Baculovirus-based production of biopharmaceuticals free of contaminating baculoviral virions
11236307 · 2022-02-01
Assignee
Inventors
Cpc classification
C12N7/00
CHEMISTRY; METALLURGY
C12N2750/14143
CHEMISTRY; METALLURGY
C12N2710/14152
CHEMISTRY; METALLURGY
C12N15/86
CHEMISTRY; METALLURGY
C12N2710/14143
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to methods for the production of biopharmaceuticals implementing a baculovirus-based system. These methods advantageously allow the production of biopharmaceuticals with a reduced number of or without contaminating baculoviral virions.
Claims
1. A method for the production of a biopharmaceutical product, comprising: (a) infecting a biopharmaceutical-producing insect cell with at least one baculovirus, said at least one baculovirus comprising a genome coding for said biopharmaceutical product, and (b) maintaining the biopharmaceutical-producing insect cell under conditions such that the biopharmaceutical product is produced, wherein the genome of said at least one baculovirus is deficient for the p6.9 gene and, optionally, deficient for at least one gene selected from vp80, vp1054 and vp39, or wherein said biopharmaceutical-producing insect cell comprises an expression control system allowing the inactivation of the p6.9 gene and, optionally, inactivation at least one gene selected from vp80, vp1054 and vp39.
2. The method according to claim 1, wherein the p6.9 gene is made deficient in said genome by way of nucleotide substitution, insertion or deletion.
3. The method according to claim 1, wherein the biopharmaceutical-producing insect cell is a recombinant insect cell comprising a construct expressing a dsRNA specific for the p6.9 gene, the dsRNA being optionally expressed under an inducible promoter.
4. The method according to claim 1, wherein the at least one baculovirus is produced before step (a) in a baculovirus-producing cell expressing a complementing copy of the p6.9 gene.
5. The method according to claim 1, wherein the genome of said at least one baculovirus is further deficient for at least one gene selected from vp80, vp1054 and vp39 or wherein said biopharmaceutical-producing insect cell further comprises an expression control system allowing the inactivation of at least one gene selected from vp80, vp1054 and vp39.
6. The method according to claim 1, wherein the deficiency or inactivation of the p6.9 gene does not affect very late expression from said baculovirus in comparison to very late expression from wild-type baculovirus.
7. The method according to claim 1, wherein the at least one baculovirus is Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) or Bombyx mori nucleopolyhedrovirus (BmNPV).
8. The method according to claim 1, wherein the biopharmaceutical product is a recombinant protein, a recombinant virus or a virus-like particle.
9. The method according to claim 8, wherein the biopharmaceutical product is a recombinant AAV.
10. The method according to claim 1, wherein the biopharmaceutical product is coded by at least one gene introduced in the recombinant baculovirus genome under the control of the polyhedrin or p10 promoter.
11. A bacmid comprising a baculoviral genome, wherein said genome is deficient for the p6.9 gene.
12. The bacmid according to claim 11, wherein said genome is further deficient for at least one gene selected from vp80, vp1054 and vp39.
13. The bacmid according to claim 11, wherein said genome is a mutated genome of AcMNPV.
14. A recombinant baculovirus vector, wherein the genome of said baculovirus vector is deficient for the p6.9 gene.
15. The recombinant baculovirus vector according to claim 14, wherein the genome of said baculovirus is further deficient for at least one gene selected from vp80, vp1054 and vp39.
16. The recombinant baculovirus vector according to claim 14, wherein said vector is an AcMNPV baculovirus vector.
17. An insect cell infected with a recombinant baculovirus vector comprising a genome which is deficient for the p6.9 gene.
18. The insect cell according to claim 17, wherein said genome is further deficient for at least one gene selected from vp80, vp1054 and vp39.
19. The insect cell according to claim 17, wherein said genome is a mutated genome of AcMNPV.
20. A method for the production of a baculovirus deficient for the p6.9 gene, comprising the step of transfecting an insect cell comprising an expression cassette coding for the p6.9 gene with a bacmid comprising a baculoviral genome, wherein said genome is deficient for the p6.9 gene.
21. The method according to claim 20, wherein said insect cell further comprises an expression cassette coding for at least one gene selected from vp80, vp1054 and vp39 and wherein said baculoviral genome is further deficient for said at least one gene selected from vp80, vp1054 and vp39.
Description
LEGENDS TO THE FIGURES
(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication, with color drawing(s), will be provided by the Office upon request and payment of the necessary fee.
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(3) (A) Determination of very late gene expression level using fluorescent microscopy. Cells were infected at MOI=10 TCID.sub.50 units/cell and transfection with gene-specific dsRNA for vp1054, vp39, vp80, dbp and ec-27 was performed at 1 h post infection (p.i.). The level of very late gene expression was checked by EGFP-specific fluorescence at 48 h p.i. dsRNAs specific for egfp and cat sequences were used as RNAi controls. (B) Measurement of very late gene expression levels by an immunoblotting-based assay. The cells were infected with AcMNPV-EGFP at MOI=1 and transfection with gene-specific dsRNA was also performed at 1 h p.i. The level of very late gene expression was analyzed by using a rabbit anti-EGFP polyclonal antiserum at 48 h p.i. Anti-vp39 and anti-α-tubulin antibodies were used as internal controls. (C) Titration and detection of produced budded virions in dsRNA-treated cells. Budded virions were harvested at 36 hours p.i., and used either for end-point dilution assays to measure titers of infectious virions, or for PCR-based detection to check the presence of virus particles. (D) Presence of occlusion-derived virions and rod-shaped structures in vp39- and vp80-down-regulated cells. The cells were harvested 36 hours p.i., lysed, and the cell lysates were ultracentrifuged through a cushion of 40% sucrose solution (45,000 rpm for 1 hour, Beckman SW55). Pellets were resuspended in demi-water and analyzed by negative staining electron microscopy. The bars represent 100 nm.
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EXAMPLES
Example I
(20) Materials and Methods
(21) Insect Cells and Viruses
(22) Spodoptera frugiperda (Sf9) cells were maintained in SF900-II serum-free medium (Invitrogen) under standard conditions. Recombinant bacmid-derived AcMNPV virus (AcMNPV-EGFP) carrying an egfp reporter gene under control of the very late polyhedrin promoter transposed into the polyhedrin locus was obtained from Pijlman et al. (2006). The virus was propagated and its titers were determined by an end-point dilution assay in Sf9 cells.
(23) In Vitro Synthesis of dsRNA
(24) The method used to synthesize dsRNA is similar to that described by Ramadan et al. (2007) with minor modifications. All DNA templates were PCR amplified using primers with twenty-five nucleotide overhangs homologous to the T7 RNA polymerase promoter sequence 5′-gcttctaatacgactcactataggg-3′. The sequences of the primers indicated below are given in Table 1. The following primers were used for amplifying these genes: primers vp39-F and vp39-R for vp39; primers 45510 and 46235 for vp1054, primers 90292 and 90889 for vp80; primers ec-27-F and ec-27-R for odv-ec27; and primers dbp-F and dbp- for dbp. To test the efficiency of the RNAi studies we made dsRNA against egfp with primers gfp-F and gfp-R, and to have a negative control we made dsRNA with primers cat-F and cat-R for the chloramphenicol acetyl transferase (cat) gene.
(25) The PCR products were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, UK) and were used as templates for dsRNA in vitro synthesis using the T7 RiboMAX™ Express RNAi System (Promega, Madison, Wis., USA) according to manufacturer's protocol. Briefly, approximately 1 μg of purified DNA templates were used for RNA synthesis at 37° C. for 4 h. After synthesis, DNA templates were removed by digestion with DNase. Complementary RNA strands were annealed by incubation at 70° C. for 10 min followed by slow cooling to room temperature (˜30 min). Non-annealed (single-stranded) RNA molecules were degraded by RNase A treatment (30 min, 37° C.). Finally, the dsRNA was isopropanol precipitated, resuspended in DEPC-treated sterile water to a final concentration of 0.5-1 mg/ml, and its purity and integrity were checked by agarose gel electrophoresis. The dsRNA was kept at −80° C. in aliquots of 40 μl. Immediately before transfection, the dsRNA was thawed on ice.
(26) RNAi Procedure in Baculovirus-Infected Insect Cells
(27) Sf9 cells were seeded in 24-well tissue culture plates (2×10.sup.5 cells/well) in 1 ml Sf900-II culture medium without serum at 28° C. After two hours, the culture medium was removed, and the cells were infected with recombinant baculovirus AcMNPV-EGFP at a multiplicity of infection (MOI) of 10 TCID.sub.50 units/cell for 1 h, under standard conditions. One hour post infection (p.i.), dsRNA (20 μg/well) was introduced into the cells by Cellfectin™-based (Invitrogen) transfection in Grace's serum-free medium. After 4 h, the transfection mixture was replaced with Sf900-II serum-free medium. The cells were incubated for a total of 48 h p.i. at 28° C. and then harvested by centrifuging at 1000×g for 5 min for Western blot and electron microscopy analysis. However, one fifth of the culture medium was harvested at 36 h p.i., and used for titration of budded virions by end-point dilution assays or for PCR-based detection of viral DNA. In all the experiments, dsRNA corresponding to the cat gene was taken as negative control. On the other hand, egfp gene-specific dsRNA was used as positive control for the RNAi procedure.
