The HEK293 cells were maintained and propagated in complete DMEM supplemented with penicillin and streptomycin (Mediatech Inc., Herndon, VA) and 10% FBS (Gemini Bio-Products, West Sacramento, CA). Autologous peripheral blood mononuclear cells (PBMCs) and were obtained from 3 female HLA-A2 restricted healthy donors. All of the clinical materials were obtained with the patient's consent and approval by the local ethics committee.

The AAV/IE1 genome was constructed as a plasmid as previously described 2830. Briefly, the IE1 gene was amplified by PCR from plasmid pCGN-IE1, which was kindly provided by Dr. Thomas Shenk at the Department of Molecular Biology, Princeton University. PCR amplification for IE1 was carried out using the following primer pair: upstream, 5'-GGTACCATGGAGTCCTCTGCCAAGA-3'; downstream, 5'-CTCGAGGACCTTGTACTCATTACACATTG-3'. AAV/IE1 virus stocks were generated using complementary plasmids ins96-0.8 or pSH3, using HEK293 cells as described previously 283032. Lysates of HEK293 cells were used as virus-negative controls for mock infections.

HEK293 cells were spun in a cytospin column (5 × 10⁴ cells/slide), fixed with SlideRite (Fisher, USA), and air dried overnight. Each sample was permeabilized (P) in PBS 1×/0.1% Triton X-100 for 15 minutes at 4°C not permeabilized (NP). Results were analyzed using an Olympus IX71 inverted microscope equipped with a Fluoview 300 confocal laser system.

The titer of virus stocks was determined by real-time PCR as previously described 32. Briefly, we used the plasmid AAV/IE1 for the real-time PCR standards, respectively. Concentration was measured by absorbance at 260 nm.

Autologous DCs (2 × 10⁵ adherent monocytes) were generated and infected (0.5 mL virus [10⁹ eg/mL]) as previously described 2830. Recombinant granulocyte macrophage-colony-stimulating factor (GM-CSF) (R&D Systems, Minneapolis, MN, USA), at a final concentration of 800 IU/mL, was included in the medium throughout the culture. To induce monocytes into DCs, human interleukin-4 (IL-4) (R&D Systems, Minneapolis, MN, USA) at 1000 IU/mL was added on day 3, after infection.

Non-adherent PBMCs, isolated from healthy donors, were infected with AAV/IE1 virus at a multiplicity of infection of 100, 4 days before the ⁵¹Cr release assay.

The recombinant IE1 protein was made as previous described 33. Lipofection was performed using the cationic liposome-mediated transfection reagent, DOTAP (Roche Diagnostics, Indianapolis, IN). IE1 protein was mixed with the DOTAP reagent and serum-free media at ratios following the manufacturer's recommendations. The cells were then incubated in serum-free media containing the lipofection mix for 4–6 hours. Final IE1 concentration was 100 nM for the DCs and PBMCs. After 4–6 hours of incubation, serum-supplemented DMEM was added to cells. After 24 hours, all of the lipofection media was replaced with fresh growth media for cells.

CTL were generated from 3 normal donors (HLA matched). Experiments were performed in quadruplicate (experiments were preformed 4 times independently with different ratios of responders to DCs from 5:1; 10:1; 20:1; 40:1 data not provided) 2324. For each experiment, the non-adherent PBMCs were washed and re-suspended in AIM-V at 10 to 20 × 10⁶ cells per well in 6-well culture plates with AAV/IE1-loaded autologous DCs (optimal ratios of responders to DCs from 20:1). The cultures were supplemented with GM-CSF (800 U/mL) and recombinant human IL-2 (10 U/mL). After 7 days of co-culture, the cells were used for cytotoxicity assays in a 6-hour ⁵¹Cr assay, as previously described 162324. To determine the CTLs' HLA restriction, HLA-class I (W6/32) of antibodies, at a concentration of 25 μg/mL, were pre-incubated with the target cells for 30 minutes before addition of the stimulated T-cells. K562 cells were used as targets to observe natural killer (NK) cell activity. In all of these CTL killing assays, spontaneous release of chromium never exceeded 25% of the maximum release 2324.

This protocol was adapted from that described by Pala et al. and modified 2428. Cell surface marker analysis of T cells and DCs was conducted using fluorescence-activated cell scanning (FACS) (FACScan; BD Biosciences-PharMingen, Franklin Lakes, NJ), as described previously 2428.

All results are expressed as mean ± SD. Data were analyzed using nonparametric analysis of variance (ANOVA). Differences were considered significant if P < 0.05.

