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WO1999029872A1 - Molecules d'acide nucleique codant des proteines f et hn de virus parainfluenza 2 canin - Google Patents

Molecules d'acide nucleique codant des proteines f et hn de virus parainfluenza 2 canin Download PDF

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Publication number
WO1999029872A1
WO1999029872A1 PCT/US1998/025203 US9825203W WO9929872A1 WO 1999029872 A1 WO1999029872 A1 WO 1999029872A1 US 9825203 W US9825203 W US 9825203W WO 9929872 A1 WO9929872 A1 WO 9929872A1
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Prior art keywords
vector
canine
virus
vectors
interest
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Inventor
Laurent Fischer
Jill Taylor
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Virogenetics Corp
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Virogenetics Corp
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Priority to AU16066/99A priority Critical patent/AU1606699A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18711—Rubulavirus, e.g. mumps virus, parainfluenza 2,4
    • C12N2760/18722—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Canine parainfluenza virus type 2 (cPIV2) a member of the Paramyxovindae
  • cPIV2 Isolation of cPIV2 is generally associated with upper respiratory infections m dogs housed in kennel situations (Appel et al , 1970, Binn et al , 1967, Crandell et al , 1968) Experimental infection of dogs inoculated intra-nasally with cPIV2 results in pharyngitis, tonsillitis and tracheobronchitis (Lazar et al , 1970, Rosenberg et al ,
  • Paramyxoviruses are enveloped viruses containing an 18-20 kb single- stranded RNA genome of negative polarity
  • the genome encodes 5 to 7 structural proteins including a fusion (F) and either a hemagglutinin-neuraminidase (HN) or hemagglutinin (HA) glycoprotein.
  • the membrane glycoprotein hemagglutinin (HA) is responsible for hemagglutination and attachment of the virus to the host cell, and the fusion glycoprotein (F), causes membrane fusion between the virus and the infected cell or between the infected and adjacent uninfected cells.
  • Canine parainfluenza virus as a member ofthe Paramyxoviridae family, contains a negative-sense single stranded RNA genome encoding six major proteins. These are the nucleocapsid (N) protein which encapsidates the viral RNA, the phospho- (P), and Large (L)
  • RNA transcription and replication proteins associated with RNA transcription and replication, and the three integral membrane proteins, matrix (M), fusion (F) and hemagglutinin-neuraminidase (FIN) (Lamb and Kolakofsky, 1996).
  • M matrix
  • F fusion
  • FIN hemagglutinin-neuraminidase
  • the HN protein has both hemagglutininating and neuraminidase activities while the F protein is involved in virus penetration, cell fusion and hemolysis (Paterson, 1987). While it is recognized that antibodies to HN neutralize the virus, antibodies to both F and HN are required to prevent spread
  • the F glycoprotein is synthesized as an
  • inactive precursor F 0 that is cleaved by a host proteolytic enzyme to form the biologically active
  • Paramyxovirus nucleic acid molecules including canine parainfluenza (type 2) F, HN, F and HN, recombinants containing the same, compositions thereof, and methods for making and using such molecules, recombinants, and compositions, especially recombinants which can express epitopes of interest from canine parainfluenza (type 2) F, FIN, and F and HN, in vivo, e g , poxvirus, adenovirus recombinants, or DNA-based plasmid vectors, or in vitro, e g , baculovirus recombinants, which obtain improved protection in compa ⁇ son to previous vaccinia virus recombinants when the recombin
  • compositions thereof therefrom is administered, compositions thereof, and methods for making and using such recombinants, vectors and compositions
  • vector composing an exogenous nucleic acid molecule encoding a first epitope of interest from canine parainfluenza type 2 (cPIV), wherein the vector expresses the exogenous nucleic acid molecule, e g , wherein the product expressed either in vivo or in vitro is capable of inducing an
  • immunological response in a host such as a protective immunological response or a response which is evinced by a reduction of duration of virus isolation following challenge
  • a method for inducing an immunological response comprising administering the vector to a host, an
  • the present invention provides a vector comprising an exogenous nucleic acid molecule, e.g., DNA, encoding a first epitope of interest from canine parainfluenza type 2 (cPrV), wherein the vector expresses the exogenous nucleic acid molecule.
  • the product expressed either in vivo or in vitro is capable of inducing an immunological response in a
  • the vector can be any suitable or desired vector.
  • the vector can be a poxvirus, such as a vaccinia virus, e.g., a NYVAC, NYVAC.l or NYVAC.2 vaccinia virus; or a canarypox virus, e.g., an ALVAC canarypox virus.
  • the vector can be a baculovirus or a plasmid vector.
  • the vector can be an adenovirus, e.g. a canine adenovirus such as canine adenovirus type 2 (CAV2).
