+

WO1988002022A1 - Virus de la variole recombinant - Google Patents

Virus de la variole recombinant Download PDF

Info

Publication number
WO1988002022A1
WO1988002022A1 PCT/AU1987/000323 AU8700323W WO8802022A1 WO 1988002022 A1 WO1988002022 A1 WO 1988002022A1 AU 8700323 W AU8700323 W AU 8700323W WO 8802022 A1 WO8802022 A1 WO 8802022A1
Authority
WO
WIPO (PCT)
Prior art keywords
virus
gene
recombinant
foreign dna
poxvirus
Prior art date
Application number
PCT/AU1987/000323
Other languages
English (en)
Inventor
David Bernard Boyle
Barbara Elizabeth Howieson Coupar
Gerald Wayne Both
Original Assignee
Commonwealth Scientific And Industrial Research Or
The Australian National University
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 Commonwealth Scientific And Industrial Research Or, The Australian National University filed Critical Commonwealth Scientific And Industrial Research Or
Publication of WO1988002022A1 publication Critical patent/WO1988002022A1/fr

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
    • 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
    • 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
    • C12N2710/24041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This invention relates to recombinant poxviruses and their construction, and in one particular aspect this invention relates to recombinant fowlpox virus and the use thereof as a vehicle for the expression of foreign genes particularly for the expression of avian disease antigens.
  • Poxviruses are large DNA viruses that replicate within the cytoplasm of infected cells.
  • Vaccinia virus the type species of the orthopox virus group, has been widely studied because of its role as the vaccine virus for smallpox in man.
  • Such recombinant vaccinia viruses have the potential to deliver vaccine antigens to a variety of animal species (13).
  • the present invention which now makes possible a range of recombinant avian vaccines, is based on the mapping and sequencing of a non-essential region of the FPV genome.
  • This apparently non-essential region is the thymidine kinase (TK) gene, and it permits the introduction of foreign DNA into fowlpox virus (FPV) without impairing the potential for virus replication and the production of live infectious recombinant viruses.
  • TK thymidine kinase
  • the present invention provides recombinant fowlpox virus related avian poxvirus characterised by the inclusion of foreign DNA in the virus genome.
  • the foreign DNA is inserted into a non-essential region of the fowlpox virus genome, such as the TK gene or in virus DNA sequences controlling expression of the TK gene.
  • the foreign DNA may be inserted into intergenic regions of the fowlpox virus genome, for example regions preceding the TK gene or after the TK gene in such a manner as not to disrupt its function or the function of genes immediately upstream or downstream of the TK gene.
  • the foreign DNA may also be inserted between genes (intergenic) in essential regions of the fowlpox virus genome, or within genes or their regulatory sequences (intragenic) in other non-essential regions of the whole genome.
  • the invention provides an avian disease vaccine comprising recombinant fowlpox virus wherein a foreign DNA sequence encoding an antigen characteristic of the said avian disease has been inserted into the TK gene of the fowlpox virus or in virus DNA sequences controlling expression of the TK gene.
  • the FPV TK gene is characterised by an open reading frame of 183 codons commencing 279bp upstream of the central Xba 1 site of the nucleotide sequence as shown in Figure 8, and terminating 273bp downstream of this Xba 1 site. Confirmation of this sequence as the FPV TK gene is given by homologies with the vaccinia TK gene at nucleotide and amino acid levels, and the expression of TK enzyme which results from its insertion in TK ⁇ vaccinia virus.
  • this invention provides an avian disease vaccine comprising recombinant fowlpox virus wherein a foreign DNA sequence encoding an antigen characteristic of the said avian disease has been inserted in the region of the fowlpox virus commencing 279bp before and terminating 273b ⁇ after the central Xba 1 site of the nucleotide sequence shown in Fig.8, or in sequences controlling expression of this sequence.
  • vaccines will become available for protection against a wide variety of avian diseases such as those caused by viruses, bacteria, protozoa, metazoa, fungi and other pathogenic organisms, by insertion of DNA sequences encoding appropriate antigens characteristic of these diseases into the FPV genome as broadly outlined above.
  • a general method for the insertion of foreign DNA into vaccinia virus is based on the selective inactivation of the TK gene and uses 5-bromodeoxyuridine to select TK ⁇ recombinant viruses (3) . Since there are no TK ⁇ avian cell lines available, selection of TK ⁇ FPV recombinants is not possible. Other general methods for the construction of recombinant vaccinia viruses are known, and are discussed in detail hereinafter.Some of these approaches may be applicable to the insertion of foreign DNA into other poxviruses, however, none have yet been successfully applied to FPV.
  • Escherichia coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene can be used as a dominant selectable marker for the insertion of foreign genes into poxviruses and that this technique has a number of advantages over other methods for the construction of recombinant viruses.
  • the selection for the Ecogpt gene operates in a wide variety of cell types and therefore is potentially applicable as a dominant selectable marker for the insertion of foreign genes into other poxviruses (29).
  • Mycophenolic acid the antibiotic which provides the basis for selection of the Ecogpt gene (together with the required presence of xanthine, hypoxanthine, aminopterine, and thymidine), is a potent inhibitor of fowlpox virus in chicken embryo skin (CES) cell cultures. This has allowed the Ecogpt gene to be used as a dominant selectable marker for the insertion of foreign DNA into the TK gene of FPV.
  • vaccinia viruses require their being positioned near unique vaccinia virus transcriptional signals (1-4, 7-9), and it has been shown that the FPV TK promoter is similar to vaccinia promoters and that it operates in vaccinia virus (14,15). Vaccinia virus promoters have now been used to express foreign antigen coding sequences in FPV.
