and 50 mg also had increased IL-2 and TNF levels compared to those receiving Fluzone alone, Fluzone+SE, or Fluzone+GLA-SE (1 or 5 mg) boosts (Figure 5A). Thus, only animals receiving the Fluzone+GLA-SE at the highest doses (25 or 50 mg GLA) produced elevated levels of all three of the TH1-associated cytokines IFNc, TNF and IL-2. The cytokine responses 4 weeks post-boost (day 58) revealed that NHP boosted with Fluzone alone and Fluzone+SE had specific H1N1 and H3N2 responses marked by lower TH1:TH2 cytokine ratios than those immunized with Fluzone+GLA-SE at 5, 25 and 50 mg (Figure 5B). The highest IFNc:IL-5 ratios (.100) produced in response to both the H3N2 and H1N1 components of the Fluzone vaccine were observed in animals immunized with Fluzone +25 mg of GLA-SE. Interest- ingly, the addition of GLA-SE to the Fluzone vaccine increased median levels of H1N1- and H3N2-specific Granzyme B (Figure 5C), as well as several chemokines. These chemokines [(C-X-C motif) ligand 10 (CXCL10), also called IP-10) and monocyte-chemotactic-protein-3 (MCP-3; also called chemokine (C-C motif) ligand 7 (CCL7)] are selectively expressed on monocytes, endothelial cells, and fibroblasts, and all are chemotactic for and activate a great variety of inflammatory cell types including monocytes/macrophages, T cells, NK cells, and/ or dendritic cells (Figure 5D). Discussion Next generation influenza vaccines that potentiate immune responses and elicit protection against a broader range of antigenically drifted strains are urgently needed. To further advance development of such vaccines, we have analyzed the impact on immune responses of incorporating a stable oil-in-water emulsion and a synthetic TLR4 agonist into a standard influenza vaccine. The experiments described in this report are the first to evaluate an oil-in-water emulsion (SE) with various concentrations of this synthetic TLR4 agonist (GLA), all in combination with Fluzone, a licensed trivalent inactivated influenza vaccine for its safety and ability to significantly enhance immune responses in both mice and NHP. With the ever increasing need for safe ways to either improve vaccine efficacy and/or to provide dose sparing effects, most candidate adjuvants fail to reach the market place because of safety issues including toxicity or reactogenicity particularly at the site of injection. Though increased levels of pro-inflammatory cytokines were observed in the context of GLA- SE, there was no evidence of injection site reactions or systemic toxicity in the animals receiving the Fluzone+GLA-SE vaccine or adjuvant alone. Our results demonstrate that incorporating SE into Fluzone enhances Ab responses. Furthermore, the incorporation of GLA with SE enhances the same Ab responses, stimulates production of Abs that cross-react with antigenically variant influenza strains not present in the vaccine, and generates TH1 cellular immune responses. Thus, our studies clearly demonstrate that GLA-SE provides further enhancement and broadening of the immune responses already elevated by the addition of SE to Fluzone. The ability of SE to adjuvant flu vaccines, as observed in our studies, is in general agreement with those previously reported for another oil-in-water adjuvant, MF59TM [13,14]. After two immunizations, the addition of SE to the Fluzone vaccine increased HI titers to the A/H1N1/Solomon Islands and the A/ H3N2/Wisconsin components of the Fluzone vaccine to seroprotective levels HI titers of $40. In contrast to previous reports indicating the generation of cross-reactive Abs against heterovariant influenza strains by the addition of oil-in-water adjuvants [14], our results failed to demonstrate that the addition Flu Vaccine Plus GLA-SE of SE alone to Fluzone could increase HI titers against antigenically drifted influenza variants. It should be noted that we used a low dose of Fluzone vaccine and that even when administered with SE, this may not be a potent enough combination to stimulate cross-reactive antigen-specific respons- es. Regardless, our results confirm those of others which indicate that incorporation of SE in influenza vaccines is an important step in the development of more effective seasonal and pandemic influenza vaccines. Our results clearly demonstrate that, compared to vaccination with Fluzone alone or Fluzone formulated with SE, the further addition of GLA increases cross-protective HI Ab responses against antigenic variants. In this study, a single administration of Fluzone with GLA-SE leads to the generation of HI titers at seroprotective levels in a majority of NHP. GLA-SE further enhanced HI titers in NHP following two immunizations, as compared to either SE plus Fluzone or Fluzone alone. Previous studies have shown that other TLR agonists, such as the TLR9 agonist CpG, enhance cellular interactions within germinal centers, leading to the selection of B cells with a high affinity for antigen binding [15]. GLA-SE broadened HI responses and generated more virus-specific BMPCs, the hallmark of long-term humoral immunity [16], compared with SE alone. Taken together, these results suggest that a GLA-SE-adjuvanted influenza vaccine may elicit not only more durable but also more broadly reactive Abs that have the potential to extend the protective lifespan of the vaccine. Our results also show that the addition of GLA-SE to the Fluzone vaccine selectively shifts the cellular immune response toward a type-1 cellular immune response pattern, characterized by the production of IFNc. TLR4 agonists, such as MPL, have been shown to be potent adjuvants in a range of species, including humans [8,17]. NHP immunized with GLA-SE-adjuvanted Fluzone vaccine produced higher Ab titers and type-1 cytokine levels than those found in animals immunized with Fluzone alone or a Fluzone+SE formulation. As many aging individuals experience a shift from TH1 to TH2 responses [3] and a concomitant increase in susceptibility to influenza disease, a potent anti-influenza cellular response may be of particular importance in older adults. Many elderly patients respond poorly to the existing influenza vaccines [18,19]. The ability of the GLA- SE adjuvant to induce potent type-1 cellular immune responses is in marked contrast to the TH2 response induced by traditional adjuvants such as Alum and MF59 TM [20]. The anti-viral activity of type-1 cytokines such IFNc and TNF [21,22,23], and the synergistic effect of these cytokines, against human influenza viruses is well known [24]. The enhanced induction of both IFNc and TNF by Fluzone adjuvanted with GLA-SE could positively impact an influenza vaccination program aimed at the elderly population since prior studies have reported a putative correlation of cell mediated immunity and protection against influenza. The weakened immune system of elderly people presents particular challenges in the development of vaccines and adjuvants for this population. There are well recognized changes in the function of APCs, T cells and B cells in older people. Several strategies to improve vaccination in the elderly have been explored. A number of studies have shown that the use of adjuvants (e.g. emulsions and TLR agonists) could trigger more potent activation of APCs. Like other TLR4 containing adjuvants, we have preliminary data demonstrating that GLA can induce robust immune responses in aged mice and humans whose immune systems have undergone significant decline with increas- ing age. Recent studies suggest that T cell responses, rather than Ab, may be a better immune correlate of protection against PLoS ONE | www.plosone.org 8 October 2010 | Volume 5 | Issue 10 | e13677