In order to determine any inhibition of cell entry by VLPs using antibodies present in sera of vaccinated animals, Huh7 cells were Enhancement of HCV-VLP Immunogenicity PLOS ONE | www.plosone.org first incubated with PBS (10% FCS) for 15 min at 4uC to reduce non-specific binding of antibodies subsequently added. Cells were washed twice, resuspended in PBS (0.1% FCS) and incubated with FITC-labelled VLPs at 4uC for 1 hr. Serial dilutions of sera from vaccinated or non-vaccinated mice were then added and incubated for a further 1 hour at 37uC. For each reaction, 5610 5 Huh7 cells and 200 ng of FITC-labelled VLPs were used in a total volume of 500 ml. At the end of this incubation period, cells were washed with PBS (0.1% FCS) and then fixed in BD Cytofix (Becton Dickinson, USA). Inhibition of VLP entry was determined by flow cytometry and analysed using WEASEL 2.0 software (Walter and Eliza Hall Institute, Melbourne, Australia). Sera from vaccinated mice that demonstrated a decrease in specific cellular binding of 50% or more compared to sera from naïve mice were considered to contain neutralising antibodies [18] .