Fig 1.
Predicted optimal combination of factors with maximal desirability.
DOE analysis was carried out using the optimization and desirability settings (minimized with the relative importance set to 1. V-factor: maximized with the relative importance set to 0.7. Dynamic Range: maximized with the relative importance set to 1) in JMP13. The curves show the predicted interaction between the different factors. Flat lines indicates no predicted interaction. The horizontal redlines show the predicted optimal parameter and the vertical red lines show the geometric mean of the desirability measured.
Fig 2.
Viability and infectivity assessment for Z-RVP lot 229A.
The CellTiter-Glo (RLU) response is displayed on the left y-axis and the Renilla-Glo (RLU) response is shown on the right y-axis. The CellTiter-Glo RLU expressed by the cells was consistent over Z-RVP amount tested whereas the Renilla-Glo (RLU) increases as the Z-RVP amount increases.
Fig 3.
Neutralization curves of Z-RVP dilutions.
Neutralization curves of different dilutions of Z-RVP to determine the effects of Z-RVP input against Serum 1, 2 and 3. Neutralization titers in log10EC50 titers are shown as calculated by nonlinear regression in GraphPad Prism for each Z-RVP dilution.
Table 1.
Titration of Z-RVP reagent for use in the Z-RVP-384 assay.
Fig 4.
Relationship between serum dilution in assay media and neutralization activity.
To examine dilutional linearity in assay media, serum samples were prediluted, starting at 1:8 followed by 2-fold dilutions to generate eight dilutions of samples. The log10EC50 titers were determined by the RLU signal plotted against the log10-transformed serum dilution factor and then fitted with a non-linear regression curve with the lower asymptote constrained to 0 using GraphPad PRISM using RLU’s and lower asymptote constrained to 0. The log10EC50 values from each dilution were determined and plotted against the input dilutions (log10) of the serum. Shown is the best fit line and 95% confidence interval (CI) (y = 3.901–0.3154x, with root mean square error = 0.13, and R2 = 0.968 using JMP13.
Fig 5.
Dilutional linearity of the Z-RVP-384 assay in serum matrix.
(A, B) Two positive human sera were diluted in negative human serum. (C, D) Two positive cynomolgus macaque sera were diluted in negative cynomolgus macaque serum. Serial dilutions test stocks were generated and titrated 8–16 times.
Table 2.
Matrix effect observed in human serum and cynomolgus macaque serum.
Fig 6.
Typical Z-RVP-384 assay neutralization curves of three species using negative and positive sera.
Nonlinear regression neutralization curves of human, rhesus macaque, and mouse serum from vaccinated subjects (+ PIZV) or nonvaccinated subjects (Matrix; defined as naïve serum of the species). Each serum dilution was measured in duplicate and the lower asymptote of the nonlinear regression curve was constrained to 0 (A) or left unconstrained (B). Neutralization Titers in log10 are shown in (C) as calculated by nonlinear regression in GraphPad Prism. Cannot calculate = effect on curve was too low to calculate a titer.
Fig 7.
Matrix effect on the Z-RVP-384 assay by species.
Quantile plots of log10EC50 values are shown for 124 human, 84 rhesus macaque, and 69 CD-1 mouse sera. Titers below 1.5 log10 are extrapolated (only included for demonstration), and should be reported as < LOD/LLOQ.
Table 3.
Positive serum cut-off for the Z-RVP-384 assay in human, rhesus macaque and mouse serum.
Table 4.
Variance components of assay robustness assessment.
Fig 8.
Determination of the ULOQ of Z-RVP-384 assay.
(A) Neutralization curves of several dilutions of high potency rabbit serum in assay media to estimate the ULOQ for the standard assay. A rabbit hyperimmune serum generated by immunization with live ZIKV (Dakar) was diluted in assay media 1:8, 1:25, 1:50, 1:200, and 1:400. (B) log10EC50 titers generated by each serum dilution shown with Standard error (SE) of the log10EC50 estimate and % FE (SE*LN(10)*100).
Fig 9.
The Z-RVP-384 assay consistently measure neutralizing titers in human, rhesus macaque and CD-1 mouse sera.
ZIKV neutralizing titers in human, rhesus macaques (NHP) and mouse sera was measured after addition of (A) mAb 2C8; (B) mAb ZKA64; (C) ZIKV neutralizing rhesus macaque polyclonal serum. (D) differences in mean log10EC50 titers plus 95% CI between the different sera, by spiking group. For mAbs EC50 are expressed as ng/mL, and for sera EC50 are expressed as reciprocal serum dilution.
Table 5.
Titer in Z-RVP-384 assay of non-human primate sera post-immunization with five different flavivirus vaccines.
Fig 10.
Correlation of ZIKV neutralization titers in human sera using Z-PRNT and Z-RVP-384 assays.
Human sera from a clinical trial of PIZV (ZIK-101) day 29 (post dose one sera) and day 57 (post dose two sera) were tested in both Z-PRNT (Y axis) and Z-RVP-384 (X-axis) and log10-transformed titers plotted. A Spearman correlation analysis was conducted in R and showed a strong correlation of 0.94.