Evaluation of NK cell responses in response to 5mer4.

<p>(a) NK cells were isolated from the spleen of BALB/c mice using negative selection. Cells were incubated overnight in the presence of 100 U/ml recombinant IL-2, with 2.5 µg/ml of 5mer4, 5mer2 control, DMSO control. Additional groups of cells were incubated with 1000 U/ml IL2 and 1000 U/ml IL2 combined with 5mer4 (2.5 µg/ml). NK cell activation was monitored for PE-CD69 upregulation on APC-DX5⁺ cells by flow cytometry. All cells were CD3⁻. The data is representative of three independent experiments performed on different days. (b) NK depletion in BALB/c mice. Mouse NK cells were depleted by administration of anti-asialo antibody at day -2 and day -1 before vaccination. Whole blood samples were collected from the saphenous vein (50 µl) from NK-depleted and undepleted control animals. All samples were first treated with ACK lysis buffer to remove red blood cells and then stained with PE-CD69 and FITC-DX5 to detect the percentage of NK cells remaining following depletion. (c) Survival of vaccine groups following NK cell depletion. At day 0, NK-depleted BALB/c mice were immunized with H5N1-HA (1 µg) or H5N1-HA (1 µg) + 5mer4 (50 µg), or PBS control. Three groups of mice that were not depleted were immunized with H5N1-HA (1 µg), H5N1-HA (50 ug) or H5N1-HA + 5mer4 (50 µg) as controls. Animals were challenged with 100LD₅₀ of homologous H5N1-H05 virus and survival was monitored over 17 days. Groups of 8–10 BALB/c mice were evaluated.