GB2282601A - Coronavirus vaccines - Google Patents

Abstract

There are disclosed vaccines which are effective for protecting a mammal against coronaviruses without inducing antibody dependent enhancement (ADE). In particular, there is disclosed a modified Feline Infectious Peritonitis virus (FIPV) S protein wherein at least one of the A1, A2 or D regions are antigenically-inactive. The S protein may have its signal peptide modified or deleted. Proteins for a FIPV vaccine may include the SM protein and/or the M protein. The protein sequence and corresponding nucleotide sequence of the FIPV SM protein is disclosed. The protein fragments may be attached to proteins such as BSA or KLH. The proteins may be formulated as a live vaccine e.g. by utilising recombinant carriers such as vaccinia, herpes, adeno, sinbis, corona viruses or bacterial carriers.

Description

ANTIGENICALLY-ACTIVE PROTEINS/POLYPEPTIDES AND CORONAVIRUS VACCINES CONTAINING TEE SAHE The present invention relates to antigenically-active proteins/polypeptides, the cloning of the base sequences coding therefor and to the formulation of the antigenically-active proteins/polypeptides for use as vaccines for cornaviruses, and in particular, feline infectious peritonitis virus.

Feline Infectious Peritonitis (FIP) is a normally-fatal infectious feline disease. FIP is caused by a coronavirus known as the Feline Infectious Peritonitis Virus (FIPV). 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 approximately 27 to 31 kilo base pairs (kbp), making it the largest among RNA viruses. Among other members of the family, feline enteritis coronavirus (FECV) is the most closely related.

Corona viruses 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 approximately 40-50K 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.

Classical approaches attempted to develop a vaccine against coronaviruses, especially FIP, include vaccination with live attenuated FIPV and heterologous live coronaviruses. However, the humoral antibodies obtained by the use of such vaccines have largely proven to be ineffective. Indeed, 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). This phenomena finds counterparts in herpes viridae, pox viridae, rhabdo viridae, flavi viridae, alpha viridae, reo viridae and bunya viridae. This phenomenon is believed to be based upon the binding of virus antibody complexes to the Fc-receptors of macrophages. In vitro, 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).

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 PIPV. 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. Unfortunately, the high rate of mutation and natural recombination in the coronaviridae family presents a risk in the use of such mutated FIPV strains as a vaccine.

There remains a need for new antigenically-active agents against coronaviruses, and especially FIPV, and vaccines containing the same which are capable of protecting mammals (such as felines) against the disease while avoiding the phenomena of antibody dependent enhancement. There further remains a need for such a vaccine which includes a suitable live recombinant carrier.

It is a primary object of the present invention to identify and provide new antigenically-active proteins/polypeptides against coronaviruses, and in particular against FIPV.

It is a further primary object of the present invention to determine and provide base sequences coding for the new antigenically-active proteins/polypeptides and to clone the same for providing the antigenically-active proteins/polypeptides.

It is a further primary object of the present invention to formulate such antigenically-active proteins/polypeptides into vaccines for the protection of mammals against coronaviruses, and in particular for the protection of felines against FIPV.

It is a particular object of the present invention to provide such a vaccine that includes, as a part thereof, a live recombinant carrier.

In accordance with the teachings of the present invention, novel antigenically-active proteins/polypeptides are disclosed for protecting mammals (such as felines) against coronaviruses (such as FIPV) without provoking ADE. The proteins disclosed herein include the SM protein and modified S proteins in which certain fragments thereof, which we believe induce ADE, are absent.

Preferably, the new antigenically-active proteins of the present invention are modified S proteins in which one or more of the following antigenic regions are either modified (so as to no longer be antigenic), deleted or otherwise absent: Al and/or A2 and/or D, as those regions are defined herein. As such, such regions are antigenically-inactive (are not antigenic).

It is further preferred that both the Al and the A2 regions be modified, deleted or absent, as discussed above. It is still further preferred that the D region also be modified, deleted or absent (in addition to the Al and A2 regions).

In another preferred embodiment, the S protein has its signal peptide removed (by, for example, cleaving) therefrom.

As used herein, the term "M protein" is used to refer to the same protein designated as the M protein in European Patent Application No. 411,684. The complete amino acid sequence, as well as the complete nucleotide sequence which codes therefor are also described in European Patent Application No. 411,684.

As used herein the term "SM protein" refers to the small membrane protein of FIPV whose complete amino acid sequence, as well as the complete nucleotide sequence which codes therefor are set forth in figure 1.

As used herein the term "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.

As used herein, the term "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.

As used herein, the term "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.

As used herein, the term "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.

The proteins to which the invention relates each further comprise at least one region which is immunogenic, 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.

