GLA-SE, a Synthetic Toll-like Receptor 4 Agonist, Enhances T-Cell Responses to Influenza Vaccine in Older Adults

Hayedeh Behzad 2 3 Anke L. W. Huckriede 0 2 Laura Haynes 2 5 Beth Gentleman 2 3 Krysta Coyle 2 3 Jan C. Wilschut 0 2 Tobias R. Kollmann 1 2 Steven G. Reed 2 4 Janet E. McElhaney 2 3 0 University Medical Center , Groningen , the Netherlands 1 Children and Families Research Institute, University of British Columbia , Vancouver , Canada 2 Received 23 June 2011; accepted 23 September 2011; electronically published 5 December 2011. 186-828 W 10th Ave, Vancouver, BC , Canada V7E 5S4 3 Vancouver Coastal Health Research Institute 4 Immune Design, Seattle , Washington 5 Trudeau Institute , Saranac Lake , New York Background. The decline in influenza vaccine efficacy in older adults is associated with a limited ability of current split-virus vaccines (SVVs) to stimulate cytotoxic T lymphocyte (CTL) responses required for clinical protection against influenza. Methods. The Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) was combined with SVV to stimulate peripheral blood mononuclear cells (PBMCs) in vitro to determine the cytokine response in dendritic cell subsets. Stimulated PBMCs were then challenged with live influenza virus to mimic the response to natural infection following vaccination, using previously identified T-cell correlates of protection. Results. GLA-SE significantly increased the proportion of myeloid dendritic cells that produced tumor necrosis factor a, interleukin 6, and interleukin 12. When combined with SVV to stimulate PBMCs in vitro, this effect of GLA-SE was shown to regulate a T-helper 1 cell response upon challenge with live influenza virus; interleukin 10 production was suppressed, thus significantly increasing the interferon c to interleukin 10 ratio and the cytolytic (granzyme B) response to influenza virus challenge, both of which have been shown to correlate with protection against influenza in older adults. Conclusions. Our findings suggest that a novel adjuvant, GLA-SE, combined with standard SVV has the potential to significantly improve vaccine-mediated protection against influenza in older adults. Influenza virus infection can cause life-threatening complications in older adults. Annual vaccination is considered the best strategy to contain the virus and prevent its associated morbidity and mortality in this vulnerable population. However, the current seasonal split-virus influenza vaccines (SVVs) have limited effectiveness among people aged .65 years [1]. The activation of innate immune mechanisms is critical to stimulating adaptive immune responses against intracellular pathogens such as influenza virus. The wellknown defects in innate and adaptive immune responses 466 d JID 2012:205 (1 February) d Behzad et al - in older adults are major contributors to the poor vaccine efficacy in this population [2]. The age-related decline in innate immune function reduces antigen presentation by dendritic cells [3] and can substantially alter the activation of an already compromised adaptive immune response; defects in both memory and effector T-cell function further contribute to diminished T-cell responses to vaccination in older adults [4, 5]. A practical approach to designing new vaccines is to add adjuvants to the existing SVVs to overcome the defects in the aged immune system. Toll-like receptor (TLR) agonists are currently being explored as vaccine adjuvants to enhance immunogenicity [6]. TLRs expressed on innate immune cells, including dendritic cells, recognize and bind to the conserved molecular patterns of viruses, bacteria, and fungi, a process that initiates the innate immune response. This response can be triggered by TLR agonists, which mimic these pathogens and stimulate dendritic cells to produce T-helper 1 (Th1) cell promoting cytokines, tumor necrosis factor a (TNF-a), interleukin 1b (IL-1b), interleukin 6 (IL-6), and interleukin 12 (IL-12) [7]. TLR agonists have been shown to activate dendritic cells from aged mice and to enhance their ability to stimulate priming of aged naive CD41 T cells and their subsequent proliferation and differentiation to effector T cells [8]. Polyinosinic:polycytidylic acid (poly I:C), a TLR3 agonist, has been shown to enhance the maturation of dendritic cells, which promote Th1-cell responses and antigen-specific cytotoxic T lymphocyte (CTL) activation [9, 10]. The TLR4 agonist monophosphoryl lipid A has also been shown to promote a Th1-cell response in mice [11, 12]. Because there is a shift from Th1 to Th2 cytokine responses with aging, TLR adjuvants offer the possibility of reversing this defect when added to influenza vaccine. A Th1 cellmediated increase in the CTL response to vaccination could contribute to improved clinical protection against severe disease when antibodies fail to provide sterilizing immunity and prevent infection in older adults [13]. We have shown in this population that higher ratios of Th1:Th2 cytokines (interferon c [IFN-c]:IL-10) and higher levels of CTL (granzyme B [GrzB]) activity in influenza A/H3N2 stimulated peripheral blood mononuclear cells (PBMCs) provide more-sensitive correlates of protection against influenza, compared with serologic responses to vaccination [14, 15]. To facilitate the translation of preclinical studies in animal models to phase I vaccine trials in older adults, we have developed a novel model for preclinical testing of adjuvants combined with SVV to determine their effect on T-cell responses to influenza virus challenge. In this study, we evaluated a novel TLR4 agonist, glucopyranosyl lipid adjuvantstable emulsion (GLA-SE), for its potential to enhance the Th1 cellmediated CTL response to influenza virus in older adults. We found that GLA-SE can activate myeloid dendritic cells (mDCs) to produce high levels of Th1 cellpromoting cytokines, including TNF-a, IL-6, and IL-12. We further show that stimulation of PBMCs with the GLA-SEadjuvanted SVV enhances the Th1-cell response to live influenza virus challenge by suppressing IL-10 production, thereby increasing the ratio of IFN-c:IL-10 and GrzB activity produced in response to influenza virus challenge in young and older adults. MATERIAL AND METHODS Study Design and Participants Healthy adult volunteers, of whom 12 were young (aged 2030 years) and 15 were older (aged .65 years), were recruited by written informed consent from the community of Greater Vancouver, Canada. Subjects were studied before and after influenza vaccination to evaluate the in vitro effects of different adjuvants on cytokine production by dendritic cells and their combined effect with SVV on the response to influenza virus challenge. Blood samples obtained from study participants were de-identified at the time of collection. The study protocol and informed consent form were approved by the Clinical Research Ethics Board, University of British Columbia. Procedures The study participants were recruited to the center in the fall of 2009. Blood samples (35 cm3 heparinized whole blood) were d (...truncated)