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WO1993023422A1 - Compositions et procedes de vaccination contre les coronavirus - Google Patents

Compositions et procedes de vaccination contre les coronavirus Download PDF

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Publication number
WO1993023422A1
WO1993023422A1 PCT/US1993/004384 US9304384W WO9323422A1 WO 1993023422 A1 WO1993023422 A1 WO 1993023422A1 US 9304384 W US9304384 W US 9304384W WO 9323422 A1 WO9323422 A1 WO 9323422A1
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thr
val
asn
ser
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Inventor
Timothy J. Miller
Sharon Klepfer
Albert Paul Reed
Elaine V. Jones
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SmithKline Beecham Corp
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SmithKline Beecham Corp
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Priority to EP93911185A priority Critical patent/EP0640097A4/fr
Priority to AU42410/93A priority patent/AU678971B2/en
Priority to CA002134898A priority patent/CA2134898A1/fr
Priority to JP6503668A priority patent/JPH08501931A/ja
Publication of WO1993023422A1 publication Critical patent/WO1993023422A1/fr
Anticipated expiration legal-status Critical
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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates generally to compositions useful for vaccination against, and treatment of, coronavirus infections, as well as methods for using these compositions.
  • Feline Infectious Peritonitis Virus is a coronavirus which causes a highly lethal immune complexing disease in cats.
  • the exact mechanism(s) of FIP disease pathogenesis is not understood, but lesions are found in all major internal organs due to the deposition of immune complexes.
  • Several strains of FIPV have been isolated from diseased cats. Some are highly virulent and cause disease after a primary infection (Type II). Other virulent strains (Type I) cannot initiate disease unless the cat has been previously exposed to FIPV.
  • Feline enteric coronavirus was isolated from cats but differs in pathogenesis from FIPV.
  • FIPV Feline enteric coronavirus
  • FECV in contrast to FIPV, specifically infects intestinal epithelial cells and causes only a mild enteritis in cats.
  • FECV has not been reported to sensitize cats to disease following challenge with FIPV.
  • coronaviruses have been isolated from other animal species, i.e., transmissible gastroenteritis virus of swine (TGEV), porcine respiratory coronavirus (PRCV), bovine coronavirus (BCV), avian coronavirus (ACV), murine coronavirus (MCV) and human coronavirus (HCV).
  • TGEV transmissible gastroenteritis virus of swine
  • PRCV porcine respiratory coronavirus
  • BCV bovine coronavirus
  • ACMV avian coronavirus
  • MCV murine coronavirus
  • HCV human coronavirus
  • Coronaviruses encode three structural proteins: S - surface glycoprotein or spike; M - the envelope matrix protein; and N - the nucleoprotein which interacts with the RNA genome to make the viral capsid.
  • S protein induces an immune response including the production of neutralizing antibodies following infection. It is also responsible for attachment to infected cells and mediates cell fusion, aiding spread of the virus.
  • the S protein is the primary focus for effective coronavirus vaccine development because it is the target of virus neutralizing antibodies and it contains sensitizing epitopes.
  • no other FIPV gene products M or N have been found to stimulate effective immunity to that virus.
  • Type II wild-type FIPV strains are highly virulent in cats; the S gene alone of a virulent FIPV can sensitize cats to disease. A strong humoral response to the S gene appears responsible for the immune complexes found in diseased cats. However, little is known about the location of antigenic/neutralizing epitopes on the FIPV S gene.
  • Recombinant vaccines have been engineered which consist of FIPV gene products produced in a heterologous expression system.
  • Vennema et al, J. Virol., 64:1407-1409 (1990) constructed vaccinia virus recombinants expressing the S, M, and N genes of FIPV (Type II, WSU 1146 strain).
  • Cats immunized with the recombinant vaccinia virus expressing the S gene developed FIP and died sooner than non-vaccinated animals following FIPV challenge.
  • the vaccinia recombinants expressing the FIPV M or N proteins neither conclusively protected nor sensitized cats to FIPV. While no protection from challenge was observed, these results clearly illustrate that the S gene is implicated in FIP disease development.
  • the present invention provides chimeric coronavirus spike (S) proteins which are useful in treating and preventing coronavirus infections.
  • S coronavirus spike
  • a chimeric S protein of this invention can induce an enhanced response to a single coronavirus strain or can elicit an immune response to more than one coronavirus strain and/or species.
  • the present invention provides a polynucleotide sequence which encodes a chimeric S protein or S fragment of the invention.
  • the present invention provides a recombinant polynucleotide molecule comprising a chimeric S gene polynucleotide sequence in operative association with regulatory sequences capable of directing the replication and expression thereof in a selected host cell.
  • the present invention provides a host cell transformed with a recombinant polynucleotide molecule as described above.
  • the invention provides a pharmaceutical or vaccinal composition
  • a pharmaceutical or vaccinal composition comprising an effective or immunogenic amount of one or more of the chimeric coronavirus S proteins or S protein fragments of the invention or fragments thereof in a suitable carrier.
  • These chimeric coronavirus S proteins are useful in the treatment or prophylaxis of a disease caused by the coronavirus from which its fragments are derived and may induce cross-strain and cross-species responses.
  • the invention provides a method for vaccinating a naive animal against a coronavirus, or treating an infected animal for coronavirus infection, both by administering an effective dosage of a pharmaceutical composition of the invention to the animal.
  • the present invention provides a method and a diagnostic kit for use in clinical laboratories, for distinguishing between animals vaccinated with a chimeric S protein of the invention and an animal which has been naturally exposed to the coronavirus or coronaviruses which comprise the chimeric S protein.
  • the present invention provides novel chimeric coronavirus S proteins useful for vaccination against, and treatment of, coronavirus infection, as well as methods for producing these proteins and pharmaceutical compositions containing them. Methods for using these novel chimeric proteins in the identification of the immune sensitizing and protective epitopes of the coronavirus spike protein are also disclosed.
  • chimeric coronavirus S proteins include proteins composed of an amino acid sequence encoded by a whole or a partial S gene sequence from a selected coronavirus fused in any order to one or more additional amino acid sequence (s) encoded by a whole or partial S gene sequence from a selected coronavirus.
  • these chimeric proteins may also contain one or more non-S protein or non-coronavirus protein sequences fused to the chimeric protein, S protein amino acid sequences or fragments thereof.
  • a chimeric coronavirus S protein according to this invention may contain a first coronavirus s protein fragment from a selected coronavirus and at least one additional coronavirus S protein fragment from a selected coronavirus, with the fragments fused in any desired order by an optional amino acid linker, the protein fragments forming the desired chimeric protein being isolated or derived from at least two different strains or species of coronavirus.
  • a chimeric coronavirus S protein according to this invention contains a first coronavirus S protein fragment from a selected coronavirus fused in any order to at least one discontinuous coronavirus S protein fragment (i.e., the fragments are encoded by different regions of the S gene) from the same selected coronavirus, the coronavirus contributing the fragments being a Type I FIPV strain, a Type II FIPV strain, a FECV strain, a CCV strain, a PRCV strain, an avian coronavirus strain, a human coronavirus strain, a bovine coronavirus strain and a murine coronavirus strain.
  • these amino acid sequences forming the chimeric protein are fused in such a way that they are translated in the same reading frame and form a protein, i.e. fused in frame. See, for example, A. Shatzman and M. Rosenberg, Hepatology, 7(1):30S-35S (1987) and G.M. Weinstock et al, Proc. Natl. Acad. Sci.. 80:4432-4436 (July 1983). Fusion can be direct, i.e., S protein amino acid sequence to S protein amino acid sequence, or indirect, i.e., through another amino acid sequence which can serve as a spacer or linker.
