Crimean-congo haemorrhagic fever virus antigenic composition

Abstract

The present invention provides a viral vector or bacterial vector, said vector comprising a nucleic acid sequence encoding a Crimean-Congo Haemorrhagic Fever Virus (CCHFV) glycoprotein or antigenic fragment thereof; wherein said vector is capable of inducing an immune response in a subject. The present invention also provides compositions and uses of the vector in methods of medical treatment.

Claims

1. A non-replicating poxvirus vector for inducing a protective immune response in a subject against Crimean-Congo Haemorrhagic Fever Virus (CCHFV), wherein said vector comprises a nucleic acid sequence encoding a CCHFV glycoprotein or antigenic fragment thereof and wherein the non-replicating poxvirus vector comprises a Modified Vaccinia virus Ankara (MVA) vector and wherein the nucleic acid sequence encoding a CCHFV glycoprotein encodes one or more of: a CCHFV G.sub.N glycoprotein, a CCHFV G.sub.C glycoprotein, and a CCHFV M segment polyprotein.

2. The vector of claim 1, wherein the nucleic acid sequence encoding a CCHFV glycoprotein or antigenic fragment thereof comprises a nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1, 2, and 3.

3. A composition comprising a vector according to claim 1, and a pharmaceutically-acceptable carrier.

4. The composition of claim 3, further comprising an adjuvant.

5. A non-replicating poxvirus vector comprising a Modified Vaccinia virus Ankara (MVA) vector for inducing a protective immune response in a subject against Crimean-Congo Haemorrhagic Fever Virus (CCHFV), wherein said vector comprises a nucleic acid sequence encoding a CCHFV glycoprotein or antigenic fragment thereof and wherein the nucleic acid sequence encoding a CCHFV glycoprotein or antigenic fragment thereof comprises a nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1, 2, and 3.

6. A non-replicating poxvirus vector comprising a Modified Vaccinia virus Ankara (MVA) vector for inducing a protective immune response in a subject against Crimean-Congo Haemorrhagic Fever Virus (CCHFV), wherein said vector comprises a nucleic acid sequence encoding a CCHFV glycoprotein or antigenic fragment thereof and wherein the nucleic acid sequence encoding a CCHFV glycoprotein encodes one or more of: a CCHFV G.sub.N glycoprotein, a CCHFV G.sub.C glycoprotein, and a CCHFV M segment polyprotein.

7. A nucleic acid sequence encoding a viral vector according to claim 1.

8. A method of making a viral vector, comprising: providing a nucleic acid, wherein the nucleic acid comprises a nucleic acid sequence encoding a vector according to claim 1; transfecting a host cell with the nucleic acid; culturing the host cell under conditions suitable for the propagation of the vector; and obtaining the vector from the host cell.

9. A host cell comprising the nucleic acid sequence of claim 8.

10. A method of inducing an immune response to a patient in need thereof comprising: administering the composition of claim 1.

11. A method of inducing an immune response in a subject against CCHFV, said method comprising administering to said subject a composition according to claim 3.

12. The method according to claim 11, wherein the immune response comprises a T cell response.

13. A method of preventing or treating a CCHFV infection in a subject, said method comprising administering to said subject a composition according to claim 3.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A-C. Example MVA Vector Construction.

(2) FIG. 1A. Cassette used in plasmid pLW44. Green Fluorescent Protein (GFP) is driven by poxvirus promoter p11. A multi-cloning site is downstream of poxvirus promoter mH5. MVA flanks L and R allow recombination with MVA genome.

(3) FIG. 1B. Cassette used in plasmid pLW44 following modification (renamed as plasmid pDEST44-TPA-V5).

(4) FIG. 1C. Recombination between pDEST44-TPA-V5 and pENTR-GP results in pTP-GP, containing the depicted cassette (SEQ ID NO: 7).

(5) FIG. 2. MVA-GP immunogenicity in Balb/c mice. IFN-? ELISpot on splenocytes 8 or 14 days post-boost showed T-cell immunogenicity of GP

(6) FIG. 3. Experimental protocol for Example 2.

(7) FIG. 4A-B. IFN-? ELISPOT results.

(8) FIG. 5A-B. IFN-? ELISPOT results subdivided into peptide pools (1 for TPA/V5, 7 for GP and 2 for NP).

(9) FIG. 6. Animal survival rates following CCHFV challenge (Example 3).

(10) FIG. 7A-B. Weight loss in study animals (Example 3).

(11) FIG. 8A-B. Temperature rise in study animals (Example 3).

(12) FIG. 9. Experimental protocol for Example 4.

(13) FIG. 10. Animal survival rates following CCHFV challenge (Example 4).

(14) FIG. 11. Animal clinical scores (Example 4).

(15) FIG. 12. Animal temperature scores (Example 4).

(16) FIG. 13. Animal weight change scores (Example 4).

(17) FIG. 14A-B. Animal immune responses (Example 4).

(18) FIG. 15.

(19) Western Blot Analysis of Glycoproteins Expressed by MVA-GP and CCHFv.

(20) (A) Western blotting of MVA-GP with anti-V5 antibody indicates a protein of approximately 75 kDa, confirming recombinant protein expression, and consistent with cleavage of the predicted 76.6 kDa G.sub.C?V5 fusion protein.

(21) (B) Western blotting of MVA-1974 (lane 1), MVA-GP (lane 2), CCHFv-infected SW13 cells (lane 3) and uninfected SW13 cells (lane 4) with polyclonal serum from rabbits vaccinated with peptides derived from the CCHF viral glycoprotein. Major products expressed by MVA-GP correspond with those expressed by CCHFv, indicating the recombinant protein undergoes similar post-translational cleavages as the native protein.

(22) FIG. 16.

(23) Antibody Responses from Vaccinated A129 and 129Sv/Ev Mice.

(24) Sera from vaccinated mice were tested for reactivity with CCHFv-infected (lane 3) or uninfected (lane 2) SW13 cells by Western blotting. Lane 1 shows a molecular weight marker. Blots show proteins reactive with serum from representative individual animals 7 days after booster vaccination (A-E) or representative pooled serum 14 days after booster vaccination (F). Secondary antibody used was specific for mouse IgG (A-C, F), or mouse IgG, IgA and IgM (D-E). Arrows indicate CCHFv-specific proteins, indicating specific antibody responses in both mouse strains.

(25) (A) 129Sv/Ev mouse vaccinated with MVA-GP. (B, E) A129 mouse vaccinated with MVA-GP. (C) A129 mouse vaccinated with MVA 1974. (D) A129 mouse vaccinated with MVA-GP. (F) Pooled serum from A129 mice vaccinated with MVA-GP.

(26) FIG. 17.

(27) Tissue Histology of A129 Mice, 4 Days after Challenge with CCHFv.

(28) A129 mice were challenged with double the minimum lethal dose of CCHFv, 14 days after booster vaccination with MVA 1974 (A-B) or MVA-GP (C-D). Four days after challenge, sections of spleen (A, C) and liver (B, D) were fixed, HE stained, and examined for pathology. More severe pathology was found in mice that received MVA 1974, compared to those that received MVA-GP. (A) Marked lymphocyte loss with prominent apoptotic bodies, and infiltration by macrophages. (B) Marked, multifocally extensive hepatocyte necrosis (arrows). (C) A single infiltration of macrophages in the white pulp (asterisk) (scored minimal). (D) Scattered, multifocal areas of hepatocellular necrosis with a mixed inflammatory cell infiltrate (arrows) (scored moderate).

(29) FIG. 18.

