AU621934B2 - Insertion of heterologous gene from a pathogen to produce a recombinant capripoxvirus - Google Patents

Description

OPI DATE 23/03/90 APPLN. 4 0 8 c P AOJP DATE 26/04/90 PCT NuMB P2/G 9 6 4 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 90/02190 C12N 15/86, A61K 39/275 //C12N 15/30, 15/31, A61K 39/002 Al A61K 39/02, 39/12, 39/295 (43) International Publication Date: 8 March 1990 (08.03.90) (C12N 15/00, C12R 1/91) (21) International Application Number: (22) International Filing Date: PCT/GB89/00965 18 August 1989 (18.08.89) Priority data: 8819836.1 20 August 1988 (20.08.88) GB (71) Applicant (for all designated States except US): INSTITUTE FOR ANIMAL HEALTH LIMITED [GB/GB]; Compton, Nr Newbury, Berkshire RG16 ONN (GB).

(72) Inventors; and Inventors/Applicants (for US only) BLACK, Donald, Norman [GB/GB]; BOSTOCK, Christopher, John [GB/ GB]; GERSHON, Paul, David [GB/GB]; AFRC Institute for Animal Health Limited, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 ONF (GB).

(74) Agent: BASSETT, Richard, Simon; Eric Potter Clarkson, 14 Oxford Street, Nottingham NG1 5BP (GB).

(81) Designated States: AT (European patent), AU, BE (European patent), BF (OAPI patent), BJ (OAPI patent), CF (OAPI patent), CG (OAPI patent), CH (European patent), CM (OAPI patent), DE (European patent), FR (European patent), GA (OAPI patent), GB (European patent), IT (European patent), LU (European patent), ML (OAPI patent), MR (OAPI patent), NL (European patent), SE (European patent), SN (OAPI patent), TD (OAPI patent), TG (OAPI patent), US.

Published With international search report.

Before the eipiration of the time limit for amending the claims and to be republished in the event of the receipt of amendments.

(54) Title: (57) Abstract "Insertion of a heterologous gene from a pathogen to produce a recombinant capripoxvirus"

I

A heterologous coding sequence, for example for an antigen associated with a viral or other disease, is inserted in a non-essential region of the capripoxvirus genome, for example the TK (thymidine kinase) coding region or the open reading frames designated herein as CT3C, CT4 or Q2.

A-c 14 1' The present invention relates to vaccines in which a viral vector is used to express a heterologous gene.

Capripoxvirus causes sheep pox and goat pox, diseases which can kill over 50% of young animals in those areas, principally Africa and the Middle and Far East, where the disease is enzootic. Different strains :i of the virus show a preference for either sheep or goats but none have so far been found to be totally speciesspecific. Some strains also infect, and cause lumpy skin o. disease in, cattle but (apart from buffalo) capripoxviruses do not infect any other domestic animals. A vaccine has been developed which comprises ai attenuated strain of the strain known as "the Kenya isolate" (Kitching, Rev. Sci. Tech. Off. Epiz., 1986, 503- 9 It is known that the vaccinia virus genome can be altered by insertion of a heterologous gene from a pathogen, such that the product of that gene will be expressed in infected cells. Protection against diseases Isl 2 associated with the pathogen has been induced in this way. A review of such work may be found in Mackett Smith, J. Gen. Virol. 67. 2067-2082 (1986).

At a U.N. Food and Agriculture Organisation meeting in Madrid in October 1986, Mowat disclosed that capripoxvirus represented a possible alternative to vaccinia as a vector for foot and mouth disease vaccines.

In the 1987 Annual Report of the Institute for Animal Health, Compton, (published April 1,988) Black and Gershon disclosed that work was in progress on the use of i: capripoxvirus as a vector for heterologous antigens and that the thymidine kinase gene was a candidate insertion S site.

o We have now shown that capripoxvirus can indeed be used as a vector for heterologous genes and that there are several possible insertion sites for such genes.

One aspect of the invention provides a recombinant capripoxvirus capable of infecting and replicating in capripoxvirus-susceptible cells and carrying at least one heterologous gene encoding an antigen of a pathogen such that the gene will be expressed during transcription and translation from the capripoxvirus genome in the said cell, i!

I-.

i 3 wherein the heterologous gene is inserted in one of the following regions of the capripoxvirus genome: the CT3C region, the C4 region, or the Q2 region.

By "thymidine kinase gene region" (the term is used below) we mean the genetic unit, including coding and non-coding sequences, for the expression of thymidine kinase.

By "CT3C region" and "CT4 region", we mean the respective regions of the capripox virus genome S identified by these designations in the attached figures, or variations thereof. By "Q2 region", we mean the approximately 2.4 kb fragment bounded by the respective HindIII sites approximately 6.6 and 9.0 kb from the right-hand end of the KS-1 isolate genome shown in Figure 1 and identified thereon as Q2, and variations thereof.

Such variations of any of these regions will include: minor polymorphic changes associated with different WO 90/02190 PCT/GB89/00965 strains of capripoxvirus; any other changes which might occur or be created in the nucleic acid of capripoxvirus but which do not alter the non-essential nature of the said region or the nature of the gene product encoded by that region; and any other nucleotide sequences which encode a gene product with substantially the same structure or function as the gene product of the said region or which correspond to the said region but are non-coding. In general, nucleotide sequences with at least 80% homology to the said regions are regarded as variations thereof. Preferably, the extent of homology is 90%, 95% or 99%.

By "antigen" we mean any structure, part thereof or mimic thereof associated with a pathogen and capable of giving rise to an immune response in a host when the immune system of that host is exposed to the antigen.

Preferably, the immune response is a protective response.

