Nine-valent human papillomavirus vaccine and its application

Abstract

The present application relates to the field of medicine, and specifically discloses a nine-valent human papillomavirus vaccine and its application. The nine-valent human papillomavirus vaccine includes truncated L1-VLP antigens of HPV6, 11, 16, 18, 31, 33, 45, 52, and 58, and an aluminum hydroxide adjuvant. The antigen components in the nine-valent HPV vaccine provided by the present invention are all derived from the main capsid protein L1 of HPV and are truncated. The truncated L1 protein expressed by the Escherichia coli system increases the yield of the L1 protein and reduces the production cost of the vaccine. The present application optimizes the particle size of each type of VLP antigen and the particle size of the aluminum hydroxide adjuvant, and controls the ratio of each type of VLP antigen and the aluminum hydroxide adjuvant, so that the obtained nine-valent HPV vaccine induces a high level of immune response, especially showing obvious effects in clinical practice, which is better than the effects of related vaccines currently on the market.

Description

Technical Field

[0001] The present application relates to the field of medicine, specifically to a human papillomavirus vaccine, and in particular to a nine-valent human papillomavirus vaccine and its application. Background Art

[0002] Human papillomavirus (HPV) is a small, non-enveloped DNA virus that can infect human epidermal and mucosal squamous epithelial cells. The relationship between HPV infection and cervical cancer was first proposed in 1974, and it was eventually proved that HPV infection is the main cause of cervical cancer. HPV infection is the most common genital tract viral infection. Human infection with HPV may not only cause cervical cancer, but also anogenital cancer and genital warts. In addition, the occurrence of oropharyngeal cancer and other head and neck cancers, colon cancer, and 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, etc., of which 90% of genital warts are caused by HPV6 and 11 infections; the World Health Organization (WHO) International Agency for Research on Cancer (International Agency for Research on The latest high-risk HPV definitions by the International Institute for Cancer (IARC) include 12 types that are clearly associated with human cancer (HPV16 / 18 / 31 / 33 / 35 / 39 / 45 / 51 / 52 / 56 / 58 / 59) and 2 types with limited evidence of carcinogenicity (HPV66 / 68). High-risk HPV is carcinogenic and is the cause of almost all cervical cancers. In addition, it also causes 88% of anal cancer, 78% of vaginal cancer, 15% to 48% of vulvar cancer (age-related), 51% of penile cancer, and 13% to 60% of oropharyngeal cancer.

[0003] HPV vaccination is the most economical and effective means to prevent persistent HPV infection and related diseases. Currently, there is only one nine-valent HPV vaccine on the market at home and abroad: the recombinant nine-valent HPV vaccine (HPV6 / 11 / 16 / 18 / 31 / 33 / 45 / 52 / 58) produced by Merck in the United States (trade name: Gardasil 9). Gardasil 9 is expressed by eukaryotic cells (Saccharomyces cerevisiae). The eukaryotic cell production technology has the problem of low yield and high cost due to the disassembly and reassembly of VLPs. Summary of the invention

[0004] Based on the needs of the prior art, the present invention has obtained a preventive nine-valent human papillomavirus vaccine through in-depth research, covering 2 types of antigens clearly associated with genital warts and 7 types of antigens clearly associated with cervical cancer (HPV6, 11 and HPV16, 18, 31, 33, 45, 52, 58).

[0005] The present invention first provides a nine-valent human papillomavirus vaccine, which includes truncated L1-VLP antigens of HPV6, 11, 16, 18, 31, 33, 45, 52, and 58, and an aluminum hydroxide adjuvant;

[0006] Preferably, the weight ratio of HPV6, 11, 16, 18, 31, 33, 45, 52, 58 L1-VLP 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); Preferably, the weight ratio of HPV6, 11, 16, 18, 31, 33, 45, 52, 58 L1-VLP antigens is 1.5:2:3:2:1:1:1:1:1;

[0007] More specifically, the dosage of each antigen in every 0.5 ml is 10-100 μg, and more preferably the HPV6, 11, 16, 18, 31, 33, 45, 52, and 58 L1-VLP antigens are 30 μg, 40 μg, 60 μg, 40 μg, 20 μg, 20 μg, 20 μg, 20 μg, and 20 μg, respectively; the dosage of aluminum hydroxide adjuvant is 0.5-1.0 mg, preferably 0.75 mg.

[0008] More specifically, HPV6, 11, 16, 18, 31, 33, 45, 52, and 58 L1-VLP antigens are expressed by the Escherichia coli system.

[0009] In a specific embodiment, the particle size of each type of L1-VLP antigen is 45 to 80 nm, more preferably the particle size of HPV6 L1-VLP antigen is 45 to 60 nm, the particle size of HPV11 L1-VLP antigen is 45 to 60 nm, the particle size of HPV16 L1-VLP antigen is 45 to 60 nm, the particle size of HPV18 L1-VLP antigen is 55 to 75 nm, the particle size of HPV31 L1-VLP antigen is 55 to 75 nm, the particle size of HPV33 L1-VLP antigen is 45 to 60 nm, the particle size of HPV45 L1-VLP antigen is 50 to 65 nm, the particle size of HPV52 L1-VLP antigen is 50 to 65 nm, and the particle size of HPV58 L1-VLP antigen is 45 to 60 nm;

[0010] More specifically, the particle size of the aluminum hydroxide adjuvant is 3 to 10 μm.

[0011] Preferably, each type of L1-VLP protein antigen is assembled by mixing each type of L1 pentamer protein with an assembly solution (pH 4.5-5.5, NaCl concentration 2.0-5.0 M), and the particle size of the L1 pentamer protein is 10-15 nm.

