WO1988002022A1

WO1988002022A1 - Recombinant poxviruses

Info

Publication number: WO1988002022A1
Application number: PCT/AU1987/000323
Authority: WO
Inventors: David Bernard Boyle; Barbara Elizabeth Howieson Coupar; Gerald Wayne Both
Original assignee: Commonwealth Scientific And Industrial Research Or; The Australian National University
Priority date: 1986-09-22
Filing date: 1987-09-22
Publication date: 1988-03-24

Abstract

Recombinant fowlpox virus or related avian poxvirus is characterised by the inclusion of foreign DNA in the virus genome. The foreign DNA sequence may comprise the Ecogpt gene coupled with a second gene and be inserted into a non-essential region of the virus genome.

Description

"RECOMBINANT POXVIRUSES" This invention relates to recombinant poxviruses and their construction, and in one particular aspect this invention relates to recombinant fowlpox virus and the use thereof as a vehicle for the expression of foreign genes particularly for the expression of avian disease antigens.

Poxviruses are large DNA viruses that replicate within the cytoplasm of infected cells. Vaccinia virus, the type species of the orthopox virus group, has been widely studied because of its role as the vaccine virus for smallpox in man. In recent years, recognition that the TK gene was a non-essential region of the vaccinia virus genome, followed by mapping and nucleotide sequence determination, has made it possible to insert and express a wide variety of foreign genes through the medium of recombinant vaccinia viruses (1-12). Such recombinant vaccinia viruses have the potential to deliver vaccine antigens to a variety of animal species (13). However, the risk of spread to man and the potential for disease problems from the widespread use of vaccinia virus in animals makes the construction of recombinants based on host specific poxviruses desirable, e.g. fowlpox virus for poultry or Orf for sheep vaccines. To achieve this requires considerable understanding of the molecular biology of these viruses, including the identification of non-essential regions into which foreign DNA might be inserted.

In one aspect, the present invention, which now makes possible a range of recombinant avian vaccines, is based on the mapping and sequencing of a non-essential region of the FPV genome. This apparently non-essential region is the thymidine kinase (TK) gene, and it permits the introduction of foreign DNA into fowlpox virus (FPV) without impairing the potential for virus replication and the production of live infectious recombinant viruses. According to this aspect, the present invention provides recombinant fowlpox virus related avian poxvirus characterised by the inclusion of foreign DNA in the virus genome.

In a preferred embodiment of this aspect, the foreign DNA is inserted into a non-essential region of the fowlpox virus genome, such as the TK gene or in virus DNA sequences controlling expression of the TK gene. In other embodiments, the foreign DNA may be inserted into intergenic regions of the fowlpox virus genome, for example regions preceding the TK gene or after the TK gene in such a manner as not to disrupt its function or the function of genes immediately upstream or downstream of the TK gene. The foreign DNA may also be inserted between genes (intergenic) in essential regions of the fowlpox virus genome, or within genes or their regulatory sequences (intragenic) in other non-essential regions of the whole genome. In a further aspect, the invention provides an avian disease vaccine comprising recombinant fowlpox virus wherein a foreign DNA sequence encoding an antigen characteristic of the said avian disease has been inserted into the TK gene of the fowlpox virus or in virus DNA sequences controlling expression of the TK gene.

By mapping and nucleotide sequence analysis (14,15) it has been shown that the FPV TK gene is characterised by an open reading frame of 183 codons commencing 279bp upstream of the central Xba 1 site of the nucleotide sequence as shown in Figure 8, and terminating 273bp downstream of this Xba 1 site. Confirmation of this sequence as the FPV TK gene is given by homologies with the vaccinia TK gene at nucleotide and amino acid levels, and the expression of TK enzyme which results from its insertion in TK^~ vaccinia virus.

According to another aspect, this invention provides an avian disease vaccine comprising recombinant fowlpox virus wherein a foreign DNA sequence encoding an antigen characteristic of the said avian disease has been inserted in the region of the fowlpox virus commencing 279bp before and terminating 273bρ after the central Xba 1 site of the nucleotide sequence shown in Fig.8, or in sequences controlling expression of this sequence.

It is envisaged that in accordance with this invention vaccines will become available for protection against a wide variety of avian diseases such as those caused by viruses, bacteria, protozoa, metazoa, fungi and other pathogenic organisms, by insertion of DNA sequences encoding appropriate antigens characteristic of these diseases into the FPV genome as broadly outlined above.

A general method for the insertion of foreign DNA into vaccinia virus is based on the selective inactivation of the TK gene and uses 5-bromodeoxyuridine to select TK^~ recombinant viruses (3) . Since there are no TK^~ avian cell lines available, selection of TK~ FPV recombinants is not possible. Other general methods for the construction of recombinant vaccinia viruses are known, and are discussed in detail hereinafter.Some of these approaches may be applicable to the insertion of foreign DNA into other poxviruses, however, none have yet been successfully applied to FPV. As described in detail below, it has now been demonstrated that the Escherichia coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene can be used as a dominant selectable marker for the insertion of foreign genes into poxviruses and that this technique has a number of advantages over other methods for the construction of recombinant viruses.