(30) Immunohistochemistry of Tissues from A129 Mice, 4 Days after Challenge with CCHFv.

(31) A129 mice were challenged with double the minimum lethal dose of CCHFv, 14 days after booster vaccination with MVA 1974 (A-C) or MVA-GP (D-E). Four days after challenge, sections of spleen (A, D) and liver (B-C, E) were fixed, immunohistochemically stained with CCHFv-specific antibody, and examined microscopically. Tissues in panels A, B, D and E were from the same individuals as shown in FIGS. 5A, 5B, 5C and 5D, respectively. A diffuse staining pattern of viral proteins was found in tissues from animals that received the MVA 1974 negative control. However, in MVA-GP vaccinated animals, the only staining found was of a minimal degree, in liver from one individual.

(32) (A) A few, scattered cells with cytoplasmic staining within the parenchyma. (B) Frequent, diffuse, positively stained hepatocytes. (C) Scattered, small, elongated cells consistent with Kupffer cells, with cytoplasmic staining. (D) Normal parenchyma. (E) A few, positively stained cells within an inflammatory cell focus.

(33) FIG. 19.

(34) Viral Load Analysis of CCHFv RNA Copy Number by RT-PCR.

(35) A129 mice (n=9) were challenged with double the minimum lethal dose of CCHFv, 14 days after booster vaccination with MVA-GP, MVA 1974, or saline. Four days post-challenge (day 32), 3 randomly selected animals from each group were killed humanely and analysed by RT-PCR for copy number of CCHFv S segment in various tissues. Fourteen days post-challenge (day 42), all surviving animals were killed humanely and also analysed. Each bar represents the mean?standard deviation in an individual animal.

(36) In all tissues tested, viral load was significantly lower in MVA-GP vaccinated mice than in control groups. Within the MVA-GP group, there was no significant difference in viral load between 4 days and 14 days post-challenge. (A) Blood. (B) Spleen. (C) Liver.

(37) FIG. 20.

(38) Normalised Viral Load Analysis of CCHFv RNA by RT-PCR.

(39) A129 mice (n=9) were challenged with double the minimum lethal dose of CCHFv, 14 days after booster vaccination with MVA-GP, MVA 1974, or saline. Four days post-challenge (day 32), 3 randomly selected animals from each group were killed humanely and analysed by RT-PCR for CCHFv gene expression, normalised to mouse HPRT gene expression. Fourteen days post-challenge (day 42), all surviving animals were killed humanely and also analysed. Each point represents the mean value of triplicate measurements in an individual animal. Lines show mean?standard deviation.

(40) In all tissues tested, viral load was significantly lower in MVA-GP vaccinated mice than in control groups. Within the MVA-GP group, there was no significant difference in viral load between 4 days and 14 days post-challenge. (A) Blood. (B) Spleen. (C) Liver.

KEY TO SEQ ID NOS

(41) SEQ ID NO: 1 Full length CCHFV M segment open reading frame nucleic acid sequence (from Accession Number U39455).

(42) SEQ ID NO: 2 CCHFV G.sub.N glycoprotein nucleic acid sequence.

(43) SEQ ID NO: 3 CCHFV G.sub.C glycoprotein nucleic acid sequence.

(44) SEQ ID NO: 4 Amino acid sequence of protein encoded by CCHFV M segment.

(45) SEQ ID NO: 5 CCHFV G.sub.N glycoprotein amino acid sequence.

(46) SEQ ID NO: 6 CCHFV G.sub.C glycoprotein amino acid sequence.

(47) SEQ ID NO: 7 MVA-GP nucleic acid sequence (representing the section shown in FIG. 1C).

