# Patent

## Title
Baculovirus system for expressing proteins forming virus-like particles

## Abstract

The present invention may be included in the field of biotechnology and it covers the improved production of recombinant proteins in insect cells or insect larvae as biofactories by a novel expression cassette. This expression cassette comprises nucleic acid sequences such as promoters, homologous regions (hr) as enhancers, and sequences encoding transcriptional regulators, for example, the baculovirus Ac-ie-01 cDNA, or any combination thereof, which are able to increase the quality and production efficiency of the recombinant proteins. Moreover, the present invention is also directed to the vectors themselves comprising the above mentioned nucleic acid sequences of the invention, cells or insects infected, transformed or transfected with those sequences or vectors, and methods for producing the recombinant proteins by using the aforesaid sequences, vectors, cells or insects.

## Background
RELATED APPLICATIONS
This application is a 35 U.S.C. §371 national stage filing of International Application No. PCT/EP2013/075812, filed Dec. 6, 2013, which claims priority to European Patent Application No. 12196120.5, filed Dec. 7, 2012. The entire contents of each of the foregoing applications are incorporated herein by reference.
FIELD OF THE INVENTION
The present invention may be included in the field of biotechnology and it covers the improved production of recombinant proteins in insect cells or insect larvae as biofactories by a novel expression cassette. This expression cassette comprises nucleic acid sequences such as promoters, homologous regions (hr) as enhancers, and sequences encoding transcriptional regulators, for example, the baculovirus Ac-ie-01 cDNA, or any combination thereof, which are able to increase the quality and production efficiency of the recombinant proteins, in particular those forming virus-like particles. Moreover, the present invention is also directed to the vectors themselves comprising the above mentioned nucleic acid sequences of the invention, cells or insects infected, transformed or transfected with those sequences or vectors, and methods for producing the recombinant proteins by using the aforesaid sequences, vectors, cells or insects.
STATE OF THE ART
The baculovirus expression vector system (BEVS) is a well-established method for the production of recombinant proteins to be used as vaccines, therapeutic molecules or diagnostic reagents. With its potential for over-expression and rapid speed of development, BEVS is one of the most attractive choices for producing recombinant proteins for any purpose. The most employed baculovirus used in industry for recombinant protein expression is based on Autographa californica multiple nucleopolyhedrovirus (AcMNPV) with Spodoptera frugiperda 9 (Sf9) or 21 (Sf21) insect cells as suitable expression hosts (1), as well as Trichoplusia ni (T. ni) insect larvae as living biofactories (2). Since the BEVS was developed in the 80's (3), hundreds of recombinant proteins, ranging from cytosolic enzymes to membrane-bound proteins, have been successfully produced in baculovirus-infected insect cells.
Efforts have been made to increase BEVS productivity (4). A variety of transfer vectors are available for the construction of recombinant baculoviruses, encoding resident fusion proteins, which have been reported to improve protein expression, including maltose binding protein, glutathione S transferase, SUMO and KDEL retention signal. Other attempts related to improve the stability of expressed proteins have been investigated focusing on two genes in the baculovirus genome, which are not essential for growth of the virus in cell culture, namely chiA (chitinase) and cath (cathepsin). ChiA deletion appears to improve the production of secreted proteins by accumulating the protein in the endoplasmic reticulum and processing the proteins through the secretory pathway of the cells. Additionally, the prevention of the formation of cathepsin protease may also contribute to improved product stability from chiA− viruses. Novel insect cell lines, such as High-FiveTM (Hi-5) or BTI-TnAo38 cell lines from T. ni, have recently been developed to increase the baculovirus productivity with significant improvements in the final amount of heterologous protein recovery (5, 6).
Accelerating recombinant protein expression, so that protein expression takes place before the machinery of insect cells is severely impaired by the baculovirus infection, would be an important improvement of the BEVS. Late expression, driven by the conventional strong virus promoters of polyhedrin (polh) or p10 genes, has serious disadvantages in the foreign protein post-translational modifications. Baculovirus promoters that allow for earlier expression than the conventionally used polh or p10 promoters have been characterized and been used for heterologous protein production, but showed a reduced productivity (7).
Another possibility for improving the BEVS would be to increase preservation of cell integrity at late times post-infection by reducing the virus-induced cell death. Reduction in the severe impairment of the insect cell machinery at late times post-infection caused by BEVS should not only increase the time frame for producing and accumulating recombinant proteins (secreted or not), but also allow more time for the folding of complex proteins or any post-translational modification of the produced proteins.
Some baculovirus DNA elements have been determined to be involved in the activation of late expression factor genes, which are necessary for virus propagation. One of them is the immediate early (ie) protein IE-1 and its splice variant IE-0 from AcMNPV (8). Translation of the AcMNPV mRNAs, encoded by the Ac-ie-01 gene, results in both IE-0 and IE-1 expression due to internal translation initiation. Both are thought to be critical mediators of baculovirus gene expression due to their potency as transcriptional regulators (9). Synthesized very early during infection, AcMNPV IE-1 is a 67-kDa dimeric DNA-binding protein that stimulates transcription in plasmid transfection assays through the activity of its N-terminal acidic domain (10, 11). IE-1 accumulates within the nucleus, where it is maintained through late times (12). Transactivation by IE-1 is enhanced by its binding as a homodimer to the baculovirus homologous region (hr) sequences, which function as transcriptional enhancers and origins of viral DNA replication. AcMNPV IE-0 is a 72.6-kDa 636 amino acid protein composed of 38 amino acids encoded by orf141 (exon0), 16 amino acids encoded by the upstream nontranslated leader of ie1, and the entire 582 amino acid IE-1 protein. The final product is therefore identical to IE-1 except for the additional 54 amino acids fused to the N-terminus. Presumably due to their common sequences, IE-0 and IE-1 share biochemical activities, including hr enhancer binding and transcriptional regulation.
SUMMARY OF THE INVENTION
The present invention is based to a large extent on the unexpected properties of the expression cassette of the invention.
In particular, it was discovered that the expression cassette of the invention drives the expression of recombinant proteins markedly higher than the expression obtained by conventional promoters, such as polh, and thus to unprecedented levels.
Furthermore, cells and insects infected with a recombinant baculovirus containing an expression cassette that expresses the IE-1/IE-0 proteins above endogenous levels have an increased viability and an increase in the integrity of the molecular cell machinery and cell morphology.
The present invention thus provides products and methods for the improved expression of recombinant proteins and, in particular, those forming virus-like particles.
The following items are preferred embodiments for allowing this improved expression:

    
    
        1. An expression cassette comprising nucleic acid sequences that allow for the expression of the transcriptional regulators IE-1 and/or IE-0 above the endogenous levels obtained during baculovirus infection and the expression of a recombinant protein,
        
            wherein the expression of the recombinant protein is driven by a promoter comprising the baculoviral p10 promoter and
            wherein the recombinant protein is selected from the group consisting of a virus like particle protein, vaccine protein, subunit monomeric vaccine protein, subunit multimeric vaccine protein, therapeutic protein, antibody, enzyme, cytokine, blood clotting factor, anticoagulant, receptor, hormone and diagnostic protein.
        
        
        2. An expression cassette comprising nucleic acid sequences that allow for the expression of the transcriptional regulators IE-1 and/or IE-0 above the endogenous levels obtained during baculovirus infection and the expression of a recombinant protein,
        
            wherein the recombinant protein is selected from the group consisting of virus-like particle protein, vaccine protein, subunit monomeric vaccine protein, subunit multimeric vaccine protein, therapeutic protein, antibody, enzyme, cytokine, blood clotting factor, anticoagulant, receptor, hormone and diagnostic protein.
        
        
        3. The expression cassette according to item 1 or 2, wherein the recombinant protein is any virus-like particle protein selected from the group consisting of:
        
            (a) Porcine circovirus capsid protein,
            (b) Foot and mouth disease virus VP1, VP3 or VP0 protein,
            (c) Canine parvovirus VP1 and VP2 proteins,
            (d) Porcine parvovirus VP1 and VP2 proteins,
            (e) Human norovirus (genogroup I or II) capsid protein,
            (f) Calicivirus capsid protein,
            (g) Human papillomavirus L1 protein,
            (h) Hepatitis E protein E2,
            (i) Infectious bursal disease virus VP1, VP2 and VP3 proteins,
            (j) Astrovirus ORF2-encoded proteins,
            (k) Influenza virus HA, NA and M1 proteins,
            (l) Hepatitis B core and surface antigens,
            (m) Parvovirus VP1 and VP2 proteins, and
            (n) Rabbit calicivirus VP60 protein.
        
        
        4. The expression cassette according to item 3, wherein the porcine circovirus capsid protein is the capsid protein from porcine circovirus type 2, the human papillomavirus L1 protein is the L1 protein from human papillomavirus 16 and the rabbit calicivirus VP60 protein is the VP60 protein from rabbit haemorrhagic disease virus.
        5. The expression cassette according to item 1 or 2, wherein the recombinant protein is any virus-like particle protein, vaccine protein or diagnostic protein selected from the group consisting of:
        
            (a) Porcine circovirus capsid protein,
            (b) Foot and mouth disease virus VP1, VP3 or VP0 protein,
            (c) Canine parvovirus VP1 and VP2 proteins,
            (d) Porcine parvovirus VP1 and VP2 proteins,
            (e) Human parvovirus VP1 and VP2 proteins
            (f) Human norovirus (genogroup I or II) capsid protein,
            (g) Calicivirus capsid protein,
            (h) Human papillomavirus L1 protein,
            (i) Hepatitis E protein E2,
            (j) Classical swine fever E2 protein,
            (k) Bovine viral diarrhoea virus (BVDV) E2 protein,
            (l) Influenza A virus HA, NA and M1 proteins,
            (m) Infectious bursal disease virus VP1, VP2 and VP3 proteins,
            (n) Astrovirus ORF2-encoded proteins,
            (o) Hepatitis B core and surface antigens,
            (p) West Nile virus E protein,
            (q) African swine fever virus p30, p54, p72 and CD2-like proteins,
            (r) Single domain antibody (VHH) from camelids,
            (s) Parvovirus VP1 and VP2 proteins,
            (t) Rabbit calicivirus VP60 protein, and
            (u) Full-length mammalian antibodies or derived fragments thereof.
        
        
        6. The expression cassette according to any of the items 1-5, comprising a nucleic acid sequence encoding the IE-1 and/or IE-0 proteins selected from the group consisting of:
        
            (a) nucleic acid sequence indicated in any of SEQ ID NO: 1-5;
            (b) nucleic acid sequence having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90% and most preferably at least 95% with the nucleic acid sequence indicated in any of SEQ ID NO: 1-5 and encoding a protein functioning as a transcriptional regulator in a baculovirus;
            (c) nucleic acid sequence encoding a protein with the amino acid sequence indicated in any of SEQ ID NO: 6-9; and
            (d) nucleic acid sequence encoding a protein functioning as a transcriptional regulator in a baculovirus and having a sequence similarity of at least 70%, preferably at least 80%, more preferably at least 90% and most preferably at least 95% with the amino acid sequence indicated in any of SEQ ID NO: 6-9.
        
