Nine-valent human papillomavirus vaccine and use thereof

Abstract

Provided are a nine-valent human papillomavirus vaccine and use thereof. The nine-valent human papillomavirus vaccine comprises HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58 type truncated L1-VLP antigens. All antigen components in the provided nine-valent HPV vaccine are derived from L1, a major capsid protein of the HPV, and truncated. The yield of the L1 protein is improved by means of a truncated L1 protein expressed by an Escherichia coli system, thereby reducing the production cost of the vaccine. Optimizing the particle size of each type of VLP antigens and the particle size of the aluminum hydroxide adjuvant, combined with control over the ratio of each type of VLP antigens to the adjuvant and the buffer system and the like, enables the obtained nine-valent HPV vaccine to induce a high-level immune response, especially to demonstrate a significant clinical effect.

Description

Nine-valent human papillomavirus vaccine and its application 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 infects the squamous epithelial cells of the human epidermis and mucous membranes. The relationship between HPV infection and cervical cancer was first proposed in 1974, and it was ultimately proven that HPV infection is the main cause of cervical cancer. HPV infection is the most common viral infection of the reproductive tract. In humans, HPV infection 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. Currently, more than 200 types of HPV have been identified, which can be divided into low-risk and high-risk types based on their carcinogenicity. Low-risk HPV types mainly include HPV6 / 11 / 30 / 42 / 43 / 44 / 61, among others. Among them, 90% of genital warts are caused by infection with HPV6 and 11. The World Health Organization (WHO) International Agency for Research on Cancer (IARC) The latest definition of high-risk HPV by the International Institute for Cancer (IARC) includes 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 (related to age), 51% of penile cancer and 13% to 60% of oropharyngeal cancer.

[0003] HPV vaccination is the most economical and effective means of preventing persistent HPV infection and related diseases. Currently, only one nine-valent HPV vaccine is commercially available domestically and internationally: 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 in eukaryotic cells (Saccharomyces cerevisiae). The eukaryotic cell production process suffers from the problem of disassembly and reassembly of VLPs, resulting in low yield and high cost.

[0004] In addition, in the development process of vaccine formulations, it is very important and challenging to ensure the stability of proteins. This is because, as a biological macromolecule, the stability of proteins depends on the integrity of more complex higher-level structures (secondary structure, tertiary structure and quaternary structure), and these higher-level structures are easily disturbed and destroyed by the external environment during protein purification, storage and formulation production. After the protein is subjected to various environmental stresses (temperature, storage time, vibration, freeze-thaw, freeze-drying), a series of chemical amino acid residue degradations will occur, such as asparagine deamination, methionine oxidation and peptide bond hydrolysis; in addition, physical aggregation or disaggregation is also one of the challenges faced by protein biological products, such as partial structural unfolding, protein aggregation or adsorption and protein precipitation. Such chemical or physical changes in the antigen protein will lead to a reduction in the immunogenicity of the antigen. Summary of the Invention

[0005] Based on the needs of the existing technology, the present invention has obtained a preventive nine-valent human papillomavirus vaccine through in-depth research, covering 2 types of antigens that are clearly related to genital warts and 7 types that are clearly related to cervical cancer (HPV6, 11 and HPV16, 18, 31, 33, 45, 52, 58).

[0006] 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 adjuvant;

[0007] 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); 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;

[0008] More specifically, the dosage of each antigen per 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.

[0009] More specifically, HPV6, 11, 16, 18, 31, 33, 45, 52, and 58L1-VLP antigens are expressed in an Escherichia coli system; preferably, each type of VLP antigen is matured at 30°C to 40°C for 6 to 48 hours to obtain mature VLP proteins, which are then used to prepare the vaccine; more preferably, the maturation treatment is performed at 35°C to 38°C for 12 to 36 hours to obtain mature VLP proteins; and the vaccine uses a histidine-hydrochloric acid buffer system.

[0010] The present inventors found in their research that when using a histidine-hydrochloric acid buffer system, the histidine system is prone to VLP disaggregation, resulting in some unassembled pentamers; by maturing the human papillomavirus virus-like particles (VLPs) assembled from HPV L1 protein pentamers at 30°C to 40°C for 6 to 48 hours, preferably at 35°C to 38°C for 12 to 36 hours, and more preferably at 37°C for 24 hours; the stability and immunogenicity of the vaccine preparation are improved.

[0011] 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 the HPV6 L1-VLP antigen is 45 to 60 nm, the particle size of the HPV11 L1-VLP antigen is 45 to 60 nm, the particle size of the HPV16 L1-VLP antigen is 45 to 60 nm, the particle size of the HPV18 L1-VLP antigen is 55 to 75 nm, the particle size of the HPV31 L1-VLP antigen is 55 to 75 nm, the particle size of the HPV33 L1-VLP antigen is 45 to 60 nm, the particle size of the HPV45 L1-VLP antigen is 50 to 65 nm, the particle size of the HPV52 L1-VLP antigen is 50 to 65 nm, and the particle size of the HPV58 L1-VLP antigen is 45 to 60 nm.

[0012] More specifically, the adjuvant is an aluminum hydroxide adjuvant; preferably, the particle size of the aluminum hydroxide adjuvant is 3 to 10 μm, more preferably 5 to 8 μm.

[0013] 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.

[0014] 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.

[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 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 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 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 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 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 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 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 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 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, an osmotic pressure regulator and a surfactant are added to the histidine-hydrochloric acid buffer system. 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 5-7, more preferably 5.5-6.5; the histidine-hydrochloric acid concentration is 5-20 mM, preferably 10-15 mM; the sodium chloride concentration is 154-500 mM, more preferably 320-400 mM; the concentration of polysorbate 80 is 0.005%-0.025%, preferably 0.008%-0.02%, more preferably 0.01%.

[0019] 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°C-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 reaction is carried out 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.

[0020] 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.

