WO2002090550A2

WO2002090550A2 - Method for preparing recombinant poxvirus

Info

Publication number: WO2002090550A2
Application number: PCT/GB2002/002190
Authority: WO
Inventors: Sarah Gilbert
Original assignee: Isis Innovation Limited
Priority date: 2001-05-10
Filing date: 2002-05-10
Publication date: 2002-11-14
Also published as: WO2002090550A3; GB0111442D0; AU2002307938A1

Abstract

The invention relates to use of fluorescence activated cell sorting in the production of recombinant poxvirus, in particular to the use of fluorescence activated cell sorting in the separation of cells containing recombinant poxvirus from cells not containing recombinant poxvirus.

Description

METHOD FOR PREPARING RECOMBINANT VIRUS

Field of the invention

The invention relates to a method for preparing recombinant poxvirus.

Background to the invention

Recombinant non-replicating poxviruses such as, for example, attenuated fowlpox and modified vaccinia virus (MVA) are safe vaccine delivery systems that may be used to elicit T cell responses against heterologous antigens. T cell responses are important in protection against a wide range of infectious and non-infectious disease conditions, including malaria, HIV, tuberculosis and cancer. Although many types of delivery system can prime the desired immune response, only recombinant viruses (Vaccinia, MVA, fowlpox, adenovirus) can boost it, and non-replicating viruses are more effective as well as safe. The strongest T cell responses obtained so far in the mouse malaria model have been obtained using fowlpox to prime and MVA to boost.

Present methods for making recombinant poxviruses involve using beta-galactosidase as a marker gene. A shuttle vector is prepared consisting of short sections of viral DNA flanking the coding sequence of interest with a strong early viral promoter to drive expression of the coding sequence, plus beta-galactosidase expressed from a late viral promoter. This shuttle vector is then used to transform host cells, most commonly chicken embryo fibroblast cells (CEFs) , which have previously been infected with the wild type pox virus. In some of these cells recombination between the virus and shuttle vector will take place, resulting in the formation of the desired recombinant virus. After transfection the mixture of recombinant and non-recombinant viruses is harvested and replated on CEFs which are then overlaid with agarose containing X-gal so that cells infected with recombinant virus turn blue, can be identified and 'picked' .

Unfortunately the percentage of recombinant virus at this stage is so small that the method often fails at this point. Cells infected with non-recombinant virus can not be subsequently infected with recombinant virus. It is necessary to plate a high concentration of virus to identify the very low percentage of recombinants, but if too much virus is used recombinants may also be missed. If blue cells can be identified it is not possible to recover them without also taking up some of the surrounding cells which are infected with non-recombinant virus. It is then necessary to release all the isolated virus from the CEF cells and replate on fresh CEF monolayers. As the proportion of recombinant virus in the mixture should have increased, this time the size of the blue stained plaques should be increased and it becomes slightly easier to pick the plaques. However dead cells can also be stained blue, and it can be difficult to differentiate these from genuine blue plaques.

Plaque picking is repeated until a pure preparation of recombinant virus is obtained, usually around six or more times. For MVA passaged CEFs may be used, that is fibroblasts isolated from a chicken embryo and grown in tissue culture for a number of cell divisions. Fowlpox will only form plaques on unpassaged CEFs, that is cells isolated from a chicken embryo and plated straight into the dish which will then be infected with fowlpox. It is therefore a slow and extremely labour intensive process to produce recombinant viruses by this method, and even in labs where the method is used continually it can sometimes fail completely.

The present inventors have now developed a method of preparing recombinant poxvirus which avoids the disadvantages of the prior art methods. This method is based on the use of conventional techniques for generating the recombinant virus in combination with the use of fluorescent activated cell sorting (FACS sorting) to select host cells containing recombinant virus from amongst a mixed population of host cells.

Description of the invention

The invention relates to the use of FACS sorting in the production of recombinant poxviruses. In particular the invention relates to the Use of FACS sorting in a method for the production of recombinant poxvirus, wherein FACS sorting is used for the separation of cells containing a recombinant poxvirus from cells not containing the said recombinant poxvirus.

The term "recombinant poxvirus" generally encompasses poxviruses which have been genetically engineered to express one or more heterologous polypeptide (s) which are not naturally encoded by the poxvirus. Examples include recombinant poxvirus vaccines. Also encompassed by the term "recombinant poxvirus" are poxviruses which have been manipulated to remove one or more viral genes, for example viral genes which have immunomodulatory activity. The technique is of general utility in the production/selection of recombinant poxviruses, hence the precise nature of the "recombinant poxvirus", or of any heterologous polypeptide (s) which it encodes are not material to the invention.

In this context the term "production of recombinant poxvirus" refers to the steps of constructing the recombinant by manipulation of the poxvirus genome, and isolation of host cells containing the desired recombinant virus which may then be used to produce a stock of recombinant virus.

Depending on the method chosen to construct the recombinant poxvirus and on what stage in the method FACS sorting is used (see below) , the "cells containing recombinant poxvirus" may include cells containing only recombinant poxvirus or cells containing a mixture of recombinant and non- recombinant poxvirus. The "cells not containing recombinant poxvirus" may include cells which do not contain any poxvirus or cells containing only non- recombinant poxvirus. The term "non-recombinant poxviruses" generally refers to poxviruses which do not express at least one heterologous polypeptide expressed by the recombinant poxvirus. Examples of "non-recombinant poxviruses" include poxviruses used in the production of the recombinant virus, for example poxviruses introduced into a host cell in order to provide a genetic background for homologous recombination with a shuttle vector (as described below) , or helper poxviruses used in the packaging of recombinant poxvirus genomes generated in vitro . The non-recombinant poxviruses may themselves be genetically modified but will be distinguishable from the desired recombinant virus on the basis of expression of at least one heterologous protein.

In a preferred embodiment cells containing recombinant poxvirus are distinguished from cells not containing recombinant poxvirus by the presence of a selection marker which is directly or indirectly detectable by FACS. There are several ways in which cells containing recombinant virus may be differentially marked, as discussed below.

In a particularly preferred embodiment the invention provides a method of preparing recombinant poxvirus which comprises:

(i) providing a shuttle vector comprising a first gene expression cassette positioned between flanking viral DNA sequences corresponding to fragments of the genome of a poxvirus, the first gene expression cassette comprising a DNA sequence encoding a heterologous polypeptide operably linked to a promoter sequence;

(ii) introducing the shuttle vector into host cells which have previously been infected with a pox virus and incubating the host cells under conditions which allow for homologous recombination between the pox virus and the flanking viral DNA sequences present in the shuttle vector;

(iii) selecting for host cells containing recombinant virus using FACS sorting; and

(iv) isolating recombinant virus from one or more host cells selected in step (iii) .

The above method combines the "shuttle vector" approach to preparing recombinant viral vectors with a novel and inventive use of FACS sorting for selection of recombinants. The use of FACS for selection of recombinants provides significant technical advantages over the conventional plaque picking approach, in particular simplicity, speed and reliability.

The steps of constructing a suitable shuttle vector and introducing (e.g. by transfection or equivalent) the shuttle vector into host cells previously infected with a pox virus under conditions which allow for homologous recombination between the shuttle vector and the pox virus genome present in the host cells may be carried out according to standard techniques known in the art. Standard protocols for construction of recombinant poxviruses are described in the section by Earl, Moss, Wyatt and Carroll in Current Protocols in Molecular Biology, F.M. Ausubel, et al . , eds., John Wiley & Sons Inc., New York.

FACS sorting is used in the method of the invention in order to select cells containing recombinant virus. One or more separate rounds of FACS sorting may be carried out in order to isolate cells containing only recombinant virus. In a preferred embodiment a first round of FACS sorting is carried out immediately after transfection of the shuttle vector into the host cells in order to isolate all recombinants. The cells collected after FACS are lysed to release virus particles, which are a mixture of recombinant and wild type (i.e. identical to the pox virus initially used to infect the host cells before introduction of the shuttle vector) . These viruses are used to infect fresh host cells and a further round of FACS is carried out to select cells containing recombinant virus. It is, however, within the scope of the invention to omit the FACS sorting step following transfection with the shuttle vector.

In order to use FACS sorting to separate cells containing recombinant virus it is necessary to differentially mark cells containing recombinant virus and cells not containing recombinant virus using one or more selection markers which are directly or indirectly detectable using FACS. There are several ways in which cells containing recombinant virus may be differentially marked. In one embodiment differential marking of cells containing recombinant virus and cells not containing recombinant virus is achieved by engineering the recombinant virus to express a positive selection marker protein. This may be achieved using the shuttle vector approach by including in the shuttle vector a second gene expression cassette, comprising a sequence encoding a positive selection marker protein operably linked to a second promoter. The second gene expression cassette is also positioned between the flanking viral DNA sequences in the shuttle vector, such that it is included in recombinant viral genomes generated by homologous recombination between the shuttle vector and the virus present in the host cells. In a variation of this embodiment the heterologous polypeptide and the marker protein may be expressed as a fusion protein. In a further variation the heterologous polypeptide and the marker protein may be encoded on a single mRNA molecule but not translationally fused. For example, the heterologous polypeptide and marker protein may be encoded by separate open reading frames on a bicistronic mRNA, such as an mRNA containing an internal ribosome binding site.

