EP0656949A1 - Virus de la vaccine b15r utilise pour traiter un etat impliquant l'interleukine b1 - Google Patents

Virus de la vaccine b15r utilise pour traiter un etat impliquant l'interleukine b1

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Publication number
EP0656949A1
EP0656949A1 EP93905517A EP93905517A EP0656949A1 EP 0656949 A1 EP0656949 A1 EP 0656949A1 EP 93905517 A EP93905517 A EP 93905517A EP 93905517 A EP93905517 A EP 93905517A EP 0656949 A1 EP0656949 A1 EP 0656949A1
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Prior art keywords
cells
interleukin
receptor
binding
virus
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German (de)
English (en)
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Geoffrey Lilley Smith
Antonio Javier Alcami
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Oxford University Innovation Ltd
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Oxford University Innovation Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to vaccinia virus proteins, uses and products relating thereto.
  • IL-l ⁇ receptor is used in the text as referring to the vaccinia virus B15R polypeptide or allele or functionally equivalent fragment, derivative or variant thereof, the word "receptor” should be interpreted as referring to a substantially soluble binding protein.
  • VV Vaccinia virus
  • the cytoplasmic site of replication requires that vaccinia virus encodes many enzymes and protein factors necessary for transcription and replication of its genome.
  • the virus also encodes a variety o£ factors which modulate virus replication in the multicellular host and aid evasion of the host immune system (Moss, B. (1990a)).
  • 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 humoral and cell-mediated immune responses, the stability of the viral vaccine without refrigeration and the practicality of inserting multiple foreign genes from different organisms into vaccinia virus, to construct polyvalent vaccines effective against multiple pathogens.
  • a disadvantage of this approach is the use of a virus vaccine that has been recognised as causing rare vaccine-related complications. It would therefore be of considerable value to produce a vaccinia virus with an attenuated phenotype as a basis for the production of a new generation of live recombinant vaccinia virus vaccines.
  • Vaccinia virus contains two genes designated B15R and B18R from near the right inverted terminal repeat (ITR) which are each predicted to encode a soluble glycoprotein that has three immunoglobulin-like (Ig) domains, but no transmembrane anchor sequence of cytoplasmic tail (Smith, G.L. and Chan Y.S. 1991).
  • ITR inverted terminal repeat
  • the product of B18R is apparently expressed on the surface of the infected cell early during infection and antibodies directed against a mixture of it and other proteins confer resistance to virus infection without directly neutralizing infectivity (Ueda, Y. , Morikawa, S. and Matsuura, Y. (1990); Ueda and Tagaya, I. (1973) J. Exp. Med.
  • this binding is specific for IL- ⁇ as IL- ⁇ and the IL-1 receptor antagonist protein (IL-lra) do not bind to the B15R gene product.
  • one or more of the gene sequences B15R and B18R may be changed to alter the function of a protein product encoded by the nucleotide sequence.
  • Mutation of the nucleotide sequence may " be effected by the deletion, addition, substitution or inversion of one or more nucleotides.
  • 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.
  • a heterologous peptide is one not normally coded for by wild type vaccinia virus.
  • the heterologous nucleotide sequence will encode an immunogen or a desirable polypeptide product.
  • An immunogenic polypeptide will be substantially homologous to an epitope expressed by a pathogenic organism during infection, and which is seen by the infected individual as foreign.
  • the recombinant vaccinia viruses as described have the potential for enhanced immunogenicity. This may result from the deletion of vaccinia genes B15R and/or B18R which cause immunosuppression.
  • the recombinant vaccinia vectors as described 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 term antibody as used above should be construed as also covering antibody fragments and derivatives of a parent antibody and which have the same specificity as the parent antibody.
  • the monoclonal antibodies, polyclonal antibodies, antisera and/or T-cells produced by the use of the recombinant virus vectors hereof can be used in the diagnostic tests and procedures, for example, in detecting antigen in a clinical sample. They can also be used therapeutically or prophylactically for administration by way of passive immunisation.
  • Diagnostic test kits may comprise monoclonal antibodies, polyclonal antibodies, antisera and/or T- cells obtained by the use of the recombinant vaccinia vectors described.
  • Vaccines and medicaments may comprise a recombinant vaccinia virus as described. These may have enhanced safety and immunogenicity over current vaccinia virus strains for the reasons indicated.
  • a method of attenuating a vaccinia virus vector may comprise: 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) B15R, ii) B18R.
  • a vaccinia virus vector as described may be used to prepare a vaccine or a medicament.
  • the translation products encoded by the nucleotide sequences B15R and B18R may have pharmaceutical utility. In particular, they may have utility as anti-inflammatory medicaments. These genes and/or translation products thereof may be used in processes relating to the preparation of an anti-inflammatory medicament.
  • the gene product B15R binds IL-1, though not IL-6. Surprisingly, this binding is specific for IL-l ⁇ as IL-l ⁇ and IL-lra do not bind to the B15R gene product. Therefore, the B15R translation product or functional equivalents thereof will have useful pharmaceutical applications.
  • IL-l ⁇ is implicated in chromic inflammatory conditions such as rheumatoid arthritis and it has the following functions in relation to inflammation: i) it acts as a T-cell activator; ii) it induces fibroblast proliferation; iii) it increases the production of mediators of the inflammatory response e.g. prostaglandin-E2 from fibroblasts and iv) it increases the production of collagenase from chondrocytes (Digiovine F.S. et al, 1990, Immunol. Today Vol.11 pl3-20). Therefore the gene product of B15R which can bind to IL-l ⁇ provides a means of blocking the activity of IL-l ⁇ in order to control and/or reduce the symptoms of inflammation.
  • IL-l ⁇ acts as a mediator in septic shock and it is a growth factor for certain malignant cells e.g. leukaemic cells. Therefore the gene product of B15R provides a means for controlling septic shock and growth of certain tumour cells.
  • IL-l ⁇ receptor may be used to control disease mediated by excessive fever.
  • the present invention provides use of the nucleotide sequence designated herein as B15R, or of a nucleotide sequence coding for an allele of, or functionally equivalent fragment, derivative or variant of the polypeptide encoded by the B15R nucleotide sequence, in a process relating to the manufacture of a medicament for the treatment of a condition in which IL-l ⁇ is involved in the mediation of one or more symptoms associated with the condition.
  • polypeptide which is encoded by the nucleotide sequence designated herein as B15R or of an allele of, or functionally equivalent fragment, derivative or variant of the polypeptide, to manufacture a medicament for the treatment of a condition in which IL-l ⁇ is involved in the mediation of one or more symptoms associated with the condition.
  • An allele of, or functionally equivalent fragment, derivative or variant of the B15R polypeptide may be any polypeptide with substantial homology to part or all of the B15R polypeptide and which also has the ability to bind IL-l ⁇ .
  • Conditions may be fever, inflammation, diseases such as rheumatoid arthritis in which inflammation is presented as a symptom, septic shock, and certain leukaemias and/or cancers, inflammatory bowl disease, graft versus host disease and diabetes.
  • diseases such as rheumatoid arthritis in which inflammation is presented as a symptom, septic shock, and certain leukaemias and/or cancers, inflammatory bowl disease, graft versus host disease and diabetes.
  • the results disclosed hereafter also demonstrate that surprisingly the cytokine IL-l ⁇ is the primary mediator of fever.
