Abstract
A highly sensitive, automated, purely add-on, high-throughput pseudovirion-based neutralization assay (HT-PBNA) with excellent repeatability and run-to-run reproducibility was developed for human papillomavirus types (HPV) 16, 18, 31, 45, 52, 58 and bovine papillomavirus type 1. Preparation of 384 well assay plates with serially diluted sera and the actual cell-based assay are separated in time, therefore batches of up to one hundred assay plates can be processed sequentially. A mean coefficient of variation (CV) of 13% was obtained for anti-HPV 16 and HPV 18 titers for a standard serum tested in a total of 58 repeats on individual plates in seven independent runs. Natural antibody response was analyzed in 35 sera from patients with HPV 16 DNA positive cervical intraepithelial neoplasia grade 2+ lesions. The new HT-PBNA is based on Gaussia luciferase with increased sensitivity compared to the previously described manual PBNA (manPBNA) based on secreted alkaline phosphatase as reporter. Titers obtained with HT-PBNA were generally higher than titers obtained with the manPBNA. A good linear correlation (R2 = 0.7) was found between HT-PBNA titers and anti-HPV 16 L1 antibody-levels determined by a Luminex bead-based GST-capture assay for these 35 sera and a Kappa-value of 0.72, with only 3 discordant sera in the low titer range. In addition to natural low titer antibody responses the high sensitivity of the HT-PBNA also allows detection of cross-neutralizing antibodies induced by commercial HPV L1-vaccines and experimental L2-vaccines. When analyzing the WHO international standards for HPV 16 and 18 we determined an analytical sensitivity of 0.864 and 1.105 mIU, respectively.
Highlights
Human papillomaviruses (HPV) are causally involved in the induction of cervical cancer and its precursor lesions
Built on the recognition of the HPV causality in cervical cancer development, two commercial vaccines, GardasilH and CervarixH targeting the two most prevalent carcinogenic HPV types 16 and 18 were licensed in the EU in 2006 and 2007, respectively [5,6]. Both vaccines employ the major capsid protein L1 in form of virus-like particles (VLPs) as antigen and are highly effective in preventing infections by HPV types 16 and 18 as well as cervical intraepithelial neoplasias induced by these viruses [7,8]
Human Sera Base line sera from 35 women with HPV 16 mono-infected high grade cervical intraepithelial neoplasia (CIN 2/3) participating in a vaccination trial with chimeric HPV 16 L1-E7 virus-like particles [17] were used as examples of naturally acquired HPV 16 antibodies
Summary
Human papillomaviruses (HPV) are causally involved in the induction of cervical cancer and its precursor lesions. Built on the recognition of the HPV causality in cervical cancer development, two commercial vaccines, GardasilH and CervarixH targeting the two most prevalent carcinogenic HPV types 16 and 18 were licensed in the EU in 2006 and 2007, respectively [5,6]. Both vaccines employ the major capsid protein L1 in form of virus-like particles (VLPs) as antigen and are highly effective in preventing infections by HPV types 16 and 18 as well as cervical intraepithelial neoplasias induced by these viruses [7,8]
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