Thus, surface carbohydrates, glycoproteins and proteins of viruses and other pathogens are included in this definition, as are peptides which are homologous to part of such whole molecules, and peptides which mimic the tertiary structure of such molecules. In the case of antigens which are glycoproteins, the heterologous gene S. will encode they amino acid sequence of the glycoprotein i_

I

,WO 90/02190 PCT/GB89/00965 and/or one of more enzymes which cause appropriate glycosylation of that amino acid sequence. In the case of antigens which are carbohydrates, the heterologous gene or genes will encode one or more enzymes which catalyse the synthesis of such carbohydrates.

By selecting appropriate heterologous genes from other pathogens, it is possible to use the vectors of the invention in vaccines effective in preventing a wide variety of diseases in sheep, goats and cattle. The following is a non-exhaustive list of such diseases: Viral disease Rotavirus infection Bluetongue Ephemeral fever Foot-and-mouth disease Infectious bovine rhinotracheitis Infectious pustular vulvovaginitis Malignant catarrh Mucosal disease/bovine viral diarrhoea Border disease Rabies Host Sheep, cattle Sheep, goats, cattle Cattle Sheep, cattle, goats wild ruminants Cattle Cattle Sheep, cattle Cattle Sheep Sheep, cattle, I-s L ~-il PC/GB89/00965 WO 90/02190 Rift Valley fever Rinderpest Peste des petits ruminants Vesicular stomatitis Nairobi Sheep disease Scrapie Lentivirus infection Akabane Ricketsial disease Anaplasmosis Bovine petechial fever Heartwater Jembrana disease Enzootic abortion Bacterial disease Anthrax Brucella abortus infection Brucella melitensis infection Brucella ovis infection goats, wild ruminants Sheep, Cattle, goats, wild ruminants Cattle, goats Sheep, goats Sheep, goats, cattle Sheep Sheep Sheep, goats Cattle Host Cattle Cattle Cattle Cattle Sheep Host Sheep, goats, cattle, wild ruminants, buffalo Buffalo, cattle Cattle, sheep, goats, buffalo Sheep 46 Si i i i 11 IC- l** WO 90/02190 PCr/GB9/00965 Clostridium septicum infection blackleg malignant oedema Sheep, cattle, goats, braxy buffalo Clostridium chauvoei infection blackquarter Sheep, cattle, goats, buffalo Clostridium novyi (type A) infection Big head Sheep Clostridium perfringens Sheep, cattle, goats.

infection buffalo Clostridium novyi (type B) infection Black disease Sheep, cattle, goats Clostridium novyi (type D) infection Bacillary Haemoglobinuria Cattle, sheep, goats Clostridium perfringens (type B) infection Lamb dysentery Sheep Clostridium perfringens (type C) infection Sheep, cattle Clostridium perfringens (type D) infection- Pulpy kidney disease Sheep, goats Clostridium tetani infection Sheep, goats, cattle, Tetanus wild ruminants i i WO 90/02190 PPr/GB89/0096- Clostridium botulinum infection Botulism S] Mycoplasma agalactia infection Contagious agalactin S] Mycoplasma mycoides infection Contagious bovine pleuro- B pneumonia Contagious caprine pleuropneumonia S Pasteurella multocida infection Haemorrhagic septiceamia C Mycobacterium johnei infection Johne's disease C Leptospira sps. infections C Salmonella sps. infections C Mycobacterium tuberculosis C infection, Pasteurella sps. infection S heep, goats, cattle, wide ruminants heep, goats uffalo, cattle, wild ruminants heep, goats attle, buffalo attle, sheep, buffalo attle, sheep, buffalo attle, sheep, buffalo attle, sheep, buffalo heep, goats goats, goats, goats, goats,

'I'

S''

WO 90/02190 PC/G89/00965 Protozoal disease Babesia sps. infections Theileria sps. infections Toxoplasma gondii infection Trypanosoma sps. infections Helminth disease Bunostomum sps. infection Chabertia sps. infection Taenia sps. infection Trichuris sps. infection Dicrocoelium infection Dictyocaulus sps. infection Echinococcus sps. infection Fasciola sps. infection Host Cattle, buffalo, sheep, goats, wild ruminants Cattle, buffalo, sheep, goats, wild ruminants Sheep Cattle, buffalo, sheep, goats, wild ruminants Host Sheep, goats, cattle, buffalo, wild ruminants Sheep, goats, cattle, buffalo, wild ruminants Sheep, goats, cattle, buffalo, wild ruminants Sheep, goats, cattle, buffalo, wild ruminants Sheep, goats, cattle, buffalo, wild ruminants Sheep, goats, cattle, buffalo, wild ruminants Sheep, goats, cattle, buffalo, wild ruminants Sheep, goats, cattle, buffalo, wild ruminants i WO 90/02190 Haemonchus sps. infection Oesophagostomum sps. infection PCT/GB89/00965 Onchocerca sps.

Ostertagia sps.

infection infection Paramphistoma sps. infection Schistosoma sps. infection Stephanofilaria sps. infection Strongylus sps. infection Thelazia sps. infection Trichostrongylus sps. infection Cooperia sps. infection Nematodirus sps. infection Sheep, goats, cattle, buffalo, wild ruminants Sheep, goats, cattle, buffalo, wild ruminants Cattle, buffalo Sheep, goats, cattle, buffalo, wild ruminants Sheep, goats, cattle, buffalo, wild ruminants Sheep, goats, cattle, buffalo, wild ruminants Cattle, buffalo Sheep, goats, cattle, buffalo, wild ruminants Sheep, goats, cattle, buffalo, wild ruminants Sheep, goats, cattle, buffalo, wild ruminants Sheep, goats, cattle, buffalo, wild ruminants Sheep, goats, cattle, buffalo, wild ruminants

P

ii &i r, L- B *I WO90/02190 PCT/GB89/00965 ii Genes have already been isolated for antigens in those viruses marked with an asterisk or a cross.

