Stable formulation of human papillomavirus virus-like particle vaccine

Abstract

Provided is a stable formulation of a human papillomavirus virus-like particle vaccine. The stable formulation is composed of a human papillomavirus virus-like particle, a buffer solution, an osmotic pressure regulator, a surfactant and an aluminum adjuvant, wherein the components of the vaccine comprise HPV virus-like particles assembled by L1 proteins of HPV types 6, 11, 16, 18, 31, 33, 45, 52 and 58, and one or more HPV virus-like particles assembled by L1 proteins of other pathogenic HPV types. The formulation can enhance the stability of the vaccine and prolong the validity period of the vaccine in an aqueous formulation.

Description

A stable formulation of human papillomavirus virus-like particle vaccine

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Chinese patent application No. 202110049777.7 filed on January 14, 2021, the contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biopharmaceutical preparations, and in particular to a stable human papillomavirus virus-like particle vaccine preparation. Background Art

[0004] Cervical cancer is one of the most common female malignancies, with approximately 500,000 new cases worldwide each year, making it the second most common cancer in women. Over 95% of cervical cancers are closely linked to infection with the human papillomavirus (HPV). In addition to directly causing cervical cancer, HPV is also significantly linked to bronchial, lung, colorectal, oral, and skin cancers. Furthermore, HPV is a major cause of skin and mucous membrane warts.

[0005] Over 100 HPV types have been identified, and different types can cause different diseases. HPV can be categorized into high-risk, suspected oncogenic, and low-risk types based on their association with cervical cancer. High-risk and suspected oncogenic types can cause cancers such as cervical cancer; high-risk types include types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59; suspected oncogenic types include types 26, 53, 66, 68, 73, and 82. Low-risk types, often associated with genital warts and condyloma acuminata, include types 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, and 89. HPV types 6, 11, and 16 are the most frequently detected subtypes in patients with genital lesions.

[0006] HPV vaccines are an effective way to prevent papillomavirus infection. Virus-like particle (VLP) vaccines are the most advantageous among various vaccine formats. However, VLP-based HPV vaccines are type-restricted, meaning they offer strong protection only against the same HPV type as the VLPs within the vaccine. To provide broad protection, the development of a multivalent HPV vaccine is essential.

[0007] However, before administration, human papillomavirus vaccine preparations will undergo storage and transportation processes, during which the antigens will undergo physical and chemical degradation. These instabilities may reduce the immunogenicity and / or safety of the antigens. Therefore, a stable preparation is needed to ensure that the antigens still have the immunogenicity and safety required for prevention before administration.

[0008] Summary of the Invention

[0009] In one aspect, the present invention provides a stable formulation of a multivalent human papillomavirus virus-like particle vaccine for preventing HPV-related diseases or infections, comprising a plurality of papillomavirus virus-like particles adsorbed on an adjuvant; a physiologically acceptable concentration of a buffer, an osmotic pressure regulator, and optionally a surfactant.

[0010] The human papillomavirus virus-like particles are selected from HPV types 6, 11, 16, 18, 31, 33, 45,

[0011] HPV virus-like particles assembled from L1 proteins of types 51, 52, and 58; and

[0012] HPV virus-like particles assembled from one or more L1 proteins of other pathogenic HPV types.

[0013] In one embodiment,

[0014] The buffer is selected from one or more of a citrate buffer, an acetate buffer or a histidine buffer;

[0015] The osmotic pressure regulator is selected from one or more of sodium chloride, sodium phosphate or sodium sulfate;

[0016] The surfactant is a polysorbate 80, preferably polysorbate 80;

[0017] The adjuvant is selected from one or more of aluminum hydroxyphosphate (AlPO4), amorphous aluminum hydroxyphosphate sulfate (AAHS) or aluminum hydroxide (Al(OH3)), preferably aluminum hydroxyphosphate (AlPO4).

[0018] In one embodiment,

[0019] (a) the total concentration of all types of papillomavirus virus-like particles is 40 μg / mL to 740 μg / mL; (b) the concentration of the buffer is 10 mM to 26 mM, preferably 10 mM, 18 mM or 26 mM;

[0020] (c) the concentration of the osmotic pressure regulator is 150 mM to 320 mM, preferably 150 mM or 320 mM;

[0021] (d) the concentration of the surfactant is 0 to 0.02% by weight;

[0022] (e) the concentration of the adjuvant is about 1.0 mg / mL;

[0023] (f) The pH value of the preparation is 5.9-6.5, preferably 5.9, 6.2 or 6.5.

[0024] In one embodiment, the concentration of any type of papillomavirus virus-like particles constituting the multivalent papillomavirus virus-like particles is 40 μg / mL to 120 μg / mL.

[0025] In one embodiment, the formulation contains a total of 0.74 mg / mL papillomavirus virus-like particles, 1.0 mg / mL aluminum phosphate adjuvant, 18 mM histidine buffer, 320 mM sodium chloride, and the pH value of the formulation solution is 6.2; optionally, the concentration of polysorbate 80 is not higher than 0.3 mg / mL.

[0026] In one embodiment, the one or more other pathogenic HPV types are selected from HPV types 35, 39, 51, 56 and 59.

[0027] In one embodiment, at least one of the HPV virus-like particles is a chimeric HPV virus-like particle, wherein the chimeric HPV virus-like particle comprises a chimeric HPV L1 protein; the chimeric HPV L1 protein comprises, from its N-terminus to its C-terminus:

[0028] a. An N-terminal fragment derived from a first type of papillomavirus L1 protein, wherein the N-terminal fragment retains the immunogenicity of the type L1 protein, wherein the first type of papillomavirus is selected from HPV types 6, 11, 16, 18, 31, 33, 45, 52 and 58 and one or more other pathogenic HPV types; and

[0029] b. a C-terminal fragment derived from a second type of papillomavirus L1 protein, wherein the second type of papillomavirus L1 protein has better expression and solubility than other types of L1 proteins;

[0030] The chimeric HPV L1 protein has the immunogenicity of the first type of papillomavirus L1 protein.

