Hantavirus antigen composition

Abstract

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

Description

[0001] The present invention relates to viral and bacterial vectors containing hantavirus antigens and their use in immunogenic and antigenic compositions. The present invention also relates to prophylactic uses of such compositions. The present invention also relates to immunogens for generating therapeutic antibodies and methods for producing such immunogens.

[0002] Hantavirus is an emerging zoonotic virus with a widespread distribution. There are many strains of hantavirus, which are broadly divided into three serogroups. Hantavirus is the causative agent of hantavirus pulmonary syndrome (HPS), a severe respiratory illness in humans that is typically fatal in 36% of cases, with mortality rates of 50% recorded during some outbreaks. It is also the causative agent of hemorrhagic fever with renal syndrome (HFRS), a group of clinically similar illnesses that can be fatal in up to 15% of cases. Hantavirus is usually transmitted to humans through exposure to aerosolized body fluids or feces of infected small mammals, usually rodents. Human-to-human transmission has also been reported.

[0003] According to the Centers for Disease Control and Prevention (CDC), symptoms associated with hantavirus infection include fever, headache, muscle aches, and severe breathing difficulties. Symptoms associated with HPS can also include fatigue, chills, dizziness, nonproductive cough, nausea, vomiting, and other gastrointestinal symptoms, as well as malaise, diarrhea, mild headache, joint pain, back pain, and abdominal pain. Symptoms associated with HFRS include severe headache, back pain and abdominal pain, fever, chills, nausea, blurred vision, flushing, irritation or redness of the eyes, and rash. Late-stage symptoms of HFRS can include low blood pressure, acute shock, vascular leakage, and acute kidney failure, which can lead to severe fluid overload.

[0004] There is currently no licensed vaccine for hantavirus. According to the CDC, there is no specific treatment or cure for hantavirus infection, HPS, or HFRS. Patients with HPS are admitted to the intensive care unit and treated with intubation and oxygen therapy to help the patient during severe respiratory distress. The success of HPS treatment depends on the severity of the respiratory distress and early detection of the infection. Treatment of HFRS may involve managing the patient's fluid and electrolyte levels, oxygen and blood pressure levels, dialysis to correct severe fluid overload, and treating any secondary infections. The antiviral drug ribavirin has been shown to reduce illness and death if used very early in the clinical course of HFRS. However, no benefit of ribavirin has been found for patients with HPS.

[0005] Therefore, there is a great need for a protective vaccine against hantavirus infection. There is also an urgent need for other therapeutic agents for the prevention, treatment and suppression of hantavirus infection.

[0006] The present invention solves one or more of the above problems by providing viral vectors and bacterial vectors encoding Hantavirus nucleoprotein (NP) or antigenic fragments thereof, as well as corresponding compositions and uses of the vectors and compositions in preventing and treating Hantavirus infection.

[0007] The vectors and compositions of the present invention are capable of stimulating (ie, inducing) an immune response against Hantavirus in an individual (ie, a subject) and provide improved immunogenicity and efficacy.

[0008] In one aspect, the present invention provides a viral or bacterial vector comprising a nucleic acid sequence encoding hantavirus NP or an antigenic fragment thereof; wherein the vector is capable of inducing an immune response in an individual. The present inventors have discovered that by using a viral or bacterial vector to deliver a nucleic acid sequence encoding hantavirus NP (or an antigenic fragment thereof) to a subject, a highly effective immune response against hantavirus can be generated in the individual.

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

[0010] Hantavirus is a genus of enveloped, single-stranded, three-segmented, negative-sense RNA viruses belonging to the family Bunyaviridae. More than 20 strains of hantavirus that are pathogenic to humans have been described, each of which is adapted to a single rodent species. Hantavirus strains are broadly classified as either Old World or New World. Old World strains include Seoul virus ("SEOV," distributed worldwide), Puumala virus (primarily distributed in Europe), Hantaan virus ("HNT," distributed primarily in Asia), and Dobrava virus (primarily distributed in Europe), and are commonly associated with causing HFRS. New World strains include Sin Nombre virus (primarily distributed in North America) and Andes virus (primarily distributed in Latin America), and are commonly associated with HPS.

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

[0012] Hantavirus glycoproteins Gn and Gc play an important role in target cell infection through interactions with specific entry receptors, such as integrins. Hantavirus NP forms a ribonucleoprotein complex with the viral polymerase and plays multiple roles in viral proliferation. NP has also been reported to enhance host cell translation of viral RNA, downregulate apoptosis, inhibit interferon signaling responses, and block TNFα-induced NF-κB activation.

[0013] Seoul virus can be used as a reference hantavirus strain. GenBank accession number KM948598.1 provides a reference nucleic acid sequence of Hantavirus NP (see SEQ ID NO: 1) and a reference polypeptide sequence of Hantavirus NP (SEQ ID NO: 4).

[0014]

[0015] The coding sequence of SEQ ID NO: 1 corresponds to nucleic acid residues 43-1332 thereof and is represented by SEQ ID NO: 2:

[0016]

[0017] The inventors have generated a nucleic acid sequence encoding Hantavirus NP that is optimized for expression in Homo sapiens (see SEQ ID NO: 3):

[0018]

[0019] Translation of the nucleic acid sequence of SEQ ID NO:2 or SEQ ID NO:3 produces the Hantavirus NP polypeptide sequence represented by (SEQ ID NO:4):

[0020] MATMEEIQREISAHEGQLVIARQKVKDAEKQYEKDPDDLNKRALHDRESVAASIQSKIDELKRQLADRIAAGKNIGQDRDPTGVEPGDHLKERSALSYGNTLDLNSLD IDEPTGQTADWLTIIVYLTSFVVPIILKALYMLTTRGRQTSKDNKGMRIRFKDDSSYEDVNGIRKPKHLYVSMPNAQSSMKAEEITPGRFRTAVCGLYPAQIKARNMV SPVMSVVGFLALAKDWTSRIEEWLGAPCKFMAESPIAGSLSGNPVNRDYIRQRQGALAGMEPKEFQALRQHSKDAGCTLVEHIESPSSIWVFAGAPDRCPPTCLFVGG MAELGAFFSILQDMRNTIMASKTVGTADEKLRKKSSFYQSYLRRTQSMGIQLDQRIIVMFMVAWGKEAVDNFHLGDDMDPELRSLAQILIDQKVKEISNQEPMKL(SEQ ID NO:4).

[0021] Hantavirus can be used as a reference hantavirus strain. GenBank accession number KC570390.1 provides a reference nucleic acid sequence of Hantavirus NP (see SEQ ID NO: 5) and a reference polypeptide sequence of Hantavirus NP (see SEQ ID NO: 7).

[0022]

[0023] The coding sequence of SEQ ID NO:5 corresponds to nucleic acid residues 37-1323 thereof and is represented by SEQ ID NO:6.

[0024]

[0025] Translation of the nucleic acid sequence of SEQ ID NO:6 produces the Hantavirus NP polypeptide sequence represented by (SEQ ID NO:7):

[0026] MATMEELQREINAHEGQLVIARQKVRDAEKQYEKDPDELNKRALTDREGVAVSIQAKIDELKRQLADRIATGKNLGKEQDPTGVEPGDHLKERSMLSYGNVLDLNHLD IDEPTGQTADWLGIVIYLTSFVVPILLKALYMLTTRGRQTTKDNKGTRIRFKDDSSFEDVNGIRKPKHLYVSLPNAQSSMKAEEITPGRYRTAICGLYPAQIKARQMI SPVMSVIGFLALAKDWSDRIEQWLSEPCKLLPDTAAVSLLGGPATNRDYLRQRQVALGNMETKESKAIRQHAEAAGCSMIEDIESPSSIWVFAGAPDRCPPTCLFIAG MAELGAFFSILQDMRNTIMASKTVGTSEEKLRKKSSFYQSYLRRTQSMGIQLDQRIIVLFMVAWGKEAVDNFHLGDDMDPELRTLAQSLIDVKVKEISNQEPLKL(SEQ ID NO:7).

[0027] A reference nucleic acid sequence of Hantavirus NP can be provided by SEQ ID NO:8, which corresponds to nucleic acid residues 319-1323 of SEQ ID NO:5.

[0028]

[0029] The inventors have generated a nucleic acid sequence encoding Hantavirus NP that is optimized for expression in Homo sapiens (see SEQ ID NO: 9):

[0030]

[0031] The nucleic acid sequence comprising SEQ ID NO: 8 or 9 is particularly suitable for use in vectors of the present invention that also encode nucleoproteins from Hantaan virus strains other than Hantaan virus, such as the nucleoprotein from Seoul virus. The inventors determined that the 94 N-terminal amino acids of the wild-type Hantaan virus nucleoprotein exhibit high sequence similarity to the N-terminus of the wild-type nucleoprotein from Seoul virus, and where sequence differences exist within this region, the inventors determined that both sequences contain closely related amino acids. Residue 95 of the wild-type Hantaan virus nucleoprotein sequence was identified as the first residue that differs significantly from the corresponding residue in the wild-type nucleoprotein sequence from Seoul virus. The inventors believe that a nucleic acid encoding the 94 N-terminal amino acids of the wild-type Hantaan virus nucleoprotein is essentially antigenically redundant when present in a vector that also encodes a nucleoprotein from Seoul virus (or at least the 94 N-terminal amino acids of the wild-type nucleoprotein from Seoul virus, or an antigenic fragment thereof). Therefore, the inventors believe that the nucleic acid encoding the 94 N-terminal amino acids of the wild-type Hantaan virus nucleoprotein can be omitted from a vector that also encodes the nucleoprotein from Seoul virus (or at least the 94 N-terminal amino acids of the wild-type nucleoprotein from Seoul virus, or an antigenic fragment thereof) without sacrificing antigenic diversity. Removal of unnecessary nucleic acid sequences is generally advantageous in the design of vector constructs (e.g., MVA constructs) because it can enhance vector stability.

[0032] For the reasons described above, the inventors believe that similar advantages can be achieved when the nucleic acid encoding the 94 N-terminal amino acids of the Seoul virus nucleoprotein is omitted, particularly when omitted from a vector encoding the Hantaan virus nucleoprotein (or at least the 94 N-terminal amino acids of the wild-type nucleoprotein from the Hantaan virus, or an antigenic fragment thereof).

[0033] Translation of the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:9 produces the Hantavirus NP polypeptide sequence represented by (SEQ ID NO:10):

[0034] MLSYGNVLDLNHLDIDEPTGQTADWLGIVIYLTSFVVPILLKALYMLTTRGRQTTKDNKGTRIRFKDDSSFEDVNGIRKPKHLYVSLPNAQSSMKAEEITPGRYRTAICGLYPAQIKARQMISPVMSVIGFLALAKDWSDRIEQWLSEPCKLLPDTAAVSLLGGPATNR DYLRQRQVALGNMETKESKAIRQHAEAAGCSMIEDIESPSSIWVFAGAPDRCPPTCLFIAGMAELGAFFSILQDMRNTIMASKTVGTSEEKLRKKSSFYQSYLRRTQSMGIQLDQRIIVLFMVAWGKEAVDNFHLGDDMDPELRTLAQSLIDVKVKEISNQEPLKL(SEQ ID NO:10).

[0035] As used herein, the term "antigenic fragment" refers to a peptide or protein fragment of a hantavirus NP that retains the ability to induce an immune response in an individual compared to a reference hantavirus NP. Thus, the antigenic fragment may include at least one epitope of a reference protein. For example, the antigenic fragment of the present invention may comprise (or consist of) a peptide sequence having at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300 amino acids, wherein the peptide sequence has at least 70% sequence homology to the corresponding peptide sequence of (contiguous) amino acids of the reference protein. An antigenic fragment may comprise (or consist of) at least 10 consecutive amino acid residues from a reference protein sequence (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, or 300 consecutive amino acid residues of the reference protein).

[0036] Antigenic fragments of a reference protein may have a common antigenic cross-reactivity and / or substantially the same in vivo biological activity as the reference protein. For example, an antibody that is capable of binding to an antigenic fragment of a reference protein may also be capable of binding to the reference protein itself. As a further example, a reference protein and its antigenic fragments may have a common ability to induce a "recall response" in T lymphocytes (e.g., CD4+, CD8+, effector T cells, or memory T cells, such as TEMs or TCMs) that have previously been exposed to antigenic components of a hantavirus infection.

[0037] In one aspect, the present invention provides a viral vector or a bacterial vector comprising a nucleic acid sequence encoding a Hantavirus nucleoprotein or an antigenic fragment thereof; wherein the vector is capable of inducing an immune response in a subject.

[0038] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to a nucleic acid sequence selected from SEQ ID NO: 1, 2 and 3.

[0039] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity with the nucleic acid sequence of SEQ ID NO:3.

[0040] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to a nucleic acid sequence selected from SEQ ID NO: 5, 6, 8 and 9.

[0041] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO:9.

[0042] "Peptide library 4" induced a very strong antigen-specific T cell response (see Examples). The amino acid sequence represented by peptide library 4 corresponds to SEQ ID NO:11.

[0043] LYPAQIKARNMVSPVMSVVGFLALAKDWTSRIEEWLGAPCKFMAESPIAGSLSGNPVNRDYIRQRQGALAGMEPKEFQA(SEQ ID NO:11)

[0044] The amino acid sequence of SEQ ID NO:11 is encoded by nucleic acid residues 655 - 891 of SEQ ID NO:1 (see SEQ ID NO:15); residues 613 - 849 of SEQ ID NO:2 (see SEQ ID NO:16); and residues 613 - 849 of SEQ ID NO:3 (see SEQ ID NO:17).

[0045] CTATACCCTGCACAGATAAAGGCAAGGAACATGGTAAGCCCTGTCATGAGTGTAGTTGGGTTTTTGGCACTGGCAAAAGACTGGACATCTAGAATTGAAGAATGGCTTGGTGCACCCTGCAAGTTCATGGCAGAGTCTCCCATTGCCGGGAGCTTATCTGGGAATCCTGTGAATCGTGATTATATCAGACAGAGACAAGGTGCACTTGCAGGGATGGAGCCAAAAGAATTTCAAGCT(SEQ ID NO:15)

[0046] CTATACCCTGCACAGATAAAGGCAAGGAACATGGTAAGCCCTGTCATGAGTGTAGTTGGGTTTTTGGCACTGGCAAAAGACTGGACATCTAGAATTGAAGAATGGCTTGGTGCACCCTGCAAGTTCATGGCAGAGTCTCCCATTGCCGGGAGCTTATCTGGGAATCCTGTGAATCGTGATTATATCAGACAGAGACAAGGTGCACTTGCAGGGATGGAGCCAAAAGAATTTCAAGCT(SEQ ID NO:16)

[0047] CTGTACCCCGCTCAGATCAAGGCCAGAAACATGGTGTCCCCAGTGATGAGCGTCGTGGGATTTCTGGCCCTGGCTAAGGACTGGACCAGCAGGATTGAGGAATGGCTGGGAGCCCCTTGCAAGTTTATGGCCGAGTCTCCTATCGCCGGCAGCCTGTCTGGCAACCCCGTGAATAGAGACTACATCAGACAGAGGCAGGGCGCTCTGGCCGGAATGGAACCCAAAGAATTTCAGGCC(SEQ ID NO:17)

[0048] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 15, 16 or 17.

[0049] In one embodiment, the nucleic acid sequence encoding a Hantavirus nucleoprotein or an antigenic fragment thereof comprises (or consists of) at least 10 consecutive nucleic acid residues from a sequence of SEQ ID NO: 15, 16 or 17 (e.g., at least 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or 65 nucleic acids of SEQ ID NO: 15, 16 or 17).

[0050] "Peptide library 9" also induced a very strong antigen-specific T cell response. The amino acid sequence represented by peptide library 9 corresponds to SEQ ID NO:12.

[0051] IKARQMISPVMSVIGFLALAKDWSDRIEQWLSEPCKLLPDTAAVSLLGGPATNRDYLRQRQVALGNMETKESKAIRQHA (SEQ ID NO: 12).

[0052] The amino acid sequence of SEQ ID NO:12 is encoded by nucleic acid residues 664-900 of SEQ ID NO:5 (see SEQ ID NO:18); residues 628-864 of SEQ ID NO:6 (see SEQ ID NO:19); residues 346-582 of SEQ ID NO:8 (see SEQ ID NO:20); and residues 346-582 of SEQ ID NO:9 (see SEQ ID NO:21).

[0053] ATTAAGGCAAGACAGATGATTAGTCCAGTCATGAGTGTAATCGGATTCTTGGCTTTGGCAAAAGATTGGAGTGACCGCATTGAGCAGTGGTTAAGTGAACCGTGTAAGCTTCTTCCAGATACAGCAGCAGTTAGCCTTCTTGGTGGTCCTGCAACCAACAGGGACTATTTACGGCAGCGACAAGTAGCATTGGGCAACATGGAAACAAAAGAGTCTAAGGCTATACGCCAACATGCA (SEQ ID NO:18)

[0054] ATTAAGGCAAGACAGATGATTAGTCCAGTCATGAGTGTAATCGGATTCTTGGCTTTGGCAAAAGATTGGAGTGACCGCATTGAGCAGTGGTTAAGTGAACCGTGTAAGCTTCTTCCAGATACAGCAGCAGTTAGCCTTCTTGGTGGTCCTGCAACCAACAGGGACTATTTACGGCAGCGACAAGTAGCATTGGGCAACATGGAAACAAAAGAGTCTAAGGCTATACGCCAACATGCA (SEQ ID NO:19)

[0055] ATTAAGGCAAGACAGATGATTAGTCCAGTCATGAGTGTAATCGGATTCTTGGCTTTGGCAAAAGATTGGAGTGACCGCATTGAGCAGTGGTTAAGTGAACCGTGTAAGCTTCTTCCAGATACAGCAGCAGTTAGCCTTCTTGGTGGTCCTGCAACCAACAGGGACTATTTACGGCAGCGACAAGTAGCATTGGGCAACATGGAAACAAAAGAGTCTAAGGCTATACGCCAACATGCA (SEQ ID NO:20)

[0056] ATCAAAGCCCGCCAGATGATCAGCCCCGTGATGTCCGTTATCGGATTCCTGGCTCTGGCCAAAGATTGGAGCGACAGGATCGAGCAGTGGCTGAGCGAGCCTTGCAAGCTGCTTCCTGATACAGCCGCTGTGTCACTGCTTGGCGGCCCTGCCACAAACAGAGATTACCTGAGACAGAGACAGGTGGCACTGGGCAACATGGAAACAAAAGAGAGCAAGGCCATCCGGCAGCATGCC(SEQ ID NO:21)

[0057] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 18, 19, 20 or 21.

[0058] In one embodiment, the nucleic acid sequence encoding a Hantavirus nucleoprotein or an antigenic fragment thereof comprises (or consists of) at least 10 consecutive nucleic acid residues from a sequence of SEQ ID NO: 18, 19, 20 or 21 (e.g., at least 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or 65 nucleic acids of SEQ ID NO: 18, 19, 20 or 21).

[0059] As demonstrated herein, peptide pools 4 and 9 induce very strong antigen-specific T cell responses. By aligning the polypeptide sequence represented by "peptide pool 4" with the polypeptide sequence represented by "peptide pool 9," the inventors identified regions of high sequence identity, represented by SEQ ID NO: 13 and SEQ ID NO: 14, respectively. Without wishing to be bound by theory, the inventors believe that the amino acid sequences of SEQ ID NO: 13 and 14 play an important role in eliciting the particularly strong antigen-specific T cell responses observed with peptide pools 4 and 9, respectively.

[0060] SPVMSVVGFLALAKD (SEQ ID NO: 13)

[0061] PVMSVIGFLALAKDW (SEQ ID NO: 14)

[0062] SEQ ID NO: 13 is encoded by, inter alia, nucleic acid residues 691-735 of SEQ ID NO: 1 (see SEQ ID NO: 22); residues 649-693 of SEQ ID NO: 2 (see SEQ ID NO: 23); and residues 649-693 of SEQ ID NO: 3 (see SEQ ID NO: 24).

[0063] AGCCCTGTCATGAGTGTAGTTGGGTTTTTGGCACTGGCAAAAGAC(SEQ ID NO:22)

[0064] AGCCCTGT CAT GAGT GT AGTT GGGTTTTTGGC ACTGGC A AAAGAC (SEQ ID NO:23)

[0065] TCCCCAGTGATGAGCGTCGTGGGATTTCTGGCCCTGGCTAAGGAC (SEQ ID NO: 24)

[0066] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 22, 23 or 24.

[0067] SEQ ID NO: 14 is encoded by, among other things, nucleic acid residues 688-732 of SEQ ID NO: 5 (see SEQ ID NO: 25); residues 652-696 of SEQ ID NO: 6 (see SEQ ID NO: 26); residues 370-414 of SEQ ID NO: 8 (see SEQ ID NO: 27); and residues 370-414 of SEQ ID NO: 9 (see SEQ ID NO: 28).

[0068] CCAGTCATGAGTGTAATCGGATTCTTGGCTTTGGCAAAAGATTGG(SEQ ID NO:25)

[0069] CCAGTCATGAGTGTAATCGGATTCTTGGCTTTGGCAAAAGATTGG(SEQ ID NO:26)

[0070] CCAGTCATGAGTGTAATCGGATTCTTGGCTTTGGCAAAAGATTGG(SEQ ID NO:27)

[0071] CCCGTGATGTCCGTTATCGGATTCCTGGCTCTGGCCAAAGATTGG(SEQ ID NO:28)

[0072] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 25, 26, 27 or 28.

[0073] In one embodiment, the nucleic acid sequence encoding a Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0074] (A) the first nucleic acid sequence is provided by a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99, or 100%) sequence identity to SEQ ID NO: 1, 2, or 3; and

[0075] (B) The second nucleic acid sequence is provided by a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to SEQ ID NO: 5, 6, 8 or 9.

[0076] In one embodiment, the nucleic acid sequence encoding a Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0077] (A) the first nucleic acid sequence is provided by a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99, or 100%) sequence identity to SEQ ID NO: 15, 16, 17, 22, 23, or 24; and

[0078] (B) The second nucleic acid sequence is provided by a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to SEQ ID NO: 18, 19, 20, 21, 25, 26, 27 or 28.

[0079] In one embodiment, the nucleic acid sequence encoding a Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0080] (A) the first nucleic acid sequence has at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NO: 15, 16, 17, 22, 23, or 24; and

[0081] (B) The second nucleic acid sequence has at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NO: 18, 19, 20, 21, 25, 26, 27 or 28.

[0082] In one embodiment, the nucleic acid sequence encoding a Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0083] (A) the first nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 24; and

[0084] (B) The second nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO:28.

[0085] In one embodiment, the nucleic acid sequence encoding a Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0086] (A) the first nucleic acid sequence has at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NO: 22 or 23; and

[0087] (B) The second nucleic acid sequence is provided by a nucleic acid sequence having at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 25, 26 or 27.

[0088] In one embodiment, the nucleic acid sequence encoding a Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0089] (A) the first nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 17; and

[0090] (B) The second nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO:21.

[0091] In one embodiment, the nucleic acid sequence encoding a Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0092] (A) the first nucleic acid sequence has at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NO: 15 or 16; and

[0093] (B) The second nucleic acid sequence has at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NO: 18, 19 or 20.

[0094] In one embodiment, the nucleic acid sequence encoding a Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0095] (A) the first nucleic acid sequence has at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NO: 1, 2 or 3; and

[0096] (B) The second nucleic acid sequence has at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NO: 5, 6, 8 or 9.

[0097] In one embodiment, the nucleic acid sequence encoding a Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0098] (A) the first nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 3; and

[0099] (B) The second nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO:9.

[0100] In one embodiment, the nucleic acid sequence encoding a Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0101] (A) the first nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 2; and

[0102] (B) The second nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO:8.

[0103] In one embodiment, the nucleic acid sequence encoding a Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0104] (A) the first nucleic acid has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 2; and

[0105] (B) The second nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO:6.

[0106] In one embodiment, the nucleic acid sequence encoding a Hantavirus nucleoprotein or an antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein:

[0107] (A) the first nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO: 1; and

[0108] (B) The second nucleic acid sequence has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO:5.

[0109] 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.

[0110] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to SEQ ID NO:29.