[0012] In a specific embodiment, the particle size of the HPV6L1 pentamer protein is 12 to 15 nm, the particle size of the HPV11L1 pentamer protein is 12 to 15 nm, the particle size of the HPV16L1 pentamer protein is 11 to 14 nm, the particle size of the HPV18L1 pentamer protein is 11 to 14 nm, the particle size of the HPV31L1 pentamer protein is 12 to 15 nm, the particle size of the HPV33L1 pentamer protein is 12 to 15 nm, the particle size of the HPV45L1 pentamer protein is 12 to 15 nm, the particle size of the HPV52L1 pentamer protein is 12 to 15 nm, and the particle size of the HPV58L1 pentamer protein is 11 to 14 nm.

[0013] In a specific embodiment, the preparation method of the aluminum hydroxide adjuvant is as follows: take aluminum chloride and sodium hydroxide, according to Al 3+ and OH - The reaction is carried out at a molar ratio of 1:(2-5), without adding sodium chloride to the reaction system, and the reaction is carried out at room temperature or 50-70°C, and then the temperature is raised to 80°C to maintain the pH at about 6.0-7.0 for 50-70 minutes. After being placed in a tank, the mixture is washed with a sodium chloride solution (resuspended at a mass ratio of ≥6.5:1) for more than 4 times to obtain an aluminum hydroxide adjuvant.

[0014] Furthermore, the washed aluminum hydroxide adjuvant is resuspended with injection water or 0.9% sodium chloride solution according to the feed amount until the aluminum content reaches 7 mg / ml, and then packaged and sterilized by high-pressure steam at 121° C. for 20 min.

[0015] In a preferred embodiment, the L1 protein in each type of L1-VLP antigen has the following truncations based on the wild-type sequence: the N-terminal truncation of the wild-type HPV6 L1 is no more than 10 amino acids, and the C-terminal truncation is no more than 30 amino acids; the N-terminal truncation of the wild-type HPV11 L1 is no more than 10 amino acids, and the C-terminal truncation is no more than 30 amino acids; the N-terminal truncation of the wild-type HPV16 L1 is no more than 10 amino acids, and the C-terminal truncation is no more than 30 amino acids; the N-terminal truncation of the wild-type HPV18 L1 is no more than 10 amino acids, and the C-terminal truncation is no more than 30 amino acids. ; The N-terminal truncation of the wild-type HPV31L1 is no more than 10 amino acids, and the C-terminal truncation is no more than 30 amino acids; the N-terminal truncation of the wild-type HPV33L1 is no more than 10 amino acids, and the C-terminal truncation is no more than 30 amino acids; the N-terminal truncation of the wild-type HPV45L1 is no more than 10 amino acids, and the C-terminal truncation is no more than 30 amino acids; the N-terminal truncation of the wild-type HPV52L1 is no more than 10 amino acids, and the C-terminal truncation is no more than 30 amino acids; the N-terminal truncation of the wild-type HPV58L1 is no more than 10 amino acids, and the C-terminal truncation is no more than 30 amino acids.

[0016] More preferably, the amino acid sequences of the various types of L1 proteins are shown as SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, and SEQ ID NO.17, respectively.

[0017] In another embodiment, the vaccine adopts a histidine-hydrochloric acid buffer system, and adds an osmotic pressure regulator and a surfactant. Preferably, the osmotic pressure regulator is selected from one or more of sodium chloride, sodium phosphate or sodium sulfate, preferably sodium chloride; and the surfactant is polysorbate 80.

[0018] Preferably, the pH of the histidine-hydrochloric acid buffer system is 5 to 7, more preferably the pH is 5.5 to 6.5; the concentration of histidine-hydrochloric acid is 5 to 20 mM, preferably 10 to 15 mM; the concentration of sodium chloride is 300 to 600 mM, more preferably 420 to 480 mM; the concentration of polysorbate 80 is 0.005% to 0.025%, preferably 0.01% to 0.025%, more preferably 0.02%.

[0019] In a specific embodiment, purified HPV L1 proteins of various types are self-assembled in vitro to form VLPs, and the VLPs are further subjected to column chromatography, liquid exchange, sterilization and filtration to prepare protein stock solutions, which are diluted to the required concentrations using buffer solutions; then, the protein dilutions of various types are respectively mixed with aluminum hydroxide adjuvant dilutions in proportion to prepare monovalent adsorption products, and finally, the required amount of the monovalent adsorption products of various types are taken and completely mixed to obtain the finished vaccine preparation;

[0020] Alternatively, the various types of protein stock solution dilutions can be mixed in proportion to prepare a nine-valent protein dilution, and then the required amount of the nine-valent protein dilution is taken and thoroughly mixed with the aluminum hydroxide adjuvant dilution to obtain a finished vaccine preparation;

[0021] Furthermore, it also includes filling of finished products, specifically starting a prefilled syringe filling machine for filling, preferably, the filling speed is 30-40 rpm, more preferably 0.55 ml / tube is filled and packaged and plugged, and stored in a cold storage at 2-8°C for future use.

[0022] The present invention provides use of the nine-valent human papillomavirus vaccine in preparing a medicament for preventing or treating diseases caused by human papillomavirus.

[0023] The present invention also provides a method for storing the vaccine, which is to store the vaccine at 2-8°C.

[0024] Finally, the present invention provides use of the vaccine in preparing medicines for preventing or treating diseases caused by human papillomavirus.

[0025] The aluminum adjuvant used in the commercially available nine-valent HPV vaccine Gardasil 9 is aluminum phosphate sulfate adjuvant (a type of aluminum phosphate adjuvant), and the aluminum adjuvant used in the nine-valent HPV vaccine of the present invention is aluminum hydroxide. The various types of proteins in the vaccine of the present invention are expressed and prepared after truncation of the N / C termini, removing the positively charged part. The protein is negatively charged as a whole in an environment with a pH close to neutral, and is more easily adsorbed by the positively charged aluminum hydroxide adjuvant. Gardasil 9 uses the full-length sequence to express and prepare the protein. The protein is positively charged as a whole in an environment with a pH close to neutral, and is not easily adsorbed by the aluminum hydroxide adjuvant. Most importantly, in actual human clinical trials, the nine-valent HPV vaccine of the present invention is significantly better than commercially available vaccines.

