Classifications

• • 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 15 compositions.
• FIP virus Feline Infectious Peritonitis Virus
• Feline enteric coronavirus was isolated from cats but differs in pathogenesis from FIPV.
• FECV Feline enteric coronavirus
• coronavirus 30 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.
• Other 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
• ACV 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 -25 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
• 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.
• the term "chimeric coronavirus S proteins", “chimeric proteins of the invention”, “chimerics”, and the like 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. Shatz an and M. Rosenberg, Hepatoloqy. 2(1) :30S-35S (1987) and G.M. Weinstock et al, Proc. Natl. Acad. Sci.. _ ⁇ _: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.
• 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
• 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 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.
• FIPV DF2-HP, TS, and TS-BP
• FECV FECV
• other coronaviruses are believed to contain an antigenic site in these regions.
• S protein fragment for use in a chimeric protein of this invention.
• other suitable fragments of a feline, canine or a related coronavirus may be used in a chimeric molecule of the invention. Because these sites are antigenic and were mapped by monoclonal antibodies, they are suspected to be B cell.sites.
• VN virus neutralization
• induction of VN antibodies may be important for clearing infecting virus and may not contribute to sensitization.
• Antibodies to the non-neutralizing antigenic epitopes then, 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) .
• DF2-HP sequences are identical to DF2 insofar as DF2-HP has been sequenced
• 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
• coronavirus strain or species 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
• chimeric S protein 20 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 - 25 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. In such fusions, amino acid 311 is
• 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.
• Another currently preferred embodiment of the chimeric protein of the invention comprises the fusion of desirable B and T cell sites, as identified 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. As above, 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 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.
• thes chimerics include:
• FECV amino acids 1-542 fused to FIPV 594-1454 e.g. , DF2 or TS
• FIPV 594-1454 e.g. , DF2 or TS
• preferred 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.
• 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 l 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.
• 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. IV. Modified Amino Acid and Nucleotide Sequences
• 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 which code for protein are also included in the present invention, as well as analogs or derivatives thereof.
• DNA sequences which code for protein are also included in the present invention, as well as analogs or derivatives thereof.
• analogs may typically include analogs that differ by only 1 to about 4 codon changes at the nucleotide level.
• 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,
• amino acids 15 conservative amino acid replacements.
• non-polar alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
• uncharged polar glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are
• the chimeric proteins or coronavirus fragments of the invention can be prepared by chemical synthesis techniques. Preferably, however, they are prepared by using known 5 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 by chemical synthesis techniques. Preferably, however, they are prepared by using known 5 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
• 10 be.prepared synthetically or can be derived from viral RNA by known techniques, or from available cDNA-containing plasmids.
• 15 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. Also useful are
• Another preferred expression system includes mammalian cells, such as Chinese Hamster ovary cells (CHO) or COS-1 cells.
• Another desirable 5 expression system includes insect cell systems, such as the baculovirus expression system- The selection of other suitable host cells and methods for transformation, culture, amplification, screening and product production and purification can be performed by one of skill in the
• 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
• 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.. 61:1407-1409 (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.
• vaccine composition 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 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.
• each dose will comprise
• 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 capable of protecting cats against infection with one or more coronaviruses. Further, 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 capable of protecting cats against infection with one or more coronaviruses. Further, these vaccine compositions are expected to be capable of protecting cats against infection with one or more coronaviruses. Further, these vaccine compositions
• Another vaccine agent of the present invention is an anti-sense RNA sequence generated to a sequence of a
• 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
• 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
• bioluminescence or chemiluminescence can be detected using, respectively, NAD oxidoreductase with luciferase and substrates NADH and FMN or peroxidase with luminol and substrate peroxide.
• NAD oxidoreductase with luciferase and substrates NADH and FMN
• peroxidase with luminol and substrate peroxide.
• 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
• 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
• 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 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 15 (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
• Vaccinia virus strain WR was received from Dr. Bernard 20 Moss, NIH.
• the DF2 FIPV spike gene sequence contained a PstI site
• 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 Smal site in the
• 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 Xmal. This first 6 bp are a restriction site and are non-specific sequence. The seventh bp is a spacer to ensure that the 0 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 5 ⁇ -CCCGGGCATG ATTGTGCTCG TAACTTGCC TCTTGTTGTTA TGC.
• a top/left strand PCR primer specific for FECV 5 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 5'-GTGCCCCGGG CATGATTGTG CTCGTAACTT GCCTCTTGTT GTTATGC.
• GTGC stabilizing sequence
• 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. 5
• 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 5 SEQ ID NO: 23: 5'-CTGCAGATGT ACAATCTGGT ATGGGTGCT, and is used with the StuI bottom primer.
• the bottom strand primer containing the PstI site is SEQ ID NO: 24: 5'- CTGCAGTGAA ATTAAGATTG AATCTAATA, and is used with the Xmal top primer. 5.. 2. Primers for Cloning AA 1-311 /AA 311-872 /AA
• 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 5 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].
