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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • 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
  • A61K39/12—Viral antigens
  • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
• • 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
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  • 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
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  • 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
  • C07K14/08—RNA viruses
  • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/16011—Herpesviridae
  • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
  • C12N2710/16741—Use of virus, viral particle or viral elements as a vector
  • C12N2710/16743—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • 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
• • 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
  • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• the present invention relates to proteins/polypeptides which are antigenically-active so as to be capable of stimulating the immunity of cats (Felidae) against coronaviruses, and in parti ⁇ cular, feline infectious peritonitis virus, the preparation and cloning of base sequences coding for such antigenically-active proteins/polypeptides, cotransfection vectors having the base sequences coding for such proteins/polypeptides, live recombinant carriers for expressing such antigenically-active protein/- peptides and to vaccines having such Live Recombinant Carriers.
• Feline Infectious Peritonitis is a normally-fatal infectious feline disease.
• FIP Feline Infectious Peritonitis Virus
• Infection occurs by one of two routes: in utero transmission; and oral/nasaloral ingestion.
• FIPV belongs to the coronavidae family of viruses.
• the genomic plus-strand RNA of the coronavidae family is approxi ⁇ mately 27 to 31 kilo base pairs (kbp), making it the largest among RNA viruses.
• feline enteritis coronavirus FECV
• Coronaviruses are spherical particles having a spiral-like nucleocapsid which is enveloped by a lipid-containing envelope.
• These viruses contain four proteins of interest herein. These proteins are: the nucleocapsid (N) protein, which is approxi ⁇ mately 40-50 (kilodaltons) in size; a membrane protein, known as the spike (S) protein, which is approximately 180-200K in size; a membrane protein, known as the matrix (M) protein, which is approximately 25-30K in size; and another membrane protein known as the small membrane (SM) protein, which is approximately 10K in size.
• N nucleocapsid
• S spike
• M matrix protein
• SM small membrane
• Classical approaches attempted to develop a vaccine against coronaviruses, especially FIP include vaccination with live attenuated FIPV and heterologous live coronaviruses.
• the humoral antibodies obtained by the use of such vaccines have largely proven to be ineffective.
• felines which have developed antibodies against FIPV as a result of earlier infection will often develop clinical phenomena and lesions much sooner, and will survive the onset of the infection for a much shorter period (in a phenomena known as "early death” syndrome) than those felines which have not been so treated.
• "Early-death” syndrome is, presumably, the result of a phenomena known as antibody dependent enhancement (ADE).
• ADE antibody dependent enhancement
• This phenomena finds counterparts in herpesviridae, poxviridae, rhabdoviridae, flaviviridae, alphaviridae, reoviridae and bunyaviridae. This phenomenon is believed to be based upon the binding of virus antibody complexes to the Fc-receptors of macrophages.
• ADE of feline macrophage infectivity has been demonstrated to involve the formation of a ternary complex: virus/anti-virus antibody/macrophage fragment c receptor (FcR).
• FcR virus/anti-virus antibody/macrophage fragment c receptor
• Such binding is said to be more efficient than binding between macrophages and virus without the intermediary of antibodies.
• the result is that infection occurs more rapidly and more efficiently when the virus binds in a complexed form than when the virus binds in a non-complexed form.
• This complex enhances the uptake of virus by the macrophages and its further replication, suggesting the mediation, in vivo, of ADE of FIP.
• These macrophages then behave like vectors for dissemination of the virus in the feline.
• European Patent Application No. 411,684 in the name of DUPHAR INTERNATIONAL RESEARCH discloses recombinant vaccines whose antigen is constituted by the M protein or the N protein of FIPV.
• the vaccines are prepared by coupling the protein to a suitable carrier.
• the use of various suitable live recombinant carriers is also disclosed therein.
• European Patent Application No. 264,979 in the name of DUPHAR INTERNATIONAL RESEARCH discloses a recombinant vaccine for FIPV whose antigen is constituted by the S protein or certain fragments thereof.
• the vaccines are prepared with the S protein or fragment thereof being coupled to a suitable carrier.
• the use of live recombinant carriers is also disclosed.
• PCT Patent Application No. 92/08487 discloses the use of the FIPV S protein in a recombinant vaccine introduced by this virus.
• European Patent Application No. 510,773 discloses a vaccine for canine coronavirus which includes therein a polypeptide having at least one antigenic determinant of the S protein of the canine coronavirus. It is mentioned therein that that canine coronavirus vaccine also protects cats against infectious peritonitis.
• European Patent Application No. 310,362 discloses a temperature sensitive FIP virus, a vaccine containing that virus and the use thereof in immunizing felines against FIP infections.
• the vaccine confers partial protection in specified pathogen-free cats.
• proteins/polypeptides which stimulate the immunity of cats (Felidae) against coronaviruses, and in particular FIPV.
• proteins/poly ⁇ peptides in vaccines, including those having live recombinant carriers, which are capable of protecting mammals (such as felines) against the disease while avoiding the phenomena of antibody dependent enhancement.
• novel proteins/polypeptides are disclosed herein. These proteins/polypeptides are antigenically-active for protecting mammals (such as felines) against coronaviruses (such as FIPV) without provoking ADE.
• the proteins disclosed herein include modified coronavirus S proteins [proteins in which certain regions (fragments) thereof, which can include regions (fragments) which we believe induce ADE, are either modified and/or absent, having been deleted therefrom].
• modified coronavirus S proteins have at least one of the Al, A2 or D antigenic regions modified (so as to be ADE-inactive) or removed (deleted) therefrom, so that said regions are ADE-inactive.
• the modified coronavirus S proteins have the Al and the A2 antigenic regions modified or removed therefrom, so that said regions are ADE-inactive.
• the modified coronavirus S proteins have the Al and the D antigenic regions modified or removed therefrom, so that said regions are ADE-inactive.
• the modified coronavirus S proteins have the A2 and the D antigenic regions thereof modified or removed therefrom, so that said regions are ADE-inactive.
• the modified coronavirus S proteins of the present invention have the Al, A2 and the D antigenic regions modified or removed therefrom, so that said regions are ADE-inactive.
• modified coronavirus proteins of the present invention are a modified S protein.
• modified FIPV S protein wherein at least one of the Al, A2 or D antigenic regions have been modified or removed therefrom, so that said modified regions are ADE-inactive.
• anigenically-active what is meant is that the substance or entity spoken of (such as a protein and/or poly- peptide, and/or a portion thereof, which includes one or more epitopes) induces or stimulates a host's immune system to make a humoral and/or cellular antigen-specific response (against, for example, FIPV or FHV-1).
• antigenic region(s) amino acid sequences (including one or more epitopes) of, for example, a protein/polypeptide which induce or stimulate a host's immune system to make a humoral and/or cellular antigen-specific response (against, for example, FIPV or FHV-1).
• antigen what is meant is a molecule (such as, for example, a protein/polypeptide) having one or more epitopes that will stimulate a host's immune system to make a humoral and/or cellular antigen-specific response.
• AD-inactive what is meant is that the substance or entity spoken of (such as a modified protein and/or polypeptide and/or region of a modified protein and/or poly- peptide) does not provoke (induce) antibody dependent enhancement (ADE).
• this term may refer to substances or entities which may or may not still be antigenically-active.
• this term specifically refers to the region(s) of the S protein in which the ADE-epitopes (of the Al and/or A2 and/or D antigenic regions) have been either modified or removed therefrom, so that such regions do not provoke ADE.
