Method for enhancing immunogenicity of recombinant protein antigen and use thereof

Abstract

Provided are a method for enhancing the immunogenicity of a recombinant protein antigen and use thereof. The method comprises conjugating a polysaccharide with a recombinant protein antigen to form a nano-scale protein antigen. The polysaccharide is selected from sodium hyaluronate, chitosan, glucan, fucoidan, and sodium alginate. The recombinant protein antigen is a protein antigen derived from a bacterium. The bacterium belongs to the genus of Staphylococcus, Neisseria, Klebsiella, Escherichia, Clostridium, Salmonella, Shigella, Pseudomonas, Acinetobacter, Bordetella, Enterococcus, Haemophilus, Mycobacterium, or Streptococcus. The method not only improves the immunogenicity of the recombinant protein antigen, but also overcomes the defects of competitive immunoregulation and poor immune enhancement effects caused by bacterial capsular polysaccharide modification. The method also has the advantages of high clinical application value, simple starting material acquisition, low cost, good process stability, ease in scale expansion, high safety, and the like.

Description

A method for enhancing the immunogenicity of recombinant protein antigens and its application

[0001] This application claims priority to Chinese patent application 2024106853923, filed on May 30, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biomedicine, specifically to a method and application for enhancing the immunogenicity of recombinant protein antigens. Background Technology

[0003] As of early 2023, there were 966 vaccines in clinical trials worldwide. The main technologies employed included recombinant protein vaccines, nucleic acid vaccines, conjugate vaccines, viral vector vaccines, inactivated vaccines, and live attenuated vaccines, with recombinant protein vaccines accounting for the largest share (22%). Recombinant protein vaccines have shown excellent application prospects among various vaccine technologies due to their safety, high efficacy, mature technology platforms, and ease of large-scale production.

[0004] Recombinant protein vaccines, also known as genetically engineered recombinant subunit vaccines, are vaccines made using genetic engineering technology by extracting immunogenic components from pathogenic microorganisms. These immunogenic components typically include pathogenic microbial surface antigens, toxin antigens with host cell-killing effects, and antigens that bind to recipient cells. These protein antigens usually have a molecular weight of 10–100 kDa. Protein antigens with smaller molecular weights or fewer epitopes often exhibit weak immunogenicity, failing to induce a high level of immune response and thus failing to provide effective immune protection.

[0005] Polysaccharide conjugate vaccines utilize the conjugation of protein antigens with T-cell epitopes to polysaccharide antigens, overcoming the limitation of polysaccharide antigens alone failing to induce T-cell immune responses. This process stimulates immune responses from both T and B cells, converting low-affinity IgM antibodies into high-affinity IgG antibodies and generating memory B cells and plasma cells. Inspired by this principle, the applicant discovered that conjugating a truncated S protein (RBD protein) of SARS-CoV-2 using serotype 9V pneumococcal capsular polysaccharide significantly enhances its immunogenicity (CN113150084A); CN116113644A also found that serotypes 14 and 7F pneumococcal capsular polysaccharides and dextran enhance the immunogenicity of the RBD protein.

[0006] The inventors speculate that the main principle behind the enhancement of recombinant protein immunogenicity by polysaccharide antigens is that they increase the molecular size of the protein antigen and improve the antigen presentation efficiency, thereby enhancing the immune system's response level. However, when using exogenous polysaccharide antigens such as pneumococcal capsular polysaccharide, meningococcal capsular polysaccharide, and Haemophilus influenzae capsular polysaccharide as the immune-enhancing matrix for recombinant protein vaccines, there are several shortcomings: (1) It will compete with the protein antigen for immune regulation. As an exogenous substance, capsular polysaccharide itself has a certain immunogenicity. When it is coupled with the protein and simultaneously immunized into the body, it will cause a competitive immune regulation phenomenon, which is especially evident in multi-component vaccines, and cannot exert the ideal immune enhancement effect. (2) It cannot share the vaccination population with commercially available vaccines containing the polysaccharide antigen. If the same age group is vaccinated with commercially available polysaccharide vaccines or polysaccharide conjugate vaccines, there will be phenomena such as repeated vaccination with polysaccharide antigens, excessively high immune doses, and interference with the immunization program, which will have a great impact on the immune effect of commercially available polysaccharide vaccines or polysaccharide conjugate vaccines, and will also bring great safety risks to the vaccinated population.

[0007] Therefore, given the shortcomings of the aforementioned technologies, developing an immune enhancement method that can significantly enhance the immunogenicity of recombinant protein antigens without affecting the safety and efficacy of already marketed vaccines is a pressing issue that needs to be addressed.

[0008] Summary of the Invention

[0009] To address the aforementioned technical problems, this invention discloses a method and its application for enhancing the immunogenicity of recombinant protein antigens.

[0010] Specifically, a first aspect of the present invention provides a method for enhancing the immunogenicity of recombinant protein antigens, the method comprising coupling polysaccharides with recombinant protein antigens to form nanoscale protein antigens.

[0011] In some embodiments, the polysaccharide is selected from sodium hyaluronate (HA), chitosan, dextran, fucose, and sodium alginate.

[0012] In some specific implementations, the polysaccharide is sodium hyaluronate.

[0013] In some embodiments, the recombinant protein antigen is a bacterial protein antigen, wherein the bacteria belong to the genera Staphylococcus, Neisseria, Klebsiella, Escherichia, Clostridium, Clostridium, Salmonella, Shigella, Pseudomonas, Acinetobacter, Bordetella, Enterococcus, Haemophilus, Mycobacterium, Corynebacterium, or Streptococcus.

[0014] In some specific embodiments, the bacteria are selected from one or more of the following: Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Neisseria meningitidis, Neisseria gonorrhoeae, Klebsiella pneumoniae, Escherichia coli, Clostridium tetani, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, and Salmonella typhi. (S. typhi), Salmonella paratyphi, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Acinetobacter baumanii, Bordetella pertussis, Enterococcus faecium, Enterococcus faecalis, Haemophilus influenzae, Mycobacterium tuberculosis, Mycobacterium bovis, Corynebacterium diphtheriae, Group A Streptococcus, Group B Streptococcus, and Streptococcus pneumoniae.

[0015] In some specific embodiments, the bacteria are selected from one or more of the following: Staphylococcus aureus, Group B meningococcus, Corynebacterium diphtheriae, Streptococcus pneumoniae, and Haemophilus influenzae.

