Tetracycline-Inducible Packaging Cell Line for Production of Flavivirus Replicon Particles

Tracey J. Harvey, Wen Jun Liu, Xiang Ju Wang, Richard Linedale, Michael Jacobs, Andrew Davidson, Thuy T T Le, Itaru Anraku, Andreas Suhrbier, Pei-Yong Shi, Alexander A. Khromykh

Research output: Contribution to journalArticle

56 Citations (Scopus)

Abstract

We have previously developed replicon vectors derived from the Australian flavivirus Kunjin that have a unique noncytopathic nature and have been shown to direct prolonged high-level expression of encoded heterologous genes in vitro and in vivo and to induce strong and long-lasting immune responses to encoded immunogens in mice. To facilitate further applications of these vectors in the form of virus-like particles (VLPs), we have now generated a stable BHK packaging cell line, tetKUNCprME, carrying a Kunjin structural gene cassette under the control of a tetracycline-inducible promoter. Withdrawal of tetracycline from the medium resulted in production of Kunjin structural proteins that were capable of packaging transfected and self-amplified Kunjin replicon RNA into the secreted VLPs at titers of up to 1.6 × 10 9 VLPs per ml. Furthermore, secreted KUN replicon VLPs from tetKUNCprME cells could be harvested continuously for as long as 10 days after RNA transfection, producing a total yield of more than 1010 VLPs per 106 transfected cells. Passaging of VLPs on Vero cells or intracerebral injection into 2- to 4-day-old suckling mice illustrated the complete absence of any infectious Kunjin virus. tetKUNCprME cells were also capable of packaging replicon RNA from closely and distantly related flaviviruses, West Nile virus and dengue virus type 2, respectively. The utility of high-titer KUN replicon VLPs was demonstrated by showing increasing CD8+-T-cell responses to encoded foreign protein with increasing doses of KUN VLPs. A single dose of 2.5 × 107 VLPs carrying the human respiratory syncytial virus M2 gene induced 1,400 CD8 T cells per 106 splenocytes in an ex vivo gamma interferon enzyme-linked immunospot assay. The packaging cell line thus represents a significant advance in the development of the noncytopathic Kunjin virus replicon-based gene expression system and may be widely applicable to the basic studies of flavivirus RNA packaging and virus assembly as well as to the development of gene expression systems based on replicons from different flaviviruses.

Original languageEnglish (US)
Pages (from-to)531-538
Number of pages8
JournalJournal of Virology
Volume78
Issue number1
DOIs
StatePublished - Jan 2004
Externally publishedYes

Fingerprint

Flavivirus
replicon
Replicon
virus-like particles
Product Packaging
Tetracycline
tetracycline
Virion
packaging
cell lines
Cell Line
West Nile virus
Flaviviridae
RNA
T-lymphocytes
Human respiratory syncytial virus
cells
Genes
T-Lymphocytes
Gene Expression

ASJC Scopus subject areas

  • Immunology

Cite this

Harvey, T. J., Liu, W. J., Wang, X. J., Linedale, R., Jacobs, M., Davidson, A., ... Khromykh, A. A. (2004). Tetracycline-Inducible Packaging Cell Line for Production of Flavivirus Replicon Particles. Journal of Virology, 78(1), 531-538. https://doi.org/10.1128/JVI.78.1.531-538.2004

Tetracycline-Inducible Packaging Cell Line for Production of Flavivirus Replicon Particles. / Harvey, Tracey J.; Liu, Wen Jun; Wang, Xiang Ju; Linedale, Richard; Jacobs, Michael; Davidson, Andrew; Le, Thuy T T; Anraku, Itaru; Suhrbier, Andreas; Shi, Pei-Yong; Khromykh, Alexander A.

In: Journal of Virology, Vol. 78, No. 1, 01.2004, p. 531-538.

