Abstract:
A vaccine against feline leukaemia virus (FeLV) comprises a protein formed of two proteins derived from FeLV gp70 which are not adjacent in the native protein, particularly VR1 and VR5 and effective fragments thereof. VR5 is preferably attached to the carboxy terminus of VR1. A co-protein such as GST or β-galactosidase may be attached to the terminus of the fused protein, such as to stabilize and solubilize the fused protein. Attachments to the carboxy terminus may reduce the immunogenicity.

Description:
FIELD OF THE INVENTION 
     The present invention relates to an immunogenic fused recombinant protein derived from feline leukaemia virus (FeLV), and to a protective vaccine comprising the immunogenic protein. 
     BACKGROUND OF THE INVENTION 
     FeLV is a retrovirus of the sub-family oncovirinae. The virus has a simple genomic organisation with three genes, gag, pol and env, encoding vital proteins. 
     FeLV occurs in three subgroups, A, B or C, which differ in their env genes. FELV-A is present in every isolate and is efficiently transmitted between cats in nature. Viruses of the other two subgroups arise as variants of FeLV-A in individual cats by recombination of endogenous FeLV env genes (FeLV-B) or mutation within the env gene (FeLV-C). Hence the occurrence of subgroups B or C is dependent upon natural infection of cats with FeLV-A. 
     FeLV is responsible for several fatal diseases in domestic cats including lymphoid and myeloid leukaemias, fibrosarcomas, anaemia, immunodeficiency, enteritis and reproductive failure. The virus is common in nature: a recent survey in the U.K. indicated that the overall prevalance of infection is approximately 15% of sick cats and 6% in healthy cats. These cats have a persistent infection with viraemia and are a source of infection for susceptible animals. FeLV is transmitted either by contact through the transfer of infectious saliva or prenatally across the placenta. 
     Following natural or experimental exposure of cats to FeLV, there are two possible outcomes. Cats either develop a persistent, life-long infection or recover. Persistently infected cats have a viraemia while recovered cats have no virus in their blood. Some cats which recover may harbour a latent infection in the bone marrow for several weeks or months before it is eliminated. Recovered cans are immune to FeLV as evidenced by their resistance to subsequent challenge with virus. 
     Two factors influence the outcome of infection. The first is the age an which a cat is exposed to virus. Kittens up to 14 weeks of age are very susceptible and essentially all can be affected by virus challenge. Older kittens are more resistant so that by 16 weeks of age only about 20% can be infected. The other factor governing susceptibility is dose of virus: high doses lead to persistent viraemia while lower does immunise. The consequences of persistent infection are severe and essentially all viraemic cats die within four years of infection. By contrast recovered cats have a normal life span. 
     Recovery is due to an immune responsa to the virus. Only antibody responses have been examined in detail. However, it is clear that resistance to reinfection is mediated by virus neutralising antibodies which are directed at the envelope surface glycoprotein the virus, gp70. Attempts have been made to immunise cats against FeLV infection with vaccines containing gp70, some of which have been successful. 
     European patent specification EP 0377842 describes the production of replication-defective FeLV viral sequences. U.S. Pat. No. 4,794,168 relates to the use of immunogens from FeLV envelope protein P15E in vaccines. European patent specifications 0247904, 0173997 and 0216564, International patent publication WO85/02625 and U.S. Pat. No. 4,789,702 all describe the use of the FeLV envelope protein gp70 or fragments thereof as antigens. 
     SUMMARY OF THE INVENTION 
     The present invention is based on the discovery that a fused recombinant protein comprising two regions of the FeLV molecule which are normally non-adjacent, when combined produced an immunogenic determinant capable of inducing anti FeLV antibodies on inoculation into cats. The immunised cats are resistant to challenge with live FeLV. 
     Thus, the present invention provides an immunogenic fused recombinant protein which comprises: 
     a first protein which is feline leukaemia virus (FeLV) gp70 VR1 protein (SEQ ID NO:4) or effective fragment thereof, having fused thereto; 
     a second non-adjacent FeLV protein or effective fragment thereof different from the first protein and capable of stabilising the first protein, such that said fused recombinant protein is immunogenic and protective against FeLV infection. 
