FR2767335A1 - Production of recombinant CELO adenovirus vectors - Google Patents

Abstract

Method for preparing recombinant CELO chicken embryo lethal orphan adenovirus vectors comprises inserting a DNA sequence encoding a heterologous polypeptide into a nonessential region of the CELO adenovirus genome. Also claimed are: (1) a vector prepared as above; (2) a vaccine containing the vector; (3) a cell transformed with the vector.

Description

 The invention relates to methods for the preparation of recombinant CELO avian adenoviruses and their uses as a vector for expression of heterologous proteins for the preparation of vaccines, in particular for the protection of avian species against common infectious diseases, or as of expression vector for heterologous proteins involved in metabolism.

The presentation of an effective and lasting antigen, capable of mobilizing short-term and long-term immunological memories, remains the major concern of any vaccine-based prophylaxis. For a long time, the latter were constituted by the presentation, in the case of viruses with pathogenic effects, of the entire virion, either dead (inactivated vaccine), or more or less defective (live attenuated vaccine). In recent years, several strategies have emerged to overcome the shortcomings of these conventional vaccines, such as more or less persistent pathogenicity,
The impossibility of obtaining the virion by in vitro culture to obtain sufficient vaccinating antigens. Two of these strategies are predominant at present: one is the presentation of synthetic peptides with antigenic properties, derived from knowledge of the immunogenic proteins of the viruses or bacteria studied; The other is the use of viral vectors capable of carrying genes coding for immunogenic proteins derived from totally different viruses or for other infectious agents. The viral vector is used for its ability to penetrate target cells in order to penetrate the foreign antigen and, thereby, trigger humoral or cell-mediated immune reactions. In addition, the vaccinating viral vector can, in certain options, replicate and thus increase the in situ production of antigenic determinants and lead to stimulation of the immune system in the longer term.

Among the recombinant viral particles which can be used as a vector for expression of heterologous proteins and in particular as a vaccine vector, there are herpesviruses, retroviruses and adenoviruses. Herpesviruses are viruses
Double-stranded DNA with capsid and envelope, and are, for example, responsible for two important diseases: Aujesky's disease in pigs and Marek's disease in chicken. These viruses have complex genomes (150 kb on average) and obtaining the high titers necessary for industrial application is not always easy. Retroviruses are capsid and envelope RNA viruses which, as recombinant vectors, are essentially studied and used for gene transfer in mammals, human gene therapy, or in avian species. Their use as a recombinant vaccine vector is limited by their low capacity of transmissible exogenous material (approximately 3 kb of DNA), by the difficulty of obtaining high titers, by their instability (recombination of reconstructions). On the other hand, since the retrovirus is integrated into the genome of the retrovirus, it requires that the target cell is a dividing cell and imposes a high degree of safety without its use taking into account the possible passage of the construction in the germinal lines.

 Adenoviruses are double-stranded DNA viruses 30 to 44 kb in length. They are capsid viruses, not enveloped. In practice, one can insert, without any deletion, 1.5 kb in addition of exogenous material into an original genome of adenovirus without disturbing replication. Adenovirus has the following advantages for it: it is, in general, a pathogenic agent, responsible for mild damage to the upper airways. It can infect a wide range of cells, whether or not they are dividing. It is thus possible to modify cells that divide little, such as lung cells or muscle cells. In addition, this virus multiplies very easily, which allows the production of recombinant viruses at very high titers (10lut to 1013 viruses per milliliter), much higher than those of recombinant retroviruses and therefore to obtain a better rate of genetic transformation. target cells. An additional advantage of the adenoviral vector is that its genome does not integrate into the cellular genome, minimizing the risks of activation of oncogenes, it remains in the state of episomes. Finally, given the resistance of the capsid to external agents, nebulization or spraying by means of aerosols of recombinant virions, associated with high titers, should allow mass vaccinations, particularly advantageous for avian species.

 Today there is indeed a real need for inexpensive, effective and easy-to-administer vaccine vectors, making it possible in particular to protect industrial poultry farms against infectious diseases, in particular of viral origin, which are responsible each year. losses estimated at several hundred million dollars. Recombinant viral vaccine vectors have already been described such as poxviruses (AU-A-34353/93) or recombinant avian adenoviruses of serotypes FAV -4, -9 or -10 (WO 94 24268), serotype 1 -FAV (serotype CELO) not having been retained in this document because presented at this date as a vector of potentially oncogenic nature (SARMA, PS, et al., 1965, Science, 149, 1108) despite the results published subsequently showing the safety of l infection of chickens with this adenovirus (COWEN, BRW, et al., 1978, Avian Diseases, 22, 459-470).

The complete nucleotide sequence as well as the general organization of the genome of the avian adenovirus CELO (serotype 1-FAV) have been published recently by
CHIOCCA (CHIOCCA, S., 1996, J. Virol. 70, 5, 2939-2949) (see Figure 1). In this document, the comparative analysis between the complete nucleotide sequence of the avian adelovirus CELO and those known from other adenoviruses has made it possible to establish on the one hand, by homology, the presence or absence of a certain number of genes coding for viral proteins already characterized and on the other hand, based on the position of the codons
ATG and arrest, the presence of at least a number of open reading frames (ORF). From this information, it is not possible to predict which genes are actually transcribed and expressed by the virus, and therefore essential for essential functions of the virus such as that of replication. The generation of recombinant avian adenovirus as a recombinant vector for its application as a recombinant vaccine or for gene transfer requires the insertion of heterologous (exogenous) DNA of interest in nonessential regions of the viral genome. When the size of the heterologous DNA of interest to be inserted is greater than a value of approximately 1.5 kb, the insertion must be preceded by the deletion of one or more nonessential regions of the viral genome. This is why, only the precise knowledge of these nonessential regions of the genome of the adenovirus can make it possible to define a strategy in the preparation of such recombinant vectors.

 One of the essential aims of the present invention is to obtain expression vectors of heterologous proteins for the preparation in particular of avian vaccine from preparation methods based on the discovery of nonessential and essential regions of the avian adelovirus CELO.

The present invention relates to methods for preparing a recombinant CELO avian adenovirus, characterized in that a DNA sequence coding for all or part of a polypeptide heterologous to the adenovirus is inserted into a non-essential part of its genome. avian CELO, said DNA sequence possibly further comprising elements capable of ensuring the expression of said polypeptide in a cell infected with said recombinant adenovirus, preferably said nonessential part corresponding to a non-coding intergenic zone of avian adenovirus
CELO.

 The invention also relates to preparation methods according to the invention, characterized in that said methods are preceded by a deletion step of at least one non-essential part of the genome of the avian adelovirus CELO.

 The invention further relates to preparation methods according to the invention, characterized in that said methods are preceded by a deletion step of at least one essential region of the genome of the avian adelovirus CELO.

 The invention also relates to methods according to the invention, characterized in that said DNA sequence codes for a polypeptide of interest, preferably an antigenic polypeptide of an infectious agent responsible for disease in animals such as species avians.

 The invention further relates to methods according to the invention, characterized in that the polypeptide of interest is a polypeptide involved in animal or human metabolism.

 The vectors, characterized in that they comprise a recombinant CELO avian adenovirus obtained by a method according to the invention, also form part of the invention.

 Also included in the invention are the vaccines, characterized in that they contain a vector according to the invention for the protection of avian species against infectious diseases.

