WO1999009194A1 - Adenovirus aviaire celo recombinant et son utilisation comme vecteur vaccinant - Google Patents
Adenovirus aviaire celo recombinant et son utilisation comme vecteur vaccinant Download PDFInfo
- Publication number
- WO1999009194A1 WO1999009194A1 PCT/FR1998/001803 FR9801803W WO9909194A1 WO 1999009194 A1 WO1999009194 A1 WO 1999009194A1 FR 9801803 W FR9801803 W FR 9801803W WO 9909194 A1 WO9909194 A1 WO 9909194A1
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- WO
- WIPO (PCT)
- Prior art keywords
- celo
- avian
- recombinant
- polypeptide
- adenovirus
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10211—Aviadenovirus, e.g. fowl adenovirus A
- C12N2710/10241—Use of virus, viral particle or viral elements as a vector
- C12N2710/10243—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10211—Aviadenovirus, e.g. fowl adenovirus A
- C12N2710/10251—Methods of production or purification of viral material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2840/00—Vectors comprising a special translation-regulating system
- C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
- C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
Definitions
- the invention relates to methods for the preparation of recombinant CliLO 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 of heterologous proteins involved in metabolism.
- the presentation of an effective and durable 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).
- Herpes viruses are double-stranded DNA viruses with capsids and envelopes, and are, for example, responsible for two important diseases: Aujesky's disease in pigs and Marck'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 ⁇ RN viruses which, as recombinant vectors, are essentially studied and used for gene transfer in mammals, human gene therapy, or in avian species.
- Adenovi s 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 which divide little, such as lung cells or muscle cells.
- this virus multiplies very easily, which allows the production of recombinant viruses at very high titers (10 11 to 10 13 viruses per milliliter), much higher than those of recombinant retroviruses and therefore to obtain a better rate of genetic transformation of 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.
- the nebulization or aerosol spraying of the recombinant virions associated with strong titles, should allow mass vaccinations, particularly interesting for avian species.
- 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.
- 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 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 which is 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.
- 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.
- the subject of the invention is processes for the preparation of recombinant polypeptides of interest, characterized in that they use the culture of cells transformed 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 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.
- the adenovirus family is divided into two groups: Mastadenoviradea, isolated from mammalian cells and Aviadenoviradea, isolated from bird cells.
- the genus Aviadenovirus is itself divided into 5 species groups including FAV (“Fowl Adeno Virus”) poultry adenoviruses.
- FAV Flural Adeno Virus
- the CELO avian adenovirus (“Chickcn Embryo Lethal Orphan") represents serotype 1 (FAV-1) among the 1 1 distinct serotypes of FAV recognized.
- the CELO adenovirus is a 43,804 bp long double-stranded DNA virus (CHIOCCA, S., et al.) ( Figure I). Its capsid is made up of elements of 3 types: hexons, pentons and free (proteins II, III, IV respectively according to their increasingly low molecular weight). Antigenic motifs (neutralizing epitopes) are carried mainly by hexons and fibers. The free is fixed by its NH 2 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 preventing lysc by the endosomes, as normally occurs for any infectious agent picked up by endocytosis by the cell.
- the CELO adenovirus 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).
- Each adenovirus consists of double stranded DNA, terminated by reverse repetitions of 50 to 150 bp (ITR: "Inverted Terminal Repeat").
- ITR Inverted Terminal 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 for 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.
- TP terminal protein is associated
- the adenovirus genes are distributed (more or less arbitrarily) into two groups:
- adenovirus 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.
- 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).
- E l . (1.3 to 1 1.2 mu): 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: E l A and E l 13, the first of which is essential for the establishment of viral infection.
- EI ⁇ and E l 13 allow, by combined effects, cell multiplication and immortalization transformation of cells receiving them jointly by trans ection (if E l A allows immortalization, E1B avoids apoptosis and thus allows maintaining the immortalized phenotype).
- E2 (3 1- 1 1 and 66.6-61.7 mu): its transcription is dependent on E 1 A (although the protein El A is not a DBP, "DNA binding Protein", protein which binds to the 'DNA).
- This gene codes among others 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 ; 55 kDa mature protein).
- 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. of the lyric action of TNF ("Tumor Necrosis Factor").
