DE3890874C2 - Recombinant viruses - Google Patents

Abstract

Described is a method for eliciting a vertebrate immune response to a pathogen by vaccinating the vertebrate with a synthetic recombinant avipox virus generated by the presence of DNA from any source that encodes and expresses an antigen of the pathogen in a non-essential region of the Avipoxvirus genome, modified. Also described is a synthetic recombinant avipox virus modified by the insertion of DNA from any source, particularly from a non-avipoxvirus source, into a non-essential portion of the avipoxvirus genome.

Description

This is a CIP (continuation-in-part) of the application Ser. No. 186,054, filed on April 25, 1988, which in turn a CIP of the application Ser. No. 110,335 is filed on October 20, 1987, which in turn is a CIP of the application Ser. No. 090,711, filed on August 28, 1987.

The present invention relates  a recombinant virus that when introduced into a verte fry an immune response against a specific pathogen triggers, characterized in that the re Combination virus containing an exogenous DNA insert, the at a non-essential region of the virus genome and inserted downstream of a suitable promoter sequence is, wherein the exogenous DNA insertion is an antigen of the Pathogens encoded and its expression under the Kon control the promoter sequence in vivo in the vertebrate allowed, and no replicative propagation of the virus in the vertebrate takes place.

The present invention also relates to recombinant virus that when introduced into a bird to trigger an immune response to a bird pathogen ver may be, this recombinant virus being a recombinant Avipox virus comprising an exogenous DNA insert stops in a non-essential region of Avi poxvirus genome and downstream of a suitable promo is inserted, wherein the insertion is a Se comprising an antigen of the pathogen, and the recombinant virus expresses the antigen in vivo, when introduced into the bird.  

Technical background

Avipox or Avipoxvirus is a bird-infecting, close related genus of poxviruses. The genus Avipox includes the species of fowlpox, canarypox, snow chicken pox, pigeon pox, quail pox, spatzenpocken, Starpox and turkey pox. The kind of chicken pox infects chickens. It must not be with human Er illness chickenpox be confused. The genus Avipox has many common features with other smallpox viruses and is a member of the same subfamily, poxviruses of Vertebrates, like vaccinia. Poxviruses, including Vac cinia and avipox, replicate within eukaryotic Host cells. These viruses are characterized by their Size, complexity and their replication in the cytoplasm. However, Vaccinia and Avipox belong to different Gat and are in their respective molecular weights, their antigenic determinants and in their host specificity different; see. Intervirology, Vol. 17, pages 42-44, Fourth Report of the International Committee on Taxonomy of Viruses (1982).

Avipoxviruses do not infect non-avian under reproduction vertebrates, such as mammals, including humans. Furthermore Avipoxvirus does not increase when sowing acid inoculated (including human) cell cultures. In such mammalian cell cultures infected with Avipox are, the cells die because of a cytotoxic effect but show no evidence of viral infection under propagation.

Vaccination of a non-avian vertebrate such. B. of a mammal with live avipoxes leads to formation a lesion at the vaccination site following vaccination with vaccinia  remind. But it does not lead to productive viral Infection. However, it has now been found that one on This manner of vaccinating mammal immunologically on the Avi poxvirus reacts. This is an unexpected result.

Vaccines made from killed pathogen or purified antigenic components of such pathogens are in larger quantities than live virus vaccines be injected to produce an effective immune response fen. The reason is that vaccination with live virus is a lot more efficient method of vaccination. A comparatively small inoculum can produce an effective immune response because the antigen in question during replication of the virus is increased. From a medical point of view, Le Bendvirus vaccines a more effective and longer-lasting Immunity, as a vaccine with a killed pathogen or purified antigen vaccine. Therefore, vaccine require substances derived from killed pathogen or purified anti components of such pathogens are composed, a production of larger quantities of the vaccine material when used with live virus.

From the previous discussion it is clear that it medizi niche and economic benefits for the use of Le bendvirus vaccines. Such a live virus vaccine substance contains vaccinia virus. It is known that this Vi can be used to recombinant DNA metho to insert the DNA, the genetic sequences of the antigens represents mammalian pathogens.

Therefore, the prior art has developed methods that the production of recombinant vaccinia viruses by the Insertion of DNA from any source (eg viral, prokaryotic, eukaryotic, synthetic) into a non- allow essential region of the vaccinia genome, including of DNA sequences encoding antigenic determinants of a encode pathogenic organism. Certain recombinant vaccinia viruses,  which were produced by means of these methods, were used to provide specific immunity in mammals to elicit a variety of mammalian pathogens that are known in US-A-4,603,112, the contents of which are hereby incorporated by reference Reference is made.

Unchanged vaccinia virus has a long history of one relatively safe and effective use for vaccination against smallpox. Before the eradication of smallpox, however, as unmodified vaccinia virus was widely administered There is a small but real risk of complications Form of generalized vaccinia infection, especially at Patients who had eczema or immunosuppression. A another rare but possible complication arising from the Vaccinia vaccination can be followed by postvacinar encepha litis. Most of these reactions occurred in the Inoculation of persons with skin diseases, such as eczema, or with a weakened immune system, or from individuals in Households where others have an eczema or a weakened one Had immune response. Vaccinia is a live virus and is usually harmless for a healthy person. Indeed It can be between individuals for several weeks after the Be transferred vaccine. If a person with a Weakening of the normal immune response either by vaccination or by contagion from a newly vaccinated person in The consequences can be serious.

Thus, it is understood that a method used in the Take the advantages of live virus vaccination closes, but vermin the previously mentioned problems ders or excludes, a highly desirable fort would be above the current state of the art. This is today all the more with the advance of the as "acquired immune deficiency syndrome "(AIDS) known disease of Meaning. Victims of this disease suffer from severe immune dysfunction and can be easily si by a normally which live virus preparation will be taken if they are either  directly or through contact with a person who just before with such a live virus vaccine immu has been in contact with such a virus men.

Subject of the invention

The invention relates to a recombinant Virus that when introduced into a verte fry an immune response against a specific pathogen triggers, characterized in that the re Combination virus containing an exogenous DNA insert, the at a non-essential region of the virus genome and inserted downstream of a suitable promoter sequence is, wherein the exogenous DNA insertion is an antigen of the Pathogens encoded and its expression under the Kon control the promoter sequence in vivo in the vertebrate allowed, and no replicative propagation of the virus in the Vertegraten takes place.

The invention is also a recombinant Virus that when introduced to a bird to trigger an immune response to a bird pathogen ver may be, this recombinant virus being a recombinant Avipox virus comprising an exogenous DNA insert stops in a non-essential region of Avi poxvirus genome and downstream of a suitable promo is inserted, wherein the insertion is a Se comprising an antigen of the pathogen, and the recombinant virus expresses the antigen in vivo, when introduced into the bird.

The present invention also relates to a vaccine, which contains a recombinant virus according to the invention.

Finally, the present invention relates to a method for expression of a gene product in vitro, in which Vertebrate cells of an in vitro cell culture Recombinant virus according to the invention is introduced.  

Synthetically modified Avipoxvirus recombinants containing exogenous (i.e., non-avipox) Carry genes that encode and express an antigen and which in a vertebrate host an immune response to the on and therefore cause the exogenous pathogen who the invention especially for vaccination non-avian Vertebrates used as new vaccines that have the disadvantages common, killed or attenuated living organisms avoid using vaccines.

It must be remarked again that avipox viruses are only replicative propagation can be in or passaged by bird species or bird cell lines. The recombinant, out Bird host cells obtained avipox viruses call an imp lung lesion without replicative propagation of the avipox virus when non-avian vertebrates, such as mammals, in one of the vaccination of mammals with vaccinia virus ent vaccinating manner. Despite the lack of a replikati multiplication of the avipox virus in such a vaccinated Non-avian vertebrates do enough expression of the virus, so that the vaccinated animal immunologically on the antigenic determinants of the recombinant avipox virus and also responds to the antigenic determinants for which the encode exogenous genes contained in the virus.

When vaccinating a bird calls such a synthetic recombinant avipox virus not only an immune response to the antigens that appear on the exogenous DNA from a be may be included, but also leads to replicative propagation of the virus in the host under trigger tion of an expected immune response to the avipox vector se.

Various researchers have suggested recombinant Ge wingpox specifically for use as a veterinary vaccine for the protection of poultry herds; see.  Boyle and Coupar, J. Gen. Virol. Bd. 67 (1986), pages 1591- 1600, and Binns et al., Isr. J. Vet. Med., Vol. 42 (1986), Pages 124-127. But there are neither suggestions nor fact Recent reports on the use of recombinant avipox viruses for generating specific immunity in mammals has become known.

Stickl and Mayr, Fortschr. Med. 97 (40) (1979), pages 1781- In 1788, the injection of avipox, especially Ge wingpox viruses in humans. However, they are related these investigations only on the use of ordinary Fowl pox to the non-specific immunity in Pati to strengthen the ducks due to the side effects of cancer suffer from mother therapy. There are no recombinant DNA techniques thereby applied. There is no indication of an avi poxvirus into which antigens of vertebrate pathogens co DNA has been inserted, or by a method to induce specific immunity in vertebrates. Instead of whose state of the art depends on a general and non-specific stimulating effect on the human host.

In U.S. Patent No. 4,603,112, although modified vaccinia viruses are However, these replicate after introduction to the Host cells. The same applies to the methods described in Paoletti et al. Biotechnology: Potentials and Limitations, Dahlem Conferences 1986, Berlin, Heidelberg, New York, Tokyo (Springer Verlag), page 155- 164, described recombinant poxviruses. In Boyle et al., Virology 156 (1987), 355-365, although recombinant vaccinia viruses containing the TK gene of a fowlpox virus, however, no poultry pox viruses are described here a nonessential region expressing a foreign Antigens allow, and it was not known whether Fowlpox viruses are even suitable as a vaccine.

A more complete discussion of the fundamentals of geneti Recombination helps to understand how the modifi modified recombinant viruses of the present invention have been made.

Genetic recombination is generally the substitution of homolo ger sections of deoxyribonucleic acid (DNA) between two strands of DNA. (In certain viruses, Ribo nucleic acid (RNA) replace the DNA). Homologous sections a nucleic acid are portions of the nucleic acid (RNA or DNA) having the same sequence of nucleotide bases.

Genetic recombination can occur naturally during replication or generation of new viral Ge nome within the infected host cell. Therefore, gene table recombination between viral genes during vi ral replication cycle in one with two or more ver different viruses or other genetic constructions co infected host cell take place. A DNA section out a first genome is used in the construction of the section of the Ge noms of a second, coinfecting virus in which the DNA homologous to that from the first viral genome is out exchanges.

However, recombination can also occur between sections of DNA in different, not completely homologous genomes occur. If such a section from a first genome is homologous with a portion of another genome (with Exception, for example, the presence of a genetic Marker or a gene that is responsible for an antigenic, in an Ab coded homologous DNA inserted determinant) There can always be recombination. The products of this Recombination can then be due to the presence of that prove genetic marker or gene.

Successful expression of the genetic introduced as DNA Sequence of the modified infectious virus requires two Conditions:

First, the insertion into a non-essential region of the virus for the modified virus to live remains capable. Neither for chicken pox nor for other avi poxviruses have been analogous to nonessential regions so far to those described for vaccinia virus. Entspre were not essential for the present invention Regions of avian pox due to fission poultry ken genome into fragments, then separating the Frag according to size and insertion of these fragments in plasmid Constructs found to propagate. Plasmids are small, circular DNA molecules acting as extrachromosomal elements can be found in many bacteria including E. coli. Method for insertion of DNA sequences, such as genes for antigenic  Determinants or other genetic markers in Plas Mides are well known to the skilled person and in detail be in Maniatis et al., Molecular Cloning: A Laboratory Tory Manual, Cold Spring Harbor Laboratory, New York (1982). Analog was used in the insertion of genetic markers and / or antigen coding genes in the cloned fl u gelpockenfragmente proceeded. The successfully recombined fragments (as by successful recovery of the genetic marker or antigen), were the those who live in a non-essential region of poultry contained poxvirus-inserted DNA.

The second condition for the expression of inserted DNA is the Presence of a promoter in the correct relationship to the inserted DNA. The promoter must be arranged so that lies in front of the DNA sequence to be expressed. Because Avipoxvi are not well characterized and avipox promoters hitherto unidentified are known promoters of other pox viruses successful as part of the present the invention inserted in front of the DNA to be expressed wor the. Fowlpox promoters may also be successful be used to perform the method according to the invention and produce the products. According gefun was that with chicken pox promoters, vaccine promoters and entomopox promoters transcription into recombinant Poc kenviren is started.

Boyle and Coupar, J. gen. Virol., Vol. 67 (1986), page 1591, suggest that vaccinia promoters can be expected to that they work in (fowlpox) virus. "The authors lo calved and cloned a chicken pox TK gene (Boyle et al., Virology, Vol. 156 (1987), pp. 355-365) and inse put it in vaccinia virus. This TK gene was probably because of the detection of the chicken pox TK promoter sequence expressed by vaccinia polymerase. Despite her guess However, the authors did not advertise any Vaccinia promoter in the fowlpox virus was still observed  she any expression of a stranger, in the poultry gelpox genome existing DNA sequence. Before the present The invention was not known that promoters from others Poxviruses such. Vaccinia promoters, in fact the Drive expression of a gene in an avipox genome.

