For in vitro evaluation of IVTT products, splenocytes were stimulated with unpurified rPyCSP IVTT; rPyCSP IVTT associated to Polybeads or ProteinG beads; or rPyCSP IVTT purified using NI-NTA resin or MagneHis Ni-particles. Unpurified IVTT products were used at a dilution of 1:200 or 1:1000 and purified IVTT products were used at a dilution of 1:100 or 1:200 for evaluation of cytokines in the supernatant using the Cytometric Bead Array assay or IFN-c secreting cells via ELIspot, respectively. Pilot experiments were carried out to find out the optimal dilutions of IVTT products for these assays (data not shown). An IVTT reaction using the empty pIVEX HisHA vector was used as negative control. A pool of synthetic peptides representing defined CD4 + and CD8 + T cell epitopes from PyCSP (residues 57-70, sequence KIYNRNIVNRLLGD [52] ; residues 280-288, sequence SYVPSAEQI [53] ; and resides 280-295, sequence SYVPSAEQILEFVKQI [54] ) at 10 mg/ml of each peptide was used as a positive control. ConA (Sigma-Aldrich, St. Louis, MO) at 5 mg/ml was used as a mitogen control. All stimuli were added in association with 1610 5 cells/well of A20/2 J cells (ATCC clone HB-98) irradiated at 1600 rads, as antigen presenting cells.