- 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.
- early genes indicates a predominance of certain genes at some point in the replication cycle of the virus.
- a so-called early gene such as El A for example, can express itself throughout the viral cycle, or a gene known as late, like the promoter MLP ("Major Late Promoter"), can be expressed, although weakly, from the first hours following the infection.
- Gjinciia.t iXs There are five of them: L1 to L5, coding for the various structural proteins (mainly capsid): L2 for the penton, L3 for the hexon, L5 for the fiber, etc.
- RNAs 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: I, 2 and 3.
- TPL TriPartite Leader
- adenovirus fragments into plasmids, and by cotransfection of overlapping fragments representing the whole genome, by homologous recombination, to obtain recombinants reproducing the original virion.
- 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.
- the deletion of the region E l may, with the previous ones, allow, a priori, insertions up to 7 kb.
- Line 293 can be called “helpcr” line by analogy with what is happening for retroviruses, the principle remains the same, providing in trans genes that have been previously removed to make room for the exogenous genes that l 'we want to express in its viral construction.
- the first case is more favorable allowing on the one hand the multiplication, therefore more numerous copies of the gene to be expressed.
- 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.
- the E3 promoter is chosen, which can also comprise the MLP promoter.
- 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 ⁇ RNs.
- Viral RNAs are also preferentially translated, thanks to two
- RNAs of the adenovirus transcribed by the RNA polymerase III of the host cell named: VAI and VAII (for "virus associated RNA”). These RNAs promote the translation of other viral RNAs by preventing the phosphorylation of the elf2 ⁇ elongation factor, phosphorylation which inhibits the activity of the latter.
- helpcr a complementation cell line
- E 1 A and EIB human kidney embryonic line
- the recombinant adenoviruses obtained such as the recombinant retroviruses obtained from transcomplementing lines (Isolde type in avian, see Nigon, Verdier, Associated CNRS-INRA, Lyon-Villeurbanne) are incapable of reproducing by themselves, but are capable infect the target cells and synthesize the heterologous protein there, encoded 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 power of autonomous replication.
- transcomplementing lines Isolde type in avian, see Nigon, Verdier, Associated CNRS-INRA, Lyon-Villeurbanne
- 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.
- 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 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.
- essential parts of the genome of the avian adelovirus CELO is meant the parts of said genome consisting of at least one strictly essential part of said genome and which may also comprise one or more non-essential parts of said genome.
- strictly essential part of said genome is meant a part in which the insertion of a heterologous DNA sequence disrupts the transcription of one or other of the two strands of genomic DNA, no longer allowing the autonomous replication of said genome.
- 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.
- 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.
- heterologous polypeptides of interest whose nucleic sequence can be incorporated in the nonessential parts of the genome of the avian adenovirus 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 infectious agent, viral or other types, which are responsible for infectious diseases in animals and in particular in avian species such as, for example, Marek's disease, infectious bronchitis, larynchotracheitis, gumboro, Ncwcastlc disease, mycoplasma infections, l anemia of chicken, avian encephalomyelitis, avian influenza or bursa disease of Fabricus of viral origin Gumboro. It is also possible to use the recombinant avian adelovirus CELO as a vaccine vector for mammals by replacing the original free ones (MICHAEL, SI, 1995, Gene Therapy, 660-668).
- 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.
- the present invention also includes the methods according to the invention into which several DNA sequences are each coded for a heterologous polypeptide of interest. b) The polypeptides involved in metabolism
- the invention also aims to produce in vivo, in particular in poultry, polypeptide factors involved in the metabolism of the animal infected with the recombinant CELO avian adenovirus.
- polypeptide factors involved in the metabolism of the animal infected with the recombinant CELO avian adenovirus include, for example, of the polypeptides involved in the metabolism of fats such as ⁇ 9 desaturase, acetyl coA carboxylase, lipase, etc., growth hormones or their inducing factor (such as GRF). or immunopotentiators such as cytokines, interleukins, etc.
- the polypeptide of interest can be an intracellular, membrane polypeptide present on the surface of the host cell or secreted out of 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.