[0016] In some specific embodiments, the Staphylococcus aureus antigen includes one or more of the following: recombinant enterotoxin A (rSEA), enterotoxin B (rSEB), enterotoxin C2 (rSEC2), recombinant toxic shock syndrome toxin-1 (rTSST1), recombinant α-hemolysin (rHlα), and recombinant Panton-Valentine leucocidin S subunit (rLukS).

[0017] In some specific implementations, the Group B meningococcal antigen includes recombinant factor H binding protein variant 1 (rfHBPv1).

[0018] In some specific implementations, the Corynebacterium diphtheriae antigen includes recombinant diphtheria toxin fragment B (rDTB).

[0019] In some specific embodiments, the pneumococcal antigen includes recombinant pneumolysin (rPly) and / or recombinant pneumococcal surface protein A (rPspA).

[0020] In some specific implementations, the Haemophilus influenzae antigen includes outer membrane protein D (recombinant Haemophilus influenza protein D, rPD);

[0021] In some embodiments, the Staphylococcus aureus antigen includes one or more of Staphylococcus aureus enterotoxin A (rSEA), enterotoxin B (rSEB), enterotoxin C2 (rSEC2), and toxic shock toxin syndrome toxin 1 (rTSST1).

[0022] In some specific implementations, the Staphylococcus aureus antigen is Staphylococcus aureus enterotoxin A (rSEA), enterotoxin B (rSEB), enterotoxin C2 (rSEC2), and toxic shock toxin syndrome toxin 1 (rTSST1).

[0023] In some implementations, the coupling method includes one or more of the following:

[0024] (1) Polysaccharides are directly linked to recombinant proteins;

[0025] (2) The polysaccharide activated by 1-cyano-4-(dimethylamino)pyridine tetrafluoroborate (CDAP) was modified with adipic dihydrazide (ADH), and then bridging was performed with recombinant protein using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDAC);

[0026] (3) The CDAP-activated polysaccharide was modified with ADH, the recombinant protein was modified with succinic anhydride, and then EDAC was used for bridging.

[0027] (4) The CDAP-activated polysaccharide is bridged with the ADH-modified recombinant protein.

[0028] In some specific implementations, the coupling method involves the direct linking of polysaccharides to recombinant proteins.

[0029] In some specific implementations, the method includes the following:

[0030] (1) The mass ratio of polysaccharide to protein antigen is 1:(1-5);

[0031] (2) The reaction pH for the coupling of the polysaccharide with the protein antigen is 6.5–8.5;

[0032] (3) The reaction time is 1 to 8 hours.

[0033] In some implementations, the polysaccharide in (1) is a CDAP-activated polysaccharide.

[0034] In some specific implementations, the activation ratio of the polysaccharide to CDAP in (1) is 1:(0.05~0.5).

[0035] In some embodiments, the method further includes a step of purifying the obtained nanosized protein antigen;

[0036] In some specific embodiments, the step of purifying the nanoscale protein antigen includes ultrafiltration replacement of the nanoscale protein antigen with a 300 kDa ultrafiltration membrane, wherein the replacement buffer is a 0.85% NaCl solution, collecting the ultrafiltration replacement retentate, and filtering it through a 0.2 μm filter to obtain the purified nanoscale protein antigen.

[0037] In some specific embodiments, the method includes: hydrolyzing injectable grade sodium hyaluronate to obtain hydrolyzed sodium hyaluronate with a molecular weight of 200kDa to 400kDa. The activation concentration of the hydrolyzed sodium hyaluronate is 5mg / mL, the activation time is 2min, and the activation pH is 8.3. The mass ratio of hydrolyzed sodium hyaluronate to CDAP is 1:0.1, 1:0.2, or 1:0.3. The mass ratio of the activated hydrolyzed sodium hyaluronate to recombinant protein is 1:1, 1:2, or 1:3, and the reaction time is 2 to 8h to obtain the coupling reaction product. The coupling reaction product is ultrafiltered using a 300kDa ultrafiltration membrane, the replacement buffer being 0.85% NaCl solution, and the ultrafiltration retentate is collected. The retentate is filtered through a 0.2μm filter to obtain nano-sized antigen.

[0038] In some specific implementations, the polysaccharide is sodium hyaluronate.

[0039] In some specific implementations, the molecular weight of the sodium hyaluronate is 1000kDa to 4000kDa.

[0040] In some specific implementations, the molecular weight of the sodium hyaluronate is 1800kDa to 2000kDa.

[0041] In some specific implementations, the polysaccharide is hydrolyzed sodium hyaluronate.

[0042] In some specific implementations, the molecular weight of the hydrolyzed sodium hyaluronate is 200kDa-400kDa.

[0043] In some specific implementations, the hydrolyzed sodium hyaluronate has a molecular weight of 300 kDa.

[0044] The second aspect of the present invention provides nanoscaled protein antigens prepared by the method described in any one of the first aspects of the present invention.

[0045] A third aspect of the invention provides the use of nano-sized protein antigens as described in the second aspect of the invention in the preparation of bacterial vaccines.

[0046] In some embodiments, the bacteria belong to the genera Staphylococcus, Neisseria, Klebsiella, Escherichia, Clostridium, Clostridium, Salmonella, Shigella, Pseudomonas, Acinetobacter, Bordetella, Enterococcus, Haemophilus, Mycobacterium, Corynebacterium, or Streptococcus.

[0047] The fourth aspect of the present invention provides the use of nano-sized protein antigens as described in the second aspect of the present invention in the treatment or prevention of bacterial infections.

[0048] In some embodiments, the bacteria belong to the genera Staphylococcus, Neisseria, Klebsiella, Escherichia, Clostridium, Clostridium, Salmonella, Shigella, Pseudomonas, Acinetobacter, Bordetella, Enterococcus, Haemophilus, Mycobacterium, Corynebacterium, or Streptococcus.

[0049] A fifth aspect of the invention provides a method for treating or preventing bacterial infection, the method comprising administering a therapeutically effective amount of a nanoparticle protein antigen as described in a second aspect of the invention to a subject in need.

[0050] The therapeutically effective dose of the pharmaceutical composition of the present invention can initially be estimated in successful animal models, such as rodents, rabbits, dogs, pigs, and / or primates, using cell culture experiments. Animal models can also be used to determine suitable concentration ranges and routes of administration. These can then be used to determine the effective dose and route of administration in humans. Generally, the determination and adjustment of the effective amount or dose, and the assessment of when and how to make such adjustments, are known to those skilled in the art.

