Chicken embryo lethal orphan (CELO) virus

Abstract

A CELO virus obtained by in vitro manipulation of a plasmid-cloned CELO virus DNA is suitable for the production of vectors for gene therapy and as a vaccine against infectious diseases in humans and animals, particularly birds.

Description

[0001] The invention relates to adenoviruses. The large family of adenoviruses is subdivided according to its host into adenoviruses which infect mammals (the mastadenoviridae) and adenoviruses which infect birds (the aviadenoviridae). The CELO virus (Chicken Embryo Lethal Orphan; article by Mcferran, et al., 1977; McCracken and Adair, 1993) was identified as an infectious agent in 1957 (Yates and Fry, 1957). CELO virus is classified as a poultry adenovirus type 1 (FAV-1) and first aroused interest because of its property of being tumorigenic in baby hamsters. However, since infection with the CELO virus does not have any serious health and economic consequences, the interest in this virus disappeared in recent years. The FAV-1 adenoviruses can be isolated from healthy chickens and do not cause any disease when reintroduced experimentally into chickens (Cowen, et al., 1978). Their isolation from sick birds is presumably the result of adenovirus replication in a host which has an immune system weakened by other influences.

[0002] The general structural organisation of CELO virus, with an icosahedral capsid of 70-80 nm, made up of hexon and penton structures, is similar to that of the mammalian adenoviruses (Layer, et al., 1971). The CELO virus genome is a linear, double-stranded DNA molecule, the DNA being condensed inside the virion by virus-coded core proteins (Layer et al., 1971; Li, et al., 1984b). The CELO virus genome has covalently bound terminal proteins (Li, et al., 1983) and the genome has inverted terminal repeats (ITRs), although they are shorter than the mammalian ITRs (Aleström, et al., 1982b; Sheppard and Trist, 1992). The CELO virus codes a protease with 61-69% homology for the mammalian adenovirus proteases (Cai and Weber, 1993).

[0003] There are significant differences between CELO virus and the mastadenoviruses. CELO virus has a larger genome, with sequence homology with Ad5 which can only be detected in two short regions of the CELO virus genome (by hybridisation) (Aleström, et al., 1982a). The CELO virion has been reported to have two fibres of different lengths at each vertex. The CELO virus cannot complement the E1A functions of Ad5, and the replication of CELO virus is not made easier by the activity of Ad5E1 (Li, et al., 1984c).

[0004] Within the scope of the present invention, total sequence analysis of the CELO virus was carried out; on the one hand because it is useful for understanding the biology of adenoviruses to clarify the genomic organisation of an adenovirus which is very remote from the mammalian adenoviruses generally studied. Since the conditions for transmission and survival for a virus which infects a type of bird are presumably different than for mammalian viruses, it is possible that the bird adenoviruses have acquired new virus functions or exhibit a higher degree of variability than the mastadenoviridae. The complete CELO virus sequence also permits changes in the CELO virus genome with respect to functional analysis.

[0005] Since adenovirus vectors have proved highly effective vectors for gene transfer (see the summarising article by Graham, 1990; Kozarsky and Wilson, 1993; Trapnell and Gorziglia, 1994), the complete CELO virus sequence, on the other hand, is particularly interesting as the basis for preparing new recombinant vectors for gene transfer.

[0006] Sequence analysis has shown that the CELO virus genome has 43.8 kb, being more than 8 kb longer than the human subtypes Ad2 and Ad5. The genes for the main structural proteins (hexon, penton based, IIIa, fibres, pVI, pVII, pVIII) are on the one hand both present and also located at the corresponding sites in the genome. The genes of the early region 2 (E2; DNA binding protein, DNA polymerase and terminal protein) are also present. However, the CELO virus lacks sequences homologous to the regions E1, E3 and E4 of the mammalian adenoviruses.

[0007] There are approximately 5 kb at the left hand end and 15 kb at the right hand end of the CELO virus genome, where there is only restricted homology or no homology at all with the mastadenovirus genomes. These new sequences contain a number of open reading frames, and it can be assumed that these code for functions which replace the missing E1, E3 and possibly E4 regions.

[0008] Parts of the CELO virus sequence have already been published; they are listed in Table 1, as are the differences between the sequence known from the databank and the sequence determined within the scope of the present invention. From studies concentrating on specific viral genes, a homolog of the VA RNA gene of mastadenovirus was known (Larsson, et al., 1986) and part of the genome sequence which carries the endoprotease has been described (Cai and Weber, 1993). In addition, fragments of the CELO virus genome have been published (Akopian, et al., 1990; Akopian, et al., 1992; Hess, et al., 1995). The sequence of the penton base of the related virus FAV-10 has also been reported (Sheppard and Trist, 1992). Some other sequence fragments have been deposited in the databank and are also shown in Table 1. In all, about 50% of the CELO virus genome is available in the form of fragments (total about 24 kb). The sequence obtained within the scope of the present invention is complete and has the advantage of having been obtained from a single isolated material.

[0009] The total sequence of the CELO virus is shown in the sequence listing (in the sequence listing the word “complementary” indicates that the open reading frames are present in the reverse arrangement). It shows a large number of striking differences between Ad2 and the CELO virus. The organisation of the recognisable open reading frames (ORFs) of the CELO virus genome based on the sequence analysis, compared with Ad2, is shown in FIG. 1A: the Figure shows an overview of the genomic organisation of Ad2/5 and CELO virus. The arrows indicate the position of the coding regions but not the exact cleavage patterns of the gene products. The pattern of the CELO virus also (in the first 6,000 bp and in the last 13,000 bp) gives all the non-associated open reading frames which begin with a methionine and which code for more than 99 amino acid groups. The central region of the two genomes which show homology on the basis of dot matrix analysis (cf. FIG. 3) and the regions at the ends of the CELO virus genome which have no homology with other adenoviruses (“unique to CELO”) are given. The abbreviations in the Figure, which also correspond to those in the Tables, have the following meanings: PB, penton base; EP, endoproteinase; DBP, DNA binding protein; bTP, pre-terminal protein; pol, DNA polymerase.

[0010] The sequenced CELO virus genome has a length of 43,804 bp and has a content of G+C of 54.3%. It had already been presumed at an earlier stage that the CELO virus genome is much larger than the mastadenovirus genome with 34-36 kb; it has been found that the CELO virus DNA has a weight of 30×106 Daltons, determined according to its sedimentation coefficient (Layer, et al., 1971), compared with 24×106 Daltons for Ad2 (Green, et al., 1967). The size of the CELO virus genome determined by the addition of the restriction fragments is about 43 kb (Cai and Weber, 1993; Denisova, et al., 1979). A Pulsed Field Gel Analysis of the CELO virus genome isolated from purified virions is shown in FIG. 2A and is compared with the DNA isolated from Ads dl1014 (34,600 bp; Bridge and Ketner, 1989) or Wild-type Ad5-virions (35,935 bp; vt300; Chroboczek, et al., 1992; Jones and Shenk, 1978); a mixture of uncleaved bacteriophage λ-DNA and γ-DNA cleaved with five different restriction enzymes (Biorad) was used as the size marker (tracks 1 and 7 show the molecular weight markers, track 2 shows the DNA of Ad5 dl1014, track 3 shows the DNA of Ad5 wt300, track 4 shows the CELO virus DNA, track 5 shows the DNA of OTE, track 6 shows the DNA of Indiana C). FIG. 2A shows that the CELO virus genome has a length of 44 kb. From this analysis it is clear that the CELO virus genome is actually substantially larger than the genome of the mammalian virus. Calculations based on the migration of fragments of the lambda bacteriophage give a size of 43 kb for the CELO virus genome. The DNA extracted from two other FAV-1 isolates, Indiana C and OTE, co-migrates with the CELO virus species, which is further evidence of the size of the CELO virus genome. FIG. 2B shows that the CELO virus sequence contained in the bacterial plasmid pBR327 has the same size.

[0011] There is no identifiable E1 region. No significant homology could be found between the CELO virus genome and the first 4,000 bp of Ad2. There are a few small open reading frames in the first 5,000 bp of CELO virus which might possibly perform some of the E1 tasks. An open reading frame at the right hand end of the virus genome (GAM-1) may replace E1B 19K in functional assays without there being any significant homology between GAM-1 and E1B 19K. In order to confirm that the left hand end originates from the Wild type CELO virus genome and is not the sequence of a cloned variant, various tests were carried out; comparison of the direct sequence analysis of CELO virions at three different sites with the corresponding sites of the cloned sequences; Southern analyses with DNA from various virus isolates which yielded the same restriction fragments; pulsed field gel electrophoresis of various virus genomes which showed no heterogenicity.

[0012] There is no identifiable E3 region; the two small open reading frames in the corresponding region of the CELO virus have no significant homology with the E3 functions described.

[0013] There is a group of small open reading frames between 36,000 and 31,000 the position of which indicates the mammalian virus E4 region, but with additional 8 kb sequence at the right hand end of the CELO virus.

[0014] Nor was any sequence resembling protein IX identified (protein IX is essential for the hexon-hexon interactions and the stability of the mammalian adenovirus virions).

[0015] A protein V gene was not identified either.