  • an amino acid fragment as it relates to amino acid sequences of the S protein refers to any amino acid sequence from at least about 8 amino acids in length up to about the full-length S gene protein (about 1454 amino acids).
  • a nucleotide fragment as it relates to nucleotide sequences of a coronavirus S gene defines a nucleotide sequence which encodes from at least about 8 amino acids in length up to about the full-length S gene protein. Preferably, these fragments are immunogenic.
  • immunogenic means any molecule, protein, peptide, carbohydrate, virus, or region thereof which is capable of eliciting a protective immune response in a host into which it is introduced.
  • antigenic refers only to the ability of a molecule, protein, peptide, carbohydrate, virus, or region thereof to elicit antibody formation in a host (not necessarily protective).
  • region refers to all or a portion of a gene or protein. Such a region may contain one or more fragments as defined above.
  • epitope refers to a region of a protein which is involved in its immunogenicity, and can include regions which induce B cell and/or T cell responses.
  • B cell site or T cell site defines a region of the protein which is a site for B cell or T cell binding. Preferably this term refers to sites which are involved in the immunogenicity of the protein.
  • a sensitizing region is defined as a region of the coronavirus S gene which, when used as an immunogen, results in exacerbating disease following challenge, theoretically by causing a strong humoral response.
  • S protein fragments from a desired coronavirus can be selected from a desired coronavirus to construct a chimeric protein of this invention.
  • Particularly desired coronaviruses for use in supplying S protein, fragments for the chimeric proteins of this invention include FECV, and FIPV strains referred to as DF2, DF2-HP, TS-BP, TS, TN406, UCD-2, UCD-1, UCD-3, UCD-4.
  • FIPV strains may also be useful in providing S protein fragments.
  • other coronavirus species including CCV, TGEV, PRCV, BCV, ACV, MCV, or HCV are expected to be useful in supplying S protein fragments for the chimeric S proteins of this invention.
  • CCV CCV
  • TGEV CCV
  • PRCV PRCV
  • BCV BCV
  • ACV ACV
  • MCV MCV
  • HCV HCV
  • S protein sequences or partial S protein sequences and the corresponding nucleotide sequences of several of the above-identified coronaviruses and their respective SEQ ID NOS are provided in Table I below.
  • Amino acid 1 refers to the first amino acid of the S protein.
  • Those proteins having 1454 or (for canine CCV, 1452) amino acids are full-length S proteins. The remaining proteins are partial S proteins. TABLE I
  • Canine CV 1-1452 18 17 The inventors have defined several immunogenic sites on the DF2 FIPV S protein for use in the chimeric proteins of this invention and have identified corresponding regions in other related coronaviruses which are anticipated to be useful in the same manner.
  • the following information, including the sequence listing and tables herein, provide predicted immunogenic regions of the coronaviruses spike genes illustrated herein.
  • One of skill in the art, provided with this disclosure and the predicted regions could readily select suitable immunogenic regions for use in the chimeric proteins of the invention from any desirable coronavirus.
  • the fragments based on amino acid position and the associated SEQ ID NOS from the S proteins from which they are derived are set out in Table II for B cell site and T cell site fragments.
  • the sensitizing regions on the coronavirus S gene for feline as well as other species coronaviruses are predicted to lie within the regions of antigenicity mapped between avirulent FECV and virulent FIPV. If antibodies to such regions are found to be protective, these regions can be useful in vaccine compositions.
  • One antigenic region is a major neutralizing site in the area of amino acid residues #525-650 of DF2 FIPV.
  • This sequence and its analogous sequences on FECV, FIPV (DF2-HP, TS, and TS-BP), CCV and other coronaviruses is a desirable S protein fragment for use in a chimeric" protein of this invention.
  • Another major antigenic site was mapped to amino acids #350-550 and #1170-1190 of DF2-FIPV. Again, FIPV (DF2-HP, TS, and TS-BP), FECV and other coronaviruses are believed to contain an antigenic site in these regions. Thus, these sequences are another desirable region for an S protein fragment for use in a chimeric protein of this invention.
  • VN virus neutralization
  • Antibodies to the non-neutralizing antigenic epitopes may be responsible for exacerbation of disease. Any sites, neutralizing or non-neutralizing, which contribute to sensitization are desirable regions for deletion from a chimeric S gene of the invention.
  • Certain chimeric proteins of the present invention can also contain epitopes which do not stimulate antibody production but rather induce T cell immunity, i.e., T cell sites.
  • T cell sites The majority of the T cell sites predicted by the inventors and identified in Table II below lie within the C-terminus of the surface glycoprotein. The other coronaviruses are expected to have T cell sites in approximately these regions.
  • S fragments suitable for use in chimeric S proteins of this invention may be selected using the information provided in the present disclosure and conventional computer programs which predict T cell sites on a protein using several parameters. Suitable exemplary programs include EpiPlot, (Louis Menendez-Arias, University of Madrid), the GCG Program, and a T Cell predictive program available from Robert Lew (University of Massachusetts). As can be seen from the attached Sequence Listing with reference to Table II, in the listed S protein T cell sites, DF2-HP sequences are identical to DF2 insofar as DF2-HP has been sequenced; and TS-BP sequences are identical to TS insofar as TS-BP has been sequenced.
  • the amino acid positions provided correlate identically to the S protein and the sequence listing number.
  • S protein fragment reported in SEQ ID NO: 10 actually spans amino acids 102-223 of the S protein.
  • those amino acid positions are reported as 1-122. Therefore, the fragment identified as amino acid 38-50 of SEQ ID NO: 10 corresponds to amino acid 139-151 of the TN406 S protein.
  • the fragment identified as amino acid 59-72 of SEQ ID NO: 10 corresponds to amino acid 160-173 of the TN406 S protein.
  • additional B and T cell sites can be predicted based upon analogy to the sequences herein.
  • these fragments may be selected from previously amplified regions of feline coronaviruses.
  • the amplification procedure and suitable fragments are described in more detail in co-owned, co-pending U.S. Patent Application Ser. No. 07/698,927, and its corresponding published International Application No. WO92/08487, published May 29, 1992 which is incorporated by reference herein.
  • Additional exemplary fragments useful in making chimeric proteins of the invention include those fragments identified in the examples, and other fragments, all of which are set forth in Table III below. Fragments similar to the fragments of Table III can be identified in other related coronaviruses.
  • chimeric proteins of the invention are capable of eliciting an enhanced immune response against one coronavirus strain and/or an immune response to more than one coronavirus strain or species. These chimeric proteins are also believed to be capable of eliciting an immune response in more than one animal species.
  • the chimeric protem be a full length S protein.
  • the chimeric proteins of this invention may be as small as about 16 amino acids in length, or may be as large as at least 2 full length S proteins. It is anticipated that useful chimeric proteins will be within the above stated lower and upper amino acid lengths.
  • a chimeric protein can comprise one or more protein or peptide segments of FIPV S protein fused to one or more protein or peptide segments of FECV S protein to form a full length, chimeric S protein. See Tables I, II or III for relevant SEQ ID NOS.
  • One specific chimeric protein is formed by the fusion of an S gene protein fragment from amino acid #1-311 of a selected strain of FIPV S protein to the carboxy terminal amino acids #311-1454 of FECV S protein.