  • This invention therefore also offers methods of constructing recombinant fowlpox viruses or related avian poxviruses, which methods are characterised by the introduction of foreign DNA into the TK gene of the virus or into virus DNA sequences controlling expression of the TK gene.
  • vaccinia virus promoter sequences P7.5 and PL11 have been used to express foreign genes in FPV.
  • Recombinant FPVs have been constructed which contain the Ecogpt gene (demonstrated by hybridization) and which express the Ecogpt gene since they are able to grow in MXHAT (mycophenolic acid, resume xanthine, hypoxanthine, aminopterine, thymidine) selective conditions.
  • MXHAT mycophenolic acid, resume xanthine, hypoxanthine, aminopterine, thymidine
  • This gene is under the control of the P7.5 promoter which clearly demonstrates that this and possibly other vaccinia virus promoters can be used to express foreign genes in FPV.
  • the PL11 promoter expresses the influenza HA gene. The level of expression by this promoter may be modulated in these recombinants since the translational initiation codon of the llkd protein gene is present as well as the inserted HA gene
  • FPV TK gene is not essential for FPV growth i vitro. This establishes the FPV TK gene as a non-essential region into which foreign DNA can be inserted and expressed. Further it provides the means to construct TK ⁇ FPVs which by analogy with TK ⁇ vaccinia viruses (32) would be expected to have reduced virulence in poultry and thus be useful as vaccines against FPV themselves.
  • FPV the Ecogpt gene and the influenza HA gene
  • Vaccinia virus recombinants carrying three vaccine antigens have been constructed and shown to simultaneously induce antibody responses to all three vaccine antigens when inoculated into animals (33).
  • Additional poxvirus promoters and vaccine antigen genes in tandem with the Ecogpt can be inserted within the FPV TK gene in the general insertion vector pDB22 described herein. In this manner, recombinant FPVs can be constructed which will vaccinate poultry simultaneously against two, three or more diseases. Recombinant FPVs carrying multiple vaccine antigens would be a very cost effective way to deliver vaccines to poultry.
  • TK production would be inactivated by the introduction of foreign DNA into the TK gene or controlling sequences, or by the excision of DNA essential for TK gene expression.
  • TK ⁇ fowlpox viruses would have reduced virulence and pathogenic potential in poultry and other avian species, and correspondingly enhanced usefulness as fowlpox vaccines and vehicles for the delivery of other vaccine antigens.
  • recombinant fowlpox viruses is not limited to the delivery of antigens. It is envisaged that they might also be useful for the delivery of a variety of other proteinaceous materials, e.g. growth hormones or immunomodulators, capable of being produced by expression of an appropriate gene.
  • vaccinia virus has been the subject of interest as a vehicle for the expression of various foreign genes and thus as a basis for recombinant vaccines.
  • a variety of techniques have been developed for the construction of recombinant poxviruses based on vaccinia virus. In general, they rely on homologous recombination between vaccinia virus flanking sequences bounding the foreign gene of interest and the virus genome during simultaneous infection of cells with virus and transfection with recombinant plasmid. From the progeny virus, recombinant viruses representing less than 0.1% or 0.01% of the population have to be selected and plaque purified.
  • TK ⁇ recombinants when the foreign gene has been inserted into the TK gene of the virus; or by the co-expression of a marker gene product eg. ⁇ -galactosidase; or by selection for a dominant marker eg. HSV TK or neomycin resistance, concurrently inserted into the virus genome (28) .
  • the proportion of the progeny virus as recombinants can be increased by alternative transfection protocols involving the use of temperature sensitive mutants (5) or single stranded recombinant DNA molecules (34) .
  • TK ⁇ recombinants are selectable under BUdR, the mutagenic effect of BUdR and the resulting background of TK ⁇ mutants does not allow selection at the recombination step or enrichment at subsequent passage.
  • the neomycin resistance gene can be used as a dominant selectable marker, however, high concentrations of G418 antibiotic are required with 48hr pretreatment for the selection to operate (35) .
  • the herpes simplex virus TK (HSV-TK) gene has been inserted into non-essential regions in the vaccinia virus genome by positive selection protocols.
  • HSV-TK herpes simplex virus TK
  • positive selection protocols for the construction of recombinant vaccinia viruses expressing other genes have been achieved.
  • This approach requires the availability of a TK ⁇ cell line which will survive for sufficient time under the methotrexate (TK + ) selective conditions to allow growth and plaquing of TK + vaccinia viruses.
  • the Ecogpt gene can be used as a dominant selectable marker for insertion into poxvirus such as vaccinia virus.
  • poxvirus such as vaccinia virus.
  • the antibiotic mycophenolic acid which, together with the required presence of xanthine, hypoxanthine, aminopterine and -, c thymidine, provides the basis for selection using the Ecogpt gene is a very potent inhibitor of poxvirus growth. Since the Ecogpt gene has been used as a dominant selectable maker in a variety of mammalian cells, this obviates the need for TK ⁇ cell lines 0 for the construction of recombinant poxviruses (36).
  • the Ecogpt gene has previously been expressed from Simian Virus 40 - pBR322 hybrid plasmid vectors, and has been used as a dominant selectable marker in mammalian cells. Selection is 5 based on the ability of the Ecogpt gene to counteract the inhibitory effect of mycophenolic acid on the growth of mammalian cells. Since mycophenolic acid is also a very strong inhibitor of poxvirus growth, this provides a marker for the insertion of the 0 Ecogpt gene into poxviruses, so that when the Ecogpt gene is coupled in tandem with another gene of interest, recombinant poxviruses can be constructed and positively selected for the presence of both genes. The Ecogpt gene thus provides a dominant 5 selectable marker for insertion into any non-essential region of the poxvirus genome.
