JPH10509593A - Expression of hemagglutinin / neuraminidase (HN) glycoprotein of bovine parainfluenza virus type 3 in two heterologous systems - Google Patents

Abstract

  (57) [Summary] A new vaccine to combat bovine respiratory disease is to convert the cDNA encoding the hemagglutinin / neuraminidase (HN) glycoprotein of bovine parainfluenza virus into bovine herpes virus so that the protein is expressed by the corresponding virus. -Produced by insertion into type 1 (BHV-1) or baculovirus.

Description

DETAILED DESCRIPTION OF THE INVENTION Expression of Hemagglutinin / Neuraminidase (HN) Glycoprotein of Bovine Parainfluenza Virus Type 3 in Two Heterologous Systems Background of the Invention Field of the invention The present invention relates to the field of Bovine Parainfluenza Virus Type 3 and vaccines for its treatment. Disclosure of information U.S. Pat. No. 4,847,081 [(John M. Rice, issued Jul. 11, 1989)] U.S. Pat. No. 4,703,011 [(Maron and Sole Kit ( Malon and Saul Kit)), issued October 27, 1987] U.S. Pat. No. 4,992,051 [(Malon and Saul Kit), issued February 12, 1991] RW Morgan et al., Avian Diseases, 36: 858-870 (1992); Y. Sakai et al., J. Virol. .), 63: 3661-8 (1989); T. Shioda et al., Virology, 162: 338-96 (1988); S. Suzu et al., Nucleic Acids.・ Research (Nucleic Acids Res.), 15: 2945-58 1987), Car El van Wyck, Soerinfu (K.L.Van Wyke Coelingh), et al., Bairoroji (Virology), 160: 465-472 (1987). background Bovine parainfluenza virus-3 (BPIV-3) belongs to the genus Paramyxovirus of the family Paramyxoviridae. Virions are shell particles that come in a variety of forms and contain nucleocapsids that encompass single-stranded RNA genomes. The genome acts as a transcription template for six unique structural proteins: nuclear protein (NP), matrix protein (M), fusion protein (F), hemagglutinin-neuramidase protein (HN), and large protein (L). . The HN and F proteins protrude from the virion surface as spike-like structures and are also expressed on the surface of infected cells. The HN protein is strongly hydrophobic at the amino terminus, and this domain is important in anchoring the protein in the virion envelope and host cell membrane. The second domain is near the C-terminus and mediates the attachment of sialic acid-containing host cell receptors to virions during the early stages of infection. (RM Chanock et al., Virology, edited by B. Fields (1990)). Hemagglutination is widely used in serodiagnosis and experimental laboratory studies, but is also the manifestation of this type of interaction, which is why this glycoprotein was named hemagglutinin. Later in infection, the HN protein also cleaves sialic acid residues from the virion surface by neuraminidase activity. After attachment to the cell, the virus must transport the nucleocapsid to the cytoplasm, which is mediated by the second surface glycoprotein, the F protein. This causes a fusion of the viral lipoprotein envelope with the lipoprotein surface of the host cell. The F protein is also expressed on the surface of infected cells, which allows fusion of contact infected cells with uninfected cells, leading to syncytia formation and spread of infection. The crucial role these two proteins play in the infection process and their presence on the surface of infected cells makes them an obvious target of the immune system, and animals that have recovered from infection have intermediates to both of these glycoproteins. Studies have shown that they have a sum antibody. The importance of BPIV-3 as a pathogen has been implicated in the bovine respiratory disease complex (BRDC). BRDC is a major cause of bovine morbidity and mortality, leading to significant economic losses. . A common view is that viruses, bacteria and stress interact to cause disease. The most commonly associated viruses are bovine herpesvirus type-1 (BHV-1), BPIV-3, bovine viral diarrhea virus. It is believed that viral pathogens cause early damage to the tracheal epithelium due to stress that leads to increased susceptibility to infection, which in turn causes bacterial invasion and further exacerbates clinical signs [RJ Yancy ( R. J. Yancey), Journal of Daly Sci., 76: 2418-36 (1993)]. It is often presumed that more than one viral pathogen is involved at a time, and in fact, virus isolation and serological evidence in BRDC supports this view. For this reason, most viral vaccines developed to prevent this disease include antigens for each of these viruses. Despite the availability of a wide range of vaccine combinations against the viral pathogens associated with BDRC, this disease conjugate remains a major source of economic loss for the bovine industry. Commonly used vaccine preparations consist of various combinations of inactivated virus (IV) and modified live virus (MLV), each of which individually adjusts safety and efficiency. However, in all cases, combination vaccines require separate preparation of various viral vaccines, which adds considerable complexity and cost to production. In addition, differences in optimal storage and administration protocols for the various components of many viral vaccine preparations can compromise the potency and stability of some components. The present invention provides users with a vaccine effective for the prevention of BPIV-3 and / or BHV-1, caused by components of the BDRC complex. SUMMARY OF THE INVENTION The present invention consists of a novel vaccine that fights respiratory disease in cattle. The vaccine comprises a cDNA encoding the hemagglutinin / neuraminidase (HN) glycoprotein of bovine parainfluenza virus, such as bovine herpesvirus type 1 (BHV-), such that the protein is expressed by the corresponding virus. 