WO2017157172A1 - Protéine chimère de papillomavirus et son application - Google Patents

Protéine chimère de papillomavirus et son application Download PDF

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WO2017157172A1
WO2017157172A1 PCT/CN2017/075401 CN2017075401W WO2017157172A1 WO 2017157172 A1 WO2017157172 A1 WO 2017157172A1 CN 2017075401 W CN2017075401 W CN 2017075401W WO 2017157172 A1 WO2017157172 A1 WO 2017157172A1
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protein
hpv16
chimeric
amino acid
papillomavirus
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PCT/CN2017/075401
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Chinese (zh)
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许雪梅
陈雪
王志荣
刘洪洋
望硕
张婷
胡美丽
徐雁英
周艳
乔良
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中国医学科学院基础医学研究所
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Publication of WO2017157172A1 publication Critical patent/WO2017157172A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K14/01—DNA viruses
    • C07K14/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
    • C—CHEMISTRY; METALLURGY
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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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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 a papillomavirus chimeric protein or papillomavirus chimeric virus-like particle for 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 HPV 5/6/45/52/58 (Gambhira R, Karanam B, et al. J. Virol.
  • the monoclonal antibody RG-1 of the polypeptide also has cross-neutralizing activity (Gambhira R, Karanam B, et al. J. Virol. 2007; 81(24): 13927–13931), therefore, the a2 of the L2 protein and HPV16 L2.
  • the 17-36 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.
  • HPV16RG-1 epitope is the most immunogenic, and the broad-spectrum neutralizing antibody reaction can be induced by inserting HPV and phage VLP surface; HPV31/45/51 Possibly, other types are unknown (lack of reports); it is worth noting that 8 types (HPV1/5/6/11/16/18/45/58) truncated peptides in the RG-1 region ( L2 aa.17-31) Antiserum obtained by phage cVLP mixed immunization, the type of which is not much, suggesting that some types of truncated polypeptides may be inactive or weak.
  • HPV58 is a dominant strain in China, Southeast Asia, and Latin America. It has a high detection rate in HPV infection-related lesions and tumors, second only to HPV16 or second only to HPV16/18 (X.Castellsagué et al. Vaccine 25S ( 2007) C1–C26).
  • HPV58 L1VLP the immunogenicity of the RG-1 epitope region of HPV58 L2 protein is lacking. Whether the epitope can induce neutralization antibody and the neutralizing range and characteristics of the induced antibody are not clear. Based on current research progress and knowledge, the immunological activity of HPV58 RG-1 epitope vaccine is unpredictable.
  • HPV16 L1VLP is a promising epitope vaccine vector. It can be displayed on the surface of VLP after insertion into the HPV16 RG-1 epitope in a specific position on the surface of HPV16 L1 protein. The spectrum neutralizes antibodies and protects the reaction.
  • a chimeric VLP Chomeric VLP, cVLP
  • a broad-spectrum neutralizing antibody response neutralizing at least 14 HPV type (Schellenbacher C, Roden R, et al 2009; J. Virol.
  • the HPV58 RG-1 epitope and the truncated RG-1 epitope were selected for the HPV16cVLP study, and the results showed that the HPV58 RG-1 long epitope and the short epitope cVLP obtained by the present invention were obtained.
  • It is highly immunogenic (can neutralize at least 10 HPV types) and is comparable to the HPV16 RG-1 cVLP reported in the literature, but the range of neutralization 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 (PV) chimeric protein for use in the preparation of a vaccine for preventing papillomavirus infection and infection-induced diseases.
  • PV papillomavirus
  • the present inventors have unexpectedly discovered that insertion of HPV58 L2 polypeptide into the surface region of full-length or truncated HPV16 L1 protein can increase the immunogenicity of HPV58 L2 polypeptide, and the obtained chimeric protein is expressed in Escherichia coli or insect cell expression system. Highly expressed in the middle, the chimeric protein can be assembled into a VLP, and the needle can be induced A broad-spectrum protective immune response to 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 HPV58 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 HPV58 L2 protein (amino acid sequence as set forth in SEQ ID No. 8). Further preferably, the polypeptide is the HPV58 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 shown in SEQ ID No. 2.