[0031] In one embodiment, the N-terminal fragment is a fragment obtained by truncating the C-terminus of the native sequence of the first type papillomavirus L1 protein at any amino acid site within its α5 region, and a fragment having at least 98% identity thereto; and the C-terminal fragment is a fragment obtained by truncating the N-terminus of the native sequence of the second type papillomavirus L1 protein at any amino acid site within its α5 region, and a functional variant produced by further mutation, deletion and / or addition of the fragment.

[0032] In one embodiment, the C-terminal fragment contains one or more nuclear localization sequences.

[0033] In one embodiment, the first type of papillomavirus L1 protein is selected from HPV types 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, 56 or 58; preferably, the native sequence thereof is the amino acid sequence encoded by the encoding gene shown in SEQ ID No: 30, SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 33, SEQ ID No: 34, SEQ ID No: 35, SEQ ID No: 36, SEQ ID No: 37, SEQ ID No: 38, SEQ ID No: 39, SEQ ID No: 40, or SEQ ID No: 41, respectively;

[0034] The second type of papillomavirus L1 protein is selected from HPV type 16, 28, 33, 59, or 68 L1 protein;

[0035] More preferably, the second type of papillomavirus L1 protein is selected from HPV type 33 or HPV type 59 L1 protein.

[0036] In one embodiment, the C-terminal fragment is SEQ ID No: 1; or a fragment thereof with a length of m1 amino acids, preferably a fragment covering amino acids 1 to m1 of SEQ ID No: 1; wherein m1 is an integer from 8 to 26; or the C-terminal fragment is SEQ ID No: 2; or a fragment thereof with a length of m2 amino acids, preferably a fragment covering amino acids 1 to m2 of SEQ ID No: 2; wherein m2 is an integer from 13 to 31.

[0037] In one embodiment, the C-terminal fragment is SEQ ID No: 3; or a fragment thereof having a length of n amino acids, preferably a fragment covering amino acids 1 to n of SEQ ID No: 3; wherein n is an integer of 16-38.

[0038] In one embodiment, the N-terminal fragment of the HPV type 6 L1 protein is 98%, 98.5%, 99%, 99.5%, 99% or 100% identical to a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 4 at any amino acid position within the α5 region thereof;

[0039] The N-terminal fragment of the HPV type 11 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 5 at any amino acid position within the α5 region thereof;

[0040] The N-terminal fragment of the HPV type 16 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 6 at any amino acid position within the α5 region thereof;

[0041] The N-terminal fragment of the HPV type 18 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 7 at any amino acid position within the α5 region thereof;

[0042] The N-terminal fragment of the HPV type 31 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 8 at any amino acid position within the α5 region thereof;

[0043] The N-terminal fragment of the HPV type 35 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 9 at any amino acid position within the α5 region thereof;

[0044] The N-terminal fragment of the HPV type 39 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 10 at any amino acid position within the α5 region thereof;

[0045] The N-terminal fragment of the HPV type 45 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 11 at any amino acid position within the α5 region thereof;

[0046] The N-terminal fragment of the HPV type 51 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 12 at any amino acid position within the α5 region thereof;

[0047] The N-terminal fragment of the HPV type 52 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 13 at any amino acid position within the α5 region thereof;

[0048] The N-terminal fragment of the HPV 56 type L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 14 at any amino acid position in the α5 region thereof; and

[0049] The N-terminal fragment of the HPV 58 type L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with the fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 15 at any amino acid position in its α5 region.

[0050] In one embodiment, the C-terminus of the N-terminal fragment is directly linked to the N-terminus of the C-terminal fragment or is linked via a linker.

[0051] In one embodiment, when the C-terminus of the N-terminal fragment is connected to the N-terminus of the C-terminal fragment, the following continuous amino acid sequence exists within the range of plus or minus 4 amino acid sites of the connection point: RKFL; preferably, the following continuous amino acid sequence exists within the range of plus or minus 6 amino acid sites of the connection point: LGRKFL.

[0052] In one embodiment, the chimeric HPV L1 proteins of HPV types 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, 56, and 58 are 98%, 98.5%, 99%, 99.5%, or 100% identical to SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21, SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25, SEQ ID No: 26, and SEQ ID No: 27, respectively; and the HPV L1 protein of type 33 and type 59 are 98%, 98.5%, 99%, 99.5%, or 100% identical to SEQ ID No: 28 and SEQ ID No: 29, respectively.

[0053] In one embodiment, the preparation comprises chimeric HPV L1 proteins of HPV types 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, 56, and 58 having amino acid sequences shown in SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21, SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25, SEQ ID No: 26, and SEQ ID No: 27, respectively; and

[0054] HPV type 33 L1 protein and HPV type 59 L1 protein having the amino acid sequences set forth in SEQ ID No: 28 and SEQ ID No: 29, respectively. In one aspect, the present invention provides a method for preventing an HPV-related disease or infection, comprising administering to a subject a stable formulation of the multivalent human papillomavirus virus-like particle vaccine formulation. The terms "prevention" and "treatment" may be considered treatment, and the two terms are used interchangeably. In one embodiment, the subject is a human.

[0055] In one aspect, the formulation is stable for at least 24 months at 2-8°C and for at least 16 weeks at 25°C.

[0056] In one aspect, the present invention provides a use of the human papillomavirus virus-like particle vaccine preparation in preparing a vaccine for preventing HPV-related diseases or infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG1 is the adsorption test result of each formula sample in Example 1.

[0058] Figure 2 shows the results of antigen content analysis of the formulation samples in Example 1. T0: 37°C, week 0; 37°C_1W: 37°C, week 1; 37°C_2W: 37°C, week 2; 37°C_4W: 37°C, week 4.

[0059] FIG3 is the adsorption test results of each formula sample in Example 2.

