WO2017157173A1 - 一种乳头瘤病毒嵌合蛋白及其用途 - Google Patents

一种乳头瘤病毒嵌合蛋白及其用途 Download PDF

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WO2017157173A1
WO2017157173A1 PCT/CN2017/075402 CN2017075402W WO2017157173A1 WO 2017157173 A1 WO2017157173 A1 WO 2017157173A1 CN 2017075402 W CN2017075402 W CN 2017075402W WO 2017157173 A1 WO2017157173 A1 WO 2017157173A1
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protein
hpv16
type
chimeric
amino acid
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French (fr)
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许雪梅
陈雪
王志荣
刘洪洋
望硕
张婷
胡美丽
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中国医学科学院基础医学研究所
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Publication of WO2017157173A1 publication Critical patent/WO2017157173A1/zh

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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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    • A61K2039/5258—Virus-like particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to the field of biotechnology, in particular to a papillomavirus chimeric protein, and virus-like particles formed therefrom, and papillomavirus chimeric protein or papillomavirus chimeric virus-like particles in preparing for preventing papillomavirus infection And use in vaccines for infection-induced diseases.
  • HPV human papillomavirus
  • the induced lesions can be divided into high-risk types that induce malignant tumors (including HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, etc.); HPV26, 30, 53, 66, 67, 69, 70, 73, 82, 85, etc.); not yet determined (HPV34, 42, 43, 54, 71, 81, 83, 97, 102, 114, etc.); Low-risk type of benign lesions such as verrucous hyperplasia (HPV6, 7, 11, 13, 32, 40, 42, 44, 61, 62, 72, 74, 81, 83, 84, 86, 87, 89, 90, 91 , 106, etc.).
  • the skin-skinned group mainly infects skin tissue other than the above-mentioned parts, induces skin-like hyperplasia, and is closely related to the occurrence of certain skin cancers.
  • Cervical cancer Malignant tumors associated with high-risk HPV infection have been identified: cervical cancer, vaginal cancer, labial cancer, penile cancer, anal perianal cancer, oropharyngeal cancer, tonsil cancer, oral cancer, among which cervical cancer is the most harmful. Cervical cancer is the third most common malignant tumor in women in the world, with an annual incidence of about 527,000, including 285,000 in Asia; and an annual incidence of 75,000 in China.
  • HPV16 is the dominant strain in the world, and the detection rate of HPV-related tumors such as cervical cancer, perianal cancer, penile cancer, vulvar cancer and other related cancers and precancerous lesions is the highest.
  • cervical cancer associated with HPV16 infection accounts for 58.7% of the total number of cervical cancers in China, accounting for 53.5% of the total incidence of cervical cancer in the world, and the remaining 41.3%-46.5% of cervical cancers are caused by the merger of about 19 high-risk HPV infections.
  • VLP HPV L1 protein-like particle
  • GlaxoSmithKline's bivalent vaccine Cervarix HPV16/18
  • Merck's tetravalent seedling Gardasil HPV6/11/16/18
  • the nine-valent seedling Gardasil 9 HPV6/11/16/18/31/33/35/45/52/58. Since the immune protection responses induced by such vaccines are mainly directed at the vaccine type, most of these vaccines are HPV multivalent vaccines, and the prevention of broad-spectrum vaccines needs to continue to increase the price of vaccines.
  • the viral minor capsid protein L2 induces cross-neutralizing antibodies and has in vivo cross-protective activity.
  • the immune serum of amino acid (aa.) 17-36 polypeptide of HPV16 L2 protein can neutralize HPV16 with high titer. /18, It also effectively neutralizes HPV5/6/45/52/58 (Gambhira R, Karanam B, et al. J. Virol.
  • the monoclonal antibody RG-1 also has cross-neutralizing activity (Gambhira R, Karanam B, et al. J. Virol. 2007; 81(24): 13927–13931), therefore, the a2.17- of the L2 protein with HPV16 L2
  • the 36 polypeptide homology region is also referred to as the RG-1 epitope.
  • thioredoxin Trx
  • phage VLP phage VLP
  • plant virus VLP viral VLPs infected with mammals
  • viral VLPs infected with mammals adeno-associated virus, bovine papillomavirus-1, HPV16
  • HPV16L2 aa.17-36 A peptide-based fusion protein vaccine that significantly increases the immunogenicity of the polypeptide, increases the titer of neutralizing antibodies, and cross-neutralizes or protects the range (Christina S, Richard R, et al. J. Virol. 2009; 83 (19 ): 10085-10095; Seitz H, Canali E, et al.
  • the HPV L2 aa.17-36 polypeptide region has high amino acid sequence homology between different species of papillomaviruses.
  • the existing vaccines based on different HPV type RG-1 epitopes are as follows: The HPV31/51 type RG-1 epitope is inserted into the surface of the bacterial protein Trx, and the obtained immune serum has cross-neutralizing activity, but is neutralized. The type is relatively small (Seitz H, Canali E, et al. Vaccine 2014; 32: 2610–2617); the HPV16/31 RG-1 epitope is inserted into the surface of the adeno-associated virus VLP, and the obtained immune sera is neutralized.
  • truncated polypeptides of various types of RG-1 regions were inserted into the surface of phage VLPs, respectively.
  • Mice after mixed immunization with VLP (Chimeric VLP, cVLP) can produce immunoprotective responses against 8 types of viruses (Tumban E, Peabody J, et al. PLoS One 2011; 6(8): e23310) due to lack
  • VLP Chimeric VLP
  • cVLP Chimeric VLP, cVLP
  • Tumban E Peabody J, et al. PLoS One 2011; 6(8): e23310
  • HPV16 type RG-1 epitope is the most immunogenic, and the broad-spectrum neutralizing antibody reaction can be induced by inserting HPV and phage VLP surface; HPV31/45/ The 51 type may be followed, the other types are unknown (lack of reports); it is worth noting that the 8 types (HPV1/5/6/11/16/18/45/58) RG-1 region is truncated Anti-serum obtained by mixed immunization with peptide (L2aa.17-31) phage cVLP, the type of neutralization is not much, suggesting that some types of truncated polypeptides may be inactive or weakly active, and the specific analysis is different.
  • the immunogenicity of the RG-1 epitope polypeptide and the truncated polypeptide is expected to clarify the difference in immunogenicity and immunogenicity. Therefore, in addition to HPV16, there is currently little or no research on the immunogenicity of other different types of RG-1 epitope regions; and there is a lack of comparative analysis of immunogenicity for different types of RG-1 epitope regions; Importantly, in the relevant vector vaccine studies, the choice of RG-1 epitope type is not based on its immunogenicity, but on other factors, such as whether the type of virus is dominant and infection-related. Whether the degree of harm is serious. Therefore, the existing vaccine research based on RG-1 epitope needs to be improved.
  • HPV33 and the detection rate of HPV infection-related tumors are high.
  • the detection rate of cervical cancer specimens is 2.6% in the world; 3.8% in Asia and China, second only to HPV16.
  • Type /18/58 X.Castellsagué et al. Vaccine 25S (2007) C1–C26.
  • the immunogenicity of the HPV33 L2 protein RG-1 epitope is lacking, and whether the epitope can induce neutralizing antibodies and the neutralizing range and characteristics of the induced antibodies are not clear. Based on current research progress and knowledge, the immunological activity of the HPV33 RG-1 epitope vaccine is unpredictable.
  • the HPV16 L2 RG-1 epitope vaccine vector was found to be a promising HPV16 L2 RG-1 epitope vaccine. It can be displayed on the VLP surface after insertion into the HPV16 L2 RG-1 epitope in a specific location on the surface of the HPV16 L1 protein. After immunization, broad-spectrum neutralizing antibodies and protective responses can be induced. For example, at the DEV loop (aa 136/137) site of HPV16L1 protein, the chimeric VLP (cVLP) formed by direct insertion of HPV16 L2 aa.17-36 can induce a broad-spectrum neutralizing antibody response, neutralizing at least 14 HPV types. (Schellenbacher C, Roden R, et al 2009; J.
  • the present invention selects the HPV33 type RG-1 epitope and the truncated RG-1 epitope for the study of HPV type 16 cVLP, and the results show that the HPV33 type RG-1 long epitope and short obtained by the present invention are short.
  • Epitope cVLP is highly immunogenic (can neutralize at least 10 HPV types) and is comparable to the HPV16 RG-1 VLP reported in the literature, but the range of neutralizing types is different (Schellenbacher C, Roden R, et al 2009; J. Virol. 2009; 83(19): 10085–10095; Schellenbacher C, Kwak K, et al. J. Invest. Derma. 2013; doi: 10.1038/jid. 2013.253).
  • an object of the present invention is to provide a papillomavirus chimeric protein for use in the preparation of a vaccine for preventing papillomavirus infection and infection-induced diseases.
  • the present inventors have unexpectedly discovered that insertion of the HPV33 L2 protein polypeptide into the surface region of the full-length or truncated HPV16 L1 protein can increase the immunogenicity of the HPV33 L2 polypeptide, and the obtained chimeric protein is expressed in Escherichia coli or insect cells. Highly expressed in the system, the chimeric protein can be assembled into VLPs and can be tempted A broad-spectrum protective immune response against multiple types of HPV from different genera/subgenus.
  • the present invention has been completed based on the above findings, and data is provided in the examples herein.
  • the present invention provides a papillomavirus chimeric protein, the skeleton of which is a L1 protein of a papillomavirus or a mutant of a L1 protein of a papillomavirus, wherein at least one of the backbone is chimeric from HPV33 A polypeptide of the L2 protein.
  • polypeptide is selected from any of the consecutive 8-33 amino acid fragments within the aa. 1-200 region of the HPV33 L2 protein (amino acid sequence as set forth in SEQ ID No. 7). Further preferably, the polypeptide is the HPV33 L2 protein RG-1 epitope region.
  • amino acid sequence of the polypeptide is shown in SEQ ID No. 1.
  • the polypeptide is a polypeptide obtained by lengthening or truncating 1-5 amino acids at the N-terminus of the amino acid sequence shown in SEQ ID No. 1, and/or prolonging or truncating 1-5 amino acids at the C-terminus. .
  • amino acid sequence of the polypeptide is as shown in SEQ ID No. 2 or SEQ ID No. 3.
  • the polypeptide may also be a polypeptide having greater than 60% homology with the amino acid sequence shown in SEQ ID No. 1, preferably a polypeptide having a homology greater than 70%, and a polypeptide having a homology greater than 80%.
  • the backbone is a mutant of HPV16 L1 protein or HPV16 L1 protein.
