WO2000020600A1 - Vaccin contre le pneumovirus aviaire et agent diagnostique - Google Patents

Vaccin contre le pneumovirus aviaire et agent diagnostique Download PDF

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Publication number
WO2000020600A1
WO2000020600A1 PCT/EP1999/007400 EP9907400W WO0020600A1 WO 2000020600 A1 WO2000020600 A1 WO 2000020600A1 EP 9907400 W EP9907400 W EP 9907400W WO 0020600 A1 WO0020600 A1 WO 0020600A1
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acid sequence
polypeptide
nucleic acid
avian pneumovirus
amino acid
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PCT/EP1999/007400
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English (en)
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Jane Kathleen Alexandra Cook
Ian Tarpey
Michael Brian Huggins
Phil Davis
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Akzo Nobel N.V.
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Priority to AU62003/99A priority Critical patent/AU6200399A/en
Publication of WO2000020600A1 publication Critical patent/WO2000020600A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • 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 is concerned with a nucleic acid sequence encoding an avian pneumovirus related polypeptide, a recombinant nucleic acid molecule comprising such a nucleic acid sequence, a recombinant vector virus comprising said nucleic acid sequence, a host cell transformed with such a nucleic acid sequence, an avian pneumovirus related polypeptide encoded by the nucleic acid sequence and antibodies reactive with the polypeptide as well as a vaccine against avian pneumovirus infection.
  • the invention also relates to an immunochemical reagent .and a test kit comprising said reagent.
  • Avian pneumoviruses form one of the two genera of the Pneumoviridae, which in turn is one of the two subfamilies belonging to the family Paramyxoviridae.
  • the Paramyxoviridae are members of the Order Mononegavirales, which are viruses containing nonsegmented, negative sense, single-stranded RNA.
  • TRT virus turkey rhinotracheitis virus (Cavanagh D. & Barrett T., Virus Research, 11, 241-256, [1988]). This virus causes a highly contagious upper respiratory tract infection in turkeys resulting in high morbidity and variable mortality. In laying turkeys, TRT virus can cause a substantial drop in egg production and poor shell quality.
  • TRT virus has also been incriminated as a pathogen in chickens, and TRT infection has also been demonstrated in pheasants and guinea fowl.
  • TRT virus has two surface glycoproteins, F and G, which are important for the induction of protective immunity.
  • monoclonal antibodies which recognise the G glycoprotein and molecular sequencing of the G glycoprotein (Juhasz & Easton, 1994, Journal of General Virology, 75, 2873-2880), two subgroups, A and B, within the single serotype have been recognised.
  • TRT virus Following isolation in tracheal organ cultures, serial passage of TRT virus in a variety of cell cultures, including chick embryo fibroblasts and VERO cells, has resulted in rapid attenuation of the virus.
  • TRT vaccines comprise chemically inactivated or live attenuated viruses (Cook, J.K.A. et al, [1989] Avian Pathology, 18, 511-522 & 523-534; Williams, R.A., [1991], Avian Pathology, 20, 45-55 & 585-596; Cook, J.K.A., [1996], Avian Pathology, 25, 231-243).
  • Inactivated vaccines require additional immunizations, disadvantageously contain adjuvants, are expensive to produce and are laborious to administer. Furthermore, some infectious particles may survive the inactivation process and may cause disease after administration to the bird.
  • Attenuated live virus vaccines are preferred because they evoke an immune response often based on both humoral and cellular reactions.
  • vaccines based on TRT strains can only be prepared by serial passage of virulent strains in cell culture.
  • uncontrolled mutations can be introduced into the viral genome, resulting in a population of virus particles heterogeneous with regard to virulence and immunizing properties.
  • it is well known that such traditional attenuated live virus vaccines can revert to virulence resulting in disease outbreaks in inoculated birds and the possible spread of the pathogen to other birds.
  • the causal agent of the Colorado outbreak was isolated from swabs collected from the infected turkeys by passaging the material 3 times via the yolk sac of embryonated chicken eggs, followed by several passages in chick embryo fibroblasts (Senne, D. A. et al, Proc. Amer. Vet.
  • the Colorado isolate was compared to known TRT strains in in vitro serum neutralisation tests.
  • the Colorado isolate was tested against monospecific polyclonal antisera to recognised TRT isolates.
  • the Colorado isolate was not neutralised by monospecific antisera to any of the TRT strains. It was, however, neutralised by a hyperimmune antiserum raised against a subgroup A strain. This antiserum neutralised the homologous virus to a titre of 1 :400 and the Colorado isolate to a titre of 1 :80.
  • TRT virus strain subgroup B it has been found that the Colorado strain of avian pneumovirus does not protect SPF chicks against challenge with either a subgroup A or a subgroup B strain of TRT virus.