[0111]

[0112] In one embodiment, the nucleic acid sequence encoding the Hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to SEQ ID NO:30.

[0113]

[0114] The inventors have discovered that the hantavirus NP encoded by the nucleic acid sequences of the present invention can be used to generate an effective immune response against hantavirus in an individual. In particular, the inventors have discovered that when the hantavirus NP is delivered to a subject using a bacterial vector or a viral vector (such as a non-replicating poxvirus vector or an adenovirus vector), an effective immune response against hantavirus is obtained.

[0115] Vectors are tools that can be used as carriers for delivering genetic material into target cells. For example, viral vectors act as antigen delivery vehicles and also have the ability to activate the innate immune system by binding to cell surface molecules that recognize viral elements. Recombinant viral vectors carrying nucleic acids encoding given antigens can be produced. Viral vectors can then be used to deliver nucleic acids to target cells, where the encoded antigens are produced, which are then presented to the immune system by the target cell's own molecular machinery. As "non-self," the antigens produced produce an acquired immune response in the target subject. Advantageously, it has been demonstrated herein that the vectors of the present invention provide a protective immune response.

[0116] Viral vectors suitable for use in the present invention include poxvirus vectors (such as non-replicating poxvirus vectors), adenovirus vectors, and influenza virus vectors.

[0117] In certain embodiments, a "viral vector" can be a virus-like particle (VLP). VLP is a lipid-coated particle containing viral proteins. Certain viral proteins have inherent self-assembly capabilities and, in this process, bud from the cell membrane as independent envelope particles. VLP is easy to purify and can, for example, be used to present viral antigens. Therefore, VLP is suitable for use in immunogenic compositions, such as those described below. In certain embodiments, a viral vector is not a virus-like particle.

[0118] Bacterial vectors can also be used as antigen delivery media. Recombinant bacterial vectors carrying nucleic acids encoding given antigens can be produced. Recombinant bacterial vectors can express antigens on their surfaces. After being administered to a subject, the bacterial vector colonizes antigen-presenting cells (e.g., dendritic cells or macrophages). Induce antigen-specific immune responses. The immune response can be a cellular (T cell) immune response, or can include humoral (e.g., B cells) and cellular (T cell) immune responses. Examples of bacterium suitable for use as recombinant bacterial vectors include Escherichia coli, Shigella, Salmonella (e.g., Salmonella typhimurium) and Listeria bacteria. In one embodiment, the carrier of the present invention is a bacterial vector, wherein the bacterium is a Gram-negative bacterium. In one embodiment, the carrier of the present invention is a bacterial vector selected from Escherichia coli vectors, Shigella vectors, Salmonella vectors and Listeria vectors.

[0119] Without wishing to be bound by any particular theory, the inventors believe that antigen delivery using the vectors of the present invention stimulates T cell responses and other responses in the subject. Thus, the inventors believe that one way the present invention provides protection against Hantavirus infection is by stimulating T cell responses and the cell-mediated immune system. Additionally, humoral (antibody)-based protection may also be achieved.

[0120] The viral vector of the present invention may be a non-replicating viral vector.

[0121] As used herein, a non-replicating viral vector is a viral vector that lacks effective replication ability after infecting a target cell. Therefore, the ability of a non-replicating viral vector to produce its own copy after infecting a target cell (such as a human target cell in an individual inoculated with a non-replicating viral vector) is highly reduced or absent. This viral vector may also be referred to as attenuated or replication-deficient. The reason may be the loss / deletion of genes necessary for replication in the target cell. Therefore, a non-replicating viral vector cannot effectively produce its own copy after infecting a target cell. Therefore, compared with a viral vector with replication ability, a non-replicating viral vector can advantageously have an improved safety profile. A non-replicating viral vector can retain the ability to replicate in cells that are not target cells, thereby allowing viral vectors to be produced. For example, a non-replicating viral vector (such as a non-replicating poxvirus vector) may lack the ability to productively replicate in a target cell (such as a mammalian cell (such as a human cell)), but retains the ability to replicate (and therefore allow vector production) in avian cells (such as chicken embryo fibroblasts or CEF cells).

[0122] The viral vector of the present invention may be a non-replicating poxvirus vector. Therefore, in one embodiment, the viral vector encoding Hantavirus NP or an antigenic fragment thereof is a non-replicating poxvirus vector.

[0123] In one embodiment, the non-replicating poxvirus vector is selected from the group consisting of a modified vaccinia Ankara (MVA) vector, a NYVAC vaccinia vector, a canarypox (ALVAC) vector, and a fowlpox (FPV) vector. MVA and NYVAC are both attenuated derivatives of vaccinia virus. Compared to vaccinia virus, MVA lacks approximately 26 of the approximately 200 open reading frames.

[0124] In one embodiment, the non-replicating poxvirus vector is an FPV vector.

[0125] In a preferred embodiment, the non-replicating poxvirus vector is an MVA vector.

[0126] The viral vector of the present invention may be an adenoviral vector. Therefore, in one embodiment, the viral vector encoding Hantavirus NP or an antigenic fragment thereof is an adenoviral vector.

[0127] In one embodiment, the adenovirus vector is a non-replicating adenovirus vector (wherein non-replicating is defined as above). Adenoviruses can become non-replicating by deleting E1 or both E1 and E3 gene regions. Alternatively, by changing E1 or E1 and E3 gene regions so that the gene regions are non-functional, adenoviruses can be made non-replicating. For example, a non-replicating adenovirus may lack a functional E1 region or may lack functional E1 and E3 gene regions. In this way, adenoviruses cannot replicate in most mammalian cell lines and do not replicate in immune mammals. Most preferably, both E1 and E3 gene region deletions are present in adenoviruses, thereby allowing the insertion of larger transgenes. This is particularly important for allowing the expression of larger antigens, or when expressing multiple antigens in a single vector, or when using large promoter sequences (such as CMV promoters). The deletion of E3 and E1 regions is particularly advantageous for recombinant Ad5 vectors. Optionally, the E4 region can also be engineered.

[0128] In one embodiment, the adenoviral vector is selected from the group consisting of: a human adenoviral vector, a simian adenoviral vector, a group B adenoviral vector, a group C adenoviral vector, a group E adenoviral vector, an adenovirus 6 vector, a PanAd3 vector, an adenovirus C3 vector, a ChAdY25 vector, an AdC68 vector, and an Ad5 vector.

[0129] The viral vector of the present invention may be a measles virus vector. Therefore, in one embodiment, the viral vector encoding Hantavirus NP or an antigenic fragment thereof is a measles virus vector.

[0130] In one embodiment, the expression cassette comprising the nucleic acid sequence encoding Hantavirus NP (or an antigenic fragment thereof) is less than 9 kb (e.g., less than 9.0, 8.5, 8.0, 7.5, 7.0, 6, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3 ,4.2,4.1,4.0,3.9,3.8,3.7,3.6,3.5,3.4,3.3,3.2,3.1,3.0,2.9,2.8,2.7,2.6 ,2.5,2.4,2.3,2.2,2.1,2.0,1.9,1.8,1.7,1.6,1.5,1.4,1.3,1.2,1.1,1.0kb).

[0131] In one embodiment, the expression cassette comprising the nucleic acid sequence encoding Hantavirus NP (or an antigenic fragment thereof) is less than 8 kb (e.g., less than 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0 kb).

[0132] In one embodiment, the expression cassette comprising the nucleic acid sequence encoding the Hantavirus NP (or an antigenic fragment thereof) is less than 7 kb (e.g., less than 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4. 4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0kb).

[0133] In one embodiment, the expression cassette comprising the nucleic acid sequence encoding Hantavirus NP (or an antigenic fragment thereof) is less than 6 kb (e.g., less than 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0 kb).

[0134] In one embodiment, the expression cassette comprising the nucleic acid sequence encoding Hantavirus NP (or an antigenic fragment thereof) is less than 5 kb (e.g., less than 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0 kb).

[0135] In one embodiment, the expression cassette comprising the nucleic acid sequence encoding hantavirus NP (or an antigenic fragment thereof) is less than 4.5 kb (e.g., less than 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0 kb).

[0136] In one embodiment, wherein the vector is a viral vector, the virus (ie, the viral vector) is not a pseudotyped virus. Thus, in one embodiment, the envelope of the viral vector does not comprise exogenous glycoproteins (ie, glycoproteins that are not native to the viral vector).

[0137] In one embodiment, wherein the vector is a non-replicating poxvirus vector (such as an MVA vector), the nucleic acid sequence encoding hantavirus NP or an antigenic fragment thereof comprises a nucleic acid sequence encoding a hantavirus glycoprotein.

[0138] In one embodiment, wherein the vector is a non-replicating poxvirus vector (such as an MVA vector), the nucleic acid sequence encoding hantavirus NP or an antigenic fragment thereof comprises a nucleic acid sequence encoding a hantavirus glycoprotein (GP) epitope.

[0139] In one embodiment, wherein the vector is a non-replicating poxvirus vector (such as an MVA vector), the nucleic acid sequence encoding hantavirus NP or an antigenic fragment thereof does not include a nucleic acid sequence encoding hantavirus glycoprotein (GP).

[0140] In one embodiment, wherein the vector is a non-replicating poxvirus vector (such as an MVA vector), the nucleic acid sequence encoding hantavirus NP or an antigenic fragment thereof does not include a nucleic acid sequence encoding a hantavirus glycoprotein (GP) epitope.

[0141] In one embodiment, the hantavirus nucleoprotein or antigenic fragment thereof is the only hantavirus nucleic acid sequence in the vector.

[0142] In one embodiment, wherein the vector is a non-replicating poxvirus vector, the vector is stable, expresses the hantavirus NP product, and induces a protective immune response in the subject.

[0143] In one embodiment, wherein the vector is an adenoviral vector, the vector is stable, expresses the hantavirus NP product, and induces a protective immune response in the subject.

[0144] The nucleic acid sequence described above may comprise a nucleic acid sequence encoding a hantavirus NP, wherein the NP comprises a fusion protein. The fusion protein may comprise a hantavirus NP polypeptide fused to one or more other polypeptides, such as an epitope tag, another antigen, or a protein that increases immunogenicity, such as flagellin.

[0145] In one embodiment, the nucleic acid sequence encoding the Hantavirus NP (as described above) also encodes a tissue plasminogen activator (tPA) signal sequence and / or a V5 fusion protein sequence. In certain embodiments, the presence of the tPA signal sequence can provide increased immunogenicity; the presence of the V5 fusion protein sequence can provide identification of the expressed protein by immunolabeling.

[0146] In one embodiment, the vector (as described above) further comprises a nucleic acid sequence encoding an adjuvant (eg, cholera toxin, E. coli lethal toxin, or flagellin).

[0147] In one embodiment, the vector does not comprise a nucleic acid sequence encoding an adjuvant.In one embodiment, the vector does not comprise a nucleic acid sequence encoding Hsp70.

[0148] The bacterial vectors of the present invention can be produced by using any technique known in the art for manipulating and producing recombinant bacteria.

[0149] In another aspect, the present invention provides a nucleic acid sequence encoding a viral vector as described above. Thus, the nucleic acid sequence may encode a non-replicating poxvirus vector as described above. Alternatively, the nucleic acid sequence may encode an adenovirus vector as described above.

[0150] Nucleic acid sequences encoding viral vectors (as described above) can be produced by using any of the techniques known in the art for the manipulation and production of recombinant nucleic acids.

[0151] In one aspect, the present invention provides a method for preparing a viral vector (as described above), comprising providing a nucleic acid, wherein the nucleic acid comprises a nucleic acid sequence encoding a vector (as described above); transfecting a host cell with the nucleic acid; culturing the host cell under conditions suitable for vector proliferation; and obtaining the vector from the host cell.

[0152] As used herein, "transfection" may refer to any non-viral method for introducing a nucleic acid into a cell. The nucleic acid may be any nucleic acid suitable for transfecting a host cell. Thus, in one embodiment, the nucleic acid is a plasmid. The host cell may be any cell in which a vector (e.g., a non-replicating poxvirus vector or adenovirus vector as described above) can grow. As used herein, "culturing host cells under conditions suitable for vector propagation" means using any cell culture conditions and techniques known in the art that are suitable for the selected host cell and capable of producing the vector in the host cell. As used herein, "obtaining a vector" means using any technique known in the art that is suitable for isolating the vector from the host cell. Thus, the host cells may be lysed to release the vector. The vector may then be isolated and purified using any suitable method or methods known in the art.

[0153] In one aspect, the invention provides a host cell comprising a nucleic acid sequence encoding a viral vector as described above. The host cell can be any cell in which a viral vector (e.g., a non-replicating poxvirus vector or adenovirus vector as described above) can grow or reproduce. In one embodiment, the host cell is selected from: 293 cells (also referred to as HEK or human embryonic kidney cells), CHO cells (Chinese hamster ovary), CCL81.1 cells, Vero cells, HELA cells, Per.C6 cells, BHK cells (baby hamster kidney), primary CEF cells (chicken embryo fibroblasts), duck embryo fibroblasts, DF-1 cells, or rat IEC-6 cells.

[0154] The present invention also provides a composition comprising the carrier described above.

[0155] In one aspect, the invention provides a composition comprising a carrier (as described above) and a pharmaceutically acceptable carrier.

[0156] Materials suitable for use as pharmaceutically acceptable carriers are known in the art. Non-limiting examples of pharmaceutically acceptable carriers include water, saline, and phosphate buffered saline (PBS). However, in some embodiments, the composition is in lyophilized form, in which case it may include a stabilizer such as bovine serum albumin (BSA). In some embodiments, it may be necessary to formulate the composition with a preservative such as thimerosal or sodium azide to facilitate long-term storage. Examples of buffers include, but are not limited to, sodium succinate (pH 6.5) and phosphate buffered saline (PBS, pH 7.4).

[0157] In addition to a pharmaceutically acceptable carrier, the composition of the present invention may be combined with one or more of salts, excipients, diluents, adjuvants, immunomodulators, and / or antimicrobial compounds.

[0158] Advantageously, the vectors of the present invention have been shown to provide a protective immune response even in the absence of an adjuvant. Thus, in one embodiment, the composition of the present invention does not comprise an adjuvant.

[0159] The composition can be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids (such as, for example, hydrochloric acid or phosphoric acid) or with organic acids (such as acetic acid, oxalic acid, tartaric acid, maleic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, as well as such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0160] In one embodiment, the composition (as described above) further comprises at least one hantavirus NP antigen (i.e., an antigen present in the composition in polypeptide form). Thus, the composition may comprise both a carrier and a polypeptide. In one embodiment, the polypeptide antigen is hantavirus NP. In one embodiment, the polypeptide antigen is hantavirus GP. In one embodiment, the presence of the polypeptide antigen means that upon administration of the composition to a subject, improved T cell and antibody responses may be achieved. In one embodiment, the T cell and antibody responses achieved exceed those achieved when the carrier or polypeptide antigen is used alone.

[0161] In one embodiment, the polypeptide antigen is not associated with the carrier. In one embodiment, the polypeptide antigen is a separate component of the carrier. In one embodiment, the polypeptide antigen is provided separately from the carrier.

[0162] In one embodiment, the polypeptide antigen is a variant of an antigen encoded by a vector. In one embodiment, the polypeptide antigen is a fragment of an antigen encoded by a vector. In one embodiment, the polypeptide antigen comprises at least a portion of a polypeptide sequence encoded by the nucleic acid sequence of the vector. Thus, the polypeptide antigen may correspond to at least a portion of an antigen encoded by a vector.

[0163] In one embodiment, the polypeptide antigen is hantavirus NP, which comprises (or consists of) an amino acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to an amino acid sequence selected from SEQ ID NO: 4, 7 and 10.

[0164] In one embodiment, the polypeptide antigen is hantavirus NP, which comprises (or consists of) an amino acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity with an amino acid sequence selected from SEQ ID NO: 11 and 12.

[0165] In one embodiment, the polypeptide antigen is a hantavirus NP comprising (or consisting of) an amino acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99, or 100%) sequence identity to an amino acid sequence selected from SEQ ID NOs: 13 and 14.

[0166] In one embodiment, the polypeptide antigen is hantavirus NP, which comprises (or consists of) an amino acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity with an amino acid sequence selected from SEQ ID NO: 31 and 32.

[0167] MATMEEIQREISAHEGQLVIARQKVKDAEKQYEKDPDDLNKRALHDRESVAASIQSKIDELKRQLADRIAAGKNIGQDRDPTGVEPGDHLKERSALSYGNTLDLNSLDIDEPTGQTADWLTIIVYLTSFVVPIILKALYMLTTRGRQTSKDNKGMRIRFKDDSSYEDVNGIRKPKHLYVSMPNAQSSMKAEEITPGRFRTAVCGLYPAQIKARNMVSPVMSVVGFLALAKDWTSRIEEWLGAPCKFMAESPIAGSLSGNPVNRDYIRQRQGALAGMEPKEFQALRQHSKDAGCTLVEHIESPSSIWVFAGAPDRCPPTCLFVGGMAELGAFFSILQDMRNTIMASKTVGTADEKLRKKSSFYQSYLRRTQSMGIQLDQRIIVMFMVAWGKEAVDNFHLGDDMDPELRSLAQILIDQKVKEISNQEPMKLMLSYGNVLDLNHLDIDEPTGQTADWLGIVIYLTSFVVPILLKALYMLTTRGRQTTKDNKGTRIRFKDDSSFEDVNGIRKPKHLYVSLPNAQSSMKAEEITPGRYRTAICGLYPAQIKARQMISPVMSVIGFLALAKDWSDRIEQWLSEPCKLLPDTAAVSLLGGPATNRDYLRQRQVALGNMETKESKAIRQHAEAAGCSMIEDIESPSSIWVFAGAPDRCPPTCLFIAGMAELGAFFSILQDMRNTIMASKTVGTSEEKLRKKSSFYQSYLRRTQSMGIQLDQRIIVLFMVAWGKEAVDNFHLGDDMDPELRTLAQSLIDVKVKEISNQEPLKL(SEQ ID NO:31)

[0168] (SEQ ID NO:32)

[0169] The polypeptide antigen can be identical (or similar) to the polypeptide antigen encoded by the nucleic acid sequence of the carrier of the composition. Therefore, administering a composition comprising a carrier and a polypeptide antigen can be used to achieve an enhanced immune response to a single antigen, wherein the enhanced immune response comprises combined T cell and antibody responses, as described above.

[0170] In one embodiment, the composition of the invention (as described above) further comprises at least one naked DNA (i.e., a DNA molecule that is separate from, and not part of, the viral vector of the invention) encoding a hantavirus NP or antigenic fragment thereof. In one embodiment, the naked DNA comprises (or consists of) a nucleic acid sequence that 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 to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 5, 6, 8, 9, and 15-30. In one embodiment, the naked DNA encodes hantavirus NP, which comprises (or consists of) an amino acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity with an amino acid sequence selected from SEQ ID NO: 4, 7, 10-14, 31 and 32.

[0171] In one embodiment, the composition of the present invention (as described above) further comprises an adjuvant. Non-limiting examples of adjuvants suitable for use in the compositions of the present invention include aluminum phosphate, aluminum hydroxide, and related compounds; monophosphoryl lipid A and related compounds; outer membrane vesicles from bacteria; oil-in-water emulsions, such as MF59; liposomal adjuvants, such as virosomes, Freund's adjuvant, and related mixtures; polylactic-co-glycolic acid (PLGA) particles; cholera toxin; E. coli lethal toxin; and flagellin.

[0172] The vectors and compositions of the present invention (as described above) may be used as vaccines. Thus, the compositions of the present invention may be vaccine compositions.

[0173] As used herein, a vaccine is a formulation that, when administered to an animal subject, such as a mammal (e.g., a human, bovine, porcine, ovine, caprine, equine, cervical, canine, or feline subject, particularly a human subject), stimulates a protective immune response against an infectious disease. The immune response can be humoral and / or cell-mediated. Thus, a vaccine can stimulate B cells and / or T cells.

[0174] The term "vaccine" is used herein interchangeably with the terms "therapeutic / prophylactic composition," "immunogenic composition," "formulation," "antigenic composition," or "medicament."

[0175] In one aspect, the invention provides a vector (as described above) or a composition (as described above) for use in medicine.

[0176] In one aspect, the present invention provides vectors (as described above) or compositions (as described above) for use in methods of inducing an immune response in a subject. The immune response can be directed against a hantavirus antigen (e.g., hantavirus NP) and / or a hantavirus infection. Thus, the vectors and compositions of the present invention can be used to induce an immune response against hantavirus NP in a subject (e.g., as an immunogenic composition or as a vaccine).

[0177] In one embodiment, the immune response comprises a T cell response.

[0178] In one embodiment, a method of inducing an immune response in a subject comprises administering to the subject an effective amount of a vector (as described above) or a composition (as described above).

[0179] In one aspect, the invention provides a vector (as described above) or a composition (as described above) for use in a method of preventing or treating a hantavirus infection in a subject.

[0180] In one embodiment, the invention provides a vector (as described above) or a composition (as described above) for use in a method of preventing or treating HFRS in a subject.

[0181] The vectors and compositions of the present invention are ideally suited for the prevention or treatment of HFRS, particularly when the hantavirus nucleoprotein or antigenic fragment thereof is derived from Seoul virus. As mentioned above, Seoul virus is commonly associated with causing HFRS.

[0182] The vectors and compositions of the present invention are ideally suited for the prevention or treatment of HFRS, particularly when the Hantavirus nucleoprotein or antigenic fragment thereof is derived from Hantavirus. As mentioned above, Hantavirus is often associated with causing HFRS.

[0183] The vectors and compositions of the present invention are ideally suited for the prevention or treatment of HFRS, particularly when the Hantavirus nucleoprotein or antigenic fragment thereof is a chimeric sequence comprising a chimera of the Seoul virus nucleoprotein (or antigenic fragment thereof) and the Hantavirus nucleoprotein (or antigenic fragment thereof), for example as demonstrated in the Examples.

[0184] As used herein, the term "prevention" includes preventing the onset of a hantavirus infection and / or reducing the severity or intensity of a hantavirus infection. Thus, "prevention" includes vaccination.

[0185] As used herein, the term "treatment" includes therapeutic and preventative / prophylactic measures (including post-exposure prophylaxis), and includes post-infection treatment and amelioration of hantavirus infection.

[0186] 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.

[0187] Each of the above methods can include the step of administering to the subject an effective amount (eg, a therapeutically effective amount) of a vector or composition of the present invention.

[0188] In this respect, as used herein, an effective amount is a dosage or content sufficient to achieve a desired biological result. As used herein, a therapeutically effective amount is a content that is effective for treating, preventing, inhibiting a cure, delaying, reducing the severity of a disorder or a recurrent disorder, improving at least one symptom of a disorder or a recurrent disorder, or prolonging the survival of the subject beyond the expected survival in the absence of such treatment, after single or multiple dose administration to a subject (such as a mammalian subject, particularly a human subject).

[0189] Thus, the amount of active ingredient to be administered depends on the subject to be treated, the ability of the subject's immune system to produce a protective immune response, and the degree of protection desired. The precise amount of active ingredient required to be administered can depend on the judgment of the practitioner and can be specific to each subject.

[0190] Administration to a subject can include administering a vector (as described above) or a composition (as described above) to the subject, wherein the composition is administered multiple times in sequence (e.g., wherein the composition is administered two, three, or four times). Thus, in one embodiment, a vector (as described above) or a composition (as described above) is administered to a subject, and then the same vector or composition (or a substantially similar vector or composition) is administered again at a different time.

[0191] In one embodiment, administering to a subject comprises administering to the subject a vector (as described above) or a composition (as described above), wherein the composition is administered substantially before, simultaneously with, or after another immunogenic composition.

[0192] Prior, simultaneous, and sequential administration regimens are discussed in more detail below.

[0193] In certain embodiments, the above method further comprises administering to the subject a second vector, wherein the second vector comprises a nucleic acid sequence encoding a hantavirus NP. Preferably, the second vector is a vector of the present invention as described above (e.g., a viral vector, e.g., a non-replicating poxvirus vector or an adenovirus vector as described above).