1) or by insertion into a baculovirus. The present invention consists of a replicating non-pathogenic virus, which inserts and expresses the hemagglutinin / neuraminidase (NH 2) glycoprotein at the appropriate locus of bovine herpesvirus type 1 (BHV-1). A gene or combination of genes encoding the hemagglutinin / neuraminidase (NH) glycoprotein or a virus wherein the gene or combination of genes is from bovine parainfluenza virus type 3 (BPIV-3); The gene or combination of genes encoding the neuraminidase (HN) glycoprotein is inserted into the BHV-1 thymidine kinase locus and consists of the virus expressed therein. A vaccine for the prevention of disease caused by bovine parainfluenza virus type-3 (BHV-3), which is inserted into the appropriate locus of bovine herpes virus type 1 (BHV-1) and expressed there. A vaccine comprising a replicating non-pathogenic virus comprising a gene or gene combination encoding a hemagglutinin / neuraminidase (NH) glycoprotein, wherein the gene or gene combination encoding a hemagglutinin / neuraminidase (HN) glycoprotein is bovine A vaccine from parainfluenza virus type 3 (BPIV-3), a gene or combination of genes encoding hemagglutinin / neuraminidase (HN) glycoprotein is inserted into the thymidine kinase locus of BHV-1 where The vaccine to be expressed. A vaccine comprising a gene or gene combination encoding a hemagglutinin / neuraminidase (NH) glycoprotein inserted into and expressed in a baculovirus expression system, a gene encoding a hemagglutinin / neuraminidase (HN) glycoprotein or A vaccine wherein the gene combination is from bovine parainfluenza type-3 (BPIV-3). Pharmaceutical compositions, carriers, diluents, adjuvants suitable for viral vaccines are also provided or will be appreciated by those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS FIG. Construction of a shuttle vector for expression of the BPIV-3 HN gene (and other genes) at the BHV-1 tk locus. FIG. Construction of a recombinant baculovirus transfer vector for expression of the BPIV-3HN gene in a baculovirus expression system. FIG. Immunoprecipitation of infection-cell lysates showing expression of BPIV-3 HN from BHV-1 and baculovirus. FIG. Neuraminidase activity in BHV-1 and baculovirus recombinants. Further description of the invention and preferred embodiments Definition All terms used below will be readily understood by those skilled in the art. In many cases, the name of the manufacturer of the device or reagent will be provided in parentheses after the name of the device or manufacturer is listed. Commonly used terms such as reagents such as "plasmid", "Klenow fragment", "blunt ends", "Tris" and chelating agents such as EDTA are mentioned without further explanation. In defining the construction of the compounds used in the present invention, standard molecular biology techniques are used and are shortly named herein. A detailed description of these techniques can be found in the standard laboratory manuals of FM Ausubel et al. (1993) and J. Sambrook et al. (1989). All abbreviations, such as "BV", "BT cells", "BDRC", "BPIV-3" and "BHV-1" that are not always readily understood by those skilled in the art, are often defined based on their first use, Used thereafter without further consideration. Composition and administration The pharmaceutically effective amount of the vaccine according to the invention, together with a pharmaceutically acceptable carrier, diluent or adjuvant, is used as a vaccine against BHV-1 and BPIV-3 in animals such as cows, sheep and goats. Examples of pharmaceutically acceptable carriers or diluents that are useful in the present invention include those that are free of antibodies to BHV-1, ie, about 2.5 to 15% serologically negative for BHV-1. Includes any physiologically buffered medium of about pH 7.0 to 7.4, including serum. Serum without gamma albumin is preferred over serum with gamma globulin. Examples of sera used in the present invention include fetal bovine serum, lamb serum, horse serum, porcine serum, and goat serum. Serum proteins, such as porcine albumin or bovine serum albumin (hereinafter "BSA"), in amounts between about 0.5 and 3.0% can be used as a serum replacement. However, it is desirable to avoid using foreign proteins as carriers or diluents that will induce an allergic reaction in the vaccinated animal. Examples of pharmaceutically acceptable adjuvants are those commonly used to enhance non-replicating antigens, such as oil-in-water, water-in-oil, saponins, and lipopolysaccharide (LPS). The virus contains phosphate buffer, glutamate, cacitone, and