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Background Newborns suffer frequent infection and manifest impaired vaccine responses, motivating a search for neonatal vaccine adjuvants. Alum is a neonatal adjuvant, but may confer a Th2 bias. Toll-like receptor (TLR) agonists are candidate adjuvants, but human neonatal cord blood monocytes (Mos) demonstrate impaired Th1-polarizing responses to many TLR agonists due to plasma adenosine acting via cAMP. TLR8 agonists, including imidazoquinolines (IMQs) such as the small synthetic 3M-002, induce adult-level TNF from neonatal Mos, but the scope and mechanisms of IMQ-induced activation of neonatal Mos and Mo-derived dendritic cells (MoDCs) have not been reported. Objectives To characterize IMQ-induced activation of neonatal Mos and MoDCs. Methods Neonatal cord and adult peripheral blood Mos and MoDCs were cultured in autologous plasma; Alum- and TLR agonist-induced cytokines and co-stimulatory molecules were measured. TLR8 and inflammasome function were assayed using siRNA and western blotting/caspase-1 inhibitory peptide, respectively. The ontogeny of TLR8 agonist–induced cytokine responses was defined in Rhesus macaque whole blood ex vivo. Results IMQs were more potent and effective than Alum at inducing TNF and IL-1β from Mos. 3M-002 induced robust TLR pathway transcriptome activation and Th1-polarizing cytokine production in neonatal and adult Mos and MoDCs, signaling via TLR8 in an adenosine/cAMP- refractory manner. Newborn MoDCs displayed impaired LPS/ATP-induced caspase-1-mediated IL-1β production, but robust 3M-002-induced caspase-1-mediated inflammasome activation independent of exogenous ATP. TLR8-IMQs induced robust TNF and IL-1β in whole blood of Rhesus macaques at birth and infancy. Conclusions IMQ TLR8 agonists engage adenosine-refractory TLR8 and inflammasome pathways to induce robust Mo and MoDC activation and represent promising neonatal adjuvants.

Summary Based upon the recognition of antiviral compounds and single stranded viral RNA the Toll‐like receptors TLR7 and TLR8 are suggested to play a significant role in initiating antiviral immune responses. Here we report the molecular characterization of the chicken TLR7/8 loci which revealed an intact TLR7 gene and fragments of a TLR8‐like gene with a 6‐kilobase insertion containing chicken repeat 1 (CR1) retroviral‐like insertion elements. The chicken TLR7 gene encodes a 1047‐amino‐acid protein with 62% identity to human TLR7 and a conserved pattern of predicted leucine‐rich repeats. Highest levels of chicken TLR7 mRNA were detected in immune‐related tissues and cells, especially the spleen, caecal, tonsil and splenic B cells. Alternative spliced forms of TLR7 mRNA were identified in chicken, mouse and human and expressed in similar tissues and cell types to the major form of chicken TLR7. The chicken TLR7+ HD11 cell line and fresh splenocytes produced elevated levels of interleukin‐1β (IL‐1β) mRNA after exposure to the agonists R848 and loxoribine. Interestingly, none of the TLR7 agonists stimulated increased type I interferon (IFN) mRNA whereas poly(I:C) (a TLR3 agonist) up‐regulated both chicken IFN‐α and chicken IFN‐β mRNA. In contrast, TLR7 agonists, particularly R848 and poly(U) stimulated up‐regulation of chicken IL‐1β, and chicken IL‐8 mRNAs more effectively than poly(I:C). Stimulation of chicken TLR7 with R848 was chloroquine sensitive, suggesting signalling within an endosomal compartment, as for mammalian TLR7. The deletion of TLR8 in galliforms, accompanied with the differential response after exposure to TLR7 agonists, offers insight into the evolution of vertebrate TLR function.

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