One of these is that the selection for the Ecogpt gene operates in a wide variety of cell types and therefore is potentially applicable as a dominant selectable marker for the insertion of foreign genes into other poxviruses (29). Mycophenolic acid, the antibiotic which provides the basis for selection of the Ecogpt gene (together with the required presence of xanthine, hypoxanthine, aminopterine, and thymidine), is a potent inhibitor of fowlpox virus in chicken embryo skin (CES) cell cultures. This has allowed the Ecogpt gene to be used as a dominant selectable marker for the insertion of foreign DNA into the TK gene of FPV. Expression of foreign genes by vaccinia viruses requires their being positioned near unique vaccinia virus transcriptional signals (1-4, 7-9), and it has been shown that the FPV TK promoter is similar to vaccinia promoters and that it operates in vaccinia virus (14,15). Vaccinia virus promoters have now been used to express foreign antigen coding sequences in FPV.

This invention therefore also offers methods of constructing recombinant fowlpox viruses or related avian poxviruses, which methods are characterised by the introduction of foreign DNA into the TK gene of the virus or into virus DNA sequences controlling expression of the TK gene.

At this stage the vaccinia virus promoter sequences P7.5 and PL11 have been used to express foreign genes in FPV. Recombinant FPVs have been constructed which contain the Ecogpt gene (demonstrated by hybridization) and which express the Ecogpt gene since they are able to grow in MXHAT (mycophenolic acid,„ xanthine, hypoxanthine, aminopterine, thymidine) selective conditions. This gene is under the control of the P7.5 promoter which clearly demonstrates that this and possibly other vaccinia virus promoters can be used to express foreign genes in FPV. The PL11 promoter expresses the influenza HA gene. The level of expression by this promoter may be modulated in these recombinants since the translational initiation codon of the llkd protein gene is present as well as the inserted HA gene initiation codon.

The insertion of the Ecogpt and HA genes into FPV and the recovery of viable virus able to grow in tissue culture clearly demonstrates that the FPV TK gene is not essential for FPV growth i vitro. This establishes the FPV TK gene as a non-essential region into which foreign DNA can be inserted and expressed. Further it provides the means to construct TK^~ FPVs which by analogy with TK^~ vaccinia viruses (32) would be expected to have reduced virulence in poultry and thus be useful as vaccines against FPV themselves.

Using the techniques described herein, two foreign genes have been inserted into FPV (the Ecogpt gene and the influenza HA gene) . Vaccinia virus recombinants carrying three vaccine antigens have been constructed and shown to simultaneously induce antibody responses to all three vaccine antigens when inoculated into animals (33). Additional poxvirus promoters and vaccine antigen genes in tandem with the Ecogpt can be inserted within the FPV TK gene in the general insertion vector pDB22 described herein. In this manner, recombinant FPVs can be constructed which will vaccinate poultry simultaneously against two, three or more diseases. Recombinant FPVs carrying multiple vaccine antigens would be a very cost effective way to deliver vaccines to poultry.

Included in the recombinant fowlpox viruses of this invention are thymidine kinase negative fowlpox viruses, which in their own right would be vaccines against fowlpox. In such viruses, TK production would be inactivated by the introduction of foreign DNA into the TK gene or controlling sequences, or by the excision of DNA essential for TK gene expression. By analogy with TK^~ vaccinia and herpes viruses, TK^~ fowlpox viruses would have reduced virulence and pathogenic potential in poultry and other avian species, and correspondingly enhanced usefulness as fowlpox vaccines and vehicles for the delivery of other vaccine antigens.

It is to be understood that the utility of recombinant fowlpox viruses according to this invention is not limited to the delivery of antigens. It is envisaged that they might also be useful for the delivery of a variety of other proteinaceous materials, e.g. growth hormones or immunomodulators, capable of being produced by expression of an appropriate gene.

As mentioned above, in recent years vaccinia virus has been the subject of interest as a vehicle for the expression of various foreign genes and thus as a basis for recombinant vaccines. A variety of techniques have been developed for the construction of recombinant poxviruses based on vaccinia virus. In general, they rely on homologous recombination between vaccinia virus flanking sequences bounding the foreign gene of interest and the virus genome during simultaneous infection of cells with virus and transfection with recombinant plasmid. From the progeny virus, recombinant viruses representing less than 0.1% or 0.01% of the population have to be selected and plaque purified. This is achieved by plaque hybridization with an appropriate radiolabelled probe; or by selection for TK^~recombinants when the foreign gene has been inserted into the TK gene of the virus; or by the co-expression of a marker gene product eg. β-galactosidase; or by selection for a dominant marker eg. HSV TK or neomycin resistance, concurrently inserted into the virus genome (28) . The proportion of the progeny virus as recombinants can be increased by alternative transfection protocols involving the use of temperature sensitive mutants (5) or single stranded recombinant DNA molecules (34) .

Many of these approaches rely on the TK phenotypes selection requiring TK^~cell lines. They are therefore not adaptable to other poxviruses such as fowlpox virus for which suitable TK^~ cell lines are unavailable. Although TK^~ recombinants are selectable under BUdR, the mutagenic effect of BUdR and the resulting background of TK^~ mutants does not allow selection at the recombination step or enrichment at subsequent passage. The neomycin resistance gene can be used as a dominant selectable marker, however, high concentrations of G418 antibiotic are required with 48hr pretreatment for the selection to operate (35) . The herpes simplex virus TK (HSV-TK) gene has been inserted into non-essential regions in the vaccinia virus genome by positive selection protocols. By placing another gene under the control of a vaccinia virus promoter in tandem with the HSV-TK gene, positive selection protocols for the construction of recombinant vaccinia viruses expressing other genes have been achieved. This approach, however, requires the availability of a TK^~ cell line which will survive for sufficient time under the methotrexate (TK⁺) selective conditions to allow growth and plaquing of TK⁺ vaccinia viruses.