  • this invention provides a method of constructing a recombinant virus such as a poxvirus, adenovirus, herpesvirus, or the like, which comprises inserting into the non-essential region of a virus genome, a foreign DNA sequence comprising the Ecogpt gene coupled with a second gene, and selecting the virus 0 recombinants by their ability to reverse the inhibition of virus growth by mycophenolic acid.
  • a recombinant virus such as a poxvirus, adenovirus, herpesvirus, or the like, which comprises inserting into the non-essential region of a virus genome, a foreign DNA sequence comprising the Ecogpt gene coupled with a second gene, and selecting the virus 0 recombinants by their ability to reverse the inhibition of virus growth by mycophenolic acid.
  • the Ecogpt/mycophenolic acid system provides a dominant selectable marker for insertion into any non-essential region of the genome 5 of any virus where Ecogpt selection operates in the cell type in which the virus will grow. Not only will this facilitate the construction of recombinant vaccinia viruses, but it will also provide a method for the construction of recombinant virus vaccines Q including poxvirus vaccines based on host-specific poxviruses such as fowlpox virus for poultry or Orf virus for sheep, which are desirable because of the risk of disease spreading back to man from the use of vaccinia virus recombinants in animals.
  • the present invention also provides recombinant virus such as a poxvirus, adenovirus, herpesvirus or the like, characterised in that it has a foreign DNA sequence comprising the Ecogpt gene coupled with a second gene Q inserted into a non-essential region of the virus genome.
  • recombinant virus such as a poxvirus, adenovirus, herpesvirus or the like, characterised in that it has a foreign DNA sequence comprising the Ecogpt gene coupled with a second gene Q inserted into a non-essential region of the virus genome.
  • the second gene included within the foreign DNA sequence is preferably a gene which is expressed as a antigenic polypeptide which is foreign to the 5 virus. Operation of the Ecogpt gene as a dominant selectable marker for construction of recombinant poxviruses offers the additional advantages of allowing selective enrichment of recombinant viruses prior to plaque purification and characterization. It is also important in that it obviates the need for TK ⁇ cell lines in the construction of recombinant viruses.
  • the ability to select at both the recombination and subsequent steps allows the construction of recombinants where the frequencies of recombination may be low e.g. the insertion of very large fragments of foreign DNA.
  • Those skilled in the art will be able to adapt known virus recombination techniques for insertion of specific Ecogpt/antigen gene couples into poxviruses. Essentially, the procedures involve insertion of a DNA sequence comprising the Ecogpt gene under the control of a poxvirus promoter plus a second gene under the control of the same or another poxvirus promoter, into a non-essential region of a poxvirus genome.
  • More than one promoter plus second gene may be coupled with the Ecogpt gene, thus enabling insertion into poxvirus of multiple antigens or multiple serotypes of one antigen.
  • Plasmids constructed with a second gene in tandem with the Ecogpt gene having flanking poxvirus sequences from non-essential regions could be used in the marker rescue recombination protocol.
  • Recombinant virus containing the Ecogpt and second gene could be amplified and plaque-purified under mycophenolic acid selective conditions; all recombinants containing the Ecogpt gene would also contain the second gene, and this would be expressed provided it had been placed under the control of a poxvirus promoter.
  • suitable promoters include P7.5, PF or PL 11.
  • Ecogpt gene product is required for the recombinant viruses to grow under the mycophenolic acid selective conditions.
  • the Ecogpt enzyme activity in virus-infected cell extracts can be demonstrated and measured using the conversion of 14C-xanthine to 14C-xanthine monophosphate and 14C-xanthosine as described by Chu and Berg (24) .
  • Expression of the second gene inserted in tandem with the Ecogpt gene can be demonstrated by immuno chemical techniques, e.g. antibody and 125I protein A binding to plaques for a cell surface - expressed antigen like the influenza HA gene.
  • DNA can be purified by known techniques from the recombinant viruses.
  • the purified DNA may then be analysed by restriction enzyme digests and DNA:DNA hybridisations to demonstrate the place and orientation in which the Ecogpt gene and second gene have been inserted.
  • the effectiveness of the recombinant viruses of this invention containing one or more foreign antigen genes as vaccines can be demonstrated by antibody and cell-mediated immume responses in animals infected with the recombinant viruses. Protection against disease can be demonstrated by challenge with the organism(s) from which the antigen gene(s) were derived.
  • This Example illustrates the construction of recombinant FPV wherein the TK gene of the fowlpox virus is interrupted by the Ecogpt gene under control of a poxvirus promoter in tandem with a gene of interest under the control of another poxvirus promoter.
  • Figure 1 shows construction of a plasmid, pDB16, containing multiple unique restriction enzyme sites within the TK gene of FPV.
  • a Hindlll-Clal fragment of the FPV genome containing the TK gene was cloned into pUC9 which had been digested with EcoRI, mung bean nuclease treated and then digested with Hindlll. The Hindlll site was then deleted by Hindlll digestion, Klenow Poll fill in and religation.
  • a synthetic oligonucleotide was inserted into the unique Ncol site within the TK gene (pDBl ⁇ ). Unique sites for H, Hindlll, C, Clal, E, EcoRI and S, Smal were inserted with this polylinker. Other restriction enzyme sites are N, Ncol and X, Xbal.
  • Figure 2 shows construction of plasmids for insertion of the Ecogpt and influenza genes into the TK gene of FPV.
  • the Ecogpt gene with attached P7.5 vaccinia virus promoter (as an EcoRI-Ahalll fragment) was subcloned from pGpt07/14 into pDBl ⁇ which had been digested with EcoRI and Smal (pDB18) .
  • the HA gene under the control of the PL11 vaccinia virus promoter was cloned into pDB18 as a Clal fragment from pBCB08/HA.
  • Plasmids ⁇ DB19/l and pDB19/6 have the FPV TK gene interrupted by the P7.5-Ecogpt and PL11-HA gene fragments. Abbreviations for restriction enzyme sites; C, Clal, E, EcoRI, H, Hindlll, N, Ncol, X, Xbal. CIAP, calf intestinal alkaline phosphatase.
