Mutant of L1 protein of human papillomavirus type 11

Abstract

Disclosed are a mutated HPV11 L1 protein (or a variant thereof), a sequence encoding the same, a method for preparing the same, and a virus-like particle comprising the same, wherein the protein (or a variant thereof) and the virus-like particle can induce the generation of neutralizing antibodies against at least two HPV types (e.g. HPV11 and HPV6), and therefore can be used to prevent infection by said at least two HPV types, and a disease caused by said infection, such as cervical cancer and condyloma acuminatum. Also disclosed is use of the protein and the virus-like particle in the manufacture of a pharmaceutical composition or a vaccine for preventing infection by said at least two HPV types, and a disease caused by said infection, such as cervical cancer and condyloma acuminatum.

Claims

1. A mutated HPV11 L1 protein or a variant thereof, wherein as compared with a wild type HPV11 L1 protein, the mutated HPV11 L1 protein has the following mutations: (1) N-terminal truncation of 3, 4, 5 or 6 amino acids; and (2) (a) substitution of amino acid residues at positions 346-351 of the wild type HPV11 L1 protein with amino acid residues at the corresponding positions of a L1 protein of a wild-type HPV6; or (b) substitution of amino acid residues at positions 119-140 of the wild type HPV11 L1 protein with amino acid residues at the corresponding positions of a L1 protein of a wild type HPV6.

2. An isolated nucleic acid, encoding the mutated HPV11 L1 protein according to claim 1.

3. A vector comprising the isolated nucleic acid according to claim 2.

4. A host cell comprising the isolated nucleic acid according to claim 2 and/or a vector comprising the isolate nucleic acid.

5. A HPV virus-like particle, comprising the mutated HPV11 L1 protein according to claim 1.

6. A composition, comprising any of the following: (i) the mutated HPV11 L1 protein according to claim 1, or (ii) an isolated nucleic acid encoding the mutated HPV11 L1 protein as described in part (i), or (iii) a vector comprising the isolated nucleic acid as described in part (ii), or (iv) a host cell comprising the isolated nucleic acid as described in part (ii) or the vector as described in part (iii), or (v) an HPV virus-like particle comprising the mutated HPV11 L1 protein as described in part (i).

7. A pharmaceutical composition or vaccine, comprising the HPV virus-like particle according to claim 5, and optionally a pharmaceutically acceptable carrier and/or excipient.

8. A method for preparing the mutated HPV11 L1 protein according to claim 1, comprising expressing the mutated HPV11 L1 protein in a host cell, and then recovering the mutated HPV11 L1 protein from a culture of the host cell.

9. A method for preparing a vaccine, comprising combining the HPV virus-like particle according to claim 5 with a pharmaceutically acceptable carrier and/or excipient.

10. A method for preventing HPV infection or a disease caused by HPV infection, comprising administering to a subject a prophylactically effective amount of the HPV virus-like particle according to claim 5 or a pharmaceutical composition or vaccine comprising the HPV virus-like particle according to claim 5 and optionally a pharmaceutically acceptable carrier and/or excipient.

11. The mutated HPV11 L1 protein according to claim 1, wherein the mutated HPV11 L1 protein has 4 amino acids truncated from the N-terminal, as compared with the wild type HPV11 L1 protein.

12. The mutated HPV11 L1 protein according to claim 1, wherein: the amino acid residues at the corresponding positions as described in (2) (a) are amino acid residues at positions 345-350 of a wild type HPV6 L1 protein; and/or, the amino acid residues at the corresponding positions as described in (2) (b) are amino acid residues at positions 119-139 of a wild type HPV6 L1 protein.

13. The mutated HPV11 L1 protein according to claim 1, wherein: the wild type HPV11 L1 protein has an amino acid sequence as set forth in SEQ ID NO: 1; and/or, the wild type HPV6 L1 protein has an amino acid sequence as set forth in SEQ ID NO: 2.

14. The mutated HPV11 L1 protein according to claim 1, wherein the mutated HPV11 L1 protein has an amino acid sequence selected from the group consisting of: SEQ ID NOs: 6 and 9.

15. The pharmaceutical composition or vaccine according to claim 7, wherein the HPV virus-like particle is present in an amount effective for preventing HPV infection or a disease caused by HPV infection.

16. The pharmaceutical composition or vaccine according to claim 15, wherein: the HPV infection is HPV11 infection and/or HPV6 infection; and/or, the disease caused by HPV infection is selected from the group consisting of cervical cancer and condyloma acuminatum.

17. The method according to claim 8, wherein the host cell is E. coli.

18. The method according to claim 8, wherein the method comprises the steps of: expressing the mutated HPV11 L1 protein in E. coli, and then obtaining the mutated HPV11 L1 protein by purifying a lysate supernatant of the E. coli.

19. The method according to claim 10, wherein: the HPV infection is an HPV11 infection and/or an HPV6 infection; and/or, the disease caused by the HPV infection is selected from the group consisting of cervical cancer and condyloma acuminatum.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the SDS-PAGE result of the purified mutated proteins in Example 1. Lane M: protein molecular weight marker; Lane 1: HPV11N4 (HPV11 L1 protein having 4 amino acids truncated at N-terminal); Lane 2: H11N4-6T1; Lane 3: H11N4-6T2; Lane 4: H11N4-6T3; Lane 5: H11N4-6T4; Lane 6: H11N4-6T5. The result showed that after chromatographic purification, the proteins H11N4-6T1, H11N4-6T2, H11N4-6T3, H11N4-6T4 and H11N4-6T5 reached a purity of above 95%.

(2) FIGS. 2A-2F show the results of molecular sieve chromatographic analysis of the samples comprising the protein HPV11N4, H11N4-6T1, H11N4-6T2, H11N4-6T3, H11N4-6T4 and H11N4-6T5, respectively. FIG. 2A: HPV11N4; FIG. 2B: H11N4-6T1; FIG. 2C: H11N4-6T2; FIG. 2D: H11N4-6T3; FIG. 2E: H11N4-6T4; FIG. 2F: H11N4-6T5. The results showed that the first protein peak of the samples comprising H11N4-6T1, H11N4-6T2, H11N4-6T3, H11N4-6T4 and H11N4-6T5 respectively appeared at about 12 min, which was comparable to that of HPV11N4 VLP. This showed that all these mutated proteins were able to assemble into VLPs.

(3) FIGS. 3A-3F show the results of sedimentation velocity analysis of HPV11N4 VLP, H11N4-6T1 VLP, H11N4-6T2 VLP, H11N4-6T3 VLP, H11N4-6T4 VLP and H11N4-6T5 VLP. FIG. 3A, HPV11N4 VLP; FIG. 3B, H11N4-6T1 VLP; FIG. 3C, H11N4-6T2 VLP; FIG. 3D, H11N4-6T3 VLP; FIG. 3E, H11N4-6T4 VLP; FIG. 3F, H11N4-6T5 VLP. The results showed that the sedimentation coefficient of H11N4-6T1 VLP, H11N4-6T2 VLP, H11N4-6T3 VLP, H11N4-6T4 VLP and H11N4-6T5 VLP was 140S, 138S, 111S, 139S and 139S, respectively. This indicated that the 5 mutated HPV11 L1 proteins as prepared above were able to assemble into virus-like particles that were similar to wild type VLP (HPV11N4 VLP, 136.3S) in terms of size and morphology.

(4) FIGS. 4A-4F show the transmission electron microscopy (TEM) photographs (taken at 100,000 magnification, Bar=0.1 m) of various VLP samples. FIG. 4A, HPV11N4 VLP; FIG. 4B, H11N4-6T1 VLP; FIG. 4C, H11N4-6T2 VLP; FIG. 4D, H11N4-6T3 VLP; FIG. 4E, H11N4-6T4 VLP; FIG. 4F, H11N4-6T5 VLP. The results showed that H11N4-6T1, H11N4-6T2, H11N4-6T3, H11N4-6T4 and H11N4-6T5 were similar to HPV11N4, and were able to assemble into VLPs with a radius of about 25 nm.

(5) FIGS. 5A-5F show the results of thermostability evaluation of VLPs formed by HPV11N4, H11N4-6T1, H11N4-6T2, H11N4-6T3, H11N4-6T4 and H11N4-6T5 respectively. FIG. 5A, HPV11N4 VLP; FIG. 5B, H11N4-6T1 VLP; FIG. 5C, H11N4-6T2 VLP; FIG. 5D, H11N4-6T3 VLP; FIG. 5E, H11N4-6T4 VLP; FIG. 5F, H11N4-6T5 VLP. The results showed that all the VLPs formed by these proteins had very high thermostability.

(6) FIGS. 6A-6D show the cryo-electron microscopy (cryoEM) photographs and the analyzed structures of H11N4-6T3 VLP and H11N4-6T5 VLP; wherein, FIG. 6A and FIG. 6C show the cryo-electron microscopy (cryoEM) photographs of H11N4-6T3 VLP and H11N4-6T5 VLP, respectively; FIG. 6B and FIG. 6D show the three-dimensional structures of H11N4-6T3 VLP and H11N4-6T5 VLP as analyzed by using cryo-electron microscopy (cryoEM), at a resolution of 17.38 and 20.48 , respectively.

(7) FIG. 7 shows the evaluation result of immune protection of the Experimental groups H11N4-6T1, H11N4-6T2, H11N4-6T3, H11N4-6T4 and H11N4-6T5, and the Control groups HPV11N4 VLP, HPV6N5 VLP and a mixed HPV11/HPV6 VLP in mice. The result showed that either of H11N4-6T3 VLP and H11N4-6T5 VLP could induce the generation of high-titer neutralizing antibodies against HPV11 and HPV6 in mice; and their protective effects against HPV11 were comparable to that of HPV11N4 VLP alone or the mixed HPV11/HPV6 VLP, and were significantly higher than that of HPV6N5 VLP alone; and their protective effects against HPV6 were comparable to that of HPV6N5 VLP alone or the mixed HPV11/HPV6 VLP, and were significantly higher than that of HPV11N4 VLP alone. H11N4-6T2 VLP could also induce the generation of high-titer neutralizing antibodies against HPV11 and HPV6 in mice, but its ability of inducing the generation of neutralizing antibodies against HPV6 was weaker than that of H11N4-6T3 VLP and H11N4-6T5 VLP. These results showed that H11N4-6T2 VLP, H11N4-6T3 VLP and H11N4-6T5 VLP could be used as effective vaccines for preventing HPV11 infection and HPV6 infection, and could be used in place of a mixed vaccine comprising HPV11 VLP and HPV6 VLP.

