WO2019233400A1 - 一种人乳头瘤病毒66型l1蛋白的突变体 - Google Patents

一种人乳头瘤病毒66型l1蛋白的突变体 Download PDF

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WO2019233400A1
WO2019233400A1 PCT/CN2019/089940 CN2019089940W WO2019233400A1 WO 2019233400 A1 WO2019233400 A1 WO 2019233400A1 CN 2019089940 W CN2019089940 W CN 2019089940W WO 2019233400 A1 WO2019233400 A1 WO 2019233400A1
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protein
hpv66
type
hpv
amino acid
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PCT/CN2019/089940
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English (en)
French (fr)
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李少伟
柳欣林
杨与柔
陈洁
王大宁
夏宁邵
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厦门大学
厦门万泰沧海生物技术有限公司
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Priority to BR112020024687-5A priority Critical patent/BR112020024687A2/pt
Priority to JP2021517891A priority patent/JP7337352B2/ja
Priority to EP19815012.0A priority patent/EP3805252A4/en
Priority to US15/734,750 priority patent/US11464846B2/en
Priority to KR1020207038061A priority patent/KR20210019465A/ko
Publication of WO2019233400A1 publication Critical patent/WO2019233400A1/zh

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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
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P17/12—Keratolytics, e.g. wart or anti-corn preparations
    • 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
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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    • A61K2039/5258—Virus-like particles
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
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    • 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
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    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention relates to the fields of molecular virology and immunology.
  • the present invention relates to a mutant HPV66 L1 protein (or a variant thereof), a coding sequence and a preparation method thereof, and a virus-like particle comprising the same, which protein (or a variant thereof) and the virus-like particle are capable of inducing Neutralizing antibodies against at least two types of HPV (eg, HPV66 and HPV56, or HPV66, HPV56, and HPV53), and thus can be used to prevent the at least two types of HPV infections and diseases caused by the infections Examples include cervical cancer and genital warts.
  • the present invention also relates to the use of the above-mentioned protein and virus-like particles for the preparation of a pharmaceutical composition or vaccine, which can be used to prevent the at least two types of HPV infections and diseases caused by the infections Examples include cervical cancer and genital warts.
  • HPV Human Papillomavirus
  • HPV Human Papillomavirus
  • the high-risk HPV infection has been proven to be the main cause of genital cancers including cervical cancer in women.
  • the low-risk type mainly causes genital warts.
  • the most effective way to prevent and control HPV infection is to administer an HPV vaccine, especially a vaccine against high-risk HPV that can cause cervical cancer.
  • HPV VLP Virus-Like Particles
  • HPV VLP is a 20-hedral stereosymmetric structure consisting of pentamers of 72 major capsid proteins L1 (Doorbar, J. and P.H. Gallimore. 1987. J. Virol, 61 (9): 2793-9).
  • the structure of HPVVLP is highly similar to that of natural HPV. It retains most of the neutralizing epitopes of natural viruses and can induce high titer neutralizing antibodies (Kirnbauer, R., F.Booy, et al. 1992 Proc Natl Acad Sci U.S.A. 89 (24): 12180-4).
  • HPV VLP mainly induces neutralizing antibodies against homotype HPV, and produces protective immunity against homotype HPV, while there is only a low cross-protection effect between some types with high homology (Sara L. Bissett, Giada Mattiuzzo, et al. 2014 Vaccine. 32: 6548-6555). Therefore, the scope of protection of existing HPV vaccines is very limited. In general, one type of HPV VLP can only be used to prevent HPV infection of that type. In this case, if the scope of protection of the HPV vaccine is to be expanded, more types of HPV VLPs can only be added to the vaccine.
  • HPV vaccines currently on the market including Merck's (It is a tetravalent vaccine against HPV 16, 18, 6, and 11) (Which is a bivalent vaccine against HPV 16, 18) and Merck's 9 (which is a nine-valent vaccine against HPV 6, 11, 16, 18, 31, 33, 45, 52, 58) is made by mixing multiple types of HPV VLPs.
  • Merck's It is a tetravalent vaccine against HPV 16, 18, 6, and 11
  • Which is a bivalent vaccine against HPV 16, 18
  • Merck's 9 which is a nine-valent vaccine against HPV 6, 11, 16, 18, 31, 33, 45, 52, 58
  • HPV virus-like particles capable of inducing protective neutralizing antibodies against multiple types of HPV in order to more economically and effectively prevent multiple types of HPV infections and diseases such as cervical cancer And genital warts.
  • the present invention is based at least in part on the inventor's unexpected discovery that a specific segment in the human papilloma virus (HPV) type 66 L1 protein is replaced with a corresponding segment of the second type of HPV (eg, HPV56) L1 protein Later, the obtained mutant HPV66 L1 protein can induce the body to produce high titer neutralizing antibodies against HPV66 and type II HPV (such as HPV56), and its protective effect is similar to that of mixed HPV66 and VLP and type II HPV.
  • VLP is equivalent
  • its protective effect against HPV66 is equivalent to that of HPV66 alone
  • its protective effect against the second type of HPV for example, HPV56
  • one or two specific segments of the HPV66 L1 protein can be further replaced with corresponding segments of the third type HPV (eg, HPV53) L1 protein.
  • the double substitution mutant HPV66 L1 protein can induce the body to produce high titer neutralizing antibodies against HPV66, type II HPV (such as HPV56) and type III HPV (such as HPV53), and its protective effect is mixed with HPV66, VLP of type II, VLP of type II, and VLP of type III of HPV; and its protection effect against HPV66 is equivalent to that of HPV66 alone, and protection effect of type II HPV (such as HPV56) It is equivalent to the HPV VLP of the second type alone, and the protection effect against the HPV of the third type (for example, HPV53) is equivalent to the HPV of the third type alone.
  • the present invention provides a mutant HPV66 L1 protein or a variant thereof, wherein the mutant HPV66 L1 protein has the following mutations compared to the wild-type HPV66 L1 protein:
  • N-terminal is truncated by 1-20 amino acids, such as 1-20 amino acids, such as 1-5, 3-8, 5-10, 9-13, 10-15, 12-18 Or 15-20 amino acids; and
  • the variant differs from the mutant HPV66 L1 protein by only one or a few (e.g., one, two, three, four, five, six, seven, eight, nine (10, 11 or 11) amino acid substitutions (preferably conservative substitutions), additions or deletions, and the function of the mutated HPV66 L1 protein is retained, that is, capable of inducing HPV targeting at least two types (eg, HPV66) And HPV56, or HPV66, HPV56 and HPV53).
  • a few e.g., one, two, three, four, five, six, seven, eight, nine (10, 11 or 11) amino acid substitutions (preferably conservative substitutions), additions or deletions, and the function of the mutated HPV66 L1 protein is retained, that is, capable of inducing HPV targeting at least two types (eg, HPV66) And HPV56, or HPV66, HPV56 and HPV53).
  • the mutated HPV66 L1 protein has the mutation defined in (2) (b), and optionally also has the following mutations:
  • the mutated HPV66 L1 protein has the mutations defined in (2) (b) and (4), and optionally also has the following mutations:
  • the mutant HPV66 L1 protein is truncated by three, five, eight, ten, twelve, fifteen, eighteen, N-terminally compared to the wild-type HPV66 l1 protein. Amino acid.
  • the mutant HPV66 L1 protein is truncated by 5 amino acids at the N-terminus compared to the wild-type HPV66 L1 protein.
  • the second type of wild-type HPV is HPV56.
  • the amino acid residues at the corresponding positions described in (2) (a) are amino acid residues at positions 265-283 of the wild-type HPV56 L1 protein.
  • the amino acid residues at the corresponding positions described in (2) (b) are the amino acid residues at positions 347-357 of the wild-type HPV56 L1 protein.
  • the third type of wild-type HPV is HPV53.
  • the amino acid residues at the corresponding positions described in (3) are the amino acid residues at positions 51-59 of the wild-type HPV53 L1 protein.
  • the amino acid residues at the corresponding positions described in (4) are the amino acid residues at positions 113-149 of the wild-type HPV53 L1 protein.
  • the amino acid residues at the corresponding positions described in (5) are amino acid residues at positions 258-282 of the wild-type HPV53 L1 protein.
  • the amino acid residues at the corresponding positions described in (6) are amino acid residues 171-180 of the wild-type HPV53 L1 protein
  • the wild-type HPV66 L1 protein has an amino acid sequence as shown in SEQ ID NO: 1 or 28.
  • the wild-type HPV56 L1 protein has an amino acid sequence as shown in SEQ ID NO: 2.
  • the wild-type HPV53 L1 protein has an amino acid sequence as shown in SEQ ID NO: 3 or 31.
  • sequence of amino acid residues 347-357 of the wild-type HPV56 L1 protein is shown in SEQ ID NO: 23.
  • sequence of amino acid residues 51 to 59 of the wild-type HPV53 L1 protein is shown in SEQ ID NO: 24.
  • sequence of amino acid residues 113-149 of the wild-type HPV53 L1 protein is shown in SEQ ID NO: 25.
  • sequence of amino acid residues 171-180 of the wild-type HPV53 L1 protein is shown in SEQ ID NO: 65.
  • sequence of amino acid residues 258-282 of the wild-type HPV53 L1 protein is shown in SEQ ID NO: 26.
  • the mutated HPV66 L1 protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 7, 8, 9, 10, 11, and 63.
  • the invention provides an isolated nucleic acid encoding a mutant HPV66 L1 protein or a variant thereof as described above.
  • the invention provides a vector comprising the isolated nucleic acid.
  • the isolated nucleic acid of the invention has a nucleotide sequence selected from the group consisting of SEQ ID NO: 18, 19, 20, 21, 22, and 64.
  • Vectors useful for inserting a polynucleotide of interest include, but are not limited to, cloning vectors and expression vectors.
  • the vector is, for example, a plasmid, cosmid, phage, and the like.
  • the invention also relates to a host cell comprising an isolated nucleic acid or vector as described above.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).
  • the host cell of the invention may also be a cell line, such as a 293T cell.
  • the present invention relates to an HPV virus-like particle, wherein the virus-like particle contains the mutant HPV66 L1 protein or a variant thereof of the invention, or is composed or formed of the mutant HPV66 L1 protein or a variant thereof of the invention .
  • the HPV virus-like particles of the invention comprise a mutated HPV66 L1 protein, which is truncated by 1-20 amino acids at the N-terminus, such as 1-20 amino acids, compared to the wild-type HPV66 L1 protein. , Such as 1-5, 3-8, 5-10, 9-13, 10-15, 12-18, or 15-20 amino acids, and are located at positions 347-357 of the wild-type HPV66 L1 protein Amino acid residues were replaced with amino acid residues 347-357 of the wild-type HPV56 L1 protein.
  • the HPV virus-like particles of the invention comprise a mutated HPV66 L1 protein, which is truncated by 1-20 amino acids at the N-terminus, such as 1-20 amino acids, compared to the wild-type HPV66 L1 protein. , Such as 1-5, 3-8, 5-10, 9-13, 10-15, 12-18, or 15-20 amino acids, and are located at positions 347-357 of the wild-type HPV66 L1 protein Amino acid residues were replaced with wild-type HPV56 amino acid residues at positions 347-357 of wild-type HPV66 and wild-type HPV66 amino acid residues at positions 51-60 of wild-type HPV53 were replaced with wild-type HPV53 proteins at 51-59 Amino acid residues.
  • the HPV virus-like particles of the invention comprise a mutated HPV66 L1 protein, which is truncated by 1-20 amino acids at the N-terminus, such as 1-20 amino acids, compared to the wild-type HPV66 L1 protein. , Such as 1-5, 3-8, 5-10, 9-13, 10-15, 12-18, or 15-20 amino acids, and are located at positions 347-357 of the wild-type HPV66 L1 protein Amino acid residues of wild-type HPV56 L1 protein at positions 347-357, and amino acid residues of wild-type HPV66 L1 protein at positions 114-150 were replaced by wild-type HPV53 L1 protein 113-149 Amino acid residues.
  • the HPV virus-like particles of the invention comprise a mutated HPV66 L1 protein, which is truncated by 1-20 amino acids at the N-terminus, such as 1-20 amino acids, compared to the wild-type HPV66 L1 protein. , Such as 1-5, 3-8, 5-10, 9-13, 10-15, 12-18, or 15-20 amino acids, and are located at positions 347-357 of the wild-type HPV66 L1 protein Amino acid residues were replaced with amino acid residues 347-357 of the wild-type HPV56 L1 protein, and amino acid residues located at positions 259-283 of the wild-type HPV66 L1 protein were replaced with wild-type HPV53 L1 protein 258-282 Amino acid residues.
  • the HPV virus-like particles of the invention comprise a mutated HPV66 L1 protein, which is truncated by 1-20 amino acids at the N-terminus, such as 1-20 amino acids, compared to the wild-type HPV66 L1 protein.
  • the HPV virus-like particle of the present invention comprises a mutated HPV66 L1 protein having the sequences shown in SEQ ID NOs: 7, 8, 9, 10, 11, and 63.
  • the present invention also relates to a composition
  • a composition comprising the mutant HPV66 L1 protein or a variant thereof, or the isolated nucleic acid or vector or host cell or HPV virus-like particle described above.
  • the composition comprises a mutant HPV66 L1 protein of the invention or a variant thereof.
  • the composition comprises an HPV virus-like particle of the invention.
  • the invention also relates to a pharmaceutical composition or vaccine comprising the HPV virus-like particles of the invention, optionally further comprising a pharmaceutically acceptable carrier and / or excipient.
  • the pharmaceutical composition or vaccine of the present invention can be used to prevent HPV infection or diseases caused by HPV infection such as cervical cancer and genital warts.
  • the HPV virus-like particles are present in an effective amount to prevent HPV infection or a disease caused by HPV infection.
  • the HPV infection is one or more types of HPV infections (eg, HPV56 infection, HPV66 infection, and / or HPV53 infection).
  • the disease caused by HPV infection is selected from cervical cancer and genital warts.
  • composition or vaccine of the present invention can be administered by methods known in the art, such as, but not limited to, oral administration or injection.
  • a particularly preferred mode of administration is injection.
  • the pharmaceutical composition or vaccine of the invention is administered in a unit dosage form.
  • the amount of HPV virus-like particles contained in each unit dose is 5 ⁇ g to 80 ⁇ g, preferably 20 ⁇ g to 40 ⁇ g.
  • the present invention relates to a method for preparing a mutant HPV66 L1 protein or a variant thereof as described above, which comprises expressing the mutant HPV66 L1 protein or a variant thereof in a host cell, and The mutant HPV66 L1 protein or a variant thereof is recovered from the host cell culture.
  • the host cell is E. coli.
  • the method includes the steps of: expressing the mutant HPV66 L1 protein or a variant thereof in E. coli, and then purifying the lysed supernatant of the E. coli to obtain the mutant HPV66 L1 protein or a variant thereof.
  • the mutant HPV66L1 is recovered from the lysed supernatant of the E. coli by chromatography (e.g., cation exchange chromatography, hydroxyapatite chromatography, and / or hydrophobic interaction chromatography). Protein or a variant thereof.
  • the invention in another aspect, relates to a method of preparing a vaccine comprising mixing an HPV virus-like particle of the invention with a pharmaceutically acceptable carrier and / or excipient.
  • the present invention relates to a method for preventing HPV infection or a disease caused by HPV infection, comprising administering to a subject a prophylactically effective amount of an HPV virus-like particle or a pharmaceutical composition or vaccine according to the present invention.
  • the HPV infection is one or more types of HPV infection (eg, HPV66 infection, HPV56 infection, and / or HPV53 infection).
  • the diseases caused by HPV infection include, but are not limited to, cervical cancer and genital warts.
  • the subject is a mammal, such as a human.
  • the present invention also relates to the use of the mutated HPV66 L1 protein or a variant thereof or HPV virus-like particles of the present invention in the preparation of a pharmaceutical composition or vaccine for preventing HPV infection or Diseases caused by HPV infection.
  • the HPV infection is one or more types of HPV infection (eg, HPV66 infection, HPV56 infection, and / or HPV53 infection).
  • the diseases caused by HPV infection include, but are not limited to, cervical cancer and genital warts.
  • the invention also relates to the mutated HPV66 L1 protein of the invention or a variant or HPV virus-like particle thereof for use in the prevention of HPV infection or a disease caused by HPV infection.
  • the HPV infection is one or more types of HPV infection (eg, HPV66 infection, HPV56 infection, and / or HPV53 infection).
  • the diseases caused by HPV infection include, but are not limited to, cervical cancer and genital warts.
  • wild type HPV of the second type refers to another type of wild type HPV different from HPV66.
  • the wild type HPV of the second type is preferably wild type HPV56.
  • wild type HPV of the third type refers to another type of wild type HPV different from HPV66 and different from the wild type HPV of the second type.
  • the third type of wild-type HPV is preferably wild-type HPV53.
  • the expression "corresponding position” refers to the equivalent position in the compared sequences when the sequences are optimally aligned, that is, when the sequences are aligned to obtain the highest percentage identity.
  • wild-type HPV66 L1 protein refers to the major capsid protein L1 naturally occurring in human papillomavirus type 66 (HPV66).
  • HPV66 human papillomavirus type 66
  • the sequence of the wild-type HPV66 L1 protein is well known in the art, and can be found in various public databases (for example, NCBI database access ABO76865.1, Q80961.1, ABO76858.1, ALT54961.1, etc.).
  • amino acid sequence of the wild-type HPV66 L1 protein when referring to the amino acid sequence of the wild-type HPV66 L1 protein, the description is made with reference to the sequence shown in SEQ ID NO: 1.
  • amino acid residues 53-56 of the wild-type HPV66 L1 protein refers to amino acid residues 53-56 of the polypeptide represented by SEQ ID NO: 1.
  • wild-type HPV66 may include a variety of isolates, and there may be differences between the amino acid sequences of the L1 proteins of the various isolates.