  • the HPV16 L1 protein is selected from a mutant of a high-risk HPV L1 protein or a high-risk HPV L1 protein; further preferably, the chimeric protein backbone involved in the present invention is selected from the group consisting of HPV16 L1 protein (eg NCBI database AAC09292.1) Sequence) or HPV16 L1 protein mutant.
  • the HPV16 L1 protein backbone can be derived from, but not limited to, the L1 protein of HPV16 Phi1, Tha7, Alg1, Sen32, Fra25, Fra63, 114K, 114B, Z-1194 and other variants (Touze A, Mehdaoui SE, et al. J. Clin. Micr .1998;36(7):2046-2051).
  • the amino acid sequence of the HPV16 L1 protein is shown in SEQ ID No. 4.
  • the mutant of the HPV16 L1 protein is a protein obtained by truncating a 0-9 amino acid at the N-terminus of the HPV16 L1 protein and/or a C-terminal truncation of 0-34 amino acids.
  • the polypeptide derived from the HPV33 type L2 protein is chimeric to the surface region of the HPV16 type L1 protein or the C-terminally truncated 31 amino acid mutant of the HPV16 L1 protein, preferably chimeric The HPV16 L1 protein or the DE loop of the C-terminally truncated 31 amino acid mutant of the HPV16 L1 protein, more preferably chimeric to the HPV16 L1 protein or C-terminal truncation by direct insertion.
  • the HPV16 L1 protein of the mutant of the HPV16 L1 protein of the amino acid, or the HPV16 L1 protein, or the C-terminally truncated 31 amino acids, is interspersed between the amino acid 136 and the amino acid 137 by a non-equal length substitution.
  • a region of amino acid 135-138 of a mutant of a protein, wherein in the non-equal length substitution mode, the N-terminal and/or C-terminus of the polypeptide from the HPV33 L2 protein contains 1-3 amino acid linkages child.
  • the linker is composed of any combination of amino acids selected from the group consisting of glycine (G), serine (S), alanine (A), and valine (P).
  • G glycine
  • S serine
  • A alanine
  • P valine
  • the amino acid sequence from the HPV33 L2 polypeptide is SEQ ID No. 1 or SEQ ID No. 2, and the insertion site is the HPV16 L1 protein or C-terminal truncation.
  • a 31 amino acid mutant of the HPV16 L1 protein is between amino acid 136 and amino acid 137.
  • the HPV16 L1 protein or the C-terminally truncated 31 amino acids of the HPV16 L1 protein mutant amino acid 135-138 region is deleted in the HPV16 L1 protein
  • the amino acid sequence shown in SEQ ID No. 5 or SEQ ID No. 6 is inserted between amino acids 134 and 139 of the mutant of the HPV16 L1 protein having a C-terminal truncation of 31 amino acids.
  • the polypeptide from the HPV33 L2 protein is chimeric to the surface region of the HPV16 L1 protein or the C-terminally truncated 31 amino acid mutant of the HPV16 L1 protein, preferably chimeric The h4 region of the HPV16 L1 protein or the C-terminally truncated 31 amino acid mutant of the HPV16 L1 protein, more preferably chimeric to the HPV16 L1 protein or the C-terminal truncated 31 amino acids by non-equal length replacement.
  • amino acid 431-432 region of the mutant of the HPV16 L1 protein optionally, in the non-equal length substitution manner, the N-terminus and/or C-terminus from the HPV33 L2 polypeptide contains 1-3 A linker for amino acids.
  • the HPV16 L1 protein or the amino acid 431-433 region of the mutant of the HPV16 L1 protein having a C-terminally truncated 31 amino acids is deleted, and the HPV16 L1 protein is deleted.
  • the amino acid sequence shown in SEQ ID No. 2 or SEQ ID No. 3 is inserted between amino acids 430 and 434 of the mutant of the HPV16 L1 protein having a C-terminal truncation of 31 amino acids.
  • Another aspect of the invention relates to polynucleotides encoding the papillomavirus chimeric proteins described above.
  • the present invention also provides a vector comprising the above polynucleotide, and a cell comprising the vector.
  • polynucleotide sequences encoding the above-described papillomavirus chimeric proteins of the present invention are applicable to different expression systems.
  • these nucleotide sequences are fully genetically optimized using E. coli codons and can be expressed at high levels in E. coli expression systems; or whole cell optimization using insect cell codons can be expressed at high levels in insect cell expression systems.
  • the present invention also provides a papillomavirus coat protein multimer, preferably the polymer is a papillomavirus chimeric pentameric or chimeric virus-like particle comprising the above-described papillomavirus chimeric protein, Or formed by the above-described papillomavirus chimeric protein.
  • the present invention also provides the use of the above-described papillomavirus chimeric protein, papillomavirus chimeric pentamer or the above-described papillomavirus chimeric virus-like particle in preparing a vaccine for preventing papillomavirus infection and infection-induced diseases. .
  • the present invention also provides a vaccine for preventing papillomavirus infection and infection-induced diseases comprising the above-described papillomavirus chimeric pentameric or chimeric virus-like particles, an adjuvant, and a vaccine excipient Or a carrier, preferably a virus-like particle or chimeric virus-like particle of HPV comprising at least one mucosal group and/or skin-skin group.
  • a vaccine excipient Or a carrier, preferably a virus-like particle or chimeric virus-like particle of HPV comprising at least one mucosal group and/or skin-skin group.
  • the content of these virus-like particles is an effective amount capable of inducing a protective immune response.
  • the adjuvant is a human adjuvant, preferably an aluminum adjuvant, an oil-in-water emulsion or a water-in-oil emulsion and Adjuvant composition of TLR stimulating agent, composition of aluminum hydroxide adjuvant or aluminum phosphate adjuvant with polyinosinic acid-polycytidine adjuvant and stabilizer or MF59 adjuvant and polyinosinic acid-polycytidine A combination of an acid adjuvant and a stabilizer.
  • TLR stimulating agent composition of aluminum hydroxide adjuvant or aluminum phosphate adjuvant with polyinosinic acid-polycytidine adjuvant and stabilizer or MF59 adjuvant and polyinosinic acid-polycytidine A combination of an acid adjuvant and a stabilizer.
  • insect cell expression system includes insect cells, recombinant baculovirus, recombinant Bacmid and expression vectors.
  • the insect cells are derived from commercially available cells, such as but not limited to: Sf9, Sf21, High Five.
  • prokaryotic expression system includes, but is not limited to, an E. coli expression system.
  • expression host bacteria are derived from commercially available strains, such as but not limited to: BL21 (DE3), BL21 (DE3) plysS, C43 (DE3), Rosetta-gami B (DE3).
  • full length HPV type 16 L1 protein examples include, but are not limited to, a full length L1 protein of the same length as AAC09292.1 in the NCBI database.
  • the gene fragment of the "truncated HPV16 type L1 protein” refers to a nucleoside encoding one or more amino acids deleted at its 5' end and/or 3' end compared to the wild type HPV type 16 L1 protein gene.
  • the full length sequence of the acid, wherein the "wild type HPV type 16 L1 protein” is, for example but not limited to, the following sequences in the NCBI database: AAC09292.1, AIQ82817.1, AAC61736.1, and the like.
  • the term "vaccine excipient or carrier” means selected from one or more, including but not limited to: pH adjusting agents, surfactants, ionic strength enhancers.
  • pH adjusting agents are exemplified, but not limited to, phosphate buffers
  • surfactants include cationic, anionic or nonionic surfactants such as, but not limited to, polysorbate 80 (Tween-80), ionic strength enhancers are exemplified but not Limited to sodium chloride.
  • adjuvant for human means an adjuvant that is clinically applicable to the human body, including various adjuvants that are currently approved and may be approved in the future, such as, but not limited to, aluminum adjuvant, MF59. And various forms of adjuvant compositions.
  • the term "emulsion” means a heterogeneous liquid dispersion system formed by emulsification of an aqueous phase component, an oil phase component and an emulsifier in an appropriate ratio.
  • the aqueous phase components include, but are not limited to, buffer buffers such as phosphate buffers and HEPES buffers;
  • the oil phase components are metabolizable lipids including, but not limited to, vegetable oils, fish oils, animal oils, synthetic oils, and other lipid components (eg, but not Limited to squalene, tocopherol);
  • emulsifiers are suitable surfactants such as, but not limited to, sorbitan trioleate (Span-85), polysorbate 80 (Tween-80).
  • the term "stabilizer” refers to a component which binds to polyinosyl-polycytidine in an adjuvant and which acts as a stabilizing agent, including but not limited to antibiotics such as, but not limited to, kanamycin, Neomycin, gentamicin), inorganic salts (such as, but not limited to, calcium chloride, magnesium chloride, calcium phosphate), cationic organic complexes (such as, but not limited to, calcium stearate, calcium gluconate).
  • antibiotics such as, but not limited to, kanamycin, Neomycin, gentamicin
  • inorganic salts such as, but not limited to, calcium chloride, magnesium chloride, calcium phosphate
  • cationic organic complexes such as, but not limited to, calcium stearate, calcium gluconate.
  • the vaccine of the present invention may be in a form acceptable to the patient including, but not limited to, oral or injection, preferably injection.
  • the vaccine of the invention is preferably used in unit dosage form, wherein the dosage of the protein virus-like particles in the unit dosage form is from 5 ⁇ g to 100 ⁇ g, preferably from 30 ⁇ g to 60 ⁇ g.
  • 1A-1B Expression and identification of chimeric proteins in E. coli and insect cells in Example 5 of the present invention. The results showed that all 12 chimeric proteins can be expressed at high levels in E. coli or insect cells.
  • Figure 1A Identification of chimeric proteins in E. coli: 1 for HPV16 L1DE 136-137 /33dE; 2 for HPV16 L1DE 136-137 /33dEs; 3 for HPV16 L1DE 135-138 /33dE; 4 for HPV16L1DE 135-138 /33dEs; 5 is HPV16 L1h4/33dE; 6 is HPV16 L1h4/33dEs;
  • Figure 1B Identification of chimeric proteins in insect cells: 1 for HPV16 L1 ⁇ CDE 136-137 /33dE; 2 for HPV16 L1 ⁇ CDE 136-137 /33dEs; 3 for HPV16 L1 ⁇ CDE 135-138 /33dE; 4 for HPV16L1 ⁇ CDE 135-138 /33dEs; 5 is HPV16 L1 ⁇ Ch4/33dE; 6 is HPV16 L1 ⁇ Ch4/33dEs.
  • 2A-2B are results of dynamic light scattering analysis of cVLP obtained after purification in Example 6 of the present invention.
