Figure 5: (a) DNA molecules bind to VLPs in the DNA/VLP mixture. HIV 89.6 Env-DNA, SHIV 89.6 VLPs, or their mixtures were loaded onto a two-layer sucrose cushion, with 500 30% sucrose at bottom and 2 mL 20% sucrose above, followed by ultracentrifugation. After centrifugation, 10 from the 30% cusion of each sample was loaded onto a 1% agarose gel, and after electrophoresis, the gel was stained with Ethidium Bromide followed by destaining and then visualization of DNA by UV light. Marker: DNA molecular weight marker (lambda DNA Hind III digest); VLP: 50 SHIV 89.6 VLPs; DNA: 50 HIV Env-DNA; DNA/VLP: 50 HIV Env-DNA mixed with 50 SHIV: 89.6 VLPs; DNA/VLP (lysed): 50 HIV Env-DNA mixed with 50 SHIV 89.6 VLPs that were lysed with 1% Triton X-100. (b) SHIV 89.6 VLPs stimulate cytokine secretion by BMDCs. BMDCs were prepared as described in Section 2 and incubated with different stimulants in triplicates. DCs incubated with culture medium only were used as negative controls and DCs incubated with LPS (10 ng/mL) were used as positive controls. To ensure that stimulation of DC by DNA or VLPs is not due to contamination of endotoxin, the DCs were also incubated with the same stimulants that have been heat-treated at 100°C for 30 minutes. Cell-free supernatants were harvested 24 hours after incubation at 37°C in 5% and assayed for the levels (pg/mL) of IL-6, IL-12, and TNF-alpha by ELISA. Error bars represent standard deviations and the results shown represent typical results obtained from two different stimulation experiments. Medium, cell culture medium (negative control): LPS, 10 ng/mL (positive control); DNA: HIV Env-DNA (50 /mL); VLP: SHIV 89.6 VLPs (10 /mL); DNA-VLP: a mixture of HIV Env-DNA (50 /mL) and SHIV 89.6 VLPs (10 /mL). (c) Maturation of BMDCs after stimulation by SHIV 89.6 VLP vaccines. BMDCs were incubated with different stimulants as described above. After stimulation, the BMDCs were stained for surface expression of CD11c, CD80, and CD86 and then analyzed by flow cytometry. The results are presented as histograms for CD80 (upper panel) and CD86 (lower panel) for CD11c positive cells. Medium: cell culture medium (negative control); LPS: 10 ng/mL (positive control); DNA: HIV Env-DNA (50 /mL); VLP: SHIV 89.6 VLPs (10 /mL); DNA-VLP: a mixture of HIV Env-DNA (50 /mL) and SHIV 89.6 VLPs (10 /mL).