  • wild-type HPV66 L1 protein includes not only the protein shown in SEQ ID NO: 1, but also the L1 protein of various HPV66 isolates (for example, ABO76865.1, Q80961.1, ABO76858. 1 and ALT54961.1 and other HPV66 L1 proteins).
  • sequence fragments of the wild-type HPV66 L1 protein when describing the sequence fragments of the wild-type HPV66 L1 protein, it includes not only the sequence fragments of SEQ ID NO: 1 but also the corresponding sequence fragments in the L1 protein of various HPV66 isolates.
  • amino acid residues 53-56 of the wild-type HPV66 L1 protein includes amino acid residues 53-56 of SEQ ID NO: 1 and corresponding fragments in the L1 protein of various HPV66 isolates.
  • wild-type HPV56 L1 protein refers to the major capsid protein L1 naturally present in human papillomavirus type 56 (HPV56).
  • HPV56 human papillomavirus type 56
  • the sequence of the wild-type HPV56 L1 protein is well known in the art and can be found in various public databases (for example, NCBI database accession numbers ALT54892.1, ALT54864.1, P36743.1, and ABO76830.1, etc.).
  • amino acid sequence of the wild-type HPV56 L1 protein when referring to the amino acid sequence of the wild-type HPV56 L1 protein, the description is made with reference to the sequence shown in SEQ ID NO: 2.
  • amino acid residues 347-357 of the wild-type HPV56 L1 protein refers to amino acid residues 347-357 of the polypeptide shown by SEQ ID NO: 2.
  • wild-type HPV56 may include various isolates, and there may be differences between the amino acid sequences of the L1 proteins of the various isolates.
  • sequence differences the L1 proteins of different isolates of HPV56 have extremely high amino acid sequence identity (usually higher than 95%, such as higher than 96%, higher than 97%).
  • wild-type HPV56 L1 protein includes not only the protein shown in SEQ ID NO: 2 but also the L1 protein of various HPV56 isolates (for example, ALT54892.1, ALT54864.1, P36743. 1 and HPV56 L1 protein shown in ABO76830.1, etc.). Furthermore, when describing the sequence fragments of the wild-type HPV56 L1 protein, it includes not only the sequence fragments of SEQ ID NO: 2 but also the corresponding sequence fragments in the L1 protein of various HPV56 isolates. For example, the expression "amino acid residues 347-357 of the wild-type HPV56 L1 protein” includes amino acid residues 347-357 of SEQ ID NO: 2 and corresponding fragments in the L1 protein of various HPV56 isolates.
  • wild-type HPV53 L1 protein refers to the major capsid protein L1 naturally occurring in human papillomavirus type 53 (HPV53).
  • HPV53 human papillomavirus type 53
  • sequence of the wild-type HPV53 L1 protein is well known in the art, and can be found in various public databases (for example, NCBI database accession numbers NP041848.1, ANY26596.1, ABU54090.1, ALJ32506.1, etc.).
  • amino acid residues 51 to 59 of the wild-type HPV53 L1 protein means amino acid residues 51 to 59 of the polypeptide represented by SEQ ID NO: 3.
  • wild-type HPV53 may include a variety of isolates, and there may be differences between the amino acid sequences of the L1 proteins of the various isolates.
  • wild-type HPV53 L1 protein includes not only the protein shown in SEQ ID NO: 3, but also L1 proteins of various HPV53 isolates (for example, NP041848.1, ANY26596.1, ABU54090. 1 and HPV53 L1 protein shown by ALJ32506.1).
  • sequence fragments of the wild-type HPV53 L1 protein when describing the sequence fragments of the wild-type HPV53 L1 protein, it includes not only the sequence fragments of SEQ ID NO: 3, but also the corresponding sequence fragments in the L1 protein of various HPV53 isolates.
  • amino acid residues 51-59 of the wild-type HPV53L1 protein includes amino acid residues 51-59 of SEQ ID NO: 3 and corresponding fragments in the L1 protein of various HPV53 isolates.
  • corresponding sequence fragment or “corresponding fragment” means that when the sequences are optimally aligned, that is, when the sequences are aligned to obtain the highest percent identity, the compared sequences are located at equivalent positions Fragment.
  • the expression "the N-terminus is truncated by X amino acids” means that the methionine residue encoded by the start codon (for initiating protein translation) is used to replace the amino acids 1-X at the N-terminus of the protein. Residues.
  • HPV66 L1 protein truncated by 5 amino acids at the N-terminus is obtained by replacing the amino acid residues 1 to 5 at the N-terminus of wild-type HPV66 L1 protein with a methionine residue encoded by the start codon. protein.
  • the term "variant” refers to a protein whose amino acid sequence is the same as that of the mutated HPV66 L1 protein of the present invention (such as the proteins shown in SEQ ID NO: 7, 8, 9, 10, and 11).
  • substitutions preferably conservative substitutions
  • the term "function of mutated HPV66 L1 protein” refers to the ability to induce the body to produce neutralizing antibodies against at least two types of HPV (eg, HPV66 and HPV56, or HPV66, HPV56, and HPV53).
  • identity is a measure of the similarity of a nucleotide sequence or an amino acid sequence. Sequences are usually arranged for maximum matching. "Identity” itself has a well-known meaning in the art and can be calculated using published algorithms such as BLAST.
  • the term "identity" is used to refer to a sequence match between two polypeptides or between two nucleic acids.
  • a position in two compared sequences is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or two Each position of the polypeptide is occupied by lysine)
  • the molecules are identical at that position.
  • the "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of compared positions x 100. For example, if 6 of the 10 positions of two sequences match, the two sequences are 60% identical.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 positions out of a total of 6 positions match).
  • comparisons are made when two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc).
  • Align program DNAstar, Inc.
  • the algorithm of E.Meyers and W.Miller Comput.Appl.
  • the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithm integrated into the GAP program of the GCG software package can be used, using the Blossom 62 matrix or PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences .
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the necessary properties of a protein / polypeptide comprising an amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as those that are physically or functionally similar to the corresponding amino acid residue (e.g., have similar size, shape, charge, chemical properties, including The ability to form covalent or hydrogen bonds, etc.).
  • a family of amino acid residues with similar side chains has been defined in the art.
  • These families include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), and uncharged polar side chains (e.g., glycine). , Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g.
  • a conservative substitution generally refers to replacing the corresponding amino acid residue with another amino acid residue from the same side chain family.
  • Methods for identifying conservative substitutions of amino acids are well known in the art (see, eg, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12 (10): 879-884 (1999) ; And Burks et al. Proc. Natl. Acad. Set USA 94: 412-417 (1997), which is incorporated herein by reference).
  • E. coli expression system refers to an expression system composed of E. coli (strain) and a vector, wherein E. coli (strain) is derived from a commercially available strain, such as, but not limited to: ER2566, BL21 ( DE3), B834 (DE3), BLR (DE3).
  • the term "vector” refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • a vector When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector.
  • a vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic material elements carried by the vector can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phages; cosmids and the like.
  • the term "pharmaceutically acceptable carrier and / or excipient” refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient and is in the art It is well known (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th Ed., Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers.
  • pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or non-ionic surfactants, such as Tween-80; adjuvants include, but are not limited to, aluminum adjuvants (such as hydroxide Aluminum), Freund's adjuvant (eg, complete Freund's adjuvant); ionic strength enhancers include, but are not limited to, sodium chloride.
  • surfactants include, but are not limited to, cationic, anionic, or non-ionic surfactants, such as Tween-80
  • adjuvants include, but are not limited to, aluminum adjuvants (such as hydroxide Aluminum), Freund's adjuvant (eg, complete Freund's adjuvant); ionic strength enhancers include, but are not limited to, sodium chloride.
  • an effective amount refers to an amount effective to achieve the intended purpose.
  • an effective amount for preventing a disease e.g., HPV infection
  • an effective amount effective to prevent, prevent, or delay the occurrence of a disease e.g., HPV infection. Determining such an effective amount is well within the capabilities of those skilled in the art.
  • chromatographic chromatography includes, but is not limited to: ion exchange chromatography (such as cation exchange chromatography), hydrophobic interaction chromatography, adsorption chromatography (such as hydroxyapatite chromatography), gel filtration (gel chromatography Resistance) chromatography, affinity chromatography.
  • the term "lysed supernatant” refers to a solution produced by the steps of: disrupting a host cell (eg, E. coli) in a lysate, and then insoluble matter in the lysate containing the disrupted host cell Remove.
  • a host cell eg, E. coli
  • Various lysates are well known to those skilled in the art, including but not limited to Tris buffer, phosphate buffer, HEPES buffer, MOPS buffer and the like.
  • host cell disruption can be achieved by various methods well known to those skilled in the art, including, but not limited to, homogenizer disruption, homogenizer disruption, ultrasonic treatment, grinding, high-pressure extrusion, lysozyme treatment, and the like. Methods for removing insolubles in the lysate are also well known to those skilled in the art, including but not limited to filtration and centrifugation.
  • the invention provides a mutated HPV66 L1 protein and HPV virus-like particles formed therefrom.
  • the HPV virus-like particles of the present invention are capable of providing significant cross-protection capabilities between HPV66 and other types of HPV, such as HPV56 and HPV53.
  • the HPV virus-like particles of the present invention can induce the body to produce high titer neutralizing antibodies against at least two types of HPV (eg, HPV66 and HPV56, or HPV66, HPV56 and HPV53), And the effect is equivalent to a mixture of multiple types of HPV VLP (for example, a mixture of HPV66 VLP and HPV56 VLP, or a mixture of HPV66 VLP, HPV56 VLP, and HPV53 VLP).
  • the HPV virus-like particles of the present invention can be used to prevent infection of at least two types of HPV (for example, HPV66 and HPV56, or HPV66, HPV56, and HPV53) and related diseases at the same time, and have significant advantageous technical effects. .
  • HPV66 and HPV56 for example, HPV66 and HPV56, or HPV66, HPV56, and HPV53
  • HPV53 HPV66, HPV56, and HPV53
  • FIG. 1 shows the results of SDS polyacrylamide gel electrophoresis of the purified mutein in Example 1.
  • FIG. Lane 1 Protein molecular weight marker
  • Lane 2 HPV66N5 (HPV66 L1 protein with N-terminal truncated 5 amino acids);
  • Lane 3 HPV56N0 (full-length HPV56 L1 protein);
  • Lane 4 H66N5-56T1;
  • Lane 5 H66N5 -56T2;
  • lane 6 H66N5-56T3;
  • lane 7 H66N5-56T4;
  • lane 8 H66N5-56T5;
  • lane 9 protein molecular weight marker;
  • lane 10 H66N5-56T5;
  • lane 11 HPV53N5 (N-terminal truncated by 5 Amino acid HPV53L1 protein);
  • lane 12 H66N5-56T5-53S1;
  • lane 13 H66N5-56T5-53S2
  • Figure 2 shows detection of the mutant proteins H66N5-56T1, H66N5-56T2, H66N5-56T3, H66N5-56T4, H66N5-56T5, H66N5-56T5-53S1, H66N5-56T5-, and HPV L1 broad-spectrum antibody 4B3. Results of Western blot detection of 53S2, H66N5-56T5-53S4.
  • Lane 1 HPV66N5 (HPV66 L1 protein with N-terminal truncated 5 amino acids); Lane 2: HPV56N0 (full-length HPV56 L1 protein); Lane 3: H66N5-56T1; Lane 4: H66N5-56T2; Lane 5: H66N5 -56T3; Lane 6: H66N5-56T4; Lane 7: H66N5-56T5; Lane 8: H66N5-56T5; Lane 9: HPV53N5 (HPV53L1 protein with N-terminal truncated 5 amino acids); Lane 10: H66N5-56T5-53S1 Lane 11: H66N5-56T5-53S2; Lane 12: H66N5-56T5-53S4 The results show that the mutant proteins H66N5-56T1, H66N5-56T2, H66N5-56T3, H66N5-56T4, H66N5-56T5, H66N5-56T5-53S
  • Figure 3 shows molecular sieve chromatography analysis of samples containing proteins H66N5-56T1, H66N5-56T2, H66N5-56T3, H66N5-56T4, H66N5-56T5, HPV56N0, and HPV66N5 (HPV66L1 protein with N-terminal truncated 5 amino acids) result.
  • the results showed that the first protein peaks in the samples containing proteins H66N5-56T1, H66N5-56T2, H66N5-56T3, H66N5-56T4, and H66N5-56T5 were about 13-14min, which were comparable to HPV56N0 and HPV66N5 VLP. This indicates that these proteins can be assembled into VLPs.
  • Figure 4 shows the results of molecular sieve chromatography analysis of samples containing proteins H66N5-56T5, HPV53N5, H66N5-56T5-53S1, H66N5-56T5-53S2, H66N5-56T5-53S4, H66N5-56T5-53S2-53S3.
  • the results showed that the first protein peak of the samples containing the proteins H66N5-56T5 and H66N5- 56T5-53S4 was about 13-14 min, which was equivalent to HPV53N5.
  • the peak shapes of H66N5-56T5-53S1 and H66N5-56T5-53S2 are asymmetric, and the peak time of H66N5-56T5-53S1 is about 15 minutes, indicating that the diameter of VLP particles formed by H66N5-56T5-53S1 is smaller, and H66N5-56T5- The particles formed by 53S2 and H66N5-56T5-53S2-53S3 are not uniform.
  • Figure 5 shows the results of sedimentation rate analysis of H66N5-56T1 VLP, H66N5-56T2 VLP, H66N5-56T3 VLP, H66N5-56T4 VLP, H66N5-56T5 VLP, HPV56N0VLP, and HPV66N5 VLP.
  • VLP settlement coefficients of H66N5-56T1 VLP, H66N5-56T2, VLP, H66N5-56T3, VLP, H66N5-56T4, VLP, H66N5-56T5, VLP are 126S, 125S, 126S, 127S, and 128S, respectively, and the settlement of HPV56N0VLP and HPV66N5VLP
  • the coefficients are 134S and 141S, respectively.
  • VLPs can be assembled into virus-like particles that are similar in size and shape to wild-type VLPs.
  • Figure 6 shows the results of the sedimentation rate analysis of H66N5-56T5 VLP, HPV53N5 VLP, H66N5-56T5-53S1 VLP, H66N5-56T5-53S2 VLP, H66N5-56T5-53S4 VLP, and H66N5-56T5-53S2-53S3.
  • H66N5-56T5-53S4 VLPs can be assembled into virus-like particles that are similar in size and shape to wild-type VLPs; while H66N5-56T5-53S1, H66N5-56T5-53S2VLP and H66N5-56T5-53S2-53S3VLP are virus-like particles.
  • the diameter is small and the particles formed by H66N5-56T5-53S2 and H66N5-56T5-53S2-53S3 are not uniform.
  • Figure 7A VLP assembled by HPV66N5
  • Figure 7B VLP assembled by HPV56N0
  • Figure 7C VLP assembled by H66N5-56T1
  • Figure 7D VLP formed by H66N5-56T2
  • Figure 7E VLP assembled by H66N5-56T3
  • Figure 7F VLP assembled by H66N5-56T4
  • Figure 7G VLP assembled by H66N5-56T5
  • Figure 7H VLP assembled by HPV53N5
  • Figure 7I VLP assembled by H66N5-56T5-53S1, with smaller particle diameter
  • Figure 7J VLP assembled by H66N5-56T5-53S2
  • Figure 7K VLP assembled by H66N5-56T5-53S4
  • Figure 7L VLP assembled by H66N5-56T5-53S2-53S3; The results
  • Figures 8A-8C show experimental groups H66N5-56T1 VLP, H66N5-56T2 VLP, H66N5-56T3 VLP, H66N5-56T4 VLP, H66N5-56T5 VLP, and control groups HPV56N0 VLP and HPV66N5 VLP and mixed HPV56 / HPV66 Results of evaluation of immunoprotection in mice.
  • H66N5-56T4 VLP and H66N5-56T5 VLP can induce high titers of neutralizing antibodies against HPV66 and HPV56 in mice; and their protection against HPV66 is comparable to that of HPV66N5 VLP alone and significantly higher than HPV56N0VLP alone; and its protective effect against HPV56 is comparable to that of HPV56N0VLP alone, and significantly higher than that of HPV66N5 VLP alone.
  • H66N5-56T4 VLP and H66N5-56T5 VLP can be used as effective vaccines to prevent HPV66 infection and HPV56 infection, and can be used to replace the mixed vaccine containing HPV66VLP and HPV56VLP.
  • Figures 9A-9B show the experimental group H66N5-56T5-53S1 VLP, H66N5-56T5-53S2, VLP and H66N5-56T5-53S4 VLP, and the control group HPV66N5, VLP, HPV56N0VLP, HPV53N5, VLP and mixed HPV66 / 56/53 VLP in Results of evaluation of immunoprotection in mice.
  • H66N5-56T5-53S1 VLP, H66N5-56T5-53S2, VLP and H66N5-56T5-53S4 VLP can induce high titers of neutralizing antibodies against HPV66, HPV56 and HPV53 in mice; and its protection against HPV66
  • the effect is equivalent to HPV66N5 VLP alone, mixed HPV66 / 56/53 VLP, and significantly higher than HPV56N0VLP alone and HPV53N5VLP; and its protection against HPV56 is similar to HPV56N0 VLP alone, mixed HPV66 / 56/53 VLP Equivalent and significantly higher than HPV66N5 VLP and HPV53N5 VLP alone; and its protective effect against HPV53 is comparable to HPV53N5 VLP alone, mixed HPV66 / 56/53 VLP, and significantly higher than HPV66N5 VLP alone and HPV56N0 VLP.
  • H66N5-56T5-53S1 VLP, H66N5-56T5-53S2, VLP, and H66N5-56T5-53S4 VLP can be used as an effective vaccine to prevent HPV66 infection, HPV56 infection, and HPV53 infection, and can be used instead of HPV66VLP, HPV56, VLP, and HPV53 VLP Mixed vaccine.