  • 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 backbone of the chimeric protein of 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. 3.
  • 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 from the HPV58 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 HPV16 L1 protein or the C-terminally truncated 31 amino acids of the DEV loop of the mutant of the HPV16 L1 protein more preferably by direct insertion into the HPV16 L1 protein or the C-terminal truncated 31 amino acids
  • the HPV16 L1 protein mutant has amino acid 136 and amino acid 137, or is conjugated to the HPV16 L1 protein or the C-terminally truncated 31 amino acid of the HPV16 L1 protein by non-equal length replacement.
  • the amino acid 135-138 region of the mutant, wherein the polypeptide of the HPV58 L2 protein has a linker of 1-3 amino acids at the N-terminus and/or C-terminus in the non-equal-length substitution manner.
  • 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 of the polypeptide derived from HPV58 L2 protein is SEQ ID No. 1 or SEQ ID No. 2, and the insertion site is the HPV16 L1 protein or C-terminus.
  • a 31 amino acid mutant of the HPV16 L1 protein was truncated 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. 4 or SEQ ID No. 5 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 HPV58 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.
  • 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
  • P valine
  • a P (valine) G (glycine) linker is used at the N-terminus.
  • 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. 6 or SEQ ID No. 7 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 of HPV comprising at least one mucosal group and/or skin-skin group or Chimeric virus-like particles.
  • a vaccine excipient Or a carrier, preferably, a virus-like particle of HPV comprising at least one mucosal group and/or skin-skin group or Chimeric virus-like particles.
  • 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 adjuvant of aluminum adjuvant, oil-in-water emulsion or water-in-oil emulsion and TLR stimulating agent, aluminum hydroxide adjuvant or aluminum phosphate adjuvant and poly
  • a composition of inosine mono-polycytidine adjuvant and a stabilizer or a combination of a MF59 adjuvant and a polyinosinic acid-polycytidine adjuvant and a stabilizer a human adjuvant, preferably an adjuvant of aluminum adjuvant, oil-in-water emulsion or water-in-oil emulsion and TLR stimulating agent, aluminum hydroxide adjuvant or aluminum phosphate adjuvant and poly
  • inosine mono-polycytidine adjuvant and a stabilizer or a combination of a MF59 adjuvant and a polyinosinic acid-polycytidine 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 HPV16 L1 protein examples include, but are not limited to, the full length L1 protein of the same length as AAC09292.1 in the NCBI database.
  • a gene fragment of "truncated HPV16 L1 protein” refers to a nucleotide lacking one or more amino acids deleted at its 5' end and/or 3' end compared to the wild type HPV16 L1 protein gene, wherein The full length sequence of the "wild-type HPV16 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 /58dE; 2 for HPV16 L1DE 136-137 /58dEs; 3 for HPV16 L1DE 135-138 /58dE; 4 for HPV16L1DE 135-138 /58dEs; 5 is HPV16 L1h4/58dE; 6 is HPV16 L1h4/58dEs;
  • Figure 1B Identification of chimeric proteins in insect cells: 1 for HPV16 L1 ⁇ CDE 136-137 /58dE; 2 for HPV16 L1 ⁇ CDE 136-137 /58dEs; 3 for HPV16 L1 ⁇ CDE 135-138 /58dE; 4 for HPV16L1 ⁇ CDE 135-138 /58dEs; 5 is HPV16 L1 ⁇ Ch4/58dE; 6 is HPV16 L1 ⁇ Ch4/58dEs.
  • 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 hydration kinetics of the virus-like particles formed by HPV16 L1 ⁇ CDE 135-138 /58dE and HPV16 L1 ⁇ CDE 135-138 /58dEs recombinant proteins were 91.8 nm and 93.4 nm, respectively, and the percentage of particle assembly was 100%.