  • the vector can be a he ⁇ esvirus, such as a canine he ⁇ esvirus.
  • the exogenous nucleic acid molecule can encode cPIV fusion (F), Hemagglutinin-Neuraminidase (HN), or F and HN, proteins.
  • the vector can additionally
  • nucleic acid molecule e.g., DNA
  • a second epitope of interest for instance DNA encoding a second epitope of interest from an antigen of a canine pathogen, e.g., from a Morbillivirus (for instance canine distemper virus, measles virus and the like), a rabies virus,
  • a Morbillivirus for instance canine distemper virus, measles virus and the like
  • rabies virus for instance canine distemper virus, measles virus and the like
  • canine he ⁇ esvirus parvovirus (e.g., canine parvovirus), canine adenovirus, coronavirus (e.g.,
  • the vector can include a nucleic acid molecule, e g , DNA encoding a biological response modifier or modulator, a growth factor, a recognition sequence, a therapeutic gene, or a fusion protein, or a combination thereof (in addition to an exogenous nucleic acid molecule
  • the vector can compose an exogenous nucleic acid molecule encodmg a cPIV antigen or epitope of interest and optionally one or more of a second antigen or epitope of interest, a biological response modifier or modulator, a growth factor, a recognition
  • the invention provides an immunological composition composing a earner and the expression product of one or more of the recombinants ofthe present invention.
  • the invention provides a method for inducing an immunological response composing administeong the recombinant to a host. A vaoation of this
  • embodiment is where the expression product of the recombinant is administered to the host
  • the invention further comprehends products from the in vitro expression, as well as products from the methods for inducing an immunological response, and uses for such products. Also, the invention comprehends methods for making the
  • nucleic acid molecules the vectors, and the compositions.
  • Fig. 1 shows a map of pJT008
  • Fig. 2 shows a map of pJT007
  • Fig. 3 shows a map of pJTOlO; and Fig. 4 shows a map ofpJT009.
  • the present invention pertains to paramyxovirus nucleic acid molecules, expression products therefrom, e.g., epitopes of interest.
  • acid molecules can be canine parainfluenza (type 2) ("PIV2" or "cPIV2”) nucleic acid molecules, for instance such molecules encoding an epitope of interest from PIV2, or an antigen from PIV2
  • the present invention further pertains to vectors which contain and express the nucleic acid molecules, especially vectors which express products which elicit an immunological response, preferably a protective immunological response, e.g., a
  • compositions comprising the
  • vectors or expression products therefrom and to methods for making and using such compositions, vectors, expression products, and nucleic acid molecules.
  • the patent and scientific literature includes various vector systems such as virus-
  • recombinant poxvirus e.g., vaccinia, avipox virus
  • exogenous poxvirus e.g., vaccinia, avipox virus
  • exogenous poxvirus e.g., vaccinia, avipox virus
  • Particularly useful poxvirus vectors are NYVAC, NYVAC.l, NYVAC.2,
  • ALVAC, TROVAC and NYVAC were deposited under the terms ofthe Budapest Treaty with the American Type Culture
  • ALVAC host-restricted avipox vector
  • avipoxviruses causing a productive infection in any non-avian species, including man.
  • This host restriction provides an inherent safety barrier to transmission of the virus to other species and makes the use of avipoxvirus-based vaccine vectors in veterinary and human applications an attractive proposition.
  • ALVAC-recombinants expressing the rabies glycoprotein (Taylor et al., 1991, 1995), feline leukemia virus antigens (Tartaglia et al 1993), equine influenza virus hemagglutinin (Taylor et al 1992), and measles virus or canine distemper virus hemagglutinin and neuraminidase glycoproteins (Taylor et al, 1992, Stephensen et al, 1997 respectively).
  • ALVAC recombinants expressing the rabies glycoprotein (Taylor et al., 1991, 1995), feline leukemia virus antigens (Tartaglia et al 1993), equine influenza virus hemagglutinin (Taylor et al 1992), and measles virus or canine distemper virus hemagglutinin and neuraminidase glycoproteins (Taylor et al, 1992, Stephensen
  • U.S. Patent No. 4,769,331 relates to he ⁇ esvirus as a vector. See also Roizman,
  • Epstein-Barr virus vectors are also known. See Robertson et al.
  • the heterologous or exogenous nucleic acid molecule in vectors of the instant invention, preferably encodes an expression product comprising: an epitope of interest or antigen from a paramyxovirus such as PIV2, e.g., an epitope of interest from F, FIN or F and HN,
  • an antigen such as F, HN or F and HN.