 The invention also relates to cells transformed with a vector according to the invention.

 Another aspect of the invention relates to methods for the in vivo preparation of recombinant polypeptides of interest, characterized in that they involve the infection of an animal by a vector according to the invention, of which said sequence of inserted DNA encodes said polypeptide of interest.

 Finally, the subject of the invention is processes for the preparation of recombinant polypeptides of interest, characterized in that they use the culture of transformed cells according to the invention.

 The invention relates to a method for preparing a recombinant CELO avian adenovirus, characterized in that a DNA sequence coding for all or part of a polypeptide heterologous to the adenovirus is inserted into a non-essential part of its genome. CELO avian.

 The adenovirus family is divided into two groups: Mastadenowradea, isolated from mammalian cells and Aviadenowradea, isolated from bird cells. The genus Aviadenovirus is itself divided into 5 groups of species including the poultry adenoviruses FAV ("Fowl AdenoVirus"). The bird adenovirus CELO ("Chicken Embryo Lethal Orphan") represents serotype 1 (FAV-1) among the 11 distinct serotypes of FAV recognized.

 The CELO adenovirus is a 43,804 bp long double-stranded DNA virus (CHIOCCA, S., et al.) (Figure 1). Its capsid is made up of elements of 3 types: hexons, pentons and fibers (proteins II, III, IV respectively according to their increasingly low molecular weight). Antigenic motifs (neutralizing epitopes) are carried mainly by hexons and fibers. The fiber is fixed by its NH2 terminal end at the base of the penton, its COOH carboxyl end ending in a button zone, responsible for the attachment of the virion to a specific receptor. Between these two ends is the fiber shaft, made of a number of repeating patterns (from 20 to 26 amino acids depending on the adenovirus).

The particularly resistant capsid allows very good protection of viral DNA, which explains the persistence of this virus in the environment and its ubiquitous presence. This capsid also has an essential role in the penetration of the virion inside the host cell by avoiding lysis by the endosomes, as normally occurs for any infectious agent picked up by endocytosis by the cell. Adenovirus
CELO, like the Aviadenoviruses, has two fibers whose role is not yet explained today, and is distinguished from mammalian adenoviruses (including human adenoviruses) which have a single fiber (except for human adenoviruses 40 and 41 which also have two fibers).

Genome and gene transcription in adenovirus
For a better understanding, the description below has the essential object of introducing the concepts and vocabulary which will be used subsequently in the present invention, relating to the organization of the adenovirus genome, the transcription of genes and their function, from known mammalian adenoviruses.

 In mammals, it seems that the structure of the genomes of the various adenoviruses is similar, with significant homologies of genes coding for given proteins (essentially of structure): hexon, penton, fiber, endopeptidase, etc. For avian adenoviruses, weak counterparts, 30 to 50% at best, exist with mammalian adenoviruses.

 Each adenovirus consists of double-stranded DNA, terminated by reverse repetitions of 50 to 150 bp (ITR: "InverteiTerminal Repeat"). The genome length varies from 30 to 44 kb (avian adenoviruses are generally longer than mammalian adenoviruses). These inverted terminal repeats, containing the origins of virion DNA replication, are fundamental to virion replication. In any construction of recombinant adenovirus, it is imperative to keep at least one of these ends in order to ensure replication of the recombinant adenovirus. In vivo, with these ITRs, a terminal protein is associated (TP) of 87 kDa in the terminal preprotein state, of 55 kDa in the mature state (packaged inside the mature virion). The presence of this TP makes it possible to improve the replication of the virion by a factor of 100 to 1000.

As with many viruses, the adenovirus genes are distributed (more or less arbitrarily) into two groups:
- so-called early genes expressing themselves before DNA replication;
- so-called late genes (Late genes) expressed after DNA replication and imperatively requiring the prior transcription of early genes.

 Whatever the adenovirus and its size, it is customary to partition its genome into 100 units (map unit: m. U.) And to position the genes according to these units.

Early genes:
For human adenoviruses, there are four: E1 to E4, each with their own promoter, sometimes located on one strand, sometimes on the other. These genes are involved in the regulation of viral transcription, the transformation and replication of viral DNA (conversely, late genes essentially produce the virion's structural proteins).

 El (1.3 to 11.2 m. U.): Essential gene, because it is the initiator of the entire viral cycle.

Its expression starts 20 to 30 minutes after the virion enters the cell, and its transcription depends on cellular factors specific to the host (host specificity of adenoviruses). This gene is broken down into two genes: ElA and E1B, the first of which is essential for the establishment of viral infection. These two genes E1A and E1B allow, by combined effects, cell multiplication and immortalization transformation of cells receiving them jointly by transfection (if E1A allows immortalization, E1B avoids apoptosis and thus allows the maintenance of the immortalized phenotype) .

E2 (31-11 and 66.6-61.7 mu): its transcription is dependent on E1A (although the protein E1A is not a DBP, "DNA binding Protein", protein which binds to DNA). This gene codes, among other things, for a DNA polymerase of 140 kDa, a
DBP of 58 kDa and TP ("Terminal Protein"), whose role has already been mentioned above for the efficiency of viral replication (precursor of 80 kDa; mature protein of 55 kDa).

 E3 (76.6-86 m. U.): Gene not essential for viral multiplication in vitro. Its role in vivo would be to protect the virion from the host's immune defenses. It codes for several proteins, including a 19 kDa protein which blocks at the endoplasmic reticulum the exit of type I antigens from the MHC (Major Histocompatibility Complex), and also a 14.7 kDa protein which protects the infected cell. lytic action of TNF ("Tumor Necrosis Factor").

 E4 (99-91.3 mu): gene which codes for proteins involved in the regulation of late genes, and in stopping the synthesis of cellular proteins, thus allowing the diversion of the possibilities of cell biosynthesis in favor of virus.

 The distinction between early genes and late genes indicates a predominance of certain genes at some point in the replication cycle of the virus. A so-called early gene, such as E1A for example, can express itself throughout the viral cycle, or a late gene, such as the Major Late Promoter, can express itself, albeit weakly, from the first hours following infection.

Late genes
There are five of these: L1 to L5, coding for the various structural proteins (mainly capsid): L2 for the penton, L3 for the hexon, L5 for the fiber, etc.

All these transcripts start from the same promoter, the MLP (for
Major Late Promoter), and all have the same 5 'end (TPL: "TriPartite Leader"), made of the first three exons: 1, 2 and 3. These RNAs are produced by alternative splicing. Their transcription increases by 1,000 during the late replication phase of the virion. This extraordinary increase in transcription carried out by the MLP makes it a privileged promoter in any construction of recombinant adenovirus for its efficiency.

It should be noted in this late phase, the transcription of the protein gene
IX, transcriptional unit just 3 'from E1B, the synthesis of which is essential for the assembly of the virion (entry of DNA into the capsid).

 It is possible to subclone adenovirus fragments into plasmids, and by cotransfection of overlapping fragments representing the whole genome, by homologous recombination, to obtain recombinants reproducing the original virion.

To succeed, two minimum constraints must be met in the constructions: i) a fragment must have a terminal repetition (ITR, necessary for the replication of the virus), and ii) the two fragments to be joined must have at least one unit (360 to 440 nucleotides) to allow homologous recombination. Two circular plasmids, each carrying two complementary fragments, having at least 400 to 500 nucleotides in common, which together represent the entire genome, can thus recombine to produce recombinant adenoviruses.

