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.

The first avian pneumovirus to be described was turkey rhinotracheitis (TRT) 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.

Turkey rhinotracheitis was first described in South Africa in the late 1970s, where it had a devastating effect on the turkey industry. Within a few years TRT had spread to Israel and parts of Europe. It is now a major disease threat to the poultry industry worldwide. TRT infections have continued to be reported in many countries, but until now, not in North America.

(Senne, D. A. et al, Proc. Amer. Vet. Med. Assoc. 134th Annual Congress, Reno, Nevada, July 1997, pl90).

Using neutralisation tests in tracheal organ cultures (TOC), all TRT isolates studied from many countries, and obtained from both turkeys and chickens, have been found to belong to a single serotype (Cook, J. K. A. et al, [1993], Avian Pathology, 22,257-273). TRT virus has two surface glycoproteins, F and G, which are important for the induction of protective immunity. Based on the results of both neutralisation tests in TOC, using 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.

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.

Current 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, however, 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.

In general, attenuated live virus vaccines are preferred because they evoke an immune response often based on both humoral and cellular reactions. Up to now, such vaccines based on TRT strains can only be prepared by serial passage of virulent strains in cell culture. However, because of this treatment uncontrolled mutations can be introduced into the viral genome, resulting in a population of virus particles heterogeneous with regard to virulence and immunizing properties. In addition, 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. Using recombinant DNA technology, a fowl pox

vector expressing the F gene of TRT virus has been demonstrated to protect turkeys against homologous challenge (Yu, Q. et al, [1994], Vaccine, 12,569-573), but this approach has not been used commercially.

In 1996 a disease in turkeys was reported in the State of Colorado, USA, which was characterised by coughing, sneezing and tracheal rales. Clinically the disease appeared to be similar to TRT as seen in other countries. Examination of sera taken from infected birds has revealed a low incidence of antibodies to TRT virus when tested by ELISA.

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.

Med. Assoc. 134th Annual Congress, Reno, Nevada, July 1997, pl90). The cytopathic effect seen was characterised by syncytial and giant cell formation. Preliminary characterisation as a pneumovirus was done using a TRT fluorescent antibody.

The Colorado isolate was compared to known TRT strains in in vitro serum neutralisation tests. First, 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.

Using the above method, the Colorado isolate was then tested against TRT monoclonal antibodies (Cook et al., 1993, Avian Pathology, 22,257-273). In each case, the reciprocal neutralisation titre was <10.

Monospecific antiserum raised to the Colorado isolate was also tested against TRT strains of both subgroups. None of the TRT strains tested were neutralised by the antiserum to the Colorado isolate.

By inoculating specific pathogen free (SPF) chickens via the oculonasal route with viral inoculum, and subsequently challenging the birds with either a TRT virus strain subgroup A or a 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.

This evidence, together with results of the neutralisation tests in which the Colorado isolate and TRT strains of both subgroups A and B were compared, suggests that the Colorado isolate may be the first example of a second serotype of avian pneumovirus.

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.

According to the present invention there is provided a 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 SEQ. ID No. 2 or is a functional variant thereof.

"Nucleic acid sequence"as used herein 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 single-stranded DNA, as well as double and single-stranded RNA and modifications thereof.

In general, the term"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 phosphorylation; thus inter dia, peptides, oligopeptides and proteins are included.

SEQ. ID. No. 1 shows the nucleotide sequence of the Colorado isolate APV F gene (this sequence is given in the mRNA sense) and the amino acid sequence of the Colorado isolate APV F protein.

SEQ. ID. No. 2 shows the amino acid sequence of the Colorado isolate APV F protein encoded by the gene shown in SEQ. ID. No. 1.

Also included within the scope of the present invention are nucleic acid sequences which encode a fuctional variant of the polypeptide having the amino acid sequence shown in SEQ. ID.

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. ID. No. 2 Several techniques are bred routinely to clone, sequence and express the F gene from the Colorado isolate of avian pneumovirus. Such molecular biology methods are standard and are described in Molecular Cloning. A Laboratory Manual. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Cold Spring Harbour, New York, and also in Current Protocols in Molecular Biology (Eds. Anfubel, Brent, Kingston, More, Friedman, Smith and Stuhl, Greene Publishing Associates; Wiley Interscience, NY (1992). These include making cDNA from isolated mRNA, performing in vitro mRNA translations and several ways of transcript mapping, like Sl nuclease-protection assays and primer extension analyses.

It will be understood that for the particular polypeptide shown in SEQ. ID. No. 2, derived from the Colorado isolate of APV, natural variations can exist between individual viruses.

These variations may be demonstrated by (an) amino acid difference (s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid (s) in said sequence. Amino acid substitutions from which can be expected that they do not essentially alter biological and immunological activites, have been described. Amino acid replacements between related amino acids or replacements which have occurred frequently in evolution are, inter alia Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M. D., Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington D. C., 1978, vol 5, suppl. 3).