- 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:
- cytokine and in particular an interleukinc, an interferon, a tissue necrosis factor and a growth factor and in particular hematopoietic (G-CSF, GM-CSF),
- factor VIII a factor or cofactor involved in coagulation and in particular factor VIII, von Willebrand factor, antithrombin III, protein C, thrombin and hirudin,
- an enzyme inhibitor such as viral protease inhibitors
- thimidine kinase of the HSV virus (herpes virus) type 1 an expression product of a suicide gene such as thimidine kinase of the HSV virus (herpes virus) type 1,
- a polypeptide whose absence, modification or deregulation of expression is responsible for a genetic disease, such as the CFTR protein, dystrophin or minidystrophin, insulin, FADA (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 P53 genes,
- - 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 likely to enter into competition with native viral proteins.
- 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 n ⁇ o gene (neomycin) conferring resistance to the antibiotic G418, by the dhfr gene (dihydrofolate reductase), by the CAT gene (Chloramphcnicol Trans ferase) or also by the gpt gene (xanthinc phosphoribosyl).
- polypeptide of interest is also meant the recombinant polypeptides of industrial or therapeutic interest which may be prepared either // vitro by culturing cells, in particular avian, transformed with the recombinant adenovirus CELO, or either / '// vivo the animal.
- the nucleic sequence can code for a polypeptide of interest corresponding to all or part of a native protein as found in nature. It can 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.
- promoters or leader sequences there may be mentioned those of human adenoviruses, SV40 virus, cytomegalo or avian adenoviruses, particularly preferred is the MLP promoter of avian adenovirus CELO, the TPL leader sequence (here bipartite: L 1 + 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 adelovirus CELO, preferably said non-essential part is chosen from the following sites, identified by coordinates numerical values (bp) relating to the published sequence of 43,804 bp (CHIOCCA, S.
- 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) relating to the sequence as defined in Figure I: 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.
- bp numerical coordinates
- 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 (pb) relative to the sequence as defined in Figure 1: a) part between 300 and 5 300, b) part between 10 170 and 1 1280, 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 included 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
- the invention also relates to methods according to the invention, characterized in that the deletion step results in 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 an packaging signal M'.
- Such vectors are especially preferred as a vector for heterologous gene transfer into 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, R.J. et al., 1996, PNAS, 93, 13565-13570, WANG, P. et al., 1995, Somalia
- the principle is based on Crc-rccombinase which recognizes the LoxP sequence and makes it possible to excise 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 ⁇ ), which in the case of avian adelovirus CELO, leaves room for approximately 43 kb of exogenous DNA.
- LMH line (Kawaguchi T., Nomura K., Hirayama Y., Kitagawa T. (1987): Establishment and characterization of a chicken hepatocellular carcinoma cell Une, 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 ⁇ 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 packaging 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 transcompliant line LMI I / Crc-rccoinbinase.
- the helper virus is, apart from the LoxP sites, identical to the wild-type adelovirus CELO.
- 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.
- 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.
- Marek's disease infectious bronchitis
- larynchotracheitis larynchotracheitis
- gumboro Newcastle disease
- 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.
- 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.
- a vaccine according to the invention comprises between 10 1 1 and 10 1 adenoviral particles / ml, preferably between 10 9 and 10 12 pfu / ml (plate forming unit).
- the invention also relates to cells transformed with a vector according to the invention.
- cells transformed with a vector according to the invention are preferred.
- cells characterized in that they are eukaryotic cells allowing the replication of a defective recombinant avian adelovirus CELO are preferred.
- transcomplementing cells 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 the knowledge of the essential and nonessential parts of the genome of the virus which one wants 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 inol ⁇ ensive, because more or less deleted, and incapable of dissemination in animals and between animals.
- GMO genetically modified organism
- it is possible to free up more and more space, even all of the genes of the virus to provide a maximum of exogenous information ("gutless viais", virus "disoyoyed” and Cre-recombinase). This is an advantage that can more than offset the disadvantages of non-autonomous replication.
- the effectiveness of the first vaccination is essential, but it is not necessarily necessary to increase the number of injections.
- CELO Defective recombinant CELO, transcomplementing cell lines of avian origin, LMH or QT6 type are preferred (ATCC CRL 1708-fibrosarcoma-Moscovici et al. 1977).
- 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).
