HANTAVIRUS ANTIGENIC COMPOSITION

Abstract

The present invention provides a viral vector or bacterial vector, said vector comprising a nucleic acid sequence encoding a Hantavirus nucleoprotein or antigenic fragment thereof; wherein said vector is capable of inducing a protective immune response in a subject. The present invention also provides compositions and uses of the vector in methods of medical treatment.

Claims

1. A viral vector or bacterial vector, said vector comprising a nucleic acid sequence encoding a Hantavirus nucleoprotein or antigenic fragment thereof; wherein said vector is capable of inducing an immune response in a subject.

2. The vector of claim 1, wherein the nucleic acid sequence encoding a Hantavirus nucleoprotein 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. The vector of claim 1 or claim 2, wherein the nucleic acid sequence encoding a Hantavirus nucleoprotein or antigenic fragment thereof comprises a nucleic acid sequence having at least 70% sequence identity to the nucleic acid sequence of SEQ ID NOs: 22, 23 or 24.

4. The vector of any one of claims 1 to 3, wherein the nucleic acid sequence encoding a Hantavirus nucleoprotein or antigenic fragment thereof comprises a nucleic acid sequence having at least 70% sequence identity to the nucleic acid sequence of SEQ ID NOs: 15, 16 or 17.

5. The vector of any one of the preceding claims, wherein the nucleic acid sequence encoding a Hantavirus nucleoprotein 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: 5, 6, 8 and 9.

6. The vector of any one of the preceding claims, wherein the nucleic acid sequence encoding a Hantavirus nucleoprotein or antigenic fragment thereof comprises a nucleic acid sequence having at least 70% sequence identity to the nucleic acid sequence of SEQ ID NOs: 25, 26, 27 or 28.

7. The vector of any one of the preceding claims, wherein the nucleic acid sequence encoding a Hantavirus nucleoprotein or antigenic fragment thereof comprises a nucleic acid sequence having at least 70% sequence identity to the nucleic acid sequence of SEQ ID NOs: 18, 19, 20 or 21.

8. The vector of any one of the preceding claims, wherein the nucleic acid sequence encoding a Hantavirus nucleoprotein or antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein: (A) the first nucleic acid sequence has at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 15, 16, 17, 22, 23 or 24; and (B) the second nucleic acid sequence has at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 18, 19, 20, 21, 25, 26, 27 or 28.

9. The vector of any one of the preceding claims, wherein the nucleic acid sequence encoding a Hantavirus nucleoprotein or antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein: (A) the first nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 24; and (B) the second nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 28.

10. The vector of any one of the preceding claims, wherein the nucleic acid sequence encoding a Hantavirus nucleoprotein or antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein: (A) the first nucleic acid sequence has at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 22 or 23; and (B) the second nucleic acid sequence is provided by a nucleic acid sequence has at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 25, 26 or 27.

11. The vector of any one of the preceding claims, wherein the nucleic acid sequence encoding a Hantavirus nucleoprotein or antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein: (A) the first nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 17; and (B) the second nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 21.

12. The vector of any one of the preceding claims, wherein the nucleic acid sequence encoding a Hantavirus nucleoprotein or antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein: (A) the first nucleic acid sequence has at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 15 or 16; and (B) the second nucleic acid sequence has at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 18, 19 or 20.

13. The vector of any one of the preceding claims, wherein the nucleic acid sequence encoding a Hantavirus nucleoprotein or antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein: (A) the first nucleic acid sequence has at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1, 2 or 3; and (B) the second nucleic acid sequence has at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 5, 6, 8 or 9.

14. The vector of any one of the preceding claims, wherein the nucleic acid sequence encoding a Hantavirus nucleoprotein or antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein: (A) the first nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 3; and (B) the second nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 9.

15. The vector of any one of the preceding claims, wherein the nucleic acid sequence encoding a Hantavirus nucleoprotein or antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein: (A) the first nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 2; and (B) the second nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 8.

16. The vector of any one of the preceding claims, wherein the nucleic acid sequence encoding a Hantavirus nucleoprotein or antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein: (A) the first nucleic acid has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 2; and (B) the second nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 6.

17. The vector of any one of the preceding claims, wherein the nucleic acid sequence encoding a Hantavirus nucleoprotein or antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein: (A) the first nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 1; and (B) the second nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 5.

18. The vector of any one of the preceding claims, wherein the nucleic acid sequence encoding a Hantavirus nucleoprotein or antigenic fragment thereof comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 29.

19. The vector of any one of claims 1 to 17, wherein the nucleic acid sequence encoding a Hantavirus nucleoprotein or antigenic fragment thereof comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 30.

20. The vector of any one of the preceding claims, wherein the vector is a viral vector.

21. The vector of claim 20, wherein the vector is a non-replicating poxvirus vector.

22. The vector of claim 21, wherein the non-replicating poxvirus vector is selected from: a Modified Vaccinia virus Ankara (MVA) vector, a NYVAC vaccinia virus vector, a canarypox (ALVAC) vector, and a fowlpox (FPV) vector.

23. The vector of claim 21 or claim 22, wherein the non-replicating poxvirus vector is an MVA vector.

24. The vector of claim 21 or claim 22, wherein the non-replicating poxvirus vector is a fowlpox vector.

25. The vector of claim 20, wherein the vector is an adenovirus vector.

26. The vector of claim 25, wherein the adenovirus vector is a non-replicating adenovirus vector.

27. The vector of claim 25 or claim 26, wherein the adenovirus vector is selected from: a human adenovirus vector, a simian adenovirus vector, a group B adenovirus vector, a group C adenovirus vector, a group E adenovirus vector, an adenovirus 6 vector, a PanAd3 vector, an adenovirus C3 vector, a ChAdY25 vector, an AdC68 vector, and an Ad5 vector.

28. The vector of claim 20, wherein the vector is a measles virus vector.

29. The vector of claim 28, wherein the measles virus vector is a non-replicating measles virus vector.

30. The vector of any preceding claim, wherein the Hantavirus nucleoprotein comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 4, or an antigenic fragment thereof.

31. The vector of claim 30, wherein the antigenic fragment comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 13.

32. The vector of claim 30 or claim 31, wherein the antigenic fragment comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 11.

33. The vector of any preceding claim, wherein the Hantavirus nucleoprotein comprises an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 7 and 10, or an antigenic fragment thereof.

34. The vector of claim 33, wherein the antigenic fragment comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 14.

35. The vector of any one of claims 30 to 34, wherein the antigenic fragment comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 12.