  • a vaccine against this new serotype of avian pneumovirus might be constructed based on recombinant DNA technology. Such a vaccine would only contain the necessary and relevant avian pneumovirus immunogenic material which is capable of eliciting a protective response against the Colorado strain, or the genetic information encoding said material, and would not display the above-mentioned disadvantages of live or inactivated TRT vaccines.
  • nucleic acid sequence encoding a polypeptide comprising at least part of the protein expressed by the F gene of an avian pneumovirus characterised in that the polypeptide has an amino acid sequence shown in o SEQ. ID No. 2 or is a functional variant thereof.
  • Nucleic acid sequence refers to a polymeric form of nucleotides of any length, both to ribonucleic acid sequences and to deoxyribonucleic acid sequences. In principle, this term refers to the primary structure of the molecule. Thus, this term includes double and 5 single-stranded DNA, as well as double and single-stranded RNA and modifications thereof.
  • polypeptide refers to a molecular chain of amino acids with a biological activity, it does not refer to a specific length of the product and if required can be modified in vivo or in vitro, for example by glycosylation, amidation, carboxylation or o phosphorylation; thus inter dia, peptides, oligopeptides and proteins are included.
  • SEQ. LD. No. 1 shows the nucleotide sequence of the Colorado isolate APV F gene (this sequence is given in the rnRNA sense) and the amino acid sequence of the Colorado isolate APV F protein.
  • SEQ. LD. No. 2 shows the amino acid sequence of the Colorado isolate APV F protein encoded by the gene shown in SEQ. ID. No. 1.
  • nucleic acid sequences which encode a fuctional variant of the polypeptide having the amino acid sequence shown in SEQ. LD. No. 2.
  • Such functional variant polypeptides exhibit at least 80% homology with said sequence, preferably at least 90% homology, most preferably at least 95% homology, and are able to induce antibodies in poultry which react with the polypetide shown in SEQ. LD. No. 2
  • SEQ. ID. Nos. 1 and 2 allows a person skilled in the art to isolate and identify nucleic acid sequences encoding the various functional variant polypeptides mentioned above having corresponding immunological characteristics with the Colorado isolate APV protein specifically disclosed herein.
  • the generally applied Southern Blotting technique or colony hybridization can be used for that purpose (Experiments in Molecular Biology, ed. R.J. Slater, Clifton, U.S.A., 1986); Singer-Sam, J. et al, Proc. Natl. Acad. Sci. 80, 802-806, 1983; Maniatis T. et al, Molecular Cloning, A Laboratory Manual, Cold Spring Harbour Laboratory Press, 1989).
  • restriction enzyme digested DNA fragments derived from the Colorado isolate APV is electrophoresed and transferred, or "blotted” thereafter onto a piece of nitrocellulose filter. It is now possible to identify the nucleic sequences encoding the functional variant polypeptides on the filter by hybridization to a defined labelled DNA or "probe" back translated from the amino acid sequence shown in SEQ. ID. No. 2 under specific conditions of salt concentration and temperature that allow hybridization of the probe to any homologous DNA sequences present on the filter. After washing the filter, hybridized material may be detected by autoradiography. Once having identified the relevant sequence, DNA fragments that encode a functionally variant polypeptide to the polypeptide disclosed in SEQ. ID. No. 2 can now be recovered after agarose gel electrophoresis by elution and used to direct the synthesis of a polypeptide functionally equivalent to a polypeptide disclosed in SEQ. ID. No. 2.
  • APV cDNA may be cloned into a phage such as ⁇ gt 11 and expressed in a bacterial host. Recombinant phages can then be screened with polyclonal serum raised against the purified Colorado isolate APV polypeptide disclosed in SEQ. ID. No. 2, determining the presence of corresponding regions of the variant polypeptide.
  • the degeneracy of the genetic code permits substitution of bases in a codon resulting in another codon but still coding for the same amino acid e.g. the codon for the amino acid glutamic acid is both GAT and GAA. Consequently, it is clear that for the expression of a polypeptide with the amino acid sequence shown in SEQ. ID. No. 2 use can be made of a derivative nucleic acid sequence with such an alternative codon composition different from the nucleic acid sequence shown in SEQ. ID. No. 1.
  • fragments of the nucleic acid sequence encoding the Colorado isolate APV polypeptide or functional equivalents thereof as mentioned above are included in the present invention.
  • fragment means a DNA or amino acid sequence comprising a subsequence of a nucleic acid sequence or polypeptide defined above. Said fragment is or encodes a polypeptide having one or more immunogenic determinants of the Colorado isolate APV polypeptide defined above, i.e. has one or more epitopes of the APV polypeptide which are capable of inducing antibodies in poultry which react with the polypetide disclosed in SEQ. ID. No. 2 to a greater extent as with the F proteins of subgroup A and B TRT viruses. Methods for determining usable polypeptide fragments are outlined below.
  • Fragments can inter alia be produced by enzymatic cleavage of precursor molecules, using restriction endonucleases for the DNA and proteases for the polypeptides. Other methods include chemical synthesis of the fragments or the expression of polypeptide fragments by DNA fragments.
  • a nucleic acid sequence according to the present invention can be ligated to various replication effecting DNA sequences with which it is not associated or linked in nature resulting in a so called recombinant vector molecule which can be used for the transformation of a suitable host.
  • Useful recombinant vector molecules are preferably derived from, for example plasmids, bacteriophages, cosmids or viruses.
  • vectors or cloning vehicles which can be used to clone nucleic acid sequences according to the invention are known in the art and include inter alia plasmid vectors such as pBR322, the various pUC, pGEM and Bluescript plasmids, bacteriophages, e.g. ⁇ gt-Wes- ⁇ B, Charon 28 and the Ml 3 derived phages or viral vectors such as SV40, adenovirus or polyoma o virus (see also Rodriquez, R.L. and D.T. Denhardt, ed., Vectors: A survey of molecular cloning vectors and their uses, Butterworths, 1988; Lenstra, J.A.
  • the APV cDNA may be inserted into a recombinant virus vector such as turkey herpesvirus (HVT), infectious laryngeotracheitis (ILT) or Fowlpox.
  • HVT turkey herpesvirus
  • ILT infectious laryngeotracheitis
  • Fowlpox a virus vector
  • This can be achieved by cloning the sequence into the vector downstream from a promoter to drive expression.
  • a vector would also contain flanking regions homologous to the virus into which it is to be inserted. Co-transfection of suitable cells with the vector and viral DNA can lead to homologous recombination so that a proportion of viruses contain the inserted gene.
  • the APV cDNA may be inserted into a DNA vaccination vector such as pi.18 which may then be used directly for vaccination.
  • the insertion of the nucleic acid sequence according to the invention into a cloning vector can easily be achieved when both the genes and the desired cloning vehicle have been cut with the same restriction enzyme(s) as complementary DNA termini are thereby produced.
  • blunt end ligation with an enzyme such as T4 DNA ligase may be carried out.
  • any restriction site may be produced by ligating linkers onto the DNA termini.
  • linkers may comprise specific oligonucleotide sequences that encode restriction site sequences.
  • the restriction enzyme cleaved vector and nucleic acid sequence may also be modified by homopolymeric tailing.
  • "Transformation" as used herein, refers to the introduction of a heterologous nucleic acid sequence into a host cell, irrespective of the method used, for example direct uptake or transduction.
  • the heterologous nucleic acid sequence may be maintained through autonomous replication or alternatively, may be integrated into the host genome.
  • the recombinant vector molecules are provided with appropriate control sequences compatible with the designated host which can regulate the expression of the inserted nucleic acid sequence.
  • culture of cells derived from multicellular organisms may also be used as hosts.
  • the recombinant vector molecules according to the invention preferably contain one or more marker activities that may be used to select for desired transformants, such as ampicillin and tetracycline resistance in pBR322, ampicillin resistance and ⁇ -galactosidase activity in pUC8.
  • a suitable host cell is a pro- or eukaryotic cell which can be transformed by a nucleic acid sequence encoding a polypeptide or by a recombinant vector molecule comprising such a nucleic acid sequence and which can if desired be used to express said polypeptide encoded by said nucleic acid sequence.
  • the host cell can be of prokaryotic origin, e.g. bacteria such as Escherichia coli, Bacillus subtilis and Pseudomonas species; or of eukaryotic origin such as yeasts, e.g. Saccharomyces cerevisiae or higher eukaryotic cells such as insects, plant or mammalian cells, including HeLa cells and Chinese hamster ovary (CHO) cells.
  • Insects cells include the Sf9 cell line of Spodoptera frugiperda (Luckow et al, Bio-technology 6, 47-55, 1988). Information with respect to the cloning and expression of the nucleic acid sequence of the present invention in eukaryotic cloning systems can be found in Esser, K. et al. (Plasmids of Eukaryotes, Springer- Verlag, 1986).
  • prokaryotes are preferred for cloning of DNA sequences in constructing the vectors useful in the invention.
  • E.coli K12 and derivative strains such as DH5 ⁇ or JM101 are particularly useful.
  • nucleic acid sequences of the present invention are introduced into an expression vector, such as yeast, (baculo) virus or bacterial systems. Such sequences are operably linked to expression control sequences which may comprise promoters, enhancers, operators, inducers, ribosome binding sites etc. Therefore, the present invention provides a recombinant vector molecule comprising a nucleic acid sequence encoding the Colorado isolate APV polypeptide as defined above operably linked to expression control sequences, capable of expressing the DNA sequences contained therein in (a) transformed host cell(s).
