AU7587300A - Recombinant eggs and gene cloning and expression vectors based on avian adenoviruses - Google Patents

Description

WO 01/19968 -1- PCT/USOO/25489 RECOMBINANT EGGS AND GENE CLONING AND EXPRESSION VECTORS BASED ON AVIAN ADENOVIRUSES 5 FIELD OF THE INVENTION This invention is in the field of molecular biology. In particular, this invention relates to eggs containing recombinant protein and/or recombinant DNA and to vectors and genes useful for recombinant protein production in eggs. 10 BACKGROUND OF THE INVENTION Transforming genetic information into market-ready products is an emerging paradigm in the pharmaceuticals industry. The market for biological products is expected to approach $150 billion by the year 2000, largely driven by genomics and drug discovery. This growth reflects a huge demand for 15 molecular characterization of newly discovered genes whose functions might offer cures for disease. Once such genes have been identified, there is often a need to produce larger quantities for research, and clinical trials leading to a market ready product. There are very few recombinant protein production technologies that can be used to produce clinical quality products. Making 20 bacterial, yeast or insect cell culture systems the basis of a manufacturing process is still difficult, unpredictable, and expensive. Thus, there is a need to develop a highly productive recombinant protein expression system that is simple to use with a low cost of scale up to manufacturing production levels. SUMMARY OF THE INVENTION 25 In order to meet these needs, the present invention is directed to a chicken adenovirus expression vector (AdCEV), a recombinant protein expression vector based on the Fowl Adenovirus Type 1 (Ad FAV1). The Ad FAV1 host is fertilized chicken eggs which for many years, have provided a simple way to grow diverse viruses that can be used for vaccinating people or 30 animals. For example, influenza vaccines for humans and animals, along with WO 01/19968 -2- PCT/USOO/25489 many poultry vaccines, are still manufactured in fertilized eggs. The present invention extends the range of useful, high-value products that can be produced in a fertilized egg to any protein that can be stably expressed by the AdCEV system of this invention. AdCEV transfection transforms each egg into 5 a miniature factory producing large amounts of recombinant protein in the egg fluids. Our methods eliminate the need for a screening step, yielding a high percentage of recombinant cells after transfection. The AdCEV/Egg system of this invention offers the following benefits: 1. Simplicity and low cost; 2. high productivity - milligrams of protein/egg; 3. short production process - 72 hr 10 incubation; 4. correct processing of eukaryotic proteins; 5. simple purification procedures from egg fluid; 6. easy scaleability for mass production; and 7. high level of biosafety. The invention is further directed to an egg comprising a recombinant protein. The egg may be a fowl egg. The egg may be the egg of any avian 15 species that is susceptible to infection by adenoviruses. The invention is further directed to an egg comprising recombinant DNA. The invention is further directed to an egg comprising recombinant RNA. The invention is also directed to a method of preparing a recombinant avian adenovirus vector containing a heterologous gene, comprising the steps 20 of: (a) preparing a plasmid, cosmid, or phage containing avian adenovirus DNA, said DNA comprising: (i) at least one transcriptional regulatory sequence next to at least one restriction endonuclease site, and; (ii) DNA from a non-essential region of Ad FAVI genome flanking said transcriptional regulatory sequence and said restriction endonuclease site; and (b) inserting at 25 least one protein coding sequence from a foreign gene into said restriction endonuclease site next to said transcriptional regulatory sequence. The invention is further directed to a method for genetically modifying an avian adenovirus, comprising the steps of: (a) preparing a vector comprising plasmid, cosmid, or phage containing avian adenovirus DNA, said 30 DNA comprising: (i) at least one transcriptional regulatory sequence next to at least one restriction endonuclease site, and (ii) DNA from a non-essential region of avian adenovirus flanking said transcriptional regulatory sequence and said restriction endonuclease site; and (b) inserting at least one protein coding sequence from a foreign gene into said restriction endonuclease site WO 01/19968 -3- PCT/USOO/25489 next to said transcriptional and translational regulatory sequences; (c) providing at least one cell infected with a recombinant avian adenovirus; (d) isolating from said cell a recombinant avian adenovirus capable of expressing said protein coding sequence. 5 The invention is further directed to a method of using a recombinant avian adenovirus as a vaccine, comprising the steps of: (a) preparing an infectious avian adenovirus containing therein a chimeric gene comprising at least one avian adenovirus transcriptional and translational regulatory sequences and at least one protein coding sequence from a foreign gene, said 10 chimeric gene being flanked by DNA from a non-essential region of avian adenovirus genome, such that said infectious avian adenovirus is capable upon infection of a cell of expressing said protein coding sequence. In a second step an animal or human is inoculated with an inoculant containing a concentration of said recombinant protein sufficient to elicit an immunological 15 response in the animal or human. The invention is further directed to a vector comprising: (a) a plasmid, cosmid, or phage; (b) a chimeric gene which comprises at least one avian adenovirus transcriptional and translational regulatory sequences from a foreign gene; and (c) DNA from a non-essential region of avian adenovirus 20 genome, said DNA flanking said chimeric gene. The invention is further directed to an infectious avian adenovirus containing therein a chimeric gene comprising at least one avian adenovirus transcriptional and translational regulatory sequences and at least one protein coding sequence from a foreign gene, said chimeric gene being flanked by 25 DNA from a non-essential region of avian adenovirus genome, such that said infectious avian adenovirus is capable upon infection of a cell of expressing said protein coding sequence. In an alternative embodiment, the invention is directed to an infectious avian adenovirus recombinant produced by a process comprising the steps of: 30 (a) preparing a vector comprising a bacterial or yeast plasmid, cosmid, or phage containing avian adenovirus DNA, said DNA comprising: (i) at least one transcriptional and translational regulatory sequences next to at least one restriction endonuclease site, and (ii) DNA from a non essential region of avian adenovirus genome flanking said transcriptional regulatory sequence and said WO 01/19968 -4~ PCT/USOO/25489 restriction site: (b) inserting at least one protein coding sequence from a foreign gene into said restriction endonuclease site next to said transcriptional and translational regulatory sequences. In an alternative embodiment, the invention is directed to producing 5 large quantities of a recombinant protein in the egg fluids from an expressed gene. Sometimes a gene of interest encodes a protein that is poorly expressed i.e. is not produced in abundance and/or is only transiently produced under natural physiological conditions. In other cases, the size of the encoding gene or genes which it is desired to express exceeds the capacity of the "non 10 essential" regions of the virus that can be replaced without affecting the required infectivity of the recombinant virus. One approach to achieving enhanced protein production is the use of transient cell expression systems wherein cells are transfected with "mini chromosomes" that are not expected to integrate in the host cell genome. The 15 mini-chromosomes used in transient cell expression systems can also be modified to further enhance their copy numbers during replication in infected cells. The invention includes the incorporation of two cloned inverted adenovirus termini sequences in a vector DNA molecule and co-transfection 20 of cells with both vector and non-defective adenovirus DNA as a helper. In these systems, a helper adenovirus DNA provides in trans all of the protein functions required for the packaging of the vector which contains only those cis-acting elements required for viral DNA replication and packaging, mainly the inverted terminal repeat sequences (including origins of viral replication) 25 and packaging signal sequences. In the present invention, the vector system is composed of a DNA mini chromosome comprising a transcription sequence (promoter) that can be activated in trans, an RNA stabilizing sequence that enhances translation, at least one protein coding sequence from a foreign gene and inverted 30 adenovirus termini required for vector replication, and non-defective adenovirus DNA as a helper. In an alternative embodiment, the vector system is composed of a DNA mini-chromosome comprising a transcription sequence (promoter) that can be activated in trans, an RNA stabilizing sequence that enhances WO 01/19968 -5- PCT/USOO/25489 translation, at least one protein coding sequence from a foreign gene and inverted adenovirus termini required for vector replication, and non-defective adenovirus particles as a helper. Thus, in view of this disclosure, skilled genetic engineers can construct 5 transfectants which overcome the production problems associated with size limitation of expression vectors and certain low expression genes. Specifically, those skilled in recombinant DNA techniques can design appropriate DNA vectors encoding a protein of interest, adenovirus origins of DNA replication from both the right and left ends of the genome, transcription trans-activators 10 and translation stimulators and adenovirus DNA as a helper and then use the methods of manufacturing transfectants disclosed herein to obtain large quantities of a desired protein through production of transgenic eggs. Such proteins can be in their native forms or truncated analogs as well as fusion proteins or other engineered constructs capable of mimicking the biological 15 activity of a protein of interest.