- transcomplementing cell lines of avian origin preferably require the use of avian retroviruses making it possible to obtain avian CELO-transcomplementing 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. The transcomplementing cells are obtained quickly and then selected on their efficiency in producing the genes of the avian adenovirus CELO.
- the invention further comprises methods of preparing / '// vivo a recombinant polypeptide of interest, characterized in that they implement the infection of an animal with a vector according to the invention, which vector said inserted DNA sequence 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.
- Transformation techniques / '; / vivo or /' // vitro cells from recombinant adenovirus vectors to prepare the recombinant polypeptides are well known to those skilled in the art and will not be described.
- Figure 1 complete nucleotide sequence of the CELO viral genome.
- the 5 ′ to 3 ′ strand of the genome is also represented by SEQ ID No. 1.
- Figure 2 construction of the plasmid pPOLY II / CELO ends.
- the plasmid ppoly II ext celo of 3964 bp is obtained by carrying out a triple ligation between ppoly II, the Bamhl-Spel fragment, and the Spel-Hindlll fragment; These two fragments ⁇ being previously amplified by PCR and purified.
- FIG. 3 construction of the plasmid ppoly 1I / CELO recombining with the entire genome of the adenovirus celo, by homologous recombination between the total DNA of the CELO virus and the plasmid ppoly II / CELO ends.
- Figure 4 ppoly II / CELO plasmid recombining with the entire adenovirus celo genome with its 4 constituent fragments A, B, C and D.
- Figure 5 Potential sites of insertion or deletion of the viral genome of the avian adenovirus CELO.
- 1 cm corresponds approximately to 2kb.
- the arrows on the top diagram represent the potential insertion sites and the arrows on the bottom diagram frame the potential areas of deletion.
- FIG. 6A represents the homologous recombination between identical regions of the plasmid ZZ and of the recombinant adenovirus ppoly / celo rec 45.8 Kb.
- FIG. 6B represents the plasmid pPolyllCelo Rec with a gene of interest introduced by homologous recombination as indicated in FIG. 6A.
- Figure 7 the four plasmids A, B, C and D representing the entire CELO genome and allowing the construction of recombinants (deleted and / or by insertion) by successive subcloning.
- Figure 8 diagram of the four plasmids A, B, C and D as well as of the recombinant ppoly 1I / CELO plasmid carrying the entire viral CELO genome.
- Figure 8A shows the presence of fragment A of celo in pBS / KS + Amp Resistant.
- Figure 8B shows the presence of fragment B of celo in pBS / KS + Amp Resistant.
- Figure 8C shows the presence of fragment C of celo in pBS / KS + Amp Resistant.
- Figure 8D shows the presence of fragment D of celo in pBS / KS + ligand.
- FIG. 8E shows the plasmid p Polyll Amp R recombining with the entire genome of celo.
- FIG. 9A corresponds to the recombinant L2 retrovirus
- FIG. 9B corresponds to the recombinant L3 retrovirus
- FIG. 9C corresponds to the recombinant L4 retrovirus
- FIG. 9D corresponds to the recombinant L5 retrovirus.
- the rectangles with black and white tiles and those with a tile pattern represent the Neo gene and the hygromycin gene respectively
- the striated rectangles and those with up arrows define the promoter CMV and SV40 respectively.
- CELO recombinant vaccines I) The X and Y genes of the celo adenovirus are integrated on each side of an IRE (Entry
- Neo gene Internal Ribosomal
- CMV promoter Internal Ribosomal
- LMH line Cells of the LMH line are infected with retroviruses carrying the X and Y genes produced by the cells of step 2.
- a LMH line transcomplementing for the X and Y genes is selected.
- the LMH line isolated in 3) is transfected with the recombinant Celo DNA carrying the genes of interest 1 and 2.
- viruses produced carrying the genes of interest are purified.
- Figure 1 1 creation of CELO “gutless” recombinant through CRE-recombinase.
- An Isolde line is transfected with a retrovirus carrying the CRE recombinase gene.
- the rectangle with diagonal lines represents an inducible promoter.
- the grid rectangle is an NLS (Nuclear Location Signal), the rectangle with diamonds is an IRE and the Neo gene is represented as in Figure 9.
- An LMH line is infected with the retrovirus expressing Cre recombinase produced by a line of step 1).
- An LMH line expressing the CRE recombinase protein is selected.
- the selected LMH line is co-transfected with a Celo Helper DNA comprising two LoxP sites flanking the viral packaging signal and a recombinant celo DNA in which genes of interest of type X, Y, Z are inserted in a region available from 43 kb. 4)
- the LMH line possessing CRE recombinase produces non-replicative vias and carriers of genes of interest.
- EOPS embryos 19-day chicken kidney embryo cells (EOPS embryos) are 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% CO 2 .
- FCS medium fetal calf serum
- RNAnow is used at a rate of 1 ml / 10 cm 2 (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 a Tris-EDTA 10: 0.1 (RNAse free). Each extraction is followed by an analysis on agarose gel of the quality of the RNAs.
- the first library is constructed in the plasmid pSPORT1 from the kit
- the cDNAs with oligo (dT) primers associated with primers-adapters with four restriction sites Spel.Xbal, NruI, Notl, in the presence of SuperScript II RT were 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 with NotI and subcloned into pSPORTl by SalI / NotI.
- the second library is constructed in the plasmid pBluescript BS / KS +
- the first strand is synthesized in the presence of oligo (dT) 1 primers and hcxanucleotides.
- 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.
- the DIG high Prime DNA labeling and detection starter kit 11 from Boehringer was used to detect positive clones in XL 1 blue.
- 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. Hybridization is done in a 50% formamide buffer at a temperature of 42 ° C overnight. The revelation is made by chemiluminiscence. Clones found positive on autoradiographs are screened a second time. Positive disorders are amplified and then sequenced.
- RNA-PCR Kinetics by RT-PCR (transcriptasc reverse and PCR)
- An RNA-PCR is carried out with a treatment prior to DNase I.
- the cDNAs are synthesized in a reaction of 20 ⁇ l (Huang, 1996) containing for 1 ⁇ g RNA: 1 x PCR buffer II (Boehringer), 5 mM MgC12, 1 mM dNTP mix, 1U DNAsel 20U RNase inhibitor, 2.5 ⁇ M oligo (dt) 15, 1 ⁇ M hexanucleotides.
- the DNAsel reaction is placed for 2 hours at 37 ° C and then 5 minutes at 75 ° C to destroy the enzyme.
- a microliter of 50U / ⁇ l 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.
- reverse transcriptase is replaced by 2 ⁇ l of RNase free H2O.
- Two microliters of this stock diluted to 1: 30 are added to 23 ⁇ l of PCR reaction, for a final volume of 25 ⁇ l containing: lx PCR bu proud 11, 2 mM MgCI2, 25 pmol of each primer, I mM dNTP mix, and 0.5U Taq polymerase (Boehringer).
- the amplification conditions vary depending on the primers chosen. The number of cycles varies between 25 and 34.
- the Clontech Marathon cDNA amplification kit was used. First, single-stranded cDNAs from 1 ⁇ g of RNA extracted from infected cells were produced. O! Igo (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 specific Notl-Sfrl-Xmal adapter is attached to the ends. Amplifications were then carried out by PCR (polymerase chain reaction) with a primer sense A I specific for this adapter and an antisense primer chosen 5 ′ of our ORFs (generally in the ATG region). The amplification products are then sequenced and analyzed on MacDNasis.
- PCR polymerase chain reaction
- Example 1 The genomic DNA of the avian adenovirus CELO (serotype 1), double-stranded DNA virus, is completely sequenced (cf. FIG. 1). The DNA is obtained after culture on embryonic S.PF eggs (free of specific pathogen) and harvested a few days after infection with allantoic fluid. It is purified by conventional virus purification protocols. Approximately 300 ⁇ g of viral DNA (approximately 150 infected embryonic eggs) are isolated for one liter of infected allantoic fluid. The DNA of the avian adelovirus CELO (serotype 1) thus comprises 43,804 bp and codes for at least forty genes.
- avian adenovirus CELO embryonic chicken kidney cells SPF
- the messenger RNAs produced by these infected cells are extracted at various post-infection times and used to build cDNA libraries. Screening is then carried out using a kit digoxygenin hybridization. Under these conditions, a transcriptional model is established for the avian adenovirus CELO.
