In spite of advances in conventional four-plasmid transient transfection methods and development of inducible stable production cell lines, production of replication-defective lentiviral vectors in clinical scale has been challenging. Baculovirus technology offers an alternative to scalable virus production as a result of fast and easy production of baculoviruses, efficient transduction of mammalian cells and safety of the baculoviruses. As a first step toward scalable lentiviral production system, we have constructed four recombinant baculoviruses: the BAC-transfer virus expresses green fluorescent protein (GFP) as a transgene and BAC-gag-pol, BAC-vesicular stomatitis virus glycoprotein G and BAC-rev express all elements required for a safe lentivirus vector generation. Following 293T cell transduction with recombinant baculoviruses functional lentiviruses were produced. Different baculovirus concentrations, mediums and transduction times were used to find optimal conditions for lentivirus production. The unconcentrated lentiviral titers in cell culture mediums were on average 2.5 × 106 TU ml−1, which are comparable to titers of the lentiviruses produced by conventional four-plasmid methods. Lentiviruses produced by baculovirus method transduced HeLa cells and showed sustained GFP expression. No evidence of the formation of replication competent lentiviruses was detected by p24 enzyme-linked immunosorbent assay. Our results show that baculoviruses are an attractive alternative for the production of lentiviruses in mammalian cells.
The production of replication-defective lentiviral vectors for a large-scale clinical use is challenging. Lentiviral vectors are normally produced by cotransfecting 293T human embryonic kidney cells with several different plasmid constructs. The first clinical lentiviral vector production was based on a two-plasmid system.1 To further improve the safety of the system, lentivirus genome can be separated into four plasmids. The plasmids are a self-inactivating transfer vector, a packaging plasmid containing gag-pol, a rev plasmid and an envelope glycoprotein plasmid, which usually encodes vesicular stomatitis virus glycoprotein G (VSV-G)2, 3 To scale up the lentivirus production, the growth of adherent cells has been changed to cell factories.4 Recently, further improvement was achieved when lentiviral vectors were transiently produced in suspension cultures using 3 l bioreactors in serum-free conditions.5
As transient transfection system for virus production may be problematic and time consuming, attempts have been made to develop stable large-scale production systems6, 7, 8, 9 However, the toxicity of lentiviral protease,10 and the fusogenic envelope protein VSV-G11, 12 have prohibited constitutive vector production. The only suitable means at present has been to use inducible packaging cell lines. Inducible production has been controlled either by tetracycline-6, 7, 9, 13 or ecdysome-inducible8 systems. Another approach to obtain a large-scale production has been to replace the toxic VSV-G protein with a less-toxic glycoprotein. A variety of different envelope glycoproteins like those from gammaretroviruses (for example, feline endogenous retrovirus RD114 env, modified gibbon-ape leukemia virus, moloney murine leukemia virus) alphaviruses, lyssaviruses or baculoviruses were shown to pseudotype lentiviral vectors14, 15, 16, 17 Pseudotyping broadens the transduction range and can strengthen the otherwise fragile lentivirus (reviewed in Cronin et al.11). Toward safer lentivirus production system one approach has been to develop a codon optimized systems, which is based on reducing the sequence homology with vectors containing gag-pol genes allowing rev-independent expression of gag-pol and the use of cell lines lacking endogenous homologous sequences to the virus.18, 19 Another approach is to split gag/gag-pol sequences into several parts.20
Baculoviruses have been widely used for a large-scale protein production in insect cells.21 Baculoviruses are capable of transducing mammalian cells22 and have been used previously for the production of several viruses23, 24, 25, 26 and viral-like particles (VLP).27 Thus, methods for the culturing and handling of baculoviruses are well developed. In order to ease and improve the lentivirus production, we constructed four recombinant baculoviruses which encode all elements needed for the lentiviral vector generation in mammalian cells. All four baculoviruses used were derived from Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Optimized protocol allowed straightforward high-titer lentivirus generation. Sustained transgene expression was achieved after lentivirus transduction of HeLa cells. No replication competent lentivirus (RCL) formation was detected.