WO 01/19968 -6- PCT/USOO/25489 BRIEF DESCRIPTION OF THE DRAWINGS The invention will be better understood by reference to the figures, in which: Figure 1 shows a schematic diagram representing plasmid pSKII-DC5' 5 MLP-2bLD. Figures 2 show a schematic diagram representing plasmid psKIl-DC5' MLP-2bLD-RGSV40-p(A). Figure 3 shows a schematic diagram representing plasmid pSKII-DC5' MLP-2bLD-RG-p(A)-DC(Xbal-Notl). 10 Figure 4 shows a schematic diagram of construction steps for a recombinant Ad CELO (AdCEV) genome. Figure 5 shows a schematic diagram of the rCRP construct. Figure 6 shows the 660 base pairs CRP PCR product used in the construction of the CRP clone (Fig.5) 15 Figure 7 shows expression of human CRP in eggs. 7ul of allantoic fluid from eggs infected with CRP construct was loaded on a 4-20% gradient SDS PAGE gel. Half the gel was stained with Coumassie blue stain and the other half was transferred to nitrocellulose and reacted with anti-CRP antiserum. The contents of the lanes are as follows: 1. rCRP clone; 2. CRP control 20 protein isolated from human fluids; 3. N/A; 4. FAV1 infected allantoic fluid; 5. Uninfected allantoic fluid. Pre-stained molecular weight markers are designated MW. Figure 8 shows expression of recombinant CRP in the allantoic fluids of recombinant adenovirus (AdCEV) infected chick embryos. 25 Allantoic fluid and control samples were incubated with 0 phosphorylethanolamine Sepharose (Sigma, USA) according to the manufacturers protocol. The samples were eluted by boiling with PAGE loading buffer containing 1% SDS and loaded on the gel. 1. FAV1 infected allantoic fluid; 2. Uninfected allantoic fluid; 3. recombinant CRP 30 virus infected; 4. recombinant CRP virus infected; 5. CRP purified from human fluids; 6. Uninfected allantoic fluid; 7. Molecular Weight WO 01/19968 - PCTIUSOO/25489 Standards; 8. CRP purified from human fluids. Figure 9 shows a schematic diagram representing the preparation of the plasmid pSKIlI-DC5'-MLP-28LD-GRV-pA-DC3' Figure 10 shows expression of rabies glycoprotein G in allantoic fluid of 5 infected (R) and control (C) eggs. In the control, wild-type virus was used to infect the eggs and one of the components of the adenovirus can be seen (A). In the rabies clone a 68kd protein can be seen corresponding to the predicted size of the rabies construct. MW is molecular weight markers. Figure 11 Shows expression of rabies glycoprotein G in allantoic fluids 10 of chick embryos transfected with recombinant adenovirus vector pKSII-DC5' MLP-2BLD-GRV-p(A)-DC3' and helper CELO virus DNA. 1. MW standards 2. Allantoic fluid from pKSII-DC5'-MLP-2BLD-GRV-p(A)-DC3' and helper CELO virus DNA transfected embryos (protein sample boiled before loading). 3. Allantoic fluid from pKSII-DC5'-MLP-2BLD-GRV-p(A)-DC3' and helper CELO 15 virus DNA transfected embryos (protein sample boiled before loading) 4. Allantoic fluid from CELO virus DNA transfected embryos; 5. Allantoic fluid from untransfected embryos; WO 01/19968 -8- PCTIUSOO/25489 DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS To ensure a complete understanding of the invention, the following definitions are provided: 5 Adenovirus: Any virus belonging to the family Adenoviridae. The large adenovirus family (45 types) is divided by natural host range into adenoviruses that infect mammals (the Mastadenoviridae) and adenoviruses that infect avian species( Aviadenoviridae). Avian Adenovirus: An adenovirus of the Aviadenoviridae family. There 10 are at least 10 different serotypes of avian adenoviruses, commonly infecting chickens as well as other avian species such as ducks, quails, and turkeys as disclosed in Fenner, F. et al. Veterinary Virology, Academic Press, Orlando Florida 1987, pp329-337) which is hereby incorporated by reference. CELO: Chicken Embryo Lethal Orphan virus. CLEO is synonymous 15 with Fowl Adenovirus stereotype 1 (FAV-1) or Avian Adenovirus stereotype 1 (AAV1). CELO is an avian adenovirus of the Adenoviridae family. The general structural organization of CELO virus is an icosahedral capsid 70-80nm in diameter, made up of hexon and penton structures. The CELO virus genome is a linear double stranded DNA molecule with the DNA condensed within the 20 virion by virus encoded core proteins. The CELO virus has covalently attached terminal proteins and has inverted terminal repeats. Ad CEV: A recombinant adenoviral vector derived from Chicken Embryo Lethal Orphan virus. Promoter: A promoter is the minimal DNA sequence sufficient to direct 25 transcription. Promoters can render transcription controllable for cell-type specific, tissue-specific, organ specific, or inducible expression. Promoter elements may be located in the 5' or 3' regions of the native gene. Poly-Adenylation Site: A poly-Adenylation site is a nucleotide sequence which causes certain enzymes to cleave mRNA at a specific site and 30 to add a sequence of adenylic acid residues to the 3'-end of the mRNA.

