PRODUCTION OF RECOMBINANT EXPRESSION VECTORS

Abstract

The present invention relates to a method for the production of recombinant expression vectors, a kit adapted to carrying out the method, a vector used in the context of the method, a cell containing such vector and the use of the vector.

Claims

1. A method for the production of recombinant expression vectors, comprising the following steps: (1) providing a cell population containing a starting vector, said cell population expresses a first gene product encoded by the starting vector in such a manner that it is accessible to a first binding molecule that binds to said first gene product, whereas in the starting vector the coding nucleotide sequence for the first gene product is upstreamly and downstreamly flanked by first recombination nucleotide sequences; (2) transfecting the cell population obtained from step (1) with a transfer vector encoding a second gene product, whereas in the transfer vector the coding nucleotide sequence for the second gene product is upstreamly and downstreamly flanked by second recombination nucleotide sequences which are homologous to said first recombination nucleotide sequences; (3) incubating the cell population obtained from step (2) under conditions which allow an exchange of the coding nucleotide sequence for said first gene product against the coding nucleotide sequence for said second gene product by homologous recombination of said first and second recombination nucleotide sequences and the formation of a recombinant expression vector; (4) incubating the cell population obtained from step (3) under conditions which allow the expression of said second gene product, where applicable in such a manner that it is accessible to a second binding molecule that binds to said second gene product, (5) contacting the cell population obtained from step (4) with said first binding molecule that binds to said first gene product under conditions which allow the formation of complexes of said first binding molecule and said first gene product; (6) separating said complexes of first binding molecule and first gene product from the cell population, and (7) isolation of the recombinant expression vectors from the cell population;

2. The method of claim 1, comprising the following steps: (5′) contacting said cell population obtained from step (4) with said second binding molecule that binds to said second gene product under conditions which allow the formation of complexes of said second binding molecule and second gene product; (6′) separating said complexes of second binding molecule and second gene product, and (7′) isolation of the recombinant expression vectors from said complexes of second binding molecule and second gene product.

3. The method of claim 1, wherein said first gene product is selected from the group consisting of: protein, protein fragment, membrane protein, cell surface protein, CD4.

4. The method of claim 3, wherein in step (5) the cell population obtained from step (4) is contacted with said binding molecule which binds to said first gene product under conditions which allow the formation of complexes of first binding molecule and cell, and in step (6) said complexes of first binding molecule and cell are separated from cells which are not bound to said first binding molecule (negative cells), and in step (7) the recombinant expression vectors are isolated from the negative cells.

5. The method of claim 4, wherein after step (6) and before step (7) the following further steps occur: (6.1) disintegrating the negative cells to obtain a cell lysate containing said recombinant expression vectors; (6.2) incubating the cell lysate obtained from step (6.1) with non transfected cells under conditions which allow a transfection of the cells with the recombinant expression vectors, to obtain a transfected cell population; (6.3) contacting said transfected cell population obtained from step (6.2) with said first binding molecule which binds to the first gene product under conditions which allow the formation of complexes of first binding molecule and cell, (6.4) separating said complexes of first binding molecule and cell from cells which are not bound to the binding molecule (negative cells), and (6.5) repeating the steps (6.1) to (6.4) at least one time.

6. The method of claim 1, wherein after step (7) the following further steps occur: (8) incubating of the recombinant expression vectors with non-transfected cells under conditions which allow a transfection of the cells with said recombinant expression vectors, to obtain a further transfected cell population; (9) contacting said further transfected cell population with said binding molecule which binds to the second gene product under conditions which allows the binding of complexes of second binding molecule and second gene product; (10) separating said complexes of second binding molecule and second gene product, and (11) repeating the steps (8) to (10) at least one time.

7. The method of claim 1, wherein said first and said second binding molecule is an antibody.

8. The method of any of claim 1, wherein said first and second binding molecule is bound to a magnetic entity.

9. The method of claim 7, wherein said second gene product is selected from the group consisting of: antigen, viral antigen, tumor antigen, tumor associated antigen, viral tumor antigen, viral tumor associated antigen, HPV selective viral tumor antigen, HPV selective viral tumor associated antigen.

