JPH02500879A - Recombinant avipoxvirus - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Recombinant avipoxvirus This application is filed under U.S. Patent Application No. 186.054, CIP, filed April 25, 1988. This CIP application is also a U.S. patent application filed on October 20, 1987. CIP application no. tto, ass, which was further filed on August 28, 1987 No. 090.711, CIP Application No. 090.711.

The present invention uses a synthetic recombinant Avibogges virus (lVipOXv'1rus). and methods for inducing immune responses in vertebrates, including non-avian vertebrates. . More specifically, the present invention provides methods for treating vertebrate pathogens in vertebrates, particularly mammals. Synthetic recombinant Avipods containing DNA encoding and expressing antigenic determinants of the body. Immunization against vertebrate pathogens can be achieved by inoculating vertebrates with virus. Methods for inducing biological responses and vaccines comprising such modified avipoxviruses Regarding the types. Furthermore, the present invention provides a modified avipoxvirus, its production and use. Method of use, produced in the production of modified avipoxvirus, or as an intermediate The present invention relates to DNA sequences involved in this invention and methods for producing such sequences.

Background of the invention Avipoxvirus, or avipoxvirus, is a pox that infects poultry. It is a genus closely related to viruses.

The genus Avibox includes the fowl pox and the canakubok. Canary pox, Jininko box Uunc.

pox)・Vibyon Box(pigeon I)OX)% Quailpox( quail pox) s sparrow box (sparrow pox) s Starling pox and turkey box urkey pox) species. Foulbox species infect chickens It should not be confused with the human disease called chickenpox. Other Avibox genus It shares many characteristics with the poxviruses of other animals, and belongs to the same subfamily as vaccinia. Pock containing vaccinia and avibox Viruses replicate within the eukaryotic cells of the host. These viruses are Characterized by being large, complex, and replicated in a cytoplasmic site It will be done.

However, vaccinia and Avivox are different genera, and the International Committee on Taxonomy of Viruses (International Coa+m1ttee onTaxonomy of Viruses) 4th Report, Interbio.

Reported in Intervlology, Vol. 17, pp. 42-44 (19H). As reported, each species differs in molecular weight, antigenic determinants, and host species. ing.

Avipoxviruses have a tendency to proliferate in non-avian animals such as mammals including humans. I don't dye it. In addition, Avivoc can be used even when inoculated into mammalian (including human) cultured cells. The seeds do not proliferate. Cytotoxicity in cultured mammalian cells inoculated with Avivox Cells die due to the effects, but there is no evidence of productive viral infection.

When a non-avian animal such as a mammal is inoculated with a live Avibox, the inoculated area will have a scar. Lesions similar to those of Cucilia inoculation are formed. However, a productive infection of the virus doesn't happen. Nevertheless, mammals vaccinated in this way do not become infected. It has now been discovered that the virus responds immunologically to the virus. this is unexpected This is the result.

Vaccines consisting of killed pathogens or purified antigenic components of such pathogens produce effective immune responses. In order to induce a response, a larger amount must be injected than a live virus vaccine. do not have.

This is because live virus inoculation is a much more efficient method of vaccination. Ru. Even with a relatively small amount of inoculation, the antigen in question can be amplified during virus replication. , can induce an effective immune response. From a medical point of view, viruses Live vaccines are more effective than vaccination with killed pathogens or purified antigen vaccines. Moreover, it provides immunity that lasts for a longer period of time. Therefore, killing pathogens or such diseases Vaccines consisting of bulk purified antigen components contain less vaccine material than live viruses. It needs to be manufactured in large quantities.

As is clear from what has already been said, the use of live virus vaccines is There are medical and economic advantages. One such live virus vaccine Examples include vaccines consisting of vaccinia virus. This virus is Inserting DNA representing the gene sequence of the antigen of the mammalian pathogen using transgenic DNA methods It is known in the prior art to be one of the useful viruses for.

Therefore, any origin that contains a DNA sequence encoding an antigenic determinant of a pathogenic organism Source (e.g., viral, prokaryotic, eukaryotic, synthetic) DNA into vaccinia Generate recombinant vaccinia viruses by inserting into nonessential regions of the genome. Methods have been developed in the prior art that can. produced by these methods Certain recombinant vaccinia viruses have specificity in mammals against various mammalian pathogens. These are all described in U.S. Patent No. 4. ．． No. 603,112, which is incorporated herein by reference.

Unmodified vaccinia virus is relatively safe and effective for smallpox vaccination. It has a long history of being used as However, before smallpox eradication, unmodified vaccinia When Niavirus was widely administered, it posed a moderate risk but did not pose a systemic risk. There is a real risk of complications in the form of senior infections, especially those with eczema or immunosuppression. There was a risk associated with people suffering from. Possible consequences of vaccinia vaccination One complication, although rare, is encephalitis after vaccination. these reactions Most people with a skin disease like eczema, or with a compromised immune system, or who have a family history of eczema or This was caused by vaccination of people with compromised immune systems. Vaccinia is It is a live virus and is usually harmless to healthy people. However, the number after vaccination It is contagious between individuals over a period of weeks. People who have a deficient normal immune response should be vaccinated or If infected through contact transmission from someone who has recently been vaccinated, serious consequences may occur. There is.

Therefore, while having the advantages of live virus inoculation in this technique, it overcomes the problems already mentioned. Such mitigation or elimination methods would provide desirable benefits far beyond the current state of the art. You can see that it has points. This is known as acquired immunodeficiency syndrome (AIDS). This is extremely important now that many known diseases are occurring. The cost of this disease The victim had a severe immunodeficiency and was exposed directly to live virus, which would be safe for others. Close contact or contact with someone who has recently received a vaccination consisting of a live virus preparation They are easily injured by such viral preparations when infected.

Purpose of invention The object of the present invention is to provide a vaccine capable of immunizing animals against pathogenic organisms. to immunize non-avian animals, especially non-avian animals, with the advantages of live viral vaccines. The drawbacks of live virus vaccines, such as those already listed, are that when used for The objective is to provide a vaccine that also has few or no disadvantages.

The present invention further provides synthetic recombinant avipoxviruses for use in such vaccines. The purpose is to provide

The present invention further provides for promoting non-avian species such as mammals in avian and non-avian species. In the case of animals, productive replication accompanied by the production of infectious virus within the animal body. Inoculating vertebrates with synthetic recombinant avipoxviruses that cannot The object of the present invention is to provide a method for inducing an immune response. It's a mammal. When vertebrates are inoculated with non-avian species such as eels, such viruses are transmitted to non-vaccinated hosts. Self-limiting and reducing the possibility of expansion.

The present invention further provides a method for inducing an immune response against an antigen in a vertebrate. a synthetic recombinant product containing and expressing antigenic determinants of a pathogen for vertebrates; A method comprising inoculating a vertebrate with a vaccine containing an avipoxvirus. The purpose is to provide

It also encodes a gene product and is capable of productively replicating the virus in an animal. A recombinant virus containing DNA that expresses a gene product is injected into a vertebrate. It also provides a method for expressing a gene product in a propagating animal by seeding. This is the object of the present invention.

Furthermore, the virus encodes an antigen and does not allow the virus to undergo productive replication in the animal. Inoculating a vertebrate with a recombinant virus containing DNA that expresses a specific antigen. The present invention also provides a method for inducing an immune response to antigens in vertebrates. This is the object of the invention.

Description of the invention One embodiment of the invention provides antigens of pathogens in non-essential regions of the Avivox genome. modified by the presence of DNA of any origin encoding and expressing By inoculating vertebrates with recombinant avipoxvirus, the pathogen can be The present invention relates to a method for inducing an immune response in a mammal in a mammal.

In another embodiment, the present invention provides a method for producing a vertebrate that does not replicate proliferatively in cells of a vertebrate, but that in animals using recombinant viruses that express gene products or antigens in cells that expressing a gene product in a vertebrate or inducing an immune response to an antigen in a vertebrate Regarding the method.

In another embodiment, the invention provides D Synthetic antibodies modified by inserting NA into non-essential regions of the Avivox genome Concerning vipoxvirus.

A person with a foreign (i.e., non-avibox) gene that encodes and expresses an antigen The engineered recombinant avipoxvirus has a vertebrate host that responds to the antigen. induce the development of an immune response against a foreign pathogen. However, in the present invention, such recombinants are treated as dead or attenuated living organisms. The disadvantages of conventional vaccines that use living organisms can be overcome by inoculating non-avian animals into vertebrate animals. It can be used to create new vaccines when needed.

Avipoxviruses can only replicate productively in birds or avian cell lines. , it should be mentioned here again that it can also be passaged. bird inn The recombinant avipoxvirus collected from the chief cells is caused by vaccinia virus. When non-avian animals such as mammals are inoculated into vertebrates in the same manner as inoculating mammals, the inoculation lesions occur, but productive replication of the avipoxvirus does not occur. this Avipoxviruses replicate productively in non-avian vertebrates that have been inoculated with Despite the lack of The substances are the antigenic determinants of this recombinant avipoxvirus and the foreign genes of the virus. immunologically responds to antigenic determinants encoded by

When inoculated into birds, such synthetic recombinant avipoxviruses are present in them. elicit an immune response against antigens encoded by foreign DNA of any origin Not only that, but the virus also undergoes productive replication within the host, resulting in Avivox vector. triggering the expected immunological response against the target itself.

Several researchers have developed a recombinant Ffururbox, specifically a movement to protect poultry. It has been proposed to produce lylum for use as a commercial vaccine. Bo Boy 1e and Coupar, Journal of the Genetic Pyrology (J, Gen, Virol), 87.1591 -1800 (19118) and Blnns et al. Le of: Veterinacue Medicine (Isr, J, Yet.

Med, ) 42.124-127 (198El), specific immunity in mammals. Regarding the use of recombinant avipoxvirus as a method for inducing Nothing was said about it, and it was never actually reported.

Step 4? Stiekl and Mayer at Fort Sinilitte ・Der Medizin (Fortscher, Med) 97(40), 178 1-1788 (1979), Avibox, specifically Foulbox Will. This paper describes the injection of this drug into humans. However, these studies have regular famine to enhance non-specific immunity in patients suffering from the sequelae of the It's only about using the foul box. No recombinant DNA technology used It has not been done. Insert the DNA encoding the antigen of the vertebrate pathogen into the Avibox. There are no suggestions as to how to induce specific immunity in vertebrates. It has not been. Instead, this prior art focuses on general and non-specific virulence of the human host. Based on the powerful effect.

Modifications of the present invention by adding a more complete review on the basis of genetic recombination It will be helpful in understanding how recombinant viruses are produced.

Genetic recombination generally involves combining homologous portions of deoxyribonucleic acid (DNA) into two DNAs. It is to exchange between the A helices.

(In some viruses, riboenzyme (RNA) can replace DNA.

) Homologous portions of nucleic acids are nucleic acids with the same sequence of nucleotide bases (RNA or D NA) part.

Genetic recombination occurs during the replication or production of new viral genomes within infected host cells. Sometimes it happens naturally.

Therefore, genetic recombination between viral genes can occur between two or more different viruses or other during the viral replication cycle that occurs in host cells co-infected with the genetic constructs of can also happen. The DNA portion of the first genome is compatible with the first viral genome DNA. in assembling parts of a co-infected second viral genome with the same DNA. Used interchangeably.

However, recombination also affects DNA in different genomes that are not completely homologous. It can also occur between A parts. For example, if such a portion is a gene map within a portion of the first genome, A gene encoding a marker or antigenic determinant is inserted into a portion of homologous DNA. If it originates from a first genome that is otherwise homologous to another genome part, then Recombination can still occur, and the products of this recombination may still be linked to their genetic markers or genes. Can be detected by the presence of children.

In order for the inserted DNA gene sequence to be expressed well by the modified infectious virus, Two conditions are required.

First of all, in order for the modified virus to remain viable, the insertion must be must be in a non-essential area of the space. Foul box is also avipoxwill It has also been suggested that a description of vaccinia virus It does not have areas similar to the non-essential areas that have been considered. Therefore, the present invention The non-essential regions of the foul box are generated by cutting the foul box genome into fragments. separated by size and inserted into plasmid constructs for amplification of these fragments. It was discovered by It is a small circular DNA molecule discovered as a color body element. antigenic determinant gene or Methods for inserting NA sequences into plasmids, such as other genetic markers, are in the art. Manlatls et al., Molecular cronin Molecular Clonlng: Experiment Manual (A Labo ratory Manual), Cold Spring a/%-Bar Lab Laboratory (Cold Spring Harbor Laboratory) 2 (described in detail in K. K. [19H]). Next, genetic markers and/or inserted the gene encoding the antigen into the cloned foul box fragment. . Fragments that have been successfully recombined in a specified manner should have good recovery of genetic markers or antigens. It can be demonstrated that such fragments are inserted into non-essential regions of the foul box genome. It was a fragment consisting of DNA.

The second condition for expression of the inserted DNA is the presence of a protein in the appropriate relationship with the inserted DNA. The existence of a lomotor.

The promoter must be placed upstream of the DNA sequence to be expressed. No. The properties of Avipoxvirus are not fully understood, and Avipoxvirus is not fully understood. Lomotors have not previously been identified in the art, and part of the present invention Expression promoters of known viral promoters can also be used in the methods of the present invention. have been successfully implemented and used to make the products of the present invention. According to the present invention, Foulbox promoter, vaccinia promoter and Entomobox The promoter was found to promote transcription of recombinant poxviruses.

