Human papillomavirus vaccine and uses thereof

Abstract

The application relates to the field of medicine, in particular to a human papilloma virus vaccine. The vaccine comprises L1 protein antigens of HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 types. The recombinant fifteen-valent human papilloma virus vaccine of the application comprises the above-mentioned 12 types of human cancer-related types, and two most common low-risk types, HPV6 and 11, and one suspicious cancer-causing type, HPV68. In China, the prevention effect of the vaccine on female cervical cancer is expected to be improved to above 97.2%, and in the world, the prevention effect of the vaccine on female cervical cancer is expected to be improved to above 94.1%, and the vaccine has great application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to vaccines for human papillomavirus, especially to vaccines for fifteen-valent human papillomavirus. BACKGROUND

[0002] Human papillomavirus (HPV) is a small DNA virus without envelope, which can infect human epidermis and mucosal squamous epithelial cells. In 1974, the relationship between HPV infection and cervical cancer was first proposed, and it was finally proved that HPV infection is the main cause of the occurrence of cervical cancer. HPV infection is the most common genital viral infection, and 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 and other head and neck cancer, colon cancer, rectal cancer is also related to HPV infection. At present, more than 200 types of HPV have been identified, which can be divided into low-risk and high-risk types according to carcinogenicity. Low-risk HPV mainly includes HPV6 / 11 / 30 / 42 / 43 / 44 / 61 type, etc., among which 90% of genital warts are caused by HPV6 and 11 type infection; the World Health Organization (WHO) International Agency for Research on Cancer (IARC) defines the high-risk HPV as 12 types of HPV (HPV16 / 18 / 31 / 33 / 35 / 39 / 45 / 51 / 52 / 56 / 58 / 59 type) and 2 types of HPV (HPV66 / 68 type) with limited evidence of carcinogenicity, which have carcinogenicity and are the cause of almost all cervical cancers, in addition, also cause 88% of anal cancer, 78% of vaginal cancer, 15% to 48% of vulvar cancer (related to age), 51% of penile cancer and 13% to 60% of oropharyngeal cancer.

[0003] HPV vaccination is the most economical and effective means to prevent HPV persistent infection and related diseases. Currently, there are three kinds of HPV vaccines on the market abroad: the bivalent HPV vaccine (HPV16 / 18 type) produced by GSK company in the United Kingdom (trade name: Cervarix), the quadrivalent HPV vaccine (HPV6 / 11 / 16 / 18 type) produced by Merck company in the United States (trade name: Gardasil) and the recombinant nine-valent HPV vaccine (HPV6 / 11 / 16 / 18 / 31 / 33 / 45 / 52 / 58 type) (trade name: Gardasil9). These three kinds of HPV preventive vaccines all use DNA recombinant technology to express and purify HPV L1 structural proteins, and then form HPV genotype-specific VLPs through self-assembly. Because VLPs do not contain any viral nucleic acid components and have no protein conversion effect similar to prion protein, they are not infectious and are only for preventive use.

[0004] So far, there is no broad-spectrum preventive HPV vaccine on the market that covers all 12 types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 type) clearly related to human cancer and two types with limited evidence of carcinogenicity (HPV66 / 68 type) and HPV6 and 11 types causing genital warts. Therefore, a more broad-spectrum HPV vaccine is urgently needed to improve the prevention effect of cervical cancer in women. SUMMARY

[0005] Based on the needs of the prior art, the present application provides a fifteen-valent vaccine for preventing HPV that covers all 12 types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 type) clearly related to human cancer.

[0006] Firstly, the present application provides a vaccine for human papillomavirus, which comprises one or more combinations of L1 antigens of HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 types, such as combinations of 15, 9, 6 and 3 kinds;

[0007] Preferably, the HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 antigens are present in a ratio of 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: 0.5-2 by weight; preferably, the HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 antigens are present in a ratio of 1.5: 2: 3: 2: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1 by weight; or 1.5: 2: 3: 2: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1 by weight; or 3: 4: 6: 4: 2: 2: 2: 2: 2: 1: 1: 1: 1: 1: 1 by weight.

[0008] More specifically, each protein is present in an amount of 10-100 μg, more preferably 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 per 0.5 ml for HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 respectively.

[0009] Further preferably, the L1 antigens of each type are truncated based on the wild-type sequence as follows: N-terminal truncation of 2 amino acids and C-terminal truncation of 29 amino acids for wild-type HPV6 L1, N-terminal truncation of 3 amino acids and C-terminal truncation of 29 amino acids for wild-type HPV11 L1, N-terminal truncation of 4 amino acids and C-terminal truncation of 29 amino acids for wild-type HPV16 L1, N-terminal truncation of 4 amino acids and C-terminal truncation of 30 amino acids for wild-type HPV18 L1, N-terminal truncation of 4 amino acids and C-terminal truncation of 27 amino acids for wild-type HPV31 L1, N-terminal truncation of 4 amino acids and C-terminal truncation of 24 amino acids for wild-type HPV33 L1, N-terminal truncation of 4 amino acids and C-terminal truncation of 28 amino acids for wild-type HPV35 L1, N-terminal truncation of 9 amino acids and C-terminal truncation of 29 amino acids for wild-type HPV39 L1, N-terminal truncation of 4 amino acids and C-terminal truncation of 30 amino acids for wild-type HPV45 L1, N-terminal truncation of 4 amino acids and C-terminal truncation of 28 amino acids for wild-type HPV51 L1, N-terminal truncation of 4 amino acids and C-terminal truncation of 23 amino acids for wild-type HPV52 L1, N-terminal truncation of 4 amino acids and C-terminal truncation of 25 amino acids for wild-type HPV56 L1, N-terminal truncation of 4 amino acids and C-terminal truncation of 23 amino acids for wild-type HPV58 L1, N-terminal truncation of 4 amino acids and C-terminal truncation of 31 amino acids for wild-type HPV59 L1, N-terminal truncation of 4 amino acids and C-terminal truncation of 28 amino acids for HPV68 L1.