(28) SDS-Polyacrylamide Electrophoresis and Western Blotting
(29) For immuno-detection, the Sf9 cells were disrupted in 125 mM Tris-HCl, 2% sodium dodecyl sulfate (SDS), 5% 2-mercapthoethanol, 10% glycerol, 0.001% bromophenol blue, pH 6.8 at 95° C. for 10 min. Proteins were separated in 10% SDS-polyacrylamide gels, and subsequently transferred to Immobilon-P membranes (Millipore) by semi-dry electroblotting. Membranes were blocked for 30 min in 1×PBS containing 2% fat-extracted milk powder, followed by incubation for 1 h at room temperature with either rabbit polyclonal anti-GFP antiserum (Molecular Probes), rabbit polyclonal anti-VP39 antiserum, or monoclonal anti-α-tubulin antibody (Sigma-Aldrich), all diluted 1/2000 in 1×PBS containing 0.2% milk power. After washing (3×10 min) in 1×PBS, the membranes were incubated with 1/4000 dilution of either goat anti-rabbit IgG or rabbit anti-mouse IgG antibodies conjugated with alkaline phosphatase (Sigma). After final washing (3×10 min) in AP buffer (100 mM Tris-Cl [pH 9.5], 100 mM NaCl, 5 mM MgCl.sub.2), the blots were developed with 5-bromo-4-chloro-3-indolyl phosphate nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolylphosphate (BCIP) (Bio-Rad) according to the manufacturer's instructions.
(30) Preparation of Viral Genomic DNA and its PCR-Based Detection
(31) Two-hundred microliters of cell culture medium were collected at 36 h p.i. and used for preparation of viral DNA. The cells and cell debris were removed from samples by centrifuging at 1000×g for 5 min. Supernatants containing budded virions were quantitatively transferred to new sterile tubes and centrifuged again at 12000×g for 90 min. Pelleted BVs were re-suspended in 200 μl TE buffer (10 mM Tris-HCl [pH 7.5], 1 mM EDTA) containing Proteinase K (540 μg/ml), and incubated at 55° C. for 2 h. A phenol:chloroform:isoamyl alcohol (25:24:1) and a chloroform extraction were subsequently performed. The DNA was precipitated by adding an equal amount of isopropanol and the pellet was washed with 70% ethanol. The DNA pellet was dissolved in 15 μl sterile water, and 2 μl of the final DNA solution was applied to PCR-based detection of the vp39 gene sequence using primers mentioned above. All PCR reactions were performed in 25 μl volumes including: 2 μl DNA, 200 μM dNTPs, 10 pmol of each primer, 1.5 mM MgCl.sub.2 and 1.5 U GoTaq DNA polymerase (Promega). Amplification conditions were as follows: an initial denaturation at 94° C. for 2 min, after which 30 cycles of denaturation (30 s at 94° C.), primer annealing (20 s at 60° C.) and primer extension (25 s at 72° C.). The termination cycle was 7 min at 72° C. Negative controls were included in all PCR amplifications to test for contaminants in the reagents. Aliquots (3.0 μl) of the PCR products were analysed by electrophoresis in 1.2% (w:v) agarose gels, with 1×TAE buffer, stained with ethidium bromide (0.5 μg/ml).
(32) Generation of an Antibiotic Resistance Gene-Free AcMNPV Vp80-Null Bacmid
(33) To determine whether the VP80 protein has an essential role in the context of viral progeny production, we constructed an AcMNPV bacmid (derived from bMON14272 (from Invitrogene)) with a deletion of the vp80 ORF by homologous recombination in E. coli. To accomplish this, a cat gene flanked by mutant LoxP sites (Suzuki et al., 2005) was amplified using PCR primers vp80-KO-F and vp80-KO-R (see Table 1) from a plasmid comprising a cat gene flanked by mutant LoxP sites. The resulting PCR fragment, which contained the cat gene flanked by mutant LoxP sites and AcMNPV ˜50-bp homology sequences to the 5′ or 3′ proximal region of the vp80 ORF, was treated with DpnI and gel-purified to eliminate the template plasmid. The PCR product was then transformed into DH10ß E. coli cells containing bMON14272 (Invitrogen) and the Lambda RED recombinase-producing plasmid pKD46 (Datsenko & Wanner, 2000), which had been prepared in the following manner. Transformed DH10ß-bMON14272/pKD46 E. coli cells were grown in 50-ml LB (2.0% peptone, 0.5% yeast extract, 85.5 mM NaCl, [pH 7.0]) cultures with kanamycin (50 μg/ml), ampicillin (100 μg/ml) and L-arabinose (1.5 mg/ml) at 30° C. to an OD.sub.600 of ≈0.6 and then made electrocompetent by a standard procedure. The electroporated cells were incubated at 37° C. for 3 h in 3 ml LB medium and plated on LB-agar containing chloramphenicol at a concentration of 6.5 μg/ml. After 48-h incubation at 37° C., the chloramphenicol-resistant colonies were streaked to fresh LB-agar medium with 34 μg/ml chloramphenicol. The plates were incubated at 37° C. overnight, and colonies resistant to chloramphenicol were selected for further confirmation of the relevant genotype by PCR. Primers 90292 and 90889 were used to confirm the absence of the vp80 ORF, and primers cat-F and cat-R were employed to verify the presence of cat cassette into bacmid (detailed sequences in Table 1).
(34) To eliminate the introduced antibiotic resistance gene (cat) from the bacmid backbone, a Cre/LoxP recombinase system was employed. A Cre recombinase-carrying plasmid pCRE obtained from Jeanine Louwerse (LUMC Leiden, The Netherlands) was introduced into DH10b-bMON14272-vp80null E. coli cells, and CRE expression was subsequently induced by the addition of isopropyl thiogalactoside (IPTG). Briefly, the electroporated cells were incubated at 37° C. for 3 h in 3 ml of LB medium (2.0% peptone, 0.5% yeast extract, 85.5 mM NaCl, [pH 7.0]) and plated on LB-agar medium containing 50 μg/ml kanamycin, 100 μg/ml ampicillin and 2 mM IPTG. After 24-h incubation, colonies resistant to kanamycin and ampicillin were selected for further verification of the desired genotype by PCR. In PCR-based analysis, primers 89507 and 91713 (Table 1) were used to verify elimination of cat gene from bacmid backbone. Positive clones were also confirmed by DNA-sequencing.
(35) To recover transposition competence, the helper transposase-encoding plasmid pMON7124 (Invitrogen) was re-introduced into DH10ß-bMON14272-vp80null E. coli cells. Finally, the egfp reporter gene was introduced into the vp80-null bacmid to facilitate observation of its behaviour in insect cells. Briefly, the egfp reporter gene was amplified using PCR oligonucleotides gfp-NheI-F and gfp-SphI-R (Table 1) from plasmid pEGFP-N3 (Clontech). The PCR product was cloned into plasmid pJet1.2/Blunt using CloneJET™ PCR Cloning Kit (Fermentas) according to manufacturer's protocol. Subsequently, the egfp ORF was excised from error-free pJet1.2-egfp with NheI and SphI and subcloned into NheI/SphI-digested pFastBacDUAL (Invitrogen), to generate plasmid pFB-egfp. An expression cassette containing the egfp reporter gene under transcriptional control of the very late p10 promoter was transposed from pFB-egfp into polyhedrin locus of vp80-null bacmid as described in the Bac-to-Bac manual (Invitrogen). In the resulting genome, the complete vp80 ORF has been removed (see
(36) Construction of Repaired Vp80-Null Bacmids
(37) To prepare vp80 repair donor vectors, we modified plasmid pFB-egfp (noted above) by removing the polyhedrin promoter and replacing it with a fragment containing the vp80 promoter region and the vp80 ORF. First, a 2300-bp fragment containing both the vp80 promoter and ORF sequence was amplified using primers pvp80-StuI-F and vp80-XbaI-R (Table 1) from bacmid bMON14272 template, and cloned into vector pJet1.2/Blunt (Fermentas) to form pJet1.2-pvp80-vp80. After DNA sequence verification, the vp80 cassette was excised from pJet1.2-pvp80-vp80 by StuI/XbaI double digestion, and then subcloned into Bst1107I/XbaI-digested and gel-purified pFB-egfp to generate donor plasmid pFB-egfp-pvp80-vp80. Parallelly, a donor plasmid pFB-egfp-polh-vp80, where vp80 ORF is driven by the very late polyhedrin promoter (polh) was constructed. To this aim, a 2105-bp fragment carrying the vp80 ORF was amplified using primers vp80-SacI-F and vp80-XbaI-R (Table 1) and cloned into pJet1.2/Blunt, to generate pJet1.2-vp80. In the final step, the vp80 ORF was cut out (SacI/XbaI) from pJet1.2-vp80, and subcloned into SacI/XbaI-digested pFB-egfp, to create pFB-egfp-polH-vp80.
