Hantavirus antigenic composition

Abstract

To provide a protective vaccine against Hantavirus infection and a therapeutic agent for prevention, treatment, and suppression of Hantavirus infection.SOLUTION: The present invention provides a viral vector or bacterial vector, the vector comprising a nucleic acid sequence encoding a Hantavirus nucleoprotein or antigenic fragment thereof; wherein the vector is capable of inducing a protective immune response in a subject. The present invention also provides compositions and uses of the vector in methods of medical treatment.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to virus vectors and bacterial vectors containing hantavirus antigens, and their use in immunogenic and antigenic compositions. The present invention also relates to the preventive use of said compositions. The present invention also relates to an immunogen for use in the production of therapeutic antibodies and a method for generating said immunogen.

Background Art

[0002] Hantavirus is an emerging zoonotic infectious virus distributed worldwide. There are a number of hantavirus strains, roughly classified into three serotypes. Hantavirus is the causative agent of hantavirus pulmonary syndrome (HPS), a severe human respiratory disease in which typically 36% of cases are fatal, and in some epidemics a 50% fatality rate has been recorded. It is also the causative agent of clinically similar groups of diseases in which up to 15% of cases can be fatal, hemorrhagic fever with renal syndrome (HFRS). Hantavirus is typically transmitted to humans by exposure to aerosolized body fluids or feces of infected small

[0003] mammals, typically rodents. Human-to-human transmission has also been reported. According to the Centers for Disease Control and Prevention (CDC), symptoms associated with hantavirus infection include fever, headache, muscle pain, and severe respiratory distress. Symptoms associated with HPS can include fatigue, chills, dizziness, dry cough, nausea, vomiting, and other gastrointestinal Later symptoms of HFRS include hypotension, acute shock, vascular leakage, and acute renal failure, which can lead to severe fluid overload. Summary of the Invention [Problem to be solved by the invention]

[0004] There is currently no licensed vaccine against hantavirus. There is no clear treatment or cure for antiviral infections, HPS, or HFRS. Patients with HPS are admitted to intensive care units and provided with support during severe respiratory distress. HPS is treated with intubation and oxygen therapy to help relieve respiratory distress. Successful treatment of HPS depends on the severity of the respiratory distress. Treatment of HFRS depends on the severity of the illness and early detection of infection. Management of blood pressure, oxygen and blood pressure levels; dialysis to correct severe fluid overload; The antiviral drug ribavirin is used to treat HFR and any secondary infections. When used very early in the course of clinical disease due to S., it has been shown to reduce illness and mortality. However, no benefit of ribavirin has been found in patients with HPS. Not yet.

[0005] Therefore, there is a significant need for a protective vaccine against hantavirus infection. Further therapeutic agents for the prevention, treatment and control of tuberculosis virus infections are also urgently needed. There are. [Means for solving the problem]

[0006] The present invention relates to a method for the production of hantavirus nucleoprotein (NP) or an antigenic fragment thereof. The viral and bacterial vectors are provided in the corresponding compositions and for the determination of hantavirus infection. By providing, together with the use of said vectors and compositions in prevention and treatment, Addressing one or more of the issues above.

[0007] The vectors and compositions of the present invention can be used to induce an immune response against hantavirus in an individual (i.e. and (c) stimulating (i.e., inducing) the IL-16 antibody in a subject (e.g., a subject) to provide improved immunogenicity and efficacy. do.

[0008] In one aspect, the invention provides a nucleic acid sequence encoding hantavirus NP or an antigenic fragment thereof. A viral or bacterial vector that contains a sequence and is capable of inducing an immune response in an individual. The present inventors provide a vector for carrying out a gene transfer using a viral or bacterial vector. By delivering a nucleic acid sequence encoding the NP of the HIV virus (or an antigenic fragment thereof) to a subject, This has led to the development of a highly effective immune response against hantavirus in individuals. Found it.

[0009] In a preferred embodiment, the vector of the invention is a viral vector.

[0010] Hantaviruses are enveloped viruses that belong to the Bunyaviridae family. It is a genus of enveloped, single-stranded, tripartite, negative-sense RNA viruses. More than 20 strains of hantaviruses that are pathogenic to Hantavirus strains are either Old World or New World. Old World strains include Seoul virus ("SEOV"; worldwide distribution), Puerto Rico virus ("Puerto Rico virus"; worldwide distribution), and The Marburg virus (mainly distributed in Europe), the Hantaan virus ("HNT"; mainly distributed in Asia), and the Dobravavirus (mainly distributed in Europe) are included and are typically associated with causing HFRS. New World strains include the Sin Nombre virus (mainly distributed in North America) and the Andes virus (mainly distributed in Latin America) and are typically associated with HPS.

[0011] The hantavirus genome consists of three single-stranded RNA segments called small (S), medium (M), and large (L). The S segment is 1 - 3 kb and encodes the nucleocapsid protein (NP). The M segment is 3.2 - 4.9 kb and encodes the glycoproteins (GP), Gn, and Gc. The L segment is 6.8 - 12 kb and encodes the viral RNA-dependent RNA polymerase.

[0012] The hantavirus glycoproteins, Gn and Gc, play important roles in the infection of target cells through interaction with specific entry receptors, such as integrin. The hantavirus NP forms a complex with the viral polymerase and ribonucleoprotein and plays multiple roles in viral replication. NP also plays a role in enhancing the translation of viral RNA by host cells, downregulating apoptosis, inhibiting interferon signaling responses, and blocking the TNFα-induced activation of NF-κB. It has been reported.

[0013] The Seoul virus can be used as a reference hantavirus strain. The GenBank accession number KM948598.1 is the reference nucleic acid sequence for hantavirus NP. Column (see SEQ ID NO: 1) and reference polypeptide for hantavirus NP Provides the sequence (SEQ ID NO: 4). TAGTAGTAGGCTCCCTAAAGAGCTACTACACTAACAAGGA AAATGGCAACTATGGAAGAAATCCAGAGAGAAATCAGTGC GCACGAGGGGCAGCTTGTAATAGCACGCCAGAAGGTCAAG GATGCAGAAAAGCAGTATGAGAAGGATCCTGATGACCTAA ATAAGAGGGCACTGCATGATCGGGAGAGTGTCGCAGCTTC AATACAATCAAAAATTGATGAATTGAAGCGCCAACTTGCT GACAGGATTGCAGCAGGGAAGAACATCGGGCAAGACCGGG ATCCTACAGGGGTAGAGCCGGGTGATCATCTCAAGGAAAG ATCAGCACTAAGCTACGGGAATACACTGGACCTGAATAGC CTTGACATTGATGAACCTACAGGACAGACAGCTGATTGGT TGACCATAATTGTCTATTTGACATCATTCGTGGTCCCGAT CATCTTGAAGGCACTGTACATGTTGACAACAAGAGGCAGG CAGACTTCAAAGGACAACAAGGGAATGAGGATCAGATTCA AGGATGACAGCTCATATGAAGATGTCAATGGAATCAGAAA GCCCAAACATCTGTATGTGTCAATGCCAAACGCCCAATCA AGCATGAAGGCTGAAGAGATAACACCTGGAAGATTCCGCA CTGCAGTATGTGGGCTATACCCTGCACAGATAAAGGCAAG GAACATGGTAAGCCCTGTCATGAGTGTAGTTGGGTTTTTG GCACTGGCAAAAGACTGGACATCTAGAATTGAAGAATGGC TTGGTGCACCCTGCAAGTTCATGGCAGAGTCTCCCATTGC CGGGAGCTTATCTGGGAATCCTGTGAATCGTGATTATATC AGACAGAGACAAGGTGCACTTGCAGGGATGGAGCCAAAAG AATTTCAAGCTCTCAGGCAACATTCAAAGGATGCTGGATG TACACTGGTTGAACATATTGAGTCACCATCATCAATATGG GTATTTGCTGGGGCCCCTGATAGGTGCCCACCGACATGCC TGTTTGTTGGAGGGATGGCTGAGTTAGGTGCTTTCTTTTC TATACTTCAGGATATGAGGAACACAATCATGGCTTCAAAG ACTGTGGGAACAGCTGATGAAAAGCTTCGAAAGAAGTCAT CATTCTATCAATCATACCTCAGACGCACACAATCAATGGG AATACAACTGGACCAGAGGATAATTGTTATGTTTATGGTT GCCTGGGGAAAGGAGGCAGTGGACAACTTTCATCTCGGTG ATGACATGGATCCAGAGCTTCGCAGCCTGGCTCAGATCCT GATTGACCAGAAAGTGAAGGAAATCTCAAACCAGGAACCT ATGAAATTATAAGTACATAATTATGTAATCCATACTAACT ATAGGTTAAGAAATACTAATCATTAGTTAATAAGAATATA GATTTATTGAATAATCATATTAAATAATTAGGTAAGTTAA CTATTAGTTAGTTAAGTTAGCTAATTGATTTATATGATTA TCACAATTGAATGTAATCATAAGCACAATCACTGCCATGT ATAATCACGGGTATACGGGTGGTTTTCATATGGGGAACAG GGTGGGCTTAGGGCCAGGTCACCTTAAGTGACCTTTTTTG TATATATGGATGTAGATTTCAATTGATCGAGTACTAATCC TACTGTTCTCTTTTCCTTTCCTTTCTCCTTCTTTACTAAC AACAACAAACTACCTCACAACCTTCTACCTCAACACATAC TACCTCATTCAGTTGTTTCCTTTTGTCTTTTTAGGGAGCA TACTACTA (SEQ ID NO: 1)

[0014] The coding sequence of SEQ ID NO: 1 corresponds to nucleic acid residues 43 to 1332 therein and is represented by SEQ ID NO: 2. Therefore, it is represented by ATGGCAACTATGGAAGAAATCCAGAGAGAAATCAGTGCGC ACGAGGGGCAGCTTGTAATAGCACGCCAGAAGGTCAAGGA TGCAGAAAAGCAGTATGAGAAGGATCCTGATGACCTAAAT AAGAGGGCACTGCATGATCGGGAGAGTGTCGCAGCTTCAA TACAATCAAAAATTGATGAATTGAAGCGCCAACTTGCTGA CAGGATTGCAGCAGGGAAGAACATCGGGCAAGACCGGGAT CCTACAGGGGTAGAGCCGGGTGATCATCTCAAGGAAAGAT CAGCACTAAGCTACGGGAATACACTGGACCTGAATAGCCT TGACATTGATGAACCTACAGGACAGACAGCTGATTGGTTG ACCATAATTGTCTATTTGACATCATTCGTGGTCCCGATCA TCTTGAAGGCACTGTACATGTTGACAACAAGAGGCAGGCA GACTTCAAAGGACAACAAGGGAATGAGGATCAGATTCAAG GATGACAGCTCATATGAAGATGTCAATGGAATCAGAAAGC CCAAACATCTGTATGTGTCAATGCCAAACGCCCAATCAAG CATGAAGGCTGAAGAGATAACACCTGGAAGATTCCGCACT GCAGTATGTGGGCTATACCCTGCACAGATAAAGGCAAGGA ACATGGTAAGCCCTGTCATGAGTGTAGTTGGGTTTTTGGC ACTGGCAAAAGACTGGACATCTAGAATTGAAGAATGGCTT GGTGCACCCTGCAAGTTCATGGCAGAGTCTCCCATTGCCG GGAGCTTATCTGGGAATCCTGTGAATCGTGATTATATCAG ACAGAGACAAGGTGCACTTGCAGGGATGGAGCCAAAAGAA TTTCAAGCTCTCAGGCAACATTCAAAGGATGCTGGATGTA CACTGGTTGAACATATTGAGTCACCATCATCAATATGGGT ATTTGCTGGGGCCCCTGATAGGTGCCCACCGACATGCCTG TTTGTTGGAGGGATGGCTGAGTTAGGTGCTTTCTTTTCTA TACTTCAGGATATGAGGAACACAATCATGGCTTCAAAGAC TGTGGGAACAGCTGATGAAAAGCTTCGAAAGAAGTCATCA TTCTATCAATCATACCTCAGACGCACACAATCAATGGGAA TACAACTGGACCAGAGGATAATTGTTATGTTTATGGTTGC CTGGGGAAAGGAGGCAGTGGACAACTTTCATCTCGGTGAT GACATGGATCCAGAGCTTCGCAGCCTGGCTCAGATCCTGA TTGACCAGAAAGTGAAGGAAATCTCAAACCAGGAACCTAT GAAATTA (SEQ ID NO: 2)

[0015] The inventors generated a nucleic acid sequence encoding hantavirus N P optimized for expression in Homo sapiens (see SEQ ID NO: 3): ATGGCCACAATGGAAGAGATCCAGAGAGAGATCAGCGCCC ACGAGGGACAGCTGGTTATCGCCAGACAGAAAGTGAAGGA CGCCGAGAAGCAGTACGAGAAGGACCCCGACGATCTGAAC AAGAGAGCCCTGCACGACAGAGAAAGCGTGGCCGCCTCTA TCCAGAGCAAGATCGATGAGCTGAAGAGACAGCTGGCCGA CAGAATCGCCGCTGGCAAGAATATTGGCCAGGACAGAGAT CCCACAGGCGTGGAACCTGGCGATCACCTGAAAGAGAGAA GCGCCCTGTCCTATGGCAACACCCTGGACCTGAACAGCCT GGACATTGATGAGCCTACCGGCCAGACAGCCGACTGGCTG ACAATCATTGTGTACCTGACCAGCTTCGTGGTCCCCATCA TCCTGAAGGCCCTGTACATGCTGACCACCAGAGGCAGACA GACCAGCAAGGACAACAAGGGCATGAGAATCCGGTTCAAG GATGACAGCAGCTACGAGGACGTGAACGGCATTAGAAAGC CCAAGCACCTGTACGTGTCCATGCCTAACGCTCAGAGCAG CATGAAGGCCGAGGAAATCACCCCTGGCAGATTCAGAACA GCCGTGTGCGGACTGTACCCCGCTCAGATCAAGGCCAGAA ACATGGTGTCCCCAGTGATGAGCGTCGTGGGATTTCTGGC CCTGGCTAAGGACTGGACCAGCAGGATTGAGGAATGGCTG GGAGCCCCTTGCAAGTTTATGGCCGAGTCTCCTATCGCCG GCAGCCTGTCTGGCAACCCCGTGAATAGAGACTACATCAG ACAGAGGCAGGGCGCTCTGGCCGGAATGGAACCCAAAGAA TTTCAGGCCCTGCGGCAGCACTCTAAGGATGCCGGATGTA CCCTGGTGGAACACATTGAGAGCCCCAGCAGCATCTGGGT TTTCGCTGGCGCTCCTGATAGATGCCCTCCTACCTGTCTG TTTGTTGGCGGAATGGCCGAGCTGGGCGCCTTCTTTAGCA TTCTGCAGGACATGCGGAATACCATCATGGCCAGCAAGAC CGTGGGCACCGCCGATGAGAAGCTGAGAAAGAAGTCCAGC TTCTACCAGAGCTACCTGCGGAGAACCCAGAGCATGGGCA TTCAGCTGGACCAGAGAATCATCGTGATGTTCATGGTGGC CTGGGGCAAAGAAGCCGTGGACAATTTTCACCTGGGCGAC GACATGGACCCCGAGCTGAGATCTCTGGCCCAGATCCTGA TCGACCAGAAAGTCAAAGAGATCTCCAATCAAGAGCCCAT GAAGCTG (SEQ ID NO: 3).

[0016] The nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 is translated to obtain a hantavirus NP polypeptide sequence, which is represented by (SEQ ID NO: 4): MATMEEIQREISAHEGQLVIARQKVKDAEKQYEKDPDDLN KRALHDRESVAASIQSKIDELKRQLADRIAAGKNIGQDRD PTGVEPGDHLKERSALSYGNTLDLNSLDIDEPTGQTADWL TIIVYLTSFVVPIILKALYMLTTRGRQTSKDNKGMRIRFK DDSSYEDVNGIRKPKHLYVSMPNAQSSMKAEEITPGRFRT AVCGLYPAQIKARNMVSPVMSVVGFLALAKDWTSRIEEWL GAPCKFMAESPIAGSLSGNPVNRDYIRQRQGALAGMEPKE FQALRQHSKDAGCTLVEHIESPSSIWVFAGAPDRCPPTCL FVGGMAELGAFFSILQDMRNTIMASKTVGTADEKLRKKSS FYQSYLRRTQSMGIQLDQRIIVMFMVAWGKEAVDNFHLGD DMDPELRSLAQILIDQKVKEISNQEPMKL (SEQ ID NO: 4).