[0016] The following regions are conserved between CELO virus and Ad2: the central part of the CELO virus genome, from the IVa2 gene (approximately from nucleotide (nt) 5,000) on the left hand strand up to the fibre genes on the right hand strand (approximately up to nt 33,000) is organised as in the mastadenoviruses, and the majority of the important viral genes can be identified both by their position and by sequence homology. Earlier studies on the homology between CELO and Ad2 (Aleström, et al., 1982a) showed two regions of the CELO virus which cross-hybridise with the Ad2 sequence. These two fragments are nt 5,626 to 8,877 (coding for IVa2 and the carboxy terminus of DNA polymerase) and nt 17,881 to 21,607 (coding for the hexon). The dot matrix analysis shown in FIG. 3 (carried out using the UWGCG program Compare with a window of 30 and a stringency of 20; summarised in FIG. 1A) shows that the total DNA sequence homology between CELO virus and Ads is mapped in the central region of the CELO virus genome. This is to be expected because the capsid proteins are coded in this central region and the coarse structure of the CELO virion is comparable with the capsid of the mammalian adenovirus (Layer, et al., 1971; Li, et al., 1984a). The genes which code for proteins corresponding to the human adenovirus proteins hexon, IIIa, penton base, protein VI and protein VIII, are present, and indeed in the expected sequence and position (FIG. 1A and Table 2A; Table 2B shows non-associated open reading frames which code for gene products with more than 99 amino acid groups). Each vertex of the mastadenovirus virion contains a pentamer of the penton base protein in conjunction with a single fibre consisting of three copies of the fibre polypeptide. Ad2, like most mastadenoviruses, has a single fibre gene, some adenovirus types have two fibre genes. The CELO virus genome codes for two fibre polypeptides of different lengths and sequences.

[0017] DNA binding proteins were identified in the region E2 (Li, et al., 1984c); four proteins with similar peptide maps were described, indicating a single precursor which is then cleaved or decomposed. The left hand open reading frame of the CELO virus genome, starting at nt 23,224, is located in the expected DNA binding protein region. The genes coding for DNA polymerase and pTP (pre-terminal protein) are present and in the expected positions (FIG. 1A, Table 2A).

[0018] With a view to the preparation of vectors based on the CELO virus it is useful to identify the mechanisms which the CELO virus uses in order to package almost 44 kb of DNA into a virion which is of a similar size to the human adenoviruses which are subjected to considerable restrictions on their packaging capacity (Bett, et al., 1983; Caravokyri and Leppard, 1995; Ghosh-Choudhury, 1987). One possibility is that the CELO virion, although virtually identical in size to Ad2 and Ad5, has a sufficiently widened structure in order to accommodate the larger genome. An alternative hypothesis is that CELO has a different mechanism for condensing DNA and therefore has differences in its provision of core proteins which are responsible for DNA packaging. Layer et al., in 1971 identified two proteins in the nucleus of the CELO virus and noted the absence of a molecule resembling protein V. Li, et al., 1984b, used electrophoresis with a higher resolution and reported a nuclear structure with three polypeptides (20 kD, 12 kD and 9.5 kD). These two findings lead one to conclude that the CELO virus must lack the larger basic nuclear protein V (41 kD) which occurs in mammalian adenoviruses. Perhaps the absence of protein V and/or the presence of smaller basic proteins is responsible for the additional packaging capacity of the CELO virion. The smallest of the CELO virus core proteins identified by Li, et al., 1984b (9.5 kD) is most closely associated with the virus DNA, similar to protein VII of the human adenovirus. An open reading frame which leads one to expect a protein with 8,597 D having 72 amino acids is located at nt 16,679; the coded protein is rich in arginine (32.9 mol%) and contains two cleavage sites for protease (pVII of Ad2 has only one cleavage site). An open reading frame which leads one to expect a protein with 19,777 D having 188 amino acid groups is located at nt 16,929. The protein has protease cleavage sites after groups 22, 128 and 145, and the carboxy-terminal groups have homology with pX of mastadenovirus. FIG. 4 shows the amino acid sequences of protein VII and pX of various mastadenoviruses compared with the CELO virus and the core proteins Core 2 and Core 1 of FAV-10. The sequences were arranged using the UWGCG Bestfit Program with a gap weight of 3.0 and a weight and gap length (“Gap Length Weight”) of 0.1. The protease cleavage sites of adenovirus are underlined. In connection with this it is interesting that the mastadenovirus DNA binding protein designated “mu” consisting of 19 groups is formed by two protease cleavings of the pX precursor (Hosokava and Sung, 1976; Weber and Anderson, 1988; Anderson, et al., 1989). Cleaving of the protein having 188 groups after groups 128 and 145 would produce a mu-like basic protein consisting of 17 groups (41% arginine, 12% lysine). The uncleaved form of the protein is also highly basic; the uncleaved copies of this protein could correspond to the 20 kD core protein observed by Li, et al., 1984b; a third 12 kD core protein identified by these authors could not yet be assigned.

[0019] Moreover, some new or non-assigned open reading frames were found in the CELO virus genome. A summary of these open reading frames is shown in Table 2A; these open reading frames are also given in FIG. 1A. This summary was restricted to the sequences from nt 0-6,000 and 31,000-43,804 and only ORFs which contain a methionine group and code for a protein >99 amino acid groups are mentioned. As already stated, there is an ORF at nt 1999 which codes for a protein having homology with parvovirus—REP, and an ORF at nt 794 having homology with dUTPase and Ad2 E4 ORF1. The objective of the present invention was to prepare a new CELO virus.

[0020] Thus, on the basis of the complete CELO virus genome sequence, the present invention relates to a CELO virus obtained by in vitro manipulation of a plasmid-cloned CELO virus DNA.

[0021] The CELO virus according to the invention derived from the genomic DNA contains, in a preferred embodiment, the left and right terminal repeat and the packaging signal and has modifications in the form of insertions and/or deletions and/or mutations in regions of the CELO virus DNA which are different therefrom.

[0022] The left or right terminal repeat (“Inverted Terminal Repeat”, ITR) extends from nucleotides 1-68 or from nucleotides 43734-43804, the packaging signal (also referred to as “Psi”) extends from nucleotides 70-200. Modifications-in DNA sections other than these ensures that the genes affected by the modification are non-functional or are deleted.

[0023] Preferably, modifications of the CELO virus genome are undertaken which are located on a section of the CELO virus DNA which includes the nucleotides from about 201 to about 5,000 (following the left terminal repeat of the section at the left hand end, hereinafter referred to as “Section A”) and/or on a section which includes the nucleotides from about 31,800-about 43,734 (the section at the right hand end located in front of the right terminal repeat, hereinafter referred to as “Section B”) and/or on a section which includes the nucleotides from about 28,114-30,495 (the region of the fibre 1 gene, hereinafter referred to as “Section C”).

[0024] A CELO virus in which certain genes are non-functional or are deleted, e.g. genes which affect the immune response of the host, such as antagonists to genes of the E3 region of mammalian adenoviruses, can be used as a vaccine.

[0025] In one embodiment of the invention the CELO virus contains one or more foreign DNA molecules, particularly a foreign DNA which is to be expressed in a host organism. In this embodiment the CELO virus acts as a vector which is capable of transporting the foreign DNA into higher eukaryotic cells, tissue or organisms, particularly mammals and birds, and expressing it therein.

[0026] Suitable insertion sites for the foreign DNA are the sections A and/or B and/or C.

[0027] The foreign DNA preferably replaces one or more sequences from these sections.

[0028] The CELO virus according to the invention is contained on a plasmid which is replicatable in bacteria or yeast and which yields virus particles after being introduced into suitable cells. Examples of suitable cells are bird embryo kidney or liver cells.

[0029] With a view to using a recombinant CELO virus vector for gene therapy, the foreign DNA may consist of one or more therapeutically active genes. Examples of these are genes coding for immunomodulators or modulators of inflammatory processes (cytokines such as IL-2, GM-CSF, IL-1, IL-6, IL-12; interferons, tumour antigens, IKB, and derivatives of IKB which lack serine phosphorylation sites (Traenckner, et al., 1995) or which lack lysine ubiquitinisation sites; glucocorticoid receptors; enzymes such as catalase, manganese superoxide dismutase, glutathione peroxidase, LIP members of the C/EBP family such as LIP or LAP (Descombes and Schibler, 1991), ADF (Tagaya, et al., 1989)), genes which influence apoptosis (members of the Bcl-2 family such as Bcl-2, adenovirus E1B19K, Mcl-2; BAX; IRF-2; members of the ICE protease family; variants of cJun, such as TAM-67 (Brown, et al., 1991); adenovirus E1A; p53) and genes which code for other therapeutic proteins (e.g. clotting factors such as factor VIII or IX; growth factors such as erythropoetin; cystic fibrosis transmembrane regulator gene (CFTR); dystrophin and derivatives thereof; globin; the LDL receptor; genes which are absent in lysosomal storage dysfunctions such as β-glucuronidase; etc.).

[0030] With regard to the production of cellular tumour vaccines or for pharmaceutical compositions with which the immune response to tumours is to be intensified, the foreign DNA codes for immunostimulating proteins or tumour antigens or fragments thereof.

[0031] The therapeutically active DNA may also code for antisense molecules which prevent the expression of genes or the transcription of specific RNA sequences in the target cell.

[0032] With regard to the use of the recombinant CELO virus vector as a vaccine, the foreign DNA codes for one or more antigens which bring about an immune response in the individual treated.

[0033] In one embodiment of the invention the foreign DNA codes for an antigen derived from a human pathogen, particularly a pathogen of infectious diseases.

[0034] Epitopes which can be expressed by recombinant CELO viruses include epitopes derived from all kinds of human viral pathogens such as HIV, hepatitis A, B, C, hanta virus, polio virus, influenza virus, respiratory syncytial virus, measles, mumps, rubella, papilloma and many other viruses. The non-viral pathogens include trypanosomes (the causal agents of sleeping sickness and Chagas' sickness), leishmania, Plasmodium falciparum (malaria), various bacterial pathogens such as the pathogens which cause tuberculosis, leprosy, Pseudomonas aeruginosa (complications in cystic fibrosis) and many others.

[0035] A summary of vaccines based on mastadenoviruses is given in Table 3; the epitopes mentioned therein by way of example can also be used for insertion in a vector based on the CELO virus.

[0036] With regard to the use of the recombinant CELO virus vector as a vaccine in the veterinary field, e.g. for birds, particularly poultry, the foreign DNA, in another preferred embodiment, codes for an antigen derived from a protein of a pathogen of animal diseases, particularly infectious diseases in birds.

