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 PRNT50 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 PRNT50 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.

Transmission of Zika virus through breast milk and other breastfeeding-related bodily-fluids: a systematic review

“…4 compared to other flaviviruses [3]. Due to the possible cross reactivity with other members of the Flaviviridae family, the presence of IgM is not enough to rule out ZIKV infection, and the PRNT 90 will determine if the in vitro inhibition of cell growth is produced by antibodies against ZIKV [4,5]. An enzyme linked immunoassay (ELISA) for ZIKV has been developed by the Centers for Disease Control and Prevention, but is only available upon request for emergency use [6].…”

Lot-to-lot consistency of a tetravalent dengue vaccine in healthy adults in Australia: A randomised study

“…First, 76.8% of participants were eligible for inclusion in the per-protocol analysis while it had been assumed that 80% would be included. In addition, at the planning stages of the study, the intrinsic variability of the neutralising antibody assay was assumed to be 2, but was later determined to be 3 [25].…”

“…Dengue neutralising antibody titres were determined on the serum samples collected before the first injection on Day 0 and 28 days (per-protocol range 28 to 32 days) after the third injection using a 50% plaque reduction neutralisation test (PRNT 50 ) with parental dengue virus strains of the four CYD-TDV constructs as described previously (Sanofi Pasteur GCI, Swiftwater, USA) [24,25]. The assay had a lower limit of quantification titre of 10 (1/dilution): titres below the lower limit of quantification were assigned a value of 5.…”

Immunogenicity and safety of a tetravalent dengue vaccine during a five-year follow-up period

“…Baseline, post-vaccination and follow-up samples from the study were then tested with this single assay to generate a complete dataset, [12] as such the GMTs reported here for baseline and after the third vaccination differ from those reported previously [11].…”

“…Serum samples for immunogenicity analyses were taken at baseline, 28-days after each study injection and annually after the last injection. Serum neutralising antibody levels to each of the four parental wild-type dengue virus strains were determined using a 50% plaque reduction neutralisation test (PRNT 50 ) [12]. Exposure to wild-type dengue was defined as a 4-fold increase in PRNT 50 antibody titre (and with a resultant titre P40) for at least one serotype between two consecutive years (with no missing data for any serotype at both time points).…”