[0026] Therefore, the beneficial effects of the present invention are as follows: the antigen components in the nine-valent HPV vaccine of the present invention are all derived from the main capsid protein L1 of HPV and are truncated. The truncated L1 protein expressed by the E. coli system improves the yield of the L1 protein and reduces the production cost of the vaccine. The present invention optimizes the particle size of each type of VLP antigen and the particle size of the aluminum hydroxide adjuvant, and controls the ratio of each type of VLP antigen and the aluminum hydroxide adjuvant, so that the obtained nine-valent HPV vaccine induces a high level of immune response, especially showing obvious effects in clinical practice, which is better than the effects of related vaccines currently available on the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 , HPV11, HPV16 and HPV18 neutralizing antibody analysis.

[0028] Figure 2 , HPV33, HPV58, and HPV6 neutralizing antibody analysis.

[0029] Figure 3 , HPV31, HPV45 and HPV52 neutralizing antibody analysis.

[0030] in, Figure 1-Figure 3 In the table, A-4 represents that each type of antigen is placed at 4℃ in prescription A, B-4 represents that each type of antigen is placed at 4℃ in prescription B, C-4 represents that each type of antigen is placed at 4℃ in prescription C, D-4 represents that each type of antigen is placed at 4℃ in prescription D, and E-4 represents that each type of antigen is placed at 4℃ in prescription E; A-37-14d represents that each type of antigen is placed at 37℃ in prescription A for 14 days, B-37-14d represents that each type of antigen is placed at 37℃ in prescription B for 14 days, C-37-14d represents that each type of antigen is placed at 37℃ in prescription C for 14 days, D-37-14d represents that each type of antigen is placed at 37℃ in prescription D for 14 days, and E-37-14d represents that each type of antigen is placed at 37℃ in prescription E for 14 days. DETAILED DESCRIPTION

[0031] The present invention is described below through specific implementation modes in order to better understand the present invention, but it does not constitute a limitation to the present invention.

[0032] Example 1. Preparation of various types of antigens

[0033] The present invention studies and truncates each type of L1 protein based on the wild-type sequence to remove the positively charged part, thereby obtaining optimized each type of L1 protein. The specific each type of L1 protein used in this example is as follows:

[0034] The amino acid sequence of the truncated HPV6L1 is shown in SEQ ID NO. 1, and the nucleotide sequence of the codon-optimized HPV6L1 is shown in SEQ ID NO. 2. Finally, the HPV6L1-VLP protein with an average particle size of 45-60 nm and PdI < 0.1 was obtained.

[0035] The sequence of the truncated HPV11L1, the amino acid sequence of HPV11L1 is shown in SEQ ID NO. 3, and the nucleotide sequence of HPV11L1 is shown in SEQ ID NO. 4. Finally, the HPV11L1-VLP protein with an average particle size of 45-60nm and PdI<0.1 was obtained.

[0036] The truncated sequence of HPV16L1, the amino acid sequence of HPV16L1 is shown in SEQ ID NO. 5, and the nucleotide sequence of HPV16L1 is shown in SEQ ID NO. 6. Finally, HPV16L1-VLP protein with an average particle size of 45-60nm and PdI<0.1 was obtained.

[0037] The sequence of the truncated HPV18L1, the amino acid sequence of HPV18L1 is shown in SEQ ID NO. 7, and the nucleotide sequence of HPV18L1 is shown in SEQ ID NO. 8. Finally, the HPV18L1-VLP protein with an average particle size of 55-75nm and PdI<0.1 was obtained.

[0038] The sequence of the truncated HPV31L1, the amino acid sequence of HPV31L1 is shown in SEQ ID NO. 9, and the nucleotide sequence of HPV31L1 is shown in SEQ ID NO. 10. Finally, the HPV31L1-VLP protein with an average particle size of 55-75nm and PdI<0.1 was obtained.

[0039] The sequence of the truncated HPV33L1, the amino acid sequence of HPV33L1 is shown in SEQ ID NO. 11, and the nucleotide sequence of HPV33L1 is shown in SEQ ID NO. 12. Finally, the HPV33 L1-VLP protein with an average particle size of 45-60nm and PdI<0.1 was obtained.

[0040] The sequence of the truncated HPV45L1, the amino acid sequence of HPV45L1 is shown in SEQ ID NO. 13, and the nucleotide sequence of HPV45L1 is shown in SEQ ID NO. 14. Finally, the HPV45L1-VLP protein with an average particle size of 50-65nm and PdI<0.1 was obtained.

[0041] The sequence of the truncated HPV52L1, the amino acid sequence of HPV52L1 is shown in SEQ ID NO. 15, and the nucleotide sequence of HPV52L1 is shown in SEQ ID NO. 16. Finally, the HPV52L1-VLP protein with an average particle size of 50-65nm and PdI<0.1 was obtained.

[0042] The sequence of the truncated HPV58L1, the amino acid sequence of HPV58L1 is shown in SEQ ID NO. 17, and the nucleotide sequence of HPV58L1 is shown in SEQ ID NO. 18. Finally, the HPV58L1-VLP protein with an average particle size of 45-60nm and PdI<0.1 was obtained.

[0043] Taking the preparation of HPV6L1-VLP antigen as an example, the process is as follows:

[0044] The nucleotide sequence of the artificially synthesized truncated HPV6L1 is (SEQ ID NO.2). The DNA fragment of HPV6L1 was first amplified by PCR. The L1 gene PCR fragment containing NdeI and Xho1 restriction sites and the recombinant vector pKL1 were double-digested with NdeI / Xho1 and the digested fragments were recovered. Then, the recovered gene fragment was ligated with the vector fragment pKL1 containing the corresponding sticky ends using T4 DNA ligase, and the ligation was carried out at 16°C for 10 to 15 hours.