The positive selection marker may be a protein which is directly detectable by FACS by virtue of a signal-generating property of the protein. By "directly detectable" is meant that it is not necessary to add any further component prior to detection by FACS. Suitable directly detectable marker proteins include fluorescent proteins. Particularly preferred fluorescent marker proteins for use in this embodiment are GFP and similar autonomous fluorescent proteins. A wide variety of GFPs, including synthetic variants and mutant forms, and other similar fluorescent proteins, such as the red and blue fluorescent proteins are known in the art. Any such proteins which generate a fluorescence detectable using FACS are suitable for use in the methods of the invention.

In a further embodiment differential marking of cells containing recombinant virus versus cells not containing recombinant virus may be achieved with the use of a fluorescent antibody conjugate immunologically specific for an epitope on a positive selection marker encoded by the recombinant pox virus. The use of fluorescent antibody conjugates allows FACS sorting of cells expressing the selection marker protein by detection of fluorescence from the antibody conjugate bound to marker protein present in the cells. Suitable fluorescent labels include FITC. A number of FITC labelled antibodies are available from commercial suppliers.

In this approach a mixed population of cells

(i.e. cells containing recombinant virus and cells not containing recombinant virus) is contacted under cell permeabilising conditions with the fluorescent labelled antibody conjugate and the cells incubated under conditions which allow for specific binding of the antibody conjugate to any target marker protein present in the cells. FACS sorting is then carried out, selecting for cells which exhibit fluorescence from the antibody conjugate.

In this embodiment there is no need for the marker protein itself to be directly detectable by FACS. It is therefore possible to use non-fluorescent marker proteins such as, for example, β-galactosidase in combination with an appropriate fluorescent antibody conjugate. In a variation on this embodiment the positive selection marker protein may take the form of an epitope tag attached to the heterologous polypeptide expressed from the recombinant poxvirus. The inclusion of an epitope tag (or indeed any type of positive selection marker protein) may be achieved by expressing the heterologous polypeptide and the epitope tag (marker protein) as a fusion protein.

If the recombinant virus is intended for clinical use in vivo, for example as a viral vaccine, then it may be necessary to remove sequences encoding the positive selection marker protein. Removal of these sequences may be achieved in several different ways. In a first method, following initial isolation of recombinant virus expressing the desired heterologous polypeptide and the marker protein, a second transformation may be carried out using a shuttle vector which is identical to that used initially for construction of the recombinant except that the sequences encoding the marker protein are absent. Recombination between this vector and the recombinant virus results in removal of the sequences encoding the marker protein. Cells containing the new recombinant virus without the marker sequences may be separated using FACS with selection against expression of the marker protein.

Removal of the marker sequences may also be achieved using transient dominant selection.

Transient dominant selection involves the use of a shuttle vector encoding both the desired heterologous polypeptide and a positive selection marker protein. The shuttle vector is transformed into a host cell which has previously been infected with a pox virus and the entire vector is integrated into the viral genome by homologous recombination at a single site. Cells containing the resulting recombinant virus will express both the heterologous polypeptide and the positive selection marker protein and may be isolated by FACS sorting, as described above.

The shuttle vector is constructed in such a way that a second recombination event may take place between sequences flanking the gene expression cassette encoding the positive selection marker, resulting in removal of the gene expression cassette from the recombinant virus. After a period of growth in suitable host cells, during which time this second recombinant event will take place in a percentage of the cells, further FACS sorting may then be used to isolate cells containing recombinant virus which still express the heterologous polypeptide but no longer express the positive selection marker protein.

Removal of the marker sequence may still further be carried out using an approach based on the use of a short, double stranded DNA sequence which is capable of annealing to viral sequences flanking the marker sequence to be deleted. This approach is illustrated in the accompanying Example 5 and is based on techniques described by Storici et al . , Nature

Biotechnology 2001, Vol : 19(8), pp 773-6. It is particularly advantageous since in results in the targeted deletion of regions of the poxvirus genome (including a marker sequence, as required) , but does not leave behind any extraneous sequence.

In a further embodiment differential marking of cells containing recombinant virus versus cells not containing recombinant virus may be achieved with the use of a fluorescent antibody conjugate immunologically specific for an epitope on the heterologous polypeptide encoded by the recombinant pox virus. A mixed population of cells is contacted with the fluorescent labelled antibody conjugate under cell per eabilising conditions and the cells incubated under conditions which allow for specific binding of the antibody conjugate to any target heterologous polypeptide present in the cells. FACS sorting is then carried out, selecting for cells which exhibit fluorescence from the antibody conjugate. A particular advantage of this approach is that there is no need for inclusion of a separate marker gene in the recombinant virus. This is an obvious advantage when the recombinant virus is intended for use in vivo, for example as a viral vaccine.

In a still further embodiment differential marking of cells containing recombinant virus and cells not containing recombinant virus may be achieved by using a modified poxvirus which expresses a negative selection marker protein to infect the host cells used for production of the recombinant virus. The modified poxvirus includes a marker gene expression cassette inserted in the viral genome such that it is flanked by regions of the viral genome homologous with the viral DNA sequences present in the shuttle vector. Homologous recombination between the shuttle vector and the modified viral genome present in the host cells will result in loss of .the marker gene expression cassette from the latter. Hence, cells containing recombinant virus may be separated with the use of FACS sorting, selecting against expression of the negative selection marker protein.

The negative selection marker protein is preferably a fluorescent protein, expression of which is directly detectable by FACS. Suitable fluorescent proteins include GFP and similar autonomous fluorescent proteins, including synthetic variant and mutant forms thereof.

The inclusion of a negative selection marker in the modified viral genome present in the host cells used for production of the recombinant virus provides a technical advantage, since the recombinant virus produced using this approach does not contain a marker gene, hence there is no need for extra manipulation to remove the marker gene prior to clinical use in vivo .

In a still further embodiment the use of a positive selection marker in the shuttle vector and a negative selection marker in the modified viral genome initially present in the host cells can be combined to provide a dual positive/negative selection. In this embodiment homologous recombination between the shuttle vector and the modified viral genome present in the host cells will result in a recombinant virus in which a region of the modified viral genome encoding the negative selection marker is replaced with sequences from the shuttle vector encoding the heterologous polypeptide and the positive selection marker. Hence, cells containing recombinant virus may be separated with the use of FACS sorting, selecting for expression of the positive selection marker protein and against expression of the negative selection marker protein. This "dual selection" may be carried out in a single round of FACS sorting or in two rounds of FACS sorting, for example a first round of FACS sorting for expression of the positive selection marker and a second round of FACS sorting against expression of the negative selection marker.

The positive and negative selection marker proteins used in the dual selection approach are preferably fluorescent proteins which exhibit different fluorescent properties. A preferred embodiment uses GFP as the positive selection marker and red fluorescent protein as the negative selection marker. However, any combination of fluorescent proteins which are distinguishable by FACS may be used with equivalent effect.

The use of FACS to detect cells infected with recombinant viruses expressing GFP has been described in the literature (see Aran et al . , Cancer Gene Therapy, Vol 5, ppl95-206, 1998; Mar Lorenzo and Blasco, BioTechniques, Vol 24, pp 308-313, 1998) . However, there is no suggestion in the prior art of using a fluorescent marker in combination with FACS sorting in the actual preparation of the recombinant virus.

FACS sorting can only be carried out on single cells in suspension. However, the only cells permissive for replication of the poxviruses MVA and fowlpox are unpassaged primary chicken embryo fibroblasts (CEFs) which are adherent. Therefore, in order to replace conventional "plaque picking" with FACS sorting it was necessary to find conditions under which the cells may be treated to form a single cell suspension suitable for FACS analysis.

Poxviruses replicate inside a permissive cell, then when infectious virions have formed the cell lyses to release them. Thus it might have been expected that cells infected with poxviruses, and therefore in a fragile state, which were then trypsinised and resuspended would lyse either during this procedure, or during the passage through the FACS machine, thus losing the viral contents which could not then be isolated. Cells that have recently been infected and do not yet contain assembled infectious virions would be more robust and may be considered more suitable for FACS. However the process of trypsinisation and sorting, with the necessary period of time out of growth medium in a controlled environment incubator, could halt viral replication such that no infectious particles are then produced in the cell. In fact, somewhat surprisingly, it has been determined that if cells are trypsinised and sorted two days after infection, the cells are robust enough to remain intact during preparation and FACS sorting. After sorting the cells may be lysed deliberately, releasing the infectious virions from inside the cell. These virions may be collected and used to infect other cells.

