Flu Vaccine Plus GLA-SE Figure 4. GLA-SE enhances serum HI titers to antigenically drifted A/H1N1 and A/H3N2 influenza strains following a boost immunization. NHPs (N = 3/group) were immunized with Fluzone (2007–2008) alone, or formulated with emulsion (SE), or GLA-SE (1, 5, 25 or 50 mg). Serum samples were collected 4 weeks after the boost (day 58). Shown are the HI titers from individual animals (diamonds) and the GMT (indicated by the horizontal bars 6 the 95% CI) against the antigenically A/Brisbane/59/2007(H1N1), A/New Caledonia/20/99(H1N1) and A/Beijing/ 262/1995(H1N1) strains and the drifted A/Uruguay/716/2007(H3N2) strain. doi:10.1371/journal.pone.0013677.g004 of $40 to the H1N1/Brisbane and A/H1N1/New Caledonia strains). Titers to the A/H1N1/Beijing strain were also higher in the groups given GLA-SE (5–50 mg) than titers observed with Fluzone, either used alone or combined with SE or the lowest dose of GLA-SE (1 mg), although most of the animals did not achieve titers $40 (Figure 4, Table S3). In addition to testing HI responses to drifted H1N1 strains, ,HI titers were measured against the vaccine strain (A/H3N2/ Wisconsin) (Figure 3) and one antigenically drifted A/H3N2 strain included in seasonal influenza vaccines in 2008 (A/ H3N2/Uruguay) (Figure 4). Although Fluzone+SE stimulated higher HI titers to the vaccine strain than Fluzone alone, a boost was required to induce the higher titers to the drifted A/ H3N2/Uruguay strain (Figure 4). All of the NHPs that received Fluzone+GLA-SE (5–50 mg) exhibited cross-reactive HI titers well above 40 against the drifted H3N2 strain following two immunizations, and 2 of 3 NHPs given the lowest dose GLA-SE (1 mg) achieved this titer. In contrast, only 1 of 3 NHPs reached a cross reactive titer $40 following immunization with Fluzone+SE, and none of the animals given Fluzone without adjuvant responded to the drifted strain (Figure 4, Table S3). Cytokine Response in Immunized Animals Cellular immune responses, including TH1-type responses, play a critical role in the clearance of influenza viruses in animal models and are associated with protection against influenza-related illness in the elderly. We, therefore, assessed the ability of the GLA-SE- and SE-formulated Fluzone vaccines to elicit influenza-specific cellular responses. In initial studies, spleen cells from mice immunized with Fluzone alone or with either SE or GLA-SE were restimulated in vitro with the vaccine strain or with the antigenically drifted A/H3N2 influenza The levels of cytokine produced in response to stimulation by any A/H3N2 strain were comparable, suggesting that the cellular responses are most likely directed towards conserved proteins within the antigen mixture (Figure S1 (7)). Fluzone+GLA-SE vaccinated mice induced significantly greater levels of antigen-specific IFNc compared to responses from mice immunized with Fluzone+SE or Fluzone alone (Figure S1 (7)). In sharp contrast to GLA-SE, the vaccine PLoS ONE | www.plosone.org 6 October 2010 | Volume 5 | Issue 10 | e13677