  • the results showed that the virion kinetics of the virus-like particles formed by HPV16 L1 ⁇ CDE 135-138 /33dE and HPV16 L1 ⁇ CDE 135-138 /33dEs recombinant proteins were 91.6 nm and 97.9 nm, respectively, and the percentage of particle assembly was 100%.
  • FIG. 2A HPV16 L1 ⁇ CDE 135-138 /33dE
  • Figure 2B HPV16 L1 ⁇ CDE 135-138 /33dEs.
  • 3A to 3F are transmission electron microscope observation results of cVLP obtained after purification in Example 7 of the present invention. A large number of virus-like particles were visible in the field of view, and the particles were once good.
  • Figure 3A HPV16 L1 ⁇ CDE 136-137 /33dE VLP
  • Figure 3B HPV16 L1 ⁇ CDE 136-137 /33dEs VLP
  • Figure 3C HPV16 L1 ⁇ CDE 135-138 /33dE VLP
  • Figure 3D HPV16 L1 ⁇ CDE 135-138 /33dEs VLP
  • Figure 3E HPV16 L1 ⁇ Ch4/33dE VLP
  • Figure 3F HPV16 L1 ⁇ Ch4/33dEs VLP.
  • Example 1 Synthesis of a chimeric L1 protein gene and construction of its expression vector
  • the backbone is a full-length HPV16 L1 protein (sequence shown in SEQ ID No. 4), and its HPV33 L2 protein is directly inserted at its DE loop aa.136/137 site.
  • the polypeptide of aa.17-32 (the insert sequence is shown in SEQ ID No. 2).
  • the polynucleotide sequence encoding HPV16 L1DE 136-137 /33dEs was designed by E. coli codon optimization in the manner of nucleotide 408/ in the HPV16L1 E. codon-optimized gene backbone (sequence shown in SEQ ID No. 8).
  • An E. coli codon-optimized gene of aa.17-32 of HPV33 L2 protein inserted between 409 (the sequence is shown in SEQ ID No. 10);
  • the N-terminus of the HPV33 L2aa.16-37 polypeptide is fused with a G (glycine) P (valine) linker and the C-terminus is fused with a P (valine) linker.
  • the polynucleotide sequence encoding HPV16L1DE 135-138 /33dE was designed by codon optimization in E. coli by deleting nucleotides 403- of the HPV16 L1 E. codon-optimized gene backbone (sequence shown in SEQ ID No. 8). 414, and insert the sequence SEQ ID No. 11 between nucleotides 402/415;
  • Chimeric L1 protein HPV16 L1DE 135-138 /33dEs The backbone is the full-length HPV16 L1 protein (sequence shown in SEQ ID No. 4), and its aa.135-138 region is deleted, and at aa.134/139 The aa.17-32 polypeptide of the HPV33 L2 protein comprising the linker (non-equal-length insertion in the aa.135-138 region of the HPV16 L1 protein) was fused, and the amino acid sequence of the insert was as shown in SEQ ID No. 6.
  • the N-terminus of the aa.17-32 polypeptide of the HPV33 L2 protein is fused with a G (glycine) P (valine) linker and the C-terminal fusion P (valine) linker.
  • the polynucleotide sequence encoding HPV16 L1DE 135-138 /33dEs was designed by codon optimization in E. coli by deleting the nucleotide 403 of the HPV16 L1 E. codon-optimized gene backbone (sequence shown in SEQ ID No. 8). -414, and insert the sequence SEQ ID No. 12 between nucleotides 402/415;
  • Chimeric L1 protein HPV16 L1h4/33dE The backbone is a full-length HPV16 L1 protein (sequence shown in SEQ ID No. 4), and the HPV33 L2 protein is inserted in the aa.430-433 region of the h4 region.
  • the aa.17-37 polypeptide, ie, the aa.431-432 region of the HPV16 L1 protein is deleted, and the aa.17-37 polypeptide is fused between aa.430/433, and the amino acid sequence of the insert is as shown in SEQ ID No. 3.
  • Show. Code HPV16 The polynucleotide sequence of L1h4/33dE was designed by E.
  • Chimeric L1 protein HPV16 L1h4/33dEs The backbone is a full-length HPV16 L1 protein (sequence shown in SEQ ID No. 4), and the HPV33 L2 protein is inserted in the aa.430-433 region of the h4 region.
  • the aa.17-32 polypeptide, ie, the aa.431-432 region of the HPV16 L1 protein is deleted, and the aa.17-32 polypeptide is fused between aa.430/433, and the amino acid sequence of the insert is as shown in SEQ ID No. 2. Show.
  • the polynucleotide sequence encoding HPV16L1h4/33dEs was designed by codon optimization in E.
  • polynucleotide sequence encoding HPV16 L1 ⁇ CDE 136-137 /33dE was optimized by insect cell codon design in the manner of nucleotide 408 in the HPV16 L1 insect cell codon-optimized gene backbone (sequence shown in SEQ ID No. 14). Inserting the insect codon-optimized gene of aa.16-37 of HPV33 L2 protein between /409 (the sequence is shown in SEQ ID No. 15);
  • polynucleotide sequence encoding HPV16 L1 ⁇ CDE 136-137 /33dEs was optimized by insect cell codon design in the manner of nucleotide 408 in the HPV16 L1 insect cell codon-optimized gene backbone (sequence shown in SEQ ID No. 14). Inserting an insect codon optimized gene of aa.17-32 of HPV33 L2 protein between /409 (sequence shown in SEQ ID No. 16);
  • Chimeric L1 protein HPV16 L1 ⁇ CDE 135-138 /33dE The backbone is a C-terminally truncated 31 amino acid HPV16 L1 protein (ie, 31 amino acids truncated at the C-terminus of SEQ ID No. 4), and its aa is deleted. 135-138 region, and aa.16-37 polypeptide containing the HPV33 L2 protein of the linker between aa.134/139 (non-equal length substitution insertion in the aa.135-138 region of the HPV16 L1 protein), insertion
  • the amino acid sequence of the fragment is shown in SEQ ID No. 5.
  • the N-terminus of the aa.16-37 polypeptide of the HPV33 L2 protein is fused with a G (glycine) P (valine) linker and the C-terminal fusion P (valine) linker.
  • the polynucleotide sequence encoding HPV16 L1 ⁇ CDE 135-138 /33dE was designed by insect cell codon optimization in such a way as to delete the nucleotide 403 of the HPV16 L1 insect cell codon-optimized gene backbone (sequence shown in SEQ ID No. 14). -414, and insert the sequence SEQ ID No. 17 between nucleotides 402/415;
  • Chimeric L1 protein HPV16 L1 ⁇ CDE 135-138 /33dEs The backbone is a C-terminally truncated 31 amino acid HPV16 L1 protein (ie, 31 amino acids truncated at the C-terminus of SEQ ID No. 4), and its aa is deleted.
  • the amino acid sequence is shown in SEQ ID No. 6.
  • the N-terminus of the aa.17-32 polypeptide of the HPV33 L2 protein is fused with a G (glycine) P (valine) linker and the C-terminal fusion P (valine) linker.
  • the polynucleotide sequence encoding HPV16L1 ⁇ CDE 135-138 /33dEs was designed by insect cell codon optimization in such a way as to delete the nucleotide 403- of the HPV16 L1 insect cell codon-optimized gene backbone (sequence shown in SEQ ID No. 14). 414, and insert the sequence SEQ ID No. 18 between nucleotides 402/415;
  • Chimeric L1 protein HPV16 L1 ⁇ Ch4/33dE The backbone is a C-terminally truncated 31 amino acid HPV16L1 protein (ie, 31 amino acids truncated at the C-terminus of SEQ ID No. 4), aa.430 in its h4 region.
  • the -433 region non-equal length replacement inserts the aa.17-37 polypeptide of the HPV33 L2 protein, ie, the aa.431-432 region of the HPV16 L1 protein is deleted, and the aa.17- of the HPV33 L2 protein is fused between aa.430/433- 37 polypeptide, the amino acid sequence of the insert is shown in SEQ ID No. 3.
  • the polynucleotide sequence encoding HPV16 L1 ⁇ Ch4/33dE was designed by insect cell codon optimization by deleting nucleotides 1291-1296 of the HPV16 L1 insect cell codon-optimized gene backbone (sequence shown in SEQ ID No. 14). And inserting the sequence SEQ ID No. 19 between nucleotides 1290/1297;
  • the polynucleotide sequence encoding HPV16 L1 ⁇ Ch4/33dEs was optimized by insect cell codon design in such a way as to delete the nucleotides 1291-1296 of the HPV16 L1 insect cell codon-optimized gene backbone (sequence shown in SEQ ID No. 14). And the sequence SEQ ID No. 16 was inserted between nucleotides 1290/1297.
  • the chimeric L1 gene optimized according to the E. coli codon and the insect cell codon is synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd. by means of whole gene synthesis.
  • the E. coli codon-optimized chimeric protein gene was digested with NdeI/XhoI and inserted into the commercial expression vector pET22b (manufactured by Novagen).
  • the insect cell codon-optimized chimeric protein gene was digested with EcoRI/Xba I and inserted into the commercial expression vector pFastBac1 (manufactured by Invitrogen).
  • the expression vector containing the chimeric protein gene was obtained: pET22b-16L1DE 136-137 /33dE, pET22b-16L1DE 136-137 /33dEs, ET22b-16L1DE 135-138 /33dE, pET22b-16L1DE 135-138 /33dEs, pET22b -16L1h4/33dE, pET22b-16L1h4/33dEs, pFastBac1-16L1 ⁇ CDE 136-137 /33dE, pFastBac1-16L1 ⁇ CDE 136-137 /33dEs, pFastBac1-16L1 ⁇ CDE 135-138 /33dE, pFastBac1-16L1 ⁇ CDE 135-138 /33dEs, pFastBac1-16L1 ⁇ CDE 135-138 /33dE,
  • Example 2 Recombinant Bacmid of a gene encoding a L1 protein and construction of a recombinant baculovirus
  • Example 3 Expression of a gene encoding a L1 protein in Sf9 cells
  • Sf9 cells were inoculated with 6 recombinant L1 gene recombinant baculoviruses, and the expression of chimeric L1 protein was carried out. After incubation at 27 ° C for about 88 h, the fermentation broth was collected, centrifuged at 3000 rpm for 15 min, the supernatant was discarded, and the cells were washed with PBS. Expression identification and purification. Methods of infecting expression are disclosed, for example, patent CN 101148661 B.
  • Example 4 Expression of a gene encoding a L1 protein in Escherichia coli
  • Recombinant expression vectors containing the chimeric L1 gene, pET22b-16L1DE 136-137 /33dE, pET22b-16L1DE 136-137 /33dEs, pET22b-16L1DE 135-138 /33dE, pET22b-16L1DE 135-138 /33dEs, pET22b-16L1h4 were used, respectively.