The goal of this study was to determine whether rAAV-based gene loading of IE1 genes into DCs could elicit a significant CTL response against IE1-positive target cell lines. This was the first time that the gene encoding IE1 was inserted into the AAV vector. First, the IE1 gene was amplified by PCR from plasmid pCGN-IE1. The IE1 cDNA obtained from pCGN-IE1 was inserted into the gutted AAV vector to generate AAV/IE1 as described in the materials and methods section. Figure 1A shows a structural map of the AAV/IE1 vector. In this vector, the IE1 gene was expressed from the AAV p5 promoter, which is known to be active in DCs 31. After rAAV vector generation, we evaluated their ability to infect HEK293 cells. The rAAV-vector infected cells expressed the target antigens, as confirmed by immunofluorecence labeling, which showed the expression of IE1 transduced HEK293 cells. (Figure 1)

Virus stock titers were determined by real-time PCR (Figure 2). We assessed the linearity of the real-time PCR by using a dilution row of the AAV/IE1 plasmid that would serve as standard curve in all further experiments. The obtained fragments corresponded to the expected size and no additional bands could be detected by gel electrophoresis, showing the specificity and selectivity of the PCR. We did not observe signals from the template sample in either the amplification plot or the agarose gel photograph (data not shown).

Protocols for generating DCs by differentiating PBMCs usually involve the use of GM-CSF and IL-4 during adherent monocyte culturing. We modified this protocol to promote AAV vector transduction in DC precursor monocytes by treating adherent monocytes just after AAV infection with GM-CSF alone, adding IL-4 on day 3. This method allowed higher levels of AAV transduction 34. Figure 1B shows a schematic diagram of the experimental protocol. Monocyte/DC population transduction was confirmed by measuring polyadenylated RNA expression of the AAV/IE1 transgene. At day 10, polyadenylated RNA was isolated from AAV/IE1-infected and mock-infected DC cultures. The mRNA levels were analyzed by RT-PCR for AAV/IE1 expression. A cellular housekeeping gene, TF_IIB, was included as a control. IE1 mRNA expression took place only in the infected DCs (Figure 3). A PCR-only control (no RT step) failed to generate a product, indicating that there was no DNA contamination in our samples.

We analyzed the ability of the AAV/IE1 vectors to generate IE1 specific-CTLs (optimal ratio E:T; 1:20). To analyze CTL activity, we used the following 5 target cell types for the ⁵¹Cr release assays (Figures 4, 5, 6): 1) Autologous PBMCs. Because late B cells are only a small percentage of PBMCs, PBMCs served as an autologous, antigen-negative control; 2) PBMCs transfected with AAV/IE1 expression plasmid; 3) PBMCs transfected with AAV only and AAV/GFP, as a negative controls; 4) PBMCs transfected with E6, as a control; 5) PBMCs transfected with IE1 protein.

To determine the ability of AAV/IE1-transduced DCs to stimulate IE1-specific CTLs, we performed a standard 6-hour⁵¹Cr assay on day 7 using a 1:20 (ratio: Effector:Target) (Figure 5) using the T-cell populations primed in co-culture with the rAAV-transduced DCs 30. We generated autologous targets by infecting donor PBMCs with AAV/IE1 virus 4 days before the CTL assay. AAV/IE1-infected PBMCs were found to express IE1 by RT-PCR analysis, whereas unaltered PBMCs and K562 cells did not express IE1 (data not shown). T-cells incubated with AAV/IE1-loaded DCs were able to kill the IE1-positive autologous target cells. These data are consistent with a strong antigen-specific CTL response. Figure 7 shows that CTL killing activity was dose-dependent and MHC class I restricted. In this experiment, 2 different doses of AAV/IE1 vector were used for DC loading and a zero virus control (PBMC only). The cytotoxicity of the stimulated T-cells directly correlated with the amount of AAV/IE1 used to load the DCs at day 0. Alternately, the addition of anti-class I antibodies significantly inhibited the killing activity (P < 0.05), suggesting that CTLs were MHC class I restricted. The CTL stimulation performed by AAV/IE1 loaded DCs was superior to the one performed by IE1 protein lipofection (P < 0.05). The negative controls (K562 and the targets pre-incubated with anti-MHC class I antibodies) did not induce significant killing activity. These data showed CTLs to be highly AAV/IE1 specific and MHC class I restricted. Figure 7 demonstrates that the use of AAV/GFP/IE1 loading DCs resulted in a higher delivery effect (80%) than IE1 protein lipofected DCs did (15%).