WO1990012101A1 - Vaccinia vectors, vaccinia genes and expression products thereof - Google Patents

Abstract

The invention discloses recombinant vaccinia virus vectors wherein: a) part or all of one or more of the following nucleotide sequences is deleted from the viral genome; and/or b) one or more of said nucleotide sequences is inactivated by mutation or the insertion of foreign DNA; and/or c) one or more of said nucleotide sequences is changed to alter the function of the protein product encoded by said nucleotide sequence; which nucleotide sequences are sequences designated herein as i) Sal F 3R, ii) Sal F 9R, iii) Sal F 13R, iv) B 5R, v) Sal F 15 R.

Description

VACCINIA VECTORS, VACCINIA GENES AND

EXPRESSION PRODUCTS THEREOF

BACKGROUND OF INVENTION FIELD OF INVENTION

The present invention relates to recombinant vaccinia virus vectors. In particular it relates to the attenuation of the virus, to potential enhanced immunogenicity of the virus, to the provision of sites for the insertion of heterologous gene sequences into the virus, and to the use of the recombinant virus vectors thereby provided. It also relates to proteins which are the expression products of vaccinia genes. DESCRIPTION OF PRIOR ART Live vaccinia virus was used as the vaccine to immunise against, and eradicate smallpox. Vaccinia virus is the prototypical member of the poxvirus family and therefore it has been extensively studied. It is a large DNA-containing virus which replicates in the cytoplasm of the host cell. The linear double-stranded genome of approximately 185,000 base pairs has the potential to encode at least 200 proteins (Moss, B. (1985) In B.N. Fields, D.M. Knipe, J.L. Melnick, R.M. Channock, B.R. Roizman and R.E. Shope (eds.), Virology. Raven Press, New York, pp. 685-704). The cytoplasmic site of replication requires that vaccinia virus encodes many enzymes and protein factors necessary for DNA synthesis. Advances in molecular genetics have made possible the construction of recombinant vaccinia viruses that contain and express genes derived from other organisms (for review see Mackett, M. & Smith G.L. (1986), J. Gen. Virol., 67, 2067-2082). The recombinant viruses retain their infectivity and express the foreign gene (or genes) during the normal replicative cycle of the virus. Immunisation of animals with the recombinant viruses has resulted in specific immune responses against the protein(s) expressed by the vaccinia virus, including those protein(s) expressed by the foreign gene(s) and in several cases has conferred protection against the pathogenic organism from which the foreign gene was derived.

Recombinant vaccinia viruses have, therefore, potential application as new live vaccines in human or veterinary medicine. Advantages of this type of new vaccine include the low cost of vaccine manufacture and administration (because the virus is self-replicating), the induction of both humeral and cell-mediated immune responses, the stability of the viral vaccine without refrigeration and the practicality of inserting multiple foreign gengs from different organisms into vaccinia virus, to construct polyvalent vaccines effective against multiple pathogens. A disadvantage of this approach, is the re¬ use of a virus vaccine that has been recognised as causing rare vaccine-related complications. The applicants have now identified unobvious gene sequences which may be deleted from the viral genome. The applicants.propose that part or all of one or more of these gene sequences may be deleted from the viral genome to allow (i) greater attenuation of the virus; and/or (ii) enhancement of immunogenicity of recombinant vaccinia virus; and/or (iii) further gene sequence insertion sites so that more foreign DNA may be included in the virus. Where however, the gene sequences are essential for viral replication, viral attenuation can still be effected by altering the gene product (e.g. by manipulation at gene level) such that a protein function affecting pathogenicity is adversely affected whilst keeping the protein functional for virus application. SUMMARY OF INVENTION

According to one aspect of the present invention there is provided a vaccinia virus vector wherein a) part or all of one or more of the following nucleotide sequences is deleted from the viral genome; and/or b) one or more of said nucleotide sequences is inactivated by mutation or the insertion of foreign DNA; and/or c) one or more of said nucleotide sequences is changed to alter the function of a protein product encoded by said nucleotide sequence; which nucleotide sequences are sequences designated herein as i) Sal F 3R, ii) Sal F 9R, iii) Sal F 13R, iv) B5R, v) Sal F 15R.

DNA sequences encoding one or more heterologous polypeptides may be incorporated in the viral genome. The DNA sequences encoding the heterologous peptides may be inserted into one or more ligation sites created by the deletion or deletions from the viral genome.

The recombinant vaccinia viruses of the present invention have the potential for enhanced immunogenicity. This may result from either the deletion of vaccinia genes which cause immunosuppression (e.g. the complement homologue and the human FcR for IgE) or by insertion of a gene which potentiates the immune response (e.g. expressing the authentic CD23 gene in vaccinia virus). Therefore the present invention provides a vaccinia virus wherein a) part or all of one or more vaccinia nucleotide sequences causing immunosuppression are deleted from the viral genome; and/or b) one or more of said vaccinia nucleotide sequences causing immunosuppression is inactivated by mutation or the insertion of foreign DNA; and/or c) one or more of said vaccinia nucleotide sequences causing immunosuppression is changed to alter the function of a protein product encoded by said nucleotide sequence; which nucleotide sequences are sequences designated herein as i) Sal F 3R, ii) Sal F 9R, iii) Sal F 13R, iv) B5R, v) Sal F.15R.

In particular the vaccinia nucleotide sequence may be the sequence designated herein as Sal F 3R.

Where the vaccinia virus comprises a DNA sequence encoding a heterologous polypeptide which potentiates the immune response, the DNA sequence may encode CD23.

The recombinant vaccinia vectors of the present invention may be used as immunogens for the production of monoclonal and polyclonal antibodies or T-cells with specificity for heterologous peptides encoded by DNA sequences ligated into the viral genome. The invention also provides the monoclonal antibodies, polyclonal antibodies, .antisera and/or T cells obtained by use of the recombinant vaccinia vectors provided. The antibodies produced by use of the recombinant virus vectors hereof can be used in diagnostic tests and procedures, for example in detecting the antigen in a clinical sample; and they can also be used therapeutically or prophylactically for administration by way of passive immunisation. Also provided are diagnostic test kits comprising monoclonal antibodies, polyclonal antibodies, antisera and/or T cells obtained by use of the recombinant vaccinia vectors provided. Also provided are vaccines and medicaments which comprise a recombinant vaccinia virus hereof. These may have enhanced safety and immunogenicity over current vaccinia virus strains for the reasons indicated.

According to another aspect of the present invention there is provided a polypeptide encoded by a nucleotide sequence selected from those defined above and alleles and variants of said polypeptides. The polypeptide, allele or variant thereof may be encoded by the nucleotide sequence designated herein as Sal F 13R and which has activity as a DNA ligase.

The invention also includes sub-genomic DNA sequences encoding such a polypeptide, recombinant cloning and expression vectors containing such DNA, recombinant microorganisms and cell cultures capable of producing such a polypeptide.

The invention also provides a method of attenuating a vaccinia virus vector which comprises: a) deleting part or all of one or more of the following nucleotide sequences from the viral genome; and/or b) inactivating one or more of said nucleotide sequences by mutating said nucleotide sequences or by inserting foreign DNA; and/or c) changing said one or more nucleotide sequences to alter the function of a protein product encoded by said nucleotide sequence; which nucleotide sequences are sequences designated herein as: i) Sal F 3R, ii) Sal F 9R, iii) Sal F 13R, iv) B5R, v) Sal F 15R.

The invention also provides a method which comprises using a vaccinia virus vector as defined herein to prepare a vaccine or a medicament.