- the various promoter zones have been localized, mainly for the ORF1, ORF2 genes (promoter located around 350-510), for late genes with the major late promoter (major late promoter: MLP, located around 7 3 10 - 7,520) and its two "leaders" L1 and L3 (located respectively at 7,533 - 7,545 (22 bp), and 1,1285 - 1,113 (129 bp), for the ORF22 gene (promoter around 43,400 - 43,700), for OR l 8 and 1 genes (promoter around 36 460 - 36 770)
- 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 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. FIGS.
- 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 site of insertion, to artificially reconstitute the adenovirus recombinant wanted.
- 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.
- deletions are based essentially on the results concerning the study of the transcription of the genome of the avian adenovirus CELO obtained previously.
- the cellular model chosen for said study (embryonic chicken kidney cells) makes it possible to obtain the complete lyrical replicative cycle of the adenovirus, demonstrating that the non-shredded genes are not essential for the "in vitro" survival of adenovirus and that it is possible to delete them.
- Deleted replicative vectors are then obtained capable of carrying one or more genes of interest.
- this zone is more critical, because it includes the leader of IORF22 (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).
- IORF20 the leader of IORF22
- PORF5 the termination zone of IORF20
- this last gene has never been highlighted 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) .
- 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 provided by the LMH cell line, previously transformed by the retrovirus, after transfection with the recombinant vaccine vector.
- Deletion of 1ORF7 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 cDNA and RT-PCR bank (genes very early to early from 2 hours and 4 hours after injection and whose role is therefore essential for the viral cycle. Deletion of 1ORF7 can be done from 35,620 to 36,440 (or 820 bp released; with the size increase of 5%, possibility of insertion of 'around 3 kb).
- transcomplementing line a pTP transcomplementing line: pTP binds to the ends of the DNA of the avian adenovirus genome
- CELO (or any other recombinant carrying 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 of 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.
- the retrovirus carrying pTP the latter 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) .
- pTP uses the leader of 1ORF12, and the ⁇ leader of DBP, we can thus make an appropriate construction to have, with these elements, maximum production efficiency of pTP.
- the genomic DNA of the avian adenovirus CELO is released from 10,170 to 1,128, or 1.1 kb, in order not to touch the leader 3 used by the late genes.
- the recombinant CELO avian adenovirus 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 DBP transcomplementing line:
- DBP extending (on the “down” strand) from: 21,800 to 23,170, or a place of 1.37 kb on the recombinant CELO avian adenovirus and this without affecting any other gene
- L2 or L3, or L2 + L3, even L2 + L3 + D.B.P.
- entity L2 (penton at pX): 16,050 to 17,540, release of approximately 1.5 kb.
- L2 + L3 entities (penton to E.P.): 16,050 to 21,800, release of approximately 5 kb.
- L2 + L3 + DBP entities 16,050 to 23,170, release of around 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.
- L4 pVIII, 100 K, between ORF 12 and 22: 27 100 and 31 790: 4 690 bp
- L5 fibers 1 and 2
- L4 + L5 according to schemes equivalent to those used previously: 100 K start: 23,180 to 27,960 (end pVIII): 4,780 bp.
- E1A and E1B 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 E1A, promoting cell multiplication (to be sought among very early genes) and a gene preventing apoptosis such as does E1B. According to a recent publication (CHIOCCA, S. et al, 1997, J. Virol., 71, 3 168-3 177) it is known that it is known that it is IORF8 (gene with anti-apoptosis effect, of Bcl2 type in its mode of action).
- IORF8 gene with anti-apoptosis effect, of Bcl2 type in its mode of action.
- ORF4 ORF5
- ORF 16 ORF 18
- ORF20 ORF21
- ORF21 ORF20 and ORF21
- ORF4 and ORF 18 seem to be the most relevant for this.
- ORF8 + ORF4 (most relevant combination), ORF8 + ORF4, ORF8 + ORF18, ORF8 + ORF 19, ORF8 + ORF5, ORF8 + ORF 16, ORF8 + ORF20 and ORF8 + ORF21 are tested on embryonic liver cells. or chicken kidney. 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, Omony1995, J. Virol., 69, 4079-4085).