[0021] In a specific embodiment, purified HPV L1 proteins of various types are self-assembled in vitro to form VLPs, which are then further subjected to column chromatography, liquid exchange, and sterile filtration to prepare a protein stock solution, preferably matured at 30°C to 40°C for 6 to 48 hours to obtain a matured VLP protein stock solution, which is then diluted to the desired concentration using a buffer solution; then, the protein dilutions of various types are mixed with aluminum hydroxide adjuvant dilutions in proportion to prepare monovalent adsorption products, and finally, the desired amount of each type of monovalent adsorption product is thoroughly mixed to obtain a finished vaccine preparation;

[0022] 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 the finished vaccine preparation;

[0023] Furthermore, the finished product is packaged, specifically using a prefilled syringe filler or a vial filler. Preferably, the filling speed is 30-40 rpm, and more preferably, 0.55 ml per vial is filled and stoppered. After packaging, the finished vaccine preparation is stored in a cold storage at 2°C to 8°C until ready for use.

[0024] 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.

[0025] The present invention also provides a method for storing the vaccine, which is to store it at 2°C to 8°C.

[0026] Finally, the present invention provides use of the vaccine in preparing medicines for preventing or treating diseases caused by human papillomavirus.

[0027] 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). The aluminum adjuvant used in the nine-valent HPV vaccine of the present invention is aluminum hydroxide, which has a special advantage. The various types of proteins in the vaccine of the present invention are expressed and prepared after truncation of the N / C terminus, 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 for protein expression and preparation. 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.

[0028] 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 major 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 improves the stability and immunogenicity of the vaccine preparation by optimizing the particle size of each type of VLP antigen and the particle size of the aluminum hydroxide adjuvant, combining the control of the ratio of each type of VLP antigen and adjuvant such as aluminum hydroxide adjuvant, or adopting a buffer system containing histidine, and by maturing each type of VLP protein at 30°C to 40°C for 6 to 48 hours. The resulting nine-valent HPV vaccine induces a high level of immune response, especially showing significant effects in clinical practice, which is better than the effects of currently available related vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1. Analysis of neutralizing antibodies against HPV11, HPV16, and HPV18.

[0030] Figure 2. Analysis of neutralizing antibodies against HPV33, HPV58 and HPV6.

[0031] Figure 3. Analysis of neutralizing antibodies against HPV31, HPV45 and HPV52.

[0032] Among them, in Figures 1 to 3, A-4 represents that each type of antigen is placed at 4°C in prescription A, B-4 represents that each type of antigen is placed at 4°C in prescription B, C-4 represents that each type of antigen is placed at 4°C in prescription C, D-4 represents that each type of antigen is placed at 4°C in prescription D, and E-4 represents that each type of antigen is placed at 4°C in prescription E; A-37-14d represents that each type of antigen is placed at 37°C in prescription A for 14 days, B-37-14d represents that each type of antigen is placed at 37°C in prescription B for 14 days, C-37-14d represents that each type of antigen is placed at 37°C in prescription C for 14 days, D-37-14d represents that each type of antigen is placed at 37°C in prescription D for 14 days, and E-37-14d represents that each type of antigen is placed at 37°C in prescription E for 14 days.

[0033] Figure 4. Analysis of immunogenicity of nine-valent HPV vaccines placed at high temperature. Among them, 16-4 represents type 16 antigen placed at 4°C, 16-37 / 7d represents type 16 antigen placed at 37°C for 7 days, and 16-37 / 14d represents type 16 antigen placed at 37°C for 14 days; 18-4 represents type 18 antigen placed at 4°C, 18-37 / 7d represents type 18 antigen placed at 37°C for 7 days, and 18-37 / 14d represents type 18 antigen placed at 37°C for 14 days; 58-4 represents type 58 antigen placed at 4°C. ℃, 58-37 / 7d means 58 antigen is placed at 37℃ for 7 days, 58-37 / 14d means 58 antigen is placed at 37℃ for 14 days; 6-4 means 6 antigen is placed at 4℃, 6-37 / 7d means 6 antigen is placed at 37℃ for 7 days, 6-37 / 14d means 6 antigen is placed at 37℃ for 14 days; 11-4 means 11 antigen is placed at 4℃, 11-37 / 7d means 11 antigen is placed at 37 ℃ for 7 days, 11-37 / 14d means type 11 antigen is placed at 37℃ for 14 days; 31-4 means type 31 antigen is placed at 4℃, 31-37 / 7d means type 31 antigen is placed at 37℃ for 7 days, 31-37 / 14d means type 31 antigen is placed at 37℃ for 14 days; 33-4 means type 33 antigen is placed at 4℃, 33-37 / 7d means type 33 antigen is placed at 37℃ for 7 days, 33-37 / 14d means type 31 antigen is placed at 37℃ for 14 days Table 33 type antigen is placed at 37℃ for 14 days; 45-4 represents type 45 antigen placed at 4℃, 45-37 / 7d represents type 45 antigen placed at 37℃ for 7 days, 45-37 / 14d represents type 45 antigen placed at 37℃ for 14 days; 52-4 represents type 52 antigen placed at 4℃, 52-37 / 7d represents type 52 antigen placed at 37℃ for 7 days, 52-37 / 14d represents type 52 antigen placed at 37℃ for 14 days.

[0034] Figure 5. In vitro relative potency of samples from different time points after maturation treatment of HPV nine-valent L1-VLP protein solutions of various types.

[0035] Figure 6. Immunogenicity of immature (marked as "unripened" in the figure) and mature (matured for 24 hours, marked as "ripened" in the figure) samples of HPV nine-valent L1-VLP protein solutions 2 weeks after the first immunization.

[0036] Figure 7. Immunogenicity of immature (marked as "unripened" in the figure) and mature (matured for 24 hours, marked as "ripened" in the figure) samples of HPV nine-valent L1-VLP protein solutions 4 weeks after the first immunization. DETAILED DESCRIPTION

[0037] The present invention is described below through specific embodiments in order to better understand the present invention, but it does not constitute a limitation of the present invention.

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

[0039] The present invention studies and truncates each type of L1 protein based on the wild-type sequence to remove the positively charged portion, thereby obtaining optimized L1 proteins of each type. The specific L1 proteins used in this example are as follows:

[0040] 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, HPV6L1-VLP protein with an average particle size of 45-60 nm and a PdI < 0.1 was obtained.

[0041] The truncated HPV11L1 sequence, 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, HPV11L1-VLP protein with an average particle size of 45-60 nm and a PdI < 0.1 was obtained.