Description of certain preferred embodiments

Particularly poultry pox and cana were invented according to the invention pienviruses as preferred by means of recombination Incorporation of exogenous DNA modified avipox species used.

Fowlpox is a species of Avipox, which in particular Ge wings, but not mammals infected. The as FP-5 be Drewed chicken pox trunk is a commercial one Chicken embryo derived fowlpox virus vaccine strain, and from American Scientific Laboratories (Division of Sche ring Corp.), Madison, WI, United States Veterinary License No. 165, Serial No. 30321, available.

The poultry pox strain referred to herein as FP-1 is a Duvette strain for use as a vaccine in day cream ken is modified. The tribe is a commercial poultry gelpox vaccine strain identified as O DCEP 25 / CEP67 / 2309 October Designated 1980 and available from Institute Merieux, Inc. is.

Canarypox virus is another type of avipox virus. Canarypox virus is infected with chicken pox especially canaries, but not mammals. This here Canarypox strain called CP is a commercial one Canary vaccine strain designated LF2 CEP 524 24 10 75 and available from Institute Merieux, Inc.

Those in these avipox viruses by genetic recombination inserted DNA sequences include according to the invention follow of the one: the lac Z gene of prokaryotic origin; the rabies glycoprotein  (G) gene that is an antigen of a non-bird like (specific mammalian) pathogens encoded; the Turkey influenza hemagglutinin gene responsible for the antigen a pathogenic bird other than an avipox virus virus encoded; the gp51.30 envelope of bovine leukemia virus, a mammalian virus; the fusion protein gene of Newcastle Disease Virus, a bird virus; the FeLV envelope of Feline Leukemia Virus, a mammalian virus, RAV-1 env gene of Rous-associated virus containing a bird vi rus / poultry disease is; the nucleoprotein (NP) gene of Chickens / Pennsylvania / 1/83 influenza virus, a bird virus; the matrix gene and peplomerogen of infectious bronchitisvi rus (strain Mass 41), a bird virus; and the glycoprotein D gene (gD) of the herpes simplex virus, a mammalian virus.

The isolation of the lac Z gene is described by Casadaban et al., Methods in Enzymology, Vol. 100 (1983), pages 293- 308. The structure of the rabies G gene is, for example, From anilionis et al., Nature, Vol. 294 (1981), pages 275-278. Its incorporation in vaccinia and expression in this vector who Kieny et al., Nature, Vol. 312 (1984), 163-166 discussed. The turkey influenza hemagglutinin gene is be by Kawaoka et al., Virology, Vol. 158 (1987), p 218-227. The bovine leukemia virus gp51,30 env gene was described by Rice et al., Virology, Vol. 138 (1984), Sci ten 82-93. The fusion gene of Newcastle Disease Virus (Te xasstamm) is available from the Institute Merieux, Inc., as Plasmid pNDV 108. The Feline Leukemia Virus env gene was from Guilhot et al., Virology, Vol. 161 (1987), pages 252-258 wrote. Rous-associated virus type 1 is available from Institute Merieux, Inc., as two clones penVRVIPT and np19env (190). Chicken Flu Virus NP gene is available from Yoshihiro Kawaoka from St. Jude Children's Research Hospital as plasmid pNP 33. An infectious bronchitis virus cDNA He is clone of the IBV Mass 41 matrix gene and peplomer gene from the Institute Merieux, Inc. as plasmid pIBVM63.  

The herpes simplex virus gD gene is described by Watson et al., Science, Vol. 218 (1982), pages 381-384.

The recombinant avipox viruses, described in more detail below, include one of three vaccinia promoters. The Pi promoter from the Ava I H section of Vac cinia is described by Wachsman et al., J. of Inf. Dis., Vol. 155 (1987), pages 1188-1197. In particular, this promoter is derived from the Ava IH (Xho IG) fragment from the L-variant WR vaccinia strain in which the promoter directs transcription from right to left. The card position of the promoter is about 1.3 Kbp (kilobase pairs) from the left end of Ava IH, about 12.5 Kbp from the left end of the vaccinia gene and about 8.5 Kbp to the left of the Hind III C / N junction. The promoter has the following sequence:

where the symbols in parentheses are linker sequences.

The Hind III H promoter (also "HH" and "H6") was determined by standard transcription mapping techniques. He has the sequence:

The sequence is identical to that described as 5'-sided of the the reading frame H6 of Rosel et al., J. Virol., Vol. 60 (1986), pages 436-449.

The 11K promoter is described by Wittek, J. Virol., Vol. 49 (1984), Pages 371-378 and C. Bertholet et al., Proc. Natl. Acad. Sci. USA 82 (1985), pages 2096-2100.  

The recombinant avipoxviruses of the invention are described in U.S. Pat well-known way assembled in two steps and analogous to those described in the aforementioned U.S. Patent 4,603,112 for the preparation of synthetic recombinants of Vaccinia virus are described.

First, the DNA to be inserted into the virus is transformed into an E. coli plasmid into which a portion of non-human essential DNA of avipox virus homologous DNA incorporated has been. Separated from this is the DNA gene sequence to be inserted ligated with a promoter. The promoter gene verbin tion is then inserted into the plasmid construction, so that the promoter-gene connection on both sides of DNA Flanked, the homologous to a non-essential Ab cut from Avipox DNA. The resulting plas Midconstruction is then followed by growth in E. coli Bakte increased. (Plasmid DNA is used to generate exogenous genes to carry and multiply material; This methodology is known. For example, these plasmid techniques are be by Clewell, J. Bacteriol., 110 (1972), pages 667-676. The techniques of isolating amplified Plasmids from the E. coli host are also known and in For example, Clewell et al., in Proc. Natl. Acad. Sci. USA, Vol. 62 (1969), pages 1159-1166).

The amplified after growth in E. coli amplified Plasmid material is now used for the second step. For this purpose, that containing the DNA gene sequence to be inserted Plasmid in a cell culture (eg chicken embryo fibroblasts) together with the avipox virus (such as avian pox strain FP-1 or FP-5) transfected. Recombination between homolo fowlpox DNA in the plasmid and the viral genome lead to an avipox virus caused by the presence of Non-fowlpox DNA sequences in a non-essential animal len section of its genome is modified.  

A better understanding of the invention and its numerous Advantages will be found in the following examples to be given for illustration.

example 1 Transient expression experiments show the recognition of Vaccinia promoters by avian pox RNA transcriptions factors

A number of plasmid constructions were made which the coding sequence for hepatitis B virus surfaces (HBSAg) linked to vaccinia virus promoter sequences contained zen. With fifty μg of each plasmid, CEF Cells (chicken embryo fibroblasts) transfected with 10 Plaque forming units (pfu) fowlpox virus or Vaccinia virus were infected per cell. One left the Infek Continue to run 24 hours and then lysed the cells through three consecutive cycles of freezing and thawing.

The amount of HBSAg in the lysate was estimated using a commercially available AUSRIA II- ¹²⁵ J kit from Abbott Laboratories, Diagnostic Division. The presence or absence of HBSAg is expressed as the ratio of the net values (sample minus background) of the unknown sample to the negative, manufacturer-defined exclusion value: this results in a P / N (positive / negative) ratio. The results are summarized in Table I.

There were three different vaccinia promoter sequences be uses: The early in vaccinia infection before the DNA repli cation recognized Pi promoter; the 11K promoter that is late in vaccinia infection after the onset of DNA replication is recognized; and the Hind III H (HH) promoter, both early and late in vaccinia infection. These Promoters are described above.  

The data show that in the lysates of infected Cells, HBSAg produced the result of detection of Vac cinia promoters either by avian pox or vaccinia nia transcription factors.

Table I

Example 2 Construction of a recombinant lac Z gene-containing gene Fowlpox virus vFP-1

A fragment in a nonessential section of the Ge Wingpox virus was isolated and isolated as follows.

With Nuclease Bal 31, the single-stranded terminal was Hairpin structure removed from FP-5 DNA. The Klenow (big) Fragment of DNA polymerase I was used to form smooth ene the used. After removal of the hairpin structure were  Fragments made by digestion with the restriction endo nuclease Bgl II. This digestion results in a series of FP- 5 fragments separated by agarose gel electrophoresis were.

A blunt ended 8.8 Kbp BglII fragment was isolated and ligated with the commercially available plasmid pUC 9, the had been split with Bam HI and Sma I. The result The preceding plasmid was named pRW 698.

To reduce the size of the chicken pox fragment, this plasmid was cleaved with Hind III, where two white tere fragments were generated. A 6.7 Kbp fragment was discarded and the remaining 4.7 Kbp fragment was with self-ligating, so that a new, as pRW 699 be recorded plasmid.

To start a lac Z gene started at the 11K promoter in this plasmid mid pRW 699 was split with Eco RV, which was the Plasmid splits only in one place. The lac Z fragment under the control of the 11K promoter was thereafter called blunt Pst I-Bam HI fragment inserted and revealed new plasmid called pRW 702. The lac Z clone is from pMC 1871 as described by Casadaban et al., a. a. O .. The 11K promoter was added to the eighth codon of the lac Z Gene ligated by means of a Bam HI linker.

Using recombinant techniques, such as those described for vaccinia in US-A-4,603,112, plasmid pRW 702 was recombined with fowlpox virus FP-5 grown on CEF using the following methods to obtain vFP-1 manufacture. 50 μg pRW 702 DNA was mixed in a final volume of 100 μl with 0.5 μg of total genomic wingpox DNA. To this were added: 10 μl 2.5 molar CaCl ₂ and 110 μl 2 × HEBS buffer (pH 7) prepared
40 mmol Hepes
300 mmol NaCl
1.4 mmol Na ₂ HPO ₄
10 mmol KCl
12 mmol dextrose.

After 30 minutes at room temperature, 200 μl of a Ge Wingpox virus suspension (diluted to 5 pfu / cell) added and the mixture on 60 mm dishes, a pri contain CEF monolayers, inoculated. In addition, 0.7 ml Eagles medium containing 2% fetal calf serum (FBS) contained, added at the same time. The plates were Incubated for 2 hours at 37 ° C, then another 3 ml Eagles medium with 2% FBS added and the plates for 3 days incubated for a long time. The cells were lysed by three following Cycles of freezing and thawing; the virus progeny was then after the presence of recombinants examined.

Evidence of successful insertion of the 11K promoter lac Z gene by recombination into the genome of poultry poc ken FP-5 was guided by the fact that expression of the lac Z gene was tested. The lac Z gene encodes the enzyme β-Ga lactosidase, which is the chromogenic substrate 5-bromo-4-chloro-3 indolyl-β-D-galactosidase (X-gal) cleaves and enters releases blue indolyl derivative. Blue plaques were as selected positive recombinants.

In addition, the successful introduction of lac Z into the Genome of chicken pox FP-5 and its expression confirmed by immunoprecipitation of the β-galactosidase protein by means of commercially available antisera and standard methods, which vFP-1 infected CEF, BSC (monkey kidney cell line - ATCC CCL26), VERO (monkey kidney cell line - ATCC CCL81) and MRC-5 (diploid human lung cell line - ATCC CCL171) Zen.  

Furthermore, the expression of β-galactosidase by the recombinant virus vFP-1 confirmed in vivo. Ka rabbits and mice were vaccinated with the virus. The rise in the Titers of the anti-β-galactosidase protein antibody in the serum of the vaccinated animals was measured.

In particular, the recombinant vFP-1 was purified from host cell contaminants and inoculated intradermally at two sites on each side of two rabbits. Each rabbit received a total of 10 ⁸ pfu.

The animals were bled weekly. The sera were used for an ELISA test involving a com mercially available preparation of purified β-galacto sidase used as an antigen source.

Both the rabbits vaccinated with the recombinant vFP-1 as well as the mice showed a detectable in the ELISA test Immune response to the β-galactosidase protein. In both ways The response was detectable one week after vaccination.

Example 3 Construction of the rabies G gene and lac Z containing rekom Binding virus vFP-2 from fowlpox virus FP-5

A 0.9 Kbp Pvu II fragment was obtained from FP-5 and with standard procedure between the two Pvu II sites in pUC 9 inserted. The resulting construction, as gRW 688.2, is asymmetric in the Pvu II Fragment two about 30 bp apart Hinc II-Stel len and therefore forms a long and a short arm of Fragment.

Using known techniques were used between these Hinc II sites oligonucleotide adapters used to  Introduce Pst I and Bam HI sites and in this way to produce the plasmid pRW 694.

This plasmid was then cleaved with Pst I and Bam HI and that with the 11K vaccinia promoter described above linked lac Z gene inserted. The new plasmid was obtained pRW 700.

To produce a pi promoter-controlled rabies G- The gene became the Bgl II site, the 5'-proximal to the rabies gene (see Kieny et al., supra), with smooth ends at the ends make and replicate the replenished Eco RI site ligated Pi promoter described.

