Production of Furin‐Cleaved Papillomavirus Pseudovirions and Their Use for In Vitro Neutralization Assays of L1‐ or L2‐Specific Antibodies

Joshua W. Wang1, Ken Matsui1, Yuanji Pan2, Kihyuck Kwak3, Shiwen Peng4, Troy Kemp2, Ligia Pinto2, Richard B.S. Roden5

1 These authors contributed equally to this work, 2 Human Papillomavirus (HPV) Immunology Laboratory, Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, Frederick, Maryland, 3 Department of Pathology, The Johns Hopkins University, Baltimore, Maryland, 4 Department of Oncology, The Johns Hopkins University, Baltimore, Maryland, 5 Department of Gynecology and Obstetrics, The Johns Hopkins University, Baltimore, Maryland
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14B.5
DOI:  10.1002/9780471729259.mc14b05s38
Online Posting Date:  August, 2015
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Immunization with Human Papillomavirus (HPV) L1 virus‐like particles or L2 capsid protein elicits neutralizing antibodies that mediate protection. A high‐throughput and sensitive in vitro neutralization assay is therefore valuable for prophylactic HPV vaccine studies. Over several hours during infection of the genital tract, virions take on a distinct intermediate conformation, including a required furin cleavage of L2 at its N‐terminus. This intermediate is an important target for neutralization by L2‐specific antibody, but it is very transiently exposed during in vitro infection of most cell lines resulting in insensitive measurement for L2, but not L1‐specific neutralizing antibodies. To model this intermediate, we describe a protocol to generate furin‐cleaved HPV pseudovirions (fc‐PsV), which deliver an encapsidated reporter plasmid to facilitate infectivity measurements. We also describe a protocol for use of fc‐PsV in a high‐throughput in vitro neutralization assay for the sensitive measurement of both L1 and L2‐specific neutralizing antibodies. © 2015 by John Wiley & Sons, Inc.

Keywords: furin‐cleavage; papillomavirus; antibody; human papillomavirus; HPV neutralization assay; HPV L2

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Table of Contents

  • Introduction
  • Basic Protocol 1: Production of Furin‐Cleaved Pseudovirions (FC‐PSV)
  • Support Protocol 1: Assessment of Encapsidated Reporter Plasmid or Viral Genomic Equivalents (VGE)
  • Furin‐Cleaved HPV Pseudovirus Neutralization Assay (FC‐PBNA)
  • Basic Protocol 2: Performing FC‐PBNA with Secreted Alkaline Phosphatase
  • Alternate Protocol 1: Performing FC‐PBNA with Firefly Luciferase
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Production of Furin‐Cleaved Pseudovirions (FC‐PSV)

  • 293TTF cells (see Background Information)
  • 293TTF culture medium (see recipe)
  • Phosphate‐buffered saline (PBS; Invitrogen/Life Technologies, cat. no. 10010‐023)
  • 0.05% Trypsin/EDTA (Life Technologies, cat. no. 25300)
  • HPV types specific L1 and L2 vector plasmid and a reporter plasmid
  • Trypan blue
  • Mirus TransIT‐2020 transfection reagent (Mirus Bio, cat. no. MIR 5404)
  • 0.25% trypsin/EDTA
  • DPBS‐10 mM MgCl 2 with antibiotics (see recipe)
  • Lysis buffer (see recipe)
  • 1 M CaCl 2 (KD Medical, cat. no. PMS‐0614)
  • OptiPrep density gradient medium (60% w/v; Sigma, cat. no. D1556)
  • DPBS‐0.8 M NaCl (see recipe)
  • DPBS (Invitrogen/Life Technologies, cat. no. 14040)
  • Ice
  • DPBS with Mg++ and Ca++ (Invitrogen/Life Technologies, cat. no. 14287‐080)
  • 10× DPBS (Invitrogen/Life Technologies, cat. no. 14200‐075)
  • 1 M KCl (KD Medical, cat. no. PMB‐0730)
  • 1 M MgCl 2 (KD Medical, cat. no. PMS‐0630)
  • Distilled water (Invitrogen/Life Technologies, cat. no. 15230‐147)
  • 5 M NaCl (KD Medical, cat. no. RGF‐3270)
  • Brij58 (Sigma‐Aldrich, cat. no. P‐5884)
  • OptiMEM I reduced serum medium (Invitrogen/Life Technologies, cat. no. 11058‐021)
  • 225‐cm2 flasks (BD, cat. no. 353138)
  • 150‐cm2 flasks (Corning, cat. no. 430825)
  • 37°C, 5% CO 2 incubator
  • Microscope
  • 50‐ml conical tubes, sterile
  • Centrifuge (e.g.,Thermo Scientific Sorval Legend RT+)
  • Hemacytometer
  • Siliconized 1.5‐ml screw‐cap tubes (Fisher Scientific, cat. no. 05‐541‐63)
  • Microcentrifuge (e.g., Eppendorf Centrifuge 5417R); need to use it at 4°C
  • 1000‐μl pipets
  • 37°C water bath
  • Parafilm
  • Ultracentrifuge (e.g., Beckman Coulter Optima L‐80 XP Ultracentrifuge)
  • 15‐ml Polyallomer ultracentrifuge tubes (Beckman Coulter, cat. no. 331374) or 5‐ml Polyallomer ultracentrifuge tubes (Beckman Coulter, cat. no. 326819)
  • Swinging bucket ultracentrifuge rotor rated for >200,000 × g (e.g., SW55ti for 5‐ml tube or SW40ti for 15‐ml tube)
  • Siliconized pipet tips (VWR, cat. no.60828‐914)
  • Clamp ring or retort stand
  • 25‐G needles
  • 1‐ml TB syringe with 25‐G needle (BD, cat. no. 309626)
  • Pipetting needles with blunt end and standard hub (Popper and sons, cat. no. 7936)
  • Additional reagents and equipment for SDS PAGE (see ), immunoblotting (see unit ), cell counting with hemacytometer (see )
NOTE: Plasmids can be obtained from (Addgene: or