(48) Sequences

(49) TABLE-US-00003 SEQIDNO:1 atgcatatatcattaatgtatgcaatcctttgcctacagctgtgtggtct gggagagactcatggatcacacaatgaaactagacacaataaaacagaca ccatgacaacacacggtgataacccgagctctgaaccgccagtgagcacg gccttgtctattacacttgacccctccactgtcacacccacaacaccagc cagtggattagaaggctcaggggaagtctacacatcccctccgatcacca ccgggagcttgcccctgtcggagacaacaccagaactccctgttacaacc ggcacagacaccttaagcgcaggtgatgtcgatcccagcacgcagacagc cggaggcacctccgcaccaacagtccgcacaagtctacccaacagcccta gcacaccatctacaccacaagacacacaccatcctgtgagaaatctactt tcagtcacgagtcctgggccagatgaaacatcaacaccctcgggaacagg caaagagagctcagcaaccagtagccctcatccagtctccaacagaccac caacccctcctgcaacagcccagggacccactgaaaatgacagtcacaac gccactgaacaccctgagtccctgacacagtcagcaaccccaggcctaat gacctctccaacacagatagtccacccacaaagtgccacccccataaccg ttcaagacacacatcccagtccaacgaacaggtctaaaagaaaccttaag atggaaataatcttgactttatctcagggtttaaaaaagtactatgggaa aatattaaggcttctgcaactcaccttagaggaggacactgaaggtctac tggaatggtgtaagagaaatcttggtcttgattgtgatgacactttcttt caaaagagaattgaagaattctttataactggtgagggccattttaatga agttttacaatttagaacgccaggcacgttgagcaccacagagtcaacac ctgctgggctgccaacagctgaaccttttaagtcctacttcgccaaaggc ttcctctcgatagattcaggttactactcagccaaatgttactcaggaac atccaattcagggcttcaattgattaacattacccgacattcaactagaa tagttgacacacctgggcctaagatcactaacctaaagaccatcaactgc ataaacttgaaggcatcgatcttcaaagaacatagagaggttgaaatcaa tgtgcttctcccccaagttgcagttaatctctcaaactgtcacgttgtaa tcaaatcacatgtctgtgactactctttagacattgacggtgcggtgagg cttcctcacatttaccatgaaggagttttcatcccaggaacttacaaaat agtgatagataaaaaaaataagttgaatgacagatgcaccttatttaccg actgtgtgataaaaggaagggaggttcgtaaaggacagtcagttttgagg cagtacaagacggaaatcaggattggcaaggcatcaaccggctttagaag attgctttcagaagaacccagtgatgactgtgtatcaagaactcaactat taaggacagagactgcagagatccacggcgacaactatggtggcccgggt gacaaaataaccatctgcaatggctcaactattgtagaccaaagactggg cagtgaactaggatgctacaccatcaatagagtgaggtcattcaagctat gcgaaaacagtgccacagggaagaattgtgaaatagacagtgtcccagtt aaatgcaggcagggttattgcctaagaatcactcaggaagggaggggcca cgtaaaattatctaggggctcagaggttgtcttagatgcatgcgatacaa gctgtgaaataatgatacctaagggcactggtgacatcctagttgactgt tcaggtgggcagcaacattttctaaaggacaatttgatagatctaggatg ccccaaaattccattattgggcaaaatggctatttacatttgcagaatgt caaaccaccccaaaacaaccatggctttcctcttctggttcagctttggc tatgtaataacctgcatactttgcaaggctattttttacttgttaataat tgttggaacactagggagaaggctcaagcagtatagagagttgaaacctc agacttgcaccatatgtgagacaactcctgtaaatgcaatagatgctgag atgcatgacctcaattgcagttacaacatttgtccctactgtgcatctag actaacctcagatgggcttgctaggcatgtgatacaatgccctaagcgga aggagaaagtggaagaaactgaactgtacttgaacttagaaagaattcct tgggttgtaagaaagctgttgcaggtgtcagagtcaactggtgtggcatt gaaaagaagcagttggctgattgtgctgcttgtgctattcactgtttcat tatcaccagttcaatcagcacccattggtcaagggaagacaattgaggca taccgggccagggaagggtacacaagtatatgcctctttgtactaggaag tatcctatttatagtttcttgcctaatgaaagggctggttgacagtgttg gcaactccttcttccctggactgtccatttgcaaaacgtgctccataagc agcattaatggctttgaaattgagtcccataagtgctattgcagcttatt ctgttgcccctattgtaggcactgctctaccgataaagaaattcataagc tgcacttgagcatctgcaaaaaaaggaaaacaggaagtaatgtcatgttg gctgtctgcaagctcatgtgtttcagggccaccatggaagtaagtaacag agccctgtttatccgtagcatcatcaacaccacttttgttttgtgcatac tgatactagcagtttgtgttgttagcacctcagcagtggagatggaaaac ctaccagcagggacctgggaaagagaagaagacctaacaaatttctgtca tcaggaatgccaggttacagagactgaatgcctctgcccttatgaagctc tagtactcagaaagcctttattcctagatagtacagctaaaggcatgaaa aatctgctaaattcaacaagtttagaaacgagtttatcaattgaggcacc atggggagcaataaatgttcagtcaacctacaaaccaactgtgtcaactg caaacatagcactcagttggagctcagtggaacacagaggcaataagatc ttggtttcaggcagatcagaatcaattatgaagctggaagaaaggacagg aatcagctgggatctcggtgtagaagatgcctctgaatctaaactgctta cagtatctgtcatggacttgtctcagatgtactctcctgtcttcgagtac ttatcaggggacagacaggtggaagagtggcccaaagcaacttgcacagg tgactgcccagaaagatgtggctgcacatcatcaacctgtttgcacaaag aatggcctcactcaagaaattggagatgcaatcccacttggtgctggggt gtagggactggctgcacctgttgtggattagatgtgaaagacctttttac agattatatgtttgtcaagtggaaagttgaatacatcaagacagaggcca tagtgtgtgtagaacttactagtcaggaaaggcagtgtagcttgattgaa gcgggcacaaggttcaatttaggtcctgtgaccatcacactgtcagaacc aagaaacatccaacaaaaactccctcctgaaataatcacactgcatccta ggatcgaagaaggtttttttgacctgatgcatgtgcaaaaggtgttatcg gcaagcacagtgtgtaagttgcagagttgcacacatggtgtgccaggaga cctacaggtctaccacatcggaaatttattaaaaggggataaggtaaatg gacatctaattcataaaattgagccacacttcaacacctcctggatgtcc tgggatggttgtgacctagactactactgcaacatgggagattggccttc ttgcacatacacaggggtcacccaacacaatcatgcttcatttgtaaact tactcaacattgaaactgattacacaaagaacttccactttcactctaaa agggtcactgcacacggagatacaccacaactagatcttaaggcaagacc aacctatggtgcaggcgagatcactgttctggtagaagttgctgacatgg agttacatacaaagaagattgaaatatcaggcttaaaatttgcaagctta gcttgcacaggttgttatgcttgtagctctagcatctcatgcaaagttag aattcatgtggatgaaccagatgaacttacagtacatgttaaaagtgatg atccagatgtggttgcagctagctcaagtctcatggcaaggaagcttgaa tttggaacagacagtacatttaaagctttctcggccatgcctaaaacttc tctatgtttctacattgttgaaagagaacactgtaagagctgcagtgaag aagacacaaaaaaatgtgttaacacaaaacttgagcaaccacaaagcatt ttgatcgaacacaagggaactataatcggaaagcaaaacagcacttgcac ggctaaggcaagttgctggttagagtcagtcaagagttttttttatggcc taaagaacatgcttagtggcatttttggcaatgtctttatgggcattttc ttgttccttgcccccttcatcctgttaatactattctttatgtttgggtg gaggatcctattctgctttaaatgttgtagaagaaccagaggcctgttca agtatagacacctcaaagacgatgaagaaactggttatagaaggattatt gaaaaactaaacaataaaaaaggaaaaaacaaactgcttgatggtgaaag acttgctgatggaagaattgccgaactgttctctacaaaaacacacattg gctag SEQIDNO:2 agaagattgctttcagaagaacccagtgatgactgtgtatcaagaactca actattaaggacagagactgcagagatccacggcgacaactatggtggcc cgggtgacaaaataaccatctgcaatggctcaactattgtagaccaaaga ctgggcagtgaactaggatgctacaccatcaatagagtgaggtcattcaa gctatgcgaaaacagtgccacagggaagaattgtgaaatagacagtgtcc cagttaaatgcaggcagggttattgcctaagaatcactcaggaagggagg ggccacgtaaaattatctaggggctcagaggttgtcttagatgcatgcga tacaagctgtgaaataatgatacctaagggcactggtgacatcctagttg actgttcaggtgggcagcaacattttctaaaggacaatttgatagatcta ggatgccccaaaattccattattgggcaaaatggctatttacatttgcag aatgtcaaaccaccccaaaacaaccatggctttcctcttctggttcagct ttggctatgtaataacctgcatactttgcaaggctattttttacttgtta ataattgttggaacactagggagaaggctcaagcagtatagagagttgaa acctcagacttgcaccatatgtgagacaactcctgtaaatgcaatagatg ctgagatgcatgacctcaattgcagttacaacatttgtccctactgtgca tctagactaacctcagatgggcttgctaggcatgtgatacaatgccctaa gcggaaggagaaagtggaagaaactgaactgtacttgaacttagaaagaa ttccttgggttgtaagaaagctgttg SEQIDNO:3 agaaagcctttattcctagatagtacagctaaaggcatgaaaaatctgct aaattcaacaagtttagaaacgagtttatcaattgaggcaccatggggag