  • the present invention particularly provides use of the nucleotide sequence designated herein as B15R, or a nucleotide sequence coding for an allele of, or functionally equivalent fragment, derivative or variant of the polypeptide encoded by the B15R nucleotide sequence in a process relating to the manufacture of a medicament for the treatment of fever. Also provided is use of a polypeptide encoded by the nucleotide sequence designated herein as B15R or of an allele of, or functionally equivalent fragment, derivative or variant of the polypeptide to manufacture such a medicament. Also provided is a pharmaceutical which is an anti-fever medicament.
  • a pharmaceutical which is an anti-fever medicament comprising a polypeptide which is encoded by the nucleotide sequence designated herein as B15R, or an allele of, or functionally equivalent fragment derivative or variant of the polypeptide.
  • a diagnostic reagent for the detection or measurement of interleukin-l ⁇ in a sample which reagent comprises a polypeptide encoded by the nucleotide sequence designated herein as B15R, or of an allele of, or functionally equivalent fragment, derivative or variant of the polypeptide.
  • a diagnostic kit which comprises a diagnostic reagent as described above and one or more ancillary kit components for making the detection or measurement of interleukin-1 ⁇ .
  • Also provided is a method of diagnosing a patient's condition in which interleukin-l ⁇ is involved in the mediation of one or more symptoms associated with the condition which method comprises testing a sample of biological material obtained from the patient using a diagnostic reagent or kit as described above.
  • the proteins may be produced in a recombinant system according to techniques well known in the art.
  • the nucleotide sequences provided herein could be inserted into a suitable expression vector (not necessarily vaccinia, for example the baculovirus system described herein) .
  • a suitable expression vector not necessarily vaccinia, for example the baculovirus system described herein.
  • Such vectors can then be used to infect or transform a cell line suitable for the production of these particular proteins.
  • Reagents comprising polypeptides such as the B15R gene product, or alleles of or functionally equivalent fragments, derivatives or variants of that gene product may be used as research tools as a means to study the function of IL-l ⁇ versus IL-l ⁇ .
  • sequence information for B15R and B18R and identified the ' location of their nucleotide sequences in the viral genome. Having done this, it is within the capacity of one skilled in the art to either inactivate these sequences in or delete these sequences from the VV genome or change them to alter the function of the encoded protein product. All the necessary standard procedures are described in Molecular Cloning, eds. Sambrook, Fritsch and Maniatis, Cold Spring Harbour Laboratory Press 1989. Furthermore, it would be within the normal capacity of one skilled in the art to use the sequence information provided herein to develop useful pharmaceuticals and immunogens as herein provided; to use the immunogens to produce antibodies and the like; to use the antibodies in kits and pharmaceuticals. Brief Description of the Drawings
  • Figure 1(A) HindiII restriction map of the 186kb W genome.
  • the 9.8kb Sail I fragment is expanded to show the position and direction of transcription of the genes B15R and B18R and the serpin genes (Smith, G.L., Howard, S.T. and Chan, Y.S. (1989). J. Gen. Virol. 70, 2333-2343) (this nomenclature indicates the genes are the fifteenth and eighteenth ORFs starting from the left end of the HindiII B and are transcribed rightwards towards the genomic terminus) .
  • Figure 1(B) Nucleotide sequence and deduced amino acid sequence of gene B15R.
  • NXS/T N-linked carbohydrate
  • FIG. 1(C) Nucleotide sequence"and deduced amino acid sequence of gene B18R. The three amino acid positions at which the sequence differs from the published sequence of this gene from another strain of VV (Ueda, Y. , Morikawa, S. and Matsuura, Y. (1990)) are shown. Other features as marked in (B).
  • FIG. 1 Amino acid alignment of the Ig domains from B15R and B18R with the Ig-like domains of the human and murine IL-IRI, the human IL-6R, the VV haemagglutinin (VV HA), domain 1 of the fasciclin II, domain 3 of myelin-associated glycoprotein and the V-domain of Ig kappa.
  • the regions predicted to form the ⁇ -strand structures of Ig-like domains are indicated above the alignment. Residues identical in 6 or more sequences are boxed. A few residues between the ⁇ -strands B and C have been omitted. Also omitted for brevity are ⁇ -strands D and, where appropriate, C'and C". Higher numbers of residues (about 30 or more) between strands C and E are indicative of the V domains.
  • Figure 3 Amino acid alignment of B15R with the external regions of the IL-IRI from human and mouse and the signal sequence and single Ig domain of human IL-6R. Gaps have been introduced to maximise the sequence alignments and are indicated by dashes. Where 4 sequences are aligned, the boxes indicate identical amino acids in three sequences, otherwise boxes indicate complete conservation in all aligned sequences.
  • the sizes of the probes and fragments protected are indicated in bases. Indicated below the autoradiographs are the probe position relative to the ORFs (underline and asterisks) , the nucleotide and the deduced amino acid sequence at the 5' end of the ORFs, the vaccinia late promoter consensus sequence (underline), and the sites of transcriptional initiation (asterisks) .
  • FIG. 1 Structure of Recombinant Vaccinia Virus Genomes.
  • Vaccinia virus DNA was digested with HindiII (A) or Clal (B), and fragments were resolved on an agarose gel and transferred to nitrocellulose. Filters were probed with fragments containing the gene and flanking sequences of B15R (lane a) or B18R (lane b), with an internal oligonucleotide to B15R (lane c) or an internal fragment to B18R (lane d). Sizes in kilobases are indicated.
  • B15R from vaccinia virus- infected cells.
  • BS-C-1 cells were mock infected (M) or infected with WR, vB15R, or v B15R and pulse-labeled with ⁇ 5 STrans-label either from 2 to 4hr after infection in the presence of cytosine arabinoside (E) or from 6 to 8 hr after infection in the absence ( L) or presence (T) of tunicamycin.
  • E cytosine arabinoside
  • T tunicamycin
  • B Identification of B18R from vaccinia virus- infected cells.
  • BS-C-1 cells were infected with vB18R or v B18R and pulse-labeled with 35 STrans-label from 6 to 8h after infection, the cell extracts and media were immunoprecipitated with antiserum raised against B18R expressed in baculovirus-infected cells, and the samples were resolved by SDS-PAGE. A fluorograph is shown. As in (A), the quantity of sample from medium was estimated to correspond to about four times the amount analyzed from cells. Molecular size markers and the size of the B18R gene products (closed arrowhead) are indicated in kilodaltons.
  • (C) Expression of B15R and B18R in baculovirus- infected insects cells Sf cells infected with AcNPV, AcB15R, or AcB18R were pulse-labeled with 35 STrans-label for 2 hr after 24 hr of infection. Proteins present in cells and media were analyzed by SDS-PAGE and visualized by autoradiography. As in (A), the quantity of sample from medium was estimated to correspond to about four times the amount analyzed from cells.
  • the B15R (open arrowhead) and B18R (closed arrowhead) gene products and the molecular size markers are indicated in kilodaltons. The positions of ⁇ -galactosidase ( ⁇ gal) , coexpressed with B15R and B18R in the recombinant baculoviruses, and polyhedrin (P), expressed only. in AcNPV, are shown.
  • EL4 6.1 CIO cells, U266 cells, mock- or vaccinia (WR)- infected TK ⁇ 143 cells, and Sf cells infected with AcNPV, AcB15R, or AcB18R were dotted onto nitrocellulose filters, and the membranes were incubated with radioiodinated IL-l ⁇ (120 pM), IL-l ⁇ (200 pM) or IL-6
  • FIG. 8 Binding Assay in Solution to Recombinant Viruses.
