Mutants of the L1 protein of human papillomavirus type 39

Abstract

The present invention relates to a mutant HPV39 L1 protein (or a variant thereof), a sequence encoding the same, a method for preparing the same, and a virus-like particle comprising the same, wherein the protein (or a variant thereof) and the virus-like particle are capable of inducing the development of neutralizing antibodies against at least two HPV types (e.g., HPV39 and HPV68, or HPV39, HPV68, and HPV70) and can therefore be used to prevent infection with the at least two HPV types and diseases caused by such infection (such as cervical cancer and genital warts). The present invention further relates to the use of the protein and the virus-like particle in the manufacture of a pharmaceutical composition or vaccine for preventing infection with the at least two HPV types and diseases caused by such infection (such as cervical cancer and genital warts).

Description

[Technical field]

[0001] This application is based on and claims the benefit of priority from Chinese Application No. 201810563378.0, filed on June 4, 2018, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Technical Field The present invention relates to the field of molecular virology and immunology.In particular, the present invention relates to a mutant HPV39 L1 protein (or its variant), a sequence encoding it, a method for preparing it, and a virus-like particle containing it, wherein the protein (or its variant) and the virus-like particle can induce the development of neutralizing antibodies against at least two HPV types (for example, HPV39 and HPV68, or HPV39, HPV68, and HPV70), and can therefore be used to prevent infection by at least two HPV types and diseases caused by said infection (such as cervical cancer and genital warts).The present invention also relates to the use of the protein and the virus-like particle in the manufacture of a pharmaceutical composition or vaccine for preventing infection by at least two HPV types and diseases caused by said infection (such as cervical cancer and genital warts). [Background technology]

[0003] Human papillomavirus (HPV) mainly causes warts on the skin and mucous membranes. HPV types are divided into high-risk and low-risk types depending on their association with tumor formation. Among them, infection with high-risk HPV types has been shown to be the main cause of genital cancers, including cervical cancer, in women, while low-risk HPV types mainly cause genital warts. The most effective method for preventing and controlling HPV infection is HPV vaccination, especially vaccination against high-risk HPV types that cause cervical cancer.

[0004] The HPV major capsid protein L1 is characterized by its ability to self-assemble into hollow virus-like particles (VLPs). HPV VLPs have a symmetrical icosahedral structure composed of 72 L1 pentamers (Doorbar, J. and PH Gallimore. 1987. J Virol, 61(9): 2793-9). HPV VLPs are structurally very similar to native HPV, retain most of the neutralizing epitopes of the native virus, and are capable of inducing the development of high titers of neutralizing antibodies (Kirnbauer, R., F. Booy, et al. 1992 Proc Natl Acad Sci USA 89(24): 12180-4).

[0005] However, previous studies have shown that HPV VLPs mainly induce the development of neutralizing antibodies against the same type of HPV, provide protective immunity against the same type of HPV, and only have low cross-protective effects against a few highly homologous HPV types (Sara L. Bissett, Giada Mattiuzzo, et al. 2014 Vaccine. 32:6548-6555). Therefore, the protective scope of existing HPV vaccines is very limited. In general, VLPs of one type of HPV can only be used to prevent infection by the same type of HPV. In this case, if it is necessary to broaden the protective scope of the HPV vaccine, the only option is to add VLPs of more types of HPV to the vaccine. Currently, commercial HPV vaccines, including Merck's Gardasil® (a quadrivalent vaccine against HPV 16, 18, 6, and 11), GSK's Cervarix® (a bivalent vaccine against HPV 16 and 18), and Merck's Gardasil® 9 (a 9-valent vaccine against HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58), are prepared by combining VLPs of multiple HPV types. However, such a solution would significantly increase the manufacturing costs of the HPV vaccine and may also raise safety concerns due to increased immunization doses. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there is an urgent need in the art to develop HPV virus-like particles capable of inducing the development of protective neutralizing antibodies against multiple HPV types in order to more economically and effectively prevent infection with multiple HPV types and the diseases caused by this infection, such as cervical cancer and genital warts. [Means for solving the problem]

[0007] Contents of the invention The present invention is based at least in part on the surprising discovery by the inventors that after replacing a specific segment of the L1 protein of human papillomavirus (HPV) type 39 with the corresponding segment of the L1 protein of a second type of HPV (such as HPV68), the resulting mutated HPV39 L1 protein can induce the development of high titer neutralizing antibodies against HPV39 and the second type of HPV (such as HPV68) in an organism, with a protective effect equivalent to that of a mixture of HPV39 VLPs and VLPs of the second type of HPV, with a protective effect equivalent to that of HPV68 VLPs alone, and with a protective effect equivalent to that of the VLPs of the second type of HPV (such as HPV68) alone.

[0008] In addition, based on the substitutions described above, other specific segments of the HPV39 L1 protein can be further replaced with corresponding segments of the L1 protein of a third type of HPV (such as HPV70), and the resulting mutant HPV39 L1 protein with double substitutions can induce the development of high-titer neutralizing antibodies against HPV39, a second type of HPV (such as HPV68), and a third type of HPV (such as HPV70), and its protective effect is equivalent to that of a mixture of HPV39 VLPs, a VLP of a second type of HPV, and a VLP of a third type of HPV, its protective effect against HPV39 is equivalent to that of HPV39 VLPs alone, its protective effect against the second type of HPV (such as HPV68) is equivalent to that of the VLP of the second type of HPV alone, and its protective effect against the third type of HPV (such as HPV70) is equivalent to that of the VLP of the third type of HPV alone.

[0009] Thus, in one aspect, the present invention provides a mutated HPV39 L1 protein or a variant thereof, wherein, in comparison to a wild type HPV39 L1 protein, the mutated HPV39 L1 protein has the following mutations: (1) truncation of 1 to 25 amino acids at the N-terminus, e.g., 1 to 5, 1 to 10, 1 to 15, 1 to 20, 5 to 15, 10 to 15, 10 to 20, or 15 to 20 amino acids; and (2) replacement of amino acid residues at positions 269 to 288 of the wild-type HPV39 L1 protein with amino acid residues at the corresponding positions of the L1 protein of a wild-type HPV of a second type; having The variant differs from the mutant HPV39 L1 protein only by having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acids substituted (preferably conservatively substituted), added, or deleted, and retains the function of the mutant HPV39 L1 protein, i.e., the ability to induce the development of neutralizing antibodies against at least two HPV types (e.g., HPV39 and HPV68, or HPV39, HPV68, and HPV70).

[0010] In some preferred embodiments, the mutant HPV39 L1 protein optionally further comprises the following mutations: (3)(a) a substitution of amino acid residues at positions 117 to 140 of the wild-type HPV39 L1 protein with amino acid residues at the corresponding positions of the L1 protein of a wild-type HPV of a third type; or (b) a substitution of amino acid residues at positions 169 to 181 of the wild-type HPV39 L1 protein with amino acid residues at the corresponding positions of the L1 protein of a wild-type HPV of a third type; or (c) Substitution of amino acid residues at positions 347 to 358 of the wild-type HPV39 L1 protein with amino acid residues at the corresponding positions of the L1 protein of a wild-type HPV of a third type. has.

[0011] In some preferred embodiments, the mutant HPV39 L1 protein is truncated by 3, 5, 8, 10, 12, 15, 18, 20, or 22 amino acids at the N-terminus compared to the wild-type HPV39 L1 protein. In some preferred embodiments, the mutant HPV39 L1 protein is truncated by 15 amino acids at the N-terminus compared to the wild-type HPV39 L1 protein.

[0012] In some preferred embodiments, the wild-type HPV of the second type is HPV68. In some preferred embodiments, the amino acid residues at the corresponding positions described in (2) are amino acid residues at positions 270 to 289 of the wild-type HPV68 L1 protein. In some preferred embodiments, the wild-type HPV of the third type is HPV70. In some preferred embodiments, the amino acid residues at the corresponding positions described in (3)(a) are amino acid residues at positions 117 to 141 of the wild-type HPV70 L1 protein. In some preferred embodiments, the amino acid residues at the corresponding positions described in (3)(b) are amino acid residues at positions 170 to 182 of the wild-type HPV70 L1 protein. In some preferred embodiments, the amino acid residues at the corresponding positions described in (3)(c) are amino acid residues at positions 348 to 359 of the wild-type HPV70 L1 protein.

[0013] In some preferred embodiments, the wild-type HPV39 L1 protein has the amino acid sequence of SEQ ID NO:1.

[0014] In some preferred embodiments, the wild-type HPV68 L1 protein has the amino acid sequence of SEQ ID NO:2.

[0015] In some preferred embodiments, the wild-type HPV70 L1 protein has the amino acid sequence of SEQ ID NO:3.

[0016] In some preferred embodiments, amino acid residues 270 to 289 of the wild-type HPV68 L1 protein have the sequence of SEQ ID NO:25.

[0017] In some preferred embodiments, amino acid residues 117 to 141 of the wild-type HPV70 L1 protein have the sequence of SEQ ID NO:26.

[0018] In some preferred embodiments, amino acid residues 170 to 182 of the wild-type HPV70 L1 protein have the sequence of SEQ ID NO:27.

[0019] In some preferred embodiments, amino acid residues 348 to 359 of the wild-type HPV70 L1 protein have the sequence of SEQ ID NO:28.

[0020] In some preferred embodiments, the mutant HPV39 L1 protein has an amino acid sequence selected from the group consisting of SEQ ID NOs:7, 10, 11, and 12.

[0021] In another aspect, the present invention provides an isolated nucleic acid encoding the mutant HPV39 L1 protein or a variant thereof described above. In another aspect, the present invention provides a vector comprising the isolated nucleic acid. In some preferred embodiments, the isolated nucleic acid of the present invention has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 19, 22, 23, and 24.

[0022] Vectors useful for inserting a polynucleotide of interest are known in the art and include, but are not limited to, cloning vectors and expression vectors. In one embodiment, the vector is, for example, a plasmid, cosmid, phage, etc.

[0023] In another aspect, the present invention also relates to a host cell comprising the above-mentioned isolated nucleic acid or vector.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, e.g., mouse cells, human cells, etc.).The host cell of the present invention may be a cell line such as 293T cells and 293TT cells.

[0024] In another aspect, the present invention relates to an HPV virus-like particle comprising or consisting of a mutated HPV39 L1 protein or a variant thereof according to the present invention.

[0025] In some preferred embodiments, the HPV virus-like particle of the present invention comprises a mutant HPV39 L1 protein in which 1 to 25 amino acids, for example 1 to 5, 1 to 10, 1 to 15, 1 to 20, 5 to 15, 10 to 15, 10 to 20, or 15 to 20 amino acids, for example 3, 5, 8, 11, 13, 15, 18, 20, or 22 amino acids, have been truncated at the N-terminus compared to the wild-type HPV39 L1 protein, and amino acid residues at positions 269 to 288 of the wild-type HPV39 L1 protein have been replaced with amino acid residues at positions 270 to 289 of the wild-type HPV68 L1 protein.

[0026] In some preferred embodiments, the HPV virus-like particle according to the present invention comprises a mutant HPV39 L1 protein in which 1 to 25 amino acids, for example 1 to 5, 1 to 10, 1 to 15, 1 to 20, 5 to 15, 10 to 15, 10 to 20, or 15 to 20 amino acids, for example 3, 5, 8, 11, 13, 15, 18, 20, or 22 amino acids, have been truncated at the N-terminus compared to the wild-type HPV39 L1 protein, amino acid residues at positions 269 to 288 of the wild-type HPV39 L1 protein have been substituted with amino acid residues at positions 270 to 289 of the wild-type HPV68 L1 protein, and amino acid residues at positions 117 to 140 of the wild-type HPV39 L1 protein have been substituted with amino acid residues at positions 117 to 141 of the wild-type HPV70 L1 protein.

[0027] In some preferred embodiments, the HPV virus-like particle according to the present invention comprises a mutant HPV39 L1 protein in which 1 to 25 amino acids, for example 1 to 5, 1 to 10, 1 to 15, 1 to 20, 5 to 15, 10 to 15, 10 to 20, or 15 to 20 amino acids, for example 3, 5, 8, 11, 13, 15, 18, 20, or 22 amino acids, have been truncated at the N-terminus compared to the wild-type HPV39 L1 protein, amino acid residues at positions 269 to 288 of the wild-type HPV39 L1 protein have been substituted with amino acid residues at positions 270 to 289 of the wild-type HPV68 L1 protein, and amino acid residues at positions 169 to 181 of the wild-type HPV39 L1 protein have been substituted with amino acid residues at positions 170 to 182 of the wild-type HPV70 L1 protein.

[0028] In some preferred embodiments, the HPV virus-like particle according to the present invention comprises a mutant HPV39 L1 protein in which 1 to 25 amino acids, for example 1 to 5, 1 to 10, 1 to 15, 1 to 20, 5 to 15, 10 to 15, 10 to 20, or 15 to 20 amino acids, for example 3, 5, 8, 11, 13, 15, 18, 20, or 22 amino acids, have been truncated at the N-terminus compared to the wild-type HPV39 L1 protein, amino acid residues at positions 269 to 288 of the wild-type HPV39 L1 protein have been substituted with amino acid residues at positions 270 to 289 of the wild-type HPV68 L1 protein, and amino acid residues at positions 347 to 358 of the wild-type HPV39 L1 protein have been substituted with amino acid residues at positions 348 to 359 of the wild-type HPV70 L1 protein.

[0029] In a particularly preferred embodiment, the HPV virus-like particle according to the invention comprises a mutated HPV39 L1 protein having the sequence of SEQ ID NO:7, 10, 11, or 12.

[0030] In another aspect, the present invention further relates to a composition comprising the mutant HPV39 L1 protein or variant thereof, the isolated nucleic acid, the vector, the host cell, or the HPV virus-like particle. In some preferred embodiments, the composition comprises the mutant HPV39 L1 protein or variant thereof of the present invention. In some preferred embodiments, the composition comprises the HPV virus-like particle of the present invention.

[0031] In another aspect, the present invention further relates to a pharmaceutical composition or vaccine comprising the HPV virus-like particle of the present invention and, optionally, a pharma- ceutical 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).

[0032] In some preferred embodiments, the HPV virus-like particles are present in an amount effective to prevent HPV infection or a disease caused by HPV infection. In some preferred embodiments, the HPV infection is an infection with one or more HPV types (e.g., HPV39 infection, HPV68 infection, and / or HPV70 infection). In some preferred embodiments, the disease caused by HPV infection is selected from the group consisting of cervical cancer and genital warts.

[0033] The pharmaceutical composition or vaccine of the present invention may be administered by methods known in the art, such as, but not limited to, oral or injection. In the present invention, a particularly preferred administration route is injection.

[0034] In some preferred embodiments, the pharmaceutical compositions or vaccines of the present invention are administered in the form of unit dosages, for example and without intending to limit the invention, each unit dosage contains 5 μg to 80 μg, preferably 20 μg to 40 μg, of HPV virus-like particles.

[0035] In another aspect, the present invention relates to a method for preparing a mutated HPV39 L1 protein or a variant thereof as described above, comprising expressing the mutated HPV39 L1 protein or a variant thereof in a host cell and then recovering the mutated HPV39 L1 protein or a variant thereof from the culture of the host cell.

[0036] In some preferred embodiments, the host cell is E. coli. In some preferred embodiments, the method comprises expressing a mutant HPV39 L1 protein or variant thereof in E. coli and then obtaining the mutant HPV39 L1 protein or variant thereof by purifying the E. coli lysate supernatant. In some preferred embodiments, the mutant HPV39 L1 protein or variant thereof is recovered from the E. coli lysate supernatant by chromatography (e.g., cation exchange chromatography, hydroxyapatite chromatography, and / or hydrophobic interaction chromatography).