caataaatgttcagtcaacctacaaaccaactgtgtcaactgcaaacata gcactcagttggagctcagtggaacacagaggcaataagatcttggtttc aggcagatcagaatcaattatgaagctggaagaaaggacaggaatcagct gggatctcggtgtagaagatgcctctgaatctaaactgcttacagtatct gtcatggacttgtctcagatgtactctcctgtcttcgagtacttatcagg ggacagacaggtggaagagtggcccaaagcaacttgcacaggtgactgcc cagaaagatgtggctgcacatcatcaacctgtttgcacaaagaatggcct cactcaagaaattggagatgcaatcccacttggtgctggggtgtagggac tggctgcacctgttgtggattagatgtgaaagacctttttacagattata tgtttgtcaagtggaaagttgaatacatcaagacagaggccatagtgtgt gtagaacttactagtcaggaaaggcagtgtagcttgattgaagcgggcac aaggttcaatttaggtcctgtgaccatcacactgtcagaaccaagaaaca tccaacaaaaactccctcctgaaataatcacactgcatcctaggatcgaa gaaggtttttttgacctgatgcatgtgcaaaaggtgttatcggcaagcac agtgtgtaagttgcagagttgcacacatggtgtgccaggagacctacagg tctaccacatcggaaatttattaaaaggggataaggtaaatggacatcta attcataaaattgagccacacttcaacacctcctggatgtcctgggatgg ttgtgacctagactactactgcaacatgggagattggccttcttgcacat acacaggggtcacccaacacaatcatgcttcatttgtaaacttactcaac attgaaactgattacacaaagaacttccactttcactctaaaagggtcac tgcacacggagatacaccacaactagatcttaaggcaagaccaacctatg gtgcaggcgagatcactgttctggtagaagttgctgacatggagttacat acaaagaagattgaaatatcaggcttaaaatttgcaagcttagcttgcac aggttgttatgcttgtagctctagcatctcatgcaaagttagaattcatg tggatgaaccagatgaacttacagtacatgttaaaagtgatgatccagat gtggttgcagctagctcaagtctcatggcaaggaagcttgaatttggaac agacagtacatttaaagctttctcggccatgcctaaaacttctctatgtt tctacattgttgaaagagaacactgtaagagctgcagtgaagaagacaca aaaaaatgtgttaacacaaaacttgagcaaccacaaagcattttgatcga acacaagggaactataatcggaaagcaaaacagcacttgcacggctaagg caagttgctggttagagtcagtcaagagttttttttatggcctaaagaac atgcttagtggcatttttggcaatgtctttatgggcattttcttgttcct tgcccccttcatcctgttaatactattctttatgtttgggtggaggatcc tattctgctttaaatgttgtagaagaaccagaggcctgttcaagtataga cacctcaaagacgatgaagaaactggttatagaaggattattgaaaaact aaacaataaaaaaggaaaaaacaaactgcttgatggtgaaagacttgctg atggaagaattgccgaactgttctctacaaaaacacacattggctag SEQIDNO:4 MHISLMYAILCLQLCGLGETHGSHNETRHNKTDTMTTHGDNPSSEPPVST ALSITLDPSTVTPTTPASGLEGSGEVYTSPPITTGSLPLSETTPELPVTT GTDTLSAGDVDPSTQTAGGTSAPTVRTSLPNSPSTPSTPQDTHHPVRNLL SVTSPGPDETSTPSGTGKESSATSSPHPVSNRPPTPPATAQGPTENDSHN ATEHPESLTQSATPGLMTSPTQIVHPQSATPITVQDTHPSPTNRSKRNLK MEIILTLSQGLKKYYGKILRLLQLTLEEDTEGLLEWCKRNLGLDCDDTFF QKRIEEFFITGEGHFNEVLQFRTPGTLSTTESTPAGLPTAEPFKSYFAKG FLSIDSGYYSAKCYSGTSNSGLQLINITRHSTRIVDTPGPKITNLKTINC INLKASIFKEHREVEINVLLPQVAVNLSNCHVVIKSHVCDYSLDIDGAVR LPHIYHEGVFIPGTYKIVIDKKNKLNDRCTLFTDCVIKGREVRKGQSVLR QYKTEIRIGKASTGFRRLLSEEPSDDCVSRTQLLRTETAEIHGDNYGGPG DKITICNGSTIVDQRLGSELGCYTINRVRSFKLCENSATGKNCEIDSVPV KCRQGYCLRITQEGRGHVKLSRGSEVVLDACDTSCEIMIPKGTGDILVDC SGGQQHFLKDNLIDLGCPKIPLLGKMAIYICRMSNHPKTTMAFLFWFSFG YVITCILCKAIFYLLIIVGTLGRRLKQYRELKPQTCTICETTPVNAIDAE MHDLNCSYNICPYCASRLTSDGLARHVIQCPKRKEKVEETELYLNLERIP WVVRKLLQVSESTGVALKRSSWLIVLLVLFTVSLSPVQSAPIGQGKTIEA YRAREGYTSICLFVLGSILFIVSCLMKGLVDSVGNSFFPGLSICKTCSIS SINGFEIESHKCYCSLFCCPYCRHCSTDKEIHKLHLSICKKRKTGSNVML AVCKLMCFRATMEVSNRALFIRSIINTTFVLCILILAVCVVSTSAVEMEN LPAGTWEREEDLTNFCHQECQVTETECLCPYEALVLRKPLFLDSTAKGMK NLLNSTSLETSLSIEAPWGAINVQSTYKPTVSTANIALSWSSVEHRGNKI LVSGRSESIMKLEERTGISWDLGVEDASESKLLTVSVMDLSQMYSPVFEY LSGDRQVEEWPKATCTGDCPERCGCTSSTCLHKEWPHSRNWRCNPTWCWG VGTGCTCCGLDVKDLFTDYMFVKWKVEYIKTEAIVCVELTSQERQCSLIE AGTRFNLGPVTITLSEPRNIQQKLPPEIITLHPRIEEGFFDLMHVQKVLS ASTVCKLQSCTHGVPGDLQVYHIGNLLKGDKVNGHLIHKIEPHFNTSWMS WDGCDLDYYCNMGDWPSCTYTGVTQHNHASFVNLLNIETDYTKNFHFHSK RVTAHGDTPQLDLKARPTYGAGEITVLVEVADMELHTKKIEISGLKFASL ACTGCYACSSSISCKVRIHVDEPDELTVHVKSDDPDVVAASSSLMARKLE FGTDSTFKAFSAMPKTSLCFYIVEREHCKSCSEEDTKKCVNTKLEQPQSI LIEHKGTIIGKQNSTCTAKASCWLESVKSFFYGLKNMLSGIFGNVFMGIF LFLAPFILLILFFMFGWRILFCFKCCRRTRGLFKYRHLKDDEETGYRRII EKLNNKKGKNKLLDGERLADGRIAELFSTKTHIG SEQIDNO:5 RRLLSEEPSDDCVSRTQLLRTETAEIHGDNYGGPGDKITICNGSTIVDQR LGSELGCYTINRVRSFKLCENSATGKNCEIDSVPVKCRQGYCLRITQEGR GHVKLSRGSEVVLDACDTSCEIMIPKGTGDILVDCSGGQQHFLKDNLIDL GCPKIPLLGKMAIYICRMSNHPKTTMAFLFWFSFGYVITCILCKAIFYLL IIVGTLGRRLKQYRELKPQTCTICETTPVNAIDAEMHDLNCSYNICPYCA SRLTSDGLARHVIQCPKRKEKVEETELYLNLERIPWVVRKLL SEQIDNO:6 RKPLFLDSTAKGMKNLLNSTSLETSLSIEAPWGAINVQSTYKPTVSTANI ALSWSSVEHRGNKILVSGRSESIMKLEERTGISWDLGVEDASESKLLTVS VMDLSQMYSPVFEYLSGDRQVEEWPKATCTGDCPERCGCTSSTCLHKEWP HSRNWRCNPTWCWGVGTGCTCCGLDVKDLFTDYMFVKWKVEYIKTEAIVC VELTSQERQCSLIEAGTRFNLGPVTITLSEPRNIQQKLPPEIITLHPRIE EGFFDLMHVQKVLSASTVCKLQSCTHGVPGDLQVYHIGNLLKGDKVNGHL IHKIEPHFNTSWMSWDGCDLDYYCNMGDWPSCTYTGVTQHNHASFVNLLN IETDYTKNFHFHSKRVTAHGDTPQLDLKARPTYGAGEITVLVEVADMELH TKKIEISGLKFASLACTGCYACSSSISCKVRIHVDEPDELTVHVKSDDPD VVAASSSLMARKLEFGTDSTFKAFSAMPKTSLCFYIVEREHCKSCSEEDT KKCVNTKLEQPQSILIEHKGTIIGKQNSTCTAKASCWLESVKSFFYGLKN MLSGIFGNVFMGIFLFLAPFILLILFFMFGWRILFCFKCCRRTRGLFKYR HLKDDEETGYRRIIEKLNNKKGKNKLLDGERLADGRIAELFSTKTHIG SEQIDNO:7 GTTGGTGGTCGCCATGGATGGTGTTATTGTATACTGTCTAAACGCGTTAG TAAAACATGGCGAGGAAATAAATCATATAAAAAATGATTTCATGATTAAA CCATGTTGTGAAAAAGTCAAGAACGTTCACATTGGCGGACAATCTAAAAA CAATACAGTGATTGCAGATTTGCCATATATGGATAATGCGGTATCCGATG TATGCAATTCACTGTATAAAAAGAATGTATCAAGAATATCCAGATTTGCT AATTTGATAAAGATAGATGACGATGACAAGACTCCTACTGGTGTATATAA TTATTTTAAACCTAAAGATGCCATTCCTGTTATTATATCCATAGGAAAGG ATAGAGATGTTTGTGAACTATTAATCTCATCTGATAAAGCGTGTGCGTGT ATAGAGTTAAATTCATATAAAGTAGCCATTCTTCCCATGGATGTTTCCTT TTTTACCAAAGGAAATGCATCATTGATTATTCTCCTGTTTGATTTCTCTA TCGATGCGGCACCTCTCTTAAGAAGTGTAACCGATAATAATGTTATTATA TCTAGACACCAGCGTCTACATGACGAGCTTCCGAGTTCCAATTGGTTCAA GTTTTACATAAGTATAAAGTCCGACTATTGTTCTATATTATATATGGTTG TTGATGGATCTGTGATGCATGCAATAGCTGATAATAGAACTTACGCAAAT ATTAGCAAAAATATATTAGACAATACTACAATTAACGATGAGTGTAGATG CTGTTATTTTGAACCACAGATTAGGATTCTTGATAGAGATGAGATGCTCA ATGGATCATCGTGTGATATGAACAGACATTGTATTATGATGAATTTACCT GATGTAGGCGAATTTGGATCTAGTATGTTGGGGAAATATGAACCTGACAT GATTAAGATTGCTCTTTCGGTGGCTGGGTACCAGGCGCGCCTTTCATTTT GTTTTTTTCTATGCTATAAATGGTGAGCAAGGGCGAGGAGCTGTTCACCG GGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAG TTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGAC CCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCC TCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGAC CACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGT CCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCG CCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAG GGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTA CAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACG GCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTG CAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGT GCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAG ACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCC GCCGGGATCACTCTCGGCATGCACGAGCTGTACAAGTAAGCGGCCGCTGG TACCCAACCTAAAAATTGAAAATAAATACAAAGGTTCTTGAGGGTTGTGT TAAATTGAAAGCGAGAAATAATCATAAATAAGCccggtGCCACCATGgat gcaatgaagagagggctctgctgtgtgctgctgctgtgtggagcagtctt cgtttcgcccagccaggaaatccatgcccgattcagaagaggagccagat ctcccATCAAACAAGTTTGTACAAAAAAGCAGGCTcatatatcattaatg tatgcaatcctttgcctacagctgtgtggtctgggagagactcatggatc acacaatgaaactagacacaataaaacagacaccatgacaacacacggtg ataacccgagctctgaaccgccagtgagcacggccttgtctattacactt gacccctccactgtcacacccacaacaccagccagtggattagaaggctc aggggaagtctacacatcccctccgatcaccaccgggagcttgcccctgt cggagacaacaccagaactccctgttacaaccggcacagacaccttaagc gcaggtgatgtcgatcccagcacgcagacagccggaggcacctccgcacc aacagtccgcacaagtctacccaacagccctagcacaccatctacaccac aagacacacaccatcctgtgagaaatctactttcagtcacgagtcctggg ccagatgaaacatcaacaccctcgggaacaggcaaagagagctcagcaac cagtagccctcatccagtctccaacagaccaccaacccctcctgcaacag cccagggacccactgaaaatgacagtcacaacgccactgaacaccctgag tccctgacacagtcagcaaccccaggcctaatgacctctccaacacagat agtccacccacaaagtgccacccccataaccgttcaagacacacatccca gtccaacgaacaggtctaaaagaaaccttaagatggaaataatcttgact ttatctcagggtttaaaaaagtactatgggaaaatattaaggcttctgca actcaccttagaggaggacactgaaggtctactggaatggtgtaagagaa atcttggtcttgattgtgatgacactttctttcaaaagagaattgaagaa ttctttataactggtgagggccattttaatgaagttttacaatttagaac gccaggcacgttgagcaccacagagtcaacacctgctgggctgccaacag ctgaaccttttaagtcctacttcgccaaaggcttcctctcgatagattca ggttactactcagccaaatgttactcaggaacatccaattcagggcttca attgattaacattacccgacattcaactagaatagttgacacacctgggc ctaagatcactaacctaaagaccatcaactgcataaacttgaaggcatcg atcttcaaagaacatagagaggttgaaatcaatgtgcttctcccccaagt tgcagttaatctctcaaactgtcacgttgtaatcaaatcacatgtctgtg actactctttagacattgacggtgcggtgaggcttcctcacatttaccat gaaggagttttcatcccaggaacttacaaaatagtgatagataaaaaaaa taagttgaatgacagatgcaccttatttaccgactgtgtgataaaaggaa gggaggttcgtaaaggacagtcagttttgaggcagtacaagacggaaatc aggattggcaaggcatcaaccggctttagaagattgctttcagaagaacc cagtgatgactgtgtatcaagaactcaactattaaggacagagactgcag agatccacggcgacaactatggtggcccgggtgacaaaataaccatctgc aatggctcaactattgtagaccaaagactgggcagtgaactaggatgcta caccatcaatagagtgaggtcattcaagctatgcgaaaacagtgccacag ggaagaattgtgaaatagacagtgtcccagttaaatgcaggcagggttat tgcctaagaatcactcaggaagggaggggccacgtaaaattatctagggg ctcagaggttgtcttagatgcatgcgatacaagctgtgaaataatgatac ctaagggcactggtgacatcctagttgactgttcaggtgggcagcaacat tttctaaaggacaatttgatagatctaggatgccccaaaattccattatt gggcaaaatggctatttacatttgcagaatgtcaaaccaccccaaaacaa ccatggctttcctcttctggttcagctttggctatgtaataacctgcata ctttgcaaggctattttttacttgttaataattgttggaacactagggag aaggctcaagcagtatagagagttgaaacctcagacttgcaccatatgtg agacaactcctgtaaatgcaatagatgctgagatgcatgacctcaattgc agttacaacatttgtccctactgtgcatctagactaacctcagatgggct tgctaggcatgtgatacaatgccctaagcggaaggagaaagtggaagaaa ctgaactgtacttgaacttagaaagaattccttgggttgtaagaaagctg ttgcaggtgtcagagtcaactggtgtggcattgaaaagaagcagttggct gattgtgctgcttgtgctattcactgtttcattatcaccagttcaatcag cacccattggtcaagggaagacaattgaggcataccgggccagggaaggg tacacaagtatatgcctctttgtactaggaagtatcctatttatagtttc ttgcctaatgaaagggctggttgacagtgttggcaactccttcttccctg gactgtccatttgcaaaacgtgctccataagcagcattaatggctttgaa attgagtcccataagtgctattgcagcttattctgttgcccctattgtag gcactgctctaccgataaagaaattcataagctgcacttgagcatctgca aaaaaaggaaaacaggaagtaatgtcatgttggctgtctgcaagctcatg tgtttcagggccaccatggaagtaagtaacagagccctgtttatccgtag catcatcaacaccacttttgttttgtgcatactgatactagcagtttgtg ttgttagcacctcagcagtggagatggaaaacctaccagcagggacctgg gaaagagaagaagacctaacaaatttctgtcatcaggaatgccaggttac agagactgaatgcctctgcccttatgaagctctagtactcagaaagcctt tattcctagatagtacagctaaaggcatgaaaaatctgctaaattcaaca agtttagaaacgagtttatcaattgaggcaccatggggagcaataaatgt tcagtcaacctacaaaccaactgtgtcaactgcaaacatagcactcagtt ggagctcagtggaacacagaggcaataagatcttggtttcaggcagatca gaatcaattatgaagctggaagaaaggacaggaatcagctgggatctcgg tgtagaagatgcctctgaatctaaactgcttacagtatctgtcatggact tgtctcagatgtactctcctgtcttcgagtacttatcaggggacagacag gtggaagagtggcccaaagcaacttgcacaggtgactgcccagaaagatg tggctgcacatcatcaacctgtttgcacaaagaatggcctcactcaagaa attggagatgcaatcccacttggtgctggggtgtagggactggctgcacc tgttgtggattagatgtgaaagacctttttacagattatatgtttgtcaa gtggaaagttgaatacatcaagacagaggccatagtgtgtgtagaactta ctagtcaggaaaggcagtgtagcttgattgaagcgggcacaaggttcaat ttaggtcctgtgaccatcacactgtcagaaccaagaaacatccaacaaaa actccctcctgaaataatcacactgcatcctaggatcgaagaaggtttCt ttgacctgatgcatgtgcaaaaggtgttatcggcaagcacagtgtgtaag ttgcagagttgcacacatggtgtgccaggagacctacaggtctaccacat cggaaatttattaaaaggggataaggtaaatggacatctaattcataaaa ttgagccacacttcaacacctcctggatgtcctgggatggttgtgaccta gactactactgcaacatgggagattggccttcttgcacatacacaggggt cacccaacacaatcatgcttcatttgtaaacttactcaacattgaaactg attacacaaagaacttccactttcactctaaaagggtcactgcacacgga gatacaccacaactagatcttaaggcaagaccaacctatggtgcaggcga gatcactgttctggtagaagttgctgacatggagttacatacaaagaaga ttgaaatatcaggcttaaaatttgcaagcttagcttgcacaggttgttat gcttgtagctctagcatctcatgcaaagttagaattcatgtggatgaacc agatgaacttacagtacatgttaaaagtgatgatccagatgtggttgcag ctagctcaagtctcatggcaaggaagcttgaatttggaacagacagtaca tttaaagctttctcggccatgcctaaaacttctctatgtttctacattgt tgaaagagaacactgtaagagctgcagtgaagaagacacaaaaaaatgtg ttaacacaaaacttgagcaaccacaaagcattttgatcgaacacaaggga actataatcggaaagcaaaacagcacttgcacggctaaggcaagttgctg gttagagtcagtcaagagtttCttttatggcctaaagaacatgcttagtg gcatttttggcaatgtctttatgggcattttcttgttccttgcccccttc atcctgttaatactattctttatgtttgggtggaggatcctattctgctt taaatgttgtagaagaaccagaggcctgttcaagtatagacacctcaaag acgatgaagaaactggttatagaaggattattgaaaaactaaacaataaa aaaggaaaaaacaaactgcttgatggtgaaagacttgctgatggaagaat tgccgaactgttctctacaaaaacacacattggcACCCAGCTTTCTTGTA CAAAGTGGTTCGATggggatctagagggcccgcggttcgaaggtaagcct atccctaaccctctcctcggtctcgattctacgtaaGTCGACCTGCAGGG AAAGTTTTATAGGTAGTTGATAGAACAAAATACATAATTTTGTAAAAATA AATCACTTTTTATACTAATATGACACGATTACCAATACTTTTGTTACTAA TATCATTAGTATACGCTACACCTTTTCCTCAGACATCTAAAAAAATAGGT GATGATGCAACTTTATCATGTAATCGAAATAATACAAATGACTACGTTGT TATGAGTGCTTGGTATAAGGAGCCCAATTCCATTATTCTTTTAGCTGCTA AAAGCGACGTCTTGTATTTTGATAATTATACCAAGGATAAAATATCTTAC GACTCTCCATACGATGATCTAGTTACAACTATCACAATTAAATCATTGAC TGCTAGAGATGCCGGTACTTATGTATGTGCATTCTTTATGACATCGCCTA CAAATGACACTGATAAAGTAGATTATGAAGAATACTCCACAGAGTTGATT GTAAATACAGATAGTGAATCGACTATAGACATAATACTATCTGGATCTAC ACATTCACCAGAAACTAGTT