  • /33dE, pET22b-16L1h4/33dEs was transformed into E. coli BL21 (DE3).
  • the monoclonal was inoculated into 3 ml of ampicillin-containing LB medium and cultured overnight at 37 °C.
  • the culture solution of the overnight culture was added to the LB medium at a ratio of 1:100, and cultured at 37 ° C for about 3 hours, until the OD600 reached 0.8-1.0, IPTG was added to a final concentration of 0.5 ⁇ M, and cultured at 16 ° C for about 12 hours to collect the bacterial liquid. .
  • chimeric L1 proteins can be expressed at high levels in insect cells or prokaryotic expression systems, including HPV16 L1DE 136-137 /33dE, HPV16L1DE 136-137 /33dEs, HPV16 L1DE 135-138/ 33dE, HPV16 L1DE 135-138 /33dEs, HPV16L1h4/33dE, HPV16 L1h4/33dEs are about 55 kDa in size, and the remaining 6 proteins are about 50 kDa in size.
  • Methods for SDS-PAGE electrophoresis and Western blot identification are disclosed, for example, patent CN101148661 B.
  • Example 6 Purification of chimeric L1 protein and dynamic light scattering particle size analysis
  • the VLP was depolymerized by adding 4% ⁇ -mercaptoethanol (w/w) to the lysate, and then the sample was filtered using a 0.22 ⁇ m filter, followed by DMAE anion exchange chromatography or CM cation exchange chromatography (20 mM Tris, 180 mM NaCl, 4% ⁇ -ME, pH 7.9 elution), TMAE anion exchange chromatography or Q cation exchange chromatography (20 mM Tris, 180 mM NaCl, 4% ⁇ -ME, pH 7.9 elution) and hydroxyapatite chromatography (100 mM) Purified by NaH 2 PO 4 , 30 mM NaCl, 4% ⁇ -ME, eluted with pH 6.0.
  • the purified product was concentrated using a Planova ultrafiltration system and replaced with a buffer (20 mM NaH 2 PO 4 , 500 mM NaCl, pH 6.0) to facilitate VLP assembly.
  • a buffer (20 mM NaH 2 PO 4 , 500 mM NaCl, pH 6.0) to facilitate VLP assembly.
  • the above purification methods are all disclosed, for example, patents CN101293918 B, CN1976718 A, and the like.
  • the purified chimeric protein solution was subjected to DLS particle size analysis (Zetasizer Nano ZS 90 dynamic light scattering instrument, Malvern), and the results are shown in Table 1, wherein HPV16 L1 ⁇ CDE 135-138 /33dE and HPV16L1 ⁇ CDE 135-138 /33dEs
  • DLS particle size analysis Zetasizer Nano ZS 90 dynamic light scattering instrument, Malvern
  • the chimeric VLPs were separately purified according to the chromatographic purification method described in Example 6, and copper mesh was prepared using dialysis VLP, and stained with 1% uranyl acetate, and dried sufficiently to use JEM-1400 electron microscope (Olympus). ) to observe.
  • the cVLP of the chimeric L2 antigen polypeptide expressed in the DE region of the insect cell was about 50 nm in diameter
  • the cVLP of the polypeptide having the L2 antigen in the h4 region was about 35-40 nm in diameter.
  • the prokaryotic expression of cVLP is consistent with the cVLP size pattern expressed by insect cells.
  • the cVLP of the chimeric L2 antigen polypeptide in the DE region is about 50 nm in diameter, and the cVLP of the chimeric L2 antigen polypeptide in the h4 region is about 35-40 nm in diameter.
  • Methods for copper mesh preparation and electron microscopy are disclosed, for example, patent CN 101148661 B.
  • mice of 4-6 weeks old were randomly divided into groups of 5, and mice were immunized with 10 ⁇ g of cVLP, 50 ⁇ g of Al(OH) 3 and 50 ⁇ g of PIKA adjuvant. Subcutaneous injection, immunization at 0, 2, 4, 6 weeks, a total of 4 times. Blood was collected from the tail vein 2 weeks after the fourth immunization, and serum was separated.
  • the chimeric protein constructed by using the polypeptide of the L2 protein in the DE region or the h4 region in the DE region or the h4 region can form a cVLP, and the level of the cross-neutralizing antibody induced by the immunization of the mouse by the above strategy is There is no difference in the cVLP shown in Table 2.
  • the pentameric consisting of the above 12 chimeric L1 proteins, respectively, can also induce cross-neutralizing antibodies after immunizing mice with the above strategy.

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Abstract

提供了一种乳头瘤病毒嵌合蛋白，其骨架是乳头瘤病毒的L1蛋白或其突变体，所述的骨架上嵌合至少一个来自人乳头瘤病毒33型L2蛋白或其突变体多肽。该乳头瘤病毒嵌合蛋白可用于制备预防乳头瘤病毒感染及感染诱发的疾病的疫苗。

Description

一种乳头瘤病毒嵌合蛋白及其用途 技术领域
本发明涉及生物技术领域，具体涉及一种乳头瘤病毒嵌合蛋白，及由其形成的病毒样颗粒，以及乳头瘤病毒嵌合蛋白或乳头瘤病毒嵌合病毒样颗粒在制备预防乳头瘤病毒感染及感染诱发的疾病的疫苗中的用途。
背景技术
目前已分离鉴定了200多型人乳头瘤病毒(human papillomavirus，HPV)，分为嗜黏膜组和嗜皮肤组。黏膜组的HPV主要感染泌尿生殖道、肛门肛周及口咽部的黏膜及周围皮肤，诱发各种良恶性病变。根据诱发病变的性质不同，可分为诱发恶性肿瘤的高危型(包括HPV16、18、31、33、35、39、45、51、52、56、58、59、68等)；可疑高危型(HPV26、30、53、66、67、69、70、73、82、85等)；尚未确定型(HPV34、42、43、54、71、81、83、97、102、114等)；及诱发疣状增生等良性病变的低危型(HPV6、7、11、13、32、40、42、44、61、62、72、74、81、83、84、86、87、89、90、91、106等)。嗜皮肤组主要感染上述部位之外的皮肤组织，诱发皮肤疣状增生，并与某些皮肤癌的发生密切相关。