[0037] In another aspect, the present invention relates to a method for preparing a vaccine, comprising combining an HPV virus-like particle according to the present invention with a pharma- ceutically acceptable carrier and / or excipient.

[0038] In another aspect, 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 the HPV virus-like particle or pharmaceutical composition or vaccine according to the present invention. In a preferred embodiment, the HPV infection is an infection by one or more HPV types (e.g., HPV39 infection, HPV68 infection, and / or HPV70 infection). In another preferred embodiment, the disease caused by HPV infection includes, but is not limited to, cervical cancer and genital warts. In another preferred embodiment, the subject is a mammal, such as a human.

[0039] In another aspect, the present invention further relates to the use of the mutated HPV39 L1 protein or variant thereof or HPV virus-like particle according to the present invention in the manufacture of a pharmaceutical composition or vaccine for preventing HPV infection or a disease caused by HPV infection. In a preferred embodiment, the HPV infection is an infection by one or more HPV types (e.g., HPV39 infection, HPV68 infection, and / or HPV70 infection). In another preferred embodiment, the disease caused by HPV infection includes, but is not limited to, cervical cancer and genital warts.

[0040] In another aspect, the present invention further relates to a mutant HPV39 L1 protein or a variant thereof or a HPV virus-like particle according to the present invention for preventing HPV infection or a disease caused by HPV infection. In a preferred embodiment, the HPV infection is an infection by one or more HPV types (e.g., HPV39 infection, HPV68 infection, and / or HPV70 infection). In another preferred embodiment, the disease caused by HPV infection includes, but is not limited to, cervical cancer and genital warts.

[0041] Definitions of Terms Used in the Invention In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.In addition, the operations in cell culture, molecular genetics, nucleic acid chemistry, and immunology experiments used herein are common operations widely used in the corresponding fields.Here, for the purpose of better understanding the present invention, the definitions and explanations of related terms are provided below.

[0042] In the present invention, the term "a wild-type HPV of a second type" refers to a wild-type HPV type other than HPV 39. In the present invention, the wild-type HPV of a second type is preferably wild-type HPV 68.

[0043] In the present invention, the term "wild type HPV of the third type" refers to a wild type HPV type other than HPV39 and wild type HPV of the second type. In the present invention, the wild type HPV of the third type is preferably wild type HPV70.

[0044] In the present invention, the expression "corresponding positions" refers to equivalent positions in the sequences to be compared when the sequences to be compared are optimally aligned, ie when the sequences to be compared are aligned so as to maximize the percent identity.

[0045] In the present invention, the term "wild-type HPV39 L1 protein" refers to the native major capsid protein L1 of human papillomavirus type 39 (HPV39). The sequence of wild-type HPV39 L1 protein is known in the art and can be found in public databases (such as in the NCBI database under accession numbers P24838.1, ARQ82617.1, AGU90549.1, and AEP23084.1).

[0046] In the present invention, when the amino acid sequence of wild-type HPV39 L1 protein is mentioned, it is described with reference to the sequence of SEQ ID NO: 1. For example, the expression "amino acid residues 53-61 of wild-type HPV39 L1 protein" refers to amino acid residues 53-61 of the polypeptide of SEQ ID NO: 1. However, it is understood by those skilled in the art that wild-type HPV39 may include various isolates, and the amino acid sequence of L1 protein may differ among various isolates. Furthermore, it is understood by those skilled in the art that the amino acid sequence of L1 protein may differ in sequence, but has a very high identity (generally more than 95%, for example more than 96%, more than 97%, more than 98%, or more than 99%) among different HPV39 isolates, and has substantially the same biological function. Therefore, in the present invention, the term "wild-type HPV39 L1 protein" includes not only the protein of SEQ ID NO: 1, but also the L1 proteins of various HPV39 isolates (such as HPV39 L1 proteins of numbers P24838.1, ARQ82617.1, AGU90549.1, and AEP23084.1). In addition, when a sequence fragment of wild-type HPV39 L1 protein is described, this includes not only the sequence fragment of SEQ ID NO: 1, but also the corresponding sequence fragment of L1 protein of various HPV39 isolates. For example, the expression "amino acid residues 53-61 of wild-type HPV39 L1 protein" includes amino acid residues 53-61 of SEQ ID NO: 1 and the corresponding fragment of L1 protein of various HPV39 isolates.

[0047] In the present invention, the term "wild-type HPV68 L1 protein" refers to the native major capsid protein L1 of human papillomavirus type 68 (HPV68). The sequence of wild-type HPV68 L1 protein is known in the art and can be found in public databases (such as in the NCBI database under accession numbers AAZ39498.1, AGU90717.1, P4669.1, and AGU90703.1).

[0048] In the present invention, when the amino acid sequence of wild-type HPV68 L1 protein is mentioned, it is described with reference to the sequence of SEQ ID NO: 2. For example, the expression "amino acid residues 53-61 of wild-type HPV68 L1 protein" refers to amino acid residues 53-61 of the polypeptide of SEQ ID NO: 2. However, it is understood by those skilled in the art that wild-type HPV68 may include various isolates, and the amino acid sequence of L1 protein may differ among various isolates. Furthermore, it is understood by those skilled in the art that the amino acid sequence of L1 protein may differ in sequence, but has a very high identity (generally more than 95%, for example more than 96%, more than 97%, more than 98%, or more than 99%) among different HPV68 isolates, and has substantially the same biological function. Therefore, in the present invention, the term "wild-type HPV68 L1 protein" includes not only the protein of SEQ ID NO: 2, but also the L1 proteins of various HPV68 isolates (such as HPV68 L1 proteins of AAZ39498.1, AGU90717.1, P4669.1, and AGU90703.1). In addition, when a sequence fragment of wild-type HPV68 L1 protein is described, this includes not only the sequence fragment of SEQ ID NO: 2, but also the corresponding sequence fragment of L1 protein of various HPV68 isolates. For example, the expression "amino acid residues 53-61 of wild-type HPV68 L1 protein" includes amino acid residues 53-61 of SEQ ID NO: 2 and the corresponding fragment of L1 protein of various HPV68 isolates.

[0049] In the present invention, the term "wild-type HPV70 L1 protein" refers to the native major capsid protein L1 of human papillomavirus type 70 (HPV70). The sequence of the wild-type HPV70 L1 protein is known in the art and can be found in public databases (such as in the NCBI database under accession numbers AGU90846.1, AGU90854.1, AAC54879.1, and P50793.1).

[0050] In the present invention, when the amino acid sequence of wild-type HPV70 L1 protein is mentioned, it is described with reference to the sequence of SEQ ID NO: 3. For example, the expression "amino acid residues 117-141 of wild-type HPV70 L1 protein" refers to amino acid residues 117-141 of the polypeptide of SEQ ID NO: 3. However, it is understood by those skilled in the art that wild-type HPV70 may include various isolates, and that the amino acid sequence of L1 protein may differ among various isolates. Furthermore, it is understood by those skilled in the art that the amino acid sequence of L1 protein may differ in sequence, but has a very high identity (generally more than 95%, for example more than 96%, more than 97%, more than 98%, or more than 99%) among different HPV70 isolates, and has substantially the same biological function. Thus, in the present invention, the term "wild-type HPV70 L1 protein" includes not only the protein of SEQ ID NO: 3, but also the L1 proteins of various HPV70 isolates (such as HPV70 L1 proteins of AGU90846.1, AGU90854.1, AAC54879.1, and P50793.1). Also described are sequence fragments of wild-type HPV70 L1 protein, including not only the sequence fragment of SEQ ID NO: 3, but also the corresponding sequence fragments of L1 proteins of various HPV70 isolates. For example, the expression "amino acid residues 117-141 of wild-type HPV70 L1 protein" includes amino acid residues 117-141 of SEQ ID NO: 3 and the corresponding fragments of L1 proteins of various HPV70 isolates.

[0051] In the present invention, the term "corresponding sequence fragment" or "corresponding fragment" refers to a fragment that is located at the same position in the compared sequences when the compared sequences are optimally aligned, i.e., when the compared sequences are aligned to maximize the percent identity.

[0052] In the present invention, the phrase "truncated by X amino acids at the N-terminus" or "X amino acids are truncated at the N-terminus" refers to amino acid residues 1 to X at the N-terminus of a protein being replaced with methionine residues encoded by the start codon (to initiate protein translation). For example, an HPV39 L1 protein truncated by 15 amino acids at the N-terminus refers to a protein in which amino acid residues 1 to 15 at the N-terminus of the wild-type HPV39 L1 protein are replaced with methionine residues encoded by the start codon.

[0053] In the present invention, the term "variant" refers to a protein whose amino acid sequence has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (preferably conservative substitutions), additions, or deletions, or has at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the mutant HPV39 L1 protein of the present invention (e.g., the protein of SEQ ID NO: 7, 10, 11, or 12), and retains the function of the mutant HPV39 L1 protein of the present invention. In the present invention, the term "function of mutant HPV39 L1 protein" refers to its ability to induce the development of neutralizing antibodies against at least two HPV types (e.g., HPV39 and HPV68, or HPV39, HPV68, and HPV70). The term "identity" refers to a measure of similarity between nucleotide or amino acid sequences. In general, the sequences are aligned to maximize matching. "Identity" has the meaning known in the art and can be calculated by published algorithms (such as BLAST).

[0054] In the present invention, the term "identity" refers to the degree of match between two polypeptides or two nucleic acids. If the two sequences being compared have the same base or amino acid monomer subunit at a particular site (e.g., if two DNA molecules each have an adenine at a particular site, or if two polypeptides each have a lysine at a particular site), then the two molecules are identical at that site. The percentage of identity between two sequences correlates with the number of identical sites shared by the two sequences over the total number of sites compared (x100). For example, if 6 out of 10 sites are identical in two sequences, the two sequences are 60% identical. For example, the DNA sequences CTGACT and CAGGTT are 50% identical (3 out of 6 sites are identical). In general, the comparison of two sequences is performed in a manner that maximizes identity. Such sequence comparison can be carried out using a computer program such as the Align program (DNAstar) based on the method of Needleman et al. (J. Mol. Biol. 48:443-453, 1970). The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com) using either a Blossum 62 matrix or a PAM250 matrix, with a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6.

[0055] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the essential properties of the protein / polypeptide containing the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-based mutagenesis. Conservative amino acid substitutions include replacing an amino acid residue with another amino acid residue having a similar side chain, for example, a residue that is physically or functionally similar to the amino acid residue in question (e.g., similar in size, shape, charge, chemical properties including ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Thus, in general, a conservative substitution refers to the replacement of a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are known in the art (see, e.g., 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. Sci. USA 94: 412-417 (1997); which are incorporated herein by reference).

[0056] In the present invention, the term "E. coli expression system" refers to an expression system consisting of an E. coli (strain) and a vector, wherein the E. coli (strain) is derived from a commercially available strain, including, but not limited to, ER2566, BL21(DE3), B834(DE3), and BLR(DE3).

[0057] In the present invention, the term "vector" refers to a nucleic acid carrying means into which a polynucleotide can be inserted. If the vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector is capable of expressing the genetic material elements it carries in a host cell by transformation, transduction, or transfection into the host cell. Vectors are known to those skilled in the art and include, but are not limited to, plasmids, phages, cosmids, and the like.

[0058] In the present invention, the term "pharmaceutical 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, which are known in the art (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, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers, surfactants include, but are not limited to, cationic surfactants, anionic surfactants, or non-ionic surfactants, such as, for example, Tween-80, adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide) and Freund's adjuvants (e.g., Freund's complete adjuvant), and ionic strength enhancers include, but are not limited to, NaCl.

[0059] In the present invention, the term "effective amount" refers to the amount that can effectively achieve the intended purpose.For example, the amount that is effective for preventing a disease (such as HPV infection) refers to the amount that is effective for preventing, suppressing or delaying the occurrence of a disease (such as HPV infection).It is within the ability of a person skilled in the art to determine such an effective amount.

[0060] In the present invention, the term "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 chromatography (gel exclusion chromatography), and affinity chromatography.

[0061] In the present invention, the term "lysate supernatant" refers to a solution obtained by the following process: host cells (such as E. coli) are disrupted in a lysis buffer, and then insoluble material is removed from the lysis solution containing the disrupted host cells. Various lysis buffers are known in the art, including but not limited to Tris buffer, phosphate buffer, HEPES buffer, MOPS buffer, etc. In addition, host cell disruption can be performed by methods known to those skilled in the art, including but not limited to homogenizer disruption, sonication, grinding, high-pressure extrusion, lysozyme treatment, etc. Methods for removing insoluble material are also known to those skilled in the art, including but not limited to filtration and centrifugation. Effect of the Invention

[0062] Beneficial Effects of the Invention Studies have shown that while there is some cross-protection between HPV39 and other HPV types (such as HPV68 and HPV70), such cross-protection is very small, typically less than 1 percent, and one thousandth of the protective level of VLPs of the same HPV type. Thus, subjects vaccinated with the HPV39 vaccine are still at high risk of infection with other HPV types (such as HPV68 and HPV70).

[0063] The present invention provides mutated HPV39 L1 protein and HPV virus-like particles formed thereby. The HPV virus-like particles of the present invention provide significant cross-protection against HPV39 and other HPV types (such as HPV68 and HPV70). In particular, when the immunizing dose is the same, the HPV virus-like particles of the present invention can induce the development of high-titer neutralizing antibodies against at least two HPV types (e.g., HPV39 and HPV68, or HPV39, HPV68, and HPV70) in an organism, and the effect is equivalent to that of a mixture of VLPs of multiple HPV types (e.g., a mixture of HPV39 VLPs and HPV68 VLPs, or a mixture of HPV39 VLPs, HPV68 VLPs, and HPV70 VLPs). Therefore, the HPV virus-like particle according to the present invention can be used to prevent infection with at least two HPV types (e.g., HPV39 and HPV68, or HPV39, HPV68, and HPV70) and also diseases associated with said infection, which has a very beneficial technical effect, which is particularly advantageous in terms of expanding the protective scope of HPV vaccines and reducing the production costs of HPV vaccines.

