Optimization and validation of a plaque reduction neutralization test for the detection of neutralizing antibodies to four serotypes of dengue virus used in support of dengue vaccine development

Abstract

A dengue plaque reduction neutralization test (PRNT) to measure dengue serotype-specific neutralizing antibodies for all four virus serotypes was developed, optimized, and validated in accordance with guidelines for validation of bioanalytical test methods using human serum samples from dengue-infected persons and persons receiving a dengue vaccine candidate. Production and characterization of dengue challenge viruses used in the assay was standardized. Once virus stocks were characterized, the dengue PRNT(50) for each of the four serotypes was optimized according to a factorial design of experiments approach for critical test parameters, including days of cell seeding before testing, percentage of overlay carboxymethylcellulose medium, and days of incubation post-infection to generate a robust assay. The PRNT(50) was then validated and demonstrated to be suitable to detect and measure dengue serotype-specific neutralizing antibodies in human serum samples with acceptable intra-assay and inter-assay precision, accuracy/dilutability, specificity, and with a lower limit of quantitation of 10.

Figure 1.

Dengue virus growth kinetics. The dengue virus plaque assay titers in plaque-forming units (PFU)/mL are plotted against the harvest days post-infection for each dengue virus (DENV) serotype, illustrating the viral growth kinetics. The concentration of dengue virus steadily increased from day 4 to day 8 post-infection, with a maximum titer in the 10⁷ PFU/mL range at days 6–8 post-infection.

Figure 2.

Morphology of dengue virus-1 (DENV-1), DENV-2, DENV-3, and DENV-4 plaques. Immunostained DENV-1 (A), DENV-2 (B), DENV-3 (C), and DENV-4 (D) viral plaques developed after incubation with dengue serotype-specific monoclonal antibodies to the envelope protein followed by alkaline phosphatase–conjugated anti-mouse IgG (secondary antibodies) and insoluble nitro-blue tetrazolium/5-bromo-4-chloro-3′-indolyphosphate substrate. The first three wells were infected with the challenge virus dose and the fourth well was the uninfected cell control.