EP0555291A1 - Vecteur de la vaccine, genes de la vaccine et leurs produits d'expression - Google Patents

Vecteur de la vaccine, genes de la vaccine et leurs produits d'expression

Info

Publication number
EP0555291A1
EP0555291A1 EP91918806A EP91918806A EP0555291A1 EP 0555291 A1 EP0555291 A1 EP 0555291A1 EP 91918806 A EP91918806 A EP 91918806A EP 91918806 A EP91918806 A EP 91918806A EP 0555291 A1 EP0555291 A1 EP 0555291A1
Authority
EP
European Patent Office
Prior art keywords
virus
nucleotide sequences
vaccinia
gene
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP91918806A
Other languages
German (de)
English (en)
Inventor
Geoffrey Lilley Smith
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Lynxvale Ltd
Original Assignee
Lynxvale Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Lynxvale Ltd filed Critical Lynxvale Ltd
Publication of EP0555291A1 publication Critical patent/EP0555291A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • 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
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2710/24161—Methods of inactivation or attenuation

Definitions

  • the present invention relates to recombinant vaccinia virus vectors.
  • 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.
  • Live vaccinia virus was used as the vaccine to immunise against, and eradicate smallpox.
  • Vaccinia virus was used as the vaccine to immunise against, and eradicate smallpox.
  • W 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. (1990). In "Virology”. B.N. Fields Ed. pp 2079-2111.
  • vaccinia virus encodes many enzymes and protein factors necessary for transcription and replication of its genome.
  • the virus also encodes a variety of factors which modulate virus replication in the multicellular host and aid evasion of the host immune system (Moss, B. (1990). In "Virology” B.N. Fields Ed. pp 2079-2111. Raven Press, New York). 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., 6_7, 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 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.
  • one or more of the gene sequences may be inactivated, or 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.
  • 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 replication.
  • 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) B15R, ii) B18R iii) SalL4R. 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 imm urgeic 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 of the present invention have the potential for enhanced immunogenicity. This may result from either the deletion of vaccinia genes which cause immunosuppression (eg. the interleukin receptors and 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).
  • vaccinia genes which cause immunosuppression eg. the interleukin receptors and the complement homologue and the human FcR for IgE
  • a gene which potentiates the immune response e.g. expressing the authentic CD23 gene in vaccinia virus.
  • 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) B15R ii)
  • the vaccinia nucleotide sequence may be the sequence designated herein as SalL4R.
  • 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 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 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 the use of the recombinant virus vectors hereof can be used in the 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.
  • diagnostic test kits comprising monoclonal antibodies, polyclonal antibodies, antisera and/or T cells obtained by the use of the recombinant vaccinia vectors provided.
  • 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.
  • polypeptide encoded by a nucleotide sequence selected from those defined above and alleles and variants of said polypeptides.
  • 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) B15R, ii) B18R, iii) SalL4R.
  • the invention also provides a method which comprises using a vaccinia virus vector as defined herein to prepare a vaccine or a medicament.
  • the present invention also provides the translation products encoded by the nucleotide sequences B15R and B18R disclosed herein. These translation products may be utilised as anti-inflammatory medicaments. The invention also provides methods using these translation products for the preparation of an anti-inflammatory medicament. BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 A) Hindlll restriction map of the 186 kb W genome. The 9.8 kb 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 Hindlll B and are transcribed rightwards towards the genomic terminus)
  • FIG. 1 Amino acid alignment of the Ig domains from B15R and B18R with the Ig domains of the human and murine IL-1R, the human IL-6R, the W haemagglutinin (W 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 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 11 .
  • Figure 4 Shows the nucleotide sequence and amino acid sequence for the gene SalF3R.
  • FIG. 5 Shows hydrophobicity profiles of vaccinia virus genes SalL4R and SalF3R (renamed SalF2R, Smith, G.L., Chan, Y.S. & Howard, S.T. J. Gen. Virol. 72 ⁇ 1349, 1991). Vertical lines on the horizontal axis indicate blocks of 20 amino acids.
  • FIG. 6 Shows the amino acid alignment of the vaccinia virus SalL4R gene (VV L4R) with similar lectin-like molecules from vaccinia virus (W F3R encoded by SalF3R), fowlpox virus FPV 2, 8 & 11 (Tomley et al., J. gen. Virol. 6 , 1025 (1988)), the human low affinity receptor for IgE (HFcR) (Kitutani et al., Cell 47, 657, (1986)), the antifreeze polypeptide from Hemitripterus americans (AFP) Ng et al., J. Biol. Chem.
  • Cells were infected with either WT virus (lanes 2, 3, 7 £_ 8) or recombinant virus vAAl (lanes 4, 5, 9 & 10) or mock infected (lanes 1 & 6) and labelled with [35S]-methionine either early (lanes 2, 4, 7, & 9) or late (lanes 3, 5, 8, & 10) during infection.