  • nucleotide sequences inserted at the selected site of the cloning vector may include nucleotides which are not part of the actual structural gene for the desired polypeptide or may include only a fragment of the complete structural gene for the desired protein as long as transformed host will produce a polypeptide having at least one or more immunogenic determinants of the Colorado isolate APV polypeptide as defined above.
  • illustrative useful expression control sequences include the Trp promoter and operator (Goeddel, et al, Nucl. Acids Res. 8, 4057, 1980); the lac promoter and operator (Chang, et al., Nature 275, 615, 1978); the outer membrane protein promoter (Nakamura, K. and Inouge, M., EMBO J. 1, 771-775, 1982); the bacteriophage ⁇ promoters and operators (Remaut, E. et al., Nucl. Acids Res. 11, 4677-4688, 1983); the ⁇ - amylase (B. subtilis) promoter and operator, termination sequence and other expression enhancement and control sequences compatible with the selected host cell.
  • Trp promoter and operator Goeddel, et al, Nucl. Acids Res. 8, 4057, 1980
  • the lac promoter and operator Chang, et al., Nature 275, 615, 1978
  • the outer membrane protein promoter Neakamura, K
  • illustrative useful expression control sequences include, e.g., ⁇ -mating factor.
  • the polyhedrin or plO promoters of baculoviruses can be used (Smith, G. E. et al., Mol. Cell. Biol, 3, 2156-65, 1983).
  • illustrative useful expression control sequences include, e.g. the SV40 promoter (Berman, P.W. et al., Science 222, 524-527, 1983) or, e.g.
  • the invention also comprises (a) host cell(s) transformed with a nucleic acid sequence or recombinant expression vector molecule described above, capable of producing the Colorado isolate APV polypeptide as defined above by expression of the necleic acid sequence.
  • the present invention also provides a purified polypeptide having one or more immunogenic determinants of an APV protein having an amino acid sequence shown in SEQ. ID. No. 2, or a functional variant thereof, essentially free from the whole virus or other protein with which it is ordinarily associated.
  • polypeptide which can be used for immunization of poultry against APV infection or disease or for diagnostic purposes substantially comprising an immunogenic fragment of the Colorado isolate APV polypeptide is included in the present invention.
  • Various methods are known for detecting such usable polypeptide fragments within a amino acid sequence.
  • Suitable immunochemically active immunogenic fragments of a polypeptide according to the invention containing (an) epitope(s) can be found by means of the method described in Patent Application WO 86/06487, Geysen, H.M. et al. (Prod. Natl. Acad. Sci. 81, 3998-4002, 1984), Geysen, H.M. et al. (J. Immunol. Meth. 102, 259-274, 1987) based on the so-called pep- scan method, wherein a series of partially overlapping polypeptides corresponding with partial sequences of the complete polypeptide under consideration, are synthesized and their reactivity with antibodies is investigated.
  • a number of regions of the polypeptide, with the stated amino acid sequence, can be designated epitopes on the bases of theoretical considerations and structural agreement with epitopes which are now known. The determination of these regions is based on a combination of the hydrophilicity criteria according to Hopp and Woods (Proc. Natl. Acad. Sci. 78, 3824-3828, 1981) and the secondary structure aspects according to Chou and Fasman (Advances in Enzymology 47, 45-148, 1987) T-cell epitopes which may be necessary can likewise be derived on theoretical grounds with the aid of Berzofsky's amphiphilicity criterion (Science 235, 1059-62, 1987).
  • polypeptide having an amino acid sequence encoded by a nucleic acid sequence mentioned above is used.
  • Immunization of poultry against APV infection or disease can, for example be achieved by administering to the birds a polypeptide according to the invention in an immunologically relevent context as a so-called subunit vaccine.
  • the subunit vaccine according to the invention may comprise a polypeptide in a pure form, optionally in the presence of a pharmaceutically acceptable carrier.
  • the polypeptide can optionally be covalently bonded to a non-related protein, which, for example can be of advantage in the purification of the fusion product. Examples are ⁇ -galactosidase, protein A, prochymosine, blood clotting factor Xa, etc.
  • Small fragments are preferably conjugated to carrier molecules in order to raise their immunogenicity.
  • Suitable carriers for this purpose are macromolecules, such as natural polymers (proteins like key hole limpet haemocyanin, albumin, toxins), synthetic polymers like polyamino acids (polylysine, polyalanine), or micelles of amphiphilic compounds like saponins.
  • these fragments may be provided as polymers thereof, preferably linear polymers.
  • Polypeptides to be used in such subunit vaccines can be prepared by methods know in the art, e.g. by isolating said polypeptides from APV by recombinant DNA techniques or by chemical syntheses.
