Hantavirus antigen composition

Abstract

The present invention provides a viral vector or a bacterial vector comprising a nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof; wherein the vector is capable of inducing an immune response in a subject. The invention also provides the use of the composition and the carrier in a method of drug therapy.

Description

[0001] This application is a divisional application with a filing date of 2020-07-29 and application number 202080054858X.

[0002] The present invention relates to viral vectors and bacterial vectors containing Hantavirus antigens and their use in immunogenic and antigenic compositions. The present invention also relates to prophylactic uses of the compositions. The present invention also relates to immunogens for producing therapeutic antibodies and methods for producing the immunogens.

[0003] Hantavirus is an emerging zoonotic virus with widespread distribution. Many strains of hantavirus exist, broadly divided into three serogroups. Hantavirus is the causative agent of Hantavirus Pulmonary Syndrome (HPS), a severe respiratory disease in humans that is typically fatal in 36% of cases, with mortality rates of 50% recorded during some outbreaks. It is also the causative agent of hemorrhagic fever with renal syndrome (HFRS), a group of clinically similar illnesses that can be fatal in up to 15% of cases. Hantaviruses are typically transmitted to humans through exposure to aerosolized body fluids or feces of infected small mammals, usually rodents. Human-to-human transmission has also been reported.

[0004] According to the Centers for Disease Control and Prevention (CDC), symptoms associated with hantavirus infection include fever, headache, muscle pain, and severe difficulty breathing. Symptoms associated with HPS can also include fatigue, chills, dizziness, nonproductive cough, nausea, vomiting, and other gastrointestinal symptoms, as well as malaise, diarrhea, mild headache, joint pain, back pain, and abdominal pain. Symptoms associated with HFRS include severe headache, back and abdominal pain, fever, chills, nausea, blurred vision, flushed face, irritated or red eyes, and rash. Late symptoms of HFRS can include low blood pressure, acute shock, vascular leak, and acute kidney failure, which can lead to severe fluid overload.

[0005] There is currently no licensed vaccine for hantavirus. According to the CDC, there is no specific treatment or cure for hantavirus infection, HPS, or HFRS. Patients with HPS are admitted to the intensive care unit and treated with intubation and oxygen therapy to help the patient during severe respiratory distress. The success of HPS treatment depends on the severity of the respiratory distress and early detection of infection. Treatment of HFRS may involve managing the patient's fluid and electrolyte levels, oxygen and blood pressure levels, dialysis to correct severe fluid overload, and treating any secondary infections. The antiviral drug ribavirin has been shown to reduce illness and death if used very early in the clinical illness course of HFRS. However, no benefit of ribavirin has been found for patients with HPS.

[0006] Therefore, there is a great need for a protective vaccine against Hantavirus infection. There is also an urgent need for other therapeutic agents for the prevention, treatment and suppression of Hantavirus infection.

[0007] The present invention solves one or more of the above problems by providing viral vectors and bacterial vectors encoding Hantavirus nucleoprotein (NP) or antigenic fragments thereof, as well as corresponding compositions and uses of the vectors and compositions in preventing and treating Hantavirus infection.

[0008] The vectors and compositions of the present invention are capable of stimulating (ie, inducing) an immune response against Hantavirus in an individual (ie, subject) and provide improved immunogenicity and efficacy.

[0009] In one aspect, the present invention provides a viral vector or a bacterial vector comprising a nucleic acid sequence encoding a Hantavirus NP or an antigenic fragment thereof; wherein the vector is capable of inducing an immune response in an individual. The inventors have discovered that by using a viral vector or a bacterial vector to deliver a nucleic acid sequence encoding a Hantavirus NP (or an antigenic fragment thereof) to a subject, an efficient immune response against a Hantavirus can be generated in an individual.

[0010] In a preferred embodiment, the vector of the present invention is a viral vector.

[0011] The genus Hantavirus is an enveloped, single-stranded, three-segmented, negative-sense RNA virus genus belonging to the family Bunyaviridae. More than 20 strains of Hantavirus pathogenic to humans have been described, each of which is adapted to a single rodent species. Hantavirus strains are broadly classified as Old World or New World. Old World strains include Seoul virus ("SEOV", distributed worldwide), Puumala virus (mainly distributed in Europe), Hantaan virus ("HNT", mainly distributed in Asia) and Dobrava virus (mainly distributed in Europe), and are often associated with causing HFRS. New World strains include Sin Nombre virus (mainly distributed in North America) and Andes virus (mainly distributed in Latin America), and are often associated with HPS.

[0012] The Hantavirus genome consists of three single-stranded RNA segments, called small (S), medium (M), and large (L). The S segment is between 1 and 3 kb and encodes the nucleocapsid protein (NP). The M segment is between 3.2 and 4.9 kb and encodes the glycoprotein (GP), Gn, and Gc. The L segment is between 6.8 and 12 kb and encodes the viral RNA-dependent RNA polymerase.

[0013] Hantavirus glycoproteins Gn and Gc play an important role in target cell infection through interaction with specific entry receptors (e.g., integrins). Hantavirus NP forms a ribonucleoprotein complex with the viral polymerase and plays multiple roles in viral proliferation. NP has also been reported to play a role in enhancing host cell translation of viral RNA, downregulating apoptosis, inhibiting interferon signaling responses, and blocking TNFα-induced NF-κB activation.

[0014] Seoul virus can be used as a reference hantavirus strain. GenBank Accession No. KM948598.1 provides a reference nucleic acid sequence of Hantavirus NP (see SEQ ID NO: 1) and a reference polypeptide sequence of Hantavirus NP (SEQ ID NO: 4).

[0015] TAGTAGTAGGCTCCCTAAAGAGCTACTACACTAACAAGGAAAATGGCAACT

[0016] ATGGAAGAAATCCAGAGAGAAATCAGTGCGCACGAGGGGCAGCTTGTAAT

[0017] AGCACGCCAGAAGGTCAAGGATGCAGAAAAGCAGTATGAGAAGGATCCTG

[0018] ATGACCTAAATAAGAGGGCACTGCATGATCGGGAGAGTGTCGCAGCTTCAA

[0019] TACAATCAAAAATTGATGAATTGAAGCGCCAACTTGCTGACAGGATTGCAG

[0020] CAGGGAAGAACATCGGGCAAGACCGGGATCCTACAGGGGTAGAGCCGGGT

[0021] GATCATCTCAAGGAAAGATCAGCACTAAGCTACGGGAATACACTGGACCTG

[0022] AATAGCCTTGACATTGATGAACCTACAGGACAGACAGCTGATTGGTTGACC

[0023] ATAATTGTCTATTTGACATCATTCGTGGTCCCGATCATCTTGAAGGCACTGTA

[0024] CATGTTGACAACAAGAGGCAGGCAGACTTCAAAGGACAACAAGGGAATG

[0025] AGGATCAGATTCAAGGATGACAGCTCATATGAAGATGTCAATGGAATCAGA

[0026] AAGCCCAAACATCTGTATGTGTCAATGCCAAACGCCCAATCAAGCATGAAG

[0027] GCTGAAGAGATAACACCTGGAAGATTCCGCACTGCAGTATGTGGGCTATAC

[0028] CCTGCACAGATAAAGGCAAGGAACATGGTAAGCCCTGTCATGAGTGTAGTT

[0029] GGGTTTTTGGCACTGGCAAAAGACTGGACATCTAGAATTGAAGAATGGCTT

[0030] GGTGCACCCTGCAAGTTCATGGCAGAGTCTCCCATTGCCGGGAGCTTATCT

[0031] GGGAATCCTGTGAATCGTGATTATATCAGACAGAGACAAGGTGCACTTGCA

[0032] GGGATGGAGCCAAAAGAATTTCAAGCTCTCAGGCAACATTCAAAGGATGC

[0033] TGGATGTACACTGGTTGAACATATTGAGTCACCATCATCAATATGGGTATTT

[0034] GCTGGGGCCCCTGATAGGTGCCCACCGACATGCCTGTTTGTTGGAGGGATG

[0035] GCTGAGTTAGGTGCTTTCTTTTCTATACTTCAGGATATGAGGAACACAATCA

[0036] TGGCTTCAAAGACTGTGGGAACAGCTGATGAAAAGCTTCGAAAGAAGTCA

[0037] TCATTCTATCAATCATACCTCAGACGCACACAATCAATGGGAATACAACTGG

[0038] ACCAGAGGATAATTGTTATGTTTATGGTTGCCTGGGGAAAGGAGGCAGTGG

[0039] ACAACTTTCATCTCGGTGATGACATGGATCCAGAGCTTCGCAGCCTGGCTC

[0040] AGATCCTGATTGACCAGAAAGTGAAGGAAATCTCAAACCAGGAACCTATG

[0041] AAATTATAAGTACATAATTATGTAATCCATACTAACTATAGGTTAAGAAATAC

[0042] TAATCATTAGTTAATAAGAATATAGATTTATTGAAATAATCATATTAAATAATTA

[0043] GGTAAGTTAACTATTAGTTAGTTAAGTTAGCTAATTGATTTATATGATTATCAC

[0044] AATTGAATGTAATCATAAGCACAATCACTGCCATGTATAATCACGGGTATAC

[0045] GGTGGTTTTCATATGGGGAACAGGGTGGGCTTAGGGCCAGGTCACCTTAA

[0046] GTGACCTTTTTTGTATATATGGATGTAGATTTCAATTGATCGAGTACTAATCCT

[0047] ACTGTTCTCTTTTCCTTTCCTTTCTCCTTCTTTACTAACAACAACAAACTACC

[0048] TCACAACCTTCTACCTCAACACATACTACCTCATTCAGTTGTTTCCTTTTGTCTTTTTAGGGAGCATACTACTA(SEQ ID NO:1)。

[0049] The coding sequence of SEQ ID NO:1 corresponds to nucleic acid residues 43 - 1332 therein and is represented by SEQ ID NO:2:

[0050] ATGGCAACTATGGAAGAAATCCAGAGAGAAATCAGTGCGCACGAGGGGCA

[0051] GCTTGTAATAGCACGCCAGAAGGTCAAGGATGCAGAAAAGCAGTATGAGA

[0052] AGGATCCTGATGACCTAAATAAGAGGGCACTGCATGATCGGGAGAGTGTCG

[0053] CAGCTTCAATACAATCAAAAATTGATGAATTGAAGCGCCAACTTGCTGACA

[0054] GGATTGCAGCAGGGAAGAACATCGGGCAAGACCGGGATCCTACAGGGGTA

[0055] GAGCCGGGTGATCATCTCAAGGAAAGATCAGCACTAAGCTACGGGAATAC

[0056] ACTGGACCTGAATAGCCTTGACATTGATGAACCTACAGGACAGACAGCTGA

[0057] TTGGTTGACCATAATTGTCTATTTGACATCATTCGTGGTCCCGATCATCTTGA

[0058] AGGCACTGTACATGTTGACAACAAGAGGCAGGCAGACTTCAAAGGACAAC

[0059] AAGGGAATGAGGATCAGATTCAAGGATGACAGCTCATATGAAGATGTCAAT

[0060] GGAATCAGAAAGCCCAAACATCTGTATGTGTCAATGCCAAACGCCCAATCA

[0061] AGCATGAAGGCTGAAGAGATAACACCTGGAAGATTCCGCACTGCAGTATGT

[0062] GGGCTATACCCTGCACAGATAAAGGCAAGGAACATGGTAAGCCCTGTCATG

[0063] AGTGTAGTTGGGTTTTTGGCACTGGCAAAAGACTGGACATCTAGAATTGAA

[0064] GAATGGCTTGGTGCACCCTGCAAGTTCATGGCAGAGTCTCCCATTGCCGGG

[0065] AGCTTATCTGGGAATCCTGTGAATCGTGATTATATCAGACAGAGACAAGGT

[0066] GCACTTGCAGGGATGGAGCCAAAAGAATTTCAAGCTCTCAGGCAACATTC

[0067] AAAGGATGCTGGATGTACACTGGTTGAACATATTGAGTCACCATCATCAATA

[0068] TGGGTATTTGCTGGGGCCCCTGATAGGTGCCCACCGACATGCCTGTTTGTTG

[0069] GAGGGATGGCTGAGTTAGGTGCTTTCTTTTCTATACTTCAGGATATGAGGAA

[0070] CACAATCATGGCTTCAAAGACTGTGGGAACAGCTGATGAAAAGCTTCGAA

[0071] AGAAGTCATCATTCTATCAATCATACCTCAGACGCACACAATCAATGGGAAT

[0072] ACAACTGGACCAGAGGATAATTGTTATGTTTATGGTTGCCTGGGGAAAGGA

[0073] GGCAGTGGACAACTTTCATCTCGGTGATGACATGGATCCAGAGCTTCGCAG

[0074] CCTGGCTCAGATCCTGATTGACCAGAAAGTGAAGGAAATCTCAAACCAGGAACCTATGAAATTA (SEQ ID NO: 2).

[0075] The inventors have generated a nucleic acid sequence encoding Hantavirus NP that is optimized for expression in Homo sapiens (see SEQ ID NO:3):

[0076] ATGGCCACAATGGAAGAGATCCAGAGAGAGATCAGCGCCCACGAGGGACA

[0077] GCTGGTTATCGCCAGACAGAAAGTGAAGGACGCCGAGAAGCAGTACGAG

[0078] AAGGACCCCGACGATCTGAACAAGAGAGCCCTGCACGACAGAGAAAGCG

[0079] TGGCCGCCTCTATCCAGAGCAAGATCGATGAGCTGAAGAGACAGCTGGCC

[0080] GACAGAATCGCCGCTGGCAAGAATATTGGCCAGGACAGAGATCCCACAGG

[0081] CGTGGAACCTGGCGATCACCTGAAAGAGAGAAGCGCCCTGTCCTATGGCA

[0082] ACACCCTGGACCTGAACAGCCTGGACATTGATGAGCCTACCGGCCAGACA

[0083] GCCGACTGGCTGACAATCATTGTGTACCTGACCAGCTTCGTGGTCCCCATC

[0084] ATCCTGAAGGCCCTGTACATGCTGACCACCAGAGGCAGACAGACCAGCAA

[0085] GGACAACAAGGGCATGAGAATCCGGTTCAAGGATGACAGCAGCTACGAGG

[0086] ACGTGAACGGCATTAGAAAGCCCAAGCACCTGTACGTGTCCATGCCTAACG

[0087] CTCAGAGCAGCATGAAGGCCGAGGAAATCACCCCTGGCAGATTCAGAACA

[0088] GCCGTGTGCGGACTGTACCCCGCTCAGATCAAGGCCAGAAACATGGTGTC

[0089] CCCAGTGATGAGCGTCGTGGGATTTCTGGCCCTGGCTAAGGACTGGACCAG

[0090] CAGGATTGAGGAATGGCTGGGAGCCCCTTGCAAGTTTATGGCCGAGTCTCC

[0091] TATCGCCGGCAGCCTGTCTGGCAACCCCGTGAATAGAGACTACATCAGACA

[0092] GAGGCAGGGCGCTCTGGCCGGAATGGAACCCAAAGAATTTCAGGCCCTGC

[0093] GGCAGCACTCTAAGGATGCCGGATGTACCCTGGTGGAACACATTGAGAGC

[0094] CCCAGCAGCATCTGGGTTTTCGCTGGCGCTCCTGATAGATGCCCTCCTACCT

[0095] GTCTGTTTGTTGGCGGAATGGCCGAGCTGGGCGCCTTCTTTAGCATTCTGC

[0096] AGGACATGCGGAATACCATCATGGCCAGCAAGACCGTGGGCACCGCCGAT

[0097] GAGAAGCTGAGAAAGAAGTCCAGCTTCTACCAGAGCTACCTGCGGAGAAC

[0098] CCAGAGCATGGGCATTCAGCTGGACCAGAGAATCATCGTGATGTTCATGGT

[0099] GGCCTGGGGCAAAGAAGCCGTGGACAATTTTCACCTGGGCGACGACATGG

[0100] ACCCCGAGCTGAGATCTCTGGCCCAGATCCTGATCGACCAGAAAGTCAAAGAGATCTCCAATCAAGAGCCCATGAAGCTG (SEQ ID NO: 3).

[0101] Translation of the nucleic acid sequence of SEQ ID NO:2 or SEQ ID NO:3 produces a Hantavirus NP polypeptide sequence represented by (SEQ ID NO:4):

[0102] MATMEEIQREISAHEGQLVIARQKVKDAEKQYEKDPDDLNKRALHDRESVAA

[0103] SIQSKIDELKRQLADRIAAGKNIGQDRDPTGVEPGDHLKERSALSYGNTLDLN

[0104] SLDIDEPTGQTADWLTIIVYLTSFVVPIILKALYMLTTRGRQTSKDNKGMRIRFK

[0105] DDSSYEDVNGIRKPKHLYVSMPNAQSSMKAEEITPGRFRTAVCGLYPAQIKAR

[0106] NMVSPVMSVVGFLALAKDWTSRIEEWLGAPCKFMAESPIAGSLSGNPVNRD

[0107] YIRQRQGALAGMEPKEFQALRQHSKDAGCTLVEHIESPSSIWVFAGPDRCPP

[0108] TCLFVGGMAELGAFFSILQDMRNTIMASKTVGTADEKLRKKSSFYQSYLRRT

[0109] QSMGIQLDQRIIVMFMVAWGKEAVDNFHLGDDMDPELRSLAQILIDQKVKEISNQEPMKL (SEQ ID NO: 4).

[0110] Hantaan virus can be used as a reference hantavirus strain. GenBank Accession No. KC570390.1 provides a reference nucleic acid sequence of Hantavirus NP (see SEQ ID NO: 5) and a reference polypeptide sequence of Hantavirus NP (see SEQ ID NO: 7).