sucrose or sorbose, or is diluted in any of the usual stabilizing solutions containing phosphate buffer, lactose, dextran, and glutamate. Is also good. The vaccine virus of the present invention has at least 10 ° C at 70 ° C to -90 ° C. ^Five Or 10 ⁶ It is preferably stored at a titer of PFU / ml or in a lyophilized state at 2 ° C to 7 ° C. The lyophilized virus can be reconstituted for use in sterile distilled water or using an aqueous diluent with a preservative such as gentamicin and amphotericin B or streptomycin. Useful doses to be administered will depend on the age, weight and species of the animal to be vaccinated and the mode of administration. A suitable dose is, for example, about 10 ^4.5 Or 10 ⁷ PFU / animal, preferably 10 ^4.5 Or 10 ^5.5 It may be a PFU. The vaccine of the present invention can be administered intranasally, intravaginally, and intramuscularly. Nasal administration is a more preferred mode of administration for the BHV-1 component and intramuscular administration is a more preferred mode of administration for the baculovirus component. Compounds of the Invention and Preparation of the Compounds Specific methods used in the preparation of the compounds of the present invention are set forth below. These methods will enable any person skilled in the art to reproduce the claimed invention. Alternative methods are possible for these various procedures, including cloning, transformation, tissue culture and immunological detection, and are described in standard laboratory manuals [FM Earthbell (FM) Ausubel et al. (1993) and J. Sambrook et al. (1989)]. These alternatives will also enable those skilled in the art to reproduce the invention. CDNA clones of the BPI-3 HN gene can be obtained by methods well known in the art or by Y. Sakai et al., Journal of Biology (J. Virol.), 63: 3661-8 (1989). And H. Shibata et al., Virology, 155: 688-96 (1986) (hereby incorporated by reference). it can. This cDNA can be obtained by the techniques described above or from Dr. Hiroshi Shibata of the University of Tokyo, but is a shuttle vector for recombination and expression in BHV-1 and baculovirus (BV). Inserted inside. For introduction into BH V-1, the HN gene was transferred to the shuttle vector pHAS4DB X _ex- 1 (this construction is described in this section). The insertion is such that the HN gene disrupts the BHV-1 thymidine kinase gene. For baculovirus expression, the HN gene was transformed with a standard, commercially available shuttle vector (PVL1392, Pharmingen, San Diego, California, CA) under the control of the polyhedron promoter. Insert Following co-transfection of the shuttle plasmid with its respective viral genome, tk-negative BHV-1 and polyhedron-negative BV were isolated, the presence of the HN gene was screened by Southern hybridization, and HN expression was determined by radioimmunoprecipitation. Screen. Cells and viruses Bovine turbinate (BT) cells are available from the American Type Culture Collection (ATCC, Rodkville, MD). BT cells are grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with penicillin, streptomycin, non-essential amino acids and 10% horse serum. Thymidine kinase deficient rabbit skin cells (RabBU) were provided by Saul Kit and Malon Kit; however, any other thymidine kinase deficient cell line that supports the growth of BHV-1 Can also be used to reproduce the invention. tk-deficient cells were grown in DMEM supplemented with penicillin, streptomycin, 10% fetal calf serum, and routinely grown at 100 μg / ml 5-bromo-2′-deoxyuridine (BDUR, Sigma, St. Louis). (St. Louis), Missouri (MO). Spodoptera frugiperda (SF9) cells were grown in Grace's Media (complete) supplemented with penicillin, streptomycin, fungizone and 10% fetal bovine serum. Tissue culture media, supplements and serum were all obtained from Gibco / BRL (Grand Island, NY). The bovine herpesvirus type 1 (BHV-1) strain "Iowa" is a National Animal Diasease Center of the USDA (Ames). ), Iowa). Other strains of BHV-1 can also be used. An SF-4 challenge strain of bovine parainfluenza virus type 3 (BHIV-3) was obtained from the National Veterinary Services Laboratory (Ames, Iowa). The effects of the present invention, however, can be shown against any effective attack model for BPIV-3 disease. Virus was propagated by standard methods well known to those skilled in the art and titers were calculated. Initial plaque isolation was performed under 0.8% agarose and plaque purification was achieved by limiting dilution in 96-well plates. Molecular Cloning and DNA Purification Techniques The methods used for the described molecular cloning techniques are described in standard literature [FM Ausubel et al. (1993) and J. Sambrook. (1989)]. Restriction and modification enzymes are available from New England Biolabs (Beverly, Mass., Mass.), Gibco / BRL, Takara (TaKaRa) Madison, Wis. (WI)) or Boehringer Mannheim Biochemicals (BMB), Indianapolis, Indiana (IN) and can be purchased from suppliers and used as described by the manufacturer. The recombinant plasmid is transformed into an acceptable E. coli, such as DH5α, Frozen acceptable cells are from Gibco / BRL