[0051] In some embodiments, the bacteria belong to the genera Staphylococcus, Neisseria, Klebsiella, Escherichia, Clostridium, Clostridium, Salmonella, Shigella, Pseudomonas, Acinetobacter, Bordetella, Enterococcus, Haemophilus, Mycobacterium, Corynebacterium, or Streptococcus.

[0052] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0053] The reagents and raw materials used in this invention are all commercially available.

[0054] The positive and progressive effects of this invention are as follows:

[0055] This invention obtains nanoscale antigens by conjugating recombinant protein antigens derived from bacterial pathogens with polysaccharides. The immunogenicity of the polysaccharide-conjugated recombinant protein antigens is significantly enhanced, overcoming the shortcomings of poor immunogenicity enhancement caused by competitive immune regulation between capsular polysaccharides and recombinant protein antigens in vivo. It also solves the safety and efficacy issues arising after the market launch of products using capsular polysaccharide antigens as a nanoscale matrix due to repeated or excessive inoculation with the capsular polysaccharide antigen, or interference with immunization programs. Furthermore, this invention has advantages such as simple and low-cost raw material acquisition, good process stability and easy scale-up, and high safety. Attached Figure Description

[0056] Figure 1 shows a comparative study of the immunogenicity of antigens modified with different nanostructured matrices. Detailed Implementation

[0057] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0058] The following embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the following embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0059] Example 1: Construction of recombinant protein antigen expression strain

[0060] According to the method shown in Table 1, the amino acid sequences of each recombinant protein antigen are as shown in SEQ ID NO: 1-11. The biotechnology company constructed the recombinant expression strains. In short, the amino acid sequence of the recombinant protein antigen was converted into a nucleotide sequence, optimized according to the codon preference of *E. coli*, and restriction enzyme sites were added to the N-terminus and C-terminus for full-fragment chemical synthesis. The synthesized nucleotide sequence was ligated into a T vector and transformed into DH5α competent cells. The correctly sequenced bacterial culture was used to extract plasmids using a kit, and the plasmids were then ligated into the pET28a / pET30a vector, transformed into BL21(DE3) competent cells, and the plasmids were extracted again to confirm correct sequencing.

[0061] Table 1. Construction information of four recombinant superantigen expression strains

[0062] Example 2: Fermentation of recombinant protein antigen expression strain

[0063] Prepare the first-generation seed culture medium according to Table 2. After preparation, add 0.5 mL of 30 mg / mL kanamycin sulfate stock solution.

[0064] Table 2. Formulation of seed culture medium for first-generation seed culture

[0065] Note: Kanamycin sulfate in Table 2 is a pre-prepared stock solution with a concentration of 30 mg / mL.

[0066] Take one vial of frozen glycerol bacteria, thaw it completely, and then inoculate 1.0 mL into an Erlenmeyer flask containing 0.5 L of seed culture medium. Incubate overnight at 32°C and 140 rpm on a shaker. OD 600 It reaches between 1 and 3.

[0067] Prepare the basic culture medium according to Table 3. After preparation, set the temperature to 35±0.5℃. Under flame protection and sterile conditions, add the defoaming agent, 2.5mL of kanamycin sulfate stock solution, K2HPO4 solution, MgSO4·7H2O solution in sequence, and then add the first-generation seed culture. The inoculation ratio of seed culture to culture medium is 1:2.5.

[0068] Table 3. Basic Culture Medium Formulation

[0069] Note: The defoamer in the table above is added before inoculation.

[0070] The fermentation process parameters were set as follows: pH 7.0, rotation speed controlled between 100 and 600 rpm, deep aeration set at 50 L / h, tank pressure controlled at 0.05 MPa, and dissolved oxygen ≥30%.

[0071] When OD 600 Once the value reaches 1 or higher, begin replenishing material in batches, once per hour, in stages, completing the replenishment during the logarithmic phase; perform OD (Original Discharge) analysis every hour. 600 Measurement, staining and microscopic examination.

[0072] When OD 600 When the value reaches around 10, start cooling to 20-32℃, add IPTG inducer to a final concentration of 0.1 mmol / L, and induce fermentation for 4-18 hours to complete the fermentation.

[0073] Stop fermentation, release the material, and harvest the fermentation liquid.

[0074] Example 3: Crude purification of engineered coli fermentation broth

[0075] 2.5L of fermentation broth was harvested and crushed using a homogenizer at 900 bar three times.

[0076] The rupture fluid was treated by membrane filtration using a 500kDa membrane pack (Millipore), and the clarified flowthrough was collected.

[0077] The clarified flow-through was concentrated using a 3kDa or 10kDa membrane pack (Cobate). After concentration to a certain volume, the concentrated retentate was washed and replaced with a replacement solution. After replacement, the sample was collected. After collection, a certain volume of replacement solution was added to rinse the membrane pack once, and the coarse sample was collected.

[0078] The replacement solutions mentioned above are not entirely consistent due to different antigens. The principle is to keep them consistent with the initial purification equilibration buffer used in column chromatography.

[0079] Example 4: Column chromatography purification of recombinant protein antigen

[0080] 4.1 Preparation of rSEA, rSEB, rSEC2 and rTSST1 protamine

[0081] (1) The initial purification packing material was DEAE Bestarose FF from BorgLone, with a sample loading volume of 5-10 mg / ml gel, and purification was performed using flow-through mode. The crude sample obtained in Example 3 was then loaded onto the gel and washed with the equilibration buffer for 3CV using 50 mmol / L Tris-HCl, pH 8.0, and 1 mmol / L EDTA. The loaded flow-through and the washed flow-through were collected and combined.

[0082] (2) The medium-purification packing material was Phenyl Bestarose HP from BorgL, and purification was performed using the dissociation mode. The combined flow-through solution from step (1) was equilibrated for 3CV with 50 mmol / L Tris-HCl, pH 8.0, containing 1 mmol / L EDTA and 1.8 mol / L (NH4)2SO4. The conductivity of the combined flow-through solution was adjusted to match that of the equilibration solution with 3.5 mol / L (NH4)2SO4 solution. Then, the sample was loaded and washed with the equilibration solution for 3CV. Then, the impurities were removed with 50 mmol / L Tris-HCl, pH 8.0, containing 1 mmol / L EDTA and 148 mS / cm to 165 mS / cm (NH4)2SO4 buffer for 2CV. Then, the solution was eluted with 50 mmol / L Tris-HCl, pH 8.0, containing 1 mmol / L EDTA and 119 mS / cm to 129 mS / cm (NH4)2SO4 buffer for 4CV. The eluent was collected.