  • Figure 3 shows construction of insertion vectors for the selection of FPV recombinants using the Ecogpt gene and having foreign genes under the control of the PL11 vaccinia virus promoter.
  • pDB20 the size of the flanking FPV genome sequences has been increased by subcloning the P7.5-Ecogpt- PL11-HA fragment into the 5.5 EcoRI FPV genome fragment at the unique Ncol site contained in pDBl.
  • pDB22 is a general vector into which foreign DNA can be inserted downstream of the PL11 vaccinia virus promoter and recombinants selected on the basis of growth in Ecogpt selective conditions.
  • FIG. 4 shows antibody and 125I protein A binding to FPV plaques.
  • A normal mouse serum; B, hyperimmune mouse antiserum to influenza virus A/PR/8/34.
  • FPV fowlpox virus; FPV-HA, fowlpox virus recombinant containing the Ecogpt-P7.5 promoter and the influenza HA-PLll promoter constructed using pDB20.
  • Figure 5 shows the structure of the TK region of the parent, FPV-M3, and the recombinant, FPV-HA, genomes.
  • the recombinant, FPV-HA was constructed using ⁇ DB20 (Fig.4). Abbreviations for restriction enzyme sites: E, EcoRI; N, Ncol; P, Pstl; S, Sail. Not all EcoRI and Ncol sites are shown.
  • Figure 6 shows analysis of DNAs from the parent, FPV-M3 (1) and the recombinant FPV-HA (2) viruses.
  • DNAs were digested with Sail (S) and Pstl (P) and separated by electrophoresis through 0.6% agarose gels containing 0.5 ⁇ g/ml ethidium bromide (EtBr) . The gel was photographed with UV illumination then blotted onto Gene-Screen Plus. The membrane was then hybridized with various 32P labelled DNA fragments (pDBl; HA; Ecogpt; P7.5;
  • Figure 7 shows the HI antibody response of chickens inoculated with FPV of FPV-HA (5 birds/group) , 15 day old SPV chickens were inoculated with 10 6 to 107 pfu of virus and reinoculated 21 days later. For birds inoculated with FPV-HA titers for individual birds are shown by the dots.
  • the dots show the HI antibody response of chickens inoculated with FPV of FPV-HA (5 birds/group) , 15 day old SPV chickens were inoculated with 10 6 to 107 pfu of virus and reinoculated 21 days later.
  • Figure 8 shows the location, nucleotide
  • Hindlll, Xbal, and Clal sites present in the 5,5-kb EcoRI fragment are shown.
  • the position of the FPV TK gene was confirmed by its homology to the vaccinia virus TK gene.
  • the derived amino acid sequence is
  • pDBl to pDBll have been described previously (14).
  • pBCBO ⁇ a multiple cloning site plasmid for the construction of recombinant vaccinia viruses, contains the late vaccinia virus promoter PL11 (26,30).
  • the A/PR/8/34 influenza HA gene in pJZ102 was provided by Dr.P.N.Graves, The Mount Sinai Medical Center (27).
  • the P7.5 vaccinia virus promoter in pGS20 was provided by Dr.B.Moss, National Institutes of Allergy and Infectious Diseases, Bethesda, Md. (2).
  • CES Primary and secondary chick embryo skin (CES) cell cultures were prepared from specific pathogen free embryonated eggs (CSIRO, SPF Poultry Unit, Maribyrnong, Victoria) as described by Silim et.al. (18) with the modification that collagenase at lOO ⁇ g/ml (Sigma C2139) was used to digest the skin of the embryos in place of trypsin.
  • Fowlpox virus. (Mild Vaccine Strain: Arthur Webster Pty.Ltd., Northmead, NSW, 2152, Australia) was adapted to chick embryo cell cultures by passage at low multiplicity and plaque purified twice. This virus, designated FPV-M3, contains an approximately lOkb deletion from its genome.
  • Plaque assays were performed on CES monolayers overlayed with Eagle's minimum essential medium with Earle's salts containing 1% agar and 5% foetal bovine serum. Plaques were stained with MTT tetrazolium (19) on the fifth or sixth day after inoculation. All virus stocks were disaggregated by digestion for 30min at 37°C with lmg/ml trypsin immediately prior to dilution for assays or infection of cell cultures. Marker Rescue.
  • CES cells seeded overnight at 5x10 2 cells/25cm flask were infected with FPV-M3 at 0.01 plaque forming units per cell. Virus was adsorbed for lhr at 37 ⁇ C then culture medium added. 7hr later the monolayers were washed four times with medium without serum and buffered with 0.05M Tris pH7.4, then once with Tris buffered saline (25mM Tris pH7.4, 137mM NaCl, 5mM KC1, 0.7mM CaCl-,, 0.5mM gCl 2 , 0.6mM Na 2 HPO.; TBS; (31).
  • MXHAT 1 or 2-5 ⁇ g/ml mycophenolic acid, 250 ⁇ g/ml xanthine, lOO ⁇ M hypoxanthine, 0.4 ⁇ M aminopterine and 30 ⁇ M thymidine
  • the cultures were incubated for 6 or 7 days at which time the cells were scraped into the medium, pelleted by centrifugation and resuspended in 1ml of phosphate buffered saline. This virus stock was then passaged two or three times on CES cells under selective conditions. At the second and subsequent passages plaques grew out from marker rescues performed with plasmids containing the Ecogpt gene. Recombinant viruses containing the Ecogpt gene were plaque purified under agar without selection then amplified under selection in liquid medium in 24 well plates. Viruses containing the Ecogpt gene and gene of interest were identified by dot blot hybridization with 32P-labelled recombinant DNA probes (2,3).
  • FPV was purified from CES cultures (21) and the DNA extracted as described by Nakano et.al.
  • influenza haemagglutinin by recombinant viruses was demonstrated by binding of anti-influenza antibodies and 125I-protein A to FPV plaques. The solid overlay was removed from plaques then the monolayer fixed for 5mins at room temperature with methanol. Antibodies and
  • 125 I-protein A were then bound to the plaques as previously described (1,11). Responses of Poultry to recombinant FPVs.
  • SPF poultry 15 days old, were inoculated subcutaneously/intradermally (s.c./i.d.) into the breast skin with 0.05ml of virus containing 10 6-107 pfu. Serum was collected at 7 day
  • the FPV TK gene is contained within a 2.2kb Hindlll-Clal fragment of the FPV genome (14). This fragment was subcloned from pDBlO (14) between the Hindlll-EcoRI sites of ⁇ UC9 (Fig.l; pDB14) . The remaining Hindlll site was deleted by digestion, fill in with Klenow fragment of polymerase I and religation (pDB15). Inspection of the TK gene nucleotide sequence revealed a unique Ncol site located in the centre of the TK gene and about 20bp away from the Xbal site also in this gene (15). Restriction enzyme analysis revealed that the Ncol site was unique to the 5.5kb EcoRI fragment of the FPV genome originally shown to contain the TK gene (15).
