Nine-valent human papillomavirus vaccine formulations and their applications

Abstract

The present invention relates to the field of biomedicine, and specifically discloses a nine-valent human papillomavirus vaccine preparation and its application. The nine-valent human papillomavirus vaccine preparation includes truncated L1-VLP protein antigens of HPV6, 11, 16, 18, 31, 33, 45, 52, and 58 types that have been matured, adjuvants, a histidine buffer system, and an osmotic regulator and a surfactant are added. The antigen components in the nine-valent HPV vaccine preparation 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 invention optimizes the buffer system and combines the post-maturation treatment of the preparation process of each type of VLP antigen, so that the obtained nine-valent HPV vaccine preparation induces a high level of immune response.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically to a human papillomavirus vaccine preparation, and in particular to a nine-valent human papillomavirus vaccine preparation and application thereof. 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 proven that HPV infection is the main cause of cervical cancer. HPV infection is the most common viral infection of the reproductive tract. Human infection with HPV may not only cause cervical cancer, but also anogenital cancer and genital warts. In addition, oropharyngeal cancer and other head and neck cancers, colon cancer, and rectal cancer are also related to HPV infection. Vaccination with HPV vaccine is the most economical and effective means to prevent persistent HPV infection and related diseases.

[0003] 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 antigenic proteins will lead to a decrease in the immunogenicity of the antigen. 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 preparation 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] First, the nine-valent human papillomavirus vaccine preparation provided by the present invention includes L1-VLP protein antigens of HPV6, 11, 16, 18, 31, 33, 45, 52, and 58, and an adjuvant;

[0006] The weight ratio of HPV6, 11, 16, 18, 31, 33, 45, 52, 58 protein 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 amount of each protein in every 0.5 ml is 10-100 μg, and the amount of adjuvant is 0.5-1.0 mg;

[0007] Each type of VLP protein is matured at 30-40°C for 6-48 hours to obtain mature VLP protein, which is then used to prepare the vaccine preparation; and the vaccine preparation adopts a histidine buffer system, and an osmotic pressure regulator and a surfactant are added.

[0008] The inventors found in their research that when using a histidine buffer system, the histidine system is prone to VLP disaggregation, with some unassembled pentamers; the human papillomavirus virus-like particles (VLPs) assembled from HPV L1 protein pentamers are matured at 30-40° C. for 6-48 h, preferably at 35-38° C. for 12-36 h, and more preferably at 37° C. for 24 h; the stability and immunogenicity of the vaccine preparation are improved.

[0009] Preferably, the pH of the histidine buffer system is 5-7, more preferably pH 5.5-6.5; the histidine concentration is 5-20 mM, preferably 10-15 mM; the sodium chloride concentration is 300-600 mM, more preferably 420-480 mM; the concentration of polysorbate 80 is 0.005%-0.025%, preferably 0.01%-0.025%.

[0010] Further preferably, the osmotic pressure regulator is selected from one or more of sodium chloride, sodium phosphate or sodium sulfate; the surfactant is polysorbate 80;

[0011] In a preferred embodiment, the weight ratio of L1-VLP protein antigens of HPV6, 11, 16, 18, 31, 33, 45, 52, and 58 is 1.5:2:3:2:1:1:1:1:1; in every 0.5 ml, the L1-VLP protein antigens of HPV6, 11, 16, 18, 31, 33, 45, 52, and 58 are 30 μg, 40 μg, 60 μg, 40 μg, 20 μg, 20 μg, 20 μg, 20 μg, and 20 μg, respectively; and the amount of adjuvant is 0.75 mg.

[0012] More specifically, the L1-VLP protein antigens of HPV6, 11, 16, 18, 31, 33, 45, 52, and 58 are expressed by the E. coli system.

[0013] In a specific embodiment, the adjuvant is an aluminum adjuvant, preferably an aluminum hydroxide adjuvant.