It has now been discovered that the use of the Escherichia coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene in novel combination with another gene, offers a route to recombinant poxviruses which does not suffer the aforesaid disadvantages of the prior art. In particular, it

10 has been found that the Ecogpt gene can be used as a dominant selectable marker for insertion into poxvirus such as vaccinia virus. The antibiotic mycophenolic acid which, together with the required presence of xanthine, hypoxanthine, aminopterine and -, c thymidine, provides the basis for selection using the Ecogpt gene is a very potent inhibitor of poxvirus growth. Since the Ecogpt gene has been used as a dominant selectable maker in a variety of mammalian cells, this obviates the need for TK~ cell lines 0 for the construction of recombinant poxviruses (36).

The Ecogpt gene has previously been expressed from Simian Virus 40 - pBR322 hybrid plasmid vectors, and has been used as a dominant selectable marker in mammalian cells. Selection is 5 based on the ability of the Ecogpt gene to counteract the inhibitory effect of mycophenolic acid on the growth of mammalian cells. Since mycophenolic acid is also a very strong inhibitor of poxvirus growth, this provides a marker for the insertion of the 0 Ecogpt gene into poxviruses, so that when the Ecogpt gene is coupled in tandem with another gene of interest, recombinant poxviruses can be constructed and positively selected for the presence of both genes. The Ecogpt gene thus provides a dominant 5 selectable marker for insertion into any non-essential region of the poxvirus genome.

Accordingly, in another aspect, this invention provides a method of constructing a recombinant virus such as a poxvirus, adenovirus, herpesvirus, or the like, which comprises inserting into the non-essential region of a virus genome, a foreign DNA sequence comprising the Ecogpt gene coupled with a second gene, and selecting the virus 0 recombinants by their ability to reverse the inhibition of virus growth by mycophenolic acid.

Potentially the Ecogpt/mycophenolic acid system provides a dominant selectable marker for insertion into any non-essential region of the genome ₅ of any virus where Ecogpt selection operates in the cell type in which the virus will grow. Not only will this facilitate the construction of recombinant vaccinia viruses, but it will also provide a method for the construction of recombinant virus vaccines _Q including poxvirus vaccines based on host-specific poxviruses such as fowlpox virus for poultry or Orf virus for sheep, which are desirable because of the risk of disease spreading back to man from the use of vaccinia virus recombinants in animals. _g In this aspect, therefore, the present invention also provides recombinant virus such as a poxvirus, adenovirus, herpesvirus or the like, characterised in that it has a foreign DNA sequence comprising the Ecogpt gene coupled with a second gene _Q inserted into a non-essential region of the virus genome.

The second gene included within the foreign DNA sequence is preferably a gene which is expressed as a antigenic polypeptide which is foreign to the ₅ virus. Operation of the Ecogpt gene as a dominant selectable marker for construction of recombinant poxviruses offers the additional advantages of allowing selective enrichment of recombinant viruses prior to plaque purification and characterization. It is also important in that it obviates the need for TK~ cell lines in the construction of recombinant viruses. This is of particular value in the construction of recombinant fowlpox viruses, since TK~ avian cell lines are not currently available and so it is not possible to adopt the general method for insertion of foreign DNA into vaccinia virus, which is based on the selective inactivation of the TK gene and the use of BUdR to select TK^~ recombinants. Furthermore, use of the Ecogpt gene as a marker operates in most if not all eukaryotic cell- types. The ability of confluent monolayers to maintain for extended periods and plaque poxviruses in the presence of MXHAT make it useful for the construction of recombinants based on slow growing host specific poxviruses. Further the ability to select at both the recombination and subsequent steps allows the construction of recombinants where the frequencies of recombination may be low e.g. the insertion of very large fragments of foreign DNA. Those skilled in the art will be able to adapt known virus recombination techniques for insertion of specific Ecogpt/antigen gene couples into poxviruses. Essentially, the procedures involve insertion of a DNA sequence comprising the Ecogpt gene under the control of a poxvirus promoter plus a second gene under the control of the same or another poxvirus promoter, into a non-essential region of a poxvirus genome. More than one promoter plus second gene may be coupled with the Ecogpt gene, thus enabling insertion into poxvirus of multiple antigens or multiple serotypes of one antigen. Plasmids constructed with a second gene in tandem with the Ecogpt gene having flanking poxvirus sequences from non-essential regions could be used in the marker rescue recombination protocol. Recombinant virus containing the Ecogpt and second gene could be amplified and plaque-purified under mycophenolic acid selective conditions; all recombinants containing the Ecogpt gene would also contain the second gene, and this would be expressed provided it had been placed under the control of a poxvirus promoter. In the case of vaccinia and fowlpox virus, suitable promoters include P7.5, PF or PL 11.

Expression of the Ecogpt gene product is required for the recombinant viruses to grow under the mycophenolic acid selective conditions. The Ecogpt enzyme activity in virus-infected cell extracts can be demonstrated and measured using the conversion of 14C-xanthine to 14C-xanthine monophosphate and 14C-xanthosine as described by Chu and Berg (24) . Expression of the second gene inserted in tandem with the Ecogpt gene can be demonstrated by immuno chemical techniques, e.g. antibody and 125I protein A binding to plaques for a cell surface - expressed antigen like the influenza HA gene.

In order to demonstrate that the Ecogpt gene and second gene have been inserted into the poxvirus genome in the correct position, DNA can be purified by known techniques from the recombinant viruses. The purified DNA may then be analysed by restriction enzyme digests and DNA:DNA hybridisations to demonstrate the place and orientation in which the Ecogpt gene and second gene have been inserted. The effectiveness of the recombinant viruses of this invention containing one or more foreign antigen genes as vaccines can be demonstrated by antibody and cell-mediated immume responses in animals infected with the recombinant viruses. Protection against disease can be demonstrated by challenge with the organism(s) from which the antigen gene(s) were derived.