  • Fig. 10 shows the results of the immunoprotective evaluation of the experimental group H66N5-56T5-53S2-53S3 and the control group HPV66N5VLP, HPV56N0VLP, HPV53N5VLP, and mixed HPV66 / 56/53 VLP in mice.
  • H66N5-56T5-53S2-53S3 VLP can induce high titers of neutralizing antibodies against HPV66, HPV56 and HPV53 in mice; and its protective effect against HPV66 is comparable to HPV66N5 VLP alone and mixed HPV66 / 56 / 53 VLP is equivalent and significantly higher than HPV56N0VLP and HPV53N5VLP alone; its protection against HPV56 is slightly weaker than HPV56N0 VLP alone, HPV66N5 VLP alone, mixed HPV66 / 56/53 VLP, but significantly higher than VLP alone HPV53N5 VLP; its protection against HPV53 is slightly weaker than HPV53N5 VLP alone, mixed HPV66 / 56/53 VLP, but significantly higher than HPV66N5 VLP alone and HPV56N0 VLP alone.
  • H66N5-56T5-53S2-53S3 VLP can be used as an effective vaccine to prevent HPV66 infection, HPV56 infection, and HPV53 infection, and can be used instead of a mixed vaccine containing HPV66VLP, HPV56 VLP, and HPV53 VLP.
  • Sequence 7 (SEQ ID NO: 7):
  • Sequence 8 (SEQ ID NO: 8):
  • Sequence 9 (SEQ ID NO: 9):
  • the molecular biology experimental methods and immunoassays used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FMAusubel et al., Compilation of a Guide to Molecular Biology Experiments, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes was in accordance with conditions recommended by the product manufacturer.
  • restriction enzymes was in accordance with conditions recommended by the product manufacturer.
  • a multi-point mutation PCR reaction was used to construct an expression vector encoding the HPV66 L1 protein containing a mutation derived from the HPV56 L1 protein segment.
  • the initial template used was the pTO-T7-HPV66N5C plasmid (which encodes a truncated N-terminus) 5 amino acid HPV66 L1 protein; abbreviated 66L1N5 in Table 2).
  • the templates and primers used for each PCR reaction are shown in Table 2, and the amplification conditions of the PCR reaction are set to: 94 ° C denaturation for 2 minutes; 30 cycles (98 ° C denaturation for 10 seconds, specified temperature annealing for a certain time, 58 ° C extension) 6 minutes and 30 seconds); the last 68 ° C extension for 10 minutes.
  • the annealing temperatures and times are listed in Table 2.
  • the specific sequences of the PCR primers used are listed in Table 3.
  • coli was coated on a solid LB medium containing kanamycin (final concentration 25 ⁇ g / mL, hereinafter the same) (LB medium components: 10 g / L peptone, 5 g / L yeast powder, 10 g / L chlorine Sodium sulphate, the same below), and allowed to stand at 37 ° C for 10-12 hours until a single colony is clearly distinguishable. A single colony was picked into a test tube containing 4 mL of liquid LB medium (containing kanamycin), and cultured with shaking at 37 ° C and 220 rpm for 10 hours. Subsequently, 1 mL of the bacterial solution was stored at -70 ° C. The plasmid was extracted from E.
  • LB medium components 10 g / L peptone, 5 g / L yeast powder, 10 g / L chlorine Sodium sulphate, the same below
  • nucleotide sequence of the target fragment inserted in the plasmid was sequenced using T7 primers. Sequencing results showed that the nucleotide sequence of the target fragment inserted in each constructed plasmid (expression vector) was SEQ ID NO: 15, 17 and its encoded amino acid sequence was SEQ ID NO: 4, 6 (the corresponding proteins Named H66N5-56T1 and H66N5-56T3 respectively.
  • mutant protein H66N5-56T1 and HPV66N5 are replaced with amino acid residues at positions 53-56 of the wild-type HPV66L1 protein.
  • mutant protein H66N5-56T3 and HPV66N5 is that the amino acid residues at positions 178-180 of the wild-type HPV66L1 protein are replaced with amino acid residues at positions 178-180 of the wild-type HPV56L1 protein.
  • the templates and primers used for each PCR reaction are shown in Table 2, and the amplification conditions for PCR reactions used to amplify short fragments were set as follows: denaturation at 94 ° C for 10 minutes; 25 cycles (denaturation at 94 ° C for 50 seconds, specified temperature) Anneal for a certain time, extending at 72 ° C for 1 minute); finally, extending at 72 ° C for 10 minutes.
  • the amplification conditions for the PCR reaction for amplifying long fragments are set as follows: denaturation at 94 ° C for 10 minutes; 25 cycles (denaturation at 94 ° C for 50 seconds, annealing at a specified temperature for a certain time, and extension at 72 ° C for 7 minutes and 30 seconds); finally 72 Extend for 10 minutes.
  • the specific sequences of the PCR primers used are listed in Table 3.
  • the amplified product was subjected to electrophoresis, and then a target fragment was recovered using a DNA recovery kit (BEYOTIME, article number: D0033) and its concentration was measured.
  • the assembled product (6 ⁇ L) was used to transform 40 ⁇ L of competent E. coli ER2566 (purchased from New England Biolabs) prepared by the calcium chloride method.
  • the transformed E. coli was spread on a kanamycin-containing solid LB medium, and cultured at 37 ° C for 10-12 hours until a single colony was clearly distinguishable.
  • a single colony was picked into a test tube containing 4 mL of liquid LB medium (containing kanamycin), and cultured with shaking at 37 ° C and 220 rpm for 10 hours. Subsequently, 1 mL of the bacterial solution was stored at -70 ° C.
  • the plasmid was extracted from E.
  • nucleotide sequence of the target fragment inserted in the plasmid was sequenced using T7 primers. Sequencing results showed that the nucleotide sequences of the target fragments inserted in each constructed plasmid (expression vector) were SEQ ID NO: 16, 18, 19, 20, 21, 22, and the encoded amino acid sequence was SEQ ID NO : 5, 7, 8, 9, 10, 11 (the corresponding proteins are named H66N5-56T2, H66N5-56T4, H66N5-56T5, H66N5-56T5-53S1, H66N5-56T5-53S2, H66N5-56T5-53S4) .
  • the H66N5-56T5-53S2-53S3 gene was synthesized by Shanghai Shenggong Biological Company.
  • the synthesized plasmid containing the HPV66N5-56T5-53S2-53S3 gene was digested with NdeI and SalI (37 ° C, 12 hours).
  • the gene was recovered using a DNA recovery kit (BEYOTIME, article number: D0033).
  • the H66N5-56T5-53S2-53S3 gene was digested with the pTO-T7 vector at 16 ° C for 12 hours, and then transformed into competent cells.
  • E. coli ER2566 purchased from New England Biolabs
  • the transformed Escherichia coli was spread on a solid LB medium containing kanamycin, and cultured at 37 ° C for 10-12 hours until a single colony was clearly distinguishable.
  • the plasmid was extracted from E. coli, and the nucleotide sequence of the target fragment inserted in the plasmid was sequenced using T7 primers.
  • mutant protein H66N5-56T2 and HPV66N5 are replaced with amino acid residues at positions 130-150 of the wild-type HPV66 L1 protein.
  • mutant protein H66N5-56T4 and HPV66N5 is that the amino acid residues at positions 265-283 of the wild-type HPV66 L1 protein are replaced with amino acid residues at positions 265-283 of the wild-type HPV56 L1 protein.
  • mutant protein H66N5-56T5 and HPV66N5 is that the amino acid residues at positions 347-357 of the wild-type HPV66 L1 protein are replaced with amino acid residues at positions 347-357 of the wild-type HPV56 L1 protein.
  • mutant protein H66N5-56T5-53S1 and HPV66N5 is that the amino acid residues at positions 347-357 of the wild-type HPV66 L1 protein are replaced with amino acid residues at positions 347-357 of the wild-type HPV56 L1 protein and are located in the wild-type
  • amino acid residues 51 to 60 of the HPV66 L1 protein were replaced with amino acid residues 51 to 59 of the wild-type HPV53 L1 protein.
  • mutant protein H66N5-56T5-53S2 and HPV66N5 is that the amino acid residues at positions 347-357 of the wild-type HPV39 L1 protein are replaced with amino acid residues at positions 347-357 of the wild-type HPV56 L1 protein and are located in the wild type
  • amino acid residues at positions 114-150 of the HPV66 L1 protein were replaced with amino acid residues at positions 113-149 of the wild-type HPV53 L1 protein.
  • mutant protein H66N5-56T5-53S4 and HPV66N5 is that the amino acid residues at positions 347-357 of the wild-type HPV66 L1 protein are replaced with amino acid residues at positions 347-357 of the wild-type HPV56 L1 protein and are located in the wild-type
  • amino acid residues at positions 259-283 of the HPV66 L1 protein were replaced with amino acid residues at positions 258-282 of the wild-type HPV53 L1 protein.
  • mutant protein H66N5-56T5-53S2-53S3 and HPV66N5 is that the amino acid residues at positions 347-357 of the wild-type HPV66L1 protein are replaced with amino acid residues at positions 347-357 of the wild-type HPV56 L1 protein, and are located at The amino acid residues at positions 114-150 of the wild-type HPV66 L1 protein were replaced with amino acid residues at positions 113-149 of the wild-type HPV53 L1 protein, and the amino acid residues at positions 172-181 of the wild-type HPV66 L1 protein were replaced by Replaced with amino acid residues 171-180 of the wild-type HPV53 L1 protein.
  • coli bacterial solution In 100ml kanamycin-containing LB liquid medium, incubate at 200rpm, 37 ° C for about 8 hours; then transfer to 500ml kanamycin-containing LB medium (1ml bacterial solution), and continue Cultivate. When the bacterial concentration reached an OD 600 of about 0.6, the culture temperature was lowered to 25 °C, and each culture flask was added 500 ⁇ L IPTG, incubation was continued for 8 hours. After the culture was completed, the cells were collected by centrifugation.
  • the cells obtained above were resuspended in a proportion of 1 g of cells corresponding to 10 mL of lysate (20 mM Tris buffer, pH 7.2, 300 mM NaCl).
  • the bacterial cells were disrupted with an ultrasonic instrument for 30 min.
  • the lysate containing the disrupted bacterial cells was centrifuged at 13,500 rpm (30000 g) for 15 min, and the supernatant was taken (that is, the bacterial cell broken supernatant).
  • Instrument system AKTA explorer 100 preparative liquid chromatography system produced by GE Healthcare (formerly Amershan Pharmacia).
  • Chromatographic media SP Sepharose 4 Fast Flow (GE Healthcare), CHT-II (purchased from Bio-RAD) and Butyl Sepharose 4 Fast Flow (GE Healthcare).
  • Buffer Buffer A (20 mM phosphate buffer, pH 8.0, 20 mM DTT); and Buffer B (20 mM phosphate buffer, pH 8.0, 20 mM DTT, 2M NaCl).
  • Buffer A (20 mM phosphate buffer, pH 8.0, 20 mM DTT)
  • Buffer B (20 mM phosphate buffer, pH 8.0, 20 mM DTT, 2M NaCl).
  • the buffers containing different concentrations of NaCl used in the following elution procedure are prepared by mixing buffers A and B in proportion.
  • H66N5-56T4 H66N5-56T5, H66N5-56T5-53S1, H66N5-56T5-53S2,
  • H66N5-56T5-53S4 H66N5-56T5-53S2-53S3 shattered supernatant.
  • step (3) Take 150 ⁇ L of the eluted fraction obtained in step (3), add 30 ⁇ L of 6X Loading buffer (1M contains 1M TB, 6.8300ml, 100% glycerol, 600ml, SDS, 120g, bromophenol blue, 6g, ⁇ -mercaptoethanol, 50ml), and mix well. And incubate for 10 min in a water bath at 80 ° C. Then 10 ⁇ l of the sample was electrophoresed in a 10% SDS-polyacrylamide gel at 120 V for 120 min; then electrophoretic bands were displayed by Coomassie blue staining. The electrophoresis results are shown in FIG. 1.
  • HPV66N5 protein was prepared and purified using E. coli and pTO-T7-HPV66N5 plasmid;
  • HPV56N0 protein was prepared and purified using E. coli and pTO-T7-HPV56L1N0 plasmid; and
  • E. coli and pTO-T7-HPV53N5 plasmid were used to prepare and purify And purified HPV53N5 protein.
  • H66N5-56T1, H66N5-56T2, H66N5-56T3, H66N5-56T4, H66N5-56T5, H66N5-56T5-53S1, H66N5-56T5-53S2, H66N5-56T5-53S4 proteins were electrophoresed as described above. After the end of the electrophoresis, Western Blot detection was performed using a broad-spectrum antibody 4B3 against HPV L1 protein, and the results are shown in FIG. 2.
  • mutant proteins H66N5-56T1, H66N5-56T2, H66N5-56T3, H66N5-56T4, H66N5-56T5, H66N5-56T5-53S1, H66N5-56T5-53S2, and H66N5-56T5-53S4 were all specific to the broad-spectrum antibody 4B3. Sexual recognition.
  • Example 2 Assembly and particle morphology detection of HPV virus-like particles
  • HPV66N5, HPV56N0 and HPV53N5 proteins were assembled into HPV66N5, VLP, HPV56N0, VLP and HPV53N5, respectively.
  • the 1120Compact LC high-performance liquid chromatography system of Agilent Corporation was used to perform molecular sieve chromatography analysis on the dialyzed samples.
  • the analytical column used was TSK Gel PW5000xl7.8x300mm.
  • the analysis results are shown in Figures 3 and 4. The results showed that the protein peaks of the samples containing the proteins H66N5-56T1, H66N5-56T2, H66N5-56T3, H66N5-56T4, H66N5-56T5, H66N5-56T5-53S4 all appeared at about 13-14 minutes, which were similar to HPV66N5 VLP and HPV56N0.
  • VLP and HPV53N5 and VLP are comparable, which indicates that these proteins can be assembled into VLPs; H66N5-56T5-53S2-53S3; the peak shapes of VLP, H66N5-56T5-53S1, and H66N5-56T5-53S2 are asymmetric, and the shape of H66N5-56T5-53S1 is asymmetric.
  • the peak time was about 15 minutes, indicating that the diameter of VLP particles formed by H66N5-56T5-53S1 was smaller, and the particles formed by H66N5-56T5-53S2 and H66N5-56T5-53S2-53S3 were not uniform.
  • the instrument used for sedimentation rate analysis is a Beckman XL-A analytical ultracentrifuge, which is equipped with an optical detection system and An-50Ti and An-60Ti rotors.
  • the results are shown in Figure 5 and Figure 6.
  • the results show that H66N5-56T1 VLP, H66N5-56T2 VLP, H66N5-56T3 VLP, H66N5-56T4 VLP, H66N5-56T5 VLP, H66N5-56T5-53S1 VLP, H66N5-56T5-53S2
  • the settlement coefficients of VLP and H66N5-56T5-53S4 are VLPs of 126S, 125S, 126S, 127S, 128S, 93S, 106S, and 116S.
  • the settlement coefficients of VLP are 53S and 107S, HPV56N0, VLP, HPV66N5, VLP.
  • the settlement coefficients of HPV53N5 and VLP are 134S and 141S and 130S, respectively.
  • H66N5-56T1 VLP, H66N5-56T2, VLP, H66N5-56T3, VLP, H66N5-56T4, VLP, H66N5-56T5, VLP, and H66N5-56T5-53S4 VLP can be assembled into size, shape, and wild-type VLP (HPV56N0VLP, HPV66N5, VLP and HPV53N5 (VLP) similar virus-like particles; while H66N5-56T5-53S1, H66N5-56T5-53S2, VLP and H66N5-56T5-53S2-53S3, VLPs have smaller diameters, and H66N5-56T5-53S2, VLP and H66N5-56T5- 53S2-53S3 The particle size of VLP is not uniform.
  • a 100 ⁇ L sample containing VLP was observed by transmission electron microscopy.
  • the instrument used is a 100kV transmission electron microscope produced by Japan Electronics Corporation, with a magnification of 100,000 times. Briefly, a 13.5 ⁇ L sample was taken, negatively stained with 2% phosphotungstic acid, pH 7.0, and fixed on a carbon sprayed copper mesh, followed by transmission electron microscope observation. The observation results are shown in Figures 7A-7L.
  • H66N5-56T1, H66N5-56T2, H66N5-56T3, H66N5-56T4, H66N5-56T5, H66N5-56T5-53S2, H66N5-56T5-53S4, H66N5-56T5-53S2-53S3 can be assembled into virus-like particles.
  • the results show that the particles formed by the assembly of H66N5-56T1, H66N5-56T2, H66N5-56T3, H66N5-56T4, H66N5-56T5, H66N5-56T5-53S4 have a radius of about 25nm and a uniform size, which indicates that these The mutant proteins are similar to the L1 proteins of HPV66, HPV56, and HPV53, and can form VLPs of uniform size.
  • H66N5-56T5-53S2 and H66N5-56T5-53S2-53S3 can also form virus-like particles with a radius of about 25nm, but they contain small particles with a diameter of about 20nm; while H66N5-56T5-53S1 can form a radius of about 10-20nm. VLP with irregular particles.
  • Example 3 Evaluation of neutralizing antibody titers in mouse serum after immunization with virus-like particles 1
  • virus-like particles used were: H66N5-56T1 VLP, H66N5-56T2 VLP, H66N5-56T3 VLP, H66N5-56T4 VLP, H66N5-56T5 VLP.
  • mice (6-week-old BalB / c female mice) were divided into 3 groups: aluminum adjuvant group 1 (immunized dose of 5 ⁇ g, using aluminum adjuvant), aluminum adjuvant group 2 (immunized dose of 1 ⁇ g, using Aluminum adjuvant), and aluminum adjuvant group 3 (immunized dose of 0.2 ⁇ g, using aluminum adjuvant). Each group was subdivided into 8 subgroups.
