RECOMBINANT VIRUS

Abstract: A nucleic acid which encodes a polypeptide which produces a protective immune response against an alpha-virus such as a Venezuelan Equine Encephalitis Virus, in a mammal to which it is administered, said nucleic acid lacking a competant nuclear targeting signal in the capsid gene. The nucleic acids of the invention are expressed at enhanced levels in vectors such as adenovirus vectors. In particular there is described a deletion mutant of an alpha-virus. Vectors such as recombinant adenoviruses which express the deletion mutant are potentially of use as vaccines.

Description: Recombinant Virus The present invention relates to the production of vectors which express alpha-virus genes, such as recombinant viral vectors like adenovirus. The invention further relates to prophylactic and therapeutic vaccines which are protective against these alpha-viruses, such Venezuelan Equine Encephalitis Virus (VEEV) , as well as nucleic acids which are used in the vectors, and methods of treatment using the vaccines . The structural proteins of the alphaviruses are translated from a 26s RNA. The genes encoding these proteins are contained within a single open reading frame in the order: capsid-E3-E2-6K-El . The capsid protein is also known as the "core" and both terms are used herein. Each protein is either co- or post-translationally cleaved from the poly-protein precursor. Many prophylactic and therapeutic vaccines rely on the use of recombinant viruses such as vaccinia virus, or adenovirus including replication competant and replication defective adenovirus, for effective delivery of the immunogens. However, there are sometimes difficulties associated with the expression of alpha viruses in such vectors. The applicants have found that deletion of a region of an alphavirus gene improves expression in certain vectors and allows expression in other vectors which could otherwise not be made. This is useful in vaccine production. Thus according to the present invention there is provided a nucleic acid which encodes a polypeptide which produces a protective immune response against an alpha-virus in a mammal to which it is administered, said nucleic acid lacking a competant nuclear targeting signal from a capsid gene thereof. The nucleic acid as described above may be expressed at enhanced levels, for example in an adenovirus. As used herein, the term "polypeptide" encompasses short polypeptides as well as proteins. The expression "enhanced" means that the expression level of the polypeptide is increased as compared to that which would occur if an otherwise similar nucleic acid including the said portion of the nuclear targeting signal were present. The nuclear targeting signal (NTS) of any particular alphavirus is either known or it can be determined by alignment with sequences which resemble known nuclear targeting signals in other alphaviruses. For instance, Jakob, Preparative Biochemistry (1995) 2j 99-117 shows the nuclear targeting signal in the Semliki forest virus. An example of a nuclear targeting sequence of VEEV is described below. These sequences are present in the gene encoding the core or capsid protein. In general, the nuclear targeting signal will be located in a lysine rich area of the genome and will comprise a region which has at least 3 and generally 4 adjacent lysines such as described by Chelsky et al . , 1989, Mol + Cell Biology, 9, p2487-2492. In the nucleic acids of the invention, the NTS is inactivated either by complete or partial deletion, or by mutation, for example to alter at least some of the lysine residues. The nucleic acid of the invention may be a DNA or an RNA molecule, suitably a cDNA. Furthermore it suitably encodes a polypeptide which comprises at least one structural protein of said alpha-virus, and most preferably all of these. Thus, in a preferred embodiment, the recombinant nucleic acid of the invention encodes at least the capsid- E3-E2 proteings of the alpha-virus and more preferably the capsid-E3-E2-6K-El proteins of an alpha-virus, provided that the region which encodes the capsid protein lacks a competant nuclear targeting domain. One alphavirus which has been found to benefit particularly from the present invention is a Venezulan Equine Encephalitis Virus (VEEV) . This virus, is a mosquito-borne alphavirus which is an important cause of epidemic disease in humans and of epizootics in horses, donkeys and mules in certain parts of the world, in particular the South Americas. The existing VEE vaccine, TC-83, was initially produced by attenuation of the Trinidad donkey strain (TRD) of VEE by sequential passage in guinea pig heart cell cultures. However, this vaccine is generally regarded as being inadequate for human vaccination. This is mainly due to the high incidence of side effects in vaccinees and the large proportion of vaccinees who fail to develop neutralising antibodies (Monath et al. 1992, Vaccine Research, 1, 55-68) . A vaccinia-based vaccine against VEE has been constructed (Kinney et al. J. Gen. Virol. 