• epitope refers to the site on an antigen to which a specific antibody molecule binds.
• the new antigenically-active proteins of the present invention also include modified coronavirus S proteins in which the S protein has had its signal peptide removed therefrom (by, for example, cleaving).
• modified coronavirus S proteins in which the S protein has had its signal peptide removed therefrom (by, for example, cleaving).
• Such proteins/polypeptides are variously referred to herein as “deleted S proteins” and "del S proteins”.
• modified coronavirus S protein is a modified FIPV S protein.
• the new antigenically-active proteins of the present invention further include a substantially-pure coronavirus SM protein having an amino acid sequence being substantially homo ⁇ logous with the amino acid sequence of Figure 1.
• the novel antigenically-active proteins include a substantially pure FIPV SM protein.
• the preferred antigenically-active proteins to be used in the live recombinant carriers and the vaccines of the present invention are all derived from the nucleic acid sequence of FIPV.
• the SM and the M proteins of the present invention are derived from the FIPV nucleic acid sequence of plasmid B12 and the S (spike) protein is derived from the FIPV nucleic acid sequence of pUCE2.
• M protein is used to refer to the same protein designated as the M protein in European Patent
• SM protein refers to the novel small membrane protein of FIPV whose isolation cloning and structure are described herein.
• SEQ ID NO: 3 The complete amino acid sequence of this SM protein (SEQ ID NO: 3), the complete nucleotide sequence which codes therefor (SEQ ID NO: 1) and the translation of that nucleic acid sequence into amino acids (SEQ ID NO: 2) are set forth in figure 1.
• S protein is used to refer to the same protein designated as the S protein in European Patent Application No. 264,979.
• the complete amino acid sequence, as well as the complete nucleotide sequence which codes therefor, are also described in European Patent Application No. 264,979.
• the coding sequence of the S-gene begins with an ATG codon in positions 367-369 as designated therein and ends with the stop codon in the positions 4723-4725 as designated therein.
• the coding part of the S-gene thus comprises 4356 base pairs and codes for a protein of 1452 amino acids.
• the entirety of European Patent Application is incorporated herein by reference.
• Al antigenic region is used to refer to amino acid residues coded for by the base sequences of the FIPV S protein as designated in European Patent Application No. 264,979 begining with the GCT codon in positions 1963-1965 through the CTA codon in positions 2029-2031.
• A2 antigenic region is used to refer to amino acid residues coded for by the base sequences of the FIPV S protein as designated in European Patent Application No. 264,979 begining with the ACT codon in positions 2116-2118 through the GCT codon in positions 2176-2178.
• D antigenic region is used to refer to amino acid residues coded for by the base sequences of the FIPV S protein as designated in European Patent Application No. 264,979 begining with the TCA codon in positions 1489-1491 through the TAC codon in positions 1570-1572.
• ADE-epitope-deleted S protein is used to refer to the S protein having at least one of the Al antigenic region, the A2 antigenic region and/or the D antigenic region thereof either modified, deleted or absent therefrom, so that such region is ADE-inactive.
• the term "dels protein” is used to refer to amino acid residues coded for by the base sequences of the FIPV S protein as designated in European Patent Application No. 264,979 (begining with an ATG codon in positions 367-369 as designated therein and ends with the stop codon in the positions 4723-4725 as designated therein) but in which the amino acid sequence begining with the ATT codon in positions 370-372 through (and including) the AGT codon in positions 421-423, has been deleted therefrom.
• the dels protein is the same as the S protein but without the signal sequence thereof.
• ADE-epitope-deleted is generally used to refer to proteins (including the ADE-epitope-deleted S protein) wherein at least one epitope thereof which may result in antibody dependent enhancement (ADE) is either modified or deleted therefrom, so that said epitope is ADE-inactive.
• ADE antibody dependent enhancement
• the proteins to which the invention relates each further have at least one region which is antigenically-active, but which does not provoke ADE.
• the proteins/polypeptide according to the present invention may be prepared according to standard methods known for the preparation of peptides and proteins. Such methods include the synthetic preparation thereof by means of known techniques starting from the individual amino acids or smaller peptide fragments.
• the antigenically-active protein(s)/polypeptide(s) may also be obtained biosynthetically while using recombinant DNA techniques and expression systems, for example, by: a) transformation of host cells with an appropriate (i.e., expression) vector having a nucleotide sequence coding for the antigenically-active proteins/polypeptides of the present invention; b) expressing the nucleotide sequence coding for the antigeni ⁇ cally-active proteins/polypeptides; c) harvesting the cell culture; and d) isolating the expressed antigenically-active proteins/poly ⁇ peptides.
• the invention therefore also relates to a method of preparing a DNA molecule which codes for the antigenically-active (to coronaviruses) modified S protein(s)/polypeptides according to the invention.
• a method comprises the steps of: a) isolating an expression cassette coding for the desired coro ⁇ navirus S protein (such as the FIPV S protein); b) determining the location of the nucleotide base sequences coding for the region(s) of interest of the the S protein which are desired to modified or deleted (i.e., at least one antigenic region) ; and c) modifying or deleting the nucleotide base sequences coding for the at least one region of interest.
• the region(s) of interest is the signal peptide sequence of the S protein.
• the nucleotide base sequence coding for the signal peptide sequence is deleted therefrom, so that no signal peptide is coded for by the nucleotide sequences.
• the region(s) of interest are at least one of the antigenic regions.
• the nucleotide base sequences coding for at least one of the antigenic regions of interest have been modified or deleted therefrom, so that the corresponding region of any protein coded for thereby is ADE-inactive.
• these antigenically- active region(s) of interest are at least one of the Al, A2 and D antigenic regions. Most preferred is that the antigenically-active regions are all of the Al, A2 and D regions.
• protein of interest refers to those proteins which are desired to be expressed and/or modified and/or deleted as the case may be.
• antigenic regions of interest refers to those antigenic regions which are desired to be modified and/or deleted from the protein/polypeptide to be expressed.
• coronavirus vaccines such as FIPV vaccines
• FIPV vaccines are disclosed herein which are effective for protecting a mammal (such as a feline) against a coronavirus (such as FIPV) in the absence of (while avoiding inducing or not provoking) ADE.
• the vaccines include the new antigenically-active coronavirus protein(s)/polypeptide(s) (against coronaviruses) in a suitable carrier.
• these antigenically-active protein(s)/polypeptides(s) are derived from FIPV.
• FIPV fragments
• fragments may be used coupled to suitable known carriers in manners well-known to those skilled in the art. Examples of such carriers are KLH or BSA.
• the vaccines include the coronavirus S protein.
• the coronavirus S protein is a modified coronavirus S protein.
• the modified coronavirus S protein be modified so as to have at least one of the Al, A2 or D antigenic regions thereof modified or deleted therefrom so as to be ADE-inactive.
• the modified coronavirus S protein be modified so as to have the signal peptide sequence deleted therefrom.
• the vaccine includes the novel coronavirus SM protein of the present invention or the coronavirus M protein.
• the vaccines may include any combination of two or more of the above antigenically-active coronavirus proteins/polypeptides.
• the vaccines are formulated to include a live recombinant carrier, whereby expression of the antigenically-active protein(s)/polypeptide(s), in vivo, in a host organism in need thereof, is obtained.
• the DNA molecule obtained as described above may be inserted in a manner known per se into an expression vector as a result of which a recombinant expression vector is formed.
• This vector may then be inserted into a suitable host cell, for example, by transformation or cotransfection.
