WO2021013071A1 - 人***瘤病毒多价免疫原性组合物 - Google Patents

人***瘤病毒多价免疫原性组合物 Download PDF

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WO2021013071A1
WO2021013071A1 PCT/CN2020/102601 CN2020102601W WO2021013071A1 WO 2021013071 A1 WO2021013071 A1 WO 2021013071A1 CN 2020102601 W CN2020102601 W CN 2020102601W WO 2021013071 A1 WO2021013071 A1 WO 2021013071A1
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type
hpv
protein
terminal
seq
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PCT/CN2020/102601
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English (en)
French (fr)
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罗春霞
张伟
索晓燕
庞琳
胡萍
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神州细胞工程有限公司
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Priority to EP20843624.6A priority Critical patent/EP4001298A4/en
Priority to MX2022000777A priority patent/MX2022000777A/es
Priority to AU2020317321A priority patent/AU2020317321B2/en
Priority to JP2022503540A priority patent/JP2022540950A/ja
Priority to CA3147849A priority patent/CA3147849A1/en
Priority to KR1020227005633A priority patent/KR20220074855A/ko
Priority to CN202080051322.2A priority patent/CN114127092B/zh
Priority to BR112022001070A priority patent/BR112022001070A2/pt
Publication of WO2021013071A1 publication Critical patent/WO2021013071A1/zh
Priority to US17/579,459 priority patent/US20220370590A1/en

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    • 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
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • 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
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/70—Multivalent vaccine
    • 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 present invention relates to a multivalent immunogenic composition for preventing human papillomavirus (HPV) related diseases or infections and uses thereof.
  • HPV human papillomavirus
  • Papilloma virus belongs to the family of papillomaviruses (Papillomaviridae) and can cause papilloma in humans, cattle, dogs, rabbits, etc. Its member human papillomavirus (Human Papillomavirus, HPV) is a non-enveloped DNA virus.
  • the genome of the virus is a double-stranded closed-loop DNA with a size of about 7.2-8 kb and 8 open reading frames, which can be divided into three regions according to their functions: (1) early region (E), about 4.5 kb, encoding E1, E2 E4-E7 are 6 non-structural proteins related to virus replication, transcription and transformation; (2) Late region (L), about 2.5kb, encoding major capsid protein L1 and minor capsid protein L2; (3) long
  • the regulatory region (LCR) located between the end of the L region and the beginning of the E region, is about 800-900 bp in length, does not encode any protein, but has DNA replication and expression regulatory elements.
  • L1 and L2 proteins are synthesized in the middle and late stages of the HPV infection cycle.
  • the L1 protein is the main capsid protein and has a molecular weight of 55-60 kDa.
  • the L2 protein is a minor capsid protein.
  • 72 L1 protein pentamers constitute the outer shell of icosahedral HPV virus particles (45-55nm in diameter), which wraps the closed-loop double-stranded DNA.
  • the L2 protein is located inside the L1 protein (Structure of Small Virus-like Particles Assembled from the L1 Protein of Human Papillomavirus 16 Chen, X.S., R.L. Garcea, Mol. Cell. 5(3): 557-567, 2000).
  • the ORF of L1 protein is the most conserved gene in PV genome and can be used to identify new PV types. If the complete genome is cloned, and the DNA sequence of the L1 ORF differs by more than 10% from the closest known PV type, it is considered to have isolated a new PV type. Differences between 2% and 10% homology are defined as different subtypes, and differences less than 2% are defined as different variants of the same subtype (E.-M.de V Amsterdam et al./Virology 324(2004) 17- 27).
  • the newly synthesized L1 protein in the cytoplasm is transported to the terminally differentiated keratin cell nucleus. Together with the L2 protein, the copied HPV genomic DNA is packaged to form an infectious virus (Nelson, LM, et al. 2002. Nuclear import strategies of high risk HPV16 L1 major capsid protein. J.Biol.Chem.277:23958-23964). This indicates that the nuclear introduction of L1 protein plays a very important role in HPV infection and production.
  • the ability of the virus to enter the nucleus is determined by the nuclear localization signal (NLS) at the C-terminal of the HPV L1 protein.
  • NLS nuclear localization signal
  • a feature of the nuclear localization signal is that it is rich in basic amino acids (Garcia-Bustos, J., et al. 1991. Nuclear protein localization. Biochimica et al. Biophysica Acta 1071:83-101).
  • HPV-15 high-risk (HR) HPV types can cause cancer of the cervix, anus, penis, vagina, vulva, and oropharynx.
  • HR-HPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82) caused.
  • HPV-16 accounts for about 95% of HPV-positive oropharyngeal carcinomas (OPCs).
  • HPV-6 and HPV-11 cause most anogenital warts and respiratory papilloma, but they are rarely associated with cancer (Human Papillomavirus in Cervical Cancer and Oropharyngeal Cancer: One Cause, Two Diseases Tara A. Berman and John T. Schiller, PhD2 Cancer 2017; 123:2219-29).
  • VLP virus-like particle
  • VLP can induce neutralizing antibodies in vaccinated animals and protect laboratory animals from subsequent attacks by infectious viruses. Therefore, VLP seems to be an excellent candidate for papillomavirus vaccine (Structure of Small Virus-like Particles Assembled from the L1 Protein of Human Papillomavirus 16 Chen, XS, RL Garcea, Mol. Cell. 5(3): 557-567, 2000).
  • Glaxo's It is a bivalent recombinant HPV vaccine. It contains the HPV 16 type recombinant L1 protein and the HPV 18 type recombinant L1 protein obtained by the recombinant baculovirus expression vector system in the insect cells of Trichoplusia ni.
  • L1 protein self-assembles into virus-like particles, which are used to prevent cervical cancer caused by HPV types 16 and 18 in women aged 9-25, grade 2 or 3 cervical intraepithelial neoplasia and adenocarcinoma in situ, and grade 1 cervical cancer Intraepithelial neoplasia (carcinogenic) (https://www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/ApprovedProducts/UCM186981.pdf).
  • HPV 16 and 18 are the cause of about 70% of cervical cancers, and the remaining 20% of cases are attributed to types 31, 33, 45, 52, and 58. It can prevent 90% of cervical cancers (https://www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedProducts/ucm426445.htm).
  • virus-like particles can be produced in large quantities.
  • the more common systems for producing virus-like particles are mainly divided into eukaryotic expression systems and prokaryotic expression systems.
  • eukaryotic expression systems include poxvirus expression system, insect baculovirus expression system, and yeast expression system.
  • the HPV L1 protein expressed in the eukaryotic expression system has less natural conformation damage and can assemble spontaneously to form virus-like particles, but the yield is low.
  • the prokaryotic expression system is mainly Escherichia coli expression system, with high yield but mostly in the form of inclusion bodies, which is not conducive to purification and the production process is complicated.
  • HPV multivalent vaccines can be obtained to prevent HPV-related diseases or infections with a broad spectrum, including HPV-related diseases caused by HPV types that are not currently covered by commercial vaccines. infection.
  • the present invention provides a multivalent HPV immunogenic composition for preventing HPV-related diseases or infections, comprising: HPV type 6, type 11, type 16, type 18, type 31, type 33, type 45 HPV virus-like particles assembled from L1 proteins of, 52 and 58 types; and one or more HPV virus-like particles assembled from L1 proteins of other pathogenic HPV types.
  • the present invention provides a method for preventing HPV-related diseases or infections, which comprises: administering a multivalent HPV immunogenic composition to a subject.
  • the present invention provides the use of a multivalent HPV immunogenic composition for preparing a vaccine or medicine for preventing HPV-related diseases or infections.
  • FIG. 1A HPV 6 L1: 33C L1 protein expression.
  • M Marker
  • L cell lysate
  • E-S supernatant collected after centrifugation of the lysate.
  • FIG. 1B HPV 11 L1: 33C L1 protein expression.
  • M Marker
  • L cell lysate
  • E-S supernatant collected after centrifugation of the lysate.
  • FIG. 1C HPV 16 L1: 33C L1 protein expression.
  • M Marker
  • L cell lysate
  • E-S supernatant collected after centrifugation of the lysate.
  • FIG. 1D HPV 18 L1: 33C L1 protein expression.
  • M Marker
  • L cell lysate
  • E-S supernatant collected after centrifugation of lysate.
  • FIG. 1E HPV 31 L1: 33C L1 protein expression.
  • M Marker
  • L cell lysate
  • E-S supernatant collected after centrifugation of the lysate.
  • Figure 1F HPV 33 L1 L1 protein expression.
  • M Marker
  • L cell lysate
  • E-S supernatant collected after centrifugation of the lysate.
  • FIG. 1G HPV 35 L1: 33C L1 protein expression.
  • M Marker
  • L cell lysate
  • E-S supernatant collected after centrifugation of the lysate.
  • FIG. 1J HPV 51 L1: 33C L1 protein expression.
  • M Marker; L: cell lysate; E-S: supernatant collected after centrifugation of the lysate.
  • M Marker; L: cell lysate; E-S: supernatant collected after centrifugation of the lysate.
  • M Marker; L: cell lysate; E-S: supernatant collected after centrifugation of the lysate.
  • FIG. 1 HPV 58 L1: 33C L1 protein expression.
  • M Marker; L: cell lysate; E-S: supernatant collected after centrifugation of the lysate.
  • FIG. 1N HPV 59 L1 expression of L1 protein.
  • M Marker
  • L cell lysate
  • E-S supernatant collected after centrifugation of the lysate.
  • Figure 4 The expression of HPV16L1 (1-474) truncated at the C-terminal.
  • M Marker
  • L cell lysate
  • E-S supernatant collected after centrifugation of the lysate.
  • the present invention provides a multivalent immunogenic composition for preventing papillomavirus-related diseases or infections.
  • the papillomavirus may be human papillomavirus.
  • the papillomavirus can be canine papillomavirus or rabbit papillomavirus.
  • the present invention provides a multivalent HPV immunogenic composition for preventing HPV-related diseases or infections, comprising: HPV type 6, type 11, type 16, type 18, type 31, type 33, type 45 HPV virus-like particles assembled from L1 proteins of, 52 and 58 types; and one or more HPV virus-like particles assembled from L1 proteins of other pathogenic HPV types.
  • the L1 protein of each type of HPV may be a naturally occurring L1 protein, or a non-naturally occurring L1 protein, or a chimeric HPV L1 protein.
  • the HPV virus-like particle can be assembled from a single type of HPV L1 protein to form a monovalent HPV virus-like particle, or it can be assembled from multiple types of HPV L1 protein to form a multivalent HPV virus-like particle. Particles.
  • the one or more other pathogenic HPV types are selected from HPV type 35, 39, 51, 56 and 59.
  • At least one of the HPV virus-like particles is a chimeric HPV virus-like particle, and the chimeric HPV virus-like particle comprises one or more chimeric HPV L1 proteins; the chimeric HPV
  • the L1 protein includes from its N-terminal to C-terminal direction: a. N-terminal fragment derived from the L1 protein of the first type papillomavirus, wherein the first type of papilloma L1 protein is selected from HPV types 6, 11, Type 16, type 18, type 31, type 35, type 39, type 45, type 51, type 52, type 56 or type 58, the N-terminal fragment maintains the immunogenicity of the L1 protein of the corresponding type of HPV; and b.
  • the L1 protein of the second type papillomavirus has the characteristics of better expression and solubility than other types of L1 protein; wherein the chimeric The HPV type 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, 56 or 58 L1 protein has the immunogenicity of the L1 protein of the corresponding HPV type .
  • the chimeric HPV virus-like particle can be assembled from a single type of chimeric HPV L1 protein to form a monovalent HPV virus-like particle, or it can be assembled from multiple types of chimeric HPV L1 protein. The formation of multivalent HPV virus-like particles.
  • the C-terminal segment and the N-segment segment can be freely combined as needed.
  • the chimeric HPV L1 protein may include one or more C-terminal fragments. The multiple C-terminal fragments may be the same or different.
  • the N-terminal fragment is a fragment obtained by truncating the C-terminal of the natural sequence of the L1 protein of the first type papillomavirus to any amino acid position in its ⁇ 5 region, and the A fragment having at least 98% identity;
  • the C-terminal fragment is a fragment obtained by truncating the N-terminus of the natural sequence of the second type papilloma virus L1 protein to any amino acid position in its ⁇ 5 region , And functional variants resulting from further mutation, deletion and/or addition of the fragment.
  • the N-terminal fragment and the fragment obtained by truncating the C-terminus of the natural sequence of the L1 protein of the first type papillomavirus to any amino acid position in its ⁇ 5 region have at least 98.5%, 99%, 99.5% or 100% identity.
  • the C-terminal fragment contains one or more nuclear localization sequences.
  • the L1 protein of the second type of papillomavirus is selected from HPV type 1, type 2, type 3, type 4, type 6, type 7, type 10, type 11, type 13, type 16.
  • the L1 protein of the second type of papillomavirus is selected from HPV type 16, type 28, type 33, type 59, or type 68 L1 protein;
  • the L1 protein of the second type of papillomavirus is selected from HPV type 33 or HPV type 59 L1 protein.
  • the second type of papillomavirus L1 protein is HPV type 33 L1 protein
  • the C-terminal fragment is SEQ ID No: 2; or a fragment with a length of m1 amino acids, preferably covering SEQ ID A fragment of amino acids 1-m1 of No: 2; wherein m1 is an integer from 8 to 26; or the C-terminal fragment is SEQ ID No: 135; or a fragment of m2 amino acids in length, preferably covering SEQ ID No : A fragment of amino acids 1-m2 of 135; wherein m2 is an integer from 13 to 31.
  • the C-terminal fragment of HPV type 33 L1 protein has a nuclear localization sequence. In another embodiment, the C-terminal fragment of HPV type 33 L1 protein has two nuclear localization sequences. In one embodiment, the amino acid sequence (KR) of amino acid number 7-8 of SEQ ID No: 2 and the amino acid sequence of amino acid sequence number 20-23 (KRKK) are the nuclear localization sequence of the C-terminal fragment of HPV 33 type L1 protein .
  • the L1 protein of the second type of papillomavirus is HPV type 59 L1 protein, and the C-terminal fragment is SEQ ID No: 13; or a fragment of n amino acids in length, preferably covering SEQ ID No: 13 is a fragment of amino acids 1-n; where n is an integer from 16 to 38.
  • the C-terminal fragment of HPV type 59 L1 protein has a nuclear localization sequence. In another embodiment, the C-terminal fragment of HPV type 59 L1 protein has two nuclear localization sequences. In some embodiments, the chimeric HPV L1 protein comprises one or more C-terminal fragments of HPV type 59 L1 protein. The C-terminal fragments of the multiple HPV type 59 L1 proteins may be the same or different. In one embodiment, the amino acid sequence (RKR) of amino acid number 14-16 of SEQ ID No: 13 and the amino acid sequence of amino acid sequence number 28-34 (KRVKRRK) are the nuclear localization sequence of the C-terminal fragment of HPV type 59 L1 protein .
  • the chimeric HPV L1 protein includes both the C-terminal fragment of HPV type 33 L1 protein and the C-terminal fragment of HPV type 59 L1 protein.
  • the N-terminal fragment of the HPV type 6 L1 protein and the fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No:1 to any amino acid position in its ⁇ 5 region have 98% , 98.5%, 99%, 99.5%, 99% or 100% identity;
  • N-terminal fragment of the HPV type 11 L1 protein and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No: 14 to any amino acid position in its ⁇ 5 region have 98%, 98.5% and 99%. , 99.5%, 99% or 100% identity;
  • N-terminal fragment of the HPV type 16 L1 protein and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No: 27 to any amino acid position in its ⁇ 5 region have 98%, 98.5%, 99% , 99.5%, 99% or 100% identity;
  • N-terminal fragment of the HPV type 18 L1 protein and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No: 40 to any amino acid position in its ⁇ 5 region have 98%, 98.5%, 99% , 99.5%, 99% or 100% identity;
  • N-terminal fragment of the HPV type 31 L1 protein and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No: 53 to any amino acid position in its ⁇ 5 region have 98%, 98.5%, 99% , 99.5%, 99% or 100% identity;
  • N-terminal fragment of the HPV type 35 L1 protein and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No: 69 to any amino acid position in its ⁇ 5 region have 98%, 98.5% and 99% , 99.5%, 99% or 100% identity;
  • N-terminal fragment of the HPV type 39 L1 protein and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No: 82 to any amino acid position in its ⁇ 5 region have 98%, 98.5%, 99% , 99.5%, 99% or 100% identity;
  • N-terminal fragment of the HPV type 45 L1 protein and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No: 95 to any amino acid position in its ⁇ 5 region have 98%, 98.5% and 99%. , 99.5%, 99% or 100% identity;
  • N-terminal fragment of the HPV type 51 L1 protein and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No: 108 to any amino acid position in its ⁇ 5 region have 98%, 98.5%, and 99% , 99.5%, 99% or 100% identity;
  • N-terminal fragment of the HPV 52 L1 protein and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No: 121 to any amino acid position in its ⁇ 5 region have 98%, 98.5%, and 99% , 99.5%, 99% or 100% identity;
  • N-terminal fragment of the HPV type 56 L1 protein and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No: 134 to any amino acid position in its ⁇ 5 region have 98%, 98.5% and 99% , 99.5%, 99% or 100% identity;
  • N-terminal fragment of the HPV type 58 L1 protein and the fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 147 to any amino acid position in its ⁇ 5 region have 98%, 98.5%, 99% , 99.5%, 99% or 100% identity.
  • the C-terminus of the N-terminal fragment and the N-terminus of the C-terminal fragment are directly connected or connected via a linker.
  • the linker does not affect the immunogenicity of the N-terminal fragment, and does not affect the expression or solubility of the protein.
  • the N-terminal fragment and the C-terminal fragment are connected by a linker consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
  • the linker is an artificial sequence.
  • the linker is a sequence naturally occurring in the HPV L1 protein.
  • the linker may be a partial sequence of HPV type 33 L1 protein.
  • the linker may be a partial sequence of HPV type 59 L1 protein.
  • the following continuous amino acid sequence exists within the range of plus and minus 4 amino acid positions of the connection point: RKFL; preferably Ground, the following continuous amino acid sequence exists in the range of plus or minus 6 amino acid positions of the connection point: LGRKFL.
  • the L1 protein is respectively associated with SEQ ID No: 3, SEQ ID No: 16, SEQ ID No: 29, SEQ ID No: 42, SEQ ID No: 55, SEQ ID No: 71, SEQ ID No: 84, SEQ ID No: 97.
  • SEQ ID No: 110, SEQ ID No: 123, SEQ ID No: 136, and SEQ ID No: 149 have 98%, 98.5%, 99%, 99.5% or 100% identity; and HPV type 33 L1 protein And HPV type 59 L1 protein has 98%, 98.5%, 99%, 99.5% or 100% identity with SEQ ID No: 66 and SEQ ID No: 160, respectively.
  • At least one of the HPV virus-like particles is composed of a single type of chimeric HPV L1 protein, preferably, the single type of chimeric HPV L1 protein having the same amino acid sequence.
  • the chimeric HPV virus-like particle is an icosahedron composed of 72 pentamers of the chimeric HPV L1 protein. In one embodiment, the chimeric HPV virus-like particles have correctly formed disulfide bonds and thus have a good natural conformation. In one embodiment, the chimeric HPV virus-like particles self-assemble in an in vivo expression system.
  • the multivalent HPV immunogenic composition further comprises a physiologically acceptable carrier and optionally, an adjuvant.
  • the adjuvant includes one or more of aluminum salt, lipid A derivative, and ISCOM.
  • the adjuvant is an aluminum phosphate adjuvant.
  • the present invention provides a method for preventing HPV-related diseases or infections, which comprises: administering a multivalent HPV immunogenic composition to a subject.
  • the prevention can be considered a treatment, and the two can be used interchangeably.
  • the subject is a human.
  • the present invention provides the use of the aforementioned multivalent HPV immunogenic composition in the preparation of vaccines or drugs for preventing HPV-related diseases or infections.
  • the papillomavirus L1 protein expressed by the eukaryotic expression system can spontaneously assemble into virus-like particles, but has the disadvantage of low expression and difficult mass production.
  • the sequence of the L1 protein of each type of HPV can be conveniently obtained from https://www.uniprot.org.