Construction of baculoviruses
We cloned all third-generation lentivirus elements into four baculoviruses (Figure 1a). Baculovirus plasmid constructs were verified by restriction analysis and four baculoviruses were produced in insect cells. To monitor consistency of the baculovirus production, we made immunoblot analysis from each batch with anti-gp64 antibody that detects the major envelope protein of baculovirus (data not shown). End-point titer determination (IU ml−1) for concentrated baculoviruses was done in insect cells. High titers (>1010 IU ml−1) were obtained for all produced viruses. These titers were used to control multiplicity of infection (MOI) in lentivirus production.
Production of lentiviruses
Lentiviruses were produced by transduction of 293T cells with four baculoviruses. Transduction was performed using different mediums and incubation times. The transduction efficacy (over 90%) was monitored 20 h after the transduction by flow cytometer and fluorescent microscopy (Figures 1b and c). Lentivirus-containing supernatants were collected 48 h after the transduction and titers were determined in HeLa cells as transducing units (TU ml−1).
Four baculoviruses at MOI 500 each yielded lentivirus titers with an average of 6.0 × 105 TU ml−1 (Figure 2a) when transduction was performed for 4 h in serum-free conditions. Baculovirus concentration at MOI 750 produced higher lentivirus titers with an average of 1.2 × 106 TU ml−1. A decrease in the titer was detected when higher baculovirus concentrations (four baculoviruses at MOI 1000 each) were used. Four baculoviruses at MOI 250 yielded the highest titers with an average of 2.5 × 106 TU ml−1 when baculovirus transduction was performed overnight (Figure 2b). When RPMI medium was used in transduction, the highest titers were an average of 5.9 × 105 TU ml−1 already at MOI 50 (Figure 2c). The titers are comparable to those produced with a conventional four-plasmid transduction method (t-test P<0.05) (Figure 2d) and are in line with published information.2
Differences in the ratios of lentivirus plasmids can influence titers in the plasmid-based lentivirus production. When we used the same ratios of baculoviruses as is commonly used in the lentivirus plasmid production,2 the lentivirus titers were 0.6-fold lower. We also studied whether higher lentivirus titers could be obtained by doubling the BAC-transfer virus. However, there was no significant difference in the titers compared to the production with equal amounts of baculoviruses. Titers obtained with the doubled amounts of the BAC-transfer virus were on an average of 1.4 × 106 TU ml−1.
As negative controls, we performed lentivirus production omitting one of the baculoviruses (BAC-gag-pol, BAC-Rev or BAC-VSV-G) at the time. Collected mediums were used to transduce HeLa cells and the number of green fluorescent protein (GFP)-positive cells (%) was analyzed 4 days after the transduction with a flow cytometer. No GFP-positive cells could be detected in these experiments (data not shown).
A frequently used titration method alongside with the biological titer (TU ml−1) of the lentiviruses measures p24 concentration (pg ml−1) by enzyme-linked immunosorbent assay (ELISA). The p24 concentrations in the media were 191±105 ng ml−1 that is in line with the values for the representative virus preparations.2 However, p24 concentration does not identify biologically active virus particles. To compare infectious particles and p24 titers, we measured both of these parameters from several preparations produced with different amounts and ratios of baculoviruses (Figure 3). The results showed a good correlation between these measurements (r=0.598; P<0.01).
Characterization of transgene expression
Residual baculoviruses in the collected lentivirus media were evaluated by end-point titering and the titers were 0.1–0.5% of the dose used for the 293T cell transductions. To confirm that the transgene expression originated from the produced lentiviruses, and not from the residual baculoviruses, 293T cells were transduced only with the BAC-transfer baculovirus. HeLa cells were then transduced using this medium collected in a similar way than in the lentivirus production, and GFP-positive cells were analyzed with a flow cytometer 4 days after the transduction. No GFP-positive cells were detected.
