Diseases (Jan 2024)

Plaque Reduction Neutralization Test (PRNT) Accuracy in Evaluating Humoral Immune Response to SARS-CoV-2

  • Ingrid Siciliano Horbach,
  • Adriana de Souza Azevedo,
  • Waleska Dias Schwarcz,
  • Nathalia dos Santos Alves,
  • Brenda de Moura Dias,
  • Bruno Pimenta Setatino,
  • Luma da Cruz Moura,
  • Ariane Faria de Souza,
  • Caio Bidueira Denani,
  • Stephanie Almeida da Silva,
  • Thiago Goes Pimentel,
  • Victor de Oliveira Silva Ferreira,
  • Tamiris Azamor,
  • Ana Paula Dinis Ano Bom,
  • Maria da Penha Gomes Gouvea,
  • José Geraldo Mill,
  • Valéria Valim,
  • Jessica Polese,
  • Ana Carolina Campi-Azevedo,
  • Vanessa Peruhype-Magalhães,
  • Andréa Teixeira-Carvalho,
  • Olindo Assis Martins-Filho,
  • Sheila Maria Barbosa de Lima,
  • Ivanildo Pedro de Sousa Junior

DOI
https://doi.org/10.3390/diseases12010029
Journal volume & issue
Vol. 12, no. 1
p. 29

Abstract

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Massive vaccination positively impacted the SARS-CoV-2 pandemic, being a strategy to increase the titers of neutralizing antibodies (NAbs) in the population. Assessing NAb levels and understanding the kinetics of NAb responses is critical for evaluating immune protection. In this study, we optimized and validated a PRNT50 assay to assess 50% virus neutralization and evaluated its accuracy to measure NAbs to the original strain or variant of SARS-CoV-2. The optimal settings were selected, such as the cell (2 × 105 cells/well) and CMC (1.5%) concentrations and the viral input (~60 PFU/well) for PRNT-SARS-CoV-2 with cut-off point = 1.64 log5 based on the ROC curve (AUC = 0.999). The validated PRNT-SARS-CoV-2 assay presented high accuracy with an intraassay precision of 100% for testing samples with different NAb levels (low, medium, and high titers). The method displays high selectivity without cross-reactivity with dengue (DENV), measles (MV), zika (ZIKV), and yellow fever (YFV) viruses. In addition, the standardized PRNT-SARS-CoV-2 assay presented robustness when submitted to controlled variations. The validated PRNT assay was employed to test over 1000 specimens from subjects with positive or negative diagnoses for SARS-CoV-2 infection. Patients with severe COVID-19 exhibited higher levels of NAbs than those presenting mild symptoms for both the Wuhan strain and Omicron. In conclusion, this study provides a detailed description of an optimized and validated PRNT50 assay to monitor immune protection and to subsidize surveillance policies applied to epidemiologic studies of COVID-19.

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