[0014] In another preferred embodiment, the particle size of each type of L1-VLP protein antigen is 45 to 80 nm, and the particle size of the adjuvant is 3 to 10 μm; more preferably, the particle size of the HPV6 L1-VLP protein antigen is 45 to 60 nm, the particle size of the HPV11 L1-VLP protein antigen is 45 to 60 nm, the particle size of the HPV16 L1-VLP protein antigen is 45 to 60 nm, and the particle size of the HPV18 L1-VLP protein antigen is 55 to 60 nm. 75nm, the particle size of HPV31L1-VLP protein antigen is 55-75nm, the particle size of HPV33L1-VLP protein antigen is 45-60nm, the particle size of HPV45L1-VLP protein antigen is 50-65nm, the particle size of HPV52L1-VLP protein antigen is 50-65nm, the particle size of HPV58L1-VLP protein antigen is 45-60nm, and the particle size of the adjuvant is 5-8μm.

[0015] 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.

[0016] 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.

[0017] In a specific embodiment, the L1 protein sequence in each type of L1-VLP protein antigen is truncated as follows 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 not more than 10 amino acids, and the C-terminal truncation is not more than 30 amino acids; the N-terminal truncation of the wild-type HPV33L1 is not more than 10 amino acids, and the C-terminal truncation is not more than 30 amino acids; the N-terminal truncation of the wild-type HPV45L1 is not more than 10 amino acids, and the C-terminal truncation is not more than 30 amino acids; the N-terminal truncation of the wild-type HPV52L1 is not more than 10 amino acids, and the C-terminal truncation is not more than 30 amino acids; the N-terminal truncation of the wild-type HPV58L1 is not more than 10 amino acids, and the C-terminal truncation is not more than 30 amino acids.

[0018] 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.

[0019] The present invention also provides a method for preparing the vaccine preparation, which comprises the following steps:

[0020] 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 sterilization filtration to prepare VLP protein stock solutions, which are matured at 30-40°C for 6-48 hours to obtain mature VLP protein stock solutions, which are diluted to the required concentrations using buffer solutions; then, the various types of protein dilutions are respectively mixed with aluminum hydroxide adjuvant dilutions, osmotic regulators, and surfactants in proportion to prepare monovalent adsorption products, and finally, the required amounts of the various types of monovalent adsorption products are completely mixed to obtain the finished vaccine preparations;

[0021] 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 and the aluminum hydroxide adjuvant dilution are taken and thoroughly mixed to obtain the finished vaccine preparation.

[0022] Optionally, the method further comprises filling of the finished product, specifically, starting a prefilled syringe filling machine for filling. Preferably, the filling speed is 30 to 40 revolutions per minute, more preferably, 0.55 ml / vial is filled and subpackaged and a rubber stopper is added, and the product is stored in a cold storage at 2 to 8°C for future use.

[0023] Finally, the present invention provides the use of the vaccine preparation in preparing medicines for preventing or treating diseases caused by human papillomavirus.

[0024] Beneficial effects of the present invention:

[0025] 1. The nine-valent human papillomavirus vaccine preparation of the present invention uses a buffer containing histidine and matures various types of VLP proteins at 30 to 40° C. for 6 to 48 hours, thereby improving the stability and immunogenicity of the vaccine preparation.

[0026] 2. The antigen components of the nine-valent human papillomavirus vaccine preparation of the present invention are all derived from the major capsid protein L1 of human papillomavirus and are truncated. The truncated L1 protein is expressed by the E. coli system, which increases the yield of the L1 protein and reduces the production cost of the vaccine preparation. 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] 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.

[0031] Figure 4, Analysis of immunogenicity of nine-valent HPV vaccine 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 ℃, 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.

[0032] Figure 5 , the in vitro relative potency of samples from different time points after maturation treatment of HPV nine-valent L1-VLP protein solutions of various types.

[0033] Figure 6 , the immunogenicity of the 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.

[0034] Figure 7 , the immunogenicity of the 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

[0035] 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.