Baculovirus vectors neither replicate in vertebrate cells nor can they integrate into the host genome.22 Gene expression from these vectors is transient and usually diminishes in 2 weeks. However, transgene expression from an integrated lentivirus is relatively stable assuming no silencing of the transgene expression occurs.28 Transduction with the baculovirus-produced lentiviruses led to an efficient GFP expression which could still be observed 43 days post-transduction (Figure 4a). Expression was also detected by fluorescent microscopy in HeLa cells at day 3 (Figures 4c and d). No morphologic changes in the transduced cells were detected during the experiments by microscopy or flow cytometry. For comparison, baculovirus-mediated GFP expression achieved using MOIs 100 and 1000 (18.7±1.9 and 11.5±0.4% at day 3, respectively) were undetectable 17 days post-transduction (Figure 4b).
Replication competent lentiviruses
Replication competent lentivirus were tested by p24 ELISA assay. HeLa cells were transduced with lentivirus-containing media. Transduction efficiencies were verified by flow cytometer. Cells were cultured for 4 weeks and concentration of p24 in the supernatant was repeatedly measured. An increasing concentration of p24 would indicate an ongoing viral replication,29 but no such increase was detected. Media collected from the transduced HeLa cells after 2.5 weeks were further used to transduce naive HeLa cells but no GFP expression was detected neither with fluorescent microscopy nor a flow cytometer.
Baculoviruses possess several advantages for gene delivery applications.22 They have an extraordinary large insert capacity (>100 kb), capable of transducing most mammalian cell lines22 and can be cultured in large-scale suspension cell cultures under serum-free conditions.30 In addition to, baculoviruses are easy to produce in large scale and high titers, and they posses minimal safety problems as they cannot replicate in mammalian cells.31 No cytotoxicity has been detected even with high MOIs.32 Baculoviruses have been widely used for a large-scale protein production in insect cells21 and for the production of VLP, such as hepatitis VLP.27 Intact viruses have been also produced using hybrid baculoviruses. Production of adeno-associated viruses (AAV) vector in insect cells by baculoviruses has gained much interest due to difficulties to produce enough AAV for clinical trials by conventional plasmid transfection methods.33 Baculovirus-mediated production of recombinant influenza viruses,23 adenoviruses24 and AAV26 in mammalian cells has been also described.
In the current study, we demonstrated for the first time a successful generation of functional lentiviruses using hybrid baculoviruses. Lentivirus titers produced by the baculoviruses were comparable to those produced using the conventional four-plasmid transfection methods. Good lentivirus titers were achieved when optimal doses of the baculoviruses and extended transduction times were used. Decreased lentivirus titers and packaging cell death were observed when very high MOIs of baculoviruses were used. This was due to the toxicity of VSV-G11, 12 to the packaging cells since no problems were observed when the VSV-G-expressing baculovirus was left out while keeping the total number of baculovirus particles constant (data not shown). By replacing Dulbecco's modified Eagle's medium (DMEM) with RPMI 1640, the lowest baculovirus dose (MOI 50) gave already the best lentivirus titers. This is in line with the fact that the transduction medium affects baculovirus-mediated gene expression in vertebrate cells.34, 35 To confirm the functionality of the generated lentiviruses, HeLa cells were transduced and sustained GFP expression was observed for 6 weeks. On the contrary, with the control baculovirus the GFP expression was lost in 17 days. If lentivirus generation was performed by omitting either BAC-gag-pol, BAC-Rev or BAC-VSV-G, no lentivirus was produced.
Although baculoviruses are safe, contamination of the lentivirus preparation with baculoviruses is not desirable. The amount of residual baculoviruses in the lentivirus preparations was in the range of 0.1–0.5% of the baculovirus dose used in the lentivirus production. The residual baculoviruses can probably be further reduced by simply adding an extra washing step and using improved downstream purification methods.36
One of the major concerns associated with the use of lentivirus vectors is the probability of generating pathogenic human viruses. To avoid this, the lentivirus genome has been separated into four different production plasmids in order to minimize the risk of RCL formation by recombination.37, 38 No RCL was detected in the baculovirus-mediated lentivirus preparations in this study. p24 levels were not increased after prolonged cultures and no GFP expression was detected.
