EP1232248A1 - Recombinant eggs and gene cloning and expression vectors based on avian adenoviruses - Google Patents

Abstract

We describe a new technology for producing eggs containing recombinant proteins based on the novel use of avian adenoviruses (AAV). Adenovirus infected embryonated eggs are used as the host organism. The proteins synthesized are isolated in high amounts per egg and purified excluding time-consuming steps of purification. As far as is known, the mechanisms of translation and post-translational modification of proteins in avian species fully resembles those of other higher eukaryotes such as humans, veterinary animals, and plants. In contrast with bacterial, yeast and plant host systems for producing recombinant proteins, avian embryos allow isolation of proteins in biologically active form free of endotoxins. The combination of high productivity and possibility of obtaining proteins in native form makes the system a unique choice for biotechnology, much more favorable than other hosts currently being used for this purpose.

Description

RECOMBINANT EGGS AND

GENE CLONING AND EXPRESSION VECTORS BASED ON AVIAN ADENOVIRUSES

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.

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 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 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

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 animals. For example, influenza vaccines for humans and animals, along with many poult^ry 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 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 incubation; 4. correct processing of eukaryotic proteins; 5. simple purification procedures from egg fluid; 6. easy scaleability for mass proαuction; 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 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, compπsing 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; (ii) DNA from a non-essential region of Ad FAV1 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 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 DNA comprising: (i) at least one transcriptional regulatory sequence next to at least one restriction endonuclease site, and (n) 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 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 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 chimeπc gene comprising at least one avian adenovirus transcriptional and translational regulatory sequences and at least one protein coding sequence from a foreign gene, said chimeπc 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 response in the animal or human.

The invention is further directed to a vector comprising: (a) a plasmid, cosmid, or phage; (b) a chimeπc 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 DNA flanking said chimeπc gene.

The invention is further directed to an infectious avian adenovirus containing therein a chimeπc gene comprising at least one avian adenovirus transcriptional and translational regulatory sequences and at least one protein coding sequence from a foreign gene, said chimeπc gene being flanked by DNA from a non-essential region of avian adenovirus genome, such that said infectious avian adenovirus is capable upon infection ot 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" (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 (u) DNA from a non essential region of avian adenovirus genome flanking said transcriptional regulatory sequence and 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

In an alternative embodiment the invention is directed to producing 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- essential" regions of the virus that can be replaced without affecting the required mfectivity 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 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 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 c/s-acting elements required for viral DNA replication and packaging, mainly the inverted terminal repeat sequences (including origins of viral replication) 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 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 translation at least one protein coding sequence from a foreign gene and inverted adenovirus termini required for vector rβDlication, and non-defective adenovirus particles as a helper

Thus, in view of this disclosure, skilled genetic engineers can construct 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 frans-activators 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 activity of a protein of interest

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'- MLP-2bLD.

Figures 2 show a schematic diagram representing plasmid psKII-DC5'- MLP-2bLD-RGSV40-p(A).

Figure 3 shows a schematic diagram representing plasmid pSKII-DC5'- MLP-2bLD-RG-p(A)-DC(Xbal-Notl). 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) 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 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. Allantoic fluid and control samples were incubated with O- 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 virus infected; 4. recombinant CRP virus infected; 5. CRP purified from human fluids; 6. Uninfected allantoic fluid; 7. Molecular Weight Standards; 8. CRP purified from human fluids.

Figure 9 shows a schematic diagram representing the preparation of the plasmid pSKII-DC5'-MLP-2BLD-GRV-pA-DC3'

Figure 10 shows expression of rabies glycoprotein G in allantoic fluid of 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 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 virus DNA transfected embryos (protein sample boiled before loading) 4.