WO 01/19968 -9~ PCTIUSOO/25489 Polypeptide: Polypeptide means any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation) and includes proteins. Polypeptides that are useful in the invention include but are not limited to HIV glycoproteins; Rabies glycoprotein; 5 Hanta virus glycoproteins; Ebola virus glycoproteins; Human Papilloma virus glycoproteins; Hepatitis B surface antigen; Hepatitis A envelope proteins; Hepatitis C envelope proteins; Hepatitis E envelope proteins; Human cytomegalovirus major envelope glycoprotein; Pseudorabiesvirus glycoprotein; Vesicular stomatitis virus glycoprotein; respiratory syncitial virus envelope 10 proteins; Rubella virus glycoproteins; measles virus envelope proteins; Yellow fever virus envelope proteins; Influenza virus proteins; tick borne encephalitis virus envelope proteins; parainfluenza virus envelope proteins; osteocalcin; osteonectin; chymase; thyroid peroxidase; Interleukins; caspases; calpains; apoptosis proteins; insulin; tumor necrosis factors; granulocyte macrophage 15 colony stimulation factor; epidermal growth factor; erithropoetin; Interferons; prostaglandins; thrombolysis proteins (plasminogen, urokinase, plasminogen tissue activator, etc); eosinophil-derived neurotoxin (major eosinophil ribonuclease); morphogenetic proteins (human jagged, mouse jagged, integrin, fibronectin; vitronectin; osteopontin, cadherin, lavendustin A; receptor tyrosine 20 kinase; pituitary proteins; endoglin; beta-2-mocroglobulin antigen; human C reactive protein; fatty acid binding protein; Human chorionic gonadotrophin; neuron specific enolase, Human growth hormone; Cytomegalovirus envelope proteins, Epstein Bar virus proteins, hepatitis core antigen; Dilofilaria immitis glycoprotein; Bovine Leukemia virus glycoprotein; mumps virus envelope 25 proteins; human parvovirus envelope proteins, Rotavirus glycoproetins, verotoxins; parathyroid hormone. Substantially Identical: For a polypeptide, substantially identical means a polypeptide exhibiting at least 50%, preferably 70%, more preferably 90%, and most preferably 95% identity to a reference polypeptide. For a 30 nucleic acid substantially identical means a nucleic acid sequence exhibiting at least 85%, preferably 90%, more preferably 95%, and most preferably 97% identity to a reference nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most 35 preferably 35 amino acids. For nucleic acids, the length of comparison WO 01/19968 -10- PCTIUSOO/25489 sequences will generally be at least 30 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics 5 Computer Group (GCG), University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, substitutions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; 10 valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, and tyrosine. Substantially Pure Polypeptide: Substantially pure polypeptide means a polypeptide which has been separated from components which naturally accompany it. Typically, the polypeptide is substantially pure when it 15 is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight polypeptide of interest. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel 20 electrophoresis, or HPLC analysis. Substantially Pure DNA: Substantially pure DNA means DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA which is incorporated into 25 the vectors of the invention. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. Transformed Cell: Transformed cell means a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding (as used herein) a polypeptide. 30 Transformed Egg: Transformed egg means an egg into which has been introduced, by means of recombinant DNA techniques, DNA encoding (as used herein) a polypeptide.

WO 01/19968 -11- PCT/USOO/25489 Positioned for Expression: Positioned for expression means that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of, e.g., a recombinant polypeptide or RNA molecule). 5 Operably Linked: Operably linked mean that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s). Mini-chromosome: A DNA molecule comprised of at least the 5' 10 terminal and 3'-terminal repeat sequences of the adenovirus genome which contain origins of replication and packaging sequences. Such a DNA molecule may be autonomously replicated in a cell infected with a helper virus that provides all the necessary replication functions in trans. 15 DETAILED DESCRIPTION OF THE INVENTION Taking into account these definitions, the present invention is directed to a recombinant protein production system based upon avian adenovirus infected chicken eggs. The large adenovirus family is divided by host range into adenoviruses 20 that infect mammals (the Mastadenoviridae) and adenoviruses that infect avian species (the Aviadenoviridae). Chicken embryo lethal orphan (CELO) virus (McCracken, R.M., et al., Viral infections of vertebrates, vol.3. Viral infections of birds. Elsevier Scientific Publishers, Amsterdam., 1993; McFerran, J.B., and B.M. Adair, Avian Pathol. v.6, 189-217, 1977), was first identified as an 25 adventitious contaminant of embryonated eggs during efforts to propagate a bovine skin disease agent (Van den Ende, M., et al., J. Gen. Microbiol.,v.3, 174-183, 1949) and was identified as an infectious agent in 1957 (Yates, V.J., and D.E. Fry, Am. J. Vet. Res.,v.18,657-660,1957). CELO virus is classified as a fowl adenovirus type 1 (FAV-1) and was the major subject of avian 30 adenovirology for a number of years. The FAV-1 adenoviruses can be isolated from healthy chickens and do not cause disease when experimentally reintroduced into chickens (Cowen, B., et al., Avian Dis.,v.22, 459-470, 1978). The general structural organization of CELO virus is similar to that of the WO 01/19968 -12- PCTIUSOO/25489 mammalian adenoviruses, with an icosahedral capsid 70 to 80 nm in diameter, made up of hexon and penton structures. The CELO virus genome is a linear, double-stranded DNA molecule with the DNA condensed within the virion by virus-encoded core proteins. The CELO virus genome has covalently attached 5 terminal proteins and has inverted terminal repeats (ITRs), although they are shorter than the mammalian ITRs. CELO virus encoded a protease with 61 to 69% homology to the mammalian adenovirus proteases. The DNA sequence and the genomic organization of CELO virus are reported (Chiocca, S., et al., J. Virol., v.70,N5, 2939-2949, 1996). The 10 sequence indicates a viral genome of 43.8 kb, nearly 8 kb longer than the 35.9-kb genome of human subgenus C adenoviruses Ad2 and Ad5. The genes for major viral structural proteins (hexon, penton base, Illa, fiber, pVI, pVIl, and pVIllI) are present and in the expected locations in the genome. The early region 2 (E2) genes (encoding DNA-binding protein, DNA polymerase, 15 and terminal protein) are also present. However, CELO virus lacks sequences homologous to the mammalian adenovirus El, E3, and E4 regions. There is approximately 5 kb of sequence at the left end and 15 kb of sequence at the right end of the CELO virus genome with limited or no homology to the mastadenovirus genomes. These new sequences contain a number of open 20 reading frames (ORFs), and it is likely that these encode functions that replace the missing El, E3, and perhaps E4 regions. Deproteinized DNA of AD CELO (strain Phelps) introduced into allantoic cavities of 9-days chick embryos induces the reproduction of infectious viruses Ad CELO (Grabko, V. I., Acta Virologica - 1987, v.31,No 1 25 2,pp.97-102). Using the method of heteroduplex analysis of virion DNA, we localized a non-essential region to the EcoRI-B fragment of the Ad CELO genome. As a test of the potential for viral vector construction, we inserted the plasmid pUC 19 into the non-essential region (2686bp), assembled CELO DNA fragments in vitro by ligation, and transfected 9-day old chick embryos with the 30 overlapping fragments of CELO DNA. As a result of viral DNA recombination in vivo we obtained AdCELO virions containing the genome of the plasmid pUC 19 in the non-essential region (Grabko V.I., Construction of recombinant adenovirus vector by method of insertion heterologous DNA in genome of Adenovirus CELO, Patent of Russia, N5061908/13, 1993). Thus a DNA 35 fragment up to 2700 n.b. can be inserted into a native genome of Ad CELO WO 01/19968 -13- PCT/USOO/25489 (into a non-essential region) without affecting the success of viral replication. If the non-essential region (about 6% of the genome) is deleted however, the genome capacity can presumably be increased by 2000-3000 additional base pairs. 5 A major advantage of prokaryotic systems is the ease with which genetic and phenotypic markers can be employed as tools for selecting the correct recombinants. In eukaryotic systems, this process is usually difficult and is especially so in the Chick embryo system since engineering an efficient screening system for recombinants is a major problem in vector design. In the 10 present invention, we have developed a powerful technique for assembling recombinant Ad CELO from two fragments in vitro. Using the assembly scheme of this invention, labor and time-consuming selection and purification of the recombinant clone using phenotypic markers is not required. In the scheme of this invention, each fragment is not infectious. It is only after the 15 assembly in vitro that a recombinant infectious DNA formed, which induces the reproduction of infectious recombinant virions during transfection (Celis, E., et al., J. Immunol., v.136, 629-697, 1986). Virtually any polypeptide of interest can be expressed using the expression system of this invention. An examples of such polypeptides is the 20 rabies virus glycoprotein G from the vaccine strain Vnukovo-32. Vaccination against rabies continues to be the only effective means to prevent disease following rabies infection. Recombinant viruses expressing rabies glycoproteins (RGs) used as vaccines have considerable potential for overcoming some of these problems. RG can induce protective virus 25 neutralizing antibodies (Wiktor, T.J., et al., J. Am. Med. Assoc. v.224, 1170 1171, 1973) and MHC class I- or class l-restricted cytotoxic T cells (Celis, E., et al., J. Immunol.,v.136, 629-697, 1986) and vaccinia virus vectors expressing this antigen have been evaluated as oral vaccines (Kieny, M.P., Nature, v.312,163-166, 1984). Adenovirus-vectored vaccines will be a useful adjunct 30 vaccine with vaccinia virus vectored vaccines (Hruby, D.E. Clin. Microbiol. Rev. v.3, 153-170, 1990) and may be a suitable alternative vaccine in some situations (Kaplan, C., Archs. Virol., v.106, 127-139, 1989). The cloning of the rabies virus glycoprotein G in embryonated eggs is described in the example section below.