  • the radioactivity bound to soluble receptor present in supematants is represented.
  • FIG. 10 Competition Experiments to EL4 6.1 CIO and U266 Cells.
  • Different cell equivalents of medium from baculovirus-infected cells expressing B15R (AcB15R; open circle) or B18R (AcBl ⁇ R; closed circle) were incubated with 130 pM of 125 I-IL-l ⁇ (A), 180 pM of 12 I-IL-l ⁇ (B), or 100 pM of 125 I-IL-6 (C) for 1 hr at 4°C.
  • the percentages refer to the binding in the absence of competitor, which was 3720 cpm for 125 I-IL-l ⁇ and EL4 6.1 CIO cells (A), 2040 cpm for 125 I-IL-l ⁇ and EL4 6.1 CIO cells (B), and 4963 cpm for 125 I-IL-6 and U266 cells (C).
  • FIG. 11 Effect of the Deletion of B15R from Vaccinia Virus on the Infection of Mice.
  • A Groups of five mice were intransally infected with 3 x 10 7 (panels a), 10 7 (panels b), 10 6 (panels c), 10-- * (panels d) or 10 4 (panels e) pfu of WR (open circle) or v B15R (closed circle) and examined daily for- symptoms of illness or death. The number of animals that presented strong symptoms of illness (including death) and the accumulated number of mortalities are represented for each dose of virus at different days of infection. No differences were observed between day 12 and day 17.
  • the (f) panels summarize the onset of symptoms (left) and the number of mortalities (right) that occurred at different days after infection.
  • mice were intransally infected with 10 * -- * (panels a) of 10 4 (panels b) pfu of WR (open circle) or v B15R (closed circle) . Symptoms of illness were scored from zero to four, and the mean value of each group was represented. Animals were weighed individually each day and the mean group weight was expressed as the percentage of the mean weight of that group of animals immediately prior to infection. No mortalities occurred at these doses of virus.
  • FIG. 12 Effect of Expression of the Vaccinia IL- l ⁇ Receptor on Mice Infected with Vaccinia Virus.
  • Cowpox Tissue culture medium (1 x 10 * ⁇ cell equivalents) from TK ⁇ 143 cells infected with the indicated viruses was incubated in a binding assay in solution with 100 pM of radioiodinated IL-l ⁇ or mIL-l ⁇ , expressed in femtomoles, is shown.
  • One femtomole corresponded to 935 or 535 cpm for IL-l ⁇ or mIL-l ⁇ , respectively.
  • FIG. 14 Kinetics of symptoms of illness and mortality in mice infected with recombinant vaccinia viruses.
  • BALB/c mice were intranasally infected with WR, v B15R or v B18R as described in Table 2.
  • the nucleotide sequence of the Sail I restriction fragment of the vaccinia virus genome were determined by established methods (Sanger, F. et al.
  • the 9.8kb Sail I fragment of vaccinia virus (strain WR) was isolated from cosmid 6, which ' contains virus DNA derived from a rifampicin resistant mutant (Baldick, CJ. & Moss, B. (1987) Virology 156, 138-145), and was cloned into Sail cut pUC13 to form plasmid pSall I.
  • 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 [ ⁇ S]-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 6.3 kb Sail L fragment was similarly treated.
  • 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 verion 14 and. against the applicant's own database of vaccinia amino acid sequences using programme FASTP (Lipman, D.J. & Pearson, W.R.
  • IL-1 a cytokine produced in response to infection and tissue injury
  • IL-1 a cytokine produced in response to infection and tissue injury
  • the two forms of IL-1, IL-l ⁇ and IL-l ⁇ produce similar biological effects that are mediated by interaction with specific receptors in different cells.
  • the 80 kDa type I IL-1 receptor is found on T cells and fibroblasts (Bird, T.A.
  • Binding studies have shown heterogeneity in the IL-1 receptor on B cells concerning the affinity of IL-l ⁇ or IL-l ⁇ (Benjamin, D. , et al, (1990)).
  • There are different natural inhibitors that modulate the biological effect of IL-1 (Larrick, J.W. (1989); Shields, J. , and Mazzei, G.J. (1991)).
  • IL-1 receptor antagonist IL-1 receptor antagonist
  • Genes B15R and B18R (Figure 1) from near the right hand inverted terminal repeat (ITR) are predicted to encode proteins of 36.5 kDa and 40.7 kDa, respectively, that have an N-terminal hydrophobic sequence, possible attachment sites for N-linked carbohydrate and hydrophobic residues near the C-terminus. These properties are consistent with the mature proteins being either virion, cell-surface or secretory glycoproteins.
  • the nucleotide sequence and deduced amino acid sequence around the gene designated B15R is shown in figure IB.
  • nucleotide sequence shown is 11462-12664 nucleotides from the left end of the vaccinia virus Hindlll B fragment and the coding region for B15R is at nucleotide positions 11584-12561 (or at nucleotides 815 to 1792 from the left end of the Sail I fragment).
  • the nucleotide sequence and deduced amino acid sequence around the gene designated B18R is shown in figure lc.
  • the nucleotide sequence shown is 15448-16741 nucleotides from the left end of the vaccinia virus HindiII B fragment and the coding region for B18R is at nucleotide positions 15568-16621 (or at nucleotides 4799 to 5851 from the left end of the Sail I fragment).
  • the single letter code is used for the designation of amino acids.
  • B15R and B18R each possess three domains with characteristics of the immunoglobulin ( Ig) superfamily (Williams, A.F. and Barclay, A.N. (1988). Ann. Rev.
  • B15R, B18R and the IL-lRs located near the beginning of ⁇ -strands A and G in domain 1 and at similar positions in domain 2 of the IL-lRs and B18R.
  • These cysteines lie within the 3-dimensional structure of an Ig C domain in positions probably allowing another intradomain disulphide bond.
  • Domain 3 does not contain additional cysteines and is longer than 1 and 2 in B15R, B18R and the IL-lRs.
  • VV proteins are related to each other (22.5% identity), to the human and murine IL-1R, the human IL-6R (Yamasaki, K., Taga, T., Hirat, Y. , Yawata, H., Kawanishi, Y. , Seed, B., Taniguchi, T., Hirano, T. and Kishismoto, T. (1988). Proc. Jpn. Acad, 64, 209-211) and the immunoglobulin (Ig) superfamily (Williams, A.F. and Barclay, A.N. (1988). Ann. Rev. Immunol. 6, 381-405).
  • VV haemagglutinin is another member of this superfamily (Jin, D. , Li, Z., Jin, Q. , Yuwen, H. and Hou, Y. (1989). J. Exp. Med. 170, 571-576).
  • the B18R sequence from VV strain IHD was recently reported but the relationship to interleukin receptors and the Ig superfamily was not described (Ueda, Y., Morikawa, S. and Matsuura, Y. (1990). Virology 177, 588- 594).
  • Cytokines IL-1 and IL-6 by binding to their respective natural receptors mediate immune responses against an invading pathogen and cause inflammation. VV may be combatting this part of the immune response by producing proteins which mimic the receptors for IL-1 and IL-6.
  • B15R and B18R ORFs are actively transcribed, translated, and secreted to the medium during the vaccinia virus replication cycle.