高危型HPV感染相关的恶性肿瘤目前已确定的有：宫颈癌、阴道癌、阴唇癌、阴茎癌、肛门肛周癌、口咽癌、扁桃体癌、口腔癌，其中以宫颈癌的危害最大。宫颈癌是世界范围第三高发的妇女恶性肿瘤，年发病约52.7万，其中亚洲地区28.5万；中国的年发病数7.5万。
HPV16是全球范围内的优势流行株，在HPV相关的肿瘤如宫颈癌、肛周癌、阴茎癌、外阴癌等相关癌及癌前病变中的检出率都是最高的。例如，HPV16感染相关的宫颈癌约占中国宫颈癌总数的58.7％，占世界宫颈癌发病总数的53.5％，其余41.3％-46.5％的宫颈癌由其余约19种高危型HPV感染合并引起。目前已经上市了多种以HPV L1蛋白的病毒样颗粒(VLP)为基础的混合性预防疫苗，如葛兰素史克公司的二价苗Cervarix(HPV16/18)，默沙东公司的四价苗Gardasil(HPV6/11/16/18)及九价苗Gardasil 9(HPV6/11/16/18/31/33/35/45/52/58)。由于这类疫苗诱发的免疫保护反应主要是针对疫苗构成型别的，因此这类疫苗多为HPV多价疫苗，如要获得广谱疫苗的预防效果需要继续扩大疫苗的价次。鉴于HPV的型别目前已鉴定了200多型，高危型已鉴定多达20型，因此通过单纯扩大VLP型别种类研发广谱疫苗在经济成本及人体所能承受的最大接种量方面都带来了许多挑战。
病毒次要衣壳蛋白L2可诱发交叉中和抗体，且具有体内交叉保护活性。研究发现，诱发交叉保护活性的中和抗体表位主要分布在L2蛋白的N端多个保守区域，如HPV16 L2蛋白的氨基酸(aa.)17-36多肽的免疫血清可高滴度中和HPV16/18， 同时还可有效中和HPV5/6/45/52/58(Gambhira R,Karanam B,et al.J.Virol.2007；81(24):13927–13931)，针对HPV16 L2 aa.17-36的单抗RG-1亦具有交叉中和活性(Gambhira R,Karanam B,et al.J.Virol.2007；81(24):13927–13931)，因此，L2蛋白的与HPV16 L2的aa.17-36多肽同源区又被称为RG-1表位。
采用不同疫苗载体，如硫氧还蛋白(Trx)、噬菌体VLP、植物病毒VLP及感染哺乳类的病毒VLP(腺相关病毒、牛乳头瘤病毒-1、HPV16)，构建以HPV16L2 aa.17-36多肽为基础的融合蛋白疫苗，可显著提高多肽的免疫原性，提高中和抗体的滴度及交叉中和或保护范围(Christina S,Richard R,et al.J.Virol.2009；83(19):10085-10095；Seitz H,Canali E,et al.Vaccine 2014；32:2610–2617；Tumban E,Peabody J,et al.PLoS One 2011；6(8):e23310；Nieto K,Weghofer M,et al.PLoS One 2012；7(6):e39741)。
HPV L2 aa.17-36多肽区域在不同种属的乳头瘤病毒之间，氨基酸序列同源性很高。现有的以不同HPV型别RG-1表位为基础的疫苗研究如下：将HPV31/51型RG-1表位插入细菌蛋白Trx的表面，获得的免疫血清有交叉中和活性，但中和型别相对较少(Seitz H,Canali E,et al.Vaccine 2014；32:2610–2617)；将HPV16/31型RG-1表位联合插入腺相关病毒VLP表面，获得的免疫血清合计中和6个HPV型别(Nieto K,Weghofer M,et al.PLoS One 2012；7(6):e39741)；将HPV45型RG-1表位插入HPV18VLP表面，获得的免疫血清合计中和4个HPV型别(Huber B,Schellenbacher C,et al.PLoS One 2015；10(3):e0120152)。这些结果表明，上述型别的RG-1表位均有诱发交叉中和抗体的活性。此外，多种型别的RG-1区域的截短型多肽(HPV1/5/6/11/16/18/45/58 L2 aa.17-31)分别插入噬菌体VLP表面，形成的8种嵌合VLP(Chimeric VLP,cVLP)混合免疫后的小鼠可产生针对8种型别病毒的免疫保护反应(Tumban E,Peabody J,et al.PLoS One 2011；6(8):e23310)，由于缺乏对每种RG-1截短多肽cVLP免疫活性的具体分析，因此不能判断各型截短型的RG-1表位多肽的免疫活性。从上述文献报道的中和型别分析来看，HPV16型RG-1表位的免疫原性最强，无论是插入HPV及噬菌体的VLP表面均可诱发广谱中和抗体反应；HPV31/45/51型的可能次之，其他型别的不明(缺乏报道)；值得注意的是8种型别(HPV1/5/6/11/16/18/45/58)RG-1区域的截短型多肽(L2aa.17-31)噬菌体cVLP混合免疫获得的抗血清，其中和的型别不多，提示其中某些型别截短多肽可能是没有活性的或是活性很弱，具体比较分析不同型别RG-1表位多肽及截短型多肽免疫原性，可望明确其免疫原性及免疫原性的差异。因此，除HPV16外，目前对其他不同型别的RG-1表位区的免疫原性研究欠深入或者缺乏研究；而且缺乏针对不同型别RG-1表位区的免疫原性的比较分析；重要的是，在相关的载体疫苗研究中，RG-1表位型别的选择不是依据其免疫原性的强弱，而是依据其他因素，如该型别的病毒是否优势流行及感染相关疾病的危害程度是否严重。因此现有以RG-1表位为基础的疫苗研究有待提高。
HPV33的感染率及在HPV感染相关肿瘤中的检出率较高，在世界范围内，其在宫颈癌标本中的检出率为2.6％；在亚洲和中国均为3.8％，仅次于HPV16/18/58型(X.Castellsagué et al.Vaccine 25S(2007)C1–C26)。但是HPV33 L2蛋白RG-1表位的免疫原性缺乏研究，该表位是否能诱发中和抗体及诱发抗体的中和范围及特点都不明确。基于目前的研究进展及认知，HPV33型RG-1表位疫苗的免疫活性是无法预测的。
研究发现，HPV16 L1VLP是一个极具研发前景的HPV16 L2 RG-1表位疫苗载体，在HPV16 L1蛋白表面区的特定位置按照一定的方式插入HPV16 L2 RG-1表位后，可在VLP表面展示，免疫后可诱发广谱中和抗体及保护反应。如在HPV16L1蛋白DE环(aa 136/137)位点，直接插入HPV16 L2 aa.17-36形成的嵌合VLP(cVLP)可诱发广谱中和抗体反应，可中和至少14个HPV型别(Schellenbacher C,Roden R,et al 2009；J.Virol.2009；83(19):10085–10095)；在h4区域的430/433位点，采用非等长置换法插入HPV16 L2 aa.18-38，可以诱发交叉中和HPV18及31型的中和抗体(Kondo K,Ochi H,et al.J.Med.Virol.2008；80:841–846)。目前尚未见有将HPV33 L2蛋白表位嵌合在乳头瘤病毒VLP表面的报道。由于HPV33RG-1表位与HPV16 RG-1表位的氨基酸序列有一定差异(同源性约80％)，在上述位点插入后是否能形成VLP尚不清楚。另外，采用其他不同的插入方式，如在DE环135-138区域，采用非等长置换合并插入片段两端引入氨基酸修饰，获得的融合蛋白是否能形成VLP，插入的表位是否能在表面有效呈递尚不清楚，插入后是否影响骨架自身的主要中和抗体表位也不清楚。最后，在上述区域插入截短型的HPV33 L2多肽，是否能形成VLP，形成VLP以后是否具有诱发交叉保护反应的活性，是否影响骨架自身的主要中和抗体表位，对其表达量的影响如何，同样也是不清楚的。上述不清楚的问题是无法预测的。
因此，本发明选用了HPV33型RG-1表位及截短型的RG-1表位，用于HPV 16型cVLP的研究，结果显示，本发明获得的HPV33型RG-1长表位及短表位的cVLP的免疫原性很强(可中和至少10个HPV型别)，可与文献报道的HPV16型RG-1VLP的相媲美，但中和型别的范围有所不同(Schellenbacher C,Roden R,et al 2009；J.Virol.2009；83(19):10085–10095；Schellenbacher C,Kwak K,et al.J.Invest.Derma.2013；doi:10.1038/jid.2013.253)。
发明内容
有鉴于此，本发明的目的在于提供一种乳头瘤病毒嵌合蛋白，用于制备预防乳头瘤病毒感染及感染诱发的疾病的疫苗。
本发明人经研究出人意料地发现，在全长或截短型HPV16 L1蛋白的表面区插入HPV33 L2蛋白多肽，可提高HPV33 L2多肽的免疫原性，获得的嵌合蛋白在大肠杆菌或昆虫细胞表达系统中可高水平表达，该嵌合蛋白可组装成VLP，并可诱 发针对来自不同属/亚属的多种型别HPV的广谱保护性免疫反应。本发明基于以上发现，现已完成，在本文实施例中提供数据。
基于上述目的，本发明一方面提供了一种乳头瘤病毒嵌合蛋白，其骨架是乳头瘤病毒的L1蛋白或乳头瘤病毒的L1蛋白的突变体，所述的骨架上嵌合至少一个来自HPV33 L2蛋白的多肽。
可选地，所述的多肽选自HPV33 L2蛋白(氨基酸序列如SEQ ID No.7所示)的aa.1-200区域内的任意连续8-33个氨基酸的片段。进一步优选地，所述的多肽为HPV33 L2蛋白RG-1表位区。
优选地，所述的多肽的氨基酸序列如SEQ ID No.1所示。
可选地，所述的多肽是在SEQ ID No.1所示的氨基酸序列的N端延长或截短1-5个氨基酸和/或C端延长或截短1-5个氨基酸所获得的多肽。
优选地，所述的多肽的氨基酸序列如SEQ ID No.2或者SEQ ID No.3所示。
可选地，所述的多肽还可以是与SEQ ID No.1所示的氨基酸序列同源性大于60％的多肽，优选是同源性大于70％的多肽、同源性大于80％的多肽、同源性大于90％的多肽，甚至更优选是同源性大于95％的多肽。
可选地，所述的骨架是HPV16 L1蛋白或HPV16 L1蛋白的突变体。
优选地，所述的HPV16 L1蛋白选自高危型HPV L1蛋白或高危型HPV L1蛋白的突变体；进一步优选地，本发明涉及的嵌合蛋白骨架选自HPV16 L1蛋白(例如NCBI数据库AAC09292.1序列)或HPV16 L1蛋白突变体。HPV16 L1蛋白骨架可来自但不限于HPV16 Phi1、Tha7、Alg1、Sen32、Fra25、Fra63、114K、114B、Z-1194等变异株的L1蛋白(Touze A,Mehdaoui SE,et al.J.Clin.Micr.1998；36(7):2046-2051)。优选地，所述的HPV16 L1蛋白的氨基酸序列如SEQ ID No.4所示。