WO 01/19968 -14- PCT/USOO/25489 Vaccinia virus, the most thoroughly studied member of the poxvirus family, was successfully used as a live vaccine to eradicate smallpox. Medical interest in vaccinia virus subsequently declined but was re-stimulated when live vaccinia recombinants were shown to be capable of expressing foreign genes 5 (Mackett, M., et al., Proc. NatI. Acad. Sci. USA, v.79, 7415-7419, 1982) and of protectively immunizing animals against infections with rabies virus (Wiktor, T.J., et al., Proc. NatI. Acad. Sci. USA v.81, 7194-7198, 1984) and many other viruses. Vaccinia virus may also be used as a cloning and expressing vehicle 10 (Grabko, V.I., et al., VI Conference of Russian Federation-New Direction Biotechnology, p.102-103, 1994). A recombinant vaccinia virus expressing RG from strain Vnukovo-32 induced protective antibodies against rabies virus in mice. The rabies glycoprotein DNA coding sequence may also be followed by 15 a polyadenylation (poly(A)) sequences, such as an SV40 early poly(A) region. The poly(A) region which is a signal for the polyadenylation of RNA transcripts appears to play a role in stabilizing transcription. A similar poly(A) region can be derived from a variety of genes in which it is naturally present. This region can also be modified to alter its sequence provided that polyadenylation and 20 transcript stabilization functions are not significantly adversely affected. The recombinant DNA molecule comprising the left terminal of the Ad CELO genome, major late promoter, bipartite leader, rabies glycoprotein DNA coding sequence and poly(A) SV40 is ligated with the right large fragment of Ad CELO genome. Together, these two ligated pieces of DNA encompass all 25 of the adenovirus genome and contain the information for an infectious virus. The next step is to introduce the recombinant viral vector DNA into an avian egg or avian cell cultures. Transcription and expression of the heterologous protein coding sequences in the above described systems can be monitored. For example, 30 Southern blot analysis can be used to determine copy number of the RG gene. Northern blot analysis can be used to determine copy number of the RG gene. Northern blot analysis provides information regarding the size of the transcribed gene sequence (see, e.g. Maniatis et al., cited above). The level of transcription can also be quantified. Expression of the selected RG protein in WO 01/19968 -15- PCT/USOO/25489 the avian cells or the allantoic fluid of an avian egg can be further verified through western blot analysis and activity tests on the resulting glycoprotein As another example, an expression system employing the avian egg, consisting of the vector pSKII-DC5'-MLP-2BLD-RG-poly(A)-DC3' and DNA Ad 5 CELO as a helper. The vector pSKil- DC5'-MLP-2BLD-RG-poly(A)-DC3' comprises the left terminal of Ad CELO genome, major late promoter, bipartite leader, rabies glycoprotein DNA coding sequence, poly(A) SV40 and the right terminal of the AdCELO genome. Embryonated eggs are co-transfected with this vector and purified Ad CELO DNA, yielding expression of recombinant 10 rabies glycoprotein that accumulates in the allantoic fluid. The following examples will render these and other embodiments of the present invention readily apparent to those of skill in the art. While the example often refers to Fowl adenovirus type 1 (FAV1), it should be understood that this is for the purpose of illustration and that the same features 15 apply to Fowl adenovirus of the other types, specifically Types 1, 2,3,4, 5, 6, 7, 8,9,10,11 and 12 and the invention described herein is intended to cover all of these avian adenoviruses. In addition, it should be understood that for purposes of this invention, any protein can be produced using these various adenoviruses in eggs. 20 Example 1 Using the results of Swedish researchers (J.Virology-1982,v.42,N1,306 310) that describe regions of homology between hexon genes of the human Ad2 and Ad CELO, we synthesized several primers, which uniquely identify 25 sequences of hexon genes of human Ad2. The specificity of primers was determined by sequencing of cloned fragments from Ad CELO and human Ad5. One of the primers was used to synthesize an Ad CELO fragment that hybridized to the human Ad2 hexon gene. Reverse primer from human Ad 2 genome positions: 21164-21185bp 30 5' AGGAACCAGTCTTTGGTCATGT-3' SEQ ID NO: 27 This primer has been used for cDNA synthesis of the Ad CELO hexon gene. Synthesis of the first strand cDNA was carried out with AMV reverse transcriptase. For synthesis of the second strand, RNA-ase H, DNA-pol 1 and WO 01/19968 -16- PCT/USOO/25489 T4 DNA-pol were used. The double stranded cDNA (approximately 2500-3500 nucleotide base pairs), was eluted from agarose gel and cloned into pBluescript 1i SK(+) vector. Clones were selected by molecular weight and by hybridization with 32P-labeled CELO DNA. The most likely sized clones were 5 sequenced from each end of the cloned fragments. Clones all had the primer sequence at their 3' ends while the 5' ends of the cloned fragments had sequences homologous among the tested clones. Hybridization of these clones with 3Pilabeled fragments of Ad CELO (Xbal - B fragment and EcoR I A fragment) has shown, that they contain part of the hexon gene, and the bi 10 partite leader sequences. The promoter for the CELO hexon gene (MLP) is useful in the vectors of invention for the expression of foreign proteins. Example 2 This example describes the generation of the plasmid 15 pSKII-DC5'-MLP-2bLd, which is depicted in figure 1 The plasmid pSKII-DC5' MLP-2bld, was prepared by directional cloning in three sequential steps. 1. Cloning of the 5' terminal of the FAV1 genome First, using oligonucleotide primers and the polymerase chain reaction (PCR), 20 up to 538 base pairs (bp) of the left end of the Ad CELO genome (located between 0 and 538 bp DNA sequence on the Avian adenovirus CELO genome) were amplified and isolated. The primers used were: 5'-CAAGTGGTACCGGCCAAATTGGCCGATGATGTATAATAACCTCA-3' [SEQ ID 25 NO:1] 5'-CAACCAAGCTTCTCTTCCGAAGTCATCTG-3' [SEQ ID NO:2} 30 The 538 bp amplified sequence is presented in Table I [SEQ ID NO:21].