  • Another such chimeric protein is formed by the fusion of the N terminal fragment of the FECV S protein (from about amino acid #1-311) to the carboxy terminal protein sequence (amino acid #311-1454) of a selected FIPV strain.
  • amino acid 311 is present only once (i.e., the total chimeric is 1454 amino acids in length).
  • the chimeric proteins of the invention consist of a fragment of the FECV S protein inserted into the FIPV S protein to replace a homologous fragment thereof, so that both the amino and carboxy terminal regions are FIPV S protein fragments.
  • the reverse construct can be made, so that both the amino and carboxy terminal S protein fragments are FECV S gene protein fragments.
  • Example 4 Such chimeric constructs are illustrated in Example 4 below.
  • Another currently preferred embodiment of the chimeric protein of the invention comprises the fusion of desirable B and T cell sites, as identified above.
  • Particularly suitable regions of a feline or canine coronavirus include the B cell sites provided in Table II above.
  • an exemplary chimeric S gene may comprise FECV amino acids 1-542 fused to a selected FIPV strain amino acids 542-597, fused to FECV 598-1454.
  • the chimeric is 1454 amino acids in length;
  • chimerics may be constructed by inserting one or more of the above-identified amino acid regions from one coronavirus into the corresponding region of another coronavirus.
  • Other chimeric S proteins of the invention can be made using any of the feline coronavirus strains identified herein or by substituting any of the other species coronaviruses for either coronavirus S protein fragment of the constructs described above.
  • a chimeric S protein of the invention may comprise a selected strain of FIPV amino acids 1-311 fused to one or more of the following T cell amino acid regions identified in Table II. Because these T cell regions are derived largely from the conserved C terminus, it is expected that a strong cross-species T cell response will be elicited, particularly when the T cell site is fused to a B cell site. Thus, it is expected that even if these B and T cell sites are from the same coronavirus strain, the chimeric will elicit a cross-species response.
  • chimerics of the invention which comprise less than full length S proteins are those containing a deletion corresponding to one or more B cell sites, sensitizing regions, or other non-critical fragments of the S protein identified herein.
  • a chimeri ⁇ S protein of the invention may be an S protein which has been truncated at the amino or carboxyl termini, provided that the immunogenicity of the S gene protein is not lost.
  • these chimerics include:
  • FIPV 594-1454 e.g., DF2 or TS
  • chimeric coronavirus S proteins are provided by the fusion of FIPV (Type I , Type II or both) and FECV S protein fragments for use in compositions to be administered to felines.
  • FIPV Type I , Type II or both
  • FECV S protein fragments for use in compositions to be administered to felines.
  • Another composition which is anticipated to be useful is a chimeric feline/canine coronavirus S protein.
  • Such a chimeric may comprise FIPV and/or FECV proteins fused in frame to CCV protein(s).
  • chimerics which may be useful in preventing disease in animals, and particularly in more than one animal (cross-species protection), include fusions of S protein fragments from FIPV/TGEV, FECV/TGEV, FECV/CCV, FIPV/FECV/ TGEV, FIPV/TGEV/PRCV, ACV/TGEV, PRCV/ACV/TGEV, or BCV/ACV/ TGEV.
  • FIPV/TGEV FIPV/TGEV
  • FIPV/TGEV/PRCV FIPV/TGEV/PRCV
  • ACV/TGEV PRCV/ACV/TGEV
  • BCV/ACV/TGEV BCV/ACV/ TGEV
  • the chimeric S proteins of the invention may be fused to a fusion partner, e.g, a larger, carrier molecule.
  • the fusion partner may be a preferred signal sequence, a sequence which is characterized by enhanced secretion in a selected host cell system, or a sequence which enhances the stability of the S-derived peptide.
  • exemplary fusion partners include, without limitation, ubiquitin and ⁇ - mating factor for yeast expression systems, and ⁇ -galactosidase and influenza NS-1 protein for bacterial systems.
  • One of skill in the art can readily select an appropriate fusion partner.
  • the present invention is not limited to the use of any particular fusion partner.
  • any of the above-identified S protein fragments which can be useful in a chimeric protein may optionally be fused to each other or to a fusion partner through a conventional linker sequence, i.e., containing about 1 to 50 amino acids, and more preferably, about 2 to about 20 amino acids in length.
  • This optional linker may provide space between the two linked sequences.
  • this linker sequence may encode, if desired, a polypeptide which is selectively cleavable or digestible by conventional chemical or enzymatic methods.
  • the selected cleavage site may be an enzymatic cleavage site, including sites for cleavage by a proteolytic enzyme, such as enterokinase, factor Xa, trypsin, collagenase and thrombin.
  • a proteolytic enzyme such as enterokinase, factor Xa, trypsin, collagenase and thrombin.
  • the cleavage site in the linker may be a site capable of being cleaved upon exposure to a selected chemical, e.g., cyanogen bromide or hydroxylamine.
  • the cleavage site if inserted into a linker useful in the fused sequences of this invention, does not limit this invention. Any desired cleavage site, of which many are known in the art, may be used for this purpose.
  • nucleic acid sequences include those sequences capable of hybridizing to the relevant S gene fragments disclosed herein under conditions of at least 85% stringency, i.e. having at least 85% homology to the selected S gene fragment encoding a portion of the chimeric protein, more preferably at least 90% homology, and most preferably at least 95% homology.
  • allelic variations naturally-occurring base changes in the species population which may or may not result in an amino acid change
  • DNA sequences encoding the various S gene amino acid or DNA sequences from the illustrated coronaviruses are also included in the present invention, as well as analogs or derivatives thereof.
  • DNA sequences which code for protein sequences of the invention but which differ in codon sequence due to the degeneracies of the genetic code or variations in the DNA sequence encoding these proteins which are caused by point mutations or by induced modifications to enhance the activity, half-life or production of the peptide encoded thereby are also encompassed in the invention.
  • Variations in the amino acid sequences of this invention may typically include analogs that differ by only 1 to about 4 codon changes at the nucleotide level.
  • Other examples of analogs include polypeptides with minor amino acid variations from the natural amino acid sequence of S gene proteins and/or the fusion partner; in particular, conservative amino acid replacements. Conservative replacements are those that take place within a family of amino acids that are related in their side chains.
  • the chimeric proteins or coronavirus fragments of the invention can be prepared by chemical synthesis techniques. Preferably, however, they are prepared by using known recombinant DNA techniques by cloning and expressing within a host microorganism or cell a DNA fragment carrying a coding sequence for the selected chimeric protein. Coding sequences for the S gene protein fragments and other viral proteins, e.g., fusion partners, of the coronaviruses can be. prepared synthetically or can be derived from viral RNA by known techniques, or from available cDNA-containing plasmids.
  • vaccinal polypeptide in various viral vectors, microorganisms and cells, are known and available from private and public laboratories and depositories and from commercial vendors.
  • the most preferred expression systems include those involving viral vectors, particularly poxvirus vectors, such as vaccinia or swinepox.
  • viral vectors particularly poxvirus vectors, such as vaccinia or swinepox.
  • feline herpes virus vectors See, e.g. R. Wardley et al, J. Gen. Virol., 73(7):1811-1818 (1992) and J. Nunberg et al, Vaccines, 91:191-196 (1991)].
  • Another preferred expression system includes mammalian cells, such as Chinese Hamster ovary cells (CHO) or COS-1 cells.
  • Another desirable expression system includes insect cell systems, such as the baculovirus expression system.
  • insect cell systems such as the baculovirus expression system.