36. A nucleic acid sequence encoding a viral vector according to any one of claims 1-35.

37. 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 any one of claims 1-35; 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.

38. A host cell comprising the nucleic acid sequence of claim 36.

39. A composition comprising a vector according to any one of claims 1-35, and a pharmaceutically-acceptable carrier.

40. The composition of claim 39, further comprising an adjuvant.

41. A vector according to any one of claims 1-35 or a composition according to claim 39 or claim 40, for use in medicine.

42. A vector according to any one of claims 1-35 or a composition according to claim 27 or claim 28, for use in a method of inducing an immune response in a subject.

43. The vector for use according to claim 42, wherein the immune response comprises a T cell response.

44. A vector according to any one of claims 1-35, or a composition according to claim 39 or claim 40, for use in a method of preventing or treating a Hantavirus infection in a subject.

45. A vector according to claim 44, or a composition according to 44, for use in a method of preventing or treating hemorrhagic fever with renal syndrome in a subject.

Description

FIGURE LEGENDS

[0223] FIG. 1A-B. Example MVA vector construction. FIG. 1A provides a schematic representation of cassette “MVAHantaNP”. FIG. 1B provides a schematic representation of plasmid 17ACNHBP_MVA-SEOV-HNT-NP_pMS-RQ (pMVAHantaNP).

[0224] FIG. 2. PCR confirmation of pure recombinant Nucleoprotein-agarose gel confirming the presence of the MVAHantaNP construct. Flank to flank primers (SEQ ID NOs: 46 and 47) cover the entire insert and run from the MVA flanking regions at either end of the vaccine insert yielding an expected amplification product size. Contents of wells are as follows (numbered left to right): 1. Ladder; 2. Positive control (MVA-HantaNP plasmid) GFP to flank primers—expected size 3260 bp; 3. MVA-HantaNP “Batch 1”; 4. MVA-HantaNP “Batch 2+3”; 5. MVA-HantaNP “Batch 4+5+6”; 6. Negative control; 7. Ladder; 8. Positive control (MVA-HantaNP plasmid) flank to flank primers—expected size 3788 bp; 9. MVA-HantaNP “Batch 1”; 10. MVA-HantaNP “Batch 2+3”; 11. MVA-HantaNP “Batch 4+5+6”; 12. Negative control; 13. Ladder.

[0225] FIG. 3. Western blot confirming expression of the NP/Flag tag. The expected size of the protein is 89 kDa. Contents of wells are as follows (numbered left to right): 1. Ladder; 2. “Passage 3” P3 (1.1.1); 3. P3(4.1.1); 4.P3(4.1.2); 5. Vaccine Batch 1; 6. Vaccine batch 2+3 combined; 7. Vaccine batch 4+5+6 combined.

[0226] FIG. 4. Clinical scores (% daily weight gain) during the immunisation study. (a) Weight and (b) temperature of mice following prime immunisation (first arrow, day 0) and boost immunisation (second arrow, day 14).

[0227] FIG. 5. Total ELISPOT response from vaccinated and unvaccinated mice.

[0228] FIG. 6. Splenocyte IFN-γ ELISPOT re-stimulation responses to individual peptide pools (“NP1”-“NP11” therein). i) Group 1 indicates mice vaccinated with MVA-HantaNP prime and boost; ii) Group 2 indicates mice vaccinated with a single dose of MVA-HantaNP; iii) Group 3 indicates mice vaccinated with empty MVA wild-type prime and boost; and iv) Group 4 indicates PBS controls, prime and boost.

[0229] FIG. 7. IgG response to Hantavirus NP in mouse sera. Absorbance readings provide a readout of antibody binding activity to recombinant Hantavirus NP.

[0230] FIG. 8. Weight and temperature of mice following intramuscular challenge (left column) or intranasal challenge (right column) with Hantavirus.

[0231] FIG. 9. Viral load in the blood, lung, kidney, spleen and liver of mice at (a) day 5 following intramuscular challenge; (b) day 5 following intranasal challenge; (c) day 14 following intranasal challenge.

[0232] FIG. 10. Viral load in the kidney, lung and spleen of mice at day 5 following intranasal challenge. Results relating to immunisation with empty MVA wild-type vector are represented by circles; results relating to immunisation with MVA-HantaNP are represented by triangles.

EXAMPLES

Example 1. Preparation of an Example MVA-NP (Nucleoprotein) Vector

[0233] A cassette for MVAHantaNP (denoted “MVAHantaNP”) was generated by GeneArt (Thermofisher) to contain a P11 promotor, Green fluorescence Protein (GFP) and MH5 promotor followed by a kozak sequence upstream of the NP sequence. The nucleoprotein sequence is a chimeric sequence containing two distinct sequences of Seoul and Hantaan. Downstream is a 24 residue linker sequence followed by a Flagtag epitope and stop codon. A schematic representation of MVAHantaNP is provided in FIG. 1(A).

[0234] The cassette was inserted into an Sfil/Sfil cloning site of plasmid pMS-RQ-Bb to produce plasmid 17ACNHBP_MVA-SEOV-HNT-NP_pMS-RQ (pMVAHantaNP).

[0235] A schematic representation of pMVAHantaNP is provided in FIG. 1(B), and the nucleic acid sequence of pMVAHantaNP is provided in SEQ ID NO: 33.