  • Each type of HPV L1 can be derived from different strains, so its amino acid sequence has multiple versions, and any one version of the natural sequence can be used in the present invention.
  • a certain The sequence of the HPV L1 protein of a given type may be different from the sequence used in the examples, but this difference does not affect the judgment and conclusion of the inventor.
  • HPV16 L1 protein C The end truncation of 1-34 amino acids, preferably 26 amino acids, declares that the production of VLP is increased many times, preferably at least 10 times, especially about 10 to 100 times.
  • HPV16 L1 the inventors tried to shorten the C-terminus of HPV type 16 L1 by 31 amino acids and named it HPV16 L1 (1-474).
  • its protein expression is high but the protein solubility is poor, and it is difficult to extract and purify (see comparative example).
  • the poor protein solubility caused by this truncation may be caused by the deletion of the C-terminal nuclear localization sequence, and the present invention is not limited to this speculation.
  • the inventors found that the expression levels of HPV type 16 L1 protein, HPV type 28 L1 protein, HPV type 33 L1 protein, HPV type 59 L1 protein, and HPV type 68 L1 protein are compared with other types of L1 protein. The solubility is better.
  • the inventors replaced the C-terminus of the HPV type that is difficult to extract or low-expression with the C-terminus of the L1 protein with better expression and solubility.
  • the inventors constructed such a chimeric protein which contains the N-terminal fragment derived from the first type of papillomavirus L1 protein (such as HPV L1 protein) and the second type of papillae from the N-terminal to the C-terminal direction.
  • the C-terminal fragment of oncovirus L1 protein (such as HPV L1 protein).
  • the former provides the immunogenicity of the first type of papillomavirus (such as HPV), and the latter provides the characteristics of better expression and solubility.
  • the two can be connected directly or through a joint.
  • protein secondary structure prediction software that can be used for prediction includes but is not limited to:
  • the inventors determined the length of the N-terminal fragment of the L1 protein derived from the first type of HPV in the following manner: truncated the natural sequence of the L1 protein in its ⁇ 5 region and its vicinity, and kept the length from The sequence from the N-terminal to the C-terminal newly generated in the ⁇ 5 region. Such a truncated sequence can ensure that it has the immunogenicity of this type and can form a VLP.
  • the N-terminal fragment derived from the HPV L1 protein of the first type can be further modified to ensure that it has the immunogenicity of this type and can form a VLP.
  • the inventors determined the length of the C-terminal fragment derived from the second type of HPV L1 protein in the following manner.
  • the natural sequence of L1 protein was truncated in its ⁇ 5 region and its vicinity, and the newly generated N-terminal to C-terminal sequence from its ⁇ 5 region was retained. Such a truncated sequence does not have a main neutralizing epitope and does not interfere with the immunogenicity of the chimeric protein formed.
  • the C-terminal fragment derived from the second type of HPV L1 protein may be further mutated, deleted and/or added, preferably retaining at least one nuclear localization sequence.
  • Yang et al. predicted the nuclear localization sequence of 107 HPV subtypes (Yang et al. Predicting the nuclear localization signals of 107 types of HPV L1 proteins by bioinformatic analysis.Geno.Prot.Bioinfo.Vol. 4 No. 1 2006 by reference All are incorporated herein), the nuclear localization sequence of each type of HPV L1 protein can be easily determined by sequence analysis software commonly used in this field.
  • the ligation of the aforementioned N-terminal fragment and the C-terminal fragment occurs at the newly generated C-terminus of the former and the newly generated N-terminus of the latter. It can be directly connected or connected through a joint. Regarding the connection point as the origin, the N terminal side of the origin is negative, and the C terminal side is positive.
  • HPV type 45 some HPV type 45 strains have an additional 26 amino acids at the N-terminus of the L1 protein, while in other HPV type 45 strains There is no such additional 26 amino acids at the N-terminus of the L1 protein, so it is expressed as (478)+26.
  • the inventors conveniently completed the C-terminal replacement of the L1 protein between the different types with the help of the sequence similarity of the ⁇ 5 region and its surrounding regions between multiple HPV types.
  • each type of HPV L1 protein has a tetrapeptide RKFL in a similar position, and a more favorable situation is a hexapeptide LGRKFL.
  • the inventor cleverly used this highly conserved sequence to design the connection point of the chimeric protein at any amino acid position of this oligopeptide.
  • the sequence from the N-terminus of the chimeric protein to RKFL or LGRKFL is the same as the sequence of the N-terminal fragment derived from the first type of HPV L1 protein, while on the other hand, it is from RKFL or LGRKFL to the chimeric protein.
  • the C-terminal end of the synthin has the same sequence as the C-terminal fragment derived from the second type of L1 protein.
  • the chimeric protein thus produced maintains a high degree of similarity with the natural HPV L1 protein, and it can be expected that it will perform well in the production and subsequent medical or preventive processes.
  • the N-terminal fragment derived from the first type of HPV L1 protein will extend more amino acids to the C-terminal.
  • Residues, or the C-terminal fragment derived from the HPV L1 protein of the second type extends more amino acid residues to the N-terminal, and it is also possible that the same or similar amino acids at the corresponding positions form the structure of the present invention Consistent chimeric protein.
  • the chimeric protein thus formed also falls into the present invention.
  • variants of the chimeric protein may be formed through mutation, deletion and/or addition of amino acid residues. These variants may have the immunogenicity of the first type of HPV L1 protein, can form VLPs, and have good yield and solubility.
  • the chimeric protein thus formed also falls into the present invention.
  • the expression systems commonly used for the production of virus-like particles are divided into eukaryotic expression systems and prokaryotic expression systems.
  • the papillomavirus L protein expressed by the eukaryotic expression system can spontaneously assemble into virus-like particles, but it has the disadvantage of low expression and difficult mass production.
  • the natural conformation of the papillomavirus L protein expressed by the prokaryotic expression system is often destroyed, and later in vitro processing is required to obtain virus-like particles, and the yield is low, making it difficult to industrialize.
  • the present invention transforms the C-terminus of the L protein of papillomavirus (such as human papillomavirus), for example, replacing it with HPV type 16 L1 protein, HPV type 28 L1 protein, HPV type 33 L1 protein, HPV type 59 L1 protein, or HPV 68
  • the C-terminal fragment in the type L1 protein can increase the expression and solubility of the papillomavirus L protein in an expression system (for example, host cells, such as insect cells). This can be used for large-scale production of vaccines such as HPV vaccines.
  • HPV type 16 L1 protein, HPV type 28 L1 protein, HPV type 33 L1 protein, HPV type 59 L1 protein, and HPV type 68 L1 protein are better in expression and solubility than other types of L1 protein, and found The increased protein expression and solubility depend on the C-terminal sequence of the HPV L1 protein. In the 107 type HPV L1 protein, most of them have a nuclear localization sequence at the C-terminal, and the C-terminal sequence has a certain similarity.
  • the expression level is very low, or the expression is insoluble, replace its C-terminal fragment with HPV type 16 L1 protein, HPV type 28 L1 protein, HPV type 33 L1 protein, HPV type 59 L1
  • the C-terminal fragment of the protein or HPV type 68 L1 protein makes it possible to soluble expression and subsequent purification of papilloma L protein, which is originally very low or insoluble. This can be used for the large-scale production of more valent vaccines (such as HPV vaccines), making it possible to prevent multiple papillomaviruses, especially HPV infections more comprehensively.
  • HPV L1 protein In order to achieve the large-scale production of vaccines, there is also a need to increase the expression and solubility of HPV L1 protein in insect cells.
  • yeast cells the virus-like particles assembled by HPV L1 protein lack a good conformation because disulfide bonds cannot be formed correctly.
  • HPV L1 protein with low expression and poor solubility in insect cells after the C-terminal fragment is transformed into the C-terminal fragment of HPV 33 or 59 L1 protein, the expression and solubility are significantly improved, which can be used for large-scale HPV vaccines. Scale production.
  • HPV L1 proteins that are better expressed and soluble in insect cells than other types of L1 proteins, such as HPV type 16, HPV type 28 L1 protein, HPV type 68 L1 protein, etc., in order to achieve large-scale production of vaccines, There is also a need to further improve the expression level and solubility.
  • the expression and solubility of the transformed chimeric HPV 16 type L1 protein are improved, which is beneficial to Mass production of HPV vaccine.
  • the expression and solubility of the chimeric HPV L1 protein in insect cells are greatly improved compared to the unmodified HPV L1 protein. It can be used for large-scale production of HPV vaccine.
  • the chimeric HPV L1 protein can correctly form disulfide bonds in insect cells and assemble into HPV virus-like particles with a good conformation. This can improve the immunogenicity of HPV virus-like particles and produce a better immune response.
  • the multivalent vaccine of the present invention can be used to prevent a variety of HPV-related diseases or infections, including HPV types that cannot be prevented at present.
  • immunogenicity refers to the ability of a substance, such as a protein or polypeptide, to stimulate an immune response, that is, the ability to stimulate the production of antibodies, especially the production of body fluids or to stimulate a cell-mediated response.
  • antibody refers to an immunoglobulin molecule capable of binding an antigen.
  • Antibodies can be polyclonal mixtures or monoclonal.
  • the antibody may be a whole immunoglobulin derived from a natural source or a recombinant source or may be an immunoreactive part of a whole immunoglobulin.
  • Antibodies can exist in a variety of forms, including, for example, Fv, Fab', F(ab')2, and as a single chain.
  • antigenicity refers to the ability of a substance, such as a protein or polypeptide, to produce antibodies that specifically bind to it.
  • epitope includes any protein determinant capable of specifically binding to an antibody or T cell receptor.
  • Epitope determinants usually consist of chemically active surface groups of molecules (for example, amino acids or sugar side chains, or combinations thereof), and usually have specific three-dimensional structural characteristics and specific charge characteristics.
  • subtype or “type” are used interchangeably herein, and refer to a genetic variant of the viral antigen so that a subtype can be recognized by the immune system by distinguishing it from a different subtype.
  • HPV 16 can be distinguished from HPV 33 in immunology.
  • HPV L1 protein is used herein, and the terms “HPV” and "human papilloma virus” refer to non-enveloped double-stranded DNA viruses of the papillomavirus family. Their genomes are round and are about 8 kilobase pairs in size. Most HPV encode eight major proteins, six are located in the “early” region (E1-E2), and two are located in the “late” region (L1 (major capsid protein) and L2 (minor capsid protein)). More than 120 HPV types have been identified and they are numbered (for example, HPV-16, HPV-18, etc.).
  • HPV or "HPV virus” refers to the papillomavirus of the papillomavirus family. It is a non-enveloped DNA virus.
  • the viral genome is a double-stranded closed-loop DNA with a size of about 8kb. It can usually be divided into three regions: 1Early region (E), containing 6 open reading frames encoding E1, E2, E4 ⁇ E7 virus replication, transcription and transformation related non-structural proteins, and E3 and E8 open reading frames; 2Late region (L) contains codes The reading frame of the major capsid protein L1 and the minor capsid protein L2; 3Long regulatory region (LCR) does not encode any protein, but has the origin of replication and multiple transcription factor binding sites.
  • HPV L1 protein and HPV L2 protein refer to proteins encoded by the late region (L) of the HPV gene and synthesized late in the HPV infection cycle.
  • the L1 protein is the main capsid protein and has a molecular weight of 55-60 kDa.
  • L2 protein is the minor capsid protein.
  • 72 L1 pentamers constitute the outer shell of icosahedral HPV virus particles, which enclose the closed-loop double-stranded DNA microchromosomes.
  • the L2 protein is located inside the L1 protein.
  • virus-like particle is a hollow particle containing one or more structural proteins of a certain virus without viral nucleic acid.
  • HPV pseudovirus utilizes the characteristic of HPV VLP to wrap nucleic acid non-specifically, and is formed by wrapping free DNA or introducing foreign plasmids into VLP composed of HPV L1 and L2 expressed in cells. It is an ideal HPV neutralization experimental model in vitro.
  • Pseudovirus neutralization method is a method to evaluate the neutralizing activity of antibodies. After immunized animal serum is incubated with a certain amount of pseudovirus, the cells will be infected. The cells will increase with the increase of neutralizing antibodies in the serum. Decrease, there may be a linear negative correlation within a certain range, so the neutralizing activity of antibodies in serum can be evaluated by detecting changes in the number of expressing cells.
  • fragment thereof or “variant thereof” means that a part of the nucleotide or amino acid sequence according to the present invention is deleted, inserted and/or substituted.
  • the fragments or variants of the polypeptides provided by the present invention can trigger humoral and/or cellular immune responses in animals or humans.
  • chimeric means that polypeptides or nucleotide sequences derived from different parent molecules are linked together via amide bonds or 3', 5'-phosphodiester bonds, respectively. Preferably, they are not separated by additional linker sequences, but are directly adjacent to each other.
  • truncated means by removing one or more amino acids from the N and/or C-terminus of the polypeptide or deleting one or more amino acids within the polypeptide.
  • nuclear localization sequence is an amino acid sequence that can guide a protein into the nucleus.
  • two close basic residue clusters ie, nuclear localization sequence
  • nuclear localization sequence for example, one is KRKR, KRKK, KRKRK, KRKKRK, KRVKRRK, etc., and the other is KR, RKR, KRK, etc.
  • Spacer of 10-14 amino acids for example, one is KRKR, KRKK, KRKRK, KRKKRK, KRVKRRK, etc.
  • the above-mentioned basic residue cluster belongs to the nuclear localization sequence.
  • the nuclear localization sequence is a compact cluster of basic residues formed by arginine and/or lysine.
  • Nuclear localization sequences include, but are not limited to, examples of basic residue clusters as described above.
  • the term "functional variant” refers to a version of a polypeptide or protein that has been truncated, mutated, deleted, and/or added and still retains the desired activity or characteristics.
  • sequence identity between two polypeptide or nucleic acid sequences means the number of identical residues between the sequences as a percentage of the total number of residues, and is calculated based on the size of the smaller of the compared molecules.
  • sequences being compared are aligned in a way that produces the largest match between the sequences, and the gaps in the alignment (if any) are resolved by a specific algorithm.
  • Preferred computer program methods for determining the identity between two sequences include, but are not limited to, the GCG program package, including GAP, BLASTP, BLASTN, and FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-410) .
  • the above program can be publicly obtained from the International Center for Biotechnology Information (NCBI) and other sources.
  • NCBI International Center for Biotechnology Information
  • Smith Waterman algorithm can also be used to determine identity.
  • Non-critical amino acids can be conservatively substituted without affecting the normal function of the protein.
  • Conservative substitution means replacing an amino acid with a chemically or functionally similar amino acid. It is well known in the art to provide conservative substitution tables for similar amino acids. For example, in some embodiments, the amino acid groups provided in Tables 1-3 are considered to be mutually conservative substitutions.
  • amino acid means twenty common naturally occurring amino acids.
  • Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C ); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine ( Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).
  • Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspart
  • a “physiologically acceptable carrier” is non-toxic to cells or mammals at the dose and concentration used.
  • a pH buffered aqueous solution non-limiting examples of which include buffers; antioxidants; oligopeptides; proteins; hydrophilic polymers; amino acids; monosaccharides, disaccharides and other carbohydrates; chelating agents; sugar alcohols; salt formation Counterions such as sodium; and/or nonionic surfactants.
  • adjuvant refers to a compound or mixture that enhances the immune response.
  • the vaccine may contain adjuvants.
  • the adjuvant used in the present invention may include, but is not limited to, one or more of the following: mineral adjuvant compositions, oil-milk adjuvants, saponin adjuvant preparations, bacteria or microbial derivatives.
  • vector means a nucleic acid molecule capable of multiplying another nucleic acid to which it is linked.
  • the term includes a vector as a self-replicating nucleic acid structure and as a vector integrated into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids operably linked by such vectors.
  • host cell means a cell into which exogenous nucleic acid has been introduced, and the progeny of such a cell.
  • Host cells include “transformants” (or “transformed cells”), “transfectants” (or “transfected cells”), or “infectants” (or “infected cells”), each of which includes primary transformation, transfection, or Infected cells and their descendants.
  • Such offspring may not be exactly the same as the parent cell in nucleic acid content, and may contain mutations.
  • the amount of administration is preferably a "prophylactically effective amount" (prevention can be regarded as a treatment herein, and the two are used interchangeably), which is sufficient to show a benefit to the individual.
  • Example 1.1 Construction of a chimeric gene in which HPV6L1 C-terminal is replaced with HPV33L1 C-terminal
  • Thermo Fisher company [former Yingwei Jieji (Shanghai) Trading Co., Ltd.] to synthesize HPV6L1 gene, and the synthesized sequence has KpnI and XbaI restriction sites at both ends, and the sequence is shown in SEQ ID NO: 5.
  • the synthesized gene fragment was ligated with pcDNA3 vector (seller Thermo Fisher) through KpnI and XbaI restriction sites to obtain a plasmid pcDNA3-HPV6-L1 containing a nucleotide sequence encoding HPV6L1 and 1-500 amino acids.
  • the obtained pcDNA3-HPV6-L1 plasmid was digested with KpnI and XbaI to obtain the HPV6L1(1-500) gene fragment. Then the fragment was ligated with the pFastBac TM 1 vector (seller Thermo Fisher) that was digested with KpnI and XbaI to obtain a bacmid vector containing the HPV6L1 (1-500) gene fragment, named pFB-HPV6L1.
  • Entrusted Thermo Fisher Company [former Yingweijieji (Shanghai) Trading Co., Ltd.] to synthesize the HPV33L1 gene, and the synthetic sequence has KpnI and XbaI restriction sites at both ends, and the gene fragment sequence is shown in SEQ ID NO: 6.
  • the synthesized gene fragment was ligated with pcDNA3 vector (seller Thermo Fisher) through KpnI and XbaI restriction sites to obtain a plasmid pcDNA3-HPV33-L1 containing a nucleotide sequence encoding HPV33L1 and 1-499 amino acids.
  • the obtained pcDNA3-HPV33-L1 plasmid was digested with KpnI and XbaI to obtain the HPV33L1 (1-499) gene fragment. Then the fragment was ligated with the pFastBacTM1 vector (seller Thermo Fisher) double digested with KpnI and XbaI to obtain a bacmid vector containing the HPV33L1 (1-499) gene fragment, named pFB-HPV33L1.
  • This gene fragment includes a gene fragment encoding 1-469 amino acids of HPV6L1, 10 bases overlapping with the gene fragment of 474-499 amino acids of HPV33L1, and a KpnI restriction site (GGTAC ⁇ C) segment.
  • the amplified sequence is as SEQ ID No: 9 shows:
  • PCR amplification parameters pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 seconds, annealing at 69°C for 15 seconds, 72°C for 1kb/1min, and 30 cycles; extension at 72°C for 5 minutes; ending at 16°C.
  • primers F2 and R2 were used to amplify a gene fragment with a length of 101 bp.
  • the primer sequence F2 is shown in SEQ ID No: 10
  • R2 is shown in SEQ ID No: 11.
  • This gene fragment contains the 26 (474-499) amino acid gene fragment of HPV33L1 C-terminal, the 10bp base overlapping with the 1-469 amino acid C-terminal gene fragment of HPV6L1 and the XbaI (T ⁇ CTAGA) restriction site, amplified
  • the sequence is shown in SEQ ID No: 12.
  • PCR amplification parameters pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 seconds, annealing at 69°C for 15 seconds, 72°C for 1kb/1min, and 30 cycles; extension at 72°C for 5 minutes; ending at 16°C.
  • the splicing primers are F1 and R2 respectively, and the fragments amplified by the above primers (fragments amplified by F1 and R1, and fragments amplified by F2 and R2) are used as templates.
  • PCR splicing parameters 94°C pre-denaturation 5min; 98°C denaturation 10s, 52°C annealing 15s, 72°C 1kb/1min, for 5 cycles; 98°C denaturation 10s, 68°C annealing 15s, 72°C 1kb/1min, for 25 cycles Cycle; extend at 72°C for 5 min; end at 16°C.
  • SEQ ID NO: 4 encodes a nucleotide sequence consisting of amino acids 1-469 of HPV6L1 and 26 (474-499) amino acids of the C-terminal of HPV33L1, with KpnI and XbaI restriction sites on both ends (hereinafter referred to as splicing sequence).
