Development of HEK-293 Cell Lines Constitutively Expressing Flaviviral Antigens for Use in Diagnostics

Abstract

Flaviviruses are important human pathogens worldwide. Diagnostic testing for these viruses is difficult because many of the pathogens require specialized biocontainment. To address this issue, we generated 39 virus-like particle (VLP)- and nonstructural protein 1 (NS1)-secreting stable cell lines in HEK-293 cells of 13 different flaviviruses, including dengue, yellow fever, Japanese encephalitis, West Nile, St. Louis encephalitis, Zika, Rocio, Ilheus, Usutu, and Powassan viruses. Antigen secretion was stable for at least 10 cell passages, as measured by enzyme-linked immunosorbent assays and immunofluorescence assays. Thirty-five cell lines (90%) had stable antigen expression over 10 passages, with three of these cell lines (7%) increasing in antigen expression and one cell line (3%) decreasing in antigen expression. Antigen secretion in the HEK-293 cell lines was higher than in previously developed COS-1 cell line counterparts. These antigens can replace current antigens derived from live or inactivated virus for safer use in diagnostic testing. IMPORTANCE Serological diagnostic testing for flaviviral infections is hindered by the need for specialized biocontainment for preparation of reagents and assay implementation. The use of previously developed COS-1 cell lines secreting noninfectious recombinant viral antigen is limited due to diminished antigen secretion over time. Here, we describe the generation of 39 flaviviral virus-like particle (VLP)- and nonstructural protein 1 (NS1)-secreting stable cell lines in HEK-293 cells representing 13 medically important flaviviruses. Antigen production was more stable and statistically higher in these newly developed cell lines than in their COS-1 cell line counterparts. The use of these cell lines for production of flaviviral antigens will expand serological diagnostic testing of flaviviruses worldwide.

Keywords: arbovirus; diagnostics; flavivirus; recombinant protein production.

FIG 1

Workflow for production and selection of stable cell lines in the absence of selective pressure.

FIG 2

Predicted mean EPT curves over 10 passages for stable HEK-293 cell lines. Predicted mean values of EPTs (black line) from each cell line were plotted as log (P/N) versus log₂ (dilution) with confidence bands shown as dashed black lines. P/N represents the ratio of the OD of positive antigen over the ratio of negative antigen tested in duplicate by ELISA. The reference line in blue for determining the EPT is shown at log₂. The estimated EPT (95% CI) is indicated with vertical red lines with confidence bands shown as dashed red lines.

FIG 3

EPT against passage by cell line. Statistically significant trends (P < 0.05) are shown for the LAD regression test, with the fit drawn as a red line. Statistically nonsignificant LAD regression fits are shown as their corresponding regression lines in gray.

FIG 4

Comparison of antigen production from constitutively expressing flaviviral antigen COS-1 and HEK-293 cell lines. EPTs from antigen produced in COS-1 (blue) or HEK-293 (red) cell lines measured by ELISA were plotted as log₂ (P/N) versus log₂ (dilution), with confidence bands shown as dashed lines. P/N represents the ratio of the OD of positive antigen over the ratio of negative antigen tested in duplicate. The reference line shown in gray represents the P/N of 2 used to determine EPTs for each cell line.

FIG 5

Intracellular expression of recombinant antigen from three representative HEK-293 and COS-1 VLP and NS1 constitutively expressing cell lines. Cells expressing recombinant antigen were harvested at the 1st and 10th passage, fixed, and stained with mouse hyperimmune ascitic fluid (MHIAF) and goat anti-mouse antibody conjugated to FITC. Nuclei of cells were stained with DAPI.

FIG 6

Recombinant antigen expression in HEK-293 and COS-1 cells was measured by flow cytometry over 10 passages. Significance in antigen expression between HEK-293 and COS-1 cell lines was measured by a two-tailed Mann-Whitney U test; ****, P < 0.0001. The median percent recombinant antigen expression (line and percentage) with 95% confidence interval is depicted for each cell line.