WO2021013067A1 - 嵌合的人乳头瘤病毒6型l1蛋白 - Google Patents

嵌合的人乳头瘤病毒6型l1蛋白 Download PDF

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WO2021013067A1
WO2021013067A1 PCT/CN2020/102584 CN2020102584W WO2021013067A1 WO 2021013067 A1 WO2021013067 A1 WO 2021013067A1 CN 2020102584 W CN2020102584 W CN 2020102584W WO 2021013067 A1 WO2021013067 A1 WO 2021013067A1
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protein
hpv
hpv type
chimeric
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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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    • 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
    • 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
    • C07K14/01—DNA viruses
    • C07K14/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/04—Inactivation or attenuation; Producing viral sub-units

Definitions

  • the present invention relates to human papillomavirus (HPV) L1 protein and polynucleotide encoding the protein, and also relates to HPV virus-like particles and a preparation method 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).
  • L1 protein is produced by Saccharomyces cerevisiae. Fermentation production, self-assembly into VLP.
  • 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. 9Can prevent 90% of cervical cancers (https://www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedPro ducts/ucm426445.htm).
  • the key factor in HPV vaccine development is that 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.
  • Commonly used 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.
  • the present invention provides a chimeric human papillomavirus (HPV) type 6 L1 protein, comprising a. N-terminal fragment derived from HPV type 6 L1 protein from its N-terminal to C-terminal direction, said N The terminal fragment maintains the immunogenicity of the HPV type 6 L1 protein; and b.
  • the C-terminal fragment derived from the L1 protein of the second type papillomavirus, the second type of papillomavirus L1 protein is compared with other types Other L1 protein expression and solubility are better; wherein the chimeric HPV type 6 L1 protein has the immunogenicity of HPV type 6 L1 protein.
  • the present invention provides a HPV type 6 virus-like particle, which comprises a chimeric HPV type 6 L1 protein.
  • the present invention provides an immunogenic composition for preventing HPV-related diseases or infections, which comprises HPV type 6 virus-like particles and an adjuvant.
  • the present invention provides an isolated polynucleotide encoding a chimeric HPV type 6 L1 protein.
  • the present invention provides a vector comprising a polynucleotide encoding a chimeric HPV type 6 L1 protein.
  • the present invention provides a baculovirus comprising a polynucleotide encoding a chimeric HPV type 6 L1 protein.
  • the present invention provides a host cell comprising the polynucleotide, vector, or baculovirus as described above.
  • the present invention provides a method for preparing HPV type 6 virus-like particles, which comprises culturing the host cell described above to express the chimeric HPV type 6 L1 protein and assemble it into virus-like particles; and purification The HPV type 6 virus-like particles.
  • HPV 6 L1 33C L1 protein expression.
  • M Marker; L: cell lysate; E-S: supernatant collected after centrifugation of the lysate.
  • FIG. 3 Expression of HPV16L1 (1-474) with C-terminal truncation.
  • M Marker;
  • L cell lysate;
  • E-S supernatant collected after centrifugation of the lysate.
  • the present invention provides a chimeric human papillomavirus (HPV) type 6 L1 protein, from its N-terminal to C-terminal direction comprising: a. an N-terminal fragment derived from HPV type 6 L1 protein, said The N-terminal fragment maintains the immunogenicity of the HPV type 6 L1 protein; and b.
  • the C-terminal fragment derived from the L1 protein of the second type papillomavirus, the second type of papillomavirus L1 protein has more Types of L1 protein expression and solubility are good; wherein the chimeric HPV type 6 L1 protein has the immunogenicity of HPV type 6 L1 protein.
  • the N-terminal fragment is a fragment obtained by truncating the C-terminus of the natural sequence of HPV type 6 L1 protein to any amino acid position in its ⁇ 5 region, and is at least 98% identical to it.
  • sexual fragment the C-terminal fragment is a fragment obtained by truncating the N-terminus of the natural sequence of the second type papillomavirus L1 protein to any amino acid position in its ⁇ 5 region, and the fragment is further mutated , Deletions and/or additions and functional variants.
  • the N-terminal fragment and the fragment obtained by truncating the C-terminal of the natural sequence of HPV type 6 L1 protein 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 L1 protein of the second type of papillomavirus is HPV type 33 L1 protein, and the C-terminal fragment is SEQ ID No: 2; or a fragment of m amino acids in length, preferably covering SEQ ID No: 2 is a fragment of amino acid at position 1-m; where m is an integer from 8-26.