  • Lanes 1-5 show cell extracts S. 6-10 supernatants. Samples were run on a polyacrylamides gel and an autoradiograph prepared. The position of the Mr 50 K protein in the supernatant of vAAl infected cells is indicated by an arrow. Figure 10. Plaque size in the presence or absence of IPTG.
  • BSC-1 monolayers were infected with WT, vSAD7 or vSAD9 in the presence or absence of IPTG. 2 days later the cells were stained with crystal violet and photographed. Figure 11. BSC-1 cells were infected with either vSAD7 or vSAD9 in the presence or absence of IPTG. Infections were at either 10 plaque forming units (pfu) per cell (panels A & B) or 0.001 pfu / cell (panels C & D). Virus present within infected cells at the indicated times after infection was determined by plaque assay on fresh duplicate monolayers of BSC-1 cells.
  • FIG. 12 The morphogenesis of vSAD9. BSC-1 cells infected at 25 pfu/cell with vSAD9 with or without 5 mM IPTG were incubated for 6, 12 and 24 hours before being fixed with 2% gluteraldehyde, embedded, sectioned and observed by electron microscopy.
  • A Section of a cell 24 hpi in the absence of IPTG showing virus factories and INV particle (4). 1-3 as for
  • B Magnification 5800 X.
  • B Detail of vSAD9 virus factory without IPTG at 24 hpi: (1) partially formed lipid crescents, (2) early immature particle completely surrounded by lipid, (3) immature particle containing condensed nucleoid. Magnification 36,000 X.
  • FIG. 13 Immunoblot analysis of the SalL4R glycoproteins.
  • Cells were infected for 24 hours with WR (lanes 1 and 2), or vSAD9 (lanes 3-6), or mock infected (lanes 7 and 8), in the presence (+) or absence (-) of 5 mM IPTG and/or 1 ⁇ g/ml tunicamycin as indicated. Extracts were resolved by SDS-PAGE, transferred to nitrocellulose, incubated with anti-SalL4R serum and immune complexes detected using alkaline phosphatase-conjugated donkey anti-rabbit Ig (materials and methods). The molecular weights of the observed proteins are shown in kDa. Figure 14.
  • Carbon coated copper 400 mesh grids were floated on 4 x 107 pfu of IHD-J INV (panel D) or EEV (panels A-C, E & F) purified virus particles in 96 well tissue culture dishes. Grids were washed with PBS and TBS for 2 mins, and then in 50% ethanol for 30 sees.
  • Virus-coated grids were then incubated for 15 mins in TBG (TBS pH 8.2, 0.1% BSA [Fraction V], 1% gelatin), before being transferred to wells containing either anti-SalL4R (Panels A & C-E), or an unrelated rabbit serum diluted 1/50 in TBS containing 1% BSA (panel F), or affinity purified anti-SalL4R specific Ig diluted 1/5 in the same (panel B).
  • virion-coated grids were washed for 10 mins in TBG, and bound Ig detected by incubation for 30 mins in colloidal gold-conjugated goat anti-rabbit Ig diluted 1/10 in TBS containing 1% BSA. Grids were washed for 5 mins with TBG and then TBS, before proteins were fixed in TBS containing 2% gluteraldehyde. Virus particles were finally negatively stained using 2% uranyl acetate.
  • the nucleotide sequence of the Sail I and Sal I L restriction fragment of the vaccinia virus genome 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.
  • the 9.8 kb Sail I 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.
  • Genes B15R and B18R 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 of the gene designated B15R is shown in figure IB.
  • the nucleotide sequence shown for B15R is 11462-12664 nucleotides from the left end of the vaccinia virus Hindlll B fragment and the coding region 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 of the gene designated B18R is shown in figure lc.
  • the nucleotide sequence shown for B18R is 15448-16741 nucleotides from the left end of the vaccinia virus Hindlll B fragment and the coding region is at nucleotide positions 15448-16741 (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. Immunol. 6, 381-405) namely a pair of cysteines forming an intradomain disulphide bridge, sequences predicted to form ⁇ -strand structures and an invariant tryptophan in ⁇ -strand C.
  • Ig immunoglobulin
  • the distance between these cysteine pairs in B15R suggest the first two domains are C regions while the third maybe a V-domain.
  • the distances suggest these are C-domains. These regions are aligned with selected Ig domains of IL-1R (interleukin 1 receptor and interleukin 6 receptor), W haemagglutinin and Ig kappa (Hilschman, N. and Hoppe-Seyer's, Z. (1967) Physiol. Chem. 348, 1077-1080), fasciclin II (Harrelson, A.L. and Goodman, C.S. (1988).
  • cysteines There are additional conserved cysteines in 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.
  • B18R domain 1 also contains proline at this position.
  • a glycosylation site is conserved in domain 1, ⁇ -strand F of IL-lRs and B15R despite divergence of amino acid sequence.
  • Domain 3 does not contain additional cysteines and is longer than 1 and 2 in B15R, B18R and the IL-lRs.
  • mRNA corresponding to these genes was analysd by SI nuclease protection experiments.
  • the radiolabelled probe used for detection of B15R mRNA was prepared by cloning the Sall-Xbal fragment from the left end of the vaccinia virus Sail I restriction fragment into pUC118. This plasmid was used as a template for polymerase chain reaction using an oligonucleotide complementary to the coding strand of B15R and the universal primer complementry to pUCll ⁇ .
  • the PCR product was purified, labelled with y[32]-ATP using polynucleotide kinase, digested with Sail I, and a 1024 bp fragment isolated. This was hybridized to early or late virus mRNA, hybrids mechanism employed by VV against the host immune response. Other mechanisms, proposed or proven, include the interference with the complement system by a secretory homologue of C4b binding protein (Kotwal, G.J. and Moss, B. (1988). Nature 335, 176-178) the expression; of serine protease inhibitors which may block the presentation of peptides to cytotoxic T cells (Boursnell, M.E.G., Foulds, I.J., Campbell, J.