[0064] The embodiments of the present invention will be further described in detail with reference to the drawings and examples. However, those skilled in the art will understand that the following drawings and examples are intended to illustrate the present invention only, and do not define the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following drawings and detailed description of the preferred embodiments. [Brief description of the drawings]

[0065] [Figure 1]1 shows the results of SDS-PAGE for the purified mutant proteins in Example 1. Lane 1, protein molecular weight marker; lane 2, HPV39N15 (HPV39 L1 protein truncated by 15 amino acids at the N-terminus); lane 3, HPV68N0 (HPV68 L1 protein truncated by 0 amino acids at the N-terminus, i.e., full-length wild-type HPV68 L1 protein); lane 4, H39N15-68T1; lane 5, H39N15-68T2; lane 6, H39N15-68T3; lane 7, H39N15-68T4; lane 8, H39N15-68T5; lane 9, protein molecular weight marker; lane 10, H39N15-68T4; lane 11, HPV70N10 (HPV70 L1 protein truncated by 10 amino acids at the N-terminus, i.e., full-length wild-type HPV68 L1 protein); lane 12, H39N15-68T1; lane 13, H39N15-68T2; lane 14, H39N15-68T3; lane 15, H39N15-68T4; lane 16, H39N15-68T3; lane 17, H39N15-68T4; lane 18, H39N15-68T5; lane 19, protein molecular weight marker; lane 20, H39N15-68T4; lane 21, HPV70N10 (HPV70 L1 protein truncated by 10 amino acids at the N-terminus, lane 10, H39N15-68T4-70S1; lane 11, H39N15-68T4-70S2; lane 12, H39N15-68T4-70S1; lane 13, H39N15-68T4-70S2; lane 14, H39N15-68T4-70S3; lane 15, H39N15-68T4-70S5. The results showed that the purity of H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 proteins was more than 90% after purification by chromatography. [Diagram 2]1 shows the results of Western blot for mutant proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 prepared in Example 1 (performed using broad spectrum antibody 4B3). Lane 1, HPV39N15; lane 2, HPV68N0; lane 3, H39N15-68T1; lane 4, H39N15-68T2; lane 5, H39N15-68T3; lane 6, H39N15-68T4; lane 7, H39N15-68T5; lane 8, H39N15-68T4; lane 9, HPV70N10; lane 10, H39N15-68T4-70S1; lane 11, H39N15-68T4-70S2; lane 12, H39N15-68T4-70S3; lane 13, H39N15-68T4-70S5. The results showed that the mutant proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 could be specifically recognized by the broad spectrum antibody 4B3. [Diagram 3] The results of analysis by molecular sieve chromatography of samples containing proteins HPV39N15, H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, and H39N15-68T5 are shown. The results showed that the first protein peak of samples containing proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, or H39N15-68T5 appeared at approximately 13-14 minutes, which is similar to that of HPV39N15. This indicated that all of these proteins were able to assemble to form VLPs. [Figure 4]1 shows the results of analysis by molecular sieve chromatography of samples containing proteins HPV39N15, HPV68L1N0, HPV70N10, H39N15-68T4, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5. The results showed that the first protein peaks for samples containing proteins H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, or H39N15-68T4-70S5 appeared at approximately 13-14 min, which was similar to that for HPV39N15, HPV68L1N0, HPV70N10, and H39N15-68T4 VLPs, indicating that all of these proteins were able to assemble into VLPs. [Figure 5A]4 shows the results of sedimentation velocity analysis of HPV39N15 VLP, HPV68L1N0 VLP, HPV70N10 VLP, H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP. Figure 5A, HPV39N15 VLP; Figure 5B, HPV68L1N0 VLP; Figure 5C, H39N15-68T1 VLP; Figure 5D, H39N15-68T2 VLP; Figure 5E, H39N15-68T3 VLP; Figure 5F, H39N15-68T4 VLP; Figure 5G, H39N15-68T5 VLP; Figure 5H, HPV70N10 VLP; Figure 5I, H39N15-68T4-70S1 VLP; Figure 5J, H39N15-68T4-70S2 VLP; Figure 5K, H39N15-68T4-70S3 VLP; Figure 5L, H39N15-68T4-70S5 VLP. The results showed that the sedimentation coefficients of H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP were 136S, 151S, 138S, 145S, 135S, 124S, 108S, 99S, and 127S, respectively. This indicated that the mutant proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were able to assemble to form virus-like particles similar in size and morphology to wild-type VLPs (HPV39N15 VLP, 115S; HPV68N0 VLP, 153S; HPV70N10 VLP, 144S). [Figure 5B]4 shows the results of sedimentation velocity analysis of HPV39N15 VLP, HPV68L1N0 VLP, HPV70N10 VLP, H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP. Figure 5A, HPV39N15 VLP; Figure 5B, HPV68L1N0 VLP; Figure 5C, H39N15-68T1 VLP; Figure 5D, H39N15-68T2 VLP; Figure 5E, H39N15-68T3 VLP; Figure 5F, H39N15-68T4 VLP; Figure 5G, H39N15-68T5 VLP; Figure 5H, HPV70N10 VLP; Figure 5I, H39N15-68T4-70S1 VLP; Figure 5J, H39N15-68T4-70S2 VLP; Figure 5K, H39N15-68T4-70S3 VLP; Figure 5L, H39N15-68T4-70S5 VLP. The results showed that the sedimentation coefficients of H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP were 136S, 151S, 138S, 145S, 135S, 124S, 108S, 99S, and 127S, respectively. This indicated that the mutant proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were able to assemble to form virus-like particles similar in size and morphology to wild-type VLPs (HPV39N15 VLP, 115S; HPV68N0 VLP, 153S; HPV70N10 VLP, 144S). [Figure 5C]4 shows the results of sedimentation velocity analysis of HPV39N15 VLP, HPV68L1N0 VLP, HPV70N10 VLP, H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP. Figure 5A, HPV39N15 VLP; Figure 5B, HPV68L1N0 VLP; Figure 5C, H39N15-68T1 VLP; Figure 5D, H39N15-68T2 VLP; Figure 5E, H39N15-68T3 VLP; Figure 5F, H39N15-68T4 VLP; Figure 5G, H39N15-68T5 VLP; Figure 5H, HPV70N10 VLP; Figure 5I, H39N15-68T4-70S1 VLP; Figure 5J, H39N15-68T4-70S2 VLP; Figure 5K, H39N15-68T4-70S3 VLP; Figure 5L, H39N15-68T4-70S5 VLP. The results showed that the sedimentation coefficients of H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP were 136S, 151S, 138S, 145S, 135S, 124S, 108S, 99S, and 127S, respectively. This indicated that the mutant proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were able to assemble to form virus-like particles similar in size and morphology to wild-type VLPs (HPV39N15 VLP, 115S; HPV68N0 VLP, 153S; HPV70N10 VLP, 144S). [Figure 5D]4 shows the results of sedimentation velocity analysis of HPV39N15 VLP, HPV68L1N0 VLP, HPV70N10 VLP, H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP. Figure 5A, HPV39N15 VLP; Figure 5B, HPV68L1N0 VLP; Figure 5C, H39N15-68T1 VLP; Figure 5D, H39N15-68T2 VLP; Figure 5E, H39N15-68T3 VLP; Figure 5F, H39N15-68T4 VLP; Figure 5G, H39N15-68T5 VLP; Figure 5H, HPV70N10 VLP; Figure 5I, H39N15-68T4-70S1 VLP; Figure 5J, H39N15-68T4-70S2 VLP; Figure 5K, H39N15-68T4-70S3 VLP; Figure 5L, H39N15-68T4-70S5 VLP. The results showed that the sedimentation coefficients of H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP were 136S, 151S, 138S, 145S, 135S, 124S, 108S, 99S, and 127S, respectively. This indicated that the mutant proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were able to assemble to form virus-like particles similar in size and morphology to wild-type VLPs (HPV39N15 VLP, 115S; HPV68N0 VLP, 153S; HPV70N10 VLP, 144S). [Figure 5E]4 shows the results of sedimentation velocity analysis of HPV39N15 VLP, HPV68L1N0 VLP, HPV70N10 VLP, H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP. Figure 5A, HPV39N15 VLP; Figure 5B, HPV68L1N0 VLP; Figure 5C, H39N15-68T1 VLP; Figure 5D, H39N15-68T2 VLP; Figure 5E, H39N15-68T3 VLP; Figure 5F, H39N15-68T4 VLP; Figure 5G, H39N15-68T5 VLP; Figure 5H, HPV70N10 VLP; Figure 5I, H39N15-68T4-70S1 VLP; Figure 5J, H39N15-68T4-70S2 VLP; Figure 5K, H39N15-68T4-70S3 VLP; Figure 5L, H39N15-68T4-70S5 VLP. The results showed that the sedimentation coefficients of H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP were 136S, 151S, 138S, 145S, 135S, 124S, 108S, 99S, and 127S, respectively. This indicated that the mutant proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were able to assemble to form virus-like particles similar in size and morphology to wild-type VLPs (HPV39N15 VLP, 115S; HPV68N0 VLP, 153S; HPV70N10 VLP, 144S). [Figure 5F]4 shows the results of sedimentation velocity analysis of HPV39N15 VLP, HPV68L1N0 VLP, HPV70N10 VLP, H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP. Figure 5A, HPV39N15 VLP; Figure 5B, HPV68L1N0 VLP; Figure 5C, H39N15-68T1 VLP; Figure 5D, H39N15-68T2 VLP; Figure 5E, H39N15-68T3 VLP; Figure 5F, H39N15-68T4 VLP; Figure 5G, H39N15-68T5 VLP; Figure 5H, HPV70N10 VLP; Figure 5I, H39N15-68T4-70S1 VLP; Figure 5J, H39N15-68T4-70S2 VLP; Figure 5K, H39N15-68T4-70S3 VLP; Figure 5L, H39N15-68T4-70S5 VLP. The results showed that the sedimentation coefficients of H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP were 136S, 151S, 138S, 145S, 135S, 124S, 108S, 99S, and 127S, respectively. This indicated that the mutant proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were able to assemble to form virus-like particles similar in size and morphology to wild-type VLPs (HPV39N15 VLP, 115S; HPV68N0 VLP, 153S; HPV70N10 VLP, 144S). [Figure 5G]4 shows the results of sedimentation velocity analysis of HPV39N15 VLP, HPV68L1N0 VLP, HPV70N10 VLP, H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP. Figure 5A, HPV39N15 VLP; Figure 5B, HPV68L1N0 VLP; Figure 5C, H39N15-68T1 VLP; Figure 5D, H39N15-68T2 VLP; Figure 5E, H39N15-68T3 VLP; Figure 5F, H39N15-68T4 VLP; Figure 5G, H39N15-68T5 VLP; Figure 5H, HPV70N10 VLP; Figure 5I, H39N15-68T4-70S1 VLP; Figure 5J, H39N15-68T4-70S2 VLP; Figure 5K, H39N15-68T4-70S3 VLP; Figure 5L, H39N15-68T4-70S5 VLP. The results showed that the sedimentation coefficients of H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP were 136S, 151S, 138S, 145S, 135S, 124S, 108S, 99S, and 127S, respectively. This indicated that the mutant proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were able to assemble to form virus-like particles similar in size and morphology to wild-type VLPs (HPV39N15 VLP, 115S; HPV68N0 VLP, 153S; HPV70N10 VLP, 144S). [Figure 5H]4 shows the results of sedimentation velocity analysis of HPV39N15 VLP, HPV68L1N0 VLP, HPV70N10 VLP, H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP. Figure 5A, HPV39N15 VLP; Figure 5B, HPV68L1N0 VLP; Figure 5C, H39N15-68T1 VLP; Figure 5D, H39N15-68T2 VLP; Figure 5E, H39N15-68T3 VLP; Figure 5F, H39N15-68T4 VLP; Figure 5G, H39N15-68T5 VLP; Figure 5H, HPV70N10 VLP; Figure 5I, H39N15-68T4-70S1 VLP; Figure 5J, H39N15-68T4-70S2 VLP; Figure 5K, H39N15-68T4-70S3 VLP; Figure 5L, H39N15-68T4-70S5 VLP. The results showed that the sedimentation coefficients of H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP were 136S, 151S, 138S, 145S, 135S, 124S, 108S, 99S, and 127S, respectively. This indicated that the mutant proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were able to assemble to form virus-like particles similar in size and morphology to wild-type VLPs (HPV39N15 VLP, 115S; HPV68N0 VLP, 153S; HPV70N10 VLP, 144S). [Figure 5I]4 shows the results of sedimentation velocity analysis of HPV39N15 VLP, HPV68L1N0 VLP, HPV70N10 VLP, H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP. Figure 5A, HPV39N15 VLP; Figure 5B, HPV68L1N0 VLP; Figure 5C, H39N15-68T1 VLP; Figure 5D, H39N15-68T2 VLP; Figure 5E, H39N15-68T3 VLP; Figure 5F, H39N15-68T4 VLP; Figure 5G, H39N15-68T5 VLP; Figure 5H, HPV70N10 VLP; Figure 5I, H39N15-68T4-70S1 VLP; Figure 5J, H39N15-68T4-70S2 VLP; Figure 5K, H39N15-68T4-70S3 VLP; Figure 5L, H39N15-68T4-70S5 VLP. The results showed that the sedimentation coefficients of H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP were 136S, 151S, 138S, 145S, 135S, 124S, 108S, 99S, and 127S, respectively. This indicated that the mutant proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were able to assemble to form virus-like particles similar in size and morphology to wild-type VLPs (HPV39N15 VLP, 115S; HPV68N0 VLP, 153S; HPV70N10 VLP, 144S). [Figure 5J]4 shows the results of sedimentation velocity analysis of HPV39N15 VLP, HPV68L1N0 VLP, HPV70N10 VLP, H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP. Figure 5A, HPV39N15 VLP; Figure 5B, HPV68L1N0 VLP; Figure 5C, H39N15-68T1 VLP; Figure 5D, H39N15-68T2 VLP; Figure 5E, H39N15-68T3 VLP; Figure 5F, H39N15-68T4 VLP; Figure 5G, H39N15-68T5 VLP; Figure 5H, HPV70N10 VLP; Figure 5I, H39N15-68T4-70S1 VLP; Figure 5J, H39N15-68T4-70S2 VLP; Figure 5K, H39N15-68T4-70S3 VLP; Figure 5L, H39N15-68T4-70S5 VLP. The results showed that the sedimentation coefficients of H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP were 136S, 151S, 138S, 145S, 135S, 124S, 108S, 99S, and 127S, respectively. This indicated that the mutant proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were able to assemble to form virus-like particles similar in size and morphology to wild-type VLPs (HPV39N15 VLP, 115S; HPV68N0 VLP, 153S; HPV70N10 VLP, 