Figure 1.
Viral vectors used in this study (A) and their titers (B).
(A) A schematic representation of the engineered viruses used in this study. All viruses are based on E1-delated human Ad5. The transgenes (GFP or pp65) are under control of the human cytomegalovirus (CMV) immediate early promoter. The sequence encoding Tat-PTD was inserted into the hexon HVR5 region. The gene encoding the adenovirus fiber shaft and knob was either kept from Ad5 or replaced with the Ad35 counterparts. (B) Titers of the viruses used in this study established by three methods of determination: encapsidated virus genomes (evg) based on quantitative PCR, fluorescent forming units (FFU) based on infection of 911 cells and virus particles (vp) based on optical density measurements.
Table 1.
pAdEasy backbone vectors for production of recombinant adenovirus.
Figure 2.
Transduction efficiency of Ad5(GFP), Ad5PTD(GFP), Ad5f35(GFP) and Ad5PTDf35(GFP) in human primary cell cultures.
Primary human cells were isolated as described in the materials and methods section and transduced with the four GFP-encoding adenoviral vectors using fixed evg/cell and fixed total volumes. GFP expression was then analyzed by flow cytometry and transduction level is presented as the percentage of GFP-positive cells. The numbers under the x-axes indicate the evg/cell used for transduction. Experiments were repeated on cell cultures from 4–8 different donors except for MSCs, which was repeated 3 times. Non-act.: non-activated. Act.: activated. DCs: dendritic cells. Error bar represents standard deviation. Non-parametric student t-test was used for comparison between different groups. *:p<0.05, **:p<0.01, ***:p<0.001.
Figure 3.
Transduction efficiency of Ad5(GFP), Ad5PTD(GFP), Ad5f35(GFP) and Ad5PTDf35(GFP) in human primary tumor cells.
Primary human tumor cells were transduced with the four GFP-encoding adenoviral vectors using fixed evg/cell and fixed total volumes. GFP expression was then analyzed by flow cytometry and transduction level is presented as the percentage of GFP-positive cells. The numbers under the x-axes indicate the evg/cell used for transduction. Experiments were repeated three times for the two primary glioma cell cultures (U3013MG and U3054MG) and performed once for each individual sample for primary prostatic cells (due to low numbers of cells). CaP: prostate cancer. GL: Gleason score. BPH: benign prostatic hyperplasia. n.d.: not done. Prostate cancer initiating cells were sorted as the CD133+ αvβ1high population of primary prostate cancer cells. Error bar represents standard deviation. Non-parametric student t-test was used for comparison between different groups. *:p<0.05, **:p<0.01, ***:p<0.001.
Figure 4.
Biodistribution of surface-modified adenoviruses in mice after intravenous injection.
Ad5(GFP), Ad5PTD(GFP), Ad5f35(GFP) and Ad5PTDf35(GFP) virus vectors (evg = 5×109) were injected intravenously (tail-vein) into female balb/c mice and organs were harvested 48 hours post-virus injection. Viral genome DNA was isolated from the various organs and quantified by quantitative PCR in triplicates. Viral genome copy per tissue weight (mg) are shown as mean+SD (n = 3). One-way ANOVA with Tukey post test was used for comparison between different groups. #: Blood values are based on 100 µl blood from sacrificed mice and bone marrow (BM) values are based on 5×106 cells. *:p<0.05.
Figure 5.
Ex vivo expansion of CMV pp65-reactive T cells using adenovirus-transduced DCs.
DCs generated from four CMV-seropositive, HLA-A2-positive blood donors were transduced with 100 evg/cell of Ad5PTDf35(pp65) or Ad5(pp65). After expression of the pp65 protein from the viral vector inside the DC the protein is cleaved by proteasomes and the pp65459–503 peptide is presented by HLA-A2 on the surface of the DCs. The pp65-modified DCs were co-cultured with autologous T cells for 11 days to specifically stimulate and expand CMV pp65459–503-reactive CD8+ T cells. The frequency of CMV-pp65-specific T cells (CD3+) were evaluated by HLA-A*0201/pp65459–503 tetramer staining before (pre-stim) and 11 days after stimulation (post-stim).