[0010] In one specific embodiment, the amino acid sequences of the L1 antigens of HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 are shown in SEQ ID NOs: 1-15, respectively.

[0011] Further preferably, 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, preferably 0.7-0.85:1.

[0012] Further preferably, the buffer used by the vaccine is an acetic acid-sodium acetate buffer system, and a supplementary material containing histidine, sodium chloride, and polysorbate 80 is added.

[0013] More preferably, the acetic acid-sodium acetate buffer system has a pH of 5-7, more preferably 5.3-6.5, and a concentration of 5-15 mM, preferably 10 mM; the sodium chloride concentration is set to 100-400 mM, more preferably 320-335 mM; and the polysorbate 80 concentration is 0.005%-0.02%, preferably 0.01%.

[0014] The application also provides a preparation method of the vaccine, comprising the following steps:

[0015] The purified HPV L1 proteins of different types are self-assembled in vitro to form VLPs, and the VLPs are further prepared into protein stock solutions through column chromatography and sterilization filtration, and then diluted to the required concentrations using buffers; then, the protein dilution solutions of different types are mixed in proportion to obtain a fifteen-valent protein dilution solution, which is sterilized and ready for use;

[0016] The prescription amount of aluminum hydroxide adjuvant is diluted and uniformly mixed (preferably using a magnetic stirrer at 300-340 rpm for not less than 30 minutes), filtered through a capsule filter, and ready for use;

[0017] The required amount of fifteen-valent protein dilution solution and aluminum hydroxide adjuvant dilution solution are mixed until completely mixed to obtain a semi-finished product;

[0018] Optionally, the finished product is filled: a pre-filled syringe filling machine is started to fill, preferably at a filling speed of 30-40 revolutions / min, more preferably at 0.55 mL / branch filling and dispensing with a rubber plug, and stored in a refrigerator for standby.

[0019] In a specific embodiment, the preparation method of the purified HPV L1 proteins of different types is as follows:

[0020] The fermentation product of the recombinant bacteria of different types of L1 proteins is resuspended, the bacterial cells are broken by a high-pressure homogenizer, and the supernatant is collected by centrifugation after breaking the cell slurry;

[0021] Ammonium sulfate powder is added to the centrifugal supernatant to a saturation degree of 25%-45%, and slowly stirred until completely dissolved;

[0022] The precipitate is collected by continuous centrifugation, and the supernatant is collected again after the precipitate is completely resuspended;

[0023] The clarified supernatant is filtered, preferably using 2-stage deep filtration, more preferably with a filter membrane pore size of 3.0-6.0 μm and 0.2-0.4 μm;

[0024] Then, the EQ anion exchange chromatography→SQ anion exchange chromatography→gel filtration chromatography are used for optimization; preferably, the specific operation is as follows:

[0025] (1) EQ anion exchange chromatography: after column equilibration, the sample collected after deep filtration is loaded; after loading, it is eluted, and the EQ ion exchange flow-through solution with OD280 greater than 50 mAU is collected;

[0026] (2) SQ anion exchange chromatography: After column equilibration, take the EQ anion exchange flow-through solution from the previous step and load the sample; after loading the sample, rinse; after rinsing, elute, and collect the absorption peak of the target protein with an OD280 of not less than 40 mAU during elution;

[0027] (3) Gel filtration chromatography: After equilibration of the chromatography column until the baseline is stable, load the target protein sample collected by SQ anion exchange chromatography and collect the HPV L1 protein absorption peak with OD280 greater than 50mAU.

[0028] The present invention also provides a method for preserving the vaccine, wherein it is stored at 2-8°C.

[0029] Finally, this invention provides the use of the vaccine in the preparation of medicaments for the prevention or treatment of diseases caused by human papillomavirus.

[0030] The beneficial effects of this invention are: This vaccine adds 6 new valence antigens, providing a broader spectrum of immunity. Furthermore, the optimized antigen dosage does not cause immune interference between antigen types. The recombinant 15-valent human papillomavirus vaccine of this invention includes the aforementioned 12 types clearly associated with human cancers, as well as the two most common low-risk types: HPV6 and 11, and one suspected carcinogenic type: HPV68. In China, the vaccine's preventive efficacy against cervical cancer in women is expected to reach over 97.2%, while globally, its preventive efficacy against cervical cancer in women is expected to reach over 94.1%.

[0031] The commercially available 9-valent HPV vaccine Gardasil 9 uses aluminum hydroxyphosphate sulfate adjuvant (a type of aluminum phosphate adjuvant), while the vaccine of this invention uses aluminum hydroxide as the aluminum adjuvant. The proteins of each type in the vaccine of this invention are prepared by expression after C-terminal truncation, removing the positively charged portion. The proteins are negatively charged overall in a near-neutral pH environment, making them more easily adsorbed by the positively charged aluminum hydroxide adjuvant. In contrast, Gardasil 9 uses the full-length sequence for protein expression, resulting in a positively charged protein overall in a near-neutral pH environment, making it less susceptible to adsorption by aluminum hydroxide. Attached Figure Description

[0032] Figure 1 : Results of Tm determination by differential scanning fluorescence method.