  • the B15R gene product is shown to bind IL-l ⁇ when expressed from vaccinia or from recombinant baculovirus.
  • the role of the IL-l ⁇ binding activity in the biology of vaccinia virus was investigated by deleting the gene from the virus genome and analyzing the biological effects on infected mice. The biological effects on infected mice were also investigated for vaccinia virus without the B18R coding sequence. The presence of the binding activity in other orthopoxviruses is also presented.
  • the cell line EL4 6.1 CIO a subclone of the mouse thymoma EL4 that expresses a high number of IL-1 binding sites (MacDonald et al, 1985), was a gift of H.R. MacDonald (Ludwig Institute for Cancer Research, Lausanne, Switzerland).
  • U266 cells overexpressing IL-6 receptors (Taga, et al 1987) were obtained from the Cell Bank of the Sir William Dunn School of Pathology
  • Sf 21 insect cells and AcNPV were obtained from R. Possee (Natural Environmental Research Council Institute of Virology and Environmental Microbiology, Oxford) and were cultured in TC100 medium (GIBCO) containing 10% fetal calf serum (Brown and Faulkner, 1977).
  • Vaccinia virus strain WR and recombinants derived from it were grown in CV-1 or BS-C-1 cells. TK ⁇ 143 and HeLa D98 cells were used for the selection of recombinants.
  • the WR strain was obtained from B. Moss (National Institute of Health, Bethesda, Maryland) and cells were obtained from the American Type Culture Collection. Cells were grown in minimal essential medium (GIBCO) supplemented with 10% fetal calf serum. Purified virus stocks were prepared by sedimentation through a sucrose cushion (Mackett et al, 1985). The Tashkent, IHD-J, and IHD-W strains of vaccinia virus and cowpox virus were obtained from M.
  • Copenhagen strain were obtained from J. Zhou (Princess Alexandra Hospital, Brisbane, Australia) and R. Drillien (University Louis Pasteur, France), respectively. Reagents
  • Radioiodinated human recombinant IL-6 was purchased from Amersham. IL-l ⁇ had been, radioiodinated using the chloramine-T procedure to a specific activity of 70-120 ⁇ Ci/ ⁇ g. IL-l ⁇ and IL-6 had been labeled with Bolton Hunter reagent to a specific activity of 80-180 ⁇ Ci/ ⁇ g and 800-1200 Ci/mmol, respectively. Unlabeled human recombinant IL-l ⁇ (code 86/632), IL-l ⁇ - (code 86/680), and IL-6 (code 88/514) were obtained from the National Institute for Biological Standards and Control (South Mimms, Hertfordshire, England).
  • pAAl was digested with EcoRV and Xbal, and the largest fragment was gel purified, end filled with Klenow fragment, ' and self-ligated to form pAA3, which contains 348 bp of the 3' flanking region.
  • a PCR copy of 5' region of the ORF was constructed, using plasmid pAAl as template and an oligonucleotide containing the sequence of the first 21 nt of B15R and the recognition sequence for BamHI and Ncol (B15R-1; 5'-CCCGGATCCACCATGGGTATACTACCTGTTATA-3 ' ) and an oligonucleotide that hybridizes to an internal sequence of B15R, corresponding to nucleotides 1008-1025 of the Sail I fragment (B15R-2; 5' -CCGCTCCTCGTTTTTCCC-3' ) .
  • the fourth nucleotide of B15R in the PCR fragment was"G instead of A to create a Ncol recognition sequence, giving rise to a serine to glycine substitution in the second amino acid of the protein.
  • Th 222 bp PCR fragment was digested with AccI, forming a 20 bp fragment containing the BamHI and Ncol restriction sites bound to the first 8 nt of B15R, and was treated with polynucleotide kinase.
  • This fragment was cloned into pAA3 digested with Hindi and AccI, which removed the 5' flanking region, to render plasmid pAA4. This construct was confirmed by DNA sequencing. Cloning of B18R into pUC4K
  • the transfer vector used for overexpression of B15R and B18R in vaccinia virus was pRK19 (Kent, R.K. 1988), which contains the vaccinia virus 4b promoter to control the transcription of the inserted gene, flanked by sequences of the TK gene that allow insertion in the TK locus of the virus genome.
  • BamHI fragments containing the ORFs were excised from pAA4 and pAA5 and cloned into the BamHI site of pRK19, and the resulting plasmids were named pAAlO and pAAll, respectively.
  • the transfer vector for construction of baculovirus recombinants was pAcDZl, which uses the polyhedrin promoter to drive the transcription of foreign genes and coexpresses Escherichia coli ⁇ -galactosidase for selection of the recombinants ( Zuidema et al, 1990).
  • This vector was provided by J.M. Vlak (Department of Virology, Agricultural University, Wageningen, The Netherlands) .
  • the genes were excised from pAA4 and ⁇ AA5 with BamHI and inserted into BamHI-cut pAcDZl, forming pAA14 and pAA15, respectively.
  • flanking sequences of B15R and B18R were excised from pAAl or clones from a M13 library containing random subfragments of the SailI fragment of vaccinia DNA, which were used to sequence this region of the vaccinia virus genome (Smith et al, 1991a), and were cloned into pSJH7 (Hughes et al, 1991).
  • the 5' flanking region of B15R was obtained by digestion of the replicative form of the M13 clone SalII.144 with EcoRI and SphI, and the 3' flanking region was excised from pAAl by digestion with SphI and BamHI.
  • Both fragments were, cloned in one step into EcoRI- and BamHI-cut pSJH7.
  • the resultant plasmid, called pAA16 contained 360 and 1316 nt of the 5' and 3' flanking sequence, including 17 and 252 nt of the coding sequence, respectively, so that 72% of the B15R coding sequence was deleted.
  • DNA fragments containing the flanking sequences of B18R were obtained by BamHI and EcoRI digestion of the replicative form of the M13 clones SalII.44 and SalII.81.
  • PCR fragment was obtained with the oligonucleotide B15R-2 (above) and the 17-mer sequencing primer (-20), using pAAl as template.
  • the PCR product was purified, labeled with [ - 2 P]ATP and polynucleotide kinase, and subsequently digested with Sail, giving rise to a 1024 bp fragment containing 211 nt of the 5 ' coding region of B15R.
  • pAA2 was digested with EcoRI, a band of 505 bp was purified and dephosphorylated with calf intestinal alkaline phosphatase, and the 5' ends were labeled with [ - 3 P]ATP using polynucleotide kinase. After digestion with Dral, a fragment of 379 bp was isolated that contained 98 nt corresponding to the 5' coding region of B18R.
  • Both 3 P-labeled fragments specific for B15R and B18R were hybridized to lO ⁇ g of vaccinia virus RNA obtained at 8 hr after infection from cells infected in the presence (early) ' or absence (late) of cycloheximide or yeast transfer RNA.
  • the hybrids were digested with SI nuclease, and the protected fragments were separated on 6% polyacrylamide sequencing gel and detected by autoradiography as described (Moore and Smith, 1992). An M13 sequencing ladder was used as size markers.
  • Sf cells were cotransfected with purified AcNPV DNA and pAA14 or pAA15 using the calcium phosphate precipitation technique, and the recombinant viruses were identified by staining with X-Gal as described ( Zuidema et al, 1990).