The recombinant viruses of the invention may be prepared by constructing a DNA construct comprising the heterologous gene and two sequences which flank the heterologous gene and which are homologous to respective parts of the capripoxvirus DNA, such that recombination of the construct with capripoxvirus may occur, (ii) infecting cells with capripoxvirus and introducing the said construct into the said cells, (iii) screening the infected cells for recombinant capripoxvirus, (iv) isolating the recombinants from step (iii) and (v) growing the isolated viruses in a suitable host.

The construct may comprise a promoter for the heterologous gene, which will be any promoter which is functional in the cytoplasm of the capripoxvirus-infected cells. Thus, the promoter may interact with a polymerase which is also heterologous to both the capripoxvirus and the infected cell and which is encoded by another heterologous gene which is inserted in the recombinant capripoxvirus, as with the bacterial T7 system. Suitably, the promoter is derived from capripoxvirus itself but it may alternatively be derived from vaccinia virus, for i l t WO 90/02190 PCT/GB89/00965 12 example it may be the promoter for the P7.5 protein (Mackett et al (1984), J. Virol. 49, 857-864; and Mackett Smith, loc. cit.). Alternatively, the construct may not comprise a promoter at all but is so constructed as to recombine with the capripoxvirus at a site close to, and in correct reading frame with, an endogenous capripoxvirus promoter.

The flanking sequences may be synthesised by reference to the sequence of the insertion region or may be obtained from such a region and cloned. Typically, a length of about 100 nucleotides for each flanking sequence is sufficient to ensure recombination, but 120 nucleotides or more may be used.

In step the infection of the cells with the capripoxvirus may be before, after or at the same time as introduction of the DNA construct. The cells may be any which are permissive for capripoxvirus, such as most ovine, bovine and caprine derived cells, for example lamb testis, bovine palate, or ovine kidney cells. The DNA construct is preferably provided as a plasmid. The plasmid may be based on commercially available bacterial plasmids, such as pUC9, which may be cloned in bacteria and which have suitable markers for selection, for WO 90/02190 PCT/GPS9/00965 13 example Ampr. Transfection of the capripoxvirussusceptible cells with the plasmid may be facilitated by known techniques involving calcium phosphate or electroporosis.

The plasmid or other transfection vehicle is so chosen as to be non-infectious. Following introduction of the transfection vehicle into the host cell in step (ii), the vehicle is not propagated between cells. Hence, screening in step (iii) for the product of the heterologous gene effectively screens for recombinant viruses. Such screening may, for example, comprise an immunological assay for the antigen expressed by that gene or nucleotide hybridisation using sequences homologous to the heterologous gene. In one screening approach, the heterologous gene is in the first instance the E.coli beta-galactosidase gene (lac thus allowing screening in step (iii) by the well-known technique using "X-gal" (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) in an agar overlay on the cell culture. Blue plaques are formed by recombinants which express the lacZ Sgene. Insertion of a further heterologous gene into the recombinant lac Z region, with concomitant deletion of the beta-galactosidase function, then allows selection by isolating colourless plaques against the parental blue 1'I.

1 1 WO 90/02190 PCT/GB89/00965 14 plaques. This approach may be useful where a suitable assay for the product of the second heterologous gene is not available.

Step (iv) will usually involve successive rounds of purification from plaques or cell-foci of infection, or end-point dilutions, and screening for recombinants in known ways. Amplification may be desirable during such procedures.

In step the host may be any suitable cell line or, less preferably, any suitable living animal. Lamb testis and the other cell lines mentioned above are suitable.

The recombinant viruses may then be used to make vaccines in known ways, for example as described in Kitching, loc.cit. The virus is preferably included in the vaccine in an attenuated form, rather than killed.

The vaccines may be used to prevent the particular Sdisease or diseases associated with the pathogen(s) of which an antigen or antigens are encoded by the virus of S the invention, and may be used in sheep, goats, cattle (B.os bovis and Bos indicus) buffalo, llamas, alpacas and

I.-

*L i i P .i~i I WO 90/02190 PPr/G9/00965 S. vicunas. Generally speaking, the vaccines will be most suitable for use in areas where capripoxvirus-associated diseases are endemic and/or where the animals have all been vaccinated against capripoxvirus.

The vaccines may be used to prevent the particular disease or diseases associated with the pathogen(s) of which an antigen or antigens are encoded by the virus of the invention, and may be used in sheep, goats, cattle (Bos bovis and Bos indicus), buffalo, llamas, alpacas and vicunas. Generally speaking, the vaccines will be most suitable for use in areas where capripoxvirus-associated diseases are endemic and/or where the animals have all be vacpinated against capripoxvirus.

The vaccines may be administered by any suitable route, including intra-nasally (as a spray), orally (for example in the feed or drinking water), intra-muscularly, sub-cutaneously, intra-venously or intra-dermally.

However, experience with existing capripoxvirus vaccines shows that the intra-dermal route is not preferred and that the sub-cutaneous or intra-muscular routes are preferred.

WO 90/02190 PCT/GB89/00965 16 For at least some time, typically 10 days, after the vaccine is administered to the animal, especially when the virus is attenuated rather than killed, the virus will be detectable in the animal's tissues, thus enabling an animal which has been vaccinated in accordance with the invention to be identified. The viral vectors of the invention may further include markers to aid such identification, if desired. In addition, the immunity of an animal which has been vaccinated in accordance with the invention may be distinguished from that of an animal which has been exposed to the corresponding pathogen since the former will include antibodies only to the particular antigen expressed by the modified viral genome and not to other antigens of the pathogen.