(38) To overcome a problem associated with the unavailability of anti-VP80 antibody, FLAG tag decoration (N- and C-terminus fusion) of VP80 was performed to facilitate immunodetection. The N-terminally fused FLAG-vp80 sequence was generated by a double-step PCR strategy, a so-called fusion PCR. First, a 259-bp fragment containing the vp80 promoter and the FLAG tag was PCR amplified using primers pvp80-StuI-F and vp80-FLAG-R1 from the bMON14272 bacmid template. After gel-purification and DNA quantification, the 259-bp fragment was used as forward primer in a second step PCR amplification with the reverse primer vp80-XbaI-R on the bMON14272 bacmid template. The final PCR product (2324 bp) was cloned into vector pJet1.2/Blunt (Fermentas) to form pJet1.2-pvp80-FLAG-vp80. After DNA sequence verification, the FLAG-vp80 cassette was excised from pJet1.2-pvp80-FLAG-vp80 by StuI/XbaI double digestion, and then subcloned into Bst1107I/XbaI-digested and gel-purified pFB-egfp to generate donor plasmid pFB-egfp-pvp80-FLAG-vp80. The C-terminally fused vp80-FLAG cassette was amplified using pvp80-StuI-F and vp80-FLAG-R from the bMON14272 bacmid template. The 2324-bp fragment was cloned into pJet1.2/Blunt, and subsequently transferred into pFB-egfp in a similar way as previous constructs.
(39) The inserts of all developed donor plasmids were transposed into the vp80-null bacmid following the Bac-to-Bac protocol (Invitrogen). Screening of transposition-positive constructs into the polh locus was done by a triplex PCR-based assay employing a M13 forward and reverse primers and a gentamicin resistance gene-specific primer GenR (Table 1).
(40) Transfection-Infection Assay
(41) Bacmid DNAs were prepared from 1.5-ml over-night bacterial cultures of 2 to 3 independent colonies carrying the bacmid with the inserted heterologous gene according to the Bac-to-Bac manual (Invitrogen) and were analyzed in parallel. For transfections, 1 μg of each bacmid DNA preparation was used to transfect 1×10.sup.6 Sf9 cells in a 6-well plate by the Cellfectin™-based transfection protocol as described in the Bac-to-Bac (Invitrogen) manual. From 72 h to 120 h post transfection (p.t.), viral propagation was checked by fluorescence microscopy. At 120 h p.t., the cell culture medium was centrifuged for 5 min at 2000×g to remove cell debris, and this clarified supernatant was used to infect 1.5×10.sup.6 Sf9 cells in 6-well plates. After 72 h p.i., the spread of virus infection was again monitored by fluorescence microscopy. In all experiments, a wild-type bMON14272 bacmid carrying the egfp reporter gene under control of the p10 promoter was used as positive control. A bMON14272-gp64null bacmid also carrying the egfp reporter gene under control of the p10 promoter served as negative control, since it has lost the ability of cell-to-cell movement of the infection (Lung et al., 2002).
(42) Time-Course Characterization of Viral Propagation in Cell Culture
(43) Time course analyses were performed to compare budded virus production of the AcMNPV-vp80null virus and the various repair constructs in comparison to the wild type AcMNPV bacmid (Ac-wt) all containing egfp. Briefly, the Sf9 cells were seeded in 6-well tissue culture plates (1×10.sup.6 cells/well in 1 ml Sf900-II culture medium without serum at 28° C.). After two hours, the culture medium was removed, and the cells were transfected with 5 μg bacmid DNA, under standard conditions as recommended in the Bac-to-Bac manual (Invitrogen). Cell culture supernatants were harvested at 24, 48, 72, 96 and 120 h p.t., and analysed for the production of infectious budded virus by an end-point dilution assay to determine the tissue culture infective dose 50 (TCID.sub.50). Infection was determined by monitoring egfp expression (from the p10 promoter). The average values of infectious titers derived from three independent transfections were calculated and plotted into graphs.
(44) Transmission Electron Microscopy
(45) Insect Sf9 cells were seeded in 25 T flask (3.5×10.sup.6 cells/flask), and transfected with 20 μg either the Ac-Δvp80, rescue Ac-Δvp80-vp80 or Ac-wt bacmid construct. After 48 h p.t., the cells were harvested and prepared for transmission electron microscopy as described previously (van Lent et al., 1990). Samples were examined and photographed with a Philips CM12 electron microscope.
(46) Budded Virus Production Assay
(47) Insect Sf9 cells were seeded in two 25 T flasks (3.5×10.sup.6 cells/flask), and transfected with 20 μg either Ac-Δvp80, Ac-Δvp80-vp80, Ac-Δvp80-pH-vp80, Ac-Δvp80-FLAG-vp80, Ac-Δvp80-vp80-FLAG, or Ac-wt bacmid construct. Five days p.t., the BV-enriched cell culture supernatants were harvested, and ultracentrifuged through a cushion of 10% sucrose solution (25,000 rpm for 1.5 hour, Beckman SW32). Pelleted budded virions were resuspended in sterile demi-water, and prepared for either negative staining electron microscopy, SDS-polyacrylamide electrophoresis, or PCR-based detection (as mentioned above).
(48) Purification of ODVs and Rod-Shaped Structures from Infected Cells
(49) The presence of ODVs and rod-like structures in infected/transfected insect cells was analyzed by electron microscopy (EM). For this purpose, insect cells were harvested 48 h p.i., lysed and the cell lysates were ultracentrifuged through a 40% sucrose cushion in TE (1 mM Tris-HCl pH 7.4, 0.1 mM EDTA) buffer (45,000 rpm for 1 hour, Beckman SW55). Pellets were resuspended in sterile demi-water and analyzed by negative staining EM as described previously (van Lent et al., 1990).
(50) Development of Transgenic Sf9-Derived Cell Line Expressing Vp80
(51) To develop a cell line, which produces the VP80 protein, a 2105-bp fragment carrying the vp80 ORF was amplified using primers vp80-SacI-F and vp80-XbaI-R (Table 1) and cloned into pJet1.2/Blunt, to generate pJet1.2-vp80. In the next step, the vp80 ORF was cut out (SacI/XbaI) from pJet1.2-vp80, and subcloned into SacI/XbaI-digested pIZ (Invitrogen), to create pIZ-vp80. The resulting plasmid vector pIZ-vp80 was linearized with Eco57I, and gel-purified. Sf9 cells were seeded in a six-well plate (1×10.sup.6 cells/well), and transfected with 10 μg of the linearized vector. After 24 hours post-transfection, cells were selected by cell culture medium containing Zeocin™ (300 μg/ml) for 2 to 3 weeks, until no control Sf9 cells survived under the same conditions. Cells were then propagated as an uncloned cell line.
(52) Generation and Characterization of a AcMNPV Vp39-Null Bacmid
(53) To study the role of the vp39 gene in the context of viral progeny production and the nucleocapsid assembly process, we constructed an AcMNPV bacmid (bMON14272) with a deletion of vp39 by homologous recombination in E. coli according to the same procedure as noted above for the AcMNPV vp80null bacmid construct. Since the sequence of the vp39 ORF is overlapping with promoter sequences of both flanking ORFs (cg-30 and lef-4), only an internal part of the vp39 ORF could be deleted, to avoid de-regulations of cg-30 and lef-4 expression. To reach this, a cat gene flanked by mutant LoxP sites was amplified using PCR primers vp39-KO- and vp39-KO-R (Table 1) from a plasmid comprising a cat gene flanked by mutant LoxP sites. The resulting PCR fragment, which contained the cat gene flanked by mutant LoxP sites and ˜50-bp sequences homologous to an internal region of the vp39 ORF, was treated with DpnI and gel-purified to eliminate the template plasmid. The PCR product was then transformed into DH10ß E. coli cells containing bacmid bMON14272 (Invitrogen) and Lambda RED recombinase-producing plasmid pKD46 (Datsenko & Wanner, 2000) prepared in the above mentioned manner. In the final step, colonies resistant to kanamycin were subjected to PCR-based analysis using primers 75834 and 76420 (Table I) to verify insertion/elimination of the cat gene from the bacmid backbone. Positive clones were further verified by DNA-sequencing of the obtained PCR products. According to this protocol, an internal part (498 nt=166 aa) of the vp39 ORF was removed, coordinates: 75894-76391 as indicated in
(54) Construction and Analysis of Repaired Vp39-Null Bacmids
(55) To prepare a vp39 repair donor vector, we modified plasmid pFB-egfp (noted above) by introduction of the vp39 ORF under control of the polyhedrin promoter. Initially, a 1073-bp fragment was amplified using primers vp39-SacI-F and vp39-XbaI-R (see Table I for primer sequences) from the bMON14272 template, and cloned into vector pJet1.2/Blunt (Fermentas) to form pJet1.2-vp39. After DNA sequence verification, the vp39 ORF was excised from pJet1.2-vp39 by SacI/XbaI double digestion, and then subcloned into SacI/XbaI-digested and gel-purified pFB-egfp to generate donor plasmid pFB-egfp-vp39. After an unsuccessful attempt to rescue AcMNPV vp39null with pFB-egfp-vp39, a set of novel donor plasmids was prepared. First, a 2498-bp fragment containing vp39 and lef-4 ORFs was PCR-generated using primers vp39-StuI-F and lef-4-XbaI-R from bacmid bMON14272 template, and cloned into vector pJet1.2/Blunt (Fermentas) to form pJet1.2-vp39-lef-4. After DNA sequence confirmation, the fragment containing vp39 and lef-4 ORFs was excised from pJet1.2-vp39-lef-4 by StuI/XbaI double digestion, and then subcloned into StuI/XbaI-digested and gel-purified pFB-egfp to generate donor plasmid pFB-egfp-vp39-lef-4.