WO 01/19968 -17- PCT/USOO/25489 The amplified DNA contained the essential origin (ori) and packaging sequences (pkg). This kPCR fragment (Kpnl-ori/pkg-Hindlll) was digested only with Hindli and inserted into the pSKil vector which was digested with Hindlll and Smal. This construct was used to delete an EcoRi site from the DC5' 5 fragment (Kpnl-ori/pkg-Hindlll). The plasmid (pSKII-DC5' was digested with EcoRI and the site was filled using T4 DNA polymerase. The vector was then re-ligated. The deleted pSKIl-DC5' EcoRl[-]) was digested with Kpnl-Hindlll. The resulting fragment of DC5' EcoRl[-] was isolated and ligated to the Kpnl Hindlll cut pSkll vector. The region located between 538 bp and 1988 bp 10 genome of Ad CELO was thereby deleted. 2. Cloning of the Major Late Promoter MLP Second, a region of Major late promoter is found near 7000 bp, with a TATA box et 7488 bp (Chiocca, S., et al., J. Virol., v.70,N5, 2939-2949, 1996) was generated by PCR using the following primers: 15 5'-CAACTAAGCTTGAGCTGTACGTGTCACTTCC-3' [SEQ ID NO:3] 5'-CAACAGAATTCCTGGAAGTCGAGGCGACC-3' [SEQ ID NO:3] 20 The amplified DNA contained the major late promoter (MLP). The pSKII vector was then opened with Hindill-EcoRi, and the PCR fragment Hindill MLP-EcoR1 was inserted therein. The sequence is presented in Table I [SEQ ID NO:22). 25 3. Isolation and cloning the bipartite leader sequence. Nine-day-old chicken embryos were inoculated with 0.1 ml CELO virus on concentration 108 virions/ml on one embryo. The inoculation procedure followed was that described in (Rev. Roum. Med. Virol. (1985) 36,4, 235-240). 30 Eighteen to twenty hours after infection, the two embryos were cooled on ice. Chorioallantoic membranes were isolated as described and rinsed with cold phosphate-buffered saline. The washed membranes were then blended in WO 01/19968 -18- PCT/USOO/25489 Potter-Elvehjem homogenizer in buffer A (10 mM Hepes pH 7,5: 25 mM NaCl; 5 mM MgCl2 ). The homogenate was lysed by adding 5% Triton X-100 in buffer A. Nuclei were pelleted by centrifugation for 10 min at 3000 rpm. The supernatants were added to 3% SDS and then subjected to phenol extraction 5 (2x), chloroform extraction (2x), and ethanol precipitation. Total cytoplasmic RNA were dissolved in 4M guanidinium isothiocyanate in 40 mM Tris-HCI (pH 7,4), containing 20 mM NaCl, and the RNA was sedimented through 5.7 M CsCl cushions by centrifugation for 20 h at 150,000 x g (+4C). Pellets were re suspended in 6 M Guanidine hydrochloride. 0.025 volume of 1 M acetic acid 10 and 0.5 volume of ethanol were then added and the RNA was precipitated as above. The RNA pellet was dissolved in a minimum volume of DEP-treated water and used for to produce double stranded cDNA by RT/PCR as described by Grabko. (Grabko, V.I., FEBS Letters, v.387, pp.189- 192, 1996): Reverse transcriptase (RT) mixture (20 ul) contained 67 mM Tris-Cl (pH 15 8,8), 16.6 mM (NH 4

)

2

SO

4 , 0.25 mM each out of four deoxynucleoside triphosphates (dATP, dCTP, dGTP, dTTP), 2 mM MnCl 2 , or 1,5 mM MgCl 2 , 20 pM Xbal primer (reverted), 5 units of Taq or Tth DNA polymerases and variable amounts of virion RNA (0.2 ug to 2 pg). RNA template was added to RT mixture heated to 600C, 40 ul of mineral oil was then overlaid and 20 incubated for 3 min at 600C, the process continued for 15 min at 700C. Following the RT reaction there were added 80 ul of PCR mixture containing 67 mM Tris-HCI pH 8.8, 16.6 mM (NH4) 2

SO

4 , 0.01% Tween-20, 0.75 mM EGTA, 0.25 mM each out of four deoxynucleoside triphosphates, 2 mM MgCl 2 and finally 20 pM EcoRI of the primer (direct). The general incubation mixture 25 (100ul) was then amplified in a DNA Thermal Cycler (Perkin-Elmer Cetus Instruments) as follows: 1 min at 940C,1 min at 560C , and 1.5 min at 72*C for 35 cycles. Aliquots (5 ul) were analyzed by electrophoresis on 1% agarose gel. RT/PCR incubation mixture containing the cDNA amplified fragment was extracted with chloroform and precipitated with 3 ethanol volumes. The 30 amplified fragment and plasmid pBluescript il SK(+) were hydrolyzed with restriction enzymes Xbal and EcoRI, and ligated by means of T4 DNA ligase. E. coli DH5 cells were transformed and the recombinant clones were screened by amplification of the inserted fragment by PCR and subsequent electrophoresis on 1 % agarose gel.

WO 01/19968 -19- PCT/USOO/25489 The primers used were: 5'-CATGGAATTCCAGGTCTACGCCGACGAGAGGATCG-3' [SEQ ID NO:5] 5 5'-CAACTCTAGAGCCTGAATTTGTTTTTCAAGTC-3' [SEQ ID NO:6] 10 The amplified DNA contained the bipartite leader sequence (2bld) and the sequence of hexon mRNA. The pSKII vector was then opened with EcoRI-Xbal, and the PCR fragment EcoRI-2bld-5'hex-Xbal was inserted therein. Only the bipartite leader was obtained by PCR using the following 15 primers: 5'-CATGGAATTCGACTTCCAGGTCTACGGCGACGAGAGG-3' [SEQ ID NO:7] 20 5'-CCTGGATCCGATGTGTTCCTTGAACCAAAC-3' [SEQ ID NO:8] The amplified DNA contained only the bipartite leader sequence (2bld). The pSKII vector was then opened with EcoRl-BamHl, and the PCR fragment 25 EcoRI-2bld-BamHI was inserted therein. The pSKII vector contained the essential origin (ori) and packaging sequences (pkg) (Kpni-ori/pkg-Hindlll) was opened with Hindill - EcoRi, and the Major late promoter (MLP) was ligated to the ori and pkg elements. Then the pSKIl vector contained (Kpnl-ori/pkg HindIll-MLP-EcoRI) was opened with EcoRI - BamHI, and the bipartite leader 30 sequence was ligated to the ori/pkg and MLP elements.

