Figure 1.
DENV RVPs are infectious and antigenically equivalent to live dengue virus.
(A) Dengue Reporter Virus Particles (RVPs) are composed of the proteins capsid (C), premembrane/membrane (prM/M) and envelope (E) from defined DENV strains, and an RNA reporter genome made from a DENV genomic replicon. (B) Serial dilutions of DENV RVPs representing each serotype were used to infect Raji DC-SIGN-R cells. Forty-eight hours after infection, cells were analyzed for GFP expression by flow cytometry. Regression analysis shows a linear relationship (R2>0.98 for DENV-1; R2>0.99 for DENV-2; R2>0.99 for DENV-3; R2>0.99 for DENV-4) with the amount (µl) of RVPs used for infection (n = 4, error bars represent the standard deviation). (C) Neutralization assays using DENV RVPs were performed with serially diluted monoclonal antibodies 4G2, 3H5, or the control monoclonal antibody 15F3 for 1 hour prior to infection of Raji DC-SIGN-R cells. Forty-eight hours after infection, cells were quantified for GFP expression by flow cytometry. All neutralization results are shown normalized to the maximum (uninhibited) infection achieved, defined as 100% (n = 3, error bars represent the standard deviation). The infectious titers of the lots of RVPs used here, calculated based on the number of infected cells using an RVP input of 50 µl, are as follows: DENV-1: 773,325 infectious units/ml, DENV-2: 422,970 infectious units/ml, DENV-3: 347,292 infectious units/ml, and DENV-4: 353,995 infectious units/ml.
Figure 2.
DENV RVPs are infectious under conditions commonly used in neutralization assays and following long-term cryopreservation.
DENV RVPs representing all 4 serotypes were incubated at 4°C (A), 25°C (B), or at 37°C (C) for up to 10 hours, followed by infection of Raji DC-SIGN-R cells. Forty-eight hours post-infection, cells were analyzed for GFP expression by flow cytometry. At all temperatures, DENV RVPs remain highly infectious after a 2-hour incubation (a typical incubation period used in neutralization assays, vertical dashed line). (D) A single preparation of DENV-2 RVPs was aliquoted and frozen at −80°C from 2 days up to 1 year prior to infection of Raji DC-SIGN-R cells. Forty-eight hours after infection, cells were analyzed for GFP expression by flow cytometry. DENV RVPs showed little or no substantial loss of infectivity after long-term cryopreservation (n = 2, error bars represent the range).
Figure 3.
DENV RVPs can be used to derive reproducible antibody neutralization titers.
(A) Serial dilutions of a single lot of DENV-2 RVPs were pre-incubated with the monoclonal antibody 4G2 at room temperature for 1 hour followed by infection of Raji DC-SIGN-R cells. Forty-eight hours after infection, cells were analyzed for GFP expression by flow cytometry (n = 8 for each dilution, error bars represent the standard deviation). (B) NT50 values for each replicate for the indicated RVP input were calculated and plotted (the bar represents the mean NT50, boxes show the mean and standard deviation for each RVP dilution tested). The mean NT50 values for each volume of RVPs tested for neutralization were not statistically different (ANOVA, p>0.05). (C) A single lot of DENV-2 RVPs was used for independent neutralization experiments performed on four different days in duplicate (mean and range shown). Mean NT50 values are not statistically different (ANOVA, p>0.05). (D) Independently derived lots of DENV-2 RVPs were used for neutralization experiments performed in duplicate (mean and range shown). Mean NT50 values are not statistically different across different lots (ANOVA, p>0.05).
Figure 4.
Neutralization of DENV RVPs using WHO human reference sera.
DENV RVPs representing each of the four serotypes were pre-incubated for 1 hour with a WHO reference panel of human primary sera reactive to each RVP serotype (monovalent, squares: anti-DENV-1 serum in A; anti-DENV-2 in B; anti-DENV-3 in C; anti-DENV-4 in D) or reactive to all serotypes (tetravalent, triangles in all four panels). Naïve serum was used as a negative control. After incubation, DENV RVPs were used to infect Raji DC-SIGN-R cells. Forty-eight hours after infection, cells were quantified for GFP expression by flow cytometry (n = 3, error bars represent the standard deviation).
Table 1.
Comparison of neutralization titers for human sera obtained by PRNT or RVPs.
Table 2.
Application of DENV RVPs to clinical serum samples.