HK1161304A - Bovine viral diarrhea virus with a modified erns protein - Google Patents

Description

Bovine viral diarrhea virus with modified ERNS protein

Technical Field

The present invention relates to novel chimeric pestiviruses and their use in immunogenic compositions and vaccines. The invention also relates to methods and kits for treating or preventing the spread of bovine viral diarrhea virus infection. The invention also relates to the use of chimeric pestiviruses in methods and kits for differentiating between vaccinated animals and animals infected with wild-type virus.

Background

Pestiviruses, including bovine viral diarrhea virus (BVD virus or BVDV), have been isolated from several species of animals, both bred and wild. Hosts for BVDV identified include buffalo, antelope, reindeer and various deer species, and unique pestivirus species have been identified in giraffes and pronghorn antelope. BVDV is a small RNA virus of the flaviviridae family. It is closely related to other pestiviruses, which are causative agents of border disease in sheep and swine fever (classical swine farm) in pigs. Recently, a different pestivirus, called the bungowanah pestivirus, was identified as the causative agent of australian piglet foetal infection.

Diseases caused by BVDV, especially in cattle, are very widespread and can bring about economic damage. BVDV infection in cattle can cause breeding problems and can lead to abortion or premature birth. BVDV is able to cross the placenta of pregnant cows and can Persistently Infect (PI) calves, making them immune-tolerant to the virus and sustaining viremia throughout the life thereafter. Infected cattle may also exhibit "mucosal disease" characterized by elevated body temperature, diarrhea, coughing, and ulceration of the digestive mucosa. These persistently infected animals provide a source of transmission of viruses in herds that cause further outbreaks of mucosal disease and are highly susceptible to infection by microorganisms that cause intestinal disease or pneumonia.

BVDV is divided into two biotypes. BVDV of the "cp" biotype induces cytopathic effects in cultured cells, whereas viruses of the non-cytopathic or "ncp" biotype do not. In addition, two major genotypes (type 1 and type 2) have been identified, both of which have been shown to cause a variety of clinical symptoms.

The BVDV virions have a diameter of 40-60 nm. The nucleocapsid of BVDV consists of a single RNA molecule and capsid protein C. The nucleocapsid is surrounded by a lipid membrane to which two glycoproteins, E1 and E2, are anchored. Glycoprotein E of type 3^rnsLoosely associated with the envelope. The BVDV genome is approximately 12.5kb in length, comprising a single open reading frame located between the 5 'and 3' untranslated regions (NTRs). A polyprotein of approximately 438kD is translated from this open reading frame and processed by cellular and viral proteases into at least 11 viral structural and non-structural (NS) proteins (Tautz, et al, J.Virol.71: 5415-. The genomic order of BVDV is p20/N^pro、p14/C、gp48/E^rnsGp25/E1, gp53/E2, p54/NS2, p80/NS3, p10/NS4A, p32/NS4B, p58/NS5A and p75/NS 5B. Three envelope proteins gp48/E^rnsGp25/E1 and gp53/E2 are highly glycosylated. E^rns(previously referred to as E0 or gp48) form homodimers covalently linked by disulfide bonds. Absence of the hydrophobic Membrane anchoring region illustrates E^rnsLoosely associated with the envelope. E^rnsHigh antibody titers were induced in infected cattle, but antisera had limited virus-neutralizing activity.

Currently available BVDV vaccines are: a vaccine comprising a chemically inactivated wild-type virus. These vaccines typically require multiple doses to be administered and generate a short-term immune response; in addition, they do not provide protection against fetal transmission of the virus. In sheep, subunit vaccines based on purified E2 protein have been reported. Although this vaccine appears to protect the fetus from infection, protection is limited to homologous strains of the virus, and there is no correlation between antibody titers and protection.

Modified Live (ML) BVDV vaccines have been produced using viruses that have been attenuated by repeated passages in bovine or porcine cells or by chemical induction of mutations that confer a temperature sensitive phenotype on the virus. It has been demonstrated that a single dose of MLV BVDV vaccine is sufficient to provide protection against infection, and the duration of immunity in vaccinated cattle can extend for years. In addition, cross-protection using MLV vaccines has been reported (Martin et al, see "Proceedings of the Conference of research works in Animal Diseases", 75: 183 (1994)). However, existing MLV vaccines do not distinguish between vaccinated and naturally infected animals.

Thus, there is a clear need for new vaccines for controlling BVDV transmission. Such vaccines would be invaluable in future national or regional BVDV clearance projects, and could also be combined with other bovine vaccines, which represent a substantial advance in the industry. A more effective vaccine for controlling and monitoring BVDV transmission is a "marked" vaccine. Such vaccines may comprise additional antigenic determinants that are not present in the wild-type virus, or lack antigenic determinants that are present in the wild-type virus. In the former case, vaccinated animals produce an immune response against the "marker" immunogenic determinant, whereas non-vaccinated animals do not. By using immunological tests against the marker determinant, vaccinated animals can be distinguished from non-vaccinated naturally infected animals by the presence or absence of antibodies against the marker determinant. In the case of the latter strategy, animals infected with the wild-type virus develop an immune response against the marker determinant, as the determinant is not present in the labeled vaccine, whereas non-infected vaccinated animals do not. Infected animals can be distinguished from vaccinated, uninfected animals by using immunological tests for marker determinants. In both cases, by culling infected animals, the herd may become a BVDV-free herd over time. In addition to the benefit of removing the threat of BVDV disease, the evidence of BVDV-free herd has direct free-trade economic benefits.

Disclosure of Invention

In one embodiment, the present invention provides a chimeric pestivirus, wherein said chimeric pestivirus comprises no E homologous thereto^rnsA bovine viral diarrhea virus of protein, and wherein said chimeric pestivirus expresses a heterologous E derived from another pestivirus^rnsProtein, or said heterologous E^rnsNatural, synthetic or genetic variants of the protein.

In another embodiment, the present invention provides a chimeric pestivirus as described above, wherein said chimeric pestivirus is heterologous to E^rnsProtein or said heterologous E^rnsA natural, synthetic or genetic variant of a protein is derived from a pestivirus selected from the group consisting of reindeer pestivirus, giraffe pestivirus and pronghorn antelope pestivirus.

In a different embodiment, the present invention provides a chimeric pestivirus as described above, wherein said chimeric pestivirus is heterologous to E^rnsThe protein has at least one E which is not present in wild type bovine viral diarrhea virus^rnsAn epitope.

In a separate embodiment, the present invention provides a chimeric pestivirus as described above, wherein said chimeric pestivirus is heterologous to E^rnsProtein deletion of at least one E present in wild-type bovine viral diarrhea virus^rnsAn epitope.

In one embodiment, the present invention provides a culture of the chimeric pestivirus as described above.

In another embodiment, the present invention provides a cell line or host cell comprising the chimeric pestivirus as described above.

In another embodiment, the present invention provides a polynucleotide molecule encoding a chimeric pestivirus as described above.

In various embodiments, the present invention provides an immunogenic composition comprising the chimeric pestivirus as described above and a veterinarily acceptable carrier.

In a separate embodiment, the present invention provides the immunogenic composition as described above, wherein the veterinarily acceptable carrier is an adjuvant.

In another embodiment, the present invention provides an immunogenic composition as described above, wherein said chimeric pestivirus is live attenuated.

In another embodiment, the present invention provides an immunogenic composition as described above, wherein said chimeric pestivirus is inactivated.

In various embodiments, the invention provides an immunogenic composition as described above, further comprising one or more additional antigens for treating or preventing the spread of one or more additional pathogenic microorganisms in an animal.

In a separate embodiment, the present invention provides an immunogenic composition comprising a polynucleotide molecule encoding a chimeric pestivirus as described above and a veterinarily acceptable carrier.

In one embodiment, the present invention provides a vaccine comprising the chimeric pestivirus as described above and a veterinarily acceptable carrier.

In another embodiment, the present invention provides a vaccine as described above, wherein the veterinarily acceptable carrier is an adjuvant.

In another embodiment, the present invention provides a vaccine as described above, wherein said chimeric pestivirus is live attenuated.

In another embodiment, the present invention provides a vaccine as described above, wherein said chimeric pestivirus is inactivated.

In another embodiment, the invention provides a vaccine as described above, further comprising one or more additional antigens for treating or preventing the spread of one or more additional pathogenic microorganisms in an animal.

In a separate embodiment, the present invention provides a vaccine comprising a polynucleotide molecule encoding a chimeric pestivirus as described above and a veterinarily acceptable carrier.

In one embodiment, the present invention provides a kit comprising, in at least one container, a vaccine comprising a chimeric pestivirus as described above.

In another embodiment, the invention provides a method of treating or preventing the spread of a bovine viral diarrhea virus infection, wherein a vaccine comprising the chimeric pestivirus as described above is administered to an animal.

In various embodiments, the present invention provides a method of vaccinating an animal, wherein a DIVA pestivirus vaccine is administered to said animal, wherein said DIVA pestivirus vaccine comprises a chimeric pestivirus as described above, and wherein said chimeric pestivirus has at least one E that is not present in wild-type bovine viral diarrhea virus^rnsAn epitope.

In a separate embodiment, the present invention provides a method of vaccinating an animal, wherein a DIVA pestivirus vaccine is administered to said animal, wherein said DIVA vaccine comprises a chimeric pestivirus as described above, and wherein said chimeric pestivirus lacks at least one E present in wild-type bovine viral diarrhea virus^rnsAn epitope.

In another embodiment, the present invention provides a method of differentiating vaccinations comprisingA method for vaccinating an animal against said chimeric pestivirus against an animal infected with wild-type bovine viral diarrhea virus, wherein said vaccinated animal produces antibodies against at least one E present in the chimeric pestivirus of said vaccine but not present in wild-type bovine viral diarrhea virus^rnsAn epitope, the method comprising the steps of:

a) obtaining a serum sample from an animal;

b) determining the sample for the presence or absence of the antibody;

c) identifying an animal having the antibody as having been vaccinated with the vaccine; and

d) animals not containing the antibody were identified as having been infected with wild-type BVDV.