Boyle and Coupar, Journal of Gen. Tick Bioogy (J, Gen, Virol,) 67.1591 (19 8B), the vaccinia promoter acts on the (foul box) virus. It is predicted that this will happen.'' The authors are using Foulbox Locating and cloning the TK gene (Boyle et al., pyrology) (Virology) 15B, 855J85 [1987]) and this was inserted into the vaccinia virus. This TK gene was expressed, but it is probably , this foul box TK promoter by the function of vaccinia polymerase This is probably because the sequence of - was recognized. But despite their speculation , the authors insert a vaccinia promoter into a foul box virus There was no evidence that foreign DNA sequences were expressed in the foul box genome. I didn't even observe that. Promoters from other poxviruses, e.g. The Cucnia promoter actually drives gene transcription in the Avivox genome. This was not known prior to the present invention.

Description of the preferred embodiment Foulbox and Kanazybox viruses take up the foreign DNA of the present invention. It was specifically used as a preferred Avivox species to be modified by integrated recombination.

Foulbox is a type of fowlbox that particularly infects chickens, but it also affects mammals. It does not infect milk. The foulbox strain, referred to herein as PP-5, is American and Scientific Laboratories (AmericanScl) entiflc Laboratores) (Schering tngCorp, a division of ), Madlson% Wyoming Chicken, available from Gu., U.S. Veterinary License No. 165, Serial No. 30321. This is a commercially available foul box virus vaccine strain derived from embryos.

The foul box strain, referred to herein as PP-1, is a 1-day-old chicken. This is an improved Duvette strain for use in vaccines. This stock is called ODCP:P25/C1l:P67/2309 October 1980 It is a commercially available foul box virus vaccine strain. Available from Merleuxx Inc. be.

Kanazy Box is a species of Abi Box.

Like foul box, canary box infects canaries specifically, but it also infects mammals. It does not infect milk.

The Kanakubox strain, referred to herein as CP, is LF2 CEP52424. It is a commercially available Kanakuvox vaccine strain called 1075, and Available from Tomerix.

DN inserted into these avipoxviruses by genetic recombination of the present invention The A gene sequence includes the LacZ gene derived from prokaryotes, non-avian (especially mammalian) disease Rabies glycoprotein (G) gene, one of the antigens of the original substance, Avipotsuvirus Turkey influenza hemagglutinin, an antigen of pathogenic avian viruses other than influenza gp51.30 gene of bovine leukemia virus, a mammalian virus. Newcastle disease virus (Texas virus), which is an avian virus feline leukemia, a mammalian virus. Disease virus PeLV envelope gene, avian virus/poultry disease La RAY-1 env gene of mouse-related virus, chicken/pen virus of avian virus Sylvania (Chicken pathogen (NP) gene, an infectious avian virus Matrix gene and veplomer inheritance of bronchitis virus (Mass 41 strain) The glycoprotein D gene (gD) of herpes simplex virus, a mammalian virus. It is included.

The isolation of the LacZ gene was carried out using the method described by Casadaban et al. Enzymology (Methods 1nEnzymology), Lu, 293-3011 (1911:l). Structure of the rabies G gene For example, Anilionfs et al., Nat. ure) 294.275-278 (1981).

Integration into vaccinia and expression within this vector are carried out using Key 4 ([1eny ) et al., Nature 312.1B:l-1e6 (1984). Therefore, it is being considered. Turkey influenza hemagglutinin gene is cute Kavaoka et al., Pyrology (VirologF) 158, 218- 227 (19117). bovine leukemia virus gp51. 30 The env gene was developed by Rice et al. in pyrology (VlrologF ) 13g, 82-91 (19114, ). newcastle disease w rus (Texas strain) was injected as a plasmid pNDV10g. Available from Tichinito Merricks. feline leukemia virus env The gene was described in pyrology 1 by Gullhot et al. 81.

252-258 (1987). There are two types of Rous-associated virus type 1. Clone, install as pen VRVIPT and mp19env (190) Available from Chinito Merricks.

The chicken influenza NP gene was isolated as plasmid pNP33. ・Jude Children's Research Hospital (St, Jude Children's Re5ea Yoshlhlro Ka of rch Ho5pital) Available from vaoka). IBV Mass41W trix gene and The infectious bronchitis virus cDNA clone of the bromer gene was cloned into a plasmid. It is available from Institignito Merricks as plBVM83.

The herpes simplex virus gD gene was developed by Watson et al. (Science) 218, 381-384 (1982).

The recombinant avipoxviruses described in more detail below are derived from vaccinia promoters. Incorporate one of three types of tar. P derived from the Ava IH region of vaccinia 1 promoter is Wachsman et al. J, or Inf, Dlg.

5, 118g-1197 (1987). In more detail , this promoter is the AvalH (Xhol) of the L mutant WR vaccinia strain. G) derived from a fragment in which the promoter directs transcription from right to left.

The location of this promoter on the map is approximately 1.8K from the left end of Ava IH bp (kilobase bear), approximately 12.5 Kbp from the left end of the vaccinia genome. It is located approximately 8.5 Kbp of the HlndllI C/N bond.

The sequence of this promoter is (GGATCC)-A(WAAAAATAGAAAACTATAAπATATA ATAGTCTAm-AG'rAGGGrAflATrAATrTrATrGπ AAA(TrG-(AATTC)), and the symbol in parentheses in the above sequence is the linker. It is an array.

The former ndmH promoter (also referred to herein as rHI and rllBJ) ) was revealed using standard transcriptional mapping methods. This has the following array: do.

ATrCTITATTrCTATAACTTrAAAAAATGiAAAATAAAT ACAAACXXrcr'rGA2AAATrGAAAGCGAGAAATAA TCATA-ATr AffATrATCGCGATATCCGTT frost ■ nail painting Mπ.

This sequence was published by Rosel et al., Journal of Biology, upstream of the open reading frame by Vlrol, ) 80.48B-449 (198B) It is the same as that described as the sequence.

11, the promoters are Vlttek, Journal of Piro Rosie (J, Vlrol,) 49.371J78 (19B4) and Partre Bertholet, C et Bros. of National Academy of Sciences USA (Proc, Natl, Acad, Sci, USA) 82.209B-2100 (1985).

The recombinant avipoxviruses of the present invention are synthetic vaccinia viruses known in the art. The method disclosed in the aforementioned U.S. Pat. No. 4.803.112 for producing recombinants Similarly, it was made in two steps.

First, the DNA gene sequence to be inserted into the virus is Escherichia coli (E. eoli) plasmid construction with homologous DNA inserted into the non-essential region Put it in the structure. Separately, the DNA gene sequence to be inserted is linked to the promoter. Match. The promoter-gene combination is then placed into a plasmid construct and It is flanked on both ends by DNA homologous to the non-essential region of the box DNA. obtained The plasmid construct is grown and amplified in E. coli. (Plasmid DNA is Used to retain and amplify foreign genetic material, this method is well known in the art. be. For example, for these plasmid technologies, see Frewell (C1evel). l) is Journal of Bacteriology (J, Bacteriol,) 1 10.667-676 (1972). Amplification plus from host E. coli Techniques for isolating mids are also well known in the art, for example, as described by Flewell et al. Rosseedings Φ of National Academy of Sciences USA 82.1159-1188 (1969). ) after multiplication in E. coli. The released amplified plasmid material is then used in the second step. In other words, the DNA residue to be inserted The plasmid containing the gene sequence was isolated from avipoxvirus (foulbox strain P). P-1 or PP-5), such as chicken embryo fibroblasts (CEF). Transfect cultured cells. Homologous foul-bocks in plasmids Through recombination between the viral DNA and the viral genome, non-essential regions of this genome are Avipoxvirus modified by the presence of non-foul box DNA sequences I can do this.

The invention and its many advantages will be better understood from the following illustrative examples. It would be perfect.

Example 1 Recognition of vaccinia promoter by foul box RNA transcription factor Assays for transient expression suggestive of It binds to the vaccinia virus promoter sequence and generates hepatitis B virus surface antigen (H Several plasmid constructs containing sequences encoding BSAg) were generated. . 50 μg of each plasmid was added to a foul box at 1 Opfu per cell. transfection into CEP cells infected with virus or vaccinia virus. I set it. Infection was allowed to proceed for 24 hours and then lysed by repeated freezing and thawing three times in a row. I set it.

The amount of IBSAg (in this solute) was determined by Laboratories) Diagnostic Reagent Department (DlagnostlcPivlsl) Austrian CAUSRIA) ll-125I kit commercially available from on) It was evaluated using Negative cut preset by the manufacturer for the presence or absence of HBSAg The ratio of the net count of unknown specimens (sample value minus bag grout value) to the off value and and show. This is expressed as the P/N (positive/negative) ratio.

The results are shown in Table 1.

Different vaccinia promoter sequences 3 [i.e., vaccinia promoter sequence before DNA replication] P1 promoter seen early in infection, late in vaccinia infection after initiation of DNA replication Promoter found in 11, found in early and late rainy season of vaccinia infection The old ndnI H (HH) promoter was used. About these promoters have already been described herein.

From the above data, it is clear that HBSAg produced in the lysate of infected cells is Result of recognition of vaccinia promoter by vaccinia or vaccinia transcription factor It is suggested that

pMPK 22.189 Foul Box IIK Promoter 14 wax SAg2 bound to near pPDK 22.5 Foul Box IIK Promoter 92.6 wax SAg bound to near 5.8pRv668 7y? 'L'yNy9X　11 ! 2°-1-y-Hy, SAg bound to 4 vaccinia (No plasmid Foul Box 1.1 (No plasmid Vaccinia) 1.8MMPK 22.1BS (No virus 1.8 Example 2 LACZ genetics Production of child-containing recombinant foul box virus vFP-1 A fragment in the non-essential region of the foul box virus can be extracted as follows. was located and isolated.

Using nuclease Ba1ll, the hairpin loop of the single-stranded end of FP-5 DNA was isolated. was removed. Plasma using the Flenow (large) fragment of DNA polymerase caused an end. After removing the loop, restriction enzyme digestion with Bgl II The above fragment was produced. This digestion produced a series of PP-5 fragments, which They were separated by agarose gel electrophoresis.

A fragment with an 8.8 Kbp Bglm plant end was isolated, and Ba-old and S It was ligated with commercially available plasmid pUC9, which had been cut with s+aI. The generated program The lasmid was named PRVB9g.

In order to reduce the size of the foul box fragment, this plasmid was [was cut with [] to generate two further fragments. Discard the 6.7Kbp fragment and remain Two 4 and 7 Kbp fragments were ligated to create a new plasmid pRV699.

The LacZ gene whose transcription is promoted by the IIK promoter was inserted into this plasmid. In order to insert the gene, pRV899 was cut with EeoRV. This EcoRV is Cut the sumid in only one place. The transcription of La is promoted by this IIK promoter. c Insert the Z segment as the Pst l-Ba5+ old fragment at the plant end. , created a new plasmid pRV702. This LacZ clone is pMc1 as described by Casadaban et al. It is derived from 871. The promoter in 11 above is %Bas old linker. e It was linked to the 8th codon of the Z gene.

Using the recombinant technology for vaccinia described in U.S. Pat. No. 4.803.112, This pRV702 plasmid was then propagated in chicken embryo fibroblasts (CEF). The virus was recombined into the foul box virus FP-5. At this time, create vFP-1. The following procedure was used to 50 μg of pRw7020NA was added to the whole genome foul box. The final volume was 100μ4. 2.5M C aCJ! 2 for 10uls and 40mM Hepess300mM NaCJ2. Prepared from 1.4mM Na2HPO4, 1hM KCI, 12+eM dextrose. 110 μ4 of adjusted 2X HEBS buffer (pH 7) was added thereto. to room temperature After incubation for 30 minutes, a foul box solution diluted to 5 pfu/cell was added. After adding 200 μi of Illsbourg, this mixture was mixed with 6 It was inoculated on the 0m display. At this time, Eagle containing 2% fetal bovine serum <FBS) (Eagles) medium 0.7 ml was also added. Incubate the plate at 87°C for 2 hours. After incubation, add Eagle's medium 3 containing 2% PBS and incubate the plate for 3 days. Incubated for a while. Cells are frozen and thawed three times in succession. After lysis, newly formed virus particles were assayed in the presence of recombinants.

11, the LacZ gene whose transcription is promoted by the promoter is foul box pp -The evidence of successful insertion by recombination into the s genome is % Lac This was obtained by testing the expression of the Z gene. Lac Z gene is enzyme β- The enzyme encodes galactosidase, which is a chromogen, 5-bromo-4-chloride. Rolo-3-indolyl-β-D-galactoside (X-gal) is cleaved to produce a blue color. O blue plaques releasing ndryl derivatives were selected as positive recombinants.

The successful insertion of LacZ into the foul box FP-6 genome and its expression PP-1 infected CEP, BSC (monkey kidney cell line-ATCCCCCL2G), VE RO (SaJl, kidney cell line-ATCCCCCL 81), and MRC-5 (human double Standard method using coelomic cell line - ATCCCCL 171) and β with commercially available antiserum. - Also confirmed by galactosidase immunoprecipitation.

Expression of β-galactosidase by recombinant virus vFP-1 was further tested in rabbits. This virus was inoculated into animals and mice, and β-galactosidase in the serum of the inoculated animals was Measure the increase in the titer of antibodies against the protein after inoculation and confirm that the increase is positive. It was also confirmed in vivo.

In particular, the recombinant vFP-1 was purified from host cell impurities and tested on both sides of two rabbits. Cynic inoculation was carried out at two locations in each area.

Each rabbit was inoculated with a statistical 10" pfu.

- Collect blood from animals every week and use the serum as a commercially available purified β-galactosidase preparation. was detected by ELISA assay using as antigen.

Both rabbits and mice inoculated with this recombinant vFP-1 showed β-galactin It was detected by ELISA that an immune response was generated against the sidase protein. Ta. In both species this response was detectable one week after inoculation.

Example 3 Rabies G gene and LAC from foul box virus FP-5 Preparation of Z-containing recombinant virus vFP-2 The 0.9 Kbp Pvu II fragment was -5 and inserted between the two Pvu II clusters of pUc9 using standard techniques.