[0037] Examples of pathogens of bird diseases are Avian Infectious Bronchitis Virus (IBV, a corona virus; Jia, et al., 1995; Ignjatovic and McWaters, 1991; Kusters, et al., 1990; Lenstra, et al., 1989; Cavanagh, et al., 1988; Cunningham, 1975), Avian Influenza Virus (Orthomyxovirus Type A; Kodihalli, et al., 1994; Treanor, et al., 1991; Tripathy and Schnitzlein, 1991), Fowlpox-Virus (McMillen, et al., 1994), Avian Infectious Laryngotracheitis Virus (Guo, et al., 1994; Scholz, et al., 1993; Keeler, et al., 1991), Mycoplasma Gallisepticum (Nascimento, et al., 1993), Avian Pasteurella Multocida (Wilson, et al., 1993; Lee, et al., 1991; Hertman, et al., 1980; Hertman, et al., 1979), Avian Reovirus (Ni and Kemp, 1992; Huang, et al., 1987), Marek's Disease Virus (MDV; Malkinson, et al., 1992; Scott, et al., 1989), Herpes virus of turkeys (HVT, Herpes virus of Turkeys), Newcastle Disease Virus (NDV; Cosset, et al., 1991; Morrison, et al., 1990), Avian Paramyxovirus Type 1 (Jestin, et al., 1989), Avipoxvirus Isolates (Schnitzlein, et al., 1988), such as Juncopox, Pigeon Pox, and Field—(Field) and vaccine strains of bird pox viruses, Avian Encephalomyelitis Virus (Shafren and Tannock, 1991; Nicholas, et al., 1987; Deshmukh, et al., 1974), Avian Sarcoma Virus, Rotavirus (Estes and Graham, 1985), Avian Reovirus (Haffer, 1984; Gouvea, et al., 1983; Gouvea and Schnitzer, 1982), H7 Influenza Virus (Fynan, et al., 1993).

[0038] Apart from DNA sequences which code for therapeutically active gene products or for antigens, the foreign DNA may code for proteins or protein fragments which change the behaviour of the CELO virus, particularly its ability to bind to cells, with regard to the use of mammals, particularly on mammalian cells. Examples of such proteins are fibre or penton base proteins from mammalian and other adenoviruses, surface proteins of other viruses and ligands which have the ability to bind to mammalian cells and transport the CELO virus into the cells. Suitable ligands include transferrin from various mammalian species, lectins, antibodies or antibody fragments, etc. The skilled person will be aware of ligands of this kind, other examples can be found in WO 93/07283.

[0039] The recombinant CELO virus may contain one or more foreign DNA molecules. These may be inserted either in tandem or spaced apart in different sections of the CELO virus sequence.

[0040] The foreign DNA is under the control of regulatory sequences; suitable promoters include for example the CMV immediate early promoter/enhancer, the Rous sarcoma virus LTR, the adenovirus major late promoter and the CELO virus major late promoter.

[0041] The suitability of the CELO virus for preparing vectors and the advantages of these vectors and their applications are based particularly on the following properties of the CELO virus:

[0042] i) Safety: Naturally, the CELO virus does not replicate in mammalian cells. Therefore, vectors based on this virus can be used in humans without any danger of a subsequent infection with a Wild type human adenovirus complementing the vector and possibly allowing replication. This is an advantage over the Ad2 and Ads vectors used at present.

[0043] ii) Increased packaging capacity: The CELO virus genome is about 44 kb long, compared with the 36 kb of the Ad5 genome. Both viruses have comparable virion sizes, so that with a CELO virus vector it is possible to expand the strict packaging limit of 35 kb which is available with Ads. On the basis of the sequencing carried out within the scope of the present invention, DNA-packaging core proteins of the CELO virus were identified, and striking differences were found from Ad2 which could be responsible for the increased packaging capacity. There are about 13 kb at both ends of the CELO virus which would appear not to code for structural proteins (e.g. capsid components) or for proteins required directly for the virus replication (e.g. DNA polymerase). For the production of vectors, these sequences on the CELO virus genome can be removed and if necessary replaced by complementing cell lines. These sequences can be assumed to code for the immune functions or the apoptotic functions (e.g. GAM-1) of the host cell or to be involved in the activation of the host cell for virus replication (antagonists to the E1, E3 and E4 regions of Ad2). These are the gene types which are either non-essential for virus growth in the cell culture, such as the E3 genes of Ad2 (Wold and Gooding, 1991; Gooding, 1992), or which are easily removed from the virus and can be expressed by a complementing cell line, such as the E1 region in 293 cells (Graham, et al., 1977) and the E4 region in W162 cells (Weinberg and Ketner, 1983).

[0044] iii) Stability: The CELO virion is remarkably stable. Its infectivity and its ability to transport DNA withstand a 30 minute treatment at 60° C. As a comparison, Ad5 loses two powers of ten of its infectivity at 48° C. and is completely inactivated at 52° C. Presumably, the CELO virus did not develop its heat stability naturally, rather this heat stability would indicate a reaction to another type of selective pressure on the virion. The natural route of CELO virus infection is a faecal-oral route, which requires the virion to survive contact with a chemically aggressive environment with extreme pH values and with proteases. For particular applications in gene therapy, a more resistant virus would be desirable which would survive, for example, in the digestive tract or in the lungs of a patient suffering from cystic fibrosis.

[0045] iv) Targeted use: The CELO virus binds only slightly to mammalian cells on its own and for efficient entry into the cell requires the addition of a ligand (transferrin or lectin; Cotten, et al., 1993). Therefore, recombinant CELO virions are unable to penetrate into human cells, resulting in the following possible applications:

[0046] The virus may be genetically modified as stated above in order to express, on its surface, ligands which enable targeted transportation, such as for example specific peptides or fibres and/or penton bases of human adenoviruses.

[0047] Another possibility is the chemical modification of the virus in order to couple specific ligands such as transferrin thereto, as proposed for example in WO 94/24299 and additionally the virus may be biotinylated (W93/07283) and bound via streptavidin to biotinylated ligands such as wheatgerm agglutinin or other lectins (WO 93/07283). CELO virus vectors thus do not have the disadvantages of human adenoviruses which have good binding ability to human cells but have to be masked for a specific, targeted use mediated by the ligand.

[0048] v) Possibility of use for vaccines: The CELO virus is seldom connected to diseases in birds, indicating that it provokes a strong protective immune response in bird hosts. The CELO virus vector can easily be adapted for the expression of new vaccine epitopes.

[0049] With regard to the removal of regions of the CELO virus DNA, it was concluded from the results obtained with the mastadenovirus that, when the central sections of the CELO genome are removed, these sections have to be made available in trans, e.g. by a packaging cell line. This restriction is based on the large quantity of virion components which are necessary for the assembly of the virus and the need to produce a cell line which is capable of producing the corresponding amounts of these proteins without any toxicity.

[0050] The approaches which have hitherto proved more successful in this respect with mastadenoviruses consisted of the production of cell lines which express regulatory proteins (E1 and E4 regions) or enzymatic proteins (DNA polymerase, DNA binding protein), because these proteins are not required in large amounts during a productive virus infection.

[0051] Starting from the analysis of the known CELO genes, the sections of the genome from nt about 12,000 to about 33,000 which code for structural components of the virus are preferably not interrupted. The region from about nt 5,000 to about 12,000 codes for the E2 genes IVa2 (a viral transcription factor), viral DNA polymerase (POL) and the terminal virus protein (viral terminal protein; pTP). These genes are essential for the function of mastadenoviruses; they ought therefore to be essential for the CELO virus as well. However, it is theoretically also possible to have deletions of essential genes of this kind provided that they can be produced in trans, e.g. by a packaging line. For example, a packaging cell line was prepared which produces Ad5 DNA polymerase, thus making it possible for this gene to be deleted from the virus genome. A similar procedure may be used in the construction of CELO virus vectors, by removing sections or the entire region from nt 5,000 to 12,000 from the CELO virus and having the corresponding functions controlled in trans by a packaging cell line.

[0052] Another possible restriction exists with regard to the presumed major late promoter, which was provisionally assigned to the region at about nt 7,000 (TATA box at nt 7,488). In the mastadenoviruses, this promoter is essential for driving late gene expression. Therefore, any change in the region at nt 7,000 of the CELO genome must be carried out in a way which maintains the promoter function of this region.

[0053] Table 4 lists the sequence elements of the CELO virus genome and is divided into various categories with regard to their deletion and/or mutation in the production of CELO virus vectors (in Table 4 L1, L2, etc. denote “late message 1, 2, etc., corresponding to the nomenclature normally used for mastadenoviruses):

[0054] Category 1 includes sequence elements which are required in cis and therefore cannot be made available in trans by a complementing cell line or by a complementing plasmid. These sections are necessary and are therefore present on the CELO virus according to the invention; these are the left and right terminal repeats and the packaging signal.

[0055] Sequences of category 2 code for proteins which are required in large amounts for virion production. These proteins may optionally be produced by a gene contained in a complementing cell line or on a complementing plasmid. Other sequences of category 2 are the major later promoter, the tripartite leader sequence and also the splice acceptor sites (SA) or the polyadenylation sites (poly A sites) of genes which are essential and which cannot be made available in trans.

[0056] Fundamentally, care must be taken to ensure that, in modifications of the CELO virus DNA which are carried out at the boundaries of genes, any control signals present, e.g. polyA sites are not interrupted or affected in any other way if possible.

[0057] The genes deleted from the CELO virus or non-functional genes therein may be prepared in trans, e.g. by complementing cell lines.