(8) FIGS. 8A-8C show the evaluation results of neutralizing antibody titer in mouse serum after vaccination of mice with H11N4-6T3 VLP and H11N4-6T5 VLP respectively. FIG. 8A: Aluminum adjuvant group 1 (at an immunizing dose of 10 g, using aluminum adjuvant); FIG. 8B: Aluminum adjuvant group 2 (at an immunizing dose of 1 g, using aluminum adjuvant); FIG. 8C: Aluminum adjuvant group 3 (at an immunizing dose of 0.1 g, using aluminum adjuvant). The results showed that either of H11N4-6T3 VLP and H11N4-6T5 VLP could induce the generation of high-titer neutralizing antibodies against HPV11 in mice, and their protective effects were comparable to that of HPV11N4 VLP alone or the mixed HPV11/HPV6 VLP at the same dose, and were significantly superior to that of HPV6N5 VLP alone at the same dose; and they could induce the generation of high-titer neutralizing antibodies against HPV6 in mice, and their protective effects were comparable to that of HPV6N5 VLP alone or the mixed HPV11/HPV6 VLP at the same dose, and were significantly superior to that of HPV11N4 VLP alone at the same dose. This showed that H11N4-6T3 VLP and H11N4-6T5 VLP had good cross-immunogenicity and cross-protection against HPV11 and HPV6.

(9) FIG. 9 shows the evaluation result of neutralizing antibodies in cynomolgus monkey serum after vaccination of cynomolgus monkeys with H11N4-6T3 VLP or H11N4-6T5 VLP. The result showed that either of H11N4-6T3 VLP and H11N4-6T5 VLP could induce the generation of high-titer neutralizing antibodies against HPV11 and HPV6 in cynomolgus monkeys, and their protective effects were comparable to that of the mixed HPV11/HPV6 VLP. This showed that H11N4-6T3 VLP and H11N4-6T5 VLP had good cross-immunogenicity and cross-protection against HPV11 and HPV6.

SEQUENCE INFORMATION

(10) Some of the sequences involved in the invention are provided in the following Table 1.

(11) TABLE-US-00001 TABLE 1 Description of sequences SEQ ID NO: Description 1 wild type HPV11 L1 protein 2 wild type HPV6 L1 protein 3 the HPV11 L1 protein having 4 amino acids truncated at N-terminal, HPV11N4 4 the HPV6 L1 protein having 5 amino acids truncated at N-terminal, HPV6N5 5 the mutated HPV11 L1 protein comprising Segment 1 of HPV6 L1 protein, H11N4-6T1 6 the mutated HPV11 L1 protein comprising Segment 2 of HPV6 L1 protein, H11N4-6T2 7 the mutated HPV11 L1 protein comprising Segment 3 of HPV6 L1 protein, H11N4-6T3 8 the mutated HPV11 L1 protein comprising Segment 4 of HPV6 L1 protein, H11N4-6T4 9 the mutated HPV11 L1 protein comprising Segment 5 of HPV6 L1 protein, H11N4-6T5 10 the DNA sequence encoding SEQ ID NO: 3 11 the DNA sequence encoding SEQ ID NO: 4 12 the DNA sequence encoding SEQ ID NO: 5 13 the DNA sequence encoding SEQ ID NO: 6 14 the DNA sequence encoding SEQ ID NO: 7 15 the DNA sequence encoding SEQ ID NO: 8 16 the DNA sequence encoding SEQ ID NO: 9 35 the sequence of amino acid residues at positions 169 to 178 of wild type HPV6 L1 protein 36 the sequence of amino acid residues at positions 345 to 350 of wild type HPV6 L1 protein 37 the sequence of amino acid residues at positions 119 to 139 of wild type HPV6 L1 protein