  • the pFastBac TM 1 vector and the splicing sequence fragments were digested with KpnI+XbaI, and the splicing sequence was cloned into the pFastBac TM 1 vector to obtain the recombinant plasmid pFB-HPV6L1:33C. It is a chimeric gene in which the C-terminus of HPV6L1 is replaced with the C-terminus of HPV33L1.
  • Example 1.3 Construction of a chimeric gene in which HPV16L1 C-terminal is replaced with HPV33L1 C-terminal
  • Example 1.4 Construction of a chimeric gene in which HPV18L1 C-terminal is replaced with HPV33L1 C-terminal
  • Example 1.5 Construction of a chimeric gene in which HPV31L1 C-terminal is replaced with HPV33L1 C-terminal
  • the HPV33L1 gene was constructed through gene synthesis, and was commissioned by Thermo Fisher Company [formerly Yingwei Jieji (Shanghai) Trading Co., Ltd.] for gene synthesis.
  • the synthetic sequence has KpnI and XbaI restriction sites at both ends, and the gene fragment sequence is shown in SEQ ID No: 68.
  • the synthesized gene fragment was ligated with pcDNA3 vector (seller Thermofisher) through KpnI and XbaI restriction sites to obtain a plasmid pcDNA3-HPV33-L1 containing a nucleotide sequence encoding HPV33L1 and 1-499 amino acids.
  • the obtained pcDNA3-HPV33-L1 plasmid was subjected to KpnI and XbaI double enzyme digestion to obtain the HPV33L1 (1-499) gene fragment. Then the fragment was ligated with the pFastBac TM 1 vector (seller Thermofisher) that was digested with KpnI and XbaI to obtain a bacmid vector containing the HPV33L1 (1-499) gene fragment, named pFB-HPV33L1.
  • Example 1.7 The construction of a chimeric gene in which HPV35L1 C-terminal is replaced with HPV33L1 C-terminal
  • Example 1.8 Construction of a chimeric gene in which HPV39L1 C-terminal is replaced with HPV59L1 C-terminal
  • Entrusted Thermo Fisher Company [former Yingwei Jieji (Shanghai) Trading Co., Ltd.] to gene synthesis of HPV39L1 gene, and the synthesized sequence has KpnI and XbaI restriction sites at both ends, and its sequence is shown in SEQ ID NO: 86.
  • the synthesized gene fragments were ligated with pcDNA3 vector (seller Thermo Fisher) through KpnI and XbaI restriction sites to obtain a plasmid pcDNA3-HPV39-L1 containing a nucleotide sequence encoding HPV39L1 1-505 amino acids.
  • the obtained pcDNA3-HPV39-L1 plasmid was subjected to double enzyme digestion with KpnI and XbaI to obtain a fragment of the HPV39L1 (1-505) gene. Then the fragment was ligated with the pFastBac TM 1 vector (seller Thermo Fisher) double digested with KpnI and XbaI to obtain a bacmid vector containing the HPV39L1 (1-505) gene fragment, named pFB-HPV39L1.
  • Entrusted Thermo Fisher Company [formerly Yingwei Jieji (Shanghai) Trading Co., Ltd.] to synthesize the HPV59L1 gene, and the synthetic sequence has KpnI and XbaI restriction sites at both ends, and the gene fragment sequence is shown in SEQ ID NO: 87.
  • the synthesized gene fragment was ligated with pcDNA3 vector (seller Thermo Fisher) through KpnI and XbaI restriction sites to obtain a plasmid pcDNA3-HPV59-L1 containing a nucleotide sequence encoding HPV59L1 and 1-508 amino acids.
  • the obtained pcDNA3-HPV59-L1 plasmid was digested with KpnI and XbaI to obtain a fragment of the HPV59L1 (1-508) gene. Then the fragment was ligated with the pFastBacTM1 vector (seller ThermoFisher) digested with KpnI and XbaI to obtain a bacmid vector containing the HPV59L1 (1-508) gene fragment, named pFB-HPV59L1.
  • the gene fragment contains a gene fragment encoding 1-469 amino acids of HPV39L1, 12 bases overlapping with the gene fragment of 471-508 amino acids of HPV59L1, and a KpnI restriction site (GGTAC ⁇ C) segment.
  • the amplified sequence is as SEQ ID No: 90 shows:
  • PCR amplification parameters pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 seconds, annealing at 69°C for 15 seconds, 72°C for 1kb/1min, and 30 cycles; extension at 72°C for 5 minutes; ending at 16°C.
  • primers F2 and R2 were used to amplify a gene fragment of 139 bp in length.
  • the primer sequence F2 is shown in SEQ ID No: 91
  • R2 is shown in SEQ ID No: 92.
  • This gene fragment contains 38 (471-508) amino acid gene fragments at the C-terminal of HPV59L1, 12 bp bases overlapping with the 1-469 amino acid C-terminal gene fragments of HPV39L1, and XbaI (T ⁇ CTAGA) restriction sites, amplified
  • the sequence is shown in SEQ ID No: 93.
  • PCR amplification parameters pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 seconds, annealing at 69°C for 15 seconds, 72°C for 1kb/1min, and 30 cycles; extension at 72°C for 5 minutes; ending at 16°C.
  • the splicing primers are F1 and R2 respectively, and the fragments amplified by the above primers (fragments amplified by F1 and R1, and fragments amplified by F2 and R2) are used as templates.
  • PCR splicing parameters 94°C pre-denaturation 5min; 98°C denaturation 10s, 52°C annealing 15s, 72°C 1kb/1min, for 5 cycles; 98°C denaturation 10s, 68°C annealing 15s, 72°C 1kb/1min, for 25 cycles Cycle; extend at 72°C for 5 min; end at 16°C.
  • SEQ ID NO: 85 encodes a nucleotide sequence consisting of amino acids 1-469 of HPV39L1 and 38 (471-508) amino acids of HPV59L1 C-terminal, with KpnI and XbaI restriction sites at both ends (hereinafter referred to as splicing sequence).
  • the pFastBac TM 1 vector and the splicing sequence fragments were digested with KpnI+XbaI, and the splicing sequence was cloned into the pFastBac TM 1 vector to obtain the recombinant plasmid pFB-HPV39L1:59C. It is a chimeric gene in which HPV39L1 C-terminal is replaced with HPV59L1 C-terminal.
  • Example 1 Construction of a chimeric gene in which HPV51L1 C-terminal is replaced with HPV33L1 C-terminal
  • Example 1.13 The construction of a chimeric gene with HPV58L1 C-terminal replaced with HPV33L1 C-terminal
  • the HPV59L1 gene was constructed through gene synthesis, and was commissioned by Thermo Fisher Company [formerly Yingwei Jieji (Shanghai) Trading Co., Ltd.] for gene synthesis.
  • the synthetic sequence has KpnI and XbaI restriction sites at both ends, and the gene fragment sequence is shown in SEQ ID No: 162.
  • the synthesized gene fragments were ligated with pcDNA3 vector (seller Thermofisher) through KpnI and XbaI restriction sites to obtain a plasmid pcDNA3-HPV59-L1 containing a nucleotide sequence encoding HPV59L1 and 1-508 amino acids.
  • the obtained pcDNA3-HPV59-L1 plasmid was digested with KpnI and XbaI to obtain the HPV59L1 (1-508) gene fragment. Then the fragment was ligated with the pFastBac TM 1 vector (seller Thermofisher) double digested with KpnI and XbaI to obtain a bacmid vector containing the HPV59L1 (1-508) gene fragment, named pFB-HPV59L1.
  • DH10Bac bacterial competent cells The kit, purchased from Thermo Fisher, was cultured and amplified at 37°C, and streaked on a plate. White plaque was selected and amplified. After culturing overnight, the bacterial solution was collected and the recombinant bacmid DNA was extracted by alkaline lysis.
  • the virus supernatant is collected after the cells have obvious lesions, and the culture is generally 7-11 days. Collect the virus supernatant aseptically with a pipette, which is the HPV6L1:33C P1 generation virus seed. Use HPV6L1: 33C P1 generation virus to infect SF9 cells at a ratio of 1:50 (V/V). The infection density of SF9 cells is 2 ⁇ 10 6 cells/mL. Culture and expand at 27°C for 3 days. Centrifuge at 1000g ⁇ 200g for 10min at room temperature. , The collected virus supernatant is the P2 generation virus, which can be used for infection production.
  • Example 2.2 HPV 11L1: 33C Recombinant Baculovirus Packaging
  • Example 2.6 HPV 33L1 recombinant baculovirus packaging
  • Example 2.8 HPV 39L1: 59C Recombinant Baculovirus Packaging
  • Example 2.14 HPV 59L1 recombinant baculovirus packaging
  • the baculovirus containing the HPV 6L1:33C recombinant gene obtained in Example 2 was used to infect High Five cells, the infection ratio was 1:200 (V/V), and the cell pellet was collected by centrifugation at 1000g ⁇ 100g at room temperature.
  • Use PBS or MOPS buffer ( (pH 6.0-7.0, salt concentration 100mM-1M) ultrasonically lyse the cell pellet, sonicate at low temperature for 3min, centrifuge at a centrifugal force greater than 10000g for 10 minutes, collect the centrifuged supernatant, and detect by SDS-PAGE electrophoresis.
  • Lane 1 Marker (Marker is seven purified proteins with a molecular weight ranging from 14.4 to 116 kDa, and the manufacturer is Thermo Scientific); Lane 2: Cell lysate; Lane 3: Supernatant collected after centrifugation of the lysate.
  • HPV 6L1:33C L1 protein produced by this method has a protein yield greater than 100 mg/L and a protein size of about 56KD, which can be used for large-scale production.
  • HPV 11L1:33C L1 protein produced by this method has a protein yield of more than 100 mg/L and a protein size of about 56 KD, which can be used for large-scale production.
  • HPV 16L1:33C L1 protein produced by this method has a protein yield greater than 100 mg/L and a protein size of about 56KD, which can be used for large-scale production.
  • HPV 18L1:33C L1 protein produced by this method has a yield of greater than 100 mg/L and a protein size of about 56KD, which can be used for large-scale production.
  • HPV 31L1:33C L1 protein produced by this method has a yield of more than 100 mg/L and a protein size of about 56KD, which can be used for large-scale production.
  • HPV 33L1 L1 protein produced by this method has a yield of greater than 100 mg/L and a protein size of about 56 KD, which can be used for large-scale production.
  • HPV 35L1:33C L1 protein produced by this method has a protein yield of more than 100 mg/L and a protein size of about 56KD, which can be used for large-scale production.
  • HPV 39L1:59C L1 protein produced by this method has a protein yield greater than 100 mg/L and a protein size of about 56KD, which can be used for large-scale production.
  • HPV 45L1:33C L1 protein produced by this method has a protein yield of more than 100 mg/L and a protein size of about 56KD, which can be used for large-scale production.
  • HPV 51L1:33C L1 produced by this method has a protein yield greater than 100 mg/L and a protein size of about 56 KD, which can be used for large-scale production.
  • HPV 52L1:33C L1 protein produced by this method has a protein yield greater than 100mg/L and a protein size of about 56KD, which can be used for large-scale production.
  • the results are shown in Figure 1L.
  • the HPV 56L1:33C L1 produced by this method has a protein yield of greater than 100 mg/L and a protein size of about 56 KD, which can be used for large-scale production.
  • the results are shown in Figure 1M.
  • the HPV 58L1:33C L1 produced by this method has a protein yield of more than 100 mg/L and a protein size of about 56KD, which can be used for large-scale production.
  • the results are shown in Figure 1N.
  • the protein yield of HPV 59L1 L1 prepared by this method is greater than 100 mg/L, and the protein size is about 56KD, which can be used for large-scale production.
  • Example 4.1 Purification and preparation of HPV 6L1: 33C virus-like particles
  • the HPV 6L1: 33C virus-like particle purification method is a two-step chromatography method, namely the HS-MMA method.
  • the supernatant collected in Example 3 is purified to obtain high-purity virus-like particles.
  • Medium volume medium volume 150mL, linear flow rate 30mL/min.
  • the column is first equilibrated with 5CV buffer and then loaded. After loading the sample, 5CV equilibration buffer and washing buffer were used to elute the contaminated proteins.
  • Elution conditions pH 6.2, elution salt concentration of 1.25 M sodium chloride, elution with 50 mM phosphate buffer containing 50 mM arginine hydrochloride.
  • MMA ion exchange medium produced by Shanghai Boglong Company.
  • Medium volume medium volume 150mL, linear flow rate 30mL/min.
  • Chromatography conditions balance buffer 50mM PB, 1.25M NaCl, pH 6.2.
  • the column is first equilibrated with 4CV equilibration buffer and then loaded. After loading the sample, wash the mixed protein with 5CV equilibration buffer, and then use the elution buffer to elute the target protein to collect the protein.
  • Elution conditions 100mM NaAC, 150mM NaCl, 0.01% Tween 80, pH 4.5.
  • Example 4.2 Purification and preparation of HPV 11L1: 33C virus-like particles
  • Example 4.4 Purification and preparation of HPV 18L1: 33C virus-like particles
  • Example 4.5 Purification and preparation of HPV 31L1: 33C virus-like particles
  • Example 4.8 Purification and preparation of HPV 39L1:59C virus-like particles
  • Example 4.9 Purification and preparation of HPV 45L1: 33C virus-like particles
  • Example 5.2 Morphological detection of HPV 11L1: 33C virus-like particles
  • Example 5.6 Morphological detection of HPV 33L1 virus-like particles
  • HPV is difficult to culture in vitro and has strong host specificity, it is difficult to reproduce in organisms other than the human body, and there is a lack of suitable animal models. Therefore, it is necessary to establish a suitable and effective in vitro neutralization experimental model for the evaluation of vaccine immunity.
  • HPV pseudovirus is an ideal HPV in vitro neutralization experimental model: HPV VLP has the characteristic of non-specifically encapsulating nucleic acid, and the VLP composed of HPV L1 and L2 expressed in cells wraps free DNA or introduces foreign plasmid to form HPV pseudovirus.
  • HPV6 virus-like particle samples can produce neutralizing antibodies against HPV6 after immunizing animals, which can neutralize HPV6 pseudoviruses.
  • the cells that can express GFP fluorescence will decrease with the increase of neutralizing antibodies in the serum. There may be a linear negative correlation within a certain range, so The neutralizing activity of antibodies in serum can be evaluated by detecting changes in the number of cells expressing GFP.
  • HPV 6L1 33C virus-like particles were adsorbed on aluminum phosphate adjuvant, and 200 ⁇ L was used to immunize mice after mixing.
  • the immunization dose per mouse was 0.15 ⁇ g, and 10 mice were immunized on day 0 and day 7 of the experiment.
  • the diluted samples were used to immunize the mice.
  • a blank serum control group was set up.
  • the mice’s eyeballs were taken for blood, and the serum was separated for pseudovirus neutralization titer detection.
  • mouse serum After the mouse serum was inactivated at 56°C for 30 minutes, it was centrifuged at 6000 g, and the supernatant was taken for detection after 5 minutes. 4-8 hours before the detection, 293FT cells were plated in a 96-well plate at a density of 15000 cells/well and cultured in a carbon dioxide incubator at 37°C and 5% CO 2 . After immunization, mouse serum and blank control serum were serially diluted with neutralization medium and mixed with the HPV6 pseudovirus prepared in 6.1 at a volume ratio of 1:1.
  • HPV6 serum pseudovirus neutralization titer The detection results of HPV6 serum pseudovirus neutralization titer are shown in Table 4.
  • GMT Greenwich Mean Titer
  • HPV 6L1:33C virus-like particles prepared in the present invention have good immunogenicity, can produce high-titer neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.
  • the experimental method and steps are the same as in Example 6.1.
  • the L1 sequence is derived from Uniprot P04012, and the L2 sequence is derived from Uniprot P04013.
  • HPV11 serum pseudovirus neutralization titer The detection results of HPV11 serum pseudovirus neutralization titer are shown in Table 5.
  • HPV 11L1:33C virus-like particles prepared by the present invention have good immunogenicity, can produce high-titer neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.
  • the experimental method and steps are the same as in Example 6.1.
  • the L1 sequence is derived from Uniprot P03101, and the L2 sequence is derived from Uniprot P03107.
  • HPV16 serum pseudovirus neutralization titer The detection results of HPV16 serum pseudovirus neutralization titer are shown in Table 6.
  • HPV 16L1:33C virus-like particles prepared by the present invention have good immunogenicity, can produce high-titer neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.
  • the experimental method and steps are the same as in Example 6.1.
  • the L1 sequence is derived from Uniprot Q80B70, and the L2 sequence is derived from Uniprot P06793.
  • HPV18 serum pseudovirus neutralization titer The detection results of HPV18 serum pseudovirus neutralization titer are shown in Table 7.
  • HPV 18L1:33C virus-like particles prepared in the present invention have good immunogenicity, can produce high-titer neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.
  • Example 6.5 Evaluation of animal immunogenicity of HPV 31L1: 33C virus-like particles
  • the experimental method and steps are the same as in Example 6.1.
  • the L1 sequence is derived from Uniprot P17388, and the L2 sequence is derived from Uniprot P17389.
  • HPV31 serum pseudovirus neutralization titer The detection results of HPV31 serum pseudovirus neutralization titer are shown in Table 8.
  • HPV 31L1:33C virus-like particles prepared by the present invention have good immunogenicity, can produce high-titer neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.
  • the experimental method and steps are the same as in Example 6.1.
  • the L1 sequence is derived from Uniprot P06416, and the L2 sequence is derived from Uniprot P06418.
  • HPV33 serum pseudovirus neutralization titer The detection results of HPV33 serum pseudovirus neutralization titer are shown in Table 9.
  • HPV 33L1 virus-like particles prepared by the present invention have good immunogenicity, can produce high-titer neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.
  • the experimental method and steps are the same as in Example 6.1.
  • the L1 sequence is derived from Uniprot P27232, and the L2 sequence is derived from Uniprot P27234.
  • HPV35 serum pseudovirus neutralization titer The detection results of HPV35 serum pseudovirus neutralization titer are shown in Table 10.
  • HPV 35L1:33C virus-like particles prepared by the present invention have good immunogenicity, can produce high-titer neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.
  • the experimental method and steps are the same as in Example 6.1.
  • the L1 sequence is derived from Uniprot P24838, and the L2 sequence is derived from Uniprot P24839.
  • HPV39 serum pseudovirus neutralization titer The detection results of HPV39 serum pseudovirus neutralization titer are shown in Table 11.
  • HPV 39L1:59C virus-like particles prepared in the present invention have good immunogenicity, can produce high-titer neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.
  • the experimental method and steps are the same as in Example 6.1.
  • the L1 sequence is derived from Uniprot P36741, and the L2 sequence is derived from Uniprot P36761.
  • HPV45 serum pseudovirus neutralization titer The detection results of HPV45 serum pseudovirus neutralization titer are shown in Table 12.
  • HPV 45L1:33C virus-like particles prepared in the present invention have good immunogenicity, can produce high-titer neutralizing antibodies in animals, and can be used to prepare vaccines to prevent HPV infection.
  • the experimental method and steps are the same as in Example 6.1.
  • the L1 sequence is derived from Uniprot P26536, and the L2 sequence is derived from Uniprot P26539.
  • HPV51 serum pseudovirus neutralization titer The detection results of HPV51 serum pseudovirus neutralization titer are shown in Table 13.
  • HPV 51L1:33C virus-like particles prepared in the present invention have good immunogenicity, can produce high-titer neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.
  • the experimental method and steps are the same as in Example 6.1.
  • the L1 sequence is from Uniprot Q05138, and the L2 sequence is from Uniprot F8S4U2.
  • HPV52 serum pseudovirus neutralization titer The detection results of HPV52 serum pseudovirus neutralization titer are shown in Table 14.
  • HPV 52L1:33C virus-like particles prepared by the present invention have good immunogenicity, can produce high-titer neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.
  • the experimental method and steps are the same as in Example 6.1.
  • the L1 sequence is derived from Uniprot P36743, and the L2 sequence is derived from Uniprot P36765.
  • HPV56 serum pseudovirus neutralization titer The detection results of HPV56 serum pseudovirus neutralization titer are shown in Table 15.
  • HPV 56L1:33C virus-like particles prepared in the present invention have good immunogenicity, can produce high-titer neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.
  • the experimental method and steps are the same as in Example 6.1.
  • the L1 sequence is derived from Uniprot P26535, and the L2 sequence is derived from Uniprot B6ZB12.
  • HPV 58L1:33C virus-like particles prepared by the present invention have good immunogenicity, can produce high-titer neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.
  • Example 6.14 Evaluation of animal immunogenicity of HPV 59L1 virus-like particles
  • the experimental method and steps are the same as in Example 6.1.
  • the L1 sequence is from Uniprot Q81971, and the L2 sequence is from Uniprot Q81970.
  • HPV59 serum pseudovirus neutralization titer The test results of HPV59 serum pseudovirus neutralization titer are shown in Table 17.
  • HPV 59L1 virus-like particles prepared in the present invention have good immunogenicity, can produce high-titer neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.
  • Example 6.15 Evaluation of animal immunogenicity of 14-valent virus-like particle immune composition
  • mice On day 0, day 7, and day 21 of the experiment, the diluted samples A group of mice were immunized, a group of mice were immunized with samples, and a blank serum control group was set up. On the 28th day of the experiment, the mice’s eyeballs were taken to get blood, and the serum was separated for pseudovirus neutralization titer detection.
  • mice serum After the mouse serum was inactivated at 56°C for 30 minutes, it was centrifuged at 6000 g, and the supernatant was taken for detection after 5 minutes. 4-8 hours before the detection, 293FT cells were plated in a 96-well plate at a density of 15000 cells/well and cultured in a carbon dioxide incubator at 37°C and 5% CO 2 . After immunization, the mouse serum and the blank control serum were serially diluted with neutralization medium and compared with the pre-diluted 14 pseudoviruses in a volume ratio of 1:1 (type 14 HPV pseudoviruses include 6, 11, 16, 18, 31). , 33, 35, 39, 45, 51, 52, 56, 58, and 59 types, see Examples 6.1-6.14 for their preparation respectively) mixing.
  • type 14 HPV pseudoviruses include 6, 11, 16, 18, 31.
  • the 14-valent virus-like particle immune composition can produce very good neutralizing antibodies and can be used to prepare a vaccine to prevent HPV infection.
  • HPV16L1(1-474) SEQ ID NO: 27
  • HPV16L1(1-474) SEQ ID NO: 27
  • the truncated HPV16L1(1-474) protein has high expression level but poor protein solubility, and it is difficult to extract and purify.
  • the specific expression and extraction results are shown in Figure 4.
  • Appendix 1 Sequence Listing-Chimeric Human Papillomavirus Type 6 L1 Protein
  • Appendix 2 Sequence Listing-Chimeric Human Papillomavirus Type 11 L1 Protein
  • Appendix 3 Sequence Listing-Chimeric Human Papillomavirus Type 16 L1 Protein
  • Appendix 4 Sequence Listing-Chimeric Human Papillomavirus Type 18 L1 Protein
  • Appendix 5 Sequence Listing-Chimeric Human Papillomavirus Type 31 L1 Protein
  • Appendix 6 Sequence Listing-Human Papillomavirus Type 33 L1 Protein
  • Appendix 7 Sequence Listing-Chimeric Human Papillomavirus Type 35 L1 Protein
  • Appendix 8 Sequence Listing-Chimeric Human Papillomavirus Type 39 L1 Protein
  • Appendix 9 Sequence Listing-Chimeric Human Papillomavirus Type 45 L1 Protein
  • Appendix 10 Sequence Listing-Chimeric Human Papillomavirus Type 51 L1 Protein
  • Appendix 11 Sequence Listing-Chimeric Human Papillomavirus Type 52 L1 Protein
  • Appendix 12 Sequence Listing-Chimeric Human Papillomavirus Type 56 L1 Protein
  • Appendix 13 Sequence Listing-Chimeric Human Papillomavirus Type 58 L1 Protein
  • Appendix 14 Sequence Listing-Human Papillomavirus Type 59 L1 Protein

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Abstract

本发明公开了预防人***瘤病毒(HPV)相关疾病或感染的多价HPV免疫原性组合物及其用途。所述多价HPV免疫原性组合物包含：由HPV 6型、11型、16型、18型、31型、33型、45型、52型和58型的L1蛋白组装而成的HPV病毒样颗粒；和一种或多种由其他致病的HPV型别的L1蛋白组装而成的HPV病毒样颗粒。在一个实施方式中，所述一种或多种其他致病的HPV型别选自HPV 35型、39型、51型、56型和59型。在一个实施方式中，至少一种所述HPV病毒样颗粒为嵌合的HPV病毒样颗粒，所述嵌合的HPV病毒样颗粒包含一种或多种嵌合HPV L1蛋白。

Description

人***瘤病毒多价免疫原性组合物 技术领域
本发明涉及预防人***瘤病毒(HPV)相关疾病或感染的多价免疫原性组合物及其用途。
背景技术
***瘤病毒(papilloma virus，PV)属于***瘤病毒科(Papillomaviridae)，能引起人、牛、狗、兔等的***瘤。其成员人***瘤病毒(Human Papillomavirus,HPV)为无包膜DNA病毒。该病毒的基因组为双链闭环DNA，大小约7.2-8kb，具有8个开放阅读框，按照功能可分为三个区域：(1)早期区(E),约4.5kb，编码E1、E2、E4-E7共6个与病毒复制、转录及转化有关的非结构蛋白；(2)晚期区(L)，约2.5kb，编码主要衣壳蛋白L1和次要衣壳蛋白L2；(3)长调控区(LCR)，其位于L区末端与E区起始端之间，长约800-900bp，不编码任何蛋白，但具有DNA复制和表达调控元件。
L1和L2蛋白在HPV感染周期的中晚期合成。L1蛋白是主要衣壳蛋白并且具有55-60kDa的分子量。L2蛋白是次要衣壳蛋白。72个L1蛋白五聚体构成二十面体HPV病毒粒子的外壳(直径为45-55nm),其包裹闭环双链DNA。L2蛋白质位于L1蛋白质内侧(Structure of Small Virus-like Particles Assembled from the L1 Protein of Human Papillomavirus 16 Chen,X.S.,R.L.Garcea,Mol.Cell.5(3):557-567,2000)。
L1蛋白的ORF是PV基因组中最保守的基因，可以用于鉴别新的PV型。如果克隆了完整的基因组，并且L1 ORF的DNA序列与最接近的已知PV型相差超过10％，则被认定为分离出新的PV型。差异在2％和10％同源性被定义为不同的亚型，差异小于2％被定义为同一亚型的不同变种(E.-M.de Villiers et al./Virology 324(2004) 17–27)。
在HPV感染的后期，细胞质中新合成的L1蛋白被输送到终端分化的角蛋白细胞核中，与L2蛋白一起，包装复制的HPV基因组DNA形成传染性病毒(Nelson,L.M,et al.2002.Nuclear import strategies of high risk HPV16 L1 major capsid protein.J.Biol.Chem.277:23958-23964)。这表明L1蛋白的核导入在HPV感染和生产中起着非常重要的作用。病毒进入细胞核的能力由HPV L1蛋白C端的核定位信号(NLS)决定，核定位信号的一个特征是富含碱性氨基酸(Garcia-Bustos,J.,et al.1991.Nuclear protein localization.Biochimica et Biophysica Acta 1071:83-101)。
15种高风险(HR)HPV型可导致宫颈、***、***、***、外阴和口咽癌。其中，HPV-16和HPV-18型是迄今最为常见的癌症起因，约占***的70％，其余为其他HR-HPV型(31、33、35、39、45、51、52、56、58、59、68、73和82)引起。HPV-16约占HPV阳性口咽癌(OPCs)的95％。持续低风险基因型HPV-6和HPV-11导致大多数***生殖器疣和呼吸道***状瘤，但很少与癌症相关(Human Papillomavirus in Cervical Cancer and Oropharyngeal Cancer:One Cause,Two Diseases Tara A.Bermanand John T.Schiller,PhD2 Cancer 2017；123:2219-29)。
使用痘苗病毒、杆状病毒或酵母***重组表达L1蛋白，L1蛋白可自我装配形成病毒样颗粒(VLP)，大约含有72个L1蛋白，与病毒体外壳相似。VLP没有适应症。VLP可以在接种动物中诱导中和抗体，保护实验动物免受感染性病毒的随后攻击。因此，VLP似乎是***瘤病毒疫苗的优秀候选者(Structure of Small Virus-like Particles Assembled from the L1 Protein of Human Papillomavirus 16 Chen,X.S.,R.L.Garcea,Mol.Cell.5(3):557-567,2000)。
葛兰素公司的
Figure PCTCN2020102601-appb-000001
是双价重组HPV疫苗。其中含有由重组杆状病毒表达载体***在夜蛾(Trichoplusia ni)昆虫细胞中表达获得的HPV 16型重组L1蛋白和HPV 18型重组L1蛋白。L1蛋白 自组装成病毒样颗粒，用于预防9-25岁的妇女由16和18型HPV引起的***，2级或3级宫颈上皮内瘤样变和原位腺癌，和1级宫颈上皮内瘤样病变(致癌)(https://www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/ApprovedProducts/UCM186981.pdf)。
Figure PCTCN2020102601-appb-000002
是默克公司生产的人***状瘤病毒四价(6、11、16和18型)重组疫苗，用于9-26岁的女孩和妇女用于预防***生殖器疣(***)和由HPV 6、11、16、18型引起癌前或增生异常病变；以及9-26岁的男孩和男人用于预防***癌、生殖器疣(***)和由HPV 6、11、16、18型引起的癌前期或发育异常病变(https://www.fda.gov/vaccines-blood-biologics/vaccines/gardasil)。
Figure PCTCN2020102601-appb-000003
是默克公司生产的人***状瘤病毒九价重组疫苗，包含HPV 6、11、16、18、31、33、45、52和58型L1蛋白的病毒样颗粒，该L1蛋白由酿酒酵母发酵生产，自组装为VLP。用于9-45岁的女孩和妇女用于预防HPV16、18、31、33、45、52和58型引起的***、外阴癌、***癌和***癌，HPV6和11引起的生殖器疣(***)和由HPV 6、11、16、18、31、33、45、52和58型引起癌前或或增生异常病变；以及9-45岁的男孩和男人用于预防16、18、31、33、45、52和58型引起的***癌，HPV 6和11引起的生殖器疣(***)和由HPV 6、11、16、18、31、33、45、52和58型引起的癌前期或发育异常病变(https://www.fda.gov/vaccines-blood-biologics/vaccines/gardasil-9)。
Figure PCTCN2020102601-appb-000004
的说明书中声称HPV16和18型是约70％的***的发病缘由，其余的20％病例归责于31、33、45、52和58型，由是
Figure PCTCN2020102601-appb-000005
可以预防90％的***的发生(https://www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedProducts/ucm426445.htm)。
HPV疫苗研制的关键因素是病毒样颗粒可进行大量生产。目前较为普遍的生产病毒样颗粒的***主要分为真核表达***和原核表 达***。
常用的真核表达***有痘病毒表达***、昆虫杆状病毒表达***、酵母表达***。在真核表达***中表达的HPV L1蛋白的天然构像破坏较少，可自发装配形成病毒样颗粒，但产量较低。原核表达***主要大肠杆菌表达***，产量高但大多以包涵体形式存在，不利于纯化，生产工艺复杂。
因此，在本领域仍然存在获得高产量的HPV病毒样颗粒，从而获得HPV多价疫苗以广谱地预防HPV相关疾病或感染，包括目前商业化疫苗尚未涵盖的HPV型别引起的HPV相关疾病或感染。
发明内容
在一个方面，本发明提供一种预防HPV相关疾病或感染的多价HPV免疫原性组合物，其包含：由HPV 6型、11型、16型、18型、31型、33型、45型、52型和58型的L1蛋白组装而成的HPV病毒样颗粒；和一种或多种由其他致病的HPV型别的L1蛋白组装而成的HPV病毒样颗粒。
在另一个方面，本发明提供一种预防HPV相关疾病或感染的方法，其包括：向受试者施用多价HPV免疫原性组合物。
在另一个方面，本发明提供多价HPV免疫原性组合物在用于制备用于预防HPV相关疾病或感染的疫苗或药物中的用途。
附图说明
图1A HPV 6 L1：33C的L1蛋白的表达。M：Marker；L：细胞裂解液；E-S：裂解液离心后收集的上清液。
图1B HPV 11 L1：33C的L1蛋白的表达。M：Marker；L：细胞裂解液；E-S：裂解液离心后收集的上清液。
图1C HPV 16 L1：33C的L1蛋白的表达。M：Marker；L：细胞裂解液；E-S：裂解液离心后收集的上清液。
图1D HPV 18 L1：33C的L1蛋白的表达。M：Marker；L：细 胞裂解液；E-S：裂解液离心后收集的上清液。
图1E HPV 31 L1：33C的L1蛋白的表达。M：Marker；L：细胞裂解液；E-S：裂解液离心后收集的上清液。
图1F HPV 33 L1的L1蛋白的表达。M：Marker；L：细胞裂解液；E-S：裂解液离心后收集的上清液。
图1G HPV 35 L1：33C的L1蛋白的表达。M：Marker；L：细胞裂解液；E-S：裂解液离心后收集的上清液。
图1H HPV 39 L1：59C的L1蛋白的表达。M：Marker；L：细胞裂解液；E-S：裂解液离心后收集的上清液。
图1I HPV 45 L1：33C的L1蛋白的表达。M：Marker；L：细胞裂解液；E-S：裂解液离心后收集的上清液。
图1J HPV 51 L1：33C的L1蛋白的表达。M：Marker；L：细胞裂解液；E-S：裂解液离心后收集的上清液。
图1K HPV 52 L1：33C的L1蛋白的表达。M：Marker；L：细胞裂解液；E-S：裂解液离心后收集的上清液。
图1L HPV 56 L1：33C的L1蛋白的表达。M：Marker；L：细胞裂解液；E-S：裂解液离心后收集的上清液。
图1M HPV 58 L1：33C的L1蛋白的表达。M：Marker；L：细胞裂解液；E-S：裂解液离心后收集的上清液。
图1N HPV 59 L1的L1蛋白的表达。M：Marker；L：细胞裂解液；E-S：裂解液离心后收集的上清液。
图2A透射电镜观察HPV 6 L1：33C病毒样颗粒。
图2B透射电镜观察HPV 11 L1：33C病毒样颗粒。
图2C透射电镜观察HPV 16 L1：33C病毒样颗粒。
图2D透射电镜观察HPV 18 L1：33C病毒样颗粒。
图2E透射电镜观察HPV 31 L1：33C病毒样颗粒。
图2F透射电镜观察HPV 33 L1病毒样颗粒。
图2G透射电镜观察HPV 35 L1：33C病毒样颗粒。
图2H透射电镜观察HPV 39 L1：59C病毒样颗粒。
图2I透射电镜观察HPV 45 L1：33C病毒样颗粒。
图2J透射电镜观察HPV 51 L1：33C病毒样颗粒。
图2K透射电镜观察HPV 52 L1：33C病毒样颗粒。
图2L透射电镜观察HPV 56 L1：33C病毒样颗粒。
图2M透射电镜观察HPV 58 L1：33C病毒样颗粒。
图2N透射电镜观察HPV 59 L1病毒样颗粒。
图3 14型病毒样颗粒组合物免疫小鼠后的假病毒中和效价。动物数，N＝10。GMT(Geometric Mean Titer)：几何平均滴度。
图4C端截短的HPV16L1(1-474)的表达。M：Marker；L：细胞裂解液；E-S：裂解液离心后收集的上清液。
具体实施方式
在一个方面，本发明提供一种预防***瘤病毒相关的疾病或感染的多价免疫原性组合物。在一个实施方式中，***瘤病毒可以为人***瘤病毒。在另一个实施方式中，***瘤病毒可以为狗***瘤病毒或兔***瘤病毒。
在一个方面，本发明提供一种预防HPV相关疾病或感染的多价HPV免疫原性组合物，其包含：由HPV 6型、11型、16型、18型、31型、33型、45型、52型和58型的L1蛋白组装而成的HPV病毒样颗粒；和一种或多种由其他致病的HPV型别的L1蛋白组装而成的HPV病毒样颗粒。
在一个实施方式中，所述HPV各型别的L1蛋白可以为天然存在的L1蛋白，或非天然存在的L1蛋白，或嵌合HPV L1蛋白。在一个实施方式中，所述HPV病毒样颗粒可以由单一型别的HPV L1蛋白装配形成单价的HPV病毒样颗粒，也可以由多个型别的HPV L1蛋白共同装配形成多价的HPV病毒样颗粒。
在一个实施方式中，所述一种或多种其他致病的HPV型别选自HPV 35型、39型、51型、56型和59型。
在一个实施方式中，至少一种所述HPV病毒样颗粒为嵌合的 HPV病毒样颗粒，所述嵌合的HPV病毒样颗粒包含一种或多种嵌合HPV L1蛋白；所述嵌合HPV L1蛋白自其N末端至C末端方向包含：a.衍生于第一型别***瘤病毒L1蛋白的N端片段，其中所述第一型别***瘤L1蛋白选自HPV 6型、11型、16型、18型、31型、35型、39型、45型、51型、52型、56型或58型，所述N端片段保持HPV相应型别L1蛋白的免疫原性；和b.衍生于第二型别***瘤病毒L1蛋白的C端片段，所述第二型别***状瘤病毒L1蛋白具有相较于其他型别的L1蛋白表达量和可溶性较好的特性；其中嵌合的HPV 6型、11型、16型、18型、31型、35型、39型、45型、51型、52型、56型或58型L1蛋白具有HPV相应型别L1蛋白的免疫原性。
在一个实施方式中，所述嵌合的HPV病毒样颗粒可以由单一型别的嵌合HPV L1蛋白装配形成单价的HPV病毒样颗粒，也可以由多个型别的嵌合HPV L1蛋白共同装配形成多价的HPV病毒样颗粒。
在一个实施方式中，所述C端片段和N段片段可以根据需要自由组合。在一个实施方式中，嵌合HPV L1蛋白可以包含一个或多个C端片段。所述多个C端片段可以相同或不同。
在一个实施方式中，所述N端片段为将所述第一型别***瘤病毒L1蛋白的天然序列的C端截短于其α5区内的任一氨基酸位点而得到的片段，以及与其具有至少98％的同一性的片段；所述C端片段为将第二型别***状瘤病毒L1蛋白的天然序列的N末端截短于其α5区内的任一氨基酸位点而得到的片段，以及该片段进一步突变、缺失和/或添加而产生的功能性变体。
在另一个实施方式中，所述N端片段与将所述第一型别***瘤病毒L1蛋白的天然序列的C端截短于其α5区内的任一氨基酸位点而得到的片段具有至少98.5％、99％、99.5％或100％的同一性。
在一个实施方式中，所述C端片段含有一个或多个核定位序列。
在一个实施方式中，所述第二型别***瘤病毒L1蛋白选自HPV 1型、2型、3型、4型、6型、7型、10型、11型、13型、16型、 18型、22型、26型、28型、31型、32型、33型、35型、39型、42型、44型、45型、51型、52型、53型、56型、58型、59型、60型、63型、66型、68型、73型或82型L1蛋白；
优选地，所述第二型别***瘤病毒L1蛋白选自HPV 16型、28型、33型、59型、或68型L1蛋白；
更优选地，所述第二型别***瘤病毒L1蛋白选自HPV 33型或HPV 59型L1蛋白。
在一个实施方式中，所述第二型别***瘤病毒L1蛋白为HPV 33型L1蛋白,所述C端片段为SEQ ID No:2；或其长度为m1个氨基酸的片段，优选涵盖SEQ ID No:2的第1-m1位氨基酸的片段；其中m1为8-26的整数；或所述C端片段为SEQ ID No:135；或其长度为m2个氨基酸的片段，优选涵盖SEQ ID No:135的第1-m2位氨基酸的片段；其中m2为13-31的整数。
在一个实施方式中，HPV 33型L1蛋白的C端片段具有一个核定位序列。在另一个实施方式中，HPV 33型L1蛋白的C端片段具有两个核定位序列。在一个实施方式中，SEQ ID No:2的氨基酸编号7-8的氨基酸序列(KR)和氨基酸序列编号20-23的氨基酸序列(KRKK)为HPV 33型L1蛋白的C端片段的核定位序列。
在一个实施方式中，所述第二型别***瘤病毒L1蛋白为HPV 59型L1蛋白,所述C端片段为SEQ ID No:13；或其长度为n个氨基酸的片段，优选涵盖SEQ ID No:13的第1-n位氨基酸的片段；其中n为16-38的整数。
在一个实施方式中，HPV 59型L1蛋白的C端片段具有一个核定位序列。在另一个实施方式中，HPV 59型L1蛋白的C端片段具有两个核定位序列。在一些实施方式中，嵌合HPV L1蛋白包含一个或多个HPV 59型L1蛋白的C端片段。所述多个HPV 59型L1蛋白的C端片段可以相同也可以不同。在一个实施方式中，SEQ ID No:13的氨基酸编号14-16的氨基酸序列(RKR)和氨基酸序列编号28-34的氨基酸序列(KRVKRRK)为HPV 59型L1蛋白的C端片段的核 定位序列。
在一个实施方式中，所述嵌合HPV L1蛋白包含HPV 33型L1蛋白的C端片段和HPV 59型L1蛋白的C端片段两者。
在一个实施方式中，所述HPV 6型L1蛋白的N端片段与将SEQ ID No:1所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
所述HPV 11型L1蛋白的N端片段与将SEQ ID No:14所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
所述HPV 16型L1蛋白的N端片段与将SEQ ID No:27所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
所述HPV 18型L1蛋白的N端片段与将SEQ ID No:40所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
所述HPV 31型L1蛋白的N端片段与将SEQ ID No:53所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
所述HPV 35型L1蛋白的N端片段与将SEQ ID No:69所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
所述HPV 39型L1蛋白的N端片段与将SEQ ID No:82所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
所述HPV 45型L1蛋白的N端片段与将SEQ ID No:95所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
所述HPV 51型L1蛋白的N端片段与将SEQ ID No:108所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有 98％、98.5％、99％、99.5％、99％或100％的同一性；
所述HPV 52型L1蛋白的N端片段与将SEQ ID No:121所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
所述HPV 56型L1蛋白的N端片段与将SEQ ID No:134所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；和
所述HPV 58型L1蛋白的N端片段与将SEQ ID No:147所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性。
在一个实施方式中，所述N端片段的C末端与所述C端片段的N末端直接连接或通过接头连接。
接头不影响所述N端片段的免疫原性，且不影响蛋白的表达量或可溶性。在一个实施方式中，所述N端片段和所述C端片段通过由1、2、3、4、5、6、7、8、9或10个氨基酸组成的接头连接。在一个实施方式中，接头是人工序列。在另一个实施方式中，接头是HPV L1蛋白中天然存在的序列。在另一个实施方式中，接头可以是HPV 33型L1蛋白的部分序列。在另一个实施方式中，接头可以是HPV 59型L1蛋白的部分序列。
在一个实施方式中，当所述N端片段的C末端与所述C端片段的N末端连接时，在连接点的正负4个氨基酸位点的范围内存在以下连续氨基酸序列：RKFL；优选地，在连接点的正负6个氨基酸位点的范围内存在以下连续氨基酸序列：LGRKFL。
在一些实施方式中，所述嵌合的HPV 6型、11型、16型、18型、31型、35型、39型、45型、51型、52型、56型和58型嵌合HPV L1蛋白分别与SEQ ID No:3、SEQ ID No:16、SEQ ID No:29、SEQ ID No:42、SEQ ID No:55、SEQ ID No:71、SEQ ID No:84、SEQ ID No:97、SEQ ID No:110、SEQ ID No:123、SEQ ID No:136和SEQ ID No:149具有98％、98.5％、99％、99.5％或100％的同一性；以及 HPV 33型L1蛋白和HPV 59型L1蛋白分别与SEQ ID No:66和SEQ ID No:160具有98％、98.5％、99％、99.5％或100％的同一性。
在一个实施方式中，至少一种所述HPV病毒样颗粒由单一型别的嵌合HPV L1蛋白组成，优选地，由具有相同的氨基酸序列的所述单一型别的嵌合HPV L1蛋白组成。
在一个实施方式中，嵌合的HPV病毒样颗粒为由72个所述嵌合HPV L1蛋白的五聚体构成的二十面体。在一个实施方式中，嵌合的HPV病毒样颗粒具有正确形成的二硫键，因而具有良好的天然构象。在一个实施方式中，嵌合的HPV病毒样颗粒在体内表达***中自行装配。
在一个实施方式中，所述多价HPV免疫原性组合物还包含生理学上可接受的载体以及任选地，还包含佐剂。在一个实施方式中，佐剂包含铝盐、脂质A衍生物和ISCOM中的一种或多种。
在一个实施方式中，所述佐剂为磷酸铝佐剂。
在一个方面，本发明提供一种预防HPV相关疾病或感染的方法，其包括：向受试者施用多价HPV免疫原性组合物。所述预防可被认为是治疗，两者可互换使用。在一个实施方式中，受试者是人。
在一个方面，本发明提供如前所述的多价HPV免疫原性组合物在用于制备用于预防HPV相关疾病或感染的疫苗或药物中的用途。
真核表达***表达的***瘤病毒L1蛋白能自发装配成病毒样颗粒，但具有表达量低不易规模化生产的缺点。
各型别HPV的L1蛋白的序列可以从https://www.uniprot.org方便地获得。每一型别的HPV L1可以来源于不同的毒株，因而其氨基酸序列有多个版本，其中任何一个版本的天然序列都可以用于本发明，本发明的构思和设计过程中，所用某一给定型别的HPV L1蛋白序列有可能不同于实施例中使用的序列，但是这种差异不影响发明人的判断和结论。
本领域技术人员普遍认为L1蛋白的C端不含有主要中和抗原表位，因此试图通过截短HPV L1蛋白的C端提高表达量，例如葛兰 素公司的美国专利US6361778B1中，HPV16 L1蛋白C端截短1-34个氨基酸，优选26个氨基酸，声明VLP的产量增加许多倍,最好至少增加10倍,特别是大约10到100倍。受此启发，发明人尝试将HPV 16型L1的C端截短31个氨基酸，命名为HPV16 L1(1-474)。但其蛋白表达量高但蛋白可溶性差，难以提取纯化(见对比例)。
这种截短引起的蛋白可溶性差有可能是C端的核定位序列的缺失造成的,本发明并不受限于此推测。发明人在研究和生产过程中发现HPV 16型L1蛋白、HPV 28型L1蛋白、HPV 33型L1蛋白、HPV 59型L1蛋白和HPV 68型L1蛋白相较于其他型别的L1蛋白表达量和可溶性较好，受此启发，发明人用表达量和可溶性较好型别的L1蛋白C端替换不易提取或表达量低的HPV型别的C端。即发明人构建了这样一种嵌合蛋白：自其N末端至C末端方向包含衍生于第一型别***瘤病毒L1蛋白(例如HPV L1蛋白)的N端片段和衍生于第二型别***瘤病毒L1蛋白(例如HPV L1蛋白)的C端片段，前者提供第一型别***瘤病毒(例如HPV)的免疫原性，后者提供表达量和可溶性较好的特性。两者可以直接连接也可以通过接头连接。
为保持第一型别HPV L1蛋白的免疫原性，以及保证其能够形成VLP，发明人确定了合适的HPV L1蛋白的N端片段的长度。以下报道涉及常见HPV亚型的表位研究：
Sunanda Baidya等人报道,L1蛋白的表位48EEYDLQFIFQLCKITLTA65,45RHGEEYDLQFIFQLCKITLTA65,63LPDPNKF69,79PETQRLVWAC88,36PVPGQYDA43,77YNPETQRLVWAC88,188DTGYGAMD195,36PVPGQYDATK45,45KQDIPKVSAYQYRVFRV61,130RDNVSVDYKQTQLCI144 and 49YSRHVEEY DLQFIF62可以用作设计HPV16和18型疫苗的工具(参见Epitope design of L1 protein for vaccine production against Human Papilloma Virus types 16 and 18，Bioinformation 13(3):86-93March 2017,通过引用全部并入本文)。
Katharina Slupetzky等人报道HPV-16的aa 282–286及351–355 附近的区域对于中和表位有贡献，而且后者是免疫优势位点(参见Chimeric papillomavirus-like particles expressing a foreign epitope on capsid surface loops，Journal of General Virology(2001),82,2799–2804，通过引用全部并入本文)。
Brooke Bishop等人制备了HPV11、16、18和35 L1蛋白的以下3种变体：其N端9个氨基酸缺失、α4(对应于HPV16的404–436位氨基酸残基)缺失、其C端31个氨基酸缺失，报道前两者不能组装成VLP，但是未报道后者有此现象
Figure PCTCN2020102601-appb-000006
(Crystal Structures of Four Types of Human Papillomavirus L1 Capsid Proteins UNDERSTANDING THE SPECIFICITY OF NEUTRALIZING MONOCLONAL ANTIBODIES，The Journal of Biological Chemistry，282,31803-31811。通过引用全部并入本文)。各型别的HPV L1蛋白的α螺旋、β折叠片各个Loop区都可以通过本领域常用的序列分析软件方便地确定。其中α螺旋区包含α1区、 α2区、α3区、α4区和α5区。
发明人对14种型别(6型、11型、16型、18型、31型、33型、35型、39型、45型、51型、52型、56型、58型和59型)的HPV L1蛋白进行序列比对，然后根据如上引用的文献(Crystal Structures of Four Types of Human Papillomavirus L1 Capsid Proteins UNDERSTANDING THE SPECIFICITY OF NEUTRALIZING MONOCLONAL ANTIBODIES，The Journal of Biological Chemistry，282,31803-31811)进行二级结构预测，结果如下所示，其中向下的箭头之间的部分对应于该文献中涉及的为制备变体而缺失的区域。
Figure PCTCN2020102601-appb-000007
Figure PCTCN2020102601-appb-000008
除发明人所用的序列对比的方法之外，可用于预测的蛋白质二 级结构预测软件包括但不限于：
1.JPred: http://www.compbio.dundee.ac.uk/jpred/index.html
2.ProtPredicct: http://predictprotein.org
3.PsiPred: http://bioinf.cs.ucl.ac.uk/psipred
4.SCRATCH-1D: http://download.igb.uci.edu
5.Nnpredict： http://www.cmpharm.ucsf.edu/～nomi/nnpredict
6.Predictprotein： http://www.embl-heidelberg.de/predictprotein/SOPMA  http://www.ibcp.fr/predict.html
7.SSPRED： http://www.embl-heidelberg.de/sspred/ssprd_info.html。
在本发明的一个实施方式中，发明人以以下方式确定衍生于第一型别的HPV L1蛋白的N端片段的长度：将L1蛋白天然序列在其α5区及其附近区域截短，保留从其N末端至α5区域新产生的C末端的序列。如此截短的序列可以保证其具有本型别的免疫原性，且能够形成VLP。
衍生于第一型别的HPV L1蛋白的N端片段还可以进一步改造，以保证其具有本型别的免疫原性，且能够形成VLP为限。
发明人以以下方式确定了衍生于第二型别的HPV L1蛋白的C端片段的长度。将L1蛋白天然序列在其α5区及其附近区域截短、保留从其α5区域新产生的N末端至C末端序列。如此截短的序列不具有主要中和抗原表位，不干扰形成的嵌合蛋白的免疫原性。
衍生于第二型别的HPV L1蛋白的C端片段还可以进一步突变、缺失和/或添加，优选保留其至少一个核定位序列。Yang等人预测了107种HPV亚型的核定位序列(Yang et al.Predicting the nuclear localization signals of 107 types of HPV L1 proteins by bioinformatic analysis.Geno.Prot.Bioinfo.Vol.4 No.1 2006通过引用全部并入本文)，各型别的HPV L1蛋白的核定位序列可以通过本领域常用的序列分析软件方便地确定。
上述N端片段和C端片段的连接发生在前者的新产生的C末端和后者的新产生的N末端。可以是直接连接也可以是通过接头连接。将连接点视为原点，则在原点的N端侧为负，而其C端侧为正。
如下示出HPV6 L1蛋白的453-469位氨基酸序列、以及多个型别HPV L1蛋白的与之相对应的一段序列。可以看出这些序列高度相似。这段序列和α5区有重合。括号内数字表示所列出序列的最后一位氨基酸的位置，其中对于HPV 45型，一些HPV 45型毒株的L1蛋白的N端存在额外的26个氨基酸，而在另一些HPV 45型毒株的L1蛋白的N端不存在所述额外的26个氨基酸，所以以(478)+26表示。
HPV6  ELDQYPLGRKFLLQSGY(469)
HPV11 ELDQFPLGRKFLLQSGY(470)
HPV16 DLDQFPLGRKFLLQAGL(474)
HPV18 DLDQYPLGRKFLVQAGL(475)
HPV31 DLDQFPLGRKFLLQAGY(475)
HPV35 DLDQFPLGRKFLLQAGL(472)
HPV39 ELDQFPLGRKFLLQARV(474)
HPV45 DLDQYPLGRKFLVQAGL(478)+26
HPV51 DLDQFALGRKFLLQVGV(474)
HPV52 DLDQFPLGRKFLLQAGL(478)
HPV56 DLDQFPLGRKFLMQLGTRS(474)
HPV58 DLDQFPLGRKFLLQSGL(473)
HPV33 DLDQFPLGRKFLLQAGL(473) KAKPKLKRAAPTSTRTSSAKRKKVKK其中，480-481的KR和493-496 位的KRKK是核定位序列。
HPV59  DLDQFPLGRKFLLQLGA(475)RPKPTIGPRKRAAPAPTSTPSPKRVKRR KSSRK，其中，484-486的RKR和498-504的KRVKRRK是核定位序 列。
在本发明的一个实施方式中，发明人借助多个HPV型别之间的α5区及其附近区域的序列相似性，便利地完成了不同型别之间的L1蛋白的C端替换。
在本发明的最优选的实施方式中，发明人注意到各个型别的HPV L1蛋白都在相似的位置具有一段四肽RKFL，更有利的情形是一段六肽LGRKFL。发明人巧妙地利用这一高度保守的序列,将嵌合蛋白的连接点设计在这一段寡肽的任一氨基酸位点。自一个方面看来，自嵌合蛋白N末端起至RKFL或LGRKFL止与衍生于第一型别的HPV L1蛋白的N端片段的序列相同，而从另一方面看来自RKFL或LGRKFL起至嵌合蛋白的C末端止，与衍生于第二型别的L1蛋白的C端片段的序列相同。
如此产生的嵌合蛋白保持与天然HPV L1蛋白高度相似性，可以预期在生产乃至此后的医疗或预防过程中，都会有良好的表现。
本领域的技术人员会理解，因为同一型别的HPV有不同的毒株，因此其天然序列不同，利用不同毒株构建而成的嵌合蛋白亦落入本发明。
本领域的技术人员会理解，因为不同型别HPV L1的高度相似性，如果在嵌合蛋白构建过程中，将衍生于第一型别HPV L1蛋白的N端片段向C末端延伸更多的氨基酸残基，或者是将衍生于第二型别的HPV L1蛋白的C端片段向N末端延伸更多的氨基酸残基，亦有可能因相应位点上氨基酸的相同或相似，形成与本发明结构一致的嵌合蛋白。如此形成的嵌合蛋白亦落入本发明。
本领域的技术人员会理解，在以上描述的实施方式的嵌合蛋白的基础上，会通过氨基酸残基的突变、缺失和/或添加形成嵌合蛋白的变体。这些变体有可能具有第一型别的HPV L1蛋白的免疫原性、可以形成VLP，且具有良好的产量和可溶性。如此形成的嵌合蛋白亦落入本发明。
发明的有益效果
目前普遍用于生产病毒样颗粒的表达***分为真核表达***和原核表达***。真核表达***表达的***瘤病毒L蛋白能自发装配成病毒样颗粒，但具有表达量低不易规模化生产的缺点。原核表达***表达的***瘤病毒L蛋白的天然构象往往被破坏，需要后期进行体外处理才能得到病毒样颗粒，而且产量较低，很难进行产业化。
本发明将***瘤病毒(例如人***瘤病毒)L蛋白的C端进行改造，例如替换为HPV 16型L1蛋白、HPV 28型L1蛋白、HPV 33型L1蛋白、HPV 59型L1蛋白或HPV 68型L1蛋白中的C端片段，可以在表达***(例如宿主细胞，例如昆虫细胞)中提高***瘤病毒L蛋白的表达量和可溶性。这可用于疫苗例如HPV疫苗的大规模生产。
发明人自行发现HPV 16型L1蛋白、HPV 28型L1蛋白、HPV 33型L1蛋白、HPV 59型L1蛋白和HPV 68型L1蛋白相较于其他型的L1蛋白表达量和可溶性较好，且发现所述增加的蛋白表达量和可溶性取决于所述HPV L1蛋白的C端序列。在107型HPV L1蛋白中，大部分在C端具有核定位序列，且C端序列具有一定的相似性。