or other donors. The DNA fragment excised from the agarose gel was purchased from Qiaex (Qiagen, Chatworth, CA, CA). )) Or can be purified by other methods.Plasmid DNA can be purified by Qiagen columns (Qiagen) or other methods.BHV-1 DNA is substantially purified Can be purified from infected BT cells on a sodium iodide gradient by the method described by Wallboomers and Ter Shegett (JM Wallboomers et al., Virology, 74: 256-258 (1976)), or can be purified on a small scale. For the preparation of the "mini-prep" method described by Engel et al. (JP Engel et al., Virology, 192: 112-120 (1993)). The DNA hybridization of the viral DNA restriction digests was performed using the digoxigenin / anti-digoxigenin reagent provided by the Genius Kit (BMB). The previously cloned DNA fragment Plasmid DNA was obtained from the BHV-1 HindIII "A" fragment (from Cooper strain) cloned into vector pUC18. This is a subfragment of 0.7 kb. This plasmid can be obtained by methods well known in the art, or by JE Mayfield et al., Incorporated herein by reference. It can be assembled according to the method described by J. Virol, 47: 259-264 (1983). This SalI fragment contains the complete thymidine kinase (tk) gene, the BHV-1 homologue of the HSV-1 UL24 gene, and a small portion of the BHV-1 glycoprotein H gene (LJBello et al. Virology, 189: 407-414 (1992); JG Jacobson et al., Virology, 63: 1839-1843 (1989); ALMayer et al., Biosimica. -Biochim Biophys Acta, 1090: 267-9 (1991), JC Whitbeck et al., Virology, 200: 263-270 (1994)). The 424 bp deletion is made by digesting with BglII and XhoI and blunting the filled ends. This procedure regenerates the BglII site and the resulting plasmid is named pHAS4DBX. Creation of Shuttle Vectors for Expression of Foreign Genes at BHV-1 tk Locus _ex-1 To enable recombination of a foreign gene into the viral thymidine kinase locus. The construction of this vector is described in detail in FIG. Starting at pHAS4, a 2.7 kb BHV-1 SalI fragment containing the complete tk gene and flanking regions on either side. One HindIII restriction enzyme site present in the pHAS4 (cloned into the pUC18 vector, a DNA cloning vector well known to those skilled in the art (J. Sambrook et al., (1989))) was replaced with HindIII. Digested, filled in ends and removed by religation. A 424 bp deletion is made in the tk gene by digesting with the enzymes BglII and XhoI, filling the cohesive ends and religating the resulting blunt ends (this operation regenerates the BglII restriction site), resulting in the plasmid pHAS4DBX . This BglII restriction site is separated from the immediate early promoter of human cytomegalovirus (HCMV) and the polyadenylation signal from bovine growth hormone (these are the only HindIII restriction sites for insertion of the foreign gene to be expressed). (RJ J. Brideau et al., Journal of Genevirol, 74: 471-474 (1993)). This shuttle vector is pHAS4DBX _ex-1 Called. As shown in FIG. 1, the promoter / polyadenylation signal cassette in this vector is inserted in the direction of transcription opposite to that of the original tk gene. The HN gene was a cloned MR2-9tr from the MR strain (Y. Sakai et al., J. Virol., 63: 3661-8 (11989), H. H. Shibuta et al., Virology, 155: 688-96 (198)). This cDNA was cloned completely into the Okayama and Berg type cloning vectors (H. Okayama et al., Molecular and Cellular Biology, 2: 261-170 (1982)). HN mRNA. This HN gene clone can be obtained by methods known in the art or by methods described in Y. Sakai et al., Nucleic Acids Research, 15: 2927-44 (1987). Can be obtained or constructed. Creation of Recombinant BHV-1 and Baculovirus To obtain recombinant BHV-1, a unit length of viral DNA and plasmid containing the sequence of interest was prepared using Graham and Van der Eb et al. RL Graham et al., Virology, 52: 456-467 (1973)) and described by Lawrence et al. (WCLawrence et al., J. Virol). ), 60: 405-14 (1986). Co-transfect BT cells by the calcium phosphate method.Prior to co-transfection, the shuttle plasmid was linearized by digestion with Sse8387I and the resulting lines The denatured fragments were prepared using Qiaex glass matris (Qiagen). Purified from enzyme by use according to the method provided by the manufacturer tk-negative virus resulting from co-transfection is selected by passage twice with RabBU in the presence of 100 ug / ml BDUR. The BDUR-resistant virus is then plaque purified three times by limiting dilution on BT cells (in the absence of BDUR) BaculoGold System (Pharminogen, San Diego) (California, Calif.) Can be used to create a recombinant baculovirus (BV) using the protocol provided by the manufacturer, in which the shuttle plasmid carrying the foreign gene of interest is replaced with a shuttle plasmid. Only genomes rescued by recombination of flanking regions on plasmids are wild-type cells The virus is co-transfected with the defective genome of AcNPV so that it is viable, so that the viral progeny from the transfection need only be plaque purified and screened for expression of the gene of interest. For immunoprecipitation (described later in this section), cells can be infected