[0194] In one embodiment, the first and second vectors are of the same vector type. In one embodiment, the first and second vectors are of different vector types. In one embodiment, the first vector is an adenoviral vector (as described above) and the second vector is a non-replicating poxviral vector (as described above). In one embodiment, the first vector is a non-replicating poxviral vector (as described above) and the second vector is an adenoviral vector (as described above).

[0195] In one embodiment, the first and second vectors are administered sequentially in any order. Thus, the first ("1") and second ("2") vectors can be administered to the subject in a 1-2 order or a 2-1 order.

[0196] As used herein, "sequential administration" has the meaning of "sequential administration" as defined below. Thus, the first and second vectors are administered sequentially at (substantially) different times.

[0197] In one embodiment, the first and second vectors are administered as part of a prime-boost administration regimen. Thus, the first vector can be administered to a subject as a "prime," and the second vector can subsequently be administered to the same subject as a "boost." Prime-boost regimens are discussed below.

[0198] In one embodiment, each of the above methods further comprises the step of administering a hantavirus polypeptide antigen to the subject. In one embodiment, the hantavirus polypeptide antigen is a hantavirus NP (or an antigenic fragment thereof) as described above. In one embodiment, the hantavirus polypeptide antigen is a hantavirus NP comprising an amino acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99, or 100%) sequence identity to an amino acid sequence selected from SEQ ID NOs: 4, 7, 10-14, 31, and 32.

[0199] In one embodiment, the polypeptide antigen is administered separately from the administration of the vector; preferably, 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 VP or in the order of PV.

[0200] In one embodiment, each of the above methods further comprises the step of administering to the subject naked DNA encoding hantavirus NP or an antigenic fragment thereof. In one embodiment, the naked DNA comprises (or consists of) a nucleic acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99, or 100%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1, 2, 3, 5, 6, 8, 9, and 15-30. In one embodiment, the naked DNA encodes hantavirus NP, and the hantavirus NP comprises (or consists of) an amino acid sequence having at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99, or 100%) sequence identity to an amino acid sequence selected from SEQ ID NOs: 4, 7, 10-14, 31, and 32.

[0201] In one embodiment, naked DNA is administered separately from the administration of the vector; preferably, naked DNA and vector are administered sequentially. In one embodiment, vector ("V") and naked DNA ("D") can be administered in the order of VD or in the order of DV.

[0202] In one embodiment, naked DNA (as described above) is administered to a subject as part of a prime-boost regimen.

[0203] Heterologous prime-boost approaches can improve immune responses by allowing repeated vaccination without increasing anti-vector immunity. Hantavirus NP or antigenic fragments thereof can be delivered sequentially via different vectors (as described above) or naked DNA vectors (as described above). In any heterologous prime-boost vaccination regimen, NP-specific antibody responses are increased, NP-specific T cell responses are increased, and / or clinical disease is reduced compared to the use of a single vector. Suitable combinations of vectors include, but are not limited to:

[0204] DNA prime, MVA boost

[0205] DNA primary vaccination, fowlpox boost

[0206] Fowlpox primary vaccination, MVA booster

[0207] MVA primary immunization, fowlpox booster immunization

[0208] DNA prime, fowlpox boost, MVA boost

[0209] MVA priming, adenovirus boosting

[0210] As used herein, the term polypeptide includes peptides and proteins.

[0211] In certain embodiments, the above method further comprises administering an adjuvant to the subject. The adjuvant can be administered together with one, two, three or all four of the first carrier, the second carrier, the polypeptide antigen and the naked DNA.

[0212] The immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions and prophylactic formulations (e.g., vaccines) of the present invention can be administered in a single dose regimen (i.e., substantially a full dose is administered at one time). Alternatively, the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions and prophylactic formulations (e.g., vaccines) of the present invention can be administered in a multiple dose regimen.

[0213] A multiple dose regimen is one in which the primary course of treatment (e.g., vaccination) may be 1-6 divided doses, followed by additional doses at subsequent intervals as required to maintain and or enhance the immune response, e.g. (for human subjects) a second dose at 1-4 months and subsequent doses after a further 1-4 months if required.

[0214] The dosage regimen will be determined, at least in part, by the individual's needs and will depend on the judgment of the practitioner (eg, a physician or veterinarian).

[0215] Simultaneous administration means (substantially) simultaneous administration.

[0216] Sequential administration of two or more compositions / therapeutics / vaccines means that the compositions / therapeutics / vaccines are administered one after the other at (substantially) different times.

[0217] For example, sequential administration can include administering two or more compositions / therapeutics / vaccines at different times, with varying intervals of days (e.g., at least 1, 2, 5, 10, 15, 20, 30, 60, 90, 100, 150, or 200 days).

[0218] For example, in one embodiment, the vaccines of the invention can be administered as part of a "prime-boost" vaccination regimen.

[0219] In one embodiment, the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions and prophylactic formulations (e.g., vaccines) of the present invention can be administered to a subject, such as a mammal (e.g., a human, bovine, porcine, ovine, goat, horse, deer, bear, canine or feline subject) in combination with one or more immunomodulators selected from, for example, immunoglobulins, antibiotics, interleukins (e.g., IL-2, IL-12) and / or cytokines (e.g., IFNγ) (simultaneously or sequentially).

[0220] Immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions and prophylactic formulations (e.g., vaccines) may contain 5% to 95% active ingredient, such as at least 10% or 25% active ingredient, or at least 40% active ingredient or at least 50, 55, 60, 70 or 75% active ingredient.

[0221] Immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (eg, vaccines) are administered in a manner compatible with the dosage formulation, and in such amount as to be prophylactically and / or therapeutically effective.

[0222] Administration of immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions and prophylactic formulations (e.g., vaccines) is typically by conventional routes, such as intravenous, subcutaneous, intraperitoneal or mucosal routes. Administration can be by parenteral administration; for example, subcutaneous or intramuscular injection.

[0223] Thus, the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions and prophylactic formulations (e.g., vaccines) of the present invention can be prepared as injectables, as liquid solutions or suspensions. Alternatively, solid forms suitable for dissolution or suspension in liquids prior to injection can be prepared. The formulations can also be emulsified, or the peptides encapsulated in liposomes or microcapsules.

[0224] The active ingredient is typically mixed with a pharmaceutically acceptable and compatible excipient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the immunogenic composition, therapeutic formulation, medicament, pharmaceutical composition, and prophylactic formulation (e.g., vaccine) may contain small amounts of auxiliary substances, such as wetting agents or emulsifiers and / or pH buffers.

[0225] Typically, the carrier is a pharmaceutically acceptable carrier. Non-limiting examples of pharmaceutically acceptable carriers include water, saline, and phosphate buffered saline. However, in some embodiments, the composition is in lyophilized form, in which case it may include a stabilizer, such as bovine serum albumin (BSA). In some embodiments, it may be necessary to formulate the composition with a preservative (such as thimerosal or sodium azide) to facilitate long-term storage.

[0226] 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).

[0227] Other formulations suitable for other modes of administration include suppositories, and in some cases, oral formulations or formulations suitable for distribution as aerosols. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.

[0228] Oral formulations include such commonly used excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders.

[0229] It may be desirable to direct the compositions of the invention (as described above) to the respiratory system of the subject.Effective delivery of therapeutic / prophylactic compositions or medicaments to sites of lung infection can be achieved by oral or intranasal administration.

[0230] Formulations for intranasal administration may be in the form of nasal drops or nasal sprays. Intranasal formulations may comprise droplets having an approximate diameter in the range of 100-5000 μm, such as 500-4000 μm, 1000-3000 μm, or 100-1000 μm. Alternatively, in terms of volume, the droplets may be in the range of about 0.001-100 μl, such as 0.1-50 μl or 1.0-25 μl, or such as 0.001-1 μl.

[0231] Alternatively, the therapeutic / prophylactic formulation or drug can be an aerosol formulation. The aerosol formulation can take the form of a powder, a suspension or a solution. The size of the aerosol particles is related to the delivery ability of the aerosol. Smaller particles can travel further down the respiratory airways toward the alveoli than larger particles. In one embodiment, the aerosol particles have a diameter distribution that facilitates delivery along the entire length of the bronchi, bronchioles and alveoli. Alternatively, the particle size distribution can be selected to target a specific part of the respiratory airways, such as the alveoli. In the case of aerosol delivery of a medicament, the diameter of the particles can be in the approximate range of 0.1 μm-50 μm, preferably 1 μm-25 μm, more preferably 1 μm-5 μm.

[0232] Aerosol particles can be delivered using a nebulizer (e.g., through the mouth) or a nasal spray. Aerosol formulations may optionally contain a propellant and / or a surfactant.

[0233] In one embodiment, the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions and prophylactic formulations (e.g., vaccines) of the invention comprise a pharmaceutically acceptable carrier, and optionally one or more of salts, excipients, diluents and / or adjuvants.

[0234] In one embodiment, the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions and prophylactic formulations (e.g., vaccines) of the present invention may comprise one or more immunomodulators selected from, for example, immunoglobulins, antibiotics, interleukins (e.g., IL-2, IL-12) and / or cytokines (e.g., IFNγ).

[0235] The present invention includes polypeptides that are substantially homologous to polypeptides based on any one of the polypeptide antigens identified in this application (including fragments thereof).The terms "sequence identity" and "sequence homology" are considered synonymous in this specification.

[0236] For example, the subject polypeptide can comprise an amino acid sequence having at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99, or 100% amino acid sequence identity to the amino acid sequence of the reference polypeptide.

[0237] Many established algorithms can be used for comparing two amino acid sequences.Usually, a sequence serves as a reference sequence, and a test sequence can be compared thereto.The sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters specified.The comparison for the amino acid sequence compared can be carried out, for example, by an algorithm (such as GAP, BESTFIT, FASTA or TFASTA) or BLAST and BLAST 2.0 algorithms implemented by a computer.

[0238] The BLOSLIM62 table shown below is an amino acid substitution matrix derived from approximately 2,000 local multiple alignments of protein sequence segments representing highly conserved regions of over 500 groups of related proteins (Henikoff and Henikoff, Proc. Natl. Acad. Sci. LISA 89:10915-10919, 1992; incorporated herein by reference). Amino acids are represented by the standard single-letter code. Percent identity is calculated as follows:

[0239]

[0240] BLOSUM62 table

[0241] ARNDCQEGHILKMFPSTWYV

[0242] A-4

[0243] R-15

[0244] N-2 0 6

[0245] D-2-2 1 6

[0246] C 0-3-3-3 9

[0247] Q-1 1 0 0-3 5

[0248] E-1 0 0 2-4 2 5

[0249] G 0-2 0-1-3-2-2 6

[0250] H-2 0 1-1-3 0 0-2 8

[0251] I-1-3-3-3-1-3-3-4-3 4

[0252] L-1-2-3-4-1-2-3-4-3 2 4

[0253] K-1 2 0-1-3 1 1-2-1-3-2 5

[0254] M-1-1-2-3-1 0-2-3-2 1 2-1 5

[0255] F-2-3-3-3-2-3-3-3-1 0 0-3 0 6

[0256] P-1-2-2-1-3-1-1-2-2-3-3-1-2-4 7

[0257] S 1-1 1 0-1 0 0 0-1-2-2 0-1-2-1 4

[0258] T 0-1 0-1-1-1-1-2-2-1-1-1-1-2-1 15

[0259] W-3-3-4-4-2-2-3-2-2-3-2-3-11-4-3-2 11

[0260] Y-2-2-2-3-2-1-2-3 2-1-1-2-1 3-3-2-2-2 7

[0261] V 0-3-3-3-1-2-2-3-3 3 1-2 1-1-2-2 0-3-1 4

[0262] In a homology comparison, identity can exist over a region of the sequence that is at least 10 amino acid residues in length (e.g., at least 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550 or 570 amino acid residues in length - e.g., up to the entire length of the reference sequence).

[0263] Substantially homologous polypeptides have one or more amino acid substitutions, deletions or additions. In many embodiments, these changes are of a smaller nature, for example, only involving conservative amino acid substitutions. Conservative substitutions are those made by replacing one amino acid with another within the following groups: basic: arginine, lysine, histidine; acidic: glutamic acid, aspartic acid; polar: glutamine, asparagine; hydrophobic: leucine, isoleucine, valine; aromatic: phenylalanine, tryptophan, tyrosine; small: glycine, alanine, serine, threonine, methionine. Substantially homologous polypeptides also encompass those comprising other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically 1 to about 30 amino acids (e.g., 1-10 or 1-5 amino acids); and small amino or carboxyl terminal extensions, such as amino-terminal methionine residues, small linker peptides of up to about 20-25 residues, or affinity tags.

[0264] 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.

[0265] The polynucleotide sequences of the present invention include nucleic acid sequences that have been removed from their naturally occurring environment, recombinant or cloned DNA isolates, and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.

[0266] Polynucleotides of the present invention can be prepared by any means known in the art. For example, a large amount of polynucleotides can be produced by replicating in a suitable host cell. The natural or synthetic DNA fragment encoding the required fragment will be incorporated into a recombinant nucleic acid construct (generally a DNA construct) that can be introduced into a prokaryotic or eukaryotic cell and replicated therein. Usually, the DNA construct will be suitable for autonomous replication in a unicellular host (such as yeast or bacterium), but can also be used to introduce and be integrated into the genome of cultivated insects, mammals, plants or other eukaryotic cell lines.

[0267] The polynucleotides of the present invention can also be produced by chemical synthesis, for example by the phosphoramidite method or the triester method, and can be carried out on a commercial automated oligonucleotide synthesizer. Double-stranded fragments can be obtained from chemically synthesized single-stranded products by synthesizing complementary strands and annealing the strands together under appropriate conditions or by adding complementary strands using a DNA polymerase with appropriate primer sequences.

[0268] As applied to nucleic acid sequences, the term "isolated" in the context of the present invention means that the polynucleotide sequence has been removed from its natural genetic environment and is therefore free of other extraneous or unwanted coding sequences (but may include naturally occurring 5' and 3' untranslated regions, such as promoters and terminators), and is in a form suitable for use in genetically engineered protein production systems. Such isolated molecules are those separated from their natural environment.

[0269] Given the degeneracy of the genetic code, considerable sequence variation is possible in the polynucleotides of the present invention. The degenerate codons comprising all possible codons for a given amino acid are shown below:

[0270]

[0271]

[0272] Those skilled in the art will appreciate that there is flexibility in determining degenerate codons, which represent all possible codons encoding each amino acid. For example, some polynucleotides comprised by a degenerate sequence may encode variant amino acid sequences, but those skilled in the art can readily identify such variant sequences by reference to the amino acid sequences of the present invention.

[0273] A "variant" nucleic acid sequence has substantial homology or substantial similarity to a reference nucleic acid sequence (or a fragment thereof). A nucleic acid sequence or a fragment thereof is "substantially homologous" (or "substantially identical") to a reference sequence if, when optimally aligned (with appropriate nucleotide insertions or deletions) with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98% or 99% of the nucleotide bases. Methods for determining homology of nucleic acid sequences are known in the art.

[0274] Alternatively, a "variant" nucleic acid sequence is substantially homologous (or substantially identical) to a reference sequence (or fragment thereof) if the "variant" and reference sequence are capable of hybridizing under stringent (e.g., highly stringent) hybridization conditions. As will be readily appreciated by those skilled in the art, nucleic acid sequence hybridization will be affected by conditions such as salt concentration (e.g., NaCl), temperature, or organic solvents, in addition to base composition, the length of the complementary strand, and the number of nucleotide base mismatches between the hybridizing nucleic acids. Stringent temperature conditions are preferably used, and typically include temperatures exceeding 30° C., typically exceeding 37° C., and preferably exceeding 45° C. Stringent salt conditions are typically less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. The pH is typically between 7.0 and 8.3. The combination of parameters is much more important than any single parameter.

[0275] Methods for determining nucleic acid sequence identity percentages are known in the art. For example, when assessing nucleic acid sequence identity, a sequence having a limited number of consecutive nucleotides can be compared with a nucleic acid sequence (having the same number of consecutive nucleotides) from the corresponding portion of the nucleic acid sequence of the present invention. Tools known in the art for determining nucleic acid sequence identity percentages include nucleotide BLAST.

[0276] It is understood by those of ordinary skill in the art that different species exhibit "preferred codon usage". As used herein, the term "preferred codon usage" refers to the codons that are most frequently used in the cells of certain species, thus favoring one or more 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; in other species, different Thr codons may be preferred. The preferred codons of a particular host cell species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introducing a preferred codon sequence into recombinant DNA can enhance protein production, for example, by making protein translation more efficient within a particular cell type or species.

[0277] Therefore, in one embodiment of the present invention, the codons of the nucleic acid sequence are optimized for expression in a host cell.

[0278] A "fragment" of a polynucleotide of interest comprises a series of consecutive nucleotides from the sequence of the full-length polynucleotide. For example, a "fragment" of a polynucleotide of interest can comprise (or consist of) at least 30 consecutive nucleotides from the sequence of the polynucleotide (e.g., at least 35, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, or 1710 consecutive nucleic acid residues of the polynucleotide). A fragment may include at least one antigenic determinant and / or may encode at least one antigenic epitope of the corresponding polypeptide of interest and / or may have a common antigenic cross-reactivity and / or substantially the same in vivo biological activity as the polypeptide of interest.

[0279] legend

[0280] Figure 1 AB. Example MVA vector construction. Figure 1 A provides a schematic diagram of the cassette "MVAHantaNP". Figure 1B provides a schematic diagram of plasmid 17ACNHBP_MVA-SEOV-HNT-NP_pMS-RQ (pMVAHantaNP).

[0281] Figure 2 PCR confirmation of pure recombinant nucleoprotein - Agarose gel confirmed the presence of the MVAHantaNP construct. Flanking to flank primers (SEQ ID NOs: 46 and 47) covered the entire insert and ran from the MVA flanking regions at either end of the vaccine insert, yielding the expected amplification product size. The contents of the wells are as follows (numbered from left to right): 1. Ladder; 2. Positive control (MVA-HantaNP plasmid) GFP to flanking primers - expected size 3260bp; 3. MVA-HantaNP "Batch 1"; 4. MVA-HantaNP "Batch 2+3"; 5. MVA-HantaNP "Batch 4+5+6"; 6. Negative control; 7. Ladder; 8. Positive control (MVA-HantaNP plasmid) flanking to flanking primers - expected size 3788bp; 9. MVA-Hantanp "Batch 1"; 10. MVA-HantaNP "Batch 2+3"; 11. MVA-HantaNP "Batch 4+5+6"; 12. Negative control; 13. Ladder.

[0282] Figure 3 Western blot confirmed expression of the NP / Flag tag. The expected size of the protein is 89 kDa. The contents of the wells are as follows (numbered from left to right): 1. Ladder; 2. "Passage 3" P3 (1.1.1); 3. P3 (4.1.1); 4. P3 (4.1.2); 5. Vaccine batch 1; 6. Vaccine batches 2+3 combined; 7. Vaccine batches 4+5+6 combined.

[0283] Figure 4 Clinical scores (% daily weight gain) during the immunization study: (a) weight and (b) temperature of mice after the primary immunization (first arrow, day 0) and booster immunization (second arrow, day 14).

[0284] Figure 5 Total ELISPOT responses from vaccinated and unvaccinated mice.

[0285] Figure 6 Splenocyte IFN-γ ELISPOT restimulation responses to individual peptide pools ("NP1"-"NP11" therein). i) Group 1 represents mice primed and boosted with MVA-HantaNP; ii) Group 2 represents mice vaccinated with a single dose of MVA-HantaNP; iii) Group 3 represents mice primed and boosted with empty MVA wild-type; and iv) Group 4 represents PBS control, prime, and boost.

[0286] Figure 7 IgG responses to Hantavirus NP in mouse sera. Absorbance readings provide a readout of antibody binding activity to recombinant Hantavirus NP.

[0287] Figure 8 Weight and temperature of mice after intramuscular (left column) or intranasal (right column) challenge with hantavirus.

[0288] Figure 9 . Viral loads in the blood, lungs, kidneys, spleen, and liver of mice at (a) day 5 after intramuscular challenge; (b) day 5 after intranasal challenge; and (c) day 14 after intranasal challenge.

[0289] Figure 10 Viral loads in the kidneys, lungs, and spleens of mice on day 5 after intranasal challenge. Results associated with immunization with empty MVA wild-type vector are represented by circles; results associated with immunization with MVA-HantaNP are represented by triangles. Example

[0290] Example 1: Preparation of an exemplary MVA-NP (nucleoprotein) vector

[0291] The MVA HantaNP cassette (denoted "MVA HantaNP") was generated by GeneArt (Thermofisher) and contains the p11 promoter, green fluorescent protein (GFP), and the MH5 promoter, followed by the Kozak sequence upstream of the NP sequence. The nucleoprotein sequence is a chimeric sequence containing two different sequences, Seoul and Hantan. Downstream is a 24-residue linker sequence, followed by the Flag tag epitope and a stop codon. Figure 1 A schematic diagram of MVA HantaNP is provided in (A).

[0292] This cassette was inserted into the Sfil / Sfil cloning site of plasmid pMS-RQ-Bb to generate plasmid 17ACNHBP_MVA-SEOV-HNT-NP_pMS-RQ (pMVAHantaNP).

[0293] Figure 1 A schematic diagram of pMVAHantaNP is provided in (B), and the nucleic acid sequence of pMVAHantaNP is provided in SEQ ID NO:33.

[0294]

[0295] pMVAHantaNP contains:

[0296] DelIII left wing:

[0297] GTTGGTGGTCGCCATGGATGGTGTTATTGTATACTGTCTAAACGCGTTAGTAAAACATGGCGAGGAAATAAATCATATAAAAAATGATTTCATGATTAAACCATGTTGTGAAAAAGTCAAGAACGTTCACATTGGCGGACAATCTAAAAACAATACAGTGATTGCAGATTTGCCATATATGGATAATGCGGTATCCGATGTATGCAATTCACTGTATAAAAAGAATGTATCAAGAATATCCAGATTTGCTAATTTGATAAAGATAGATGACGATGACAAGACTCCTACTGGTGTATATAATTATTTTAAACCTAAAGATGCCATTCCTGTTATTATATCCATAGGAAAGGATAGAGATGTTTGTGAACTATTAATCTCATCTGATAAAGCGTGTGCGTGTATAGAGTTAAATTCATATAAAGTAGCCATTCTTCCCATGGATGTTTCCTTTTTTACCAAAGGAAATGCATCATTGATTATTCTCCTGTTTGATTTCTCTATCGATGCGGCACCTCTCTTAAGAAGTGTAACCGATAATAATGTTATTATATCTAGACACCAGCGTCTACATGACGAGCTTCCGAGTTCCAATTGGTTCAAGTTTTACATAAGTATAAAGTCCGACTATTGTTCTATATTATATATGGTTGTTGATGGATCTGTGATGCATGCAATAGCTGATAATAGAACTTACGCAAATATTAGCAAAAATATATTAGACAATACTACAATTAACGATGAGTGTAGATGCTGTTATTTTGAACCACAGATTAGGATTCTTGATAGAGATGAGATGCTCAATGGATCATCGTGTGATATGAACAGACATTGTATTATGATGAATTTACCTGATGTAGGCGAATTTGGATCTAGTATGTTGGGGAAATATGAACCTGACATGATTAAGATTGCTCTTTCGGTGGCTGG(SEQ ID NO:34)

[0298] First adapter:

[0299] GTACAGGCGCGCC(SEQ ID NO:35)

[0300] P11:

[0301] TTTCATTTTGTTTTTTTCTATGCTATAA(SEQ ID NO:36)

[0302] GFP:

[0303] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA(SEQ ID NO:37)

[0304] Second adapter:

[0305] GAGCTCCGGCCCGCTCGAGGCCGCTGGTACCCAACCT(SEQ ID NO:38)

[0306] MH5 promoter:

[0307] AAAAATTGAAAATAAATACAAAGGTTCTTGAGGGTTGTGTTAAATTGAAAGCGAGAAATAATCATAAATA (SEQ ID NO: 39)

[0308] Third connector:

[0309] AGCCCGGT

[0310] Kozak sequence:

[0311] GCCACCATGG (SEQ ID NO: 41). The 3' end of the Kozak sequence overlaps with four nucleic acids at the 5' end of SEQ ID NO: 29.