Methods for making recombinant poxviruses by in vivo recombination have previously relied on plaque formation to separate stable from unstable recombinants before isolating the recombinants from the wild type virus. Immediately after transfection of a host cell infected with a wild type virus with a shuttle vector containing regions of viral homology flanking a heterologous polypeptide, it is likely that most recombination events will result in a single crossover producing an unstable intermediate which may resolve to form either the desired recombinant, or release the shuttle vector and reform wild type virus. Also, all cells containing recombinant virus, whether stable or unstable, will also contain wild type virus. After allowing viral replication to occur, the virus is then released from the cells and used to infect fresh host cells, in order to separate the wild type and recombinant. These host cells are then incubated to allow plaque formation. If the recombinant virus is stable, it will spread from one cell to another and form a plaque of recombinant virus all expressing the marker gene from the shuttle vector, which may then be seen by eye after e.g. adding Xgal if the marker is beta-galactosidase. If the recombinant is unstable, during replication the shuttle vector will be lost and recombinant plaques will not form. Thus it would be logical to conclude that any method for isolating recombinants which does not include plaque formation would result in the isolation of unstable recombinants, which are unsuitable for bulking up into a stock of recombinant virus.

The method described herein does not rely on plaque formation prior to isolation of recombinant viruses. FACS sorting is carried out on the cells immediately after transfection in order to isolate all recombinants, both stable and unstable. When the cells collected by FACS are lysed the viruses released from them are a mixture of recombinant and wild type viruses, but the proportion of recombinants is significantly enriched over that which would have been produced had all the cells been lysed after transfection and plated out in order to see plaque formation. The viruses released from the cells collected after FACS are, in the preferred embodiment, replated and sorted again two days later. This second selection step separates recombinant from non-recombinant virus and, perhaps surprisingly, the vast majority of recombinant viruses after this second selection step are stable.

FACS sorting may also be employed in the generation of recombinant poxviruses by removal of one or more viral genes, referred to hereinafter as the "knock-out" method. In one approach the viral gene(s) which it is desired to remove could be replaced with a marker DNA sequence encoding a marker protein which is detectable by FACS, or the marker sequence may be inserted into the gene to be removed. Replacement of one or more viral genes with a marker sequence may be achieved using a shuttle vector which includes appropriate viral sequences flanking the marker sequence. Homologous recombination between this shuttle vector and a viral genome from which it is desired to remove one or more viral genes will result in replacement of the viral gene(s) with the marker sequence. Cells containing the recombinant virus may then be isolated using FACS sorting. If required the marker sequence may subsequently be removed, e.g. using one of the approaches outlined above.

When using the knock-out method to generate recombinants it is particularly advantageous to remove the marker sequence using a technique which does not leave behind any extraneous sequence in the viral genome. A particularly preferred approach is therefore that based on the use of a short double- stranded nucleic acid which anneals to the viral genome on either side of the marker sequence (and any viral sequences) to be removed (as illustrated in the accompanying example 5) . A method has been described (Scheiflinger, F., Dorner, F. and Falkner, F.G. Transient marker stabilisation: a general procedure to construct marker-free recombinant vaccinia virus. Arch. Virol. 143: 467-474 (1998)) to create marker- free recombinants, but this results in a 300bp sequence remaining in the genome after the marker sequence has been removed. This technique could not be used many times over to sequentially remove a number of viral genes, since the addition of multiple copies of the 300bp sequence would result in instability of the genome. In contrast, the method outlined above which does not leave behind any extraneous sequence may be used many times over without introducing instability.

The knock-out method may be used to remove one or more viral genes from or poxvirus which is a "wild type" virus, or from a recombinant virus which expresses one or more heterologous genes, e.g. a viral vaccine expressing an antigen. In a particularly useful application the method may be used to remove one or more immunomodulatory genes from a viral vaccine in order to enhance the immunogenicity of the viral vaccine.

The utility of FACS sorting as a method of selecting cells containing recombinant virus from a mixed population of cells is not limited to use with the shuttle vector approach for generating recombinants. FACS sorting may be used in conjunction with other methods of generating recombinants including, for example, in vi tro cloning approaches to construction of the recombinant poxvirus. In this context "construction of recombinant poxvirus" encompasses removal or knock-out of one or more viral genes as well as insertion of one or more sequences encoding heterologous polypeptides.

Therefore, in a further embodiment the invention also provides method of preparing recombinant poxvirus which comprises:

(i) constructing a recombinant pox virus .genome expressing a heterologous polypeptide;

(ii) introducing the recombinant pox virus genome into host cells which are capable of packaging the recombinant poxvirus genome into infectious virions;

(iii) infecting further host cells with infectious virions released from the host cells of step (ii) ;

(iv) selecting cells containing recombinant virus using FACS sorting; and

(v) isolating recombinant virus from one or more cells selected in step (iv) .

The use of FACS selection in connection with in vitro cloning provides the same technical advantages as with the shuttle vector approach, in particular simplicity, speed and reliability.

Construction of a recombinant pox virus genome expressing the desired heterologous polypeptide may be carried out in vi tro using standard molecular biology techniques, for example direct ligation of a heterologous DNA sequence into a poxvirus genome cleaved with restriction endonuclease. Techniques for construction of recombinant poxvirus genomes by direct cloning in vi tro are known in the art and described, for example, in EP 0 561 034, the contents of which are incorporated herein by reference.

The recombinant viral genome is introduced into host cells capable of packaging the genome into infectious virions. These host cells will generally be cells that are infected by a helper virus containing within the virion viral proteins required for the replication of viral genomes. The helper poxvirus may be a different type of poxvirus to the recombinant viral genome. For example, recombinant MVA genomes may use fowlpox as the helper virus.

Infectious virions packaged in the host cells are released and may be used to infect fresh (host) cells permissive for replication of the recombinant virus. These fresh host cells may the same type of cell as those used for initial packaging of the recombinant virus (but not infected with helper virus) or may be a different type of cell permissive for replication of the recombinant virus. If the recombinant virus can replicate in host cells in which the helper virus cannot replicate, such as for example Vaccinia recombinant with fowlpox as helper, then different types of cells may be used. However, if the recombinant can only grow on CEF cells, as is the case with fowlpox, CEFs are used both for the initial packaging of recombinant virus and for further rounds of viral replication.

Techniques for use in the preparation of recombinant poxvirus by in vi tro cloning are also well known in the art and described, for example, in EP 0 561 034. A number of variations are known in the art and described in EP 0 561 034, including variations which prevent packaging of the helper virus genome and/or prevent infection of the second host cells with infectious virions comprising the helper genome. It is within the scope of the invention to use any of these known variations in combination with FACS sorting.

FACS sorting is used in the method of the invention in order to isolate cells containing the recombinant genome of interest. In one embodiment a single round of FACS sorting is used. Host cells previously infected with a helper virus are transfected with the recombinant genome. All of these cells are then harvested, the packaged virions released and used to infect fresh host cells. Following this second round of infection the host cells are FACS sorted to separate cells infected with recombinant virus from cells infected with helper virus . In a further embodiment two rounds of FACS sorting may be used. Host cells previously infected with a helper virus are transfected with the recombinant genome. All of these cells are then subjected to a first round of FACS sorting in order to remove cells which only contain the helper virus. The packaged virions are released from the remaining cells, i.e. those cells which contain both recombinant virus and helper virus, and used to infect fresh host cells. Following this second round of infection the host cells are FACS sorted to separate cells infected with recombinant virus from cells infected with helper virus. In this two-sort method the first sort removes cells which contain helper virus only and thus increases the proportion of recombinant to helper virus present when the viruses are replated onto fresh host cells.

The most preferred method for selecting recombinant MVA and recombinant fowlpox by direct in vi tro cloning comprises the following steps:

(i) constructing a recombinant poxvirus genome expressing a heterologous polypeptide by in vi tro manipulation;

(ii) introducing the recombinant poxvirus genome into host cells (e.g. CEFs) infected with a helper virus which will package the recombinant genome into infectious virions;

(iii) selecting cells containing recombinant virus (but also the helper virus) by FACS sorting;

(iv) infecting fresh host cells (i.e. CEFs not infected with the helper virus) with viruses released from the sorted cells. The viral mixture is diluted and plated such that only one virion infects each host cell;

(v) selecting cells containing recombinant virus (but not helper virus) by FACS sorting; and

(vi) isolating recombinant virus from one or more cells isolated in step (v) .

In order to use FACS sorting to separate host cells containing the recombinant genome of interest it is essential to distinguish cells which containing the recombinant virus from cells which do not contain the recombinant virus using one or more markers which are detectable by FACS. This may be achieved using analogous approaches to those described above in connection with the "shuttle vector" approach.

Thus, in one embodiment, differential marking of host cells containing the recombinant virus may be achieved using a fluorescent labelled antibody conjugate specific for an epitope on the heterologous polypeptide encoded by the recombinant virus.

In a further embodiment the recombinant viral genome may be engineered to encode a positive selection marker protein. The positive selection marker protein may be a protein which is directly detectable by FACS, for example a fluorescent protein, in which case cells containing recombinant virus are FACS sorted on the basis of fluorescence from the marker protein. Alternatively, FACS sorting may be based on the use of a labelled antibody conjugate specific for an epitope on the marker protein.