[0042] The truncated HPV16L1 sequence, 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 45-60 nm and a PdI < 0.1 was obtained.

[0043] The truncated HPV18L1 sequence, 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, HPV18L1-VLP protein with an average particle size of 55-75 nm and PdI < 0.1 was obtained.

[0044] The truncated HPV31L1 sequence, 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, HPV31L1-VLP protein with an average particle size of 55-75 nm and PdI < 0.1 was obtained.

[0045] The truncated HPV33L1 sequence, 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, HPV33 L1-VLP protein with an average particle size of 45-60 nm and PdI < 0.1 was obtained.

[0046] The truncated sequence of 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, HPV45L1-VLP protein with an average particle size of 50-65 nm and PdI < 0.1 was obtained.

[0047] The truncated sequence of 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, HPV52L1-VLP protein with an average particle size of 50-65 nm and PdI < 0.1 was obtained.

[0048] The truncated sequence of 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, HPV58L1-VLP protein with an average particle size of 45-60 nm and PdI < 0.1 was obtained.

[0049] Taking the preparation of HPV6L1-VLP antigen as an example, the process is as follows:

[0050] The nucleotide sequence of the artificially synthesized truncated HPV6L1 is (SEQ ID NO.2). The HPV6L1 DNA fragment 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. The recovered gene fragment was then ligated with the vector fragment pKL1 containing the corresponding sticky ends using T4 DNA ligase. The ligation was carried out at 16°C for 10 to 15 hours.

[0051] The ligation system was as follows: 6 μl of the pKL1 vector fragment, 2 μl of the HPV6 L1 gene fragment, 1 μl of T4 DNA ligase, and 1 μl of T4 DNA Ligase buffer. After the ligation reaction, the ligation product was transformed into E. coli DH5α for recombinant screening. The selected monoclonal colonies were expanded and the plasmids were extracted and verified by sequencing to obtain the recombinant expression vector pKL1-HPV6L1.

[0052] The recombinant vector identified by sequencing was transformed into Escherichia coli BL21 host cells and used as a recombinant protein expression strain to express the HPV6 L1 protein. The recombinant protein was expressed in 2YT medium (10 g / L tryptone; 5 g / L yeast extract; 10 g / L NaCl). A single bacterial plaque containing the recombinant plasmid was placed in 10 ml of 2YT medium (containing 100 μg / ml ampicillin) and cultured overnight at 37°C with shaking at 230 rpm. Five ml of the overnight culture was transferred to 500 ml of 2YT liquid medium (containing 100 μg / ml ampicillin) and cultured at 37°C with shaking until the recombinant bacteria reached an OD600nm of ≈0.4-1. IPTG was then added to induce expression of the recombinant protein at a final concentration of 0.2 mM at 28°C for at least 6 hours. The cells were harvested and disrupted, and the supernatant was collected.

[0053] Affinity chromatography of GST-tagged recombinant protein: 5 ml of GST agarose affinity chromatography medium was loaded into the affinity column. The column was equilibrated with buffer L (pH 8.0, 50 mM Tris, 200 mM NaCl, 5 mM DTT), and the GST-tagged protein solution 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. The eluate was collected, and the affinity chromatography was completed.

[0054] 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 (GE) molecular sieve gel filtration medium without the ion exchange chromatography step. The molecular sieve mobile phase is pH 8.0, 10 mM Tris, 100 mM NaCl, and the sample corresponding to the ultraviolet absorption peak of the HPV6L1 pentamer protein is collected.

[0055] The above steps can be used to purify HPV6 L1 pentamer proteins with a purity exceeding 98%, an average particle size of 10-15 nm, and a PdI <0.1. Each type of L1 pentamer protein is mixed with an assembly solution (pH 4.5-5.5, NaCl concentration 2.0-5.0 M) and further assembled to produce HPV6 L1-VLP proteins with an average particle size of 45-60 nm and a PdI <0.1.

[0056] The HPV6L1 pentameric protein obtained above self-assembled to form VLPs. After being placed for stabilization, the particle size and particle size distribution were measured using a Malvern Zetasizer Nano ZS 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 was assembled to obtain VLPs with uniform particle size (PdI < 0.1).

[0057] HPV11 L1-VLP, HPV16 L1-VLP, HPV18 L1-VLP, HPV31 L1-VLP, HPV33 L1-VLP, HPV45 L1-VLP, HPV52 L1-VLP, and HPV58 L1-VLP were prepared according to the above method, except that the nucleotide sequence of the synthesized human papillomavirus coat protein L1 was different.

[0058] Example 2: Experiment on the application scope of adjuvant

[0059] Preparation process of aluminum hydroxide adjuvant: aluminum chloride and sodium hydroxide are selected. 3+ and OH - The reaction is carried out in a molar ratio of 1:(2-5), without adding sodium chloride to the reaction system. After the reaction at room temperature or 50°C to 70°C, 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 by 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 divided into 500ml blue-capped reagent bottles (about 400ml / bottle) and sterilized by high-pressure steam at 121°C for 20 minutes.

[0060] The aluminum hydroxide adjuvant obtained in this example was fully tested according to the pharmacopoeia method, and the test results were in compliance with the corresponding standard requirements.

[0061] The test results of the aluminum hydroxide adjuvant used in the following experimental studies are shown in the following table.

[0062] Table 1 Test results of aluminum hydroxide adjuvant obtained in this example

[0063] 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, the following is an example of HPV16. Other types of HPV were also tested and corresponding results were obtained.

[0064] Adjuvant preparation: Prepare a 0.9% sodium chloride solution with water for injection and sterilize by filtration through a 0.22μm sterile microporous filter or autoclaving. Each adjuvant was diluted with 0.9% sodium chloride solution to an aluminum content of 2mg / ml. The adjuvant was then further diluted to intermediate concentrations of 400μg / ml, 200μg / ml, and 100μg / ml for the preparation of the respective immunization samples. Each batch of adjuvant dilution was mixed with 0.5ml of 20μg / ml and 4μg / ml HPV16 L1-VLP protein dilutions at a 1:1 ratio to prepare the final immunization samples. Each group of experimental mice was immunized intramuscularly with 100μl per mouse. The immunization results are shown below.

