influenza in older adults but that this population overall may have impaired TH1 responses [3,19]. The GLA-SE-adjuvanted Fluzone vaccine described in this study elicits high IFNc:IL-10 and IFNc:IL-5 ratios that clearly show a dominant type-1 cellular immune response. Potent virus- specific IFNc responses are also thought to be capable of reversing the age-related impairments of CTL activity considered essential for viral clearance [25]. Therefore, the robust type-1 cellular immune response elicited by GLA-SE may have implications for the development of influenza vaccines better suited for the elderly. Conversely, the oil-in-water-based adjuvant we evaluated clearly shifted responses towards a type-2 cellular immune response, dominated by IL-5 (and IL-10), results consistent with other studies evaluating oil-in-water-based adjuvants, including MF59TM [26]. We show that, in the absence of the TLR4 agonist, the oil-in-water emulsion adjuvant potentiates TH2 responses reported to adversely affect viral clearance in animal models [27]. Thus, the data described herein suggests that influenza vaccines combined with GLA-SE could provide an additional benefit to the oil-in-water emulsion adjuvants by generating more broadly cross- reactive Ab responses, and by potentiation of TH1 responses. Materials and Methods Animals Female BALB/c mice 6–7 weeks (Charles River Laboratories, Wilmington, MA) were maintained under SPF conditions by IDRI Animal Care and Use Committee (IACUC). Twenty-one confirmed healthy male Cynomologus macaques ages 4.7 to 6.6 years old and weights of 4.81 to 6.47 kg were housed at SNBL USA (Everett, WA) in accordance with SNBL USA’s IACUC. Monkeys were pre-screened for their influenza status, randomized by body weights and assigned into study groups. Immunizations The 2006–2007 Fluzone influenza vaccine formulated for the Northern Hemisphere (Sanofi Pasteur, Swiftwater, PA), containing antigens from A/New Caledonia/20/99(H1N1), A/Wisconsin/ 67/2005(H3N2) and B/Malaysia/2506/2004 was used for the murine studies. The 2007–2008 Northern Hemisphere Fluzone influenza vaccine containing antigens from A/Solomon Islands/ 3/2006(H1N1), A/Wisconsin/67/2005(H3N2) and B/Malaysia/ 2506/2004, was used for the NHP studies. Fluzone was combined with either IDRI’s oil-in-water emulsion (SE) or SE containing a synthetic TLR4 agonist (GLA-SE), before administration to animals. Adjuvant formulations were prepared as previously described [28]. Mice were immunized IM twice, 3 weeks apart. Mice received a low dose (0.2 mg total HA) of the 2006–2007 Fluzone vaccine with or without SE, or GLA-SE (20 mg). NHP (N = 3/group) were immunized by IM injection on days 1 and 30 with the pediatric dose, which included 7.5 mg HA/strain resulting in a total HA dose of 22.5 mg, of the 2007–2008 Fluzone vaccine alone, or adjuvanted with SE, or escalating doses (1, 5, 25, or 50 mg) of GLA-SE. Influenza Strains and Proteins used for murine HI and cytokine assays To evaluate the ability of the vaccines to induce serum Ab responses against heterovariant influenza strains, vaccine and non-vaccine drifted influenza A strains were selected for use in HI assays and splenocyte cultures. A/Wisconsin/67/ 2005(H3N2)(Wis/05), A/Wyoming/03/2003(H3N2)(Wyo/03) Flu Vaccine Plus GLA-SE and A/Panama/2007/99(H3N2)(Pan/99) strains were provid- ed by the CDC and Prevention (Atlanta, GA) and purified viral protein from A/Brisbane/10/2007(H3N2)(Bris/07) was pur- chased from ProSpec (Rehovot, Israel). All of the viruses used were grown in the allantoic cavity of embryonated eggs and were stored at 280uC until use. Clinical Assessments of NHP Injection sites were evaluated prior to each inoculation, at 2 hours 630 minutes and again at 24, 48, 72, 96, 120 and 14461 hour after each dose, using a Draize dermal irritation scoring system [29]. Clinical observations to establish the general safety of vaccine administration were collected throughout the investigation. Limb use impairment and food consumption were evaluated for each monkey. The following parameters were also measured: (1) body weight (weekly throughout the study), (2) rectal temperature (at intervals prior to and following each dose, and at 6, 12 and 24 hours630 minutes after each dose), (3) urinalysis, (4) hematological parameters, (5) coagulation, and (6) serum chem- istry (shown in Table S1)). Blood samples were collected 5 days before, on the day of, and 2, 30 and 58 days after the initial inoculation. Serum was stored at 260uC until analysis. Influenza strains used for NHP HI assays For the NHP HI assays, the 2007 vaccine components, including A/Solomon Islands/3/2006(H1N1), A/Wisconsin/67/ 2005(H3N2) and B/Malaysia/2506/2004 plus all 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/Beijing/262/1995(H1N1) and the drifted A/Uruguay/716/2007(H3N2) influenza strains were evaluated. All viruses were provided by the Centers for Disease Control and Prevention (Atlanta, GA). Bone Marrow ELISPOT Antigen-specific mouse bone marrow plasma cells were quantified by ELISPOT as previously described, five weeks after the second immunization (7). Purified and concentrated A/Wisconsin/67/2005(H3N2)(Wis/05), A/Brisbane/10/2007 (H3N2)(Bris/07) and A/Panama/2007/99(H3N2)(Pan/99) viral antigens were purchased from ProSpec for use in the ELISPOT. Cellular Immune Responses Seven weeks after the second immunization, mice were euthanized and spleens (N = 4/group) were harvested. Spleno- cytes were cultured with 5 HAU of inactivated A/Wisconsin/67/ 2005(H3N2)(Wis/05), A/Brisbane/10/2007(H3N2)(Bris/07), A/Wyoming/03/2003(H3N2)(Wyo/03) or A/Panama/2007/ 99(H3N2)(Pan/99) influenza virus at 37uC in 5% CO2 for 72 hrs following a method adapted from Lu et al [30]. Culture supernatants were then collected and cytokine levels were measured by ELISA according to the manufacturer’s instructions (IFNa˜ and IL-10, eBioscience, San Diego, CA). Monkeys were bled at day 0 and 58 to evaluate cellular immune responses by whole blood assay (WBA). Heparinized blood (450 mL) was dispensed into 48-well plates (ICN Biomedicals, Inc). Phytohemagglutinin (PHA; 100 mg/mL), Fluzone (9 mg/mL), purified rH3 from A/Wisconsin/67/2005(H3N2) (10 mg/mL) (Protein Sciences Corp., Meriden, CT), inactivated A/Solomon Islands/3/2006(H1N1) virus (10HAU/mL) (provided by the Centers for Disease Control and Prevention) or saline was then added to provide stimulation in a final volume of 500 mL/well in PLoS ONE | www.plosone.org 9 October 2010 | Volume 5 | Issue 10 | e13677