Allantoic fluid from CELO virus DNA transfected embryos; 5. Allantoic fluid from untransfected embryos;

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

To ensure a complete understanding of the invention, the following definitions are provided Adenovirus Any virus belonging to the family Adenoviπdae. The large adenovirus family (45 types) is divided by natural host range into adenoviruses that infect mammals (the Mastadenovindae) and adenoviruses that infect avian specιes( Aviadenoviπdae)

Avian Adenovirus: An adenovirus of the Aviadenoviπdae family There 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 with Fowl Adenovirus stereotype 1 (FAV-1) or Avian Adenovirus stereotype 1

(AAV1) CELO is an avian adenovirus of the Adenoviπdae 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 vinon 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 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 to add a sequence of adenyhc acid residues to the 3'-end of the mRNA. 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; 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 cytomegaiovirus major envelope glycoprotein; Pseudorabiesvirus glycoprotein; Vesicular stomatitis virus glycoprotein; respiratory syncitial virus envelope 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 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 kinase; pituitary proteins; endoglin; beta-2-mocroglobulin antigen; human C- reactive protein; fatty acid binding protein; Human chorionic gonadotrophin; neuron specific eπolase, Human growth hormone; Cytomegaiovirus envelope proteins, Epstein Bar virus proteins, hepatitis core antigen; Dilofilaria immitis glycoprotein; Bovine Leukemia virus glycoprotein; mumps virus envelope 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 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 preferably 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 30 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleot'des, and most preferably 1 10 nucleotides Sequence identity is typically measured using sequence analysis software (e g , Sequence Analysis Software Package of the Genetics 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; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; senne, 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 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 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 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. 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. Positioned for Expression Positioned for express'on means that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i e , facilitates tne production of e g , a recombinant polypeptide or RNA molecule) 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'- terminal and 3'-termιnal 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

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 that infect mammals (the Mastadenovindae) and adenoviruses that infect avian species (the Aviadenovindae). 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 adventitious contaminant of embryonated eggs during efforts to propagate a bovine skin disease agent (Van den Ende, M., et al., J. Gen. Mιcrobιol.,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 adenovirology for a number of yea^rs. 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 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 viπon by virus-encoded core proteins. The CELO virus genome has covalently attached 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 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, Ilia, fiber, pVI, pVII, and pVIII) are present and in the expected locations in the genome. The early region 2 (E2) genes (encoding DNA-binding protein, DNA polymerase, and terminal protein) are also present. However, CELO virus lacks sequences homologous to the mammalian adenovirus E1 , 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 reading frames (ORFs), and it is likely that these encode functions that replace the missing E1 , 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- 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 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 fragment up to 2700 n.b. can be inserted into a native genome of Ad CELO (into a non-essential region) without affecting the success o* viral replication If the non-essential region (about 6% of the genome) is deleted however, the genome caDacity can presumably be increased by 2000-3000 additional base pairs 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 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 assembly in vitro that a recombinant infectious DNA formed, which induces the reproduction of infectious recombinant vinons 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 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 neutralizing antibodies (Wiktor, T.J., et al., J. Am. Med. Assoc. v.224, 1170-

1171 , 1973) and MHC class I- or class ll-restncted cytotoxic T cells (Celis, E., et al., J. lmmunol.,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 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 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 cf expressing foreign genes (Mackett, M., et al., Proc. Natl. Acad. Sci. USA, v.79, 7415-7419, 1982) and of protectively immunizing animals against infections with rabies virus (Wiktor, T.J., et al., Proc. Natl. Acad. Sci. USA v.81 , 7194-7198, 1984) and many other viruses.

Vaccinia virus may also be used as a cloning and expressing vehicle (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 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 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 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, 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 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 CELO as a helper. The vector pSKII- 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 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 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.