In another embodiment, the invention provides a method of differentiating between an animal infected with wild-type bovine viral diarrhea virus and an animal vaccinated with a vaccine comprising the chimeric pestivirus as described above, wherein the animal infected with wild-type bovine viral diarrhea virus produces antibodies to at least one E present in wild-type bovine viral diarrhea virus but not present in the chimeric pestivirus of the vaccine^rnsAn epitope, the method comprising the steps of:

a) obtaining a serum sample from an animal;

b) determining the sample for the presence or absence of the antibody;

c) identifying an animal having the antibody as having been infected with wild-type BVDV; and

d) identifying an animal that does not contain the antibody as having been vaccinated with the vaccine.

In one embodiment, the invention provides a diagnostic kit for differentiating between an animal vaccinated with a vaccine comprising a chimeric pestivirus as described above and an animal infected with wild-type bovine viral diarrhea virus, said kit comprising a nucleic acid sequence capable of detecting a viral diarrhea virus directed against at least one of the viruses of bovineAn E which is present in the chimeric pestivirus of said vaccine but which is not present in wild-type bovine viral diarrhea virus^rnsAn antibody to the epitope.

In another embodiment, the invention provides a diagnostic kit for differentiating between an animal infected with wild-type bovine viral diarrhea virus and an animal vaccinated with a vaccine comprising the chimeric pestivirus as described above, said kit comprising means capable of detecting a viral pathogen that is not present in the chimeric pestivirus of said vaccine but is present in the wild-type bovine viral diarrhea virus^rnsAn antibody to the epitope.

In another embodiment, the present invention provides a method of identifying E present in a chimeric pestivirus as described above but absent from wild-type bovine viral diarrhea virus^rnsAn antibody to the epitope.

In various embodiments, the invention provides antibodies that recognize an epitope that is present in wild-type bovine viral diarrhea virus but is not present in the chimeric pestivirus as described above.

In another embodiment, the chimeric pestivirus as described herein is used in the manufacture of a medicament for the prevention or treatment of an infection by BVDV.

Detailed Description

The following definitions apply to terms used in the description of embodiments of the present invention. The following definitions supersede any conflicting definition contained in each individual reference incorporated herein by reference.

Unless otherwise indicated herein, scientific and technical terms used in the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

The term "amino acid" as used herein refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that have been subsequently modified, e.g., hydroxyproline, carboxyglutamic acid, and O-phosphoserine. Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), unnatural amino acids such as alpha and alpha-disubstituted amino acids, N-alkyl amino acids, lactic acid and other unconventional amino acids may also be suitable components of the polypeptides of the invention. Examples of unconventional amino acids include: 4-hydroxyproline, gamma-carboxyglutamic acid, epsilon-N, N, N-trimethyllysine, epsilon-N-acetyl lysine, O-phosphoserine, N-acetyl serine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, sigma-N-methylarginine and other similar amino acids and imino acids.

Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., have a carbon atom bound to a hydrogen, a carboxyl group, an amino group, and an R group. Exemplary amino acid analogs include, for example, homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain substantially the same chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by the well-known three-letter or one-letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission.

The term "animal" as used herein is intended to include any animal susceptible to BVDV, including, but not limited to, the following species: cattle, sheep, goats and pigs, including raised and wild.

The term "antibody" as used herein refers to an immunoglobulin molecule capable of binding an antigen by means of recognizing an epitope. The antibodies may be polyclonal mixtures or monoclonal. The antibody may beAn intact immunoglobulin from a natural source or from a recombinant source, or may be an immunoreactive portion of an intact immunoglobulin. Antibodies can exist in a variety of forms, including, for example, Fv, Fab ', F (ab')₂And single stranded forms.

The term "antigen" as used herein refers to a molecule comprising one or more epitopes (linear, conformational or both) that, when exposed to a subject, will induce an immune response specific for that antigen. The term "antigen" may refer to an attenuated, inactivated or modified live bacterium, virus, fungus, parasite or other microorganism. The term "antigen" as used herein may also refer to a subunit antigen that is separate and isolated from the whole organism to which the antigen binds in its natural state. The term "antigen" may also refer to antibodies, such as anti-idiotypic antibodies or fragments thereof, as well as synthetic peptidomimetic positions that can mimic an antigen or antigenic determinant (epitope). The term "antigen" may also refer to oligonucleotides or polynucleotides that express an antigen or antigenic determinant in vivo, e.g., for DNA immunization applications.

The term "BVDV", "BVDV isolate" or "BVDV strain" as used herein refers to bovine viral diarrhea virus, including but not limited to type I and type II, which consists of the viral genome, associated proteins and other chemical components (e.g., lipids). A variety of bovine viral diarrhea viruses type I and I I are known to those of skill in the art and may be obtained, for example, from the American type culture CollectionAnd (4) obtaining. Bovine viral diarrhea virus has a genome in the form of RNA. RNA can be reverse transcribed into DNA for cloning. Thus, reference herein to nucleic acid and bovine viral diarrhea virus sequences includes viral RNA sequences and DNA sequences derived from viral RNA sequences.

The term "cell line" or "host cell" as used herein means a prokaryotic or eukaryotic cell in which a virus can replicate and/or be maintained.

The term "chimeric" or "chimera" as used herein means a microorganism, such as a virus, that comprises genetic or physical components derived from more than one ancestor.

The term "culture" as used herein means a population of cells or microorganisms that grow in the absence of other species or types.

The term "DIVA" as used herein means a vaccine capable of distinguishing infected from vaccinated animals.

An "epitope" is a specific site of an antigen that binds to a T cell receptor or specific antibody, and typically comprises from about 3 amino acid residues to about 20 amino acid residues.

The term "heterologous" as used herein means derived from a different species or strain.

The term "homologous" as used herein means derived from the same species or strain.

The term "immunogenic composition" as used herein means a composition that, when administered to an animal, alone or in combination with a pharmaceutically acceptable carrier, generates an immune response (i.e., has immunogenic activity). The immune response may be a cellular immune response mediated primarily by cytotoxic T cells, or a humoral immune response mediated primarily by helper T cells, which in turn activate B cells, resulting in the production of antibodies.

The term "pathogen" or "pathogenic microorganism" as used herein means a microorganism, such as a virus, bacterium, fungus, protozoan or parasite, capable of inducing or causing a disease, illness or abnormal state in its host animal.

The term "pestivirus" as used herein means an RNA virus from the genus pestivirus (pestivirus) of the family Flaviviridae (Flaviviridae). Pestiviruses include, but are not limited to BVDV (type 1 and type 2), swine fever virus (CSFV) and Border Disease Virus (BDV), as well as pestiviruses isolated from, for example, the following species: boars, buffalos, great antelopes (eland), bison, alpaca, pudu, agogue antelope (bongo), various deer, giraffe, reindeer, antelope, and pronghorn antelope (Vilcek and Netteton; Vet Microbiol.116: 1-12 (2006)).

The term "polynucleotide molecule" as used herein means an organic polymer molecule consisting of nucleotide monomers covalently bonded into a chain. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are examples of polynucleotides with unique biological functions.

The terms "prevent", "preventing" or "prevention" and the like as used herein mean inhibiting the replication of a microorganism, inhibiting the spread of a microorganism, or inhibiting the self-establishment of a microorganism in its host. These terms and the like as used herein may also mean inhibiting or blocking one or more signs or symptoms of infection.

The term "therapeutically effective amount" as used herein means an amount of a microbial or subunit antigen or polypeptide or polynucleotide molecule or combination thereof sufficient to elicit an immune response in a subject to which it is administered. The immune response may include, but is not limited to, induction of cellular and/or humoral immunity.

The terms "treatment", "treating" or "treatment" and the like as used herein mean reducing or eliminating infection by a microorganism. These terms and the like as used herein may also mean reducing replication of the microorganism, reducing spread of the microorganism, or reducing the ability of the microorganism to self-establish in its host. These terms and the like as used herein may also mean reducing, alleviating or eliminating one or more signs or symptoms of a microbial infection, or accelerating recovery from a microbial infection.

The terms "vaccine" and "vaccine composition" as used herein mean a composition that prevents or reduces infection, or prevents or reduces one or more signs or symptoms of infection. The protective effect of a vaccine composition against a pathogen is typically achieved by inducing an immune response (which may be cell-mediated or humoral or a combination of both) in the subject. In general, elimination or reduction of the incidence of infection, alleviation of signs or symptoms, or accelerated clearance of microorganisms from infected subjects is indicative of the protective effect of the vaccine composition. The vaccine compositions of the present invention provide a protective effect against BVDV-caused infections.

The term "variant" as used herein refers to a derivative of a given protein and/or gene sequence, wherein the derived sequence is substantially identical to the given sequence, but differs by mutation. The difference may be naturally occurring, or produced synthetically or genetically.

The term "veterinarily acceptable carrier" as used herein refers to a substance which is, within the scope of sound medical judgment, suitable for use in contact with the tissues of animals without excessive toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for its intended use.

The following description is provided to assist those skilled in the art in carrying out the invention. Even so, this description should not be construed as unduly limiting this invention since modifications and variations to the embodiments discussed herein may be made by those of ordinary skill in the art without departing from the spirit or scope of this invention.

Viruses, immunogenic compositions and vaccines

The present invention provides immunogenic compositions and vaccines comprising one or more chimeric pestiviruses, wherein said chimeric pestivirus comprises an E that does not express its homologue^rnsA bovine viral diarrhea virus of protein, and wherein said chimeric pestivirus expresses a heterologous E derived from another pestivirus^rnsProtein, or said heterologous E^rnsNatural, synthetic or genetic variants of the protein. The chimeric pestivirus may be selected from, but is not limited to, pestivirus chimeras of BVDV/reindeer pestivirus, BVDV/giraffe pestivirus and BVDV/pronghorn antelope pestivirus.

In one embodiment, the BVDV/Giraffe chimeric pestivirus is deposited with the American type culture Collection(10801 University Boulevard, Manassas, VA20110-2209, USA) strain UC 25547, whichThe product is deposited as PTA-9938. In one embodiment, the BVDV/pronghorn chimeric pestivirus is deposited withStrain UC 25548 of (1), whichThe deposit number is PTA-9939. In one embodiment, the BVDV/reindeer chimeric pestivirus is deposited withStrain UC 25549 of (1), whichThe product is deposited as PTA-9940.

The chimeric pestiviruses of the invention can be propagated in cells, cell lines and host cells. The cells, cell lines, and host cells can be, for example, but are not limited to, mammalian cells and non-mammalian cells, including insect and plant cells. Cells, cell lines and host cells useful for propagation of the chimeric pestiviruses of the invention are readily known and available to those of ordinary skill in the art.

The chimeric pestiviruses of the invention may be attenuated or inactivated prior to use in an immunogenic composition or vaccine. Methods of attenuation and inactivation are well known to those skilled in the art. Methods for attenuation include, but are not limited to, serial passage in cell culture in a suitable cell line, ultraviolet radiation, and chemical mutagenesis. Methods for inactivation include, but are not limited to, treatment with formalin, Beta Propiolactone (BPL), or diethylene imine (BEI), or other methods known to those skilled in the art.

Inactivation using formalin may be performed as follows: the virus suspension was mixed with 37% formaldehyde to reach a final formaldehyde concentration of 0.05%. The virus-formaldehyde mixture was mixed at room temperature for about 24 hours with constant stirring. The inactivated virus mixture is then tested for residual live virus by measuring growth on a suitable cell line.

Inactivation using BEI may be performed as follows: the viral suspension of the invention was mixed with 0.1M BEI (2-bromo-ethylamine in 0.175N NaOH) to reach a final BEI concentration of 1 mM. The virus-BEI mixture was mixed at room temperature for approximately 48 hours with constant stirring, and then 1.0M sodium thiosulfate was added to a final concentration of 0.1 mM. Mixing for another 2 hours. Residual live virus in the inactivated virus mixture was detected by measuring growth on a suitable cell line.

The immunogenic compositions and vaccines of the invention may include one or more veterinarily acceptable carriers. As used herein, "veterinarily acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and the like. Diluents may include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents may include, inter alia, sodium chloride, glucose, mannitol, sorbitol, lactose and the like as known to those skilled in the art. Stabilizers include, inter alia, albumin and the like known to those skilled in the art. Preservatives include, inter alia, thimerosal, and the like, known to those skilled in the art.

Adjuvants include, but are not limited to, RIBI adjuvant system (Ribi Inc.), alum, aluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as Freund's complete adjuvant and Freund's incomplete adjuvant, block copolymers (CytRx, Atlanta Ga.), SAF-M (Chiron, Emeryville Calif.), among others as known to those skilled in the art,Adjuvants, saponins, Quil A, QS-21(Cambridge Bio)tech inc., Cambridge Mass.), GPI-0100 (galenicals, inc., Birmingham, AL) or other saponin fractions, monophosphoryl lipid a, an avridine lipid-amine adjuvant, heat-labile enterotoxin (recombinant or otherwise) from e. The amounts and concentrations of adjuvants and additives used in the context of the present invention can be readily determined by the skilled person. In one embodiment, the invention relates to immunogenic compositions and vaccines comprising about 50 μ g to about 2000 μ g of adjuvant. In another embodiment, the amount of adjuvant included is from about 100 μ g to about 1500 μ g, or from about 250 μ g to about 1000 μ g, or from about 350 μ g to about 750 μ g. In another embodiment, the amount of adjuvant included is about 500 μ g/2ml dose of the immunogenic composition or vaccine.

The immunogenic compositions and vaccines may also comprise an antibiotic. Such antibiotics include, but are not limited to, the following classes of antibiotics: aminoglycosides, carbapenems, cephalosporins, glycopeptides, macrolides, penicillins, polypeptides, quinolones, sulfonamides, and tetracyclines. In one embodiment, the invention relates to immunogenic compositions and vaccines comprising from about 1 μ g/ml to about 60 μ g/ml antibiotic. In another embodiment, the immunogenic compositions and vaccines comprise from about 5 μ g/ml to about 55 μ g/ml antibiotic, or from about 10 μ g/ml to about 50 μ g/ml antibiotic, or from about 15 μ g/ml to about 45 μ g/ml antibiotic, or from about 20 μ g/ml to about 40 μ g/ml antibiotic, or from about 25 μ g/ml to about 35 μ g/ml antibiotic. In another embodiment, the immunogenic compositions and vaccines comprise less than about 30 μ g/ml antibiotic.

The immunogenic compositions and vaccines of the invention may also comprise one or more other immunostimulants, for example interleukins, interferons or other cytokines, the appropriate amount of which may be determined by the skilled person.

The immunogenic compositions and vaccines of the invention can comprise one or more polynucleotide molecules encoding the chimeric pestivirus. The immunogenic composition or vaccine may comprise a DNA or RNA molecule encoding the entire genome or one or more open reading frames of the chimeric pestivirus. The DNA or RNA molecule can be administered in the absence of other agents, or can be administered with agents that promote cellular uptake (e.g., liposomes or cationic lipids). The total polynucleotides in the immunogenic composition or vaccine is generally about 0.1. mu.g/ml to about 5.0 mg/ml. In another embodiment, the total polynucleotides in the immunogenic composition or vaccine is about 1 μ g/ml to about 4.0mg/ml, or about 10 μ g/ml to about 3.0mg/ml, or about 100 μ g/ml to about 2.0 mg/ml. Vaccines and vaccination procedures using nucleic acids (DNA or mRNA) are widely described in the art, for example, U.S. Pat. No. 5,703,055, U.S. Pat. No. 5,580,859, U.S. Pat. No. 5,589,466, which are all incorporated herein by reference.

The immunogenic compositions and vaccines of the invention may also comprise additional BVDV antigens, such as those described in U.S. patent 6,060,457, U.S. patent 6,015,795, U.S. patent 6,001,613, and U.S. patent 5,593,873, all of which are incorporated herein by reference.

In addition to one or more chimeric pestiviruses, the immunogenic compositions and vaccines can comprise other antigens. The antigen may be in the form of an inactivated whole or partial preparation of the microorganism, or may be in the form of an antigenic molecule obtained by genetic engineering techniques or chemical synthesis. Other antigens suitable for use in the present invention include, but are not limited to, antigens derived from pathogenic bacteria, for example, Haemophilus somnus, Haemophilus parasuis (Haemophilus parasuis), Bordetella bronchiseptica (Bordetella bronopolica), Bacillus anthracis (Bacillus antrhacis), Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae), Pasteurella multocida (Pasteurella multocida), Salmonella mannheimica (Mannheimia haemolytica), Mycoplasma bovis (Mycoplasma bovis), Mycobacterium bovis (Mycobacterium bovis), Mycobacterium paratuberculosis (Mycobacterium paratuberculosis), Clostridium (Clostridium clostridia), Streptococcus uberis (Streptococcus uberculosis), Staphylococcus aureus (Staphylococcus aureus), Salmonella suis (Salmonella typhi), Salmonella typhi, and Salmonella typhi. Antigens may also be derived from pathogenic fungi such as Candida (Candida), protozoa such as Cryptosporidium parvum (Cryptosporidium parvum), Neospora caninum (Neospora canium), Toxoplasma gondii (Toxoplasma gondii), Eimeria spp (Eimeria spp.), Babesia spp, Giardia spp (Giardia spp.), or parasites such as oesophagus spp (Ostertagia), Cooperia spp (Cooperia), Haemonchus spp (Haemonchus) and schistosoma spp (Fasciola). Additional antigens include pathogenic viruses, e.g., bovine coronavirus, bovine herpes virus-1, 3, 6, bovine parainfluenza virus, bovine respiratory syncytial virus, bovine leukemia virus, rinderpest virus, foot and mouth disease virus, rabies virus, and influenza virus.

Form, dosage, route of administration

The immunogenic compositions and vaccines of the invention can be administered to animals to induce an effective immune response against BVDV. Accordingly, the present invention provides methods of stimulating an effective immune response against BVDV by administering to an animal a therapeutically effective amount of an immunogenic composition or vaccine of the invention described herein.

The immunogenic compositions and vaccines of the present invention can be prepared in a variety of forms depending on the route of administration. For example, immunogenic compositions and vaccines can be prepared in the form of sterile aqueous solutions or dispersions suitable for injectable use, or in lyophilized form using freeze-drying techniques. Lyophilized immunogenic compositions and vaccines are typically maintained at about 4 ℃, and can be reconstituted in a stabilizing solution such as saline or and HEPES (with or without adjuvant). The immunogenic compositions and vaccines can also be prepared in the form of suspensions or emulsions.

The inventionThe immunogenic compositions and vaccines of (a) comprise a therapeutically effective amount of one or more chimeric pestiviruses as described above. The purified virus may be used directly in an immunogenic composition or vaccine, or may be further attenuated or inactivated. Immunogenic compositions or vaccines typically comprise about 1 × 10²To about 1X 10¹²Individual virus particles, or about 1X 10³To about 1X 10¹¹Individual virus particles, or about 1X 10⁴To about 1X 10¹⁰Individual virus particles, or about 1X 10⁵To about 1X 10⁹Individual virus particles, or about 1X 10⁶To about 1X 10⁸And (c) viral particles. The precise amount of virus in an immunogenic composition or vaccine effective to provide a protective effect can be determined by the skilled artisan.

Immunogenic compositions and vaccines typically comprise a veterinarily acceptable carrier in a volume of about 0.5ml to about 5 ml. In another embodiment, the volume of the carrier is from about 1ml to about 4ml, or from about 2ml to about 3 ml. In another embodiment, the volume of the carrier is about 1ml or about 2ml, or about 5 ml. Veterinary acceptable carriers suitable for use in the immunogenic compositions and vaccines can be any of those described above.

One skilled in the art can readily determine whether a virus requires attenuation or inactivation prior to administration. In another embodiment of the invention, the chimeric pestivirus can be administered directly to the animal without additional attenuation. The therapeutically effective amount of the virus may vary depending on the particular virus used, the condition of the animal, and/or the extent of infection, and may be determined by the skilled artisan.

According to the methods of the invention, a single dose may be administered to the animal, or alternatively, 2 or more vaccinations may be performed at intervals of about 2 to about 10 weeks. A booster regimen may be required and the dosage regimen may be adjusted to provide optimal immunization. Optimal dosing regimens can be readily determined by one skilled in the art.

The immunogenic compositions and vaccines can be administered directly into the bloodstream, into muscle, or into internal organs. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) syringes, needleless injectors and infusion techniques.

Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffers (preferably to a pH of from about 3 to about 9, or from about 4 to about 8, or from about 5 to about 7.5, or from about 6 to about 7.5, or from about 7 to about 7.5), but for some applications may more suitably be formulated as a sterile non-aqueous solution or in dry form for use with a suitable medium such as sterile, pyrogen-free water.

The preparation of parenteral formulations under sterile conditions (e.g., by lyophilization) can be readily carried out using standard pharmaceutical techniques well known to those skilled in the art.

The solubility of the compounds used to prepare the parenteral solutions can be increased by using suitable formulation techniques known to the skilled person, for example incorporating solubility enhancing agents such as buffers, salts, surfactants, liposomes, cyclodextrins and the like.

Formulations for parenteral administration may be formulated for immediate and/or modified release. Modified release formulations include delayed release, sustained release, pulsatile release, controlled release, targeted release and programmed release. Thus, the compounds of the present invention may be formulated as solids, semi-solids or thixotropic liquids for application as an implant depot, which provides for modified release of the active compound. Examples of such formulations include drug-coated stents and poly (dl lactic-co-glycolic acid) copolymer (PGLA) microspheres.

The immunogenic compositions and vaccines of the invention may also be administered topically, i.e., transdermally or transdermally, to the skin or mucosa. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, pastes, dusting powders, dressings, foams, films, patches, wafers, implants, sponges, fibers, bandages and microemulsions. Liposomes may also be used. Typical carriers include alcohols, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol, and propylene glycol. Penetration enhancers may be incorporated. See, e.g., Finnin and Morgan, j.pharm Sci, 88 (10): 955-958(1999).

Other topical application modes include: by electroporation, iontophoresis, phonophoresis, sonophoresis and microneedles or needleless (e.g. Powderject)^TM、Bioject^TMEtc.) delivery by injection.

Formulations for topical administration may be formulated for immediate and/or modified release. Modified release formulations include delayed release, sustained release, pulsatile release, controlled release, targeted release and programmed release.

Immunogenic compositions and vaccines can also be administered intranasally or by inhalation, typically in the form of a dry powder from a dry powder inhaler (alone or as a mixture, e.g. dry blended with lactose, or as a mixed component particle, e.g. blended with a phospholipid such as phosphatidylcholine), or as an aerosol spray from a pressurised container, pump, nebuliser (preferably one using electrohydrodynamics to provide a fine mist) or nebuliser, with or without the use of a suitable propellant, e.g. 1, 1, 1, 2-tetrafluoroethane or 1, 1, 1, 2, 3, 3, 3-heptafluoropropane. For intranasal administration, the powder may comprise a bioadhesive agent, such as chitosan or cyclodextrin.

Pressurized containers, pumps, sprayers, atomizers or sprinklers contain a solution or suspension of a compound of the invention comprising, for example, ethanol, aqueous ethanol or a suitable alternative agent for dispersing, dissolving or delaying the release of the active, a booster as solvent and optionally a surfactant, such as sorbitan trioleate, oleic acid or oligomeric lactic acid.

Prior to use in dry powder or suspension formulations, the drug product is typically micronized to a size suitable for delivery by inhalation (typically less than about 5 microns). This may be achieved by any suitable comminution method, for example, spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization or spray drying.

Capsules (e.g. prepared from gelatin or hydroxypropylmethyl cellulose), blister packs and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention, a suitable powder base such as lactose or starch and a performance modifying agent such as 1-leucine, mannitol or magnesium stearate. Lactose can be in anhydrous or monohydrate form. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.

Suitable solution formulations for nebulizers (which use electrohydrodynamics to generate fine mist) may contain from about 1 μ g to about 20mg of the compound of the invention per actuation, and the actuation volume may be from about 1 μ l to about 100 μ l. In another embodiment, the amount of compound per actuation may be from about 100 μ g to about 15mg, or from about 500 μ g to about 10mg, or from about 1mg to about 10mg, or from about 2.5 μ g to about 5 mg. In another embodiment, the priming volume may be from about 5. mu.l to about 75. mu.l, or from about 10. mu.l to about 50. mu.l, or from about 15. mu.l to about 25. mu.l. A typical formulation may comprise a compound of the invention, propylene glycol, sterile water, ethanol and sodium chloride. Alternative solvents that may be used in place of propylene glycol include glycerol and polyethylene glycol.

Formulations for inhalation/intranasal administration may be formulated for immediate and/or modified release using, for example, PGLA. Modified release formulations include delayed release, sustained release, pulsatile release, controlled release, targeted release and programmed release.

In the case of dry powder inhalers and aerosols, the dosage unit is generally determined by delivering a metered number of valves. The units according to the invention are generally arranged to administer a metered dose or "puff" comprising from about 10ng to about 100. mu.g of a compound of the invention. In another embodiment, the amount of compound administered in a metered dose is from about 50ng to about 75 μ g, or from about 100ng to about 50 μ g, or from about 500ng to about 25 μ g, or from about 750ng to about 10 μ g, or from about 1 μ g to about 5 μ g. The total daily dose is typically from about 1 μ g to about 100mg, which may be administered as a single dose, or, more typically, as separate doses throughout the day. In another embodiment, the total daily dose may be from about 50 μ g to about 75mg, or from about 100 μ g to about 50mg, or from about 500 μ g to about 25mg, or from about 750 μ g to about 10mg, or from about 1mg to about 5 mg.

The immunogenic compositions and vaccines of the invention may also be administered orally or perorally, i.e., through the mouth or via the mouth into the subject, including swallowing or transport through the oral mucosa (e.g., sublingual or buccal absorption) or both. Suitable flavoring agents, such as menthol and levomenthol, or sweetening agents, such as saccharin or saccharin sodium, may be added to the formulations of the invention intended for oral or peroral administration.

The immunogenic compositions and vaccines of the invention may be administered rectally or vaginally, for example in the form of suppositories, pessaries or enemas. Cocoa butter is a traditional suppository base, but various alternatives may be used as desired. Formulations for rectal/vaginal administration may be formulated for immediate and/or modified release. Modified release formulations include delayed release, sustained release, pulsatile release, controlled release, targeted release and programmed release.

The immunogenic compositions and vaccines of the invention may also be administered directly to the eye or ear, typically in the form of drops of micronized suspension or solution in isotonic pH adjusted sterile saline. Other formulations suitable for ocular and otic administration include ointments, biodegradable (e.g. absorbable gel sponges, collagen) and non-biodegradable (e.g. silicone) implants, wafers, lenses and particulate or vesicular systems, such as non-ionic surfactant liposomes (niosomes) or liposomes. Polymers may be incorporated, for example cross-linked polyacrylic acid, polyvinyl alcohol, hyaluronic acid, cellulosic polymers such as hydroxypropylmethylcellulose, hydroxyethylcellulose or methylcellulose or isopolysaccharide polymers, for example gelan gum; and together with a preservative, such as benzalkonium chloride. Such formulations may also be delivered by iontophoresis.

Formulations for ocular/otic administration may be formulated for immediate release and/or modified release. Modified release formulations include delayed release, sustained release, pulsatile release, controlled release, targeted release and programmed release.

The immunogenic compositions and vaccines of the invention can be used in the preparation of a medicament for treating or preventing the spread of bovine viral diarrhea virus infection in an animal.

The immunogenic compositions and vaccines of the invention are useful for the preparation of a medicament for administration to an animal, wherein the medicament is a DIVA pestivirus vaccine comprising a chimeric pestivirus comprising an E which does not express its homology^rnsA bovine viral diarrhea virus of protein, and wherein said chimeric pestivirus expresses a heterologous E derived from another pestivirus^rnsProtein, or said heterologous E^rnsNatural, synthetic or genetic variants of the protein. In one embodiment, the chimeric pestivirus has at least one E which is not present in wild-type bovine viral diarrhea virus^rnsAn epitope. In another embodiment, said chimeric pestivirus lacks at least one E present in wild-type bovine viral diarrhea virus^rnsAn epitope.

Detection and diagnosis method

The present invention provides methods for determining the source of pestiviruses present in an animal subject.

Vaccination with a DIVA vaccine provides a method for determining the source of pestiviruses present in an animal subject, wherein the DIVA vaccine is capable of distinguishing infected from vaccinated animals. This differentiation can be achieved by any of a variety of diagnostic methods, including, but not limited to, ELISA, Western blot, and PCR. These and other methods are readily known and appreciated by those of ordinary skill in the art.

The chimeric pestiviruses of the invention are distinguishable from wild-type BVDV strains by their genomic composition and the proteins expressed. Such a distinction allows to distinguish between vaccinated and infected animals. For example, it can be determined whether an animal tested positive for BVDV in certain laboratory tests carries a wild-type BVDV strain or a chimeric pestivirus of the invention obtained by a previous vaccination.

A variety of assays may be used to make the determination. For example, a virus can be isolated from an animal that tests positive for BVDV, and the presence of the chimeric pestivirus genome (which indicates a previous vaccination) can be determined using a nucleic acid-based assay. Nucleic acid-based assays include Southern or Northern blot analysis, PCR, and sequencing. Alternatively, protein-based assays may be used. In protein-based assays, cells or tissues suspected of being infected may be isolated from animals tested positive for BVDV. Cell extracts can be prepared from such cells or tissues and can be subjected to, for example, Western blotting using suitable antibodies directed against viral proteins that are capable of discriminatively identifying the presence of previously inoculated chimeric pestiviruses versus wild-type BVDV.