[0060] Figure 4 shows the results of antigen content analysis of the formulation samples in Example 2. T0: 37°C, week 0; 37°C_1W: 37°C, week 1; 37°C_2W: 37°C, week 2; 37°C_4W: 37°C, week 4.

[0061] FIG5 is the adsorption test results of each formula sample in Example 3.

[0062] Figure 6 shows the results of antigen content analysis of the formulation samples in Example 3. T0: 37°C, week 0; 37°C_1W: 37°C, week 1; 37°C_2W: 37°C, week 2; 37°C_4W: 37°C, week 4.

[0063] FIG7 is the adsorption test results of each formula sample in Example 4.

[0064] Figure 8 shows the results of antigen content analysis of the formulation samples in Example 4. T0: 37°C, week 0; 37°C_1W: 37°C, week 1; 37°C_2W: 37°C, week 2; 37°C_4W: 37°C, week 4.

[0065] Detailed Description of the Invention

[0066] The present invention provides a stable formulation of human papillomavirus vaccine, which solves the problem of antibody stability during storage and transportation, and ensures that the antigen still has the immunogenicity and safety required for prevention before administration.

[0067] The term "formulation" refers to a composition that is effective in maintaining the biological activity of the active ingredient and does not contain other ingredients that are unacceptably toxic to the subject. Such formulations are sterile. "Sterile" means free of viable bacteria or free or substantially free of all viable microorganisms and their spores.

[0068] As used herein, a "stable" formulation refers to a formulation in which the active ingredient substantially retains its physical stability and / or chemical stability and / or biological activity after storage. Preferably, the formulation substantially retains its physical and chemical stability, as well as its biological activity, after storage.

[0069] The terms "patient" or "subject" are used interchangeably to refer to any mammal, preferably a human, suffering from a condition or disease according to the present invention.

[0070] As used herein, "physiologically acceptable" refers to a concentration or ionic strength of a buffer, excipient, or salt that makes the formulation biologically compatible with the intended host to be immunized, such as a human.

[0071] The stable preparation of the present invention contains human papillomavirus virus-like particles, a buffer, an osmotic pressure regulator and an aluminum adjuvant.

[0072] The terms "comprising" and "including" mean that additional ingredients may be included in addition to the listed ingredients.

[0073] As used herein and in the appended claims, the singular forms "a," "an," "another," and "the" include plural referents unless the context clearly dictates otherwise.

[0074] As used herein, "buffer" refers to a buffered solution that resists pH changes through the action of its acid-base complexing components. In one embodiment of the present invention, a histidine buffer is selected, and the pH of the formulation solution is preferably around 5.9-6.5, more preferably 6.2.

[0075] As used herein, "surfactant" refers to a surface active agent. In one embodiment, the surfactant herein is polysorbate 80.

[0076] The term "osmotic pressure regulator" means a pharmaceutically acceptable osmotic pressure regulator. Suitable osmotic pressure regulators include, but are not limited to, salts, and in one embodiment of the present invention, sodium chloride (NaCl) at a concentration of about 150 mM to 320 mM.

[0077] The term "adjuvant" refers to a compound or mixture that enhances an immune response. In particular, a vaccine may contain an adjuvant. The adjuvant used in the present invention is selected from one or more of aluminum hydroxyphosphate (AlPO4), amorphous aluminum hydroxyphosphate sulfate (AAHS) or aluminum hydroxide (Al(OH3)), preferably aluminum hydroxyphosphate (AlPO4).

[0078] The "stability" of a protein after storage at a selected temperature for a selected period of time can be qualitatively and / or quantitatively assessed in a variety of different ways. In an embodiment of the present invention, the content of active antigen that can bind to recombinant human papillomavirus neutralizing antibodies is obtained by enzyme-linked immunosorbent assay (ELISA), and the ratio of the active antigen content at each time point to T0 is compared to compare the stability of each preparation; the enzyme-linked immunosorbent assay (ELISA) method is used to obtain antigens that are not adsorbed to the aluminum phosphate adjuvant by centrifugation and perform content analysis to calculate the adsorption degree; the EC values ​​of the human papillomavirus vaccine preparation, the positive control, and the recombinant human papillomavirus neutralizing antibodies are respectively measured. 50 , calculate the EC of vaccine preparation and positive control 50 The relative efficacy of the vaccine in vitro was determined by comparing the two values.

[0079] The term "immunogenicity" refers to the ability of a substance, such as a protein or polypeptide, to stimulate an immune response, ie, the production of antibodies, particularly a humoral or cell-mediated response.

[0080] The term "HPV" or "HPV virus" refers to the papillomavirus of the Papillomaviridae family. It is a non-enveloped DNA virus with a double-stranded closed circular DNA genome of approximately 8kb in size. It can usually be divided into three regions: ① The early region (E), which contains 6 open reading frames encoding E1, E2, E4 to E7 non-structural proteins related to viral replication, transcription and transformation, as well as E3 and E8 open reading frames; ② The late region (L) contains reading frames encoding the major capsid protein L1 and the minor capsid protein L2; ③ The long regulatory region (LCR) does not encode any protein, but has the origin of replication and multiple transcription factor binding sites.

[0081] The terms "HPV L1 protein" and "HPV L2 protein" refer to proteins encoded by the late region (L) of the HPV gene and synthesized late in the HPV infection cycle. The L1 protein is the major capsid protein with a molecular weight of 55-60 kDa. The L2 protein is the minor capsid protein. Seventy-two L1 pentamers form the outer shell of the icosahedral HPV virion, encapsidating the closed-circular double-stranded DNA minichromosome. The L2 protein is located internally to the L1 protein.

[0082] The term "virus-like particle" refers to a hollow particle containing one or more structural proteins of a virus, but without viral nucleic acid.