In a further embodiment, a negative selection marker may be included in the helper virus genome. This allows for separation of host cells containing recombinant virus from host cells containing helper virus by FACS sorting against expression of the negative selection marker.

In a further embodiment, the use of a negative selection marker in the helper virus and a positive selection marker in the recombinant virus may be combined to provide a dual selection system, as described above. Dual selection against expression of the negative selection marker protein and for expression of the positive selection marker protein may be carried out in a single round of FACS sorting or in two rounds of FACS sorting. In a preferred embodiment a first round of FACS sorting for expression of the positive selection marker is carried out after transfection of the recombinant genome into the host cells in step (ii) . Viruses released from the sorted cells are used to infect fresh host cells and a second round of FACS sorting against expression of the negative selection marker is carried out on these cells in order to separate cells infected with recombinant virus from cells infected with helper virus .

Recombinant poxviruses expressing heterologous polypeptides are useful as viral vaccine vectors and as laboratory research tools in vaccine development. The term "heterologous polypeptide" refers to a polypeptide which is not naturally expressed by the virus in its wild-type form. The precise nature of heterologous polypeptide is not material to the invention. In the case of recombinant viruses intended for use as viral vaccines the heterologous polypeptide may be an antigenic polypeptide capable of eliciting a T cell and/or B cell immune response. In certain embodiments, for example when the method in used in order to construct "recombinant virus" by removal of one or more viral genes, the heterologous polypeptide may be a marker protein which is directly detectable by FACS.

The methods of the invention are preferred for preparation of recombinant poxviruses, particularly recombinant non-replicating poxviruses for use as vaccines and a tools in vaccine research. However, the use of FACS sorting may be extended to the production of other types of recombinant virus.

The method of the invention may be used to prepare recombinant forms of a wide range of poxviruses including, inter alia , vaccinia (including modified or attenuated forms) , fowlpox, canary pox, ALVAC, NYVAC, TROVAC, camelpox, cowpox, extromelia, monkeypox, racoonpox, skunkpox, taterapox, Uasin Gishu, variola, volepox, Auzduk disease virus, chamois contagious ecthyma, orf, pseudocowpox, parapox of deer, sealpox, juncopox, mynapox, pigeonpox, psittacinepox, quailpox, peacockpox, penguipox, sparrowpox, starlingpox, turkeypox, goatpox, lumpy skin disease, sheeppox, hare fibroma, myxoma, rabbit fibroma, squirrel fibroma, molluscum contagiosum, tanapox, Yaba monkey tumor, Melolontha melolontha, Amsacta moorei, Chironimus luridus .

The most preferred types of poxvirus for vaccine development include non-replicating poxviruses, which are poxviruses that do not replicate in mammalian cells, such as modified forms of vaccinia, e.g. Modified Vaccinia Virus Ankara (MVA) , and also attenuated fowlpox.

The methods of the invention are particularly advantageous in the preparation of recombinant forms of poxviruses which will only form plaques on primary cells, such as unpassaged chicken embryo fibroblasts. A particular example of this type of virus is- attenuated fowlpox. Production of recombinant attenuated fowlpox vectors by conventional plaque picking has proven extremely difficult, even in the hands of highly technically skilled individuals. With use of the methods of the invention, the success rate in the construction of attenuated fowlpox vectors is considerably improved.

The invention still further provides a method for preparing a vaccine composition comprising a recombinant poxvirus. This method comprises preparing a recombinant poxvirus with the use of FACS sorting, according to any one of methods described herein, and formulating the recombinant poxvirus into a vaccine composition with one or more diluents, carriers or excipients. For use in a vaccine composition the heterologous polypeptide encoded by the recombinant poxvirus will generally be an antigenic polypeptide capable of eliciting a T cell and/or B cell immune response.

The skilled reader will appreciate that once a recombinant poxvirus has been made according to one of the methods of the invention then it may be necessary to prepare a stock of the poxvirus for formulation into a vaccine composition. This may be achieved by using the recombinant virus to infect host cells, allowing replication of the poxvirus, and collecting the stock of poxvirus produced in the host cells . For commercial production of vaccines for human or veterinary use large scale production techniques may be used. It is generally known in the art to produce recombinant poxviruses on a large scale for vaccine use, and any of the known techniques may be used in order to scale-up production of recombinant viruses isolated using the method of the invention.

For vaccine use the recombinant virus will generally be formulated with at least one pharmaceutically acceptable carrier, diluent or excipient. Again, techniques for formulating poxvirus for in vivo vaccine use are generally known and any of the known techniques may be applied to the formulation of recombinant viruses isolated using the method of the invention.

The invention still further relates to a number of kits, vectors etc which may be used in the production of recombinant poxviruses using the methods of the invention.

Therefore, the invention provides a shuttle vector comprising a promoter sequence operably linked to a cloning site positioned between flanking viral DNA sequences corresponding to fragments of the genome of a poxvirus, characterised in that the shuttle vector further comprises a DNA sequence encoding a marker protein which is directly detectable by FACS, which DNA sequence is also positioned between the said flanking viral DNA sequences.

In one embodiment the DNA sequence encoding the marker protein may form part of separate marker gene expression cassette, still positioned between the flanking viral DNA sequences, comprising the said DNA sequence operably linked to a promoter which is distinct from the promoter operably linked to the cloning site.

In a further embodiment the DNA sequence encoding a marker protein may be in close juxtaposition with the cloning site such that a heterologous protein- encoding DNA fragment may be inserted into the cloning site in-frame with the DNA sequence encoding the marker protein.

The cloning site may be any site which facilitates the insertion of a heterologous DNA fragment into the vector, for example a restriction site or multi-cloning site comprising two or more restriction sites.

The promoter is "operably linked" to the cloning site, meaning that it is positioned to direct expression of heterologous DNA fragments introduced into the cloning site.

The "marker protein" encoded by the shuttle vector may be any marker protein which is directly detectable by FACS but will most preferably be a fluorescent protein, for example GFP, redFP or similar autonomous fluorescent protein.

The shuttle vectors according to the invention may be used in the generation of recombinant poxviruses expressing any heterologous DNA fragment of choice using the "shuttle vector" approach, described above. The chose heterologous DNA fragment is simply inserted into the cloning site of the shuttle vector. The inclusion of a marker protein in the shuttle vector facilitates separation of host cells containing a recombinant from cells not containing a recombinant using FACS sorting, as described above.

The invention further relates to a modified poxvirus, the genome of which contains a marker gene expression cassette comprising a DNA sequence encoding a marker protein operably linked to a promoter. Most preferably the marker protein included in the modified poxvirus will be a marker which is directly detectable by FACS, more preferably a fluorescent protein.

Such modified poxviruses may be used in the generation of recombinant poxviruses using the "shuttle vector" approach. Briefly, the modified poxvirus is used to infect host cells and a shuttle vector is then introduced into the host cells and allowed to recombine with homologous sequences in the modified poxvirus in order to generate the desired recombinant virus. The inclusion of a marker protein sequence in the modified virus may facilitate separation of host cells containing a recombinant from cells not containing a recombinant using FACS sorting, as described above.

In one embodiment the modified poxvirus may be a "helper virus" which may be used to infect a host cell and package recombinant poxvirus genomes prepared by in vi tro cloning techniques.

The invention still further relates to a kit for use in the preparation of recombinant poxvirus, the kit comprising a shuttle vector and a modified poxvirus, wherein the shuttle vector comprises a promoter sequence operably linked to a cloning site positioned between flanking viral DNA sequences corresponding to fragments of the genome of a poxvirus and further comprises a DNA sequence encoding a positive selection marker protein which is directly detectable by FACS, which DNA sequence is also positioned between the said flanking viral DNA sequences, and wherein the genome of the modified poxvirus contains a marker gene expression cassette flanked by regions of the viral genome homologous with the viral genome regions present in the shuttle vector, the marker gene expression- cassette comprising a DNA sequence encoding a negative selection marker protein operably linked to a promoter.

The components of this kit may be used in the construction of recombinant poxvirus expressing any heterologous DNA fragment of choice using the "shuttle vector" approach. The inclusion of positive and negative selection markers in the shuttle vector and modified poxvirus facilitates the isolation of host cells containing a recombinant by FACS sorting with "dual marker selection", as described above.

The invention further provides a kit for use in the preparation of recombinant poxvirus, the kit comprising a shuttle vector and a fluorescent labelled antibody conjugate, wherein the shuttle vector comprises a promoter sequence operably linked to a cloning site positioned between flanking viral DNA sequences corresponding to fragments of the genome of a poxvirus and further comprises a DNA sequence encoding a marker protein, which DNA sequence is also positioned between the said flanking viral DNA sequences, and wherein the fluorescent labelled antibody conjugate is immunologically specific for the marker protein.