• the vaccines disclosed herein include the DNA sequences (molecule) coding for the antigenically-active protein(s)/polypeptide(s) of the invention incorporated into a live recombinant carrier (LRC) with the aim of producing a vaccine capable of expression of said peptide(s) or protein(s) in susceptible host cells and/or host organisms.
• LRC live recombinant carrier
• the antigenically-active protein(s)/poly- peptide(s) of the present invention are expressed in the target animal, in vivo.
• the nucleotide base sequences which code for the SM protein and/or the modified S protein (or parts thereof) may be incorporated in live recombinant carriers (LRC).
• LRC's examples include vaccinia LRC, herpes-LRC, adeno-LRC, adeno-associated-LRC, Sindbis-LRC, corona-LRC and bacterial-LRC.
• a live recombinant carrier comprising FHV-1, the genome of which has DNA sequences coding for a modified coronavirus S protein, wherein at least one DNA sequences coding for the Al, A2 or D antigenic regions have been modified or removed therefrom, so that said regions of the protein coded for thereby are ADE-inactive.
• the DNA sequences coding for the Al, A2 and D antigenic regions have all been modified or removed therefrom, so that said Al, A2 and D regions of the modified S protein coded for thereby are all ADE-inactive.
• the modified coronavirus S protein is a modified FIPV S protein.
• a live recombinant carrier comprising FHV-1 whose genome has DNA sequences coding for a modified coronavirus S protein, and more preferably, a modified FIPV S protein, whose signal peptide has been modified or removed therefrom.
• a live recombinant carrier comprising FHV-1 whose genome has DNA sequences coding for a coronavirus SM protein, and more particularly an FIPV SM protein.
• the live recombinant carrier expresses the coronavirus SM protein.
• a live recombinant carrier comprising FHV-1 whose genome has DNA sequences coding for a coronavirus M protein, and more preferably an FIPV M protein.
• the live recombinant carrier expresses the coronavirus SM protein.
• a live recombinant carrier for in vivo expression of at least one protein/polypeptide of interest in a target mammal is disclosed. This live recombinant carrier includes a viral strain.
• This live recombinant carrier further has DNA expression sequences (signals) derived from RSV or HCMV and SV40 which have been incorporated into the genome of the viral strain.
• this live recombinant carrier further has a nucleotide coding sequence coding for a protein of interest (such as a coronavirus protein), which has also been incorporated into the genome of the viral strain, for a protein of interest to be expressed by the live recombinant carrier.
• the viral strain is FHV-1 and the nucleotide coding sequence codes for an antigenically-active coronavirus protein.
• the present invention further includes cotransfection vectors useful for cotransfection with viral DNA (of, for example, FHV-1) for the production of live recombinant carriers capable of expressing proteins/polypeptides of interest, in susceptible target host cells and/or host organisms (animals).
• viral DNA of, for example, FHV-1
• a cotransfection plasmid for cotransfection with viral DNA.
• This cotransfection plasmid includes at least a portion of the DNA sequences which flank (the flanking sequences) the insertion site in the viral genome.
• This cotransfection plasmid further includes expression sequences (signals) derived from RSV or HCMV and SV40.
• this cotransfection plasmid has the coding sequences for a protein of interest to be expressed by the live recombinant carrier to be produced using this cotransfection plasmid.
• the protein of interest is an FIPV protein.
• insertion site is that site of the viral genome into which nucleotide sequences including at least those nucleotide sequences coding for the proteins/polypeptides of interest (such as the coronavirus proteins) are inserted.
• flanking sequences refers to a portion of those nucleotide sequences which are located adjacent to, and upstream and downstream from, the insertion site.
• the inclusion of the flanking sequences permits the cotransfection plasmid to sucessfully recombine with the viral DNA (FHV-1) which is cotransfected therewith.
• both the viral DNA (to be cotransfected) and the flanking sequences of the cotransfection plasmid are derived from FHV-1 DNA. It is most preferred that the flanking sequences include nucleotide sequences derived from the thymidine kinase gene of the viral strain (i.e., FHV-1) to be transfected therewith.
• the proteins/polypeptides of interest are antigenically-active coronavirus proteins/polypeptides and, more particularly, antigenically-active FIPV proteins/polypeptides.
• the coding sequences for the protein of interest code for a modified coronavirus S protein and, more particularly, a modified FIPV S protein.
• the coding sequences for the modified coronavirus S protein code for a modified coronavirus S protein which has had the signal peptide deleted therefrom.
• especially preferred are the cotransfection plasmids pdTKHCMVSIG and pdTKSIGLAC and pdTKRSVSSIG and pdTKRSVSSIGLAC.
• the coding sequences for the modified coronavirus S protein (the protein of interest) code for a modified coronavirus S protein which has had at least one cf the antigenic regions thereof either modified or deleted therefrom, so that said regions of the protein coded for thereby are ADE-inactive.
• the preferred antigenic regions of interest are at least one of the Al, A2 or D antigenic regions thereof either modified or deleted therefrom, so that said regions are ADE-inactive.
• especially preferred are the cotransfection plasmids pdTKHCMVSDAD and pdT SDADLAC and pdTKRSVSDAD and pd TKRSVSDADLAC.
• the coding sequences for the protein of interest codes for the coronavirus S protein and, more preferably, the FIPV S protein.
• the cotransfection plasmids pdTKHCMVS and pdT SLAC and pdT RSMVS and pdTKRSVSLAC are especially preferred.
• the coding sequences for the protein of interest codes for the coronavirus SM protein and, more preferably, the FIPV SM protein.
• the coding sequences for the protein of interest code for the coronavirus M protein and, more preferably, the FIPV M protein.
• especially preferred are the cotransfection plasmids pdTKHCMVM and pdTKMLAC and pdTKRSVM and pdTKRSVMLAC.
• a method of preparing the vaccines of the present invention by isolating and purifying the antigenically-active protein(s)/polypeptide(s), including the new antigenically-active protein(s)/polypeptide(s) of the present invention, and then formulating the purified protein(s)/polypeptide(s) in a pharmaceutically-acceptable carrier.
• Still further disclosed herein is a method of preparing the vaccines of the present invention comprised of isolating and purifying the DNA nucleotide base sequences coding for the desired antigenically-active protein(s)/polypeptide(s), including those of the present invention, and then incorporating said nucleotide base sequences into viral DNA for production of a live carrier.
• a method of preparing a coronavirus vaccine including the steps of: a) isolating and purifying a DNA sequence coding for an antigenically-active coronavirus protein; b) forming a cotransfection plasmid having the DNA sequence coding for the antigenically-active coronavirus protein; c) cotransfecting the cotransfection plasmid and FHV-1 viral DNA, so as to form a live recombinant carrier capable of coding for the antigenically-active coronavirus protein for expressing the antigenically-active coronavirus protein in the target animal iji vivo; and d) combining the live recombinant carrier with a pharmaceutically-acceptable carrier.
• a method of preparing an FIPV vaccine including the steps of: a) isolating a DNA sequence coding for an FIPV S protein being antigenically-active; b) modifying DNA sequences coding for the Al, A2 or D antigenic regions of the FIPV S protein, so that at least one of the said modified antigenic regions of the protein coded for thereby is ADE-inactive; c) forming a cotransfection plasmid having the DNA sequence coding for the modified FIPV S protein; d) cotransfecting FHV-1 viral DNA with the cotransfection plasmid, so as to form a live recombinant carrier capable of coding for the antigenically-active modified FIPV S protein for expressing the antigenically-active modified FIPV S protein in the target animal ii vivo; and e) combining the live recombinant carrier with a pharmaceutically-acceptable carrier.