The extent and nature of the immune response induced in an animal can be assessed by using a variety of techniques. For example, serum can be collected from the vaccinated animal and assayed for the presence of antibodies specific for the chimeric virus, e.g., in a conventional virus neutralization assay. Detection of responsive Cytotoxic T Lymphocytes (CTL) in lymphoid tissues can be carried out by assays such as T cell proliferation assays to indicate induction of a cellular immune response. Related techniques are well described in the art, such as Coligan et al Current Protocols in Immunology, John Wiley & Sons Inc. (1994).

Reagent kit

Since it may be desirable to administer an immunogenic composition or vaccine in combination with additional compounds, for example for the purpose of treating a particular disease or condition, the scope of the invention includes: the immunogenic composition or vaccine may conveniently be included or combined in the form of a kit suitable for administration or co-administration of the composition.

Thus, the kit of the invention may comprise one or more separate pharmaceutical compositions, at least one of which is an immunogenic composition or vaccine according to the invention, and means for separately containing the compositions, e.g. a container, a split bottle or a split aluminium foil pouch. Examples of such kits are syringes and needles, etc. The kits of the invention are particularly suitable for administering different dosage forms (e.g., oral or parenteral), for administering separate compositions at different dosing intervals, or for titrating separate compositions against each other. To aid in the administration of the compositions of the invention, the kit will generally contain instructions for administration.

Another kit of the invention may comprise one or more reagents for detecting and differentiating BVDV infected animals from chimeric pestivirus vaccinated animals. The kit may comprise reagents for assaying a sample for the presence of an intact BVDV or BVDV polypeptide, epitope or polynucleotide sequence that is not present in the chimeric pestivirus of the immunogenic composition or vaccine. Alternatively, the kit of the invention may comprise reagents for assaying a sample for the presence of a chimeric pestivirus or polypeptide, epitope or polynucleotide sequence which is not present in wild-type BVDV. The presence of a virus, polypeptide, or polynucleotide sequence can be determined using antibody, PCR, hybridization, and other detection methods known to those skilled in the art.

Another kit of the invention may provide reagents for detecting antibodies directed against a particular epitope. The epitope is present in the chimeric pestivirus of the invention and not in the wild-type BVDV; alternatively, it is present in wild-type BVDV and not in the chimeric pestivirus of the invention. Such reagents can be used to analyze a sample for the presence of antibodies and are readily known and available to those of ordinary skill in the art. Standard detection methods known to those skilled in the art can be used to determine the presence of the antibody.

In some embodiments, the kit may comprise a set of printed instructions or labels to indicate that the kit can be used to detect and distinguish BVDV-infected animals from chimeric pestivirus-vaccinated animals.

Antibodies

The antibody may be monoclonal, polyclonal or recombinant. Antibodies to the immunogen or portion thereof may be conveniently prepared. For example, synthetic peptides based on the amino acid sequence of the immunogen, or peptides recombinantly produced by cloning techniques, or natural gene products and/or portions thereof, can be isolated and used as immunogens. Antibodies can be raised using immunogens by standard antibody production techniques well known to those skilled in the art, a general description of which is found, for example, in Harlow and Lane, "Antibodies: a Laboratory Manual ", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1988) and Borrepeeck," antibody engineering-A Practical Guide ", W.H.Freeman and Co. (1992). Antibody fragments, including Fab, F (ab')₂And Fv.

In preparing antibodies, screening for the desired antibody can be accomplished by standard immunological methods known in the art. Techniques not described in detail generally follow standards, et al (eds), "Basic and Clinical Immunology" (8 th edition), Appleton and Lange, Norwalk, CT (1994), and Mishell and shiigi (eds), "Selected methods in Cellular Immunology", w.h.freeman and co.w.york (1980). In general, ELISA and Western blotting are preferred types of immunoassays. Both assays are well known to those skilled in the art. Both polyclonal and monoclonal antibodies can be used for these assays. The antibody may be bound to a solid support substrate or conjugated to a detectable moiety, or both, as is well known in the art. (for a general discussion of conjugation of fluorescent or enzymatic moieties, see Johnstone and Thorpe, "Immunochemistry in Practice", Blackwell ScScientific Publications, Oxford (1982)). The binding of antibodies to solid support substrates is also well known in the art (for general discussion, see Harlow and Lane (1988) and Borebaeck (1992)). Detectable moieties useful in the invention can include, but are not limited to, fluorescent, metallic, enzymatic, and radioactive markers such as biotin, gold, ferritin, alkaline phosphatase, b-galactosidase, peroxidase, urease, fluorescein, rhodamine, tritium, and the like,¹⁴C and iodine.

The invention is further illustrated by the following examples, without being limited thereto.

Example 1: construction and serological characterization of chimeric pestiviruses

Escherichia coli K12 GM2163[ F-ara-14, leuB6, thi-1, fhuA31, lacY1, tsx-78, galK2, galT22, supE44, hisG4, rpsL136, (Str)^r)，xyl-5，mtl-1，dam13::Tn9(Cam^r)，dcm-6，mcrB1，hsdR2(rk^-mk^-)，mcrA]A plasmid containing the full-length genomic cDNA comprising the bovine viral diarrhea virus strain NADL (BVDV-NADL) obtained from doctor r.

RD cells (bovine testis cells transformed with SV 40; obtained from r. donis) were maintained in OptiMEM supplemented with 3% horse serum, 1% non-essential amino acids (NEAA) (in Modified Eagle Medium (MEM)), 2mM GlutaMax and 10ug/ml gentamicin. BK-6 cells were obtained from Pfizer Global Manufacturing (PGM). Cells were grown in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 5% horse serum or donor bovine serum (PGM), 2mM Glutamax and 1% antibiotics and antimycotics. All media components were purchased from Invitrogen (Carlsbad, CA) except as indicated. All cells were maintained at 37 ℃ and 5% CO₂In the environment of (2).

Specific for BVDV E^rnsMonoclonal antibody (MAb)15C5 of (a) was purchased from IDEXX (Westbrook, ME). MAb 20.10.6 against BVDV NS3 was complimentary by doctor e.dubovi (CornellUniversity). Having a viral response against Border Disease Virus (BDV) E^rnsProtein specificity of MAbWS 363, WS 373, and WS 371 were obtained from Veterinary Laboratories Agency (Surrey, UK). Bovine serum samples #77, #816, #1281 and #1434 were provided internally by Pfizer.

By using the overlapping PCR method, by using E of BVDV-NADL strain^rnsGene replacement of Giraffe E^rnsGene (G-E)^rns) Reindeer E^rnsGene (R-E)^rns) Or E of pronghorn antelope^rnsGene (P-E)^rns) To produce chimeric pestiviruses. Using Pfuultra^TMII fusion with HS DNA polymerase (Stratagene; La Jolla, CA) orTaq DNA Polymerase High Fidelity (Invitrogen). Oligonucleotide primers (and corresponding SEQ ID NOs) used for overlap PCR and for generating full length viral DNA are listed in table 1.

TABLE 1 oligonucleotide primers for PCR amplification

A plasmid containing the full-length cDNA of BVDV-NADL was extracted from dam-E.coli K12 GM 2163. This plasmid was methylated in vitro using dam methyltransferase and S-adenosylmethionine (New England Biolabs; Ipshoch, Mass.). Synthesis of G-E^rns、R-E^rnsAnd P-E^rnsGenes (GenBank accession numbers NC-003678, NC-003677, and AY781152, respectively) were cloned into cloning vectors.

To construct a chimeric BVDV-NADL/G-E^rnsDNA, a fragment encoding BVDV-NADL from the 5 'UTR to the 3' end of the C gene was amplified by PCR from the methylated plasmid using primers Oligo B-5 and Oligo 127. G-E-containing DNA from DNA containing G-E by PCR using primers Oligo 128 and Oligo 129^rnsAmplification of G-E in plasmid DNA of genes^rnsA gene. Amplification of the code from the methylated plasmid by PCR Using primers Oligo 130 and Oligo 84BVDV fragment from E1 to 3' UTR. PCR products were Gel purified using the QIAquick Gel Extraction Kit (Qiagen; Valencia, Calif.). The purified PCR product was treated with Dpn I and Exonaclease 1(New England Biolabs). Assembly of the treated PCR products by PCR using Oligo B-5 and Oligo 84 to generate full-length chimeric BVDV-NADL/G-E^rnsA genome.

To construct a chimeric BVDV-NADL/R-E^rnsDNA, a fragment encoding BVDV-NADL from the 5 'UTR to the 3' end of the C gene was amplified by PCR from the methylated plasmid using primers Oligo B-5 and Oligo 131. From the vector containing R-E by PCR using primers Oligo 132 and Oligo 133^rnsAmplification of R-E in plasmid of Gene^rnsA gene. BVDV fragments encoding E1 to 3' UTR were amplified from methylated plasmids by PCR using primers Oligo 134 and Oligo 84. The PCR product was Gel purified using the QIAquick Gel Extraction Kit. The purified PCR product was treated with Dpn I and Exonaclease 1. Assembly of the treated PCR products by PCR using Oligo B-5 and Oligo 84 to generate full-length chimeric BVDV-NADL/R-E^rnsA genome.

To construct a chimeric BVDV-NADL/P-E^rnsDNA, a fragment encoding BVDV-NADL from the 5 'UTR to the 3' end of the C gene was amplified by PCR from the methylated plasmid using primers Oligo B-5 and Oligo 135. PCR from a plasmid containing P-E using primers Oligo 136 and Oligo 137^rnsAmplification of P-E in plasmid DNA of genes^rnsA gene. BVDV fragments encoding E1 to 3' UTR were amplified from methylated plasmids by PCR using primers Oligo 138 and Oligo 84. The PCR product was Gel purified using the QIAquick Gel Extraction Kit. The purified PCR product was treated with Dpn I and Exonaclease 1. Assembly of the treated PCR products by PCR using Oligo B-5 and Oligo 84 to generate full-length chimeric BVDV-NADL/P-E^rnsA genome.