        
        7. The expression cassette according to any of the items 1-6, wherein the promoter driving the expression of the recombinant protein comprises a nucleic acid sequence selected from the group consisting of:
        
            (a) nucleic acid sequence indicated in any of SEQ ID NO: 10-16; and
            (b) nucleic acid sequence functioning as a promoter in a baculovirus and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90% and most preferably at least 95% with the nucleic acid sequence indicated in any of SEQ ID NO: 10-16.
        
        
        8. The expression cassette according to item 7, wherein the promoter driving the expression of the recombinant protein comprises a nucleic acid sequence selected from the group consisting of:
        
            (a) nucleic acid sequence indicated in any of SEQ ID NO: 11, 12, 13, 15 and 16; and
            (b) nucleic acid sequence functioning as a promoter in a baculovirus and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90% and most preferably at least 95% with the nucleic acid sequence indicated in any of SEQ ID NO: 11, 12, 13, 15 and 16.
        
        
        9. The expression cassette according to item 7 or 8, wherein the promoter driving the expression of the recombinant protein comprises a nucleic acid sequence selected from the group consisting of:
        
            (a) nucleic acid sequence indicated in SEQ ID NO: 11; and
            (b) nucleic acid sequence functioning as a promoter in a baculovirus and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90% and most preferably at least 95% with the nucleic acid sequence indicated in SEQ ID NO: 11.
        
        
        10. The expression cassette according to any of the items 1-9, comprising at least one recombinant homologous region (hr) as enhancer region, operably linked to the promoter that drives the expression of the recombinant protein.
        11. The expression cassette according to item 10, wherein the recombinant homologous region (hr) is selected from the group of nucleic acid sequences consisting of:
        
            (a) nucleic acid sequence indicated in SEQ ID NO: 27; and
            (b) nucleic acid sequence functioning as an enhancer homologous region in a baculovirus and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90% and most preferably at least 95% with the nucleic acid sequence indicated in SEQ ID NO: 27.
        
        
        12. The expression cassette according to any of the items 1-11, comprising a nucleic acid sequence that is operably linked to the expression of the recombinant protein and selected from the group consisting of:
        
            (a) nucleic acid sequence containing the nucleic acid sequence indicated in any of SEQ ID NO: 17-22, 25 and 26 and
            (b) nucleic acid sequence substantially retaining the activity of the functional elements and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90% and most preferably at least 95% with the nucleic acid sequence indicated in any of SEQ ID NO: 17-22, 25 and 26.
        
        
        13. The expression cassette according to item 12, comprising a nucleic acid sequence that is operably linked to the expression of the recombinant protein and selected from the group consisting of:
        
            (a) nucleic acid sequence containing the nucleic acid sequence indicated in any of SEQ ID NO: 17-19 and 25; and
            (b) nucleic acid sequence substantially retaining the activity of the functional elements and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90% and most preferably at least 95% with the nucleic acid sequence indicated in any of SEQ ID NO: 17-19 and 25.
        
        
        14. The expression cassette according to any one of items 1-11, comprising a nucleic acid sequence selected from the group consisting of:
        
            (a) nucleic acid sequence containing the nucleic acid sequence indicated in any of SEQ ID NO: 51-56; and
            (b) nucleic acid sequence substantially retaining the activity of the functional elements and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90% and most preferably at least 95% with the nucleic acid sequence indicated in any of SEQ ID NO: 51-56.
        
        
        15. A cloning vector comprising the expression cassette of any of the items 1-14.
        16. A transfer vector comprising the expression cassette of any of the items 1-14 and further a nucleic acid sequence suitable for integration or transposition in a baculovirus genome.
        17. The transfer vector according to item 16, characterized in that the transfer vector is derived from any of the baculovirus expression systems “Bac-to-Bac®” (InvitrogenTM), “BacPAKTM” (ClontechTM), “FlashBACTM” (Oxford Expression TechnologiesTM), “BacuVanceTM” (GenScriptTM), “Bac-N-Blue DNATM” (InvitrogenTM), “BaculoDirectTM” (InvitrogenTM), “BacVector®” 1000, 2000, 3000 (Novagen®), “DiamondBacTM” (Sigma-Aldrich®) or “BaculoGoldTM” (BD BiosciencesTM).
        18. A bacmid comprising the expression cassette of any of the items 1-14.
        19. A recombinant baculovirus comprising the expression cassette of any of the items 1-14.
        20. A cell comprising the expression cassette of any of the items 1-14.
        21. The cell according to item 20, infected, transfected, transduced or transformed with the expression cassette, cloning vector, transfer vector, bacmid or recombinant baculovirus of any of the items 1-19.
        22. The cell according to item 20 or 21, characterized in that it is of insect origin.
        23. The cell according to any of the items 20-22, characterized in that it is derived from an insect belonging to the Lepidoptera or Diptera genus.
        24. The cell according to any of the items 20-23, characterized in that it is derived from Trichoplusia ni, Spodoptera frugiperda, Ascalapha odorata, Bombyx mori, Drosophila melanogaster, Stigmene acrea or Aedes aegypti. 
        25. The cell according to any of the items 20-24, characterized in that it is a cell line selected from the group consisting of Hi-5TM, Sf9, Sf21, BTI-Tn5B-1, Tn368, ExpresSf+®, BTI-TnAo38, ATC-10, MimicTM Sf9, SfSWT-1, SfSWT-3, SfSWT-5, TriEXTM and Schneider's Drosophila Line 2.
        26. An insect comprising the expression cassette of any of the items 1-14.
        27. The insect according to item 26, infected, transfected, transduced or transformed with the expression cassette, cloning vector, transfer vector, bacmid or recombinant baculovirus of any of the items 1-19.
        28. The insect according to item 26 or 27, wherein the insect is derived from the genus Lepidoptera. 
        29. The insect according to any of the items 26-28, wherein the insect is selected from the group consisting of Trichoplusia ni, Spodoptera frugiperda, Spodoptera exigua, Ascalapha odorata, Bombyx mori, Rachiplusia ni and Stigmene acrea. 
        30. The insect according to any of the items 26-29, wherein the expression cassette is introduced into the insect by a recombinant baculovirus, preferably AcMNPV, SeNPV or BmNPV.
        31. A culture medium comprising the expression cassette, cloning vector, transfer vector, bacmid or recombinant baculovirus of any of the items 1-19.
        32. A method for producing a recombinant protein comprising the use of the expression cassette, cloning vector, transfer vector, bacmid, recombinant baculovirus, cell or insect according to any of the items 1-30 and the extraction and purification of the recombinant protein by conventional means.
        33. Recombinant protein obtainable by the method according to item 32.
        34. Recombinant protein according to item 33 for use in a method of treatment, therapy or diagnostic.
        35. Method of treatment, therapy or diagnostic using the recombinant protein according to item 33.
        36. A use of the expression cassette according to any of the items 1-14 for producing a cloning vector, transfer vector, bacmid, recombinant baculovirus, cell, insect or culture medium according to any of the items 15-31.
        37. A use of the cloning vector according to item 15 for producing a transfer vector, bacmid, recombinant baculovirus, cell, insect or culture medium according to any of the items 16-31.
        38. A use of the transfer vector according to any of the items 16-17 for producing a bacmid, recombinant baculovirus, cell, insect or culture medium according to any of the items 18-31.
        39. A use of the bacmid according to item 18 for producing a recombinant baculovirus, cell, insect or culture medium according to any of the items 19-31.
        40. A use of the recombinant baculovirus according to item 19 for producing a cell, insect or culture medium according to any of the items 20-31.
    
    





BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Sf9 insect cells were cultured in suspension and infected by a baculovirus overexpressing the Ac-ie-01 cDNA under the control of polh. or by a conventional baculovirus expressing the GFP protein under the control of polh promoter to assess the cell density (A) and viability (B) of these cells. The insect cells were infected in suspension at a MOI of 0.1. (A) The cells were counted at different times post-infection (0, 24 and 48 hours) to calculate the cell density. A more detailed analysis of the precise moment in which cell proliferation is produced by the overexpression of the Ac-ie-01 cDNA is shown in the insert for cells infected with polhGFP or polhAc-ie-01. (B) Cell viability was assessed by Trypan blue staining (dilution 1:1 of suspended cells and colorant at 0.4% in PBS buffer). This staining allows the differentiation between live and death cells. Cell viability was calculated by the percentage of living cells with respect to the total number of cells at different times post-infection (from 0 to 120 hours). Micrographs of Hi-5TM insect cell monolayers infected at a MOI of 5 with a control conventional baculovirus overexpressing the reporter protein GFP under the polh promoter (C) or with a baculovirus overexpressing the Ac-ie-01 cDNA under the control of the polh promoter (D). Micrographs were obtained at 96 h post-infection at a 20× magnification in an inverted microscope LeicaTM DMILTM.
FIG. 2: A) Percentage of T. ni larvae surviving 96 h post-infection using 5×104 PFUs as the infectious dose of the baculovirus overexpressing the Ac-ie-01 cDNA under the polh promoter (polhAc-ie-01) or using a conventional baculovirus expressing GFP under the polh promoter (polhGFP). B) Insect biomass at the time of infection with the same baculoviruses as in panel A and the recovered biomass at 96 h post-infection. The infectious dose was 5×104 PFUs.
FIG. 3: The effect of the Ac-ie-01 cDNA in combination with different promoters on the expression of a reporter GFP protein was analyzed. Sf21 insect cells were infected in monolayer at a MOI of 5 with the respective recombinant baculoviruses and the increase or decrease in fluorescence was subsequently measured 96 h post-infection. The fluorescence obtained with a conventional baculovirus expressing the GFP protein under the control of polh promoter was considered as the 100% value.
FIG. 4: Schematic representation of baculovirus recombinant DNA elements of the invention: a sequence encoding for transcriptional regulators (e.g. IE-0 and IE-1, for instance, encoded by the cDNA Ac-ie-01), which expression is driven by a promoter (e.g. polh); an enhancer homologous region (hr) sequence (e.g. hr1) upstream of the promoters (e.g. p6.9p10) driving the expression of the foreign gene, coding for a recombinant capsid protein from a porcine circovirus type 2 (ORF2 PCV2), or for the capsid protein from rabbit haemorrhagic disease virus (VP60 RHDV), or for the capsid protein from the human papillomavirus 16 (L1 HPV16).
FIG. 5: A) SDS-PAGE and Coomassie blue staining analysis or Western blot analysis using a monoclonal antibody against the Cap protein of extracts from Sf9 cells cultured in suspension and infected with a conventional baculovirus expressing the Cap protein from PCV2 under the control of the polh promoter (polhCap) or with a baculovirus vector engineered with the expression cassette of the invention containing the elements polhAc-ie-01/hr1p6.9p10Cap. Cells were sampled at different times post-infection (0 to 120 hours). B) Comparison of recombinant Cap protein productivity in mg/L in Sf9 insect cells grown in suspension at the time of maximum expression obtained by a conventional baculovirus engineered with the expression cassette polhCap (light grey) or by a baculovirus of the invention with the expression cassette polhAc-ie-01/hr1p6.9p10Cap (black). The productivity yields of each baculovirus was determined by Western blot with a Cap-specific monoclonal antibody and a standard curve of purified Cap protein. Quantification of Western blot reactions was carried out by a ChemiDocTM XRS Gel Imaging System (Bio-RadTM, USA). Cells were infected at a MOI of 0.1 with each virus. C) Electron micrographs of purified VLPs obtained from insect cells infected in suspension at a MOI of 0.1 with a baculovirus expressing the Cap protein under the control of polh promoter or by the expression cassette polhAc-ie-01/hr1p6.9p10. VLPs are shown at two magnifications. The inserts in the lower left corners of the upper panels show Coomassie blue staining protein profiles of purified VLP preparations resolved by SDS-PAGE electrophoresis.
FIG. 6: A) SDS-PAGE and Coomassie blue staining analysis or Western blot analysis using a monoclonal antibody against the VP60 protein of extracts from Sf9 cells cultured in suspension and infected with a conventional baculovirus expressing the VP60 protein from RHDV under the control of the polh promoter (polhVP60) or with a baculovirus vector engineered with the expression cassette of the invention containing the elements polhAc-ie-01/hr1p6.9p10VP60. Cells were sampled at different times post-infection (0 to 120 hours). B) Comparison of recombinant VP60 protein productivity in mg/L in Sf9 insect cells grown in suspension at the time of maximum expression obtained by a conventional baculovirus engineered with the expression cassette polhVP60 (light grey) or by a baculovirus of the invention with the expression cassette polhAc-ie-01/hr1p6.9p10VP60 (black). The productivity yields of each baculovirus was determined by Western blot with a VP60-specific monoclonal antibody and a standard curve of purified VP60 protein. Quantification of Western blot reactions was carried out by a ChemiDocTM XRS Gel Imaging System (Bio-RadTM, USA). Cells were infected at a MOI of 0.1 with each virus. C) Electron micrographs of purified VLPs obtained from insect cells infected in suspension at a MOI of 0.1 with a baculovirus expressing the VP60 protein under the control of polh promoter or by the expression cassette polhAc-ie-01/hr1p6.9p10. VLPs are shown at two magnifications. The inserts in the lower left corners of the upper panels show the Coomassie blue staining protein profiles of purified VLP preparations resolved by SDS-PAGE electrophoresis.
FIG. 7: A) SDS-PAGE and Western blot analysis using a monoclonal antibody against the L1 protein of extracts from Sf9 cells cultured in suspension and infected with a conventional baculovirus expressing the L1 protein from the human papillomavirus 16 (HPV 16) under the control of the polh promoter (polhL1) or with a baculovirus vector engineered with the expression cassette of the invention containing the elements polhAc-ie-01/hr1p6.9p10L1. Cells were sampled at different times post-infection (0 to 120 hours). B) Comparison of recombinant L1 protein productivity expressed in arbitrary units in Sf9 insect cells grown in suspension at the time of maximum expression obtained by a conventional baculovirus engineered with the expression cassette polhL1 (light grey) or by a baculovirus of the invention with the expression cassette polhAc-ie-01/hr1p6.9p10L1 (black). The productivity yields of each baculovirus was determined by quantification of Western blot reactions using a ChemiDocTM XRS Gel Imaging System (Bio-RadTM, USA). Cells were infected at a MOI of 0.1 with each virus. C) Immunofluorescence of Sf21 insect cells infected with each of the above baculoviruses with a monoclonal antibody against L1 protein and a secondary fluorescence labeled ant-mouse IgG commercial antibody. The fluorescence intensity observed in each case had a correlation with the level of expressed protein detected by other techniques.
FIG. 8: Differences in cell density and viability at different times post-infection observed in Sf9 cells infected (MOI 0.1) by different baculoviruses expressing recombinant proteins under the control of polh promoter or with baculovirus vectors engineered with the expression cassette of the invention containing the elements polhAc-ie-01/hr1p6.9p10. A) polhAc-ie-01/hr1p6.9p10Cap, B) polhAc-ie-01/hr1p6.9p10VP60 and C) polhAc-ie-01/hr1p6.9p10L1.
FIG. 9: A) Comparison of the recombinant Cap protein expressed by a conventional baculovirus under the control of the polh promoter (polhCap) or by a baculovirus containing the expression cassette of the present invention polhAc-ie-01/hr1p6.9p10Cap. Protein extracts from larvae infected with 5×105 PFUs of every baculovirus were obtained at 72 and 96 h post-infection and analyzed by SDS-PAGE electrophoresis and Coomassie blue staining. The same extracts were analyzed by Western blot and reacting bands were quantified by the ChemiDocTM XRS Gel Imaging System (Bio-RadTM, USA). B) Representation of the quantification of Western blots. The baculovirus containing the expression cassette polhAc-ie-01/hr1p6.9p10Cap increased the productivity of Cap protein by about 325% at 72 h post-infection and by about a 24% at 96 h post-infection compared to the expression by the conventional baculovirus.
FIG. 10: Percentage of larvae surviving at 96 h post-infection with 5×105 PFU of the baculovirus containing the expression cassette of the present invention polhAc-ie-01/hr1p6.9p10Cap or the conventional baculovirus (polhCap).
FIG. 11: Comparison of recombinant Cap protein productivity in mg in 100 Trichoplusia ni larvae 72 and 96 h post-infection with a conventional baculovirus engineered with the expression cassette polhCap (light grey) or with a baculovirus of the invention with the expression cassette polhAc-ie-01/hr1p6.9p10Cap (dark grey). Productivities were measured by microfluidic protein analysis (ExperionTM; BioRadTM, USA). Insects were infected with 5×105 PFU of each baculovirus.



DETAILED DESCRIPTION OF THE INVENTION
The present invention improves the expression of recombinant proteins by combining the recombinant DNA elements of the invention in a novel expression cassette.
Expression Cassette
An “expression cassette” comprises recombinant DNA elements that are involved in the expression of a certain gene, such as the gene itself and/or elements that control the expression of this gene (e.g. the promoter).
“Recombinant DNA” refers to a form of artificial DNA that is engineered through the combination or insertion of one or more DNA strands, thereby combining DNA that would normally not occur together.
“Recombinant DNA element” refers to a functional element within recombinant DNA, such as a promoter, enhancer or gene (such as a gene encoding a recombinant protein or a transcriptional regulator).
The expression cassette of the invention comprises the following recombinant DNA elements:

    
    
        1. a nucleic acid sequence that allows expression of a recombinant protein, which comprises the nucleic acid sequence encoding the recombinant protein and preferably nucleic acid sequences controlling its expression (at least a promoter), and
        2. a nucleic acid sequence that allows expression of baculovirus transcriptional regulators, such as IE-1 and IE-0, above the normal, i.e. endogenous, levels of said regulators that are obtained during baculovirus infection of an insect cell or an insect.
    
    


Most preferably, the expression cassette of the invention further comprises the p10 promoter (chimeric or not) that is driving the expression of the recombinant protein.
In some embodiments, the expression cassette of the invention further comprises an enhancer homologous region (hr), such as hr1, operably linked to the promoter of said sequence encoding the recombinant protein.
In a preferred embodiment, the recombinant DNA elements forming part of the expression cassette of the invention are present in a single nucleic acid molecule.
In another preferred embodiment, the recombinant DNA elements forming part of the expression cassette of the invention are present in distinct nucleic acid molecules. Preferably, the distinct nucleic acid molecules are present within the same cell.
The recombinant protein of the invention is selected from the group consisting of virus-like particle protein, vaccine protein, subunit monomeric vaccine protein, subunit multimeric vaccine protein, therapeutic protein, antibody, enzyme, cytokine, blood clotting factor, anticoagulant, receptor, hormone and diagnostic protein.
Preferably, the recombinant protein is selected from the following group of virus like-particle proteins:

    
    
        Porcine circovirus capsid protein, which is, for example, represented by the amino acid sequence of SEQ ID NO: 32 or encoded by the nucleic acid sequence of SEQ ID NO: 31.
        Foot and mouth disease virus (FMDV) VP1, VP3 and VP0 protein, the sequence of which is indicated or can be derived, for example, from the following sequences:
        
            FMDV serotype 0 complete genome: GenBank JX570650.1
            FMDV serotype A complete genome: GenBank HQ832592.1
            FMDV serotype C complete genome: GenBank AY593810.1
            FMDV serotype SAT 1 complete genome: GenBank AY593846.1
            FMDV serotype SAT 2 complete genome: GenBank JX014256.1
            FMDV serotype ASIA 1 complete genome: GenBank HQ631363.1
        
        
        Human parvovirus VP1 and VP2 protein, the sequence of which is indicated or can be derived, for example, from the following sequences:
        
            Human parvovirus B19 isolate Vn115. GenBank: DQ357065.1
            Human parvovirus 4 isolate VES065 CSF. GenBank: HQ593532.1
        
        
        Porcine parvovirus VP1 and VP2 protein, the sequence of which is indicated or can be derived, for example, from the following sequences:
        
            Porcine parvovirus strain 693a. GenBank: JN400519.1
            Porcine parvovirus strain 8a. GenBank: JN400517.1
        
        
        Canine parvovirus VP1 and VP2 protein, the sequence of which is indicated or can be derived, for example, from the following sequences:
        
            Canine parvovirus type 1 proteins VP1 and VP2: GenBank AB518883.1
            Canine parvovirus type 2a VP1 and VP2. GenBank: M24003.1
            Canine parvovirus type 2b VP2: GenBank FJ005265.1
            Canine parvovirus Type 2C VP2: GenBank FJ005248.1
        
        
        Human norovirus (genogroup I or II) capsid protein, the sequence of which is indicated or can be derived, for example, from the following sequences:
        
            Norwalk virus: GenBank M87661, NP056821
            Southampton virus: GenBank L07418
            Mexico virus: GenBank U22498
            Seto virus: GenBank AB031013
            Chiba virus: GenBank AB042808
            Lordsdale virus: GenBank X86557
            Snow Mountain virus: GenBank U70059
            Hawaii virus: GenBank U07611
        
        
        Rabbit haemorrhagic disease virus VP60 protein, the sequence of which is indicated or can be derived, for example, from the following sequence:
        
            NCBI Reference Sequence: NC_001543.1
        
        
        Human papillomavirus L1 protein, the sequence of which is indicated or can be derived, for example, from the following sequences:
        
            HPV 6: GenBank: JN252323.1
            HPV 11: GenBank: JQ773411.1
            HPV 16: GenBank DQ155283.1
            HPV 18: GenBank FJ528600.1
        
        
        Hepatitis E virus E2 protein, the sequence of which is indicated or can be derived, for example, from the following sequences:
        
            Hepatitis E virus, complete genome NCBI Reference Sequence: NC_001434.1
            Swine hepatitis E virus isolate ITFAE11 capsid protein gene. GenBank: JN861806.1
        