  • the vector can also contain a nucleic acid molecule encoding, and express, an additional epitope of interest or antigen, e.g., an epitope of interest or antigen of a pathogen of a host of PIV2 in addition to an antigen or epitope of interest from PIV2, such as an epitope of
  • the vector for instance, the vector
  • rabies antigen or epitope of interest can also encode a Morbillivirus antigen or epitope of interest.
  • CDV canine distemper virus
  • MV measles virus
  • CHV canine he ⁇ esvirus
  • a coronavirus antigen or epitope of interest or a leptospira bacterium antigen or epitope of interest, or any combination thereof
  • the vector encoding a canine adenovirus antigen or epitope of interest can be an adenovirus such as CAV2; and, reference is made to U.S. application Serial No. 08/675,566 and 08/675,556, filed July 3, 1996 and to PCT WO91/1 1525.
  • the vector itself can be a he ⁇ esvirus.
  • compositions comprising vectors expressing rabies antigen(s) or epitope(s) of interest and may
  • a biological response modulator modulates biological activity
  • a biological response modulator is a modulatory component such as a high molecular weight protein associated with non-NMDA excitatory amino acid receptors and which allosteocally regulates affinity of AMPA binding (See Kendrev. , supra)
  • Biological response modulators or modifiers can enhance the lmmunogenicity of the cPIV2 antigen or epitope of
  • Cytokines or costimulators can be considered biological response modulators or biological response modifiers
  • a growth factor can be defined as multifunctional, locally acting intercellular signalling peptides which control both ontogeny and maintenance of tissue and function (see Kendrew, supra, especially at page 455 et seq )
  • the growth factor or therapeutic gene can encode a disease-fighting protein, a molecule for treating cancer, a tumor suppressor, a cytokine, a tumor associated antigen, or interferon, and, the growth factor or therapeutic gene can, for example, be selected from the group consisting of a gene encoding alpha-globin, beta-globm, gamma-globin, granulocyte macrophage-colony stimulating factor, tumor necrosis factor, an mterleukin (e g , an mterleukin selected from interleukins 1 to 14, or 1 to 1 1, or any combination thereof), macrophage colony stimulating factor, granulocyte colony stimulating factor, erythropoietin,
  • a gene encoding alpha-globin, beta-globm, gamma-globin, granulocyte macrophage-colony stimulating factor, tumor necrosis factor, an mterleukin (e g , an mterleukin selected from interleukins
  • a cPIV2 antigen or epitope of interest and/or an additional antigen or epitope of interest as a fusion protein can be a useful way to enhance lmmunogenicity, e g , expression as a fusion lipoprotein with the lipidation enhancing immunogenicity and from a source other than cPIV2 such as OspA o ⁇ Borrelia.
  • co-expression with cholera toxin b (ctxb) is known as useful for enhancing the immunogenicity of an antigen or epitope of
  • vector which contains and expresses an epitope of interest from a paramyxovirus such as cPIV2, e.g., from cPIV2 F, HN or F and HN, and optionally at least one of a second epitope of interest, a biological response modifier or modulator, a growth factor, a recognition sequence, a therapeutic gene, or a fusion protein; or for the skilled artisan to use such a recombinant or vector.
  • a paramyxovirus such as cPIV2
  • a paramyxovirus such as cPIV2 F, HN or F and HN
  • the cells are disrupted and the protein of interest is released into an aqueous "extract".
  • extract There are many methods of cellular disintegration, which vary from relatively
  • Animal tissues vary from the very easily broken erythrocytes to tough collagenous material such as found in blood vessels and other smooth-muscle containing tissue.
  • Bacteria vary from fairly fragile organisms that can be broken up by digestive enzymes or osmotic shock to more resilient species with thick cell walls, needing vigorous mechanical
  • the extract is prepared by centrifuging off insoluble material. At this stage, one may proceed with the purification method, as an extract containing as much of the protein of interest as possible has been prepared, and, where appropriate,
  • Standard techniques of protein purification may be employed to further purify the protein of interest, including: precipitation by taking advantage of the solubility of the protein of interest at varying salt concentrations, precipitation with organic solvents, polymers and other materials, affinity precipitation and selective denaturation; column chromatography, including high performance liquid chromatography (HPLC), ion-exchange, affinity, immuno-affinity or dye-ligand chromatography; immunoprecipitation and the use of gel filtration, electrophoretic
  • the invention further relates to an immunogenic, immunological or vaccine composition containing the recombinant and an acceptable carrier or diluent (e.g. acceptable to the veterinary profession or pharmaceutically acceptable).
  • An immunological composition containing the recombinant (or an expression product thereof) elicits an immunological response - local or systemic. The response can, but need not be protective.
  • An immunogenic composition containing the recombinant (or an expression product thereof) elicits an immunological response - local or systemic. The response can, but need not be protective.