TABLE-US-00023 (SEQ ID NO: 33) GTTGGTGGTCGCCATGGATGGTGTTATTGTATACTGTCTAAACGCGTTAGTAAAA CATGGCGAGGAAATAAATCATATAAAAAATGATTTCATGATTAAACCATGTTGTG AAAAAGTCAAGAACGTTCACATTGGCGGACAATCTAAAAACAATACAGTGATTG CAGATTTGCCATATATGGATAATGCGGTATCCGATGTATGCAATTCACTGTATAA AAAGAATGTATCAAGAATATCCAGATTTGCTAATTTGATAAAGATAGATGACGA TGACAAGACTCCTACTGGTGTATATAATTATTTTAAACCTAAAGATGCCATTCCT GTTATTATATCCATAGGAAAGGATAGAGATGTTTGTGAACTATTAATCTCATCTG ATAAAGCGTGTGCGTGTATAGAGTTAAATTCATATAAAGTAGCCATTCTTCCCAT GGATGTTTCCTTTTTTACCAAAGGAAATGCATCATTGATTATTCTCCTGTTTGATT TCTCTATCGATGCGGCACCTCTCTTAAGAAGTGTAACCGATAATAATGTTATTAT ATCTAGACACCAGCGTCTACATGACGAGCTTCCGAGTTCCAATTGGTTCAAGTTT TACATAAGTATAAAGTCCGACTATTGTTCTATATTATATATGGTTGTTGATGGATC TGTGATGCATGCAATAGCTGATAATAGAACTTACGCAAATATTAGCAAAAATAT ATTAGACAATACTACAATTAACGATGAGTGTAGATGCTGTTATTTTGAACCACAG ATTAGGATTCTTGATAGAGATGAGATGCTCAATGGATCATCGTGTGATATGAACA GACATTGTATTATGATGAATTTACCTGATGTAGGCGAATTTGGATCTAGTATGTT GGGGAAATATGAACCTGACATGATTAAGATTGCTCTTTCGGTGGCTGGGTACCAG GCGCGCCTTTCATTTTGTTTTTTTCTATGCTATAAATGGTGAGCAAGGGCGAGGA GCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGG CCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCT GACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTC GTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGA AGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCA CCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCG AGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGG ACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCT ATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCC ACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCC CCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTC CGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTT CGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGAGCT CCGGCCCGCTCGAGGCCGCTGGTACCCAACCTAAAAATTGAAAATAAATACAAA GGTTCTTGAGGGTTGTGTTAAATTGAAAGCGAGAAATAATCATAAATAAGCCCG GTGCCACCATGGCCACAATGGAAGAGATCCAGAGAGAGATCAGCGCCCACGAG GGACAGCTGGTTATCGCCAGACAGAAAGTGAAGGACGCCGAGAAGCAGTACGA GAAGGACCCCGACGATCTGAACAAGAGAGCCCTGCACGACAGAGAAAGCGTGG CCGCCTCTATCCAGAGCAAGATCGATGAGCTGAAGAGACAGCTGGCCGACAGAA TCGCCGCTGGCAAGAATATTGGCCAGGACAGAGATCCCACAGGCGTGGAACCTG GCGATCACCTGAAAGAGAGAAGCGCCCTGTCCTATGGCAACACCCTGGACCTGA ACAGCCTGGACATTGATGAGCCTACCGGCCAGACAGCCGACTGGCTGACAATCA TTGTGTACCTGACCAGCTTCGTGGTCCCCATCATCCTGAAGGCCCTGTACATGCT GACCACCAGAGGCAGACAGACCAGCAAGGACAACAAGGGCATGAGAATCCGGT TCAAGGATGACAGCAGCTACGAGGACGTGAACGGCATTAGAAAGCCCAAGCACC TGTACGTGTCCATGCCTAACGCTCAGAGCAGCATGAAGGCCGAGGAAATCACCC CTGGCAGATTCAGAACAGCCGTGTGCGGACTGTACCCCGCTCAGATCAAGGCCA GAAACATGGTGTCCCCAGTGATGAGCGTCGTGGGATTTCTGGCCCTGGCTAAGG ACTGGACCAGCAGGATTGAGGAATGGCTGGGAGCCCCTTGCAAGTTTATGGCCG AGTCTCCTATCGCCGGCAGCCTGTCTGGCAACCCCGTGAATAGAGACTACATCAG ACAGAGGCAGGGCGCTCTGGCCGGAATGGAACCCAAAGAATTTCAGGCCCTGCG GCAGCACTCTAAGGATGCCGGATGTACCCTGGTGGAACACATTGAGAGCCCCAG CAGCATCTGGGTTTTCGCTGGCGCTCCTGATAGATGCCCTCCTACCTGTCTGTTTG TTGGCGGAATGGCCGAGCTGGGCGCCTTCTTTAGCATTCTGCAGGACATGCGGAA TACCATCATGGCCAGCAAGACCGTGGGCACCGCCGATGAGAAGCTGAGAAAGAA GTCCAGCTTCTACCAGAGCTACCTGCGGAGAACCCAGAGCATGGGCATTCAGCT GGACCAGAGAATCATCGTGATGTTCATGGTGGCCTGGGGCAAAGAAGCCGTGGA CAATTTTCACCTGGGCGACGACATGGACCCCGAGCTGAGATCTCTGGCCCAGATC CTGATCGACCAGAAAGTCAAAGAGATCTCCAATCAAGAGCCCATGAAGCTGATG CTGAGCTACGGCAACGTGCTGGATCTGAACCACCTGGATATCGACGAGCCAACA GGACAGACCGCTGATTGGCTGGGCATCGTGATCTACCTGACCTCCTTTGTGGTGC CTATTCTGCTCAAAGCCCTCTATATGCTGACAACACGCGGAAGGCAGACCACCA AAGATAACAAAGGCACCCGGATCAGGTTTAAGGACGACAGCTCCTTTGAGGATG TCAACGGCATCCGGAAACCTAAGCACCTCTATGTGTCTCTGCCCAATGCACAGTC CTCCATGAAGGCAGAAGAGATCACACCAGGCCGGTACAGAACCGCCATCTGTGG ACTGTATCCTGCACAAATCAAAGCCCGGCAGATGATCAGCCCCGTGATGTCCGTT ATCGGATTCCTGGCTCTGGCCAAAGATTGGAGCGACAGGATCGAGCAGTGGCTG AGCGAGCCTTGCAAGCTGCTTCCTGATACAGCCGCTGTGTCACTGCTTGGCGGCC CTGCCACAAACAGAGATTACCTGAGACAGAGACAGGTGGCACTGGGCAACATGG AAACAAAAGAGAGCAAGGCCATCCGGCAGCATGCCGAAGCTGCTGGCTGTAGCA TGATCGAGGATATCGAGTCCCCTAGCTCCATTTGGGTGTTCGCAGGGGCCCCAGA TAGATGTCCACCAACATGCCTGTTCATTGCCGGCATGGCTGAACTGGGAGCTTTT