[0083] During the moderate purification process, no impurity removal steps were performed on rSEB and rSEC2 proteins.

[0084] (3) The fine purification packing material was Polar MC30 HIC Butyl from Saifen Technology Co., Ltd., and purification was performed using the dissociation mode. The eluent from step (2) was equilibrated with 50 mmol / L Tris-HCl, pH 8.0, containing 1 mmol / L EDTA and 1.8 mol / L (NH4)2SO4 for 3CV. The conductivity of the eluent was adjusted to match that of the equilibration buffer with 3.5 mol / L (NH4)2SO4 solution. Then the sample was loaded and washed with the equilibration buffer for 3CV. The eluent was then purified with 50 mmol / L Tris-HCl, pH 8.0, containing 1 mmol / L EDTA and 148 mS / cm to 158 mS / cm (NH4)2SO4 buffer for 4CV. The eluent was then washed with 50 mmol / L Tris-HCl, pH 8.0, containing 1 mmol / L EDTA and 119 mS / cm to 185 mS / cm (NH4)2SO4 buffer for 4CV. The eluent was collected.

[0085] During the fine purification process, no impurity removal steps were required for rSEB and rSEC2 proteins.

[0086] (4) Ultrafiltration replacement: Using a 3kDa ultrafiltration membrane pack from Cobot, the finely purified eluent obtained in step (3) was subjected to ultrafiltration replacement. The replacement solution was 0.2mol / L borate, pH 8.4, containing 0.85% NaCl. The protein solution after replacement was filtered with a 0.2μm syringe filter. The filtered protein was protamine, which was frozen at -80℃ for later use.

[0087] 4.2 Preparation of rHla protamine

[0088] (1) The initial purification packing material was Cytiva's DEAE Sepharose FF, with a sample loading volume of 5-10 mg / ml gel, and purification was performed using flow-through mode. The sample was equilibrated with 50 mmol / L Tris-HCl, pH 9.0, containing 1 mmol / L EDTA and 2.25 mS / cm NaCl for 3CV. The crude sample obtained in Example 3 was then loaded onto the gel, followed by rinsing with the equilibration buffer for 3CV. The loaded flow-through and the rinse flow-through were collected and combined.

[0089] (2) The medium-purification packing material was Cytiva's Q Sepharose FF, and purification was performed using the flow-through mode. The purification method was the same as in step (1).

[0090] (3) The fine purification packing material was Phenyl Bestarose HP from BorgL, and purification was performed using the dissociation mode. The flow-through solution in step (2) was equilibrated with 50 mmol / L Tris-HCl, pH 8.5, containing 1 mmol / L EDTA and 1.5 mol / L (NH4)2SO4 for 3CV. The conductivity of the flow-through solution was adjusted to match that of the equilibration solution with 3.5 mol / L (NH4)2SO4 solution before loading the sample. Then, the flow-through solution was rinsed with the equilibration solution for 3CV. The flow-through solution was then eluted with 50 mmol / L Tris-HCl, pH 8.5, containing 1 mmol / L EDTA and 104 mS / cm (NH4)2SO4 buffer for 4CV. The eluent was collected.

[0091] (4) Ultrafiltration replacement is the same as step (4) in Example 4.1.

[0092] 4.3 Preparation of rLukS protamine

[0093] (1) The initial purification packing material was Cytiva's SP Sepharose FF, with a sample loading volume of 5-10 mg / ml gel, and purification was performed using the dissociation mode. The 3CV was equilibrated with 50 mmol / L Tris-HCl, pH 7.5, containing 1 mmol / L EDTA and 11 mS / cm NaCl buffer. The crude sample obtained in Example 3 was then loaded onto the gel, and the 3CV was washed with the equilibration buffer. The 3CV was then eluted with 50 mmol / L Tris-HCl, pH 7.5, containing 1 mmol / L EDTA and 17.5 mS / cm NaCl buffer, and the eluent was collected.

[0094] (2) The medium-purification packing material was Diamond Butyl Mustang from BorgLone, and purification was performed using the dissociation mode. The eluent from step (1) was equilibrated with 50 mmol / L Tris-HCl, pH 7.5, containing 1 mmol / L EDTA and 1.0 mol / L (NH4)2SO4 for 3CV. The conductivity of the eluent was adjusted to match that of the equilibration buffer with 3.5 mol / L (NH4)2SO4 solution. Then the sample was loaded, and the eluent was rinsed with the equilibration buffer for 3CV. Then the eluent was eluted with 50 mmol / L Tris-HCl, pH 7.5, containing 1 mmol / L EDTA and 61 mS / cm (NH4)2SO4 buffer for 4CV. The eluent was collected.

[0095] (3) The fine purification packing material was Cytiva's Q Sepharose FF, and purification was performed using flow-through mode. The 3CV was equilibrated with 50 mmol / L Tris-HCl, pH 8.5, containing 1 mmol / L EDTA. The eluent from step (2) was replaced by ultrafiltration with the equilibration buffer. The replacement membrane pore size was 3 kDa until the conductivity was consistent with the equilibration buffer. Then, the sample was loaded, and the 3CV was washed with the equilibration buffer. The flow-through was collected.

[0096] (4) Ultrafiltration replacement is the same as step (4) in Example 4.1.

[0097] 4.4 Preparation of rDTB protamine

[0098] (1) The initial purification packing material was Cytiva's SP Sepharose FF, and purification was performed using the dissociation mode. The crude sample obtained in Example 3 was equilibrated with 50 mmol / L PB, pH 7.0, containing 1 mmol / L EDTA and 1 mmol / L DTT buffer for 3CV. The crude sample was then loaded onto the buffer and washed with the equilibration buffer for 3CV. The crude sample was then washed with 50 mmol / L PB, pH 7.0, containing 1 mmol / L EDTA, 1 mmol / L DTT and 10 mS / cm NaCl buffer for 3CV to remove impurities. The crude sample was then washed with 50 mmol / L PB, pH 7.0, containing 1 mmol / L EDTA, 1 mmol / L DTT and 18 mS / cm NaCl buffer for 3CV. The eluent was collected.