with BHV-1 or BPIV-3 and SF9 cells can be infected with recombinant BV at a multiplicity of infection of 5-10. For infection of BT cells, normal medium is treated 8% post-infection with 10% cold methionine and 100 uCi / ml. ³⁵ The medium is replaced with a medium containing S-methionine (50 mCI / mMol), Amersham, Arlington Heights, Ill. (IL), and the cells are labeled for an additional 24 hours. For SF9 infected cells, 24 hours post-infection, the medium is 100 UCi / ml. ³⁵ Replace with methionine-free Grace's media containing S-methionine and continue labeling for an additional 24 hours. Neuraminidase Assay Neuraminidase activity was determined by the method of Meyers et al. (RW Meyers et al., RW Meyers), Anal Biochem, 101: 166-174 (1980)). Analyze using '-(4-methylumbelliferyl) -a-DN-acetylneuraminic acid (Sigma). The analysis was performed at pH 5.1, which was performed by Shibuta et al. (H. Shibuta et al., Infect Immun, 41: 780-788 (1983)). HN protein has been shown to be optimal. 225cm in short ^Two BT cells in a flask (about 6 × 10 ⁶ Cells) are infected with the BHV-1 virus of interest at an MOI of 1 and infection is allowed to proceed until 100% CPE is observed (about 36 hours). Spinner flask of SF9 cells (about 5 × 10 ⁷ Cells) with the baculovirus of interest at an MOI of 1 and the infection is allowed to proceed for 48 hours. After harvesting, the various harvested cell pellets were resuspended in 1.0 ml of cold PBS and subjected to three freeze / thaw cycles in a dry ice / ethanol bath, then at 12000 × g for 15 minutes at 4 ° C. Centrifuge at. The resulting supernatant is adjusted to 1 mg / ml using the BCA protein assay (Pierce, Rockford, Ill.). The protein samples (50 ug each) are then incubated with 80 mmole of substrate in 100 μl of a total volume of 50 mM sodium acetate, pH 5.1 at 37 ° C. for 0-60 minutes. The reaction was stopped by the addition of 3.0 mM 250 mM glycine (pH 10.4), and the intensity of the fluorescence was measured using a Perkin Elmer LS50-V luminescence spectrophotometer (Perkin Elmer LS50-V luminescence spectrophotometer). Excitation at 360 nm, slit width of 10.0 nm, emission, 440 nm, 2.5 nm slit width), or with any other suitable instrument. The sodium salt of 4-methylumbelliferone (Sigma) can be used as a standard, and under the conditions described above, a linear range between 0.2 nmole and 10 nmole is obtained. To ³⁵ Infected or mock-infected cells labeled with S-methionine were washed with RIPA buffer (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% deoxycholic acid. (Sodium and 1 mg / ml ovalbumin) for 1 hour at room temperature, then the insoluble fraction is removed by centrifugation at 15 K × g for 10 minutes at room temperature in a microfuge. The clarified lysate was then shaken at 4 ° C. for 1 hour to give 100 μl of protein A Sepharose 4B (Pharmacia, 100 mg / ml Tris-buffered saline, (pH 8.0). After removal of the pre-adsorbed matrix, the lysate is mixed with an appropriate amount of antiserum (usually 20-25 μl, undiluted) and incubated with shaking at 4 ° C. for 2 hours. For each sample, 50 μl of Protein A Sepharose is added and shaking at 4 ° C. is continued for another hour. Thus, the immune complex was adsorbed to protein A sepharose and washed three times with HO buffer (10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1% NP40, and sodium deoxycholate) The immune complex is then dissociated from the matrix by resuspending in 50 μl of SDS sample buffer containing 2-mercaptoethanol (Sigma) and boiling for 5 minutes. The samples are analyzed by SDS-polyacrylamide gel electrophoresis and fluorography. Example The BPIV-3 HN gene is expressed in two heterologous systems; recombinant herpesvirus and recombinant baculovirus are described. The recombinant protein is very similar in antigenicity and electrophoretic mobility to native HN, and retains the expected neuraminidase activity of native NH, as exemplified in the Examples below. Embodiment 1 FIG. Introduction of BPIV-3 HN into BHV-1 and Selection of Recombinant Virus A map of the BPIV-3 HN cDNA clone and its insertion into the BHV-1 shuttle vector is shown in FIG. For cloning into BHV-1, the HN gene is removed from the cDNA cloning vector with HindIII and BamHI (this fragment contains the 3 'polyA tail retained from the cloning of the cDNA and is present in the vector. (Including a small portion of the SV40 polyadenylation region). The HindIII site is 80 bp upstream of the ATG start codon. FIG. 1 After filling the protruding single-stranded ends of the insert generated by BamHI and HindIII, the HN gene was replaced with pHAS4DBX. _ex-1 To the similarly filled HindIII site. Transformation and selection of the insert containing the plasmid determines the proper orientation of the insert relative to the CMV promoter by restriction mapping of the asymmetric site in the HN gene and vector. The linearized BHV-1 shuttle vector containing the BPIV-3 HN gene was transferred to the wild-type BHV-1 tk locus via co-transfection of the plasmid into BT cells and unit length BHV-1 strain "Iowa" DNA. Rearrange. "Io wa" is a highly toxic disease isolate of