  • suitable host cells and methods for transformation, culture, amplification, screening and product production and purification can be performed by one of skill in the art by reference to known techniques. See, e.g., Gething and Sambrook, Nature, 293:620-625(1981).
  • the chimeric proteins When produced by conventional recombinant means, the chimeric proteins may be isolated either from the cellular contents by conventional lysis techniques or from cell medium by conventional methods, such as chromatography. See, e.g., Sambrook et al. Molecular Cloning. A Laboratory Manual.. 2d Edit., Cold Spring Harbor Laboratory, New York (1989).
  • the resulting chimeric proteins may be screened for efficacy as a vaccine or therapeutic agent in an animal model system involving challenging animals immunized with vaccinia recombinants expressing a selected coronavirus gene products with the virulent FIPV strain [Vennema et al, J. Virol.. 64:1407-1409 (1990)].
  • the present invention provides vaccine compositions containing an immunogenic amount of a chimeric protein together with a carrier suitable for administration as a composition for prophylactic treatment of coronavirus infections.
  • the protein of the invention can be employed alone, or in a vaccine composition containing additional antigens, e.g. other chimeric or recombinant coronavirus S gene proteins of the invention, other proteins from the applicable coronavirus, or other proteins or peptides from other pathogens.
  • the selected coronavirus chimeric S gene or fragment may be incorporated into a live vector, e.g., adenovirus, vaccinia virus and the like.
  • a live vector e.g., adenovirus, vaccinia virus and the like.
  • the expression of vaccinal proteins in such live vectors are well-known to those in the art [See, e.g., U. S. Patent No. 4,920,209]. See, also. Examples 7 through 9 below.
  • a vaccine composition containing a FIPV/FECV chimeric S protein may be formulated to further contain the temperature sensitive FIPV vaccine described in detail in co-owned, co-pending U.S. Patent Application Ser. No. 07/428,796 filed October 30, 1989, incorporated by reference herein.
  • a vaccine may also contain, e.g. antigens directed against feline leukemia.
  • suitable feline antigens include rabies, feline calici virus, chlamydia, feline immunodeficiency virus, feline parvovirus and feline rhinotracheitis.
  • Suitable antigens include, for example, pseudorabies, porcine parvovirus, and bacterial antigens.
  • other suitable antigens include, among others, rabies, canine distemper, Borrelia burgdorferi , canine Bordetell a, canine parvovirus, canine rotavirus, canine parainfluenza, canine adenovirus, and Leptos-pira species.
  • vaccines having appropriate pH isotonicity, stability and other conventional characteristics is within the skill of the art.
  • vaccines may optimally contain other conventional components, such as adjuvants and/or carriers, e.g. aqueous suspensions of aluminum and magnesium hydroxides, liposomes and the like.
  • the vaccine composition containing a coronavirus chimeric S protein according to the invention can be employed to vaccinate naive animals (i.e., animals not previously exposed to the coronavirus) against at least the clinical symptoms associated with the coronaviruses from which the chimeric protein was derived.
  • an FIPV/CCV chimeric is useful in vaccinating against infection with FIP or canine coronavirus.
  • the chimerics of the invention will also provide cross-species protection against coronavirus infection.
  • the vaccines according to the present invention can be administered by an appropriate route, e.g., by the oral, intranasal, subcutaneous, intraperitoneal or intramuscular routes.
  • the presently preferred methods of administration are the subcutaneous and intranasal routes.
  • the amount of the chimeric coronavirus S protein of the invention present in each vaccine dose is selected with regard to consideration of the animal's age, weight, sex, general physical condition and the like.
  • the amount required to induce an immunoprotective response in the animal without significant adverse side effects may vary depending upon the recombinant protein employed as immunogen and the optional presence of an adjuvant.
  • each dose will comprise 0.05-5000 micrograms of protein per mL, and preferably 0.1- 100 micrograms per mL of a sterile solution of an immunogenic amount of a protein or peptide of this invention.
  • Initial doses may be optionally followed by repeated boosts, where desirable.
  • the vaccine compositions of the invention may be used to protect a naive animal against coronavirus infection.
  • these vaccine compositions are expected to be capable of protecting cats against infection with one or more coronaviruses. Further, these vaccine compositions are expected to be able to protect against coronavirus-related disease in other species, particularly dogs and pigs.
  • Another vaccine agent of the present invention is an anti-sense RNA sequence generated to a sequence of a chimeric protein.
  • This sequence may easily be generated by one of skill in the art either synthetically or recombinantly. Suitable techniques are known [See, e.g. S.
  • RNA sequence when administered to an infected animal should be capable of binding to the RNA of a selected coronavirus, thereby preventing viral replication in the cell.
  • the invention also provides a pharmaceutical composition useful in the treatment of animals infected with a coronavirus comprising the chimeric coronavirus S proteins prepared according to the present invention and a pharmaceutically effective carrier.
  • these pharmaceutical compositions may contain a recombinant S gene protein as defined herein, or as described in co-pending U.S. application Ser. No. 07/698,927, and its corresponding published PCT application, WO 9208487.
  • Suitable pharmaceutically effective carriers for internal administration are known to those skilled in the art.
  • One selected carrier is sterile saline.
  • Other anti-viral agents can also be employed in compositions for the treatment of coronavirus infection.
  • the pharmaceutical composition can be adapted for administration by any appropriate route, but is designed preferentially for administration by injection or intranasal administration. VIII. Antibodies of the Invention
  • the present invention provides antibodies to a selected chimeric S protein or S protein fragment. These antibody may be generated using conventional techniques for production of monoclonal [W. D. Huse et al. Science, 246:1275-1281 (1989); Kohler and Milstein] or polyclonal antibodies.
  • the antibodies may be associated with individual labels, and where more than one antibody is employed in a diagnostic method, the labels are desirably interactive to produce a detectable signal. Most desirably, the label is detectable visually, e.g. colorimetrically. Detectable labels for attachment to antibodies useful in the diagnostic assays of this invention may also be easily selected by one skilled in the art of diagnostic assays. Labels detectable visually are preferred for use in clinical applications due to the rapidity of the signal and its easy readability. For colorimetric detection, a variety of enzyme systems have been described in the art which will operate appropriately.
  • Colorimetric enzyme systems include, e.g., horseradish peroxidase (HRP) or alkaline phosphatase (AP).
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • Other proximal enzyme systems are known to those of skill in the art, including hexokinase in conjunction with glucose-6-phosphate dehydrogenase.
  • bioluminescence or chemiluminescence can be detected using, respectively, NAD oxidoreductase with luciferase and substrates NADH and FMN or peroxidase with luminol and substrate peroxide.
  • Other conventional label systems that may be employed include fluorescent compounds, radioactive compounds or elements, or immunoelectrodes. These and other appropriate label systems and methods for coupling them to antibodies or peptides are known to those of skill in the art.
  • Antibodies may also be used therapeutically as targeting agents to deliver virus-toxic or infected cell-toxic agents to infected cells. Rather than being associated with labels for diagnostic uses, a therapeutic agent can employ the antibody linked to an agent or ligand capable of disabling the replicating mechanism of the virus or of destroying the virally-infected cell. The identity of the toxic ligand does not limit the present invention. It is expected that preferred antibodies to peptides encoded by the chimeric S proteins identified herein can be screened for the ability to internalize into the infected cell and deliver the ligand into the cell. IX. Diagnostic Kit
  • the present invention also provides a diagnostic kit which enables distinction between native coronavirus exposure and animals vaccinated with the chimeric or recombinant molecules of the invention.