[0017] Hantaan virus can be used as a reference hantavirus strain. GenBan The accession number KC570390.1 is the reference nucleic acid for the hantavirus NP sequence (see SEQ ID NO: 5) and the reference polypeptide sequence (see SEQ ID NO: 7) for the hantavirus NP are provided. TAGTAGTAGACTCCCTAAAGAGCTACTAGAACAACGATGG CAACTATGGAGGAATTGCAGAGGGAAATCAATGCCCATGA GGGTCAACTGGTGATAGCCAGGCAGAAGGTGAGGGATGCA GAAAAGCAGTATGAAAAGGATCCAGATGAGTTAAACAAGA GAGCATTGACAGATCGAGAGGGTGTTGCAGTATCCATTCA AGCAAAGATTGATGAGTTAAAGAGGCAATTGGCAGATAGG ATTGCAACCGGGAAGAACCTTGGAAAGGAACAAGACCCAA CAGGGGTAGAACCTGGAGATCATCTGAAAGAGAGATCAAT GCTCAGTTATGGAAATGTTCTTGACTTAAACCACCTGGAT ATTGATGAGCCAACAGGACAGACAGCAGACTGGCTGGGCA TTGTTATCTATCTCACATCCTTTGTTGTCCCGATACTTCT GAAAGCCCTGTACATGTTAACAACAAGAGGGAGGCAGACC ACCAAGGACAATAAAGGAACTCGGATTCGATTCAAGGATG ATAGCTCCTTCGAGGATGTCAATGGCATTCGGAAGCCGAA ACATCTATATGTGTCCTTACCAAATGCACAGTCAAGTATG AAAGCAGAAGAGATTACACCTGGTAGATATAGAACAGCAA TTTGTGGACTTTACCCTGCACAAATTAAGGCAAGACAGAT GATTAGTCCAGTCATGAGTGTAATCGGATTCTTGGCTTTG GCAAAAGATTGGAGTGACCGCATTGAGCAGTGGTTAAGTG AACCGTGTAAGCTTCTTCCAGATACAGCAGCAGTTAGCCT TCTTGGTGGTCCTGCAACCAACAGGGACTATTTACGGCAG CGACAAGTAGCATTGGGCAACATGGAAACAAAAGAGTCTA AGGCTATACGCCAACATGCAGAAGCAGCAGGCTGTAGTAT GATTGAGGACATTGAGTCACCATCATCAATATGGGTGTTT GCTGGGGCACCGGACCGCTGTCCACCAACATGTCTCTTTA TTGCAGGTATGGCTGAGCTTGGGGCATTTTTTTCCATCCT GCAGGACATGCGAAATACAATTATGGCATCCAAGACAGTT GGAACCTCTGAGGAGAAGCTACGGAAGAAATCCTCATTCT ATCAGTCTTATCTCAGGAGAACACAATCAATGGGAATACA ACTGGATCAGAGGATAATTGTGCTCTTCATGGTAGCCTGG GGGAAAGAAGCAGTGGATAACTTCCACCTAGGAGATGATA TGGACCCTGAGCTGCGAACACTAGCACAGAGCCTGATTGA TGTTAAAGTGAAGGAAATTTCCAACCAAGAGCCTTTAAAA CTATAATCAGTGAATGTATAACCCTCATTATGTGATTATT ATATACTACTGAATCATTATCAATCATATTTGCACTATTA TTATCAGGGGAATTAGTATATCAGGGTAAGGGCACATTTA TGGGTGGGAATCATTACTCAGAGGGTGGGTCAGTTAATCC GTTGTGGGTGGGTTTAGTTCCTGGCTGCCTTAAGTAGCCT TTTTTTGTATATATGGATGTAGATTTCATTTGATCTTTAA ACTAATCTTGCTCTTTTTCCTTTTCCTCCTGCTTTCTCTG CTTACTAACAACAACATTCTACCTCAACACACAACTACCT CAACTAAACTACCTCATTTGATTGCTCCTTGATTGTCTCT TTAGGGAGTCTACTACTA (SEQ ID NO: 5).

[0018] The coding sequence of SEQ ID NO: 5 corresponds to nucleic acid residues 37 to 1323 therein and is represented by SEQ ID NO: 6. Thus represented. ATGGCAACTATGGAGGAATTGCAGAGGGAAATCAATGCCC ATGAGGGTCAACTGGTGATAGCCAGGCAGAAGGTGAGGGA TGCAGAAAAGCAGTATGAAAAGGATCCAGATGAGTTAAAC AAGAGAGCATTGACAGATCGAGAGGGTGTTGCAGTATCCA TTCAAGCAAAGATTGATGAGTTAAAGAGGCAATTGGCAGA TAGGATTGCAACCGGGAAGAACCTTGGAAAGGAACAAGAC CCAACAGGGGTAGAACCTGGAGATCATCTGAAAGAGAGAT CAATGCTCAGTTATGGAAATGTTCTTGACTTAAACCACCT GGATATTGATGAGCCAACAGGACAGACAGCAGACTGGCTG GGCATTGTTATCTATCTCACATCCTTTGTTGTCCCGATAC TTCTGAAAGCCCTGTACATGTTAACAACAAGAGGGAGGCA GACCACCAAGGACAATAAAGGAACTCGGATTCGATTCAAG GATGATAGCTCCTTCGAGGATGTCAATGGCATTCGGAAGC CGAAACATCTATATGTGTCCTTACCAAATGCACAGTCAAG TATGAAAGCAGAAGAGATTACACCTGGTAGATATAGAACA GCAATTTGTGGACTTTACCCTGCACAAATTAAGGCAAGAC AGATGATTAGTCCAGTCATGAGTGTAATCGGATTCTTGGC TTTGGCAAAAGATTGGAGTGACCGCATTGAGCAGTGGTTA AGTGAACCGTGTAAGCTTCTTCCAGATACAGCAGCAGTTA GCCTTCTTGGTGGTCCTGCAACCAACAGGGACTATTTACG GCAGCGACAAGTAGCATTGGGCAACATGGAAACAAAAGAG TCTAAGGCTATACGCCAACATGCAGAAGCAGCAGGCTGTA GTATGATTGAGGACATTGAGTCACCATCATCAATATGGGT GTTTGCTGGGGCACCGGACCGCTGTCCACCAACATGTCTC TTTATTGCAGGTATGGCTGAGCTTGGGGCATTTTTTTCCA TCCTGCAGGACATGCGAAATACAATTATGGCATCCAAGAC AGTTGGAACCTCTGAGGAGAAGCTACGGAAGAAATCCTCA TTCTATCAGTCTTATCTCAGGAGAACACAATCAATGGGAA TACAACTGGATCAGAGGATAATTGTGCTCTTCATGGTAGC CTGGGGGAAAGAAGCAGTGGATAACTTCCACCTAGGAGAT GATATGGACCCTGAGCTGCGAACACTAGCACAGAGCCTGA TTGATGTTAAAGTGAAGGAAATTTCCAACCAAGAGCCTTT AAAACTA (SEQ ID NO: 6).

[0019] Translation of the nucleic acid sequence of SEQ ID NO: 6 yields the hantavirus NP polypeptide sequence, and the sequence is represented by (SEQ ID NO: 7): MATMEELQREINAHEGQLVIARQKVRDAEKQYEKDPDELN KRALTDREGVAVSIQAKIDELKRQLADRIATGKNLGKEQD PTGVEPGDHLKERSMLSYGNVLDLNHLDIDEPTGQTADWL GIVIYLTSFVVPILLKALYMLTTRGRQTTKDNKGTRIRFK DDSSFEDVNGIRKPKHLYVSLPNAQSSMKAEEITPGRYRT AICGLYPAQIKARQMISPVMSVIGFLALAKDWSDRIEQWL SEPCKLLPDTAAVSLLGGPATNRDYLRQRQVALGNMETKE SKAIRQHAEAAGCSMIEDIESPSSIWVFAGAPDRCPPTCL FIAGMAELGAFFSILQDMRNTIMASKTVGTSEEKLRKKSS FYQSYLRRTQSMGIQLDQRIIVLFMVAWGKEAVDNFHLGD DMDPELRTLAQSLIDVKVKEISNQEPLKL (SEQ ID NO: 7).

[0020] The reference nucleic acid sequence for the hantavirus NP is provided by SEQ ID NO: 8, which corresponds to nucleic acid residues 319 to 1323 of SEQ ID NO: 5. This corresponds to nucleic acid residues 319 to 1323 of SEQ ID NO: 5. ATGCTCAGTTATGGAAATGTTCTTGACTTAAACCACCTGG ATATTGATGAGCCAACAGGACAGACAGCAGACTGGCTGGG CATTGTTATCTATCTCACATCCTTTGTTGTCCCGATACTT CTGAAAGCCCTGTACATGTTAACAACAAGAGGGAGGCAGA CCACCAAGGACAATAAAGGAACTCGGATTCGATTCAAGGA TGATAGCTCCTTCGAGGATGTCAATGGCATTCGGAAGCCG AAACATCTATATGTGTCCTTACCAAATGCACAGTCAAGTA TGAAAGCAGAAGAGATTACACCTGGTAGATATAGAACAGC AATTTGTGGACTTTACCCTGCACAAATTAAGGCAAGACAG ATGATTAGTCCAGTCATGAGTGTAATCGGATTCTTGGCTT TGGCAAAAGATTGGAGTGACCGCATTGAGCAGTGGTTAAG TGAACCGTGTAAGCTTCTTCCAGATACAGCAGCAGTTAGC CTTCTTGGTGGTCCTGCAACCAACAGGGACTATTTACGGC AGCGACAAGTAGCATTGGGCAACATGGAAACAAAAGAGTC TAAGGCTATACGCCAACATGCAGAAGCAGCAGGCTGTAGT ATGATTGAGGACATTGAGTCACCATCATCAATATGGGTGT TTGCTGGGGCACCGGACCGCTGTCCACCAACATGTCTCTT TATTGCAGGTATGGCTGAGCTTGGGGCATTTTTTTCCATC CTGCAGGACATGCGAAATACAATTATGGCATCCAAGACAG TTGGAACCTCTGAGGAGAAGCTACGGAAGAAATCCTCATT CTATCAGTCTTATCTCAGGAGAACACAATCAATGGGAATA CAACTGGATCAGAGGATAATTGTGCTCTTCATGGTAGCCT GGGGGAAAGAAGCAGTGGATAACTTCCACCTAGGAGATGA TATGGACCCTGAGCTGCGAACACTAGCACAGAGCCTGATT GATGTTAAAGTGAAGGAAATTTCCAACCAAGAGCCTTTAA AACTA (SEQ ID NO: 8).

[0021] The inventors generated a nucleic acid sequence encoding hantavirus N P optimized for expression in Homo sapiens (see SEQ ID NO: 9): ATGCTGAGCTACGGCAACGTGCTGGATCTGAACCACCTGG ATATCGACGAGCCAACAGGACAGACCGCTGATTGGCTGGG CATCGTGATCTACCTGACCTCCTTTGTGGTGCCTATTCTG CTCAAAGCCCTCTATATGCTGACAACACGCGGAAGGCAGA CCACCAAAGATAACAAAGGCACCCGGATCAGGTTTAAGGA CGACAGCTCCTTTGAGGATGTCAACGGCATCCGGAAACCT AAGCACCTCTATGTGTCTCTGCCCAATGCACAGTCCTCCA TGAAGGCAGAAGAGATCACACCAGGCCGGTACAGAACCGC CATCTGTGGACTGTATCCTGCACAAATCAAAGCCCGGCAG ATGATCAGCCCCGTGATGTCCGTTATCGGATTCCTGGCTC TGGCCAAAGATTGGAGCGACAGGATCGAGCAGTGGCTGAG CGAGCCTTGCAAGCTGCTTCCTGATACAGCCGCTGTGTCA CTGCTTGGCGGCCCTGCCACAAACAGAGATTACCTGAGAC AGAGACAGGTGGCACTGGGCAACATGGAAACAAAAGAGAG CAAGGCCATCCGGCAGCATGCCGAAGCTGCTGGCTGTAGC ATGATCGAGGATATCGAGTCCCCTAGCTCCATTTGGGTGT TCGCAGGGGCCCCAGATAGATGTCCACCAACATGCCTGTT CATTGCCGGCATGGCTGAACTGGGAGCTTTTTTCAGCATC CTCCAGGATATGCGCAACACGATTATGGCCTCCAAGACAG TGGGAACCAGCGAGGAAAAGCTGCGGAAGAAAAGCAGCTT TTACCAGTCTTACCTGAGGCGGACCCAGTCCATGGGGATC CAACTGGATCAGCGGATCATTGTGCTGTTTATGGTCGCTT GGGGAAAAGAGGCTGTCGATAACTTCCACCTGGGAGATGA TATGGATCCTGAACTGCGGACCCTGGCTCAGTCCCTGATC GATGTGAAAGTGAAAGAAATTAGTAATCAAGAACCCCTCA AGCTG (SEQ ID NO: 9).

[0022] The nucleic acid sequence containing SEQ ID NO: 8 or 9 is derived from a hantavirus strain other than Hantaan virus The vector of the present invention which also encodes the core protein, for example, the core protein derived from Seoul virus, is particularly suitable for use in it. The inventors have determined that the 94 N-terminal amino acids of the wild-type hantavirus core protein show high sequence similarity with the N-terminus of the wild-type core protein derived from Seoul virus, and if sequence differences exist within this region, the inventors have determined that both sequences contain closely related amino acids. The 95th residue of the wild-type hantavirus core protein sequence was identified as the first residue that is significantly different from the corresponding residue of the wild-type core protein sequence derived from Seoul virus. The inventors have found that the nucleic acid encoding the 94 N-terminal amino acids of the hantavirus wild-type core protein is present in a vector that also encodes the Seoul virus-derived core protein (or at least 94 N-terminal amino acids of the wild-type core protein derived from Seoul virus, or an antigenic fragment thereof), is considered to be substantially antigenically redundant. Therefore, the inventors believe that without sacrificing antigenic diversity, the nucleic acid encoding the 94 N-terminal amino acids of the wild-type hantavirus core protein can be omitted from a vector that also encodes the Seoul virus-derived core protein (or at least 94 N-terminal amino acids of the wild-type core protein derived from Seoul virus, or an antigenic fragment thereof). The removal of unnecessary nucleic acid sequences can enhance vector stability and is generally advantageous in the design of vector constructs (e.g., MVA constructs). For the above reasons, the inventors have used the nucleic acid encoding the 94 N-terminal amino acids of the Seoul virus core protein, especially in the hantavirus core protein (or hantavirus derived from Seoul virus, or an antigenic fragment thereof). is also considered to be substantially antigenically redundant. Therefore, the inventors believe that without sacrificing antigenic diversity, the nucleic acid encoding the 94 N-terminal amino acids of the wild-type hantavirus core protein can be omitted from a vector that also encodes the Seoul virus-derived core protein (or at least 94 N-terminal amino acids of the wild-type core protein derived from Seoul virus, or an antigenic fragment thereof). The removal of unnecessary nucleic acid sequences can

[0023] For the reasons described above, the inventors have used the nucleic acid encoding the 94 N-terminal amino acids of the Seoul virus core protein, especially in the hantavirus core protein (or hantavirus derived from Seoul virus, or an antigenic fragment thereof). At least 94 N-terminal amino acids of the wild-type nuclear protein from which it is derived, or an antigenic fragment thereof It is considered that similar advantages can be achieved when omitted from the vector encoding ().

[0024] Hantavirus NP polypeptide by translation of the nucleic acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9 A sequence is obtained, and the sequence is represented by (SEQ ID NO: 10): MLSYGNVLDLNHLDIDEPTGQTADWLGIVIYLTSFVVPIL LKALYMLTTRGRQTTKDNKGTRIRFKDDSSFEDVNGIRKP KHLYVSLPNAQSSMKAEEITPGRYRTAICGLYPAQIKARQ MISPVMSVIGFLALAKDWSDRIEQWLSEPCKLLPDTAAVS LLGGPATNRDYLRQRQVALGNMETKESKAIRQHAEAAGCS MIEDIESPSSIWVFAGAPDRCPPTCLFIAGMAELGAFFSI LQDMRNTIMASKTVGTSEEKLRKKSSFYQSYLRRTQSMGI QLDQRIIVLFMVAWGKEAVDNFHLGDDMDPELRTLAQSLI DVKVKEISNQEPLKL (SEQ ID NO: 10).

[0025] As used herein, the term "antigenic fragment" refers to a peptide of hantavirus NP that retains the ability to induce an immune response in an individual as compared to the reference hantavirus NP or protein fragment. Thus, an antigenic fragment may contain at least one epitope of the reference protein. By way of example, an antigenic fragment of the invention has at least 70% sequence homology across the corresponding peptide sequence of (consecutive) amino acids of the reference protein and may contain at least one epitope of the reference protein. By way of example, an antigenic fragment of the invention has at least 70% sequence homology across the corresponding peptide sequence of (consecutive) amino acids of the reference protein having properties of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, and may comprise (or consist of) a peptide sequence having 275, 300 amino acids ). The antigenic fragment is at least 10 contiguous amino acid residues derived from the sequence of the reference protein (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 contiguous amino acid residues of the reference protein) and may comprise ( or consist of).

[0026] The antigenic fragment of the reference protein may have antigen cross-reactivity common to the reference protein and / or substantially the same in vivo biological activity. For example, an antibody capable of binding to the antigenic fragment of the reference protein can also bind to the reference protein itself. As a further example, the reference protein and its antigenic fragment may share the common ability to induce a "recall response" of T lymphocytes (e.g., CD4+, CD8+, effector T cells or memory T cells, e.g., TEM or TCM) previously exposed to the antigenic components of hantavirus infection.

[0027] In one aspect, the invention provides a viral vector or a bacterial vector comprising a nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof, which can induce an immune response in a subject.

[0028] In one embodiment, a nucleic acid encoding a hantavirus nucleoprotein or an antigenic fragment thereof The array comprises a nucleic acid sequence selected from nucleic acid sequences of array numbers 1, 2 and 3 and 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.

[0029] In one embodiment, the nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof has at least 70% (e.g., at least 70, 75, 8 0, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity with the nucleic acid sequence of array number 3.

[0030] In one embodiment, the nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof has at least 70% (e.g., at least 70, 75, 8 0, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity with a nucleic acid sequence selected from nucleic acid sequences of array numbers 5, 6, 8 and 9.

[0031] In one embodiment, the nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof has at least 70% (e.g., at least 70, 75, 8 0, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity with the nucleic acid sequence of array number 9.

[0032] "Peptide pool 4" induced a very strong antigen-specific T cell response (see Examples for details). The amino acid sequence represented by peptide pool 4 corresponds to array number 11. . LYPAQIKARNMVSPVMSVVGFLALAKDWTSRIEEWLGAPC KFMAESPIAGSLSGNPVNRDYIRQRQGALAGMEPKEFQA( Sequence number 11).

