TY - JOUR
T1 - Development of HEK-293 Cell Lines Constitutively Expressing Flaviviral Antigens for Use in Diagnostics
AU - Powers, Jordan A.
AU - Skinner, Benjamin
AU - Davis, Brent S.
AU - Biggerstaff, Brad J.
AU - Robb, Lucy
AU - Gordon, Elizabeth
AU - de Souza, William M.
AU - Fumagalli, Marcilio Jorge
AU - Calvert, Amanda E.
AU - Chang, Gwong Jen
N1 - Funding Information:
Financial support for J.A.P., B.S.D., L.R., and E.G. and the work presented here was provided to G.-J.C. by an interagency agreement with BARDA (IAA750117PR09000036). W.M.S. acknowledges support from the Global Virus Network Fellowship and was supported by São Paulo Research Foundation #2017/13981-0. M.J.F. was supported by São Paulo Research Foundation #2018/09383-3. This project was supported, in part, by an appointment to the Research Participation Program at the Centers for Disease Control and Prevention administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the Centers for Disease Control and Prevention.
Funding Information:
Financial support for J.A.P., B.S.D., L.R., and E.G. and the work presented here was provided to G.-J.C. by an interagency agreement with BARDA (IAA750117PR09000036). W.M.S. acknowledges support from the Global Virus Network Fellowship and was supported by São Paulo Research Foundation #2017/13981-0. M.J.F. was supported by São Paulo Research Foundation #2018/09383-3. This project was supported, in part, by an appointment to the Research Participation Program at the Centers for Disease Control and Prevention administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the Centers for Disease Control and Prevention. We thank Li-Kung Chen, College of Medicine, Tzu Chi University, Hualien, Taiwan, for the MAb Flo221. We appreciate John Lee, BARDA project officer, and William Kramp for their lively discussion and constant encouragement. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the CDC.
Publisher Copyright:
© 2022 American Society for Microbiology. All rights reserved.
PY - 2022/6
Y1 - 2022/6
N2 - 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.
AB - 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.
KW - arbovirus
KW - diagnostics
KW - flavivirus
KW - recombinant protein production
UR - http://www.scopus.com/inward/record.url?scp=85133214647&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85133214647&partnerID=8YFLogxK
U2 - 10.1128/spectrum.00592-22
DO - 10.1128/spectrum.00592-22
M3 - Article
C2 - 35532242
AN - SCOPUS:85133214647
SN - 2165-0497
VL - 10
JO - Microbiology spectrum
JF - Microbiology spectrum
IS - 3
ER -