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 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 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 T4 DNA-pol were used The double stranded cDNA (app^rcxιmately 2500-3500 nucleotide base pairs), was eluted from agarose gel and cloned into pBluescnpt II SK(+) vector Clones were selected by molecular weight and by hybridization with ³²P-labeled CELO DNA The most likely sized clones were 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 ³²P labeled 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- 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 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), 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 NO:1]

δ'-CAACCAAGCTTCTCTTCCGAAGTCATCTG-S' [SEQ ID NO.2]

The 538 bp amplified sequence is presented in Table I [SEQ ID

NO:21 ] The amplified DNA contained the essential origin (on) and packaging sequences (pkg). This kPCR fragment (Kpnl-oπ/pkg-Hindlll) was digested only with Hindu and inserted into the pSKII vector which was digested with Hindlll and Smal. This construct was used to delete an EcoRI site from the DC5' 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 pSKII-DC5' EcoRI[-]) was digested with Kpnl-Hindlll. The resulting fragment of DC5' EcoRI[-] was isolated and ligated to the Kpnl- Hindlll cut pSkli vector. The region located between 538 bp and 1988 bp 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:

5'-CAACTAAGCTTGAGCTGTACGTGTCACTTCC-3' [SEQ ID NO:3]

5'-CAACAGAATTCCTGGAAGTCGAGGCGACC-3' [SEQ ID NO:3]

The amplified DNA contained the major late promoter (MLP). The pSKII vector was then opened with Hindlll-EcoRI, and the PCR fragment Hindlll- MLP-EcoRI was inserted therein. The sequence is presented in Table II [SEQ ID NO:22].

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). 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 Potter-Elvehjem homogenizer in buffer A (10 mM Hepes pH 7,5, 25 mM NaCl, 5 mM MgCI₂ ) The homogenate was lysed by adding 5% Triton X-100 in buffer A Nuclei were pelleted by centnfugation for 10 mm at 3000 rpm The supernatants were added to 3% SDS and then subjected to phenol extraction (2x), chloroform extraction (2x), and ethanol precipitation Total cytoplasmic

RNA were dissolved in 4M guamdinium isothiocyanate in 40 mM Tns-HCI (pH 7,4), containing 20 mM NaCl, and the RNA was sedimented through 5.7 M CsCI cushions by centnfugation for 20 h at 150,000 x g (+4C). Pellets were resuspended in 6 M Guanidine hydrochloπde. 0.025 volume of 1 M acetic acid 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 Tns-CI (pH 8,8), 16.6 mM (NH₄ )₂S0₄ , 0.25 mM each out of four deoxynucleoside tπphosphates (dATP, dCTP, dGTP, dTTP), 2 mM MnCI₂, or 1 ,5 mM MgCI₂ , 20 pM Xbal primer (reverted), 5 units of Taq or Tth DNA polymerases and variable amounts of vinon RNA (0.2 ug to 2 pg). RNA template was added to RT mixture heated to 60°C, 40 ul of mineral oil was then overlaid and incubated for 3 mm at 60°C, the process continued for 15 m at 70°C.

Following the RT reaction there were added 80 ul of PCR mixture containing 67 mM Tns-HCI pH 8.8, 16.6 mM (NH^SO.,, 0.01 % Tween-20, 0.75 mM EGTA, 0.25 mM each out of four deoxynucleoside tπphosphates, 2 mM MgCI₂ and finally 20 pM EcoRI of the primer (direct). The general incubation mixture (100ul) was then amplified in a DNA Thermal Cycler (Perkin-Elmer Cetus

Instruments) as follows: 1 m at 94°C,1 mm at 56°C , and 1.5 min at 72°C for 35 cycles. A quots (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 amplified fragment and plasmid pBluescnpt II 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. The primers used were:

5'-CATGGAATTCCAGGTCTACGCCGACGAGAGGATCG-3' [SEQ ID NO:5]

S'-CAACTCTAGAGCCTGAATTTG I I I I I CAAGTC-3' [SEQ ID NO:6]

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 primers:

5'-CATGGAATTCGACTTCCAGGTCTACGGCGACGAGAGG-3' [SEQ ID NO:7]

S'-CCTGGATCCGATGTGTTCCTTGAACCAAAC-S' [SEQ ID NO:8]