[0111] TAGTAGTAGACTCCCTAAAGAGCTACTAGAACAACGATGGCAACTATGGAG

[0112] GAATTGCAGAGGGAAATCAATGCCCATGAGGGTCAACTGGTGATAGCCAG

[0113] GCAGAAGGTGAGGGATGCAGAAAAGCAGTATGAAAAGGATCCAGATGAGT

[0114] TAAACAAGAGCATTGACAGATCGAGAGGGTGTTGCAGTATCCATTCAAG

[0115] CAAAGATTGATGAGTTAAAGAGGCAATTGGCAGATAGGATTGCAACCGGG

[0116] AAGAACCTTGGAAAGGAACAAGACCCAACAGGGGTAGAACCTGGAGATC

[0117] ATCTGAAAGAGAGATCAATGCTCAGTTATGGAAATGTTCTTGACTTAAACC

[0118] ACCTGGATATTGATGAGCCAACAGGACAGACAGCAGACTGGCTGGGCATT

[0119] GTTATCTATCTCACATCCTTTGTTGTCCCGATACTTCTGAAAGCCCTGTACAT

[0120] GTTAACAACAAGAGGGAGGCAGACCACCAAGGACAATAAAGGAACTCGG

[0121] ATTCGATTCAAGGATGATAGCTCCTTCGAGGATGTCAATGGCATTCGGAAGC

[0122] CGAAACATCTATATGTGTCCTTACCAAATGCACAGTCAAGTATGAAAGCAG

[0123] AAGAGATTACACCTGGTAGATATAGAACAGCAATTTGTGGACTTTACCCTGC

[0124] ACAAATTAAGGCAAGACAGATGATTAGTCCAGTCATGAGTGTAATCGGATT

[0125] CTTGGCTTTGGCAAAAGATTGGAGTGACCGCATTGAGCAGTGGTTAAGTGA

[0126] ACCGTGTAAGCTTCTTCCAGATACAGCAGCAGTTAGCCTTCTTGGTGGTCC

[0127] TGCAACCAACAGGGACTATTTACGGCAGCGACAAGTAGCATTGGGCAACAT

[0128] GGAAACAAAAGAGTCTAAGGCTATACGCCAACATGCAGAAGCAGCAGGCT

[0129] GTAGTATGATTGAGGACATTGAGTCACCATCATCAATATGGGTGTTTGCTGG

[0130] GGCACCGGACCGCTGTCCACCAACATGTCTCTTTATTGCAGGTATGGCTGA

[0131] GCTTGGGGCATTTTTTTCCATCCTGCAGGACATGCGAAATACAATTATGGCA

[0132] TCCAAGACAGTTGGAACCTCTGAGGAGAAGCTACGGAAGAAATCCTCATT

[0133] CTATCAGTCTTATCTCAGGAGAACACAATCAATGGGAATACAACTGGATCA

[0134] GAGGATAATTGTGCTCTTCATGGTAGCCTGGGGGAAAGAAGCAGTGGATAA

[0135] CTTCCACCTAGGAGATGATATGGACCCTGAGCTGCGAACACTAGCACAGAG

[0136] CCTGATTGATGTTAAAGTGAAGGAAATTTCCAACCAAGAGCCTTTAAAACT

[0137] ATAATCAGTGAATGTATAACCCTCATTATGTGATTATTATATACTACTGAATCA

[0138] TTATCAATCATATTTGCACTATTATTATCAGGGGAATTAGTATATCAGGGTAA

[0139] GGGCACATTTATGGGTGGGAATCATTACTCAGAGGGTGGGTCAGTTAATCC

[0140] GTTGTGGGTGGGTTTAGTTCCTGGCTGCCTTAAGTAGCCTTTTTTTGTATATA

[0141] TGGATGTAGATTTCATTTGATCTTTAAACTAATCTTGCTCTTTTTCCTTTTCCT

[0142] CCTGCTTTCTCTGCTTACTAACAACAACATTCTACCTCAACACACAACTACC

[0143] TCAACTAAACTACCTCATTTGATTGCTCCTTGATTGTCTCTTTAGGGAGTCTACTACTA(SEQ IDNO:5).

[0144] The coding sequence of SEQ ID NO:5 corresponds to nucleic acid residues 37 - 1323 therein and is represented by SEQ ID NO:6.

[0145] ATGGCAACTATGGAGGAATTGCAGAGGGAAATCAATGCCCATGAGGGTCA

[0146] ACTGGTGATAGCCAGGCAGAAGGTGAGGGATGCAGAAAAGCAGTATGAAA

[0147] AGGATCCAGATGAGTTAAACAAGAGAGCATTGACAGATCGAGAGGGTGTT

[0148] GCAGTATCCATTCAAGCAAAGATTGATGAGTTAAAGAGGCAATTGGCAGAT

[0149] AGGATTGCAACCGGGAAGAACCTTGGAAAGGAACAAGACCCAACAGGGG

[0150] TAGAACCTGGAGATCATCTGAAAGAGAGATCAATGCTCAGTTATGGAAATG

[0151] TTCTTGACTTAAACCACCTGGATATTGATGAGCCAACAGGACAGACAGCAG

[0152] ACTGGCTGGGCATTGTTATCTATCTCACATCCTTTGTTGTCCCGATACTTCTG

[0153] AAAGCCCTGTACATGTTAACAACAAGAGGGAGGCAGACCACCAAGGACAA

[0154] TAAAGGAACTCGGATTCGATTCAAGGATGATAGCTCCTTCGAGGATGTCAA

[0155] TGGCATTCGGAAGCCGAAACATCTATATGTGTCCTTACCAAATGCACAGTCA

[0156] AGTATGAAAGCAGAAGAGATTACACCTGGTAGATATAGAACAGCAATTTGT

[0157] GGACTTTACCCTGCACAAATTAAGGCAAGACAGATGATTAGTCCAGTCATG

[0158] AGTGTAATCGGATTCTTGGCTTTGGCAAAAGATTGGAGTGACCGCATTGAG

[0159] CAGTGGTTAAGTGAACCGTGTAAGCTTCTTCCAGATACAGCAGCAGTTAGC

[0160] CTTCTTGGTGGTCCTGCAACCAACAGGGACTATTTACGGCAGCGACAAGTA

[0161] GCATTGGGCAACATGGAAACAAAAGAGTCTAAGGCTATACGCCAACATGC

[0162] AGAAGCAGCAGGCTGTAGTATGATTGAGGACATTGAGTCACCATCATCAAT

[0163] ATGGGTGTTTGCTGGGGCACCGGACCGCTGTCCACCAACATGTCTCTTTATT

[0164] GCAGGTATGGCTGAGCTTGGGGCATTTTTTTCCATCCTGCAGGACATGCGA

[0165] AATACAATTATGGCATCCAAGACAGTTGGAACCTCTGAGGAGAAGCTACGG

[0166] AAGAAATCCTCATTCTATCAGTCTTATCTCAGGAGAACACAATCAATGGGAA

[0167] TACAACTGGATCAGAGGATAATTGTGCTCTTCATGGTAGCCTGGGGGAAAG

[0168] AAGCAGTGGATAACTTCCACCTAGGAGATGATATGGACCCTGAGCTGCGAA

[0169] CACTAGCACAGAGCCTGATTGATGTTAAAGTGAAGGAAATTTCCAACCAAGAGCCTTTAAAACTA(SEQ ID NO:6).

[0170] Translation of the nucleic acid sequence of SEQ ID NO:6 yields the Hantavirus genus NP polypeptide sequence, which is represented by (SEQ ID NO:7):

[0171] MATMEELQREINAHEGQLVIARQKVRDAEKQYEKDPDELNKRALTDREGVAV

[0172] SIQAKIDELKRQLADRIATGKNLGKEQDPTGVEPGDHLKERSMLSYGNVLDL

[0173] NHLDIDEPTGQTADWLGIVIYLTSFVVPILLKALYMLTTRGRQTTKDNKGTRIR

[0174] FKDDSSFEDVNGIRKPKHLYVSLPNAQSSMKAEEITPGRYRTAICGLYPAQIKA

[0175] RQMISPVMSVIGFLALAKDWSDRIEQWLSEPCKLLPDTAAVSLLGGPATNRDY

[0176] LRQRQVALGNMETKESKAIRQHAEAAGCSMIEDIESPSSIWVFAGAPDRCPPT

[0177] CLFIAGMAELGAFFSILQDMRNTIMASKTVGTSEEKLRKKSSFYQSYLRRTQS

[0178] MGIQLDQRIIVLFMVAWGKEAVDNFHLGDDMDPELRTLAQSLIDVKVKEISNQEPLKL (SEQ ID NO: 7).

[0179] A reference nucleic acid sequence of Hantavirus NP can be provided by SEQ ID NO:8, which corresponds to nucleic acid residues 319-1323 of SEQ ID NO:5.

[0180] ATGCTCAGTTATGGAAATGTTCTTGACTTAAACCACCTGGATATTGATGAGC

[0181] CAACAGGACAGACAGCAGACTGGCTGGGCATTGTTATCTATCTCACATCCT

[0182] TTGTTGTCCCGATACTTCTGAAAGCCCTGTACATGTTAACAACAAGAGGGA

[0183] GGCAGACCACCAAGGACAATAAAGGAACTCGGATTCGATTCAAGGATGAT

[0184] AGCTCCTTCGAGGATGTCAATGGCATTCGGAAGCCGAAACATCTATATGTGT

[0185] CCTTACCAAATGCACAGTCAAGTATGAAAGCAGAAGAGATTACACCTGGTA

[0186] GATATAGAACAGCAATTTGTGGACTTTACCCTGCACAAATTAAGGCAAGAC

[0187] AGATGATTAGTCCAGTCATGAGTGTAATCGGATTCTTGGCTTTGGCAAAAGA

[0188] TTGGAGTGACCGCATTGAGCAGTGGTTAAGTGAACCGTGTAAGCTTCTTCC

[0189] AGATACAGCAGCAGTTAGCCTTCTTGGTGGTCCTGCAACCAACAGGGACTA

[0190] TTTACGGCAGCGACAAGTAGCATTGGGCAACATGGAAACAAAAGAGTCTA

[0191] AGGCTATACGCCAACATGCAGAAGCAGCAGGCTGTAGTATGATTGAGGACA

[0192] TTGAGTCACCATCATCAATATGGGTGTTTGCTGGGGCACCGGACCGCTGTC

[0193] CACCAACATGTCTCTTTATTGCAGGTATGGCTGAGCTTGGGGCATTTTTTTC

[0194] CATCCTGCAGGACATGCGAAATACAATTATGGCATCCAAGACAGTTGGAAC

[0195] CTCTGAGGAGAAGCTACGGAAGAAATCCTCATTCTATCAGTCTTATCTCAG

[0196] GAGAACACAATCAATGGGAATACAACTGGATCAGAGGATAATTGTGCTCTT

[0197] CATGGTAGCCTGGGGGAAAGAAGCAGTGGATAACTTCCACCTAGGAGATG

[0198] ATATTGGACCCTGAGCTGCGAACACTAGCACAGAGCCTGATTGATGTTAAAGTGAAGGAAATTTCCAACCAAGAGCCTTTAAAACTA (SEQ ID NO: 8).

[0199] The inventors have generated a nucleic acid sequence encoding Hantavirus NP that is optimized for expression in Homo sapiens (see SEQ ID NO:9):

[0200] ATGCTGAGCTACGGCAACGTGCTGGATCTGAACCACCTGGATATCGACGAG

[0201] CCAACAGGACAGACCGCTGATTGGCTGGGCATCGTGATCTACCTGACCTCC

[0202] TTTTGTGGTGCCTATTCTGCTCAAAGCCCTCTATATGCTGACAACACGCGGAA

[0203] GGCAGACCACCAAAGATAACAAAGGCACCCGGATCAGGTTTAAGGACGAC

[0204] AGCTCCTTTGAGGATGTCAACGGCATCCGGAAACCTAAGCACCTCTATGTG

[0205] TCTCTGCCCAATGCACAGTCCTCCATGAAGGCAGAAGAGATCACACCAGG

[0206] CCGGTACAGAACCGCCATCTGTGGACTGTATCCTGCACAAATCAAAGCCCG

[0207] GCAGATGATCAGCCCCGTGATGTCCGTTATCGGATTCCTGGCTCTGGCCAA

[0208] AGATTGGAGCGACAGGATCGAGCAGTGGCTGAGCGAGCCTTGCAAGCTGC

[0209] TTCCTGATACAGCCGCTGTGTCACTGCTTGGCGGCCCTGCCACAAACAGAG

[0210] ATTACCTGAGACAGAGACAGGTGGCACTGGGCAACATGGAAACAAAAGA

[0211] GAGCAAGGCCATCCGGCAGCATGCCGAAGCTGCTGGCTGTAGCATGATCG

[0212] AGGATATCGAGTCCCCTAGCTCCATTTGGGTGTTCGCAGGGGCCCCAGATA

[0213] GATGTCCACCAACATGCCTGTTCATTGCCGGCATGGCTGAACTGGGAGCTT

[0214] TTTTCAGCATCCTCCAGGATATGCGCAACACGATTATGGCCTCCAAGACAGT

[0215] GGGAACCAGCGAGGAAAAGCTGCGGAAGAAAAGCAGCTTTTACCAGTCTT

[0216] ACCTGAGGCGGACCCAGTCCATGGGGATCCAACTGGATCAGCGGATCATTG

[0217] TGCTGTTTATGGTCGCTTGGGGAAAAGAGGCTGTCGATAACTTCCACCTGG

[0218] GAGATGATATGGATCCTGAACTGCGGACCCTGGCTCAGTCCCTGATCGATGTGAAAGTGAAAGAATTAATCAAGAACCCCTCAAGCTG(SEQ ID NO:9)。

[0219] The nucleic acid sequence comprising SEQ ID NO: 8 or 9 is particularly suitable for use in the vector of the present invention, which also encodes a nucleoprotein from a Hantaan virus strain other than Hantaan virus, such as a nucleoprotein from Seoul virus. The inventors determined that the 94 N-terminal amino acids of the wild-type Hantaan virus nucleoprotein showed high sequence similarity to the N-terminus of the wild-type nucleoprotein from Seoul virus, and when there were sequence differences in this region, the inventors determined that both sequences contained closely related amino acids. The 95th residue of the wild-type Hantaan virus nucleoprotein sequence was identified as the first residue that was significantly different from the corresponding residue in the wild-type nucleoprotein sequence from Seoul virus. The inventors believe that the nucleic acid encoding the 94 N-terminal amino acids of the wild-type nucleoprotein of Hantaan virus is essentially antigenically redundant when present in a vector that also encodes a nucleoprotein from Seoul virus (or at least the 94 N-terminal amino acids of the wild-type nucleoprotein from Seoul virus, or an antigenic fragment thereof). Therefore, the inventors believe that the nucleic acid encoding the 94 N-terminal amino acids of the wild-type Hantaan virus nucleoprotein can be omitted from a vector that also encodes the nucleoprotein from Seoul virus (or at least the 94 N-terminal amino acids of the wild-type nucleoprotein from Seoul virus, or an antigenic fragment thereof) without sacrificing antigenic diversity. Removal of unnecessary nucleic acid sequences is generally advantageous in the design of vector constructs (e.g., MVA constructs) because it can enhance vector stability.

[0220] For the reasons stated above, the inventors believe that similar advantages can be achieved when the nucleic acid encoding the 94 N-terminal amino acids of the Seoul virus nucleoprotein is omitted, particularly when omitted from a vector encoding the Hantaan virus nucleoprotein (or at least the 94 N-terminal amino acids of the wild-type nucleoprotein from the Hantaan virus, or an antigenic fragment thereof).

[0221] Translation of the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:9 produces a Hantavirus NP polypeptide sequence represented by (SEQ ID NO:10):

[0222] MLSYGNVLDLNHLDIDEPTGQTADWLGIVIYLTSFVVPILLKALYMLTTRGRQ

[0223] TTKDNKGTRIRFKDDSSFEDVNGIRKPKHLYVSLPNAQSSMKAEEITPGRYRT

[0224] AICGLYPAQIKARQMISPVMSVIGFLALAKDWSDRIEQWLSEPCKLLPDTAAVS

[0225] LLGGPATNRDYLRQRQVALGNMETKESKAIRQHAEAAGCSMIEDIESPSSIWV

[0226] FAGAPDRCPPTCLFIAGMAELGAFFSILQDMRNTIMASKTVGTSEEKLRKKSSF

[0227] YQSYLRRTQSMGIQLDQRIIVLFMVAWGKEAVDNFHLGDDMDPELRTLAQSLIDVKVKEISNQEPLKL (SEQ ID NO: 10).

[0228] As used herein, the term "antigenic fragment" refers to a peptide or protein fragment of a Hantavirus NP that retains the ability to induce an immune response in an individual compared to a reference Hantavirus NP. Thus, an antigenic fragment may include at least one epitope of a reference protein. For example, an antigenic fragment of the invention may comprise (or consist of) a peptide sequence having at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300 amino acids, wherein the peptide sequence has at least 70% sequence homology with the corresponding peptide sequence of (contiguous) amino acids of the reference protein. An antigenic fragment may comprise (or consist of) at least 10 consecutive amino acid residues from a reference protein sequence (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, or 300 consecutive amino acid residues of the reference protein).

