WO2014086973A1

WO2014086973A1 - Enhanced production of the porcine circovirus capsid protein by a baculovirus vector expression system

Info

Publication number: WO2014086973A1
Application number: PCT/EP2013/075799
Authority: WO
Inventors: Silvia Gomez Sebastian; Javier Lopez Vidal; José Angel MARTINEZ ESCRIBANO
Original assignee: Alternative Gene Expression S.L.
Priority date: 2012-12-07
Filing date: 2013-12-06
Publication date: 2014-06-12

Abstract

The present invention may be included in the field of biotechnology and it covers the improved production of a subunit vaccine against porcine circovirus based on the capsid protein (Cap) 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 a recombinant Cap protein from a porcine circovirus. 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 Cap protein by using the aforesaid sequences, vectors, cells or insects.

Description

ENHANCED PRODUCTION OF THE PORCINE CIRCOVIRUS CAPSID PROTEIN BY A BACULOVIRUS VECTOR EXPRESSION SYSTEM FIELD OF THE INVENTION

The present invention may be included in the field of biotechnology and it covers the improved production of a subunit vaccine against porcine circovirus based on the capsid protein (Cap) 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 a recombinant Cap protein from a porcine circovirus. 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 Cap protein 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 ( cMNPV) with Spodoptera frugiperda 9 (5/9) or 21 (5/21) 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-Five™ (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 plO genes, has serious disadvantages in the foreign protein post-translational modifications. Baculovirus promoters that allow for earlier expression than the conventionally used polh or plO promoters have been characterized and been used for heterologous protein production, but showed a reduced productivity i

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, lcMNPV 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 (fir) 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 orfl41 (exonO), 16 amino acids encoded by the upstream nontranslated leader of iel, 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.

A virus-like particle consists of a protein or a group of proteins that self- assemble forming structures similar to virus particles but lacking the nucleic acid material. These virus-like particles are excellent alternatives to inactivated viruses as vaccines because they have the spatial and epitope structure of the infective virus but they are not infective and completely safe for individuals that become vaccinated. One of the marketed vaccines based on virus-like particles is that based on the capsid protein (Cap), encoded by the ORF2 (open reading frame 2) from porcine circovirus type 2 (PCV2). PCV2 is a circular single- stranded DNA virus that causes the postweaning multisystemic wasting syndrome (PMWS), a multifactorial disease affecting weaned piglets and characterized by growth retardation, loss of weight and death (13).

The Cap protein from PCV2 is a leading animal vaccine produced by BEVS. There is thus a need for novel alternative BEVSs that produce this specific baculovirus- based vaccine more efficiently. In particular, there is a need for novel BEVSs that allow a) stronger expression than the commercial BEVS that uses the polh promoter and/or b) reduction of the virus-induced cell damage during the production process in order to increase the virus-like particle formation avoiding protein degradation.

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 capsid protein from porcine circovirus 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-l/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 capsid protein. 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 expression of a recombinant capsid protein from a porcine circovirus.

The expression cassette according to item 1, comprising a nucleic acid sequence encoding the capsid protein selected from the group consisting of:

(a) nucleic acid sequence indicated in SEQ ID NO: 31;

(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 SEQ ID NO: 31 and encoding a protein functioning as a subunit of a virus-like particle;

(c) nucleic acid sequence encoding a protein with the amino acid sequence indicated in SEQ ID NO: 32; and

(d) nucleic acid sequence encoding a protein functioning as a subunit of a virus-like particle 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 SEQ ID NO: 32.

The expression cassette according to item 1 or 2, 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.

The expression cassette according to any of the items 1-3, comprising a promoter driving the expression of the capsid protein 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.

The expression cassette according to item 4, comprising a promoter driving the expression of the capsid protein 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.