[0083] The term "concentration of any type of papillomavirus virus-like particles" refers to the content of any type of papillomavirus virus-like particles in the preparation, and the term "total concentration of all types of papillomavirus virus-like particles" is the sum of the concentrations of each type of papillomavirus virus-like particles contained in the preparation.

[0084] In one embodiment of the present invention, a human papillomavirus multivalent immunogenic composition described in patent application PCT / CN2020 / 102601 filed on July 17, 2020 is used, and PCT / CN2020 / 102601 is introduced into this specification and claims by reference.

[0085] In a particularly preferred embodiment of the present invention, the formulation contains 0.74 mg / mL of papillomavirus virus-like particles, 1.0 mg / mL of aluminum phosphate adjuvant, 18 mM histidine buffer, 320 mM sodium chloride, and a pH of 6.2. Due to process residues during the preparation of the formulation, the vaccine may contain polysorbate 80 at a concentration of no more than 0.3 mg / mL. The formulation exhibits excellent stability, being stable for at least 24 months at 2-8°C and for at least 16 weeks at 25°C.

[0086] The formulations of the present invention may be provided in liquid form or in lyophilized form. Lyophilized formulations may be reconstituted prior to administration. Example

[0087] The present invention will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the present invention. All documents, patents, and patent applications are incorporated herein by reference.

[0088] In the following examples, the preparation, characterization and performance identification of the various types of papillomavirus virus-like particles used are described in patent application PCT / CN2020 / 102601 filed on July 17, 2020.

[0089] In the following examples, the detection method used is as follows:

[0090] 1) Antigen content analysis (Enzyme-linked Immunosorbent Assay, ELISA)

[0091] Use desorption buffer to completely dissolve the positive control (human papillomavirus virus-like particle standard, source: Shenzhou Cell Engineering Co., Ltd., which are chimeric HPV L1 proteins of HPV types 6, 16, 18, 31, 35, 30, 45, 51, 52, and 56, as well as HPV33 and HPV59 L1 proteins, corresponding to amino acid sequences SEQ ID NO: 16-29, respectively, the same below) and the test sample as the positive control and test sample to be tested.

[0092] Recombinant human papillomavirus neutralizing antibodies (Source: Beijing Sino-Bio Technologies Co., Ltd., hereinafter referred to as "Sino-Bio Technologies") are bound to a solid-phase carrier to form a solid-phase antibody. The positive control and test sample to be tested are diluted with sample diluent and then bound to the solid-phase antibody to form a solid-phase antigen-antibody complex. An enzyme-labeled antibody is then added, and a substrate is added for color development. The color product is read at a wavelength of 450 nm. A linear regression is performed using a series of positive control concentrations and the corresponding absorbance values. Substituting the measured absorbance values ​​of the test sample into the linear regression equation, the antigen content in the test sample can be calculated (M. Shank-Retzlaff, F. Wang, T. Morley et al. Correlation between Mouse Potency and In Vitro Relative Potency for Human Papillomavirus Type 16 Virus-Like Particles and Gardasil Vaccine Samples. Human Vaccines, 1:5, 191-197).

[0093] 2) Adsorption analysis (Enzyme-linked Immunosorbent Assay, ELISA)

[0094] Recombinant human papillomavirus neutralizing antibodies are bound to a solid-phase carrier to form solid-phase antibodies. The sample to be tested is centrifuged, and the supernatant is used as the test sample. The positive control and test sample are diluted accordingly with sample diluent and then bound to the solid-phase antibody to form a solid-phase antigen-antibody complex. An enzyme-labeled antibody is then added, and a substrate is added for color development. The color product is read at a wavelength of 450 nm. A linear regression is performed using a series of positive control concentrations and the corresponding absorbances. The absorbance values ​​measured for the test sample are substituted into the linear regression equation to determine the antigen concentration in the supernatant. The adsorption of the sample to be tested is then calculated using the following formula (Michael J. Caulfield, Li Shi, Su Wang et al. Effect of Alternative Aluminum Adjuvants on the Absorption and Immunogenicity of HPV16 L1 VLPs in Mice. Human Vaccines 3:4,139-146).

[0095] Adsorption degree (%) = (1-antigen concentration in the supernatant / antigen concentration in the sample to be tested)%.

[0096] 3) In Vitro Relative Potency (IVRP)

[0097] Use desorption buffer to completely dissolve the positive control (14-valent human papillomavirus vaccine (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59) control, source: Beijing Shenzhou Cell Biotechnology Group Co., Ltd., sequence is the same as the protein sequence in the test sample) and the test sample as the positive control and test sample to be tested. Dilute the recombinant human papillomavirus neutralizing antibody to a final concentration of 2μg / mL and add 100μL / well to a 96-well plate. Tap the plate gently to mix the sample and coat overnight at 4°C; wash the plate once with 200μL / well of washing solution and dry the ELISA plate; then block the ELISA plate with 300μL / well of blocking solution and block at room temperature for one hour; wash twice with 300μL / well of washing solution and add 100μL of treated blank control (corresponding buffer for the test sample) to each well. Wash the plate with 200 μL / well of washing solution), the positive control to be tested, and the test sample, and react at room temperature for 1 hour; wash the plate 3 times with washing solution at 200 μL / well, then add the diluted enzyme-labeled recombinant human papillomavirus neutralizing antibody at 100 μL / well; react at room temperature for 1 hour, wash the plate 3 times with washing solution at 200 μL / well, add the color development solution at 200 μL / well, and let it stand at room temperature for 20±5 minutes; add the stop solution at 50 μL / well to terminate the reaction; detect the absorbance at 450 nm with a microplate reader. Use the computer program Origin or the four-parameter fitting method to process, with the positive control or test sample concentration as the horizontal axis and the average absorbance intensity as the vertical axis to obtain the EC values ​​of the test sample and positive control. 50 , based on the EC of the test article 50 Divide by the EC of the positive control 50 The in vitro relative potency of the test article was obtained (M. Shank-Retzlaff, F. Wang, T. Morley et al. Correlation between Mouse Potency and In Vitro Relative Potency for Human Papillomavirus Type 16 Virus-Like Particles and Gardasil Vaccine Samples. Human Vaccines, 1:5, 191-197).

