title: Reactivity of human sera in a sensitive, high-throughput pseudovirus-based papillomavirus neutralization assay for HPV16 and HPV18.
abstract: Sensitive high-throughput neutralization assays, based upon pseudoviruses carrying a secreted alkaline phosphatase (SEAP) reporter gene, were developed and validated for human papillomavirus (HPV)16, HPV18, and bovine papillomavirus 1 (BPV1). SEAP pseudoviruses were produced by transient transfection of codon-modified papillomavirus structural genes into an SV40 T antigen expressing line derived from 293 cells, yielding sufficient pseudovirus from one flask for thousands of titrations. In a 96-well plate format, in this initial characterization, the assay was reproducible and appears to be as sensitive as, but more specific than, a standard papillomavirus-like particle (VLP)-based enzyme-linked immunosorbent assay (ELISA). The neutralization assay detected type-specific HPV16 or HPV18 neutralizing antibodies (titers of 160-10240) in sera of the majority of a group of women infected with the corresponding HPV type, but not in virgin women. Sera from HPV16 VLP vaccinees had high anti-HPV16 neutralizing titers (mean: 45000; range: 5120-163840), but no anti-HPV18 neutralizing activity. The SEAP pseudovirus-based neutralization assay should be a practical method for quantifying potentially protective antibody responses in HPV natural history and prophylactic vaccine studies.
authors.affiliation: Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, MD 20892-4263, USA.
authors.forename: Diana V
authors.initials: DV
authors.lastname: Pastrana
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Buck
authors.forename: Yuk-Ying S
authors.initials: YY
authors.lastname: Pang
authors.forename: Cynthia D
authors.initials: CD
authors.lastname: Thompson
authors.forename: Philip E
authors.initials: PE
authors.lastname: Castle
authors.forename: Peter C
authors.initials: PC
authors.lastname: FitzGerald
authors.forename: Susanne
authors.initials: S
authors.lastname: Krüger Kjaer
authors.forename: Douglas R
authors.initials: DR
authors.lastname: Lowy
authors.forename: John T
authors.initials: JT
authors.lastname: Schiller
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis / Q000235:genetics; D000914:Antibodies, Viral / Q000032:analysis*; D052688:Bovine papillomavirus 1 / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D005260:Female; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D009500:Neutralization Tests / Q000379:methods*; D027383:Papillomaviridae / Q000235:genetics* / Q000276:immunology; D030361:Papillomavirus Infections / Q000097:blood / Q000276:immunology / Q000517:prevention & control; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014611:Vaccination; D015678:Viral Structural Proteins / Q000235:genetics; D014765:Viral Vaccines / Q000008:administration & dosage / Q000276:immunology; D014771:Virion / Q000235:genetics / Q000276:immunology
chemical_list: D000914:Antibodies, Viral; D008164:Luminescent Proteins; D015678:Viral Structural Proteins; D014765:Viral Vaccines; D049452:Green Fluorescent Proteins; D000469:Alkaline Phosphatase