        
    
    


Preferably, the recombinant protein is selected from the above group of virus-like particle proteins except for the capsid protein from porcine circovirus.
In an alternative preferred embodiment, the recombinant protein is the virus-like particle protein selected from the group consisting of the capsid protein from a porcine circovirus, the L1 protein from human papillomavirus and the VP60 protein from rabbit calicivirus. Preferably, the capsid protein is from porcine circovirus type 2, the L1 protein is from human papillomavirus 16 and the VP60 protein is from rabbit haemorrhagic disease virus.
In another preferred embodiment, the recombinant protein is a vaccine protein and/or diagnostic protein selected from the following group of proteins:

    
    
        Classical swine fever virus E2 protein, the sequence of which is indicated or can be derived, for example, from the following sequences:
        
            complete genome strain HNLY-2011: GenBank JX262391.1
            complete genome strain Alfort/187: GenBank X87939.1
            complete genome strain BRESCIA: GenBank AF091661.1
        
        
        Bovine viral diarrhoea virus (BVDV) E2 protein, the sequence of which is indicated or can be derived, for example, from the following sequences:
        
            BVDV type 1 polyprotein strain Osloss: GenBank M96687.1
            BVDV type 1 polyprotein strain KS86-1cp: GenBank AB078952.1
            BVDV type 1 polyprotein strain KS86-1ncp: GenBank AB078950.1
        
        
        Influenza A virus HA protein, the sequence of which is indicated or can be derived, for example, from the following sequences:
        
            H4 influenza A virus (A/shorebird/Delaware Bay/589/2008(H4N6)) hemagglutinin (HA) gene: GenBank: CY126622.1
            H1 Influenza A virus (A/England/93760756/2009(H1N1) hemagglutinin (HA) gene: GenBank: JX625498.1
            H3 Influenza A virus (A/mallard-black duck hybrid/New Brunswick/02657/2007(H3N8)) hemagglutinin (HA) gene: GenBank: CY129382.1
            H7 Influenza A virus (A/American black duck/New Brunswick/02493/2007(H7N3)) hemagglutinin (HA) gene: GenBank: CY128714.1
            H5 Influenza A virus (A/chicken/Giza/CAI26/2011(H5)) hemagglutinin (HA) gene: GenBank: CY126001.1
        
        
        West Nile virus E protein, the sequence of which is indicated or can be derived, for example, from the following sequence:
        
            West Nile virus, complete genome NCBI Reference Sequence: NC_001563.2
        
        
        African swine fever virus p30, p54, p72 and CD2-like protein, the sequence of which is indicated or can be derived, for example, from the following sequences:
        
            Protein p30: GenBank: M88336.1
            Protein p54: GenBank: JQ771683.1
            Protein p72: GenBank: AY578708.1
            Protein CD2-like (8DR): GenBank: AF050111.1
        
        
        Single domain antibody (VHH) from camelids, such as 3B2 and 2KD1, which are specific for the VP6 protein from rotavirus A
        
            VHH 3B2 is, for example, represented by the amino acid sequence of SEQ ID NO: 44 or encoded by the nucleic acid sequence of SEQ ID NO: 43
            VHH 2KD1 is, for example, represented by the amino acid sequence of SEQ ID NO: 46 or encoded by the nucleic acid sequence of SEQ ID NO: 45
        
        
        Full-length mammalian antibodies or derived fragments thereof
    
    


The recombinant proteins are preferably encoded by the above indicated nucleic acid sequences (or the respective ORF in case of the genomic sequence) or represented by the respective amino acid sequences. The recombinant proteins may also be encoded or represented by variants of said sequences, for instance, representing different virus types and subtypes.
The sequence of the nucleic acid sequence variants is preferably at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95% identical to the respective nucleic acid sequences (or the respective ORF in case of the genomic sequence).
The sequence of the amino acid sequence variants is preferably at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95% similar to the respective amino acid sequences.
“Promoter” refers to a DNA sequence to which RNA polymerase can bind to initiate transcription. The sequence may further contain binding sites for various proteins that regulate transcription, such as transcription factors. The promoter sequence may be composed of different promoter fragments (either different or the same fragments) that are localized closely in the DNA sequence and may be separated by linkers or spacers. Such promoters are referred to as chimeric promoters.
The expression of the recombinant protein of the invention is preferably driven by a promoter selected from the group consisting of SEQ ID NO: 10-16 and variants thereof that are still functioning as a promoter in a baculovirus and have preferably at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95% identity with the nucleic acid sequence indicated in any of SEQ ID NO: 10-16.
In another preferred embodiment, the expression of the recombinant protein of the invention is driven by a promoter selected from the group consisting of SEQ ID NO: 12-16 and variants thereof that are still functioning as a promoter in a baculovirus and have preferably at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95% identity with the nucleic acid sequence indicated in any of SEQ ID NO: 12-16.
Most preferably, the expression of the recombinant protein of the invention is driven by a promoter that comprises SEQ ID NO: 11, i.e. the p10 promoter, or variants thereof that are still functioning as a promoter in a baculovirus and have preferably at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95% identity with the nucleic acid sequence indicated in SEQ ID NO: 11. The promoter comprising SEQ ID NO: 11 may also comprise further promoter fragments and thus form a chimeric promoter.
The promoter comprising SEQ ID NO: 11 is preferentially selected from the group consisting of SEQ ID NO: 11, 12, 13, 15 and 16 and variants thereof that are still functioning as a promoter in a baculovirus and have preferably at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95% identity with the nucleic acid sequence indicated in any of SEQ ID NO: 11, 12, 13, 15 and 16.
In a preferred embodiment, the polyadenylation signal from the nucleic acid encoding the recombinant protein is the p10 or SV40 polyadenylation signal. Most preferably, it is the p10 polyadenylation signal. The most preferred expression cassettes comprising the polyadenylation signal from the nucleic acid encoding the recombinant protein are represented by SEQ ID NO: 51-56 (or variants of these sequences retaining the activity of the functional elements and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90% and most preferably at least 95% with the nucleic acid sequence indicated in any of SEQ ID NO: 51-56).
As described above, a further recombinant DNA element that is present in the expression cassette of the invention is a nucleic acid sequence that allows for the expression above endogenous levels of baculovirus transcriptional regulators. Preferably this nucleic acid sequence is operably linked to the expression of the recombinant protein.
“Transcriptional regulator” refers to a regulatory protein that has the ability to modulate the transcription of specific genes by, for example, binding to enhancer or repressor regions and/or recruiting further proteins that are involved in transcription.
“Endogenous expression level” refers to the ground level of expression of a protein that is obtained during the infection of an insect cell or insect with a baculovirus that has not been altered in its expression of said protein by, for example, artificial means, such as introduction of a recombinant DNA sequence.
The “expression above endogenous levels” is also referred to as “overexpression”.
Expression above endogenous levels can be achieved through, for example, introducing further copies of the endogenous gene encoding the transcriptional regulator or manipulating the expression of the promoter of the endogenous gene. Further, copies of the endogenous genes can be introduced as transgenes under the control of a suitable promoter such as polh or pB29.
The expression level can be determined at both the mRNA and the protein level with methods conventionally known to the person skilled in the art, such as quantitative PCR and Western Blot analysis.
“Being operably linked” refers to two nucleic acid sequences that are connected in a way that one influences the other in terms of, for example, transcriptional regulation.
IE-1 and its splice variant IE-0 are transcriptional regulators that are endogenously expressed by baculoviruses.
In a preferred embodiment, the baculovirus transcriptional regulators of the invention are IE-1 and/or IE-0.
In another preferred embodiment, the expression level of IE-1 and/or IE-0 reaches expression levels above those obtained by wild-type AcMNPV, such as the AcMNPV clone C6 (genomic sequence: GenBank accession no. NC_001623.1). In another preferred embodiment, the expression level of IE-1 and/or IE-0 reaches more than twofold the amount that can be obtained with wild-type AcMNPV, such as the AcMNPV clone C6.
IE-1 and/or IE-0 are preferably encoded by any of the nucleic acid sequences of SEQ ID NO: 1-5 or represented by any of the corresponding amino acid sequences of SEQ ID NO: 6-9. IE-1 and/or IE-0 may also be encoded or represented by any of the variants of said sequences.
SEQ ID NO: 1 is the Ac-ie-01 cDNA that encodes both IE-1 and IE-0, SEQ ID NO: 2 is the coding sequence (CDS) of IE-1 and SEQ ID NO: 3 is the CDS of IE-0. SEQ ID NO: 4 and 5 are the CDSs of the N-terminal domains of IE-1 and IE-0 respectively that substantially retain the transcriptional regulator activity. The proteins that are encoded by SEQ ID NO: 2-5 are represented by SEQ ID NO: 6-9 respectively.
The variants of SEQ ID NO: 1-9 are or encode amino acids that substantially retain their function as a transcriptional regulator.
The sequence of the variants of SEQ ID NO: 1-5 is preferably at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95% identical to the sequences of SEQ ID NO: 1-5.
The sequence of the variants of SEQ ID NO: 6-9 is preferably at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95% similar to the sequences of SEQ ID NO: 6-9.
In a preferred embodiment, the above sequences are limited to those encoding or representing the IE-1 protein, i.e. SEQ ID NO: 1, 2, 4, 6 and 8 or variants thereof as defined above.
In another preferred embodiment, the above sequences are limited to those encoding or representing the IE-0 protein, i.e. SEQ ID NO: 1, 3, 5, 7 and 9 or variants thereof as defined above.
In yet another preferred embodiment, IE-1 and/or IE-0 are encoded by the nucleic acid sequence of SEQ ID NO: 1.
In some embodiments, a recombinant homologous region (hr) that can enhance the expression of the recombinant protein by being operably linked to the promoter(s) of the same may further be present in the expression cassette of the invention, in addition to the nucleic acid sequences that allow for the expression of the recombinant protein and the expression above endogenous levels of the transcriptional regulators.
“Enhancer region” refers to a control sequence, whose binding by transcriptional regulators increases the level of transcription of associated genes.
Homologous regions, hr, are comprised of repeated units of about 70-bp with an imperfect 30-bp palindrome near their center. Homologous regions are repeated at eight locations in the AcMNPV genome with 2 to 8 repeats at each side. Homologous regions have been implicated as both transcriptional enhancers and origins of baculovirus DNA replication.
The enhancer homologous region sequence hr upstream of the promoter(s) is preferably hr1 (SEQ ID NO: 27) or a sequence that is able to function as an enhancer homologous region and has preferably at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95% identity with the nucleic acid sequence indicated in SEQ ID NO: 27.
In a preferred embodiment, the expression cassette of the invention comprises combinations of recombinant promoters, sequences encoding transcriptional regulators and enhancer regions, which are operably linked to the expression of the recombinant protein, wherein these combinations are represented by any of SEQ ID NO: 17-22, 25 and 26 or variants thereof that substantially retain the activities of the functional elements and have preferably at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95% identity with the nucleic acid sequences indicated in any of SEQ ID NO: 17-22, 25 and 26.
More preferably, the above mentioned combinations are represented by any of SEQ ID NO: 17-19 and 25 or variants thereof that substantially retain the activities of the functional elements and have preferably at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95% identity with the nucleic acid sequences indicated in any of SEQ ID NO: 17-19 and 25.
The expression cassette of the invention can preferably be used to produce the cloning vector, transfer vector, bacmid, recombinant baculovirus, cell, insect or culture medium of the invention.
Cloning Vector
“Cloning vector” refers to any vector that is suitable for cloning, which generally involves the presence of restriction sites, an origin of replication for bacterial propagation and a selectable marker.
The cloning vector of the invention comprises the expression cassette of the invention and can preferably be used to produce the transfer vector, bacmid, recombinant baculovirus, cell, insect or culture medium of the invention.
The cloning vector comprising an expression cassette is also known as a “donor vector”.
Transfer Vector
“Transfer vector” (or “baculovirus transfer vector”) refers to a vector that is suitable for integration or transposition in a baculovirus genome. The transfer vector thus generally permits the insertion of genetic information into a baculovirus.
The transfer vector of the invention comprises the expression cassette of the invention and can preferably be used to produce the bacmid, recombinant baculovirus, cell, insect or culture medium of the invention.
In a further preferred embodiment, the transfer vector is derived from any of the commercially available baculovirus expression systems “Bac-to-Bac®” (InvitrogenTM), “BacPAKTM” (ClontechTM), “FlashBACTM” (Oxford Expression TechnologiesTM), “BacuVanceTM” (GenScriptTM), “Bac-N-Blue DNATM” (InvitrogenTM), “BaculoDirectTM” (InvitrogenTM), “BacVector®” 1000, 2000, 3000 (Novagen®), “DiamondBacTM” (Sigma-Aldrich®) or “BaculoGoldTM” (BD BiosciencesTM).
Bacmid
“Bacmid” refers to a plasmid construct which contains the nucleic acid sequence that is sufficient for generating a baculovirus when transfected into a cell.
The bacmid of the invention comprises the expression cassette of the invention and can preferably be used to produce the recombinant baculovirus, cell, insect or culture medium of the invention.
Baculovirus
“Baculovirus” refers to a family of infectious viruses for invertebrates, mainly infecting insects and arthropods. A “recombinant baculovirus” has further introduced recombinant DNA through, for example, homologous recombination or transposition.
The recombinant baculovirus of the invention comprises the expression cassette of the invention and can preferably be used to produce the cell, insect or culture medium of the invention.
The recombinant baculovirus preferably originates from AcMNPV.
In another preferred embodiment, the recombinant baculovirus originates from Bombyx mori nucleopolyhedrovirus (BmNPV) or Spodoptera exigua nucleopolihedrovirus (SeNPV).
Cell
The cell of the invention comprises the expression cassette of the invention. Inside this cell, the recombinant DNA elements of the expression cassette may be present on different molecules.
In a preferred embodiment, the cell is infected, transfected, transduced or transformed with the expression cassette, cloning vector, transfer vector, bacmid or recombinant baculovirus of the invention, most preferably with the recombinant baculovirus.
In a preferred embodiment, the cell is kept in cell culture.
The cell is preferably an insect cell line, more preferably a cell line derived from an insect belonging to the Lepidoptera or Diptera genus, more preferably the cell is derived from the group consisting of Trichoplusia ni, Spodoptera frugiperda, Ascalapha odorata, Bornbyx mori, Drosophila melanogaster, Stigmene acrea and Aedes aegypti and most preferably it is selected from the group of insect cell lines consisting of Hi-5TM, Sf9, Sf21, BTI-Tn5B-1, Tn368, ExpresSf+®, BTI-TnAo38, ATC-10, MimicTM Sf9, SfSWT-1, SfSWT-3, SfSWT-5, TriExTM and Schneider's Drosophila Line 2. The cell of the invention may be cultured in monolayer or in suspension.
Insect
The insect of the invention comprises the expression cassette of the invention. Inside this insect, the recombinant DNA elements of the expression cassette may be present on different molecules.
In a preferred embodiment the insect is infected, transfected, transduced or transformed with the expression cassette, cloning vector, transfer vector, bacmid or recombinant baculovirus of the invention.
The expression cassette of the invention is preferably introduced into the insect by a recombinant baculovirus. Preferably, this baculovirus is AcMNPV, SeNPV or BmNPV and the insect is an insect larva or insect pupa. The baculovirus is administered to the insect by oral administration (per os) or more preferably by injection.
In a further preferred embodiment, the insect is a transgenic insect.
The insect is preferably a lepidopter and more preferably an insect selected from the group consisting of Trichoplusia ni, Spodoptera frugiperda, Spodoptera exigua, Ascalapha odorata, Bombyx mori, Rachiplusia ni and Stigmene acrea. In a preferred embodiment, the insect is a larva or a pupa.
Preferably, the insect larvae are reared in a rearing module, such as the one described in the patent application ES 2 232 308.
Culture Medium
The culture medium of the invention comprises the expression cassette, cloning vector, transfer vector, bacmid or recombinant baculovirus of the invention.
In a preferred embodiment, the culture medium comprises the baculovirus of the invention.
Methods for Producing the Recombinant Protein
In a further aspect, the invention discloses methods for producing the recombinant protein of the invention.
In a preferred embodiment, the production of the recombinant protein comprises use of the expression cassette, cloning vector, transfer vector, bacmid, recombinant baculovirus, cell or insect of the invention. After expression of the recombinant protein, extraction and purification of said protein is made by conventional means.
Most preferably, said production method comprises use of the cell or insect of the invention.
In another preferred embodiment of the method for producing the recombinant protein, the cells of the invention are cultured in suspension (bioreactors), at densities between 2×106 to 8×106 cells per ml, depending on the cell line and the fermentation procedure used. Furthermore, cells are preferably infected at a MOI of 0.05 to 10.
In a preferred embodiment for the recombinant protein production, insect larvae or insect pupa are infected by injecting a high virus dose (higher than 104 Plaque Forming Units) of the recombinant baculovirus of the invention. 3-4 days after infection, the infected insects are processed and the whole soluble protein extract is obtained by the use of appropriate extraction buffers. Extracts are centrifuged and the lipid fraction eliminated. Then, the recombinant protein is purified by conventional means.
In a preferred embodiment, the recombinant protein of the invention is used in a method of treatment, therapy or diagnostic. For example, the recombinant protein of the invention may be used for vaccination.
SUMMARY OF SEQUENCES











