1. Volume 130, Issue 2, Pages 453-464 (February 2006)
Vaccination With Protein-Transduced Dendritic Cells Elicits a Sustained Response to Hepatitis C Viral Antigens 
Noriyoshi Kuzushita, Stephen H. Gregory, Nola A. Monti, Rolf Carlson, Stephan Gehring, Jack R. Wands 
Gastroenterology 
Volume 130, Issue 2, Pages (February 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association Terms and Conditions

2. Figure 1
Determination of transfection efficiency of core- or NS5-transduced DCs. The CD11c+ splenocytes were obtained by positive selection with magnetic beads and transduced with HCV core or NS5. The percentage of core+ or NS5+ cells was determined by indirect immunofluorescence analysis. The HCV core/fluorescein isothiocyanate (FITC) uptake (shaded area) after core protein transduction was compared with the FITC control (A). The HCV core transduction efficiency was approximately 60%. The NS5/FITC uptake (shaded area) after NS5 protein transduction was compared with the FITC control (B). The NS5 transduction efficiency was approximately 90%.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

3. Figure 2
Characterization of core- or NS5-transduced DCs. The CD11c+ splenocytes were derived from mice after hydrodynamic delivery of Flt3L and transduced with core or NS5, followed by staining for intracellular core or NS5 and the indicated cell-surface markers. The cells were analyzed by 2-color flow cytometry. The cells transduced with core and NS5 composed a relatively mature DC population based on the basis of the high percentage of cells that expressed MHC class II molecules. The results of NS5-transduced DCs showed a substantial proportion of cells expressing costimulatory molecules (ie, CD40, CD80, and CD86, as well as CD11b) (B). In contrast, the CD40 and CD4 expression in core-transduced DCs was relatively decreased, even considering the lower transfection efficiency of 60% (A). FITC, fluorescein isothiocyanate.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

4. Figure 3
Splenocytes derived from mice vaccinated with core-transduced DCs show antigen-specific CTL activity. Four mice in each group were inoculated subcutaneously 3 times at 2-week intervals with 200 μL of medium alone or medium that contained 1 × 106 DCs transduced or not transduced with HCV core protein. Spleen cells were obtained from mice 2 weeks after inoculation with core-transduced DCs, nontransduced DCs, or medium alone and cultured 3 days in the presence of 0.3 μg/mL recombinant core protein. Subsequently, the cells were cocultured with 51Cr-labeled, core-expressing target cells at the effector to target (E:T) cell ratio listed (A). Values are the means ± SD (percentage specific cytotoxicity calculated from quadruplicate wells in a single experiment representative of 2 experiments). *Significantly greater CTL activity than expressed by splenocytes derived from vaccinated control mice (P < .05). In panels B and C, splenocytes (2 × 106 per well in 24-well plates) obtained from vaccinated and control animals were cultured 2 days with recombinant core protein at the concentrations indicated. The culture supernates were collected; IFN-γ and IL-4 were quantified by ELISA. Values are the means ± SD calculated from triplicate wells in a single experiment representative of 2 similar experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

5. Figure 4
Antigen-specific responses of splenocytes derived from NS5-DC–vaccinated mice. Four mice in each group were inoculated subcutaneously 3 times at 2-week intervals with 200 μL of medium alone or medium that contained 1 × 106 DCs transduced or not transduced with HCV NS5 protein. Splenocytes obtained from mice at 2 weeks after inoculation with NS5-transduced DCs, nontransduced DCs, or medium alone were cultured 3 days in the presence of 0.3 μg/mL recombinant NS5. Subsequently, the cells were cocultured with 51Cr-labeled, NS5-expressing (———) or nonexpressing (-----) target cells at the effector to target (E:T) cell ratio listed (A). Values are the means ± SD (percentage specific cytotoxicity calculated from quadruplicate wells in a single experiment representative of 2 experiments). *Splenocytes derived from mice vaccinated with NS5-transduced DCs and incubated with NS5-expressing target cells show significantly greater cytolytic activity (P < .05). In panels B and C, splenocytes (2 × 106 per well in 24-well plates) derived from control or vaccinated mice at 2 weeks after immunization were cultured in the presence of recombinant NS5 at the concentration indicated. The culture supernates were collected after 48 hours of incubation, and the quantities of IFN-γ and IL-4 were determined by ELISA. Values are the means ± SD calculated from triplicate wells in a single experiment representative of 2 similar experiments. *Significantly increased when cells were incubated in the presence of recombinant protein (P < .05). (D) Splenocytes (2 × 105 per well in 96-well plates) derived from mice vaccinated with NS5-transduced DCs were cultured in the absence or presence of 1.0 μg/mL recombinant NS5 and 100 μg/mL chromatographically purified normal rat immunoglobulin (Ig)G, monoclonal rat IgG2b anti-mouse CD4 (GK1.5 hybridoma; American Type Culture Collection, Manassas, VA), or monoclonal rat IgG2b anti-mouse CD8 (2.43 hybridoma; American Type Culture Collection). Values are the means ± SD calculated from triplicate wells in a single experiment representative of 2 similar experiments. *Significantly less than other groups incubated with NS5 (P < .05).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

6. Figure 5
Prolonged expression of cellular immunity by NS5-DC–vaccinated mice in vitro. Four mice in each group were inoculated subcutaneously 3 times at 2-week intervals with 200 μL of medium alone or medium that contained 1 × 106 DCs transduced or not transduced with HCV NS5 protein. Splenocytes were obtained from mice at 10 weeks after final inoculation with NS5-transduced DCs. CTL activity (A) and IFN-γ production (B) were assessed. Values are the means ± SD calculated from multiple wells in single experiments; results obtained in repeat experiments were comparable. *Splenocytes derived from mice vaccinated with NS5-transduced DCs produced significantly more IFN-γ and showed greater cytolytic activity (P < .05). E:T, effector to target ratio.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

7. Figure 6
Vaccination with NS5-transduced DCs induces strong and sustained NS5-specific immunity in vivo. Groups of animals were inoculated 3 times at 2-week intervals with an empty or an NS5-expressing plasmid (100 μg) (A) or with 200 μL medium alone or 200 μL containing 1 × 106 DCs transduced or not transduced with NS5 proteins; there was no difference between groups (B). Vaccinated animals received 1 × 106 stably expressing HCV NS5 (———) or nonexpressing (-----) murine myeloma cells at 10 weeks after the last inoculation. *Mice vaccinated with NS5-transduced DCs showed significantly less NS5-specific tumor cell growth than all other groups (P < .05). On day 15 of growth, the tumors (SP2/21) were dissected from mice vaccinated with NS5-transduced DCs, weighed, and compared with those growing in mice immunized with conventional NS5-DNA constructs (C). Data are the mean ± SE weights of tumors obtained from 9–10 mice in each group. *Significantly less than mice vaccinated with NS5-expressing DNA (P < .01).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions