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WO1998004710A2 - Vaccin recombine vivant contre l'herpes virus bovin/le virus respiratoire syncytial bovin - Google Patents

Vaccin recombine vivant contre l'herpes virus bovin/le virus respiratoire syncytial bovin Download PDF

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WO1998004710A2
WO1998004710A2 PCT/IB1997/001040 IB9701040W WO9804710A2 WO 1998004710 A2 WO1998004710 A2 WO 1998004710A2 IB 9701040 W IB9701040 W IB 9701040W WO 9804710 A2 WO9804710 A2 WO 9804710A2
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brsv
gene
bhv
recombinant
synthetic
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PCT/IB1997/001040
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WO1998004710A9 (fr
WO1998004710A3 (fr
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Gunther M. Keil
Franciscus A. M. Rijsewijk
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Akzo Nobel N.V.
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Priority to AU37819/97A priority Critical patent/AU3781997A/en
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Publication of WO1998004710A3 publication Critical patent/WO1998004710A3/fr
Publication of WO1998004710A9 publication Critical patent/WO1998004710A9/fr

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    • 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
    • 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
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16741—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16743—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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention refers to synthetic Bovine Respiratory Syncytium virus genes, live attenuated Bovine Herpesvirus recombinants, live attenuated Bovine Herpesvirus recombinants carrying such genes, vaccines based on these live attenuated recombinants, methods for the preparation of such live attenuated recombinants and to methods for the preparation of such vaccines.
  • Bovine Respiratory Syncytium virus (BRSV), a member of the paramyxoviruses, is a cause of respiratory tract infections in cattle.
  • BRSV infection occurs world-wide and can cause severe disease especially in the lower respiratory tract, similar to the disease caused by human respiratory syncytial virus (HRSV) in children (Kimman and Westenb ⁇ nk, Archives of Virology, 1990, 112, 1-25).
  • HRSV human respiratory syncytial virus
  • vector viruses mimic the natural infection in the sense that they do infect host cells and express, next to their own genetic material, the additional genetic information cloned into their genome.
  • vaccinia virus An obvious vector for essaying the possible expression of both F and G protein under laboratory conditions is vaccinia virus. This virus has successfully been used as an expression vector for a multitude of different genes for many years.
  • mice vaccinated with the live HRSV gF vaccinia recombinant vector were also protected against infection with wild-type HRSV.
  • vaccinia virus As a live recombinant vector virus is attractive, because much is known about the virus and many tools for making vaccinia recombinants are available.
  • Vaccinia virus is known to have an extremely broad host range. It is known to be infectious for all mammalian species tested so far, ranging from rabbits, mice, raccoons, sheep, goats and camels to humans (Jolly, D.J.; Semin-lmmunol. 1990 Sep; 2(5): 329-339
  • Vaccinia virus although a bovine pathogen, would therefore certainly not be the vector of preference for use in animal vaccines for the protection of bovine animals against BRSV for the following reason:
  • Bovine herpesvirus infection • they protect against two different diseases: Bovine herpesvirus infection and BRSV- infection.
  • Live attenuated recombinant BHV-recombinants are known to be good potential expression vectors, and vaccine viruses at the same time.
  • BHV-1 has e.g. been used for the expression of LacZ (Schmitt et al., J. Virol. 70: 1091-1099
  • BHV-4 has been used e.g. for the expression of the LacZ-gene (Vanderplasschen et al.,
  • BRSV-genes cloned in BHV as a recombinant carrier virus, let alone showing in vivo protection by such a BHV-recombinant.
  • Two main problems are encountered when expression of BRSV-genes is tested in BHV-1:
  • the objective of the present invention is, to provide a BHV/BRSV-recombinant virus that overcomes the problems mentioned above.
  • the synthetic gene of the present invention can be efficiently expressed in BHV- recombinant viruses under the control of eukaryotic promoters, and the so obtained BHV/BRSV-recombinant viruses can be propagated in a stable manner.
  • a naturally occurring nucleic acid sequence is understood to be a nucleotide sequence as it is found in naturally occurring viruses, such as the viruses isolated from the field.
  • the synthetic gene according to the present invention has a nucleotide sequence that, albeit different from the original nucleotide sequence (i.e. the nucleotide sequence found in virus isolated from the field), still encodes exactly the same amino acid sequence, and thus encodes the native protein.
  • This principle is applicable to any gene, e.g. the BRSV-F- or G-gene.
  • the BRSV-gene encodes the BRSV-G protein. This protein, as motivated above, is known to play an important role in the induction of an immunological response against BRSV-infection. In an equally preferred form of the invention, the BRSV-gene encodes the BRSV-F protein. This protein, as also motivated above, also plays an important role in the induction of an immunological response against BRSV-infection.
  • the degeneracy of the genetic code also allowed e.g. the introduction of a number of restriction enzyme cleavage sites in the nucleic acid sequence encoding the G-protein gene, without modifying the amino acid sequence of the G-protein.
  • Synthetic DNA is defined as a DNA that is made by synthesis, instead of being isolated from a natural source.
  • each and every nucleotide of the DNA is synthesized. It is also possible to modify an existing DNA by replacing part thereof by a part with another nucleic acid sequence using recombinant DNA technology. The result of this modification is also considered to be a synthetic DNA
  • the synthetic DNA can be made in a number of different ways, all known in the art.
  • One useful method for modifying a nucleotide sequence is e.g. site-directed mutagenesis. With this generally known method, modifications are made deliberately at predetermined sites. It is also possible to replace small or longer fragments by fragments with an alternative sequence. Many different techniques for DNA-manipulation are currently available.
  • One possible method of replacing (parts of) a naturally found sequence with a synthetically made sequence is e.g. to cut the nucleic acid sequence with restriction enzymes to remove the sequence to be replaced, followed by ligation of a synthetically made fragment with the same restriction sites. Alternatively, the whole gene can be replaced by synthetic DNA-fragments. Techniques for site-directed mutagenesis, and for DNA-synthesis are known in the art. Also, DNA can be made fully synthetically in a DNA-synthesizer.
  • RNA from synthetic BRSV-genes is so stable.
  • RNA synthesized in the cytoplasm, as is the case with BRSV-genes after natural infection, is not subjected to the process of RNA-splicing. RNA-splicing is restricted to RNAs synthesized in the nucleus, such as the nuclear RNAs.
  • BRSV normally replicating in the cytoplasm, may lead to the formation of unstable (BRSV-
  • Splice-sites have small consensus sequences for both splice-donor sites (AGGU as a consensus sequence) and splice acceptor sites (a UC-rich region).
  • AGGU a consensus sequence
  • splice acceptor sites a UC-rich region
  • At least one potential splice-acceptor site is located from position 268 on, where a UC-rich region is found.
  • the BRSV-gene is modified in such a way, that at least one possible splice-donor or acceptor site, found in the naturally occurring nucleic acid sequence, is removed in the synthetic BRSV-gene.
  • Splice-sites can be removed by replacing one or more nucleotides of the potential splice-site within the framework of what is allowed by the degeneracy of the genetic code. This can be done by e.g. site-directed mutagenesis, or replacement of small or longer fragments by fragments with an alternative sequence. Alternatively, the whole gene can be replaced by synthetic DNA-fragments.
  • BRSV-RNA even if this is correctly transcribed and not destroyed due to splicing-events, can not be correctly translated.
  • the synthetic BRSV-gene has been modified in such a way, that the GC-content is at least 50%.
  • the GC-content is at least 60 %
  • the synthetic BRSV-gene has the sequence presented in table 4, second line.
  • BRSV-gene in order to be expressed, must be placed under the control of a promoter.
  • promoters are e.g. the Pseudorabies gX-promoter, the Pseudorabies TK-promoter, the Adenovirus Major Late promoter, the Retroviral Long Terminal Repeat, the SV40 Early and Late promoters, the MCMViel promoter, the MCMVel promoter, the HCMViel promoter, and the BHV-gE promoter.
  • the MCMViel promoter is preferred promoters.
  • the synthetic BRSV-gene is placed under the control of one of the promotors of the group of promotors consisting of the MCMViel promoter, the MCMVel promoter, the HCMViel promoter, and the BHV-gE promoter.
  • Another embodiment of the invention relates to live attenuated BHV-recombinant viruses carrying a synthetic BRSV-gene according to the present invention.
  • BHV-viruses are the carriers of choice for BRSV-genes.
  • the BHV-1 virus is used as a BHV-recombinant.
  • This virus is a very commonly found pathogen in cattle, also (as is the case with BRSV) causing high economical losses.
  • Bovine Herpes Virus-I infection The most common manifestation of Bovine Herpes Virus-I infection is bovine rhinotracheitis which varies from a mild respiratory disease to a severe infection of the entire respiratory tract. From an economical point of view, IBR is also the most dramatic manifestation of BHV-I infection.
  • Morbidity rate in IBR is usually close to 100%.
  • BHV- live attenuated recombinant virus for carrying and expressing a BRSV-gene is very efficient: vaccines based on such a live attenuated recombinant virus protect against both BRSV and BHV-1. Animals so vaccinated are protected against the two most frequently found causes of respiratory disease in cattle.
  • BRSV-gene in the BHV-recombinant The most suitable technique for such an insertion is homologous recombination, known in the art and frequently used.
  • the BRSV-gene to be inserted should be cloned between left and right fragments of a non-essential BHV-gene, since in that case, a viable recombinant is obtained that is not disturbed in essential functions.
  • BHV BHV
  • the genes coding for the (glyco)proteins gE, gl, gG, and US2 are e.g. very suitable as integration sites.
  • the BRSV-gene to be expressed is integrated in the gE-gene of
  • the BRSV-gene to be expressed is integrated in the gl- gene of BHV-1.
  • a BHV-I recombinant according to the invention may, next to a BRSV-gene, comprise other genes encoding antigens from microorganisms or viruses that are pathogenic for cattle.
  • a very attractive live attenuated BHV-recombinant is e.g. a BHV-recombinant that comprises both the BRSV-G-gene and the BRSV-F-gene, or one BRSV-gene and a non- BRSV-gene.
  • a BHV-recombinant comprising and expressing both the BRSV-G-gene and the BRSV-F- gene is also within the scope of the invention.
  • the BHV-recombinant comprises, next to one or more BRSV-genes, genes encoding antigens from other microorganisms or viruses that are pathogenic for cattle.
  • the present invention provides BHV-recombinants comprising, next to a BRSV-gene, a gene encoding an antigen from microorganisms or viruses that are pathogenic for cattle.
  • the gene is chosen from the group of cattle pathogens, consisting of Bovine Rotavirus, Bovine Viral Diarrhoea virus, Parainfluenza type 3 virus, Bovine Paramyxovirus, Foot and Mouth Disease virus, Bovine Respiratory Syncytium virus and Pasteurella haemolytica.
  • a gene may be introduced into the BHV-recombinant according to the invention, that encodes a cytokine.
  • cytokines e.g. interferons are known to play an important role as immune modulators.
  • Still another embodiment of the present invention refers to vaccines for the protection of cattle against virus infection, based upon live attenuated BHV-recombinants expressing a
  • Vaccines based thereon have the advantage that they mimic the natural infection of not only BHV, but to a large extend also of BRSV, as motivated above.
  • Vaccination is in many cases at least a two-step process: a first immunisation with an antigen triggers the immune response, and a second immunisation; the booster, actually enhances both the speed and the strength of the immune response.
  • a first vaccination immunity against both the carrier itself and the heterologous gene carried by the carrier is triggered. This is the result of the fact that the recombinant carrier infects a cell and during viral replication both the viral proteins and the heterologous protein of the carried gene are expressed and become presented at the cell membrane. There they are detected by the immune system.
  • viruses as live recombinant carriers
  • antibodies against the recombinant carrier raised during first immunisation prevent a successful second round of infection with the recombinant carrier.
  • the immune system will not see the encoded proteins a second time.
  • the carrier virus this problem can be circumvented by just giving a higher dose of virus particles, since the viral proteins are perse present on the virus particle.
  • the virus particles act as an inactivated vaccine and as such stimulates the immune system.
  • Infection is however necessary for the heterologous gene to be expressed. Therefore, since no second round of infection occurs, the heterologous gene carried by the carrier is not expressed a second time. Thus no booster immunisation against the heterologous gene product will be generated
  • BRSV gG protein is incorporated into the envelope of the BHV-1 virus particles during the maturation of the virus.
  • Class II membrane glycoproteins are characteristic in that they have an N- terminal membrane anchor.
  • Membrane glycoproteins having an N-terminal membrane anchor are e.g. the hemagglutinin-neuraminidase of the paramyxovirus Simian Virus 5, the Influenza virus neuraminidase and the G-protein of the Human Respiratory Syncytial virus.
  • BRSV gG lacks any herpesspecific targeting signals, BRSV gG could not be expected to be incorporated in the envelope of the BHV-1 particle.
  • BRSV gG in the envelope of BHV means that the problem mentioned above can be circumvented: a booster immunisation with a high dose of BHV virus particles carrying the BRSV gG protein on their envelope causes a second immune response to be triggered against both the BHV envelope proteins and the BRSV gG protein on the envelope, without infection being necessary.
  • BRSV gG in the envelope to be dependent on the presence of the BRSV gG membrane anchor.
  • a membrane anchor has the same characteristics in all class II membrane glycoproteins (C-ll MGs). This means that in general class II membrane glycoproteins for which the gene is carried by BHV, will be incorporated into the envelope of the BHV-particles.
  • BHV as a carrier for C-ll MGs thus circumvents the problem addressed above concerning the inefficiency of booster-reactions against heterologous genes in carriers.
  • One embodiment of the invention therefore relates to live attenuated BHV-recombinant virus particles, carrying a heterologous class II membrane glycoprotein.
  • heterologous non-Class-ll-proteins into the envelope of BHV virus particles can also be obtained, provided that these proteins are coupled to a C-ll MG membrane anchor.
  • This BHV-recombinant can easily be used for targeting a non-C-ll protein into the envelope of BHV virus particles, by simply replacing all of the cloned gG-gene except for the first 213 base pairs encoding the membrane anchor, with the coding sequence of a non-C-ll protein-encoding gene. Therefore, still another embodiment of the invention relates to live attenuated BHV- recombinants carrying a heterologous gene fused to a class II membrane glycoprotein membrane anchor. In a preferred form the invention relates to live attenuated BHV-recombinants carrying a heterologous gene fused to the BRSV-gG membrane anchor
  • the membrane anchor of the gG protein comp ⁇ ses the region from ammo acid number 1 to 71
  • the whole membrane anchor sequence should be present in order to assure a stable integration into the virus envelope It is however possible to use only part of the membrane anchor, i e less than the above-mentioned 71 ammo acids, provided that integration of the final construct into the envelope can still be obtained
  • the present invention relates to method for the preparation of live attenuated recombinant BHV particles carrying a heterologous protein incorporated in the particle envelope Such methods comprise growing a live attenuated BHV-recombinant carrying a heterologous gene that encodes a class II membrane glycoprotein membrane anchor or is fused to a class II membrane glycoprotein membrane anchor
  • the above-mentioned live attenuated BHV-recombinants are very suitable as a basis for vaccines for combating at the same time both BHV-infection and infections caused by any microorganisms having a protein that plays an important role in raising protective immunity when fused to a class II membrane glycoprotein membrane anchor
  • the vaccine according to the present invention may compnse a pharmaceutically acceptable earner
  • a pharmaceutically acceptable earner One possible earner is a physiological salt-solution
  • Another pharmaceutically acceptable earner is for instance the solution in which an adjuvant is provided
  • an adjuvant and possibly one or more emulsrfiers such as Tween( ⁇ ) and
  • Span(R) are also incorporated in the live or inactivated vaccine according to the invention
  • Suitable adjuvants are for example vitamin-E acetate solubi sate, aluminum hydroxide, - phosphate or -oxide, (mineral) oil emulsions such as Bayol( ⁇ ) and Marcol52(R), and saponins. Incorporation of the antigens in Iscoms is also a possible way of adjuvation.
  • a stabilizer to live or inactivated viruses, particularly if a dry composition of live viruses is prepared by lyophilisation.
  • Suitable stabilizers are, for example, SPGA (Bovamik et al., J. Bacteriology 59, 509, 1950), carbohydrates (such as sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose), proteins (such as albumin or casein), or degradation products thereof, and buffers (such as alkali metal phosphates). If desired, one or more compounds with adjuvant activity as described above can also be added.
  • a vaccine according to the invention may be administered by intramuscular or subcutaneous injection or via intranasal, intratracheal, oral, cutane, percutane or intracutane administration.
  • the vaccine is administered intranasally
  • the DNA isolated from the live attenuated BHV-recombinant according to the present invention can be administered.
  • Vaccination methods using naked DNA instead of viruses have been described i.a. by Cohen (Science 259: 1691-1692 (1993)), by Pardoll (Immunity 3: 165-169 (1995)) and by Montgomery (Current Biology 5: 505-510 (1994))
  • the useful effective amount to be administered will vary depending on the age, weight, and mode of administration.
  • a suitable dosage can be for example about 10 3 - 10 10 pfu/animal.
  • Another embodiment of the present invention relates to methods for the preparation of vaccines according to the invention.
  • Such methods comprise admixing a live attenuated BHV-recombinant according to the present invention and a pharmaceutically acceptable carrier.
  • a live vaccine the BHV-I mutant according to the present invention can be grown on susceptible cells.
  • susceptible cells are e.g. Madin Darby Bovine Kidney cells (MDBK-cells) or bovine embryonic cells.
  • the viruses thus grown can be harvested by collecting the tissue cell culture fluids and/or cells.
  • the live vaccine may be prepared in the form of a suspension or may be lyophilized.
  • Still another embodiment of the present invention refers to methods for the preparation of live attenuated BHV-recombinants according to the invention.
  • Such methods comprise bringing together in a suitable host cell BHV-DNA and a vector comprising the BRSV-gene to be expressed, placed under the control of a suitable promoter and flanked by 3' and 5' flanking regions that share homology with BHV- sequences, in order to facilitate homologous recombination.
  • both the vector and the isolated viral DNA may transfected together into a suitable host cell.
  • PrV belongs just like BHV-1 to the alpha-he ⁇ esviruses, a group of herpesviruses of which the members show extensive structural and functional homology.
  • Gori was cloned under the control of the gp50-promoter as follows: Plasmid pRSV02 (see figure 1, and kindly provided by Dr. P. Sondermeijer, Intervet Int., Boxmeer, The Netherlands) was cleaved with EcoRI, the ends filled with Klenow- polymerase, and the fragment containing Gori was isolated using gel-electrophoresis. Plasmid Tn77 (Klupp et al.; Virology 182: 732-741 (1991)) as depicted in figure 2, containing the PrV gp50 promoter was cleaved with Xbal (in the multicloning site downstream the promoter) and the ends filled with Klenow-polymerase. Subsequently the Gori fragment was ligated into the cleaved Tn77. See figure 3 for cloning scheme.
  • Fori was cloned under the control of the gp50-promoter as follows: Plasmid pRSV02 (see figure 1) was cleaved with Bglll, the ends filled with Klenow- polymerase, and the fragment containing Fori was isolated using gel-electrophoresis. Plasmid Tn77 was digested with Xbal and the ends filled with Klenow-polymerase. Subsequently the Gori fragment was ligated into the cleaved Tn77. See figure 3 for cloning scheme.
  • Vector pAT-glV has been described extensively in EP 0.663.403. It should be mentioned here that the vector pAT-glV is also referred to as pAT-gD, due to the new nomenclature of the he ⁇ esvirus (glycoprotein-)genes.
  • Madin Darby Bovine Kidney (MDBK) cells were transfected with 5 ⁇ g DNA of each of the above-mentioned plasmids 24 hours after seeding using the calcium phosphate precipitation technique.
  • the cells were harvested and the cell culture supernatant was used for the infection of MDBK cells. Under these conditions only genotypically gIV positive virions can grow productively and form plaques on the monolayers.
  • virions from single plaques were isolated and DNA from MDBK cells was purified, cleaved with Hindlll, size separated by agarose gel electrophoresis and transferred to nitrocellulose filters. Filters were hybridized with 32 P-labeled DNA either from the MCMV-ie2 polyadenylation signal, the ⁇ -galactosidase-ORF, Gori and Fori. The results demonstrate that the isolated virions lack the ⁇ -galactosidase sequences, and that the plasmids mentioned above are integrated.
  • RNA obtained from cells infected with the various recombinants were probed with BHV-specific and BRSV-G- or BRSV-F-specific DNA probes.
  • the cDNA sequence of the wild-type BRSV-G gene was determined by Lerch (J. Virol. 64: 5559-5569 (1990)). This sequence was used as the guiding sequence to compose a synthetic gene called BRSV Gsyn ORF or shortly Gsyn, that has many more convenient restriction sites compared to the wild-type BRSV-G gene, but still codes for a gG-protein that has an amino acid sequence that is fully identical to the wild-type gG-protein (further called Gori).
  • the synthetic gene has 10 different restriction-sites, regularly divided over the whole gene, whereas the original gene has only three restriction-sites, two of which are moreover located almost at the same site .
  • the synthetic gene therefore, contrary to the original gene, facilitates the construction of a whole range of smaller and larger deletion mutants. This allows the determination of sites that negatively interfere with the expression of BRSV- genes in vector viruses that replicate in the nucleus. To reach this goal, the following steps were taken:
  • Plasmid pF6 was digested with Pstl and BstEII and after insertion of fragment 5 the obtained pF5 was treated with Pstl and Clal, after which fragment 4 was integrated, resulting in plasmid pF4-7.
  • pUC19-F4-7 was digested with Xbal, filled with Klenow-Polymerase, and cleaved with Aflll.
  • Plasmid pUC18-F1-3 was also digested with Xbal, filled with Klenow-Polymerase, and cleaved with Aflll, whereafter the insert was isolated trough gel-electrophoresis. Finally, the insert was cloned in the cleaved pUC19-F4-7, to yield pBRSV Gsyn-ORF, containing the reconstructed BRSV Gsyn-ORF, shortly called Gsyn, flanked by EcoRI sites. This is depicted in figure 6.
  • AAC GAA ACC CAA AAC AGA AAA ATC AAA GGC CAA TCC ACT CTA CCC GCC ACC AGA 432 G ..G ..G ... C.C ..G GAG. ..G ..G ..G ...G CC
  • Plasmid pAMB33 (Dorsch-Hasler et al., Proc. Natl. Acad. Sci 82: 8325-8329 (1985) was digested with Hpal and Xbal. Fragments were filled with Klenow- polymerase and isolated from agarose gels. A 1.4 kB fragment comprising the MCMV ie1 promoter was obtained.
  • the plasmid pAT-gD PLUS was cleaved in its polylinker site with EcoRV. In this site, the isolated MCMV ie1 fragment was cloned, to yield pROMI (See figure 7).
  • Plasmid MM354 comprising the MCMV e1 promoter (sequence of the Murine Cytomegalovirus early 1 gene is present in the EMBL Gene Bank at Heidelberg, Germany) was digested with Smal and Hindlll. Fragments were filled with Klenow- polymerase and isolated from agarose gels. A 1.1 kB fragment comprising the MCMV e1 promoter was obtained.
  • the plasmid pAT-gD PLUS was cleaved in its polylinker site with EcoRV. In this site, the isolated MCMV e1 fragment was cloned, to yield pROME (See figure 7).
  • Plasmid pRSV02 was cleaved with Bglll, and a 0.8 kB fragment was isolated from agarose gel.
  • Plasmid BRSV Gsyn ORF was cleaved with EcoRI and the resulting fragments were filled with Klenow-polymerase. A 0.8 kB fragment comprising Gsyn was isolated from agarose gel.
  • Both the plasmids pROME and pROMI were digested with Bglll and the isolated 0.8 kB fragment was cloned to yield pROME-Gsyn and pROMI-Gsyn.
  • Plasmid pRSV02 was cleaved with Bglll, and a 2.0 kB fragment comprising Fori was isolated from agarose gel.
  • Both the plasmids pROME and pROMI were digested with Bglll and the isolated 2.0 kB fragment was cloned to yield pROME-Fori and pROMI-Fori.
  • MDBK cells were transfected with 5 ⁇ g DNA of each of the above-mentioned plasmids 24 hours after seeding using the calcium phosphate precipitation technique.
  • Transfected cells were shocked with glycerol 4 h after addition of the DNA and cultures were infected with phenotypically complemented gIV" virus BHV-1/80-221 (Fehler et al, J. of Virol., 66: 831- 839 (1992)) with a multiplicity of 10 PFU per cell.
  • the cells were harvested and the cell culture supernatant was used for the infection of MDBK cells. Under these conditions only genotypically gIV positive virions can grow productively and form plaques on the monolayers.
  • virions from single plaques were isolated and DNA from MDBK cells was purified, cleaved with Hindlll, size separated by agarose gel electrophoresis and transferred to nitrocellulose filters. Filters were hybridized with 32 P-labeled DNA either from the MCMV-ie2 polyadenylation signal, the ⁇ -galactosidase-ORF, ORF-1 or the sequences downstream ORF-1 presented for homologous recombination. The results demonstrate that the isolated virions lack the ⁇ - galactosidase sequences, that ORF-1 is deleted and that the plasmids mentioned above are integrated.
  • BHV-1/eGsyn the BHV-1 recombinant in which the Gsyn gene is integrated under the control of the MCMVel promoter
  • BHV-1/eGori the BHV-1 recombinant in which the Gsyn gene is integrated under the control of the MCMVel promoter
  • Cytoplasmic RNA was isolated at 6 h p.i., size separated by agarose gel electrophoresis, and transferred to nitrocellulose.
  • a "P-labeled DNA probe representing the BRSV Gsyn ORF detected an RNA of 1.3 kb after infection with BHV-1/eGsyn ( Figure 11 , lane 1) and, after extended exposure, a transcript of 1.9 kb ( Figure 11, lane 5) whose synthesis is initiated approximately 600 bp upstream the MCMV e1 promoter (Grzimek and Keil unpublished). Even after longer exposure no transcripts were unequivocally detected by 32 P-iabeled DNA from the BRSV Gori ORF ( Figure 11 , lanes 2 and 6).
  • RNA synthesized in nuclei isolated at 6 h p.i. was hybridized to BRSV Gori, BRSV Gsyn, BHV-1gD and plasmid vector pSP73 sequences, dotted on nitrocellulose membranes ( Figure 12).
  • BRSV-G specific monoclonal antibody 20 MAb 20
  • anti-VacGsyn a polyclonal antiserum
  • the anti- VacGsyn serum was raised in rabbits after infection with VacGsyn, a recombinant vaccinia virus which expresses the BRSV Gsyn ORF.
  • MDBK cells were infected with wild type BHV- 1 strain Sch ⁇ nb ⁇ ken (BHV-1/Sch ⁇ ), BHV-1/eGsyn or BHV-1/eGori.
  • This antibody did not specifically bind to proteins from cells infected with BHV-I/Scho ( Figure 14, lane 1 , ) and BHV-1/eGori ( Figure 14, lane 2), proteins from purified BHV-1/Sch ⁇ and BHV-1/eGori virions ( Figure 14, lanes 5 and 6) and proteins released into the culture medium of BHV-1/eGsyn infected cells ( Figure 14, lane 8) but strongly reacted with proteins with apparent molecular masses of 38 and 43 kDa among BHV-1/eGsyn infected cell proteins ( Figure 14, lanes 3). In addition, several weaker bands ranging in size from 30 to 100 kDa were detected.
  • the synthetic BRSV-G gene Gsyn has also been cloned behind the BHV1 gE-promoter.
  • the glycoprotein E (gE) gene has been chosen because it is not essential for virus replication and because gE deletion mutants have good vaccine properties (Van Engelenburg et al., Journal of Virology 75: 2311-2318 (1994); Patent Application WO92/21751).
  • the Gsyn gene has first been cloned into BHV1 recombination cassette p175.
  • Recombination cassette p175 is constructed by cloning the flanking regions of the BHV1 gE gene into pUC18 using standard methods (Maniatis, T. et al, in "Molecular cloning; a laboratory manual,(1982) ISBN 0-87969-136-0).
  • This 1.1 kb Pstl-BstBI fragment contains the gE promoter that directs the transcription of the gE gene.
  • the right hand gE flanking region starts at the EcoNI site at the stop codon of the gE open reading frame and ends at the first Smal site downstream of the gE open reading frame that is located in the terminal repeat region. (The left and right flanking region have been described in WO92/21751.)
  • the Gsyn gene has been liberated from plasmid BRSV Gsyn ORF (see figure 6) using restriction enzymes Accl and Ncol and has been ligated in the proper orientation between the BstBI and EcoNI site, using standard methods (Maniatis, T. et al, in "Molecular cloning; a laboratory manual,(1982) ISBN 0-87969-136-0).
  • the promoter of the immediate early 1 gene of human cytomegalovirus (hCMVie-promoter: Peeters et al.,; J. Virol. 66: 894-905 (1992)) has been cloned upstream of the BRSV-G gene.
  • hCMVie-promoter Peeters et al.,; J. Virol. 66: 894-905 (1992)
  • the BRSV-G gene has first been cloned into the phCMV175 recombination cassette.
  • the phCMV175 recombination cassette has been derived from the cassette p175 by inserting a 720 bp Asel fragment, containing most of the hCMVie promoter cis-regulatory signals, between the BstBI site and the EcoNI site of p175.
  • the 720 bp Asel fragment has downstream of the hCMVie promoter sequences a polylinker region and a polyadenylation signal.
  • the synthetic BRSV-G gene has been liberated with restriction enzymes Accl and Ncol and cloned into the Smal site in this polylinker region using standard methods (Maniatis, T.
  • the cultured EBTr cells have been freeze-thawed to liberate the emerged wild type and recombinant viruses.
  • the freeze-thawed cell debris has been pelleted and 100 microliters of the supernatant has been used to infect monolayers of Ebtr cells on a 96 well plate to enrich for recombinant virus.
  • After three days of virus replication the culture medium of each well has been transferred to a second 96 well plate and stored at -20°C.
  • IPMA immuno-peroxidase monolayer assay
  • MAb20 anti-BRSV-G monoclonal antibody 20
  • the culture medium of Mab20 positive wells has been used to infect another 96 well plate with EBTr monolayers and the procedure has been repeated until virtually all BHV1 infected cells express BRSV-G.
  • the virus stocks enriched for recombinant virus have been used for three rounds of plaque purification and the resulting BHV-1-BRSV-G recombinants have been named respectively 531 and 608 (see Figures 17 and 18).
  • MDBK cells were infected with BHV-1/Sch ⁇ , BHV-1/G 0r i and BHV- 1/G ⁇ y ⁇ . (See fig. 25). Proteins from infected cells, harvested at 10 h.p.i. (lanes 1-4), from purified virions (lanes 5-7) and cell culture medium (lane 8) were analysed by immunoblotting with BRSV G-specific MAb 20. Proteins shown in lane 4 were from cells incubated with cycloheximide (100 ⁇ g/ml) for 2 h before lysis.
  • BHV-1/Gsyn virus particles contain the G glycoprotein and therefore should be susceptible to inactivation by gG specific antibodies.
  • BHV-1/G 0 and BHV-1/G syn virions were tested for complement dependent and independent neutralisation by an anti-VacG ⁇ yn serum raised against vaccinia virus expressing G 8yn and an anti-BRSV serum, raised in gnotobiotic calves after infection with BRSV.
  • G glycoprotein influences the entry of BHV-1/G ⁇ yn virions into the target cells.
  • Fig. 27 shows the result of a representative experiment.
  • BHV-1/G 8 yand required approximately 5 min more than BHV-1/Sch ⁇ and BHV-1/G on
  • the plasmid contains a 2.8 kB cDNA fragment that comprises the native BRVS-G-gene and the BRSV-F-gene
  • This plasmid comprises a 0 24 kb Ndel-Nlalll fragment from
  • Pseudorabies virus comprising the PrV gp50 promoter
  • Gsyn-ORF which was constructed from pUC18-F1-3 and pUC19-F4-7
  • FIG. 8 Physical map of the plasmids pROME-Gori and pROMI-Gori. The construction of these plasmids from pROME and pROMI, and the stepwise cloning of fragments constituting the Gori-genes are cloned is indicated.
  • RNA from cells infected with BHV-1/eGsyn (lanes 1 , 3, 5 and 7) and BHV-1/eGori (lanes 2, 4, 6 and 8) was prepared at 6 h p.i. and 5 ⁇ g were transferred to nitrocellulose after 1% agarose gel electrophoresis. Filters were hybridized to 32 P-labeled DNA from the BRSV Gsyn ORF (lanes 1 and 5), the BRSV Gori ORF (lanes 2 and 6) and the BHV- gD ORF (lanes 3, 4, 7 and 8). Bound radioactivity was visualized by autoradiography. Lanes 5-8 show longer exposures of lanes 1-4. Transcript sizes are indicated in kb.
  • MDBK cell cultures were infected with approximately
  • BHV-1/eGsyn expressed BRSV-G MDBK cells were infected with BHV- 1/Scho (lanes 1 and 5), BHV-1/eGor ⁇ (lanes 2 and 6) and BHV-1/eGsyn (lanes 3, 4, 7 and 8) Proteins from infected cells, harvested at 10 h p i (lanes 1-4) from purified vi ⁇ ons (lanes 5-7) and cell culture medium (lane 8) were analyzed by immunoblottmg with the BRSV-G specific MAb 20 Proteins shown in lane 4 were from cells incubated with cycloheximide (100 ⁇ g/ml) for 2 h before lysis
  • the BRSV-G gene has been isolated from the BRSV-Gsyn-ORF vector using restnction enzymes Accl and Ncol Shown at the top
  • This Accl-Ncol fragment has been cloned between the BstBI and the EcoNI site of recombination cassette p175 Shown in the middle Recombination cassette p175 contains the flanking regions of the BHV1-gE gene
  • the nght hand 1 1 kb Pstl -BstBI fragment covers the C-terminal portion of the gl gene and the gE promoter region
  • the left hand 1 kb EcoNI-Smal fragment covers the US9 gene and part of the terminal repeat region (TR)
  • the resulting construct has been named p531 and is shown at the bottom
  • the top line represents the BHV1 genome with its two segments: a long unique segment (L) and a short segment (S).
  • the S segment has a central unique short region that is bordered by a direct repeat indicated by the black boxes.
  • the gE locus is located in the unique short region.
