This analysis differed from that in 2002 in two important ways: it used the improved EpiMatrix algorithm and drew from a database of HIV sequences that had expanded four-fold since 2002. Thirteen new highly conserved HLA-A2 epitopes were identified and selected for validation studies, including two peptides from ENV, four from REV, three from VIF, and one each from GAG, POL, NEF, and VPU. Fourteen epitopes from the 2002 epitope Ku0059436 set were reselected in 2009 for validation in Mali in in vitro studies based on updated

EpiMatrix scores and peptide availability. The complete list of peptides tested in this report is shown in Table 1. Peptides corresponding to the 2002 epitope selections were prepared by 9-fluorenylmethoxycarbonyl (Fmoc) synthesis on an automated Rainin Symphony/Protein Technologies synthesizer (Synpep, Dublin, CA). The peptides were delivered 90% pure as ascertained by HPLC. Peptides corresponding to the 2009 epitope selections were prepared by solid-phase Fmoc synthesis on an Applied Biosystems/Perceptive Model Pioneer peptide synthesizer (New England Peptide, Gardner, MA). The peptides were Protein Tyrosine Kinase inhibitor delivered >80% pure as ascertained by HPLC, matrix-assisted

laser desorption/ionization (MALDI) mass spectrometry, and UV scan at wavelengths of 220 and 280 (ensuring purity, mass, and spectrum, respectively). The MHC class I binding assays were performed as previously described [56]. The HLA class I molecule ADAMTS5 was incubated at an active concentration of 2 nM together with 25 nM human β2 microglobulin (β2 m) and an increasing concentration of the test peptide at 18 °C for 48 h. The HLA molecules were then captured on an ELISA plate coated with the pan-specific anti-HLA antibody W6/32, and HLA-peptide complexes were detected with an anti-β2 m specific polyclonal serum conjugated with horseradish peroxidase (Dako P0174), followed by a signal enhancer (Dako Envision). The plates were developed,

and the colorimetric reaction was read at 450 nm using a Victor2 Multilabel ELISA reader. Using a standard, these readings were converted to the concentration of HLA-peptide complexes generated and plotted against the concentration of test peptide offered. The concentration of peptide required to half-saturate (EC50) the HLA was determined. At the limiting HLA concentration used in the assay, the EC50 approximates the equilibrium dissociation constant, KD. The relative affinities of peptides, based on a comparison of known HLA-A2 ligands, were categorized as high binders (KD < 50 nM), medium binders (50 nM < KD > 500 nM), low binders (500 nM < KD > 5000 nM), and non-binders (KD > 5000 nM). Binding scores for each of the selected peptides can be found in Table 1. Interferon gamma ELISpot assays were performed using peripheral blood mononuclear cells (PBMCs) separated by Ficoll density gradient centrifugation of whole blood.