The invention will now be illustrated by way of the following non-limiting ex~nples, with reference to the accompanying drawings, in which:- Figure 1 shows the distribution of HindIII ,s restriction enzyme recognition sites on the genome of capripoxvirus strain KS-1, together with a scale rprpresenting length in numbers of kilobase pairs; o

V

I: i. WO 90/02190 PC1F/GB89/00965 3 c ,d:T;:i Figure 2 shows the nucleotide sequence of the S fragment identified in Figure 1, with the region labelled as CF8 corresponding to the thymidine kinase (TK) region; Figure 3 shows the sequence from the terminal region of the genome of capripoxvirus isolate KS-1, reading in a telomere-proximal telomere-distal direction. The terminal HindIII site (AAGCTT) of the genome occurs in the sequence. This site, and the Sau3Al (GATC) site defining the proximal end of clone pPG64, are underlined and emboldened; and Figure 4 shows the distribution of stop-codons in the 6 possible reading frames for the sequence between approximately 700 and 2,400 bp from the apex of the terminal hairpin loops of capripoxvirus strain KS-1 DNA, the telomere proximal and distal ends bring on the left and right, respectively'.

EXAMPLE 1: Insertion into the TK region The thymidine kinase (TK) gene of capripoxvirus was localized -on the genome of isolate KS-1 (previously referred to as 'Kenya Sheep and Goat Pox') as described

L

-i 1 WO 90/02190 Pcr/GB89/00965 18 in reference 1. The gene was found to be present within HindIII fragment S (as shown on Figure and HindIII S was completely sequenced.

To insert foreign DNA into the TK gene of capripoxvirus, we used a variation of the previously described method (Mackett Smith, loc. cit.) of: cloning a construct comprising the genomic region containing the candidate insertion site in a bacterial plasmid and (ii) a foreign gene attached to a poxvirus promoter at the site in the plasmid clone equivalent to the intended insertion site in the virus genome; and transfecting the recombinant plasmid into poxvirus infected cells.

In the experiments performed here, the genomic region containing the intended insertion site was the KS- 1 genomic DNA fragment HindIII S, which was cloned in the plasmid vector pUC9. The plasmid was linearized by incubating with the restriction enzyme KpnI, for which a single site is present in the clone, and is located within the KS-1 TK gene.

The bacterial lacZ gene coupled to the vaccinia virus promoter P7.5 (P7.5-lacZ), was inserted at the KonI Ssite in the above plasmid using T4 ligase, after cleaving I. I 1 1 1 ill 1 1 11 17 7 Z i7 1 z' 1 L/ 1 1 1 11 1 i- I i ii-: WO 90/02190 PCT/G89/00965 19 from another plasmid with the restriction enzyme BamHI, and treating both the BamHI and the KpnI fragments with Klenow enzyme. The resulting plasmid was named Lamb testis (LT) cell cultures infected with capripoxvirus isolate KS-1 were transfected with the and the cultures were incubated at 37oC for days. Further LT cell cultures were infected with the harvested virus and after seven days incubation these cultures were overlaid with low gelling temperature (LGT) agarose containing the substrate X-gal (5-bromo-4-chloro- 3-indolyl-beta-D-galactopyranoside) ('X-gal overlay').

After a further 24 hours blue foci were visualised through the X-gal overlay, and virus was picked through the agarose and subjected to further rounds of plaque purification.

EXAMPLE 2: Identification of Further Insertional Sites A library of capripoxvirus KS-1 DNA in plasmid pPG2 was produced by partial digestion of the capripoxvirus DNA with Sau3A, and cloning of the resulting fragments into BamH1 linearised pPG2. pPG2 was derived from the commercially available pUC9 by removal of the multiple cloning site by digestion with HindIII and EcoR1, filling

I

i i WO '90/02190 P(7r/B89/00965 in of the single-stranded ends with Klenow enzyme, and ligation (using T4 ligase) of a synthetic BamHI linker (GGGATCCC) to recircularise the plasmid DNA. The ligation mixture was used to transform E.coli strain TG1 and the bacteria plated onto LB agar containing ampicillin.

Individual bacterial colonies were picked at random and their plasmid DNAs prepared. Plasmid DNas containing HindIII sites were identified by digestion with HindIII and analysis by agarose gel electrophoresis. 40 clones carrying HindIII sites located at different positions on the capripoxvirus genome were digested with HindIII and the HindIII P7.5-lacZ fragment was inserted into each using T4 ligase to create a series of trial insertional vectors for at least 10 different sites in the capripoxvirus genome. Following cloning in E.coli and preparation of purified plasmid DNA, each trial insertional vector was transfected into capripoxvirus KS-1 infected lamb testis cells and the presence of blue plaques/foci was screened for. The presence of blue plaques/foci identified those insertional vectors carrying sequences which directed insertion by recombination at sites whick are not essential for capripoxvirus replication and infection in lamb testis cells. In this way we have identified three HindIII sites within non-essential regions of capripox-virus DNA into which heterologous DNA 1 1 1 1 1 1 1 1

I

K 1 1 1 1 1 1 1 1 1 WO 90/02190 PCT/GB89/00965 21 can be inserted.

This represents a general method for identifying non-essential insertion sites in the capripoxvirus genome. Other restriction enzymes may be used instead of HindIII, preferably those which (for the capripoxvirus genome) cut at about the same frequency, namely about times, preferably about 30 times. Infrequent cutting results in insertion sites being missed, whereas too frequent cutting yields fragments which are too small to be practicable. Generally, one would use enzymes which recognise a sequence of 5 to 7 nucleotides, preferably 6.

However, as the capripoxvirus genome is rich in A's and T's, an enzyme recognising a 4-nucleotide sequence rich in G's or C's might also be suitable.

Sau3A is a suitable enzyme for the initial digestion as it recognises frequently occurring sites (about 600, i.e. an average distribution of about one per 250 a partial, rather than complete, digest then yields virtually random, conveniently-sized fragments likely to contain a HindIII site. Other enzymes, such as HaeIII, may be used for differing frequencies of occurrence of INTERNATIONAL SEARCH REPORT -Intefrmtional Application No PCT/GB 89/00965 O i a- n_ y--a--l l, I- ULASSIFICATION1 OF SUBJECTt MAXTTER(it seveal classfiction symbols 8001Y, indicate: all) 6 According to International Patent Classification (IPC) or to both National Clasification and IPC I

I

WO 90/02190 PCT/GB89/00965 the recognition sites. The partial digests may be pooled.

