Utility of an In-Vitro Micro-Neutralizing Test in Comparison to a Plaque Reduction Neutralization Test for Dengue Virus, Japanese Encephalitis Virus, and Zika Virus Serology and Drug Screening

Abstract

Flavivirus infections, including dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV), present significant global public health challenges. For successful vaccine design, the assessment of neutralizing antibody activity requires reliable and robust methodologies for determining antibody titers. Although the plaque reduction neutralization test (PRNT) is commonly acknowledged as the gold standard, it has limitations in terms of time and cost, and its usage may be limited in resource-limited settings. To address these challenges, we introduced the micro-neutralization test (MNT) as a simplified alternative to the PRNT. The MNT employs a 96-well plate format, conducts microscale neutralization assays, and assesses cell viability by dissolving cells to create a uniform color solution, which is measured with a spectrometer. In this study, we evaluated the utility of the MNT by contrasting the end-point titers of the MNT and PRNT using 4 monoclonal antibodies, 15 non-human primate serum samples, and 2 therapeutic drug candidates across flaviviruses. The results demonstrated a strong correlation between the MNT and PRNT titers, affirming the robustness and reproducibility of the MNT for evaluating control measures against flaviviruses. This research contributes valuable insights toward the development of a cost-effective antibody titer testing approach that is particularly suitable for resource-limited settings.

Keywords: Japanese encephalitis virus; Zika; dengue; flavivirus; microneutralization test; neutralizing antibodies.

Figure 1

The procedure for the PRNT is presented in this flowchart: (A) entails the cultivation of Vero-9013 cells on a 12-well plate, incubated at 37 °C for 24 h in a pre-prepared medium, while (B) corresponds to the neutralization phase. The virus and serially diluted testing samples are pre-incubated on ice for 30 min. The sample virus mixtures are then introduced to the pre-cultured cells. After 1 h of virus adsorption, overlay medium is added and the plate is incubated for 5 to 7 days. The cells then undergo fixation with methanol, staining with methylene blue, and the formed plaques are counted by the naked eye.

Figure 2

The procedure for the MNT is presented in this flowchart: (A) entails the cultivation of Vero-9013 cells on a 96-well plate, incubated at 37 °C for 24 h in a pre-prepared medium, while (B) corresponds to the neutralization phase. The virus and serially diluted testing samples are pre-incubated under specified conditions (refer to the protocol for details). The sample virus mixtures are then introduced to the pre-cultured cells. Following 1 h of virus adsorption and 5–7 days of incubation, the cells undergo fixation with methanol, staining with crystal violet, dissolution with 1% SDS, and measurement of absorbance at 595 nm using a spectrometer.

Figure 3

Vero-9013 cells were exposed to varying virus titers of DENV-1, JEV, and ZIKV, ranging from 10 pfu/well to 10⁵ pfu/well using a 10-fold dilution series. OD_{595 nm} measurements were performed for the experimental group and control group, from which the percentage of viable cells was subsequently calculated. Each data point represents the geometric mean derived from three replicate measurements, with the error bars denoting the standard deviation among these replicates.

Figure 4

Ribavirin (A) and glycyrrhizin (B) were used to test the antiviral effects against DENV-1, JEV, and ZIKV through the MNT using Vero-9013 cells. Both ribavirin and glycyrrhizin were diluted in 2-folds ranging from 1000 $\mathsf{\mu }\mathrm{g}/\mathrm{m}\mathrm{L}$ to 0.975 $\mathsf{\mu }\mathrm{g}/\mathrm{m}\mathrm{L}$. The percent reduction is calculated by the OD_{595 nm} values of the experimental group, positive control group, negative group, and control group as shown in Equation (2). Each data point represents the geometric mean derived from six replicate measurements, with the error bars denoting the 95% confidence intervals among these replicates.

Figure 5

Correlation between the neutralizing titers measured using the MNT and PRNT. MNT₅₀ and PRNT₅₀ of 4 anti-flavivirus mouse monoclonal antibodies and 15 serum samples are plotted in a logistic scale, and the correlation between the two is calculated ($r=0.916737$), with a p value significantly smaller than 0.05. The 95% confidence interval of the linear regression is shown in the shaded area.