[0033] The amino acid sequence of Sequence number 11 is encoded by nucleic acid residues 655 - 891 of Sequence number 1 (see Sequence number 15 for reference), by residues 613 - 849 of Sequence number 2 (see Sequence number 16 for reference), and by residues 613 - 849 of Sequence number 3 (see Sequence number 17 for reference). CTATACCCTGCACAGATAAAGGCAAGGAACATGGTAAGCC CTGTCATGAGTGTAGTTGGGTTTTTGGCACTGGCAAAAGA CTGGACATCTAGAATTGAAGAATGGCTTGGTGCACCCTGC AAGTTCATGGCAGAGTCTCCCATTGCCGGGAGCTTATCTG GGAATCCTGTGAATCGTGATTATATCAGACAGAGACAAGG TGCACTTGCAGGGATGGAGCCAAAAGAATTTCAAGCT(Sequence number 15) CTATACCCTGCACAGATAAAGGCAAGGAACATGGTAAGCC CTGTCATGAGTGTAGTTGGGTTTTTGGCACTGGCAAAAGA CTGGACATCTAGAATTGAAGAATGGCTTGGTGCACCCTGC AAGTTCATGGCAGAGTCTCCCATTGCCGGGAGCTTATCTG GGAATCCTGTGAATCGTGATTATATCAGACAGAGACAAGG TGCACTTGCAGGGATGGAGCCAAAAGAATTTCAAGCT(Sequence number 16)​ CTGTACCCCGCTCAGATCAAGGCCAGAAACATGGTGTCCC CAGTGATGAGCGTCGTGGGATTTCTGGCCCTGGCTAAGGA CTGGACCAGCAGGATTGAGGAATGGCTGGGAGCCCCTTGC AAGTTTATGGCCGAGTCTCCTATCGCCGGCAGCCTGTCTG GCAACCCCGTGAATAGAGACTACATCAGACAGAGGCAGGG CGCTCTGGCCGGAATGGAACCCAAAGAATTTCAGGCC(sequence No. 17)

[0034] In one embodiment, a nucleic acid encoding a hantavirus nucleoprotein or an antigenic fragment thereof sequence has 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.

[0035] In one embodiment, a nucleic acid encoding a hantavirus nucleoprotein or an antigenic fragment thereof sequence comprises (or consists of) at least 10 consecutive nucleic acid residues derived from the 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).

[0036] "Peptide pool 9" also induced a very strong antigen-specific T cell response. The amino acid sequence represented by peptide pool 9 corresponds to SEQ ID NO: 12. ​IKARQMISPVMSVIGFLALAKDWSDRIEQWLSEPCKLLPD TAAVSLLGGPATNRDYLRQRQVALGNMETKESKAIRQHA( Sequence number 12)

[0037] The amino acid sequence of Sequence number 12 is encoded by nucleic acid residues 664 - 900 of Sequence number 5 (see Sequence number 18 for reference), by residues 628 - 864 of Sequence number 6 (see Sequence number 19 for reference), by residues 346 - 582 of Sequence number 8 (see Sequence number 20 for reference ), and by residues 346 - 582 of Sequence number 9 (see Sequence number 21 for reference ). ATTAAGGCAAGACAGATGATTAGTCCAGTCATGAGTGTAA TCGGATTCTTGGCTTTGGCAAAAGATTGGAGTGACCGCAT TGAGCAGTGGTTAAGTGAACCGTGTAAGCTTCTTCCAGAT ACAGCAGCAGTTAGCCTTCTTGGTGGTCCTGCAACCAACA GGGACTATTTACGGCAGCGACAAGTAGCATTGGGCAACAT GGAAACAAAAGAGTCTAAGGCTATACGCCAACATGCA(Sequence number 18) ATTAAGGCAAGACAGATGATTAGTCCAGTCATGAGTGTAA TCGGATTCTTGGCTTTGGCAAAAGATTGGAGTGACCGCAT TGAGCAGTGGTTAAGTGAACCGTGTAAGCTTCTTCCAGAT ACAGCAGCAGTTAGCCTTCTTGGTGGTCCTGCAACCAACA GGGACTATTTACGGCAGCGACAAGTAGCATTGGGCAACAT GGAAACAAAAGAGTCTAAGGCTATACGCCAACATGCA (Sequence No. 19) ATTAAGGCAAGACAGATGATTAGTCCAGTCATGAGTGTAA TCGGATTCTTGGCTTTGGCAAAAGATTGGAGTGACCGCAT TGAGCAGTGGTTAAGTGAACCGTGTAAGCTTCTTCCAGAT ACAGCAGCAGTTAGCCTTCTTGGTGGTCCTGCAACCAACA GGGACTATTTACGGCAGCGACAAGTAGCATTGGGCAACAT GGAAACAAAAGAGTCTAAGGCTATACGCCAACATGCA (Sequence No. 20) ATCAAAGCCCGGCAGATGATCAGCCCCGTGATGTCCGTTA TCGGATTCCTGGCTCTGGCCAAAGATTGGAGCGACAGGAT CGAGCAGTGGCTGAGCGAGCCTTGCAAGCTGCTTCCTGAT ACAGCCGCTGTGTCACTGCTTGGCGGCCCTGCCACAAACA GAGATTACCTGAGACAGAGACAGGTGGCACTGGGCAACAT GGAAACAAAAGAGAGCAAGGCCATCCGGCAGCATGCC (Sequence No. 21)

[0038] In one embodiment, a nucleic acid encoding a hantavirus nucleoprotein or an antigenic fragment thereof sequence 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.

[0039] In one embodiment, a nucleic acid encoding a hantavirus nucleoprotein or an antigenic fragment thereof sequence comprises at least 10 consecutive nucleic acid residues derived from the 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 6 5 nucleic acids) (or consists of).

[0040] As demonstrated herein, peptide pools 4 and 9 induced very strong antigen-specific T cell responses. The inventors aligned the polypeptide sequences represented by "peptide pool 4" and the polypeptide sequence represented by "peptide pool 9" to identify 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 1 4 play an important role in inducing the particularly strong antigen specific T cell responses observed by peptide pools 4 and 9, respectively. SPVMSVVGFLALAKD (SEQ ID NO: 13) PVMSVIGFLALAKDW (SEQ ID NO: 14)

[0041] SEQ ID NO: 13 is particularly encoded by nucleic acid residues 691-735 of SEQ ID NO: 1 (see SEQ ID NO: 22), by residues 649-693 of SEQ ID NO: 2 (see SEQ ID NO: 23), and by residues 649-693 of SEQ ID NO: 3 (see SEQ ID NO: 24). ) AGCCCTGTCATGAGTGTAGTTGGGTTTTTGGCACTGGCAA AAGAC (SEQ ID NO: 22) AGCCCTGTCATGAGTGTAGTTGGGTTTTTGGCACTGGCAA AAGAC (SEQ ID NO: 23) TCCCCAGTGATGAGCGTCGTGGGATTTCTGGCCCTGGCTA AGGAC (SEQ ID NO: 24)

[0042] In one embodiment, a nucleic acid encoding a hantavirus nucleoprotein or an antigenic fragment thereof The sequence 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.

[0043] SEQ ID NO: 14 is specifically encoded by nucleic acid residues 688 - 732 of SEQ ID NO: 5 (see SEQ ID NO: 25), by residues 652 - 696 of SEQ ID NO: 6 (see SEQ ID NO: 26), by residues 370 - 414 of SEQ ID NO: 8 (see SEQ ID NO: 27), and by residues 370 - 414 of SEQ ID NO: 9 (see SEQ ID NO: 28). SEQ ID NO: 14 is specifically encoded by nucleic acid residues 688 - 732 of SEQ ID NO: 5 (see SEQ ID NO: 25), by residues 652 - 696 of SEQ ID NO: 6 (see SEQ ID NO: 26), by residues 370 - 414 of SEQ ID NO: 8 (see SEQ ID NO: 27), and by residues 370 - 414 of SEQ ID NO: 9 (see SEQ ID NO: 28). SEQ ID NO: 14 is specifically encoded by nucleic acid residues 688 - 732 of SEQ ID NO: 5 (see SEQ ID NO: 25), by residues 652 - 696 of SEQ ID NO: 6 (see SEQ ID NO: 26), by residues 370 - 414 of SEQ ID NO: 8 (see SEQ ID NO: 27), and by residues 370 - 414 of SEQ ID NO: 9 (see SEQ ID NO: 28). SEQ ID NO: 14 is specifically encoded by nucleic acid residues 688 - 732 of SEQ ID NO: 5 (see SEQ ID NO: 25), by residues 652 - 696 of SEQ ID NO: 6 (see SEQ ID NO: 26), by residues 370 - 414 of SEQ ID NO: 8 (see SEQ ID NO: 27), and by residues 370 - 414 of SEQ ID NO: 9 (see SEQ ID NO: 28). SEQ ID NO: 14 is specifically encoded by nucleic acid residues 688 - 732 of SEQ ID NO: 5 (see SEQ ID NO: 25), by residues 652 - 696 of SEQ ID NO: 6 (see SEQ ID NO: 26), by residues 370 - 414 of SEQ ID NO: 8 (see SEQ ID NO: 27), and by residues 370 - 414 of SEQ ID NO: 9 (see SEQ ID NO: 28). CCAGTCATGAGTGTAATCGGATTCTTGGCTTTGGCAAAAG ATTGG (SEQ ID NO: 25) CCAGTCATGAGTGTAATCGGATTCTTGGCTTTGGCAAAAG ATTGG (SEQ ID NO: 26) CCAGTCATGAGTGTAATCGGATTCTTGGCTTTGGCAAAAG ATTGG (SEQ ID NO: 27) CCCGTGATGTCCGTTATCGGATTCCTGGCTCTGGCCAAAG ATTGG (SEQ ID NO: 28)

[0044] In one embodiment, a nucleic acid encoding a hantavirus nucleoprotein or an antigenic fragment thereof sequence 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.

[0045] In one embodiment, a nucleic acid encoding a hantavirus nucleoprotein or an antigenic fragment thereof sequence comprises a first nucleic acid sequence and a second nucleic acid sequence, (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 with the nucleic acid sequence of SEQ ID NO: 1, 2 or 3; and (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 with the nucleic acid sequence of SEQ ID NO: 5, 6, 8 or 9. is provided.

[0046] In one embodiment, a nucleic acid encoding a hantavirus nucleoprotein or an antigenic fragment thereof sequence comprises a first nucleic acid sequence and a second nucleic acid sequence, (A) the first nucleic acid sequence has 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, 17, 22, 23 or 24; and is provided by a nucleic acid sequence; and and (B) The second nucleic acid sequence is at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) identical to the nucleic acid sequence of SEQ ID NO: 18, 19, 20, 21, 25, 26, 27 or 28. or 28 and is provided by a nucleic acid sequence having sequence identity.

[0047] 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, (A) the first nucleic acid sequence has at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 15, 16, 17, 22, 23 or 24; and (B) the second nucleic acid sequence has at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 18, 19, 20, 21, 25, 26, 27 or 28. (B) the second nucleic acid sequence has at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 18, 19, 20, 21, 25, 26, 27 or 28.

[0048] 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, (A) the first nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO: 24; and (B) the second nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO: 28. (B) the second nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO: 28.

[0049] 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, (A) the first nucleic acid sequence has at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 22 or 23; and (B) the second nucleic acid sequence has at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 25, 26 or 27. (B) the second nucleic acid sequence has at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 25, 26 or 27. and provided by a nucleic acid sequence having at least 70% sequence identity.

[0050] In one embodiment, a nucleic acid encoding a hantavirus nucleoprotein or an antigenic fragment thereof sequence comprises a first nucleic acid sequence and a second nucleic acid sequence, (A) the first nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO: 17; and and (B) the second nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO: 21.

[0051] In one embodiment, a nucleic acid encoding a hantavirus nucleoprotein or an antigenic fragment thereof sequence comprises a first nucleic acid sequence and a second nucleic acid sequence, (A) the first nucleic acid sequence has at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 15 or 16; and and (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.

[0052] In one embodiment, a nucleic acid encoding a hantavirus nucleoprotein or an antigenic fragment thereof sequence comprises a first nucleic acid sequence and a second nucleic acid sequence, (A) the first nucleic acid sequence has at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 1, 2 or 3; and and (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.

[0053] In one embodiment, a nucleic acid encoding a hantavirus nucleoprotein or an antigenic fragment thereof sequence comprises a first nucleic acid sequence and a second nucleic acid sequence, (A) The first nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO: 3 ; and (B) The second nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO: 9 .

[0054] 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, (A) The first nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO: 2 ; and (B) The second nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO: 8 .

[0055] 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, (A) The first nucleic acid has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO: 2 ; and (B) The second nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO: 6 .

[0056] 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, (A) The first nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO: 1 ; and (B) The second nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO: 5 .

[0057] 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.

[0058] In one embodiment, a nucleic acid encoding a hantavirus nucleoprotein or an antigenic fragment thereof sequence 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 SEQ ID NO: 29. ATGGCCACAATGGAAGAGATCCAGAGAGAGATCAGCGCCC ACGAGGGACAGCTGGTTATCGCCAGACAGAAAGTGAAGGA CGCCGAGAAGCAGTACGAGAAGGACCCCGACGATCTGAAC AAGAGAGCCCTGCACGACAGAGAAAGCGTGGCCGCCTCTA TCCAGAGCAAGATCGATGAGCTGAAGAGACAGCTGGCCGA CAGAATCGCCGCTGGCAAGAATATTGGCCAGGACAGAGAT CCCACAGGCGTGGAACCTGGCGATCACCTGAAAGAGAGAA GCGCCCTGTCCTATGGCAACACCCTGGACCTGAACAGCCT GGACATTGATGAGCCTACCGGCCAGACAGCCGACTGGCTG ACAATCATTGTGTACCTGACCAGCTTCGTGGTCCCCATCA TCCTGAAGGCCCTGTACATGCTGACCACCAGAGGCAGACA GACCAGCAAGGACAACAAGGGCATGAGAATCCGGTTCAAG GATGACAGCAGCTACGAGGACGTGAACGGCATTAGAAAGC CCAAGCACCTGTACGTGTCCATGCCTAACGCTCAGAGCAG CATGAAGGCCGAGGAAATCACCCCTGGCAGATTCAGAACA GCCGTGTGCGGACTGTACCCCGCTCAGATCAAGGCCAGAA ACATGGTGTCCCCAGTGATGAGCGTCGTGGGATTTCTGGC CCTGGCTAAGGACTGGACCAGCAGGATTGAGGAATGGCTG GGAGCCCCTTGCAAGTTTATGGCCGAGTCTCCTATCGCCG GCAGCCTGTCTGGCAACCCCGTGAATAGAGACTACATCAG ACAGAGGCAGGGCGCTCTGGCCGGAATGGAACCCAAAGAA TTTCAGGCCCTGCGGCAGCACTCTAAGGATGCCGGATGTA CCCTGGTGGAACACATTGAGAGCCCCAGCAGCATCTGGGT TTTCGCTGGCGCTCCTGATAGATGCCCTCCTACCTGTCTG TTTGTTGGCGGAATGGCCGAGCTGGGCGCCTTCTTTAGCA TTCTGCAGGACATGCGGAATACCATCATGGCCAGCAAGAC CGTGGGCACCGCCGATGAGAAGCTGAGAAAGAAGTCCAGC TTCTACCAGAGCTACCTGCGGAGAACCCAGAGCATGGGCA TTCAGCTGGACCAGAGAATCATCGTGATGTTCATGGTGGC CTGGGGCAAAGAAGCCGTGGACAATTTTCACCTGGGCGAC GACATGGACCCCGAGCTGAGATCTCTGGCCCAGATCCTGA TCGACCAGAAAGTCAAAGAGATCTCCAATCAAGAGCCCAT GAAGCTGATGCTGAGCTACGGCAACGTGCTGGATCTGAAC CACCTGGATATCGACGAGCCAACAGGACAGACCGCTGATT GGCTGGGCATCGTGATCTACCTGACCTCCTTTGTGGTGCC TATTCTGCTCAAAGCCCTCTATATGCTGACAACACGCGGA AGGCAGACCACCAAAGATAACAAAGGCACCCGGATCAGGT TTAAGGACGACAGCTCCTTTGAGGATGTCAACGGCATCCG GAAACCTAAGCACCTCTATGTGTCTCTGCCCAATGCACAG TCCTCCATGAAGGCAGAAGAGATCACACCAGGCCGGTACA GAACCGCCATCTGTGGACTGTATCCTGCACAAATCAAAGC CCGGCAGATGATCAGCCCCGTGATGTCCGTTATCGGATTC CTGGCTCTGGCCAAAGATTGGAGCGACAGGATCGAGCAGT GGCTGAGCGAGCCTTGCAAGCTGCTTCCTGATACAGCCGC TGTGTCACTGCTTGGCGGCCCTGCCACAAACAGAGATTAC CTGAGACAGAGACAGGTGGCACTGGGCAACATGGAAACAA AAGAGAGCAAGGCCATCCGGCAGCATGCCGAAGCTGCTGG CTGTAGCATGATCGAGGATATCGAGTCCCCTAGCTCCATT TGGGTGTTCGCAGGGGCCCCAGATAGATGTCCACCAACAT GCCTGTTCATTGCCGGCATGGCTGAACTGGGAGCTTTTTT CAGCATCCTCCAGGATATGCGCAACACGATTATGGCCTCC AAGACAGTGGGAACCAGCGAGGAAAAGCTGCGGAAGAAAA GCAGCTTTTACCAGTCTTACCTGAGGCGGACCCAGTCCAT GGGGATCCAACTGGATCAGCGGATCATTGTGCTGTTTATG GTCGCTTGGGGAAAAGAGGCTGTCGATAACTTCCACCTGG GAGATGATATGGATCCTGAACTGCGGACCCTGGCTCAGTC CCTGATCGATGTGAAAGTGAAAGAAATTAGTAATCAAGAA CCCCTCAAGCTG (SEQ ID NO: 29)

