The HPV Serology Laboratory leads an initiative to standardize and harmonize human papillomavirus serology assays

Abstract

The HPV Serology Laboratory is leading a global partnership initiative aiming for standardization and harmonization of current serology assay platforms being used to assess immune responses to HPV vaccines. Serology standardization is particularly important given the increasing number of immunobridging trials relying on serology data for approval of new vaccine dosing schedules or vaccine formulations. The initiative was established in 2017 to enable comparisons of data between different vaccines and relevant studies as well as expedite the implementation of new vaccines and vaccine indications. The HPV Serology Laboratory has held or attended several meetings with partnering laboratories, including international meetings in 2017, 2018, and 2021.

Manuscript

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.

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

	Title	Version
10023	Good Documentation Practices	1.0
15006	Reagent Preparation	4.0
Forms:	* 15006–01_Reagent Preparation 15006–02_HPV Plate Coating Form	4.0
15010	Equipment Qualification and Calibration	2.0
Forms:	15010–01: Equipment Calibration Documentation Form
15013	Rounding and Significant Figures	1.0
20000	Bacterial Culture Maintenance	3.0
Forms:	20000–01_Bacterial Plasmid Culture Form
20001	HEK293TT Cell Culturing and Maintenance	7.0
Forms:	20001–02_293TT Cell Thaw Form 20001–03_Cell Culture Maintenance Form 20001–04_293TT Cell Culture Freezing Form
20002	Preparation of Bacterial Glycerol Stock for Plasmid Purification	1.0
Forms:	20002–01_Preparation of Bacterial Glycerol Stock for Plasmid Purification Form
20003	Procedure for Separating Serum and Plasma from Whole Blood	1.0
Forms:	20003–01_Serum and Plasma Separation Procedure Form
20005	Plasmid DNA Transfection in HEK293TT for VLP Production and Purification	5.0
Forms:	20005–01: HEK293TT Transfection Form, Day 1–4 20005–02: HEK293 Transfection Form, Day 20005–03: Fraction Pool Form
20006	Ripcord pDNA Transfection in HEK293TT for Pseudoviruses Production and Purification	1.0
Forms:	20006–01_HEK293TT Transfection Form, Day 1–4 20006–02_HEK293TT Transfection Form, Day 5 20006–03_Fraction Pool Form
20007	Bacterial Transformation	1.0
20010	Plasmid Purification Using a Zymo Research Kit	1.0
Forms:	20010–01_ZymoPure II Maxiprep Kit Plasmid Purification Form
20011	HPV VLP Conjugation to Luminex-Based Microspheres	1.0
Forms:	HPV VLP Coupling to Luminex Magnetic Beads Data Capture Form
26000	Biosafety Cabinet (BSC) Use and Maintenance	2.0
Forms:	26000–01_BSC Daily Use Form 26000–02_BSC Maintenance Form
26001	Operation, Use and Maintenance of CO2 Incubators	3.0
Forms:	26001–01_CO2 Incubator Weekly Maintenance Form 26001–02_CO2 Incubator Maintenance form
26002	Use and Maintenance of a BioTek Plate Washer	4.0
Forms:	26002–01_Microplate Washer Carryover Check Form 26002–02_Plate Washer Maintenance Form 26002–03: Plate Washer Daily Use Maintenance Form
26003	Use and Maintenance of a Molecular Devices M5 Plate Reader	3.0
Forms:	26003–01_Molecular Devices Plate Reader Monthly Maintenance Form 26003–02_Molecular Devices Plate Reader Plate Calibration Form
26004	Use and Maintenance of the Cellometer Cell Counter	4.0
Forms:	26004–01_Cellometer Monthly Maintenance Form 26004–02_Cellometer Monthly Quality Check Form
26005	Use and Maintenance of a 2-8C Refrigerator	4.0
Forms:	26005–01_Rrefrigerator Maintenance Form 26005–02_Rrefrigerator Temperature Monitor Log Form
26006	Use and Maintenance of the Liquid Nitrogen Freezer	3.0
Forms:	26006–01_Liquid Nitrogen Freezer Maintenance Form
26007	Use and Maintenance of the Water Bath	2.0
Forms:	26007–01_Waterbath Monthly Maintenance Form
26008	Use and Maintenance of Incubator Shakers	2.0
Forms:	26008–01_Maintenance of Incubator Shaker Form
26009	Use and Maintenance of Pipettes	1.0
Forms:	26009–01_Pipette Accuracy Confirmation Form
26010	Use and Maintenance of a Thermomixer	4.0
Forms:	26010–01_Thermomixer Maintenance Form
26011	Use and Maintenance of a pH Meter	3.0
Forms:	26011–01_pH Daily Use Calibration Form 26011–02_pH Maintenance Form
26012	Use and Maintenance of an Analytical & Precision Balance	3.0
Forms:	26012–01_Analytical Balance Maintenance Form 26012–02_Precision Balance Maintenance Form
26013	Use and Maintenance of -20C Freezers	3.0
Forms:	26013–01_-20°C Freezer Maintenance Form 26013–02_-20°C Freezer Temperature Monitor Log Form
26014	Use and Maintenance of a Laboratory Convection Incubators	3.0
Forms:	26014–01_Convection Incubator Maintenance Form 26014–02_Convection Incubator Weekly Maintenance Form
26015	Use and Maintenance of an Inverted Microscope	3.0
Forms:	26015–01_Inverted Microscope Use and Maintenance Form
26016	Operation, Use and Maintenance of the Water Purification Systems	1.0
