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. © 2015 by John Wiley & Sons, Inc.