[0036] Example 1. Preparation of various types of antigens

[0037] The present invention studies and truncates various types of L1 proteins based on the wild-type sequence, thereby obtaining optimized various types of L1 proteins. The specific various types of L1 proteins used in this example are as follows:

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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 HPV33L1-VLP protein with an average particle size of 45-60nm and PdI<0.1 was obtained.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] Taking the preparation of HPV6L1 protein antigen as an example, the process is as follows:

[0048] 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.

[0049] The ligation system is as follows: 6μl of pKL1 vector fragment, 2μl of HPV6L1 gene fragment, 1μl of T4 DNA ligase, and 1μl of T4DNA 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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 nanoparticle 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).

[0055] 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.

[0056] Example 2: Study on the buffer system of the preparation

[0057] The present invention studies the effects of different buffer salt concentrations, pH, NaCl concentrations and their combination conditions (see Table 1) of the nine-valent HPV various types L1-VLP protein stock solutions 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 nine-valent HPV various types of protein stock solution samples were placed under different storage temperatures (4°C, 25°C, 37°C, -80°C (repeated freeze-thaw)) for investigation, 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 2 and 3. HPLC was used to detect the protein purity, and the detection results of HPV6L1-VLP are shown in Tables 4 and 5.

[0058] Table 1. Prescription of HPVL1-VLP protein stock solution buffer

[0059]

[0060] From the results in Tables 2 and 3, it can be seen that the particle sizes of the samples in the two buffer systems of acetic acid-sodium acetate and histidine-hydrochloric acid are slightly different. Overall, under the conditions of each prescription in the histidine-hydrochloric acid buffer, the particle size of HPV6L1-VLP is smaller and the particles are more compact. After three freeze-thaw treatments of the samples in the two buffer systems, the particle size and PdI of the samples in the acetic acid-sodium acetate buffer system are larger than those in the histidine-hydrochloric acid buffer system, indicating that the histidine-hydrochloric acid buffer system is more conducive to the freeze-thaw storage of L1-VLP samples. After the samples in the two buffer systems were stored at 4°C, 25°C and 37°C, the fluctuation range of the particle size of HPV6L1-VLP in the histidine-hydrochloric acid buffer system was smaller than that in the acetic acid-sodium acetate body, and the histidine-hydrochloric acid buffer system was more conducive to maintaining the stability of HPV6L1-VLP.

[0061] Table 2. Summary of HPV6L1-VLP particle size (nm) results

[0062]

[0063] Note: Average is the average value of the particle size measurement of HPV6L1-VLP samples; SD is the standard deviation.

[0064] Table 3. Summary of results of HPV6L1-VLPPdI

[0065]

[0066]

[0067] Note: Average is the average value of the particle size measurement of HPV6L1-VLP samples; SD is the standard deviation.

[0068] From the results of Tables 4 and 5, it can be seen that the plate number of HPV6L1-VLP test results under each prescription condition in the histidine-hydrochloric acid buffer system is high and the fluctuation range is small, and the value after the symmetry factor -1 is closer to zero, indicating that each prescription of the histidine-hydrochloric acid buffer system can maintain the stability of HPV6L1-VLP under freeze-thaw conditions. From the overall data, the plate number and symmetry factor fluctuation of the HPV6L1-VLP test results in the histidine-hydrochloric acid buffer system are smaller than those in the acetic acid-sodium acetate buffer system, and there is no obvious difference between the prescriptions in the histidine-hydrochloric acid buffer system (except H7), indicating that the histidine-hydrochloric acid buffer system is more conducive to the stability of HPV6L1-VLP.

[0069] Table 4. HPV6L1-VLP stock solution HPLC plate number USP

[0070]

[0071] Table 5. (A)symmetry factors of the main peak of HPV6 L1-VLP stock solution HPLC

[0072]

[0073]

[0074] Furthermore, the research results of the HPV11L1-VLP, HPV16L1-VLP, HPV18L1-VLP, HPV31L1-VLP, HPV33L1-VLP, HPV45L1-VLP, HPV52L1-VLP and HPV58L1-VLP protein stock solutions all showed that the histidine-hydrochloric acid buffer system is beneficial to the stability of various types of HPV L1-VLP. In summary, the nine-valent HPV L1-VLP stock solutions of various types use a histidine buffer system.

