Data from the in vitro and in vivo tests in mice were analyzed by the Student t-test and two-way ANOVA, respectively, where a p value,0.05 was considered significant. Data from human PBMC ELIspot and CBA were analyzed using two-way ANOVA and one-way ANOVA, respectively. A p value,0.05 was considered significant. Figure S1 Recombinants produced using E. coli cellfree IVTT system. Western Blot of (A) viral antigens FluM (32 kDa), FluHA (68 kDa), CMVpp65 (68 kDa) and EBNA3A (108 kDa), and parasite protein PyCSP (44 kDa), probed with anti-HA antibody, and (B) viral antigens FluM, FluHA, CMVpp65 and EBNA3A probed with anti-His antibody. Whole IVTT extracts (5 ml) of each antigen were run on a 12% NUPAGE gel, transferred to a PVDF membrane, and probed with anti-HA HRP antibody (1:500 dilution) or anti-His HRP antibody (1:5000 dilution). The western probed with anti-HA also detected a cross reactive band of 56 kDa in all expression extracts. The western probed with anti-His mAb showed the presence of partial products probably due to early termination of translation. (TIF) Figure S2 Antigen-specific TNF-a and IL-10 responses of mouse splenocytes stimulated in vivo or in vitro with IVTT-proteins. (A) and (B): splenocytes of mice immunized with VR2516 PyCSP plasmid DNA or VR1020 control DNA were cultured in vitro with unpurified rPyCSP IVTT; rPyCSP IVTT associated to Polybeads or ProteinG beads; rPyCSP IVTT purified using NI-NTA resin, MagneHis Ni-particles, or anti-HIS; or synthetic peptides representing defined T cell epitopes from PyCSP (positive control), as indicated. (C) and (D): splenocytes of mice immunized with VR2516 PyCSP plasmid DNA and boosted in vivo with unpurified rPyCSP IVTT; rPyCSP IVTT associated to Polybeads or ProteinG beads; or rPyCSP IVTT purified using NI-NTA resin or MagneHis Ni-particles; all formulated with Alum adjuvant. Parallel groups of mice were boosted with either Alum only or VR2516 as controls. Splenocytes were cultured in vitro with A20 cells transfected with VR2516 PyCSP plasmid DNA or A20 cells pulsed with synthetic peptides representing defined PyCSP T cell epitopes, as indicated. Secreted TNF-a (A and C) or IL-10 (B and D) in culture supernatant was measured by Cytometric Bead Array (CBA) after 48 hrs stimulation. *P,0.05, **P,0.01 and ***P,0.001 compared to negative controls. (TIF) Figure S3 Antigen-specific TNF-a responses by human PBMC stimulated with IVTT-proteins. PBMCs were cultured with IVTT-produced FluM, FluHA, CMVpp65 and EBNA3A purified using (A) NI-NTA nickel resin or (B) MagneHis Ni-particles; (C) unpurified; associated to (D) ProteinG beads or (E) Polybeads; or (F) added to wells precoated with Anti-His; IVTT products were diluted 1:100, 1:1000, or 1:10,000. Secreted TNF-a in the supernatant of cultured PBMCs was analyzed by Cytometric Bead Array after 48 hrs stimulation. * P,0.05 compared to negative controls. (TIF) Porcine major histocompatibility complex (MHC) class I molecules and analysis of their peptide-binding specificities In all vertebrate animals, CD8(+) cytotoxic T lymphocytes (CTLs) are controlled by major histocompatibility complex class I (MHC-I) molecules. These are highly polymorphic peptide receptors selecting and presenting endogenously derived epitopes to circulating CTLs. The polymorphism of the MHC effectively individualizes the immune response of each member of the species. We have recently developed efficient methods to generate recombinant human MHC-I (also known as human leukocyte antigen class I, HLA-I) molecules, accompanying peptide-binding assays and predictors, and HLA tetramers for specific CTL staining and manipulation. This has enabled a complete mapping of all HLA-I specificities (“the Human MHC Project”). Here, we demonstrate that these approaches can be applied to other species. We systematically transferred domains of the frequently expressed swine MHC-I molecule, SLA-1*0401, onto a HLA-I molecule (HLA-A*11:01), thereby generating recombinant human/swine chimeric MHC-I molecules as well as the intact SLA-1*0401 molecule. Biochemical peptide-binding assays and positional scanning combinatorial peptide libraries were used to analyze the peptide-binding motifs of these molecules. A pan-specific predictor of peptide–MHC-I binding, NetMHCpan, which was originally developed to cover the binding specificities of all known HLA-I molecules, was successfully used to predict the specificities of the SLA-1*0401 molecule as well as the porcine/human chimeric MHC-I molecules. These data indicate that it is possible to extend the biochemical and bioinformatics tools of the Human MHC Project to other vertebrate species. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00251-011-0555-3) contains supplementary material, which is available to authorized users. Major histocompatibility complex class I (MHC-I) molecules are found in all vertebrate animals where they play a crucial role in generating specific cellular immune responses against viruses and other intracellular pathogens. They are highly polymorphic proteins that bind 8-11 amino acid long peptides derived from the intracellular protein metabolism. The resulting heterotrimeric complexes-consisting of the MHC-I heavy chain, the monomorphic light chain, beta-2 microglobulin (β 2 m), and specifically bound peptides-are translocated to the cell surface where they displayed as target structures for peptide-specific, MHC-Irestricted CTLs. If a peptide of foreign origin is detected, the T cells may become activated and kill the infected target cell.