Flu Vaccine Plus GLA-SE Figure 2. GLA-SE enhances IgG-secreting BMPCs to vaccine and drifted A/H3N2 strains and induces cross-reactive type-1 immunity. Mice were immunized Fluzone (2006–2007) either alone ( - ), or formulated with emulsion (SE), or GLA-SE. (A) Shown are virus-specific [vaccine (Wis/ 05) or drifted H3N2 strains] IgG-secreting BMPC responses five weeks after boost from individual mice (diamonds; N = 4/group). The group mean is represented by the horizontal bar 6 standard deviation (s.d.) per million cells. Statistical significance was determined and compared to Fluzone (*p,0.05) or Fluzone+SE (**p,0.05). (B) IFNc:IL-10 ratios from immunized mice (N = 4/group) seven weeks after the boost immunization. Splenocytes were harvested and cultured with 5 HAU of inactivated Wis/05, Bris/07, Wyo/03 or Pan/99 influenza virus strains for 3 days. Supernatants were analyzed by ELISA. Results represent the mean IFNc:IL-10 ratio. doi:10.1371/journal.pone.0013677.g002 less, following the boost, the inclusion of GLA-SE in the Fluzone vaccine formulations enhanced HI titers in a GLA-SE dose dependent manner. HI titers were increased by, on average, 2–28 fold compared with titers achieved in vaccine groups lacking GLA- SE. After the first immunization, Fluzone formulated with GLA-SE at 5–50 mg doses elicited higher mean serum HI titers to the A/ H1N1/Solomon Islands and A/H3N2/Wisconsin components of the vaccine compared with titers elicited by Fluzone alone. Following the boost, animals that received Fluzone+SE, and Fluzone plus GLA- SE, showed a dose-dependent enhancement in the mean serum HI titers to all three components of the vaccine (Figure 3). A desirable feature of an adjuvanted seasonal influenza vaccine is the ability to induce more broadly cross-reactive Abs against antigenically drifted variants or homosubtypic heterovariant influenza strains that are not included in the vaccine for a given season [12]. To address the ability of SE and GLA-SE to enhance NHP Ab responses against antigenically drifted influenza viruses when formulated with the Fluzone vaccine, we compared the HI PLoS ONE | www.plosone.org 4 October 2010 | Volume 5 | Issue 10 | e13677