     It has been found that the use of gp70 VR1 protein alone, whilst producing antibodies, is not protective against FeLV infection. 
     It has however been found that the fusion of a second non-adjacent FeLV protein (or fragment thereof) may result in a fused recombinant protein which is both antigenic and protective against FeLV infection. The second protein is preferably also derived from gp70. In particular, the gp70 VR5 protein (SEQ ID NO:6) (or fragment thereof), which may not by itself produce antibodies, is capable of stabilising the first protein and producing a fused protein which surprisingly has immunogenic and protective properties. 
     Preferably, the second protein is fused to the carboxy terminus of VR1. 
     Usually, the fused recombinant protein will be free of glycosylation. 
     The fused recombinant protein, preferably further comprises an antigenic co-protein, such as Glutathione-S-transferase (GST) or beta-galactosidase. relatively large co-proteins solubilise the protein and facilitate production and purification thereof. Moreover, the co-protein may act as an adjuvant in the sense of providing a generalised stimulation of the immune system. Preferably the co-protein is attached to the amino terminus of the fused protein. 
     Surprisingly, it has been found that co-proteins (such as GST or β-galactosidase) may be attached to the amino terminus of the fused protein without adversely affecting immunogenicity thereof, and imparting desirable solubility and stability properties. However, it has been found that the attachment of other proteins to the carboxy terminus (whilst being useful in stabilising the fusion protein against egradation during preparation) may adversely affect its immunogenicity. It is postulated that this may be due to conformational changes brought about by the presence of the co-protein attached at the carboxy terminus. Therefore any co-protein attached at the carboxy terminus should be relatively small such as not to disturb the antigenic conformation of the fusion protein. 
     The present invention also provides a vaccine formulation which comprises the immunogenic fused recombinant protein together with a suitable carrier. Since the protein may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc. injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient: and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. 
     The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. 
     The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation. 
     The present invention also provides a method of constructing a fused recombinant protein which comprises: 
     introducing a DNA sequence coding for the first protein (SEQ ID NO:3) into a plasmid; 
     opening the plasmid and introducing a DNA sequence coding for the second protein (SEQ ID NO:5) adjacent the first DNA sequence, and 
     expressing the recombinant protein. 
     In particular, the DNA sequences may be amplified using polymerase chain reaction (PCR). The amplified sequences may then be inserted one after another into adjacent positions in the plasmid. The plasmid may also include a gone coding for GST or similar co-protein. 
     The plasmid thus constructed may be expressed in any suitable bacteria, such as E. coil. 
     Whilst the invention has been described with reference to certain named proteins, such as gp70 VR1 and VR5 proteins, it is to be understood that this covers fragments of the naturally occurring protein and similar proteins (for example, having sequence homologies of 50% or greater) with additions, deletions or substitutions which do not substantially affect the immunogenic properties of the fused recombinant protein. 
     The second protein is one which does not normally lie adjacent to the first protein in the native FeLV gp70 protein. Non-adjacent means that it is not adjacent in the linearised amino acid sequence. 
     DESCRIPTION OF PREFERRED EMBODIMENTS 
     Embodiments of the present invention will now be described by way of example only with reference to following figures and examples. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 shows the plasmid pGEX-2T employed in the construction of the fused recombinant protein; 
     FIG. 2 shows a terminal sequence including restriction sites (SEQ ID NO:1); 
     FIG. 3 shows the native FeLV gp70 VR1 sequence (SEQ ID NO:3); 
     FIG. 4 shows the native FeLV gp70 VR5 sequence (SEQ ID NO:5); and 
     FIG. 5 is a diagrammatic representation of the variable regions in the FeLV gp70 gene. 
    