 The deletion in the E3 region, not essential for in vitro replication, can allow insertions up to 4 kb. Finally, the deletion of the El region can, with the previous ones, allow, a priori, insertions up to 7 kb.

 Four insertion sites are possible 1) after MLP, 2) in E1, 3) in E3 or 4) to the right of E4, about 150 nucleotides from the right ITR (between E4 and ITR). Most often the insertion is done in the El region, with conservation of the 50 terminal bases which represent the left origin of DNA replication, of the packaging signal (between 194 and 358 nucleotides) and of the sequence on the right. of E1B which codes for protein IX allowing the adjustment of DNA at the capsid. In the latter case, the functions of E1A and E1B are provided in trans in type 293 lines in which the E1A and E1B genes have been transfected and are expressed permanently. The recombinant adenoviral construct is then in turn transfected into such transcomplementing lines and the recombinant virion can then be produced in such lines. The 293 line can be called helper line by analogy with what happens for retroviruses, the principle remains the same, providing in trans genes that have been previously removed to make room for the exogenous genes that we wants to express in its viral construction.

Two possibilities of vectors exist:
i) Replicative or ii) non-replicative (defective). A priori, the first case is more favorable allowing on the one hand the multiplication, therefore more numerous copies of the gene to be expressed. On the other hand, it is known that the intervention of cis elements of replication promotes the transcription of late genes, thus, if the gene to be expressed is dependent on the MLP promoter, this also promotes the transcription of the latter.

Choice of promoters:
Usually, for a gene inserted in the E3 region, the promoter of
E3, which may also include the MLP promoter. For a gene inserted in the region
El, one can choose either a completely different promoter, or more generally a combinatorial artificially recreated in this place: MLP + TLP ("tripartite leader" formed of three exons present in 5 'of all late transcripts), TLP allowing the recognition of adenoviral transcripts and their transfer from the nucleus to the cytoplasm, for their translation, transfer inhibited for all other cellular RNAs.

Translation control:
Viral RNAs are also preferentially translated, thanks to two
RNAs of the adenovirus transcribed by the RNA polymerase III of the host cell, called VAI and VAII (for "virus associated RNA"). These RNAs promote the translation of other viral RNAs by preventing the phosphorylation of the elongation factor elF2y, phosphorylation which inhibits the activity of the latter.

 The development of these adenoviral vectors started in 1977 with the establishment of a complementation cell line (helper cell) which contains the genes essential for viral replication (11% of the genome of human adenovirus type 5, mainly ElA and E1B genes, human kidney embryonic line, 293, GRAHAM, FL et al., 1977, J. Gen. Virol., 36, 59-72). The recombinant adenoviruses obtained, such as the recombinant retroviruses obtained from transcomplementing lines (Isolde type in avian, see Nigon, Verdier, Associated CNRS-TNRA, Lyon-Villeurbanne) are incapable of reproducing by themselves, but are capable infect the target cells and synthesize there the heterologous protein, coded by the gene introduced in place of El. If the introduced gene is placed in the E3 region, the transcomplementing line is not necessary, the recombinant vector retaining all autonomous replication power.

 The term “non-essential part of the genome of the avian adelovirus CELO into which a sequence coding for a heterologous polypeptide can be inserted, is meant in particular the regions of the genome in which this insertion does not prevent the replication of said recombinant adenovirus. The regions of the genome which after deletion do not prevent the replication of said recombinant adenovirus are also non-essential parts of the genome. Among these nonessential parts of the genome, we prefer the areas called non-coding intergene areas, which are naturally silent from a transcriptional point of view and where the insertion of a heterologous DNA sequence does not disturb the transcription of l either of the two strands of genomic DNA.

The deletion of these non-coding intergenic zones does not affect the replication of said recombinant adenovirus either.

 The term “essential parts of the genome of the avian adelovirus CELO” means the parts of said genome consisting of at least one strictly essential part of said genome and which can also comprise one or more non-essential parts of said genome. By strictly essential part of said genome is meant a part in which the insertion of a heterologous DNA sequence disturbs the transcription of one or other of the two strands of genomic DNA, no longer allowing the autonomous replication of said recombinant adenovirus. By strictly essential part of said genome is also meant the parts which are essential for the autonomous replication of said adenovirus, the deletion of at least one of said strictly essential parts preventing the autonomous replication of said recombinant adenovirus. These nonessential and essential parts, the discovery of which is the basis of the present invention, are described in the examples of the present description.

 The term “heterologous polypeptide” means any polypeptide or any protein, polypeptide and protein having the same meaning in the present description, not expressed naturally by the avian adenovirus CELO or any polypeptide whose coding nucleic sequence is not included in the genome of said adenovirus.

Among the heterologous polypeptides of interest whose nucleic sequence can be incorporated into the nonessential parts of the genome of the avian adelovirus CELO, there may be mentioned:
a) Antigenic polypeptides
Antigenic polypeptides are polypeptides corresponding to antigenic determinants of organisms against which it is desirable to generate immunity by cell mediation or by the production of antibodies. The antigenic determinants are preferably the antigenic determinants of an infectious agent, of the viral type or others, and which are responsible for infectious diseases in animals and in particular in avian species such as Marek's disease, infectious bronchitis, larynchotracheitis , gumboro, Newcastle disease, mycoplasma infections, chicken anemia, avian encephalomyelitis, avian influenza or Gumboro viral Fabricus bursa disease. It is also possible to use the recombinant CELO avian adenovirus as a vaccine vector for mammals by substituting the original fibers (MICHAEL, SI, 1995, Gene
Therapy, 660-668).

 Among these infectious agents, particular preference is given to the infectious agents responsible for Marek's disease, infectious bronchitis, larynchotracheitis, gumboro or Newcastle disease.

 The present invention also includes the methods according to the invention in which the DNA sequence coding for a heterologous polypeptide of interest results from the combination of several DNA sequences each coding for a heterologous polypeptide of interest.

It is understood that the present invention also comprises the methods according to the invention in which several DNA sequences are inserted, each coding for a heterologous polypeptide of interest.
b) The polypeptides involved in metabolism
In another aspect, the invention also aims to produce in vivo, in particular in poultry, polypeptide factors involved in the metabolism of the animal infected by the recombinant avian adelovirus CELO. Among these polypeptides of interest, mention may, for example, be made of the polypeptides involved in the metabolism of fats such as A9 desaturase, acetyl coA carboxylase, lipase, etc., growth hormones or their inducing factor (such as GRF). or immunopotentiators such as cytokines, interleukins, etc.

 In the context of the present invention, the polypeptide of interest can be an intracellular, membrane polypeptide present on the surface of the host cell or secreted outside the host cell. Its nucleic acid sequence can therefore also comprise appropriate additional elements such as, for example, a sequence coding for a secretion signal, these signals being known to those skilled in the art.