[0058] Complementing cell lines (“helper cells”) can be prepared by the manner known from the literature, analogously to helper cells which complement the functions of mammalian adenoviruses. To do this, the relevant CELO virus gene on a plasmid, preferably combined with a selectable marker, is introduced into cells which permit the replication of CELO virus, preferably into immortalised cell lines such as LMH (Kawaguchi, et al., 1987) or immortalised quail cell lines, as described for example by Guilhot et al., 1993. In helper cells which express the relevant CELO virus genes, optionally stably integrated, the defective CELO viruses are able to replicate.

[0059] Instead of making the regions of the CELO virus deleted in the vector available by means of a cell line, the deletions may also be complemented by a copy of the relevant gene contained on a plasmid. For this, the method described in WO 96/03517, that described by Cotten et al., 1994a and 1994 b) or the one described by Wagner et al., 1992, may be used, for example, in which a CELO virus vector containing a deletion is inserted, as a component of a transfection complex, containing a conjugate of polylysine and a UV/psoralen-inactivated adenovirus (human or CELO) and optionally transferrin-polylysine, into embryonic chicken kidney cells or liver cells, embryonic or immortalised quail cells, e.g. liver or kidney cells, and the transfection complex also contains a plasmid which carries a copy of the gene or genes which lack the CELO virus vector. The combination of genes contained on the vector and genes carried by the plasmid results in a normal virus replication cycle. (Similar approaches were used in mastadenovirus systems in order to complement E1-deficient adenoviruses (Goldsmith, et al., 1994) and E4-deficient adenoviruses (Scaria, et al., 1995)). The subsequent amplification of the virus may be carried out by using the defective virus as a carrier which is dependent on the complementing plasmid using the methods described above.

[0060] Another possible way of replacing the genes missing from the CELO virus is by using helper viruses.

[0061] The helper virus used may be a CELO virus (Wild type or partly defective). In this embodiment the CELO plasmid carrying the mutation (e.g. a derivative of pCELO7), is introduced into chicken cells, e.g. using the method described in WO 96/03517 or described by Cotten et al., 1994, using as the carrier for the derivative, for example, psoralen/UV-inactivated adenovirus (human or CELO) together with an adenovirus (human or CELO) as a carrier for the plasmid or plasmids with the genes which complement the defect. Alternatively, a Wild type CELO virus may be used both as a carrier and as source for complementing gene functions. The subsequent amplification of the defective CELO viruses obtained is carried out by co-infection of the defective CELO virus with a complementing adenovirus (e.g. Wild-type CELO or a CELO which has mutations at other points of the genome).

[0062] CELO virus genes of category 3 include the sequences on Sections A, B and C. These are sequences which code for a protein or an RNA molecule which is necessary for the interaction with the host cell machinery or with the host immune system. These proteins should be required in fairly low concentrations or may be dispensable for cultivation of the virus in the tissue culture.

[0063] Thus, preferably, in the CELO virus vectors according to the invention the genes of category 3 are replaced by the gene in question; if necessary, complementary cell lines or plasmids or helper viruses can be prepared which produce the corresponding gene products.

[0064] In one embodiment of the invention the vectors according to the invention contain the gene in question instead of one of the fibre genes. The CELO virus has two fibre proteins (Layer, et al., 1971; Gelderblom and Maichle-Lauppe, 1982; Li, et al., 1984a). It can be assumed that one of the fibres of the CELO virus is not necessary for the assembly of the virion and the infectivity. This assumption is backed up by electron microscopic observations that the longer fibre (fibre 1) should associate with the penton base along the side of the complex, whilst the shorter fibre (fibre 2) projects out of the middle of the penton base, similarly to the penton/fibre complexes in the mastadenoviruses (Hess, et al., 1995). In adenoviruses with only a single fibre, the fibre molecule is required for the assembly of the virus; in the absence of fibres no stable mature viruses are formed. The CELO virion should therefore require fibre 2 for stability and as a ligand, whereas fibre 1 acts only as a ligand. Within the scope of the present invention the assumption that, of the two fibre genes of the CELO virus, fibre gene 1 located in region C is superfluous and can be replaced by the gene in question was proved correct by removing the fibre 1 gene and replacing it with a luciferase expression unit.

[0065] Other examples are inserts in region A and/or B.

[0066] Within the scope of the present invention it was found that destruction of the reading frame at nt 794 (region A) which codes for dUTPase yields viable viruses. The dUTPase gene is thus a gene which is not necessary for growth in cell culture.

[0067] In one embodiment of the invention, the recombinant CELO virus thus contains a foreign gene which is inserted in the region of the reading frame coding for dUTPase.

[0068] According to another aspect the present invention relates to a process for preparing recombinant CELO virus.

[0069] The process is characterised in that the CELO virus genome contained on a plasmid or sections thereof is or are genetically manipulated.

[0070] In one aspect of the invention the genetic manipulation consists of insertion and/or deletion. Insertions and/or deletions can be carried out by using restriction enzyme cutting sites which occur naturally in the CELO virus DNA in these sections, e.g. the FseI cutting site occurring at position 35,693 in Section B. The insertion may be carried out directly into this cutting site or beyond this cutting site or close to this cutting site, or this cutting site may be used to allow recombination in the surrounding area.

[0071] In one preferred embodiment the manipulation consists of carrying out insertions and/or deletions using standard methods of molecular biology (Maniatis, 1989). The naturally occurring restriction cutting sites can be used for this, e.g. sites located in regions of the genome which are non-essential for cultivation of the virus in the host cell, e.g. the FseI cutting site which occurs at position 35,693 in Section B. The insertion can be made directly into this cutting site or beyond this cutting site or in the vicinity of this cutting site, or the cutting site may be used to facilitate recombination in the surrounding area. Foreign DNA sequences can be inserted, e.g. marker genes or genes coding for therapeutically active proteins.

[0072] An alternative possibility is to remove CELO sequences which are flanked by two restriction sites and replace them with new sequences. (An example of this is dUTPase mutation as carried out in Example 7.) In these cases the manipulation is carried out with the entire CELO virus genome. In the event that restriction sites are present, the deletion/insertion can alternatively be carried out on a subfragment which is then re-incorporated in the entire genome by ligation and optionally recloning in bacteria.

[0073] Another possibility is to insert the foreign gene in artificial restriction enzyme cutting sites produced by conventional methods of recombinant DNA technology (Maniatis, 1989).

[0074] In one embodiment the process is characterised in that manipulations are carried out in a plasmid DNA which contains the CELO virus genome, in CELO DNA sequences, with the exception of the left and right inverted terminal repeats and the packaging signal.

[0075] In another preferred method, the manipulation of the CELO genome is carried out by recombination. For this, a subfragment of the CELO genome is manipulated in order to introduce mutations and/or new sequences. Subfragments can be prepared by various methods, specifically by PCR (polymerase chain reaction), by ligation between PCR products or between restriction fragments or by subcloning in bacteria (as described in the Examples of the previous invention; see also Chartier et al., 1996). Examples of suitable bacteria strains for recombination are BJ 5183 (Hanahan, 1983) or JC 8679 (Gillen et al., 1974) or JC 5176 (Capado-Kimball and Barbour, 1971).

[0076] For recombination using PCR products, the sequence to be inserted into the CELO genome is prepared by PCR (Oliner et al., 1993) using primers which flank the sequence plus about 15 nucleotides of the sequence complementary to the insertion site in the CELO genome. In a second round of PCR, another 15 nucleotides are hung from the sequence complementary to CELO, resulting in a PCR product which consists of the sequence to be inserted with 30 nucleotides of the CELO sequence at each end. This fragment is mixed with a plasmid which contains the CELO DNA (e.g. the plasmid pCELO7 prepared within the scope of the present invention) and which has been linearised with a restriction enzyme which cuts only between the two flanking sequences hung from the sequence by PCR.

[0077] For recombination using ligation reaction products (prepared by conventional techniques as described for example by Maniatis et al., 1989), in principle the same procedure is used as in the recombination with cloned fragments, except that the intermediate cloning step is omitted.

[0078] In every case, the manipulated product obtained is characterised and used to prepare virus by transfecting avian cells (e.g. using the method described by Wagner et al., 1992; Cotten et al., 1994; or Cotten et al., 1993) and then cultivating them, after which the virus is harvested.

[0079] For preparing recombinant CELO virus using cloned fragments, the method preferably comprises subcloning a small fragment from the relevant region of CELO virus into which the foreign gene is to be inserted on a bacterial plasmid in order to ensure that restriction sites which occur several times on the CELO virus genome occur only once on the plasmid. These restriction sites are used to remove a region from the small fragment. For preparing the CELO virus vector this region is replaced by foreign DNA. The foreign DNA may consist solely of a linker with a restriction site occurring only once or of a sequence coding for a protein or for an antigen. The sequence may also code for a reporter gene with a restriction site occurring only once. This makes further manipulation of the CELO virus easier by inserting the foreign DNA, which codes for a therapeutically active gene product or for an antigen, into this restriction site, and at the same time the reporter gene permits rapid information as to the efficiency of the vector, by introducing the plasmid into cells and monitoring the expression of the reporter gene.

SUMMARY OF THE FIGURES

[0080] FIG. 1A: Comparison of the genomic organisation of Ad2/5 with the CELO virus

[0081] FIG. 1B: Restriction map of the CELO virus genome

[0082] FIGS. 2A and B: Pulsed Field gel-electrophoretic analysis of the genome size of adenoviruses

[0083] FIG. 2C: Characterisation of plasmid-cloned copies of the CELO virus genome by means of restriction endonucleases

[0084] FIG. 3: Dot matrix analysis of the DNA sequence homology between CELO virus and Ad2

[0085] FIG. 4: Amino acid sequences of protein VII and pX from various mastadenoviruses compared with CELO virus and the core proteins core 2 and core 1

[0086] FIG. 5: Construction of a plasmid which contains the entire length of the CELO genome

[0087] FIG. 6: Preparation of a CELO vector from a copy of the CELO virus genome contained on a plasmid

[0088] FIG. 7: Identification of bacterial clones which contain a deletion in the dUTPase gene

[0089] FIG. 8: Comparison of Wild-type CELO and CELO containing a deletion in the dUTPase gene by Western blot analysis.

