EP0432196A1 - Vakzin - Google Patents

Vakzin

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Publication number
EP0432196A1
EP0432196A1 EP89909820A EP89909820A EP0432196A1 EP 0432196 A1 EP0432196 A1 EP 0432196A1 EP 89909820 A EP89909820 A EP 89909820A EP 89909820 A EP89909820 A EP 89909820A EP 0432196 A1 EP0432196 A1 EP 0432196A1
Authority
EP
European Patent Office
Prior art keywords
capripoxvirus
sheep
cattle
disease
goats
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Withdrawn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
EP89909820A
Other languages
English (en)
French (fr)
Inventor
Donald Norman Afrc Institute For Animal Black
C. J. Afrc Inst. For Animal Healthltd. Bostock
P. D. Afrc Inst. For Animal Health Ltd. Gershon
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.)
INSTITUTE FOR ANIMAL HEALTH Ltd
Pirbright Institute
Original Assignee
INSTITUTE FOR ANIMAL HEALTH Ltd
Pirbright Institute
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 INSTITUTE FOR ANIMAL HEALTH Ltd, Pirbright Institute filed Critical INSTITUTE FOR ANIMAL HEALTH Ltd
Publication of EP0432196A1 publication Critical patent/EP0432196A1/de
Withdrawn legal-status Critical Current

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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
    • 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
    • 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
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/60—Vector systems having a special element relevant for transcription from viruses