• cytoplasmic RNA was prepared from the infected monolayers by guanidine isothiocyanate extraction [Chirgwin et al, Biochem.. 18.:5294 (1979)].
• 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.
• TE buffer 10 mM Tris-HCl, 1 mM EDTA, pH 7.5
• reverse transcriptase Moloney MuLV, Bethesda Research Labs, Gaithersburg, MD
• 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 wild ⁇ type (WT) DF2 FIPV S protein was employed as a positive control for sensitization; and a full length FECV S protein was also tested.
• pSCll 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 Smal 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 , and an antitermination mechanism to ensure efficient transcription across any gene insert, high vector stability r 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 cl + 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.
• Insert-bearing clones were identified by BamHI digest of mini-prep DNA. Full-length f clones were oriented with respect to vector by digestion
• the fusions of the individual fragments are performed so that overlapping amino acids are entered only once.
• SEQ ID 10 NO:. 45 of Example 3 the overlapping amino acid position 311 of FIPV and FECV appears only once in the chimeric protein, as does the amino acid position 872 of FECV and FIPV, resulting in a chimeric protein of 1454 amino acids in length.
• DNA fragments were isolated and eluted using GeneClean (BiolOl 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; (2) 311-1454 amino acid FIPV, Xbal; (3) 1-311 amino acid FECV, Xbal: and (4) 311-1454 amino acid FECV, X nl. Restriction enzymes were purchased from New England Biolabs (Beverly, MA) or Bethesda Research Labs (Gaithersburg, MD) and used according to manu cturer's specifications.
• FECV fused to AA 872-1454 FIPV are identified by SEQ ID NO:
• MscI/BstEII AA311-872 amino acid insert fragments and MscI/BstEII 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 Smal/BstEII 1-872 amino acid insert 0 and S al/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 5 BamHI digest of mini prep DNA.
• a FIPV Smal/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 5 Inc., La olla, CA) according to the manufacturer's instructions. Purified insert was ligated with pSCll Smal- 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
• 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
• 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.
• 25 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 pSCll Smal-digested, dephos ⁇ phorylated vector as above. Full length clones were
• plasmid DNA prepared from partial * (Example 6) , full-length (Example 2) or chimeric S
• Examples 3-5 /pSCll clones was used to transfect CV-1 '30 [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.
• First- round plaques were plaque-purified twice more on HuTK- cells and then used to infect CV-1 and HuTK- monolayers in T25 flasks. Infected monolayers were harvested at 2 days p.i. and frozen at -20°C for use as crude virus stocks.
• 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) , or FIPV/FECV chimeric S genes (v/FIPV + FECV S) are used to immunize kittens.
• 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 x 10 4 PFU (low) or 1 x 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.
• Example 10 - Chimeric FECV/FIPV Genes 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). To evaluate the role of the neutralizing epitope in immunity to FIPV, full-length 'chimeric' S proteins were constructed in which amino acids 311-872 FIPV DF2 are replaced by the analogous region of FECV or conversely, amino acids 311-872 of FECV are interchanged for amino acids 311-872 of FIPV DF2.
• 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. Hgal) to excise the middle portion of the S gene encompassing the predicted VN epitope(s) .
• BstEII i.e., PvuII. Hgal
• 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 Xmal 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- x g for 30 min (JA20) .
• the supernatant was discarded and the pellet resuspended by vortexing for 10 minutes in 200 is 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. Following centrifugation 27,000 x g, 20 minutes) , the pellet was resuspended by vortexing and brief homogenization in 5-10 mis of PBS (1.15 g dibasic sodium phosphate, 0.2 g monobasic sodium phosphate and 8.5 g NaCl per liter pH 7.4). This completes the pellet-3 antigen. The amount of specific galK/TS FIPV fusion protein in each pellet-3 lysate was quantitated by Western blot against a galK protein standard.
• 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 x 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 (LPS) , and pokeweed mitogen and were used as controls.
• ConA concanavalin A
• LPS lipopolysaccharide
• pokeweed mitogen pokeweed mitogen and were used as controls.
• the cultures were incubated at 37°C in an atmosphere of 5% C0 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 Schrec , Patrica A.
• GGA ACA AAA ATC TAT GGT CTT GAG TGG AAT GAT GAC TTT GTT ACA GCT 576 Gly Thr Lys He Tyr Gly Leu Glu Trp Asn Asp Asp Phe Val Thr Ala 180 185 190
• MOLECULE TYPE DNA (genomic)
• GGA ACA AAA ATC TAT GGT CTT GAG TGG AAT GAT GAC TTT GTT ACA GCT 576 Gly Thr Lys He Tyr Gly Leu Glu Trp Asn Asp Asp Phe Val Thr Ala 180 185 190

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• 1993
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