[0229] Antigenic fragments of a reference protein may have common antigenic cross-reactivity and / or substantially the same in vivo biological activity as the reference protein. For example, an antibody that is capable of binding to an antigenic fragment of a reference protein may also be capable of binding to the reference protein itself. As a further example, a reference protein and its antigenic fragment may have a common ability to induce a "recall response" in T lymphocytes (e.g., CD4+, CD8+, effector T cells, or memory T cells, such as TEMs or TCMs) that have previously been exposed to antigenic components of a hantavirus infection.

[0230] In one aspect, the present invention provides a viral vector or a bacterial vector comprising a nucleic acid sequence encoding a Hantavirus nucleoprotein or an antigenic fragment thereof; wherein the vector is capable of inducing an immune response in a subject.

[0231] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or its antigenic fragment comprises a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity with a nucleic acid sequence selected from SEQ ID NO: 1, 2 and 3.

[0232] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity with the nucleic acid sequence of SEQ ID NO:3.

[0233] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity with a nucleic acid sequence selected from SEQ ID NO: 5, 6, 8 and 9.

[0234] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity with the nucleic acid sequence of SEQ ID NO:9.

[0235] "Peptide library 4" induces a very strong antigen-specific T cell response (see Examples). The amino acid sequence represented by peptide library 4 corresponds to SEQ ID NO:11.

[0236] LYPAQIKARNMVSPVMSVVGFLALAKDWTSRIEEWLGAPCKFMAESPIAGSLSGNPVNRDYIRQRQGALAGMEPKEFQA(SEQ ID NO:11)

[0237] The amino acid sequence of SEQ ID NO: 11 is encoded by nucleic acid residues 655-891 of SEQ ID NO: 1 (see SEQ ID NO: 15); residues 613-849 of SEQ ID NO: 2 (see SEQ ID NO: 16); and residues 613-849 of SEQ ID NO: 3 (see SEQ ID NO: 17).

[0238] CTATACCCTGCACAGATAAAGGCAAGGAACATGGTAAGCCCTGTCATGAGT

[0239] GTAGTTGGGTTTTTGGCACTGGCAAAAGACTGGACATCTAGAATTGAAGAA

[0240] TGGCTTGGTGCACCCTGCAAGTTCATGGCAGAGTCTCCCATTGCCGGGAGC

[0241] TTATCTGGGAATCCTGTGAATCGTGATTATATCAGACAGAGACAAGGTGCACTTGCAGGGATGGAGCCAAAAGAATTTCAAGCT(SEQ ID NO:15)

[0242] CTATACCCTGCACAGATAAAGGCAAGGAACATGGTAAGCCCTGTCATGAGT

[0243] GTAGTTGGGTTTTTGGCACTGGCAAAAGACTGGACATCTAGAATTGAAGAA

[0244] TGGCTTGGTGCACCCTGCAAGTTCATGGCAGAGTCTCCCATTGCCGGGAGC

[0245] TTATCTGGGAATCCTGTGAATCGTGATTATATCAGACAGAGACAAGGTGCACTTGCAGGGATGGAGCCAAAAGAATTTCAAGCT(SEQ ID NO:16)

[0246] CTGTACCCCGCTCAGATCAAGGCCAGAAACATGGTGTCCCCAGTGATGAGC

[0247] GTCGTGGGATTTCTGGCCCTGGCTAAGGACTGGACCAGCAGGATTGAGGA

[0248] ATGGCTGGGAGCCCCTTGCAAGTTTATGGCCGAGTCTCCTATCGCCGGCAG

[0249] CCTGTCTGGCAACCCCGTGAATAGAGACTACATCAGACAGAGGCAGGGCGCTCTGGCCGGAATGGAACCCAAAGAATTTCAGGCC (SEQ ID NO: 17)

[0250] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity with the nucleic acid sequence of SEQ ID NO:15, 16 or 17.

[0251] In one embodiment, the nucleic acid sequence encoding a Hantavirus nucleoprotein or an antigenic fragment thereof comprises (or consists of) at least 10 consecutive nucleic acid residues from a sequence of SEQ ID NO: 15, 16 or 17 (e.g., at least 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or 65 nucleic acids of SEQ ID NO: 15, 16 or 17).

[0252] "Peptide library 9" also induced a very strong antigen-specific T cell response. The amino acid sequence represented by peptide library 9 corresponds to SEQ ID NO:12.

[0253] IKARQMISPVMSVIGFLALAKDWSDRIEQWLSEPCKLLPDTAAVSLLGGPATNRDYLRQRQVALGNMETKESKAIRQHA (SEQ ID NO: 12).

[0254] The amino acid sequence of SEQ ID NO:12 is encoded by nucleic acid residues 664-900 of SEQ ID NO:5 (see SEQ ID NO:18); residues 628-864 of SEQ ID NO:6 (see SEQ ID NO:19); residues 346-582 of SEQ ID NO:8 (see SEQ ID NO:20); and residues 346-582 of SEQ ID NO:9 (see SEQ ID NO:21).

[0255] ATTAAGGCAAGACAGATGATTAGTCCAGTCATGAGTGTAATCGGATTCTTGG

[0256] CTTTGGCAAAAGATTGGAGTGACCGCATTGAGCAGTGGTTAAGTGAACCG

[0257] TGTAAGCTTCTTCCAGATACAGCAGCAGTTAGCCTTCTTGGTGGTCCTGCA

[0258] ACCAACAGGGACTATTTACGGCAGCGACAAGTAGCATTGGGCAACATGGAAACAAAAGAGTCTAAGGCTATACGCCAACATGCA(SEQ ID NO:18)ATTAAGGCAAGACAGATGATTAGTCCAGTCATGAGTGTAATCGGATTCTTGGCTTTGGCAAAAGATTGGAGTGACCGCATTGAGCAGTGGTTAAGTGAACCGTGTAAGCTTCTTCCAGATACAGCAGCAGTTAGCCTTCTTGGTGGTCCTGCAACCAACAGGGACTATTTACGGCAGCGACAAGTAGCATTGGGCAACATGGAAACAAAAGAGTCTAAGGCTATACGCCAACATGCA(SEQ ID NO:19)ATTAAGGCAAGACAGATGATTAGTCCAGTCATGAGTGTAATCGGATTCTTGGCTTTGGCAAAAGATTGGAGTGACCGCATTGAGCAGTGGTTAAGTGAACCGTGTAAGCTTCTTCCAGATACAGCAGCAGTTAGCCTTCTTGGTGGTCCTGCAACCAACAGGGACTATTTACGGCAGCGACAAGTAGCATTGGGCAACATGGAAACAAAAGAGTCTAAGGCTATACGCCAACATGCA(SEQ ID NO:20)ATCAAAGCCCGGCAGATGATCAGCCCCGTGATGTCCGTTATCGGATTCCTGGCTCTGGCCAAAGATTGGAGCGACAGGATCGAGCAGTGGCTGAGCGAGCCTTGCAAGCTGCTTCCTGATACAGCCGCTGTGTCACTGCTTGGCGGCCCTGCCACAAACAGAGATTACCTGAGACAGAGACAGGTGGCACTGGGCAACATGGAAACAAAAGAGAGCAAGGCCATCCGGCAGCATGCC(SEQ ID NO:21)

[0259] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity with the nucleic acid sequence of SEQ ID NO:18, 19, 20 or 21.

[0260] In one embodiment, the nucleic acid sequence encoding a Hantavirus nucleoprotein or an antigenic fragment thereof comprises (or consists of) at least 10 consecutive nucleic acid residues from a sequence of SEQ ID NO: 18, 19, 20 or 21 (e.g., at least 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or 65 nucleic acids of SEQ ID NO: 18, 19, 20 or 21).

[0261] As demonstrated herein, peptide libraries 4 and 9 induce very strong antigen-specific T cell responses. By aligning the polypeptide sequence represented by "peptide library 4" with the polypeptide sequence represented by "peptide library 9", the inventors identified regions of high sequence identity, represented by SEQ ID NO: 13 and SEQ ID NO: 14, respectively. Without wishing to be bound by theory, the inventors believe that the amino acid sequences of SEQ ID NO: 13 and 14 play an important role in eliciting the particularly strong antigen-specific T cell responses observed with peptide libraries 4 and 9, respectively.

[0262] SPVMSVVGFLALAKD (SEQ ID NO: 13)

[0263] PVMSVIGFLALAKDW (SEQ ID NO: 14)

[0264] SEQ ID NO: 13 is encoded, among other things, by nucleic acid residues 691-735 of SEQ ID NO: 1 (see SEQ ID NO: 22); residues 649-693 of SEQ ID NO: 2 (see SEQ ID NO: 23); and residues 649-693 of SEQ ID NO: 3 (see SEQ ID NO: 24).

[0265] AGCCCTGTCATGAGTGTAGTTGGGTTTTTGGCACTGGCAAAAGAC(SEQ ID NO:22)

[0266] AGCCCTGT CAT GAGT GT AGTT GGGTTTTTGGC ACTGGC A AAAGAC (SEQ ID NO:23)

[0267] TCCCCAGTGATGAGCGTCGTGGGATTTCTGGCCCTGGCTAAGGAC (SEQ ID NO: 24)

[0268] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity with the nucleic acid sequence of SEQ ID NO:22, 23 or 24.

[0269] SEQ ID NO: 14 is encoded, among other things, by nucleic acid residues 688-732 of SEQ ID NO: 5 (see SEQ ID NO: 25); residues 652-696 of SEQ ID NO: 6 (see SEQ ID NO: 26); residues 370-414 of SEQ ID NO: 8 (see SEQ ID NO: 27); and residues 370-414 of SEQ ID NO: 9 (see SEQ ID NO: 28).

[0270] CCAGTCATGAGTGTAATCGGATTCTTGGCTTTGGCAAAAGATTGG(SEQ ID NO:25)

[0271] CCAGTCATGAGTGTAATCGGATTCTTGGCTTTGGCAAAAGATTGG(SEQ ID NO:26)

[0272] CCAGTCATGAGTGTAATCGGATTCTTGGCTTTGGCAAAAGATTGG(SEQ ID NO:27)

[0273] CCCGTGATGTCCGTTATCGGATTCCTGGCTCTGGCCAAAGATTGG(SEQ ID NO:28)

[0274] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity with the nucleic acid sequence of SEQ ID NO:25, 26, 27 or 28.

[0275] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0276] (A) the first nucleic acid sequence is provided by a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to SEQ ID NO: 1, 2 or 3; and

[0277] (B) The second nucleic acid sequence is provided by a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to SEQ ID NO:5, 6, 8 or 9.

[0278] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0279] (A) the first nucleic acid sequence is provided by a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to SEQ ID NO: 15, 16, 17, 22, 23 or 24; and

[0280] (B) The second nucleic acid sequence is provided by a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to SEQ ID NO:18, 19, 20, 21, 25, 26, 27 or 28.

[0281] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0282] (A) the first nucleic acid sequence has at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NO: 15, 16, 17, 22, 23 or 24; and

[0283] (B) the second nucleic acid sequence has at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NO: 18, 19, 20, 21, 25, 26, 27 or 28.

[0284] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0285] (A) the first nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 24; and

[0286] (B) The second nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO:28.

[0287] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0288] (A) the first nucleic acid sequence has at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NO: 22 or 23; and

[0289] (B) The second nucleic acid sequence is provided by a nucleic acid sequence having at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 25, 26 or 27.

[0290] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0291] (A) the first nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 17; and

[0292] (B) The second nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO:21.

[0293] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0294] (A) the first nucleic acid sequence has at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NO: 15 or 16; and

[0295] (B) The second nucleic acid sequence has at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 18, 19 or 20.

[0296] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0297] (A) the first nucleic acid sequence has at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NO: 1, 2 or 3; and

[0298] (B) The second nucleic acid sequence has at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 5, 6, 8 or 9.

[0299] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0300] (A) the first nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 3; and

[0301] (B) The second nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO:9.

[0302] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0303] (A) the first nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 2; and

[0304] (B) The second nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO:8.

[0305] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0306] (A) the first nucleic acid has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 2; and

[0307] (B) The second nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO:6.

[0308] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0309] (A) the first nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 1; and

[0310] (B) The second nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO:5.

[0311] In one embodiment, the first nucleic acid sequence is located 5' to the second nucleic acid sequence. In one embodiment, the second nucleic acid sequence is located 5' to the first nucleic acid sequence.

[0312] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to SEQ ID NO:29.

[0313] ATGGCCACAATGGAAGAGATCCAGAGAGAGATCAGCGCCCACGAGGGACA

[0314] GCTGGTTATCGCCAGACAGAAAGTGAAGGACGCCGAGAAGCAGTACGAG

[0315] AAGGACCCCGACGATCTGAACAAGAGAGCCCTGCACGACAGAGAAAGCG

[0316] TGGCCGCCTCTATCCAGAGCAAGATCGATGAGCTGAAGAGACAGCTGGCC

[0317] GACAGAATCGCCGCTGGCAAGAATATTGGCCAGGACAGAGATCCCACAGG

[0318] CGTGGAACCTGGCGATCACCTGAAAGAGAAGCGCCCTGTCCTATGGCA

[0319] ACACCCTGGACCTGAACAGCCTGGACATTGATGAGCCTACCGGCCAGACA

[0320] GCCGACTGGCTGACAATCATTGTGTACCTGACCAGCTTCGTGGTCCCCATC

[0321] ATCCTGAAGGCCCTGTACATGCTGACCACCAGAGGCAGACAGACCAGCAA

[0322] GGACAACAAGGGCATGAGAATCCGGTTCAAGGATGACAGCAGCTACGAGG

[0323] ACGTGAACGGCATTAGAAAGCCCAAGCACCTGTACGTGTCCATGCCTAACG

[0324] CTCAGAGCAGCATGAAGGCCGAGGAAATCACCCCTGGCAGATTCAGAACA

[0325] GCCGTGTGCGGACTGTACCCCGCTCAGATCAAGGCCAGAAACATGGTGTC

[0326] CCCAGTGATGAGCGTCGTGGGATTTCTGGCCCTGGCTAAGGACTGGACCAG

[0327] CAGGATTGAGGAATGGCTGGGAGCCCCTTGCAAGTTTATGGCCGAGTCTCC

[0328] TATCGCCGGCAGCCTGTCTGGCAACCCCGTGAATAGAGACTACATCAGACA

[0329] GAGGCAGGGCGCTCTGGCCGGAATGGAACCCAAAGAATTTCAGGCCCTGC

[0330] GGCAGCACTCTAAGGATGCCGGATGTACCCTGGTGGAACACATTGAGAGC

[0331] CCCAGCAGCATCTGGGTTTTCGCTGGCGCTCCTGATAGATGCCCTCCTACCT

[0332] GTCTGTTTGTTGGCGGAATGGCCGAGCTGGGCGCCTTCTTTAGCATTCTGC

[0333] AGGACATGCGGAATACCATCATGGCCAGCAAGACCGTGGGCACCGCCGAT

[0334] GAGAAGCTGAGAAAGAAGTCCAGCTTCTACCAGAGCTACCTGCGGAGAAC

[0335] CCAGAGCATGGGCATTCAGCTGGACCAGAGAATCATCGTGATGTTCATGGT

[0336] GGCCTGGGGCAAAGAAGCCGTGGACAATTTTCACCTGGGCGACGACATGG

[0337] ACCCCGAGCTGAGATCTCTGGCCCAGATCCTGATCGACCAGAAAGTCAAA

[0338] GAGATCTCCAATCAAGAGCCCATGAAGCTGATGCTGAGCTACGGCAACGTG

[0339] CTGGATCTGAACCACCTGGATATCGACGAGCCAACAGGACAGACCGCTGAT

[0340] TGGCTGGGCATCGTGATCTACCTGACCTCCTTTGTGGTGCCTATTCTGCTCA

[0341] AAGCCCTCTATATGCTGACAACACGCGGAAGGCAGACCACCAAAGATAAC

[0342] AAAGGCACCCGGATCAGGTTTAAGGACGACAGCTCCTTTGAGGATGTCAA

[0343] CGGCATCCGGAAACCTAAGCACCTCTATGTGTCTCTGCCCAATGCACAGTC

[0344] CTCCATGAAGGCAGAAGAGATCACACCAGGCCGGTACAGAACCGCCATCT

[0345] GTGGACTGTATCCTGCACAAATCAAAGCCCGGCAGATGATCAGCCCCGTGA

[0346] TGTCCGTTATCGGATTCCTGGCTCTGGCCAAAGATTGGAGCGACAGGATCG

[0347] AGCAGTGGCTGAGCGAGCCTTGCAAGCTGCTTCCTGATACAGCCGCTGTGT

[0348] CACTGCTTGGCGGCCCTGCCACAAACAGAGATTACCTGAGACAGAGACAG

[0349] GTGGCACTGGGCAACATGGAAACAAAAGAGAGCAAGGCCATCCGGCAGC

[0350] ATGCCGAAGCTGCTGGCTGTAGCATGATCGAGGATATCGAGTCCCCTAGCT

[0351] CCATTTGGGTGTTCGCAGGGGCCCCAGATAGATGTCCACCAACATGCCTGT

[0352] TCATTGCCGGCATGGCTGAACTGGGAGCTTTTTTTCAGCATCCTCCAGGATAT

[0353] GCGCAACACGATTATGGCCTCCAAGACAGTGGGAACCAGCGAGGAAAAGC

[0354] TGCGGAAGAAAAGCAGCTTTTACCAGTCTTACCTGAGGCGGACCCAGTCC

[0355] ATGGGGATCCAACTGGATCAGCGGATCATTGTGCTGTTTATGGTCGCTTGGG

[0356] GAAAAGAGGCTGTCGATAACTTCCACCTGGGAGATGATATGGATCCTGAAC

[0357] TGCGGACCCTGGCTCAGTCCCTGATCGATGTGAAAGTGAAAGAAATTAGTA

[0358] ATCAAGAACCCCTCAAGCTG(SEQ ID NO:29)

[0359] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to SEQ ID NO:30.