[0065] Table 2 Immune effect of aluminum hydroxide adjuvant

[0066] As can be seen from the results in Table 2, based on the HPV16 neutralizing antibody levels at different time points, the neutralizing antibody level induced by the aluminum hydroxide adjuvant of the present invention is higher than that of the commercially available adjuvant. Neutralizing antibodies are the key to the immune protection effect of HPV vaccines. Therefore, the immune enhancement effect of the aluminum hydroxide adjuvant of the present invention is not lower than that of the commercially available aluminum hydroxide adjuvant. The aluminum hydroxide adjuvant of the present invention 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 invention is slightly higher than that of the commercially available adjuvant, and has a good immune enhancement effect.

[0067] Example 3: Study on the buffer system of the preparation

[0068] The present invention studies the effects of different buffering conditions on sodium chloride concentration, pH, NaCl concentration and their combination conditions (see Table 3) of the nine-valent HPV various types L1-VLP protein stock solution in the acetic acid-sodium acetate buffer system and the histidine-hydrochloric acid buffer system on the thermal stability of the HPV L1-VLP protein. The samples of the nine-valent HPV various types of protein stock solution were placed under different storage temperatures (4°C, 25°C, 37°C, -80°C (repeated freeze-thaw)) and the particle size and other stability indicators of the sample solution VLP under different conditions were detected. Dynamic light scattering (DLS) was used to detect the particle size, and the detection results of HPV6L1-VLP are shown in Tables 4 and 5. HPLC was used to detect the protein purity, and the detection results of HPV6L1-VLP are shown in Tables 6 and 7.

[0069] Table 3. Prescription of HPVL1-VLP protein stock solution buffer

[0070] The results in Tables 4 and 5 show that the particle sizes of the samples in the acetic acid-sodium acetate and histidine-hydrochloric acid buffer systems varied slightly. Overall, the HPV6 L1-VLP particles were smaller and more compact under the conditions of the histidine-hydrochloric acid buffer system. After three freeze-thaw cycles, the particle size and PdI of the samples in the acetic acid-sodium acetate buffer system were larger than those in the histidine-hydrochloric acid buffer system, indicating that the histidine-hydrochloric acid buffer system is more conducive to freeze-thaw storage of L1-VLP samples. After storage at 4°C, 25°C, and 37°C, the fluctuation in the particle size of HPV6 L1-VLP in the histidine-hydrochloric acid buffer system was smaller than that in the acetic acid-sodium acetate system, indicating that the histidine-hydrochloric acid buffer system is more conducive to maintaining the stability of HPV6 L1-VLP.

[0071] Table 4. Summary of HPV6L1-VLP particle size (nm) Note: Average is the average value of HPV6L1-VLP sample particle size measurement; SD is the standard deviation.

[0072] Table 5. Summary of results of HPV6L1-VLPPdI Note: Average is the average value of HPV6L1-VLP sample particle size measurement; SD is the standard deviation.

[0073] The results in Tables 6 and 7 show that the plate counts of HPV6L1-VLP assays under various formulation conditions in the histidine-hydrochloric acid buffer system were high and had small fluctuations, and the values ​​after the symmetry factor -1 were closer to zero, indicating that the histidine-hydrochloric acid buffer system formulations can maintain the stability of HPV6L1-VLP under freeze-thaw conditions. Overall, the fluctuations in the plate counts and symmetry factors of HPV6L1-VLP assays in the histidine-hydrochloric acid buffer system were smaller than those in the acetic acid-sodium acetate buffer system, and there were no significant differences between the formulations in the histidine-hydrochloric acid buffer system (except for H7), indicating that the histidine-hydrochloric acid buffer system is more conducive to the stability of HPV6L1-VLP.

[0074] Table 6. HPV6L1-VLP stock solution HPLC plate number USP

[0075] Table 7. HPV6 L1-VLP stock solution HPLC main peak (asymmetry factor)

[0076] Furthermore, research results on the formulations of HPV11 L1-VLP, HPV16 L1-VLP, HPV18 L1-VLP, HPV31 L1-VLP, HPV33 L1-VLP, HPV45 L1-VLP, HPV52 L1-VLP, and HPV58 L1-VLP protein stock solutions all demonstrated that a histidine-hydrochloric acid buffer system facilitated the stability of each type of HPV L1-VLP. In summary, the histidine-hydrochloric acid buffer system was used for the L1-VLP stock solutions of each type of nine-valent HPV.

[0077] Since the L1-VLP stock solutions of each type of nine-valent HPV vaccine use a histidine-hydrochloric acid buffer system, the nine-valent HPV vaccine formulation of the present invention also uses a histidine-hydrochloric acid buffer system. The main purpose of the formulation buffer system research is to determine the pH range and the preferred concentration of sodium chloride. The Tm value of each type of L1-VLP protein antigen under different formulations (different pH and sodium chloride concentration) was determined by differential scanning fluorimetry. The higher the Tm value, the more stable the antigen under this formulation. The results of the measurement of each type and formulation were comprehensively considered to select the parameter range that is conducive to antigen stability.

[0078] The results (as shown in Table 9) show that HPV types 6 and 11, both classified as belonging to the α10 genus, have high Tm values, while HPV types 18 and 45, both classified as belonging to the α7 genus, have high or intermediate Tm values. HPV types 16, 31, 33, 52, and 58, both classified as belonging to the α9 genus, have low or intermediate Tm values. These results suggest a correlation between Tm values ​​and the evolutionary relationships of these virus types.

[0079] From the results in Tables 8 and 9, it can be seen that when the pH of the nine-valent HPV vaccine preparation of the present invention is 5.5-6.5 and the sodium chloride concentration is in the range of 154-500 mM, the Tm values ​​of each type of VLP in the preparation prescription are relatively high (H1-H13). When the pH or sodium chloride concentration is too high or too low, the Tm values ​​of each type of VLP are reduced (H14-H17).

[0080] Based on the above results, it was preliminarily determined that the pH of the nine-valent HPV vaccine preparation of the present invention was 5.5-6.5, and the sodium chloride concentration was 154-500 mM.