BHV-1. In addition, the plasmid pHAS4DBX (containing the deleted version of the BHV-1 tk gene without the inserted promoter or gene) is co-transfected with BHV-1 Iowa to obtain a tk deletion control virus without the inserted gene. . Tk-negative progeny from these co-transfections are selected and isolated by passage in RabBU cells in the presence of BDUR. DNA from these viruses is isolated for restriction digestion and Southern hybridization using either the BPIV-3 HN gene or pHAS4 as a probe. Two BHV-1 virus clones, HN16 and HN21, individually hybridized with the HN gene were isolated from BDUR-resistant transfected progeny. This latter virus is BHVD _tk It is referred to as HN in animal experiments (see "Utility of the present invention" below). Some BDUR-resistant transfected progeny containing the tk deletion were also isolated, as assessed by the reduction in the size of the pHAS4 fragment in these clones. A representative virus was isolated and called IowaDtk (BH VD _tk In animal experiments). Embodiment 2. FIG. Transfer of BPIV-3 HN into Baculovirus and Selection of Recombinant Virus For expression in the BV system, we cloned the HN gene into pVL1392, a polyhedrin-based transfer vector. FIG. 2 shows the insertion of NH into this vector. The HN gene is digested with HindIII, the single-stranded protruding ends are filled with Klenow, and the insert is then excised from plasmid vector pSP72 by digestion with BamHI. Plasmid pVL1392 is digested with BglII, the ends are filled with Klenow fragment, and the plasmid is digested with BamHI. The HN insert is ligated through one blunt end and one sticky end to the digested pVL1392 vector, ensuring the correct orientation for expression. The HN gene was properly cloned into pVL1392, but recombines into the AcNPV genome via co-transfection of BaculoGold DNA into SF9 cells. Embodiment 3 FIG. Demonstration of HN protein expression and biological activity in BHV-1 and baculovirus BT cells infected with viruses HN21 and HN16 and SF9 cells infected with baculo-HN show high levels of HN protein as judged by radioimmunoprecipitation. Was expressed. Metabolically labeled infected cell lysates were incubated with polyclonal bovine antiserum to BPIV-3 (NVSL, obtained from Ames, Iowa (IA)). As shown in FIG. 3, along with a representative virus clone purified from BV / HN transfection (lane 6, designated baculo HN), HN21, HN16 (lanes 3 and 4) were native BPIV-3. Expressed large amounts of about 70 KD BPIV-3 immunoreactive protein that appeared to co-migrate with HN (lane 2). This protein is not present in wild-type BHV-1 infected (lane 1) or mock infected BT cells (data not shown), but is also present in SF9 cells infected with inappropriate recombinant baculovirus. No (lane 5). The abundance of NH antigens expressed on BHV-1 and baculovirus made it difficult to accurately size these glycoproteins relative to native NH. However, subsequent radioimmunoprecipitation with BHV-1 and baculovirus expression material showed that BHV-1 HN protein actually migrated in SDS-PAGE with the native HN protein, while baculovirus expression material Showed slightly faster migration (data not shown). We did not perform any kind of glycoconjugate analysis, but we expect that PI-3 and BHV-1 expressing proteins grown in the same cell line will be similarly modified. The smaller size of the baculovirus expressed material is easily explained. This is because insect cells recognize and modify N-linked glycosylation sites, but do not synthesize complex oligosaccharides found in vertebrate cells. To test the biological activity of the BPIV-3 HN protein expressed on BHV-1 and BV, we analyzed infected cell lysates for neuraminidase (NA) activity. Both virus HN21 and baculo HN showed significant NA activity as measured by hydrolysis of 2 '-(4-methylumbelliferyl) -a-DN-acetylneuraminic acid (Figure 4). . This activity was absent in negative controls consisting of BHV-1 IowaDtk virus and recombinant baculovirus expressing BHV-1 gIII protein. Under the conditions we used to isolate the viral proteins and analyze the NA, the SF-4 BPIV-3 strain, which we used as a negative control, did not No activity could be detected. Although the strain MR is known to have significantly higher NA activity compared to other BPIV-3 strains (HS hibuta et al., Infect Immun 41: 780-788 (1983)), This would explain the higher levels of NA activity in our recombinants compared to SF-4. In fact, previous studies have shown that it is very difficult to detect NA activity in some strains of BPIV-3 (H. Shibuta et al., Infect Immun). 