  • In the bottom line shows a part of the unique short region and a bordering repeat has been shown in more detail. Indicated are the position of the glycoprotein I (gl ) gene, the gE promoter (gE-P), the synthetic BRSV-G gene and the US9 gene.
  • the top line represents the BHV1 genome with its two segments: a long unique segment (L) and a short segment (S).
  • the S segment has a central unique short region that is bordered by a direct repeat indicated by the black boxes.
  • the gE locus is located in the unique short region.
  • the bottom line shows a part of the unique short region and a bordering repeat has been shown in more detail. Indicated are the position of the glycoprotein I (gl) gene, the promoter of the human cytomegalovirus (hCMV-P), the synthetic BRSV-G gene, a polyadenylation signal (Poly-A sign) and the US9 gene.
  • Genomic DNA has been isolated according to standard methods (see Van Engelenburg et al., 1994, Journal of Virology 75, 2311-2318) and digested with restriction enzyme Hindlll. The obtained DNA fragments have been separated on a 0.7% agarose gel according to standard methods (Maniatis, T. et al, in "Molecular cloning; a laboratory manual,(1982) ISBN 0-87969-136-0), stained with ethidium bromide using an UV transilluminator and photographed using a Polaroid camera with a 667 Polaroid film.
  • genomic DNA of phage lambda digested with Hindlll has been separated as a molecular size marker.
  • 3 and 4 digests of genomic DNA of respectively BHV1 parent strain Lam BHV1-BRSV-G recombinant 531 and BHV1-BRSV-G recombinant 608 have been separated.
  • Figure 24 Excretion of BRSV virus from nasal and tracheal tissues from BRSV challenged calves as recovered in nasopharyngeal swabs.
  • the recBHV-G group had been vaccinated twice with the BHV1 -BRSV-G recombinant 608.
  • the control group has been twice mock infected.
  • MDBK cells were infected with BHV- 1/Sch ⁇ (lanes 1 and 5), BHV-1/G W i (lanes 2 and 6) and BHV-1/G ⁇ yr , (lanes 3, 4, 7 and 8). Proteins from infected cells, harvested at 10 h.p.i. (lanes 1-4), from purified virions (lanes 5- 7) and cell culture medium (lane 8) were analysed by immunoblotting with BRSV G-specific MAb 20. Proteins shown in lane 4 were from cells incubated with cycloheximide (100 ⁇ g/ml) for 2 h before lysis.
  • BHV-1/G,y n virions are susceptible to neutralisation by antibodies against the G glycoprotein.
  • Approximately 200 PFU of BHV-1/eG or i were incubated with 20% fetal calf serum and complement or serial dilutions of anti-VacGsyn with complement (open squares) or the anti-BRSV hyperimmune serum with complement (open circles) and about 160 PFU of BHV-1/G 8 y n were incubated with 20% fetal calf serum and complement or with serial dilutions of anti-VacGsyn with complement (closed squares) the anti BRSV hyperimmune serum without complement (triangles, no complement added to the FCS control) or the anti- BRSV hyperimmune serum with complement (closed circles).

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Abstract

La présente invention a trait à des gènes du virus respiratoire syncytial bovin synthétiques. L'invention concerne également des produits recombinés vivants atténués d'herpès virus bovin portant ces gènes synthétiques. De plus, l'invention concerne des vaccins à base de ces produits recombinés atténués vivants destinés à la protection du bétail contre les infections de type herpès virus bovin et les infections dues au virus respiratoire syncytial bovin. L'invention a également trait à des procédés de préparation de ces produits recombinés atténués vivants ainsi qu'à des procédés de préparation de ces vaccins. En outre, l'invention concerne des particules recombinées d'herpès virus bovin vivantes portant une glycoprotéine de membrane de classe II, des produits recombinés d'herpès virus bovin vivants portant un gène hétérologue fusionné à une ancre membranaire de glycoprotéine de membrane de classe II, des vaccins à base de ceux-ci, ainsi que des procédés de préparation de ces produits recombinés, de ces particules et de ces vaccins.
PCT/IB1997/001040 1996-07-26 1997-07-28 Vaccin recombine vivant contre l'herpes virus bovin/le virus respiratoire syncytial bovin WO1998004710A2 (fr)

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AU37819/97A AU3781997A (en) 1996-07-26 1997-07-28 Live recombinant bhv/brsv vaccine

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US68788196A 1996-07-26 1996-07-26
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1026252A1 (fr) * 1999-02-02 2000-08-09 Akzo Nobel N.V. Gène synthétique du virus de la diarrhée virale des bovins

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WO1992007940A2 (fr) * 1990-11-05 1992-05-14 Siba Kumar Samal Genes de virus syncytiaux respiratores chez les bovins
US5869312A (en) * 1992-01-13 1999-02-09 Syntro Corporation Recombinant swinepox virus
EP0663403A1 (fr) * 1993-11-23 1995-07-19 Akzo Nobel N.V. Vaccins du type I de l'herpesvirus bovin

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1026252A1 (fr) * 1999-02-02 2000-08-09 Akzo Nobel N.V. Gène synthétique du virus de la diarrhée virale des bovins

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