Microbiology Spectrum (Dec 2021)

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

  • Emelissa J. Valcourt,
  • Kathy Manguiat,
  • Alyssia Robinson,
  • Yi-Chan Lin,
  • Kento T. Abe,
  • Samira Mubareka,
  • Altynay Shigayeva,
  • Zoë Zhong,
  • Roxie C. Girardin,
  • Alan DuPuis,
  • Anne Payne,
  • Kathleen McDonough,
  • Zhen Wang,
  • Romain Gasser,
  • Annemarie Laumaea,
  • Mehdi Benlarbi,
  • Jonathan Richard,
  • Jérémie Prévost,
  • Sai Priya Anand,
  • Kristina Dimitrova,
  • Clark Phillipson,
  • David H. Evans,
  • Allison McGeer,
  • Anne-Claude Gingras,
  • Chen Liang,
  • Martin Petric,
  • Inna Sekirov,
  • Muhammad Morshed,
  • Andrés Finzi,
  • Michael Drebot,
  • Heidi Wood

DOI
https://doi.org/10.1128/Spectrum.00886-21
Journal volume & issue
Vol. 9, no. 3

Abstract

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ABSTRACT 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. IMPORTANCE 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.

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