  • 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 some embodiments, the chimeric HPV type 6 L1 protein comprises one or more C-terminal fragments of HPV type 33 L1 protein. The C-terminal fragments of the multiple HPV type 33 L1 proteins may be the same or different. 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 second type of papillomavirus L1 protein 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 encompassing SEQ ID No: A fragment of amino acids 1-n of 13; 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 type 6 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 type 6 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 and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No:1 to any amino acid position in its ⁇ 5 region have 98%, 98.5%, and 99% , 99.5% 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 6 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 chimeric HPV type 6 L1 protein has 98%, 98.5%, 99%, 99.5%, or 100% identity with SEQ ID No: 3.
  • the present invention provides a HPV type 6 virus-like particle, which comprises the chimeric HPV type 6 L1 protein as described above.
  • the HPV type 6 virus-like particle is an icosahedron composed of 72 pentamers of the chimeric HPV type 6 L1 protein.
  • HPV type 6 virus-like particles have correctly formed disulfide bonds and thus have a good natural conformation.
  • HPV type 6 virus-like particles self-assemble in an in vivo expression system.
  • the present invention provides an immunogenic composition for preventing HPV-related diseases or infections, which comprises the HPV type 6 virus-like particles as described above and an adjuvant.
  • the prevention can be considered a treatment, and the two can be used interchangeably.
  • the above immunogenic composition is administered to a subject.
  • the subject is a human.
  • the present invention provides an isolated polynucleotide encoding the chimeric HPV type 6 L1 protein as described above.
  • the polynucleotide is a polynucleotide that has been codon optimized for different expression systems.
  • the polynucleotide is a polynucleotide that has been codon optimized for the insect baculovirus expression system.
  • the present invention provides an isolated polynucleotide having the sequence shown in SEQ ID No: 4.
  • the present invention provides a vector comprising the polynucleotide as described above.
  • the vector is a baculovirus vector.
  • the vector may be a transfer vector used in a baculovirus expression system.
  • the vector may be an expression vector used in a baculovirus expression system.
  • the vector may be a recombined vector used in a baculovirus expression system.
  • the present invention provides a baculovirus comprising the polynucleotide as described above.
  • the present invention provides a host cell comprising the polynucleotide, vector, or baculovirus as described above.
  • the host cell is an insect cell.
  • the insect cell is selected from Sf9 cell, Sf21 cell, Hi5 cell and S2 cell.
  • the present invention provides a method for preparing HPV type 6 virus-like particles as described above, which comprises: culturing a host cell as described above to express the chimeric HPV type 6 L1 protein and assembling Into virus-like particles; and purifying the HPV type 6 virus-like particles.
  • the host cell is an insect cell. In one embodiment, the host cell is a Hi5 cell. In one embodiment, the chimeric HPV type 6 L1 protein self-assembles into HPV type 6 virus-like particles in the host cell. In one embodiment, the chimeric HPV type 6 L1 protein self-assembles into HPV type 6 virus-like particles in the host cell, which has a two-dimensional structure composed of 72 pentamers of the chimeric HPV type 6 L1 protein. Decahedron. In one embodiment, HPV type 6 virus-like particles have correctly formed disulfide bonds and thus have a good natural conformation.
  • the purification uses cation exchange chromatography. In one embodiment, strong cation exchange chromatography is used for purification. In another embodiment, the purification uses weak cation exchange chromatography. In one embodiment, the purification uses a combination of multiple cation exchange chromatography. In one embodiment, HS strong cation exchange chromatography is used for purification. In another embodiment, MMA ion exchange chromatography is used for purification. In another embodiment, HS-MMA two-step chromatography is used for purification.
  • 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 yield 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 solubility of the protein caused by this truncation may be caused by the defect 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 type 6 L1 protein has low expression in insect cells and poor solubility, which is not conducive to subsequent purification and vaccine production.
  • yeast cells the virus-like particles assembled from HPV type 6 L1 protein lack a good conformation because disulfide bonds cannot be formed correctly.
  • the expression level and solubility of the chimeric HPV type 6 L1 protein of the present invention in insect cells are greatly improved compared with the HPV type 6 L1 protein before modification. It can be used for large-scale production of HPV 6 vaccine.