144S). [Figure 5K]4 shows the results of sedimentation velocity analysis of HPV39N15 VLP, HPV68L1N0 VLP, HPV70N10 VLP, H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP. Figure 5A, HPV39N15 VLP; Figure 5B, HPV68L1N0 VLP; Figure 5C, H39N15-68T1 VLP; Figure 5D, H39N15-68T2 VLP; Figure 5E, H39N15-68T3 VLP; Figure 5F, H39N15-68T4 VLP; Figure 5G, H39N15-68T5 VLP; Figure 5H, HPV70N10 VLP; Figure 5I, H39N15-68T4-70S1 VLP; Figure 5J, H39N15-68T4-70S2 VLP; Figure 5K, H39N15-68T4-70S3 VLP; Figure 5L, H39N15-68T4-70S5 VLP. The results showed that the sedimentation coefficients of H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP were 136S, 151S, 138S, 145S, 135S, 124S, 108S, 99S, and 127S, respectively. This indicated that the mutant proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were able to assemble to form virus-like particles similar in size and morphology to wild-type VLPs (HPV39N15 VLP, 115S; HPV68N0 VLP, 153S; HPV70N10 VLP, 144S). [Figure 5L]4 shows the results of sedimentation velocity analysis of HPV39N15 VLP, HPV68L1N0 VLP, HPV70N10 VLP, H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP. Figure 5A, HPV39N15 VLP; Figure 5B, HPV68L1N0 VLP; Figure 5C, H39N15-68T1 VLP; Figure 5D, H39N15-68T2 VLP; Figure 5E, H39N15-68T3 VLP; Figure 5F, H39N15-68T4 VLP; Figure 5G, H39N15-68T5 VLP; Figure 5H, HPV70N10 VLP; Figure 5I, H39N15-68T4-70S1 VLP; Figure 5J, H39N15-68T4-70S2 VLP; Figure 5K, H39N15-68T4-70S3 VLP; Figure 5L, H39N15-68T4-70S5 VLP. The results showed that the sedimentation coefficients of H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP were 136S, 151S, 138S, 145S, 135S, 124S, 108S, 99S, and 127S, respectively. This indicated that the mutant proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were able to assemble to form virus-like particles similar in size and morphology to wild-type VLPs (HPV39N15 VLP, 115S; HPV68N0 VLP, 153S; HPV70N10 VLP, 144S). [Figure 6A]Transmission electron microscopy (TEM) images of various VLP samples are shown (taken at 100,000x magnification, bar = 0.1 μm). Figure 6A, VLPs assembled from HPV39N15; Figure 6B, VLPs assembled from HPV68L1N0; Figure 6C, VLPs assembled from HPV70N10; Figure 6D, VLPs assembled from H39N15-68T1; Figure 6E, VLPs assembled from H39N15-68T2; Figure 6F, VLPs assembled from H39N15-68T3; Figure 6G, VLPs assembled from H39N15-68T1; Figure 6F, VLPs assembled from H39N15-68T2; Figure 6G, VLPs assembled from H39N15-68T3; Figure 6H, VLPs assembled from H39N15-68T1; Figure 6H, VLPs assembled from H39N15-68T2; Figure 6H, VLPs assembled from H39N15-68T3 ... 4 assembly; Figure 6H, VLPs assembled from H39N15-68T5; Figure 6I, VLPs assembled from H39N15-68T4-70S1; Figure 6J, VLPs assembled from H39N15-68T4-70S2; Figure 6K, VLPs assembled from H39N15-68T4-70S3; Figure 6L, VLPs assembled from H39N15-68T4-70S5. The results showed that H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were similar to HPV39N15, HPV68L1N0, and HPV70N10 and could assemble to form VLPs with a radius of approximately 25 to 30 nm. [Figure 6B]Transmission electron microscopy (TEM) images of various VLP samples are shown (taken at 100,000x magnification, bar = 0.1 μm). Figure 6A, VLPs assembled from HPV39N15; Figure 6B, VLPs assembled from HPV68L1N0; Figure 6C, VLPs assembled from HPV70N10; Figure 6D, VLPs assembled from H39N15-68T1; Figure 6E, VLPs assembled from H39N15-68T2; Figure 6F, VLPs assembled from H39N15-68T3; Figure 6G, VLPs assembled from H39N15-68T1; Figure 6F, VLPs assembled from H39N15-68T2; Figure 6G, VLPs assembled from H39N15-68T3; Figure 6H, VLPs assembled from H39N15-68T1; Figure 6H, VLPs assembled from H39N15-68T2; Figure 6H, VLPs assembled from H39N15-68T3 ... 4 assembly; Figure 6H, VLPs assembled from H39N15-68T5; Figure 6I, VLPs assembled from H39N15-68T4-70S1; Figure 6J, VLPs assembled from H39N15-68T4-70S2; Figure 6K, VLPs assembled from H39N15-68T4-70S3; Figure 6L, VLPs assembled from H39N15-68T4-70S5. The results showed that H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were similar to HPV39N15, HPV68L1N0, and HPV70N10 and could assemble to form VLPs with a radius of approximately 25 to 30 nm. [Figure 6C]Transmission electron microscopy (TEM) images of various VLP samples are shown (taken at 100,000x magnification, bar = 0.1 μm). Figure 6A, VLPs assembled from HPV39N15; Figure 6B, VLPs assembled from HPV68L1N0; Figure 6C, VLPs assembled from HPV70N10; Figure 6D, VLPs assembled from H39N15-68T1; Figure 6E, VLPs assembled from H39N15-68T2; Figure 6F, VLPs assembled from H39N15-68T3; Figure 6G, VLPs assembled from H39N15-68T1; Figure 6F, VLPs assembled from H39N15-68T2; Figure 6G, VLPs assembled from H39N15-68T3; Figure 6H, VLPs assembled from H39N15-68T1; Figure 6H, VLPs assembled from H39N15-68T2; Figure 6H, VLPs assembled from H39N15-68T3 ... 4 assembly; Figure 6H, VLPs assembled from H39N15-68T5; Figure 6I, VLPs assembled from H39N15-68T4-70S1; Figure 6J, VLPs assembled from H39N15-68T4-70S2; Figure 6K, VLPs assembled from H39N15-68T4-70S3; Figure 6L, VLPs assembled from H39N15-68T4-70S5. The results showed that H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were similar to HPV39N15, HPV68L1N0, and HPV70N10 and could assemble to form VLPs with a radius of approximately 25 to 30 nm. [Figure 6D]Transmission electron microscopy (TEM) images of various VLP samples are shown (taken at 100,000x magnification, bar = 0.1 μm). Figure 6A, VLPs assembled from HPV39N15; Figure 6B, VLPs assembled from HPV68L1N0; Figure 6C, VLPs assembled from HPV70N10; Figure 6D, VLPs assembled from H39N15-68T1; Figure 6E, VLPs assembled from H39N15-68T2; Figure 6F, VLPs assembled from H39N15-68T3; Figure 6G, VLPs assembled from H39N15-68T1; Figure 6F, VLPs assembled from H39N15-68T2; Figure 6G, VLPs assembled from H39N15-68T3; Figure 6H, VLPs assembled from H39N15-68T1; Figure 6H, VLPs assembled from H39N15-68T2; Figure 6H, VLPs assembled from H39N15-68T3 ... 4 assembly; Figure 6H, VLPs assembled from H39N15-68T5; Figure 6I, VLPs assembled from H39N15-68T4-70S1; Figure 6J, VLPs assembled from H39N15-68T4-70S2; Figure 6K, VLPs assembled from H39N15-68T4-70S3; Figure 6L, VLPs assembled from H39N15-68T4-70S5. The results showed that H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were similar to HPV39N15, HPV68L1N0, and HPV70N10 and could assemble to form VLPs with a radius of approximately 25 to 30 nm. [Figure 6E]Transmission electron microscopy (TEM) images of various VLP samples are shown (taken at 100,000x magnification, bar = 0.1 μm). Figure 6A, VLPs assembled from HPV39N15; Figure 6B, VLPs assembled from HPV68L1N0; Figure 6C, VLPs assembled from HPV70N10; Figure 6D, VLPs assembled from H39N15-68T1; Figure 6E, VLPs assembled from H39N15-68T2; Figure 6F, VLPs assembled from H39N15-68T3; Figure 6G, VLPs assembled from H39N15-68T1; Figure 6F, VLPs assembled from H39N15-68T2; Figure 6G, VLPs assembled from H39N15-68T3; Figure 6H, VLPs assembled from H39N15-68T1; Figure 6H, VLPs assembled from H39N15-68T2; Figure 6H, VLPs assembled from H39N15-68T3 ... 4 assembly; Figure 6H, VLPs assembled from H39N15-68T5; Figure 6I, VLPs assembled from H39N15-68T4-70S1; Figure 6J, VLPs assembled from H39N15-68T4-70S2; Figure 6K, VLPs assembled from H39N15-68T4-70S3; Figure 6L, VLPs assembled from H39N15-68T4-70S5. The results showed that H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were similar to HPV39N15, HPV68L1N0, and HPV70N10 and could assemble to form VLPs with a radius of approximately 25 to 30 nm. [Figure 6F]Transmission electron microscopy (TEM) images of various VLP samples are shown (taken at 100,000x magnification, bar = 0.1 μm). Figure 6A, VLPs assembled from HPV39N15; Figure 6B, VLPs assembled from HPV68L1N0; Figure 6C, VLPs assembled from HPV70N10; Figure 6D, VLPs assembled from H39N15-68T1; Figure 6E, VLPs assembled from H39N15-68T2; Figure 6F, VLPs assembled from H39N15-68T3; Figure 6G, VLPs assembled from H39N15-68T1; Figure 6F, VLPs assembled from H39N15-68T2; Figure 6G, VLPs assembled from H39N15-68T3; Figure 6H, VLPs assembled from H39N15-68T1; Figure 6H, VLPs assembled from H39N15-68T2; Figure 6H, VLPs assembled from H39N15-68T3 ... 4 assembly; Figure 6H, VLPs assembled from H39N15-68T5; Figure 6I, VLPs assembled from H39N15-68T4-70S1; Figure 6J, VLPs assembled from H39N15-68T4-70S2; Figure 6K, VLPs assembled from H39N15-68T4-70S3; Figure 6L, VLPs assembled from H39N15-68T4-70S5. The results showed that H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were similar to HPV39N15, HPV68L1N0, and HPV70N10 and could assemble to form VLPs with a radius of approximately 25 to 30 nm. [Figure 6G]Transmission electron microscopy (TEM) images of various VLP samples are shown (taken at 100,000x magnification, bar = 0.1 μm). Figure 6A, VLPs assembled from HPV39N15; Figure 6B, VLPs assembled from HPV68L1N0; Figure 6C, VLPs assembled from HPV70N10; Figure 6D, VLPs assembled from H39N15-68T1; Figure 6E, VLPs assembled from H39N15-68T2; Figure 6F, VLPs assembled from H39N15-68T3; Figure 6G, VLPs assembled from H39N15-68T1; Figure 6F, VLPs assembled from H39N15-68T2; Figure 6G, VLPs assembled from H39N15-68T3; Figure 6H, VLPs assembled from H39N15-68T1; Figure 6H, VLPs assembled from H39N15-68T2; Figure 6H, VLPs assembled from H39N15-68T3 ... 4 assembly; Figure 6H, VLPs assembled from H39N15-68T5; Figure 6I, VLPs assembled from H39N15-68T4-70S1; Figure 6J, VLPs assembled from H39N15-68T4-70S2; Figure 6K, VLPs assembled from H39N15-68T4-70S3; Figure 6L, VLPs assembled from H39N15-68T4-70S5. The results showed that H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were similar to HPV39N15, HPV68L1N0, and HPV70N10 and could assemble to form VLPs with a radius of approximately 25 to 30 nm. [Figure 6H]Transmission electron microscopy (TEM) images of various VLP samples are shown (taken at 100,000x magnification, bar = 0.1 μm). Figure 6A, VLPs assembled from HPV39N15; Figure 6B, VLPs assembled from HPV68L1N0; Figure 6C, VLPs assembled from HPV70N10; Figure 6D, VLPs assembled from H39N15-68T1; Figure 6E, VLPs assembled from H39N15-68T2; Figure 6F, VLPs assembled from H39N15-68T3; Figure 6G, VLPs assembled from H39N15-68T1; Figure 6F, VLPs assembled from H39N15-68T2; Figure 6G, VLPs assembled from H39N15-68T3; Figure 6H, VLPs assembled from H39N15-68T1; Figure 6H, VLPs assembled from H39N15-68T2; Figure 6H, VLPs assembled from H39N15-68T3 ... 4 assembly; Figure 6H, VLPs assembled from H39N15-68T5; Figure 6I, VLPs assembled from H39N15-68T4-70S1; Figure 6J, VLPs assembled from H39N15-68T4-70S2; Figure 6K, VLPs assembled from H39N15-68T4-70S3; Figure 6L, VLPs assembled from H39N15-68T4-70S5. The results showed that H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were similar to HPV39N15, HPV68L1N0, and HPV70N10 and could assemble to form VLPs with a radius of approximately 25 to 30 nm. [Figure 6I]Transmission electron microscopy (TEM) images of various VLP samples are shown (taken at 100,000x magnification, bar = 0.1 μm). Figure 6A, VLPs assembled from HPV39N15; Figure 6B, VLPs assembled from HPV68L1N0; Figure 6C, VLPs assembled from HPV70N10; Figure 6D, VLPs assembled from H39N15-68T1; Figure 6E, VLPs assembled from H39N15-68T2; Figure 6F, VLPs assembled from H39N15-68T3; Figure 6G, VLPs assembled from H39N15-68T1; Figure 6F, VLPs assembled from H39N15-68T2; Figure 6G, VLPs assembled from H39N15-68T3; Figure 6H, VLPs assembled from H39N15-68T1; Figure 6H, VLPs assembled from H39N15-68T2; Figure 6H, VLPs assembled from H39N15-68T3 ... 4 assembly; Figure 6H, VLPs assembled from H39N15-68T5; Figure 6I, VLPs assembled from H39N15-68T4-70S1; Figure 6J, VLPs assembled from H39N15-68T4-70S2; Figure 6K, VLPs assembled from H39N15-68T4-70S3; Figure 6L, VLPs assembled from H39N15-68T4-70S5. The results showed that H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were similar to HPV39N15, HPV68L1N0, and HPV70N10 and could assemble to form VLPs with a radius of approximately 25 to 30 nm. [Figure 6J]Transmission electron microscopy (TEM) images of various VLP samples are shown (taken at 100,000x magnification, bar = 0.1 μm). Figure 6A, VLPs assembled from HPV39N15; Figure 6B, VLPs assembled from HPV68L1N0; Figure 6C, VLPs assembled from HPV70N10; Figure 6D, VLPs assembled from H39N15-68T1; Figure 6E, VLPs assembled from H39N15-68T2; Figure 6F, VLPs assembled from H39N15-68T3; Figure 6G, VLPs assembled from H39N15-68T1; Figure 6F, VLPs assembled from H39N15-68T2; Figure 6G, VLPs assembled from H39N15-68T3; Figure 6H, VLPs assembled from H39N15-68T1; Figure 6H, VLPs assembled from H39N15-68T2; Figure 6H, VLPs assembled from H39N15-68T3 ... 