Protein expression was confirmed by western blot. IVTT products were diluted 1:1 in 2x reducing sample buffer (125 mM Tris pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 2% beta-mercaptoethanol) and denatured at 95uC for 5 mins. Samples (2.5 ml of E. coli IVTT whole extract or supernatant, and 5 ml of insect cell IVTT whole extract or supernatant) were run in parallel with molecular weight standard (BenchMark pre-stained protein marker, Invitrogen) on a 4-12% NuPage SDS Page gel (Invitrogen, Carlsbad, CA, USA) at 120 V for 70 min. Samples were transferred onto a PVDF membrane (Bio-Rad, Hercules, CA) pre-wet with 100% methanol, using the BioRad Mini Trans-Blot system at 100 V for 1 hr, according to manufacturer's instructions. Membranes were blocked with PBS containing 5% skim milk powder overnight at 4uC with shaking, washed 3 times with PBS containing 0.05% Tween20, then probed with anti-HIS HRP (1:5000; Roche Diagnostics) or anti-HA HRP (1:500) (Roche Diagnostics) for 1 hr at room temperature. Membranes were Immunization of mice using the VR2516 PyCSP DNA vaccine and PyCSP IVTT product Mice (n = 5/group) were immunized intramuscularly in the tibialis anterior muscle with 50 mg of VR2516 PyCSP DNA vaccine or VR1020 negative control plasmid, two times at 3 week intervals. Splenocytes were harvested from DNA immunized mice or naïve control mice (n = 5) at 3 weeks post-boost for in vitro T cell assays using IVTT products. Serum was collected from select groups for antibody assays. The potential toxicity of IVTT products was assessed prior to use for in vitro stimulation of T cell responses by culturing splenocytes (5610 5 cells/well) of naïve BALB/c mice with IVTT-produced PyCSP either unpurified or purified using NI-NTA resin (Qiagen) or MagneHis particles (Promega) at dilutions of 1:100, 1:1000 and 1:10000, or media alone, and monitoring cell growth for 48 hours. No toxicity due to the unpurified or purified IVTT preparations was apparent, as indicated by cell death.