10. The method of claim 1, wherein the starting vector is selected from: virus derived vectors, virus vectors which derive from: pox viruses, Parapoxvirus ovis viruses (ORFV), ORFV D1701 strain; adeno associated viruses (AAV); adeno viruses; vaccinia viruses; baculo viruses; toga viruses; alpha viruses; arteri viruses; rubi viruses; influenza viruses; human papilloma viruses; herpes viruses, CMV, RhCMV; arena viruses, LCMV; bacterial vectors, bacterial vectors which originate from: Salmonella sp., Shigella sp., L. monocytogenesis, S. gordonii; plasmids.

11. A kit for the selection of recombinant expression vectors, comprising the following: a starting vecor encoding a first gene product, wherein the coding nucleotide sequence for said first gene product is upstreamly and downstreamly flanked by first recombination nucleotide sequences; a transfer vector comprising a cloning site, wherein said cloning site is upstreamly and downstreamly flanked by second recombination nucleotide sequences which are homologous to said first recombination nucleotide sequences.

12. The kit of claim 11, wherein said first gene product is selected from the group consisting of: protein, protein fragment, membrane protein, cell surface protein, CD4.

13. The kit of claim 11, wherein it further comprises a binding molecule which binds to said first gene product, wherein said binding molecule is selected from the group consisting of: antibody, antibody bound to a magnetic entity; and which further comprises a magnetic field column.

14. A vector comprising a coding nucleotide sequence encoding a first gene product, which is upstreamly and downstreamly flanked by first recombination nucleotide sequences, wherein said first gene product is expressible in a cell in such a manner that it is accessible to a first binding molecule which binds to said first gene product.

15. The vector of claim 14, wherein said first transgene product is selected from the group consisting of: protein, protein fragment, membrane protein, cell surface protein, CD4.

16. A cell containing the vector of claim 14.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0087] FIG. 1 schematic illustration of the MACS based selection;

[0088] FIG. 2 schematic illustration of the transfer plasmid pdV-CD4;

[0089] FIG. 3 production of the recombinant D1701-V-CD4-P2Cherry;

[0090] FIG. 4 selection of the recombinant D1701-V-CD4-P2Cherry;

[0091] FIG. 5 MACS based selection oft he recombinant D1701-V-RabG-P2Cherry.

EXAMPLES

1. New Selection Method for the Isolation of Recombinant Orf Viruses

[0092] Usually recombinant viruses such as pox viruses, are generated in permissive cells by means of homologous recombination of a transfer plasmid and the genome of the replicating virus. To ensure that the foreign DNA will be integrated into the desired position of the viral genome, the transfer plasmid contains homologous viral sequence sections which flank the foreign DNA to be inserted. Because recombination events occur very seldom, the recombination frequencies are usually between 1:1000 and 1:10.000, the selection of new recombinant viruses is mostly a quite difficult, expensive, and long lasting process which requires a high degree of technical know-how. As a result, different strategies were developed to facilitate a targeted selection of recombinant viruses. An interchangeable marker gene is often incorporated into the transfer plasmid and integrated into the viral genome together with the foreign gene or the “gene of interest”. This technology has the decisive disadvantage that besides the foreign gene at the same time the marker gene is expressed in the recombinant viruses. Potential risks by an unwanted influence of the marker gene cannot be excluded and are in contradiction to a use that has to be approved. For this reason the production of marker-free recombinants is desirable. Different methods such as a visual selection by means of the expression of the β-galactosidase, β-glucuronidase or of fluorescence proteins and a transient “host range” selection, a transient dominant selection, or removable marker systems are described in the state of the art.

[0093] Often the common blue white selection is used for the production of recombinant Orf viruses, which is based on the hydrolysis of X-Gal by the β-galactosidase of the lacZ gene. However this method is very time-consuming and labor-intensive so that establishing a new more simple selection method is desirable.

[0094] For this reason in the first instance a fluorescence based selection method was developed wherein the lacZ gene was replaced by a fluorescence gene. New recombinants should be generated by replacing the fluorescence gene by a foreign gene and then visually selected via the loss of fluorescence.

[0095] Furthermore the method according to the invention was developed which, according to a preferred further development, is based on the use of the MACS technology.