The generated construct pR11168ft, 2 Hlnc 11 sites at two locations, approx. aobp distant position 1: carried asymmetrically within the Pvun fragment, thereby Form the long and short arms from this fragment.

Oligonucleotide adapters were used to introduce the PStl and Bag R1 sites. using known techniques to insert between these H1ncn sites and create plasmid pR. V894 was produced.

Next, this plasmid was cut with PstI and Bag old, and the ligation llK A new plasmid p was created by inserting the LacZ gene with a vaccinia promoter. RW700 was produced.

In order to create a rabies G gene whose transcription is promoted by the P1 promoter, the rabies G gene was BgJ close to the 5' end of the gene! 11 sites (KlenF et al., supra) (see cited references) to the plant end, and the single chain of the P1 promoter mentioned above. It was ligated to the Eco R1 site filled in.

This construct was inserted into the PstI site of pRW700, and the foreign gene sequence was inserted into it. pRV735 with P1-Rabies CIIK-LacZ. Create t plus and mid did. This insertion indicates that the PvuII-former nelI long arm of the FP-5 donor sequence is c. Position in the plasmid so that it is on the 3' side to the Z gene.

The final construct obtained was the previously described recombinant foulpoxvirus vFP-2. By infecting/confecting chicken embryo fibroblasts using the production method, It was recombined with the foul box virus FP-5. This recombinant virus is -selected by gal staining.

Both the Lac Z gene and the rabies G gene were properly inserted and expressed. This was confirmed using the various additional methods described below.

When the location of the rabies antigen was determined by immunofluorescence using a specific antibody, vPP- 2. Rabies virus is successfully transmitted on the surface of avian or non-avian cells infected with the virus. It was shown that it was expressed.

As already mentioned, rabies in avian or non-avian cells infected with vPP-2 virus The expression of disease antigen and β-galactosidase was confirmed by immunoprecipitation.

An embodiment of the present invention regarding vFP-2 includes the rabies G gene and β-galactosidin. Another proof that this is a successful example of a recombinant virus harboring the ze gene is Obtained by inoculating two rabbits with vPP-2 virus.

Ironically using vFP-2 with IX 10” pfu per rabbit for two rabbits. Inoculated. Blood was collected from rabbits every other week and the serum was tested by ELISA to detect rabies. The presence of specific antibodies for glycoprotein and β-galactosidase protein was detected. .

As shown in Table ■ below, rabbit 205 showed anti-β-galactin resistance one week after inoculation. Sidase antibodies were shown in detectable amounts in an ELISA test. This is 40 in the second week The titer increased to 1/000 and persisted until 5 weeks after inoculation. Antigen capture ELISA assay Using Sei, rabbit 205 serum has detectable levels from 3 to 10 weeks after inoculation. It showed anti-rabies antibodies.

Table ■ Pre-blood collection for rabies antigen and β-galactosidase protein anti-β-galactosidase ese 0 1 week 500 2-5 weeks (each week) 4000 6 weeks 500 9 weeks 250 Pre-blood collection Anti-rabies 0 3 weeks 200 6 weeks 200 10 weeks 100 This example shows that the rabies G gene is a foul box strain other than FP-5, especially F Expressed entirely by another strain of foul box virus called P-1 This shows that.

As in Example 3, a Pvu II fragment of 0.9 KbP was used as in pp-s. This fragment was obtained from FP-1 after the assumption that the fragment contained non-essential regions.

This fragment was inserted into the two Pvu II sites of pUC9, and pRV731.15R A plasmid named .

This plasmid contains two asymmetric locations approximately 30 bP apart within the Pvu II fragment. The former ncII site forms the long and short arms of this fragment.

Commercially available Pst linker-1 (5’)-CCTGCAGG-(3’) Plasmid pRW741 was created by inserting into the nc11 site.

The rabies G gene whose transcription is promoted by the HH promoter is expressed in this platinum at the Pst1 site. A new plasmid, pRV742B, was created by inserting it into Sumid. Infection of CEP cells /transfection to recombine this plasmid with FP-1. Thus, virus vPP-3 was obtained.

ATG translation initiation code of the open reading frame whose transcription is promoted by the HH promoter The EcoRV site in the HH promoter and the former ndI site in the rabies G gene The initiation cod of the rabies G gene using synthetic oligonucleotides that connect the stacked on top of each other. Rabies G gene whose transcription is promoted by such HH promoter The 5' end of was modified using known techniques to contain one Pst I site. After that, this construct was ligated to the pR141) Pst 1 site and 1) RV742B was Created. The location of this structure in this plasmid was already described in Example 3. pRV735.1.

Recombination was performed as described in Example 2. The generated recombinant was called vFP-3. Bu.

Rabies antigen production by both avian and non-avian cells infected with vPP-3 virus This was confirmed by the immunoprecipitation method and immunofluorescence method described above.

An embodiment of the present invention relating to vPP-3 is a recombinant virus expressing the rabies G gene. Another proof of the success of this system is that the recombinant virus was injected into two rabbits. Obtained by meat inoculation. 2 rabbits IX 108pf per rabbit was inoculated with vPP-3 of u. Both of these rabbits are typical bock The lesions developed, which reached their maximum size 5 to 6 days after contact. weekly from rabbit Blood was drawn and serum was tested by ELISA to determine the presence of antibodies specific to the rabies glycoprotein. detected.

Five rats each were inoculated intradermally with 5×107 pru of vPP-3. All of animals remained with lesions.

Both rabbits and rats have detectable levels of rabies specificity by 2 weeks after vaccination. produced antibodies. Two control rabbits subcutaneously inoculated with the parental PP-1 virus , did not show any detectable levels of anti-rabies antibodies. This antibody response was tested de novo synthesis of rabies antigens in animals using recombinant viruses. The virus may have been introduced with the inoculum or the recombinant virus. The rabies antigen was introduced by chance by being incorporated into the Urvox virus membrane. This vPP-3 virus was chemically inactivated to rule out the possibility that and inoculated rabbits.

The purified virus was incubated overnight at 4°C in the presence of 0.001% β-propiolactone. After activation, it was pelleted by centrifugation. 10m of pelleted virus M) Collect it in Squirrel buffer, treat it with ultrasound, and assay the titer to confirm that there is no infectious virus. I made sure there were none left. Two rabbits were subcutaneously inoculated with inactivated vPP-3 and given the same amount. The untreated recombinant was subcutaneously inoculated to two other animals. The size of the lesions was monitored.

Both rabbits that received untreated vFP-3 showed a typical 4-5+ grade at 5 days post-inoculation. A lesion developed. Rabbits inoculated with inactivated virus also developed lesions, but these was graded 2+ 5 days after inoculation.

Rabbits were bled every other week, and the serum was tested by ELISA to detect rabies-specific antibodies and The presence of specific antibodies in the foul box was investigated. The results are shown in Table ■ below.

In this study, the endpoint titer (expressed as the reciprocal of the serum dilution) was determined before challenge. After subtracting the serum absorption value of , it was arbitrarily determined to be 0.2. Rabbit 295 and 318 rabies glycoprotein and foul box virus were both administered live virus. generated an immune response against the Rus antigen. Rabbits 303 and 320 are also foul box cows Although an immune response was generated against the virus antigen, the titer was low. What is this rabbit? Neither produced a detectable response to rabies glycoprotein.

This finding suggests that the immune response generated in the rabbits is affected by the rabies sugar protein carried by the recombinant virus. Due to de novo expression of protein genes, sugar tags accidentally introduced into the inoculated virus This suggests that it is not a reaction to the protein.

Expression of foreign genes inserted into the foulpox genome by recombination involves Requires the presence of a promoter. vP except for lacking the HH promoter This was demonstrated by producing a recombinant vFP-5 identical to P-3. This recombinant The presence of the rabies gene was confirmed by nucleic acid hybridization. deer However, no rabies antigen was detected in the culture medium of CEP cells infected with this virus. In vitro subculture experiments to determine whether or not to produce 1 bird species and 2 non-avian species Experiments were conducted by inoculating seed cell lines with the FP-1 parent strain or recombinant vPP-3.

CEP.

NRC-5 and VERO (7) So, put PP-1 or vFP- on two plates each. 3 were inoculated at a multiplicity of inoculation of 10 pfu per cell.

After 3 days, collect the cells from one plate for each plate, freeze, and thaw three times in a row. The virus was released twice and then re-inoculated into fresh monolayers of the same cell line. . This was serially passaged 6 times, and at the end of the experiment, the titer of the sample from each passage was assayed. Viral infectivity on EP monolayers was investigated.

The results are shown in Table IVA. From this result, PP-1 and vPP- 3. However, serial passage of both of the above two non-avian cell lines is possible. It was also suggested that this is impossible. Infectious viruses are VERO or Not detectable after 3 or 4 passages in NCR-5 cells.

In the second dish, virus that was undetectable by direct titration was detectable in permissive CEP cells after amplification. It was used to decide whether or not to

After 3 days, scrape the cells from the second dish and lyse one-third of the cells. Inoculated onto fresh CEP monolayers.

Lyse the cells at maximum cytopathic effect (CPE) or 7 days after inoculation. The yield of virus was titrated. The results are shown in Table IVB. Passage in CEP cells When all of the existing viruses are amplified using multiple generations, this virus is present after 4 or 5 generations. could not be detected.

Attempts to establish constantly infected cells were unsuccessful.

In addition, we hope to detect evidence of continuous viral expression in non-avian cells. However, the sample used for the above virus titer assay was tested using a standard immunodot assay. I used it for This assay uses anti-foul box antibodies and anti-rabies antibodies. were used to detect the presence of each antigen. The results of these assays are used for titration Check the result ftl 11” 4.g 4.1 u 5.4 1.!! @, t ! , 1 1J ・J 4.1! ’, 1ko@, 41.41.0141.14. 44 1, IN, Dbll, 0 1. ! 11.0 1. +15 @4 N, D 110 [H, D H, D passage 1 @, 4” @, 1 14 @, 5 1. 1 @, 4! 7J IJ @, 0 1J 1.1 1.83 1! @, I S, $5.9 1.1 1141 @4JL, 5Sj4.111 SL, S 4.1 11. OLl 4.1 4.7 Example 6 Other foul bowls xFP-1 recombinants: vFP-6, vFP-7, vFP-8 and vPP-9 Recombinant viruses vPP-6 and vPP-7 were produced by the following procedure.

The 5.5 Kbp PvuII fragment of FP-1 was inserted into the two PvuII sites of pUc9. Plasmid PR111731,13 was prepared by inserting the plasmid PR111731 and PR111731. Then this plus cleaved at its unique old ncn site, containing the l1B-promoter and platinum Plasmids pRV748A and pR were created by inserting the rabies G gene with V748B was produced. These plasmids have opposite orientations of insertion It is shown that. Plasmid I) RV748A and B were then used separately to -1 virus into CEP cells and recombinantly transfect it into CEP cells. vFP-6 and vPP-7 were produced, respectively. This locus was named locus f7. .

The 10Kbp Pvu-II fragment of FP-1 is combined with the two Pvu-II parts of ptlc9. pRV781.15 was created by inserting the pRV781.15 between the two positions. This plasmid is then transformed into a unique Ba Cut at the old site and then insert the LacZ gene fragment containing the IIK promoter. pRV749A and pRV749B were created in which the insertion direction was reversed. . These donor plasmids were recombined with PP-1 to produce vFP-8 and vFP-8, respectively. FP-9 was obtained. This locus was named locus f8.

vPP-8 and vFP-9 are LacZ genes detected by X-gal. was expressed. vPP-6 and vPP-7 are detected by rabies-specific antiserum The rabies G gene was expressed.

Example 7 vPP-3 for protecting animals from attack by live rabies virus immunity Groups of 20 female SPF mice (4-B weeks old) were treated with 0.0. vPP-350μ4 was applied to the foot pads at doses ranging from 7 to 1f, 7 TCID5o. The tissue culture infective dose (TCID5) was determined by the cytopathic effect on 50% of the tissue culture cells. (the amount received). 144 days after inoculation, 10 mice in each group were sacrificed and serum samples were analyzed. The bodies were harvested for Rppt test assay. For the remaining 10 mice, mad dogs Number of survivors on day 144 after challenge by inoculating the cerebrum with 078 strain at a dose of 10LD50 I counted.

The results are shown in Table VA below.

6.7 1.9 8710 4.7 1.8 0/l.

2.7 0.4 0/10 Inhibition) Test, Laboratory Technique R ABLES 3rd edition, measured by WHO Geneva.

This experiment was carried out in 12. The challenge was repeated with SL, 5o dose of rabies virus. results It is shown in Table VB below.

4.7 2.1 2/10 2.7 0. B O/8 0.7 0.8 018 10 gl of recombinant vPP-3 for 62 dogs and cats.

Immunization was carried out by intravenously inoculating once 8 times the amount of TCID5o. In addition, age and weight Two dogs and four cats were used as a non-vaccinated control group. - from all animals every other week Blood was drawn. On day 94, each dog received a Apply the obtained salivary gland homogenate of rabies virus NY strain 0.5 times twice to the temporalis muscle. Inoculated and attacked. The total dose is equivalent to 10,000 times the mouse LD5o via the cerebral route. Ta. Six cats were treated with the same virus suspension at 0.551i! twice, neck was inoculated into the muscle of the patient. The total dose per mouse is the cerebral route mouse L. It was equivalent to 40,000 times that of D5o.

Animals were observed daily. All non-vaccinated animals should show signs of rabies. He died on the date indicated. Vaccinated animals survive and respond to challenge. Observations were made until 3 weeks after the last animal died. The results are shown in the table below. Ta.