    
     The following examples illustrate the construction and immunogenic properties of the fused recombinant protein GST-VR1-VR5. 
     EXAMPLE 1 
     Construction of a Plasmid Expressing Recombinant gp70 Fusion Protein 
     The cloning vector used for these experiments is pGEX-2T (Smith &amp; Johnston, Gene 67 (1988) 31-40). The FeLV gp70 gone fragment is cloned into this plasmid to produce a polypeptide fused to the --COOH terminus of the enzyme Glutathione-S-transferase (GST). Previous experiments have demonstrated that large fragments of gp70 (over 20kd) often result in problems of insolubility and degradation. In an attempt to circumvent these problems it was decided to express a range of smaller gp70 fragments. Furthermore, the regions chosen for expression are some of the &#34;variable&#34; regions of gp70, i.e. those regions showing the greatest variation between sub-groups A, B and C. 
     The region of the FeLV gp70 gone containing VR1 was amplified using the following primers. The cloned envelope gene was the template used. 
     
         __________________________________________________________________________1)  5&#39; GAG GGATCC CTA CAT GTT GAC TTA TG 3&#39; (SEQ ID NO: 7)  BamHI2)  5&#39; TAG GAATTC ACA ATG TGT TCC CTT TG 3&#39; (SEQ ID NO: 8)  EcoRI__________________________________________________________________________ 
    
     The PCR product was digested with the restriction enzymes BamHI and EcoRI the sites for which had been incorporated into the primers as shown above. There are no sites for these two restriction enzymes within the amplified fragment. 
     The digested PCR product was separated, by electrophoresis on an agarose gel, from any contaminating by products of the PCR reaction. The section of the agarose gel containing the DNA fragment was excised and the DNA removed by electroelution. After ethanol precipitation the DNA fragment was cloned into BamHI/EcoRI cut pGEX-2T. 
     After transformation of a recipient E.coli strain, clones harboring a recombinant pGEX-2T/VR1 plasmid were isolated. 
     The following primers were used to amplify a DNA fragment spanning the VR5 region: 
     
         __________________________________________________________________________5&#39; GAC GAATTC CAG GCT TTG TGC AAT AAG ACA CAA 3&#39; (SEQ ID NO: 9)EcoRI5&#39; TAG GAATTCGCATGC GGT GAG TCC AGT GTT ACA 3&#39; (SEQ ID NO: 10)EcoRI SphI__________________________________________________________________________ 
    