The pool itself may be cloned or one may select a particular size class of fragment for cloning.

EXAMPLE 3: Insertion into the CT4 Region The work outlined in this Example is based on clones covering a region within the terminal repeats of the genome of capripoxvirus isolate KS-1. This region covers two open reading frames (ORFs) named CT3C and CT4. Figure 3 shows the 1651 b.p. nucleotide sequence of the above mentioned region. Figure 4 shows the positions of the stop codons in the sequence. ORFs CT3C and CT4 are marked; CT3C lies between nucleotide 252 and.647 and CT4 lies between nucleotide 839 and 1621 in this sequence.

We constructed a plasmid, pPG64, to enable to be inserted within CT3C; more specifically at the Hind III site between nucleotides 561 and 566 of the sequence shown in Figure 3. This HindIII site divides HindIII fragments W1 and 11 at the left-hand end of the genome and fragments F and W2 at the right-hand end (see Figure Clone pPG64 was made by ligating a Sau3A partial digest fragment of KS-1 DNA of size 1648 starting at nucleotide 440 on the sequence shown in Figure 3, in a d if' Fr :u 1 WO 90/02190 PCT/GB89/00965 23 derivative of the commercially available plasmid cloning vehicle pUC9, named pPG2 (see Example The resulting plasmid, in which P7.5-lacZ is cloned at the HindIII site of pPG64, is called Recombinant capripoxvirus KS-1 was generated using by the method described in the last paragraph of Example 1. In the experiments using pPG65, virus obtained from blue viral foci was propagated on LT cells, and gave a further round of blue foci. These in turn were propagated by several additional rounds of plaque purification and shown to maintain the ability to express blue foci.

The capripoxvirus isolates used in the work described herein are typical of wild-type capripoxvirus and the techniques describeed and claimed herein do nut depend upon the particular strain or isolate which is used. Other isolates may have minor variations in their genomes, but it will be well within the capabilities of the man skilled in the art to adapt the techniques to .4 such other isolates. For example, it is believed that the region of another isolate (InS; Gershon Black (1987) Virol. 160, 473-476) which corresponds to the ORF in KS-1 l •j i WO 90/02190 PCT/GB89/00965 labelled herein as CT4 is a non-coding region. Such a region would clearly be a suitable candidate for insertion of the heterologous gene.

Claims (7)

1. ai capripoxvirus capable of infecting and replicating in capripoxvirus-susceptible cells and carrying at least one heterologous gene encoding an antigen of a pathogen such that the gene will be expressed during transcription and translation from the capripoxvirus genome in the said cell wherein at least one said heterologous gene is inserted in one of the following regions of the capripoxvirus genome: the CT3C region, the CT4 region, or the Q2 region the regions being as defined herein.

2. A capripoxvirus according to Claim 1 wherein the antigen is associated with bluetongue disease, ephemeral fever, foot-and-mouth disease, mucosal disease, border disease, rabies, Rift Valley fever, rinderpest, peste des petits ruminants, vesicular stomatitis, Nairobi Sheep AYc-i MLte 4rUnitedHF nirt I I J-r-- 1 i rt L.JBS T I T S] "9 J:.i 7 1990 19 07 26 disease, lentivirus infections, trypanosomiasis, theileriosis, clostridial infections, tuberculosis, haemonchiosis, schistosomiasis or Brucella infections.

3. A vaccine comprising a capripoxvirus according to any one of the preceding claims and a carrier and/or adjuvant.

4. A vaccine according to Claim 3 wherein the vaccine is suitable for sub-cutaneous delivery. A process for preparing a capripoxvirus according to Claim 1 or 2 by constructing a DNA construct comprising the heterologous gene and two sequences which flank the heterologous gene and which are homologous to respective parts of the capripoxvirus DNA such that recombination of the construct with capripox-virus may occur and (ii) infecting cells with capripox-virus and introducing the said construct into the said cells, (iii) screening the infected cells for recombinant capripoxvirus, (iv) isolating the recombinants from step (iii) and growing the isolated viruses in a suitable host. ,PC f ot_ 27

6. A process according to Claim 5 wherein the construct comprises a promoter for the heterologous gene, the promoter being derived from capripoxvirus.

