Flu Vaccine Plus GLA-SE formulated with SE induced a strong virus-specific TH2 cytokine response characterized by significantly greater levels of IL-5 compared to Fluzone alone or Fluzone+GLA-SE (Figure S1 (7)). Greater levels of IL-10 were induced in response to the vaccine with SE compared to GLA-SE (Figure S1 (7)). Comparison of IFNc to IL-10 levels, measured after the second immunization as shown in Figure 2B, shows that GLA-SE directs type-1 cellular responses, characterized by the production of IFNc. These data suggest that the virus-specific recall responses can be modulated by formulating Fluzone; inclusion of an oil-in-water emulsion induces a TH2 response whereas the addition of a TLR4 agonist to the emulsion directs a TH1 response. In additional studies we used whole blood assays (WBA) to assess antigen-specific cytokine responses of immunized and non- immunized monkeys (Figure 5A). To evaluate T cell responsive- ness to the H1N1 and H3N2 components of the Fluzone vaccine, the levels of IFNc, TNF, IL-2, IL-6, IL-5 and IL-4 secreted during WBA were evaluated by a TH1/TH2 Cytokine Bead Array. All of the NHPs immunized with any of the Fluzone+GLA-SE (5-25 mg) vaccines produced increased H1N1- and H3N2-specific IFNc responses four weeks after the second immunization compared to NHPs that were immunized with the Fluzone alone, Fluzone with SE, or Fluzone with 1 mg GLA-SE (Figure 5A). All animals that received the booster immunizations with Fluzone+GLA-SE at 25 Figure 5. Cellular responses after injection with saline, Fluzone alone, or combined with SE or GLA-SE. NHP (N = 3/group) were given 2 injections, 4 weeks apart with saline, Fluzone, Fluzone+SE or Fluzone+GLA-SE (1, 5, 25 and 50 mg). (A) IFNc, IL-2, TNF and IL-5 (pg/mL) recall responses after stimulation of whole blood with H1N1 or H3N2 were evaluated by CBA at day 0 and day 58. (B) IFNc:IL-5 ratios of cytokine recall responses after stimulation of whole blood with H1N1 or H3N2, were evaluated by CBA at day 58. (C) Granzyme B levels (pg/mL) responses in whole blood following stimulation with H1N1 or H3N2 was evaluated by Luminex at day 58. Horizontal lines represent median Granzyme B concentrations. (D) Chemokine responses to Fluzone antigens after immunization. MCP-3 and IP-10 (pg/mL) responses in whole blood following stimulation with H1N1 or H3N2 were evaluated by Luminex at day 58. Horizontal lines represent median chemokine concentrations. doi:10.1371/journal.pone.0013677.g005 PLoS ONE | www.plosone.org 7 October 2010 | Volume 5 | Issue 10 | e13677