[0098] Example 1: Screening study of surfactant concentration

[0099] The formula of the papillomavirus virus-like particle vaccine preparation of this embodiment is shown in the following table:

[0100] Table 1 Preparations with different surfactant concentrations

[0101] Preparation formula number Papillomavirus virus-like particles aluminum phosphate adjuvant histidine buffer sodium chloride polysorbate 80 pH F1 0.74 mg / mL 1.0 mg / mL 18 mM 320 mM 0.00 wt% 6.5 F2 0.74 mg / mL 1.0 mg / mL 18 mM 320 mM 0.02 wt% 6.5

[0102] Preparation method of papillomavirus virus-like particle vaccine preparation:

[0103] Take a certain amount of HPV 18 virus-like particles, adjust its formula, and then mix it with aluminum phosphate adjuvant. Adsorb it at 4°C overnight so that the pH, the corresponding concentrations of papillomavirus virus-like particles, aluminum phosphate adjuvant, histidine, sodium chloride and polysorbate 80 in the papillomavirus virus-like particle vaccine preparation meet the requirements of Table 1. Sterile package, number them accordingly, and place them in a 37°C constant temperature box. Take them out at week 0 for adsorption and antigen content analysis, and take them out at weeks 1, 2, and 4 for antigen content analysis.

[0104] Analytical testing methods:

[0105] Adsorption analysis: The detection principle is to obtain the antigen that is not adsorbed to the aluminum phosphate adjuvant by centrifugation and perform content analysis to calculate the adsorption degree.

[0106] Antigen content analysis: The detection principle is to obtain the active antigen content that can bind to recombinant human papillomavirus neutralizing antibodies through ELISA. The stability of each preparation is compared by comparing the active antigen content at each time point with T0. The higher the ratio, the higher the active antigen content in the preparation and the better the activity is maintained.

[0107] The test results are shown in Table 2-3 and Figure 1-2.

[0108] Table 2 Adsorption of HPV 18 virus-like particles

[0109] Num% HPV18 VLPs F199.8 F299.9 bound to aluminum adjuvant

[0110] Table 3 Antigen content of HPV 18 virus-like particle preparations

[0111]

[0112] The test results showed that the adsorption rates of the two papillomavirus virus-like particle vaccine preparations were both above 99%, and there was no significant difference in the changing trend of the antigen content of the papillomavirus virus-like particle vaccine in F1 and F2, that is, the stability of F1 and F2 was comparable.

[0113] Example 2: pH screening study

[0114] The formula of the papillomavirus virus-like particle vaccine preparation of this embodiment is shown in the following table:

[0115] Table 4 Preparations at different pH

[0116] Preparation formula number Papillomavirus virus-like particles Aluminum phosphate adjuvant Histidine buffer Sodium chloride Polysorbate 80 pH F1 0.74 mg / mL 1.0 mg / mL 18 mM 320 mM 0.00 wt% 5.9 F2 0.74 mg / mL 1.0 mg / mL 18 mM 320 mM 0.00 wt% 6.2 F3 0.74 mg / mL 1.0 mg / mL 18 mM 320 mM 0.00 wt% 6.5

[0117] Preparation method of papillomavirus virus-like particle vaccine preparation:

[0118] Take a certain amount of HPV 18 virus-like particles, adjust its formula, and then mix it with aluminum phosphate adjuvant. Adsorb it overnight at 4°C so that the pH, the corresponding concentrations of papillomavirus virus-like particles, aluminum phosphate adjuvant, histidine, sodium chloride and polysorbate 80 in the papillomavirus virus-like particle vaccine preparation meet the requirements of Table 4. Sterile package, number them accordingly, and place them in a 37°C constant temperature box. Take them out at week 0 for adsorption and antigen content analysis, and take them out at weeks 1, 2, and 4 for antigen content analysis.

[0119] Analytical testing methods:

[0120] Adsorption analysis: The detection principle is to obtain the antigen that is not adsorbed to the aluminum phosphate adjuvant by centrifugation and perform content analysis to calculate the adsorption degree.

[0121] Antigen content analysis: The detection principle is to obtain the active antigen content that can bind to recombinant human papillomavirus neutralizing antibodies through ELISA. The stability of each preparation is compared by comparing the active antigen content at each time point with T0. The higher the ratio, the higher the active antigen content in the preparation and the better the activity is maintained.

[0122] The test results are shown in Table 5-6 and Figure 3-4.

[0123] Table 5 Adsorption of HPV 18 virus-like particles

[0124] Num% HPV18 VLPs bound to aluminum adjuvant F199.4 F299.4 F399.8

[0125] Table 6 Antigen content of HPV 18 virus-like particle preparations

[0126]

[0127] The test results showed that the adsorption degrees of the three papillomavirus virus-like particle vaccine preparations were all above 99%, and the antigen content change trend of the papillomavirus virus-like particle vaccine in F1 and F2 was better than that in the F3 preparation, that is, the stability of F1 and F2 was better than that of F3.

[0128] Example 3: Screening study of osmotic pressure regulator concentration

[0129] The formula of the papillomavirus virus-like particle vaccine preparation of this embodiment is shown in the following table:

[0130] Table 7 Preparations with different osmotic pressure regulator concentrations

[0131] Preparation formula number Papillomavirus virus-like particles aluminum phosphate adjuvant histidine buffer sodium chloride polysorbate 80 pH F10.74 mg / mL1.0 mg / mL18 mM320 mM0.00 wt%6.2 F20.74 mg / mL1.0 mg / mL18 mM150 mM0.00 wt%6.2

[0132] Preparation method of papillomavirus virus-like particle vaccine preparation:

[0133] Take a certain amount of HPV 18 virus-like particles, adjust its formula, and then mix it with aluminum phosphate adjuvant. Adsorb it overnight at 4°C so that the pH, the corresponding concentrations of papillomavirus virus-like particles, aluminum phosphate adjuvant, histidine, sodium chloride and polysorbate 80 in the papillomavirus virus-like particle vaccine preparation meet the requirements of Table 7. Sterile package, number them accordingly, and place them in a 37°C constant temperature box. Take them out at week 0 for adsorption and antigen content analysis, and take them out at weeks 1, 2, and 4 for antigen content analysis.