This kit may again be used in the preparation of recombinant poxvirus using a shuttle vector approach. In this case, the fluorescent labelled antibody conjugate may be used to label host cells containing recombinant poxvirus, which may then be isolated by FACS sorting. In this instance there is no requirement for the marker protein itself to be directly detectable by FACS. The marker protein could, for example be an epitope tag which is expressed as a fusion protein with the heterologous polypeptide of interest.

The kits provided by the invention may further include a supply of suitable host cells for use in the preparation of the recombinant poxvirus. Depending on the nature of the poxvirus, these host cells may be, for example, CEFs or a cell line, such as BHK cells. The kit may further include protocols or instructions for construction of the recombinant poxvirus and/or isolation of the recombinant using FACS sorting.

The invention will be further understood with reference to the following, non-limiting Examples and the accompanying drawings in which:

Fig. 1 illustrates the construction of FP-GFP

Fig. 2 is a plasmid map of FP-GFP

Fig. 3 illustrates the construction of MVA-GFP

Fig. 4 is a plasmid map of MVA-GFP

Fig. 5. illustrates selection of all cells expressing green fluorescent protein (GFP) from a mixture expressing red fluorescent protein (RedFP) and GFP using a Cytomation Mo Flo cytometer. Forward scatter (FSC) and side scatter (SSC) were first used to select a population of single cells only, with no cellular debris. Pulse width was then used to select only the single cells. GFP (FLl) and RedFP (FL2) expression were then examined in the remaining cells and all cells expressing GFP (High FLl, region 9) were collected.

Fig. 6. further illustrates selection of cells expressing GFP only from a mixture expressing GFP and RedFP. Gates to select intact single cells were used as in figure 5. Cells expressing GFP but not RedFP (high FLl, low FL2, region 4) were then collected.

Fig. 7. illustrates selection of cells not expressing GFP. Single intact cells were selected as before (see Fig. 4.), then cells expressing no GFP (low FLl, low FL2, region 7, shown in white box) were collected.

Experimental Methods

Cell culture Primary chicken embryo fibroblasts were prepared from 10 day chicken embryos from a pathogen free flock by opening the shell, removing the embryo and decapitating it, then passing the embryo through a plastic 50 ml syringe into a sterile tissue culture flask. The cells were then separated using trypsin and EDTA, stirring for 5 minutes, then removing the cell suspension and adding fresh trypsin to the remaining cell clumps, stirring again for 5 minutes. The trypsinised cells were then filtered through gauze, centrifuged and resuspended in growth medium (Dulbecco's Modified Eagle Medium - DMEM- containing 10% foetal bovine serum - FBS) centrifuged again, resuspended in growth medium again and plated in T150 tissue culture flasks containing 30 ml DMEM 10% FBS, with the cells from one embryo divided between six flasks. The flasks were incubated at 37°C, 5% C0₂ overnight. Next day when the cells are confluent they were trypsinised and divided 1:3 into T150 flasks. Passaged cells were used for transfections, plating viruses prior to FACS sorting, MVA virus production and titration. Fowlpox FP9 only forms plaques in unpassaged cells so 6 well plates were seeded with 2 million cells per well immediately after they were prepared from the embryos, incubated overnight at 37°C, 5% C0₂ and used the next day for fowlpox titrations. Unpassaged cells in T150 flasks were used for fowlpox virus production.

Agarose overlay medium used in titrations consists of 2% low melting point (LMP) agarose in water, autoclaved, cooled to 40°C, mixed 1:1 with 2xMEM without phenol red, 4% FCS, pen/strep, warmed to 37°C.

Construction of shuttle vectors

Construction of FP-GFP is shown as Fig. 1. A plasmid map of FP-GFP is shown as Fig. 2.

FP-GFP

This consists of four fragments of DNA (A to D) assembled in a pUCl9 backbone (Ampicillin resistant)

Preparation and assembly of the four fragments are described below.

A: consists of a Pstl fragment from pEFL29 (Qingzhong et al . , 1994, Vaccine, Vol: 12(6), pp569-73) containing flank 1 for homologous recombination, joined to the Vaccinia P7.5 promoter, with a Smal site for antigen insertion after the promoter.

B: A fragment containing the fowlpox late promoter FP4b plus the N-terminus of the coding sequence for FP4b, obtained by PCR using pEFL29 as a template and adding a Sail site before the promoter and a BamHI site within the coding sequence by incorporating these sites into the PCR primers. The BamHI site is positioned to allow an in-frame fusion to the GFP coding sequence in fragment C.

C: A BamHI to Not I restriction fragment containing the coding sequence for eGFP taken from the plasmid pEGFP-Nl supplied by Clontech.

D: A fragment obtained by PCR using pEFL29 as a template, consisting of a Notl site, flank 2 for homologous integration, a Bglll site and a Notl site.

Construction of FP-GFP is illustrated in Fig. 1. A plasmid map of FP-GFP is shown as Fig. 2. PCR products B and D were obtained using the proof-reading polymerase combination Expand (Roche Molecular Biochemicals) to minimise the introduction of errors, cloned into plasmid pGEM-T (Promega) and sequenced to check that no errors had been introduced. B was then isolated from pGEM-T as a Sall-BamHI fragment and ligated into pEGFP-NI cut with Sail and BamHI, to join the FP4b promoter and N-terminal sequence as an in-frame fusion to eGFP (intermediate plasmid named B+GFP) . This plasmid was then linearised with Notl after the stop codon of eGFP, treated with CIP to prevent religation, and fragment D was ligated in, having prepared the fragment by a Not I digest of pGEM-T containing PCR product D. This resulted in the addition of flank 2 (intermediate named BD+GFP) . Fragment D can insert in either orientation but in BD+GFP the orientation is such that flank 2 is in the same orientation relative to FP4b as in pEFL29.

A Sail - Bglll fragment containing the FP4b promoter, eGFP and flank 2 was then isolated from BD+GFP and ligated into plasmid pIC20H (Marsh, Erfle and Wykes 1984 Gene 32 481-5) cut with Sail and Bglll . This plasmid consists of a pUC19 backbone with a longer polylinker than in pUCl9 (intermediate plasmid named BD+GFP-20H) . This was then linearised at the unique Pstl site upstream of the FP4b promoter, treated with CIP and fragment A was ligated in. This fragment can insert in either orientation but in the final plasmid, FP-GFP, the orientation of flank 1 relative to FP4b is the same as in pEFL29.

MVA-GFP

Construction of MVA-GFP is illustrated in Fig. 3. A plasmid map of MVA-GFP is shown as Fig. 4.

MVA-GFP was derived from FP-GFP by replacing the FP flank 1 with the right portion of the MVA TK locus (TKR) and replacing the FP flank 2 with the left portion of the MVA TK locus (TKL) .

TKR was obtained as a PCR product using pSCll

(Chakrabarti et al . , 1985, Mol Cell Biol, vol: 5(12), pp3403-9) as a template (Expand polymerase as above) incorporating a Sphl site into one of the primers and a Bglll site into the other. The PCR product was cloned into pGEM-T and sequenced, then the plasmid was linearised at the unique Ncol site in the pGEM-T polylinker which was filled in with Klenow polymerase to make a blunt end, then cut with Sphl to release the TKR fragment which was ligated into FP-GFP between the Sphl and Smal sites to replace flank 1 with TKR. This created the intermediate plasmid FP-GFP-TKR. The Smal site (used in FP-GFP to insert antigen coding sequences next to the P7.5 promoter) was therefore destroyed but a new Bglll cloning site was created.

TKL was obtained as a PCR product using pSCll as a template with Expand polymerase, incorporating Notl and Sad sites into the PCR primers. After sequencing the TKL fragment was obtained by a Notl-Sacl digest and ligated into FP-GFP-TKR cut with Notl and Sad such that flank 2 was replaced by TKL, creating plasmid MVA-GFP. Transfeetions

Passaged CEF cells (passage number 2 to 5) were plated in 6 well tissue culture plates at 0.5 million per well, in DMEM 10% FBS, and grown overnight at 37°C, 5% C0₂. The medium was removed from the cells and they were rinsed in phosphate buffered saline (PBS) and then 1 ml DMEM 2% FBS containing either MVA (obtained from Anton Mayr, Institute of Medical Microbiology, Infectious and Epidemic Diseases, Veterinary Faculty, Ludwig-Maximilians Universitat, Munchen, Germany) or FP (FP9 obtained from Mike Skinner, Institute of Animal Health, Compton, UK) was added, at a multiplicity of infection of 0.05. The cells were incubated at 37°C, 5% C0₂ for 90 minutes. The required shuttle vector (1 μg) was mixed with 90 μl of DMEM with no additions, plus 10 μl Superfect transfection reagent (Qiagen) and vortexed. The mix was allowed to stand at room temperature for 10 minutes for transfection complexes to form. The medium was then removed from the infected cells and the transfection mix was added along with 2 ml DMEM 2% FBS. The plates were incubated for a further two hours before removing the medium, replacing it with fresh DMEM 2% FCS and incubating for two days.