  • Ig domains structurally similar domains
  • the W 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 ) .
  • the present applicants have identified herein the nucleotide and amino acid sequence data for B15R and B18R and identified the surprising homology between the gene products of B15R and B18R to human and murine IL-1R, human IL-6R and the immunoglobulin (Ig) superfamily.
  • Cytokines IL-1 and IL-6 by binding to their respective natural receptors mediate immune responses against an invading pathogen and cause inflammation. W appears to be combatting this part of the immune response by producing proteins which mimic the receptors for IL-1 and IL-6. Thus the function of these W proteins appears to be one of binding the cytokines to prevent them reaching their natural receptors. In this way the virus reduces the inflammatory response directed against it. Thus the virus is able to replicate more effectively.
  • the applicants proposal is to use this surprising observation to render vaccinia virus less harmful, so that problems associated with its use as a vaccine and/or other problems are ameliorated.
  • Virology 179, 247-2666 indicates that WR genes B15R and B18R are included in the non-essential regions. However, it remains to be established whether either or both of these genes are non-essential for replication of the WR strain of virus. Deletion of these ORFs could provide a suitable means of virus attenuation. Since the translation products encoded by the nucleotide sequences B15R and B18R appear likely to bind interleukin 1 or interleukin 6, these protein products will be useful as antiinflammatory agents. The proteins may be produced in a recombinant system according to techniques well known in the art. Thus the nucleotide sequences provided herein could be inserted into a suitable expression vector (not necessarily vaccinia). That vector can then be used to transform a cell line suitable for the production of these particular proteins. SalL4R
  • SalF3R (renamed SalF2R, Smith et al., J. Gen Virol. 72, 1349-1376, 1991), a gene related to SalL4R.
  • the nucleotide sequence and deduced amino acid sequence of the gene designated SalF3R is shown in figure 4.
  • the single letter code is used for the designation of amino acids.
  • the coding region of the SalF3R gene maps between nucleotides 595 and 1071 from the left end of the SallF fragment.
  • the molecular weight of the primary translation product is predicted to be 18.1 kiloDaltons (kD).
  • SalL4R which shows homology to, and appears to be structurally related to SalF3R, to C-type animal lectins in general and to CD23.
  • the gene SalL4R maps 1755-2498 from the left hand end of SallL fragment of the vaccinia virus strain WR.
  • SalF3R 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.
  • FcR human low affinity Fc receptor
  • SalF3R and SalL4R show sequence homology with respect to each other (Fig 6) and similar hydrophobicity profiles (Fig. 5).
  • the homologies suggest that the proteins encoded by these genes function as lectins or as homologues 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 activated B lymphoctyes which is of central importance in regulating B cell growth (Gordon, J. & Guy G.R. (1987), Immunol. Today, _8, 339).
  • the vaccinia virus protein encoded by SalF3R or by SalL4R may 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.
  • the protein may play a role in the attachment of virus to the target cell.
  • 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 the SalF3R 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 SallF fragment into pUC13 that had been digested with EcoRI and Sail.
  • This plasmid contains the entire coding region of SalF3R and was digested with Nsil, which cuts twice only, within the coding sequence ( Figure 6).
  • 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.
  • Ecogpt E.coli xanthine guanine phosphoribosyl transferase
  • 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.
  • a plasmid, pSAD3G was isolated in which lOObp of the SalF3R 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 hypoxan hine. These drugs permit the replication only of recombinant viruses which contain and express the Ecogpt gene (Boyle & Coupar 1988 supra; Falkner & Moss, J. Virol, 62, 1849-54, 1988). After three rounds of plaque purification, the virus was amplified in larger cultures of CV-1 cells.
  • a single-stranded radio-labelled DNA probe complementary only to the coding strand of SalF3R 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 SalF3R promoter or from further upstream. This data allows the conclusion that the gene SalF3R is certainly transcribed early and possibly also late during infection.
  • SalL4R Similar studies have been carried out with SalL4R. Analysis (by SI nuclease protection experiments) of mRNA isolated from virus infected cells demonstrated that late during infection RNA is transcribed from the TAAATG motif at the 5' end of the SalL4R gene. A virus expressing only an IPTG-inducible form of SalL4R has been constructed in a similar manner to that described for the vaccinia virus 14K protein (Rodriguez and Smith, Nucleic Acids Reserach 18_, 5347-5351, 1990).
  • a copy of the SalL4R open reading frame was constructed by polymerase chain reaction and cloned into plasmid pPR34 (Rodriguez and Smith, Virology 177, 239-250, 1990) downstream of the IPTG-inducible vaccinia virus 4b promoter.