The expression cassette according to item 4 or 5, comprising a promoter driving the expression of the capsid protein 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. The expression cassette according to any of the items 1-6, comprising at least one recombinant homologous region (hr) as enhancer region, operably linked to the promoter that drives the expression of the capsid protein. The expression cassette according to item 7, 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. The expression cassette according to any of the items 1-8, comprising a nucleic acid sequence that is operably linked to the expression of the capsid 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. The expression cassette according to item 9, comprising a nucleic acid sequence that is operably linked to the expression of the capsid 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. The expression cassette according to any one of items 1-8, 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: 43-44; 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: 43-44. A cloning vector comprising the expression cassette of any of the items 1- 11. A transfer vector comprising the expression cassette of any of the items 1- 11 and further a nucleic acid sequence suitable for integration or transposition in a baculovirus genome. The transfer vector according to item 13, characterized in that the transfer vector is derived from any of the baculovirus expression systems "Bac-to-Bac®" (invitrogen™), "BacPAK™" (Clontech™), "FlashBAC™" (Oxford Expression Technologies™), "BacuVance™" (GenScript™), "Bac- N-Blue D A™" (invitrogen™), "BaculoDirect™" (invitrogen™), "BacVector®" 1000, 2000, 3000 (Novagen®), "DiamondBac™" (Sigma- Aldrich®) or "BaculoGold™" (BD biosciences™). A bacmid comprising the expression cassette of any of the items 1-11. 16. A recombinant baculovirus comprising the expression cassette of any of the items 1-11.

17. A cell comprising the expression cassette of any of the items 1-11.

18. The cell according to item 17, infected, transfected, transduced or transformed with the expression cassette, cloning vector, transfer vector, bacmid or recombinant baculovirus of any of the items 1-16. 19. The cell according to item 17 or 18, characterized in that it is of insect origin.

20. The cell according to any of the items 17-19, characterized in that it is derived from an insect belonging to the Lepidoptera or Diptera genus.

21. The cell according to any of the items 17-20, characterized in that it is derived from T choplusia ni, Spodoptera frugiperda, Ascalapha odorata, Bombyx mori, Drosophila melanogaster, Stigmene acrea or Aedes aegypti. 22. The cell according to any of the items 17-21, characterized in that it is a cell line selected from the group consisting of Hi-5™, 5/9, /21, BTI-Tn5B- 1, Tn368, ExpresSf+®, BTI-TnAo38, ATC-10, Mimic™ Sf9, SfSWT-1, SfSWT-3, SfSWT-5, TriEx™ and Schneider's Drosophila Line 2. 23. An insect comprising the expression cassette of any of the items 1-11.

24. The insect according to item 23, infected, transfected, transduced or transformed with the expression cassette, cloning vector, transfer vector, bacmid or recombinant baculovirus of any of the items 1-16.

25. The insect according to item 23 or 24, wherein the insect is derived from the genus Lepidoptera.

26. The insect according to any of the items 23-25, wherein the insect is selected from the group consisting of T choplusia ni, Spodoptera frugiperda, Spodoptera exigua, Ascalapha odorata, Bombyx mori, Rachiplusia ni and Stigmene acrea.

27. The insect according to any of the items 23-26, wherein the expression cassette is introduced into the insect by a recombinant baculovirus, preferably AcMNPV, SeNPV or B NPV.

28. A culture medium comprising the expression cassette, cloning vector, transfer vector, bacmid or recombinant baculovirus of any of the items 1- 16.

29. A method for producing a capsid protein from a porcine circovirus comprising the use of the expression cassette, cloning vector, transfer vector, bacmid, recombinant baculovirus, cell or insect according to any of the items 1-27 and the extraction and purification of the capsid protein by conventional means.

30. Capsid protein obtainable by the method according to item 29. 31. Capsid protein according to item 30 for use as a vaccine.

32. Capsid protein according to item 30 for use in a therapy to induce an immune response in an animal body against said protein. 33. Capsid protein according to item 30 for use in a vaccine therapy to prevent postweaning multisystemic wasting syndrome in animals.

34. Method of vaccination to prevent postweaning multisystemic wasting syndrome in animals using the capsid protein according to item 30.

35. A use of the expression cassette according to any of the items 1-11 for producing a cloning vector, transfer vector, bacmid, recombinant baculovirus, cell, insect or culture medium according to any of the items 12-28. 36. A use of the cloning vector according to item 12 for producing a transfer vector, bacmid, recombinant baculovirus, cell, insect or culture medium according to any of the items 13-28. 37. A use of the transfer vector according to any of the items 13-14 for producing a bacmid, recombinant baculovirus, cell, insect or culture medium according to any of the items 15-28.