[0134] Analytical testing methods:

[0135] Adsorption analysis: The detection principle is to obtain the antigen that is not adsorbed to the aluminum phosphate adjuvant by centrifugation and perform content analysis to calculate the adsorption degree.

[0136] Antigen content analysis: The detection principle is to obtain the active antigen content that can bind to recombinant human papillomavirus neutralizing antibodies through ELISA. The stability of each preparation is compared by comparing the active antigen content at each time point with T0. The higher the ratio, the higher the active antigen content in the preparation and the better the activity is maintained.

[0137] The test results are shown in Table 8-9 and Figure 5-6.

[0138] Table 8 Adsorption of HPV 18 virus-like particles

[0139] Num% HPV18 VLPs F199.4 F299.6 bound to aluminum adjuvant

[0140] Table 9 Antigen content of HPV 18 virus-like particle preparations

[0141]

[0142] The test results showed that the adsorption rates of the two papillomavirus virus-like particle vaccine preparations were both above 99%. After 4 weeks at 37°C, there was no significant difference in the change in the active antigen content in the F1 and F2 preparations, indicating that the stability of F1 and F2 was comparable.

[0143] Example 4: Screening study of buffer concentration

[0144] The formula of the papillomavirus virus-like particle vaccine preparation of this embodiment is shown in the following table:

[0145] Table 10 Preparations with different buffer concentrations

[0146] Preparation formula number Papillomavirus virus-like particles Aluminum phosphate adjuvant Histidine buffer Sodium chloride Polysorbate 80 pH F10.74 mg / mL1.0 mg / mL10 mM320 mM0.00 wt% 6.2 F20.74 mg / mL1.0 mg / mL18 mM320 mM0.00 wt% 6.2 F30.74 mg / mL1.0 mg / mL26 mM320 mM0.00 wt% 6.2

[0147] Preparation method of papillomavirus virus-like particle vaccine preparation:

[0148] Take a certain amount of HPV 18 virus-like particles, adjust its formula, and then mix it with aluminum phosphate adjuvant. Adsorb it at 4°C overnight so that the pH, the corresponding concentrations of papillomavirus virus-like particles, aluminum phosphate adjuvant, histidine, sodium chloride and polysorbate 80 in the papillomavirus virus-like particle vaccine preparation meet the requirements of Table 10. Sterile package, number them accordingly, and place them in a 37°C constant temperature box. Take them out at week 0 for adsorption and antigen content analysis, and take them out at weeks 1, 2, and 4 for antigen content analysis.

[0149] Analytical testing methods:

[0150] Adsorption analysis: The detection principle is to obtain the antigen that is not adsorbed to the aluminum phosphate adjuvant by centrifugation and perform content analysis to calculate the adsorption degree.

[0151] Antigen content analysis: The detection principle is to obtain the active antigen content that can bind to recombinant human papillomavirus neutralizing antibodies through ELISA. The stability of each preparation is compared by comparing the active antigen content at each time point with T0. The higher the ratio, the higher the active antigen content in the preparation and the better the activity is maintained.

[0152] The test results are shown in Tables 11-12 and Figures 7-8.

[0153] Table 11 Adsorption of HPV 18 virus-like particles

[0154] Num% HPV18 VLPsF199.6 bound to aluminum adjuvant

[0155] F299.4F399.6

[0156] Table 12 Antigen content of HPV 18 virus-like particle preparations

[0157]

[0158] The test results showed that the adsorption degrees of the three papillomavirus virus-like particle vaccine preparations were all above 99%, and the antigen content change trend of the papillomavirus virus-like particle vaccine in F1 and F2 was better than that in the F3 preparation, that is, the stability of F1 and F2 was better than that of F3.

[0159] Example 5: Study on the Confirmation of the Prescription of Papillomavirus Virus-like Particle Vaccine Preparations of Various Types (Types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59)

[0160] The stability of various types (6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59) of papillomavirus virus-like particle vaccine formulations (formulation formula: 0.74 mg / mL papillomavirus virus-like particles + 1.0 mg / mL aluminum phosphate adjuvant + 18 mM histidine buffer + 320 mM sodium chloride, pH 6.2) was investigated at 2-8°C. Due to process residues during the preparation of the formulation, the vaccine will contain polysorbate 80 at a concentration not exceeding 0.3 mg / mL. The investigation time points were 3 months (3 months), 6 months (6 months), 9 months (9 months), and 12 months (12 months), and the investigation items were adsorption and in vitro relative efficacy.

[0161] Analytical testing methods:

[0162] Adsorption degree: The detection principle is to obtain the antigen that is not adsorbed to the aluminum phosphate adjuvant by centrifugation and perform content analysis to calculate the adsorption degree.

[0163] In vitro relative potency: The detection principle of this method is to detect the EC of the test sample and positive control with the recombinant human papillomavirus neutralizing antibody. 50 , calculate the EC of the test article and the positive control 50 The higher the value, the higher the in vitro relative potency of the test article and the better the quality of the test article.

[0164] The experimental results are shown in Table 13.

[0165] Experimental results show that the various types of papillomavirus virus-like particle vaccine preparations disclosed in the present invention have good stability and can be stably stored at 2-8°C for at least 12 months.

