Expanding on the beneficial effects of MPLH, we have developed a series of formulations containing a synthetic molecule called Glucopyranosyl Lipid A (GLA), which is optimized for binding to the human MD2 molecule of the TLR4 complex. GLA is active on mouse and human antigen presenting cells (APC) ([9]; Coler et al., manuscript submitted), resulting in the induction of cytokines such as IL-12 and TNF. Here we evaluate the safety and immunogenicity of GLA in a stable-emulsion (GLA-SE) combined with seasonal influenza vaccines in mice and cynomolgus monkeys. The results demon- strate that the adjuvanted Fluzone vaccine had increased humoral and cellular immunogenicity (characterized by a T-helper 1 (TH1)- biased immune response). The GLA-SE adjuvanted vaccine caused no adverse side effects in animals and enhanced the potency of the Fluzone vaccine both in terms of Ab and T cell responses and in broadened serological responses against drifted A/H1N1 and an A/H3N2 influenza variant(s) compared to vaccination with Fluzone alone. Results Safety and Reactogenicity af Fluzone+ GLA-SE To address the safety of GLA-SE in NHPs, we observed the site of injection for reactogenicity and examined several markers indicative of systemic inflammatory responses. Safety and toxicity data were collected during the course of non-human primate (NHP) dose escalation studies examining the immunogenicity and efficacy of Fluzone+GLA-SE. Two IM administrations of 1 to 50 mg of GLA-SE in combination with Fluzone over 30 days were well tolerated by cynomolgus macaques (Table S1). There was no evidence of injection site reactions or systemic toxicity in the animals receiving the Fluzone+GLA-SE vaccine or adjuvant alone. No definitive test-article-related changes in hematology were identified. Serum chemistry changes were limited to a mild display of a hepatic acute phase response (mild increases in both CRP and circulating fibrinogen concentrations) after each injection in groups injected with Fluzone+GLA-SE (Table S1). The Fluzone+GLA-SE Vaccine Increases and Broadens Hi Ab Levels We recently reported the immunological effects of combining Fluzone with a research-grade GLA in an oil-in-water emulsion in BALB/c mice [7]. To assess the ability of SE and GLA-SE to enhance Ab activity against influenza strains when formulated with the Fluzone vaccine, we first compared the serum HI titers induced against A/H3N2 influenza variants following immuniza- tion in mice. HI titers were measured against the vaccine strain A/ Wisconsin/67/2005(H3N2)(Wis/05) and three antigenic variants which represent the A/H3N2 components of past seasonal influenza vaccines: A/Brisbane/10/2007(H3N2), A/Wyoming/ 02/2003(H3N2) and A/Panama/2007/99(H3N2) (Bris/07, Wyo/ 03 and Pan/99, respectively). The phylogenetic relationship among all of the A/H3N2 strains except Pan/99 has been recently described [10]. In mice administered a single immunization, only Fluzone formulated with GLA-SE significantly increased HI titers against the vaccine and drifted A/H3N2 strains compared to immuniza- tion with Fluzone alone [2 to 6.5-fold increase in titer (p,0.05); Figure 1]. Furthermore, the group given Fluzone+GLA-SE induced significantly higher HI Ab titers against the drifted Bris/07 and Pan/99 strains compared to immunization with Fluzone formulated with SE [(p,0.05); Figure 1]. Following a vaccine boost, Fluzone+SE elicited significantly higher HI titers Flu Vaccine Plus GLA-SE against the vaccine strain (Wis/05; 16-fold increase in HI titer over Fluzone alone) and the drifted H3N2 strains [(Bris/07,Wyo/03 and Pan/99); 16- and 14-, and 2.7-fold increase in HI titer over Fluzone alone, respectively] [(p,0.05); Figure 1]). The addition of GLA to Fluzone+SE further enhanced HI titers over those achieved either by vaccine alone [21-fold, (p,0.05); Figure 1] or by Fluzone+SE (Figure 1). Fold-increase in HI titers in mice receiving Fluzone+GLA-SE were 47-, 35- and 10.7-fold higher compared with titers elicited by Fluzone alone when tested against the drifted Bris/07, Wyo/03, and Pan/99 strains, respectively [(p,0.05); Figure 1B]. These data demonstrate that combining the trivalent influenza vaccine with either SE or GLA-SE not only enhances vaccine potency, but also induces broader Ab responses against antigenically drifted influenza strains. To examine the potential of SE and GLA-SE to induce cells associated with long-lived humoral immunity, we enumerated bone marrow plasma cells (BMPC) that secreted IgG specific for the Wis/05, Bris/07 and Pan/99 influenza strains. When compared to vaccination with Fluzone alone, both the Fluzo- ne+SE and Fluzone+GLA-SE vaccines significantly enhanced virus-specific BMPC levels against these A/H3N2 strains [,11- fold for the vaccine strain Wis/05, (p,0.001); and ,16-fold for the drifted Bris/07 strain, (p,0.01), shown in Figure 2A]. Although the Ab levels were lower than for Bris/07 and Wis/ 05, Fluzone+GLA-SE also enhanced virus-specific BMPC levels against the drifted Pan/99 strain about 10-fold [(p,0.01); Figure 2A]. As expected from the serum IgG results, the GLA- SE-adjuvanted Fluzone vaccine generated significantly more Wis/ 05-specific BMPC than immunization with Fluzone+SE [,2 fold; (p,0.01)]. These data indicate that formulating Fluzone with GLA-SE induces a long-lasting humoral response against not only the vaccine strain but also antigenically drifted influenza variants. IFNc:IL-10 ratios were increased in in vitro-stimulated splenocytes collected from mice immunized with GLA-SE and Fluzone compared with those from mice administered Fluzone alone, demonstrating a clear Th1-type immune response (Figure 2B). Next, we examined whether results observed in mice could be reproduced in a higher animal species. NHPs were immunized with the 2007-08 Fluzone vaccine and geometric mean Ab titers (GMT) against vaccine components [A/Solomon Islands/3/ 2006(H1N1), A/Wisconsin/67/2005(H3N2), and B/Malaysia/ 2506/2004] and antigenically drifted strains [A/Brisbane/59/ 2007(H1N1), A/New Caledonia/20/1999(H1N1), A/Beijing/ 262/1995(H1N1) and A/Uruguay/716/2007(H3N2)] were mea- sured. In human clinical studies, the criteria commonly used to assess vaccine immunogenicity include a) rates of seroconversion (a four- fold or greater increase in post-vaccination HI titer compared with pre-vaccination titer) or b) rates of achievement of an HI titer of $40. An HI titer of $40 is a serologic endpoint generally considered to be associated with at least a 50% reduction in the risk of infection or disease within a population [11]. In this study we measured serum HI titers four weeks after both the prime and boost immunizations. Following a single immunization, the Fluzone vaccine induced HI titers of $40 against all 3 components of the vaccine in only 1 of 3 animals (Table S2). The addition of emulsion to the Fluzone vaccine resulted in titers of $40 against A/H1N1/ Solomon Islands, A/H3N2/Wisconsin, and B/Malaysia compo- nents in 2 of 3, 3 of 3, and 1 of 3 animals, respectively. Formulation of Fluzone with GLA-SE (at 25 mg) resulted in titers of $40 to the type A components of the vaccine in all 3 animals and against B/ Malaysia virus in 2 of 3 animals (Table S2). After the boost, all animals achieved titers of $40 titers against all three vaccine components with either Fluzone+SE vaccine or Fluzone+GLA-SE PLoS ONE | www.plosone.org 2 October 2010 | Volume 5 | Issue 10 | e13677