To perform the tabling E^rnsSequence confirmation of regions, fragments corresponding to the 5' UTR to E1 region of each assembled full-length chimeric genome were amplified by PCR using Oligo B-5 and Oligo 175, and the PCR products were sequenced and analyzed.

From full-length cDNA containing BVDV-NADL or chimeric BVDV-NADL/E using the mMessage mMachine T7 Ultra kit (Ambion; Austin, TX)^rnsThe plasmid of DNA produces a full-length viral genomic RNA transcript. The quality and quantity of each RNA transcript was determined on RNA gels and a Nanodrop spectrophotometer (Nanodrop; Wilmington, DE). Overnight cultures of RD cells in wells of 6-well plates were transfected with viral RNA using Li-fectin reagent (Invitrogen) according to the manufacturer's instructions. After transfection, cells were incubated at 37 ℃ for 3 days. The supernatant was collected and stored at-80 ℃.

MagMax was used according to the manufacturer's instructions^TMAI/ND Viral RNA IsolationKit (Ambion) extracted Viral RNA from the collected supernatant. Primers Oligo 177 and Oligo 175 (Table 1) and Thermoscript were used according to the manufacturer's instructions^TMRT-PCRSystem (Invitrogen) reverse transcribing RNA and amplifying the code N of each chimera^proTo the region of E1. The RT-PCR products were then sequenced.

Cell monolayers from viral RNA transfection or viral infection were fixed in 80% acetone. BVDV-or BDV-specific monoclonal antibodies (mabs) and anti-mouse-IgG peroxidase ABC Elite kit (Vector Laboratories; Burlingame, Calif.) were used. Color development was performed using VIP peroxidase substrate (Vector Laboratories).

The titer of the chimeric virus was determined by limiting dilution method. Virus samples were serially diluted 10-fold and transferred to 96-well plates (100 μ l/well), each dilution being 4-6 replicates. Then, 100. mu.l of a suspension of BK-6 cells was added to each well, and the plates were incubated at 37 ℃ for 4-5 days. Viral infection was determined by both cytopathic effect (CPE) and MAb staining. Use of Spearman-The method calculates the virus titer.

To obtain the biological clones of each chimera, the virus samples were first diluted 100-fold and then serially diluted 10-fold. 100 μ l of diluted virus was transferred to each well of a 96-well plate in quadruplicate for each dilution. Then, 100. mu.l BK-6 cells were added to each well and the plates were incubated at 37 ℃ for 4 days. The supernatant was collected and transferred to a new plate and stored at-80 ℃. Cells were fixed and stained. Supernatants were collected from wells containing single viral foci and amplified as viral stocks.

Growth kinetics studies were performed in T-25 flasks containing BK-6 cells. When the cells reached approximately 90% confluence, the cells were infected with each chimera at an MOI of 0.02. After 1 hour of adsorption, the inoculum was removed. Cells were washed 3 times with PBS and then 3ml of fresh growth medium was added. Samples were then collected at various time points from 0-144 hours for titer determination.

For the virus neutralization test, 3 BVDV-NADL/E were tested^rnsFrozen stock solutions of chimeras, parental BVDV-NADL and BVDV-CM5960 (BVDV type I) were diluted to approximately 4,000TCID in DMEM₅₀And/ml. Sera from cattle immunized with Bovi-Shield Gold (Pfizer; New York, NY) with predetermined titers against type I and type II BVDV were serially diluted at 2 fold DMEM. 50 μ l of virus (200 TCID)₅₀) Mixed with an equal volume of diluted bovine serum in 96-well tissue culture plates (each dilution in quadruplicate) and incubated at 37 ℃ for 60 minutes. Then, 100. mu.l BK-6 cells were added to each well and the plates were incubated at 37 ℃ for 3-6 days. As a control, BVDV antibody negative sera were also included in each plate. Endpoint neutralization titers of sera were determined at day 3 and day 6 using both CPE and Immunohistochemistry (IHC) methods.

As a result, a chimeric BVDV-NADL/E was constructed^rnsDNA, wherein NADL E^rnsGene/protein replacement by Giraffe E^rns(G-E^rns) Reindeer E^rns(R-E^rns) Or E of pronghorn antelope^rns(P-E^rns). For each chimeric E^rnsPlasmid DNA of the regions was sequenced to confirm sequence authenticity. The following chimeric pestiviruses were deposited at the American type culture Collection on 4.2.200910801 University blvd, Manassas, VA, 20110, USA, andsurvival was confirmed at 23 days 4-month 2009: BVDV-NADL/G-E^rns(PTA-9938)、BVDV-NADL/P-E^rns(PTA-9939) and BVDV-NADL/R-E^rns(PTA-9940)。

BVDV-NADL/E recovery from RD cells following transfection with in vitro transcribed viral RNA^rnsA chimeric virus. In use with BVDV-NADL/G-E^rnsOr BVDV-NADL/R-E^rnsSevere cytopathic effect (CPE) was observed in RD cells 48-72 hours after RNA transcript transfection. However, with BVDV-NADL/P-E^rnsCPE was not apparent from virus transfected cells. Culture supernatants were collected from each well and the remaining cells were fixed and stained with BVDV NS 3-specific MAb antibody 20.10.6. Cells infected with one of the 3 chimeric pestiviruses were incubated with MAb. Viral RNA was extracted from the collected supernatants and sequenced to confirm E of all 3 chimeras^rnsA gene.

3 BVDV-NADL/E were tested^rnsChimeras for several E specific for BVDV or BDV^rnsReactivity of each of the mabs. The results are shown in Table 2. BVDV-NADL/R-E^rnsChimera to all 3 BDV E^rnsMabs all reacted, whereas BVDV-NADL/G-E^rns、BVDV-NADL/P-E^rnsAnd BVDV-NADL parental virus is not BDV E^rnsAnd (5) identifying the Mab. BVDV-NADL/G-E^rns、BVDV-NADL/R-E^rnsAnd NADL parental virus with broad spectrum-BVDV E^rnsMAb15C5 reacted. E specific for BDV or BVDV^rnsMab and BVDV-NADL/P-E of^rnsThe chimera did not respond.

TABLE 2 BVDV-NADL/E^rnsReactivity of chimeras with mabs

To determine chimerism E in viruses^rnsWhether the protein has an effect on the recognition of virus-neutralizing epitopes by antibodies from BVDV-vaccinated cattle, 3 BVDV-NADL/E were used^rnsVirus neutralization assays were performed with chimeras, BVDV-NADL and BVDV-CM5960 (BVDV type I). Sera from 4 cattle were used with neutralizing antibody titers ranging from 0 to greater than 40,000 (previously determined against BVDV-CM 5960). The results (Table 3) show that the titers against all 3 chimeras are essentially comparable to the titers against the parental BVDV-NADL and BVDV-CM 5960. For BVDV-NADL/P-E^rnsWas slightly lower than the titers for the other two chimeras, BVDV-NADL and BVDV-CM 5960.

TABLE 3 bovine antisera against BVDV-NADL/E^rnsNeutralizing titer of chimeras

3 BVDV-NADL/E were diluted by limiting dilution method^rnsThe chimera was subcloned twice. BVDV-NADL/G-E was obtained^rns3 clones of (5), BVDV-NADL/R-E^rns4 clones of (5) and BVDV-NADL/P-E^rns3 clones of (4). These clones were each amplified 1-3 times. The titration results show that: amplified BVDV-NADL/G-E^rnsClone 1, BVDV-NADL/R-E^rnsClones 3 and 5 and BVDV-NADL/P-E^rnsClone 2 produced the highest titer.

With BVDV-NADL/G-E^rnsClone 1, BVDV-NADL/R-E^rnsClone 3, BVDV-NADL/P-E^rnsClone 2 and uncloneable BVDV-NADL/P-E^rnsGrowth kinetics studies were performed. The growth curves generated from these clones were compared to the parental BVDV-NADL. BVDV-NADL/G-E^rnsAnd BVDV-NADL/R-E^rnsThe chimeras have growth kinetics similar to the parental BVDV-NADL, whereas BVDV-NADL/P-E^rnsSlower growth relative to parental virus and the other two chimerasAnd lower titers at each time point.

3 BVDV-NADL/E were generated^rnsChimeric virus, wherein NADL E^rnsE with gene/protein replaced by Giraffe, reindeer or pronghorn antelope pestivirus^rns. All 3 chimeras were viable and infectious in RD and BK-6 cells. In vitro data demonstrate chimerism E^rnsThe protein did not affect the neutralization of the chimeras by antisera from BVDV vaccinated cattle. This indicates that neutralizing epitopes on chimeric viruses, wherever they are located, are not affected by E^rnsThe effect of the replacement.

Chimeric viruses have different growth kinetics and are directed against BVDV or BDV E^rnsDifferential reactivity of monoclonal antibodies. BVDV-NADL/G-E^rnsAnd BVDV-NADL/R-E^rnsHas growth kinetics similar to that of the parental virus, and BVDV-NADL/P-E^rnsSlower than the parental virus and have lower titers. BVDV-NADL/G-E^rnsAnd BVDV-NADL/R-E^rnsAre all in agreement with BVDV E^rnsMonoclonal antibody 15C5 response, and BVDV-NADL/P-E^rnsDoes not react with it. The sequence comparison results show that: G-E^rnsAnd R-E^rnsSequence similarity to BVDV NADL (75.8% and 76.2%, respectively) was higher than that of P-E^rnsSequence similarity to BVDV NADL (59%). These data, together with the Mab reactivity results, illustrate that G-E^rnsAnd R-E^rnsAntigenically identical to parent E^rnsMay have a higher similarity than P-E^rnsWith parent E^rnsThe similarity of (c).