7. A method of treating or preventing pathogen-caused disease in cattle, buffalo, sheep or goats comprising administering to the cattle, buffalo, sheep or goats a non-toxic disease-protective amount of vaccine according to Claim 3 or 4. I S j 8. A cow, buffalo, sheep or goat treated by the method j of Claim 7. Dated this 7th day of January 1992 INSTITUTE FOR ANIMAL HEALTH LIMITED S. By their Patent Attorneys GRIFFITH HACK CO L 1 'I WO 90/02190 PCr/GB89/00965 1/4 iI Pi Wl Y ElU DI 11 11 1 1 1 R u P2 R TI IBH 82 S Z 18 I.E OIL D2 C I I I 1 Q2 6 12 E2 K 3T2 F142, I I I I I I 1 1 8 18 29 38 48 58 68 78 88 98 .1.8 118 129 139 140 158 kb I I I I I I I I I I I-I I I I I 'Ffgure 1 C F6 49r( ;F7. II D If I AAI;C-TlTTTTAT-TA(GOTTA(:(:T'AATA(:TAT(2 IA'(-AI(AIfA:IA'ATAAAA(AA(GTA-AIAAAAT(AT;T:'TiAATCIAArAC(A; Q) V 2 I I I H Y L T I F 1, E If 1 S F F HC Y 1. S E Q O 0 K T -A if ND 1 1 T I V T Y1 I. F' r 121 CAAC;TAITCTATTAArrATATTCTTACAAAATI:A1 (;A('CCTTTTTAAArIACT1AI:A(;AACAA2A1 I2ATC.AAA(CA((:AT(CT(ICIATATC((;AAACTAT'TGTAArATATTI'AAA 7 1'TT-TT 241 ATTUTCATTG-TTAATTAC)ATCAAAC(-ATAAA1 TArA(UTCCA VAI2U;TTAI rA rTATC2AA:CACTGT(:T'CAACAATOGTAAAACAT'TArTT(ATTTITTC:AA1 ATU;AAAAATITTT'AGU(A1 KIT F 11 IC 1 V I N 1 I, Ii H C) 1 T V N$ H C Y L S; 1 F V T T I, If 11 1. C HC 1, 1. F 1. E 5 P E 361 ATITVAATAACA TTCCTATAAATATAC-TAAATFAAAI,:AAAI AAC I UTAAA1 AAA(;IAc rTATCAC;AT1TTTTAC(;ArCATUATGA(;ATTAAAAAAA(AACI1TTTTTTAAATCACCA;A r I T Y I D) P fl It O P1-T F 1. N1 I 1. 5 1 1 11 E K I' 0 y G) y I i 481 CCGCATAACATATAT-AU-ACCC-TAC;AAAA(;ATC:CAACATI TrTAAACATT FTAI1CAA-IATTCW(-AcrAAAAATAATTUAAC-AAATATTTTl TTITAAAAAAAAATUU(ACTATC.GATATA-1AC-A L I I G P H F S G K 5 T E 1. 1i I I V n Yi Q I A Q) Y H C C V V K y U K 0 1 II y G N S V Y T 11 0 N Nt 11 V 5 A I S T T 1, L. Y C V V D K If I N F D) I I C I D C G 0 F F 721 TA-CCT-TTC-CTAATACTTTTCAACAACTATTT;~TCTTAA-ACA1T(AATTC!TTCAG~GCAT~ K0D I V S F S IF IfIfAN ?I ifCK I I I I A A L D S T F 9 K R K F N1 t t K LI P FL 841 TACTTCACTTCGAAAUCATTOAAAATAATUTCCAAACCTTACAArATATAAATAATAACT S E H V T K L Nt A V C If E C Y K 0 A A F 5 K Rl I T H E K K I E L. I G r, K K K Y K 96 1 -CCAA~TAAATAAC;CAGAOATITTAGCCUATTCACGACCAAAAGAAAACCTOOOAGAAAAA CF-8al S V C R K C Y F L E M G JIn If K L 0 I L L V S E Nf I, A L H Nt V E L L R G 0 S Y G C T I NM I R V N 0QC) K K L 0 F I I L n P F V T E V R N1 V RKH I Nt If V C N) C; V V 1 0 T T L I I( K S F Y E E V Y 132 1 ATATAATG(CCAT;AAAGArATTTAAATATTGAGACCTTTTT'ATCAATACAAACTTACAGA-AIT S S S V T V F Q N1 T T V E F r s 0 T S K K Y K E K Y F I V It I N T I X R Y Y F 1 144 1 CTAC-TACCTTTAATCACTAATTTGATCATAAAAAAAGAACCTGTAAAAACTAGTTTAGAA K D S Il 11 T C I N F E S P' I S 0 Y 0 Q V N Y L K D Y I N I S D 0 Y Y L. Y D A C D 156 1 ArTAAATAAGAAATTATATAAT(ATT-ACAAATATAAGTAAAAAA(TATATTACGAAGCTCT D C I I S 5 V 0 0 0 Nt D N A D D 0 E E 0 0 D E V N1 D 1 E 0 D Y E 1681 Tc-ACT~-ACAc TACAAIf TATCG G GACGAACAG~A(AGTA AAA (CTCCAr TATTTT~AAACAT; CF9 11 E A V 5 11 0 K F F I] Y F D E I D Nt E L F V 0 F K T S 1801 TAAATAAATAAATTCTTAAT;A(C(TTTTGAAACATACATTAAATATAGATGAAACCAACTAA E K F K H L F Y Q G 9 1. K I, 1 I C E L F FI S R U~ C) I 11 I L 0 G C T I V Y V G 1921 AAAcrAAAATCAACA;;CATAATCTTTT;ATTTTCGGAGTCAGCTGAATOTOTCCAATTTTO S A F G T 11 1 L it I C If F 1, s it I' V I L I D G Ri Q If D T I LIfN G L n 0 2041 ATCACCTCCACGAACACATATTAAATACTTAACACATIrfATTTTTTATCTATCCCGA T1ACT'ATAAATC(ATATTAATAATCACCACAACATCACACAATTTTAAATGCGCTrACACA v T L I T K F V V E 8 Y I PI V L K K Q) L Y Q 5 K I V L, I SD V R S K nC C C V K P' S T F 0 L L S N1 Y A 1, Q I1 1 if V S J It P A A 5 S L K W II C F F 11 0 9 K~ H IC F 2281 TAGCA CATTTU;ATTTATTAA CCAATTAC(;CA TTACAA AATA TA ATOCTAACGTA TAT TAA, ~ACCACIACATCTAGTTTA AAAT-(CCATUTCCATTCCCTUACCA A Tr(.