[0090] In the Examples, the following materials and methods were used unless otherwise specified:

[0091] a) Virus and Virus DNA

[0092] A plate-purified isolate from CELO virus (FAV-1, Phelps strain) which was used as starting material for the DNA both for direct sequencing and for the formation of bacterial plasmid clones, was grown in 9 day old pathogen-free chicken embryos, as described by Cotten, et al., 1993. The FAV-1 isolates OTE (Kawamura, et al., 1963) and Indiana C (Calnek and Cowen, 1975; Cowen, et al., 1978) were cultivated in chicken embryo kidney cells. The virus was purified from the allantoic fluid or from infected embryo kidney cells by separation in CsCl gradients, as described by Layer, et al., 1971, and Cotten, et al., 1993. Virus DNA was isolated by treating the purified virions with proteinase K (0.1 mg/ml) and SDS (0.2%) at 56° C. for 45 min and subsequent equilibrium centrifugation of the DNA in a CsCl gradient in the presence of ethidium bromide. After the second gradient, the ethidium bromide was removed by extraction with CsCl-saturated isopropanol and the virus DNA was exhaustively dialysed against 10 mM Tris, 0.1 mM EDTA, pH 8.

[0093] b) Chicken Embryo Kidney Cells

[0094] The kidneys of 14 day old chicken embryos were collected, washed in PBS and digested with pancreas trypsin (2.5 mg/ml in PBS) at 37° C. The dispersed cells were mixed with an equal volume of foetal calf serum, the cells were collected by centrifugation, washed once with FCK medium and taken up in the same medium again. (The FCK medium is medium 199 with Earle's salts (Sigma M2154) supplemented with 10% tryptose phosphate (Sigma T8159), with 10% foetal calf serum, 2 mM glutamine, 100 μg/ml streptomycin and 100 IU/ml penicillin.) The cells were plated out in 175 cm2 tissue culture flasks (2 embryo kidneys per flask), stored at 37° C. under 5% CO2 and infected 24 to 48 hours later. The cells were infected with about 1,000 virus particles per cell and harvested 3 to 4 days after infection when the cytopathic effect was complete.

[0095] c) Pulsed Field Electrophoresis

[0096] Aliquots of purified adenovirus DNA (10-20 ng) were loaded onto a 1% agarose gel (BioRad, PFC agarose) and separated using a BioRad CHEF Mapper Pulsed Field Electrophoresis system (FIGE mode) for 24 hours in 0.5×TBE cooled to 14° C. The switching time, both in the forward direction and in reverse, was changed logarithmically from 0.22 seconds to 0.92 seconds with a ramp factor of 0.357 (21%). The forward voltage gradient was 9 V/cm (300 V), the reverse voltage gradient was 6 V/cm (200 V). After the run the gel was stained for 25 minutes in 0.5 μg/ml of ethidium bromide solution in water and then destained for 1 hour before the DNA pattern was made visible under UV light.

[0097] d) Sequencing Methods, Data Analysis

[0098] For the sequencing, EcoRI and HindIII restriction fragments of CELO virus DNA were cloned in pBlueScript SK(−). Three of the EcoRI clones containing the EcoRI fragments C, D and E (see FIG. 1B) and five of the HindIII clones containing the HindIII fragments F, A, G, B and E (cf. FIG. 1B, were selected for the preparation of deletions in one direction using exonuclease III (in FIG. 1B the cleavage sites for the restriction enzymes EcoRI, HindIII, BamHI and BglII are given; the alphabetical names for the EcoRI and HindIII fragments, on the basis of their relative sizes, are also given). These deletion clones were sequenced using the Taq Dyedeoxy Terminator System with the automatic sequencing apparatus ABI 373 according to the manufacturer's instructions. The sequence analysis of the terminal 2,000 bp at the left hand end and the 1,000 bp at the right hand end of the CELO virus genome, the sequencing to close the gaps between the fragments EcoRI C/HindIII G and the fragments HindIII B/EcoRI D and the sequencing to confirm the sequence at various points of the genome were carried out by direct sequencing of the viral DNA. All the sequence data are the results of at least three sequence reactions. The sequence data were combined using the programs SeqEd (ABI) and SeqMan (Lasergene). The sequence analysis was carried out using the program GCG of the University of Wisconsin.

EXAMPLE 1

[0099] Preparation of a Recombinant Bacterial Plasmid Clone of the CELO Virus Genome

[0100] a) Preparation of a Plasmid Vector with a Low Copy Number for Cloning the CELO Virus

[0101] The bacterial vector pBR327 (ATCC No. 37516) was chosen for this because it is retained in bacterial host strains at relatively low copy numbers (instead of this plasmid any other plasmid with a low copy number such as pBR322 could be used equally well). It was essential to create on the vector a restriction site which occurs only once and which does not appear in the CELO virus sequence. As described hereinafter, the virus sequence has to be cut from the plasmid vector sequences in order to inject a productive infection; therefore, restriction sites which flank the CELO sequence (but which are not present within the CELO sequence) have to be incorporated in the vector. In the experiments carried out, the restriction enzyme SpeI was used; however, any other enzymes which do not have recognition sites in the CELO sequence, such as AscI, PacI and SfiI, may be used instead.

[0102] The plasmid p327SpeI was prepared by ligating an SpeI linker (New England Biolabs) into the Klenow-treated EcoRI site of pBR327, thereby destroying the EcoRI site and creating an SpeI site which occurs only once.

[0103] b) Cloning the Ends of CELO

[0104] The two terminal HindIII fragments were cloned. In order to do this, CsCl-purified genomic CELO DNA was digested with HindIII and separated on a low-melting agarose gel (0.7% low melting agarose in TAE). The 1601 bp left hand end fragment and the 959 bp right hand end fragment were cut from the gel, and each gel fragment was suspended in 300 μl of 10 mM Tris, 1 mM EDTA pH 7.4 and heated to 70° C. for 10 min to melt the agarose. The terminal peptides were eliminated by the addition of NaOH to 0.3 N and heating to 37° C. for 90 min. (Hay, et al., 1984). The solutions were then cooled to ambient temperature, then Tris pH 7.4 (to 0.1 M) and HCl (to 0.3 M) were added in order to neutralise the NaOH. The fragments were heated to 56° C. for 20 min. and slowly cooled (over 1 hour) to ambient temperature in order to facilitate re-annealing. Then the DNA was purified over a Qiaquick column and ligated for 4 hours at 16° C. using a Pharmacia T4 ligase reaction (New England Biolabs) to an SpeI linker (New England Biolabs). The ligase was inactivated by heating to 70° C. for 10 minutes, excess linker was removed (and an overhang complementary to SpeI was formed) by digesting for 2 hours with restriction endonuclease SpeI. The DNA fragments were in turn purified by Quiquick column chromatography and ligated to p327SpeI treated with SpeI/HindIII/calf alkaline phosphatase. The ligation product was transformed into the bacterial strain DH5alpha, and plasmid clones were identified which carried either the 1601 bp left hand end fragment or the 959 bp right hand end fragment (both released by SpeI/HindIII digestion). In order to confirm the terminal 300 bp of both fragments, DNA sequence analysis was carried out.

[0105] c) Cloning of both CELO Ends on the same Plasmid

[0106] The 1601 bp left hand end and the 959 bp right hand end fragment were cut from their vectors by HindIII/SpeI digestion, separated by gel electrophoresis and purified by Qiaquick chromatography. The two fragments were mixed in approximately equimolar amounts and ligated for 30 minutes using the Pharmacia T4 ligase reaction. An aliquot of SpeI/CIP-treated p327SpeI was added and ligation was continued for 4 hours. The ligation mixture was transformed in DH5alpha and plasmid clones were identified which carried the correct double insert (pwu#1 and pWu#3).

[0107] The second HindIII site was removed by cleaving pWu#3 with ClaI and BamHI, treating with Klenow enzyme, religation, transforming DH5alpha and selecting a clone which

[0108] the ClaI/BamHI (which had contained a HindIII site). The resulting plasmid designated pWu-H35 now contained a single HindIII site between the left and right hand CELO end fragments.

[0109] d) Cloning the Entire CELO Genome

[0110] The plasmid pWu-H35 obtained in c) was treated with HindIII and CIP and purified on a low melting agarose gel following by Qiaquick chromatography. The linearised vector pWu-H35 was mixed with 0.3 μg of purified CELO virus DNA, then 30 μl of electro-competent bacterial strain JC8679 (Gillen, et al., 1974; Oliner, et al., 1993) were added to the DNA mixture on ice. 10 Minutes later the mixture was transferred into a BioRad Electroporation chamber and pulsed with an electric charge of 2.4 kV (BioRad Gene Pulser; Oliner, et al., 1993). The bacteria were then plated onto LB ampicillin plates and the ampicillin-resistant colonies were investigated for their plasmid content. Recombination between the terminal CELO sequences on pWu-H35 and the ends of the genomic CELO DNA re-establishes the circularity of the linearised plasmid and allows growth on ampicillin. A plasmid which contains the CELO genome over its full length was identified, and this plasmid, referred to as pCEL07, was used for the subsequent investigations. FIG. 2C shows the characterisation of plasmid-cloned copies of the CELO virus genome. Plasmid DNA from clones designated pCEL07, 8, 9 and 13 or DNA isolated from purified CELO virus, was digested either with BglII (tracks 2-6) or HindIII (tracks 7-11) and separated on a 0.6% agarose gel, and the DNA was shown up by ethidium bromide staining. The molecular weight marker (tracks 1 and 12) was bacteriophage λ-DNA cut with HindIII and EcoRI. The sizes of some molecular weight fragments (in base pairs) are shown on the right of the Figure. For each enzyme, the two CELO end fragments which are bound to the bacterial plasmid during cloning (and which are therefore not released after restriction digestion) are given on the left of the Figure (in base pairs). These are the fragments with 5832 and 5102 bp with BglII or 1601 and 959 with HindIII.