[0081] Table 8. List of preparation buffer systems

[0082] Table 9. Summary of Tm values ​​of various types of VLPs in the established formulations determined by DSF method

[0083] Furthermore, the monovalent HPV6, HPV11, HPV 16, HPV18, HPV 31. Five prescriptions were set for each of the nine types of HPV33, HPV45, HPV52, and HPV58 (A, B, C, D, and E, the histidine concentration of each prescription was 10 mM, and the polysorbate 80 concentration was 0.01%, prescription A: pH 5.5, sodium chloride concentration 327 mM, prescription B: pH 6.0, sodium chloride concentration 154 mM, prescription C: pH 6.0, sodium chloride concentration 327 mM, prescription D: pH 6.0, sodium chloride concentration 500 mM, and prescription E: pH 6.5, sodium chloride concentration 327 mM), the VLP concentration of each type was 80 μg / ml, and immunization experiments were carried out on samples of each prescription preparation that were placed at 37°C for 14 days; and control samples of each prescription that were placed at 4°C; and samples of the first batch of filled nine-valent HPV vaccines that were placed at 4°C, 37°C for 7 days, and 37°C for 14 days.

[0084] The results of neutralizing antibody titer determination in each group are shown in Figures 1 to 3 below. The results indicate that there were no significant differences in the antibody levels induced by each type of VLP in vivo within the range of the designed prescription (there were no significant differences in the antibody levels induced by each type of VLP within the range of the designed prescription (data from each group were subjected to multiple comparison analysis, p>0.05), indicating that each type of VLP had good stability within this prescription range. After being stored at 37°C for 14 days, the immunogenicity of the VLP did not change, further demonstrating the good stability of the prescription.

[0085] On this basis, the present invention verifies the storage stability of the vaccine produced in the pilot test. The pH in the preparation prescription is 6.02 and the sodium chloride concentration is 327 mM. As can be seen from the results in Figure 4, after the finished product is placed at a high temperature of 37°C for 14 days, the neutralizing antibody titers of various types of VLPs do not change significantly, indicating that the immunogenicity of the VLPs does not change after high-temperature placement, further verifying the stability of the preparation prescription.

[0086] Example 4: Study on HPV L1-VLP maturation

[0087] The present invention uses a constant temperature water tank to perform maturation treatment on the HPV nine-valent L1-VLP protein solution of each type. The maturation treatment temperature is 25°C or 37°C. Samples are taken at four time points (0h, 12h, 24h and 36h). The particle size and PdI of the samples of the HPV nine-valent L1-VLP protein solution at different maturation temperatures and time points are detected using a dynamic light scattering nanoparticle size analyzer (DLS). The test results are shown in Tables 10 and 11.

[0088] Table 10. Particle size and PdI of HPV 9-valent L1-VLP protein solutions at different time points after maturation at 37°C

[0089] Table 11. Particle size and PdI of HPV 9-valent L1-VLP protein solutions at different time points after maturation at 25°C

[0090] The results in Tables 10 and 11 indicate that the particle size of most HPV L1-VLP protein solutions decreases with increasing maturation time at 37°C. Compared to unmatured samples, the particle size of matured HPV samples decreased by approximately 2 nm across all HPV types. The PdI data show that the PdI values ​​for most types decrease with increasing maturation time, indicating greater uniformity among these samples. Maturation at 25°C, on the other hand, had little effect on sample particle size and PdI at all time points. These results preliminarily indicate that 37°C maturation has a positive impact on HPV L1-VLP protein samples.

[0091] Furthermore, the HPV nine-valent L1-VLP protein solutions of various types were matured using a constant temperature water bath at 37°C, and samples were taken at four time points (0h, 12h, 24h, and 36h) for purity and in vitro relative potency testing. The purity of the nine-valent HPV vaccine protein was tested using HPLC, and the test results are shown in Table 12. The relative potency of samples of each type, batch, and maturation time point was determined using a double antibody sandwich (monoclonal antibody) enzyme-linked immunosorbent assay, and the relative potency of each sample is shown in Figure 5.

[0092] Table 12. HPLC purity of HPV 9-valent L1-VLP protein solutions at different time points after maturation

[0093] The results in Table 12 show that purity increases with increasing maturation time at 37°C. The HPLC purity of all HPV L1 VLP proteins after maturation reached over 99%. Based on these experimental results, the optimal maturation conditions are: 37°C for 12 to 36 hours.

[0094] As shown in Figure 5, with the exception of HPV11 and HPV31, the in vitro relative potency of HPV L1-VLP protein solutions of all other types (6, 16, 18, 33, 45, 52, and 58) increased with the extension of maturation time. Compared with the unmatured sample (0 hour), the in vitro relative potency of samples at the three maturation time points (12, 24, and 36 hours) increased significantly. The in vitro relative potency of the HPV31 L1-VLP protein solution decreased with the extension of maturation time, and the in vitro relative potency of the HPV11 L1-VLP protein solution samples after maturation decreased slightly compared to those before maturation.

[0095] Furthermore, L1-VLP protein solutions of nine HPV types were matured at 37°C for 2 hours and then adsorbed onto aluminum hydroxide adjuvant. Animals were immunized with a dose of 1 μg antigen and 25 μg aluminum adjuvant. Blood samples were collected 2 and 4 weeks after immunization to measure neutralizing antibody titers and compare the effects of maturation on antigen immunogenicity. The results in Figures 6 and 7 show that maturation at 37°C, especially for 24 hours, significantly enhances the immunogenicity of the nine HPV types' L1-VLP protein antigens. In particular, the matured antigens can induce the production of specific neutralizing antibodies earlier in the early post-immunization period (e.g., 2 weeks after immunization). At 4 weeks after immunization, while neutralizing antibody production by immature and matured antigens was similar, the matured antigens produced higher levels of neutralizing antibodies for most types. HPV type 11 L1-VLP protein, whose relative titer decreased in vitro after maturation, still demonstrated good immunogenicity in mice.