[0312] Nucleoprotein (SEQ ID NO: 29)

[0313] Fourth connector:

[0314] GACCTGGAAGGCCCTAGATTCGAG(SEQ ID NO:42)

[0315] Flag tag:

[0316] GACTACAAGGACGAT GACGAC AAG(SEQ ID NO:43)

[0317] termination:

[0318] TGA

[0319] Fifth connector:

[0320] CTCGACCTGCAGTTTTATG(SEQ ID NO:44)

[0321] Del III right wing:

[0322] (SEQ ID NO:45)

[0323] From transformed bacteria (Escherichia coli K12 DH10B TM Plasmid DNA was purified from T1R) and the concentration was determined by UV spectroscopy using GeneArt (Thermofisher).

[0324] BHK-21 cells were infected with MVA 1974 at a multiplicity of infection of 0.05. According to the manufacturer's instructions, cells infected with pMVAHantaNP were transfected using lipofectamine (Life Technologies). Based on GFP expression, the resulting recombinant MVAHantaNP was continuously plaque-purified 4 times in chicken embryo fibroblasts ("CEF") cells. MVAHantaNP was amplified on CEF cells, purified by centrifugation with a sucrose cushion, and titrated by plaque assay on CEF cells before use in vivo. GFP fluorescence was used and plaques were visualized by immunostaining with rabbit anti-vaccinia antibodies (AbD Serotec, UK) and Vectastain Universal ABC-AP kits (Vector laboratories, USA). Wizard SV genomic DNA purification system (Promega, USA) was used to extract genomic DNA from infected cells and used as a template in PCR to carry out genotyping analysis with KAPA2G FastHotStart PCR kits (KAPABiosystems, USA).

[0325] The presence of the MVA HantaNP construct was confirmed by polymerase chain reaction (PCR). A set of primers was designed specifically to detect the HantaNP construct within the MVA flanking region of an expected size of 3260 bp - this is Figure 2 Shown in.

[0326] Sequencing of the expressed protein confirmed very high sequence fidelity. The recombinant purified MVA HantaNP was then filled into flasks of increasing size in stages by tissue culture. Initially, MVA HantaNP was grown in vials of chicken embryo fibroblast (CEF) cells and harvested before infection into slightly larger bottles of CEF cells. The process was repeated in larger and larger flasks until MVA HantaNP successfully infected 10× large flasks of CEF cells. Sucrose cushion centrifugation was performed; the viral pellet was resuspended in PBS and prepared for immunogenicity studies. A total of six batches were produced. Batches 2+3 and 4+5+6 were merged into a single sample and the viral concentration was titrated.

[0327] The purified vaccine batches were compared to a positive control (the original received plasmid from Geneart). A second set of primers was designed to identify the entire insert from both MVA flanking regions. The results showed the presence of pure recombinant MVA (MVA containing the insert) in all vaccine batches. The original plasmid was again used as a positive control, and all vaccine batches had the same expected size product as the positive control.

[0328] Primer details are as follows:

[0329] SEQ ID NO:46:CGGCACCTCTCTTAAGAAGT (Fwd target Del III left flank)

[0330] SEQ ID NO:47:GTGTAGCGTATACTAATGATATTAG (Rev target Del III right flank)

[0331] SEQ ID NO:48: GGAGTACAACTACAACAGCCACAACG (Fwd target GFP)

[0332] The GFP Fwd primer binds to the GFP sequence and, when used in combination with the Rev Del III right flank primer, covers GFP to the right MVA flank by the nucleoprotein and specifically identifies the presence of the NP gene.

[0333] Detection of protein expression

[0334] CEF cells were infected with MVAHantaNP at a multiplicity of infection of 0.05 and incubated at 37°C in modified Eagle's medium (MEM) supplemented with 2% FBS (Sigma-Aldrich, UK). Once good GFP fluorescence and CPE were observed under a microscope, the medium was removed after 48 hours. 1×LDS was used to treat the cells. Reducing sample buffer (containing Sample reduction buffer LDS sample buffer (Thermofisher, UK) lysed cells, transferred to Eppendorf tubes and heated at 70 ° C for 10 minutes. Uninfected cells were treated in the same manner as the negative control. MVAHantaNP lysate was subjected to SDS-PAGE on 4-12% Bis-Tris gel (Life technologies) and the protein was transferred to a nitrocellulose membrane. Nitrocellulose membrane was blocked using 5% milk powder (Merck Millipore), then incubated with shaking for 1-2 hours in the presence of a primary antibody (rabbit anti-V5 polyclonal (Invitrogen) at 1 / 1000 in PBS-0.05% Tween), and then washed 3 times in PBS containing 0.05% Tween-20 (Sigma-Aldrich). The membrane was incubated with shaking for 1 hour in the presence of an HRP-conjugated secondary antibody (anti-rabbit IgG peroxidase (Sigma-Aldrich) at 1 / 1000 in PBS-0.05% Tween), and washed as described above. Protein expression was determined by detecting bound antibodies using the Pierce ECL Western blotting substrate kit (Thermofisher) according to the manufacturer's instructions and visualized in a chemiluminescent imager (Syngene). Molecular weights were determined using MagicMark XP Western protein markers (Invitrogen) as a reference.

[0335] Western blot analysis (see Figure 3 ) confirmed the expression of the flag tag located downstream of NP. The expected 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 the 3rd generation pick to the vaccine batch (the inventors observed a low level of protein degradation, which is not considered significant). The target band is at the expected size of the protein, which again indicates good expression.

[0336] (SEQ ID NO:49)

[0337] The amino acid sequence of SEQ ID NO: 49 corresponds to the amino acid sequence of SEQ ID NO: 31 plus a fourth linker and a flag tag for expression.

[0338] Example 2. Immunogenicity of MVA HantaNP in A129 mice

[0339] Eighty male 6-8 week old A129 mice were randomly divided into four groups and ear-marked before inoculation.

[0340] Group 1 received 1 × 10 7 Two doses of vaccination with PFU of MVAhant ANPI in endotoxin-free phosphate-buffered saline (PBS).

[0341] Group 2 received 1×10 7 A single vaccine injection of 100 plaque forming units (pfu) of MVA HantaNP in endotoxin-free PBS was performed.

[0342] Group 3 received 1 × 10 7 Two doses of vaccination with PFU of MVA empty vector in endotoxin-free PBS.

[0343] Group 4 received two doses of vaccination with endotoxin-free PBS on days 0 and 14 as a negative control.

[0344] All mice were injected intramuscularly into the tail thigh. 100 μ l was administered at each inoculation (50 μ l was administered in each thigh). Animal weights were recorded every day throughout the study. 5 animals per group were euthanized, and spleen tissue and blood were collected on the 28th day after the initial inoculation. All efforts were made to minimize animal suffering. These studies were approved by the ethical review process of the PHE in Porton Down, UK, and the UK Home Office through project license number 30 / 2993. Work was carried out according to the Animals (Scientific Procedures) Act 1986 and the Animal Care Practice Specification (1989) used in the Home Office (UK) scientific procedures.

[0345] Throughout the study, no clinical signs related to vaccination were observed, and all mice gained weight as expected (see Figure 4 As expected, all four groups gained weight throughout the study, and Group 4 consistently weighed less than Groups 1-3. At the end of the study, similar % weight gain was observed in all groups. These clinical data indicate that the mice tolerated the vaccine without side effects.

[0346] To determine T cell responses in immunized animals, an interferon-γ ELISPOT assay was used to measure the frequency of responding T cells after stimulation with hantavirus-specific peptides.

[0347] Spleens from test animals were collected aseptically, homogenized, and erythrocytes 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). Antigen recall responses of splenocytes were assessed by IFN-γ ELISPOT (Mabtech, Sweden) according to the manufacturer's instructions. Cells were seeded in PVDF microtiter plates at 2 × 10e6 per well and restimulated with a peptide library (JPT, Berlin).

[0348] The peptides spanning the Hanta NP protein sequence were 15 residues long, with an 11 residue overlap between the peptides. A total of 189 peptides were generated and tested in 11 peptide pools (see Table 1).

[0349] Table 1: Peptide library (the starting amino acid ("AA") number corresponds to the amino acid number in SEQ ID NO: 31)

[0350]

[0351]

[0352]

[0353]

[0354]

[0355] They were applied to the cells at a final concentration of 2.5 μg / ml per peptide, with 17 peptides in each of pools 1 to 10 and 19 peptides in pool 11. The plates were developed after 18 hours in a humidified incubator at 37°C, 5% CO2. Spots were visually counted on an automated ELISPOT reader (Cellular Technologies Limited, USA). Background values from wells containing cells and medium but no peptide were subtracted, and data were presented as responses to individual pools or as a sum across target proteins. Results are expressed as 10 6 spot-forming units (SFU) per cell.

[0356] When stimulated with all Hanta NP pools, the MVA-WT and PBS groups (Groups 3 and 4) were negative. In the prime / boost and prime groups, IFN-γ responses were detected to several peptide pools, and particularly strong responses were directed against two different regions of the NP (corresponding to pools 4 and 9).

[0357] The inventors found that for SEQ ID NOs: 11 and 12, T cell (IFN-γ) stimulation was greatly increased.

[0358] Increased responses to pools 2, 3, 5, 7, 8, and 10 were also detected for both the prime / boost and prime groups compared to the control group. Figure 5 Total ELISPOT responses from vaccinated and unvaccinated mice are provided in; and Figure 6 ELISPOT responses to individual peptide pools are shown.

[0359] To measure the antibody response in immunized mice, ELISA analysis was performed to evaluate the binding of antibodies to hantavirus-specific proteins. Recombinant Hanta NP (Native Antigen Company, UK) as crude lysate was diluted in 0.2 M carbonate-bicarbonate buffer pH 9.4 (Thermo Scientific) and used to coat Maxisorp 96-well plates (Nunc, Denmark) at 10 μg / ml in 100 μL, incubated overnight at 4°C, then washed with PBS + 0.01% Tween-20 (Sigma-Aldrich), and blocked with 100 μL of 5% milk powder (Merck, Millipore) in PBS + 0.01% Tween-20 for 1 hour at 37°C, followed by another wash in PBS + 0.01% Tween-20. The samples were diluted 1:50 in 5% milk powder in PBS + 0.01% Tween-20 buffer, added to the plates in triplicate (100 μl / well) and incubated at 37°C for 1 hour. Normal mouse serum (Sigma-Aldrich) and polyclonal anti-hantavirus hyperimmune mouse ascites samples (BEIResources, USA) were used as positive and negative control samples, respectively. The plates were washed with PBS + 0.01% Tween-20, and 100 μl of polyclonal anti-mouse HRP conjugate (Sigma-Aldrich) diluted 1:20,000 in 5% milk PBS + 0.01% Tween-20 was added to each well. After incubation for another 1 hour at 37°C, the plates were washed with PBS + 0.01% Tween-20, and 100 μl of TMB substrate (Surmodics) was added to each well, followed by incubation at 20°C for 1 hour. The reaction was stopped by adding 100 μl of stop solution (Surmodics) prepared according to the manufacturer's instructions and the plate was read at 450 nm using a Molecular Devices plate reader and Softmax Pro version 5.2 software (Molecular Devices). Background absorbance values were subtracted from sample values and results were reported as absorbance (450 nm) of a 1:50 dilution. Data were interpreted and analyzed using Graph Pad Prism 7 (see Figure 7 ).

[0360] The MVA-WT and PBS control groups showed very little absorbance, with values similar to those in the blank wells. The responses of all mice in the prime and prime / boost vaccination groups were significantly higher. The average absorbance recorded in the prime group alone was ~2.3, while the average OD recorded in the prime / boost group was ~1.5.

[0361] Thus, the vectors of the present invention demonstrate highly desirable induction of both cellular and humoral immune responses.

[0362] Example 3. Efficacy test

[0363] Sixty male A129 mice weighing 19-21 g were pre-randomized into four groups and then ear-tagged and microchipped for identification, weight monitoring, and temperature monitoring.

[0364] The remaining mice that were not culled on day 28 for immunogenicity studies were challenged with Hanta SEOV on day 28. In each group, 1.36 × 10 6 TCID50 / dose n=10 animals were challenged via the intranasal route and n=5 animals were challenged via the intramuscular route.

[0365] The animals that were attacked intramuscularly were euthanized on the 33rd day. The mice that were attacked intranasally were euthanized on the 33rd day (5 per group) or the 42nd day (5 per group). Blood, saliva, liver, kidney, lung and spleen were collected for histological and viral load analysis. All efforts were made to minimize the suffering of the animals. These studies were approved by the ethical review process of the PHE in Porton Down, UK and the UK Home Office through project license number 30 / 2993. Work was carried out according to the Animals (Scientific Procedures) Act 1986 and the Animal Care Practice Code (1989) used in the Home Office (UK) scientific procedures.

[0366] Clinical signs:

[0367] Animal weights and temperatures were recorded daily throughout the study. All challenged animals remained healthy and no clinical signs were observed after challenge with hantavirus. Temperatures and body weights throughout the study are reported in Figure 8 middle.

[0368] Viral load:

[0369] Viral load was assessed 5 and 14 days after challenge. Figure 9 As shown, immunization with MVA HantaNP achieved a reduction or complete clearance of hantavirus from the tissues tested already on day 5. Highly favorable reductions in viral loads were also observed in most tissues 14 days after challenge.

[0370] Viral Load-Follow-up Study:

[0371] In the follow-up study, 28 female A129 mice were pre-randomized into two groups and then ear-tagged and microchipped for identification, weight monitoring, and temperature monitoring.

[0372] Of these 28 mice, 16 were primed with GLP-grade MVA HantaNP on day 0 and then boosted on day 14 ("Group A"), and 12 mice received prime and boost immunizations with an empty MVA wild-type vector on day 0 and day 14, respectively ("Group B"). Immunizations were performed according to Example 2 above.

[0373] On day 28, 3×10 6 Dosage of TCID50 / mouse Eight mice in group A and eight mice in group B were challenged intranasally with Hanta SEOV.

[0374] In this follow-up study, viral load was assessed 5 days after challenge. Figure 10 As shown, immunization with MVA HantaNP achieved a favorable reduction of hantavirus in the tested tissues, even when the challenge dose exceeded two-fold.

[0375] Example 4: Preparation of an exemplary adenoviral vector

[0376] A non-replicating adenovirus is engineered to express the Hantavirus NP nucleic acid or fragment thereof of the present invention. The genetic sequence of Hantavirus NP is inserted into the genome of the adenoviral vector. Expression of Hantavirus NP is indicated by reactivity between NP-specific antibodies and adenoviral products by Western blot or ELISA as follows:

[0377] Cell lysates from cells infected with the recombinant adenovirus were subjected to SDS-PAGE and Western blotting using a hantavirus NP-specific antibody, which showed specific reactivity compared to a negative control.

[0378] Alternatively, products from cells infected with recombinant adenovirus are used to coat ELISA plates. Hantavirus-specific antibodies bind to the coating and are detected by a chemical reaction.

[0379] Example 5: Hantavirus vaccine provides cross-strain protection

[0380] A vaccine expressing the hantavirus NP nucleic acid or fragment thereof of the present invention in an adenoviral or non-replicating poxvirus vector is delivered parenterally to mice susceptible to diseases caused by hantavirus. They are challenged with a lethal dose of Hantavirus from a strain different from the strain on which the vaccine is based. The challenged animals show no or mild clinical signs of disease and do not require euthanasia. Control animals that receive the same challenge dose of Hantavirus but do not receive the vaccine show severe signs of disease, reach humane clinical endpoints, and require euthanasia.

[0381] Example 6. Preparation and efficacy of recombinant influenza virus vectors

[0382] Reverse genetics was used to construct a recombinant influenza virus carrying a protective epitope of hantavirus NP in the neuraminidase stalk. Following intranasal or parenteral administration, hantavirus-specific cytotoxic T lymphocytes (CTLs) were induced in mice. These CTLs provided a reduction in viral load and clinical disease after challenge with hantavirus.

