title: Production of Furin-Cleaved Papillomavirus Pseudovirions and Their Use for In Vitro Neutralization Assays of L1- or L2-Specific Antibodies.
abstract: 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.
authors.affiliation: Department of Pathology, The Johns Hopkins University, Baltimore, Maryland.|These authors contributed equally to this work.
authors.forename: Joshua W
authors.initials: JW
authors.lastname: Wang
authors.affiliation: Human Papillomavirus (HPV) Immunology Laboratory, Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, Frederick, Maryland.|These authors contributed equally to this work.
authors.forename: Ken
authors.initials: K
authors.lastname: Matsui
authors.affiliation: Human Papillomavirus (HPV) Immunology Laboratory, Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Yuanji
authors.initials: Y
authors.lastname: Pan
authors.affiliation: Department of Pathology, The Johns Hopkins University, Baltimore, Maryland.
authors.forename: Kihyuck
authors.initials: K
authors.lastname: Kwak
authors.affiliation: Department of Oncology, The Johns Hopkins University, Baltimore, Maryland.
authors.forename: Shiwen
authors.initials: S
authors.lastname: Peng
authors.affiliation: Human Papillomavirus (HPV) Immunology Laboratory, Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Troy
authors.initials: T
authors.lastname: Kemp
authors.affiliation: Human Papillomavirus (HPV) Immunology Laboratory, Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Ligia
authors.initials: L
authors.lastname: Pinto
authors.affiliation: Department of Pathology, The Johns Hopkins University, Baltimore, Maryland.|Department of Oncology, The Johns Hopkins University, Baltimore, Maryland.|Department of Gynecology and Obstetrics, The Johns Hopkins University, Baltimore, Maryland.
authors.forename: Richard B S
authors.initials: RB
authors.lastname: Roden
mesh_terms: D057134:Antibodies, Neutralizing / Q000276:immunology*; D000914:Antibodies, Viral / Q000276:immunology*; D045683:Furin / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D006801:Humans; D009500:Neutralization Tests / Q000379:methods*; D027383:Papillomaviridae / Q000235:genetics / Q000276:immunology*; D022701:Virosomes / Q000235:genetics / Q000276:immunology*
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D022701:Virosomes; D045683:Furin
keywords: HPV L2; HPV neutralization assay; antibody; furin-cleavage; human papillomavirus; papillomavirus