  • a diagnostic kit which enables distinction between native coronavirus exposure and animals vaccinated with the chimeric or recombinant molecules of the invention.
  • Such a kit may contain a sufficient amount of at least one chimeric S protein or at least one recombinant S protein of the invention and such components as are necessary to practice the assay. It is anticipated that of the chimeric S proteins, those comprising less than a full length S protein and having a deletion of one or more epitopes will be particularly useful in such a kit.
  • Such assays are conventional, and the necessary reagents and other components of such a kit are well known to those of skill in the art.
  • kit, and other diagnostic formats using a chimeric S protein or recombinant S protein fragment of the invention are useful for distinguishing between animals vaccinated with a chimeric protein of the invention and an animal which has been naturally exposed to the coronavirus or coronaviruses which comprise the chimeric S protein.
  • DMEM Dulbecco's modified Eagle's medium
  • the DF2 wildtype FIPV virus was originally isolated at SBAH, Lincoln, from a cat liver explant. After several passages of tissue homogenates in specific pathogen free (SPF) cats, the virus was adapted to NLFK cells by cocultivation with infected primary spleens and later plaque-purified.
  • a feline enteric coronavirus, FECV WSU- 1683
  • FECV WSU- 1683
  • the DF2 FIPV spike gene sequence contained a PstI site at amino acid 352 while the FECV and CCV spike genes did not.
  • Oligonucleotide primers were specifically designed to allow PCR amplification of FECV and CCV spike protein amino acid regions 1-352 and 352-1454.
  • PCR primers were 30-40 base pairs in length and included a SmaI site in the upstream (5') primer and a PstI site in the downstream (3') primer (1-352 amino acids) and a PstI in the upstream primer and a StuI in the downstream primer (352-1454 amino acids).
  • Oligonucleotide primers were synthesized on an Applied Biosystem Model 380B DNA Synthesizer using the phosphor- amidite method. The primers were gel-purified prior to use. The primers used are as follows.
  • the top/left strand PCR primer specific for amplification of fuli length FECV S contains sequence for amino acids 1-9 fused to the recognition site for XmaI. This first 6 bp are a restriction site and are non-specific sequence. The seventh bp is a spacer to ensure that the primer is correctly translated.
  • the FECV specific sequence starts at the 8th bp position of the primer, which is the following SEQ ID NO: 19
  • a top/left strand PCR primer specific for FECV contains additional stabilizing sequence (GTGC from the published sequence upstream of the ATG) upstream of the Smal/Xmal site to help with digestion, which primer is SEQ ID NO: 20
  • the following two primers are FECV Pst primers and are used to regenerate the PstI site in FECV and CCV which does not result- in an amino acid change when compared to the WT WSU 1146 published strain. These primers contain the PstI site in the middle and contain one FECV specific base pair.
  • the top strand primer is SEQ ID NO: 21:
  • the bottom strand primer is SEQ ID NO: 22:
  • the following FECV Pst primers are also useful in regenerating the PstI site in FECV and CCV which does not result in an amino acid change when compared to the WSU strain.
  • the top strand primer containing the PstI site is SEQ ID NO: 23: 5'-CTGCAGATGTACAATCTGGTATGGGTGCT, and is used with the StuI bottom primer.
  • the bottom strand primer containing the PstI site is SEQ ID NO: 24:
  • Table IV of primers which were identified in the parent application (U. S. Ser. No. 07/698,927).
  • the following Table IV primers were used to PCR amplify the full length S gene from DF2 FIPV and FECV (See Table I).
  • the top strand primer for amplification of full length DF2 FIPV S gene contained the nucleotide sequence encoding amino acids 1-9 fused to the recognition site for Xmal [SEQ ID NO: 25].
  • the top strand primer for FECV S was SEQ ID NO: 19, identified above.
  • the bottom strand primer for full length FIPV S and for FECV S contained sequence for amino acids 1450-1454 fused to the recognition site for StuI for each S sequence [SEQ ID NO: 42].
  • Roller bottles of confluent NLFK cells were infected with either FIPV or FECV virus using the following protocol.
  • FIPV infections were performed in serum-free medium.
  • the virus was absorbed for 2 hours and then 250 ml of growth medium added.
  • the cultures were monitored for cytopathic effect (CPE) and typically harvested at 24-36 hours post- infection.
  • Total cytoplasmic RNA was prepared from the infected monolayers by guanidine isothiocyanate extraction [Chirgwin et al, Biochem., 18:5294 (1979)].
  • PCR reagents were produced by Perkin Elmer-Cetus (Norwalk, CT). PCR amplified fragments were generated using the following procedure. Synthesis of cDNA from total RNA isolated from cells infected with a specific coronavirus was performed for each virus.
  • RNAse free siliconized 500 ⁇ l microcentrifuge tubes 1.0 mM of each dNTP, 20 units of RNAsin (Promega Corporation, Madison, WI), 100 picomoles of random hexamer oligonucleotides (Pharmacia, Milwaukee, WI), 100 picomoles/ ⁇ l solution in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5), 200 units of reverse transcriptase (Moloney MuLV, Bethesda Research Labs, Gaithersburg, MD) and 1.0 ⁇ g of respective RNA isolated as described above.
  • IX PCR buffer 100 mM Tris-HCl, 500 mM KCl, 15 mM MgCl 2 , 0.01% (w/v) gelatin
  • Amplification of the cDNA was performed essentially according to the method of Saiki et al. Science. 230:1350- 1354 (1985) using the Taq polymerase. Briefly, to the 20 ⁇ l CDNA reaction mix from above was added: 8.0 ⁇ l 10X PCR buffer, 1.0 ⁇ l of each upstream and downstream primer previously diluted in water to 30 picomoles per microliter and 5.0 units of Taq polymerase (Perkin-Elmer Cetus, Norwalk, CT). Final volume was made up to 100 ⁇ l of mineral oil. As above, master mixes were prepared to avoid contamination.
  • the reaction was performed in the Perkin-Elmer Cetus thermal cycler for one cycle by denaturing at 95°C for 1 minute, annealing at 37°C for 2' followed by an extension at 72°C for 40 minutes. This initial cycle increased i;he likelihood of first strand DNA synthesis.
  • a standard PCR profile was then performed by a 95°C-1 minute denaturation, 37°C-2 minutes annealing, 72°C-3 minutes extension for 40 cycles.
  • a final extension cycle was done by 95°C-1 minute denaturation, 37°C-2 minutes annealing, 72°C-15 minutes extension and held at 4°C until analyzed.
  • PCR products were analyzed by electrophoresing 5.0 ⁇ l of the reaction on a 1.2% agarose gel run 16-17 hours. Amplified fragments were purified prior to use for cloning by column chromatography with the PrimeErase Quik method (Stratagene, LaJolla, CA). Bands were visualized by ethidium bromide staining the gel and fluorescence by UV irradiation at 256 nm. Photography using Polaroid type 55 film provided a negative that could be digitized for sample distance migration and comparison against markers run on each gel. The actual sizes of the bands were then calculated using the Beckman Microgenie software running on an IBM AT.
  • Example 2 Cloning of DF2 FIPV and FECV S Proteins
  • a full length wildtype (WT) DF2 FIPV S protein was employed as a positive control for sensitization; and a full length FECV S protein was also tested.
  • pSC11 is a vector designed to allow expression of a cloned gene of interest inserted into an unique Smal site downstream of the p7.5 promoter of vaccinia virus.