[0033] Figure 2 Thermograph of Tm value determined by differential scanning fluorescence method.

[0034] Figure 3 The vaccine neutralizes antibody titers when stored at 4°C for 2 weeks.

[0035] Figure 4 The vaccine neutralizes antibody titers when stored at 37°C for one week.

[0036] Figure 5 Neutralizing antibody titers of the vaccine after being placed at 37℃ for 2 weeks. DETAILED DESCRIPTION

[0037] The present application is described below by way of specific embodiments, so as to better understand the present application, but does not constitute a limitation on the present application.

[0038] Example 1, Preparation of antigens of various types

[0039] The amino acid sequences and preparation methods of the L1 proteins of various types used as antigens in the embodiments of the present application are as follows:

[0040] The amino acid sequence of the HPV6 L1 protein is shown in SEQ ID NO: 1, and the preparation steps of the antigen protein are shown in the patent CN201410685769.1, Example 1, Example 2, Example 10, Example 11, and Example 12.

[0041] The amino acid sequence of the HPV11 L1 protein is shown in SEQ ID NO: 2, and the preparation steps of the antigen protein are shown in the patent CN201410672159.8, Example 1, Example 2, Example 10, Example 11, and Example 12.

[0042] The amino acid sequence of the HPV16 L1 protein is shown in SEQ ID NO: 3, and the preparation steps of the antigen protein are shown in the patent CN201410683185.0, Example 1, Example 2, Example 10, Example 11, and Example 12.

[0043] The amino acid sequence of the HPV18 L1 protein is shown in SEQ ID NO: 4, and the preparation steps of the antigen protein are shown in the patent CN201410672158.3, Example 1, Example 2, Example 10, Example 11, and Example 12.

[0044] The amino acid sequence of the HPV31 L1 protein is shown in SEQ ID NO: 5, and the preparation steps of the antigen protein are shown in the patent CN201510490172.6, Example 1, Example 2, Example 10, Example 11, and Example 12.

[0045] The amino acid sequence of the HPV33 L1 protein is shown in SEQ ID NO: 6, and the preparation steps of the antigen protein are shown in the patent CN201510490177.9, Example 1, Example 2, Example 10, Example 11, and Example 12.

[0046] The amino acid sequence of HPV35 L1 protein is shown as SEQ ID NO: 7, and the preparation steps of the antigen protein are shown in patent CN202211702926.6, embodiments 1, 2, 3, and 4.

[0047] The amino acid sequence of HPV39 L1 protein is shown as SEQ ID NO: 8, and the preparation steps of the antigen protein are shown in patent CN202211702935.5, embodiments 1, 2, 3, and 4.

[0048] The amino acid sequence of HPV45 L1 protein is shown as SEQ ID NO: 9, and the preparation steps of the antigen protein are shown in patent CN201510490367.0, embodiments 1, 2, 10, 11, and 12.

[0049] The amino acid sequence of HPV51 L1 protein is shown as SEQ ID NO: 10, and the preparation steps of the antigen protein are shown in patent CN202310020457.8, the entire content of the experimental method in the specific embodiment.

[0050] The amino acid sequence of HPV52 L1 protein is shown as SEQ ID NO: 11, and the preparation steps of the antigen protein are shown in patent CN201510490149.7, embodiments 1, 2, 10, 11, and 12.

[0051] The amino acid sequence of HPV56 L1 protein is shown as SEQ ID NO: 12, and the preparation steps of the antigen protein are shown in patent CN202310019679.8, the entire content of the experimental steps 1, 2, and 3 in the specific embodiment.

[0052] The amino acid sequence of HPV58 L1 protein is shown as SEQ ID NO: 13, and the preparation steps of the antigen protein are shown in patent CN201410672161.5, embodiments 1, 2, 10, 11, and 12.

[0053] The amino acid sequence of HPV59 L1 protein is shown as SEQ ID NO: 14, and the preparation steps of the antigen protein are shown in patent CN202211702927.0, embodiments 1, 2, 3, and 4.

[0054] The amino acid sequence of HPV68 L1 protein is shown as SEQ ID NO: 15, and the preparation steps of the antigen protein are shown in patent CN202211339029.3, embodiments 1, 3, 4, and 5. However, the truncated HPV68 L1 protein coding nucleotide sequence 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 a recombinant expression.

[0055] The sequences of each type of antigen can also be seen in patent CN202310114443.2 SEQ ID NO: 1 to SEQ ID NO: 15, and the overall construction steps can be seen in patent CN202310114443.2 embodiments 1 and 2.

[0056] Among them, the preparation method of each type of antigen is as follows (see the description in the above-mentioned related patent applications),

[0057] 1. Resuspension of bacterial cells: Take the fermentation product (containing wet bacterial cells 2-5 kg), add the broken bacteria buffer to resuspend at a ratio of 1:4-1:10 of bacterial cells to resuspension liquid. Resuspend the bacterial cells with a high-pressure homogenizer, set the pressure to 80 MPa, and crush the cells 3 times. The cell slurry after crushing is collected by tubular centrifugation (15760g, feed flow rate 200-250mL / min).

[0058] 2. Ammonium sulfate precipitation: Add ammonium sulfate powder to the centrifugal supernatant to 25%-45% saturation, slowly stir at room temperature until completely dissolved. Collect the precipitate by tubular centrifugation (15760g, feed flow rate 200-250mL / min). Resuspend the precipitate by adding resolubilization buffer at a ratio of 1:5-1:10 of mass, and then collect the supernatant by centrifugation.

