Tumorvirus-specific Vaccination Strategies

High-Throughput Pseudovirion-Based Neutralization assay for Human Papillomavirus

Need to measure HPV neutralizing antibodies

To study natural immunity to HPV but also to monitor humoral immune responses after prophylactic HPV vaccination, measurement of anti-L1 or anti-L2 antibodies in sera is of importance. A number of non-functional assays such as ELISA have been applied, but these assays are not able to discriminate neutralizing from non-neutralizing responses.The current ‘gold standard’ for measuring neutralizing anti-
HPV 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.  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.

Neutralization assay: principle

© dkfz.de

Papillomavirus pseudovirions are produced by transfection of expression plasmids encoding codon-adapted L1 and L2 genes together with a reporter plasmid encoding for gaussia luciferase. After extraction and purification, the pseudovirion can be used to transduce target cells. Luciferase activity is then a measure for transduction (A). In the presence of neutralizing antibodies, e.g. derived from human sera, the transduction will be inhibited, i.e. 'neutralized'.

© dkfz.de

HT-PBNA protocol. Assay plate preparation (A) and neutralization assay (B) are separated. In a first step, serial dilutions of serum samples are performed on one dilution plate. Identical assay plates are generated by transferring the dilutions to multiple replica-assay plates which can be stored. In the second step, the neutralization assay is carried out in a add-on format using the previously prepared assay plates. A) Assay plate preparation. Sera are transferred from a 96 well storage in SBS standard to a 384 well polypropylene V-bottom plate for serial dilution with a pipetting robot in cell culture medium. Finally the serially diluted sera are transferred with the same pipetting robot to each of 3*n white 384 well cell culture assay plates (n = number of PV-types). The plates are sealed immediately with a cover foil and stored at 220uC until their use in the PBNA. B) Assay assembly and read out. Assay plates are thawed and pseudovirions followed by reporter cells are added with a bulk dispenser. Three
identical assay plates originating from the same serum dilution plate are used for each PV- type. After 2 days of incubation the luminescence from the Gaussia reporter is read directly in the assay plates. Inhibition (%) is calculated by normalization of the luminescence to the mean of the negative control wells without serum present on each plate. 

Collaboration project with DKFZ-EMBL small molecule screening facility.

Sehr et al. Plos (One) 2013.

Publications using HT-PBNA

Immunogenicity and HPV infection following one, two and three doses of quadrivalent vaccine; Early results from a multi-center cohort study in India.
Rengaswamy Sankaranarayanan, Priya R. Prabhu,  Michael Pawlita, Tarik Gheit, Neerja Bhatla, Richard Muwonge, Bhagwan M. Nene, Pulikottil O. Esmy, Smita Joshi, Usha Rani Reddy Poli, Parimal Jivarajani, Yogesh Verma, Eric Zomawia, Maqsood Siddiqi, Surendra S. Shastri, Kasturi Jayant, Sylla G Malvi, Eric Lucas, Angelika Michel, Julia Butt, Janki Mohan Babu, Subha Sankaran, Kannan TR2, Rintu Varghese, Uma Divate, Shila Thomas, Geeta Joshi, Martina Willhauck-Fleckenstein, Tim Waterboer, Martin Müller, Peter Sehr, Sanjay Hingmire, Alka Kriplani, Gauravi Mishra, Sharmila Pimple, Radhika Jadhav, Catherine Sauvaget, Massimo Tommasino, and M. Radhakrishna Pillai for the Indian HPV vaccine study group.  (2015). Lancet Oncology doi;10.1016.S1470-2045(15)00414-3.

Human Papillomavirus neutralizing and cross-reactive antibodies induced in HIV-positive subjects after vaccination with quadrivalent and bivalent HPV vaccines.
Helena Faust; Lars Toft; Peter Sehr; Martin Müller; Jesper Bonde; Ola Forslund; Lars Østergaard; Martin Tolstrup; Joakim Dillner, M.D. (2016). Vaccine. in press

Concordance assessment between a multiplexed competitive Luminex immunoassay, a multiplexed IgG Luminex immunoassay, and a pseudovirion-based neutralization assay for detection of human papillomaviruses types 16 and 18.
Brown D, Müller M, Sehr P, Pawlita M, Seitz H, Rubio I, Antonello J, Radley D, Roberts C, Saah A. (2014) Vaccine. 7; 5880-5887

Comparison of the Immunogenicity of Cervarix® and Gardasil® Human Papillomavirus Vaccines for oncogenic non-Vaccine Serotypes HPV-31, HPV-33 and HPV-45 in HIV-infected Adults.
Lars Toft, Martin Tolstrup, Martin Müller,Peter Sehr, Jesper Bonde, Merete Storgaard, Lars Østergaard and Ole S. Søgaard (2014)

Comparison of the Immunogenicity and Reactogenicity of Cervarix® and Gardasil® Human Papillomavirus Vaccines in HIV-infected Adults; A Randomized, Double-Blind, Clinical Trial. 
Toft L, Storgaard M, Müller M, Sehr P, Bonde J, Tolstrup M, Ostergaard L, Søgaard OS (2013) J Infect Dis. 2013 Dec 26

High-Throughput Pseudovirion-Based Neutralization Assay for Analysis of Natural and Vaccine-Induced Antibodies against Human Papillomaviruses
Peter Sehr, Ivonne Rubio, Hanna Seitz, Kerstin Putzker, Lis Ribeiro-Müller, Michael Pawlita, Martin Müller (2013) PLoS ONE 8(10); e75677. doi;10.1371.journal.pone.0075677

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