  • Control subgroups 1 and 2 were immunized with separate HPV66N5 VLP and HPV56N0 VLP, respectively, and control subgroup 3 was mixed with HPV66 / HPV56 VLP (that is, HPV66N5 VLP and HPV56N0 VLP). Mixture, where each VLP is administered at a specified immunization dose).
  • the experimental subgroups 1, 2, 3, 4, and 5 were immunized with H66N5-56T1VLP, H66N5-56T2VLP, H66N5-56T3VLP, H66N5-56T4VLP, and H66N5-56T5 VLP, respectively.
  • mice / subgroup were immunized by intraperitoneal injection, the immunization doses were 5 ⁇ g, 1 ⁇ g, 0.2 ⁇ g, and the injection volume was 1 ml. All mice were initially immunized at week 0 and then boosted once at weeks 2 and 4 each. Orbital blood was collected from the mice at week 6, and the titers of anti-HPV66 and HPV56 antibodies in the serum were analyzed. The analysis results are shown in Figures 8A-8C.
  • H66N5-56T4 VLP and H66N5-56T5 VLP can induce high titer neutralizing antibodies against HPV66 in mice, and its protective effect is equivalent to that of HPV66N5 VLP alone at the same dose, and significantly better than that of the same dose alone.
  • HPV56N0 VLP and it can induce mice to produce high titers of neutralizing antibodies against HPV56, its protective effect is equivalent to the same dose of HPV56N0 VLP alone, and significantly better than the same dose of HPV66N5 VLP alone.
  • H66N5-56T4 VLP and H66N5-56T5 VLP have good cross-immunogenicity and cross-protection against HPV66 and HPV56.
  • Example 4 Evaluation of particle-induced seroconversion ED 50 of a virus-like
  • virus-like particles used were H66N5-56T4 VLP and H66N5-56T5 VLP.
  • 6-week-old BalB / c female rats (8) were immunized with aluminum adjuvant and single intraperitoneal injection.
  • the experimental group used H66N5-56T4 VLP and H66N5-56T5 VLP
  • the control group used HPV56N0 VLP or HPV66N5 VLP alone or mixed HPV66 / HPV56 VLP (ie, a mixture of HPV66N5 VLP and HPV56N0 VLP);
  • the immune dose was 0.900 ⁇ g, 0.300 ⁇ g, 0.100 ⁇ g, 0.033 ⁇ g, or 0.011 ⁇ g; the immune volume was 1 mL.
  • the dilution used to dilute the vaccine was used as a blank control.
  • mice Eight mice were immunized in each group, and in the fifth week after immunization, ocular venous blood was drawn to detect HPV antibodies in the serum, and the Reed-Muench method (Reed LJ MH.A simple method of estimating fifty percent endpoints. Am J Hyg.1938; 27: 493-7) calculates ED induce seroconversion of each sample (i.e., an antibody induced in mice) of 50. The results are shown in Table 5-9.
  • H66N5-56T5 VLP mice induce anti-HPV66 antibodies ED 50 is quite HPV66N5 VLP alone, and significantly better than single HPV56N0 VLP; and that anti-HPV56 induced in mice ED 50 antibody alone, although weaker in HPV56N0 VLP, but significantly better than separate HPV66N5 VLP. This indicates that the H66N5-56T5 VLP has good cross-immunogenicity and cross-protection against HPV66 and HPV56.
  • Example 5 Evaluation of neutralizing antibody titers in mouse serum after immunization with virus-like particles 2
  • virus particles used were H66N5-56T5-53S1 VLP, H66N5-56T5-53S2 VLP, and H66N5-56T5-53S4 VLP.
  • mice (6-week-old BalB / c female mice) were divided into 2 groups: aluminum adjuvant group 1 (immunized dose of 5 ⁇ g, using aluminum adjuvant) and aluminum adjuvant group 2 (immunized dose of 1 ⁇ g, using Aluminum adjuvant). Each group was further subdivided into 7 subgroups. Control subgroups 1, 2, and 3 were immunized with HPV66N5, VLP, HPV56N0, VLP, and HPV53N5, respectively. Control subgroup 4 was mixed with HPV66 / 56/53.
  • VLPs ie, a mixture of HPV66N5, VLP, HPV56N0, VLP, and HPV53N5, where each VLP is administered at a specified immunization dose
  • mice / subgroup were immunized by intraperitoneal injection, the immunization dose was 5 ⁇ g, 1 ⁇ g, and the injection volume was 1 ml. All mice were initially immunized at week 0 and then boosted once at weeks 2 and 4 each. Orbital blood was collected from the mice at week 6 and the serum titers of anti-HPV66, HPV56 and HPV53 antibodies were analyzed. The analysis results are shown in Figures 9A-9B.
  • H66N5-56T5-53S1, H66N5-56T5-53S2 and H66N5-56T5-53S4 can all induce mice to produce high titers of neutralizing antibodies against HPV66, although the protective effect is slightly weaker than the same dose of HPV66N5 alone VLP and mixed HPV66 / 56/53 VLP, but still significantly better than the same dose of HPV56N0 VLP alone or HPV53N5 VLP alone; and it can induce high titer neutralizing antibodies against HPV56 in mice, and its protective effect It is equivalent to the same dose of HPV56N0 VLP alone and mixed HPV66 / 56/53 VLP, and is significantly better than the same dose of HPV66N5 VLP alone or HPV53N5 VLP alone; and it can induce mice to produce high titers against HPV53
  • the protective effect of neutralizing antibodies is equivalent to that of HPV53N5 VLP alone and mixed HPV66 / 56/53 VLP at the same dose, and
  • H66N5-56T5-53S1, VLP, H66N5-56T5-53S2, VLP and H66N5-56T5-53S4, VLP have good cross-immunogenicity and cross-protection against HPV66, HPV56 and HPV53.
  • Example 6 Evaluation of neutralizing antibody titers in mouse serum after immunization with virus-like particles 3
  • virus particles used were H66N5-56T5-53S2-53S3 VLP.
  • mice (6-week-old BalB / c female mice) were divided into two groups: an experimental group (immunized dose of 5 ⁇ g, using an aluminum adjuvant) and a control group (immunized dose of 5 ⁇ g, using an aluminum adjuvant).
  • the control group was divided into 4 subgroups.
  • the control subgroups 1, 2 and 3 were immunized with HPV66N5, HPV56N0, VLP, and HPV53N5, respectively.
  • the control subgroup 4 was mixed with HPV66 / 56/53.
  • VLPs ie, a mixture of HPV66N5, VLP, HPV56N0, VLP, and HPV53N5, where each VLP is administered at a specified immunization dose
  • the experimental group was immunized with H66N5-56T5-53S2-53S3 VLP.
  • mice / subgroups were immunized by intraperitoneal injection.
  • the immunization doses were 5 ⁇ g and the injection volume was 1 ml. All mice were initially immunized at week 0 and then boosted once at weeks 2 and 4 each. Orbital blood was collected from the mice at week 6 and the serum titers of anti-HPV66, HPV56 and HPV53 antibodies were analyzed. The analysis results are shown in Fig. 10.
  • the results show that H66N5-56T5-53S2-53S3 VLP can induce high titer neutralizing antibodies against HPV66 in mice, and its protective effect is comparable to the same dose of HPV66N5 VLP alone and mixed HPV66 / 56/53 VLP.
  • HPV66N5 VLP and mixed HPV66 / 56/53 VLP but significantly better than the same dose of HPV53N5 VLP alone; and it can induce mice to produce high titers of neutralizing antibodies against HPV53, although its protective effect is slightly weaker than the same Dose of HPV53N5 alone VLP and mixed HPV66 / 56/53 VLP, but significantly better than the same dose of HPV66N5 alone VLP or HPV56N0 alone VLP.
  • H66N5-56T5-53S2-53S3, VLP has good cross-immunogenicity and cross-protection to HPV66, HPV56 and HPV53.
  • H66N5-56T5-53S2-53S3 VLP can be used as an effective vaccine to prevent HPV66 infection, HPV56 infection and HPV53 infection, and can be used to replace the mixed vaccine containing HPV66VLP, HPV56 VLP and HPV53 VLP.

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Abstract

本发明涉及一种突变的HPV66L1蛋白(或其变体)，其编码序列和制备方法，以及包含其的病毒样颗粒，所述蛋白(或其变体)和病毒样颗粒能够诱发抗至少两个型别的HPV(例如，HPV66和HPV56，或者HPV66、HPV56和HPV53)的中和抗体，从而可用于预防所述至少两个型别的HPV感染以及由所述感染所导致的疾病例如宫颈癌和尖锐湿疣。本发明还涉及上述蛋白和病毒样颗粒用于制备药物组合物或疫苗的用途，所述药物组合物或疫苗可用于预防所述至少两个型别的HPV感染以及由所述感染所导致的疾病例如宫颈癌和尖锐湿疣。

Description

一种人乳头瘤病毒66型L1蛋白的突变体
本申请是以CN申请号为201810563504.2，申请日为2018年6月4日的申请为基础，并主张其优先权，该CN申请的公开内容在此作为整体引入本申请中。
技术领域
本发明涉及分子病毒学和免疫学领域。具体地，本发明涉及一种突变的HPV66 L1蛋白(或其变体)，其编码序列和制备方法，以及包含其的病毒样颗粒，所述蛋白(或其变体)和病毒样颗粒能够诱发抗至少两个型别的HPV(例如，HPV66和HPV56，或者HPV66、HPV56和HPV53)的中和抗体，从而可用于预防所述至少两个型别的HPV感染以及由所述感染所导致的疾病例如宫颈癌和尖锐湿疣。本发明还涉及上述蛋白和病毒样颗粒用于制备药物组合物或疫苗的用途，所述药物组合物或疫苗可用于预防所述至少两个型别的HPV感染以及由所述感染所导致的疾病例如宫颈癌和尖锐湿疣。
背景技术
人乳头瘤病毒(Human Papillomavirus，HPV)主要引起皮肤和粘膜的疣状病变。根据其与肿瘤发生的关系，HPV可分为高危型与低危型，其中高危型的HPV感染被证实是诱发包括女性宫颈癌在内的生殖器癌症的主要原因；低危型则主要引起尖锐湿疣。预防与控制HPV感染的最有效方式是施用HPV疫苗，特别是针对能引起宫颈癌的高危型HPV的疫苗。
HPV的主要衣壳蛋白L1具有自组装为空心病毒样颗粒(Virus-Like Particle,VLP)的特性。HPV VLP是由72个主要衣壳蛋白L1的五聚体构成的20面体立体对称结构(Doorbar,J.and P.H.Gallimore.1987.J Virol,61(9):2793-9)。HPV VLP的结构与天然HPV高度相似，保留了天然病毒的绝大多数中和表位，可诱导高滴度的中和抗体(Kirnbauer，R.，F.Booy，et al.1992 Proc Natl Acad Sci U S A 89(24):12180-4)。
