Synthetic Virus-Like Particles Target Dendritic Cell Lipid Rafts for Rapid Endocytosis Primarily but Not Exclusively by Macropinocytosis

Abstract

DC employ several endocytic routes for processing antigens, driving forward adaptive immunity. Recent advances in synthetic biology have created small (20–30 nm) virus-like particles based on lipopeptides containing a virus-derived coiled coil sequence coupled to synthetic B- and T-cell epitope mimetics. These self-assembling SVLP efficiently induce adaptive immunity without requirement for adjuvant. We hypothesized that the characteristics of DC interaction with SVLP would elaborate on the roles of cell membrane and intracellular compartments in the handling of a virus-like entity known for its efficacy as a vaccine. DC rapidly bind SVLP within min, co-localised with CTB and CD9, but not caveolin-1. In contrast, internalisation is a relatively slow process, delivering SVLP into the cell periphery where they are maintained for a number of hrs in association with microtubules. Although there is early association with clathrin, this is no longer seen after 10 min. Association with EEA-1+ early endosomes is also early, but proteolytic processing appears slow, the SVLP-vesicles remaining peripheral. Association with transferrin occurs rarely, and only in the periphery, possibly signifying translocation of some SVLP for delivery to B-lymphocytes. Most SVLP co-localise with high molecular weight dextran. Uptake of both is impaired with mature DC, but there remains a residual uptake of SVLP. These results imply that DC use multiple endocytic routes for SVLP uptake, dominated by caveolin-independent, lipid raft-mediated macropinocytosis. With most SVLP-containing vesicles being retained in the periphery, not always interacting with early endosomes, this relates to slow proteolytic degradation and antigen retention by DC. The present characterization allows for a definition of how DC handle virus-like particles showing efficacious immunogenicity, elements valuable for novel vaccine design in the future.

Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence

“…SK-6 cells were transfected with the expression vectors using Lipofectamine 3000 (Life Technologies) and Opti-MEM (Life Technologies), according to the manufacturer's protocols. At the time indicated, SK-6 cells grown on an eight-well chamber slide (Matsunami) were fixed and stained as described by Sharma et al (2012) and Yamasaki et al (2012). Anti-GFP mAb as the primary antibody was used at a concentration of 1 mg ml 21 .…”

Functional RNA Delivery Targeted to Dendritic Cells By Synthetic Nanoparticles

“…This has been observed with DC internalizing synthetic virus-like particles. Clathrinmediated uptake was only witnessed early; the accumulation of synthetic virus-like particles in the cells was related to macropinocytic and lipid raft-mediated responses [79]. Hassane et al reviewed the evidence that cells may employ at least three pathways for endocytosis of cationic peptides -macropinocytosis, clathrin-mediated endocytosis and lipid raft-dependent endocytosis (FiguRe 4) [65].…”

Dendritic Cell Endocytosis Essential for Viruses and Vaccines

“…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].…”

“…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)).…”