- FIG. 11 shows as an example the general diagram of the process for the preparation of recombinant defective CELO vaccine vectors according to the Cre-recombinase system, from Isolde type retroviral 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 co-transfection with the recombinant vaccine vector and an assistant adenovirus.
Abstract
Description
Claims
Priority Applications (1)
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AU90760/98A AU9076098A (en) | 1997-08-14 | 1998-08-13 | Recombinant celo avian adenovirus and use as vaccinating vector |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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FR9710386A FR2767335B1 (fr) | 1997-08-14 | 1997-08-14 | Adenovirus aviaire celo recombinant comme vecteur vaccinant |
FR97/10386 | 1997-08-14 |
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WO1999009194A1 true WO1999009194A1 (fr) | 1999-02-25 |
WO1999009194B1 WO1999009194B1 (fr) | 1999-03-25 |
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PCT/FR1998/001803 WO1999009194A1 (fr) | 1997-08-14 | 1998-08-13 | Adenovirus aviaire celo recombinant et son utilisation comme vecteur vaccinant |
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AU (1) | AU9076098A (fr) |
FR (1) | FR2767335B1 (fr) |
WO (1) | WO1999009194A1 (fr) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000017373A1 (fr) * | 1998-09-22 | 2000-03-30 | Boehringer Ingelheim International Gmbh | Virus celo de recombinaison et son adn |
EP1092780A1 (fr) * | 1999-10-13 | 2001-04-18 | Boehringer Ingelheim International GmbH | Virus CELO recombinant et incapable de replication ainsi que l'ADN du virus CELO |
CN103060376A (zh) * | 2012-12-11 | 2013-04-24 | 上海实验动物研究中心 | 一种禽腺病毒转移载体及其制备方法 |
US8940534B2 (en) | 2003-11-03 | 2015-01-27 | Probiogen Ag | Immortalized avian cell lines for virus production |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0473210A2 (fr) * | 1990-07-30 | 1992-03-04 | Akzo Nobel N.V. | Virus recombinant de la maladie de Marek |
WO1994024268A1 (fr) * | 1993-04-14 | 1994-10-27 | Arthur Webster Pty. Ltd. | Vecteur d'adenovirus de recombinaison avien |
DE19615803A1 (de) * | 1996-04-20 | 1997-10-23 | Boehringer Ingelheim Int | CELO-Virus |
-
1997
- 1997-08-14 FR FR9710386A patent/FR2767335B1/fr not_active Expired - Lifetime
-
1998
- 1998-08-13 AU AU90760/98A patent/AU9076098A/en not_active Abandoned
- 1998-08-13 WO PCT/FR1998/001803 patent/WO1999009194A1/fr active Application Filing
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0473210A2 (fr) * | 1990-07-30 | 1992-03-04 | Akzo Nobel N.V. | Virus recombinant de la maladie de Marek |
WO1994024268A1 (fr) * | 1993-04-14 | 1994-10-27 | Arthur Webster Pty. Ltd. | Vecteur d'adenovirus de recombinaison avien |
DE19615803A1 (de) * | 1996-04-20 | 1997-10-23 | Boehringer Ingelheim Int | CELO-Virus |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000017373A1 (fr) * | 1998-09-22 | 2000-03-30 | Boehringer Ingelheim International Gmbh | Virus celo de recombinaison et son adn |
AU774399B2 (en) * | 1998-09-22 | 2004-06-24 | Boehringer Ingelheim International Gmbh | Recombinant CELO virus and celo virus DNA |
EP1092780A1 (fr) * | 1999-10-13 | 2001-04-18 | Boehringer Ingelheim International GmbH | Virus CELO recombinant et incapable de replication ainsi que l'ADN du virus CELO |
US8940534B2 (en) | 2003-11-03 | 2015-01-27 | Probiogen Ag | Immortalized avian cell lines for virus production |
CN103060376A (zh) * | 2012-12-11 | 2013-04-24 | 上海实验动物研究中心 | 一种禽腺病毒转移载体及其制备方法 |
Also Published As
Publication number | Publication date |
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AU9076098A (en) | 1999-03-08 |
WO1999009194B1 (fr) | 1999-03-25 |
FR2767335A1 (fr) | 1999-02-19 |
FR2767335B1 (fr) | 2001-09-28 |
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