[0059] In one embodiment, a nucleic acid encoding a hantavirus nucleoprotein or an antigenic fragment thereof The sequence 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 SEQ ID NO: 30. ATGCTGAGCTACGGCAACGTGCTGGATCTGAACCACCTGG ATATCGACGAGCCAACAGGACAGACCGCTGATTGGCTGGG CATCGTGATCTACCTGACCTCCTTTGTGGTGCCTATTCTG CTCAAAGCCCTCTATATGCTGACAACACGCGGAAGGCAGA CCACCAAAGATAACAAAGGCACCCGGATCAGGTTTAAGGA CGACAGCTCCTTTGAGGATGTCAACGGCATCCGGAAACCT AAGCACCTCTATGTGTCTCTGCCCAATGCACAGTCCTCCA TGAAGGCAGAAGAGATCACACCAGGCCGGTACAGAACCGC CATCTGTGGACTGTATCCTGCACAAATCAAAGCCCGGCAG ATGATCAGCCCCGTGATGTCCGTTATCGGATTCCTGGCTC TGGCCAAAGATTGGAGCGACAGGATCGAGCAGTGGCTGAG CGAGCCTTGCAAGCTGCTTCCTGATACAGCCGCTGTGTCA CTGCTTGGCGGCCCTGCCACAAACAGAGATTACCTGAGAC AGAGACAGGTGGCACTGGGCAACATGGAAACAAAAGAGAG CAAGGCCATCCGGCAGCATGCCGAAGCTGCTGGCTGTAGC ATGATCGAGGATATCGAGTCCCCTAGCTCCATTTGGGTGT TCGCAGGGGCCCCAGATAGATGTCCACCAACATGCCTGTT CATTGCCGGCATGGCTGAACTGGGAGCTTTTTTCAGCATC CTCCAGGATATGCGCAACACGATTATGGCCTCCAAGACAG TGGGAACCAGCGAGGAAAAGCTGCGGAAGAAAAGCAGCTT TTACCAGTCTTACCTGAGGCGGACCCAGTCCATGGGGATC CAACTGGATCAGCGGATCATTGTGCTGTTTATGGTCGCTT GGGGAAAAGAGGCTGTCGATAACTTCCACCTGGGAGATGA TATGGATCCTGAACTGCGGACCCTGGCTCAGTCCCTGATC GATGTGAAAGTGAAAGAAATTAGTAATCAAGAACCCCTCA AGCTGATGGCCACAATGGAAGAGATCCAGAGAGAGATCAG CGCCCACGAGGGACAGCTGGTTATCGCCAGACAGAAAGTG AAGGACGCCGAGAAGCAGTACGAGAAGGACCCCGACGATC TGAACAAGAGAGCCCTGCACGACAGAGAAAGCGTGGCCGC CTCTATCCAGAGCAAGATCGATGAGCTGAAGAGACAGCTG GCCGACAGAATCGCCGCTGGCAAGAATATTGGCCAGGACA GAGATCCCACAGGCGTGGAACCTGGCGATCACCTGAAAGA GAGAAGCGCCCTGTCCTATGGCAACACCCTGGACCTGAAC AGCCTGGACATTGATGAGCCTACCGGCCAGACAGCCGACT GGCTGACAATCATTGTGTACCTGACCAGCTTCGTGGTCCC CATCATCCTGAAGGCCCTGTACATGCTGACCACCAGAGGC AGACAGACCAGCAAGGACAACAAGGGCATGAGAATCCGGT TCAAGGATGACAGCAGCTACGAGGACGTGAACGGCATTAG AAAGCCCAAGCACCTGTACGTGTCCATGCCTAACGCTCAG AGCAGCATGAAGGCCGAGGAAATCACCCCTGGCAGATTCA GAACAGCCGTGTGCGGACTGTACCCCGCTCAGATCAAGGC CAGAAACATGGTGTCCCCAGTGATGAGCGTCGTGGGATTT CTGGCCCTGGCTAAGGACTGGACCAGCAGGATTGAGGAAT GGCTGGGAGCCCCTTGCAAGTTTATGGCCGAGTCTCCTAT CGCCGGCAGCCTGTCTGGCAACCCCGTGAATAGAGACTAC ATCAGACAGAGGCAGGGCGCTCTGGCCGGAATGGAACCCA AAGAATTTCAGGCCCTGCGGCAGCACTCTAAGGATGCCGG ATGTACCCTGGTGGAACACATTGAGAGCCCCAGCAGCATC TGGGTTTTCGCTGGCGCTCCTGATAGATGCCCTCCTACCT GTCTGTTTGTTGGCGGAATGGCCGAGCTGGGCGCCTTCTT TAGCATTCTGCAGGACATGCGGAATACCATCATGGCCAGC AAGACCGTGGGCACCGCCGATGAGAAGCTGAGAAAGAAGT CCAGCTTCTACCAGAGCTACCTGCGGAGAACCCAGAGCAT GGGCATTCAGCTGGACCAGAGAATCATCGTGATGTTCATG GTGGCCTGGGGCAAAGAAGCCGTGGACAATTTTCACCTGG GCGACGACATGGACCCCGAGCTGAGATCTCTGGCCCAGAT CCTGATCGACCAGAAAGTCAAAGAGATCTCCAATCAAGAG CCCATGAAGCTG (SEQ ID NO: 30)

[0060] The inventors of the present invention have found 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 found 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, a very effective immune response against hantavirus can be obtained.

[0061] ​​​​​A vector can be used as a vector for delivering genetic material to a target cell and is a tool. As an example, a viral vector functions as an antigen delivery vehicle and also has the ability to activate the innate immune system by binding to cell surface molecules that recognize viral elements . It is possible to prepare a recombinant viral vector carrying a nucleic acid encoding a given antigen . Subsequently, the viral vector can be used to deliver the nucleic acid to the target cell, where the antigen encoded therein is produced and then presented to the immune system by the molecular machinery of the target cell itself . As "non-self", the produced antigen generates an adaptive immune response in the target subject . Advantageously, the vectors of the present invention have been demonstrated herein to provide a protective immune response .

[0062] Viral vectors suitable for use in the present invention include poxvirus vectors (such as non-replicating poxvirus vectors), adenovirus vectors, and influenza virus vectors .

[0063] In certain embodiments, the "viral vector" is a virus-like particle (VLP) . A VLP is a lipid envelope particle containing viral proteins. Certain viral proteins have the inherent ability to self-assemble, and in this process, they bud from the cell membrane as independent membrane envelope particles . VLPs are easy to purify and can be used, for example, to present viral antigens. Therefore, VLPs are suitable for use in immunogenic compositions such as those described below . In certain embodiments, the "viral vector" is not a virus-like particle .

[0064] ​​​​​A bacterial vector can also be used as an antigen delivery vehicle. A recombinant bacterial vector carrying a nucleic acid encoding a given antigen can be prepared. The recombinant bacterial vector can express the antigen on its surface. After administration to a subject, the bacterial vector colonizes antigen-presenting cells (e.g., dendritic cells or macrophages). An antigen-specific immune response is induced. The immune response can be a cellular (T cell) immune response or can include both a humoral (e.g., B cell) and a cellular (T cell) immune response. Examples of bacteria suitable for use as recombinant bacterial vectors include Escherichia coli, Shigella, Salmonella (e.g., Salmonella typhimurium), and Listeria. In one embodiment, the vector of the present invention is a bacterial vector and the bacterium is a gram-negative bacterium. In one embodiment, the vector of the present invention is a bacterial vector selected from an Escherichia coli vector, a Shigella vector, a Salmonella vector, and a Listeria vector. It is not desired to be bound by any particular theory, but the inventors believe that antigen delivery using the vectors of the present invention stimulates a T cell response in a subject among several responses. Thus, the inventors believe that one way the present invention provides protection against hantavirus infection is by stimulating the T cell response and the cell-mediated immune system. Additionally, humoral (antibody)-based protection can also be achieved. The viral vector of the present invention can be a non-replicating viral vector. As used herein, a non-replicating viral vector does not productively after infection of the target cell

[0065]

[0066]

[0067] It is a viral vector lacking the ability to replicate. Therefore, the ability of a non-replicating viral vector to produce its own copies after infecting a target cell (e.g., a human target cell of an individual vaccinated with the non-replicating viral vector) is highly reduced or absent. Such viral vectors are sometimes referred to as attenuated or replication-deficient. The cause can be the loss / deletion of genes essential for replication in the target cell. Therefore, a non-replicating viral vector cannot effectively produce its own copies after infecting a target cell. Therefore, non-replicating viral vectors may advantageously have an improved safety profile compared to replicable viral vectors. Non-replicating viral vectors can retain the ability to replicate in cells other than target cells, enabling the production of viral vectors. As an example, a non-replicating viral vector (e.g., a non-replicating poxvirus vector) lacks the ability to productively replicate in target cells such as mammalian cells (e.g., human cells), but may retain the ability to replicate in avian cells (e.g., chicken embryo fibroblasts or CEF cells) (therefore enabling vector production). After infecting a target cell (e.g., a human target cell of an individual vaccinated with the non-replicating viral vector), the ability to produce its own copies is highly reduced or absent Such viral vectors are sometimes also called attenuated or replication-deficient. The cause can be the loss / deletion of genes essential for replication in the target cell. Therefore, non-replicating viral vectors cannot effectively produce their own copies after infecting target cells Therefore, non-replicating viral vectors may advantageously have an improved safety profile compared to replicable viral vectors. Non-replicating viral vectors can retain the ability to replicate in cells other than target cells, enabling the production of viral vectors As an example, a non-replicating viral vector (e.g., a non-replicating poxvirus vector ) lacks the ability to productively replicate in target cells such as mammalian cells (e.g., human cells), but may retain the ability to replicate in avian cells (e.g., chicken embryo fibroblasts or CEF cells) and thus enable vector production

[0068] The viral vector of the present invention can be a non-replicating poxvirus vector. Therefore in one embodiment, the viral vector encoding the hantavirus NP or an antigenic fragment thereof is a non-replicating poxvirus vector

[0069] In one embodiment, the non-replicating poxvirus vector is a modified vaccinia virus Ankara (MVA) vector, NYVAC vaccinia virus vector, canarypox (ALVA virus vector ​​​​C) It is selected from vectors and fowlpox (FPV) vectors. MVA and NYVAC are both attenuated derivatives of vaccinia virus. Compared with vaccinia virus, MVA lacks about 26 out of about 200 open reading frames.

[0070] In one embodiment, the non-replicating poxvirus vector is an FPV vector.

[0071] In a preferred embodiment, the non-replicating poxvirus vector is an MVA vector.

[0072] The viral vector of the present invention can be an adenovirus vector. Thus, in one embodiment, the viral vector encoding the hantavirus NP or an antigenic fragment thereof is an adenovirus vector.

[0073] In one embodiment, the adenovirus vector is a non-replicating adenovirus vector (non-replicating is defined as above). Adenovirus can be made non-replicating by deletion of both the E1 gene region or both the E1 and E3 gene regions. Alternatively, adenovirus can be made non-replicating by modification of the E1 gene region or both the E1 and E 3 gene regions such that the gene regions are non-functional. For example, the non-replicating adenovirus may lack a functional E1 region or may lack both functional E1 and E3 gene regions. In this way, adenovirus is rendered non-replicable in most mammalian cell lines and does not replicate in immunized mammals. Most preferably, both the E1 and E3 gene regions are deleted in the adenovirus, and thus, a larger size of the introduced gene can be accommodated. gene can be accommodated. A transgene can be inserted. This is to express a larger antigen or when expressing multiple antigens with a single vector, or when using a large promoter sequence such as the CMV promoter. Deletion of the E3 region as well as the E1 region is particularly preferred for recombinant Ad5 vectors. Optionally, the E4 region can also be manipulated .

[0074] In one embodiment, the adenovirus vector is a human adenovirus vector, a simian adeno virus 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 selected from.

[0075] The viral vector of the present invention can be a measles virus vector. Thus, in one embodiment the viral vector encoding the hantavirus NP or an antigenic fragment thereof is a measles virus vector.

[0076] In one embodiment, the expression cassette containing the nucleic acid sequence encoding the hantavirus NP (or an antigenic fragment thereof) is less than 9 kb (e.g., 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 is less than 0 kb).

[0077] In one embodiment, a nucleic acid sequence encoding hantavirus NP (or an antigenic fragment thereof) The expression cassette containing is less than 8 kb (e.g., 8.0, 7.9, 7.8, 7.7, 7.6 , 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 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, 1.0 kb less).

[0078] In one embodiment, a nucleic acid sequence encoding hantavirus NP (or an antigenic fragment thereof) The expression cassette containing is less than 7 kb (e.g., 7.0, 6.9, 6.8, 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 , less than 1.5, 1.4, 1.3, 1.2, 1.1, 1.0 kb).

[0079] In one embodiment, a nucleic acid sequence encoding hantavirus NP (or an antigenic fragment thereof) The expression cassette containing is less than 6 kb (e.g., 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, less than 1.4, 1.3, 1.2, 1.1, 1.0 kb).

[0080] In one embodiment, a nucleic acid sequence encoding hantavirus NP (or an antigenic fragment thereof) The expression cassette containing is less than 5 kb (e.g., 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 , less than 1.5, 1.4, 1.3, 1.2, 1.1, 1.0 kb).

[0081] In one embodiment, a nucleic acid sequence encoding hantavirus NP (or an antigenic fragment thereof) The expression cassette containing is less than 4.5 kb (e.g., 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, less than 1.0 kb).

[0082] In one embodiment where the vector is a viral vector, the virus (i.e., the viral vect tor) is not a pseudotyped virus. Thus, in one embodiment, the envelope of the viral vector does not contain foreign glycoproteins (i.e., glycoproteins that are not native to the viral vector).

[0083] In one embodiment where the vector is a non-replicating poxvirus vector (such as an MVA vector), the nucleic acid sequence encoding the hantavirus NP or an antigenic fragment thereof comprises a nucleic acid sequence encoding the hantavirus glycoprotein.

[0084] In one embodiment where the vector is a non-replicating poxvirus vector (such as an MVA vector), the nucleic acid sequence encoding the hantavirus NP or an antigenic fragment thereof comprises a nucleic acid sequence encoding an epitope of the hantavirus glycoprotein (GP).

[0085] In one embodiment where the vector is a non-replicating poxvirus vector (such as an MVA vector), the nucleic acid sequence encoding the hantavirus NP or an antigenic fragment thereof does not comprise a nucleic acid sequence encoding the hantavirus glycoprotein (GP).

[0086] In one embodiment where the vector is a non-replicating poxvirus vector (such as an MVA vector), the nucleic acid sequence encoding the hantavirus NP or an antigenic fragment thereof does not comprise a nucleic acid sequence encoding an epitope of the hantavirus glycoprotein (GP).

[0087] ​​​​​ In one embodiment, the hantavirus nucleoprotein or an antigenic fragment thereof is the only hantavirus nucleic acid sequence in the vector.

[0088] In one embodiment where 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.

[0089] In one embodiment where the vector is an adenovirus vector, the vector is stable, expresses the hantavirus NP product, and induces a protective immune response in a subject.

[0090] The nucleic acid sequence described above may include a nucleic acid sequence encoding a hantavirus NP that includes a fusion protein. The fusion protein may include a hantavirus NP polypeptide fused to one or more additional polypeptides, such as an epitope tag, another antigen, or a protein that increases immunogenicity (e.g., flagellin ).

[0091] In one embodiment, the nucleic acid sequence encoding a hantavirus NP (as described above) further 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, and the presence of the V5 fusion protein sequence can provide identification of the expressed protein by immunolabeling.

[0092] In one embodiment, the vector (as described above) further includes a nucleic acid sequence encoding an adjuvant (e.g., cholera toxin, E. coli lethal toxin, or flagellin).

[0093] In one embodiment, the vector does not contain a nucleic acid sequence encoding an adjuvant. In some embodiments, the vector does not include a nucleic acid sequence encoding Hsp70.

[0094] The bacterial vectors of the present invention can be used in any manner known in the art for engineering and generating recombinant bacteria. The method may be performed using the techniques described above.

[0095] In another aspect, the present invention provides a nucleic acid sequence encoding the above-described viral vector. Thus, the nucleic acid sequence may encode a non-replicating poxvirus vector as described above. Thus, the nucleic acid sequence may encode an adenoviral vector as described above.

[0096] Nucleic acid sequences encoding viral vectors (as described above) can be used in recombinant vectors known in the art. It may be produced by use of any technique for manipulating and producing acids.

[0097] In one aspect, the invention provides a method for preparing a viral vector (as described above), comprising the steps of: providing a nucleic acid comprising a nucleic acid sequence encoding a vector (as described above); and rendering said host cell suitable for propagation of said vector. and obtaining the vector from the host cell. Provide.

[0098] As used herein, "transfection" refers to any non-invasive method of introducing nucleic acid into a cell. The nucleic acid may be any suitable vector for transfecting a host cell. Thus, in one embodiment, the nucleic acid is a plasmid. The host cell is , a vector (e.g., a non-replicating poxvirus vector or an adenovirus vector as described above) a cell in which the () can grow. As used herein, "culturing a host cell under conditions suitable for the growth of the vector" means any cell culture conditions and techniques known in the art that are suitable for the selected host cell and allow the vector to be produced within the host cell. As used herein, "obtaining a vector" means using any technique known in the art suitable for separating the vector from the host cell. Thus, the host cell can be lysed to release the vector. The vector can then be isolated and purified using any one or more suitable methods known in the art. "Culturing a host cell under conditions suitable for the growth of the vector" means using any cell culture conditions and techniques known in the art that are suitable for the selected host cell and allow the vector to be produced within the host cell. "Obtaining a vector" means using any technique known in the art suitable for separating the vector from the host cell. Thus, the host cell can be lysed to release the vector. The vector can then be isolated and purified using any one or more suitable methods known in the art.

[0099] In one aspect, the invention provides a host cell comprising a nucleic acid sequence encoding the viral vector described above. The host cell can be any cell in which the viral vector (e.g., the non-replicating poxvirus vector or adenovirus vector described above) can grow or proliferate. In one embodiment, the host cell is selected from 293 cells (also known 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.

[0100] The invention also provides a composition comprising the vector described above.

[0101] In one aspect, the invention provides a composition comprising a vector (as described above) and a pharmaceutically acceptable carrier.

[0102] ​​​​​​​​​​ Substances suitable for use as pharmaceutically acceptable carriers are known in the art. Pharmaceutically Non-limiting examples of acceptable carriers include water, physiological saline, and phosphate-buffered saline can be mentioned. However, in some embodiments, the composition is in lyophilized form, in which case , stabilizers such as bovine serum albumin (BSA) may be included. In some embodiments, for ease of long-term storage, it may be desirable to formulate the composition with preservatives such as thimerosal or sodium azide . Examples of buffers include, but are not limited to, sodium succinate (pH 6.5), and phosphate-buffered saline (P BS; pH 7.4).