然而，现有的研究显示，HPV VLP主要诱导针对同型HPV的中和抗体，产生针对同型HPV的保护性免疫，而仅在一些同源性高的型别之间 存在低的交叉保护作用(Sara L.Bissett,Giada Mattiuzzo,et al.2014 Vaccine.32:6548-6555)。因此，现有的HPV疫苗的保护范围非常有限。通常，一个型别的HPV VLP只能用于预防该型别的HPV感染。在这种情况下，如果要扩大HPV疫苗的保护范围，那就只能在疫苗中增加更多型别的HPV VLP。目前已上市的HPV疫苗，包括Merck公司的
Figure PCTCN2019089940-appb-000001
(其为针对HPV16，18，6和11的四价疫苗)，GSK公司的
Figure PCTCN2019089940-appb-000002
(其为针对HPV16，18的二价疫苗)和Merck公司的
Figure PCTCN2019089940-appb-000003
9(其为针对HPV6，11，16，18，31，33，45，52，58的九价疫苗)，均是通过混合多个型别的HPV VLP而制成的。然而，这种方案将导致HPV疫苗的生产成本大大提高，并且可能因为免疫剂量的增加而导致潜在的安全性问题。
因此，本领域需要开发能够诱导针对多个型别的HPV的保护性中和抗体的HPV病毒样颗粒，以更经济、有效地预防多个型别的HPV感染和由此导致的疾病例如宫颈癌和尖锐湿疣。
发明内容
本发明至少部分基于发明人的下述出人意料的发现：将人乳头瘤病毒(HPV)66型L1蛋白中的一个特定区段置换为第二型别的HPV(例如HPV56)L1蛋白的相应区段后，所获得的突变的HPV66 L1蛋白能够诱导机体产生针对HPV66和第二型别的HPV(例如HPV56)的高滴度中和抗体，其保护效果与混合的HPV66 VLP和第二型别的HPV VLP相当，并且其针对HPV66的保护效果与单独的HPV66 VLP相当，且针对第二型别的HPV(例如HPV56)的保护效果与单独的第二型别的HPV VLP相当。
此外，在上述置换的基础上，还可以将HPV66 L1蛋白中的另一个或两个特定区段进一步置换为第三型别的HPV(例如HPV53)L1蛋白的相应区段，由此所获得的含有双置换的突变的HPV66 L1蛋白能够诱导机体产生针对HPV66、第二型别的HPV(例如HPV56)和第三型别的HPV(例如HPV53)的高滴度中和抗体，其保护效果与混合的HPV66 VLP、第二型别的HPV VLP和第三型别的HPV VLP相当；并且，其针对 HPV66的保护效果与单独的HPV66 VLP相当，针对第二型别的HPV(例如HPV56)的保护效果与单独的第二型别的HPV VLP相当，且针对第三型别的HPV(例如HPV53)的保护效果与单独的第三型别的HPV VLP相当。
因此，在一个方面，本发明提供了一种突变的HPV66 L1蛋白或其变体，其中，所述突变的HPV66 L1蛋白与野生型HPV66 L1蛋白相比，具有下述突变：
(1)N端截短了1-20个氨基酸，例如1-20个氨基酸，例如1-5个、3-8个、5-10个、9-13个、10-15个、12-18个或15-20个氨基酸；和
(2)(a)位于野生型HPV66 L1蛋白第265-283位的氨基酸残基被替换为第二型别的野生型HPV的L1蛋白的相应位置的氨基酸残基；或
(b)位于野生型HPV66 L1蛋白第347-357位的氨基酸残基被替换为第二型别的野生型HPV的L1蛋白的相应位置的氨基酸残基；
并且，所述变体与所述突变的HPV66 L1蛋白相异仅在于一个或几个(例如，1个、2个、3个、4个、5个、6个、7个、8个、9个、10个或11个)氨基酸的置换(优选保守置换)、添加或缺失，且保留了所述突变的HPV66 L1蛋白的功能，即，能够诱导针对至少两个型别的HPV(例如，HPV66和HPV56，或者HPV66、HPV56和HPV53)的中和抗体。
在某些优选的实施方案中，所述突变的HPV66 L1蛋白具有(2)(b)中所定义的突变，并且任选地，还具有下述突变：
(3)位于野生型HPV66 L1蛋白第51-60位的氨基酸残基被替换为第三型别的野生型HPV L1蛋白的相应位置的氨基酸残基；或
(4)位于野生型HPV66 L1蛋白第114-150位的氨基酸残基被替换为第三型别的野生型HPV L1蛋白的相应位置的氨基酸残基；或
(5)位于野生型HPV66 L1蛋白第259-283位的氨基酸残基被替换为第三型别的野生型HPV L1蛋白的相应位置的氨基酸残基。
在某些优选的实施方案中，所述突变的HPV66 L1蛋白具有(2)(b)和(4)中所定义的突变，并且任选地，还具有下述突变：
(6)位于野生型HPV66 L1蛋白第172-181位的氨基酸残基被替换为第三型别的野生型HPV L1蛋白的相应位置的氨基酸残基。
在某些优选的实施方案中，所述突变的HPV66 L1蛋白与野生型HPV66 L1蛋白相比，N端截短了3个、5个、8个、10个、12个、15个、18个氨基酸。
在某些优选的实施方案中，所述突变的HPV66 L1蛋白与野生型HPV66 L1蛋白相比，N端截短了5个氨基酸。
在某些优选的实施方案中，所述第二型别的野生型HPV为HPV56。在某些优选的实施方案中，(2)(a)中所述的相应位置的氨基酸残基为野生型HPV56 L1蛋白第265-283位的氨基酸残基。在某些优选的实施方案中，(2)(b)中所述的相应位置的氨基酸残基为野生型HPV56 L1蛋白第347-357位的氨基酸残基。
在某些优选的实施方案中，所述第三型别的野生型HPV为HPV53。在某些优选的实施方案中，(3)中所述的相应位置的氨基酸残基为野生型HPV53 L1蛋白第51-59位的氨基酸残基。在某些优选的实施方案中，(4)中所述的相应位置的氨基酸残基为野生型HPV53 L1蛋白第113-149位的氨基酸残基。在某些优选的实施方案中，(5)中所述的相应位置的氨基酸残基为野生型HPV53 L1蛋白第258-282位的氨基酸残基。在某些优选的实施方案中，(6)中所述的相应位置的氨基酸残基为野生型HPV53 L1蛋白第171-180位的氨基酸残基
在某些优选的实施方案中，所述野生型HPV66 L1蛋白具有如SEQ ID NO:1或28所示的氨基酸序列。
在某些优选的实施方案中，所述野生型HPV56 L1蛋白具有如SEQ ID NO:2所示的氨基酸序列。
在某些优选的实施方案中，所述野生型HPV53 L1蛋白具有如SEQ ID NO:3或31所示的氨基酸序列。
在某些优选的实施方案中，所述野生型HPV56 L1蛋白第347-357位的氨基酸残基的序列如SEQ ID NO:23所示。
在某些优选的实施方案中，所述野生型HPV53 L1蛋白第51-59位的氨基酸残基的序列如SEQ ID NO:24所示。
在某些优选的实施方案中，所述野生型HPV53 L1蛋白第113-149位的氨基酸残基的序列如SEQ ID NO:25所示。
在某些优选的实施方案中，所述野生型HPV53 L1蛋白第171-180位的氨基酸残基的序列如SEQ ID NO:65所示。
在某些优选的实施方案中，所述野生型HPV53 L1蛋白第258-282位的氨基酸残基的序列如SEQ ID NO:26所示。
在某些优选的实施方案中，所述突变的HPV66 L1蛋白具有选自下列的氨基酸序列：SEQ ID NO:7、8、9、10、11和63。
在另一个方面，本发明提供了一种分离的核酸，其编码如上所述的突变的HPV66 L1蛋白或其变体。在另一个方面，本发明提供了一种载体，其包含所述分离的核酸。在某些优选的实施方案中，本发明的分离的核酸具有选自下列的核苷酸序列：SEQ ID NO:18、19、20、21、22和64。
可用于插入目的多核苷酸的载体是本领域公知的，包括但不限于克隆载体和表达载体。在一个实施方案中，载体是例如质粒，粘粒，噬菌体等等。
在另一个方面，本发明还涉及包含上述分离的核酸或载体的宿主细胞。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。本发明的宿主细胞还可以是细胞系，例如293T细胞。
在另一个方面，本发明涉及一种HPV病毒样颗粒，其中该病毒样颗粒含有本发明的突变的HPV66 L1蛋白或其变体，或者由本发明的突变的HPV66 L1蛋白或其变体组成或形成。
在某些优选的实施方案中，本发明的HPV病毒样颗粒包含突变的HPV66 L1蛋白，其与野生型HPV66 L1蛋白相比，N端截短了1-20个氨基酸，例如1-20个氨基酸，例如1-5个、3-8个、5-10个、9-13个、10-15个、12-18个或15-20个氨基酸，并且位于野生型HPV66 L1蛋白第347-357位的氨基酸残基被替换为野生型HPV56 L1蛋白第347-357位的氨基酸残基。
在某些优选的实施方案中，本发明的HPV病毒样颗粒包含突变的HPV66 L1蛋白，其与野生型HPV66 L1蛋白相比，N端截短了1-20个氨基酸，例如1-20个氨基酸，例如1-5个、3-8个、5-10个、9-13个、10-15 个、12-18个或15-20个氨基酸，并且位于野生型HPV66 L1蛋白第347-357位的氨基酸残基被替换为野生型HPV56 L1蛋白第347-357位的氨基酸残基，并且位于野生型HPV66 L1蛋白第51-60位的氨基酸残基被替换为野生型HPV53 L1蛋白第51-59位的氨基酸残基。
在某些优选的实施方案中，本发明的HPV病毒样颗粒包含突变的HPV66 L1蛋白，其与野生型HPV66 L1蛋白相比，N端截短了1-20个氨基酸，例如1-20个氨基酸，例如1-5个、3-8个、5-10个、9-13个、10-15个、12-18个或15-20个氨基酸，并且位于野生型HPV66 L1蛋白第347-357位的氨基酸残基被替换为野生型HPV56 L1蛋白第347-357位的氨基酸残基，并且位于野生型HPV66 L1蛋白第114-150位的氨基酸残基被替换为野生型HPV53 L1蛋白第113-149位的氨基酸残基。
在某些优选的实施方案中，本发明的HPV病毒样颗粒包含突变的HPV66 L1蛋白，其与野生型HPV66 L1蛋白相比，N端截短了1-20个氨基酸，例如1-20个氨基酸，例如1-5个、3-8个、5-10个、9-13个、10-15个、12-18个或15-20个氨基酸，并且位于野生型HPV66 L1蛋白第347-357位的氨基酸残基被替换为野生型HPV56 L1蛋白第347-357位的氨基酸残基，并且位于野生型HPV66 L1蛋白第259-283位的氨基酸残基被替换为野生型HPV53 L1蛋白第258-282位的氨基酸残基。
在某些优选的实施方案中，本发明的HPV病毒样颗粒包含突变的HPV66 L1蛋白，其与野生型HPV66 L1蛋白相比，N端截短了1-20个氨基酸，例如1-20个氨基酸，例如1-5个、3-8个、5-10个、9-13个、10-15个、12-18个或15-20个氨基酸，并且位于野生型HPV66 L1蛋白第347-357位的氨基酸残基被替换为野生型HPV56 L1蛋白第347-357位的氨基酸残基，并且位于野生型HPV66 L1蛋白第114-150位的氨基酸残基被替换为野生型HPV53 L1蛋白第113-149位的氨基酸残基，并且位于野生型HPV66 L1蛋白第172-181位的氨基酸残基被替换为野生型HPV53 L1蛋白第171-180位的氨基酸残基。
在一个特别优选的实施方案中，本发明的HPV病毒样颗粒包含突变的HPV66 L1蛋白，其具有SEQ ID NO:7、8、9、10、11和63所示的序列。
在另一个方面，本发明还涉及包含上述突变的HPV66 L1蛋白或其变体，或上述分离的核酸或载体或宿主细胞或HPV病毒样颗粒的组合物。在某些优选的实施方案中，所述组合物包含本发明的突变的HPV66 L1蛋白或其变体。在某些优选的实施方案中，所述组合物包含本发明的HPV病毒样颗粒。
在另一个方面，本发明还涉及一种药物组合物或疫苗，其包含本发明的HPV病毒样颗粒，任选地还包含药学可接受的载体和/或赋形剂。本发明的药物组合物或疫苗可以用于预防HPV感染或由HPV感染所导致的疾病例如宫颈癌和尖锐湿疣。
在某些优选的实施方案中，所述HPV病毒样颗粒以预防HPV感染或由HPV感染导致的疾病的有效量存在。在某些优选的实施方案中，所述HPV感染是一个或多个型别的HPV感染(例如，HPV56感染、HPV66感染和/或HPV53感染)。在某些优选的实施方案中，所述由HPV感染所导致的疾病选自宫颈癌和尖锐湿疣。
本发明的药物组合物或疫苗可通过本领域公知的方法进行施用，例如但不限于通过口服或者注射进行施用。在本发明中，特别优选的施用方式是注射。
在某些优选的实施方案中，本发明的药物组合物或疫苗以单位剂量形式进行施用。例如但不意欲限定本发明，每单位剂量中包含的HPV病毒样颗粒的量为5μg－80μg，优选20μg－40μg。
在另一个方面，本发明涉及一种制备如上所述的突变的HPV66 L1蛋白或其变体的方法，其包括，在宿主细胞中表达所述突变的HPV66 L1蛋白或其变体，然后从所述宿主细胞的培养物中回收所述突变的HPV66 L1蛋白或其变体。
在某些优选的实施方案中，所述宿主细胞为大肠杆菌。
在某些优选的实施方案中，所述方法包括步骤：在大肠杆菌中表达所述突变的HPV66 L1蛋白或其变体，然后从所述大肠杆菌的裂解上清中纯化得到所述突变的HPV66 L1蛋白或其变体。在某些优选的实施方案中，通过色谱法(例如，阳离子交换色谱，羟基磷灰石色谱和/或疏水相互作用色谱)，从所述大肠杆菌的裂解上清中回收所述突变的HPV66 L1蛋白或 其变体。
在另一个方面，本发明涉及一种制备疫苗的方法，其包括将本发明的HPV病毒样颗粒与药学可接受的载体和/或赋形剂混合。
在另一个方面，本发明涉及一种预防HPV感染或由HPV感染所导致的疾病的方法，其包括将预防有效量的根据本发明的HPV病毒样颗粒或药物组合物或疫苗施用给受试者。在一个优选的实施方案中，所述HPV感染是一个或多个型别的HPV感染(例如，HPV66感染、HPV56感染和/或HPV53感染)。在另一个优选的实施方案中，所述由HPV感染所导致的疾病包括但不限于，宫颈癌和尖锐湿疣。在另一个优选的实施方案中，所述受试者是哺乳动物，例如人。
在另一个方面，本发明还涉及本发明的突变的HPV66 L1蛋白或其变体或HPV病毒样颗粒在制备药物组合物或疫苗中的用途，所述药物组合物或疫苗用于预防HPV感染或由HPV感染所导致的疾病。在一个优选的实施方案中，所述HPV感染是一个或多个型别的HPV感染(例如，HPV66感染、HPV56感染和/或HPV53感染)。在另一个优选的实施方案中，所述由HPV感染所导致的疾病包括但不限于，宫颈癌和尖锐湿疣。
在另一个方面，本发明还涉及本发明的突变的HPV66 L1蛋白或其变体或HPV病毒样颗粒，其用于预防HPV感染或由HPV感染所导致的疾病。在一个优选的实施方案中，所述HPV感染是一个或多个型别的HPV感染(例如，HPV66感染、HPV56感染和/或HPV53感染)。在另一个优选的实施方案中，所述由HPV感染所导致的疾病包括但不限于，宫颈癌和尖锐湿疣。
本发明中相关术语的说明及解释
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
根据本发明，术语“第二型别的野生型HPV”是指，不同于HPV66的另一型别的野生型HPV。在本发明中，第二型别的野生型HPV优选为野生型HPV56。
根据本发明，术语“第三型别的野生型HPV”是指，不同于HPV66，且不同于第二型别的野生型HPV的另一型别的野生型HPV。在本发明中，第三型别的野生型HPV优选为野生型HPV53。
根据本发明，表述“相应位置”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，进行比较的序列中的等同位置。
根据本发明，术语“野生型HPV66 L1蛋白”是指，天然存在于人乳头瘤病毒66型(HPV66)中的主要衣壳蛋白L1。野生型HPV66 L1蛋白的序列是本领域公知的，并且可参见各种公共数据库(例如NCBI数据库登录ABO76865.1、Q80961.1、ABO76858.1和ALT54961.1等)。
在本发明中，当提及野生型HPV66 L1蛋白的氨基酸序列时，参照SEQ ID NO：1所示的序列来进行描述。例如，表述“野生型HPV66 L1蛋白的第53-56位氨基酸残基”是指，SEQ ID NO：1所示的多肽的第53-56位氨基酸残基。然而，本领域技术人员理解，野生型HPV66可包括多种分离株，并且各种分离株的L1蛋白的氨基酸序列之间可能存在着差异。进一步，本领域技术人员理解，尽管可能存在着序列差异，但是HPV66的不同分离株的L1蛋白在氨基酸序列上具有极高的同一性(通常高于95％，例如高于96％，高于97％，高于98％，或高于99％)，并且具有实质上相同的生物学功能。因此，在本发明中，术语“野生型HPV66 L1蛋白”不仅包括SEQ ID NO:1所示的蛋白，而且应包括各种HPV66分离株的L1蛋白(例如ABO76865.1、Q80961.1、ABO76858.1和ALT54961.1等所示的HPV66 L1蛋白)。并且，当描述野生型HPV66 L1蛋白的序列片段时，其不仅包括SEQ ID NO：1的序列片段，还包括各种HPV66分离株的L1蛋白中的相应序列片段。例如，表述“野生型HPV66 L1蛋白的第53-56位氨基酸残基”包括，SEQ ID NO：1的第53-56位氨基酸残基，以及各种HPV66分离株的L1蛋白中的相应片段。
根据本发明，术语“野生型HPV56 L1蛋白”是指，天然存于人乳头瘤病毒56型(HPV56)中的主要衣壳蛋白L1。野型HPV56 L1蛋白的序列是本领域公知的，并且可参见各种公共数据库(例如NCBI数据库登录号ALT54892.1、ALT54864.1、P36743.1和ABO76830.1等)。
在本发明中，当提及野生型HPV56 L1蛋白的氨基酸序列时，参照 SEQ ID NO：2所示的序列来进行描述。例如，表述“野生型HPV56 L1蛋白的第347-357位氨基酸残基”是指，SEQ ID NO：2所示的多肽的第347-357位氨基酸残基。然而，本领域技术人员理解，野生型HPV56可包括多种分离株，并且各种分离株的L1蛋白的氨基酸序列之间可能存在着差异。进一步，本领域技术人员理解，尽管可能存在着序列差异，但是HPV56的不同分离株的L1蛋白在氨基酸序列上具有极高的同一性(通常高于95％，例如高于96％，高于97％，高于98％，或高于99％)，并且具有实质上相同的生物学功能。因此，在本发明中，术语“野生型HPV56 L1蛋白”不仅包括SEQ ID NO:2所示的蛋白，而且应包括各种HPV56分离株的L1蛋白(例如ALT54892.1、ALT54864.1、P36743.1和ABO76830.1等所示的HPV56 L1蛋白)。并且，当描述野生型HPV56 L1蛋白的序列片段时，其不仅包括SEQ ID NO：2的序列片段，还包括各种HPV56分离株的L1蛋白中的相应序列片段。例如，表述“野生型HPV56 L1蛋白的第347-357位氨基酸残基”包括，SEQ ID NO：2的第347-357位氨基酸残基，以及各种HPV56分离株的L1蛋白中的相应片段。
根据本发明，术语“野生型HPV53 L1蛋白”是指，天然存在于人乳头瘤病毒53型(HPV53)中的主要衣壳蛋白L1。野生型HPV53 L1蛋白的序列是本领域公知的，并且可参见各种公共数据库(例如NCBI数据库登录号NP041848.1、ANY26596.1、ABU54090.1和ALJ32506.1等)。
