Fig 1.
Diagram of the pSinRep5 plasmid and transcribed viral RNA encoding the CHIKV-181/25 luciferase genome.
The chikungunya virus-181/25 nonstructural proteins (blue), luciferase (red), and structural polyprotein open reading frame (green) locations are shown within the pSinRep5 DNA plasmid. The luciferase transgene was inserted just downstream of the 26S subgenomic promoter.
Fig 2.
Flow Diagram of the CHIKV-luc NT80 Assay.
Vero cell monolayers are first established in 96-well culture plates (Day 1). Dilutions of serum are incubated with CHIKV-luc virus and the mixtures added to the Vero cells (Day 2). Luciferase activity is detected by adding luciferin substrate then measuring relative light unit (RLU) luminescence signal on a plate reader luminometer (Day 3).
Fig 3.
Virus titration curves of luciferase activity after infecting Vero cells with 2- (panel A) and 3-fold (panel B) dilutions of CHIKV-luc reporter virus. Ten total dilutions were tested for each dilution series.
Fig 4.
Flow Diagram of Assay and Vaccine Development Pathways.
Development of the CHIKV-luc NT80 assay started with generation of the luciferase-expressing CHIKV-181/25 reporter virus. The assay was developed using samples collected from epidemiological cohort studies and further optimized to test sera from preclinical and clinical samples. Qualification and validation of the assay enabled testing of phase 2 and 3 clinical serum samples, respectively.
Table 1.
Summary of the CHIKV neutralization assays.
Table 2.
Comparison of CHIKV-specific antibody titers obtained from PRNT80, SFV/CHIK OPY1-GFP IC80, and CHIKV-luc NT80 assay methods.
Fig 5.
Pairwise comparison of CHIKV-specific antibody titers from the PRNT80, SFV/CHIKV OPY1 IC80, and CHIKV-luc NT80 assays.
Scatter plots of each of three pairwise assay comparisons of data generated from testing VRC311 samples are shown, including 1) PRNT80 versus SFV/CHIKV IC80 (left panel), 2) CHIKV-luc NT80 versus SFV/CHIKV IC80 (middle panel), and 3) PRNT80 versus CHIKV-luc NT80 (right panel). The correlation coefficient (r), number of samples measured by both assays (n), and statistical significance (p) are listed within each panel.
Fig 6.
The neutralization curve shows percent virus neutralization versus serum dilution. NT50 and NT80 titers are marked on the curve to demonstrate the shift in antibody concentration required to achieve 80% vs 50% virus neutralization (inhibition of infection). The curve shows that higher antibody concentration is required to achieve 80% neutralization compared to 50%.
Fig 7.
Linear fit of NT50 versus NT80, one-way analysis of variance, and bivariate fit.
A. Linear fit of NT50 versus NT80 shows the linear relationship of NT50 and NT80 data with slope (red line) and the ratio of the best fit indicating a 3.03- fold difference. B. One-way analysis of NT50 and NT80 data shows the NT50/NT80 ratio for each sample (dots) in relation to the overall geometric mean ratio of 2.98 (black line). C. Bivariate fit of NT50/NT80 by NT80 shows the ratio stays the same over the NT80 range. The black line is the arithmetic mean and the green line shows that the slope CI overlaps 0, so no effect.
Table 3.
CHIKV-luc assay overall precision.
Table 4.
Summary of CHIKV-luc NT80 assay validation parameters, acceptance criteria, and results.
Table 5.
Clinical trials using the CHIKV-luc NT80 assay.