[0103] In addition to pharmaceutically acceptable carriers, the compositions of the present invention can be further combined with one or more of salts, excipients, diluents, adjuvants, immunomodulators, and / or antibacterial compounds .

[0104] Advantageously, the vectors of the present invention have been demonstrated to provide a protective immune response without the use of adjuvants . Thus, in one embodiment, the compositions of the present invention do not contain adjuvants .

[0105] The composition can be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, succinic acid , tartaric acid, maleic acid, etc.). Salts formed with free carboxyl groups also include inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, and organic bases such as isopropyl amine . ​​​Luramine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc. can be derived from.

[0106] In one embodiment, the composition (such as described above) further comprises at least one hantavirus NP antigen in the form of a polypeptide, i.e., an antigen present in the composition). Thus, the composition can comprise both a vector 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, an improved simultaneous response of T cells and antibodies can be achieved. In one embodiment, the T cell and antibody responses achieved are greater than those achieved when either the vector or the polypeptide antigen is used alone.

[0107] In one embodiment, the polypeptide antigen is not bound to the vector. In one embodiment, the polypeptide antigen is a separate component from the vector. In one embodiment, the polypeptide antigen is provided separately from the vector.

[0108] In one embodiment, the polypeptide antigen is a variant of the antigen encoded by the vector. In one embodiment, the polypeptide antigen is a fragment of the antigen encoded by the vector. In one embodiment, the polypeptide antigen comprises at least a portion of the polypeptide sequence encoded by the nucleic acid sequence of the vector. Thus, the polypeptide antigen can correspond to at least a portion of the antigen encoded by the vector.

[0109] 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 with an amino acid sequence selected from SEQ ID NOs: 4, 7 and 10. and 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 NOs: 11 and 12.

[0110] 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 with an amino acid sequence selected from SEQ ID NOs: 13 and 14.

[0111] 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 with an amino acid sequence selected from SEQ ID NOs: 31 and 32.

[0112] 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 with an amino acid sequence selected from SEQ ID NOs: 31 and 32. MATMEEIQREISAHEGQLVIARQKVKDAEKQYEKDPDDLN ​​​KRALHDRESVAASIQSKIDELKRQLADRIAAGKNIGQDRD PTGVEPGDHLKERSALSYGNTLDLNSLDIDEPTGQTADWL TIIVYLTSFVVPIILKALYMLTTRGRQTSKDNKGMRIRFK DDSSYEDVNGIRKPKHLYVSMPNAQSSMKAEEITPGRFRT AVCGLYPAQIKARNMVSPVMSVVGFLALAKDWTSRIEEWL GAPCKFMAESPIAGSLSGNPVNRDYIRQRQGALAGMEPKE FQALRQHSKDAGCTLVEHIESPSSIWVFAGAPDRCPPTCL FVGGMAELGAFFSILQDMRNTIMASKTVGTADEKLRKKSS FYQSYLRRTQSMGIQLDQRIIVMFMVAWGKEAVDNFHLGD DMDPELRSLAQILIDQKVKEISNQEPMKLMLSYGNVLDLN HLDIDEPTGQTADWLGIVIYLTSFVVPILLKALYMLTTRG RQTTKDNKGTRIRFKDDSSFEDVNGIRKPKHLYVSLPNAQ SSMKAEEITPGRYRTAICGLYPAQIKARQMISPVMSVIGF LALAKDWSDRIEQWLSEPCKLLPDTAAVSLLGGPATNRDY LRQRQVALGNMETKESKAIRQHAEAAGCSMIEDIESPSSI WVFAGAPDRCPPTCLFIAGMAELGAFFSILQDMRNTIMAS KTVGTSEEKLRKKSSFYQSYLRRTQSMGIQLDQRIIVLFM VAWGKEAVDNFHLGDDMDPELRTLAQSLIDVKVKEISNQE PLKL (Sequence No. 31) MLSYGNVLDLNHLDIDEPTGQTADWLGIVIYLTSFVVPIL LKALYMLTTRGRQTTKDNKGTRIRFKDDSSFEDVNGIRKP KHLYVSLPNAQSSMKAEEITPGRYRTAICGLYPAQIKARQ MISPVMSVIGFLALAKDWSDRIEQWLSEPCKLLPDTAAVS LLGGPATNRDYLRQRQVALGNMETKESKAIRQHAEAAGCS MIEDIESPSSIWVFAGAPDRCPPTCLFIAGMAELGAFFSI LQDMRNTIMASKTVGTSEEKLRKKSSFYQSYLRRTQSMGI QLDQRIIVLFMVAWGKEAVDNFHLGDDMDPELRTLAQSLI DVKVKEISNQEPLKLMATMEEIQREISAHEGQLVIARQKV KDAEKQYEKDPDDLNKRALHDRESVAASIQSKIDELKRQL ADRIAAGKNIGQDRDPTGVEPGDHLKERSALSYGNTLDLN SLDIDEPTGQTADWLTIIVYLTSFVVPIILKALYMLTTRG RQTSKDNKGMRIRFKDDSSYEDVNGIRKPKHLYVSMPNAQ SSMKAEEITPGRFRTAVCGLYPAQIKARNMVSPVMSVVGF LALAKDWTSRIEEWLGAPCKFMAESPIAGSLSGNPVNRDY IRQRQGALAGMEPKEFQALRQHSKDAGCTLVEHIESPSSI WVFAGAPDRCPPTCLFVGGMAELGAFFSILQDMRNTIMAS KTVGTADEKLRKKSSFYQSYLRRTQSMGIQLDQRIIVMFM VAWGKEAVDNFHLGDDMDPELRSLAQILIDQKVKEISNQE PMKL (Accession No. 32)

[0113] The polypeptide antigen can be the same as ( or similar to) that encoded by the nucleic acid sequence of the vector of the composition. Thus, the administration of the composition comprising the vector and the polypeptide antigen can be used to achieve an enhanced immune response against a single antigen, and the above-mentioned enhanced immune response includes, as described above, the combined T cell and antibody responses. .

[0114] In one embodiment, the composition of the present invention (as described above) further comprises at least one naked DNA encoding the hantavirus NP or an antigenic fragment thereof (i.e., a DNA molecule separate from and not part of the viral vector of the present invention). 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 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, 9 , 5, 96, 97, 98, 99 or 100%) sequence identity with an amino acid sequence selected from SEQ ID NOs: 4, 7, 10-14, 31 and 32 and encodes a hantavirus NP. In one embodiment, the composition of the present invention (as described above) further comprises an adjuvant. Non-limiting examples of adjuvants suitable for use with the composition of the present invention include aluminum phosphate e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 9 , 5, 96, 97, 98, 99 or 100%) sequence identity and encodes a hantavirus NP comprising (or consisting of) an amino acid sequence. In one embodiment, the composition of the present invention (as described above) further comprises an adjuvant. Non-limiting examples of adjuvants suitable for use with the

[0115] composition of the present invention include aluminum phosphate phosphate Aluminum, aluminum hydroxide and related compounds; monophosphoryl lipid A and related compounds; outer membrane vesicles derived from bacteria; oil-in-water emulsions such as MF59; liposome adjuvants such as virosomes, Freund's adjuvant and related mixtures; polylactide co-glycolic acid (PLGA) particles; cholera toxin; Escherichia coli lethal toxin; and flagellin are exemplified.

[0116] The vectors and compositions (as described above) of the present invention can be used as vaccines. Therefore, the compositions of the present invention can be vaccine compositions.

[0117] As used herein, a vaccine is a preparation that stimulates a protective immune response against an infectious disease when administered to an animal subject such as a mammal (e.g., a human, bovine, porcine, ovine, caprine, equine, deer, canine or feline subject; particularly a human subject). The immune response can be a humoral and / or cell-mediated immune response. Therefore, a vaccine can stimulate B cells and / or T cells.

[0118] The term "vaccine" is used interchangeably herein with the terms "therapeutic / preventive composition", "immunogenic composition", "preparation", "antigenic composition", or "pharmaceutical".

[0119] In one aspect, the present invention provides a vector (as described above) or a composition (as described above) for use in medicine.

[0120] In one aspect, the present 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 is a hantavirus The Lassa antigen (e.g., hantavirus NP) and / or those against hantavirus infection can be. Therefore, the vectors and compositions of the present invention are used (e.g., as an immunogenic composition or as a vaccine) to induce an immune response against hantavirus NP in a subject .

[0121] In one embodiment, the immune response includes a T cell response.

[0122] In one embodiment, the method of inducing an immune response in a subject includes administering to the subject an effective amount of the vector (as described above) or the composition (as described above).

[0123] In one aspect, the present invention provides a vector (as described above) or a composition (as described above) for use in a method of preventing or treating hantavirus infection in a subject .

[0124] In one embodiment, the present 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 .

[0125] The vectors and compositions of the present invention are particularly ideal for use in the prevention or treatment of HFRS, especially when the hantavirus nucleoprotein or its antigenic fragment is derived from Seoul virus. As described above, Seoul virus is typically associated with the cause of HFRS .

[0126] The vectors and compositions of the present invention are particularly ideal for use in the prevention or treatment of HFRS, especially when the hantavirus nucleoprotein or its antigenic fragment is derived from Hantan virus ​is suitable. As described above, hantaviruses are typically associated with the cause of HFRS. associated.

[0127] The vectors and compositions of the present invention are particularly suitable for use in the prevention or treatment of HFRS when the hantavirus nucleoprotein or an antigenic fragment thereof, for example as demonstrated in the examples, is a chimeric sequence comprising a chimera of the Seoul virus nucleoprotein (or an antigenic fragment thereof) and the hantavirus nucleoprotein (or an antigenic fragment thereof). is ideally suitable for use in the prevention or treatment of HFRS. end.

[0128] As used herein, the term "prevent" includes preventing the onset of hantavirus infection and / or reducing the severity of the intensity of hantavirus infection. Thus, "prevent" includes vaccination. end.

[0129] As used herein, the term "treat" includes therapeutic and prophylactic / preventive measures (including post-exposure prophylaxis) and includes post-infection therapy and improvement of hantavirus infection. end.

[0130] In one embodiment, the hantavirus infection is a Seoul virus infection. In one embodiment the hantavirus infection is a hantaan virus infection. In one embodiment, the hantavirus infection is a Seoul virus infection and / or a hantaan virus infection. end. end.

[0131] Each of the above methods may include administering to a subject an effective amount, for example a therapeutically effective amount, of a vector or composition of the present invention. end.

[0132] In this regard, as used herein, an effective amount is an amount that achieves a desired biological result. As used herein, a therapeutically effective amount is a dose or amount sufficient to treat a subject (e.g., The non-invasiveness of such treatments upon single or multiple administration to mammalian subjects, particularly human subjects. Treatment of at least one symptom of the disorder or a recurrence of the disorder in excess of that expected in the presence of the disorder , effective in preventing, suppressing, curing, delaying, reducing the severity of, ameliorating, or prolonging the survival of a subject The quantity.

[0133] Thus, the amount of active ingredient administered will depend on the subject being treated, the target against which a protective immune response is to be generated, The amount of active ingredient required depends on the capacity of the elephant's immune system and the degree of protection required. Precise amounts will depend on the judgment of the practitioner and may be peculiar to each subject.

[0134] Administration to a subject can include administering a vector (as described above) or a composition (as described above) to a subject. The composition can include administering the composition multiple times in succession (e.g., the composition can be administered 2, Thus, in one embodiment, a subject is administered a vector (as described above). a vector or composition (as described above), followed by administration of the same vector or composition (or or a substantially similar vector or composition) is administered again at a different time point.

[0135] In one embodiment, administration to a subject comprises administering a vector (as described above) or administering the composition to a subject, said composition being administered substantially prior to another immunogenic composition. , simultaneously therewith, or subsequently.

[0136] Prior, concurrent, and sequential administration regimens are discussed in more detail below.

[0137] In certain embodiments, the method further comprises administering to a subject a second vector comprising a nucleic acid sequence encoding a hantavirus NP. Preferably, the second vector is a vector of the invention as described above (such as a viral vector, e.g., the non-replicating poxvirus vector or adenovirus vector described above). In certain embodiments, the method further comprises administering to a subject a second vector comprising a nucleic acid sequence encoding a hantavirus NP. Preferably, the second vector is a vector of the invention as described above (such as a viral vector, e.g., the non-replicating poxvirus vector or adenovirus vector described above). In certain embodiments, the method further comprises administering to a subject a second vector comprising a nucleic acid sequence encoding a hantavirus NP. Preferably, the second vector is a vector of the invention as described above (such as a viral vector, e.g., the non-replicating poxvirus vector or adenovirus vector described above). In certain embodiments, the method further comprises administering to a subject a second vector comprising a nucleic acid sequence encoding a hantavirus NP. Preferably, the second vector is a vector of the invention as described above (such as a viral vector, e.g., the non-replicating poxvirus vector or adenovirus vector described above).

[0138] 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 adenovirus vector (such as described above) and the second vector is a non-replicating poxvirus vector (such as described above). In one embodiment, the first vector is a non-replicating poxvirus vector (such as described above) and the second vector is an adenovirus vector (such as described above). 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 adenovirus vector (such as described above) and the second vector is a non-replicating poxvirus vector (such as described above). In one embodiment, the first vector is a non-replicating poxvirus vector (such as described above) and the second vector is an adenovirus vector (such as described above). 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 adenovirus vector (such as described above) and the second vector is a non-replicating poxvirus vector (such as described above). In one embodiment, the first vector is a non-replicating poxvirus vector (such as described above) and the second vector is an adenovirus vector (such as described above). 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 adenovirus vector (such as described above) and the second vector is a non-replicating poxvirus vector (such as described above). In one embodiment, the first vector is a non-replicating poxvirus vector (such as described above) and the second vector is an adenovirus vector (such as described above). 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 adenovirus vector (such as described above) and the second vector is a non-replicating poxvirus vector (such as described above). In one embodiment, the first vector is a non-replicating poxvirus vector (such as described above) and the second vector is an adenovirus vector (such as described above). 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 adenovirus vector (such as described above) and the second vector is a non-replicating poxvirus vector (such as described above). In one embodiment, the first vector is a non-replicating poxvirus vector (such as described above) and the second vector is an adenovirus vector (such as described above).

[0139] In one embodiment, the first and second vectors are administered sequentially in either order. Thus, the first ("1") and second ("2") vectors can be administered to the subject in the order 1 to 2 or 2 to 1. In one embodiment, the first and second vectors are administered sequentially in either order. Thus, the first ("1") and second ("2") vectors can be administered to the subject in the order 1 to 2 or 2 to 1. In one embodiment, the first and second vectors are administered sequentially in either order. Thus, the first ("1") and second ("2") vectors can be administered to the subject in the order 1 to 2 or 2 to 1.

[0140] As used herein, "administered sequentially" has the meaning of "sequential administration" as defined below. Thus, the first and second vectors are administered (substantially) at different times, one after the other. As used herein, "administered sequentially" has the meaning of "sequential administration" as defined below. Thus, the first and second vectors are administered (substantially) at different times, one after the other. As used herein, "administered sequentially" has the meaning of "sequential administration" as defined below. Thus, the first and second vectors are administered (substantially) at different times, one after the other.

[0141] In one embodiment, the first and second vectors are administered as part of a prime-boost administration protocol. Thus, the first vector is administered to the subject as the "prime". In one embodiment, the first and second vectors are administered as part of a prime-boost administration protocol. Thus, the first vector is administered to the subject as the "prime". Alternatively, the second vector can subsequently be administered to the same subject as a "boost". Prime- The prime-boost protocol will be described later.

[0142] In one embodiment, each of the above methods further comprises administering a hantavirus polypeptide antigen to a 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 selected from SEQ ID NOs: 4, 7, 10-14, 31 and 32 and 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 amino acid sequence.

[0143] In one embodiment, the polypeptide antigen is administered separately from the administration of the vector, preferably and the polypeptide antigen and the vector are administered sequentially. In one embodiment, the vector ( "V") and the polypeptide antigen ( "P") can be administered in the order of V to P or in the order of P to V.

[0144] In one embodiment, each of the above methods further comprises administering naked DNA encoding a hantavirus NP or an antigenic fragment thereof to a subject. In one embodiment, the naked DNA has 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. nucleic acid having comprising (or consisting of) an array. In one embodiment, the naked DNA has an amino acid sequence selected from SEQ ID NOs: 4, 7, 10 - 14, 31, and 32 and has 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 and encodes a hantavirus NP comprising (or consisting of) the amino acid sequence.

[0145] In one embodiment, the naked DNA is administered separately from the administration of the vector, and preferably, the naked DNA and the vector are administered sequentially. In one embodiment, the vector (''V'') and the naked DNA (''D'') can be administered in the order of V to D or in the order of D to V.

[0146] In one embodiment, the naked DNA (as described above) is administered as part of a prime - boost protocol.

[0147] Heterologous prime - boost approaches can improve the immune response by enabling repeated vaccination without increasing anti - vector immunity. The hantavirus NP or its antigenic fragment can be delivered sequentially via different vectors (as described above) or naked DNA vectors (as described above). In any heterologous prime - boost vaccination regimen, compared to the use of a single vector, the NP - specific antibody response is increased, the NP - specific T - cell response is increased, and / or the clinical disease is reduced. Suitable combinations of vectors include, but are not limited to: DNA prime, MVA boost DNA prime, fowlpox boost Fowlpox prime, MVA boost MVA Prime, Fowlpox Boost DNA Prime, Fowlpox Boost, MVA Boost MVA Prime, Adenovirus Boost

[0148] As used herein, the term polypeptide includes peptides and proteins and encompasses them.

[0149] In certain embodiments, the above methods further comprise administering an adjuvant to a subject. The adjuvant can be administered with one, two, three, or all four of the first vector, the second vector, the polypeptide antigen, and naked DNA A.

