Evaluating Humoral Immunity against SARS-CoV-2: Validation of a Plaque-Reduction Neutralization Test and a Multilaboratory Comparison of Conventional and Surrogate Neutralization Assays

• Published in: Microbiology spectrum Vol. 9; no. 3; p. e0088621
• Main Authors: Valcourt, Emelissa J, Manguiat, Kathy, Robinson, Alyssia, Lin, Yi-Chan, Abe, Kento T, Mubareka, Samira, Shigayeva, Altynay, Zhong, Zoë, Girardin, Roxie C, DuPuis, Alan, Payne, Anne, McDonough, Kathleen, Wang, Zhen, Gasser, Romain, Laumaea, Annemarie, Benlarbi, Mehdi, Richard, Jonathan, Prévost, Jérémie, Anand, Sai Priya, Dimitrova, Kristina, Phillipson, Clark, McGeer, Allison, Gingras, Anne-Claude, Liang, Chen, Petric, Martin, Sekirov, Inna, Morshed, Muhammad, Finzi, Andrés, Drebot, Michael, Wood, Heidi
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 22-12-2021
• Subjects: Angiotensin-Converting Enzyme 2; Animals; Antibodies, Neutralizing; Antibodies, Viral - immunology; Chlorocebus aethiops; Clinical Microbiology; COVID-19; COVID-19 - diagnosis; COVID-19 - immunology; COVID-19 Serological Testing - methods; COVID-19 Vaccines - immunology; Diagnostic Tests, Routine; Enzyme-Linked Immunosorbent Assay - methods; HEK293 Cells; Humans; Immunity; Immunity, Humoral - immunology; immunoserology; Neutralization Tests - methods; neutralizing antibodies; Research Article; SARS-CoV-2; SARS-CoV-2 - immunology; SARS-CoV-2 - isolation & purification; Sensitivity and Specificity; Vero Cells; COVID-19; immunoserology; neutralizing antibodies; SARS-CoV-2; immunity
• ISSN: 2165-0497; 2165-0497
• DOI: 10.1128/Spectrum.00886-21

The evaluation of humoral protective immunity against SARS-CoV-2 remains crucial in understanding both natural immunity and protective immunity conferred by the several vaccines implemented in the fight against COVID-19. The reference standard for the quantification of antibodies capable of neutralizing SARS-CoV-2 is the plaque-reduction neutralization test (PRNT). However, given that it is a laboratory-developed assay, validation is crucial in order to ensure sufficient specificity and intra- and interassay precision. In addition, a multitude of other serological assays have been developed, including enzyme-linked immunosorbent assay (ELISA), flow cytometry-based assays, luciferase-based lentiviral pseudotype assays, and commercially available human ACE2 receptor-blocking antibody tests, which offer practical advantages in the evaluation of the protective humoral response against SARS-CoV-2. In this study, we validated a SARS-CoV-2 PRNT to assess both 50% and 90% neutralization of SARS-CoV-2 according to guidelines outlined by the World Health Organization. Upon validation, the reference-standard PRNT demonstrated excellent specificity and both intra- and interassay precision. Using the validated assay as a reference standard, we characterized the neutralizing antibody response in specimens from patients with laboratory-confirmed COVID-19. Finally, we conducted a small-scale multilaboratory comparison of alternate SARS-CoV-2 PRNTs and surrogate neutralization tests. These assays demonstrated substantial to perfect interrater agreement with the reference-standard PRNT and offer useful alternatives to assess humoral immunity against SARS-CoV-2.
SARS-CoV-2, the causal agent of COVID-19, has infected over 246 million people and led to over 5 million deaths as of October 2021. With the approval of several efficacious COVID-19 vaccines, methods to evaluate protective immune responses will be crucial for the understanding of long-term immunity in the rapidly growing vaccinated population. The PRNT, which quantifies SARS-CoV-2-neutralizing antibodies, is used widely as a reference standard to validate new platforms but has not undergone substantial validation to ensure excellent inter- and intraassay precision and specificity. Our work is significant, as it describes the thorough validation of a PRNT, which we then used as a reference standard for the comparison of several alternative serological methods to measure SARS-CoV-2-neutralizing antibodies. These assays demonstrated excellent agreement with the reference-standard PRNT and include high-throughput platforms, which can greatly enhance capacity to assess both natural and vaccine-induced protective immunity against SARS-CoV-2.