[0045] The ligation system is as follows: 6 μl of pKL1 vector fragment, 2 μl of HPV6L1 gene fragment, 1 μl of T4 DNA ligase, and 1 μl of T4 DNA Ligase buffer. After the ligation reaction, the ligation product is transformed into E. coli DH5α for recombinant screening. The screened monoclonal colonies are expanded and the plasmid is extracted, and then sequenced and verified to obtain the recombinant expression vector pKL1-HPV6L1.

[0046] The recombinant vector with the correct sequencing results was transformed into Escherichia coli BL21 host cells, and used as an engineered bacterium to express the HPV6L1 protein. The engineering bacterium culture medium was 2YT medium (10g / L tryptic peptone; 5g / L yeast powder; 10g / L NaCl). Pick a single bacterial plaque containing the recombinant plasmid in 10ml 2YT medium (containing 100μg / ml ampicillin), 230 rpm, 37℃ shaking culture overnight. Transfer 5ml of overnight bacteria to 500ml (containing 100μg / ml ampicillin) 2YT liquid culture medium, shake culture at 37℃ until the recombinant engineering bacteria grow to OD600nm≈0.4~1, add IPTG induction with a final concentration of 0.2mM, and induce the expression of the recombinant protein for more than 6h at 28℃. Collect and crush the bacteria and collect the supernatant.

[0047] Affinity chromatography of recombinant protein with GST tag: 5 ml of GST agarose affinity chromatography medium was loaded into the affinity column, and the column was equilibrated with buffer L (pH 8.0, 50 mM Tris, 200 mM NaCl, 5 mM DTT), and then the protein solution with GST tag was loaded. After completion, 3C enzyme was added for enzymatic digestion. After enzymatic digestion, the column was washed with buffer L until no protein flowed out, and the eluate was collected. The affinity chromatography was completed.

[0048] Molecular sieve chromatography purification: The HPV6L1 pentamer protein collected after affinity chromatography purification in the previous step is further purified. The HPV6L1 pentamer protein can be first collected by ion exchange chromatography, or further molecular sieve chromatography can be performed directly using Superdex200 (produced by GE) molecular sieve gel filtration medium without ion exchange chromatography step. The molecular sieve mobile phase is pH 8.0, 10mM Tris, 100mM NaCl, and the sample corresponding to the ultraviolet absorption peak of the HPV6L1 pentamer protein is collected.

[0049] After the above steps, the HPV6L1 pentamer protein with a purity of more than 98% and an average particle size of 10-15nm PdI<0.1 can be purified. After each type of L1 pentamer protein is mixed with the assembly solution (pH 4.5-5.5, NaCl concentration 2.0-5.0M), the HPV6L1-VLP protein with an average particle size of 45-60nm PdI<0.1 is further assembled.

[0050] The HPV6L1 pentameric protein obtained above self-assembles to form VLPs. After being placed to stabilize, the particle size and particle size distribution are measured using a Malvern ZetasizerNanoZS dynamic light scattering particle size analyzer (the particle size distribution coefficient PdI value is an index of particle size dispersion, less than 0.05 is a highly uniform sample; 0.05-0.1 is a quasi-uniform sample, 0.1-0.3 is a sample with poor uniformity, and greater than 0.3 is an inhomogeneous sample). The HPV6L1 pentameric protein is assembled to obtain VLPs with uniform particle size (PdI < 0.05).

[0051] HPV11L1-VLP, HPV16L1-VLP, HPV18L1-VLP, HPV31L1-VLP, HPV33L1-VLP, HPV45L1-VLP, HPV52L1-VLP and HPV58L1-VLP were prepared according to the above method, the only difference being that the nucleotide sequence of the synthesized human papillomavirus coat protein L1 was different.

[0052] Example 2: Experiment on the application scope of adjuvant

[0053] Preparation process of aluminum hydroxide adjuvant: aluminum chloride and sodium hydroxide are selected. 3+ and OH - The reaction is carried out at a molar ratio of 1:(2-5), without adding sodium chloride to the reaction system, and the reaction is carried out at room temperature or 50-70°C, and then the temperature is raised to 80°C to maintain the pH at about 6.0-7.0 for 50-70 minutes. After the adjuvant is placed in the tank, it is washed with 0.9% sodium chloride solution (resuspended mass ratio ≥6.5:1) for more than 4 times. The washed adjuvant is resuspended with injection water or 0.9% sodium chloride solution according to the feed amount to an aluminum content of 7 mg / ml, and then dispensed into 500ml blue-capped reagent bottles (about 400ml / bottle) and sterilized by high-pressure steam at 121°C for 20 minutes.

[0054] The aluminum hydroxide adjuvant obtained in this example was fully tested according to the pharmacopoeia method, and the test results all met the requirements of the corresponding standards.

[0055] Among them, the test results of the aluminum hydroxide adjuvant used in the following experimental studies are shown in the following table.

[0056] Table 1 Test results of aluminum hydroxide adjuvant obtained in this example

[0057] Test items Test results Aluminum content 6.99mg / ml pH 6.68 Particle size 7.96μm Osmolarity 292mOsmol / kg Endotoxin content Less than 5.0EU / mg Sterility test Sterile growth

[0058] Next, the immune effect of the aluminum hydroxide adjuvant obtained above was further studied. Using the commercially available aluminum hydroxide adjuvant (Alhydrogel) produced by Brenntag, Denmark as a control, HPV16 was used as an example for explanation, and experiments were also conducted on other types to obtain corresponding results.

[0059] Adjuvant preparation: Prepare 0.9% sodium chloride solution with injection water, filter and sterilize with 0.22μm sterile microporous filter or autoclave; dilute each adjuvant with 0.9% sodium chloride solution to an aluminum content of 2mg / ml, and further dilute the adjuvant to 400μg / ml, 200μg / ml and 100μg / ml intermediate samples on this basis, which are used to prepare each immunization sample. Each batch of adjuvant dilution was mixed with 0.5mL of 20μg / ml and 4μg / ml HPV16L1-VLP protein dilution in a ratio of 1:1 to prepare the final immunization sample. Each group of experimental mice was immunized by leg muscle injection, 100μL / mouse, and the mouse immunization results are as follows.