38. A use of the bacmid according to item 15 for producing a recombinant baculovirus, cell, insect or culture medium according to any of the items

16-28.

39. A use of the recombinant baculovirus according to item 16 for producing a cell, insect or culture medium according to any of the items 17-28.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. 5/9 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-5™ 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 hours post-infection at a 2 OX magnification in an inverted microscope Leica™ DMIL™.

Figure 2: A) Percentage of T. ni larvae surviving 96 hours post-infection using 5 x 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 hours post-infection. The infectious dose was 5 x 10⁴ PFUs.

Figure 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 96h 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. Figure 4: Schematic representation of baculovirus recombinant DNA elements of the invention: a sequence encoding for transcriptional regulators (A; e.g. IE-0 and IE-1], which expression is driven by a promoter (B; e.g. polh); an enhancer homologous region (hr) sequence (C; e.g. hrl) upstream of the promoters (D; e.g. p6.9pl0) driving the expression of the foreign gene (ORF2) coding for a recombinant capsid protein from a porcine circovirus type 2 (PCV2). The scheme shows the theoretical mechanism of interaction between the recombinant DNA elements of the present invention that results in the unprecedented overexpression of the PCV2 capsid protein Cap. Figure 5: SDS-PAGE and Coomassie blue staining analysis of extracts from S 21 and S 9 cells cultured in monolayer and suspension respectively and infected with a conventional baculovirus expressing the Cap protein 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/hrlp6.9plOCap. Additionally, a Western blot analysis of the same extracts using an anti-tubulin antiserum revealed a higher degradation degree of this protein at late post-infection times when the conventional baculovirus polhCap was used to infect the cells.

Figure 6: 5/21 and 5/9 insect cells, cultured in monolayer and suspension respectively, were infected with a conventional baculovirus engineered with the expression cassette polhCap or with a baculovirus of the invention with the expression cassette polhAc-ie-01/hrlp6.9plOCap. Cells were sampled at different times post-infection (0 to 120 hours) and cell extracts analysed by SDS-PAGE and Western blot with a monoclonal antibody against the Cap protein.

Figure 7: A) and B) Time course study of the expression levels of Cap protein obtained in 5/21 cells infected in monolayer at a MOI of 5 with a conventional baculovirus engineered with the expression cassette polhCap or with a baculovirus of the invention with the expression cassette polhAc-ie- 01/hrlp6.9plOCap, measured by the ChemiDoc™ XRS Gel Imaging System (Bio- Rad™, USA). C) and D) A similar experiment was conducted in 5/9 cells cultured in suspension that were infected at a MOI of 0.1. The expression of the recombinant Cap protein under these conditions was also measured by the same system at different times post-infection. The figure shows the arithmetic media of three independent experiments. Figure 8: Comparison of recombinant Cap protein productivity in mg/L in 5/9 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 po!hAc-ie-01/hrlp6.9plOCap (dark grey). 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 ChemiDoc™ XRS Gel Imaging System (Bio- Rad™, USA). Cells were infected at a MOI of 0.1 with each virus. Figure 9; The differences in cell density (A) and viability (B) of 5/9 cells cultured in suspension and infected at a MOI of 0.1 with a baculovirus containing the expression cassette polhCap or polhAc-ie-01/hrlp6.9plOCap were analyzed up to 120 h post-infection.

Figure 10: 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 the polh promoter or the expression cassette polhAc- ie-01/hrlp6.9pl0. VLPs are shown at two magnifications. The inserts in the lower left corners of the upper panels show Coomassie blue staining protein profile of purified VLPs preparations resolved by SDS-PAGE electrophoresis.

Figure 11; 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/hrlp6.9plOCap. Protein extracts from larvae infected with 5 x 10⁵ PFUs of every baculovirus were obtained at 72 and 96h 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 ChemiDoc™ XRS Gel Imaging System (Bio-Rad™, USA). B) Representation of the quantification of Western blots. The baculovirus containing the expression cassette polhAc-ie-01/hrlp6.9plOCap increased the productivity of Cap protein by about 325% at 72h post-infection and by about a 24% at 96h post-infection compared to the expression by the conventional baculovirus.