The invention also provides the use of part or all of the nucleotide sequence designated herein as Sal F 13R or part or all of the amino acid sequence encoded by said nucleotide sequence in the identification of polypeptides with activity as a DNA ligase. Furthermore the polypeptide represented by the amino acid sequence 7625 to 9280 (inclusive) of Fig. 11 hereof, or an allele or variant thereof, may be used as an enzyme in the manipulation of DNA in recombinant technology. BRIEF DESCRIPTION OF THE DRAWINGS

In order that the present invention.may be understood more clearly, the identified gene sequences will be described more fully with reference to the Figures wherein:

Figure 1 shows the location and direction of transcription of DNA ligase gene within the vaccinia virus genome. A. Vaccinia virus Hindlll restriction map. B. The 13.4 kb Sail F restriction fragment is expanded and the positions of EcoRI(E) Smal(S) restriction sites are indicated. CA 3300bp EcoRI - S al fragment is expanded showing the position of the DNA ligase gene (arrow), and the positions of EcoRI(E), Clal(C), Bell(Be), BglII(B) and Smal(S) restriction sites;

Figure 2 shows the location and direction of transcription of a thymidylate kinase gene within the vaccinia virus genome. A. Hind III restriction map. B. Expanded 13.4 kb Sail F fragment with the position of Sail F 13R (0RF13) shown as filled box. C. Expanded 2.4 kb Dra I fragment with position and direction of transcription of 0RF13 shown. The scale refers to this fragment. Letters in B and C indicate restriction enzyme sites: EcoRI(E), Dral(D), BamHI (B) and BclI(BC).

Figure 3 shows the nucleotide sequence of a 1776 nucleotide region of the 13.4 kb Sail F fragment. The deduced sequence of a 552 amino acid open reading frame is shown. The open reading frame is designated Sal F 15R;

Figure 4 shows the nucleotide sequence of 800 bp region of the vaccinia virus Sail F fragment. The deduced amino acid sequence of a 227 amino acid open reading frame designated Sal F 13R is shown. Numbers on the upper and lower lines refer to amino acids from the.beginning of the ORF or to the nucleotides from start of DNA fragment, respectively. Underlined nucleotides represent potential early transcriptional termination sequences and asterisks represent the 5' ends of early mRNA determined by SI nuclease protection.

Figure 5 shows the nucleotide sequence and amino acid sequence for the gene Sal F 3R;

Figure 6 shows the amino acid sequence homology between the protein encoded by gene Sal F 3R and i) the human low affinity Fc receptor for IgE, (huFcR(IgE) ); ii) the antifreeze polypeptide (ANP) from Hemitripterus americans; and iii) a lectin (LEC) from Megabalanus rosa; Figure 7 shows the construction of plasmid pSAD3G for the deletion of Sal F 3R from the virus genome;

Figure 8 shows a Southern blot analysis of virus vSAD3. Virus DNA was extracted from purified WT or vSAD3 virus and digested with Spel. DNA fragments were resolved on an agarose gel, transferred to nitrocellulose and probed with a radio-labelled DNA fragment from the Ecogpt gene. The band of 7 kb is as predicted and there is no hybridization with DNA from WT virus;

Figure 9 shows a Northern blot of mRNA from mock- infected (lane 1) or WT virus-infected cells early (lane 3) or late (lane 2) after infection. RNAs were resolved on an agarose gel transferred to nitrocellulose and probed with a sijigle stranded, radio-labelled DNA fragment complementary only to the Sal F 3R open reading frame; Figure 10 shows the nucleotide and amino acid sequence for the gene Sal F 9R;

Figure 11 shows the amino acid sequence homology between the protein encoded by gene Sal F 9R (Sail F 0RF9) and i) cow; and ii) human, superoxide dismutase (SOD) (Cu- Zn) proteins;

Figure 12 shows the amino acid sequence homology between the protein encoded by gene Sal F 13R and yeast thymidylate kinase (TmpK);

Figure 13 shows aligned amino acid sequences of vaccinia virus Sal F 13R (W) and Saccharomyces cerevisiae (SC) TmpK. Identical amino acid residues are boxed. Numbers above or below the aligned sequences refer to amino acid positions of W or SC respectively; Figure 14 shows : A. the aligned amino acid sequences for the presumed ATP binding site of vaccinia (W) and Saccharomyces cerevisiae (SC) TmpK, HSV TK/TmpK and human and W TK. Residues identical in all 5 sequences are boxed. Numbers indicate the amino acids between the amino terminus and the region shown; and B. Amino acid sequences for region of HSV TK/TmpK involved in nucleoside/nucleotide binding, aligned with corresponding regions of vaccinia virus (W) or Saccharomyces cerevisiae (SC) TmpK proteins. Amino acids conserved between two or all, of the sequences are boxed.

Figure 15 shows the biochemical pathway of dTTP synthesis in which thymidylate kinase is active;

Figure 16 shows the construction of plasmids pACVl and pACV2; Figure 17 shows a Southern blot analyses of viruses vACHB and vACl. Virus DNA was extracted from purified WT, vACl or vACHB viruses and digested with Sail. DNA fragments were resolved on an agarose gel, transferred to nitrocellulose and probed with a radio-labelled DNA fragment from entirely within the TmpK gene. Sail digest gives a 13.4kb band with WT virus but bands of 8.8 and 6.7 kb for recombinants VACHB and VACl (due to an extra Sail site introduced at 3' end of Ecogpt cassette);

Figure 18 shows the nucleotide and amino acid sequence for the gene B5R;

Figure 19 shows the amino acid sequence homology between the protein encoded by gene B5R (Sail G ORF10) and i) coagulation factor XIII B chain (F13 B); ii) complement factor H precursor (CFAH); iii) complement C2 precursor (C02); and iv) complement C4B-binding protein precursor (C4BP);

Figure 20 shows the hydrophobicity profiles for B5R (Sail G ORF10) and H3C 28K proteins; Figure 21 shows Northern blot of mRNA from virus infected cells early (E) or late (L) during infection. RNAs were resolved on an agarose gel transferred to nitrocellulose and probed with a single stranded, radio- labelled, DNA fragment complementary only to the B5R gene. The position of molecular weight size markers is shown in kb;

Figure 22 shows the amino acid sequence homology between the vaccinia virus (W) protein encoded by gene Sal F 15R and amino acid sequences of yeast DNA ligases from S.pombe (sp) and S.cerevisiae (sc) made using programme MULTALIGN;

Figure 23 shows the identification of vaccinia virus DNA ligase protein. Crude extracts were prepared from mock infected or vaccinia virus infected (100 pfu/cell) CV1 cells by Dounce homogenisation in 100 mM NaCl buffer as described in Kerr and Smith. Vaccinia virus infected early (lane 2), late (lane 3) or mock infected (lane 1) CV1 cell extracts and purified calf thymus DNA ligase I (a gift from T. Lindahl) (lane 5) were incubated with α-(³²P) ATP (Methods). Reactions were terminated by trichloroacetic acid and covalently labelled polypeptides analysed by SDS PAGE on a 12.5% gel;

Figure 24 shows the 61 kD polypeptide is a DNA ligase. A DNA ligase preparation partially purified from vaccinia virus infected cells late (15h) post infection was labelled with α-(³P) ATP (lane 3). Preparations of calf thymus DNA ligase (a gift from T.Lindahl, ICRF) (lane 1) and bacteriophage T4 DNA ligase (New England Biolabs) (lane 2) were labelled in parallel. The vaccinia sample was divided into four equal parts. One part was analysed without further manipulation (lane 3) and the remainder centrifuged through a column to remove unincorporated ATP as described in (25) except that Sephadex-G25 was used. The excluded volume was divided into three equal parts and incubated at 37°C for 30 minutes with either no addition (lane 4), cold poly (dA):oligo (dT) DNA ligase substrate (lane 5) or lOOμM sodium pyrophosphate (lane 6). The products were analysed as in Figure 1; Figure 25 shows DNA ligase activity in vaccinia virus infected cells. Crude extracts from- CV-.l cells infected with vaccinia virus early (3h), late (17h) post infection or mock infected were assayed for DNA ligase activity (Kerr and Smith, Nucleic Acids Res. , 17, 9039 (1989)). The 30mer (³²P) oligo dT:poly dA substrate is shown in lane 1 and corresponds to the monomer n = 1. Four units (lane 2), 0.4 units (lane 3) and 0.04 units (lane 4) of bacteriophage T4 DNA ligase (New England Biolabs) were assayed in parallel and provide markers (n = 2, n = 3, n = 4). Lanes 5, 6 and 7 represent the supernatant fractions from early, mock and late samples respectively after Dounce homogenisation and centrifugation at 10K for 20 minutes. Lanes 8, 9 and 10 are the pellet fractions from early, mock and late samples. An autoradiograph of the dried gel is shown;