(12) TABLE-US-00002 Sequence1(SEQIDNO:1): MWRPSDSTVYVPPPNPVSKVVATDAYVKRTNIFYHASSSRLLAVGHPYYSIKKVNKTVVPKVSGYQY RVFKVVLPDPNKFALPDSSLFDPTTQRLVWACTGLEVGRGQPLGVGVSGHPLLNKYDDVENSGGYG GNPGQDNRVNVGMDYKQTQLCMVGCAPPLGEHWGKGTQCSNTSVQNGDCPPLELITSVIQDGDMV DTGFGAMNFADLQTNKSDVPLDICGTVCKYPDYLQMAADPYGDRLFFYLRKEQMFARHFFNRAGTV GEPVPDDLLVKGGNNRSSVASSIYVHTPSGSLVSSEAQLFNKPYWLQKAQGHNNGICWGNHLFVTVV DTTRSTNMTLCASVSKSATYTNSDYKEYMRHVEEFDLQFIFQLCSITLSAEVMAYIHTMNPSVLEDW NFGLSPPPNGTLEDTYRYVQSQAITCQKPTPEKEKQDPYKDMSFWEVNLKEKFSSELDQFPLGRKFLL QSGYRGRTSARTGIKRPAVSKPSTAPKRKRTKTKK Sequence2(SEQIDNO:2): MWRPSDSTVYVPPPNPVSKVVATDAYVTRTNIFYHASSSRLLAVGHPYFSIKRANKTVVPKVSGYQY RVFKVVLPDPNKFALPDSSLFDPTTQRLVWACTGLEVGRGQPLGVGVSGHPFLNKYDDVENSGSGGN PGQDNRVNVGMDYKQTQLCMVGCAPPLGEHWGKGKQCTNTPVQAGDCPPLELITSVIQDGDMVDT GFGAMNFADLQTNKSDVPIDICGTTCKYPDYLQMAADPYGDRLFFFLRKEQMFARHFFNRAGEVGEP VPDTLIIKGSGNRTSVGSSIYVNTPSGSLVSSEAQLFNKPYWLQKAQGHNNGICWGNQLFVTVVDTTR STNMTLCASVTTSSTYTNSDYKEYMRHVEEYDLQFIFQLCSITLSAEVMAYIHTMNPSVLEDWNFGLS PPPNGTLEDTYRYVQSQAITCQKPTPEKEKPDPYKNLSFWEVNLKEKFSSELDQYPLGRKFLLQSGYR GRSSIRTGVKRPAVSKASAAPKRKRAKTKR Sequence3(SEQIDNO:3): MSDSTVYVPPPNPVSKVVATDAYVKRTNIFYHASSSRLLAVGHPYYSIKKVNKTVVPKVSGYQYRVF KVVLPDPNKFALPDSSLFDPTTQRLVWACTGLEVGRGQPLGVGVSGHPLLNKYDDVENSGGYGGNP GQDNRVNVGMDYKQTQLCMVGCAPPLGEHWGKGTQCSNTSVQNGDCPPLELITSVIQDGDMVDTG FGAMNFADLQTNKSDVPLDICGTVCKYPDYLQMAADPYGDRLFFYLRKEQMFARHFFNRAGTVGEP VPDDLLVKGGNNRSSVASSIYVHTPSGSLVSSEAQLFNKPYWLQKAQGHNNGICWGNHLFVTVVDTT RSTNMTLCASVSKSATYTNSDYKEYMRHVEEFDLQFIFQLCSITLSAEVMAYIHTMNPSVLEDWNFGL SPPPNGTLEDTYRYVQSQAITCQKPTPEKEKQDPYKDMSFWEVNLKEKFSSELDQFPLGRKFLLQSGY RGRTSARTGIKRPAVSKPSTAPKRKRTKTKK Sequence4(SEQIDNO:4): MDSTVYVPPPNPVSKVVATDAYVTRTNIFYHASSSRLLAVGHPYFSIKRANKTVVPKVSGYQYRVFK VVLPDPNKFALPDSSLFDPTTQRLVWACTGLEVGRGQPLGVGVSGHPFLNKYDDVENSGSGGNPGQ DNRVNVGMDYKQTQLCMVGCAPPLGEHWGKGKQCTNTPVQAGDCPPLELITSVIQDGDMVDTGFG AMNFADLQTNKSDVPIDICGTTCKYPDYLQMAADPYGDRLFFFLRKEQMFARHFFNRAGEVGEPVPD TLIIKGSGNRTSVGSSIYVNTPSGSLVSSEAQLFNKPYWLQKAQGHNNGICWGNQLFVTVVDTTRSTN MTLCASVTTSSTYTNSDYKEYMRHVEEYDLQFIFQLCSITLSAEVVAYIHTMNPSVLEDWNFGLSPPP NGTLEDTYRYVQSQAITCQKPTPEKQKPDPYKNLSFWEVNLKEKFSSELDQYPLGRKFLLQSGYRGRS SIRTGVKRPAVSKASAAPKRKRAKTKR Sequence5(SEQIDNO:5): MSDSTVYVPPPNPVSKVVATDAYVKRTNIFYHASSSRLLAVGHPYFSIKRANKTVVPKVSGYQYRVF KVVLPDPNKFALPDSSLFDPTTQRLVWACTGLEVGRGQPLGVGVSGHPLLNKYDDVENSGGYGGNP GQDNRVNVGMDYKQTQLCMVGCAPPLGEHWGKGTQCSNTSVQNGDCPPLELITSVIQDGDMVDTG FGAMNFADLQTNKSDVPLDICGTVCKYPDYLQMAADPYGDRLFFYLRKEQMFARHFFNRAGTVGEP VPDDLLVKGGNNRSSVASSIYVHTPSGSLVSSEAQLFNKPYWLQKAQGHNNGICWGNHLFVTVVDTT RSTNMTLCASVSKSATYTNSDYKEYMRHVEEFDLQFIFQLCSITLSAEVMAYIHTMNPSVLEDWNFGL SPPPNGTLEDTYRYVQSQATTCQKPTPEKEKQDPYKDMSFWEVNLKEKFSSELDQFPLGRKFLLQSGY RGRTSARTGIKRPAVSKPSTAPKRKRTKTKK Sequence6(SEQIDNO:6): MSDSTVYVPPPNPVSKVVATDAYVKRTNIFYHASSSRLLAVGHPYYSIKKVNKTVVPKVSGYQYRVF KVVLPDPNKFALPDSSLFDPTTQRLVWACTGLEVGRGQPLGVGVSGHPFLNKYDDVENSGSGGNPG QDNRVNVGMDYKQTQLCMVGCAPPLGEHWGKGTQCSNTSVQNGDCPPLELITSVIQDGDMVDTGF GAMNFADLQTNKSDVPLDICGTVCKYPDYLQMAADPYGDRLFFYLRKEQMFARHFFNRAGTVGEPV PDDLLVKGGNNRSSVASSIYVHTPSGSLVSSEAQLFNKPYWLQKAQGHNNGICWGNHLFVTVVDTTR STNMTLCASVSKSATYTNSDYKEYMRHVEEFDLQFIFQLCSITLSAEVMAYIHTMNPSVLEDWNFGLS PPPNGTLEDTYRYVQSQAITCQKPTPEKEKQDPYKDMSFWEVNLKEKFSSELDQFPLGRKFLLQSGYR GRTSARTGIKRPAVSKPSTAPKRKRTKTKK Sequence7(SEQIDNO:7): MSDSTVYVPPPNPVSKVVATDAYVKRTNIFYHASSSRLLAVGHPYYSIKKVNKTVVPKVSGYQYRVF KVVLPDPNKFALPDSSLFDPTTQRLVWACTGLEVGRGQPLGVGVSGHPLLNKYDDVENSGGYGGNP GQDNRVNVGMDYKQTQLCMVGCAPPLGEHWGKGKQCTNTPVQAGDCPPLELITSVIQDGDMVDTG FGAMNFADLQTNKSDVPLDICGTVCKYPDYLQMAADPYGDRLFFYLRKEQMFARHFFNRAGTVGEP VPDDLLVKGGNNRSSVASSIYVHTPSGSLVSSEAQLFNKPYWLQKAQGHNNGICWGNHLFVTVVDTT RSTNMTLCASVSKSATYTNSDYKEYMRHVEEFDLQFIFQLCSITLSAEVMAYIHTMNPSVLEDWNFGL SPPPNGTLEDTYRYVQSQAITCQKPTPEKEKQDPYKDMSFWEVNLKEKFSSELDQFPLGRKFLLQSGY RGRTSARTGIKRPAVSKPSTAPKRKRTKTKK Sequence8(SEQIDNO:8): MSDSTVYVPPPNPVSKVVATDAYVKRTNIFYHASSSRLLAVGHPYYSIKKVNKTVVPKVSGYQYRVF KVVLPDPNKFALPDSSLFDPTTQRLVWACTGLEVGRGQPLGVGVSGHPLLNKYDDVENSGGYGGNP GQDNRVNVGMDYKQTQLCMVGCAPPLGEHWGKGTQCSNTSVQNGDCPPLELITSVIQDGDMVDTG FGAMNFADLQTNKSDVPLDICGTVCKYPDYLQMAADPYGDRLFFYLRKEQMFARHFFNRAGEVGEP VPDTLIIKGSGNRTSVGSSIYVHTPSGSLVSSEAQLFNKPYWLQKAQGHNNGICWGNHLFVTVVDTTR STNMTLCASVSKSATYTNSDYKEYMRHVEEFDLQFIFQLCSITLSAEVMAYIHTMNPSVLEDWNFGLS PPPNGTLEDTYRYVQSQAITCQKPTPEKEKQDPYKDMSFWEVNLKEKFSSELDQFPLGRKFLLQSGYR GRTSARTGIKRPAVSKPSTAPKRKRTKTKK Sequence9(SEQIDNO:9): MSDSTVYVPPPNPVSKVVATDAYVKRTNIFYHASSSRLLAVGHPYYSIKKVNKTVVPKVSGYQYRVF KVVLPDPNKFALPDSSLFDPTTQRLVWACTGLEVGRGQPLGVGVSGHPLLNKYDDVENSGGYGGNP GQDNRVNVGMDYKQTQLCMVGCAPPLGEHWGKGTQCSNTSVQNGDCPPLELITSVIQDGDMVDTG FGAMNFADLQTNKSDVPLDICGTVCKYPDYLQMAADPYGDRLFFYLRKEQMFARHFFNRAGTVGEP VPDDLLVKGGNNRSSVASSIYVHTPSGSLVSSEAQLFNKPYWLQKAQGHNNGICWGNHLFVTVVDTT RSTNMTLCASVTTSSTYTNSDYKEYMRHVEEFDLQFIFQLCSITLSAEVMAYIHTMNPSVLEDWNFGL S PPPNGTLEDTYRYVQSQATTCQKPTPEKEKQDPYKDMSFWEVNLKEKFSSELDQFPLGRKFLLQSGYR GRTSARTGIKRPAVSKPSTAPKRKRTKTKK Sequence10(SEQIDNO:10): ATGAGCGACAGCACAGTATATGTGCCTCCTCCCAACCCTGTATCCAAGGTTGTTGCCACGGATGC GTATGTTAAACGCACCAACATATTTTATCACGCCAGCAGTTCTAGACTCCTTGCTGTGGGACATCC ATATTACTCTATCAAAAAAGTTAACAAAACAGTTGTACCAAAGGTGTCTGGATATCAATATAGAG TGTTTAAGGTAGTGTTGCCAGATCCTAACAAGTTTGCATTACCTGATTCATCTCTGTTTGACCCCA CTACACAGCGTTTAGTATGGGCGTGCACAGGGTTGGAGGTAGGCAGGGGTCAACCTTTAGGCGTT GGTGTTAGTGGGCATCCATTGCTAAACAAATATGATGATGTAGAAAATAGTGGTGGGTATGGTGG TAATCCTGGTCAGGATAATAGGGTTAATGTAGGTATGGATTATAAACAAACCCAGCTATGTATGG TGGGCTGTGCTCCACCGTTAGGTGAACATTGGGGTAAGGGTACACAATGTTCAAATACCTCTGTA CAAAATGGTGACTGCCCCCCGTTGGAACTTATTACCAGTGTTATACAGGATGGGGACATGGTTGA TACAGGCTTTGGTGCTATGAATTTTGCAGACTTACAAACCAATAAATCGGATGTTCCCCTTGATAT TTGTGGAACTGTCTGCAAATATCCTGATTATTTGCAAATGGCAGCAGACCCTTATGGTGATAGGT TGTTTTTTTATTTGCGAAAGGAACAAATGTTTGCTAGACACTTTTTTAATAGGGCCGGTACTGTGG GGGAACCTGTGCCTGATGACCTGTTGGTAAAAGGGGGTAATAATAGGTCATCTGTAGCTAGTAGT ATTTATGTACATACACCTAGTGGATCCTTGGTGTCTTCAGAGGCTCAATTATTTAATAAACCATAT TGGCTTCAAAAGGCTCAGGGACATAACAATGGTATTTGCTGGGGAAACCACTTGTTTGTTACTGT GGTAGATACCACACGCAGTACAAATATGACACTATGTGCATCTGTGTCTAAATCTGCTACATACA CTAATTCAGATTATAAGGAATATATGCGCCATGTGGAGGAGTTTGATTTACAGTTTATTTTTCAAT TGTGTAGCATTACATTATCTGCAGAAGTCATGGCCTATATACACACAATGAATCCTTCTGTTTTGG AGGACTGGAACTTTGGTTTATCGCCTCCACCAAATGGTACACTGGAGGATACTTATAGATATGTA CAGTCACAGGCCATTACCTGTCAGAAACCCACACCCGAAAAAGAAAAACAGGACCCCTATAAGG ATATGAGTTTTTGGGAGGTTAACTTAAAAGAAAAGTTTTCTTCTGAATTAGATCAGTTTCCCCTTG GACGTAAGTTTTTATTGCAAAGTGGATATCGAGGACGGACGTCTGCTCGTACAGGTATAAAGCGC CCAGCTGTGTCTAAGCCCTCTACAGCCCCCAAACGAAAACGTACCAAAACCAAAAAGTAA Sequence11(SEQIDNO:11): ATGGACAGCACAGTATATGTGCCTCCTCCTAACCCTGTATCCAAAGTTGTTGCCACGGATGCTTAT GTTACTCGCACCAACATATTTTATCATGCCAGCAGTTCTAGACTTCTTGCAGTGGGTCATCCTTAT TTTTCCATAAAACGGGCTAACAAAACTGTTGTGCCAAAGGTGTCAGGATATCAATACAGGGTATT TAAGGTGGTGTTACCAGATCCTAACAAATTTGCATTGCCTGACTCGTCTCTTTTTGATCCCACAAC ACAACGTTTGGTATGGGCATGCACAGGCCTAGAGGTGGGCAGGGGACAGCCATTAGGTGTGGGT GTAAGTGGACATCCTTTCCTAAATAAATATGATGATGTTGAAAATTCAGGGAGTGGTGGTAACCC TGGACAGGATAACAGGGTTAATGTTGGTATGGATTATAAACAAACACAATTATGCATGGTTGGAT GTGCCCCCCCTTTGGGCGAGCATTGGGGTAAAGGTAAACAGTGTACTAATACACCTGTACAGGCT GGTGACTGCCCGCCCTTAGAACTTATTACCAGTGTTATACAGGATGGCGATATGGTTGACACAGG CTTTGGTGCTATGAATTTTGCTGATTTGCAGACCAATAAATCAGATGTTCCTATTGATATATGTGG CACTACATGTAAATATCCAGATTATTTACAAATGGCTGCAGACCCTTATGGTGATAGATTATTTTT TTTTCTACGGAAGGAACAAATGTTTGCCAGACATTTTTTTAACAGGGCTGGCGAGGTGGGGGAAC CTGTGCCTGATACTCTTATAATTAAGGGTAGTGGAAATCGAACGTCTGTAGGGAGTAGTATATAT GTTAACACCCCAAGCGGCTCTTTGGTGTCCTCTGAGGCACAATTGTTTAATAAGCCATATTGGCTA CAAAAAGCCCAGGGACATAACAATGGTATTTGTTGGGGTAATCAACTGTTTGTTACTGTGGTAGA TACCACACGCAGTACCAACATGACATTATGTGCATCCGTAACTACATCTTCCACATACACCAATT CTGATTATAAAGAGTACATGCGTCATGTGGAAGAGTATGATTTACAATTTATTTTTCAATTATGTA GCATTACATTGTCTGCTGAAGTAGTGGCCTATATTCACACAATGAATCCCTCTGTTTTGGAAGACT GGAACTTTGGGTTATCGCCTCCCCCAAATGGTACATTAGAAGATACCTATAGGTATGTGCAGTCA CAGGCCATTACCTGTCAAAAGCCCACTCCTGAAAAGCAAAAGCCAGATCCCTATAAGAACCTTA GTTTTTGGGAGGTTAATTTAAAAGAAAAGTTTTCTAGTGAATTGGATCAGTATCCTTTGGGACGC AAGTTTTTGTTACAAAGTGGATATAGGGGACGGTCCTCTATTCGTACCGGTGTTAAGCGCCCTGC TGTTTCCAAAGCCTCTGCTGCCCCTAAACGTAAGCGCGCCAAAACTAAAAGGTAA