[0383] Example 7. Preparation and efficacy of recombinant bacterial vectors

[0384] The hantavirus NP nucleic acid or fragments thereof of the present invention are expressed on the surface of genetically attenuated Gram-negative bacteria. Following intranasal or parenteral administration to mice, the bacterial vectors colonize antigen-presenting cells (e.g., dendritic cells or macrophages), inducing humoral and cellular hantavirus-specific immune responses. These immune responses provide a reduction in viral load and clinical disease following challenge with hantavirus. Sequence Listing <110> Secretary of State for Health and Social Care <120> Hantavirus antigen composition <130> P60420WO <150> GB 1910804.2 <151> 2019-07-29 <160> 235 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1768 <212> DNA <213> Seoul virus <400> 1 tagtagtagg ctccctaaag agctactaca ctaacaagga aaatggcaac tatggaagaa 60 atccagagag aaatcagtgc gcacgagggg cagcttgtaa tagcacgcca gaaggtcaag 120 gatgcagaaa agcagtatga gaaggatcct gatgacctaa ataagagggc actgcatgat 180 cgggagagtg tcgcagcttc aatacaatca aaaattgatg aattgaagcg ccaacttgct 240 gacaggattg cagcagggaa gaacatcggg caaccggg atcctacagg ggtagagccg 300 ggtgatcatc tcaaggaaag atcagcacta agctacggga atacactgga cctgaatagc 360 cttgacattg atgaacctac aggagagaca gctgattggt tgaccataat tgtctatttg 420 acatcattcg tggtcccgat catcttgaag gcactgtaca tgttgacaac aagaggcagg 480 540 gatgtcaatg gaatcagaaa gcccaaacat ctgtatgtgt caatgccaaa cgcccaatca 600 agcatgaagg ctgaagagat aacacctgga agattccgca ctgcagtatg tgggctatac 660 cctgcacaga taaaggcaag gaacatggta agccctgtca tgagtgtagt tgggtttttg 720 gcactggcaa aagactggac atctagaatt gaagaatggc ttggtgcacc ctgcaagttc 780 atggcagagt ctcccattgc cgggagctta tctgggaatc ctgtgaatcg tgattatatc 840 agacagagac aaggtgcact tgcagggatg gagccaaaag aatttcaagc tctcaggcaa 900 cattcaagg atgctggatg tacactggtt gaacatattg agtcaccatc atcaatgg 960 gtatttgctg gggcccctga taggtgccca ccgacatgcc tgttgttgg agggatggct 1020 gagttaggtg ctttcttttc tatacttcag gattgagga acacaatcat ggcttcaaag 1080 actgtgggaa cagctgatga aaagcttcga aagaagtcat cattctatca atcatacctc 1140 agacgcacac atcaatggg atacaactg gaccagagga taattgttat gtttatggtt 1200 gcctggggaa aggaggcagt ggacaacttt catctcggtg atgacatgga tccagctt 1260 cgcagcctgg ctcagatcct gattgaccag aaagtgaagg aaatctcaa ccaggaacct 1320 atgaattat aagtacata ttgtaatc catactact atagttaag aattactaat 1380 help windows gatttattga atatcatat taaatta ggtagttaa 1440 ctattagtta gttaagttag ctattgatt tattatgatta tcacaattga atgtaatcat 1500 aagcacaatc actgccatgt ataatcacgg gtatacggt gttttcata tggggaacag 1560 ggtgggctta gggccaggtc accttaagtg accttttg tatatatgga tgtagatttc 1620 aattgatcga gtactaatcc tactgttctc ttttcctttc ctttctcctt ctttactaac 1680 aacaacaaac tacctcacaa ccttctacct caacacatac tacctcattc agttgtttcc 1740 ttttgtcttt ttagggagca tactacta 1768 <210> 2 <211> 1287 <212> DNA <213> Seoul virus <400> 2 atggcaacta tggaagaaat ccagagagaa atcagtgcgc acgaggggca gcttgtaata 60 gcacgccaga aggtcaagga tgcagaaaag cagtatgaga aggatcctga tgacctaaat 120 aagagggcac tgcatgatcg ggagagtgtc gcagcttcaa tacaatcaaa aattgatgaa 180 ttgaagcgcc aacttgctga caggattgca gcagggaaga acatcgggca agaccgggat 240 cctacagggg tagagccggg tgatcatctc aaggaaagat cagcactaag ctacgggaat 300 acactggacc tgaatagcct tgacattgat gaacctacag gacagacagc tgattggttg 360 accataattg tctatttgac atcattcgtg gtcccgatca tcttgaaggc actgtacatg 420 ttgacaacaa gaggcaggca gacttcaaag gacaacaagg gaatgaggat cagattcaag 480 gatgacagct catatgaaga tgtcaatgga atcagaaagc ccaaacatct gtatgtgtca 540 atgccaaacg cccaatcaag catgaaggct gaagagataa cacctggaag attccgcact 600 gcagtatgtg ggctataccc tgcacagata aaggcaagga acatggtaag ccctgtcatg 660 agtgtagttg ggtttttggc actggcaaaa gactggacat ctagaattga agaatggctt 720 ggtgcaccct gcaagttcat ggcagagtct cccattgccg ggagcttatc tgggaatcct 780 gtgaatcgtg attatatcag acagagacaa ggtgcacttg cagggatgga gccaaaagaa 840 tttcaagctc tcaggcaaca ttcaaaggat gctggatgta cactggttga acatattgag 900 tcaccatcat caatatgggt atttgctggg gcccctgata ggtgcccacc gacatgcctg 960 tttgttggag ggatggctga gttaggtgct ttctttcta tacttcagga tatgaggaac 1020 acaatcatgg cttcaaagac tgtgggaaca gctgatgaaa agcttcgaaa gaagtcatca 1080 ttctatcaat catacctcag acgcacacaa tcaatgggaa tacaactgga ccagaggata 1140 attgttatgt ttatggttgc ctggggaaag gaggcagtgg acaactttca tctcggtgat 1200 gacatggatc cagagcttcg cagcctggct cagatcctga ttgaccagaa agtgaaggaa 1260 atctcaaacc aggaacctat gaaatta 1287 <210> 3 <211> 1287 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <223> Codon - optimized <400> 3 atggccacaa tggaagagat ccagagagag atcagcgccc acgagggaca gctggttatc 60 gccagacaga aagtgaagga cgccgagaag cagtacgaga aggaccccga cgatctgaac 120 aagagagccc tgcacgacag agaaagcgtg gccgcctcta tccagagcaa gatcgatgag 180 ctgaagagac agctggccga cagaatcgcc gctggcaaga atattggcca ggacagagat 240 cccacaggcg tggaacctgg cgatcacctg aaagagagaa gcgccctgtc ctatggcaac 300 accctggacc tgaacagcct ggacattgat gagcctaccg gccagacagc cgactggctg 360 acaatcattg tgtacctgac cagcttcgtg gtccccatca tcctgaaggc cctgtacatg 420 ctgaccacca gaggcagaca gaccagcaag gacaacaagg gcatgagaat ccggttcaag 480 gatgacagca gctacgagga cgtgaacggc attagaaagc ccaagcacct gtacgtgtcc 540 atgcctaacg ctcagagcag catgaaggcc gaggaaatca cccctggcag attcagaaca 600 gccgtgtgcg gactgtaccc cgctcagatc aaggccagaa acatggtgtc cccagtgatg 660 agcgtcgtgg gatttctggc cctggctaag gactggacca gcaggattga ggaatggctg 720 ggagcccctt gcaagtttat ggccgagtct cctatcgccg gcagcctgtc tggcaacccc 780 gtgaatagag actacatcag acagaggcag ggcgctctgg ccggaatgga acccaaagaa 840 tttcaggccc tgcggcagca ctctaaggat gccggatgta ccctggtgga acacattgag 900 agccccagca gcatctgggt tttcgctggc gctcctgata gatgccctcc tacctgtctg 960 tttgttggcg gaatggccga gctgggcgcc ttctttagca ttctgcagga catgcggaat 1020 accatcatgg ccagcaagac cgtgggcacc gccgatgaga agctgagaaa gaagtccagc 1080 ttctaccaga gctacctgcg gagaacccag agcatgggca ttcagctgga ccagagaatc 1140 atcgtgatgt tcatggtggc ctggggcaaa gaagccgtgg acaattttca cctgggcgac 1200 gacatggacc ccgagctgag atctctggcc cagatcctga tcgaccagaa agtcaaagag 1260 atctccaatc aagagcccat gaagctg 1287 <210> 4 <211> 429 <212> PRT <213> Seoul virus <400> 4 Met Ala Thr Met Glu Glu Ile Gln Arg Glu Ile Ser Ala His Glu Gly 1 5 10 15 Gln Leu Val Ile Ala Arg Gln Lys Val Lys Asp Ala Glu Lys Gln Tyr 20 25 30 Glu Lys Asp Pro Asp Asp Leu Asn Lys Arg Ala Leu His Asp Arg Glu 35 40 45 Ser Val Ala Ala Ser Ile Gln Ser Lys Ile Asp Glu Leu Lys Arg Gln 50 55 60 Leu Ala Asp Arg Ile Ala Ala Gly Lys Asn Ile Gly Gln Asp Arg Asp 65 70 75 80 Pro Thr Gly Val Glu Pro Gly Asp His Leu Lys Glu Arg Ser Ala Leu 85 90 95 Ser Tyr Gly Asn Thr Leu Asp Leu Asn Ser Leu Asp Ile Asp Glu Pro 100 105 110 Thr Gly Gln Thr Ala Asp Trp Leu Thr Ile Ile Val Tyr Leu Thr Ser Note: There was a small error in the original text where the sequence "Ser Val Ala Ala Ser Ile Gln Ser Lys Ile Asp Glu Leu Lys Arg Gln" was split into two lines in the original. I have combined it into one line in the translation for better readability while maintaining the integrity of the sequence. If you have any specific requirements regarding this, please let me know. 115 120 125 Phe Val Val Pro Ile Ile Leu Lys Ala Leu Tyr Met Leu Thr Thr Arg 130 135 140 Gly Arg Gln Thr Ser Lys Asp Asn Lys Gly Met Arg Ile Arg Phe Lys 145 150 155 160 Asp Asp Ser Ser Tyr Glu Asp Val Asn Gly Ile Arg Lys Pro Lys His 165 170 175 Leu Tyr Val Ser Met Pro Asn Ala Gln Ser Ser Met Lys Ala Glu Glu 180 185 190 Ile Thr Pro Gly Arg Phe Arg Thr Ala Val Cys Gly Leu Tyr Pro Ala 195 200 205 Gln Ile Lys Ala Arg Asn Met Val Ser Pro Val Met Ser Val Val Gly 210 215 220 Phe Leu Ala Leu Ala Lys Asp Trp Thr Ser Arg Ile Glu Glu Trp Leu 225 230 235 240 Gly Ala Pro Cys Lys Phe Met Ala Glu Ser Pro Ile Ala Gly Ser Leu 245 250 255 Ser Gly Asn Pro Val Asn Arg Asp Tyr Ile Arg Gln Arg Gln Gly Ala 260 265 270 Leu Ala Gly Met Glu Pro Lys Glu Phe Gln Ala Leu Arg Gln His Ser 275 280 285 Lys Asp Ala Gly Cys Thr Leu Val Glu His Ile Glu Ser Pro Ser Ser 290 295 300 Ile Trp Val Phe Ala Gly Ala Pro Asp Arg Cys Pro Pro Thr Cys Leu 305 310 315 320 Phe Val Gly Gly Met Ala Glu Leu Gly Ala Phe Phe Ser Ile Leu Gln 325 330 335 Asp Met Arg Asn Thr Ile Met Ala Ser Lys Thr Val Gly Thr Ala Asp 340 345 350 Glu Lys Leu Arg Lys Lys Ser Ser Phe Tyr Gln Ser Tyr Leu Arg Arg 355 360 365 Thr Gln Ser Met Gly Ile Gln Leu Asp Gln Arg Ile Ile Val Met Phe 370 375 380 Met Val Ala Trp Gly Lys Glu Ala Val Asp Asn Phe His Leu Gly Asp 385 390 395 400 Asp Met Asp Pro Glu Leu Arg Ser Leu Ala Gln Ile Leu Ile Asp Gln 405 410 415 Lys Val Lys Glu Ile Ser Asn Gln Glu Pro Met Lys Leu 420 425 <210> 5 <211> 1698 <212> DNA <213> Hantaan virus <400> 5 tagtagtaga ctccctaaag agctactaga acaacgatgg caactatgga ggaattgcag 60 agggaaatca atgcccatga gggtcaactg gtgatagcca ggcagaaggt gagggatgca 120 gaaaagcagt atgaaaagga tccagatgag ttaaacaaga gagcattgac agatcgagag 180 ggtgttgcag tatccattca agcaaagatt gatgagttaa agaggcaatt ggcagatagg 240 attgcaaccg ggaagaacct tggaaaggaa caagacccaa caggggtaga acctggagat 300 catctgaaag agagatcaat gctcagttat ggaaatgttc ttgacttaaa ccacctggat 360 attgatgagc caacaggaca gacagcagac tggctgggca ttgttatcta tctcacatcc 420 tttgttgtcc cgatacttct gaaagccctg tacatgttaa caacaagagg gaggcagacc 480 accaaggaca ataaaggaac tcggattcga ttcaaggatg atagctcctt cgaggatgtc 540 aatggcattc ggaagccgaa acatctatat gtgtccttac caaatgcaca gtcaagtatg 600 aaagcagaag agattacacc tggtagat agaacagcaa ttgtggact ttaccctgca 660 aaattaagg caacagat gattagtcca gtcatgagtg taatcggatt cttggctttg 720 gcaaagatt ggagtgaccg cattgagcag tggttaagtg aaccgtgtaa gcttcttcca 780 gatacagcag cagttagcct tcttggtggt cctgcaacca acagggacta tttacggcag 840 cgacaagtag cattgggcaa catggaaca aaagagtcta aggctatacg ccacatgca 900 gaagcagcag gctgtagtat gattgaggac attgagtcac catcatcaat atgggtgttt 960 gctggggcac cggaccgctg tccaccaca tgtctcttta ttgcaggtat ggctgagctt 1020 ggggcatttt tttccattct gcaggacatg cgaaataca ttggcattc gcagacagtt 1080 ggaacctctg aggagaagct acggaaaa tcctcattct atcagtctt tctcaggaga 1140 acacaatcaa tgggaataca actggatcag aggatattg tgcttcat ggtagcctgg 1200 gggaaagaag cagtggataa cttccaccta ggagatgata tggaccctga gctgcgaaca 1260 ctagcacaga gcctgattga tgttaaagtg aaggaattt ccaaccaga gcctttaaaa 1320 ctataatcag tgaatgtata accctcatta tgtgattatt atatactact gaatcattat 1380 caatcatatt tgcactatta ttatcagggg aattagtata tcagggtaag ggcacattta 1440 tgggtgggaa tcattactca gagggtgggt cagttaatcc gttgtgggtg ggtttagttc 1500 ctggctgcct taagtagcct ttttttgtat atatggatgt agatttcatt tgatctttaa 1560 actaatcttg ctctttttcc ttttcctcct gctttctctg cttactaaca acaacattct 1620 acctcaacac acaactacct caactaaact acctcatttg attgctcctt gattgtctct 1680 ttagggagtc tactacta 1698 <210> 6 <211> 1287 <212> DNA <213> Hantaan virus <400> 6 atggcaacta tggaggaatt gcagagggaa atcaatgccc atgagggtca actggtgata 60 gccaggcaga aggtgaggga tgcagaaaag cagtatgaaa aggatccaga tgagttaaac 120 aagagagcat tgacagatcg agagggtgtt gcagtatcca ttcaagcaaa gattgatgag 180 ttaaagaggc aattggcaga taggattgca accgggaaga accttggaaa ggaacaagac 240 ccaacagggg tagaacctgg agatcatctg aaagagagat caatgctcag ttatggaaat gttcttgact taaaccacct ggatattgat gagccaacag gacagacagc agactggctg ggcattgtta tctatctcac atcctttgtt gtcccgatac ttctgaaagc cctgtacatg 420 ttaacaacaa gagggaggca gaccaccaag gacaataag gaactcggat tcgattcaag gatgatagct ccttcgagga tgtcaatggc attcggaagc cgaaacatct atatgtgtcc ttaccaaatg cacagtcaag tatgaaagca gaagagatta cacctggtag atatagaaca gcaatttgtg gactttaccc tgcacaaatt aaggcaagac agatgattag tccagtcatg agtgtaatcg gattcttggc tttggcaaaa gattggagtg accgcattga gcagtggtta agtgaaccgt gtaagcttct tccagataca gcagcagtta gccttcttgg tggtcctgca 780 accaacaggg actatttacg gcagcgacaa gtagcattgg gcaacatgga aacaaaagag tctaaggcta tacgccaaca tgcagaagca gcaggctgta gtatgattga ggacattgag tcaccatcat caatatgggt gtttgctggg gcaccggacc gctgtccacc aacatgtctc tttattgcag gtatggctga gcttggggca tttttttcca tcctgcagga catgcgaaat 1020 acaattatgg catccaagac agttggaacc tctgaggaga agctacggaa gaaatcctca 1080 ttctatcagt cttatctcag gagaacacaa tcaatgggaa tacaactgga tcagaggata 1140 attgtgctct tcatggtagc ctgggggaaa gaagcagtgg ataacttcca cctaggagat 1200 gatatggacc ctgagctgcg aacactagca cagagcctga ttgatgttaa agtgaaggaa 1260 atttccaacc aagagccttt aaaacta 1287 <210> 7 <211> 429 <212> PRT <213> Hantaan virus <400> 7 Met Ala Thr Met Glu Glu Leu Gln Arg Glu Ile Asn Ala His Glu Gly 1 5 10 15 Gln Leu Val Ile Ala Arg Gln Lys Val Arg Asp Ala Glu Lys Gln Tyr 20 25 30 Glu Lys Asp Pro Asp Glu Leu Asn Lys Arg Ala Leu Thr Asp Arg Glu 35 40 45 Gly Val Ala Val Ser Ile Gln Ala Lys Ile Asp Glu Leu Lys Arg Gln 50 55 60 Leu Ala Asp Arg Ile Ala Thr Gly Lys Asn Leu Gly Lys Glu Gln Asp 65 70 75 80 Pro Thr Gly Val Glu Pro Gly Asp His Leu Lys Glu Arg Ser Met Leu 85 90 95 Ser Tyr Gly Asn Val Leu Asp Leu Asn His Leu Asp Ile Asp Glu Pro 100 105 110 Thr Gly Gln Thr Ala Asp Trp Leu Gly Ile Val Ile Tyr Leu Thr Ser 115 120 125 Phe Val Val Pro Ile Leu Leu Lys Ala Leu Tyr Met Leu Thr Thr Arg 130 135 140 Gly Arg Gln Thr Thr Lys Asp Asn Lys Gly Thr Arg Ile Arg Phe Lys 145 150 155 160 Asp Asp Ser Ser Phe Glu Asp Val Asn Gly Ile Arg Lys Pro Lys His 165 170 175 Leu Tyr Val Ser Leu Pro Asn Ala Gln Ser Ser Met Lys Ala Glu Glu 180 185 190 Ile Thr Pro Gly Arg Tyr Arg Thr Ala Ile Cys Gly Leu Tyr Pro Ala 195 200 205 Gln Ile Lys Ala Arg Gln Met Ile Ser Pro Val Met Ser Val Ile Gly 210 215 220 Phe Leu Ala Leu Ala Lys Asp Trp Ser Asp Arg Ile Glu Gln Trp Leu 225 230 235 240 Ser Glu Pro Cys Lys Leu Leu Pro Asp Thr Ala Ala Val Ser Leu Leu Leu 245 250 255 Gly Gly Pro Ala Thr Asn Arg Asp Tyr Leu Arg Gln Arg Gln Val Ala 260 265 270 Leu Gly Asn Met Glu Thr Lys Glu Ser Lys Ala Ile Arg Gln His Ala 275 280 285 Glu Ala Ala Gly Cys Ser Met Ile Glu Asp Ile Glu Ser Pro Ser Ser 290 295 300 Ile Trp Val Phe Ala Gly Ala Pro Asp Arg Cys Pro Pro Thr Cys Leu 305 310 315 320 Phe Ile Ala Gly Met Ala Glu Leu Gly Ala Phe Phe Ser Ile Leu Gln 325 330 335 Asp Met Arg Asn Thr Ile Met Ala Ser Lys Thr Val Gly Thr Ser Glu 340 345 350 Glu Lys Leu Arg Lys Lys Ser Ser Phe Tyr Gln Ser Tyr Leu Arg Arg 355 360 365 Thr Gln Ser Met Gly Ile Gln Leu Asp Gln Arg Ile Ile Val Leu Phe 370 375 380 Met Val Ala Trp Gly Lys Glu Ala Val Asp Asn Phe His Leu Gly Asp 385 390 395 400 Asp Met Asp Pro Glu Leu Arg Thr Leu Ala Gln Ser Leu Ile Asp Val 405 410 415 Lys Val Lys Glu Ile Ser Asn Gln Glu Pro Leu Lys Leu 420 425 <210> 8 <211> 1005 <212> DNA <213> Hantaan virus <400> 8 atgctcagtt atggaaatgt tcttgactta aaccacctgg atattgatga gccaacagga 60 cagacagcag actggctggg cattgttatc tatctcacat cctttgttgt cccgatactt 120 ctgaaagccc tgtacatgtt aacaacaaga gggaggcaga ccaccaagga caataaagga 180 actcggattc gattcaagga tgatagctcc ttcgaggatg tcaatggcat tcggaagccg 240 aaacatctat atgtgtcctt accaaatgca cagtcaagta tgaaagcaga agagattaca 300 cctggtagat atagaacagc aatttgtgga ctttaccctg cacaaattaa ggcaagacag 360 atgattagtc cagtcatgag tgtaatcgga ttcttggctt tggcaaaaga ttggagtgac 420 cgcattgagc agtggttaag tgaaccgtgt aagcttcttc cagatacagc agcagttagc 480 cttcttggtg gtcctgcaac caacagggac tatttacggc agcgacaagt agcattgggc 540 aacatggaaa caaaagagtc taaggctata cgccaacatg cagaagcagc aggctgtagt 600 atgattgagg acattgagtc accatcatca atatgggtgt ttgctggggc accggaccgc 660 tgtccaccaa catgtctctt tattgcaggt atggctgagc ttggggcatt tttttccatc 720 ctgcaggaca tgcgaaatac aattatggca tccaagacag ttggaacctc tgaggagaag 780 ctacggaaga aatcctcatt ctatcagtct tatctcagga gaacacaatc aatgggaata 840 caactggatc agaggataat tgtgctcttc atggtagcct gggggaaaga agcagtggat 900 aacttccacc taggagatga tatggaccct gagctgcgaa cactagcaca gagcctgatt 960 gatgttaaag tgaaggaaat ttccaaccaa gagcctttaa aacta 1005 <210> 9 <211> 1005 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <223> Codon-optimized <400> 9 atgctgagct acggcaacgt gctggatctg aaccacctgg atatcgacga gccaacagga 60 cagaccgctg attggctggg catcgtgatc tacctgacct cctttgtggt gcctattctg 120 ctcaaagccc tctatatgct gacaacacgc ggaaggcaga ccaccaaaga taacaaaggc 180 acccggatca ggtttaagga cgacagctcc tttgaggatg tcaacggcat ccggaaacct 240 aagcacctct atgtgtctct gcccaatgca cagtcctcca tgaaggcaga agagatcaca 300 ccaggccggt acagaaccgc catctgtgga ctgtatcctg cacaaatcaa agcccggcag 360 atgatcagcc ccgtgatgtc cgttatcgga ttcctggctc tggccaaaga ttggagcgac 420 aggatcgagc agtggctgag cgagccttgc aagctgcttc ctgatacagc cgctgtgtca 480 ctgcttggcg gccctgccac aaacagagat tacctgagac agagacaggt ggcactgggc 540 aacatggaaa caaaagagag caaggccatc cggcagcatg ccgaagctgc tggctgtagc 600 atgatcgagg atatcgagtc ccctagctcc atttgggtgt tcgcaggggc cccagataga 660 tgtccaccaa catgcctgtt cattgccggc atggctgaac tgggagcttt tttcagcatc 720 ctccaggata tgcgcaacac gattatggcc tccaagacag tgggaaccag cgaggaaaag 780 ctgcggaaga aaagcagctt ttaccagtct tacctgaggc ggacccagtc catggggatc 840 caactggatc agcggatcat tgtgctgttt atggtcgctt ggggaaaaga ggctgtcgat 900 aacttccacc tgggagatga tatggatcct gaactgcgga ccctggctca gtccctgatc 960 gatgtgaaag tgaaagaaat tagtaatcaa gaacccctca agctg 1005 <210> 10<00已翻译内容1138><211> 335 <212> PRT <213> Hantaan virus[[ID=已翻译内容17]] <400> 10 Met Leu Ser Tyr Gly Asn Val Leu Asp Leu Asn His Leu Asp Ile Asp 1 5 10 15 Glu Pro Thr Gly Gln Thr Ala Asp Trp Leu Gly Ile Val Ile Tyr Leu 20 25 30 Thr Ser Phe Val Val Pro Ile Leu Leu Lys Ala Leu Tyr Met Leu Thr 35 40 45 Thr Arg Gly Arg Gln Thr Thr Lys Asp Asn Lys Gly Thr Arg Ile Arg[[ID=已翻译内容33]] 50 55 60 Phe Lys Asp Asp Ser Ser Phe Glu Asp Val Asn Gly Ile Arg Lys Pro 65 70 75 80 请注意,你提供的原始文本中存在一些重复的标签编号,我按照原样保留了。同时,对于一些不太明确的标签含义(如<210>、<211>、<212>、<213>、<400>等),我直接进行了翻译,你可以根据实际情况进一步确认其确切含义。 Lys His Leu Tyr Val Ser Leu Pro Asn Ala Gln Ser Ser Met Lys Ala 85 90 95 Glu Glu Ile Thr Pro Gly Arg Tyr Arg Thr Ala Ile Cys Gly Leu Tyr 100 105 110 Pro Ala Gln Ile Lys Ala Arg Gln Met Ile Ser Pro Val Met Ser Val 115 120 125 Ile Gly Phe Leu Ala Leu Ala Lys Asp Trp Ser Asp Arg Ile Glu Gln 130 135 140 Trp Leu Ser Glu Pro Cys Lys Leu Leu Pro Asp Thr Ala Ala Val Ser 145 150 155 160 Leu Leu Gly Gly Pro Ala Thr Asn Arg Asp Tyr Leu Arg Gln Arg Gln 165 170 175 Val Ala Leu Gly Asn Met Glu Thr Lys Glu Ser Lys Ala Ile Arg Gln 180 185 190 His Ala Glu Ala Ala Gly Cys Ser Met Ile Glu Asp Ile Glu Ser Pro 195 200 205 Ser Ser Ile Trp Val Phe Ala Gly Ala Pro Asp Arg Cys Pro Pro Thr 210 215 220 Cys Leu Phe Ile Ala Gly Met Ala Glu Leu Gly Ala Phe Phe Ser Ile 225 230 235 240 Leu Gln Asp Met Arg Asn Thr Ile Met Ala Ser Lys Thr Val Gly Thr 245 250 255 Ser Glu Glu Lys Leu Arg Lys Lys Ser Ser Phe Tyr Gln Ser Tyr Leu 260 265 270 Arg Arg Thr Gln Ser Met Gly Ile Gln Leu Asp Gln Arg Ile Ile Val 275 280 285 Leu Phe Met Val Ala Trp Gly Lys Glu Ala Val Asp Asn Phe His Leu 290 295 300 Gly Asp Asp Met Asp Pro Glu Leu Arg Thr Leu Ala Gln Ser Leu Ile 305 310 315 320 Asp Val Lys Val Lys Glu Ile Ser Asn Gln Glu Pro Leu Lys Leu 325 330 335 <210> 11 <211> 79 <212> PRT <213> artificial sequence(Artificial) <220> <221> PEPTIDE <223> Peptides 4 <400> 11 Leu Tyr Pro Ala Gln Ile Lys Ala Arg Asn Met Val Ser Pro Val Met 1 5 10 15 Ser Val Val Gly Phe Leu Ala Leu Ala Lys Asp Trp Thr Ser Arg Ile 20 25 30 Glu Glu Trp Leu Gly Ala Pro Cys Lys Phe Met Ala Glu Ser Pro Ile 35 40 45 Ala Gly Ser Leu Ser Gly Asn Pro Val Asn Arg Asp Tyr Ile Arg Gln 50 55 60 Arg Gln Gly Ala Leu Ala Gly Met Glu Pro Lys Glu Phe Gln Ala 65 70 75 <210> 12 <211> 79 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <223> Peptide Library 9 <400> 12 Ile Lys Ala Arg Gln Met Ile Ser Pro Val Met Ser Val Ile Gly Phe 1 5 10 15 Leu Ala Leu Ala Lys Asp Trp Ser Asp Arg Ile Glu Gln Trp Leu Ser 20 25 30 Glu Pro Cys Lys Leu Leu Pro Asp Thr Ala Ala Val Ser Leu Leu Gly 35 40 45 Gly Pro Ala Thr Asn Arg Asp Tyr Leu Arg Gln Arg Gln Val Ala Leu 50 55 60 Gly Asn Met Glu Thr Lys Glu Ser Lys Ala Ile Arg Gln His Ala 65 70 75 <210> 13 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library subregion <400> 13 Ser Pro Val Met Ser Val Val Gly Phe Leu Ala Leu Ala Lys Asp 1 5 10 15 <210> 14 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library subregion <400> 14 Pro Val Met Ser Val Ile Gly Phe Leu Ala Leu Ala Lys Asp Trp 1 5 10 15 <210> 15 <211> 237 <212> DNA <213> Seoul virus <400> 15 ctataccctg cacagataaa ggcaaggaac atggtaagcc ctgtcatgag tgtagttggg 60 tttttggcac tggcaaaaga ctggacatct agaattgaag aatggcttgg tgcaccctgc 120 aagttcatgg cagagtctcc cattgccggg agctttatctg ggaatcctgt gaatcgtgat 180 tatatcagac agagacaagg tgcacttgca gggatggagc caaaagaatt tcaagct 237 <210> 16 <211> 237 <212> DNA <213> Seoul virus <400> 16 ctataccctg cacagataaa ggcaaggaac atggtaagcc ctgtcatgag tgtagttggg 60 tttttggcac tggcaaaaga ctggacatct agaattgaag aatggcttgg tgcaccctgc 120 aagttcatgg cagagtctcc cattgccggg agcttatctg ggaatcctgt gaatcgtgat 180 tatatcagac agagacaagg tgcacttgca gggatggagc caaaagaatt tcaagct 237 <210> 17 <211> 237 <212> DNA <213> Artificial <220> <221> misc_feature <223> Codon-optimized <400> 17 ctgtaccccg ctcagatcaa ggccagaaac atggtgtccc cagtgatgag cgtcgtggga 60 tttctggccc tggctaagga ctggaccagc aggattgagg aatggctggg agccccttgc 120 aagtttatgg ccgagtctcc tatcgccggc agcctgtctg gcaaccccgt gaatagagac 180 tacatcagac agaggcaggg cgctctggcc ggaatggaac ccaaagaatt tcaggcc 237 <210> 18 <211> 237 <212> DNA <213> Hantaan virus <400> 18 attaaggcaa gacagatgat tagtccagtc atgagtgtaa tcggattctt ggctttggca 60 aaagattgga gtgaccgcat tgagcagtgg ttaagtgaac cgtgtaagct tcttccagat 120 acagcagcag ttagccttct tggtggtcct gcaaccaaca gggactattt acggcagcga 180 caagtagcat tgggcaacat ggaaacaaaa gagtctaagg ctatacgcca acatgca 237 <210> 19 <211> 237 <212> DNA <213> Hantaan virus <400> 19 attaaggcaa gacagatgat tagtccagtc atgagtgtaa tcggattctt ggctttggca 60 aaagattgga gtgaccgcat tgagcagtgg ttaagtgaac cgtgtaagct tcttccagat 120 acagcagcag ttagccttct tggtggtcct gcaaccaaca gggactattt acggcagcga 180 caagtagcat tgggcaacat ggaaacaaaa gagtctaagg ctatacgcca acatgca 237 <210> 20 <211> 237 <212> DNA <213> Hantaan virus <400> 20 attaaggcaa gacagatgat tagtccagtc atgagtgtaa tcggattctt ggctttggca 60 aaagattgga gtgaccgcat tgagcagtgg ttaagtgaac cgtgtaagct tcttccagat 120 acagcagcag ttagccttct tggtggtcct gcaaccaaca gggactattt acggcagcga 180 caagtagcat tgggcaacat ggaaacaaaa gagtctaagg ctatacgcca acatgca 237 <210> 21 <211> 237 <212> DNA <213> Artificial sequence <220> <221> misc_feature <223> Codon-optimized <400> 21 atcaaagccc ggcagatgat cagccccgtg atgtccgtta tcggattcct ggctctggcc 60 aaagattgga gcgacaggat cgagcagtgg ctgagcgagc cttgcaagct gcttcctgat 120 acagccgctg tgtcactgct tggcggccct gccacaaaca gagattacct gagacagaga 180 caggtggcac tgggcaacat ggaaacaaaa gagagcaagg ccatccggca gcatgcc 237 <210> 22 <211> 45 <212> DNA <213> Seoul virus <400> 22 agccctgtca tgagtgtagt tgggtttttg gcactggcaa aagac 45 <210> twenty three <211> 45 <212> DNA <213> Seoul virus <400> twenty three agccctgtca tgagtgtagt tgggtttttg gcactggcaa aagac 45 <210> twenty four <211> 45 <212> DNA <213> Artificial <220> <221> misc_feature <223> Codon-optimized <400> twenty four tccccagtga tgagcgtcgt gggatttctg gccctggcta aggac 45 <210> 25 <211> 45 <212> DNA <213> Hantaan virus <400> 25 ccagtcatga gtgtaatcgg attcttggct ttggcaaaag attgg 45 <210> 26 <211> 45 <212> DNA <213> Hantaan virus <400> 26 ccagtcatga gtgtaatcgg attcttggct ttggcaaaag attgg 45 <210> 27 <211> 45 <212> DNA <213> Hantaan virus <400> 27 ccagtcatga gtgtaatcgg attcttggct ttggcaaaag attgg 45 <210> 28 <211> 45 <212> DNA <213> Artificial <220> <221> misc_feature <223> Codon-optimized <400> 28 cccgtgatgt ccgttatcgg attcctggct ctggccaaag attgg 45 <210> 29 <211> 2292 <212> DNA <213> Artificial <220> <221> misc_structure <223> chimeric sequence <400> 29 atggccacaa tggaagagat ccagagagag atcagcgccc acgagggaca gctggttatc 60 gccagacaga aagtgaagga cgccgagaag cagtacgaga aggaccccga cgatctgaac 120 aagagagccc tgcacgacag agaaagcgtg gccgcctcta tccagagcaa gatcgatgag 180 ctgaagagac agctggccga cagaatcgcc gctggcaaga atattggcca ggacagagat 240 cccacaggcg tggaacctgg cgatcacctg aaagagagaa gcgccctgtc ctatggcaac 300 accctggacc tgaacagcct ggacattgat gagcctaccg gccagacagc cgactggctg 360 acaatcattg tgtacctgac cagcttcgtg gtccccatca tcctgaaggc cctgtacatg 420 ctgaccacca gaggcagaca gaccagcaag gacaacaagg gcatgagaat ccggttcaag 480 gatgacagca gctacgagga cgtgaacggc attagaaagc ccaagcacct gtacgtgtcc 540 atgcctaacg ctcagagcag catgaaggcc gaggaaatca cccctggcag attcagaaca 600 gccgtgtgcg gactgtaccc cgctcagatc aaggccagaa acatggtgtc cccagtgatg 660 agcgtcgtgg gatttctggc cctggctaag gactggacca gcaggattga ggaatggctg 720 ggagcccctt gcaagtttat ggccgagtct cctatcgccg gcagcctgtc tggcaacccc 780 gtgaatagag actacatcag acagaggcag ggcgctctgg ccggaatgga acccaaagaa 840 tttcaggccc tgcggcagca ctctaaggat gccggatgta ccctggtgga acacattgag 900 agccccagca gcatctgggt tttcgctggc gctcctgata gatgccctcc tacctgtctg 960 tttgttggcg gaatggccga gctgggcgcc ttctttagca ttctgcagga catgcggaat 1020 accatcatgg ccagcaagac cgtgggcacc gccgatgaga agctgagaaa gaagtccagc 1080 ttctaccaga gctacctgcg gagaacccag agcatgggca ttcagctgga ccagagaatc 1140 atcgtgatgt tcatggtggc ctggggcaaa gaagccgtgg acaattttca cctgggcgac 1200 gacatggacc ccgagctgag atctctggcc cagatcctga tcgaccagaa agtcaaagag 1260 atctccaatc aagagcccat gaagctgatg ctgagctacg gcaacgtgct ggatctgaac 1320 cacctggata tcgacgagcc aacaggacag accgctgatt ggctgggcat cgtgatctac 1380 ctgacctcct ttgtggtgcc tattctgctc aaagccctct atatgctgac aacacgcgga 1440 aggcagacca ccaaagataa caaaggcacc cggatcaggt ttaaggacga cagctccttt 1500 