  • the chimeric HPV type 6 L1 protein can correctly form disulfide bonds in insect cells and assemble into HPV type 6 virus-like particles with a good conformation. This can improve the immunogenicity of HPV type 6 virus-like particles and produce a better immune response.
  • 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)). Over 120 HPV types have been identified and they are numbered (for example, HPV16, HPV18, 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
  • 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 Construction of a chimeric gene where 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 Yingwei Jieji (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 5min; denaturation at 98°C for 10s, annealing at 69°C for 15s, 72°C 1kb/1min, for 30 cycles; extension at 72°C for 5min; 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 5min; denaturation at 98°C for 10s, annealing at 69°C for 15s, 72°C 1kb/1min, for 30 cycles; extension at 72°C for 5min; 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, 5 cycles; 98°C denaturation 10s, 68°C annealing 15s, 72°C 1kb/1min, 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 2 HPV 6L1: 33C recombinant baculovirus packaging
  • DH10Bac bacterial competent cells In the kit, the seller Thermo Fisher, cultured and amplified at 37°C and streaked the plate. White plaque was selected and amplified. After overnight culture, 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.
  • 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 100 mM-1M) ultrasonically lyse the cell pellet, sonicate at low temperature for 3 minutes, centrifuge at a centrifugal force greater than 10,000 g for 10 minutes, collect the supernatant after centrifugation, 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 100mg/L and a protein size of about 56KD, which can be used for large-scale production.
  • 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.
  • 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.
  • HPV16L1(1-474) SEQ ID NO: 14
  • HPV16L1(1-474) SEQ ID NO: 14
  • 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 3.

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Abstract

提供了一种嵌合的人乳头瘤病毒6型L1蛋白和编码其的多核苷酸，还提供了HPV 6型病毒样颗粒和其制备方法。所述嵌合的人乳头瘤病毒6型L1蛋白包含衍生于HPV 6型L1蛋白的N端片段，所述N端片段保持HPV 6型L1蛋白的免疫原性；和衍生于第二型别乳头瘤病毒L1蛋白的C端片段，所述第二型别乳头状瘤病毒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；其中所述嵌合的HPV 6型L1蛋白具有HPV 6型L1蛋白的免疫原性。所述嵌合的人乳头瘤病毒6型L1蛋白具有较高的表达量和可溶性，可用于疫苗的大规模生产。

Description

嵌合的人乳头瘤病毒6型L1蛋白 技术领域
本发明涉及人乳头瘤病毒(HPV)L1蛋白和编码该蛋白的多核苷酸，还涉及HPV病毒样颗粒及其制备方法。
背景技术
乳头瘤病毒(papilloma virus，PV)属于乳头瘤病毒科(Papillomaviridae)，能引起人、牛、狗、兔等的乳头瘤。其成员人乳头瘤病毒(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、肛门、阴茎、阴道、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，约占宫颈癌的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导致大多数肛门生殖器疣和呼吸道乳头状瘤，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、杆状病毒或酵母系统重组表达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似乎是乳头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 PCTCN2020102584-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系统在夜蛾(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引起的宫颈癌，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 PCTCN2020102584-appb-000002