对于目前无法表达、表达量非常低或表达后不可溶的***瘤病毒L蛋白，将其C端片段替换为HPV 16型L1蛋白、HPV 28型L1蛋白、HPV 33型L1蛋白、HPV 59型L1蛋白或HPV 68型L1蛋白中的C端片段，使得原本表达量极低或不可溶的***瘤L蛋白的可溶性表达和后续纯化成为可能。这可以用于更多价疫苗(例如HPV疫苗)的大规模生产，使得更全面地预防多种***瘤病毒，特别是HPV的感染成为可能。
为了实现疫苗的大规模生产，还存在提高HPV L1蛋白在昆虫细胞中的表达量和可溶性的需求。此外，在酵母细胞中，因为无法正确形成二硫键，HPV L1蛋白装配成的病毒样颗粒缺乏良好的构象。
对于在昆虫细胞中表达量低且可溶性差的HPV L1蛋白，将其C端片段改造为HPV 33型或59型L1蛋白的C端片段后，表达量和可溶性显著提高，可用于HPV疫苗的大规模生产。
对于相较于其他型的L1蛋白在昆虫细胞中表达量和可溶性较好的HPV L1蛋白，例如HPV 16型、HPV 28型L1蛋白、HPV 68型L1蛋白等，为了实现疫苗的大规模生产，还存在进一步提高表达量和可溶性的需求。在本发明中，例如，将HPV 16型L1蛋白的C端片段改造为HPV 33型L1蛋白的C端片段后，改造后的嵌合的HPV16型L1蛋白表达量和可溶性均得到改善，有利于HPV疫苗的大规模生产。
总之，嵌合HPV L1蛋白相比于未改造之前的HPV L1蛋白在昆虫细胞中的表达量和可溶性大大提高。可用于HPV疫苗的大规模生产。此外，嵌合HPV L1蛋白在昆虫细胞中可以正确形成二硫键而装配为具有良好构象的HPV病毒样颗粒。这可以提高HPV病毒样颗粒的免疫原性，产生更好的免疫应答。
本发明的多价疫苗可以用于预防多种HPV相关疾病或感染，包括目前尚无法预防的HPV型别。
定义
除非另有说明，本文使用的所有技术和科学术语具有本发明所属的技术领域的普通技术人员通常理解的含义。为方便地理解本发明，以下引述下列术语的通常含义。
当用于本文和所附权利要求书中时，单数形式“一个/种”、“另一个/种”和“所述/该”包括复数指代对象，除非上下文明确地另有指示。除非另有明确说明，否则术语“包括/包含/具有”、“例如”等旨在传达包含而非限制。
术语“免疫原性”是指某种物质，例如蛋白质或多肽刺激免疫应答的能力，即刺激产生抗体，尤其是产生体液或者刺激细胞介导的应答的能力。
术语“抗体”指能结合抗原的免疫球蛋白分子。抗体可以是多克隆混合物或单克隆。抗体可以是源于天然来源或源于重组来源的完整的免疫球蛋白或可以是完整的免疫球蛋白的免疫反应性部分。 抗体可以存在于多种形式，包括例如Fv、Fab’、F(ab’)2以及以单链存在。
术语“抗原性”是指某种物质，例如蛋白质或多肽产生与其特异性结合的抗体的能力。
术语“表位”包括能够特异性结合至抗体或T细胞受体的任何蛋白质决定簇。表位决定簇通常由分子的化学活性表面基团(例如氨基酸或糖侧链，或其组合)组成，并且通常具有特定三维结构特征以及特定的电荷特征。
术语“亚型”或“型别”可在本文中互换使用，表示所述病毒抗原的遗传变体以使得一个亚型区别于一个不同亚型地被免疫***识别。例如，HPV 16在免疫学上可区别于HPV 33。
术语“HPV L1蛋白”如本文所用，术语“HPV”和“人***状瘤病毒”是指***状瘤病毒科的无包膜双链DNA病毒。它们的基因组是圆形的，并且大小约为8千碱基对。大多数HPV编码八种主要蛋白，六种位于“早期”区域(E1-E2)，并且两种位于“晚期”区域(L1(主要衣壳蛋白)和L2(次要衣壳蛋白))。已经鉴定了超过120种HPV类型，并且它们由数字标出(例如，HPV-16、HPV-18等)。
术语“HPV”或“HPV病毒”指***状瘤病毒科的***状瘤病毒，为无包膜DNA病毒，该病毒基因组为双链闭环DNA，大小约为8kb,通常可以分为三个区域：①早期区(E)，含有编码E1、E2、E4～E7病毒复制，转录及转化有关的非结构蛋白的6个开放阅读框，以及E3和E8开放阅读框；②晚期区(L)含有编码主要衣壳蛋白L1和次要衣壳蛋白L2的阅读框；③长调控区(LCR)不编码任何蛋白，但具有复制的起源以及多个转录因子结合位点。
术语“HPV L1蛋白”及“HPV L2蛋白”指由HPV基因的晚期区(L)编码，在HPV感染周期中晚期合成的蛋白。L1蛋白质是主要的衣壳蛋白并且具有55-60kDa的分子量。L2蛋白质是次要的衣壳蛋白质。72个L1五聚体构成二十面体HPV病毒粒子的外壳,包裹闭环双链DNA微染色体。L2蛋白质位于L1蛋白质内侧。
术语“病毒样颗粒”是含有某种病毒的一个或多个结构蛋白的空心颗粒，没有病毒核酸。
“HPV假病毒”系利用HPV VLP的非特异包裹核酸的特性，通过细胞内表达的HPV L1和L2组成的VLP包裹游离的DNA或导入外源质粒形成。是理想的HPV体外中和实验模型。
“假病毒中和法”是评价抗体的中和活性的一种方法，将免疫后的动物血清与一定量的假病毒孵育后再侵染细胞，细胞会随着血清中中和抗体的增加而减少，在一定的范围内可存在线性负相关，因此可以通过检测表达细胞数的变化来评价血清中抗体的中和活性。
术语“其片段”或“其变体”指根据本发明的部分核苷酸或氨基酸序列被缺失、***和/或取代。优选地，本发明提供的多肽的片段或变体能在动物或人体中引发体液和/或细胞免疫应答。
术语“嵌合”意指，源自不同的亲本分子的多肽或核苷酸序列分别经由酰胺键或3’，5’－磷酸二酯键连接在一起。优选的，不被额外的接头序列分隔开，而是直接彼此相邻。
术语“截短”意指通过从多肽的N和/或C-末端除去一个或多个氨基酸或者缺失一个或多个多肽内部的氨基酸。
术语“核定位序列”为可引导蛋白质进入细胞核的氨基酸序列。在一些HPV L1蛋白中，两个紧密的碱性残基簇(即核定位序列)(例如一个是KRKR、KRKK、KRKRK、KRKKRK、KRVKRRK等，另一个是KR、RKR、KRK等)之间具有10-14个氨基酸的间隔区。上述碱性残基簇属于核定位序列。在另一些HPV L1蛋白中，核定位序列为精氨酸和/或赖氨酸形成的紧密的碱性残基簇。核定位序列包括但不限于如上所述碱性残基簇的实例。参见Jun Yang等，Predicting the Nuclear Localization Signals of 107 Types of HPV L1 Proteins by Bioinformatic Analysis，Genomics,Proteomics&Bioinformatics Volume 4,Issue 1,2006,Pages 34-41，其全部内容通过引用并入本文。
术语“功能性变体”为某一多肽或蛋白经截短、突变、缺失和/ 或添加后仍然保持所需要的活性或特征的版本。
两条多肽或核酸序列之间的“序列同一性”表示所述序列之间相同的残基的数目占残基总数的百分比，且基于比较的分子中较小者的大小来计算。在计算同一性百分数时，将正在比较的序列以产生序列之间最大匹配的方式比对，通过特定算法解决比对中的空位(如果存在的话)。确定两个序列之间同一性的优选计算机程序方法包括，但不限于，GCG程序包，包括GAP、BLASTP、BLASTN和FASTA(Altschul等人，1990，J.Mol.Biol.215：403-410)。上述程序可以公开地从国际生物技术信息中心(NCBI)和其他来源得到。熟知的Smith Waterman算法也可用于确定同一性。
可以保守性置换非关键的氨基酸而不影响蛋白质的正常功能。保守性置换意指用化学或功能相似的氨基酸置换氨基酸。提供相似氨基酸的保守性置换表是本领域熟知的。举例来说，在一些实施方式中，表1-3中提供的氨基酸组被认为是相互的保守性置换。
表1在某些实施方式中，被认为是相互保守性置换的氨基酸的所选组
酸性残基 D和E
碱性残基 K、R和H
亲水性不带电荷的残基 S、T、N和Q
脂肪族不带电荷的残基 G、A、V、L和I
非极性不带电荷的残基 C、M和P
芳香族残基 F、Y和W
表2在某些实施方式中，被认为是相互的保守性置换的氨基酸的其他所选组
组1 A、S和T
组2 D和E
组3 N和Q
组4 R和K
组5 I、L和M
组6 F、Y和W
表3在某些实施方式中，被认为是相互的保守性置换的氨基酸的其他所选组
组A A和G
组B D和E
组C N和Q
组D R、K和H
组E I、L、M、V
组F F、Y和W
组G S和T
组H C和M
术语“氨基酸”意指二十种常见的天然存在的氨基酸。天然存在的氨基酸包括丙氨酸(Ala；A)、精氨酸(Arg；R)、天冬酰胺(Asn；N)、天冬氨酸(Asp；D)、半胱氨酸(Cys；C)；谷氨酸(Glu；E)、谷氨酰胺(Gln；Q)、甘氨酸(Gly；G)、组氨酸(His；H)、异亮氨酸(Ile；I)、亮氨酸(Leu；L)、赖氨酸(Lys；K)、蛋氨酸(Met；M)、苯丙氨酸(Phe；F)、脯氨酸(Pro；P)、丝氨酸(Ser；S)、苏氨酸(Thr；T)、色氨酸(Trp；W)、酪氨酸(Tyr；Y)和缬氨酸(Val；V)。
如本文所用的“生理学上可接受的载体”，其对所用剂量和浓度的细胞或哺乳动物是无毒的。通常为pH缓冲含水溶液，其非限制性实例包括缓冲剂；抗氧化剂；寡肽；蛋白质；亲水性聚合物；氨基酸；单糖、二糖和其它碳水化合物；螯合剂；糖醇；成盐的抗衡离子，例如钠；以及/或非离子表面活性剂。
术语“佐剂”指一种增强免疫应答的化合物或混合物。特别的，疫苗可以包含佐剂。用于本发明的佐剂可以包括但不限于以下的一种或多种：含矿物佐剂组合物、油-乳佐剂、皂素佐剂制剂、细菌或微生物衍生物。
术语“载体”意指能够增殖与其连接的另一核酸的核酸分子。 该术语包括作为自我复制核酸结构的载体以及作为整合至已引入载体的宿主细胞基因组中的载体。某些载体能够引导该类载体以可操作方式连接的核酸的表达。
术语“宿主细胞”意指已引入外源核酸的细胞，以及这样的细胞的后代。宿主细胞包括“转化体”(或“转化细胞”)、“转染体”(或“转染细胞”)或“感染体”(或“感染细胞”)，其各自包括初代转化、转染或感染的细胞和由其衍生的后代。这样的后代在核酸含量上可能不与亲本细胞完全相同，并且可能含有突变。
施用量优选的为“预防性有效量”(本文预防可以被认为是治疗，两者可互换使用)，其足以对个体显示出益处。
实施例
实施例1基因的构建
实施例1.1：HPV6L1 C端替换为HPV33L1 C端的嵌合基因的构建
1.1.1用作模板的pFB-HPV6L1的构建
委托Thermo Fisher公司[原英潍捷基(上海)贸易有限公司]基因合成HPV6L1基因，且合成的序列两端分别具有KpnⅠ和XbaI酶切位点，其序列见SEQ ID NO:5。通过KpnⅠ和XbaI酶切位点将合成的基因片段与pcDNA3载体(销售商Thermo Fisher)连接，得到含有编码HPV6L1 1-500个氨基酸的核苷酸序列的质粒pcDNA3-HPV6-L1。
将得到的pcDNA3-HPV6-L1质粒进行KpnⅠ和XbaI双酶切得到HPV6L1(1-500)的基因的片段。再将该片段与KpnⅠ和XbaI双酶切的pFastBac TM1载体(销售商Thermo Fisher)进行连接，得到含HPV6L1(1-500)基因片段的杆粒载体，命名为pFB-HPV6L1。
1.1.2用作模板的pFB-HPV33L1的构建
委托Thermo Fisher公司[原英潍捷基(上海)贸易有限公司]基因合成HPV33L1基因，且合成序列两端分别具有KpnI和XbaI酶切 位点，基因片段序列见SEQ ID NO:6。通过KpnI和XbaI酶切位点将合成的基因片段与pcDNA3载体(销售商Thermo Fisher)连接，得到含有编码HPV33L1 1-499位氨基酸的核苷酸序列的质粒pcDNA3-HPV33-L1。
将得到的pcDNA3-HPV33-L1质粒进行KpnI和XbaI双酶切得到HPV33L1(1-499)的基因的片段。再将该片段与KpnI和XbaI双酶切的pFastBacTM1载体(销售商Thermo Fisher)进行连接，得到含HPV33L1(1-499)基因片段的杆粒载体，命名为pFB-HPV33L1。
1.1.3 pFB-HPV6L1：33C的构建
HPV6L1 C端替换为HPV33L1 C端的嵌合基因：以构建成功的重组质粒pFB-HPV6L1为基因模板，用引物F1和R1扩增长度为1426bp基因片段,引物序列F1如SEQ ID No:7所示，R1如SEQ ID No:8所示。
该基因片段包含编码HPV6L1的1-469氨基酸的基因片段、与HPV33L1的474-499氨基酸的基因片段重叠的10个碱基以及KpnI酶切位点(GGTAC^C)段，扩增的序列如SEQ ID No:9所示：
PCR扩增参数：94℃预变性5min；98℃变性10s、69℃退火15s、72℃1kb/1min、进行30个循环；72℃延伸5min；16℃结束。
以重组质粒pFB-HPV33L1为基因模板，用引物F2和R2，扩增长度101bp的基因片段，引物序列F2如SEQ ID No:10所示，R2如SEQ ID No:11所示。
该基因片段含HPV33L1 C端的26个(474-499)氨基酸的基因片段、与HPV6L1的1-469氨基酸C端基因片段重叠的10bp碱基以及XbaI(T^CTAGA)酶切位点，扩增的序列如SEQ ID No:12所示。
PCR扩增参数：94℃预变性5min；98℃变性10s、69℃退火15s、72℃1kb/1min、进行30个循环；72℃延伸5min；16℃结束。
PCR拼接序列：
拼接引物分别为F1和R2，以上述引物扩增得到的片段(F1和R1扩增得到的片段，F2和R2扩增得到的片段)为模板。
PCR拼接参数：94℃预变性5min；98℃变性10s、52℃退火15s、72℃1kb/1min、进行5个循环；98℃变性10s、68℃退火15s、72℃1kb/1min、进行25个循环；72℃延伸5min；16℃结束。
最终得到SEQ ID NO:4，编码由HPV6L1的1-469氨基酸和HPV33L1 C端的26个(474-499)氨基酸组成的核苷酸序列，两端带有KpnI和XbaI酶切位点(下称拼接序列)。
用KpnI+XbaI双酶切pFastBac TM1载体和拼接序列片段，将拼接序列克隆到pFastBac TM1载体上，获得重组质粒pFB-HPV6L1：33C。即为HPV6L1 C端替换为HPV33L1 C端的嵌合基因。
实施例1.2 HPV11L1 C端替换为HPV33L1 C端的嵌合基因的构建
实验方法和步骤与实施例1.1相同，相关序列参见附录2。
实施例1.3 HPV16L1 C端替换为HPV33L1 C端的嵌合基因的构建
实验方法和步骤与实施例1.1相同，相关序列参见附录3。
实施例1.4 HPV18L1 C端替换为HPV33L1 C端的嵌合基因的构建
实验方法和步骤与实施例1.1相同，相关序列参见附录4。
实施例1.5 HPV31L1 C端替换为HPV33L1 C端的嵌合基因的构建
实验方法和步骤与实施例1.1相同，相关序列参见附录5。
实施例1.6 HPV33L1基因的构建
1.6.1 pFB-HPV33L1基因的制备
HPV33L1基因通过基因合成的方法构建而成，委托Thermo Fisher公司[原英潍捷基(上海)贸易有限公司]基因合成，合成序列 两端分别具有KpnⅠ和XbaI酶切位点，基因片段序列见SEQ ID No:68。通过KpnⅠ和XbaI酶切位点将合成的基因片段与pcDNA3载体(销售商Thermofisher)连接，得到含有编码HPV33L1 1-499个氨基酸的核苷酸序列的质粒pcDNA3-HPV33-L1。
将得到的pcDNA3-HPV33-L1质粒进行KpnⅠ和XbaI双酶切得到HPV33L1(1-499)的基因的片段。再将该片段与KpnⅠ和XbaI双酶切的pFastBac TM1载体(销售商Thermofisher)进行连接，得到含HPV33L1(1-499)基因片段的杆粒载体，命名为pFB-HPV33L1。
实施例1.7 HPV35L1 C端替换为HPV33L1 C端的嵌合基因的构建
实验方法和步骤与实施例1.1相同，相关序列参见附录7。
实施例1.8 HPV39L1 C端替换为HPV59L1 C端的嵌合基因的构建
1.8.1用作模板的pFB-HPV39L1的构建
委托Thermo Fisher公司[原英潍捷基(上海)贸易有限公司]基因合成HPV39L1基因，且合成的序列两端分别具有KpnⅠ和XbaI酶切位点，其序列见SEQ ID NO:86。通过KpnⅠ和XbaI酶切位点将合成的基因片段与pcDNA3载体(销售商Thermo Fisher)连接，得到含有编码HPV39L1 1-505个氨基酸的核苷酸序列的质粒pcDNA3-HPV39-L1。
将得到的pcDNA3-HPV39-L1质粒进行KpnⅠ和XbaI双酶切得到HPV39L1(1-505)的基因的片段。再将该片段与KpnⅠ和XbaI双酶切的pFastBac TM1载体(销售商Thermo Fisher)进行连接，得到含HPV39L1(1-505)基因片段的杆粒载体，命名为pFB-HPV39L1。
1.8.2用作模板的pFB-HPV59L1的构建
委托Thermo Fisher公司[原英潍捷基(上海)贸易有限公司]基因合成HPV59L1基因，且合成序列两端分别具有KpnI和XbaI酶切 位点，基因片段序列见SEQ ID NO:87。通过KpnI和XbaI酶切位点将合成的基因片段与pcDNA3载体(销售商Thermo Fisher)连接，得到含有编码HPV59L1 1-508位氨基酸的核苷酸序列的质粒pcDNA3-HPV59-L1。
将得到的pcDNA3-HPV59-L1质粒进行KpnI和XbaI双酶切得到HPV59L1(1-508)的基因的片段。再将该片段与KpnI和XbaI双酶切的pFastBacTM1载体(销售商Thermo Fisher)进行连接，得到含HPV59L1(1-508)基因片段的杆粒载体，命名为pFB-HPV59L1。
1.8.3 pFB-HPV39L1：59C的构建
HPV39L1 C端替换为HPV59L1 C端的嵌合基因：以构建成功的重组质粒pFB-HPV39L1为基因模板，用引物F1和R1扩增长度为1428bp基因片段,引物序列F1如SEQ ID No:88所示，R1如SEQ ID No:89所示。
该基因片段包含编码HPV39L1的1-469氨基酸的基因片段、与HPV59L1的471-508氨基酸的基因片段重叠的12个碱基以及KpnI酶切位点(GGTAC^C)段，扩增的序列如SEQ ID No:90所示：
PCR扩增参数：94℃预变性5min；98℃变性10s、69℃退火15s、72℃1kb/1min、进行30个循环；72℃延伸5min；16℃结束。
以重组质粒pFB-HPV59L1为基因模板，用引物F2和R2，扩增长度139bp的基因片段，引物序列F2如SEQ ID No:91所示，R2如SEQ ID No:92所示。
该基因片段含HPV59L1 C端的38个(471-508)氨基酸的基因片段、与HPV39L1的1-469氨基酸C端基因片段重叠的12bp碱基以及XbaI(T^CTAGA)酶切位点，扩增的序列如SEQ ID No:93所示。
PCR扩增参数：94℃预变性5min；98℃变性10s、69℃退火15s、72℃1kb/1min、进行30个循环；72℃延伸5min；16℃结束。
PCR拼接序列：
拼接引物分别为F1和R2，以上述引物扩增得到的片段(F1和R1扩增得到的片段，F2和R2扩增得到的片段)为模板。
PCR拼接参数：94℃预变性5min；98℃变性10s、52℃退火15s、72℃1kb/1min、进行5个循环；98℃变性10s、68℃退火15s、72℃1kb/1min、进行25个循环；72℃延伸5min；16℃结束。
最终得到SEQ ID NO:85，编码由HPV39L1的1-469氨基酸和HPV59L1 C端的38个(471-508)氨基酸组成的核苷酸序列，两端带有KpnI和XbaI酶切位点(下称拼接序列)。
用KpnI+XbaI双酶切pFastBac TM1载体和拼接序列片段，将拼接序列克隆到pFastBac TM1载体上，获得重组质粒pFB-HPV39L1：59C。即为HPV39L1 C端替换为HPV59L1 C端的嵌合基因。
实施例1.9 HPV45L1 C端替换为HPV33L1 C端的嵌合基因的构建
实验方法和步骤与实施例1.1相同，相关序列参见附录9。
实施例1.10 HPV51L1 C端替换为HPV33L1 C端的嵌合基因的构建
实验方法和步骤与实施例1.1相同，相关序列参见附录10。
实施例1.11 HPV52L1 C端替换为HPV33L1 C端的嵌合基因的构建
实验方法和步骤与实施例1.1相同，相关序列参见附录11。
实施例1.12 HPV56L1 C端替换为HPV33L1 C端的嵌合基因的构建
实验方法和步骤与实施例1.1相同，相关序列参见附录12。
实施例1.13 HPV58L1 C端替换为HPV33L1 C端的嵌合基因的构建
实验方法和步骤与实施例1.1相同，相关序列参见附录13。
实施例1.14 HPV59L1基因的构建
实施例1.14.1 pFB-HPV59L1基因的制备
HPV59L1基因通过基因合成的方法构建而成，委托Thermo Fisher公司[原英潍捷基(上海)贸易有限公司]基因合成，合成序列两端分别具有KpnⅠ和XbaI酶切位点，基因片段序列见SEQ ID No:162。通过KpnⅠ和XbaI酶切位点将合成的基因片段与pcDNA3载体(销售商Thermofisher)连接，得到含有编码HPV59L1 1-508个氨基酸的核苷酸序列的质粒pcDNA3-HPV59-L1。
将得到的pcDNA3-HPV59-L1质粒进行KpnⅠ和XbaI双酶切得到HPV59L1(1-508)的基因的片段。再将该片段与KpnⅠ和XbaI双酶切的pFastBac TM1载体(销售商Thermofisher)进行连接，得到含HPV59L1(1-508)基因片段的杆粒载体，命名为pFB-HPV59L1。
