Human papillomavirus vaccine and its use

Abstract

A human papillomavirus (HPV) vaccine is offered. This vaccine contains L1 protein antigens of HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. The recombinant 15-valent human papillomavirus vaccine contains the 12 types clearly associated with human cancer, two common low-risk types (HPV6 and 11), and one suspected carcinogenic type (HPV68). In China, this vaccine is expected to improve the preventive effect against cervical cancer in women by more than 97.2%, and worldwide, it is expected to improve the preventive effect against cervical cancer in women by more than 94.1%, indicating high application value.

Description

Technical Field

[0001] The present invention relates to the field of medicine, specifically to a vaccine against human papillomavirus, particularly a 15-valent human papillomavirus vaccine.

Background Art

[0002] Human papillomavirus (HPV) is a small DNA virus without an envelope and can infect human epidermal and mucosal squamous epithelial cells. In 1974, the relationship between HPV infection and cervical cancer was first proposed, and finally it was proved that HPV infection is the main cause of cervical cancer. HPV infection is the most common genital virus infection. Human infection with HPV can not only cause cervical cancer, but also cause anal-genital cancer and genital warts. In addition, the occurrence of oropharyngeal cancer, other head and neck cancers, colon cancer, and rectal cancer is also related to HPV infection. Currently, more than 200 types of HPV have been identified and divided into low-risk types and high-risk types according to carcinogenicity. Low-risk types of HPV mainly include HPV6 / 11 / 30 / 42 / 43 / 44 / 61 types, etc. Among them, 90% of genital warts are caused by infection with HPV6 and 11 types. High-risk types of HPV recently defined by the World Health Organization (WHO) International Agency for Research on Cancer (IARC) include types clearly related to 12 human cancers (HPV16 / 18 / 31 / 35 / 35 / 39 / 51 / 52 / 56 / 58 / 59 types) and two types with evidence of carcinogenicity (HPV 66 / 68 types). High-risk types of HPV have carcinogenicity and are the cause of almost all uterine cancers. In addition, they cause 88% of anal cancers, 78% of vaginal cancers, 15%-48% of vulvar cancers (age-related), 51% of penile cancers, and 13%-60% of oropharyngeal cancers.

[0003] Vaccination with the HPV vaccine is the most economical and effective way to prevent persistent HPV infection and related diseases. Currently, there are three types of HPV vaccines commercially available overseas: a bivalent HPV vaccine (HPV types 16 / 18) manufactured by GSK in the UK (trade name: Cervarix), a quadrivalent HPV vaccine (HPV types 6 / 11 / 16 / 18) manufactured by Merck in the US (trade name: Gardasil), and a recombinant 9-valent HPV vaccine (HPV types 6 / 11 / 16 / 11 / 31 / 45 / 52 / 52 / 58) (trade name: Gardasil9). All three of these HPV preventive vaccines use DNA recombination technology to express and purify the HPVL1 structural protein, which then self-assembles to form HPV genotype-specific VLPs. Since VLPs do not contain viral nucleic acid components and do not have protein conversion activity like proteins, they are not infectious and are used only for prevention.

[0004] To date, there are no broad-spectrum HPV vaccines available in China or outside of China that cover 12 types of HPV clearly associated with human cancer (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59), 2 types with limited evidence of carcinogenicity (HPV types 66 / 68), and HPV types 6 and 11, which cause genital warts. Therefore, a broader-spectrum HPV vaccine is needed to improve the preventive effect against cervical cancer in women. [Overview of the project]

[0005] Based on the demands of the prior art, the present invention, through diligent research, has obtained a 15-valent HPV vaccine that provides protection against all 12 types (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59) that are clearly associated with human cancer.

[0006] The human papillomavirus vaccine provided in the present invention comprises one or more L1 antigens from HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68, for example, 15, 9, 6, and 3. Preferably, the weight ratio of HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 antigens is 1-3:1-5:2-7:1-5:0.5-3:0.5-3:0.5-3:0.5-3:0.5-3:0.5-2:0.5-2:0.5-2:0.5-2:0.5-2, and preferably, HP The weight ratio of the doses of antigens V6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 is 1.5:2:3:2:1:1:1:1:1:1:1:1:1:1:1, or 1.5:2:3:2:1:1:1:1:1:1:1:1:1:1:1, or 3:4:6:4:2:2:2:2:1:1:1:1:1:1, More specifically, the dose of each protein is 10-100 μg, and more preferably, HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 contain 30 μg, 40 μg, 60 μg, 40 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, and 20 μg, respectively, per 0.5 ml.

[0007] More preferably, the L1 antigen of each type is shortened from the wild-type sequence as follows: In wild-type HPV6 L1, the N-terminus is shortened by 2 amino acids and the C-terminus by 29 amino acids; in wild-type HPV11 L1, the N-terminus is shortened by 3 amino acids and the C-terminus by 29 amino acids; in wild-type HPV16 L1, the N-terminus is shortened by 4 amino acids and the C-terminus by 29 amino acids; in wild-type HPV18 L1, the N-terminus is shortened by 4 amino acids and the C-terminus by 30 amino acids; in wild-type HPV31 L1, the N-terminus is shortened by 4 amino acids and the C-terminus by 27 amino acids; in wild-type HPV33 L1, the N-terminus is shortened by 4 amino acids and the C-terminus by 24 amino acids; in wild-type HPV35 L1, the N-terminus is shortened by 4 amino acids and the C-terminus by 28 amino acids; in wild-type HPV39 L1, the N-terminus is shortened by 9 amino acids and the C-terminus by 29 amino acids; and in wild-type HPV45 L1. In L1, the N-terminus is shortened by 4 amino acids and the C-terminus by 30 amino acids; in wild-type HPV51 L1, the N-terminus is shortened by 4 amino acids and the C-terminus by 28 amino acids; in wild-type HPV52 L1, the N-terminus is shortened by 4 amino acids and the C-terminus by 23 amino acids; in wild-type HPV56 L1, the N-terminus is shortened by 4 amino acids and the C-terminus by 25 amino acids; in wild-type HPV58 L1, the N-terminus is shortened by 4 amino acids and the C-terminus by 23 amino acids; in wild-type HPV59 L1, the N-terminus is shortened by 4 amino acids and the C-terminus by 31 amino acids; and in wild-type HPV68 L1, the N-terminus is shortened by 4 amino acids and the C-terminus by 28 amino acids.

[0008] In one embodiment, the amino acid sequences of the L1 antigens of HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 are shown in SEQ ID NOs. 1 to 15, respectively.

[0009] Preferably, the adjuvant used is an aluminum adjuvant, more preferably an aluminum hydroxide adjuvant, and more preferably the mass ratio of the antigen protein to the aluminum adjuvant is 0.5-1:1, more preferably 0.7-0.85:1.

[0010] More preferably, the vaccine is prepared using an acetate-sodium acetate buffer system as a buffer, and an adjuvant containing histidine, sodium chloride, and polysorbate 80 is added.

[0011] More preferably, the acetate-sodium acetate buffer system has a pH of 5-7, more preferably 5.3-6.5, a concentration of 5-15 mM, preferably 10 mM, the concentration of sodium chloride is set to 100-400 mM, more preferably 320-335 mM, and the concentration of polysorbate 80 is 0.005%-0.02%, preferably 0.01%.

[0012] In the vaccine preparation method provided in the present invention, Purified HPV L1 proteins of each type are self-assembled in vitro to form VLPs. The VLPs are then subjected to column chromatography with liquid exchange and sterile filtration to obtain the protein stock solution. Each stock solution is then diluted to the required concentration with buffer, and then all of the protein dilutions of each type are mixed in a predetermined ratio to obtain a 15-valent protein dilution. This is then sterile filtered and prepared for use. Dilute the prescribed amount of aluminum hydroxide adjuvant and mix it uniformly (preferably by mixing with a magnetic stirrer at 300-340 rpm for 30 minutes or more), filter it through a capsule filter, and prepare it for use. By taking the required amounts of the 15-valent protein dilution filtration sample and the aluminum hydroxide adjuvant dilution filtration sample and mixing them completely and homogeneously, i.e., obtaining a semi-finished product, The selection further includes filling the finished product, which involves starting a pre-filled syringe filling machine to fill the syringes, preferably at a filling speed of 30-40 rpm / min, more preferably dispensing 0.55 mL / syringe, sealing it with a rubber stopper, and storing it in a refrigerator for use.