[0075] Since the L1-VLP stock solution of each type of nine-valent HPV adopts a histidine buffer system, the nine-valent HPV vaccine preparation of the present invention also adopts a histidine buffer system. The main purpose of the preparation buffer system study 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 salt concentration) was determined by differential scanning calorimetry. The higher the Tm value, the more stable the antigen is under this formulation. The results of the determination of each type and each formulation were combined to select the parameter range that is conducive to antigen stability.

[0076] Table 6. List of preparation buffer systems

[0077] Group Histidine / mM Sodium chloride / mM pH Polysorbate 80 / % H1 10 204.67 5.65 0.01 H2 10 204.67 6.35 0.01 H3 10 449.33 5.65 0.01 H4 10 449.33 6.35 0.01 H5 10 327.00 5.50 0.01 H6 10 327.00 6.50 0.01 H7 10 154.00 6.00 0.01 H8 10 500.00 6.00 0.01 H9 10 327.00 6.00 0.01 H10 10 327.00 6.00 0.01 H11 10 327.00 6.00 0.01 H12 10 327.00 6.00 0.01 H13 10 327.00 6.00 0.01 H14 10 327.00 4.50 0.01 H15 10 327.00 7.50 0.01 H16 10 550.00 6.00 0.01 H17 10 100.00 6.00 0.01

[0078] Table 7. Summary of Tm values ​​of various types of VLPs in the set formulations determined by DSF method

[0079]

[0080]

[0081] The results show (as shown in Table 7) that the Tm values ​​of HPV6 and 11, which belong to the α10 genus in virus taxonomy, are relatively high, the Tm values ​​of HPV18 and 45, which belong to the α7 genus, are relatively high or medium, and the Tm values ​​of HPV16, 31, 33, 52, and 58, which belong to the α9 genus, are relatively low or medium. The results show that there is a certain correlation between the Tm value and the evolutionary relationship of each type of virus.

[0082] From the results of Tables 6 and 7, 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 salt 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 salt concentration is too high or too low, the Tm values ​​of each type of VLP are reduced (H14-H17).

[0083] 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 salt concentration was 154-500 mM.

[0084] Furthermore, the monovalent HPV6, HPV11, HPV 16, HPV18, HPV 31. Five prescriptions were set up for each of the nine types of HPV33, HPV45, HPV52, and HPV58 (A, B, C, D, and E, each prescription had a histidine concentration of 10 mM and a polysorbate 80 concentration of 0.01%, prescription A: pH 5.5, sodium chloride concentration of 327 mM, prescription B: pH 6.0, sodium chloride concentration of 154 mM, prescription C: pH 6.0, sodium chloride concentration of 327 mM, prescription D: pH 6.0, sodium chloride concentration of 500 mM, prescription E: pH 6.5, sodium chloride concentration of 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 vaccine at 4°C, 37°C for 7 days, and 37°C for 14 days.

[0085] The results of neutralizing antibody titer determination in each group are as follows Figures 1 to 3 As shown, the results showed that there was no significant difference in the antibody level induced by each type of VLP in vivo within the designed prescription range (there was no significant difference in the antibody level induced by each type of VLP within the designed prescription range (multiple comparison analysis of data between groups p>0.05), indicating that each type of VLP has good stability within this prescription range. After being placed at 37°C for 14 days, the immunogenicity of the VLP did not change, further proving the good stability of the prescription.

[0086] On this basis, the present invention verifies the shelf stability of the vaccine produced in pilot test, and the pH in the formulation is 6.02, the salt concentration is 327mM, and the Figure 4 The results show that after the finished product was placed at 37°C for 14 days, the neutralizing antibody titers of various types of VLPs did not change significantly, indicating that the immunogenicity of VLPs did not change after high-temperature placement, further verifying the stability of the formulation of this preparation.