可选地，所述的HPV16 L1蛋白的突变体是在所述的HPV16 L1蛋白的N端截短0-9个氨基酸和/或C端截短0-34个氨基酸的所获得的蛋白。
可选地，所述的来自HPV33型L2蛋白的多肽嵌合于所述的HPV16型L1蛋白或C端截短31个氨基酸的所述HPV16 L1蛋白的突变体的表面区，优选为嵌合于所述的HPV16 L1蛋白或C端截短31个氨基酸的所述HPV16 L1蛋白的突变体的DE环，更优选为通过直接插入的方式嵌合于所述的HPV16 L1蛋白或C端截短31个氨基酸的所述HPV16 L1蛋白的突变体的氨基酸136和氨基酸137之间，或者通过非等长置换的方式嵌合于所述的HPV16 L1蛋白或C端截短31个氨基酸的所述HPV16 L1蛋白的突变体的氨基酸135-138区域，其中，在所述的非等长置换的方式中，所述的来自HPV33 L2蛋白的多肽的N端和/或C端含有1-3个氨基酸的连接子。
可选地，所述的连接子由选自甘氨酸(G)、丝氨酸(S)、丙氨酸(A)及脯氨酸(P)的氨基酸任意组合构成。优选地，N端选用G(甘氨酸)P(脯氨酸)连接子，C端选用P(脯氨酸)连接子。
可选地，在所述直接插入的方式中，所述来自HPV33 L2多肽的氨基酸序列是SEQ ID No.1或SEQ ID No.2，插入位点为所述的HPV16 L1蛋白或C端截短31个氨基酸的所述HPV16 L1蛋白的突变体的氨基酸136和氨基酸137之间。
可选地，在所述非等长置换的方式中，删除所述HPV16 L1蛋白或C端截短31个氨基酸的所述HPV16 L1蛋白的突变体的氨基酸135-138区域后，在HPV16 L1蛋白或C端截短31个氨基酸的所述HPV16 L1蛋白的突变体的氨基酸134及139之间插入如SEQ ID No.5或SEQ ID No.6所示的氨基酸序列。
可选地，所述的来自HPV33 L2蛋白的多肽嵌合于所述的HPV16 L1蛋白或C端截短31个氨基酸的所述HPV16 L1蛋白的突变体的表面区，优选为嵌合于所述的HPV16 L1蛋白或C端截短31个氨基酸的所述HPV16 L1蛋白的突变体的h4区，更优选为通过非等长置换的方式嵌合于HPV16 L1蛋白或C端截短31个氨基酸的所述HPV16 L1蛋白的突变体的氨基酸431-432区域，可选地，在所述的非等长置换的方式中，所述的来自HPV33 L2多肽的N端和/或C端含有1-3个氨基酸的连接子。
可选地，在所述非等长置换的方式中，删除所述HPV16 L1蛋白或C端截短31个氨基酸的所述HPV16 L1蛋白的突变体的氨基酸431-433区域后，在HPV16 L1蛋白或C端截短31个氨基酸的所述HPV16 L1蛋白的突变体的氨基酸430及434之间插入如SEQ ID No.2或SEQ ID No.3所示的氨基酸序列。
本发明的另一方面涉及编码上述的乳头瘤病毒嵌合蛋白的多核苷酸。
本发明还提供了包含上述的多核苷酸的载体，以及包含所述的载体的细胞。
本发明涉及的编码上述的乳头瘤病毒嵌合蛋白的多核苷酸序列适用于不同的表达系统。可选地，这些核苷酸序列采用大肠杆菌密码子进行全基因优化，可在大肠杆菌表达系统中高水平表达；或采用昆虫细胞密码子进行全基因优化，可在昆虫细胞表达系统中高水平表达。
本发明还提供了一种乳头瘤病毒外壳蛋白多聚物，优选地，该多聚物为乳头瘤病毒嵌合五聚体或嵌合病毒样颗粒，其含有上述的乳头瘤病毒嵌合蛋白，或者由上述的乳头瘤病毒嵌合蛋白所形成。
本发明还提供了上述的乳头瘤病毒嵌合蛋白、乳头瘤病毒嵌合五聚体或上述的乳头瘤病毒嵌合病毒样颗粒在制备预防乳头瘤病毒感染及感染诱发的疾病的疫苗中的用途。
本发明还提供了一种用于预防乳头瘤病毒感染及感染诱发的疾病的疫苗，其包含上述的乳头瘤病毒嵌合五聚体或嵌合病毒样颗粒、佐剂、以及疫苗用赋形剂或载体，优选地，还包含至少一种嗜黏膜组和/或嗜皮肤组的HPV的病毒样颗粒或嵌合病毒样颗粒。其中，这些病毒样颗粒的含量分别为能诱发保护性免疫反应的有效量。
可选地，所述佐剂为人用佐剂，优选是铝佐剂、水包油乳剂或油包水乳剂及 TLR刺激剂的佐剂组合物、氢氧化铝佐剂或磷酸铝佐剂与聚肌苷酸-聚胞苷酸佐剂及稳定剂的组合物或者MF59佐剂与聚肌苷酸-聚胞苷酸佐剂及稳定剂的组合物。
发明中相关术语的说明及解释
根据本发明，术语“昆虫细胞表达系统”包括昆虫细胞、重组杆状病毒、重组Bacmid及表达载体。其中昆虫细胞来源于市场上可得到的细胞，在此举例但不限于：Sf9，Sf21，High Five。
根据本发明，术语“原核表达系统”包括但不限于大肠杆菌表达系统。其中表达宿主菌来源于市场上可得到的菌株，在此举例但不限于：BL21(DE3)，BL21(DE3)plysS，C43(DE3)，Rosetta-gami B(DE3)。
根据本发明，术语“全长HPV16型L1蛋白”的例子包括但不限于NCBI数据库中编号为AAC09292.1的蛋白等长的全长L1蛋白。
“截短型HPV16型L1蛋白”的基因片段指的是其与野生型HPV 16型L1蛋白基因相比，在其5’端和/或3’端缺失编码1个或多个氨基酸的核苷酸，其中“野生型HPV16型L1蛋白”的全长序列例如但不限于NCBI数据库中的如下序列:AAC09292.1、AIQ82817.1、AAC61736.1等。
根据本发明，术语“疫苗用赋形剂或载体”是指选自一种或多种，包括但不限于：pH调节剂，表面活性剂，离子强度增强剂。例如，pH调节剂举例但不限于磷酸盐缓冲液，表面活性剂包括阳离子、阴离子或非离子型表面活性剂，举例但不限于聚山梨酯80(Tween-80)，离子强度增强剂举例但不限于氯化钠。
根据本发明，术语“人用佐剂”是指在临床上可应用于人体的佐剂，包括当前已获得批准的和将来可能获得批准的各种佐剂，例如但不限于铝佐剂、MF59及各种形式的佐剂组合物。
根据本发明，术语“乳剂”是指由水相成分、油相成分及乳化剂按适当比例混合，经乳化后形成的非均相液体分散体系。其中水相成分包括但不限于磷酸盐缓冲液、HEPES缓冲液等缓冲系统；油相成分为可代谢脂类，包括但不限于植物油、鱼油、动物油、合成油及其他脂类成分(例如但不限于角鲨烯、生育酚)；乳化剂为适宜的表面活性剂，例如但不限于山梨醇酐三油酸酯(Span-85)、聚山梨酯80(Tween-80)。
根据本发明，术语“稳定剂”是指可与佐剂中的聚肌苷酸-聚胞苷酸结合并起到稳定作用的成分，包括但不限于抗生素(例如但不限于卡那霉素、新霉素、庆大霉素)、无机盐(例如但不限于氯化钙、氯化镁、磷酸钙)、阳离子的有机复合物(例如但不限于硬脂酸钙、葡萄糖酸钙)。
根据本发明，本发明的疫苗可采用患者可接受的形式，包括但不限于口服或者注射，优选注射。
根据本发明，本发明疫苗优选单位剂型使用，其中单位剂型中蛋白病毒样颗粒的剂量为5μg-100μg，优选30μg-60μg。
附图说明
图1A-图1B：本发明实施例5中嵌合蛋白在大肠杆菌及昆虫细胞中的表达鉴定。结果显示，12种嵌合蛋白均可在大肠杆菌或昆虫细胞中高水平表达。
图1A：嵌合蛋白在大肠杆菌中的表达鉴定：1为HPV16 L1DE136-137/33dE；2为HPV16 L1DE136-137/33dEs；3为HPV16 L1DE135-138/33dE；4为HPV16L1DE135-138/33dEs；5为HPV16 L1h4/33dE；6为HPV16 L1h4/33dEs；
图1B：嵌合蛋白在昆虫细胞中的表达鉴定：1为HPV16 L1ΔCDE136-137/33dE；2为HPV16 L1ΔCDE136-137/33dEs；3为HPV16 L1ΔCDE135-138/33dE；4为HPV16L1ΔCDE135-138/33dEs；5为HPV16 L1ΔCh4/33dE；6为HPV16 L1ΔCh4/33dEs。
图2A-图2B：本发明实施例6中纯化后获得的cVLP的动态光散射分析结果。结果显示HPV16 L1ΔCDE135-138/33dE及HPV16 L1ΔCDE135-138/33dEs重组蛋白形成的病毒样颗粒水化动力学直径分别为91.6nm和97.9nm，颗粒组装的百分比均为100％。
图2A：HPV16 L1ΔCDE135-138/33dE；图2B：HPV16 L1ΔCDE135-138/33dEs。
图3A-图3F：本发明实施例7中纯化后获得的cVLP的透射电镜观察结果。视野中可见大量的病毒样颗粒，颗粒均一度好。其中在DE区域插入L2蛋白的多肽的cVLP直径为50nm左右，与L1蛋白的VLP的大小相似；在h4区域插入L2蛋白的多肽的cVLP直径较小，在35-40nm之间。Bar＝200nm。
图3A：HPV16 L1ΔCDE136-137/33dE VLP；图3B：HPV16 L1ΔCDE136-137/33dEs VLP；图3C：HPV16 L1ΔCDE135-138/33dE VLP；图3D：HPV16 L1ΔCDE135-138/33dEs VLP；图3E：HPV16 L1ΔCh4/33dE VLP；图3F：HPV16 L1ΔCh4/33dEs VLP。
具体实施方式
下面将通过下述非限制性实施例进一步说明本发明，本领域技术人员公知，在不背离本发明精神的情况下，可以对本发明做出许多修改，这样的修改也落入本发明的范围。下面的实施例仅用于说明本发明，而不应视为限定本发明的范围，因为实施方案必然是多样的。本说明书中使用的用语仅是为了阐述特定的实施方案，而非作为限制，本发明的范围已界定在所附的权利要求中。
除非特别说明，本说明书中所使用的所有技术和科学用语均和本案所属技术领域的技术人员所普遍明了的意义相同。下面就本发明的优选方法和材料加以叙述，但是与本说明书中所述方法和材料类似或等效的任何方法和材料均可用以实施或测试本发明。下述实验方法如无特别说明，均为常规方法或产品说明书所描述的方法，所使用的实验材料如无特别说明，均可容易地从商业公司获取。本说明书中所提到的所有公开文献均被并入于此作为参考，以揭示并说明所述公开文献中的方法和/或材料。
实施例1：嵌合L1蛋白的基因的合成及表达载体构建
12种嵌合L1蛋白，分别为：
1)嵌合L1蛋白HPV16 L1DE136-137/33dE：骨架为全长HPV16 L1蛋白(序列如SEQ ID No.4所示)，在其DE环aa.136/137位点，直接插入HPV33 L2蛋白的aa.16-37多肽(插入片段序列如SEQ ID No.1所示)。编码HPV16 L1DE136-137/33dE的多核苷酸序列经大肠杆菌密码子优化设计，构建方式为在HPV16 L1大肠杆菌密码子优化基因骨架(序列如SEQ ID No.8所示)的核苷酸408/409之间插入HPV33型L2蛋白的aa.16-37的大肠杆菌密码子优化基因(序列如SEQ ID No.9所示)；
2)嵌合L1蛋白HPV16L1DE136-137/33dEs：骨架为全长的HPV16 L1蛋白(序列如SEQ ID No.4所示)，在其DE环aa.136/137位点，直接插入HPV33 L2蛋白的aa.17-32的多肽(插入片段序列如SEQ ID No.2所示)。编码HPV16 L1DE136-137/33dEs的多核苷酸序列经大肠杆菌密码子优化设计，构建方式为在HPV16L1大肠杆菌密码子优化基因骨架(序列如SEQ ID No.8所示)的核苷酸408/409之间插入HPV33 L2蛋白的aa.17-32的大肠杆菌密码子优化基因(序列如SEQ ID No.10所示)；