TTCAGCATCCTCCAGGATATGCGCAACACGATTATGGCCTCCAAGACAGTGGGA ACCAGCGAGGAAAAGCTGCGGAAGAAAAGCAGCTTTTACCAGTCTTACCTGAGG CGGACCCAGTCCATGGGGATCCAACTGGATCAGCGGATCATTGTGCTGTTTATGG TCGCTTGGGGAAAAGAGGCTGTCGATAACTTCCACCTGGGAGATGATATGGATC CTGAACTGCGGACCCTGGCTCAGTCCCTGATCGATGTGAAAGTGAAAGAAATTA GTAATCAAGAACCCCTCAAGCTGGACCTGGAAGGCCCTAGATTCGAGGACTACA AGGACGATGACGACAAGTGACTCGACCTGCAGTTTTTATGGAAAGTTTTATAGGT AGTTGATAGAACAAAATACATAATTTTGTAAAAATAAATCACTTTTTATACTAAT ATGACACGATTACCAATACTTTTGTTACTAATATCATTAGTATACGCTACACCTTT TCCTCAGACATCTAAAAAAATAGGTGATGATGCAACTTTATCATGTAATCGAAAT AATACAAATGACTACGTTGTTATGAGTGCTTGGTATAAGGAGCCCAATTCCATTA TTCTTTTAGCTGCTAAAAGCGACGTCTTGTATTTTGATAATTATACCAAGGATAA AATATCTTACGACTCTCCATACGATGATCTAGTTACAACTATCACAATTAAATCA TTGACTGCTAGAGATGCCGGTACTTATGTATGTGCATTCTTTATGACATCGCCTAC AAATGACACTGATAAAGTAGATTATGAAGAATACTCCACAGAGTTGATTGTAAA TACAGATAGTGAATCGACTATAGACATAATACTATCTGGATCTACACATTCACCG GAAACTAGTTG pMVAHantaNP comprises: DelIII Left flank: (SEQ ID NO: 34) GTTGGTGGTCGCCATGGATGGTGTTATTGTATACTGTCTAAACGCGTTAGTAAAA CATGGCGAGGAAATAAATCATATAAAAAATGATTTCATGATTAAACCATGTTGTG AAAAAGTCAAGAACGTTCACATTGGCGGACAATCTAAAAACAATACAGTGATTG CAGATTTGCCATATATGGATAATGCGGTATCCGATGTATGCAATTCACTGTATAA AAAGAATGTATCAAGAATATCCAGATTTGCTAATTTGATAAAGATAGATGACGA TGACAAGACTCCTACTGGTGTATATAATTATTTTAAACCTAAAGATGCCATTCCT GTTATTATATCCATAGGAAAGGATAGAGATGTTTGTGAACTATTAATCTCATCTG ATAAAGCGTGTGCGTGTATAGAGTTAAATTCATATAAAGTAGCCATTCTTCCCAT GGATGTTTCCTTTTTTACCAAAGGAAATGCATCATTGATTATTCTCCTGTTTGATT TCTCTATCGATGCGGCACCTCTCTTAAGAAGTGTAACCGATAATAATGTTATTAT ATCTAGACACCAGCGTCTACATGACGAGCTTCCGAGTTCCAATTGGTTCAAGTTT TACATAAGTATAAAGTCCGACTATTGTTCTATATTATATATGGTTGTTGATGGATC TGTGATGCATGCAATAGCTGATAATAGAACTTACGCAAATATTAGCAAAAATAT ATTAGACAATACTACAATTAACGATGAGTGTAGATGCTGTTATTTTGAACCACAG ATTAGGATTCTTGATAGAGATGAGATGCTCAATGGATCATCGTGTGATATGAACA GACATTGTATTATGATGAATTTACCTGATGTAGGCGAATTTGGATCTAGTATGTT GGGGAAATATGAACCTGACATGATTAAGATTGCTCTTTCGGTGGCTGG First linker: (SEQ ID NO: 35) GTACCAGGCGCGCC p11: (SEQ ID NO: 36) TTTCATTTTGTTTTTTTCTATGCTATAA GFP: (SEQ ID NO: 37) ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAG CTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGC GATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTG CCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCA GCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGA AGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGAC CCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAA GGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAA CTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAA GGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGA CCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAA CCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGA TCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGAC GAGCTGTACAAGTAA Second linker: (SEQ ID NO: 38) GAGCTCCGGCCCGCTCGAGGCCGCTGGTACCCAACCT MH5 promoter: (SEQ ID NO: 39) AAAAATTGAAAATAAATACAAAGGTTCTTGAGGGTTGTGTTAAATTGAAAGCGA GAAATAATCATAAATA Third linker: AGCCCGGT Kozak sequence: (SEQ ID NO: 41) GCCACCATGG. The 3′ end of the Kozak sequence overlaps with the four nucleic acids at the 5′ end of SEQ ID NO: 29. Nucleoprotein (SEQ ID NO: 29) Fourth linker: (SEQ ID NO: 42) GACCTGGAAGGCCCTAGATTCGAG Flag tag: (SEQ ID NO: 43) GACTACAAGGACGATGACGACAAG STOP: TGA Fifth linker: (SEQ ID NO: 44) CTCGACCTGCAGTTTTTATG DelIII Right flank: (SEQ ID NO: 45) GAAAGTTTTATAGGTAGTTGATAGAACAAAATACATAATTTTGTAAAAATAAATC ACTTTTTATACTAATATGACACGATTACCAATACTTTTGTTACTAATATCATTAGT ATACGCTACACCTTTTCCTCAGACATCTAAAAAAATAGGTGATGATGCAACTTTA TCATGTAATCGAAATAATACAAATGACTACGTTGTTATGAGTGCTTGGTATAAGG AGCCCAATTCCATTATTCTTTTAGCTGCTAAAAGCGACGTCTTGTATTTTGATAAT TATACCAAGGATAAAATATCTTACGACTCTCCATACGATGATCTAGTTACAACTA TCACAATTAAATCATTGACTGCTAGAGATGCCGGTACTTATGTATGTGCATTCTTT ATGACATCGCCTACAAATGACACTGATAAAGTAGATTATGAAGAATACTCCACA GAGTTGATTGTAAATACAGATAGTGAATCGACTATAGACATAATACTATCTGGAT CTACACATTCACCGGAAACTAGTTG