[0013] In a specific embodiment, in a method for preparing purified HPV L1 proteins of each type, The fermentation products of recombinant bacteria with each type of L1 protein are taken, the bacterial cells are resuspended, and the cells are disrupted using a high-pressure homogenizer. The resulting cell homogenates are then centrifuged and the supernatant is collected. Add ammonium sulfate powder to the centrifugated supernatant until it reaches a saturation level of 25% to 45%, and stir slowly until completely dissolved. After collecting the precipitate by continuous centrifugation and completely resuspending the precipitate, collect the supernatant by centrifugation again. The supernatant is filtered and clarified, preferably by two-stage deep filtration, and more preferably by a filtration membrane with pore sizes of 3.0 to 6.0 μm and 0.2 to 0.4 μm. Furthermore, the product is purified by EQ anion exchange chromatography → SQ anion exchange chromatography → gel filtration chromatography, and preferably, the specific procedure is as follows: (1) EQ anion exchange chromatography: After equilibrating the column, deep filtration is performed, the sample is collected and loaded, and after loading, rinse is performed, and the EQ ion exchange phosphate with an OD280 higher than 50 mAU is collected. (2) SQ anion exchange chromatography: After equilibrating the column, take the EQ anion exchange perfusion from step (1) and load it. After loading, rinse, and after rinsing, elute. When eluting, collect the eluted fraction corresponding to the absorption peak with an OD280 of 40 mAU or more to obtain the target protein. (3) Gel filtration chromatography: After equilibrating the chromatography column and stabilizing the baseline, load the target protein sample eluted and collected from SQ anion exchange chromatography, collect the eluted fraction corresponding to the absorption peak with an OD280 higher than 50 mAU to obtain the HPV L1 protein.

[0014] The present invention further provides a method for storing the vaccine, which is stored at 2-8°C.

[0015] The present invention further provides, lastly, the use of the vaccine in a drug for preventing or treating a disease caused by the human papillomavirus.

[0016] The beneficial effects of the present invention are as follows: The vaccine of the present invention provides a broader spectrum by adding six types of antigens. Furthermore, after optimizing the antigen dose, it does not cause immune interference between antigenic groups. The recombinant 15-valent human papillomavirus vaccine of the present invention contains the 12 types clearly associated with human cancer, two common low-risk types (HPV6 and 11), and one suspected oncogenic type (HPV68). In China, the preventive effect of this vaccine against cervical cancer in women is expected to improve by more than 97.2%, and worldwide, the preventive effect against cervical cancer in women is expected to improve by more than 94.1%.

[0017] The aluminum adjuvant used in the commercially available 9-valent vaccine Gardasil 9 is aluminum sulfate hydroxyphosphate adjuvant (a type of aluminum phosphate adjuvant), while the aluminum adjuvant for the vaccine of the present invention is aluminum hydroxide. The proteins of each type of vaccine of the present invention are prepared by expression after shortening the C-terminus, removing the positively charged portion, and the proteins become negatively charged overall in an environment with a near-neutral pH, making them readily adsorbed by positively charged aluminum hydroxide adjuvants. Gardasil 9, on the other hand, is prepared for protein expression using the full-length sequence, and the proteins become positively charged overall in an environment with a near-neutral pH, making them less readily adsorbed by aluminum hydroxide. [Brief explanation of the drawing]

[0018] [Figure 1] This is the Tm measurement result using differential scanning fluorescence. [Figure 2] This is a heatmap of Tm values ​​measured by differential scanning fluorescence. [Figure 3] This is the neutralizing antibody titer after leaving the vaccine at 4°C for two weeks. [Figure 4] This is the neutralizing antibody titer after leaving the vaccine at 37°C for one week. [Figure 5] This is the neutralizing antibody titer after leaving the vaccine at 37°C for two weeks.

Best Mode for Carrying Out the Invention

[0019] Hereinafter, the present invention will be described with specific embodiments in order to better understand the present invention, but this does not limit the present invention.

[0020] Example 1: Preparation of Each Type of Antigen The relevant amino acid sequences and preparation methods of each type of L1 protein used as an antigen in the examples of the present invention are as follows. <N

[0021] The amino acid sequence of the HPV6 L1 protein is shown in SEQ ID NO: 1. For the preparation procedure of its antigen protein, please refer to Examples 1, 2, 10, 11, and 12 in CN201410685769.1.

[0022] The amino acid sequence of the HPV11 L1 protein is shown in SEQ ID NO: 2. For the preparation procedure of its antigen protein, please refer to Examples 1, 2, 10, 11, and 12 in Chinese Patent Application CN201410672159.8.

[0023] The amino acid sequence of the HPV16 L1 protein is shown in SEQ ID NO: 3. For the preparation procedure of its antigen protein, please refer to Examples 1, 2, 10, 11, and 12 in Chinese Patent Application CN201410683185.0.

[0024] The amino acid sequence of the HPV18 L1 protein is shown in SEQ ID NO: 4. For the preparation procedure of its antigen protein, please refer to Examples 1, 2, 10, 11, and 12 in Chinese Patent Application CN201410672158.3.

[0025] The amino acid sequence of the HPV31 L1 protein is shown in SEQ ID NO: 5. For the preparation procedure of its antigen protein, please refer to Examples 1, 2, 10, 11, and 12 in Chinese Patent Application CN201510490172.6.

[0026] The amino acid sequence of the HPV33 L1 protein is shown in Sequence ID No. 6, and the procedure for preparing its antigen protein is shown in Examples 1, 2, 10, 11, and 12 of Chinese Patent Application CN201510490177.9.

[0027] The amino acid sequence of the HPV35 L1 protein is shown in Sequence ID No. 7, and the procedure for preparing the antigen protein is shown in Examples 1, 2, 3, and 4 of Chinese Patent Application CN202211702926.6.

[0028] The amino acid sequence of the HPV39 L1 protein is shown in Sequence ID No. 8, and the procedure for preparing its antigen protein is shown in Examples 1, 2, 3, and 4 of Chinese Patent Application CN202211702935.5.

[0029] The amino acid sequence of the HPV45 L1 protein is shown in Sequence ID No. 9, and the procedure for preparing its antigen protein is shown in Examples 1, 2, 10, 11, and 12 of Chinese Patent Application CN201510490367.0.

[0030] The amino acid sequence of the HPV51 L1 protein is shown in Sequence ID No. 10, and the procedure for preparing the antigen protein is described in the entirety of the experimental method in the embodiment of Chinese Patent Application CN202310020457.8.

[0031] The amino acid sequence of the HPV52 L1 protein is shown in Sequence ID No. 11, and the procedure for preparing the antigen protein is shown in Examples 1, 2, 10, 11, and 12 of Chinese Patent Application CN201510490149.7.

[0032] The amino acid sequence of the HPV56 L1 protein is shown in Sequence ID No. 12, and the procedure for preparing its antigen protein is described in detail in experimental steps 1, 2, and 3 of the embodiment in Chinese patent application CN202310019679.8.

[0033] The amino acid sequence of the HPV58 L1 protein is shown in Sequence ID No. 13, and the procedure for preparing the antigen protein is shown in Examples 1, 2, 10, 11, and 12 of Chinese Patent Application CN201410672161.5.

[0034] The amino acid sequence of the HPV59 L1 protein is shown in Sequence ID No. 14, and the procedure for preparing the antigen protein is shown in Examples 1, 2, 3, and 4 of Chinese Patent Application CN202211702927.0.

[0035] The amino acid sequence of the HPV68 L1 protein is shown in SEQ ID NO: 15, and the procedure for preparing its antigen protein is described in Examples 1, 3, 4, and 5 of Chinese Patent Application CN202211339029.3. The coding nucleotide sequence of the shortened HPV68 L1 protein is optimized as shown in SEQ ID NO: 16, and the vector used is pKL30 (SD sequence: 5'-AGGAGGAATTA-3'), which is used to construct recombinant expression.

[0036] For each antigen sequence, please refer to Sequence IDs 1 to 15 in Chinese Patent Application CN202310114443.2, and for the overall construction procedure, please refer to Examples 1 and 2 in Chinese Patent Application CN202310114443.2.

[0037] The preparation methods for each type of antigen are as follows (see the description in the above patent application). 1. Cell resuspension: Fermentation product (containing 2-5 kg ​​of wet cells) was taken, and rupture buffer was added to resuspend the cells so that the mass ratio of cells to resuspension was 1:4-1:10. The resuspended cells were lysed using a high-pressure homogenizer (pressure 80 MPa), and the cells were lysed three times. The resulting cell homogenate was centrifuged using a tube centrifuge (15760 g, feed rate 200-250 mL / min), and the supernatant was collected.

