Bicistronic vectors (pXsheLL, where X is the Papillomavirus type from which the codon optimized L1 and L2 genes were derived) representing the HPV types

16, 18, 31, 45, 52, 58 and Bovine Papillomavirus (BPV) were obtained from J.T. Schiller, National Cancer Institute, Bethesda, MD, USA, while p33sheLL and p68sheLL were obtained from H. Faust and J. Dillner, Malmö University Hospital, Malmö, Sweden. Constructs representing HPV35 (p35sheLL), HPV39 (p39sheLL) and HPV59 (p59sheLL) were generated by the insertion of codon optimized genes (Blue Heron, Inc., Bothell, WA, USA) based upon consensus L1 and L2 amino acid sequences into p5shell (http://home.ccr.cancer.gov/lco/default.asp). The consensus sequences were derived from NCBI database sequences (HPV35: M74117, X74477; HPV39: M62849; HPV59: X77858, EU918767) and contemporary sequences from anonymous, HPV-infected cytology samples (HPV35 L1: JN104062–64; HPV35 L2: JN104065–67; HPV39 L1: JN104068–70;

PD0332991 HPV39 L2: JN104071–72; HPV59 L1: JN104073–74; HPV59 L2: JN104075–77). The production of L1L2 pseudovirus stocks was performed as described elsewhere [24] using the alternative protocol (http://home.ccr.cancer.gov/lco/ripcord.htm), developed to reduce the inclusion of excess non-reporter-containing ‘cold capsids’, and by using luciferase (pGL4.51 [luc2/CMV/Neo]; Promega, Madison, WI) as the encapsidated reporter. Briefly, 293TT cells were transfected with equal amounts of pXsheLL and pGL4.51 [luc2/CMV/Neo] plasmids (Lipofectamine 2000; Invitrogen, Carlsbad, CA) and the encoded PFI-2 mw proteins expressed for 48 h before the cells were lysed, the capsids matured overnight in the presence of ribonucleases (RNase Cocktail; Applied Biosystems/Ambion, Austin, TX) and the double-clarified supernatant subjected to iodixanol gradient fractionation. Purified pseudovirus stocks were titrated on 293TT cells in quadruplicate, five-fold serial dilutions and the equivalent of a Tissue Culture Infectious Dose 50% Ergoloid (TCID50) was estimated using the Spearman–Karber equation. The average of three such estimations was made for each pseudovirus

stock used in this study. Pseudovirus-mediated reporter gene transduction of target cells in both the infectivity and neutralization assays was measured using the Steady-Glo Luciferase Assay Reagent (Promega) and the Glomax Multi Detection System (Promega) according to manufacturer’s instructions. The HPV pseudovirus neutralization assay was performed as originally described [25] with some modification. For the present study, heat-inactivated (56 °C, 30 min) serum samples were initially screened against all pseudoviruses (at a final serum dilution of 1/20 with pseudovirus) and any serum that demonstrated ≥80% reduction in the luciferase signal (RLU) relative to the pseudovirus and cell only controls was subsequently titrated and an 80% reciprocal neutralization titer estimated by interpolation.