The amplified DNA contained only the bipartite leader sequence (2bld). The pSKII vector was then opened with EcoRI-BamHI, and the PCR fragment EcoRI-2bld-BamHI was inserted therein. The pSKII vector contained the essential origin (ori) and packaging sequences (pkg) (Kpnl-ori/pkg-Hindlll) was opened with Hindi 11 - EcoRI, and the Major late promoter (MLP) was ligated to the ori and pkg elements. Then the pSKII vector contained (Kpnl-ori/pkg- Hindlll-MLP-EcoRI) was opened with EcoRI - BamHI, and the bipartite leader sequence was ligated to the ori/pkg and MLP elements. Example 3

This example describes the generation o^f 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 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:

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 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 and isolated. The primers used were:

5'-CAATCTGCAGATCATAATCAGCCATACCAC-3' [SEQ ID NO:11]

S'-CAACTCTAGATCCAGACATGATAAGATACATTG-S" [SEQ ID NO 12]

The amplified DNA contained the poly(A) site of SV40 . The pSKII vector was then opened with BamHI-Xbal, and the fragment BamHI-RG-Pstl and also the PCR fragment Pstl-p(A)-Xbal was inserted therein. Third, the pSKII vector contained (Kpnl-oπ/pkg-Hindlll-MLP-EcoRI- 2bld-BamHI) was opened with BamHI-Xbal, and the fragment BamHI-RG-Pstl- p(A)-Xbal was inserted therein. The sequence of the poly (A) SV40 is presented in Table V [SEQ ID No:25j.

Example 4 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 pSKII-DC5'- MLP-2bld-RG-p(A)-DC(Xbal-Notl) was prepared by directional cloning in one step. The pSKII vector contained (Kpnl-ori/pkg-Hindlll-MLP- EcoRI-2bld-BamHI- RG-Pstl-p(A)-Xba) was opened with Xbal-Notl, and the 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- EcoRI-2bld-BamHI-RG-Pstl- p(A)-Xbal-Ad CELO-Notl) was opened with Sfil-Notl, and were ligated with the 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°C in a humidified incubator, the allantoic fluid was harvested and were assayed for rabies glycoprotein recombinant protein.

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 the crystallized molecule as described in the structure data entry PDB GNH 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 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 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 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. PCR Primers

The design of primers for introducing restriction sites and constructing the recombinant CRP (rCRP) molecule described above was done using the Genbank (Ace. No. X56692) sequence as a guide. The primers introduce restriction sites that are used for building the construct (figure 5).

5' pπmer

5'- CGTTAGGATCCATGCAGACAGACATGTCGAGGAAGGC-3' [SEQ ID NO:13]

3' pπmeπ 3'- CCAAGCGGCCGCTGCAGTCATCAGGGCCACAGCTGGGGTTTGGT-3¹

[SEQ IDNO:14]

I primer-For (37 mer) 5'-CGTTAGGATCCATGTCGAGGAAGGCTTTTGTGTTTCC-3' [SEQ ID NO:15]

II primer-Rev (38 mer) 5'-CCAATCTGCAGTCATCAGGGCCACAGCTGGGGTTTGGT-3' [SEQ ID NO:16] 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 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 the PCR insert for our recombinant human CRP protein is shown in Table III

[SEQ ID NO:23].

The PCR fragment was incoφorated 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 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 pentameπze and its immunochemical cross-reactivity with CRP specific antisera. The results are presented in Figure 7. In another experiment, the recombinant

CRP was assayed for it's ability to bind to 0-phophorylethanolomme sepharose, a matrix commonly used for the biospecific absoφtion of CRP. The results suggest that the recombinant CRP forms a pentamer since this is required for efficient binding to the matrix (Figure 8).

Example 6

Cloning of Rabies Virus Glycoprotein

The nucleotide and deduced am o acid sequence of the glycoprotein 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 ultrafiltration and viral particles concentrated by ultraceπtπfugation. 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'-GG ATCCAGGAAAGATGGTTCCTCAGGCTCTCCTGTTTG-3' [SEQ ID NO: 17],

which overlaps the translation initiation (ATG) codon using reverse transcriptase (Amersham). The cDNA was amplified using PCR (GeneAmp, Perkin Elmer Cetus), the 3'- and a 5'-specific primer.