[0111] The construction of pCEL07 is shown in FIG. 5.

[0112] e) Initiation of a CELO Virus Infection by a Cloned CELO Genome

[0113] pCELO7 was cleaved with SpeI (which cleaves at the sites flanking the adenovirus termini), extracted with phenol/chloroform and passed over an HBS-equilibrated gel filtration column (Pharmacia Nick column) to remove any impurities. The cleaved DNA was then incorporated in streptavidin-polylysine/transferrin-polylysine/biotin-adenovirus (UV/psoralen-inactivated) as described in WO 93/07283. Complexes containing 0.5 μg of SpeI-cleaved pCEL07 plus 5.5 μg of carrier DNA (pSP65; Boehringer Mannheim) were used to transfect primary embryonic chicken kidney cells (the complexes contain 4 mg of DNA per 180 cm2 flask, containing about 3×106 cells), and the cells were investigated for the cytopathic effect caused by virus replication. Five days after transfection, when the majority of the transfected cells had been rounded off and detached from the surface of the plate, the cells were obtained by centrifuging and the CELO virus was purified as described by Cotten, et al., 1993. The virus yield from plasmid cloned CELO virus is comparable with the yields obtained by using pure CELO virus DNA (purified from virions).

EXAMPLE 2

[0114] Preparation of a CELO Mutant which Lacks the Sequences from nt 35,870 to 42,373 at the Right Hand End

[0115] There are no identifiable viral structural genes beyond the two fibre genes with the L5-polyadenylation site at position 31771. (There is a cryptic VA-gene at positions 39,841 to 39,751.) Investigations were therefore carried out to see whether the sequences between about 32,000 and the right ITR are necessary for the growth of the virus in cell culture. For this an accumulation of seven AseI sites were used at positions 35,870, 36,173, 38,685, 38,692, 39,015, 42,348 and 42,373, which does not appear anywhere else in the CELO virus genome. pCELO7 was digested with AseI, religated and a plasmid which lacked the inner AseI fragments was identified and designated pALMCELO—35870-42373. In connection with this it should be noted that the plasmid vector also has an AseI site; however, this is located in the ampicillin resistance gene, and selection for ampicillin resistance requires that all positive colonies have at least the two fragments which carry the right and left hand halves of the amp gene.

[0116] To aid further manipulations of the virus with the missing left hand end of the genome, pALMCELO—35870-42373 was digested with AseI (which cuts once in the ampicillin resistance gene of the plasmid and once at position 35,870) and ligated to a linker oligonucleotide TACCCTTAATTAAGGG which codes for a cutting site for the restriction endonuclease PacI and for ends which are complementary to those formed during AseI digestion. Religation, followed by selection for ampicillin resistance, identified plasmids which did not integrate the oligonucleotide at the AseI site of the ampicillin resistance gene. Restriction digestion identified a plasmid which carried a PacI site at the earlier AseI site of CELO at position 35,870. The plasmid was designated pALMCELO—35870-42373P.

EXAMPLE 3

[0117] Preparation of a CELO Virus Vector in which a Fibre Gene is Missing which is Replaced by a Gene of Interest

[0118] The CELO fibre genes are contained on a HindIII fragment which extends from nt 27,060 to 33,920 (the HindIII B-fragment, cf. the restriction map in FIG. 1B). On this fragment the sequence coding for fibre 1 extends from nt 1,054 to 3,435. The 5H3 fragment was digested with BglII (which cuts at nt 1,168) and HpaI (which cuts at 3,440), the BglII end was filled with Klenow enzyme and ligated to a blunt CMV/luciferase/β-globin cleavage/polyadenylation signal fragment from the plasmid pCMVL (Plank et al., 1992) to form the plasmid p5H—28227-30502(luc) which lacks almost the entire fibre 1 sequence which is replaced by a luciferase expression unit.

[0119] The relevant restriction cutting sites in CELO are as follows:

BglII A′GATC_T
Cuts at: 0 5102 15979 23472 28227 37972 43804
Size     5102 10877  7493  4755  9745  5832
HindIII A′AGCT_T
Cuts at: 0 1601 5626 17881 23327 27060 33920 38738 42845
         42845 43804
Size:    1601 4025 12255 5446  3733  6860  4818  4107
         959
HpaI GTT′AAC
Cuts at: 0 5503 20673 23355 30502 43804
Size:    5503  15170  2682  7147  13302
NotI GC′GGCC_GC
Cuts at: 0 17389 43804
Size:    17389  26415
XbaI T′CTAG_A
Cuts at: 0 1659 1988 28608 39268 41746 43804
Size:    1659  329  26620  10660  2478  2058

[0120] The modifications which were carried out on p5H—28227-30502(luc) were introduced into the entire CELO genome in the following manner: The CELO/luciferase/CELO fragment was cut from p5H—28227-30502(luc) as a HindIII fragment. This fragment was recombined with the 26kb XbaI fragment (CELO nucleotides 1988-28608) and the terminal HpaI fragments derived from pCELO7 (obtained by cutting with HpaI, containing the left hand end of the CELO virus and pBR327 sequences, defined by the HpaI sites). The three DNA fragments (each about 50 ng) were mixed in water and electroporated in JC8679 cells as described above.

EXAMPLE 4

[0121] Insertion of a Reporter Gene (Luciferase) in the CELO Genome

[0122] i) Preparation of a Left Hand End Fragment Containing a CMV Luciferase Construct

[0123] The EcoRI fragment designated 7R1 (nucleotides from positions 79 to 8877) was cloned into a pSP65 derivative designated pAAALM (described in WO 95/33062). The plasmid was transformed into the DAM methylase negative bacterial strain JM110 in order to allow cleavage of the ClaI sites in the fragment. The plasmid was purified, cut with ClaI (at position 1083) and NcoI (at position 4334), treated with Klenow enzyme to fill the overhanging ends and ligated to a blunt CMV/luciferase/β-globin cleavage/polyadenylation signal (Plank et al., 1992). The resulting plasmid was designated p7R1—1083-4334Luc.

[0124] ii) Recombination of the Luciferase Left Hand End Fragment into a Complete (Full Length) CELO Sequence

[0125] The plasmid p7R1—1083-4334Luc was cleaved with Eco47 III, which cleaves at the CELO nucleotides 937, 1292, 2300 and 8406 (the sites at nucleotide 1292 and 2300 are absent from p7R1—1083-4334Luc) in order to release a large fragment containing the sequence CELOnt937-1083/CMVLusPA/CELOnt4334-8406.

[0126] This fragment was recombined in pCELO7. pCELO7 was cleaved at the single PmeI site at CELO nt7433 and exhaustively dephosphorylated with calf intestinal phosphatase. The linearised pCEL07 was mixed with an approximately 3 to 5 molar excess of CELOnt937-1083/CMVLucPA/CELOnt4334-8406. The mixture was electroporated into the bacterial strain JC8679 and ampicillin-resistant colonies were examined on plasmids which contain the desired recombinant DNA. The correct plasmid was identified, characterised by restriction enzyme analysis and designated pCELOLucI.

[0127] iii) A CELO Virus Expressing Luciferase was Prepared by Transfecting pCELOLucI into Primary Embryonic Chicken Kidney Cells as Described above.

EXAMPLE 5

[0128] Preparation of a CELO Vector from a Copy of the CELO Virus Genome Contained on a Plasmid

[0129] The region between the DraIII site (originally contained at nt 34,426 in the CELO virus genome) and the XhoI site (originally contained at nt 36,648 in CELO virus genome) were removed from the plasmid pAALMH3 which contains the HindIII fragment from nt 33,920 to nt 38,738, cloned in pAALM. Then it was treated with T4-DNA polymerase to produce blunt ends and ligated with the CMV/luciferase/β-globin fragment (cf. Example 4). In this way the plasmid p7H3Δ34426-36648 Luc was obtained. The CELO/luciferase/CELO fragment was cut on a HindIII fragment and inserted into the CELO genome of pCELO7 by recombination via the FseI site occurring only once at position 35,694. This yielded the plasmid PCELOΔ34426-36648Luc. Digestion with SpeI and transfection into embryonic chicken kidney cells yielded a virus CELOΔ34426-36648Luc. Then further insertions were carried out replacing the luciferase sequence with other genes of interest, using the once occurring PacI site which was introduced with the luciferase sequence.

[0130] FIG. 6 shows the cloning strategy used in this Example in general form: a small CELO fragment is subcloned into a plasmid (containing restriction site C); the restriction sites A and B which occur only once in this plasmid are used to replace the sequence with foreign DNA. As the next step, the entire fragment containing the foreign DNA between CELO sequence is cut from the plasmid and mixed with the plasmid which contains the entire CELO DNA and which has been cut with a restriction enzyme (D) which cleaves the CELO-DNA only once. With this mixture, bacteria (e.g. of the strain JC8679; Oliner et al., 1993; or another bacterial strain with a similar capacity for recombination) are transformed; recombination yields the desired plasmid containing the foreign DNA as an insert in the CELO virus genome.

EXAMPLE 6

[0131] Preparation of a Quail Cell Line which Complements the 7R1 Deletions and/or the 9R1 Deletions in CELO

[0132] The plasmids pX7R1 and pX9R1 (described in WO 95/33062) were introduced into primary embryonic quail kidney or liver cells by transferrinfection as described in WO 93/07283. Four days after transfection the cells were trypsinised and seeded at ⅕ of the original density. The cells were fed twice a week with FCK medium. Clonal lines were expanded and clones which carried either the 7R1, 9R1 plasmid or both plasmids were identified by PCR analysis. The RNA expression of the integrated plasmids was determined by Northern analysis.