SEQ ID NO:
Name:
















1
Complete Ac-ie-01 cDNA


2
Coding DNA sequence (CDS) of IE-1


3
CDS of IE-0


4
CDS of the IE-1 N-terminal domain


5
CDS of the IE-0 N-terminal domain


6
IE-1 protein


7
IE-0 protein


8
IE-1 N-terminal domain protein


9
IE-0 N-terminal domain protein


10
polh (promoter)


11
p10 (promoter)


12
pB29p10 (promoter)


13
p6.9p10 (promoter)


14
pB29 (promoter)


15
pB2p10 (promoter)


16
polhp10 (promoter)


17
polhAc-ie-01/hr1p10


18
polhAc-ie-01/hr1pB29p10


19
polhAc-ie-01/hr1p6.9p10


20
pB29Ac-ie-01/hr1p10


21
pB29Ac-ie-01/hr1pB29p10


22
pB29Ac-ie-01/hr1p6.9p10


23
polhAc-ie-01/hr1polh


24
pB29Ac-ie-01/hr1polh


25
polhAc-ie-01/hr1polhp10


26
pB29Ac-ie-01/hr1polhp10


27
Homologous region enhancer hr1


28
polhAc-ie-01


29
polhGFP


30
polhAc-ie-01/hr1p6.9p10Cap


31
ORF2 from porcine circovirus type 2


32
Capsid protein (Cap) from porcine



circovirus type 2


33
polhCap


34
polhAc-ie-01/hr1p6.9p10GFP


35
polhAc-ie-01/hr1polhp10GFP


36
polhAc-ie-01/hr1pB29p10GFP


37
polhAc-ie-01/hr1p6.9GFP


38
polhAc-ie-01/hr1p10GFP


39
polhAc-ie-01/hr1polhGFP


40
p6.9GFP


41
pB29GFP


42
p10GFP


43
Nucleic acid sequence encoding VHH



3B2


44
Amino acid sequence of VHH 3B2


45
Nucleic acid sequence encoding VHH



2KD1


46
Amino acid sequence of VHH 2KD1


47
Nucleic acid sequence encoding the L1



protein of the human papilloma virus 16


48
Amino acidic sequence of the L1 protein



of the human papilloma virus 16


49
Nucleic acid sequence of the VP60



encoding gene from RHDV


50
Amino acidic sequence of the VP60



protein from RHDV


51
polhAc-ie-01/hr1p6.9p10Cap



(polyadenylation signal from p10 gene)


52
polhAc-ie-01/hr1p6.9p10L1



(polyadenylation signal from p10 gene)


53
polhAc-ie-01/hr1p6.9p10VP60



(polyadenylation signal from p10 gene)


54
polhAc-ie-01/hr1p6.9p10L1 (SV40



polyadenylation signal)


55
polhAc-ie-01/hr1p6.9p10VP60 (SV40



polyadenylation signal)


56
polhAc-ie-01/hr1p6.9p10Cap (SV40



polyadenylation signal)