[0059] 3. Microfiltration clarification: The centrifugal supernatant is further clarified by 2-stage deep filtration. The filter membrane pore size is 3.0-6.0μm and 0.2-0.4μm.

[0060] 4. Chromatography: The chromatography process includes the following 3 steps: EQ anion exchange chromatography→SQ anion exchange chromatography→gel filtration chromatography, the operation is briefly described as follows:

[0061] (1) EQ anion exchange chromatography. After column equilibration, collect the sample after deep filtration and load it; after loading, elute and collect the EQ anion exchange flow-through liquid with OD280 greater than 50mAU.

[0062] (2) SQ anion exchange chromatography. After equilibration of the column, the flow-through of the previous step was loaded. After completion of the loading, the column was washed. After completion of the washing, the elution was performed, and the HPV L1 protein of interest was collected in the OD280 of more than 40 mAU.

[0063] (3) Gel filtration chromatography. After equilibration of the column, the HPV L1 protein of interest collected from the SQ anion exchange chromatography was loaded. The HPV L1 protein of interest was collected in the OD280 of more than 50 mAU.

[0064] 5. VLP assembly, buffer exchange, and stock preservation. The HPV L1 protein purified was self-assembled in vitro to form VLPs. The VLPs were further subjected to buffer exchange by column chromatography and sterilized filtration to prepare a protein stock solution, which was stored at -80°C for later use. The stock solution of each type of protein was stored at a concentration of at least 1.56 mg / mL, and the buffer pH range was 4.3-5.8.

[0065] Example 2. Study of the buffer system of the formulation

[0066] The main purpose of the study of the buffer system of the formulation was to determine the pH range and the optimal concentration of sodium chloride. Two methods were selected. One was to determine the Tm value of each type of L1-VLP antigen under different formulation conditions (different pH and salt concentrations) by differential scanning fluorimetry. The higher the Tm value, the more stable the antigen under the condition. Based on the results of each type of antigen under each condition, the parameter range that was beneficial to the stability of the antigen was selected. The other method was to prepare fifteen-valent HPV vaccines with different formulation conditions (different pH and salt concentrations). After being placed at 37°C for 1-2 weeks, animals were immunized with an appropriate dose (e.g., 1 / 20x of the human dose), and the neutralizing antibody level was determined. The neutralizing antibody level after immunization with the vaccine placed in the buffer system that was beneficial to the stability of the antigen should be higher, so as to determine the appropriate formulation pH range and sodium chloride concentration.

[0067] The formulation of each type of L1-VLP stock solution of the vaccine of the present application was 20 mM acetic acid-sodium acetate, 500-700 mM sodium chloride, 0.02% polysorbate 80, and pH 4.8-5.3. In the process of vaccine preparation, histidine was introduced to adjust the pH to be slightly neutral to increase the body compliance. The formulation buffer system was determined to be 10 mM histidine, 10 mM acetic acid-sodium acetate, 327 mM sodium chloride, and 0.01% polysorbate 80.

[0068] In this study, 20 formulation buffer systems were set, of which 15 contained histidine (pH 5.3, 5.6, 5.9, 6.2, 6.5, and sodium chloride 154, 327, 500 mM), and 5 did not contain histidine (pH 4.4, 4.7, 5.0, 5.3, 5.6, and sodium chloride 327 mM). See Table 1.

[0069] Table 1 Formulation buffer system list

[0070]

[0071] ① Differential scanning fluorimetry (DSF)

[0072] The Tm values of fifteen types of HPV L1-VLP were determined one by one under 20 formulations by DSF method.

[0073] The results showed (as shown in Figure 1 , HPV6 and HPV11 of the same taxonomy of the alpha 10 genus had higher Tm values, HPV16, HPV31, HPV33, HPV35, HPV52 and HPV58 of the same taxonomy of the alpha 9 genus had lower or medium Tm values, HPV18, HPV39, HPV45, HPV59 and HPV68 of the same taxonomy of the alpha 7 genus had higher or medium Tm values, and HPV51 and HPV56 belonged to the alpha 5 genus and the alpha 6 genus respectively. The results showed that the Tm values were related to the evolutionary relationship of each type of virus.

[0074] In the buffer containing histidine and acetic acid, the effects of salt concentration and pH on Tm values can be divided into three categories: ① HPV6, HPV11, HPV16, HPV31, HPV33, HPV35, HPV39, HPV52, HPV58, these nine types showed high Tm values at low salt concentration and low pH; ② HPV18, HPV45 and HPV59, these three types showed high Tm values at low salt concentration and medium pH; HPV51, HPV56 and HPV68, these three types were not sensitive to salt concentration, and showed high Tm values at medium pH.

[0075] In order to further analyze the formulation of fifteen-valent HPV vaccine, the Tm values obtained by DSF method were plotted into a heat map, and the darker the color, the higher the Tm value (see Figure 2 ). Under the same salt concentration (327mM) and the same pH (5.3 or 5.6), the Tm value of the histidine-containing acetate buffer was significantly higher than that of the histidine-free acetate buffer. Therefore, the formulation range was locked in the histidine-containing acetate buffer. In the histidine-containing acetate buffer, pH 5.3-6.2 was significantly better than pH 6.5, so the formulation range was preliminarily determined as pH 5.3-6.2. Under the condition of pH 5.3-6.2, the salt concentration of 154mM and 327mM was significantly better than 500mM.