[0096] Example 5. Development of a nine-valent HPV vaccine

[0097] 1. The vaccine preparation steps of the present invention are as follows:

[0098] 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. The VLPs are further subjected to chromatography column liquid exchange: the liquid exchange chromatography column is fully equilibrated in advance with a buffer solution (10-30mM histidine-hydrochloric acid, 400-1500mM NaCl, 0.001%-0.3% polysorbate 80, pH 5.5-6.5), and then the assembled VLPs are loaded onto the equilibrated chromatography column. After loading, the sample is eluted with a buffer solution to collect the fractions containing the target protein. The collected fractions 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;

[0099] 2) Dilute each type of matured HPVL1-VLP protein stock solution with buffer to the desired concentration; finally, mix all the VLP dilutions of each type in proportion to obtain a nine-valent protein dilution solution, sterilize it with a filter, and set aside;

[0100] 3) Dilute and mix the aluminum hydroxide adjuvant prepared in Example 2 in the prescribed amount, filter through a capsule filter, and set aside;

[0101] 4) Take the required amount of the filtered sample of the nine-valent protein dilution solution and the filtered sample of the aluminum hydroxide adjuvant dilution solution and mix them until they are completely mixed to obtain the finished vaccine preparation.

[0102] 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 thoroughly mixed to obtain the finished vaccine preparation.

[0103] 5) For finished vaccine preparations that are not used in time, the finished product packaging is also included: pre-filled syringe filling machines or vial filling machines are used for packaging. After packaging, the finished vaccine preparations are stored in a cold storage at 2℃~8℃ for future use.

[0104] 2. Dosage study of antigen and aluminum adjuvant

[0105] A series of nine-valent HPV vaccines (see Table 13) were formulated with varying antigen dose ratios and aluminum hydroxide adjuvant doses and used to immunize BALB / c mice to investigate immunogenicity. Specifically, the vaccine was administered with a monovalent antigen to investigate immune interference; Gardasil 9 was used as a control vaccine, and different aluminum hydroxide adjuvant dose groups were established to determine the adjuvant dose in the vaccine; and different antigen dose ratios were used to determine the antigen dose ratio for each type. Animal grouping information, antigen dose ratios, and aluminum hydroxide adjuvant dose settings are shown in Table 13.

[0106] The immunization schedule involved immunizations at weeks 0 and 4, with neutralizing antibody titers measured 4 weeks after the first immunization and 3 weeks after the second immunization. Intergroup comparisons were performed to determine the dose ratio of each antigen type in the vaccine and the dosage of aluminum adjuvant.

[0107] Table 13 Antigen dose ratio and aluminum adjuvant dose study animal grouping

[0108] The geometric mean values ​​of neutralizing antibody detection 4 weeks after the first vaccination and 3 weeks after the second vaccination are shown in Table 14. The results showed that:

[0109] (1) Immune interference

[0110] Using the same aluminum adjuvant dose, mice were immunized with the monovalent and nine-valent mixed antigens listed in Table 13 (same dose of antigens of the same type), and the immune response levels were compared to investigate immune interference. The results showed that neutralizing antibody titers for all types of antigens prepared in this invention were significantly higher when immunized in a monovalent form than in the nine-valent HPV vaccine, and were also greater than those for the corresponding types in the control vaccine (Gardasil 9).

[0111] (2) Aluminum hydroxide adjuvant dosage

[0112] The fixed antigen dose was 1 / 20× of the human dose (for example, the human dose is 0.5 ml, and 1 / 20 of the human dose (0.5 ml) was used to immunize mice). Four aluminum hydroxide adjuvant doses were set: 37.5 μg (1 / 20× of the nine-valent HPV vaccine), 0 μg, 25 μg, and 50 μg. Mice were immunized (n=10 mice per group) to investigate the differences between the aluminum hydroxide adjuvant doses and to compare with the control vaccine (Gardasil 9, with aluminum phosphate as the adjuvant). From the results in Table 14, it can be seen that the neutralizing antibody level in the aluminum-free adjuvant group was 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 level of antigen immune response. 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 produced a certain superiority. 37.5 μg (corresponding to a human dose of 750 μg) of aluminum hydroxide adjuvant can reach or approach immune saturation, and is comparable to the immune response level of the control vaccine.

[0113] Table 14 Summary of immune doses and neutralizing antibody geometric mean titers

[0114] (3) Antigen dosage ratio

[0115] By adjusting the dose of HPV6 antigen by 10 μg each and HPV16 and HPV18 antigens by 20 μg each, or by increasing the dose of HPV16 and HPV18 antigens by 20 μg each and HPV31, 33, 45, 52, and 58 antigens by 10 μg each, the researchers investigated whether reducing or increasing the dose of these antigens could significantly increase the immune response level to these antigens, ensuring that the vaccine's efficacy was no less than that of the marketed vaccine, Gardasil 9. The results in Table 14 show that, compared with the initially determined antigen dose, increasing the dose of these antigens did not produce a significant immune-enhancing effect, indicating that the initial dose ratio of the antigens in the mice had already reached immune saturation, and that increasing the dose of these antigens was not necessary.

[0116] 3. Study on the particle size of antigen

[0117] According to the various types of L1-VLP antigens obtained in Example 1, antigens with different average particle sizes were prepared by adjusting different sodium chloride concentrations and pH values ​​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 30 μg, 40 μg, 60 μg, 40 μg, 20 μg, 20 μg, 20 μg, 20 μg, and 0.75 mg of the above-mentioned aluminum hydroxide adjuvant, and then different nine-valent HPV vaccines were prepared. Gardasil 9 and aluminum hydroxide adjuvant were used as controls, and BALB / c mice were immunized at a 1 / 50× human dose 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 15. Inter-group comparisons were made to optimize and control the particle size of each type of antigen in the vaccine.

[0118] Table 15 Summary of geometric mean titers of neutralizing antibodies

[0119] As shown in Table 15, the vaccine of the present invention comprising various antigen types within the selected particle size range can achieve a good immune response level. The immune response level to types 6, 11, 16, 18, 31, 45, and 58 is higher than that of the marketed Gardasil 9.

[0120] 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:

[0121] 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.