EXAMPLES

Example 1. Preparation of an Example MVA-GP (Glycoprotein) Vector (FIG. 1A-1C)

(50) The sequence of the CCHFV M segment was taken from published sequence data on the IbAr10200 CCHFV strain.

(51) The M segment sequence was modified for improved compatibility with MVA expression. M segment untranslated regions were deleted, start and stop codons were deleted, and 2?TTTTTNT sequences (poxvirus transcription stop signals) silently mutated. attB1 and attB2 sequences were added for compatibility with the proprietary cloning system used (Gateway Cloning, by Invitrogen).

(52) Tissue Plasminogen Activator (tPA) signal sequence (for increased immunogenicity and intracellular transport) and V5 fusion protein sequence (for identification of expressed protein by immunolabeling) were added.

(53) The construct was synthesised and recombined into a pDONR vector to generate Entry Clone plasmid (pENTR-GP).

(54) The plasmid was then recombined with Destination vector pDEST44-TPA-V5 to generate pTP-GP plasmid.

(55) This resulted in the gene for tPA-GP-V5 fusion protein downstream of the poxvirus mH5 promoter. This promoter was chosen for increased stability and strong early expression, to drive a cytotoxic T lymphocyte response.

(56) pTP-GP was transfected into MVA-infected cells. Recombination occurs between the MVA flanks on the plasmid and in the MVA genome, inserting the GFP and GP cassette into the MVA genome. The MVA strain used was MVA 1974/NIH clone 1.

(57) MVA-GP was plaque-purified based on GFP expression and underwent quality checks: confirmation of purity by PCR, confirmation of GP expression by Western blot (FIG. 15A), and sequencing of the insertion site.

(58) SDS-PAGE of MVA-GP and Western blotting with anti-V5 antibody (FIG. 15A) indicated a single protein of approximately 75 kDa, confirming expression and consistent with cleavage of the predicted 76.6 kDa Gc-V5 fusion protein from the GP precursor at RKPL sequence.

(59) SDS-PAGE and Western blotting with anti-glycoprotein polyclonal serum were performed, in order to compare glycoprotein expression by MVA-GP with CCHFv (FIG. 15B). Several products were expressed by CCHFv (lane 3). Some of these were also detected in uninfected SW13 cells (lane 4), suggesting a possible cross-reaction with cellular proteins. CCHFv-specific products were detected at approximately 109, 60, 34 and <20 kDa. Similarly, sucrose cushion-purified MVA-GP expressed major proteins at approximately 92-136, 64 and 35 kDa (lane 2). Sucrose cushion-purified MVA-1974 (a negative control) did not show any products of a similar size (lane 1), confirming all products in lane 2 as specific to the recombinant vaccine.

(60) The appropriate clone of MVA-GP was amplified, purified by sucrose cushion centrifugation and titrated by plaque assay.

Example 2. MVA-GP Immunogenicity in Balb/c Mice

(61) 11 Balb/c mice were injected intramuscularly with 10.sup.7 plaque-forming units (pfu) per animal of MVA-GP, prepared according to Example 1. A volume of 100 ?l was delivered, split into two sites at 50 ?l each. Animals received 2 vaccinations, spaced 2 weeks apart. Control animals (n=9) received saline.