gaggatgtca acggcatccg gaaacctaag cacctctatg tgtctctgcc caatgcacag 1560 tcctccatga aggcagaaga gatcacacca ggccggtaca gaaccgccat ctgtggactg 1620 tatcctgcac aaatcaaagc ccggcagatg atcagccccg tgatgtccgt tatcggattc 1680 ctggctctgg ccaaagattg gagcgacagg atcgagcagt ggctgagcga gccttgcaag 1740 ctgcttcctg atacagccgc tgtgtcactg cttggcggcc ctgccacaaa cagagattac 1800 ctgagacaga gacaggtggc actgggcaac atggaaacaa aagagagcaa ggccatccgg 1860 cagcatgccg aagctgctgg ctgtagcatg atcgaggata tcgagtcccc tagctccatt 1920 tgggtgttcg caggggcccc agatagatgt ccaccaacat gcctgttcat tgccggcatg 1980 gctgaactgg gagctttttt cagcatcctc caggatatgc gcaacacgat tatggcctcc 2040 aagacagtgg gaaccagcga ggaaaagctg cggaagaaaa gcagctttta ccagtcttac 2100 ctgaggcgga cccagtccat ggggatccaa ctggatcagc ggatcattgt gctgtttatg 2160 gtcgcttggg gaaaagaggc tgtcgataac ttccacctgg gagatgatat ggatcctgaa 2220 ctgcggaccc tggctcagtc cctgatcgat gtgaaagtga aagaaattag taatcaagaa 2280 cccctcaagc tg 2292 <210> 30 <211> 2292 <212> DNA <213> Artificial Sequence <220> <221> misc_structure <223> Chimeric Sequence <400> 30 atgctgagct acggcaacgt gctggatctg aaccacctgg atatcgacga gccaacagga 60 cagaccgctg attggctggg catcgtgatc tacctgacct cctttgtggt gcctattctg 120 ctcaaagccc tctatatgct gacaacacgc ggaaggcaga ccaccaaaga taacaaaggc 180 acccggatca ggtttaagga cgacagctcc tttgaggatg tcaacggcat ccggaaacct 240 aagcacctct atgtgtctct gcccaatgca cagtcctcca tgaaggcaga agagatcaca 300 ccaggccggt acagaaccgc catctgtgga ctgtatcctg cacaaatcaa agcccggcag 360 atgatcagcc ccgtgatgtc cgttatcgga ttcctggctc tggccaaaga ttggagcgac 420 aggatcgagc agtggctgag cgagccttgc aagctgcttc ctgatacagc cgctgtgtca 480 ctgcttggcg gccctgccac aaacagagat tacctgagac agagacaggt ggcactgggc 540 aacatggaaa caaaagagag caaggccatc cggcagcatg ccgaagctgc tggctgtagc 600 atgatcgagg atatcgagtc ccctagctcc atttgggtgt tcgcaggggc cccagataga 660 tgtccaccaa catgcctgtt cattgccggc atggctgaac tgggagcttt tttcagcatc 720 ctccaggata tgcgcaacac gattatggcc tccaagacag tgggaaccag ccgagaaaag 780 ctgcggagaaa aaagcagctt ttaccagtct tacctgaggc ggacccagtc catggggatc 840 caactggatc agcggatcat tgtgctgttt atggtcgctt ggggaaaaga ggctgtcgat 900 aacttccacc tgggagatga tatggatcct gaactgcgga ccctggctca gtccctgatc 960 gatgtgaaag tgaaagaaat tagtaatcaa gaacccctca agctgatggc cacaatggaa 1020 gagatccaga gagagatcag cgcccacgag ggacagctgg ttatcgccag agaaagtg 1080 aagcagccg agaagcagta cgagaaggac cccgacgatc tgaacaagag agccctgcac 1140 gacgagaa gcgtggccgc ctctatccag agcaagatcg atgagctgaa gagagagctg 1200 gccgacagaa tcgccgctgg caagaatatt ggcaggaca gagatcccac aggcgtggaa 1260 cctggcgatc acctgaaaga gagaagcgcc ctgtcctatg gcaacaccct ggacctgaac 1320 agcctggaca ttgatgagcc taccggccag acagccgact ggctgacaat cattgtgtac 1380 ctgaccagct tcgtggtccc catcatcctg aaggccctgt acatgctgac caccagaggc 1440 agacagacca gcaaggacaa caagggcatg agaatccggt tcaaggatga cagcagctac gaggacgtga acggcattag aaagcccaag cacctgtacg tgtccatgcc taacgctcag agcagcatga aggccgagga aatcacccct ggcagattca gaacagccgt gtgcggactg taccccgctc agatcaaggc cagaaacatg gtgtccccag tgatgagcgt cgtgggattt ctggccctgg ctaaggactg gaccagcagg attgaggaat ggctggggagc cccttgcaag tttatggccg agtctcctat cgccggcagc ctgtctggca accccgtgaa tagagactac atcagacaga ggcagggcgc tctggccgga atggaaccca aagaatttca ggccctgcgg cagcactcta aggatgccgg atgtaccctg gtggaacaca ttgagagccc cagcagcatc tgggttttcg ctggcgctcc tgatagatgc cctcctacct gtctgtttgt tggcggaatg 1980. gccgagctgg gcgccttctt tagcattctg caggacatgc ggaataccat catggccagc aagaccgtgg gcaccgccga tgagaagctg agaaagaagt ccagcttcta ccagagctac ctgcggagaa cccagagcat gggcattcag ctggaccaga gaatcatcgt gatgttcatg gtggcctggg gcaaagaagc cgtggacaat tttcacctgg gcgacgacat ggaccccgag 2220 ctgagatctc tggcccagat cctgatcgac cagaaagtca aagagatctc caatcaagag 2280 cccatgaagc tg 2292 <210> 31 <211> 764 <212> PRT <213> Artificial <220> <221> DOMAIN <223> Chimeric protein <400> 31 Met Ala Thr Met Glu Glu Ile Gln Arg Glu Ile Ser Ala His Glu Gly 1 5 10 15 Gln Leu Val Ile Ala Arg Gln Lys Val Lys Asp Ala Glu Lys Gln Tyr 20 25 30 Glu Lys Asp Pro Asp Asp Leu Asn Lys Arg Ala Leu His Asp Arg Glu 35 40 45 Ser Val Ala Ala Ser Ile Gln Ser Lys Ile Asp Glu Leu Lys Arg Gln 50 55 60 Leu Ala Asp Arg Ile Ala Ala Gly Lys Asn Ile Gly Gln Asp Arg Asp 65 70 75 80 Pro Thr Gly Val Glu Pro Gly Asp His Leu Lys Glu Arg Ser Ala Leu 85 90 95 Ser Tyr Gly Asn Thr Leu Asp Leu Asn Ser Leu Asp Ile Asp Glu Pro 100 105 110 Thr Gly Gln Thr Ala Asp Trp Leu Thr Ile Ile Val Tyr Leu Thr Ser 115 120 125 Phe Val Val Pro Ile Ile Leu Lys Ala Leu Tyr Met Leu Thr Thr Arg 130 135 140 Gly Arg Gln Thr Ser Lys Asp Asn Lys Gly Met Arg Ile Arg Phe Lys 145 150 155 160 Asp Asp Ser Ser Tyr Glu Asp Val Asn Gly Ile Arg Lys Pro Lys His 165 170 175 Leu Tyr Val Ser Met Pro Asn Ala Gln Ser Ser Met Lys Ala Glu Glu 180 185 190 Ile Thr Pro Gly Arg Phe Arg Thr Ala Val Cys Gly Leu Tyr Pro Ala 195 200 205 Gln Ile Lys Ala Arg Asn Met Val Ser Pro Val Met Ser Val Val Gly 210 215 220 Phe Leu Ala Leu Ala Lys Asp Trp Thr Ser Arg Ile Glu Glu Trp Leu 225 230 235 240 Gly Ala Pro Cys Lys Phe Met Ala Glu Ser Pro Ile Ala Gly Ser Leu 245 250 255 Ser Gly Asn Pro Val Asn Arg Asp Tyr Ile Arg Gln Arg Gln Gly Ala 260 265 270 Leu Ala Gly Met Glu Pro Lys Glu Phe Gln Ala Leu Arg Gln His Ser 275 280 285 Lys Asp Ala Gly Cys Thr Leu Val Glu His Ile Glu Ser Pro Ser Ser 290 295 300 Ile Trp Val Phe Ala Gly Ala Pro Asp Arg Cys Pro Pro Thr Cys Leu 305 310 315 320 Phe Val Gly Gly Met Ala Glu Leu Gly Ala Phe Phe Ser Ile Leu Gln 325 330 335 Asp Met Arg Asn Thr Ile Met Ala Ser Lys Thr Val Gly Thr Ala Asp 340 345 350 Glu Lys Leu Arg Lys Lys Ser Ser Phe Tyr Gln Ser Tyr Leu Arg Arg 355 360 365 Thr Gln Ser Met Gly Ile Gln Leu Asp Gln Arg Ile Ile Val Met Phe 370 375 380 Met Val Ala Trp Gly Lys Glu Ala Val Asp Asn Phe His Leu Gly Asp 385 390 395 400 Asp Met Asp Pro Glu Leu Arg Ser Leu Ala Gln Ile Leu Ile Asp Gln 405 410 415 Lys Val Lys Glu Ile Ser Asn Gln Glu Pro Met Lys Leu Met Leu Ser 420 425 430 Tyr Gly Asn Val Leu Asp Leu Asn His Leu Asp Ile Asp Glu Pro Thr 435 440 445 Gly Gln Thr Ala Asp Trp Leu Gly Ile Val Ile Tyr Leu Thr Ser Phe 450 455 460 Val Val Pro Ile Leu Leu Lys Ala Leu Tyr Met Leu Thr Thr Arg Gly 465 470 475 480 Arg Gln Thr Thr Lys Asp Asn Lys Gly Thr Arg Ile Arg Phe Lys Asp 485 490 495 Asp Ser Ser Phe Glu Asp Val Asn Gly Ile Arg Lys Pro Lys His Leu 500 505 510 Tyr Val Ser Leu Pro Asn Ala Gln Ser Ser Met Lys Ala Glu Glu Ile 515 520 525 Thr Pro Gly Arg Tyr Arg Thr Ala Ile Cys Gly Leu Tyr Pro Ala Gln 530 535 540 Ile Lys Ala Arg Gln Met Ile Ser Pro Val Met Ser Val Ile Gly Phe 545 550 555 560 Leu Ala Leu Ala Lys Asp Trp Ser Asp Arg Ile Glu Gln Trp Leu Ser 565 570 575 Glu Pro Cys Lys Leu Leu Pro Asp Thr Ala Ala Val Ser Leu Leu Gly 580 585 590 Gly Pro Ala Thr Asn Arg Asp Tyr Leu Arg Gln Arg Gln Val Ala Leu 595 600 605 Gly Asn Met Glu Thr Lys Glu Ser Lys Ala Ile Arg Gln His Ala Glu 610 615 620 Ala Ala Gly Cys Ser Met Ile Glu Asp Ile Glu Ser Pro Ser Ser Ile 625 630 635 640 Trp Val Phe Ala Gly Ala Pro Asp Arg Cys Pro Pro Thr Cys Leu Phe 645 650 655 Ile Ala Gly Met Ala Glu Leu Gly Ala Phe Phe Ser Ile Leu Gln Asp 660 665 670 Met Arg Asn Thr Ile Met Ala Ser Lys Thr Val Gly Thr Ser Glu Glu 675 680 685 Lys Leu Arg Lys Lys Ser Ser Phe Tyr Gln Ser Tyr Leu Arg Arg Thr 690 695 700 Gln Ser Met Gly Ile Gln Leu Asp Gln Arg Ile Ile Val Leu Phe Met 705 710 715 720 Val Ala Trp Gly Lys Glu Ala Val Asp Asn Phe His Leu Gly Asp Asp 725 730 735 Met Asp Pro Glu Leu Arg Thr Leu Ala Gln Ser Leu Ile Asp Val Lys 740 745 750 Val Lys Glu Ile Ser Asn Gln Glu Pro Leu Lys Leu 755 760 <210> 32 <211> 764 <212> PRT <213> Artificial <220> <221> DOMAIN <223> Chimeric protein <400> 32 Met Leu Ser Tyr Gly Asn Val Leu Asp Leu Asn His Leu Asp Ile Asp 1 5 10 15 Glu Pro Thr Gly Gln Thr Ala Asp Trp Leu Gly Ile Val Ile Tyr Leu 20 25 30 Thr Ser Phe Val Val Pro Ile Leu Leu Lys Ala Leu Tyr Met Leu Thr 35 40 45 Thr Arg Gly Arg Gln Thr Thr Lys Asp Asn Lys Gly Thr Arg Ile Arg 50 55 60 Phe Lys Asp Asp Ser Ser Phe Glu Asp Val Asn Gly Ile Arg Lys Pro 65 70 75 80 Lys His Leu Tyr Val Ser Leu Pro Asn Ala Gln Ser Ser Met Lys Ala 85 90 95 Glu Glu Ile Thr Pro Gly Arg Tyr Arg Thr Ala Ile Cys Gly Leu Tyr 100 105 110 Pro Ala Gln Ile Lys Ala Arg Gln Met Ile Ser Pro Val Met Ser Val 115 120 125 Ile Gly Phe Leu Ala Leu Ala Lys Asp Trp Ser Asp Arg Ile Glu Gln 130 135 140 Trp Leu Ser Glu Pro Cys Lys Leu Leu Pro Asp Thr Ala Ala Val Ser 145 150 155 160 Leu Leu Gly Gly Pro Ala Thr Asn Arg Asp Tyr Leu Arg Gln Arg Gln 165 170 175 Val Ala Leu Gly Asn Met Glu Thr Lys Glu Ser Lys Ala Ile Arg Gln 180 185 190 His Ala Glu Ala Ala Gly Cys Ser Met Ile Glu Asp Ile Glu Ser Pro 195 200 205 Ser Ser Ile Trp Val Phe Ala Gly Ala Pro Asp Arg Cys Pro Pro Thr 210 215 220 Cys Leu Phe Ile Ala Gly Met Ala Glu Leu Gly Ala Phe Phe Ser Ile 225 230 235 240 Leu Gln Asp Met Arg Asn Thr Ile Met Ala Ser Lys Thr Val Gly Thr 245 250 255 Ser Glu Glu Lys Leu Arg Lys Lys Ser Ser Phe Tyr Gln Ser Tyr Leu 260 265 270 Arg Arg Thr Gln Ser Met Gly Ile Gln Leu Asp Gln Arg Ile Ile Val 275 280 285 Leu Phe Met Val Ala Trp Gly Lys Glu Ala Val Asp Asn Phe His Leu 290 295 300 Gly Asp Asp Met Asp Pro Glu Leu Arg Thr Leu Ala Gln Ser Leu Ile 305 310 315 320 Asp Val Lys Val Lys Glu Ile Ser Asn Gln Glu Pro Leu Lys Leu Met 325 330 335 Ala Thr Met Glu Glu Ile Gln Arg Glu Ile Ser Ala His Glu Gly Gln 340 345 350 Leu Val Ile Ala Arg Gln Lys Val Lys Asp Ala Glu Lys Gln Tyr Glu 355 360 365 Lys Asp Pro Asp Asp Leu Asn Lys Arg Ala Leu His Asp Arg Glu Ser 370 375 380 Val Ala Ala Ser Ile Gln Ser Lys Ile Asp Glu Leu Lys Arg Gln Leu 385 390 395 400 Ala Asp Arg Ile Ala Ala Gly Lys Asn Ile Gly Gln Asp Arg Asp Pro 405 410 415 Thr Gly Val Glu Pro Gly Asp His Leu Lys Glu Arg Ser Ala Leu Ser 420 425 430 Tyr Gly Asn Thr Leu Asp Leu Asn Ser Leu Asp Ile Asp Glu Pro Thr 435 440 445 Gly Gln Thr Ala Asp Trp Leu Thr Ile Ile Val Tyr Leu Thr Ser Phe 450 455 460 Val Val Pro Ile Ile Leu Lys Ala Leu Tyr Met Leu Thr Thr Arg Gly 465 470 475 480 Arg Gln Thr Ser Lys Asp Asn Lys Gly Met Arg Ile Arg Phe Lys Asp 485 490 495 Asp Ser Ser Tyr Glu Asp Val Asn Gly Ile Arg Lys Pro Lys His Leu 500 505 510 Tyr Val Ser Met Pro Asn Ala Gln Ser Ser Met Lys Ala Glu Glu Ile 515 520 525 Thr Pro Gly Arg Phe Arg Thr Ala Val Cys Gly Leu Tyr Pro Ala Gln 530 535 540 Ile Lys Ala Arg Asn Met Val Ser Pro Val Met Ser Val Val Gly Phe 545 550 555 560 Leu Ala Leu Ala Lys Asp Trp Thr Ser Arg Ile Glu Glu Trp Leu Gly 565 570 575 Ala Pro Cys Lys Phe Met Ala Glu Ser Pro Ile Ala Gly Ser Leu Ser 580 585 590 Gly Asn Pro Val Asn Arg Asp Tyr Ile Arg Gln Arg Gln Gly Ala Leu 595 600 605 Ala Gly Met Glu Pro Lys Glu Phe Gln Ala Leu Arg Gln His Ser Lys 610 615 620 Asp Ala Gly Cys Thr Leu Val Glu His Ile Glu Ser Pro Ser Ser Ile 625 630 635 640 Trp Val Phe Ala Gly Ala Pro Asp Arg Cys Pro Pro Thr Cys Leu Phe 645 650 655 Val Gly Gly Met Ala Glu Leu Gly Ala Phe Phe Ser Ile Leu Gln Asp 660 665 670 Met Arg Asn Thr Ile Met Ala Ser Lys Thr Val Gly Thr Ala Asp Glu 675 680 685 Lys Leu Arg Lys Lys Ser Ser Phe Tyr Gln Ser Tyr Leu Arg Arg Thr 690 695 700 Gln Ser Met Gly Ile Gln Leu Asp Gln Arg Ile Ile Val Met Phe Met 705 710 715 720 Val Ala Trp Gly Lys Glu Ala Val Asp Asn Phe His Leu Gly Asp Asp 725 730 735 Met Asp Pro Glu Leu Arg Ser Leu Ala Gln Ile Leu Ile Asp Gln Lys 740 745 750 Val Lys Glu Ile Ser Asn Gln Glu Pro Met Lys Leu 755 760 <210> 33 <211> 4695 <212> DNA <213> Artificial sequence <220> <221> misc_feature <223> pMVAHantaNP <400> 33 gttggtggtc gccatggatg gtgttattgt atactgtcta aacgcgttag taaaacatgg 60 cgaggaaata aatcatataa aaaatgattt catgattaaa ccatgttgtg aaaaagtcaa 120 gaacgttcac attggcggac aatctaaaaa caatacagtg attgcagatt tgccatatat 180 ggataatgcg gtatccgatg tatgcaattc actgtataaa aagaatgtat caagaatatc 240 cagatttgct aatttgataa agatagatga cgatgacaag actcctactg gtgtatataa 300 ttattttaaa cctaaagatg ccattcctgt tattatatcc ataggaaagg atagagatgt 360 ttgtgaacta ttaatctcat ctgataaagc gtgtgcgtgt atagagttaa attcatataa 420 agtagccatt cttcccatgg atgtttcctt ttttaccaaa ggaaatgcat cattgattat 480 tctcctgttt gatttctcta tcgatgcggc acctctctta agaagtgtaa ccgataataa 540 tgttatta tctagacacc agcgtctaca tgacgagctt ccgagttcca attggttcaa 600 gttttacata agtataaagt ccgactattg ttctatatta tatatggttg ttgatggatc 660 tgtgatgcat gcaatagctg ataatagaac ttacgcaaat attagcaaaa atatattaga 720 caatactaca attaacgatg agtgtagatg ctgttatttt gaaccacaga ttaggattct 780 tgatagagat gagatgctca atggatcatc gtgtgatatg aacagacatt gtattatgat 840 gaatttacct gatgtaggcg aatttggatc tagtatgttg gggaaatatg aacctgacat 900 gattaagatt gctctttcgg tggctgggta ccaggcgcgc cttcatttt gttttttct 960 atgctataaa tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc 1020 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat 1080 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc 1140 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac 1200 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc 1260 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc 1320 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc 1380 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag 1440 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg 1500 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc 1560 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat 1620 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg 1680 tacaagtaag agctccggcc cgctcgaggc cgctggtacc caacctaaaa attgaaaata 1740 atacaagg ttctgagggg ttgtgttaaa ttgaaagcga gaataatca taaataagcc 1800 cggtgccacc atggccaca tggagagat ccagagagag atcagcgccc 1860 gctggttatc gccagacaga aagtgaagga cgccgagaag cagtacgaga aggaccccga 1920 cgatctgaac agagagccc tgcacgacag agaagcgtg gccgccctcta tccagagcaa 1980 gatcgatgag ctgagagac agctggccga cagaatcgcc gctggcaaga atattggcca 2040 ggacagagat cccacaggcg tggaacctgg cgatcacctg aaagagagaa gcgccctgtc 2100 ctatggcaac accctggacc tgacagcct ggacattgat gagcctaccg gccagacagc 2160 cgactggctg acaatcattg tgtacctgac cagctcgtg gtcccatca tcctgaaggc 2220 cctgtacatg ctgaccacca gaggcagaca gaccagacaag gacaacagg gcatgagaat 2280 ccggttcaag gatgacagca gctacgagga cgtgaacggc attagaagc ccaagcacct 2340 gtacgtgtcc atgcctaacg ctcagagcag catgaaggcc gaggaatca cccctggcag 2400 attcagaaca gccgtgtgcg gactgtaccc cgctcagatc aaggccagaa acatgtgtc 2460 cccagtgatg agcgtcgtgg gatttctggc cctggctaag gactggacca gcaggattga 2520 ggaatggctg ggagcccctt gcaagtttat ggccgagtct cctatcgccg gcagcctgtc 2580 tggcaacccc gtgaatagag actacatcag acagaggcag ggcgctctgg ccggaatgga 2640 acccaaagaa tttcaggccc tgcggcagca ctctaaggat gccggatgta ccctggtgga 2700 acacattgag agccccagca gcatctgggt tttcgctggc gctcctgata gatgccctcc 2760 tacctgtctg tttgttggcg gaatggccga gctgggcgcc ttctttagca ttctgcagga 2820 catgcggaat accatcatgg ccagcaagac cgtgggcacc gccgatgaga agctgagaaa 2880 gaagtccagc ttctaccaga gctacctgcg gagaacccag agcatgggca ttcagctgga 2940 ccagagaatc atcgtgatgt tcatggtggc ctggggcaaa gaagccgtgg acaattttca 3000 cctgggcgac gacatggacc ccgagctgag atctctggcc cagatcctga tcgaccagaa 3060 agtcaaagag atctccaatc aagagcccat gaagctgatg ctgagctacg gcaacgtgct 3120 ggatctgaac cacctggata tcgacgagcc aacaggacag accgctgatt ggctgggcat 3180 cgtgatctac ctgacctcct tgtgtgcc tattctcctc aaagccctct atatgctgac 3240 aacacgcgga aggcagacca cxaagata aaggcacc cggatcaggt ttaggacga 3300 cagctccttt gaggatgtca acggcatccg gaaactaag cacctag tgtctctgcc 3360 caatgcacag tcctccatga aggcagaga gatcacacca ggccggtaca gaaccgccat 3420 ctgtggactg tatcctgcac aaatcaaagc ccggcagatg atcagccccg tgatgtccgt 3480 tatcggattc ctggctctgg ccaagattg gagcgacagg atcgagcagt ggctgagcga 3540 gccttgcaag ctgcttccctg atacagccgc tgtcactg cttggcggcc ctgccacaaa 3600 cagagattac ctgagacaga gagggtggc actgggcaac atgaaaaaagagaaa 3660 ggccatccgg cagcatgccg aagctgctgg ctgtagcatg atcgaggata tcgagtcccc 3720 tagctccatt tgggtgttcg caggggcccc agatagatgt ccaccacat gcctgttcat 3780 tgccggcatg gctgaactgg gagcttttt cagcatccctc caggatatgc gcacacgat 3840 tatggcctcc aagacagtgg gaaccagcga ggaaagctg cggaaaaa gcagcttta 3900 ccagtcttac ctgaggcgga cccagtccat ggggatccaa ctggatcagc ggatcattgt 3960 gctgtttatg gtcgcttggg gaaaagaggc tgtcgataac ttccacctgg gagatgatat 4020 ggatcctgaa ctgcggaccc tggctcagtc cctgatcgat gtgaaagtga aagaaattag 4080 taatcagaa cccctcaagc tggacctgga aggccctga tcgaggact ahaggacga 4140 tgacgacag tgactcgacc tgcagttttt tgaggtag tgatagac 4200 aaatacata atttgtaaa aaaatcac tttttatact aatatgacac gattaccaat 4260 acttttgtta ctaatatcat tagtatacgc tacacctttt cctcagacat ctaaaaaaat 4320 aggtgatgat gcaactttat catgtaatcg aaataataca atgactacg ttgttatgag 4380 tgcttggtat aaggagccca attccattat tctttagct gctaaaagcg acgtcttgta 4440 ttttgataat ataccaagg aaaataatc ttacgactct ccatacgatg atctagttac 4500 aactatcaca attaaatcat tgactgctag agatgccggt acttatgtat gtgcattctt 4560 tatgacatcg cctacaatg acactgataa agtagatt gagaatact ccacagagtt 4620 gattgtaaat acagatagtg aatcgactat agacataata ctatctggat ctacacattc 4680 accggaaact agttg 4695 <210> 34 <211> 927 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <223> Left Flank of DelIII <400> 34 gttggtggtc gccatggatg gtgttattgt atactgtcta aacgcgttag taaaacatgg 60 cgaggaaata aatcatataa aaaatgattt catgattaaa ccatgttgtg aaaaagtcaa 120 gaacgttcac attggcggac aatctaaaaa caatacagtg attgcagatt tgccatatat 180 ggataatgcg gtatccgatg tatgcaattc actgtataaa aagaatgtat caagaatatc 240 cagatttgct aatttgataa agatagatga cgatgacaag actcctactg gtgtatataa 300 ttattttaaa cctaaagatg ccattcctgt tattatatcc ataggaaagg atagagatgt 360 ttgtgaacta ttaatctcat ctgataaagc gtgtgcgtgt atagagttaa attcatataa 420 agtagccatt cttcccatgg atgtttcctt ttttaccaaa ggaaatgcat cattgattat 480 tctcctgttt gatttctcta tcgatgcggc acctctctta agaagtgtaa ccgataataa 540 tgttattata tctagacacc agcgtctaca tgacgagctt ccgagttcca attggttcaa 600 gttttacata agtataaagt ccgactattg ttctatatta tatatggttg ttgatggatc 660 tgtgatgcat gcaatagctg ataatagaac ttacgcaaat attagcaaaa atatattaga 720 caatactaca attaacgatg agtgtagatg ctgttatttt gaaccacaga ttaggattct 780 tgatagagat gagatgctca atggatcatc gtgtgatatg aacagacatt gtattatgat 840 gaatttacct gatgtaggcg aatttggatc tagtatgttg gggaaatatg aacctgacat 900 gattaagatt gctctttcgg tggctgg 927 <210> 35 <211> 14 <212> DNA <213> Artificial sequence <220> <221> misc_feature <223> First linker <400> 35 gtaccaggcg cgcc 14 <210> 36 <211> 28 <212> DNA <213> Artificial sequence <220> <221> promoter <223> p11 <400> 36 tttcattttg tttttttcta tgctataa 28 <210> 37 <211> 720 <212> DNA <213> Artificial sequence <220> <221> mat_peptide <223> Green fluorescent protein <400> 37 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720 <210> 38 <211> 37 <212> DNA <213> Artificial <220> <221> misc_feature <223> Second connector <400> 38 gagctccggc ccgctcgagg ccgctggtac ccaacct 37 <210> 39 <211> 70 <212> DNA <213> Artificial <220> <221> promoter <223> MH5 promoter <400> 39 aaaaattgaa aataaataca aaggttcttg agggttgtgt taaattgaaa gcgagaaata 60 atcataaata 70 <210> 40 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 40 Asp Val Lys Val Lys Glu Ile Ser Asn Gln Glu Pro Leu Lys Leu 1 5 10 15 <210> 41 <211> 10 <212> DNA <213> Artificial <220> <221> primer_bind <223> Kozak sequence <400> 41 gccaccatgg 10 <210> 42 <211> twenty four <212> DNA <213> Artificial <220> <221> misc_feature <223> Fourth connector <400> 42 gacctggaag gccctagatt cgag 24 <210> 43 <211> twenty four <212> DNA <213> Artificial <220> <221> misc_binding <223> Flag Tags <400> 43 gactacaagg acgatgacga caag 24 <210> 44 <211> 20 <212> DNA <213> Artificial <220> <221> misc_feature <223> Fifth joint <400> 44 ctcgacctgc agtttttatg 20 <210> 45 <211> 522 <212> DNA <213> Artificial sequence <220> <221> misc_feature <223> Right flank of DelIII <400> 45 gaaagtttta taggtagttg atagaacaaa atacataatt ttgtaaaaat aaatcacttt 60 ttatactaat atgacacgat taccaatact tttgttacta atatcattag tatacgctac 120 accttttcct cagacatcta aaaaaatagg tgatgatgca actttatcat gtaatcgaaa 180 taatacaaat gactacgttg ttatgagtgc ttggtataag gagcccaatt ccattattct 240 tttagctgct aaaagcgacg tcttgtattt tgataattat accaaggata aaatatctta 300 cgactctcca tacgatgatc tagttacaac tatcacaatt aaatcattga ctgctagaga 360 tgccggtact tatgtatgtg cattctttat gacatcgcct acaaatgaca ctgataaagt 420 agattatgaa gaatactcca cagagttgat tgtaaataca gatagtgaat cgactataga 480 cataatacta tctggatcta cacattcacc ggaaactagt tg 522 <210> 46 <211> 20 <212> DNA <213> Artificial <220> <221> primer_bind <223> Primers <400> 46 cggcacctct cttaagaagt 20 <210> 47 <211> 25 <212> DNA <213> Artificial <220> <221> primer_bind <223> Primers <400> 47 gtgtagcgta tactaatgat attag 25 <210> 48 <211> 26 <212> DNA <213> Artificial <220> <221> primer_bind <223> Primers <400> 48 ggagtacaac tacaacagcc acaacg 26 <210> 49 <211> 780 <212> PRT <213> Artificial <220> <221> DOMAIN <223> Chimeric protein <400> 49 Met Ala Thr Met Glu Glu Ile Gln Arg Glu Ile Ser Ala His Glu Gly 1 5 10 15 Gln Leu Val Ile Ala Arg Gln Lys Val Lys Asp Ala Glu Lys Gln Tyr 20 25 30 Glu Lys Asp Pro Asp Asp Leu Asn Lys Arg Ala Leu His Asp Arg Glu 35 40 45 Ser Val Ala Ala Ser Ile Gln Ser Lys Ile Asp Glu Leu Lys Arg Gln 50 55 60 Leu Ala Asp Arg Ile Ala Ala Gly Lys Asn Ile Gly Gln Asp Arg Asp 65 70 75 80 Pro Thr Gly Val Glu Pro Gly Asp His Leu Lys Glu Arg Ser Ala Leu 85 90 95 Ser Tyr Gly Asn Thr Leu Asp Leu Asn Ser Leu Asp Ile Asp Glu Pro 100 105 110 Thr Gly Gln Thr Ala Asp Trp Leu Thr Ile Ile Val Tyr Leu Thr Ser 115 120 125 Phe Val Val Pro Ile Ile Leu Lys Ala Leu Tyr Met Leu Thr Thr Arg 130 135 140 Gly Arg Gln Thr Ser Lys Asp Asn Lys Gly Met Arg Ile Arg Phe Lys 145 150 155 160 Asp Asp Ser Ser Tyr Glu Asp Val Asn Gly Ile Arg Lys Pro Lys His 165 170 175 Leu Tyr Val Ser Met Pro Asn Ala Gln Ser Ser Met Lys Ala Glu Glu 180 185 190 Ile Thr Pro Gly Arg Phe Arg Thr Ala Val Cys Gly Leu Tyr Pro Ala 195 200 205 Gln Ile Lys Ala Arg Asn Met Val Ser Pro Val Met Ser Val Val Gly 210 215 220 Phe Leu Ala Leu Ala Lys Asp Trp Thr Ser Arg Ile Glu Glu Trp Leu 225 230 235 240 Gly Ala Pro Cys Lys Phe Met Ala Glu Ser Pro Ile Ala Gly Ser Leu 245 250 255 Ser Gly Asn Pro Val Asn Arg Asp Tyr Ile Arg Gln Arg Gln Gly Ala 260 265 270 Leu Ala Gly Met Glu Pro Lys Glu Phe Gln Ala Leu Arg Gln His Ser 275 280 285 Lys Asp Ala Gly Cys Thr Leu Val Glu His Ile Glu Ser Pro Ser Ser 290 295 300 Ile Trp Val Phe Ala Gly Ala Pro Asp Arg Cys Pro Pro Thr Cys Leu 305 310 315 320 Phe Val Gly Gly Met Ala Glu Leu Gly Ala Phe Phe Ser Ile Leu Gln 325 330 335 Asp Met Arg Asn Thr Ile Met Ala Ser Lys Thr Val Gly Thr Ala Asp 340 345 350 Glu Lys Leu Arg Lys Lys Ser Ser Phe Tyr Gln Ser Tyr Leu Arg Arg 355 360 365 Thr Gln Ser Met Gly Ile Gln Leu Asp Gln Arg Ile Ile Val Met Phe 370 375 380 Met Val Ala Trp Gly Lys Glu Ala Val Asp Asn Phe His Leu Gly Asp 385 390 395 400 Asp Met Asp Pro Glu Leu Arg Ser Leu Ala Gln Ile Leu Ile Asp Gln 405 410 415 Lys Val Lys Glu Ile Ser Asn Gln Glu Pro Met Lys Leu Met Leu Ser 420 425 430 Tyr Gly Asn Val Leu Asp Leu Asn His Leu Asp Ile Asp Glu Pro Thr 435 440 445 Gly Gln Thr Ala Asp Trp Leu Gly Ile Val Ile Tyr Leu Thr Ser Phe 450 455 460 Val Val Pro Ile Leu Leu Lys Ala Leu Tyr Met Leu Thr Thr Arg Gly 465 470 475 480 Arg Gln Thr Thr Lys Asp Asn Lys Gly Thr Arg Ile Arg Phe Lys Asp 485 490 495 Asp Ser Ser Phe Glu Asp Val Asn Gly Ile Arg Lys Pro Lys His Leu 500 505 510 Tyr Val Ser Leu Pro Asn Ala Gln Ser Ser Met Lys Ala Glu Glu Ile 515 520 525 Thr Pro Gly Arg Tyr Arg Thr Ala Ile Cys Gly Leu Tyr Pro Ala Gln 530 535 540 Ile Lys Ala Arg Gln Met Ile Ser Pro Val Met Ser Val Ile Gly Phe 545 550 555 560 Leu Ala Leu Ala Lys Asp Trp Ser Asp Arg Ile Glu Gln Trp Leu Ser 565 570 575 Glu Pro Cys Lys Leu Leu Pro Asp Thr Ala Ala Val Ser Leu Leu Gly 580 585 590 Gly Pro Ala Thr Asn Arg Asp Tyr Leu Arg Gln Arg Gln Val Ala Leu 595 600 605 Gly Asn Met Glu Thr Lys Glu Ser Lys Ala Ile Arg Gln His Ala Glu 610 615 620 Ala Ala Gly Cys Ser Met Ile Glu Asp Ile Glu Ser Pro Ser Ser Ile 625 630 635 640 Trp Val Phe Ala Gly Ala Pro Asp Arg Cys Pro Pro Thr Cys Leu Phe 645 650 655 Ile Ala Gly Met Ala Glu Leu Gly Ala Phe Phe Ser Ile Leu Gln Asp 660 665 670 Met Arg Asn Thr Ile Met Ala Ser Lys Thr Val Gly Thr Ser Glu Glu 675 680 685 Lys Leu Arg Lys Lys Ser Ser Phe Tyr Gln Ser Tyr Leu Arg Arg Thr 690 695 700 Gln Ser Met Gly Ile Gln Leu Asp Gln Arg Ile Ile Val Leu Phe Met 705 710 715 720 Val Ala Trp Gly Lys Glu Ala Val Asp Asn Phe His Leu Gly Asp Asp 725 730 735 Met Asp Pro Glu Leu Arg Thr Leu Ala Gln Ser Leu Ile Asp Val Lys 740 745 750 Val Lys Glu Ile Ser Asn Gln Glu Pro Leu Lys Leu Asp Leu Glu Gly 755 760 765 Pro Arg Phe Glu Asp Tyr Lys Asp Asp Asp Asp Lys 770 775 780 <210> 50 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 50 Met Ala Thr Met Glu Glu Ile Gln Arg Glu Ile Ser Ala His Glu 1 5 10 15 <210> 51 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 51 Glu Glu Ile Gln Arg Glu Ile Ser Ala His Glu Gly Gln Leu Val 1 5 10 15 <210> 52 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 52 Arg Glu Ile Ser Ala His Glu Gly Gln Leu Val Ile Ala Arg Gln 1 5 10 15 <210> 53 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 53 Ala His Glu Gly Gln Leu Val Ile Ala Arg Gln Lys Val Lys Asp 1 5 10 15 <210> 54 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 54 Gln Leu Val Ile Ala Arg Gln Lys Val Lys Asp Ala Glu Lys Gln 1 5 10 15 <210> 55 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 