A Synthetic Adjuvant to Enhance and Expand Immune Responses to Influenza Vaccines Rhea N. Coler1., Susan L. Baldwin1., Narek Shaverdian1, Sylvie Bertholet1, Steven J. Reed 1 , Vanitha S. Raman1, Xiuhua Lu2, Joshua DeVos2, Kathy Hancock2, Jacqueline M. Katz2, Thomas S. Vedvick1, Malcolm S. Duthie1, Christopher H. Clegg 3 , Neal Van Hoeven3, Steven G. Reed1,3* 1 Infectious Disease Research Institute, Seattle, Washington, United States of America, 2 National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America, 3 Immune Design Corporation, Seattle, Washington, United States of America Abstract Safe, effective adjuvants that enhance vaccine potency, including induction of neutralizing Abs against a broad range of variant strains, is an important strategy for the development of seasonal influenza vaccines which can provide optimal protection, even during seasons when available vaccines are not well matched to circulating viruses. We investigated the safety and ability of Glucopyranosyl Lipid Adjuvant-Stable Emulsion (GLA-SE), a synthetic Toll-like receptor (TLR)4 agonist formulation, to adjuvant FluzoneH in mice and non-human primates. The GLA-SE adjuvanted Fluzone vaccine caused no adverse reactions, increased the induction of T helper type 1 (TH1)-biased cytokines such as IFNc, TNF and IL-2, and broadened serological responses against drifted A/H1N1 and A/H3N2 influenza variants. These results suggest that synthetic TLR4 adjuvants can enhance the magnitude and quality of protective immunity induced by influenza vaccines. Citation: Coler RN, Baldwin SL, Shaverdian N, Bertholet S, Reed SJ, et al. (2010) A Synthetic Adjuvant to Enhance and Expand Immune Responses to Influenza Vaccines. PLoS ONE 5(10): e13677. doi:10.1371/journal.pone.0013677 Editor: Robert E. Means, Yale Medical School, United States of America Received June 11, 2010; Accepted September 25, 2010; Published October 27, 2010 Copyright: 2010 Coler et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by National Institutes of Health grant AI-044373 and contract AI-25479, by grant 42387 from the Bill and Melinda Gates Foundation, and by Immune Design Corporation. Immune Design Corporation had a role in study design and data collection and review of the manuscript. Competing Interests: Dr. Steven G. Reed is a founder of, and holds an equity interest in, Immune Design Corporation, a licensee of certain rights associated with GLA. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: sreed@idri.org . These authors contributed equally to this work. Introduction Seasonal influenza epidemics are responsible for an average 36,000 deaths and 226,000 hospitalizations annually in the United States alone [1]. Worldwide 3–5 million cases of severe influenza infection occur each year, resulting in between 250,000–500,000 deaths [2]. Although influenza infection rates are highest in children, increased mortality occurs in those older than 65 years, in children less than 2 years of age, and in patients with medical conditions [2]. Vaccination is the best preventative measure against influenza, and acts by increasing neutralizing Abs directed against the HA, the major surface glycoprotein [3]. Currently licensed trivalent seasonal influenza vaccines include antigens from two circulating type A strains and one type B influenza strain. One limitation to seasonal vaccine efficacy is that selected influenza strains are not always representative of the circulating virus, and as a conse- quence the resulting protection afforded by the vaccine may be reduced [4]. Even for years in which components are antigenically well matched to circulating influenza strains, vaccines are only 60– 80% effective at preventing death in older adults aged 65 years and over and are only effective at preventing 70–90% of influenza- specific illnesses in younger adults [2,4,5]. When the vaccine is mismatched, efficacy against serologically confirmed influenza illness in adults (18–64 years of age) can be as low as 50% [5]; in those .65 years, efficacy is as low as 35%[4]. Adjuvants provide an important means of improving influenza vaccines. To be effective, these adjuvants should increase the levels of hemagglutination-inhibition (HI) Ab titers against the HA components included in the vaccine and ideally also against variant viruses possessing drifted HA proteins. Emulsion-based adjuvants are currently used in certain licensed seasonal influenza vaccines. MF59 TM (an oil-in-water adjuvant) combined with a seasonal influenza vaccine, (FluadH, Novartis Vaccines and Diagnostics) has been approved in some European countries since the late 1990’s [6]. In addition, two H5N1 vaccines have recently been approved in Europe: FocetriaH (MF59 + H5N1 vaccine; Novartis), and PrepandrixTM (an AS03+H5N1 vaccine; GlaxoS- mithKline) [6]. The use of formulations containing oil-in-water emulsions plus Toll-like receptor (TLR) agonists [7] have been successfully used in combination with influenza vaccine and shown to enhance humoral and cellular immunity in animal models compared to using the vaccine alone. The most advanced TLR4 agonist is an adjuvant derived from the LPS of Salmonella minnesota which is known as Monophosphoryl Lipid A (MPLH). MPLH in a variety of formulations has been licensed in Europe as a component of the hepatitis B vaccine FendrixH [8] and HPV vaccine CervarixH; both containing MPL/ alum. The inclusion of GSK’s AS04 Adjuvant System in CervarixH, recently approved in the US, is believed to significantly broaden immune responses against HPV serotypes not contained in the vaccine. PLoS ONE | www.plosone.org 1 October 2010 | Volume 5 | Issue 10 | e13677