[0060] Table 2 Immune effect of aluminum hydroxide adjuvant

[0061]

[0062] It can be seen from the results in Table 2 that, based on the HPV16 neutralizing antibody levels at different time points, the neutralizing antibody level induced by the aluminum hydroxide adjuvant of the present application is higher than that of the commercially available adjuvant, and neutralizing antibodies are the key to the HPV vaccine's immune protection effect. Therefore, the immune enhancement effect of the aluminum hydroxide adjuvant of the present application is not lower than that of the commercially available aluminum hydroxide adjuvant, and the aluminum hydroxide adjuvant of the present application also has a good immune enhancement effect at low antigen doses, and can also induce a high level of immune response faster in the early stage of immunization. The results of the neutralizing antibody levels of other types at different time points show that the neutralizing antibody level induced by the aluminum hydroxide adjuvant of the present application is slightly higher than that of the commercially available adjuvant, and has a good immune enhancement effect.

[0063] Example 3: Development of nine-valent HPV vaccine

[0064] 1. The steps for preparing the vaccine of the present invention are as follows:

[0065] 1) The various types of HPV L1 proteins obtained according to the method of Example 1 are purified and self-assembled in vitro to form VLPs, and the VLPs are further subjected to chromatography column liquid exchange: the liquid exchange chromatography column is fully equilibrated with a buffer solution (10-30 mM histidine-hydrochloric acid, 400-1500 mM NaCl, 0.001%-0.3% polysorbate 80, pH 5.5-6.5) in advance, and then the assembled VLPs are loaded onto the equilibrated chromatography column. After loading, the buffer solution is used for elution, and the components containing the target protein are collected. The collected components are sterilized and filtered through a sterile filter to obtain an HPVL1-VLP protein stock solution; the obtained HPVL1-VLP protein stock solution is stored below -65°C, or directly matured at 37°C for 12-36 hours to obtain a matured HPVL1-VLP protein stock solution;

[0066] 2) Using a buffer solution, dilute each type of matured HPVL1-VLP protein stock solution to the required concentration; finally, mix all types of VLP dilutions in proportion to obtain a nine-valent protein dilution solution, and sterilize it after filtering for later use;

[0067] 3) taking the prescribed amount of aluminum hydroxide adjuvant prepared according to Example 2, diluting and mixing, filtering through a capsule filter and setting aside;

[0068] 4) Take the required amount of the sample filtered by the nine-valent protein dilution solution and the sample filtered by the aluminum hydroxide adjuvant dilution solution and mix them until they are completely mixed to obtain the finished vaccine preparation.

[0069] Alternatively, each type of protein dilution is mixed with an aluminum hydroxide adjuvant dilution in proportion to prepare a monovalent adsorption product, and finally the required amount of each type of monovalent adsorption product is taken and mixed thoroughly to obtain a finished vaccine preparation.

[0070] 5) For finished vaccine preparations that are not used in time, the filling of finished products is also included: start the pre-filled syringe filling machine for filling, the filling speed is 30-40 rpm, fill and package at 0.55 ml / vial and add rubber stoppers, and store in a cold storage at 2-8°C for future use.

[0071] 2. Applicability of buffer system

[0072] This experiment involves the applicability of the buffer system. The VLP concentration in the experimental immunization preparations is 80μg / ml, and each of the nine monovalent HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58 types has 5 buffer system formulas, namely formula A, B, C, D, and E. The histidine-hydrochloric acid concentration of each formula is 10mM, and the polysorbate 80 concentration is 0.01%, among which formula A: pH5.5, sodium chloride concentration 327mM, formula B: pH6.0, sodium chloride concentration 154mM, formula C: pH6.0, sodium chloride concentration 327mM, formula D: pH6.0, sodium chloride concentration 500mM, formula E: pH6.5, sodium chloride concentration 327mM. The samples of the above prescriptions were placed at a high temperature of 37°C for 14 days; and a control sample of each formula placed at 4°C was set, with a total of 10 samples for each type and a total of 90 samples for nine types, and BALB / c mouse immunization experiments were carried out.

[0073] The results of neutralizing antibody titer determination in each group are as follows Figures 1 to 3 As shown, the results show that: the antibody levels induced by each type of VLP in vivo have no significant difference within the designed prescription range (data between groups were analyzed for multiple comparisons p>0.05), indicating that the vaccine of the present invention has good stability within this prescription range. After being placed at 37°C for 14 days, its immunogenicity did not change, further proving the good stability of the preparation prescription.

[0074] 3. Dosage study of antigen and aluminum adjuvant

[0075] A series of nine-valent HPV vaccines (see Table 3) were prepared with different antigen dose ratios and aluminum hydroxide adjuvant doses, which were used to immunize BALB / c mice to investigate immunogenicity. Specifically, the following steps were used: setting up a monovalent antigen immunization group to investigate immune interference; using Gardasil 9 as a control vaccine, setting up different aluminum adjuvant dose groups to determine the adjuvant dose in the vaccine; and setting up different antigen dose ratios to determine the antigen dose ratios of each type. Animal grouping information, antigen dose ratios, and aluminum adjuvant dose settings are shown in Table 3.

[0076] The immunization program used 0 and 4 weeks of immunization, and the neutralizing antibody titer was measured 4 weeks after the first immunization and 3 weeks after the second immunization. Inter-group comparison was performed to determine the dosage ratio of each type of antigen in the vaccine and the dosage of aluminum adjuvant.

[0077] Table 3 Antigen dose ratio and aluminum adjuvant dose study animal grouping table

[0078]

[0079] The geometric mean of neutralizing antibody detection 4 weeks after the first vaccination and 3 weeks after the second vaccination is shown in Table 4. The results show that:

[0080] (1) Immune interference

[0081] Using the same aluminum adjuvant dose, mice were immunized with monovalent antigens and nine-valent mixed antigens in Table 3 (same dose of antigens of the same type), and the difference in immune response levels between the two was compared to investigate immune interference. The results showed that the neutralizing antibody titers of all types of antigens prepared by the present invention were significantly higher than those of the nine-valent HPV vaccine when immunized in a monovalent form, and were all greater than the corresponding types of the control vaccine (Gardasil 9).

