Abstract
BackgroundNewborns display distinct immune responses that contribute to susceptibility to infection and reduced vaccine responses. Toll-like receptor (TLR) agonists may serve as vaccine adjuvants, when given individually or in combination, but responses of neonatal leukocytes to many TLR agonists are diminished. TLR8 agonists are more effective than other TLR agonists in activating human neonatal leukocytes in vitro, but little is known about whether different TLR8 agonists may distinctly activate neonatal leukocytes. We characterized the in vitro immuno-stimulatory activities of a novel benzazepine TLR8 agonist, VTX-294, in comparison to imidazoquinolines that activate TLR8 (R-848; (TLR7/8) CL075; (TLR8/7)), with respect to activation of human newborn and adult leukocytes. Effects of VTX-294 and R-848 in combination with monophosphoryl lipid A (MPLA; TLR4) were also assessed.MethodsTLR agonist specificity was assessed using TLR-transfected HEK293 cells expressing a NF-κB reporter gene. TLR agonist-induced cytokine production was measured in human newborn cord and adult peripheral blood using ELISA and multiplex assays. Newborn and adult monocytes were differentiated into monocyte-derived dendritic cells (MoDCs) and TLR agonist-induced activation assessed by cytokine production (ELISA) and co-stimulatory molecule expression (flow cytometry).ResultsVTX-294 was ∼100x more active on TLR8- than TLR7-transfected HEK cells (EC50, ∼50 nM vs. ∼5700 nM). VTX-294-induced TNF and IL-1β production were comparable in newborn cord and adult peripheral blood, while VTX-294 was ∼ 1 log more potent in inducing TNF and IL-1β production than MPLA, R848 or CL075. Combination of VTX-294 and MPLA induced greater blood TNF and IL-1β responses than combination of R-848 and MPLA. VTX-294 also potently induced expression of cytokines and co-stimulatory molecules HLA-DR and CD86 in human newborn MoDCs.ConclusionsVTX-294 is a novel ultra-potent TLR8 agonist that activates newborn and adult leukocytes and is a candidate vaccine adjuvant in both early life and adulthood.
Highlights
Newborns display distinct immune responses that leave them vulnerable to higher rates of infections as compared to older children and adults [1], with over 2,000,000 deaths per year worldwide due to infection in those less than 6 months of age [2]
We find that VentiRx benzazepine compounds (VTXs)-294 is an ultra-potent TLR8-selective agonist with greater potency and activity than IMQs in both neonatal and adult leukocytes and monocyte-derived dendritic cells (MoDCs), suggesting that it may be a attractive candidate as an adjuvant towards neonatal leukocytes
Specificity of VTX Agonists for Human TLR7 and TLR8 To assess the selectivity of novel VTX benzazepine compounds, we characterized VTX-induced NF-kB activation in human embryonic kidney (HEK)-293 cells expressing human TLR7 or TLR8 in comparison to the imidazoquinolines R848 (TLR7/8) and CL075 (TLR8) Figure 1 (C–D)
Summary
Newborns display distinct immune responses that leave them vulnerable to higher rates of infections as compared to older children and adults [1], with over 2,000,000 deaths per year worldwide due to infection in those less than 6 months of age [2]. Lack of effective immunization strategies in the neonatal period (first 28 days of life) leads to a window of susceptibility to infections in newborns and infants lasting several months. The coming decade is likely to focus on developing technologies that overcome or circumvent these immunological obstacles to the development of more effective early life vaccines [2]. Toll-like receptor (TLR) agonists may serve as vaccine adjuvants, when given individually or in combination, but responses of neonatal leukocytes to many TLR agonists are diminished. Effects of VTX-294 and R-848 in combination with monophosphoryl lipid A (MPLA; TLR4) were assessed
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