[0038] 2. Ammonium sulfate precipitation: Ammonium sulfate powder was added to the supernatant of the centrifugation solution to a saturation level of 25% to 45%, and the solution was slowly stirred at room temperature until completely dissolved. The precipitate was collected by centrifugation using a tube centrifuge (15760g, feed rate 200-250mL / min). The precipitate was resuspended by adding redissolution buffer in a mass ratio of 1:5 to 1:10, and after complete dissolution, the supernatant was collected again by centrifugation.

[0039] 3. Microfiltration clarification: The supernatant from centrifugal separation was further clarified by two-stage deep filtration. The pore sizes of the filtration membranes were 3.0-6.0 μm and 0.2-0.4 μm.

[0040] 4. Chromatography: The chromatography process consists of three steps: EQ anion exchange chromatography → SQ anion exchange chromatography → gel filtration chromatography. The procedure is as follows:

[0041] (1) EQ anion exchange chromatography: After equilibrating the column and performing deep filtration, the sample was collected and loaded, rinsed, and the EQ anion exchange phosphate with an OD280 higher than 50 mAU was collected. (2) SQ anion exchange chromatography: After equilibrating the column, the EQ anion exchange perfusion from step (1) was taken and loaded, rinsed, and eluted after rinsing. The eluted fraction corresponding to the absorption peak with an OD280 of 40 mAU or more during elution was collected to obtain the target protein. (3) Gel filtration chromatography: After equilibrating the chromatography column and stabilizing the baseline, the target protein sample eluted and collected from SQ anion exchange chromatography was loaded, and the eluted fraction corresponding to the absorption peak with an OD280 higher than 50 mAU was collected to obtain the HPV L1 target protein.

[0042] 5. VLP assembly, solution exchange, and stock solution storage: Purified HPV L1 protein was self-assembled in vitro to form VLPs. The VLPs were then subjected to solution exchange by column chromatography and sterile filtration to prepare the protein stock solution. The protein stock solution was stored at -80°C for use. The minimum storage concentration of each type of protein stock solution was 1.56 mg / mL, and the pH range of the buffer used was 4.3–5.8.

[0043] Example 2: Study of the buffering system of the formulation The main objective of formulation buffer system research is to determine the preferred pH range and sodium chloride concentration. There are two research methods. One is to measure the Tm value of each type of L1-VLP antigen using differential scanning fluorescence (Tm) in different formulations (different pH and salt concentration). A higher Tm value indicates greater antigen stability in that formulation, and a parameter range favorable to antigen stability is selected based on the measurement results for each formulation of each type. The other is to prepare 15-valent HPV vaccines in different formulations (different pH and salt concentration), leave them at 37°C for 1-2 weeks, select immunized animals with an appropriate dose (e.g., 1 / 20× of the human dose), measure the neutralizing antibody level, and immunize them with the vaccine left in a buffer system favorable to antigen stabilization. The neutralizing antibody level should be higher after this, and this determines the appropriate formulation pH range and sodium chloride concentration.

[0044] The formulation of the L1-VLP stock solution for each type of the vaccine of the present invention was 20 mM sodium acetate, 500-700 mM sodium chloride, 0.02% polysorbate 80, and pH 4.8-5.3. During the vaccine preparation process, histidine was introduced to adjust the pH to neutral in order to improve biocompatibility. The formulation buffer system consisted of 10 mM histidine, 10 mM sodium acetate, 327 mM sodium chloride, and 0.01% polysorbate 80.

[0045] In this study, a total of 20 different formulation buffering systems were established. Of these, 15 contained histidine (pH 5.3, 5.6, 5.9, 6.2, 6.5, sodium chloride 154, 327, 500 mM), and 5 did not contain histidine (pH 4.4, 4.7, 5.0, 5.3, 5.6, sodium chloride 327 mM). These are shown in Table 1.

[0046] Table 1: Formulation buffer systems [Table 1]

[0047] (1) Differential scanning fluorescence (DSF) The Tm values ​​for 20 different formulations targeting HPV type 15 L1-VLP were measured using the DSF method.

[0048] As can be seen from the results (Figure 1), the Tm values ​​of HPV types 6 and 11, which belong to the α10 genus in virological classification, are high, while the Tm values ​​of HPV types 16, 31, 33, 35, 52, and 58, which belong to the α9 genus, are relatively low or moderate, while the Tm values ​​of HPV types 18, 39, 45, 59, and 68, which belong to the α7 genus, are relatively high or moderate, and HPV types 51 and 56 belong to the α5 and α6 genus, respectively. The results indicate that there is a certain correlation between Tm values ​​and the evolutionary relationship of each type of virus.

[0049] In buffer solutions containing histidine and acetate, the effects of salt concentration and pH on the Tm value can be divided into the following three categories: (1) Nine types of HPV (HPV6, HPV11, HPV16, HPV31, HPV33, HPV35, HPV39, HPV52, HPV58) have high Tm values ​​at low salt concentrations and low pH. (2) Three types of HPV (HPV18, HPV45, HPV59) have high Tm values ​​at low salt concentrations and medium pH. (3) Three types of HPV (HPV51, HPV56, HPV68) are not sensitive to salt concentration and have high Tm values ​​at medium pH.

[0050] To further analyze the formulations of the 15-valent HPV vaccine, a heatmap was created using Tm values ​​obtained by the DSF method. Darker colors indicate higher Tm values ​​(Figure 2). Under the same salt concentration (327 mM) and pH (5.3 or 5.6) conditions, the Tm value of the acetate buffer containing histidine was significantly higher than that of the buffer without histidine. Therefore, the formulation range was set to the acetate buffer containing histidine. In the acetate buffer containing histidine, pH 5.3-6.2 was clearly superior to pH 6.5, so the formulation range was set to pH 5.3-6.2. Under the pH 5.3-6.2 conditions, salt concentrations of 154 mM and 327 mM were significantly superior to 500 mM.

[0051] Based on the analysis results of antigen Tm values ​​under each formulation prescription condition, the stable range of the formulation was pH 5.3-6.2 and NaCl concentration 154-327 mM.

[0052] (2) Neutralizing antibody method Of the vaccines used in this example, the L1-VLP stock solution formulation for type 13 contained 20 mM sodium acetate, 500 mM sodium chloride, 0.02% polysorbate 80, and a pH of 5.0. Based on this, the HPV type 31 stock solution formulation had an optimal pH of 4.8 and a salt concentration of 700 mM. The HPV type 56 formulation had an optimal pH of 5.3. 15-valent HPV vaccines with or without histidine were prepared, the prepared vaccine samples were aliquoted (15 tubes containing histidine and 5 tubes without histidine), centrifuged, the supernatant was removed, the precipitate was resuspended in the same volume of 20 formulation solutions, thoroughly mixed, and then each tube was evenly aliquoted into three small tubes and sealed. For each formulation, one tube was taken and left at 4°C as a control, while two other small tubes were placed in a 37°C incubator. On the 7th day, one tube was removed and stored at 4°C, and on the 14th day, the other tube was stored at 4°C.

[0053] After standing, 0.3 mL of the vaccine sample was taken, diluted fourfold to 1.2 mL with the corresponding formulation buffer, and used for immunization of mice. Blood was collected 4 weeks after immunization, serum was separated, and the HPV15 neutralizing antibody titer was measured using a neutralizing antibody detection method based on HPV pseudotype viruses.

[0054] Neutralizing antibody titers were calculated using the Reed-Muench method, and geometric mean titer calculations and intergroup comparisons (Mann-Whitney test) were performed using Graphpad Prism software to evaluate the impact of different formulations on vaccine stability and determine appropriate formulation parameter ranges.

[0055] The results of neutralizing antibody detection are shown in Figures 3, 4, and 5. The 15-valent HPV vaccines prepared according to 20 different formulations were left at 4°C for 2 weeks, and at 37°C for 1 and 2 weeks, respectively. Afterward, mice were immunized with 1 / 20 of the human dose, and blood was collected 4 weeks post-immunization. The measured neutralizing antibody titers did not show clear differences between the different formulations, and there was no discernible regularity. This indicated that the buffer system pH (histidine-sodium acetate system 5.3-6.5, acetate-sodium acetate system 4.4-5.6) and sodium chloride concentration (histidine-sodium acetate system 154, 327, 500 mM) did not significantly affect the stability of the vaccine's immunogenicity within the study range. Furthermore, there was no clear difference between the neutralizing antibody titers measured after the 37°C accelerated reaction and those measured after leaving the vaccine at 4°C for 2 weeks (without the accelerated reaction), indicating that the stability of each vaccine antigen was good across all 20 formulations.