Further features of the present invention will be apparent from the detailed description in the Examples hereinafter and in the accompanying drawings.

EXAMPLE 1

This Example illustrates the construction of recombinant FPV wherein the TK gene of the fowlpox virus is interrupted by the Ecogpt gene under control of a poxvirus promoter in tandem with a gene of interest under the control of another poxvirus promoter. By simultaneous infection of cells with FPV and transfection with recombinant plasmid in which the FPV TK gene was interrupted by the appropriate foreign genes and promoters, recombination occurs between the FPV TK flanking sequences of the plasmid and homologous sequences in the FPV genome. Selection of viruses expressing the Ecogpt gene simultaneously selects recombinants carrying both the Ecogpt gene and the gene of interest.

In Figures 1 to 8:

Figure 1 shows construction of a plasmid, pDB16, containing multiple unique restriction enzyme sites within the TK gene of FPV. A Hindlll-Clal fragment of the FPV genome containing the TK gene was cloned into pUC9 which had been digested with EcoRI, mung bean nuclease treated and then digested with Hindlll. The Hindlll site was then deleted by Hindlll digestion, Klenow Poll fill in and religation. A synthetic oligonucleotide was inserted into the unique Ncol site within the TK gene (pDBlβ). Unique sites for H, Hindlll, C, Clal, E, EcoRI and S, Smal were inserted with this polylinker. Other restriction enzyme sites are N, Ncol and X, Xbal.

Figure 2 shows construction of plasmids for insertion of the Ecogpt and influenza genes into the TK gene of FPV. The Ecogpt gene with attached P7.5 vaccinia virus promoter (as an EcoRI-Ahalll fragment) was subcloned from pGpt07/14 into pDBlδ which had been digested with EcoRI and Smal (pDB18) . Then the HA gene under the control of the PL11 vaccinia virus promoter was cloned into pDB18 as a Clal fragment from pBCB08/HA. Plasmids ρDB19/l and pDB19/6 have the FPV TK gene interrupted by the P7.5-Ecogpt and PL11-HA gene fragments. Abbreviations for restriction enzyme sites; C, Clal, E, EcoRI, H, Hindlll, N, Ncol, X, Xbal. CIAP, calf intestinal alkaline phosphatase.

Figure 3 shows construction of insertion vectors for the selection of FPV recombinants using the Ecogpt gene and having foreign genes under the control of the PL11 vaccinia virus promoter. In pDB20 the size of the flanking FPV genome sequences has been increased by subcloning the P7.5-Ecogpt- PL11-HA fragment into the 5.5 EcoRI FPV genome fragment at the unique Ncol site contained in pDBl. pDB22 is a general vector into which foreign DNA can be inserted downstream of the PL11 vaccinia virus promoter and recombinants selected on the basis of growth in Ecogpt selective conditions. Abbreviations for restriction enzyme sites; B, BamHI; C, Clal; E, EcoRI; H, Hindlll; N, Ncol; P, Pstl; S, Smal and X, Xbal. Figure 4 shows antibody and 125I protein A binding to FPV plaques. A, normal mouse serum; B, hyperimmune mouse antiserum to influenza virus A/PR/8/34. FPV, fowlpox virus; FPV-HA, fowlpox virus recombinant containing the Ecogpt-P7.5 promoter and the influenza HA-PLll promoter constructed using pDB20.

Figure 5 shows the structure of the TK region of the parent, FPV-M3, and the recombinant, FPV-HA, genomes. The recombinant, FPV-HA, was constructed using ρDB20 (Fig.4). Abbreviations for restriction enzyme sites: E, EcoRI; N, Ncol; P, Pstl; S, Sail. Not all EcoRI and Ncol sites are shown.

Figure 6 shows analysis of DNAs from the parent, FPV-M3 (1) and the recombinant FPV-HA (2) viruses. DNAs were digested with Sail (S) and Pstl (P) and separated by electrophoresis through 0.6% agarose gels containing 0.5μg/ml ethidium bromide (EtBr) . The gel was photographed with UV illumination then blotted onto Gene-Screen Plus. The membrane was then hybridized with various 32P labelled DNA fragments (pDBl; HA; Ecogpt; P7.5;

PL11) .

Figure 7 shows the HI antibody response of chickens inoculated with FPV of FPV-HA (5 birds/group) , 15 day old SPV chickens were inoculated with 10 6 to 107 pfu of virus and reinoculated 21 days later. For birds inoculated with FPV-HA titers for individual birds are shown by the dots. The

5 geometric mean titer for these birds is shown by the stars. For the birds inoculated with the vaccine virus or FPV-M3 the titers were never greater than 10 and are shown by the solid squares.

Figure 8 shows the location, nucleotide

10 sequence, and derived amino acid sequence of the FPV TK gene and flanking regions. The nucleotide sequence was determined as described by Boyle and Coupar (14) and Boyle et.al. (15). Restriction enzyme cleavage sites are indicated as follows: E,

₁₅ EcoRI; H, Hindlll; X, Xbal; C Clal. Not all

Hindlll, Xbal, and Clal sites present in the 5,5-kb EcoRI fragment are shown. The position of the FPV TK gene was confirmed by its homology to the vaccinia virus TK gene. The derived amino acid sequence is

2₀ presented using the single-letter amino acid code. The Sau96I-DraI fragment used as primer for 5' end mapping of mRNA is indicated by the line above the squence. The initiation and termination codons of the second open reading frame identified in this

₂-. fragment are similarly indicated. * Termination codon.