This construction was placed in the Pst I site of pRW 700 and gave the plasmid pRW 735.1, which thus the foreign gene sequence Pi rabies G-11K-lac Z contains. This In Sert is located within the plasmid so that the long Pvu II Hinc II arm of the FP-5 donor sequence on the 3 'side of the lac Z gene.

The finished construction thus produced was treated with poultry poc kenvirus FP-5 by infection / transfection of chicken embryo fibroblasts (by methods as described above) recombined. Recombinant chicken pox was obtained rus vFP-2. This recombinant virus was after staining selected with X-gal.

The correct insertion and expression of both the lac Z-Mar Ker gene such as the rabies G gene was with a number too additional procedure described below.

Immunofluorescence detection of rabies antigen by spe zifische antibody successfully showed the expression of the Rabies antigen on the surface of birdlike and non-avian cells infected with vFP-2 virus.  

As before, the expression of the rabies antigen and the β-galactosidase by avian and non-avian, with vFP-2 virus infected cells by immunoprecipitation procedure confirmed.

Further evidence that the embodiment vFP-2 of this invention is a successful recombinant virus carrying the genes for rabies G and β-galactosidase was achieved by vaccinating two rabbits with vFP-2 virus. Both rabbits were inoculated intradermally with 1 x 10 ⁸ pfu vFP-2 per rabbit. Both rabbits developed typical pubic lesions. The rabbits were bled weekly; the sera were tested by ELISA to detect the presence of the antibodies specific for the rabies glycoprotein and the β-galactosidase protein.

As shown below in Table II, rabbit showed 205 detectable levels of anti-β-galactosidase antibody in the ELISA test 1 week after vaccination. This amount rose after 2 weeks to a titer of 1 in 4000, the 5 weeks was maintained long after vaccination. Used one an antigen-capture ELISA test, showed rabbit sera 205 detectable levels of anti-rabies antibodies zwi 3 and 10 weeks after vaccination.  

Table II Rabbit antibody production against rabies antigen and β-galactosidase protein

Time Antibody titer (reciprocal of serum dilution) Pre-bleeding anti-β-galactosidase 0 week 1 500 Weeks 2-5 (each) 4000 Week 6 500 Week 9 250 Pre-bleeding anti-rabies 0 Week 3 200 Week 6 200 Week 10 100

Example 4A Construction of a recombinant virus vFP-3 with a Toll Raises G gene under promoter control from fowlpox virus FP-1

This embodiment shows that the rabies G gene is complete dig also by other strains of poultry pox than FP-5, in particular by another, designated as FP-1 Strain of fowlpox virus is expressed.

As in Example 3, a 0.9 Kbp Pvu II fragment from FP-1 on the assumption that, as in FP-5, the fragment would contain a nonessential section.

This fragment was inserted between the two Pvu II sites of pUC 9 and the plasmid designated pRW 731.15R generated.

This plasmid is asymmetric within the pvu II Fragments two Hinc II bugs spaced about 30 bp apart. Make and form such a long and a short arm this fragment.  

A commercially available Pst I linker
(5 ') - CCTGCAGG - (3')
was inserted between the two Hinc II sites and he gave the plasmid pRW 741.

A rabies G gene dependent on the promoter HH has been generated in inserted and generated this plasmid in the Pst I site the new plasmid pRW 742B. By recombination of this plas mids with FP-1 after infection / transfection of CEF cells the virus was given vFP-3.

The ATG translation start codon of the HH promoter ge piloted open reading frames became the start codon of the Toll G-gene superimposed by placing an Eco RV site in the HH promoter and the Hind III site in the rabies G gene superimposing synthetic oligonucleotide was used. The 5 'end of this rabies gene was under the HH promoter modified in a conventional manner so that there is a Pst I site contained. The construction was then with the Pst I site from pRW 741 ligated to give pRW 742B. The Orientation of the construction in the plasmid is the same as in the pRW 735.1 previously discussed in Example 3.

The recombination was carried out according to Example 2. The resulting recombinant was named vFP-3.

The expression of rabies antigen in both birdlike and in non-avian cells infected with vFP-3 virus was determined by immunoprecipitation and immunofluorescence techniques (as described above) confirmed.

Further evidence that the vFP-3 embodiment of this invention is a successful recombinant virus expressing rabies G genes was obtained by intradermal inoculation of pairs of rabbits with the recombinant virus. Two rabbits were vaccinated intradermally with 1 x 10 ⁸ pfu of vFP-3 each. Both rabbits developed typical smallpox lesions that reached a maximum size 5 to 6 days after vaccination. The rabbits were bled at weekly intervals and sera tested by ELISA to detect the presence of antibodies specific for rabies glycoprotein.

In addition, 5 rats were inoculated intradermally each with 5 x 10 ⁷ pfu vFP-3. All animals developed lesions.

Both the rabbits and the rats evolved detectable levels of rabies specific antibodies in within 2 weeks after vaccination. Two control channels inoculated intradermally with the parent FP-1 virus showed no detectable levels of anti-rabies Antibodies.

To rule out the possibility that the antibody response due to the advent of rabies antigen, that was accidentally carried by the vaccine virus or in the Membrane of the recombinant fowlpox virus integrated was, instead of, as proposed, de novo synthesis of Rabies antigen by the recombinant virus in the animal, the vFP-3 virus was chemically inactivated and thus Rabbits vaccinated.

The purified virus was incubated overnight at 4 ° C in the presence of 0.001% β-propiolactone inactivated and then by Zen centrifuged pelleted. The pelleted virus was in with 10 mM Tris buffered saline, ultra sonicated and titrated to ensure that no infectious virus was left over. Two rats were intradermally with inactivated vFP-3 and two with an ent speaking amount of untreated recombinant virus inoculated. The size of the lesions was determined.  

Both rabbits that had received untreated vFP-3 developed typical smallpox lesions 5 days after the Inoculation was classified as 4-5 +. Rabbit with Inactivated virus were also inoculated, developed Lä but these became 2+ 5 days after vaccination classified.

The rabbits were bled weekly men and the sera by ELISA on the presence of for Rabies specific antibodies and for poultry pox spe tested on specific antibodies. The results are in Ta belle III summarized.

Table III

In this test, the titer final value (expressed as Reciprocal of the serum dilution arbitrarily set as 0.2,  after the absorption values of all sera before the "Bela rabbits 295 and 318, who received the live virus developed one Immune response to the rabies glycoprotein and poultry poxvirus antigenic. Rabbits 303 and 320 developed also an immune response to fowlpox virus antigens; however, the titer was lower. None of these rabbits ent Wrapped a demonstrable response to the rabies glycopro ton.

This finding confirms that the result in the rabbit called immune response to the de novo expression of the in the rabies glycoprotein gene containing recombinant virus is not an answer to any lig glycoprotein carried in the vaccine virus.

Example 4B Construction of the recombinant virus vFP-5, which controlled rabies contains G genes from poultry cams virus FP-1

The expression of a foreign, by recombination into the Ge wingpox genome inserted gene, requires the estate a promoter. This was shown by the manufacturer tion of another recombinant vFP-5, which with vFP-3 with Exception of the omission of the HH promoter is identical. The Presence of the rabies gene in this recombinant was by Confirmed nucleic acid hybridization. No rabies antigen however, was found in CEF cell cultures infected with the virus detected.  

Example 5 Experiments with in vitro passengers to prove whether poultry poxvirus replicated in non-avian cells

An experiment was carried out in which three cell systems (one birdlike and two non-birdlike) with the paren ternal FP-1 strain or inoculated with recombinant vFP-3 the. Two trays each were made with CEF, MRC-5 or VERO with FP-1 or vFP-3 with an input multiplicity of 10 pfu Inoculated per cell.

After 3 days, one dish was harvested in each case. The virus was through three consecutive cycles of freezing and thawing released and onto a fresh monolayer the same cell line again. This was about six repeated passages repeated; at the end of the expe Riments were samples of each passage for viral infectivity titrated on CEF monolayers. The results are in table IVa summarized. They show that a series passage both FP-1 and vFP-3 are possible in CEF cells, but in neither of the two non-avian cell lines. Infectious virus failed in VERO or MRC-5 cells be detected after 3 or 4 passages.

The second shell was used to determine if virus, that was not detectable by direct titration after Amplification can be detected in permissive CEF cells could. After 3 days, cells were removed from the second dish harvested by scraping, one third of these cells lysed and inoculated on a fresh CEF monolayer. When the full cytopathic effect (CPE) was reached or 7 days after Infection, the cells were lysed. The virus yield was titrated. The results are summarized in Table IVB caught. Passage in CEF cells was used to approximate virus could not replicate virus after four or five passages are detected.  

Attempts to establish a permanently infected cell, Failed.

In another attempt, hints of a persistent vira len expression in non-avian cells, were used above for virus titer determination Samples used in a standard immunodotest in which anti-fowlpox antibodies and anti-rabies antibodies were used to detect the presence of each antigen demonstrated. The results of these tests confirm the Titration.

Table IVA

passenger trial

Table IVB

propagation test

Example 6 Additional recombinants of fowlpox FP-1: vFP-6, vFP-7, vFP-8 and vFP-9

The recombinant viruses vFP-6 and vFP-7 were followed produced the method.

A 5.5 Kbp Pvu II fragment from FP-1 was inserted between the inserted into pUC 9 Pvu II sites and gave the plasmid pRW 731.13. This plasmid was then added to a sin cleft Hinc II site and stumped at the ends inserted, the HH promoter-dependent rabies G gene, which gave the plasmids pRW 748A and B, the two entge represent oriented orientations of the insert. The Plasmids pRW 748A and B were then used separately to CEF cells together with FP-1 virus infect and yielded by recombination vFP-6 or vFP-7. This genome will now designated as locus f7.

A 10 Kbp Pvu II fragment from FP-1 was inserted between the inserted Pvu II sites of pUC 9, yielding pRW 731.15. This plasmid was then cloned on a singular Bam HI site cleaved and then one from the 11K promoter dependent lac Z gene fragment, resulting in pRW 749A and B which produced the opposite orientations of the insert represent. Recombination of these donor plasmids with FP-1 led to vFP-8 and vFP-9, respectively. This locus is now called gene designated location f8.

vFP-8 and vFP-9 expressed (as detected by X-gal sen) the lac Z gene. vFP-6 and vFP-7 expressed rabies G gene, as demonstrated by a rabies-specific antiserum was meadows.  

Example 7 Immunization with vFP-3 to protect animals against Be pollution with live rabies viruses

Groups of 20 female 4 to 6-week-old SPF mice were inoculated with 50 μl vFP-3 on the paw surface at dose levels ranging from 0.7 to 6.7 TCID ₅₀ per mouse. (The TCID ₅₀ or tissue culture infectious dose is the dose at which 50% of tissue culture cells have a cytopathic effect). Ten days after vaccination, 10 mice from each group were sacrificed and serum samples were collected for RFFI assays. The remaining 10 mice were exposed to challenge infection by inoculation with 10 LD _{50 of} the rabies strain CVS via the intracerebral route and the survivors were calculated 14 days after the challenge infection.

The results are summarized in Table VA.

Table VA

The experiment was repeated with 12.5 LD _{50 of} the rabies virus as loading infection. The results are summarized in Table VB.

Table VB

Two dogs and two cats were immunized with a single subcutaneous vaccination with 8 log ₁₀ TCID ₅₀ of recombinant vFP-3. In addition, 2 dogs and 4 cats of appropriate age and weight were considered unvaccinated controls. All animals were bled weekly. On day 94, each dog was challenged by inoculation into the temporal muscle with two doses of 0.5 ml of a salivary gland homogenate of the NY strain of rabies virus (available from the Institute Merieux, Inc.). The total dose corresponded to 10,000 mice LD ₅₀ via an intracerebral route. The six cats were similarly exposed to stress infection by inoculation into the neck muscle with two 0.5 ml doses of the same virus suspension. The total dose per animal was equivalent to 40,000 LD ₅₀ mice via an intracerebral route. The animals were observed daily. All non-vaccinated animals became rabies symptoms on the day indicated in Table VI. The vaccinated animals survived the challenge infection and were observed for 3 weeks after the death of the last control animal. The results are summarized in Table VI.

Table VI

In further experiments, the recombinant viruses were subjected to vFP 2 and vFP-3 for inoculating cattle over several different used their ways.

Vaccinated animals were tested for anti-rabies antibodies on the Ta tested at 6, 14, 21, 28 and 35. All animals show one  Serological answer to the rabies antigen. This is in Ta Belle VII A shown.

Table VIIA

Antibody titers in vFP-3 vaccinated mammals (bovine) anti-rabies neutralizing antibody RFFI assay log ₁₀ dilution

All cattle were re-vaccinated on day 55 post-vaccination with 8 log ₁₀ TCID ₅₀ . They showed an anamnestic response to the rabies antigen. In booster vaccination, all cattle were vaccinated subcutaneously except No. 1421, which was again vaccinated intramuscularly. RFFI titers were determined on days 55, 57, 63, 70, 77 and 86. The results are summarized in Table VIIB.

Table VIIB

All data refer to vFP-3 except at Tier 1423, where they refer to vFP-2.

Cattle, cats and rabbits were also intradermally with be knew quantities of avianpox virus vaccinated. scab was collected from the animals after about 1 week. This was ground, suspend in physiological saline titrated and titrated to determine the amount of virus.