Preparation for FACS sorting

Transfected cells were observed with a fluorescent inverted microscope to check for GFP expression. The medium was then removed and the cells were rinsed in PBS. Trypsin/EDTA was then added (0.5 ml per well) and the plate was gently agitated at room temperature for approximately five minutes until the cells became detached from the plastic, as observed with an inverted microscope. The cells were then resuspended in 2 ml PBS containing 2% FBS, placed in a 15 ml

Falcon centrifuge tube at centrifuged at 1000 rpm for 5 minutes. The cells were then resuspended in 0.5 ml PBS 2% FBS and FACS sorted.

FACS

Cells were FACS sorted using a Becton Dickinson FACSVantage, either into a tube containing 200 μl lO M Tris pH 9.0 or a 96 well plate containing a CEF monolayer, at one cell per well. The CEF monolayer was prepared by seeding 5 x 10⁴ cells per well in DMEM 10% FBS and incubating overnight at 37°C, 5% C0₂, then removing the medium, rinsing with PBS and adding 50 μl DMEM (serum free) per well.

After transfection 0.5 to 1% of the CEFs were found to express GFP. Any cells in which GFP expression was above background were collected.

Example 1

Production of a recombinant fowlpox using GFP as a selectable marker.

The following methods use primary chicken embryo fibroblast (CEF) cells throughout. However, other host cells permissive for the relevant poxvirus could have been used. For example, MVA may also be propagated in the baby hamster kidney BHK cell line. Staib et al . (Biotechniques, Vol 28(6), pp 1137-42, 2000) described the use of transient dominant selection of recombinants by using the host range gene K1L as a selectable marker, since this enables the recombinant virus to replicate in rabbit kidney RK-13 cells. The marker is flanked by repeated sequences which allow the marker to be lost when the virus is then propagated in CEF cells. However, use of cells other than CEFs is seen as problematic by regulatory agencies governing the use of recombinant viruses in clinical trials. Poxviruses undergo changes to their genome in tissue culture. MVA and FP9 were both produced by hundreds of passages in CEFs and a number of deletions to the genome occurred. If these viruses are then passaged in other cells such as BHK and RK-13 it is likely that further changes will occur and these will need to be fully characterized before using those viruses in clinical trials. Therefore to produce vaccines for clinical trials rather than animal experiments, CEFs should be used in the production of the virus.

The gene encoding Myco acterium tuberculosis antigen 85A was ligated into FP-GFP. CEFs were infected with fowlpox FP9 and transfected with this plasmid using two wells of a six-well plate, each with 0.5 x 10⁶ CEF cells and 1 μg DNA. Two days after transfection green cells were visible with the fluorescent inverted microscope. The cells were trypsinised, taken up in PBS 2% FBS, spun down and resuspended in 0.5 ml PBS 2% FBS before FACS sorting. Single green cells were sorted into each well of two 96 well plates containing a CEF monolayer with 50 μl DMEM (serum-free) per well. The plates were incubated for 5 days and then examined under the fluorescent inverted microscope. One well on one of the plates contained green cells. The cells and medium from this well were harvested and replated on CEF monolayers in 6 well plates. After 2 days incubation a- well containing five areas (they were not well-defined plaques as the cells had been passaged) of green cells was trypsinised and FACS sorted again, with one cell per well of a single 96 well plate. After 5 days incubation the majority of wells in the plate were green. Cells and medium from 6 wells were harvested and replated on unpassaged CEF cells to allow plaque formation and visualisation. The medium was replaced with agarose after two hours incubation, and the plate was incubated for a further five days . White plaques were visible when the plate was held up to the light, and these were ringed in a well containing well separated plaques (10 - 30 per well) . The fluorescent inverted microscope was then used to ascertain if these plaques expressed GFP. From one of the isolates all the plaques examined (11/11) were green. Six of the plaques were picked from this plate and replated on unpassaged cells. Again, all plaques expressed GFP, therefore the recombinant virus was free of wild type, and was bulked up using unpassaged CEF cells.

Example 2

Production of a recombinant MVA using GFP as a selectable marker.

The gene encoding Plasmodium cynomolgi circumsporozoite protein was ligated into the shuttle vector MVAGFP. CEFs were infected with MVA and transfected with this plasmid using two wells of a six-well plate, each with 0.5 x 10⁶ CEF cells and 1 μg DNA. Two days after transfection green cells were visible with the fluorescent inverted microscope. The cells were trypsinised, taken up in PBS 2% FBS, spun down and resuspended in 0.5 ml PBS 2% FBS before FACS sorting. The fraction of the population collected was 0.85% and 7661 cells were collected into a tube containing 200 μl lOmM Tris pH 9.0. This tube was frozen and thawed three times, then sonicated to break open the cells and release the virus, which will be a mixture of wild-type and recombinant MVA.

Half of this mixture was then replated into a six well plate of CEF cells, adding the virus to the first well and making serial 10 fold dilutions into the rest of the plate. The plate was incubated for two hours to allow the virus to infect the CEFs, then the medium was removed and replaced with agarose overlay. After three days incubation approximately 30 green staining areas involving 5-20 cells were visible with the fluorescent inverted microscope in the first well and three in the second well.

The cells in the second well were trypsinised and FACS sorted a second time, this time collecting one cell per well of a 96 well plate containing 25 μl 10 mM Tris pH 9.0 per well. The plate was frozen and thawed to release virus from the sorted cells and 5 x 10" CEF cells suspended in 200μl DMEM containing 2 % FCS were added to each well. The plate was incubated at 37°C, 5% C0₂ for three days and then examined under a fluorescent microscope. The majority of the wells contained green cells. Cells and medium from one of the positive wells were taken up in a pipette, frozen and thawed three times to release the virus from the cells, sonicated and plated out on passaged CEF monolayers in a 6 well plate. The monolayer was grown overnight from 1 x 10⁶ cells in DMEM with 10% FCS, then the medium was removed, the cells were rinsed in PBS and 1 ml DMEM containing 2% FCS was added per well. The entire cell/virus mix was added to the first well of the plate and 5 serial 10-fold dilutions were made within the plate. The plate was incubated for 2 hours, then the medium was replaced with an agarose overlay. The plate was incubated for 5 days after which time white plaques were visible when the plate was held up to the light. Plaques were ringed with a marker pen and the fluorescent inverted microscope was used to confirm that all the ringed plaques expressed GFP, indicating that all were recombinant. A plaque was then picked and used to bulk up the recombinant virus.

Example 3

Production of .a recombinant MVA using beta-galactosidase in combination with a FITC-conjugated anti-beta-galactosidase antibody. A synthetic gene encoding Plasmodi um falciparum circumsporozoite protein, codon-optimised for mammalian cell expression, was ligated into pSCll for expression by the P7.5 promoter. CEF cells were infected with MVA and transfected with this shuttle vector as described above, using two wells of a six-well plate, each with 0.5 x 10⁶ CEF cells and 1 μg DNA. Two days after transfection cells were trypsinised, taken up in PBS 2% FBS, washed and resuspended in 1 ml PBS containing 2% FBS, 1% saponin (to permeabilise the cells and allow the antibody to enter) and a 1:200 dilution of a FITC-conjugated anti beta-galactosidase antibody. The cells were incubated in the antibody solution for 30 minutes at room temperature without agitation, then FACS sorted, collecting any cells in which FITC content was above background. In total 1510 cells were collected, representing 0.15% of the population, into a tube containing 200 μl lOmM Tris pH 9.0. This tube was frozen and thawed three times, then sonicated to break open the cells and release the virus, which will be a mixture of wild-type and recombinant MVA.

The virus mixture was plated on two six well plates, with 100 μl added to the first well and 10 fold serial dilutions made into the remainder of the plate. The plate was incubated for two hours to allow the virus to infect the CEFs, then the medium was removed and replaced with agarose overlay. After 5 days incubation white plaques were visible when the plate was held up to the light, indicating the presence of MVA plaques. The plate was then overlaid with a second layer of agarose containing X-gal, which is a substrate for beta-galactosidase and turns blue when cleaved by the enzyme. After overnight incubation two blue plaques were visible in the first well of one of the plates, but none in the second plate. The first plate contained 50 MVA plaques in the second well, indicating that in the first well there were two recombinant plaques out of a total of 500 MVA plaques. These two plaques were then picked, subjected to freeze/thawing and sonication as before and replated. After the second plating there were 7 blue plaques out of a total of 25. Three of the well-isolated blue plaques were picked and replated, and at the next stage one plaque proved to be a pure recombinant which was then bulked up.

Example 4 Replacement of Red FP with a recombinant antigen and GFP marker

Red fluorescent protein (Clontech) was ligated into the shuttle vector pSCll (Chakrabarti et al . , 1985, Mol Cell Biol, vol: 5(12), pp3403-9) such that expression of RedFP was driven by the Vaccinia P7.5 promoter. CEFs were infected with wild type MVA and transfected with pSCHRedFP using the conditions described above. FACS sorting was then used to isolate a pure recombinant virus expressing RedFP.