All sequences of the invention include variants thereof that substantially retain the functional activity of the parental sequence.
“Variants” are nucleic or amino acids whose nucleic or amino acid sequence differs in one or more positions from the parental nucleic or amino acid sequence, whereby differences might be additions, deletions and/or substitutions of nucleic acids or amino acid residues.
The variants of the invention have preferably at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95% identity (nucleic acid sequences) or similarity (amino acid sequences) to the parental sequence.
In another preferred embodiment, the variants of the invention are fragments of the nucleic acid or amino acid sequence that substantially retain their functional activity.
Nucleic and amino acid sequences of the present invention can be distinguished from other nucleic and amino acid sequences by their degree of sequence identity or similarity respectively as determined using, for example, EMBOSS Needle with the default parameters (http://www.ebi.ac.uk/Tools/psa/emboss_needle/). Methods for the generation of such variants include random or site directed mutagenesis, site-saturation mutagenesis, PCR-based fragment assembly, DNA shuffling, homologous recombination in vitro or in vivo, and methods of gene-synthesis.
Deposition of Microorganisms According to the Budapest Treaty
The plasmid containing the expression cassette polhAc-ie-01/hr1p6.9p10Cap was deposited in the Spanish Type Culture Collection (CECT) (www.cect.org); University of Valencia, Parc Cientific Universitat de València; Catedrático Agustín Escardino, 9; 46980 Paterna (Valencia), Spain, with the accession number CECT 8228 on Nov. 6, 2012.
EXAMPLES
Example 1. The Baculovirus Expression Cassette of the Invention Induces Cell Proliferation and Increases Cell Viability Through the Transcriptional Regulators Encoded by the Ac-ie-01 cDNA
We observed by microscopy that recombinant baculoviruses incorporating a baculovirus expression cassette with the Ac-ie-01 cDNA have interesting properties related to a decrease in the virus-induced cytopathic effects and an increase of the cell density in cultures. To quantify these phenomena and to determine the DNA element/s responsible for such interesting properties, we generated a recombinant baculovirus expressing the transcriptional regulators encoded by the Ac-ie-01 cDNA under the control of polh promoter. As a control, the conventional recombinant baculovirus expressing the GFP protein under the control of the polh promoter was used. These baculoviruses were used to infect Sf9 cells in suspension at a low multiplicity of infection (MOI) of 0.1. The increase in cell number was studied until 48 h post-infection and cell viability was studied between 24 to 120 h post-infection. At 24 h post-infection, insect cells infected by the baculovirus overexpressing the Ac-ie-01 cDNA encoded transcriptional regulators, i.e. IE-1 and IE-0, presented an increase in cell number higher than 10% with respect to cultures infected by the control recombinant baculovirus (FIG. 1A). These differences in cell number were observed as early as 6 h post-infection (FIG. 1A). A more detailed analysis by flow cytometry of the time required for these factors to induce the observed differences in cell proliferation revealed an increase of insect cells in S phase at 3 h post-infection and then at 6 h post-infection, an increase in the number of insect cells in G1 was observed. These data imply a very early increment of the mitosis in those cultures infected by the baculovirus overexpressing the Ac-ie-01 cDNA encoding proteins (data not shown).
Fluorescence measurement was performed on a FACSCaliburTM (BD BiosciencesTM) flow cytometer. Cells were fixed in 70% EtOH, resuspended and incubated in the staining solution (50 μg/ml propidium iodide in PBS, 5 ug/ml RNAse). The data were gated to eliminate particles with a distinct size from cells and analyzed by plotting the cell number vs the red fluorescence from propidium iodide. 50,000 cells were counted per assay. Data analysis of the total number of cells per cell cycle phase (G1, S and G2) was made using Modfit software.
Infected cell cultures were also analyzed by Trypan blue staining to determine cell viability at different times post-infection. Interestingly, at very late times post-infection (96-120 hours), insect cells infected by the virus overexpressing the transcriptional regulators showed an increase (50-60% increase) of cell viability and integrity (FIG. 1B). This suggests that the overexpression of the transcriptional regulators of the present invention protects the cells from the baculovirus-induced cytopathic effect, allowing long-term expression. Both cell proliferation and increased cell viability after infection have important consequences in the recombinant protein productivity of the BEVS. Similar results were obtained when the overexpression of the transcriptional regulators was driven by both the pB29 or polh promoters (data not shown). Results observed in Sf9 insect cells infected in suspension were confirmed in Sf21 cells cultured in monolayer (data not shown) and also in Hi-5TM cells cultured in monolayer (FIGS. 1C and D). These figures demonstrate how the overexpression of the transcriptional regulators improves the cell integrity at late times post-infection (96 hours).
Example 2. The Baculovirus Expression Cassette of the Invention Increases the Baculovirus-Infected Insect Larvae Surviving Rates and Insect Biomass Recovered Using High Infectious Doses Through the Transcriptional Regulators Encoded by the Ac-ie-01 cDNA
In the previous example, an advantage of baculoviruses expressing recombinant GFP protein in the context of the baculovirus cassette expressing the transcriptional regulators IE-1 and IE-0 above endogenous levels was shown in terms of viability and proliferation of insect cells.
Using the same baculovirus constructs with the expression cassette polhAc-ie-01 or polhGFP, T. ni larvae were infected with a high infectious dose of 5×104 plaque forming units (PFU). Similarly to the cells infected with baculoviruses with these expression cassettes, larvae infected with the baculovirus overexpressing the Ac-ie-01 cDNA (polhAc-ie-01) also showed increased survival rates when compared to larvae infected with a conventional baculovirus expressing the GFP reporter protein under the control of the same promoter (polhGPF) (FIG. 2A). This strongly suggests that the overexpression of the transcriptional regulators used in the baculovirus expression cassette of the present invention reduces the mortality of baculovirus-infected insect larvae, allowing long-term expression (more recombinant protein production) and increasing the insect biomass recovery using high infectious doses (maximum productivity) (FIG. 2B).
Example 3. The Overexpression of the Transcriptional Regulator Proteins Encoded by Ac-ie-01 cDNA in a Baculovirus Increases Production Yields of Recombinant Proteins when their Expression is Driven by the Promoter p10 or any Chimeric Promoter Containing p10
To analyze the effect of the transcriptional regulators IE-1/IE-0 in combination with different promoters on protein expression, recombinant AcMNPV baculoviruses with the following expression cassettes were prepared:

    
    
        polhAc-ie-01/hr1polhp10GFP
        polhAc-ie-01/hr1p6.9p10GFP
        polhAc-ie-01/hr1pB29p10GFP
        polhAc-ie-01/hr1p6.9GFP
        polhAc-ie-01/hr1p10GFP
        polhAc-ie-01/hr1polhGFP
        p6.9GFP
        pB29GFP
        p10GFP
        polhGFP
    
    