Research output: Contribution to journalArticle

Harvey, TJ, Liu, WJ, Wang, XJ, Linedale, R, Jacobs, M, Davidson, A, Le, TTT, Anraku, I, Suhrbier, A, Shi, P-Y & Khromykh, AA 2004, 'Tetracycline-Inducible Packaging Cell Line for Production of Flavivirus Replicon Particles', Journal of Virology, vol. 78, no. 1, pp. 531-538. https://doi.org/10.1128/JVI.78.1.531-538.2004
Harvey, Tracey J. ; Liu, Wen Jun ; Wang, Xiang Ju ; Linedale, Richard ; Jacobs, Michael ; Davidson, Andrew ; Le, Thuy T T ; Anraku, Itaru ; Suhrbier, Andreas ; Shi, Pei-Yong ; Khromykh, Alexander A. / Tetracycline-Inducible Packaging Cell Line for Production of Flavivirus Replicon Particles. In: Journal of Virology. 2004 ; Vol. 78, No. 1. pp. 531-538.
@article{9d21bd703a5042efac59c2661cf53409,
title = "Tetracycline-Inducible Packaging Cell Line for Production of Flavivirus Replicon Particles",
abstract = "We have previously developed replicon vectors derived from the Australian flavivirus Kunjin that have a unique noncytopathic nature and have been shown to direct prolonged high-level expression of encoded heterologous genes in vitro and in vivo and to induce strong and long-lasting immune responses to encoded immunogens in mice. To facilitate further applications of these vectors in the form of virus-like particles (VLPs), we have now generated a stable BHK packaging cell line, tetKUNCprME, carrying a Kunjin structural gene cassette under the control of a tetracycline-inducible promoter. Withdrawal of tetracycline from the medium resulted in production of Kunjin structural proteins that were capable of packaging transfected and self-amplified Kunjin replicon RNA into the secreted VLPs at titers of up to 1.6 × 10 9 VLPs per ml. Furthermore, secreted KUN replicon VLPs from tetKUNCprME cells could be harvested continuously for as long as 10 days after RNA transfection, producing a total yield of more than 1010 VLPs per 106 transfected cells. Passaging of VLPs on Vero cells or intracerebral injection into 2- to 4-day-old suckling mice illustrated the complete absence of any infectious Kunjin virus. tetKUNCprME cells were also capable of packaging replicon RNA from closely and distantly related flaviviruses, West Nile virus and dengue virus type 2, respectively. The utility of high-titer KUN replicon VLPs was demonstrated by showing increasing CD8+-T-cell responses to encoded foreign protein with increasing doses of KUN VLPs. A single dose of 2.5 × 107 VLPs carrying the human respiratory syncytial virus M2 gene induced 1,400 CD8 T cells per 106 splenocytes in an ex vivo gamma interferon enzyme-linked immunospot assay. The packaging cell line thus represents a significant advance in the development of the noncytopathic Kunjin virus replicon-based gene expression system and may be widely applicable to the basic studies of flavivirus RNA packaging and virus assembly as well as to the development of gene expression systems based on replicons from different flaviviruses.",
author = "Harvey, {Tracey J.} and Liu, {Wen Jun} and Wang, {Xiang Ju} and Richard Linedale and Michael Jacobs and Andrew Davidson and Le, {Thuy T T} and Itaru Anraku and Andreas Suhrbier and Pei-Yong Shi and Khromykh, {Alexander A.}",
year = "2004",
month = "1",
doi = "10.1128/JVI.78.1.531-538.2004",
language = "English (US)",
volume = "78",
pages = "531--538",
journal = "Journal of Virology",
issn = "0022-538X",
publisher = "American Society for Microbiology",
number = "1",

}

TY - JOUR

T1 - Tetracycline-Inducible Packaging Cell Line for Production of Flavivirus Replicon Particles

AU - Harvey, Tracey J.

AU - Liu, Wen Jun

AU - Wang, Xiang Ju

AU - Linedale, Richard

AU - Jacobs, Michael

AU - Davidson, Andrew

AU - Le, Thuy T T

AU - Anraku, Itaru

AU - Suhrbier, Andreas

AU - Shi, Pei-Yong

AU - Khromykh, Alexander A.