First of all, the peptides may be prepared synthetically 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 expression vector which comprises a DNA molecule coding for the antigenically-active proteins/polypeptides of the present invention; expressing the genome inserted in the expression vector; c) harvesting the cell culture; and d) isolating the synthesized peptide (protein).

The invention therefore also relates to a method of preparing a DNA molecule which codes for the antigenically-active protein(s)/polypeptides according to the invention. Such a method comprises the steps of a) isolating the expression cassette coding for the S protein and/or SM protein of the target coronavirus (such as FIPV); b) determining the location of the nucleotide base sequences coding for the antigenic region(s) of interest of the the S protein which are desired to modified or deleted; and c) modifying or deleting the nucleotide base sequences coding for the antigenic regions of interest.

In further accordance with the teachings of the present invention, vaccines are disclosed herein which are effective for protecting a mammal (such as a feline) against coronaviruses (such as FIPV) while avoiding inducing ADE.

In a preferred embodiment, the vaccines include the new antigenically-active protein(s)/polypeptide(s) in a suitable carrier. When fragments are used instead of whole proteins, they may be used coupled to suitable known carriers. Examples of such carriers are KLH or BSA.

If desired, the vaccine may include the antigenically-active M protein in a suitable carrier.

In another preferred embodiment, the vaccines include the new antigenically-active protein(s)/polypeptide(s) which are formulated in a live vaccines.

In this respect, 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. The vector may be inserted into a suitable host cell, for example, by transformation.

It is especially preferred that the vaccines disclosed herein include the DNA molecule coding for the antigenicallyactive protein(s)/polypeptide(s) of the invention incorporated into a live recombinant carrier (LRC) with the aim of expression of said peptide(s) or protein(s) in susceptible host cells and/or host organisms. In this manner, the antigenically-active protein(s)/polypeptide(s) of the present invention are expressed in the target animal, in vivo. For this purpose, 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). Examples of such suitable LRC's are vaccinia LRC, herpes-LRC, adeno-LRC, adeno-associated-LRC, sindbis-LRC, corona-LRC and bacterial-LRC.

Further disclosed herein is 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 a live carrier.

In another aspect of the present invention, disclosed herein is a method for immunizing mammals, and in particular felines, against coronaviruses and, in particular, against FIPV, which comprises preparing the 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.

Administration to the target animal of the vaccines of the present invention which include the live recombinant carriers leads to expression of the inserted genes, including the SM protein and the modified S proteins of the present invention in all infected cells.

Figure 1 is the amino acid sequence of the SM protein and the corresponding nucleotide base sequence that codes for the SM protein.

The DNA clonings and sequencing techniques and procedures employed herein e those which are described by Sambrook et al, in Molecular Cloning -- A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989).

The cell & Tissue culturing techniques and procedures utilized herein are those which are described by Doyle et al, in Cell & Tissue Culture: Laboratory Procedures, John Wiley Publishing (1993).

The invention will now be described in more detail with reference to the following specific examples.

EXAMPLE 1 - PROPAGATION OF FELINE HERPESVIRUS 1 (FEV-1).

The FHV-1 viral strain utilized is 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).

The virus is grown on Crandell feline kidney (CRFK) cells (deposited in the American Type Culture Collection under Accession Number CCL 94). Cells are grown at 370C with 3% of C02 in culture media consisting of medium 199 with Earle's salts, 2.2 g/L NaHC03 and L-glutamine (GIBCO, 500 ml), Ham's F12 with L-glutamine (GIBCO, 500 ml), LAH (lactalbumin hydrolysate, GIBCO, 25 ml), FCS (fetal calf serum, GIBCO, 25 ml) and fructose (5 ml of a solution at 200 g/l). 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 105 cells/cm2 of plate).

EXAHPLE 2 - PURIFICATION OF TEE FHV-1 VIRAL DNA.

Supernatants of CRFK cells infected with FHV-1, as described above in Example 1, were collected when cytopathic effect was almost complete, as determined by visual observation.

Supernatants were first clarified by centrifugation at 500 g for 10 min then centrifuged at 25000 rpm (SW 28 BECKMAN rotor) and 40C for 1 hour. The pellet containing viral particles was resuspend in 10 mM Tris pH 7.5, 1 mM EDTA, 0.1% (v/v) NONIDET P-40 (SIGMA). The suspension was then loaded on a cushion of 30% (w/v) sucrose in Tris 10 mM pH 7.5, EDTA lmM and centrifuged at 25000 RPM t-T; 28 rotor) and 40 C for 2 hours. The capsid pellet was resuspended in Tris 10 mM pH 8.3, EDTA lmM, 1% (w/v) SDS (Sodium Dodecyl Sulphate), 500 ug proteinase K (BOEHRINGER)/ml of the suspension and incubated at 500C for 2 hours. Viral DNA was then purified by successive phenol/chloroform extractions and ethanol precipitation.