Sequence12(SEQIDNO:12): ATGAGCGACAGCACAGTATATGTGCCTCCTCCCAACCCTGTATCCAAGGTTGTTGCCACGGATGC GTATGTTAAACGCACCAACATATTTTATCACGCCAGCAGTTCTAGACTCCTTGCTGTGGGACATCC ATATTTTTCCATAAAACGGGCTAACAAAACTGTTGTGCCAAAGGTGTCAGGATATCAATATAGAG TGTTTAAGGTAGTGTTGCCAGATCCTAACAAGTTTGCATTACCTGATTCATCTCTGTTTGACCCCA CTACACAGCGTTTAGTATGGGCGTGCACAGGGTTGGAGGTAGGCAGGGGTCAACCTTTAGGCGTT GGTGTTAGTGGGCATCCATTGCTAAACAAATATGATGATGTAGAAAATAGTGGTGGGTATGGTGG TAATCCTGGTCAGGATAATAGGGTTAATGTAGGTATGGATTATAAACAAACCCAGCTATGTATGG TGGGCTGTGCTCCACCGTTAGGTGAACATTGGGGTAAGGGTACACAATGTTCAAATACCTCTGTA CAAAATGGTGACTGCCCCCCGTTGGAACTTATTACCAGTGTTATACAGGATGGGGACATGGTTGA TACAGGCTTTGGTGCTATGAATTTTGCAGACTTACAAACCAATAAATCGGATGTTCCCCTTGATAT TTGTGGAACTGTCTGCAAATATCCTGATTATTTGCAAATGGCAGCAGACCCTTATGGTGATAGGT TGTTTTTTTATTTGCGAAAGGAACAAATGTTTGCTAGACACTTTTTTAATAGGGCCGGTACTGTGG GGGAACCTGTGCCTGATGACCTGTTGGTAAAAGGGGGTAATAATAGGTCATCTGTAGCTAGTAGT ATTTATGTACATACACCTAGTGGATCCTTGGTGTCTTCAGAGGCTCAATTATTTAATAAACCATAT TGGCTTCAAAAGGCTCAGGGACATAACAATGGTATTTGCTGGGGAAACCACTTGTTTGTTACTGT GGTAGATACCACACGCAGTACAAATATGACACTATGTGCATCTGTGTCTAAATCTGCTACATACA CTAATTCAGATTATAAGGAATATATGCGCCATGTGGAGGAGTTTGATTTACAGTTTATTTTTCAAT TGTGTAGCATTACATTATCTGCAGAAGTCATGGCCTATATACACACAATGAATCCTTCTGTTTTGG AGGACTGGAACTTTGGTTTATCGCCTCCACCAAATGGTACACTGGAGGATACTTATAGATATGTA CAGTCACAGGCCATTACCTGTCAGAAACCCACACCCGAAAAAGAAAAACAGGACCCCTATAAGG ATATGAGTTTTTGGGAGGTTAACTTAAAAGAAAAGTTTTCTTCTGAATTAGATCAGTTTCCCCTTG GACGTAAGTTTTTATTGCAAAGTGGATATCGAGGACGGACGTCTGCTCGTACAGGTATAAAGCGC CCAGCTGTGTCTAAGCCCTCTACAGCCCCCAAACGAAAACGTACCAAAACCAAAAAGTAA Sequence13(SEQIDNO:13): ATGAGCGACAGCACAGTATATGTGCCTCCTCCCAACCCTGTATCCAAGGTTGTTGCCACGGATGC GTATGTTAAACGCACCAACATATTTTATCACGCCAGCAGTTCTAGACTCCTTGCTGTGGGACATCC ATATTACTCTATCAAAAAAGTTAACAAAACAGTTGTACCAAAGGTGTCTGGATATCAATATAGAG TGTTTAAGGTAGTGTTGCCAGATCCTAACAAGTTTGCATTACCTGATTCATCTCTGTTTGACCCCA CTACACAGCGTTTAGTATGGGCGTGCACAGGGTTGGAGGTAGGCAGGGGACAGCCATTAGGTGT GGGTGTAAGTGGACATCCTTTCCTAAATAAATATGATGATGTTGAAAATTCAGGGAGTGGTGGTA ACCCTGGACAGGATAACAGGGTTAATGTTGGTATGGATTATAAACAAACCCAGCTATGTATGGTG GGCTGTGCTCCACCGTTAGGTGAACATTGGGGTAAGGGTACACAATGTTCAAATACCTCTGTACA AAATGGTGACTGCCCCCCGTTGGAACTTATTACCAGTGTTATACAGGATGGGGACATGGTTGATA CAGGCTTTGGTGCTATGAATTTTGCAGACTTACAAACCAATAAATCGGATGTTCCCCTTGATATTT GTGGAACTGTCTGCAAATATCCTGATTATTTGCAAATGGCAGCAGACCCTTATGGTGATAGGTTG TTTTTTTATTTGCGAAAGGAACAAATGTTTGCTAGACACTTTTTTAATAGGGCCGGTACTGTGGGG GAACCTGTGCCTGATGACCTGTTGGTAAAAGGGGGTAATAATAGGTCATCTGTAGCTAGTAGTAT TTATGTACATACACCTAGTGGATCCTTGGTGTCTTCAGAGGCTCAATTATTTAATAAACCATATTG GCTTCAAAAGGCTCAGGGACATAACAATGGTATTTGCTGGGGAAACCACTTGTTTGTTACTGTGG TAGATACCACACGCAGTACAAATATGACACTATGTGCATCTGTGTCTAAATCTGCTACATACACT AATTCAGATTATAAGGAATATATGCGCCATGTGGAGGAGTTTGATTTACAGTTTATTTTTCAATTG TGTAGCATTACATTATCTGCAGAAGTCATGGCCTATATACACACAATGAATCCTTCTGTTTTGGAG GACTGGAACTTTGGTTTATCGCCTCCACCAAATGGTACACTGGAGGATACTTATAGATATGTACA GTCACAGGCCATTACCTGTCAGAAACCCACACCCGAAAAAGAAAAACAGGACCCCTATAAGGAT ATGAGTTTTTGGGAGGTTAACTTAAAAGAAAAGTTTTCTTCTGAATTAGATCAGTTTCCCCTTGGA CGTAAGTTTTTATTGCAAAGTGGATATCGAGGACGGACGTCTGCTCGTACAGGTATAAAGCGCCC AGCTGTGTCTAAGCCCTCTACAGCCCCCAAACGAAAACGTACCAAAACCAAAAAGTAA Sequence14(SEQIDNO:14): ATGAGCGACAGCACAGTATATGTGCCTCCTCCCAACCCTGTATCCAAGGTTGTTGCCACGGATGC GTATGTTAAACGCACCAACATATTTTATCACGCCAGCAGTTCTAGACTCCTTGCTGTGGGACATCC ATATTACTCTATCAAAAAAGTTAACAAAACAGTTGTACCAAAGGTGTCTGGATATCAATATAGAG TGTTTAAGGTAGTGTTGCCAGATCCTAACAAGTTTGCATTACCTGATTCATCTCTGTTTGACCCCA CTACACAGCGTTTAGTATGGGCGTGCACAGGGTTGGAGGTAGGCAGGGGTCAACCTTTAGGCGTT GGTGTTAGTGGGCATCCATTGCTAAACAAATATGATGATGTAGAAAATAGTGGTGGGTATGGTGG TAATCCTGGTCAGGATAATAGGGTTAATGTAGGTATGGATTATAAACAAACCCAGCTATGTATGG TGGGCTGTGCTCCACCGTTAGGTGAACATTGGGGTAAAGGTAAACAGTGTACTAATACACCTGTA CAGGCTGGTGACTGCCCGCCCTTGGAACTTATTACCAGTGTTATACAGGATGGGGACATGGTTGA TACAGGCTTTGGTGCTATGAATTTTGCAGACTTACAAACCAATAAATCGGATGTTCCCCTTGATAT TTGTGGAACTGTCTGCAAATATCCTGATTATTTGCAAATGGCAGCAGACCCTTATGGTGATAGGT TGTTTTTTTATTTGCGAAAGGAACAAATGTTTGCTAGACACTTTTTTAATAGGGCCGGTACTGTGG GGGAACCTGTGCCTGATGACCTGTTGGTAAAAGGGGGTAATAATAGGTCATCTGTAGCTAGTAGT ATTTATGTACATACACCTAGTGGATCCTTGGTGTCTTCAGAGGCTCAATTATTTAATAAACCATAT TGGCTTCAAAAGGCTCAGGGACATAACAATGGTATTTGCTGGGGAAACCACTTGTTTGTTACTGT GGTAGATACCACACGCAGTACAAATATGACACTATGTGCATCTGTGTCTAAATCTGCTACATACA CTAATTCAGATTATAAGGAATATATGCGCCATGTGGAGGAGTTTGATTTACAGTTTATTTTTCAAT TGTGTAGCATTACATTATCTGCAGAAGTCATGGCCTATATACACACAATGAATCCTTCTGTTTTGG AGGACTGGAACTTTGGTTTATCGCCTCCACCAAATGGTACACTGGAGGATACTTATAGATATGTA CAGTCACAGGCCATTACCTGTCAGAAACCCACACCCGAAAAAGAAAAACAGGACCCCTATAAGG ATATGAGTTTTTGGGAGGTTAACTTAAAAGAAAAGTTTTCTTCTGAATTAGATCAGTTTCCCCTTG GACGTAAGTTTTTATTGCAAAGTGGATATCGAGGACGGACGTCTGCTCGTACAGGTATAAAGCGC CCAGCTGTGTCTAAGCCCTCTACAGCCCCCAAACGAAAACGTACCAAAACCAAAAAGTAA Sequence15(SEQIDNO:15): ATGAGCGACAGCACAGTATATGTGCCTCCTCCCAACCCTGTATCCAAGGTTGTTGCCACGGATGC GTATGTTAAACGCACCAACATATTTTATCACGCCAGCAGTTCTAGACTCCTTGCTGTGGGACATCC ATATTACTCTATCAAAAAAGTTAACAAAACAGTTGTACCAAAGGTGTCTGGATATCAATATAGAG TGTTTAAGGTAGTGTTGCCAGATCCTAACAAGTTTGCATTACCTGATTCATCTCTGTTTGACCCCA CTACACAGCGTTTAGTATGGGCGTGCACAGGGTTGGAGGTAGGCAGGGGTCAACCTTTAGGCGTT GGTGTTAGTGGGCATCCATTGCTAAACAAATATGATGATGTAGAAAATAGTGGTGGGTATGGTGG TAATCCTGGTCAGGATAATAGGGTTAATGTAGGTATGGATTATAAACAAACCCAGCTATGTATGG TGGGCTGTGCTCCACCGTTAGGTGAACATTGGGGTAAGGGTACACAATGTTCAAATACCTCTGTA CAAAATGGTGACTGCCCCCCGTTGGAACTTATTACCAGTGTTATACAGGATGGGGACATGGTTGA TACAGGCTTTGGTGCTATGAATTTTGCAGACTTACAAACCAATAAATCGGATGTTCCCCTTGATAT TTGTGGAACTGTCTGCAAATATCCTGATTATTTGCAAATGGCAGCAGACCCTTATGGTGATAGGT TGTTTTTTTATTTGCGAAAGGAACAAATGTTTGCTAGACACTTTTTTAACAGGGCTGGCGAGGTGG GGGAACCTGTGCCTGATACTCTTATAATTAAGGGTAGTGGAAATCGAACGTCTGTAGGGAGTAGT ATATATGTACATACACCTAGTGGATCCTTGGTGTCTTCAGAGGCTCAATTATTTAATAAACCATAT TGGCTTCAAAAGGCTCAGGGACATAACAATGGTATTTGCTGGGGAAACCACTTGTTTGTTACTGT GGTAGATACCACACGCAGTACAAATATGACACTATGTGCATCTGTGTCTAAATCTGCTACATACA CTAATTCAGATTATAAGGAATATATGCGCCATGTGGAGGAGTTTGATTTACAGTTTATTTTTCAAT TGTGTAGCATTACATTATCTGCAGAAGTCATGGCCTATATACACACAATGAATCCTTCTGTTTTGG AGGACTGGAACTTTGGTTTATCGCCTCCACCAAATGGTACACTGGAGGATACTTATAGATATGTA CAGTCACAGGCCATTACCTGTCAGAAACCCACACCCGAAAAAGAAAAACAGGACCCCTATAAGG ATATGAGTTTTTGGGAGGTTAACTTAAAAGAAAAGTTTTCTTCTGAATTAGATCAGTTTCCCCTTG GACGTAAGTTTTTATTGCAAAGTGGATATCGAGGACGGACGTCTGCTCGTACAGGTATAAAGCGC CCAGCTGTGTCTAAGCCCTCTACAGCCCCCAAACGAAAACGTACCAAAACCAAAAAGTAA Sequence16(SEQIDNO:16): ATGAGCGACAGCACAGTATATGTGCCTCCTCCCAACCCTGTATCCAAGGTTGTTGCCACGGATGC GTATGTTAAACGCACCAACATATTTTATCACGCCAGCAGTTCTAGACTCCTTGCTGTGGGACATCC ATATTACTCTATCAAAAAAGTTAACAAAACAGTTGTACCAAAGGTGTCTGGATATCAATATAGAG TGTTTAAGGTAGTGTTGCCAGATCCTAACAAGTTTGCATTACCTGATTCATCTCTGTTTGACCCCA CTACACAGCGTTTAGTATGGGCGTGCACAGGGTTGGAGGTAGGCAGGGGTCAACCTTTAGGCGTT GGTGTTAGTGGGCATCCATTGCTAAACAAATATGATGATGTAGAAAATAGTGGTGGGTATGGTGG TAATCCTGGTCAGGATAATAGGGTTAATGTAGGTATGGATTATAAACAAACCCAGCTATGTATGG TGGGCTGTGCTCCACCGTTAGGTGAACATTGGGGTAAGGGTACACAATGTTCAAATACCTCTGTA CAAAATGGTGACTGCCCCCCGTTGGAACTTATTACCAGTGTTATACAGGATGGGGACATGGTTGA TACAGGCTTTGGTGCTATGAATTTTGCAGACTTACAAACCAATAAATCGGATGTTCCCCTTGATAT TTGTGGAACTGTCTGCAAATATCCTGATTATTTGCAAATGGCAGCAGACCCTTATGGTGATAGGT TGTTTTTTTATTTGCGAAAGGAACAAATGTTTGCTAGACACTTTTTTAATAGGGCCGGTACTGTGG GGGAACCTGTGCCTGATGACCTGTTGGTAAAAGGGGGTAATAATAGGTCATCTGTAGCTAGTAGT ATTTATGTACATACACCTAGTGGATCCTTGGTGTCTTCAGAGGCTCAATTATTTAATAAACCATAT TGGCTTCAAAAGGCTCAGGGACATAACAATGGTATTTGCTGGGGAAACCACTTGTTTGTTACTGT GGTAGATACCACACGCAGTACAAATATGACACTATGTGCATCTGTAACTACATCTTCCACATACA CCAATTCTGATTATAAGGAATATATGCGCCATGTGGAGGAGTTTGATTTACAGTTTATTTTTCAAT TGTGTAGCATTACATTATCTGCAGAAGTCATGGCCTATATACACACAATGAATCCTTCTGTTTTGG AGGACTGGAACTTTGGTTTATCGCCTCCACCAAATGGTACACTGGAGGATACTTATAGATATGTA CAGTCACAGGCCATTACCTGTCAGAAACCCACACCCGAAAAAGAAAAACAGGACCCCTATAAGG ATATGAGTTTTTGGGAGGTTAACTTAAAAGAAAAGTTTTCTTCTGAATTAGATCAGTTTCCCCTTG GACGTAAGTTTTTATTGCAAAGTGGATATCGAGGACGGACGTCTGCTCGTACAGGTATAAAGCGC CCAGCTGTGTCTAAGCCCTCTACAGCCCCCAAACGAAAACGTACCAAAACCAAAAAGTAA Sequence35(SEQIDNO:35): KQCTNTPVQA Sequence36(SEQIDNO:36): TTSSTY Sequence37(SEQIDNO:37): FLNKYDDVENSGSGGNPGQDN