  • inventive recombinants or an expression product thereof likewise elicits a local or systemic immunological response which can, but need not be, protective.
  • composition elicits a local or systemic protective response. Accordingly, the terms “immunological composition” and “immunogenic composition” include a “vaccine composition”
  • the invention therefore also provides a method for inducing an immunological response in a host vertebrate comprising administering to the host an immunogenic,- immunological or vaccine composition composing the inventive recombinant virus or vector and an acceptable earner or diluent
  • animal includes all vertebrate species, except humans, and “vertebrate” includes all vertebrates, including animals (as “animal” is used herein) and humans
  • vertebrate includes all vertebrates, including animals (as “animal” is used herein) and humans
  • a subset of "animal” is "mammal”
  • compositions of the invention such as immunological, antigenic or vaccine compositions or therapeutic compositions
  • parenteral route mtradermal, intramuscular or subcutaneous
  • administration enables a systemic immune response
  • mucosal route e g , oral, nasal, genital, etc
  • inventive antigenic, immunological or vaccine compositions or therapeutic compositions can be prepared in accordance with standard techniques well known to those skilled in the pharmaceutical, medical or veteonary arts Such compositions can be administered in dosages and by techniques well known to those skilled in the medical or veteonary arts taking into consideration such factors as the breed or species, age, sex, weight and condition of the particular patient, and the route of administration
  • the compositions can be administered alone, or can be co-admmistered or sequentially administered with other compositions of the invention or with other immunological, antigenic or vaccine or therapeutic
  • compositions Such other compositions can include pu ⁇ fied native antigens or epitopes. or
  • compositions ofthe invention include liquid preparations for o ⁇ fice.
  • recombinant or vector may be in admixture with a suitable earner, diluent, or excipient such as sterile water, physiological saline, buffered saline, glucose or the like, with or without a preservative.
  • the earner may also be a polymeoc delayed release system, e.g., microcapsules or microencapsulation. See Kreuter, J., Microcapsules and Nanoparticles in Medicine and Pharmacology. M Donbrow (Ed). CRC Press, p. 125-148; Eldridge, J.H., et al
  • Antigenic, immunological or vaccine compositions typically can contain an adjuvant and an amount of the recombinant or vector or expression product to elicit the desired
  • alum aluminum phosphate or aluminum hydroxide
  • Saponin and its puofied component Quil A, Freund's complete adjuvant and other adjuvants used in research and veteonary applications have toxicities which limit their potential use in human vaccines
  • Chemically defined preparations such as muramyl dipeptide, monophosphoryl hpid A, phosphohpid conjugates such as those descobed by Goodman-Snitkoff et al. J. Immunol 147.410-415 (1991) and inco ⁇ orated by reference herein, encapsulation of the protein within a proteohposome as descobed by Miller et al , J Exp Med. 176.1739-1744 (1992) and inco ⁇ orated by reference herein, and encapsulation of the protein in hpid vesicles
  • composition may be packaged in a single dosage form for immunization by
  • parenteral i.e., intramuscular, mtradermal or subcutaneous
  • parenteral i.e., intramuscular, mtradermal or subcutaneous
  • oofice admimstration e g., perlmgual (i.e., oral), intragastoc, mucosal including intraoral, intraanal, lntravagmal, and the like administration
  • the effective dosage and route of administration are determined by the nature of the composition, by the nature of the expression product, by expression level if the recombinant is directly used, and by known factors, such as breed or species, age, sex, weight, condition and nature of host, as well as LD 50 and other screening procedures which are known and do not require undue expe ⁇ mentation
  • Dosages of expressed product can range from a few to a few hundred micrograms. e.g., 5 to 500 ⁇ g.
  • inventive recombinant or vector can be administered in any suitable amount
  • the viral recombinants of the invention can be administered in an amount of about 10 3 5 pfu or greater, thus, the inventive viral recombinant is
  • inventive plasmid or naked DNA in plasmid or naked DNA compositions can be 1 wg to 100 mg, preferably 0 1 to 10 mg. but lower
  • levels such as 0 1 to 2 mg or preferably 1-10 w may be employed
  • the expression product or recombinant or vector may be lyophihzed for resuspension at the time of administration or can be in solution.
  • solid including solid-containing-liquid, liquid, and gel (including "gel
  • compositions are envisioned.
  • the recombinants can be used in any desired immunization or administration regimen; e.g., as part of periodic vaccinations such as annual, semiannual, quarterly, vaccinations as in the veterinary arts or as in periodic vaccinations as in the human
  • an inventive vector or recombinant is administered either before or after the administration ofthe same or of a different epitope of interest or recombinant or vector expressing such a same or different epitope of interest (including a recombinant expressing such a same or different epitope of interest), see, e.g., documents cited herein such as U.S. application Serial Nos. 08/746,668, 08/815,809, 08/816,155, 08/675,556 and 08/675,566.