[0087] Example 3: Study on HPV L1-VLP maturation

[0088] The present invention uses a constant temperature water tank to perform maturation treatment on the HPV nine-valent L1-VLP protein solutions of various types. The temperature of the maturation treatment is 25°C or 37°C. Samples are taken at four time points (0h, 12h, 24h and 36h), and a dynamic light scattering nanoparticle size analyzer (DLS) is used to detect the particle size and PdI of the samples of the HPV nine-valent L1-VLP protein solutions of various types at different maturation temperatures and time points. The test results are shown in Tables 8 and 9.

[0089] Table 8. Particle size and PdI of HPV 9-valent L1-VLP protein solutions at different time points after maturation at 37°C

[0090]

[0091] Table 9. Particle size and PdI of HPV 9-valent L1-VLP protein solutions at different time points after aging at 25°C

[0092]

[0093] From the results in Tables 8 and 9, it can be seen that with the extension of the maturation time at 37°C, the particle size of most HPV L1-VLP protein solutions tends to decrease: compared with the unmatured samples, the particle size of the HPV types after maturation is reduced by about 2nm. From the PdI data, with the extension of the maturation time, the PdI of most types tends to decrease, indicating that these samples are more uniform. Maturation at 25°C has little effect on the sample particle size and PdI at each time point. The above results preliminarily show that the 37°C maturation treatment has a positive effect on the HPV L1-VLP protein samples.

[0094] Furthermore, the HPV nine-valent L1-VLP protein solutions of various types were matured using a 37°C constant temperature water bath, and samples were taken at four time points (0h, 12h, 24h and 36h) for purity and in vitro relative potency testing. The nine-valent HPV vaccine protein purity test was performed using HPLC, and the test results are shown in Table 10. The double antibody sandwich (monoclonal antibody) enzyme-linked immunosorbent assay was used to determine the relative potency of samples of various types, batches, and maturation time points. The relative potency of each sample is shown in Table 10. Figure 5 shown.

[0095] Table 10. HPLC purity of samples at different time points after maturation of HPV 9-valent L1-VLP protein solutions

[0096]

[0097] From the results in Table 10, it can be seen that as the maturation time at 37°C increases, the purity also increases. The HPLC purity of all types of HPV L1-VLP proteins after maturation can reach more than 99%. Based on the above experimental results, the maturation treatment conditions are: 37°C, maturation treatment for 12 to 36 hours.

[0098] Depend on Figure 5 The results show that, except for HPV11 and HPV31, the in vitro relative titers of HPV L1-VLP protein solutions of other types (6, 16, 18, 33, 45, 52, 58) tend to increase with the extension of maturation time. Compared with the immature samples (0 hours), the in vitro relative titers of the samples at the three maturation time points (12, 24, 36h) were significantly increased. The in vitro relative titer of the HPV31 type L1-VLP protein solution decreased with the extension of maturation time, and the in vitro relative titer of the HPV11 type L1-VLP protein solution after maturation was slightly lower than that before maturation.

[0099] Furthermore, the L1-VLP protein solutions of nine HPV types were matured at 37°C for 2 hours, then adsorbed on aluminum hydroxide adjuvant, and 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 the neutralizing antibody titer, and the effect of maturation on the immunogenicity of the antigen was compared. Figure 6 and Figure 7 The results show that maturation treatment at 37°C, especially for 24 hours, has a significant enhancing effect on the immunogenicity of the nine types of HPV L1-VLP protein antigens, especially in the early stage after immunization (such as 2 weeks after immunization). The matured antigens can induce the body to produce specific neutralizing antibodies earlier. At 4 weeks after immunization, although the neutralizing antibodies induced by immature and mature antigens tended to be at similar levels, most types showed that the neutralizing antibodies produced by matured antigens were higher. The HPV11 type L1-VLP protein, whose relative titer in vitro decreased after maturation, still showed good immunogenicity in mice.