Figure 26 shows: A. Immune-precipitation of (³⁵S)- methionine labelled polypeptides from vaccinia virus infected cells. TK cells infected with vaccinia virus (30 pfu/cell) or mock infected were labelled with (³⁵S)- methionine_.1.5 - 4h post infection. Cell extracts were prepared ad immune-precipitated with pEX LIG antiserum (Kerr and Smith 1989). Lane 1 represents uninfected cells and lane 2 vaccinia virus infected cells. Molecular weight markers are shown to the right of the gel; and B. Co- migration of (³⁵S)-methionine and α-(³²P)-ATP labelled proteins. A (³²P)-labelled DNA ligase-AMP adduct from vaccinia virus infected cells (lane 1) and cell extracts labelled with (³⁵S)-methionine 2.5 - 6h p.i. from either vaccinia virus infected (lane 2) or mock infected cells (lane 3), immune-precipitated with pEX LIG antiserum as described in Part A, were electrophoresed through a 12.5% polyacrylamide gel;

Figure 27 shows immune-precipitation of labelled vaccinia virus DNA ligase. Calf thymus DNA ligase (lane 1 ), T4 DNA ligase (lane 2) and a phosphocellulose column fraction from vaccinia virus infected cells (lane 3) were incubated with α-(³²P)-ATP (Kerr and Smith 1989). Each sample was divided into four equal parts and either analysed directly by TCA precipitation and SDS-PAGE (lanes 1, 2 and 3) or, in the case of extract from vaccinia virus infected cells, immune-precipitated with either pre-immune serum (lane 4), pEX LIG serum (lane 5) or a non-specific pEX immune serum (lane 6), followed by SDS-PAGE;

Figure 28 shows the cloning of the vaccinia virus DNA ligase gene by PCR to form plasmid pSK17;

Figure 29 (i) shows the expression of the DNA ligase gene in E.coli. Bacteria harbouring either parent vector pGMT7 or plasmid pSK18, were incubated with (+) or without (-) IPTG and the total cell protein run on an SS- polyacrylamide gel 4 hours after induction. The presence of an additional band of roughly 63 kDa is evident in bacteria containing pSK18 after addition of IPTG; (ii) timecourse of induction of DNA ligase after addition of IPTG. Bacterial cultures containing plasmid psK18 were inbucated for 0, 1, 2 or 4 hours after addition of IPTG and the total bacterial protein run on a polyacrylamide gel. The DNA ligase protein appears as a 63 kDa protein; (iii) the cell extracts shown in Figure 4b(i) were incubated with alpha-labelled ³²P-ATP and run on a pσlyacrylamide gel and an autoradiograph produced. The DNA ligase binds AMP and appears as a 63 kDa protein;

Figure 30 shows a Southern blot of virus DNAs from viruses derived from cells infected with WT vaccinia virus and transfected with pSK14. DNA was digested with Sail, run on an agarose gel and probed with the region of the DNA ligase gene deleted from pSK14. Isolates 3, 6, 7 and 8 lack the DNA ligase sequence but replicate efficiently in tissue culture; and

Figure 31 shows covalent binding of alpha-labelled ³P- ATP to extracts of cells infected with viruses 1, 5, 7 and 8 described in Figure 30. Viruses 7 and 8 lack a DNA ligase protein consistent with the lack of DNA for the gene product shown in Figure 30. DESCRIPTION OF EMBODIMENTS

All the genetic manipulations described below were carried out according to standard procedures (Molecular Cloning, eds. Sambrook, Fritsch & Maniatis, Cold Spring Harbor Laboratory Press, 1989) and the conditions used for enzymatic reactions were as recommended by the manufacturer (GIBCO-BRL Life Technologies). Determination of the Nucleotide and Amino Acid Sequences. The nucleotide sequence of the Sail F and Sail G restriction fragments of the vaccinia virus genome (strain WR) were determined by established methods (Sanger, F. et al. (1980), J. Mol. Biol., 143, 161-178) and Bankier, A. and Barrell, B.G. (1983) Techniques in Life Sciences B508., 1-34, Elsevier.

For example, the 13.4 kb Sail F fragment of vaccinia virus (strain WR) was isolated from cosmid 6, which contains virus DNA derived from a rifampicin resistant mutant (Baldick, C.J. & Moss, B. (1987) Virology 156, 138- 145), and was cloned into Sail cut pUC13 to form plasmid pSall F. The Sail fragment was separated from plasmid sequences and self-ligated with T4 DNA ligase. Circular molecules were randomly sheared by sonication, end-repaired with T4 DNA polymerase and Klenow enzyme and fragments of greater than 300 nucleotides cloned into Smal cut M13mpl8. Single stranded DNA was prepared and sequenced using the dideoxynucleotide chain termination method (Sanger, F. , Nicklen, S. & Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA, 74, 5463-5467), using [³⁵S]-dATP and buffer gradient polyacrylamide gels (Biggin, M.D., Gibson, T.J. & Hong, G.F. (1983), Proc. Natl. Acad. Sci. USA, 80, 3693-3695). For further details see (Bankier, A.T., Western, K.M. & Barrell, B.G. (1987) in Wu R. (ed. ) Methods in Enzymology 155, 51-93. Academic Press, London). The 12.6 Sail G fragment was similarly treated. Computer analysis

Nucleotide sequence data were read from autoradiographs by sonic digitiser and assembled into contiguous sequences using programmes DBAUTO and DBUTIL (Staden, R. (1980) Nucleic Acids Res. 8, 3673-3694; Staden, R. (1982) Nucleic Acids Res. 10, 4731-4751) on a VAX 8350 computer. The consensus sequence was translated in 6 frames using programmes ORFFILE and DELIB (M. Boursnell, Institute of Animal Health, Houghton, UK.). Open reading frames were compared against SWISSPROT protein database and against the applicants own database of vaccinia amino acid sequences using programme FASTP (Lipman, D.J. & Pearson, W.R. (1985) Science 227, 1435-1441). Alignments of multiple protein sequences were performed using programme MULTALIGN (Barton, G.J. & Sternberg, M.J.E. (1987) J. Mol. Biol. 198, 327-337).

There follows a description of the individual gene sequences the applicants have identified. Genes are named by (i) the restriction fragment from which they derive (i.e. SalF means Sail F fragment, or B means Hindlll B fragmen ), (ii) the number of the open reading frame initiating within the restriction fragment starting from _j the left end and (iii) the direction of transcription leftwards (L) or rightwards (R).

1. Sal F 3R

The nucleotide sequence and deduced amino acid sequence of the gene designated Sal F 3R is shown in Figure 5. The single letter code is used for the designation of amino acids. The coding region of the gene maps between nucleotides 595 and 1071 from the left end of the Sail F fragment. The molecular weight of the primary translation product is predicted to be 18.1 kiloDaltons (kD). Near the amino terminus there is a string of hydrophobic amino acids thought to cause the protein to be associated with, or secreted through, the cell membrane. Near the carboxy terminus there are three potential N-linked glycosylation sites, indicating that the mature gene product is a glycoprotein.

Comparisons of the deduced amino acid sequence with the protein database SWISSPROT established several significant homologies. Three of these are shown in Figure 6. The amino acid sequence encoded by the gene Sal F 3R shows^" sequence homology to a variety of lectins and the nearest homologue is human CD23 (see later). In particular, the amino acid sequence encoded by the gene

■Sal F 3R shows sequence homology with the amino acid sequence of the human low affinity Fc receptor for IgE (Kitutani, H. et al. (1986), Cell, 47, 657), the amino acid sequence of an antifreeze polypeptide from Hemitripterus americans (see Ng, N.F. et al, (1986), J. Biol. Chem. , 261, 15690-5) (5) and the amino acid sequence of a lectin from Megabalanus rosa (acorn barnacle) (Maramoto, K. & Kamiya, H. (1986), Biochem. ^' Biophys. Acta., 874, 285-295.). Sal F 3R has a 26.1% amino acid identity over a 92 amino acid region of the human low affinity Fc receptor (FcR) for IgE, 22.4% amino acid identity over a 98 amino acid region of the antifreeze polypeptide from Hemitripterus americans, and a 27.0% amino acid identity over a 63 amino acid region of the lectin from Megabalanus rosa.