[0166] Table 13 Stability data of 14 types (6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59) of papillomavirus virus-like particles adjuvant adsorption liquid formulations

[0167]

[0168]

[0169] Example 6: Preparation and formulation verification experiments of 14-valent human papillomavirus vaccines (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59)

[0170] Preparation method of 14-valent human papillomavirus vaccine (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59):

[0171] Take each type (6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59) of papillomavirus virus-like particle vaccine preparation (preparation formula is 0.74 mg / mL papillomavirus virus-like particles + 1.0 mg / mL aluminum phosphate adjuvant + 18 mM histidine buffer + 320 mM sodium chloride, pH 6.2) according to a certain volume ratio (6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 type = 1.5: 2: 3: 2: 1: 1: 1: 1: 1: 1: 1: 1: 1) were aseptically mixed to obtain a semi-finished product of a 14-valent human papillomavirus vaccine (6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 type). The concentrations of virus-like particles of each type are 0.06 mg / mL, 0.08 mg / mL, 0.12 mg / mL, 0.08 mg / mL, 0.04 mg / mL, 0.04 mg / mL, 0.04 mg / mL, 0.04 mg / mL, 0.04 mg / mL, 0.04 mg / mL, 0.04 mg / mL, 0.04 mg / mL, 0.04 mg / mL, 0.04 mg / mL, 0.04 mg / mL, and then filled into vials, stoppered, capped, and labeled to prepare recombinant fourteen-valent human papillomavirus vaccine (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59). Due to process residues in the preparation process, the vaccine will contain polysorbate 80 at a concentration not higher than 0.3 mg / mL. Then, stability studies were conducted at 2-8°C (time points: 3, 6, 9, 12, 18, and 24 months) and 25±2°C (time points: 2 weeks, 4 weeks, 8 weeks, and 16 weeks), and the study items were in vitro relative efficacy and adsorption.

[0172] Analytical testing methods:

[0173] Adsorption degree: The detection principle is to obtain the antigen that is not adsorbed to the aluminum phosphate adjuvant by centrifugation and perform content analysis to calculate the adsorption degree.

[0174] In vitro relative potency: The detection principle of this method is to detect the EC of the test sample and positive control with the recombinant human papillomavirus neutralizing antibody. 50 , calculate the EC of the test article and the positive control 50 The higher the value, the higher the in vitro relative potency of the test article and the better the quality of the test article.

[0175] The experimental results are shown in Tables 14-17.

[0176] Table 14 In vitro relative efficacy of recombinant 14-valent human papillomavirus vaccines (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59) (25°C)

[0177]

[0178] Table 15 Adsorption of recombinant 14-valent human papillomavirus vaccine (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59) (25°C)

[0179]

[0180] Table 16 In vitro relative efficacy of recombinant 14-valent human papillomavirus vaccines (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59) (4°C)

[0181]

[0182] Table 17 Adsorption of recombinant 14-valent human papillomavirus vaccine (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59) (4°C)

[0183]

[0184]

[0185] Experimental results show that the fourteen-valent human papillomavirus vaccine (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59) preparation disclosed in the present invention has good stability and can be stably stored for at least 24 months at 2-8°C and for at least 16 weeks at 25°C.

[0186] Sequence Listing

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

Claims

1. A stable preparation of a multivalent human papillomavirus virus-like particle vaccine for preventing HPV-related diseases or infections, comprising (a) multiple human papillomavirus virus-like particles; (b) an adjuvant; (c) a buffer at a physiologically acceptable concentration; (d) an osmotic pressure regulator at a physiologically acceptable concentration; and optionally (e) a surfactant at a physiologically acceptable concentration, wherein the human papillomavirus virus-like particles are selected from HPV virus-like particles assembled from the L1 proteins of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58; and one or more HPV virus-like particles assembled from the L1 proteins of other pathogenic HPV types, wherein the human papillomavirus virus-like particles are adsorbed on the adjuvant.

2. The preparation according to claim 1, characterized in that the buffer is selected from one or more of citrate buffer, acetate buffer, or histidine buffer; the osmotic pressure regulator is selected from one or more of sodium chloride, sodium phosphate, or sodium sulfate; the surfactant is a polyethoxy ether, preferably polysorbate 80; The adjuvant is preferably an aluminum adjuvant, more preferably one or more of aluminum hydroxyphosphate (AlPO 4 ), amorphous aluminum hydroxyphosphate sulfate (AAHS), or aluminum hydroxide (Al(OH 3 ))), and most preferably aluminum hydroxyphosphate (AlPO 4 ).

3. The preparation according to claim 1 or 2, characterized in that (a) the total concentration of all types of papillomavirus virus-like particles is 40 - 740 μg / mL; (b) the concentration of the buffer is 10 mM - 26 mM, preferably 10 mM, 18 mM, or 26 mM; (c) the concentration of the osmotic pressure regulator is 150 mM - 320 mM, preferably 150 mM or 320 mM; (d) the concentration of the surfactant is 0 - 0.02 wt%; (e) the concentration of the adjuvant is about 1.0 mg / mL; (f) the pH of the preparation is 5.9 - 6.5, preferably 5.9, 6.2, or 6.

5.

4. The preparation according to claim 3, characterized in that the concentration of any type of papillomavirus virus-like particle constituting the multivalent papillomavirus virus-like particles is 40 μg / mL - 120 μg / mL.

5. The preparation according to any one of claims 1 - 4, which contains a total of 0.74 mg / mL papillomavirus virus-like particles, 1.0 mg / mL aluminum phosphate adjuvant, 18 mM histidine buffer, 320 mM sodium chloride, and a pH value of 6.2; optionally, polysorbate 80 at a concentration not higher than 0.3 mg / mL.

6. The preparation according to claim 5, wherein the one or more other pathogenic HPV types are selected from HPV types 35, 39, 51, 56, and 59.