MATERIALS AND METHODS Enzymes and Plasmids.

Restriction endonucleases and DNA-modifying _.„ enzymes were obtained from several commercial suppliers and used according to the manufacturers' instructions or those described in detail by Maniatis et.al. (17). Recombinant plasmids were prepared from the pUC (25) series and DNA purified in CsCl-,

35 gradients. pDBl to pDBll have been described previously (14). pBCBOβ, a multiple cloning site plasmid for the construction of recombinant vaccinia viruses, contains the late vaccinia virus promoter PL11 (26,30). The A/PR/8/34 influenza HA gene in pJZ102 was provided by Dr.P.N.Graves, The Mount Sinai Medical Center (27). The P7.5 vaccinia virus promoter in pGS20 was provided by Dr.B.Moss, National Institutes of Allergy and Infectious Diseases, Bethesda, Md. (2).

Cell cultures and viruses.

Primary and secondary chick embryo skin (CES) cell cultures were prepared from specific pathogen free embryonated eggs (CSIRO, SPF Poultry Unit, Maribyrnong, Victoria) as described by Silim et.al. (18) with the modification that collagenase at lOOμg/ml (Sigma C2139) was used to digest the skin of the embryos in place of trypsin. Fowlpox virus. (Mild Vaccine Strain: Arthur Webster Pty.Ltd., Northmead, NSW, 2152, Australia) was adapted to chick embryo cell cultures by passage at low multiplicity and plaque purified twice. This virus, designated FPV-M3, contains an approximately lOkb deletion from its genome. Plaque assays were performed on CES monolayers overlayed with Eagle's minimum essential medium with Earle's salts containing 1% agar and 5% foetal bovine serum. Plaques were stained with MTT tetrazolium (19) on the fifth or sixth day after inoculation. All virus stocks were disaggregated by digestion for 30min at 37°C with lmg/ml trypsin immediately prior to dilution for assays or infection of cell cultures. Marker Rescue.

CES cells seeded overnight at 5x10 2 cells/25cm flask were infected with FPV-M3 at 0.01 plaque forming units per cell. Virus was adsorbed for lhr at 37^βC then culture medium added. 7hr later the monolayers were washed four times with medium without serum and buffered with 0.05M Tris pH7.4, then once with Tris buffered saline (25mM Tris pH7.4, 137mM NaCl, 5mM KC1, 0.7mM CaCl-,, 0.5mM gCl₂, 0.6mM Na₂HPO.; TBS; (31). To each flask was added 2ml medium without serum containing 20μg of plasmid in DEAE-Dextran (MW=2xlO , Pharmacia) at a final concentration of 200μg/ml. The DNA solution was prepared by dissolving 20μg of plasmid (purified by twice banding in CsCl- gradients) in 125μl of TBS, adding 40μl of DEAE-Dextran at lOmg/ml and then adding 2ml of culture medium without serum (31) . The cultures were then incubated for a further 16hr at 37°C at which time they were washed twice with TBS. Medium with 5% foetal bovine serum and selective conditions were then added (MXHAT= 1 or 2-5μg/ml mycophenolic acid, 250μg/ml xanthine, lOOμM hypoxanthine, 0.4μM aminopterine and 30μM thymidine) .

The cultures were incubated for 6 or 7 days at which time the cells were scraped into the medium, pelleted by centrifugation and resuspended in 1ml of phosphate buffered saline. This virus stock was then passaged two or three times on CES cells under selective conditions. At the second and subsequent passages plaques grew out from marker rescues performed with plasmids containing the Ecogpt gene. Recombinant viruses containing the Ecogpt gene were plaque purified under agar without selection then amplified under selection in liquid medium in 24 well plates. Viruses containing the Ecogpt gene and gene of interest were identified by dot blot hybridization with 32P-labelled recombinant DNA probes (2,3).

Characterisation of recombinant FPVs.

FPV was purified from CES cultures (21) and the DNA extracted as described by Nakano et.al.

(22) . DNA was subjected to restriction enzyme digestion and separated by agarose gel electrophoresis. After blotting to Gene-Screen Plus membrane (NEN) , 32P-labelled DNA probes were hybridized to the membrane to analyse the arrangement of the virus genome.

Expression of influenza haemagglutinin by recombinant viruses was demonstrated by binding of anti-influenza antibodies and 125I-protein A to FPV plaques. The solid overlay was removed from plaques then the monolayer fixed for 5mins at room temperature with methanol. Antibodies and

125 I-protein A were then bound to the plaques as previously described (1,11). Responses of Poultry to recombinant FPVs.

SPF poultry, 15 days old, were inoculated subcutaneously/intradermally (s.c./i.d.) into the breast skin with 0.05ml of virus containing 10 6-107 pfu. Serum was collected at 7 day

-intervals. The birds were reinoculated s.c./i.d. into the wing web 21 days later. Sera were tested against 4 haemagglutinating units (HAU) of A/PR/8/34 virus. Serum dilutions were two-fold beginning at

1:5. End points were recorded as the dilution giving complete inhibition of 4HAU and the titre was expressed as the reciprocal of that dilution.

Construction of insertion vectors.