The homologies suggest that the protein encoded by the Sal F 3R functions as a lectin or as a homologue of the human low affinity FcR for IgE. The latter homology is particularly important, as the human low affinity FcR for IgE is the same as CD23, a cell surface protein expressed on B lymphocytes which is of central importance in regulating B cell growth (Gordon, J. & Guy G.R. (1987), Immunol. Today, 8, 339).

Thus, the vaccinia virus protein encoded by Sal F 3R is thought to function as an agonist of the normal CD23 molecule, to restrict the growth and/or differentiation of B cells and thereby reduce the host immune response to infection by the virus. Therefore, deletion of this gene from the virus genome would enhance the host immune response to the virus. The consequence of this could be restriction of virus growth and hence attenuation. It is also possible that the immune response to foreign proteins expressed by recombinant vaccinia viruses lacking this gene would be enhanced and the efficacy of such candidate vaccines improved. Expression of the authentic human CD23 protein in vaccinia recombinants that do or do not contain the vaccinia homologue of CD23 may also enhance the immunogenicity of recombinant vaccinia virus vaccines that express antigens from heterologous pathogens.

If the protein has alternative or additional functions as a lectin, it may play a role in the attachment of virus to the target cell. Thus, deletion of the functioning gene in this capacity results in virus attenuation since the ability of virus particles to infect cells would be diminished. A mutant virus with the coding region of this gene interrupted and partially deleted has been constructed. A plasmid, pPROF was constructed by the ligation of the leftmost 3524 bp (Sall-EcoRI DNA fragment) of the vaccinia virus Sail F fragment into pUC13 that had been digested with EcoRI and Sail. This plasmid contains the entire coding region of Sal F 3R and was digested with Nsil, which cuts twice only, within the coding sequence (Figure 7). The digested DNA was treated with bacteriophage T4 DNA polymerase to create blunt ends, and the larger of the two fragments was purified by agarose gel electrophoresis. This fragment was ligated with a gel-purified DNA fragment containing the E.coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene joined to the vaccinia virus 7.5K promoter sequence. The latter fragment was obtained by digestion of plasmid pGpt07/14 (Boyle, D.B. and Coupar, B.E. H. Gene 65, 123-8 (1988)) with EcoRI, followed by treatment of the digested DNA with DNA polymerase (Klenow fragment) to create blunt ends and isolation of a 2.1kb DNA fragment. The ligated DNA was cloned into E.coli, and the resulting bacterial colonies screened for the presence of the desired plasmid with appropriate restriction enzymes. Through this procedure, (outlined in Figure 7) a plasmid, pSAD3G, was isolated in which lOObp of the Sal F 3R coding sequence was replaced by a functional copy of the Ecogpt gene under the control of the vaccinia virus 7.5K promoter.

Plasmid pSAD3G was transfected into CV-1 cells that were infected with wild type (WT) vaccinia virus and the virus progeny derived from these cells after 48 hours at 37°C were then plated on fresh CV-1 cells in the presence of mycophenolic acid (MPA), xanthine and hypoxanthine. These drugs permit the replication only of recombinant viruses which contain and express the Ecogpt gene (Boyle & Coupar 1988 supra; Falkner & Moss, J. Virol, j52, 1849- 54, 1988). After three rounds of plaque purification, the virus was amplified in larger cultures of CV-1 cells. Southern blot analysis of virus DNA confirmed that the Ecogpt gene was present at the predicted location in the virus genome, that no functional copy of the Sal F 3R.gene remained and that no other virus genomic DNA re¬ arrangements had occurred (see Figure 8). Since a virus lacking the Sal F 3R gene is viable, these data established that the gene SalF 0RF3 is non-essential for virus replication in vitro. The likelihood that the inactivation of the Sal F 3R coding region would generate an attenuated virus, depends on whether or not the region is expressed during normal virus replication. To address this point, virus mRNA transcribed from this region during the early and late phase of infection was analysed by Northern blotting. The results are shown in Figure 9. A single-stranded radio- labelled DNA probe complementary only to the coding strand of Sal F 3R detected an early mRNA species of about 600 nucleotides. Late during infection, this mRNA was replaced by some RNA species of heterogeneous length which appear as a smear on the Northern blot. Due to the heterogeneous length,of late vaccinia virus mRNA, it is possible that this represents either mRNA initiating from the Sal F 3R promoter or from further upstream. This data allows the conclusion that the gene Sal F 3R is certainly transcribed early and possibly also late during infection.

2. Sal F 9R The nucleotide and amino acid sequence of this gene is shown in Figure 10. The coding* region of the gene resides between nucleotides 4447 and 4821 from the left end of the Sail F fragment. The encoded protein has a predicted molecular weight of 13.6kD. Figu e 11 shows the amino acid sequence homology between the protein encoded by gene Sal F 9R and two superoxide . dismutase (Cu-Zn) proteins from cow (i) (Steinman et al„ (1974), J. Biol. Chem. , 249, 7326-38) and man (ii) (Sherman, L. et al (1983), PNAS, 80, 5465-9). The protein encoded by Sail F ORF9 has a 36.8% amino acid identity over a 57 amino acid region of bovine superoxide dismutase, and a 37.3% amino acid identity over a 59 amino acid region of human superoxide dismutase. Superoxide dismutase (SOD) is an enzyme that converts toxic oxidative free radicals (0₂ ^") into oxygen and hydrogen peroxide. Following engulfment of microorganisms, phagocytic cells undergo an oxidative burst which produces 0₂ ^~ to cause destruction of the microorganism. The presence of SOD in the structure of a virus, or its expression shortly after infection, would provide a defence mechanism against this toxic radical 0₂ ^" (by converting it into oxygen and hydrogen peroxide) and thereby enhance the survival and replication of vaccinia virus in host macrophages, a site in which poxviruses can survive and which facilitate the systemic spread of the virus (Fenner F. (1985), in "Virology". B.N. Field (Ed) pp. 661-684, Raven Press, New York). However, a thorough analysis of the amino acid structure of the predicted protein encoded by the gene Sal F 9R, shows that it lacks some critical amino acid residues that are involved in binding of the copper and zinc divalent cations in other SOD enzymes. On the basis of this it seems unlikely that the vaccinia virus SOD homologue has SOD- like enzyme activity, and novel virus-induced enzyme activity has not been detected in infected cells. However, the presence of this gene in the virus genome remains interesting, and suggests that other poxviruses might retain a functional SOD enzyme. For example, African Swine Fever Virus (ASFV) is a likely candidate to contain this enzyme as it replicates efficiently in swine macrophages. Deletion of this gene from the viruses containing the identified gene sequence and which retain superoxide dismutase enzyme activity would result in virus attenuation due to.a reduced ability of the virus to replicate within, and be disseminated by, macrophages.

3. Sal F 13R

Figure 4 shows the deduced sequence of the 227 amino acid ORF designated Sal F 13R. Figure 2 shows the position of Sail F 0RF13 within the vaccinia virus genome.

Approximately 40 nucleotides upstream of the ATG codon at the beginning of the ORF and 20 nucleotides downstream of the termination codon there are sequences TTTTTGT and TTTTTAT, respectively, which represent termination signals for early transcription (Yuen, L. and Moss, B. (1987) Proc. Natl. Acad. Sci. USA., 84, 6417-6421). The next downstream T₅NT motif is located a further 540 nucleotides away within the promoter region of the DNA ligase gene and contains two overlapping termination signals within the sequence TTTTTTTAT. The location of these early transcriptional termination signals and the absence of the sequence TAAAT(G) (a late transcription initiation site (Rosel, J.L., Earl, P.L. and Moss, B. (1986) J. Virol., 60, 436- 449; Hanggi, M., Bannwarth, W. and Stunnenberg, H.G. (1986) EMBO J., 5, 1071-1076)) at the 5' ends of Sal F 13R suggests that the gene may be transcribed early during infection. The coding region of the Sal F 13R gene maps between nucleotides 6313 and 7113 from the left end of the Sail F fragment. The encoded protein has a predicted molecular weight of 26.1kD. The deduced amino acid sequence of Sal F 13R was compared against protein database SWISSPROT and our own database of vaccinia virus proteins using programme FASTP (Lipman, D.J. and Pearson, W.R. (1985) Science, 227, 1435- 1441). No strong matches were found against other vaccinia proteins but the deduced amino acid sequence of SalF 13R had a high FASTP homology score (371) against thymidylate kinase (TmpK) of Saccharomyces cerevisiae (Jong, A.Y.S., Kuo, C.L. and Campbell, J.L. (1984) J. Biol. Chem., 259, 11052-11059; Rothstein, R., Helms, C and Rosenberg, N. (1987) Mol. Cell Biol. 7, 1198-1207).