[0056] From the perspective of human acceptance, when the pH of the vaccine preparation is neutral (pH 7.0) and the salt concentration is close to 0.9% (physiological saline concentration), the irritation to the human body is less. Therefore, it is more appropriate to select a histidine-sodium acetate buffer system (pH 5.3-6.5), which is closer to neutral pH, as the buffer component of the 15-valent HPV vaccine preparation. Since the salt concentrations of the individual protein stock solutions are relatively high (500, 700 mM) and the initial salt concentration of the aluminum hydroxide adjuvant is 0.9% (154 mM), setting the sodium chloride salt concentration to 327 mM during the vaccine preparation process is more convenient for the formulation process. Furthermore, as can be seen from the stock solution preparation process and formulation studies, high salt concentrations are advantageous for maintaining the uniform 72 pentameric L1-VLP conformations of the antigen, and are advantageous for the stability of the antigenic structure.

[0057] Example 3: Study of antigen dose-to-combination ratio and aluminum adjuvant dose Based on the VLP antigen proteins of each type obtained in Example 1, 15-valent HPV vaccines with different antigen dose ratios and aluminum adjuvant doses were prepared according to the subsequent preparation process (Table 2), and immunogenicity was investigated by immunizing BALB / c mice. Specifically, a monovalent antigen immunization group was established, and the immune interference status was examined. Gardasil 9 was used as a control vaccine, and different aluminum adjuvant dose groups were established, the adjuvant dose in the vaccine was determined, different antigen dose ratios were established, and the antigen dose ratios for each type were determined. Table 2 shows the animal grouping information, antigen dose ratios, and aluminum adjuvant dose settings. The immunization program employed 0-week immunization and 4-week immunization, and neutralizing antibody titers were measured 4 weeks after primary immunization and 4 weeks after secondary immunization. Intergroup comparisons were performed to determine the antigen dose ratios and aluminum adjuvant doses for each type in the vaccine.

[0058] Table 2: Animal group classification for antigen-dose ratio and aluminum adjuvant dose studies [Table 2] TIFF2026509820000004.tif112170 Note: Intramuscular injection was used, and 100 μl was injected into each BALB / c mouse. Note: In Groups 1-15, aluminum adjuvants were adsorbed onto single-type antigens (i.e., HPV6, 11, 16, 16, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 6), and 10 mice were immunized. In Groups 16-19, different aluminum adjuvants were adsorbed onto the same antigen dose of the 15-valent antigen, and 10 mice were immunized. In Groups 20-21, the same dose of adjuvant was adsorbed onto different antigen doses of each type, and 10 mice were immunized. In Group 22, 10 mice were immunized with the antigen and adjuvant doses of Marsalton's commercially available Gardasil 9-valent vaccine as a comparative study. In Group 23, 5 mice were immunized as an aluminum adjuvant control group.

[0059] Table 3 shows the geometric mean values ​​of neutralizing antibody detection 4 weeks after primary immunization and 4 weeks after secondary immunization. The following can be seen from the results. (1) Immune interference Mice were immunized with monovalent and pentavalent antigens using the same aluminum adjuvant dose (the dose of the same type of antigen was the same), and the differences in immune response levels between the two groups were compared to investigate the state of immune interference. As can be seen from the results, when all types of antigens were immunized in monovalent form, the neutralizing antibody titers were significantly higher than those obtained with the pentavalent HPV vaccine, and in all cases, they were higher than those corresponding to the control vaccine (Gardasil9).

[0060] (2) Aluminum adjuvant dosage The antigen dose was set to 1 / 20 × the human dose, and four aluminum adjuvant doses (37.5 μg (1 / 20 × of the 9-valent vaccine), 0, 25, and 50 μg) were established. Mice (n=10 mice in each group) were immunized, and the differences between the aluminum adjuvant doses were examined, along with a comparison to the control vaccine (Gardasil9). As can be seen from the results, the neutralizing antibody level in the group without aluminum adjuvant was clearly lower than in the group with aluminum adjuvant, indicating the need to add aluminum adjuvant to the vaccine. The effect of aluminum adjuvant on improving the level of antigen immune response was clear, and for most of the three doses in the group with aluminum adjuvant, there was no significant difference between types. Compared to the control vaccine, the increase in aluminum adjuvant was not significant enough to show a remarkable superiority. The results indicate that 25 μg of aluminum adjuvant (corresponding to the human dose of 500 μg) reached or came close to immune saturation, corresponding to the immune response level of the control vaccine.

[0061] (3) The ratio of antigen doses Initially, regarding the antigen dose of the 15-valent HPV vaccine, six new types of antigens were increased by 20 μg each compared to the 9-valent vaccine (i.e., containing 30, 40, 60, 40, 20, 20, 20, 20, 20, 20, 20, 20, 20, 20, 20, 20, and 20 μg of HPV types 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, 56, 58, 59, and 68 antigens, respectively). Based on this, adjustments were made to increase the HPV 16 and 18 antigens by 10 μg each, or to increase the HPV types 16, 18, 31, 33, 45, 52, and 58 by 10 μg each, and it was investigated whether increasing the antigen doses of these types could significantly increase the level of the immune response for these types in order to ensure that the vaccine effect was greater than or equal to that of the commercially available vaccine Gardasil 9. As can be seen from the results (Table 3), increasing the dose of each type of antigen by 10 μg compared to the initial antigen dose did not produce a significant immune-enhancing effect, indicating that the initial formulation antigen ratio reached immunosaturation in mice, and there is no need to increase the dose of these types of antigens.

[0062] Table 3: Table of Geometric Mean Potency of Immunodoses and Neutralizing Antibodies [Table 3] TIFF2026509820000006.tif45170

[0063] Example 4: Preparation process for 15-valent HPV vaccine HPV types 6 / 11 / 16 / 18 / 31 / 33 / 35 / 39 / 45 / 51 / 52 / 56 / 58 / 59 / 68 VLP protein stock solutions were taken from a refrigerator at -65℃--80℃. The storage concentration of each type of protein stock solution was a minimum of 1.56 mg / mL, and the pH range of the buffer used was 4.3-5.8 (Example 1). Buffer formulation: 20 mM HAc-NaAc, 500 mM-700 mM NaCl, 0.02% Tween 80. After standing at room temperature for 10 minutes, the solutions were thawed in a 37℃ water bath until completely thawed. Aluminum hydroxide adjuvant was diluted to 4 mg / mL, and bacterial endotoxins, sterility, osmotic molar concentration, and aluminum content were detected. The density of 4.0 mg / mL aluminum hydroxide adjuvant was 1.01 g / mL. 3120 mL of the type 15 protein diluent (density 1.02 g / mL) and 3120 mL of the aluminum hydroxide adjuvant diluent (density 1.01 g / mL), respectively, were mixed in 10 L glass vertical bottles (magnetic stirrer rotor diameter 1.39 cm, length 8.6 cm) and mixed uniformly at 300-340 rpm for at least 30 minutes. A pre-filled syringe filling machine was started and set up and operated according to the instrument's operating manual. 0.55 mL was dispensed per syringe and sealed with a rubber stopper at a filling speed of 30-40 revolutions / min. The bottles were tagged and stored.

[0064] Example 5: Re-examination of antigen dose-to-combination ratios To obtain more research data on antigen dose-to-body ratios and provide reference for vaccine doses for clinical trials, antigen dose-to-body ratios were re-examined. 15-valent HPV vaccines with different antigen dose-to-body ratios were prepared, and BALB / c mice were immunized with 1 / 50 × human dose. Differences in immunogenicity at each antigen dose-to-body ratio were compared. Animal grouping information and antigen dose-to-body ratio information are shown in Table 4. The immunization program employed 0-week and 4-week immunization, and neutralizing antibody titers were measured 4 weeks after primary and 4 weeks after secondary immunity. Intergroup comparisons were performed to determine the antigen dose-to-body ratios for each type in the vaccine.

[0065] Table 4: Animal group classification for antigen-dose-combination ratio restudies. [Table 4]

[0066] Table 5: Immunodose and Geometric Mean Titer of Neutralizing Antibody [Table 5]

[0067] Table 5 shows the geometric mean values ​​of neutralizing antibodies 4 weeks after primary immunity and 4 weeks after secondary immunity.

[0068] (1) Results of neutralizing antibodies 4 weeks after primary immunity Compared to the same HPV types included in Gardasil 9, the types showing a significant difference in the low-dose group were HPV11, 33, and 45. Of these, Gardasil 9 was more effective than the 15-valent HPV vaccine for HPV11, and the 15-valent HPV vaccine was more effective than Gardasil 9 for HPV33 and 45. In the medium-dose group, the types showing a significant difference were HPV33 and 45, with the 15-valent HPV vaccine being more effective than Gardasil 9 in both cases. In the high-dose group, the types showing a significant difference were HPV11 and 33. Gardasil 9 was more effective than the 15-valent HPV vaccine for HPV11, and the 15-valent HPV vaccine was more effective than Gardasil 9 for HPV33. For other types in the three dose groups, the levels were comparable and there were no significant differences. The six HPV types (HPV35, 39, 51, 56, 59, and 68) added to the vaccine of the present invention can induce a strong immune response in low, intermediate, and high-dose groups.