(56) Parallelly, donor plasmid pFB-egfp-vp39-cg30 was constructed, where both vp39 and cg-30 ORFs are driven from the very late polyhedrin promoter, and the cg-30 ORF can also use its native promoter situated inside the 3′-end of the vp39 ORF. Briefly, a 1868-bp fragment carrying both vp39 and cg-30 ORFs was amplified using primers cg30-XbaI-F and vp39-XbaI-R (noted above) and cloned into pJet1.2/Blunt, to generate pJet1.2-vp39-cg30. The vp39/cg-30 cassette was subcloned as SacI/Xba into pFB-egfp, to create pFB-egfp-vp39-cg30. Additionally, a similar donor vector pFB-egfp-FLAG-vp39-cg30 was constructed, where vp39 ORF is N-terminally FLAG-tagged. The same strategy was employed to develop this vector, only the reverse primer vp39-FLAG-SacI-R was used to amplify vp39/cg-30 cassette instead of the vp39-XbaI-R primer.
(57) All developed donor plasmids were transposed into vp39-null bacmid following the Bac-to-Bac kit protocol (Invitrogen) and screened as detailed above for vp80 repair bacmids. The functional analysis was performed as described above for the vp80 constructs.
(58) Generation and Analysis of AcMNPV Vp1054-Null Bacmid
(59) To verify the essential role of the vp1054 gene in the context of viral progeny production and nucleocapsid assembly, we constructed an AcMNPV bacmid (bMON14272) with a deletion of vp1054 by homologous recombination in E. coli according to the same procedure as for the vp80null bacmid construct with minor alternations. Since the vp1054 ORF is overlapping with the essential lef-10 ORF, we could not remove the whole vp1054 ORF, but only a 955-bp nucleotide 3′-end part of the ORF. To prevent translation of the C-truncated VP1054 mutant in insect cells, we decided to mutate the first translation codon ATG.fwdarw.Met to ACG.fwdarw.Thr. This single nucleotide substitution also changed an internal codon no. 32 (AAT) to AAC of lef-10 ORF, however, both are encoding the same amino acid (Asn). To accomplish this, we first amplified the 5′-end of the vp1054 ORF using primers vp1054-KO-F and vp1054-KO-R1 from bacmid bMON14272 (Invitrogen). The 214-bp PCR product contained a mutation of the ATG start codon of the vp1054 ORF, introduced a synthetic stop/poly-A signal sequence for the lef-10 ORF, and has a 3′-end sequence homology overhang to the cat cassette to facilitate the second PCR, and a 49-bp homology sequence to the 5′-end of vp1054 ORF to mediate Lambda RED-directed homologous recombination in E. coli. After gel-purification and DNA quantification, the 214-bp fragment was used as forward primer in a second step PCR with reverse primer vp1054-KO-R2 with a plasmid comprising a cat gene flanked with mutant LoxP sites as template. The resulting 1230-bp PCR fragment, which contained the cat gene flanked by mutant LoxP sites, a mutated 5′-end of the vp1054 ORF and ˜50-bp sequences homologous to the 5′ or 3′ proximal region of the vp1054 ORF, was treated with DpnI and gel-purified to eliminate the template plasmid. Recombination of this PCR product with the bMON14272 bacmid was performed as described above for the vp80 mutant. Kanamycin resistant colonies were verified by PCR with primer pairs cat-F/cat-R, 45510/46235, and 45122 and 46441 to check the insertion/elimination of the cat gene from the bacmid backbone. Insertion sites were also confirmed by DNA-sequencing. This method resulted in the deletion of 955 bp from nucleotide positions 45365 to 46319 in the AcMNPV clone C6 genome provided in SEQ ID NO: 1. All primer sequences are given in Table 1.
(60) Construction of a Repaired Vp1054-Null Bacmid Construct
(61) To prepare vp1054 repair donor vector, we modified plasmid pFB-egfp (noted above) by removing the polyhedrin promoter and replacing it with a fragment containing the vp1054 promoter region and the vp1054 ORF. First, a 1714-bp fragment containing both the vp1054 promoter and ORF sequence was amplified using primers vp1054-Rep-F and vp1054-Rep-R from bacmid bMON14272 template, and cloned into vector pJet1.2/Blunt (Fermentas) to form pJet1.2-pvp1054-vp1054. After DNA sequence verification, the vp1054 cassette was excised from pJet1.2-pvp1054-vp1054 by StuI/XbaI double digestion, and then subcloned into Bst1107I/XbaI-digested and gel-purified pFB-egfp to generate donor plasmid pFB-egfp-pvp1054-vp1054. The developed donor plasmids were transposed into the vp1054-null bacmid following the Bac-to-Bac protocol (Invitrogen) and screened. Recombinant bacmids were analyzed as detailed above for vp80 bacmids.
(62) Generation and Analysis of AcMNPV p6.9-Null Bacmid
(63) To verify the essential role of p6.9 in the context of viral progeny production, we constructed an AcMNPV bacmid (bMON14272) with a deletion of p6.9 by homologous recombination in E. coli. To accomplish this, a chloramphenicol resistance gene (cat) flanked by mutant LoxP sites was amplified using PCR primers p6.9-KO-F and p6.9-KO-R from a plasmid comprising a cat gene flanked by mutant LoxP sites. Mutant viruses were obtained following the same procedure as for the other mutants. For the PCR-based analysis of the finally obtained mutant clones the primer pairs cat-F and cat-R and 86596 and 86995 were used to check insertion/elimination of cat gene from bacmid backbone. Positive clones were also confirmed by DNA-sequencing. This method results in the deletion of 164 bp from nucleotide positions 86716 to 86879 in the AcMNPV clone C6 genome provided in SEQ ID NO: 1. Table 1 for primer sequences.
(64) Construction and Functional Analysis of Repaired p6.9-Null Bacmids
(65) To prepare p6.9 repair donor vectors, the pFB-GFP-p6.9 vector was used, which was constructed by Marcel Westenberg (Wageningen University). To make this vector, the AcMNPV p6.9 promoter sequence was amplified from the plasmid pAcMP1 (Hill-Perkins & Possee, 1990) with primers pp6.9-F and pp6.9-R using the high-fidelity Expand long-template PCR system (Roche). The PCR product was cloned as SalI fragment into pFastBac1 (Invitrogen), from which the polyhedrin promoter was deleted in advance by fusing the Bst1107I to the StuI site, to obtain pFB1-p6.9. The p6.9 promoter from pFB1-p6.9 was recloned as SnaBI/BamHI fragment into the Bst1107I and BamHI sites of pFastBacDUAL (Invitrogen), thereby deleting the polyhedrin promoter. Subsequently, the egfp reporter gene was cloned downstream of the p10 promoter into the XmaI site to obtain pFB-GFP-p6.9. Finally, the p6.9 genes of AcMNPV and Spodoptera exigua (Se)MNPV were PCR amplified from either the AcMNPV bacmid (bMON14272) or SeMNPV genomic DNA by using the high-fidelity Expand long-template PCR system and primers generating EcoRI and NotI at the 5′ and 3′ ends, respectively (Table 1). The PCR products were cloned downstream of the p6.9 promoter in the EcoRI/NotI sites of pFB-GFP-p6.9. All generated clones were sequenced to verify the incorporated p6.9 sequences.
(66) The expression cassettes of both developed donor plasmids were transposed into the p6.9-null bacmid following the Bac-to-Bac protocol (Invitrogen). Screening of transposition-positive constructs into the polh locus was done by the triplex PCR-based assay as described above for the vp80 constructs. The analysis was performed as for the vp80 constructs.
(67) Results
(68) Silencing of AcMNPV Vp80 does not Affect Baculovirus Very Late Gene Expression
(69) We explored the effect of transfecting Sf9 cells with different dsRNAs during infection with AcMNPV-GFP. To trigger dsRNA-induced silencing of selected baculoviral genes (vp1054, vp39, vp80, dbp and odv-ec27), we generated gene-specific dsRNAs using in vitro T7 RNA polymerase-based synthesis. However, when we began these studies it was not clear what amount and time point of dsRNA transfection is the most effective to silence baculoviral genes. To determine an optimal amount of dsRNA for RNAi assay purposes in baculovirus-infected cells, we first attempted to silence reporter egfp gene with different amounts of dsRNA. These pilot assays showed that the most potent RNAi effect is achieved using 100 pg dsRNA per cell (data not shown). At the same time, it was also proved that RNAi treatment has no negative effect on the production of infectious budded virions progeny. We also tried to transfect dsRNA into the cells at two different time points, 24 h prior to infection or 1 h p.i. The results proved that transfection performed at 1 h p.i. is more efficient in silencing of genes expressed at late/very late phases of baculoviral infection in contrast to transfection carried out at 24 h prior to infection (data not shown). In addition, to ensure that knock-down was gene-specific, dsRNA corresponding to the cat gene was transfected as an RNAi negative control. Herein, we could observe a moderate inhibition of baculovirus infection propagation in comparison to untransfected insect cells. However, the same phenomenon was also observed when insect cells were treated only with transfection reagents. Therefore, we could conclude that the effect can be explained by a negative impact (cytotoxicity) of the presence of transfection reagents on cell viability.