Figure 12 shows the amino acid sequence homology between the protein encoded by gene Sal F 13R and thymidylate kinase (TmpK) from yeast (Jong et al, (1984), J. Biol. Chem. 259, 11052-9). The two proteins share 42% amino acid identity over a 200 amino acid region and there are many additional conservative changes. The aligned amino acid sequences are very similar in length, (yeast 216 amino acids versus vaccinia virus 204 amino acids), and are almost colinear. The computer predicted extra amino acid residues at the amino terminus of Sal F 13R which are upstream of the 5' end of early mRNA, have no homology with yeast TmpK. This is consistent with these amino acids not being part of the vaccinia TmpK enzyme. An alignment of the two amino acid sequences is shown in Figure 13 with identical amino acids boxed.

Amino acids residues 11-18 of the putative vaccinia TmpK enzyme fit the consensus motif for ATP binding proteins GxxGxGKS/T (Otsuka, M. and Kit, S. (1984) Virology, 135, 316-330) except for the second glycine, where there is lysine. An alignment of this region with the presumed ATP binding sites of yeast TmpK (Jong, A.Y.S., Kuo, C.L. and Campbell, J.L. (1984) J. Biol. Chem., 259, 11052-11059; Rothstein, R., Helms, C. and Rosenberg, N. (1987) Mol. Cell Biol. 7, 1198-1207) HSV thymidine kinase (TK) TmpK (Otsuka, M. and Kitt, S. (1984) Virology, 135, 316-330; McKnight, S.L. (1980) Nucleic Acids Res. 8, 5949- 5964; Wagner, M.J. Sharp, J.A. and Summers, W.C. (1981) Proc. Natl. Acad. Sci. USA., 78, 1441-1445; Gompels, U. and Minson, A.C. (1986) Virology, 153, 23-247; Darby, C, Larder, B.A. and Inglis, M.M. (1986) J. Gen. Virol., 67, 753-758; Kit, S., Kit. M., Qavi, H., Trkula, D and Otsuka, H. (1983) Biochem. Biophys. Acta, 741, 158-170; Swain, M.A. and Galloway, D.A. (1983) J. Virol, 46, 1045-1050) vaccinia TK (Weir, J.P. and Moss. B. (1983) J.- Virol. 46, 530-537) and human TK (Bradshaw, H.D. and Deininger, P.L. (1984) Mol. Cell Biol., 4, 2316-2320) is shown in Figure 14A. In all these sequences the glycine residues at positions five and ten, lysine at position eleven and threonine at position thirteen are invariant. Only herpes simplex virus (HSV) TK/TmpK contains the second glycine of the ATP binding site consensus (above). The alignment of this region also shows that the highly homologous yeast and vaccinia TmpK sequences and the more divergent HSV TK/TmpK, differ from TK sequences, of which vaccinia and man are representative examples, in several respects. First, immediately preceding the first glycine all with TmpK enzymes contain an acidic residue while TKs contain a hydrophobic residue. Second, at positions six to eight all poxvirus (Weir, J.P. and Moss. B. (1983) J. Virol, 46, 530- 537; Boyle, D.B., Coupar, B.E.H., Gibbs, A.J., Seigman, L.J. and Both. G.W. (1987) Virology, 156, 335-367; Esposito, J.J. and Knight, J.C. (1984) Virology, 135, 561- 567; Upton. C. and McFadden, G. (1986) J. Virol., 60, 920- 927) and cellular (Bradshaw, H.D. and Deininger, P.L. (1984) Mol., Cell Biol., 4, 2316-2320; Lin. P.F. Lieberman, H.B., Yeh, D.B., Xu T., Zhao. S.Y. and Ruddle. F.H. (1985) Mol. Cell Biol,. 5, 3149-3156; Kwoh, T.J. and Engler, J.A. (1984) Nucleic Acids Res., 12, 3959-3971) and cellular TK enzymes contain PMF residues while yeast and vaccinia TmpK sequences contain LDK/R. Here the HSV enzyme fits neither pattern and this may reflect its broader substrate specificity. Third, as position fourteen poxvirus and cellular TKs contain glutamic acid while vaccinia and yeast TmpK contain glutamine and HSV has threonine.

Outside the ATP binding site there is no detectable homology between the vaccinia TmpK and TK sequences. However, homology exists between vaccinia TmpK and HSV TmpK/TK at a second nucleotide/nucleoside binding region. The alignment of the sequences from yeast TmpK, vaccinia TmpK and HSV TK/TmpK in this region is shown in Figure 14B. Although the yeast and vaccinia enzymes are clearly more homologous, a TLI triplet is conserved between vaccinia and HSV (positions three to five).

The gene encoding TK has been mapped and sequenced. It is a nonessential gene for in vitro replication and has been widely used as a site for insertion of foreign DNA into recombinant vaccinia viruses (Mackett, M. and Smith. G.L. (1986) J. Gen. Virol. 67, 2067-2082). It is also a determinant of virus pathogenicity for both vaccinia (Buller, R.M.L., Smith. G.L., Cremer, K., Notkins, A.L. and Moss. B. (1985) Nature, 317, 813-815) and HSV (Field, H.J. and Wildy, P. (1978) J. Hyg. Camb. 81, 267-277; Kit S., Qavi, H., Dubbs, D.R. and Otsuka, H. (1983) J. Med. Virol., 12, 25-36). Deletion of the TK gene results in virus attenuation (Buller et al (1985) Nature, 317, 813-5). The enzyme TmpK converts thymidine monophosphate

(thymidylate or dTMP) into thymidine diphosphate (dTDP) within the biochemical pathway illustrated in Figure 15. Vaccinia virus encodes a separate enzyme, thymidine kinase (TK) that acts to convert thymidine into thymidine monophosphate in the first part of this pathway. Given that TK and TmpK perform sequential steps in the same biochemical pathway the present applicants have realised that very probably the vaccinia TmpK gene is also not essential for virus replication and that its deletion would also cause virus attenuation. This gene would therefore provide an additional site for insertion of foreign DNA into vaccinia virus and be a target for effecting virus attenuation.

Two vaccinia virus mutants have been constructed in which the Sal F 13R gene has been inactivated. The Ecogpt gene joined to the vaccinia virus promoter p7.5K was inserted into a region of the TmpK gene predicted to be involved in nucleoside/nucleotide binding and, therefore, likely to be essential for enzyme activity (Smith et al. Nucleic Acids Res. , 17, 7581, (1989)). The strategy followed that described above for the Sal F 13R gene (Figure 16). A plasmid, pACVl, was constructed by the ligation of a 2392 bp Dral DNA fragment, derived by Dral digestion of the Sail F fragment, into Smal cut pUC13. pACVl contains the entire Sal F 13R coding sequence and was digested with restriction enzyme Mlul which cuts pACVl only once and within the coding region of TmpK. The Ecogpt gene joined to the vaccinia virus promoter p7.5K was isolated as an EcoRI fragment (as above), made blunt-ended by treatment with DNA polymerase (Klenow fragment), and ligated with pACVl that had been digested with Mlul. The resultant plasmid, pACV2, contained the TmpK gene interrupted by Ecogpt. The procedure is outlined in Figure 16. This plasmid was used to transfect CV-1 cells infected with either WT vaccinia virus or a TK" recombinant virus which expresses the hepatitis B virus surface antigen gene (Smith et al., Nature 302, 490-5, 1983). Recombinant viruses expressing the Ecogpt gene were selected by plaque assay in the presence of MPA and stocks grown. The virus derived from WT virus was called vACl and the virus derived from vHBs4 was called vACHB. Their genomic DNAs were analysed by Southern blotting (Figure 17). These data showed that both viruses contain the Ecogpt gene integrated at the predicted location, that no other genomic alterations had occurred and established that the product of SalF 13R is non-essential for virus replication in vitro. Transcriptional mapping by Northern blotting and SI nuclease protection demonstrated that the SalF 13R gene is transcribed early but not late during infection. An early mRNA of approximately 850 nucleotides was detected with a probe specific for the coding strand of SalF 13R. This size corresponds to the size of the mRNA predicted if transcription initiates just upstream of the ORF and terminated 50 nucleotides downstream of the first downstream early transcription termination signal. SI nuclease mapping precisely located the 5' end of the early mRNA to just upstream of the second inframe ATG codon. This is roughly 65 nucleotides downstream of the first ATG codon and the protein is therefore 23 amino acids shorter than that previously predicted. (Smith et al., Nucl. Acids Res. 17, 7581-90). Assays for TmpK. activity in vaccinia virus-infected cells have been performed and enzyme activity has been detected. . The assays consist of incubating extracts of mock or virus-infected cells with tritiated thymidylate, resolving the reaction products by thin layer chromatography (TLC) (to separate TMP, TDT and TTP) and counting the areas of the tritium in TLC corresponding to these compounds (Jong et al., J.B.C. 259, 11052-9 (1984).