实施例2重组杆状病毒的包装
实施例2.1：HPV 6L1：33C重组杆状病毒包装
实施例1构建的pFB-HPV 6L1：33C的重组质粒经鉴定和测序正确后，将其转化至DH10Bac细菌感受态细胞(
Figure PCTCN2020102601-appb-000009
试剂盒，购于Thermo Fisher)中，37℃培养扩增，并进行平皿划线培养，挑选白色菌斑并扩增，培养过夜后收集菌液，使用碱裂解法提取重组杆粒DNA。
用阳离子转染试剂(购于Sino Biological)将其转染至昆虫细胞SF9中进行重组杆状病毒毒种包装。具体操作如下：
a.取对数生长期的SF9细胞按照0.6×10 6cell/dish的密度接种dish，将接种有SF9细胞的dish室温放置2h，贴壁。
b.提取的质粒20μL Bacmid DNA加至200μL Grace’s Medium(无血清，无添加物，购于Gibico)混和颠倒5次。
c.25μL 0.2x TF1(转染试剂，购于Sino Biological)滴加至200μL Grace’s Meduim轻轻混匀。
d.将b和c混合。室温孵育15-45min。
e.当DNA与cellfectin(购于Sino Biological)孵育时，弃细胞上清，添加无血清添加物的Grace Medium 0.8mL/dish。
f.将d中孵育好的DNA与转染试剂复合物滴加到dish中。
g.27℃孵育2hr。
h.弃细胞培养液，加2.5mL/dish完全生长培养基(SCD6 SF+10％FBS)(SCD6 SF购于Sino Biological，FBS购于Gibico)。
i.27℃培养7天观察是否有病毒感染。
转染后待细胞产生明显的病变后收集病毒上清，一般培养7-11天。用移液器无菌收取病毒上清液，即为HPV6L1：33C P1代毒种。使用HPV6L1：33C P1代毒种按照1:50(V/V)比例感染SF9细胞，SF9细胞的感染密度为2×10 6cells/mL，27℃培养扩增3天，1000g±200g室温离心10min，收集的病毒上清液即为P2代病毒，可用于感染生产。
实施例2.2：HPV 11L1：33C重组杆状病毒包装
实验方法和步骤与实施例2.1相同。
实施例2.3：HPV 16L1：33C重组杆状病毒包装
实验方法和步骤与实施例2.1相同。
实施例2.4：HPV 18L1：33C重组杆状病毒包装
实验方法和步骤与实施例2.1相同。
实施例2.5：HPV 31L1：33C重组杆状病毒包装
实验方法和步骤与实施例2.1相同。
实施例2.6：HPV 33L1重组杆状病毒包装
实验方法和步骤与实施例2.1相同。
实施例2.7：HPV 35L1：33C重组杆状病毒包装
实验方法和步骤与实施例2.1相同。
实施例2.8：HPV 39L1：59C重组杆状病毒包装
实验方法和步骤与实施例2.1相同。
实施例2.9：HPV 45L1：33C重组杆状病毒包装
实验方法和步骤与实施例2.1相同。
实施例2.10：HPV 51L1：33C重组杆状病毒包装
实验方法和步骤与实施例2.1相同。
实施例2.11：HPV 52L1：33C重组杆状病毒包装
实验方法和步骤与实施例2.1相同。
实施例2.12：HPV 56L1：33C重组杆状病毒包装
实验方法和步骤与实施例2.1相同。
实施例2.13：HPV 58L1：33C重组杆状病毒包装
实验方法和步骤与实施例2.1相同。
实施例2.14：HPV 59L1重组杆状病毒包装
实验方法和步骤与实施例2.1相同。
实施例3嵌合蛋白或蛋白的表达
实施例3.1：HPV 6L1：33C表达生产
用实施例2中获得的含有HPV 6L1：33C重组基因的杆状病毒感染High Five细胞，感染比例1:200(V/V),1000g±100g室温离心收集细胞沉淀，使用PBS或MOPS缓冲液(pH6.0-7.0，盐浓度100mM-1M)超声裂解细胞沉淀，低温超声破碎3min，大于10000g的离心力离心10分钟，收集离心后上清液，SDS-PAGE电泳检测。泳道1:Marker(Marker为7种纯化后的蛋白，分子量大小包含14.4至116kDa，生产商为Thermo Scientific)；泳道2：细胞裂解液；泳道3：裂解液离心后收集的上清液。
结果如图1A所示，该方法制备的HPV 6L1：33C L1蛋白产量大于100mg/L，蛋白大小约56KD，可以用于大规模生产。
实施例3.2：HPV 11L1：33C表达生产
实验方法和步骤与实施例3.1相同。
结果如图1B所示，该方法制备的HPV 11L1：33C L1蛋白产量大于100mg/L，蛋白大小约56KD，可以用于大规模生产。
实施例3.3：HPV 16L1：33C表达生产
实验方法和步骤与实施例3.1相同。
结果如图1C所示，该方法制备的HPV 16L1：33C L1蛋白产量大于100mg/L，蛋白大小约56KD，可以用于大规模生产。
实施例3.4：HPV 18L1：33C表达生产
实验方法和步骤与实施例3.1相同。
结果如图1D所示，该方法制备的HPV 18L1：33C L1蛋白产量大于100mg/L，蛋白大小约56KD，可以用于大规模生产。
实施例3.5：HPV 31L1：33C表达生产
实验方法和步骤与实施例3.1相同。
结果如图1E所示，该方法制备的HPV 31L1：33C L1蛋白产量 大于100mg/L，蛋白大小约56KD，可以用于大规模生产。
实施例3.6：HPV 33L1表达生产
实验方法和步骤与实施例3.1相同。
结果如图1F所示，该方法制备的HPV 33L1 L1蛋白产量大于100mg/L，蛋白大小约56KD，可以用于大规模生产。
实施例3.7：HPV 35L1：33C表达生产
实验方法和步骤与实施例3.1相同。
结果如图1G所示，该方法制备的HPV 35L1：33C L1蛋白产量大于100mg/L，蛋白大小约56KD，可以用于大规模生产。
实施例3.8：HPV 39L1：59C表达生产
实验方法和步骤与实施例3.1相同。
结果如图1H所示，该方法制备的HPV 39L1：59C L1蛋白产量大于100mg/L，蛋白大小约56KD，可以用于大规模生产。
实施例3.9：HPV 45L1：33C表达生产
实验方法和步骤与实施例3.1相同。
结果如图1I所示，该方法制备的HPV 45L1：33C L1蛋白产量大于100mg/L，蛋白大小约56KD，可以用于大规模生产。
实施例3.10：HPV 51L1：33C表达生产
实验方法和步骤与实施例3.1相同。
结果如图1J所示，该方法制备的HPV 51L1：33C L1蛋白产量大于100mg/L，蛋白大小约56KD，可以用于大规模生产。
实施例3.11：HPV 52L1：33C表达生产
实验方法和步骤与实施例3.1相同。
结果如图1K所示，该方法制备的HPV 52L1：33C L1蛋白产量大于100mg/L，蛋白大小约56KD，可以用于大规模生产。
实施例3.12：HPV 56L1：33C表达生产
实验方法和步骤与实施例3.1相同。
结果如图1L所示，该方法制备的HPV 56L1：33C L1蛋白产量大于100mg/L，蛋白大小约56KD，可以用于大规模生产。
实施例3.13：HPV 58L1：33C表达生产
实验方法和步骤与实施例3.1相同。
结果如图1M所示，该方法制备的HPV 58L1：33C L1蛋白产量大于100mg/L，蛋白大小约56KD，可以用于大规模生产。
实施例3.14：HPV 59L1表达生产
实验方法和步骤与实施例3.1相同。
结果如图1N所示，该方法制备的HPV 59L1 L1蛋白产量大于100mg/L，蛋白大小约56KD，可以用于大规模生产。
实施例4病毒样颗粒的纯化制备
实施例4.1：HPV 6L1：33C病毒样颗粒的纯化制备
HPV 6L1：33C病毒样颗粒纯化方法为两步层析法，即HS-MMA法，纯化实施例3中收集的上清液，最终可得到高纯度的病毒样颗粒。
第一步层析：
介质：采用Thermo Fisher公司生产的
Figure PCTCN2020102601-appb-000010
50 HS强阳离子交换介质。
介质体积：介质体积150mL，线性流速30mL/min。
层析条件：平衡缓冲液(pH6.2，盐浓度为50mM磷酸盐，0.5M氯化钠)；清洗缓冲液(盐浓度为50mM磷酸盐，0.75M氯化钠， pH6.2；)
层析柱先用5CV平衡缓冲液，然后上样。上样结束后，之后分别用5CV的平衡缓冲液和清洗缓冲液洗脱杂蛋白。
洗脱条件：pH6.2，洗脱盐浓度为1.25M氯化钠采用含有50mM盐酸精氨酸的50mM磷酸盐缓冲液进行洗脱。
第二步层析：
介质：采用上海博格隆公司生产的MMA离子交换介质。
介质体积：介质体积150mL，线性流速30mL/min。
层析条件：平衡缓冲液50mM PB,1.25M NaCl,pH6.2。层析柱先用4CV平衡缓冲液平衡，然后上样。上样结束后，用5CV平衡缓冲液冲洗杂蛋白后，然后用洗脱缓冲液洗脱目标蛋白收集蛋白。
洗脱条件：100mM NaAC,150mM NaCl,0.01％Tween 80,pH4.5。
实施例4.2：HPV 11L1：33C病毒样颗粒的纯化制备
实验方法和步骤与实施例4.1相同。
实施例4.3：HPV 16L1：33C病毒样颗粒的纯化制备
实验方法和步骤与实施例4.1相同。
实施例4.4：HPV 18L1：33C病毒样颗粒的纯化制备
实验方法和步骤与实施例4.1相同。
实施例4.5：HPV 31L1：33C病毒样颗粒的纯化制备
实验方法和步骤与实施例4.1相同。
实施例4.6：HPV 33L1病毒样颗粒的纯化制备
实验方法和步骤与实施例4.1相同。
实施例4.7：HPV 35L1：33C病毒样颗粒的纯化制备
实验方法和步骤与实施例4.1相同。
实施例4.8：HPV 39L1：59C病毒样颗粒的纯化制备
实验方法和步骤与实施例4.1相同。
实施例4.9：HPV 45L1：33C病毒样颗粒的纯化制备
实验方法和步骤与实施例4.1相同。
实施例4.10：HPV 51L1：33C病毒样颗粒的纯化制备
实验方法和步骤与实施例4.1相同。
实施例4.11：HPV 52L1：33C病毒样颗粒的纯化制备
实验方法和步骤与实施例4.1相同。
实施例4.12：HPV 56L1：33C病毒样颗粒的纯化制备
实验方法和步骤与实施例4.1相同。
实施例4.13：HPV 58L1：33C病毒样颗粒的纯化制备
实验方法和步骤与实施例4.1相同。
实施例4.14：HPV 59L1病毒样颗粒的纯化制备
实验方法和步骤与实施例4.1相同。
实施例5病毒样颗粒的形态学检测
实施例5.1：HPV 6L1：33C病毒样颗粒的形态学检测
取10μL样品用于透射电镜观察。将样品固定到碳喷铜网上吸附2min，残余液体用滤纸吸掉，再使用磷钨酸(北京中镜科仪技术有限公司，浓度2％，pH6.5)染色两次，每次30秒，残余染色液用滤纸吸掉，晾干后即可在透射电子显微镜下观察。透射电子显微镜(品 牌：日立，型号：H-7650)为80KV，放大倍数为80，000倍。
电镜观察结果见图2A，由图2A可见，C端改造的HPV 6L1:33C可以形成大小均一的病毒样颗粒，平均直径在60nm左右。
实施例5.2：HPV 11L1：33C病毒样颗粒的形态学检测
实验方法和步骤与实施例5.1相同。
电镜观察结果见图2B，由图2B可见，C端改造的HPV 11L1:33C可以形成大小均一的病毒样颗粒，平均直径在60nm左右。
实施例5.3：HPV 16L1：33C病毒样颗粒的形态学检测
实验方法和步骤与实施例5.1相同。
电镜观察结果见图2C，由图2C可见，C端改造的HPV 16L1:33C可以形成大小均一的病毒样颗粒，平均直径在60nm左右。
实施例5.4：HPV 18L1：33C病毒样颗粒的形态学检测
实验方法和步骤与实施例5.1相同。
电镜观察结果见图2D，由图2D可见，C端改造的HPV 18L1:33C可以形成大小均一的病毒样颗粒，平均直径在60nm左右。
实施例5.5：HPV 31L1：33C病毒样颗粒的形态学检测
实验方法和步骤与实施例5.1相同。
电镜观察结果见图2E，由图2E可见，C端改造的HPV 31L1:33C可以形成大小均一的病毒样颗粒，平均直径在60nm左右。
实施例5.6：HPV 33L1病毒样颗粒的形态学检测
实验方法和步骤与实施例5.1相同。
电镜观察结果见图2F，由图2F可见，C端改造的HPV 33L1可以形成大小均一的病毒样颗粒，平均直径在60nm左右。
实施例5.7：HPV 35L1：33C病毒样颗粒的形态学检测
实验方法和步骤与实施例5.1相同。
电镜观察结果见图2G，由图2G可见，C端改造的HPV 35L1:33C可以形成大小均一的病毒样颗粒，平均直径在60nm左右。
实施例5.8：HPV 39L1：59C病毒样颗粒的形态学检测
实验方法和步骤与实施例5.1相同。
电镜观察结果见图2H，由图2H可见，C端改造的HPV 39L1:59C可以形成大小均一的病毒样颗粒，平均直径在60nm左右。
实施例5.9：HPV 45L1：33C病毒样颗粒的形态学检测
实验方法和步骤与实施例5.1相同。
电镜观察结果见图2I，由图2I可见，C端改造的HPV 45L1:33C可以形成大小均一的病毒样颗粒，平均直径在60nm左右。
实施例5.10：HPV 51L1：33C病毒样颗粒的形态学检测
实验方法和步骤与实施例5.1相同。
电镜观察结果见图2J，由图2J可见，C端改造的HPV 51L1:33C可以形成大小均一的病毒样颗粒，平均直径在60nm左右。
实施例5.11：HPV 52L1：33C病毒样颗粒的形态学检测
实验方法和步骤与实施例5.1相同。
电镜观察结果见图2K，由图2K可见，C端改造的HPV 52L1:33C可以形成大小均一的病毒样颗粒，平均直径在60nm左右。
实施例5.12：HPV 56L1：33C病毒样颗粒的形态学检测
实验方法和步骤与实施例5.1相同。
电镜观察结果见图2L，由图2L可见，C端改造的HPV 56L1:33C可以形成大小均一的病毒样颗粒，平均直径在60nm左右。
实施例5.13：HPV 58L1：33C病毒样颗粒的形态学检测
实验方法和步骤与实施例5.1相同。
电镜观察结果见图2M，由图2M可见，C端改造的HPV 58L1:33C可以形成大小均一的病毒样颗粒，平均直径在60nm左右。
实施例5.14：HPV 59L1病毒样颗粒的形态学检测
实验方法和步骤与实施例5.1相同。
电镜观察结果见图2N，由图2N可见，C端改造的HPV 59L1:33C可以形成大小均一的病毒样颗粒，平均直径在60nm左右。
实施例6病毒样颗粒动物免疫原性评价
实施例6.1：HPV 6L1：33C病毒样颗粒动物免疫原性评价
6.1.1假病毒中和细胞的模型建立
由于HPV很难进行体外培养，又具有较强的宿主特异性，很难在除人体以外的生物体进行繁殖，缺乏合适的动物模型。所以需要建立合适有效的体外中和实验模型，用于疫苗免疫保护性的评估。
HPV假病毒是理想的HPV体外中和实验模型：利用HPV VLP具有非特异包裹核酸的特性，细胞内表达的HPV L1和L2组成的VLP包裹游离的DNA或导入外源质粒形成HPV假病毒。
采用假病毒中和法对样品免疫后动物血清样品进行免疫原性分析。HPV6病毒样颗粒样品免疫动物后能产生针对HPV6的中和抗体，能中和HPV6型的假病毒。将免疫后的动物血清与一定量的假病毒孵育后再侵染细胞，可表达GFP荧光的细胞会随着血清中中和抗体的增加而减少，在一定的范围内可存在线性负相关，因此可以通过检测表达GFP的细胞数的变化来评价血清中抗体的中和活性。
假病毒构建方法：将HPV6型的pCMV3-3-HPV6L1+L2(L1序列来源于Uniprot P69898，L2序列来源Uniprot Q84297)质粒(购于Sino Biological)以及荧光质粒(PSEU-GFP Spark，购于Sino  Biological)共转染至293FT贴壁细胞(购于Thermo Fisher)。具体方法参考文献(Pastrana D V,Buck C B,Pang Y S,Thompson C D,Castle P E,FitzGerald P C,Kjaer S K,Lowy D R,Schiller J T.Reactivity of human sera in a sensitive,high-throughput pseudovirus-based papillomavirus neutralization assay for HPV16 and HPV18.[J]Virology 2004,321:205-216.)。收集假病毒上清液并进行分装，置于-80℃冰箱中保存备用。
6.1.2 HPV 6L1：33C病毒样颗粒动物免疫保护性评价
小鼠免疫程序：
HPV 6L1：33C病毒样颗粒吸附于磷酸铝佐剂上，经混合后取200μL用于免疫小鼠，每只小鼠免疫剂量0.15μg，免疫10只小鼠，于实验第0天、第7天、第21天分别用稀释后样品对小鼠进行免疫，同时设立空白血清对照组，于实验第28天摘取小鼠眼球取血，分离出血清进行假病毒中和滴度检测。
小鼠EC50检测：
小鼠血清在56℃灭活30分钟后，离心6000g，5分钟后取上清进行检测。检测前4-8小时，将293FT细胞以15000细胞/孔的密度铺板于96孔板中，培养于37℃，5％CO 2的二氧化碳培养箱中。免疫后小鼠血清、空白对照血清均用中和培养基系列稀释后按照体积比1:1分别与6.1中制备的HPV6假病毒混合。2～8℃冰箱中孵育1小时后按照100μL/孔加入到提前4-8小时铺板的293FT细胞上，每个样品2个复孔，同时设立空白血清对照孔、假病毒阳性对照孔和阴性对照孔。加样后的细胞继续在37℃，5％CO 2的二氧化碳培养箱中培养62-96小时后，在酶联斑点分析仪中(型号：S6 Universal-V Analyzer，厂家：CTL)进行荧光扫描拍照以及计数。通过计算每个小鼠血清样品的中和抑制率，依据Reed-Muench法计算得到血清中和抑制率为50％时血清最大稀释倍数，即半数有效稀释倍数EC 50。
HPV6血清假病毒中和滴度检测结果详见表4。
表4小鼠血清中和滴度检测结果EC 50(GMT±SEM)
Figure PCTCN2020102601-appb-000011
备注：
1.动物数，N＝10；
2.GMT(Geometric Mean Titer)：几何平均滴度；
3.SEM(Standard Error of Mean)：标准误差。
上述检测结果显示，本发明制备的HPV 6L1：33C病毒样颗粒具有较好的免疫原性，可在动物体内产生高滴度的中和抗体，可以用于制备成预防HPV感染的疫苗。
实施例6.2：HPV 11L1：33C病毒样颗粒动物免疫原性评价
实验方法和步骤与实施例6.1相同。L1序列来源于Uniprot P04012，L2序列来源Uniprot P04013。
HPV11血清假病毒中和滴度检测结果详见表5。
表5小鼠血清中和滴度检测结果EC 50(GMT±SEM)
Figure PCTCN2020102601-appb-000012
上述检测结果显示，本发明制备的HPV 11L1：33C病毒样颗粒具有较好的免疫原性，可在动物体内产生高滴度的中和抗体，可以用于制备成预防HPV感染的疫苗。
实施例6.3：HPV 16L1：33C病毒样颗粒动物免疫原性评价
实验方法和步骤与实施例6.1相同。L1序列来源于Uniprot P03101，L2序列来源Uniprot P03107。
HPV16血清假病毒中和滴度检测结果详见表6。
表6小鼠血清中和滴度检测结果EC 50(GMT±SEM)
Figure PCTCN2020102601-appb-000013
上述检测结果显示，本发明制备的HPV 16L1：33C病毒样颗粒 具有较好的免疫原性，可在动物体内产生高滴度的中和抗体，可以用于制备成预防HPV感染的疫苗。
实施例6.4：HPV 18L1：33C病毒样颗粒动物免疫原性评价
实验方法和步骤与实施例6.1相同。L1序列来源于Uniprot Q80B70，L2序列来源Uniprot P06793。
HPV18血清假病毒中和滴度检测结果详见表7。
表7小鼠血清中和滴度检测结果EC 50(GMT±SEM)
Figure PCTCN2020102601-appb-000014
上述检测结果显示，本发明制备的HPV 18L1：33C病毒样颗粒具有较好的免疫原性，可在动物体内产生高滴度的中和抗体，可以用于制备成预防HPV感染的疫苗。
实施例6.5：HPV 31L1：33C病毒样颗粒动物免疫原性评价
实验方法和步骤与实施例6.1相同。L1序列来源于Uniprot P17388，L2序列来源Uniprot P17389。
HPV31血清假病毒中和滴度检测结果详见表8。
表8小鼠血清中和滴度检测结果EC 50(GMT±SEM)
Figure PCTCN2020102601-appb-000015
上述检测结果显示，本发明制备的HPV 31L1：33C病毒样颗粒具有较好的免疫原性，可在动物体内产生高滴度的中和抗体，可以用于制备成预防HPV感染的疫苗。
实施例6.6：HPV 33L1病毒样颗粒动物免疫原性评价
实验方法和步骤与实施例6.1相同。L1序列来源于Uniprot P06416，L2序列来源Uniprot P06418。
HPV33血清假病毒中和滴度检测结果详见表9。
表9小鼠血清中和滴度检测结果EC 50(GMT±SEM)
Figure PCTCN2020102601-appb-000016
上述检测结果显示，本发明制备的HPV 33L1病毒样颗粒具有较好的免疫原性，可在动物体内产生高滴度的中和抗体，可以用于制备成预防HPV感染的疫苗。
实施例6.7：HPV 35L1：33C病毒样颗粒动物免疫原性评价
实验方法和步骤与实施例6.1相同。L1序列来源于Uniprot P27232，L2序列来源Uniprot P27234。
HPV35血清假病毒中和滴度检测结果详见表10。
表10小鼠血清中和滴度检测结果EC 50(GMT±SEM)
Figure PCTCN2020102601-appb-000017
上述检测结果显示，本发明制备的HPV 35L1：33C病毒样颗粒具有较好的免疫原性，可在动物体内产生高滴度的中和抗体，可以用于制备成预防HPV感染的疫苗。