The FPV TK gene is contained within a 2.2kb Hindlll-Clal fragment of the FPV genome (14). This fragment was subcloned from pDBlO (14) between the Hindlll-EcoRI sites of ρUC9 (Fig.l; pDB14) . The remaining Hindlll site was deleted by digestion, fill in with Klenow fragment of polymerase I and religation (pDB15). Inspection of the TK gene nucleotide sequence revealed a unique Ncol site located in the centre of the TK gene and about 20bp away from the Xbal site also in this gene (15). Restriction enzyme analysis revealed that the Ncol site was unique to the 5.5kb EcoRI fragment of the FPV genome originally shown to contain the TK gene (15).

To facilitate the insertion of foreign DNA sequences within the TK gene, a synthetic oligonucleotide linker with Ncol cohesive ends and restriction enzyme sites for Smal, EcoRI, Clal and Hindlll was inserted into this unique Ncol site (pDB16) . The orientation of the linker with, respect to the TK gene was determined by restriction enzyme digests and polyacrylamide gel electrophoresis. pDB16 was then used as the backbone for insertion of foreign sequences within the FPV TK gene.

The Ecogpt gene from pSV2A-gρt has previously been cloned into ρBCB07 (29). A 900bp fragment, derived by EcoRI-Ahalll digestion, containing the Ecogpt gene with the vaccinia virus promoter P7.5 attached was cloned into pDB16 digested with EcoRI-Smal. The resulting plasmid pDB18 contains the TK gene interrupted by the Ecogpt gene under the control of the P7.5 promoter.

The influenza A/PR/8/34 haemagglutinin gene had been inserted previously into pBCB08 at the Hindlll site of the multiple cloning site (30). A Clal fragment from pBCB08/HA containing the PL11 promoter and HA gene was subcloned into pDB18 which had been digested with Clal and treated with calf intestinal alkaline phosphatase. This placed the influenza HA gene under the control of the PL11 promoter in tandem with the Ecogpt gene all contained within the FPV TK gene (pDB19/l and pDB19/6) (Fig.2).

In an attempt to increase the frequency of recombination by having larger flanking sequences, the Ecogpt in tandem with the HA gene was subcloned from pDB19/6 into the unique Ncol site of pDBl (Fig.3; pDB20). pDBl contains a 5.5kb EcoRI fragment of the FPV genome in which the TK gene is contained. To construct a general insertion vector, the

HA gene in pDB19/l was removed by Hindlll digestion and religation. pDB22 contains the FPV TK gene interrupted by the P7.5 promoter-Ecogpt gene plus the PL11 promoter. Attached to the PL11 promoter is a multiple cloning site with unique restriction enzyme sites for Smal, BamHI, Sail, Pstl and Hindlll (Fig.3). This vector is suitable for the insertion of a variety of genes into the FPV genome using the Ecogpt gene to select for the recombinants. Construction and characterization of FPV recombinants expressing influenza haemagglutinin.

Preliminary experiments showed that FPV plaquing and growth in CES cells was strongly inhibited by mycophenolic acid using the MXHAT selective conditions. This was similar to the inhibition of vaccinia virus by MXHAT (29) .

Insertion of the influenza HA gene into FPV in tandem with the Ecogpt gene was achieved using pDB20. Virus plaques which grew in the presence of MXHAT were plaque purified three times and confirmed as containing the Ecogpt and HA genes by dot blot hybridization. Subsequently recombinant FPVs were successfully constructed using pDB18 and ρDB19 which have shorter FPV flanking sequences.

Expression of influenza haemagglutinin by the recombinant FPV-HA was demonstrated by the binding of antibodies and 125I-protein A to plaques. Only plaques of FPV-HA bound antibodies to influenza virus whilst the wild type virus, FPV-M3, failed to do so. Neither virus bound antibodies from normal serum (Fig.4).

The structure of the recombinant, FPV-HA, genome was analysed by restriction enzyme digestion and Southern hybridization in comparison with the parent virus, FPV-M3 (Figs. 5 and 6). The 5.5kb EcoRI fragment which contains the-TK gene spans the junction of two Pstl fragments of 26kb and 13kb (Fig.5). The TK gene is contained wholly within the 26kb fragment. Pstl digestion of the FPV-M3 genome showed in ethidium bromide stained gels that the 26kb fragment was replaced by 23 and 6kb fragments. With Sail digestion a new 15.5kb fragment was present (Figs. 5 and 6) . The agarose gel separated fragments were transferred to Gene-Screen-Plus membrane (NEN) and successively hybridized with various 32P-labelled

DNA fragments (Fig.6). After each hybridization and autoradiography the 32P-labelled probe was removed

(as per manufacturers instructions) and the membrane hybridized with a different 32P-labelled fragment.

By insertion of the foreign DNA in ρDB20 into the FPV genome an additional Pstl site within the PL11 multiple cloning site would be present. Hybridization with the EcoRI, 5.5kb, fragment in pDBl showed that wild type pattern of 26 and 13kb fragments changed to new fragments of 23 and 6kb with the 13 kb fragment unchanged. The Sail pattern was also changed with a large unresolved fragment replaced by a smaller fragment and the 15.5kb fragment present in the ethidium bromide stained gel (Fig.5). A Sail site had been inserted as part of the multiple cloning site of the PL11 promoter. When hybridized with the HA or Ecogpt genes or the P7.5 promoter, the patterns were the same. As expected all hybridized with the new 6kb Pstl fragment and the large Sail fragment. None hybridized with the genome of the parent virus, FPV-M3. As predicted by the restriction enzyme map in Fig.5 the PL11 promoter hybridized with the 15.5kb Sail and the 23kb Pstl fragments.