[0122] Example 6: Study on the immunogenicity of the nine-valent HPV vaccine of the present invention in Wistar rats

[0123] The nine-valent HPV vaccine used in this example was prepared according to the steps for preparing the nine-valent HPV vaccine in Example 5 above: each 0.5 ml of the finished vaccine formulation contained 30 μg, 40 μg, 60 μg, 40 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, and 20 μg of HPV6, 11, 16, 18, 31, 33, 45, 52, and 58 antigens, respectively, and 0.75 mg of aluminum hydroxide adjuvant. The buffer solution consisted of 10 mM histidine, 0.01% polysorbate 80, a pH of 5.5-6.5, and a sodium chloride concentration of 154-500 mM.

[0124] The present invention used three dose groups for immunization: 1 / 10×, 1 / 20×, and 1 / 40× the human dose (0.5 ml). Merck's Gardasil 9 (1 / 40× the human dose), which has the same antigenic components, was used as a positive control. Two immunization schedules, one single dose and one three-dose schedule (two weeks apart), were used to assess the vaccine's immunogenicity.

[0125] The present invention determined the immunogenicity and dose effect of the nine-valent HPV vaccine in Wistar rats. The animal grouping, immunization sample preparation, immunization schedule, and immunization route of the Wistar rats are shown in Table 16.

[0126] Table 16 Animal grouping

[0127] Wistar rats were immunized once with the three dose groups of the nine-valent HPV vaccine of the present invention and a positive control group (Gardasil 9). The neutralizing antibody titers measured at each time point are shown in Table 17. In addition, Wistar rats were immunized three times (2 weeks apart) with the three dose groups of the nine-valent HPV vaccine and a positive control group (Gardasil 9). The neutralizing antibody titers measured at each time point are shown in Table 18.

[0128] The results in Table 17 show that the dose-response relationship between the three immunization doses of the vaccine of the present invention is significant for HPV types 6, 11, 18, 31, 52, and 58, but less pronounced for HPV types 16, 33, and 45. This suggests that increasing the immunization dose can still enhance the immune response level for most types in Wistar rats. Compared with the positive control, the immunogenicity of HPV type 33 was significantly higher than that of the positive control, while the immunogenicity of the other eight types was comparable or close to that of the positive control vaccine.

[0129] Combined with the results in Table 18, it can be seen that compared to a single dose, three doses significantly increased neutralizing antibody levels by several times, or even more than 10 times. In addition to boosting antibody levels to higher levels, the triple dose also demonstrated a clear dose-effect relationship. Compared to the positive control, the immunogenicity of the present invention against HPV33 was significantly higher than that of the positive control, and the immunogenicity of the other eight types was at the same level or even better.

[0130] Table 17. Summary of geometric mean titers of neutralizing antibodies in Wistar rats after one injection

[0131] Table 18. Summary of geometric mean titers of neutralizing antibodies in Wistar rats after three immunizations (0, 2, and 4 weeks)

[0132] Example 7: Evaluation of the immunogenicity of the nine-valent HPV vaccine of the present invention in 120 healthy women aged 18-26 years

[0133] According to the preparation steps of the nine-valent HPV vaccine in Example 5 above, the nine-valent HPV vaccine used in this example was prepared, wherein the nine-valent HPV vaccine contains HPV6, 11, 16, 18, 31, 33, 45, 52, 58 L1-VLP antigens and aluminum hydroxide adjuvant, the amount of each antigen in each 0.5 ml is 20-80 μg, the amount of aluminum hydroxide adjuvant is 0.75 mg; the particle size of HPV6 antigen is 45-60 nm, the particle size of HPV11 antigen is The particle size of the HPV16 antigen is 45-60 nm, the particle size of the HPV18 antigen is 55-75 nm, the particle size of the HPV31 antigen is 55-75 nm, the particle size of the HPV33 antigen is 45-60 nm, the particle size of the HPV45 antigen is 50-65 nm, the particle size of the HPV52 antigen is 50-65 nm, and the particle size of the HPV58 antigen is 45-60 nm. The particle size of the aluminum hydroxide adjuvant is 3-10 μm. The buffer comprises: 10 mM histidine, 0.01% polysorbate 80, a pH of 5.5-6.5, and a sodium chloride concentration of 154-500 mM.

[0134] 120 subjects were divided into three dose groups: low, medium and high. The doses were implemented step by step from low dose to high dose according to the principle of sequence. Each group had 40 subjects (including 10 positive controls). After enrollment, the subjects 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 sequentially enrolled in four batches of 5, 10, 10 and 15 subjects, and were randomly vaccinated with the first dose of the low-dose test vaccine or the positive control vaccine at a ratio of 3:1. The enrollment interval for each batch was no less than 3 days. Secondly, after the 40 subjects in the low-dose group had been enrolled for a full week and their safety had been confirmed, 40 subjects in the medium-dose group were enrolled, and the enrollment batches and time intervals were the same as those in the low-dose group. Finally, after the 40 subjects in the medium-dose group had been enrolled for a full week and their safety had been confirmed, 40 subjects in the high-dose group were enrolled, and the enrollment batches and time intervals were the same as those in the low-dose group.

[0135] 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);

[0136] The vaccines administered in the medium-dose group were: V_270: HPV-6 / 11 / 16 / 18 / 31 / 33 / 45 / 52 / 58 (30 μg, 40 μg, 60 μg, 40 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg);

[0137] The vaccine administered in the high-dose group was: V_360: HPV-6 / 11 / 16 / 18 / 31 / 33 / 45 / 52 / 58 (30 μg, 40 μg, 80 μg, 60 μg, 30 μg, 30 μg, 30 μg, 30 μg, 30 μg).

[0138] Other parameters of the vaccine in the low-dose group, medium-dose group and high-dose group refer to the research results obtained in Example 3 above.

[0139] The mean age (standard deviation) of the subjects in the low-dose group (N=30) was 22.60 (2.18) years, with a minimum age of 18 years and a maximum age of 26 years; the mean age (standard deviation) of the subjects in the medium-dose group (N=30) was 22.93 (2.36) years, with a minimum age of 18 years and a maximum age of 26 years; the mean age (standard deviation) of the subjects in the high-dose group (N=30) was 22.73 (1.72) years, with a minimum age of 19 years and a maximum age of 26 years; the mean age (standard deviation) of the subjects in the positive control group (N=30) was 22.73 (2.41) years, with a minimum age of 18 years and a maximum age of 26 years.

