Dendritic Cell Sensing of Hydrophobic Di- and Triacylated Lipopeptides Self-Assembled within Synthetic Virus-like Particles

Abstract

Dendritic cells (DCs) play critical roles in developing immune defenses. One important aspect is interaction with pathogen-associated molecular patterns (PAMPs)/danger-associated molecular patterns, including di- and triacylated lipopeptides. Isolated or synthetic lipopeptides are potent vaccine adjuvants, interacting with cell surface TLR2 heterodimers. In contrast, deep embedment within bacteria cell walls would impair lipopeptide interaction with cell surface TLR2, requiring degradation for PAMP recognition. Accordingly, DC processing in the absence of surface TLR2 ligation was defined using synthetic virus-like particles (SVLPs) carrying hydrophobic TLR2 PAMPs within di- and triacylated lipopeptide cores (P2Cys-SVLPs and P3Cys-SVLPs) compared with SVLPs lacking immunomodulatory lipopeptides. DCs rapidly and efficiently internalized SVLPs, which was dominated by slow endocytic processing via macropinocytosis, although some caveolar endocytosis was implicated. This delivered SVLPs primarily into macropinosomes often interacting with EEA-1 early endosomes. Although endoplasmic reticulum association was occasionally noted, association with recycling/sorting structures was not observed. Involvement of LysoTracker structures slowly increased with time, with SVLPs present in such structures ultimately dominating. Only SVLPs carrying di- and triacylated lipopeptide cores induced DC activation and maturation independently of surface TLR2 ligation. Intracellular recognition of SVLP TLR2 ligands was confirmed by observing SVLPs' association with internal TLR2, which had similar kinetics to SVLP association with LysoTracker. This related to inflammatory cytokine induction by SVLP DCs, with adaptive immune response activation ex vivo/in vivo. Importantly, particular DCs, not monocytes, recognized intracellular exposure of the TLR2 PAMPs carried by di- and triacylated SVLP cores, which indicates subset-distinct recognition of functional internal TLR2 ligands. Thus, vaccines carrying hydrophobic TLR2 ligands would interact with particular DCs for efficient induction of specific immunity in the absence of additional adjuvant.

Dendritic Cell Endocytosis Essential for Viruses and Vaccines

“…Vesicular TLR2 can detect lipopeptides buried deep within bacterial cell walls following endosomal degradation of the latter to expose the lipopeptides. This was proven using SVLPs carrying TLR2 ligands within their hydrophobic cores [15]. By employing such SVLPs, there was no influence of interaction with cell surface TLR2 heterodimers, as is the case with bacteria and yeast particles through the lipoteichoic acid and peptidoglycan moieties in their cell walls.…”

“…Combining studies on both virus infection of and vaccine delivery to DCs have led to the creation of new vaccine formulations, such as SVLP vaccines [14,15] and the self-amplifying RepRNA vaccines [9,[17][18][19]33]. While clathrin-mediated endocytosis has often been implicated with both virus infection and vaccine delivery [9,22,25,[55][56][57], the rapidity of the process and levels of enzymatic activity therein would favour a more degradative pathway, rather than one promoting antigen processing or RNA-release for translation.…”

“…Using SVLPs, DCs primarily endocytose these vaccines via macropinocytosis, but an underlying additional endocytic process is also active [14]. While a dominant processing towards MHC Class II presentation is evident, crosspresentation pathways also exist, directing the processing towards MHC Class I presentation [15]. Importantly, these SVLPs do not activate the DC family to promote DC maturation which is essential for efficient induction of adaptive immunity.…”

“…Importantly, these SVLPs do not activate the DC family to promote DC maturation which is essential for efficient induction of adaptive immunity. By modifying the lipopeptide monomers of the SVLPs to carry TLR2 ligands, certain SVLPs are directed into internal TLR2-containing sites for induction of cytokines that are important for DC maturation [15].…”

“…Resolution of this situation is showing promise from the more recent application of synthetic biology to create both synthetic virus-like particles (SVLPs) [14][15][16] and self-amplifying/replicating RNA (replicon or RepRNA) vaccines [9,13,[17][18][19][20], but also from advances in studies on virus infections. It has been observed that the majority of endocytosed material may well traverse rapid clathrin-mediated pathways, which is more likely favouring degradation of the internalised material [9,[21][22][23][24][25] (Figure 3, pathway (a)).…”