  • a synthetic oligonucleotide linker with Ncol cohesive ends and restriction enzyme sites for Smal, EcoRI, Clal and Hindlll was inserted into this unique Ncol site (pDB16) .
  • the orientation of the linker with, respect to the TK gene was determined by restriction enzyme digests and polyacrylamide gel electrophoresis. pDB16 was then used as the backbone for insertion of foreign sequences within the FPV TK gene.
  • the Ecogpt gene from pSV2A-g ⁇ t has previously been cloned into ⁇ BCB07 (29).
  • a 900bp fragment, derived by EcoRI-Ahalll digestion, containing the Ecogpt gene with the vaccinia virus promoter P7.5 attached was cloned into pDB16 digested with EcoRI-Smal.
  • the resulting plasmid pDB18 contains the TK gene interrupted by the Ecogpt gene under the control of the P7.5 promoter.
  • influenza A/PR/8/34 haemagglutinin gene had been inserted previously into pBCB08 at the Hindlll site of the multiple cloning site (30).
  • a Clal fragment from pBCB08/HA containing the PL11 promoter and HA gene was subcloned into pDB18 which had been digested with Clal and treated with calf intestinal alkaline phosphatase. This placed the influenza HA gene under the control of the PL11 promoter in tandem with the Ecogpt gene all contained within the FPV TK gene (pDB19/l and pDB19/6) (Fig.2).
  • pDBl contains a 5.5kb EcoRI fragment of the FPV genome in which the TK gene is contained.
  • HA gene in pDB19/l was removed by Hindlll digestion and religation.
  • pDB22 contains the FPV TK gene interrupted by the P7.5 promoter-Ecogpt gene plus the PL11 promoter. Attached to the PL11 promoter is a multiple cloning site with unique restriction enzyme sites for Smal, BamHI, Sail, Pstl and Hindlll (Fig.3). This vector is suitable for the insertion of a variety of genes into the FPV genome using the Ecogpt gene to select for the recombinants. Construction and characterization of FPV recombinants expressing influenza haemagglutinin.
  • Insertion of the influenza HA gene into FPV in tandem with the Ecogpt gene was achieved using pDB20.
  • Virus plaques which grew in the presence of MXHAT were plaque purified three times and confirmed as containing the Ecogpt and HA genes by dot blot hybridization. Subsequently recombinant FPVs were successfully constructed using pDB18 and ⁇ DB19 which have shorter FPV flanking sequences.
  • FPV-HA expression of influenza haemagglutinin by the recombinant FPV-HA was demonstrated by the binding of antibodies and 125I-protein A to plaques. Only plaques of FPV-HA bound antibodies to influenza virus whilst the wild type virus, FPV-M3, failed to do so. Neither virus bound antibodies from normal serum (Fig.4).
  • FPV-HA The structure of the recombinant, FPV-HA, genome was analysed by restriction enzyme digestion and Southern hybridization in comparison with the parent virus, FPV-M3 (Figs. 5 and 6).
  • the 5.5kb EcoRI fragment which contains the-TK gene spans the junction of two Pstl fragments of 26kb and 13kb (Fig.5).
  • the TK gene is contained wholly within the 26kb fragment.
  • Pstl digestion of the FPV-M3 genome showed in ethidium bromide stained gels that the 26kb fragment was replaced by 23 and 6kb fragments. With Sail digestion a new 15.5kb fragment was present (Figs. 5 and 6) .
  • the agarose gel separated fragments were transferred to Gene-Screen-Plus membrane (NEN) and successively hybridized with various 32P-labelled
  • Figure 9 shows plaquing of vaccinia virus on CV-1 monolayers in the presence of MXHAT (1 or 25 ⁇ g/ml of mycophenolic acid). 1, 2 and 3, W-WR; 4, W-Ecogpt; 3, guanine at 25 ⁇ g/ml was able to reverse the inhibition of plaque formation by MXHAT.
  • Figure 10 shows construction of plasmids for the insertion of the Ecogpt gene into the TK gene of vaccinia virus.
  • Figure 11 shows the effect of selection and moi at the recombination step on the output of recombinant viruses expressing the Ecogpt gene.
  • Mycophenolic acid has previously been used at 25 ⁇ g/ml to inhibit mammalian cell growth and to select for cells carrying the Ecogpt gene (36,37).
  • Preliminary experiments established that confluent monolayers of CV-1 cells used for the vaccinia virus plaque assay survived for more than 7 days in mycophenolic acid up to 25 ⁇ g/ml.
  • mycophenolic acid MXHAT
  • doses from 1 to 25 ⁇ g/ml completely inhibited plaque formation and even at O.l ⁇ g/ml there was significant reduction in plaque size (Fig. 9).
  • Inhibition of mammalian cell growth by mycophenolic acid is reversible by guanine. Addition of guanine, " 25 ⁇ g/ml, to plaque assays in the presence of MXHAT (M from 0.1 to 25 ⁇ g/ml) reversed the inhibition of plaque formation and plaque sizes approached normal (Fig. 9).
  • mycophenolic acid was inhibiting vaccinia virus growth by acting as an inhibitor of inosine monophosphate dehydrogenase preventing the formation of xanthine monophosphate and therefore guanine monophosphate in a manner similar to its action on mammalian cells (37) .
  • Mycophenolic acid alone failed to inhibit vaccinia virus plaque formation on CV-1 cells.
  • HAT hypoxanthine, aminopterine and thymidine
  • HAT was required to inhibit de novo synthesis of inosine monophosphate to allow expression of the mycophenolic acid inhibition.
  • HAT alone had no effect on vaccinia virus plaquing on CV-1 cells.
  • the conditions used to inhibit completely vaccinia virus plaquing even at very high virus input were mycophenolic acid, 1 or 2.5 ⁇ g/ml, xanthine, 250 ⁇ g/ml, hypoxanthine, l ⁇ O ⁇ M, aminopterine, 0.4 ⁇ M, thymidine, 30 ⁇ M (MXHAT) .
  • the cell monolayers do not require pretreatment with MXHAT for inhibition of virus plaquing and preformed monolayers maintain for long periods (7 to 10 days) under MXHAT selection.
  • the translation initiation codon of the Ecogpt gene is located 200b ⁇ from the Hindlll site.
  • a unique Bglll site is located 120bp closer to the translation initiation codon (38) .
  • pBCB07 39; Fig.10
  • Recombinant plasmids pGpt07/14 and 15 were selected by restriction enzyme digests as having the Ecogpt gene in the correct and incorrect orientation with respect to the vaccinia virus promoter and flanked by the TK gene sequences.
  • a recombinant vaccinia virus has been constructed using ⁇ Gpt07/14 and the insertion of the Ecogpt gene into the TK region confirmed by restriction enzyme analysis and Southern hybridization analysis.
  • This recombinant, W-Ecogpt is able to plaque under MXHAT on CV-1 monolayers whilst plaquing of the wild type virus, W-WR, is completely inhibited (Fig. 9).

