duplicate. Following incubation at 37uC, 5% CO2 for 24 hours, 200 mL plasma was collected and IFNc,? TNF, IL-2, IL-6, IL-5, IL-4, Granzyme B, MCP3 and IP-10 levels measured using a TH1/TH2 cytometric bead array (CBA) kit (Becton Dickinson) or a custom Luminex panel (Panomics). HI Assay Sera were collected from mice one week after each immuniza- tion. Anti-HI Abs specific to the A/H3N2 component of the 2006–2007 Northern Hemisphere Fluzone formulation, A/ Wisconsin/67/2005(H3N2)(Wis/05), and to the A/H3N2 com- ponent of other seasonal influenza vaccines used since 2001 including A/Brisbane/10/2007(H3N2)(Bris/07), A/Wyoming/ 03/2003(H3N2)(Wyo/03) or A/Panama/2007/99(H3N2)(Pan/ 99) were determined as previously described using 0.5% turkey RBC [31]. Mouse sera were treated with receptor destroying enzyme followed by heat treatment (56uC, 30 min) before use in the HI assay. The HI titer was defined as the reciprocal of the highest dilution of sera which completely inhibits the agglutination of the turkey RBCs. Sera were collected from NHP on Day 1, 30 and 58 and evaluated for the presence of hemagglutination inhibiting antibodies specific to the 2007–2008 Northern Hemisphere Fluzone components including A/Solomon Islands/3/ 2006(H1N1), A/Wisconsin/67/2005(H3N2) and B/Malaysia/ 2506/2004 and also against drifted A (H1N1) strains incorporated in seasonal influenza vaccines since 1998 including A/Brisbane/ 59/2007(H1N1), A/New Caledonia/20/99(H1N1) and A/Beij- ing/262/1995(H1N1) and the drifted A/Uruguay/716/ 2007(H3N2) influenza strain by means of established methods, using four hemaglutinating units (HAU) of virus and 0.5% turkey RBCs). NHP sera were treated with receptor destroying enzyme followed by heat treatment (56uC, 30 min) before use in the HI assay. 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Supporting Information Figure S1 Virus-specific cytokine responses 7 weeks post boost with the 2006-2007 Fluzone influenza vaccine alone (-) or formulated with either an oil and water emulsion (SE) or a synthetic TLR4 agonist formulation (GLA-SE). Splenocytes were harvested from 4 mice/vaccine group and cultured with 5 HAU of inactived Wis/05, Bris/07, Wyo/03 or Pan/99 influenza strains. Supernatants were collected 72 hours post culture and analyzed by ELISA. Values represent the average virus-specific cytokine responses detected in cultures +SD. One asterisk represents significantly greater responses to Fluzone alone (p,0.05). Two asterisks represents significantly greater responses to Fluzone+SE (p,0.05). Three asterisks represents significantly greater responses to Fluzone +GLA-SE (p,0.05). Found at: doi:10.1371/journal.pone.0013677.s001 (0.06 MB PDF) Table S1 Found at: doi:10.1371/journal.pone.0013677.s002 (0.05 DOC) Table S2 Found at: doi:10.1371/journal.pone.0013677.s003 (0.04 DOC) Table S3 Found at: doi:10.1371/journal.pone.0013677.s004 (0.04 DOC) MB MB MB Acknowledgments We are grateful to Tara Evers, Farah Mompoint, Alison Bernard, Elsa Laughlin, Ines Tucakovic, Jeff Guderian and Tim Dutill for excellent technical assistance. We appreciate the thoughtful discussions with Dr. Martin Friede. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control. Author Contributions Conceived and designed the experiments: RC SB CHC SGR. Performed the experiments: NS SJR VSR XL JD MSD NVH. Analyzed the data: RC SB NS SB. Contributed reagents/materials/analysis tools: TSV. Wrote the paper: RC SB NS SB. Edited the manuscript: KH JMK SGR. 10. 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