(R.S., unpublished data); of particular note is that unlike most non- replicating vaccines, the SVLP do not require formulation with adjuvant. It was considered that analysis of how DC handle a more complex virus-like structure would be informative; by using SVLP, the analyses of DC function would not be complicated by any adjuvant-formulation effect. SVLP were bound rapidly by DC, even on ice, more efficiently than for high molecular dextran, ovalbumin or transferrin. Internalisation of the SVLP was initially determined in terms of PK-resistance (internalised). Although the PK did not remove the DC receptors binding SVLP (whereas pronase did), the PK did efficiently destroy any SVLP still bound to the DC surface. Following binding to the DC on ice, then washing to remove unbound SVLP, further incubation at 39uC showed that the SVLP gradually became resistant to the PK-treatment, but in contrast with the rapid binding, internalisation was more gradual. This was reflected by cell fractionation analyses. Association with the cytoskeletal fraction was strongest at earlier time points, whereas association with the cytoplasmic fraction was not noted until after 30 min. Membrane association was observed throughout the 4 hrs observation period, probably reflecting the initial interaction with the plasma membrane, followed by vesicular membranes relating to internalisation. The SVLP signal faded at later time points, but the loss of signal was still gradual over a number of days. This kinetics of uptake and probable processing is consistent with the report that DC process antigen slowly [9]. However, it was still unclear if the slow kinetics were related to intracellular processing or simply the rate of internalisation. Primary DC and monocyte-derived DC are inefficient at functional DNA transfection [23], possibly due to the imperme- ability and resistance of their nuclear membranes. It was considered inappropriate to employ cell lines, because the aim of the work was to characterize how primary DC handled a complex antigen with proven in vivo efficacy. Accordingly, the intracellular compartments with which the SVLP associated were analysed using ligands and antibodies for their identification. SVLP interacted with lipid rafts on the DC surface. This was concluded due to their patchy association with the cell surface on ice, accumulation at the leading edge of the cell on shifting to 39uC, and co-localisation with CTB, which is known to interact with lipid rafts [21]. Co-localisation of the SVLP with the CD9 marker associated with lipid rafts of human monocytes [22], and sensitivity to MBCD treatment, known to interfere with cholesterol restoration, confirmed that SVLP endocytosis by DC was dominated by lipid raft-dependent processes. These were caveo- lin-independent, because there was no co-localisation of SVLP- containing structures with caveolin-1 (data not shown). Following internalisation, there was an association with EEA-1+ structures, which would relate to macropinosomes interacting with early endosomes initiating antigen processing [5,6,7,8]. The association of the internalised SVLP with EEA-1+ structures was a gradual process, the frequency increasing as time progressed. Moreover, not all SVLP-containing structures co-localised with EEA-1+ structures, even at later time points. The nature of this EEA-1 association was studied further using SVLP in which the fluorochrome signal was self-quenched, and therefore required degradation for de-quenching. When the processed SVLP was de- quenched, the signal did co-localise with EEA-1, but this only became apparent between 30 and 60 min (depending on the experiment), more frequent by 1 to 2 hrs. This kinetics followed that of DQ-Ova, a self-quenched entity employed for detecting endosomal-processing events, the high degree of overlap indicating similar processing pathways. This confirmed slow enzymatic processing, but also indicated that the SVLP-containing vesicles DC Endocytosis of SVLP not associated with EEA-1 had probably not interacted with early endosomes. In contrast to the strong association with EEA-1+ structures, even at later time points it was difficult to visualise association with MHCII+ structures. Yet, the SVLP signal was aligning with microtubules, confirming that intracellular transport was probable. Nevertheless, the SVLP-containing vesicles, even when EEA-1+, tended to remain more peripheral rather than trafficking towards a more perinuclear region of the cell. The occasional SVLP-MHC interaction that was noted, tended to be more at the peripheral edge of region containing MHCII+ structures (intermediate between the peripheral area occupied by most of the SVLP and a more perinuclear region, also EEA-1+). With the peripheral SVLP signal not declining rapidly, the endocytic process was apparently maintaining the SVLP-containing structures in this region, even after interaction with EEA-1+ vesicles. Being indicative of macropinocytosis, with the advancement of processing appearing gradual, this could explain why the observed interaction with the MHCII+ structures was a rare event. Visualisation of further processing may have proven difficult due to a slow processing, when the amount of SVLP moving forward in the processing chain was low, perhaps often too low to be visualised. The same argument may be true for association with the ER. This can also occur by translocation for macropinosomes [4], before the acidification and proteolytic processing is too far advanced. Although it was difficult to observe an association of SVLP with the ER, there may have been a low level of translocation, the number of events or amount of material involved being too low for detection. Although the results were indicating an important role for lipid- raft mediated macropinocytosis, there was evidence of an early involvement for a clathrin-dependent route. Nevertheless, most SVLP were not associated with clathrin+ structures, and the latter were only visible during the first 10 min. This implies that clathrin-dependent endocytosis is of minor relevance to the processing of SVLP for antigen presentation; indeed, the early clathrin-dependent uptake may be leading the SVLP into a rapid degradative process, rather than the slower and more progressive antigen-processing route. The results with transferrin would also argue in this direction. While transferrin was continually observed translocating from the periphery, the SVLP tended to remain more peripheral than the transferrin. Some peripheral vesicles did contain both SVLP and transferrin, but this was rather late – at the 60-min time point. This may reflect diversion of some SVLP- containing vesicles into sorting endosomes. Although such structures were in the minority, such a possibility relates to the sorting of antigen for delivery to B-lymphocytes [1,29]. The importance of macropinocytosis in SVLP internalisation by DC was confirmed using high molecular weight dextran [27], which should remain blocked within macropinosomes. Allowing dextran to accumulate in macropinosomes, SVLP-containing vesicles entering at the leading edge of the DC were not associated with dextran, confirming their early endocytic stage. By 20 min at 39uC, most of the SVLP co-localised with the dextran+ vesicles, strongly indicative of SVLP accumulation in macropinosomes. This would certainly relate to the importance of macropinocytosis for the entry of viruses into DC [26]. Use of maturing DC for their reduced macropinocytic activity [28] further confirmed the importance of macropinocytosis during SVLP uptake. Mature DC were inferior to immature DC for endocytosis of SVLP; in contrast to high molecular weight dextran, the uptake of which was abrogated in mature DC, there was a residual uptake of SVLP. This internalisation by mature DC was much slower than with immature cells, and never reached the levels obtained with PLOS ONE | www.plosone.org 13 August 2012 | Volume 7 | Issue 8 | e43248