  • the insertion of foreign genes (B15R and B18R) into the baculovirus genome was confirmed by Southern blot hybridization of 2 P-labeled specific probes on viral DNA digested with Hindlll (data not shown).
  • the recombinant viruses expressing B15R and B18R were plaque purified five times and called AcAA3 and AcAA4, respectively, and are referred to here as AcB15R and AcB18R.
  • Recombinant vaccinia viruses were constructed by standard procedures (Mackett et al, 1985). The genomes of viruses containing a second copy of B15R or B18R in the TK locus of the vaccinia DNA were analyzed by Southern blotting, using viral DNA extracted from virus cores (Esposito et al, 1981). These structures were confirmed by PCR using oligonucleotides that hybridized to the 5' and 3' ends of the TK gene, which gives rise to a longer PCR product in the recombinant viruses compared with WR owing to the insertion of foreign DNA in the TK locus (data not shown).
  • vaccinia viruses containing a second copy of B15R and B18R were called vAAl and vAA4, respectively, and are referred to here as vB15R and vB18R.
  • Vaccinia virus deletion mutants were constructed by transient dominant selection as described elsewhere ( Falkner and Moss, 1990; Isaacs et al, 1990).
  • Vaccinia viruses containing deleted versions of B15R and B18R were termed vAA5 and vAA6, respectively, and are referred to here as v B15R and v B18R.
  • the B15R-specific probe containing the ORF and 348 bp of the 3 ' flanking region was excised from pAA4 by digestion with BamHI and used for Southern blot hybridization.
  • the oligonucleotide B15R-2 was used as an internal probe for B15R ORF.
  • the internal probe for B18R was obtained by excision of a 424 bp EcoRI fragment from pAA2.
  • Sf or BS-C-1 cells were infected with the baculovirus or vaccinia virus recombinants, respectively, at high multiplicity of infection (20-40 pfu/cei-1).
  • infected cells were pulse-labeled with 750 ⁇ Ci/ml 35 STrans-label (ICN Biomedicals; a mixture of -80% [ S]methionine and -20% [ 35 S]cysteine, 1200 Ci/mmol) in methionine-free TC100 medium or methionine- and cysteine-free minimal essential medium, respectively, in the absence of serum.
  • Cytosine arabinoside 40 ⁇ g/ml
  • tunicamycin (1 ⁇ g/ml
  • Sf cells and TK ⁇ 143 cells grown in 175 cm 2 or 80 cm 2 flasks, were infected at a density of 1.5 x 10 ** - to 2 x 10 ⁇ cells/cm 2 with a multiplicity of infection of 5-10 pfu per cell in serum-free medium.
  • Cells and medium were harvested from vaccinia- or baculovirus-infected cells at 1 or 3 days after infection, respectively.
  • the final concentration of the supematants was 1 x 10° to 5 x 10° cell equivalents per millilitre.
  • the medium was centrifugated at 3000 rpm for 10 min at 4°C, the pellet discarded, and supematants made 20 mM HEPES (pH 7.4) and 0.1% sodium azide.
  • Supematants were stored at -70°C until used in binding assays in solution or concentrated and dialized against phosphate-buffered saline (PBS) at 4°C in a Micro-ProDiCon (Bio-Molecular Dynamics) with PA- 10 ProDiMen dialysis membranes (MW 10,000) to a-final concentration of 5 x 10 7 cell equivalents per millilitre.
  • the concentrated medium was made 1% in sodium azide and stored at -70°C Cells were detached from the plastic by incubation with 0.5 mM EDTA in PBS and washed twice with PBS, and the pellet was resuspended in 1% Triton X-100 in PBS containing 1 mM phenylmethylsulfonyl fluoride to a final concentration of 1 x 10* ⁇ cells per millilitre. Samples were incubated on ice for 15 min and centrifuged at 12,000 x g for 30 min at 4°C as described (Urdal et al, 1988).
  • the cell extracts were made 1% in sodium azide and stored at -70°C Detergent-solubilized lysates of EL4 6.1 CIO and U266 cells were " prepared in the same way to a final concentration of 4 x 10° cells * per millilitre.
  • the supematants were harvested from cells seeded at a cellular density of 5 x 10 ** - * cells per millilitre and grown in culture over a period of 3 days.
  • the medium was concentrated in a Micro-ProDiCon to 5 x 10 7 cell equivalents per millilitre.
  • Sf and TK ⁇ 143 cells were harvested for binding assays to intact cells by treatment with PBS containing 0.5 mM EDTA.
  • EL4 6.1 CIO, U266, Sf, and TK " 143 cells were washed twice in serum-free medium and resuspended in binding medium. Binding Assays
  • the binding medium used in the different assays was RPMI 1640 containing 20 mM HEPES ( pH 704), 1% bovine serum albumin, and 0.1% sodium azide. Solid phase binding assays on nitrocellulose were performed as described (Urdal et al, 1988). Binding to intact cells was carried out in duplicate in 150 ⁇ l of binding medium for 2 hr at 4°C, and bound *J - 2 ⁇ I-IL was determined by phthlate oil centrifugation as described (Dower et al, 1985).
  • Soluble receptor binding assays were performed by precipitating the ligand-receptor complexes with polyethylene glycol and filtration through Whatman GF/C filters as described by Symons et al (1990). Supematants were incubated in duplicate with labeled ILs in a final volume of 150 ⁇ l for 2 hr at room temperature. Background radioactivity precipitated in the presence of binding medium was subtracted. Kinetics experiments of 125 I-IL-l ⁇ binding to vaccinia virus supematants showed that maximum binding was reached after 5 min at room temperature (data not shown) . The saturation experiments to soluble receptor from WR, vB15R,a nd AcB15R were performed in a final volume of 75 ⁇ l in the same conditions.
  • mice were weighed daily and monitored for signs of illness or death (Turner, 1967; Williamson et al, 1990).
  • an aliquot of the dilutions of v B15R or WR used to inculate the animals was grown in TK ⁇ 143 cells, and the absence or the presence of IL-l ⁇ binding activity in the medium at 24 hr after infection was confirmed in a binding assay in solution (data not shown) .
  • RNAs messenger RNAs
  • Figure 4A shows that the B15R-specific probe was partially protected from SI nuclease digestion by late viral RNA, and the size of the protected fragment mapped the transcriptional start site to the TAAAAT motif at the 5' end of B15R.
  • TAAAT(G) has been shown to constitute a late promoter consensus sequence for vaccinia virus, a few exceptions have been found that possess an additional A TMOSS, 1990b).
  • the B18R-specific probe was protected from SI nucflease digestion by early viral RNA that initiated 16-18 nt upstream of the ORF (Figure 4B) .
  • This is consistent with the presence of vaccinia virus early transcriptional terminator signal TTTTTNT 13 nt downstream of the ORF (Smith and Chan, 1991) and is in agreement with results obtained by primer extension in the Lister strain (Ueda et al., 1990), although analysis of late RNA was not included in the previous report.
  • the weak signal detected at late times probably corresponds to early transcripts still present in the late viral RNA sample since, for ccnstitutively expressed genes, the early and late transcripts initiate at different positions (Moss, 1990b).
  • vB15R and vB18R The genomic structure of the recombinant viruses, called vB15R and vB18R, was confirmed by Southern blot hybridization ( Figure 5A) and by polymerase chain reaction (PCR) using oligonucleotides specific for the 5' and 3' ends of the TK gene (data not shown). Deletion of 72% of B15R or 92% of B18R from the viral genome was carried out by transient dominant selection (Falkner and Moss, 1990; Isaacs et al., 1990). This method allows construction of a virus that only differs from the wild type in the deleted sequence and does not contain any selectable marker that could affect the new phenotype.