Based on this information Lipman and Pearson developed a method for rapid and sensitive

protein comparison (Science 227,1435-1441,1985) and determining the functional similarity between homologous polypeptides. Nucleic acid sequences encoding such homologous functional equivalents to the F protein specifically disclosed herein are included within the scope of this invention. Moreover, the potential exists to use recombinant DNA technology of the preparation of nucleic acid sequences encoding these various functional equivalents.

The information provided in 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). For example, 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.

In another way APV cDNA may be cloned into a phage such as kgt 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.

As is well known in the art, 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.

In addition, 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.

The term"fragment"as used herein 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.

Specific 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. kgt-Wes-kB, Charon 28 and the M13 derived phages or viral vectors such as SV40, adenovirus or polyoma

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. et al., Arch. Virol. 110,1-24,1990).

The methods to be used for the construction of a recombinant vector molecule according to the invention are known to those of ordinary skill in the art and are inter alia set forth in Maniatis, T. et al (Molecular Cloning A Laboratory Manual, second edition; Cold Spring Harbor Laboratory 1989).

Alternatively, the APV cDNA may be inserted into a recombinant virus vector such as turkey herpesvirus (HVT), infectious laryngeotracheitis (ILT) or Fowlpox. This can be achieved by cloning the sequence into the vector downstream frorn a promoter to drive expression. Such 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.

In yet another alternative the APV cDNA may be inserted into a DNA vaccination vector such as pI. 18 which may then be used directly for vaccination.

For example, 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.

Alternatively, it may be necessary to modify the restriction sites that are produced into blunt ends either by digesting the single-stranded DNA or by filling in the single-stranded termini with an appropriate DNA polymerase. Subsequently, blunt end ligation with an enzyme such as T4 DNA ligase may be carried out.

If desired, any restriction site may be produced by ligating linkers onto the DNA termini.

Such 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. If desired, 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. In addition to micro-organisms, 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 P-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).

In general, prokaryotes are preferred for cloning of DNA sequences in constructing the vectors useful in the invention. For example E. coli K12 and derivative strains such as DH5a or JM101 are particularly useful.

For expression, 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).

It should, of course, be understood that the 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.

When the host cells are bacteria, 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 X promoters and operators (Remaut, E. et al., Nucl. Acids Res. 11,4677-4688,1983); the a- amylase (B. subtilis) promoter and operator, termination sequence and other expression enhancement and control sequences compatible with the selected host cell. When the host cell is yeast, illustrative useful expression control sequences include, e. g., a-mating factor. For insect cells the polyhedrin or plO promoters of baculoviruses can be used (Smith, G. E. et al., Mol.

Cell. Biol, 3,2156-65,1983). When the host cell is of mammalian origin 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 metallothionein promoter (Brinster, R. L., Nature 296,39-42,1982) or a heat shock promoter (Voellmy et al., Proc. Natl. Acad. Sci. USA 82,4949-53,1985).

Alternatively, also expression control sequences present in APV, may be applied. For maximizing gene expression, see also Roberts and Leuer (Methods in Enzymology 68,473, 1979).

Therefore, 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.

It will be understood that derivatives of said amino acid sequences displaying the same immunological properties in essence, i. e. functional variants, are also within the scope of the present invention.

In addition a 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.

In addition, 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).

In another embodiment of the invention a 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 P-galactosidase, protein A, prochymosine, blood clotting factor Xa, etc.

In some cases the ability to raise neutralizing antibodies against these polypeptides per se may be low. 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.

Alternatively 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.

If required the 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.

An alternative to subunit vaccines are live vector vaccines. 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 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. The term"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.

Furthermore, 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.

First, 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 rechniques. 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, W088/07088).

Second, if desired, 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.

Third, 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. Thereafter, 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.

Next, 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 Disease viruses such as HVT (WO 88/07088) or 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. In this context fusion of the said polypeptide with synthetic signal and anchor sequences or with an antigen from another viral or bacterial pathogen, e. g. the haemagglutinin- neuraminidase protein form Newcastle disease virus, are conceivable. It is also possible that the said immunogenic polypeptide, if desired as part of a larger whole, is released inside the animal to be immunized. In all of these cases it is also possible that one or more immunogenic products will find expression which generate protection against various pathogens and/or against various antigens of a given pathogen.

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.

In order to purify the polypeptides produced, host cells transformed with a recombinant vector 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. salt fractionation, centrifugation, ultrafiltration, chromatography, gel filtration or immuno affinity chromatography, whereas inter cellular polypeptides can be isolated by first collecting said cells, disrupting the cell, for example by sonication or by other mechanically disruptive means such as French press followed by separation of the polypeptides from the other inter cellular components and forming the polpeptides to a vaccine. Cell disruption could also be accomplished by chemical (e. g. EDTA treatment) or enzymatic means such as lysozyme digestion.

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 Colorado isolate APV polypeptide as characterized above can be used to produce antibodies by methods well known in the art.