     Cloned FeLV gp70 gene was again the template used, the PCR product was digested with EcoRI prior to purification on agarose. This PCR product was cloned into the pGEX-2T/VR1 plasmid described above. Clones carrying VR5 in the correct orientation with respect to VR1 were identified both by DNA sequence analysis and restriction enzyme analysis. 
     EXAMPLE 2 
     Preparation of GST-VR1VR5 Recombinant Protein 
     The glutathione-S-transferase (GST) of Schistosoma japonicum is a small, very soluble enzyme which is tolerant of insertions at its carboxyl end. The gene (Sj26) encoding for this enzyme has been placed under the control of an inducible promoter and introduced into a plasmid together with an ampicillin resistance marker on it. A multiple restriction site has been introduced at the 3&#39; end of Sj26, in tandem with a region coding for a thrombin cleavage site. The resulting plasmid (pGEX-2T) can produce large quantities of a fusion protein, which is extractable using affinity chromatography. The fusion protein is cleavable and the resulting peptide can be purified by repeating the affinity chromatography. 
     The following materials and methods are part of routine laboratory practice and can be found in &#34;Molecular Cloning, A Laboratory Manual&#34; second edition, Eds. Sambrook, Fritsch and Maniatis. and also in &#34;Current Protocols in Molecular Biology.&#34; Below is a detailed description of the materials and methods used. 
     Materials 
     Enriched L Broth 
     100 ml L Broth 
     1 ml 2M glucose 
     1 ml 1M mgCl 2   
     200x IPTG 
     1 g isopropyl D thio-galactopyranoside (IPTG) 
     8 ml water make up to 10 ml 
     Filter sterilise, dispense into aliquots of 0.5 ml, freeze at -20° C. 
     1000x PMSF in isopropanol 
     1 mg/ml phenyl methyl sulphonyl fluoride 
     20% TX100 
     2 ml TX100 
     8 ml water 
     Filter sterilise. Easier to handle at this concentration. 
     Glutathione/Tris 
     250 ml 1.0M Tris 
     4.75 ml water 
     30.73 mg glutathione 
     Filter sterilise. 
     a) Preparation of glutathione beads 
     A 15 ml polypropylene tube is taken and 70 mg of dehydrated glutathione agarose beads are swollen with 10 mls of PBS/1% TX100 using gentle agitation for 30 minutes. 
     They are then precipitated at 2000 rpm for 5 minutes. The beads are resuspended in 5 mls of PBS to wash them, this procedure is repeated three times. (PBS/1%TX100 or PBS/tween are valid alternatives). 
     b) Production of Fusion Protein 
     1. Bacterial cells, transfected with a recombinant pGEX plasmid containing the DNA coding for the protein of interest, are cultured in 100 mls of enriched L Broth overnight in the presence of ampicillin. 
     2. The overnight culture is inoculated into 900 mls of enriched L Broth (with ampicillin). The bacterial suspension is grown up with vigorous shaking to an O.D of approximately 0.6 (550 nm) (about 2 hours) then IPTG is added to a final concentration of 50ug/ml. The culture is left for a further 90 minutes. Whilst inducing the culture, stare to prepare the swollen, washed glutathione beads for use in stage 5 below. 
     3. The culture is then centrifuged at 6000 rpm for 15 minutes. The supernatant is discarded. The pellets are resuspended in 10 mls of PBS/1% TX100. 10 ul of PMSF in isopropanol is added. 
     4. The concentrated bacterial suspension is sonicated in ice for 30 seconds and then centrifuged at 18000 rpm for 15 minutes. The supernatant is retained. 
     5. The beads are then resuspended in the supernatant from step 4 above and 20% TX100 added to a final concentration of 1% TX100. The mixture is gently agitated for 1 hour. 
     6. The mixture is centrifuged at 2000 rpm for 15 minutes and the beads washed 3 tiles as before. One ml of the solution of 20 mM glutathione/50 mM Tris is added to the beads and left for 30 minutes. The beads are again centrifuged out (the supernatant being retained) and washed once using another 1 ml of the above glutathione solution. The washing is added to the supernatant. 
     7. The supernatant contains the fusion protein. An aliquot should be retained to verify this. The supernatant is then dialysed overnight in PBS to remove the excess glutathione. It may then be frozen at -200° C. 
     The preparation exhibited a degree of degradation and/or incomplete synthesis. Nevertheless sufficient material from a 2 liter culture was obtained to conduct these experiments (4 mg. total protein). 
     EXAMPLE 3 
     Vaccination 
     Since young kittens are most at risk of FeLV infection, the immunisation regime is designed to test the capacity of a vaccine to protect kittens at an early age, and to be compatible with the timing of vaccination against other feline viruses. 
     The route of challenge is by the oronasal route to simulate natural horizontal transmission of FeLV. A dose of virus is used which will cause persistent viraemia in over 90% of 14 week old kittens. 
     The schedule of vaccination and monitoring is in Table 1 . The response of the kittens to vaccination challenge is given in Table 2. 
     The VR1-VR5 preparation elicited an immune response. A large proportion of the antibodies raised are directed against the GST portion of the recombinant as measured by ELISA. However, a competition ELISA using native GST and the recombinant protein also indicates that the immunised cats also responded to the FeLV component of the recombinant protein. 
     Western blot analysis using FeLV as antigen shows that three out of the four VR1-VR5 treated cats (59,61,62) produced easily detectable anti-gp70 antibodies and cat 60 gave a weak response on Western analysis. All controls were negative. 
     Virus neutralising antibodies were found in one cat prior to challenge and in a further 2 of the 4, four cats 6 weeks post challenge. 
     Three of the four vaccinated cats (59,61,62) were also found to be negative for virus isolation 3, 6 and 9 weeks post challenge, whilst all 4 control animals developed viraemia. 
     The cats responses to immunisation and challenge show that the VR1-VR5 preparation is able to produce an anti-gp70 antibody response and protect the animals from virus challenge. 
     