[0082] (2) Aluminum hydroxide adjuvant dosage

[0083] The fixed antigen dose is 1 / 20× of the human dose (for example, the human dose is 0.5ml, and 1 / 20 of the human dose (0.5ml) is used to immunize mice). Four aluminum hydroxide adjuvant doses are set: 37.5μg (1 / 20× of the nine-valent HPV vaccine), 0μg, 25μg, and 50μg. Mice are immunized (n=10 mice per group), and the differences between the aluminum hydroxide adjuvant doses are investigated, and compared with the control vaccine (Gardasil 9, the adjuvant is aluminum phosphate). As shown in Table 4, the neutralizing antibody level in the aluminum-free adjuvant group is significantly lower than that in the aluminum-containing adjuvant group, indicating that it is necessary to add aluminum hydroxide adjuvant to the vaccine. The aluminum hydroxide adjuvant has a significant effect on increasing the antigen immune response level. There is no significant difference in most types between the three doses of the aluminum-containing adjuvant group. Compared with the control vaccine, the addition of aluminum hydroxide adjuvant has a certain superiority. 37.5μg (corresponding to the human dose of 750μg) of aluminum hydroxide adjuvant can reach or approach immune saturation, and the immune response level is equivalent to that of the control vaccine.

[0084] (3) Antigen dosage ratio

[0085] By adjusting the HPV6 antigen to reduce 10μg each, HPV16, 18 antigens to reduce 20μg each; or adjusting the HPV16, 18 antigens to increase 20μg each, HPV31, 33, 45, 52, 58 antigens to increase 10μg each, we investigated whether reducing or increasing the dose of these types of antigens could significantly increase the immune response level of these types, so as to ensure that the vaccine effect is not lower than the marketed vaccine Gardasil 9. As shown in Table 4, compared with the initial antigen dose, increasing the dose of these types of antigens did not produce a significant immune enhancement effect, indicating that the initial dose ratio of the antigens in mice has reached immune saturation, and there is no need to increase the dose of these types of antigens.

[0086] Table 4 Summary of immune doses and neutralizing antibody geometric mean titers

[0087]

[0088] 4. Study on the particle size of antigen

[0089] According to the various types of L1-VLP antigens obtained in Example 1, antigens with different average particle sizes were prepared by adjusting different NaCl salt concentrations and pH according to the preparation process, and dynamic light scattering (DLS) was used to measure the antigen particle size. Each 0.5 ml of HPV6, 11, 16, 18, 31, 33, 45, 52, and 58 antigens contained 30μg, 40μg, 60μg, 40μg, 20μg, 20μg, 20μg, 20μg, 20μg and 0.75mg of the above-mentioned aluminum hydroxide adjuvant for mixed adsorption, and different nine-valent HPV vaccines were prepared, and Gardasil 9 and aluminum hydroxide adjuvant were used as controls. BALB / c mice were immunized at 1 / 50× human doses to compare the immunogenicity differences of each nine-valent HPV vaccine. The immunization program used 0 and 4 weeks of immunization, and the neutralizing antibody titer was determined 3 weeks after the second immunization. The results are shown in Table 5. Comparisons between groups were made to optimize and control the particle size of each type of antigen in the vaccine.

[0090] Table 5 Summary of geometric mean titers of neutralizing antibodies

[0091]

[0092] From the results in Table 5, it can be seen that the vaccine of the present invention comprising various types of antigens in the selected particle size range can achieve a good immune response level, and the immune response level to types 6, 11, 16, 18, 31, 45 and 58 is higher than that of the marketed Gardasil 9.

[0093] Through the above research, the present invention finally determined that the key parameters for the preparation of the optimal nine-valent HPV vaccine are as follows:

[0094] Each 0.5 ml vaccine contains 30 μg of HPV6, 40 μg of HPV11, 60 μg of HPV16, 40 μg of HPV18, 20 μg of HPV31, 20 μg of HPV33, 20 μg of HPV45, 20 μg of HPV52, 20 μg of HPV58 antigens, and 0.75 mg of aluminum hydroxide adjuvant; the particle size of HPV6 antigen is 45-60 nm, the particle size of HPV11 antigen is 4 The particle size of HPV16 antigen is 45-60nm, the particle size of HPV18 antigen is 55-75nm, the particle size of HPV31 antigen is 55-75nm, the particle size of HPV33 antigen is 45-60nm, the particle size of HPV45 antigen is 50-65nm, the particle size of HPV52 antigen is 50-65nm, and the particle size of HPV58 antigen is 45-60nm; the particle size of aluminum hydroxide adjuvant is 3-10μm.

[0095] Embodiment 4

[0096] The immunogenicity of the nine-valent HPV vaccine of the present invention was evaluated in 120 healthy women aged 18-26 years old.

[0097] The preparation process of the nine-valent HPV vaccine described in this embodiment is as follows:

[0098] 1) Purifying various types of HPVL1 proteins obtained according to the method of Example 1 and forming VLPs through in vitro self-assembly, and further subjecting the VLPs to chromatography column liquid exchange and sterilization filtration to obtain HPVL1-VLP protein stock solution (for specific operations, refer to step 1 of Example 3); subjecting the obtained HPVL1-VLP protein stock solution to maturation treatment at 37° C. for 12 to 36 h to obtain a matured HPVL1-VLP protein stock solution;

[0099] 2) using a buffer solution to dilute each type of matured HPVL1-VLP protein stock solution to the optimal concentration obtained in the above study; finally, all types of VLP dilutions were mixed according to the optimal ratio obtained in the above study to obtain a nine-valent protein dilution solution, which was sterile filtered for later use;

[0100] 3) diluting and mixing the aluminum hydroxide adjuvant prepared according to Example 2 in the optimal prescription amount of the above study, filtering through a capsule filter and setting aside;

[0101] 4) Take the required amount of the 9-valent protein dilution filtered sample and the aluminum hydroxide adjuvant dilution filtered sample and mix them until they are completely mixed to obtain the finished vaccine preparation. The parameters of the finished vaccine preparation are the same as those in Example 3, and the optimal key parameters for the preparation of the 9-valent HPV vaccine are finally determined.