Figure 12: Percentage of larvae surviving at 96h post-infection with 5 x 10⁵ PFU of the baculovirus containing the expression cassette of the present invention polhAc-ie-01/hrlp6.9plOCap or the conventional baculovirus [polhCap).

Figure 13: Comparison of recombinant Cap protein productivity in mg in 100 Trichoplusia ni larvae 72 and 96h 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/hrlp6.9plOCap (dark grey). Productivities were measured by microfluidic protein analysis (Experion™; BioRad™, USA). Insects were infected with 5 x 10^s PFU of each baculovirus.

DETAILED DESCRIPTION OF THE INVENTION

The present invention improves the expression of recombinant porcine circovirus capsid protein 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 capsid 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 capsid protein, which comprises the nucleic acid sequence encoding the capsid protein from a porcine circovirus (ORF2) 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.

In some embodiments, the expression cassette of the invention further comprises an enhancer homologous region (hr), such as hrl, operably linked to the promoter of said sequence encoding the capsid 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 capsid protein of the invention is a recombinant capsid protein from a porcine circovirus.

The capsid protein of the invention can function as a subunit of a virus-like particle and may thus be used as a vaccine. It can be used for vaccination of animals against the postweaning multisystemic wasting syndrome (PMWS) and may have industrial, commercial, therapeutic or preventive application.

The capsid protein of the invention is preferably encoded by the nucleic acid sequence of SEQ ID NO: 31 (also referred to as Cap) or represented by the corresponding amino acid sequence of SEQ ID NO: 32. The capsid protein may also be encoded or represented by variants of said sequences.

SEQ ID NO: 31 corresponds to the 0RF2 gene from porcine circovirus type 2. The variants of SEQ ID NO: 31-32 are or encode amino acids still functioning as subunits of virus-like particles.

The sequence of the variants of SEQ ID NO: 31 is preferably at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95% identical to the sequence of SEQ ID NO: 31.

The sequence of the variants of SEQ ID NO: 32 is preferably at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95% similar to the sequence of SEQ ID NO: 32.

"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 capsid 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 capsid 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 capsid protein of the invention is driven by a promoter that comprises SEQ ID NO: 11, i.e. the pl O 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 sequence encoding the capsid protein is the plO or SV40 polyadenylation signal. Most preferably, it is the pl O polyadenylation signal. The most preferred expression cassettes comprising the polyadenylation signal from the nucleic acid sequence encoding the capsid protein are represented by SEQ ID NO: 43 [pl O polyadenylation signal) and SEQ ID NO: 44 (SV40 polyadenylation signal) (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: 43-44).

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 porcine circovirus capsid 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 pB2 . The expression level can be determined at both the mR A 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 capsid 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 porcine circovirus capsid 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 ;4cMNPV 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 hrl (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 capsid 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^®" (invitrogen™), "BacPAK™" (Clontech™), "FlashBAC™" (Oxford Expression Technologies™), "BacuVance™" (GenScript™), "Bac-N-Blue DNA™" (invitrogen™), "BaculoDirect™" (invitrogen™), "BacVector^®" 1000, 2000, 3000 (Novagen^®), "DiamondBac™" (Sigma-Aldrich^®) or "BaculoGold™" (BD biosciences™).

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 J CMNPV.

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, Bombyx mori, Drosophila melanogaster, Stigmene acrea and Aedes aegypti and most preferably it is selected from the group of insect cell lines consisting of Hi-5™, 5/9, 5/21, BTI-Tn5B-l, Tn368, ExpresSf+^®, BTI-TnAo38, ATC-10, Mimic™ Sf9, SfSWT-1, SfSWT-3, SfSWT-5, TriEx™ 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 ^cMNPV, 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 porcine circovirus capsid protein

In a further aspect, the invention discloses methods for producing the porcine circovirus capsid protein.

In a preferred embodiment, the production of the capsid 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 capsicl 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 porcine circovirus capsid protein, the cells of the invention are cultured in suspension (bioreactors), at densities between 2xl0⁶ to 8xl0⁶ 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 capsid 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 porcine circovirus capsid protein is purified by conventional means.

In a preferred embodiment, the porcine circovirus capsid protein that is produced according to the method of the invention is used as a vaccine.