Definitions

  • the present invention relates to vaccines in which a viral vector is used to express a heterologous gene.
  • Capripoxvirus causes sheep pox and goat pox, diseases which can kill over 50% of young animals in those areas, principally Africa and the Middle and Far East, where the disease is enzootic. Different strains of the virus show a preference for either sheep or goats but none have so far been found to be totally species- specific. Some strains also infect, and cause lumpy skin disease in, cattle but (apart from buffalo) capripox- viruses do not infect any other domestic animals .
  • a vaccine has been developed which comprises an attenuated strain of the strain known as "the Kenya isolate” (Kitching, Rev. Sci . Tech. Off. Epiz. , 1986, 5(2) , 503- 511) .
  • vaccinia virus genome can be altered by insertion of a heterologous gene from a pathogen, such that the product of that gene will be expressed in infected cells. Protection against diseases associated with the pathogen has been induced in this way.
  • a review of such work may be found in Mackett & Smith, J. Gen. Virol. 67 . . 2067-2082 (1986).
  • capripoxvirus represented a possible alternative to vaccinia as a vector for foot and mouth disease vaccines.
  • Black and Gershon disclosed that work was in progress on the use of capripoxvirus as a vector for heterologous antigens and that the thymidine kinase gene was a candidate insertion site .
  • capripoxvirus can indeed be used as a vector for heterologous genes and that there are several possible insertion sites for such genes.
  • One aspect of the invention provides a capripoxvirus capable of infecting and replicating in capripoxirus- susceptible cells and carrying at least one heterologous gene encoding an antigen of a pathogen such that the gene will be expressed during transcription and translation from the capripoxvirus genome in the said cell.
  • the heterologous gene is inserted in one of the following regions of the capripoxvirus genome:
  • thymidine kinase gene region we mean the genetic unit, including coding and non-coding sequences, for the expression of thymidine kinase.
  • C3C region and "CT4 region” we mean the respective regions of the capripox virus genome identified by these designations in the attached figures, or variations thereof.
  • Q2 region we mean the approximately 2.4 kb fragment bounded by the respective Hindlll sites approximately 6.6 and 9.0 kb from the right-hand end of the KS-1 isolate genome shown in Figure 1 and identified thereon as Q2 , and variations thereof.
  • any of these regions will include: minor polymorphic changes associated with different strains of capripoxvirus ; any other changes which might occur or be created in the nucleic acid of capripoxvirus but which do not alter the non-essential nature of the said region or the nature of the gene product encoded by that region; and any other nucleotide sequences which encode a gene product with substantially the same structure or function as the gene product of the said region or which correspond to the said region but are non-coding.
  • nucleotide sequences with at least 80% homology to the said regions are regarded as variations thereof.
  • the extent of homology is 85%, 90%, 95% or 99%.
  • anigen we mean any structure, part thereof or mimic thereof associated with a pathogen and capable of giving rise to an immune response in a host when the immune system of that host is exposed to the antigen.
  • the immune response is a protective response.
  • surface carbohydrates, glycoproteins and proteins of viruses and other pathogens are included in this definition, as are peptides which are homologous to part of such whole molecules , and peptides which mimic the tertiary structure of such molecules.
  • the heterologous gene will encode the amino acid sequence of the glycoprotein and/or one of more enzymes which cause appropriate glycosylation of that amino acid sequence.
  • the heterologous gene or genes will encode one or more enzymes which catalyse the synthesis of such carbohydrates.
  • Clostridium chauvoei infection blackquarter Sheep, cattle, goats, buffalo Clostridium novyi (type A) infection - Big head Sheep
  • Clostridium novyi (type B) infection - Black disease Sheep, cattle, goats
  • Clostridium novyi (type D) infection - Bacillary Haemo- globinuria Cattle, sheep, goats
  • Clostridium perfringens (type B) infection - Lamb dysentery Sheep Clostridium perfringens (type C) infection Sheep, cattle
  • Clostridium perfringens (type D) infection- Pulpy kidney disease Sheep, goats Clostridium tetani infection - Sheep, goats, cattle, Tetanus wild ruminants Clostridium botulinum infection -
  • Mycoplasma mycoides infection - Contagious bovine pleuro- Buffalo, cattle, wild pneumonia ruminants
  • the recombinant viruses of the invention may be prepared by (i) constructing a DNA construct comprising the heterologous gene and two sequences which flank the heterologous gene and which are homologous to respective parts of the capripoxvirus DNA, such that recombination of the construct with capripoxvirus may occur, (ii) infecting cells with capripoxvirus and introducing the said construct into the said cells, (iii) screening the infected cells for recombinant capripoxvirus, (iv) isolating the recombinants from step (iii) and (v) growing the isolated viruses in a suitable host.
  • the construct may comprise a promoter for the heterologous gene, which will be any promoter which is functional in the cytoplasm of the capripoxvirus-infected cells .
  • the promoter may interact with a polymerase which is also heterologous to both the capripoxvirus and the infected cell and which is encoded by another heterologous gene which is inserted in the recombinant capripoxvirus, as -with the bacterial T7 system.
  • the promoter is derived from capripoxvirus itself but it may alternatively be derived from vaccinia virus, for example it may be the promoter for the P7.5 protein (Mackett et al (1984) , J. Virol.
  • the construct may not comprise a promoter at all but is so constructed as to recombine with the capripoxvirus at a site close to, and in correct reading frame with, an endogenous capripox ⁇ virus promoter.
