Optimization of 8-oxoadenines with toll-like-receptor 7 and 8 activity.

Toll-like receptors 7 and 8 (TLR7/8) agonists are potent immunostimulants that are attracting considerable interest as vaccine adjuvants. We recently reported the synthesis of a new series of 2-O-butyl-8-oxoadenines substituted at the 9-position with various linkers and N-heterocycles, and showed that TLR7/8 selectivity, potency and cytokine induction could be modulated by varying the alkyl linker length and the N-heterocyclic ring. In the present study, we further optimized the oxoadenine scaffold by investigating the effect of different substituents at the 2-position of the oxoadenine on TLR7/8 potency/selectivity, cytokine induction and DC maturation in human PBMCs. The results show that introducing a 1-(S)-methylbutoxy group at the 2-position of the oxoadenine significantly increased potency for TLR7/8 activity, cytokine induction and DC maturation.

Novel Lipidated Imidazoquinoline TLR7/8 Adjuvants Elicit Influenza-Specific Th1 Immune Responses and Protect Against Heterologous H3N2 Influenza Challenge in Mice.

Most licensed seasonal influenza vaccines are non-adjuvanted and rely primarily on vaccine-induced antibody titers for protection. As such, seasonal antigenic drift and suboptimal vaccine strain selection often results in reduced vaccine efficacy. Further, seasonal H3N2 influenza vaccines demonstrate poor efficacy compared to H1N1 and influenza type B vaccines. New vaccines, adjuvants, or delivery technologies that can induce broader or cross-seasonal protection against drifted influenza virus strains, likely through induction of protective T cell responses, are urgently needed. Here, we report novel lipidated TLR7/8 ligands that act as strong adjuvants to promote influenza-virus specific Th1-and Th17-polarized T cell responses and humoral responses in mice with no observable toxicity. Further, the adjuvanted influenza vaccine provided protection against a heterologous H3N2 influenza challenge in mice. These responses were further enhanced when combined with a synthetic TLR4 ligand adjuvant. Despite differences between human and mouse TLR7/8, these novel lipidated imidazoquinolines induced the production of cytokines required to polarize a Th1 and Th17 immune response in human PBMCs providing additional support for further development of these compounds as novel adjuvants for the induction of broad supra-seasonal protection from influenza virus.

Co-encapsulation of synthetic lipidated TLR4 and TLR7/8 agonists in the liposomal bilayer results in a rapid, synergistic enhancement of vaccine-mediated humoral immunity.

To increase vaccine immunogenicity, modern vaccines incorporate adjuvants, which serve to enhance immune cross-protection, improve humoral and cell-mediated immunity, and promote antigen dose sparing. Pattern recognition receptors (PRRs), including the Toll-like receptor (TLR) family are promising targets for development of agonist formulations for use as vaccine adjuvants. Combinations of co-delivered TLR4 and TLR7/8 ligands have been demonstrated to have synergistic effects on innate and adaptive immune response. Here, we create liposomes that stably co-encapsulate CRX-601, a synthetic TLR4 agonist, and UM-3004, a lipidated TLR7/8 agonist, within the liposomal bilayer in order to achieve co-delivery, allow tunable physical properties, and induce in vitro and in vivo immune synergy. Co-encapsulation demonstrates a synergistic increase in IL-12p70 cytokine output in vitro from treated human peripheral blood mononuclear cells (hPBMCs). Further, co-encapsulated formulations give significant improvement of early IgG2a antibody titers in BALB/c mice following primary vaccination when compared to single agonist or dual agonists delivered in separate liposomes. This work demonstrates that co-encapsulation of TLR4 and lipidated TLR7/8 agonists within the liposomal bilayer leads to innate and adaptive immune synergy which biases a Th1 immune response. Thus, liposomal co-encapsulation may be a useful and flexible tool for vaccine adjuvant formulation containing multiple TLR agonists.

Synthetic Toll-like Receptors 7 and 8 Agonists: Structure-Activity Relationship in the Oxoadenine Series.

Toll-like receptors 7 and 8 (TLR7/8) are broadly expressed on antigen-presenting cells, making TLR7/8 agonists likely candidates for the development of new vaccine adjuvants. We previously reported the synthesis of a new series of 8-oxoadenines substituted at the 9-position with a 4-piperidinylalkyl moiety and demonstrated that TLR7/8 selectivity and potency could be modulated by varying the length of the alkyl linker. In the present study, we broadened our initial structure-activity relationship study to further evaluate the effects of N-heterocycle ring size, chirality, and substitution on TLR7/8 potency, receptor selectivity, and cytokine (IFNα and TNFα) induction from human peripheral blood mononuclear cells (PBMCs). TLR7/8 activity correlated primarily to linker length and to a lesser extent to ring size, while ring chirality had little effect on TLR7/8 potency or selectivity. Substitution of the heterocyclic ring with an aminoalkyl or hydroxyalkyl group for subsequent conjugation to phospholipids or antigens was well tolerated with the retention of both TLR7/8 activity and cytokine induction from human PBMCs.