WO 01/19968 -20- PCT/US00/25489 Example 3 This example describes the generation of the plasmid pSKII-DC5'-MLP 2BLD-RG-p(A), which is depicted in Figure 2. The plasmid pSKII-DC5'-MLP 2BLD-RG-p(A), was prepared by directional cloning in three sequential steps. 5 First, the glycoprotein gene of the rabies virus vaccine strain Vnukovo-32 was made from the virus RNA using oligonucleotide primers, reverse transcriptase (Amersham) and PCR, up to 1640 bp DNA sequence of the glycoprotein gene of Rabies virus were amplified and isolated. The primers used were: 10 5'-GGATCCAGGAAAGATGGTTCCTCAGGCTCTCCTGTTTG-3' [SEQ ID NO:9] 5'-GCTGCAGCAAGGGGAGGTGATCTTCAGACTTGGATCGT-3' [SEQ ID NO:10] The amplified DNA contained the glycoprotein gene of the rabies virus 15 vaccine strain Vnukovo-32 sequence (RG). The pSKII vector was then opened with BamHI-Pstl, and the PCR fragment BamHI-RG-Pstl was inserted therein. Second, using oligonucleotide primers and the polymerase chain reaction (PCR), up to 240 base pairs (bp) of the genome SV40 (located between 2530 and 2770 bp DNA sequence on the simian virus 40 genome) were amplified 20 and isolated. The primers used were: 5'-CAATCTGCAGATCATAATCAGCCATACCAC-3' [SEQ ID NO: 11] 25 5'-CAACTCTAGATCCAGACATGATAAGATACATTG-3' [SEQ ID NO:12] The amplified DNA contained the poly(A) site of SV40 . The pSKII vector was then opened with BamHl-Xbal, and the fragment BamHl-RG-Pstl and also the PCR fragment PstI-p(A)-Xbal was inserted therein. Third, the 30 pSKII vector contained (Kpnl-ori/pkg-Hindlll-MLP-EcoRl- 2bld-BamHl) was opened with BamHI-Xbal, and the fragment BamHI-RG-PstI- p(A)-Xbal was WO 01/19968 -21- PCTIUSOO/25489 inserted therein. The sequence of the poly (A) SV40 is presented in Table V [SEQ ID No:25]. Example 4 5 This example describes the generation of the plasmid pSKII-DC5'-MLP 2bLD- RG-p(A)-DC(Xbal-Notl), which is depicted in figure 3. The plasmid pSKIl-DC5'- MLP-2bld-RG-p(A)-DC(Xbal-Notl) was prepared by directional cloning in one step. The pSKIl vector contained (Kpni-ori/pkg-Hindlll-MLP EcoRl-2bld-BamHl- RG-Pstl-p(A)-Xba) was opened with Xbal-Notl, and the 10 fragment Ad CELO genome Xbal-(located between 2 to 17.4 kb)-Notl was inserted therein. The collection of recombinant Ad CELO genome was prepared by in vitro ligation in one step, which is depicted in figure 4.The pSKII vector contained (Kpnl- Sfil-ori/pkg-Hindlll-MLP- EcoRl-2bld-BamHl-RG-Pstl p(A)-Xbal-Ad CELO-Notl) was opened with Sfil-Not, and were ligated with the 15 fragment Ad CELO genome Notl-(located between 17.4 to 48.3 kb)-3' end Ad CELO genome. This ligation mixture (about 1 ug/one embryo) were injected into the allantoic cavity of nine-day-old chicken embryos. After a 72-h incubation at 37 0 C in a humidified incubator, the allantoic fluid was harvested and were assayed for rabies glycoprotein recombinant protein. 20 Example 5 Construction of a CRP expressing clone We constructed a clone that would express the subunit in the mature form with the N-terminal leader peptide removed-mimicking the form found in 25 the crystallized molecule as described in the structure data entry PDB:1GNH of the Protein Data Bank our recombinant construct therefore encodes a protein beginning with the sequence (NH- GLN THR ASP MET SER ARG ...... ) [SEQ ID NO:23]. We considered the fact that though the embryonated egg system could, in principle, correctly cleave the precursor form of CRP, this 30 might not yield the same localization result in the CELO infected cells. Viral proteins are specifically exported into the allantoic fluid by CELO infected cells WO 01/19968 -22- PCTIUSOO/25489 and we would like the same thing to occur with the CRP subunits or pentamers. In order to make a correct protein, we therefore artificially added a methionine to the N-terminal end of the construct protein (NH- MET GLN THR 5 ASP MET SER ARG ....... ) [SEQ ID NO:13] by introducing a methionine codon in the correct reading frame. Since most proteins in eukaryotic cells are processed non-specifically to remove the N-terminal methionine by cellular enzymes, we believe the addition could actually enhance the chance of a correct product. 10 PCR Primers The design of primers for introducing restriction sites and constructing the recombinant CRP (rCRP) molecule described above was done using the Genbank (Acc.No. X56692) sequence as a guide. The primers introduce restriction sites that are used for building the construct (figure 5). 15 5' primer 5'- CGTTAGGATCCATGCAGACAGACATGTCGAGGAAGGC-3' [SEQ ID NO:13] 3' primer 20 3'- CCAAGCGGCCGCTGCAGTCATCAGGGCCACAGCTGGGGTTTGGT-3' [SEQ IDNO:14] I primer-For (37 mer) 5'-CGTTAGGATCCATGTCGAGGAAGGCT1TGTGTTTCC-3' [SEQ ID NO:15] 25 11 primer-Rev (38 mer) 5'-CCAATCTGCAGTCATCAGGGCCACAGCTGGGGTTTGGT-3' [SEQ ID NO:16] 30 WO 01/19968 -23- PCT/USOO/25489 Plasmid DNA was isolated from overnight cultures of the ATCC 324978 clone and digested with EcoRI and Xhol restriction enzymes. The predicted fragment size of about 1.8 kb was observed on the gels. Plasmid DNA was 5 used as the template for a PCR reaction with the primers described in the materials and methods section. Consistent with the published sequence data, a fragment of about 650 bp was observed. In the photograph of an agarose gel shown in Figure 6, the product of PCR amplification with the CRP primers is shown along side size markers. The sequence of coding sequence encoded by 10 the PCR insert for our recombinant human CRP protein is shown in Table IlIl [SEQ ID NO:23]. The PCR fragment was incorporated in a construct pSKII-DC5'-MLP 2bld-hCRP-p(A)-DC3'. Selection of the recombinants at each stage was carried out by restriction digests and fragment size determinations on agarose 15 gels. The terminal repeat sequence of the virus (TR), essential for viral genome replication is also included in the construct . The cloned human C-reactive protein in the egg resulted in a recombinant that is identical to the normal protein in its ability to pentamerize and its immunochemical cross-reactivity with CRP specific antisera. The 20 results are presented in Figure 7. In another experiment, the recombinant CRP was assayed for it's ability to bind to O-phophorylethanolomine sepharose, a matrix commonly used for the biospecific absorption of CRP. The results suggest that the recombinant CRP forms a pentamer since this is required for efficient binding to the matrix (Figure 8). 25 Example 6 Cloning of Rabies Virus Glycoprotein The nucleotide and deduced amino acid sequence of the glycoprotein 30 gene of rabies virus vaccine strain Vnukovo-32 is known (Fodor, I., Grabko, V.I., et al., Arch. Virol. v.135, N3-4, 451-459, 1994). The rabies virus vaccine strain Vnukovo-32 was propagated on primary hamster kidney cells or monkey kidney cell line 4647. After infection, supernatants were clarified by WO 01/19968 -24- PCTIUSOO/25489 ultrafiltration and viral particles concentrated by ultracentrifugation. Pellets were re-suspended in 4M guanidinium thiocyanate and the viral RNA was isolated by phenol: chloroform extraction and ethanol precipitation. The cDNA was made from the RNA using a 3' end-specific primer: 5 5'-GGATCCAGGAAAGATGGTTCCTCAGGCTCTCCTGTTTG-3' [SEQ ID NO:17], which overlaps the translation initiation (ATG) codon using reverse transcriptase (Amersham). The cDNA was amplified using PCR (GeneAmp, 10 Perkin Elmer Cetus), the 3'- and a 5-specific primer. The 5'-specific primer had the following sequence: 5'-GCTGCAGCAAGGGGAGGTGATCTTCAGA CTTGGATCGT-3' [SEQ ID NO:18], 15 The 5'-primer contained the stop-codon. The PCR product was electrophoresed on a 1% agarose gel and the band of expected size of double stranded DNA was excised from the gel and subsequently cleaved with BamHI and Pstl. The cleaved product was ligated with a similarly treated pUC19 vector. Clones were obtained after transformation of E.coli DH5 bacterial cells 20 on plates treated with X-gal. Plasmid DNAs containing insert were identified by agarose gel electrophoresis after cleavage with BamHl and Pstl. Both strands of cDNA were sequenced by the dideoxynucleotide termination method with the SEquenase kit (USB, USA). DNA sequences were analyzed by computer using "GCG" package version 7.1. The nucleotide 25 and deduced amino acid sequences for the glycoprotein have been submitted to EMBL Date Library and have been assigned accession no. X71879. The deduced sequence of the polypeptide of 524 amino acids is identical in size and organization to most previously characterized rabies glycoproteins. When the nucleotide sequences of eight gp G genes were compared, Vnukovo-32 30 strain had the greatest homology with ERA (99.4%) and with SAD B1 9 (99.1%). Similar results were obtained when the deduced amino acid sequences were analyzed. The functional activity of the glycoprotein G gene WO 01/19968 -25- PCTIUSOO/25489 was investigated in bacterial cells (Grabko V... Fragment of DNA coding synthesis glycoprotein G of Rabies virus, recombinant plasmid DNA coding glycoprotein G of rabies virus, strain of bacteria E.coli - Production of Glycoprotein G of Rabies virus. Patent of Russia, N2008355, 1994). The gene 5 glycoprotein G inserted in pUC18 vector under lacZ promoter. Recombinant plasmid DNA transformed the E.coli strain DH5. Clone PVG18-1 has shown high immunogenic activity of rabies glycoprotein G . Example 7 10 This example describes the generation of the plasmid DC5'-GRV-DC3' (pSKIl-DC5'-MLP-2BLD-GRV-p(A)SV40-DC3'-pSKII), which is depicted in Figures 9. The plasmid pSKIl-DC5'-MLP-2BLD-GRV-p(A)SV40-DC3'-pSKII was prepared by directional cloning in two sequential steps. 1. Cloning of the 3' terminal of the FAV 1 genome 15 First, using oligonucleotide primers, DNA Ad CELO and the polymerase chain reaction (PCR), up to 830 base pairs (bp) of the right end of the Ad CELO genome (located between 42995 bp and 43804 bp DNA sequence on the Avian adenovirus CELO genome) were amplified and isolated. The primers used were: 20 5'-CAACCTCTAGACATCACCATAGCAATCATTGG-3' [SEQ ID No:19] Xbal 42995 - 43016 bp Ad CELO 5'-CATTCGCGGCCGCGATGATGTATAATAACCTCAAAAACTAACG-3' NotI 43804 - 43774 bp Ad CELO [SEQ ID No20] 25 The 830 bp amplified sequence is presented in Table VI.