[0069] Significant differences between the low, intermediate, and high-dose groups were observed for HPV type 45 (intermediate dose was higher than high dose) and HPV type 59 (high dose was higher than low dose). In the low-dose group, the doses of HPV types 35, 39, 51, 56, 59, and 68 antigens were halved compared to the intermediate-dose group, and there was no significant difference in neutralizing antibody levels. In the high-dose group, the HPV type 16 antigen increased from 60 μg / agent to 80 μg / agent compared to the intermediate-dose group, but there was no significant difference in neutralizing antibody titer levels.

[0070] (2) Results of neutralizing antibodies 2 weeks after secondary immunity Compared to the same types included in Gardasil 9, HPV45 showed a significant difference in the low-dose group, with the 15-valent HPV vaccine showing a higher level than Gardasil 9. In the medium-dose group, HPV33 showed a significant difference, with the 15-valent HPV vaccine showing a higher level than Gardasil 9. In the high-dose group, HPV33 showed a significant difference, with the 15-valent HPV vaccine showing a higher level than Gardasil 9. For the other types in the three dose groups, the levels were comparable and there were no significant differences. The six types added to the vaccine of this invention (HPV35, 39, 51, 56, 59, 68) can induce a strong immune response and produce high levels of neutralizing antibodies in all low, medium, and high-dose groups.

[0071] Significant differences between the low, medium, and high-dose groups were observed for HPV type 56 (higher in high-dose groups than low-dose groups) and HPV type 59 (higher in medium-dose groups than low-dose groups). In the low-dose group, the doses of HPV types 35, 39, 51, 56, 59, and 68 antigens were halved compared to the medium-dose group, and the levels of induced neutralizing antibodies were lower in all cases than in the medium-dose group, although there was a significant difference for HPV type 59. In the high-dose group, compared to the medium-dose group, the HPV type 16 antigen increased from 60 μg / agent to 80 μg / agent, and the level of neutralizing antibody titers improved, but not to a significant degree.

[0072] Reducing the doses of HPV types 35, 39, 51, 56, 59, and 68 antigens based on a moderate dose of the 15-valent HPV vaccine reduces the immune response level for these six types to some extent, but does not significantly affect the immune response to the other nine types.

[0073] By increasing the dose of HPV type 16 antigen to 80 μg / dose based on the intermediate dose of the 15-valent HPV vaccine, the level of the immune response to this type was improved to some extent, bringing it closer to that of Gardasil 9, but no statistically significant difference was observed. In subsequent clinical trials, it will be necessary to determine the formulation ratio of the test vaccine dose by comprehensively considering the results of major preclinical pharmacodynamic studies and safety evaluations.

[0074] The properties related to the formulation of the present invention, as determined by the above research, are as follows: The adjuvant used is a commonly used aluminum hydroxide adjuvant. When its particle size distribution is measured with a laser particle size analyzer, it is found to be between approximately 1 and 20 μm. In aqueous solution, it appears milky white, and precipitation occurs after standing for a certain period of time. After protein antigens are adsorbed onto the surface of the aluminum hydroxide adjuvant, the particle size may increase slightly, and its appearance is almost similar to that of aluminum hydroxide adjuvant without protein adsorption.

[0075] During the preparation of the formulation, adding 10 mM histidine adjusts the pH from approximately 5.0 in the stock solution to approximately 6.0 in the formulation, making it more palatable to the human body and reducing irritation. Stability studies have shown that when the pH of this formulation was controlled to 5.3-6.5 (histidine-containing formulation) and left at 37°C for two weeks, there was no significant difference in vaccine immunogenicity.

[0076] Setting the sodium chloride concentration in the formulation to 327 mM, as can be seen from the results of studies on its stability, does not significantly affect the vaccine's immunogenicity when the sodium chloride concentration in this formulation is set between 154 and 500 mM.

[0077] The amount of aluminum adjuvant used in this formulation is 0.5 mg / drug, which is the same as the aluminum content of the commercially available 9-valent vaccine Gardasil 9. The difference is that the aluminum adjuvant used in Gardasil 9 is aluminum hydroxyphosphate sulfate adjuvant (a type of aluminum phosphate adjuvant) instead of aluminum hydroxide. The selection of the type of aluminum adjuvant is mainly related to the properties of the charge on the antigen. The proteins of each type of vaccine in this product are prepared by expressing them after shortening the C-terminus, removing the positively charged portion, and the protein becomes negatively charged overall in an environment with a near-neutral pH. In contrast, Gardasil 9 expresses the protein in its full sequence, and the protein becomes positively charged overall in an environment with a near-neutral pH. Negatively charged antigens are easily adsorbed by positively charged aluminum hydroxide adjuvants, and positively charged antigens are easily adsorbed by negatively charged aluminum phosphate adjuvants. In the vaccine formulation of the present invention, the adsorption rate of aluminum hydroxide adjuvant to each type of antigen exceeds 95% and is close to 100%. After the antigen is adsorbed to the surface by the aluminum adjuvant, aggregation of antigens is less likely to occur, which is advantageous for stability.

[0078] The vaccine of the present invention belongs to the category of sterile formulations and must be stored at 2-8°C. It cannot be frozen, and during the freezing process, the crystalline structure of the aluminum adjuvant is destroyed, affecting its precipitation properties, antigen adsorption capacity, and further affecting the efficacy of the vaccine.

[0079] The foregoing are merely preferred embodiments of the present invention and do not limit it. While the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical proposals described in the above embodiments or replace some of their technical features with equivalent ones. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be within the scope of protection of the present invention.