  • An exemplified dosage regiment is twice within a three week period.
  • the recombinants can reduce the duration of virus isolation following challenge, the recombinants or expression products therefrom can be employed in
  • hosts infected with canine parainfluenza virus (type 2) for treatment e.g., to stimulate the immune system against the virus.
  • type 2 canine parainfluenza virus
  • the skilled artisan can determine dosages and regimens for such treatment from this disclosure and the knowledge in the art, taking into account typical factors such as the breed or species, sex, age, weight, and condition ofthe host to be treated, without undue experimentation.
  • expression products can provide an antigenic, immunological, or protective
  • the invention further relates to uses of the expression products and products therefrom; namely, uses of the expression products as antigens and to antibodies and uses thereof. More in particular, the expression products can elicit antibodies by administration of those products or ofthe recombinants or vectors expressing the products.
  • the antibodies can be monoclonal antibodies; and, the antibodies or expression products can be used in kits, assays, tests, and the like involving binding, so that the invention relates to these uses too.
  • Monoclonal antibodies can be employed in well known antibody binding assays, diagnostic kits or tests to determine the presence or absence of antigen(s) or the presence or absence ofthe natural causative agent ofthe antigen or, to determine whether an immune response to that agent or to the antigen(s) has simply been stimulated, see, e.g., U.S. Patent Nos. 4,376,110 and 4,486,530. Monoclonal antibodies have also been used to recover materials by immunoadso ⁇ tion chromatography, e.g. Milstein, C, 1980, Scientific American 243:66, 70, inco ⁇ orated herein by reference. As regards the use of immunochromatography methods, the skilled artisan can be employed in well known antibody binding assays, diagnostic kits or tests to determine the presence or absence of antigen(s) or the presence or absence ofthe natural causative agent ofthe antigen or, to determine whether an immune response to that agent or to the antigen(s) has simply been stimulated, see, e.g., U.S. Patent Nos.
  • EP-A-299 428, EP-A-291 194, EP-A-294 232 U.S. Patents Nos. 5,120,643, 5,030,558, 5,266,497, 4,740,468, 5,266,497, 4,855,240, 5,451,504, 5,141,850, 5,232,835 and 5,238,652.
  • the invention relates to the inventive recombinants as vectors and methods for
  • the resultant DNA can be used as probes or primers, e.g., to detect a pathogen in a sample, or for amplification.
  • inventive recombinants or vectors or expression products therefrom can be used to stimulate a response in cells in vitro or ex vivo for subsequent reinfusion into a patient.
  • the reinfusion is to stimulate an immune response, e.g., an immunological or antigenic response such as active immunization.
  • the reinfusion is to stimulate or boost the immune system against a pathogen.
  • MDCK cells (ATCC #CCL34) were infected with cPIV2 (strain D-008) which is
  • Vaccine Recombitek C4 a vaccine strain marketed as Vaccine Recombitek C4 by Merial, Lyon, France. Infected cells were extracted for total RNA using the TRI reagent protocol (Molecular Research Center,
  • Primer CPIV6 (SEQ ID NO: 1) covers 19 base pairs at the 5' end of the cPIV F gene and the 28 most 3' bases of the H6 promoter ending at the Nrul site.
  • Primer CPIV 13 (SEQ ID NO: 2) encompasses 21 base pairs at the 3' end of the F coding sequence and a Xhol site. Derivation of the H6 promoter has been described (Perkus et al, 1989).
  • Primer pairs CPIV6 and CPIV 13 were used in a PCR reaction using the first strand cDNA as template to amplify a 1600 base pair fragment.
  • the PCR fragment pooled from four separate reactions, was digested with Nrwl and Xhol and ligated into the insertion plasmid
  • H6CP3LSA-2 (see USS ⁇ 08/816,155, filed March 12, 1997) which directs insertion into the C3 locus and which had been digested with the same enzymes.
  • the ligation of the PCR fragment (which had been digested with Nrul and Xhol) into the C3 insertion plasmid (H6Cp3LSA-2) digested with the same enzymes resulted in the generation of plasmid pC3F32 (SEQ ID NO: 3).
  • Primer cPIV-1 (SEQ ID NO: 4) spans the region from the EcoRV site of the H6 promoter through 19 bases at the 5' end of the cPIV HN
  • Primer cPrV-11 (SEQ ID NO: 5) encompasses 21 bases at the 3' end of the HN coding
  • Primer pairs 1 and 11 were used in a PCR reaction using the first-strand DNA as a template to amplify a 1700 bp fragment.