[0360] ATGCTGAGCTACGGCAACGTGCTGGATCTGAACCACCTGGATATCGACGAG

[0361] CCAACAGGACAGACCGCTGATTGGCTGGGCATCGTGATCTACCTGACCTCC

[0362] TTTGTGGTGCCTATTCTGCTCAAAGCCCTCTATATGCTGACAACACGCGGAA

[0363] GGCAGACCACCAAAGATAACAAAGGCACCCGGATCAGGTTTAAGGACGAC

[0364] AGCTCCTTTGAGGATGTCAACGGCATCCGGAAACCTAAGCACCTCTATGTG

[0365] TCTCTGCCCAATGCACAGTCCTCCATGAAGGCAGAAGAGATCACACCAGG

[0366] CCGGTACAGAACCGCCATCTGTGGACTGTATCCTGCACAAATCAAAGCCCG

[0367] GCAGATGATCAGCCCCGTGATGTCCGTTATCGGATTCCTGGCTCTGGCCAA

[0368] AGATTGGAGCGACAGGATCGAGCAGTGGCTGAGCGAGCCTTGCAAGCTGC

[0369] TTCCTGATACAGCCGCTGTGTCACTGCTTGGCGGCCCTGCCACAAACAGAG

[0370] ATTACCTGAGACAGAGACAGGTGGCACTGGGCAACATGGAAACAAAAGA

[0371] GAGCAAGGCCATCCGGCAGCATGCCGAAGCTGCTGGCTGTAGCATGATCG

[0372] PRICETCGAGTCCCCTAGCTCCATTTGGGTGTTCGCAGGGGCCCCAGATA

[0373] GATGTCCACCAACATGCCTGTTCATTGCCGGCATGGCTGAACTGGGAGCTT

[0374] TTTTCAGCATCCTCCAGGATATGCGCAACACGATTATGGCCTCCAAGACAGT

[0375] GGGAACCAGCGAGGAAAAGCTGCGGAAGAAAAGCAGCTTTTACCAGTCTT

[0376] ACCTGAGGCGGACCCAGTCCATGGGGATCCAACTGGATCAGCGGATCATTG

[0377] TGCTGTTTATGGTCGCTTGGGGAAAAGAGGCTGTCGATAACTTCCACCTGG

[0378] GAGATGATATGGATCCTGAACTGCGGACCCTGGCTCAGTCCCTGATCGATG

[0379] TGAAAGTGAAAGAAATTAGTAATCAAGAACCCCTCAAGCTGATGGCCACA

[0380] ATGGAAGAGATCCAGAGAGAGATCAGCGCCCACGAGGGACAGCTGGTTAT

[0381] CGCCAGACAGAAAGTGAAGGACGCCGAGAAGCAGTACGAGAAGGACCCC

[0382] GACGATCTGAACAAGAGAGCCCTGCACGACAGAGAAAGCGTGGCCGCCT

[0383] CTATCCAGAGCAAGATCGATGAGCTGAAGAGACAGCTGGCCGACAGAATC

[0384] GCCGCTGGCAAGAATATTGGCCAGGACAGAGATCCCACAGGCGTGGAACC

[0385] TGGCGATCACCTGAAAGAGAGAAGCGCCCTGTCCTATGGCAACACCCTGG

[0386] ACCTGAACAGCCTGGACATTGATGAGCCTACCGGCCAGACAGCCGACTGG

[0387] CTGACAATCATTGTGTACCTGACCAGCTTCGTGGTCCCCATCATCCTGAAGG

[0388] CCCTGTACATGCTGACCACCAGAGGCAGACAGACCAGCAAGGACAACAAG

[0389] GGCATGAGAATCCGGTTCAAGGATGACAGCAGCTACGAGGACGTGAACGG

[0390] CATTAGAAAGCCCAAGCACCTGTACGTGTCCATGCCTAACGCTCAGAGCAG

[0391] CATGAAGGCCGAGGAAATCACCCCTGGCAGATTCAGAACAGCCGTGTGCG

[0392] GACTGTACCCCGCTCAGATCAAGGCCAGAAACATGGTGTCCCCAGTGATGA

[0393] GCGTCGTGGGATTTCTGGCCCTGGCTAAGGACTGGACCAGCAGGATTGAG

[0394] GAATGGCTGGGAGCCCCTTGCAAGTTTATGGCCGAGTCTCCTATCGCCGGC

[0395] AGCCTGTCTGGCAACCCCGTGAATAGAGACTACATCAGACAGAGGCAGGG

[0396] CGCTCTGGCCGGAATGGAACCCAAAGAATTTCAGGCCCTGCGGCAGCACT

[0397] CTAAGGATGCCGGATGTACCCTGGTGGAACACATTGAGAGCCCCAGCAGCA

[0398] TCTGGGTTTTCGCTGGCGCTCCTGATAGATGCCCTCCTACCTGTCTGTTTGT

[0399] TGGCGGAATGGCCGAGCTGGGCGCCTTCTTTAGCATTCTGCAGGACATGCG

[0400] GAATACCATCATGGCCAGCAAGACCGTGGGCACCGCCGATGAGAAGCTGA

[0401] GAAAGAAGTCCAGCTTCTACCAGAGCTACCTGCGGAGAACCCAGAGCATG

[0402] GGCATTCAGCTGGACCAGAGAATCATCGGTGATGTTCATGGTGGCCTGGGGC

[0403] AAAGAAGCCGTGGACAATTTTCACCTGGGCGACGACATGGACCCCGAGCT

[0404] GAGATCTCTGGCCCAGATCCTGATCGACCAGAAAGTCAAAGAGATCTCCAATCAAGAGCCCATGAAGCTG (SEQ ID NO: 30)

[0405] The inventors have discovered that the Hantavirus NP encoded by the nucleic acid sequence of the present invention can be used to generate an effective immune response against Hantavirus in an individual. In particular, the inventors have discovered that when Hantavirus NP is delivered to a subject using a bacterial vector or a viral vector (such as a non-replicating poxvirus vector or an adenovirus vector), an efficient immune response against Hantavirus is obtained.

[0406] Vector is a tool that can be used as a carrier to deliver genetic material into target cells. For example, viral vectors act as antigen delivery media and also have the ability to activate the innate immune system by binding to cell surface molecules that recognize viral elements. Recombinant viral vectors carrying nucleic acids encoding a given antigen can be produced. Viral vectors can then be used to deliver nucleic acids to target cells, where the encoded antigens are produced, which are then presented to the immune system by the molecular mechanisms of the target cells themselves. As "non-self", the antigens produced produce an acquired immune response in the target subject. Advantageously, it has been demonstrated herein that the vectors of the present invention provide a protective immune response.

[0407] Viral vectors suitable for use in the present invention include poxvirus vectors (such as non-replicating poxvirus vectors), adenovirus vectors, and influenza virus vectors.

[0408] In certain embodiments, a "viral vector" can be a virus-like particle (VLP). VLP is a lipid-coated particle containing viral proteins. Some viral proteins have inherent self-assembly capabilities and bud from the cell membrane as independent coated particles in this process. VLP is easy to purify and can be used, for example, to present viral antigens. Therefore, VLP is suitable for use in immunogenic compositions, such as those described below. In certain embodiments, a viral vector is not a virus-like particle.

[0409] Bacterial vectors can also be used as antigen delivery media. Recombinant bacterial vectors carrying nucleic acids encoding given antigens can be produced. Recombinant bacterial vectors can express antigens on their surfaces. After being applied to a subject, bacterial vectors colonize antigen presenting cells (e.g., dendritic cells or macrophages). Induce antigen-specific immune responses. The immune response can be a cellular (T cell) immune response, or can include humoral (e.g., B cells) and cellular (T cell) immune responses. Examples of bacteria suitable for use as recombinant bacterial vectors include Escherichia coli, Shigella, Salmonella (e.g., Salmonella typhimurium) and Listeria bacteria. In one embodiment, the carrier of the present invention is a bacterial vector, wherein the bacterium is a gram-negative bacterium. In one embodiment, the carrier of the present invention is a bacterial vector selected from Escherichia coli vectors, Shigella vectors, Salmonella vectors and Listeria vectors.

[0410] Without wishing to be bound by any one particular theory, the inventors believe that antigen delivery using the vectors of the present invention stimulates T cell responses as well as other responses in the subject. Thus, the inventors believe that one way the present invention provides protection against Hantavirus infection is by stimulating T cell responses and the cell-mediated immune system. Additionally, humoral (antibody) based protection may also be achieved.

[0411] The viral vector of the present invention may be a non-replicating viral vector.

[0412] As used herein, a non-replicating viral vector is a viral vector that lacks effective replication ability after infecting a target cell. Therefore, the ability of a non-replicating viral vector to produce its own copy after infecting a target cell (such as a human target cell in an individual inoculated with a non-replicating viral vector) is highly reduced or absent. This viral vector may also be referred to as attenuated or replication-deficient. The reason may be the loss / deletion of genes necessary for replication in the target cell. Therefore, a non-replicating viral vector cannot effectively produce its own copy after infecting a target cell. Therefore, compared with a viral vector with replication ability, a non-replicating viral vector may advantageously have improved safety features. A non-replicating viral vector may retain the ability to replicate in a cell that is not a target cell, thereby allowing a viral vector to be produced. For example, a non-replicating viral vector (such as a non-replicating poxvirus vector) may lack the ability to productively replicate in a target cell (such as a mammalian cell (such as a human cell)), but retains the ability to replicate (and therefore allow vector production) in avian cells (such as chicken embryo fibroblasts or CEF cells).

[0413] The viral vector of the present invention may be a non-replicating poxvirus vector. Therefore, in one embodiment, the viral vector encoding Hantavirus NP or an antigenic fragment thereof is a non-replicating poxvirus vector.

[0414] In one embodiment, the non-replicating poxvirus vector is selected from the group consisting of: a modified vaccinia Ankara virus (MVA) vector, a NYVAC vaccinia virus vector, a canarypox (ALVAC) vector, and a fowlpox (FPV) vector. Both MVA and NYVAC are attenuated derivatives of vaccinia viruses. Compared to vaccinia viruses, MVA lacks approximately 26 of the approximately 200 open reading frames.

[0415] In one embodiment, the non-replicating poxvirus vector is an FPV vector.

[0416] In a preferred embodiment, the non-replicating poxvirus vector is an MVA vector.

[0417] The viral vector of the present invention may be an adenoviral vector. Therefore, in one embodiment, the viral vector encoding Hantavirus NP or an antigenic fragment thereof is an adenoviral vector.

[0418] In one embodiment, the adenoviral vector is a non-replicating adenoviral vector (wherein non-replicating is defined as above). An adenovirus can become non-replicating by deleting two gene regions, E1 or E1 and E3. Alternatively, an adenovirus can be made non-replicating by changing the E1 or E1 and E3 gene regions so that the gene regions are non-functional. For example, a non-replicating adenovirus may lack a functional E1 region or may lack a functional E1 and E3 gene region. In this way, the adenovirus cannot replicate in most mammalian cell lines and does not replicate in immune mammals. Most preferably, both E1 and E3 gene region deletions are present in the adenovirus, thereby allowing the insertion of larger transgenes. This is particularly important for allowing the expression of larger antigens, or when expressing multiple antigens in a single vector, or when using large promoter sequences (such as CMV promoters). The deletion of E3 and E1 regions is particularly advantageous for recombinant Ad5 vectors. Optionally, the E4 region can also be engineered.

[0419] In one embodiment, the adenoviral vector is selected from the group consisting of: a human adenoviral vector, a simian adenoviral vector, a group B adenoviral vector, a group C adenoviral vector, a group E adenoviral vector, an adenovirus 6 vector, a PanAd3 vector, an adenovirus C3 vector, a ChAdY25 vector, an AdC68 vector, and an Ad5 vector.

[0420] The viral vector of the present invention may be a measles virus vector. Therefore, in one embodiment, the viral vector encoding Hantavirus NP or an antigenic fragment thereof is a measles virus vector.

[0421] In one embodiment, the expression cassette comprising the nucleic acid sequence encoding Hantavirus NP (or an antigenic fragment thereof) is less than 9 kb (e.g., less than 9.0, 8.5, 8.0, 7.5, 7.0, 6, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3 ,4.2,4.1,4.0,3.9,3.8,3.7,3.6,3.5,3.4,3.3,3.2,3.1,3.0,2.9,2.8,2.7,2.6 ,2.5,2.4,2.3,2.2,2.1,2.0,1.9,1.8,1.7,1.6,1.5,1.4,1.3,1.2,1.1,1.0kb).

[0422] In one embodiment, the expression cassette comprising the nucleic acid sequence encoding Hantavirus NP (or an antigenic fragment thereof) is less than 8 kb (e.g., less than 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0 kb).

[0423] In one embodiment, the expression cassette comprising the nucleic acid sequence encoding the Hantavirus NP (or an antigenic fragment thereof) is less than 7 kb (e.g., less than 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4. 4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0kb).

[0424] In one embodiment, the expression cassette comprising the nucleic acid sequence encoding Hantavirus NP (or an antigenic fragment thereof) is less than 6 kb (e.g., less than 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0 kb).

[0425] In one embodiment, the expression cassette comprising the nucleic acid sequence encoding Hantavirus NP (or an antigenic fragment thereof) is less than 5 kb (e.g., less than 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0 kb).

[0426] In one embodiment, the expression cassette comprising a nucleic acid sequence encoding Hantavirus NP (or an antigenic fragment thereof) is less than 4.5 kb (e.g., less than 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0 kb).

[0427] In one embodiment, wherein the vector is a viral vector, the virus (ie, the viral vector) is not a pseudotyped virus. Thus, in one embodiment, the envelope of the viral vector does not comprise exogenous glycoproteins (ie, glycoproteins that are not native to the viral vector).

[0428] In one embodiment, wherein the vector is a non-replicating poxvirus vector (eg, an MVA vector), the nucleic acid sequence encoding hantavirus NP or an antigenic fragment thereof comprises a nucleic acid sequence encoding a hantavirus glycoprotein.

[0429] In one embodiment, wherein the vector is a non-replicating poxvirus vector (eg, an MVA vector), the nucleic acid sequence encoding hantavirus NP or an antigenic fragment thereof comprises a nucleic acid sequence encoding a hantavirus glycoprotein (GP) epitope.

[0430] In one embodiment, wherein the vector is a non-replicating poxvirus vector (eg, an MVA vector), the nucleic acid sequence encoding hantavirus NP or an antigenic fragment thereof does not include a nucleic acid sequence encoding hantavirus glycoprotein (GP).

[0431] In one embodiment, wherein the vector is a non-replicating poxvirus vector (eg, an MVA vector), the nucleic acid sequence encoding hantavirus NP or an antigenic fragment thereof does not include a nucleic acid sequence encoding a hantavirus glycoprotein (GP) epitope.

[0432] In one embodiment, the hantavirus nucleoprotein or antigenic fragment thereof is the only hantavirus nucleic acid sequence in the vector.

[0433] In one embodiment, wherein the vector is a non-replicating poxvirus vector, the vector is stable, expresses the hantavirus NP product, and induces a protective immune response in a subject.

[0434] In one embodiment, wherein the vector is an adenoviral vector, the vector is stable, expresses the hantavirus NP product, and induces a protective immune response in the subject.

[0435] The nucleic acid sequence described above may include a nucleic acid sequence encoding a Hantavirus NP, wherein the NP comprises a fusion protein. The fusion protein may include a Hantavirus NP polypeptide fused to one or more other polypeptides, such as an epitope tag, another antigen, or a protein that increases immunogenicity, such as flagellin.

[0436] In one embodiment, the nucleic acid sequence encoding Hantavirus NP (as described above) also encodes a tissue plasminogen activator (tPA) signal sequence and / or a V5 fusion protein sequence. In certain embodiments, the presence of the tPA signal sequence can provide increased immunogenicity; the presence of the V5 fusion protein sequence can provide identification of the expressed protein by immunological labeling.

[0437] In one embodiment, the vector (as described above) further comprises a nucleic acid sequence encoding an adjuvant (eg, cholera toxin, E. coli lethal toxin, or flagellin).

[0438] In one embodiment, the vector does not comprise a nucleic acid sequence encoding an adjuvant. In one embodiment, the vector does not comprise a nucleic acid sequence encoding Hsp70.

[0439] The bacterial vectors of the present invention can be produced by using any technique known in the art for the manipulation and production of recombinant bacteria.

[0440] In another aspect, the present invention provides a nucleic acid sequence encoding a viral vector as described above. Thus, the nucleic acid sequence may encode a non-replicating poxvirus vector as described above. Alternatively, the nucleic acid sequence may encode an adenovirus vector as described above.

[0441] Nucleic acid sequences encoding viral vectors (as described above) can be produced by using any technique known in the art for the manipulation and production of recombinant nucleic acids.

[0442] In one aspect, the present invention provides a method for preparing a viral vector (as described above), which comprises providing a nucleic acid, wherein the nucleic acid comprises a nucleic acid sequence encoding a vector (as described above); transfecting a host cell with the nucleic acid; culturing the host cell under conditions suitable for vector proliferation; and obtaining the vector from the host cell.

[0443] As used herein, "transfection" may mean any non-viral method of introducing a nucleic acid into a cell. The nucleic acid may be any nucleic acid suitable for transfecting a host cell. Thus, in one embodiment, the nucleic acid is a plasmid. The host cell may be any cell in which a vector (e.g., a non-replicating poxvirus vector or an adenovirus vector as described above) can grow. As used herein, "culturing host cells under conditions suitable for vector propagation" means using any cell culture conditions and techniques known in the art that are suitable for the selected host cell and capable of producing the vector in the host cell. As used herein, "obtaining a vector" means using any technique known in the art that is suitable for separating a vector from a host cell. Thus, the host cell may be lysed to release the vector. The vector may then be isolated and purified using any suitable method or methods known in the art.