4 assembly; Figure 6H, VLPs assembled from H39N15-68T5; Figure 6I, VLPs assembled from H39N15-68T4-70S1; Figure 6J, VLPs assembled from H39N15-68T4-70S2; Figure 6K, VLPs assembled from H39N15-68T4-70S3; Figure 6L, VLPs assembled from H39N15-68T4-70S5. The results showed that H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were similar to HPV39N15, HPV68L1N0, and HPV70N10 and could assemble to form VLPs with a radius of approximately 25 to 30 nm. [Figure 6K]Transmission electron microscopy (TEM) images of various VLP samples are shown (taken at 100,000x magnification, bar = 0.1 μm). Figure 6A, VLPs assembled from HPV39N15; Figure 6B, VLPs assembled from HPV68L1N0; Figure 6C, VLPs assembled from HPV70N10; Figure 6D, VLPs assembled from H39N15-68T1; Figure 6E, VLPs assembled from H39N15-68T2; Figure 6F, VLPs assembled from H39N15-68T3; Figure 6G, VLPs assembled from H39N15-68T1; Figure 6F, VLPs assembled from H39N15-68T2; Figure 6G, VLPs assembled from H39N15-68T3; Figure 6H, VLPs assembled from H39N15-68T1; Figure 6H, VLPs assembled from H39N15-68T2; Figure 6H, VLPs assembled from H39N15-68T3 ... 4 assembly; Figure 6H, VLPs assembled from H39N15-68T5; Figure 6I, VLPs assembled from H39N15-68T4-70S1; Figure 6J, VLPs assembled from H39N15-68T4-70S2; Figure 6K, VLPs assembled from H39N15-68T4-70S3; Figure 6L, VLPs assembled from H39N15-68T4-70S5. The results showed that H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were similar to HPV39N15, HPV68L1N0, and HPV70N10 and could assemble to form VLPs with a radius of approximately 25 to 30 nm. [Figure 6L]Transmission electron microscopy (TEM) images of various VLP samples are shown (taken at 100,000x magnification, bar = 0.1 μm). Figure 6A, VLPs assembled from HPV39N15; Figure 6B, VLPs assembled from HPV68L1N0; Figure 6C, VLPs assembled from HPV70N10; Figure 6D, VLPs assembled from H39N15-68T1; Figure 6E, VLPs assembled from H39N15-68T2; Figure 6F, VLPs assembled from H39N15-68T3; Figure 6G, VLPs assembled from H39N15-68T1; Figure 6F, VLPs assembled from H39N15-68T2; Figure 6G, VLPs assembled from H39N15-68T3; Figure 6H, VLPs assembled from H39N15-68T1; Figure 6H, VLPs assembled from H39N15-68T2; Figure 6H, VLPs assembled from H39N15-68T3 ... 4 assembly; Figure 6H, VLPs assembled from H39N15-68T5; Figure 6I, VLPs assembled from H39N15-68T4-70S1; Figure 6J, VLPs assembled from H39N15-68T4-70S2; Figure 6K, VLPs assembled from H39N15-68T4-70S3; Figure 6L, VLPs assembled from H39N15-68T4-70S5. The results showed that H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 were similar to HPV39N15, HPV68L1N0, and HPV70N10 and could assemble to form VLPs with a radius of approximately 25 to 30 nm. [Figure 7A]The results of neutralizing antibody titers in mouse serum after inoculation of mice with H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, or H39N15-68T5 VLP are shown in Figure 7A, aluminum adjuvant group 1 (immunization dose 5 μg, aluminum adjuvant used); Figure 7B, aluminum adjuvant group 2 (immunization dose 1 μg, aluminum adjuvant used); Figure 7C, aluminum adjuvant group 3 (immunization dose 0.2 μg, aluminum adjuvant used). The results showed that H39N15-68T4 VLP could induce the development of high titer neutralizing antibodies against HPV39 in mice, the protective effect of which was slightly weaker than that of the same dose of HPV39N15 VLP alone, but significantly better than that of the same dose of HPV68N0 VLP alone; and it could induce the development of high titer neutralizing antibodies against HPV68 in mice, the protective effect of which was slightly weaker than that of the same dose of HPV68N0 VLP alone, but significantly better than that of the same dose of HPV39N15 VLP alone. This indicated that H39N15-68T4 VLP had good cross-immunogenicity and cross-protection against HPV39 and HPV68. [Figure 7B]The results of neutralizing antibody titers in mouse serum after inoculation of mice with H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, or H39N15-68T5 VLP are shown in Figure 7A, aluminum adjuvant group 1 (immunization dose 5 μg, aluminum adjuvant used); Figure 7B, aluminum adjuvant group 2 (immunization dose 1 μg, aluminum adjuvant used); Figure 7C, aluminum adjuvant group 3 (immunization dose 0.2 μg, aluminum adjuvant used). The results showed that H39N15-68T4 VLP could induce the development of high titer neutralizing antibodies against HPV39 in mice, the protective effect of which was slightly weaker than that of the same dose of HPV39N15 VLP alone, but significantly better than that of the same dose of HPV68N0 VLP alone; and it could induce the development of high titer neutralizing antibodies against HPV68 in mice, the protective effect of which was slightly weaker than that of the same dose of HPV68N0 VLP alone, but significantly better than that of the same dose of HPV39N15 VLP alone. This indicated that H39N15-68T4 VLP had good cross-immunogenicity and cross-protection against HPV39 and HPV68. [Figure 7C]The results of neutralizing antibody titers in mouse serum after inoculation of mice with H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, or H39N15-68T5 VLP are shown in Figure 7A, aluminum adjuvant group 1 (immunization dose 5 μg, aluminum adjuvant used); Figure 7B, aluminum adjuvant group 2 (immunization dose 1 μg, aluminum adjuvant used); Figure 7C, aluminum adjuvant group 3 (immunization dose 0.2 μg, aluminum adjuvant used). The results showed that H39N15-68T4 VLP could induce the development of high titer neutralizing antibodies against HPV39 in mice, the protective effect of which was slightly weaker than that of the same dose of HPV39N15 VLP alone, but significantly better than that of the same dose of HPV68N0 VLP alone; and it could induce the development of high titer neutralizing antibodies against HPV68 in mice, the protective effect of which was slightly weaker than that of the same dose of HPV68N0 VLP alone, but significantly better than that of the same dose of HPV39N15 VLP alone. This indicated that H39N15-68T4 VLP had good cross-immunogenicity and cross-protection against HPV39 and HPV68. [Figure 8A]The results of neutralizing antibody titers in mouse serum after inoculation of mice with H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP are shown in Figure 8A, aluminum adjuvant group 1 (immunization dose 5 μg, aluminum adjuvant used); Figure 8B, aluminum adjuvant group 2 (immunization dose 1 μg, aluminum adjuvant used); Figure 8C, aluminum adjuvant group 3 (immunization dose 0.2 μg, aluminum adjuvant used). The results showed that H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 VLPs could induce the development of high-titer neutralizing antibodies against HPV39 in mice, with protective effects slightly weaker than those of the same dose of HPV39N15 VLP alone and mixed HPV39 / HPV68 / HPV70 VLPs, but significantly better than those of the same dose of HPV68N0 VLP alone and HPV70N10 VLPs alone; and they could induce the development of high-titer neutralizing antibodies against HPV68 in mice, with protective effects comparable to those of the same dose of HPV68N0 VLP alone and mixed HPV39 / HPV68 / HPV70 VLPs, and comparable to those of the same dose of HPV39N15 VLP alone and HPV70N10 VLPs. were able to induce the development of high titer neutralizing antibodies against HPV70 in mice, with protective efficacy comparable to that of the same dose of HPV70N10 VLP alone and mixed HPV39 / HPV68 / HPV70 VLPs, and significantly superior to that of the same dose of HPV39N15 VLP alone and HPV68N0 VLP alone, indicating that H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP had good cross-immunogenicity and cross-protection against HPV39, HPV68, and HPV70. [Figure 8B]The results of neutralizing antibody titers in mouse serum after inoculation of mice with H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP are shown in Figure 8A, aluminum adjuvant group 1 (immunization dose 5 μg, aluminum adjuvant used); Figure 8B, aluminum adjuvant group 2 (immunization dose 1 μg, aluminum adjuvant used); Figure 8C, aluminum adjuvant group 3 (immunization dose 0.2 μg, aluminum adjuvant used). The results showed that H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 VLPs could induce the development of high-titer neutralizing antibodies against HPV39 in mice, with protective effects slightly weaker than those of the same dose of HPV39N15 VLP alone and mixed HPV39 / HPV68 / HPV70 VLPs, but significantly better than those of the same dose of HPV68N0 VLP alone and HPV70N10 VLPs alone; and they could induce the development of high-titer neutralizing antibodies against HPV68 in mice, with protective effects comparable to those of the same dose of HPV68N0 VLP alone and mixed HPV39 / HPV68 / HPV70 VLPs, and comparable to those of the same dose of HPV39N15 VLP alone and HPV70N10 VLPs. were able to induce the development of high titer neutralizing antibodies against HPV70 in mice, with protective efficacy comparable to that of the same dose of HPV70N10 VLP alone and mixed HPV39 / HPV68 / HPV70 VLPs, and significantly superior to that of the same dose of HPV39N15 VLP alone and HPV68N0 VLP alone, indicating that H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP had good cross-immunogenicity and cross-protection against HPV39, HPV68, and HPV70. [Figure 8C]The results of neutralizing antibody titers in mouse serum after inoculation of mice with H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP are shown in Figure 8A, aluminum adjuvant group 1 (immunization dose 5 μg, aluminum adjuvant used); Figure 8B, aluminum adjuvant group 2 (immunization dose 1 μg, aluminum adjuvant used); Figure 8C, aluminum adjuvant group 3 (immunization dose 0.2 μg, aluminum adjuvant used). The results showed that H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 VLPs could induce the development of high-titer neutralizing antibodies against HPV39 in mice, with protective effects slightly weaker than those of the same dose of HPV39N15 VLP alone and mixed HPV39 / HPV68 / HPV70 VLPs, but significantly better than those of the same dose of HPV68N0 VLP alone and HPV70N10 VLPs alone; and they could induce the development of high-titer neutralizing antibodies against HPV68 in mice, with protective effects comparable to those of the same dose of HPV68N0 VLP alone and mixed HPV39 / HPV68 / HPV70 VLPs, and comparable to those of the same dose of HPV39N15 VLP alone and HPV70N10 VLPs. were able to induce the development of high titer neutralizing antibodies against HPV70 in mice, with protective efficacy comparable to that of the same dose of HPV70N10 VLP alone and mixed HPV39 / HPV68 / HPV70 VLPs, and significantly superior to that of the same dose of HPV39N15 VLP alone and HPV68N0 VLP alone, indicating that H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP had good cross-immunogenicity and cross-protection against HPV39, HPV68, and HPV70. [Figure 9]1 shows the results of detection of the thermal stability of HPV39N15 VLP, HPV68N0 VLP, HPV70N10 VLP, H39N15-68T4 VLP, H39N15-68T4-70S2 VLP, and H39N15-68T4-70S5 VLP, where A shows the results of detection of the thermal stability of HPV39N15 VLP, B shows the results of detection of the thermal stability of HPV68L1N0 VLP, C shows the results of detection of the thermal stability of HPV70N10 VLP, D shows the results of detection of the thermal stability of H39N15-68T4 VLP VLP, E shows the results of detection of the thermal stability of H39N15-68T4-70S2 VLP, and F shows the results of detection of the thermal stability of H39N15-68T4-70S5 VLP. The results showed that all VLPs formed by these proteins had very high thermal stability. [Figure 10]1 shows cryo-electron microscope (cryo-EM) photographs of H39N15-68T4-70S2 VLP and H39N15-68T4-70S5 VLP, and a reconstruction of their three-dimensional structure. A shows a cryo-electron microscope (cryo-EM) photograph of H39N15-68T4-70S2 VLP, B shows a reconstruction of the three-dimensional structure of H39N15-68T4-70S2 VLP, C shows a cryo-electron microscope (cryo-EM) photograph of H39N15-68T4-70S5 VLP, and D shows a reconstruction of the three-dimensional structure of H39N15-68T4-70S5 VLP. The reconstructed three-dimensional structures showed that both H39N15-68T4-70S2 VLP and H39N15-68T4-70S5 VLP had a T=7 icosahedral structure (h=1, k=2) consisting of 72 capsomeres (morphological subunits, pentamers). Unlike conventional icosahedral viral capsids that conform to the quasi-equivalence principle, all constituent subunits in the structures of H39N15-68T4-70S2 VLP and H39N15-68T4-70S5 VLP were pentamers, with no hexamers. The outer diameters of these two VLPs were approximately 55 nm. These were similar to previously reported three-dimensional structures of native HPV virus particles and HPV VLPs prepared by eukaryotic expression systems (e.g., poxvirus expression systems) (Baker TS, Newcomb WW, Olson NH. et al. Biophys J. (1991), 60(6): 1445-1456. Hagensee ME, Olson NH, Baker TS, et al. J Virol. (1994), 68(7):4503-4505. Buck CB, Cheng N, Thompson CD. et al. J Virol. (2008), 82(11): 5190-7). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] Sequence information Some of the relevant sequences in the present invention are shown in Table 1 below.