1988, 69, 3005-3013). In this recombinant, 26S RNA encoding structural genes of VEE were inserted into the NYCBH strain of vaccinia. The recombinant virus protected against sub-cutaneous challenge but had limited efficacy against aerosol challenge with VEE. Attempts to express the full length sequence of this virus in adenovirus, a particularly useful virus for vaccine production, failed completely. However, certain deletion mutants could be successfully expressed. It has been found that the virus proteins will be expressed from a recombinant adenovirus which lacks at least some, and suitably all of nucleotides 7749-7887 within the VEEV genome. All DNA sequence co-ordinants on the VEEV TC-83 strain followed those of the Trinidad Donkey virus strain (R.M. Kinney et al. Virology (1989) 170, 19-30). The cDNA of the 26s mRNA encoding the TC-83 structural region is that reported by Kinney et al (1988) supra, the content of which is incorporated herein by reference. The virulent Trinidad donkey strain of VEE and the attenuated strain TC-83 have both been cloned and sequenced and the amino acid and nucleotide numbering system used in this reference will be used hereinafter. This work revealed that there are seven amino acid changes between TRD and TC-83. The majority (five) of these changes occur within the gene encoding the glycoprotein E2. The applicants have found a further three changes over and above those described by Kinney as detailed below. The deletion or omission of a nuclear targeting domain has also been found to improve the expression of other VEEV encoded proteins in other vectors such as plasmids. Deletion of corresponding regions in other alphaviruses should produce similar enhancements in expression. The recombinant nucleic acids of the invention may be prepared by any of the well known techniques used in recombinant DNA technology. They may be prepared ab initio using the available chemical methods, for example the automated chemical synthesisers. Alternatively, they may be prepared from wild-type alpha viruses, using known recombinant DNA techniques to generate deletion mutants. Thus in a further aspect the invention provides a deletion mutant of an alpha-virus, which mutant lacks a nuclear targeting domain, such as a region corresponding to nucleotides 7749-7887 of VEEV. Corresponding regions in other alphaviruses could be readily be determined by comparing sequences and determining analogous regions as is understood in the art. Such comparisons can be made by computer programs . Confirmation of the nature of the nuclear targeting domain can be confirmed, for example using labelled fragments which may or may not include the purported nuclear targeting domain, and examining where the label appears when the fragments are located in cells transfected with the fragments. The labels may be radiolabels or fluorescent labels as are well known in the art. Preferably the deletion mutant of the invention is a deletion mutant of VEEV. A further aspect of the invention provides a recombinant nucleic acid which encodes a deletion mutant of an alpha- virus as described above. Nucleic acids of the invention are suitably incorporated into vectors, in particular virus vectors like adenovirus (which may be either replication competant or replication defective) , vaccinia virus, or in other expression plasmids where they are under the control of a suitable promoter as understood in the art. Preferably the nucleic acids of the invention is incorporated into an adenovirus and most preferably a replication defective adenovirus. These viruses or plasmids can form vaccines. For this purpose, they will suitably be combined with a pharmaceutically acceptable carrier. Virus vectors are preferably combined with a liquid carrier in an injectable formulation. Plasmid vectors may also be made into an injectable formulation or they may alternatively be bound onto a solid carrier such as a gold bead, which are suitable for administration by means of a gene gun, to the skin of a patient. In yet a further aspect, the invention provides a method of producing a protective immune response to an