  • the PCR fragment was pooled from 4 separate reactions, gel purified, digested with EcoRV and Kpnl and ligated into the C5 insertion plasmid (see U.S. Patent No. 5,494,807 and allowed application Serial No. 08/184,009, filed January 19,
  • oligonucleotides cPIV35 S ⁇ Q ID NO: 7
  • cPIV 36 S ⁇ Q ID NO: 8
  • Primer cPIV35 spans the EcoRV site in the H6 promoter through 80 bases at the 5' end of the cPIV HN gene. The sequence changes a T to an A in the T5NT signal (underlined in primer cPIV35) without changing the amino acid composition, and creates an Sspl site to aid in screening.
  • Primer cPIV36 spans a unique Bell site 1 140 bases
  • T5NT sequence has been removed was obtained by taking appropriate fragments from two
  • Plasmid pC5CPIVHN was digested with Pad and Kpnl to isolate a 1700bp fragment containing the HN gene linked to the H6 promoter. This fragment was then blunt-ended using the Klenow fragment
  • plasmid pC3F32 contains the F and HN genes in a tail to tail configuration in the C3 insertion locus.
  • Plasmid pC3F32 was used in recombination with ALVAC as the rescuing virus to derive recombinant vCP1472 expressing the cPIV2 (canine parainfluenza (type 2)) F gene and
  • plasmid pC3CPIVFHN was used in recombination with ALVAC to derive recombinant vCP1485 expressing both F and HN genes using methods described in Piccini et al (1987). Expression of appropriate gene products was confirmed by immunoprecipitation analysis in the A-72 canine tumor cell line (ATCC CRL 1542) as described in Tartaglia et al (1992). Briefly, cells were lysed and cPIV2-specific gene products were immunoprecipitated using a polyclonal immune serum from dogs (VMRD Inc.
  • ALVAC-cPIV2 F+HN (vCP1485) was demonstrated as follows. Groups of two or three dogs with or without prior exposure to cPIV2 were vaccinated by the subcutaneous route with either 5.5 log, 0 pfu or 7.0 log )0 pfu of vCP1485, using two
  • MDCK cells were infected w ith cPIY2 (strain D-00S). a vaccine strain marketed as Vaccine Recombitek C4 by Merial, Lyon. France. Infected cells were extracted for total RN ⁇ using the TRI reagent protocol (Molecular Research Center. Cincinnati. OH) Using random hexamers (Perkin Elmer. Foster . CA) as primers, a first strand cDNA preparation w as made
  • Primer TAV 1 P (SEQ ID ⁇ 0 ' 9) cov ers I base pairs at the 5' end of the cPIV F gene and a Sail restriction site
  • Primer TAY 1 I S (SEQ ID NO: 10) encompasses 18 base pairs at the 3 * end of the F coding sequence and a ⁇ ' o/1 restriction site.
  • Primer pairs TAY 117 and TAY 1 18 were used in a PCR reaction using the first strand cDNA as template to amplify a 1600 base pair fragment.
  • the PIV2 F coding sequence begins at position 300 and ends at position 1889.
  • pCR2.1 sequences are located between positions 293 and 1898.
  • Primer TAY 119 (SEQ ID NO: 12 ) covers 18 bases at the 5' end of the cPIV HN gene and a Sail restriction site.
  • Primer TAY 120 (SEQ ID NO: 13) encompasses 18 base pairs at the 3' end of the HN coding sequence and a NotI restriction site.
  • Primer pairs TAY 119 and TAY 120 were used in a PCR reaction using the first strand DNA as a template to amplify a 1700 bp fragment.
  • the PCR fragment was pooled from 4 separate reactions and cloned into the TA vector system using plasmid pCR2.1. Following transformation and screening of minipreps, four colonies were amplified, sequenced and a consensus obtained in plasmid pJT007 (Fig. 2; SEQ ID NO: 14).
  • the PIV2 HN coding sequence begins at position 300 and ends at position 1997.
  • the pCR2.1 sequences are located between positions 293 and 2006.
  • pJT008 (Fig. 3; SEQ ID NO: 11) is digested with Sail and Notl and the 1597 base pair fragment is isolated and cloned
  • Baculovirus recombinants are generated using the BAC-TO-BAC system (Gibco- BRL, Gaithersburg, MD). Briefly, the pFastBac donor plasmids described above, pJT009 and pJTOlO, are transfected into an E. coli host containing a circular baculovirus genome (bacmid).