  • the protein encoded by B18R was detected in vaccinia virus-infected cells when overexpressed at late times of infection under the strong 4b promoter but was not detected in the deletion mutant ( Figure 6B ⁇ .
  • Two forms of the protein 52kd and 60-65kd) were detected in cell extracts, and only the 60-65 kd protein, presumably containing a higher degree of glycosylation, was secreted to the medium. Since translation of B18R-specific mRNA in rabbit reticuloyte lysates produces a protein of the predicted size of 40 kd (Ueda et al., 1990), the carbohydrate component of the secreted B18R gene product accounts for 33%-38% of the size of the protein.
  • Spodoptera frugiperda insect cells infected with Autographa califomica nuclear polyhedrosis virus (AcNPV) under the control of the polyhedrin promoter.
  • AcNPV Autographa califomica nuclear polyhedrosis virus
  • the recombinant viruses constructed were termed AcB15R and AcB18R.
  • both B15R and B18R proteins were secreted to the medium by insect cells as 40-44 kd and 48 kd polypeptides, respectively.
  • An incomplete glycosylation of the polypeptides in insect cells might explain the lower size of the proteins when compared with the vaccinia virus expression.
  • the 45 kd protein in AcB15R-infected insect cell extracts might correspond to a glycosylated form with a signal sequence still bound to the polypeptide, possibly owing to the inability of insect cells to process properly the high amount of B15R protein expressed under the strong polyhedrin promoter.
  • IL-l ⁇ binding activity was clearly found in vaccinia virus-infected cells and supematants harvested at 24 hr after infection and in the baculovirus recombinant expressing B15R 3 days after infection.
  • the low binding to EL4 6.1C10 cells at this dose of 125 I-IL-l ⁇ probably reflects a 6-fold lower affinity for the ⁇ form compared with IL-l ⁇ , described for the type I receptor expressed in this cell line (Sims et al., 1988).
  • the cell-associated binding activity may correspond to protein present in the secretory pathway or in the plasma membrane.
  • the first possibility was supported by comparing • ⁇ " ⁇ "I-IL-l ⁇ binding with detergent-solubilised cell extracts ( Figure 7B ) and intact cells in suspension, which showed that only 6%-9% of the cell-associated binding activity is detected on the cell surface (data not shown) .
  • IL-l ⁇ receptor The kinetics of production of soluble IL-l ⁇ receptor from vaccinia virus-infected cells was examined by soluble binding assay and showed that no IL-l ⁇ receptor was detected above the background attributable to virus inoculum, in the presence of cytosine arabinoside. In contrast, in the absence of the drug, IL-l ⁇ accumulated in the supernatant and reached 80% of total by 24 hr (data not shown). These data are consistent with the transcriptional and polypeptide analyses and show that the IL-l ⁇ receptor is expressed late during infection.
  • Figure 8 shows the binding of radioiodinated IL-l ⁇ to medium from different recombinants using 1 ⁇ -10 4 cell equivalents, conditions that allowed a better quantitation of the binding activity.
  • the fact that the - ⁇ - ⁇ "I-IL-l ⁇ binding increased in the vaccinia recombinant containing two copies of B15R (vB15R) and was absent in the mutant containing a deleted version of the gene (v B15R) demonstrates that B15R is the only gene product from vaccinia virus responsible for the IL-l ⁇ binding activity.
  • the natural competitor IL-IRA did not block the binding of labelled IL-l ⁇ to vaccinia IL-1 receptor, even when added at higher concentrations that are required to compete the binding of 12 * ⁇ I-IL-1 to the type II IL-1 receptor on polymorphonuclear leukocytes or a pre-B lymphocyte line (Dripps et al., 1991; Granowitz et al., 1991; Mclntyre et al., 1991).
  • the doses of unlabelled ILs used competed the binding of the corresponding . radioiodinated IL to its natural receptor on EL4 6.1 CIO or U266 cells (data not shown).
  • the receptor expressed in the baculovirus system showed similar properties.
  • the binding of lOOpM of 125 I-IL-l ⁇ to 8 x 10 3 cell - equivalents of medium from AcB15R-infected cells (6610 cpm) was 94.1%, 7.4%, 99.0% and 110.1% in the presence of lOnM of IL-l ⁇ , lOnM of IL-l ⁇ , lOOnM of IL-lra, and lOnM of IL-6, respectively.
  • IL-l ⁇ The specificity for IL-l ⁇ was also tested in competition experiments of binding of labelled cytokines to their natural receptors on cell lines overexpressing IL-1 and IL-6 receptors. As shown in Figure 10, the binding of labelled IL-l ⁇ to EL4 6.1 CIO cells (A) and of IL-6 to U266 cells (C) was not competed by medium from baculovirus-infected insect cells expressing B15R or
  • Figure 11A shows that, although there were not significant differences in the final number of mortalities following infection with different doses of WR or v B15R, 70% of the mortalities on v B15R-infected animals occurred 1 day sooner than control (panels f ) .
  • This unexpected and enhanced pathogenicity of y B15R was also demonstrated by the clearly accelerated onset of symptoms with doses of virus from lO * --* to 3 x 10 7 plaque- forming units (pfu) ( Figure 11A, panels a-d) .
  • Figure 11A panels a-d
  • the IL-l ⁇ , IL-l ⁇ , and IL-6 binding activity was investigated in a soluble receptor binding assay on supematants from cultures infected with different strains of vaccinia virus (Copenhagen, IHD-J, IHD-W, Wyeth, Lister, Tian-Tan, and Tashkent) and the related orthopoxviruses rabbitpox and cowpox compared with the WR strain.
  • the binding to labelled murine IL-l ⁇ (mIL-l ⁇ ) was also investigated to confirm that B15R is able to sequester IL-l ⁇ in infected mice. No binding to human 12**** I-IL-l ⁇ or -"--"I-IL-6 was detected (data not shown).
  • B15R protein is shown to suppress the temperature of the infected mouse over the first six days of infection.
  • Animals infected with the wild-type (WT ) virus (B15R +ve ) have a reduced temperature compared with those infected with the deletion mutant (B15R -ve ) and re-insertion of the B15R gene into the deletion mutant virus restored the temperature profile of the infected animals to that typical of WT virus.
  • WT wild-type virus
  • B15R -ve deletion mutant
  • re-insertion of the B15R gene into the deletion mutant virus restored the temperature profile of the infected animals to that typical of WT virus.
  • the applicants have compared the temperatures of mice infected with different strains of vaccinia virus, which had been characterised previously with respect to the expression or non-expression of the IL-l ⁇ receptor (Alcami and Smith, Cell 71, 153-167,
  • mice infected with the Tian- Tan strain which expresses the IL-l ⁇ receptor, have reduced temperatures following infection, while those animals infected with strains lacking the receptor (Copenhagen and Tashkent) develop fever.
  • ORFs Two vaccinia virus ORFs, B15R and B18R, that encode proteins of the immunoglobulin superfamily related to the extracellular domains of the IL-1 and IL-6 receptors have been characterized. Both ORFs are transcribed, but at different phases of the virus replication cycle, suggesting functional differences. The gene products are glycosylated and secreted from infected cells, in agreement with the absence of transmembrane anchor sequences that are present in the cellular IL-1 and IL-6 receptors. The dispensability for virus replication in tissue culture, the secretion to the extracellular space, and the predicted receptor-like structure of the proteins suggest that both are involved in interference with host defense mechanisms in vivo.