[0102] 120 subjects were divided into three dose groups: low, medium and high. The dose was implemented step by step from low dose to high dose according to the principle of sequence. There were 40 subjects in each group (including 10 positive controls). After the subjects were enrolled, they were randomly vaccinated with the test vaccine or the positive control vaccine at a ratio of 3:1. First, the 40 subjects in the low-dose group were enrolled in four batches of 5, 10, 10 and 15 people, and the first low-dose test vaccine or positive control vaccine was randomly vaccinated at a ratio of 3:1. The interval between each batch of enrollment was no less than 3 days. Secondly, under the condition that the 40 subjects in the low-dose group had been enrolled for a full week and the safety was confirmed, 40 subjects in the medium-dose group were enrolled, and the batches and time intervals were the same as those in the low-dose group. Finally, under the condition that the 40 subjects in the medium-dose group had been enrolled for a full week and the safety was confirmed, 40 subjects in the high-dose group were enrolled, and the batches and time intervals were the same as those in the low-dose group.

[0103] Among them, the vaccines administered in the low-dose group were: V_220: ​​HPV-6 / 11 / 16 / 18 / 31 / 33 / 45 / 52 / 58 (20μg, 40μg, 40μg, 20μg, 20μg, 20μg, 20μg, 20μg, 20μg, 20μg);

[0104] The vaccines administered in the medium dose group were: V_270: HPV-6 / 11 / 16 / 18 / 31 / 33 / 45 / 52 / 58

[0105] (30μg, 40μg, 60μg, 40μg, 20μg, 20μg, 20μg, 20μg, 20μg);

[0106] The vaccines administered in the high-dose group were: V_360: HPV-6 / 11 / 16 / 18 / 31 / 33 / 45 / 52 / 58

[0107] (30μg, 40μg, 80μg, 60μg, 30μg, 30μg, 30μg, 30μg, 30μg).

[0108] The other parameters of the vaccine in the low-dose group, medium-dose group and high-dose group refer to the research results obtained in the above Example 3.

[0109] The mean age (standard deviation) of the subjects in the low-dose group (N=30) was 22.60 (2.18) years old, with a minimum age of 18 years old and a maximum age of 26 years old; the mean age (standard deviation) of the subjects in the medium-dose group (N=30) was 22.93 (2.36) years old, with a minimum age of 18 years old and a maximum age of 26 years old; the mean age (standard deviation) of the subjects in the high-dose group (N=30) was 22.73 (1.72) years old, with a minimum age of 19 years old and a maximum age of 26 years old; the mean age (standard deviation) of the subjects in the positive control group (N=30) was 22.73 (2.41) years old, with a minimum age of 18 years old and a maximum age of 26 years old.

[0110] The immunization schedule for this trial is based on the 0, 2, and 6 month immunization schedule, with three injections. The injection site is the deltoid muscle on the outer side of the upper arm, with the deltoid muscle of the non-dominant hand being the preferred site; vaccination in the buttocks and other sites is prohibited.

[0111] Immunogenicity results are usually evaluated by calculating the geometric mean titres (GMT), geometric mean intensity (GMI) and antibody positive conversion rate of neutralizing antibodies and IgG antibodies in different groups and their 95% CI.

[0112] The immunogenicity results in Tables 6 and 7 show that the positive conversion rates of binding antibody levels and neutralizing antibody levels were all 100%. For subjects with negative antibodies before immunization and based on PPS analysis, the binding antibody GMT and GMI of the HPV18 medium-dose group were higher than those of the low-dose group, high-dose group and control group 3 months after immunization (P<0.05), and the HPV58 high-dose group was higher than the control group (P<0.05); the neutralizing antibody GMT and GMI of the HPV33 low-dose group and high-dose group were higher than the control group (P<0.05) 3 months after immunization, and the HPV52 high-dose group was higher than the medium-dose group (P<0.05). There were statistical differences in the binding antibody GMT and GMI among the HPV type groups 7 months after immunization, and the medium and high-dose groups were generally higher; the neutralizing antibody GMT and GMI of the HPV33 low-dose group, medium-dose group and high-dose group were higher than the control group 7 months after immunization (P<0.05).

[0113] Table 6 Geometric mean antibody titer and positive conversion rate of neutralizing antibodies against HPV6 / 11 / 16 / 18 / 31 / 33 / 45 / 52 / 58 in different groups

[0114]

[0115]

[0116]

[0117] Among them, GMT: mean antibody titer calculated for all subjects; 95% CI: 95% confidence interval; N: number of subjects in each group; LL: lower limit; UL: upper limit; RRE: before vaccination; M3 3 months after vaccination; M7 7 months after vaccination;

[0118] V_220: ​​HPV-6 / 11 / 16 / 18 / 31 / 33 / 45 / 52 / 58 (20μg, 40μg, 40μg, 20μg, 20μg, 20 μg, 20μg, 20μg, 20μg); V_270: HPV-6 / 11 / 16 / 18 / 31 / 33 / 45 / 52 / 58 (30μg, 40μg ,60μg,40μg,20μg,20μg,20μg,20μg,20μg); V_360: HPV-6 / 11 / 16 / 18 / 31 / 33 / 45 / 52 / 58 (30μg, 40μg, 80μg, 60μg, 30μg, 30μg, 30μg, 30μg, 30μg); Gardasil 9: Jiadaxiu, HPV-6 / 11 / 16 / 18 / 31 / 33 / 45 / 52 / 58 (30μg, 40μg, 60μg, 40μg, 20μg, 20μg, 20μg, 20μg, 20μg).