(13) Specific Modes for Carrying Out the Invention

(14) The present invention is further described by reference to the examples as follows, wherein the examples are used only for the purpose of illustrating the present invention, rather than limiting the present invention.

(15) Unless indicated otherwise, the molecular biological experimental methods and immunological assays used in the present invention are carried out substantially in accordance with the methods as described in Sambrook J et al., Molecular Cloning: A Laboratory Manual (Second Edition), Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; and restriction enzymes are used under the conditions recommended by the manufacturers. Those skilled in the art understand that the examples are used for illustrating the present invention, but not intended to limit the protection scope of the present invention.

Example 1

Expression and Purification of the Mutated HPV11 L1 Proteins

(16) Construction of Expression Vectors

(17) An expression vector encoding the mutated HPV11 L1 protein (H11N4-6T1) comprising a specific segment from HPV6 L1 protein was constructed by PCR for multi-site mutagenesis, wherein the initial template used was the plasmid pTO-T7-HPV11N4C (encoding the HPV11 L1 protein having 4 amino acids truncated at N-terminal; abbreviated as 11L1N4 in Table 2). The templates and primers for each PCR were shown in Table 2, and the amplification conditions for PCR were as followed: denaturation at 94 C. for 10 min; 25 cycles (denaturation at 94 C. for 50 sec, annealing at a given temperature for a certain period of time, and extension at 72 C. for 7.5 min); and finally extension at 72 C. for 10 min. The sequences of the PCR primers used were listed in Table 3.

(18) To the amplification product (50 L), 2 L restriction endonuclease DpnI was added, and the resultant mixture was incubated at 37 C. for 60 min. 10 L of the product of digestion was used to transform 40 L competent E. coli ER2566 (purchased from New England Biolabs) prepared by the Calcium chloride method. The transformed E. coli was spread onto solid LB medium (the components of the LB medium: 10 g/L peptone, 5 g/L yeast powder, 10 g/L NaCl, the same hereinafter) containing kanamycin (at a final concentration of 25 mg/mL, the same hereinafter), and was subjected to static culture at 37 C. for 10-12 h until single colonies could be observed clearly. Single colony was picked and inoculated into a tube containing 4 mL liquid LB medium (containing kanamycin), and cultured with shaking at 220 rpm for 10 h at 37 C., and then 1 ml bacterial solution was taken and stored at 70 C. Plasmids were extracted from E. coli, and T7 primer was used to sequence the nucleotide sequence of the fragment of interest inserted into the plasmids. The sequencing result showed that the nucleotide sequence of the fragment of interest inserted into the constructed plasmids (expression vectors) was SEQ ID NO: 12, and its encoded amino acid sequence was SEQ ID NO: 5 (the corresponding protein was designated as H11N4-6T1). The mutated protein H11N4-6T1 differs from HPV11N4 by: the substitution of the amino acid residues from positions 49 to 63 of wild type HPV11 L1 protein with the amino acid residues from positions 49 to 63 of wild type HPV6 L1 protein.

(19) Gibson assembly (Gibson D G, Young L, Chuang R Y, Venter J C, Hutchison C A, Smith H O. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods. 2009; 6:343-5. doi: 10.1038/nmeth.1318) was used to construct the expression vector encoding other mutated HPV11 L1 proteins, wherein the mutated HPV11 L1 proteins comprised a specific segment from HPV6 L1. In brief, a short fragment comprising mutations and a long fragment comprising no mutation were obtained by PCR, and Gibson assembly system was then used to ligate the two fragments to form a ring. The initial template used comprised the plasmid pTO-T7-HPV11N4C and the plasmid pTO-T7-HPV6N5C (encoding the HPV6 L1 protein having 5 amino acids truncated at N-terminal; abbreviated as 6L1N5 in Table 2). The templates and primers for each PCR were shown in Table 2, and, the amplification conditions for PCR for amplifying the short fragment were as followed: denaturation at 94 C. for 10 min; 25 cycles (denaturation at 94 C. for 50 sec, annealing at a given temperature for a certain period of time, and extension at 72 C. for 1 min); and finally extension at 72 C. for 10 min. The amplification conditions for PCR for amplifying the long fragment were as followed: denaturation at 94 C. for 10 min; 25 cycles (denaturation at 94 C. for 50 sec, annealing at a given temperature for a certain period of time, and extension at 72 C. for 7.5 min); and finally extension at 72 C. for 10 min. The sequences of the PCR primers used were listed in Table 3. The amplification product was subjected to electrophoresis, the fragment of interest was then recovered by using DNA Extraction Kit, and its concentration was determined. The short fragment and long fragment obtained by amplification were mixed at a molar ratio of 2:1 (a total volume of 3 L), and 3 L of 2 Gibson Assembly Master Mix (purchased from NEB, containing T5 exonuclease, Phusion DNA polymerase, Taq DNA ligase) was then added, and reacted at 50 C. for 1 h.