The invention further relates to the use of the recombinant CELO avian adenovirus as a vector for expression of heterologous protein in the context of treatment by gene therapy applied to humans, the recombinant CELO avian adenovirus being in fact very unlikely to recombine with the human adenoviruses usually used. Among the polypeptides of interest which can be expressed in the context of gene therapy treatment applied to humans, very particularly preferred are those capable of inhibiting or delaying the establishment and / or the development of a genetic or acquired disease such as for example cystic fibrosis, hemophilia A or B, Duchenne or Becker's myopathy, cancer, AIDS or infectious diseases caused by a pathogenic organism. The following polypeptides of interest are particularly preferred.
- a cytokine and in particular an interleukin, an interferon, a tissue necrosis factor and a growth factor and in particular hematopoietic (G-CSF,
GM-CSF),
- a factor or cofactor involved in coagulation and in particular the factor
VIII, von Willebrand factor, antithrombin III, protein C, thrombin and hirudin
- an expression product of a suicide gene such as thimidine kinase of the virus
HSV (herpes virus) type 1,
- an ion channel activator or inhibitor,
- a polypeptide whose absence, modification or deregulation of expression is responsible for a genetic disease, such as the CFTR protein, dystrophin or minidystrophin, insulin, ADA (adenosine diaminose), glucocerebrosidase and phenylhydroxylase,
a polypeptide capable of inhibiting the initiation or progression of cancers, such as the expression products of tumor suppressor genes, for example the genes
P53,
- a polypeptide capable of stimulating an immune response or an antibody,
- a polypeptide capable of inhibiting a viral infection or its development, for example the antigenic epitopes of the virus in question or altered variants of viral proteins capable of entering into competition with the native viral proteins.

Furthermore, a polypeptide of interest in use in the present invention can also be a selection marker making it possible to select or identify the host cells transfected with a vector according to the invention. Mention may be made, for example, of the polypeptide encoded by the neo gene (neomycin) which confers resistance to the antibiotic.
G418, by the dhfr gene (dihydrofolate reductase), by the CAT gene (Chloramphenicol
Transferase) or by the gpt (xanthine phosphoribosyl) gene.

 The term “polypeptide of interest” also means the recombinant polypeptides of industrial or therapeutic interest which can be prepared either in vitro by culture of cells, in particular avian cells, transformed by the recombinant CELO adenovirus, or either in vivo in animals.

 In accordance with the aims pursued by the present invention, the nucleic acid sequence can code for a polypeptide of interest corresponding to all or part of a native protein as found in nature. It may also be a chimeric protein, for example originating from the fusion of polypeptides of various origins or a mutant exhibiting improved and / or modified biological properties. Such a mutant can be obtained by conventional biological techniques by substitution, deletion and / or addition of one or more amino acid residues.

 Part of the polypeptide is understood to mean a biologically active fragment of the polypeptide capable of fully or partially exercising the biological activity of the polypeptide from which it is derived.

 The invention also relates to a method according to the invention, characterized in that said DNA sequence further comprises the elements capable of ensuring the expression of said heterologous polypeptide in a cell infected with said recombinant adenovirus.

 The DNA sequence encoding a polypeptide of interest can be associated with a promoter or a leader sequence in order to express the DNA sequence with the best possible efficiency. Any promoter which makes it possible to express the DNA sequence efficiently can be used. Among the preferred promoters or leader sequences, mention may be made of those of human adenoviruses, of SV40 virus, of cytomegalovirus or of avian adenoviruses, particularly preferred is the MLP promoter of avian adenovirus CELO, the TPL leader sequence (here bipartite: L1 + L3).

The invention also relates to methods according to the invention, characterized in that the non-essential part corresponds to a non-coding intergenic zone of the avian adenovirus CELO, preferably said non-essential part is chosen from the following sites, identified by coordinates numbers (bp) relating to the published sequence of 43,804 bp (CHIOCCA, S. et al., 1996) and as defined in Figure 1:
a) between 600 and 650,
b) between 750 and 780,
c) between 3,380 and 3,520,
d) between 10,160 and 10,260,
e) between 12,160 and 12,180,
f) between 21,775 and 21,818,
g) between 26,650 and 27,060,
h) between 27,970 and 28,170,
i) between 30500 and 30510,
j) between 32,480 and 32,730,
k) between 33,720 and 33,880, I) between 36,210 and 36,340,
m) between 36,640 and 36,740,
n) between 37,220 and 37,380, o) between 38,280 and 38,650,
p) between 42,380 and 43,040.

The invention also includes methods according to the invention characterized in that they comprise a step of deletion of at least one non-essential part of the genome of the avian adenovirus CELO, preferably said non-essential part is chosen from among the parts following, identified in numerical coordinates (bp) relative to the sequence as defined in Figure 1
a) part between 1,970 and 2,850,
b) part between 1,970 and 3,000,
c) part between 3 130 and 3 520,
d) part between 33 210 and 33 450.

The invention also includes methods according to the invention, characterized in that they comprise a step of deletion of at least one essential part of the genome of the avian adenovirus CELO, preferably said essential part is chosen from the following parts, identified in numerical coordinates (bp) relative to the sequence as defined in Figure 1:
a) part between 300 and 5,300,
b) part between 10 170 and 11 280,
c) part between 15,100 and 17,560,
d) part between 15,100 and 21,800,
e) part between 16,050 and 17,540,
f) part between 16,050 and 21,800,
g) part between 16,050 and 23,170,
h) part between 17,540 and 21,800,
i) part between 21,800 and 23,170,
j) part between 23,180 and 27,960,
k) part between 23,670 and 27,050,
l) part between 24,800 and 27,060,
m) part between 27,100 and 31,790,
n) part between 28,100 and 31,800,
o) part between 35,620 and 36,440.

 The invention also relates to methods according to the invention, characterized in that the deletion step leads to the production of a recombinant CELO avian adenovirus comprising at least 2 nucleic acid sequences coding for an ITR and a sequence d nucleic acid coding for a T packaging signal

The preparation of such vectors, based in particular on the localization of the packaging signals of the avian adenovirus CELO, makes it possible to generate vectors with high capacity for incorporating heterologous DNA, safer and with reduced immunogenicity. Such vectors are especially preferred as a heterologous gene transfer vector in mammalian cells in the context of treatment by gene therapy. The principle of obtaining such vectors is known to those skilled in the art (PARKS, RJ et al., 1996, PNAS, 93, 13565-13570, WANG, P. et al., 1995, Somatic Cell and Mol Gen. 21, 6, 429-441). The principle is based on Cre-recombinase which recognizes the sequence
LoxP and allows excision of any gene between 2 LoxP sites. Recombinant defective adenovirus vectors are thus created, which can be reduced to their simplest expression (2 ITR (inverted terminal repeat) and an encapsidation signal w), which in the case of the avian adelovirus CELO, leaves room for approximately 43 kb of exogenous DNA.

The principle is described opposite. An LMH line (Kawaguchi T., Nomura K.,
Hirayama Y., Kitagawa T. (1987): Establishment and characterization of a chicken hepatocellular carcinoma cell line, LMH. Cancer Research, 47, pp. 4460-4464) is obtained with Cre-recombinase by conventional recombinant retrovirus, the helper virus having on both sides of the packaging site g LoxP sequences.

The recombinant virion is then deleted from the chosen number of non-essential and essential parts of the genome with the exception of the following elements: the two terminal ITRs and the packaging sequence (the first allowing replication of the recombinant avian adenovirus and the second sequence allowing the encapsulation of said recombinant adenovirus).