Table ■ Cat 7016 vFP-3” 0 2.2b 2.4 2.4 1.5 +TI Oc 0 0 0 0 0 d/12T41 c 0 0 0 0 0 d/1 3T42 c 0 0 0 0 0 d/12 dog 426 vFP-300,8 1,01,11,2+427 vFP-30L,S 2.82.21.9+5 5 c, o OO00d/15 Cats vaccinated with 8240c000000d/16a-vFP-3, Both dogs received a dose of 81 ogxoTCIDso intravenously.

d - Dead animals/number of days until death after challenge.

Furthermore, recombinant viruses vFP-2 and vFP-3 were introduced into cattle by several different routes. We conducted an experiment to inoculate them.

Inoculated animals were tested for anti-rabies antibodies at 6.14.21.2 g and on day 35.

All animals showed serological responses to rabies antigens, as shown in Table 1A below.

+No significant difference The animal was re-inoculated with rabies and showed a history of reaction to this rabies antigen.

All cows except No. 1421 were given a booster vaccination by revaccination. 1 Cow #421 was revaccinated intramuscularly.

RFPI titers were measured after 55.57.63.70.77 and 88E].

The results are shown in Table ■B.

Table ■B Days 55 57 83 70 77 118 Cattle number 1419 1.7 1.5 2.9 2.9 2.8 2.91420 1.0 0.5 1.9. 2.3 2.2 2.01421 1.3 1.2 2. 9 2.7 2.5 2.5142B 1.0 0.72.4 2.5 2.5 2. All data are from vFP-2 except for the data for animal number 21423. It is FP-3.

Cattle, cats, and rabbits can also be subtly inoculated with a known amount of foul box virus, resulting in approximately 1. The scabs were collected from the animals a week later. After crushing these, suspend them in physiological saline, Titers were assayed to determine viral load.

Only the remaining amount of infectious virus could be recovered. From this, in vivo there is no This suggests that productive infection does not occur.

Example 8 Inoculation of chickens with vPP-3 recombinant Furvox virus v Inoculating chickens with PP-3 in a system that allows productive replication of this vector. Expression of foreign DNA by recombinant foul box virus is shown.

For white Leghorn chickens, 10 g l of vPP-3.

TC! From muscle at 9 times the dose of D5o or vPP-3 at logloTCID5° It was inoculated by true penetration at 3 times the volume. Blood samples were collected 21 days after vaccination. Rabies antibody titer values were examined by RFFI test. The titer of the inoculated chicken on the 21st day is , which was much higher than the titer on day 21 of the control group. That is, the average of the uninfected control group The average titer was 0.8, but the average titer for chickens inoculated intramuscularly was 1.9; The average number of chickens tested was 1.2. Birds can be immunized against similar pathogens.

Therefore, A/Turkey/Irula is derived from the foul box virus FP-1. The old ndn[H promoter of 711/113 (TYHA) A new plasmid pRV759 containing the hemagglutinin gene (H5) (see below) ) to CEF cells co-infected with the parental virus PP-1. Transfected. The recombinant foul box virus vFP-11 herein This was obtained using the techniques already mentioned in the book.

The synthesis of hemagglutinin molecules by vPP-11-infected cells was labeled with a specific anti-H5 antibody. Confirmed by immunoprecipitation from metabolically radiolabeled infected cell lysates using standard techniques. Approved. Specific immunology of a hemagglutinin precursor with a molecular weight of approximately 63 Kd (kilodaltons) Epidemiological precipitation and two degradation products with molecular weights of 44 and 23 Kd were revealed. insensitivity Such proteins were detected from the lysate of infected CEP cells or cells infected with the parent virus FP-1. Paku did not settle at all.

HA molecules produced in cells infected with recombinant Foulbox vPP-11 Immunofluorescence studies were performed to examine expression on the cell surface. set CEP cells infected with the modified foul box virus vFP-11 have a strong surface area. Fluorescently stained. No fluorescence was detected in cells infected with the parent virus PP-1. won.

Plasmid pRV769 was constructed as follows.

pRV742B (see Example 4) was partially digested with PstI to make it linear. The fragment was re-cut with EcoRV to remove the rabies G gene, resulting in a remaining approximately 3.4 Kb. The OH promoter was left on the fragment. This is treated with alkaline phosphatase, A synthetic oligonucleotide was used to bind this H1l promoter and TYHA in ATG. Cleotide was inserted to create pRV744.

This plasmid was partially digested with Dra I to make it linear, and this linear fragment was The larger fragment was isolated again and treated with alkaline phosphatase. ・Finally, Katsairiki et al. opened in Pyrology, 158.21g-227 (1987). As shown, this isolated TYHA Sal I-Dra I coding sequence was pRV759 was created by inserting into the pRV744 vector.

To investigate the immunogenicity of the recombinant foul box virus vFP-11, chicken Vaccination and challenge experiments were conducted with chickens and turkeys.

For sterile white Leghorns, poultry is fed a foul box at 2 days of age and 5 weeks of age. The double’ dose used in commercial vaccination against virus Vaccination was performed by puncturing red sea bream with a needle. 6x1 vFP-11 Approximately 2 μi containing 0.05 pfu was administered to each chicken. Older chickens are vaccinated All chickens were bled before challenge and 2 weeks later. Ta.

For comparison, another group of chickens was treated with a water-in-oil emulsion of the inactivated H3N2 strain. The conventional H5 vaccine consisting of:

The inactivated H3N2 vaccine is an A/? vaccine grown in 11-day-old embryonic chicken eggs. lard (Mal 1ard)/NY/189782 (H5N2). HA power Price 80010.1 jti! and infected plasma with an infectivity value of 108.510.1 m Urine fluid was inactivated with 0.1% propiolactone and Avlan 018% 22,666-874 (197 g) and Brugh et al., Proceedings on Second. International Symposium on Evian Influenza (Proc. , 5econd Inter, Sym, on Avian Influe nza), 28B-292 (1986), in water-in-oil emulsions. suspended in. A volume of 0.2 m of vaccine was administered intravenously to 2-day-old and 5-week-old SPPs. It was administered to the inner skin of the thigh muscle of white leghorn chickens.

Groups 3 and 4 of chickens were administered parent virus pp-t or not vaccinated at all. Chin was not administered.

Administer 0.1 m into the external nostril of each chicken, and Land 7187g/H (H5NIi) or A/Chick/Pen ( Pen) 8370/118 (H5N2) influenza virus about 1 in LD Chickens were challenged with 0" dose. Two-day-old chickens were challenged 6 weeks after vaccination. Five suitable age chickens were challenged 5 weeks after vaccination. The chicken has a face and crest swelling and cyanosis, as well as bleeding in the legs (these chickens often have Symptoms of illness suggested by symptoms such as being unable to stand (often unable to stand), paralysis and Observations were made daily for mortality. Most deaths occurred 4 to 7 days after infection. After infection On the third day, samples were taken from the trachea and cloaca of each surviving chicken using a wire rod, and embryonic development Virus screening was performed by inoculating eggs. Wild cow type or recombinant foul Chickens inoculated with box virus developed lesions typical of red sea bream. needle A pus is formed at the puncture site by the third day, and cellular infiltration continues with scab formation. The patient recovered by the seventh day. No secondary lesions are formed and non-vaccinated contacts There was no evidence of transmission to the chickens. The results of the attack experiment are shown in Table■ The relevant serological findings are shown in Table ■.

The chickens were protected for over a month.

H5 expressed by vFP-11 also elicits a protective immune response in turkeys. wake up Outbred white turkeys were grown at 2 days of age and 4 weeks of age, as described above. Vaccination was done by sea bream inoculation. The results are shown in Table X.

Significant survival was seen in both age groups upon challenge with the homologous Ty/Ire virus. It was. Non-vaccinated contact turkeys were housed together with vaccinated turkeys and recombinant Tested for viral transmission. These turkeys did not survive the attack. Ta.

Example 11 Chicken influenza nucleoprotein (NP) ffi gene expression factor Construction of Urbox FP-1 recombinant vPP-12 The plasmid pNP$3 is Fluenza virus chicken/Pennsylvania/l/83 nucleoprotein gene ( NP) cDNA clone. Approximately 1.6 Kbp 5' of the NP gene Only the 3' end was determined. 5’ C1a with NP as the plant end As a I-XhoI 3' fragment, p digested with SrinaI from PNP33 It was transferred to the EcoR1 site at the 3' end of UC9 to create pR1714. NP's The translation initiation codon (ATO) has an Aha n site underlined below. was: ATGGCGTC, the vaccinia H6 promoter mentioned above was duplicated. A chain synthesis oligonucleotide was attached to this NP. This synthetic oligonucleotide contains the He sequence from the Eco RV site to its ATC, and the Aha II n-site into the NP coding sequence. To insert the oligonucleotide into pUC9 was synthesized to be available for both Bag+old and EcoR1 cleavage, and pR v755 was created. Double-stranded synthetic oligonucleotides starting from Bag old compatible ends The sequence of codes (ATC is underlined) is: G A T CCG A T A T CCG T T A A G T T T G T A T CG T A I G CG T Cf GCTATAGGCAATTCAAACATAGCATTACCGCAGCTT It was AA.

The linear partial digestion product of pRv755 with Aha n was isolated and digested with Eco R1. Amputated. At ATG, cut once with Aha II and then again with Eco R1. The following pRV714 fragment was isolated and treated with phosphatase. It was used as a vector for the digestion products.

The Aha II linear partial digest of pRV714 was recut with Eco R1. N The approximately t, exbp AhaII-Eco R1 isolated fragment containing the P coding sequence was The vector was inserted into the pRV755 vector described above to create pRW757.

Complete H6 promoter adds sequences upstream (3' side) of the Eco RV site It was formed by Plasmid pRW742B (described in Example 4) is pU Downstream (3' side) of Eco RV site cleaved together with C9' Nde1 site It had the H6 sequence. pRV742 B Eco RV-Nde IK piece was treated with phosphatase and used as a vector for the pRV757 fragment described below.

After isolating the linear part of pRV767 Eco RV digestion product, it was digested with NdeI and re-digested. and isolated. Insert this pRV757 fragment into pR111742B vector and pR V75g was formed. The Eco R1 fragment of pRV75g is driven by the H8 promoter. plant protein with the Flenow fragment of DNA polymerase I. After changing to pRW731.13 Hln c IIn site, pRW7BO was created.

The pRW7B1.8 Hlnc n site was used in Example 6 for vPP-6 and vPP-7. This is the FP-1 locus used for production.

Using Foulbox FP-1 as a rescue virus, plasmid pRwyeo was used in in vitro recombination studies. Assay progeny plaques in situ Plaques were purified using plaque hybridization. Goat gene expression This was confirmed by immunoprecipitation experiments using a polyclonal anti-NP antiserum. vPP-12 The size of this protein, which was specifically precipitated from the lysate of CEP cells infected with Approximately 55% D, which is the range of reported influenza virus nucleoproteins. It was within the limits.

About the hemagglutinin (HA) gene from A/Tyr/Ire/137g/83 was already described in the preparation of vFP-11 (Example 9). Creating a double recombinant First, the IA gene was transformed into vPP- It was transferred to the locus f8 defined in the preparation of No. 8.

The plasmid used to create vFP-11 was pRV759.

The hemagglutinin gene bound to the H6 promoter was isolated by partial PstI digestion. It was transferred from this plasmid. This fragment is then treated with DNA polymerase I. Fragment with plant end and pRw7B1.15 plant end Bag pRV771 was created by inserting it into the old site.

Plasmid PRV771 was then incubated with vPP-12 as the rescue virus. Used for in vitro recombination test. vPP-12 recombinant virus is plasmid pRV A nuclear protein linked to the He promoter at locus f7 defined in 731.13. Contains quality genes. Select recombinant plaques with both inserts and in situ C. Purify plaques by hybridization to protect the surface expression of hemagglutinin. Confirmed by A-β-galactosidase binding immunoassay. expression of both genes The present invention was determined by immunoprecipitation from double recombinant virus VFP-15 and infected cell lysate. confirmed.

Example 13 Preparation of recombinant Kanaku box virus In the following example, Kanaku box virus Confirmation of four non-essential insertion loci in the Kanaji box genome and recombinant Kanazy box virus Regarding the production of four types of vCP-16, vCP-17, vCP-19 and vCP-20 It shows.

Recombinant Kanakubox vCP-16 was produced as follows.

Clone the 3.4Kbp Pvu If Canarycoux DNA fragment into pUC9. and generated pRV7B4.2. Eco R1 site is asymmetric within this fragment Only one location was found in , with a short arm of 700 bp and a long arm of 2.7 Kbp. This program The lasmid was digested with Eco R1 and the Flenow fragment of DNA polymerase I was extracted. The plant end was made using Plant End He/Rabies G Gene Next was ligated to this site and used to transform Escherichia coli (E, colt). Living The resulting plasmid pRV775 was used for in vitro recombination tests. immune scree Positive progeny plaques were selected by scanning, and the plaques were purified. Generated recombinant was named vcp-to, and the insertion locus was named 03.

The plasmid pRI+l764.2 used for the above construction has the above Eco R1 site. It contained only one Bgl II site approximately 2.4 KM away from the site. Same black The H6/rabies G gene was inserted into plasmid pRV7 at this site using a 84.2 to create pRIJ774. Using this plasmid, C4 and A recombinant vCP-17 having the named insertion locus was produced.

Plasmid pRV784.5 is Kanacupo-/RiX DNA f) 850 It has a 400 bp Pvu n fragment from one end with an asymmetric unique fragment. It has 11 Bgl sites. Using the same cloning method as described above pR1 was used to insert the rabies G gene linked to the He promoter into this site. 77 was produced. The produced stable recombinant virus was named VCP-19 and inserted The seat was named 05.

Plasmid pRV7134.7 contains only one Bg 300 bases away from one end. 1.2 Kbp Pvul with II site [contains fragment.