  • polypeptides according to the invention to be used in a vaccine can be modified in vitro or in vivo, for example by glycosylation, amidation, carboxylation or phosphorylation.
  • a nucleic acid sequence according to the invention is introduced by recombinant DNA techniques into a micro-organism (e.g. a bacterium or virus) in such a way that the recombinant micro-organism is still able to replicate thereby expressing a polypeptide coded by the inserted nucleic acid sequence and eliciting an immune response in the infected host animal.
  • a micro-organism e.g. a bacterium or virus
  • a preferred embodiment of the present invention is a recombinant vector virus comprising a heterologous nucleic acid sequence described above, capable of expressing the DNA sequence in (a) host cell(s) or host animal infected with the recombinant vector virus.
  • heterologous indicates that the nucleic acid sequence according to the invention is not normally present in the nature in the vector virus or is not present in the same genetic context as in the naturally occurring vector virus.
  • the invention also comprises (a) host cell(s) or cell culture infected with the recombinant vector virus, capable of producing the APV protein by expression of the nucleic acid sequence.
  • the well know technique of in vivo homologous recombination can be used to introduce a heterologous nucleic acid sequence, e.g. a nucleic acid sequence according to the invention into the genome of the vector virus.
  • a DNA fragment corresponding with an insertion region of the vector genome i.e. a region which can be used for the incorporation of a heterologous sequence without disrupting essential functions of the vector such as those necessary for infection or replication, is inserted into a cloning vector according to standard recDNA Techniques. Insertion-regions have been reported for a large number of micro-organisms (e.g. EP 80,806, EP 110,385, EP 83,286, EP 314,569, WO88/07088).
  • a deletion can be introduced into the insertion region present in the recombinant vector molecule obtained from the first step. This can be achieved for example by appropriate exonuclease 111 digestion or restriction enzyme treatment of the recombinant vector molecule from the first step.
  • the heterologous nucleic acid sequence is inserted into the insertion-region present in the recombinant vector molecule of the first step or in place of the DNA deleted from said recombinant vector molecule.
  • the insertion region DNA sequence should be of appropriate length as to allow homologous recombination with the vector genome to occur.
  • suitable cells can be infected with wild-type vector virus or transformed with vector genomic DNA in the presence of the recombinant vector molecule containing the insertion flanked by appropriate vector DNA sequences whereby recombination occurs between the corresponding regions in the recombinant vector molecule and the vector genome.
  • Recombinant vector progeny can now be produced in cell culture and can be selected for example genotypically or phenotypically, e.g. by hybridization, detecting enzyme activity encoded by a gene co-integrated along with the heterologous nucleic acid sequence, or detecting the antigenic heterologous polypeptide expressed by the recombinant vector immunologically.
  • this recombinant micro-organism can be administered to the host animal for immunization after which it maintains itself for some time, or even replicates in the body of the inoculated animal, expressing in vivo a polypeptide coded for by the inserted nucleic acid sequence according to the invention resulting in the stimulation of the immune system of the inoculated animal.
  • Suitable vectors for the incorporation of a nucleic acid sequence according to the invention can be derived from viruses such as (avian) poxviruses, e.g. vaccinia virus or fowl poxvirus (EP 314,569 and WO 88/02022), herpesviruses, for example vaccine strains of Marek's
  • HVT HVT
  • MDV MDV strains of serotype 1 or 2
  • adenovirus or influenza virus or bacteria such as E. coli or specific Salmonella species.
  • These vectors can be designed in a way to obtain an efficient recognition of the polypeptide by the immune system of the host animal.
  • fusion of the said polypeptide with synthetic signal and anchor sequences or with an antigen from another viral or bacterial pathogen, e.g. the haemaggmtinin- neuraminidase protein form Newcastle disease virus, are conceivable.
  • the said immunogenic polypeptide if desired as part of a larger whole, is released inside the animal to be immunized.
  • a vaccine according to the invention can be prepared by culturing a host cell infected with a recombinant vector virus comprising a nucleic acid sequence according to the invention, after which virus containing cells and/or recombinant vector viruses grown in the cells can be collected, optionally in a pure form.
  • Another possibility to produce a vector vaccine is to incorporate a nucleic acid sequence according to the invention in an infectious bacteria or parasite and to culture such a modified infectious agent. After collection, optionally in a pure form, a vaccine, optionally in a lyophilized form, can be produced.
  • Host cells transformed with a recombinant vector molecule according to the invention can also be cultured under conditions which are favourable for the expression of a polypeptide coded by said nucleic acid sequence.
  • Vaccines may be prepared using samples of the crude culture, host cell lysates or host cell extracts, although in another embodiment more purified polypeptides according to the invention are formed to a vaccine, depending on its intended use.