[0076] According to the analysis results of the Tm values of the antigens under each formulation condition, the stable range of the formulation was determined as pH 5.3-6.2 and NaCl concentration of 154-327mM.

[0077] ② Neutralizing antibody method

[0078] The prescription of the thirteen type Ll-VLP stock in the vaccine of the present example is 20 mM acetic acid-sodium acetate, 500 mM sodium chloride, 0.02% polysorbate 80, pH 5.0. On this basis, the optimal pH of the HPV31 type stock is 4.8, and the salt concentration is 700 mM. The optimal pH of the HPV56 type is 5.3. The fifteen-valent HPV vaccine with or without histidine is prepared first, and the prepared vaccine sample is divided into 15 tubes with histidine and 5 tubes without histidine. After centrifugation, the supernatant is removed, and the precipitate is resuspended with the same volume of 20 preparation prescription solutions, evenly divided into 3 small tubes, and sealed. For each preparation prescription, 1 tube is placed at 4°C as a control, and the other 2 small tubes are placed in a 37°C incubator. On the 7th day, one of the tubes is taken out and temporarily stored at 4°C, and on the 14th day, the other tube is taken out and temporarily stored at 4°C.

[0079] 0.3 mL of the placed vaccine sample is taken, diluted 4-fold with the corresponding preparation buffer to 1.2 mL, and used for mouse immunization. Four weeks after immunization, blood is collected, and serum is separated. The neutralizing antibody titers of the fifteen types of HPV are determined by using the HPV pseudovirus-based neutralizing antibody detection method.

[0080] The neutralizing antibody titers are calculated by using the Reed-Muench method, and the geometric mean titers of the neutralizing antibodies are calculated and compared between groups (Mann-Whitney Test) by using the Graphpad Prism software. The effects of different preparation prescriptions on the stability of the vaccine are evaluated, and the appropriate preparation prescription parameter range is determined.

[0081] The neutralizing antibody detection results show that Figure 3 、 Figure 4 、 Figure 5 The fifteen-valent HPV vaccines prepared according to the 20 preparation prescriptions are placed at 4°C for 2 weeks, at 37°C for 1 week and 2 weeks, respectively, and then used to immunize mice at a dose of 1 / 20 of the human dose. Four weeks after immunization, blood is collected, and the neutralizing antibody titers are determined. There is no obvious difference between the neutralizing antibody titers of different preparation prescriptions, and there is no regular trend, indicating that the buffer system pH (histidine-acetic acid sodium system 5.3-6.5, acetic acid-acetic acid sodium system 4.4-5.6) and sodium chloride concentration (histidine-acetic acid sodium system 154, 327, 500 mM) have little effect on the stability of the immunogenicity of the vaccine in the studied range. Moreover, there is no obvious difference between the neutralizing antibody titers determined after the 37°C accelerated reaction and the neutralizing antibody titers determined after 2 weeks of placement at 4°C (without accelerated reaction), indicating that the stability of the antigens of each type of the vaccine in the 20 preparation prescriptions is very good.

[0082] From the human body acceptance, the vaccine formulation pH tends to neutral (pH 7.0), the salt concentration close to 0.9% (physiological saline concentration), the human body stimulation is smaller. So choose the pH more biased to the neutral histidine-sodium acetate buffer system (pH 5.3-6.5) as the fifteen valent HPV vaccine formulation buffer component is more appropriate. Because the type of protein stock salt concentration is higher (500, 700 mM), and the initial salt concentration of aluminum hydroxide adjuvant is 0.9% (154 mM), the sodium chloride salt concentration is set to 327 mM during the preparation of the vaccine formulation, which is more convenient for the preparation process operation. Moreover, the stock preparation process and prescription research results show that high salt concentration is more conducive to the maintenance of uniform 72 pentamer L1-VLP conformation of the antigen, which is conducive to the stability of the antigen structure.

[0083] Example Three, Antigen Dose Ratio and Aluminum Adjuvant Dose Study

[0084] According to the antigen protein of each type VLP obtained in Example 1, the fifteen valent HPV vaccine with different antigen dose ratio and aluminum adjuvant dose was prepared according to the subsequent preparation process (see Table 2), and BALB / c mice were immunized to investigate the immunogenicity. Specifically, a single antigen immunization group was set up to investigate the immunization interference. Gardasil 9 was used as a control vaccine to set up different aluminum adjuvant dose groups to determine the adjuvant dose in the vaccine, and different antigen dose ratios were set up to determine the antigen dose ratio of each type. See Table 2 for animal grouping information, antigen dose ratio, and aluminum adjuvant dose setting. The immunization program was 0, 4 weeks, and the neutralizing antibody titer was determined 4 weeks after the first immunization and 4 weeks after the second immunization. Group comparison was performed to determine the antigen dose ratio and aluminum adjuvant dose in the vaccine.

[0085] Table 2 Animal grouping table for antigen dose ratio and aluminum adjuvant dose study

[0086]

[0087] Note: Intramuscular injection was used, and each BALB / c mouse was injected with 100 μl.

[0088] Note: Group 1-Group 15 is a single type antigen (i.e. HPV6, 11, 16, 16, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 6) adsorbed with aluminum adjuvant, and 10 mice were immunized. Group 16-Group 19 is the same type of antigen in 15 valent antigen, and different aluminum adjuvants are adsorbed, and 10 mice are immunized. Group 20-Group 21 is each type of different antigen dose adsorbed with the same dose of adjuvant, and 10 mice are immunized. Group 22 is the antigen and adjuvant dose of Merck's commercial product Gardasil 9-valent vaccine, and 10 mice are immunized as a comparative study. Group 23 is an aluminum adjuvant control group, and 5 mice are immunized.