[0236] The plasmid DNA was purified from transformed bacteria (E. coli K12 DH10B™ T1R) and concentration determined by UV spectroscopy by GeneArt (Thermofisher).

[0237] BHK-21 cells were infected with MVA 1974 at a multiplicity of infection of 0.05. Infected cells were transfected with pMVAHantaNP using lipofectamine (Life Technologies) as directed by the manufacturer. The resulting recombinant MVAHantaNP was serially plaque-purified 4 times in Chick Embryo Fibroblast (“CEF”) cells, based on GFP expression. MVAHantaNP was amplified on CEF cells, purified by sucrose cushion centrifugation and titrated by plaque assay on CEF cells prior to in vivo use. Plaques were visualised using GFP fluorescence and by immunostaining with rabbit anti-vaccinia antibody (AbD Serotec, UK) and Vectastain Universal ABC-AP kit (Vector laboratories, USA). Genomic DNA from infected cells was extracted using Wizard SV genomic DNA purification system (Promega, USA) and used as a template in PCR with KAPA2G Fast HotStart PCR Kit (KAPABiosystems, USA) for genotype analysis.

[0238] Polymerase chain reaction (PCR) confirmed presence of the MVAHantaNP construct. One set of primers was designed specifically to check for the Hanta NP to the MVA flanking region with an expected size of 3260 bp—this is shown in FIG. 2.

[0239] Sequencing of the expressed protein confirmed very high sequence fidelity. Recombinant purified MVAHantaNP was then bulked-up in stages, by tissue culture into increasing sized flasks. Initially the MVAHantaNP was grown in a small flask of Chicken Embryo Fibroblast (CEF) cells and harvested before infecting into a slightly larger flask of CEF cells. This process was repeated into increasingly larger flasks until the MVAHantaNP successfully infected 10× large flasks of CEF cells. Sucrose cushion centrifugation was performed; viral pellets were re-suspended in PBS ready for immunogenicity studies. A total of six batches was produced. Batches 2+3 and 4+5+6 were pooled into single samples and titrated for viral concentration.

[0240] The purified vaccine batches align to a positive control (the original received plasmid from Geneart). A second set of primers were designed to identify the entire insert, from both MVA flanking regions. The results indicate presence of pure recombinant MVA (MVA containing the insert) in all vaccine batches. Again the original plasmid was used as a positive control and all vaccine batches have the same expected size product as the positive control.

[0241] Primer details are as follows:

TABLE-US-00024 SEQ ID NO: 46: CGGCACCTCTCTTAAGAAGT (Fwd targets Del III Left flank) SEQ ID NO: 47: GTGTAGCGTATACTAATGATATTAG (Rev targets Del III Right flank) SEQ ID NO: 48: GGAGTACAACTACAACAGCCACAACG (Fwd targets GFP)

[0242] The GFP Fwd primer binds to the GFP sequence and, when used in combination with the Rev Del III Right flank primer, covers the GFP through the nucleoprotein to the right MVA flank, and specifically identifies presence of the NP gene.

[0243] Detection of Protein Expression

[0244] CEF cells were infected with MVAHantaNP at a multiplicity of infection of 0.05 and incubated at 37° C. in Modified Eagle Medium (MEM) supplemented with 2% FBS (Sigma-Aldrich. UK). The medium was removed after 48 hours once good GFP fluorescence and CPE was observed microscopically. Cells were lysed with 1×LDS Nupage® reducing sample buffer (Nupage® LDS sample buffer containing 1× Nupage® sample reducing buffer) (Thermofisher, UK), transferred to Eppendorf tubes and heated at 70° C. for 10 minutes. Uninfected cells were treated in the same manner as a negative control. MVAHantaNP lysates were subjected to SDS-PAGE on a 4-12% Bis-Tris gel (Life technologies) and proteins transferred to a nitrocellulose membrane. The nitrocellulose membrane was blocked using 5% milk powder (Merck Millipore), then incubated in the presence of a primary antibody (Rabbit anti-V5 polyclonal (Invitrogen) at 1/1000 in PBS-0.05% Tween) for 1-2 hours rocking, before washing in PBS containing 0.05% Tween-20 (Sigma-Aldrich) 3 times. Membranes were incubated in the presence of a HRP-conjugated secondary antibody (anti-rabbit IgG peroxidase (Sigma-Aldrich) at 1/1000 in PBS-0.05% Tween) for 1 hour rocking and washed as before. Protein expression was determined by detection of bound antibody using Pierce ECL WB substrate kit (Thermofisher) according to the manufacturer's instructions and visualised in a Chemi-Illuminescent Imager (Syngene). Molecular weights were determined using molecular ladder MagicMark XP Western Protein Standard (Invitrogen) as a reference.

[0245] Western blot analysis (see FIG. 3) confirms expression of the flag tag located downstream from the NP. The expected size of the protein (the NP+linker and flag tag) is 89 kDa and the protein sequence is provided in SEQ ID NO: 49. Expression is observed from the passage 3 picks through to the vaccine batches (the inventors observed low level protein degradation, which is not believed to be significant). The band of interest is located at the expected size of the protein which again suggests good expression.

TABLE-US-00025 (SEQ ID NO: 49) MATMEEIQREISAHEGQLVIARQKVKDAEKQYEKDPDDLNKRALHDRESV AASIQSKIDELKRQLADRIAAGKNIGQDRDPTGVEPGDHLKERSALSYGN TLDLNSLDIDEPTGQTADWLTIIVYLTSFVVPIILKALYMLTTRGRQTSK DNKGMRIRFKDDSSYEDVNGIRKPKHLYVSMPNAQSSMKAEEITPGRFRT AVCGLYPAQIKARNMVSPVMSVVGFLALAKDWTSRIEEWLGAPCKFMAES PIAGSLSGNPVNRDYIRQRQGALAGMEPKEFQALRQHSKDAGCTLVEHIE SPSSIWVFAGAPDRCPPTCLFVGGMAELGAFFSILQDMRNTIMASKTVGT ADEKLRKKSSFYQSYLRRTQSMGIQLDQRIIVMFMVAWGKEAVDNFHLGD DMDPELRSLAQILIDQKVKEISNQEPMKLMLSYGNVLDLNHLDIDEPTGQ TADWLGIVIYLTSFVVPILLKALYMLTTRGRQTTKDNKGTRIRFKDDSSE EDVNGIRKPKHLYVSLPNAQSSMKAEEITPGRYRTAICGLYPAQIKARQM ISPVMSVIGFLALAKDWSDRIEQWLSEPCKLLPDTAAVSLLGGPATNRDY LRQRQVALGNMETKESKAIRQHAEAAGCSMIEDIESPSSIWVFAGAPDRC PPTCLFIAGMAELGAFFSILQDMRNTIMASKTVGTSEEKLRKKSSFYQSY LRRTQSMGIQLDQRIIVLFMVAWGKEAVDNFHLGDDMDPELRTLAQSLID VKVKEISNQEPLKLDLEGPRFEDYKDDDDK

[0246] The amino acid sequence of SEQ ID NO 49 corresponds to the amino acid sequence of SEQ ID NO: 31 plus the expressed fourth linker and flag tag.

Example 2. MVAHantaNP Immunogenicity in A129 Mice

[0247] 80 male 6-8 week old A129 mice were randomly divided into 4 groups and ear tagged prior to vaccinations.

[0248] Group 1 received a two dose vaccination of MVAHantaNP in endotoxin free phosphate buffered saline (PBS) at 1×10.sup.7 pfu per animal on days 0 and 14.