Scalability of the virus production for clinical studies in adherent cells remains difficult.39 Thus, adaptation of lentivirus production to suspension cell cultures would be advantageous. Our preliminary results in suspension-adapted HEK293 cells in serum-free conditions showed a very efficient baculovirus transduction (>95% GFP-positive cells). Production of lentiviruses in insect cells would also be an attractive alternative. However, expression of Gag has resulted in an assembly of only immature VLPs40, 41, 42 making lentivirus production in insect cells challenging.
In conclusion, our study demonstrates that hybrid baculoviruses can be used for the efficient production of lentiviral vectors. Baculovirus technology offers thus an attractive possibility for a scalable lentivirus production because baculoviruses are safe and fast to produce in high titers, and can transduce mammalian cells in suspension cultures under serum-free conditions.
Materials and methods
Cloning of plasmids for the production of baculoviruses
All necessary elements for the production of third-generation lentiviral vectors in mammalian cells were subcloned into baculovirus donor vector pFastBac1 (Invitrogen, Carlsbad, CA, USA) to construct four recombinant baculoviruses, BAC-transfer, BAC-gag-pol, BAC-VSV-G and BAC-rev, derived from AcMNPV (Figure 1a). First, we cloned a polylinker containing multiple cloning sites (PmlI/NheI/PstI/SalI/AflII/PacI/SpeI/MluI/PmeI/EcoRI/ApaI/SwaI/AscI) into the unique AvrII site of pFastBac1. The sequence of the polylinker was 5′-IndexTermCACGTGGCTAGCCTGCAGGTCGACCTTAAGTTAATTAAACTAGTACGCGTGTTTAAACGAATTCGGGCCCATTTAAATGGCGCGCC-3′. The donor vector contained also the red fluorescent protein marker gene (DsRed) under the control of a polyhedrin promoter for convenient baculovirus titer determination.
To generate the third-generation self-inactivating lentivirus transfer construct (LV1-GFP) ΔNGFP from plasmid LV-hPGK-ΔNGFP-WPRE-SIN43 was replaced by GFP. In this construct, the GFP marker gene is driven by the phosphoglycerate kinase promoter. The pBAC-transfer vector was constructed by subcloning the relevant sequence from LV1-GFP into the pFastBac1 donor vector polylinker in two stages. To clone the first part of the sequence, LV1-GFP was digested with BsrBI and AscI and subcloned into SwaI/AscI site of the donor vector polylinker. The second part of the sequence was cloned by digesting LV1-GFP with AscI and AvrII and inserting the fragment into the AscI and AvrII sites of the modified pFastBac1 plasmid.
The packaging construct (pBAC-gag-pol) expressing gag and pol driven by a CMV promoter was derived from the plasmid pMDLg/pRRE2, 37 by ApaLI digestion and subcloned into the SmiI site of the donor vector. Prior to ligation, ApaLI ends were blunted with T4 DNA Polymerase (Finnzymes, Helsinki, Finland).
The VSV-G envelope construct from the plasmid pCMV-VSV-G (kindly supplied by Dr T Friedmann, USCD, La Jolla CA, USA) was subcloned into the pFastBac1 vector in two stages. First, pCMV-VSV-G was digested with NotI and blunted using T4 DNA polymerase prior to digestion with EcoRI. This fragment was subcloned to the SmiI/EcoRI site of the polylinker. The second part of the sequence was digested from pCMV-VSV-G with EcoRI and subcloned into the polylinker EcoRI site.
Rev cDNA was obtained by PCR from the plasmid pRSV-REV37 using forward and reverse primers: 5′-IndexTermCGAAGGAATTC GTCGCCACC ATG GCAGGAAGAAGCGGA-3′ (sequence for nucleotides 1–18 of the rev gene in bold, Kozak consensus sequence in italic, EcoRI site underlined) and 5′-IndexTermAGCTAGCTAGC GTATTCTCCTGACTCCAATATTGT-3′ (sequence for nucleotides 349–325 of the rev gene, NheI site underlined), respectively. The amplified PCR product was digested with EcoRI and NheI, purified using a Wizard Clean up kit (Promega, Madison, WI, USA), and subcloned into the EcoRI/NheI site of the pFastBac1 polylinker to form pBAC-rev. Rev cDNA was under the control of the CMV promoter which was previously subcloned as a NruI/EcoRI fragment from the pcDNA3 vector (Invitrogen) into the SwaI/EcoRI site of the pFastBac1 polylinker.