EXAMPLE 7

[0133] a) Preparation of a CELO Virus Genome with a Mutation in the ORF794 DUTPase Gene

[0134] A plasmid designated pWuΔdut was produced by removing a 540 bp AflIII-SacI fragment from the ORF794 in pWu-H35 (see Example 1 c). In order to product pCELOΔdut, pWuΔdut was linearised with HindIII and dephosphorylated using alkaline shrimp phosphatase. After gel purification the DNA was mixed with purified CELO DNA and used to transform E. coli BJ5183 (Degryse, 1996) to ampicillin resistance. From the ampicillin resistant bacterial colonies obtained the DNA was extracted and E. coli DH5a was transformed therewith. DNA extracted from these bacteria was analysed by restriction mapping in order to identify recombinant virus plasmids. The identity of the clones was determined by restriction mapping (FIG. 7; pWu-H35 is designated “pWu” in the Fig. ). The digestion of the Wild type plasmid pWu-H35 with HindIII and SpeI yields fragments of 2944 bp, 1607 bp and 961 bp (track 2). The deletion which changes the dUTPase converts the 1607 bp fragment into a 1071 bp fragment (track 3; the modified fragments are marked with an asterisk). The plasmids which contain the complete sequence coding for CELO or the complete CELO sequence plus the dUTPase mutation were analysed by SpeI/HindIII digestion and showed the same change of the 1607 bp fragment into a 961 bp fragment (tracks 4 and 5).

[0135] b) Preparation of Recombinant CELO Clones from Chicken Cells

[0136] Either 6 μg pCELO7 (see Example 1 d) or 6 μg of pCELOΔdut (see above, digested with SpeI) were used to transfect primary embryonic chicken kidney cells (approximately 500,000 cells in a 2.5 cm well) using polyethylene amine (PEI)/adenovirus complex. For this, the Qiagen-purified DNA was extracted with Triton X-114 in order to remove lipopolysaccharide as described by Cotten et al., 1994. Transfection complexes were prepared by diluting 6 μg of digested DNA in 250 μl of 20 mM HEPES, pH 7.4. 20 μl of 10 mM PEI (molecular weight 2,000, pH 7) were diluted in 250 μl of 20 mM HEPES, pH 7.4. The PEI solution was added dropwise to the DNA solution, incubated for 20 minutes at ambient temperature and then mixed with 1.5 μl of an adenovirus preparation (psoralen/UV-inactivated adenovirus type 5, cf. WO 1719, 1.5×1012 particles/ml). After another 20 minutes the complex was added to the cells in DMEM without serum (250 μl of complex to 1.25 ml of medium). The medium was changed for normal growth medium (with serum) and 4 to 5 days later the cells were harvested, taken up again in 100 μl of HBS and sonicated for 2 minutes. A 10 μl aliquot of this sonicate (virus in passage 1) was used to infect the same number of primary embryonic chicken kidney cells in a 2.5 cm well in a cell culture plate. After another 4 to 5 days the cells were counted in order to determine the cytopathic effect (CPE end point assay or plaque assay, Precious and Russel, 1985). The cells were harvested (virus in passage 2) as in the first step and used to infect fresh chicken cells; harvesting these cells yielded viruses of the third passage, the analysis of which is shown in FIG. 8.

[0137] c) Western Blots

[0138] The virus infected cells were harvested, taken up in HBS and sonicated. Aliquots were mixed with 5x charging buffer (250 mM Tris-Cl, pH 6.8, 500 mM DTT, 10% SDS, 0.5% bromophenol blue, 50% glycerol), heated to 95° C. for 3 minutes and then allowed to run in a 10% polyacrylamide gel. The proteins were transferred onto nitrocellulose, blocked overnight in 5% skimmed milk/TBST (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.05% Tween 20). Viral proteins were shown up using anti-CELO-antisera from rabbits (1:1000) and anti-rabbit-horseradish peroxidase (DAKO; 1:20000) and made visible by ECL (Amersham). CELO virus (2.5×1012 virus particles/ml) was used as the control. The rabbit serum was prepared using CsCl-purified CELO virus and heat-inactivated at 60° C. for 30 minutes.

[0139] d) Extraction of Virus DNA

[0140] Virus infected cells were harvested and taken up in 100 μl of HBS/0.1% SDS/1 g/ml proteinase K, incubated for 1 hour at 56° C. and extracted with phenol/chloroform. The DNA was precipitated with ethanol.

[0141] The analyses carried out showed that recombination between the linearised plasmid pWuΔdut and the CELO DNA yielded two types of plasmids. Recombination to the left hand end of the dUTPase mutation yielded a Wild type CELO genome; recombination on the right hand side of the dUTPase mutation yielded a CELO genome which carried the dUTPase mutation.

[0142] The infection of primary embryonic chicken cells both with CELO DNA and with CELO-Δdut-DNA produced cytopathic effects which took the form of swollen, detached cells after 36 hours, whereas control cells (treated with lysates of cells which had been transfected with an empty vector (Bluescript pBS, Stratagene)) remained healthy in their morphology.

[0143] Western blot analysis showed that CELO viruses and CELO Δdut viruses produced by plasmid DNA are indistinguishable from Wild type virus grown in fertilised, 9 day old hens eggs.

[0144] In all, the tests carried out in this example show that pCEL07 codes a viable CELO virus genome. After excision with SpeI and transfection into primary embryonic chicken kidney cells this DNA yields infectious, passagable virus. Lysates of viruses of the 1st and 2nd passage produce a cytopathic effect on primary embryonic chicken kidney cells. It should be noted that these lysates were produced by sonication, which rules out the possibility that the CPE in the secondary and tertiary infections can be put down to the expression of viral genes which originates from the residual plasmid DNA in the lysates; plasmid DNA cannot be expected to withstand the method used to produce the lysates. In addition, it was found that with each round of infection there is a 100 fold amplification of the agent which causes CPE; this conforms to the amplification of a virus but not to the single passage of residual plasmid DNA from the first transfection.

[0145] The deletion of 540 bp in the CELO genome with which sequences between an AflIII site at bp 609 and a SacI site at bp 1145 were removed and the open reading frame coding for dUTPase was destroyed, yielded a virus genome which is viable even in primary embryonic chicken cells. With the UTPase gene a virus gene was thus identified which is not necessary for growth in cell culture.

TABLE 1
CELO virus Sequences, published or from data banks
				differences between
data bank				published sequences and
accession			coordinates in	new sequence (GenBank
#	authors, publication	size	the sequence	Accession #U46933)	remarks
	Aleström, et al, 1982b	101 bp	1-101	7 bases different	5′ITR
K00939	Shinagawa, et al, 1983	68 bp	1-68	7 bases different	5′ITR
Z17216,	Akopian, et al., 1992	3576 bp	1-3576	3 bases different	92-100% labelled
S61107				1 missing base	slight differences in
				3 additional bases	these 2 versions
Z48167	unpublished	3433 bp	13597-17033	6 bases different	contains genes for
				4 missing bases	penton base and core
				1 additional base	proteins
L13161	Cai, et al., 1993	900 bp	21023-21922	no differences	contains protease gene
X84724	Hess, et al., 1995	7359 b	27060-34299	2 bases different	contains genes for
				6 missing bases	pVIII, fibre 1, fibre 2
				3 additional bases
				11 (GCA) repeats (new
				sequence shows 9)
				6 doubtful bases
M12738	Larsson, et al., 1986	440 bp	39584-40023	no differences	contains VA gene
Z22864	unpublished	3670 bp	35235-38905	2 bases different	assigned by the
				4 missing bases	authors: 11.2-19.2%
				2 additional bases
X17217	Akopian, et al., 1990	4898 bp	38906-43804	2 bases different	assigned by the
					authors: 11.2%
K00940	Shinagawa, et al, 1983	68 bp	4374-43804	7 bases different	3′ITR
	Aleström, et al.,	124 bp	43680-43804	2 bases different	3′ITR
	1982b			1 missing base

[0146]

TABLE 2A
Organisation of the CELO Virus Genome
			Cap,
			cleav-
			age,		Amino
			polyA	MW	acid
Protein	ATG	STOP	sites	Dalton	groups	Remarks
L1
52K	12193	13329		42094	378
IIla	13316	15043		63771	575	Protease
						cleavage site
						at amino acid
						551
L2
			15080			Penton base SA
Penton	15110	16657		56719	515	noRGD
base			16196			poly A-site
pVII	16679	16897		8562	72	Protease
						cleavage sites
						at amino
						acids 27, 40
mu	16929	17495		19787	188	Protease
(pX,						cleavage sites
11K)						at amino acids
						125, 144
			17526			poly A-site
L3
pVI	17559	18230		23890	223	Protease
						cleavage sites
						at amino acids
						28, 212
Hexon	18289	21117		106704	942
			18261			Hexon SA
Pro-	21134	21754		23763	206
tease			21102			Protease SA
			or
			21123
			21767,			L3 poly A-site
			21836
L4
			23608			100K SA
			or
			23649
100K	23680	26634		109905	984
pVIII	27149	27886		26876	245	Protease
						cleavage sites
						at amino acids
						40, 115, 130,
						141, 166
			27920			L4 poly A-site
L5
			28315			fibre SA
			or
			28341
fibre 1	28114	30495		81526	793
			30509			[GCA]9 repeat
			30511			fibre SA
fibre 2	30536	31768		42939	410
			31771			L5 poly A-site
VA			39841
RNA			to
			39751
E2 and
IVa2
IVa2	6685	5366		50366	439
E2b	10268	6501		144984	1255
pol
E2b	11996	10269		66089	575	Protease
pTP						cleavage sites
						at amino acids
						116, 141, 260,
						264
DBP	23224	21899		49272	441
			23292			DBP cap site
			21824			DBP poly A
			or			site
			21882

[0147]