SUMMARY OF SEQUENCES

7 IE-0 protein

8 IE-1 N-terminal domain protein

9 IE-0 N-terminal domain protein

10 polh (promoter)

11 plO (promoter)

12 pB29plO (promoter)

13 p6.9pl0 (promoter)

14 pB2₉ (promoter)

15 pB2plO (promoter)

16 polhplO (promoter)

17 polhAc-ie-01 /hrl pi 0

18 polhAc-ie-01/hrlpB2₉plO

19 polhAc-ie-01/hrl p6.9pl 0

20 pB2₉Ac-ie-01/hrlplO

21 pB2₉Ac-ie-01/hrlpB2₉plO

22 pB2₉Ac-ie-01/hrl p6.9pl 0

23 polhAc-ie-01 /hrl polh

24 pB2₉Ac-ie-01/hrlpolh

25 polhAc-ie-01/hrlpolhplO

26 pB2₉Ac-ie-01 /hrl polhpl 0

27 Homologous region enhancer hrl

28 polhAc-ie-01

29 po!hGFP

30 polhAc-ie-01/hrlp6.9plOCap

31 ORF2 from porcine circovirus type 2

32 Capsid protein (Cap) from porcine circovirus type 2

33 polhCap

34 polhAc-ie-01/hrlp6.9plOGFP

35 polhAc-ie-01/hrlpolhplOGFP

36 polhAc-ie-01 /hrl pB2₉pl 0GFP

37 polhAc-ie-01 /hrlp6, 9GFP 38 polhAc-ie-01/hrlplOGFP

39 poJhAc-ie-01/hrlpolhGFP

40 p6.9GFP

41 pB2₉GFP

42 plOGFP

43 polhAc-ie-01 /hrl p6.9pl OCap (including the polyadenylation signal from the plO gene after the Cap gene)

44 polhAc-ie-01 /hrlp6.9pl OCap (including the SV40 polyadenylation signal after the Cap gene)

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/hrlp6.9plOCap was deposited in the Spanish Type Culture Collection (CECT) (www.cect.org); University of Valencia, Pare Cientific Universitat de Valencia; Catedratico Agustin Escardino, 9; 46980 Paterna (Valencia), Spain, with the accession number CECT 8228 on November 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 S 9 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 120h 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 (Figure 1A). These differences in cell number were observed as early as 6h postinfection (Figure 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 Gl 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 FACSCalibur™ (BD Biosciences™) flow cytometer. Cells were fixed in 70% EtOH, resuspended and incubated in the staining solution (50 μ /ιτι1 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 (Gl, 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 (Figure IB). 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 pB2g or polh promoters (data not shown). Results observed in S/ insect cells infected in suspension were confirmed in 5/21 cells cultured in monolayer (data not shown) and also in Hi-5™ cells cultured in monolayer (Figure 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 poJhAc-ie-01 or polhGFP, T. ni larvae were infected with a high infectious dose of 5 x 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 (polh Ac-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) (Figure 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) (Figure 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 plO or any chimeric promoter containing plO

To analyze the effect of the transcriptional regulators IE-l/IE-0 in combination with different promoters on protein expression, recombinant ^cMNPV baculoviruses with the following expression cassettes were prepared: polhAc-ie-01 /hrl polhpl OGFP

polhAc-ie-01 /hrl p6.9pl OGFP

polhAc-ie-01 /hrl pB2₉pl OGFP

polhAc-ie-01 /hrl p6.9GFP

polhAc-ie-01/hrlplOGFP

polhAc-ie-01 /hrlpolhGFP

p6.9GFP

pB2₉GFP

plOGFP

polhGFP

As a control, a conventional ^lcMNPV 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 Figure 3, the presence of the plO 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 a recombinant Cap protein in a baculovirus vector expression system (BEVS).