  • Recombinants are formed by Tn7 transposition, which inserts the foreign sequence into the baculovirus DNA within the bacteria. Bacteria are screened for correct insertion of foreign sequences and recombinant bacmid DNA is isolated. Insect cells are transfected with the recombinant bacmid DNA to generate recombinant baculoviruses, vBACCPIVF and vBACCPIVHN, containing F and HN coding sequences, respectively. Recombinants are purified by several cycles of plaque purification on insect cells followed by amplification to produce a stock virus.
  • SF9 cells are infected with vBACCPIVF and vBACCPIVHN at a multiplicity of 5 pfu per cell. After 48 hours infection, infected cells are lysed and cPIV2-
  • HN-specific monoclonal antibodies designated PI2 2018 H7K
  • PI2 1015 E5B and P12 208 A3 obtained from Merial, Lyon, France. These reagents are used to demonstrate expression ofthe fusion FI and F2 cleavage products and the formation of a
  • hemagglutinin-neuraminidase protein of appropriate molecular weight.
  • Recombinants containing and expressing F or HN can be analogously obtained.
  • va ⁇ ety of infectious diseases see also McClements et al , "Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of he ⁇ es simplex v ⁇ rus-2 disease," PNAS US A 93.11414- 11420,
  • NYVAC, NYVAC.l, NYVAC.2 and TROVAC recombinants and plasmid DNA recombinants, containing and expressing cPIV2 F, HN, and F and HN and DNA encoding at least one or more
  • At least one epitope of interest e.g., from an antigen of a pathogen of a host of PIV, e.g., DNA encoding at least one antigen or epitope of interest from rabies, Morbillivirus (CDV and or MV), CHV, canine adenovirus, parvovirus, coronavirus, and leptospira bacterium; a biological response modifier or modulator, a growth factor, a recognition sequence, a therapeutic gene, or a fusion protein are generated; e.g., recombinants containing and expressing cPIV2 F, HN, and F and HN with one or more of DNA encoding at least one antigen or epitope of interest from rabies, Morbillivirus (CDV and or MV), CHV, canine adenovirus, parvovirus, coronavirus, and leptospira bacterium, and optionally with a cytokine and/or costimulator or
  • combination compositions comprising a vector or vectors expressing rabies ant ⁇ gen(s) or ep ⁇ tope(s) and cPIV2 F, HN or F and HN, and optionally one or more of: at least one antigen or epitope of interest from a Morbillivirus (CDV and or MV), CHV, canine adenovirus, parvovirus, coronavirus, and
  • CDV and or MV Morbillivirus
  • CHV canine adenovirus
  • parvovirus coronavirus
  • leptospira bacterium a biological response modifier or modulator, a growth factor, a recognition sequence, a therapeutic gene, or a fusion protein are generated; e g., recombinants containing and expressing cPIV2 F, FIN, and F and HN with one or more of DNA encoding at least one antigen or epitope of interest from rabies, Morbillivirus (CDV and/ or MV), CHV, canine adenovirus.
  • CDV Morbillivirus
  • CHV canine adenovirus
  • parvovirus, coronavirus, leptospira bacteoum, and optionally with a cytokine and or costimulator or a lipidation region (i.e., as a fusion protein lipoprotein) to enhance immunogenicity are generated and used in a combination composition, or a ⁇ ector expressing rabies ant ⁇ gen(s) or ep ⁇ tope(s) and recombinantly expressed cPIV2 F, HN, and F and HN and optionally one or more of.
  • an antigen or epitope of interest from one or more of Morbillivirus (CDV and/or MV), CHV,
  • a growth factor or modulator, a growth factor, a recognition sequence, a therapeutic gene, or a fusion protein
  • antigens or epitopes of interest are generated; e g , recombinants containing and expressing rabies antigen(s) or epitopes and recombinantly expressed cPIV2 F, HN, and F and HN with one or more of at least one antigen or epitope of interest from Morbillivirus (CDV and/or MV), CHV, canine adenovirus, parvovirus, coronavirus, leptospira bacteoum, and optionally a cytokine and/or costimulator or a lipidation region to enhance immunogenicity, are generated and employed in a combination composition.
  • the combination compositions are administered to a suitable host, e.g., a canine such as a pup or dog, in a suitable dosage, and can induce an immunological response in a suitable host against cPIV and the pathogen of the at least one
  • antigen or epitope of interest therein, e.g., rabies, Morbillivirus (CDV and/or MV),
  • CHV canine adenovirus, parvovirus, coronavirus, leptospira bacteoum.
  • vaccine an alternative poxvirus vector system. Vaccine 14 (5): 428-434.
  • CDV Canine distemper virus infection of ferrets as a model for testing Morbillivirus vaccine strategies: NYVAC- and ALVAC-based CDV recombinants protect against symptomatic infection. J. Virology. 71 : 1506-1513.
