High-Throughput Pseudovirion-Based Neutralization Assay for Analysis of Natural and Vaccine-Induced Antibodies against Human Papillomaviruses

et al. (2013) High-Throughput Pseudovirion-Based Neutralization Assay for Analysis of Natural and Vaccine-Induced Antibodies against Human Papillomaviruses. PLoS ONE 8(10): e75677. doi:10.1371/journal.pone.0075677 High-Throughput Pseudovirion-Based Neutralization Assay for Analysis of Natural and Vaccine-Induced Antibodies against Human Papillomaviruses Martin Mu ller 0 Peter Sehr 0 Ivonne Rubio 0 Hanna Seitz 0 Kerstin Putzker 0 Lis Ribeiro-Mu ller 0 Michael Pawlita 0 Suryaprakash Sambhara, Centers for Disease Control and Prevention, United States of America 0 1 EMBL-DKFZ Chemical Biology Core Facility, European Molecular Biology Laboratory , Heidelberg, Germany , 2 Infections and Cancer Program, German Cancer Research Center (DKFZ) , Heidelberg , Germany 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 cellbased 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. - Funding: Funding from the German Cancer Research Center. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Martin Mu ller receives royalties from sales of Cervarix vaccine. Other authors have declared that no competing interests exist. This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials. Human papillomaviruses (HPV) are causally involved in the induction of cervical cancer and its precursor lesions. Currently, 12 HPV types are classified as carcinogenic to humans and an additional 8 types as probably or possibly carcinogenic to human [1]. Worldwide, the ten HPV types identified most frequently in cervical cancer are HPV 16, 18, 33, 45, 31, 58, 52, 35, 59 and 56 [2]. HPV infection is recognized as an absolute requirement for the transformation process in cervical cancer [3,4], but host cell cofactors also play a role. 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]. The mode of action of both vaccines is considered to be the induction of neutralizing antibodies directed against L1 surface loops of the viral capsid. With more than six years history on papillomavirus prophylactic vaccination, monitoring long term development of protective titers of neutralizing antibodies is of increasing importance. Thus, there is a need for the evaluation of such antibody responses, specifically for functional assays analyzing neutralizing antibodies. Papillomaviruses cannot be replicated in simple cell culture systems. Therefore, in the past a number of functional assays have been developed to measure antibody-mediated neutralization of papillomaviruses. These assays involved the use of authentic viruses [9] [10] and so called pseudovirions with an encapsidated reporter construct [11,12,13]. In addition, neutralizing antibodies have been measured more indirectly e.g. by a hemagglutination inhibition assay [14] or by competition of binding of a neutralizing monoclonal antibody [15]. The current gold standard for measuring neutralizing antiHPV antibodies is a manually performed pseudovirion-based neutralization assay (manPBNA; [16]) using secreted alkaline phosphatase (SEAP) as reporter. Although infectious pseudovirions of different PV types can be easily produced, the manPBNA remains tedious and variable, restricting its applicability mainly to small sample numbers. Several arguments make a case for the need of a highthroughput neutralization assay with improved sensitivity: (i) requirement of larger serum sample numbers for follow-up studies on current vaccines, (ii) detection of cross-neutralizing antibodies induced by the commercial vaccines, and (iii) monitoring the effect of simplified vaccination schemes. Also, induction of neutralizing antibodies by second generation vaccines, e.g. based on the L2 protein needs to be assessed. Finally, large scale neutralization assays would allow addressing questions on naturally occurring protective immunity against HPV infections. Especially in respect to antibody responses against natural papillomavirus infections there are high demands for sensitivity and reproducibility in a neutralization assay. To date, high-throughput detection of HPV capsid-specific antibodies has been possible only with the aid of surrogate detection assays such as ELISA and competitive Luminex immunoassay (cLIA) using VLPs as antigen. Here we describe the adaptation of the PBNA to a high-throughput (HT) setting. We developed a purely add-on system in which the serial dilution of serum samples is separated from the cell-based assay, providing a high degree of flexibility. The high-throughput assay demonstrates high robustness with little intra- and inter-assay day variability. Also, the HT-PBNA shows higher sensitivity compared to the manually performed assay using SEAP as reporter. In its current format, the neutralization titer of 110 serum samples for seven HPV types can be determined in a single run. The HTPBNA will allow the execution of larg (...truncated)