[0067] [Table 1]

[0068] Sequence 1 (SEQ ID NO:1): MALWRSSDSMVYLPPPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVGMNGGRKQDIPKVSAYQYRVFRVTLPDPNKFSIPDASLYNPETQRLVWACVGVEVGRGQPLGVGISGHPLYNRQ DDTENSPFSSTTNKDSRDNVSVDYKQTQLCIIGCVPAIGEHWGKGKACKPNNVSTGDCPPLELVNTPIEDGDMIDTGYGAMDFGALQETKSEVPLDICQSICKYPDYLQMSADVYGDSMFFCLRRE QLFARHFWNRGGMVGDAIPAQLYIKGTDIRANPGSSVYCPSPSGSMVTSDSQLFNKPYWLHKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTSIESSIPSTYDPSKFKEYTRHVEEYDLQFIFQL CTVTLTTDVMSYIHTMNSSILDNWNFAVAPPPSASLVDTYRYLQSAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQARVRRRPTIGPRKRPAASTSSSSATKHKRKRVSK Sequence 2 (SEQ ID NO:2): MALWRASDNMVYLPPPSVAKVVNTDDYVTRTGMYYYAGTSRLLTVGHPYFKVPMSGGRKQGIPKVSAYQYRVFRVTLPDPNKFSVPESTLYNPDTQRMVWACVGVEIGRGQPLGVGLSGHPLYNRL DDTENSPFSSNKNPKDSRDNVAVDCKQTQLCIIGCVPAIGEHWAKGKSCKPTNVQQGDCPPLELVNTPIEDGDMIDTGYGAMDFGTLQETKSEVPLDICQSVCKYPDYLQMSADVYGDSMFFCLRR EQLFARHFWNRGGMVGDTIPTDMYIKGTDIRETPSSYVYAPSPSGSMVSSDSQLFNKPYWLHKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTTTDSTVPAVYDSNKFKEYVRHVEEYDLQFIFQ LCTITLSTDVMSYIHTMNPAILDDWNFGVAPPPSASLVDTYRYLQSAAITCQKDAPAPVKKDPYDGLNFWNVDLKEKFSSELDQFPLGRKFLLQAGVRRRPTIGPRKRTATAATTSTSKHKRKRVSK Sequence 3 (SEQ ID NO:3): MALWRSSDNTVYLPPPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVPVNGGRKQEIPKVSAYQYRVFRVSLPDPNKFGLPDPSLYNPDTQRLVWACIGVEIGRGQPLGVGVSGHPLYNRL DDTENSHFSSAVNTQDSRDNVSVDYKQTQLCIIGCVPAMGEHWAKGKACKSTTVQQGDCPPLELVNTAIEDGDMIDTGYGAMDFRTLQETKSEVPLDICQSVCKYPDYLQMSADVYGDSMFFCLRKE QLFARHFWNRGGMVGDTIPSELYIKGTDIRDRPGTHVYSPSPSGSMVSSDSQLFNKPYWLHKAQGHNNGICWHNQLFITVVDTTRSTNFTLSACTETAIPAVYSPTKFKEYTRHVEEYDLQFIFQL CTITLTADVMAYIHTMNPAILDNWNIGVTPPPSASLVDTYRYLQSAAIACQKDAPAPEKKDPYDDLKFWNVDLKEKFSTELDQFPLGRKFLLQVGARRRPTIGPRKRPASAKSSSSASKHKRKRVSK Sequence 4 (SEQ ID NO:4): MPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVPMSGGRKQGIPKVSAYQYRVFRVTLPDPNKFSIPDASLYNPETQRLVWACVGVEVGRGQPLGVGISGHPLYNRQDDTENSPFSS TTNKDSRDNVSVDYKQTQLCIIGCVPAIGEHWGKGKACKPNNVSTGDCPPLELVNTPIEDGDMIDTGYGAMDFGALQETKSEVPLDICQSICKYPDYLQMSADVYGDSMFFCLRREQLFARHF WNRGGMVGDAIPAQLYIKGTDIRANPGSSVYCPSPSGSMVTSDSQLFNKPYWLHKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTSIESSIPSTYDPSKFKEYTRHVEEYDLQFIFQLCTVT LTTDVMSYIHTMNSSILDNWNFAVAPPPSASLVDTYRYLQSAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQARVRRRPTIGPRKRPAASTSSSSATKHKRKRVSK Sequence 5 (SEQ ID NO:5): MPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVGMNGGRKQDIPKVSAYQYRVFRVTLPDPNKFSIPDASLYNPETQRLVWACVGVEVGRGQPLGVGLSGHPLYNRLDDTENSPFSSN KNPKDSRDNVAVDCKQTQLCIIGCVPAIGEHWGKGKACKPNNVSTGDCPPLELVNTPIEDGDMIDTGYGAMDFGALQETKSEVPLDICQSICKYPDYLQMSADVYGDSMFFCLRREQLFARHF WNRGGMVGDAIPAQLYIKGTDIRANPGSSVYCPSPSGSMVTSDSQLFNKPYWLHKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTSIESSIPSTYDPSKFKEYTRHVEEYDLQFIFQLCTVT LTTDVMSYIHTMNSSILDNWNFAVAPPPSASLVDTYRYLQSAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQARVRRRPTIGPRKRPAASTSSSSATKHKRKRVSK Sequence 6 (SEQ ID NO:6): MPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVGMNGGRKQDIPKVSAYQYRVFRVTLPDPNKFSIPDASLYNPETQRLVWACVGVEVGRGQPLGVGISGHPLYNRQDDTENSPFSS TTNKDSRDNVSVDYKQTQLCIIGCVPAIGEHWAKGKSCKPTNVQQGDCPPLELVNTPIEDGDMIDTGYGAMDFGALQETKSEVPLDICQSICKYPDYLQMSADVYGDSMFFCLRREQLFARHF WNRGGMVGDAIPAQLYIKGTDIRANPGSSVYCPSPSGSMVTSDSQLFNKPYWLHKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTSIESSIPSTYDPSKFKEYTRHVEEYDLQFIFQLCTVT LTTDVMSYIHTMNSSILDNWNFAVAPPPSASLVDTYRYLQSAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQARVRRRPTIGPRKRPAASTSSSSATKHKRKRVSK Sequence 7 (SEQ ID NO:7): MPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVGMNGGRKQDIPKVSAYQYRVFRVTLPDPNKFSIPDASLYNPETQRLVWACVGVEVGRGQPLGVGISGHPLYNRQDDTENSPFSS TTNKDSRDNVSVDYKQTQLCIIGCVPAIGEHWGKGKACKPNNVSTGDCPPLELVNTPIEDGDMIDTGYGAMDFGALQETKSEVPLDICQSICKYPDYLQMSADVYGDSMFFCLRREQLFARHF WNRGGMVGDTIPTDMYIKGTDIRETPSSYVYCPSPSGSMVTSDSQLFNKPYWLHKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTSIESSIPSTYDPSKFKEYTRHVEEYDLQFIFQLCTVT LTTDVMSYIHTMNSSILDNWNFAVAPPPSASLVDTYRYLQSAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQARVRRRPTIGPRKRPAASTSSSSATKHKRKRVSK Sequence 8 (SEQ ID NO:8): MPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVGMNGGRKQDIPKVSAYQYRVFRVTLPDPNKFSIPDASLYNPETQRLVWACVGVEVGRGQPLGVGISGHPLYNRQDDTENSPFSS TTNKDSRDNVSVDYKQTQLCIIGCVPAIGEHWGKGKACKPNNVSTGDCPPLELVNTPIEDGDMIDTGYGAMDFGALQETKSEVPLDICQSICKYPDYLQMSADVYGDSMFFCLRREQLFARHF WNRGGMVGDAIPAQLYIKGTDIRANPGSSVYCPSPSGSMVTSDSQLFNKPYWLHKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTSTDSTVPAVYDSNKFKEYTRHVEEYDLQFIFQLCTVT LTTDVMSYIHTMNSSILDNWNFAVAPPPSASLVDTYRYLQSAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQARVRRRPTIGPRKRPAASTSSSSATKHKRKRVSK Sequence 9 (SEQ ID NO:9): MPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVPVNGGRKQEIPKVSAYQYRVFRVTLPDPNKFSIPDASLYNPETQRLVWACVGVEVGRGQPLGVGISGHPLYNRQDDTENSPFSS TTNKDSRDNVSVDYKQTQLCIIGCVPAIGEHWGKGKACKPNNVSTGDCPPLELVNTPIEDGDMIDTGYGAMDFGALQETKSEVPLDICQSICKYPDYLQMSADVYGDSMFFCLRREQLFARHF WNRGGMVGDTIPTDMYIKGTDIRETPSSYVYCPSPSGSMVTSDSQLFNKPYWLHKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTSIESSIPSTYDPSKFKEYTRHVEEYDLQFIFQLCTVT LTTDVMSYIHTMNSSILDNWNFAVAPPPSASLVDTYRYLQSAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQARVRRRPTIGPRKRPAASTSSSSATKHKRKRVSK Sequence 10 (SEQ ID NO:10): MPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVGMNGGRKQDIPKVSAYQYRVFRVTLPDPNKFSIPDASLYNPETQRLVWACVGVEVGRGQPLGVGVSGHPLYNRLDDTENSHFSSA VNTQDSRDNVSVDYKQTQLCIIGCVPAIGEHWGKGKACKPNNVSTGDCPPLELVNTPIEDGDMIDTGYGAMDFGALQETKSEVPLDICQSICKYPDYLQMSADVYGDSMFFCLRREQLFARHF WNRGGMVGDTIPTDMYIKGTDIRETPSSYVYCPSPSGSMVTSDSQLFNKPYWLHKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTSIESSIPSTYDPSKFKEYTRHVEEYDLQFIFQLCTVT LTTDVMSYIHTMNSSILDNWNFAVAPPPSASLVDTYRYLQSAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQARVRRRPTIGPRKRPAASTSSSSATKHKRKRVSK Sequence 11 (SEQ ID NO:11): MPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVGMNGGRKQDIPKVSAYQYRVFRVTLPDPNKFSIPDASLYNPETQRLVWACVGVEVGRGQPLGVGISGHPLYNRQDDTENSPFSS TTNKDSRDNVSVDYKQTQLCIIGCVPAIGEHWAKGKACKSTTVQQGDCPPLELVNTPIEDGDMIDTGYGAMDFGALQETKSEVPLDICQSICKYPDYLQMSADVYGDSMFFCLRREQLFARHF WNRGGMVGDTIPTDMYIKGTDIRETPSSYVYCPSPSGSMVTSDSQLFNKPYWLHKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTSIESSIPSTYDPSKFKEYTRHVEEYDLQFIFQLCTVT LTTDVMSYIHTMNSSILDNWNFAVAPPPSASLVDTYRYLQSAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQARVRRRPTIGPRKRPAASTSSSSATKHKRKRVSK Sequence 12 (SEQ ID NO:12): MPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVGMNGGRKQDIPKVSAYQYRVFRVTLPDPNKFSIPDASLYNPETQRLVWACVGVEVGRGQPLGVGISGHPLYNRQDDTENSPFSS TTNKDSRDNVSVDYKQTQLCIIGCVPAIGEHWGKGKACKPNNVSTGDCPPLELVNTPIEDGDMIDTGYGAMDFGALQETKSEVPLDICQSICKYPDYLQMSADVYGDSMFFCLRREQLFARHF WNRGGMVGDTIPTDMYIKGTDIRETPSSYVYCPSPSGSMVTSDSQLFNKPYWLHKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTSTETAIPAVYSPTKFKEYTRHVEEYDLQFIFQLCTVT LTTDVMSYIHTMNSSILDNWNFAVAPPPSASLVDTYRYLQSAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQARVRRRPTIGPRKRPAASTSSSSATKHKRKRVSK Sequence 13 (SEQ ID NO:13): Sequence 14 (SEQ ID NO:14): Sequence 15 (SEQ ID NO:15): Sequence 16 (SEQ ID NO:16): Sequence 17 (SEQ ID NO:17): Sequence 18 (SEQ ID NO:18): Sequence 19 (SEQ ID NO:19): Sequence 20 (SEQ ID NO:20): Sequence 21 (SEQ ID NO:21): Sequence 22 (SEQ ID NO:22): Sequence 23 (SEQ ID NO:23): Sequence 24 (SEQ ID NO:24): Sequence 25 (SEQ ID NO:25): TIPTDMYIKGTDIRETPSSY Sequence 26 (SEQ ID NO:26): VSGHPLYNRLDDTENSHFSSAVNTQ Sequence 27 (SEQ ID NO:27): AKGKACKSTTVQQ Sequence 28 (SEQ ID NO:28): TETAIPAVYSPT Sequence 29 (SEQ ID NO:29): MPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVGMNGGRKQDIPKVSAYQYRVFRVTLPDPNKFSIPDASLYNPETQRLVWACVGVEVGRGQPLGVGISGHPLYNRQDDTENSPFSS TTNKDSRDNVSVDYKQTQLCIIGCVPAIGEHWGKGKACKPNNVSTGDCPPLELVNTPIEDGDMIDTGYGAMDFGALQETKSEVPLDICQSICKYPDYLQMSADVYGDSMFFCLRREQLFARHF WNRGGMVGDAIPAQLYIKGTDIRANPGSSVYCPSPSGSMVTSDSQLFNKPYWLHKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTSIESSIPSTYDPSKFKEYTRHVEEYDLQFIFQLCTVT LTTDVMSYIHTMNSSILDNWNFAVAPPPSASLVDTYRYLQSAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQARVRRRPTIGPRKRPAASTSSSSATKHKRKRVSK Sequence 30 (SEQ ID NO:30): Sequence 31 (SEQ ID NO:31): MVYLPPPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVPVNGGRKQEIPKVSAYQYRVFRVSLPDPNKFGLPDPSLYNPDTQRLVWACIGVEIGRGQPLGVGVSGHPLYNRLDDTENSH FSSAVNTQDSRDNVSVDYKQTQLCIIGCVPAMGEHWAKGKACKSTTVQQGDCPPLELVNTAIEDGDMIDTGYGAMDFRTLQETKSEVPLDICQSVCKYPDYLQMSADVYGDSMFFCLRKEQLFA RHFWNRGGMVGDTIPSELYIKGTDIRDRPGTHVYSPSPSGSMVSSDSQLFNKPYWLHKAQGHNNGICWHNQLFITVVDTTRSTNFTLSACTETAIPAVYSPTKFKEYTRHVEEYDLQFIFQLCT ITLTADVMAYIHTMNPAILDNWNIGVTPPPSASLVDTYRYLQSAAIACQKDAPAPEKKDPYDDLKFWNVDLKEKFSTELDQFPLGRKFLLQVGARRRPTIGPRKRPASAKSSSSASKHKRKRVSK Sequence 32 (SEQ ID NO:32): Sequence 33 (SEQ ID NO:33): PMSGGRKQG Sequence 34 (SEQ ID NO:34): LSGHPLYNRLDDTENSPFSSNKNPKDSRDNVAVDC Sequence 35 (SEQ ID NO:35): AKGKSCKPTNVQQ Sequence 36 (SEQ ID NO:36): TDSTVPAVYDSN Sequence 37 (SEQ ID NO:37): PVNGGRKQE EXAMPLES

[0069] Specific Mode for Carrying Out the Invention The present invention will be further described below with reference to examples, which are used only to illustrate the present invention and are not intended to limit the present invention.

[0070] Unless otherwise specified, the molecular biological experimental methods and immunological assays used in the present invention are substantially performed according to the methods described in Sambrook J et al., Molecular Cloning: A Laboratory Manual (Second Edition), Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995, and restriction enzymes are used under the conditions recommended by the manufacturer. Those skilled in the art will understand that the examples are used to illustrate the present invention and are not intended to limit the scope of protection of the present invention.

[0071] Example 1. Expression and purification of mutant HPV39 L1 proteins Construction of expression vectors For the purpose of multi-site mutagenesis, an expression vector encoding a mutant HPV39 L1 protein containing a segment derived from HPV68 L1 protein was constructed by PCR, and the initial template used was the plasmid pTO-T7-HPV39N15C (encoding HPV39 L1 protein truncated by 15 amino acids at the N-terminus; abbreviated as 39L1N15 in Table 2). The templates and primers in each PCR were as shown in Table 2, and the PCR amplification conditions were as follows: denaturation at 94°C for 10 minutes; 25 cycles (denaturation at 94°C for 50 seconds, annealing at a given temperature for a specific time, extension at 72°C for 7.5 minutes); final extension at 72°C for 10 minutes. The annealing temperature and time are shown in Table 2. The sequences of the PCR primers used are shown in Table 3.

[0072] To the amplified product (50 μL), 2 μL of restriction endonuclease DpnI (Fermentas (MBI), Cat. No. FD1704, 2500 U / tube) was added, and the resulting mixture was incubated at 37°C for 60 minutes. 10 μL of the digested product was used to transform 40 μL of competent E. coli ER2566 (purchased from New England Biolabs) prepared by the calcium chloride method. The transformed E. coli was spread on a solid LB medium (LB medium components: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, hereinafter) containing kanamycin (final concentration 25 μg / mL, hereinafter the same) and cultured at 37°C for 10 to 12 hours until a single colony was clearly observed. A single colony was selected and inoculated into a tube containing 4 mL of liquid LB medium (containing kanamycin), and cultured at 37°C for 10 hours with shaking at 220 rpm, after which 1 mL of the bacterial solution was taken and stored at -70°C. A plasmid was extracted from E. coli, and the nucleotide sequence of the target fragment inserted into the plasmid was sequenced using a T7 primer. The sequencing results showed that the nucleotide sequence of the target fragment inserted into the constructed plasmid (expression vector) was SEQ ID NO: 16, and the amino acid sequence encoded thereby was SEQ ID NO: 4 (the corresponding protein was named H39N15-68T1). The mutant protein H39N15-68T1 differs from HPV39N15 in that the amino acid residues from positions 53 to 61 of the wild-type HPV39 L1 protein are replaced with amino acid residues from positions 53 to 61 of the wild-type HPV68 L1 protein.

[0073] Using Gibson assembly (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), we constructed an expression vector encoding another mutant HPV39 L1 protein, where the mutant HPV39 L1 protein contains a specific segment derived from HPV68 L1 and a specific segment derived from HPV70L1. Briefly, a short fragment containing a mutation and a long fragment not containing a mutation were obtained by PCR, and then the two fragments were ligated to form a circle using the Gibson assembly system. Initial templates used included plasmid pTO-T7-HPV39N15 (encoding the HPV39 L1 protein truncated by 15 amino acids at its N-terminus; abbreviated in Table 2 as 39L1N15), plasmid pTO-T7-HPV68L1 (encoding the HPV68 L1 protein; abbreviated in Table 2 as 68L1N0), plasmid pTO-T7-H39N15-68T4 (encoding the mutant protein H39N15-68T4; abbreviated in Table 2 as H39N15-68T4), and plasmid pTO-T7-HPV70N10 (encoding the HPV70 L1 protein truncated by 10 amino acids at its N-terminus; abbreviated in Table 2 as 70L1N10). The templates and primers in each PCR were as shown in Table 2, and the PCR amplification conditions for amplifying the short fragments were as follows: denaturation at 94° C. for 10 minutes; 25 cycles (denaturation at 94° C. for 50 seconds, annealing at a given temperature for a specific time, extension at 72° C. for 1 minute); final extension at 72° C. for 10 minutes. The PCR amplification conditions for amplifying the long fragments were as follows: denaturation at 94° C. for 10 minutes; 25 cycles (denaturation at 94° C. for 50 seconds, annealing at a given temperature for a specific time, extension at 72° C. for 7.5 minutes); final extension at 72° C. for 10 minutes.The sequences of the PCR primers used are shown in Table 3. The amplified products were subjected to electrophoresis, and then the target fragments were recovered using a DNA Extraction Kit (BEYOTIME, Cat. No. D0033), and their concentrations were determined. The short and long fragments obtained by amplification were mixed in a molar ratio of 2:1 (total volume 3 μL), and then 3 μL of 2× Gibson Assembly Master Mix (purchased from NEB; containing T5 exonuclease, Phusion DNA polymerase, and Taq DNA ligase) was added, and the mixture was allowed to react at 50° C. for 1 hour.