• expression cassette refers to discrete DNA sequences which include those regions of DNA (nucleotide sequences), including a structural gene and expression sequences required for transcription and translation of the protein/polypeptide coded for by the structural gene in a host.
• expression sequences refers collectively to promoter sequences, ribosome binding sites and polyadenylation signals which collectively provide for the transcription and translation of a coding sequence in a host cell.
• promoter sequences are sequences upstream of the structural gene's coding sequence (the nucleotide sequence which codes for the protein/polypeptide of interest) which permit the binding of RNA polymerase and transcription of the coding sequence to occur.
• structural gene is a nucleotide sequence which serves as a template for the synthesis of RNA and which allows the synthesis of the protein of interest by a host thereof.
• a method for immunizing mammals, and in particular felines, against coronavirus infection and, in particular, against FIPV which comprises preparing the coronavirus vaccine of the present invention and administering a therapeutically-effective quantity of the vaccine to a mammal and, in particular, a feline in need thereof.
• a method of protecting a feline from FIPV including the steps of preparing the FIPV vaccine disclosed herein and administering a therapeutically-effective quantity of the vaccine to a feline in need thereof.
• Figure 1 is the amino acid sequence of the SM protein and the corresponding nucleotide base sequence that codes for the SM protein.
• Figure 2 is a restriction map of pdTK.
• Figure 3 is a restriction map of pSV40polyAE.
• Figure 4 is a restriction map of pSV40polyAL.
• Figure 5 is a restriction map of pHCMVpolyAE.
• Figure 6 is a restriction map of pHCMVpolyAL.
• Figure 7 is a restriction map of pRSVpolyAL.
• Figure 8 is a restriction map of pRSVpolyAE.
• Figure 9 is a restriction map of pSVLACE.
• Figure 10 is a restriction map of pHCMVLACE.
• Figure 11 is a restriction map of pdTKSVLAC.
• Figure 12 is a restriction map of pdTKCMVLAC.
• Figure 13 is a restriction map of pHCMVMS.
• Figure 14 is a restriction map of pHCMVM.
• Figure 15 is a restriction map of pRSVMS.
• Figure 16 is a restriction map of pRSVM.
• Figure 17 is a restriction map of pdTKHCMVM.
• Figure 18 is a restriction map of pdTKHCMVMS.
• Figure 19 is a restriction map of pdTKMLAC.
• Figure 20 is a restriction map of pdTKMSLAC.
• Figure 21 is a restriction map of pdTKRSVM.
• Figure 22 is a restriction map of pdTKRSVMS.
• Figure 23 is a restriction map of pdTKRSVMLAC.
• Figure 24 is a restriction map of pdTKRSVMSLAC.
• Figure 25 is a restriction map of pHCMVS.
• Figure 26 is a restriction map of pRSVS.
• Figure 27 is a restriction map of pdTKHCMVS.
• Figure 28 is a restriction map of pdTKSLAC.
• Figure 29 is a restriction map of pdTKRSVS.
• Figure 30 is a restriction map of pdTKRSVSLAC.
• Figure ' 31 is a restriction map of pHCMVSIG.
• Figure 32 is a restriction map of pRSVSIG.
• Figure 33 is a restriction map of pdTKHCMVSIG.
• Figure 34 is a restriction map of pdTKSIGLAC.
• Figure 35 is a restriction map of pdTKRSVSIG.
• Figure 36 is a restriction map of pdTKRSVSIGLAC.
• Figure 37 is a restriction map of pHCMVSDAD.
• Figure 38 is a restriction map of pRSVSDAD.
• Figure 39 is a restriction map of pdTKHCMVSDAD.
• Figure 40 is a restriction map of pdTKSDADLAC.
• Figure 41 is a restriction map of pdTKRSVSDAD.
• Figure 42 is a restriction map of pdTKRSVSDADLAC.
• the FHV-1 viral strain utilized was a live attenuated vaccine strain that was isolated from a bivalent calici-herpes feline vaccine for nasal administration, marketed by SOLVAY DUPHAR under the trademark DOHYVAC CH using the method described in, and the parameters disclosed by (1).
• CCL 94 Crandell Feline Kidney cells
• CCL 94 Crandell Feline Kidney cells
• Cells were grown at 37°C in an atmosphere having 3% of CO2 and in culture media of : (1) 500 ml of medium 199 with Earle's salts, 2.2 g/1 NaHC0 3 and L-glutamine (GIBC0); (2) 500 ml of Ham's F12 with L-glutamine (GIBC0); (3) 25 ml of lactalbumin hydrolysate (GIBC0); 25 ml of fetal calf serum (GIBC0); and 5 ml of a fructose solution (cone, of 200 grams of fructose/1).
• GIBC0 Crandell Feline Kidney
• the cells were then infected with approximately 0.01 virus particles per cell at a cell confluence of about 50% to 80%, as visually observed (as used in the Examples herein, 100% confluence is defined as 10- ⁇ cells/cm ⁇ of plate).
• the infected CRFK cells were then incubated for two to three days at 37°C in an atmosphere having 3% CO2 and in culture media having the same composition as that described above.
• Viral DNA was then purified by successive phenol/chloroform extractions and ethanol precipitation.
• FHV-1 Sail A fragment contains the thymidine kinase (TK) gene (2).
• TK thymidine kinase
• Example 2 was digested by Sail and the resulting fragments cloned in the cosmid pHC79 (B0EHRINGER cat.# 567 795).
• cosmid pHC79 B0EHRINGER cat.# 567 795.
• the cosmid containing the 19 kbp Sail A fragment was then subjected to double digestion with Sail and BamHI and a 5.6 kbp Sall-BamHI internal fragment of the Sail A fragment, having the thymidine kinase gene, was isolated.
• This 5.6 kbp Sall-BamHI fragment was then subcloned in pHC79, resulting in cosmid pFHVAl.
• pFHVAl was then subjected to digestion with Sail and Hindlll and a 3.8 kbp Sall-Hindlll fragment isolated therefrom.
• pFHVA-1 cosmids were subjected to digestion with Hindlll and BamHI and a 1.8 kbp Hindlll-BamHI fragment of pFHVAl was isolated therefrom.
• the TK gene is split between the said 3.8 kbp Sall-Hindlll fragment and the 1.8 kbp Hindlll-BamHI fragment.
• the 3.8 kbp Sall-Hindlll and the 1.8 kbp Hindlll-BamHI fragments were then subcloned in respective pBSLK2 (described in European Patent Application No.
• plasmids giving, respectively, plasmid pTKNl (having the 3.8 kbp Sall-Hindlll fragment) and plasmid pTKCl (having the 1.8 kbp Hindlll-BamHI fragment).
• pBSLKl also described in European Patent Application No. 517,292
• pTKNl A 1.5 kbp Xbal-EcoRV fragment of pTKNl was then obtained and cloned in pBSLKl, which had been treated as described above. The resulting plasmid was pTKN2. pTKCl was then subjected to a double digestion with Smal and Fspl and a 1 kbp Smal-Fspl fragment was isolated therefrom.
• the pdTK transfer plasmid was then obtained by insertion of the 1 kbp Smal-Fspl fragment into the Smal site of pTKN2 (which had previously been digested with Smal).
• a restriction map of pdTK can be seen by reference to Figure 2.