[0444] In one aspect, the invention provides a host cell comprising a nucleic acid sequence encoding a viral vector as described above. The host cell can be any cell in which a viral vector (e.g., a non-replicating poxvirus vector or an adenovirus vector as described above) can grow or propagate. In one embodiment, the host cell is selected from: 293 cells (also referred to as HEK or human embryonic kidney cells), CHO cells (Chinese hamster ovary), CCL81.1 cells, Vero cells, HELA cells, Per.C6 cells, BHK cells (baby hamster kidney), primary CEF cells (chicken embryo fibroblasts), duck embryo fibroblasts, DF-1 cells, or rat IEC-6 cells.

[0445] The present invention also provides a composition comprising the carrier as described above.

[0446] In one aspect, the invention provides a composition comprising a carrier (as described above) and a pharmaceutically acceptable carrier.

[0447] The material suitable for use as a pharmaceutically acceptable carrier is known in the art. Non-limiting examples of pharmaceutically acceptable carriers include water, saline and phosphate buffered saline. However, in some embodiments, the composition is a lyophilized form, in which case it may include a stabilizer, such as bovine serum albumin (BSA). In some embodiments, it may be necessary to formulate the composition with a preservative (such as thimerosal or sodium azide) to facilitate long-term storage. Examples of buffers include, but are not limited to, sodium succinate (pH 6.5) and phosphate buffered saline (PBS, pH 7.4).

[0448] In addition to a pharmaceutically acceptable carrier, the composition of the present invention may also be combined with one or more of salts, excipients, diluents, adjuvants, immunomodulators and / or antimicrobial compounds.

[0449] Advantageously, it has been demonstrated that the vectors of the present invention provide a protective immune response even in the absence of an adjuvant. Thus, in one embodiment, the composition of the present invention does not comprise an adjuvant.

[0450] The composition can be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids (such as, for example, hydrochloric acid or phosphoric acid) or with organic acids (such as acetic acid, oxalic acid, tartaric acid, maleic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases, such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, and such organic bases, such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0451] In one embodiment, the composition (as described above) further comprises at least one Hantavirus NP antigen (i.e., an antigen present in the composition in polypeptide form). Thus, the composition may comprise both a carrier and a polypeptide. In one embodiment, the polypeptide antigen is Hantavirus NP. In one embodiment, the polypeptide antigen is Hantavirus GP. In one embodiment, the presence of the polypeptide antigen means that upon administration of the composition to a subject, improved simultaneous T cell and antibody responses may be achieved. In one embodiment, the T cell and antibody responses achieved exceed those achieved when the carrier or polypeptide antigen is used alone.

[0452] In one embodiment, the polypeptide antigen is not associated with the carrier. In one embodiment, the polypeptide antigen is a separate component of the carrier. In one embodiment, the polypeptide antigen is provided separately from the carrier.

[0453] In one embodiment, the polypeptide antigen is a variant of an antigen encoded by a vector. In one embodiment, the polypeptide antigen is a fragment of an antigen encoded by a vector. In one embodiment, the polypeptide antigen comprises at least a portion of a polypeptide sequence encoded by a nucleic acid sequence of a vector. Therefore, the polypeptide antigen can correspond to at least a portion of an antigen encoded by a vector.

[0454] In one embodiment, the polypeptide antigen is Hantavirus NP, which comprises (or consists of) an amino acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity with an amino acid sequence selected from SEQ ID NO:4, 7 and 10.

[0455] In one embodiment, the polypeptide antigen is Hantavirus NP, which comprises (or consists of) an amino acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity with an amino acid sequence selected from SEQ ID NO: 11 and 12.

[0456] In one embodiment, the polypeptide antigen is a Hantavirus NP comprising (or consisting of) an amino acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to an amino acid sequence selected from SEQ ID NOs: 13 and 14.

[0457] In one embodiment, the polypeptide antigen is Hantavirus NP, which comprises (or consists of) an amino acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity with an amino acid sequence selected from SEQ ID NO:31 and 32.

[0458] MATMEEIQREISAHEGQLVIARQKVKDAEKQYEKDPDDLNKRALHDRESVAA

[0459] SIQSKIDELKRQLADRIAAGKNIGQDRDPTGVEPGDHLKERSALSYGNTLDLN

[0460] SLDIDEPTGQTADWLTIIVYLTSFVVPIILKALYMLTTRGRQTSKDNKGMRIRFK

[0461] DDSSYEDVNGIRKPKHLYVSMPNAQSSMKAEEITPGRFRTAVCGLYPAQIKAR

[0462] NMVSPVMSVVGFLALAKDWTSRIEEWLGAPCKFMAESPIAGSLSGNPVNRD

[0463] YIRQRQGALAGMEPKEFQALRQHSKDAGCTLVEHIESPSSIWVFAGAPDRCPP

[0464] TCLFVGGMAELGAFFSILQDMRNTIMASKTVGTADEKLRKKSSFYQSYLRRT

[0465] QSMGIQLDQRIIVMFMVAWGKEAVDNFHLGDDMDPELRSLAQILIDQKVKEIS

[0466] NQEPMKLMLSYGNVLDLNHLDIDEPTGQTADWLGIVIYLTSFVVPILLKALYM

[0467] LTTRGRQTTKDNKGTRIRFKDDSSFEDVNGIRKPKHLYVSLPNAQSSMKAEEI

[0468] TPGRYRTAICGLYPAQIKARQMISPVMSVIGFLALAKDWSDRIEQWLSEPCKLL

[0469] PDTAAVSLLGGPATNRDYLRQRQVALGNMETKESKAIRQHAEAAGCSMIEDIE

[0470] SPSSIWVFAGAPDRCPPTCLFIAGMAELGAFFSILQDMRNTIMASKTVGTSEEK

[0471] LRKKSSFYQSYLRRTQSMGIQLDQRIIVLFMVAWGKEAVDNFHLGDDMDPELRTLAQSLIDVKVKEISNQEPLKL(SEQ ID NO:31)

[0472] MLSYGNVLDLNHLDIDEPTGQTADWLGIVIYLTSFVVPILLKALYMLTTRGRQ

[0473] TTKDNKGTRIRFKDDSSFEDVNGIRKPKHLYVSLPNAQSSMKAEEITPGRYRT

[0474] AICGLYPAQIKARQMISPVMSVIGFLALAKDWSDRIEQWLSEPCKLLPDTAAVS

[0475] LLGGPATNRDYLRQRQVALGNMETKESKAIRQHAEAAGCSMIEDIESPSSIWV

[0476] FAGAPDRCPPTCLFIAGMAELGAFFSILQDMRNTIMASKTVGTSEEKLRKKSSF

[0477] YQSYLRRTQSMGIQLDQRIIVLFMVAWGKEAVDNFHLGDDMDPELRTLAQSLI

[0478] DVKVKEISNQEPLKLMATMEEIQREISAHEGQLVIARQKVKDAEKQYEKDPD

[0479] DLNKRALHDRESVAASIQSKIDELKRQLADRIAAGKNIGQDRDPTGVEPGDHL

[0480] KERSALSYGNTLDLNSLDIDEPTGQTADWLTIIVYLTSFVVPIILKALYMLTTRG

[0481] RQTSKDNKGMRIRFKDDSSYEDVNGIRKPKHLYVSMPNAQSSMKAEEITPGR

[0482] FRTAVCGLYPAQIKARNMVSPVMSVVGFLALAKDWTSRIEEWLGAPCKFMAE

[0483] SPIAGSLSGNPVNRDYIRQRQGALAGMEPKEFQALRQHSKDAGCTLVEHIESP

[0484] SSIWVFAGAPDRCPPTCLFVGGMAELGAFFSILQDMRNTIMASKTVGTADEKL

[0485] RKKSSFYQSYLRRTQSMGIQLDQRIIVMFMVAWGKEAVDNFHLGDDMDPELRSLAQILIDQKVKEISNQEPMKL(SEQ ID NO:32)

[0486] The polypeptide antigen can be identical (or similar) to the polypeptide antigen encoded by the nucleic acid sequence of the carrier of the composition. Therefore, administration of a composition comprising a carrier and a polypeptide antigen can be used to achieve an enhanced immune response to a single antigen, wherein the enhanced immune response comprises a combined T cell and antibody response, as described above.

[0487] In one embodiment, the composition of the invention (as described above) further comprises at least one naked DNA (i.e., a DNA molecule that is separate from the viral vector of the invention and not part of it) encoding a Hantavirus NP or an antigenic fragment thereof. In one embodiment, the naked DNA comprises (or consists of) a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 1, 2, 3, 5, 6, 8, 9 and 15-30. In one embodiment, the naked DNA encodes Hantavirus NP, which comprises (or consists of) an amino acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity with an amino acid sequence selected from SEQ ID NO:4, 7, 10-14, 31 and 32.

[0488] In one embodiment, the composition of the invention (as described above) further comprises an adjuvant. Non-limiting examples of adjuvants suitable for use in the composition of the invention include aluminum phosphate, aluminum hydroxide, and related compounds; monophosphoryl lipid A and related compounds; outer membrane vesicles from bacteria; oil-in-water emulsions, such as MF59; liposomal adjuvants, such as virosomes, Freund's adjuvant, and related mixtures; polylactic-co-glycolic acid (PLGA) particles; cholera toxin; E. coli lethal toxin; and flagellin.

[0489] The vectors and compositions of the invention (as described above) may be used as vaccines. Thus, the compositions of the invention may be vaccine compositions.

[0490] As used herein, a vaccine is a formulation that, when administered to an animal subject, such as a mammal (e.g., a human, bovine, porcine, ovine, goat, equine, deer, canine or feline subject, particularly a human subject), stimulates a protective immune response against an infectious disease. The immune response may be a humoral and / or cell-mediated immune response. Thus, a vaccine may stimulate B cells and / or T cells.

[0491] The term "vaccine" is used herein interchangeably with the terms "therapeutic / prophylactic composition," "immunogenic composition," "formulation," "antigenic composition," or "medicament."

[0492] In one aspect, the invention provides a vector (as described above) or a composition (as described above) for use in medicine.

[0493] In one aspect, the invention provides a vector (as described above) or a composition (as described above) for use in a method of inducing an immune response in a subject. The immune response can be directed against a hantavirus antigen (e.g., hantavirus NP) and / or a hantavirus infection. Thus, the vectors and compositions of the invention can be used to induce an immune response in a subject against hantavirus NP (e.g., as an immunogenic composition or as a vaccine).

[0494] In one embodiment, the immune response comprises a T cell response.

[0495] In one embodiment, a method of inducing an immune response in a subject comprises administering to the subject an effective amount of a vector (as described above) or a composition (as described above).

[0496] In one aspect, the invention provides a vector (as described above) or a composition (as described above) for use in a method of preventing or treating a hantavirus infection in a subject.

[0497] In one embodiment, the invention provides a vector (as described above) or a composition (as described above) for use in a method of preventing or treating HFRS in a subject.

[0498] The vectors and compositions of the present invention are ideally suited for the prevention or treatment of HFRS, particularly when the Hantavirus nucleoprotein or antigenic fragment thereof is from Seoul virus. As mentioned above, Seoul virus is often associated with causing HFRS.

[0499] The vectors and compositions of the present invention are ideally suited for the prevention or treatment of HFRS, particularly when the Hantavirus nucleoprotein or antigenic fragment thereof is from Hantaan virus. As mentioned above, Hantaan virus is often associated with causing HFRS.

[0500] The vectors and compositions of the present invention are ideally suited for the prevention or treatment of HFRS, particularly when the Hantavirus nucleoprotein or antigenic fragment thereof is a chimeric sequence comprising a chimera of the Seoul virus nucleoprotein (or antigenic fragment thereof) and the Hantavirus nucleoprotein (or antigenic fragment thereof), for example as demonstrated in the Examples.

[0501] As used herein, the term "prevention" includes preventing the onset of a hantavirus infection and / or reducing the severity or intensity of a hantavirus infection. Thus, "prevention" includes vaccination.

[0502] As used herein, the term "treatment" includes therapeutic and preventative / preventative measures (including post-exposure prophylaxis), and includes post-infection treatment and amelioration of hantavirus infection.

[0503] In one embodiment, the Hantavirus infection is a Seoul virus infection. In one embodiment, the Hantavirus infection is a Hantan virus infection. In one embodiment, the Hantavirus infection is a Seoul virus infection and / or a Hantan virus infection.

[0504] Each of the above methods may include the step of administering to a subject an effective amount (eg, a therapeutically effective amount) of a vector or composition of the present invention.

[0505] In this respect, as used herein, an effective amount is a dosage or content sufficient to achieve a desired biological result. As used herein, a therapeutically effective amount is a content that is effective for treating, preventing, inhibiting cure, delaying, reducing the severity of a disorder or recurrent disorder, improving at least one symptom of a disorder or recurrent disorder, or prolonging the survival of a subject beyond the expected survival in the absence of such treatment after single or multiple dose administration to a subject (such as a mammalian subject, particularly a human subject).

[0506] Thus, the amount of active ingredient to be administered depends on the subject to be treated, the ability of the subject's immune system to produce a protective immune response, and the degree of protection desired. The precise amount of active ingredient required to be administered may depend on the judgment of the practitioner and may be specific to each subject.

[0507] Administration to a subject may include administering a vector (as described above) or a composition (as described above) to the subject, wherein the composition is administered multiple times in sequence (e.g., wherein the composition is administered twice, three times, or four times). Thus, in one embodiment, a vector (as described above) or a composition (as described above) is administered to a subject, and then the same vector or composition (or a substantially similar vector or composition) is administered again at a different time.

[0508] In one embodiment, administering to a subject comprises administering to the subject a vector (as described above) or a composition (as described above), wherein the composition is administered substantially before, simultaneously with, or after another immunogenic composition.

[0509] Prior, simultaneous, and sequential administration regimens are discussed in more detail below.

[0510] In certain embodiments, the above method further comprises administering to the subject a second vector, wherein the second vector comprises a nucleic acid sequence encoding a Hantavirus NP. Preferably, the second vector is a vector of the present invention as described above (such as a viral vector, e.g., a non-replicating poxvirus vector or an adenovirus vector as described above).

[0511] In one embodiment, the first and second vectors are of the same vector type. In one embodiment, the first and second vectors are of different vector types. In one embodiment, the first vector is an adenoviral vector (as described above) and the second vector is a non-replicating poxviral vector (as described above). In one embodiment, the first vector is a non-replicating poxviral vector (as described above) and the second vector is an adenoviral vector (as described above).

[0512] In one embodiment, the first and second vectors are administered sequentially in any order. Thus, the first ("1") and second ("2") vectors can be administered to a subject in a 1-2 order or in a 2-1 order.

[0513] As used herein, "sequential administration" has the meaning of "sequential administration" as defined below. Thus, the first and the second vector are administered sequentially at (substantially) different times.

[0514] In one embodiment, the first and second vectors are administered as part of a prime-boost administration regimen. Thus, the first vector can be administered to a subject as a "prime," and the second vector can then be administered to the same subject as a "boost." The prime-boost regimen is discussed below.

[0515] In one embodiment, each of the above methods further comprises the step of administering a hantavirus polypeptide antigen to the subject. In one embodiment, the hantavirus polypeptide antigen is a hantavirus NP (or an antigenic fragment thereof) as described above. In one embodiment, the hantavirus polypeptide antigen is a hantavirus NP comprising an amino acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99, or 100%) sequence identity to an amino acid sequence selected from SEQ ID NOs: 4, 7, 10-14, 31, and 32.

[0516] In one embodiment, the polypeptide antigen is administered separately from the administration of the carrier; preferably, the polypeptide antigen and the carrier are administered sequentially. In one embodiment, the carrier ("V") and the polypeptide antigen ("P") can be administered in the order of VP or in the order of PV.

[0517] In one embodiment, each of the above methods further comprises the step of administering to the subject naked DNA encoding hantavirus NP or an antigenic fragment thereof. In one embodiment, the naked DNA comprises (or consists of) a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99, or 100%) sequence identity with a nucleic acid sequence selected from SEQ ID NO: 1, 2, 3, 5, 6, 8, 9, and 15-30. In one embodiment, the naked DNA encodes hantavirus NP, and the hantavirus NP comprises (or consists of) an amino acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99, or 100%) sequence identity with an amino acid sequence selected from SEQ ID NO: 4, 7, 10-14, 31, and 32.

[0518] In one embodiment, naked DNA is administered separately from the administration of the vector; preferably, naked DNA and vector are administered sequentially. In one embodiment, the vector ("V") and naked DNA ("D") can be administered in the order of VD or in the order of DV.

[0519] In one embodiment, naked DNA (as described above) is administered to a subject as part of a prime-boost regimen.

[0520] Heterologous prime-boost approaches can improve immune responses by allowing repeated vaccinations without increasing anti-vector immunity. Hantavirus NP or antigenic fragments thereof can be delivered sequentially via different vectors (as described above) or naked DNA vectors (as described above). In any heterologous prime-boost vaccination regimen, NP-specific antibody responses are increased, NP-specific T cell responses are increased, and / or clinical disease is reduced compared to the use of a single vector. Suitable combinations of vectors include, but are not limited to:

[0521] DNA prime, MVA boost

[0522] DNA primary immunization, fowlpox booster

[0523] Fowlpox primary vaccination, MVA booster

[0524] MVA primary immunization, fowlpox booster immunization

[0525] DNA primary, fowlpox boost, MVA boost

[0526] MVA priming, adenovirus boosting

[0527] As used herein, the term polypeptide includes peptides and proteins.