[0089] The geometric mean of the neutralizing antibody titer at 4 weeks after the first and second immunization is shown in Table 3. The results show that:

[0090] (1) Immunological interference

[0091] The mice were immunized with the same dose of aluminum adjuvant in the form of monovalent antigen and fifteen-valent antigen (the same dose of homologous antigen), and the difference in the level of immune response between the two was compared to investigate the immunological interference. The results showed that the neutralizing antibody titers of all types of antigens immunized in monovalent form were significantly higher than those in the fifteen-valent HPV vaccine form, and were all higher than those of the corresponding types of the control vaccine (Gardasil9).

[0092] (2) Aluminum adjuvant dose

[0093] The antigen dose was fixed at 1 / 20x of the human dose, and four aluminum adjuvant doses were set: 37.5 (1 / 20x of the nine-valent vaccine), 0, 25, and 50 μg. The mice were immunized (n = 10 mice per group), and the differences between the aluminum adjuvant doses were investigated, and the control vaccine (Gardasil9) was also compared. The results showed that the neutralizing antibody level in the group without aluminum adjuvant was significantly lower than that in the group with aluminum adjuvant, indicating that it is necessary to add aluminum adjuvant to the vaccine. The effect of aluminum adjuvant on increasing the antigen immune response level is obvious. There is no significant difference between the three doses in the aluminum adjuvant-containing group. Compared with the control vaccine, the increase in aluminum adjuvant did not produce a significant advantage, indicating that 25 μg (corresponding to 500 μg of the human dose) of aluminum adjuvant can achieve or approach immunization saturation, and the immune response level is comparable to that of the control vaccine.

[0094] (3) Antigen dose ratio

[0095] The initial antigen dose of the fifteen-valent HPV vaccine was set as 20 μg for each of the six new types of antigens added to the nine-valent vaccine (i.e., the HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 types of antigens were 30, 40, 60, 40, 20, 20, 20, 20, 20, 20, 20, 20, 20, 20, and 20 μg, respectively). On this basis, the HPV16 and 18 antigens were each increased by 10 μg, or the HPV16, 18, 31, 33, 45, 52, and 58 types were each increased by 10 μg, to investigate whether increasing the dose of these types of antigens can significantly increase the immune response level of these types of antigens to ensure that the vaccine effect is not lower than that of the marketed vaccine Gardasil9. The results (Table 3) show that compared with the initial antigen dose, increasing the dose of these types of antigens by 10 μg does not produce a significant immune enhancement effect, indicating that the initial formulation antigen amount ratio has reached immunization saturation in mice, and there is no need to increase the antigen dose of these types.

[0096] Table 3 Summary of immunization dose and neutralizing antibody geometric mean titer

[0097]

[0098]

[0099] Example 4, Preparation process of fifteen-valent HPV vaccine

[0100] Take HPV6 / 11 / 16 / 18 / 31 / 33 / 35 / 39 / 45 / 51 / 52 / 56 / 58 / 59 / 68 type VLP protein stock from -65℃ to -80℃ refrigerator, each type protein stock storage concentration minimum 1.56mg / mL, buffer pH range 4.3-5.8 (see example 1). Buffer prescription 20mM HAc-NaAc, 500mM-700mM NaCl, 0.02% Tween 80. After 10min at room temperature, thaw completely in 37℃ water bath. Dilute aluminum hydroxide adjuvant to 4mg / mL, conduct bacterial endotoxin, sterility, osmolality, aluminum content detection. 4.0mg / mL aluminum hydroxide adjuvant density 1.01g / mL. Take fifteen type protein diluent 3120mL (density 1.02g / mL) and aluminum hydroxide adjuvant diluent 3120mL (density 1.01g / mL) respectively, mix in 10L glass cylinder (magnetic stirrer rotor diameter 1.39cm, length 8.6cm), mix at 300-340rpm for not less than 30 minutes. Start pre-filled syringe filling machine, set and operate filling machine according to equipment operation and use procedures. Fill and sub-pack with 0.55mL / branch and rubber stopper, filling speed 30-40 turns / min. Label and store.

[0101] Example 5, Further study of antigen dose ratio

[0102] In order to obtain more research data of antigen dose ratio and provide reference for vaccine dose for clinical trial, further study of antigen dose ratio was conducted. Prepare fifteen-valent HPV vaccine with different antigen dose ratio, immunize BALB / c mice according to 1 / 50x human dose, compare immunogenicity difference under different antigen dose ratio. Animal grouping information and antigen dose ratio information are shown in Table 4. Immunization program uses 0, 4 weeks immunization, determine neutralizing antibody titer at 4 weeks after first immunization and 4 weeks after second immunization. Conduct inter-group comparison to determine antigen dose ratio of each type in vaccine.

[0103] Table 4 Animal grouping table of further study of antigen dose ratio

[0104]

[0105] Table 5 Summary table of immunization dose and neutralizing antibody geometric mean titer

[0106]

[0107]

[0108] The geometric mean of the neutralizing antibody detection in the first 4 weeks and the second 4 weeks is shown in Table 5.