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Un virus de la variole de la volaille recombinant ou un virus qui se rapporte à la variole aviaire se caractérise par l'inclusion d'ADN étranger dans le génome du virus. La séquence d'ADN étranger peut comprendre le gène Ecogpt couplé à un second gène et peut être inséré dans une région non essentielle du génome du virus.
PCT/AU1987/000323 1986-09-22 1987-09-22 Virus de la variole recombinant WO1988002022A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
AUPH8109 1986-09-22
AU811086 1986-09-22
AU810986 1986-09-22
AUPH8110 1986-09-22

Publications (1)

Publication Number Publication Date
WO1988002022A1 true WO1988002022A1 (fr) 1988-03-24

Family

ID=25612752

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU1987/000323 WO1988002022A1 (fr) 1986-09-22 1987-09-22 Virus de la variole recombinant

Country Status (1)

Country Link
WO (1) WO1988002022A1 (fr)

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0308220A1 (fr) * 1987-09-16 1989-03-22 Nippon Zeon Co., Ltd. Avipoxvirus recombinant
FR2621487A1 (fr) * 1987-08-28 1989-04-14 Health Research Inc Virus avipox recombinant
EP0314569A1 (fr) * 1987-10-29 1989-05-03 Transgene S.A. Virus du fowlpox recombinant, vecteurs d'expression de protéines héterologues et vaccins pour la volaille dérivés de ce virus
WO1989003879A1 (fr) * 1987-10-23 1989-05-05 National Research Development Corporation Promoteurs du virus de la variole aviaire
GB2220941A (en) * 1988-06-24 1990-01-24 Nat Res Dev Fowlpox virus non-essential regions
GB2222165A (en) * 1988-08-20 1990-02-28 Animal Health Inst Recombinant capripox virus
JPH02131593A (ja) * 1988-04-19 1990-05-21 Immuno Ag Chem Med Prod 組換体アビポックスウィルスベクターを用いた単離された蛋白様物質の産生
WO1990007581A1 (fr) * 1988-12-29 1990-07-12 Nippon Zeon Co., Ltd. Promoteur, plasmide le contenant, et avipoxvirus de recombinaison
EP0404835A4 (en) * 1988-03-18 1991-05-08 Us Health Novel recombinant vaccinia virus expression vectors and method of selecting same
EP0443335A1 (fr) * 1990-01-29 1991-08-28 IMMUNO Aktiengesellschaft Plasmide recombinant pour un vecteur pox-viral comportant une cassette ADN multifonctionnelle
WO1992000372A1 (fr) * 1990-07-02 1992-01-09 Smithkline Beecham Corporation Vaccin recombine contre l'avipoxvirose du pigeon
US5093258A (en) * 1988-08-26 1992-03-03 Therion Biologics Corporation Recombinant fowlpox virus and recombination vector
US5174993A (en) * 1981-12-24 1992-12-29 Health Research Inc. Recombinant avipox virus and immunological use thereof
US5286639A (en) * 1987-09-16 1994-02-15 Nippon Zeon Co., Ltd. Recombinant avipoxvirus
US5387519A (en) * 1987-03-27 1995-02-07 Nippon Zeon Co., Ltd. Recombinant avipoxvirus
US5631154A (en) * 1988-06-10 1997-05-20 Therion Biologics, Incorporated Self assembled, defective, non-self-propagating lentivirus particles
EP0775746A2 (fr) 1995-07-03 1997-05-28 Akzo Nobel N.V. Vaccin contre la coccidiose aviaire
US5651972A (en) * 1989-04-21 1997-07-29 University Of Florida Research Foundation, Inc. Use of recombinant swine poxvirus as a live vaccine vector
US5670367A (en) * 1991-08-26 1997-09-23 Immuno Aktiengesellschaft Recombinant fowlpox virus
RU2130971C1 (ru) * 1991-05-27 1999-05-27 Димминако АГ/СА/Лтд. Рекомбинантный вектор на основе вируса оспы птиц, рекомбинантный вектор для вакцинации домашней птицы
US5925358A (en) * 1993-02-26 1999-07-20 Syntro Corporation Recombinant fowlpox viruses and uses thereof
US5942235A (en) * 1981-12-24 1999-08-24 Health Research, Inc. Recombinant poxvirus compositions and methods of inducing immune responses
US6001369A (en) * 1993-02-26 1999-12-14 Syntro Corporation Recombinant fowlpox viruses and uses thereof
US6136318A (en) * 1993-02-26 2000-10-24 Cochran; Mark D. Recombinant fowlpox viruses and uses thereof
US7767449B1 (en) 1981-12-24 2010-08-03 Health Research Incorporated Methods using modified vaccinia virus
US8506947B2 (en) 1999-05-28 2013-08-13 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Vaccinia virus expression vector for selective replication in a tumor cell and introduction of exogenous nucleotide sequence into a tumor cell