Only residual amounts of infectious virus could be detected become. This shows that no infection is propagated in vivo.

Example 8 Vaccination of chickens with vFP-3

Chickens were treated with recombinant avipox virus vFP-3 vaccinated to the expression of foreign DNA by a rekom to show binant avipox virus in a system that allows a replicative propagation of the vector.

White leghorn chickens were inoculated intramuscularly with 9 log ₁₀ TCID ₅₀ vFP-3 or by wing piercing with 3 log ₁₀ TCID ₅₀ vPP-3. Blood samples were taken for an RFFI test for rabies antibody titer 21 days after vaccination. Day 21 titers in vaccinated chickens were significantly higher than those in controls on day 21. The mean titer of the uninfected controls was 0.6, that of the intramuscularly vaccinated birds 1.9, that of the wing-pierced birds 1.2.

Example 9 Turkey influenza H5 HA antigen-expressing recombinant Poultry pox vFP-11

Bird species can interact with the recombinant avipox viruses of Er be immunized against bird pathogens.

Thus, the new plasmid described below pRW 759, which derives from the fowlpox virus FP-1 and a Hemagglutinin (H5) from A / Turkey / Ireland / 1378/83 (TYHA) under the control of the Hind III H promoter, for Transfection of simultaneously with the parent virus FP-1 infi graced CEF cells used. Recombinant chicken pox vi rus vFP-11 was replaced by those described further above Obtained procedure.

The synthesis of a hemagglutinin molecule in with VFP-11 infi ciliated cells were isolated by immunoprecipitation Metabolism radiolabeled, infected cell lysates under Use of a specific anti-H5 antibody and Standard procedure confirmed. The specific immunoprecipitation tion of precursor hemagglutinin with a molecular weight of about 63 kd (kilodaltons) and two fission products with one Molecular weight of 44 and 23 kd was shown. None of the Such proteins were non-infected from a lysate CEF cells or infected with parental virus FP-1 Cells precipitated.

To prove that in the with the recombinant poultry pox vFP-11 infected cells produced HA molecule cell surface expressed immunofluores zenz examinations. With the recombinant Ge wingpox virus vFP-11 infected CEF cells showed one  strong fluorescence staining on the surface. In with the El Tern virus FP-1 infected cells did not fluoresce measured.

The plasmid pRW 759 was prepared as follows:
pRW 742B (see Example 4) is linearized by partial digestion with Pst I and the fragment is cleaved with Eco RV to remove the rabies G gene with the HH pro-motor remaining on the remaining fragment of approximately 3.4 Kbp , After treatment with alkaline phosphatase, a synthetic oligonucleotide is inserted to allow the HH promoter to be linked to TYHA at the ATG resulting in pRW 744.

This plasmid was prepared by partial digestion with Dra I li linearised; the linearized fragment was extracted with Sal I split and the larger fragment recovered and with al treated with potassium phosphatase.

Finally, pRW 759 was generated by inserting the iso Sal I-Dra I fragment produced, containing the codie In the pRW 744 vector, such as by Kawaoka et al., Virology, Vol. 158 (1987), pages 218-227 was shown.

Example 10 Immunization with vFP-11 to protect birds against Stress infection with live influenza virus

To demonstrate the immunogenic effect of recombinant Ge fowlpox virus vFP-11, have been vaccinated and debilitated tion experiments in chickens and turkeys.

Specific pathogen - free (SPF) white leghorn chickens were found in the Age of 2 days and 5 weeks by piercing the Flü Peeled with a double needle as usual Vaccination of chickens with fowlpox virus is used.  

About 2 μl of vFP-11 at 6x10 ⁵ pfu was added to each bird. The older birds were bled before vaccination, and all birds were bled before the challenge and 2 weeks later.

For comparison purposes, a second group of chickens was added inoculated with a standard H5 vaccine made from an inak tivated H5N2 strain in a water-in-oil emulsion.

Inactivated H5N2 vaccine was obtained from A / Mallard / NY / 189/82 (H5N2) influenza virus grown in 11 day old berry eggs; the infected allanoid fluid with an HA titre of 800 / 0.1 ml and an infectious titre of 10 ^8.5 / 0.1 ml was inactivated with 0.1% propionolactone and dissolved in a water-in-oil emulsion, as in Stone et al., Avian Dis., Vol. 22 (1978) pp. 666-674 and Brugh et al., Proc. Second Inter. Sym. on Avian Influenza (1986), pages 283-292. In 0.2 ml volume, the vaccine was administered by the subcutaneous route to the inside of the thigh muscle 2 days and 5 weeks old of SPF white leghorn chickens.

A third and fourth group of chickens received parenteral original virus FP-1 or no vaccine.

Chickens were challenged with about 10 ³ LD _{50 of} the highly pathogenic A / Turkey / Ireland / 1378/83 (H5N8) or A / Chick / Penn / 1370/83 (H5N2) immune virus by administration of 0.1 ml to the Nostrils of each bird. Two-day-old birds were exposed to a challenge infection 6 weeks after vaccination and 5-week-old birds were exposed to a challenge infection 5 weeks after vaccination. The birds were daily observed for symptoms of disease, which included swelling and cyanosis of the face and crest and bleeding on the feet (such birds often could not stand), paralysis and death. Most deaths occurred between 4 and 7 days after infection. Three days after infection, trachea and cloaca smears from each live chicken were tested for virus by inoculation of fertilized eggs. The chickens vaccinated with either wild type or recombinant poultry pox virus developed typical lesions on the winged web. A pustule formation until the 3rd day at each needle puncture followed by cellular infiltration with scab formation and recovery at 7 days. No secondary lesions were formed and there was no evidence of spread to unvaccinated chickens that came into contact. The results of the challenge infection experiment are summarized in Table VIII and the associated serological findings in Table IX.

Table VIII

H5-mediated protection of chickens expressed in chicken pox

(a) The 5-week-old birds were bled prior to vaccination, which was tested in HI and neutralization tests: none contained detectable levels of antibody; the results are not shown. The 2 day old chickens were bled 6 weeks after vaccination (post-1) and the 5 week old birds were bled 5 weeks after vaccination (post-1); Both groups were bled 2 weeks after challenge (post-2). The numbers represent averaged antibody titers from the same group of chickens as described in Table I.
(b) The numbers in parentheses indicate how much the stress infection survived.
<= less than 10
(c) Hemagglutination inhibition (HI) tests were carried out in microtiter plates using receptor-disrupting enzyme-treated sera (4 HA units of Ty / Irish virus, and 0.5% chicken erythrocytes as described in Palmer et al., Immun Series No. 6, 51-52, US Dept. Health, Education and Welfare (1975)). The infec tivitäts-neutralization assays were carried out by 10 ³ EID ₅₀ of Ty / Ire virus were incubated with dilutions of the sera for 30 minutes at room temperature, followed by a vaccination with aliquots into fertilized eggs. Virus growth was determined by hemagglutination tests after incubation of the eggs for 2 days at 33 ° C.

Chickens infected with recombinant chicken pox H5 (vFP-11) or the inactivated H5N2 influenza vaccine in adjuvant were inoculated before the load infection with the homologous Ty / Irish (H5N8) influenza virus and the related, but distinguishable Ck / Penn (H5N2) influenza virus protects. In contrast, the majority of birds showed who were vaccinated with the parent FPV or no vaccine had received clinical signs of highly pathogenic Influenza including swelling and cyanosis  on the face and crest, bleeding on the feet and paralysis. The majority of these birds died. The vaccinated birds no detectable levels of Ty / Ire, but Ck / Penn out.

Both the inactivated and the recombinant Vaccines induced HI and neutralizing antibodies on Ty / Ire, but the levels of antibody produced by the Fowlpox H5 recombinant vFP-11 before Induced stress infection did not inhibit HA or neutralized the heterologous Ck / Penn H5. Regardless of the chickens were both with a stress infection Ty / Ire as well as protected with Ck / Penn influenza viruses.

Immunization against H5- induced by vaccination with vFP-11 Influenza lasted at least 4 to 6 weeks and was cross-reactive. To prove the duration and specificity of The answer was a group of 4 week old chickens in the Wing mesh tissue was inoculated with vFP-11 as previously described and at monthly intervals with the cross-reacting Ck / Penn virus exposed to a stress infection. In turn were no HI antibodies before the challenge infection detectable. Regardless, the birds were also after 4 Months protected.

In addition, H5 expressed by vFP-11 induces a protective immune response in turkeys. White Outbread Turkeys were transmitted at the age of 2 days and 4 weeks Inoculation on the wing network as described inoculated. The Results are summarized in Table X.  

In both age groups was a significant survival advise against a stress infection with the homologous Ty / Irish virus observed. Unvaccinated contact birds were kept together with the vaccinated birds to one To examine proliferation of the recombinant virus. These birds did not survive the stress infection.

Example 11 Construction of the chicken fluke nucleoprotein (NP) gene expressing fowlpox virus FP-1 recombinant vFP-12

The plasmid pNP33 contains a cDNA clone of influenza virus chickens / Pennsylvania / 1/83 nucleoprotein gene (NP). Only the 5 'and 3' ends of the approximately 1.6 Kbp NP gene were sequenced. NP was transferred from pNP 33 into Sma I digested pUC 9 as a blunt ended 5 'Cla I-Xho I 3' fragment with the pUC 9 Eco RI site at the 3 'end, creating pRW 714. The translational initiation codon (ATG) of NP contains the following underlined Aha II site: AT GGCGTC . The previously described vaccinia H6 promoter was joined to NP by a double-stranded synthetic oligonucleotide. The synthetic oligonucleotide contained the H6 sequence from the Eco RV site to the ATG and to the NP coding sequence at the Aha II site. The oligonucleotide was synthesized with ends compatible with Bam HI and Eco RI for insertion into pUC 9 and resulted in pRW 755. Starting at the Bam HI compatible end, with which ATG underlined, is the sequence of the double-stranded synthetic oligonucleotide the following:

The linear product of a partial digest of pRW 755 with Aha II was isolated and post-cleaved with Eco RI. The pRW 755 fragment showing a single Aha II cut at the ATG contained and post-split with Eco RI was isolated,  treated with phosphatase, and as a vector for digestion product of pRW 714 (see below).

The isolated, linear product of a partial digestion with Aha II of pRW 714 was cleaved with Eco RI. A co dative sequence for NP containing isolated Aha II Eco RI fragment of about 1.6 Kbp was used in the pRW 755 vector and led to pRW 757. The complete H6 promo Tor was formed by the sequences above (5 ') of the Eco RV site were added. The plasmid pRW 742B (be written in Example 4), the H6 sequence had below (3 ') the Eco RV site with the sequences up to the Nde I site removed in pUC 9. The phosphatase-treated Eco RV-Nde Fragment from pRW 742B was used as a vector for the pRW 757- Fragment (see below) used. The isolated linear Product of a partial digest of pRW 757 with Eco RV was recovered after a split with Nde I; this question ment contains the H6 promoter from the Eco RV site via NP to the Nde I site in pUC 9. The pRW 757 fragment was inserted into the pRW 742B vector and formed pRW 758. The Eco RI fragment of pRW 758, which depended on the H6 promoter gige complete NP was included with the Klenow fragment the DNA polymerase I made smooth at the ends and in the Hinc II site of pRW 731.13 and yielded pRW 760. The Hinc II site of pRW 731.13 is the FP-1 locus, the used in Example 6 to construct vFP-6 and vFP-7 has been.

Using poultry pox FP-1 as a collecting Vi rus was the plasmid pRW 760 for an in vitro recombina tion test used. The plaques of the offspring were tested and purified by in situ plaque hybridization. Expression of the gene was assessed by immunoprecipitation studies using a polyclonal goat anti-NP antiserum approved. The size of the protein, in particular a lysate of vFP-12 infected CEF cells  was about 55 kD, which was published within the range of influenza nucleoproteins.

Example 12 Production of a Bird Flu Nucleoprotein (NP) - and Hemagglutinin (HA) gene expressing double recombinant vFP-15 of fowlpox virus

The hemagglutinin (HA) gene from A / Tyr / Ire / 1378/83 was used in Related to the construction of vFP-11 (Example 9) described. In the preparation of the double recombinant The HA gene was first used in the preparation of vFP-8 using plasmid pRW 731.15 Locus f8 brought.

The plasmid used to construct vFP-11 was pRW 759. Hemagglutination associated with the H6 promoter was from this plasmid by partial digestion with Pst I removed. This fragment was then attached to the ends made the Klenow fragment of DNA polymerase I smooth, in the smoothed Bam HI site advertised by pRW 731.15 and resulted in pRW 771.

The plasmid pRW 771 was then recombined in an in vitro tion test using vFP-12 as a catching virus used. The recombinant virus vFP-12 contains this nucleoproteins linked to the H6 promoter at the locus f7, as defined in plasmid pRW 731.13. recombinant Plaques now containing both inserts were knocked out selects and by means of in situ hybridization and Oberflächenex Pressing the hemagglutinin plaque-purified, causing a protein A-β-galactosidase-linked immunoassay bestä was taken. The expression of both genes was by Immunprä zipitation with the double recombinant virus vFP-15 in detected cell lysates.  