This plasmid was digested with Bglm, and Frenow flag of DNA polymerase I was added. Plant end with ment. Transcription is promoted by the IIK promoter at the plant end. Plasmid pRV778 was created by inserting the LacZ gene to be developed. this The stable recombinant virus created using the plasmid was named vCP-20, and the inserted The seat was named CB.

Plasmid pNDV108 is a cDNA clone of the fusion gene of the NDV Texas strain. It consists of an approximately 3.3 Kbp Hpa I cDNA fragment, and is a fusion protein. NOV cloned into the Sea 1 site of pBR322 Contains another sequence encoding Below are the steps for creating an insertion plasmid. state

(1) Production of plasmid pcE11 The ppv insertion vector pcEit is inserted into the old nc11 site of pRW731.13. pRV73, created by inserting a relinker (named locus f7) 1.13 contains a 5.5 Kbp Pvu fragment of FP-I DNA. non-essential In the production of stable recombinant vFP-6, which was already described in Example 6, dust was Defined by location. The polylinker inserted into the HIncI site is the following restriction enzyme site. has the highest rank. In other words, Nru　I　Eco　R1%5acI%Kpn ISSma ISBag old, Xba Is HlncI [, 5all.

Ace Is Pst I, Sph Is HlndI[[and Hpa It is I.

(2) Production of plasmid pcE19 This plasmid is a further modified version of pcEll and is used for vaccinia virus transfer. Transcription end signal ATTTTTTNT (L, Yuen and B, Mo ss), Journal of Obvirology, 60.320-323 [1986]) (,: (7) Case N HA Teal) KapcE11's Sac I and Eco R 1 site, and as a result, the Eco R1 site has disappeared.

(3) Insertion of NDV code sequence 1 containing all but the 5'-terminal 22 nucleotides of the fusion protein gene , insert the 8Kbp gel-purified Bag old fragment into the Ba old site of puctg. , formed pcElB.

This plasmid is inserted into the vector 12 bases upstream of the 5' end of the coding sequence. Digested with SalI, which cuts.

The ends were filled in with the Klenow fragment and the plasmid was further inserted into the Sal I site. Digested with old ndI[I, which cuts 18 bases upstream of. In the preferred embodiment vaccinia virus H6 promoter and polylinker sequences at both ends as already described in The gel-purified 146-base SIa I-Hlnd m fragment containing It was ligated to this vector and transformed into E. coli cells. The generated plasmid is pcEl It was named B.

Place the start ATG codon of the NDV fusion protein gene at the 3' end of the H6 promoter. The 22 nucleotides missing from the 5' end of NDV were added to pCE. To complement 16, complementary synthetic oligonucleotides were added to EcoRV and Kpn1. It was designed so that the position is the end. This oligonucleotide sequence is 5' ATC-C GT-TAA-GTT-TGT-ATC-GTA-ATG-GGC-TCC-A GA-TCT-TCT-ACC-AGG-ATC-CCG-GTA-C3' It was.

The prepared pcElB was then digested with EcoRV and KpnI.

The Eco RV site occurs 24 bases upstream of the initiating ATG within the He promoter. . The Kpn1 site occurs 29 bases downstream of the ATG within the NDV coding sequence. .

Anneal the oligonucleotide, phosphorylate it, and ligate it to the linearized plasmid described above. I let it happen. The resulting DNA was used to transform E. coli cells. This plasmid The code was named pcElg.

To insert this NDV coding sequence into the FPV insertion vector, 1.9 Kbp SmaI-Hlnd purified by gel (cleaved at relinker region) m fragment to the 7JKbpSea I-old nd III fragment of pcElB mentioned above. concluded. A transcription termination signal occurs 16 bases downstream of the Sa1 site. The obtained plastic The sumid was named pcE20.

Plasmid pc820 was used as rescue virus, and furbox virus FP-1 was used as rescue virus. It was used for in vitro recombination tests. Apply the resulting progeny to a CEF monolayer The plaques were then treated with β-galactyl chloride using polyclonal anti-NDV chicken serum. It was subjected to a sidase-linked protein A immunoscreen. Select positively stained plaques After four rounds of plaque purification, this recombinant was transformed into a single population with vPP- It was named 29.

FeLV env gene is a sequence encoding p704P15E polyprotein Contains. The FeLV gene first contains vaccinia H6 protein on the 5' side of this gene. The motors were inserted into plasmid psD467vc in juxtaposition. plasmid p sD467vc contains the vaccinia hemagglutinin (HA) gene. Insert the bp SalI/H1ndm fragment into the 9001g vector first. It was induced by this. Regarding the location of the HA gene, 451-482, [ 198g]). Most of the open reading frames encoding the HA gene products are missing ( nucleotide 443 to nucleotide 1311), Bgln・SIaI Multiple cloning site containing aPst I and Ega I restriction enzyme sites inserted. The resulting ps04B7vc plasmid has a multiple cloning site contains the vaccinia flanking arm 491 bp downstream of these restriction sites at 442 bp of ing. These adjacent arms insert the genetic material into the multiple cloning site and create the vaccine. The virus can be recombined into the IA site of the Copenhagen strain of the senior virus. obtained The recombinant progeny were HA negative.

The He promoter, in a preferred embodiment, has four promoters consisting of the complete sequence previously described. Synthesized by annealing burst oligonucleotides. obtained The 132-base fragment has a Bgl II restriction site at the 5' end and a Bgl II restriction site at the 3' end. It had a Sma I site at the end. via Bgl II and 5Ila I restriction sites. and inserted this into psD467vc. The obtained plasmid was called pPT15. I named it. The only Pst1 site in pPT15 immediately downstream of the H6 promoter This FeLV env gene was inserted into. The generated plasmid was transformed into pFeLVI Named it A.

For the production of FP-1 recombinant, the 2.4 Kbp He/PeLV env sequence was It was separated from pPeLVIA by Bglm digestion and Pst1 partial digestion.

The Bgln site is at the 5' border of the [1 promoter sequence.

The Pst1 site is at 420 of the translation termination signal of the envelope glycoprotein open reading frame. bp downstream.

Delete this 2.4Kbp He/PeLV env sequence from Bam old and PstI. pcEl. The FP-1 insertion vector pcE11 is a multiclonal from pRW731.13 by inserting a coding site into the non-essential ncII site. Obtained. This insertion vector contains F A P-1 recombinant can be produced. Here is the recombinant PP-1/PeLV insertion plasmid. It was named pFeLVFI. The second structure provides a complete ATG for ATC substitution. No.

Complete ATC: To create an ATG structure, approximately 1.4 Kbp of Nru I/Sst One II fragment was derived from the vaccinia virus insertion vector pPeLV1c.

The Nru1 site is generated in the He promoter located 24 bp upstream from AT(i). Jiru. The Sst II site is located 1.4 Kbp downstream from ATG and contains the translation termination signal tK. Located bp upstream. This Nru I/Sst■ fragment was digested with Sst II. It was ligated to a 9.9 Kbp fragment prepared by partial Nru I digestion. This 9.9Kbp The fragment consists of the 5.5 Kbp FP-1 flanking arm, which is the pUC vector sequence, and the env gene. FeLV sequence (1.4 Kbl) corresponding to the downstream part of the child, and the H6 promoter Contains most of the 5' sequence (approximately 100 bp). Obtained plasmid was named pFeFLVP2.

This ATG for ATG construction was confirmed by nucleotide sequence analysis.

Another FP-1 insertion vector, pFeLVF3, contains a putative immunosuppressive region C1anc1o1o et al., Science 230 , 453-455 [19851) (: from nucleotide 1548 of the l-do sequence pP by removing the PeLV env sequence corresponding to It was derived from eLVF2. This is a SstIf/PstI disconnection of approximately IKbp. The fragment (sites described above) is isolated from the vaccinia virus insertion vector pFeLVID. This plasmid pFeLVID, completed by The enV sequence corresponding to cleotides 1548 to 1628) is the oligonucleotide. pPeLVlc, except that it was deleted by mutagenesis with (Mandecki, Proceedings of ・National φ Academy of Sciences USA%83゜7177-71 81 [1987]). 1 lacking nucleotides 1548 to 1828 The Sstn/PstI fragment of Kbp was derived from the remaining pFeLVF2 H8: 1O, 4Kbp Sstn/Ps containing FeLV any gene t　I　Insert into the fragment.

Insertion plasmids pFeLVP2 and pFeLVP8 are clones of rescue virus FP-1. used for in vitro recombination tests. The progeny of this recombinant was smeared onto a CEP monolayer, and the recombinant Viruses were selected by plaque hybridization on CEP monolayers. Yes Recombinant progeny identified by hybrid analysis were selected and subjected to four rounds of plaque purification to ensure homogeneity. As a group. The FP-1 recombinant carrying all FeLV env genes was transformed into vFP- A PP-1 recombinant containing the entire gene lacking the immunosuppressive region was named 25. It was named vFP-32. These two recombinants are both bovine anti-PeLV polychrome Naru Serum (Antlbodeles% Inc.), De by immunoprecipitation using Davis, California). It was shown that this gene product was expressed. These PP-1 recombinants are CR The above foreign FeLV env gene in FK cell line (^TCC11CCL94) It is also important to express this cell line, which is of feline origin.

For the production of Kanakubox (CP) recombinant, the above H8:PeLV en A 2.2 Kbl) fragment containing the v sequence was digested with Sla 1 and Hpa I. It was separated from pFeLVF2. This SIa 1 site is the H6 promoter sequence It is on the 5' boundary of

The Hpa1 site is located downstream of the translation termination signal of the envelope glycoprotein open reading frame. It is located at 180 bp.

Plasmid pRV764.2 inserting 2.2Kbp He/FeLV env sequence into the non-essential Eco R1 site of I made it. This insertion vector carries a foreign gene in locus C4 of the CP genome. Produce recombinants. This recombinant CP insertion plasmid is now pFeLVcP It was named 2.

This structure provides one complete ATG for ATG replacement.

The insertion plasmid pFeLVcP2 was tested in vitro using CP as the rescue virus. (b) Used for recombination testing. After applying the progeny of this recombinant to a CEP monolayer, the bovine anti- FeLV commercially available polyclonal serum (Antibodies, Davis, Califol) By β-galactosidase-linked protein A immunoscreen method using , selected recombinant viruses.

Positive staining plaques were selected and purified four times to obtain a homogeneous population.

The recombinant expressing the complete PeLV env gene was named vcP-36.

Example IB Rous-associated virus type 1 (RAY-1) envelope v) Production of recombinant foul box virus vFP-22 expressing the gene The RAV-1 envelope gene clone penvRVIPT is Ml:1Bl The encoding sequence was cloned as a [pnI-8acI fragment in B. Contains 1.1 Kbp of RAY-1 env DNA. This fragment is the 5' end is intact, but part of the sequence on the 3' side is missing, and is used in the following operations. Ta. EcoR1 derived from gel-purified 1, IKbp penvRV I - Insert the Pst I fragment into the Eco R1 and Pst 1 sites of pUC9, and If75G was produced. This plasmid was digested with KpnI and )11ndI [[ and cleaved 59 bases upstream of ATG in the vector. Vaccinia H6 already mentioned Insert the 14B base pair KpnI-Hlndm fragment containing the promoter Then, plasmid pc8B was created.

The starting ATG of the RAY env gene is located in the H6 promoter lacking the foreign sequence. To confirm that they are adjacent to the 3′ end, the terminal Eco RV and Ban Two complementary synthetic oligonucleotides were created at the n site. This oligonucleo The sequence is A　TC-CGT-TAA-GTT-TGT-ATC-CTA-ATG-AGO -CG A-GCC-3'.

Cut plasmid pcE8 within the H6 promoter 24 bases upstream of ATG A cut is made 7 bases downstream of ATG within the Eco RV and RAY env encoding sequences. Digested with Bann. These DNA fragments are ligated to transform E. coli cells. Using. The resulting plasmid pcE7 contains the He promoter and gave the correct 5' sequence.

Clone ■p19env (190) was mapped with restriction enzymes to generate all RAY- It was found that it contains 1 env gene. 5p19env containing all genes The 1.9 Kbp KpnI-3acI fragment of (190) was converted into Kp of pUclg. It was inserted into the nI and SacI sites to form pcIl:3. This plasmid , Hp that cleaves 132 bases downstream of the initiating ATG within the RAV-1 encoding sequence. It was digested with aI and SacI, which cuts at the 3' end of this gene. already mentioned The FPV insertion vector pcE11 was digested with Sma I and Sac I, and this plus The mido was cut at the polylinker region. pc with Hpa l-8ac fragment of pcE3 They were ligated at Ell to create pcE14.

Plasmid pCE7 was then digested with XhoI and old ndn[, and the He promoter was A 332 base pair fragment containing the correct 5' sequence was generated. Plasmid pCE 1 4 with the old ndI cutting in the polylinker region of this vector [and with the coding sequence Digested with XhoI. This DNA was obtained from pCE7 as the old ndIII- Xh.

I fragment to form the final RAV-1 envelope gene construct, pcE15. did.

This plasmid was tested in vitro using Foulbox pp-t as a rescue virus. Used for recombination testing. Recombinant progeny were smeared onto a CEP monolayer and anti-RAV-1 polycrystalline β-galactosidase-linked protein A immunoassay using local serum Screened for plaque. Purification of plaques 4 times after selection of positive staining plaques A single population was obtained. The produced recombinant was named vPP-22. vP Immunoprecipitation experiments using P-22-infected CEP lysate revealed that the envelope gene remains. Gene product 2 (2 with apparent molecular weights 76.5 Kd and 30 Kd corresponding to Il) It was shown that two proteins were specifically precipitated. The precursor gene product is completely I couldn't see it.

Preliminary tests showed that this RAV-1 envelope gene production in chickens inoculated with vPP-22 An immune response to the substance was elicited.