  • Nonreplicatmg viral vectors as potential vaccines recombinant canarypox virus expressing measles virus fusion (F) and hemagglutinin (HA) glycoproteins

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Abstract

L'invention concerne des séquences nucléiques isolées codant les protéines visées du virus parainfluenza 2 canin (par exemple, F, HN, F & HN), des vecteurs contenant et exprimant lesdites séquences, des compositions à base de ces vecteurs et/ou produits d'expression, et des procédés relatifs à l'élaboration et à l'utilisation des séquences, des vecteurs et des compositions en question. Il peut s'agir de n'importe quel vecteur approprié, y compris le poxvirus des canaris ALVAC, un baculovirus, un vecteur à base de plasmide d'ADN, ou un adénovirus comme l'adénovirus canin de type 2 (CAV2). Les produits d'expression issus des vecteurs (par exemple, expression in vivo ou in vitro) réduisent la durée de l'isolation du virus après test de provocation, ce qui donne une amélioration surprenante, inattendue et importante. Les vecteurs ou produits d'expression résultants peuvent aussi être utilisés dans des compositions de combinaison. Par exemple, les vecteurs peuvent exprimer de manière additionnelle un ou plusieurs antigènes ou épitopes visés dans un virus morbilleux (virus de la maladie de Carré chez le chien, virus de la rougeole et autres), un virus de la rage, un herpèsvirus canin, un parvovirus (parvovirus canin, entre autres), un adénovirus canin, un coronavirus (coronavirus canin, entre autres), ou une bactérie de leptospirose; et, éventuellement, une cytokine ou un costimulateur ou une région dotée de lipides, afin d'améliorer l'immunogénicité. Les vecteurs considérés peuvent être utilisés dans des compositions immunologiques, des compositions immunogènes ou des compositions de vaccins.
PCT/US1998/025203 1997-12-09 1998-12-04 Molecules d'acide nucleique codant des proteines f et hn de virus parainfluenza 2 canin Ceased WO1999029872A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002086068A3 (fr) * 2001-04-20 2003-05-30 Univ North Carolina Procedes de production de coronavirus recombinant
US7618802B2 (en) 2003-07-21 2009-11-17 The University Of North Carolina At Chapel Hill Compositions of coronaviruses with a recombination-resistant genome
US7682619B2 (en) 2006-04-06 2010-03-23 Cornell Research Foundation, Inc. Canine influenza virus
CN110302374A (zh) * 2019-08-09 2019-10-08 中国农业科学院特产研究所 犬用疫苗及其制备方法和应用
CN118406158A (zh) * 2024-04-26 2024-07-30 泰州博莱得利生物科技有限公司 一种犬瘟热病毒抗原表位融合蛋白及其免疫球蛋白制备方法和应用

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Title
BATY DU ET AL: "Sequence comparison between the haemagglutinin-neuraminidase genes of simian, canine and human isolates of simian virus 5", JOURNAL OF GENERAL VIROLOGY, vol. 72, no. 12, December 1991 (1991-12-01), READING GB, pages 3103 - 3107, XP002099274 *
PAOLETTI E ET AL: "Higly attenuated poxvirus vectors: NYVAC, ALVAC and TROVAC", DEVELOPMENTS IN BIOLOGICAL STANDARDIZATION, vol. 84, 1995, pages 159 - 163, XP002048184 *
STEPHENSEN CB ET AL: "Canine Distemper Virus (CDV) infection of ferrets as a model for testing Morbillivirus strategies: NYVAC- and ALVAC-based CDV recombinants protect against symptomatic infection", JOURNAL OF VIROLOGY, vol. 71, no. 2, February 1997 (1997-02-01), AMERICAN SOCIETY FOR MICROBIOLOGY US, pages 1506 - 1513, XP002099275 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002086068A3 (fr) * 2001-04-20 2003-05-30 Univ North Carolina Procedes de production de coronavirus recombinant
US7618802B2 (en) 2003-07-21 2009-11-17 The University Of North Carolina At Chapel Hill Compositions of coronaviruses with a recombination-resistant genome
US7682619B2 (en) 2006-04-06 2010-03-23 Cornell Research Foundation, Inc. Canine influenza virus
CN110302374A (zh) * 2019-08-09 2019-10-08 中国农业科学院特产研究所 犬用疫苗及其制备方法和应用
CN110302374B (zh) * 2019-08-09 2023-04-14 黑龙江八一农垦大学 犬用疫苗及其制备方法和应用
CN118406158A (zh) * 2024-04-26 2024-07-30 泰州博莱得利生物科技有限公司 一种犬瘟热病毒抗原表位融合蛋白及其免疫球蛋白制备方法和应用

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