[0528] In certain embodiments, the above method further comprises administering an adjuvant to the subject. The adjuvant can be administered together with one, two, three or all four of the first carrier, the second carrier, the polypeptide antigen and the naked DNA.

[0529] The immunogenic compositions, therapeutic preparations, medicaments, pharmaceutical compositions and prophylactic preparations (e.g., vaccines) of the present invention can be administered in a single dose regimen (i.e., substantially a full dose is administered at one time). Alternatively, the immunogenic compositions, therapeutic preparations, medicaments, pharmaceutical compositions and prophylactic preparations (e.g., vaccines) of the present invention can be administered in a multiple dose regimen.

[0530] A multiple dose regimen is one in which the primary course of treatment (e.g., vaccination) may be 1-6 divided doses, followed by additional doses at subsequent intervals as required to maintain and or enhance the immune response, e.g. (for human subjects) a second dose at 1-4 months, and subsequent doses after a further 1-4 months if necessary.

[0531] The dosage regimen will be determined, at least in part, by the individual need and will be subject to the judgment of the practitioner (eg, a physician or veterinarian).

[0532] Simultaneous administration means (substantially) simultaneous administration.

[0533] Sequential administration of two or more compositions / therapeutics / vaccines means that the compositions / therapeutics / vaccines are administered one after the other at (substantially) different times.

[0534] For example, sequential administration can include administering two or more compositions / therapeutics / vaccines at different times, with different intervals of several days (e.g., at least 1, 2, 5, 10, 15, 20, 30, 60, 90, 100, 150, or 200 days).

[0535] For example, in one embodiment, the vaccines of the invention may be administered as part of a "prime-boost" vaccination regimen.

[0536] In one embodiment, the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions and prophylactic formulations (e.g., vaccines) of the present invention can be co-administered (simultaneously or sequentially) to a subject, such as a mammal (e.g., a human, bovine, porcine, ovine, goat, horse, deer, bear, canine or feline subject) with one or more immunomodulators selected from, for example, immunoglobulins, antibiotics, interleukins (e.g., IL-2, IL-12) and / or cytokines (e.g., IFNγ).

[0537] Immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions and prophylactic formulations (e.g., vaccines) may contain 5% to 95% active ingredient, such as at least 10% or 25% active ingredient, or at least 40% active ingredient or at least 50, 55, 60, 70 or 75% active ingredient.

[0538] Immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (eg, vaccines) are administered in a manner compatible with the dosage formulation, and in such amount as to be prophylactically and / or therapeutically effective.

[0539] Administration of immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions and prophylactic formulations (e.g., vaccines) is typically by conventional routes, such as intravenous, subcutaneous, intraperitoneal or mucosal routes. Administration can be by parenteral administration; for example, subcutaneous or intramuscular injection.

[0540] Thus, the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions and prophylactic formulations (e.g., vaccines) of the invention can be prepared as injectables, as liquid solutions or suspensions. Alternatively, solid forms suitable for dissolution or suspension in liquids prior to injection can be prepared. The formulations can also be emulsified, or the peptides encapsulated in liposomes or microcapsules.

[0541] The active ingredient is usually mixed with a pharmaceutically acceptable and compatible excipient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, etc. and combinations thereof. In addition, if desired, immunogenic compositions, therapeutic preparations, medicaments, pharmaceutical compositions and preventive preparations (e.g., vaccines) may contain small amounts of auxiliary substances, such as wetting agents or emulsifiers and / or pH buffers.

[0542] Typically, the carrier is a pharmaceutically acceptable carrier. Non-limiting examples of pharmaceutically acceptable carriers include water, saline, and phosphate buffered saline. However, in some embodiments, the composition is in lyophilized form, in which case it may include a stabilizer, such as bovine serum albumin (BSA). In some embodiments, it may be necessary to formulate the composition with a preservative, such as thimerosal or sodium azide, to facilitate long-term storage.

[0543] Examples of buffers include, but are not limited to, sodium succinate (pH 6.5) and phosphate buffered saline (PBS, pH 6.5 and 7.5).

[0544] Other formulations suitable for other modes of administration include suppositories, and in some cases, oral formulations or formulations suitable for distribution as aerosols. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing active ingredients in the range of 0.5% to 10%, preferably 1%-2%.

[0545] Oral formulations include such commonly used excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

[0546] It may be desirable to introduce the compositions of the invention (as described above) into the respiratory system of the subject.Effective delivery of therapeutic / prophylactic compositions or medicaments to sites of lung infection may be achieved by oral or intranasal administration.

[0547] Formulations for intranasal administration may be in the form of nasal drops or nasal sprays. Intranasal formulations may contain droplets having an approximate diameter in the range of 100-5000 μm, such as 500-4000 μm, 1000-3000 μm or 100-1000 μm. Alternatively, in terms of volume, the droplets may be in the range of about 0.001-100 μl, such as 0.1-50 μl or 1.0-25 μl or such as 0.001-1 μl.

[0548] Alternatively, the therapeutic / prophylactic formulation or drug can be an aerosol formulation. The aerosol formulation can take the form of a powder, a suspension or a solution. The size of the aerosol particles is related to the delivery ability of the aerosol. Smaller particles can travel farther down the respiratory airway toward the alveoli than larger particles. In one embodiment, the aerosol particles have a diameter distribution that facilitates delivery along the entire length of the bronchi, bronchioles and alveoli. Alternatively, the particle size distribution can be selected to target a specific part of the respiratory airway, such as the alveoli. In the case of aerosol delivery of a medicament, the diameter of the particle can be in the approximate range of 0.1 μm-50 μm, preferably 1 μm-25 μm, more preferably 1 μm-5 μm.

[0549] Aerosol particles may be delivered using a nebulizer (eg, through the mouth) or a nasal spray. Aerosol formulations may optionally contain a propellant and / or a surfactant.

[0550] In one embodiment, the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions and prophylactic formulations (eg, vaccines) of the invention comprise a pharmaceutically acceptable carrier and, optionally, one or more of a salt, an excipient, a diluent and / or an adjuvant.

[0551] In one embodiment, the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions and prophylactic formulations (e.g., vaccines) of the present invention may contain one or more immunomodulators selected from, for example, immunoglobulins, antibiotics, interleukins (e.g., IL-2, IL-12) and / or cytokines (e.g., IFNγ).

[0552] The present invention includes polypeptides that are substantially homologous to polypeptides based on any one of the polypeptide antigens identified in this application (including fragments thereof).The terms "sequence identity" and "sequence homology" are considered synonymous in this specification.

[0553] For example, a polypeptide of interest can comprise an amino acid sequence having at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99, or 100% amino acid sequence identity to the amino acid sequence of a reference polypeptide.

[0554] There are many established algorithms that can be used for comparing two amino acid sequences. Usually, a sequence serves as a reference sequence, and a test sequence can be compared with it. The sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the specified program parameters. The comparison of the amino acid sequence for comparison can be performed, for example, by a computer-implemented algorithm (such as GAP, BESTFIT, FASTA or TFASTA) or BLAST and BLAST 2.0 algorithms.

[0555] The BLOSLIM62 table shown below is an amino acid substitution matrix derived from approximately 2,000 local multiple alignments of protein sequence segments representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Natl. Acad. Sci. LISA 89: 10915-10919, 1992; incorporated herein by reference). Amino acids are represented by the standard single-letter code. Percent identity is calculated as follows:

[0556] BLOSUM62 table

[0557] ARNDCQEGHILKMFPSTWYV

[0558] A-4

[0559] R-15

[0560] N-2 0 6

[0561] D-2-2 1 6

[0562] C 0-3-3-3 9

[0563] Q-1 1 0 0-3 5

[0564] E-1 0 0 2-4 2 5

[0565] G 0-2 0-1-3-2-2 6

[0566] H-2 0 1-1-3 0 0-2 8

[0567] I-1-3-3-3-1-3-3-4-3 4

[0568] L-1-2-3-4-1-2-3-4-3 2 4

[0569] K-1 2 0-1-3 1 1-2-1-3-2 5

[0570] M-1-1-2-3-1 0-2-3-2 1 2-1 5

[0571] F-2-3-3-3-2-3-3-3-1 0 0-3 0 6

[0572] P-1-2-2-1-3-1-1-2-2-3-3-1-2-4 7

[0573] S1-1 1 0-1 0 0 0-1-2-2 0-1-2-1 4

[0574] T 0-1 0-1-1-1-1-2-2-1-1-1-1-2-1 15

[0575] W-3-3-4-4-2-2-3-2-2-3-2-3-11-4-3-2 11

[0576] Y-2-2-2-3-2-1-2-3 2-1-1-2-1 3-3-2-2-2 7

[0577] V 0-3-3-3-1-2-2-3-3 3 1-2 1-1-2-2 0-3-1 4

[0578] In a homology comparison, identity can exist over a region of the sequence that is at least 10 amino acid residues in length (e.g., at least 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550 or 570 amino acid residues in length - e.g., up to the entire length of the reference sequence).

[0579] Substantially homologous polypeptides have one or more amino acid substitutions, deletions or additions. In many embodiments, these changes are of a smaller nature, for example, only involving conservative amino acid substitutions. Conservative substitutions are those made by replacing one amino acid with another amino acid within the following group: basic: arginine, lysine, histidine; acidic: glutamic acid, aspartic acid; polar: glutamine, asparagine; hydrophobic: leucine, isoleucine, valine; aromatic: phenylalanine, tryptophan, tyrosine; small: glycine, alanine, serine, threonine, methionine. Substantially homologous polypeptides also encompass those polypeptides that contain other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically 1 to about 30 amino acids (e.g., 1-10 or 1-5 amino acids); and small amino or carboxyl terminal extensions, such as amino terminal methionine residues, small linker peptides up to about 20-25 residues, or affinity tags.

[0580] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably and do not imply any length limitation. As used herein, the terms "nucleic acid" and "nucleotide" are used interchangeably. The terms "nucleic acid sequence" and "polynucleotide" include DNA (including cDNA) and RNA sequences.

[0581] The polynucleotide sequences of the present invention include nucleic acid sequences that have been removed from their naturally occurring environment, recombinant or cloned DNA isolates, as well as chemically synthesized analogs or analogs biologically synthesized by heterologous systems.

[0582] Polynucleotides of the present invention can be prepared by any means known in the art. For example, a large amount of polynucleotides can be produced by replication in a suitable host cell. The natural or synthetic DNA fragment encoding the desired fragment will be incorporated into a recombinant nucleic acid construct (usually a DNA construct) that can be introduced into a prokaryotic or eukaryotic cell and replicated therein. Usually, the DNA construct will be suitable for autonomous replication in a unicellular host (such as yeast or bacteria), but can also be used to introduce and be integrated into the genome of cultivated insects, mammals, plants or other eukaryotic cell lines.

[0583] The polynucleotides of the present invention can also be produced by chemical synthesis, for example by the phosphoramidite method or the triester method, and can be carried out on a commercial automated oligonucleotide synthesizer. Double-stranded fragments can be obtained from chemically synthesized single-stranded products by synthesizing complementary chains and annealing the chains together under appropriate conditions or by using a DNA polymerase with an appropriate primer sequence to add complementary chains.

[0584] When applied to nucleic acid sequences, the term "isolated" in the context of the present invention means that the polynucleotide sequence has been removed from its natural genetic environment and therefore does not contain other foreign or unwanted coding sequences (but may include naturally occurring 5' and 3' untranslated regions, such as promoters and terminators), and is in a form suitable for genetically engineered protein production systems. Such isolated molecules are those separated from their natural environment.

[0585] In view of the degeneracy of the genetic code, considerable sequence variation is possible in the polynucleotides of the present invention. The degenerate codons comprising all possible codons for a given amino acid are shown below:

[0586]

[0587]

[0588] Those of ordinary skill in the art will appreciate that there is flexibility in determining degenerate codons, which represent all possible codons for encoding each amino acid. For example, some polynucleotides comprised by a degenerate sequence may encode variant amino acid sequences, but those of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequences of the present invention.

[0589] A "variant" nucleic acid sequence has substantial homology or substantial similarity to a reference nucleic acid sequence (or a fragment thereof). A nucleic acid sequence or a fragment thereof is "substantially homologous" (or "substantially identical") to a reference sequence if, when optimally aligned (with appropriate nucleotide insertions or deletions) to another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99% of the nucleotide bases. Methods for determining homology of nucleic acid sequences are known in the art.

[0590] Alternatively, a "variant" nucleic acid sequence is substantially homologous (or substantially identical) to a reference sequence (or fragment thereof) if the "variant" and reference sequences are capable of hybridizing under stringent (e.g., highly stringent) hybridization conditions. As will be readily appreciated by those skilled in the art, in addition to base composition, the length of complementary strands, and the number of nucleotide base mismatches between hybridizing nucleic acids, nucleic acid sequence hybridization will be affected by conditions such as salt concentration (e.g., NaCl), temperature, or organic solvents. Stringent temperature conditions are preferably used, and typically include temperatures exceeding 30° C., typically exceeding 37° C., and preferably exceeding 45° C. Stringent salt conditions are typically less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. The pH is typically between 7.0 and 8.3. The combination of parameters is much more important than any single parameter.

[0591] Methods for determining nucleic acid sequence identity percentage are known in the art. For example, when assessing nucleic acid sequence identity, a sequence with a limited number of continuous nucleotides can be compared with a nucleic acid sequence (with the same number of continuous nucleotides) from the corresponding portion of the nucleic acid sequence of the present invention. Tools known in the art for determining nucleic acid sequence identity percentage include nucleotide BLAST.

[0592] It is understood by those of ordinary skill in the art that different species exhibit "preferred codon usage". As used herein, the term "preferred codon usage" refers to the codons that are most frequently used in cells of certain species, and therefore favors one or several representatives of possible codons encoding each amino acid. For example, the amino acid threonine (Thr) can be encoded by ACA, ACC, ACG, or ACT, but in mammalian host cells, ACC is the most commonly used codon; in other species, different Thr codons may be preferred. Preferred codons for a particular host cell species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introducing a preferred codon sequence into recombinant DNA can enhance protein production, for example, by making protein translation more efficient within a particular cell type or species.

[0593] Therefore, in one embodiment of the present invention, the codons of the nucleic acid sequence are optimized for expression in a host cell.

[0594] A "fragment" of a polynucleotide of interest comprises a series of consecutive nucleotides from the sequence of the full-length polynucleotide. For example, a "fragment" of a polynucleotide of interest may comprise (or consist of) at least 30 consecutive nucleotides from the sequence of the polynucleotide (e.g., at least 35, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, or 1710 consecutive nucleic acid residues of the polynucleotide). A fragment may include at least one antigenic determinant and / or may encode at least one antigenic epitope of the corresponding polypeptide of interest and / or may have a common antigenic cross-reactivity and / or substantially the same in vivo biological activity as the polypeptide of interest.

[0595] legend

[0596] Figure 1 AB. Example MVA vector construction. Figure 1 A provides a schematic diagram of the cassette "MVAHantaNP". Figure 1B provides a schematic diagram of plasmid 17ACNHBP_MVA-SEOV-HNT-NP_pMS-RQ (pMVAHantaNP).

[0597] Figure 2 PCR confirmation of pure recombinant nucleoprotein - Agarose gel confirmed the presence of the MVAHantaNP construct. Flanking to flank primers (SEQ ID NO: 46 and 47) covered the entire insert and ran from the MVA flanking regions at either end of the vaccine insert, yielding the expected amplification product size. The contents of the wells are as follows (numbered from left to right): 1. Ladder; 2. Positive control (MVA-HantaNP plasmid) GFP to flanking primers - expected size 3260bp; 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) flanking to flanking primers - expected size 3788bp; 9. MVA-Hantanp "Batch 1"; 10. MVA-HantaNP "Batch 2+3"; 11. MVA-HantaNP "Batch 4+5+6"; 12. Negative control; 13. Ladder.

[0598] Figure 3 .Western blot confirmed the expression of NP / Flag tag. The expected size of the protein is 89 kDa. The contents of the wells are as follows (numbered from left to right): 1. Ladder; 2. "Generation 3" P3 (1.1.1); 3. P3 (4.1.1); 4. P3 (4.1.2); 5. Vaccine batch 1; 6. Vaccine batch 2+3 combination; 7. Vaccine batch 4+5+6 combination.

[0599] Figure 4 Clinical scores (% daily weight gain) during the immunization study: (a) weight and (b) temperature of mice after the primary immunization (first arrow, day 0) and booster immunization (second arrow, day 14).

[0600] Figure 5 Total ELISPOT responses from vaccinated and unvaccinated mice.

[0601] Figure 6 .Splenocyte IFN-γ ELISPOT restimulation responses to individual peptide pools ("NP1"-"NP11" therein). i) Group 1 represents mice primed and boosted with MVA-HantaNP; ii) Group 2 represents mice vaccinated with a single dose of MVA-HantaNP; iii) Group 3 represents mice primed and boosted with empty MVA wild-type; and iv) Group 4 represents PBS control, prime, and boost.

[0602] Figure 7 IgG responses to Hantavirus NP in mouse sera. Absorbance readings provided a readout of antibody binding activity to recombinant Hantavirus NP.

[0603] Figure 8 .Weight and temperature of mice after intramuscular challenge (left column) or intranasal challenge (right column) with hantavirus.