This virus may then be used as the starting point for the generation of new recombinants. When CEFs are infected with MVA-Red and transfected with the shuttle vector MVA-GFP containing a novel antigen, recombination between the viral genome and the shuttle vector results in the replacement of RedFP with the new antigen expressed from P7.5 plus GFP expressed from FP4b as a marker gene for FACS sorting.

Using the method as described in example 2, despite selecting for GFP-expressing cells using two rounds of FACS sorting, occasionally wild type virus is present along with the recombinant and it is necessary to rule out the presence of wild type virus by plating virus on unpassaged cells to observe plaque formation, or harvesting the virus from one well of the 96 well plate and extracting DNA for PCR analysis from half the material (see example 5 for details of the method) . Cells infected with the wild type virus cannot be detected by FACS analysis and plaques may only be seen when the virus is plated on unpassaged CEFs which are then incubated for 5 days under agarose. The wild type virus may be present as a consequence of double infection of CEFs, which therefore contain GFP-expressing virus and wild-type virus, or it may be present in the liquid surrounding the CEFs having been released from infected CEFs that lysed during preparation for sorting.

Using MVA-Red as the starting point for production of a novel recombinant enables the operator to sort for GFP-expressing cells and against RedFP expressing cells at the second round of FACS sorting. Also when single cells are collected in a 96 well plate and incubated with CEFs, a fluorescence microscope may be used to check for the presence of GFP expression and absence of RedFP expression in individual wells. Wells containing only green but not red cells may then be used to isolate the recombinant virus with no contamination from the starting virus. Finally the virus may be plated on passaged CEFs (unpassaged CEFs not necessary) and plaques visualized by fluorescence microscopy. The starting virus forms red plaques and the recombinant forms green plaques. Thus if no cloning facility (i.e. collecting single cells into individual wells of a multi-well plate) is available, the cells from the second sort may be collected together and plated on passaged CEFs. Since both the starting virus and the novel recombinant may be visualized, green plaques that are well isolated from red plaques may be identified for plaque picking with no risk of contamination of the picked plaque with the starting virus.

Gene "X" (a protein-encoding DNA of known sequence used by way of example only) fused to a CD4 epitope from tetanus toxoid was ligated into MVA-GFP. The shuttle vector was recombined with MVA-RedFP in CEFs, using 4 wells of a 6 well plate each containing 5x 10⁵ CEFs, infected with MVA-Red at a multiplicity of infection of 1. After incubation for 90 minutes the shuttle vector was introduced using (per well) 1 μg DNA, 10 μl Superfect in 100 μl serum-free DMEM, vortexed and incubated at room temperature for 10 minutes . The DNA mix was then made up to 1 ml per well using DMEM with 2% FCS and this was used to replace the medium on the infected CEFs. After two days incubation the cells were trypsinised and sorted on a Cytomation Mo Flo cell sorter, collecting all cells expressing GFP (see Fig. 5) in one tube. Collection was stopped after 5313 cells had been collected.

The cells were then frozen and thawed once to release virus from the cells, and the mixture replated on fresh passaged CEF monolayers in a 6 well plate. The cells from two wells were trypsinised and sorted after two days for cells expressing GFP but not RedFP (see Fig. 6) , collecting individual cells in a 96well plate that contained 50 μl PBS per well. The plate was frozen and thawed once to release virus from the cells, then CEFs suspended in DMEM 2% FCS were added (5 x 10⁴ per well), and the plate was incubated at 37 °C, 5%C0₂ for 2 days. Examination with a UV microscope demonstrated that cells in 15 of the wells expressed GFP and not RedFP. Cells from these wells were then harvested to isolate the recombinant virus.

Example 5

Removal of the GFP marker from a recombinant virus .

The GFP marker does not interfere with the use of recombinant virus in animal experiments. However for clinical use it is desirable that the marker is removed. Another application of removing the GFP marker is in manipulating the viral genome. Genes may be inserted or deleted using GFP as a marker to select the recombinant virus. The marker may then be deleted leaving only the required addition or deletion. No other sequences remain in the genome so the same technique may be used sequentially multiple times with the same viral genome.

A recombinant FP9 expressing a codon-optimised version of Plasmodium falciparum circumsporozoite protein was generated using the shuttle vector FP-GFP and FACS sorting. The GFP and promoter associated with it were then removed using a technique based on that described by (Storici et al . , Nature Biotechnology 2001, Vol: 19(8), pp 773-6) .

Oligonucleotides DPI and DP2 were designed to anneal either side of the promoter and GFP in the viral genome, and extended by 10 bp at the 3' end to produce a 20bp region of homology between the 3' ends of the two oligonucleotides.

DPI:

ACATTACCCACATGATAAGAGATTGTATCAGTTTCGTAGTCTTGAGTATTGGTA TTACTATATAGTATATTTATTCGCGG (SEQ ID NO: 1)

DP2: TTTAGAGATTCAAGAGATCCCGCCAGACGGGGAACCTGGGTCAACGACTGGTGC GAAGATCCGCGAATAAATATACTATA (SEQ ID NO: 2)

The oligonucleotides were synthesized and purified by HPLC. They were resuspended in purified water at O.lnmol/μl and annealed and extended in the following manner to produce a linear double stranded DNA molecule. Five μl each oligonucleotide, 4 units Expand High Fidelity PCR enzyme (Boehringher) , 5 μl lOx Expand buffer including MgCl, 0.2mM dNTPs, water to a final volume of 50 μl, placed in a thin walled PCR tube and overlaid with 20 μl mineral oil, placed in a PCR machine and heated to 94 °C for 1 minute, then 68 °C for 3 minutes, cooled to room temperature, precipitated with ethanol and sodium acetate, then resuspended in 24 μl water.

CEF monolayers in a 6 well plate (4 wells used) were infected with FP9-GFP-CSO at an m.o.i. of 0.05 in DMEM 2% FCS, and 90 minutes later transfected with the annealed and extended oligonucleotides using 1 μl per well, mixed with 10 μl Superfect (Qiagen) in 100 μl final volume of serum-free DMEM. The mix was vortexed, incubated at room temperature for 10 minutes, made up to 1 ml per well with DMEM 2% FCS and used to replace the medium on the infected CEFs. The medium was replaced with fresh DMEM 2% FCS after overnight incubation. Five days after transfection the cells and medium were harvested, frozen and thawed to release the virus from the cells, and 1 ml of the undiluted viral mixture plus a series of 10-fold dilutions were plated on fresh CEF monolayers in a 6 well plate and incubated for 2 days.

The monolayers were examined by UV microscope. In the 10^"2 dilution, approximately 10 green cells per microscope field were visible. In the 10^"3 dilution approximately 1 green cell per field was visible. Cells from these wells were trypsinised and prepared for sorting, keeping the two samples separate. Sorting was carried out on a Cytomation Mo Flo cell sorter, collecting non-green cells (see Fig. 7). Approximately 50% of the cell population was collected, with a total of 3.8 x 10⁵ cells for the 10^~2 well and 3.7 x 10⁵ for the 10^"3 well. The collected cells were frozen and thawed to release the virus, plated as a series on 10-fold dilutions on unpassaged CEFs and overlaid with agarose. After 5 days incubation white plaques were visible by eye. These were also examined by UV microscopy. Using the cells from the 10^"3 dilution, one well with 1 white and 2 green plaques, plus another well with 4 white and 1 green plaques were identified. The white plaques were well separated from the green plaques. The white plaques were picked into 200 μl 10 mM Tris pH 9.0 and vortex mixed. One green plaque was also picked as a control .

Half of the picked plaque was used to prepare DNA for PCR analysis using a Qiagen mini genomic column and following the manufacturer's protocol with the exception that the purified DNA was eluted into 50 μl water. Three separate PCR reactions were carried out on each sample using 1 μl DNA per PCR reaction. The first PCR reaction uses primers annealing- either side of the required deletion and yields a product of 468 bp if the deletion has been made, and greater than 1.3 kb if GFP and the associated promoter are still present. The green plaque gave a product greater than 1.3 kb, and the 5 white plaques a product of 468 bp demonstrating that the required deletion had been made. PCR was also used to rule out the presence of wild type FP9 and detect presence of the CSO antigen in the white plaques, thus confirming that a recombinant FP9 containing the CSO antigen but not the GFP marker had been produced. The remaining portion of the picked plaque was used to bulk up the recombinant virus.

Claims

CLAIMS :

1. Use of FACS sorting in- a method for the production of recombinant poxvirus, wherein FACS sorting is used for the separation of cells containing a recombinant poxvirus from cells not containing the said recombinant poxvirus.

2. Use according to claim 1 wherein cells containing the recombinant poxvirus are distinguished from cells not containing the recombinant poxvirus by the presence of a selection marker which is directly or indirectly detectable by FACS.

3. Use according to claim 1 or claim 2 wherein the poxvirus is a non-replicating poxvirus.

. Use according to claim 3 wherein the poxvirus is modified vaccinia Ankara or attenuated fowlpox.