The presence of two LoxP sites on either side of the packaging sequence in the helper virus prevents it from being packaged in the transcomplementing line
LMH / Cre-recombinase. The helper virus is, apart from the LoxP sites, identical to the wild-type adelovirus CELO.

 Such constructions are obtained with the ppoly II plasmid / CELO ends (Figures 2, 3 and 4), as well as with the CELO / rec plasmid.

 The transcomplementing cell lines necessary for the replication of defective CELO avian adenoviruses, obtained by the preparation methods according to the invention, make it possible to construct safer recombinant helper viruses.

 In this way, it is possible to envisage producing a recombinant avian adelovirus CELO carrying genes of vaccine interest, in a configuration identical to wild genes. promoters, leaders, splices, etc., in order to have maximum efficiency.

 The invention also relates to methods according to the invention, characterized in that the DNA sequence codes for a polypeptide of interest, preferably an antigenic polypeptide corresponding to an antigenic determinant of an infectious agent responsible for infectious diseases in animals, especially in avian species.

 Among the infectious diseases, Marek's disease, infectious bronchitis, larynchotracheitis, gumboro or Newcastle disease are preferred.

 The invention also includes methods according to the invention, characterized in that the polypeptide of interest is a polypeptide intervening in animal, preferably avian, or human metabolism.

 Also forming part of the invention are the vectors, characterized in that they comprise a recombinant CELO avian adenovirus obtained by a method according to the invention.

 The invention also relates to vaccines characterized in that they contain a vector according to the invention, in particular vaccines for the protection of avian species against infectious diseases.

 A vaccine according to the invention can be manufactured in a conventional manner. In particular, a therapeutically effective amount of such a vector is associated with an acceptable support, diluent or adjuvant. It can be administered by any route of administration and this in a single or repeated dose after a certain period of interval. Nebulization, aerosol spraying or administration via solid or liquid food are the preferred modes of administration, in particular for avian species. The amount to be administered will be chosen according to certain criteria, in particular. the route of administration, the animal, the type of disease to be treated and its state of evolution, the duration of the treatment, etc. As a guide, a vaccine according to the invention comprises between 1011 and 1014 of adenoviral particles / ml, preferably between 109 and 1012 pfu / ml (plate forming unit).

 The invention also relates to cells transformed with a vector according to the invention. Among said cells, cells characterized in that they are eukaryotic cells allowing replication of a defective recombinant avian CELO avian adenovirus are preferred.

 The eukaryotic cells allowing the replication of a defective recombinant avian adelovirus CELO, that is to say normally incapable of replicating, are called transcomplementing cells. These cells make it possible to obtain sufficiently high titers of defective recombinant avian CELO adenovirus having integrated the DNA coding for the heterologous polypeptide of interest. The methods for obtaining such transcomplementing cells are known to those skilled in the art and are based in particular on knowledge of the essential and non-essential parts of the genome of the virus which it is desired to replicate.

 The defective recombinant adenovirus is a vector capable of triggering an initial immune reaction when it enters the target cell, without the possibility of reaching other neighboring cells. The vector is harmless, since it is more or less deleted, and incapable of dissemination in animals and between animals. In addition to the harmlessness of this GMO (genetically modified organism), more and more space can be freed, even all of the virus genes to provide maximum exogenous information (gutless virus, disoyoyed virus and Cre-recombinase ). This is an advantage that can more than offset the disadvantages of non-autonomous replication. In addition, in vaccination, the effectiveness of the first vaccination is essential, but it is not necessarily necessary to increase the number of injections.

Among eukaryotic cells allowing the replication of an avian adenovirus
Defective recombinant CELO, transcomplementing cell lines of avian origin, type LMH or QT6 are preferred (ATCC CRL 1708-fibrosarcoma-Moscovici et al.

1977).

 Preference is also given to transcomplementing cell lines of avian origin such as the LMH or QT6 line or those whose immortalization is made at the base by the T antigen of SV40 (Institut Mérieux). Such transcomplementing cell lines of avian origin preferably require the use of avian retroviruses making it possible to obtain avian CELOtranscomplementing lines.

 Avian retroviruses can carry 3 to 7 kb of exogenous DNA. These retroviruses easily infect LMH lines (possibly carrying the avian retrovirus receptor to facilitate their infectability) and integrate into the genome of the host cell. Transcomplementing cells are obtained quickly and then selected for their efficiency in producing the genes of the avian adenovirus CELO.

 The invention furthermore comprises methods for the in vivo preparation of a polypeptide of recombinant interest, characterized in that they involve the infection of an animal by a vector according to the invention, vector of which said sequence d DNA inserted codes for said polypeptide of interest.

 The invention finally relates to methods for preparing a recombinant polypeptide of interest, characterized in that they implement the culture of cells according to the invention.

 Techniques for in vivo or in vitro transformation of cells from recombinant adenovirus vectors aimed at preparing recombinant polypeptides are well known to those skilled in the art and will not be described.

 Other characteristics and advantages of the invention appear in the following description with the examples and the figures, the legends of which are shown below.

Legends of the figures Figure 1: complete nucleotide sequence of the CELO viral genome, Figure 2: construction of the ppoly II plasmid / CELO ends; . Figure 3: Construction of the recombinant ppoly II / CELO plasmid by recombination
homolog between the total DNA of the CELO virus and the ppoly II plasmid / ends
CELO; . Figure 4: ppoly II plasmid: CELO recombinant with its 4 constituent fragments A, B, CetD; . Figure 5: potential sites of insertion or deletion on the viral genome of
the CELO avian adenovirus,. Figure 6: principle of homologous recombination between two plasmids for the
creation of recombinant CELO adenoviruses; . Figure 7: the four plasmids A, B, C and D representing the entire genome of the
CELO and allowing the construction of recombinants (deleted and / or by insertion) by
under successive cloning; . Figure 8: diagram of the four plasmids A, B, C and D as well as the ppoly plasmid
It / recombinant CELO carrying the entire CELO viral genome; Figure 9: creation of transcomplementing lines for late CELO genes. Figure 10: Use of transcomplementing lines for the manufacture of viruses
CELO recombinant vaccines; . Figure 11: creation of recombinant gutless CELO through CRE
recombinase.

EXAMPLES
Material and methods
Cell cultures and infections
Chicken kidney embryo cells (EOPS embryos) of 19 days are cultured in 2. cultured in 75 cm 2 flasks at the rate of 106 cells / ml in 20 ml of BHK21 / 5% FCS medium (fetal calf serum) at 37 "C and 2% CO2.

A day later, the cells are washed and infected with 20 pfu 5PLAGES
FORMING UNIT2SO per cell in a minimum volume (= 4 ml) for 1 hour at 37 "C 2% CO2. The medium is then adjusted to 20 ml.

Extraction of total RNAs
The RNAnow protocol with phenol from Biogentex was followed. RNAnow is used at a rate of 1 ml / 10 cm2 (or 1 to 5,106 cells). The homogenate is coupled to 0.2 volumes of chloroform. The RNAs are precipitated in the presence of isopropanol (V / V) then washed with 70% ethanol and taken up in Tris-EDTA 10: 0.1 (RNAse free). Each extraction is followed by an analysis on agarose gel of the quality of the RNAs.