是默克公司生产的人乳头状瘤病毒四价(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宫颈癌生殖器疣(尖锐湿疣)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肛门癌、生殖器疣(尖锐湿疣)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 PCTCN2020102584-appb-000003
9是默克公司生产的人乳头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型引起的宫颈癌、外阴癌、阴道癌和肛门癌，HPV6和11引起的生殖器疣(尖锐湿疣)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型引起的肛门癌，HPV 6和11引起的生殖器疣(尖锐湿疣)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 PCTCN2020102584-appb-000004
9的说明书中声称HPV16和18型是约70％的宫颈癌的发病缘由，其余的20％病例归责于31、33、45、52和58型，由是
Figure PCTCN2020102584-appb-000005
9可以预防90％的宫颈癌的发生(https://www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedPro ducts/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系统主要分为真核表达系统和原核表达系统。常用的真核表达系统有痘病毒表达系统、昆虫杆状病毒表达系统、酵母表达系统。在真核表达系统中表达的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。原核表达系统主要大肠杆菌表达系统，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病毒样颗粒的需求。
发明内容
在一个方面，本发明提供一种嵌合的人乳头瘤病毒(HPV)6型L1蛋白，自其N末端至C末端方向包含a.衍生于HPV 6型L1蛋白的N端片段，所述N端片段保持HPV 6型L1蛋白的免疫原性；和b.衍生于第二型别乳头瘤病毒L1蛋白的C端片段，所述第二型别乳头状瘤病毒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；其中所述嵌合的HPV 6型L1蛋白具有HPV 6型L1蛋白的免疫原性。
在另一个方面，本发明提供一种HPV 6型病毒样颗粒，其包含嵌合的HPV 6型L1蛋白。
在另一个方面，本发明提供一种预防HPV相关疾病或感染的免疫原性组合物，其包含HPV 6型病毒样颗粒和佐剂。
在另一个方面，本发明提供一种分离的多核苷酸，其编码嵌合的HPV 6型L1蛋白。
在另一个方面，本发明提供一种载体，其包含编码嵌合的HPV 6型L1蛋白的多核苷酸。
在另一个方面，本发明提供一种杆状病毒，其包含编码嵌合的HPV 6型L1蛋白多核苷酸。
在另一个方面，本发明提供一种宿主细胞，其包含如上所述的多核苷酸、载体、或杆状病毒。
在另一个方面，本发明提供一种制备HPV 6型病毒样颗粒的方 法，其包括培养如上所述的宿主细胞以表达所述嵌合的HPV 6型L1蛋白并组装成病毒样颗粒；以及纯化所述HPV 6型病毒样颗粒。
附图说明
图1 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2透射电镜观察HPV 6 L1：33C病毒样颗粒。
图3 C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，本发明提供一种嵌合的人乳头瘤病毒(HPV)6型L1蛋白，自其N末端至C末端方向包含：a.衍生于HPV 6型L1蛋白的N端片段，所述N端片段保持HPV 6型L1蛋白的免疫原性；和b.衍生于第二型别乳头瘤病毒L1蛋白的C端片段，所述第二型别乳头状瘤病毒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；其中所述嵌合的HPV 6型L1蛋白具有HPV 6型L1蛋白的免疫原性。
在一个实施方式中，所述N端片段为将HPV 6型L1蛋白的天然序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段，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乳头状瘤病毒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HPV 6型L1蛋白的天然序列的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至少98.5％、99％、99.5％或100％的同一性。
在另一个实施方式中，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，所述第二型别乳头瘤病毒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，所述第二型别乳头瘤病毒L1蛋白选自HPV 16型、28型、33型、59型、或68型L1蛋白；
更优选地，所述第二型别乳头瘤病毒L1蛋白选自HPV 33型或HPV 59型L1蛋白。
在一个实施方式中，所述第二型别乳头瘤病毒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m个氨基酸的片段，优选涵盖SEQ ID No:2的第1-m位氨基酸的片段；其中m为8-26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。在一些实施方式中，嵌合的HPV 6型L1蛋白包含一个或多个HPV 33型L1蛋白的C端片段。所述多个HPV 33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: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。
在另一个实施方式中，所述第二型别乳头瘤病毒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，嵌合的HPV 6型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，所述嵌合的HPV 6型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％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。在一个实施方式中，接头可以是HPV 6型L1蛋白的部分序列。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。