Example 13 Construction of recombinant canarypox viruses

The following example shows the identification of four nonessential insertion loci in the canarypox gene nom and the construction of four recombinant canary Pok Kenvir vCP-16, vCP-17, vCP-19 and vCP-20.

The canarypox recombinant vCP-16 was prepared as follows posed.

A 3.4 Kbp Pvu II fragment of canarypox DNA was added in pUC 9 cloned and resulted in pRW 764.2. It became a singu Eco RI site found asymmetrically in the Frag ment with a short arm of 700 bp and a long arm of 7.2 kbp. The plasmid was digested with Eco RI and using Klenow fragment of DNA polymerase I made flush. The blunt-ended H6 / rabies G gene was then ligated with this site and used to transform E. coli used. The resulting plasmid pRW 775 was used for an in vitro recombination test. In an immunoscreen procedure, positive progeny plaques were obtained the selected and plaque-cleaned. The result The present recombinant was called vCP-16 and the insertion locus referred to as C3.

The plasmid pRW used in the above construction 764.2 also contained a unique Bgl II site, for example 2.4 Kbp from the Eco RI site. Using the same cloning strategy was the H6 / rabies G gene with ligated to the plasmid pRW 764.2 at this point and led to pRW 774. This plasmid was used to construct the Recom binant vCP-17 with the insertion locus designated C4 used.  

Plasmid pRW 764.5 contains an 850 bp Pvu II fragment of Canarypox DNA with a unique Bgl II site, the asymmetric in the fragment is 400 bp from one end. Using the cloning as described previously strategy was the rabies associated with the H6 promoter G gene inserted at this point and gave the plasmid pRW 777. The stable recombinant virus produced therewith was designated as vCP-19 and the insertion locus as C5.

Plasmid pRW 764.7 contains a 1.2 Kbp Pvu II fragment with a singular Bgl II site 300 bases from one end. This plasmid was digested with Bgl II and probed with Klenow's Fragment of DNA polymerase I made flush at the ends. The lac Z gene blunt-ended with the 11K promo was inserted and resulted in plasmid pRW 778. The under Using this plasmid produced stable recombinant Virus was labeled as VCP-20 and the insertion locus as C6 records.

Example 14 Construction of a fusion protein from Newcastle Disease Virus-expressing recombinant avipox virus vFP-29

Plasmid pNDV 108, the cDNA clone of the fusion gene of NDV- Texas strain, consists of an Hpa I cDNA fragment of approximately 3.3 Kbp containing both the coding sequence for fusion protein as well as additional, into the Sca I site of pBR contains 322 cloned NDV coding sequences. Steps for the preparation of the insertion plasmid are after standing described.

(1) Preparation of Plasmid pCE 11

An FPV insertion vector, pCE 11, was prepared by inserting Polylinkern made at the Hinc II site of pRW 731.13 (referred to as locus f7). pRW 731.13 contains a 5.5 Kbp  Pvu II fragment of FP-1 DNA. A nonessential locus has been defined above at the Hinc II site by Kon Construction of the stable Rekom described in Example 6 binant vFP-6. The poly introduced at the Hinc II site Left contain the following restriction enzyme cut Nru I, Eco RI, Sac I, Kpn I, Sma I, Bam HI, Xba I, Hinc II, Sal I, Acc I, Pst I, Sph I, Hind III and Hpa I.

(2) Preparation of plasmid pCE19

This plasmid is another modification of pCE 11, in that the vaccinia virus transcriptional stop signal ATTTTTNT (L. Yuen and B. Moss, J. Virology, Vol. 60 (1986), pages 320- 323) (in this case N is an A) between the sac I and Eco RI sites of pCE 11 with loss of the Eco RI site has been added.

(3) Insertion of coding NDV sequences

A gel-purified 1.8 Kbp Bam HI fragment, the with the exception of 22 nucleotides from the 5 'end of the fusion pro teingens contained all nucleotides, was added to the Bam HI Inserted site of pUC 18 and formed pCE 13. This plas mid was digested with Sal I, which in the vector was 12 bases cleaves above the 5 'end of the coding sequence. The Ends were terminated with the Klenow fragment of DNA polymerase I filled in and then the plasmid with Hind III digests 18 bases above the Sal I site. On gel-purified 146 bp Sma I - Hind III fragment, that described in the preferred embodiments Vaccinia virus H6 promoter as well as polylinker sequences each end was ligated to the vector and expressed in E. coli cells. The resulting plasmid was designated pCE16.

In order to read the ATG start codon of the NDV fusion protein gene with the 3 'end of the H6 promoter and replace the 22 nucleotides missing in the pCE 16 from the NDV 5' end, a complementary synthetic oligonucleotide with Eco RV and Kpn was prepared I developed sites at the ends. The oligo nucleotide sequence was:

The construction pCE 16 was subsequently treated with Eco RV and Kpn I digested. The Eco RV site is located in the H6 promoter 24 Bases above the initiation ATG. The Kpn I site is located in the coding NDV sequence 29 bases below the ATG.

The oligonucleotides were hybridized with each other, phos phorylated and linked to the linearized plasmid. The resulting DNA was used to transform E. coli cells used. This plasmid was as pCE 18 be records.

To insert coding NDV sequences into an FPV insert tion vector was a gel purified 1.9 Kbp Sma I-Hind III fragment of pCE 18 (where in the polylinkerre gion) with a 7.8 Kbp Sma I-Hind III Frag ment of pCE 19 (as described above). The Transcription start signal is 16 bases below the Sma I site. The resulting plasmid was named pCE20 designated.

The plasmid pCE 20 was in an in vitro recombination used fowlpox virus FP-1 as auffan virus was used. The resulting after was plated on CEF monolayers and the Pla ques were subjected to a β-galactosidase protein A immunoassay Use of a polyclonal chicken anti-NDV serum under worfen. Positive plaques in the staining were selected and 4 rounds of a plaque cleanup to make a homogeneous  To get population. The recombinant became vFP-29 called.

Example 15 Construction of Feline Leukemia Virus (FeLV) Envelope (ENV) Glycoprotein-expressing avipoxvirus recombinant

The FeLV env gene contains the sequences encoding the p70 + encode p15E polyprotein. This gene was initially in the Plasmid pSD467vC preceded by the FeLV env gene Vaccinia H6 promoter inserted. Previously, the plasmid pSD467vC by the insertion of a 1802 bp Sal I / Hind III Fragment containing the vaccinia hemagglutinin (HA) gene, obtained in a pUC 18 vector. The location of the HA gene was previously determined (Shida, Virology, vol. 150 (1988), pages 451- 462). The majority of the HA gene product coding open Reading frame was deleted (nucleotide 443 to nucleotide 1311) and a multiple cloning site, the Bgl II, Sma I, Pst I and Eag I restriction endonuclease sites contained, inserted. The resulting plasmid pSD467vC ent holds flanking arms from vaccinia from 442 bp up 5'- from the mutant cloning site and 491 bp 3'-be tig from these restriction interfaces. This flankie The resulting arms allow multiple cloning inserted into the HA region of the Copenhagen Strain of vaccinia virus can be recombined. The result Resulting recombinant progeny is HA negative.

The H6 promoter had been synthesized by the aneinan derhängen of four overlapping oligonucleotides, the together in the preferred embodiments described complete sequence included. The result The preceding 132 bp fragment contained a Bgl II residue tion interface at the 5 'end and a Sma I site at the 3' The End. This was reported in pSD467vC via the Bgl II and Sma I-Re Striction interfaces inserted. The resulting  Plasmid was designated pPT15. The FeLV env gene was located in the immediate 3 'side of the H6 promoter Inserted singular Pst I site of pPT15. The result The resulting plasmid was designated pFeLV1A.

To construct the FP-1 recombinant, the 2.4 Kbp H6 / FeLV env sequence from pFeLV1A by cleavage with Bgl II and partial cleavage with Pst 1 cut out. The Bgl II Site is located at the 5 'border of the H6 promoter sequence. The Pst I place is 420 bp after the termination signal of Translation for the open reading frame of the envelope glycopro teins.

The 2.4 Kbp H6 / FeLV env sequence was ligated in with Bam HI and Pst I split pCE 11 inserted. The FP-1 insertion vector, pCE 11, is derived from pRW 731.13 by inserting a multiple cloning site into the nonessential Hinc II site. This insertion vector allows the preparation of FP-1 recombinants expressing foreign genes at locus f7 of FP-1 Contain genome. The recombinant FP-1 / FeLV insertion plas mid was named pFeLVFI. This construction offers no complete ATG for ATG substitution.

To achieve a complete ATG: ATG construction, was a Nru I / Sst II fragment of about 1.4 Kbp from the Derived vaccinia virus insertion vector pFeLVIC. The Nru I- Site is in position within the H6 promoter of 24 bp above the ATG. The Sst II site is 1.4 Kbp 3'side the ATG and 1 Kbp 5'side of the termina tion signal of translation. This Nru I / Sst II fragment was ligated with a 9.9 Kbp fragment by digestion with Sst II and partial digestion with Nru I was generated. the This 9.9 Kbp fragment contains the 5.5 Kbp of the FP-1 flankie arms, the pUC vector sequences, 1.4 kbp at the 3 'end of the env gene corresponding sections of FeLV Se sequence and the sequence at the very 5 'end of the H6 promoter (ca. 100 bp). The resulting plasmid was named pFeFLVF2  designated. The ATG: ATG construction was by nucleotide Sequence analysis confirmed.

Another FP-1 insertion vector, pFeLVF3, was knocked out pFeLVF2 by removing the probable immunosup corresponding FeLV env sequences Tet (Cianciolo et al., Science, Vol. 230 (1985), pages 453- 455) (nucleotides 1548 to 1628 of the coding sequence). the This was achieved by isolating a Sst II / Pst I-Frag ment (digits are described above) of about 1 kbp from the Vaccinia virus insertion vector pFeLV1D. The plasmid pFeLV1D is similar to the plasmid pFeLV1C with the exception that the the immunosuppressive region corresponding env sequences (Nucleotides 1548 to 1628) by oligonucleotide-directed Mutagenesis (Mandecki, Proc. Natl. Acad Sci. USA, Vol. 83 (1987), pages 7177-7181). The 1 kbp Sst II / Pst I fragment without nucleotides 1548 to 1628 was inserted into a 10.4 Kbp Sst II / Pst I fragment the remainder of the pFeLVF2-derived H6: FeLV env gene.

The insertion plasmids, pFeLVF2 and pFeLVF3, were reported to be in vitro recombination tests with FP-1 as the intercepting Vi used. Progeny of recombination was on CEF monolayer plated and recombinant virus were used Plaque hybridization on CEF monolayers selected. through Hybridization analysis identified recombinant desc The team was selected and 4 rounds of a plaque session subjected to homogeneous population. An FP-1 recombinant containing the complete FeLV env gene was described as vFP-25 and an FP-1 recombinant the complete gene except immunosuppressive reichs, referred to as vFP-32. For both Rekom binants were used by immunoprecipitation of a polyclonal bovine anti-FeLV polyclonal serum (An Tibodies, Inc., Davis, Ca) have shown that they are the right one Express gene product. It is important that these FP  1 recombinants the foreign FeLV env gene in cats expressing CRFK cell line (ATCC # CCL94).

For the construction of canarypox (CP) recombinants, became a 2.2 Kbp fragment containing H6: FeLV env sequences ment from pFeLVF2 by cleavage with Sma I and Hpa I out cut. The Sma I site is located at the 5 'boundary of the H6 Promoter sequence. The Hpa I site is 180 bp 3 'side of the termination signal of the translation for the open Le the envelope of the envelope glycoprotein.

The 2.2 Kbp H6: FeLV env sequence was inserted into the non-essenti elle Eco RI site of the insertion plasmid pRW 764.2 sets after the ends of the Eco RI site were made smooth This insertion vector allows the generation of CP Recombinants, the foreign genes in locus C4 of the CP genome contain. The recombinant CP insertion plasmid was designated as pFeLVCP2. This construction offers a perfect ATG: ATG substitution.

The insertion plasmid, pFeLVCP2, was expressed in an in vitro Re combination test with CP as the catching virus puts. Progeny of the recombinant was on CEF mono layer plated and recombinant virus by means of a Protein A-linked β-galactosidase immunoassay under Ver use of a commercial polyclonal bovine anti-FeLV Serums (Antibodies, Inc., Davis, CA.). In the Staining positive plaques were selected and 4 rounds subjected to a plaque-cleaning to a homogeneous popula tion. A recombinant that covers the entire FeLV env gene was expressed as vCP-36.  

Example 16 Constriction of Rous-associated virus TyP 1- (RAV-1) - Envelope (env) gene expressing recombinant fowlpox virus vFP-22

The clone penvRV1PT of the RAV-1 envelope gene contains 1.1 Kbp of coding sequence of RAV-1 env DNA, cloned as Kpn I- Sac I fragment in M13mp18. This at the 5'-end complete Fragment lacking part of the sequence at the 3'-end, was used in the following manipulations. One over one Gel purified 1.1 Kbp Eco RI-Pst I fragment from penvRVIPT was inserted into the Eco RI and Pst I sites of pUC9 to give pRW 756. This plasmid was then co-infected with Kpn I and Hind III cleaved, giving the vector 59 bases was split above the ATG. A 146 bp Kpn I - Hind III fragment containing the previously described vaccinia H6 pro engine was used to construct plasmid pCE6 inserted.