Construction of cDNA libraries
The first library is constructed in the plasmid pSPORTl from the kit
Life's SuperScript Plasmid System for cDNA synthesis and plasmid cloning
Technologies. CDNAs with oligo primers (dT) associated with primersaptors with four restriction sites SpeI, XbaI, NruI, NotI, in the presence of
SuperScript II RT have been synthesized. The second strand is synthesized using the E. coli DNApol + RNAse H + DNA ligase cocktail at 16 "C. A SalI adapter is attached to the ends after treatment with T4 DNApol. The cDNAs are then digested by
NotI and subcloned into SPORT1 by SalI / NotI.

 The second library is constructed in the plasmid pBluescript BS / KS + (Stratagene). The first strand is synthesized in the presence of oligo (dT) primers 15 and hexanucleotides. The second strand is made according to the previous protocol. An EcoRi / NotI adapter is attached to the ends of the cDNAs which are then subcloned into pBS / KS + without orientation.

 The plasmids are then amplified in competent XL1 Blue bacteria.

Screening of banks
Boehringer's DIG high Prime DNA labeling and detection starter kit II was used to detect positive clones in XLlblue. The probe was produced by labeling total CELO DNA digested with Hind III or SalI with digoxige-nine-dUTP.

The clones are transferred and fixed on special nylon membranes for colonies (Boehringer). The hybridization and detection steps are carried out under the conditions described by the manufacturer. The hybridization is done in a 50% formamide buffer at a temperature of 42 "C. overnight. The revelation is made by chemiluminiscence. The clones identified positive on the autoradiographs are re-screened a second time. The positive disorders are amplified then sequenced.

Sequence analysis
The nucleic sequences are analyzed using MacDNASIS version 3.5 Hitachi Software by comparison with the sequence of CELO (Chiocca, 1996, u46933) shown in Figure 1.

Kinetics by RT-PCR (reverse transcriptase and PCR)
An RNA-PCR is carried out with a treatment prior to DNase I. First, the cDNAs are synthesized in a reaction of 20 μl (Huang, 1996) containing for 1 μg of RNA: 1 x PCR buffer II (Boehringer), 5 mM MgCl2, 1 mM dNTP mix, 1U DNAseI 20U RNase inhibitor, 2.5 UM oligo (dt) 15, 1 I1M hexanucleotides. The DNAseI reaction is placed for 2 hours at 37 "C and then 5 minutes at 75" C to destroy the enzyme. A microliter of 50U / ll murine leukemia virus (MuLV) reverse transcriptase is added followed by a cycle of 42 "C for 30 minutes of RT, 5 minutes at 90" C and a return to 4 "C. negative controls the reverse transcriptase is replaced by 2 μl of H2O RNase free. Two microliters of this stock diluted at 1:30 are added to 23 μl of PCR reaction, for a final volume of 25 μl containing: 1 x PCR buffer II , 2 mM MgC12, 25 pmol of each primer, 1 mM dNTP mix, and 0.5U Taq polymerase (Boehringer) The amplification conditions vary according to the primers chosen.The number of cycles varies between 25 and 34.

5 'race
The Clontech Marathon cDNA amplification kit was used. First, single-stranded cDNAs from 1 .mu.g of RNA extracted from infected cells were produced. Oligo (dT) primers ligated to an EcoRi-NotI adapter and MuLV reverse transcriptase were used. After the synthesis of the second strand, the cDNAs are made blunt-ended by means of T4DBA polymerase. A NotI-SfrI adapter
Specific XmaI is attached to the ends. Amplifications were then carried out by
PCR (polymerase chain reaction) with an AP 1 sense primer specific for this adapter and an antisense primer chosen 5 'from our ORFs (generally in the ATG region). The amplification products are then sequenced and analyzed on
MacDNasis.

Example 1
The genomic DNA of the avian adenovirus CELO (serotype 1), a double-stranded DNA virus, is completely sequenced (cf. FIG. 1). The DNA is obtained after culture on embryonic SPF (free of specific pathogen) eggs and harvested a few days after infection with allantoic fluid. It is purified by conventional virus purification protocols. Approximately 300 μg of viral DNA (approximately 150 infected embryonic eggs) are isolated for one liter of infected allantoic fluid. The DNA of the CELO avian adenovirus (serotype 1) thus comprises 43,804 bp and codes for at least forty genes.

 The transcription of its genome is studied in detail. For this, a cell culture model infected in vitro with the avian adenovirus CELO (embryonic chicken kidney cells S.P.F.) is used. The messenger RNAs produced by these infected cells are extracted at various post-infection times and used to build cDNA libraries. The screening is then carried out using a digoxygenin hybridization kit. Under these conditions, a transcriptional model is established for the avian adenovirus CELO.

Various promoter zones have been localized, mainly for genes
ORF1, ORF2 (promoter located around 350-510), late gene pools with the major late promoter (major late promoter: MLP, located around 7 310 - 7 520) and its two leaders L1 and L3 (located respectively at 7 533 - 7 545 (22 bp); and 11,285 - 1,413 (129 bp), for the ORF22 gene (promoter around 43,400 - 43,700), for ORF18 and 19 genes (promoter around 36,460 - 36,770)
The precise knowledge of the splicing of messenger RNAs deduced from the transcriptional model makes it possible to locate the nonessential and essential parts of the genome of the avian adelovirus CELO and to predict the zones of insertion of exogenous DNA and / or deletion of adenoviral DNA. . This precise knowledge makes it possible to establish the methods for preparing the following recombinant avian adenoviral vectors
A) Insertions, replicating recombinant CELO avian adenovirus (cf. FIG. 5).

 The results obtained previously on the transcription make it possible to determine the putative sites of insertion of exogenous DNA (of 2.5 kb maximum). The following sites are identified in numerical coordinates (bp) relative to the sequence in Figure 1.

1 / to ORFI: between 600 and 650,
between 750 and 780, no disturbance on the other strand,
2 / towards ORF3: between 3,380 and 3,520, no disturbance for the transcription of ORF3,
3 / after DNA polymerase: between 10,160 and 10,260 (possibly avoiding a poly A signal),
4 / between pTP and 52K: 12,160 to 12,180,
5 / between the two polyadenylation signals and DBP: 21,775 to 21,818,
6 / between the stop codon 100K and the reinitiation of pVIII (respecting the leader of ORF12): 26,650 to 27,060,
7 / between poly A pVIII (27,970) and start of fiber 1 initiation (28,170),
8 / between 30,500 and 30,510 (between the two fibers; antisense with respect to the two fibers), 9 / between OFR22 start and ORF21 stop: 32,480 to 32,730,
10 / between ORF2O initiation: 33 720 to 33 880,
11 / between a / 36 210 and 36 340: area not transcribed,
b / 36640 and 36740,
12 / between end of ORF7 and start of ORF8: 37,220 to 37,380,
13 / between 38,280 to 38,650; nothing transcribed in this area,
14 / between 42,380 to 43,040.

 If it is assumed that the encapsidation capacity is 105%, for the avian adenovirus CELO of 43,804 kb, it is possible to insert a maximum of 2.4 kb of exogenous DNA, either a gene of vaccinating or therapeutic interest, or an enzyme involved in a metabolic pathway and approximately 400 bp of a specific promoter (placed in the direction or antisense of the natural transcription in this zone).