However, because TmpK is an essential cellular enzyme the applicants have not yet demonstrated a difference between the endogenous activity in uninfected cells and that present in vaccinia virus-infected cells. To overcome this difficulty the applicants have reconstructed the gene by polymerase chain reaction (PCR) using synthetic oligonucleotides, re-sequenced and cloned into plasmid pEMBLyex4 (Dente et al, Nuc. Acids Res. 11., 1645-55, 1983) designed for expression of genes in Saccharomyces cerevisiae. The plasmid is used to complement a yeast mutant, CDC8, that is deficient in TmpK activity (Jong et al 1984). Complementation of this yeast strain directly shows that the Sal F 13R gene encodes TmpK enzyme activity, and since the parent yeast strain has no endogenous TmpK activity, it is straightforward to demonstrate enzyme activity in vitro using extracts of these yeast cells.

4. B5R

The nucleotide and amino acid sequence of gene B5R are shown in Figure 18. The encoded protein has a predicted molecular weight of 35.1 kD and its coding region maps between nucleotides 6654 and 7604 from the left end of the Sail G fragment. The protein .contains hydrophobic amino acid sequences near the amino- and carboxy-termini, indicating that the protein associates with cell membranes of the infected cell or virus particle. There are also three potential sites for N-linked glycosylation indicating the mature product is a glycoprotein.

Comparisons of the amino acid sequence with the SWISSPROT protein database established significant homologies with several proteins that belong to the superfamily of complement control proteins and blood coagulation factors. The alignments of Figure 19 show the amino acid sequence homology between the protein encoded by gene B5R (Sail G ORF10) and coagulation factor XIII B chain, complement factor H precursor, complement C2 precursor, and complement C4B-binding protein precursor. The protein encoded by B5R has a 27.2% amino acid identity with a 246 amino acid region of coagulation factor XIII B chain, a 27.2% amino acid identity with a 125 amino acid region of complement factor H precursor, a 26.4% amino acid identity with a 178 amino acid region of complement C2 precursor, and a 24.6% amino acid identity with a 175 amino acid region of complement C4B-binding protein precursor. Within the proteins of this superfamily, there are repeated domains of roughly 60 amino acids. The vaccinia protein encoded by gene B5R possesses four such domains.

Vaccinia virus contains a gene encoding another protein, H3C 28K (Kotwal, G. & Moss, B. (1988), Nature, 335, 176) which shows homology with this superfamily of complement and blood coagulation proteins and which is non- essential for virus replication. The protein encoded by gene B5R is related to, but distinct from, this protein, with a 29% amino acid homology. The H3C 28K protein is more closely related to the complement C4B-binding protein than the protein encoded by gene B5R. Conversely, the protein encoded by gene B5R is more closely related to coagulation factor XIII than the H3C 28K protein is. Another significant difference between the proteins Sail G 0RF10 and H3C 28K is illustrated by the hydrophobicity profiles shown in Figure 20. The presence of an extra hydrophobic domain near the carboxy-terminus of the protein encoded by B5R, and which is not shown H3C 28K, indicates that the former would remain cell associated whilst the latter is known to be secreted (Kotwal, G. & Moss, B. (1988), Nature 335, 176.

The homologies given above, indicate that the protein encoded by B5R is likely to interfere with the normal processes of complement activation (the H3C 28K protein is also known to do this) or blood coagulation. Interference in complement-mediated cell lysis would enhance the virus survival. Similarly, the prevention of blood clotting around the site of infection would prevent containment of the infection and enhance virus dissemination. Attempts to construct a virus deletion mutant by insertional inactivation with Ecogpt have proved unsuccessful and it seems likely, but not proven, that this gene is essential for virus replication.

Where a gene is essential for virus replication, the virus may still be attenuated by altering the gene product. Thus, since the encoded protein binds complement factors, the region of the protein specific for the binding can be altered whilst keeping the protein functional for virus replication. Transcriptional analysis of the B5R gene by Northern blotting (Figure 21) showed the presence of an early mRNA of 1850 nucleotides. This size corresponds to the size of the mRNA predicted if transcription initiates just upstream of B5R and terminates 50 nucleotides downstream of the first downstream early transcription termination signal. There are also late RNAs of heterogeneous length from this region. ^' SI nuclease analysis has shown that the B5R promoter is expressed both early and late during infection. Unlike the .constitutively active 7.5K promoter which also has early and late transcriptional initiation sites, the B5R promoter has the early RNA start site upstream of the late start site. The late start site maps to within a conserved motif TAAAT. The protein has been expressed as a fusion protein with " β-galactosidase in E.coli and is currently being expressed in the authentic form in CHO cells driven by the human cytomegalovirus immediate early promoter-enhancer.

If the vaccinia protein functions as an anti- coagulation factor it is possible that this protein, or a form from which the carboxy hydrophobic domain has been deleted, would be a useful reagent in preventing blood coagulation.

5. Sal F 15R

The nucleotide and amino acid sequence of this gene are shown in Figure 3. The coding region of the gene maps between nucleotides 7625 and 9280 from the left end of the Sail F fragment. The encoded protein has a predicted molecular weight of approximately 63.3KD.

Transcriptional mapping of the Sail 0RF15 gene by Northern blotting, SI nuclease protection and primer extension have demonstrated that the gene is expressed early during infection (Smith et al., Nuc. Acids Res. 17, 9051-62). Surprisingly, the 5' end of the mRNA maps to a sequence TAAATG that is a characteristic of late transcription start sites. (Rosel, J.L., Earl, P.L., Weir, J.P. & Moss, B. (1986) J. Virol. 60, 436-449; Hanggi, M., Bannwarth, W. & Stunnenberg, H.G. (1986) EMBO J. 5, 1071- 1076). The 5' end of the mRNA determined by primer extension maps 5 nucleotides upstream of the 5' end determined by SI nuclease protection. It is possible that there are 5' oligo-adenylate residues on this early mRNA, which hitherto have been considered solely as a characteristic of late mRNA's.

Comparison of the amino acid sequence of Sal F 15R with our database of vaccinia virus proteins using programme FASTP (Lipman, D.J. & Pearson, W.R. (1985) Science 227, 1435-1441) found no strong matches. However, a search of the protein database SWISSPROT revealed extensive homology to DNA ligase of Saccharomyces cerevisiae (Barker, D.G., White, J.H.M. & Johnson, L.H. (1985) Nucleic Acids Res. 13, 8323-8337). An optimised FASTP score of 527 was obtained (KTUP of 1) and the two proteins had 30% amino acid identity over a 412 amino acid region. A similar degree of homology exists between Sail F 0RF15 and Saccharomyces pombe DNA ligase (Barker, D.G. White, J.H.M., Johnston, L.H. (1987) Eur. J. Biochem. 162, 659-667) although fission yeast S.pombe and the budding yeast S.cerevisiae are evolutionarily divergent. Only weak homology was detected with bacteriophage T4 and T7 and E.coli DNA ligases. An alignment of the amino acid sequences of DNA ligases from yeasts and vaccinia virus is shown in Figure 22. This alignment shows that the amino- terminal region of the vaccinia protein is divergent from both yeast sequences and there are regions which are absent in vaccinia but present in both yeasts. The latter point is reflected in the predicted sizes of the proteins, with vaccinia DNA ligase (63.3 kD) being considerably smaller than DNA ligases of S♦pombe (86.2 kD) and S.cerevisiae (84.8 kD). The yeast DNA ligases are also least conserved in the amino terminal region. In contrast, in the carboxy- terminal region the three sequences are almost colinear and have extensive amino acid identity and conservative changes. The presumed catalytic lysine at the ATP binding site (marked with asterisk) is conserved in all these sequences as well as in T4 and T7 DNA ligases. In the E.coli enzyme, which uses NAD rather than ATP as cofactor, this site is less conserved. The most highly conserved region is very close to the carboxy terminus and is rich in basic amino acids. Over a 16 amino acid region the vaccinia protein shares identity with S. ombe at 15 positions with S.cerevisiae at 14 positions with a conservative isoleucine to valine change at one of the two divergent amino acids. This region is also well conserved in T4 with 6 identical residues and several conservative changes. The high conservation of this region suggests it plays some critical role in DNA ligase function, and its basic composition is consistent with an interaction with the DNA substrate.