  • This plasmid was transfected into cells infected with WT vaccinia virus strain WR and a thymidine kinase negative virus (vSAD7) isolated that contains the IPTG-inducible copy of the SalL4R gene within the TK locus.
  • vSAD7 thymidine kinase negative virus
  • This DNA fragment was then ligated with a DNA fragment containing the E.coli gpt gene linked to the vaccinia virus 7.5K promoter that had been isolated and rendered blunt-ended as described above for gene SalF3R.
  • the resultant plasmid (termed pSAD8) has 415 bp of Sal4R deleted and replaced with the Ecogpt gene.
  • This plasmid was amplified in E. coli, purified and then transfected into CV-1 cell infected with vaccinia virus vSAD7. Recombinant viruses were selected in the presence of IPTG and mycophenolic acid, and susequently plaque purified and amplified.
  • vSAD9 A recombinant virus which had the IPTG-inducible form of the SalL4R gene within the TK gene locus and the endogenous copy of SalL4R replaced by Ecogpt was called vSAD9. Analysis of the genomic DNA of vSAD7 and vSAD9 by Southern blotting confirmed these viruse had the predicted structures.
  • FIG 11 shows that in cells infected at high multiplicity of infection (moi) the production of intracellular virus was unaffected by the presence or absence of IPTG, and the growth kinetics were indistinguishable from vSAD7.
  • mSAD7 multiplicity of infection
  • the SalL4R gene product was identified by raising a polyclonal antisera to a region of SalL4R expressed as a TrpE-fusion protein in E.coli.
  • a 321 bp AccI fragment was isolated from within the Sal4R gene and cloned into plasmid pATH3 (Koerner et al. , Meth. Enzymol. 194, 477-490, 1991) to form pSAD24.
  • Expression of the fusion protein was induced in bacteria harbouring pSAD24 with indoleacrylic acid.
  • the digested with SI nucleae and the products run on a sequencing gel against an M13 ladder.
  • the result ( Figure 8A) shows that the B15R gene is transcribed late during infection from a TAAAATG motif at the beginning of the open reading frame.
  • a probe for analysis of B18R mRNA analysis was produced by digesting pUC118 containing the Sall-Xbal region of vaccinia virus Sail fragment (above) with EcoRI and purifying a 500 bp fragment. This was dephosphorylated with calf intestinal alkaline phosphatase, labelled with g[32]-ATP using polynucleotide kinase, digested with Xbal and a 474 bp fragment isolated. This was hydridized with virus mRNA as for the B15R probe above. The result ( Figure 8B) shows that the B18R gene is transcribed early during infection from upstream of the B18R gene. These data are consistent with primer extension analyses of the mRNA from this gene (Ueda et al., Virology 177, 577-588, 1990).
  • B15R encodes a secretory glycoprotein of Mr 50 K.
  • a recombinant vaccinia virus (vAAl) was constructed that contained a second copy of the B15R ORF driven from the late p4b promoter. This virus was formed by cloning the B15R ORF into plasmid pRK19 (Kent, R.K. Isolation and analysis of the vaccinia virus p4b gene promoter. PhD Thesis, University of Cambridge, 1988) downstream of the strong late vaccinia 4b promoter.
  • the resultant plasmid was transfected into cells infected with wild type (WT) vaccinia virus (strain WR) and a recombinant virus, that contained the second copy of B15R inserted into the TK gene locus, selected by plaque assay on human thymidine kinase negative cells in the presence of bromodeoxyuridine.
  • WT wild type
  • WT vaccinia virus
  • a recombinant virus that contained the second copy of B15R inserted into the TK gene locus, selected by plaque assay on human thymidine kinase negative cells in the presence of bromodeoxyuridine.
  • BSC-1 cells infected with either vAAl or WT virus were labelled with [35S]-methionine early (2-4 hours post infection in the presence of cytosine arbinoside) or late (6-8 hours post infection) and the radiolabelled proteins resolved by polyacylamide gel electrphoresis and detected by autoradiography.
  • Figure 9 shows
  • B18R protein can be found on the cell surface early during infection, (Ueda, Y., Morikawa, S. and Matsuura, Y. (1990). Virology 177, 588-594) despite the C-terminal hydrophobic residues being predicted to form the final ⁇ -strand of an Ig domain and therefore unlikely to function as a transmembrane anchor. This localization may be mediated by protein-protein interactions possibly involving intermolecular disulphide bonds between additional cysteines (as occurs with other members of the Ig superfamily eg. the immunoglobulins). Alternatively, these cysteines may form intradomain disulphide bridges between the A and G ⁇ -strands. Similar arguments apply for B15R.
  • B15R and B18R proteins Another likely function for B15R and B18R proteins, is to facilitate virus spread by mediating the interaction of virus particles or infected cells with other cells.
  • Ig superfamily members of the Ig superfamily (eg. MHC antigens, NCAM (Hemperley, J.J., Murray, B.A., Edelman, G.M. and Cunningham, B.A. (1986). Proc. Natl. Acad Sci. USA 83, 3037-3041) and the intercellular protein amalgam (Seeger, M.A. Haffley, L. and Kaufman, T.C. (1988). Cell 55, 589-600). Nonetheless, the closer homology to IL-IR and IL-6R make the binding of cytokines more likely.
  • SUBSTITUTESHEET genome is within the capability of one skilled in the art to either inactivate these sequences in or delete these sequences from the W 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.
  • CHNCAM 1 4.12 4.01 4.07 3.19 5.54 3.28
  • 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-IR precursor (Sims, J.E., March, C.J., Widmer, M.B., MacDonald, H.R., McMahan, D.J., Grubin, C.E., Wignall, J.M., Jackson, J.L., Call, S.M. Friend, D., Alpert, A.R.
  • CHNCAM chicken neural cell adhesion molecule
  • MAG myelin-associated glycoprotein
  • PDGFR platelet derived growth factor receptor