[0074] The ligated product (6 μL) was used to transform 40 μL of competent E. coli ER2566 (purchased from New England Biolabs) prepared by the calcium chloride method. The transformed E. coli was spread on a solid LB medium containing kanamycin and cultured stationarily at 37°C for 10 to 12 hours until a single colony was clearly observed. A single colony was selected and inoculated into a tube containing 4 mL of liquid LB medium (containing kanamycin) and cultured at 37°C for 10 hours with shaking at 220 rpm, and then 1 mL of the bacterial solution was stored at -70°C. Plasmids were extracted from E. coli, and the nucleotide sequence of the target fragment inserted into the plasmid was sequenced using a T7 primer. The sequencing results showed that the nucleotide sequences of the target fragments inserted into the constructed plasmids (expression vectors) were SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, and 24, respectively, and the amino acid sequences encoded thereby were SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, and 12, respectively (the corresponding proteins were named H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5, respectively).

[0075] The mutant protein H39N15-68T2 differs from HPV39N15 in that the amino acid residues from positions 117-150 of the wild-type HPV39 L1 protein are replaced by amino acid residues from positions 117-151 of the wild-type HPV68 L1 protein. The mutant protein H39N15-68T3 differs from HPV39N15 in that the amino acid residues from positions 169-181 of the wild-type HPV39 L1 protein are replaced by amino acid residues from positions 170-182 of the wild-type HPV68 L1 protein. The mutant protein H39N15-68T4 differs from HPV39N15 in that the amino acid residues from positions 269-288 of the wild-type HPV39 L1 protein are replaced by amino acid residues from positions 270-289 of the wild-type HPV68 L1 protein. The mutant protein H39N15-68T5 differs from HPV39N15 by a substitution of amino acid residues from positions 347-358 of the wild-type HPV39 L1 protein with amino acid residues from positions 348-359 of the wild-type HPV68 L1 protein.

[0076] The mutant protein H39N15-68T4-70S1 differs from HPV39N15 in that amino acid residues from positions 269 to 288 of the wild-type HPV39 L1 protein are replaced by amino acid residues from positions 270 to 289 of the wild-type HPV68 L1 protein, and amino acid residues from positions 53 to 61 of the wild-type HPV39 L1 protein are replaced by amino acid residues from positions 53 to 61 of the wild-type HPV70 L1 protein. The mutant protein H39N15-68T4-70S2 differs from HPV39N15 in that amino acid residues from positions 269 to 288 of the wild-type HPV39 L1 protein are replaced by amino acid residues from positions 270 to 289 of the wild-type HPV68 L1 protein, and amino acid residues from positions 117 to 140 of the wild-type HPV39 L1 protein are replaced by amino acid residues from positions 117 to 141 of the wild-type HPV70 L1 protein. The mutant protein H39N15-68T4-70S3 differs from HPV39N15 in that amino acid residues from positions 269 to 288 of the wild-type HPV39 L1 protein are replaced by amino acid residues from positions 270 to 289 of the wild-type HPV68 L1 protein, and amino acid residues from positions 169 to 181 of the wild-type HPV39 L1 protein are replaced by amino acid residues from positions 170 to 182 of the wild-type HPV70 L1 protein. The mutant protein H39N15-68T4-70S5 differs from HPV39N15 in that amino acid residues from positions 269 to 288 of the wild-type HPV39 L1 protein are replaced by amino acid residues from positions 270 to 289 of the wild-type HPV68 L1 protein, and amino acid residues from positions 347 to 358 of the wild-type HPV39 L1 protein are replaced by amino acid residues from positions 348 to 359 of the wild-type HPV70 L1 protein.

[0077] [Table 2]

[0078] [Table 3]

[0079] Large-scale expression of mutant proteins The E. coli solutions containing the recombinant plasmids pTO-T7-H39N15-68T1, pTO-T7-H39N15-68T2, pTO-T7-H39N15-68T3, pTO-T7-H39N15-68T4, pTO-T7-H39N15-68T5, pTO-T7-H39N15-68T4-70S1, pTO-T7-H39N15-68T4-70S2, pTO-T7-H39N15-68T4-70S3, and pTO-T7-H39N15-68T4-70S5 were taken out of a -70°C refrigerator, inoculated into 100 mL of kanamycin-containing LB liquid medium, and incubated at 37°C for about 8 hours at 200 rpm. The culture was then transferred to 500 mL of kanamycin-containing LB medium (1 ml of bacterial solution was transferred) and further incubated. 600 Once the ratio reached approximately 0.6, the culture temperature was reduced to 25°C and 500 μL of IPTG was added to each culture bottle. Incubation was continued for an additional 8 hours. After the end of incubation, bacteria were collected by centrifugation. Bacteria expressing H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 proteins, respectively, were obtained.

[0080] Destruction of bacteria expressing mutant proteins The resulting bacteria were resuspended in a ratio of 10 mL of lysis buffer (20 mM Tris buffer, pH 7.2, 300 mM NaCl) per gram of bacteria. The bacteria were disrupted using an ultrasonic device for 30 minutes. The lysis solution containing the disrupted bacteria was centrifuged at 13500 rpm (30000 g) for 15 minutes to obtain the supernatant (i.e., the supernatant of the disrupted bacteria).

[0081] Purification of mutant proteins by chromatography Equipment: AKTA Explorer 100 preparative liquid chromatography system from GE Healthcare (i.e., formerly Amershan Pharmacia). Chromatography media: SP Sepharose 4 Fast Flow (GE Healthcare), CHT-II (purchased from Bio-RAD), and Butyl Sepharose 4 Fast Flow (GE Healthcare) Buffers: 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, 2 M NaCl). The buffers containing different concentrations of NaCl used in the following elution protocol were prepared by mixing Buffer A and Buffer B in specific ratios.

[0082] Samples: Supernatants of disrupted bacteria containing H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5, respectively, obtained as described above.

[0083] Elution Protocol: (1) Cation exchange purification of the supernatant of disrupted bacteria by SP Sepharose 4 Fast Flow: The sample was loaded onto the column, then undesired proteins were eluted with a buffer containing 400 mM NaCl (80% Buffer A + 20% Buffer B), followed by elution of the desired protein with a buffer containing 800 mM NaCl (60% Buffer A + 40% Buffer B), and the eluted fraction was collected with a buffer containing 800 mM NaCl.

[0084] (2) Chromatographic purification of the eluted fraction obtained in step (1) by CHTII (hydroxyapatite chromatography): The eluted fraction obtained in step (1) was diluted to reduce the NaCl concentration to 0.5 M, the sample was loaded onto the column, and then undesired proteins were eluted using a buffer containing 500 mM NaCl (75% buffer A + 25% buffer B), followed by elution of the desired protein using a buffer containing 1000 mM NaCl (50% buffer A + 50% buffer B), and the eluted fraction was collected using a buffer containing 1000 mM NaCl.

[0085] (3) Chromatographic purification of the eluted fraction obtained in step (2) by HIC (hydrophobic interaction chromatography): the sample was loaded onto a column, and then undesired proteins were eluted using a buffer containing 1000 mM NaCl, followed by elution of the desired protein using a buffer containing 200 mM NaCl (90% buffer A + 10% buffer B), and the eluted fraction was collected using a buffer containing 200 mM NaCl.

[0086] 150 μL of the eluted fraction obtained in step (3) was added to 30 μL of 6× loading buffer (1 L of which contains 300 ml of 1M TB 6.8, 600 ml of 100% glycerin, 120 g of SDS, 6 g of bromophenol blue, and 50 ml of β-mercaptoethanol). The resulting solution was mixed thoroughly and incubated in a water bath at 80° C. for 10 minutes. Then, 10 μl of the resulting sample was subjected to 10% SDS-PAGE at 120 V for 120 minutes, and the electrophoretic bands were stained with Coomassie Brilliant Blue. The results of the electrophoresis are shown in FIG. 1. The results showed that after the purification steps described above, the purity of H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 was greater than 90%.

[0087] Using similar methods, HPV39N15 protein was prepared and purified using E. coli and plasmid pTO-T7-HPV39N15, HPV68N0 protein was prepared and purified using E. coli and plasmid pTO-T7-HPV68L1N0, and HPV70N10 protein was prepared and purified using E. coli and plasmid pTO-T7-HPV70N10.

[0088] Western blot assay of mutant proteins The H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3 and H39N15-68T4-70S5 proteins purified by the above-mentioned method were subjected to electrophoresis.After electrophoresis, a Western blot assay was performed using the broad spectrum antibody 4B3 against HPV L1 protein, and the results are shown in Figure 2. The results showed that H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 could be specifically recognized by broad-spectrum antibody 4B3.

[0089] Example 2: Assembly of HPV virus-like particles and morphological detection of the particles Assembly of HPV virus-like particles A given volume (approximately 2 ml) of protein H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, or H39N15-68T4-70S5 was dissolved in (1) 2 L of storage buffer (20 mM sodium phosphate buffer (pH 6.5), 0.5 M NaCl), (2) 2 L of renaturation buffer (50 mM sodium phosphate buffer (pH 6.0), 2 mM CaCl2, 2 mM MgCl2, 0.5 M NaCl), and (3) 20 mM sodium phosphate buffer (pH 7.0, 0.5 M The solution was dialyzed successively into NaCl, for 12 hours against each of the three buffers.

[0090] Using a similar method, HPV39N15, HPV68N0, and HPV70N10 proteins were assembled into HPV39N15 VLPs, HPV68N0 VLPs, and HPV70N10 VLPs, respectively.

[0091] Analysis by molecular sieve chromatography The dialyzed sample was subjected to analysis by molecular sieve chromatography using an 1120 Compact LC High Performance Liquid Chromatographic System (Agilent Technologies) (the analytical column used was a TSK Gel PW5000×1 7.8×300 mm). The analysis results are shown in FIG. 3 and FIG. 4. The results showed that the first protein peak for samples containing proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, or H39N15-68T4-70S5 appeared at approximately 13-14 minutes, which was similar to that for HPV39N15 VLPs, HPV68N0 VLPs, and HPV70N10 VLPs, indicating that all of these proteins were able to assemble into VLPs.

[0092] Sedimentation velocity analysis The apparatus used for sedimentation velocity analysis was a Beckman XL-A Analytical Ultracentrifuge equipped with an optical inspection system and An-50Ti and An-60Ti rotors. The sedimentation coefficients of HPV39N15 VLP, HPV68N0 VLP, HPV70N10 VLP, H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP were analyzed by sedimentation velocity method. The results are shown in Figure 5A to Figure 5L. The results showed that the sedimentation coefficients of H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP were 136S, 151S, 138S, 145S, 135S, 124S, 108S, 99S, and 127S, respectively. This indicated that the mutant proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 prepared above were able to assemble to form virus-like particles similar in size and morphology to wild-type VLPs (HPV39N15 VLP, 115S; HPV68N0 VLP, 153S; HPV70N10 VLP, 144S).

[0093] Morphological examination of virus-like particles 100 μL of the sample containing VLPs was observed by transmission electron microscopy (TEM). The equipment used was a 100 kV transmission electron microscope (magnification 100,000 times) provided by JEOL. Briefly, 13.5 μL of the sample was negatively stained with 2% phosphotungstic acid (pH 7.0), fixed on a carbon-coated copper grid, and then observed by TEM. The results are shown in Figures 6A to 6L. The results showed that the mutant proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 could assemble to form virus-like particles, and the virus-like particles produced by the assembly of these mutant proteins were uniform in size with a radius of about 25-30 nm. The virus-like particles produced by the assembly of wild-type HPV39N15, HPV68N0, and HPV70N10 were also uniform in size with a radius of about 25-30 nm. This indicated that these mutant proteins were similar to the wild-type HPV39, HPV68, and HPV70 L1 proteins and were able to assemble into VLPs of uniform size.

[0094] Example 3: Evaluation of neutralizing antibody titers in the serum of mice inoculated with virus-like particles 1 In this experiment, the virus-like particles used were H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, and H39N15-68T5 VLP.

[0095] The vaccination schedule in this experiment is shown in Table 4. All mice (6-week-old BalB / c female mice) were divided into three groups: aluminum adjuvant group 1 (immunization dose 5 μg, with aluminum adjuvant), aluminum adjuvant group 2 (immunization dose 1 μg, with aluminum adjuvant), and aluminum adjuvant group 3 (immunization dose 0.2 μg, with aluminum adjuvant). Each group was further divided into eight subgroups. Control subgroups 1 and 2 were inoculated with HPV39N15 VLPs alone and HPV68N0 VLPs alone, respectively, and control subgroup 3 was inoculated with mixed HPV39 / HPV68 VLPs (i.e., a mixture of HPV39N15 VLPs and HPV68N0 VLPs, each VLP at a given immunization dose). Experimental subgroups 1, 2, 3, 4, and 5 were inoculated with H39N15-68T1 VLPs, H39N15-68T2 VLPs, H39N15-68T3 VLPs, H39N15-68T4 VLPs, and H39N15-68T5 VLPs, respectively.

[0096] In aluminum adjuvant groups 1-3, five mice per subgroup were inoculated by intraperitoneal injection with immunization doses of 5 μg, 1 μg, and 0.2 μg, respectively, in an injection volume of 1 mL. All mice received a first inoculation at week 0, followed by booster inoculations at weeks 2 and 4, respectively. At week 8, blood samples were collected by orbital bleeding, and the serum was analyzed for titers of antibodies against HPV39 and HPV68. The analysis results are shown in Figures 7A-C. The results showed that H39N15-68T4 VLP could induce the development of high titer neutralizing antibodies against HPV39 in mice, the protective effect of which was slightly weaker than that of the same dose of HPV39N15 VLP alone, but significantly better than that of the same dose of HPV68N0 VLP alone; and it could induce the development of high titer neutralizing antibodies against HPV68 in mice, the protective effect of which was slightly weaker than that of the same dose of HPV68N0 VLP alone, but significantly better than that of the same dose of HPV39N15 VLP alone. This indicated that H39N15-68T4 VLP had good cross-immunogenicity and cross-protection against HPV39 and HPV68.

[0097] [Table 4]

[0098] Example 4: ED of virus-like particles that induce seroconversion 50 Rating 1 In this experiment, the virus-like particles used were H39N15-68T4 VLPs.

[0099] Six-week-old BalB / c female mice (8 mice) were inoculated with aluminum adjuvant by a single intraperitoneal injection, where experimental groups were administered H39N15-68T4 VLPs (immunizing dose of 0.900 μg, 0.300 μg, 0.100 μg, 0.033 μg, or 0.011 μg), HPV68N0 VLPs alone (immunizing dose of 0.900 μg, 0.300 μg, 0.100 μg, 0.033 μg, or 0.011 μg), HPV39N15 VLPs alone (immunizing dose of 0.900 μg, 0.300 μg, 0.100 μg, 0.033 μg, or 0.011 μg), or mixed HPV39 / HPV68 VLPs (i.e., HPV39N15 VLPs and HPV68N0 VLPs). A mixture of VLPs, each VLP in an immunization dose of 0.900 μg, 0.300 μg, 0.100 μg, 0.033 μg, or 0.011 μg; the immunization volume was 1 mL. In addition, the diluent used to dilute the vaccine was used as a blank control. Eight mice from each group were inoculated, and venous blood was collected from the eyeball 5 weeks after inoculation. Antibodies against HPV were detected in the serum, and the ED that induced seroconversion (i.e., induced the development of antibodies in mice) was determined by the Reed-Muench method (Reed LJ MH. A simple method of estimating fifty percent endpoints. Am J Hyg. 1938; 27:493-7). 50 was calculated for each sample, and the results are shown in Tables 5 to 8.