EXAMPLE 3 - CONSTRUCTION OP TEE pdTR TRANSFER PLASHED FOR INSERTION IN FHV-1 THYMIDINE ENCASE GENE It has previously been published that the FHV-1 Sall A fragment contains the thymidine kinase gene (2).

Purified FHV-1 viral DNA was digested by Sall restriction enzyme and the resulting fragments cloned in the cosmid pHC79 (BOEHRINGER). One cosmid containing a 19 kilo basepair (kbp) Sall A fragment was identified by restriction analysis and used for further constructions.

The 5.6 kbp Sall-BamH1 internal fragment of Sall A was subcloned in pHC79, resulting in cosmid pFHVA1. The 3.8 kbp Sall-Hind3 and 1.8 kbp Hind3-BamH1 fragments of pFHVA1 were then obtained and subcloned in plasmid pBSLK2 (described in European Patent Application No. 517,292) giving, respectively, plasmid pTKN1 and plasmid pTKC1.

Plasmid pBSLK1 (described in European Patent Application No.

517,292), was digested with Bcll, treated with T4 DNA polymerase and then digested with Xbal. A 1.5 kbp Xbal-EcoRV fragment of pTKN1 was then obtained and cloned in pBSLK1 treated as decribed above. The resulting plasmid was pTKN2. The transfer plasmid, called pdTK, was then obtained by insertion of the 1 kbp Smal-Fspl pTKC1 fragment into pTKN2 which had previously been digested with Smal. The insertion of the 1 kbp Smal-Fspl fragment resulted in a 456 bp internal EcoRV-Smal thymidine kinase gene deletion in the transfer plasmid pdTK. Plasmid pdTK contains a unique BamH1 site introduced in the thymidine kinase coding sequence. This site is used for insertion of foreign genes.Sequences around the insertion site are BamH1 5 ACTATCCACAATAACAGGATGATCAGCCCCCGGGAGCTCTCCGACC 3' o o 352 808 Bold nucleotides are derived from the thymidine kinase gene sequence. Nucleotide coordinates 352 and 808 refer to those mentionned in the sequence published by (2).

EXAMPLE 4 - CONSTRUCTION OF TEE pdgG TRANSFER PLASMID FOR INSERTIOtM IN THE FHV-1 gG GENE Purified FHV-1 viral DNA, obtained as described above in Example 1, was digested with EcoR1 as described in (5) and the resulting fragments (as designated by (5)) were cloned in the cosmid pHC79 (Boehringer). One cosmid containing the 4.3 kbp EcoR1 M fragment was identified by restriction enzyme analysis and used for further constructions. We suspected that the FHV-1 gG coding sequence was located on this EcoR1 M fragment.

The EcoR1 M fragment was subcloned in the plasmid pBSLK2 (whose construction is described in European Patent Application No. 517,292), resulting in plasmid pECOM. The 1 kbp EcoR1-Sacl fragment of pECOM was then obtained and subcloned in plasmid pBSLK1 (see Example 3), giving plasmid pECOMdl.

The transfer plasmid pdgG was obtained as follows: pECOM was digested with Sacl and Aval, treated with T4 DNA polymerase and then digested with BamH1. The 1.1 kbp Sacl-BamH1 pECOM fragment was then cloned in plasmid pECOMdl, which had been previously previously digested with BamH1, treated with T4 DNA polymerase and digested with Bgl2. The pdgG transfer plasmid contains a unique BamH1 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.

EXAMPLE 5 - CONSTRUCTION OF PLASMIDS FOR EXPRESSION OF FOREIGN GENES IN THE RECOMBINANT FHV-1 VIRUSES Four different expression plasmids were constructed: pSV40polyAE, pSV40polyAL, pHCMVpolyAE and pHCMVpolyAL.

Plasmids pSV40polyAE and pSV40polyAL contain simian virus 40 (SV40) early promoter sequences. As a polyadenylation signal sequence, pSV40polyAE contains the SV40 early transcript adenylation sequences, and pSV40polyAL contains the SV40 late transcript polyadenylation sequences.

Plasmids pHCMVpolyAE and pHCMVpolyAL contain human cytomegalovirus major immediate early promoter (HCMVIE) sequences and, respectively, the SV40 early and late polyadenylation signals. SV40 and HCMVIE sequences were obtained from commercial plasmids pSVK3 (PHARMACIA) and pOG44 (STRATAGENE), respectively.

The expression plasmids contain a unique Bgl2 site, between the promoter and the polyadenylation sequences, for insertion of foreign genes. The gene expression cassettes can then be isolated on a Bcll-BamH1 fragment and inserted in the BamH1 site of the transfer plasmid pdTK or pdgG.