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Communicable Diseases (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Toxicology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne des vecteurs de la vaccine et elle est basée sur la découverte de trois séquences de gènes non évidentes dans le génome viral. L'une ou plusieurs de ces séquences de gènes peut(peuvent) être neutralisée(s), ou une partie de l'ensemble de l'une ou plusieurs de ces séquences de gènes peut être supprimée du génome viral pour (i) atténuer davantage le virus et/ou (ii) accroître l'immunogénicité du virus recombinant de la vaccine; et/ou (iii) insérer d'autres séquences de gènes dans des sites, de manière à intégrer davantage d'ADN étranger dans ledit virus. Là où toutefois les séquences de gènes sont essentielles à la réplication virale, l'atténuation virale peut tout de même être réalisée par la modification du produit génique (par exemple par une manipulation au niveau du gène), de telle sorte que la fonction protéique à l'origine de la pathogénicité est contrecarrée, tandis que la fonction protéique pour la réplication du virus est conservée.
EP91918806A 1990-10-26 1991-10-28 Vecteur de la vaccine, genes de la vaccine et leurs produits d'expression Withdrawn EP0555291A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB909023352A GB9023352D0 (en) 1990-10-26 1990-10-26 Vaccinia vectors,vaccinia genes and expression products thereof
GB9023352 1990-10-26