[0099] (2) The fine purification packing material was Cytiva's Capto Q ImpRes, and purification was performed using the dissociation mode. The eluent from step (1) was equilibrated with a 50 mmol / L Tris-HCl, pH 8.5, containing 1 mmol / L EDTA, 1 mmol / L DTT, and 6 mS / cm NaCl for 3CV. The eluent from step (1) was replaced by ultrafiltration with the equilibration buffer. The replacement membrane pore size was 10 kDa until the conductivity was consistent with the equilibration buffer. Then, the sample was loaded, and the membrane was washed with the equilibration buffer for 3CV. Then, the membrane was eluted with 50 mmol / L Tris-HCl, pH 8.5, containing 1 mmol / L EDTA, 1 mmol / L DTT, and 12 mS / cm NaCl for 4CV. The eluent was collected.

[0100] (3) Ultrafiltration replacement is the same as step (4) in Example 4.1.

[0101] 4.5 Preparation of rfHBPv1 protamine

[0102] (1) The initial purification packing material was Cytiva's Q Sepharose FF, and purification was performed using the dissociation mode. The crude sample obtained in Example 3 was equilibrated with 50 mmol / L Tris-HCl, pH 8.0, containing 1 mmol / L EDTA buffer for 3 CVs. The crude sample was then loaded onto the buffer and washed with the equilibration buffer for 3 CVs. The crude sample was then washed with 50 mmol / L Tris-HCl, pH 8.0, containing 1 mmol / L EDTA, containing 12.5 mS / cm NaCl buffer for 2 CVs to remove impurities. The crude sample was then washed with 50 mmol / L Tris-HCl, pH 8.0, containing 1 mmol / L EDTA, containing 17.5 mS / cm NaCl buffer for 3 CVs. The eluent was collected.

[0103] (2) The fine purification packing material was Phenyl Bestarose HP from BorgL, and purification was performed using the dissociation mode. The eluent from step (1) was equilibrated with 50 mmol / L Tris-HCl, pH 8.5, containing 1 mmol / L EDTA and 1.8 mol / L (NH4)2SO4 for 3CV. The conductivity of the eluent was adjusted to match that of the equilibration buffer with 3.5 mol / L (NH4)2SO4 solution. Then the sample was loaded, and the eluent was rinsed with the equilibration buffer for 3CV. Then the eluent was eluted with 50 mmol / L Tris-HCl, pH 8.5, containing 1 mmol / L EDTA and 176 mS / cm (NH4)2SO4 buffer for 4CV. The eluent was collected.

[0104] (3) Ultrafiltration replacement is the same as step (4) in Example 4.1.

[0105] 4.6 Preparation of rPly protamine

[0106] (1) The initial purification packing material was Cytiva's DEAE Sepharose FF, with a sample loading volume of 5-10 mg / ml gel, and purification was performed using the elution mode. The crude sample obtained in Example 3 was then loaded onto the gel and eluted with the equilibration buffer containing 20 mmol / L Tris-HCl, pH 8.0, 1 mmol / L EDTA, and 0.062 mol / L NaCl for 3CV. The elution buffer was then used to elute the crude sample.

[0107] (2) The medium-purification packing material was Phenyl Bestarose HP from BorgL, and purification was performed using the dissociation mode. The 3CV was equilibrated with 20 mmol / L PB, pH 7.5, containing 1 mmol / L EDTA and 0.6 mol / L (NH4)2SO4. The combined flow-through solution from step (1) was adjusted to have the conductivity consistent with the equilibration solution using 3.5 mol / L (NH4)2SO4 solution. Then, the sample was loaded, and the 3CV was washed with the equilibration solution. The 3CV was then eluted with 20 mmol / L PB, pH 7.5, containing 1 mmol / L EDTA buffer. The eluent was collected.

[0108] (3) The fine purification packing material was Cytiva's Q Sepharose FF, and the purification was performed using the dissociation mode. The eluent from step (2) was diluted with 100 mmol / L Tris-HCl, pH 8.5, containing 1 mmol / L EDTA to equilibrate for 3CV. The conductivity of the eluent was adjusted to match that of the equilibration buffer by diluting it with the equilibration buffer. Then, the sample was loaded and washed with the equilibration buffer for 3CV. The eluent was then purified with 100 mmol / L Tris-HCl, pH 8.5, containing 1 mmol / L EDTA, containing 0.12 mol / L NaCl buffer for 4CV. The eluent was then washed with 50 mmol / L Tris-HCl, pH 8.0, containing 1 mmol / L EDTA, containing 119 mS / cm to 185 mS / cm (NH4)2SO4 buffer for 3CV. The eluent was collected.

[0109] (4) Ultrafiltration replacement is the same as step (4) in Example 4.1.

[0110] 4.7 Preparation of rPspA protamine

[0111] (1) The initial purification packing material was Cytiva's DEAE Sepharose FF, with a sample loading volume of 5-10 mg / ml gel, and purification was performed using the elution mode. The crude sample obtained in Example 3 was then loaded onto the gel and eluted with the equilibration buffer containing 20 mmol / L PB, pH 6.5, 1 mmol / L EDTA, and 0.2 mol / L NaCl for 3CV. The eluent was then collected.

[0112] (2) The medium-purification packing material was Phenyl Bestarose HP from BorgL, and purification was performed using the dissociation mode. The 3CV was equilibrated with 20 mmol / L PB, pH 7.5, containing 1 mmol / L EDTA and 1.4 mol / L (NH4)2SO4. The combined flow-through solution from step (1) was adjusted to have the conductivity consistent with the equilibration solution using 3.5 mol / L (NH4)2SO4 solution. Then, the sample was loaded, and the 3CV was washed with the equilibration solution. The 3CV was then eluted with 20 mmol / L PB, pH 7.5, containing 1 mmol / L EDTA and 0.5 mol / L (NH4)2SO4 buffer. The eluent was collected.

[0113] (3) The fine purification packing material was Cytiva's SP Sepharose FF, and the purification was performed using the dissociation mode. The 3CV was equilibrated with 50 mmol / L NaAC, pH 4.8, containing 1 mmol / L EDTA. The eluent from step (2) was diluted and replaced with the equilibration buffer until the conductivity was consistent with that of the equilibration buffer. Then the sample was loaded, and the 3CV was washed with the equilibration buffer. The 3CV was then eluted with 50 mmol / L NaAC, pH 4.8, containing 1 mmol / L EDTA, containing 0.097 mol / L NaCl. The eluent was collected.

