It has been documented that upregulation of SPI-6 in mice or PI-9 in humans or keeping their levels stable is at least partly involved in biological pathways by which mature dendritic cells resist CTL-mediated killing. To investigate whether adenovirus sensitizes dendritic cells through the PI-9 pathway, two different experimental strategies were performed: (i) Western blot analysis and / or reverse transcription PCR to determine whether adenovirus was or was not exposed to adenovirus Expression levels of PI-9 protein and / or RNA in human dendritic cells of the vector. (ii) Generation of adenoviral vector carrying PI-9 cDNA followed by infection of mature and immature dendritic cells to determine the effect of PI-9 overexpression on CTL-mediated killing.
[0058] To determine expression levels of PI-9, mature and immature dendritic cells from human monocytes were obtained a...
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