[0150] The immunogenic compositions, therapeutic agents, medicaments, pharmaceutical compositions, and prophylactic agents (e.g., vaccines) of the present invention can be administered on a single-dose schedule (i.e., the entire dose is administered substantially at once). The immunogenic compositions, therapeutic agents, medicaments, pharmaceutical compositions, and prophylactic agents (e.g., vaccines) of the present invention can be administered on a multiple-dose schedule.

[0151] A multiple-dose schedule can be such that the primary course of treatment (e.g., vaccination) can be 1 to 6 individual administrations, and subsequent administrations are given at intervals as needed to maintain and / or enhance the immune response, e.g., for a human subject, the second administration is given at 1 to 4 months and subsequent other administrations are given at 1 to 4 months thereafter as needed, in a schedule where subsequent administrations are given. This is the schedule.

[0152] The dosing regimen is determined at least in part by the needs of the individual and depends on the judgment of the practitioner (e.g., a physician or veterinarian).

[0153] Concurrent administration means administration (substantially) at the same time.

[0154] Sequential administration of two or more compositions / therapeutics / vaccines means that the compositions / therapeutics / vaccines are administered (substantially) sequentially at different times.

[0155] For example, sequential administration can include administration of two or more compositions / therapeutics / vaccines at different times separated by several days (e.g., at least 1, 2, 5, 10, 15, 20, 30 , 60, 90, 100, 150 or 200 days). For example, in one embodiment, the vaccine of the invention can be administered as part of a "prime-boost" vaccination

[0156] regimen. In one embodiment, the immunogenic composition, therapeutic agent, pharmaceutical, pharmaceutical composition, and prophylactic

[0157] agent (e.g., vaccine) of the invention can be administered to a subject, such as a mammalian subject (e.g., a human, bovine, porcine, ovine, caprine, equine, deer, bear, dog or cat subject), either (simultaneously or sequentially) in combination with one or more immunomodulatory agents selected from, for example, immunoglobulins, antibiotics, interleukins (e.g., IL-2, IL-12), and / or cytokines (e.g., IFNγ). The immunogenic composition, therapeutic agent, pharmaceutical, pharmaceutical composition, and prophylactic agent (e.g., vaccine ) can contain 5% to 95% active ingredient, for example at least 10% or 25% active ingredient, or

[0158] at least 40% active ingredient, or at least 50, 55, 60, 70 or 7 5% active ingredient. For example, in one embodiment, the vaccine of the invention can be administered as part of a "prime-boost" vaccination regimen.

[0159] Immunogenic compositions, therapeutic agents, pharmaceuticals, pharmaceutical compositions, and prophylactic agents (e.g., vaccines ) are administered in an amount that is prophylactically and / or therapeutically effective in a manner compatible with the dosage form administered.

[0160] Immunogenic compositions, therapeutic agents, pharmaceuticals, pharmaceutical compositions, and prophylactic agents (e.g., vaccines ) are generally administered by conventional routes, such as intravenous, subcutaneous, intraperitoneal, or mucosal routes . Administration can be parenteral, for example, by subcutaneous or intramuscular injection.

[0161] The immunogenic compositions, therapeutic agents, pharmaceuticals, pharmaceutical compositions, and prophylactic agents (e.g., vaccines) of the present invention can be prepared as either a liquid solution or suspension for injection . Alternatively, a solid form suitable for dissolving or suspending in a liquid before injection may be prepared. The preparation may also be emulsified or the peptide may be encapsulated in liposomes or microcapsules .

[0162] The active ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient . Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like, and combinations thereof. Additionally, if necessary, the immunogenic compositions, therapeutic agents, pharmaceuticals, pharmaceutical compositions, and prophylactic agents (e.g., vaccines) may contain small amounts of auxiliary substances such as wetting agents or emulsifying agents, and / or pH buffering agents.

[0163] Generally, the carrier is a pharmaceutically acceptable carrier. Non-limiting examples of pharmaceutically acceptable carriers include water, saline, and phosphate-buffered saline. However, several ​In one embodiment, the composition is in lyophilized form, in which case it may contain stabilizers such as bovine serum albumin (BS A). In some embodiments, in order to facilitate long-term storage it may be desirable to formulate the composition with preservatives such as thimerosal or sodium azide .

[0164] 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).

[0165] Additional formulations suitable for other modes of administration include suppositories and, optionally, oral formulations or formulations suitable for aerosolized delivery. In the case of suppositories, conventional binders and carriers such as, for example, polyalkylene glycols or triglycerides can be mentioned and such suppositories can be formed from a mixture containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2% .

[0166] Oral formulations include, for example, pharmaceutically grade mannitol, lactose, starch, stear magnesium phosphate, sodium saccharin, cellulose, magnesium carbonate and the like such commonly used excipients. These compositions can take the form of solutions, suspensions, tablets, pills, capsules , sustained release formulations or powders.

[0167] It may be desirable to direct the composition of the present invention (as described above) to the respiratory system of the subject . Efficient delivery of a therapeutic / prophylactic composition or pharmaceutical to the site of infection in the lung can be achieved by oral administration or intranasal administration.

[0168] Formulations for nasal administration can be in the form of nasal drops or nasal sprays. Nasal formulations can contain droplets having an approximate diameter in the range of 100 - 5000 μm, such as, for example, 500 - 4000 μm, 1000 - 3000 μ m or 100 - 1000 μm. Alternatively, in terms of volume, the droplets can be in the range of about 0.001 - 100 μl, such as 0.1 - 50 μl or 1.0 - 25 μl, or, for example, 0.001 - 1 μl.

[0169] Alternatively, the therapeutic / prophylactic formulation or pharmaceutical can be an aerosol formulation. The aerosol formulation can be in the form of a powder, suspension or solution. The size of the aerosol particles is related to the delivery ability of the aerosol. Smaller particles can move further down the respiratory tract towards the alveoli than larger particles. In one embodiment, the aerosol particles have a diameter distribution to facilitate 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 tract, such as the alveoli. In the case of aerosol delivery of a pharmaceutical, the particles can have a diameter in the range of approximately 0.1 - 50 μm, preferably 1 - 25 μm, more preferably 1 - 5 μm. The aerosol particles can be for delivery using a nebulizer (e.g., via the mouth) or a nasal spray. The aerosol formulation can optionally contain a propellant and / or a surfactant. In one embodiment, the immunogenic composition, therapeutic formulation, pharmaceutical, pharmaceutical composition, and prophylactic

[0170] agent (e.g., vaccine) of the present invention comprises a pharmaceutically acceptable carrier, and optionally, a salt, excipient and / or other additives.

[0171] In one embodiment, the immunogenic composition, therapeutic formulation, pharmaceutical, pharmaceutical composition, and prophylactic agent (e.g., vaccine) of the present invention comprises a pharmaceutically acceptable carrier, and optionally, a salt, excipient It contains one or more of an agent, a diluent and / or an adjuvant.

[0172] In one embodiment, the immunogenic composition, therapeutic agent, pharmaceutical, pharmaceutical composition, and preventive agent (e.g., vaccine) of the present invention may contain, for example, immunoglobulins, antibiotics, interleukins (e.g., IL-2, IL-12), and / or cytokines (e.g., IFNγ). It may contain one or more immunomodulators selected from

[0173] The present invention encompasses a polypeptide substantially homologous to a polypeptide based on any one of the polypeptide antigens (including fragments thereof) identified in the present application. The terms "sequence identity" and " sequence homology" are considered synonymous herein. As an example, the polypeptide of interest may contain an amino acid sequence having at least 70

[0174] , 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 1 00% amino acid sequence identity to the amino acid sequence of the reference polypeptide. There are many established algorithms available for aligning two amino acid sequences. Typically, one sequence acts as a reference sequence to which the test sequence can be compared. The sequence comparison algorithm calculates the percent sequence identity of the test sequence to the reference sequence based on the specified program parameters. Alignment of amino acid sequences for comparison can be performed, for example, by computer-implemented algorithms (e.g., GAP, BESTFIT

[0175] , FASTA or TFASTA), or by BLAST and BLAST 2.0 algorithms. (e.g., GAP, BESTFIT, FASTA or TFASTA), or by the BLAST and BLAST 2.0 algorithms.

[0176] The BLOSUM62 matrix shown below is derived from local multiple alignments of approximately 2,000 protein sequence segments representing highly conserved regions of groups of over 500 related proteins (Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992; incorporated herein by reference). Amino acids are represented by the standard single-letter code. The percent identity is calculated as follows. Total number of exact matches _____________________________ x100 [Length of the longer sequence + number of gaps introduced into the longer sequence to align the two sequences] _____________________________ x100 [Length of the longer sequence + number of gaps introduced into the longer sequence to align the two sequences] BLOSUM62 Matrix A R N D C Q E G H I L K M F P S T W Y V A 4 R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6​​​ P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4

[0177] In the homology comparison, identity can be present over regions of sequences that are 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., the full length of the largest reference sequence).

[0178] Substantially homologous polypeptides have one or more amino acid substitutions, deletions or additions. In many embodiments, these changes are of a minor nature, including, for example, only conservative amino acid substitutions. Conservative substitutions are substitutions made by replacing one amino acid in one of the following groups with another amino acid: basic: arginine, lysine, histidine; acidic: glutamic acid, aspartic acid; polar: glutamine, asparagine; hydrophobic: acid: by replacing arginine, lysine, histidine; acidic: glutamic acid, aspartic acid; polar: glutamine, asparagine; hydrophobic: acid: glutamine, asparagine; hydrophobic: ​Leucine, isoleucine, valine; aromatic: phenylalanine, tryptophan, tyrosine ; small molecules: glycine, alanine, serine, threonine, methionine. Substantially identical polypeptides include 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 to 10 or 1 to 5 amino acids) ; and small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20 - 25 residues, or a polypeptide containing an affinity tag is also included.

[0179] 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.

[0180] The polynucleotide sequences of the present invention include nucleic acid sequences transferred from their naturally occurring environment , recombinant or cloned DNA isolates, and chemically synthesized analogs or biologically synthesized analogs produced by heterologous systems.

[0181] The polynucleotides of the present invention can be prepared by any means known in the art. For example , large amounts of polynucleotides can be produced by replication in a suitable host cell. A natural or synthetic DNA fragment encoding the desired fragment is incorporated into a recombinant nucleic acid construct, typically a DNA construct, that can be introduced into and replicated in a prokaryotic or eukaryotic cell . ​. Usually, DNA constructs are suitable for autonomous replication in unicellular hosts such as yeast or bacteria, but can also be intended for introduction into and integration within the genome of cultured insect, mammalian, plant, or other eukaryotic cell lines.

[0182] The polynucleotides of the present invention can also be generated by chemical synthesis, for example, by the phosphoramidite method or the triester method, and can be carried out on commercially available automated oligonucleotide synthesizers. Double-stranded fragments can be obtained from chemically synthesized single-stranded products by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with appropriate primer sequences.

[0183] When applied to nucleic acid sequences, the term "isolated" in the context of the present invention indicates that the polynucleotide sequence has been removed from its natural genetic environment and thus does not contain other exogenous or undesirable coding sequences (although it may contain naturally occurring 5' and 3' untranslated regions such as promoters and terminators), and is in a form suitable for use in a genetically engineered protein production system. Such isolated molecules are molecules separated from their natural environment.

[0184] Considering the degeneracy of the genetic code, significant sequence variation is possible among the polynucleotides of the present invention. The degenerate codons encompassing all possible codons for a given amino acid are shown below as: [Table 1]

[0185] ​​​One of ordinary skill in the art will determine degenerate codons that represent all possible codons encoding each amino acid and will appreciate that flexibility exists when doing so. For example, some of the polynucleotides included in the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can readily identify such variant sequences by referring to the amino acid sequence of the present invention.

[0186] 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 the reference sequence when, upon optimal alignment (using appropriate nucleotide insertions or deletions) with another nucleic acid (or its complementary strand), there is nucleotide sequence identity of at least about 70%, 75%, 80%, 82%, 84%, 86%, 88%, 9 0%, 92%, 94%, 96%, 98% or 99% of the nucleotide bases. Methods for determining nucleic acid sequence homology are known in the art. Alternatively, a "variant" nucleic acid sequence is substantially homologous (or substantially identical) to a reference sequence (or a fragment thereof) if the "variant" and the reference sequence can hybridize under stringent (e.g., highly stringent) hybridization conditions. As readily understood by one of ordinary skill in the art, nucleic acid sequence hybridization

[0187] is affected by conditions such as salt concentration (e.g., NaCl), temperature, or organic solvents, in addition to base composition, length of the complementary strand, and the number of nucleotide base mismatches between the hybridizing nucleic acids. Preferably, stringent temperature conditions, e.g., generally above 30°C and, for example, highly stringent hybridization conditions, are used. As readily understood by one of ordinary skill in the art, nucleic acid sequence hybridization is affected by conditions such as salt concentration (e.g., NaCl), temperature, or organic solvents, in addition to base composition, length of the complementary strand, and the number of nucleotide base mismatches between the hybridizing nucleic acids. Preferably, stringent temperature conditions, e.g., generally above 30°C and, for example, highly stringent hybridization conditions, are used. As readily understood by one of ordinary skill in the art, nucleic acid sequence hybridization is affected by conditions such as salt concentration (e.g., NaCl), temperature, or organic solvents, in addition to base composition, length of the complementary strand, and the number of nucleotide base mismatches between the hybridizing nucleic acids. Preferably, stringent temperature conditions, e.g., generally above 30°C and, for example, highly stringent hybridization conditions, are used. As readily understood by one of ordinary skill in the art, nucleic acid sequence hybridization is affected by conditions such as salt concentration (e.g., NaCl), temperature, or organic solvents, in addition to base composition, length of the complementary strand, and the number of nucleotide base mismatches between the hybridizing nucleic acids. Preferably, stringent temperature conditions, e.g., generally above 30°C , a temperature typically above 37°C, preferably above 45°C, is used. Stringent salts conditions are usually less than 1000 mM, typically less than 500 mM, 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.

[0188] Methods for determining the percentage of nucleic acid sequence identity are known in the art. By way of example, when evaluating nucleic acid sequence identity, a sequence having a defined number of contiguous nucleotides is aligned with a nucleic acid sequence from the corresponding portion of the nucleic acid sequence of the present invention (having the same number of contiguous nucleotides). Tools known in the art for determining the percentage of nucleic acid sequence identity include Nucleotide BLAST.

[0189] Those skilled in the art will recognize that different species exhibit "preferred codon usage frequencies." As used herein, the term "preferred codon usage frequency" refers to the codons most frequently used in the cells of a particular species and thus favors one or a few representatives of the 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, and 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 various methods known in the art. Introduction of preferred codon sequences into recombinant DNA can enhance protein production, for example, by making protein translation more efficient within a particular cell type or species. ​

[0190] Thus, in one embodiment of the present invention, the nucleic acid sequence is codon-optimized for expression in a host cell.

[0191] A "fragment" of a polynucleotide of interest comprises a series of contiguous nucleotides derived from the sequence of said full-length polynucleotide. By way of example, a "fragment" of a polynucleotide of interest comprises at least 30 contiguous nucleotides derived from the sequence of said 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, 12 50, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 16 50, 1700 or 1710 contiguous nucleic acid residues) of said polynucleotide). The fragment may comprise at least one antigenic determinant and / or may encode at least one antigenic epitope of the corresponding polypeptide of interest and / or may have common antigen cross reactivity and / or substantially the same in vivo biological activity as the polypeptide of interest. BRIEF DESCRIPTION OF THE DRAWINGS

[0192]

Figure 1

[0193]

Figure 2

[0194]

Figure 3

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Figure 4

[0196]

Figure 5

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Figure 6

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Figure 7

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Figure 8

[0200]

Figure 9

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Figure 10

Mode for Carrying Out the Invention

Examples

[0202] Example 1 Preparation of the Example MVA-NP (nucleoprotein) Vector A cassette for MVAHantaNP (referred to as "MVAHantaNP") was prepared by GeneArt (Thermofisher 1 promoter, green fluorescent protein (GFP) and MH5 promoter, followed by NP to contain a Kozak sequence upstream of the array. The nuclear protein sequence is a chimeric sequence containing two different sequences from Seoul and Hantaan. ) and contains two different sequences from Seoul and Hantaan. There is a 24-residue linker sequence downstream, followed by a Flag tag epitope and a stop codon. A schematic diagram of MVAHantaNP is provided in Figure 1(A).

[0203] The cassette was inserted into the Sfil / Sfil cloning site of plasmid pMS-RQ-Bb to prepare plasmid 17ACNHBP_MVA-SEOV-HNT-NP_pMS-RQ (pMVAHantaNP).

