Temperature-dependent production of pseudoinfectious dengue reporter virus particles by complementation

Abstract

Dengue virus (DENV) is a mosquito-borne flavivirus responsible for 50 to 100 million human infections each year, highlighting the need for a safe and effective vaccine. In this study, we describe the production of pseudoinfectious DENV reporter virus particles (RVPs) using two different genetic complementation approaches, including the creation of cell lines that release reporter viruses in an inducible fashion. In contrast to studies with West Nile virus (WNV), production of infectious DENV RVPs was temperature-dependent; the yield of infectious DENV RVPs at 37 degrees C is significantly reduced in comparison to experiments conducted at lower temperatures or with WNV. This reflects both a significant reduction in the rate of infectious DENV RVP release over time, and the more rapid decay of infectious DENV RVPs at 37 degrees C. Optimized production approaches allow the production of DENV RVPs with titers suitable for the study of DENV entry, assembly, and the analysis of the humoral immune response of infected and vaccinated individuals.

Fig. 1

Production of DENV RVPs by complementation. (A) DENV RVPs were produced by transfection of HEK-293T cells with plasmids encoding a DENV sub-genomic replicon and the structural proteins of DENV1 (Western Pacific strain) or DENV2 (16681 strain). Supernatants were harvested at the indicated times and titered on Raji-DCSIGNR cells. Infection was measured as a function of the percentage of GFP expressing cells at 72 h post-infection by flow-cytometry. (B) DENV RVPs were produced by transfection of a T-REx-293 cell line (T-REx-293-DGZ) that stably propagates a DENV2 sub-genomic replicon. Supernatants were harvested 72 h post-transfection and analyzed as in panel A. (C) DENV RVPs were produced using an inducible cell line that stably propagates a DENV2 replicon and expresses DENV structural proteins in a tetracycline dependent fashion. Parental T-REx-293 cells, T-REx-293-DGZ, and T-REx-293-DGZ-WP cells were induced in fresh low glucose media in the presence (+) or absence (−) of 1 μg/ml tetracycline. Supernatants were harvested 72 h post-transfection and analyzed as in panel A.

Fig. 2

Temperature-dependent production of RVPs. (A) DENV1 RVPs were produced by induction of T-REx-293-DGZ-WP cells with tetracycline, followed by culture at the indicated temperatures. Culture supernatants were collected at the indicated times post-induction and titered using Raji-DCSIGNR cells. Infection was measured as a function of the percentage of GFP expressing cells at 72 h post-infection by flow-cytometry. (B) WNV RVPs were produced by transfection of HEK-293T cells with plasmids encoding a sub-genomic WNV replicon and WNV C-prM-E. Supernatants were collected at the indicated times post-transfection and titered as described above. (C) DENV1 RVPs that encapsidate a WNV genome were produced by transfection of HEK-293T cells with plasmids encoding a sub-genomic WNV replicon and DENV1 C-prM-E (Western Pacific strain). DENV RVPs were harvested at the indicated times and titered as described above. (D–F) To obtain a more quantitatively rigorous understanding of the temperature-dependent differences in DENV and WNV RVP production, RVPs were produced in replicate wells of a 6-well plate as described for A–C. RVP-containing supernatants were collected at 24 h and 48 h after induction and titered using Raji-DCSIGNR cells in triplicate. Infection is expressed relative to the titer achieved at 48 h post-infection at 37 °C. Error bars reflect the standard error of two independent production experiments.

Fig. 3

Temperature-dependent changes in the rate of DENV RVP production. The rate of infectious DENV and WNV RVP production at 24 and 48 h post-induction/transfection was investigated. DENV RVPs were produced by induction of T-REx-293-DGZ-WP cells (A) and WNV RVPs were produced by transfection of HEK-293T cells (B) as described in Fig. 2 and the Materials and methods. Culture supernatants were removed from duplicate wells at 22 h (d1) or 46 h (d2) post-induction and immediately replaced with conditioned media pre-equilibrated at the indicated temperature. Supernatant was collected 4 h later and titered in duplicate using Raji-DCSIGNR cells. Infection was measured as a function of the percent of GFP expressing cells at 72 h (A) or 48 h (B) post-infection by flow-cytometry. The data presented is representative of three independent experiments.

Fig. 4

Stability of WNV and DENV RVPs. (A) DENV RVPs were produced using T-REx-293-DGZ-WP cells and aliquoted (500 μl) into wells of a 48-well plate. Plates were incubated at the indicated temperatures. At the indicated times, RVPs were removed from duplicate wells and frozen at −80 °C. The titer of RVPs from each time point was determined simultaneously by infecting Raji-DCSIGNR cells with serial two-fold dilutions of RVPs in complete RPMI-1640 medium. Infection was measured as a function of GFP expression at 72 h post-infection by flow-cytometry. Non-linear regression analysis was performed to generate one-phase exponential decay curves using GraphPad Prism version 4.0b. Dotted lines indicate the 95% confidence interval for the regression analysis. (B) The average half-life of WNV and DENV RVPs at 28 °C or 37 °C is displayed. Error bars indicate the standard error of multiple independent measurements of the stability of DENV and WNV at each temperature (n=6 and n=7 for DENV and WNV at 28 °C; n=7 and n=4 for DENV and WNV at 37 °C). Statistical comparisons were made using GraphPad Prism version 4.0b (unpaired t test).

Fig. 5

Infection of a panel of cell lines with DENV RVPs. (A) WNV and DENV RVPs were produced in HEK-293T cells transfected with a WNV sub-genomic replicon and plasmids encoding either WNV (blue) or DENV2 16881 (red) structural genes. Serial four-fold dilutions of RVPs were used to infect each cell line in quadruplicate as described in the Materials and methods section. Infection was scored as a function of Renilla luciferase activity 48 h post-infection. The signal obtained with undiluted RVPs is shown, with error bars indicating the standard error. (B) The infectivity of each population of RVPs was normalized as a function of the relative genomic RNA content of each preparation. Genomic RNA was measured using real-time qRT-PCR as described (Geiss et al., 2005; Houng et al., 2001).

Fig. 6

Measuring antibody-mediated neutralization using DENV RVPs. (A) To demonstrate that RVPs allow for the study of the functional properties of antibodies under conditions that satisfy the law of mass action (Andrewes, 1933; Klasse and Sattentau, 2001; Pierson et al., 2006), nine serial four-fold dilutions of DENV1-immune primate sera were incubated for 1 h at room temperature with serial dilutions of DENV1 RVPs. RVP-antibody complexes were then added to Raji-DCSIGNR cells in triplicate. Infection was measured as a function of GFP expression using flow-cytometry. Error bars display the standard error of triplicate infections. Non-linear regression analysis was used to calculate the EC50. (B) RVPs produced from T-REx-293-DGZ-WP cells and by plasmid transfection are neutralized equivalently. Dose response curves were generated using DENV1 or DENV2 (data not shown) immune sera as described above. (C) The neutralization titer of antibody present in the pooled DENV1 or DENV2-immune sera is shown. The error bar represents the average reciprocal dilution calculated from independent dose response curves composed of nine dilutions of sera performed in duplicate (DENV1, n=12 and DENV2, n=4, respectively). The error bar represents the standard error.