(62) Eight days after the final vaccination, 5 (MVA-GP) or 4 (saline) mice were sacrificed for T cell immunogenicity testing. The remaining animals were sacrificed 14 days after the final vaccination.

(63) Animals immunised with MVA-GP showed a significant antigen response to CCHF glycoprotein peptides as compared to control animals (FIG. 2).

(64) Examples 3 and 4 (described below) utilise an animal model that replicates lethal disease in mice that are infected with CCHF virus. In vaccine efficacy experiments, all animals that were vaccinated with MVA-GP survived a lethal challenge dose of CCHFV. Those animals that received a mock immunisation with (i) saline or (ii) a simple MVA preparation all succumbed to disease and met humane clinical endpoints by five days post-challenge.

Example 3. MVA-GP Vaccine Efficacy Study in Mouse Model (Mouse Strains A129 and 129Sv/Ev)

(65) This in vivo efficacy study used both type-1 interferon receptor knockout (A129) and wild-type (129Sv/Ev) mice which were immunised with MVA containing CCHF virus glycoprotein (MVA-GP), construct alone (MVA-empty) and saline. A129 mice are susceptible to CCHF virus infection, and the 129Sv/Ev are the wild-type parent strain. Mice were immunised at day 0, and then boosted at day 14. 7 days after the last immunisation, 5 animals from each group were used to assess immune response. 14 days after the last immunisation, A129 mice from each immunisation group were challenged with 10.sup.2 TCID.sub.50 CCHF virus, delivered intradermally. 6 challenged animals from each group were observed for up to 10 days post-challenge for survival studies.

(66) Experimental protocol is depicted in FIG. 3.

(67) Results: IFN-? ELISPOT results showed that MVA-GP immunised mice generated immune responses specific to the CCHFv glycoproteins that they were immunised with. Statistically similar results were observed between both strains after MVA-GP immunisation (P<0.05, Mann-Whitney statistical test). T-cell responses to peptides derived from the TPA/V5 fusion proteins, or from an irrelevant antigen (NP) were negligible, indicating specificity of the response. (FIG. 4).

(68) When the responses shown in FIG. 4 were subdivided into the peptide pools (1 for TPA/V5, 4 for GP, 3 for GC and 2 for NP) the same pools were optimal between strains. Some peptide pools were consistently non-immunogenic. (FIG. 5).

(69) After challenge with CCHF virus, control groups of saline and MVA-empty showed 66% survival and 33% survival, respectively. 100% survival was observed with 2?MVA-GP group. (FIG. 6).

(70) Weight loss was seen in animals that reached humane clinical endpoints in control groups 2 and 3. Some of the vaccinated animals in the 100% survival group showed evidence of weight loss, indicative of disease/illness. (FIG. 7).

(71) Animals that reached humane clinical endpoints in groups 2 (MVA-empty) and 3 (saline control) also exhibited a rise in temperature at days 5-7 post-challenge. Temperatures fluctuated in the MVA-GP immunised animals, but no trend was observed. (FIG. 8).

(72) Conclusions: MVA-GP is immunogenic, producing protein-specific immune responses. No differences were observed in the immune response to MVA-GP between wild-type mice (129Sv/Ev) and those with a knockout in the type-1 interferon receptor (A129). All MVA-GP immunised animals survived a challenge with 10.sup.2 TCID.sub.50 CCHF virus (strain IbAr10200). Animals which reached humane clinical endpoints in the MVA-empty and saline groups exhibited a rise in temperature and weight loss.

Example 4. MVA-GP Vaccine Efficacy Study in Mouse Model (Mouse Strain A129)

(73) The study described above in Example 3 found 10.sup.2 TCID.sub.50/100 ?l delivered intradermally (i.d.) to have 34% lethality in saline vaccinated 10 week old IFN-?/?R.sup.?/? mice. Therefore this study used 2?10.sup.2 TCID.sub.50/100 ?l of strain IbAr10200 delivered i.d. as the challenge dose to IFN-?/?R.sup.?/? mice.

(74) Prior to challenge, animals were vaccinated with MVA-GP derived from strain IbAr10200. Vaccinations were given intramuscularly (i.m.) into the caudal thigh at a dose of 10.sup.7 plaque-forming units (pfu) per animal. A volume of 100 ?l was delivered, split into two sites at 50 ?l each. Animals received 2 vaccinations, spaced 2 weeks apart. Two weeks elapsed between final vaccination and challenge.

(75) Control groups were vaccinated with saline (groups 1 & 6), or MVA-empty (group 2), which is unmodified, non-recombinant MVA.

(76) Representatives from each vaccination group were sacrificed 7 days post-final vaccination, for histology and immunogenicity (T-cell and antibody) testing. Further animals were sacrificed 4 days post-challenge, for histology and viral load testing. After challenge, animals were weighed and temperature monitored daily, and observed for signs of illness twice daily.

(77) Animals showing moderate symptoms (such as loss of 10% body weight, shaking or paralysis) were euthanised. Spleen and liver samples were collected from animals that reached humane clinical endpoints. At 14 days post-challenge, all remaining animals were culled and samples collected for histology and viral load testing.

(78) Experimental protocol is depicted in FIG. 9.

(79) ResultsSurvival. 100% protection was achieved with MVA-GP (survival 2 weeks post-challenge). All other groups died 4-5 days post-challenge. (FIG. 10).

(80) ResultsClinical Data. MVA-GP showed no signs of disease. Control groups showed severe illness and were euthanized. (FIG. 11). MVA-GP temperature was stable whilst other groups were succumbing, but slight spike in temperature at 9 days post-challenge. Control groups showed spike in temperature, then sharp reduction as succumbed to disease. (FIG. 12). MVA-GP showed some weight loss at days 4-5, then stable, but did not regain peak weight. Control groups lost 5-10% of body weight. (FIG. 13).

(81) ResultsImmune Responses (ELISpot) IFN-? ELISpot on splenocytes 7 days post-boost showed T-cell immunogenicity of GP. Low immunogenicity from TPA & V5 fusion tags (FIG. 14A). GP\ peptide pools showed that density of epitopes varied across the GP protein. (FIG. 14B).

(82) Western Blotting

(83) Immunised animals (including those used in Example 3) were also tested for induction of a CCHFv-specific humoral response by MVA-GP, using Western Blotting. IgG antibody reacting with a protein of approximately 114 kDa was detected in 3 out of 5 vaccinated animals' sera from 129Sv/Ev mice collected on day 21 of the vaccination schedule (FIG. 16A). In A129 mice, a CCHFv-specific IgG antibody response was detectable by Western Blot in only 1 out of 8 individual animals, which recognised a 79 kDa protein (FIG. 16B). A randomly selected A129 mouse that received the MVA-1974 negative control was tested, and no CCHFv-specific antibody response was seen (FIG. 16C).

(84) Sera from the 8 A129 animals vaccinated with MVA-GP were also assessed by Western Blot for an early phase immune response, using a detector antibody specific for mouse IgG, IgA and IgM. Antibodies specific for a 79 kDa CCHFv protein were detected in the same individual animal in this assay, as had been detected by anti-IgG only (FIG. 16E). Broadening the sensitivity to additional antibody subclasses, detected antibodies specific for a protein of approximately 112 kDa (FIG. 16D) in a further 4 animals.

(85) Histopathology

(86) Histopathological findings in immunised, CCHFv challenged mice are shown in FIGS. 17-18 and Tables 1-2 (below).