[0140] The immunization schedule for this trial is three doses, administered at 0, 2, and 6 months. 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 or other areas is prohibited.

[0141] Immunogenicity results are usually evaluated by calculating the geometric mean titers (GMT), geometric mean intensity (GMI), and antibody positive conversion rates of neutralizing antibodies and IgG antibodies in different groups and their 95% CI.

[0142] The immunogenicity results in Tables 19 and 20 show that both the binding antibody and neutralizing antibody positivity rates were 100%. For subjects with negative pre-immunization antibodies, based on PPS analysis, three months after immunization, the medium-dose HPV18 group had higher binding antibody GMT and GMI than the low-dose, high-dose, and control groups (P < 0.05), and the high-dose HPV58 group had higher binding antibody GMT and GMI than the control group (P < 0.05). Three months after immunization, the low-dose and high-dose HPV33 groups had higher neutralizing antibody GMT and GMI than the control group (P < 0.05), and the high-dose HPV52 group had higher neutralizing antibody GMT and GMI than the medium-dose group (P < 0.05). Seven months after immunization, statistically significant differences in binding antibody GMT and GMI were observed among all HPV type groups, with the medium- and high-dose groups showing higher levels overall. Seven months after immunization, the low-dose, medium-dose, and high-dose HPV33 groups had higher neutralizing antibody GMT and GMI than the control group (P < 0.05).

[0143] Table 19 Geometric mean titer and positive conversion rate of neutralizing antibodies against HPV6 / 11 / 16 / 18 / 31 / 33 / 45 / 52 / 58 in different groups

[0144] Wherein, 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;

[0145] 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).

[0146] Table 20 Comparison of various types of binding antibodies and neutralizing antibodies in different groups after immunization

[0147] Finally, it should be noted that the foregoing description is merely 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 foregoing embodiments, those skilled in the art will be able to modify the technical solutions described in the foregoing embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A nine-valent human papillomavirus vaccine comprising truncated L1-VLP antigens of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58, and an adjuvant; in, The weight ratio of HPV6, 11, 16, 18, 31, 33, 45, 52, 58L1-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).

2. The nine-valent human papillomavirus vaccine according to claim 1, wherein 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; preferably, the amount of each antigen per 0.5 ml is 10-100 μg, 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 amount of adjuvant is 0.5-1.0 mg, preferably 0.75 mg; More specifically, HPV6, 11, 16, 18, 31, 33, 45, 52, and 58 L1-VLP antigens were expressed in an E. coli system; Preferably, each type of VLP antigen is matured at 30°C to 40°C for 6 to 48 hours to obtain mature VLP protein, which is then used to prepare the vaccine; more preferably, the maturation treatment is carried out at 35°C to 38°C for 12 to 36 hours to obtain mature VLP protein; and the vaccine adopts a histidine-hydrochloric acid buffer system.

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

4. The nine-valent human papillomavirus vaccine according to claim 1, wherein 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; more preferably, 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.

5. The nine-valent human papillomavirus vaccine according to claim 1, wherein The L1 proteins in the various types of L1 pentamer proteins are truncated as follows based on the wild-type sequence: the N-terminal truncation of 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 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 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 wild-type HPV18 L1 is no more than 10 amino acids, and the C-terminal truncation is no more than 30 amino acids; 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 PV31L1 is no more than 10 amino acids, and the C-terminal truncation is no more than 30 amino acids; the N-terminal truncation of 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 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 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 wild-type HPV58L1 is no more than 10 amino acids, and the C-terminal truncation is no more than 30 amino acids.

6. The nine-valent human papillomavirus vaccine according to claim 1, wherein The amino acid sequences of the various types of L1 proteins 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.

7. The nine-valent human papillomavirus vaccine according to claim 1, wherein Adding an osmotic pressure regulator and a surfactant to the histidine-hydrochloric acid buffer system; Preferably, the pH of the histidine-hydrochloric acid buffer system is 5 to 7, more preferably 5.5 to 6.5; the concentration of histidine-hydrochloric acid is 5 to 20 mM, preferably 10 to 15 mM; The osmotic pressure regulator is selected from one or more of sodium chloride, sodium phosphate or sodium sulfate, and the surfactant is polysorbate 80. Preferably, the sodium chloride concentration is 154-500mM, more preferably 320-400mM; the polysorbate 80 concentration is 0.005%-0.025%, preferably 0.008%-0.02%.

8. The nine-valent human papillomavirus vaccine according to claim 1, wherein The preparation method of the adjuvant is as follows: take aluminum chloride and sodium hydroxide, press 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°C to 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 reaction is carried out, the mixture is washed with sodium chloride solution (resuspended at a mass ratio of ≥6.5:1) for more than 4 times to obtain an aluminum hydroxide adjuvant; 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.

9. The method for preparing the nine-valent human papillomavirus vaccine according to any one of claims 1 to 8, comprising the following steps: Purified HPV L1 proteins of various types are self-assembled in vitro to form VLPs. The VLPs are further subjected to column chromatography, liquid exchange, and sterile filtration to prepare a protein stock solution. The VLPs are preferably matured at 30°C to 40°C for 6 to 48 hours to obtain a matured VLP protein stock solution, which is then diluted with a buffer solution to the desired concentration. The protein dilutions of various types are then mixed with the adjuvant dilutions in proportion to prepare monovalent adsorption products. Finally, the desired amount of the monovalent adsorption products of various types are thoroughly mixed to obtain the finished vaccine preparation. Alternatively, the various types of protein stock solution dilutions can be mixed in proportion to prepare the nine-valent protein dilution, and then the required amount of the nine-valent protein dilution is taken and thoroughly mixed with the adjuvant dilution to obtain the finished vaccine preparation.

10. The method for preparing the nine-valent human papillomavirus vaccine according to claim 9, wherein: It also includes finished product packaging, specifically using a pre-filled syringe filling machine or a vial filling machine for packaging. After packaging, the finished vaccine preparation is stored in a cold storage at 2°C to 8°C.

11. Use of the nine-valent human papillomavirus vaccine according to any one of claims 1 to 8 in the preparation of a medicament for preventing or treating diseases caused by human papillomavirus.

Images