PY - 2004/1

Y1 - 2004/1

N2 - We have previously developed replicon vectors derived from the Australian flavivirus Kunjin that have a unique noncytopathic nature and have been shown to direct prolonged high-level expression of encoded heterologous genes in vitro and in vivo and to induce strong and long-lasting immune responses to encoded immunogens in mice. To facilitate further applications of these vectors in the form of virus-like particles (VLPs), we have now generated a stable BHK packaging cell line, tetKUNCprME, carrying a Kunjin structural gene cassette under the control of a tetracycline-inducible promoter. Withdrawal of tetracycline from the medium resulted in production of Kunjin structural proteins that were capable of packaging transfected and self-amplified Kunjin replicon RNA into the secreted VLPs at titers of up to 1.6 × 10 9 VLPs per ml. Furthermore, secreted KUN replicon VLPs from tetKUNCprME cells could be harvested continuously for as long as 10 days after RNA transfection, producing a total yield of more than 1010 VLPs per 106 transfected cells. Passaging of VLPs on Vero cells or intracerebral injection into 2- to 4-day-old suckling mice illustrated the complete absence of any infectious Kunjin virus. tetKUNCprME cells were also capable of packaging replicon RNA from closely and distantly related flaviviruses, West Nile virus and dengue virus type 2, respectively. The utility of high-titer KUN replicon VLPs was demonstrated by showing increasing CD8+-T-cell responses to encoded foreign protein with increasing doses of KUN VLPs. A single dose of 2.5 × 107 VLPs carrying the human respiratory syncytial virus M2 gene induced 1,400 CD8 T cells per 106 splenocytes in an ex vivo gamma interferon enzyme-linked immunospot assay. The packaging cell line thus represents a significant advance in the development of the noncytopathic Kunjin virus replicon-based gene expression system and may be widely applicable to the basic studies of flavivirus RNA packaging and virus assembly as well as to the development of gene expression systems based on replicons from different flaviviruses.

AB - We have previously developed replicon vectors derived from the Australian flavivirus Kunjin that have a unique noncytopathic nature and have been shown to direct prolonged high-level expression of encoded heterologous genes in vitro and in vivo and to induce strong and long-lasting immune responses to encoded immunogens in mice. To facilitate further applications of these vectors in the form of virus-like particles (VLPs), we have now generated a stable BHK packaging cell line, tetKUNCprME, carrying a Kunjin structural gene cassette under the control of a tetracycline-inducible promoter. Withdrawal of tetracycline from the medium resulted in production of Kunjin structural proteins that were capable of packaging transfected and self-amplified Kunjin replicon RNA into the secreted VLPs at titers of up to 1.6 × 10 9 VLPs per ml. Furthermore, secreted KUN replicon VLPs from tetKUNCprME cells could be harvested continuously for as long as 10 days after RNA transfection, producing a total yield of more than 1010 VLPs per 106 transfected cells. Passaging of VLPs on Vero cells or intracerebral injection into 2- to 4-day-old suckling mice illustrated the complete absence of any infectious Kunjin virus. tetKUNCprME cells were also capable of packaging replicon RNA from closely and distantly related flaviviruses, West Nile virus and dengue virus type 2, respectively. The utility of high-titer KUN replicon VLPs was demonstrated by showing increasing CD8+-T-cell responses to encoded foreign protein with increasing doses of KUN VLPs. A single dose of 2.5 × 107 VLPs carrying the human respiratory syncytial virus M2 gene induced 1,400 CD8 T cells per 106 splenocytes in an ex vivo gamma interferon enzyme-linked immunospot assay. The packaging cell line thus represents a significant advance in the development of the noncytopathic Kunjin virus replicon-based gene expression system and may be widely applicable to the basic studies of flavivirus RNA packaging and virus assembly as well as to the development of gene expression systems based on replicons from different flaviviruses.

UR - http://www.scopus.com/inward/record.url?scp=10744227302&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=10744227302&partnerID=8YFLogxK

U2 - 10.1128/JVI.78.1.531-538.2004

DO - 10.1128/JVI.78.1.531-538.2004

M3 - Article

VL - 78

SP - 531

EP - 538

JO - Journal of Virology

JF - Journal of Virology

SN - 0022-538X

IS - 1

ER -