The expression of Cap protein was compared between a conventional baculovirus and a baculovirus of the invention. The conventional baculovirus expressed the Cap protein under the control of the polh promoter [polhCap). The baculovirus of the invention expressed the Cap protein under the control of the p6.9pl0 chimeric promoter that was previously synthesized. This chimeric promoter was operatively linked with the enhancer sequence homologous region hrl. 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 cassette polhAc-ie-01/hrlp6.9plOCap (Figure 4). The expression of Cap protein was then analyzed in protein extracts from S/9 and 5/21 cells cultured in monolayer and suspension at different times post-infection with these baculoviruses. Extracts were subjected to SDS-PAGE electrophoresis and the resolved proteins were stained by Coomassie blue. A more intensely stained protein band corresponding to Cap protein expressed by the baculovirus modified by the expression cassette of the present invention was observed at the different times post-infection in both the S/9 and S 21 cells (Figure 5). This clearly demonstrated that the novel expression cassette is more efficient in expressing the Cap protein in both monolayer and suspension cells independently of the multiplicity of infection used (MOI 5 or MOI 0.1].

The above extracts were also used for Western blot analysis with a specific antibody against the constitutive cellular protein tubulin, in order to observe its integrity in infected cells. Interestingly, the Western blots revealed that the integrity of tubulin is better in cells infected by the baculovirus modified by the expression cassette of the present invention than in cells infected by the conventional baculovirus (Figure 5). This indicated an improved integrity of the cells infected with virus of the invention at late times post-infection.

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/hrlp6.9plOCap). The amount of protein was analyzed using an ECL western blotting detection system and a ChemiDoc™ XRS Gel Imaging System (Bio-Rad™, USA). Western blot reactions were studied at different times post-infection in SJ9 and 5/21 cells cultured in monolayer and suspension as described above (Figure 6).

Quantification data of this analysis with the different baculoviruses obtained by the ChemiDoc™ XRS Gel Imaging System was expressed as arbitrary expression units. At 72h 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 5/21 monolayer (MOI of 5) and 5/9 suspension cultures (MOI of 0.1) respectively with the baculovirus modified by the expression cassette of the invention (polhAc-ie-01/hrlp6.9plOCap) (Figure 7). These differences in protein accumulation were also observed in Hi- 5™ 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 (Figure 7). Maximum levels of expression were detected at 72h 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 postinfection (Figure 7).

A more precise quantification of the productivity of Cap protein was made by Western blots 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/hrlp6.9plOCap). The quantification was carried out with a standard curve of purified Cap protein and subsequent analyses by the ChemiDoc™ XRS Gel Imaging System (Bio-Rad™, USA), Insect cells were cultured in suspension at a density of 2xl0⁶ 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 57mg/L, the insect cells infected with the baculovirus of the invention were able to produce about 198mg/L (Figure 8). The maximum productivities of recombinant baculoviruses were obtained at different times after infection (48 and 72 h post-infection respectively). Example 5. The baculovirus expression cassettes of the invention induce cell proliferation and increase cell viability.

The baculoviruses of Example 4 containing the polhCap and polhAc-ie- 01/hrlp6.9plOCap 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 a baculovirus containing the expression cassette of the invention, i.e. polhAc-ie-01/hrlp6.9plOCap (Figure 9).

Example 6. The expression cassette of the invention induces the formation of more Cap VLPs than a conventional baculovirus, but with the same size and shape.

5/9 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/hrlp6.9plOCap). 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 (Figure 10). The higher number of VLPs produced by the cells infected with the baculovirus of the invention correlated with the amount of Cap protein detected in the purified VLPs preparations by SDS-PAGE electrophoresis and Coomassie blue staining (Figure 10, upper panels, inserts in the lower left corners).

Example 7. The expression cassette of the present invention potentiates the productivity of recombinant Cap protein 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 x 10⁵ PFU of the baculovirus with the expression cassette po!hCop or polhAc-ie-01/hrlp6.9plOCap 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 (Figure 11A). The expressed Cap protein in the different extracts was also quantified by ChemiDoc™ XRS Gel Imaging System (Bio-Rad™, USA) (Figure 11B). 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 72h post-infection and by 24% at 96h post-infection (Figure 11B). Additionally, larvae infected with the baculovirus modified by the expression cassette of the present invention presented a 30% increase in survival (Figure 12). 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 (Experion™; BioRad™, USA) and the insect biomass recovered after infection. The productivity was studied at 72 and 96h post-infection with 5 x 10⁴ PFU of the respective baculovirus. The baculovirus containing the expression cassette of the invention, i.e. polhAc-ie-01/hrlp6.9plOCap, 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 (Figure 13). One hundred infected larvae with the baculovirus containing the expression cassette polhAc-ie-01/hrlp6.9plOCap were able to produce about 300mg of recombinant Cap protein at 96 h post-infection, whereas the larvae infected with the conventional baculovirus only produced 150 mg (Figure 13).