(20) The assembled product (6 L) was used to transform 40 L competent E. coli ER2566 (purchased from New England Biolabs) prepared by the Calcium chloride method. The transformed E. coli were spread onto solid LB medium containing kanamycin, and were subjected to static culture at 37 C. for 10-12 h until single colonies could be observed clearly. Single colony was picked and inoculated into a tube containing 4 mL liquid LB medium (containing kanamycin), and cultured with shaking at 220 rpm for 10 h at 37 C., and then 1 ml bacterial solution was taken and stored at 70 C. Plasmids were extracted from E. coli, and T7 primer was used to sequence the nucleotide sequences of the fragments of interest inserted into the plasmids. The sequencing result showed that the nucleotide sequences of the fragments of interest inserted into the constructed plasmids (expression vectors) were SEQ ID NO: 13, 14, 15 and 16, respectively, and their encoded amino acid sequences were SEQ ID NO: 6, 7, 8 and 9, respectively (the corresponding proteins were designated as H11N4-6T2, H11N4-6T3, H11N4-6T4, and H11N4-6T5, respectively).

(21) The mutated protein H11N4-6T2 differs from HPV11N4 by: the substitution of the amino acid residues from positions 119 to 140 of wild type HPV11 L1 protein with the amino acid residues from positions 119 to 139 of wild type HPV6 L1 protein. The mutated protein H11N4-6T3 differs from HPV11N4 by: the substitution of the amino acid residues from positions 170 to 179 of wild type HPV11 L1 protein with the amino acid residues from positions 169 to 178 of wild type HPV6 L1 protein. The mutated protein H11N4-6T4 differs from HPV11N4 by: the substitution of the amino acid residues from positions 257 to 288 of wild type HPV11 L1 protein with the amino acid residues from positions 256 to 287 of wild type HPV6 L1 protein. The mutated protein H11N4-6T5 differs from HPV11N4 by: the substitution of the amino acid residues from positions 346 to 351 of wild type HPV11 L1 protein with the amino acid residues from positions 345 to 350 of wild type HPV6 L1 protein.

(22) TABLE-US-00003 TABLE 2 PCR templates and primes for constructing expression vectors Template Upstream primer Downstream primer Product 11L1N4 H11N4-6T1-F H11N4-6T1-R H11N4-6T1 6L1N5 G-H11N4-6T2-F G-H11N4-6T2-R H11N4-612- short fragment 11L1N4 G-V-H11N4-6T2-F G-V-H11N4-6T2-R H11N4-6T2- long fragment 6L1N5 G-H11N4-6T3-F G-H11N4-6T3-R H11N4-6T3- short fragment 11L1N4 G-V-H11N4-6T3-F G-V-H11N4-6T3-R H11N4-6T3- long fragment 6L1N5 G-H11N4-6T4-F G-H11N4-6T4-R H11N4-6T4- short fragment 11L1N4 G-V-H11N4-6T4-F G-V-H11N4-6T4-R H11N4-6T4- long fragment 6L1N5 G-H11N4-6T5-F G-H11N4-6T5-R H11N4-6T5- short fragment 11L1N4 G-V-H11N4-6T5-F G-V-H11N4-6T5-R H11N4-6T5- long fragment

(23) TABLE-US-00004 TABLE3 Sequencesoftheprimersused (SEQIDNOs:17-34) SEQ ID Primer NO: name Primersequence(5-3) 17 H11N4- GTGGGACATCCATATTTTTCTATCAAACGGGCTAACAA 6T1-F AACAGTTGTAC 18 H11N4- GTACAACTGTTTTGTTAGCCCGTTTGATAGAAAAATAT 6T1-R GGATGTCCCAC 19 G-H11N4- TCAATATAGAGTGTTTAAGGTAGTGTTACCAGATCCTA 6T2-F ACAAATTTGC 20 G-H11N4- CCCACCATACATAGCTGGGTTTGTTTATAATCCATACC 6T2-R AACAT 21 G-V- AAACAAACCCAGCTATGTATGGTGG H11N4- 6T2-F 22 G-V- CACTACCTTAAACACTCTATATTGAT H11N4- 6T2-R 23 G-H11N4- CTGTACAGAATGGTGACTGCCCGCCCTTAG 6T3-F 24 G-H11N4- AACACTGTGTACCTTTACCCCAATGCTCGC 6T3-R 25 G-V- TAATACATCTGTACAGAATGGTGACTGCCCGCCCTTAG H11N4- 6T3-F 26 G-V- GATGTATTAGAACACTGTGTACCTTTACCCCAATGCTC H11N4- G 6T3-R 27 G-H11N4- GGAACAAATGTTTGCTAGACACTTTTTTAACAGGGCTG 6T4-F GCGAGGTGG 28 G-H11N4- ACCAAGGATCCACTAGGTGTATGAACATATATACTACT 6T4-R CCCTACAG 29 G-V- CATACACCTAGTGGATCCTTGG H11N4- 6T4-F 30 G-V- AAAGTGTCTAGCAAACATTTGTTCCT H11N4- 6T4-R 31 G-H11N4- TGGTATTTGCTGGGGAAACCACCTGTTTGTTACTGTGG 6T5-F TAGATAC 32 G-H11N4- GAAAAATAAACTGTAAATCAAACTCTTCCACATGACG 6T5-R CATGTACTC 33 G-V- TTTGATTTACAGTTTATTTTTC H11N4- 6T5-F 34 G-V- GTGGTTTCCCCAGCAAATACCATTG H11N4- 6T5-R

(24) Expression of the Mutated Proteins on a Large Scale

(25) The E. coli solutions comprising the recombinant plasmid pTO-T7-H11N4-6T1, pTO-T7-H11N4-6T2, pTO-T7-H11N4-6T3, pTO-T7-H11N4-6T4, and pTO-T7-H11N4-6T5, respectively, were taken from 70 C. refrigerator, seeded in 100 mL LB liquid medium containing kanamycin, and incubated at 200 rpm and 37 C. for about 8 h, respectively. Then, the culture was transferred to 500 mL LB medium containing kanamycin (1 ml bacterial solution was transferred), and was further incubated. When the bacterial concentration reached an OD.sub.600 of about 0.6, the culturing temperature was lowered to 25 C. and 500 L IPTG was added to each culture bottle. The incubation was further performed for 8 h. After the incubation was finished, the bacteria were collected by centrifugation. The bacteria expressing H11N4-6T1, H11N4-6T2, H11N4-6T3, H11N4-6T4 and H11N4-6T5 protein, were obtained, respectively.

(26) Disruption of Bacteria Expressing the Mutated Proteins

(27) The bacteria obtained were re-suspended at a ratio of 1 g bacteria to 10 mL lysis buffer (20 mM Tris buffer, pH7.2, 300 mM NaCl). The bacteria were disrupted by using an ultrasonic apparatus for 30 min. The lysis solution containing the disrupted bacteria were centrifuged at 13500 rpm (30000 g) for 15 min, and the supernatant (i.e. the supernatant of disrupted bacteria) was obtained.

(28) Chromatographic Purification of the Mutated Proteins

(29) Equipment: AKTA Explorer 100 preparative liquid chromatography system produced by GE Healthcare (i.e. the original Amershan Pharmacia Co.)

(30) Chromatographic media: SP Sepharose 4 Fast Flow (GE Healthcare Co.), CHT-II (purchased from Bio-RAD) and Butyl Sepharose 4 Fast Flow (GE Healthcare Co.)

(31) Buffer: 20 mM phosphate buffer, pH8.0, 20 mM DTT; and, 20 mM phosphate buffer, pH8.0, 20 mM DTT, 2M NaCl.

(32) Sample: the supernatants of disrupted bacteria containing H11N4-6T1, H11N4-6T2, H11N4-6T3, H11N4-6T4 and H11N4-6T5, respectively, as obtained above.

(33) Elution Protocol:

(34) (1) Cation exchange purification of the supernatant of disrupted bacteria by SP Sepharose 4 Fast Flow: the sample was loaded on the column, undesired proteins were then eluted with a buffer containing 400 mM NaCl, followed by the elution of the protein of interest with a buffer containing 800 mM NaCl, and the fraction eluted with the buffer containing 800 mM NaCl was collected;

(35) (2) Chromatographic purification of the elution fraction obtained in the step (1) by CHT-II (hydroxyapatite chromatography): the elution fraction obtained in the step (1) was diluted so that the NaCl concentration was decreased to 0.5 M; the sample was loaded on the column, undesired proteins were then eluted with a buffer containing 500 mM NaCl, followed by the elution of the protein of interest with a buffer containing 1000 mM NaCl, and the fraction eluted with the buffer containing 1000 mM NaCl was collected;

(36) (3) Chromatographic purification of the elution fraction obtained in the step (2) by HIC (hydrophobic interaction chromatography): the sample was loaded on the column, undesired proteins were then eluted with a buffer containing 1000 mM NaCl, followed by the elution of the protein of interest with a buffer containing 200 mM NaCl, and the fraction eluted with the buffer containing 200 mM NaCl was collected.

(37) 150 L elution fraction obtained in the step (3) was added to 30 L, of 6 Loading Buffer. The resultant solution was mixed homogeneously and incubated in 80 C. water bath for 10 min. 10 l of the resultant sample was then subjected to 10% SDS-PAGE at 120V for 120 min; and the electrophoretic bands were stained by Coomassie brilliant blue. The electrophoretic result was shown in FIG. 1. The result showed that after said purification steps, H11N4-6T1, H11N4-6T2, H11N4-6T3, H11N4-6T4 and H11N4-6T5 protein had a purity of above 95%.

(38) By similar methods, HPV11N4 protein was prepared and purified by using E. coli and the plasmid pTO-T7-HPV11N4C; HPV6N5 protein was prepared and purified by using E. coli and the plasmid pTO-T7-HPV6N5C.