5. A method of preparing recombinant poxvirus which comprises:

(i) providing a shuttle vector comprising a heterologous gene expression cassette positioned between flanking viral DNA sequences corresponding to fragments of the genome of a poxvirus, the heterologous gene expression cassette comprising a DNA sequence encoding a heterologous polypeptide operably linked to a promoter sequence;

(ii) introducing the shuttle vector into host cells which have previously been infected with a pox virus and incubating the host cells under conditions which allow for homologous recombination between the pox virus and the flanking viral DNA sequences present in the shuttle vector; (iii) selecting for host cells containing recombinant virus using FACS sorting; and

6. A method according to claim 5 wherein cells containing recombinant virus are selected in step (iii) by contacting the cells under cell permeabilising conditions with a fluorescent labelled antibody conjugate immunologically specific for an epitope on the heterologous polypeptide, incubating the cells to allow for specific binding of the antibody conjugate to any target heterologous polypeptide present in the cells and selecting for cells which exhibit fluorescence from the antibody conjugate using FACS sorting.

7. A method according to claim 5 wherein the heterologous polypeptide is a positive selection marker protein.

8. A method according to claim 5 wherein the shuttle vector further comprises a DNA sequence encoding a positive selection marker protein, which DNA sequence is also positioned between the flanking viral DNA sequences.

9. A method according to claim 8 wherein the DNA sequence encoding the positive selection marker protein forms part of a marker gene expression cassette comprising the said DNA sequence operably linked to a promoter.

10. A method according to claim 5 wherein the heterologous gene expression cassette comprises a protein encoding DNA sequence operably linked to a promoter sequence, wherein the protein encoding DNA sequence encodes a fusion protein consisting of a heterologous polypeptide fused in-frame to a positive selection marker protein.

11. A method according to any one of claims 7 to 10 wherein the positive selection marker protein is a fluorescent protein and cells containing recombinant virus are selected in step (iii) by selecting for cells which exhibit fluorescence from the marker protein using FACS sorting.

12. A method according to any one of claims 7 to 10 wherein cells containing recombinant virus are selected in step (iii) by contacting the cells under cell permeabilising conditions with a fluorescent labelled antibody conjugate immunologically specific for an epitope on the positive selection marker protein, incubating the cells to allow for specific binding of the antibody conjugate to any target marker protein present in the cells and selecting cells which exhibit fluorescence from the antibody conjugate using FACS sorting.

13. A method according to claim 5 wherein the pox virus used to infect the host cells is a modified pox virus, wherein the genome of the modified poxvirus contains a marker gene expression cassette flanked by regions of the viral genome homologous with the viral genome regions present in the shuttle vector, the marker gene expression cassette comprising a DNA sequence encoding a negative selection marker protein operably linked to a promoter, and wherein cells containing recombinant virus are selected in step (iii) by selecting against expression of the negative selection marker protein using FACS sorting.

14. A method according to any one of claims 7 to 10 wherein the pox virus used to infect the host cells is a modified pox virus, wherein the genome of the modified poxvirus contains a marker gene expression cassette flanked by regions of the viral genome homologous with the viral genome regions present in the shuttle vector, the marker gene expression cassette comprising a DNA sequence encoding a negative selection marker protein operably linked to a promoter, and wherein cells containing recombinant virus are selected in step (iii) by selecting for expression of the positive selection marker protein and against expression of the negative selection marker protein using FACS sorting.

15. A method of preparing recombinant poxvirus which comprises:

(i) constructing a recombinant poxvirus genome capable of expressing a heterologous polypeptide;

(ii) introducing the recombinant poxvirus genome into host cells which are capable of packaging the recombinant poxvirus genome into infectious virions;

(iii) infecting further host cells permissive for replication of the desired recombinant poxvirus with infectious virions released from the host cells of step (ii) ;

(iv) selecting cells containing recombinant virus using FACS sorting; and

(v) isolating recombinant virus from one or more cells selected in step (iv) .

16. A method according to claim 15 which includes a further step of FACS sorting the host cells from step (ii) to select cells containing recombinant virus.

17. A method according to claim 15 or claim 16 wherein selection of cells containing recombinant virus is performed by contacting the cells under cell permeabilising conditions with a fluorescent labelled antibody conjugate immunologically specific for an epitope on the heterologous polypeptide, incubating the cells to allow for specific binding of the antibody conjugate to any target heterologous polypeptide present in the cells and selecting for cells which exhibit fluorescence from the antibody conjugate using FACS sorting.

18. A method according to claim 15 or claim 16 wherein the heterologous polypeptide is a positive selection marker protein.

19. A method according to claim 15 or claim 16 wherein is further capable of expressing a positive selection marker protein in addition to the heterologous polypeptide.

20. A method according to claim 18 or claim 19 wherein the positive selection marker protein is a fluorescent protein and selection of cells containing recombinant virus is performed by selecting for cells which exhibit fluorescence from the marker protein using FACS sorting.

21. A method according to claim 18 or claim 19 wherein cells containing recombinant virus are selected by contacting the cells under cell permeabilising conditions with a fluorescent labelled antibody conjugate immunologically specific for an epitope on the positive selection marker protein, incubating the cells to allow for specific binding of the antibody conjugate to any target marker protein present in the cells and selecting cells which exhibit fluorescence from the antibody conjugate using FACS sorting.

22. A method according to claim 15 or claim 16 wherein the host cell of step (ii) which is capable of packaging the poxvirus genome into infectious virions is a cell previously infected with a helper poxvirus, the genome of which includes a marker gene expression cassette comprising a DNA sequence encoding a negative selection marker protein operably linked to a promoter, and wherein cells containing recombinant virus are selected by selecting against expression of the negative selection marker protein using FACS sorting.

23. A method according to claim 19 wherein the host cell of step (ii) which is capable of packaging the poxvirus genome into infectious virions is a cell previously infected with a helper poxvirus, the genome of which includes a marker gene expression cassette comprising a DNA sequence encoding a negative selection marker protein operably linked to a promoter, and wherein cells containing recombinant virus are selected by selecting for expression of the positive selection marker protein and against expression of the negative selection marker protein using FACS sorting.

24. any one of claims 5 to 23 wherein the poxvirus is a non-replicating poxvirus.

25. A method according to claim 24 wherein the poxvirus is modified vaccinia Ankara or attenuated fowlpox.

26. A method for preparing a vaccine composition comprising a recombinant poxvirus, which method comprises preparing a recombinant poxvirus according to the method of any one of claims 5 to 24, wherein the heterologous polypeptide is an antigenic polypeptide, and formulating the recombinant poxvirus into a vaccine composition with one or more diluents, carriers or excipients.

27. A shuttle vector comprising a promoter sequence operably linked to a cloning site positioned between flanking viral DNA sequences corresponding to fragments of the genome of a poxvirus, characterised in that the shuttle vector further comprises a DNA sequence encoding a marker protein which is directly detectable by FACS, which DNA sequence is also positioned between the said flanking viral DNA sequences .

28. A shuttle vector according to claim 27 wherein the DNA sequence encoding the marker protein forms part of a marker gene expression cassette comprising the said DNA sequence operably linked to a promoter.

29. A shuttle vector according to claim 27 wherein the DNA sequence encoding a marker protein is in close juxtaposition with the cloning site such that a heterologous protein-encoding DNA fragment may be inserted into the cloning site in-frame with the DNA sequence encoding the marker protein.

30. A shuttle vector according to any one of claims 27 to 29 wherein the marker protein is a fluorescent protein.

31. A modified poxvirus, the genome of which contains a marker gene expression cassette comprising a DNA sequence encoding a marker protein operably linked to a promoter.

32. A modified poxvirus according to claim 31 wherein the marker protein is directly detectable by FACS.

33. A kit for use in the preparation of recombinant poxvirus, the kit comprising a shuttle vector and a modified poxvirus, wherein the shuttle vector comprises a promoter sequence operably linked to a cloning site positioned between flanking viral DNA sequences corresponding to fragments of the genome of a poxvirus and further comprises a DNA sequence encoding a positive selection marker protein which is directly detectable by FACS, which DNA sequence is also positioned between the said flanking viral DNA sequences, and wherein the genome of the modified poxvirus contains a marker gene expression cassette flanked by regions of the viral genome homologous with the viral genome regions present in the shuttle vector, the marker gene expression cassette comprising a DNA sequence encoding a negative selection marker protein operably linked to a promoter.

34. A kit for use in the preparation of recombinant poxvirus, the kit comprising a shuttle vector and a fluorescent labelled antibody conjugate, -wherein the shuttle vector comprises a promoter sequence operably linked to a cloning site positioned between flanking viral DNA sequences corresponding to fragments of the genome of a poxvirus and further comprises a DNA sequence encoding a marker protein, which DNA sequence is also positioned between the said flanking viral DNA sequences, and wherein the fluorescent labelled antibody conjugate is immunologically specific for the marker protein.

35. A kit according to claim 33 or claim 34 which further comprises a supply of host cells.

36. A kit according to any one of claims 33 to 35 which further includes protocols for construction of recombinant poxvirus and/or isolation of recombinant poxvirus by FACS sorting.