(70) Silencing screening of baculovirus genes revealed that down-regulation of vp1054, vp39, dbp and odv/ec-27 is also associated with a reduction or inhibition of very late gene expression measured by EGFP detection (
(71) Knock-Down of Vp80 Totally Prevents Production of BVs and Normally Appearing ODVs
(72) To determine the roles of selected candidate genes (vp1054, vp39, vp80, dbp and odv/ec-27) in production of budded virions progeny, cell culture medium (36 h p.i.) from dsRNA-treated cells was examined for the presence of BVs. End-point dilution-based titrations confirmed that all tested genes are essential for infectious budded virus progeny production (
(73) The AcMNPV Vp80 Gene is Essential for Viral Replication
(74) An AcMNPV deletion virus was constructed as detailed in
(75) Moreover, to characterize the exact effect of deletion of the vp80 gene on AcMNPV infection, the viral propagation in transfected Sf9 cells was compared between Ac-wt, Ac-Δvp80, Ac-Δvp80-vp80Rep, Ac-Δvp80-polh-vp80Rep, Ac-Δvp80-FLAG-vp80Rep and Ac-Δvp80-vp80-FLAGRep. Cell culture supernatants of all the above bacmid constructs were analysed at indicated time points for BV production (
(76) These results indicate that the vp80 gene is essential for infectious BV production. It has clearly been proven that the whole sequence of vp80 ORF can completely be deleted from the bacmid backbone and adequately rescued by introduction of the vp80 ORF into a heterologous site (polyhedrin locus) of the genome. We also showed that vp80 gene expression can be driven by the heterologous polyhedrin promoter sequence with no negative effect on viral replication in cell culture. Additionally, we observed that the N-terminus in contrast to the C-terminus of VP80 is permissive to gene modifications (epitope tag-labeling). We noted that the kinetics of the C-terminally FLAG-tagged VP80 virus was significantly delayed when compared with all other rescue or wild-type viruses, indicating the functional importance of the VP80 C-terminus.
(77) VP80 is Required for Production of Both BV and ODV
(78) The results described above indicated that the Ac-vp80null mutant is completely defective in production of infectious budded virus. However, there was also a possibility that the mutant can still produce non-infectious budded particles. To investigate the ability, Sf9 cells were transfected with either the knock-out, repair or wild-type bacmid constructs and 7 days p.t. cell culture mediums were ultracentrifuged to pellet budded viruses. The formed pellets were either analyzed by negative staining electron microscopy or by Western blot- and PCR-based detection to confirm the presence of the budded viruses. No intact budded virus, virus-like particles, nor its structures (such as major capsid protein VP39 and viral genome sequence) were revealed in the pellet from the cells transfected with the Ac-vp80null mutant (
(79) To further characterize deletion of the vp80 gene on baculovirus life cycle, electron microscopy was performed with ultra-thin sections generated from bacmid-transfected cells. The Ac-vp80null-transfected cells developed the typical phenotype of baculovirus-infected cells with an enlarged nucleus, a fragmented host chromatin, an electron-dense virogenic stroma, etc. (
(80) VP80 Function can be Complemented by the Trans-Acting Vp80 Gene
(81) To prove that VP80 function can be complemented by the trans-acting vp80 ORF, a complementation assay was performed with a transgenic cell line, Sf9-vp80, that was stably transformed with the vp80 gene expressed under control of an early baculovirus Orgyia pseudotsugata ie-2 promoter. In the assay, both Sf9 and Sf9-vp80 cells were transfected with the Ac-vp80null bacmid mutant (
(82) Generation and Characterization of Vp39-Null Bacmid
(83) To study the functionality of the AcMNPV vp39 gene during virus infection, a vp39-null AcMNPV bacmid was constructed by partial deletion of the vp39 gene. The deletion construct was selected by its resistance to chloramphenicol indicating that site-specific deletion of the vp39 gene had occurred. In the resulting vp39-null AcMNPV bacmid, the internal part of vp39 gene was correctly replaced by the cat gene. Subsequently, the cat was eliminated by Cre/LoxP recombination (
(84) Functional mapping of vp39 ORF indicates a presumable functional relationship between vp39 and cg-30 ORFs
(85) The repair constructs were designed in such a way that the wild-type vp39 ORF under control of the polyhedrin promoter sequence was inserted into the polyhedrin locus along with the egfp gene controlled by the p10 promoter (
(86) These results indicate that the Ac-vp39null construct is able to reach the very late phase of infection as shown by the p10 promoter-driven EGFP expression. Unexpectedly, no viral propagation could be seen in insect cell monolayers that were transfected with the vp39 repair (vp39 driven from polyhedrin, Ac-Δvp39-polh-vp39Rep) constructs (
(87) At 7 days p.t., cell culture supernatants were collected and added to freshly plated Sf9 cells, which were then incubated for 3 days to detect infection by virus generated from cells transfected with all bacmids mentioned here (
(88) Since the vp39 ORF sequence overlaps with the promoter sequences of the two flanking ORFs (lef-4 and cg-30), we could not delete the whole vp39 ORF in our vp39null bacmid construct. It may therefore also be that C- and/or N-truncated mutant(s) of vp39 may be expressed which may interfere as a competitive inhibitor with the normal VP39 protein.
(89) Construction and Analysis of Vp1054-Null Bacmid
(90) To study the functionality of the AcMNPV vp1054 gene during virus infection, a vp1054-null AcMNPV bacmid was constructed by partially deleting the vp1054 gene from AcMNPV bacmid (bMON14272) by homologous recombination in E. coli. The deletion construct was selected by its resistance to chloramphenicol that indicated that site-specific deletion of the vp1054 gene had occurred. In the resulting vp1054-null AcMNPV bacmid, the 955-bp 3′-end part of the vp1054 gene was correctly replaced by the cat gene. Subsequently, the antibiotic resistance cassette (cat) was eliminated from bacmid backbone using Cre/LoxP recombination system (
(91) AcMNPV Vp1054 Gene is Essential for Viral Replication
(92) The repair construct was designed such that the AcMNPV vp1054 ORF with its native promoter region was inserted into the polyhedrin locus along with the egfp gene under the control of the p10 promoter (
(93) These results indicate that the vp1054 gene is essential for infectious BV production. It has clearly been proven that the 955-bp 3′-end sequence part of the vp1054 ORF can completely be deleted from the bacmid backbone and adequately rescued by introduction of the AcMNPV vp1054 ORF into a heterologous site (polyhedrin locus) of the genome. In addition, the results proved that deletion of the vp1054 gene does not affect very late gene expression, as demonstrated by EGFP-positive cells in cells transfected with Ac-vp1054null bacmid mutant (
(94) Generation and Characterization of p6.9-Null Bacmid
(95) To study the functionality of the AcMNPV p6.9 gene during virus infection, a vp80-null AcMNPV bacmid was constructed by deleting the p6.9 gene from AcMNPV bacmid (bMON14272) by homologous recombination in E. coli. The deletion construct was selected by its resistance to chloramphenicol that indicated that site-specific deletion of the p6.9 gene had occurred. In the resulting p6.9-null AcMNPV bacmid, the p6.9 gene was correctly replaced by the cat gene. Subsequently, the antibiotic resistance cassette (cat) was eliminated from bacmid backbone using Cre/LoxP recombination system (
(96) AcMNPV p6.9 Gene is Essential for Viral Replication
(97) The repair constructs were designed such that the wild-type AcMNPV or SeMNPV p6.9 ORFs with AcMNPV p6.9 promoter region were inserted into the polyhedrin locus along with the egfp gene under the p10 promoter (
(98) These results indicate that the p6.9 gene is essential for infectious BV production. It has clearly been proven that the whole sequence of p6.9 ORF can completely be deleted from the bacmid backbone and adequately rescued by introduction of the AcMNPV vp80 ORF into a heterologous site (polyhedrin locus) of the genome. We also showed that p6.9 gene can be complemented efficiently by the SeMNPV-derived p6.9 ORF (M. Westenberg). In addition, the results proved that deletion of the p6.9 gene does not affect very late gene expression, as demonstrated by EGFP-positive cells in cells transfected with Ac-p6.9null bacmid mutant (
Example II
(99) The inventors have amended the best mode of the present invention in the following example.