The incorporation of this exogenous DNA is done according to methods well known to those skilled in the art. We can quote for example:
a) or by homologous recombination (cf. FIG. 6): the entire genome of the adelovirus CELO was subcloned into a plasmid by this type of method (CHARTRIER, C. et al., 1996, J. of Virology, 70 , 7, 4805-4810). Likewise, the exogenous DNA is inserted at the chosen site, taking into account the unique restriction sites around the insertion zone (CHIOCCA, S. et al., 1996);
b) or by conventional construction: from plasmids carrying the insertion region, then reintegration in successive stages in increasingly large plasmids. The last step consists in replacing the wild fragment in the plasmid carrying the entire genome of the avian adenovirus CELO with the new one. This route is also possible from 4 plasmids which represent the entire genome of the avian adelovirus CELO divided into 4 fragments (cf. Figures 7 and 8):
fragment A (Pacl / Pmel): 7,430 bp
fragment B (Pmel / Notl): 9,956 bp fragment C (Notl / Fsel) 18,298 bp
fragment D (Fsel / Pacl): 8,116 bp,
allowing each time, whatever the insertion site, to reconstruct artificially
the desired recombinant adenovirus.

 The M zone to be modified is surrounded by two unique restriction sites in the fragment: X and Y.

 The XY subfragment is subcloned into a plasmid and rectified by mutation, deletion or insertion.

 After the modification XY is reintegrated into pBS / KS + fragment A.

 Possibility of homologous recombination with the plasmid ppolyII / celo rec which contains the entire CELO genome (cf. Figures 2, 3 and 4).

 Possibility to replace fragment A of ppolyII / celo rec with modified fragment A.

 All of these insertion cases are based on the results of the previous transcription study of the avian adenovirus CELO genome.

 The promoter used for the exogenous construction is chosen from promoters strong enough to express the exogenous DNA. The recombinant adenovirus remaining replicated

1 / Elimination of ORF2: from 1,970 to 2,850 (880 bps freed)
This deletion makes it possible with a 5% increase in size to theoretically have approximately 3 kb for an insertion); this gene on the up strand has no counterpart on the down strand. It seems to be one of the preferred positions for deletions, even if the polyadenylation signals for pTP, DNA pol. and IV a2 can pass through this area. We would only increase the size of the corresponding messenger RNAs in their 3 'untranslated part.

 2 / Elimination of ORF6: this area is more critical, because it includes the leader of ORF22 (from 33,070 to 33,204: 135 bp) to be respected so that its transcription is accomplished normally (important gene, because it belongs to the early genes appearing as early as 4 hours post-infection). There is also the termination zone of ORF2O, and part of ORF5 (in its coding part, nevertheless this last gene has never been demonstrated with the various technical means used until now). Taking these various elements into account, we can delete the area between 33,210 and 33,450 (i.e. 240 bp, which, along with the increase in size, allows the addition of approximately 2.5 kb of exogenous DNA) .

 3 / ORF14: this gene located on the down strand was not detected by our techniques (cDNA banks and RT-PCR). We could thus remove an area ranging from 1,970 to 3,520 (1,550 bp). However, it seems that the 3,000 to 3,120 zone serves as a promoter for ORF3. Taking into account the deletion on ORF2, we can therefore delete two zones: from 1,970 to 3,000 and from 3,130 to 3,520, freeing up 1,030 bp and 390 bp respectively.

C) Transcomplementing lines and defective virus, Cre-recombinase system
With these techniques well known to those skilled in the art (see for example WANG,
QJX et al., 1995, Gene Therapie, 2, 775-783 and ZHOU, B., 1996, J. Virol., 70, 70307038) and knowledge of the location of essential and non-essential parts on the genome of the CELO avian adenovirus, it is possible to obtain defective recombinant avian CELO adenovirus vectors, of sufficient titre, with high capacity for insertion of exogenous DNA up to 43 kb, in particular by the Crerecombinase method of gene excision.

 The genes to be replaced in the transcomplementing cells are more or less long, depending on the deletion chosen and the length of the exogenous DNA to be inserted. FIG. 10 shows as an example the general diagram of the process for the preparation of recombinant defective CELO vaccine vectors from Isolde-type retroviral transcomplementing cells, the replication of the vaccine vector being ensured by the LMH cell line, previously transformed by the retrovirus, after transfection by the recombinant vaccine vector.

 We can cite the example below, as an example of deletion of essential parts requiring the use of transcomplementing cells to obtain replication of the recombinant CELO avian adenovirus.

Deletion of ORF7: it is then necessary to supplement with the products of the ORF 18 and ORF 19 genes located on the opposite strand (strand down) highlighted in the bank
CDNA and RT-PCR (very early to early genes from 2 hours and 4 hours after injection and whose role is therefore essential for the viral cycle. The suppression of ORF7 can be done from 35,620 to 36,440 ( i.e. 820 bp freed up; with the size increase of 5%, possibility of insertion of approximately 3 kb).

Examples of transcomplementing lineage
a) pTP transcomplementing line
PTP binds to the ends of the DNA of the avian adenovirus genome
CELO (or any other recombinant carrier of ITR at its ends) and by its presence promotes replication of the viral matrix, by promoting the binding of DNA polymerase and DBP (DNA Binding Protein, vital for elongation with pol DNA from the viral matrix). This line can therefore in this way promote the production of the recombinant CELO avian adenovirus constructs to provide very high titers. In the retrovirus carrying pTP, it can be placed under the control of an inducible promoter (heat-shock promoter, hormone-dependent or dependent tetracycline; in the latter case, the promoter will function in the presence of tetracycline in the medium) . We have shown that pTP uses the leader of ORFî2, and the leader y of DBP, so we can make an appropriate construction to have, with these elements, maximum production efficiency of pTP. In this way, the genomic DNA of the avian adenovirus CELO is released from 10,170 to 11,280, or 1.1 kb, in order not to touch leader 3 used by the late genes. However, avian adenovirus
Recombinant CELO can use the entire coding part of pTP (from 10,170 to 12,050 or 1,880 bp), provided that leader 3 is reinserted into the construction of the recombinant so that late genes can express themselves without problem.

DBP transcomplementing line:
The exact knowledge of the transcription of the DBP allows to consider 3 possible constructions: the ORF DBP alone, the ORF DBP + leaders g and 7 and, the ORF DBP with all its leaders: a, ss, e and y . The stability of the corresponding mRNA could be higher with the leaders. In all cases, we release the corresponding zone in the
DBP extending (on the donated strand) from: 21,800 to 23,170, i.e. a place of 1.37 kb on the recombinant avian adelovirus CELO and this without affecting any other gene (just between EP endopeptidase and 100 K) .

Transcomplementing lines and late genes
Late units are completely transcomplemented using retroviruses, the constructions of which are shown according to FIG. 9 the diagrams opposite. L2 entities, or
L3, or L2 + L3, even L2 + L3 + DBP

 - In the first case: entity L2 (penton at pX) 16,050 to 17,540, release of approximately 1.5 kb.

 - In the second case: entities L2 + L3 (penton to E.P.) 16,050 to 21,800, release of approximately 5 kb.

 - In the third case: L2 + L3 + DBP entities 16 050 to 23 170, release of approximately 7 kb.

 (In fact, from the actual start of the penton around 15,100 to E.P.: 21,800, there are 6,700 bp, or about 7 kb). The L2 entity (15,100 to 17,560) is 2,460 bp, the L3 entity (17,540 to 21,800) is 4,260 bp.