As a control, a conventional AcMNPV baculovirus without any foreign gene, denominated BacNi (no insert) was used.
Sf21 cells were infected with the different baculoviruses at a MOI of 5 and the increase in fluorescence was measured at 96 h post-infection. The values were normalized to the fluorescence obtained with a conventional baculovirus vector expressing the GFP under the control of the promoter polh, which was considered as 100%.
As can be seen from FIG. 3, the presence of the p10 promoter (chimeric or not) is crucial for the increased expression mediated by the overexpression of Ac-ie-01 cDNA. Quite contrary, when the Ac-ie-01 cDNA is combined in the expression cassette with the promoters polh or p6.9 alone, there is either no significant increase or even a decrease in expression.
Example 4. Overexpression of Baculovirus Transcriptional Regulators IE-1 and IE-0 Potentiates the Enhancer Function of a Homologous Region Hr Functionally Linked to a Promoter Increasing the Expression of Recombinant Proteins Forming Virus-Like Particles in a Baculovirus Vector Expression System (BEVS)
The expression of different proteins forming virus-like particles was compared between a conventional baculovirus and a baculovirus of the invention. The conventional baculovirus expressed the Cap, VP60 and L1 proteins under the control of the polh promoter (polhCap; polhVP60 and polhL1). The baculovirus of the invention expressed the Cap, VP60 and L1 proteins under the control of the p6.9p10 chimeric promoter that was previously synthesized. This chimeric promoter was operatively linked with the enhancer sequence homologous region hr1. The baculovirus of the invention further contained the Ac-ie-01 cDNA cloned under the control of the polh promoter to obtain the baculovirus expression cassettes polhAc-ie-01/hr1p6.9p10Cap, polhAc-ie-01/hr1p6.9p10VP60 and polhAc-ie-01/hr1p6.9p10L1 (FIG. 4). The expression of Cap, VP60 and L1 proteins were then analyzed in protein extracts from Sf9 cells cultured in suspension at different times post-infection (MOI of 0.1) with these baculoviruses. Extracts were subjected to SDS-PAGE electrophoresis and the resolved proteins were stained by Coomassie blue and/or analysed by Western blot.
Cap Protein
Results obtained for the Cap protein were that a more intensely stained protein band corresponding to Cap protein from porcine circovirus type 2, expressed by the baculovirus modified by the expression cassette of the present invention was observed at the different times post-infection (FIG. 5A). This clearly demonstrated that the novel expression cassette is more efficient in expressing the Cap protein. This result was found in both monolayer (Sf21) and suspension (Sf9) cells independently of the multiplicity of infection used (MOI 5 or MOI 0.1) (data not shown).
A Western blot with a monoclonal antibody against Cap protein of the same infected cell extracts also corroborated a more abundant presence of Cap protein in cells infected with the baculovirus of the invention (polhAc-ie-01/hr1p6.9p10Cap). The amount of protein was analyzed using an ECL western blotting detection system and a ChemiDocTM XRS Gel Imaging System (Bio-RadTM, USA). Western blot reactions were studied at different times post-infection in Sf9 cells cultured in suspension as described above.
Quantification data of this analysis with the different baculoviruses obtained by the ChemiDocTM XRS Gel Imaging System was expressed as arbitrary expression units. At 72 h post-infection, compared to a conventional baculovirus expressing the Cap protein under the control of the polh promoter, the expression level of Cap was about 4.8 times and 3.5 times higher in Sf21 monolayer (MOI of 5) and Sf9 suspension cultures (MOI of 0.1) respectively with the baculovirus modified by the expression cassette of the invention (polhAc-ie-01/hr1p6.9p10Cap) (data not shown). These differences in protein accumulation were also observed in Hi-5TM cells (data not shown), suggesting that the baculovirus expression cassette of the invention could be used to produce the recombinant protein Cap in different insect cell lines used in research and industry.
Importantly, the recombinant Cap protein expression levels mediated by the baculovirus expression cassette of the present invention were higher at any of the times post-infection analyzed (FIG. 5A). Maximum levels of expression were detected at 72 h post-infection with the baculovirus genetically modified with the expression cassette of the present invention, while the maximum level of expression with the conventional baculovirus (polhCap) was at 48 h post-infection (FIG. 5A).
A more precise quantification of the productivity of Cap protein was made by Western blot analysis with a Cap-specific monoclonal antibody of extracts from Sf9 insect cells cultured in suspension and infected by a conventional baculovirus (polhCap) or the baculovirus of the present invention (polhAc-ie-01/hr1p6.9p10Cap). The quantification was carried out with a standard curve of purified Cap protein and subsequent analyses by the ChemiDocTM XRS Gel Imaging System (Bio-RadTM, USA). Insect cells were cultured in suspension at a density of 2×106 cells/ml and were infected at a MOI of 0.1 with each baculovirus. This demonstrated that while the productivity of Cap protein in cells infected with the conventional baculovirus was about 57 mg/L, the insect cells infected with the baculovirus of the invention were able to produce about 198 mg/L (FIG. 5B). The maximum productivities of recombinant baculoviruses were obtained at different times after infection (48 and 72 hours post-infection respectively).
Sf9 insect cells cultured in suspension were infected with a conventional baculovirus expressing the Cap protein (polhCap) or the baculovirus of the present invention expressing the Cap protein (polhAc-ie-01/hr1p6.9p10Cap). The VLPs that were formed after the expression of the Cap protein were purified. To this end, identical volumes of cell cultures were used in both cases. The cells were disrupted by a mild treatment with a non-ionic detergent and submitted after clarification to a high centrifugation speed in a sucrose gradient to purify the VLPs. Then, those VLPs were analyzed by electron microscopy by negative staining. VLPs formed by both baculoviruses were identical in size and shape, but the concentration of pseudoparticles observed reflected the differences in the Cap expression levels previously detected between the two baculoviruses. The number of VLPs produced by cells infected with the baculovirus modified with the expression cassette of the invention was higher than for the cells infected with the baculovirus expressing the Cap protein using the polh promoter (FIG. 5C). The higher number of VLPs produced by the cells infected with the baculovirus of the invention correlated with an increased expression of Cap protein in these cells, and the amount of Cap protein detected in purified VLPs analyzed by SDS-PAGE electrophoresis and Coomassie blue staining (FIG. 5C, upper panels, inserts in the lower left corners).
VP60 Protein
Results obtained for the VP60 protein were that a more intensely stained protein band corresponding to VP60 protein from Rabbit hameorrhagic disease virus. expressed by the baculovirus modified by the expression cassette of the present invention was observed at the different times post-infection (FIG. 6A). This clearly demonstrated that the novel expression cassette is more efficient in expressing the VP60 protein. This result was found in both monolayer (Sf21) and suspension (Sf9) cells independently of the multiplicity of infection used (MOI 5 or MOI 0.1) (data not shown).
A Western blot with a monoclonal antibody against VP60 protein of the same infected cell extracts also corroborated a more abundant presence of VP60 protein in cells infected with the baculovirus of the invention (polhAc-ie-01/hr1p6.9p10VP60). The amount of protein was analyzed using an ECL western blotting detection system and a ChemiDocTM XRS Gel Imaging System (Bio-RadTM, USA). Western blot reactions were studied at different times post-infection in Sf9 cells cultured in suspension as described above.
Quantification data of this analysis with the different baculoviruses obtained by the ChemiDocTM XRS Gel Imaging System was expressed as arbitrary expression units. At 72 h post-infection, compared to a conventional baculovirus expressing the VP60 protein under the control of the polh promoter, the expression level of VP60 was more than 3 times higher in Sf21 monolayer (MOI of 5) and Sf9 suspension cultures (MOI of 0.1) respectively with the baculovirus modified by the expression cassette of the invention (polhAc-ie-01/hr1p6.9p10VP60) (data not shown). These differences in protein accumulation were also observed in Hi-5TM cells (data not shown), suggesting that the baculovirus expression cassette of the invention could be used to produce the recombinant protein VP60 in different insect cell lines used in research and industry.
Importantly, the recombinant VP60 protein expression levels mediated by the baculovirus expression cassette of the present invention were higher at any of the times post-infection analyzed (FIG. 6A). Maximum levels of expression were detected at 120 h post-infection with the baculovirus genetically modified with the expression cassette of the present invention, while the maximum level of expression with the conventional baculovirus (polhCap) was at 72 h post-infection (FIG. 6A).
A more precise quantification of the productivity of VP60 protein was made by Western blot analysis with a VP60-specific monoclonal antibody of extracts from Sf9 insect cells cultured in suspension and infected by a conventional baculovirus (polhVP60) or the baculovirus of the present invention (polhAc-ie-01/hr1p6.9p10VP60). The quantification was carried out with a standard curve of purified VP60 protein and subsequent analyses by the ChemiDocTM XRS Gel Imaging System (Bio-RadTM, USA). Insect cells were cultured in suspension at a density of 2×106cells/ml and were infected at a MOI of 0.1 with each baculovirus. This demonstrated that while the productivity of VP60 protein in cells infected with the conventional baculovirus was about 630 mg/L, the insect cells infected with the baculovirus of the invention were able to produce about 1,950 mg/L (FIG. 6B). The maximum productivities of recombinant baculoviruses were obtained at different times after infection (72 and 120 hours post-infection respectively).
Sf9 insect cells cultured in suspension were infected with a conventional baculovirus expressing the VP60 protein (polhVP60) or the baculovirus of the present invention expressing the VP60 protein (polhAc-ie-01/hr1p6.9p10VP60). The VLPs that were formed after the expression of the VP60 protein were purified. To this end, identical volumes of cell cultures were used in both cases. The cells were disrupted by a mild treatment with a non-ionic detergent and submitted after clarification to a high centrifugation speed in a sucrose gradient to purify the VLPs. Then, those VLPs were analyzed by electron microscopy by negative staining. VLPs formed by both baculoviruses were identical in size and shape but the concentration of pseudoparticles observed reflected the differences in the VP60 expression levels previously detected between the two baculoviruses. The number of VLPs produced by cells infected with the baculovirus modified with the expression cassette of the invention was higher than for the cells infected with the baculovirus expressing the VP60 protein using the polh promoter (FIG. 6C). The higher number of VLPs produced by the cells infected with the baculovirus of the invention correlated with an increased expression of VP60 protein in these cells, and the amount of VP60 protein detected in purified VLPs analyzed by SDS-PAGE electrophoresis and Coomassie blue staining (FIG. 6C, upper panels, inserts in the lower left corners).
L1 Protein
Results obtained for the L1 protein by Western blot analysis with a monoclonal antibody against L1 were that more intensely reacting bands corresponding to L1 protein from Human papillomavirus (HPV16) were found in cells infected by the baculovirus modified by the expression cassette of the present invention at the different times post-infection (FIG. 7A). This clearly demonstrated that the novel expression cassette (polhAc-ie-01/hr1p6.9p10L1) is more efficient in expressing the L1 protein. This result was found in both monolayer (Sf21) and suspension (Sf9) cells independently of the multiplicity of infection used (MOI 5 or MOI 0.1) (data not shown).
The amount of L1 protein was analyzed using an ECL western blotting detection system and a ChemiDocTM XRS Gel Imaging System (Bio-RadTM, USA). Western blot reactions were studied at different times post-infection in Sf9 cells cultured in suspension as described above. Quantification data of this analysis with the different baculoviruses obtained by the ChemiDocTM XRS Gel Imaging System was expressed as arbitrary expression units. At the moment of maximum expression levels of L1 from different baculoviruses analysed (96 and 120 hpi respectively), the expression level of L1 VP60 was more than 3.5 times higher in Sf9 suspension cultures (MOI of 0.1) with the baculovirus modified by the expression cassette of the invention (polhAc-ie-01/hr1p6.9p10L1) (FIG. 7B). These differences in protein accumulation were also observed in Hi-5TM cells (data not shown), suggesting that the baculovirus expression cassette of the invention could be used to produce the recombinant protein L1 in different insect cell lines used in research and industry.
Importantly, the recombinant L1 protein expression levels mediated by the baculovirus expression cassette of the present invention were higher at any of the times post-infection analyzed (FIG. 7A). Maximum levels of expression were detected at 120 h post-infection with the baculovirus genetically modified with the expression cassette of the present invention, while the maximum level of expression with the conventional baculovirus (polhCap) was at 96 h post-infection (FIGS. 7A and B).
Immunofluorescence staining with a L1-specific monoclonal antibody of Sf21 cells infected in monolayer at a MOI of 5 by both baculoviruses revealed clear differences in fluorescence intensity of the infected cells. Higher immunofluorescence intensities were found in cells infected by the baculovirus modified by the expression cassette of the invention (polhAc-ie-01/hr1p6.9p10L1), indicating higher L1 expression levels (FIG. 7C).
Example 5. The Baculovirus Expression Cassettes of the Invention Induce Cell Proliferation and Increase Cell Viability
The baculoviruses of Example 4 containing the polhCap, polhVP60, polhL1, polhAc-ie-01/hr1p6.9p10Cap, polhAc-ie-01/hr1p6.9p10VP60 and polhAc-ie-01/hr1p6.9p10L1 expression cassettes were assessed in terms of their effect on cell growth and viability.
The experiment was conducted as described in Example 1 and likewise an increase in cell number, as well as an increase in viability, could be observed for the cells infected with the baculoviruses containing the expression cassette of the invention, i.e. polhAc-ie-01/hr1p6.9p10Cap (FIG. 8A), polhAc-ie-01/hr1p6.9p10VP60 (FIG. 8B) and polhAc-ie-01/hr1p6.9p10L1 (FIG. 8C).
Example 6. The Expression Cassette of the Present Invention Potentiates the Productivity of Recombinant Proteins Forming Virus-Like Particles in Baculovirus-Infected Trichoplusia ni Insect Larvae
The expression of Cap protein mediated by the different baculoviruses (with the conventional expression cassette and with the expression cassette of the present invention) was analyzed in infected Trichoplusia ni larvae. To this end, larvae were infected with 5×104 PFU of the baculovirus with the expression cassette polhCap or polhAc-ie-01/hr1p6.9p10Cap and the extracts were analyzed at 72 and 96 h post-infection by Coomassie blue staining and Western blot analysis using a monoclonal antibody against the Cap protein (FIG. 9A). The expressed Cap protein in the different extracts was also quantified by ChemiDocTM XRS Gel Imaging System (Bio-RadTM, USA) (FIG. 9B). The expression level of Cap was increased in larval extracts by the baculovirus containing the above mentioned expression cassette of the present invention by about 325% at 72 h post-infection and by 24% at 96 h post-infection (FIG. 9B).
Additionally, larvae infected with the baculovirus modified by the expression cassette of the present invention presented a 30% increase in survival (FIG. 10). This represents a significant increase of insect biomass recovery during the production process.
Subsequently, the productivity of 100 larvae infected with the above conventional baculovirus and the baculovirus of the invention was studied considering both the Cap production yield determined by microfluidic protein analysis (ExperionTM; BioRadTM, USA) and the insect biomass recovered after infection. The productivity was studied at 72 and 96 h post-infection with 5×105 PFU of the respective baculovirus. The baculovirus containing the expression cassette of the invention, i.e. polhAc-ie-01/hr1p6.9p10Cap, increased the productivity of Cap protein in infected insect larvae with respect to the conventional baculovirus about 3.5 times at 72 h post-infection and 2 times at 96 h post-infection (FIG. 11). One hundred infected larvae with the baculovirus containing the expression cassette polhAc-ie-01/hr1p6.9p10Cap were able to produce about 300 mg of recombinant Cap protein at 96 h post-infection, whereas the larvae infected with the conventional baculovirus only produced 150 mg (FIG. 11).
Example 7. Cell Culture and Viruses
The Spodoptera frugiperda Sf21 or Sf9 cell lines were cultured in 6-well tissue culture plates (1×106 cells/well) in TNM-FH insect medium (Pan BiotechTM, Germany) containing 10% heat-inactivated fetal bovine serum (Pan BiotechTM Germany) at 27° C.
Confluent Sf9 or Sf21 cells in monolayer (1×106 cells/well) were infected with the baculoviruses at different multiplicities of infection (from 0.01 to 10). In suspension, Sf9 cells (2×106 cells/ml) were infected equally at different multiplicities of infection. Infected cells were analysed from 16 to 120 h post-infection.
Example 8. Generation of the Cloning and Donor Vectors of the Invention
A pUC57 plasmid containing the baculovirus expression cassette of the present invention was used as the cloning vector. The gene encoding the Cap protein (ORF2 from porcine circovirus type 2), the gene encoding the VP60 protein (rabbit haemorrhagic disease virus) or the gene encoding the L1 protein (human papillomavirus 16) were cloned into the MCS of a cloning plasmid using the Xho I and Nco I restriction sites. After introduction of the VLP-forming protein encoding genes, the cloning vector becomes the donor vector.
The baculovirus expression cassette in the donor vector is flanked by specific restriction sites (for example BglII and BstZ17l at the 5′-terminal end and Bgl II and Sgf I at the 3′-terminal end) to facilitate subcloning into a transfer vector of a commercial baculovirus generation system (for example, based on transposition such the “Bac-to-Bac®” system; InvitrogenTM).
Example 9. Generation of the Transfer Vectors of the Invention
The transfer vectors were generated by digesting the above donor vectors with BstZ17l at the 5′-terminal end of the expression cassette and with Hind III at the 3′-terminal end of the expression cassette. In this case, as a result of the subcloning, the SV40 polyadenylation signal of the baculovirus expression cassette is exchanged by the SV40 polyadenlation signal from the transfer vector. They were then cloned into the transfer vector pFastBacTM1 that was also digested with the same enzymes. Apart from this, all the elements of the expression cassette are included in the pFastBac transfer vector, substituting the polh promoter and MCS of the original commercial transfer vector.
Example 10. Generation of the Baculovirus Expression Vectors of the Invention Using the “Bac-to-Bac®” System
The modified transfer vectors pFastBacTM1 of Example 9 were used to generate recombinant baculoviruses by the “Bac-to-Bac®” Baculovirus Expression System. More specifically, the modified transfer vectors were used to transform the E. coli host strain DH10BacTM that contains a baculovirus shuttle vector (bacmid) and a helper plasmid, and allows the generation of the recombinant bacmids following transposition of the expression cassette. The DNA of the recombinant bacmids containing the baculovirus expression cassette of the present invention were then used to transfect insect Sf21 cells using Cellfectin®. 72 h post-transfection, cells were harvested and the first recombinant baculoviruses generation were obtained. These recombinant baculoviruses could then be further amplified and/or titered following conventional protocols.
For the baculoviruses of the invention that were used in Examples 4, 5 and 6, i.e. polhAc-ie-01/hr1p6.9p10Cap, polhAc-ie-01/hr1p6.9p10VP60 and polhAc-ie-01/hr1p6.9p10L1 the Ac-ie-01 cDNA was cloned under the control of the polh promoter. In the same baculoviruses, but in another locus, the Cap, VP60 or L1 encoding genes were cloned downstream of the hr1p6.9p10 chimeric promoter that was previously synthesized and contains the homologous region hr1 operatively linked to the promoters p6.9 and p10. A schematic representation of the resulting baculovirus expression cassettes of the present invention is shown in FIG. 4. The expression cassette of this baculovirus, i.e. polhAc-ie-01/hr1p6.9p10Cap, is represented by SEQ ID NO: 30. Specifically, a version of this expression cassette with a Cap gene polyadenylation signal from SV40 was used in the Examples (SEQ ID NO: 56). Also the expression cassettes polhAc-ie-01/hr1p6.9p10VP60 and polhAc-ie-01/hr1p6.9p10L1 of the Examples were used with a SV40 polyadenylation signal and are represented by SEQ ID NO: 55 and 54, respectively.
Likewise, the other baculoviruses were generated by the same methodology of Examples 8-10.
Example 11. Protein Sample Preparation
Infected cells from each time point (1×106) were harvested and centrifuged at 14000×g for 5 min. at 4° C. The supernatants were removed and the cell pellets were resuspended in PBS and subjected to three cycles of freezing (−196° C.) and thawing (37° C.). Cellular debris was removed by centrifugation.
Example 12. Time-Course Study of Protein Expression
Sf9, Sf21 or Hi-5TM cells were infected with the different recombinant baculoviruses expressing Cap protein under the control of different regulatory, enhancer and promoter elements, using a MOI of 5 or 0.1 as indicated. Cell cultures were harvested at various time points (24, 48, 72, 96 and 120 h post-infection) and the recombinant proteins' expression was analyzed by SDS-PAGE followed by Coomassie blue staining and/or Western blot.
Quantification of the recombinant proteins was carried out by two methodologies. One involved the use of a quantitative Western blot with a specific monoclonal antibody and subsequent analysis by the ChemiDocTM XRS Gel Imaging System (Bio-RadTM, USA) using purified counterpart proteins to carry out a standard quantification curve. A second technique involved the use of Pro260 chips (Bio-RadTM) and capillary electrophoresis using the ExperionTM system (Bio-RadTM), according to the manufacturer's instructions. The electrophoresis of the samples was made through microchannels by controlling the applied voltage and electric power. The microfluidic chip allowed several sequential procedures including separation, staining, destaining, detection and basic data analysis without any need of user's intervention. The ExperionTM system resolved and quantified protein samples from 10 to 260 kDa in size, with a high sensitivity, comparable to colloidal Coomassie blue SDS-PAGE gel staining. For quantification, a Pro260 ladder was used in the ExperionTM system, which is a modified version of the Precision Plus ProteinTM standard that has been optimized for use in that system.
Example 13. Rearing and Infection of Insect Larvae
Trichoplusia ni (cabbage looper) larvae were reared under level 2 biosafety conditions. Eggs were placed into specially designed larva developmental cages containing an artificial insect diet and were kept in growth chambers at 22° C. under controlled humidity (50%) and light period (8 h/day) conditions.
Trichoplusia ni (Cabbage looper) fifth-instar larvae (last instar larvae before pupation), were used for all experiments. The standard weight of each larva was approximately 120-130 mg and larvae were injected near the proleg (anterior to the body cavity) with 5 μl of recombinant baculoviruses diluted to reach the number of PFU per dose selected. Larvae were processed at 72 or 96 h post-infection. The collected larvae were frozen immediately to be stored at −20° C. until they were processed for recombinant protein quantification. Total soluble, non-denatured proteins (TSNDPs) from frozen T. ni larvae infected by the baculoviruses were obtained by homogenization using a Bag Mixer® blender (InterscienceTM, France) for 2 min. Extraction buffer was composed of PBS 1×, Triton X-100 at 0.01%, Complete protease inhibitor cocktail (RocheTM, Germany), and DTT 25 mM.
Example 14. Western Blot Analysis
Total soluble protein fractions (10 μg) from cells infected with the recombinant baculoviruses were resolved in 15% SDS-PAGE gels. Gels were stained by the Coomassie blue staining method or transferred to nitrocellulose membranes. Western blots were probed with the anti-Cap monoclonal antibody (I36A; IngenasaTM, Spain) at 1:1000 or with the anti L1 HPV16 monoclonal antibody (Camvir1; AbCam, USA) at 1:1000, and the immunocomplexes were visualized with anti-mouse IgG-horseradish peroxidase (HRP)-labeled conjugate (KPLTM, UK), diluted 1:2,000 or by an anti-rabbit IgG-horseradish peroxidase (HRP)-labeled conjugate (KPLTM, UK), diluted 1:2,000 respectively as a secondary antibody. Protein bands were detected using an ECL western blotting detection system and analyzed by the ChemiDocTM XRS Gel Imaging System (Bio-RadTM, USA).
BIBLIOGRAPHY