Forms:	26016–01_Maintenance of the Water Purification Systems Form
26017	Use and Maintenance of the Eppendorf microcentrifuge	3.0
Forms:	26017–01_Eppendorf Microcentrifuge Maintenance Form
26018	Use and Maintenance of NanoDrop Spectrophotometers	3.0
Forms:	26018–01_ NanoDrop 1000 Spectrophotometer Maintenance 26018–02_ NanoDrop One c Spectrophotometer Maintenance Form 26018–03_NanoDrop Spectrophotometer Calibration Check Form
26019	Controlled-Rate Cell Freezing Using a CoolCell Device	2.0
Forms:	26019–01_CoolCell Maintenance Form
26020	Use and Maintenance of a Microplate Shaker	3.0
Forms:	26020–01_Microplate Shaker Maintenance Form
26021	Use and Maintenance of the Optima XPN Ultracentrifuge System	2.0
Forms:	26021–01_Optima XPN Ultracentrifuge Use Form 26021–02_Optima XPN Ultracentrifuge Maintenance Form
26022	Use and Maintenance of the Combination Refrigerator and Freezer	2.0
Forms:	26022–01_Combination Refrigerator and Freezer Use and Maintenance Form
26023	Use and Maintenance of the Agilent Bioanalyzer	2.0
Forms:	26023–01_Bioanalyzer Maintenance Form
26024	FlexMap Use and Maintenance_v. 1.0	1.0
Forms:	26024–01_Use and Weekly Maintenance of the FLEXMAP 3D 26024–02_FLEXMAP 3D Performance Maintenance Form
26025	Use and Maintenance of LI-COR Imager	1.0
Forms:	26025–01_LI-COR Maintenance Form
26027	Use and Maintenance of the FYRITE Gas Analyzer	1.0
Forms:	26027–01_Maintenance of the FYRITE Gas Analyzer Form
26028	General Use and Maintenance of the MESO QuickPlex SQ 120 Instrument	1.0
Forms:	26028–01_MESO QuickPlex SQ 120 Instrument Monthly Maintenance Form
26029	Verification and Use of Electronic Thermometer	1.0
Forms:	26029–01_Thermometer Maintenance Form
26030	Use and Maintenance of -80C Freezers	3.0
Forms:	26030–01_-80C Freezer Maintenance Form
26031	epMotion 96 Use and Maintenance	1.0
Forms:	26031–01_epMotion 96 Maintenance Form
26033	Use and Maintenance of the Thermo Fisher Sorvall XTR Centrifuge	4.0
Forms:	26033–01_Sorvall XTR Centrifuge Maintenance Form
26034	General Use and Maintenance of a Fume Hood	1.0
Forms:	26034–01_Fume Hood Maintenance Form
26035	Verification and Use of Laboratory Timers	1.0
Forms:	26035–01_Timer Calibration Verification Form	2.0
26037	Use and Maintenance of the Gel Tank and Power Supply_v.1.0_Employee	1.0
Forms:	Gel Tank and Power Supply Maintenance Form
30000	HPV Neutralization Assay for Titer Determination	5.0
Forms:	30000–01_HPV Neutralization Assay, Sample Preparation Form 30000–02_HPV Neutralization Assay, Substrate Development Form 30000–03_HPV Neutralization Assay, Titer Substrate Development Form
30001	HPV Antibody ELISA	4.0
Forms:	30001–01_HPV_Antibody_ELISA_Data_Capture_Form 30001–02_HPV_Standard_Preparation_Form
30002	HPV-16 and HPV-18 Avidity ELISA	1.0
	HPV Avidity ELISA Form	1.0
30009	BCA Protein Assay	4.0
Forms:	30009–01_BCA Data Capture Form
30010	Acrylamide Protein Gel Analysis of HPV Virus-Like Particles (VLPs)	6.0
Forms:	30010–01: Acrylamide Protein Gel Data Capture Form
30011	Protein Analysis of Virus-Like Particles (VLPs) Using the Agilent 2100 Bioanalyzer	3.0
Forms:	30011–01_Bioanalyzer Data Capture Form
30012	ELISA to Determine VLP Specificity	3.1
Forms:	30012–01_ELISA to Determine VLP Specificity Data Capture Form
30013	HPV Multiplex Assay_Version 1.0	1.0
Forms:	30013–01_HPV Multiplex Assay Data Capture Form
40000	Identity and Access Management for LabVantage(IAM)	3.0
Forms:	40000–01_ LIMS_Request_for_System_Access_Form
40001	Introduction to LabVantage	1.0
40002	Performing HPV ELISA Testing Using LES Worksheet	1.0
40005	Prepare Mixed Consumables Using LES Worksheet	2.0
40006	Receiving and Managing Consumables	1.0
40007	Receiving and Managing Samples in LIMS	2.0
Forms:	40007–01_Sample Receipt Form
40008	Performing Plasmid DNA Production using LES Worksheet	1.0
40009	Performing Cell Maintenance using LES Worksheet	1.0
40010	Performing Plasmid DNA Transfection and VLP Purification using LES Worksheet	1.0
40011	Performing VLP Fraction analysis using LES Worksheet	1.0
40012	Performing VLP Fraction Pooling and final VLP lot analysis using LES Worksheet	1.0

*All forms are included in the above SOP and are provided for standardized documentation of experimental conditions and results.

All documents are available upon request and all hyperlinked/underlined SOPs are available at our website (https://frederick.cancer.gov/research/vaccine-immunity-and-cancer-directorate/hpv-serology-laboratory).

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].

Funding Statement

This project has been funded in part with Federal funds from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services, under Contract Numbers 75N91019D00024 and HHSN261200800001E. Bill and Melinda Gates Foundation in part funded this project, under Grant Agreement No. OPP1157239. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. The funders did not play any role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.