In a second step, the amplified DNA contained the essential origin (ori). The pSKII vector (Stratagen cloning Systems, La Jola, CA) was then digested with Xbal-Notl, and the PCR fragment DC3' (Xbal-ori-NotI) was inserted therein. 30 The vector pSKIl-DC3' (Xbal-ori-Notl) was cut at the Xbal, Notl sites and Xbal-Notl fragment containing the 3' terminal of the FAVI genome was WO 01/19968 PCT/USOO/25489 -26 isolated. This fragment containing the 3' terminal of the FAV1 genome (Xbal Notl) was inserted into the Xbal-Notl digested vector pSKII-DC5'-MLP-2bid GRV-p(A)SV40. The ligated DNA was transformed into E.coli and the correct plasmid was identified by restriction with the enzymes Xbal, HindIll, Kpnl, 5 EcoRI, BamHl, Notl. Conditions for all restrictions were as recommended by manufacturer (New England BioLabs). Example 8 Expression of the Rabies G Protein in embryonated eggs The rabies glycoprotein can also be cloned in the vectors of the 10 invention. The sequence is presented in SEQ ID NO:24. The complete CELO (FAVI) virus genome sequence is presented in Table VIII [SEQ ID NO:27]. The expression of rabies glycoprotein G in allantoic fluid of infected (R) and control (C) eggs is shown in Figure 10. Based on these results, the system of this invention could produce enough material for multiple doses of 15 vaccine in each egg. Example 9 This example describes the transfections of avian eggs which were done by recombinant adenovirus vector DNA (DC5'-GRV-DC3') and DNA Ad 20 CELO as a helper. Transfections were done according Grabko V.I. (Acta Virologica 1987,v.31,N1 -2,pp.97-102). Typically, deproteinized virion DNA Ad CELO preparations were diluted in sterile saline (1-5ug per embryo) and vector DNA DC5'-GRV-DC3' (pSKIl-DC5'-MLP-2bd-RG-p(A)SV40-DC3'-pSKll) which 25 preliminary was cuted Sfil-Notl also was diluted in same sterile saline (5-10 ug per embryo). This mixture introduced via individual glass capillaries into the allantoic cavity of 9-day-old chicken embryos. The embryos were incubated for 72-96 hr at 37C. After the embryos were chilled, the allantoic fluid was collected and assayed for protein production.

WO 01/19968 -27- PCT/USOO/25489 The Bio-Rad Protein assay, based on the method of Bradford (Anal. Biochem.-1976, 72, 248-) was used as a simple and accurate procedure for determining concentration of solubilized protein. It involves the addition of an acidic dye to protein solution, and subsequent measurement at 595 nm with a 5 spectrophotometer. Comparison to a standard curve provides a relative measurement of protein concentration. Protein levels may be measured by Western blots (immunoblots) using the anti-RGP mouse monoclonal antibody was provided by "Capricon Products, Inc" (USA) and Blotting grade affinity purified Goat anti-mouse IgG 10 (H+L) Alkaline phosphatase conjugate was provided by "Bio-Rad". The purity of the samples was analyzed by SDS-PAGE (4-20% gels) followed by staining with Gradipure electrophoresis gel stain (Figure 11). Table 7 summarizes the actual experimental transfections performed to determine the effect of the above described vectors (alone and in combination with DNA Ad CELO) on 15 rabies glycoprotein G production. All references, including publications and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. While this invention has been described with an 20 emphasis upon preferred embodiments, it will be apparent to those of ordinary skill in the art that the preferred embodiments can be varied. In particular, it is noted that any protein can be expressed in the transgenic egg system of this invention. Similarly, it is intended that the invention can be practiced otherwise than as specifically described herein. Accordingly, this invention includes all 25 modifications encompassed within the spirit and scope of the appended claims.

Claims (43)

1. An egg comprising a recombinant protein.

2. The egg of claim 1 wherein said egg is a fowl egg. 5

3. The egg of claim 1 wherein said egg is any egg of an avian species.

4. An egg comprising recombinant DNA. 10

5. The egg of claim 4 wherein said egg is a fowl egg.

6 The egg of claim 4 wherein said egg is any egg of an avian species.

7. A method of preparing a recombinant avian adenovirus vector 15 containing a heterologous gene, comprising the steps of: (a) preparing a plasmid, cosmid, or phage containing avian adenovirus DNA, said DNA comprising: (i) at least one transcriptional regulatory sequence next to at least one restriction endonuclease site, and; 20 (ii) DNA from a non-essential region of Ad CELO genome flanking said transcriptional regulatory sequence and said restriction endonuclease site; and (b) inserting at least one protein coding sequence from a foreign gene into said restriction endonuclease site next to said transcription! 25 regulatory sequence.

8. The method according to Claim 7, wherein the Ad CELO DNA further comprises DNA preceding and including the site at which RNA synthesis starts. WO 01/19968 -29- PCT/USOO/25489

9. The method according to Claim 7, wherein the transcriptional regulatory sequence regulates all viral mRNAs processed from major late transcripts. 5

10. The method according to Claim 7, wherein said avian adenovirus DNA is Ad CELO DNA.

11. The method according to Claim 7, wherein said protein coding sequence from a foreign gene comprises the site corresponding to initiation of 10 translation of said foreign gene and DNA extending beyond the translational termination site of said foreign gene.

12. The method according to Claim 11, wherein the protein coding sequence is obtained from a DNA copy of a DNA gene or a DNA copy of a 15 RNA gene.

13. The method according to Claim 7, wherein said non-essential region comprises the region Ad CELO from 1% to 8% map of Ad CELO. 20

14. The method according to Claim 7, wherein the plasmid formed is one of the group consisting of Kpnil-DC5'-Hindill, Hindill-MLP-EcoRl, EcoRI-2bld BamHl, BamHI-GRV-Pstl,Pstl-p(A)SV40-Xbal, Xbal-DC -Notl.