[0080] Sequence List Sequence ID 1: HPV6L1 N / C-terminal shortening (469aa, Theoretical pI / Mw: 5.94 / 52079.77Da, N-terminus shortened by 2 amino acids, C-terminus shortened by 29 amino acids) 1 MPSDSTVYVP PPNPVSKVVA TDAYVTRTNI FYHASSSRLL AVGHPYFSIK 51 RANKTVVPKV SGYQYRVFKV VLPDPNKFAL PDSSLFDPTT QRLVWACTGL 101 EVGRGQPLGV GVSGHPFLNK YDDVENSGSG GNPGQDNRVN VGMDYKQTQL 151 CMVGCAPPLG EHWGKGKQCT NTPVQAGDCP PLELITSVIQ DGDMVDTGFG 201 AMNFADLQTN KSDVPIDICG TTCKYPDYLQ MAADPYGDRL FFFLRKEQMF 251 ARHFFNRAGE VGEPVPDTLI IKGSGNRTSV GSSIYVNTPS GSLVSSEAQL 301 FNKPYWLQKA QGHNNGICWG NQLFVTVVDT TRSTNMTLCA SVTTSSTYTN 351 SDYKEYMRHV EEYDLQFIFQ LCSITLSAEV MAYIHTMNPS VLEDWNFGLS 401 PPPNGTLEDT YRYVQSQAIT CQKPTPEKEK PDPYKNLSFW EVNLKEKFSS 451 ELDQYPLGRK FLLQSGYRG Sequence ID 2 HPV11L1 N / C-terminal shortening (470aa, Theoretical pI / Mw: 6.08 / 52274.02Da, N-terminus shortened by 3 amino acids, C-terminus shortened by 29 amino acids) 1 MPSDSTVYVP PPNPVSKVVA TDAYVKRTNI FYHASSSRLL AVGHPYYSIK 51 KVNKTVVPKV SGYQYRVFKV VLPDPNKFAL PDSSLFDPTT QRLVWACTGL 101 EVGRGQPLGV GVSGHPLLNK YDDVENSGGY GGNPGQDNRV NVGMDYKQTQ 151 LCMVGCAPPL GEHWGKGTQC SNTSVQNGDC PPLELITSVI QDGDMVDTGF 201 GAMNFADLQT NKSDVPLDIC GTVCKYPDYL QMAADPYGDR LFFYLRKEQM 251 FARHFFNRAG TVGEPVPDDL LVKGGNNRSS VASSIYVHTP SGSLVSSEAQ 301 LFNKPYWLQK AQGHNNGICW GNHLFVTVVD TTRSTNMTLC ASVSKSATYT 351 NSDYKEYMRH VEEFDLQFIF QLCSITLSAE VMAYIHTMNP SVLEDWNFGL 401 SPPPNGTLED TYRYVQSQAI TCQKPTPEKE KQDPYKDMSF WEVNLKEKFS 451 SELDQFPLGR KFLLQSGYRG Sequence ID 3 HPV16L1 N / C-terminal shortening (472aa, Theoretical pI / Mw: 6.08 / 52548.71Da, 4 amino acids shortened at the N-terminus and 29 amino acids shortened at the C-terminus) 1 MPSEATVYLP PVPVSKVVST DEYVARTNIY YHAGTSRLLA VGHPYFPIKK 51 PNNNKILVPK VSGLQYRVFR IHLPDPNKFG FPDTSFYNPD TQRLVWACVG 101 VEVGRGQPLG VGISGPLLN KLDDTENASA YAANAGVDNR ECISMDYKQT 151 QLCLIGCKPP IGEHWGKGSP CTNVAVNPGD CPPLELINTV IQDGDMVDTG 201 FGAMDFTTLQ ANKSEVPLDI CTSICKYPDY IKMVSEPYGD SLFFYLRREQ 251 MFVRHLFNRA GAVGENVPDD LYIKGSGSTA NLASSNYFPT PSGSMVTSDA 301 QIFNKPYWLQ RAQGHNNGIC WGNQLFVTVV DTTRSTNMSL CAAISTSETT 351 YKNTNFKEYL RHGEEYDLQF IFQLCKITLT ADVMTYIHSM NSTILEDWNF 401 GLQPPPGGTL EDTYRFVTSQ AIACQKHTPP APKEDPLKKY TFWEVNLKEK 451 FSADLDQFPL GRKFLLQAGL KA Sequence ID 4 HPV18L1 N / C-terminal shortening (473aa, Theoretical pI / Mw: 5.94 / 52722.42Da, N-terminus shortened by 4 amino acids, C-terminus shortened by 30 amino acids) 1 MPSDNTVYLP PPSVARVVNT DDYVTRTSIF YHAGSSRLLT VGNPYFRVPA 51 GGGNKQDIPK VSAYQYRVFR VQLPDPNKFG LPDNSIYNPE TQRLVWACAG 101 VEIGRGQPLG VGLSGHPFYN KLDDTESHA ATSNVSEDVR DNVSVDYKQT 151 QLCILGCAPA IGEHWAKGTA CKSRPLSQGD CPPLELKNTV LEDGDMVDTG 201 YGAMDFSTLQ DTKCEVPLDI CQSICKYPDY LQMSADPYGD SMFFCLRREQ 251 LFARHFWNRA GTMGDTVPQS LYIKGTGMRA SPGSCVYSPS PSGSIVTSDS 301 QLFNKPYWLH KAQGHNNGVC WHNQLFVTVV DTTRSTNLTI CASTQSPVPG 351 QYDATKFKQY SRHVEEYDLQ FIFQLCTITL TADVMSYIHS MNSSILEDWN 401 FGVPPPPTTS LVDTYRFVQS VAITCQKDAA PAENKDPYDK LKFWNVDLKE 451 KFSLDLDQYP LGRKFLVQAG LRR Sequence ID 5 HPV31L1 N / C-terminal shortening (473aa, Theoretical pI / Mw: 6.27 / 52869.89Da, N-terminus shortened by 4 amino acids, C-terminus shortened by 27 amino acids) 1 MPSEATVYLP PVPVSKVVST DEYVTRTNIY YHAGSARLLT VGHPYYSIPK 51 SDNPKKIVVP KVSGLQYRVF RVRLPDPNKF GFPDTSFYNP ETQRLVWACV 101 GLEVGRGQPL GVGISGHPLL NKFDDTENSN RYAGGPGTDN RECISMDYKQ 151 TQLCLLGCKP PIGEHWGKGS PCSNNAITPG DCPPLELKNS VIQDGDMVDT 201 GFGAMDFTAL QDTKSNVPLD ICNSICKYPD YLKMVAEPYG DTLFFYLRRE 251 QMFVRHFFNR SGTVGESVPT DLYIKGSGST ATLANSTYFP TPSGSMVTSD 301 AQIFNKPYWM QRAQGHNNGI CWGNQLFVTV VDTTRSTNMS VCAAIANSDT 351 TFKSSNFKEY LRHGEEFDLQ FIFQLCKITL SADIMTYIHS MNPAILEDWN 401 FGLTTPPSGS LEDTYRFVTS QAITCQKTAP QKPKEDPFKD YVFWEVNLKE 451 KFSADLDQFP LGRKFLLQAG YRA Sequence ID 6 HPV33L1 N / C-terminal shortening (471aa, Theoretical pI / Mw: 5.88 / 52765.94Da, N-terminus shortened by 4 amino acids, C-terminus shortened by 24 amino acids) 1 MPSEATVYLP PVPVSKVVST DEYVSRTSIY YYAGSSRLLA VGHPYFSIKN 51 PNNAKKLLVP KVSGLQYRVF RVRLPDPNKF GFPDTSFYNP DTQRLVWACV 101 GLEIGRGQPL GVGISGHPLL NKFDDTETSN KYPGQPGADN RECLSMDYKQ 151 TQLCLLGCKP PTGEHWGKGV ACTNAAPAND CPPLELINTI IEDGDMVDTG 201 FGCMDFKTLQ ANKSDVPIDI CGSTCKYPDY LKMTSEPYGD SLFFFLRREQ 251 MFVRHFFNRA GKLGEAVPDD LYIKGSGTTA SIQSSAFFPT PGSMSVTSES 301 QLFNKPYWLQ RAQGHNNGIC WGNQVFVTVV DTTRSTNMTL CTQVTSDSTY 351 KNENFKEYIR HVEEYDLQFV FQLCKVTLTA EVMTYIHAMN PDILEDWQFG 401 LTPPPSASLQ DTYRFVTSQA ITCQKTVPPK EKEDPLGKYT FWEVDLKEKF 451 SADLDQFPLG RKFLLQAGLK A Sequence ID 7 HPV35L1 N / C-terminal shortening (470aa, Theoretical pI / Mw: 6.10 / 52494.45Da, N-terminus shortened by 4 amino acids, C-terminus shortened by 28 amino acids) 1 MSNEATVYLP PVSVSKVVST DEYVTRTNIY YHAGSSRLLA VGHPYYAIKK 51 QDSNKIAVPK VSGLQYRVFR VKLPDPNKFG FPDTSFYDPA SQRLVWACTG 101 VEVGRGQPLG VGISGPLLN KLDDTENSNK YVGNSGTDNR ECISMDYKQT 151 QLCLIGCRPP IGEHWGKGTP CNANQVKAGE CPPLELLNTV LQDGDMVDTG 201 FGAMDFTTLQ ANKSDVPLDI CSSICKYPDY LKMVSEPYGD MLFFYLRREQ 251 MFVRHLFNRA GTVGETVPAD LYIKGTTGTL PSTSYFPTPS GSMVTSDAQI 301 FNKPYWLQRA QGHNNGICWS NQLFVTVVDT TRSTNMSVCS AVSTSDSTYK 351 NDNFKEYLRH GEEYDLQFIF QLCKITLTAD VMTYIHSMNP SILEDWNFGL 401 TPPPSGTLED TYRYVTSQAV TCQKPSAPKP KDDPLKNYTF WEVDLKEKFS 451 ADLDQFPLGR KFLLQAGLKA Sequence ID 8 HPV39L1 N / C-terminal shortening (467aa, Theoretical pI / Mw: 6.16 / 52393.22Da, N-terminus shortened by 9 amino acids, C-terminus shortened by 29 amino acids) 1 MVYLPPSVA KVVNTDDYVT RTGIYYYAGS SRLLTVGHPY FKVGMNGGRK 51 QDIPKVSAYQ YRVFRVTLPD PNKFSIPDAS LYNPETQRLV WACVGVEVGR 101 GQPLGVGISG HPLYNRQDDT ENSPFSSTTN KDSRDNVSVD YKQTQLCIIG 151 CVPAIGEHWG KGKACKPNNV STGDCPPLEL VNTPIEDGDM