[0114] (4) Ultrafiltration replacement is the same as step (4) in Example 4.1.

[0115] 4.8 Preparation of rPD protamine

[0116] (1) The initial purification packing material was Cytiva's DEAE Sepharose FF, with a sample loading volume of 5-10 mg / ml gel, and purification was performed using flow-through mode. The crude sample obtained in Example 3 was then loaded onto a 3CV buffer containing 50 mmol / L PB, pH 6.5, and 1 mmol / L EDTA. The sample was then washed with the equilibration buffer for 3CV, and the flow-through was collected.

[0117] (2) The medium-purification packing material was Cytiva's SP Sepharose FF, and purification was performed using the dissociation mode. The 3CV was equilibrated with 50 mmol / L PB, pH 6.5, containing 1 mmol / L EDTA buffer. The combined flow-through solution from step (1) was loaded onto the sample, and then the 3CV was washed with the equilibration buffer. The 3CV was then eluted with 20 mmol / L PB, pH 7.5, containing 1 mmol / L EDTA, containing 0.14 mol / L NaCl buffer, and the eluent was collected.

[0118] (3) The fine purification packing material was Phenyl Bestarose HP from Borglon, and purification was performed using flow-through mode. The 3CV was equilibrated with 50 mmol / L PB, pH 6.5, containing 1 mmol / L EDTA and 1.0 mol / L (NH4)2SO4. The conductivity of the 3.5 mol / L (NH4)2SO4 solution from step (2) was adjusted to match that of the equilibration solution. Then the sample was loaded, and the 3CV was washed with the equilibration solution. The flow-through solution was collected.

[0119] (4) Ultrafiltration replacement is the same as step (4) in Example 4.1.

[0120] 4.9 Quality Characterization of Recombinant Protein Antigens

[0121] Endotoxin content was characterized for each protamine antigen using horseshoe crab reagent, and purity was characterized using reducing SDS-PAGE and non-reducing SDS-PAGE. Results showed that the purity of each protein antigen was above 95%, and the endotoxin content was below 0.1 EU / μg.

[0122] Example 5: Preparation of Pneumococcal Capsular Polysaccharide

[0123] Streptococcal strains producing capsular polysaccharides of types 3 (Pn3), 6B (Pn6B), 9V (Pn9V), 14 (Pn14), and 15B (Pn15B) were cultured in tryptone medium at 37°C for 10 h. The obtained bacterial culture was inactivated with 0.5% sodium deoxycholate, centrifuged at 8000g for 30 min, and the supernatant was collected. The supernatant was concentrated by ultrafiltration with a 100 kDa membrane and fractionated with 25-80% ethanol. The precipitate was collected to obtain crude polysaccharides, which were then lyophilized. The crude polysaccharides were dissolved in sterile water for injection, and then 100 mmol / L Tris-HCl solution (pH 8.0, containing 3.0-4.25 mol / L NaCl) was added. After thorough mixing, the sample was prepared. The Diamond Mix A mustang (XK50 / 30) chromatography column was equilibrated for 2 CVs at a flow rate of 30 cm / h using 100 mmol / L Tris-HCl, pH 8.0, containing 3.0–4.25 mol / L NaCl solution. The sample was loaded onto the Diamond Mix A mustang column. After loading, the column was washed for 1 CV with 100 mmol / L Tris-HCl, pH 8.0, containing 3.0–4.25 mol / L NaCl solution. The column was then washed with 0.5 mol / L NaOH solution for 2 CVs, and finally equilibrated for 4 CVs with 100 mmol / L Tris-HCl, pH 8.0, containing 3.0–4.25 mol / L NaCl solution. Collect the loading flow-through and rinsing flow-through, and concentrate and replace them using a 10 kDa (Millipore) ultrafiltration membrane. The replacement solutions are 0.9% NaCl solution and water for injection. Replace the membrane three times with 0.9% NaCl solution, then three times with water for injection to obtain the refined saccharide solution. Finally, sterilize the desalted refined saccharide solution using a 0.2 μm capsule filter (Cobalt). After filtration, the refined saccharide solution is placed in a lyophilization vessel, and the lyophilization temperature is controlled below -30°C, the vacuum degree below 1 mba, and the lyophilization time is at least 28 hours. After lyophilization, weigh the lyophilized refined saccharide into a 250 ml reagent bottle and store it below -20°C for later use.

[0124] Example 6: Preparation of Neisseria meningitidis capsule polysaccharide

[0125] Meningococcal strains producing group A (MenA) and group Y (MenY) capsular polysaccharides were cultured on soybean peptone medium at 35°C for 8–10 h. The resulting bacterial culture was inactivated with 0.5% formaldehyde, diluted, and precipitated with sodium deoxycholate. The supernatant was collected by centrifugation and concentrated by ultrafiltration with a 100 kDa membrane. Fractionation was performed using 25–80% ethanol, and the precipitate was collected to obtain crude polysaccharides. The crude polysaccharides were then lyophilized. The crude polysaccharides were dissolved in sterile water for injection, and then 20 mmol / L Tris-HCl solution (pH 8.0) containing 1.0–4.25 mol / L NaCl was added. After thorough mixing, the sample was prepared. The Diamond Mix A mustang (XK50 / 30) chromatography column was equilibrated for 2 CVs at a flow rate of 30 cm / h using 20 mmol / L Tris-HCl, pH 8.0, containing 1.0–4.25 mol / L NaCl solution. The sample was then loaded onto the Diamond Mix A mustang column. After loading, the column was washed for 1 CV with 20 mmol / L Tris-HCl, pH 8.0, containing 1.0–4.25 mol / L NaCl solution. The column was then washed with 0.5 mol / L NaOH solution for 2 CVs, and finally equilibrated for 4 CVs with 20 mmol / L Tris-HCl, pH 8.0, containing 1.0–4.25 mol / L NaCl solution. The sample flow-through and the rinsing flow-through were collected and concentrated using a 10 kDa (Millipore) ultrafiltration membrane. The replacement solution was 20 mmol / L Tris-HCl, pH 8.0, containing 0–1.0 mol / L NaCl solution, to obtain a preliminarily purified flow-through. A Diamond Mix A mustang (XK50 / 30) chromatography column was equilibrated for 2 CVs at a flow rate of 30 cm / h using 20 mmol / L Tris-HCl, pH 8.0, containing 0–1.0 mol / L NaCl. The pre-purified flow-through was loaded onto the Diamond Mix A mustang column. After loading, the column was washed for 3 CVs with 20 mmol / L Tris-HCl, pH 8.0, containing 0–1.0 mol / L NaCl. The column was then eluted for another 3 CVs with 20 mmol / L Tris-HCl, pH 8.0, containing 1.0–4.25 mol / L NaCl. The eluent was collected. The column was then washed for 2 CVs with 0.5 mol / L NaOH solution, and equilibrated for 4 CVs with 20 mmol / L Tris-HCl, pH 8.0, containing 1.0–4.25 mol / L NaCl.The collected eluent was concentrated and replaced using a 10 kDa (Millipore) ultrafiltration membrane. The replacement fluid consisted of 0.9% NaCl solution and water for injection. The solution was replaced three times with 0.9% NaCl solution, followed by three replacements with water for injection to obtain a refined saccharide solution. Finally, the desalted refined saccharide solution was sterilized using a 0.2 μm capsule filter (Cobalt). After filtration, the refined saccharide solution was placed in a lyophilization vessel, and the lyophilization temperature was controlled below -30°C, the vacuum degree below 1 mba, and the lyophilization time was at least 28 hours. After lyophilization, the lyophilized refined saccharide was weighed into a 250 ml reagent bottle and stored below -20°C for later use.