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986000528A1 (fr) * 1984-07-05 1986-01-30 Genex Corporation Gene clone et son procede de preparation et d'utilisation
WO1986005806A1 (fr) * 1985-03-29 1986-10-09 National Research Development Corporation Proteine "pointe" du virus de la bronchite infectieuse

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986000528A1 (fr) * 1984-07-05 1986-01-30 Genex Corporation Gene clone et son procede de preparation et d'utilisation
WO1986005806A1 (fr) * 1985-03-29 1986-10-09 National Research Development Corporation Proteine "pointe" du virus de la bronchite infectieuse

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Experimental Cell Research, Volume 166, No. 1, issued 1986, January (New York, USA), M. MEVEL-NINIO et al., "A Polyoma-Derived Plasmid Vector Maintained Episomally in Both E. Coli and Mouse Hepatoma Cells", see pages 63-76. *
Journal of General Virology, Volume 67, No. 8, issued 1986, August (Colchester, England), D.B. BOYLE et al., "Identification and Cloning of the Fowlpox Virus Thymidine Kinase Gene Using Vaccinia Virus", see pages 1591-1600. *
Molecular and Cellular Biology, Volume 3, No. 8, issued 1983, August (Washington, D.C., USA), A.B. CHAPMAN et al., "Amplification and Hormone-Regulated Expression of a Mouse Mammary Tumour Virus - Ecogpt Fusion Plasmid in Mouse 3T6 Cells", see pages 1421-1429. *
Proceedings of the National Academy of Sciences USA, Volume 81, No. 22, issued 1984, November (Washington, D.C., USA), H. STUHLMANN et al., "Introduction of a Selectable Gene into Different Animal Tissue by a Retrovirus Recombinant Vector", see pages 7151-7155. *
Virology, Volume 156, No. 2, issued 1986, January (New York, USA), D.B. BOYLE et al., "Fowlpox Virus Thymidine Kinase: Nucleotide Sequence and Relationship to Other Thymidine Kinases", see pages 355-365. *