在本发明中，当提及野生型HPV53 L1蛋白的氨基酸序列时，参照SEQ ID NO：3所示的序列来进行描述。例如，表述“野生型HPV53 L1蛋白的第51-59位氨基酸残基”是指，SEQ ID NO：3所示的多肽的第51-59位氨基酸残基。然而，本领域技术人员理解，野生型HPV53可包括多种分离株，并且各种分离株的L1蛋白的氨基酸序列之间可能存在着差异。进一步，本领域技术人员理解，尽管可能存在着序列差异，但是HPV53的不同分离株的L1蛋白在氨基酸序列上具有极高的同一性(通常高于95％，例如高于96％，高于97％，高于98％，或高于99％)，并且具有实质上相同的生物学功能。因此，在本发明中，术语“野生型HPV53 L1蛋白”不仅包括SEQ ID NO:3所示的蛋白，而且应包括各种HPV53分离株的L1蛋白(例如NP041848.1、ANY26596.1、ABU54090.1和 ALJ32506.1所示的HPV53 L1蛋白)。并且，当描述野生型HPV53 L1蛋白的序列片段时，其不仅包括SEQ ID NO：3的序列片段，还包括各种HPV53分离株的L1蛋白中的相应序列片段。例如，表述“野生型HPV53L1蛋白的第51-59位氨基酸残基”包括，SEQ ID NO：3的第51-59位氨基酸残基，以及各种HPV53分离株的L1蛋白中的相应片段。
根据本发明，表述“相应序列片段”或“相应片段”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，进行比较的序列中位于等同位置的片段。
根据本发明，表述“N端截短了X个氨基酸”是指，用起始密码子(用于起始蛋白质翻译)编码的甲硫氨酸残基置换蛋白质N末端的第1－X位氨基酸残基。例如，N端截短了5个氨基酸的HPV66 L1蛋白是指，用起始密码子编码的甲硫氨酸残基置换野生型HPV66 L1蛋白N末端的第1－5位氨基酸残基所获得的蛋白质。
根据本发明，术语“变体”是指这样的蛋白，其氨基酸序列与本发明的突变的HPV66 L1蛋白(如SEQ ID NO：7、8、9、10和11所示的蛋白)的氨基酸序列相比，具有一个或几个(例如，1个、2个、3个、4个、5个、6个、7个、8个或9个)氨基酸的置换(优选保守置换)、添加或缺失，或者具有至少90％，95％，96％，97％，98％，或99％的同一性，并且其保留了所述突变的HPV66 L1蛋白的功能。在本发明中，术语“突变的HPV66 L1蛋白的功能”是指：能够诱导机体产生针对至少两个型别的HPV(例如，HPV66和HPV56，或者HPV66、HPV56和HPV53)的中和抗体。术语“同一性”是对核苷酸序列或氨基酸序列的相似性的量度。通常将序列排列起来，以获得最大限度的匹配。“同一性”本身具有本领域公知的意义并且可用公开的算法(例如BLAST)来计算。
根据本发明，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如 果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的必要特性的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，保守置换通常是指，用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12 (10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
根据本发明，术语“大肠杆菌表达系统”是指由大肠杆菌(菌株)与载体组成的表达系统，其中大肠杆菌(菌株)来源于市场上可得到的菌株，例如但不限于：ER2566，BL21(DE3)，B834(DE3)，BLR(DE3)。
根据本发明，术语“载体(vector)”是指，可将多核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸所编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌体；柯斯质粒等等。
根据本发明，术语“药学可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；佐剂包括但不限于铝佐剂(例如氢氧化铝)，弗氏佐剂(例如完全弗氏佐剂)；离子强度增强剂包括但不限于氯化钠。
根据本发明，术语“有效量”是指能够有效实现预期目的的量。例如，预防疾病(例如HPV感染)有效量是指，能够有效预防，阻止，或延迟疾病(例如HPV感染)的发生的量。测定这样的有效量在本领域技术人员的能力范围之内。
根据本发明，术语“色谱层析”包括但不限于：离子交换色谱(例如阳离子交换色谱)、疏水相互作用色谱、吸附层析法(例如羟基磷灰石色谱)、凝胶过滤(凝胶排阻)层析、亲和层析法。
根据本发明，术语“裂解上清”是指通过下述步骤所产生的溶液：将宿主细胞(例如大肠杆菌)在裂解液中破碎，然后将含有经破碎的宿主细胞的裂解液中的不溶物去除。各种裂解液是本领域技术人员公知的，包括但不限于Tris缓冲液，磷酸盐缓冲液，HEPES缓冲液，MOPS缓冲液等等。 此外，可通过本领域技术人员熟知的各种方法来实现宿主细胞的破碎，包括但不限于匀浆器破碎、均质机破碎、超声波处理、研磨、高压挤压、溶菌酶处理等等。去除裂解液中的不溶物的方法也是本领域技术人员公知的，包括但不限于过滤和离心。
发明的有益效果
研究表明，虽然HPV66和其他型别的HPV(例如HPV56和HPV53)之间存在一定的交叉保护，但是这种交叉保护的能力很低，通常低于自身型别的VLP的保护水平的百分之一，甚至低于千分之一。因此，对于接种了HPV66疫苗的受试者来说，其感染其他型别的HPV(例如HPV56和HPV53)的风险依然很高。
本发明提供了一种突变的HPV66 L1蛋白以及由其形成的HPV病毒样颗粒。本发明的HPV病毒样颗粒能够在HPV66和其他型别的HPV(例如HPV56和HPV53)之间提供显著的交叉保护能力。特别地，在同等免疫剂量下，本发明的HPV病毒样颗粒能够诱发机体产生针对至少两个型别的HPV(例如，HPV66和HPV56，或者HPV66、HPV56和HPV53)的高滴度中和抗体，并且其效果与多个型别的HPV VLP的混合物(例如，HPV66 VLP和HPV56 VLP的混合物，或者HPV66 VLP、HPV56 VLP和HPV53 VLP的混合物)相当。因此，本发明的HPV病毒样颗粒能够用于同时预防至少两个型别的HPV(例如，HPV66和HPV56，或者HPV66、HPV56和HPV53)的感染以及与此相关的疾病，具有显著的有利技术效果。这在扩大HPV疫苗的保护范围和降低HPV疫苗的生产成本等方面具有特别显著的优势。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了实施例1中经纯化的突变蛋白的SDS聚丙烯酰胺凝胶电泳的结果。泳道1：蛋白分子量标记；泳道2:HPV66N5(N端截短了5个氨基酸的HPV66 L1蛋白)；泳道3:HPV56N0(全长的HPV56 L1蛋白)； 泳道4:H66N5-56T1；泳道5:H66N5-56T2；泳道6:H66N5-56T3；泳道7:H66N5-56T4；泳道8:H66N5-56T5；泳道9:蛋白分子量标记；泳道10:H66N5-56T5；泳道11:HPV53N5(N端截短了5个氨基酸的HPV53L1蛋白)；泳道12:H66N5-56T5-53S1；泳道13:H66N5-56T5-53S2；泳道14:H66N5-56T5-53S4；泳道15:蛋白分子量标记；泳道16:H66N5-56T5-53S2-53S3；结果显示，经过色谱纯化后，H66N5-56T1、H66N5-56T2、H66N5-56T3、H66N5-56T4、H66N5-56T5、H66N5-56T5-53S1、H66N5-56T5-53S2、H66N5-56T5-53S4蛋白的纯度均达到90％左右,H66N5-56T5-53S2-53S3蛋白的纯度75％左右。
图2显示了用HPV L1广谱抗体4B3检测实施例1中制备的突变蛋白H66N5-56T1、H66N5-56T2、H66N5-56T3、H66N5-56T4、H66N5-56T5、H66N5-56T5-53S1、H66N5-56T5-53S2、H66N5-56T5-53S4的蛋白质免疫印迹检测的结果。泳道1:HPV66N5(N端截短了5个氨基酸的HPV66 L1蛋白)；泳道2:HPV56N0(全长的HPV56 L1蛋白)；泳道3:H66N5-56T1；泳道4:H66N5-56T2；泳道5:H66N5-56T3；泳道6:H66N5-56T4；泳道7:H66N5-56T5；泳道8:H66N5-56T5；泳道9:HPV53N5(N端截短了5个氨基酸的HPV53L1蛋白)；泳道10:H66N5-56T5-53S1；泳道11:H66N5-56T5-53S2；泳道12:H66N5-56T5-53S4结果显示，突变蛋白H66N5-56T1、H66N5-56T2、H66N5-56T3、H66N5-56T4、H66N5-56T5、H66N5-56T5-53S1、H66N5-56T5-53S2、H66N5-56T5-53S4均能够被广谱抗体4B3特异性识别。
图3显示了包含蛋白H66N5-56T1、H66N5-56T2、H66N5-56T3、H66N5-56T4、H66N5-56T5、HPV56N0和HPV66N5(N端截短了5个氨基酸的HPV66L1蛋白)的样品的分子筛层析分析的结果。结果显示，包含蛋白H66N5-56T1、H66N5-56T2、H66N5-56T3、H66N5-56T4、H66N5-56T5的样品最先出现的蛋白峰均在13-14min左右，与HPV56N0和HPV66N5 VLP相当。这表明这些蛋白均可组装成VLP。
图4显示了包含蛋白H66N5-56T5、HPV53N5、H66N5-56T5-53S1、H66N5-56T5-53S2、H66N5-56T5-53S4、H66N5-56T5-53S2-53S3的样品的分子筛层析分析的结果。结果显示，包含蛋白H66N5-56T5和H66N5- 56T5-53S4的样品最先出现的蛋白峰在13-14min左右，与HPV53N5相当。而H66N5-56T5-53S1和H66N5-56T5-53S2的峰形不对称，且H66N5-56T5-53S1的出峰时间在15min左右，表明H66N5-56T5-53S1形成的VLP颗粒直径较小，H66N5-56T5-53S2和H66N5-56T5-53S2-53S3形成的颗粒不均一。
图5显示了H66N5-56T1 VLP、H66N5-56T2 VLP、H66N5-56T3 VLP、H66N5-56T4 VLP、H66N5-56T5 VLP、HPV56N0 VLP和HPV66N5 VLP的沉降速率分析的结果。结果显示，H66N5-56T1 VLP、H66N5-56T2 VLP、H66N5-56T3 VLP、H66N5-56T4 VLP、H66N5-56T5 VLP的沉降系数分别为126S、125S、126S、127S和128S，而HPV56N0 VLP和HPV66N5 VLP的沉降系数分别为134S和141S。这表明，H66N5-56T1 VLP、H66N5-56T2 VLP、H66N5-56T3 VLP、H66N5-56T4 VLP和H66N5-56T5 VLP能够组装成大小、形态与野生型VLP相似的病毒样颗粒。
图6显示了H66N5-56T5 VLP、HPV53N5 VLP、H66N5-56T5-53S1 VLP、H66N5-56T5-53S2 VLP、H66N5-56T5-53S4 VLP和H66N5-56T5-53S2-53S3的沉降速率分析的结果。结果显示，H66N5-56T5 VLP、HPV53N5 VLP、H66N5-56T5-53S1 VLP、H66N5-56T5-53S2 VLP和H66N5-56T5-53S4 VLP的沉降系数分别为128S、130S、93S、106S和116S，H66N5-56T5-53S2-53S3VLP的沉降系数为53S和107S两个组分。这表明，H66N5-56T5-53S4 VLP能够组装成大小、形态与野生型VLP相似的病毒样颗粒；而H66N5-56T5-53S1、H66N5-56T5-53S2VLP和H66N5-56T5-53S2-53S3VLP形成的病毒样颗粒直径较小，且H66N5-56T5-53S2和H66N5-56T5-53S2-53S3形成的颗粒不均一。
图7A-图7L显示了各种VLP样品的透射电镜观察结果(放大倍数为100,000倍，Bar＝0.1μm)。图7A，由HPV66N5组装的VLP；图7B，由HPV56N0组装的VLP；图7C，由H66N5-56T1组装的VLP；图7D，由H66N5-56T2形成的VLP；图7E，由H66N5-56T3组装的VLP；图7F，由H66N5-56T4组装的VLP；图7G，由H66N5-56T5组装的VLP；图7H，由HPV53N5组装的VLP；图7I，由H66N5-56T5-53S1组装的VLP， 颗粒直径较小；图7J，由H66N5-56T5-53S2组装的VLP；图7K，由H66N5-56T5-53S4组装的VLP；图7L，由H66N5-56T5-53S2-53S3组装的VLP；结果显示，H66N5-56T1、H66N5-56T2、H66N5-56T3、H66N5-56T4、H66N5-56T5、H66N5-56T5-53S2、H66N5-56T5-53S4、H66N5-56T5-53S2-53S3与HPV56N0、HPV66N5和HPV53N5类似，都能够组装成半径为25nm左右的VLP，但H66N5-56T5-53S2和H66N5-56T5-53S2-53S3形成的颗粒中也含有直径20nm左右的小颗粒；而H66N5-56T5-53S1能形成半径为10-20nm左右的颗粒不规则的VLP。
图8A-图8C显示了实验组H66N5-56T1 VLP、H66N5-56T2 VLP、H66N5-56T3 VLP、H66N5-56T4 VLP、H66N5-56T5 VLP、和对照组HPV56N0 VLP和HPV66N5 VLP以及混合的HPV56/HPV66 VLP在小鼠体内的免疫保护性的评价结果。结果显示，H66N5-56T4 VLP、H66N5-56T5 VLP各自可在小鼠体内诱导高滴度的针对HPV66和HPV56的中和抗体；并且其针对HPV66的保护效果与单独的HPV66N5 VLP相当，且显著高于单独的HPV56N0VLP；并且其针对HPV56的保护效果与单独的HPV56N0 VLP相当，且显著高于单独的HPV66N5 VLP。这表明，H66N5-56T4 VLP、H66N5-56T5 VLP可用作预防HPV66感染和HPV56感染的有效疫苗，可用于代替含有HPV66 VLP和HPV56VLP的混合疫苗。
图9A-图9B显示了实验组H66N5-56T5-53S1 VLP、H66N5-56T5-53S2 VLP和H66N5-56T5-53S4 VLP以及对照组HPV66N5 VLP、HPV56N0 VLP、HPV53N5 VLP和混合的HPV66/56/53 VLP在小鼠体内的免疫保护性的评价结果。结果显示，H66N5-56T5-53S1 VLP、H66N5-56T5-53S2 VLP和H66N5-56T5-53S4 VLP可在小鼠体内诱导高滴度的针对HPV66、HPV56和HPV53的中和抗体；并且其针对HPV66的保护效果与单独的HPV66N5 VLP、混合的HPV66/56/53 VLP相当，且显著高于单独的HPV56N0VLP和单独的HPV53N5VLP；并且其针对HPV56的保护效果与单独的HPV56N0 VLP、混合的HPV66/56/53 VLP相当，且显著高于单独的HPV66N5 VLP和单独的HPV53N5 VLP；并且其针对HPV53的保护效果与单独的HPV53N5 VLP、混合的HPV66/56/53 VLP相当，且显著高于单独的HPV66N5 VLP和单独的HPV56N0 VLP。这表明， H66N5-56T5-53S1 VLP、H66N5-56T5-53S2 VLP和H66N5-56T5-53S4 VLP可用作预防HPV66感染、HPV56感染和HPV53感染的有效疫苗，可用于代替含有HPV66VLP、HPV56 VLP和HPV53 VLP的混合疫苗。
图10显示了实验组H66N5-56T5-53S2-53S3 VLP和对照组HPV66N5 VLP、HPV56N0 VLP、HPV53N5 VLP以及混合的HPV66/56/53 VLP在小鼠体内的免疫保护性的评价结果。结果显示，H66N5-56T5-53S2-53S3 VLP可在小鼠体内诱导高滴度的针对HPV66、HPV56和HPV53的中和抗体；并且其针对HPV66的保护效果与单独的HPV66N5 VLP、混合的HPV66/56/53 VLP相当，且显著高于单独的HPV56N0VLP和单独的HPV53N5VLP；其针对HPV56的保护效果稍弱于单独的HPV56N0 VLP、单独的HPV66N5 VLP、混合的HPV66/56/53 VLP，但显著高于单独的HPV53N5 VLP；其针对HPV53的保护效果稍弱于单独的HPV53N5 VLP、混合的HPV66/56/53 VLP，但显著高于单独的HPV66N5 VLP和单独的HPV56N0 VLP。这表明，H66N5-56T5-53S2-53S3 VLP可用作预防HPV66感染、HPV56感染和HPV53感染的有效疫苗，可用于代替含有HPV66VLP、HPV56 VLP和HPV53 VLP的混合疫苗。
序列信息
本发明涉及的部分序列的信息提供于下面的表1中。
表1：序列的描述
Figure PCTCN2019089940-appb-000004
Figure PCTCN2019089940-appb-000005
序列1(SEQ ID NO:1)：
Figure PCTCN2019089940-appb-000006
序列2(SEQ ID NO:2)：
Figure PCTCN2019089940-appb-000007
序列3(SEQ ID NO:3)：
Figure PCTCN2019089940-appb-000008
序列4(SEQ ID NO:4)：
Figure PCTCN2019089940-appb-000009
序列5(SEQ ID NO:5)：
Figure PCTCN2019089940-appb-000010
序列6(SEQ ID NO:6)：
Figure PCTCN2019089940-appb-000011
序列7(SEQ ID NO:7)：
Figure PCTCN2019089940-appb-000012
序列8(SEQ ID NO:8)：
Figure PCTCN2019089940-appb-000013
序列9(SEQ ID NO:9)：
Figure PCTCN2019089940-appb-000014
Figure PCTCN2019089940-appb-000015
序列10(SEQ ID NO:10)：
Figure PCTCN2019089940-appb-000016
序列11(SEQ ID NO:11)：
Figure PCTCN2019089940-appb-000017
序列12(SEQ ID NO:12)：
Figure PCTCN2019089940-appb-000018
序列13(SEQ ID NO:13)：
Figure PCTCN2019089940-appb-000019
Figure PCTCN2019089940-appb-000020
序列14(SEQ ID NO:14)：
Figure PCTCN2019089940-appb-000021
序列15(SEQ ID NO:15)：
Figure PCTCN2019089940-appb-000022
序列16(SEQ ID NO:16)：
Figure PCTCN2019089940-appb-000023
序列17(SEQ ID NO:17)：
Figure PCTCN2019089940-appb-000024
序列18(SEQ ID NO:18)：
Figure PCTCN2019089940-appb-000025
Figure PCTCN2019089940-appb-000026
序列19(SEQ ID NO:19)：
Figure PCTCN2019089940-appb-000027
序列20(SEQ ID NO:20)：