  • host cells transformed with a recombinant vector 5 according to the invention are cultured in an adequate volume and the polypeptides produced are isolated from such cells or from the medium if the protein is excreted. Polypeptides excreted into the medium can be isolated and purified by standard techniques, e.g.
  • inter cellular polypeptides can be isolated by first collecting said cells, o disrupting the cell, for example by sonication or by other mechanically disruptive means such as
  • Antibodies or antiserum directed against a polypeptide according to the invention have potential use in passive immunotherapy, diagnostic immunoassays and generation of anti- idiotype antibodies.
  • the vaccine according to the invention can be administered in a conventional active immunization scheme: single or repeated administration in a manner compatible with the dosage formulation and in such amount as will be prophylactically and/or therapeutically effective and immunogenic, i.e. the amount of immunizing .antigen or recombinant micro- organism capable of expressing said antigen that will induce immunity in an animal against challenge by a virulent APV.
  • Immunity is defined as the induction of a higher level of protection in a population of animals after vaccination compared to an unvaccinated group.
  • the dose rate per animal may range from 1.10 ⁇ - 1.10 ⁇ infectivity units.
  • a typical subunit vaccine according to the invention comprises lO ⁇ g - lmg of the polypeptide according to the invention.
  • the administration of the vaccine can be done, e.g. intradermally, subcutaneously, intramuscularly, in ovo via spray or drinking water or by 'biolistic device' such as a gene gun, intraperitonially, intravenously, by eye-drop, orally or intranasally.
  • the vaccine may also contain an aqueous medium or a water containing o suspension, often mixed with other constituents, e.g. in order to increase the activity and/or shelf life.
  • these constituents may be salts, pH buffers, stabilizers (such as skimmed milk or casein hydrolysate), emulsifiers adjuvants to improve the immune response (e.g. oils, muramyl dipeptide, aluminiumhydroxide, saponin, polyanions and amphipatic substances) and preservatives. 5
  • a vaccine according to the invention may also contain immunogens related to other pathogens of poultry or may contain nucleic acid sequences encoding these immunogens, like antigens of Infectious Bronchitis Virus (LBV), Newcastle Disease Virus (NDV), Infectious Bursal Disease Virus (IBDV) or Marek's Disease Virus different from those o disclosed herein, to produce a multivalent vaccine.
  • the invention also relates to an "immunochemical reagent", which reagent comprises a polypeptide according to the invention.
  • immunochemical reagent signifies that the polypeptide according to the invention is bound to a suitable support or is provided with a labelling substance.
  • the supports which can be used are, for example, the inner wall of a microtest well or a cuvette, a tube or capillary, a membrane, filter, test strip or the surface of a particle such as, for example, a latex particle, an erythrocyte, a dye sol, a metal sol or metal compoimd as sol particle.
  • Labelling substances which can be used are, inter alia, a radioactive isotope, a fluorescent compound, an enzyme, a dye sol, metal sol or metal compound as sol particle.
  • Swabs were used to collect material from infected turkeys, which material was then passaged 3 times via the yolk sac of embryonated chicken eggs followed by several passages in
  • the PCR reaction was electrophoresed on a 0.8% agarose gel, a 1.6kb fragment excised and then gel purified using the Qiagen gel purification kit according to manufacturers instructions. This fragment was ligated into the pGEM T -easy vector (Promega) using the pGEM T-easy vector system (Promega) according to manufacturers instructions.
  • the ligation mixture was cleaned using the Qiagen PCR purification kit according to manufacturers instructions, resuspended in 2 ⁇ l of H2O and electroporated into electrocompetent DH5 ⁇ bacteria at 2.5kV, 200ohms, 25 ⁇ F. The bacteria were then shaken at 37°C for one hour in 1ml LB broth. Following this 0.5ml of the broth was added to a LB plate containing lOO ⁇ g/ml ampicillin, 0.5mM LPTG and 80 ⁇ g/ml X-gal. Following overnight incubation at 37°C, 20 white colonies were picked and added to 1.5ml of LB broth containing lOO ⁇ g/ml ampicillin and shaken overnight. Plasmid DNA was prepared from these cultures using standard methods and digested with Notl enzyme. Plasmid DNA that contained a 1.6kb insert fragment was selected for sequencing and clones called F/pGEM.
  • Plasmid DNA was isolated using a Qiagen maxi- prep kit according to manufacturers instructions. Samples of plasmid DNA were sent for ABI sequencing by Pat Barker at the Microchemical Facility in Babraham Institute, Babraham, Cambridge. The complete sequence of the clones was determined on each strand. Sequence data was analysed at the Elms using the Omiga software program (Oxford Molecular Ltd).