  • flanking sequences may • be synthesised by reference to the sequence of the insertion region or may be obtained from such a region and cloned. Typically, a length of about 100 nucleotides for each flanking sequence is sufficient to ensure recombination, but 120 nucleotides or more may be used.
  • the infection of the cells with the capripoxvirus may be before, after or at the same time as introduction of the DNA construct.
  • the cells may be any which are permissive for capripoxvirus , such as most ovine, bovine and caprine derived cells, for example lamb testis , bovine palate, or ovine kidney cells.
  • the DNA construct is preferably provided as a plasmid.
  • the plasmid may be based on commercially available bacterial plasmids , such as pUC9 , which may be cloned In bacteria and which have suitable markers for selection, for example Amp r . Transfection of the capripoxvirus- susceptible cells with the plasmid may be facilitated by known techniques involving calcium phosphate or electroporosis .
  • the plasmid or other transfection vehicle is so chosen as to be non-infectious. Following introduction of the transfection vehicle into the host cell in step (ii) , the vehicle is not propagated between cells.
  • screening in step ( iii ) for the product of the heterologous gene effectively screens for recombinant viruses.
  • Such screening may, for example, comprise an immunological assay for the antigen expressed by that gene or nucleotide hybridisation using sequences homologous to the heterologous gene.
  • the heterologous gene is in the first instance the E.coli beta-galactosidase gene ( lac Z), thus allowing screening in step (iii) by the well-known technique using "X-gal” ( 5-bromo-4-chloro-3-indolyl-beta-D-galactopyran- oside) in an agar overlay on the cell culture. Blue plaques are formed by recombinants which express the lacZ gene. Insertion of a further heterologous gene into the recombinant lac Z region, with concomitant deletion of the beta-galactosidase function, then allows selection by isolating colourless plaques against the parental blue plaques. This approach may be useful where a suitable assay for the product of the second heterologous gene is not available.
  • Step (iv) will usually involve successive rounds of purification from plaques or cell-foci of infection, or end-point dilutions , and screening for recombinants in known ways. Amplification may be desirable during such procedures.
  • the host may be any suitable cell line or, less preferably, any suitable living animal. Lamb testis and the other cell lines mentioned above are suitable.
  • the recombinant viruses may then be used to make vaccines in known ways , for example as described in Kitching, loc.cit.
  • the virus is preferably included in the vaccine in an attenuated form, rather than killed.
  • the vaccines may be used to prevent the particular disease or diseases associated with the pathogen(s) of which an antigen or antigens are encoded by the virus of the invention, and may be used in sheep, goats, cattle
  • the vaccines will be most suitable for use in areas where capripoxvirus-associated diseases are endemic and/or where the animals have all been vaccinated against capripoxvirus .
  • the vaccines may be used to prevent the particular disease or diseases associated with the pathogen(s) of which an antigen or antigens are encoded by the virus of the invention, and may be used in sheep, goats, cattle (Bos bovis and Bos indicus ) , buffalo, llamas, alpacas and vicunas. Generally speaking, the vaccines will be most suitable for use in areas where capripoxvirus-associated diseases are endemic and/or where the animals have all be vaccinated against capripoxvirus.
  • the vaccines may be administered by any suitable route, including intra-nasally (as a spray), orally (for example in the feed or drinking water) , intra-muscularl , sub-cutaneousl , intra-venously or intra-dermally.
  • intra-nasally as a spray
  • intra-muscularl sub-cutaneousl
  • intra-venously intra-dermally.
  • experience with existing capripoxvirus vaccines shows that the intra-dermal route is not preferred and that the sub-cutaneous or intra-muscular routes are preferred.
  • the virus will be detectable in the animal's tissues, thus enabling an animal which has been vaccinated in accordance with the invention to be identified.
  • the viral vectors of the invention may further include markers to aid such identification, if desired.
  • the immunity of an animal which has been vaccinated in accordance with the invention may be distinguished from that of an animal which has been exposed to the corresponding pathogen since the former will include antibodies only to the particular antigen expressed by the modified viral genome and not to other antigens of the pathogen.
  • Figure 1 shows the distribution of Hindlll restriction enzyme recognition sites on the genome of capripoxvirus strain KS-1, together with a scale representing length in numbers of kilobase pairs;
  • Figure 2 shows the nucleotide sequence of the S fragment identified in Figure 1 , with the region labelled as CF8 corresponding to the thymidine kinase (TK) region;
  • TK thymidine kinase
  • Figure 3 shows the sequence from the terminal region of the genome of capripoxvirus isolate KS-1 , reading in a telomere-proximal telomere-distal direction.
  • the terminal Hindlll site (AAGCTT) of the genome occurs in the sequence.
  • This site, and the Sau3Al ( GATC) site defining the proximal end of clone pPG64, are underlined and emboldened; and
  • Figure 4 shows the distribution of stop-codons in the 6 possible reading frames for the sequence between approximately 700 and 2,400 bp from the apex of the terminal hairpin loops of capripoxvirus strain KS-1 DNA, the telomere proximal and distal ends bring on the left and right, respectively.
  • TK thymidine kinase
  • the genomic region containing the intended insertion site was the KS- 1 genomic DNA fragment Hindlll S, which was cloned in the plasmid vector pUC9.
  • the plasmid was linearized by incubating with the restriction enzyme Kpnl, for which a single site is present in the clone, and is located within the KS-1 TK gene.
