For the in vivo evaluation of IVTT products, mice (n = 5/group) were immunized intramuscularly with 50 mg/100 ml of VR2516 PyCSP DNA vaccine (split between two legs) and boosted 3 weeks later with PyCSP IVTT products formulated with 100 ml Aluminium Hydroxide gel adjuvant (Brenntag Biosector, Frederikssund, Denmark) as follows: A) 15 ml of IVTT reaction (9 mg PyCSP/mouse); B) 15 ml of IVTT reaction purified through NI-NTA resin (6.6 mg PyCSP/mouse); C) 15 ml of IVTT reaction purified through MagneHis Protein Purification System (3 mg PyCSP/mouse); D) 15 ml of IVTT reaction absorbed in 10 ml of PolybeadH Poly(methyl methacrylate) microspheres (PMMA microspheres, 0.08-0.09 mm diameter) (Cat #23570, Polysciences, Inc., Warrington, PA, USA) (9 mg PyCSP/mouse); and E) 15 ml of IVTT reaction associated to 10 ml of Protein G conjugated microspheres/carboxylate beads (Cat #21106, Polyscience) (9 mg PyCSP/mouse). The beads used in the immunizations were washed 3 times with PBS pH 7.0 before addition of the IVTT product, and used without further processing. Positive and negative controls groups (n = 5) were administered 50 mg of VR2516 PyCSP DNA vaccine intramuscularly or 100 ml of Alum mixed with 100 ml of PBS pH 7.0, respectively. Three weeks after immunization mice were sacrificed and the spleens harvested for use as effectors in T cell assays.