Example 2

Assembly of HPV Virus-Like Particles and Morphological Test of Particles

(39) Assembly of HPV Virus-Like Particles

(40) A given volume (about 2 ml) of the protein H11N4-6T1, H11N4-6T2, H11N4-6T3, H11N4-6T4 or H11N4-6T5, was dialyzed to (1) 2 L storage buffer (20 mM sodium phosphate buffer pH 6.5, 0.5 M NaCl); (2) 2 L renaturation buffer (50 mM sodium phosphate buffer pH 6.0, 2 mM CaCl2, 2 mM MgCl2, 0.5 M NaCl); and (3) 20 mM sodium phosphate buffer pH 7.0, 0.5 M NaCl, successively. The dialysis was performed in each of the three buffers for 12 h.

(41) By similar methods, the HPV11N4 and HPV6N5 protein were assembled into HPV11N4 VLP and HPV6N5 VLP, respectively.

(42) Molecular Sieve Chromatographic Analysis

(43) The dialyzed sample was subjected to molecular sieve chromatographic analysis by 1120 Compact LC High Performance Liquid Chromatographic System (Agilent Technologies), wherein the analytical column used was TSK Gel PW5000xl 7.8300 mm. The analysis results were shown in FIGS. 2A-2F. The results showed that the first protein peak of the samples comprising the protein H11N4-6T1, H11N4-6T2, H11N4-6T3, H11N4-6T4 and H11N4-6T5 respectively appeared at about 12 min, which was comparable to that of HPV11N4 VLP. This showed that the proteins prepared above were able to assemble into VLPs.

(44) Sedimentation Velocity Analysis

(45) The apparatus for sedimentation velocity analysis was Beckman XL-A Analytical Ultracentrifuge, equipped with optical inspection system and An-50Ti and An-60Ti rotor. The sedimentation coefficient of HPV11N4 VLP, H11N4-6T1 VLP, H11N4-6T2 VLP, H11N4-6T3 VLP, H11N4-6T4 VLP and H11N4-6T5 VLP was analyzed by sedimentation velocity method. The results were shown in FIGS. 3A-3F. The results showed that the sedimentation coefficient of H11N4-6T1 VLP, H11N4-6T2 VLP, H11N4-6T3 VLP, H11N4-6T4 VLP and H11N4-6T5 VLP was 140S, 138S, 111S, 139S and 139S, respectively. This showed that said 5 mutated HPV11 L1 proteins were able to assemble into virus-like particles that were similar to wild type VLP (HPV11N4 VLP, 136.3S) in terms of size and morphology.

(46) Morphological Test of Virus-Like Particles

(47) A 100 L sample comprising VLP was observed by transmission electron microscope (TEM). The equipment used was a 100 kV Transmission Electron Microscope supplied by JEOL Ltd. (100,000 magnification). In brief, a 13.5 L sample was negatively stained with 2% phosphotungstic acid (pH 7.0), fixed on a carbon-coated copper grid, and then observed by TEM. The results were shown in FIGS. 4A-4F. The result showed that H11N4-6T1, H11N4-6T2, H11N4-6T3, H11N4-6T4 and H11N4-6T5 were able to assemble into virus-like particles. In addition, the results also showed that the particles assembled by these mutated proteins had a radius of about 25 nm, and were uniform in size. This indicated that these mutated proteins were similar to wild type HPV11 L1 protein (HPV11N4 VLP), and were able to assemble into VLPs with a uniform size.

Example 3

Evaluation of Thermostability of Virus-Like Particles

(48) The VLPs formed by HPV11N4, H11N4-6T1, H11N4-6T2, H11N4-6T3, H11N4-6T4 and H11N4-6T5 were evaluated for their thermostability by using differential scanning calorimeter VP Capillary DSC purchased from GE Company (i.e. the original MicroCal Co.), wherein the storage buffer for the protein was used as control, and the proteins were scanned at a heating rate of 1.5 C./min within a temperature range of 10 C.-90 C. The detection results were shown in FIGS. 5A-5F. The results showed that all these VLPs formed by the proteins had very high thermostability.

Example 4

Reconstruction of Three-Dimensional Structures of H11N4-6T3 VLP and H11N4-6T5 VLP

(49) In accordance with the previously reported method (Wolf M, Garcea R L, Grigorieff N. et al. Proc Natl Acad Sci USA. (2010), 107(14): 6298-303), the three-dimensional structures of H11N4-6T3 VLP and H11N4-6T5 VLP were reconstructed by using cryo-electron microscopy (cryoEM). In brief, cryo-electron microscopy (cryoEM) was used to observe H11N4-6T3 VLP and H11N4-6T5 VLP, and then in the cryo-electron microscopy (cryoEM) photographs of H11N4-6T3 VLP and H11N4-6T5 VLP (FIGS. 6A and 6C), 300 particles and 360 particles with an uniform size and a diameter of greater than 50 nm were selected for computer overlapping and structural reconstruction, respectively, thereby obtaining the three-dimensional structures of H11N4-6T3 VLP and H11N4-6T5 VLP. The three-dimensional structures obtained were shown in FIGS. 6B and 6D (at a resolution of 17.38 and 20.48 , respectively). The results showed that both H11N4-6T3 VLP and H11N4-6T5 VLP had a T=7 icosahedral structure (h=1, k=2) consisting of 72 capsomers (morphological subunit, pentamer). Unlike conventional icosahedral viral capsids consistent with quasi-equivalence principle, all the constitutive subunits in the structures of H11N4-6T3 VLP and H11N4-6T5 VLP were pentamers, without hexamer. Moreover, said VLPs had an external diameter of about 60 nm. These were similar to the three-dimensional structures of the previously reported natural HPV viral particles and the HPV VLP prepared by eukaryotic expression system (e.g. poxvirus expression system) (Baker T S, Newcomb W W, Olson N H. et al. Biophys J. (1991), 60(6): 1445-1456. Hagensee M E, Olson N H, Baker T S, et al. J Virol. (1994), 68(7):4503-4505. Buck C B, Cheng N, Thompson C D. et al. J Virol. (2008), 82(11): 5190-7).

Example 5

Evaluation of Immune Protection of Virus-Like Particles in Animals

(50) The immune protection of the VLPs formed by H11N4-6T1, H11N4-6T2, H11N4-6T3, H11N4-6T4 and H11N4-6T5, was evaluated in mice. Animals for vaccination were BALB/c mice (ordinary grade), 5-6 weeks old (purchased from Shanghai SLAC Laboratory Animal Co. LTD.).

(51) The HPV11N4 VLP, HPV6N5 VLP, H11N4-6T1 VLP, H11N4-6T2 VLP, H11N4-6T3 VLP, H11N4-6T4 VLP, H11N4-6T5 VLP and a mixed HPV11/HPV6 VLP (i.e. a mixture of HPV11N4 VLP and HPV6N5 VLP) as prepared above were absorbed onto aluminum adjuvant, respectively. Mice were divided into 8 groups depending on immunogen, and each group included 4 mice. Vaccination procedure was as followed: the first vaccination at Week 0, and the booster vaccination at Weeks 2 and 4, respectively. Mice were vaccinated via subcutaneous injection. The immunogens used and doses thereof were shown in Table 4. At Week 8 after the first vaccination, venous blood was collected from eyeball, and serum was separated. The titers of neutralizing antibodies in the serum were determined. The detection result was shown in FIG. 7. The result showed that either of H11N4-6T3 VLP and H11N4-6T5 VLP could induce the generation of high-titer neutralizing antibodies against HPV11 and HPV6 in mice; and their protective effects against HPV11 were comparable to that of HPV11N4 VLP alone or the mixed HPV11/HPV6 VLP, and were significantly higher than that of HPV6N5 VLP alone; and their protective effects against HPV6 were comparable to that of HPV6N5 VLP alone or the mixed HPV11/HPV6 VLP, and were significantly higher than that of HPV11N4 VLP alone. H11N4-6T2 VLP could induce the generation of high-titer neutralizing antibodies against HPV11 and HPV6 in mice, but its ability of inducing the generation of neutralizing antibodies against HPV6 was weaker than that of H11N4-6T3 VLP and H11N4-6T5 VLP. These results showed that H11N4-6T2 VLP, H11N4-6T3 VLP and H11N4-6T5 VLP could be used as effective vaccines for preventing HPV11 infection and HPV6 infection, and could be used in place of a mixed vaccine comprising HPV11 VLP and HPV6 VLP.

(52) TABLE-US-00005 TABLE 4 Vaccination schedule Vaccination Antigen for procedure vaccination Adjuvant Immunizing dose Number (week) HPV6N5 VLP aluminum 5 g 4 0, 2, 4 adjuvant HPV11N4 VLP aluminum 5 g 4 0, 2, 4 adjuvant mixed HPV11/ aluminum 10 g 4 0, 2, 4 HPV6 VLP adjuvant (5 g for each VLP) H11N4-6T1 VLP aluminum 5 g 4 0, 2, 4 adjuvant H11N4-6T2 VLP aluminum 5 g 4 0, 2, 4 adjuvant H11N4-6T3 VLP aluminum 5 g 4 0, 2, 4 adjuvant H11N4-6T4 VLP aluminum 5 g 4 0, 2, 4 adjuvant H11N4-6T5 VLP aluminum 5 g 4 0, 2, 4 adjuvant

Example 6

Evaluation of Neutralizing Antibody Titer in Serum of Mice Vaccinated with VLP

(53) In this experiment, vaccination schedule was shown in Table 5. All the mice (6-week old BalB/c female mice) were divided into 3 groups: Aluminum adjuvant group 1 (at an immunizing dose of 10 g, using aluminum adjuvant), Aluminum adjuvant group 2 (at an immunizing dose of 1 g, using aluminum adjuvant), and Aluminum adjuvant group 3 (at an immunizing dose of 0.1 g, using aluminum adjuvant). Each group was further divided into 5 subgroups. The Control subgroups 1-3 were vaccinated with HPV11N4 VLP alone, HPV6N5 VLP alone and a mixed HPV11/HPV6 VLP (i.e. a mixture of HPV11N4 VLP and HPV6N5 VLP, wherein each VLP was administered at a given immunizing dose), respectively, and the Experimental subgroups 1-2 were vaccinated with H11N4-6T3 VLP and H11N4-6T5 VLP, respectively.