  • the bacterial lacZ g-ene coupled to the vaccinia virus promoter P .5 was inserted at the Kpnl site in the above plasmid using T4 ligase, after cleaving P7.5-lacZ from another plasmid with the restriction enzyme BamHI , and treating both the BamHI and the Kpnl fragments with Klenow enzyme.
  • the resulting plasmid was named pPG55.
  • LT cell cultures infected with capripoxvirus isolate KS-1 were transfected with the pPG55, and the cultures were incubated at 37° C for 5 days. Further LT cell cultures were infected with the harvested virus and after seven days incubation these cultures were overlaid with low gelling temperature (LGT) agarose containing the substrate X-gal ( 5-bromo-4-chloro- 3-indolyl-beta-D-galactopyranoside) ( 'X-gal overlay' ). After a further 24 hours blue foci were visualised through the X-gal overlay, and virus was picked through the agarose and subjected to further rounds of plaque purification.
  • LGT low gelling temperature
  • a library of capripoxvirus KS-1 DNA in plasmid pPG2 was produced by partial digestion of the capripoxvirus DNA with Sau3A, and cloning of the resulting fragments into BamHI linearised pPG2.
  • pPG2 was derived from the commercially available pUC9 by removal of the multiple cloning site by digestion with Hindlll and EcoRl , filling in of the single-stranded ends with Klenow enzyme, and ligation (using T4 ligase) of a synthetic BamHI linker (GGGATCCC) to recircularise the plasmid DNA.
  • the ligation mixture was used to transform E.coli strain TGI and the bacteria plated onto LB agar containing ampicillin.
  • Plasmid DNas containing Hindlll sites were identified by digestion with Hindlll and analysis by agarose gel electrophoresis. 40 clones carrying Hindlll sites located at different positions on the capripoxvirus genome were digested with Hindlll and the Hindlll P7.5-lacZ fragment was inserted into each using T4 ligase to create a series of trial insertional vectors for at least 10 different sites in the capripox ⁇ virus genome.
  • each trial insertional vector was transfected into capripoxvirus KS-1 infected lamb testis cells and the presence of blue plaques/foci was screened for.
  • the presence of blue plaques/foci identified those insertional vectors carrying sequences which directed insertion by recombination at sites which are not essential for capripoxvirus replication and in-fection in lamb testis cells. In this way we have identified three Hindlll sites within non-essential regions of capripox-virus DNA into which heterologous DNA can be inserted.
  • Other restriction enzymes may be used instead of Hindlll , preferably those which (for the capripoxvirus genome) cut at about the same frequency, namely about 25- 35 times, preferably about 30 times. Infrequent cutting results in insertion sites being missed, whereas too frequent cutting yields fragments which are too small to be practicable.
  • enzymes which recognise a sequence of 5 to 7 nucleotides, preferably 6.
  • an enzyme recognising a 4-nucleotide sequence rich in G's or C's might also be suitable.
  • Sau3A is a suitable enzyme for the initial digestion as it recognises frequently occurring sites (about 600, i.e. an average distribution of about one per 250 b.p.); a partial, rather than complete, digest then yields virtually random, conveniently-sized fragments likely to contain a Hindlll site. Other enzymes, such as Haelll , may be used for differing frequencies of occurrence of the recognition sites.
  • the partial digests may be pooled. The pool itself may be cloned or one may select a particular size class of fragment for cloning.
  • the work outlined in this Example is based on clones covering a region within the terminal repeats of the genome of capripoxvirus isolate KS-1. This region covers two open reading frames (ORFs) named CT3C and CT4.
  • Figure 3 shows the 1651 b.p. nucleotide sequence of the above mentioned region.
  • Figure 4 shows the positions of the stop codons In " the sequence. ORFs CT3C and CT4 are marked; CT3C lies between nucleotide 252 and 647 and CT4 lies between nucleotide 839 and 1621 in this sequence.
  • pPG64 a plasmid, to enable P7.5-lacZ to be inserted within CT3C; more specifically at the Hind III site between nucleotides 561 and 566 of the sequence shown in Figure 3.
  • This Hindlll site divides Hindlll fragments Wl and 11 at the left-hand end of the genome and fragments F and W2 at the right-hand end (see Figure 1).
  • Clone pPG64 was made by ligating a Sau3A partial digest fragment of KS-1 DNA of size 1648 b.p., starting at nucleotide 440 on the sequence shown in Figure 3 , in a derivative of the commercially available plasmid cloning vehicle pUC9, named pPG2 (see Example 2) .
  • the resulting plasmid, in which P7.5-lacZ is cloned at the Hindlll site of pPG64 is called pPG65.
  • Recombinant capripoxvirus KS-1 was generated using pPG65 by the method described in the last paragraph of Example 1.
  • virus obtained from blue viral foci was propagated on LT cells, and gave a further round of blue foci .
  • These in turn were propagated by several additional rounds of plaque purification and shown to maintain the ability to express blue foci .
  • capripoxvirus isolates used in the work described herein are typical of wild-type capripoxvirus and the techniques describeed and claimed herein do not depend upon the particular strain or isolate which is used. Other isolates may have minor variations in their genomes, but it will be well within the capabilities of the man skilled in the art to adapt the techniques to such other isolates. For example, it is believed that the region of another isolate ( InS ; Gershon & Black (1987) Virol. 160, 473-476) which corresponds to the ORF in KS-1 labelled herein as CT4 is a non-coding region. Such a region would clearly be a suitable candidate for insertion of the heterologous gene.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
EP89909820A 1988-08-20 1989-08-18 Vakzin Withdrawn EP0432196A1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB8819836 1988-08-20
GB8819836A GB2222165B (en) 1988-08-20 1988-08-20 Recombinant capripoxvirus