[0249] Group 2 received a single vaccine shot of MVAHantaNP in endotoxin free PBS at 1×10.sup.7 plaque forming units (pfu) per animal on day 14.

[0250] Group 3 received a two dose vaccination of MVA empty vector in endotoxin free PBS at 1×10.sup.7 pfu per animal on days 0 and 14.

[0251] Group 4 received a two dose vaccination of endotoxin free PBS as a negative control on days 0 and 14.

[0252] All mice were injected intramuscularly into the caudal thigh. 100 μl was administered at each vaccination (50 μl into each thigh). Animal weights were recorded daily throughout the study. 5 animals were euthanised from each group and spleen tissue and blood collected on day 28 after the primary vaccination. All efforts were made to minimise animal suffering. These studies were approved by the ethical review process of PHE, Porton Down, UK and the Home Office, UK via project license number 30/2993. Work was performed in accordance with the Animals (Scientific procedures) Act 1986 and the Home Office (UK) Code of Practice for the Housing and Care of Animals Used in Scientific Procedures (1989).

[0253] Throughout the study, no clinical signs were observed with regards to the vaccinations and all mice gained weight as expected (see FIG. 4a). All four groups gained weight throughout the study as expected, and group 4 body weights were consistently lower than groups 1-3. By the end of the study all the groups observed a similar % weight gain. These clinical data demonstrate that the mice tolerated the vaccine without adverse effects.

[0254] To determine the T-cell responses in immunised animals, an interferon-gamma ELISPOT assay was used to measure frequencies of responsive T-cells after stimulation with Hantavirus specific peptides.

[0255] Spleens from test animals were collected aseptically, homogenised, and red blood cells lysed. Splenocytes were resuspended in RPMI medium (Sigma-Aldrich) supplemented with 5% FBS, 2 mM L-Glutamine, 100 U penicillin & 0.1 mg/ml streptomycin, 50 mM 2-mercaptoethanol and 25 mM HEPES solution (Sigma-Aldrich). Splenocytes were assessed for antigen recall response via IFN-γ ELISPOT (Mabtech, Sweden), performed as per the manufacturer's instructions. Cells were seeded in PVDF microtitre plates at 2×10e6 per well and re-stimulated with peptide pools (JPT, Berlin).

[0256] Peptides spanning the Hanta NP protein sequence were 15 residues long, with an overlap of 11 residues between peptides. 189 peptides were produced in total that were tested in eleven peptide pools (see Table 1).