The 5'-specific primer had the following sequence:

S'-GCTGCAGCAAGGGGAGGTGATCTTCAGACTTGGATCGT-S¹ [SEQ ID NO:18],

The δ'-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 on plates treated with X-gal. Plasmid DNAs containing insert were identified by agarose gel electrophoresis after cleavage with BamHI 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 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 strain had the greatest homology with ERA (99.4%) and with SAD B19

(99.1 %). Similar results were obtained when the deduced amino acid sequences were analyzed. The functional activity of the glycoprotein G gene was investigated in bacterial cells (Grabko V.I.. Fragment of DNA coding synthesis glycoprotein G of Rabies virus, recombinant plasmid DNA coding glycoprotein G of rabies virus, strain of bacteria Ξ.coli - Production of Glycoprotein G of Rabies virus. Patent of Russia, N2008355, 1994). The gene 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 This example describes the generation of the plasmid DC5'-GRV-DC3'

(pSKII-DC5'-MLP-2BLD-GRV-p(A)SV40-DC3'-pSKII), which is depicted in Figures 9. The plasmid pSKII-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 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: 5'-CAACCTCTAGACATCACCATAGCAATCATTGG-3' [SEQ ID No:19]

Xbal 42995 - 43016 bp Ad CELO

5'-CAπCGCGGCCGCGATGATGTATAATAACCTCAAAAACTAACG-3'

Notl 43804 - 43774 bp Ad CELO [SEQ ID No20] 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-Notl) was inserted therein. The vector pSKII-DC3' (Xbal-ori-Notl) was cut at the Xbal, Notl sites and Xbal-Notl fragment containing the 3' terminal of the FAVI genome was isolated. This fragment containing the 3' terminal of the FAV1 genome (Xbal- Notl) was inserted into tne Xbal-Notl digested vector pSKII-DC5'-MLP-2bld- GRV-p(A)SV40. The ligated DNA was transformed into E.coli and the correct plasmid was identified by restriction with the enzymes Xbal, Hindlll, Kpnl, EcoRI, BamHI, 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 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 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 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' (pSKII-DC5'-MLP-2bld-RG-p(A)SV40-DC3'-pSKII) which 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. 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 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 (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 rabies glycoprotein G production.

All references, including publications and patents, cited herein are hereby incoφorated by reference to the same extent as if each reference were individually and specifically indicated to be incoφorated by reference and were set forth in its entirety herein. While this invention has been described with an 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 modifications encompassed within the spirit and scope of the appended claims.

Claims

We Claim:

1. An egg comprising a recombinant protein.

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

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

4. An egg comprising recombinant DNA.

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 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; (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 transcriptional 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.

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

10 The method according to Claim 7, wherein said avian adenovirus DNA

11 The method according to Claim 7, wherein said protein coding sequence from a foreign gene comprises the site corresponding to initiation of 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 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.

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

15. A method for genetically modifying a avian adenovirus, comprising the 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 (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 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 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 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 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.

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: (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 genome, said 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.

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.

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

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 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 including the site at which RNA synthesis starts.

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 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.

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

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: (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 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 vivo manipulation of avian adenovirus DNA

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, (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 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'- Hmdlll, Hmdlll-MLP-EcoRI, EcoRI-2bld-BamHI, BamHI-GRV-Pstl, Pstl- p(A)SV40-Xbal,Xbal-DC3'-Notl.

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.

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 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 foreign DNA coding for a protein derived from an animal viral or bacterial or protozoan pathogen.

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 foreign DNA coding for cytokme 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.

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 compπsing the steps of.

(a) preparing a vector comprising a plasmid, cosmid, or phage containing avian adenovirus DNA , said DNA compπsing: (i) left and right inverted terminal repeat sequences and the viral packaging signal sequence; and

(II) at least one transcπption regulator and translation stimulator sequences; and

(MI) at least one protein coding sequence from a foreign gene 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 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.