  • pSCll contains vaccinia TK gene sequences for homologous recombination into the virus and the ampicillin gene for selection in E. coli .
  • This vector also contains the pll vaccinia virus promoter regulating the E. coli lacZ gene. Expression from this promoter results in the synthesis of beta-galactosidase protein.
  • Recombinant viruses containing the pSCll plasmid will therefore appear as blue plaques in the presence of X-gal or Bluo-gal [Sigma]. All clones are introduced into the SmaI site of this vector as blunt-ended fragments and the resulting clones oriented with respect to the p7.5 promoter.
  • pOTSKF33 The bacterial expression vector, pOTSKF33, shown schematically in Figure 1 of the parent application, is being maintained at SmithKline Beecham Laboratories and is available to the public through the company.
  • This plasmid is a derivative of pBR322 [Bethesda Research Laboratories] and carries regulatory signals from bacteriophage lambda.
  • the system provides a promoter which can be controlled ( ⁇ P L ), and an antitermination mechanism to ensure efficient transcription across any gene insert, high vector stability, antibiotic selection, and flexible sites for insertion of any gene downstream of the regulatory sequences.
  • the pOTSKF33 vector also contains the coding sequence for 52 amino acids of the enzyme galactokinase, immediately adjacent to the ⁇ P L promoter.
  • the sequence of this enzyme has been manipulated to permit insertion of foreign genes and the construction of fusion proteins.
  • Linkers containing restriction sites for fusion in any of the three reading frames, stop codons for each frame and some additional cloning sites for fusion in any of the three reading frames, have been introduced after the first 52 amino acids of galactokinase.
  • Transcription from the P L promoter is tightly controlled by maintaining the plasmid in bacteria expressing the c1 + repressor protein. Induction of foreign protein expression is obtained by removing the repressor.
  • the repressor protein is temperature-sensitive. At the permissive temperature, 32°C, the repressor functions normally to inhibit transcription from the P L regulatory sequences. An increase in growth temperature (to 42°C) results in degradation of the repressor and expression of the fusion polypeptide is induced.
  • Full-length DF2 FIPV and FECV spike protein 1-1454 amino acid inserts were isolated from established pOTSKF33 plasmid clones (as described in the above-identified parent application, incorporated by reference herein), by SmaI/ StuI digestion of plasmid DNA and the excised gene cloned into pSC11/SmaI-digested, dephosphorylated vector using conventional techniques. Insert-bearing clones were identified by BamHI digest of mini-prep DNA. Full-length clones were oriented with respect to vector by digestion with XbaI (FECV) and NcoI (FIPV).
  • Full length chimeric S genes were engineered to encode (a) 1-311 amino acid of FECV fused to 311-1454 amino acid of WT DF2 FIPV, the chimeric is- identified by SEQ ID NO: 43; and (b) 1-311 amino acid of WT DF2 FIPV fused to 311-1454 amino acid of FECV, the chimeric is identified by SEQ ID NO: 44. Amino acid 311 appears only once in the chimeric so that the S gene is 1454 amino acid in total length.
  • FECV and DF2 FIPV Smal/MscI 1-311 inserts and Smal/MscI 311-1454 amino acid plasmid DNA fragments were isolated from an established pSC11 plasmid containing the full-length DF2 FIPV or FECV spike gene. Digests were loaded onto agarose gels prepared and run in Tris-Borate-EDTA buffer. DNA fragments were isolated and eluted using GeneClean (Bio101 Inc., La Jolla, CA) according to the manufacturer's instructions. The corresponding plasmid and AA 1-311 insert fragments were ligated together overnight at 15°C. HB101 host cells [ATCC] were transformed with the ligation mixes and full length clones identified by BamHI digestion of mini prep DNA.
  • the FIPV and FECV specific spike gene regions in the chimeric clones were identified by diagnostic restriction enzyme digestions: (1) 1-311 amino acid FIPV, Ncol;
  • FECV fused to AA 872-1454 FIPV are identified by SEQ ID NO:
  • MscI/BstEII AA311-872 amino acid insert fragments and MscI/BstIII AA1-311/872-1454 plasmid fragments were isolated from established clones containing the full length
  • FIPV or FECV spike genes in pSCll FIPV or FECV spike genes in pSCll.
  • DF2 FIPV and FECV SmaI/BstEII 1-872 amino acid insert and SmaI/BstEII 872-1454 amino acid plasmid fragments were isolated from established pSCll clones containing the full length FIPV and FECV spike genes, respectively. The appropriate isolated fragments were then ligated together overnight at 15°C. Full length clones were identified by BamHI digest of mini prep DNA.
  • Chimerics having 1-872 amino acid DF2 FIPV fused to 872-1454 amino acid FECV are identified by SEQ ID NO: 47.
  • Chimerics having 1-872 amino acid FECV fused to 872-1454 amino acid DF2 FIPV are identified by SEQ ID NO: 48. These chimerics are 1454 amino acid in length.
  • a FIPV SmaI/StuI insert was isolated from an established plasmid [pOTSKF33] containing the 894-1040 amino acid region of the DF2 FIPV spike protein. The isolated fragment was purified using GeneClean (Bio 101 Inc., LaJolla, CA) according to the manufacturer's instructions. Purified insert was ligated with pSC11 SmaI-digested, dephosphorylated vector overnight at 15°C. Insert-bearing clones were identified by BamHI digest of mini-prep DNA.
  • Full-length clones were oriented with respect to the 7.5K promoter in the vector by Drain, XhoI/DraIII. and BamHI/Drain digests.
  • PCR DNAs encoding the DF2 S FIPV 894-1203 amino acid fragment were purified using Prime Erase Quik Columns (Stratagene Cloning Systems, LaJolla, CA). Purified PCR DNAs were then digested SmaI/StuI. excised from agarose gels, and purified with GeneClean. Purified inserts were ligated to pSCll/Smal- digested, dephosphorylated vector overnight at 15°C. Full length clones were identified by BamHI digest of mini-prep DNA and oriented with respect to vector by HindIII and Xhol/HindIII digests.
  • the TS FIPV 1029-1454 amino acid region expressed as a galK fusion protein and used to immunize mice, induced a strong CMI response when splenocytes from these animals were stimulated with inactivated TS FIPV virus.
  • a Smal/StuI insert was isolated from an established plasmid (pOTSKF33) containing the 1029-1454 amino acid region of the TS FIPV S gene. The fragment was purified with GeneClean and ligated to pSC11 SmaI-digested, dephosphorylated vector as above. Full length clones were identified and oriented by BamHI and HindIII digests of mini prep DNA, respectively. 2. Expression of Predicted T Cell Sites Alone
  • TS FIPV 894-1040 amino acids induced a cellular immune response in mice and protected 50% of cats from FIPV challenge.
  • Another region, TS FIPV AA 1029-1454 also elicited a strong CMI response in mice stimulated with inactivated vaccine virus (TS). Therefore, the following recombinant viruses were made to determine whether or not a cell mediated immune response could be selectively stimulated by immunizing cats with one or more of the predicted major T cell sites on the DF2 FIPV S gene-expressed in vaccinia virus recombinants.
  • Recombinant vaccinia viruses were engineered to contain selected regions of the WT DF2 S gene encoding the predicted FIPV T cell sites: (a) 894-1040 amino acids (predicted to contain a T cell site at 922-934 amino acid); (b) 894-1203 amino acids (the above clone was expanded to add strong T cell site at residues 1133-1147); and (c) TS 1029-1454 amino acids.