Publications (1)

Publication Number Publication Date
EP0432196A1 true EP0432196A1 (de) 1991-06-19

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EP89909820A Withdrawn EP0432196A1 (de) 1988-08-20 1989-08-18 Vakzin

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EP (1) EP0432196A1 (de)
AU (1) AU621934B2 (de)
GB (1) GB2222165B (de)
OA (1) OA09636A (de)
WO (1) WO1990002190A1 (de)
ZA (1) ZA896372B (de)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5833975A (en) * 1989-03-08 1998-11-10 Virogenetics Corporation Canarypox virus expressing cytokine and/or tumor-associated antigen DNA sequence
US7767449B1 (en) 1981-12-24 2010-08-03 Health Research Incorporated Methods using modified vaccinia virus
US5174993A (en) * 1981-12-24 1992-12-29 Health Research Inc. Recombinant avipox virus and immunological use thereof

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2602790B1 (fr) * 1986-08-13 1990-06-01 Transgene Sa Expression d'un antigene specifique de tumeur par un virus vecteur recombinant et utilisation de celui-ci pour le traitement preventif ou curatif de la tumeur correspondante
WO1988002022A1 (en) * 1986-09-22 1988-03-24 Commonwealth Scientific And Industrial Research Or Recombinant poxviruses
EP0261940A3 (de) * 1986-09-23 1989-07-05 Applied Biotechnology, Inc. Pseudorabies-Impfstoffe und DNS-Vektoren zur Rekombination mit Poxviren

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9002190A1 *

Also Published As

Publication number Publication date
OA09636A (en) 1993-04-30
GB8819836D0 (en) 1988-09-21
AU621934B2 (en) 1992-03-26
WO1990002190A1 (en) 1990-03-08
AU4190989A (en) 1990-03-23
ZA896372B (en) 1990-07-25
GB2222165A (en) 1990-02-28
GB2222165B (en) 1992-06-17

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