34: 262-267 (1981)). However, we can conclude from these results that HN21 and BVHN express biologically active HN proteins. Utility of the Invention The present invention seeks to provide users with an effective vaccine for the prevention of diseases caused by BPIV-3 and BHV-1. The importance of BPIV-3 as a pathogen is its association with the bovine respiratory disease complex (BRDC). The vaccines of the present invention are intended for the treatment of disease, preferably through prevention. To prevent or prevent, the applicant intends to keep the recipient away from the signs of the disease or to reduce the effects of the disease, which is to avoid typical disease states . Prevention refers to the crucial effect of stopping, preventing or delaying the onset of a disease. Prevention can include the following concepts: impede, fail; obstruct, if forbidden, prevent or prevent. "Prevent from happening" indicates that the onset of the disease state does not occur, or that the pathogenesis of the disease is very ineffective in causing the disease state. Prevent or prevent can mean that the condition of the disease is preempted, and the expected action to prevent or prevent the disease has occurred, but the condition that creates the disease is still eliminated. Means no. The utility of the present invention is illustrated by the ability of the vaccine to provide effective protection against BPIV-3 disease. Vaccination for this disease involves inducing strong neutralizing antibodies to the HN glycoprotein. The invention consists of different embodiments including recombinant baculovirus and recombinant bovine herpes virus. These recombinant viruses express the HN protein from bovine parainfluenza virus-3 and thus provide protective efficacy to vaccinated animals. Calves that were poorly fed colostrum at the age of 8 to 12 weeks were kept in separate housing facilities between vaccinations and grouped together before challenge. Prior to vaccination, animals were tested to ensure that they did not have both BVDV and BPIV-3 antibodies. Nineteen animals were divided into five groups. Group 1 (3 animals) received BHV Dtk control virus, group 2 (5 animals) received BHVDtk-HN virus, and group 3 (5 animals) infected with baculo-HN virus. SF9 cells were ingested, group 4 (3 animals) received SF9 cells infected with the negative control baculovirus, and group 5 (3 animals) served as an unvaccinated control. 2 × 10 ⁷ Of animals receiving SF9 cells were pre-infected with recombinant or control baculovirus for 72 hours at two 28-day intervals. The inoculation in a 2 ml volume was administered subcutaneously. The BHV-1 virus was administered nasally, in 2 ml volumes, in 2 ml volumes at 28 day intervals. ⁶ They were given at a TCID50 / calf ratio. All animals were 4 × 10 ⁷ Challenge was performed 42 days after nasal vaccination with BPIV-3. Animals were bled 21 and 42 days after vaccination to estimate serum neutralizing antibody levels against BPIV-3. Following challenge, vaccine efficacy was assessed by measuring the duration and amount of challenge virus released in nasal secretions (isolated from animals using a sterile swab 14 days after challenge). There were no adverse signs to vaccination. Table I shows the reciprocal of the 50% serum neutralizing titers of animals vaccinated with various preparations. Animals 2481, 2478, and 2489 received the BHV-1 control vaccine. Animals 2472, 2493, 2479, 2462, and 2435 were inoculated with the recombinant BHV-HN virus. Animals 2477, 24487, 2476 and 2494 were inoculated with the recombinant baculo-NH virus. Animals 2488, 2480 and 2482 served as baculovirus controls. Animals 2152 and 2175 served as unvaccinated controls. Animals inoculated with the HN recombinant virus show increased levels of serum neutralizing antibodies to BPIV-3, indicating the immunogenicity of both embodiments of the invention. Animals showed little or no signs of clinical illness after challenge. Also, animals inoculated with the HN recombinant virus claimed in the present invention were protected from challenge as indicated by the sustained release of the challenge virus. Table II. Virus release is illustrated by the shaded box for each day after challenge, and the percentage of virus shedding on days 7 and 14 is shown in the right two columns of the table. Three control groups, BHVΔ _tk Baculo and unvaccinated controls were 86% and 95% of the samples taken over the first 7 days with 25% isolation from recombinant baculo and 74% isolation from recombinant herpes. Isolated. This marked decrease in isolation rate was again observed at an isolation rate of 14 days. Also, similar to the reduction in virus isolation rates, the amount of challenge virus found in nasal secretions was reduced compared to controls in calves vaccinated with BHV-1Dtk HN and baculo HN viruses ( Data not shown). Complete description of the drawing FIG. Construction of a shuttle vector for expression of the BPIV-3 HN gene (and other genes) at the BHV-1 tk locus. pHAS4 is a DNA fragment from the BHV-1 genome. The gene and the fragment of the gene encoded in the fragment are found. pHAS4DBX shows a 424 bp deletion made in the tk gene. Label the various cassettes inserted into pHAS4DBX. pHAS 4DBX _ex1 Was made by insertion of the CMV-IE promoter and bovine growth hormone polyadenylation signal into the tk deletion. pHAS4DBX _ex1 :: In HN (bottom), the direction of transcription of HN is indicated by an arrow. FIG. Construction of a recombinant baculovirus transfer vector for expression of the BPIV-3 NH gene in a baculovirus expression system. Top: pVL1392, a transduction vector based on the polyhedrin promoter for expression of