Publications (1)

Publication Number Publication Date
EP0555291A1 true EP0555291A1 (fr) 1993-08-18

Family

ID=10684414

Family Applications (1)

Application Number Title Priority Date Filing Date
EP91918806A Withdrawn EP0555291A1 (fr) 1990-10-26 1991-10-28 Vecteur de la vaccine, genes de la vaccine et leurs produits d'expression

Country Status (5)

Country Link
EP (1) EP0555291A1 (fr)
JP (1) JPH06502069A (fr)
CA (1) CA2094821A1 (fr)
GB (1) GB9023352D0 (fr)
WO (1) WO1992007944A1 (fr)

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9204780D0 (en) * 1992-03-05 1992-04-15 Smith Geoffrey L Vaccinia vectors,vaccinia genes,and expression products thereof;pharmaceuticals,reagents and methods
AU694519B2 (en) * 1994-04-29 1998-07-23 Immuno Aktiengesellschaft Recombinant poxviruses with foreign polynucleotides in essential regions
US7285526B2 (en) 1995-07-14 2007-10-23 Meiogen Biotechnology Corporation Interferon antagonists useful for the treatment of interferon related diseases
US7790856B2 (en) 1998-04-07 2010-09-07 Janssen Alzheimer Immunotherapy Humanized antibodies that recognize beta amyloid peptide
TWI239847B (en) * 1997-12-02 2005-09-21 Elan Pharm Inc N-terminal fragment of Abeta peptide and an adjuvant for preventing and treating amyloidogenic disease
US20080050367A1 (en) 1998-04-07 2008-02-28 Guriq Basi Humanized antibodies that recognize beta amyloid peptide
US7964192B1 (en) 1997-12-02 2011-06-21 Janssen Alzheimer Immunotherapy Prevention and treatment of amyloidgenic disease
MY139983A (en) 2002-03-12 2009-11-30 Janssen Alzheimer Immunotherap Humanized antibodies that recognize beta amyloid peptide
AR052051A1 (es) 2004-12-15 2007-02-28 Neuralab Ltd Anticuerpos ab humanizados usados en mejorar la cognicion
RS59399B1 (sr) 2005-03-23 2019-11-29 Genmab As Antitela protiv cd38 za lečenje multiplog mijeloma
US8784810B2 (en) 2006-04-18 2014-07-22 Janssen Alzheimer Immunotherapy Treatment of amyloidogenic diseases
US8003097B2 (en) 2007-04-18 2011-08-23 Janssen Alzheimer Immunotherapy Treatment of cerebral amyloid angiopathy
JP5889529B2 (ja) 2007-07-27 2016-03-22 ヤンセン・サイエンシズ・アイルランド・ユーシー アミロイド原性疾患の処置
JO3076B1 (ar) 2007-10-17 2017-03-15 Janssen Alzheimer Immunotherap نظم العلاج المناعي المعتمد على حالة apoe
US9067981B1 (en) 2008-10-30 2015-06-30 Janssen Sciences Ireland Uc Hybrid amyloid-beta antibodies
WO2012048817A2 (fr) * 2010-10-15 2012-04-19 Bavarian Nordic A/S Vaccin contre la grippe à base d'un virus de la vaccine ankara recombinant, modifié
WO2014172309A2 (fr) * 2013-04-18 2014-10-23 The United States Of America As Represented By The Secretary Of The Army, On Behalf Of The United Compositions thérapeutiques pour neutraliser les interférons de type i, et procédés d'utilisation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9207944A1 *

