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

• Published in: Diseases Vol. 12; no. 1; p. 29
• Main Authors: Horbach, Ingrid Siciliano, de Souza Azevedo, Adriana, Schwarcz, Waleska Dias, Alves, Nathalia Dos Santos, de Moura Dias, Brenda, Setatino, Bruno Pimenta, da Cruz Moura, Luma, de Souza, Ariane Faria, Denani, Caio Bidueira, da Silva, Stephanie Almeida, Pimentel, Thiago Goes, de Oliveira Silva Ferreira, Victor, Azamor, Tamiris, Ano Bom, Ana Paula Dinis, da Penha Gomes Gouvea, Maria, Mill, José Geraldo, Valim, Valéria, Polese, Jessica, Campi-Azevedo, Ana Carolina, Peruhype-Magalhães, Vanessa, Teixeira-Carvalho, Andréa, Martins-Filho, Olindo Assis, de Lima, Sheila Maria Barbosa, de Sousa Junior, Ivanildo Pedro
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 01.01.2024
• Subjects: Accuracy; Antibodies; COVID-19; COVID-19 vaccines; Cross-reactivity; Epidemiology; Evaluation; Health aspects; Immune response; Immune response (humoral); Infections; Mutation; neutralizing antibodies; Omicron; PRNT; SARS-CoV-2; Serodiagnosis; Serology; Severe acute respiratory syndrome coronavirus 2; Vaccination; Viral infections; Viruses; Wuhan; Yellow fever; Brazil; United States > US; Rio de Janeiro Brazil; COVID-19; SARS-CoV-2; Wuhan; neutralizing antibodies; Omicron; PRNT; validation; vaccination
• ISSN: 2079-9721; 2079-9721
• DOI: 10.3390/diseases12010029

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 PRNT 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 × 10 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 log 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 PRNT assay to monitor immune protection and to subsidize surveillance policies applied to epidemiologic studies of COVID-19.