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

Figure 1. Schematic workflow representation of PRNT assay optimization steps and the tested parameters (viral input, cell, and CMC concentrations). The test begins with adding samples (15 µL) and their subsequent dilution (five-fold). Then, the SARS-CoV-2 virus is added (60, 70, or 100 PFU), followed by the first one-hour incubation at 37 °C, 5% CO2. Subsequently, the supernatant is transferred to 24-well plates containing the cell monolayer previously prepared (120,000 or 200,000 cells/well), followed by the second one-hour incubation at 37 °C, 5% CO2. The supernatant is removed, and the cell monolayer is overlayed by CMC (1.5, 2.0, or 2.5%). Afterward (3 days, 37 °C, 5% CO2), the formalin fixation (1.25%) is performed, and the plates are stained with 0.04% crystal violet solution. Figure designed at canva.com.

Figure 2. Representative images of infected or mock Vero cells, with the PRNT-SARS-CoV-2 procedure optimized. (A) Cell monolayer with 2 × 105 Vero cell/well (CC: cell control), resulting in regular-sized plaques (VC: virus control) and a good resolution. (B) Representative image of positive (PC) and negative (NC) controls of Vero cells infected with approximately 60 PFU of SARS-CoV-2.

Figure 3. SARS-CoV-2 neutralizing antibodies screening of 872 serum samples. The first and second columns show the samples considered negative (230) or positive (642). The dashed line indicates the first sera dilution (1:10 or 1.43 log5).

Figure 4. Serological panel titers (a) SARS-CoV-2 NAb levels were obtained using a sera panel composed of 40 samples (10 samples for each: negative, low, medium, and high titers). (b) Correlation of SARS-CoV-2 NAb levels obtained from a panel of 40 samples titrated by PRNT-SARS-CoV-2 in two independent assays performed on different days. Linear regression shows a positive correlation (r = 0.9655) that is statistically significant (p < 0.0001) between assay repetitions.

Figure 5. (a) ROC curve was calculated using sensitivity and 100-specificity to select the optimal cut-off point (14 in reciprocal dilution or 1.64 log5) for PRNT-SARS-CoV-2. The PRNT-SARS-CoV-2 assay showed 97.28% and 98.92% sensitivity and specificity, respectively. Area under the curve (AUC = 0.999) was statistically significant (p < 0.0001). (b) Distribution of neutralizing antibody titers against SARS-CoV-2 in the 424 samples (46 negative samples, first column; 378 positive, second column). The dashed line shows the PRNT cut-off point calculated with the ROC curve (1:14 or 1.64 log5).

Figure 6. Accuracy profile for the validation of PRNT-SARS-CoV-2: (a) obtained and expected values (log5) for each sera dilution. ND: not diluted. (b) Absolute differences (solid line with bullets) for all dilutions tested. All values obtained are below the acceptability limits (dashed lines) proposed, indicating a minimal variation between theoretical and experimental tests.

Figure 7. PRNT50 optimized and validated as a tool to evaluate immune response in individuals vaccinated to COVID-19, with or without natural SARS-CoV-2 infection. Distribution of NAb titers to SARS-CoV-2 Wuhan strain. The “before pandemic” group shows negative results for samples collected before the COVID-19 pandemic. The second group represents status before vaccination (baseline), and the third one shows the group immunized with two doses of ChAdOx1-S/nCoV-19 (AZD1222; AstraZeneca) vaccine; sera were analyzed 30 days after last immunization. Asterisks indicate a statistically significant difference (*** p < 0.001). The PRNT titer levels were expressed in log5. The dashed line shows the PRNT cut-off (1:14 or 1.64 log5) established in the validation process.

Figure 8. NAb titers to the original (Wuhan) and Omicron strains of the SARS-CoV-2 virus, separated by gender and COVID-19 severity. Asterisks indicate statistically significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001) as determined by paired t-test. The PRNT titer levels were expressed in log5. The dashed line shows the PRNT cut-off point (1:14 or 1.64 log5).