The ethidium bromide stained gel and the hybridization patterns confirmed that all the foreign DNA elements in ρDB20 were inserted into the FPV genome in the expected position and orientation. Other restriction enzyme digests and hybridization analyses with FPV-M3 DNA using BamHI,EcoRI and Hindlll were also as expected. Response of poultry to recombinant FPVs

SPF chickens inoculated at 16 days of age with the FPV mild vaccina strain or FPV-M3 failed to develop haemagglutination inhibiting antibodies (HI) to the A/PR/8/34 influenza virus during the next 43 days. All birds developed a FPV lesion at the primary inoculation site. No lesions developed at the secondary stage inoculation site.

Chickens inoculated with the recombinant FPV-HA, developed a lesion at the inoculation site and HI antibodies to the influenza virus A/PR/8/34. Antibodies were detected on day 9 after inoculation and the titers increased thereafter. Maximum titers of 40 to 320 occured 29 days after inoculation and they remained stable till day 43. Following reinoculation at day 21 there was not a marked secondary antibody response, however, titers prior to this time were continuing to rise and consequently any secondary response may have been masked by the rising antibody response (Fig. 7).

EXAMPLE 2

This Example demonstrates that insertion of the Ecogpt gene into vaccinia virus under the control of a vaccinia virus promoter leads to vaccinia virus recombinants able to grow in the presence of MXHAT. When coupled in tandem with another gene of interest vaccinia virus recombinants can be constructed and positively selected carrying both genes. !π. Figures 9 to 11:

Figure 9 shows plaquing of vaccinia virus on CV-1 monolayers in the presence of MXHAT (1 or 25 μg/ml of mycophenolic acid). 1, 2 and 3, W-WR; 4, W-Ecogpt; 3, guanine at 25 μg/ml was able to reverse the inhibition of plaque formation by MXHAT.

Figure 10 shows construction of plasmids for the insertion of the Ecogpt gene into the TK gene of vaccinia virus. H, Hindlll; .B, BamHI; E, EcoRI; MCS, multiple cloning site; CIAP, calf intestinal alkaline phosphatase.

Figure 11 shows the effect of selection and moi at the recombination step on the output of recombinant viruses expressing the Ecogpt gene.

Monolayer cultures of CV-1 cells (10 6cell/25cm2 flask) were infected with W-WR at various moi's. Two hours later the cultures were transfected with CaPO. precipitated plasmid DNA (2μg of pGpt07/14 and 20μg of calf thymus DNA) . Eight hours later the medium was changed, with or without the addition of MXHAT selection. Six days later the cells were harvested and assayed on CV-1 monolayers in the presence or absence of MXHAT. The % of the output as recombinants is plotted on a log,_Q scale. no selection applied at recombination step selection applied at recombination step.

EXPERIMENTAL

(a) Inhibition of vaccinia virus plaquing by mycophenolic acid.

Mycophenolic acid has previously been used at 25μg/ml to inhibit mammalian cell growth and to select for cells carrying the Ecogpt gene (36,37). Preliminary experiments established that confluent monolayers of CV-1 cells used for the vaccinia virus plaque assay survived for more than 7 days in mycophenolic acid up to 25μg/ml. When mycophenolic acid (MXHAT) was tested against vaccinia virus on preformed monolayers of CV-1 cells, doses from 1 to 25μg/ml completely inhibited plaque formation and even at O.lμg/ml there was significant reduction in plaque size (Fig. 9). Inhibition of mammalian cell growth by mycophenolic acid is reversible by guanine. Addition of guanine,^" 25 μg/ml, to plaque assays in the presence of MXHAT (M from 0.1 to 25μg/ml) reversed the inhibition of plaque formation and plaque sizes approached normal (Fig. 9).

This showed that the mycophenolic acid was inhibiting vaccinia virus growth by acting as an inhibitor of inosine monophosphate dehydrogenase preventing the formation of xanthine monophosphate and therefore guanine monophosphate in a manner similar to its action on mammalian cells (37) . Mycophenolic acid alone failed to inhibit vaccinia virus plaque formation on CV-1 cells. HAT (hypoxanthine, aminopterine and thymidine) was required to inhibit de novo synthesis of inosine monophosphate to allow expression of the mycophenolic acid inhibition. HAT alone had no effect on vaccinia virus plaquing on CV-1 cells. The conditions used to inhibit completely vaccinia virus plaquing even at very high virus input were mycophenolic acid, 1 or 2.5μg/ml, xanthine, 250μg/ml, hypoxanthine, lσOμM, aminopterine, 0.4μM, thymidine, 30μM (MXHAT) . The cell monolayers do not require pretreatment with MXHAT for inhibition of virus plaquing and preformed monolayers maintain for long periods (7 to 10 days) under MXHAT selection. (b) Insertion of the Ecogpt gene into vaccinia virus

The Ecogpt gene and attached SV40 polyadenylation signals from pSV2A-gpt (36) was subcloned as a Hindlll-BamHI fragment into pUC . The translation initiation codon of the Ecogpt gene is located 200bρ from the Hindlll site. A unique Bglll site is located 120bp closer to the translation initiation codon (38) . To position the Ecogpt gene as close to the vaccinia virus promoter as possible, ^a Bglll-BamHI fragment was cloned into pBCB07 (39; Fig.10). Recombinant plasmids pGpt07/14 and 15 were selected by restriction enzyme digests as having the Ecogpt gene in the correct and incorrect orientation with respect to the vaccinia virus promoter and flanked by the TK gene sequences.