Detailed description of expression plasmid pSV40polyAE :

Plasmid CoordinatelNature and origin of the sequences 11 - 22 ISV40 sequences; nucleotides 1 through 22 from |from pSVK3.

123 - 28 iTGATCA linker sequence.

129 - 350 ISV40 origin and early promoter; nucleotides 1 123 123 through 1344 from plasmid pSVK3.

351 - 356 AGATCT linker sequence.

1357 - 468 ISV40 early transcript polyadenylation sequences; nucleotides 1295 through 1406 from pSVK3.

1469 - 2981 vector sequences from pSVK3, including Inucleotides 1407 through 3919.

Detailed description of expression plasmid pSV40polyAL:

IPlasmid CoordinatelNature and origin of the sequences I 11 - 22 ISV40 sequences; nucleotides 1 through 22 from |from pSVK3.

123 - 28 ITGATCA linker sequence.

129 - 350 ISV40 origin and early promoter; nucleotidesl 23 through 344 from plasmid pSVK3. I 1351 - 356 IAGATCT linker sequence.

1357 - 450 SV40 late transcript polyadenylation I sequences; Inucleotides 1294 through 1201 from pSVK3. I 1451 - 2969 vector sequences from pSVK3, including I nucleotides 1407 through 3919.

Detailed description of expression plasmid pHCMVpolyAE: Coordinates relative to the human cytomegalovirus immediate early gene promoter refer to the sequence accessible in the EBML nucleic acid sequence data bank, accession number X03922.

Plasmid CoordinatelNature and origin of the sequences I 11 - 22 vector sequences from pSVK3, including I Inucleotides 1 through 22.

123 - 28 ITGATCA linker sequence.

129 - 616 Ihuman cytomegalovirus immediate early gene promoter sequences; nucleotides 361 through I 1948 from pOG44.

1617 - 630 IGTTTAGTGAACCGT linker sequence; human I Icytomegalovirus immediate early gene I sequences, including nucleotides 1129 through 1142.

1631 - 636 IAGATCT linker sequence.

1637 - 736 SV40 early transcript polyadenylation I sequences; nucleotides 1295 through 1406 fromj pSVK3.

737 - 3249 vector sequences from pSVK3, including I Inucleotides 1407 through 3919.

Detailed description of expression plasmid pHCMVpolyAL:

Plasmid CoordinatelNature and origin of the sequences I 11 - 22 vector sequences from pSVK3, including I Inucleotides 1 through 22.

123 - 28 1TGATCA linker sequence.

129 - 616 Ihuman cytomegalovirus immediate early gene I Ipromoter sequences; nucleotides 361 through I 1948 from pOG44.

1617 - 630 /GTTTAGTGAACCGT linker sequence; human I Icytomegalovirus immediate early gene I Isequences, including nucleotides 1129 through 11142.

1631 - 636 IAGATCT linker sequence.

1637 - 736 SV40 late transcript polyadenylation sequences; nucleotides 1295 through 1201 from IpSVK3.

737 - 3249 Ivector sequences from pSVK3, including nucleotides 1407 through 3919.

EXAMPLE 6 - CONSTRUCTION OF LACZ EXPRESSION PLASMIDS Coinsertion of a LacZ expression cassette with the FIPV genes in the FHV-1 genome will allow the screening of the recombinant viruses. Two LacZ expression plasmids were constructed. They are: pSVLACE and pHCMVLACE. They were obtained by insertion of the Bgl2-BamH1 LacZ fragment from plasmid pBSMUTLACZ2 in the Bgl2 site of respectively pSV40polyAE and pHCMVpolyAE.

Plasmid pBSMUTLACZ2 is a derivative of plasmid pBSMUTLACZ1 (described in European Patent Application No. 517,292) from which the original LacZ Bcll site has been removed by site-directed mutagenesis.

Original LacZ sequences in pBSmutLACZ1: Bcll 5' AGT GTG ATC ATC TGG 3' Ser Val Ile Ile Trp LacZ sequence in pBSmutLACZ2: 5' AGT GTT ATC ATC TGG 3' Ser Val Ile Ile Trp EXAMPLE 7 - CONSTRUCTION OF LACZ TRANSFER PLASMIDS Four LacZ transfer plasmids were constructed for use as controls in the vaccination studies to be discussed below. These LacZ transfer plasmids are: pdTKSVLAC, pdTKCMVLAC, pdgGSVLAC and pdgGCMVLAC.

Plasmids pdTKSVLAC and pdgGSVLAC were obtained by insertion of the Bcll-BamH1 LacZ expression cassette from plasmid pSVLACE in the BamH1 site of, respectively, pdTK and pdgG.

Plasmids pdTKCMVLAC and pdgGCMVLAC were obtained by insertion of the Bcll-BamHl LacZ expression cassette from plasmid pHCMVLACE in the BamH1 site of, respectively, pdTK and pdgG.