Figure PCTCN2019089940-appb-000028
序列21(SEQ ID NO:21)：
Figure PCTCN2019089940-appb-000029
Figure PCTCN2019089940-appb-000030
序列22(SEQ ID NO:22)：
Figure PCTCN2019089940-appb-000031
序列23(SEQ ID NO:23)：
Figure PCTCN2019089940-appb-000032
序列24(SEQ ID NO:24)：
Figure PCTCN2019089940-appb-000033
序列25(SEQ ID NO:25)：
Figure PCTCN2019089940-appb-000034
序列26(SEQ ID NO:26)：
Figure PCTCN2019089940-appb-000035
序列27SEQ ID NO:27)：
Figure PCTCN2019089940-appb-000036
序列28(SEQ ID NO:28)：
Figure PCTCN2019089940-appb-000037
序列29(SEQ ID NO:29)：
Figure PCTCN2019089940-appb-000038
序列30(SEQ ID NO:30)：
Figure PCTCN2019089940-appb-000039
序列31(SEQ ID NO:31)：
Figure PCTCN2019089940-appb-000040
序列32(SEQ ID NO:32)：
Figure PCTCN2019089940-appb-000041
Figure PCTCN2019089940-appb-000042
序列33(SEQ ID NO:33)：
Figure PCTCN2019089940-appb-000043
序列34(SEQ ID NO:34)：
Figure PCTCN2019089940-appb-000044
序列63(SEQ ID NO:63)：
Figure PCTCN2019089940-appb-000045
序列64(SEQ ID NO:64)：
Figure PCTCN2019089940-appb-000046
Figure PCTCN2019089940-appb-000047
序列65(SEQ ID NO:65)：
Figure PCTCN2019089940-appb-000048
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons，Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1.突变的HPV66 L1蛋白的表达与纯化
表达载体的构建
采用多点突变PCR反应来构建编码含有来源于HPV56 L1蛋白的区段的突变的HPV66 L1蛋白的表达载体，其中，所使用的初始模板为pTO-T7-HPV66N5C质粒(其编码N端截短了5个氨基酸的HPV66 L1蛋白；在表2中简写为66L1N5)。用于各个PCR反应的模板和引物见表2，并且，PCR反应的扩增条件设为：94℃变性2分钟；30个循环的(98℃变性10秒，指定温度退火一定时间，58℃延伸6分30秒)；最后68℃延伸10分钟。退火温度和时间列于表2。所使用的PCR引物的具体序列列于表3。
向扩增产物(50μL)中加入2μL DpnI限制性内切酶(Fermentas(MBI)，货号：FD1704，2500U/管)，并在37℃温育60min。取10μL酶切产物，用于转化40μL以氯化钙法制备的感受态大肠杆菌ER2566(购自新英格兰生物实验室公司)。将经转化的大肠杆菌涂布于含卡那霉素(终 浓度25μg/mL，下同)的固体LB培养基(LB培养基成分：10g/L蛋白胨，5g/L酵母粉，10g/L氯化钠，下同)，并在37℃静置培养10-12小时，直至单菌落清晰可辨。挑取单菌落至含有4mL液体LB培养基(含卡那霉素)的试管中，并在37℃ 220转/分钟下振荡培养10小时。随后，取1mL菌液于-70℃保存。从大肠杆菌中提取质粒，并利用T7引物对质粒中插入的目的片段的核苷酸序列进行测序。测序结果显示，所构建的各个质粒(表达载体)中插入的目的片段的核苷酸序列为SEQ ID NO：15、17，其编码的氨基酸序列为SEQ ID NO：4、6(所对应的蛋白分别命名为H66N5-56T1和H66N5-56T3)。
突变蛋白H66N5-56T1与HPV66N5的区别在于：位于野生型HPV66L1蛋白第53-56位的氨基酸残基被替换为野生型HPV56 L1蛋白第53-56位的氨基酸残基。突变蛋白H66N5-56T3与HPV66N5的区别在于：位于野生型HPV66 L1蛋白第178-180位的氨基酸残基被替换为野生型HPV56L1蛋白第178-180位的氨基酸残基。
采用Gibson装配(Gibson DG,Young L,Chuang RY,Venter JC,Hutchison CA,Smith HO.Enzymatic assembly of DNA molecules up to several hundred kilobases.Nat Methods.2009；6:343-5.doi:10.1038/nmeth.1318)来构建编码其他的突变HPV66 L1蛋白的表达载体，所述突变的HPV66 L1蛋白含有来源于HPV56 L1的特定区段和/或来源于HPV53 L1的特定区段。简言之，首先采用PCR反应来获得一个包含突变的短片段和一个不包含突变的长片段，然后再采用Gibson装配体系将这两个片段连接成环。所使用的初始模板包括pTO-T7-HPV66L1N5质粒(其编码N端截短了5个氨基酸的HPV66 L1蛋白；在表2中简写为66L1N5)及pTO-T7-HPV53N0质粒(其编码全长的HPV56 L1蛋白；在表2中简写为56L1N0)、pTO-T7-H66N5-56T5质粒(其编码突变蛋白H66N5-56T5；在表2中简写为H66N5-56T5)和pTO-T7-HPV53N5质粒(其编码N端截短了5个氨基酸的HPV53 L1蛋白；在表2中简写为53L1N5)。用于各个PCR反应的模板和引物见表2，并且，用于扩增短片段的PCR反应的扩增条件设为：94℃变性10分钟；25个循环的(94℃变性50秒，指定温度退火一定时间，72℃延伸1分钟)；最后 72℃延伸10分钟。用于扩增长片段的PCR反应的扩增条件设为：94℃变性10分钟；25个循环的(94℃变性50秒，指定温度退火一定时间，72℃延伸7分30秒)；最后72℃延伸10分钟。所使用的PCR引物的具体序列列于表3。将扩增产物进行电泳，随后使用DNA回收试剂盒(BEYOTIME(碧云天)，货号：D0033)回收目的片段并测定其浓度。按2:1的摩尔比将扩增得到的短片段和长片段混合(总体积3μL)，随后添加3μL 2X Gibson装配预混试剂(2X Gibson Assembly Master Mix，购自NEB，包含T5exonuclease，Phusion DNA polymerase，Taq DNA ligase)，并在50℃反应1小时。
用装配后的产物(6μL)转化40μL以氯化钙法制备的感受态大肠杆菌ER2566(购自新英格兰生物实验室公司)。将经转化的大肠杆菌涂布于含卡那霉素的固体LB培养基，并在37℃静置培养10-12小时，直至单菌落清晰可辨。挑取单菌落至含有4mL液体LB培养基(含卡那霉素)的试管中，并在37℃ 220转/分钟下振荡培养10小时。随后，取1mL菌液于-70℃保存。从大肠杆菌中提取质粒，并利用T7引物对质粒中插入的目的片段的核苷酸序列进行测序。测序结果显示，所构建的各个质粒(表达载体)中插入的目的片段的核苷酸序列分别为SEQ ID NO：16、18、19、20、21、22，其编码的氨基酸序列为SEQ ID NO：5、7、8、9、10、11(所对应的蛋白分别命名为H66N5-56T2，H66N5-56T4，H66N5-56T5，H66N5-56T5-53S1，H66N5-56T5-53S2，H66N5-56T5-53S4)。
H66N5-56T5-53S2-53S3基因由上海生工生物公司合成，将合成后含有HPV66N5-56T5-53S2-53S3基因的质粒用NdeI和SalI进行酶切(37℃，12小时)，酶切后将目的基因用DNA回收试剂盒(BEYOTIME(碧云天)，货号：D0033)进行回收，将酶切后的H66N5-56T5-53S2-53S3基因与pTO-T7载体在16℃下连接12小时，随后转化感受态大肠杆菌ER2566(购自新英格兰生物实验室公司)细菌。将经转化的大肠杆菌涂布于含卡那霉素的固体LB培养基，并37℃静置培养10-12小时，直至单菌落清晰可辨。挑取单菌落至含4mL液体LB培养基(含卡那霉素)的试管，在37℃220转/分钟下振荡培养10小时，从中取1mL菌液于-70℃保存。从大肠杆菌中提取质粒，并利用T7引物对质粒中插入的目的片段的核苷 酸序列进行测序。测序结果显示，所构建的各个质粒(表达载体)中插入的目的片段的核苷酸序列分别为SEQ ID NO：64，其编码的氨基酸序列为SEQ ID NO：63(所对应的蛋白命名为H66N5-56T5-53S2-53S3)。
突变蛋白H66N5-56T2与HPV66N5的区别在于：位于野生型HPV66 L1蛋白第130-150位的氨基酸残基被替换为野生型HPV56 L1蛋白第130-150位的氨基酸残基。突变蛋白H66N5-56T4与HPV66N5的区别在于：位于野生型HPV66 L1蛋白第265-283位的氨基酸残基被替换为野生型HPV56 L1蛋白第265-283位的氨基酸残基。突变蛋白H66N5-56T5与HPV66N5的区别在于：位于野生型HPV66 L1蛋白第347-357位的氨基酸残基被替换为野生型HPV56 L1蛋白第347-357位的氨基酸残基。
突变蛋白H66N5-56T5-53S1与HPV66N5的区别在于：位于野生型HPV66 L1蛋白第347-357位的氨基酸残基被替换为野生型HPV56 L1蛋白第347-357位的氨基酸残基，并且位于野生型HPV66 L1蛋白第51-60位的氨基酸残基被替换为野生型HPV53 L1蛋白的第51-59位的氨基酸残基。突变蛋白H66N5-56T5-53S2与HPV66N5的区别在于：位于野生型HPV39 L1蛋白第347-357位的氨基酸残基被替换为野生型HPV56 L1蛋白第347-357位的氨基酸残基，并且位于野生型HPV66 L1蛋白第114-150位的氨基酸残基被替换为野生型HPV53 L1蛋白的第113-149位的氨基酸残基。突变蛋白H66N5-56T5-53S4与HPV66N5的区别在于：位于野生型HPV66 L1蛋白第347-357位的氨基酸残基被替换为野生型HPV56 L1蛋白第347-357位的氨基酸残基，并且位于野生型HPV66 L1蛋白第259-283位的氨基酸残基被替换为野生型HPV53 L1蛋白的第258-282位的氨基酸残基。
突变蛋白H66N5-56T5-53S2-53S3与HPV66N5的区别在于：位于野生型HPV66 L1蛋白第347-357位的氨基酸残基被替换为野生型HPV56 L1蛋白第347-357位的氨基酸残基，并且位于野生型HPV66 L1蛋白第114-150位的氨基酸残基被替换为野生型HPV53 L1蛋白的第113-149位的氨基酸残基,并且位于野生型HPV66 L1蛋白第172-181位的氨基酸残基被替换为野生型HPV53 L1蛋白的第171-180位的氨基酸残基。
表2.用于构建表达载体的PCR反应的模板和引物
Figure PCTCN2019089940-appb-000049
表3：所使用的引物的具体序列(SEQ ID NO:35-62)
Figure PCTCN2019089940-appb-000050
Figure PCTCN2019089940-appb-000051
突变蛋白的大量表达
从-70℃冰箱中取出携带重组质粒pTO-T7-H66N5-56T1、pTO-T7-H66N5-56T2、pTO-T7-H66N5-56T3、pTO-T7-H66N5-56T4、pTO-T7-H66N5-56T5、pTO-T7-H66N5-56T5-53S1、pTO-T7-H66N5-56T5-53S2、pTO-T7-H66N5-56T5-53S4、pTO-T7-H66N5-56T5-53S2-53S3的大肠杆菌菌液，分别接种入100ml含卡那霉素的LB液体培养基中，在200rpm，37℃下培养大约8小时；然后分别转接入500ml含卡那霉素的LB培养基中(接入1ml菌液)，并继续进行培养。当细菌浓度达到OD 600为0.6左右时，将培养温度降至25℃，并向各培养瓶中加入500μL IPTG，继续培养8小时。培养结束后，离心收集菌体。获得表达了H66N5-56T1，H66N5-56T2，H66N5-56T3，H66N5-56T4，H66N5-56T5，H66N5-56T5-53S1，H66N5-56T5-53S2，H66N5-56T5-53S4，H66N5-56T5-53S2-53S3蛋白的菌体。
表达突变蛋白的菌体破碎
按1g菌体对应10mL裂解液(20mM Tris缓冲液，pH7.2，300mM NaCl)的比例重悬上述得到的菌体。用超声波仪破碎菌体30min。以13500rpm(30000g)离心含有经破碎的菌体的裂解液15min，留取上清(即，菌体破碎上清)。
突变蛋白的色谱纯化
仪器系统：GE Healthcare公司(原Amershan Pharmacia公司)生产的AKTA explorer 100型制备型液相色谱系统。
层析介质：SP Sepharose 4Fast Flow(GE Healthcare公司)、CHT-Ⅱ(购自Bio－RAD)和Butyl Sepharose 4 Fast Flow(GE Healthcare公司)。
缓冲液：缓冲液A(20mM磷酸盐缓冲液，pH8.0，20mM DTT)；以及缓冲液B(20mM磷酸盐缓冲液，pH8.0，20mM DTT，2M NaCl)。下面的洗脱程序中用到的含有不同浓度NaCl的缓冲液是由缓冲液A和B按比例混合配置而成。
样品：如上获得的含有H66N5-56T1，H66N5-56T2，H66N5-56T3，
H66N5-56T4，H66N5-56T5，H66N5-56T5-53S1，H66N5-56T5-53S2，
H66N5-56T5-53S4，H66N5-56T5-53S2-53S3的菌体破碎上清。
洗脱程序为：
(1)用SP Sepharose 4 Fast Flow对菌体破碎上清进行阳离子交换纯化：将样品上柱，然后用含有400mM NaCl的缓冲液(80％缓冲液A+20％缓冲液B)洗脱杂蛋白，然后用含有800mM NaCl的缓冲液(60％缓冲液A+40％缓冲液B)洗脱目的蛋白，并收集由含有800mM NaCl的缓冲液洗脱的级分；
(2)用CHTⅡ(羟基磷灰石色谱)对前一步获得的洗脱级分进行色谱纯化：对前一步骤获得的洗脱级分进行稀释，以使得NaCl的浓度降至0.5M；将样品上柱，然后用含有500mM NaCl的缓冲液(75％缓冲液A+25％缓冲液B)洗脱杂蛋白，然后用含有1000mM NaCl的缓冲液(50％缓冲液A+50％缓冲液B)洗脱目的蛋白，并收集由含有1000mM NaCl的缓冲液洗脱的级分；
(3)用HIC(疏水相互作用色谱)对前一步骤获得的洗脱级分进行色谱纯化：将样品上柱，然后用含有1000mM NaCl的缓冲液洗脱杂蛋白，然后用含有200mM NaCl的缓冲液(90％缓冲液A+10％缓冲液B)洗脱目的蛋白，并收集由含有200mM NaCl的缓冲液洗脱的级分。
取步骤(3)获得的洗脱级分150μL，加入30μL 6X Loading Buffer(1L中含有1M TB 6.8 300ml、100％甘油 600ml、SDS 120g、溴酚蓝 6g、β-巯基乙醇50ml)中，混匀，并于80℃水浴中温育10min。然后取10μl样品于10％SDS-聚丙烯酰胺凝胶中以120V电压电泳120min；然后以考马斯亮兰染色显示电泳条带。电泳结果示于图1中。结果显示，经过上述纯化步骤后，H66N5-56T1，H66N5-56T2，H66N5-56T3，H66N5-56T4，H66N5-56T5，H66N5-56T5-53S1，H66N5-56T5-53S2，H66N5-56T5-53S4蛋白的纯度均约90％，H66N5-56T5-53S2-53S3蛋白的纯度为75％左右。
通过类似的方法，使用大肠杆菌和pTO-T7-HPV66N5质粒制备和纯化了HPV66N5蛋白；使用大肠杆菌和pTO-T7-HPV56L1N0质粒制备和纯化了HPV56N0蛋白；使用大肠杆菌和pTO-T7-HPV53N5质粒制备和纯化了HPV53N5蛋白。
突变蛋白的免疫印迹实验
按上述方法对经纯化的H66N5-56T1，H66N5-56T2，H66N5-56T3，H66N5-56T4，H66N5-56T5，H66N5-56T5-53S1，H66N5-56T5-53S2，H66N5-56T5-53S4蛋白进行电泳。电泳结束后，使用抗HPV L1蛋白的广谱抗体4B3进行Western Blot检测，结果示于图2中。结果显示，突变蛋白H66N5-56T1，H66N5-56T2，H66N5-56T3，H66N5-56T4，H66N5-56T5，H66N5-56T5-53S1，H66N5-56T5-53S2，H66N5-56T5-53S4均能够被广谱抗体4B3特异性识别。
实施例2：HPV病毒样颗粒的组装与颗粒形态学检测
HPV病毒样颗粒的组装
取一定体积(约2ml)的蛋白H66N5-56T1，H66N5-56T2，H66N5-56T3，H66N5-56T4，H66N5-56T5，H66N5-56T5-53S1，H66N5-56T5-53S2，H66N5-56T5-53S4，H66N5-56T5-53S2-53S3分别依次透析至(1)2L储存缓冲液(20mM磷酸钠缓冲液pH 6.5，0.5M NaCl)；(2)2L复性缓冲液(50mM磷酸钠缓冲液pH 6.0，2mM CaCl 2，2mM MgCl 2，0.5M NaCl)；和(3)20mM磷酸钠缓冲液pH 7.0，0.5M NaCl中。在三种缓冲液中各自进行透析12h。
通过类似的方法，将HPV66N5、HPV56N0和HPV53N5蛋白分别组装为HPV66N5 VLP、HPV56N0 VLP和HPV53N5 VLP。
分子筛层析分析
用美国安捷伦公司的1120Compact LC高效液相色谱系统对经透析的样品进行分子筛层析分析，其中，所使用的分析柱为TSK Gel PW5000xl 7.8x300mm。分析结果如图3和图4所示。结果显示，包含蛋白H66N5-56T1，H66N5-56T2，H66N5-56T3，H66N5-56T4，H66N5-56T5，H66N5-56T5-53S4的样品最先出现的蛋白峰均在13-14min左右，与HPV66N5 VLP、HPV56N0 VLP以及HPV53N5 VLP相当，这表明，这些蛋白均可组装成VLP；H66N5-56T5-53S2-53S3 VLP、H66N5-56T5-53S1和H66N5-56T5-53S2的峰形不对称，且H66N5-56T5-53S1的出峰时间在15min左右，表明H66N5-56T5-53S1形成的VLP颗粒直径较小，H66N5-56T5-53S2和H66N5-56T5-53S2-53S3形成的颗粒不均一。
沉降速率分析