  • the F gene was sub-cloned by standard methods into the iiltiple cloning site of the pUC-derived mammalian expression vector pi.18 (obtainable from Dr. Jim Robertson, NLBSC, South Mimms, EN6 3QG, UK).
  • the F gene sequence was digested from the F/pGEM vector using the Not 1 enzyme, the fragment gel-purified, blunted using T4 DNA polymerase and ligated into pi.18 vector that had been digested with the EcoRV enzyme and treated with calf intestinal alkaline phosphatase. Orientation of the insert was determined by digestion with restriction enzymes.
  • Insertion of the gene of interest into the multiple clomng site of this vector brings it under the transcriptional control of the human CMV promoter and intron A sequence. Transcription is terminated by the hCMV poly A signal. Expression of the F protein was detected by transfecting secondary chicken embryo fibroblasts with the F/pI.18 plasmid by electroporation and incubating the cells in M6B8 medium containing 2% foetal calf serum for 48 hours at 37°C/5%CO2. The cells were fixed in 70% acetone, air dried and stained with antisera raised in chickens specific for the Colorado isolate.
  • the poults were bled prior to the second vaccination (26 days of age) and prior to challenge at 6 weeks of age (2 weeks after the second vaccination). The results are shown in Table 1.

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Abstract

L'invention porte sur une séquence d'acide nucléique codant un polypeptide apparenté à un pneumovirus aviaire. La protéine apparentée à un pneumovirus aviaire et codée par la séquence d'acide nucléique peut être utilisée dans la préparation d'un vaccin contre le pneumovirus aviaire ou à des fins de diagnostic. Cette invention porte également sur un kit de test utilisé dans la détection de volailles infectées par le pneumovirus aviaire.
PCT/EP1999/007400 1998-10-05 1999-09-29 Vaccin contre le pneumovirus aviaire et agent diagnostique WO2000020600A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU62003/99A AU6200399A (en) 1998-10-05 1999-09-29 Avian pneumovirus vaccine and diagnostic agent

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NL98203352.4 1998-10-05
EP98203352 1998-10-05

Publications (1)

Publication Number Publication Date
WO2000020600A1 true WO2000020600A1 (fr) 2000-04-13

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PCT/EP1999/007400 WO2000020600A1 (fr) 1998-10-05 1999-09-29 Vaccin contre le pneumovirus aviaire et agent diagnostique

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AU (1) AU6200399A (fr)
WO (1) WO2000020600A1 (fr)

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WO2002009576A2 (fr) * 2000-08-02 2002-02-07 Regents Of The University Of Minnesota Systeme de diagnostic de pneumovirus aviaire
WO2002057302A2 (fr) * 2001-01-19 2002-07-25 Vironovative B.V. Virus provoquant des maladies respiratoires chez des mammiferes y etant sensibles
WO2004096993A2 (fr) 2003-04-25 2004-11-11 Medimmune Vaccines, Inc. Souches de metapneumovirus et leur utilisation dans des preparations de vaccin et en tant que vecteurs pour l'expression de sequences d'antigenes et procedes pour propager un virus
US6977079B2 (en) 2003-01-17 2005-12-20 Regents Of The University Of Minnesota Avian pneumovirus vaccine
US7449324B2 (en) 2002-02-21 2008-11-11 Vironovative Bv Metapneumovirus strains and their use in vaccine formulations and as vectors for expression of antigenic sequences
AU2008202111B2 (en) * 2001-01-19 2012-04-19 Erasmus University Medical Center Rotterdam A virus causing respiratory tract illness in susceptible mammals
US8715922B2 (en) 2001-01-19 2014-05-06 ViroNovative Virus causing respiratory tract illness in susceptible mammals

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WO2002009576A3 (fr) * 2000-08-02 2004-02-26 Univ Minnesota Systeme de diagnostic de pneumovirus aviaire
WO2002009576A2 (fr) * 2000-08-02 2002-02-07 Regents Of The University Of Minnesota Systeme de diagnostic de pneumovirus aviaire
US7531342B2 (en) 2001-01-19 2009-05-12 Medimmune, Llc Metapneumovirus strains and their use in vaccine formulations and as vectors for expression of antigenic sequences
JP2011177173A (ja) * 2001-01-19 2011-09-15 Vironovative Bv 感受性哺乳動物において気道疾患を引き起こすウイルス
JP2004531220A (ja) * 2001-01-19 2004-10-14 ヴィロノヴァティブ，ベスローテン ヴェンノートシャップ 感受性哺乳動物において気道疾患を引き起こすウイルス
US11162148B2 (en) 2001-01-19 2021-11-02 Erasmus University Medical Center Rotterdam Virus causing respiratory tract illness in susceptible mammals