To ensure that the initiation ATG of the RAV env gene was deletion of overdue sequences adjacent to the 3 'end of the H6 promoter, two complementary synthetic oligonucleotides were made with Eco RV and Ban II sites at the ends. The oligonucleotide sequence was

The plasmid pCE 6 was cleaved with Eco RV that was in the H6 Promoter cleaves 24 bases before the ATG, and with Ban II, the in the RAV env coding sequence 7 bases after the ATG splits. The DNA sections were ligated and transfor used by E. coli cells. The resulting Plasmid, pCE 7, provided the H6 promoter and the correct 5 ' Sequence for the final construction available.

Restriction mapping revealed that clone mp19env (190) contains the complete RAV-1 env gene. On  the complete gene containing 1.9 Kbp Kpn I-Sac I-Frag mp19env (190) became the Kpn I and Sac I sites used by pUC 18 and formed pCE 3. This plasmid was with Hpa I, which was 132 bases after the initiation ATG in cleaves the RAV-1 coding sequence, and with Sac I, the am 3 'end of the gene splits, digests. The above beschrie The bene FPV insertion vector pCE 11 was labeled with Sma I and Sac I which cuts the plasmid in the polylinker region the. The Hpa I - Sac I fragment of pCE 3 was treated with pCE 11 ligated and formed pCE 14.

The plasmid pCE 7 was then treated with Xho I and Hind III cleaved to the H6 promoter and the correct 5'-Se quence containing 332 base pair fragment available put. Plasmid pCE14 was digested with Hind III, which was expressed in of the polylinker region of the vector cleaves, and with Xho I, the in the coding sequence. This DNA was with the linked from pCE 7 Hind III - Xho I fragment and formed pCE 15, the final construction with the RAV 1 envelope gene.

This plasmid was used in an in vitro recombination test with poultry pox FP-1 as the host virus puts. The progeny of recombination was on CEF Monolayer plated and plaques in an immunoassay with a protein A linked to β-galactosidase under Ver use of a polyclonal anti-RAV-1 serum. In the Staining positive plagues were selected and four rounds subjected to a plaque purification to a homogeneous popula tion. The recombinant was prepared as designated vFP-22. Immunoprecipitation experiments under Ver use of vFP-22 infected CEF lysates specific precipitation of two proteins with molecular weights of 76.7 kD and 30 kD, which are the two gene products correspond to the envelope gene. No precursor gene product was visible.  

Preliminary tests revealed an immune response to RAV-I envelope Gene product with vFP-22 inoculated chickens.

Example 17 Construction of the GP51.30 envelope (env) gene from Bovine leukemia virus (BLV) -expressing avipoxvirus recombinant (1) Construction of pBLVF 1 and pBLVF 2

The plasmids pBLVF 1 and pBLVF 2 contain the gp51.30 env Gen of BLV. In both plasmids, the BLV gene is under the Transcriptional control of the vaccinia virus H6 promoter and is between the flanking arms of fowlpox virus (Locus f7) cloned. The nucleotide sequence of the two plas mide is identical except for the codon positions 268 and 269. (pBLVF 1 encodes a protein with the amino acids Arg- Ser at these two positions, while pBLVF 2 is a protein encoded containing the amino acids Gln-Thr).

pBLVF 1 and pBLVF 2 were prepared by the following methods represents: the plasmid containing the complete BLV env gene pNS97-1 was cleaved with Bam HI and partially with Mst II. The 2.3 kbp fragment containing the complete gp51.30 gene ment was isolated from an agarose gel and survived the ends (sticky ends) with E. coli DNA polymerase I (Kle now fragment). Subsequently, Pst I linkers ligated to the ends of the fragment, which after cleavage with Pst I was ligated into the Pst I site of pTP15 (Example 15). This brings the BLV gene in addition to the vaccinia H6 promoter (pTP15 contains the non-food in a vaccinia genom the clinical locus cloned Vaccinia H6 promoter).

Subsequently, this plasmid was treated with Eco RV and partially split with Ava II. The 5.2 Kbp fragment was isolated and the oligonucleotides  5'-ATCCGTTAAGTTTGTATCGTAATGCCCAAAGAACGACG-3 'and 5'-GACCGTCGTTCTTTGGGCATTACGATACAAACTTAACGGAT-3 'used to to recircularize the plasmid. This removes unnecessary ba between the BLV gene and the H6 promoter.

The resulting plasmid was digested with Pst I and par split with Bgl II and that under the H6 promoter standing BLV gene containing 1.7 Kbp fragment was in the Bam HI-Pst I site of pGE 11, previously described Fowlpox insertion vector, using the locus f7 cloned. This brings the BLV gene next to the H6 promoter between the flanking arms of fowlpox virus. This plasmid was named pBLVF 1.

An identical procedure was used to concoct pBLVF 2 with the exception that before the BLV cloning Gens under the H6 promoter in pCE 11 an additional in in vitro mutagenesis step was performed. This mutagenesis was performed by the following procedure. plasmid pNS97-1 was cleaved with Xma I and partially with Stu I. The 5.2 Kbp fragment was isolated and the oligonucleotides 5'-CCGGGTCAGACAAACTCCCGTCGCAGCCGTGACCTTAGG-3 'and 5'-CCTAAGGTCAGGGCTGCGACGGGAGTTTGTCTGAC-3 'used to prepare the Recircularize plasmid. This changes the nucleotides sequence of codons 268 and 269 from CGC-AGT to GAA-ACT.

(2) Construction of recombinant viruses

The plasmids pBLVF 1 and pBLVF 2 were in vitro Recombination test used using FP-1 as catching virus. Recombinant offspring was off chosen via in situ plaque hybridization. As soon as the Popu lation is considered purely on the basis of these criteria was immunoassay with a β-galactosidase protein A using a preparation of one specific for BLV gp tested monoclonal antibody. Both from the plas pBLVF 1 or pBLVF 2 produced recombinant vFP  23 and vFP 24 showed a positive staining in the immune test. This shows that an immunologically detectable glyco protein expressed on the surface of the infected cells has been.

The plasmids pBLVK 4 and pBLVK 6 contain the BLV env gp51.30 gene or the BLV gp51.30 gene minus one cleavage. Both genes are in the unique Eco RI site of pRW 764.2 (Locus C3) cloned (pRW 764.2 is described in Example 13 ben) and under the transcriptional control of the vaccinia H6 Promoter.

The plasmids were obtained by the following procedure:
pBLVF1 and pBLVF 2 were cleaved with the restriction enzyme Hind III. Oligonucleotide BKL 1 (AGCTTGAATTCA) was cloned into this site to generate an Eco RI site on the 3 'side of the BLV gene. Since there is also an Eco RI site on the 5 'side of the BLV gene, these plasmids (pBLVK 1 and pBLVK 2) were cleaved with Eco RI and the fragment containing the BLV gene under the H6 promoter inserted into the Eco RI site. Site of pRW 764.2 cloned. The resulting plasmids were designated as pBLVK 4 and pBLVK 6, respectively. These plasmids were used in an in vitro recombination test with canarypox virus as the catching virus. Recombinants were selected and purified for surface expression of the glycoprotein, which was checked in an immunoassay. Recombinants were selected and purified on the basis of surface expression of the glycoprotein, as demonstrated in the immunoassay. The recombinants were designated as vCP 27 and vCP 28 of plasmids pBLVK 4 and pBLVK 6, respectively.

With the poultry pox recombinants vFP 23 and vFP 24 were Sheep and cattle are vaccinated in a number of ways. The Animals were given two vaccinations, the second 45 days after the first. Serum samples were taken 5 weeks after the first Vaccination and taken two weeks after the second vaccination.  

Antibody to gp51 was tested in a competitive ELISA determined and the titer as the reciprocal of the dilution giving a 50% reduction in repression. The Results are summarized in Table XI.

None of the species tested showed any detectable Immune response after the first vaccination. Both sheep and sheep Cattle showed a significant increase in antibodies after the second vaccination.

Table XI

Inoculation of sheep and cattle with vFP23 and vFP24

Example 18 Construction of the infectious bronchitis virus Mass 41- Matrix-expressing poultry cam virus vFP-1 Recombinant vFP-26

Plasmid pIBVM63 contains an infectious bronchitis virus (IBV) cDNA clone of the matrix gene of strain Mass 41. An 8 Kbp Eco RI fragment from pIBVM63 contains the matrix gene with the Peplomergene above (5 ') and still above an Eco RV site. Plasmid pRW 715 has an Eco RI linker containing the connects both Pvu II sites of pUC 9. The 8 Kbp Eco RI Fragment from pIBVM63 was inserted into the pRW 715 Eco RI site and resulted in pRW 763. Plasmid pRW 776 was generated, to remove the 5 'side Eco RI site from the pRW 763 and so a singular Eco RI site below (3 ') of the Matrix gene left over. The isolated linear product an Eco RI partial digest of pRW 763 was digested with Eco RV recut. The largest fragment was isolated, with the Klenow fragment of DNA polymerase I at the ends smooth ge and ligated with itself, producing pRW 776 has been. The construction pRW 776 has the complete IBV Peplomer and matrix genes followed by a singular Eco RI site.

Only the 5 'and 3' ends of the approximately 0.9 Kbp matrix gene were sequenced. The 5 'sequence of the matrix gene starting at the initiation codon (ATG) of translation contains the following underlined Rsa I site:
ATGTCCAACGAGACAAAT GTAC . The previously described H6 promoter was added to the matrix gene with a synthetic oligonucleotide. The synthetic oligonucleotide contained the H6 sequence from its Eco RV site to the ATG and into the coding sequence of the matrix gene to the first Rsa I site. The oligonucleotide was synthesized with Bam HI and Eco RI compatible ends for insertion into pUC 9 and resulted in pRW 772. The Eco RI end is on the 3 'side of the Rsa I site. Beginning with the Bam HI-compatible end, the sequence of the double-stranded synthetic oligo nucleotide is the following, with the ATG underlined:

The linear product of the Rsa I partial digest of pRW 772 was isolated and post-split with Eco RI. The pRW 772- Fragment which has only been applied once to the aforementioned Rsa I Site and split at the Eco RI site became iso treated with phosphatase and as a vector for the pro the digest of pRW 776 (see below).

The linear, isolated product of Rsa I partial digestion with Rsa I from pRW 776 was cleaved with Eco RI. The eco RI site is immediately after the 3 'end of the matrix gene. An isolated, the coding matrix sequence ab vorste containing about Rsa I site, about 0.8 Kbp lan The Rsa I Eco RI fragment was used in the above Inserted vector pRW 772 and resulted in pRW 783. The full permanent H6 promoter was formed by adding 31722 00070 552 001000280000000200012000285913161100040 0002003890874 00004 31603 the Se sequences located on the 5 'side of the Eco RV site. The 5'-end of the H6 promoter was a Hinf I site, which with smoothed ends into the Sal I site of pUC 9 added and thus became an Eco RI site; on the 5'- Side of the H6 promoter is the Hind III site of pUC 9. The Hind III Eco containing the 5 'side of the H6 promoter RV fragment was inserted between the Hind III and Eco RV sites advertised by pRW 783 and resulted in pRW 786. That's the full permanent matrix gene dependent on the H6 promoter The end Eco RI fragment of pRW 786 was attached to the ends Klenow fragment of DNA polymerase I made smooth in the at the ends, blunted Bam HI site from pRW 731.15 (Locus f8) and thus generated pRW 789. The pRW 731.15 Bam HI site is that used in Example 6 for the construction of vFP-8 used FP-1 locus.  

Plasmid pRW 789 was used to construct vFP-26 uses. Recombinant plaques were selected and one in situ plaque hybridization.

Preliminary tests revealed that an immune response to the IBV Matrix protein induced in chickens vaccinated with vFP-26 that is.

Example 19 Construction of the Infectious Bronchitis Virus (IBV) - Peplomer expressing fowlpox virus FP-1 Recombinant vFP-31

The infectious bronchitis virus (IBV) Mass 41 cDNA clone pIBVM 63 and its subclone, pRW 776, were described in the construction of vFP-26 in Example 18. Subclone pRW 776 contains the 4 Kbp IBV peplomer gene followed by the matrix gene with a unique Eco RI site at the 3 'end. Only the 5 'and 3' ends of the approximately 4 Kbp IBV peplomer gene were sequenced. A singular Xba I site separates the two genes. The 5 'end of the peplomer gene starting with the translation initiation codon (ATG) contains the following underlined Rsa I site:

The H6 promoter described above was linked to the peplogen via a synthetic oligonucleotide. The synthetic oligonucleotide contains the H6 promoter sequence from its Nru I site to the ATG and into the coding peptidomer to its first Rsa I site. The oligo nucleotide was synthesized with Bam HI and Eco RI compatible ends for insertion into pUC 9 and resulted in pRW 768. The Eco RI end is on the 3 'side of the Rsa I site. Beginning with the Ham HI-compatible end, the sequence of the double-stranded synthetic oligonucleotide is as follows, with the ATG underlined:

The isolated linear product of a partial digestion with Rsa I pRW 768 was cleaved with Eco RI. The one individual section at the aforementioned Rsa I site and a post-cut with Eco RI containing pRW 768 fragment was isolated, treated with phosphatase and used as vector for the digestion product of pRW 776 described below used.