(87) TABLE-US-00004 TABLE 1 Severity of microscopic lesions in HE stained tissues from vaccinated A129 mice, challenged with CCHFv. Group Saline MVA 1974 MVA-GP MVA-GP Severity (day 32-33) (day 32-33) (day 32) (day 42) Spleen Normal 0 0 1 6 Minimal 1 1 2 0 Mild 2 3 0 0 Moderate 2 1 0 0 Marked 4 4 0 0 Liver Normal 0 1 2 6 Minimal 0 0 0 0 Mild 2 0 0 0 Moderate 3 3 1 0 Marked 4 5 0 0 Numbers of animals in each group, according to severity rating of histological lesions.

(88) TABLE-US-00005 TABLE 2 Frequency of immunohistochemically stained cells in tissues from selected vaccinated A129 mice, challenged with CCHFv. Group Saline MVA 1974 MVA-GP MVA-GP Frequency (day 32-33) (day 32-33) (day 32) (day 42) Spleen Normal 0 0 3 1 Minimal 4 4 0 0 Moderate 2 3 0 0 Marked 1 0 0 0 Liver Normal 0 0 2 1 Minimal 1 2 1 0 Moderate 3 1 0 0 Marked 3 4 0 0 Numbers of animals in each group, according to frequency of cells stained by immunohistochemistry.

(89) Viral Load Analysis

(90) Viral load was analysed by RT-PCR of CCHFv S segment in blood, spleen, and liver from 3 animals per group at day 32, and all surviving animals at day 42. CCHFv copy number was calculated by use of a standard curve. At day 32, CCHFv copy number was significantly lower in MVA-GP vaccinated animals compared to control groups in blood, spleen and liver (p=0.05). In the blood, it was detected in only 2 of 3 animals. At day 42, CCHFv levels were not statistically different in any tissue to those in vaccinated animals at the earlier timepoint. It was detectable in the blood in only 1 out of 5 vaccinated mice (FIG. 19).

(91) Alternatively, CCHFv expression was normalised to expression of the HPRT reference gene (FIG. 20). Normalised CCHFv expression was significantly lower in MVA-GP vaccinated animals compared to control groups in blood, spleen and liver (p=0.05). There was no statistically significant difference between saline and MVA 1974 control groups, or between day 32 and day 42 samples from MVA-GP vaccinated animals.

(92) Conclusions: MVA-GP is immunogenic, producing protein-specific immune responses. MVA-GP is protective, protecting 100% of mice from a fully lethal challenge of homologous strain CCHF. Animals which reached humane clinical endpoints in the MVA-empty and saline groups exhibited signs of illness, a rise in temperature and weight loss. No such clinical signs were observed in MVA-GP vaccinated animals.

Example 5. Heterologous Prime-Boost Study

(93) Heterologous prime-boosting approaches improve immune responses by allowing repeated vaccinations without increasing anti-vector immunity. A CCHFV glycoprotein (GP) or an antigenic fragment thereof is serially delivered via different viral or DNA vectors.

(94) In a heterologous regime where the prime vaccination is delivered by a DNA vector, and the boost vaccination is delivered by a Fowlpox virus vector, GP-specific antibody response is increased, GP-specific T-cell response is increased, and/or clinical illness is reduced, as compared to where the prime and boost are delivered by the same vector.

(95) The following heterologous combinations of vectors are provided for use in prime-boosting approaches:

(96) DNA prime, MVA boost

(97) Fowlpox prime, MVA boost

(98) MVA prime, Fowlpox boost

(99) DNA prime, Fowlpox boost, MVA boost

(100) MVA prime, Adenovirus boost

Example 6. Immunogenicity Studies in Non-Human Primates

(101) Prior to use in clinical trials, immune responses to a CCHF vaccine are tested in a non-human primate model.

(102) Non-human primates (e.g. rhesus macaques or cynomolgous macaques) are inoculated with the CCHF vaccine expressing the glycoprotein gene or functional fragment thereof. Animals receive either a single dose, or multiple doses in a prime-boost regime, by a parenteral route. Subsequent immunological analysis indicates that they have generated a CCHF-specific immune response. The latter is confirmed by one or more of the following methods:

(103) CCHF-specific antibodies present in serum

(104) Neutralising antibodies present in serum

(105) Cellular response to peptides derived from the CCHF glycoprotein

(106) Cellular response to virally-infected cells.

(107) A cellular response is characterised by an increase in one or more of: proliferation, production of cytokines or chemokines, phagocytosis, and/or release of granzymes.

Example 7. Preparation of an Example Adenovirus Vector

(108) A non-replicating adenovirus is engineered to express a CCHF glycoprotein or partial fragment thereof. The genetic sequence for the CCHF glycoprotein is inserted into the genome of the adenovirus vector. Expression of the glycoprotein is indicated by reactivity between a glycoprotein-specific antibody and products from the adenovirus by Western blotting or ELISA as follows:

(109) Cellular lysate of cells infected with the recombinant adenovirus, subjected to SDS-PAGE and Western blotting with an antibody specific for the CCHF glycoprotein, show a specific reactivity compared to negative controls.

(110) Alternatively, products from cells infected with the recombinant adenovirus are used to coat an ELISA plate. CCHF-specific antibodies bind to the coating and are detected via a chemical reaction.

Example 8. CCHF Vaccine Provides Cross-Strain Protection

(111) A vaccine expressing the glycoprotein gene or functional fragment thereof, in an adenovirus or non-replicating poxvirus vector, is delivered via a parenteral route into mice that are susceptible to disease caused by CCHF virus. They are challenged with a lethal dose of CCHF virus, from a strain other than that on which the vaccine is based. The challenged animals show no or mild clinical signs of illness, and do not require euthanasia. Control animals which received the same challenge dose of CCHF, but did not receive the vaccine, show severe signs of illness, reach humane clinical endpoints and require euthanasia.

Example 9. MVA-GP Immunogenicity in A129 Mice

(112) Twenty-five A129 mice were injected intramuscularly with 10.sup.7 pfu per animal of MVA-GP, prepared according to Example 1. A volume of 100 ?l was delivered, split into two sites at 50 ?l each. Animals received 2 vaccinations, spaced 2 weeks apart. Control animals (n=25) received 10.sup.7 pfu per animal of non-recombinant MVA 1974/NIH clone 1 according to the same regime.

(113) Fourteen days after the final vaccination, all mice were sacrificed for antibody testing. Sera were prepared, heat-inactivated and pooled; each pool contained sera from 5-6 animals that received the same treatment as each other. Pools were subjected to Western blot analysis against lysate of cells infected with CCHF virus.

(114) In 4 out of 5 pools from animals that received MVA-GP, an IgG antibody response specific for a CCHF virus protein of approximately 114 kDa was detected (FIG. 16F).

Example 10. Preparation and Efficacy of a Recombinant Influenza Virus Vector

(115) Reverse genetics are used to construct a recombinant influenza virus that carries a protective epitope of CCHFv glycoprotein in the neuraminidase stalk. CCHFv specific cytotoxic T lymphocytes (CTLs) are induced in mice after intranasal or parenteral administration. These CTLs provide a reduction in viral load and clinical illness after challenge with CCHFv.

Example 11. Preparation and Efficacy of a Recombinant Bacterial Vector

(116) The CCHFv glycoprotein gene, or functional fragment thereof, is expressed on the surface of genetically attenuated, gram-negative bacteria. After intranasal or parenteral administration to mice, the bacterial vector colonises antigen-presenting cells (e.g. dendritic cells or macrophages). A humoral and cellular CCHFv-specific immune response is induced. These immune responses provide a reduction in viral load and clinical illness after challenge with CCHFv.