ABSTRACT
To date, thousands of SARS-CoV-2 samples from many vaccine developers have been tested within the CEPI-Centralized Laboratory Network. To convert data from each clinical assay to international standard units, the WHO international standard and the CEPI standard generated by the Medicines and Healthcare products Regulatory Agency were run in multiple facilities to determine the conversion factor for each assay. Reporting results in international units advances global understanding of SARS-CoV-2 immunity and vaccine efficacy, enhancing the quality, reliability, and utility of clinical assay data.
Efforts in the CEPI-Centralized Laboratory Network during the SARS-CoV-2 pandemic
During the SARS-CoV-2 pandemic, the absence of standardization in assay design, test platforms, viral targets, instrumentation, staff expertise, and quality systems posed a significant challenge. Therefore, in March 2020, the Coalition for Epidemic Preparedness Innovations (CEPI) established the Centralized Laboratory Network (CLN), the first and largest laboratory network dedicated to supporting vaccine clinical trials worldwide.Citation1 Two of the CEPI-CLN laboratories partners (Nexelis, a Q2 Solutions Company in Canada (hereafter referred to as “Nexelis-Q2 Solutions”) and the UK Health Security Agency: UKHSA, UK) developed three enzyme-linked immunosorbent assay (ELISA)(Spike (S), Receptor Binding Domain (RBD) and Nucleocapsid (N)), a live microneutralization assay (MNA), a pseudotyped virus-based neutralization assay (PNA), and an IFN-γ/IL-5 T-cell enzyme-linked immunosorbent spot (ELISpot) to assess the immunogenicity of candidate SARS-CoV-2 vaccines globally, ensuring the standardization, harmonization, and highest quality.Citation2,Citation3 To transfer harmonized and standardized assays across all facilities, the receiving laboratories within CEPI-CLN engaged in a series of activities such as conducting gap analyzes at each facility, implementing identical protocols and guidelines across all facilities, using the same key reagents and controls, conducting inter-laboratory studies, complement validation, and more.Citation2,Citation3 Generating the same key reagents, particularly reference standard and controls for assays across CEPI-CLN facilities, was one of the most important parameters in standardizing and harmonizing assays.
To generate well-characterized traceable reference materials, CEPI partnered with the Medicines and Healthcare products Regulatory Agency (MHRA, former NIBSC) and the World Health Organization (WHO) to develop an international standard (IS) for anti-SARS-CoV-2 immunoglobulin (NIBSC code 20/136). Due to the high global demand for this international standard and the testing of thousands of SARS-CoV-2 samples, this standard became depleted within 8 months, leading to the development of the second WHO IS (NIBSC code 21/340) for anti-SARS-CoV-2 immunoglobulins. The standard is a pool of plasma from seven COVID-19 convalescent individuals, infected with SARS-CoV-2 in early 2020.
Given our extensive involvement in testing a large number of clinical samples from various SARS-CoV-2 vaccine developers, we deemed it necessary to establish a specific reference standard for all our facilities. In line with this, the MHRA, a member of the CEPI-CLN network (https://cepi.net/news_cepi/two-more-laboratories-join-cepis-centralised-network-to-standardise-assessment-of-covid-19-vaccines/) produced the CEPI working standard 21/360, consisting of pooled plasma from fourteen individuals recovered from SARS-CoV-2, tailored for the CEPI-CLN. Inclusion of the CEPI working reagent 21/360 in all the assays (except ELISpot as no reference standard has been used) by all the laboratories in the network allows for consistency and harmonization of the results. To extend the comparability of data generated by CEPI-CLN to a global level, the 2nd WHO IS 21/340 was run in multiple labs in the network to generate conversion factors for the antibody-based assays to report results in the IS units, BAU/mL for binding activity and IU/mL for neutralization activity. Five facilities across diverse geographical regions were chosen based on their successful transfer of validated assays and active engagement in clinical testing. These included Nexelis/Q2 Solutions in Canada and UKHSA in UK, alongside three other facilities – Vismederi in Italy, Translational Health Science and Technology Institute (THSTI) in India, and the International Centre for Diarrheal Disease Research (Icddr,b) in Bangladesh.
Conversion factors for assaying immune responses in the CEPI-Centralized Laboratory Network
For the assessment of immune responses in clinical samples, CEPI-CLN reported S, RBD and N- ELISA in ELISA Laboratory Units (ELU/mL) extrapolated from the CEPI working standard 21/360, and neutralization activity for MNA and PNA was reported as 50% neutralization dilution (ND50) and 50% neutralization titer (NT50), respectively.
