Recent studies have shown that certain combinations of Toll-like receptor (TLR) agonists can induce synergistic immune activation. However， it remains challenging to achieve such robust responses in vivo in a manner that is effective， facile， and amenable for clinical translation. Here， we show that MPLA， a TLR4 agonist， and CpG， a TLR9 agonist， can be efficiently co-loaded into synthetic high-density lipoprotein nanodiscs， forming a potent adjuvant system (ND-MPLA/CpG) that can be readily combined with a variety of subunit antigens， including proteins and peptides. ND-MPLA/CpG significantly enhanced activation of dendritic cells， compared with free dual adjuvants or nanodiscs delivering a... More
Recent studies have shown that certain combinations of Toll-like receptor (TLR) agonists can induce synergistic immune activation. However， it remains challenging to achieve such robust responses in vivo in a manner that is effective， facile， and amenable for clinical translation. Here， we show that MPLA， a TLR4 agonist， and CpG， a TLR9 agonist， can be efficiently co-loaded into synthetic high-density lipoprotein nanodiscs， forming a potent adjuvant system (ND-MPLA/CpG) that can be readily combined with a variety of subunit antigens， including proteins and peptides. ND-MPLA/CpG significantly enhanced activation of dendritic cells， compared with free dual adjuvants or nanodiscs delivering a single TLR agonist. Importantly， mice immunized with physical mixtures of protein antigens ND-MPLA/CpG generated strong humoral responses， including induction of IgG responses against protein convertase subtilisin/kexin 9 (PCSK9)， leading to 17-30% reduction of the total plasma cholesterol levels. Moreover， ND-MPLA/CpG exerted strong anti-tumor efficacy in multiple murine tumor models. Compared with free adjuvants， ND-MPLA/CpG admixed with ovalbumin markedly improved antigen-specific CD8+ T cell responses by 8-fold and promoted regression of B16F10-OVA melanoma (P < 0.0001). Furthermore， ND-MPLA/CpG admixed with E7 peptide antigen elicited ~20% E7-specific CD8+ T cell responses and achieved complete regression of established TC-1 tumors in all treated animals. Taken together， our work highlights the simplicity， versatility， and potency of dual TLR agonist nanodiscs for applications in vaccines and cancer immunotherapy.