[0119] Table 7 Comparison of binding antibodies and neutralizing antibodies of different types after immunization in different groups

[0120]

[0121] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned various embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A nine-valent human papillomavirus vaccine comprising truncated L1-VLP antigens of HPV6, 11, 16, 18, 31, 33, 45, 52, and 58, and an aluminum hydroxide adjuvant; in, The weight ratio of HPV6, 11, 16, 18, 31, 33, 45, 52, 58 L1-VLP 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); The particle size of each type of L1-VLP antigen is 45 to 80 nm, and the particle size of the aluminum hydroxide adjuvant is 3 to 10 μm; The amino acid sequences of the truncated L1 proteins of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58 are shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, and SEQ ID NO.17, respectively.

2. The nine-valent human papillomavirus vaccine according to claim 1, characterized in that The weight ratio of HPV6, 11, 16, 18, 31, 33, 45, 52, and 58 L1-VLP antigens is 1.5:2:3:2:1:1:1:1:

1.

3. The nine-valent human papillomavirus vaccine according to claim 2, characterized in that: The dosage of each antigen in each 0.5 ml is 10-100 μg, and the dosage of aluminum hydroxide adjuvant is 0.5-1.0 mg.

4. The nine-valent human papillomavirus vaccine according to claim 3, characterized in that The HPV6, 11, 16, 18, 31, 33, 45, 52, and 58 L1-VLP antigens were 30μg, 40μg, 60μg, 40μg, 20μg, 20μg, 20μg, 20μg, and 20μg, respectively; the dosage of aluminum hydroxide adjuvant was 0.75mg.

5. The nine-valent human papillomavirus vaccine according to claim 4, characterized in that HPV6, 11, 16, 18, 31, 33, 45, 52, 58 L1-VLP antigens were expressed in the E. coli system.

6. The nine-valent human papillomavirus vaccine according to claim 1, characterized in that The particle size of HPV6L1-VLP antigen is 45-60nm, the particle size of HPV11L1-VLP antigen is 45-60nm, the particle size of HPV16L1-VLP antigen is 45-60nm, the particle size of HPV18L1-VLP antigen is 55-75nm, the particle size of HPV31L1-VLP antigen is 55-75nm, the particle size of HPV33L1-VLP antigen is 45-60nm, the particle size of HPV45L1-VLP antigen is 50-65nm, the particle size of HPV52L1-VLP antigen is 50-65nm, and the particle size of HPV58L1-VLP antigen is 45-60nm; the particle size of aluminum hydroxide adjuvant is 5-8μm.

7. The nine-valent human papillomavirus vaccine according to claim 1, characterized in that Each type of L1-VLP protein antigen is assembled from each type of L1 pentamer protein, and the particle size of the L1 pentamer protein is 10 to 15 nm.

8. The nine-valent human papillomavirus vaccine according to claim 7, characterized in that The particle size of HPV6L1 pentamer protein is 12-15nm, the particle size of HPV11L1 pentamer protein is 12-15nm, the particle size of HPV16L1 pentamer protein is 11-14nm, the particle size of HPV18L1 pentamer protein is 11-14nm, the particle size of HPV31L1 pentamer protein is 12-15nm, the particle size of HPV33L1 pentamer protein is 12-15nm, the particle size of HPV45L1 pentamer protein is 12-15nm, the particle size of HPV52L1 pentamer protein is 12-15nm, and the particle size of HPV58L1 pentamer protein is 11-14nm.

9. The nine-valent human papillomavirus vaccine according to claim 1, characterized in that The preparation method of the aluminum hydroxide adjuvant is as follows: aluminum chloride and sodium hydroxide are taken according to Al 3+ and OH - The reaction is carried out at a molar ratio of 1:(2-5), without adding sodium chloride to the reaction system, and the reaction is carried out at room temperature or 50-70°C, and then the temperature is raised to 80°C to maintain the pH value at 6.0-7.0 for 50-70 minutes. After the reaction is carried out in a tank, the aluminum hydroxide adjuvant is washed more than 4 times with a sodium chloride solution having a resuspension mass ratio of ≥6.5:

1.

10. The nine-valent human papillomavirus vaccine according to claim 9, characterized in that The washed aluminum hydroxide adjuvant is resuspended with injection water or 0.9% sodium chloride solution according to the feed amount until the aluminum content reaches 7 mg / ml, then packaged and sterilized by high-pressure steam at 121°C for 20 min.

11. A method for preparing a nine-valent human papillomavirus vaccine according to any one of claims 1 to 10, comprising the following steps: (1) Purified HPV L1 proteins of various types are self-assembled in vitro to form VLPs, and the VLPs are further subjected to column chromatography, liquid exchange, and sterile filtration to prepare protein stock solutions, which are then diluted to the desired concentrations using buffer solutions; (2) Mixing the protein dilutions of each type with the aluminum hydroxide adjuvant dilutions in proportion to prepare monovalent adsorption products, and finally taking the required amount of each type of monovalent adsorption products and mixing them thoroughly to obtain the finished vaccine preparation; Or all types of protein stock solution dilutions are first mixed in proportion to prepare nine-valent protein dilutions, and then the required amount of nine-valent protein dilutions are taken and thoroughly mixed with aluminum hydroxide adjuvant dilutions to obtain the finished vaccine preparation.

12. The method for preparing the nine-valent human papillomavirus vaccine according to claim 11, characterized in that: It also includes filling of finished products and starting the prefilled syringe filling machine for filling.

13. The method for preparing the nine-valent human papillomavirus vaccine according to claim 12, characterized in that: The filling speed is 30-40 rpm. Fill and package and add rubber stoppers, and store in a cold storage at 2-8℃ for future use.

14. Use of the nine-valent human papillomavirus vaccine according to any one of claims 1 to 10 in the preparation of a medicament for preventing diseases caused by human papillomavirus.

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