foreign genes. Bottom: insertion of the HN gene into this vector. FIG. Immunoprecipitation of infected cell lysates showing expression of BPIV-3 HN from BHV-1 and baculovirus. Immunoprecipitates were precipitated with a polycornal antiserum against BPIV-3 (from bovine). Lanes 1-4 were metabolically labeled infected (or mock infected) BT cells, and lanes 5 and 6 were metabolically labeled infected SF-9 cells. Lane 1: mock infected cells, lane 2: BPIV-3 strain SF4 (HN protein migrates at about 70 kD), lane 3: HN16 (recombinant BHV-1), lane 4: HN21 (recombinant BHV-1) , Lane 5: control baculovirus (inappropriate recombinant), and lane 6: baculo HN. FIG. HN activity in BHV-1 and baculovirus recombinants. The graph shows 4-methyl over time from 2 '-(4-methylumbelliferyl) -a-DN-acetylmeramic acid substrate catalyzed by freeze-thaw lysates of various virus preparations. Figure 2 shows the enzymatic release of umbelliferone (4-MUBF). The X-axis is time in minutes. The Y axis is nanomoles of released 4-MUBF. Key: Open square, HN21 (BHVD _tk -HN); black diamond, baculo HN; open circle, BHVDtk; solid square, baculo-gC (recombinant baculovirus expressing gC protein of BHV-1, used here as negative control); black circle, BPIV- 3. Nanomoles of MUBF released were calculated from a standard curve of 4-MUBF concentration vs. fluorescence intensity (r = 0.999). Deposit of Genetic Material The present invention should not be limited in scope by the deposited materials of the specific examples disclosed herein, which are intended as one illustration of one embodiment of the present invention, Many are functionally equivalent and are within the scope of the present invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are within the scope of the claims. It should also be understood that all base pairs and amino acid residue numbers given to nucleotides and peptides are approximate and are used for the purposes of the description. All publications are incorporated herein by reference. Those skilled in the art will be able to reconstruct all of the various embodiments of the present invention by using only the written description. However, in order to ensure operability for completeness and to provide every opportunity for others to make and use the present invention, certain genetic constructs of the present invention are known under the Budapest Treaty. Deposited at the depositary institution. The virus was the American Type Culture Collection, 12301 Parklone Drive, Rockville, Maryland, Zip Code 20852, USAA (12301, Parklawn Drive, Rockville, Maryland, zip code 20852, USA). Deposited. This deposit was designated as ATCC No. UC VR-59, designated by the Upjohn Company, and given the following number by the depository: VR2478, which corresponds to the virus described herein as "HN21" or "BHVDtk HN". The deposit was accepted by the American Type Culture Collection depositary on September 14, 1994. table

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, M C, NL, PT, SE), OA (BF, BJ, CF, CG , CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, SZ, U G), AL, AM, AT, AU, BB, BG, BR, B Y, CA, CH, CN, CZ, DE, DK, EE, ES , FI, GB, GE, HU, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LT, LU, LV, M D, MG, MK, MN, MW, MX, NO, NZ, PL , PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TT, UA, US, UZ, VN

Claims (1)

[Claims]   1. Any suitable species of bovine herpesvirus type 1 (BHV-1) Hemagglutinin / neuraminidase (HN) glycoprotein inserted and expressed in gene locus A replication non-pathogenic virus consisting of a gene or a combination of genes encoding a quality.   2. From bovine parainfluenza virus type 3 (BPIV-3) Gene or Gene Encoding Hemagglutinin / Neuraminidase (HN) Glycoprotein 2. The virus according to claim 1, comprising a combination of offspring.   3. Hemagglutination inserted and expressed at the thymidine kinase locus of BHV-1 Genes or sets of genes encoding lysin / neuraminidase (HN) glycoproteins 3. The virus according to claim 2, comprising combining.   4. Inserted into any suitable loci of bovine herpesvirus type 1 Encodes and expresses a hemagglutinin / neuraminidase (HN) glycoprotein A bovine virus consisting of a replicating non-pathogenic virus containing a gene or a combination of genes. Prevention of disease caused by influenza virus type-3 Vaccine.   5. From bovine parainfluenza virus type-3 (BPIV-3) Gene or gene encoding hemagglutinin / neuraminidase (HN) glycoprotein The vaccine according to claim 4, comprising a combination of genes.   6. Hemagglutination inserted and expressed at the thymidine kinase locus of BHV-1 Gene or combination of genes encoding elemental / neuraminidase (HN) glycoprotein 6. The vaccine according to claim 5, wherein the vaccine comprises:   7. Bovine hemagglutinin / NO inserted and expressed in the baculovirus expression system From a gene or a combination of genes encoding iraminidase (HN) glycoprotein Become a vaccine.   8. From bovine parainfluenza virus type-3 (BPIV-3) Gene or gene encoding hemagglutinin / neuraminidase (HN) glycoprotein The vaccine according to claim 7, comprising a combination of genes.