  • FIG. 2A HPV16 L1 ⁇ CDE 135-138 / 58dE VLP
  • Figure 2B HPV16 L1 ⁇ CDE 135-138 / 58dEs VLP.
  • 3A to 3F are transmission electron microscope observation results of cVLP obtained after purification in Example 7 of the present invention.
  • Figure 3A HPV16 L1 ⁇ CDE 136-137 / 58dE VLP
  • Figure 3B HPV16 L1 ⁇ CDE 136-137 / 58dEs VLP
  • Figure 3C HPV16 L1 ⁇ CDE 135-138 / 58dE VLP
  • Figure 3D HPV16 L1 ⁇ CDE 135-138 / 58dEs VLP
  • Figure 3E HPV16 L1 ⁇ Ch4/58dE VLP
  • Figure 3F HPV16 L1 ⁇ Ch4/58dEs VLP.
  • Example 1 Synthesis of a chimeric L1 protein gene and construction of its expression vector
  • Chimeric L1 protein HPV16 L1DE 136-137 /58dE The backbone is a full-length HPV16 L1 protein (sequence shown in SEQ ID No. 3), and its HPV58 L2 protein is directly inserted at its DE loop aa.136/137 site.
  • Aa.16-37 polypeptide insert sequence is shown in SEQ ID No. 1.
  • the polynucleotide sequence encoding HPV16 L1DE 136-137 /58dE was designed by E. coli codon optimization in the manner of nucleotide 408 in the HPV16 L1 codon-optimized gene backbone (sequence shown in SEQ ID No. 9).
  • An A. coli codon-optimized gene of aa.16-37 in which the HPV58L2 protein was inserted between /409 the sequence is shown in SEQ ID No. 10);
  • the backbone is a full-length HPV16 L1 protein (sequence shown in SEQ ID No. 3), inserted directly into HPV58 L2 at its DE loop aa.136/137 site.
  • the polypeptide of aa.17-31 of the protein (the insert sequence is shown in SEQ ID No. 2).
  • the polynucleotide sequence encoding HPV16L1DE 136-137 /58dEs was designed by codon optimization in E. coli by nucleotide 408/ in the HPV16 L1 codon-optimized gene backbone (sequence shown in SEQ ID No. 9).
  • An E. coli codon-optimized gene of aa.17-31 in which the HPV58 L2 protein was inserted between 409 (the sequence is shown in SEQ ID No. 11);
  • Chimeric L1 protein HPV16 L1DE 135-138 /58dE The backbone is the full-length HPV16 L1 protein (sequence shown in SEQ ID No. 3), and its aa.135-138 region is deleted, and at aa.134/139 The aa.16-37 polypeptide (in the aa. 135-138 region of the HPV16 L1 protein is inserted in a non-equal length substitution) containing the linker's HPV58 L2 protein is fused, and the amino acid sequence of the insert is shown in SEQ ID No. 4.
  • the N-terminus of the aa.16-37 polypeptide of the HPV58 L2 protein is fused to the G (glycine) P (valine) linker and the C-terminal fusion P (valine) linker.
  • the polynucleotide sequence encoding HPV16 L1DE 135-138 /58dE 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. 9). -414, and insert the sequence SEQ ID No. 12 between nucleotides 402/415;
  • Chimeric L1 protein HPV16 L1DE 135-138 /58dEs The backbone is the full-length HPV16 L1 protein (sequence shown in SEQ ID No. 3), and its aa.135-138 region is deleted, and at aa.134/139 The aa.17-31 polypeptide of the HPV58 L2 protein comprising the linker (non-equal length substitution 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. 5.
  • the N-terminus of the aa.17-31 polypeptide of the HPV58 L2 protein is fused to the G (glycine) P (valine) linker and the C-terminus is fused to the P (valine) linker.