(TA AA ACA TTT V IFP 11 0 NtE K N 1 1' F Al K HY S A K IV N Nsi I y SC N f I R 1, 240 1 CTATATTCCTCACCOAAATOAAATC;CTTCAACCATTTC;CCCCTAAATATTCTCCAGAAATAGTTAATAATATTTATACCCTAATCCTATAAACTT J L WO 90/02190 PCT/GB89/00965 3/4 GAT~ CC.ATTTC:C:AAAT ACAPAC TC;A GCeC:T C'C:TTTTTI-CAAAGPATTC:A AAAA CT A :,ACACC:TTTC:CACiC:AACC:Tr-CC:TC4AC:GCAAA.OTGC:C.ACTCATCAinC:C* A 'A,.C:AATAATC:A'CAATGGC:CCTTCTCATATC--ATC:ATC:ATC:TCAP-AAACZTT GTTTC:GGTP AACA TAGTT(C: C:CGAACA C:ATC:TTC4TAAAATCiAAC: cGiCi A m-AACTC:TCTTTTAACCC:CAAC:TTCTTATTTC:C:TCTCC:TTATTTTT:,AAA TTATTCAAC:C:ATATTC:ATC:ATC:CTTTCAAC:TAAT-C:TTTTCTAGCTC-4GTT C:C:TTTATTGTGTTTTCAo TTA"C:AGTACITATTr_:GTAGTGTTTTTT,.AATOGA TTAGCC4AATTTTAA6-TAAMAATC:'AAAAAC:TC:C:ATTA 1TC:ATTTC:-AA'TATTTAT ATCCCGr:TTCTATCTTTTTTATCTAATAC:ACTTAATC4TAGATC ATTATTTA TTTC:TTTAC:TTTCTATAGCTTTGTG-ATTAGTGTTTATATTGTCTTCGATT TTTTC:AT CCTTAT C:CAAGAC:AC AA TC:GCAACCCGATTTAGGTTTC:CAAA -C:AT GAAC4CAGAT AAGCTTTTCATTC4AA AC:A2TTAAl-TAATGAATA-C:AAA"-"CTAT ATATAATTAAATTACA!TAAT:TACTATATAAAAAAT CA:AACATACAA AC:TTTAGCCTAAGCCTATTAATG'ACTACTTG-CCAA'-AAT,_C:TTTTATTCATA'" TATC:AC:AAATrCTAAAA TCTTTA GTAT AAGAACTAATGiTCCCTC-TTACAtIC- ATC:TCC:AT A TAC:TAAACATT ATTTTATC4AA TTAATGC:AA~aAAT C:C:A C:C:AC4--- A'C:TAAAAAAAAACL:CiAAAC:,TC:C:TAACTTTC:TTATTC:ATAATTTA'"TA2TAT OT GA&,AAACAAAA T AAA T ATT A TAT C G TC:TTTT 4T AT AAC:AA T COT A TACTTCATTATGAT'3AAATATTTTTTTAATATAATTATGCTGATGTTC:AT AATATTTTTTTC:TAT:AAA3TTTCC:C:.AC:AC:TCiC:AC:CTTTTTA.AA TAATTT AC:A TTTC:AC:TAAA TTTTAC:AGC:TCAC4CAA TC:T ATTTTA TTA TT AAA C* C AAAA,-ATrTTTTTTC:C:TGTA3C4ATATA-.'TATTTTTTTTCACATTCATTTC:TA C:-TATAAC:AC:TTGAAC:TTAC:ATGTTTTAC:AC:A'ACTA~ATATC:TCTTC4TTATA AC:"1AAATTC:TAA T CA T AATC:TTTAAAAC:TTACA.C:TCT CAAC CT A TG A TTT C:ATTTCTGTAGTTAC:TTGATC:ACAAAGAAATTCAC:-ATCATATTC:TC:TTA GC:AC:ACTGT CCC:AGCCCAATAC: AA T CTTA TATTACTGCTTC AT ATTTr CC- A TA TAT ACAT ATTTAC: C:C:AATCCATA AA CTGCTTTC--TTAAC C: T AT C:TAA TATAAAGiTTGiTTTTGTCTTTCTTC:TTTCCiAACAATCATC*ATC--TC;AAA TTTTAAA TC:CA TTT C:C: CTC:TC--TT AG AA'.4TCTC.CAGCCTCCTTGiAATTTTA TAA CAGC:CAGTTTT AA T CCT CT A AAA TTTT CTGTTTGATACAT AAT CC: TAC:TTCGATTTC:CC:ATTTTATTTTCTTCTTC-CTCCATCTCTTTATTC ATATT-CCGC:TTACAC:TAC:TACTAAAAAT3AAI MTAGTTCA-AAAAGAAArATG AACAAAC TTA ATCATCAC:ATA TTGG-T GTTC TTTTCAC: GTA TTCACCGA C: Figure 3 Nor-- WO 90/02190 W090/2190PCI'/GB89/00965 4/4 Sequence length =1649 nt Full scale =1649 nt Left end inset =8 nt *31 1 111 1 1 11 1 11 111 1 1 11111 1 1 111 11 HI 111 1 j TFFT -i I I I II~I I iI i I 8588 A88 1588 n C-r A 1111 IIIIII I Ill I IIIHIIIIII I .IIII II I I II III II I IN11 1 1 1 1 1 1 1 1 3' L iq /7? Figure 4 INTERNATIONAL SEARCH REPORT International Application No PCT /GB 89 /0096 L. CLASSIFICATION OF SUBJECT MATTER (if several classification symbols apply, indicate all) According to International Patent Classification UIPC) or to both National Classification and IPC C 12 N 15/86, A 61 K 39/275,//C 12 N 15/30, C 12 N 15/31, *P :A 61 K 39/002, A 61 K 39/02, A 61 K 39/12, A 61 K 39/295, If. FIELDS SEARCHED Minimum Documentation Searched Classification System ICasfcto yol 1PC 5 C 12 N Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Fields Searched 4

