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 x 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 10(10) VLPs per 10(6) 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 x 10(7) VLPs carrying the human respiratory syncytial virus M2 gene induced 1,400 CD8 T cells per 10(6) 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.

FIG. 1.

Generation and characterization of stable packaging cell line tetKUNCprME. (A) Schematic representation of the plasmid constructs used for generation of the stable packaging cell line tetKUNCprME. The pEF-tTA-IRESpuro plasmid was used to generate a first stable BHK cell line, BHK-Tet-Off, continuously expressing the tetracycline transactivator (tTA) from the human elongation factor 1α promoter (pEF-1a). tetKUNCprME, expressing KUN structural genes C, prM, and E (KUN CprME) from the tetracycline-inducible cytomegalovirus promoter (P_minCMV), was established by transfection of pTRE2CprME-IRESNeo plasmid DNA into BHK-Tet-Off cells and selection or cells growing in the presence of G418 and puromycin (see the text). In uninduced tetKUNCprME cells, doxycycline (DOX) (a form of tetracycline with higher specific activity) binds to tTA and prevents its binding to the tetracycline-responsive element (TRE) and subsequent activation of CprME mRNA transcription from the cytomegalovirus promoter. To induce expression of KUN CprME genes, DOX is removed from the medium, resulting in the release of tTA, its binding to TRE, and activation of CprME mRNA transcription from the cytomegalovirus promoter. tetR, Tet repressor protein; VP16, herpes simplex virus VP16 activation domain; IRES, EMCV internal ribosome entry site; puro, puromycin N-acetyltransferase; Neo, neomycin resistance gene; SV40 poly A, SV40 transcription terminator/poly(A) signal; β-globin poly A, β-globin transcription terminator/poly(A) signal. (B) Production of secreted E protein and VLPs in induced and uninduced tetKUNCprME cells in the presence and absence of KUN replicon RNA. -- RNA graph (left part) shows the results of an experiment without replicon RNA transfection; + RNA graph (right part) shows the results of another experiment with electroporation of RNAleu. Results are shown for tetKUNCprME cells either electroporated with KUN replicon RNA (+ RNA) or not electroporated (- RNA) and maintained for 48 h in the medium with (+) or without (-) 0.5 μg of doxycycline/ml. Detection of secreted KUN E protein (white bars) by antigen capture ELISA and determination of VLP titers (black bars) (in infectious units per milliliter) by infectivity assay on Vero cells were performed as described in the text. Negative controls in both experiments (Cont) were culture fluids from normal BHK cells. The titers of KUN positive controls (KUN) used in each experiment were determined by plaque assay on BHK cells.

FIG. 2.

Amplification and spread of KUN replicon VLPs in tetKUNCprME cells. Coverslips of tetKUNCprME and BHK21 cells were infected with 0.1 MOI of RNAleuMpt VLPs and analyzed by IF with KUN anti-NS3 antibodies at 2 and 3 days after infection.

FIG. 3.

CD8-T-cell responses in mice immunized with high-titer KUN VLP replicons. (A) C57BL/6 mice (n = 4 per group) were immunized intraperitoneally with phosphate-buffered saline (Naive), 10⁸ IU of KUN VLPs not encoding a recombinant antigen (KUN VLP Control), or the indicated dose of KUN VLPs encoding the murine polytope (2) (KUN-Mpt VLP). After 2 weeks, splenocytes were removed and analyzed for H-2^kb-restricted SIINFEKL-specific responses by IFN-γ ELISPOT. (B and C) BALB/c mice (n = 3 per group) were immunized intraperitoneally with 2.5 × 10⁷ IU of KUN VLPs encoding respiratory syncytial virus matrix 2 protein (KUN-M2 VLP), 2.5 × 10⁷ IU of KUN VLP not encoding a recombinant antigen (KUN VLP Control), or subcutaneously with a peptide vaccine containing the H-2Kd-restricted RSV M2 epitope, SYIGSINNI, formulated with tetanus toxoid in Montanide ISA 720 (SYIGSINNI/TT/M720) as described previously (4) After 2 weeks splenocytes were removed and analyzed for SYIGSINNI-specific responses by IFN-γ ELISPOT (B) and by standard chromium release assay (C) (black squares, P815 target cells sensitized with SYIGSINNI peptide; white squares, P815 target cells without peptide) as described previously (1).