  • the polynucleotide sequence encoding HPV16 L1DE 135-138 /58dEs 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. 9). -414, and insert the sequence SEQ ID No. 13 between nucleotides 402/415;
  • Chimeric L1 protein HPV16 L1h4/58dE The backbone is a full-length HPV16 L1 protein (sequence shown in SEQ ID No. 3), and the non-equal length replacement in the aa.430-433 region of the h4 region is inserted into HPV58 L2.
  • the aa.16-37 polypeptide of the protein, ie, the aa.431-433 region of the HPV16 L1 protein is deleted, and the aa.16-37 polypeptide of the HPV58 L2 protein comprising the linker is fused between aa.430/434, the insert is The amino acid sequence is shown in SEQ ID No. 6.
  • the N-terminus of the aa.16-37 polypeptide of the HPV58 L2 protein is fused with a P (valine) G (glycine) linker.
  • the polynucleotide sequence encoding HPV16 L1h4/58dE was designed by codon optimization in E. coli by deleting the nucleotides 1291-1299 of the HPV16 L1 E. codon-optimized gene backbone (sequence shown in SEQ ID No. 9). And inserting the sequence SEQ ID No. 14 between nucleotides 1290/1300;
  • Chimeric L1 protein HPV16 L1h4/58dEs The backbone is a full-length HPV16 L1 protein (sequence shown in SEQ ID No. 3), and the HPV58 L2 protein is inserted in the aa.430-433 region of the h4 region.
  • the aa.17-31 polypeptide which deletes the aa.431-433 region of the HPV16 L1 protein, and fuses the aa.17-31 polypeptide of the HPV58 L2 protein comprising the linker between aa.430/434, the amino acid of the insert
  • the sequence is shown in SEQ ID No. 7.
  • the N-terminus of the aa.17-31 polypeptide of the HPV58 L2 protein is fused with a P (valine) G (glycine) linker.
  • the polynucleotide sequence encoding HPV16 L1h4/58dEs was designed by E. coli codon optimization by deleting the nucleotides 1291-1299 of the HPV16 L1 E. codon-optimized gene backbone (sequence shown in SEQ ID No. 9). And inserting the sequence SEQ ID No. 15 between nucleotides 1290/1300;
  • the polynucleotide sequence encoding HPV16 L1 ⁇ CDE 136-137 /58dE 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. 16). Inserting the insect codon optimization gene of aa.16-37 of HPV58 L2 protein between /409 (the sequence is shown in SEQ ID No. 17);
  • the polynucleotide sequence encoding HPV16 L1 ⁇ CDE 136-137 /58dEs 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. 16).
  • An insect codon-optimized gene of aa.17-31 in which the HPV58 L2 protein is inserted between /409 (the sequence is shown in SEQ ID No. 18);
  • the N-terminus of the aa.16-37 polypeptide of the HPV58 L2 protein is fused to the G (glycine) P (valine) linker and the C-terminal fusion P (valine) linker.
  • the polynucleotide sequence encoding HPV16 L1 ⁇ CDE 135-138 /58dE 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. 16). -414, and insert the sequence SEQ ID No. 19 between nucleotides 402/415;
  • Chimeric L1 protein HPV16 L1 ⁇ CDE 135-138 /58dEs 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. 3), and its aa is deleted.
  • the amino acid sequence is shown in SEQ ID No. 4.
  • the N-terminus of the aa.17-31 polypeptide of the HPV58 L2 protein is fused to the G (glycine) P (valine) linker and the C-terminus is fused to the P (valine) linker.
  • the polynucleotide sequence encoding HPV16L1 ⁇ CDE 135-138 /58dEs was optimized by insect cell codon design 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. 16). 414, and insert the sequence SEQ ID No. 20 between nucleotides 402/415;
  • the N-terminus of the aa.16-37 polypeptide of the HPV58 L2 protein is fused with a P (valine) G (glycine) linker.
