Briefly, rIL-5 was incubated in flat bottom 96-well plates with 2 × 104 BCL1 cells
(a B cell lymphoma line) per well and incubated for 24 h at 37 °C, 5% CO2. 1 μCi of 3H-thymidine (Hartmann Analytic, Switzerland) was added to each well and the plates incubated for 6 h at 37 °C with 5% CO2. The cells were harvested, washed and the incorporation of thymidine determined by emission-counting with a liquid scintillation counter. Commercial murine IL-5 from R&D systems (cIL-5) was used as a control. To test the neutralizing activity of serum Akt inhibitor from Qβ-IL-5 vaccinated mice, BCL1 cells (2 × 104 per well) were plated in the presence of 20 ng/ml of rIL-5. Pooled sera from Qβ-IL-5 vaccinated or naive mice was titrated with the cells (starting dilution 1/4, titration steps 1/6). After 24 h, 1 μCi of 3H-thymidine was added to the cells, which were incubated for 12 h. The incorporation
of thymidine was determined by emission-counting with a liquid scintillation counter. Murine eotaxin was expressed as a fusion protein in a vector modified from pET22b. The fusion protein (r-eotaxin) consisted of the mature form of murine eotaxin, a hexa-histidine tag and a cysteine containing linker (GGC) at its C-terminus. Expression of r-eotaxin in E. coli BL21 (DE3) was induced with 1 mM IPTG. The soluble fraction of bacterial lysate containing r-eotaxin was mixed with Ni-NTA agarose (Qiagen) in 300 mM NaCl, 50 mM NaH2PO4, 0.5% tween 20 and 20 mM imidazole (pH 8). After washing away unbound contaminants, r-eotaxin was eluted with 300 mM NaCl, 50 mM NaCl, tween 20 and 250 mM imidazole (pH 8). Semi-purified r-eotaxin was loaded onto a ABT-263 supplier SP sepharose column (Amersham) in buffer containing 20 mM Tris, 200 mM NaCl (pH 8). After washing r-eotaxin was eluted with an increasing salt gradient (20 mM Tris, 1 M NaCl, pH 8.0). VLPs derived from the bacteriophage Qβ were expressed already in E. coli containing a expression plasmid pQ10 and purified as described previously . In order to be coupled to IL-5, Qβ VLPs were first derivatized with 10-fold excess of a heterobifunctional chemical cross-liker, succinimidyl-6-(β-maleimidopropionamido) hexanoate
(SMPH). The unbound SMPH was removed by dialysis against PBS. rIL-5 was reduced for 1 h with an equimolar amount of tri (2-carboxyethyl) phosphine hydrochloride (TCEP) in PBS (pH 8.0). Reduced rIL-5 (80 μM) was incubated for 4 h at 22 °C with 40 μM of SMPH derivatized Qβ (dQβ). The reaction was dialysed 12 h against PBS pH 8.0. A slightly different protocol was used to couple r-eotaxin to Qβ⋅ Qβ VLPs were derivatized with a 2.3-fold molar excess of SMPH. A 1.2–1 molar ratio of TCEP to protein was used to reduce r-eotaxin. Reduced r-eotaxin (20 μM) was incubated for 1 h at room temperature with 24 μM of dQβ. The coupling products (Qβ-IL-5 and Qβ-Eot) were analyzed by SDS-PAGE and Western blot with anti-His and anti-Qβ antibodies. Protein concentration was measured by Bradford. The coupling efficiencies (i.e.