Synthetic Virus-Like Particles Target Dendritic Cell Lipid Rafts for Rapid Endocytosis Primarily but Not Exclusively by Macropinocytosis Rajni Sharma 1 , Arin Ghasparian2, John A. Robinson 2 , Kenneth C. McCullough 1 * 1 Institute of Virology and Immunoprophylaxis, Mittelha¨usern, Switzerland, 2 Department of Chemistry, University of Z u¨rich, Z u¨rich, Switzerland 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. Citation: Sharma R, Ghasparian A, Robinson JA, McCullough KC (2012) Synthetic Virus-Like Particles Target Dendritic Cell Lipid Rafts for Rapid Endocytosis Primarily but Not Exclusively by Macropinocytosis. PLoS ONE 7(8): e43248. doi:10.1371/journal.pone.0043248 Editor: Adriano Boasso, Imperial College London, United Kingdom Received April 16, 2012; Accepted July 18, 2012; Published August 14, 2012 Copyright: 2012 Sharma et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was funded by the Swiss National Science Foundation, project NANOVACC, project number 310000–1198828 (http://www.snf.ch/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kmc.projects1@gmail.com Introduction Dendritic cells (DC) [1] play a crucial role initiating and promoting immune response, and are essential for the develop- ment of robust, efficacious immune defences. An important characteristic is the capacity of DC to employ different endocytic pathways towards efficient antigen internalisation and processing; reviewed by Lin and co-workers [2]. DC possess the potential for selective application of endocytic pathways dependent or in- dependent of clathrin, caveolae, lipid raft mobilisation and/or macropinocytosis; antigen delivery routes can employ endosomal pathways and/or the endoplasmic reticulum [3,4,5,6,7,8]. This diversity of processing routes provides DC with a high capacity for promoting antigen delivery and processing. Yet, these same endocytic pathways are involved in degrading internalised material. It has been reported that DC display a more limited protease activity compared with macrophages; this was related to a slower in vivo degradation of internalized antigens, and antigen retention for extended periods, which favoured antigen pre- sentation [9]. DC have been labelled professional antigen presenting cells [10]. Immature DCs display more efficient endocytic processes than mature DCs [11]. While appropriate targeting of vaccines to DC would favour immune defence development, this area still requires clarification concerning the characteristics of DC endocytic uptake. With particulate delivery vehicles [12,13] being promoted as vaccine carriers targeting DC for efficient immune defence induction [14,15,16] defining endocytic uptake is impor- tant for vaccine design. Although there is information on model proteins such as ovalbumin, there is less information on how DC handle more complex vaccines. Accordingly, the aim of the present work was to characterize how DC handled Synthetic Virus-Like Particles (SVLP). These are complex antigenic structures with an already proven capacity for efficient induction of IgG responses; importantly, this is achieved without adjuvant requirement in rabbits [17] and pigs (R.S, unpublished data). SVLP are formed by lipopeptide self-assembly into spherical nanostructures in the 20–30 nm size range, which resemble small virus-like particles in their shape, size and chemical composition [18,19]. Like naturally-derived VLPs, SVLP can be exploited for multivalent presentation of antigens across their surface (Fig. 1A– S1A) [17] and several SVLP constructs have previously been shown to induce strong immune responses in experimental systems without co-administration of adjuvants [18,20]. PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e43248