Despite the work showing efficient immune response induction, there is no knowledge on SVLP interaction with DC. We hypothesised that their interaction with DC could elaborate on the characteristics of DC-antigen interactions relating to effica- cious induction of immune responses. Moreover, the SVLP should provide the means of defining how DC handle a complex virus-like structure. The efficiency of the SVLP vaccine allows these characteristics to be defined without the encumbrance of over- laying adjuvant activity. Accordingly, the present work charac- terised the manner by which DC bind and internalise SVLP, relating to the involvement of different intracellular organelles and compartments known to be important for the variety of DC functions. Results SVLP interact with DC in a time and concentration dependent manner The initial analyses sought to determine the kinetics of SVLP interaction with DC, which was performed over a 20 min period on ice to impede the internalisation processes. Flow cytometry analyses showed SVLP binding in a concentration dependent manner; optimum binding with 1 mg/ml resulting in 98% SVLP- positive cells (Fig. 1B). This was confirmed by confocal micros- copy, with which the strongest signal was obtained using 2.5 mg/ ml SVLP (data not shown). The binding to DC on ice was also time-dependent and rapid, being observed as early as the 5-second time point (plus the time for washing prior to fixation of the DC with 4% w/v p-formaldehyde to prevent further activity) (Fig. 1C). Binding appeared to peak at 5 min (data not shown). Such a rapid interaction of SVLP with DC was not unique. High molecular weight dextran, ovalbumin and transferrin also interacted rapidly (Fig. 1D), but contrasted with SVLP in requiring metabolically active cells; only a low signal was obtained for binding on ice (Fig. 1E shows ovalbumin as an example). Although SVLP binding to DC on ice was variable with time, 5 independent experiments showed that the interaction depicted in Fig. 1C was consistently rapid. This rapid SVLP binding to DC may have reflected an ionic or a receptor-mediated interaction. Consequently, pronase (titrated for its optimum concentration and incubation time; data not shown) was used to pre-treat DC at 39uC. The cells were then washed and cooled on ice prior to adding SVLP or antibody against MHC Class I (MHCI), CD172a or CD14 on ice (Fig. 1F). Labelling of the cell surface markers was to confirm the proteolytic effect of pronase on sensitive cell surface protein receptors (CD172a, CD14), but not all molecules (MHCI); the pronase sensitivity of CD172a and CD14 was enhanced by treating the cells with 3mM NaN3 to impair receptor recycling (Fig. 1F). A 90% reduction in binding was observed with SVLP, compared to cells not treated with pronase (Fig. 1F). This was considered to be indicating the removal of a putative protein receptor for SVLP binding. SVLP internalisation by DC The capacity of DC to internalise bound SVLP employed incubations at 39uC (the resting body temperature of the porcine donors of the DC). Following incubation for 30 min at 39uC, confocal microscopy showed SVLP internalisation by DC using 3– D imaging (Fig. 2A). In order to confirm that the SVLP were internalised, PK treatment was employed to remove material from the cell surface. SVLP were interacted with DC for 30 min on ice, followed by PK treatment for a further 30 min on ice. This resulted in an DC Endocytosis of SVLP abrogation of the SVLP fluorescent signal (Fig. 2B; ‘‘DC + SVLP then PK’’ compared with ‘‘SVLP(positive control)’’). If the DC were pre-treated with the PK for 30 min on ice prior to addition of the SVLP (Fig. 3B; ‘‘DC + PK then SVLP’’), there was only a minor reduction in the signal obtained. It was considered that these results demonstrated a sensitivity of the SVLP to digestion by PK, whereas the receptor(s) for SVLP binding was apparently insensitive. This sensitivity of the SVLP was confirmed by pre- treating with PK on ice prior to incubation with DC; again the fluorescence signal was lost with the treated SVLP (Fig. 2C). Subsequent experiments in which SVLP internalisation required confirmation used this sensitivity of the SVLP to the PK treatment on ice – resistance to PK was taken as indicative of internalisation. The rate of SVLP internalisation by DC was analysed by pulsing the cells with SVLP on ice for 20 min, washing to remove any unbound particles, then shifting to 39uC. After different times at 39uC, treatment with PK on ice for 30 min allowed definition of the degree of internalisation (PK activity was neutralised by addition of 10% porcine serum). Internalised SVLP, and therefore protected from the PK treatment, were observed as early as one min after shifting to 39uC (Fig. 2D). However, only a minor proportion of the cells showed evidence of internalised material at this time. There was a gradual increase with time for the number of cells displaying internalised material, reaching maximum uptake at 30 min (Fig. 2D). SVLP are non-toxic for DC Despite the apparently efficient internalisation of SVLP by DC, this did not exclude an eventual toxicity for the cells. Accordingly, DC were given 1.25, 2, 4, and 8 mg/ml of SVLP, and incubated for 24, 48 and 72 hrs at 39uC without removal of the SVLP. After harvesting at these different time points, the cells were stained with propidium iodide (PI) to quantify dead or damaged cells. No cell death was observed above that obtained with the ‘‘cell only’’ control, when analysed at 24 and 48 hrs (Fig. 2E). A slight increase in the number of PI-positive cells was seen with the SVLP after 72 hrs, but this was not related to the SVLP concentration, and with most concentrations represented ,10% cell population. Statistical analysis showed that the numbers of PI+ cells with SVLP-treated cells were not significantly different from ‘‘PBS’’ or ‘‘cells alone’’. It was therefore considered that SVLP are non-toxic for DC. Kinetics of SVLP association with DC While the above results focussed on the kinetics of uptake and internalisation, it was also important to determine if the SVLP were degraded over time. Following SVLP pulsing of DC on ice, and washing prior to the 39uC shift, it was noted that between 4 and 24 hrs the fluorescent signal declined rapidly, reaching a minimum close to the background signal by a 72 hrs (Fig. 2F). Although a few positive cells could be found at later times, this was rare and inconsistent. SVLP association with lipid rafts The association of SVLP with particular subcellular fractions gave no information on the manner of uptake; DC operate various endocytic pathways, which can determine the fate of the internalised material. An early characteristic defining particular endocytic pathways is the involvement of lipid raft mobilisation, identifiable by CTB binding to five molecules of GM1 [21]. Within 30 min of incubation with DC on ice, CTB (under predetermined optimum conditions for visualising lipid rafts) showed an irregular pattern of interaction (in patches) with the plasma membrane (Fig. 3A, ‘‘CTB’’); This pattern can be seen PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e43248