[0604] Fig. 9 . Viral load in the blood, lung, kidney, spleen and liver of mice at (a) day 5 after intramuscular challenge; (b) day 5 after intranasal challenge; (c) day 14 after intranasal challenge.

[0605] Fig.10 . Viral loads in kidneys, lungs and spleens of mice at day 5 after intranasal challenge. Results associated with immunization with empty MVA wild-type vector are represented by circles; results associated with immunization with MVA-HantaNP are represented by triangles. Example

[0606] Example 1: Preparation of an exemplary MVA-NP (nucleoprotein) vector

[0607] The cassette of MVAHantaNP (denoted as "MVAHantaNP") was produced by GeneArt (Thermofisher) and contains the p11 promoter, green fluorescent protein (GFP) and MH5 promoter, followed by the kozak sequence upstream of the NP sequence. The nucleoprotein sequence is a chimeric sequence containing two different sequences, Seoul and Hantan. Downstream is a 24-residue linker sequence, followed by a Flag tag epitope and a stop codon. Figure 1 A schematic diagram of MVAHantaNP is provided in (A).

[0608] This cassette was inserted into the SfiI / SfiI cloning site of plasmid pMS-RQ-Bb to generate plasmid 17ACNHBP_MVA-SEOV-HNT-NP_pMS-RQ (pMVAHantaNP).

[0609] Figure 1 A schematic diagram of pMVAHantaNP is provided in (B), and the nucleic acid sequence of pMVAHantaNP is provided in SEQ ID NO:33.

[0610] GTTGGTGGTCGCCATGGATGGTGTTATTGTATACTGTCTAAACGCGTTAGTA

[0611] AAACATGGCGAGGAAATAAATCATATAAAAAATGATTTCATGATTAAACCAT

[0612] GTTGTGAAAAAGTCAAGAACGTTCACATTGGCGGACAATCTAAAAACAAT

[0613] ACAGTGATTGCAGATTTGCCATATATGGATAATGCGGTATCCGATGTATGCAA

[0614] TTCACTGTATAAAAAGAATGTATCAAGAATATCCAGATTTGCTAATTTGATAA

[0615] AGATAGATGACGATGACAAGACTCCTACTGGTGTATATAATTATTTTAAACCT

[0616] AAAGATGCCATTCCTGTTATTATATCCATAGGAAAGGATAGAGATGTTTGTG

[0617] AACTATTAATCTCATCTGATAAAGCGTGTGCGTGTATAGAGTTAAATTCATAT

[0618] AAAGTAGCCATTCTTCCCATGGATGTTTCCTTTTTTACCAAAGGAAATGCAT

[0619] CATTGATTATTCTCCTGTTTGATTTCTCTATCGATGCGGCACCTCTCTTAAGA

[0620] AGTGTAACCGATAATAATGTTATTATATCTAGACACCAGCGTCTACATGACG

[0621] AGCTTCCGAGTTCCAATTGGTTCAAGTTTTACATAAGTATAAAGTCCGACTA

[0622] TTGTTCTATATTATATATGGTTGTTGATGGATCTGTGATGCATGCAATAGCTGA

[0623] TAATAGAACTTACGCAAATATTAGCAAAAATATATTAGACAATACTACAATTA

[0624] ACGATGAGTGTAGATGCTGTTATTTTGAACCACAGATTAGGATTCTTGATAG

[0625] AGATGAGATGCTCAATGGATCATCGTGTGATATGAACAGACATTGTATTATG

[0626] ATGAATTTACCTGATGTAGGCGAATTTGGATCTAGTATGTTGGGGAAATATG

[0627] AACCTGACATGATTAAGATTGCTCTTTCGGTGGCTGGGTACCAGGCGCGCC

[0628] TTTCATTTTGTTTTTTTCTATGCTATAAATGGTGAGCAAGGGCGAGGAGCTG

[0629] TTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGG

[0630] CCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCA

[0631] AGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGG

[0632] CCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTAC

[0633] CCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGG

[0634] CTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGA

[0635] CCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAG

[0636] CTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCT

[0637] GGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGA

[0638] AGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGC

[0639] AGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGG

[0640] CCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGA

[0641] GCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTG

[0642] ACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGAGCT

[0643] CCGGCCCGCTCGAGGCCGCTGGTACCCAACCTAAAAATTGAAAATAAATAC

[0644] AAAGGTTCTTGAGGGTTGTGTTAAATTGAAAGCGAGAAATAATCATAAATA

[0645] AGCCCGGTGCCACCATGGCCACAATGGAAGAGATCCAGAGAGAGATCAGC

[0646] GCCCACGAGGGACAGCTGGTTATCGCCAGACAGAAAGTGAAGGACGCCG

[0647] AGAAGCAGTACGAGAAGGACCCCGACGATCTGAACAAGAGAGCCCTGCA

[0648] CGACAGAGAAAGCGTGGCCGCCTCTATCCAGAGCAAGATCGATGAGCTGA

[0649] AGAGACAGCTGGCCGACAGAATCGCCGCTGGCAAGAATATTGGCCAGGAC

[0650] AGAGATCCCACAGGCGTGGAACCTGGCGATCACCTGAAAGAGAGAAGCG

[0651] CCCTGTCCTATGCAACACCCTGGACCTGAACAGCCTGGACATTGATGAGC

[0652] CTACCGGCCAGACAGCCGACTGGCTGACAATCATTGTGTACCTGACCAGCT

[0653] TCGTGGTCCCCATCATCCTGAAGGCCCTGTACATGCTGACCACCAGAGGCA

[0654] GACAGACCAGCAAGGACAACAAGGGCATGAGAATCCGGTTCAAGGATGA

[0655] CAGCAGCTACGAGGACGTGAACGGCATTAGAAAGCCCAAGCACCTGTACG

[0656] TGTCCATGCCTAACGCTCAGAGCAGCATGAAGGCCGAGGAAATCACCCCT

[0657] GGCAGATTCAGAACAGCCGTGTGCGGACTGTACCCCGCTCAGATCAAGGC

[0658] CAGAAACATGGTGTCCCCAGTGATGAGCGTCGTGGGATTTCTGGCCCTGGC

[0659] TAAGGACTGGACCAGCAGGAGGATTGAGGAATGGCTGGGAGCCCCTTGCAAGT

[0660] TTATGGCCGAGTCTCCTATCGCCGGCAGCCTGTCTGGCAACCCCGTGAATA

[0661] GAGACTACATCAGACAGAGGCAGGCGCTCTGGCCGGAATGGAACCCAAA

[0662] GAATTTCAGGCCCTGCGGCAGCACTCTAAGGATGCCGGATGTACCCTGGTG

[0663] GAACACATTGAGAGCCCCAGCAGCATCTGGGTTTTCGCTGGCGCTCCTGAT

[0664] AGATGCCCTCCTACCTGTCTGTTTGTTGGCGGAATGGCCGAGCTGGGCGCC

[0665] TTCTTTAGCATTCTGCAGGACATGCGGAATACCATCATGGCCAGCAAGACC

[0666] GTGGGCACCGCCGATGAGAAGCTGAGAAAGAAGTCCAGCTTCTACCAGAG

[0667] CTACCTGCGGAGAACCCAGAGCATGGGCATTCAGCTGGACCAGAGAATCA

[0668] TCGTGATGTTCATGGTGGCCTGGGGCAAAGAAGCCGTGGACAATTTTCACC

[0669] TGGGCGACGACATGGACCCCGAGCTGAGATCTCTGGCCCAGATCCTGATCG

[0670] ACCAGAAAGTCAAAGAGATCTCCAATCAAGAGCCCATGAAGCTGATGCTG

[0671] AGCTACGGCAACGTGCTGGATCTGAACCACCTGGATATCGACGAGCCAAC

[0672] AGGACAGACCGCTGATTGGCTGGGCATCGTGATCTACCTGACCTCCTTTGT

[0673] GGTGCCTATTCTGCTCAAAGCCCTCTATATGCTGACAACACGCGGAAGGCA

[0674] GACCACCAAAGATAACAAAGGCACCCGGATCAGGTTTAAGGACGACAGCT

[0675] CCTTTGAGGATGTCAACGGCATCCGGAAACCTAAGCACCTCTATGTGTCTC

[0676] TGCCCAATGCACAGTCCTCCATGAAGGCAGAAGAGATCACACCAGGCCGG

[0677] TACAGAACCGCCATCTGTGGACTGTATCCTGCACAAATCAAAGCCCGGCAG

[0678] ATGATCAGCCCCGTGATGTCCGTTATCGGATTCCTGGCTCTGGCCAAAGATT

[0679] GGAGCGACAGGATCGAGCAGTGGCTGAGCGAGCCTTGCAAGCTGCTTCCT

[0680] GATACAGCCGCTGTGTCACTGCTTGGCGGCCCTGCCACAAACAGAGATTAC

[0681] CTGAGACAGAGACAGGTGGCACTGGGCAACATGGAAACAAAAGAGAGCA

[0682] AGGCCATCCGGCAGCATGCCGAAGCTGCTGGCTGTAGCATGATCGAGGATA

[0683] TCGAGTCCCCTAGCTCCATTTGGGTGTTCGCAGGGGCCCCAGATAGATGTC

[0684] CACCAACATGCCTGTTCATTGCCGGCATGGCTGAACTGGGAGCTTTTTTCA

[0685] GCATCCTCCAGGATATGCGCAACACGATTATGGCCTCCAAGACAGTGGGAA

[0686] CCAGCGAGGAAAAGCTGCGGAAGAAAAGCAGCTTTTACCAGTCTTACCTG

[0687] AGGCGGACCCAGTCCATGGGGATCCAACTGGATCAGCGGATCATTGTGCTG

[0688] TTTATGGTCGCTTGGGGAAAAGAGGCTGTCGATAACTTCCACCTGGGAGAT

[0689] GATATGGATCCTGAACTGCGGACCCTGGCTCAGTCCCTGATCGATGTGAAA

[0690] GTGAAAGAAATTAGTAATCAAGAACCCCTCAAGCTGGACCTGGAAGGCCC

[0691] TAGATTCGAGGACTACAAGGACGATGACGACAAGTGACTCGACCTGCAGT

[0692] TTTTATGGAAAGTTTTATAGGTAGTTGATAGAACAAAATACATAATTTTGTAA

[0693] AAATAAATCACTTTTTATACTAATATGACACGATTACCAATACTTTTGTTACT

[0694] AATATCATTAGTATACGCTACACCTTTTCCTCAGACATCTAAAAAAATAGGT

[0695] GATGATGCAACTTTATCATGTAATCGAAATAATACAAATGACTACGTTGTTAT

[0696] GAGTGCTTGGTATAAGGAGCCCAATTCCATTATTCTTTTAGCTGCTAAAAGC

[0697] GACGTCTTGTATTTTGATAATTATACCAAGGATAAAATATCTTACGACTCTCC

[0698] ATACGATGATCTAGTTACAACTATCACAATTAAATCATTGACTGCTAGAGAT

[0699] GCCGGTACTTATGTATGTGCATTCTTTATGACATCGCCTACAAATGACACTGA

[0700] TAAAGTAGATTATGAAGAATACTCCACAGAGTTGATTGTAAATACAGATAGT

[0701] GAATCGACTATAGACATAATACTATCTGGATCTACACATTCACCGGAAACTAGTTG(SEQ ID NO:33)

[0702] pMVAHantaNP contains:

[0703] Left flank of DelIII:

[0704] GTTGGTGGTCGCCATGGATGGTGTTATTGTATACTGTCTAAACGCGTTAGTA

[0705] AAACATGGCGAGGAAATAAATCATATAAAAAATGATTTCATGATTAAACCAT

[0706] GTTGTGAAAAAGTCAAGAACGTTCACATTGGCGGACAATCTAAAAACAAT

[0707] ACAGTGATTGCAGATTTGCCATATATGGATAATGCGGTATCCGATGTATGCAA

[0708] TTCACTGTATAAAAAGAATGTATCAAGAATATCCAGATTTGCTAATTTGATAA

[0709] AGATAGATGACGATGACAAGACTCCTACTGGTGTATATAATTATTTTAAACCT

[0710] AAAGATGCCATTCCTGTTATTATATCCATAGGAAAGGATAGAGATGTTTGTG

[0711] AACTATTAATCTCATCTGATAAAGCGTGTGCGTGTATAGAGTTAAATTCATAT

[0712] AAAGTAGCCATTCTTCCCATGGATGTTTCCTTTTTTACCAAAGGAAATGCAT

[0713] CATTGATTATTCTCCTGTTTGATTTCTCTATCGATGCGGCACCTCTCTTAAGA

[0714] AGTGTAACCGATAATAATGTTATTATATCTAGACACCAGCGTCTACATGACG

[0715] AGCTTCCGAGTTCCAATTGGTTCAAGTTTTACATAAGTATAAAGTCCGACTA

[0716] TTGTTCTATATTATATATGGTTGTTGATGGATCTGTGATGCATGCAATAGCTGA

[0717] TAATAGAACTTACGCAAATATTAGCAAAAATATATTAGACAATACTACAATTA

[0718] ACGATGAGTGTAGATGCTGTTATTTTGAACCACAGATTAGGATTCTTGATAG

[0719] AGATGAGATGCTCAATGGATCATCGTGTGATATGAACAGACATTGTATTATG

[0720] ATGAATTTACCTGATGTAGGCGAATTTGGATCTAGTATGTTGGGGAAATATGAACCTGACATGATTAAGATTGCTCTTTCGGTGGCTGG(SEQ ID NO:34) First adapter:

[0721] GTACAGGCGCGCC(SEQ ID NO:35)

[0722] P11:

[0723] TTTCATTTTGTTTTTTTCTATGCTATAA(SEQ ID NO:36)

[0724] GFP:

[0725] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT

[0726] CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGG

[0727] GCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACC

[0728] ACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTAC

[0729] GGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTT

[0730] CTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTT

[0731] CAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGC

[0732] GACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGA

[0733] CGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACG

[0734] TCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGA

[0735] TCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAG

[0736] CAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTA

[0737] CCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATC

[0738] ACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA(SEQ ID NO:37)

[0739] Second adapter:

[0740] GAGCTCCGGCCCGCTCGAGGCCGCTGGTACCCAACCT(SEQ ID NO:38)MH5 promoter:

[0741] AAAAATTGAAAATAAATACAAAGGTTCTTGAGGGTTGTGTTAAATTGAAAGCGAGAAATAATCATAAATA (SEQ ID NO: 39)

[0742] Third connector:

[0743] AGCCCGGT Kozak sequence:

[0744] GCCACCATGG (SEQ ID NO:41). The 3' end of the Kozak sequence overlaps with four nucleic acids at the 5' end of SEQ ID NO:29.

[0745] Nucleoprotein (SEQ ID NO: 29)

[0746] Fourth connector:

[0747] GACCTGGAAGGCCCTAGATTCGAG(SEQ ID NO:42)

[0748] Flag tag: GACTACAAGGACGAT GACGAC AAG (SEQ ID NO: 43)

[0749] termination:

[0750] TGA

[0751] Fifth connector:

[0752] CTCGACCTGCAGTTTTATG(SEQ ID NO:44)

[0753] DelIII right wing:

[0754] (SEQ ID NO:45)

[0755] From transformed bacteria (Escherichia coli K12 DH10B TM Plasmid DNA was purified from 100 μL T1R (10 μL) and the concentration was determined by UV spectroscopy using GeneArt (Thermofisher).

[0756] BHK-21 cells were infected with MVA1974 at a multiplicity of infection of 0.05. According to the manufacturer's instructions, cells infected with pMVAHantaNP were transfected using lipofectamine (Life Technologies). Based on GFP expression, the resulting recombinant MVAHantaNP was continuously plaque purified 4 times in chicken embryo fibroblasts ("CEF") cells. MVAHantaNP was amplified on CEF cells, purified by centrifugation with a sucrose cushion, and titrated by plaque determination on CEF cells before use in vivo. GFP fluorescence was used and plaques were visualized by immunostaining with rabbit anti-vaccinia antibodies (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, and genotype analysis was performed using KAPA2G FastHotStart PCR kit (KAPABiosystems, USA).

[0757] The presence of the MVAHantaNP construct was confirmed by polymerase chain reaction (PCR). A set of primers was designed specifically to detect the HantaNP of the MVA flanking region with an expected size of 3260 bp - this is Figure 2 Shown in.

[0758] Sequencing of the expressed protein confirmed very high sequence fidelity. The recombinant purified MVA Hanta NP was then filled into flasks of increasing size in stages by tissue culture. Initially, MVA Hanta NP was grown in vials of chicken embryo fibroblast (CEF) cells and harvested before infection into slightly larger CEF cell bottles. The process was repeated in larger and larger flasks until MVA Hanta NP successfully infected 10× large flasks of CEF cells. Sucrose cushion centrifugation was performed; the virus pellet was resuspended in PBS and prepared for immunogenicity studies. A total of six batches were produced. Batches 2+3 and 4+5+6 were merged into a single sample and the virus concentration was titrated.

[0759] The purified vaccine batches were compared to a positive control (the original received plasmid from Geneart). A second set of primers was designed to identify the entire insert from both MVA flanking regions. The results showed the 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 had the same expected size product as the positive control.

[0760] Primer details are as follows:

[0761] SEQ ID NO:46:CGGCACCTCTCTTAAGAAGT (Fwd target Del III left flank)

[0762] SEQ ID NO:47:GTGTAGCGTATACTAATGATATTAG (Rev target Del III right flank)

[0763] SEQ ID NO:48: GGAGTACAACTACAACAGCCACAACG (Fwd target GFP)

[0764] The GFP Fwd primer binds to the GFP sequence and, when used in combination with the Rev Del III right flank primer, covers GFP to the right MVA flank via the nucleoprotein and specifically identifies the presence of the NP gene.