实施例6.8：HPV 39L1：59C病毒样颗粒动物免疫原性评价
实验方法和步骤与实施例6.1相同。L1序列来源于Uniprot P24838，L2序列来源Uniprot P24839。
HPV39血清假病毒中和滴度检测结果详见表11。
表11小鼠血清中和滴度检测结果EC 50(GMT±SEM)
Figure PCTCN2020102601-appb-000018
上述检测结果显示，本发明制备的HPV 39L1：59C病毒样颗粒具有较好的免疫原性，可在动物体内产生高滴度的中和抗体，可以用于制备成预防HPV感染的疫苗。
实施例6.9：HPV 45L1：33C病毒样颗粒动物免疫原性评价
实验方法和步骤与实施例6.1相同。L1序列来源于Uniprot P36741，L2序列来源Uniprot P36761。
HPV45血清假病毒中和滴度检测结果详见表12。
表12小鼠血清中和滴度检测结果EC 50(GMT±SEM)
Figure PCTCN2020102601-appb-000019
上述检测结果显示，本发明制备的HPV 45L1：33C病毒样颗粒具有较好的免疫原性，可在动物体内产生高滴度的中和抗体，可以用于制备成预防HPV感染的疫苗。
实施例6.10：HPV 51L1：33C病毒样颗粒动物免疫原性评价
实验方法和步骤与实施例6.1相同。L1序列来源于Uniprot P26536，L2序列来源Uniprot P26539。
HPV51血清假病毒中和滴度检测结果详见表13。
表13小鼠血清中和滴度检测结果EC 50(GMT±SEM)
Figure PCTCN2020102601-appb-000020
上述检测结果显示，本发明制备的HPV 51L1：33C病毒样颗粒具有较好的免疫原性，可在动物体内产生高滴度的中和抗体，可以用于制备成预防HPV感染的疫苗。
实施例6.11：HPV 52L1：33C病毒样颗粒动物免疫原性评价
实验方法和步骤与实施例6.1相同。L1序列来源于Uniprot Q05138，L2序列来源Uniprot F8S4U2。
HPV52血清假病毒中和滴度检测结果详见表14。
表14小鼠血清中和滴度检测结果EC 50(GMT±SEM)
Figure PCTCN2020102601-appb-000021
上述检测结果显示，本发明制备的HPV 52L1：33C病毒样颗粒具有较好的免疫原性，可在动物体内产生高滴度的中和抗体，可以用于制备成预防HPV感染的疫苗。
实施例6.12：HPV 56L1：33C病毒样颗粒动物免疫原性评价
实验方法和步骤与实施例6.1相同。L1序列来源于Uniprot P36743，L2序列来源Uniprot P36765。
HPV56血清假病毒中和滴度检测结果详见表15。
表15小鼠血清中和滴度检测结果EC 50(GMT±SEM)
Figure PCTCN2020102601-appb-000022
上述检测结果显示，本发明制备的HPV 56L1：33C病毒样颗粒具有较好的免疫原性，可在动物体内产生高滴度的中和抗体，可以用于制备成预防HPV感染的疫苗。
实施例6.13：HPV 58L1：33C病毒样颗粒动物免疫原性评价
实验方法和步骤与实施例6.1相同。L1序列来源于Uniprot P26535，L2序列来源Uniprot B6ZB12。
HPV58血清假病毒中和滴度检测结果详见表16。
表16小鼠血清中和滴度检测结果EC 50(GMT±SEM)
Figure PCTCN2020102601-appb-000023
上述检测结果显示，本发明制备的HPV 58L1：33C病毒样颗粒具有较好的免疫原性，可在动物体内产生高滴度的中和抗体，可以用于制备成预防HPV感染的疫苗。
实施例6.14：HPV 59L1病毒样颗粒动物免疫原性评价
实验方法和步骤与实施例6.1相同。L1序列来源于Uniprot Q81971，L2序列来源Uniprot Q81970。
HPV59血清假病毒中和滴度检测结果详见表17。
表17小鼠血清中和滴度检测结果EC 50(GMT±SEM)
Figure PCTCN2020102601-appb-000024
上述检测结果显示，本发明制备的HPV 59L1病毒样颗粒具有较好的免疫原性，可在动物体内产生高滴度的中和抗体，可以用于制备成预防HPV感染的疫苗。
实施例6.15：14价病毒样颗粒免疫组合物动物免疫原性评价
小鼠免疫程序
将如上所述的14型(HPV 6、11、16、18、31、33、35、39、45、51、52、56、58、59)病毒样颗粒用磷酸铝空白佐剂在无菌条件下系列稀释后(以样品中单个剂量为20μg的HPV型为标准进行稀释，将该型样品稀释80倍至0.5μg/mL，其他型样品的稀释则随着各型配比组分的变化而变化)，取200μL用于免疫小鼠，每只小鼠免疫剂量见表18。将6～8周龄SPF级雌性Balb/c小鼠按照每组10只小鼠进行分组(实验组和对照)，于实验第0天、第7天、第21天分别用稀释后样品对各组小鼠进行免疫，样品免疫一组小鼠，同时设立空白血清对照组，于实验第28天摘取小鼠眼球取血，分离出血清进行假病毒中和滴度检测。
表18各型HPV疫苗每只小鼠免疫剂量(μg)
Figure PCTCN2020102601-appb-000025
假病毒中和法检测免疫后小鼠血清中和滴度
小鼠血清在56度灭活30分钟后，离心6000g，5分钟后取上清进行检测。检测前4-8小时，将293FT细胞以15000细胞/孔的密度铺板于96孔板中，培养于37℃，5％CO 2的二氧化碳培养箱中。免疫后小鼠血清、空白对照血清均用中和培养基系列稀释后按照体积 比1:1分别与预稀释好的14种假病毒(14型HPV假病毒包括6、11、16、18、31、33、35、39、45、51、52、56、58、59型，其制备分别见实施例6.1-6.14)混合。2～8℃冰箱中孵育1小时后按照100μL/孔加入到提前4-8小时铺板的293FT细胞上，每个样品2个复孔，同时设立空白血清对照孔、假病毒阳性对照孔和阴性对照孔。加样后的细胞继续在37℃，5％CO 2的二氧化碳培养箱中培养62-96小时后，在酶联斑点分析仪(型号：S6Universal-V Analyzer，厂家：CTL)中进行荧光扫描拍照以及计数。通过计算每个小鼠血清样品的中和抑制率，依据Reed-Muench法计算得到血清中和抑制率为50％时血清最大稀释倍数，即半数有效稀释倍数EC 50。检测结果见图3。
如图3所示，14价病毒样颗粒免疫组合物能够产生很好的中和抗体，可以用于制备成预防HPV感染的疫苗。
比较例1：C端截短的HPV16L1(1-474)的表达
发明人尝试将HPV16L1的C端截短31个氨基酸，命名为HPV16L1(1-474)(SEQ ID NO:27)。但在研究中发现，截短的HPV16L1(1-474)蛋白表达量高但蛋白可溶性差，难以提取纯化，具体表达和提取结果见图4。
虽然前述已经用说明和实施例的方式对本发明进行了细节描述，但其目的在于理解方便，本领域普通技术人员显然可以对本发明的技术方案作出的各种变形和改进，而不会偏离附加的权利要求的精神或范围。
附录1：序列表-嵌合的人***瘤病毒6型L1蛋白
Figure PCTCN2020102601-appb-000026
Figure PCTCN2020102601-appb-000027
Figure PCTCN2020102601-appb-000028
Figure PCTCN2020102601-appb-000029
Figure PCTCN2020102601-appb-000030
附录2：序列表-嵌合的人***瘤病毒11型L1蛋白
Figure PCTCN2020102601-appb-000031
Figure PCTCN2020102601-appb-000032
Figure PCTCN2020102601-appb-000033
Figure PCTCN2020102601-appb-000034
Figure PCTCN2020102601-appb-000035
附录3：序列表-嵌合的人***瘤病毒16型L1蛋白
Figure PCTCN2020102601-appb-000036
Figure PCTCN2020102601-appb-000037
Figure PCTCN2020102601-appb-000038
Figure PCTCN2020102601-appb-000039
Figure PCTCN2020102601-appb-000040
附录4：序列表-嵌合的人***瘤病毒18型L1蛋白
Figure PCTCN2020102601-appb-000041
Figure PCTCN2020102601-appb-000042
Figure PCTCN2020102601-appb-000043
Figure PCTCN2020102601-appb-000044
Figure PCTCN2020102601-appb-000045
附录5：序列表-嵌合的人***瘤病毒31型L1蛋白
Figure PCTCN2020102601-appb-000046
Figure PCTCN2020102601-appb-000047
Figure PCTCN2020102601-appb-000048
Figure PCTCN2020102601-appb-000049
Figure PCTCN2020102601-appb-000050
附录6：序列表-人***瘤病毒33型L1蛋白
Figure PCTCN2020102601-appb-000051
Figure PCTCN2020102601-appb-000052
Figure PCTCN2020102601-appb-000053
附录7：序列表-嵌合的人***瘤病毒35型L1蛋白
Figure PCTCN2020102601-appb-000054
Figure PCTCN2020102601-appb-000055
Figure PCTCN2020102601-appb-000056
Figure PCTCN2020102601-appb-000057
Figure PCTCN2020102601-appb-000058
附录8：序列表-嵌合的人***瘤病毒39型L1蛋白
Figure PCTCN2020102601-appb-000059
Figure PCTCN2020102601-appb-000060
Figure PCTCN2020102601-appb-000061
Figure PCTCN2020102601-appb-000062
Figure PCTCN2020102601-appb-000063
附录9：序列表-嵌合的人***瘤病毒45型L1蛋白
Figure PCTCN2020102601-appb-000064
Figure PCTCN2020102601-appb-000065
Figure PCTCN2020102601-appb-000066
Figure PCTCN2020102601-appb-000067
Figure PCTCN2020102601-appb-000068
附录10：序列表-嵌合的人***瘤病毒51型L1蛋白
Figure PCTCN2020102601-appb-000069
Figure PCTCN2020102601-appb-000070
Figure PCTCN2020102601-appb-000071
Figure PCTCN2020102601-appb-000072
Figure PCTCN2020102601-appb-000073
附录11：序列表-嵌合的人***瘤病毒52型L1蛋白
Figure PCTCN2020102601-appb-000074
Figure PCTCN2020102601-appb-000075
Figure PCTCN2020102601-appb-000076
Figure PCTCN2020102601-appb-000077
Figure PCTCN2020102601-appb-000078
附录12：序列表-嵌合的人***瘤病毒56型L1蛋白
Figure PCTCN2020102601-appb-000079
Figure PCTCN2020102601-appb-000080
Figure PCTCN2020102601-appb-000081
Figure PCTCN2020102601-appb-000082
Figure PCTCN2020102601-appb-000083
附录13：序列表-嵌合的人***瘤病毒58型L1蛋白
Figure PCTCN2020102601-appb-000084
Figure PCTCN2020102601-appb-000085
Figure PCTCN2020102601-appb-000086
Figure PCTCN2020102601-appb-000087
Figure PCTCN2020102601-appb-000088
附录14：序列表-人***瘤病毒59型L1蛋白
Figure PCTCN2020102601-appb-000089
Figure PCTCN2020102601-appb-000090
Figure PCTCN2020102601-appb-000091

Claims (17)

  1. 一种预防HPV相关疾病或感染的多价HPV免疫原性组合物，其包含：
    由HPV 6型、11型、16型、18型、31型、33型、45型、52型和58型的L1蛋白组装而成的HPV病毒样颗粒；和
    一种或多种由其他致病的HPV型别的L1蛋白组装而成的HPV病毒样颗粒。
  2. 根据权利要求1所述的多价HPV免疫原性组合物，其中所述一种或多种其他致病的HPV型别选自HPV 35型、39型、51型、56型和59型。
  3. 根据权利要求1或2所述的多价HPV免疫原性组合物，其中至少一种所述HPV病毒样颗粒为嵌合的HPV病毒样颗粒，所述嵌合的HPV病毒样颗粒包含一种或多种嵌合HPV L1蛋白；所述嵌合HPV L1蛋白自其N末端至C末端方向包含：
    a.衍生于第一型别***瘤病毒L1蛋白的N端片段，所述N端片段保持该型别L1蛋白的免疫原性，其中第一型别的***瘤病毒选自权利要求1所述的HPV 6型、11型、16型、18型、31型、33型、45型、52型和58型和一种或多种其他致病的HPV型别；和
    b.衍生于第二型别***瘤病毒L1蛋白的C端片段，所述第二型别***状瘤病毒L1蛋白具有相较于其他型别的L1蛋白表达量和可溶性较好的特性；
    其中嵌合HPV L1蛋白具有第一型别***瘤病毒L1蛋白的免疫原性。
  4. 根据权利要求3所述的多价HPV免疫原性组合物，其中
    所述N端片段为将所述第一型别***瘤病毒L1蛋白的天然序列的C端截短于其α5区内的任一氨基酸位点而得到的片段，以及与其具有至少98％的同一性的片段；并且
    所述C端片段为将第二型别***状瘤病毒L1蛋白的天然序列的 N末端截短于其α5区内的任一氨基酸位点而得到的片段，以及该片段进一步突变、缺失和/或添加而产生的功能性变体。
  5. 根据权利要求4所述的多价HPV免疫原性组合物，其中所述C端片段含有一个或多个核定位序列。
  6. 根据权利要求3所述的多价HPV免疫原性组合物，
    其中所述第一型别***瘤L1蛋白选自HPV 6型、11型、16型、18型、31型、35型、39型、45型、51型、52型、56型或58型；
    其中所述第二型别***瘤病毒L1蛋白选自HPV 1型、2型、3型、4型、6型、7型、10型、11型、13型、16型、18型、22型、26型、28型、31型、32型、33型、35型、39型、42型、44型、45型、51型、52型、53型、56型、58型、59型、60型、63型、66型、68型、73型或82型L1蛋白；
    优选地，所述第二型别***瘤病毒L1蛋白选自HPV 16型、28型、33型、59型、或68型L1蛋白；
    更优选地，所述第二型别***瘤病毒L1蛋白选自HPV 33型或HPV 59型L1蛋白。
  7. 根据权利要求6所述的多价HPV免疫原性组合物，其中所述C端片段为SEQ ID No:2；或其长度为m1个氨基酸的片段，优选涵盖SEQ ID No:2的第1-m1位氨基酸的片段；其中m1为8-26的整数；或所述C端片段为SEQ ID No:135；或其长度为m2个氨基酸的片段，优选涵盖SEQ ID No:135的第1-m2位氨基酸的片段；其中m2为13-31的整数。
  8. 根据权利要求6所述的多价HPV免疫原性组合物，其中所述C端片段为SEQ ID No:13；或其长度为n个氨基酸的片段，优选涵盖SEQ ID No:13的第1-n位氨基酸的片段；其中n为16-38的整数。
  9. 根据权利要求3所述的多价HPV免疫原性组合物，其中
    所述HPV 6型L1蛋白的N端片段与将SEQ ID No:1所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
    所述HPV 11型L1蛋白的N端片段与将SEQ ID No:14所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
    所述HPV 16型L1蛋白的N端片段与将SEQ ID No:27所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
    所述HPV 18型L1蛋白的N端片段与将SEQ ID No:40所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
    所述HPV 31型L1蛋白的N端片段与将SEQ ID No:53所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
    所述HPV 35型L1蛋白的N端片段与将SEQ ID No:69所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
    所述HPV 39型L1蛋白的N端片段与将SEQ ID No:82所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
    所述HPV 45型L1蛋白的N端片段与将SEQ ID No:95所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
    所述HPV 51型L1蛋白的N端片段与将SEQ ID No:108所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
    所述HPV 52型L1蛋白的N端片段与将SEQ ID No:121所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
    所述HPV 56型L1蛋白的N端片段与将SEQ ID No:134所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有 98％、98.5％、99％、99.5％、99％或100％的同一性；和
    所述HPV 58型L1蛋白的N端片段与将SEQ ID No:147所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性。
  10. 根据权利要求3所述的多价HPV免疫原性组合物，所述N端片段的C末端与所述C端片段的N末端直接连接或通过接头连接。
  11. 根据权利要求3所述的多价HPV免疫原性组合物，其中当所述N端片段的C末端与所述C端片段的N末端连接时，在连接点的正负4个氨基酸位点的范围内存在以下连续氨基酸序列：RKFL；
    优选地，在连接点的正负6个氨基酸位点的范围内存在以下连续氨基酸序列：LGRKFL。
  12. 根据权利要求3所述的多价HPV免疫原性组合物，其中
    所述嵌合的HPV 6型、11型、16型、18型、31型、35型、39型、45型、51型、52型、56型和58型嵌合HPV L1蛋白分别与SEQ ID No:3、SEQ ID No:16、SEQ ID No:29、SEQ ID No:42、SEQ ID No:55、SEQ ID No:71、SEQ ID No:84、SEQ ID No:97、SEQ ID No:110、SEQ ID No:123、SEQ ID No:136和SEQ ID No:149具有98％、98.5％、99％、99.5％或100％的同一性；以及
    HPV 33型L1蛋白和HPV 59型L1蛋白分别与SEQ ID No:66和SEQ ID No:160具有98％、98.5％、99％、99.5％或100％的同一性。
  13. 根据权利要求3所述的多价HPV免疫原性组合物，其中至少一种所述HPV病毒样颗粒由单一型别的嵌合HPV L1蛋白组成，优选地，由具有相同的氨基酸序列的所述单一型别的嵌合HPV L1蛋白组成。
  14. 根据权利要求1-13中任一项所述的多价HPV免疫原性组合物，其中所述多价HPV免疫原性组合物还包含生理学上可接受的载体以及任选地，还包含佐剂。
  15. 根据权利要求14所述的多价HPV免疫原性组合物，其中所述佐剂为磷酸铝佐剂。
  16. 一种预防HPV相关疾病或感染的方法，其包括：
    向受试者施用权利要求1-15中任一项所述的多价HPV免疫原性组合物。
  17. 权利要求1-15中任一项所述的多价HPV免疫原性组合物在用于制备用于预防HPV相关疾病或感染的疫苗或药物中的用途。
PCT/CN2020/102601 2019-07-19 2020-07-17 人***瘤病毒多价免疫原性组合物 WO2021013071A1 (zh)

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