IDTGYGAMDF 201 GALQETKSEV PLDICQSICK YPDYLQMSAD VYGDSMFFCL RREQLFARHF 251 WNRGGMVGDA IPAQLYIKGT DIRANPGSSV YCPSPSGSMV TSDSQLFNKP 301 YWLHKAQGHN NGICWHNQLF LTVVDTTRST NFTLSTSIES SIPSTYDPSK 351 FKEYTRHVEE YDLQFIFQLC TVTLTTDVMS YIHTMNSSIL DNWNFAVAPP 401 PSASLVDTYR YLQSAAITCQ KDAPAPEKKD PYDGLKFWNV DLREKFSLEL 451 DQFPLGRKFL LQARVRR Sequence ID 9 HPV45L1 N / C-terminal shortening (476aa, Theoretical pI / Mw: 5.94 / 53244.30Da, N-terminus shortened by 4 amino acids, C-terminus shortened by 30 amino acids) 1 MPSDSTVYLP PPSVARVVNT DDYVSRTSIF YHAGSSRLLT VGNPYFRVVP 51 NGAGNKQAVP KVSAYQYRVF RVALPDPNKF GLPDSTIYNP ETQRLVWACV 101 GMEIGRGQPL GIGLSGHPFY NKLDDTESAH AATAVITQDV RDNVSVDYKQ 151 TQLCILGCVP AIGEHWAKGT LCKPAQLQPG DCPPLELKNT IIEDGDMVDT 201 GYGAMDFSTL QDTKCEVPLD ICQSICKYPD YLQMSADPYG DSMFFCLRRE 251 QLFARHFWNR AGVMGDTVPT DLYIKGTSAN MRETPGSCVY SPSPSGSIIT 301 SDSQLFNKPY WLHKAQGHNN GICWHNQLFV TVVDTTRSTN LTLCASTQNP 351 VPSTYDPTKF KQYSRHVEEY DLQFIFQLCT ITLTAEVMSY IHSMNSSILE 401 NWNFGVPPPP TTSLVDTYRF VQSVAVTCQK DTTPPEKQDP YDKLKFWTVD 451 LKEKFSSDLD QYPLGRKFLV QAGLRR Sequence ID 10 HPV51L1 N / C-terminal shortening (472aa, Theoretical pI / Mw: 5.88 / 52769.56Da, N-terminus shortened by 4 amino acids, C-terminus shortened by 28 amino acids) 1 MTNDSKVYLP PAPVSRIVNT EEYITRTGIY YYAGSSRLIT LGHPYFPIPK 51 TSTRAAIPKV SAFQYRVFRV QLPDPNKFGL PDPNLYNPDT DRLVWGCVGV 101 EVGRGQPLGV GLSGHPLFNK YDDTENSRIA NGNAQQDVRD NTSVDNKQTQ 151 LCIIGCAPPI GEHWGIGTTC KNTPVPPGDC PPLELVSSVI QDGDMIDTGF 201 GAMDFAALQA TKSDVPLDIS QSVCKYPDYL KMSADTYGNS MFFHLRREQI 251 FARHYYNKLG SVGEDIPNDY YIKGSGNGRD PIESYIYSAT PSGSMITSDS 301 QIFNKPYWLH RAQGHNNGIC WNNQLFITCV DTTRSTNLTI STATAAVSPT 351 FTPSNFKQYI RHGEEYELQF IFQLCKITLT TEVMAYLHTM DPTILEQWNF 401 GLTLPPSASL EDAYRFVTNA ATSCQKDTPP QAKPDPLAKY KFWDVDLKER 451 FSLDLDQFAL GRKFLLQVGV QR Sequence ID 11 HPV52L1 N / C-terminal shortening (476aa, Theoretical pI / Mw: 5.88 / 53176.26Da, N-terminus shortened by 4 amino acids, C-terminus shortened by 23 amino acids) 1 MPSEATVYLP PVPVSKVVST DEYVSRTSIY YYAGSSRLLT VGHPYFSIKN 51 TSSGNGKKVL VPKVSGLQYR VFRIKLPDPN KFGFPDTSFY NPETQRLVWA 101 CTGLEIGRGQ PLGVGISGHP LLNKFDDTET SNKYAGKPGI DNRECLSMDY 151 KQTQLCILGC KPPIGEHWGK GTPCNNNSGN PGDCPPLQLI NSVIQDGDMV 201 DTGFGCMDFN TLQASKSDVP IDICSSVCKY PDYLQMASEP YGDSLFFFLR 251 REQMFVRHFF NRAGTLGDPV PGDLYIQGSN SGNTATVQSS AFFPTPSGSM 301 VTSESQLFNK PYWLQRAQGH NNGICWGNQL FVTVVDTTRS TNMTLCAEVK 351 KESTYKNENF KEYLRHGEEF DLQFIFQLCK ITLTADVMTY IHKMDATILE 401 DWQFGLTPPP SASLEDTYRF VTSTAITCQK NTPPKGKEDP LKDYMFWEVD 451 LKEKFSADLD QFPLGRKFLL QAGLQA Sequence ID 12 HPV56L1 N / C-terminal shortening (470aa, Theoretical pI / Mw: 6.11 / 52796.70Da, N-terminus shortened by 4 amino acids, C-terminus shortened by 25 amino acids) 1 MPSENKVYLP PTPVSKVVAT DSYVKRTSIF YHAGSSRLLA VGHPYYSVTK 51 DNTKTNIPKV SAYQYRVFRV RLPDPNKFGL PDTNIYNPDQ ERLVWACVGL 101 EVGRGQPLGA GLSGHPLFNR LDDTESSNLA NNNVIEDSRD NISVDGKQTQ 151 LCIVGCTPAM GEHWTKGAVC KSTQVTTGDC PPLALINTPI EDGDMIDTGF 201 GAMDFKVLQE SKAEVPLDIV QSTCKYPDYL KMSADAYGDS MWFYLRREQL 251 FARHYFNRAG KVGETIPAEL YLKGSNGREP PPSSVYVATP SGSMITSEAQ 301 LFNKPYWLQR AQGHNNGICW GNQLFVTVVD TTRSTNMTIS TATEQLSKYD 351 ARKINQYLRH VEEYELQFVF QLCKITLSAE VMAYLHNMNA NLLEDWNIGL 401 SPPVATSLED KYRYVTSTAI TCQREQPPTE KQDPLAKYKF WDVNLQDSFS 451 TDLDQFPLGR KFLMQLGTRS Sequence ID 13 HPV58L1 N / C-terminal shortening (471aa, Theoretical pI / Mw: 5.80 / 52994.97Da, N-terminus shortened by 4 amino acids, C-terminus shortened by 23 amino acids) 1 MPSEATVYLP PVPVSKVVST DEYVSRTSIY YYAGSSRLLA VGNPYFSIKS 51 PNNNKKVLVP KVSGLQYRVF RVRLPDPNKF GFPDTSFYNP DTQRLVWACV 101 GLEIGRGQPL GVGVSGHPYF NKFDDTETSN RYPAQPGSDN RECLSMDYKQ 151 TQLCLIGCKP PTGEHWGKGV ACNNNAAATD CPPLELFNSI IEDGDMVDTG 201 FGCMDFGTLQ ANKSDVPIDI CNSTCKYPDY LKMASEPYGD SLFFFLRREQ 251 MFVRHFFNRA GKLGEAVPDD LYIKGSGNTA VIQSSAFFPT PGSMSVTSES 301 QLFNKPYWLQ RAQGHNNGIC WGNQLFVTVV DTTRSTNMTL CTEVTKEGTY 351 KNDNFKEYVR HVEEYDLQFV FQLCKITLTA EIMTYIHTMD SNILEDWQFG 401 LTPPPSASLQ DTYRFVTSQA ITCQKTAPPK EKEDPLNKYT FWEVNLKEKF 451 SADLDQFPLG RKFLLQSGLK A Sequence ID 14 HPV59L1 N / C-terminal shortening (473aa, Theoretical pI / Mw: 6.08 / 52891.89Da, N-terminus shortened by 4 amino acids, C-terminus shortened by 31 amino acids) 1 MSSDNKVYLP PPSVAKVVST DEYVTRTSIF YHAGSSRLLT VGHPYFKVPK 51 GGNGRQDVPK VSAYQYRVFR VKLPDPNKFG LPDNTVYDPN SQRLVWACVG 101 VEIGRGQPLG VGLSGHPLYN KLDDTENSHV ASAVDTKDTR DNVSVDYKQT 151 QLCIIGCVPA IGEHWTKGTA CKPTTVVQGD CPPLELINTP IEDGDMVDTG 201 YGAMDFKLLQ DNKSEVPLDI CQSICKYPDY LQMSADAYGD SMFFCLRREQ 251 VFARHFWNRS GTMGDQLPES LYIKGTDIRA NPGSYLYSPS PSGSVVTSDS 301 QLFNKPYWLH KAQGLNNGIC WHNQLFLTVV DTTRSTNLSV CASTTSSIPN 351 VYTPTSFKEY ARHVEEFDLQ FIFQLCKITL TTEVMSYIHN MNTTILEDWN 401 FGVTPPPTAS LVDTYRFVQS AAVTCQKDTA PPVKQDPYDK LKFWPVDLKE 451 RFSADLDQFP LGRKFLLQLG ARP Sequence ID 15 HPV68L1 N / C-terminal shortening (473aa, Theoretical pI / Mw: 5.75 / 53032.99Da, N-terminus shortened by 4 amino acids, C-terminus shortened by 28 amino acids) 1 MASDNMVYLP PPSVAKVVNT DDYVTRTGMY YYAGTSRLLT VGHPYFKVPM 51 SGGRKQGIPK VSAYQYRVFR VTLPDPNKFS VPESTLYNPD TQRMVWACVG 101 VEIGRGQPLG VGLSGHPLYN RLDDTENSPF SSNKNPKDSR DNVAVDCKQT 151 QLCIIGCVPA IGEHWAKGKS CKPTNVQQGD CPPLELVNTP IEDGDMIDTG 201 YGAMDFGTLQ ETKSEVPLDI CQSVCKYPDY LQMSADVYGD SMFFCLRREQ 251 LFARHFWNRG GMVGDTIPTD MYIKGTDIRE TPSSYVYAPS PSGSMVSSDS 301 QLFNKPYWLH KAQGHNNGIC WHNQLFLTVV DTTRSTNFTL STTTDSTVPA 351 VYDSNKFKEY VRHVEEYDLQ FIFQLCTITL STDVMSYIHT MNPAILDDWN 401 FGVAPPPSAS LVDTYRYLQS AAITCQKDAP APVKKDPYDG LNFWNVDLKE 451 KFSSELDQFP LGRKFLLQAG VRR Sequence ID 16 (code nucleotide sequence of the shortened HPV68 L1 protein):

