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

• Published in: Virology (New York, N.Y.) N.Y.), 2004-04, Vol.321 (2), p.205-216
• Main Authors: Pastrana, Diana V, Buck, Christopher B, Pang, Yuk-Ying S, Thompson, Cynthia D, Castle, Philip E, FitzGerald, Peter C, Krüger Kjaer, Susanne, Lowy, Douglas R, Schiller, John T
• Format: Article
• Language: English
• Subjects: Alkaline Phosphatase - analysis; Alkaline Phosphatase - genetics; Antibodies; Antibodies, Viral - analysis; Bovine papillomavirus 1; Bovine papillomavirus 1 - genetics; Bovine papillomavirus 1 - immunology; Cell Line; Female; Genetic Vectors; Green Fluorescent Proteins; HPV; Human papillomavirus; Humans; Luminescent Proteins - genetics; Neutralization; Neutralization Tests - methods; Papillomaviridae - genetics; Papillomaviridae - immunology; Papillomavirus; Papillomavirus Infections - blood; Papillomavirus Infections - immunology; Papillomavirus Infections - prevention & control; Pseudovirus; Sensitivity and Specificity; Serology; Simian virus 40; Species Specificity; Vaccination; Vaccine; Vector; Viral Structural Proteins - genetics; Viral Vaccines - administration & dosage; Viral Vaccines - immunology; Virion; Virion - genetics; Virion - immunology; Virus-like particles
• ISSN: 0042-6822; 1096-0341
• 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.