TABLE-US-00026 TABLE 1 Peptide pools (start amino acid (“AA”) numbering corresponds to the amino acid numbering in SEQ ID NO: 31) SEQ ID NO Start AA Sequence Pool number 50 1 MATMEEIQREISAHE 1 51 5 EEIQREISAHEGQLV 52 9 REISAHEGQLVIARQ 53 13 AHEGQLVIARQKVKD 54 17 QLVIARQKVKDAEKQ 55 21 ARQKVKDAEKQYEKD 56 25 VKDAEKQYEKDPDDL 57 29 EKQYEKDPDDLNKRA 58 33 EKDPDDLNKRALHDR 59 37 DDLNKRALHDRESVA 60 41 KRALHDRESVAASIQ 61 45 HDRESVAASIQSKID 62 49 SVAASIQSKIDELKR 63 53 SIQSKIDELKRQLAD 64 57 KIDELKRQLADRIAA 65 61 LKRQLADRIAAGKNI 66 65 LADRIAAGKNIGQDR 67 69 IAAGKNIGQDRDPTG 2 68 73 KNIGQDRDPTGVEPG 69 77 QDRDPTGVEPGDHLK 70 81 PTGVEPGDHLKERSA 71 85 EPGDHLKERSALSYG 72 89 HLKERSALSYGNTLD 73 93 RSALSYGNTLDLNSL 74 97 SYGNTLDLNSLDIDE 75 101 TLDLNSLDIDEPTGQ 76 105 NSLDIDEPTGQTADW 77 109 IDEPTGQTADWLTII 78 113 TGQTADWLTIIVYLT 79 117 ADWLTIINYLTSEVV 80 121 TIIVYLTSFVVPIIL 81 125 YLTSFVVPIILKALY 82 129 FVVPIILKALYMLTT 83 133 IILKALYMLTTRGRQ 84 137 ALYMLTTRGRQTSKD 3 85 141 LTTRGRQTSKDNKGM 86 145 GRQTSKDNKGMRIRF 87 149 SKDNKGMRIRFKDDS 88 153 KGMRIRFKDDSSYED 89 157 IRFKDDSSYEDVNGI 90 161 DDSSYEDVNGIRKPK 91 165 YEDVNGIRKPKHLYV 92 169 NGIRKPKHLYVSMPN 93 173 KPKHLYVSMPNAQSS 94 177 LYVSMPNAQSSMKAE 95 181 MPNAQSSMKAEEITP 96 185 QSSMKAEEITPGRFR 97 189 KAEEITPGRFRTAVC 98 193 ITPGRFRIAVCGLYP 99 197 RFRTAVCGLYPAQIK 100 201 AVCGLYPAQIKARNM 101 205 LYPAQIKARNMVSPV 4 102 209 QIKARNMVSPVMSVV 103 213 RNMVSPVMSVVGFLA 13 217 SPVMSVVGFLALAKD 104 221 SVVGFLALAKDWTSR 105 225 FLALAKDWTSRIEEW 106 229 AKDWTSRIEEWLGAP 107 233 TSRIEEWLGAPCKFM 108 237 EEWLGAPCKFMAESP 109 241 GAPCKFMAESPIAGS 110 245 KFMAESPIAGSLSGN 111 249 ESPIAGSLSGNPVNR 112 253 AGSLSGNPVNRDYIR 113 257 SGNPVNRDYIRQRQG 114 261 VNRDYIRQRQGALAG 115 265 YIRQRQGALAGMEPK 116 269 RQGALAGMEPKEFQA 117 273 LAGMEPKEFQALRQH 5 118 277 EPKEFQALRQHSKDA 119 281 FQALRQHSKDAGCTL 120 285 RQHSKDAGCTLVEHI 121 289 KDAGCTLVEHIESPS 122 293 CTLVEHIESPSSIWV 123 297 EHIESPSSIWVFAGA 124 301 SPSSIWVFAGAPDRC 125 305 IWVFAGAPDRCPPTC 126 309 AGAPDRCPPTCLFVG 127 313 DRCPPTCLFVGGMAF 128 317 PTCLEVGGMAELGAF 179 321 FVGGMAELGAFFSIL 130 325 MAELGAFFSILQDMR 131 329 GAFFSILQDMRNTIM 132 333 SILQDMRNTIMASKT 133 337 DMRNTIMASKTVGTA 134 341 TIMASKTVGTADEKL 6 135 345 SKTVGTADEKLRKKS 136 349 GTADEKLRKKSSFYQ 137 353 EKLRKKSSFYQSYLR 138 357 KKSSFYQSYLRRTQS 139 361 FYQSYLRRTQSMGIQ 140 365 YLRRTQSMGIQLDQR 141 369 TQSMGIQLDQRIIVM 142 373 GIQLDQRIIVMFMVA 143 377 DQRIIVMFMVAWGKE 144 381 IVMFMVAWGKEAVDN 145 385 MVAWGKEAVDNFHLG 146 389 GKEAVDNFHLGDDMD 147 393 VDNFHLGDDMDPELR 148 397 HLGDDMDPELRSLAQ 149 401 DMDPELRSLAQILID 150 405 ELRSLAQILIDQKVK 151 409 LAQILIDQKVKEISN 7 152 413 LIDQKKVEISNQEPM 153 417 KNKEISNQEMIKLML 154 421 ISNQEPMKLMLSYGN 155 425 EPMKLMLSYGNVLDL 156 429 LMLSYGNVLDLNHLD 157 433 YGNYLDLNHLDIDEP 158 437 LDLNHLDIDEPTGQI 159 441 HIDIDEPTGQTADWL 160 445 DEPTGQTADWLGIVI 161 449 GQTADWLGIVIYLTS 162 453 DWLGIVIYLTSFVVP 163 457 IVIYLTSFVVPILLK 164 461 LTSFVVPILLKALYM 165 465 VVPILLKALYMLTTR 166 469 LLKALYMLTTRGRQT 167 473 LYMLTTRGRQTTKDN 168 477 TTRGRQTTKDNKGTR 8 169 481 RQTTKDNKGTRIRFK 170 485 KDNKGTRIRFKDDSS 171 489 GTRIRFKDDSSFEDV 172 493 RFKDDSSFEDVNGIR 173 497 DSSFEDVNGIRKPKH 174 501 EDVNGIRKPKHLYVS 175 505 GIRKPKHLYVSLPNA 176 509 PKHLYVSLPNAQSSM 177 513 YVSLPNAQSSMKAEE 178 517 PNAQSSMKAEEITPG 179 521 SSMKAEEITPGRYRT 180 525 AEEITPGRYRTAICG 181 529 TPGRYRTAICGLYPA 182 533 YRTAICGLYPAQIKA 183 537 ICGLYPAQIKARQMI 184 541 YPAQIKARQMISPVM 185 545 IKARQMISPVMSVIG 9 186 549 QMISPVMSVIGFLAL 14 553 PVMSVIGFLALAKDW 187 557 VIGFLALAKDWSDRI 188 561 LALAKDWSDRIEQWL 189 565 KDWSDRIEQWLSEPC 190 569 DRIEQWLSEPCKLLP 191 573 QWLSEPCKLLPDTAA 192 577 EPCKLLPDTAAVSLL 193 581 LLPDTAAVSLLGGPA 194 585 TAAVSLLGGPATNRD 195 589 SLLGGPATNRDYLRQ 196 593 GPATNRDYLRQRQVA 197 597 NRDYLRQRQVALGNM 198 601 LRQRQVALGNMETKE 199 605 QVALGNMETKESKAI 200 609 GNMETKESKAIRQHA 201 613 TKESKAIRQHAEAAG 10 202 617 KAIRQHAEAAGCSMI 203 621 QHAEAAGCSMIEDIE 204 625 AAGCSMIEDIESPSS 205 629 SMIEDIESPSSIWVF 206 633 DIESPSSIWVFAGAP 207 637 PSSIWVFAGAPDRCP 208 641 WVFAGAPDRCPPTCL 209 645 GAPDRCPPTCLFIAG 210 649 RCPPICLFIAGMAEL 211 653 TCLFIAGMAELGAFF 212 657 IAGMAELGAFFSILQ 213 661 AELGAFFSILQDMRN 214 665 AFFSILQDMRNTIMA 215 669 ILQDMRNTIMASKTV 216 673 MRNTIMASKTVGTSE 217 677 IMASKTVGTSEEKLR 218 681 KTVGTSEEKLRKKSS 11 719 685 TSEEKLRKKSSFYQS 770 689 KLRKKSSFYQSYLRR 221 693 KSSFYQSYLRRTQSM 222 697 YQSYLRRTQSMGIQL 223 701 LRRTQSMGIQLDQRI 224 705 QSMGIQLDQRIIVLF 225 709 IQLDQRIIVLFMVAW 226 713 QRIIVLFMVAWGKEA 227 717 VLFMVAWGKEAVDNF 228 721 VAWGKEAVDNFHLGD 229 725 KEAVDNFHLGDDMDP 230 729 DNFHLGDDMDPELRT 231 733 LGDDMDPELRTLAQS 232 737 MDPELRTLAQSLIDV 233 741 LRTLAQSLIDVKVKE 234 745 AQSLIDVKVKEISNQ 235 749 IDVKVKEISNQEPLK 40 753 DVKVKEISNQEPLKL,

[0257] They were applied to cells at a final concentration of 2.5 μg/ml per peptide, with 17 peptides in each of pools 1 to 10, and with 19 peptides in pool 11. Plates were developed after 18 hours at 37° C., 500 CO.sub.2 in a humidified incubator. Spots were counted visually on an automated ELISPOT reader (Cellular Technologies Limited, USA). Background values from wells containing cells and medium but no peptides were subtracted and data presented as response to individual pools or summed across the target protein. Results were expressed as spot forming units (SFU) per 10.sup.6 cells.

[0258] The MVA-WT group and PBS group (groups 3&4) were negative when stimulated with all Hanta NP pools. In the prime/boost and prime groups, an IFN-γ response was detected to several peptide pools, and a particularly strong response was directed to 2 distinct regions of the NP (corresponding to pools 4 and 9).

[0259] The inventors found that T-cell (IFN-γ) stimulation increased greatly in respect of SEQ ID NOs: 11 and 12.

[0260] Increased responses were also detected against pools 2, 3, 5, 7, 8 and 10 for the prime/boost and prime groups compared to the control groups. Total ELISPOT responses from vaccinated and unvaccinated mice are provided in FIG. 5; and FIG. 6 shows ELISPOT responses to individual peptide pools.