在一个实施方式中，嵌合的HPV 6型L1蛋白与SEQ ID No:3具有98％、98.5％、99％、99.5％或100％的同一性。
在另一个方面，本发明提供一种HPV 6型病毒样颗粒，其包含如前所述的嵌合的HPV 6型L1蛋白。在一个实施方式，HPV 6型病毒样颗粒为由72个所述嵌合的HPV 6型L1蛋白的五聚体构成的二十面体。在一个实施方式中，HPV 6型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，HPV 6型病毒样颗粒在体内表达系统中自行装配。
在一个方面，本发明提供一种预防HPV相关疾病或感染的免疫原性组合物，其包含根据如前所述的HPV 6型病毒样颗粒和佐剂。所述预防可被认为是治疗，两者可互换使用。
在一个方面，将上述免疫原性组合物施用至受试者。在一个实施方式中，受试者是人。
在一个方面，本发明提供一种分离的多核苷酸，其编码如前所述所述的嵌合的HPV 6型L1蛋白。在一个实施方式中，多核苷酸为针对不同表达系统进行密码子优化后的多核苷酸。在一个实施方式中，多核苷酸为针对昆虫杆状病毒表达系统进行密码子优化后的多核苷酸。
在一个方面，本发明提供一种分离的多核苷酸，其具有SEQ ID No:4所示的序列。
在一个方面，本发明提供一种载体，其包含如前所述的多核苷酸。在一个实施方式中，载体是杆状病毒载体。在一个实施方式中，载体可以是用于杆状病毒表达系统的转移载体。在另一个实施方式中，载体可以是用于杆状病毒表达系统的表达载体。在另一个实施方式中，载体可以是用于杆状病毒表达系统的重组后的载体。
在一个方面，本发明提供一种杆状病毒，其包含如前所述的多核苷酸。
在一个方面，本发明提供一种宿主细胞，其包含如前所述的多核苷酸、载体、或杆状病毒。在一个实施方式中，宿主细胞为昆虫细胞，优选地，所述昆虫细胞选自Sf9细胞、Sf21细胞、Hi5细胞和S2细胞。
在一个方面，本发明提供一种制备根据如前所述的HPV 6型病毒样颗粒的方法，其包括：培养如前所述的宿主细胞以表达所述嵌合的HPV 6型L1蛋白并组装成病毒样颗粒；以及纯化所述HPV 6型病毒样颗粒。
在一个实施方式中，宿主细胞为昆虫细胞。在一个实施方式中，宿主细胞为Hi5细胞。在一个实施方式中，嵌合的HPV 6型L1蛋白在宿主细胞中自行装配成HPV 6型病毒样颗粒。在一个实施方式中，嵌合的HPV 6型L1蛋白在宿主细胞中自行装配成HPV 6型病毒样颗粒，其具有由72个所述嵌合的HPV 6型L1蛋白的五聚体构成的二十面体。在一个实施方式中，HPV 6型病毒样颗粒具有正确形成的二硫键，从而具有良好的天然构象。
在一个实施方式中，纯化采用阳离子交换层析。在一个实施方式中，纯化采用强阳离子交换层析。在另一个实施方式中，纯化采用弱阳离子交换层析。在一个实施方式中，纯化采用多次阳离子交换层析的组合。在一个实施方式中，纯化采用HS强阳离子交换层析。在另一个实施方式中，纯化采用MMA离子交换层析。在另一个实施方式中，纯化采用HS-MMA两步层析。
真核表达系统表达的乳头瘤病毒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乳头瘤病毒L1蛋白(例如HPV L1蛋白)的N端片段和衍生于第二型别乳头瘤病毒L1蛋白(例如HPV L1蛋白)的C端片段，前者提供第一型别乳头瘤病毒(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):86-93 March 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 PCTCN2020102584-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 PCTCN2020102584-appb-000007
Figure PCTCN2020102584-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系统分为真核表达系统和原核表达系统。真核表达系统表达的乳头瘤病毒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。原核表达系统表达的乳头瘤病毒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。
本发明将乳头瘤病毒(例如人乳头瘤病毒)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，可以在表达系统(例如宿主细胞，例如昆虫细胞)中提高乳头瘤病毒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乳头瘤病毒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乳头瘤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乳头瘤病毒，特别是HPV的感染成为可能。
HPV 6型L1蛋白在昆虫细胞中表达量低且可溶性差，不利于后续纯化和疫苗的生产。此外，在酵母细胞中，因为无法正确形成二硫键，HPV 6型L1蛋白装配成的病毒样颗粒缺乏良好的构象。
本发明的嵌合的HPV 6型L1蛋白相比于未改造之前的HPV 6型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 6型疫苗的大规模生产。此外，嵌合的HPV 6型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 6型病毒样颗粒。这可以提高HPV 6型病毒样颗粒的免疫原性，产生更好的免疫应答。
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系统识别。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“人乳头状瘤病毒”是指乳头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，HPV16、HPV18等)。
术语“HPV”或“HPV病毒”指乳头状瘤病毒科的乳头状瘤病毒，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、插入和/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)。
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：HPV6L1 C端替换为HPV33L1 C端的嵌合基因的构建
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。
1.2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。
1.3 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2：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 PCTCN2020102584-appb-000009