15. A method for genetically modifying a avian adenovirus, comprising the 25 steps of: (a) preparing a vector comprising plasmid, cosmid, or phage containing avian adenovirus DNA, said DNA comprising: (i) at least one transcriptional regulatory sequence next to at least one restriction endonuclease site, and WO 01/19968 -30- PCT/USOO/25489 (ii) DNA from a non-essential region of avian adenovirus flanking said transcriptional regulatory sequence and said restriction endonuclease site; and (b) inserting at least one protein coding sequence from a foreign gene 5 into said restriction endonuclease site next to said transcriptional and translational regulatory sequences; (c) providing at least one cell infected with a recombinant avian adenovirus; (d) isolating from said cell a recombination avian adenovirus capable of 10 expressing said protein coding sequence.

16. A method of using a recombinant avian adenovirus as a vaccine, comprising the steps of: (a) preparing an infectious avian adenovirus containing therein a 15 chimeric gene comprising at least one avian adenovirus transcriptional and translational regulatory sequences and at least one protein coding sequence from a foreign gene, said chimeric gene being flanked by DNA from a non-essential region of avian adenovirus genome, such that said infectious avian adenovirus is capable upon infection of a cell 20 of expressing said protein coding sequence; and (b) inoculating an animal or human with an inoculant containing a concentration of said recombinant protein sufficient to elicit an immunological response in said animal or human. 25

17. A method according to Claim 16, wherein said immunological response comprises the production of antibodies to at least the antigenic portion of the protein encoded by said protein coding sequence.

18. A vector comprising: 30 (a) a plasmid, cosmid, or phage; WO 01/19968 -31- PCT/USOO/25489 (b) a chimeric gene which comprises at least one avian adenovirus transcriptional and translational regulatory sequences from a foreign gene; and (c) DNA from a non-essential region of avian adenovirus genome, said 5 DNA flanking said chimeric gene.

19. A vector according to claim 18, wherein said protein coding sequence is from a foreign gene selected from the group consisting of rabies virus. 10

20. A vector according to claim 18, wherein said protein coding sequence encodes an immunogenic protein.

21. A vector according to Claim 20, wherein said protein coding sequence encodes at least the antigenic portion of said immunogenic protein. 15

22. A vector according to Claim 21, said vector being selected from the group consisting of Kpnl-DC5'-HindllI, HinIll-MLP-EcoRI,EcoRI-2bld-BamHl, BamHI-GRV-Pstl,Pstl-p(A)SV40-Xbal,DC5'-MLP-2bld-GRV-p(A)-DC(Xbal Notl). 20

23. An infectious avian adenovirus containing therein a chimeric gene comprising at least one avian adenovirus transcriptional and translational regulatory sequences and at least one protein coding sequence from a foreign gene, said chimeric gene being flanked by DNA from a non-essential region of 25 avian adenovirus genome, such that said infectious avian adenovirus is capable upon infection of a cell of expressing said protein coding sequence.

24. An infectious avian adenovirus according to Claim 23, wherein said chimeric gene further comprises avian adenovirus DNA preceding and 30 including the site at which RNA synthesis starts. WO 01/19968 -32- PCT/USOO/25489

25. An infectious avian adenovirus according to Claim 24, wherein said protein coding sequence encodes an immunogenic protein.

26. An infectious avian adenovirus according to Claim 25, wherein said 5 protein coding sequence encodes at least the antigenic portion of said immunogenic protein.

27. An infectious avian adenovirus according to Claim 26, wherein said immunogenic protein is the rabies glycoprotein G antigen. 10

28. An infectious avian adenovirus according to Claim 27, said infectious avian adenovirus being selected from the group consisting of Kpnl-DC5' Hindlil, Hindill-MLP-EcoRl,EcoRI-2bld-BamHl, BamHl-GRV-Pstl, Pstl p(A)SV40-Xbal,DC5'-MLP-2bld-GRV-p(A)-DC(Xbal-Notl). 15

29. An infectious avian adenovirus recombinant produced by a process comprising the steps of: (a) preparing a vector comprising a plasmid, cosmid, or phage containing avian adenovirus DNA, said DNA comprising: 20 (i) at least one transcriptional and translational regulatory sequences next to at least one restriction endonuclease site, and (ii) DNA from a non essential region of avian adenovirus genome flanking said transcriptional regulatory sequence and 25 said restriction site; (b) inserting at least one protein coding sequence from a foreign gene into said restriction endonuclease site next to said transcriptional and translational regulatory sequences

30. An avian adenovirus recombinant vector obtainable through in vitro and in 30 vivo manipulation of avian adenovirus DNA WO 01/19968 -33- PCT/USOO/25489

31. An avian adenovirus recombinant vector according to Claim 30 specified by the fact that it contains: (a) both left and right inverted terminal repeat sequences and at least one packaging signal sequence; 5 (b) at least one transcription regulator sequence and at least one translation stimulator sequence; (c) at least one protein coding sequence; (d) at least one other region of avian adenovirus DNA possibly containing modifications such as insertions and or deletions and or 10 mutations.

32. An avian adenovirus recombinant vector according to Claims 30, and 31, wherein the plasmid formed is one of the group consisting of Kpnl-DC5' Hindlli, Hindlll-MLP-EcoRl, EcoRI-2bld-BamHl, BamHl-GRV-Pstl, Pstl p(A)SV40-Xbal,Xbal-DC3'-Notl. 15

33. An avian adenovirus recombinant vector according to Claims 30, 31, and 32 resident in a plasmid replicable in bacteria or yeast and after introduction into an avian egg or avian cells possibly in combination with avian adenovirus DNA ,or avian adenovirus particles as a helper, capable of producing recombinant protein. 20

34. An avian adenovirus recombinant vector according to Claims 30, 31, 32, 33 containing foreign DNA.

35. An avian adenovirus recombinant vector according to Claim 34 containing foreign DNA coding for a therapeutic protein.

36. An avian adenovirus recombinant vector according to Claim 34 containing 25 foreign DNA coding for an immunostimulatory protein.

37. An avian adenovirus recombinant vector according to Claim 34 containing foreign DNA coding for a protein derived from an human viral or bacterial or protozoan pathogen.

38. An avian adenovirus recombinant vector according to Claim 34 containing 30 foreign DNA coding for a protein derived from an animal viral or bacterial or protozoan pathogen. WO 01/19968 -34- PCTIUSOO/25489

39. An avian adenovirus recombinant vector according to Claim 34 containing foreign DNA coding for a protein derived from an avian viral or bacterial or protozoan pathogen.

40. An avian adenovirus recombinant vector according to Claim 34 containing 5 foreign DNA coding for cytokine protein.

41. An avian adenovirus recombinant vector according to Claim 34 containing foreign DNA coding for a plant protein.

42. An avian adenovirus recombinant vector according to Claim 34 containing foreign DNA coding for a tumor antigen protein or fragment thereof. 10

43. A method for the production of recombinant protein in an avian egg according to one of the claims 30 through 40, characterized by a process comprising the steps of: (a) preparing a vector comprising a plasmid, cosmid, or phage containing avian adenovirus DNA, said DNA comprising: 15 (i) left and right inverted terminal repeat sequences and the viral packaging signal sequence; and (ii) at least one transcription regulator and translation stimulator sequences; and (iii) at least one protein coding sequence from a foreign gene 20 into said restriction endonuclease site next to said transcriptional and translational regulatory sequences; and (b) preparing a mixture of said vector DNA with purified adenovirus DNA or whole adenovirus particles; (c) Introducing said mixture into an embryonated avian egg or an avian 25 cell culture. (d) Incubating the avian egg so treated for a period of time. (e) Harvesting fluids from the egg after such period of time containing the specific recombinant protein molecule encoded by the said vector DNA. 30