Example 8. Cell culture and viruses.

The Spodoptera frugiperda 5/21 or 5/9 cell lines were cultured in 6-well tissue culture plates (lxlO⁶ cells/well) in TNM-FH insect medium (Pan Biotech™, Germany) containing 10% heat-inactivated fetal bovine serum (Pan Biotech™, Germany) at 27°C.

Confluent 5/9 or SJ21 cells in monolayer (lxlO⁶ cells/well) were infected with the baculoviruses at different multiplicities of infection (from 0.01 to 10). In suspension, 5/9 cells (2xl0⁶ cells/ml) were infected equally at different multiplicities of infection. Infected cells were analysed from 16 to 120h postinfection. Example 9. 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) was cloned into the MCS of a cloning plasmid using the Xho I and Nco I restriction sites. After introduction of the Cap encoding gene, the cloning vector becomes the donor vector. The baculovirus expression cassette in the donor vector is flanked by specific restriction sites (for example Bglll and BstZI A at the 5 '-terminal end and Bgl II and Sgfl at the 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; invitrogen™).

Example 10. Generation of the transfer vector of the invention

The transfer vector was generated by digesting the above donor vector with BstZl 71 at the 5 '-terminal end of the expression cassette and with Hind III at the 3 '-terminal end of the expression cassette. It was then cloned into the transfer vector pFastBac™l that was also digested with the same enzymes. 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. 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 11. Generation of the baculovirus expression vector of the invention using the "Bac-to-Bac^®" system

The modified transfer vector pFastBac™l of Example 9 was used to generate recombinant baculoviruses by the "Bac-to-Bac^®" Baculovirus Expression System. More specifically, the modified transfer vector was used to transform the E. coli host strain DHlOBac™ that contains a baculovirus shuttle vector (bacmid) and a helper plasmid, and allows the generation of the recombinant bacmid following transposition of the expression cassette. The DNA of the recombinant bacmid containing the baculovirus expression cassette of the present invention was then used to transfect insect 5/21 cells using Cellfectin^®. 72 h post-transfection, cells were harvested and the first recombinant baculovirus generation was obtained. This recombinant baculovirus could then be further amplified and/or titered following conventional protocols. For the baculovirus of the invention that was used in Examples 4, 5 and 6, i.e. poIhAc-ie-01/hrlp6.9plOCap, the Ac-ie-01 cDNA was cloned under the control of the polh promoter. In the same baculovirus, but in another locus, the Cap encoding gene was cloned downstream of the hrlp6.9pl0 chimeric promoter that was previously synthesized and contains the homologous region hrl operatively linked to the promoters p6,9 and plO. A schematic representation of the resulting baculovirus expression cassette of the present invention and the putative function of the recombinant DNA elements is shown in Figure 4. The expression cassette of this baculovirus, i.e. polhAc-ie-01/hrlp6.9plOCap, 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: 44).

Likewise, the other baculoviruses were generated by the same methodology of Examples 9-11.

Example 12. Protein sample preparation.

Infected cells from each time point (lxlO⁶) were harvested and centrifuged at 14000 x 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 13. Time-Course Study of Protein Expression.

5/9, 5/21 or Hi-5™ 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 postinfection) and the Cap protein expression was analyzed by SDS-PAGE followed by Coomassie blue staining or Western blot.

Quantification of the recombinant Cap protein 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 ChemiDoc™ XRS Gel Imaging System (Bio-Rad™, USA) using purified Cap protein to carry out a standard quantification curve. A second technique involved the use of Pro260 chips (Bio-Rad™) and capillary electrophoresis using the Experion™ system (Bio-Rad™), according to the manufacturer's instructions. The electrophoresis of the samples was made through microchanneis 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 Experion™ 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 Experion™ system, which is a modified version of the Precision Plus Protein™ standard that has been optimized for use in that system.

Example 14. 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. T choplusia 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 μΐ of recombinant baculoviruses diluted to reach the number of PFU per dose selected. Larvae were processed at 72 or 96 h postinfection. 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 (Interscience™, France) for 2 min. Extraction buffer was composed of PBS Ix, Triton X-100 at 0.01%, Complete protease inhibitor cocktail (Roche™, Germany), and DTT 25 mM.