• the insertion of the 1 kbp Smal-Fspl fragment of pTKCl into pTKN2 resulted in an approximately 0.4 kbp to 0.5 kbp (456 bp) internal EcoRV-Smal deletion in the thymidine kinase gene of the resulting transfer plasmid pdTK.
• pdTK also contains a unique BamHI site which has been introduced in the thymidine kinase coding sequence. This site is used for insertion of foreign genes. Sequences (SEQ ID NO: 4)around the insertion site are:
• Purified FHV-1 viral DNA obtained as described above in Example 2, was digested with EcoRI and the resulting fragments, including a 4.3 kbp EcoRI M fragment (as designated by (5)) were cloned in the cosmid pHC79 (Boehringer). A cosmid containing the 4.3 kbp EcoRI M fragment was then identified by restriction enzyme analysis and used for further constructions.
• gG glycoprotein G
• EHV-1 herpes simplex virus type 1
• FHV-1 gG the FHV-1 glycoprotein G
• the cosmid containing the EcoRI M fragment was then digested with EcoRI and the 4.3 kbp EcoRI-EcoRI fragment was isolated therefrom.
• the transfer plasmid pdgG was obtained as follows: pECOM was digested with Sacl and Aval, treated with T4 DNA polymerase and then digested with BamHI. A 1.1 kbp SacI-BamHI pECOM fragment was then isolated therefrom. This 1.1 kbp SacI-BamHI fragment was then cloned in plasmid pECOMdl, which had been previously digested with BamHI, treated with T4 DNA polymerase and then digested with Bglll. The resulting pdgG transfer plasmid contains a unique BamHI site for insertion of foreign genes.
• This site is located in the gG gene, as deduced from partial sequencing of the pdgG plasmid and comparison with the published gG gene sequences of other herpesviruses (such as those nucleotide sequences available in GenBank nucleic acid sequence data bank under the accession number X14112, D00317 and D00374 for HSV-1 and M86664 for EHV-1).
• EXAMPLE 5 CONSTRUCTION OF INTERMEDIATE PLASMIDS HAVING EXPRESSION SIGNALS FOR FOREIGN GENES
• pSV40polyAE Six different plasmids having expression signals were constructed: pSV40polyAE, pSV40polyAL, pHCMVpolyAE, pHCMVpolyAL, pRSVpolyAE and pRSVpolyAL.
• pSV40polyAE and pSV40polyAL contain simian virus 40 (SV40) early promoter sequences.
• SV40polyAE contains the SV40 early transcript polyadenylation sequences
• pSV40polyAL contains the SV40 late transcript polyadenylation sequences.
• pHCMVpolyAE and pHCMVpolyAL contain human cytomegalovirus major immediate early gene promoter (HCMVIE) sequences and, respectively, the SV40 early and late polyadenylation signals.
• pRSVpolyAE and pRSVpolyAL contain the Rous sarcoma retrovirus (RSV) Long Terminal Repeat (LTR) promoter and, respectively, the SV40 early and late transcript and polyadenylation sequences.
• RSV Rous sarcoma retrovirus
• LTR Rous sarcoma retrovirus
• SV40 sequences were obtained from commercial plasmid pSVK3 (PHARMACIA, cat.# 274511).
• HCMVIE sequences were obtained from commercial plasmid pOG44 (STRATAGENE, cat.#218401).
• RSV promoter sequences were obtained from commercial plasmid P0PI3CAT (STRATAGENE, LAC SWITCH TM INDUCIBLE MAMMALIAN EXPRESSION SYSTEM, Cat.# 217450).
• the plasmids that have the expression signals contain a unique Bglll site, situated between the promoter and the polyadenylation sequences, for permitting the further insertion therein of coding sequences which code for foreign peptides which are to be expressed under the control of the said expression signals.
• the gene expression cassettes having both these expression signals and the peptide coding sequences can then be isolated on a BclI-BamHI fragment and inserted in the BamHI site of either pdTK or pdgG.
• pSV40polyAE was constructed by site-directed mutagenesis performed on pSVK3 uracilated single-stranded DNA, following the supplier recommendations (BioRad Muta-Gene Phagemid In Vitro
• SV40PA2 (SEQ ID NO: 8): 5' GATGCTATTGCTTTATTTGTAACCAGATCTATTATAAGCTGCAATAAACAAGTTAAC 3'
• the resulting plasmid was pSVK3mut.
• pSVK3mut was then digested with Bglll and self-ligated in order to remove the internal, approximately 1 kbp Bglll fragment therefrom, generating pSV40polyAE (see Figure 3). Plasmid Coordinate Nature and origin of the sequences
• SV40 early transcript polyadenylation sequences (nucleotides 1295 through 1406 from pSVK3). 469 - 2981 vector sequences from pSVK3 (including nucleotides 1407 through 3919).
• pSV40polyAL pSVK3mut was digested with Bglll and self-ligated in order to inverse the internal approximately 1 kbp Bglll fragment, generating pSVK3muti.
• pSVK3muti was then digested with BamHI and self-ligated to remove the internal 1 kbp and 0.2 kbp BamHI fragments therefrom. The resulting plasmid is pSV40polyAL (see Figure 4).
• nucleotides 1294 through 1201 from pSVK3 are nucleotides 1294 through 1201 from pSVK3.
• 451 - 2969 vector sequences from pSVK3 including nucleotides 1401 through 3919.
• pOG44 was digested with Aval, treated with T4 DNA polymerase and, finally, digested with Xbal.
• This 0.9 kbp Xbal-Aval fragment was then cloned into the Smal-Xbal sites of pBSLK2 (obtained as described in European Patent Application No. 517,292), generating pHCMV.
• Uraciliated single-stranded DNA was then prepared from pHCMV and used as template for site-directed mutagenesis with the following synthetic primers: HCMVP1 ( SEQ ID NO : 9 ) :
• HCMVP2 5' GTACGGGCCAGATATACGCTGCTCAGTTGACATTGATTATTGACTAG 3' HCMVP2 (SEQ ID NO: 10):
• pHCMVmut was then digested with Bell and Bglll and an approximately 0.6 kbp Bcll-Bglll fragment was identified and isolated therefrom.
• the 0.6 kbp Bcll-Bglll fragment was then cloned in a 2.6 kbp Bcll-Bglll fragment from pSV40polyAE, generating pHCMVpolyAE (see Figure 5).
• GTTTAGTGAACCGT SEQ ID NO: 11 sequence obtained from human cytomegalovirus immediate early gene sequences (including nucleotides 1129 through 1142).
• 631 - 636 AGATCT linker sequence.
• SV40 early transcript polyadenylation sequences (nucleotides 1295 through 1406 from pSVK3). 749 - 3261 vector sequences from pSVK3 (including nucleotides
• pHCMVpolyAL was prepared by digesting pHCMVmut with Bell and Bglll and an approximately 0.6 kbp Bcll-Bglll fragment was identified and isolated therefrom. The 0.6 kbp Bcll-Bglll fragment was then cloned in a 2.6 kbp Bcll-Bglll fragment from pSV40polyAL, generating pHCMVpolyAL (see Figure 6). Plasmid Coordinate Nature and origin of the sequences
• SV40 late transcript polyadenylation sequences (nucleotides 1294 through 1201 from pSVK3). 731 - 3249 vector sequences from pSVK3 (including nucleotides 1401 through 3919). Coordinates relative to the human cytomegalovirus immediate early gene promoter refer to the sequence accessible in the EMBL nucleic acid sequence data bank, accession number X03922.
