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To date, the licensed human papillomavirus (HPV) prophylactic vaccines have demonstrated robust immunogenicity and efficacy [1–4]. Although immune correlates of protection against HPV infection are unclear due to the exceptional efficacy of the vaccines, a growing body of preclinical and clinical data suggest HPV-specific neutralizing antibodies as the primary mechanism of protection [5–8]. Thus, HPV serology measurements are being proposed as endpoints in clinical trials evaluating immunogenicity and efficacy of modified regimens of the existing vaccines and follow-on products. Unfortunately, until recently there was a lack of standardization of assays, protocols, and reagents widely available and accessible to the scientific community for the assessment of immune responses to the vaccines.

Building on serology assay standardization and harmonization efforts initiated by the World Health Organization (WHO), HPV LabNet in 2006, a joint meeting with several institutions, organized by Ligia Pinto from Frederick National Laboratory for Cancer Research (FNLCR) and Elizabeth Unger from Centers for Disease Control and Prevention (CDC), was held in March 2016 to collectively recognize the growing need for standardization and harmonization. The participants of the meeting included the Bill & Melinda Gates Foundation, NCI, CDC, United States Food and Drug Administration (FDA), WHO, GlaxoSmithKline (GSK), Merck, Innovax, National Institute for Biological Standards and Control (NIBSC), Public Health England, and several US and international universities and research institutes.

Briefly, in response to the initial convention, the HPV Serology Laboratory (HPVSL) at FNLCR was established in January 2017 to address this challenge, in partnership with the National Cancer Institute and the Bill & Melinda Gates Foundation. A follow up meeting at the 32nd International Papillomavirus Conference in Sydney, Australia from October 2 to 6, 2018, was convened to discuss the progress of main aims, challenges, and some of the future needs. The outcomes of this meeting included definition of the primary mission of the initiative, which was to enable comparisons of data between different vaccines and different relevant studies, as well as expedite the implementation of new vaccines and vaccine indications. The specific aims to drive these goals include:

1. The development of a qualified secondary assay standard and a bank of serum specimens to use as assay proficiency panels for 9 HPV types (HPV-6/11/16/18/31/33/45/52/58) included in the licensed HPV vaccines.
2. The production of qualified reference reagents (HPV Virus-Like Particles) by establishing a set of criteria or guidelines for qualification.
3. The development of a validated multiplex serology assay to enable immunogenicity monitoring in HPV vaccine trials.
4. The promotion of the use of standards through platforms such as meetings, publications, data, and protocol sharing.

We herein briefly describe the progress to each of the 4 objectives as well as provide the links to all currently developed standard operating procedures (SOPs).

First, HPVSL was able to generate qualified secondary standards calibrated against HPV-16 and HPV-18 WHO International Standards (IS). For this effort, 144 human serum samples were obtained from various sample providers, and each sample was screened and categorized depending on the specific antibody response for each HPV type. Moreover, an HPV assay proficiency panel of 80 samples covering a wide range of HPV antibody responses was established. Currently, the proficiency panel has been distributed and tested by 10 laboratories with their respective antibody binding or pseudovirion-based neutralization assays.

Second, the production of the first lot of qualified VLPs for multiple HPV types (HPV-6/11/16/18/31/33/45/52/58) was completed at the time of the follow up meeting in 2018. These reagents will be available for reference use in assays in respective laboratories.

Third, a high-throughput Luminex bead-based 9-plex immunoassay was developed and a validation plan for the HPV-16 and HPV-18 ELISAs and multiplex assay was drafted. To date, the 9-plex assay validation has been completed and guidance documents for data processing and analyses are under development.

Lastly, efforts have been made to promote the development and use of standards, SOPs, and data (Table 1). Up to 8 material transfer agreements had been established at the time of the follow up meeting in 2018. Protocol and data sharing with the greater HPV serology community are now enabled by a dedicated FNL HPV Serology Laboratory website, where investigators and researchers can access serology laboratory SOPs, guidance documents for VLP production and qualification, and multiplex assay validation protocol.

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Table 1. List of HPV Serology Laboratory standard operating procedures documents and corresponding forms.

https://doi.org/10.1371/journal.ppat.1011403.t001

As part of this initiative, the HPVSL has been collaborating with NIBSC and WHO to develop International Standards for the remaining HPV types in the nonavalent vaccine, including HPV high-risk types (HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58) and low-risk types (HPV-6 and HPV-11). These standards were recently approved by WHO and are now available for request through NIBSC’s website (https://www.nibsc.org/products/brm_product_catalogue/sub_category_listing.aspx?category=Vaccines&subcategory=Human%20Papillomavirus).

Future directions discussed by the initiative consisted of (1) implementation of high-throughput HPV antibody testing using Good Clinical Laboratory Practices (GCLP) to support ongoing and future vaccine immunogenicity trials; and (2) development of an HPV Vaccine Trial Network of laboratories involved in the testing of HPV vaccines to expand training capability as well as establish an Assay Proficiency Panel Program.

In June 2021, a virtual meeting was held on June 29 to 30, 2021, to review progress of the standardization initiative thus far and to bridge scientific gaps and outstanding questions. The main aims and outcomes of the meeting were to discuss: (1) standardization of assays and reagents; (2) International Standard Calibration procedures; (3) assay cut-off values; (4) current immunobridging clinical trials; (5) gaps and challenges in standardization of HPV serology. A total of 119 scientists and clinicians from 13 countries, representing 36 institutions and industries, participated in the meeting to update the community on the progress on the objectives of the initiative. Participants also reported on advancements made by the HPV serology community in exploring and advancing with new vaccine dosing schedules or vaccine formulations. A report summarizing the meeting has been recently published [9].

Acknowledgments

We would like to thank Drs. Douglas Lowy, John Schiller, Sean Hanlon from NCI; Dr. Peter Dull from the Bill & Melinda Gates Foundation; Drs. Elizabeth Unger and Gitika Panicker from CDC; Dr. Dianna Wilkinson from NIBSC; Drs. Joakim Dillner and Helena Faust from Karolinska Institute; Dr. Simon Beddows from Public Health England; and Mr. Brian Plikaytis from Biostat Consulting, LLC. The authors would also like to thank our collaborators from academic institutions, vaccine industry laboratories, National Institutes for Food and Drug Control (NIFDC), and WHO.