Production of lentiviruses
293T cells were plated 24 h before transduction. Cells were cultured in DMEM (Sigma-Aldrich Company, Ayrshire, UK) or RPMI 1640 (Cambrex Bio Science, Verviers, Belgium) both supplemented with 10% fetal bovine serum. Transduction was performed with varying MOI between 50 and 1000 PFU per cell in either serum-free or serum-supplemented DMEM or RPMI. After 4 h incubation in serum-free medium or 18 h in serum-supplemented DMEM or RPMI at 37 °C, the cells were washed and the medium was changed. The cell supernatant containing lentiviruses was collected 48 h post-transduction and centrifuged at 1500 r.p.m. for 10 min at room temperature. Viruses produced were compared to the viruses generated by conventional four-plasmid transient transfection.2
As controls, we made batches where each of the three baculoviruses was missing (BAC-gag-pol, BAC-Rev or BAC-VSV-G). We also prepared lentiviruses by four-plasmid transfection method in 293T cells.2 To improve the attachment of the cells to the bottom of the plates, we coated the plates with poly-L-lysine according to the manufacturer's instructions (Sigma-Aldrich Company).
Titering of lentiviruses
Transforming units of lentiviruses (TU ml−1) were determined by analyzing the number of virus particles able to transduce HeLa cells. On day 1, HeLa cells were seeded on six-well plates at 1 × 105 or on 96-well plates at 5 × 103 cells per well. The lentivirus transduction with different dilutions was carried out on day 2 with serial dilution. On day 5, the cells were visualized with fluorescent microscopy and analyzed by a flow cytometer (FACSCanto II, BD Biosciences, San Jose, CA, USA) to reveal the percentage of cells which were transduced by GFP-expressing lentiviruses. Titers were calculated as described previously.2
Long-term expression of transgene
HeLa cells (5 × 103) were seeded on 96-well plates, transduced next day with baculovirus-produced lentiviruses and cells were cultured up to 6 weeks. GFP expression and cell morphology was monitored weekly by microscopy and flow cytometry. As an additional control, HeLa cells were also transduced with baculovirus BAC-transfer-expressing GFP and the expression was monitored in a similar way.
Determination of p24 concentration
The amount of lentiviral capsid protein p24 (pg ml−1) was determined by HIV-1 p24 ELISA kit (NEN, Life Science Products, Boston, MA, USA). Testing of RCL was done by p24 ELISA determination from the cell culture supernatants. HeLa cells were transduced with lentivirus, and the transduction efficiency was monitored by flow cytometry. Cells were cultured for 4 weeks, supernatants were collected and the concentration of p24 in the supernatants was measured repeatedly as a marker of RCL. This was further confirmed by transducing naive HeLa cells with collected supernatants, and the GFP expression was monitored by fluorescent microscopy and flow cytometry.
Statistical analyses were performed by GraphPadPrism 4 (GraphPad Software Inc., San Diego, CA, USA). P<0.05 was considered statistically significant.
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We thank Miia Roschier, Tarja Taskinen, Erik Peltomaa, Riikka Eisto, Joonas Malinen, Anne Martikainen and Anneli Miettinen for technical assistance, Dr Olli Laitinen, Minna Kaikkonen and Petri Mäkinen for their invaluable discussions, and Dr Jani Räty and Dr Roseanne Girnary for reviewing the manuscript. This study was supported by Ark Therapeutics Group Plc and EU Clinigene (LSHB-CT-2006-018933) Consortium Flexibility Funds.
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Lesch, H., Turpeinen, S., Niskanen, E. et al. Generation of lentivirus vectors using recombinant baculoviruses. Gene Ther 15, 1280–1286 (2008). https://doi.org/10.1038/gt.2008.76
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