alpha-virus, said method comprising administering to a mammal a vaccine as described above. The protective immune response may be used both in prophylaxis and in therapy. Suitable doses will be determined by clinicians taking into account the nature of the patient, the nature of the alphavirus and in the case of therapeutic treatment, as well as the precise nature and form of the vaccine. However in general, when using a virus vector, dosages of the vector will be of at least 10 4 pfu. For instance, in the case of vaccinia vectors or replication competant adenovirus, dosages are suitably in the range of from 10 4 -10 12 pfu (pfu = particle forming units) . Replication defective adenovirus may have to be administered at higher dosages . The invention will now be particularly described by way of example with reference to the accompanying diagrammatic drawings in which: Figure 1 is a diagrammatic map (not to scale and not inclusive) showing the structure of VEEV cDNA including restriction sites and the coding regions for the various proteins; Figure 2 shows the sequences of various linkers and adaptors used in the constructs of the examples; and Figures 3-28 are the cloning diagrams referred to in the Examples . In the following Examples, the abbreviations used are as follows: VEEV Venezuelan Equine Encephalitis Virus TRD Trinidad Donkey Virus Strain C Core El Structural protein 1 E2 Structural protein 2 E3 Structural protein 3 6K Structural protein 6K CMV Cytomegalovirus IEP Immediate Early Promoter Term Terminator signal cDNA Complementary DNA RAd Recombinant Ela- Adenovirus NCR Non-Coding Region ATG Start Codon MCS Multiple Cloning Site NTS Nuclear Targeting Signal RBS Ribosome Binding Site nt Nucleotide nts Nucleotides PCR Polymerase Chain Reaction Δ Minus The cDNA of the 26s sub-genomic RNA encoding the TC-83 structural region is that used in the work described below and was provided on the plasmid pTC-5a as reported by Kinney et al. (1988). J.G.V. 6>9: 3005-3114. All VEEV TC-83 cDNA sequence co-ordinates described below followed those Kinney and co-workers (Kinney et al. (1989) . Virology 170: 19-33) for the VEEV strain Trinidad donkey (TRD) . Nucleotide sequence analysis of the TC-83 VEEV cDNA identified three differences in addition to those described (Kinney et al. (1989) . Virology 170: 19-33) within the open reading frames of TC-83 and TRD. These changes are; i) within the 6K gene of TC-83 an addition codon GCG is located between nucleotides (nts) 9989 and 9990, ii)within the El gene of TC-83 nt 10353 is T, and ii) in El gene of TC-83 nt 18897 is G. Example 1 Preparation of deletion mutants All constructs were cloned into the expression cassette contained within pMVlOO or pEWlOl. pMVlOO has been described previously (Wilkinson and Akrigg. (1992) N.A.R. 20: 2233-2239, Jacobs et al. (1992) J. Virol €>6: 2086-2095). The insertion at the Xbal site in pMVlOO of the palindromic adapter LI (fig 2a) which encodes an EcoRI site on an Xbal 8 fragment generated pEVVlOl (Figure 3) . Thus this modification adds an EcoRI site in between the CMV immediate early promoter (IEP) and terminator signal (Term) allowing cDNA to be cloned into the expression cassette (or to be excised from it) on a BamHI, Xbal or EcoRI restriction fragment . Recombinant Ela- adenovirus 5 (Rad) was generated using materials, including the shuttle vector pMV60, and methods described elsewhere (Wilkinson and Akrigg. (1992) N.A.R. 20: 2233-2239, Jacobs et al. (1992) J. Virol €>6: 2086-2095). The VEEV cDNA from pTC-5a was cloned into pEWlOl on an EcoRI fragment generating pEW102 (Figμre 4) . The expression cassette containing the VEEV cDNA was subcloned from pEW102 into pMV60 on a Hindlll fragment generating pEW105. RAd could not be generated with pEW105 or other plasmids similarly constructed. RAd was made, however, when the VEEV cDNA was cloned in the wrong orientation under the control of the promoter. The truncated 5' non-coding region (NCR) and core gene (5'- C) were deleted from the TC-83 VEEV cDNA to investigate whether RAd could be made containing the E3-E2-6K-E1 and the truncated 3' NCR (E3-E2-6K-E1-3' ) . pEVV105 was digested with Spel cutting the VEEV cDNA immediately downstream of nt 8389, three nucleotides into the 5' of the E3 gene. The three deleted nucleotides were restored by the insertion of the palindromic adapter L4A (fig 2b) at the Spel site generating pEWHO (Figure 5) . L4A also encoded a start codon immediately upstream of the three 5' nts of the E3 gene, which is preceded by a Kozak consensus sequence ACC (Kozak. (1984). Nature 3 8: 