The isolated linear product of a Rsa I partial digest of pRW 776 was cleaved with Eco RI. The 5 kbp pRW 776- Fragment up to the Eco RI site, which is a single Section on the aforementioned Rsa I-Stlle contained, isolated; the fragment contains IBV sequences from the aforementioned Peplomer Rsa I site to the Eco RI site at the 3 'end of the matrix gene. The insertion of the pRW 776 fragment into the The aforementioned vector pRW 768 led to pRW 788. The matrix gene was removed at the aforementioned Xba I site. The 5'- Side of the H6 promoter was by insertion of the at the Ends smoothed 4 kbp long pRW 788 Nru I-Xba I-Frag ment in the vices made smooth at the ends of Nru I-Bam HI pRW 760 at the Nru I site and resulted in pRW 790. The vector pRW 760 is described in Example 11; short said is an influenza nucleoprotein under Control of the vaccinia H6 promoter from the non-it flanked by the essential FP-1 locus f7. The vector pRW 760 was generated by removing the H6 sequences on the 3 ' Side from the Nru I site to the end of the nucleoprotein to Bam HI. pRW 790 is an IBV peplomer under the promoter H6 in the Hinc II site of pRW 731.13. Recombination of the Donor plasmids pRW 790 with FP-1 resulted in vFP-31. Immunpräzi pitation experiments using CEF lysates, the from vFP-31 infected cells  specific precipitation of a small amount of precursor protein having a molecular weight of about 180 kD and of Fission products of 90 kD.

Example 20 Construction of herpes simplex gD expressing Fowlpox virus FP-1 recombinant vFP-30

The herpes simplex virus (HSV) type 1 glycoprotein D gene (gD) from the KOS strain was inserted into the Bam HI site of pUC 9 as one at the 5'-Bam HI end with Hpa II -NruI fragment bound to the at the 3'-Bam HI end, cloned; the 5 'end is immediately adjacent to the Pst I site of pUC 9. The 5' sequence of HSV gD starting with the initiation codon (ATG) of the transla tion contains the following underlined Nco I site:

The vaccinia H6 promoter described above was linked to the HSV gD gene via a synthetic oligonucleotide. The synthetic oligonucleotide contains the 3 'portion of the H6 promoter from the Nru I site to the ATG and further to the coding sequence from gD to the Nco I site. The oligonucleotide was synthesized with a Pst I compatible end on the 5 'side. The gD clone in pUC 9 was cleaved with Pst I and Nco I and the HSV sequence removed at the 5 'end for exchange with the synthetic oligonucleotide, resulting in pRW787. The sequence of the double-stranded synthetic oligonucleotide is:

Digestion of pRW 787 with Nru I and Bam HI generates a fragment of about 1.3 kbp, which is the 3 'side of the H6 promoter from the  Nru I site through the coding sequence of HSV gD through to the Bam HI site. The one with Nru I and Bam HI digested vector pRW 760 was described in Example 11 ben. The insertion of a 1.3 Kbp fragment into the vector pRW 760 resulted in pRW 791. The vector pRW 791 contains the full Steady HSV gD gene under the vaccinia H6 promoter in the nonessential Hinc II site in FP-1 in pRW 731.13 (Locus f7).

Recombination of the donor plasmid pRW 791 with FP-1 resulted VFP 30th Surface expression of the glycoprotein was reported in re combined plaques detected using β- Galactosidase linked protein A and for HSV-1 specifi sera.

Example 21 Use of Entomopockenpromotoren for the regulation of Expression of foreign genes in poxvirus vectors (a) Background

Insect pox viruses (entomopoxes) are becoming current in the subfamily Entomopoxvirinae, which continues is divided into three genera (A, B and C), the Entomo poxviruses resulting from the insect orders Co leoptera, Lepidoptera or Orthoptera were isolated. ento moxa viruses have a tight host range in nature and it is not known that she is in any vertebra replicate.

The virus used in the present studies was originally from infected larvae of Amsacta moorei (Le pidoptera: arctildae) isolated from India; see. Roberts and Granados, J. Invertebr. Pathol. Vol. 12, (1968), pages 141- 143. The virus, referred to as AmEPV, is the leading species for Genus B.  

Wild AmEPV was developed by Dr. med. R. Granados (Boyce Thomson Institute, Cornell University) as an infectious hemolymph of infected Estigmene acrea larvae were obtained. It was gefun that the virus in the invertebrate cell line IPLB-LD652Y which disparates from ovarian tissues of Lymantria (Sponge spinner) (described by Goodwin et al., In Vitro Bd. 14 (1978) pages 485-494). These cells were grown at 28 ° C in IPL 528 medium supplemented with 4% fö calf and 4% chicken serum was supplemented.

The wild-type virus was plaques on LD652Y cells ge checks and one plaque, referred to as V1, has been added for more Experiments selected. This isolate calls numerous ones final bodies (OBs) in the cytoplasm of the infected cells towards the end of the infection cycle.

(b) promoter identification

The identification and mapping of an AmEPV promoter was achieved as follows. Total RNA from infected LD652Y Cells (48 hours after infection, late phase) isolated and used to with 32P-labeled first strand cDNA to create. The cDNA was then used to check blots containing restriction digests of AnEPV genome. This Southern Blot led to the discovery of a strong On a 2.6 kb Cla I fragment, indicating that this fragment encoded a highly expressed gene. The Fragment was cloned into a plasmid vector and its DNA sequence determined.

Analysis of the sequence data revealed an open reading frame, the was able to encode a 42 kD polypeptide. In vitro Translation of the total RNA at the time 48 hours after the Infection and separation of the products resulted via SDS-PAGE to a polypeptide of about 42 kD.  

(c) Construction of a recombinant vaccinia virus with the Expression of a foreign gene under the control of the entomo poxvirus promoter

To determine whether an entomopox virus promoter would function in a vertebrate poxvirus system, the following plasmid was constructed: an oligonucleotide was chemically synthesized which was 107 bases from the 5 'side of the translation start signal of the 42K gene (hereafter referred to as AmEPV 42K gene). Promoter) and flanked by a Bgl II site at the 5 'end and at the 3' end by the first 14 bases of the coding Eco RI site cessation region of hepatitis B virus pre-S2. The AmEPV 42K promoter sequence is described below:

The AmEPV 42K promoter was linked to the hepatitis B virus top Area antigen (HBVsAG) ligated as follows. A hepatitis B virus surface antigen and the coding region of pre-52 (type ayw as described by Galibert et al., Nature Bd. 281 (1979), pages 646-650) containing pUC plasmid was generated by the vaccinia virus arms in the non- essential region of the vaccinia virus genome heme agglutinin (HA) molecule encoded (HA arms described in Bei play 15; HA region described by Shida, Virology vol. 150 (1986), pages 451-462). The above be oligonucleotide was inserted into this plasmid, by encoding the unique Eco RI site into the HBVsAg Area and the unique Bgl II site in the HA vaccin was used niaarm. The resulting recombinant Vaccinia virus was designated VP 547.

The expression of the coding HBVsAg sequence under the control of the entomopox 42K promoter was detected by immunoassay. Corresponding cultures of mammalian cell line BSC-40 were infected with parent vaccinia virus or the recombinant vP 547. 24 hours after infection, the cells were lysed and the lysate in Reihenverdünnun gene applied to a nitrocellulose membrane. The membrane was incubated first with goat anti-HBV serum followed by ¹²⁵ J protein A. After washing, an X-ray film was exposed to the membrane. Positive signals were found in cultures infected with vP 547, but not in parent virus-infected cultures, suggesting recognition of the AmEPV 42K promoter by vaccinia virus in mammalian cells.

The above results were confirmed by Verwen an Ausria assay (see Example 1 for details) HBVsAg in infected mammalian cells. vacci niavirus recombinants that either the HBVsAg gene AmEPV42K or coupled to the vaccinia virus H6 promoter were used to infect BSC-40 cells; the level of expression of sAg was in an Ausria test determined. As shown in Table XII, the data show that that the amount of expression of HBVsAg using the 42K promoter was significant.

Table XII

Expression of HBVsAg in recombinant vaccinia virus

Further experiments were performed to determine the time dep The nature of the regulation of the AmEPV 42K promoter in one Vertebrate pox virus system to confirm. similar Cultures of BSC-40 cells were present with vP 547  or absence of 40 μg / ml cytosine arabinoside, one of the half the late viral transcription blocking inhibitor DNA replication infects. The amounts of expression were transfused 24 hours after infection in an Ausria test reviewed. The results show that the 42K promoter as a early promoter in a vaccinia virus replication system was detected.

The use of the AmEPV 42K promoter for the expression of Foreign genes in a mammalian system can be seen ver divorced from the use of the Autographa californica NPV Polyhedrin promoter for gene expression in invertebrate Systems (Luckow and Summers, Biotechnology, Vol. 6 (1988), Pages 47-55). The polyhedrin promoter is not affected by the Transcriptional apparatus detected in mammalian cells (Tjla et al., Virology, Vol. 125 (1983), pages 107-117). The Verwen The production of the AmEPV 42K promoter in mammalian cells is first shown times that an insect virus promoter for the expression of foreign the genes in a non-insect viral vector in non-insect vectors. Invertebrate cells has been used.

To determine if avipoxviruses would also recognize the 42K entomopox promoter, the following experiment was performed. Identical cultures of CEF cells were inoculated with 10 pfu per cell from either fowlpox virus, rabbit pox virus or vaccinia virus and simultaneously transfected with 25 μg of one of the following plasmids: 1) plasmid 42K.17, the HBV pre-S ₂ + sAg coding sequence or 2) plasmid pMP15.spsP containing the identical HBVsAg coding sequence linked to the vaccinia virus H6 promoter. After 24 hours, the cells were frozen and lysed, and the lysate was assayed for the presence of HBVsAg using an Ausria assay (see Example 1).

The results shown in Table XIII should be in one qualitative sense. They indicate that the  Transcriptional apparatus of both fowlpox virus and Also of canarypox virus is capable of the 42K promo to recognize and transcribe the associated gate coding HBVsAg sequence allowed. Although the quantities Expression are lower than those with the vaccinia virus H6- Promoter, the amounts are well above the background quantities obtained with the negative controls.

Table XIII

Recognition of the 42K Entomopockenviruspromotors by Avipoxvi ren

Example 22 Immunization with VCP-16 to protect mice against Stress infection with live rabies virus

Groups of 20 four to six week old mice were in the paw surface with 50 to 100 μl of a series of diluents inoculated from one of the following recombinants: (a) vFP-6 - the poultry pox described in Example 6 Rabies recombinant - and (b) vCP-16 - that in Example 13 canarypox rabies recombinant.  

After 14 days, 10 mice from each group were sacrificed and the serum collected. The anti-rabies titer in the serum was calculated using the RFFI test described in Example 7. The remaining 10 mice from each group were exposed to challenge infection by intracerebral inoculation with the CVS strain of rabies virus used in Example 7. Each mouse received 30 μl, which corresponded to 16 mouse LD ₅₀ . After 28 days, the number of surviving mice was determined and the protective dose 50 (PD ₅₀ ) was calculated. The results are summarized in Table XIV.

The level of protection found in mice vaccinated with vFP-6 confirmed the result of vaccination with recombinant avipoxviruses vFP-3 as discussed in Example 7. The level of protection achieved with vaccination with vCP-16 is considerably higher. Based on the calculated PD ₅₀ , the canine rabbit-rabies recombinant is 100 times more effective in protecting against a rabies challenge than the poultry rabies recombinant.

Table XIV

The protection afforded by two avipox rabies recombinants against a challenge infection with rabies virus

Example 23 Use of poultry pox promoter elements for expression foreign genes I. Identification of a 25.8 kilodalton (kD) gene product coding fowlpox gene

Visualization of protein types present in CEF lysates infected with fowlpox (FP-1) by Coomassie Brilliant Blue staining of SDS-polyacrylamide gels revealed an abundant type with an apparent molecular weight of 25.8 kD. This protein was absent in non-infected cell lysates. Pulse experiments using ³⁵ S-methionine to radiolabel synthesized proteins at certain times after infection again showed the large amount of FP-1-induced protein and showed that it is synthesized 6 to 54 hours after infection. At its peak, this FP-1 25.8 kD protein makes up about 5 to 10% of the total protein present in the cell lysate.

The large amount of FP-1-induced 25.8 kD protein suggested that the gene encoding this gene product of a strong FP-1 promoter element is regulated. To this Promoter element for subsequent use in expres foreign genes in poxvirus recombinants to lo calis, a polysome preparation was made with FP-1 infected CEF cells were collected 54 hours after infection. RNA from this polysome preparation was isolated and Then, if you use them to program a Kanin chenreticulocyten in vitro translation system used predominantly for the formation of 25.8.8 kD FP-1 protein.