• pRSVpolyAL p0PI3CAT (STRATAGENE, Cat.# 217450) was digested with BstXI, treated with T4 DNA polymerase and subsequently digested with Bglll and an approximately 0.6 kbp fragment, containing the RSV LTR promoter sequences was identified and isolated therefrom. This 0.6 kbp fragment was then cloned into the approximately 5.7 kbp fragment of pHCMVLACE (obtained as described below in Example 6), which had been previously digested with Spel, treated with T4 DNA polymerase and, finally, digested with Bglll. The resulting plasmid is pRSVLACE.
• pRSVLACE was then digested with Bglll and Seal and an approximately 2.2 kbp Bglll-Scal fragment was identified and isolated therefrom. This 2.2 kbp Bglll-Scal fragment was then cloned into the approximately 1.1 kbp Bglll-Scal fragment of pSV40polyAL, generating pRSVpolyAL (see Figure 7).
• nucleotides 1294 through 1201 from pSVK3 716 - 3234 vector sequences from pSVK3 (including nucleotides 1407 through 3919).
• Coordinates relative to the human cytomegalovirus immediate early gene promoter refer to the sequence accessible in the EMBL nucleic acid sequence data bank, accession number X03922.
• pRSVpolyAE pRSVLACE was digested with Bglll and Seal and an approximately 2.2 kbp Bglll-Scal fragment was identified and isolated therefrom. This 2.2 kbp Bglll-Scal fragment was then cloned into the approximately 1.1 kbp Bglll-Scal fragment of pSV40polyAE, generating pRSVpolyAE (see Figure 8). Plasmid Coordinate Nature and origin of the sequences
• pSVLACE Two intermediate plasmids having LacZ expression cassettes were constructed for facilitating screening: pSVLACE (see Figure 9) and pHCMVLACE (see Figure 10). These plasmids were obtained by insertion of the approximately 3.1 kbp Bglll-BamHI fragment containing a portion of the LacZ coding sequence (obtained by double digestion of pBSMUTLACZ2 with Bglll and BamHI and the isolation therefrom) into the unique Bglll sites of pSV40polyAE (to provide pSVLACE) and pHCMVpolyAE (to provide pHCMVLACE).
• Plasmid pBSMUTLACZ2 is a derivative of plasmid pBSMUTLACZl (described in European Patent Application No. 517,292) from which the Bell site located in the LacZ gene has been removed by site-directed mutagenesis.
• Original LacZ sequences in pBSmutLACZl Bell
• LacZ cotransfection plasmids were constructed for cotransfection with FHV-1 viral DNA for use as controls in the vaccination studies to be discussed below. These LacZ cotransfection plasmids are: pdTKSVLAC (see Figure 11), pdTKCMVLAC (see Figure 12), pdgGSVLAC and pdgGCMVLAC.
• pdTKSVLAC and pdgGSVLAC were obtained by insertion of the approximately 3.5 kbp BclI-BamHI LacZ expression cassette (derived from pSVLACE by a double digestion thereof with Bell and BamHI) into the unique BamHI sites of, respectively, pdTK (obtained as described above in Example 3) and pdgG (obtained as described above in Example 4).
• pdTKCMVLAC and pdgGCMVLAC were obtained by insertion of the approximately 3.8 kbp BclI-BamHI LacZ expression cassette
• the sequences coding for the FIPV SM protein and the FIPV M protein were obtained and isolated from plasmid B12 (described by (3) and European Patent Application No. 441684). These SM and M coding sequences were then subcloned in the either pHCMVpolyAL or pRSVpolyAL for generating the intermediate plasmids pHCMVM, pHCMVMS, pRSVM and pRSVMS which have either the SM expression cassette or the M expression cassette.
• the fragment from plasmid B12 containing the SM and M coding sequences was obtained by double digestion of plasmid B12 with Hindi and Hindlll, generating a 1.3 kbp HincII-Hindlll fragment and isolation thereof.
• the PCR products were digested with BamHI and Bglll, generating the following fragments (having restriction sites at the extremities thereof in order to facilitate their further cloning in the Bglll site of the expression plasmids), which were then isolated on an agrose gel and purified. Sequences at the 5' and 3' ends of the cloned fragments are as follows:
• BamHI > M coding sequence ⁇ BamHI pHCMVMS, pHCMVM, pRSVMS and pRSVM were then constructed using the above-described SM and M coding sequences as follows: pHCMVMS (see Figure 13) was constructed by insertion of the approximately 0.3 kbp Bglll-BamHI SM fragment into the Bglll site of pHCMVpolyAL, described above in Example 5. pHCMVM (see Figure 14) was constructed by insertion of the approximately 0.8 kbp BamHI-BamHI M fragment into the Bglll site of pHCMVpolyAL, described above in Example 5.
• pRSVMS (see Figure 15) was constructed by insertion of the approximately 0.3 kbp Bglll-BamHI SM fragment into the Bglll site of pRSVpolyAL, described above in Example 5.
• pRSVM (see Figure 16) was constructed by insertion of the approximately 0.8 kbp BamHI-BamHI M fragment into the Bglll site of pRSVpolyAL, described above in Example 5.
• EXAMPLE 9 CONSTRUCTION OF pdTKMLAC, pdTKMSLAC, pdTKRSVMLAC and pdTKRSVMSLAC COTRANSFECTION PLASMIDS
• An approximately 1.5 kbp BclI-BamHI fragment containing the M expression cassette was isolated from pHCMVM (obtained as described above in Example 8). Similarily, an approximately 0.9 kbp BclI-BamHI fragment containing the SM expression cassette was isolated from pHCMVMS (obtained as described above in Example 8).
• each of the M and SM expression cassettes were then cloned in the BamHI site of respective pdTK plasmids (obtained as described above in Example 3), resulting in, respectively, pdTKHCMVM (see Figure 17) and pdTKHCMVMS (see Figure 18). If desired, each of these plasmids may be used for cotransfection with the FHV-1 viral DNA for producing a recombinant viral vector.
• the 3.5 kbp BclI-BamHI LacZ expression cassettes were obtained by double digestion of pSVLACE (obtained as described above in Example 6) with Bell and BamHI and isolated therefrom. Respective 3.5 kbp BclI-BamHI LacZ expression cassettes were then cloned into the unique BamHI sites of pdTKHCMVM and pdTKHCMVMS, resulting in, respectively, cotransfection plasmids pdTKMLAC (see Figure 19) and pdTKMSLAC (see Figure 20).
• the approximately 1.5 kbp BclI-BamHI fragment containing the M expression cassette and the approximately 0.9 kbp BclI-BamHI fragment containing the SM expression cassette were isolated, respectively, from pRSVM and pRSVMS (obtained as described above in Example 8). These expression cassettes were then cloned in the BamHI site of respective pdTK plasmids (obtained as described above in Example 3), resulting in, respectively, pdTKRSVM (see Figure 21) and pdTKRSVMS (see Figure 22). If desired, each of these plasmids may be used for cotransfection with the FHV-1 viral DNA for producing a recombinant viral vector.
• the 3.8 kbp BclI-BamHI LacZ expression cassettes were obtained by double digestion of pHCMVLACE (obtained as described above in Example 6) with Bell and BamHI and isolated therefrom. Respective 3.8 kbp BclI-BamHI LacZ expression cassettes were then cloned into the unique BamHI sites of pdTKRSVM and pdTKRSVMS, resulting in, respectively, cotransfection plasmids pdTKRSVMLAC (see Figure 23) and pdTKRSVMSLAC (see Figure 24).