To determine a conversion factor for each of our binding assays (S, RBD, and N), the heat-inactivated WHO IS 21/340 and the CEPI 21/360 standard were tested in triplicate (from independent dilution preparations) on the same plate by at least two different analysts over different days (the values generated for the CEPI standard 21/360 were used for only informational purposes). A total of 90 results for the S-ELISA, 90 results for RBD-ELISA, and 66 results for the N-ELISA from various laboratories were obtained to calculate the geometric mean concentration. For the calculation of the conversion factor from the arbitrary units (ELU/mL) to BAU/mL, the measured WHO IS 21/340 concentration in ELU/mL was divided by the assigned unitage of WHO IS 21/340 in BAU for the S, RBD, or N viral target as defined in MHRA instructions for use (ifu: (https://nibsc.org/documents/ifu/21–340.pdf). Therefore, to convert data, ELU/mL values should be divided by the conversion factor, using the equation: Result (BAU/mL) = Result (ELU/mL)/conversion factor. For example, the geometric mean concentration of the WHO IS 21/340 for the Spike-SARS-CoV-2 was calculated as 4540.2 ELU/mL. This value was subsequently divided by the concentration of 652 BAU/mL, as defined in the MHRA ifu, resulting in a final conversion factor of 6.9635. Therefore, clinical sample results generated in ELU/mL at CEPI-CLN can be reported in BAU/ml by dividing them by 6.96.
To determine conversion factors for both the MNA (Victoria strain) and PNA, we employed a similar approach to that used in the binding assay. A total of 59 and 58 values of the WHO IS 21/340 were generated for the MNA and PNA, respectively. Furthermore, to establish the conversion factor for the MNA, heat-inactivated WHO IS 21/340 was tested against the wild type (Victoria) SARS-CoV-2, while the PNA utilized generated wild-type SARS-CoV-2 pseudoparticles with a VSV backbone. The assessed geometric mean ND50 for the MNA and NT50 titer for the PNA were divided by their concentration (1424 IU/mL as per MHRA ifu) to calculate the final conversion factors for each assay. These resulted in 2.46 for the MNA and 2.77 for the PNA. Therefore, to convert data, ND50 or NT50 values should be divided by the conversion factor, using the equation: Result (IU/mL) = Result (ND50 titer or NT50 titer)/conversion factor.
All facilities had to ensure that the controls and the reference standards met the acceptance criteria according to each CEPI laboratory’s established SOP for all the SARS-CoV-2 assays. The raw data for each assay was analyzed using SoftMax Pro GxP software (SMP). The SMP files were saved in a secure location at the Laboratory within the CLN which generated the data. The obtained conversion factors for all binding assays (S, RBD, and N) and neutralization assays (MNA Victoria strain and PNA) are presented in .
Table 1. A summary of the data obtained from all facilities for the WHO IS 21/340 is presented. The conversion factor for WHO IS 21/340 was determined for the Victoria strain.
Throughout the past 3 years, CEPI-CLN has meticulously analyzed approximately 130,000 clinical samples sourced from over 60 clinical trials, including several WHO-supported Phase I-III trials. This extensive endeavor highlights the necessity of ensuring a standardized and internationally recognized approach to evaluating assay results. Overall, the implementation of a shared working standard enhances the quality, reliability, and utility of clinical assay data, and reporting of the results in WHO IS units allows for advancement of the global understanding of SARS-CoV-2 immunity and vaccine efficacy.
Concluding remarks
The efforts of the CEPI-CLN in standardizing and harmonizing immunological assays during the SARS-CoV-2 pandemic have significantly advanced the understanding of immunity, vaccine efficacy, and supported the approval of a few vaccine candidates. Through the development of standardized assays and the utilization of conversion factors to report data in international units, the network has ensured the quality, reliability, and comparability of clinical assay data. The commitment to standardization and collaboration demonstrated by the CEPI-CLN sets a valuable precedent for future pandemic preparedness and response efforts.
Acknowledgments
We extend our gratitude to the dedicated staff at MHRA, Vismederi, THSTI, Icddr,b, and Gorman Consulting for their invaluable contributions in performing the assays. We would also like to express our appreciation to Dr Mark Manak, former consultant at CEPI; Mr Vijay Zala, the project manager at CEPI-CLN; Ms Janina Hutchens, the project coordinator at CEPI-CLN, and Dr Peter Spencer, the quality manager at CEPI-CLN, for their support throughout this study.
Disclosure statement
No potential conflict of interest was reported by the author(s).
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References
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