The data below provide direct evidence that vaccinia virus encodes a DNA ligase and supports early data (Sambrook, J. & Shatkin, A.J. (1969) J. Virol. 4, 719-726) showing a 13-fold increase in DNA ligase activity in the cytoplasm of vaccinia virus-infected cells. Spadari (Spadari, S. (1976) Nucleic Acids Res. 3, 2155-2167) concluded that the increase in DNA ligase activity was probably not virus-encoded since the enzyme had similar biochemical characteristics to cellular DNA ligase I, but may be attributable to enhanced leakage of the nuclear enzyme into the cytoplasm of virus infected cells. The amino acid sequence of this vaccinia enzyme is the first reported primary structure of a 'mammalian' DNA ligase. It is also the only example of a eukaryotic virus encoding a DNA ligase, although other large DNA viruses which replicate in the cytoplasm, such as African Swine Fever Virus, probably encode this enzyme. Although much is known of mammalian DNA ligases, the genes encoding these enzymes have not been mapped.

Vaccinia virus contains two other enzymes with DNA strand sealing activity (topoisomerase and nicking-joining enzyme) and models for virus DNA replication have been proposed which do not require a conventional DNA ligase (Moyer, R.W. & Graves, R.L. (1981) Cell 27, 391-401; Baroudy, B.M., Venkatesan, S. & Moss, B. (1982) Cell 28, 315-324). In one model the linear double stranded DNA genome with covalently closed hairpin ends is nicked on one strand near one, or both, terminal hairpins to provide a 3' OH from which polymerisation may initiate. Elongation proceeds around the terminal hairpin, down the linear genome and around the opposite hairpin to produce concatemeric DNA molecules by a strand displacement mechanism. This model does not require, but may use, lagging strand synthesis.

Figure 23 shows that extracts from vaccinia virus infected cells contain a novel radio-labelled polypeptide of molecular weight approximately 61 kD after incubation with α-(³²P) ATP. This activity is detectable in both crude and partially purified extracts, at early (lane 2) and late (lane 3) times post infection. The size estimated by SDS- PAGE is in good agreement with that predicted from the amino acid composition of Sal F 15R, 63 kD which would be consistent with a lack of extensive post-translational modification. The extent of incorporation of radioactivity is much greater than that in mock infected cells, in which only a faint band of approximately 46 kD is visible (lane 1). This polypeptide is also present at reduced intensity in extracts from vaccinia virus infected cells. The 130 kD mammalian DNA ligase I, highly purified from calf thymus, is shown in lane 5. Mock infected cells contain no polypeptide which co-migrates with the 61 kD band in infected cell extracts, suggesting the appearance of this protein is a consequence of infection with vaccinia virus.

The 61 kD polypeptide has the properties expected of a DNA ligase (Figure 24). A phosphocellulose column fraction derived from extracts of vaccinia virus infected cells late in infection was incubated with α-(³²P) ATP and then excess ATP was removed using Sephadex G-25. The excluded protein was incubated with either no addition (lane 4), DNA ligase substrate (lane 5) or sodium pyrophosphate (lane 6). The presence of DNA substrate allows the ligase reaction to proceed to completion, with a disappearance of (³²P)-AMP from the enzyme. Conversely, high concentrations of pyrophosphate drive the equilibrium back towards free enzyme and ATP, again with a consequent discharge of radioactivity from the polypeptide (Figure 24). This result indicates that the 61 kD polypeptide is a DNA ligase with DNA strand joining activity.

An assay which measures ligation of 30 mer (³²P)-dT oligodeoxynucleotides annealed to poly dA was used to determine whether an increase in DNA ligase activity could be detected upon vaccinia virus infection. Activity is represented by the appearance of labelled products corresponding to two ligated molecules of the dT oligonucleotide (n=2), trimers of dT (n=3) and further higher oligomers. The assay of bacteriophage T4 DNA ligase provides a standard for this activity. An increase in DNA ligase activity above the basal level measurable in mock infected cells is observed after vaccinia virus infection (Figure 25). The ligase activity early in infection (lanes 5 and 8) is only slightly greater than the cellular activity in mock infected cells (lanes 6 and 9), but by late in infection (lanes 7 and 10) the activity is substantially higher. Extracts prepared in this low salt extraction buffer (100 mM NaCl), have an appreciable portion of the total DNA ligase activity located in the pellet fraction after centrifugation (lanes 8-10) compared to the supernatant (lanes 5-7) but an increase in the salt concentration to IM shifts the majority of the total DNA ligase activity into the soluble fraction (data not shown). Approximately one third (183 amino acids) of the protein encoded by Sal F 15R was cloned into the bacterial expression vector pEX3 (Stanley and Luzio, EMBO J. 3_, 1429 (1984) as described before (Kerr and Smith 1989)). A rabbit polyclonal antiserum (pEX LIG) was raised against the resulting β-galactosidase/Sal F 15R fusion protein. The pEX LIG antiserum immune-precipitated two virus polypeptides from extracts of cells labelled with (³⁵S)- methionine. 1.5-4h post infection (Figure 26A, lane 2). The upper band, of molecular weight approximately 61 kD on SDS-PAGE, is more intense than the lower, of approximately 54 kD. No protein is recognised in mock infected cells (lane 1). The portion of the gene inserted into the pEX LIG construct does not include the regions of strongest amino acid sequence homology with yeast DNA ligases, therefore cross-reaction with mammalian ligases might not be expected. Both polypeptides are early virus gene products as treatment of the cells with cytosine arabinoside, an inhibitor of DNA replication, does not affect their expression (data not* shown). De novo synthesis of both proteins can be detected early in infection by pulse labelling with (³⁵S) methionine 1.5- 2.5h post infection followed by immune-precipitation, but only slightly reduced levels are observed as late as 7-8h p.i. (data not shown).

The larger of the two polypeptides detected by immune- precipitation with the pEX LIG antiserum co-migrates with the DNA ligase-AMP adduct from vaccinia virus infected cells on SDS-PAGE (Figure 26). The marginal difference in size between the (³²P) and (³⁵S)-labelled polypeptides which may be detected on electrophoresis to achieve maximum resolution^• is possibly due to the addition of the AMP moiety in the (³²P)-labelled protein.

The DNA ligase would be likely to be an essential gene if it was involved in DNA replication, in which case it would not be possible to select recombinant virus containing a specific deletion of this gene. An alternative approach to prove that Sal F 15R gene product was responsible for the increase DNA ligase activity in vaccinia virus infected cells was therefore chosen. This made use of the pEX LIG antiserum, raised against Sal F 15R encoded protein, in immune-precipitation experiments against the radio-labelled DNA ligase-AMP adduct. The pEX LIG antiserum can efficientlyprecipitate the (³²P)-labelled DNA ligase protein in extracts from vaccinia virus infected cells (Figure 27, lane 5), whereas pre-immune serum from the same rabbit (lane 4), or a non-specific immune serum raised against an unrelated pEX fusion protein^' (lane 6), do not recognise the 61 kD polypeptide (Figure 28). Control experiments indicate that neither purified calf thymus (lane 1) nor bacteriophage T4 DNA ligase (lane 2) can be immune-precipitated by the pEX LIG antiserum (data not shown). The immune-precipitation of the novel DNA ligase-AMP adduct by the antiserum raised against Sal F 15R encoded protein clearly demonstrates that this vaccinia virus gene encodes the observed DNA ligase activity.