EXAMPLE 8 - CONSTRUCTION OF FIPV M AND SM EXPRESSION PLASMIDS A plasmid, named pB12 (described by (3) and European Patent Application No. 441684), containing the FIPV SM and M coding sequences was used to subclone the genes in the different expression plasmids.

The 1.3 kbp Hinc2-Hind3 fragment from pB12 was cloned in the Hinc2-Hind3 sites of pBSLK2, giving plasmid pBSM. This plasmid contains the SM and M coding sequences.

DNA fragments containing SM or M genes were produced by polymerase amplification reaction performed on pBSM, as is discussed below. Restriction sites were introduced at the extremities of both fragments in order to facilitate their further cloning in the Bgl2 site of the expression plasmids. The primers (Eurogentec, Belgium) used are SM Fragment 5 GTGGCCATTTGAAAGTTTAGGGATCCTTACACCATATGTAATAATTTTTCATG 3' 5' ATTTTTGGTTTGAACTAAAACAAAGGATCCCCACCATGAAGTACATTTTGCTAAT 3' M Fragment 5' AATGTACTTCATTTTCTTTTACTGGATCCTCAAACCAAAAAT 3' 5' GAAGAAGAAGAACACCATAACTAGATCTCCACCATGACGTTCCCTAGGGCATTTAT 3' Sequences at the 5' and 3' ends of the cloned fragments are as follows SM fragment (0.3 kbp) 5' AGATCTCCACCATGACG TTGGTTTGAGGATCC 3' Bgl2 > SM coding sequence < BamH1 M fragment (0.8 kbp):: 5' GGATCCCCACCATGAAG ATGGTGTAAGGATCC 3' BamH1 > M coding sequence < BamH1 Expression plasmids pHCMVMS and pHCMVM were constructed by insertion of, respectively, the Bgl2-BamH1 SM fragment into the Bgl2 site and the BamH1-BamH1 M fragment in the Bgl2 site of expression plasmid pHCMVpolyAL, described above in Example 5.

EXAHPLE 9 - CONSTRUCTION OF FIPV M AND SH TRANSFER PLASHIDS FOR INSERTION IN TEE FHV-1 THYMIDINE KINASE GENE.

The M and SM Bcll-BamH1 expression cassettes were isolated, respectively, from plasmids pHCMVM and pHCMVMS (see Example 8).

These expression cassettes were then cloned in the BamH1 site of pdTK (described in Example 3), resulting in, respectively, pdTKHCMVM and pdTKHCMVMS. The Bcll-BamH1 LacZ expression cassette, from plasmid pSVLACE (see Example 6), was then cloned in the BamH1 site of plasmid pdTKHCMVM and pdTKHCMVMS, resulting in, respectively, transfer plasmids pdTKMLAC and pdTKMSLAC.

EXAMPLE 10 - CONSTRUCTION OF FIPV M AND SM TRANSFER PLASMIDS FOR INSERTION IN TEE FEV-1 gG GENE.

The M and SM Bcll-BamH1 expression cassettes were isolated, respectively from plasmids pHCMVM and pHCMVMS (see Example 8).

These expression cassettes were cloned in the BamH1 site of plasmid pdgG (described in Example 4), resulting in, respectively, pdgGHCMVM and pdgGHCMVMS. The Bcll-BamH1 LacZ expression cassette, from plasmid pSVLACE (see Example 6), was then cloned in the BamH1 site of plasmid pdgGHCMVM and pdgGHCMVMS, resulting in, respectively, transfer plasmids pdgGMLAC and pdgGMSLAC. EXAMPLE 11 - CONSTRUCTION OF FIPV S EXPRESSION PLASMID A plasmid, named pUCE2, containing the FIPV spike (S) coding sequence (described in (4) and in European Patent Application No.

264,979) was used for further constructions. A 4.3-4.4 kbp BamH1 fragment carrying the whole S coding sequence was isolated from plasmid pUCE2 and cloned in the Bgl2 site of expression plasmid pHCMVpolyAL (see Example 5), resulting in plasmid pHCMVS.

EXAHPLE 12 - CONSTRUCTION OF S TRANSFER PLASMIDS Respective Bcll-BamH1 S expression cassettes were isolated from pHCMVS (described in Example 11) and inserted in the BamH1 sites of plasmids pdTK (see Example 3) and pdgG (see Example 4), giving pdTKHCMVS and pdgGHCMVS, respectively. Respective Bcll-BamHl LacZ expression cassettes from pSVLACE (described in Example 6) were then inserted in the BamH1 sites of plasmids pdTKHCMVS and pdgGHCMVS, generating, respectively, the transfer plasmids pdTKSLAC and pdgGSLAC.