[0261] To measure the antibody responses in immunised mice, ELISA analysis was undertaken to assess binding of antibodies to Hantavirus specific protein. Recombinant Hanta NP as a crude lysate (Native Antigen Company, UK) was diluted in 0.2M carbonate-bicarbonate buffer pH 9.4 (Thermo Scientific) and used to coat Maxisorp 96-well plates (Nunc, Denmark) at 10 μg/ml in 100 μl. Plates were incubated at 4° C. overnight, then washed with PBS+0.01% Tween-20 (Sigma-Aldrich) and blocked with 100 μl of 5% Milk powder (Merck, Millipore) in PBS+0.01% Tween-20 at 37° C. for 1 hour, before re-washing in PBS+0.01% Tween-20. Samples were diluted 1:50 in 5% milk powder in PBS+0.01% Tween-20 buffer, added to the plates in triplicate (100 μl per well) and incubated at 37° C. for 1 hour. Normal mouse serum (Sigma-Aldrich) and a polyclonal Anti-Hantavirus hyper immune mouse ascetic fluid sample (BEI Resources, USA) were used as positive and negative control samples respectively. Plates were washed with PBS+0.01% Tween-20 and 100 μl of a polyclonal anti-mouse HRP conjugate (Sigma-Aldrich) at a 1:20,000 dilution in 5% milk PBS+0.01% Tween-20 was added to each well. Following a further 1 hour incubation at 37° C., plates were washed with PBS+0.01% Tween-20 and 100 μl of TMB substrate (Surmodics) added to each well then incubated at 20° C. for 1 hour. The reaction was stopped by addition of 100 μl of Stop solution (Surmodics) prepared according to the manufacturer's instructions and plates read at 450 nm using a molecular devices plate reader and Softmax Pro version 5.2 software (Molecular Devices). Background absorbance values were subtracted from the sample values and results reported as Absorbance (450 nm) at a 1:50 dilution. Data was illustrated and analysed using Graph Pad Prism 7 (see FIG. 7).

[0262] The MVA-WT and the PBS control groups showed very little absorbance with values similar to those in the blank wells. The response of all mice in both the prime and the prime/boost vaccinated groups were markedly higher. The prime only group recorded an average absorbance of ˜2.3 and the prime/boost an average OD of ˜1.5.

[0263] Therefore, vector of the invention demonstrates highly desirable induction of cellular and humoral immune responses.

Example 3. Efficacy Testing

[0264] 60 male A129 mice at a weight of 19-21 g were previously randomly divided into 4 groups prior to ear tagging and microchipping for identification, weight monitoring and temperature monitoring.

[0265] The remaining mice that were not culled on Day 28 for immunogenicity studies were challenged with Hanta SEOV on Day 28. From each group, n=10 animals were challenged via the intranasal route and n=5 animals were challenged via intramuscular route at 1.36×10.sup.6 TCID50/dose.

[0266] Intramuscularly challenged animals were euthanised at day 33. Intranasally challenged mice were euthanised at day 33 (5 per group) or day 42 (5 per group). Blood, saliva, liver, kidney, lung and spleen were collected for histology and viral burden analysis. All efforts were made to minimise animal suffering. These studies were approved by the ethical review process of PHE, Porton Down, UK and the Home Office, UK via project license number 30/2993. Work was performed in accordance with the Animals (Scientific procedures) Act 1986 and the Home Office (UK) Code of Practice for the Housing and Care of Animals Used in Scientific Procedures (1989).

[0267] Clinical Signs:

[0268] Animal weights and temperatures were recorded daily throughout the study. All challenged animals remained healthy, and no clinical signs were observed following challenge with Hantavirus. Temperature and bodyweight throughout the study are reported in FIG. 8.

[0269] Viral Loads:

[0270] Viral load was assessed at 5- and 14-days post-challenge. As shown in FIG. 9, at day 5, immunisation with MVAHantaNP had achieved a reduction or complete clearance of Hantavirus from tested tissues. The highly advantageous reduction in viral load was also observed in most tissues at 14 days post-challenge.

[0271] Viral Loads—Follow-Up Study:

[0272] In a follow-up study, 28 female A129 mice were previously randomly divided into two groups prior to ear tagging and microchipping for identification, weight monitoring and temperature monitoring.

[0273] Of these 28 mice, 16 were primed with GLP-grade MVAHantaNP at Day 0 followed by a boost immunisation at Day 14 (“Group A”); and 12 mice received prime and boost immunisations with empty MVA wild-type vector at Days 0 and 14, respectively (“Group B”). Immunisations were performed according to Example 2, above.

[0274] At Day 28, 8 of the Group A mice and 8 of the Group B mice were challenged intranasally with Hanta SEOV, at a dose of 3×10.sup.6 TCID50/mouse.

[0275] In this follow-study, viral load was assessed at 5-days post challenge. As shown in FIG. 10, immunisation with MVAHantaNP achieved advantageous reduction of Hantavirus from tested tissues, even when the challenge dose was more than doubled.

Example 4: Preparation of an Example Adenovirus Vector

[0276] A non-replicating adenovirus is engineered to express Hantavirus NP nucleic acid of the invention or a fragment thereof. The genetic sequence for the Hantavirus NP is inserted into the genome of the adenovirus vector. Expression of the Hantavirus NP is indicated by reactivity between a NP-specific antibody and products from the adenovirus by Western blotting or ELISA as follows:

[0277] Cellular lysate of cells infected with the recombinant adenovirus, subjected to SDS-PAGE and Western blotting with an antibody specific for the Hanta virus NP, show a specific reactivity compared to negative controls.

[0278] Alternatively, products from cells infected with the recombinant adenovirus are used to coat an ELISA plate. Hanta virus-specific antibodies bind to the coating and are detected via a chemical reaction.

Example 5: Hanta Virus Vaccine Provides Cross-Strain Protection

[0279] A vaccine expressing Hanta virus NP nucleic acid of the invention or a 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 Hanta virus. They are challenged with a lethal dose of Hanta 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 Hanta virus, but did not receive the vaccine, show severe signs of illness, reach humane clinical endpoints and require euthanasia.

Example 6. Preparation and Efficacy of a Recombinant Influenza Virus Vector

[0280] Reverse genetics are used to construct a recombinant influenza virus that carries a protective epitope of Hanta virus NP in the neuraminidase stalk. Hanta virus-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 Hanta virus.

Example 7. Preparation and Efficacy of a Recombinant Bacterial Vector

[0281] Hanta virus NP nucleic acid of the invention or a 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 Hanta virus-specific immune response is induced. These immune responses provide a reduction in viral load and clinical illness after challenge with Hanta virus.