[0100] [Table 5]

[0101] [Table 6]

[0102] [Table 7]

[0103] [Table 8]

[0104] The results showed that the ED of H39N15-68T4 VLP induced the development of antibodies against HPV39 in mice 5 weeks after inoculation. 50 The ED for inducing antibody development against HPV68 was comparable to that of HPV39N15 VLP alone and significantly superior to that of HPV68N0 VLP alone. 50 was slightly weaker than that of HPV68N0 VLP alone, but significantly better than that of HPV39N15 VLP alone, indicating that H39N15-68T4 VLP had good cross-immunogenicity and cross-protection against HPV68 and HPV39.

[0105] Example 5: Evaluation of neutralizing antibody titers in the serum of mice inoculated with virus-like particles 2 In this experiment, the virus-like particles used were H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP.

[0106] The vaccination schedule in this experiment is shown in Table 9. All mice (6-week-old BalB / c female mice) were divided into three groups: aluminum adjuvant group 1 (immunization dose 5 μg, with aluminum adjuvant), aluminum adjuvant group 2 (immunization dose 1 μg, with aluminum adjuvant), and aluminum adjuvant group 3 (immunization dose 0.2 μg, with aluminum adjuvant). Each group was further divided into eight subgroups. Control subgroups 1, 2, and 3 were inoculated with HPV39N15 VLPs alone, HPV68N0 VLPs alone, and HPV70N10 VLPs alone, respectively, and control subgroup 4 was inoculated with mixed HPV39 / HPV68 / HPV70 VLPs (i.e., a mixture of HPV39N15 VLPs, HPV68N0 VLPs, and HPV70N10 VLPs, each VLP at a given immunization dose). Experimental subgroups 1, 2, 3, and 4 were inoculated with H39N15-68T4-70S1 VLPs, H39N15-68T4-70S2 VLPs, H39N15-68T4-70S3 VLPs, and H39N15-68T4-70S5 VLPs, respectively.

[0107] In aluminum adjuvant groups 1-3, five mice per subgroup were inoculated by intraperitoneal injection with immunization doses of 5 μg, 1 μg, and 0.2 μg, respectively, in an injection volume of 1 mL. All mice received a first inoculation at week 0, followed by booster inoculations at weeks 2 and 4, respectively. Blood samples were collected by retro-orbital bleeding at week 8, and serum was analyzed for antibody titers against HPV39, HPV68, and HPV70. The analytical results are shown in Figures 8A-8C. The results showed that H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP could induce the development of high-titer neutralizing antibodies against HPV39 in mice, the protective effect of which was slightly weaker than that of the same dose of HPV39N15 VLP alone and mixed HPV39 / HPV68 / HPV70 VLP, but significantly better than that of the same dose of HPV68N0 VLP alone and HPV70N10 VLP alone; and the development of high-titer neutralizing antibodies against HPV68 in mice, the protective effect of which was comparable to that of the same dose of HPV68N0 VLP alone and mixed HPV39 / HPV68 / HPV70 VLP, and comparable to that of the same dose of HPV39N15 VLP alone and HPV70N10 VLP. were able to induce the development of high titer neutralizing antibodies against HPV70 in mice, with protective efficacy comparable to that of the same dose of HPV70N10 VLP alone and mixed HPV39 / HPV68 / HPV70 VLPs, and significantly superior to that of the same dose of HPV39N15 VLP alone and HPV68N0 VLP alone, indicating that H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP had good cross-immunogenicity and cross-protection against HPV39, HPV68, and HPV70.

[0108] [Table 9]

[0109] Example 6: ED of virus-like particles that induce seroconversion 50 Rating 2 In this experiment, the virus-like particles used were H39N15-68T4-70S2 VLPs, H39N15-68T4-70S3 VLPs, and H39N15-68T4-70S5 VLPs.

[0110] Six-week-old BalB / c female mice (8 mice) were inoculated with aluminum adjuvant by a single intraperitoneal injection, where experimental groups were administered H39N15-68T4-70S2 VLPs, H39N15-68T4-70S3 VLPs, and H39N15-68T4-70S5 VLPs (immunizing doses of 0.900 μg, 0.300 μg, 0.100 μg, 0.033 μg, or 0.011 μg), HPV39N15 VLPs alone, HPV68N0 VLPs alone, HPV70N10 VLPs alone (immunizing doses of 0.900 μg, 0.300 μg, 0.100 μg, 0.033 μg, or 0.011 μg) or mixed HPV39 / HPV68 / HPV70 VLPs (i.e., HPV39N15 A mixture of HPV68N0 VLPs, HPV70N10 VLPs, and HPV70N10 VLPs, each VLP in an immunization dose of 0.900 μg, 0.300 μg, 0.100 μg, 0.033 μg, or 0.011 μg; the immunization volume was 1 mL. In addition, the diluent used to dilute the vaccine was used as a blank control. Eight mice from each group were inoculated, and venous blood was collected from the eyeball 5 weeks after inoculation. Antibodies against HPV were detected in the serum, and the EDs that induced seroconversion (i.e., induced the development of antibodies in mice) were determined by the Reed-Muench method (Reed LJ MH. A simple method of estimating fifty percent endpoints. Am J Hyg. 1938; 27:493-7). 50 was calculated for each sample, and the results are shown in Tables 10 to 16.

[0111] [Table 10]

[0112] [Table 11]

[0113] [Table 12]

[0114] [Table 13]

[0115] [Table 14]

[0116] [Table 15]

[0117] [Table 16]

[0118] The results showed that the ED of H39N15-68T4-70S2 VLP and H39N15-68T4-70S5 VLP induced the development of antibodies against HPV39 in mice 5 weeks after inoculation. 50 was comparable to HPV39N15 VLPs alone and mixed HPV39 / HPV68 / HPV70 VLPs, and significantly superior to HPV68N0 VLPs alone and HPV70N10 VLPs alone; ED in eliciting antibody development against HPV68 in mice 50 was comparable to HPV68N0 VLPs alone and mixed HPV39 / HPV68 / HPV70 VLPs, and significantly superior to HPV39N15 VLPs alone and HPV70N10 VLPs alone; ED in eliciting antibody development against HPV70 in mice 50was comparable to HPV70N10 VLPs alone and mixed HPV39 / HPV68 / HPV70 VLPs, and significantly superior to HPV39N15 VLPs alone and HPV68N0 VLPs alone, indicating that H39N15-68T4-70S2 VLPs and H39N15-68T4-70S5 VLPs had good cross-immunogenicity and cross-protection against HPV39, HPV68, and HPV70.

[0119] Example 7: Assessment of thermal stability of virus-like particles The thermal stability of the VLPs formed by HPV39N15 protein, HPV68N0 protein, HPV70N10 protein, H39N15-68T4 protein, H39N15-68T4-70S2 protein, and H39N15-68T4-70S5 protein was evaluated using a differential scanning calorimeter VP Capillary DSC purchased from GE Company (i.e., former MicroCal Company), where the protein storage buffer was used as a control, and the protein was scanned at a heating rate of 1.5°C / min and a temperature range of 10°C to 90°C. The detection results are shown in Figure 9. The results showed that the VLPs formed by H39N15-68T4 protein, H39N15-68T4-70S2 protein, and H39N15-68T4-70S5 all had very high thermal stability.

[0120] Example 8: Reconstruction of the three-dimensional structure of H39N15-68T4-70S2 VLP and H39N15-68T4-70S5 VLP The three-dimensional structures of H39N15-68T4-70S2 VLP and H39N15-68T4-70S5 VLP were reconstructed by three-dimensional structure reconstruction experiments using cryo-electron microscopy (cryo-EM) (Wolf M, Garcea RL, Grigorieff N. et al. Proc Natl Acad Sci US A. (2010), 107(14): 6298-303). Briefly, in the cryo-EM photograph of H39N15-68T4-70S2 VLP (A in FIG. 10), 6410 particles with a uniform size and a diameter of more than 50 nm were selected, and the three-dimensional structure of H39N15-68T4-70S2 VLP was obtained by performing computer overlapping and structural reconstruction. The obtained three-dimensional structure is shown in FIG. 10B (resolution 14.51 Å). In addition, 617 particles with uniform size and diameter larger than 50 nm were selected from the cryo-EM photograph of H39N15-68T4-70S5 VLP (FIG. 10C), and the three-dimensional structure of H39N15-68T4-70S5 VLP was obtained by performing computer overlap and structure reconstruction. The obtained three-dimensional structure is shown in FIG. 10D (resolution 17.64 Å). The results showed that both H39N15-68T4-70S2 VLP and H39N15-68T4-70S5 VLP had a T=7 icosahedral structure (h=1, k=2) consisting of 72 capsomeres (morphological subunits, pentamers). Unlike conventional icosahedral viral capsids that conform to the quasi-equivalence principle, all constituent subunits in the structures of H39N15-68T4-70S2 VLP and H39N15-68T4-70S5 VLP were pentamers, with no hexamers, and these two VLPs had outer diameters of approximately 60 nm.These were similar to previously reported three-dimensional structures of native HPV virus particles and HPV VLPs prepared by eukaryotic expression systems (e.g., poxvirus expression systems) (Baker TS, Newcomb WW, Olson NH. et al. Biophys J. (1991), 60(6): 1445-1456. Hagensee ME, Olson NH, Baker TS, et al. J Virol. (1994), 68(7):4503-4505. Buck CB, Cheng N, Thompson CD. et al. J Virol. (2008), 82(11): 5190-7).

[0121] Although the specific embodiment of the present invention has been described in detail above, it is understood that those skilled in the art can make various modifications and changes according to the teachings disclosed herein, and such modifications and changes are also within the scope of the present invention, which is provided by the appended claims and their equivalents.

Claims

1. A mutant HPV39 L1 protein, which, in comparison to a wild-type HPV39 L1 protein, (I) the mutant HPV39 L1 protein has the following mutation: (1) a truncation of 1 to 25 amino acids at the N-terminus; and (2) substitution of amino acid residues at positions in said wild-type HPV39 L1 protein corresponding to positions 269-288 of SEQ ID NO:1 with amino acid residues at the corresponding positions in the L1 protein of wild-type HPV68; or (II) The mutant HPV39 L1 protein has the mutations defined in (1) and (2) above, and further has the following mutations: (3)(a) a substitution of amino acid residues at positions in the wild-type HPV39 L1 protein corresponding to positions 117-140 of SEQ ID NO:1 with amino acid residues at the corresponding positions in the L1 protein of wild-type HPV70; or (b) replacing amino acid residues at positions in said wild-type HPV39 L1 protein corresponding to positions 169-181 of SEQ ID NO:1 with amino acid residues at the corresponding positions in the L1 protein of wild-type HPV70; or (c) replacing amino acid residues at positions in said wild-type HPV39 L1 protein corresponding to positions 347-358 of SEQ ID NO:1 with amino acid residues at the corresponding positions in the L1 protein of wild-type HPV70. having 4, wherein the corresponding positions are determined by optimal alignment of the compared sequences and the variant HPV39 L1 protein.

2. 2. The mutant HPV39 L1 protein of claim 1, wherein the mutant HPV39 L1 protein is truncated by 3, 5, 8, 10, 12, 15, 18, 20, or 22 amino acids at the N-terminus compared to the wild-type HPV39 L1 protein.

3. The amino acid residues at the corresponding positions described in (2) are amino acid residues at positions 270 to 289 of the wild-type HPV68 L1 protein. The mutant HPV39 L1 protein of claim 1 or 2.

4. A mutant HPV39 L1 protein according to claim 1 or 2, characterized by one or more of the following: (i) the amino acid residues at the corresponding positions described in (3)(a) above are amino acid residues 117 to 141 of the wild-type HPV70 L1 protein; (ii) the amino acid residues at the corresponding positions described in (3)(b) above are amino acid residues 170 to 182 of the wild-type HPV70 L1 protein; (iii) The amino acid residues at the corresponding positions described in (3)(c) above are amino acid residues 348 to 359 of the wild-type HPV70 L1 protein.

5. The mutant HPV39 L1 protein comprises the following: (i) the wild-type HPV39 L1 protein has the amino acid sequence of SEQ ID NO:1; (ii) the wild-type HPV68 L1 protein has the amino acid sequence of SEQ ID NO:2; (iii) the wild-type HPV70 L1 protein has the amino acid sequence of SEQ ID NO:

3.

3. The mutated HPV39 L1 protein according to claim 1 or 2, characterized by one or more of the following:

6. 3. The mutant HPV39 L1 protein of claim 1 or 2, wherein the mutant HPV39 L1 protein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 10, 11, and 12.

7. An isolated nucleic acid encoding a mutant HPV39 L1 protein according to any one of claims 1 to 6.

8. The isolated nucleic acid described in claim 7, wherein the isolated nucleic acid has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 19, 22, 23, and 24.

9. A vector comprising the isolated nucleic acid of claim 7 or 8.

10. A host cell comprising an isolated nucleic acid according to claim 7 or 8 and / or a vector according to claim 9.

11. 3. An HPV virus-like particle comprising or consisting of a mutant HPV39 L1 protein according to claim 1 or 2.

12. A composition comprising a mutant HPV39 L1 protein according to claim 1 or 2, or an isolated nucleic acid according to claim 7 or 8, or a vector according to claim 9, or a host cell according to claim 10, or an HPV virus-like particle according to claim 11.

13. A pharmaceutical composition or vaccine comprising the HPV virus-like particle of claim 11, and optionally a pharma- ceutically acceptable carrier and / or excipient.

14. The pharmaceutical composition or vaccine of claim 13, wherein the HPV virus-like particles are present in an amount effective to prevent HPV infection or a disease caused by HPV infection.

15. The pharmaceutical composition or vaccine described in claim 14, wherein the HPV infection is an infection with one or more HPV types, and / or the disease caused by the HPV infection is selected from the group consisting of cervical cancer and genital warts.

16. The pharmaceutical composition or vaccine of claim 15, wherein the HPV infection is selected from HPV39 infection, HPV68 infection, HPV70 infection, and any combination thereof.

17. 2. A method for preparing the mutant HPV39 L1 protein of claim 1, comprising expressing the mutant HPV39 L1 protein in a host cell and then recovering the mutant HPV39 L1 protein from a culture of the host cell.

18. The method of claim 17, characterized by one or more of the following: (i) the host cell is Escherichia coli; (ii) the method comprises expressing the mutant HPV39 L1 protein in E. coli and then obtaining the mutant HPV39 L1 protein by purifying a lysate supernatant of the E. coli.

19. 12. A method for preparing a vaccine, comprising combining the HPV virus-like particle of claim 11 with a pharma- ceutically acceptable carrier and / or excipient.

20. A composition or vaccine for preventing HPV infection or a disease caused by HPV infection, comprising a mutant HPV39 L1 protein according to claim 1 or 2 or an HPV virus-like particle according to claim 11.

21. The composition or vaccine described in claim 20, wherein the HPV infection is an infection with one or more HPV types, and / or the disease caused by the HPV infection is selected from the group consisting of cervical cancer and genital warts.

22. The composition or vaccine of claim 21, wherein the HPV infection is selected from one or more of HPV39 infection, HPV68 infection, HPV70 infection, and any combination thereof.

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