[0204] A schematic diagram of pMVAHantaNP is provided in Figure 1(B), and the nucleotide sequence of pMVAHantaNP is provided in SEQ ID NO: 33. GTTGGTGGTCGCCATGGATGGTGTTATTGTATACTGTCTA AACGCGTTAGTAAAACATGGCGAGGAAATAAATCATATAA AAAATGATTTCATGATTAAACCATGTTGTGAAAAAGTCAA GAACGTTCACATTGGCGGACAATCTAAAAACAATACAGTG ATTGCAGATTTGCCATATATGGATAATGCGGTATCCGATG TATGCAATTCACTGTATAAAAAGAATGTATCAAGAATATC CAGATTTGCTAATTTGATAAAGATAGATGACGATGACAAG ​​​​ACTCCTACTGGTGTATATAATTATTTTAAACCTAAAGATG CCATTCCTGTTATTATATCCATAGGAAAGGATAGAGATGT TTGTGAACTATTAATCTCATCTGATAAAGCGTGTGCGTGT ATAGAGTTAAATTCATATAAAGTAGCCATTCTTCCCATGG ATGTTTCCTTTTTTACCAAAGGAAATGCATCATTGATTAT TCTCCTGTTTGATTTCTCTATCGATGCGGCACCTCTCTTA AGAAGTGTAACCGATAATAATGTTATTATATCTAGACACC AGCGTCTACATGACGAGCTTCCGAGTTCCAATTGGTTCAA GTTTTACATAAGTATAAAGTCCGACTATTGTTCTATATTA TATATGGTTGTTGATGGATCTGTGATGCATGCAATAGCTG ATAATAGAACTTACGCAAATATTAGCAAAAATATATTAGA CAATACTACAATTAACGATGAGTGTAGATGCTGTTATTTT GAACCACAGATTAGGATTCTTGATAGAGATGAGATGCTCA ATGGATCATCGTGTGATATGAACAGACATTGTATTATGAT GAATTTACCTGATGTAGGCGAATTTGGATCTAGTATGTTG GGGAAATATGAACCTGACATGATTAAGATTGCTCTTTCGG TGGCTGGGTACCAGGCGCGCCTTTCATTTTGTTTTTTTCT ATGCTATAAATGGTGAGCAAGGGCGAGGAGCTGTTCACCG GGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAA CGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGAT GCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCA CCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCAC CCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGAC CACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCG AAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGA CGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGC GACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACT TCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTA CAACTACAACAGCCACAACGTCTATATCATGGCCGACAAG CAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACA ACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCA GCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCC GACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAG ACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTT CGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTG TACAAGTAAGAGCTCCGGCCCGCTCGAGGCCGCTGGTACC CAACCTAAAAATTGAAAATAAATACAAAGGTTCTTGAGGG TTGTGTTAAATTGAAAGCGAGAAATAATCATAAATAAGCC CGGTGCCACCATGGCCACAATGGAAGAGATCCAGAGAGAG ATCAGCGCCCACGAGGGACAGCTGGTTATCGCCAGACAGA AAGTGAAGGACGCCGAGAAGCAGTACGAGAAGGACCCCGA CGATCTGAACAAGAGAGCCCTGCACGACAGAGAAAGCGTG GCCGCCTCTATCCAGAGCAAGATCGATGAGCTGAAGAGAC AGCTGGCCGACAGAATCGCCGCTGGCAAGAATATTGGCCA GGACAGAGATCCCACAGGCGTGGAACCTGGCGATCACCTG AAAGAGAGAAGCGCCCTGTCCTATGGCAACACCCTGGACC TGAACAGCCTGGACATTGATGAGCCTACCGGCCAGACAGC CGACTGGCTGACAATCATTGTGTACCTGACCAGCTTCGTG GTCCCCATCATCCTGAAGGCCCTGTACATGCTGACCACCA GAGGCAGACAGACCAGCAAGGACAACAAGGGCATGAGAAT CCGGTTCAAGGATGACAGCAGCTACGAGGACGTGAACGGC ATTAGAAAGCCCAAGCACCTGTACGTGTCCATGCCTAACG CTCAGAGCAGCATGAAGGCCGAGGAAATCACCCCTGGCAG ATTCAGAACAGCCGTGTGCGGACTGTACCCCGCTCAGATC AAGGCCAGAAACATGGTGTCCCCAGTGATGAGCGTCGTGG GATTTCTGGCCCTGGCTAAGGACTGGACCAGCAGGATTGA GGAATGGCTGGGAGCCCCTTGCAAGTTTATGGCCGAGTCT CCTATCGCCGGCAGCCTGTCTGGCAACCCCGTGAATAGAG ACTACATCAGACAGAGGCAGGGCGCTCTGGCCGGAATGGA ACCCAAAGAATTTCAGGCCCTGCGGCAGCACTCTAAGGAT GCCGGATGTACCCTGGTGGAACACATTGAGAGCCCCAGCA GCATCTGGGTTTTCGCTGGCGCTCCTGATAGATGCCCTCC TACCTGTCTGTTTGTTGGCGGAATGGCCGAGCTGGGCGCC TTCTTTAGCATTCTGCAGGACATGCGGAATACCATCATGG CCAGCAAGACCGTGGGCACCGCCGATGAGAAGCTGAGAAA GAAGTCCAGCTTCTACCAGAGCTACCTGCGGAGAACCCAG AGCATGGGCATTCAGCTGGACCAGAGAATCATCGTGATGT TCATGGTGGCCTGGGGCAAAGAAGCCGTGGACAATTTTCA CCTGGGCGACGACATGGACCCCGAGCTGAGATCTCTGGCC CAGATCCTGATCGACCAGAAAGTCAAAGAGATCTCCAATC AAGAGCCCATGAAGCTGATGCTGAGCTACGGCAACGTGCT GGATCTGAACCACCTGGATATCGACGAGCCAACAGGACAG ACCGCTGATTGGCTGGGCATCGTGATCTACCTGACCTCCT TTGTGGTGCCTATTCTGCTCAAAGCCCTCTATATGCTGAC AACACGCGGAAGGCAGACCACCAAAGATAACAAAGGCACC CGGATCAGGTTTAAGGACGACAGCTCCTTTGAGGATGTCA ACGGCATCCGGAAACCTAAGCACCTCTATGTGTCTCTGCC CAATGCACAGTCCTCCATGAAGGCAGAAGAGATCACACCA GGCCGGTACAGAACCGCCATCTGTGGACTGTATCCTGCAC AAATCAAAGCCCGGCAGATGATCAGCCCCGTGATGTCCGT TATCGGATTCCTGGCTCTGGCCAAAGATTGGAGCGACAGG ATCGAGCAGTGGCTGAGCGAGCCTTGCAAGCTGCTTCCTG ATACAGCCGCTGTGTCACTGCTTGGCGGCCCTGCCACAAA CAGAGATTACCTGAGACAGAGACAGGTGGCACTGGGCAAC ATGGAAACAAAAGAGAGCAAGGCCATCCGGCAGCATGCCG AAGCTGCTGGCTGTAGCATGATCGAGGATATCGAGTCCCC TAGCTCCATTTGGGTGTTCGCAGGGGCCCCAGATAGATGT CCACCAACATGCCTGTTCATTGCCGGCATGGCTGAACTGG GAGCTTTTTTCAGCATCCTCCAGGATATGCGCAACACGAT TATGGCCTCCAAGACAGTGGGAACCAGCGAGGAAAAGCTG CGGAAGAAAAGCAGCTTTTACCAGTCTTACCTGAGGCGGA CCCAGTCCATGGGGATCCAACTGGATCAGCGGATCATTGT GCTGTTTATGGTCGCTTGGGGAAAAGAGGCTGTCGATAAC TTCCACCTGGGAGATGATATGGATCCTGAACTGCGGACCC TGGCTCAGTCCCTGATCGATGTGAAAGTGAAAGAAATTAG TAATCAAGAACCCCTCAAGCTGGACCTGGAAGGCCCTAGA TTCGAGGACTACAAGGACGATGACGACAAGTGACTCGACC TGCAGTTTTTATGGAAAGTTTTATAGGTAGTTGATAGAAC AAAATACATAATTTTGTAAAAATAAATCACTTTTTATACT AATATGACACGATTACCAATACTTTTGTTACTAATATCAT TAGTATACGCTACACCTTTTCCTCAGACATCTAAAAAAAT AGGTGATGATGCAACTTTATCATGTAATCGAAATAATACA AATGACTACGTTGTTATGAGTGCTTGGTATAAGGAGCCCA ATTCCATTATTCTTTTAGCTGCTAAAAGCGACGTCTTGTA TTTTGATAATTATACCAAGGATAAAATATCTTACGACTCT CCATACGATGATCTAGTTACAACTATCACAATTAAATCAT TGACTGCTAGAGATGCCGGTACTTATGTATGTGCATTCTT TATGACATCGCCTACAAATGACACTGATAAAGTAGATTAT GAAGAATACTCCACAGAGTTGATTGTAAATACAGATAGTG AATCGACTATAGACATAATACTATCTGGATCTACACATTC ACCGGAAACTAGTTG (SEQ ID NO: 33) pMVAHantaNP contains the following: DelIII left adjacent region: GTTGGTGGTCGCCATGGATGGTGTTATTGTATACTGTCTA AACGCGTTAGTAAAACATGGCGAGGAAATAAATCATATAA AAAATGATTTCATGATTAAACCATGTTGTGAAAAAGTCAA GAACGTTCACATTGGCGGACAATCTAAAAACAATACAGTG ATTGCAGATTTGCCATATATGGATAATGCGGTATCCGATG TATGCAATTCACTGTATAAAAAGAATGTATCAAGAATATC CAGATTTGCTAATTTGATAAAGATAGATGACGATGACAAG ACTCCTACTGGTGTATATAATTATTTTAAACCTAAAGATG CCATTCCTGTTATTATATCCATAGGAAAGGATAGAGATGT TTGTGAACTATTAATCTCATCTGATAAAGCGTGTGCGTGT ATAGAGTTAAATTCATATAAAGTAGCCATTCTTCCCATGG ATGTTTCCTTTTTTACCAAAGGAAATGCATCATTGATTAT TCTCCTGTTTGATTTCTCTATCGATGCGGCACCTCTCTTA AGAAGTGTAACCGATAATAATGTTATTATATCTAGACACC AGCGTCTACATGACGAGCTTCCGAGTTCCAATTGGTTCAA GTTTTACATAAGTATAAAGTCCGACTATTGTTCTATATTA TATATGGTTGTTGATGGATCTGTGATGCATGCAATAGCTG ATAATAGAACTTACGCAAATATTAGCAAAAATATATTAGA CAATACTACAATTAACGATGAGTGTAGATGCTGTTATTTT GAACCACAGATTAGGATTCTTGATAGAGATGAGATGCTCA ATGGATCATCGTGTGATATGAACAGACATTGTATTATGAT GAATTTACCTGATGTAGGCGAATTTGGATCTAGTATGTTG GGGAAATATGAACCTGACATGATTAAGATTGCTCTTTCGG TGGCTGG (SEQ ID NO: 34) First linker: GTACCAGGCGCGCC (SEQ ID NO: 35) p11: TTTCATTTTGTTTTTTTCTATGCTATAA (SEQ ID NO: 36) GFP: ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGC CCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAA GTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAC GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGC TGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTA CGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAG CAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACG TCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTA CAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTG GTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGG ACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAA CAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAAC GGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGG ACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACAC CCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC TACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACG AGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGC CGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA (SEQ ID NO: 37) Second linker: GAGCTCCGGCCCGCTCGAGGCCGCTGGTACCCAACCT (SEQ ID NO: 38) MH5 promoter: AAAAATTGAAAATAAATACAAAGGTTCTTGAGGGTTGTGT TAAATTGAAAGCGAGAAATAATCATAAATA (SEQ ID NO: 39) Third linker: AGCCCGGT Kozak sequence: GCCACCATGG (SEQ ID NO: 41) The 3' end of the Kozak sequence overlaps with the 5 ' end of the 4 nucleic acids of SEQ ID NO: 29. Nuclear protein (SEQ ID NO: 29) Fourth linker: GACCTGGAAGGCCCTAGATTCGAG (SEQ ID NO: 42) Flag tag: GACTACAAGGACGATGACGACAAG (SEQ ID NO: 43) Stop codon: TGA The 5th linker: CTCGACCTGCAGTTTTTATG (SEQ ID NO: 44) DelIII right adjacent region: GAAAGTTTTATAGGTAGTTGATAGAACAAAATACATAATT TTGTAAAAATAAATCACTTTTTATACTAATATGACACGAT TACCAATACTTTTGTTACTAATATCATTAGTATACGCTAC ACCTTTTCCTCAGACATCTAAAAAAATAGGTGATGATGCA ACTTTATCATGTAATCGAAATAATACAAATGACTACGTTG TTATGAGTGCTTGGTATAAGGAGCCCAATTCCATTATTCT TTTAGCTGCTAAAAGCGACGTCTTGTATTTTGATAATTAT ACCAAGGATAAAATATCTTACGACTCTCCATACGATGATC TAGTTACAACTATCACAATTAAATCATTGACTGCTAGAGA TGCCGGTACTTATGTATGTGCATTCTTTATGACATCGCCT ACAAATGACACTGATAAAGTAGATTATGAAGAATACTCCA CAGAGTTGATTGTAAATACAGATAGTGAATCGACTATAGA CATAATACTATCTGGATCTACACATTCACCGGAAACTAGT TG (SEQ ID NO: 45)

[0205] Purify plasmid DNA from transformed bacteria (E. coli K12 DH10B™ T1R) Prepared, and the concentration was determined by UV spectroscopy by GeneArt (Thermofisher). Determined.

[0206] BHK-21 cells were infected with MVA 1974 at a multiplicity of infection of 0.05. The infected cells were transfected with pMVAHantaNP using Lipofectamine (Life Technologies) as instructed by the manufacturer. The resulting recombinant MVAHantaNP was plaque purified 4 times in chicken embryo fibroblast (「CEF」 ) cells based on GFP expression. MVAHantaNP was amplified on CEF cells and purified by sucrose cushion centrifugation, and titrated by plaque assay on CEF cells before in vivo use. Plaques were visualized by immunostaining using GFP fluorescence, rabbit anti-vaccinia antibody (AbD Serotec, UK) and the Vectastain Universal ABC-AP kit (Vector laboratories, USA). Genomic DNA was extracted from the infected cells using the Wizard SV Genomic DNA Purification System (Promega, USA) and used as a template in PCR using the KAPA2G Fast HotStart PCR kit (KAPABiosystems , USA) for genotyping analysis. The presence of the MVAHantaNP construct was confirmed by polymerase chain reaction (PCR). To check from Hanta NP with an expected size of 3260 bp to the MVA adjacent region, a set of primers was specifically designed (shown in Figure 2).

[0207] The presence of the MVAHantaNP construct was confirmed by polymerase chain reaction (PCR). To check from Hanta NP with an expected size of 3260 bp to the MVA adjacent region, a set of primers was specifically designed (shown in Figure 2). To check from Hanta NP with an expected size of 3260 bp to the MVA adjacent region, a set of primers was specifically designed (shown in Figure 2).

[0208] Sequencing of the expressed proteins confirmed very high sequence fidelity. Recombinant purified MVAHantaNP was cultured in tissue culture flasks of increasing size. First, chicken embryo fibroblasts (CE) were cultured in small flasks. F) MVAHantaNP was grown in cells, harvested, and then transferred to a slightly larger flask. The process was carried out in vitro by infecting CEF cells with MVAHantaNPs, which are 10 times larger than the original size. Repeat this process in larger and larger flasks until you successfully infect the flask of CEF cells. Sucrose cushion centrifugation was performed and the virus pellet was ready for immunogenicity testing. The cells were resuspended in PBS. A total of six batches were prepared: Batches 2+3 and 4+5+6. were pooled into a single sample and titrated for virus concentration.

[0209] The purified vaccine batches were compared with the positive control (the original plasmid obtained from Geneart) and the The second set of primers is inserted from both MVA flanking regions. The results showed that all vaccine batches contained pure recombinant genomic DNA. This shows the presence of MVA (MVA containing an insert). Again, this is the original plasmid. was used as a positive control, and all vaccine batches produced products of the same expected size as the positive control. It has.

[0210] Primer details are as follows: SEQ ID NO: 46: CGGCACCTCTCTTAAGAAGT (Fwd, Del III (targeting the left adjacent region) SEQ ID NO: 47: GTGTAGCGTATACTAATGATATTAG (Rev, De l III targeting the right adjacent region) Array number 48: GGAGTACAACTACAACAGCCACAACG (Fwd, G Targeting FP)

[0211] The GFP Fwd primer binds to the GFP sequence and, when used in combination with the Rev Del III right adjacent primer, reaches from GFP through the nuclear protein to the right MVA adjacent region and specifically identifies the presence of the NP gene.

[0212] Detection of protein expression 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). After 48 hours, when the medium was removed, good GFP fluorescence and CPE were observed by microscopy. The cells were lysed in 1× LDS Nupage® reducing sample buffer (Nupage® sample reducing buffer containing 1× Nupage® sample reducing buffer) (Thermofisher, UK), transferred to Eppendorf tubes, and heated at 70 °C for 10 minutes. Non-infected cells were treated in the same way as a negative control. The MVAHantaNP lysate was subjected to SDS-PAGE on a 4-12% Bis-Tris gel (Life technologies), and the proteins were transferred to a nitrocellulose membrane. The nitrocellulose membrane was blocked using 5% powdered milk (Merck Millipore), then incubated with rocking for 1-2 hours in the presence of the primary antibody (rabbit anti-V5 polyclonal 1 / 1000 in PBS-0.05% Tween 20 (Invitrogen)), and then washed with 0.05% Tween-20 (S Washed three times with PBS containing Sigma-Aldrich). The membrane was incubated for 1 hour with rocking in the presence of HRP-conjugated secondary antibody (anti-rabbit IgG peroxidase 1 / 1000 in PBS-0.05% Tween (Sigma-Aldrich)), and washed as before. Protein expression was determined by detection of the bound antibody using the Pierce ECL WB Substrate Kit (Th ermofisher) according to the manufacturer's instructions and visualized in a Chemi-Illuminescent Imager (Syngene). Molecular weights were determined using the molecular ladder MagicMark XP Western Pr

[0213] otein Standard (Invitrogen) as a reference. Western blot analysis (see Figure 3) confirmed the expression of the fla g tag located downstream of NP. The predicted size of the protein (NP + linker and flag tag) is 89 kDa, and the protein sequence is provided in SEQ ID NO: 49. Expression was observed from passage 3 harvests through the vaccine batch (the inventors observed low levels of protein degradation, which is considered not significant). The band of interest is located at the predicted size of the protein, which also suggests good expression. MATMEEIQREISAHEGQLVIARQKVKDAEKQYEKDPDDLN DDSSYEDVNGIRKPKHLYVSMPNAQSSMKAEEITPGRFRT AVCGLYPAQIKARNMVSPVMSVVGFLALAKDWTSRIEEWL GAPCKFMAESPIAGSLSGNPVNRDYIRQRQGALAGMEPKE FQALRQHSKDAGCTLVEHIESPSSIWVFAGAPDRCPPTCL FVGGMAELGAFFSILQDMRNTIMASKTVGTADEKLRKKSS FYQSYLRRTQSMGIQLDQRIIVMFMVAWGKEAVDNFHLGD DMDPELRSLAQILIDQKVKEISNQEPMKLMLSYGNVLDLN HLDIDEPTGQTADWLGIVIYLTSFVVPILLKALYMLTTRG RQTTKDNKGTRIRFKDDSSFEDVNGIRKPKHLYVSLPNAQ SSMKAEEITPGRYRTAICGLYPAQIKARQMISPVMSVIGF LALAKDWSDRIEQWLSEPCKLLPDTAAVSLLGGPATNRDY LRQRQVALGNMETKESKAIRQHAEAAGCSMIEDIESPSSI WVFAGAPDRCPPTCLFIAGMAELGAFFSILQDMRNTIMAS KTVGTSEEKLRKKSSFYQSYLRRTQSMGIQLDQRIIVLFM VAWGKEAVDNFHLGDDMDPELRTLAQSLIDVKVKEISNQE PLKLDLEGPRFEDYKDDDDK (SEQ ID NO: 49)

[0214] The amino acid sequence of SEQ ID NO: 49 corresponds to the amino acid sequence of SEQ ID NO: 31 with the fourth linker and flag tag added to the expressed one.