241-246, Kozak. (1986). Cell _44: 283-292, Kozak. (1987). J. Mol. Biol. 196: 947-950) and an EcoRI site. Using the EcoRI site in L4A and that at the 3' of the VEEV cDNA, the cDNA VEEV construct E3-E2-6K-E1-3' was removed from pEWHO and cloned into pEWlOl generating pEVV108 (Figure 6) . The expression cassette containing the VEEV cDNA was subcloned from pEW108 to pMV60 on a Hindlll fragment generating pEVV109. RAd was generated using pEVV109. An attempt was made to generate RAd expressing the truncated 5' NCR and core (5'-C) . To clone 5'-C, the plasmid pϋCl9 (provided by Boehringer Mannheim UK) was modified by reversing the order of the Xbal and AccI sites within the multiple cloning site (MCS) . A BamHI and SphI adapter encoding an AccI and Xbal site (L7L8, fig 2c) was inserted into the MCS of pUC19 to generate pEW118 (Figure 7). The adapter L5L6 (fig 2d) provided the 3' 26 nucleotides of core gene from the AccI site (nt 8361) to the 3' of core at nt 8386 and an appropriate stop codon on an Accl/Xbal fragment and was cloned into pEW118 generating pEW119 (Figure 8). The 5' NCR and remaining 5' of the core gene, up to and including nt8360, was removed from pEW102 on a BamHI-AccI fragment and cloned into pEW119 completing the constructing of 5'-C and generating pEW120 (Figure 9). The 5'-C fragment was removed from pEW120 on an Xbal fragment and cloned into pMVlOO generating pEW121 (Figure 10). The expression cassette containing 5'-C was subcloned from pEVV121 into pMV60 on a Hindlll fragment generating pEVV123. RAd could not be generated using pEW123. In an attempt to identify the domain responsible for the failure to generate RAd containing core, the VEEV core gene was cloned in two halves. The adapter L10L13 (fig 2e) was inserted at the Nsil site in core between nucleotides 8016 and 8017. This placed an in frame stop codon downstream of the Nsil site followed by an Xbal site, an in frame start codon and a second Nsil site. The resultant plasmid (pEVV129, Figure 11) was digested with Xbal generating two fragments containing C sequence. One containing the 5' NCR and the 5' of core up to and including nt 8016 which immediately precedes an inframe stop codon. The second containing the 3' of the core gene from nt 8017 which is immediately preceded by an in frame start codon, up to and including the stop codon inserted in the construction of 10 5'-C (i e. pEVV121) . Each fragment was cloned into pMVlOO on an Xbal fragment generating pEW125 (Figure 12) which encodes the 5' of core and pEW126 (Figure 12) which encodes the 3' of core. The expression cassette were subcloned from pEVV125 and pEVV126 into pMV60 on a Hindlll fragment generating pEW127 and pEW128 respectively. RAd could not be generated using pEW127 but was generated from the pEW128. Three domains within the 5' of core encoded by pEW125 that might be responsible for the failure to generated RAd virus containing core were identified. These domains are i) the 17 nts upstream of the start codon at nt7562 which form the truncated 5' NCR; ii) the putative ribosome binding site (RB§) between nt 7886-7930 identified by sequence alignment with* the alphaviruse Sindbis (Wengler et al. (1992). Virol ; 191: 880-888, Geigenmuller-Gnirke et al. (1993). J Virol 67: 1620-1626, Owen and Kuhn. (1996). J. Virol 7_0: 2757-2763); iii) a putative nuclear targeting signal (NTS) between nt 7751-7885 identified by sequence alignment with the alphavirus Semliki forest virus (Jakob. (1995). Preparative Biochemistry 25: 99-117). To remove the 5' NCR from core, a PCR fragment was generated with primer P2 (fig 2h) and P3 (fig 2i) using pEW123 as the template. From 5' to 3' , P2 contains a GC rich tail, BamHI site, and the 17 nts homologous to the positive strand of the 5' of the core gene from the start codon at nt 7562. From 5' to 3' , P3 contains a GC rich tail and BamHI site followed by 14 nucleotides complementary the positive strand the core gene between nt 7726-7739 including a Bsu 361 site located between nt 7733-7739. The resultant PCR product therefore contained BamHI sites which flanked sequence containing the 5' of core from the start codon at nt 7562 up to and including the internal Bsu 361 site (nt 7739) . This PCR product was digested with BamHI and cloned into pUC18 (provided by Boehringer Mannheim UK) . The BamHI fragment was then subcloned into pUC19 generating pEW135 (Figure 11 13) . Reconstruction of core gene was completed by inserting the remaining 3' of core from the Bsu 361 site (nt 7735) up to and including the stop codon following nt8386 from pEW121 on a Bsu 361 - Hindlll