EXAMPLE 13 - CONSTRUCTION OF A PLASMID FOR EXPRESSION OF TEE SIGNAL SEQUENCE DELETED FIPV S GENE.

Plasmid pHCMVSIG was prepared for the expression of the FIPV S protein without its N-terminal signal sequence as follows.

First, pHCMVS (described in Example 11) was denatured and the FIPSIG1 and FIPSIG2 primers (Eurogentec, Belgium) set forth below were hybridized to the denatured pHCMVS DNA. Starting from these primers, an approximately 0.5 kbp DNA fragment was amplified by PCR (Perkins Elmer).

FIPSIG1 primer sequence 5' CCACACAGTTTTGAGTCCGCGGCCACCATGACAACAAATAATGAATGCATACAAGTTAACG 3' Sac2 FIPSIG2 primer sequence 5' CTGTCAGCACCCGTACATGTGGAATTCCACTG 3 EcoR1 The amplified fragment was cloned in the Smal site of plasmid pBSLK1 (described in European Patent Application No.

517,292), resulting in plasmid pBSMUTS. A approximately 0.5 kbp Sac2-EcoR1 fragment of plasmid pHCMVS was then replaced with an approximately 0.4 kbp Sac2-EcoR1 fragment from plasmid pBSMUTS, generating plasmid pHCMVSIC, in which the signal peptide of the S protein is deleted.

EXAMPLE 14 - CONSTRUCTION OF TRANSFER PLASMIDS FOR TEE SIGNAL SEQUENCE DELETED S GENE.

Two transfer plasmids were constructed.

The transfer plasmid for insertion in the FHV-1 thymidine kinase was constructed as follows : the Bcll-BamH1 expression cassette from plasmid pHCMVSIG (see Example 13) was cloned in the BamH1 site of plasmid pdTK (see Example 3), generating plasmid pdTKHCMVSIG. The Bcll-BamH1 LacZ expression cassette, from plasmid pSVLACE (see Example 6), was then inserted in the BamH1 site of pdTKHCMVSIG, giving the transfer plasmid pdTKSIGLAC.

The transfer plasmid for insertion in the FHV-1 gG gene was obtained by insertion of the pHCMVSIG Bcll-BamH1 fragment in the BamH1 site of plasmid pdgG (see Example 4), resulting in plasmid pdgGHCMVSIG. The Bcll-BamH1 LacZ expression cassette from plasmid pSVLACE (see Example 6) was inserted in the BamH1 site of pdgGHCMVSIG, generating the transfer plasmid pdgGSIGLAC.

EXAHPLE 15 - CONSTRUCTION OF A PLASHID FOR EXPRESSION OF FIPV S PROTEIN WITH DELETION OF ANTIBODY-DEPENDENT-ENEANCEHENT RELATED EPITOPES Deletion of what we believe to be the antibody-dependent-enhancement (ADE) epitope coding sequences was directly performed, by site-directed mutagenesis, on uracilated single-stranded pHCMVS (see Example 11) DNA (BioRad mutagenesis kit). The primers (Eurogentec, Belgium) used for hybridisation to and elongation of the single-stranded DNA template are 1. Primer to Delete D epitope of the S protein: 5' GAAATTTCATGTTATAGTCACACAGTGGAAATCCCGTTCGGCATAACTGACGG 3' 2. Primer to Delete Al epitope of the S protein 5' GTCAATATAACCATTGATCTTGGTATGCCCATAGCCTCGACACTAAGTAAC 3, 3.Primer to Delete A2 epitope of the S protein 5' GTTCATTCCACTTGCAAAAGTTCTTTAAATCAAGACTGCACGGATGTTTTAGAGGC 3, The generated plasmid is called pHCMVSDAD.

EXAHPLE 16 - CONSTRUCTION OF TRANSFER PLASMIDS FOR TEE S GENE WITH DELETION OF ANTIBODY DEPENDENT ENEANCEMENT-RELATED EPITOPES The Bcll-BamH1 S expression cassette from plasmid pHCMVSDAD (see Example 15) was inserted in the BamH1 site of pdTK (see Example 3) and pdgG (see Example 4), resulting in, repectively, plasmids pdTKHCMVSDAD and pdgGHCMVSDAD. The Bcll-BamH1 LacZ expression cassettte from pSVLACE (see Example 6) was subsequently cloned in the BamH1 site of plasmids pdTKHCMVSDAD and pdgGHCMVSDAD giving, respectively, the transfer plasmids pdTKSDADLAC and pdgGSDADLAC.

EXAMPLE 17 - PRODUCTION OF RECOMBINANT FHV-1 VIRUSES CONTAINING THE LACZ GENE AND TEE FIPV M, SM, S AND MUTATED S GENES.