Only the plasmid pGρt07/14 having the Ecogpt gene in the correct orientation with respect to the vaccinia virus promoter was able to generate recombinant viruses which could grow in MXHAT. In a typical experiment, 0.1 to 0.2% of the output virus from the recombination step where selection was not applied at this step were recombinants. A single passage at low moi (0.01) from the original recombination step resulted in 10 to 30% of the output virus being recombinants. When MXHAT selection was applied at the recombination step, the proportion of recombinants in the output increased dramatically. At a moi of 1 the proportion of recombinants increased from 0.1% to 0.5%. When low moi's (0.01 and 0.001) were used and the incubation in MXHAT extended to 5 to 7 days for the first step, recombinants represented 10 to 40% of the output compared with the normal 0.1 to 0.2 (Fig.11; the total output of virus was reduced by 100 to 1000 fold).

The ability to positively select at the recombination and subsequent steps plus the absence of spontaneous background mutants greatly facilitates the construction and selection of recombinants. A recombinant vaccinia virus has been constructed using ρGpt07/14 and the insertion of the Ecogpt gene into the TK region confirmed by restriction enzyme analysis and Southern hybridization analysis. This recombinant, W-Ecogpt, is able to plaque under MXHAT on CV-1 monolayers whilst plaquing of the wild type virus, W-WR, is completely inhibited (Fig. 9). Large fragments of fowlpox virus genome, up to 20kb, have been inserted into vaccinia virus using the ρGpt07/14 vector as a selectable insertion vehicle. Additional genes of interest could be inserted and expressed in vaccinia virus by inserting another promoter plus gene into pGpt07 then using the Ecogpt gene to select for recombinants.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications which fall within its spirit and scope.

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Claims

CLAIMS :

1. Recombinant fowlpox virus or related avian poxvirus, characterised by the inclusion of foreign DNA in the virus genome,

2. Recombinant virus according to claim 1, wherein said foreign DNA sequence is inserted in a non-essential region of the virus genome.

3. Recombinant virus according to claim 2, wherein said foreign DNA is inserted in the TK gene of the virus or in virus DNA sequences controlling expression of the TK gene.

4. Recombinant virus according to claim 1, wherein said foreign DNA comprises a DNA sequence encoding an antigenic polypeptide.

5. Recombinant virus according to claim 4, wherein said foreign DNA comprises a DNA sequence encoding an antigen characteristic of an avian disease.

6. Recombinant virus according to claim 4 or claim 5 wherein said foreign DNA comprises a DNA sequence encoding more than one antigenic polypeptide.

7. Recombinant fowlpox virus for use as an avian disease vaccine characterised by the inclusion of a foreign DNA sequence encoding an antigen characteristic of the said avian disease, said foreign DNA sequence being inserted in the .region of the fowlpox virus commencing 279bp before and terminating 273bp after the central Xbal site of the nucleotide sequence shown in Fig.8, or in sequences controlling expression of this sequence.

8. Recombinant virus, according to any one of claims 1 to 7, wherein said foreign DNA includes the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (Ecogpt), coupled with one or more additional foreign genes.

9. Recombinant virus according to any one of claims 1 to 8, wherein said foreign DNA is under control of at least one poxvirus promoter.

10. Recombinant virus according to claim 9, wherein the or each poxvirus promoter is a fowlpox virus or related avian poxvirus or vaccinia virus promoter.

11. Recombinant virus according to claim 10, wherein the or each poxvirus promoter is selected from the vaccinia virus promoter sequences P7.5 and PL11.

12. Recombinant virus according to claim 8, wherein said foreign DNA includes the Ecogpt gene under control of the vaccinia virus P7.5 promoter, coupled with one or more additional foreign genes under control of the same or additional poxvirus promoters.

13. A method for the construction of recombinant fowlpox virus or related avian poxvirus, which comprises the introduction of foreign DNA into the TK gene of the virus or into virus DNA sequences controlling expression of the TK gene.

14. A method for inducing immunity in poultry to an avian disease, which comprises administering to said poultry an avian disease vaccine comprising recombinant fowlpox virus or related avian poxvirus characterised by inclusion of a foreign DNA sequence encoding an antigen characteristic of the said avian disease in the TK gene of the virus or in virus DNA sequences controlling expression of the TK gene.

15. A fowlpox or related avian poxvirus vaccine which comprises TK- fowlpox virus or related avian poxvirus.

16. Recombinant virus characterised, in that it has a foreign DNA sequence comprising the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (Ecogpt) coupled with a second gene inserted into a non-essential region of the virus genome.

17. Recombinant virus according to claim 16, wherein the virus is a poxvirus.

18. Recombinant poxvirus according to claim 14 or 15, wherein the poxvirus is vaccinia virus, fowlpox virus or Orf virus.

19. Recombinant virus according to claim 16, wherein the foreign DNA sequence is inserted into the TK gene of the virus.

20. Recombinant virus according to any one of claims 16 to 19, wherein said second gene is expressed as an antigenic polypeptide which is foreign to the virus.

21. Recombinant virus according to any one of claims 16 to 20, wherein in said foreign DNA sequence said Ecogpt gene is under control of a poxvirus promoter and said second gene is under control of the same or another poxvirus promoter.

22. Recombinant virus according to any one of claims 16 to 21, wherein said foreign DNA sequence contains one or more additional genes.

23. A method of constructing a recombinant virus which comprises inserting into a non-essential region of the virus genome, a foreign DNA sequence comprising the Ecogpt gene coupled with a second gene, and selecting the virus recombinants by their ability to reverse the inhibition of virus growth by mycophenolic acid.