[0765] Detection of protein expression

[0766] CEF cells were infected with MVAHantaNP at a multiplicity of infection of 0.05 and incubated at 37°C in modified Eagle's medium (MEM) supplemented with 2% FBS (Sigma-Aldrich, UK). Once good GFP fluorescence and CPE were observed under a microscope, the medium was removed after 48 hours. Reducing sample buffer (containing Sample reduction buffer LDS sample buffer) (Thermofisher, UK) lysed cells, transferred to Eppendorf tubes and heated at 70 ° C for 10 minutes. Uninfected cells were treated in the same manner as the negative control. MVAHantaNP lysate was subjected to SDS-PAGE on 4-12% Bis-Tris gel (Life technologies) and the protein was transferred to a nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% milk powder (Merck Millipore), then incubated with shaking for 1-2 hours in the presence of a primary antibody (rabbit anti-V5 polyclonal (Invitrogen) at 1 / 1000 in PBS-0.05% Tween), and then washed 3 times in PBS containing 0.05% Tween-20 (Sigma-Aldrich). The membrane was incubated with shaking for 1 hour in the presence of a HRP-conjugated secondary antibody (anti-rabbit IgG peroxidase (Sigma-Aldrich) at 1 / 1000 in PBS-0.05% Tween), and washed as described above. Protein expression was determined by detecting bound antibodies using the Pierce ECL WB Substrate Kit (Thermofisher) according to the manufacturer's instructions and visualized in a chemiluminescent imager (Syngene). Molecular weights were determined using MagicMark XP Western Protein Standards (Invitrogen) as a reference.

[0767] Western blot analysis (see Figure 3 ) confirmed the expression of the flag tag located downstream of NP. The expected size of the protein (NP+joint and flag tag) is 89kDa, and the protein sequence is provided in SEQ ID NO:49. Expression was observed from the 3rd generation pick to the vaccine batch (the inventors observed a low level of protein degradation, which is not considered significant). The target band is at the expected size of the protein, which again indicates good expression.

[0768] MATMEEIQREISAHEGQLVIARQKVKDAEKQYEKDPDDLNKRALHDRESVAA

[0769] SIQSKIDELKRQLADRIAAGKNIGQDRDPTGVEPGDHLKERSALSYGNTLDLN

[0770] SLDIDEPTGQTADWLTIIVYLTSFVVPIILKALYMLTTRGRQTSKDNKGMRIRFK

[0771] DDSSYEDVNGIRKPKHLYVSMPNAQSSMKAEEITPGRFRTAVCGLYPAQIKAR

[0772] NMVSPVMSVVGFLALAKDWTSRIEEWLGAPCKFMAESPIAGSLSGNPVNRD

[0773] YIRQRQGALAGMEPKEFQALRQHSKDAGCTLVEHIESPSSIWVFAGAPDRCPP

[0774] TCLFVGGMAELGAFFSILQDMRNTIMASKTVGTADEKLRKKSSFYQSYLRRT

[0775] QSMGIQLDQRIIVMFMVAWGKEAVDNFHLGDDMDPELRSLAQILIDQKVKEIS

[0776] NQEPMKLMLSYGNVLDLNHLDIDEPTGQTADWLGIVIYLTSFVVPILLKALYM

[0777] LTTRGRQTTKDNKGTRIRFKDDSSFEDVNGIRKPKHLYVSLPNAQSSMKAEEI

[0778] TPGRYRTAICGLYPAQIKARQMISPVMSVIGFLALAKDWSDRIEQWLSEPCKLL

[0779] PDTAAVSLLGGPATNRDYLRQRQVALGNMETKESKAIRQHAEAAGCSMIEDIE

[0780] SPSSIWVFAGAPDRCPPTCLFIAGMAELGAFFSILQDMRNTIMASKTVGTSEEK

[0781] LRKKSSFYQSYLRRTQSMGIQLDQRIIVLFMVAWGKEAVDNFHLGDDMDPELRTLAQSLIDVKVKEISNQEPLKLDLEGPRFEDYKDDDDK(SEQ ID NO:49)

[0782] 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.

[0783] Example 2. Immunogenicity of MVA Hanta NP in A129 mice

[0784] Eighty male 6-8 week old A129 mice were randomly divided into four groups and ear-marked before inoculation.

[0785] Group 1 received 1 × 10 7 PFU of MVAhantANPI in endotoxin-free phosphate buffered saline (PBS) for two doses of vaccination.

[0786] Group 2 received 1×10 7 A single vaccine injection of 100 plaque forming units (pfu) of MVA Hanta NP in endotoxin-free PBS.

[0787] Group 3 received 1 × 10 7 Two doses of vaccination with MVA empty vector at 100 PFU in endotoxin-free PBS.

[0788] Group 4 received two vaccination doses of endotoxin-free PBS on days 0 and 14 as a negative control.

[0789] All mice were injected intramuscularly into the tail thigh. 100 μ l was used at each inoculation (50 μ l was used in each thigh). Animal weights were recorded every day throughout the study. Five animals in each group were euthanized, and spleen tissue and blood were collected on the 28th day after the initial inoculation. All efforts were made to minimize animal suffering. These studies were approved by the ethical review process of the PHE of Porton Down, UK and the UK Home Office through project license number 30 / 2993. Work was carried out according to the Animals (Scientific Procedures) Act 1986 and the Animal Care Practice Code (1989) used in the Home Office (UK) scientific procedures.

[0790] Throughout the study, no clinical signs related to vaccination were observed, and all mice gained weight as expected (see Figure 4 a). As expected, all four groups gained weight throughout the study, and Group 4 was consistently lower than Groups 1-3. At the end of the study, similar % weight gain was observed in all groups. These clinical data suggest that the mice tolerated the vaccine without side effects.

[0791] To determine T cell responses in immunized animals, an interferon-γ ELISPOT assay was used to measure the frequency of responding T cells after stimulation with hantavirus-specific peptides.

[0792] Spleens from test animals were collected aseptically, homogenized, and erythrocytes were lysed. Splenocytes were resuspended in RPMI medium (Sigma-Aldrich) supplemented with 5% FBS, 2mM L-glutamine, 100U penicillin and 0.1mg / ml streptomycin, 50mM 2-mercaptoethanol, and 25mM HEPES solution (Sigma-Aldrich). Antigen recall responses of splenocytes were assessed by IFN-γ ELISPOT (Mabtech, Sweden), performed according to the manufacturer's instructions. Cells were seeded in PVDF microtiter plates at 2×10e6 per well and restimulated with a peptide library (JPT, Berlin).

[0793] Peptides spanning the Hanta NP protein sequence were 15 residues long, with an overlap of 11 residues between peptides. A total of 189 peptides were generated and tested in 11 peptide pools (see Table 1).

[0794] Table 1: Peptide library (the starting amino acid ("AA") number corresponds to the amino acid number in SEQ ID NO: 31)

[0795]

[0796]

[0797]

[0798]

[0799]

[0800] They were applied to the cells at a final concentration of 2.5 μg / ml per peptide, with 17 peptides in each of pools 1 to 10 and 19 peptides in pool 11. The plates were developed after 18 hours at 37°C, 5% CO2 in a humidified incubator. Spots were visually counted on an automated ELISPOT reader (Cellular Technologies Limited, USA). Background values ​​from wells containing cells and medium but no peptide were subtracted, and data are presented as responses to individual pools or as summed across target proteins. Results are expressed as 10 6 The spot forming units (SFU) of each cell.

[0801] The MVA-WT group and the PBS group (Groups 3 and 4) were negative when stimulated with all Hanta NP pools. In both the prime / boost and prime groups, IFN-γ responses were detected to several peptide pools, and particularly strong responses were directed to 2 different regions of the NP (corresponding to pools 4 and 9).

[0802] The inventors found that with respect to SEQ ID NOs: 11 and 12, T cell (IFN-γ) stimulation was greatly increased.

[0803] Increased responses to pools 2, 3, 5, 7, 8, and 10 were also detected for both the prime / boost and prime groups compared to the control group. Figure 5 Total ELISPOT responses from vaccinated and unvaccinated mice are provided in; and Figure 6 ELISPOT responses to individual peptide pools are shown.

[0804] To measure the antibody response in immunized mice, ELISA analysis was performed to evaluate the binding of antibodies to Hantavirus-specific proteins. Recombinant Hanta NPs (Native Antigen Company, UK) as crude lysates were 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, incubated overnight at 4°C, 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 for 1 hour at 37°C, then washed again in PBS + 0.01% Tween-20. The samples were diluted 1:50 in 5% milk powder in PBS + 0.01% Tween-20 buffer, added to the plate in triplicate (100 μl / well) and incubated at 37°C for 1 hour. Normal mouse serum (Sigma-Aldrich) and polyclonal anti-hantavirus hyperimmune mouse ascites samples (BEI Resources, USA) were used as positive and negative control samples, respectively. The plates were washed with PBS + 0.01% Tween-20, and 100 μl of polyclonal anti-mouse HRP conjugate (Sigma-Aldrich) diluted 1:20,000 in 5% milk PBS + 0.01% Tween-20 was added to each well. After incubation for another 1 hour at 37°C, the plates were washed with PBS + 0.01% Tween-20, and 100 μl of TMB substrate (Surmodics) was added to each well, followed by incubation at 20°C for 1 hour. The reaction was stopped by adding 100 μl of stop solution (Surmodics) prepared according to the manufacturer's instructions and the plate was 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 sample values ​​and results were reported as absorbance (450 nm) at a 1:50 dilution. Data were interpreted and analyzed using Graph Pad Prism 7 (see Figure 7 ).

[0805] The MVA-WT and PBS control groups showed very little absorbance, with values ​​similar to those in the blank wells. The responses of all mice in the prime and prime / boost vaccination groups were significantly higher. The average absorbance recorded in the prime group alone was ˜2.3, and the average OD recorded in the prime / boost group was ˜1.5.

[0806] Thus, the vectors of the present invention demonstrate highly desirable induction of both cellular and humoral immune responses.

[0807] Example 3. Efficacy test

[0808] Sixty male A129 mice weighing 19-21 g were pre-randomized into four groups and then ear-tagged and microchipped for identification, weight monitoring, and temperature monitoring.

[0809] The remaining mice not culled on day 28 for immunogenicity studies were challenged with Hanta SEOV on day 28. In each group, 1.36 × 10 6 TCID50 / dose n=10 animals were challenged via the intranasal route and n=5 animals were challenged via the intramuscular route.

[0810] Animals attacked intramuscularly were euthanized on the 33rd day. Mice attacked intranasally were euthanized on the 33rd day (5 per group) or the 42nd day (5 per group). Blood, saliva, liver, kidney, lung and spleen were collected for histological and viral load analysis. All efforts were made to minimize animal suffering. These studies were approved by the ethical review process of the PHE of Porton Down, UK and the UK Home Office through project license number 30 / 2993. Work was performed according to the Animals (Scientific Procedures) Act 1986 and the Animal Care Practice Code (1989) used in the Home Office (UK) scientific procedures.

[0811] Clinical signs:

[0812] Animal weights and temperatures were recorded daily throughout the study. All challenged animals remained healthy and no clinical signs were observed after challenge with hantavirus. Temperatures and body weights throughout the study are reported in Figure 8 middle.

[0813] Viral load:

[0814] Viral load was assessed 5 and 14 days after challenge. Fig. 9 As shown, immunization with MVA Hanta NPs achieved a reduction or complete clearance of Hantavirus from the tested tissues already on day 5. Highly favorable reductions in viral loads were also observed in most tissues 14 days after challenge.

[0815] Viral Load-Follow-up Study:

[0816] In the follow-up study, 28 female A129 mice were pre-randomized into two groups and then ear-tagged and microchipped for identification, weight monitoring, and temperature monitoring.

[0817] Of these 28 mice, 16 were primed with GLP-grade MVA HantaNP on day 0 and then boosted on day 14 ("Group A"); and 12 mice received prime and boost immunizations with empty MVA wild-type vectors on days 0 and 14, respectively ("Group B"). Immunizations were performed according to Example 2 above.

[0818] On day 28, 3 × 10 6 Dosage of TCID50 / mouse Eight mice in group A and eight mice in group B were challenged intranasally with Hanta SEOV.

[0819] In this follow-up study, viral load was assessed 5 days after challenge. Fig.10 As shown, immunization with MVA Hanta NPs achieved a favorable reduction of hantavirus in the tested tissues, even when the challenge dose was exceeded by two times.

[0820] Example 4: Preparation of an exemplary adenoviral vector

[0821] A non-replicating adenovirus is engineered to express the Hantavirus NP nucleic acid or fragments thereof of the present invention. The genetic sequence of the Hantavirus NP is inserted into the genome of the adenovirus vector. The expression of the Hantavirus NP is indicated by reactivity between NP-specific antibodies and adenovirus products by Western blot or ELISA as follows:

[0822] Cell lysates of cells infected with the recombinant adenovirus were subjected to SDS-PAGE and Western blotting using a hantavirus NP-specific antibody, showing specific reactivity compared to a negative control.

[0823] Alternatively, products from cells infected with recombinant adenovirus are used to coat ELISA plates. Hantavirus-specific antibodies bind to the coating and are detected by a chemical reaction.

[0824] Example 5: Hantavirus vaccines provide cross-strain protection

[0825] A vaccine expressing the Hantavirus NP nucleic acid of the present invention or a fragment thereof in an adenoviral or non-replicating poxvirus vector is delivered parenterally to mice susceptible to diseases caused by Hantavirus. They are challenged with a lethal dose of Hantavirus from a strain different from the strain on which the vaccine is based. The challenged animals show no or mild clinical signs of disease and do not require euthanasia. Control animals that received the same challenge dose of Hantavirus but did not receive the vaccine showed severe signs of disease, reached humane clinical endpoints and required euthanasia.

[0826] Example 6. Preparation and efficacy of recombinant influenza virus vector

[0827] Reverse genetics was used to construct a recombinant influenza virus carrying a protective epitope of Hantavirus NP in the neuraminidase stalk. Hantavirus-specific cytotoxic T lymphocytes (CTLs) were induced in mice following intranasal or parenteral administration. These CTLs provided a reduction in viral load and clinical disease after challenge with Hantavirus.

[0828] Example 7. Preparation and efficacy of recombinant bacterial vectors

[0829] The Hantavirus NP nucleic acid or fragments thereof of the present invention are expressed on the surface of genetically attenuated Gram-negative bacteria. After intranasal or parenteral administration to mice, the bacterial vector colonizes antigen presenting cells (e.g., dendritic cells or macrophages). Humoral and cellular Hantavirus-specific immune responses are induced. These immune responses provide a reduction in viral load and clinical disease after challenge with Hantavirus.

Claims

1. A viral vector or a bacterial vector, (A) The vector comprises: (i) a first nucleic acid sequence selected from the group consisting of SEQ ID NOs: 17 and 24; and (ii) a second nucleic acid sequence selected from SEQ ID NOs: 21 and 28; and / or (B) the vector comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 29 or 30; wherein the first and second nucleic acid sequences are capable of inducing an immune response in a subject; and wherein the nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 29 or 30 is capable of inducing an immune response in a subject.

2. The vector of claim 1(A), wherein the vector comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:29 or SEQ ID NO:

30.

3. The vector of claim 1 or claim 2, wherein the vector comprises the nucleic acid sequence of SEQ ID NO:

29.

4. The vector of claim 1 or claim 2, wherein the vector comprises the nucleic acid sequence of SEQ ID NO:

30.

5. The vector of any preceding claim, wherein the immune response is a protective immune response.

6. The vector of any preceding claim, wherein the vector is a viral vector.

7. The vector of claim 6, wherein the vector is a non-replicating poxvirus vector.

8. The vector of claim 7, wherein the non-replicating poxvirus vector is selected from the group consisting of a modified vaccinia virus Ankara (MVA) vector, a NYVAC vaccinia virus vector, a canarypox (ALVAC) vector, and a fowlpox (FPV) vector.

9. The vector of claim 7 or claim 8, wherein the non-replicating poxvirus vector is an MVA vector.

10. The vector of claim 7 or claim 8, wherein the non-replicating poxvirus vector is a fowlpox vector.

11. The vector of claim 6, wherein the vector is an adenovirus vector.

12. The vector of claim 11, wherein the adenoviral vector is a non-replicating adenoviral vector.

13. The vector of claim 11 or claim 12, wherein the adenovirus vector is selected from the group consisting of: 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.

14. The vector of claim 6, wherein the vector is a measles virus vector.

15. The vector of claim 14, wherein the measles virus vector is a non-replicating measles virus vector.

16. A nucleic acid sequence encoding a viral vector according to any one of claims 1 to 15.

17. A method for preparing 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 to 15; transfecting a host cell with the nucleic acid; Cultivating the host cell under conditions suitable for vector proliferation; and The vector is obtained from the host cell.

18. A host cell comprising the nucleic acid sequence of claim 16.

19. A composition comprising the carrier according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier.

20. The composition of claim 19, further comprising an adjuvant.

21. A carrier according to any one of claims 1 to 15 or a composition according to claim 19 or claim 20 for use in medicine.

22. A vector according to any one of claims 1 to 15 or a composition according to claim 19 or claim 20 for use in a method of inducing an immune response in a subject.

23. A vector for use according to claim 22, wherein the immune response comprises a T cell response.

24. A vector according to any one of claims 1 to 15, or a composition according to claim 19 or claim 20, for use in a method of preventing or treating a hantavirus infection in a subject.

25. The vector according to claim 24, or the composition according to 24, for use in a method for preventing or treating hemorrhagic fever with renal syndrome in a subject.

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