Similarly, according to the same diagrams, the following entities can be deleted: L4 (pVIII, 100 K, between ORF12 and 22: 27 100 and 31 790: 4 690 bp). L5 (fibers 1 and 2) and L4 + L5, according to diagrams equivalent to those used previously
100 K early 23 180 to 27 960 (late pVIII) 4 780 bp.

 Fiber 1, start: 28,100 to 31,800 (end of fiber 2): 3,700 bp, resulting in a total release of 8,480 bp.

Type 293 transcomplementing lines
Like human adenoviruses with the immortalizing genes E1A and E1B, there may be equivalents of these genes in the avian adelovirus CELO. there would therefore be an immortalizing gene equivalent to El A, promoting cell multiplication (to be sought among very early genes) and a gene preventing apoptosis as does ElB. According to a recent publication (CHIOCCA, S. et al, 1997, J. Virol., 71, 31683177), it is known that it is ORF8 (gene with anti-apoptosis effect, of type Bc12 in its mode of action).

 - Among the very early candidate genes, there are: ORF4, ORF5, ORF16, ORF18, ORF19, ORF20 and ORF21. ORF4 and ORF18 seem to be the most relevant for this.

We test the combinations ORF8 + ORF4 (most relevant combination), ORF8 + ORF4, ORF8 + ORF18, ORF8 + ORF19, ORF8 + ORF5, ORF8 + ORF16, ORF8 +
ORF20 and ORF8 + ORF21, on embryonic liver or chicken kidney cells. Then we retain the combination allowing to obtain immortalization which offers the possibility of having a system equivalent to the line 293 of GRAHAM (GRAHAM, FL et al., 1977, J. Gen. Virol., 36, 59- 72, SHAAK, O., 1995, J. Virol., 69, 4079-4085).

Other specific transcomplementing lines:
100 K: releases taking into account the leader ss of the ORF12: 24,800 to 27,060, or approximately 2.2 kb.

 By removing all of the 100 K 23 670 to 27 050, approximately 3.38 kb are released, provided that artificially recreate the leader ss of ORF12 (27 068 to 27 095 28 bp) to obtain a functional transcription of this last reading frame.

Left side of the Celo: from 300 to 5,300, approximately 5 kb freed up, complementing for ORFs 1, 2, 3, 14 and 15 with the obligation to recreate a promoter for ORF4 in construction and for ORF15, detected as early at 4 a.m. after RT injection
PCR).

Cre-recombinase system transcomplementing lines
FIG. 11 represents as an example the general diagram of the process for the preparation of recombinant defective CELO vaccine vectors according to the Crerecombinase system, using retroviral isovere-type transcomplementing cells. Replication or production of the vaccine vector is ensured by the LMH cell line, previously transformed by a recombinant Cre-recombinase retrovirus, after cotransfection with the recombinant vaccine vector and an assistant adenovirus.

Claims (21)

 CLAIMS 1. Method for preparing a recombinant CELO avian adenovirus, characterized in that one inserts into a nonessential part of its genome a DNA sequence coding for all or part of a polypeptide heterologous to the avian CELO adenovirus . 2. Method according to claim 1, characterized in that said DNA sequence further comprises the elements capable of ensuring the expression of said heterologous polypeptide in a cell infected with said recombinant adenovirus. 3. Method according to one of claims 1 to 2, characterized in that the non-essential part corresponds to a non-coding intergenic zone of the avian adenovirus CELO. 4. Method according to one of claims 1 to 3, characterized in that the non-essential part corresponds to an area chosen from the following sites, identified in numerical coordinates (bp) relating to the sequence as defined in Figure 1  a) between 600 and 650,  b) between 750 and 780,  c) between 3380 and 3520,  d) between 10,160 and 10,260,  e) between 12,160 and 12,180,  f) between 21,775 and 21,818,  g) between 26,650 and 27,060,  h) between 27,970 and 28,170, i) between 30,500 and 30,510,  j) between 32480 and 32730,  k) between 33,720 and 33,880,  l) between 36,210 and 36,340,  m) between 36,640 and 36,740, n) between 37,220 and 37,380,  o) between 38,280 and 38,650,  p) between 42,380 and 43,040. 5. Method according to one of claims 1 to 4, characterized in that it further comprises a step of deletion of at least a non-essential part of the genome of the avian adelovirus CELO. 6. Method according to claim 5, characterized in that said non-essential part is chosen from the following parts, identified in numerical coordinates (pb) relating to the sequence as defined in FIG. 1:  a) part between 1,970 and 2,850,  b) part between 1,970 and 3,000,  c) part between 3 130 and 3 520,  d) part between 33 210 and 33 450. 7. Method according to one of claims 1 to 6, characterized in that it further comprises a step of deletion of at least an essential part of the genome of the avian adelovirus CELO. 8. Method according to claim 7, characterized in that said essential part of the genome of the avian adenovirus CELO is chosen from the following parts, identified in numerical coordinates (bp) relating to the sequence as defined in FIG. 1:  a) part between 300 and 5,300,  b) part between 10 170 and 11 280,  c) part between 15,100 and 17,560,  d) part between 15,100 and 21,800,  e) part between 16,050 and 17,540,  f) part between 16,050 and 21,800,  g) part between 16,050 and 23,170,  h) part between 17,540 and 21,800,  i) part between 21,800 and 23,170,  j) part between 23,180 and 27,960,  k) part between 23,670 and 27,050,  1) part between 24,800 and 27,060,  m) part between 27,100 and 31,790,  n) part between 28,100 and 31,800,  o) part between 35,620 and 36,440. 9. Method according to claim 7, characterized in that the deletion step leads to the production of a recombinant CELO avian adenovirus comprising at least 2 nucleic acid sequences coding for an ITR and a nucleic acid sequence coding for a packaging signal T. 10. Method according to one of claims 1 to 9, characterized in that the DNA sequence codes for a heterologous polypeptide of interest. 11. Method according to claim 10, characterized in that the heterologous polypeptide of interest is an antigenic polypeptide. 12. Method according to claim 11, characterized in that the antigenic polypeptide is an antigenic determinant of an infectious agent responsible for an infectious disease in animals, preferably in avian species. 13. Method according to claim 12, characterized in that the infectious disease is chosen from Marek's disease, infectious bronchitis, larynchotracheitis, gumboro or Newcastle disease. 14. Method according to claim 10, characterized in that the heterologous polypeptide of interest is a polypeptide involved in metabolism, preferably avian or human. 15. Vector, characterized in that it comprises a recombinant CELO avian adenovirus obtained by a method according to one of claims 1 to 14. 16. Vaccine characterized in that it contains a vector according to claim 15. 17. Vaccine according to claim 16, for the protection of avian species against infectious diseases. 18. Cell transformed by a vector according to claim 15. 19. Cell according to claim 18, characterized in that it is a eukaryotic cell allowing the replication of a defective recombinant CELO avian adenovirus. 20. A process for the in vivo preparation of a polypeptide of recombinant interest, characterized in that it implements the infection of an animal by a vector according to claim 15, of which said inserted DNA sequence codes for said polypeptide of interest, and in that said polypeptide of interest is then extracted from said animal. 21. A method of preparing a recombinant polypeptide of interest, characterized in that it implements the cell culture according to claim 18.