(54) 6 mice/subgroup were vaccinated by intraperitoneal injection, at an immunizing dose of 10 g, 1 g, 0.1 g, respectively, and an injection volume of 1 ml. All the mice were subjected to the first vaccination at Week 0, and then subjected to the booster vaccination at Weeks 2 and 4, respectively. At Week 8, blood sample was collected via orbital bleeding, and the titers of antibodies against HPV11 and HPV6 in serum were analyzed. The analysis results were shown in FIGS. 8A-8C. The results showed that either of H11N4-6T3 VLP and H11N4-6T5 VLP could induce the generation of high-titer neutralizing antibodies against HPV11 in mice, and their protective effects were comparable to that of HPV11N4 VLP alone or the mixed HPV11/HPV6 VLP at the same dose, and were significantly superior to that of HPV6N5 VLP alone at the same dose; and they could induce the generation of high-titer neutralizing antibodies against HPV6 in mice, and their protective effects were comparable to that of HPV6N5 VLP alone or the mixed HPV11/HPV6 VLP at the same dose, and were significantly superior to that of HPV11N4 VLP alone at the same dose. This showed that H11N4-6T3 VLP and H11N4-6T5 VLP had good cross-immunogenicity and cross-protection against HPV11 and HPV6.

(55) TABLE-US-00006 TABLE 5 Vaccination schedule Immunizing Vaccination Group Antigen for vaccination Adjuvant dose Number procedure (week) Aluminum HPV11N4 VLP aluminum 10 g 6 0, 2, 4 adjuvant group 1 adjuvant HPV6N5 VLP aluminum 10 g 6 0, 2, 4 adjuvant a mixed HPV11/HPV6 VLP aluminum 10 g for 6 0, 2, 4 adjuvant each H11N4-6T3 VLP aluminum 10 g 6 0, 2, 4 adjuvant H11N4-6T5 VLP aluminum 10 g 6 0, 2, 4 adjuvant Aluminum HPV11N4 VLP aluminum 1 g 6 0, 2, 4 adjuvant adjuvant Group 2 HPV6N5 VLP aluminum 1 g 6 0, 2, 4 adjuvant a mixed HPV11/HPV6 VLP aluminum 1 g for 6 0, 2, 4 adjuvant each H11N4-6T3 VLP aluminum 1 g 6 0, 2, 4 adjuvant H11N4-6T5 VLP aluminum 1 g 6 0, 2, 4 adjuvant Aluminum HPV11N4 VLP aluminum 0.1 g 6 0, 2, 4 adjuvant group 3 adjuvant HPV6N5 VLP aluminum 0.1 g 6 0, 2, 4 adjuvant a mixed HPV11/HPV6 VLP aluminum 0.1 g for 6 0, 2, 4 adjuvant each H11N4-6T3 VLP aluminum 0.1 g 6 0, 2, 4 adjuvant H11N4-6T5 VLP aluminum 0.1 g 6 0, 2, 4 adjuvant

Example 7

Evaluation of ED50 of VLP for Inducing Seroconversion

(56) 6-week old BalB/c female mice (8 mice) were vaccinated with aluminum adjuvant by single intraperitoneal injection, wherein H11N4-6T3 VLP or H11N4-6T5 VLP (at an immunizing dose of 0.300 g, 0.100 g, 0.033 g or 0.011 g) was used in the Experimental groups; and HPV6N5 VLP alone or HPV11N4 VLP alone (at an immunizing dose of 0.300 g, 0.100 g, 0.033 g or 0.011 g), or a mixed HPV11/HPV6 VLP (i.e. a mixture of HPV6N5 VLP and HPV11N4 VLP, wherein the immunizing dose for each VLP was 0.300 g, 0.100 g, 0.033 g, 0.011 g or 0.004 g) was used in the Control groups; and the immunizing volume was 1 mL. In addition, the diluent for diluting a vaccine was used as blank control. 8 Mice were vaccinated in each group, and at Week 5 after vaccination, serum was collected. Later, the neutralizing antibody titer of serum was determined by neutralization test, and by Reed-Muench method (Reed L J M H. A simple method of estimating fifty percent endpoints. Am J Hyg. 1938; 27:493-7), ED.sub.50 for inducing seroconversion (i.e. inducing the generation of antibodies in mice) was calculated for each sample. The results were shown in Tables 6.1-6.5.

(57) TABLE-US-00007 TABLE 6.1 ED.sub.50 of HPV6N5 VLP for inducing antibodies against HPV6 and HPV11 (seroconversion) in mice Tota Number of Immunizing number mice with dose of positive Positive ED.sub.50 Antibody (g) mice conversion conversion rate (g) Antibody 0.300 8 7 92.31% 0.090 against 0.100 8 5 55.56% HPV6 0.033 8 0 0.00% 0.011 8 0 0.00% Antibody 0.300 8 1 22.22% >0.3 against 0.100 8 0 6.25% HPV11 0.033 8 1 4.35% 0.011 8 0 0.00%

(58) TABLE-US-00008 TABLE 6.2 ED.sub.50 of H11N4-6T3 VLP for inducing antibodies against HPV6 and HPV11 (seroconversion) in mice Total Number of Immunizing number mice with dose of positive Positive ED.sub.50 Antibody (g) mice conversion conversion rate (g) Antibody 0.300 8 8 100.00% 0.025 against 0.100 8 7 92.86% HPV6 0.033 8 6 66.67% 0.011 8 0 0.00% Antibody 0.300 8 8 100.00% 0.073 against 0.100 8 5 66.67% HPV11 0.033 8 1 9.09% 0.011 8 0 0.00%

(59) TABLE-US-00009 TABLE 6.3 ED.sub.50 of H11N4-6T5 VLP for inducing antibodies against HPV6 and HPV11 (seroconversion) in mice Number of Immunizing Total mice with dose number of positive Positive ED.sub.50 Antibody (g) mice conversion conversion rate (g) Antibody 0.300 8 4 66.67% 0.180 against 0.100 8 3 30.70% HPV6 0.033 8 1 5.88% 0.011 8 0 0.00% Antibody 0.300 8 6 81.82% 0.189 against 0.100 8 2 27.27% HPV11 0.033 8 0 5.88% 0.011 8 1 4.17%

(60) TABLE-US-00010 TABLE 6.4 ED.sub.50 of HPV11N4 VLP for inducing antibodies against HPV6 and HPV11 (seroconversion) in mice Total Number of Immunizing number mice with dose of positive Positive ED.sub.50 Antibody (g) mice conversion conversion rate (g) Antibody 0.300 8 3 44.44% >0.3 against 0.100 8 1 7.69% HPV6 0.033 8 0 0.00% 0.011 8 0 0.00% Antibody 0.300 8 8 100.00% 0.044 against 0.100 8 7 91.67% HPV11 0.033 8 2 36.36% 0.011 8 2 13.33%

(61) TABLE-US-00011 TABLE 6.5 ED.sub.50 of a mixed HPV11/HPV6 VLP for inducing antibodies against HPV6 and HPV11 (seroconversion) in mice Total Number of number mice with Positive Immunizing dose of positive conversion ED.sub.50 Antibody (g) mice conversion rate (g) Antibody 0.300 for each VLP 8 7 95.24% 0.033 against 0.100 for each VLP 8 8 92.86% HPV6 0.033 for each VLP 8 4 50.00% 0.011 for each VLP 8 1 7.69% Antibody 0.300 for each VLP 8 7 95.65% 0.023 against 0.100 for each VLP 8 8 93.75% HPV11 0.033 for each VLP 8 6 70.00% 0.011 for each VLP 8 1 9.09%

(62) The results showed that ED.sub.50 of H11N4-6T3 VLP and H11N4-6T5 VLP for inducing the generation of antibodies against HPV6 in mice was comparable to that of HPV6N5 VLP alone and that of the mixed HPV11/HPV6 VLP, and was significantly superior to that of HPV11N4 VLP alone; and, ED.sub.50 of H11N4-6T3 VLP and H11N4-6T5 VLP for inducing the generation of antibodies against HPV11 in mice was comparable to that of HPV11N4 VLP alone and that of the mixed HPV11/HPV6 VLP, and was significantly superior to that of HPV6N5 VLP alone. This further showed that H11N4-6T3 VLP and H11N4-6T5 VLP had good cross-immunogenicity and cross-protection against HPV6 and HPV11.

Example 8

Evaluation of Immune Protection of H11N4-6T3 VLP and H11N4-6T5 VLP in Cynomolgus Monkey

(63) 18 Cynomolgus monkeys with a similar body weight were randomly divided into 3 groups (6 monkeys for each group), wherein, monkeys in Group 1 were vaccinated with 5 g H11N4-6T3 VLP; monkeys in Group 2 were vaccinated with 5 g H11N4-6T5 VLP; and monkeys in Group 3 were vaccinated with 10 g mixed HPV11/HPV6 VLP (5 g HPV6N5 VLP+5 g HPV11N4 VLP). The adjuvant used was aluminum adjuvant, the injection volume was 1 ml, and the monkeys were vaccinated by intramuscular injection. Vaccination schedule was shown in Table 7.

(64) Two months after vaccination, venous blood was collected, and the neutralizing antibody titer of serum was determined by neutralization test. The experimental result was shown in FIG. 9. The result showed that either of H11N4-6T3 VLP and H11N4-6T5 VLP could induce the generation of neutralizing antibodies against HPV11 and HPV6 in cynomolgus monkeys; and the titer of the neutralizing antibodies induced by them was comparable to the titer of the neutralizing antibodies induced by the mixed HPV11/HPV6 VLP. These results showed that both of H11N4-6T3 VLP and H11N4-6T5 VLP have good immunogenicity, and could induce cross-protection against HPV6 and HPV11 in cynomolgus monkey, and their protective effects against HPV11 and HPV6 were comparable to that of the mixed HPV11/HPV6 VLP. Therefore, H11N4-6T3 VLP and H11N4-6T5 VLP could be used to prevent infection by HPV6 and HPV11.

(65) TABLE-US-00012 TABLE 7 Vaccination schedule for cynomolgus monkey Vaccination Immunogen Adjuvant Immunizing dose Number procedure a mixed aluminum 10 g 6 single injection HPV11/HPV6 adjuvant (5 g for VLP each VLP) H11N4-6T3 aluminum 5 g 6 single injection VLP adjuvant H11N4-6T5 aluminum 5 g 6 single injection VLP adjuvant

(66) Although the specific embodiments of the present invention have been described in details, those skilled in the art would understand that, according to the teachings disclosed in the specification, various modifications and changes can be made thereto, and that such modifications and changes are within the scope of the present invention. The scope of the present invention is given by the appended claims and any equivalents thereof.