55 Ala Arg Gln Lys Val Lys Asp Ala Glu Lys Gln Tyr Glu Lys Asp 1 5 10 15 <210> 56 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 56 Val Lys Asp Ala Glu Lys Gln Tyr Glu Lys Asp Pro Asp Asp Leu 1 5 10 15 <210> 57 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 57 Glu Lys Gln Tyr Glu Lys Asp Pro Asp Asp Leu Asn Lys Arg Ala 1 5 10 15 <210> 58 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 58 Glu Lys Asp Pro Asp Asp Leu Asn Lys Arg Ala Leu His Asp Arg 1 5 10 15 <210> 59 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 59 Asp Asp Leu Asn Lys Arg Ala Leu His Asp Arg Glu Ser Val Ala 1 5 10 15 <210> 60 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 60 Lys Arg Ala Leu His Asp Arg Glu Ser Val Ala Ala Ser Ile Gln 1 5 10 15 <210> 61 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 61 His Asp Arg Glu Ser Val Ala Ala Ser Ile Gln Ser Lys Ile Asp 1 5 10 15 <210> 62 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 62 Ser Val Ala Ala Ser Ile Gln Ser Lys Ile Asp Glu Leu Lys Arg 1 5 10 15 <210> 63 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 63 Ser Ile Gln Ser Lys Ile Asp Glu Leu Lys Arg Gln Leu Ala Asp 1 5 10 15 <210> 64 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 64 Lys Ile Asp Glu Leu Lys Arg Gln Leu Ala Asp Arg Ile Ala Ala 1 5 10 15 <210> 65 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 65 Leu Lys Arg Gln Leu Ala Asp Arg Ile Ala Ala Gly Lys Asn Ile 1 5 10 15 <210> 66 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 66 Leu Ala Asp Arg Ile Ala Ala Gly Lys Asn Ile Gly Gln Asp Arg 1 5 10 15 <210> 67 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 67 Ile Ala Ala Gly Lys Asn Ile Gly Gln Asp Arg Asp Pro Thr Gly 1 5 10 15 <210> 68 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 68 Lys Asn Ile Gly Gln Asp Arg Asp Pro Thr Gly Val Glu Pro Gly 1 5 10 15 <210> 69 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 69 Gln Asp Arg Asp Pro Thr Gly Val Glu Pro Gly Asp His Leu Lys 1 5 10 15 <210> 70 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 70 Pro Thr Gly Val Glu Pro Gly Asp His Leu Lys Glu Arg Ser Ala 1 5 10 15 <210> 71 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 71 Glu Pro Gly Asp His Leu Lys Glu Arg Ser Ala Leu Ser Tyr Gly 1 5 10 15 <210> 72 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 72 His Leu Lys Glu Arg Ser Ala Leu Ser Tyr Gly Asn Thr Leu Asp 1 5 10 15 <210> 73 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 73 Arg Ser Ala Leu Ser Tyr Gly Asn Thr Leu Asp Leu Asn Ser Leu 1 5 10 15 <210> 74 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 74 Ser Tyr Gly Asn Thr Leu Asp Leu Asn Ser Leu Asp Ile Asp Glu 1 5 10 15 <210> 75 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 75 Thr Leu Asp Leu Asn Ser Leu Asp Ile Asp Glu Pro Thr Gly Gln 1 5 10 15 <210> 76 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 76 Asn Ser Leu Asp Ile Asp Glu Pro Thr Gly Gln Thr Ala Asp Trp 1 5 10 15 <210> 77 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 77 Ile Asp Glu Pro Thr Gly Gln Thr Ala Asp Trp Leu Thr Ile Ile 1 5 10 15 <210> 78 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 78 Thr Gly Gln Thr Ala Asp Trp Leu Thr Ile Ile Val Tyr Leu Thr 1 5 10 15 <210> 79 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 79 Ala Asp Trp Leu Thr Ile Ile Val Tyr Leu Thr Ser Phe Val Val 1 5 10 15 <210> 80 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 80 Thr Ile Ile Val Tyr Leu Thr Ser Phe Val Val Pro Ile Ile Leu 1 5 10 15 <210> 81 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 81 Tyr Leu Thr Ser Phe Val Val Pro Ile Ile Leu Lys Ala Leu Tyr 1 5 10 15 <210> 82 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 82 Phe Val Val Pro Ile Ile Leu Lys Ala Leu Tyr Met Leu Thr Thr 1 5 10 15 <210> 83 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 83 Ile Ile Leu Lys Ala Leu Tyr Met Leu Thr Thr Arg Gly Arg Gln 1 5 10 15 <210> 84 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 84 Ala Leu Tyr Met Leu Thr Thr Arg Gly Arg Gln Thr Ser Lys Asp 1 5 10 15 <210> 85 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 85 Leu Thr Thr Arg Gly Arg Gln Thr Ser Lys Asp Asn Lys Gly Met 1 5 10 15 <210> 86 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 86 Gly Arg Gln Thr Ser Lys Asp Asn Lys Gly Met Arg Ile Arg Phe 1 5 10 15 <210> 87 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 87 Ser Lys Asp Asn Lys Gly Met Arg Ile Arg Phe Lys Asp Asp Ser 1 5 10 15 <210> 88 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 88 Lys Gly Met Arg Ile Arg Phe Lys Asp Asp Ser Ser Tyr Glu Asp 1 5 10 15 <210> 89 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 89 Ile Arg Phe Lys Asp Asp Ser Ser Tyr Glu Asp Val Asn Gly Ile 1 5 10 15 <210> 90 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 90 Asp Asp Ser Ser Tyr Glu Asp Val Asn Gly Ile Arg Lys Pro Lys 1 5 10 15 <210> 91 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 91 Tyr Glu Asp Val Asn Gly Ile Arg Lys Pro Lys His Leu Tyr Val 1 5 10 15 <210> 92 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 92 Asn Gly Ile Arg Lys Pro Lys His Leu Tyr Val Ser Met Pro Asn 1 5 10 15 <210> 93 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 93 Lys Pro Lys His Leu Tyr Val Ser Met Pro Asn Ala Gln Ser Ser 1 5 10 15 <210> 94 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 94 Leu Tyr Val Ser Met Pro Asn Ala Gln Ser Ser Met Lys Ala Glu 1 5 10 15 <210> 95 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 95 Met Pro Asn Ala Gln Ser Ser Met Lys Ala Glu Glu Ile Thr Pro 1 5 10 15 <210> 96 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 96 Gln Ser Ser Met Lys Ala Glu Glu Ile Thr Pro Gly Arg Phe Arg 1 5 10 15 <210> 97 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 97 Lys Ala Glu Glu Ile Thr Pro Gly Arg Phe Arg Thr Ala Val Cys 1 5 10 15 <210> 98 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 98 Ile Thr Pro Gly Arg Phe Arg Thr Ala Val Cys Gly Leu Tyr Pro 1 5 10 15 <210> 99 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 99 Arg Phe Arg Thr Ala Val Cys Gly Leu Tyr Pro Ala Gln Ile Lys 1 5 10 15 <210> 100 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 100 Ala Val Cys Gly Leu Tyr Pro Ala Gln Ile Lys Ala Arg Asn Met 1 5 10 15 <210> 101 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 101 Leu Tyr Pro Ala Gln Ile Lys Ala Arg Asn Met Val Ser Pro Val 1 5 10 15 <210> 102 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 102 Gln Ile Lys Ala Arg Asn Met Val Ser Pro Val Met Ser Val Val 1 5 10 15 <210> 103 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 103 Arg Asn Met Val Ser Pro Val Met Ser Val Val Gly Phe Leu Ala 1 5 10 15 <210> 104 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 104 Ser Val Val Gly Phe Leu Ala Leu Ala Lys Asp Trp Thr Ser Arg 1 5 10 15 <210> 105 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 105 Phe Leu Ala Leu Ala Lys Asp Trp Thr Ser Arg Ile Glu Glu Trp 1 5 10 15 <210> 106 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 106 Ala Lys Asp Trp Thr Ser Arg Ile Glu Glu Trp Leu Gly Ala Pro 1 5 10 15 <210> 107 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 107 Thr Ser Arg Ile Glu Glu Trp Leu Gly Ala Pro Cys Lys Phe Met 1 5 10 15 <210> 108 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 108 Glu Glu Trp Leu Gly Ala Pro Cys Lys Phe Met Ala Glu Ser Pro 1 5 10 15 <210> 109 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 109 Gly Ala Pro Cys Lys Phe Met Ala Glu Ser Pro Ile Ala Gly Ser 1 5 10 15 <210> 110 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 110 Lys Phe Met Ala Glu Ser Pro Ile Ala Gly Ser Leu Ser Gly Asn 1 5 10 15 <210> 111 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 111 Glu Ser Pro Ile Ala Gly Ser Leu Ser Gly Asn Pro Val Asn Arg 1 5 10 15 <210> 112 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 112 Ala Gly Ser Leu Ser Gly Asn Pro Val Asn Arg Asp Tyr Ile Arg 1 5 10 15 <210> 113 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 113 Ser Gly Asn Pro Val Asn Arg Asp Tyr Ile Arg Gln Arg Gln Gly 1 5 10 15 <210> 114 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 114 Val Asn Arg Asp Tyr Ile Arg Gln Arg Gln Gly Ala Leu Ala Gly 1 5 10 15 <210> 115 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 115 Tyr Ile Arg Gln Arg Gln Gly Ala Leu Ala Gly Met Glu Pro Lys 1 5 10 15 <210> 116 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 116 Arg Gln Gly Ala Leu Ala Gly Met Glu Pro Lys Glu Phe Gln Ala 1 5 10 15 <210> 117 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 117 Leu Ala Gly Met Glu Pro Lys Glu Phe Gln Ala Leu Arg Gln His 1 5 10 15 <210> 118 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 118 Glu Pro Lys Glu Phe Gln Ala Leu Arg Gln His Ser Lys Asp Ala 1 5 10 15 <210> 119 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 119 Phe Gln Ala Leu Arg Gln His Ser Lys Asp Ala Gly Cys Thr Leu 1 5 10 15 <210> 120 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 120 Arg Gln His Ser Lys Asp Ala Gly Cys Thr Leu Val Glu His Ile 1 5 10 15 <210> 121 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 121 Lys Asp Ala Gly Cys Thr Leu Val Glu His Ile Glu Ser Pro Ser 1 5 10 15 <210> 122 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 122 Cys Thr Leu Val Glu His Ile Glu Ser Pro Ser Ser Ile Trp Val 1 5 10 15 <210> 123 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 123 Glu His Ile Glu Ser Pro Ser Ser Ile Trp Val Phe Ala Gly Ala 1 5 10 15 <210> 124 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 124 Ser Pro Ser Ser Ile Trp Val Phe Ala Gly Ala Pro Asp Arg Cys 1 5 10 15 <210> 125 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 125 Ile Trp Val Phe Ala Gly Ala Pro Asp Arg Cys Pro Pro Thr Cys 1 5 10 15 <210> 126 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 126 Ala Gly Ala Pro Asp Arg Cys Pro Pro Thr Cys Leu Phe Val Gly 1 5 10 15 <210> 127 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 127 Asp Arg Cys Pro Pro Thr Cys Leu Phe Val Gly Gly Met Ala Glu 1 5 10 15 <210> 128 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 128 Pro Thr Cys Leu Phe Val Gly Gly Met Ala Glu Leu Gly Ala Phe 1 5 10 15 <210> 129 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 129 Phe Val Gly Gly Met Ala Glu Leu Gly Ala Phe Phe Ser Ile Leu 1 5 10 15 <210> 130 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 130 Met Ala Glu Leu Gly Ala Phe Phe Ser Ile Leu Gln Asp Met Arg 1 5 10 15 <210> 131 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 131 Gly Ala Phe Phe Ser Ile Leu Gln Asp Met Arg Asn Thr Ile Met 1 5 10 15 <210> 132 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 132 Ser Ile Leu Gln Asp Met Arg Asn Thr Ile Met Ala Ser Lys Thr 1 5 10 15 <210> 133 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 133 Asp Met Arg Asn Thr Ile Met Ala Ser Lys Thr Val Gly Thr Ala 1 5 10 15 <210> 134 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 134 Thr Ile Met Ala Ser Lys Thr Val Gly Thr Ala Asp Glu Lys Leu 1 5 10 15 <210> 135 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 135 Ser Lys Thr Val Gly Thr Ala Asp Glu Lys Leu Arg Lys Lys Ser 1 5 10 15 <210> 136 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 136 Gly Thr Ala Asp Glu Lys Leu Arg Lys Lys Ser Ser Phe Tyr Gln 1 5 10 15 <210> 137 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 137 Glu Lys Leu Arg Lys Lys Ser Ser Ser Phe Tyr Gln Ser Tyr Leu Arg 1 5 10 15 <210> 138 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 138 Lys Lys Ser Ser Phe Tyr Gln Ser Tyr Leu Arg Arg Thr Gln Ser 1 5 10 15 <210> 139 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 139 Phe Tyr Gln Ser Tyr Leu Arg Arg Thr Gln Ser Met Gly Ile Gln 1 5 10 15 <210> 140 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 140 Tyr Leu Arg Arg Thr Gln Ser Met Gly Ile Gln Leu Asp Gln Arg 1 5 10 15 <210> 141 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 141 Thr Gln Ser Met Gly Ile Gln Leu Asp Gln Arg Ile Ile Val Met 1 5 10 15 <210> 142 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 142 Gly Ile Gln Leu Asp Gln Arg Ile Ile Val Met Phe Met Val Ala 1 5 10 15 <210> 143 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 143 Asp Gln Arg Ile Ile Val Met Phe Met Val Ala Trp Gly Lys Glu 1 5 10 15 <210> 144 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 144 Ile Val Met Phe Met Val Ala Trp Gly Lys Glu Ala Val Asp Asn 1 5 10 15 <210> 145 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 145 Met Val Ala Trp Gly Lys Glu Ala Val Asp Asn Phe His Leu Gly 1 5 10 15 <210> 146 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 146 Gly Lys Glu Ala Val Asp Asn Phe His Leu Gly Asp Asp Met Asp 1 5 10 15 <210> 147 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 147 Val Asp Asn Phe His Leu Gly Asp Asp Met Asp Pro Glu Leu Arg 1 5 10 15 <210> 148 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 148 His Leu Gly Asp Asp Met Asp Pro Glu Leu Arg Ser Leu Ala Gln 1 5 10 15 <210> 149 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 149 Asp Met Asp Pro Glu Leu Arg Ser Leu Ala Gln Ile Leu Ile Asp 1 5 10 15 <210> 150 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 150 Glu Leu Arg Ser Leu Ala Gln Ile Leu Ile Asp Gln Lys Val Lys 1 5 10 15 <210> 151 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 151 Leu Ala Gln Ile Leu Ile Asp Gln Lys Val Lys Glu Ile Ser Asn 1 5 10 15 <210> 152 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 152 Leu Ile Asp Gln Lys Val Lys Glu Ile Ser Asn Gln Glu Pro Met 1 5 10 15 <210> 153 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 153 Lys Val Lys Glu Ile Ser Asn Gln Glu Pro Met Lys Leu Met Leu 1 5 10 15 <210> 154 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 154 Ile Ser Asn Gln Glu Pro Met Lys Leu Met Leu Ser Tyr Gly Asn 1 5 10 15 <210> 155 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 155 Glu Pro Met Lys Leu Met Leu Ser Tyr Gly Asn Val Leu Asp Leu 1 5 10 15 <210> 156 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 156 Leu Met Leu Ser Tyr Gly Asn Val Leu Asp Leu Asn His Leu Asp 1 5 10 15 <210> 157 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 157 Tyr Gly Asn Val Leu Asp Leu Asn His Leu Asp Ile Asp Glu Pro 1 5 10 15 <210> 158 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 158 Leu Asp Leu Asn His Leu Asp Ile Asp Glu Pro Thr Gly Gln Thr 1 5 10 15 <210> 159 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 159 His Leu Asp Ile Asp Glu Pro Thr Gly Gln Thr Ala Asp Trp Leu 1 5 10 15 <210> 160 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 160 Asp Glu Pro Thr Gly Gln Thr Ala Asp Trp Leu Gly Ile Val Ile 1 5 10 15 <210> 161 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 161 Gly Gln Thr Ala Asp Trp Leu Gly Ile Val Ile Tyr Leu Thr Ser 1 5 10 15 <210> 162 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 162 Asp Trp Leu Gly Ile Val Ile Tyr Leu Thr Ser Phe Val Val Pro 1 5 10 15 <210> 163 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 163 Ile Val Ile Tyr Leu Thr Ser Phe Val Val Pro Ile Leu Leu Lys 1 5 10 15 <210> 164 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 164 Leu Thr Ser Phe Val Val Pro Ile Leu Leu Lys Ala Leu Tyr Met 1 5 10 15 <210> 165 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 165 Val Val Pro Ile Leu Leu Lys Ala Leu Tyr Met Leu Thr Thr Arg 1 5 10 15 <210> 166 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 166 Leu Leu Lys Ala Leu Tyr Met Leu Thr Thr Arg Gly Arg Gln Thr 1 5 10 15 <210> 167 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 167 Leu Tyr Met Leu Thr Thr Arg Gly Arg Gln Thr Thr Lys Asp Asn 1 5 10 15 <210> 168 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 168 Thr Thr Arg Gly Arg Gln Thr Thr Lys Asp Asn Lys Gly Thr Arg 1 5 10 15 <210> 169 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 169 Arg Gln Thr Thr Lys Asp Asn Lys Gly Thr Arg Ile Arg Phe Lys 1 5 10 15 <210> 170 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 170 Lys Asp Asn Lys Gly Thr Arg Ile Arg Phe Lys Asp Asp Ser Ser 1 5 10 15 <210> 171 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 171 Gly Thr Arg Ile Arg Phe Lys Asp Asp Ser Ser Phe Glu Asp Val 1 5 10 15 <210> 172 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 172 Arg Phe Lys Asp Asp Ser Ser Phe Glu Asp Val Asn Gly Ile Arg 1 5 10 15 <210> 173 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 173 Asp Ser Ser Phe Glu Asp Val Asn Gly Ile Arg Lys Pro Lys His 1 5 10 15 <210> 174 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 174 Glu Asp Val Asn Gly Ile Arg Lys Pro Lys His Leu Tyr Val Ser 1 5 10 15 <210> 175 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 175 Gly Ile Arg Lys Pro Lys His Leu Tyr Val Ser Leu Pro Asn Ala 1 5 10 15 <210> 176 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 176 Pro Lys His Leu Tyr Val Ser Leu Pro Asn Ala Gln Ser Ser Met 1 5 10 15 <210> 177 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 177 Tyr Val Ser Leu Pro Asn Ala Gln Ser Ser Met Lys Ala Glu Glu 1 5 10 15 <210> 178 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 178 Pro Asn Ala Gln Ser Ser Met Lys Ala Glu Glu Ile Thr Pro Gly 1 5 10 15 <210> 179 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 179 Ser Ser Met Lys Ala Glu Glu Ile Thr Pro Gly Arg Tyr Arg Thr 1 5 10 15 <210> 180 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 180 Ala Glu Glu Ile Thr Pro Gly Arg Tyr Arg Thr Ala Ile Cys Gly 1 5 10 15 <210> 181 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 181 Thr Pro Gly Arg Tyr Arg Thr Ala Ile Cys Gly Leu Tyr Pro Ala 1 5 10 15 <210> 182 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 182 Tyr Arg Thr Ala Ile Cys Gly Leu Tyr Pro Ala Gln Ile Lys Ala 1 5 10 15 <210> 183 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 183 Ile Cys Gly Leu Tyr Pro Ala Gln Ile Lys Ala Arg Gln Met Ile 1 5 10 15 <210> 184 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 184 Tyr Pro Ala Gln Ile Lys Ala Arg Gln Met Ile Ser Pro Val Met 1 5 10 15 <210> 185 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 185 Ile Lys Ala Arg Gln Met Ile Ser Pro Val Met Ser Val Ile Gly 1 5 10 15 <210> 186 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 186 Gln Met Ile Ser Pro Val Met Ser Val Ile Gly Phe Leu Ala Leu 1 5 10 15 <210> 187 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 187 Val Ile Gly Phe Leu Ala Leu Ala Lys Asp Trp Ser Asp Arg Ile 1 5 10 15 <210> 188 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 188 Leu Ala Leu Ala Lys Asp Trp Ser Asp Arg Ile Glu Gln Trp Leu 1 5 10 15 <210> 189 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 189 Lys Asp Trp Ser Asp Arg Ile Glu Gln Trp Leu Ser Glu Pro Cys 1 5 10 15 <210> 190 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 190 Asp Arg Ile Glu Gln Trp Leu Ser Glu Pro Cys Lys Leu Leu Pro 1 5 10 15 <210> 191 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 191 Gln Trp Leu Ser Glu Pro Cys Lys Leu Leu Pro Asp Thr Ala Ala 1 5 10 15 <210> 192 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 192 Glu Pro Cys Lys Leu Leu Pro Asp Thr Ala Ala Val Ser Leu Leu 1 5 10 15 <210> 193 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 193 Leu Leu Pro Asp Thr Ala Ala Val Ser Leu Leu Gly Gly Pro Ala 1 5 10 15 <210> 194 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 194 Thr Ala Ala Val Ser Leu Leu Gly Gly Pro Ala Thr Asn Arg Asp 1 5 10 15 <210> 195 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 195 Ser Leu Leu Gly Gly Pro Ala Thr Asn Arg Asp Tyr Leu Arg Gln 1 5 10 15 <210> 196 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 196 Gly Pro Ala Thr Asn Arg Asp Tyr Leu Arg Gln Arg Gln Val Ala 1 5 10 15 <210> 197 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 197 Asn Arg Asp Tyr Leu Arg Gln Arg Gln Val Ala Leu Gly Asn Met 1 5 10 15 <210> 198 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 198 Leu Arg Gln Arg Gln Val Ala Leu Gly Asn Met Glu Thr Lys Glu 1 5 10 15 <210> 199 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 199 Gln Val Ala Leu Gly Asn Met Glu Thr Lys Glu Ser Lys Ala Ile 1 5 10 15 <210> 200 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 200 Gly Asn Met Glu Thr Lys Glu Ser Lys Ala Ile Arg Gln His Ala 1 5 10 15 <210> 201 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 201 Thr Lys Glu Ser Lys Ala Ile Arg Gln His Ala Glu Ala Ala Gly 1 5 10 15 <210> 202 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 202 Lys Ala Ile Arg Gln His Ala Glu Ala Ala Gly Cys Ser Met Ile 1 5 10 15 <210> 203 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 203 Gln His Ala Glu Ala Ala Gly Cys Ser Met Ile Glu Asp Ile Glu 1 5 10 15 <210> 204 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 204 Ala Ala Gly Cys Ser Met Ile Glu Asp Ile Glu Ser Pro Ser Ser 1 5 10 15 <210> 205 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 205 Ser Met Ile Glu Asp Ile Glu Ser Pro Ser Ser Ile Trp Val Phe 1 5 10 15 <210> 206 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 206 Asp Ile Glu Ser Pro Ser Ser Ile Trp Val Phe Ala Gly Ala Pro 1 5 10 15 <210> 207 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 207 Pro Ser Ser Ile Trp Val Phe Ala Gly Ala Pro Asp Arg Cys Pro 1 5 10 15 <210> 208 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 208 Trp Val Phe Ala Gly Ala Pro Asp Arg Cys Pro Pro Thr Cys Leu 1 5 10 15 <210> 209 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 209 Gly Ala Pro Asp Arg Cys Pro Pro Thr Cys Leu Phe Ile Ala Gly 1 5 10 15 <210> 210 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 210 Arg Cys Pro Pro Thr Cys Leu Phe Ile Ala Gly Met Ala Glu Leu 1 5 10 15 <210> 211 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 211 Thr Cys Leu Phe Ile Ala Gly Met Ala Glu Leu Gly Ala Phe Phe 1 5 10 15 <210> 212 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 212 Ile Ala Gly Met Ala Glu Leu Gly Ala Phe Phe Ser Ile Leu Gln 1 5 10 15 <210> 213 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 213 Ala Glu Leu Gly Ala Phe Phe Ser Ile Leu Gln Asp Met Arg Asn 1 5 10 15 <210> 214 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 214 Ala Phe Phe Ser Ile Leu Gln Asp Met Arg Asn Thr Ile Met Ala 1 5 10 15 <210> 215 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 215 Ile Leu Gln Asp Met Arg Asn Thr Ile Met Ala Ser Lys Thr Val 1 5 10 15 <210> 216 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 216 Met Arg Asn Thr Ile Met Ala Ser Lys Thr Val Gly Thr Ser Glu 1 5 10 15 <210> 217 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 217 Ile Met Ala Ser Lys Thr Val Gly Thr Ser Glu Glu Lys Leu Arg 1 5 10 15 <210> 218 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 218 Lys Thr Val Gly Thr Ser Glu Glu Lys Leu Arg Lys Lys Ser Ser 1 5 10 15 <210> 219 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 219 Thr Ser Glu Glu Lys Leu Arg Lys Lys Ser Ser Phe Tyr Gln Ser 1 5 10 15 <210> 220 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 220 Lys Leu Arg Lys Lys Ser Ser Phe Tyr Gln Ser Tyr Leu Arg Arg 1 5 10 15 <210> 221 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 221 Lys Ser Ser Phe Tyr Gln Ser Tyr Leu Arg Arg Thr Gln Ser Met 1 5 10 15 <210> 222 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 222 Tyr Gln Ser Tyr Leu Arg Arg Thr Gln Ser Met Gly Ile Gln Leu 1 5 10 15 <210> 223 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 223 Leu Arg Arg Thr Gln Ser Met Gly Ile Gln Leu Asp Gln Arg Ile 1 5 10 15 <210> 224 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 224 Gln Ser Met Gly Ile Gln Leu Asp Gln Arg Ile Ile Val Leu Phe 1 5 10 15 <210> 225 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 225 Ile Gln Leu Asp Gln Arg Ile Ile Val Leu Phe Met Val Ala Trp 1 5 10 15 <210> 226 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 226 Gln Arg Ile Ile Val Leu Phe Met Val Ala Trp Gly Lys Glu Ala 1 5 10 15 <210> 227 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 227 Val Leu Phe Met Val Ala Trp Gly Lys Glu Ala Val Asp Asn Phe 1 5 10 15 <210> 228 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 228 Val Ala Trp Gly Lys Glu Ala Val Asp Asn Phe His Leu Gly Asp 1 5 10 15 <210> 229 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 229 Lys Glu Ala Val Asp Asn Phe His Leu Gly Asp Asp Met Asp Pro 1 5 10 15 <210> 230 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 230 Asp Asn Phe His Leu Gly Asp Asp Met Asp Pro Glu Leu Arg Thr 1 5 10 15 <210> 231 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 231 Leu Gly Asp Asp Met Asp Pro Glu Leu Arg Thr Leu Ala Gln Ser 1 5 10 15 <210> 232 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 232 Met Asp Pro Glu Leu Arg Thr Leu Ala Gln Ser Leu Ile Asp Val 1 5 10 15 <210> 233 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 233 Leu Arg Thr Leu Ala Gln Ser Leu Ile Asp Val Lys Val Lys Glu 1 5 10 15 <210> 234 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 234 Ala Gln Ser Leu Ile Asp Val Lys Val Lys Glu Ile Ser Asn Gln 1 5 10 15 <210> 235 <211> 15 <212> PRT <213> Artificial <220> <221> PEPTIDE <223> Peptide library <400> 235 Ile Asp Val Lys Val Lys Glu Ile Ser Asn Gln Glu Pro Leu Lys 1 5 10 15