[0109] (1) The neutralizing antibody detection result in the first 4 weeks shows that:

[0110] Compared with the same type contained in Gardasil 9, the types with significant differences in the low-dose group are HPV11, 33, and 45, among which HPV11 is higher in Gardasil 9 than in the fifteen-valent HPV vaccine, and HPV33 and 45 are higher in the fifteen-valent HPV vaccine than in Gardasil 9. The types with significant differences in the medium-dose group are HPV33 and 45, both of which are higher in the fifteen-valent HPV vaccine than in Gardasil 9. The types with significant differences in the high-dose group are HPV11 and 33, among which HPV11 is higher in Gardasil 9 than in the fifteen-valent HPV vaccine, and HPV33 is higher in the fifteen-valent HPV vaccine than in Gardasil 9. The levels of other types in the three dose groups are equivalent, and there is no significant difference. The six new types (HPV35, 39, 51, 56, 59, and 68) of the product vaccine can induce strong immune responses in the low, medium, and high dose groups.

[0111] The significant differences between the low, medium, and high dose groups are: HPV45 type (medium dose is higher than high dose), HPV59 type (high dose is higher than low dose). Compared with the medium dose group, the antigen dose of HPV35, 39, 51, 56, 59, and 68 types is reduced by half in the low dose group, and the neutralizing antibody level has no significant difference. Compared with the medium dose group, the antigen of HPV16 type is increased from 60 μg / dose to 80 μg / dose in the high dose group, and the neutralizing antibody titer level has no significant difference.

[0112] (2) The neutralizing antibody detection result in the second 2 weeks shows that:

[0113] Compared with the same type contained in Gardasil 9, the type with significant difference in the low-dose group is HPV45, which is higher in the fifteen-valent HPV vaccine than in Gardasil 9. The type with significant difference in the medium-dose group is HPV33, which is higher in the fifteen-valent HPV vaccine than in Gardasil 9. The type with significant difference in the high-dose group is 33, which is higher in the fifteen-valent HPV vaccine than in Gardasil 9. The levels of other types in the three dose groups are equivalent, and there is no significant difference. The six new types (HPV35, 39, 51, 56, 59, and 68) of the product vaccine can induce strong immune responses in the low, medium, and high dose groups, and produce high levels of neutralizing antibodies.

[0114] Significant difference between low, medium and high dose groups were found in HPV56 (higher in high dose than in low dose) and HPV59 (higher in medium dose than in low dose). Compared with medium dose group, the low dose group had half of the antigen dose of HPV35, 39, 51, 56, 59, 68, and the induced neutralizing antibody level was lower than that of the medium dose group, but only the difference of HPV59 was significant. Compared with medium dose group, the high dose group increased the antigen dose of HPV16 from 60 μg / dose to 80 μg / dose, and the neutralizing antibody titer level was improved, but no significant difference was found.

[0115] Reducing the antigen dose of HPV35, 39, 51, 56, 59, 68 on the basis of the medium dose of the fifteen-valent HPV vaccine can reduce the immune response level of these six types to some extent, but has no obvious effect on the immune response of the other nine types.

[0116] Increasing the antigen dose of HPV16 to 80 μg / dose on the basis of the medium dose of the fifteen-valent HPV vaccine can improve the immune response level of this type to some extent, making it closer to Gardasil 9, but no significant difference was found. Subsequent clinical trials need to consider the results of preclinical pharmacodynamic studies and safety evaluation results to develop trial vaccine dose ratios.

[0117] Through the above studies, the preparation-related characteristics of the product are briefly described as follows:

[0118] The adjuvant used is the commonly used aluminum hydroxide adjuvant. Its particle size distribution is about 1-20 μm, and it appears milky white in aqueous solution and precipitates after a certain period of time. After the aluminum hydroxide adjuvant adsorbs protein antigens, the particle size may increase slightly, and the appearance is basically similar to that of aluminum hydroxide adjuvant without adsorbed proteins.

[0119] During the preparation process, the pH value is adjusted from about 5.0 in the stock solution to about 6.0 in the preparation by adding 10 mM histidine, so that it is more suitable for human body and reduces irritation. The results of the storage stability study show that the pH value of the product preparation is controlled between 5.3 and 6.5 (including the histidine prescription), and there is no significant difference in the immunogenicity of the vaccine after being placed at 37°C for 2 weeks.

[0120] The concentration of sodium chloride in the preparation is set to 327 mM, and the results of the storage stability study show that the concentration of sodium chloride in the product preparation has no obvious effect on the immunogenicity of the vaccine when set between 154 and 500 mM.

[0121] The dosage of aluminum adjuvant in the preparation is 0.5 mg / dose, which is the same as the aluminum content of the commercially available nine-valent vaccine Gardasil 9, and the difference is that the aluminum adjuvant used in Gardasil 9 is a hydroxyl phosphate aluminum sulfate adjuvant (a kind of aluminum phosphate adjuvant), not aluminum hydroxide. The selection of the type of aluminum adjuvant is mainly related to the charge properties of the antigen. The proteins of each type in the vaccine are prepared by expression after C-terminal truncation, which removes the positively charged part. The protein is negatively charged as a whole in the environment close to neutral pH, while the full-length sequence is used for protein expression preparation in Gardasil 9. The protein is positively charged as a whole in the environment close to neutral pH. Negatively charged antigens are more easily adsorbed by positively charged aluminum hydroxide adjuvants, and positively charged antigens are more easily adsorbed by negatively charged aluminum phosphate adjuvants. In the vaccine preparation of the present application, the adsorption rate of aluminum hydroxide adjuvant to each type of antigen is greater than 95%, close to 100%. After the antigen is adsorbed on the surface by the aluminum adjuvant, it is not easy to aggregate between antigens, which is beneficial to its stability.