TABLE 2B
Non-assigned open readin frame, larger than 99 amino acid groups
Right ORF's	Left ORF's
ATG	STOP	groups	ATG	STOP	groups
794	1330	178	5094	4462	210
1999	2829	276	4568	3549	339
3781	4095	104	3374	2892	160
5963	6373	136	1514	1191	107
33030	33476	148	39705	39286	139
33169	33483	104	39256	38717	179
35629	36024	131	36144	35536	202
37391	38239	282	35599	34238	453
40037	41002	321	33707	32892	271
41002	41853	283	33058	32735	107
41958	42365	135	32429	31812	251

[0148]

TABLE 3
Recombinant Adenovirus vaccines
Pathogen	Reference	Comments
Respiratory	Hsu et al., (1994)	Glycoprotein F and G,
Syncytial		inserted into the
Virus		E3 region of Ad 4, 5 or 7
Hepatitis B	Chengalvalva et al., (1994)	HBsAg, inserted into
		the E3 region of Ad
		4 oder 7
Pseudorabies	Eloit et al., (1990)	Pseudorabies Glyco-
		protein gp50, inserted
		into the E1 region of
		Ad 5
Herpes Simplex	Zheng et al., (1993)	Tandem repeats of the
		epitope of gD
Herpes Simplex	Gallichan et al., (1993).	Glycoprotein B
Rotavirus	Both et al., (1993)	Rotavirus antigen,
		inserted into the E3
		region
HIV	Natuk et al., (1993)	Ad 4, 5 or 7, HIV env,
		or gag-protease gene
SIV	Cheng et al., (1992)	SIV Env Rev, inserted
		into the E3 region of
		Ad 5
Rabies	Kalicharran et al., (1992)	Ad 5 with rabies
		glycoprotein
Rabies	Charlton et al., (1992)	Ad 5 with rabies
		glycoprotein
Human	Marshall et al., (1990)	gB in E3 region of Ad 5
Cytomegaloviras
Measles virus	Fooks et al., (1995)	N protein in AdE1
		Region

[0149]

TABLE 4
Characteristics of the CELO Virus Genome.
		Coordinates
	Characteristics	(Nucleotides)	Category
	Left terminal	0-68	1
	Repeat
	Packaging signal	70-200	1
	unknown open	0-5365	3
	reading frame
	L1
	52K	12193-13329	2
	IIIa	13316-15043	2
	L2
	penton base SA	15080	2
	enton base	15110-16657	2
	Poly A site	16196	2
	pVII	16679-16897	2
	mu	16929-17495	2
	(pX, 11K)
	Poly A site	17526	2
	L3
	pYl	17559-18230	2
	Hexon SA	18261	2
	Hexon	18289-21117	2
	Protease SA	21102 or 21123	2
	Protease	21134-21754	2
	L3 Poly A site	21767, 21836	2
	L4
	100K SA	23608 or 23649	2
	100K	23680-26634	2
	pVIII	27149-27886	2
	L4 Poly A site	27920	2
	L5
	fibre SA	28315 oder 28341	2 or 3
	fibre 1	28114-30495	2 or 3
	Major Late	7350-7650	2 or 3
	promoter	(TATA box at 7488)
	Tripartite Leader	8651-8700,
		8798-8857,
		9682-9774
	fibre SA	30511	2
	fibre 2	30536-31768	2
	L5 PolyA site	31771	2
	VA RNA	39841 to 39751	3
	E2 and IVa2
	IVa2	6685-5366	3
	E2b pol	10268-6501	3
	E2b pTP	11996-10269	3
	DBP	23224-21899	3
	DBP cap site	23292	3
	DBP PolyA site	21824 or 21882	3
	right end open	31771 to 43804	3
	reading frame	(right end)
	right terminal	43734-43804	1
	repeat	(roughly the last
		70 bp)

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Claims

1. CELO virus obtained by in vitro manipulation of a plasmid-cloned CELO virus DNA.

2. CELO virus according to claim 1, characterised in that it contains the left and right inverted terminal repeat and the packaging signal and has modifications in the form of insertions and/or deletions and/or mutations in other regions of the CELO virus DNA.

3. CELO virus according to claim 2, characterised in that it contains modifications which are located on a section of the CELO virus DNA which comprises the nucleotides from about 201-about 5,000 and/or on a section which comprises the nucleotides from about 31,800-about 43,734 and/or on a section which comprises the nucleotides from about 28,114-30,495.

4. CELO virus DNA, contained on a plasmid which is replicatable in bacteria or yeast and yields virus particles after being introduced into cells, optionally together with a plasmid which complements a gene which may be missing from the CELO virus DNA and is necessary for the formation of mature virus particles.

5. CELO virus according to one of claims 2 to 4 or CELO virus DNA derived therefrom containing foreign DNA.

6. CELO virus or CELO virus DNA according to claim 5, containing as foreign DNA a DNA which codes for a therapeutically active protein.

7. CELO virus or CELO virus DNA according to claim 6, containing as foreign DNA a DNA which codes for an immunostimulatory protein.

8. CELO virus or CELO virus DNA according to claim 7, containing a DNA coding for a cytokine.

9. CELO virus or CELO virus DNA according to claim 5, containing as foreign DNA a DNA which codes for a tumour antigen or a fragment thereof.

10. CELO virus or CELO virus DNA according to claim 5, containing as foreign DNA a DNA which codes for an antigen derived from a human pathogen.

11. CELO virus or CELO virus DNA according to claim 5, containing as foreign DNA a DNA which codes for an antigen derived from an animal pathogen.

12. CELO virus or CELO virus DNA according to claim 11, containing as foreign DNA a DNA which codes for an antigen derived from a bird pathogen.

13. CELO virus or CELO virus DNA according to claim 5, containing as foreign DNA a DNA which codes for a ligand for mammalian cells.

14. CELO virus or CELO virus DNA according to one of claims 5 to 13, characterised in that it contains the foreign DNA in the FseI cutting site at position 35,693 or beyond this cutting site or close to this cutting site.

15. CELO virus or CELO virus DNA according to one of claims 5 to 13, characterised in that it contains the foreign DNA on a section which comprises the nucleotides from about 28,114-30,495, which contains the fibre 1 gene.

16. CELO virus or CELO virus DNA according to one of claims 5 to 13, characterised in that it contains the foreign DNA in the region of the reading frame at nucleotide 794 which codes for dUTPase.

17. Process for preparing recombinant CELO virus or recombinant CELO virus DNA according to one of claims 2 to 16, characterised in that the CELO virus genome or sections thereof contained on a plasmid are genetically manipulated.

18. Process according to claim 17, characterised in that the CELO virus genome or sections thereof contained on a plasmid are manipulated in a region which is different from the left and right inverted terminal repeat and from the packaging signal.

19. Process according to claim 17 or 18, characterised in that insertions and/or deletions are carried out.

20. Process according to claim 19, characterised in that a foreign gene is inserted in a naturally occurring or artificially introduced restriction cutting site on a section of the CELO virus DNA which contains a sequence which is not necessary for the replication of the virus in the host cell or which can be complemented.

21. Process according to claim 20, characterised in that in addition to the insertion, a deletion is carried out in another region of the CELO virus DNA which contains a sequence which is not necessary for the replication of the virus in the host cell or which can be complemented.

22. Process according to claim 17 or 8, characterised in that the manipulation is carried out by recombination.

23. Process according to claim 22, characterised in that DNA molecules obtained by polymerase chain reaction are recombined.

24. Process according to claim 22, characterised in that DNA molecules obtained by ligation are recombined.

25. Process according to claim 22, characterised in that DNA molecules obtained by cloning in bacteria are recombined.

26. Process for preparing recombinant CELO virus DNA according to claim 25, characterised in that a CELO virus DNA fragment containing two restriction sites is cloned into a bacterial plasmid, the foreign DNA is inserted between these restriction sites which occur only once on the plasmid, the fragment containing the foreign DNA is excised from the plasmid and mixed with a plasmid which contains the complete CELO virus DNA and which has been cut at a restriction cutting site which occurs only once, and in that bacteria are transformed with this mixture of DNA molecules and the bacteria are grown, whilst by recombination of the DNA molecules a plasmid is obtained which contains the entire CELO virus DNA with the foreign DNA as an insert.

27. Process according to one of claims 20 to 26, characterised in that a reporter gene is inserted as the foreign DNA.

28. Process according to claim 27, characterised in that the reporter gene has a restriction cutting site into which a foreign DNA as defined in one of claims 6 to 13 is inserted in an additional step.

29. Process for preparing CELO virus, characterised in that avian cells are transformed with a plasmid containing CELO virus or DNA derived therefrom, as defined in one of claims 1 to 16, the cells are cultivated and the CELO virus particles are harvested.

30. Process according to claim 29, characterised in that the CELO virus DNA lacks sequences which are necessary for the formation of mature virus particles and the missing DNA sequences are complemented.

31. Process according to claim 30, characterised in that the avian cells used are helper cells which complement the missing gene or genes.

32. Helper cells containing CELO virus genes integrated in their genome.

33. Helper cells according to claim 32, characterised in that the cells are avian cells.

34. Process according to claim 30, characterised in that the cells are also transformed with a plasmid which complements the gene or genes missing from the CELO virus DNA and necessary for the formation of mature virus particles.

35. Process according to claim 30, characterised in that the cells are also infected with a helper virus which complements the gene or genes missing from the CELO virus DNA and necessary for the formation of mature virus particles.

36. Pharmaceutical composition for use in gene therapy, containing a CELO virus according to claim 6.

37. Vaccine against infectious diseases in humans, containing a CELO virus according to claim 10.

38. Vaccine against infectious diseases in animals, containing a CELO virus according to claim 11.

39. Vaccine against infectious diseases in birds, containing a CELO virus according to claim 12.

40. CELO virus according to claim 7, 8 or 9 for the production of cancer vaccines.