[0096] The MACS technology is usually used to isolate cells which express a specific surface molecule or antigen. In this context advantage is taken of the fact that antibodies conjugated to magnetic particles, so-called MACS beads, specifically bind to cells and are retained in a strong magnetic field, thereby can be separated from nonbound cells. In connection with the works underlying the invention the recombinant D1701-V-CD4 was developed which expresses the human CD4 antigen (hCD4) and which should serve as starting recombinant for a MACS based selection. Because ORFV permissive Vero cells (monkey kidney cells) do not express hCD4, D1701-V-CD4 infected cells can be characterized by the surface expression of hCD4 and separated from noninfected cells by the aid of the MACS technology. In recombinants to be newly generated the exchange of the hCD4 by a foreign gene can now take place. Thereby in infected Vero cells the new hCD4 free recombinants can be easily separated from the parental hDC4 expressing viruses. This allows a technically essentially easier, more rapid and efficient selection of the desired new ORFV recombinants.

[0097] Important steps of the MACS based “negative” selection according to the invention are schematically shown in the FIG. 1. In this embodiment the first gene product or the selection marker is hCD4 and the second gene product or the “gene of interest” is the rabies virus glycoprotein (RabG). Further, a fluorescence protein named “Cherry” comes into play: The selection starts from a cell mixture obtained after the transfection, which contains non-infected Vero cells (medium grey), Vero cells infected with the starting recombinant D1701-V-CD4-P2Cherry and which express hCD4 (light grey), and Vero cells infected with the newly formed recombinant D1701-V-RabGP2Cherry (dark grey) (FIG. 1, left part). After the addition of hCD4 specific MACS beads conjugated with magnetic particles the antibodies bind to cells which express hDC4 at their surface (FIG. 1, middle part). The separation of the cell mixture is made by a column in a magnetic field. MACS beads coupled cells are retained in the magnetic field whereas non-bound cells can pass the magnetic field. In the flow, therefore, non-infected and the desired D1701-V-RabG-P2Cherry infected cells (hCD4 negative cell population: below, circled) are present, whereas in the column in the magnetic field D1701-V-hCD4P2Cherry infected cells accumulate (hCD4 positive cell population: top, circled) which are discarded (FIG. 1, right part).

2. Production and Characterization of the Starting Vector D1701-V-CD4-P2Cherry

[0098] The hDC4 gene sequence from pMACS 4.1 (Miltenyi) was chemically synthesized by the company Mr. Gene, Regensburg, Germany, and cloned into the plasmid pdV-Rec 1 via the restriction cleavage sites EcoRI and HindIII. The resulting transfer plasmid pdV-CD4 is schematically shown in the FIG. 2. There, the hCD4 gene is labeled in white. It is under the control of the original former promotor P.sub.VEGF. The areas flanking the hCD4 gene which are shown in dark are downstreamly homologous to the ORFV genome area ORF-3 and upstreamly homologous to the ORFV genome area F9LF10L. These areas ensure a targeted integration of the hCD4 gene into the VEGF lokus of the D1701-V genome via homologous recombination. The restriction cleavage sites HindIII and EcoRI used for the cloning the pox specific early transcription stop motive T5NT are shown.

[0099] The newly resultant transfer plasmid pdV-CD4 was subsequently transfected into D1701-V-GFP-P2Cherry infected Vero cells, see FIG. 3. There in the partial FIG. 3A the restriction map of the D1701-V genome is shown. As shown in the partial FIG. 3B as the starting virus the double fluorescent recombinant D1701-V-GFP-P2Cherry was used (AcGFP=“Aequorea coerulescens green fluorescent protein”); (mCherry=“mCherry fluorescent protein). After the infection of Vero cells with the recombinant D1701-VGFP-P2Cherry the transfer plasmid pdV-CD4 (FIG. 2) was transfected into the cells by means of nucleofection. As shown in the partial FIG. 3C the generation of the initial vector D1701-V-CD4-P2Cherry is realized by integration of the hCD4 gene in exchange with the AcGFP-gene via homologous recombination.

[0100] New D1701-V-CD4-P2Cherry starting virus vectors can be distinguished and selected from the GFP-Cherry expressing parental virus vectors via the loss of GFP fluorescence. This is shown in the FIG. 4A: “loss of fluorescence”: selection of D1701-VCD4-P2Cherry by means of fluorescence microscopy. Non-green fluorescent plaques (circled) were identified, picked and the virus was grown from the plaques. After five plaque purifications the homogeneity of D1701-V-CD4-P2Cherry could be assured via PCR analysis.