  • plasmid DNA prepared from partial (Example 6), full-length (Example 2) or chimeric S
  • Examples 3-5 /pSC11 clones was used to transfect CV-1 [ATCC CCL 70] monolayers infected with the WR strain of vaccinia virus. Transfected monolayers were harvested 2. days post-infection (p.i.) and were plaque-assayed on HuTK- cell [ATCC CRL 8303] monolayers in 60 mm dishes. At 3 days p.i. dishes were stained with an agarose overlay containing 300 ⁇ g/ml Bluo-gal [Sigma]. After 4-6 hours, blue BGal+ plaques were identified and picked with sterile Pasteur pipettes into 0.5 ml DMEM + 5% fetal bovine serum.
  • Non-reducing sample buffer 2% SDS, 80 mM Tris, pH 6.8, 10% glycerol, 0.02% bromophenol blue
  • Filters were blocked in 2% skim milk, 1% gelatin, and TBS (20 mM Tris, pH 7.5, 500 mM NaCl) for 1-2 hours (h) at room temperature (RT), rinsed with TTBS (TBS + 0.05% Tween-20) and incubated with anti-FIPV cat serum at a 1:50 dilution in TTBS and 1% gelatin for 1-2 hours at RT.
  • Anti-FIPV serum was produced at SmithKline Beecham Animal Health, Lincoln, Iowa in cats that were vaccinated two times intranasally with Primucell (SmithKline Beecham) vaccine containing a temperature- sensitive DF2 FIPV at three week intervals and challenged orally with 1 mL DF2 FIPV two weeks following the third vaccination. Animals were then bled by conventional means.
  • Filters were washed in TTBS 3X for 10 minutes each and incubated with goat anti-cat alkaline-phosphatase labeled IgG at a 1:1000 dilution for 1 hour [Kirkegaard-Perry Laboratories, Inc.]. Filters were washed as before and incubated 5-15 minutes in BCIP/NBT substrate according to the manufacturer's instructions [Kirkegaard-Perry Laboratories, Inc.]. Filters were then rinsed in water and air-dried.
  • recombinant vaccinia viruses expressing FIPV S v/FIPV S
  • FECV S v/FECV S
  • FIPV/FECV chimeric S genes v/FIPV + FECV S
  • 14-week-old specific pathogen free kittens ten per group (5 from Liberty Labs and 5 from Sprague Dawley), are immunized subcutaneously with 5 X 10 7 PFU of the recombinant vaccinia viruses.
  • a group of ten kittens receives wild-type WR- vaccinia virus (v/WR) as a negative control. Kittens are clinically examined daily and rectal temperatures taken.
  • a second immunization with the same amount of virus is given after 3 weeks.
  • Two weeks after the second immunization kittens are challenged orally with either 1.25 ⁇ 10 4 PFU (low) or 1 ⁇ 10 6 PFU (high) of WT DF2 FIPV and survival monitored.
  • Virus-neutralization titers are determined on the day of challenge and one and two weeks post-challenge. Serum samples are taken on the days of first and second vaccination, challenge, and post-challenge days 3, 7, 14, 21, and 28.
  • a predicted location of the major neutralizing epitope on the feline coronavirus S is at about amino acids 525-650 (DF2, DF2-HP, TS, TS-BP, FECV).
  • DF2, DF2-HP, TS, TS-BP, FECV amino acids 525-650
  • Full length S proteins were constructed which contain: (a) 1-311 amino acid of FECV fused to 311-872 amino acid of WT DF2 FIPV, fused to 872-1454 amino acid of FECV, this chimeric is identified by SEQ ID NO: 46; and (b) 1-311 amino acid of WT DF2 FIPV fused to 311-872 amino acid of FECV, fused to 872-1454 amino acid of WT DF2 FIPV, this chimeric is identified by SEQ ID NO: 45, were engineered.
  • Alternate cloning sites between amino acids 650 and 872 could be used in place of BstEII (i.e., PvuII. HgaI) to excise the middle portion of the S gene encompassing the predicted VN epitope(s).
  • the DF2 FIPV S gene clone from which a 352-1454 amino acid PstI and StuI insert was isolated, was cloned by digesting a PCR DNA encoding DF2 FIPV S AA1-1454 with XmaI and StuI. Ligation products were then ligated for 4 hours at room temperature to pOTSKF33 vector [SmithKline Beecham Pharmaceuticals, Swedeland, PA], digested with Xmal/StuI and dephosphorylated. Transformants in E. coli strain AR120 [SmithKline Beecham] were screened by BamHI and SphI digests of mini prep DNAs to identify insert-bearing clones.
  • Ligation products of the CCV and DF2 FIPV ligation were ligated into pOTSK33 vectors under similar conditions to those described above.
  • AR120 transformants were screened by BamHI/SphI and PstI digests of mini prep DNAs. The presence of an unique StuI site at amino acid #128 of the CCV S gene was confirmed by StuI digest.
  • Clone #2324 was identified as containing a full length chimeric spike gene encoding AA1-352 of CCV and AA352-1454 of DF2 FIPV.
  • Example 12 In Vitro T-Cell Proliferation Assay
  • Immunizing antigens were administered as pellet-3 bacterial lysates of pOTSKF33 galK/TS FIPV S fusion proteins, as described in Example 7 of co-pending, co-owned U.S. Patent Application Ser. No. 07/698,927. A corresponding application was published on May 29, 1992, Publication No. WO 92/08487, and is incorporated by reference herein.
  • the pellet 3 fractions were obtained essentially as described below.
  • the inactivated extract was centrifuged at 27,000 ⁇ g for 30 min (JA20). The supernatant was discarded and the pellet resuspended by vortexing for 10 minutes in 200 mis of Buffer A plus 0.2% sodium deoxycholic acid and 1% Triton X-100.
  • Buffer A contains 50 mM Tris-HCl, pH 8.5, 5 mM EDTA, 1 mM DTT, and 5% glycerol.
  • the extract was centrifuged at 27,000 X g for 30 minutes and again the resulting supernatant was discarded. The pellet was resuspended by vortexing 10 minutes in 200 mis of Buffer A containing Triton X-100 (1%) and 0.5 M KCl.
  • mice (10 per group, 12 weeks of age) were immunized with 15-40 ⁇ g of pellet 3 antigens formulated in an adjuvant emulsion containing 25 ⁇ g of Quil A and 5% Alhydrogel [Superfos Biosectorals, Denmark]. These antigens represented TS FIPV S gene regions from amino acids 1-105, 94-223, 213-362, 352-748, 737-1040, 894-1040 and 1029-1454. Two immunizations were administered intraperitoneally (i.p.), 0.1 mL dose on days 1 and 21. (Mice vaccinated with AA352-748 pellet-3 antigen received 10 ⁇ g of fusion protein per dose.)
  • spleens were removed aseptically from mice immunized with the antigens identified above. Spleens from 5 mice of each group were pooled. Splenocytes were seeded (5 ⁇ 10 6 cells/mL) into each well of 96-well polystyrene plates in triplicate in a total of 200 ⁇ L RPMI 1640 [Sigma] plus 10% fetal bovine serum, 20 mm HEPES, Pen Strep (100 units/mL penicillin; 100 ⁇ g/mL streptomycin), 0.1% ITS Growth Media (insulin 5 ⁇ g/mL, transferrin 5 ⁇ g/mL, selenious acid 5 ⁇ g/mL), and 2 g sodium bicarbonate/L, with or without mitogens or specified antigens (biethylimine inactivated TS FIPV or pellet-3 antigens).