Support Protocol 1: Assessment of Encapsidated Reporter Plasmid or Viral Genomic Equivalents (VGE)

  Additional Materials (also see protocol 1)
  • HPV fcPsV sample (see protocol 1)
  • PureLink Viral RNA/DNA extraction kit (Invitrogen, cat. no. 12280‐050)
  • Master mix (see Table 14.5.3) Known quantity (e.g., 5 to 50 nanograms) of reporter DNA to be utilized for the standard curve during qPCR analysis (e.g., SEAP: pYSEAP construct, or LUCIFERASE: pcDNA‐luciferase plasmid from: Forward and Reverse Primers for SEAP/Luciferase plasmid (500 nM) including:
    • Luciferase Firefly Forward: TTG ACC GCC TGA AGT CTC TGA
    • Luciferase Firefly Reverse: ACA CCT GCG TCG AAG ATG TTG
  • 96‐well plate for qPCR
  • Optical seals for qPCR plates (Microseal “B” Adhesive Seals, Optical; BioRad, cat. no. MSB‐1001)
  • Machine able to perform qPCR
Table 4.0.3   Additional Materials (also see protocol 1)Preparation of Master Mix for qPCR for Samples and/or Standards (e.g., the Reporter Plasmid Used for the fcPsV Production)

Mastermix 1× reaction per sample 2.2× reaction per sample of Standard 3.3× reaction per sample of Standard
EVAgreen Supermix (Bio‐Rad, cat. no. 172‐5200) 10 μl 22 μl 33 μl
Reporter gene's forward & reverse primers (500 nM) 1 μl 2.2 μl 3.3 μl
Sterilized dH 2O 6 μl 13.2 μl 19.6 μl
Reporter DNA template 3 μl 6.6 μl 9.9 μl
Total volume 20 μl 44 μl 66 μl

Basic Protocol 2: Performing FC‐PBNA with Secreted Alkaline Phosphatase

  • LoVoT cells (see Background Information)
  • Assay medium (see recipe)
  • 1× PBS without Ca++ or Mg++ (Life Technologies, cat. no. 14190)
  • 0.4% trypan blue stain (Life Technologies, cat. no. 15250)
  • Furin‐cleaved pseudovirions (fcPsV) particles (see protocol 1)
  • Serum samples or antibody samples (See Critical Parameters)
  • Ziva Ultra SEAP Plus Detection kit (Jaden BioScience, cat. no. CM025) (see Background Information) containing:
    • SEAP Sample Preparation Solution (SSPS)
  • 5‐ and 10‐ml serological pipets (Costar, cat. no. 4051 and 4101)
  • Serological pipet filler (Thermo Scientific, cat. no. 9531)
  • 37°C, CO 2 incubator (Forma Scientific, Model 3110)
  • 50‐ml conical tubes (BD Falcon, cat. no. 352098)
  • Inverted light microscope (Nikkon TMS and Nikkon LBOPHOT)
  • Centrifuge (Thermo Scientific; Sorval Legend RT+)
  • Hemacytometer (Improved Neubauer 0.1‐mm deep)
  • 50‐ml reservoir troughs (Costar, cat. no. 4870)
  • Flat‐bottom 96‐well tissue culture plates (Costar, cat. no. 3596)
  • 10‐, 20‐, 100‐, 200‐, and 1000‐μl single or multichannel pipets (Rainin)
  • 10‐ to 1000‐μl pipet tips (Rainin, cat. no. SR‐L10S,‐250 S, and ‐L1000S)
  • Certified class II biological safety cabinet
  • Conical‐bottom, deep‐well, 96‐well plate (e.g., 0.5 to 2 ml; VWR, cat. nos. 40002‐022, ‐011, or ‐014); depending on the number of test‐wells, different size plates can be employed
  • Seal plate films (Thomas Scientific, cat. no. 6980A03)
  • 15‐ml conical tubes (Corning, cat. no. 430055)
  • Round‐bottom 96‐well tissue culture plates (Costar, cat. no. 3788)
  • V‐bottom 96‐well plates (Costar, cat. no. 3357)
  • Cold storage adhesive sealing foil (VWR, cat. no. 89049‐034)
  • −80°C freezer (Forma Scientific, Model 8517)
  • Plate shaker (e.g., Thermo Scientific titer shaker model 4625)
  • White opaque 96‐well microplate/OptiPlate‐96 (Perkin Elmer, cat. no. 6005290)
  • Oven capable of reaching 65° to 73°C (e.g., Thermo Scientific, Model Heratherm OMH100)
  • Aluminum foil
  • Plate reader capable of measuring luminescence (e.g., Molecular Devices, SpectraMax M5)
  • Computer and appropriate software to operate the plate reader

Alternate Protocol 1: Performing FC‐PBNA with Firefly Luciferase

  Additional Materials (also see Basic Protocols protocol 11 and protocol 32)
  • Dual‐Luciferase Reporter Assay System (Promega, cat. no E1910 or E1960) containing:
  • Cell Culture Lysis Buffer (Promega, cat. no. E1531)
  • 96‐well flat‐bottom tissue culture plates (Costar, cat. no. 3596)
  • Multichannel pipet
  • Black 96‐well optiplate (PerkinElmer, cat. no. 6005290)
  • Microplate Luminometer with an injector
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