7. The preparation according to claim 6, wherein at least one of the HPV virus-like particles is a chimeric HPV virus-like particle, and the chimeric HPV virus-like particle comprises a chimeric HPV L1 protein; the chimeric HPV L1 protein comprises, from its N-terminal to C-terminal direction: a. An N-terminal fragment derived from the L1 protein of a first type of papillomavirus, the N-terminal fragment retaining the immunogenicity of the L1 protein of this type, wherein the papillomavirus of the first type is selected from HPV type 6, 11, 16, 18, 31, 33, 45, 52 and 58 and one or more other pathogenic HPV types; and b. A C-terminal fragment derived from the L1 protein of a second type of papillomavirus, the L1 protein of the second type of papillomavirus having better expression level and solubility compared to the L1 proteins of other types; wherein the chimeric HPV L1 protein has the immunogenicity of the L1 protein of the first type of papillomavirus.

8. The preparation according to claim 7, wherein the N-terminal fragment is a fragment obtained by truncating the C-terminus of the natural sequence of the L1 protein of the first type of papillomavirus at any amino acid site within its α5 region, and a fragment having at least 98% identity therewith; and the C-terminal fragment is a fragment obtained by truncating the N-terminus of the natural sequence of the L1 protein of the second type of papillomavirus at any amino acid site within its α5 region, and a functional variant generated by further mutation, deletion and / or addition of this fragment.

9. The preparation according to claim 8, wherein the C-terminal fragment contains one or more nuclear localization sequences.

10. The preparation according to claim 7, wherein the L1 protein of the first type of papillomavirus is selected from HPV type 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, 56 or 58; preferably, its natural sequence is the amino acid sequence encoded by the coding gene shown in SEQ ID No:30, SEQ ID No:31, SEQ ID No:32, SEQ ID No:33, SEQ ID No:34, SEQ ID No:35, SEQ ID No:36, SEQ ID No:37, SEQ ID No:38, SEQ ID No:39, SEQ ID No:40, or SEQ ID No:41 respectively; wherein the L1 protein of the second type of papillomavirus is selected from the L1 proteins of HPV type 16, 28, 33, 59 or 68; more preferably, the L1 protein of the second type of papillomavirus is selected from the L1 proteins of HPV type 33 or HPV type 59.

11. The preparation according to claim 10, wherein the C-terminal fragment is SEQ ID No:1; or a fragment having a length of m1 amino acids, preferably a fragment covering the 1-m1th amino acids of SEQ ID No:1; wherein m1 is an integer from 8 to 26; or the C-terminal fragment is SEQ ID No:2; or a fragment having a length of m2 amino acids, preferably a fragment covering the 1-m2th amino acids of SEQ ID No:2; wherein m2 is an integer from 13 to 31.

12. The preparation according to claim 10, wherein the C-terminal fragment is SEQ ID No: 3; or a fragment having a length of n amino acids, preferably a fragment covering the 1st - nth amino acids of SEQ ID No: 3; where n is an integer from 16 to 38.

13. The preparation according to claim 7, wherein the N-terminal fragment of the HPV type 6 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 4 at any amino acid site within its α5 region; the N-terminal fragment of the HPV type 11 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 5 at any amino acid site within its α5 region; the N-terminal fragment of the HPV type 16 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 6 at any amino acid site within its α5 region; the N-terminal fragment of the HPV type 18 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 7 at any amino acid site within its α5 region; the N-terminal fragment of the HPV type 31 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 8 at any amino acid site within its α5 region; the N-terminal fragment of the HPV type 35 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 9 at any amino acid site within its α5 region; the N-terminal fragment of the HPV type 39 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 10 at any amino acid site within its α5 region; the N-terminal fragment of the HPV type 45 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 11 at any amino acid site within its α5 region; the N-terminal fragment of the HPV type 51 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 12 at any amino acid site within its α5 region; The N-terminal fragment of the HPV 52 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 13 at any amino acid site within its α5 region; The N-terminal fragment of the HPV 56 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 14 at any amino acid site within its α5 region; and The N-terminal fragment of the HPV 58 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No: 15 at any amino acid site within its α5 region.

14. The preparation according to claim 7, wherein the C-terminus of the N-terminal fragment is directly connected or connected via a linker to the N-terminus of the C-terminal fragment.

15. The preparation according to claim 7, wherein when the C-terminus of the N-terminal fragment is connected to the N-terminus of the C-terminal fragment, the following continuous amino acid sequence exists within the range of plus or minus 4 amino acid sites at the connection point: RKFL; Preferably, the following continuous amino acid sequence exists within the range of plus or minus 6 amino acid sites at the connection point: LGRKFL.

16. The preparation according to claim 7, wherein the chimeric HPV 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, 56 and 58 chimeric HPV L1 proteins have 98%, 98.5%, 99%, 99.5% or 100% identity with SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21, SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25, SEQ ID No: 26 and SEQ ID No: 27 respectively; and the HPV 33 L1 protein and the HPV 59 L1 protein have 98%, 98.5%, 99%, 99.5% or 100% identity with SEQ ID No: 28 and SEQ ID No: 29 respectively.

17. The preparation according to claim 16, which comprises Chimeric HPV L1 proteins of HPV types 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, 56, and 58, having the amino acid sequences shown in SEQ ID No:16, SEQ ID No:17, SEQ ID No:18, SEQ ID No:19, SEQ ID No:20, SEQ ID No:21, SEQ ID No:22, SEQ ID No:23, SEQ ID No:24, SEQ ID No:25, SEQ ID No:26, and SEQ ID No:27, respectively; and HPV type 33 L1 protein and HPV type 59 L1 protein having the amino acid sequences shown in SEQ ID No:28 and SEQ ID No:29, respectively.

18. The preparation of the papillomavirus vaccine according to claims 1-17, characterized in that it can be stably stored for at least 24 months at 2-8 °C and for at least 16 weeks at 25 °C.

19. A method for preventing HPV-related diseases or infections, which comprises: administering to a subject the preparation according to any one of claims 1-18.

20. Use of the preparation according to any one of claims 1-18 in the manufacture of a vaccine for preventing HPV-related diseases or infections.