PF-1 The HPV serology standardization initiative: Development of critical assay reagents
Authors: Schafer Elizabeth , Kemp Troy J. , Alabanza Casper , Richards Angelina C. , Pinto Ligia A. 
Published: April 2019

Introduction:
Human Papillomavirus (HPV) has been linked to numerous cancers including cervical, oropharyngeal, and additional anogenital cancers, and is believed to be associated with approximately 5% of cancers world-wide. Current licensed vaccines have shown great promise in being able to reduce persistent infection and HPV-linked cancers, and are based on the use of virus-like particles (VLPs) which are comprised of type-specific HPV L1-expressing empty capsids. While commendable strides have been made within the HPV research community to focus on understanding how the HPV vaccines work and eradicating this cancer-causing virus, there is still a lack of assay standards, reference reagents and testing guidelines for HPV serology assays. Our laboratory is leading an international HPV Serology standardization initiative to develop a series of standards, multiplex immunoassays, and procedures in order to harmonize evaluation of antibody responses to currently available vaccines in the context of HPV vaccine trials. To reduce costs associated with serology testing, we investigated performance of alternative transfection reagents typically used in critical reagent production.

Discussion:
Currently licensed HPV vaccines are based on HPV L1 virus-like particles. HPV VLPs are used extensively in HPV research and as critical reagents in the assessment of HPV-induced immune responses. There are a variety of available methods to create HPV VLPs for use in the research setting; however, transfection methods are expensive and time-consuming. Our team is currently producing HPV VLPs for the 9 HPV types included in the currently administered vaccines to be used as assay reference reagents by the serology scientific community to aid in standardization and harmonization of HPV serology testing in vaccine trials. With the aim of reducing costs and saving time in VLP production while maintaining robust HPV particle assembly, we have investigated different transfection approaches in a mammalian-based HPV L1L2 VLP production system.
Aspects of consideration include transfection reagents and efficiency, cell viability, materials, and method optimization in the context of final VLP output via HEK293TT transfection with pHPVL1L2. The VLPs are subsequently confirmed by electron microscopy and dynamic light scattering as well as epitope confirmation assays. Results demonstrate that cost-efficient reagents such as PEI and Transporter5 are as efficient as Lipofectamine in producing particles. Similarly, adaptation of various transfection methods and use of large-scale production techniques lead to faster production time with similar output.

Conclusions:
The development of HPV L1-based standardized reagents and immunoassays allow research laboratories to better evaluate and compare the vaccine-induced immune response across clinical trials. VLPs, the cornerstone of the current vaccine, are used to develop immunoassays and standards by which researchers can gauge the immune response. By reducing costs and time to produce VLPs, it becomes more feasible to create large production sets of VLPs in less time and with less financial burden for immune monitoring laboratories.
Funded by NCI Contract No. HHSN261200800001E.