111. -DOCUMENT', CONSIDERED TO BE RELEVANT' Category Citation of Document. 11 with Indication, where appropriate, of the relevant passages %a Relevant to Claim No. 13 X Annual Report of the Institute for Animal 1,4-6 Health of the Agricultural and Food Research Council, 1987, Institute for Animal Health (Compton, Newbury GB), D.N. Black: "Virus vectors page 58, see page 58, columrn 1, line 24 column 2, line 29 cited in the application A J. Gen. Virol., vol. 67, 1986 1 D.B. Boyle et al.: "Identification and cloning of the fowlpox virus thymidine kinase gene using vaccinia virus", pages 1591-1600, see the whole article A Gene, vol. 47, no. 2/3, 1986, Elsevier Science Publishers B.V. (Biomedical Division), (Amsterdam, NL), D. Panicali et al.: "'Vaccinia virus *Soogial categories of cited documenta: I* later document published after the International filing date document defining the general state of the art which is not orpirt0aeadntI ofitwt h plcto u consderd tobe f paicusr rlevncecited to undestand the principle oir theory underlying the conidre tbeopticirelaneInvention eler document but published on or after, the international document of particular relevance;, the claimed invention filing date cannot to considered novel or cannot be conridered to document Which may throw doubts on priority cilim(s) or Involve an Inventive atop which is cited to stablish the publication date of another document of particular relevance;' the claimed Invention citation or other special reason (as specified) cannot be considered to Involve an Inventive step when the document referring to an oral disclosure,. use, exhibition or document Is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled 11P. document published prior to the international filing date but In the art. later than the priority date claimed document member, of the same patent family IV. CERTIFICATION_____________ Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 23rd November 1989 28~ International Searching Authority S :;atre0 or6z-*d or EUROPEAN PATENT OFFICE V L ROSSI Form PCTIISAt21O (second sheet) (January iNS) I INTERNATIONAL SEARCH REPORT PCT/GB 89/00965 Intern~ational Acolicatlon No 1. CLASSIFICATION OF SUBJECT MATTER (it several classification symbol$ apply, indicate all) According to International Patent Classification (IPC) or to bath National Classification and IPC iPC 5 (C 12 N 15/00; C 12 R. 1:91) II. FIELDS SEARCHED Minimum Documentation Searched Classification System IClassirication Symbols 1p Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Fields Searched a 111. DOCUMENTS CONSIDERED TO BE RELEVANT' Category ICitation of Document, 11 with Indication, where appropriate, of the relevant pasages 12 Relevant to Claim No." vectors utilizing the beta-galactosidase assay for rapid selection of recombinant viruses and measurement of gene expres- sion", pages 193-199, see the whole article A Virology, vol. 164, 1988, Academic Press Inc., P.D. Gershon et al.: "A comparison of the genomes of capripoxvirus isolates of sheep, goats, and- cattle", pages 3 41-3 49 A Virology, vol. 160, 1987, Academic Press Inc., P.D. Gershon et al.: "Physical characterization of the genome of a cattle isolate of capripoxvirus", pages 473-476 A Trends in Biotechnology, vol. 6, no. 2, 'Special categories of cited documents: is IT" later document published after the lnternational filing date ocuentdefiingthegenral tat attheart hic isnotor priority date and not in Conflict with the application but A"dc nside in h b ealsae of particularirelevance cited ito understand the principle or theory underlying the consdere tobe o paticuar elevnceInvertilon earlier document but published on or alter the International document of particular relevance, the claimed Invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(*) or involve an Inventive step which Is cited to establish the publication data of another document at particular relevance;' the claimed invention citation or other special reasoo (as specified) cannot be considered to involve an Inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu. other means ments, such combination being Obvious to a person skilled document published prior to the International filing date but In the art. later than the priority dae claimed document member of the same patent family IV._CERTIFICATION_____________ Ofuts of the Actual Completion of the International Search Dat* of Mailing of this International Search Report 16riwnaotil Sear.hlng Authority 319nature ~orzrnd-Ofte EUOENPATENTOFFICE L Form PCT1ISAM0 second sheet) (January 1985) if A ii *1. PCT/GB 89/00965 International Acolleation No. 3- Ill. DOCUMENTS CONSIDERED TO 31 RELEVANT (CONTINUED FROM THE SECOND SHEET) CAloitgory, Citation of DoGurflenit. withi indir-ation, witeie aoorrate, of treo relevant Passages Relevant to Claim No A P ,A February 1988, Elsevier Publications (Cambridge, GB), J. Tartaglia et al.: "Recombinant vaccinia virus vaccines", pages 43-46 J. Gen. Virol., vol. 67, no. 10, 1986, SGM (GB), M. Mackett et al.: "'Vaccinia virus expression vectors", pages 2067-2082 cited in the application J. Gen. Virol., vol. 70, 1989, SGM (GB), P.D. Gershon et "The nucleotide sequence around the capripoxvirus thymidine kinase gene reveals a gene shared specifically with leporipox- virus", pages 525-533 <A A Form PCT tSA.,210 (extra ilel (JanM~r 16") International Application No. PCT/GB 89/00965 FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET V2OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE This International search report has not been established In respect of certain claims under Article 17(2) for the following reasons: Claim numters 8...because they (elate to subjltct matter not required to be searched by this Author"t, namely: See PCT Rule 39.l(iv): Methods for treatment of the human or animal body by surgery or therapy, as well as diagnostic methods. 2M Claim numbers because they relate to parts of the international application that do not comply with the proscribed require- menits to such an extent that no meaningful International search can De carried out. aoiciftcally: 3.]Claim nurricens.......beauec avey aio wtjoadex i iams aind are not drafted in accodanlce with trea second and mrr sorenes of I PCT Rule 6.4(a). Vl.0 O@SERVATIONS WHERE UNITY OF INVENTioN IS LACKING2 This international Searching Author"t found multiplie inventionxi In this International application as follows: LM As all requited additional search fees were timely paid by the applicant, this International search report covers all searchable claims of the International application. 2.1As only comes of the required additional search fees were timely paid by the applicant, this International search report covers only those claims of the internetilonal application for which fees were paid, specIfically claims: 3.1No required additional search fees were timely paid by the applicant. Consequently, this International search report Is restricted to the Invention first mentioned In the claims; it Is covered bv claim numbers: 4.M1 As all searchable cisi me could tie searched without effort justifying an additional fe*, the International Searching Authority did not Invite Payment of any additional to. Remark on Protest r7The additional search fees wets accompanied by spolicant's protest. 11 No Protest accompanied the payment of aditional search fee. Form PCTIISAMgO (supplemental sheet (January 1965) I L.