Cited By (39)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5174993A (en) * 1981-12-24 1992-12-29 Health Research Inc. Recombinant avipox virus and immunological use thereof
US7767449B1 (en) 1981-12-24 2010-08-03 Health Research Incorporated Methods using modified vaccinia virus
US5942235A (en) * 1981-12-24 1999-08-24 Health Research, Inc. Recombinant poxvirus compositions and methods of inducing immune responses
US5387519A (en) * 1987-03-27 1995-02-07 Nippon Zeon Co., Ltd. Recombinant avipoxvirus
BE1002134A5 (fr) * 1987-08-28 1990-07-24 Health Research Inc Avipox virus recombinant.
GB2217718B (en) * 1987-08-28 1992-05-20 Health Research Inc Recombinant viruses,vaccines containing them and in vitro cell cultures thereof
FR2621487A1 (fr) * 1987-08-28 1989-04-14 Health Research Inc Virus avipox recombinant
DE3890874C5 (de) * 1987-08-28 2005-10-20 Health Research Inc Rekombinante Viren
AT408549B (de) * 1987-08-28 2001-12-27 Health Research Inc Virushältiger impfstoff, rekombinantes virus und verfahren zum exprimieren eines genproduktes
EP0308220A1 (fr) * 1987-09-16 1989-03-22 Nippon Zeon Co., Ltd. Avipoxvirus recombinant
US5286639A (en) * 1987-09-16 1994-02-15 Nippon Zeon Co., Ltd. Recombinant avipoxvirus
US5182210A (en) * 1987-10-23 1993-01-26 National Research Development Corporation Fowlpox virus promoters
WO1989003879A1 (fr) * 1987-10-23 1989-05-05 National Research Development Corporation Promoteurs du virus de la variole aviaire
US5180675A (en) * 1987-10-29 1993-01-19 Transgene, S. A. Recombinant fowlpox virus
FR2632863A2 (fr) * 1987-10-29 1989-12-22 Transgene Sa Virus du fowlpox recombinant et vaccins derives de ces virus
EP0314569A1 (fr) * 1987-10-29 1989-05-03 Transgene S.A. Virus du fowlpox recombinant, vecteurs d'expression de protéines héterologues et vaccins pour la volaille dérivés de ce virus
EP0404835A4 (en) * 1988-03-18 1991-05-08 Us Health Novel recombinant vaccinia virus expression vectors and method of selecting same
EP0338807A3 (en) * 1988-04-19 1990-09-12 Immuno Aktiengesellschaft Fur Chemisch-Medizinische Produkte Production of isolated proteinaceous materials using recombinant avipox virus vectors
JPH02131593A (ja) * 1988-04-19 1990-05-21 Immuno Ag Chem Med Prod 組換体アビポックスウィルスベクターを用いた単離された蛋白様物質の産生
US5631154A (en) * 1988-06-10 1997-05-20 Therion Biologics, Incorporated Self assembled, defective, non-self-propagating lentivirus particles
GB2220941B (en) * 1988-06-24 1991-09-11 Nat Res Dev Fowlpox virus non-essential regions
US5310671A (en) * 1988-06-24 1994-05-10 British Technology Group Limited Fowlpox virus non-essential regions
GB2220941A (en) * 1988-06-24 1990-01-24 Nat Res Dev Fowlpox virus non-essential regions
GB2222165B (en) * 1988-08-20 1992-06-17 Animal Health Inst Recombinant capripoxvirus
GB2222165A (en) * 1988-08-20 1990-02-28 Animal Health Inst Recombinant capripox virus
US5093258A (en) * 1988-08-26 1992-03-03 Therion Biologics Corporation Recombinant fowlpox virus and recombination vector
WO1990007581A1 (fr) * 1988-12-29 1990-07-12 Nippon Zeon Co., Ltd. Promoteur, plasmide le contenant, et avipoxvirus de recombinaison
US5651972A (en) * 1989-04-21 1997-07-29 University Of Florida Research Foundation, Inc. Use of recombinant swine poxvirus as a live vaccine vector
EP0443335A1 (fr) * 1990-01-29 1991-08-28 IMMUNO Aktiengesellschaft Plasmide recombinant pour un vecteur pox-viral comportant une cassette ADN multifonctionnelle
WO1992000372A1 (fr) * 1990-07-02 1992-01-09 Smithkline Beecham Corporation Vaccin recombine contre l'avipoxvirose du pigeon
RU2130971C1 (ru) * 1991-05-27 1999-05-27 Димминако АГ/СА/Лтд. Рекомбинантный вектор на основе вируса оспы птиц, рекомбинантный вектор для вакцинации домашней птицы
SG49876A1 (en) * 1991-05-27 2002-04-16 Dimminaco Ag Sa Ltd Recombinant avipox virus, the culture of cells infected with this virus and vaccines for poultry derived from this virus
US5670367A (en) * 1991-08-26 1997-09-23 Immuno Aktiengesellschaft Recombinant fowlpox virus
US6136318A (en) * 1993-02-26 2000-10-24 Cochran; Mark D. Recombinant fowlpox viruses and uses thereof
US6123949A (en) * 1993-02-26 2000-09-26 Cochran; Mark D. Recombinant fowlpox virus S-FPV-043 and uses thereof
US6001369A (en) * 1993-02-26 1999-12-14 Syntro Corporation Recombinant fowlpox viruses and uses thereof
US5925358A (en) * 1993-02-26 1999-07-20 Syntro Corporation Recombinant fowlpox viruses and uses thereof
EP0775746A2 (fr) 1995-07-03 1997-05-28 Akzo Nobel N.V. Vaccin contre la coccidiose aviaire
US8506947B2 (en) 1999-05-28 2013-08-13 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Vaccinia virus expression vector for selective replication in a tumor cell and introduction of exogenous nucleotide sequence into a tumor cell

Similar Documents

Publication Publication Date Title
WO1988002022A1 (fr) Virus de la variole recombinant
Boyle et al. Construction of recombinant fowlpox viruses as vectors for poultry vaccines
JP3044062B2 (ja) 組換えポックスウイルス宿主選択系
US5093258A (en) Recombinant fowlpox virus and recombination vector
US5225336A (en) Recombinant poxvirus host range selection system
Chakrabarti et al. Vaccinia virus expression vector: coexpression of β-galactosidase provides visual screening of recombinant virus plaques
US5364773A (en) Genetically engineered vaccine strain
JP3826055B2 (ja) 組換えアビポックスウイルスによる免疫方法
Sutter et al. A recombinant vector derived from the host range-restricted and highly attenuated MVA strain of vaccinia virus stimulates protective immunity in mice to influenza virus
US5180675A (en) Recombinant fowlpox virus
Coupar et al. Temporal regulation of influenza hemagglutinin expression in vaccinia virus recombinants and effects on the immune response
Perkus et al. Cloning and expression of foreign genes in vaccinia virus, using a host range selection system
US6340462B1 (en) Recombinant avipox virus
US5174993A (en) Recombinant avipox virus and immunological use thereof
EP0389509B1 (fr) Promoteurs du virus de la variole aviaire
Spehner et al. Construction of fowlpox virus vectors with intergenic insertions: expression of the beta-galactosidase gene and the measles virus fusion gene
Boursnell et al. A fowlpox virus vaccine vector with insertion sites in the terminal repeats: demonstration of its efficacy using the fusion gene of Newcastle disease virus
Amano et al. Identification of the canarypox virus thymidine kinase gene and insertion of foreign genes
Moss Vaccinia virus expression vector: a new tool for immunologists
Kumar et al. Activity of a fowlpox virus late gene promoter in vaccinia and fowlpox virus recombinants
Smith et al. Host range selection of vaccinia recombinants containing insertions of foreign genes into non-coding sequences
WO2007115385A2 (fr) Vecteur plasmidique de transfert et virus canarypox recombinant
Boursnell Avipoxvirus vectors
Schnitzlein et al. Utilization of vaccinia virus promoters by fowlpox virus recombinants
Moss Genetic engineering of vaccinia virus vectors: development of live recombinant vaccines

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU JP US

AL Designated countries for regional patents

Kind code of ref document: A1

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

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载