(100) Materials and Methods
(101) Generation of an Antibiotic Resistance Gene-Free AcMNPV Vp80-Null Bacmid
(102) To determine whether the VP80 protein has an essential role in the context of viral progeny production, we constructed an AcMNPV bacmid (derived from bMON14272 (from Invitrogen)) with a deletion of the vp80 ORF by homologous recombination in E. coli. To accomplish this, a cat gene flanked by mutant LoxP sites (Suzuki et al., 2005) was amplified using PCR primers vp80-KO-F and vp80-KO-R (see Table 1) from a plasmid comprising a cat gene flanked by mutant LoxP sites. The resulting PCR fragment, which contained the cat gene flanked by mutant LoxP sites and AcMNPV ˜50-bp homology sequences to the 5′ or 3′ proximal region of the vp80 ORF, was treated with DpnI and gel-purified to eliminate the template plasmid. The PCR product was then transformed into DH101 E. coli cells containing bMON14272 (Invitrogen) and the Lambda RED recombinase-producing plasmid pKD46 (Datsenko & Wanner, 2000), which had been prepared in the following manner. Transformed DH10ß-bMON14272/pKD46 E. coli cells were grown in 50-ml LB (2.0% peptone, 0.5% yeast extract, 85.5 mM NaCl, [pH 7.0]) cultures with kanamycin (50 μg/ml), ampicillin (100 μg/ml) and L-arabinose (1.5 mg/ml) at 30° C. to an OD.sub.600 of ≈0.6 and then made electrocompetent by a standard procedure. The electroporated cells were incubated at 37° C. for 3 h in 3 ml LB medium and plated on LB-agar containing chloramphenicol at a concentration of 6.5 μg/ml. After 48-h incubation at 37° C., the chloramphenicol-resistant colonies were streaked to fresh LB-agar medium with 34 μg/ml chloramphenicol. The plates were incubated at 37° C. overnight, and colonies resistant to chloramphenicol were selected for further confirmation of the relevant genotype by PCR. Primers 90292 and 90889 were used to confirm the absence of the vp80 ORF, and primers cat-F and cat-R were employed to verify the presence of cat cassette into bacmid (detailed sequences in Table 1).
(103) To eliminate the introduced antibiotic resistance gene (cat) from the bacmid backbone, a Cre/LoxP recombinase system was employed. A Cre recombinase-carrying plasmid pCRE obtained from Jeanine Louwerse (LUMC Leiden, The Netherlands) was introduced into DH10b-bMON14272-vp80null E. coli cells, and CRE expression was subsequently induced by the addition of isopropyl thiogalactoside (IPTG). Briefly, the electroporated cells were incubated at 37° C. for 3 h in 3 ml of LB medium (2.0% peptone, 0.5% yeast extract, 85.5 mM NaCl, [pH 7.0]) and plated on LB-agar medium containing 50 μg/ml kanamycin, 100 μg/ml ampicillin and 2 mM IPTG. After 24-h incubation, colonies resistant to kanamycin and ampicillin were selected for further verification of the desired genotype by PCR. In PCR-based analysis, primers 89507 and 91713 (Table 1) were used to verify elimination of cat gene from bacmid backbone. Positive clones were also confirmed by DNA-sequencing.
(104) To recover transposition competence, the helper transposase-encoding plasmid pMON7124 (Invitrogen) was re-introduced into DH10ß-bMON14272-vp80null E. coli cells. Finally, the egfp reporter gene was introduced into the vp80-null bacmid to facilitate observation of its behaviour in insect cells. Briefly, the egfp reporter gene was amplified using PCR oligonucleotides gfp-NheI-F and gfp-SphI-R (Table 1) from plasmid pEGFP-N3 (Clontech). The PCR product was cloned into plasmid pJet1.2/Blunt using CloneJET™ PCR Cloning Kit (Fermentas) according to manufacturer's protocol. Subsequently, the egfp ORF was excised from error-free pJet1.2-egfp with NheI and SphI and subcloned into NheI/SphI-digested pFastBacDUAL (Invitrogen), to generate plasmid pFB-egfp. An expression cassette containing the egfp reporter gene under transcriptional control of the very late p10 promoter was transposed from pFB-egfp into polyhedrin locus of vp80-null bacmid as described in the Bac-to-Bac manual (Invitrogen). In the resulting genome, the complete vp80 ORF has been removed (see
(105) Construction of Repaired Vp80-Null Bacmids
(106) To prepare vp80 repair donor vectors, we modified plasmid pFB-egfp (noted above) by removing the polyhedrin promoter and replacing it with a fragment containing the vp80 promoter region and the vp80 ORF. First, a 2300-bp fragment containing both the vp80 promoter and ORF sequence was amplified using primers pvp80-StuI-F and vp80-XbaI-R (Table 1) from bacmid bMON14272 template, and cloned into vector pJet1.2/Blunt (Fermentas) to form pJet1.2-pvp80-vp80. After DNA sequence verification, the vp80 cassette was excised from pJet1.2-pvp80-vp80 by StuI/XbaI double digestion, and then subcloned into Bst1107I/XbaI-digested and gel-purified pFB-egfp to generate donor plasmid pFB-egfp-pvp80-vp80. Parallelly, a donor plasmid pFB-egfp-polh-vp80, where vp80 ORF is driven by the very late polyhedrin promoter (polh) was constructed. To this aim, a 2105-bp fragment carrying the vp80 ORF was amplified using primers vp80-SacI-F and vp80-XbaI-R (Table 1) and cloned into pJet1.2/Blunt, to generate pJet1.2-vp80. In the final step, the vp80 ORF was cut out (SacI/XbaI) from pJet1.2-vp80, and subcloned into SacI/XbaI-digested pFB-egfp, to create pFB-egfp-polH-vp80.
(107) To overcome a problem associated with the unavailability of anti-VP80 antibody, FLAG tag decoration (N- and C-terminus fusion) of VP80 was performed to facilitate immunodetection. The N-terminally fused FLAG-vp80 sequence was generated by a double-step PCR strategy, a so-called fusion PCR. First, a 259-bp fragment containing the vp80 promoter and the FLAG tag was PCR amplified using primers pvp80-StuI-F and vp80-FLAG-R1 from the bMON14272 bacmid template. After gel-purification and DNA quantification, the 259-bp fragment was used as forward primer in a second step PCR amplification with the reverse primer vp80-XbaI-R on the bMON14272 bacmid template. The final PCR product (2324 bp) was cloned into vector pJet1.2/Blunt (Fermentas) to form pJet1.2-pvp80-FLAG-vp80. After DNA sequence verification, the FLAG-vp80 cassette was excised from pJet1.2-pvp80-FLAG-vp80 by StuI/XbaI double digestion, and then subcloned into Bst1107I/XbaI-digested and gel-purified pFB-egfp to generate donor plasmid pFB-egfp-pvp80-FLAG-vp80. The C-terminally fused vp80-FLAG cassette was amplified using pvp80-StuI-F and vp80-FLAG-R from the bMON14272 bacmid template. The 2324-bp fragment was cloned into pJet1.2/Blunt, and subsequently transferred into pFB-egfp in a similar way as previous constructs.
(108) The inserts of all developed donor plasmids were transposed into the vp80-null bacmid following the Bac-to-Bac protocol (Invitrogen). Screening of transposition-positive constructs into the polh locus was done by a triplex PCR-based assay employing M13 forward and reverse primers and a gentamicin resistance gene-specific primer GenR (Table 1).
(109) Transfection-Infection Assay
(110) Bacmid DNAs were prepared from 1.5-ml overnight bacterial cultures of 2 to 3 independent colonies carrying the bacmid with the inserted heterologous gene according to the Bac-to-Bac manual (Invitrogen) and were analyzed in parallel. For transfections, 1 μg of each bacmid DNA preparation was used to transfect 1×10.sup.6 Sf9 cells in a 6-well plate by the Cellfectin™-based transfection protocol as described in the Bac-to-Bac (Invitrogen) manual. From 72 h to 120 h post transfection (p.t.), viral propagation was checked by fluorescence microscopy. At 120 h p.t., the cell culture medium was centrifuged for 5 min at 2000×g to remove cell debris, and this clarified supernatant was used to infect 1.5×10.sup.6 Sf9 cells in 6-well plates. After 72 h p.i., the spread of virus infection was again monitored by fluorescence microscopy. In all experiments, a wild-type bMON14272 bacmid carrying the egfp reporter gene under control of the p10 promoter was used as positive control. A bMON14272-gp64null bacmid also carrying the egfp reporter gene under control p10 promoter served as negative control, since it has lost the ability of cell-to-cell movement of the infection (Lung et al., 2002).
(111) Time-Course Characterization of Viral Propagation in Cell Culture
(112) Time course analyses were performed to compare budded virus production of the AcMNPV-vp80null virus and the various repair constructs in comparison to the wild type AcMNPV bacmid (Ac-wt) all containing egfp. Briefly, the Sf9 cells were seeded in 6-well tissue culture plates (1×10.sup.6 cells/well in 1 ml Sf900-II culture medium without serum at 28° C.). After two hours, the culture medium was removed, and the cells were transfected with 5 μg bacmid DNA, under standard conditions as recommended in Bac-to-Bac manual (Invitrogen). Cell culture supernatants were harvested at 24, 48, 72, 96 and 120 h p.t., and analysed for the production of infectious budded virus by an end-point dilution assay to determine the tissue culture infective dose 50 (TCID.sub.50). Infection was determined by monitoring egfp expression (from the p10 promoter). The average values of infectious titers derived from three independent transfections were calculated and plotted into graphs.