  • the mitogens included concanavalin A (ConA), lipopolysaccharide
  • the cultures were incubated at 37°C in an atmosphere of 5% CO 2 . After 72 hours (mitogens) or 96 hours (antigens) the cultures were labeled with tritiated thymidine (0.5 ⁇ Ci/well) [New England Nuclear (NEN), 2 ci/mmol]. Cultures were incubated for an additional 18 to 24 hours. Cultures were harvested onto glass fiber paper and counted on a ⁇ counter. The results were expressed as the mean of triplicate counts per minute (cpm) for each culture and are provided below in qualitative terms. (Background counts obtained from pOTSKF33 negative control pellet-3 lysate were subtracted.)
  • Table VI illustrates the reactivity of mouse splenocytes to TS FIPV inactivated by BEI treatment or pellet-3 antigens in the in vitro proliferation assay.
  • the immunizing antigens in column 1 of Table VI are galK fusion proteins containing the indicated S protein fragments of SEQ ID NO: 8, as described above.
  • the stimulating antigens in columns 3, 4, and 5 indicate S protein regions identified, in the pellet-3 antigens.
  • One plus (+) indicates 10-15,000 cpm
  • two pluses (++) indicates 15-20,000 cpm
  • +++ indicates 30,000 cpm
  • ++++ indicates 35-50,000 cpm
  • - indicates ⁇ 10 K cpm.
  • Splenocytes from mice that received the FIPV TS AA213- 362, AA894-1040 and AA1029-1454 fusion protein antigens responded well to TS FIPV antigen in a T cell proliferation assay.
  • the latter two of these antigens represent regions of the TS FIPV spike gene that are predicted to contain strong T cell epitopes.
  • the computer programs utilized to predict T cell epitopes are typically 80% correct.
  • Vaccination with the AA213-362 fusion protein clearly stimulates a proliferative T cell response and so must also contain a T cell site. Serological responses of vaccinated mice as measured by ELISA, VN and Western blot are still being evaluated.
  • NAME Schreck, Patrica A.
  • GGA ACA AAA ATC TAT GGT CTT GAG TGG AAT GAT GAC TTT GTT ACA GCT 576 Gly Thr Lys Ile Tyr Gly Leu Glu Trp Asn Asp Asp Phe Val Thr Ala
  • MOLECULE TYPE protein
  • MOLECULE TYPE DNA (genomic)
  • GGA ACA AAA ATC TAT GGT CTT GAG TGG AAT GAT GAC TTT GTT ACA GCT 576 Gly Thr Lys Ile Tyr Gly Leu Glu Trp Asn Asp Asp Phe Val Thr Ala

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Abstract

Protéine S chimère de coronarivus utilisée dans des compositions diagnostiques, vaccinales et thérapeutiques comprenant deux fragments de protéine S ou plus, lesdits fragments étant sélectionnés à partir de souches de coronavirus identiques ou différentes.
PCT/US1993/004384 1992-05-08 1993-05-07 Compositions et procedes de vaccination contre les coronavirus Ceased WO1993023422A1 (fr)

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EP93911185A EP0640097A4 (fr) 1992-05-08 1993-05-07 Compositions et procedes de vaccination contre les coronavirus.
AU42410/93A AU678971B2 (en) 1992-05-08 1993-05-07 Compositions and methods for vaccination against coronaviruses
CA002134898A CA2134898A1 (fr) 1992-05-08 1993-05-07 Compositions et methodes de vaccination contre les coronavirus
JP6503668A JPH08501931A (ja) 1992-05-08 1993-05-07 コロナウイルスに対するワクチン接種用組成物および方法

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995007987A3 (fr) * 1993-09-16 1995-06-22 Solvay Nouveaux polypeptides/proteines, plasmides de cotransfection et leurs vecteurs recombinants vivants
WO1996006934A1 (fr) * 1994-08-29 1996-03-07 Rhone Merieux Vaccin de la peritonite infectieuse feline
EP0640098A4 (fr) * 1992-05-08 1997-01-02 Smithkline Beecham Corp Gene s du coronavirus canin et ses applications.
EP0640096A4 (fr) * 1992-05-08 1997-01-15 Smithkline Beecham Corp Vaccin universel contre le coronavirus.
WO2015143335A1 (fr) * 2014-03-20 2015-09-24 The University Of North Carolina At Chapel Hill Méthodes et compositions pour protéines spike de coronavirus chimère
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EP0640096A4 (fr) * 1992-05-08 1997-01-15 Smithkline Beecham Corp Vaccin universel contre le coronavirus.
EP0640098A4 (fr) * 1992-05-08 1997-01-02 Smithkline Beecham Corp Gene s du coronavirus canin et ses applications.
WO1995007987A3 (fr) * 1993-09-16 1995-06-22 Solvay Nouveaux polypeptides/proteines, plasmides de cotransfection et leurs vecteurs recombinants vivants
GB2282601B (en) * 1993-09-16 1998-04-15 Solvay Antigenically-active proteins/polypeptides and coronavirus vaccines containing the same
US6080850A (en) * 1994-08-29 2000-06-27 Rhone Merieux Feline infectious peritonitis vaccine
FR2724385A1 (fr) * 1994-08-29 1996-03-15 Rhone Merieux Vaccin de la peritonite infectieuse feline.
WO1996006934A1 (fr) * 1994-08-29 1996-03-07 Rhone Merieux Vaccin de la peritonite infectieuse feline
WO2015143335A1 (fr) * 2014-03-20 2015-09-24 The University Of North Carolina At Chapel Hill Méthodes et compositions pour protéines spike de coronavirus chimère
US9884895B2 (en) 2014-03-20 2018-02-06 The University Of North Carolina At Chapel Hill Methods and compositions for chimeric coronavirus spike proteins
CN113248576A (zh) * 2020-02-12 2021-08-13 北京科兴中维生物技术有限公司 一种针对冠状病毒的核酸疫苗及其制备方法
CN113861278A (zh) * 2021-06-18 2021-12-31 国药中生生物技术研究院有限公司 一种产生广谱交叉中和活性的重组新型冠状病毒rbd三聚体蛋白疫苗、其制备方法和应用
CN113861278B (zh) * 2021-06-18 2022-08-05 国药中生生物技术研究院有限公司 一种产生广谱交叉中和活性的重组新型冠状病毒rbd三聚体蛋白疫苗、其制备方法和应用
CN114478716A (zh) * 2021-12-28 2022-05-13 梅州市人民医院(梅州市医学科学院) 一种多肽组合及其在新型冠状病毒抗体检测中的应用
CN114478716B (zh) * 2021-12-28 2024-05-28 梅州市人民医院(梅州市医学科学院) 一种多肽组合及其在新型冠状病毒抗体检测中的应用

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WO1993023421A1 (fr) 1993-11-25
AU678971B2 (en) 1997-06-19
EP0640097A1 (fr) 1995-03-01
AU4241093A (en) 1993-12-13
AU678970B2 (en) 1997-06-19
EP0640096A4 (fr) 1997-01-15
JPH08501931A (ja) 1996-03-05
CA2134898A1 (fr) 1993-11-25
JPH07508176A (ja) 1995-09-14
AU4240493A (en) 1993-12-13
EP0640096A1 (fr) 1995-03-01
CA2135201A1 (fr) 1993-11-25
EP0640097A4 (fr) 1997-01-15

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