• each of the M and SM expression cassettes were then cloned in the BamHI site of respective pdgG plasmids (obtained as described above in Example 4), resulting in, respectively, pdgGHCMVM and pdgGHCMVMS. If desired, each of these plasmids may be used for cotransfection with the FHV-1 viral DNA for producing a recombinant viral vector.
• FHV-1 viral DNA the approximately 3.5 kbp BclI-BamHI fragments, containing the BclI-BamHI LacZ expression cassette, were obtained by double digestion of pSVLACE (obtained as described above in
• Respective 3.5 kbp BclI-BamHI fragments were then cloned into the BamHI site of pdgGHCMVM and pdgGHCMVMS, resulting in, respectively, cotransfection plasmids pdgGMLAC and pdgGMSLAC.
• PLASMIDS HAVING S EXPRESSION CASSETTES pUCE2 (described in (4)), containing the FIPV spike (S) coding sequence (described in both (4) and in European Patent
• BclI-BamHI S expression cassettes of approximately 5.2 kbp were obtained by double digestion of pHCMVS (obtained as described above in Example 11) with Bell and BamHI and isolation therefrom. These 5.2 kbp BclI-BamHI expression cassettes were then inserted in the BamHI site of pdTK (obtained as described above in Example 3) and in the BamHI site of pdgG (obtained as described above in Example 4), giving, respectively, pdTKHCMVS (see Figure 27) and pdgGHCMVS. If desired, each of these plasmids may be used for cotransfection with the FHV-1 viral DNA for producing a recombinant viral vector.
• Another cotransfection plasmid was constructed by first inserting the 5.2 kbp BclI-BamHI S expression cassette, isolated from pRSVS (obtained as described above in Example 11), in the BamHI site of plasmid pdTK, resulting in pdTKRSVS (see Figure 29). If desired, this plasmid may be used for cotransfection with the FHV-1 viral DNA for producing a recombinant viral vector.
• the 3.8 kbp BclI-BamHI LacZ expression cassette isolated from pHCMVLACE (obtained as described above in Example 6), were then inserted into the BamHI site of pdTKRSVS, resulting in cotransfection plasmid pdTKRSVSLAC (see Figure 30).
• EXAMPLE 13 - CONSTRUCTION OF pHCMVSIG and pRSVSIG INTERMEDIATE PLASMIDS HAVING THE delS EXPRESSION CASSETTE pHCMVSIG was then prepared, as follows, for the expression of the FIPV S protein from which the signal peptide therefor has been deleted therefrom (the signal sequence-deleted S gene), so that the S protein is produced thereby without its N-terminal signal sequence.
• pHCMVS which contains the coding sequence for the FIPV spike (S) protein (obtained as described above in Example 11) was used as a template to perform a PCR amplification with the primers FIPSIGl and FIPSIG2 (Eurogentec, Belgium) described below. Starting from these primers, an approximately 0.5 kbp DNA fragment was amplified by PCR (Perkin Elmer).
• FIPSIGl SEQ ID NO:22:
• SacII FIPSIG2 (SEQ ID NO:23) : 5' CTGTCAGCACCCGTACATGTGGAATTCCACTG 3' EcoRI
• the amplified approximately 0.5 kbp fragment was cloned in the Smal site of pBSLKl (described in European Patent Application No. 517,292), generating pBSMUTS.
• An approximately 0.5 kbp SacII-EcoRI fragment of plasmid pHCMVS was then replaced with an approximately 0.4 kbp SacII-EcoRI fragment from pBSMUTS, generating pHCMVSIG (see Figure 31), in which the signal peptide of the S protein is deleted.
• a plasmid for the expression of the signal peptide-deleted S protein under the control of the RSV promoter was then also constructed. pHCMVSIG was subjected to a double digestion with
• SacII and BamHI and an approximately 4.5 kbp SacII-BamHI fragment was isolated therefrom.
• pRSVS obtained as described above in Example 11
• pRSVS was subjected to a double digestion with SacII and BamHI and an approximately 3.2 kbp SacII-BamHI fragment of pRSVS was isolated therefrom.
• the 4.5 kbp SacII-BamHI fragment was then ligated with the 3.2 kbp SacII-BamHI fragment, resulting in pRSVSIG (see Figure 32).
• EXAMPLE 14 CONSTRUCTION OF pdTKSIGLAC, pdTKRSVSIGLAC and pdgGSIGLAC COTRANSFECTION PLASMIDS Three cotransfection plasmids for the signal sequence-deleted S gene were constructed.
• a first cotransfection plasmid for insertion in the FHV-1 thymidine kinase was constructed as follows: an approximately 5.1 kbp BclI-BamHI signal sequence-deleted S gene expression cassette was isolated from pHCMVSIG (obtained as described above in
• Example 13 and cloned into the BamHI site of pdTK (obtained as described above in Example 3), generating pdTKHCMVSIG (see Figure 33). If desired, this plasmid may be used for cotransfection with the FHV-1 viral DNA for producing a recombinant viral vector.
• a 3.5 kbp BclI-BamHI LacZ expression cassette from pSVLACE obtained as described above in Example 6 was then obtained and inserted into the BamHI site of pdTKHCMVSIG, giving the cotransfection plasmid pdTKSIGLAC (see Figure 34).
• a second cotransfection plasmid for insertion in the FHV-1 thymidine kinase gene was constructed as follows: an approximately 5.1 kbp BclI-BamHI signal sequence-deleted S gene expression cassette was isolated from pRSVSIG (obtained as described above in Example 13) and cloned into the BamHI site of pdTK (obtained as described above in Example 3), generating pdTKRSVSIG (see Figure 35). If desired, this plasmid may be used for cotransfection with the FHV-1 viral DNA for producing a recombinant viral vector.
• the 3.8 kbp BclI-BamHI LacZ expression cassette from pHCMVLACE (obtained as described above in Example 6) was then inserted into the BamHI site of pdTKRSVSIG, generating the cotransfection plasmid pdTKRSVSIGLAC (see Figure 36).
• a third cotransfection plasmid for insertion in the FHV-1 gG gene was constructed as follows: an approximately 5.1 kbp BclI-BamHI signal sequence-deleted S gene expression cassette was isolated from pHCMVSIG (obtained as described above in Example 13) and cloned into the BamHI site of pdgG (obtained as described above in Example 4), resulting in pdgGHCMVSIG. If desired, this plasmid may be used for cotransfection with the FHV-1 viral DNA for producing a recombinant viral vector.
• the generated plasmid, pHCMVSDAD (see Figure 37), has the D,
• pRSVSDAD for the expression of the ADE-epitop- deleted S protein under the control of the RSV promoter was also constructed by obtaining an approximately 3.5 kbp Pstl-BamHI fragment (having the S expression cassette) by double digestion of pHCMVSDAD (obtained as described above in Example 15) with Pstl and BamHI and isolation therefrom.
• An approximately 4.2 kbp Pstl-BamHI fragment was obtained by double digestion of pRSVS (obtained as described above in Example 11) with Pstl and BamHI and isolation therefrom.
• Example 15 with Bell and BamHI and isolation of the fragment therefrom. Respective 5.1 kbp BclI-BamHI fragments from pHCMVDAD were then inserted into the BamHI site of pdTK (obtained as described above in Example 3) and the BamHI site of pdgG (obtained as described above in Example 4), resulting in, repectively, pdTKHCMVSDAD (see Figure 39) and pdgGHCMVSDAD. If desired, these plasmids may be used for cotransfection with the FHV-1 viral DNA for producing recombinant viral vectors.