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## Claims

1. An expression cassette for a baculovirus expression vector system (BEVS), comprising a first nucleic acid sequence and a second nucleic acid sequence;
wherein the first nucleic acid sequence is selected from the group consisting of:
(a) a nucleic acid sequence comprising the sequence of SEQ ID NO: 1;
(b) a nucleic acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO:1 and encoding a protein able to function as a transcriptional regulator in a baculovirus;
(c) a nucleic acid sequence encoding an amino acid sequence comprising the sequence of SEQ ID NO: 6; and
(d) a nucleic acid sequence encoding a protein able to function as a transcriptional regulator in a baculovirus and having at least 95% sequence identity to the sequence of SEQ ID NO: 6;
wherein the second nucleic acid sequence directs the expression of a recombinant protein, wherein the recombinant protein is a virus-like particle protein;

wherein the expression cassette for BEVS also comprises at least one recombinant homologous region (hr) as enhancer region operably linked to a promoter suitable for driving the expression of the recombinant protein;
wherein the promoter suitable for driving the expression of the recombinant protein comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence comprising the sequence of SEQ ID NO:13; and
(b) a nucleic acid sequence able to function as a promoter in a baculovirus and having at least 95% sequence identity to the sequence of SEQ ID NO:13.

2. The expression cassette according to claim 1, wherein the recombinant protein is selected from the group consisting of:
(a) porcine circovirus capsid protein;
(b) foot and mouth disease virus VP1, VP3 and VP0 protein;
(c) canine parvovirus VP1 and VP2 protein;
(d) porcine parvovirus VP1 and VP2 protein;
(e) human norovirus (genogroup I or II) capsid protein;
(f) calicivirus capsid protein;
(g) human papillomavirus L1 protein;
(h) hepatitis E protein E2;
(i) infectious bursal disease virus VP1, VP2 and VP3 protein;
(j) astrovirus ORF2-encoded protein;
(k) influenza virus HA, NA and M1 protein;
(l) Hepatitis B core and surface antigen;
(m) parvovirus VP1 and VP2 protein; and
(n) rabbit calicivirus VP60 protein.

3. The expression cassette according to claim 2, wherein the porcine circovirus capsid protein is derived from porcine circovirus type 2.

4. The expression cassette according to claim 2, wherein the human papillomavirus L1 protein is derived from human papillomavirus type 16.

5. The expression cassette according to claim 2, wherein the rabbit calcivirus VP60 protein is derived from rabbit hemorrhagic disease virus.

6. The expression cassette according to claim 1, wherein the recombinant homologous region (hr) is selected from the group consisting of:
(a) a nucleic acid sequence comprising SEQ ID NO: 27; and
(b) a nucleic acid sequence able to function as an enhancer homologous region in a baculovirus and having at least 70% sequence identity with SEQ ID NO: 27.

7. A cloning vector, transfer vector, bacmid, recombinant baculovirus or cell comprising the expression cassette of claim 1, wherein the transfer vector further comprises a nucleic acid sequence suitable for integration or transposition in a baculovirus genome.

8. A culture media comprising the expression cassette according to claim 1.

9. An in vitro method for producing a recombinant protein comprising expressing said protein from the expression cassette according to claim 1 and purifying said protein.

10. A vaccine comprising the recombinant protein produced by the method according to claim 9.

11. An expression cassette for a baculovirus expression vector system (BEVS), comprising a first nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence comprising the sequence of SEQ ID NO:19; and
(b) a nucleic acid sequence substantially retaining the activity of the functional elements and having at least 95% sequence identity to the sequence of SEQ ID NO:19; and

a second nucleic acid sequence that encodes a recombinant protein, wherein the recombinant protein is a virus-like particle;
wherein the first nucleic acid sequence is operably linked to the expression of the second nucleic acid sequence.

12. An expression cassette for a baculovirus expression vector system (BEVS), comprising a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence comprising the sequence of SEQ ID NO:51; and
(b) a nucleic acid sequence substantially retaining the activity of the functional elements and having at least 95% sequence identity to the sequence of SEQ ID NO:51.