Also Published As

Publication number Publication date
CA2094821A1 (fr) 1992-04-27
GB9023352D0 (en) 1990-12-05
WO1992007944A1 (fr) 1992-05-14
JPH06502069A (ja) 1994-03-10

Similar Documents

Publication Publication Date Title
Alcami et al. A soluble receptor for interleukin-1β encoded by vaccinia virus: a novel mechanism of virus modulation of the host response to infection
Kotwal et al. Vaccinia virus encodes a secretory polypeptide structurally related to complement control proteins
Upton et al. Myxoma virus expresses a secreted protein with homology to the tumor necrosis factor receptor gene family that contributes to viral virulence
Farrell et al. Inhibition of natural killer cells by a cytomegalovirus MHC class I homologue in vivo
EP0555291A1 (fr) Vecteur de la vaccine, genes de la vaccine et leurs produits d'expression
Afonso et al. Characterization of p30, a highly antigenic membrane and secreted protein of African swine fever virus
Duncan et al. Identification and characterization of an extracellular envelope glycoprotein affecting vaccinia virus egress
Barry et al. The myxoma virus M-T4 gene encodes a novel RDEL-containing protein that is retained within the endoplasmic reticulum and is important for the productive infection of lymphocytes
EP0353851B1 (fr) Régions non essentielles du virus du fowlpox
JP3506683B2 (ja) Ｈｃｍｖの糖タンパク質
Cameron et al. Myxoma virus M128L is expressed as a cell surface CD47-like virulence factor that contributes to the downregulation of macrophage activation in vivo
JP4191255B2 (ja) ケモカイン類に結合する可溶性ワクシニアウイルスタンパク質
JP2007082551A (ja) 組換えポックスウイルス−カリシウイルス［ウサギ出血疾患ウイルス（ｒｈｄｖ）］組成物および使用
EP0397560A2 (fr) ADN de la spéroidine et vecteurs d'expression d'entomopoxvirus recombinants
EP0465539A1 (fr) Vecteurs de la vaccine, genes de la vaccine et leurs produits d'expression
Chen et al. Analysis of host response modifier ORFs of ectromelia virus, the causative agent of mousepox
EP0323480A1 (fr) Vaccins obtenus par genie genetique agissant contre les mycobacteries
Smith et al. Host range selection of vaccinia recombinants containing insertions of foreign genes into non-coding sequences
Nockemann et al. Expression, characterization and serological reactivity of a 41 kDa excreted–secreted antigen (ESA) from Toxoplasma gondii
WO1993018153A1 (fr) Virus de la vaccine b15r utilise pour traiter un etat impliquant l'interleukine b1
JP2004528010A (ja) 免疫モジュレーションのための核酸およびポリペプチド
JP2883085B2 (ja) Ｈｃｍｖの糖タンパク質類、それらに対する抗体類及びｈｃｍｖワクチンの産生法、並びにそれらのための組換えベクター
AU2002301315B2 (en) A soluble vaccinia virus protein that binds chemokines
US7186408B2 (en) Viral CD30 polypeptide
EP0594764B1 (fr) Facteur de croissance hematopo etique derive de lymphocytes t et methodes d'utilisation

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19930514

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LI LU NL SE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19960501