Example 15. 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; Ingenasa™, Spain) at 1:1000 or tubulin antiserum (T5168; Sigma-Aldrich™) and the immunocomplexes were visualized with anti-mouse IgG-horseradish peroxidase (HRP)-labeled conjugate (KPL™, 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 ChemiDoc™ XRS Gel Imaging System (Bio-Rad™, USA).

BIBLIOGRAPHY

Nettleship, J.E., Assenberg, R., Diprose, J.M., Rahman-Huq, N., Owens, R.J. Recent advances in the production of proteins in insect and mammalian cells for structural biology. J. Struct. Biol. 2010, 172, 55-65. Gomez-Casado E, Gomez-Sebastian S, Niinez MC, Lasa-Covarrubias R, Martinez-Pulgarin S, Escribano JM. Insect larvae biofactories as a platform for influenza vaccine production. Protein Expr Pu f. 79: 35-43. 2011.

Smith, G.E., M.D. Summers, and M.J. Fraser. 1983. Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol. Cell Biol 3: 2156-21 65.

Hitchman RB, Possee RD, King LA. Baculovirus expression systems for recombinant protein production in insect cells. Recent Pat Biotechnol. 2009;3(l):46-54.

Hashimoto, Y., S. Zhang, Y. R. Chen and G. W. Blissard (2012). "BTI- Tnao38, a new cell line derived from Trichoplusia ni, is permissive for AcMNPV infection and produces high levels of recombinant proteins." BMC Biotechnol 12: 12.

Taticek RA, Choi C, Phan SE, Palomares LA, Shuler ML. Comparison of growth and recombinant protein expression in two different insect cell lines in attached and suspension culture. Biotechnol Prog. 2001, 17 (4), 676-684.

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Claims

CLAIMS:

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 expression of a recombinant capsid protein from a porcine circovirus.

The expression cassette according to claim 1, comprising a nucleic acid sequence encoding the capsid protein selected from the group consisting of:

(a) nucleic acid sequence indicated in SEQ ID NO: 31;

The expression cassette according to claim 1 or 2, 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

4. The expression cassette according to any of the claims 1-3, comprising a promoter driving the expression of the capsid protein selected from the group consisting of:

(a) nucleic acid sequence indicated in any of SEQ ID NO: 10-16; and

5. The expression cassette according to claim 4, comprising a promoter driving the expression of the capsid protein selected from the group consisting of:

6. The expression cassette according to claim 4 or 5, comprising a promoter driving the expression of the capsid protein 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.

The expression cassette according to any of the claims 1-6, comprising at least one recombinant homologous region (hr) as enhancer region, operably linked to the promoter that drives the expression of the capsid protein.

The expression cassette according to claim 7, 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.

The expression cassette according to any of the claims 1-8, comprising a nucleic acid sequence that is operably linked to the expression of the capsid 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, preferably 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: 17-22, 25 and 26, preferably any of SEQ ID NO: 17-19 and 25.

10. The expression cassette according to any one of claims 1-8, comprising a nucleic acid sequence selected from the group consisting of:

(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: 43-44.

11. A cloning vector, transfer vector, bacmid, recombinant baculovirus, cell or insect comprising the expression cassette of any of the claims 1-10, wherein the transfer vector further comprises a nucleic acid sequence suitable for integration or transposition in a baculovirus genome.

12. A culture medium comprising the expression cassette, cloning vector, transfer vector, bacmid or recombinant baculovirus of any of the claims 1- 11. 13. A method for producing a capsid protein from a porcine circovirus comprising the use of the expression cassette, cloning vector, transfer vector, bacmid, recombinant baculovirus, cell or insect according to any of the claims 1-11 and the extraction and purification of the capsid protein by conventional means.

14. Capsid protein obtainable by the method according to claim 13.

15. Capsid protein according to claim 14 for use in a method selected from the group consisting of use as a vaccine, use in a therapy to induce an immune response in an animal body against said protein, use in a vaccine therapy to prevent postweaning multisystemic wasting syndrome in animals and use as a diagnostic reagent.