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 unvaceinated group.

For live viral vector vaccines the dose rate per animal may range from infectivity units.

A typical subunit vaccine according to the invention comprises 10tg-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.

Additionally the vaccine may also contain an aqueous medium or a water containing 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.

It is clear that 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 (IBV), Newcastle Disease Virus (NDV), Infectious Bursal Disease Virus (IBDV) or Marek's Disease Virus different from those disclosed herein, to produce a multivalent vaccine.

The invention also relates to an"immunochemical reagent", which reagent comprises a polypeptide according to the invention.

The term"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 compound 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.

Example 1 Origin of Colorado isolate of APV 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 CEF (Senne, D. A. et al, Proc. Amer. Vet. Med. Assoc. 134th Annual Congress, Reno, Nevada, July 1997, pl90). The turkey swab material was inoculated via the yolk sac into embryonated eggs. After 9 days, fluid collected from these embryos was inoculated"blind"into further embryos, again via the yolk sac. These embryos were incubated for a further 9 days, at which time haemorrhages were observed on the embryos and some mortality was seen. Harvested material was then inoculated onto CEF cultures and three passages made at 7 day intervals. The cytopathic effect seen was characterised by syncytial and giant cell formation. Preliminary characterisation as a pneumovirus was done using a TRT fluorescent antibody.

Cloning of the viral F gene Chicken embryo fibroblasts (2x107) were infected with one ampoule of the Colorado pneumovirus isolate and incubated at 37°C. 48-72 hours later mRNA was isolated from these cells using the polyAtract 1000 kit (Promega) according to manufacturer's instructions. This mRNA was then subjected to RT-PCR using the Access RT-PCR system from Promega using an oligo-dT primer and a primer partially based on TRT gene sequences. The cycles of the RT-PCR were performed as follows: One cycle at 48°C for 45 minutes.

One cycle at 94°C for 3 minutes.

40 cycles at 94°C for 30 seconds, 30°C for 30 seconds, and 68°C for 2 minutes.

One cycle at 68°C for 7 minutes.

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.

Following overnight ligation at 14°C the ligation mixture was cleaned using the Qiagen PCR purification kit according to manufacturers instructions, resuspended in 2p1 of H20 and electroporated into electrocompetent DH5a bacteria at 2.5kV, 200ohms, 25uF. The bacteria were then shaken at 37°C for one hour in lml LB broth. Following this 0.5ml of the broth was added to a LB plate containing 100, ug/ml ampieillin, 0. 5mM IPTG and 8011g/ml X-gal.

Following overnight incubation at 37°C, 20 white colonies were picked and added to 1.5ml of LB broth containing 10011g/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.

Example 2 DNA sequence analysis Clones of F/pGEM were grown in 100ml of Luria Bertani broth containing 100pg/ml ampicillin overnight with shaking at 37°C. 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).

Example 3 Expression of the F protein For analysis of the protein the F gene was sub-cloned by standard methods into the multiple cloning site of the pUC-derived mammalian expression vector pI. 18 (obtainable from

Dr. Jim Robertson, NIBSC, 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 cloning 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% C02 The cells were fixed in 70% acetone, air dried and stained with antisera raised in chickens specific for the Colorado isolate.

Example 4 DNA vaccination of turkeys with a plasmid expressing the F protein of the Colorado isolate of avian pneumovirus Experimental Design Three groups of ten 5-day-old turkey poults, from hens in which no TRT vaccine has been used were formed. Two of the groups were immunised via the intramuscular route with plasmid DNA. Group one was immunised with lOOgg of ttheF/pI. 18 plasmid and group 2 with lOOig of the control pI. 18 plasmid. Group 3 was left uninoculated. Ten siblings were bled. By 3 weeks of age the poults were wing banded, each with a unique number.

The poults were bled three and approximately six weeks after inoculation. Three weeks after inoculation the poults were re-vaccinated as above. At six weeks of age all 3 groups were challenged wit ha virulent strain of APV. At 10 days post challenge, the birds were bled and the experiment terminated. Serum samples were tested by ELISA for antibodies to the Colorado isolate of APV.

Results Ten siblings were bled at 5 days of age and the sera tested by ELISA for antibodies to the Colorado isolate of APV. All had titres of <log2 4.6.

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.

Table 1 APV ELISA titres (GMT) in poults at 6 weeks of age and post challenge. APV antibody titre (log2) Group 6 weeks Post challenge F/pI.18 5. 2 14. 5 PI.18 <4. 6 11. 1 Challenge control <4. 6 10. 7 Prior to challenge at 6 weeks of age, an antibody response to the Colorado isolate was seen in 5/8 of the poults vaccinated with the F/pI. 18 plasmid. All poults in the other two groups were still negative at that time. By 10 days post challenge, an antibody response was detected in all three groups. It was significantly higher in the group given the plasmid containing the APV F gene than in either the group given the parent plasmid or in the unvaccinated group (p<0.001 in each case).