Example 2 construction and serological characterization of chimeric pestivirus vaccine candidates and efficacy testing

Type 1 BVDV strain CM5960 and type 2 BVDV strain CM53637 were obtained from Pfizer GlobalManufating. MagMax was used according to the manufacturer's instructions^TMThe AI/ND Viral RNAI translation Kit (Ambion) extracts Viral RNA. Thermoscript was used according to the manufacturer's instructions^TMRT-PCR System (Invitrogen) reverse transcribes RNA to produce cDNA. Using an overlapping PCR method by combining CM5960 and CM53637E^rnsE of pronghorn pestivirus with gene replacement^rnsGene (P-E)^rns) Thereby producing the chimeric pestivirus. The oligonucleotide primers used for PCR are listed in Table 1.

To construct chimeric CM5960/P-E^rnsDNA, a fragment of CM5960cDNA between the 5 'UTR and the 3' end of the C gene was amplified from CM5960cDNA by PCR using primers Oligo B-5 and Oligo 135. PCR from a plasmid containing P-E using primers Oligo 136 and Oligo 137^rnsAmplification of P-E in plasmid DNA of genes^rnsA gene. A third fragment from the start of E1 to the 3' end of E2 was amplified by PCR from CM5960cDNA using primers Oligo 138 and Oligo 237.

The above fragment was Gel purified using the QIAquick Gel Extraction Kit (Qiagen), and assembled by PCR using Oligo B-5 and Oligo 237 to generate one fragment. The fragment between the E1 region and the NS5B region was amplified from CM5960cDNA by PCR using primers Oligo P7 and Oligo P8. Another fragment between the NS5A region and the end of the 3' UTR was amplified from CM5960cDNA by PCR using primers Oligo P3 and Oligo 84. These 3 fragments were then gel purified and assembled by PCR using Oligo B-5 and Oligo 84 to generate the full-length chimeric CM5960L/P-E^rnsA genome.

To construct chimeric CM53637/P-E^rnsDNA, a CM53637cDNA fragment between the 5 'UTR to the 3' end of the C gene was amplified by PCR from CM53637cDNA using primers Oligo296-1 and Oligo 297. A second fragment from the start of E1 to the 3' end of E2 was amplified by PCR from CM53637cDNA using primers Oligo 298 and Oligo 303. These two fragments were gel purified and PCR-ligated with the coding P-E using Oligo296-1 and Oligo 303^rnsFragments of the gene (see above) are assembled together to produce one fragment.

The fragment between the E1 region and the NS3 region was then amplified from CM53637cDNA by PCR using primers Oligo 298 and Oligo 299. Another fragment between the NS3 region and the end of the 3' UTR was amplified by PCR from CM53637cDNA using primers Oligo 300 and Oligo 92-1. Will be provided withThese two fragments and one fragment above were gel purified and assembled by PCR using Oligo296-1 and Oligo92-1 to generate the full-length chimeric CM53637/P-E^rnsA genome.

Chimeric CM5960/P-E was obtained from chimeric CM5960/P-E using the mMessage mMachine T7 Ultra kit (Ambion)^rnsAnd chimeric CM53637/P-E^rnsThe DNA produces full-length viral genomic RNA transcripts. The quality and quantity of each RNA transcript was determined on an RNA gel. An overnight culture of RD cells in wells of 6-well plates was transfected with viral RNA using Lipofectin reagent (Invitrogen) according to the manufacturer's instructions. After transfection, cells were incubated at 37 ℃ for 3 days. Cells plus supernatant were passaged 1 to several times in RD and/or BK-6 cells. The supernatant was then serially passaged in BK-6 cells. The supernatant was collected and stored at-80 ℃.

To confirm the identity of the recovered recombinant virus, MagMax was used according to the manufacturer's instructions^TMAI/ND Viral RNA Isolation Kit (Ambion) extracted Viral RNA from the collected supernatant. Thermoscript was used according to the manufacturer's instructions^TMRT-PCR System (Invitrogen) reverse transcription of RNA; primers Oligo B-5 and Oligo 237 (for CM 5960/P-E)^rnsChimeras) or Oligo296-1 and Oligo 321 (for CM 53637/P-E)^rnsChimeras) (table 1) the region between the 5' UTR to E2 or p7 of each chimera was amplified by PCR. The RT-PCR products were then sequenced.

Cell monolayers from viral RNA transfection or viral infection were fixed in 80% acetone. Immunohistochemistry was performed using BVDV-specific mabs and anti-mouse-IgG peroxidase ABC Elite kit (Vector Laboratories). Color development was performed using VIP peroxidase substrate (Vector Laboratories).

As a result, chimeric CM5960/P-E was constructed and recovered^rnsAnd CM53637/P-E^rnsA virus. The region from the 5' UTR to E2 (including chimeric pronghorn-E) was confirmed by sequencing^rnsA region). Both chimeras were viable and infectious in RD and BK-6 cells. In immunohistochemical staining, two chimerismFor BVDV E^rnsNone of the specific MAb15C5 was reactive, but all were reactive against BVDVNS3 specific MAb 20.10.6.

Chimeric pestivirus (BVDV-CM5960 (type I BVDV)/P-E^rns) The sequence of (a) is shown in the sequence listing as SEQ ID NO: 31. chimeric pestivirus (BVDV-CM53637 (type II BVDV)/P-E^rns) The sequence of (a) is shown in the sequence listing as SEQ ID NO: 32.

biological cloning of CM5960/P-E by limiting dilution method^rnsChimeras (the method is as described in example 1 above).

Example 3 efficacy testing of chimeric pestivirus vaccine candidates in bovine respiratory disease models

Healthy cattle, negative for BVDV, were obtained, randomized into study groups, and kept under the supervision of a home veterinarian. The test vaccines were mixed with sterile adjuvants and administered by Intramuscular (IM) or Subcutaneous (SC) injection or by Intranasal (IN) vaccination. The vaccine is administered as 1 or 2 doses. 2 doses of vaccine were administered at intervals of 21-28 days. Animals were then challenged with type 1 or type 2 BVDV strains 21-28 days after the last vaccination. Challenge inoculum was administered intranasally in 4ml divided doses (2 ml/nostril). A control group consisting of unvaccinated, non-challenged animals and/or unvaccinated, challenged animals was also maintained throughout the study.

Clinical parameters including rectal temperature, depression, anorexia and diarrhea were monitored daily. The neutralizing titer of serum was determined by a "virus constant, serum reduction" assay in bovine cell culture using serial dilutions of serum and BVDV type 1 or type 2 strains. Post challenge isolates of BVDV from peripheral blood were obtained in bovine cell cultures. BVDV-positive cell cultures were determined by indirect immunofluorescence. To demonstrate protection after challenge, a reduction in the infection rate of the vaccinated group relative to the control group was shown.

Example 4 efficacy testing of chimeric pestivirus vaccines in a pregnant cow-calf model

BVDV-negative cows and breeding-stage heifers were obtained and randomly divided into either vaccination test or placebo (control) groups. Cows were vaccinated twice by Intramuscular (IM) or Subcutaneous (SC) injection: at intervals of 21-28 days, either vaccine or placebo was administered. After the second vaccination, all cows received IM injections of prostaglandin to synchronize estrus. Cows in heat were bred by artificial insemination with semen identified as BVDV negative. At approximately 60 days of gestation, the pregnancy status of the cow was determined by rectal palpation.

After approximately 6 weeks, cows confirmed to be pregnant were randomly selected from each test group. Each of these cows was challenged by intranasal inoculation with BVDV type 1 or type 2. Blood samples were collected on the day of challenge and at various intervals after challenge to isolate BVDV.

28 days after challenge, a left flank was opened and amniotic fluid was extracted from each cow. Immediately prior to surgery, blood samples were collected from each cow for serum neutralization assays. After caesarean section, a blood sample was collected from each fetus. The fetus was then euthanized and tissues collected under sterile conditions to isolate BVDV. In the case of spontaneous abortion, blood samples were taken from the mother at the time of detection of abortion and 2 weeks later. Paired blood samples and aborted fetuses were subjected to serological testing and virus isolation. The effectiveness of the vaccine is demonstrated by the absence of infection or reduction of infection in the fetus and late abortion.

Example 5 diagnostic assay for differentiating vaccinated from naturally infected cattle

Cattle vaccinated with the vaccine of the invention can be compared to cattle naturally infected with wild-type BVDV. Cattle of various ages were vaccinated with live or inactivated chimeric pestivirus vaccines according to the provided instructions. Serum samples were collected at 2-3 weeks or later post inoculation. To distinguish cattle receiving the chimeric pestivirus vaccine from cattle infected with field (wild type) strains of BVDV, serum samples were tested by a differential diagnostic assay. The chimeric pestivirus causes the production of specific antibodies which bind to E of the chimeric pestivirus^rnsProtein, butDoes not bind E present in wild-type BVDV^rnsA protein. In the case of wild-type BVDV, the opposite is true: specific antibodies were generated which recognize E present in wild-type BVDV^rnsProteins, but not recognizing E present in chimeric pestiviruses^rnsA protein. Methods for determining antibody binding specificity and affinity are well known in the art and include, but are not limited to, the following immunoassay formats, such as competitive ELISA, direct peptide ELISA, Western blot, indirect immunofluorescence assay, and the like.

For competitive ELISA, whole or partial wild-type or chimeric pestivirus antigens (including E) were used^rnsProteins (of natural, synthetic or recombinant origin)) as the source of the antigen. After coating the ELISA plates with antigen under alkaline conditions, bovine serum samples and dilutions were added, as well as E specific for wild-type BVDV^rnsE of a proteinaceous or chimeric pestivirus^rnsOptimal dilutions of mabs of proteins and incubations for 30-90 minutes. Mabs were conjugated to horseradish peroxidase or alkaline phosphatase to allow detection of binding by colorimetry. After washing the plates, enzyme-specific chromogenic substrate is added and, after the final incubation step, the optical density of each well is determined at a wavelength appropriate to the substrate used. The extent of inhibition of binding of the labeled mAb depends on the level of antibodies in the bovine serum that specifically recognize the protein coating the plate.