• Respective 5.1 kbp Bcl-BamHI fragments from pRSVSDAD were cloned in the BamHI site of pdTK (obtained as described above in Example 3), generating pdTKRSVSDAD (see Figure 41). If desired, this plasmid may be used for cotransfection with the FHV-1 viral DNA for producing a recombinant viral vector.
• FHV-1 viral DNA the 3.8 kbp BclI-BamHI LacZ expression cassette from pHCMVLACE (obtained as described above in Example 6) was inserted into the BamHI site of pdTKRSVSDAD, resulting in the cotransfection plasmid pdTKRSVSDADLAC (see Figure 42).
• EXAMPLE 17 - PRODUCTION OF FHV-1 LIVE RECOMBINANT CARRIER A live recombinant carrier was then obtained by cotransfection (as described below) of CRFK cells (see Example 1) with purified FHV-1 DNA (obtained as described in Example 2) and with the cotransfection plasmid pdTKCMVLAC ( Figure 12), which was obtained as described above in Example 7.
• the transfection was performed with LIPOFECTIN reagent (GIBC0 BRL) following the supplier recommendations.
• Crandel feline kidney (CRFK) cells were transfected in 25 crn * ⁇ flask, at 50% to 80% confluence (visually determined) in the medium described above in Example 1 from which the serum had been ommitted and to which had been added 20 to 25 ⁇ g LIPOFECTIN, 1 to 15 yg viral DNA and an amount of plasmid DNA necessary to provide a plasmid/virus DNA molar ratio between 1 to 20. Total volume, according to the manufacturers specifications, was about 1.8 ml of the medium. Duration of transfection was from 5 to 24 hours.
• the transfection medium of each sample was removed and replaced with about 5 ml of the CRFK culture medium.
• the cells were then incubated at 37°C for 48 hours. Cells were subsequently passed in 75 cm2 flasks after trypsination and reincubated again at 37°C, until a cytopathic effect was visually observed.
• transfection stock RNA was then harvested after one cycle of freezing-thawing. This virus stock is called transfection stock.
• CRFK cells were seeded in growth medium (see Example 1) in a 25 cm ⁇ Petri dish at about 50% confluence (visually determined) and then infected with about IO*-* to 10 ⁇ infectious virus particles of the transfection stock (described above).
• the liquid growth medium was removed and replaced with 5 ml of a mixture of one volume of a 2% (w/v) agarose melted in water (SEA PLAQUE R agarose, FMC BioProducts, USA) and one volume of 50% (v/v) Medium 199 2x (GIBC0 BRL), 2.5% (v/v) lactalbumin hydrolysate (GIBC0 BRL), 5% (v/v) fetal calf serum (GIBCO BRL) and 15 mM HEPES pH 7.2.
• the Petri dishes were then incubated for about 48 hours at 37°C with 3% CO2 until viral plaques develop.
• Petri dishes were then overlaid with 2 ml of a PBS solution (GIBCO BRL) containing 1% (w/v) melted agarose and 0.3 mg/ml X-gal (5-bromo-4 chloro-3 indolyl- ⁇ -D-galactoside, Boehringer Mannheim).
• Plaques expressing the LacZ encoded ⁇ -galactosidase enzyme turned blue.
• the blue plaques were then stabbed with a Pasteur pipette, transferred onto fresh CRFK cells and purified by successive blue plaque isolations until homogeneity was achieved.
• Large stocks of recombinant viruses were then produced by infection of CRFK cells at 50% to 80% confluence (visually observed) and a multiplicity of infection of 0.01.
• TKLAC live recombinant FHV-1 carrier
• This example was designed to evaluate the vaccinating power of FHV/LACZ recombinants using as criteria, protection against feline rhinotracheitis and immune response against ⁇ -galactosidase.
• the recombinant FHV-1 strain designated TKLAC
• TKLAC recombinant FHV-1 strain obtained as described above in Example 17, having a LacZ gene under the control of the HCMV promoter, was evaluated in a cat vaccination trial and compared with the parental strain.
• Specific-pathogen-free (SPF) cats of about 10 weeks of age were oronasally vaccinated twice three weeks apart with ca. lO- ⁇ - ⁇ TCID50 per dose of either TKLAC FHV-1 recombinant (LacZ group, 5 cats), obtained as described above in Example 17, or the parental strain (Parental group, 5 cats), obtained as described above in Example 1, by applying 0.5 ml of the viral suspension in each nostril.
• a third group (Control group, 5 cats) was innoculated in each nostril with 0.5 ml of vaccine diluent, consisting of Minimal Essential Medium with Earle's Salts (GIBCO BRL, Cat.# 21090-010) as control.
• the two groups of vaccinees showed (see Table 1) mild pyrexia (As used herein, the term “pyrexia” is defined as a temperature equal to or above 39.2°C) for the first 2 to 4 days after challenge.
• the Control group developed severe pyrexia on the third to fifth days and the seventh to eighth days post challenge.
• TKLAC recombinant substantially protects SPF cats against severe challenge with wild-type virus and that that protection is as good as that obtained with the parental vaccine strain.
• Table 3 summarizes the evolution of the seroneutralizing titers. The values represent the inverse of the highest seroneutralizing dilution for all the individual cat sera. All unvaccinated cats remained seronegative prior to the challenge. All vaccinated cats seroconverted after the first vaccination and the majority showed a small rise in titer after the second vaccination and again after challenge.
• Table 4 summarizes the ELISA analyses of serum IgA. The values represent mean optical density x 1000.
• the sera were tested for total IgA and IgA specific for FHV-1 and ⁇ -galactosidase ( ⁇ -GAL).
• ⁇ -GAL ⁇ -galactosidase
• Specific anti- ⁇ -GAL IgA showed a significant increase (serotiters against ⁇ -galactosidase) in the group vaccinated with the TKLAC recombinant, showing that the LacZ insert is expressed in vivo.
• the Live Recombinant Carrier described herein (and formed by cotransfection with FHV-1 viral DNA with the cotransfection plasmids of the present invention having the coding sequence for a protein of interest) is capable of expressing the proteins of interest ir_ ⁇ vivo. Accordingly, this live recombinant carrier would be suitable not just for use with the proteins/polypeptides described herein but other proteins/polypeptides which may be desired to be expressed ij vivo.
• Specified-pathogen-free cats of 9 to 10 weeks of age will be vaccinated. Another vaccination will be administered three weeks after the first vaccination. All vaccinations will be made by the nasal route, with IO 5 to 10 7 TCID50 per cat, of the recombinant FIPV/FHV virus from the stock obtained as described above in Example 17. A group of unvaccinated cats will serve as control. Two to four weeks after the last vaccination, cats will be challenged orally, with 10 2 to IO 5 TCID50 per cat, of a virulent FIPV strain.
• ori plasmid origin of replication (ColEl) ori Fl fl origin of replication bla ⁇ -lactamase gene, conferring resistance to ampicillin LacZ E. coli ⁇ -galactosidase coding sequence HCMVp human cytomegalovirus immediate early gene promoter sequences RSVp rous sarcoma virus long terminal repeat promoter sequences SV40p SV40 early promoter sequences Ae SV40 early transcript polyadenylation signals Al SV40 late transcript polyadenylation signals M FIPV Matrix protein coding sequence
• ORGANISM Feline Infectious Peritonitis virus
• C INDIVIDUAL ISOLATE: 79-1146
• MOLECULE TYPE protein
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)

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