[0100] Example 4. Preparation of nine-valent HPV vaccine

[0101] 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, sterilization filtration to obtain HPVL1-VLP protein stock solution, and subjecting the obtained HPVL1-VLP protein stock solution to maturation treatment at 37° C. for 24 h to obtain a mature HPVL1-VLP protein stock solution;

[0102] 2) using a buffer solution to dilute each type of matured HPVL1-VLP protein stock solution to the required concentration; finally, all types of VLP dilutions are mixed in proportion to obtain a nine-valent protein dilution solution, and the mixed nine-valent protein dilution solution is sterile filtered for later use;

[0103] 3) diluting and mixing the required amount of aluminum hydroxide adjuvant (the particle size of the aluminum hydroxide adjuvant is 5 to 8 μm), filtering through a capsule filter and setting aside;

[0104] 4) Take the required amount of the nine-valent protein dilution filtered sample and the aluminum hydroxide adjuvant dilution filtered sample and mix them until completely mixed to obtain the finished vaccine preparation. Each 0.5 ml of the finished vaccine preparation contains 30 μg, 40 μg, 60 μg, 40 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, 20 μg, 750 mg of aluminum hydroxide adjuvant, and the buffer includes: 10 mM histidine, 0.01% polysorbate 80, pH 5.5-6.5, and salt concentration of 154-500 mM.

[0105] The present invention sets three dose groups of 1 / 10×, 1 / 20× and 1 / 40× human dose (0.5 mL) for immunization, and uses Merck's Gardasil 9 (1 / 40× human dose) with the same antigen components as a positive control. Two immunization programs of one injection and three injections (2 weeks apart) are set to investigate the immunogenicity and persistence of the vaccine.

[0106] The present invention determined the immunogenicity and dose effect of the nine-valent HPV vaccine in Wistar rats. The animal grouping, immune sample preparation, immune program, immune route, etc. of Wistar rats are shown in Table 11.

[0107] Table 11 Animal grouping table

[0108]

[0109] The three dose groups of the nine-valent HPV vaccine of the present invention and the positive control group (Gardasil 9) were used to immunize Wistar rats once, and the results of the neutralizing antibody titers measured at each time point are shown in Table 12. In addition, the three dose groups of the nine-valent HPV vaccine and the positive control group (Gardasil9) were used to immunize Wistar rats three times (2 weeks apart), and the results of the neutralizing antibody titers measured at each time point are shown in Table 13.

[0110] From the results in Table 12, it can be seen that the dose-correspondence relationship between the three immunization doses of the vaccine of the present invention is obvious for HPV6, 11, 18, 31, 52, 58 and other types, but not so obvious for HPV16, 33, 45, indicating that the increase in immunization dose can still increase the immune response level of most types in Wistar rats. Compared with the positive control, HPV33 type is significantly higher than the positive control, and the immunogenicity of the other eight types is equivalent to or close to the positive control vaccine level.

[0111] Combined with the results in Table 13, it can be seen that compared with one shot immunization, three shots can significantly increase the level of neutralizing antibodies by several times or even more than 10 times. In addition to increasing the antibody level to a higher level, three shots also show a significant dose-effect relationship. Compared with the positive control, the HPV33 type of the present invention is significantly higher than the positive control, and the immunogenicity of the other eight types is at the same level or even better.

[0112] Table 12. Summary of geometric mean titers of neutralizing antibodies in Wistar rats after one injection

[0113]

[0114]

[0115] Table 13. Summary of geometric mean titers of neutralizing antibodies in Wistar rats after three immunizations (0, 2, and 4 weeks of immunization)

[0116]

[0117]