3)嵌合L1蛋白HPV16 L1DE135-138/33dE：骨架为全长的HPV16 L1蛋白(序列如SEQ ID No.4所示)，删除其aa.135-138区域，并在aa.134/139之间融合包含连接子的HPV33 L2蛋白的aa.16-37多肽(在HPV16L1蛋白的aa.135-138区域非等长置换插入)，插入片段的氨基酸序列如SEQ ID No.5所示。其中，HPV33 L2aa.16-37多肽的N端融合G(甘氨酸)P(脯氨酸)连接子且C端融合P(脯氨酸)连接子。编码HPV16L1DE135-138/33dE的多核苷酸序列经大肠杆菌密码子优化设计，构建方式为删除HPV16 L1大肠杆菌密码子优化基因骨架(序列如SEQ ID No.8所示)的核苷酸403-414，并在核苷酸402/415之间插入序列SEQ ID No.11；
4)嵌合L1蛋白HPV16 L1DE135-138/33dEs：骨架为全长HPV16 L1蛋白(序列如SEQ ID No.4所示)，删除其aa.135-138区域，并在aa.134/139之间融合包含连接子的HPV33 L2蛋白的aa.17-32多肽(在HPV16 L1蛋白的aa.135-138区域非等长置换插入)，插入片段的氨基酸序列如SEQ ID No.6所示。其中，HPV33 L2蛋白的aa.17-32多肽的N端融合G(甘氨酸)P(脯氨酸)连接子且C端融合P(脯氨酸)连接子。编码HPV16 L1DE135-138/33dEs的多核苷酸序列经大肠杆菌密码子优化设计，构建方式为删除HPV16 L1大肠杆菌密码子优化基因骨架(序列如SEQ ID No.8所示)的核苷酸403-414，并在核苷酸402/415之间插入序列SEQ ID No.12；
5)嵌合L1蛋白HPV16 L1h4/33dE：骨架为全长的HPV16 L1蛋白(序列如SEQ ID No.4所示)，在其h4区域的aa.430-433区域非等长置换插入HPV33 L2蛋白的aa.17-37多肽，即删除HPV16 L1蛋白的aa.431-432区域，并在aa.430/433之间融合aa.17-37多肽，插入片段的氨基酸序列如SEQ ID No.3所示。编码HPV16 L1h4/33dE的多核苷酸序列经大肠杆菌密码子优化设计，构建方式为删除HPV16L1大肠杆菌密码子优化基因骨架(序列如SEQ ID No.8所示)的核苷酸1291-1296，并在核苷酸1290/1297之间插入序列SEQ ID No.13；
6)嵌合L1蛋白HPV16 L1h4/33dEs：骨架为全长的HPV16 L1蛋白(序列如SEQ ID No.4所示)，在其h4区域的aa.430-433区域非等长置换插入HPV33 L2蛋白的aa.17-32多肽，即删除HPV16 L1蛋白的aa.431-432区域，并在aa.430/433之间融合aa.17-32多肽，插入片段的氨基酸序列如SEQ ID No.2所示。编码HPV16L1h4/33dEs的多核苷酸序列经大肠杆菌密码子优化设计，构建方式为删除HPV16L1大肠杆菌密码子优化基因骨架(序列如SEQ ID No.8所示)的核苷酸1291-1296，并在核苷酸1290/1297之间插入序列SEQ ID No.10；
7)嵌合L1蛋白HPV16 L1ΔCDE136-137/33dE：骨架为C端截短31个氨基酸的HPV16 L1蛋白(即在序列SEQ ID No.4的C端截短31个氨基酸)，在其DE环aa.136/137位点，直接插入HPV33 L2蛋白aa.16-37多肽(插入片段序列如SEQ ID No.1所示)。编码HPV16 L1ΔCDE136-137/33dE的多核苷酸序列经昆虫细胞密码子优化设计，构建方式为在HPV16 L1昆虫细胞密码子优化基因骨架(序列如SEQ ID No.14所示)的核苷酸408/409之间插入HPV33 L2蛋白的aa.16-37的昆虫密码子优化基因(序列如SEQ ID No.15所示)；
8)嵌合L1蛋白HPV16 L1ΔCDE136-137/33dEs：骨架为C端截短31个氨基酸的HPV16型L1蛋白(即在序列SEQ ID No.4的C端截短31个氨基酸)，在其DE环aa.136/137位点，直接插入HPV33 L2蛋白的aa.17-32多肽(插入片段序列如SEQ ID No.2所示)。编码HPV16 L1ΔCDE136-137/33dEs的多核苷酸序列经昆虫细胞密码子优化设计，构建方式为在HPV16 L1昆虫细胞密码子优化基因骨架(序列如SEQ ID No.14所示)的核苷酸408/409之间插入HPV33 L2蛋白的aa.17-32的昆虫密码子优化基因(序列如SEQ ID No.16所示)；
9)嵌合L1蛋白HPV16 L1ΔCDE135-138/33dE：骨架为C端截短31个氨基酸的HPV16 L1蛋白(即在序列SEQ ID No.4的C端截短31个氨基酸)，删除其aa.135-138区域，并在aa.134/139之间融合包含连接子的HPV33 L2蛋白的aa.16-37多肽(在HPV16型L1蛋白的aa.135-138区域非等长置换插入)，插入片段的氨基酸序列如SEQ ID No.5所示。其中，HPV33 L2蛋白的aa.16-37多肽的N端融合G(甘氨酸)P(脯氨酸)连接子且C端融合P(脯氨酸)连接子。编码HPV16 L1ΔCDE135-138/33dE的多核苷酸序列经昆虫细胞密码子优化设计，构建方式为删除HPV16 L1昆虫细胞密码子优化基因骨架(序列如SEQ ID No.14所示)的核苷酸403-414，并在核苷酸402/415之间插入序列SEQ ID No.17；
10)嵌合L1蛋白HPV16 L1ΔCDE135-138/33dEs：骨架为C端截短31个氨基酸的HPV16 L1蛋白(即在序列SEQ ID No.4的C端截短31个氨基酸)，删除其aa.135-138区域，并在aa.134/139之间融合包含连接子的HPV33 L2蛋白的aa.17-32 多肽(在HPV16 L1蛋白的aa.135-138区域非等长置换插入)，插入片段的氨基酸序列如SEQ ID No.6所示。其中，HPV33 L2蛋白的aa.17-32多肽的N端融合G(甘氨酸)P(脯氨酸)连接子且C端融合P(脯氨酸)连接子。编码HPV16L1ΔCDE135-138/33dEs的多核苷酸序列经昆虫细胞密码子优化设计，构建方式为删除HPV16 L1昆虫细胞密码子优化基因骨架(序列如SEQ ID No.14所示)的核苷酸403-414，并在核苷酸402/415之间插入序列SEQ ID No.18；
11)嵌合L1蛋白HPV16 L1ΔCh4/33dE：骨架为C端截短31个氨基酸的HPV16L1蛋白(即在序列SEQ ID No.4的C端截短31个氨基酸)，在其h4区域的aa.430-433区域非等长置换插入HPV33 L2蛋白的aa.17-37多肽，即删除HPV16 L1蛋白的aa.431-432区域，并在aa.430/433之间融合HPV33 L2蛋白的aa.17-37多肽，插入片段的氨基酸序列如SEQ ID No.3所示。编码HPV16 L1ΔCh4/33dE的多核苷酸序列经昆虫细胞密码子优化设计，构建方式为删除HPV16 L1昆虫细胞密码子优化基因骨架(序列如SEQ ID No.14所示)的核苷酸1291-1296，并在核苷酸1290/1297之间插入序列SEQ ID No.19；
12)嵌合L1蛋白HPV16 L1ΔCh4/33dEs：骨架为C端截短31个氨基酸的HPV16L1蛋白(即在序列SEQ ID No.4的C端截短31个氨基酸)，在其h4区域的aa.430-433区域非等长置换插入HPV33 L2蛋白的aa.17-32多肽，即删除HPV16型L1蛋白的aa.431-432区域，并在aa.430/433之间融合HPV33 L2蛋白的aa.17-32多肽，插入片段的氨基酸序列如SEQ ID No.2所示。编码HPV16 L1ΔCh4/33dEs的多核苷酸序列经昆虫细胞密码子优化设计，构建方式为删除HPV16 L1昆虫细胞密码子优化基因骨架(序列如SEQ ID No.14所示)的核苷酸1291-1296，并在核苷酸1290/1297之间插入序列SEQ ID No.16。
依据大肠杆菌密码子及依据昆虫细胞密码子优化的嵌合L1基因采用全基因合成的方式，由上海生工生物工程技术服务有限公司合成。
大肠杆菌密码子优化的嵌合蛋白基因经NdeI/XhoI酶切后，分别插入商业化的表达载体pET22b(Novagen公司生产)。昆虫细胞密码子优化的嵌合蛋白基因经EcoRI/Xba I酶切后，分别插入商业化表达载体pFastBac1(Invitrogen公司生产)中。得到包含嵌合蛋白基因的表达载体，分别为：pET22b-16L1DE136-137/33dE，pET22b-16L1DE136-137/33dEs，ET22b-16L1DE135-138/33dE，pET22b-16L1DE135-138/33dEs，pET22b-16L1h4/33dE，pET22b-16L1h4/33dEs，pFastBac1-16L1ΔCDE136-137/33dE，pFastBac1-16L1ΔCDE136-137/33dEs，pFastBac1-16L1ΔCDE135-138/33dE，pFastBac1-16L1ΔCDE135-138/33dEs，pFastBac1-16L1ΔCh4/33dE，pFastBac1-16L1ΔCh4/33dEs。上述酶切、连接及克隆构建的方法都是公知的，例如专利CN101293918 B。
实施例2：嵌合L1蛋白的基因的重组Bacmid及重组杆状病毒的构建
分别使用包含嵌合L1基因的重组表达载体pFastBac1-16L1ΔCDE136-137/33dE，pFastBac1-16L1ΔCDE136-137/33dEs，pFastBac1-16L1ΔCDE135-138/33dE，pFastBac1-16L1ΔCDE135-138/33dEs，pFastBac1-16L1ΔCh4/33dE，pFastBac1-16L1ΔCh4/33dEs转化大肠杆菌DH10Bac感受态，筛选获得重组Bacmid，然后用重组Bacmid转染昆虫细胞Sf9，在Sf9内扩增重组杆状病毒。重组Bacmid的筛选及重组杆状病毒的扩增方法都是公知的，例如专利CN101148661 B。
实施例3：嵌合L1蛋白的基因在Sf9细胞中的表达
Sf9细胞分别接种6种嵌合L1基因的重组杆状病毒，进行嵌合L1蛋白的表达，27℃培养约88h后收发酵液，3000rpm离心15min，弃上清，用PBS洗涤细胞后，用于表达鉴定及纯化。感染表达的方法是公开的，例如专利CN 101148661 B。
实施例4：嵌合L1蛋白的基因在大肠杆菌中的表达