                       TABLE 1______________________________________SCHEDULE OF VACCINATION AND SAMPLINGAge of Kitten       Week from(weeks)     challenge       Event______________________________________ 9          -5              Blood sample                       Vaccination-112          -2              Blood sample                       Vaccination-214          0               Blood sample                       Challenge17          3               Blood sample20          6               Blood sample23          9               Blood sample______________________________________ 
    
     
                                           TABLE 2__________________________________________________________________________(VACCINATION RESULTS)    TIME (WEEKS) RELATIVE TO CHALLENGECAT      -5        -2        0GROUP F  *A      V N E W A V N E W A V N E W__________________________________________________________________________VR-1 + 5 59 - - 0     - - 0     - - 0 + + 60 - - 0     - - 0     - - 0 - W 61 - - 0     - - 0     - - 0 + + 62 - - 0     - - 0     - - 4 + +CONT  67 - - 0     - - 0     - - 0 - - 68 - - 0     - - 0     - - 0 - - 69 - - 0     - - 0     - - 0 - - 70 - - 0     - - 0     - - 0 - -__________________________________________________________________________    TIME (WEEKS) RELATIVE TO CHALLENGECAT      3         6         9GROUP F  A V N E W A V N E W A V N  E__________________________________________________________________________VR-1 + 5 59 - -       - -     + - - 0 60 + +       + +     - + + 0 61 - -       - -     + - - 128 62 - -       - -     + - - 128CONT  67 + +       + +     + + + 0 68 + +       + +     - + + 0 69 + +       + +     + + + 0 70 + +       + +     - + + 0__________________________________________________________________________ *A = p27 antigen in plasma V = Virus isolated from plasma N = Virus neutralising antibody E = antigp70 antibody (ELISA) W = antigp70 antibody (Western blot) 
    
     
         __________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 10(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 51 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO (ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..48(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:CCAAAATCGGATCTGGTTCCGCGTGGATCCCCGGGAATTCATCGTGAC48ProLysSerAspLeuValProArgGlySerProGlyIleHisAr gAsp151015TGA51(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 16 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:ProLysSerAspLeuValProArgGlySerProGlyIleHisArgAsp151015(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 208 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..207(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:CCTACCCTACATGTTGAC TTATGTGACCTAGTGGGAGACACCTGGGAA48ProThrLeuHisValAspLeuCysAspLeuValGlyAspThrTrpGlu151015CCTATAGTCCTAAAC CCAACCAATGTAAAACACGGGGCACGTTACTCC96ProIleValLeuAsnProThrAsnValLysHisGlyAlaArgTyrSer202530TCCTCAAAGTATGGA TGTAAAACTACAGATAGAAAAAAACAGCAACAA144SerSerLysTyrGlyCysLysThrThrAspArgLysLysGlnGlnGln354045ACATACCCCTTTTACGTC TGCCCCGGACATGCCCCCTCGCTGGGGCCA192ThrTyrProPheTyrValCysProGlyHisAlaProSerLeuGlyPro505560AAGGGAACACACTGTG 208LysGlyThrHisCys65(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 69 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:ProThrLeu HisValAspLeuCysAspLeuValGlyAspThrTrpGlu151015ProIleValLeuAsnProThrAsnValLysHisGlyAlaArgTyrSer20 2530SerSerLysTyrGlyCysLysThrThrAspArgLysLysGlnGlnGln354045ThrTyrProPheTyrValCysProGlyH isAlaProSerLeuGlyPro505560LysGlyThrHisCys65(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 96 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..96(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:CAGGCTTTGTGCAATAAGACACAACAGGGACATACAGGGGCGCACTAT48GlnAlaLeuCysAsnLysThrGln GlnGlyHisThrGlyAlaHisTyr151015CTAGCCGCCCCCAACGGCACCTATTGGGCCTGTAACACTGGACTCACC96LeuAlaAlaProAsnGlyThr TyrTrpAlaCysAsnThrGlyLeuThr202530(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 32 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:GlnAlaLeuCysAsnLysThrGlnGlnGlyHisThrGlyAlaHisTyr151015LeuAlaAlaProAsnGlyThrTyrTrpAlaCysAsnThrGlyLe uThr202530(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 26 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GAGGGATCCCTACATGTTGACTTATG26(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 26 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(x i) SEQUENCE DESCRIPTION: SEQ ID NO:8:TAGGAATTCACAATGTGTTCCCTTTG26(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 33 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:GACGAATTCCAGGCTTTGTGCAATAAGACACAA33(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 33 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:TAGGAATTCGCATGCGGTGAGTCCAGTGTTACA33