[0118] 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 preparation, comprising L1-VLP protein antigens of HPV6, 11, 16, 18, 31, 33, 45, 52, 58, and an adjuvant; The weight ratio of HPV6, 11, 16, 18, 31, 33, 45, 52, 58 protein 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 amount of each protein in every 0.5 ml is 10-100 μg, and the amount of adjuvant is 0.5-1.0 mg; Each type of VLP protein is matured at 30-40° C. for 6-48 hours to obtain mature VLP protein, which is then used to prepare the vaccine preparation; and the vaccine preparation adopts a histidine buffer system, and an osmotic pressure regulator and a surfactant are added; The L1 protein sequences in the various types of L1-VLP protein antigens 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; The pH of the histidine buffer system is 5 to 7; the histidine concentration is 5 to 20 mM; The osmotic pressure regulator is sodium chloride, the surfactant is polysorbate 80, the sodium chloride concentration is 300-600 mM, and the polysorbate 80 concentration is 0.005%-0.025%; The adjuvant is aluminum hydroxide adjuvant.

2. The vaccine preparation according to claim 1, characterized in that The pH of the histidine buffer system is 5.5-6.5; the histidine concentration is 10-15 mM; the sodium chloride concentration is 420-480 mM; the polysorbate 80 concentration is 0.01%-0.025%; The maturation treatment is carried out at 35-38° C. for 12-36 hours to obtain a mature VLP protein.

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

1.

4. The vaccine preparation according to claim 3, characterized in that In every 0.5 ml, the L1-VLP protein antigens of HPV6, 11, 16, 18, 31, 33, 45, 52, and 58 are 30μg, 40μg, 60μg, 40μg, 20μg, 20μg, 20μg, 20μg, and 20μg respectively; the dosage of adjuvant is 0.75mg.

5. The vaccine preparation according to claim 4, characterized in that The L1-VLP protein antigens of HPV6, 11, 16, 18, 31, 33, 45, 52, and 58 were expressed by the E. coli system.

6. The vaccine preparation according to claim 1, characterized in that The particle size of each type of L1-VLP protein antigen is 45 to 80 nm, and the particle size of the adjuvant is 3 to 10 μm.

7. The vaccine preparation according to claim 6, characterized in that The particle size of HPV6L1-VLP protein antigen is 45-60nm, the particle size of HPV11L1-VLP protein antigen is 45-60nm, the particle size of HPV16L1-VLP protein antigen is 45-60nm, the particle size of HPV18L1-VLP protein antigen is 55-75nm, the particle size of HPV31L1-VLP protein antigen is 55-75nm, the particle size of HPV33L1-VLP protein antigen is 45-60nm, the particle size of HPV45L1-VLP protein antigen is 50-65nm, the particle size of HPV52L1-VLP protein antigen is 50-65nm, the particle size of HPV58L1-VLP protein antigen is 45-60nm, and the particle size of the adjuvant is 5-8μm.

8. The vaccine preparation according to claim 7, characterized in that The various types of L1-VLP protein antigens are assembled from various types of L1 pentamer proteins, and the particle size of the L1 pentamer protein is 10-15 nm.

9. The vaccine preparation according to claim 8, 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.

10. The method for preparing the vaccine preparation according to any one of claims 1 to 9, comprising the following steps: Purified HPVL1 proteins of various types are self-assembled in vitro to form VLPs, and the VLPs are further subjected to column chromatography, liquid exchange, and sterilization filtration to prepare VLP protein stock solutions, which are matured at 30-40°C for 6-48 hours to obtain mature VLP protein stock solutions, which are diluted to the required concentrations using buffer solutions; then, the protein dilutions of various types are respectively mixed with the adjuvant dilutions in proportion to prepare monovalent adsorption products, and finally, the required amounts of the monovalent adsorption products of various types are taken and completely mixed to obtain the finished vaccine preparations; 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 and the adjuvant dilution are mixed thoroughly to obtain the finished vaccine preparation.

11. The method for preparing the nine-valent human papillomavirus vaccine preparation according to claim 10, characterized in that: It also includes filling of finished products and starting the prefilled syringe filling machine for filling.

12. The preparation method according to claim 11, 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.

13. Use of the vaccine preparation according to any one of claims 1 to 9 in the preparation of a medicament for preventing or treating a disease caused by human papillomavirus.

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