Patent Grant
9879280
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.
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.
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-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 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.
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:
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:
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:
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:
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®” (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 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-5™, Sf9, Sf21, BTI-Tn5B-1, 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 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.
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.
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 (
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 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 (
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) (
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:
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
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 (
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 (
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 ChemiDoc™ XRS Gel Imaging System (Bio-Rad™, 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 ChemiDoc™ 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-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 (
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 ChemiDoc™ XRS Gel Imaging System (Bio-Rad™, 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 (
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 (
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 (
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 ChemiDoc™ XRS Gel Imaging System (Bio-Rad™, 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 ChemiDoc™ 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-5™ 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 (
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 ChemiDoc™ XRS Gel Imaging System (Bio-Rad™, 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 (
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 (
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 (
The amount of L1 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 Sf9 cells cultured in suspension as described above. Quantification data of this analysis with the different baculoviruses obtained by the ChemiDoc™ 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) (
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 (
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 (
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 (
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 (
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 (Experion™; BioRad™, 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 (
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 Biotech™, Germany) containing 10% heat-inactivated fetal bovine serum (Pan Biotech™ 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.
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; Invitrogen™).
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 pFastBac™1 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.
The modified transfer vectors pFastBac™1 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 DH10Bac™ 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
Likewise, the other baculoviruses were generated by the same methodology of Examples 8-10.
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.
Sf9, Sf21 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 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 ChemiDoc™ XRS Gel Imaging System (Bio-Rad™, USA) using purified counterpart proteins 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 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 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.
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 (Interscience™, France) for 2 min. Extraction buffer was composed of PBS 1×, Triton X-100 at 0.01%, Complete protease inhibitor cocktail (Roche™, Germany), and DTT 25 mM.
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 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 (KPL™, UK), diluted 1:2,000 or by an anti-rabbit IgG-horseradish peroxidase (HRP)-labeled conjugate (KPL™, 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).
| Number | Date | Country | Kind |
|---|---|---|---|
| 12196120 | Dec 2012 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2013/075812 | 12/6/2013 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2014/086981 | 6/12/2014 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5965393 | Hasnain et al. | Oct 1999 | A |
| 20090068703 | Chao et al. | Mar 2009 | A1 |
| Number | Date | Country |
|---|---|---|
| 1811027 | Jul 2007 | EP |
| 2232308 | May 2005 | ES |
| 20010074351 | Aug 2001 | KR |
| WO-2005085456 | Sep 2005 | WO |
| WO-2010025764 | Mar 2010 | WO |
| WO-2011069562 | Jun 2011 | WO |
| WO-2012168492 | Dec 2012 | WO |
| WO-2012168789 | Dec 2012 | WO |
| WO-2012169940 | Dec 2012 | WO |
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| Number | Date | Country | |
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| 20150307899 A1 | Oct 2015 | US |