Chimeric E where the test antigen is present on a chimeric pestivirus^rnsProteins (e.g. pronghorn E)^rns) In the case of (a) chimeric pestivirus E^rnsAbsence of binding of the specific mAb indicates the presence of antibodies in the bovine serum that recognize specific epitopes of the chimeric pestivirus, indicating vaccination. In contrast, sera from non-immunized but naturally infected cattle will be free of chimeric pestivirus E with coated plates^rnsProtein-bound antibodies. Thus, chimeric pestivirus E^rnsThe specific mAb will bind to the coated protein, causing the subsequent generation of color.

E when the test antigen is present on wild-type BVDV^rnsIn the case of proteins, wild-type BVDVE^rnsAbsence of binding of a specific mAb indicates the presence of antibodies in the bovine serum that recognize a specific epitope of wild-type BVDV, indicating the presence of a natural (wild-type) infection. In contrast, sera from cattle immunized with the chimeric pestivirus vaccine will be free of wild-type BVDV E with coated plates^rnsProtein-bound antibodies. Thus, wild-type BVDV E^rnsThe specific mAb will bind to the coated protein, causing the subsequent generation of color.

For such measurement, the following method was performed.

First, expression of BVDV-NADL E was constructed^rnsThe recombinant baculovirus of (4). A portion of the C protein of BVDV + full length E was amplified by PCR from a plasmid containing the full length BVDV-NADL cDNA using primers Oligo 250(SEQ ID NO: 29; 5'-CACCATGAAAATAGTGCCCAAAGAATC-3') and Oligo 252(SEQ ID NO: 30; 5'-TTAAGCGTATGCTCCAAACCACGTC-3')^rnsA gene. Cloning of the PCR product into pENTR according to the manufacturer's instructions^TM(Invitrogen) and conversion into OneCompetent E.coli (Invitrogen). Recombinant plasmids were extracted and inserts were confirmed by sequencing. This plasmid is designated pENTR-E^rns. pENTR-E was used according to the manufacturer's instructions^rnsAnd Baculodirect^TMBaculovirusexpression System (Invitrogen) to construct expression BVDV-NADL E^rnsThe recombinant baculovirus of (4). Generation of expression of BVDV-NADL E^rnsThe recombinant baculovirus of (1), was plaque purified, amplified and stored at 4 ℃ and-80 ℃. By immunofluorescence staining and Western blotting (using E against BVDV)^rnsSpecific MAb15C5 according to the conventional Western blotting method) confirmed BVDV-NADL E in recombinant baculovirus^rnsExpression of (2).

To produce ELISA antigens, SF21 cells in 100ml suspension cultures were infected with 0.5ml of recombinant baculovirus stock. Cells were harvested after 4 days incubation at 27 ℃. Cells were centrifuged at low speed (approximately 800g) for 10 minutes to harvestCells were pooled and washed 1 time with PBS. Cells were lysed with 150mM NaCl, 50mM Tris HCl pH 8.0 and 1% IGEPAL CA-630. The mixture was first incubated on ice for 10 minutes and then placed at-80 ℃ for 1 hour. After thawing, the mixture was clarified by centrifugation at 1000g for 15 minutes. The supernatant was further clarified by centrifugation at 8000g for 20 minutes at 4 ℃. The final supernatant was designated as Baculo-E^rnsLysates, aliquots were aliquoted and stored at-80 ℃.

In the assay, MAb WB210(Veterinary laboratory Agency; specific for BVDV E type 1; at 100. mu.l/well at 4 ℃ C^rns) The ELISA plates were coated overnight and the antibody was diluted 1: 1000 in carbonate/bicarbonate buffer (pH 9.0). The following day, plates were washed 3 times and blocked with blocking buffer (PBS containing 1% sodium caseinate and 0.05% Tween 20) for 1 hour at 37 ℃. The plate was then washed 3 times with blocking buffer and 100. mu.l Baculo-E was added to each well^rnsLysates (1: 3200 diluted in PBS) were incubated at 37 ℃ for 1 hour. After 3 washes with blocking buffer, 100 μ l of undiluted bovine serum sample was added to the wells; wells with 1 column not added (as a non-competitive 15C5-HRP control); incubate at 37 ℃ for 1 h. After 3 washes with blocking buffer, 100. mu.l of MAb15C5-HRP conjugate (specific for BVDV E) was added to each well^rns1: 20,000 in blocking buffer) and incubated at 37 ℃ for 1 hour. After 3 washes with blocking buffer, 100. mu.l of ABTS substrate (peroxidase substrate solution A + B; KPL, USA) was added to each well and incubated at room temperature for 20-60 minutes for color development. The Optical Density (OD) was determined at a wavelength of 405 nm. The percentage reduction in OD for each serum sample was calculated according to the following formula:

[1- (sample OD/15C 5-HRP control average OD) ] x 100%

As a result:

all serum samples tested positive by the Virus Neutralization (VN) test had an o.d. reduction of over 82%, except for sample ID #13851 (table 4). All serum samples tested negative by the virus neutralization test had an o.d. reduction of 17% or less, except for sample ID #5150 (table 4). The inconsistencies can be explained by differences in the methods by which the assays are performed, as they measure different antibodies and the proportion of specific antibodies varies between animals.

TABLE 4 BVDV positive and negative serum samples in MAb15C5 competitive ELISA

Lines 1-16: positive bovine serum sample

Lines 17-27: negative bovine serum sample

All serum samples used in ELISA were undiluted

Although the present invention has been described in considerable detail with reference to certain versions thereof, other versions are possible. Accordingly, the scope of the appended claims should not be limited to the description of the versions contained herein.

Claims (24)

1. Chimeric pestivirus, wherein said chimeric pestivirus comprises E which does not express its homologue^rnsA bovine viral diarrhea virus of protein, and wherein said chimeric pestivirus expresses a heterologous E derived from another pestivirus^rnsProtein, and said heterologous E^rnsThe protein is pronghorn pestivirus E^rnsA protein.

2. The chimeric pestivirus of claim 1, wherein the heterologous E of said chimeric pestivirus is^rnsThe protein has at least one non-existent wild speciesE in raw bovine viral diarrhea virus^rnsAn epitope.

3. The chimeric pestivirus of claim 1, wherein the heterologous E of said chimeric pestivirus is^rnsProtein deletion of at least one E present in wild-type bovine viral diarrhea virus^rnsAn epitope.

4. A culture of the chimeric pestivirus of claim 1.

5. A cell line or host cell comprising the chimeric pestivirus of claim 1.

6. A polynucleotide molecule encoding the chimeric pestivirus of claim 1.

7. An immunogenic composition comprising the chimeric pestivirus of claim 1 and a veterinarily acceptable carrier.

8. The immunogenic composition of claim 7, wherein the veterinarily acceptable carrier is an adjuvant.

9. The immunogenic composition of claim 7, wherein said chimeric pestivirus is live attenuated.

10. The immunogenic composition of claim 7, wherein said chimeric pestivirus is inactivated.

11. The immunogenic composition of claim 7, further comprising one or more additional antigens for treating or preventing the spread of one or more additional pathogenic microorganisms in cattle.

12. An immunogenic composition comprising the polynucleotide molecule of claim 6 and a veterinarily acceptable carrier.

13. A vaccine comprising the chimeric pestivirus of claim 1 and a veterinarily acceptable carrier.

14. The vaccine of claim 13, wherein the veterinarily acceptable carrier is an adjuvant.

15. The vaccine of claim 13, wherein said chimeric pestivirus is live attenuated.

16. The vaccine of claim 13, wherein said chimeric pestivirus is inactivated.

17. A vaccine comprising the polynucleotide molecule of claim 6 and a veterinarily acceptable carrier.

18. The vaccine of claim 13, further comprising one or more additional antigens for treating or preventing the spread of one or more additional pathogenic microorganisms in cattle.

19. A kit comprising the vaccine of claim 13 in at least one container.

20. Use of the vaccine of claim 13 in the manufacture of a medicament for treating or preventing the spread of bovine viral diarrhea virus infection.

Use of a DIVA pestivirus vaccine in the manufacture of a medicament for vaccinating an animal, wherein said DIVA pestivirus vaccine comprises the chimeric pestivirus of claim 1, and wherein said chimeric pestivirus has at least one E that is not present in wild-type bovine viral diarrhea virus^rnsAn epitope.

Use of a DIVA pestivirus vaccine in the manufacture of a medicament for vaccinating an animal, wherein said DIVA vaccine comprises the chimeric pestivirus of claim 1, and wherein said chimeric pestivirus lacks at least one E present in wild-type bovine viral diarrhea virus^rnsAn epitope.

23. Use of an agent capable of detecting E against at least one of the chimeric pestiviruses present in said vaccine but absent from wild-type bovine viral diarrhea virus for the manufacture of a kit for distinguishing an animal vaccinated with the vaccine of claim 13 from an animal infected with wild-type bovine viral diarrhea virus by^rnsAntibodies to an epitope, wherein an animal vaccinated with said vaccine produces antibodies against at least one E present in the chimeric pestivirus of said vaccine but not present in wild-type bovine viral diarrhea virus^rnsEpitope:

a) obtaining a serum sample from an animal;

b) determining the sample for the presence or absence of the antibody;

c) identifying an animal having the antibody as having been vaccinated with the vaccine; and

d) animals not containing the antibody were identified as having been infected with wild-type BVDV.

24. Use of an agent capable of detecting E against at least one E present in wild-type bovine viral diarrhea virus but not present in the chimeric pestivirus of the vaccine for the manufacture of a kit for distinguishing between an animal infected with wild-type bovine viral diarrhea virus and an animal vaccinated with the vaccine of claim 13 by^rnsAn antibody to an epitope, wherein an animal infected with wild-type bovine viral diarrhea virus produces an antibody against at least one E present in wild-type bovine viral diarrhea virus but not present in the chimeric pestivirus of the vaccine^rnsEpitope:

a) obtaining a serum sample from an animal;

b) determining the sample for the presence or absence of the antibody;

c) identifying an animal having the antibody as having been infected with wild-type BVDV; and

d) identifying an animal that does not contain the antibody as having been vaccinated with the vaccine.