试剂盒，销售商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3：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100 mM-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1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4：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 PCTCN2020102584-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5：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2，由图2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6：HPV 6L1：33C病毒样颗粒动物免疫原性评价
6.1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。
6.2 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：S6Universal-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 PCTCN2020102584-appb-000011
Figure PCTCN2020102584-appb-000012
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。
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:14)。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3。
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
Figure PCTCN2020102584-appb-000013
Figure PCTCN2020102584-appb-000014
Figure PCTCN2020102584-appb-000015
Figure PCTCN2020102584-appb-000016
Figure PCTCN2020102584-appb-000017
Figure PCTCN2020102584-appb-000018

Claims (23)

  1. 一种嵌合的HPV 6型L1蛋白，自其N末端至C末端方向包含：
    a.衍生于HPV 6型L1蛋白的N端片段，所述N端片段保持HPV 6型L1蛋白的免疫原性；和
    b.衍生于第二型别乳头瘤病毒L1蛋白的C端片段，所述第二型别乳头状瘤病毒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；
    其中所述嵌合的HPV 6型L1蛋白具有HPV 6型L1蛋白的免疫原性。
  2. 根据权利要求1所述的嵌合的HPV 6型L1蛋白，其中
    所述N端片段为将HPV 6型L1蛋白的天然序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段，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乳头状瘤病毒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。
  3. 根据权利要求2所述的嵌合的HPV 6型L1蛋白，其中所述C端片段含有一个或多个核定位序列。
  4. 根据权利要求1所述的嵌合的HPV 6型L1蛋白，其中所述第二型别乳头瘤病毒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，所述第二型别乳头瘤病毒L1蛋白选自HPV 16型、28型、33型、59型、或68型L1蛋白；
    更优选地，所述第二型别乳头瘤病毒L1蛋白选自HPV 33型或 HPV 59型L1蛋白。
  5. 根据权利要求4所述的嵌合的HPV 6型L1蛋白，其中所述C端片段为SEQ ID No:2；或其长度为m个氨基酸的片段，优选涵盖SEQ ID No:2的第1-m位氨基酸的片段；其中m为8-26的整数。
  6. 根据权利要求4所述的嵌合的HPV 6型L1蛋白，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。
  7. 根据权利要求1所述的嵌合的HPV 6型L1蛋白，其中所述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％或100％的同一性。
  8. 根据权利要求1所述的嵌合的HPV 6型L1蛋白，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。
  9. 根据权利要求1所述的嵌合的HPV 6型L1蛋白，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。
  10. 根据权利要求1所述的嵌合的HPV 6型L1蛋白，其与SEQ ID No:3具有98％、98.5％、99％、99.5％或100％的同一性。
  11. 一种HPV 6型病毒样颗粒，其包含权利要求1至10中任一项所述的嵌合的HPV 6型L1蛋白。
  12. 根据权利要求11所述的HPV 6型病毒样颗粒，其为由72个所述嵌合的HPV 6型L1蛋白的五聚体构成的二十面体。
  13. 一种预防HPV相关疾病或感染的免疫原性组合物，其包含根据权利要求11或12所述的HPV 6型病毒样颗粒和佐剂。
  14. 一种分离的多核苷酸，其编码权利要求1至10中任一项所述的嵌合的HPV 6型L1蛋白。
  15. 一种分离的多核苷酸，其具有SEQ ID No:4所示的序列。
  16. 一种载体，其包含根据权利要求14或15所述的多核苷酸。
  17. 根据权利要求16所述的载体，其中所述载体为杆状病毒载体。
  18. 一种杆状病毒，其包含根据权利要求14或15所述的多核苷酸。
  19. 一种宿主细胞，其包含根据权利要求14或15所述的多核苷酸、根据权利要求16或17所述的载体、或根据权利要求18所述的杆状病毒。
  20. 根据权利要求19所述的宿主细胞，其为昆虫细胞，优选地，所述昆虫细胞选自Sf9细胞、Sf21细胞、Hi5细胞和S2细胞。
  21. 一种制备根据权利要求11或12所述的HPV 6型病毒样颗粒的方法，其包括：
    培养权利要求19至20中任一项所述的宿主细胞以表达所述嵌合的HPV 6型L1蛋白并组装成病毒样颗粒；以及
    纯化所述HPV 6型病毒样颗粒。
  22. 根据权利要求21所述的方法，其中所述宿主细胞为Hi5细胞。
  23. 根据权利要求21所述的方法，其中所述纯化采用阳离子交换层析，优选地，所述阳离子交换层析为HS-MMA两步层析。
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