(113) Transmission Electron Microscopy
(114) Insect Sf9 cells were seeded in a 25 T flask (3.5×10.sup.6 cells/flask), and transfected with 20 μg either the Ac-Δvp80, rescue Ac-Δvp80-vp80 or Ac-wt bacmid construct. After 48 h p.t., the cells were harvested and prepared for transmission electron microscopy as described previously (van Lent et al., 1990). Samples were examined and photographed with a Philips CM12 electron microscope.
(115) Budded Virus Production Assay
(116) Insect Sf9 cells were seeded in two 25 T flasks (3.5×10.sup.6 cells/flask), and transfected with 20 μg either Ac-Δvp80, Ac-Δvp80-vp80, Ac-Δvp80-pH-vp80, Ac-Δvp80-FLAG-vp80, Ac-Δvp80-vp80-FLAG, or Ac-wt bacmid construct. Five days p.t., the BV-enriched cell culture supernatants were harvested, and ultracentrifuged through a cushion of 10% sucrose solution (25,000 rpm for 1.5 hour, Beckman SW32). Pelleted budded virions were resuspended in sterile demi-water, and prepared for either negative staining electron microscopy, SDS-polyacrylamide electrophoresis, or PCR-based detection (as mentioned above).
(117) Purification of ODVs and Rod-Shaped Structures from Infected Cells
(118) The presence of ODVs and rod-like structures in infected/transfected insect cells was analyzed by electron microscopy (EM). For this purpose, insect cells were harvested 48 h p.i., lysed and the cell lysates were ultracentrifuged through a 40% sucrose cushion in TE (1 mM Tris-HCl pH 7.4, 0.1 mM EDTA) buffer (45,000 rpm for 1 hour, Beckman SW55). Pellets were resuspended in sterile demi-water and analyzed by negative staining EM as described previously (van Lent et al., 1990).
(119) Purification and Fractionation of BV and ODV Virions
(120) To produce BVs, 3.0×10.sup.7 Sf9 cells were infected with Ac-Δvp80-Flag.vp80 or control Ac-wt virus at an MOI=1. Six days p.i., 72 ml of BV-enriched medium was collected and centrifuged at 1,500×g for 10 min. The supernatant was then ultracentrifuged at 80,000×g (Beckman SW28 rotor) for 60 min at 4° C. The BV pellet was resuspended in 350 μl 0.1×TE buffer, and loaded onto a linear sucrose gradient (25 to 56% (w/v)), and ultracentrifuged at 80,000×g (Beckman SW55 rotor) for 90 min at 4° C. The formed BV band was collected and diluted in 12 ml 0.1×TE. The BV preparation was concentrated at 80,000×g for 60 min at 4° C. The final virus pellet was resuspended in 150 μl of 0.1×TE.
(121) To produce ODVs, 6.0×10.sup.7 Sf9 cells were co-infected with Ac-Δvp80-Flag.vp80 (MOI=25) and AcMNPV (MOI=5) viruses (strain E2, Smith & Summers, 1979). Five days p.i., the infected cells were harvested, and ODVs were purified from viral occlusion bodies as described previously (Braunagel et al., 1994). The final ODV pellet was resuspended in 0.5 ml of 0.1×TE (10 mM Tris, 1 mM EDTA, pH=7.5).
(122) The purified BV and ODV virions were fractionated into envelope and nucleocapsid fractions as described previously (Braunagel et al., 1994). Final fractions were processed for SDS-PAGE and immunoblotted against either mouse monoclonal anti-Flag antibody (Stratagene), rabbit polyclonal anti-VP39 antiserum (kindly provided by Lorena Passarelli, Kansas State University, USA), rabbit polyclonal anti-GP64 antiserum (kindly provided by Hualin Wang and Feifei Yin, Wuhan Institute of Virology, China (Yin et al., 2008)), or rabbit polyclonal antiserum against per os infectivity factor 1 (PIF-1) (kindly provided by Ke Peng, Wageningen University, The Netherlands (Peng et al., 2010)).
(123) Development of Transgenic Sf9-Derived Cell Line Expressing Vp80
(124) To develop a cell line, which produces the VP80 protein, a 2105-bp fragment carrying the vp80 ORF was amplified using primers vp80-SacI-F and vp80-XbaI-R (Table 1) and cloned into pJet1.2/Blunt, to generate pJet1.2-vp80. In the next step, the vp80 ORF was cut out (SacI/XbaI) from pJet1.2-vp80, and subcloned into SacI/XbaI-digested pIZ (Invitrogen), to create pIZ-vp80. The resulting plasmid vector pIZ-vp80 was linearized with Eco57I, and gel-purified. Sf9 cells were seeded in a six-well plate (1×10.sup.6 cells/well), and transfected with 10 μg of the linearized vector. After 24 hours post-transfection, cells were selected by cell culture medium containing Zeocin™ (300 μg/ml) for 2 to 3 weeks, until no control Sf9 cells survived under the same conditions. Cells were then propagated as an uncloned cell line.
(125) Recombinant Protein Expression with the vp80null Virus
(126) To measure the capacity to express recombinant protein with the Ac-Δvp80 (trans-complemented) virus seed, 3.0×10.sup.7 non-transformed Sf9 cells were infected (independent triplicate assay) with Ac-wt, Ac-Δvp80-Flag.vp80 (both produced in non-transformed cell line) or Ac-Δvp80 virus (produced in the Sf9-vp80 cell line) at a MOI=10. All of these virus seeds are expressing egfp as a model heterologous gene from the baculovirus very late p10 promoter. At 48 h and 72 h p.i. cells and culture medium were harvested and used for Western blotting, enzyme-linked immunosorbent assay (ELISA) or BV titration (see above). For Western blotting the same antibodies as mentioned above were used to detect the Flag-tag, EGFP, and GP64, as well as a monoclonal mouse anti-actin antibody (ImmunO).
(127) For relative quantification, Maxisorp 96-well plates (Nunc) were coated overnight at 4° C. with 100 ng of rabbit polyclonal anti-GFP antibody (Molecular Probes) in a volume of 100 μl per well, which was followed by standard ELISA procedures as previously described (Fric et al., 2008). The percentage of EGFP production was calculated (independent triplicate assay) according to the formula: % EGFP expression=(test absorbance.sub.nh−background absorbance)/(Ac-wt EGFP.sub.72h−background absorbance)×100%, where nh represents the time point p.i. The statistical significance of the observed differences between the control Ac-wt and the experimental Ac-Δvp80-Flag.vp80 and Ac-Δvp80 genotypes was analyzed with the Student's t-test.
(128) Results
(129) The AcMNPV Vp80 Gene is Essential for Viral Replication
(130) An AcMNPV deletion virus was constructed as detailed in
(131) Moreover, to characterize the exact effect of deletion of the vp80 gene on AcMNPV infection, the viral propagation in transfected Sf9 cells was compared between Ac-wt, Ac-Δvp80, Ac-Δvp80-vp80Rep, Ac-Δvp80-polh-vp80Rep, Ac-Δvp80-FLAG-vp80Rep and Ac-Δvp80-vp80-FLAGRep. Cell culture supernatants of all the above bacmid constructs were analysed at indicated time points for BV production (
(132) These results indicate that the vp80 gene is essential for infectious BV production. It has clearly been proven that the whole sequence of vp80 ORF can completely be deleted from the bacmid backbone and adequately rescued by introduction of the vp80 ORF into a heterologous site (polyhedrin locus) of the genome. We also showed that vp80 gene expression can be driven by the heterologous polyhedrin promoter sequence with no negative effect on viral replication in cell culture. Additionally, we observed that the N-terminus in contrast to the C-terminus of VP80 is permissive to gene modifications (epitope tag-labeling). We noted that the kinetics of the C-terminally FLAG-tagged VP80 virus were significantly delayed when compared with all other rescue or wild-type viruses, indicating the functional importance of the VP80 C-terminus.