[0215] Example 2 Immunogenicity of MVA Hanta NP in A129 mice Eighty male A129 mice, 6 - 8 weeks old, were randomly divided into four groups and tagged in the ear and then vaccinated.

[0216] One group received two doses of MVA Hanta NP in endotoxin - free phosphate - buffered saline (PBS) at 1×10 7 pfu per animal on days 0 and 14 and was vaccinated.

[0217] A second group received a single dose of MVA Hanta NP in endotoxin - free PBS at 1×10 7 plaque - forming units (pfu) per animal on day 14 and was vaccinated.

[0218] A third group received two doses of the MVA empty vector in endotoxin - free PBS at 1×10 7 pfu per animal on days 0 and 14 and was vaccinated.

[0219] A fourth group received two doses of endotoxin - free PBS as a negative control on days 0 and 14 and was vaccinated.

[0220] All mice were injected intramuscularly into the caudal thigh. 100 μl was administered at each vaccination (50 μl in each thigh). The body weight of the animals was recorded daily throughout the study. Five animals from each group were euthanized and spleen tissue and blood were collected on day 28 after the primary vaccination. All efforts were made to minimize the suffering of the animals. These studies were approved by the ethical review processes of PHE, Porton Down, UK and the UK Home Office via project license number 30 / 2993. The work was carried out in accordance with the Animals (Scientific Procedures) Act and the ethical review processes of PHE, Porton Down, UK and the UK Home Office via project license number 30 / 2993. The work was carried out in accordance with the Animals (Scientific Procedures) Act and the UK Home Office ethical review process. The work was carried out in accordance with the Animals (Scientific Procedures) Act​ This was carried out in accordance with the Home Office Practice Codes (1989) on the breeding and management of animals used in scientific procedures in 1986.

[0221] Throughout the study, no clinical signs were observed with respect to vaccination and all mice gained weight as expected (see Figure 4a). All four groups gained weight throughout the study as expected, and the weights of groups 4 were consistently lower than those of groups 1 - 3. Similar percentage weight gains were observed in all groups until the end of the study. These clinical data demonstrate that the mice tolerated the vaccine without adverse effects.

[0222] To determine the T cell response in immunized animals, the frequency of responsive T cells after stimulation with hantavirus - specific peptides was measured using an interferon - gamma ELISPOT assay.

[0223] Spleens were aseptically collected from the test animals, homogenized, and red blood cells were lysed. Splenocytes were resuspended in RPMI medium (Sigma - Aldrich) supplemented with 5% FBS, 2 mM L - glutamine, 100 U penicillin, 0.1 mg / ml streptomycin, 50 mM 2 - mercaptoethanol, and 25 mM HEPES solution (Sigma - Aldrich). Splenocytes were evaluated for antigen recall responses by IFN - γ ELISPOT (Mabtech, Sweden) according to the manufacturer's instructions. Cells were seeded at 2×10e6 per well in PVDF microtiter plates and restimulated with peptide pools (JPT, Berlin).

[0224] ​​​​​​​​​​​​​​The peptide range of the Hanta NP protein sequence is 15 residues long, with a 1 residue overlap between peptides. A total of 189 peptides were generated and tested as 11 peptide pools (see Table 1). See Table 1: Peptide Pools (the numbering of the starting amino acids (“AA”) corresponds to the amino acid numbering in SEQ ID NO: 31). See Table 1: Peptide Pools (the numbering of the starting amino acids (“AA”) corresponds to the amino acid numbering in SEQ ID NO: 31). See Table 1: Peptide Pools (the numbering of the starting amino acids (“AA”) corresponds to the amino acid numbering in SEQ ID NO: 31).

Table 2

[0225] These were applied to cells at a final concentration of 2.5 μg / ml per peptide, with Pools 1 - 10 each having 17 peptides and Pool 11 having 19 peptides. The plates were developed after 18 hours at 37°C and 5% CO in a humidified incubator. The spots were visually counted using an automated ELISPOT reader (Cellular Technologies Limited, USA). The data were presented as responses to individual pools or summed over the entire target protein by subtracting the background values from wells containing cells and medium but no peptides. The results were expressed as spot forming units (SFU) per 10 in a humidified incubator. The spots were visually counted using an automated ELISPOT reader (Cellular Technologies Limited, USA). The data were presented as responses to individual pools or summed over the entire target protein by subtracting the background values from wells containing cells and medium but no peptides. The results were expressed as spot forming units (SFU) per 10 2 in a humidified incubator. The spots were visually counted using an automated ELISPOT reader (Cellular Technologies Limited, USA). The data were presented as responses to individual pools or summed over the entire target protein by subtracting the background values from wells containing cells and medium but no peptides. The results were expressed as spot forming units (SFU) per 10 in a humidified incubator. The spots were visually counted using an automated ELISPOT reader (Cellular Technologies Limited, USA). The data were presented as responses to individual pools or summed over the entire target protein by subtracting the background values from wells containing cells and medium but no peptides. The results were expressed as spot forming units (SFU) per 10 in a humidified incubator. The spots were visually counted using an automated ELISPOT reader (Cellular Technologies Limited, USA). The data were presented as responses to individual pools or summed over the entire target protein by subtracting the background values from wells containing cells and medium but no peptides. The results were expressed as spot forming units (SFU) per 10 in a humidified incubator. The spots were visually counted using an automated ELISPOT reader (Cellular Technologies Limited, USA). The data were presented as responses to individual pools or summed over the entire target protein by subtracting the background values from wells containing cells and medium but no peptides. The results were expressed as spot forming units (SFU) per 10 in a humidified incubator. The spots were visually counted using an automated ELISPOT reader (Cellular Technologies Limited, USA). The data were presented as responses to individual pools or summed over the entire target protein by subtracting the background values from wells containing cells and medium but no peptides. The results were expressed as spot forming units (SFU) per 10 6 cells. The results were expressed as spot forming units (SFU) per 10 cells.

[0226] The MVA - WT group and the PBS group (Groups 3 and 4) were negative when stimulated with all Hanta NP pools. In the prime / boost group and the prime group, IFN -γ responses were detected against several peptide pools, and particularly strong responses were directed towards two distinct regions of the NP (corresponding to pools 4 and 9).

[0227] The inventors found that T cell (IFN-γ) stimulation increased significantly with respect to SEQ ID NOs: 11 and 12.

[0228] In comparison to the control group, increased responses were also detected for pools 2, 3, 5, 7, 8 and 10 with respect to the prime / boost group and the prime group. The total ELISPOT responses from vaccinated and non-vaccinated mice are shown in FIG. 5. FIG. 6 shows the ELISPOT responses against individual peptide pools.

[0229] To measure the antibody response in immunized mice, ELISA assays were performed to evaluate the binding of antibodies to hantavirus-specific proteins. Recombinant Hant a NP (Native Antigen Company, UK) as a crude lysate was diluted in 0.2 M carbonate-bicarbonate buffer pH 9.4 (Thermo Scientific) and 100 μl was used at 1 0 μg / ml to coat Maxisorp 96-well plates (Nunc, Denmark). The plates were incubated overnight at 4 °C and then washed with PBS + 0.01% Tween-20 (Sigma-Aldrich) and blocked with 100 μl of 5% skim milk (Merck, Millipore) in PBS + 0.01% Tween-20 for 1 hour at 37 °C, followed by re-washing with PBS + 0.01% Tween- 20. Samples were diluted in 5% skim milk in PBS + 0.01% Tween-20 buffer ​​​​​​​​​​Dilute to 1:50 and add to the plate in triplicate (100 μl / well), and incubate at 37 °C for 1 hour. Normal mouse serum (Sigma-Aldrich) and polyclonal anti-hantavirus hyperimmune mouse ascetic fluid samples (BEI Resources, USA) were used as positive control sample and negative control sample respectively. Wash the plate with PBS + 0.01% Tween-20, and add 100 μl of polyclonal anti-mouse HRP conjugate (Sigma-Aldrich) diluted 1:20,000 with 5% milk PBS + 0. 01% Tween-20 to each well. After further incubation at 37 °C for 1 hour, wash the plate with PBS + 0.01% Tween- 20, add 100 μl of TMB substrate (Surmodics) to each well, and then incubate at 20 °C for 1 hour. Stop the reaction by adding 100 μl of stop solution (Surmodics) prepared according to the manufacturer's instructions, and read the plate at 450 nm using a molecular devices plate reader and Softmax Pro version 5 .2 software (Molecular Devices). Subtract the background absorbance value from the sample value, and report the result as the absorbance (450 nm) at 1:50 dilution. The data was graphed and analyzed using Graph Pad Prism 7 (see Figure 7).

[0230] The MVA-WT and PBS control groups showed very slight absorbance values similar to those of the blank wells. In both the prime and prime / boost vaccination groups, all The response of the mice was significantly high. The group with prime only recorded an average absorbance of approximately 2.3 and the prime / boost group recorded an average OD of approximately 1.5.

[0231] Therefore, the vectors of the present invention have demonstrated a highly desirable induction of cellular and humoral immune responses.

[0232] Example 3 Efficacy test Sixty male A129 mice weighing 19 - 21 g were randomly divided into four groups in advance and then tagged and microchipped in the ear for identification, and body weight monitoring and temperature monitoring were carried out.

[0233] The remaining mice not sacrificed on day 28 for the immunogenicity test were challenged with Hant a SEOV on day 28. From each group, n = 10 animals were challenged via the intranasal route and n = 5 animals were challenged via the intramuscular route at 1.36×10 6 TCID50 / dose .

[0234] The animals challenged intramuscularly were euthanized on day 33. The mice challenged intranasally were euthanized on day 33 (5 animals / group) or day 42 (5 animals / group). Blood, saliva, liver, kidney, lung and spleen were collected for histological and viral load analysis. All efforts were made to minimize the suffering of the animals. These tests were approved by the ethical review process of PHE, Porton Down, UK and the UK Home Office via project license number 30 / 2993. The work was carried out in accordance with the Animals (Scientific Procedures) Act 1986 and the UK Home Office Codes of Practice for the Housing and Care of Animals Used in It was carried out according to 989).

[0235] Clinical symptoms: The body weight and body temperature of the animals were recorded daily throughout the test. All of the challenged animals remained healthy, and no clinical symptoms were observed after challenge with the hantavirus . The body temperature and body weight throughout the test are reported in Figure 8.

[0236] Virus amount: The virus amount was evaluated 5 days and 14 days after the challenge. As shown in Figure 9, on the 5 th day, due to immunization with MVAHantaNP, a decrease or complete clearance of the hantavirus from the tested tissues was achieved. A very favorable decrease in the virus amount was also observed in most tissues 14 days after the challenge .

[0237] Virus amount - follow-up investigation: In the follow-up investigation, 28 female A129 mice were randomly divided into two groups in advance, and then tagged on the ear and microchipped for identification, and body weight monitoring and temperature monitoring were carried out.

[0238] Of these 28 mice, 16 were primed with GLP-grade MVAHanta NP on day 0 and subsequently boosted on day 14 (「Group A」), and 12 mice were primed and boosted with an empty MVA wild-type vector on days 0 and 14, respectively (「Group B」). Immunization was carried out according to Example 2 above.

[0239] On day 28, 8 mice from Group A and 8 mice from Group B were intranasally challenged with Hanta SEOV at a dose of 3 ×10 6 TCID50 / mouse. ​

[0240] In this follow-up investigation, the viral load was evaluated 5 days after the challenge. As shown in Figure 10 even when the challenge dose exceeded 2-fold, with immunization with MVA Hanta NP, a favorable reduction of Hantavirus from the tissues tested was achieved.

[0241] Example 4 Preparation of the adenovirus vector of the example A non-replicating adenovirus is engineered to express the Hantavirus NP nucleic acid of the present invention or a fragment thereof. The gene sequence of the Hantavirus NP is inserted into the genome of the adenovirus vector. The expression of the Hantavirus NP is demonstrated by the reactivity between the NP-specific antibody and the adenovirus-derived product by Western blotting or ELISA as follows: shown:

[0242] Cell lysates of cells infected with the recombinant adenovirus subjected to SDS-PAGE and Western blotting with an antibody specific for Hantavirus NP show specific reactivity compared to the negative control.

[0243] Alternatively, products derived from cells infected with the recombinant adenovirus are used to coat ELISA plates. Hantavirus-specific antibodies bind to the coating and are detected via a chemical reaction.

[0244] Example 5 The Hantavirus vaccine provides cross-strain protection A vaccine expressing the Hantavirus NP nucleic acid of the present invention or a fragment thereof in an adenovirus or non-replicating poxvirus vector is used to immunize against disease caused by Hantavirus The vaccine is delivered via the parenteral route to mice susceptible to the disease. The animals are challenged with a lethal dose of hantavirus from a strain other than the one listed above. , show no or mild clinical signs of disease and do not require euthanasia. Control animals that received a single dose of hantavirus but no vaccine developed the disease. They show severe signs, reach a humane clinical endpoint, and require euthanasia.

[0245] Example 6 Preparation and efficacy of recombinant influenza virus vectors The protective epitope of Hantavirus NP in the neuraminidase stalk Using reverse genetics to construct recombinant influenza viruses carrying the tope After intranasal or parenteral administration of hantavirus-specific cytotoxic T lymphocytes (CTLs), These CTLs were then induced in mice with hantavirus infection. This results in reduced mortality and clinical disease.

[0246] Example 7 Preparation and efficacy of recombinant bacterial vectors The hantavirus NP nucleic acid or fragment thereof of the present invention is a genetically attenuated Gram-negative The vector is expressed on the surface of the bacteria. After intranasal or parenteral administration to mice, The antigen-presenting cells (e.g., dendritic cells or macrophages) are anchored to the antigen-presenting cells. These immune responses are specific to hantaviruses. This results in a reduction in viral load and clinical disease following challenge.

Claims

1. A viral or bacterial vector, (i) a first nucleic acid sequence selected from SEQ ID NO: 17 and SEQ ID NO: 24, and (ii) comprising a second nucleic acid sequence selected from SEQ ID NO: 21 and SEQ ID NO: 28; A viral or bacterial vector, wherein the first and second nucleic acid sequences are capable of inducing an immune response in a subject.

2. The vector of claim 1 , wherein the immune response is a protective immune response.

3. The vector according to claim 1 or claim 2, wherein the vector is a viral vector.

4. The vector of claim 3 , wherein the vector is a non-replicating poxvirus vector.

5. 5. The vector of claim 4, wherein the non-replicating poxvirus vector is selected from a modified vaccinia virus Ankara (MVA) vector, a NYVAC vaccinia virus vector, a canarypox (ALVAC) vector, and a fowlpox (FPV) vector.

6. The vector of claim 4 or claim 5, wherein the non-replicating poxvirus vector is an MVA vector.

7. 6. The vector of claim 4 or claim 5, wherein the non-replicating poxvirus vector is a fowlpox vector.

8. The vector of claim 3 , wherein the vector is an adenovirus vector.

9. The vector of claim 8, wherein the adenoviral vector is a non-replicating adenoviral vector.

10. The vector of claim 8 or 9, wherein the adenovirus vector is selected from a human adenovirus vector, a simian adenovirus vector, a group B adenovirus vector, a group C adenovirus vector, a group E adenovirus vector, an adenovirus 6 vector, a PanAd3 vector, an adenovirus C3 vector, a ChAdY25 vector, an AdC68 vector, and an Ad5 vector.

11. The vector of claim 3 , wherein the vector is a measles virus vector.

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

13. A nucleic acid encoding the viral vector of any one of claims 1 to 12.

14. 1. A method for preparing a viral vector, comprising: Providing a nucleic acid comprising a nucleic acid sequence encoding the vector of any one of claims 1 to 12; transfecting the nucleic acid into a host cell; culturing the host cells under conditions suitable for propagation of the vector; Obtaining the vector from the host cell; A method comprising:

15. A host cell comprising a vector described in any one of claims 1 to 12.

16. A composition comprising the vector of any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

17. 17. The composition of claim 16, further comprising an adjuvant.

18. A vector according to any one of claims 1 to 12 for use in medicine.

19. A composition according to claim 16 or claim 17 for use in medicine.

20. A vector according to any one of claims 1 to 12 for use in a method of inducing an immune response in a subject.

21. A composition described in claim 16 or claim 17 for use in a method for inducing an immune response in a subject.

22. 21. The vector for use according to claim 20, wherein the immune response comprises a T cell response.

23. The composition for use according to claim 21, wherein the immune response comprises a T cell response.

24. 13. The vector of any one of claims 1 to 12 for use in a method for preventing or treating a hantavirus infection in a subject.

25. A composition described in claim 16 or claim 17 for use in a method for preventing or treating hantavirus infection in a subject.

26. 25. The vector of claim 24 for use in a method for preventing or treating hemorrhagic fever with renal syndrome in a subject.

27. The composition of claim 25 for use in a method for preventing or treating hemorrhagic fever with renal syndrome in a subject.

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