These data have been published in 1989 (Smith et al., Nucl. Acids Res. 7, 9051; and Kerr and Smith, Nucl. Acids Res. r7, 9039).

To assess the commercial potential of vaccinia virus DNA ligase, the applicants chose to over-express the gene in E.coli. To achieve this, the gene was precisely engineered by polymerase chain reaction (PCR) (Figure 28). An oligonucleotide representing the 5' end of the coding strand (including extra 5' nucleotides to form BamHI and Ndel sites) and an oligonucleotide complementary to the coding strand roughly 150 nucleotides downstream were used in a PCR reaction with the SalL F fragment cloned into a plasmid vector as template. The PCR fragment was digested with. Sail and Bell and cloned into pSK16 that had been cut with -SalL and Bell, to form pSK17. pSK16 contains the whole DNA ligase gene inserted into the Smal site of pUC13 and was constructed by the isolation of a Clal to Mlul fragment from the pSK13. pSK13 contains the 3.3kb EcoRI to Smal fragment of the Sail F fragment cloned into pUC13. The PCR fragment was sequenced to confirm no mutations had been introduced by PCR. Finally, the whole DNA ligase gene was excised from pSK17 with Sail and EcoRI and cloned into bacterial expression vector pGMT7 (Rosenberg et al., Gene, 56, 125-135, 1987), that had been digested with Sail and EcoRI, downstream of the T7 RNA polymerase promoter, to form pSK18. Introduction of pSK18 into E.coli strains bearing an inducible T7 RNA polymerase gene, resulted in high levels o__ DNA ligase expression in the presence of the specific inducer IPTG. Crude lysates of these induced bacteria contained a novel polypeptide of 61 kDa that bound AMP (Figure 29). The applicants conclude that the vaccinia virus DNA ligase is active in E.coli and that the bacterial strain constructed potentially provides a large supply of this commercial important enzyme. A deletion mutant of vaccinia virus lacking DNA ligase has been produced by the same procedure used for the Sal F 3R and Sal F 13R. ^' Plasmid pSK13 was digested with Nrul (which cuts just downstream of the ligase methionine initiation codon) and Bglll (which cuts 997 bp further downstream). The overhanging ends were made blunt-ended with DNA polymerase (Klenow fragment) and the larger of the two fragments ligated with the Ecogpt gene linked to the vaccinia virus 7.5K promoter. The latter had been isolated as an EcoRI fragment and made blunt-ended with DNA polymerase (Klenow fragment). The resulting plasmid in which lkb of the DNA ligase gene had been replaced with the Ecogpt gene was called pSK14 and was used to transfect WT vaccinia virus infected CV-1 cells. Ecogpt expressing viruses were isolated by growth in MPA and the DNA of several isolates analysed by Southern blot (Figure 30). These data show that some of the virus isolates have lost the internal 1 kb region of the DNA ligase gene but are still able to grow well in tissue culture. Consistent with this observation, assays of vaccinia virus DNA ligase in virus-infected cells (by the method described in Kerr & Smith, Nuc. Acids Res. _17, 9039, 1989) showed the absence of detectable DNA ligase in those viruses which had lost the DNA ligase DNA (Figure 31). These data indicate (surprisingly) that the enzyme is non-essential for virus replication in vitro and that the DNA ligase gene is an additional site into which foreign DNA may be inserted into the virus genome. It is also probable that although the DNA ligase gene is non-essential for virus replication in tissue culture, the replication of DNA ligase-deficient viruses will be impaired in vivo and that such viruses will be attenuated. Applications

In a recombinant vaccinia virus vaccine for use either in vaccination programmes or for use as an immunogen in the preparation of antibodies, the gene encoding the immunogen is isolated and introduced into the virus vector by conventional genetic engineering techniques, and the virus vector is transferred into the host, e.g. humans or animals by vaccination. Where the recombinant virus vaccine is being used for antibody production, antibodies to the immunogen are either extracted from the host antiserum (or unpurified antiserum may be used) using standard techniques well known in the art. Monoclonal antibodies may also be prepared from the cells of the immunised animals using standard techniques well known in the art.

The peptides encoded by the amino acid sequences encoded by the nucleotide sequences provided may be produced using conventional genetic engineering techniques. The gene sequences identified also provide sites for the insertion of 'foreign' gene sequences into the vaccinia virus genome and may cause virus attenuation due to inactivation of the vaccinia genes.

Claims

1. A vaccinia virus vector wherein: a) part or all of one or more of the following nucleotide sequences is deleted from the viral genome; and/or b) one or more of said nucleotide sequences is inactivated by mutation or the insertion of foreign DNA; and/or c) one or more of said nucleotide sequences is changed to alter the function of a protein product encoded by said nucleotide sequence; which nucleotide sequences are sequences designated herein as i) Sal F 3R ii) Sal F 9R iii) Sal F 13R iv) B5R v) Sal F 15R.

2. A vaccinia virus according to claim 1 which comprises DNA sequences encoding one or more heterologous polypeptides.

3. A vaccinia virus according to claim 2 wherein the DNA sequences encoding one or more heterologous polypeptides are inserted into one or more ligation sites created by deleting part or all of said one or more nucleotide sequences.

4. A vaccinia virus according to claim 1 which has enhanced immunogenicity.

5. A vaccinia virus according to claim 4 wherein a) part or all of one or more vaccinia nucleotide sequences causing immunosuppression are deleted from the viral genome; and/or b) one or more of said vaccinia nucleotide sequences causing immunosuppression is inactivated by mutation or the insertion of foreign DNA; and/or c) one or more of^*>said vaccinia nucleotide sequences causing immunosuppression is changed to alter the function of a protein product encoded by said nucleotide sequence; which nucleotide sequences are sequences designated herein as i) Sal F 3R ii) Sal F 9R iii) Sal F 13R iv) B5R v) Sal F 15R.

6. A vaccinia virus according to claim 5 wherein the vaccinia nucleotide sequence is a sequence designated herein as Sal F 3R.

7. A vaccinia virus according to claim 4 which comprises a DNA sequence encoding a heterologous polypeptide which potentiates the immune response.

8. A vaccinia virus according to claim 7 wherein the DNA sequence encodes CD23.

9. A vaccine which comprises a vaccinia virus vector according to claim 1.

10. A medicament which comprises a vaccinia virus vector according to claim 1.

11. A polypeptide encoded by part or all of any of said nucleotide sequences; which sequences are designated herein as i) Sal F 3R ii) Sal F 9R iii) Sal F 13R iv) B5R v) Sal F 15R and alleles and derivatives of said polypeptide.

12. A polypeptide according to claim 11 which is encoded by a nucleotide sequence designated herein as Sal F 15R and which has activity as a DNA ligase.

13. A method of attenuating a vaccinia virus vector which comprises: a) deleting part or all of one or more of the following nucleotide sequences from the viral genome; and/or b) inactivating one or more of said nucleotide sequences by mutating said nucleotide sequences or by inserting foreign DNA; and/or c) changing said one or more nucleotide sequences to alter the function of a protein product encoded by said nucleotide sequence; which nucleotide sequences are sequences designated herein as: i) Sal F 3R ii) Sal F 9R iii) Sal F 13R iv) B5R v) Sal F 15R.

14. A method which comprises using a vaccinia virus vector according to claim 1 to prepare a vaccine or a medicament.

15. A method of using a vaccinia virus vector according to claim 1 as an immunogen for the production of antisera, monoclonal antibodies, polyclonal antibodies or T cells with specificity for a heterologous peptide encoded by a DNA sequence inserted into the viral genome; which method comprises immunising an animal with said vaccinia virus vector.

16. Monoclonal antibodies, polyclonal antibodies, antisera and/or T cells obtained by use of the method of claim 15.

17. Diagnostic test kits comprising monoclonal antibodies, polyclonal antibodies, antisera and/or T cells according to claim 16.

18. A method of using part or all of the nucleotide sequence designated herein as Sal F 15R, or part or all of the amino acid sequence encoded by said nucleotide sequence, to identify polypeptides with activity as a DNA ligase.