The polysome RNA was also used as a template for cDNA syn the first strand using an oligo (dT) 12-18 used as a primer. The first strand of the cDNA was used as a hybridization probe in a Southern blot Analysis with genomic digests of FP-1 used. Results from these hybridization analyzes suggested that this was for the 25.8 kD protein encoding gene in 10.5 kbp Hind III fragment was included. This genomic Hind III Fragment was subsequently isolated and with a commercial vector, pBS (Stratagene, La Jolla, CA.), cloned and the clone designated pFP23k-1. Further hybridization analysis using the first strand of the cDNA as a probe against digestion of pFP23k-1 led to localization tion of the 25.8 kD gene on a 3.2 Kbp Eco RV sub-fragment.  

This fragment was subcloned into pBS and called pFP23k-2 designated.

Approximately 2.4 Kbp of this FP-1 Eco RV fragment was added to the Sanger dideoxy chain termination method (Sanger et al., Proc. Natl. Acad. Sci. USA, Vol. 74 (1977), pages 5463-5467) sequenced. The analysis of the sequence gives an open Le serahmen (ORF), which is a gene product with a Molekularge weight of 25.8 kD. In vitro run-off transcription this ORF by bacteriophage T7 polymerase (Strata gene, La Jolla, CA) in a pBS vector generates an RNA Type which, when used for programming a rabbit re ticulocyte in vitro translation system (Promega Biotec, Madison, WI) a polypeptide species with an apparent molecule large weight of 25.8 kD results. This polypeptide migrates an SDS-polyacrylamide gel together with the rich IN ANY 25.8 kD protein expressed in FPF-infected CEFs is observed. These results suggest that it is the coding gene for the FP-1 induced rich existing 25.8 kD gene product is.

II. Use of upstream promoter elements of the FP-1 25.8 kD gene for the expression of feline leukemia virus (FeLV) env gene in FP-1 and vaccinia recombinants

A 270 bp Eco RV / Eco RI fragment (FP25.8K promoter) containing the regulatory region of the FP-1 25.8 kD gene and 21 bp of the coding sequence of the 25.8 kD gene were isolated from pFP23k-2. The following is the nucleotide sequence of the FP 25.8K promoter region used to generate pFeLV25.8F1 and pFeLV25.81A. This 270 nucleotide sequence provides 249 nucleotides of the 5 'region before the initiation codon (ATG) for the 25.8 kD gene product and the first 21 bp of the coding sequence.

This fragment was made smooth at the ends and then in a Sma I digested, containing FeLV env sequences FP-1 insertion vector (pFeLVF1, see Example 15) added. This insertion vector allowed recombination with the f7 locus of the FP-1 genome. The insertion of the FP25.8K Pro engine upstream sequences on the 5 'side of the FeLV env gene and the correct orientation was by sequence analysis confirmed. This insertion does not provide a perfect ATG for the ATG substitution available since that of the 25.8 kD gene provided ATG is not in the reading frame of FeLV env ATG is located and therefore no fusion protein is formed. The FP-1 insertion plasmid containing the FP25.8 kD promoter above half of the FeLV env gene was reported as pFeLV25.8F1 records.

A similar construction was made using Verwen tion of the vaccinia virus insert containing the FeLV gene vector pFeLV1A (see Example 15). The H6 promoter was excised from pFeLV1A by digestion with Bgl II and Sma I. After the Bgl II restriction site at the end smooth ge which was the FP25.8K promoter containing Finished flattened 270 bp Eco RV / Eco RI fragment beside the 5 'end of the FeLV env gene confirmed. This construction was checked by sequence analysis. Also in this Rekom binante, there is no perfect ATG for ATG substitution since the ATG and the 25.8 kD gene are not in frame with the ATG of the FeLV gene. The next the 25.8 KD gene upstream region the vaccinia insert containing the 5 'end of the FeLV gene Vector (Copenhagen strain) was designated pFeLV25.81A.  

The insertion plasmids pFeLV25.8F1 and pFeLV25.81A were for in vitro recombination with FP-1 (pFeLV25.8F1) and the Copenhagen strain of vaccinia virus (pFeLV25.81A) as the used catching viruses. The offspring of Rekom Bination was plated on appropriate cell monolayers and re Combined viruses were immunized with β-Ga lactosidase linked protein A and a bovine anti- FeLV serum (Antibodies, Inc., Davis, CA.). before Preliminary results suggest that the FP25.8K promoter is the Expression of foreign genes in poxvirus recombinants can.

Example 24 Safety and efficacy of vFP-6 and vCP-16 in poultry

With the two avipox recombinants vFP-6 and vCP-16 (be written in Examples 6 and 13) were 18 days old Chicken embryos, day-old chicks and 28-day-old chicks are inoculated and evaluated the response of the birds according to three criteria: 1) Effect of vaccination on hatching, vaccination reactions and mortality 2) that of the rabies glycoprotein called immune response; and 3) those of the fowlpoxanti gene-induced immune response. The experiments were carried out as follows.

A. Safety tests

Groups of twenty 18-day-old embryos were inoculated into the allantoic cavity with 3.0 or 4.0 log ₁₀ TCID _{50 of} either vFP-6 or vCP-16. After hatching, the chicks were observed for 14 days, after which they were individually bled and the sera collected. The two recombinants inoculated into the chick embryos had no effect on the hatching rate of the eggs and the chicks remained healthy during the 14-day observation period.

Groups of ten day-old SPF chicks were vaccinated intramuscularly with 3.0 log ₁₀ TCID ₅₀ of each of the recombinants. The chicks were observed for 28 days and serum samples collected at 14 and 28 days post vaccination. None of the recombinants were observed to vaccinate at the injection site and the chicks remained healthy during the 28 days of observation.

Groups of ten 28 day old chicks were inoculated with one of the two recombinant viruses, receiving either 3.0 log ₁₀ TCID ₅₀ intramuscularly or 3.0 log ₁₀ TCID ₅₀ via the skin route (poultry network). The chicks were observed for 28 days and serum samples collected at 14 and 28 days post vaccination. No response to intramuscular vaccination was observed in either of the two recombinants. Vaccination into the skin resulted in a very small vaccine response to fowlpox with lesions that were heterogeneous in size. Canarypox vaccination resulted in the formation of a normal lesion on the skin at the injection site. All lesions had decreased by the end of the experiment.

B. immune response

The RFFI test described in Example 7 was used to the levels of antibodies against the rabies glycoprotein deliver. For each group, the results were reported as the geometrically averaged titres of the individual sera press that corresponding to International Units (IU) a standard serum, the 23.4 IU contained. The smallest positive value was stated as an IU and used to increase the percentage of positive birds determine. Antibodies against avipoxviruses were co-administered with a ELISA method using a fowlpox virus stem tested as an antigen. Each serum sample was 1/20 and Diluted 1/80. A standard curve was constructed using the positive and negative sera. The smallest po Positive value was calculated by the mean of the different  Values of the negative sera with twice the standard value was added.

The results of the serological observations are reported in Ta XV for vFP-6 and Table XVI for vCP-16.

Limited antibody formation on both rabies and on Fowlpox antigens have been observed in embryos that were inoculated with either vFP-6 or vCP-16. The poultry pox vector caused antibody formation on both antigens in a larger number of birds than the canaries pox vector, but the answer was still heterogeneous.

One-day-old chicks inoculated with vFP-6 showed a good antibody formation, with all the chicks on rabies and chicken pox were antigenic seropositive 28 days after vaccination. The Response to vaccination with vCP-16 was much lower where 40% of the chicks are seropositive for rabies glycoprotein after 28 days and 10% were seropositive for avipox antigens.

28-day-old chicks vaccinated intramuscularly with vFP-6 100% seroconversion to both antigens 14 days after Vaccination. Although the majority of chicks are also seroconversion after vaccination into the skin, the titers achieved were much lower for both rabies and avipox antigens. As previously demonstrated with vCP-16 both over the intramuscular as well as the cutaneous way vaccinated chicks a patchy Response with a maximum of 70% seroconversion on Rabies via intramuscular inoculation. The low value seroconversion for avipox antigens after vaccination with Ka Narkepox could related to the degree of serological between the viruses.

The results show that both vFP-6 and vCP-16 are safe for the vaccination of chickens in a wide age range are. The chicken pox vector vFP-6 seems to be more efficient at triggering an immune response in chickens. For the  both recombinant avipox viruses fowlpox and cana however, it has been shown significantly that Vaccination in ovum are useful.  

Example 25 Safety and immunogenic effects of vaccination of piglets with vFP-6

Two groups of three piglets were inoculated with the recombinant vFP-6 via one of two routes:

• a) three animals received 8.1 log ₁₀ TCID ₅₀ by intramuscular vaccination; and
• b) three animals received the same dose by oral Imp fung.

All animals were bled weekly and all animals received a booster of the same Dose the same way on day 35. The piglets were observed daily for clinical symptoms. The sera were on anti-fowlpox antibodies with an ELISA test and tested a serum neutralization test. Rabies antibody were determined in an RFFI test.

All piglets remained in good health and no lesions were observed after vaccination. The temperature curves wa Normal, with no difference between vaccinated and Unvaccinated animals came to light. Both over the intramuscular as well as oral route vaccinated piglets ent carried out antibody formation on poultry pox antigens, as determined by ELISA and serum neutralization. A second answer was obvious after the refresh fung. (Results not shown). All piglets developed also an immune response to rabies glycoprotein, as in an RFFI test and a refresh effect was included both ways obviously. These results are in table XVII summarized.

The results indicate that vaccination with poultry pox / rabies recombinant is harmless to piglets and that the recombinant is capable of producing a significant immune response  to the rabies glycoprotein after oral or intramuscular vaccination.

Table XVII

Antibodies raised against rabies glycoprotein in piglets vaccinated with vFP-6

Claims (16)

A recombinant virus capable of eliciting an immune response against a particular pathogen when introduced into a host, characterized in that the recombinant virus contains an exogenous DNA insert located at a non-essential region of the virus genome and downstream of a suitable promoter sequence wherein the exogenous DNA insert encodes an antigen of the pathogen and allows its expression under the control of the promoter sequence in vivo in the vertebrate, and no replicative propagation of the virus takes place in the vertebrate. 2. Recombinant virus according to claim 1, which is a recombinant is poxvirus. 3. Recombinant virus according to claim 2, which is a recombinant This is Avipoxvirus. 4. Recombinant virus according to claim 3, which is a recombinant fowlpox virus or canarypox virus is. 5. Recombinant virus according to one of claims 1 to 4, wherein the antigen derived from the recombinant virus ex is primed when inserted into a vertebrate is an antigen of a mammalian pathogen. 6. Recombinant virus according to one of claims 1 to 5, wherein the vertebrate is a mammal. 7. Recombinant virus according to claim 5 or 6, wherein the Antigen Rabies G-antigen, the gp51,30-envelope antigen of the  Bovine leukemia virus, the FeLV envelope antigen of cats leukemia virus or the glycoprotein D antigen of the herpes simplex virus is. 8. Recombinant virus according to claim 6 or 7, wherein the recombinant virus allows expression of the antigen, without an increase of the virus by replication he follows in mammals selected from the group consisting from dogs, cats, mice, rabbits, cattle, sheep and pigs. 9. Recombinant virus according to one of claims 6 to 8, wherein the promoter sequence is a fowlpox virus Pro includes motor sequence as part of the exogenous DNA insertion is inserted into the viral genome and upstream of the antigen coding region, and which drives the expression of the antigen or in addition drives when the virus is introduced into mammals. 10. Recombinant virus according to one of claims 6 to 8, wherein the promoter sequence is a non-avipox promoter This is part of the exogenous DNA Inser tion is inserted into the viral genome and upstream is the antigen-coding region, whereby the Driven or in addition to expression of the antigen is driven when the virus is introduced into mammals becomes. 11. Recombinant virus when introduced into a bird to trigger an immune response to a bird pathogen ver may be, this recombinant virus being a recombinant Avipox virus comprising an exogenous DNA insert stops in a non-essential region of Avi poxvirus genome and downstream of a suitable promo is inserted, wherein the insertion is a Se comprising an antigen of the pathogen, and  the recombinant virus expresses the antigen in vivo, when introduced into the bird. 12. Recombinant virus according to claim 11, wherein the antigen a bird flu hemagglutinin antigen, a fusion pro teinantigen of Newcastle Disease Virus, a RAV-1 Envelope antigen of the Rous-associated virus, a Nucleopro antigen of avian influenza virus or a matrix or Preplomerantigen of the infectious bronchitis virus is. 13. recombinant avipox virus according to claim 10, 11 or 12, the exogenous DNA insert being a non-avipoxpromo and this promoter comprises a vaccinia promoter or an entomopox promoter. 14. Recombinant virus according to claim 13, wherein the vaccine niapromotor is an HH, 11K or Pi promoter. 15. Vaccine containing a recombinant virus after one of claims 1 to 14. 16. A method of expressing a gene product in vitro, in the in vertebrate cells of an in vitro cell culture a recombinant virus according to any one of claims 1 to 14 is introduced.