  • the polynucleotide sequence encoding HPV16 L1 ⁇ Ch4/58dE was designed by insect cell codon optimization in such a way as to delete the nucleotides 1291-1299 of the HPV16 L1 insect cell codon-optimized gene backbone (sequence shown in SEQ ID No. 16). And inserting the sequence SEQ ID No. 21 between nucleotides 1290/1300;
  • the N-terminus of the aa.17-31 polypeptide of the HPV58 L2 protein is fused with a P (valine) G (glycine) linker.
  • the polynucleotide sequence encoding HPV16 L1 ⁇ Ch4/58dEs was optimized by insect cell codon design in such a way as to delete the nucleotides 1291-1299 of the HPV16 L1 insect cell codon-optimized gene backbone (sequence shown in SEQ ID No. 16). And the sequence SEQ ID No. 22 was inserted between nucleotides 1290/1300.
  • the whole gene synthesis method was used, and synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.
  • 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 /58dE; pET22b-16L1DE 136-137 /58dEs; pET22b-16L1DE 135-138 /58dE; pET22b-16L1DE 135-138 /58dEs; pET22b -16L1h4/58dE; pET22b-16L1h4/58dEs; pFastBac1-16L1 ⁇ CDE 136-137 /58dE; pFastBac1-16L1 ⁇ CDE 136-137 /58dEs; pFastBac1-16L1 ⁇ CDE 135-138 /58dE; pFastBac1-16L1 ⁇ CDE 135-138 /58dE; pFastBac1-16L1 ⁇ CDE 135-138 /58dE
  • 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 /58dE, pET22b-16L1DE 136-137 /58dEs, pET22b-16L1DE 135-138 /58dE, pET22b-16L1DE 135-138 /58dEs, pET22b-16L1h4 were used, respectively.
  • /58dE, pET22b-16L1h4/58dEs 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 /58dE, HPV16L1DE 136-137 /58dEs, HPV16 L1DE 135-138/ 58dE, HPV16 L1DE 135-138 /58dEs, HPV16L1h4/58dE, HPV16 L1h4/58dEs 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 /58dE and HPV16L1 ⁇ CDE 135-138 /58dEs
  • 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 chimeric VLP expressed by the insect cells was about 50 nm in diameter, uniform in size, and regular in shape.
  • the prokaryotic expressed cVLP is also between 45-50 nm in diameter.
  • 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 neutralizing antibody titers of the immune serum were detected using 12 HPV pseudoviruses. As shown in Table 2, the cross-neutralizing antibodies were effectively induced by cVLP immunized mice, and the neutralization range was wide.
  • the cVLP immune serum expressed by insect cells such as HPV16L1 ⁇ CDE136-137/58dE can neutralize at least 12 types of pseudoviruses. Methods for pseudovirus preparation and pseudovirus neutralization experiments are disclosed, for example, patent CN104418942A.
  • the present invention encompasses a chimeric protein constructed by linking a L2 epitope with other flexible linkers in the DE region or the h4 region, and can form a cVLP, and the level of cross-neutralizing antibody induced by immunizing the mouse using the above strategy is shown. There is no difference in cVLP shown in 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

La présente invention concerne une protéine chimère de papillomavirus, dont le squelette est une protéine L1 de papillomavirus ou un mutant de celle-ci, au moins une protéine L2 de type HPV58 ou un polypeptide muté de celle-ci étant intégré(e) dans le squelette. La protéine chimère de papillomavirus selon l'invention peut être utilisée pour préparer un vaccin destiné à prévenir les infections à papillomavirus et les maladies induites par ces infections.
PCT/CN2017/075401 2016-03-15 2017-03-02 Protéine chimère de papillomavirus et son application WO2017157172A1 (fr)

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CN114539364B (zh) * 2020-11-26 2023-10-20 中国医学科学院基础医学研究所 一种c端改造的人乳头瘤病毒6型l1蛋白及其用途
EP4273174A4 (fr) * 2021-01-04 2024-12-18 Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences Protéine chimère de papillomavirus humain type 31 et son utilisation

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