分别使用包含嵌合L1基因的重组表达载体pET22b-16L1DE136-137/33dE，pET22b-16L1DE136-137/33dEs，pET22b-16L1DE135-138/33dE，pET22b-16L1DE135-138/33dEs，pET22b-16L1h4/33dE，pET22b-16L1h4/33dEs转化大肠杆菌BL21(DE3)。
挑单克隆接种到3ml含氨苄青霉素的LB培养基中，37℃培养过夜。将过夜培养的菌液按1：100的比例加入LB培养基中，37℃培养3h左右，待OD600达到0.8-1.0之间，加IPTG至终浓度0.5μM，16℃培养约12h，收取菌液。
实施例5：嵌合L1蛋白的表达鉴定
取实施例3及实施例4中所述表达不同嵌合L1蛋白的细胞各1×106个，重悬于200μl PBS溶液中，加入6×Loading Buffer 50μl，75℃变性8分钟，分别取10μl进行SDS-PAGE电泳及Western blot鉴定。结果如图1所示，12种嵌合L1蛋白均可在昆虫细胞或原核表达系统中高水平表达，其中HPV16 L1DE136-137/33dE、HPV16L1DE136-137/33dEs、HPV16 L1DE135-138/33dE、HPV16 L1DE135-138/33dEs、HPV16L1h4/33dE、HPV16 L1h4/33dEs大小约55 kDa，其余6种蛋白大小约50 kDa。SDS-PAGE电泳及Western blot鉴定的方法是公开的，例如专利CN101148661 B。
实施例6：嵌合L1蛋白的纯化及动态光散射粒径分析
取嵌合L1的细胞发酵液适量，使用10ml PBS重悬细胞，加PMSF至终浓度1mg/ml，超声破碎(宁波新芝超声破碎仪，6#探头，200 W，超声5 s，间隔7 s，总时间10min)，取破碎上清进行纯化，纯化步骤在室温进行。在裂解液中加入4％β-巯基乙醇(w/w)对VLP进行解聚，然后使用0.22μm滤器过滤样品，依次使用DMAE阴离子交换层析或CM阳离子交换层析(20mM Tris,180mM NaCl,4％ β-ME,pH 7.9洗脱)、TMAE阴离子交换层析或Q阳离子交换层析(20mM Tris,180mM NaCl,4％β-ME,pH 7.9洗脱)及羟基磷灰石层析(100mM NaH2PO4，30mM NaCl，4％β-ME，pH 6.0洗脱)纯化。纯化产物采用Planova超滤系统进行浓缩，并更换缓冲液(20mM NaH2PO4，500mM NaCl，pH 6.0)促使VLP组装。以上纯化方法均是公开的，例如专利CN101293918 B、CN1976718 A等。
取纯化后的嵌合蛋白溶液进行DLS粒径分析(Zetasizer Nano ZS 90动态光散射仪，Malvern公司)，结果如表1所示，其中HPV16 L1ΔCDE135-138/33dE及HPV16L1ΔCDE135-138/33dEs的DLS分析图如图2所示。
表1嵌合L1蛋白DLS分析
蛋白名称 水力学直径(nm) PDI
HPV16 L1DE136-137/33dE 92.5 0.131
HPV16 L1DE136-137/33dEs 98.4 0.142
HPV16 L1DE135-138/33dE 91.4 0.133
HPV16 L1DE135-138/33dEs 95.2 0.142
HPV16 L1h4/33dE 89.4 0.176
HPV16 L1h4/33dEs 82.6 0.188
HPV16 L1ΔCDE136-137/33dE 99.4 0.142
HPV16 L1ΔCDE136-137/33dEs 98.8 0.136
HPV16 L1ΔCDE135-138/33dE 91.6 0.177
HPV16 L1ΔCDE135-138/33dEs 97.9 0.143
HPV16 L1ΔCh4/33dE 88.6 0.144
HPV16 L1ΔCh4/33dEs 90.8 0.152
实施例7：嵌合VLP的透射电镜观察
按实施例6所述的层析纯化方法，对嵌合VLP分别进行纯化，使用透析后的VLP制备铜网，并用1％醋酸铀进行染色，充分干燥后使用JEM-1400电镜(奥林巴斯)进行观察。结果如图3所示，昆虫细胞表达的在DE区嵌合L2抗原多肽的cVLP直径约为50nm，在h4区域嵌合L2抗原的多肽的cVLP直径约35-40nm。原核表达的cVLP与昆虫细胞表达的cVLP大小规律一致，在DE区嵌合L2抗原多肽的cVLP直径约为50nm，在h4区域嵌合L2抗原多肽的cVLP直径约35-40nm。铜网制备及电镜观察的方法均是公开的，例如专利CN 101148661 B。
实施例8：嵌合VLP的小鼠免疫及中和抗体滴度测定
取4-6周龄的BALB/c小鼠，随机分组，每组5只，用cVLP 10μg，Al(OH)3 50μg及PIKA佐剂50μg免疫小鼠。皮下注射，于第0，2，4，6周免疫，共4次。第4次免疫后2周尾静脉采血，分离血清。
使用12种HPV假病毒对免疫血清的中和抗体滴度进行检测，结果如表2所示，结果显示大肠杆菌及昆虫细胞表达体系生产的cVLP免疫小鼠后均可有效诱发交叉中和抗体，中和范围广。其中HPV16 L1ΔCDE136-137/33dE等昆虫细胞表达的cVLP免疫血清可中和至少12个型别的假病毒。假病毒制备及假病毒中和实验的方法均是公开的，例如专利CN 104418942A。
此外，本发明包括的在DE区域或h4区域采用其他柔性连接子连接L2蛋白的多肽构建的嵌合蛋白，均能形成的cVLP，采用上述策略免疫小鼠后，诱发的交叉中和抗体水平与表2所示的cVLP相比没有差异。由前述12种嵌合L1蛋白分别构成的五聚体，采用上述策略免疫小鼠后，也可诱发交叉中和抗体。
表2不同cVLP在小鼠中诱发的中和抗体滴度
Figure PCTCN2017075402-appb-000001

Claims (11)

  1. 一种乳头瘤病毒嵌合蛋白，其骨架是乳头瘤病毒的L1蛋白或乳头瘤病毒的L1蛋白的突变体，其特征在于，所述骨架上嵌合至少一个来自HPV33型L2蛋白或HPV33型L2蛋白突变体的多肽。
  2. 根据权利要求1所述的乳头瘤病毒嵌合蛋白，其特征在于，所述多肽来自HPV33型L2蛋白的氨基酸1-200区域内的任意连续8-33个氨基酸的片段，优选地，所述多肽的氨基酸序列如SEQ ID No.1所示；或者，所述多肽是在SEQ ID No.1所示的氨基酸序列的N端延长或截短1-5个氨基酸和/或C端延长或截短1-5个氨基酸所获得的多肽，优选地，所述多肽的氨基酸序列如SEQ ID No.2或者SEQ ID No.3所示。
  3. 根据权利要求1所述的乳头瘤病毒嵌合蛋白，其特征在于，所述骨架是HPV16型L1蛋白或HPV16型L1蛋白的突变体，优选地，所述HPV16型L1蛋白的突变体是在所述的HPV16型L1蛋白的N端截短0-9个氨基酸和/或C端截短0-34个氨基酸的所获得的蛋白，更优选地，所述骨架的氨基酸序列如SEQ ID No.4所示,或在如SEQ ID No.4所示的氨基酸序列的C端截短31个氨基酸。
  4. 根据权利要求3所述的乳头瘤病毒嵌合蛋白，其特征在于，所述来自HPV33型L2蛋白的多肽嵌合于所述HPV16型L1蛋白或C端截短31个氨基酸的所述HPV16型L1蛋白的突变体的表面区，优选为嵌合于所述HPV16型L1蛋白或C端截短31个氨基酸的所述HPV16型L1蛋白的突变体的DE环，更优选为：
    通过直接插入的方式嵌合于所述HPV16型L1蛋白或C端截短31个氨基酸的所述HPV16型L1蛋白的突变体的氨基酸136和137之间，在所述直接插入的方式中，所述来自HPV33型L2蛋白的多肽的氨基酸序列是SEQ ID No.1或SEQ ID No.2；或者，
    通过非等长置换的方式嵌合于所述HPV16型L1蛋白或C端截短31个氨基酸的所述HPV16型L1蛋白的突变体的氨基酸135-138区域；其中，在所述非等长置换的方式中，所述来自HPV33型L2蛋白的多肽的N端和/或C端含有1-3个氨基酸的连接子，优选地，所述连接子由选自甘氨酸、丝氨酸、丙氨酸及脯氨酸的氨基酸任意组合构成，更优选地，N端选用甘氨酸和脯氨酸连接子，C端选用脯氨酸连接子；可选地，在所述非等长置换的方式中，删除所述HPV16型L1蛋白或C端截短31个氨基酸的所述HPV16型L1蛋白的突变体的氨基酸135-138区域后，在HPV16型L1蛋白或C端截短31个氨基酸的所述HPV16型L1蛋白的突变体的氨基酸134及139之间插入如SEQ ID No.5或SEQ ID No.6所示的氨基酸序列。
  5. 根据权利要求3所述的乳头瘤病毒嵌合蛋白，其特征在于，所述来自HPV33型L2蛋白的多肽嵌合于所述HPV16型L1蛋白或C端截短31个氨基酸的所述HPV16型L1蛋白的突变体的表面区；优选为嵌合于所述HPV16型L1蛋白或C端截短31个氨基酸的所述HPV16型L1蛋白的突变体的h4区；更优选为通过非等长置换的方式嵌合于HPV16型L1蛋白或C端截短31个氨基酸的所述HPV16型L1蛋白的突变体的氨基酸431-432区域，可选地，所述非等长置换的方式中，所述来自HPV33型L2蛋白的多肽的N端和/或C端含有1-3个氨基酸的连接子；可选地，在所述非等长置换的方式中，删除所述HPV16型L1蛋白或C端截短31个氨基酸的所述HPV16型L1蛋白的突变体的氨基酸431-432区域后，在所述HPV16型L1蛋白或C端截短31个氨基酸的所述HPV16型L1蛋白的突变体的氨基酸430及433之间插入如SEQ ID No.2或SEQ ID No.3所示的氨基酸序列。
  6. 一种多核苷酸，其编码权利要求1-5中任一项所述的乳头瘤病毒嵌合蛋白，优选地，所述多核苷酸的序列采用大肠杆菌密码子进行全基因优化或采用昆虫细胞密码子进行全基因优化。
  7. 一种载体，其包含如权利要求6所述的多核苷酸。
  8. 一种细胞，其包含如权利要求7所述的载体。
  9. 一种乳头瘤病毒外壳蛋白构成的多聚物，优选地，该多聚物为嵌合五聚体或嵌合病毒样颗粒，其含有权利要求1至5中任一项所述的乳头瘤病毒嵌合蛋白，或者由权利要求1至5中任一项所述的乳头瘤病毒嵌合蛋白所形成。
  10. 如权利要求1-5中任一项所述的乳头瘤病毒嵌合蛋白或如权利要求9所述的乳头瘤病毒嵌合五聚体或嵌合病毒样颗粒在制备预防乳头瘤病毒感染及感染诱发的疾病的疫苗中的用途。
  11. 一种用于预防乳头瘤病毒感染及感染诱发的疾病的疫苗，其包含如权利要求9所述的乳头瘤病毒嵌合五聚体或嵌合病毒样颗粒、佐剂、以及疫苗用赋形剂或载体，优选地，还包含至少一种嗜黏膜组和/或嗜皮肤组的HPV的病毒样颗粒或嵌合病毒样颗粒，更优选地，所述佐剂为人用佐剂，甚至优选地，所述人用佐剂是铝佐剂、水包油乳剂或油包水乳剂及TLR刺激剂的佐剂组合物、氢氧化铝佐剂或磷酸铝佐剂与聚肌苷酸-聚胞苷酸佐剂及稳定剂的组合物或者MF59佐剂与聚肌苷酸-聚胞苷酸佐剂及稳定剂的组合物。
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