Recombinant viruses are obtained by cotransfection (as described below) of CRFK cells (see Example 1) with purified FHV-1 DNA (obtained as described in Example 2) and the above-mentioned transfer plasmids.

The transfections are performed with LIPOFECTIN reagent (GIBCO BRL) following the supplier recommendations. Crandel feline kidney (CRFK) cells were transfected in 25 cm2 flask, at 50% to 80% confluence (visually determined) in the medium described above in Example 1 from which the serum has been ommitted but to which 20 to 25 ug LIPOFECTIN, 1 to 15 lug 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 is about 1.8 ml of the medium. Duration of transfection was from 5 to 24 hours.

After transfection, the 1.8 ml of transfection medium was removed and replaced with about 5 ml of the CRFK culture medium.

The cells were incubated at 370C for 48 hours. Cells were subsequently passed in 75 cm2 flasks after trypsination and incubated again at 370C until cytopathic effect was visually observed.

Cells and medium are harvested after one cycle of freezing-thawing. This virus stock is called transfection stock.

Screening of the transfection stock for the presence of recombinant FHV-1 viruses is based on the expression of the LacZ gene coinserted in the viral genome with the FIPV genes. Plaques containing ecombinant viruses are visualized by a simple assay.

The chemical X-gal (5 bromo-4 chloro-3 indolyl-ss-D-galactoside, Boehringer Mannheim GmbH) was incorporated into the agarose overlay during the plaque assay. Plaques expressing the LacZ encoded $-galactosidase enzyme turn blue. The blue plaques are transferred onto fresh CRFK cells and purified by successive blue plaque isolations until homogeneity is achieved. Large stocks of recombinant viruses are produced by infection of CRFK cells at 50% to 80% confluence (visually observed) and a multiplicity of infection of 0.01.

After 48 to 72 hours, when cytopathic effect is almost complete, cells and viruses are collected after one cycle of freezing-thawing. This stock is used for further vaccination experiments.

The designation of the recombinant viruses is:

I Name t FIPV gene I LacZ gene IFHV-1 insertion site I ' r I ITKLAC I- I + I TK (a) GLAC - gG (b) |TKM M IM + TK JTKSM SM t I TK JKS IS I I TK JTKSIG S without signal peptide I + I TK lTKSDADIS without ADE epitopes T + TK IGM M + I gG IGSM SM + + I gG IGGS S IS I + I gGgG IGSIG iS without signal peptide I + I gG IGSDAD S without ADE epitopes I + I gG (a) TK: thymidine kinase gene (b) gG: glycoprotein G gene EXAMPLE 18 - VACCINATION OF CATS WITH TEE CONTROLS This vaccination experiment is designed to evaluate the vaccinating power of FHV/LACZ recombinants using as criteria, protection against feline rhinotracheitis and immune response against f2galactosidase.

Specified-pathogen-free (SPF) cats of 9 to 10 weeks of age will be vaccinated twice three weeks apart by nasal route using 105 to 107 TCID5O per dose of TRLAC or GLAC FHV-1 recombinant. A group of unvaccinated cats will be used as control. Cats will be challenged 2 to 4 weeks after the second vaccination with about 106 to 108 TCID5O per cat of virulent FHV-1 strain, given as an oronasal spray.

Clinical signs of rhinotracheitis will be evaluated for 2 weeks. Blood samples will be taken weekly from the time of the first vaccination until the end of the experiment. Sera will be analysed for seroneutralizing titers against FHV-1 and S-galactosidase specific antibodies.

The clinical observations of rhinotracheitis will not detect any clinically significant signs of rhinotracheitis in the vaccinated specimens in comparison with the control specimens.

Furthermore, the serotiters against S-galactosidase will be elevated in the vaccinated specimens in comparison with serotiters of the control specimens.

EXAHPLE 19 - VACCINATION OF CATS WITH FIPV/FHV RECOMBINANTS This experiment is designed to evaluate the efficacy of the FIPV/FHV recombinants in protecting cats after challenge with virulent FIPV.

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 105 to 107 TCID5O per cat, of the recombinant FIPV/FHV virus. A group of unvaccinated cats will serve as control. T > to four weeks after the last vaccination, cats will be challenged orally, with 102 to 105 TCID5O per cat, of a virulent FIPV strain.

Clinical signs of FIP will be evaluated for 8 consecutive weeks after the challenge. Blood samples will be taken weekly from the time of the first vaccination until the end of the experiment.

Sera will be analysed for seroneutralizing titers against FHV-1 and S-galactosidase specific antibodies.

The clinical observations will show that clinically significant signs of FIP in the vaccinated specimens will either not be present or will be ameliorated in comparison with the control specimens. The serotiters against ss-galactosidase will be elevated in the vaccinated specimens in comparison with the control unvaccinated specimens.