fragment into pEW135 generating pEW136 (Figure 14). The core gene was removed on a BamHI fragment from pEW136 and cloned into pMVlOO generating pEW133 (Figure 15) . The expression cassette containing the Core was subcloned from pEW133 into pMV60 on a Hindlll fragment generating pEW134. RAd could not be generated using pEW134 To delete the RBS from core, a Tfil site within core was used which necessitated the removal of, the Tfil sites at nt641 and 781 in pUC19. pUC19 was digested with Tfil and the ^sticky ends generated were modified with Klenow before the plasmid was re-ligated generating pEW145. Core was cloned into the MCS of pEW145 on an Xbal fragment from pEVV121 generating pEVV146 (Figure 16) . To delete either the NTS or RBS from core it was necessary to remove both signals on a Bsu 361 -Tfil fragment (nts 7735-7928) and then restore either the NTS or RBS separately. This was achieved by digesting pEW146 with Bsu 361 and Tfil and replacing the RBS-NTS fragment with either a Bsu 361-TfiI PCR fragment encoding the NTS or a Bsu 361-TfiI adapter encoding the RBS. A Bsu 361-TfiI PCR fragment containing the NTS was generated using primers L20 and L21 (fig lj and Ik) . L20 is homologous to the positive strand of core gene and encodes a Bsu 361 site followed by the 10 nt from 7735 to 7749, L21 encoded 25nts complementary to the positive strand of the core gene between nts7872-7897 followed by a Tfil site . The PCR fragment was cloned into pTAg (R & D Systems Europe LTD) using the nontemplated-dependent single A added to the 3' ends of the PCR product. The Bsu 361-TfiI fragment containing the NTS was then excised from pTAg and cloned into pEW146 replacing the Bsu 361-TfiI fragment containing the NTS and RBS and generating core minus (Δ RBS 12 (CΔRBS) (pEVV148 (Figure 17)). CΔRBS was removed from pEVV148 and cloned into pMVlOO on Xbal fragment generating pEW149 (Figure 18). The expression cassette containing Core Δ RBS was subcloned from pEW149 into pMV60 on a Hindlll fragment generating pEW150. RAd could not be generated using pEVV150. Core ΔNTS was generated by cloning the adapter L28L29 (fig 2f) into Bsu 361-TfiI digested pEW146 generating pEW151 (Figure 19) . L28L29 contains nts7735 to 7750 which are adjacent to and continuous with the 42 from ήt 7886 to 7921 The NTS is located between nt 7751 and 7885 therefore L28L29 encodes the region of the core gene between the Bsu 361 site and the NTS and the NTS and the Tfil site thus containing no sequence from the NTS and generating CΔQNTS . CΔNTS was cloned into pMVlOO from pEW151 on an Xbal fragment generating pEW152 (Figure 20) . The expression cassette containing the CΔNTS was subcloned from pEW152 into pMV60 on a Hindlll fragment generating pEW153. RAd was generated using pEW153. To confirm that the NTS encoded by core was responsible for the failure to generate recombinant adenovirus containing the VEEV core, CΔNTS was restored to the construct E3-E2-6K- El-3' using adapter L30L31 (fig 2g) . Adapter L30L31 contains the 34nt downstream of the AccI site at nt8361 in core to nt 8394 of the Spel site located in El and therefore rejoins core to E3. CDNTS was removed from pEW152 on an Xbal fragment replacing 3'-C in pEW120 generating pEW159 (Figure 21) . L30L31 on a Accl-Hindlll fragment was cloned into pEW159 between the AccI site at the 3' of core and the Hindlll site within the MCS of pEW120 generating pEW160 (Figure 22) . CDNTS +L30L31 was removed from pEW160 on Xbal-Spel fragment and cloned into pEW108 placing core- L30L31 upstream of E3 and restoring the Core-E3 junction generating pEVV161 (Figure 23) . The VEEV cDNA CΔNTS-E3-E2- 13 6K-E1-3' was removed from pEW160 on an EcoRI fragment and cloned into pEWlOl generating pEW163 (Figure 24). The expression cassette containing CΔNTS-E3-E2-6K-E1-3' was subcloned from pEW163 into pMV60 on a Hindlll fragment generating pEW162. RAd was generated using pEW162. Example 2 Confirmation of Nuclear targeting domain To determine the potential of the putative NTS to locate the Core protein to the cell nucleus, core was expressed as a fusion protein with Green Fluorescent Protein (GFP) . The start codon (ATG) was removed from core by replacing the 5' of core in pEW120 from the BamHI site' immediately upstream of the start codon to the unique Bsu361 site within the core gen with a PCR fragment containing an identical fragment of core "with the exception that the start codon had been removed. This PCR fragment was generated with the Primers P3 and P15. Within the primer P15 immediately upstream of the BamHI site and downstream of the first