  • the B18R gene product Is shown not to bind IL-l ⁇ , IL-l ⁇ or IL-6, despite the homology with the receptors for these cytokines (McMahan et al, 1991; Smith and Chan, 1991).
  • Two forms of the protein 52 kd and 60-65 kd were detected, the larger of which is found in supematants while the smaller might represent a membrane-associated molecule. This would be in agreement with previous reports showing immunofluorescence in the plasma membrane of infected cells using antiserum specific for proteins secreted at early times of infection (Ueda et al, 1972), which was attributed later to reactivity against B18R (Ueda et al, 1990).
  • B15R ORF is shown to encode an IL-l ⁇ binding activity present in the supematants of vaccinia virus-infected cells and to represent a novel soluble IL-1 receptor.
  • the high affinity for IL-l ⁇ binding (K D 234 pM) is similar to those reported..for the cellular receptors (Sims et al, 1988, 1989; McMahan et al, 1991) and is consistent with the retention of full binding activity by the extracellular domain of the IL-1 receptor (Dower et al, 1989).
  • the size (50-60 kd) and high carbohydrate content of the mature vaccinia IL-l ⁇ receptor are in agreement with those reported for the truncated and complete versions of the cellular receptor, respectively (Urdal et al, 1988; Dower et al, 1989).
  • the secretion of a biologically active 40-44 kd protein from insect cells suggests that the carbohydrate is not an essential component for the IL-1 binding.
  • the vaccinia IL-1 receptor constitutes a novel receptor for IL-1 because of the specificity,for IL-l ⁇ . This was shown in binding experiments to radioiodinated ILs and was corroborated in competition assays with unlabeled cytokines and by blocking the interaction of the ILs with the natural receptor on cells in culture.
  • B15R has a higher similarity to the cellular type II receptor than to the type I receptor (McMahan et al, 1991; Smith and Chan, 1991) and since this similarity is comparable with those found between other vaccinia virus proteins and their cellular counterparts (Smith et al, 1991a), B15R may derive from the type II IL-1 receptor or a variant thereof.
  • the comparison of the sequence of the vaccinia IL-l ⁇ receptor with the type I and type II IL-1 receptors does not permit identification of the amino acids that confer specificity for IL-l ⁇ since the sequences are quite divergent.
  • the availability of the vaccinia virus gene will allow mutagenesis studies to identify these positions.
  • sequence of B15R ORF in other vaccinia virus strains that show different affinities for the human and murine IEr-l ⁇ may provide structural information on the binding domain.
  • the comparison of the sequence may be more useful since genes from different orthopoxviruses are highly conserved.
  • the vaccinia IL-l ⁇ receptor might be useful as a tool to investigate the function of IL-l ⁇ and IL-l ⁇ in vivo in different models.
  • the other IL-1 inhibitors available IL-IRA, a soluble truncated IL-1 receptor, and monoclonal antibodies against the receptor
  • IL-IRA a soluble truncated IL-1 receptor
  • monoclonal antibodies against the receptor block the binding of both forms of IL-1 (Fanslow et al, 1990; Gershenwald et al, 1990; Ohlsson et al, 1990; Alexander et al, 1991; Mclntyre et al, 1991).
  • the molecule may regulate responses normally controlled by IL-l ⁇ , and, since it does not bind IL-l ⁇ or IL-IRA, it might offer advantages over the other inhibitors.
  • the failure of the vaccinia IL-l ⁇ receptor to bind IL-IRA illustrates the adaptation of the virus to the physiological response of the host by preventing interference with the natural antagonist.
  • the number of IL-l ⁇ binding sites secreted from vaccinia virus-in ected cells is without precedent and makes the supematants from cultures infected with vaccinia virus the most concentrated naturally occurring soluble IL-1 binding activity.
  • An excess of soluble receptors must be required to block the effects of IL-l ⁇ in vivo, since only a few cellular IL-1 receptors need to be occupied to elicit a biological response. This was illustrated in the competition of IL-l ⁇ binding to T- cells, which also indicates that the vaccinia IL-l ⁇ receptor will probably block the biological effects induced in cells expressing IL-1 receptors.
  • the blockade of IL-1 by a virus is interesting since this cytokine orchestrates the host response to infection, inducing a broad spectrum of systemic effects and playing an important role in initiating the inflammatory and immune responses. But more interesting is the specificity of the blockade for IL-l ⁇ . To date, both IL-l ⁇ and IL-l ⁇ have been found to induce similar activities in a number of model systems (Dinarello, 1989 ) . The fact that vaccinia virus secretes a protein that specifically blocks the effects of IL-l ⁇ indicates that soluble IL-l ⁇ plays a more important role than IL-l ⁇ in the host response to orthopoxvirus infections.
  • the vaccinia IL-l ⁇ receptor expressed in the wild-type virus, may thus limit the systemic acute phase response otherwise initiated by increased levels of IL- l ⁇ .
  • weight loss which can be induced by IL-1 (Di Giovine and Duff, 1990), occurred earlier in animals infected with v B15R supports this view.
  • a generalized response can contribute to host defense; for example, temperature typical of fever has been reported to enhance the proliferation of T-cells that might facilitate a T-cell-dependent immune response (Duff and Durum. 1983).
  • an increased systemic reaction to infection did not affect the outcome of infection by v B15R.
  • B15R might function as a virulence or attenuation factor for the virus. It is unclear whether the effects of deleting B15R from the WR strain of vaccinia virus, which was selected for high neurovirulence by passage in mouse brain, are representative of infections with other orthopoxviruses that also express the IL-l ⁇ binding activity.
  • B15R ORF is one of a few virus genes that has been shown to increase the pathogenicity or the severity of the infection when deleted from the virus genome (Ginsberg et al, 1989; Romanczuk and Howley, 1992). Virus attenuation can result from deletion or inactivation of genes encoding proteins that 'interfere with host defense mechanisms, but as shown here, some virus proteins might also be devised to diminish effects that the infection produces in the host. This would help host survival and thereby be beneficial for the virus.
  • IL-l ⁇ is more potent than IL-l ⁇ in the induction of fever, and the effect is mediated through different mechanisms (Busbridge et al, 1989), which correlates with the discovery of IL-l ⁇ (Breder et al, 1988) and receptors specific for IL-l ⁇ (Katsuura et al, 1988) in the brain.
  • B15R is the second soluble cytokine receptor to be identified in a virus.
  • a soluble receptor for tumor necrosis factor (TNF) has been shown to be active in Leporipoxviruses and to increase the pathogenicity of the virus (Smith et al, 1991b; Upton et al, 1991).
  • the WR and Copenhagen strains of vaccinia virus contain one and two homologs, respectively, of the TNF receptor, but the presence of frameshifts and stop codons make expression of active proteins unlikely (Howard et al, 1991; Upton et al, 1991).
  • the presence of soluble receptors for either TNF or IL-1 in different genera of poxviruses, which produce very different pattens of disease, is interesting since these cytokines share many biological properties.
