EFB Dengue Literature Review

PRNT

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PRNT

Overview

The plaque reduction neutralization test (PRNT) is the gold standard functional assay for measuring virus-neutralizing antibody titers in serum. In this assay, serial dilutions of heat-inactivated patient serum are incubated with a fixed quantity of virus, and the mixture is applied to susceptible cell monolayers (typically Vero cells for DENV). After an incubation period allowing plaque formation, the dilution at which 50% (PRNT₅₀) or 90% (PRNT₉₀) of plaques are reduced relative to a virus-only control is calculated. The result is expressed as the reciprocal of this dilution.

PRNT provides serotype-specific neutralization data that ELISA and other binding assays cannot, making it essential for determining the identity of the infecting serotype, the history of prior infections, and the functional capacity of serum antibodies. It is more labour-intensive and lower-throughput than the related focus reduction neutralization test (FRNT).

Key Points from Literature

  • No correlation between plasmablast magnitude and PRNT₅₀ in dengue: In a Brazilian cohort with primary and secondary DENV infections, PRNT₅₀ titers to DENV-1, -2, -3, and -4 did not correlate with circulating plasmablast frequency at days 4–7 post-symptom onset, regardless of infection history or severity. This disconnect was observed in both secondary DF and secondary DFC (see GarciaBates2013 - Plasmablast Response and Dengue Severity, n=84 dengue patients, Spearman rank correlation).
  • PRNT profiles identify infecting and prior serotypes: Primary infections showed low neutralizing Ab to a single serotype that increased at convalescence. Secondary infections showed rising DENV-3 titers (infecting serotype) alongside constant DENV-1 titers (prior infecting serotype), with one-third having low constant DENV-2 titers. All sera had undetectable DENV-4 neutralizing Ab (see GarciaBates2013 - Plasmablast Response and Dengue Severity).
  • Technical protocol (GarciaBates2013): Brazilian DENV-1–4 isolates expanded on Vero cells; heat-inactivated sera at 2-fold dilutions (1:20 to 1:2560); PRNT₅₀ calculated as highest dilution reducing plaques by 50%; results depicted on log₁₀ scale (see GarciaBates2013 - Plasmablast Response and Dengue Severity).

Contradictions & Debates

  • PRNT measures the net neutralizing activity of polyclonal serum, which may mask the presence of both neutralizing and enhancing antibodies at different concentrations. A negative PRNT–plasmablast correlation does not exclude the possibility that individual plasmablast-derived antibodies are neutralizing — they may be diluted by a larger pool of non-neutralizing or enhancing antibodies.
  • PRNT and FRNT measure related but not identical readouts (plaque reduction vs. focus reduction). Both show no severity-dependent increase in neutralizing titers in dengue (see also Ansari2025 - Peripheral T Helper Subset Drives B Cell Response in Dengue).

FRNT, Plasmablast, IgG, ELISpot, Extrafollicular Response

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