(133) VP80 is Required for Production of Both BV and ODV
(134) The results described above indicated that the Ac-vp80null mutant is completely defective in production of infectious budded virus. However, there was also a possibility that the mutant can still produce non-infectious budded particles. To investigate the ability, Sf9 cells were transfected with either the knock-out, repair or wild-type bacmid constructs and 7 days p.t. cell culture mediums were ultracentrifuged to pellet budded viruses. The formed pellets were either analyzed by negative staining electron microscopy or by Western blot- and PCR-based detection to confirm the presence of the budded viruses. No intact budded virus, virus-like particles, nor its structures (such as major capsid protein VP39 and viral genome sequence) were revealed in the pellet from the cells transfected with the Ac-vp80null mutant (
(135) To further characterize deletion of the vp80 gene on baculovirus life cycle, electron microscopy was performed with ultra-thin sections generated from bacmid-transfected cells. The Ac-vp80null-transfected cells developed typical phenotypes of baculovirus-infected cells with an enlarged nucleus, a fragmented host chromatin, an electron-dense virogenic stroma, etc. (
(136) VP80 is Associated with Nucleocapsids of Both BV and ODV
(137) To investigate the association of VP80 with BV preparations, BVs were collected at 48 h p. i. and nucleocapsid and envelope fractions were separated. The Flag.VP80 protein was only detected in the nucleocapsid fraction as a double-band of molecular masses ranging between 80-kDa and 95-kDa that were observed in infected Sf9 cells (
(138) To examine whether VP80 is also associated with ODVs, Sf9 cells were co-infected with the Ac-Δvp80-Flag.vp80 and occlusion body (OB)-producing wt AcMNPV viruses to provide the POLH protein. Western blot analysis showed that VP80 associates with the nucleocapsid fraction of ODVs and in this case migrates as a single band of ˜80 kDa, corresponding to the 80-kDa form produced in the very late phase of infection (
(139) The Function of VP80 can be Rescued by Genetic Trans-Complementation
(140) To verify whether a vp80 deletion in the viral genome can be complemented by a vp80 ORF offered in trans under control of a constitutive promoter, a transgenic cell line expressing Flag-tagged vp80 was constructed. In these cells VP80 was mainly produced as a protein of approximately 95-kDa as was shown by Western blot analysis with anti-Flag antibody (
(141) In trans-complementation assays, Sf9-vp80 cells were transfected with the Ac-Δvp80 bacmid, and the spread of virus infection was monitored by EGFP-specific fluorescence at 96 h and 120 h p.t. (
(142) When the culture medium of the Ac-Δvp80 transfected Sf9-vp80 cells was used to infect freshly seeded non-transgenic Sf9 cells a “single-cell infection” phenotype was observed (
(143) Trans-Complemented, Replication-Deficient Ac-vp80null Virus is Competent to Express High Levels of Recombinant Protein
(144) To assess the effect of the vp80 gene deletion on the level of recombinant protein expression, a bench-scale comparative production assay has been performed. Herein, the Sf9 cells were in parallel infected with three types of baculovirus seeds at an MOI=10, namely (i) Ac-wt, (ii) Ac-Δvp80-Flag.vp80 (both produced in Sf9 cells), and (iii) Ac-Δvp80 (produced in Sf9-vp80 cells) all encoding EGFP. Western blotting profiles showed that the EGFP protein was expressed at identical levels for all three tested baculovirus genotypes as was the GP64 glycoprotein which served here for control purposes (
(145) Also during the production culture, revertant virus genotypes carrying the vp80 gene were not detected, as no de novo expressed Flag.VP80 protein (
(146) Summary
(147) In this study we focused on the improvement of conventional baculovirus-based expression tools with the goal to eliminate contaminating baculovirus progeny from manufactured recombinant protein(s). This effort is strongly driven by pharmaceutical perspectives, since recombinant baculovirus-expressed therapeutics are being more and more used in human and veterinary medicine. Hence, we aimed to identify baculovirus gene(s) whose targeting results in a deficiency of baculovirus virion production, but does not or only mildly affects very late gene expression. In this way high level expression of heterologous genes will be safeguarded.
(148) A summarizing overview of the new technology with the vp80 gene as example is presented in
(149) TABLE-US-00001 TABLE 1 List of PCR primers in order of appearance in the text. SEQ ID Orien- # Primer name Sequence tation 2 vp39-F 5′-gcttctaatacgactcactatagggtcgtatccgctaagcgttct-3′ Forward 3 vp39-R 5′-gcttctaatacgactcactatagggacgcaacgcgttatacacag-3′ Reverse 4 45510 5′-gcttctaatacgactcactatagggacagcgtgtacgagtgcat-′3 Forward 5 46235 5′-gcttctaatacgactcactatagggatctcgagcgtgtagctggt-3′ Reverse 6 90292 5′-gcttctaatacgactcactatagggtaccgccgaacattacacc-3′ Forward 7 90889 5′-gcttctaatacgactcactatagggtctattggcacgtttgct-3′ Reverse 8 ec-27-F 5′-gcttctaatacgactcactatagggaaagcagacactcggcagat-3′ Forward 9 ec-27-R 5′-gcttctaatacgactcactatagggttgagtggcttcaacctcag-3′ Reverse 10 dbp-F 5′-gcttctaatacgactcactatagggcgctcgctagttttgttct-3′ Forward 11 dbp-R 5′-gcttctaatacgactcactatagggaaagatcggaaggtggtga-3′ Reverse 12 gfp-F 5′-gcttctaatacgactcactatagggctgaccctgaagttcatctg-3′ Forward 13 gfp-R 5′-gcttctaatacgactcactatagggaactccagcaggaccatgt-3′ Reverse 14 cat-F 5′-gcttctaatacgactcactatagggacggcatgatgaacctgaat-3′ Forward 15 cat-R 5′-gcttctaatacgactcactatagggatcccaatggcatcgtaaag-3′ Reverse 16 vp80-ko-F 5′-ctgtattgtaatctgtaagcgcacatggtgcattcgatataaccttataatgtgt- Forward gctggaatgccct-3′ 17 vp80-ko-R 5′-aaatgtactgaatataaataaaaattaaaaatattttataattttttatttaccgtt- Reverse cgtatagcatacat-3′ 18 89507 5′-agcggtcgtaaatgttaaacc-3′ Forward 19 91713 5′-tgtataaacaatatgttaatatgtg-3′ Reverse 20 gfp-NheI-F 5′-ccaaaccgctagcaacatggtgagcaagggcgag-3′ Forward 21 gfp-SphI 5′-aggaaagggcatgcttaacgcgtaccggtcttgtacagctcgtccatgc-3′ Reverse 22 pvp80-StuI-F 5′-ggaacaaaggcctgagctcaaagtaagacctttactgtcc-3′ Forward 23 vp80-XbaI-R 5′-ccttctatctagattatataacattgtagtttgcg-3′ Reverse 24 vp80-SacI-F 5′-ttatcttgagctcaatatgaacgattccaattctc-3′ Forward 25 vp80-FLAG-R1 5′-caacagagaattggaatcgttcttatcgtcgtcatccttgtaatc- Reverse catattataaggttatatcgaatg-3′ 26 vp80-FLAG-R 5′-ccttctatctagattacttatcgtcgtcatccttgtaatctataacat- Reverse tgtagtttgcgttc-3′ 27 M13-F 5′-cccagtcacgacgttgtaaaacg-3′ Forward 28 M13-R 5′-agcggataacaatttcacacagg-3′ Reverse 29 GenR 5′-agccacctactcccaacatc-3′ Reverse 30 vp39-ko-F 5′-cttcttatcgggttgtacaac-3′ Forward 31 vp39-ko-R 5′-gcgtatcatgacgatggatg-3′ Reverse 32 vp39-SacI-F 5′-aaggttctctagattagacggctattcctccac-3′ Forward 33 vp39-XbaI-R 5′-ttatcttgagctcaatatggcgctagtgcccg-3′ Reverse 34 vp39-StuI-F 5′-ggaacaaaggcctgagctcttagacggctattcctccac-3′ Forward 35 lef-4-XbaI-R 5′-ccttctatctagattaatttggcacgattcggtc-3′ Reverse 36 cg-30-XbaI-F 5′-aaggttctctagattaatctacatttattgtaacatttg-3′ Forward 37 vp39-FLAG-SacI- 5′-ttatcttgagctcaatatggattacaaggatgacgacgataaggc- Reverse R gctagtgcccgtgggt-3′ 38 vp1054-ko-F 5′-gtactgaaagataatttatttttgatagataataattacattattttaa- Forward acgtgttcgaccaagaaaccgat-3′ 39 vp1054-ko-R1 5′-agggcgaattccagcacactttattacgtggacgcgttactttgc-3′ Reverse 40 vp1054-ko-R2 5′-gataagaatgcttgtttaacaaataggtcagctgttaaatact- Reverse ggcgatgtaccgttcgtatagcatacat-3′ 41 vp1054-Rep-F 5′-ggttgtttaggcctgagctcctttggtacgtgttagagtgt-3′ Forward 42 vp1054-Rep-R 5′-tcctttcctctagattacacgttgtgtgcgtgcaga-3′ Reverse 43 p6.9-ko-F 5′-gcttcgttcattcgctactgtcggctgtgtggaatgtctggttgtt- Forward aagtgtgctggaattcgccct-3′ 44 p6.9-ko-R 5′-aatattaataaggtaaaaattacagctacataaattacacaattta- Reverse aactaccgttcgtatagcatacat-3′ 45 Ac-p6.9-F 5′-tttgaattcatggttgcccgaagctccaagac-3′ Forward 46 Ac-p6.9-R 5′-tttgcggccgcttaatagtagcgtgttctgtaac-3′ Reverse 47 Se-p6.9-F 5′-tttgaattcatgtatcgtcgtcgttcatc-3′ Forward 48 Se-p6.9-R 5′-tttgcggccgcttaatagtggcgacgtctgtatc-3′ Reverse 49 86596 5′-gggcttagtttaaaatcttgca-3′ Forward 50 86995 5′-aattcaaacgaccaagacgag-3′ Reverse 51 45122 5′-gcaatcatgacgaacgtatgg-3′ Forward 52 46441 5′-cgataatttttccaagcgctac-3′ Reverse 53 pp6.9-F 5′-ggtcgacgtaccaaattccgttttgcgacg-3′ Forward 54 pp6.9-R 5′-ggtcgacggatccgtttaaattgtgtaatttatg-3′ Reverse 55 75834 5′-cttcttatcgggttgtacaac-3′ Forward 56 76420 5′-gcgtatcatgacgatggatg-3′ Reverse
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