[0126] Example 7: Preparation of Nanoscale Protein Antigens Using Capsular Polysaccharide as the Nanoscale Matrix

[0127] (1) Hydrolysis of refined capsular polysaccharides

[0128] Weigh 500 mg of refined saccharide obtained in Example 5 or Example 6 and dissolve it in 0.85% NaCl solution to a concentration of 5 mg / mL. At the start of the reaction, add 200 μl of a degradation solution containing 100 mmol / L ascorbic acid, 10 mmol / L CuSO4·5H2O, and 10 mmol / L FeSO4·7H2O. After 1 hour of reaction, add another 200 μl of the degradation solution and continue the reaction for 2 hours until the reaction ends. Ultrafilter the reaction solution using a 300 kDa ultrafiltration membrane (Millipore) and collect the permeate. Concentrate and replace the permeate using a 10 kDa ultrafiltration membrane (Millipore) with a 0.9% NaCl solution, replacing it 10 times its volume three times. Collect the retentate after replacement and filter it through a 0.45 μm filter into a lyophilization vessel. Set the lyophilizer (CHRIST, Alpha 1-2LDplus) parameters as follows: vacuum below 0.5 mbar, lyophilization temperature below -40°C, and lyophilization time greater than 28 hours. The molecular size of hydrolyzed sugars was determined using the SEC-MALLs method.

[0129] (2) Nanofiber modification of recombinant protein antigen

[0130] Weigh 50 mg of hydrolyzed sugar and dissolve it in 0.85% NaCl solution to a concentration of 5 mg / mL. Add 90 μl of 1-cyano-4-(dimethylamino)pyridine tetrafluoroborate (CDAP) solution (100 mg / mL), and then maintain the pH at 7.0–9.0 with 3% triethylamine (Sigma) solution for 2 min. Add 10 mL of protamine solution at a concentration of 5–20 mg / mL and react for 1–8 h. After the reaction is complete, add 0.85% NaCl solution to a concentration of 1 L, concentrate and replace the solution three times using a 300 kDa ultrafiltration membrane (Millipore), and finally collect the retentate. Filter the solution through a 0.2 μm filter to obtain the nano-sized protein antigen.

[0131] (3) Quality properties of nanomaterial antigens

[0132] The obtained nanoscale superantigens were analyzed using the Lowry method to determine the protein concentration in the conjugates, the lichenol method to determine the polysaccharide concentration in the conjugates, the DOC precipitation-lichenol method to determine the free sugar concentration in the conjugates, and the SEC-MALLs method or CL-4B method to determine the molecular size of the conjugates.

[0133] Table 4. Quality attributes of nanoscale antigens using capsular polysaccharide as the nanoscale matrix.

[0134] Example 8: Preparation of Nanoscale Protein Antigens Using Sodium Hyaluronate as the Nanoscale Matrix

[0135] Injectable sodium hyaluronate (HA) was purchased from Shandong Zhongshan Biotechnology. The preparation method of the nano-sized protein antigen was the same as in Example 7. The quality properties of the obtained nano-sized protein antigen are shown in Table 5.

[0136] Table 5. Quality properties of nano-antigens using sodium hyaluronate as the nano-matrix.

[0137] Example 9: Comparative Study on the Effects of Different Nanoparticle Matrix on Immunogenicity Enhancement

[0138] This study used rTSST1 antigen as a protein antigen to compare the immune-enhancing effects of different capsular polysaccharides and sodium hyaluronate modifications on TSST1 antigen.

[0139] Based on the quality attribute information of the nanomaterial protein antigen obtained in Examples 7 and 8, the nanomaterial protein antigen was diluted with sterile 0.85% NaCl solution to prepare a concentration of 25 μg / mL. This was then mixed with an equal volume of HA201 liposome adjuvant (Beijing Huanotai Biomedical Technology Co., Ltd., whose main components are QS-21 (100 μg / mL), 3D-MLA (100 μg / mL), DOPC (2 mg / mL), and cholesterol (0.5 mg / mL)). The mixture was inverted more than ten times to ensure thorough mixing, thus forming the immunizing agent. Using BALB / c mice as an animal model, eight mice per group were administered a two-dose immunization program (two weeks apart). The immunization dose was 0.1 mL via intramuscular injection in the leg, resulting in an immunization dose of 1.25 μg / dose for each group. Blood was collected from the orbital rim two weeks after the second immunization.

[0140] The titer of rTSST1 antigen-specific IgG was detected by indirect ELISA. rTSST1 antigen was diluted to 10 μg / mL, and 100 μL / well was coated onto an ELISA plate and incubated overnight at 4°C. Antisera from each experimental group were serially diluted, and 100 μL was added to each well, incubated at 37°C for 120 min. Then, 200 μL of washing buffer was added to each well for washing, for a total of 5 washes. After washing, 4000-fold diluted goat anti-mouse alkaline phosphatase-labeled secondary antibody (Southern Biotech) was added, and the plate was incubated at 37°C for 120 min. After incubation, 200 μL of washing buffer was added to each well for washing, for a total of 5 washes. Then, 100 μL of chromogenic solution (prepared from magnesium chloride hexahydrate (Sinopharm) and diethanolamine (Sigma) at a ratio of 1 (g): 1 (ml)) was added to each well, and the plate was incubated at 20–25°C for 60 min. Finally, 50 μL of stop solution (3 mol / L) was added. NaOH solution); the value of the ELISA plate was read using an ELISA reader, and the Cutoff value was determined to be 0.2 based on the negative serum.