Example 17 Bovine leukemia virus (BLV) gp51.3Q envelope (e nv) Production of recombinant avipoxvirus expressing the gene (1) Construction of pBLVPI and pBLVF2 Plasmids pBLVF1 and pBLV F2 contains the BLV gp51.30env gene. In both plasmids , the BLV env gene is under the transcriptional control of the vaccinia virus H8 promoter. Yes, it is cloned between the foul box adjacent arms (locus f7).

The nucleotide sequences of these two plasmids, excluding codon positions 288 and 269, are are equal. (pBLVFl contains the amino acid Arg-8er at these two positions. pBLVP2 encodes a protein, while pBLVP2 encodes a protein containing the amino acids Gin-Thr. code. ) pBLVF1 and I) BLVF2 were produced by the following procedure. Plasmid pN897 -1 is a plasmid containing the entire BLV env gene, which is and partially cut with Mst II. Contains all gp51.30 genes The 2.3 Kbp fragment was isolated on an agarose gel and the sticky ends were isolated from E. coli D. Filled with NA polymerase I (Klenow fragment). Disconnect the Pst1 linker After ligation to the ends of the fragments and PstI digestion, the PstI site of pTP15 (actual Example 15). This allows the BLV gene to be linked to the vaccinia promoter. It is placed adjacent to He. (pTP15 is a non-essential locus in the vaccinia genome. Contains a cloned vaccinia H6 promoter. ) This plasmid is then cut with Eco RV and partially cut with Ava II. 5 ．． A 2 Kbp fragment was isolated and oligonucleotide 5'-ATCCGTTAAGT TTGTATCGTAATGCCCAAAGAACGACG-3' and 5'-G ACCGTCGTTCTTTGGGCATTACGATACAAAACTTAAC This plasmid was recircularized using GGAT-3'. By this, BL Remove unnecessary bases between the V gene and H6 promoter.

The obtained plasmid was cut with PstI and partially cut with BglII, and the promoter was A 1.7 Kbp fragment containing the BLV gene whose transcription is promoted by tar H6 was previously described. Bag old-Pst1 part of solid foul box virus insertion vector pcEll was cloned using locus r7 at position. This results in 1 (with 6 promoters) The BLV gene is placed between the arms adjacent to the foul box. Transfer this plasmid to pB It was named LVPl.

pBLVF2 was created using the same procedure but with BL with the H6 promoter. An in vitro mutagenesis procedure was performed before cloning the V gene into pcEl. I went to This mutagenesis was performed according to the following procedure.

Plasmid pNS97-1 was cut with XaI and partially cut with StuI. 5. 2 Kbl) fragment was isolated and oligonucleotide 5'-CCGGGTCAGACA AACTCCCGTCGCAGCCCTGACCTTAGG-3' and 5'-CC TAAGGTCAGGGCTGCGACGGGAGTTTGTCTGAC-3' This plasmid was recircularized using This results in codons 288 and 28 The nucleotide sequence of 9 changes from CGC-AGT to CAA-ACT.

(2) Production of recombinant viruses Said plasmid I) BLVFI and pBLVF2, with FP-1 as rescue virus. It was used in an in vitro recombination test. Recombinant progeny are in situ Prakha Select by hybridization, and once the population is determined to be pure by this criterion, β-galactosidase protein using a BLVgp-specific monoclonal antibody preparation. Screening was performed using Lotin A immunoassay. Plasmids pBLVF1 and p Recombinant vPP23 and vFP24, each made from BLVF2, were both immobilized. Immunologically recognizable sugar proteins on the surface of infected cells that stain positively with Noscreen. This suggested that Pak was expressed.

Plus-Mead pBLVK4 and pBLVK84t, Tsuretsure, W (7) BL V env gp51. IO gene and BLV gp51.30 cleavage minus gene child contains. Both genes are the only EcoR1 of pRV7f14.2 site (locus C3) (for pRIJ7f14.2, Example 131; It was cloned into the vaccinia H6 promoter and is under the transcriptional control of the vaccinia H6 promoter.

This plasmid was induced by the following procedure. Restrict pBLVPl and pBLVF2 It was cut with the enzyme old ndI[I. Oligonucleotide BKL 1 (AGCTTGA ATTCA) was cloned into this site, and EcoR1 was placed on the 3' side of the BLV gene. Create the part. Since there is an Eco R1 site on the 5' side of this BLV gene, These plasmids (pBLVKl and pBLVK2) were cut with EcoR1, The fragment containing the BLV gene whose transcription is promoted by the H6 promoter is pRW764. ．． It was cloned into the Eco R1 site of 2. Each of the obtained plasmids was They were named BLVK4 and pBLVK6. These plasmids were This virus was used in an in vitro recombination test as a rescue virus. Select recombinants and inject Purification based on surface expression of this glycoprotein detected by immunoassay did. The respective recombinants from plasmids pBLVK4 and pBLVK6 were vc They were named P27 and vCP2g.

Foulbox recombinants vFP2s and vPP24 were delivered to sheep by various routes. and inoculated cattle. Animals were vaccinated twice, the second dose 45 days after the first dose. serum Specimens were collected 5 weeks after the first vaccination and 2 weeks after the second vaccination. against gp51 Antibodies are measured in a competitive ELISA test, and the titer value is the serum dilution that inhibits the competition by 50%. expressed as the reciprocal of The results are shown in Table XI.

None of the species tested showed a detectable immune response after the first inoculation.

Both sheep and cattle showed a significant increase in antibodies after the second vaccination.

A sarcastic inoculation in 2 places.

Construction of foul box virus PP-1 recombinant vFP-28 expressing the virus gene made Plasmid plBVB[R3 represents the infectivity of the matrix gene of Mass41 strain. Contains a bronchitis virus (IBV) cDNA clone. plBVB83 no 8 The Kbp Eco R1 fragment is a matrix with a bebromer gene upstream (5' side). It contains the Eco RV site further upstream of the Eco RV site. plasmid PRV715 is an Eco old linker that joins the two Pvu II sites of pUC9. - has. The Eco R1 fragment of gKbp derived from plBYM63 was converted into pRv 715Eco R1 site to create pRV78B. Plasmid pRV A plasmid pRw77e lacking the 5′ EcoR1 site of 76g was created, The only EcoR1 site downstream (3' side) of this matrix gene was left. single The Eco R1 partial digested linear product of pRIf783 was reconstituted with Eco RV. It was cut off. Isolate the largest fragment and convert it to DNA polymerase I Klinow fragment. It was made into a plant end and self-ligated to produce pRW77B. Construct pRV 77B has all IBY peblomer genes and all matrix genes; The x gene is followed by a unique Eeo R1 site.

The sequence of only the 5' and 3' ends of the approximately 0.9 Kbp matrix gene was determined. was. The 5' sequence of the matrix gene starting from the translation initiation codon (ATC) contains , contains the underlined Rsa I site below.

ATGTCCAACGAGACAAAATTGTAC, the previously mentioned H6 promoter was linked to this matrix gene using synthetic oligonucleotides. This synthesis The oligonucleotide contains the H6 sequence from the Eco RV site to ATG. , inserted into the matrix coding sequence through the first Rsa1 site. This o The oligonucleotides were inserted into the Ba and Eco R1 ends so that they could be inserted into pUc9. pRV772 was created.

The Eco R1 end is on the 3' side of the Rsa 1 site. Starting from the Bag old compatible end This double-stranded synthetic oligonucleotide (ATG is underlined) The array is GAgATArAA6ATCXTYang QianAACGAGACAAAACGCIDA TA world AAT side CAAAC^TAGCATrACAmAACA■AA.

The Rsa I partial digested linear product of pRV772 was isolated and recut with Eco R1. did. This p has a point cut once at one site of Rsa and cut again at Eco R1. l? The V772 fragment was treated with phosphatase, and the following pRW77B digestion product vector was created. It was used as a tar.

The isolated linear Rsa 1 partial digestion product of pRV77B was cut again with EcoR1. I cut it off. Eco R1 site is just beyond the 3' end of the matrix gene It is in. Contains the sequence encoding the matrix from the Rsa 1 site above. The approximately 0.8 Kbp Ras I-Eco R1 isolated fragment was transformed into the above pR11772 It was inserted into a vector to create pRV783. The complete H6 promoter is Ec o Created by adding sequences 5' to the RV site. H6 promoter Make the 5' end of the plant end and insert it into the Sal 1 site of pUc9. This was the Hlnf I site that generates the R1 site, but the 5 ' is the old ndI site of pUC9. Contains this 5'H6 promoter Combine the old ndm-Eco RV fragment with pRV783 Hlndm and Eco RV site. pRV78B was created. pRW78B Eco R1 fragment is Contains a complete matrix gene with H6 promoter, pRV731.15 was made plant-end with the Klinow fragment of enzyme I. It was inserted into the old Bag site (locus f8) of the plant end to create pRW789.

This pRV731.15 Bam old site was used for the production of vPP-8 in Example 6. This is the FP-1 locus.

Plasmid pRV789 was used to create vFP-28. Screening for recombinant plaques and treated with in situ plaque hybridization.

In a preliminary study, chickens inoculated with vFP-28 received IBV matrix protein. An immune response against Park was induced.

Infectious bronchitis virus (IBV) Mass41 cDNA clone pIBV MB3 and its subclone pRW77ft are similar to vFP-26 in Example 18. We have already explained the preparation of . Subclone pRV77G is a 4Kbp IBY Contains the bromer gene, and after this gene, the only EcoR1 at the 3' end It has a matrix gene with parts. The approximately 4Kbp IBY hepromer gene Only the sequences of the 5' and 3' ends had been determined. The only Xba with 2 genes in 1 part Separated into genes. 5' of the pebromer gene starting at the translation initiation codon (ATC) The end has an Rsa 1 site underlined below.

ATCTTGGTAACACCTCTTTTACTAGTG ACTCTTT GTGTGTAC, the previously mentioned H6 promoter was synthesized with synthetic oligonucleotides. linked to the Pebromer gene. This synthetic oligonucleotide contains Nru 1 site. It contains the He promoter sequence from to ATC, and the first Rsa was inserted into the peplomer coding sequence via the I site. This oligonucleotide Synthesize ends compatible with Ras old and Eco R1 for insertion into pUc9. Then, pRW7Bg was created. Eco R1 end becomes 3' of Rsa 1 site . Sequence of double-stranded synthetic oligonucleotide starting with Bag old compatible end (ATG is ) is indicated by underline. CAT mouth grave CGATAflAAflATCGT[AACACCTCTrACCI I;CTATAtEAATrCAAACATAGCATrACAACCAflA GAATrA(TAGT[iAmACG AATGATCA-AGAAAACACACA-1. isolated pRV7 6 g of linear Rsa 1 partial digest was re-cleaved with Eco R1. R above pRVT containing a site cut at one sa site and re-cut with EcoH The 68 fragment was isolated, treated with phosphatase, and added to the vector of the pRV77B digestion product below. It was used as a vector.

The isolated linear Rsa 1 partial digestion product of pRW77B was again digested with Eco R1. Amputated. Cleaved once at the above Rsa site and contains up to the Eco R1 site A 5 Kbp pRW77B fragment was isolated.

This fragment was extracted from the peplomer Rsa I site to the 3' end of the matrix gene. Contains IBV sequences up to the Eco R1 site at the end. Transfer this pRw77B fragment to It was inserted into the pRV76g vector described above to create pR111788. This matori The Xba1 gene was transferred to the Xba1 site described above. 4Kbp pRV788Nr ul-Ba5 old plant engineering fragment to pRV780 Nru I-Bag old plant By inserting the old solant endo vector, the 5'H6 promoter was converted into Nru 1 site to create I) RV790. About vector pRVTBO is described in Example 11, and simply put, H adjacent to the non-essential FP-1 locus f7 Vaccinia influenza nucleoprotein whose transcription is promoted by 6 promoters .

This pRW760 vector consists of a Baa old vector from the Nru1 site to the end of the nuclear protein. was created by removing the 3'H6 sequence up to. pRV790 is pR V731.13 IBV pebromer carrying H6 promoter in H1ncII site It is. Donor plasmid pRV790 was recombined with PP-1 to generate vFP-31. generated. Immunoprecipitation was performed using CEF lysate prepared from vFP-31 infected cells. A small amount of the precursor protein with a molecular weight of about 18QKd and a decomposed protein of 90Kd were detected. A small amount of the product was shown to specifically precipitate.

Example 20 Foulbox virus F expressing herpes simplex virus gD Preparation of P-1 recombinant vPP-30 Herpes simplex virus (H8V) type I strain KO 8 Glycoprotein D gene (gD) into the Ba1l old site of pUc9 and the 5'Bam old link. Cloned as a fragment from Hpa II to 3'Bam old link Nru I. Ta. The 5' end is adjacent to the pUc9 Pst1 site. Translation start codon (AT The 5' sequence of H9VgD starting with G) is Neo with the following underline. Contains 1 site.

A picture (8) (8) Mouth Sosou A times Asahi GG Jauji Amτ ■ Punishment Zheng ATA punishment - (8) Black face 9 The previously mentioned vaccinia H6 promoter was transformed into H3VgD using a synthetic oligonucleotide. linked to genes. Nru I to ATG and further Nc of gD coding sequence Contains the 3' portion of the He promoter leading to the o1 site. This oligonucleo Tide was synthesized with a PstI compatible 5' end. gD clone of pUC9 Cut the clone with PstI and NeoI, remove the 5') ISV sequence, and create a synthetic oligo By replacing the nucleotides, pRV787 was obtained. This double strand synthesis The oligonucleotide sequence is 龜(8) AT＾Emachi Tate πken A Ding Amuro 8 times) ACxrrcAGC [ATAGGC AATTCAAA(:ATAGCA-ACCCCTCCAg-CAGCTAGAT rA[ATrlTATIWrAGTrATAiAGG^αcTCcATCTAA TcCACGACAATAAAAATAAACATCAATAπATCCTGAG There is GTAC.