Claims

1. A viral vector or bacterial vector comprising the nucleic acid sequence of SEQ ID NO: 29; wherein the nucleic acid sequence is capable of inducing an immune response in a subject.

2. The vector of claim 1, wherein the vector is a viral vector.

3. The vector of claim 2, wherein the vector is a non-replicating poxvirus vector.

4. The vector of claim 3, wherein the non-replicating poxvirus vector is selected from the group consisting of a modified vaccinia Ankara virus (MVA) vector, a NYVAC vaccinia virus vector, a canarypox (ALVAC) vector, and a fowlpox (FPV) vector.

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

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

7. The vector of claim 2, wherein the vector is an adenoviral vector.

8. The vector of claim 7, wherein the adenoviral vector is a non-replicating adenoviral vector.

9. The vector of claim 7, wherein the adenovirus vector is selected from the group consisting of: human adenovirus vector, simian adenovirus vector, group B adenovirus vector, group C adenovirus vector, group E adenovirus vector, adenovirus 6 vector, PanAd3 vector, adenovirus C3 vector, ChAdY25 vector, AdC68 vector and Ad5 vector.

10. The vector of claim 2, wherein the vector is a measles virus vector.

11. The vector of claim 10, wherein the measles virus vector is a non-replicating measles virus vector.

12. A method for preparing a viral vector, comprising: Providing a nucleic acid, wherein the nucleic acid comprises a nucleic acid sequence encoding a vector according to any one of claims 1 to 11; transfecting a host cell with the nucleic acid; culturing the host cell under conditions suitable for vector proliferation; and The vector is obtained from the host cell.

13. A host cell comprising the vector of any one of claims 1 to 11, wherein the host cell is not an embryonic cell.

14. A composition comprising the carrier according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

15. The composition of claim 14, further comprising an adjuvant.

16. Use of a vector according to any one of claims 1 to 11, or a composition according to claim 14 or claim 15, in the preparation of a medicament for preventing hantavirus infection in a subject.

17. Use of the vector according to any one of claims 1 to 11, or the composition according to claim 14 or claim 15, in the preparation of a medicament for preventing hemorrhagic fever with renal syndrome in a subject.

Images