[0101] The detection of the correct integration of the hCD4 gene in exchange with the AcGFP-gene in the VEGF lokus could be verified by means of specific PCR analysis and the genetic homogeneity of hCD4 positive and GFP negative ORFV recombinants was ensured, as shown in the FIG. 4B: exemplary PCR analysis after the fourth plaque purification. Recombinant D1701-V-CD4-P2Cherry is positive for the hCD4 gene and negative for the AcGFP gene contained in the starting recombinant. The detection was made via the indicated specific PCRs. The samples pos. #1-3 show homogenous recombinant starting virus vector with a specific hCD4 signal (Amplicon: 857 bp); neg. #1 shows a mixture of parental GFP containing (Amplicon: 575 bp) and recombinant CD4 containing starting virus vectors. The absence of the mCherry fluorescence gene was detected via a specific PCR (Amplicon: 1.103 bp).

[0102] In the following the starting virus vector or the resulting recombinant virus was amplified in Vero cells and enriched via ultra centrifugation to high virus titers. The correct early expression of hCD4 in infected Vero cells was demonstrated via Western Blot analysis, immuno histochemical staining and immuno fluorescence analysis, as shown in FIG. 4C: immune fluorescence of D1701-V-CD4-P2Cherry infected Vero cells. Vero cells were infected with the initial vector D1701-V-CD4-P2Cherry. 20 hours after the infection the hCD4 protein was detected with a FITC conjugated anti CD4 antibody (Miltenyi). The fluorescence microscopy allows the illustration of the hCD4 surface expression (“anti-CD4”), the Cherry expression (“mCherry”), and both fluorescences in one cell after merger (“merged”).

[0103] Growth kinetics of the cells which were transfected with the starting vector D1701-V-CD4-P2Cherry showed that the integration of the CD4 gene did not result in changes of the growth characteristics. The strength and the time course of the surface expression of CD4 in relation to the cell viability was determined by means of flow cytometry. There it could be found that already early after the transfection hCD4 is expressed on the surface of the cells and the intensity increases with progressing time. After approximately 20 to 24 hours first cytopathic effects can be seen and the cell viability decreases. Since released virus particles cannot be bound by MACS beads because of the lack of CD4 incoperation an infection period of 18 to 20 hours was established.

[0104] To obtain the most efficient separation of hCD4 positive and hCD4 negative cells in several pretests in addition different amounts of beads, incubation periods, temperatures and MACS columns were tested and the optimum selection conditions were established. [0105] 3. Generation of a New RabG Expressing Expression Vector D1701-V-RabG-P2Cherry by Means of MACS Selection

[0106] In a next step by homologous recombination in the starting vector the coding nucleotide sequence for CD4 should be changed against the coding nucleotide sequence of a “gene of interest”. The rabies virus glycoprotein (RabG) is the major antigen of the rabies virus (RV). RabG is expressed in the surface of RV and RV infected cells and is the target of virus neutralizing antibodies (VNA). The protection of an individual against rabies correlates with the level of the VNA titer. A recombinant virus or a recombinant expression vector which expresses RabG is for these reasons an appropriate tool for the production of a vaccine against the rabies virus. In this embodiment for this reason as a second gene product or “gene of interest” RabG is used.

[0107] D1701-V-CD4-P2Cherry infected Vero cells were incubated with the transfer plasmid pdV-RabG and incubated for 72 hours. After the disintegration of the viruses from the cell lysate fresh Vero cells were infected therewith. The cells were then incubated with CD4 specific MACS beads (Miltenyi) and separated by a magnetic column. hCD4 expressing cells infected with the starting vector D1701-V-CD4-P2Cherry remained in the magnetic field whereas the rest of the cells passed the column and gathered in the flow. Subsequently the hCD4 negative cell population was incubated for additional 20 hours at 37° C. and MACS selection was repeated. Altogether five of such rounds of MACS selection were carried out, whereas after each round a part of the negative cell population was used for the quantification of the efficiency of enrichment of new hCD4 negative recombinants; cf. FIG. 5A: schematic illustration of the experimental setup. After the transfection of D1701-V-CD4-P2Cherry infected Vero cells with the transfer plasmid pdV-RabG cells were incubated with the transfection lysate for 20 hours. CD4 negative cells (negative cell population) were isolated by MACS selection and after the addition of non-infected Vero cells incubated for additional 20 hours. At the same time a part of the negative cell population was used for the quantification of the efficiency of selection. In total 5 rounds of MACS selection were carried out.