Claims

1. It is a vaccine for human papillomavirus, It comprises one or more L1 antigens from HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68, for example, 15 types, 9 types, 6 types, and 3 types. Preferably, the weight ratio of the doses of HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 antigens is 1-3:1-5:2-7:1-5:0.5-3:0.5-3:0.5-3:0.5-3:0.5-3:0.5-2:0.5-2:0.5-2:0.5-2:0.5-2:0.5-2, and preferably HP The weight ratio of the doses of antigens V6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 is 1.5:2:3:2:1:1:1:1:1:1:1:1:1:1:1:1, or 1.5:2:3:2:1:1:1:1:1:1:1:1:1:1:1:1, or 3:4:6:4:2:2:2:2:2:1:1:1:1:1:1, More specifically, the dose of each protein is 10-100 μg, and more preferably, the vaccine is characterized by containing 30 μg, 40 μg, 60 μg, 40 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, and 20 μg per 0.5 ml, respectively.

2. The L1 antigen for each type is shortened based on the wild-type sequence as follows: In wild-type HPV6 L1, the N-terminus is shortened by 2 amino acids and the C-terminus is shortened by 29 amino acids. In wild-type HPV11 L1, the N-terminus is shortened by 3 amino acids and the C-terminus is shortened by 29 amino acids. In wild-type HPV16 L1, the N-terminus is shortened by 4 amino acids and the C-terminus is shortened by 29 amino acids. In wild-type HPV18 L1, the N-terminus is shortened by 4 amino acids, and the C-terminus is shortened by 30 amino acids. In wild-type HPV31 L1, the N-terminus is shortened by 4 amino acids and the C-terminus is shortened by 27 amino acids. In wild-type HPV33 L1, the N-terminus is shortened by 4 amino acids and the C-terminus is shortened by 24 amino acids. In wild-type HPV35 L1, the N-terminus is shortened by 4 amino acids and the C-terminus is shortened by 28 amino acids. In wild-type HPV39 L1, the N-terminus is shortened by 9 amino acids, and the C-terminus is shortened by 29 amino acids. In wild-type HPV45 L1, the N-terminus is shortened by 4 amino acids and the C-terminus is shortened by 30 amino acids. In wild-type HPV 51 L1, the N-terminus is shortened by 4 amino acids and the C-terminus is shortened by 28 amino acids. In wild-type HPV52 L1, the N-terminus is shortened by 4 amino acids, and the C-terminus is shortened by 23 amino acids. In wild-type HPV 56 L1, the N-terminus is shortened by 4 amino acids and the C-terminus is shortened by 25 amino acids. In wild-type HPV 58 L1, the N-terminus is shortened by 4 amino acids, and the C-terminus is shortened by 23 amino acids. In wild-type HPV 59 L1, the N-terminus is shortened by 4 amino acids and the C-terminus is shortened by 31 amino acids. The vaccine according to claim 1, characterized in that the N-terminus of wild-type HPV68 L1 is shortened by 4 amino acids and the C-terminus is shortened by 28 amino acids.

3. The vaccine according to claim 1, characterized in that the amino acid sequences of each type of the L1 antigen are shown in Sequence IDs 1 to 15, respectively.

4. The vaccine according to any one of claims 1 to 3, characterized in that the adjuvant used is an aluminum adjuvant, preferably an aluminum hydroxide adjuvant, and more preferably the mass ratio of the antigen protein to the aluminum adjuvant is 0.5-1:1, and preferably 0.7-0.85:

1.

5. The vaccine according to any one of claims 1 to 3, characterized in that the vaccine uses an acetate-sodium acetate buffer system as a buffer, and an adjuvant containing histidine, sodium chloride, and polysorbate 80 is added.

6. The vaccine according to claim 5, characterized in that the acetate-sodium acetate buffer system has a pH of 5-7, more preferably 5.3-6.5, a concentration of 5-15 mM, more preferably 10 mM, the concentration of sodium chloride is set to 100-400 mM, more preferably 320-335 mM, and the concentration of polysorbate 80 is 0.005%-0.02%, more preferably 0.01%.

7. A method for preparing a vaccine according to any one of claims 1 to 6, Purified HPV L1 proteins of each type are subjected to in vitro self-assembly to form VLPs. The VLPs are then subjected to further liquid exchange by column chromatography and sterile filtration to obtain the protein stock solution. Each stock solution is then diluted to the required concentration with buffer, and then all of the protein dilutions of each type are mixed in a predetermined ratio to obtain a 15-valent protein dilution. This is then sterile filtered and prepared for use. Dilute the prescribed amount of aluminum hydroxide adjuvant and mix it uniformly (preferably by mixing with a magnetic stirrer at 300-340 rpm for 30 minutes or more), filter it through a capsule filter and prepare it for use. The vaccine is obtained by taking the required amounts of the 15-valent protein dilution filtration sample and the aluminum hydroxide adjuvant dilution filtration sample and mixing them completely and uniformly. A preparation method characterized by further comprising filling a pre-filled syringe machine, preferably at a filling speed of 30-40 rpm / min, more preferably at 0.55 mL / syringe, sealing with a rubber stopper, and storing in a refrigerator for use.

8. In a method for preparing purified HPV L1 proteins of each type, The fermentation products of recombinant bacteria with each type of L1 protein are taken, the bacterial cells are resuspended, and the cells are disrupted using a high-pressure homogenizer. The resulting cell homogenates are then centrifuged and the supernatant is collected. Add ammonium sulfate powder to the centrifuged supernatant until it reaches a saturation level of 25% to 45%, and stir slowly until completely dissolved. After collecting the precipitate by continuous centrifugation and completely resuspending the precipitate, collect the supernatant by centrifugation again. The supernatant is filtered and clarified, preferably by two-stage deep filtration, and more preferably by a filtration membrane with pore sizes of 3.0 to 6.0 μm and 0.2 to 0.4 μm. Furthermore, purification is performed by EQ anion exchange chromatography → SQ anion exchange chromatography → gel filtration chromatography, preferably with the specific procedure described below. (1) EQ anion exchange chromatography: After equilibrating the column, deep filtration is performed, the sample is collected and loaded, and after loading, it is rinsed and the EQ ion exchange phosphate with an OD280 higher than 50 mAU is collected. (2) SQ anion exchange chromatography: After equilibrating the column, take the EQ anion exchange perfusion from step (1) and load it, rinse after loading, and after rinsing, elute, and collect the eluted fraction corresponding to the absorption peak with an OD280 of 40 mAU or more during elution to obtain the target protein. (3) Gel filtration chromatography: The preparation method according to claim 7, characterized in that the chromatography column is equilibrated and the baseline is stabilized, then the target protein sample collected by elution from SQ anion exchange chromatography is loaded, and the eluted fraction corresponding to the absorption peak with an OD280 higher than 50 mAU is collected to obtain the HPV L1 protein.

9. A method for storing a vaccine according to any one of claims 1 to 6, A storage method characterized by storing at 2°C to 8°C.

10. Use of the vaccine according to any one of claims 1 to 6 in the preparation of a drug for preventing or treating a disease caused by human papillomavirus.

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