Reactivity of human sera in a sensitive, high-throughput pseudovirus-based papillomavirus neutralization assay for HPV16 and HPV18

Virology. 2004 Apr 10;321(2):205-16. doi: 10.1016/j.virol.2003.12.027.


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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alkaline Phosphatase / analysis
  • Alkaline Phosphatase / genetics
  • Antibodies, Viral / analysis*
  • Bovine papillomavirus 1 / genetics
  • Bovine papillomavirus 1 / immunology
  • Cell Line
  • Female
  • Genetic Vectors
  • Green Fluorescent Proteins
  • Humans
  • Luminescent Proteins / genetics
  • Neutralization Tests / methods*
  • Papillomaviridae / genetics*
  • Papillomaviridae / immunology
  • Papillomavirus Infections / blood
  • Papillomavirus Infections / immunology
  • Papillomavirus Infections / prevention & control
  • Sensitivity and Specificity
  • Species Specificity
  • Vaccination
  • Viral Structural Proteins / genetics
  • Viral Vaccines / administration & dosage
  • Viral Vaccines / immunology
  • Virion / genetics
  • Virion / immunology


  • Antibodies, Viral
  • Luminescent Proteins
  • Viral Structural Proteins
  • Viral Vaccines
  • Green Fluorescent Proteins
  • Alkaline Phosphatase