[0108] The ratio of parental D1701-V-CD4-P2Cherry and newly generated recombinants 1701-V-RabG-P2Cherry was determined via single well PCR, fluorescence analysis and flow cytometry, as shown in the partial FIGS. 5A to 5B. FIG. 5B: evaluation of the efficiency of selection by means of hCD4- and RabG specific immune fluorescence staining. To determine the ratio of recombinant and parental virus after each round of selection cells of the negative population (FIG. 1, right part) were seeded into 6 well plates and 48 hours later either stained with hCD4 (exemplary illustration after the fourth round of MACS selection) or RabG specific (exemplary illustration after the fifth round of MACS selection) FITC coupled antibodies. Because all viruses express the Cherry fluorescence protein a simple distintion between parental and recombinant viruses is possible in the fluorescence microscope. In the top row (S#4 CD4-FITC) all parental viruses appear red green (continuous circle), whereas the recombinant viruses only shine in red (dashed circle); in the lower row (S#5 RabG-FITC) however recombinant viruses appear red green (dotted circled) and parental viruses are red. FIG. 5C: tabular overview of the efficiency of selection. The table shows exemplarily each proportion of parental and recombinant virus plaques after each of the five rounds of MACS selection which were carried out (S#1S#5). In addition the ratio of recombinant to parental virus plaques was determined (Ratio Rec./Par.) and the percentage of recombinant viruses resulting therefrom was calculated (Rec. in %). FIG. 5D: Identification of new MACS selected recombinants by means of PCR analysis. After each of the five rounds of MACS selection (S#1-S#5) cells of the negative population (FIG. 1, right part) were mixed in different ratios with non-infected Vero cells and seeded into 384 well plates. 72 hours after seeding wells where 1-5 individual plaques could be identified were harvested and the DNA was isolated. By means of hCD4 and RabG specific PCRs it was analyzed whether in the examined individual wells there was homogenous recombinant D1701-V-RabG-P2Cherry (e.g. S#5, lane 2; labeled by an arrow), homogenous parental 1701-V-CD4-P2Cherry (e.g. S#3, lane 15) or a mixture of both virus expression vectors (e.g. S#1, lane 8). The arrows represent wells which contain the new recombinant. The sizes of the respective PCR amplicons are indicated on the right in base pairs (bp).

[0109] Already after the third round of MACS selection individual wells could be found where the recombinant and thus the recombinant expression vector were present in a homogenous manner; see FIG. 5D. The virus culture of these homogenous D1701-VRabG-P2Cherry infected individual wells thereby allows the production of recombinant vectors already within one week. After the fourth round of selection D1701-V-RabGP2Cherry was already in superior number over the starting vector and another round of selection resulted in almost 95% of all viruses representing the new homogenous recombinant which expresses the RabG instead of the hCD4 gene; see FIG. 5B and 5C. These results could be confirmed by two further recombinants which were generated on the basis of the MACS selection system within 7 to 10 days.

4. Conclusion

[0110] Compared with the blue white selection which is usually used in the prior art and which takes approximately 3 month the selection process for the production of recombinant expression vectors by means of the method according to the invention could be accelerated by far, which besides time saving also results in lower costs. Because of the accelerated selection the method according to the invention now also provides excellent preconditions for the generation of vaccines which require a fast adaptation and/or production, such as seasonal vaccines, e.g. influenza virus vaccines or individualized vaccines, e.g. tumor vaccines. Another advantage is that the MACS system is well established and user friendly and for this reason requires little technical expertise. The described principle of selection is not only restricted to the exemplarily used Orf virus but facilitates and accelerates also the production of other recombinant expression vectors. Furthermore the new method can be used to positively select new recombinants which express a surface associated antigen by means of the use of specific MACS beads. The possibility to combine negative and positive selection as needed is another success-promising option.