When pRW787 is digested with Nru I and Baa old, HSVg is generated from the Nru 1 site. Contains the 3'H6 promoter that passes through the D coding sequence to the Baa fi1 site. A fragment of approximately 1.3 Kbp is generated. This pRV7BO vector was The cutting at the a old point was described in Example 11. This 1. :1Kbp fragment was inserted into the pRwyeo vector to create pRV791. This pRv’yat The vector contains the non-essential FP-I Hlnc If site of pRv731.18 (locus 1 In 1), H! transcription is promoted by the complete Vakushiniwa promoter. Contains 3VgD gene. have

Donor plasmid pRV791 was recombined with FP-1 to obtain vFP-80. sugar Surface expression of proteins can be detected using protein A-β-galactosidase-linked immunoassay. H9V-1 and H9V-1-specific serum were detected in recombinant plaques.

Example 21 - Control of expression of foreign genes in poxvirus vectors Use of the Entomobox promoter (a)　IJr　Insect poxviruses (poxviruses) are currently It is classified into the Entoso-poxvirinae subfamily. Cages, beetles (Celeopteta), lepidoptera (Lipidoptera) , Entomopo, isolated from insects belonging to the order of Orthoptera. The virus is further subdivided into three genera (A%B%C), each corresponding to a virus. It will be done. Originally, entomopoxvirus has a narrow host range, and it is difficult to It is also known that it does not replicate.

The entomopoxvirus used in this study was originally an infected Amsac virus from India. Ta moorei (A■ sactamoore1) (Lepidoptera: Arctyl mite (a rctlldae) (Roberts). s) and Granados, Journal of Invertille Pathology (J, Invertebr, Pathol,) 12.14 1-14:1 [198B]). This virus has been named AmEPV and is It is the species type of the genus.

Dr. Granados (Boyce Thompf) produced wild-type AsEPV. Boys Institute ), Cornell University (Cornell University), infected Infection derived from Estigmenu acrea (Estig■ene acrea) larvae Obtained as hemolymph. This virus is Mandria gysper (Lywan). trla dls er) (v imai moth) ovarian tissue derived from vertebral cell lineage It was found that it replicates in certain IPLB-LD652Y (Goodow [1978 ]). The cells were subjected to IPL-1 supplemented with 4% fetal bovine serum and 4% chicken serum. 528 medium at 28°C.

A plaque assay of the wild-type virus was performed on LD852Y cells and a plaque named NVl was obtained. One arc was selected for the following experiment.

This isolate contains numerous closed bodies in the cytoplasm of infected cells late in the infection cycle. (occlusion bodles: 0Bs) are produced.

(b) Promoter identification Identification and mapping of AmEPV promoter is as follows. It was completed as follows. Total of recently infected LD652Y cells (48 hours post infection) RNA was isolated and used to generate the first eDNA strand labeled with 132P.

This eDNA was used as a probe in a restriction digest content plot of Ag+EPV. Ta. A strong signal was detected on the 2.6 Kb C1a I fragment in this Southern blot. The results showed that this fragment encodes a strongly expressed gene. book fragment was cloned into a plasmid vector and its DNA sequence was determined.

Analysis of the sequence data yielded reads that could encode a 42Kd polypeptide. The frame has been revealed. In vitro translation of total RNA and 5DS 48 hours after infection -Separation of the product by PAGE revealed one polypeptide of approximately 42 Kd. Production of recombinant vaccinia virus expressing the original gene Entomobox Promo To test whether the vector functions in the vertebrate poxvirus system, we used the following protocol. A lasmid was created. Gene translation opening at 42 adjacent to the Bglm site at the 5' end. 107 salt on the 5′ side of the initiation signal (hereinafter referred to as promoter for AmEPV42) 3 of the coding region of hepatitis B virus BreS2 terminating at the group and Eco R1 sites. An oligonucleotide containing the first 14 bases of the ' terminus was chemically synthesized. under The promoter sequence of this AmEPV42 is described below.

TCAAAAAAATATATAAATGATTCACCATCTGATAGAAA AAAAATTTATTGGGAAGAATATGATAATATTTTTGGG ATTTCAAAATTGAAAATATATAATTAATTAAA! LffLHepatitis B virus is generated by adding a promoter to this AmEPV42 as follows. s surface antigen (HBVsAg). encodes the hemagglutinin (HA) molecule. The vaccinia virus arm (HA) in the non-essential region of the tuccinia virus genome The arm is described in Example 15; the HA region Shida is pyrology. , Dan, 451-482 [198B]) adjacent to the pre-S2 code region ( Type ayw was described by Galibert et al., Nature , 84B-650 [1979]) and hepatitis B virus surface antigen. A pUC plasmid was created.

The aforementioned oligonucleotide was added to the unique EcoR1 of the coding region of HBVsAg. site and the unique Bgln site of the HA vaccinia arm into this plasmid. Inserted. The obtained recombinant vaccinia virus was named vP547.

The code for HBVsAg inserted into Entomo box 42 under the control of the promoter. Expression of the code sequence was confirmed using immunoassay. Mammalian cell line B5C-4 0 equivalent cultures were infected with parental vaccinia virus or recombinant vP547. Feeling Cells were lysed 24 hours after staining, and the lysate was serially diluted and applied to a nitrocellulose membrane. I put it on. The membrane was first incubated with goat anti-HBV serum and then Incubated with protein A. After washing, the membrane was exposed to X-ray film. . A positive signal was detected in the vP547 g-stained culture, but the parental virus-infected culture AsEPV4 is not detected in mammalian cells and is detected by vaccinia virus in mammalian cells. 2 has a promoter! 1! It suggested that he would be recognized.

The above results can be summarized as follows: This was confirmed using an assay (see Example 1 for details). AsEPV42K or HBsAg-containing vaccinia linked to the vaccinia virus H6 promoter. B10-40 cells were infected with the recombinant Avirus, and the sAg expression level was determined. Assayed with the Austria test. As shown in Table X■, using a promoter in 42 The data show that the HBsAg expression level was significant.

vP410 Control 1.0 vP481 H [l 24.3 vP547 42K 44.9 Furthermore, the promoter of AEPV42 in the vertebrate poxvirus environment - We conducted an experiment to confirm the temporal characteristics of control. inhibits late viral transcription , of cytosine arabinoside 40 μglI11, which inhibits DNA replication. Equivalent cultures of B10-40 cells were sensitized with vP547 in the presence or absence of I dyed it. Expression levels 24 hours after infection were assayed using the Austria test. Ta. From this result, this 42 promoter is the first in the vaccinia virus replication system. It was suggested that it is recognized as a phase promoter.

Using promoters in AiEPV42 for expression of foreign genes in mammalian systems Autographa californica ( Autographa calitornica) NPV polyhedrin promoter (Lockov and Summers) , Biotechnology 6.47-55 [1 It should be noted that it is clearly different from 98jl]). polyhedrin promo is not recognized by the transcriptional machinery in mammalian cells (Tjla ), BiooG, 125.107-117 [1983]). in mammalian cells A ■Using a promoter for EPV42 makes it possible to use non-insect virus vectors. Insect virus promoters are used to express foreign genes in vertebral cells. This is the first example of its use.

Avipoxviruses similarly recognize the entomobox promoter in 42. The following experiment was conducted to find out whether the for the same culture medium of CEP cells. Kanakubox, a foul box virus with 1 opfu per cell. Inoculate the virus or vaccinia virus and at the same time 1) Inject the promoter into 42. Plasmid 4 containing the IIBV plate S2+sAg coding sequence linked to 2, 17, or 2) linked to the vaccinia virus H6 promoter mentioned above. Plasmid containing coding sequence of HBVsAg 1) MP15.5psP Transformation was performed with 25 g of either plasmid.

Cells were lysed by freezing the culture after 24 hours and lysed using the Austrian test. The presence of HBVsAg in the fluid was analyzed.

The results shown in Table X■ should be examined quantitatively. As a result, the foul box The transcription machinery of both Kanakubox and Kanakubox can recognize promoters in 42, and , is suggested to allow transcription of the linked HBVsAg coding sequence.

Although the expression level is lower than that obtained with the vaccinia virus H6 promoter, well above the background levels obtained in sex controls.

Table xm None 42K 7.8 None He 7.2 Vaccinia 7.2 Two recombinant (a) vFP-6 (performed foul box rabies recombinant described in Example 6) and (b) vcP-18 ( A predetermined amount of either Kanakubox rabies recombinant described in Example 13) 50 to 100 μl of the diluted solution was inoculated.

On the 14th, 10 mice from each group were sacrificed and serum was collected.

Anti-rabies titers in serum were calculated using the RFFr test described in Example 7. each The remaining 10 mice in the group were injected with the rabies virus CVS strain used in Example 7. Attacked with intravenous vaccination. Each mouse received 30μ, which corresponds to 16 times the mouse's LD5o. 4 was administered. At 2'8 p.m., live mice were examined and the 50% protection dose (PD5o) was calculated. I calculated it. The results are shown in Table XIV.

The level of protection of mice found from inoculation with vFP-6 was based on the factor tested in Example 7. This confirms the results shown with inoculation with Urvox recombinant vFP-3.

The level of protection afforded by vcP-18 inoculation is considerably higher. Calculated PD5゜ Foul box rabid dog protection against rabies attack The Kanakubox rabies recombinant is 100 times more effective than the disease recombinant.

Foul Box vPP-6 Kanaku Box vCP-16 Inoculation amount RFPI Viability rate Inoculation amount RFFI Viability titer Titer 5.5 1.8 571G 4.5 1.9 87108.5G, 7 0/1 0 2.5 1. f I/101.5 0.8 0/10 0.5 0.4 0 /10IPD-6,171PD-4,18a virus titer is 10g1oT Expressed as CID5o.

Foul box by staining SDS polyacrylamide gel with Coomassie blue (PP-1) by staining the protein species present in the infected CEF lysate. As a result, a major molecular species with an apparent molecular weight of 25.8 KD was revealed. This tamper protein was not present in uninfected cell lysates and was synthesized at a specific time after infection. Pulse experiments in which proteins were radiolabeled with 35s-methionine revealed that PP-1 induces It has been shown that there is an abundance of this protein, and that this protein is present from 6 hours after infection. It was suggested that it was synthesized within 54 hours. At peak level, this FP-1 The 25,8 KD protein accounts for approximately 5% of the total protein present in the cell lysate. It accounts for up to 10%.

The abundance of PP-1-induced 25.8KD protein suggests that this gene production The gene encoding the product is controlled by the strong FP-1 promoter element. It was suggested that Foreign gene expression in poxvirus recombinant bodies To determine the location of this promoter element for current use, Polysome preparations were obtained from PP-1 infected CEP cells 54 hours after staining. l1NA were isolated from this polysomal preparation and introduced into the rabbit reticulocyte in vitro translation system. When used, it mainly produced 25.8 KD FP-1 protein.

This polysomal RNA is used as a primer with oligo(dT)12-18. It was used as a template for initial cDNA strand synthesis.

This first cDNA strand was analyzed in a Southern blot analysis of the FP-1 genome digest. It was used as a hybridization probe. The results of these hybridization analyzes From the fruit, the gene encoding the 25.8KD protein is lo, 5Kbp H It was suggested that it was contained in the lndI[[ fragment. Next, this genome H1ndm The fragment was isolated and converted into a commercially available vector pBS (stratagen e), La Jolla, California), and this clone The vector was named pFP23-1.

Hybridization was performed using the first cDNA strand as a probe of the pFP23-1 digest. When we performed a code formation analysis, we found that the 25.8KD protein gene was 3.2Kbp long. Eco RV small pieces were found to be located. Clone this fragment in pBS. and named pFP23 -2.

The base sequence of this PP-I Eco RV fragment of approximately 2.4 Kbp is based on Sanger's Oxychain terminator method (Sanger, etc.) National Academy of Sciences USA, 74. 5483-541i70977]). From sequence analysis, the molecular weight was 25. An open reading frame (ORP) encoding an 8KD gene product has been revealed. this The ORF was transformed in vitro with bacteriophage T7 polymerase in a pBS vector. Transcription off trolan is performed using the rabbit reticulocyte in vitro translation system (Promega Biotranslation System). Otic (Promega Biotec), Madison (Madlson)% The apparent molecular weight of the rice consin used in rice consin was 25. Polypeptide species of IIKD An RNA species was obtained that produced . This polypeptide is 5DS-polyacryl Abundant 25.8KD observed in FP-1 infected CEPs lysates on midgels. The electrophoretic mobility is the same as that of the protein. These results indicate that this is induced by PP-1. 254K [indicated to be a gene encoding a gene product] be suggested.

■, Feline leukemia virus FeLV in FP-1 and vaccinia recombinants ) Upstream promoter of FP-125,11KD gene to express env gene Use of the FP-125,11KD gene control region (FP25. 8 promoter) and 21 bp of 25.8 KD gene code sequence. A 70 bp Eco RV/Eco R1 fragment was isolated from pFP23-2. p FP used to induce FeLV25.8FL and pFeLV25.81A The nucleotide sequence of the promoter region is shown below in 25.8. These 270 pieces The nucleotide sequence is upstream of the start codon (ATG) of the 25.8KD gene product. The 249 nucleotides and first 21 bp of the coding sequence are shown.