The above results will show that the vaccinated specimens demonstrated an immune protective response.

REFERENCES 1. Payment et al., Manuel De Techniques Virologiques, Presses de 1'Universit de Quebec (1989).

2. Nunberg et al. (1989). Journal of Virology 63, 3240-3249.

3. De Groot et al. (1988). Virology 167, 370-376.

4. De Groot et al. (1987). J. gen. Virol. 68, 2639-2646.

5. Rota et al. (1986). Virology 154, 168-179.

Claims (1)

1 - A modified coronavirus S protein, wherein at least one of the Al, A2 or D antigenic regions are antigenically-inactive.

2 - The modified coronavirus S protein, wherein the Al and the A2 antigenic regions are antigenically-inactive.

3 - The modified coronavirus S protein, wherein the Al and the D antigenic regions are antigenically-inactive.

4 - The modified coronavirus S protein, wherein the A2 and D antigenic regions are antigenically-inactive.

5 - The modified coronavirus S protein, wherein the Al, A2 and D antigenic regions are antigenically-inactive.

6 - A modified FIPV S protein, wherein at least one of the Al, A2 or D antigenic regions are antigenically-inactive.

7 - A purified coronavirus SM protein of Figure 1.

8 - A process for the preparation of a protein being antigenically-active to coronaviruses, including the steps of: a) transformation of host cells with an expression vector which comprises a DNA molecule coding for the protein being antigenically-active; b) expressing the genome inserted in the expression vector; c) harvesting the cell culture; and d) isolating the antigenically-active protein.

9 - A process for preparing a DNA molecule which codes for a protein being antigenically-active to Coronaviruses including the steps of a) isolating the expression cassette coding for a coronavirus S protein and/or coronavirus SM protein; b) determining the location of the nucleotide base sequences coding for the antigenic region(s) of interest of the the S protein; and c) modifying or deleting the nucleotide base sequences coding for the antigenic regions of interest.

10 - The process of claim 9, wherein the coronavirus S and SM proteins are FIPV S and SM proteins.

11 - The process of claim 9, further including the step of modifying or deleting the nucleotide base sequences coding for the signal peptide of the S protein.

12 - A coronavirus vaccine effective for protecting a mammal against coronaviruses in the absence of ADE.

13 - The coronavirus vaccine of claim 12, wherein the vaccines includes a coronavirus S protein in a suitable carrier, the S protein having at least one of the Al, A2 or D antigenic regions thereof being antigenically-inactive.

14 - The coronavirus vaccine of claim 13, wherein the protein is a coronavirus S protein and further wherein at least one of the Al, A2 or D antigenic regions thereof is antigeically-inactive.

15 - The coronavirus vaccine of claim 13, wherein the protein is a coronavirus SM protein.

16 - The coronavirus vaccine of claim 13, wherein the coronavirus S protein further has the signal peptide modified or deleted therefrom.

17 - The vaccine of claim 13, wherein the vaccine is an FIPV vaccine for protecting a feline against FIP and further wherein the protein is an FIPV protein.

18 - The vaccine of claim 17, wherein the FIPV protein is a FIPV S protein and further wherein at least one of the Al, A2 or D antigenic regions thereof is antigenically-inactive.

19 - The vaccine of claim 13, further including a live recombinant carrier, whereby expression of the protein in a host organism in need thereof is obtained.

20 - A method of preparing a coronavirus vaccine including the steps of a) isolating and purifying a coronavirus protein being antigeni cally-active; and b) formulating the purified protein in a pharmaceutically acceptable carrier.

21 - The method of claim 20, wherein the coronavirus protein is a coronavirus S protein and further wherein the method includes the step of modifying the purified S protein so that at least one of the Al, A2 or D antigenic regions thereof is no longer antigenically-active.

22 - The method of claim 21, further including the step of modifying or deleting the signal peptide of the S protein.

23 - The method of claim 21, further wherein the pharmaceutically-acceptable carrier is a live recombinant carrier.

24 - A method of preparing an FIPV vaccine including the steps of a) isolating and purifying an FIPV S protein being antigenically active; b) modifing the purified FIPV S protein, so that at least one of the Al, A2 or D antigenic regions thereof is no longer antigeni cally-active; and c) formulating the purified protein in a pharmaceutically acceptable carrier.

25 - The method of claim 24, further wherein the pharmaceutically-acceptable carrier is a live recombinant carrier.

26 - A method of protecting a mammal from coronavirus infection including the steps of preparing a coronavirus according to the method of claim 21 and administering a therapeuticallyeffective quantity of the vaccine to a mammal in need thereof.

27 - A method of protecting a feline from FIPV including the steps of preparing a coronavirus according to the method of claim 26, and administering a therapeutically-effective quantity of the vaccine to a feline in need thereof.