  • the soluble IL-l ⁇ receptor is one of the increasing number of activities encoded by vaccinia virus that aid evasion from the host immune system ( for references see Moore and Smith, 1992) and, in particular, is another viral-encoded protein that interferes with cytokine functions.
  • TNF receptor of leporipoxvirus Besides the TNF receptor of leporipoxvirus and the crmA protein of cowpox virus, other examples found are the 14.7 kd protein of adenovirus that inhibits cytolysis by TNF (Gooding et al, 1988), the IL-10 activity encoded by Epstein-Barr virus (Hse et al, 1990), and the presence of IL-6 binding sites in the envelope protein of hepatitis B virus (Neurath et al, 1992).
  • the vaccinia IL-l ⁇ receptor may be a useful tool to discriminate the physiological roles of IL-l ⁇ and IL-l ⁇ and might be used as an anti-inflammatory therapeutic reagent.
  • the expression of this activity by vaccinia virus and other orthopoxviruses represents a novel mechanism of virus evasion from the immune system. It is shown that the IL-l ⁇ receptor is modulating the systemic response to infection and the severity of the disease, which suggests that IL-l ⁇ , and not IL-l ⁇ , is the main mediator of the endocrine effects of the IL-1 produced in response to vaccinia virus infection in mice.
  • thermogenesis ' nd fever by interleukin-l ⁇ and interleukin-l ⁇ involves different mechanisms. Biochem.Biophys.Res.Commun. 162, 591-596.
  • Interleukin 1 the first interleukin. Immunol. Today 11, 13-20.
  • Blocking IL-1 interleukin 1 receptor antagonist in vivo and in vitro. Immunol. Today 12, 404-410.
  • Interleukin-1 receptor antagonist binds to the type II interleukin-1 receptor on B cells and neutrophils. J.Biol.Chem. 266, 20311-20315.
  • Eisenberg, S.P. Evans, R.J. , Arend, W.P., ' Verderber, E., Brewer, M.T., Hannum, CH. , and Thompson, R.C. (1990).
  • Interleukin-1 receptor antagonist competitively inhibits the binding of interleukin-1 to the type II interleukin-1 receptor. J.Biol.Chem. 266, 14147-14150.
  • Vaccinia virus homologues of the Shope fibroma virus inverted terminal repeat proteins and a discontinuous ORF related to the tumor necrosis factor receptor family Virology 180, 633-647.
  • Vaccinia virus encodes an active thymidylate kinase that complements a cdc8 mutant of Saccharomyces cerevisiae. J.Biol.Chem. 266, 20103-20109.
  • IL-1 receptor antagonist J.Exp.Med. 173, 931-939.
  • a novel IL-1 receptor cloned from B cells by mammalian expression, is expressed in many cell types. EMBO J. 10, 2821-2832.
  • LIGAND a versatile computerized approach for characterization of ligand-binding systems. Anal.Biochem. 107, 220-239.
  • cowpox virus encodes an inhibitor of the interleukin-l ⁇ converting enzyme.
  • T2 open reading frame from Shope fibroma virus encodes a soluble form of the TNF receptor. Biochem. Biophys.Res.Commun. 176, 335-342.
  • Vaccinia and cowpox viruses encode a novel secreted interleukin-1-binding protein. Cell 71, this issue.
  • a soluble binding protein specific for interleukin l ⁇ is produced by activated mononuclear cells. Cytokine 2, 190- 198.
  • Receptors for the B cell stimulatory factor 2 quantitation, specificity, distribution, and regulation of their expression. J.Exp.Med. 166, 967-981.
  • Myxoma virus expresses a secreted protein with homology to the tumor necrosis factor receptor gene family that contributes to viral virulence. Virology 184, 370-382.
  • NB 4/5 4 deaths in a group of 5
  • mice 4-6 week old Balb/c mice were infected intranasally with indicated doses of WR, B15R-deleted ( v ⁇ l5ft) or B18R-del ( v ⁇ BI8F)virus . The mortality of animals after 15 days is show
  • Values of greater than 3.1 are significant (probability 10 ⁇ 3 ), while values of 4.8, 6.0 and 7.9 indicate probabilities of 10" 6 , 10 ⁇ 9 and 10 ⁇ 15 , respectively.
  • the domains illustrated are from B15R amino acids 28-119 (1), 121-214 (2), 222-end (3): B18R 53-149 (1), 152-241 (2), 252-end (3); murine IL-1R precursor (Sims, J.E., March, CJ-, Widmer, M.B., MacDonald, H.R., McMahan, D.J. , Grubin, C.E., -Wignall, J.M. , Jackson, J.L., Call, S.M.
  • T cell receptor CD3 epsilon chain (TCRCD3) (Clevers, H. , Duiilap, S., Saito, H. , Georgopoulos, K. , Wileman, T. and Terhorst, C, (1988). Proc. Natl. Acad. Sci. USA 85, 8623-8627) 1-82; leukocyte antigen receptor protein (LAR) (Streuli, M. , Krueger, N.X., Hall, L.R., Schloss an, S.F. and Saito, H. (1988). J. Exp. Med.
  • LAR leukocyte antigen receptor protein

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  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention se rapporte à des protéines codées par le virus de la vaccine et qui fonctionne de façon à interférer avec le système immunitaire de l'hôte. L'invention concerne notamment l'utilisation de la séquence nucléotidique appelée B15R ou d'une séquence nucléotidique codant pour un allèle, ou un fragment fonctionnellement équivalent, d'un dérivé ou d'une variante de polypeptide codé par la séquence nucléotidique B15R dans un procédé se rapportant à la fabrication d'un médicament pour traiter un état dans lequel l'interleukine 1β est impliquée dans la médiation d'un ou de plusieurs symptômes associés audit état. L'invention concerne également un médicament contre la fièvre comprenant un polypeptide qui est codé par le produit du gène B15R ou un allèle, ou un fragment fonctionnellement équivalent, d'un dérivé ou d'une variante de ce produit.
EP93905517A 1992-03-05 1993-03-05 Virus de la vaccine b15r utilise pour traiter un etat impliquant l'interleukine b1 Withdrawn EP0656949A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GB9204780 1992-03-05
GB929204780A GB9204780D0 (en) 1992-03-05 1992-03-05 Vaccinia vectors,vaccinia genes,and expression products thereof;pharmaceuticals,reagents and methods
PCT/GB1993/000460 WO1993018153A1 (fr) 1992-03-05 1993-03-05 Virus de la vaccine b15r utilise pour traiter un etat impliquant l'interleukine b1

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EP0656949A1 true EP0656949A1 (fr) 1995-06-14

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EP (1) EP0656949A1 (fr)
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WO (1) WO1993018153A1 (fr)

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DE602004023096D1 (de) * 2003-08-07 2009-10-22 Hoffmann La Roche Ra antigene peptide
EP1518932A1 (fr) * 2003-09-29 2005-03-30 GSF-Forschungszentrum für Umwelt und Gesundheit GmbH Mutant du virus de la vaccine Ankara modifié (MVA) et utilisation correspondante
WO2020011754A1 (fr) * 2018-07-09 2020-01-16 Transgene Virus de la vaccine chimériques

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GB9023352D0 (en) * 1990-10-26 1990-12-05 Lynxvale Ltd Vaccinia vectors,vaccinia genes and expression products thereof

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Title
See references of WO9318153A1 *

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WO1993018153A1 (fr) 1993-09-16
GB9204780D0 (en) 1992-04-15

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