[0122] The vaccine of the present application is a sterile preparation, which needs to be stored at 2-8℃ and cannot be frozen. The freezing process will destroy the crystal structure of the aluminum adjuvant, affecting its sedimentation properties, antigen adsorption capacity, etc., and thus affecting the efficacy of the vaccine.

[0123] Finally, it should be noted that: the above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent replacements to some technical features. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A vaccine for human papillomavirus characterized in that, It includes the combination of L1 antigens of HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 types; The dosage of HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 antigens is 1.5:2:3:2:1:1:1:1:1:1:1:1:1:1:1 by weight ratio; The amino acid sequences of the L1 antigens of each type are shown in SEQ ID NO: 1 to 15, respectively; The adjuvant used is aluminum hydroxide adjuvant, and the mass ratio of antigen protein to aluminum adjuvant is 0.7-0.85:

1.

2. The vaccine of claim 1, wherein The dosage of HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 antigens is 1.5:2:3:2:1:1:1:1:1:1:1:1:1:1:1 by weight ratio; Or 3:4:6:4:2:2:2:2:2:1:1:1:1:1:

1.

3. The vaccine of claim 2, wherein the antigen is a protein or a polypeptide. The dosage of each protein is 10-100ug.

4. The vaccine of claim 3, wherein the antigen is a protein or a polypeptide. HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 contains 30ug, 40ug, 60ug, 40ug, 20ug, 20ug, 20ug, 20ug, 20ug, 20ug, 20ug, 20ug, 20ug, 20ug, 20ug, 20ug per 0.5ml.

5. The vaccine as claimed in any one of claims 1 to 4, wherein, The vaccine uses a buffer system of acetic acid-sodium acetate, and adds auxiliary materials containing histidine, sodium chloride, and polysorbate 80.

6. The vaccine of claim 5, wherein the antigen is a protein or a polypeptide. The acetic acid-sodium acetate buffer system has a pH of 5-7 and a concentration of 5-15mM; the concentration of sodium chloride is set to 100-400mM; and the concentration of polysorbate 80 is 0.005%-0.02%.

7. The vaccine as described in claim 6, characterized in that, The acetic acid-sodium acetate buffer system has a pH of 5.3-6.5 and a concentration of 10mM; the concentration of sodium chloride is set to 320-335mM; and the concentration of polysorbate 80 is 0.01%.

8. The method for preparing the vaccine according to any one of claims 1 to 7, comprising the following steps: The purified L1 proteins of each type of HPV are self-assembled in vitro to form VLPs, and the VLPs are further prepared into protein stock solutions through column chromatography and sterilization filtration, and then diluted to the required concentration using a buffer solution; then, all the protein dilution solutions of each type are mixed in proportion to obtain a fifteen-valent protein dilution solution, which is sterilized and filtered for standby; The prescribed amount of aluminum hydroxide adjuvant is diluted and mixed uniformly, filtered through a capsule filter, and then standby; The required amount of fifteen-valent protein dilution solution and aluminum hydroxide adjuvant dilution solution are mixed until uniform, and then standby.

9. The method of preparing a vaccine according to claim 8, wherein the vaccine is prepared by mixing the antigen and the adjuvant. The dilution and mixing are performed using a magnetic stirrer at 300-340rpm for not less than 30 minutes.

10. The method of preparing a vaccine as claimed in claim 8, wherein, It also includes product filling: starting the pre-filled syringe filling machine to fill.

11. The method of preparing a vaccine as claimed in claim 10, wherein, The filling speed is 30-40 rounds / min, and the filling is divided and rubberized at 0.55 mL / branch, and stored in a refrigerator for standby.

12. The method of preparing a vaccine as claimed in claim 10, wherein, The preparation method of the purified HPV L1 protein of each type is as follows: The fermentation product of the recombinant bacteria of each type of L1 protein is resuspended, the cell bodies are broken by a high-pressure homogenizer, and the supernatant is collected by centrifugation after the broken cell slurry is centrifuged; Ammonium sulfate powder is added to the centrifugal supernatant to a saturation degree of 25%-45%, and slowly stirred until completely dissolved; The precipitate is collected by continuous centrifugation, and the supernatant is collected by centrifugation after the precipitate is completely resuspended; The clarified supernatant is filtered, and 2-stage deep filtration is adopted, more preferably, the filter membrane pore size is 3.0-6.0 μm and 0.2-0.4 μm; Then, EQ anion exchange chromatography→SQ anion exchange chromatography→gel filtration chromatography is adopted for optimization.

13. The method of preparing a vaccine as claimed in claim 12, wherein, The preparation method of the purified HPV L1 protein of each type is as follows: (1) EQ anion exchange chromatography: after column equilibration, the sample collected after deep filtration is loaded; after loading, elution is performed, and the EQ ion exchange flow-through liquid with OD280 greater than 50 mAU is collected; (2) SQ anion exchange chromatography: after column equilibration, the EQ anion exchange flow-through liquid collected in the previous step is loaded; After loading, elution is performed; after elution, the target protein absorption peak with OD280 not less than 40 mAU is collected; (3) Gel filtration chromatography: after equilibrating the chromatography column to the baseline, the target protein sample collected by SQ anion exchange chromatography is loaded, and the HPV L1 protein absorption peak with OD280 greater than 50 mAU is collected.

14. A method of preserving a vaccine as claimed in any one of claims 1 to 7, wherein, It is stored at 2-8°C.

15. Use of the vaccine of any one of claims 1 to 7 in the preparation of a medicament for preventing a human papillomavirus-induced disease.

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