US10519517B2 (en) 2001-01-19 2019-12-31 Vironovative Bv Virus causing respiratory tract illness in susceptible mammals
AU2002228471B2 (en) * 2001-01-19 2008-02-14 Erasmus University Medical Center Rotterdam A virus causing respiratory tract illness in susceptible mammals
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US9803252B2 (en) 2001-01-19 2017-10-31 Erasmus University Medical Center Rotterdam Virus causing respiratory tract illness in susceptible mammals
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US9593386B2 (en) 2001-01-19 2017-03-14 Erasmus Universiteit Medical Center Rotterdam Virus causing respiratory tract illness in susceptible mammals
WO2002057302A2 (fr) * 2001-01-19 2002-07-25 Vironovative B.V. Virus provoquant des maladies respiratoires chez des mammiferes y etant sensibles
WO2002057302A3 (fr) * 2001-01-19 2002-12-27 Vironovative Bv Virus provoquant des maladies respiratoires chez des mammiferes y etant sensibles
KR101105984B1 (ko) * 2001-01-19 2012-01-18 비로노바티브 비.브이. 감응성 포유류에서 호흡계 질환을 유발하는 바이러스
AU2008202111B2 (en) * 2001-01-19 2012-04-19 Erasmus University Medical Center Rotterdam A virus causing respiratory tract illness in susceptible mammals
US9376726B2 (en) 2001-01-19 2016-06-28 Erasmus University Medical Center Rotterdam Metapneumovirus strains and their use in vaccine formulations and as vectors for expression of antigenic sequences
US9334543B2 (en) 2001-01-19 2016-05-10 Erasmus University Medical Center Rotterdam Virus causing respiratory tract illness in susceptible mammals
JP2014027930A (ja) * 2001-01-19 2014-02-13 Vironovative Bv 感受性哺乳動物において気道疾患を引き起こすウイルス
US8715922B2 (en) 2001-01-19 2014-05-06 ViroNovative Virus causing respiratory tract illness in susceptible mammals
US8722341B2 (en) 2001-01-19 2014-05-13 Vironovative B.V. Metapneumovirus strains and their use in vaccine formulations and sequences
KR101412317B1 (ko) * 2001-01-19 2014-07-24 비로노바티브 비.브이. 감응성 포유류에서 호흡계 질환을 유발하는 바이러스
US8927206B2 (en) 2001-01-19 2015-01-06 Vironovative B.V. Virus causing respiratory tract illness in susceptible mammals
US9567653B2 (en) 2002-02-21 2017-02-14 Erasmus University Medical Center Rotterdam Metapneumovirus strains and their use in vaccine formulations and as vectors for expression of antigenic sequences
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US11220718B2 (en) 2002-02-21 2022-01-11 Erasmus University Medical Center Rotterdam Metapneumovirus strains and their use in vaccine formulations and as vectors for expression of antigenic sequences
US10287640B2 (en) 2002-02-21 2019-05-14 Erasmus University Medical Center Rotterdam Metapneumovirus strains and their use in vaccine formulations and as vectors for expression of antigenic sequences
EP2327418A1 (fr) 2002-02-21 2011-06-01 MedImmune, LLC Vecteurs d'expression recombinants du virus de la parainfluenza et vaccins comprenant des antigènes hétérologues dérivés d'autres virus
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US8841433B2 (en) 2002-02-21 2014-09-23 Vironovative Bv Metapneumovirus strains and their use in vaccine formulations and as vectors for expression of antigenic sequences
US6977079B2 (en) 2003-01-17 2005-12-20 Regents Of The University Of Minnesota Avian pneumovirus vaccine
WO2004096993A3 (fr) * 2003-04-25 2009-03-26 Medimmune Vaccines Inc Souches de metapneumovirus et leur utilisation dans des preparations de vaccin et en tant que vecteurs pour l'expression de sequences d'antigenes et procedes pour propager un virus
US7704720B2 (en) 2003-04-25 2010-04-27 Medimmune, Llc Metapneumovirus strains and their use in vaccine formulations and as vectors for expression of antigenic sequences and methods for propagating virus
EP2494986A1 (fr) 2003-04-25 2012-09-05 MedImmune Vaccines, Inc. Souches de métapneumovirus et leur utilisation dans des formulations de vaccins et en tant que vecteurs pour l'expression de séquences antigéniques et procédés pour propager des virus
WO2004096993A2 (fr) 2003-04-25 2004-11-11 Medimmune Vaccines, Inc. Souches de metapneumovirus et leur utilisation dans des preparations de vaccin et en tant que vecteurs pour l'expression de sequences d'antigenes et procedes pour propager un virus
EP2494987A1 (fr) 2003-04-25 2012-09-05 MedImmune Vaccines, Inc. Souches de métapneumovirus et leur utilisation dans des formulations de vaccins et en tant que vecteurs pour l'expression de séquences antigéniques et procédés pour propager des virus

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