5'-GATATCCCCATCTCTCCAGAACAGCAGCATAG TGTrAGGGACAATCATCTAA-TGCAATATCATATATG AATCTCACTCCGATAGGATA(TTACCACAGCTATrA TA-CCTTAATGTATGTrCTATATAmAAAAAACAGAAA CAAAtIXSaeATAAGTTTrAT-ATGATGTCTATATATA GTGAGTATA CATAAGTATGCGGGAATAIII Tr-TAACAGCGTACGA ■ Punishment ATAAAGTAAATATAGGC Pigeon W ATAGCATAAAπU-TrC-3' This fragment was used as a plant end for SmaI deletion containing the PeLV env sequence. FP-1 insertion vector (pPeLVFl: see Example 15). child The insertion vector allowed recombination through the r7 locus of the FP-1 genome. This F Insert the promoter upstream sequence into the 5' side of the PeLV env gene at P25.8 Their correct orientation was confirmed by sequence analysis. This insertion However, the 25.8KD gene Since the ATG provided by the child is outside the framework of this FeLV env ATG , no fusion protein is formed. PP25.8 upstream of FeLV env gene The FP-1 insertion plasmid containing the KD promoter was transformed into pFeLV25.8Pl. It was named.

The vaccinia virus insertion vector pFeLVIA carries the FeLV gene. A similar construct was prepared using this vector (see Example 15). This H The e promoter was excised from pFeLVIA by digestion with Bgln and Sea I. I cut it off.

After setting the Bgl II restriction site to the plant end, insert the promoter at PP25.8. Plant-ended Eco RV/Eco R1 fragment (270 bases) containing It was inserted adjacent to the 5' side of the FeLV env gene. Array this structure Confirmed by analysis. Although this recombinant does not have a complete ATG for ATG substitution, The ATG of the 25.8KD gene is not in the frame with the ATG of the PeLV gene. Wa Kushina (Copenhagen strain) insertion vector is adjacent to the 5' side of the FeLV gene. It possesses the upstream region of the 25.8KD gene and is named FeLV25.81A. did.

The insertion plasmids pPeLV25.8F1 and pFeLV25.1lIA were FP-1 (pPeLV25.IIPl) and vaccinia virus as supporting viruses Used for in vitro recombination with Scopenhagen strain (pFeLV25.81A) Ta. After spreading this recombinant progeny onto a suitable cell monolayer, the recombinant virus Sidase-linked protein A immunoscreen and bovine anti-FeLV serum (Antibode Selected by Amazon.com, Davis, California). From preliminary results, this PP25. The llK promoter is used as a foreign gene in poxvirus recombinants. This suggests that gene expression can be controlled.

Two Avibox recombinants vFP-6 and vcP-16 (Example 6 and Example 13) ) was inoculated into 18-day-old chicken embryos and 1-day-old chickens and 28-day-old chickens. , the response of these chickens to three criteria: 1) 511 conversion rate, 2) Effect of vaccines on response and mortality to rabies glycoproteins, based on the immune response elicited; 3) the immune response elicited by the foul box antigen; It was evaluated. The experiments conducted are described below.

A. Safety test: Groups of 20 18-day-old animals were injected with vFP-6 or is 3.0 or 4.0 times the amount of either logloTCID5o of vcP-16 was inoculated. After cape formation, the chickens were observed for 14 days and serum was collected. to chicken embryo The two inoculated recombinants had no effect on the hatching rate of eggs, and the chickens remained healthy during the observation period of 1 day.

A group of 10 1-day-old SPF chickens was administered intramuscularly with any of the above recombinants. 3.0 times the amount of logloTCID5o was inoculated. Observe chickens for 28 days Serum samples were collected 14 and 28 days after inoculation. Any recombinant No vaccine reaction was observed at the vaccination site, and the chickens remained healthy during the 28-day observation period. there were.

Groups of 10 28-day-old chickens were inoculated with either recombinant virus. Ta. The dosage is 3°010g1oTCID5o via the intramuscular route or through the skin (red sea bream). It was set to either 8.01 og 1 o TCID 5° at the road. Observe chickens for 28 days Serum samples were collected on the 14th and 28th days after inoculation. Inject any recombinant intramuscularly However, no reaction was observed at all. Foul box is extremely small due to skin inoculation A strong vaccine response occurred, but the size of the lesions was different. Kanaku box connection Depending on the species, normal skin lesions developed at the inoculation site. All lesions were removed until the end of the experiment. It has already regressed.

B. Immune reaction Rabies glycoprotein was detected using the RPPI test described earlier in Example 7. Antibody levels against were assessed. The results of each group were compared to standard blood containing 23.41U. The geometric mean titer of each serum was determined using international units (International Units). Expressed in terms of (IU). The minimum positive level is defined as IIU, The percentage of positive chickens was used for calculation. Antibiotics against avipoxviruses The body was tested using an ELISA method using the foul box virus strain as an antigen. Ta. Each serum specimen was diluted 1:20 and 1:80. Positive and negative sera A standard curve was drawn using The minimum positive level is the average plasma value of negative sera with various values. Calculated using 2 standard deviations.

Serological test results are shown in Table Xv for vFP-6 and for vcP-18. Shown in Table XVI.

In embryos inoculated with either vPP-6 or vCP-16, rabies antigen or A limited serological response to foulbox antigens was observed. Said Fau The Luvox vector has both vectors in more chickens than the Kanakubox. Although a serological response to the antigen was elicited, this response was also variable.

Chickens inoculated with vPP-6 at 1 day of age showed a good serological response and all of chickens tested against rabies antigen and foul box antigen by 28 days after vaccination. The patient was seropositive. The response to vcP-16 inoculation was much lower, at day 28 40% of chickens are seropositive for rabies glycoprotein and Avibox antigen In contrast, 10% of the chickens were seropositive.

Chickens inoculated with vFP-6 by the intramuscular route at 28 days of age showed both signs of infection by 14 days post-inoculation. There was 100% seroconversion against both antigens. Chicken after skin inoculation The majority of the animals were also seroconverted, but the rabies antigen and Avivox The titers obtained for both originals were low. As already mentioned, muscle and skin Chickens inoculated with vcP-16 in both tracts showed variable responses, with intramuscular It showed up to 70% seroconversion against rabies vaccination. Kanasobot The low level of seroconversion to Avivox antigen after vaccination with This may reflect the degree of serological relatedness between viruses.

From the above results, it is clear that both vPP-6 and vCP-18 are effective for chickens in a certain age range. It was suggested that even if inoculated into birds, it would be a blue gold. foul box vector vF P-6 appears to be more effective in eliciting an immune response in chickens. Ru. However, the avipox viruses Foulbox and Kanakubok It is important to note that both recombinant forms of this plant are effective for immunization in eggs. Ru.

Example 25 Safety of inoculating piglets with vFP-6 and immunization from 3 piglets in group 1 Two groups were inoculated with recombinant vFP-6 by one of two routes of administration.

a) 3 piglets were administered 8.11 og oTCID5o by intramuscular inoculation; b) piglets The same amount was administered to three animals by oral inoculation.

After blood was collected from all animals at weekly intervals, they were boosted on day 35 by the same route and dose. Piglet Clinical signs were observed daily. Serum was tested for anti-facial Urvox antibody was investigated. Rabies antibodies were assayed with the RFFI test.

All piglets remained in good health and no lesions were observed after vaccination. body temperature song The lines were normal and there were no differences between inoculated and non-inoculated animals.

Vaccination by intramuscular and oral routes as determined by ELISA and serum neutralization Both recipient piglets elicited a serological response to foul box antigens. did. After the secondary inoculation, the secondary reaction was significant (results not shown). RFP All piglets developed an immunological response to the rabies glycoprotein as measured by the I test. , the additional effects of both routes are significant. These results are shown in Table X■.

From the above results, it can be concluded that inoculation with Fur7 Urbox/rabies recombinant is harmless to piglets. rabies glycoprotein by oral or intramuscular inoculation. This suggests that it is possible to induce a significant immune response against.

Table X■ Rabies antibody (RFFI titer) vaccination on the following days: Animals 14 21 28 35b42 49 route number 984 2.4a2.2 2.1 2.2 3 31, M, 985 2.5 2.7 2.6 2.4 3 398B 2.2 2.0 2.1 2J 3 3987 B 2 2.1 2 3 3 Oral 988 2.9 2.4 2.2 2.4 2.7 2.59g9 2. 8 2 1.7 1.8 2.4 2.5a titer is the fluorescence concentration in RFFI test. It was expressed as the common logarithm of the maximum dilution when the decrease in well number exceeded 50%.

b Animals received a second vaccination on day 35.

Procedural amendment export) July at date, 1999

Claims (39)

[Claims] 1. a method for expressing the gene product in a vertebrate, encoding the gene product; and allows the virus to express the gene product without productive replication in vertebrates. A method comprising inoculating a vertebrate with a recombinant virus containing the DNA. 2. 2. The method of claim 1, wherein the virus is a box virus. 3. 3. The method of claim 2, wherein the virus is Avivox virus. 4. The Avibox virus is a foul box virus and a canary box virus. 4. The method according to claim 3, wherein the method is selected from the group consisting of virus. 5. The virus is administered to the vertebrate subcutaneously, intradermally, intramuscularly, orally or intravenously. Claim: wherein the vertebrate is inoculated by introducing the vertebrate in ovum. The method described in 1. 6. Encoding antigens as a way to elicit an immune response against them in vertebrates D, and the virus expresses the antigen without productive replication in vertebrates. A method comprising inoculating a vertebrate with a recombinant virus containing NA. 7. 7. The method of claim 6, wherein the virus is a box virus. 8. 8. The method of claim 7, wherein the virus is Avivox virus. 9. The Avibox virus is a foul box virus and a canary box virus. 9. The method according to claim 8, wherein the method is selected from the group consisting of virus. 10. The virus is administered to the vertebrate subcutaneously, intradermally, intramuscularly, orally, or injected into the vertebrate. 7. The method of claim 6, wherein the vertebrate is inoculated by introduction with a bum. 11. A method for inducing an immune response against a vertebrate pathogen in a vertebrate, It encodes the antigen of the pathogen and prevents the virus from productively replicating in vertebrates. Inoculating vertebrates with a recombinant virus containing DNA that expresses the antigen of a pathogen. how to. 12. 12. The method of claim 11, wherein the virus is a box virus. 13. 13. The method of claim 12, wherein the virus is Avivox virus. 14. The Avivox virus is a Foulbox virus and a Canaryvox virus. 14. The method of claim 13, wherein the method is selected from the group consisting of xviruses. 15. the vertebrate is a mammal, and the vertebrate pathogen is a mammalian pathogen; 12. The method of claim 11. 16. The antigen is rabies G antigen, gp51.30 envelope of bovine leukemia virus. FeLV envelope antigen, FeLV envelope antigen of feline leukemia virus, and herpes simplex 16. The method of claim 15, wherein the glycoprotein D antigen of a virus is selected from the group consisting of: . 17. The mammal is from a dog, a cat, a mouse, a rabbit, a cow, a sheep, and a pig. 16. The method of claim 15, wherein the method is selected from the group consisting of: 18. The virus is administered to the vertebrate subcutaneously, intradermally, intramuscularly, orally, or injected into the vertebrate. 12. The method of claim 11, wherein the vertebrate is inoculated by introduction with a bum. . 19. A method for inducing an immune response against a vertebrate pathogen in a vertebrate, A recombinant Avibox cow containing DNA that encodes and expresses a pathogen antigen. A method of inoculating vertebrates with viruses. 20. The vertebrate is a mammal, and the vertebrate pathogen is a mammal. The method according to claim 19. 21. The antigen is rabies G antigen, bovine leukemia virus gp51, 30 envelope. FeLV envelope antigen, FeLV envelope antigen of feline leukemia virus, and herpes simplex 20. The method of claim 19, wherein the glycoprotein D antigen of a virus is selected from the group consisting of: . 22. The vertebrate is a bird, and the vertebrate pathogen is an avian pathogen. The method according to claim 19. 23. The antigen may be avian influenza hemagglutinin antigen, Newcastle disease cow virus fusion protein antigen, rus-associated virus RAV-1 envelope anti- nucleoprotein antigen of avian influenza virus, infectious bronchitis virus matrix antigens of the infectious bronchitis virus, and the peplomer antigen of the infectious bronchitis virus. 23. The method of claim 22, wherein the method is selected from: 24. The non-essential region of the Avibox genome contains DNA of non-Avibox origin. Recombinant Avibox virus. 25. further contains a non-avibox promoter for expression of said DNA. The virus according to claim 24. 26. Claim 25 wherein the non-avibox promoter is a vaccinia promoter. Virus described in. 27. The vaccinia promoter is selected from the group consisting of HH, llK, and Pi. 27. The virus according to claim 26, which is selected. 28. Claims that the non-Avivox promoter is an Entomobox promoter The virus according to item 25. 29. The promoter further contains an Avibox promoter for expression of the DNA. 25. The virus according to claim 24. 30. 25. The virus of claim 24, wherein the DNA encodes an antigen of a mammalian pathogen. vinegar. 31. The antigen is rabies antigen, rabies G antigen, gp51 of bovine leukemia virus. , 30 envelope antigen, FeLV envelope antigen of feline leukemia virus, and and herpes simplex virus glycoprotein D antigen. ilus. 32. 25. The virus according to claim 24, wherein the DNA encodes an antigen of an avian pathogen. vinegar. 33. The antigen may be avian influenza hemagglutinin antigen, Newcastle disease cow virus fusion protein antigen, rus-associated virus RAV-1 envelope anti- nucleoprotein antigen of avian influenza virus, infectious bronchitis virus matrix antigens of the infectious bronchitis virus, and the peplomer antigen of the infectious bronchitis virus. 33. The virus according to claim 32, selected from: 34. 24. The Avibox virus is a Foulbox virus. Virus described in. 35. 24. The avivox virus is a canary pox virus. Virus described in. 36. DNA of any origin and Entomobox Pro to express this DNA. Recombinant box virus containing motor inside. 37. 37. The virus according to claim 36, wherein the virus is an Avivox virus. . 38. 37. The virus of claim 36, wherein the virus is vaccinia virus. 39. DNA of any origin and an Avibox promoter to express this DNA. A recombinant vaccinia virus containing a.