沉降速率分析所使用的仪器为Beckman XL-A分析型超速离心机，其配有光学检测系统及An-50Ti和An-60Ti转头。采用沉降速率法分析H66N5-56T1 VLP、H66N5-56T2 VLP、H66N5-56T3 VLP、H66N5-56T4 VLP、H66N5-56T5 VLP、H66N5-56T5-53S1 VLP、H66N5-56T5-53S2 VLP、H66N5-56T5-53S4 VLP和H66N5-56T5-53S2-53S3 VLPHPV56N0 VLP、HPV66N5 VLP和HPV53N5 VLP的沉降系数。结果如图5和图6所示：结果显示，H66N5-56T1 VLP、H66N5-56T2 VLP、H66N5-56T3 VLP、H66N5-56T4 VLP、H66N5-56T5 VLP、H66N5-56T5-53S1 VLP、H66N5-56T5-53S2 VLP和H66N5-56T5-53S4 VLP的沉降系数分别为126S、125S、126S、127S、128S、93S、106S和116S，H66N5-56T5-53S2-53S3 VLP的沉降系数为53S和107S，HPV56N0 VLP、HPV66N5 VLP和HPV53N5 VLP的沉降系数分别为134S和141S和130S。这表明，H66N5-56T1 VLP、H66N5-56T2 VLP、H66N5-56T3 VLP、H66N5-56T4 VLP、H66N5-56T5 VLP和H66N5-56T5-53S4 VLP各自能够组装成大小、形态与野生型VLP(HPV56N0 VLP、HPV66N5 VLP和HPV53N5 VLP)相似的病毒样颗粒；而H66N5-56T5-53S1、H66N5-56T5-53S2 VLP和H66N5-56T5-53S2-53S3 VLP的直径较小，且H66N5-56T5-53S2 VLP和H66N5-56T5-53S2-53S3 VLP的颗粒大小不均一。
病毒样颗粒的形态学检测
取100μL含有VLP的样品进行透射电镜观察。所使用的仪器为日本电子公司生产的100kV透射电镜，放大倍数为100,000倍。简言之，取13.5μL样品，用2％磷钨酸pH7.0进行负染，并固定于喷碳的铜网上，然后进行透射电镜观察。观察结果如图7A-7L所示。结果显示，H66N5-56T1，H66N5-56T2，H66N5-56T3，H66N5-56T4，H66N5-56T5，H66N5-56T5-53S2，H66N5-56T5-53S4，H66N5-56T5-53S2-53S3均可组装成病毒样颗粒。此外，结果还显示，H66N5-56T1，H66N5-56T2，H66N5-56T3，H66N5-56T4，H66N5-56T5，H66N5-56T5-53S4所组装形成的颗粒的半径均在25nm左右，大小均一，这表明，这些突变蛋白与HPV66、HPV56和HPV53的L1蛋白类似，能够形成大小均一的VLP。H66N5-56T5-53S2和H66N5-56T5-53S2-53S3也能够形成半径在25nm左右病毒样颗粒，但其中含有直径为20nm左右的小颗粒；而H66N5-56T5-53S1能形成半径为10-20nm左右的颗粒不规则的VLP。
实施例3：用病毒样颗粒免疫后小鼠血清中的中和抗体滴度的评价1
本实验中，所用病毒样颗粒为：H66N5-56T1 VLP，H66N5-56T2 VLP，H66N5-56T3 VLP，H66N5-56T4 VLP，H66N5-56T5 VLP。
在本实验中，免疫方案如表4所示。将所有小鼠(6周龄BalB/c雌性小鼠)分为3个组：铝佐剂组1(免疫剂量为5μg，使用铝佐剂)，铝佐剂组2(免疫剂量为1μg，使用铝佐剂)，和铝佐剂组3(免疫剂量为0.2μg，使用铝佐剂)。各个组又细分为8个亚组，对照亚组1和2分别用单独的HPV66N5 VLP和单独的HPV56N0 VLP进行免疫，对照亚组3用混合的HPV66/HPV56 VLP(即，HPV66N5 VLP和HPV56N0 VLP的混合物，其中每种VLP均以指定的免疫剂量施用)进行免疫。实验亚组1、2、3、4、5分别用H66N5-56T1VLP、H66N5-56T2VLP、H66N5-56T3VLP、H66N5-56T4VLP、H66N5-56T5 VLP进行免疫。
对于铝佐剂组1-3，采用腹腔注射方式免疫5只小鼠/亚组，免疫剂量分别为5μg、1μg、0.2μg，注射体积为1ml。所有小鼠均在第0周进行初次免疫，然后在第2和4周各自进行加强免疫一次。在第6周对小鼠进行眼眶采血，并分析血清中的抗HPV66和HPV56抗体的滴度。分析结果如 图8A-8C所示。结果显示，H66N5-56T4 VLP和H66N5-56T5 VLP能诱导小鼠产生高滴度的针对HPV66的中和抗体，其保护效果与同剂量的单独的HPV66N5 VLP相当，且显著优于同剂量的单独的HPV56N0 VLP；并且其能诱导小鼠产生高滴度的针对HPV56的中和抗体，其保护效果与同剂量的单独的HPV56N0 VLP相当，且显著优于同剂量的单独的HPV66N5 VLP。这表明，H66N5-56T4 VLP和H66N5-56T5 VLP对HPV66和HPV56具有良好的交叉免疫原性和交叉保护性。
表4免疫方案
Figure PCTCN2019089940-appb-000052
实施例4：病毒样颗粒诱导血清转换的ED 50的评价
本实验中，所用病毒样颗粒为H66N5-56T4 VLP和H66N5-56T5 VLP。
采用铝佐剂、单次腹腔注射方式对6周龄的BalB/c雌鼠(8只)进行免疫，其中，实验组使用H66N5-56T4 VLP和H66N5-56T5 VLP，对照组使用单独的HPV56N0 VLP或单独的HPV66N5 VLP或混合的HPV66/HPV56 VLP(即，HPV66N5 VLP与HPV56N0 VLP的混合物)；免疫剂量为0.900μg、0.300μg、0.100μg、0.033μg或0.011μg；免疫体积为1mL。另外，还将用于稀释疫苗的稀释液用作空白对照。每组免疫8只小鼠，并且在免疫后第五周，抽取眼球静脉血，检测血清中的HPV抗体，并通过Reed-Muench法(Reed LJ MH.A simple method of estimating fifty percent endpoints.Am J Hyg.1938；27:493-7)来计算各个样品诱导血清转换(即，诱导小鼠产生抗体)的ED 50。结果如表5-9所示。
表5.HPV66N5 VLP诱导小鼠产生抗HPV66、抗HPV56抗体(血清转换)的ED 50
Figure PCTCN2019089940-appb-000053
表6.HPV56N0 VLP诱导小鼠产生抗HPV66、抗HPV56抗体(血清转换)的ED 50
Figure PCTCN2019089940-appb-000054
表7.H66N5-56T4 VLP诱导小鼠产生抗HPV66、抗HPV56抗体(血清转换)的ED 50
Figure PCTCN2019089940-appb-000055
表8.H66N5-56T5 VLP诱导小鼠产生抗HPV66、抗HPV56抗体(血清转换)的ED 50
Figure PCTCN2019089940-appb-000056
表9.混合的HPV66/HPV56 VLP诱导小鼠产生抗HPV66、抗HPV56抗体(血清转换)的ED 50
Figure PCTCN2019089940-appb-000057
结果显示，在免疫小鼠5周后，H66N5-56T5 VLP诱导小鼠产生抗HPV66抗体的ED 50与单独的HPV66N5 VLP相当，且显著优于单独的HPV56N0 VLP；并且，其诱导小鼠产生抗HPV56抗体的ED 50虽稍弱于单独的HPV56N0 VLP，但显著优于单独的HPV66N5 VLP。这表明， H66N5-56T5 VLP对HPV66和HPV56具有良好的交叉免疫原性和交叉保护性。
实施例5：用病毒样颗粒免疫后小鼠血清中的中和抗体滴度的评价2
本实验中，所用的病毒颗粒为H66N5-56T5-53S1 VLP、H66N5-56T5-53S2 VLP、H66N5-56T5-53S4 VLP。
在本实验中，免疫方案如表10所示。将所有小鼠(6周龄BalB/c雌性小鼠)分为2个组：铝佐剂组1(免疫剂量为5μg，使用铝佐剂)和铝佐剂组2(免疫剂量为1μg，使用铝佐剂)。各个组再细分为7个亚组，对照亚组1、2、3分别用单独的HPV66N5 VLP、单独的HPV56N0 VLP、单独的HPV53N5 VLP进行免疫，对照亚组4用混合的HPV66/56/53 VLP(即，HPV66N5 VLP、HPV56N0 VLP和HPV53N5 VLP的混合物，其中每种VLP均以指定的免疫剂量施用)进行免疫。实验亚组1、2、3分别用H66N5-56T5-53S1 VLP、H66N5-56T5-53S2 VLP、H66N5-56T5-53S4 VLP进行免疫。
对于铝佐剂组1-2，采用腹腔注射方式免疫5只小鼠/亚组，免疫剂量分别为5μg、1μg，注射体积为1ml。所有小鼠均在第0周进行初次免疫，然后在第2和4周各自进行加强免疫一次。在第6周对小鼠进行眼眶采血，并分析血清中的抗HPV66、HPV56和HPV53抗体的滴度。分析结果如图9A-9B所示。结果显示H66N5-56T5-53S1、H66N5-56T5-53S2和H66N5-56T5-53S4 VLP都能诱导小鼠产生高滴度的针对HPV66的中和抗体，其保护效果虽然略微弱于同剂量的单独的HPV66N5 VLP以及混合的HPV66/56/53 VLP，但仍显著优于同剂量的单独的HPV56N0 VLP或单独的HPV53N5 VLP；并且其能诱导小鼠产生高滴度的针对HPV56的中和抗体，其保护效果与同剂量的单独的HPV56N0 VLP以及混合的HPV66/56/53 VLP相当，且显著优于同剂量的单独的HPV66N5 VLP或单独的HPV53N5 VLP；并且其能诱导小鼠产生高滴度的针对HPV53的中和抗体，其保护效果与同剂量的单独的HPV53N5 VLP以及混合的HPV66/56/53 VLP相当，且显著优于同剂量的单独的HPV66N5 VLP或单独的HPV56N0 VLP。这表明H66N5-56T5-53S1 VLP、H66N5-56T5-53S2 VLP和H66N5-56T5-53S4 VLP对HPV66、HPV56和HPV53具有良好的 交叉免疫原性和交叉保护性。
表10免疫方案
Figure PCTCN2019089940-appb-000058
实施例6：用病毒样颗粒免疫后小鼠血清中的中和抗体滴度的评价3
本实验中，所用的病毒颗粒为H66N5-56T5-53S2-53S3 VLP。
在本实验中，免疫方案如表11所示。将所有小鼠(6周龄BalB/c雌性小鼠)分为2个组：实验组(免疫剂量为5μg，使用铝佐剂)和对照组(免疫剂量为5μg，使用铝佐剂)。其中对照组又分为4个亚组，对照亚组1、2、3分别用单独的HPV66N5 VLP、单独的HPV56N0 VLP、单独的HPV53N5 VLP进行免疫，对照亚组4用混合的HPV66/56/53 VLP(即，HPV66N5 VLP、HPV56N0 VLP和HPV53N5 VLP的混合物，其中每种VLP均以指定的免疫剂量施用)进行免疫。实验组用H66N5-56T5-53S2-53S3 VLP进行免疫。
采用腹腔注射方式免疫5只小鼠/亚组，免疫剂量分别为5μg，注射体积为1ml。所有小鼠均在第0周进行初次免疫，然后在第2和4周各自进行加强免疫一次。在第6周对小鼠进行眼眶采血，并分析血清中的抗HPV66、HPV56和HPV53抗体的滴度。分析结果如图10所示。结果显示H66N5-56T5-53S2-53S3 VLP能诱导小鼠产生高滴度的针对HPV66的中和抗体，其保护效果与同剂量的单独的HPV66N5 VLP以及混合的 HPV66/56/53 VLP相当，且显著优于同剂量的单独的HPV56N0 VLP或单独的HPV53N5 VLP；并且其能诱导小鼠产生高滴度的针对HPV56的中和抗体，其保护效果虽稍弱于同剂量的单独的HPV56N0 VLP、单独的HPV66N5 VLP以及混合的HPV66/56/53 VLP，但显著优于同剂量的单独的HPV53N5 VLP；并且其能诱导小鼠产生高滴度的针对HPV53的中和抗体，其保护效果虽稍弱于同剂量的单独的HPV53N5 VLP以及混合的HPV66/56/53 VLP，但显著优于同剂量的单独的HPV66N5 VLP或单独的HPV56N0 VLP。这表明H66N5-56T5-53S2-53S3 VLP对HPV66、HPV56和HPV53具有良好的交叉免疫原性和交叉保护性。H66N5-56T5-53S2-53S3 VLP可用作预防HPV66感染、HPV56感染和HPV53感染的有效疫苗，可用于代替含有HPV66VLP、HPV56 VLP和HPV53 VLP的混合疫苗。
表11免疫方案
Figure PCTCN2019089940-appb-000059
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解，根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (12)

  1. 一种突变的HPV66 L1蛋白或其变体，其中，所述突变的HPV66 L1蛋白与野生型HPV66 L1蛋白相比，具有下述突变：
    (1)N端截短了1-20个氨基酸，例如1-5个、3-8个、5-10个、9-13个、10-15个、12-18个或15-20个氨基酸；和
    (2)(a)位于野生型HPV66 L1蛋白第265-283位的氨基酸残基被替换为第二型别的野生型HPV的L1蛋白的相应位置的氨基酸残基；或
    (b)位于野生型HPV66 L1蛋白第347-357位的氨基酸残基被替换为第二型别的野生型HPV的L1蛋白的相应位置的氨基酸残基；
    并且，所述变体与所述突变的HPV66 L1蛋白相异仅在于一个或几个(例如，1个、2个、3个、4个、5个、6个、7个、8个或9个)氨基酸的置换(优选保守置换)、添加或缺失，且保留了所述突变的HPV66 L1蛋白的功能，即，能够诱导针对至少两个型别的HPV(例如，HPV66和HPV56，或者HPV66、HPV56和HPV53)的中和抗体；
    优选地，所述突变的HPV66 L1蛋白具有(2)(b)中所定义的突变，并且任选地，还具有下述突变：
    (3)位于野生型HPV66 L1蛋白第51-60位的氨基酸残基被替换为第三型别的野生型HPV L1蛋白的相应位置的氨基酸残基；或
    (4)位于野生型HPV66 L1蛋白第114-150的氨基酸残基被替换为第三型别的野生型HPV L1蛋白的相应位置的氨基酸残基；或
    (5)位于野生型HPV66 L1蛋白第259-283的氨基酸残基被替换为第三型别的野生型HPV L1蛋白的相应位置的氨基酸残基；
    优选地，所述突变的HPV66 L1蛋白具有(2)(b)和(4)中所定义的突变，并且任选地，还具有下述突变：
    (6)位于野生型HPV66 L1蛋白第172-181的氨基酸残基被替换为第三型别的野生型HPV L1蛋白的相应位置的氨基酸残基；
    优选地，所述突变的HPV66 L1蛋白与野生型HPV66 L1蛋白相比， N端截短了3个、5个、8个、10个、12个、15个、18个氨基酸；
    优选地，所述突变的HPV66 L1蛋白与野生型HPV66 L1蛋白相比，N端截短了5个氨基酸；
    优选地，所述第二型别的野生型HPV为HPV56；
    优选地，(2)(a)中所述的相应位置的氨基酸残基为野生型HPV56 L1蛋白第265-283位的氨基酸残基；
    优选地，(2)(b)中所述的相应位置的氨基酸残基为野生型HPV56 L1蛋白第347-357位的氨基酸残基；
    优选地，所述第三型别的野生型HPV为HPV53；
    优选地，(3)中所述的相应位置的氨基酸残基为野生型HPV53 L1蛋白第51-59位的氨基酸残基；
    优选地，(4)中所述的相应位置的氨基酸残基为野生型HPV53 L1蛋白第113-149位的氨基酸残基；
    优选地，(5)中所述的相应位置的氨基酸残基为野生型HPV53 L1蛋白第258-282位的氨基酸残基；
    优选地，(6)中所述的相应位置的氨基酸残基为野生型HPV53 L1蛋白第171-180位的氨基酸残基；
    优选地，所述野生型HPV66 L1蛋白具有如SEQ ID NO:1或28所示的氨基酸序列；
    优选地，所述野生型HPV56 L1蛋白具有如SEQ ID NO:2所示的氨基酸序列；
    优选地，所述野生型HPV53 L1蛋白具有如SEQ ID NO:3或31所示的氨基酸序列；
    优选地，所述突变的HPV66 L1蛋白具有选自下列的氨基酸序列：SEQ ID NO:7、8、9、10、11和63。
  2. 一种分离的核酸，其编码权利要求1所述的突变的HPV66 L1蛋白或其变体，
    优选地，所述分离的核酸具有选自下列的核苷酸序列：SEQ ID NO:18、19、20、21、22和64。
  3. 包含权利要求2所述的分离的核酸的载体。
  4. 包含权利要求2所述的分离的核酸和/或权利要求3所述的载体的宿主细胞。
  5. 一种HPV病毒样颗粒，其含有权利要求1所述的突变的HPV66 L1蛋白或其变体，或者由权利要求1所述的突变的HPV66 L1蛋白或其变体组成或形成。
  6. 一种组合物，其包含权利要求1所述的突变的HPV66 L1蛋白或其变体，或权利要求2的分离的核酸，或权利要求3的载体，或权利要求4的宿主细胞，或权利要求5的HPV病毒样颗粒。
  7. 一种药物组合物或疫苗，其包含权利要求5所述的HPV病毒样颗粒，任选地还包含药学可接受的载体和/或赋形剂，
    优选地，所述HPV病毒样颗粒以预防HPV感染或由HPV感染导致的疾病的有效量存在；
    优选地，所述HPV感染是一个或多个型别的HPV感染(例如，HPV66感染、HPV56感染和/或HPV53感染)；
    优选地，所述由HPV感染所导致的疾病选自宫颈癌和尖锐湿疣。
  8. 制备权利要求1所述的突变的HPV66L1蛋白或其变体的方法，其包括，在宿主细胞中表达所述突变的HPV66 L1蛋白或其变体，然后从所述宿主细胞的培养物中回收所述突变的HPV 66L1蛋白或其变体；
    优选地，所述宿主细胞为大肠杆菌；
    优选地，所述方法包括步骤：在大肠杆菌中表达所述突变的HPV66 L1蛋白或其变体，然后从所述大肠杆菌的裂解上清中纯化得到所述突变的HPV66 L1蛋白或其变体；优选地，通过色谱法(例如，阳离子交换色谱，羟基磷灰石色谱和/或疏水相互作用色谱)，从所述大肠杆菌的裂解上清中回收所述突变的HPV66 L1蛋白或其变体。
  9. 一种制备疫苗的方法，其包括将权利要求5所述的HPV病毒样颗粒与药学可接受的载体和/或赋形剂混合。
  10. 一种预防HPV感染或由HPV感染所导致的疾病的方法，其包括将预防有效量的权利要求5所述的HPV病毒样颗粒或权利要求7所述的药物组合物或疫苗施用给受试者，
    优选地，所述HPV感染是一个或多个型别的HPV感染(例如，HPV66感染、HPV56感染和/或HPV53感染)；
    优选地，所述由HPV感染所导致的疾病选自宫颈癌和尖锐湿疣。
  11. 权利要求1所述的突变的HPV66 L1蛋白或其变体或权利要求5的HPV病毒样颗粒在制备药物组合物或疫苗中的用途，所述药物组合物或疫苗用于预防HPV感染或由HPV感染所导致的疾病，
    优选地，所述HPV感染是一个或多个型别的HPV感染(例如，HPV66感染、HPV56感染和/或HPV53感染)；
    优选地，所述由HPV感染所导致的疾病选自宫颈癌和尖锐湿疣。
  12. 权利要求1所述的突变的HPV39 L1蛋白或其变体或权利要求5所述的HPV病毒样颗粒，其用于预防HPV感染或由HPV感染所导致的疾病，
    优选地，所述HPV感染是一个或多个型别的HPV感染(例如，HPV39感染、HPV68感染和/或HPV70感染)；
    优选地，所述由HPV感染所导致的疾病选自宫颈癌和尖锐湿疣。
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