nucleotide of Core and additional two nucleotides were included (AT) in order to shift the reading frame two places to the right. This cloning generated the plasmid pEW188 (Figure 25) . Core minus it' s start codon was removed from pEW188 on a BamHI - Xbal fragment and cloned into pUC19 generating pEVV187 (Figure 26) . This cloning placed a Sail site downstream of the Xbal site allowing core minus its start codon to be removed from pEW187 on a BamHI-Sall fragment and cloned into the commercially available vector pEGFP-C2 (Clontech, Palo Alto CA. USA) generating pEW186 (Figure 27). This cloning placed core minus its start codon downstream of and in frame with the GFP gene so that core is expressed as a C-terminal fusion with GFP. When pEW186 transfected cells were examined by immunofluorescence GFP was found to locate to the nucleus of the cell. 14 That targeting of GFP-core fusion protein to the nucleus was directed by the NTS encoded by core was demonstrated by generating pEW172. pEW172 (Figure 28) was generated by cloning the NTS removed from pTAg (Figure 17) on an EcoRI and Sail fragment and cloned into pEGFP-C2. The NTS was therefore expressed as a C-terminal fusion with GFP. A control plasmid pEW173 was generated by removing the NTS on a BamHI and Bglll fragment and cloning it back into pEGFP-C2 in the antisence orientation behind GFP. In cells transfected with pEW173, GFP was located throughout the cell and particularly in the cytoplasm. In cells transfected with pEW172, GFP was located to the nucleus. Example 3 Immunisation studies Groups of 10 balb/c mice were immunised with plasmids using the Helios Gene Gun (Bio-Rad, Hercules, CA. USA) . The plasmid used were pEVVlOO, pEW102 and pEVV163. Each mouse received approximately 5μg plasmid at day 0 and again after 11 weeks. Mice were then challenged 22 weeks following the original immunisation, with 30LD 50 of the virulent stain of VEEV TRD subcutaneously. The results are tabulated in table 1. This data suggests that the immune response elicited following immunisation with a plasmid pEW163 elicited protection while pEW102 did not. The protective effect seen appears not to be solely the result of an antibody response. Table 1. Plasmid Number of mice surviving 20 days post challenge Total number of mice pEWlOO 0/10 pEVV102 0/10 pEW163 3/10

Claims: 15 Claims 1. A nucleic acid which encodes a polypeptide which produces a protective immune response against an alpha-virus in a mammal to which it is administered, said nucleic acid lacking a competant nuclear targeting signal in the capsid gene. 2. A nucleic acid according to claim 1 which can be expressed in an adenovirus vector. 3. A nucleic acid according to claim 1 or claim 2 which encodes at least the capsid-E3-E2 proteins of an alphavirus, provided that the region which encodes the capsid protein lacks a nuclear targeting signal. 4. A nucleic acid according to claim 3 which encodes the capsid-E3-E2-6K-El proteins of an alpha-virus, provided that the region which encodes the capsid protein lacks a nuclear targeting signal. 5. A nucleic acid according to any one of the preceding claims wherein the alpha-virus is a Venezulan Equine Encephalitis Virus (VEEV) . 6. A deletion mutant of an alpha-virus, which mutant lacks a nuclear targeting signal from the capsid gene. 7. A deletion mutant of an alpha-virus which lacks a region corresponding to nucleotides 7749-7887 of VEEV. 8. A deletion mutant according to claim 7 wherein the alpha-virus is VEEV. 9. A nucleic acid which encodes a deletion mutant of an alpha-virus as claimed in any one of claims 6 to 8. 16 10. A recombinant adenovirus which comprises a nucleic acid according to any one of claims 1 to 5 or 9. 11. A recombinant vaccinia virus which comprises a nucleic acid according to any one or claims 1 to 5 or 9. 12. A plasmid which comprises a nucleic acid according to any one or claims 1 to 5 or 9 under the control of a promoter which allows expression thereof. 13. A vaccine which comprises a virus according to claim 10 or claim 11 or a plasmid according to claim 12. 14. A vaccine according to claim 13 which further comprises pharmaceutically acceptable carrier. 15. A method of producing a protective immune response to an alpha-virus, said method comprising administering to a mammal a vaccine according to claim 13 or claim 14.

Inventors: JACOBS SUSAN CATHERINE, JACOBS, SUSAN, CATHERINE
Applicants: JACOBS SUSAN CATHERINE, SECR DEFENCE, JACOBS, SUSAN, CATHERINE, THE SECRETARY OF STATE FOR DEFENCE
Application year: 1999
Main classification: C07K14
IPC codes: A61K39, C12N7, C12N15