[0141] Table 6 Comparative Study on the Immunogenicity Enhancement Effects of Different Nanomaterials

[0142] The results are shown in Table 6 and Figure 1. The immune groups using sodium hyaluronate as the nano-matrix were significantly higher than those conjugated with Streptococcus pneumoniae capsular polysaccharide and Neisseria meningitidis capsular polysaccharide (p<0.05, One-way ANOVA). This indicates that sodium hyaluronate, which has good biocompatibility, does not compete with protein antigens for immunomodulation after conjugation, thus having a better immune-enhancing effect.

[0143] Example 10: Study on the immunogenicity enhancement effect of sodium hyaluronate on different protein antigens.

[0144] This study used sodium hyaluronate (HA) as a nanomaterial matrix to investigate the immune-enhancing effects of sodium hyaluronate on different protein antigens.

[0145] The nano-sized protein antigen prepared in Example 8 was diluted with sterile 0.85% NaCl solution to a certain concentration, and an immunomodulator was prepared with or without adjuvant. Using BALB / c mice as an animal model, 8 mice per group were administered a two-dose immunization program (two weeks apart). 0.1 mL of immunization was injected intramuscularly into the leg. Blood was collected from the orbital sinus two weeks after the second immunization. The specific IgG titers of each protein were detected using an indirect ELISA method, the same as in Example 9.

[0146] The results are shown in Table 7. Under adjuvant-free conditions, the IgG GMT of 1 / 10 dose of nano-sized protein antigen was 5–34,896 times higher than that of recombinant protein antigen. Under AlPO4 adjuvant conditions, the IgG GMT of 1 / 2 dose of nano-sized protein antigen was 6–371 times higher than that of recombinant protein antigen. For large protein molecules, under Al(OH)3 adjuvant conditions, the IgG GMT effect of 1 / 5 dose of nano-sized protein antigen was comparable to that of the full dose of recombinant protein antigen. This study fully demonstrates the effect of sodium hyaluronate nano-modification in enhancing the immunogenicity of recombinant protein antigen.

[0147] Table 7 Comparative Study of the Immunogenicity Enhancement Effects of Sodium Hyaluronate on Different Protein Antigens.

[0148] Note: The aluminum content in the aluminum adjuvants in each group in the table above is 50 μg / dose.

[0149] Example 11 Comparative Study of Immunogenicity of Four-Component Nanoscale Antigens

[0150] This study prepared four-component protein immunotherapy drugs and four-component nanoparticle protein immunotherapy drugs by mixing four recombinant proteins and four nanoparticle proteins respectively, and compared the immune enhancement effect of sodium hyaluronate modification in multi-component vaccines.

[0151] The recombinant protein antigens rSEA, rSEB, rSEC2, and rTSST1 prepared in Example 4 were diluted with sterile 0.85% NaCl solution to prepare a mixture of four antigens with a concentration of 400 μg / mL. The mixture was then mixed with HA201 liposome adjuvant by inverting the mixture more than ten times to obtain the four-component protein vaccine.

[0152] The rSEA-HA, rSEB-HA, rSEC2-HA and rTSST1-HA nanoparticle protein antigens prepared in Example 8 were diluted with sterile 0.85% NaCl solution to prepare a mixture of four antigens with an antigen concentration of 25 μg / mL. The mixture was then mixed with HA201 liposome adjuvant by inverting the mixture more than ten times to obtain the four-component nanoparticle protein vaccine.

[0153] Using BALB / c mice as an animal model, 8 mice per group were immunized with a two-dose immunization schedule (two weeks apart). The two vaccines were administered via intramuscular injection (0.1 mL) into the leg. Four weeks after the second immunization, blood was collected from the orbital cavity. The specific IgG titers of each protein were detected using an indirect ELISA method, the same as in Example 9.

[0154] The results are shown in Table 8. The immunization dose of each antigen in the nano-protein vaccine group was 1.25 μg / dose, and the immunization dose of each antigen in the protein vaccine group was 20 μg / dose. With the same adjuvant, the results showed that the IgG GMT of each antigen in the nano-protein vaccine was 6 to 30 times higher than that in the protein vaccine group (p<0.01, One-way ANOVA). This indicates that after the recombinant protein was modified by sodium hyaluronate nano-sizing, it can induce a very high level of immune response at a lower immunization dose, and further improve the safety of the vaccine.

[0155] Table 8 Comparative Study of Immunogenicity of Four-Component Protein Vaccine and Four-Component Nanoparticle Protein Vaccine

[0156] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A method for enhancing the immunogenicity of recombinant protein antigens, characterized in that, The method includes forming a nano-sized protein antigen by coupling a polysaccharide with a recombinant protein antigen; The polysaccharide is selected from sodium hyaluronate, chitosan, dextran, fucoidan and sodium alginate, preferably sodium hyaluronate; The recombinant protein antigen is a bacterial protein antigen, and the bacteria belong to the genera Staphylococcus, Neisseria, Klebsiella, Escherichia, Clostridium, Salmonella, Shigella, Pseudomonas, Acinetobacter, Bordetella, Enterococcus, Haemophilus, Mycobacterium, or Streptococcus.

2. The method as described in claim 1, characterized in that, The bacteria are selected from one or more of the following: Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Neisseria meningitidis, Neisseria gonorrhoeae, Klebsiella pneumoniae, Escherichia coli, Clostridium tetani, Clostridium botulinum, Clostridium difficile and Clostridium perfringens, Salmonella typhi, Salmonella paratyphi, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Acinetobacter baumannii, Bordetella pertussis, Enterococcus faecalis, Haemophilus influenzae, Mycobacterium tuberculosis and Mycobacterium bovis, Group A Streptococcus, Group B Streptococcus pneumoniae, and Streptococcus pneumoniae. Preferably, the bacteria are selected from one or more of the following: Staphylococcus aureus, Neisseria meningitidis group B, Corynebacterium diphtheriae, Streptococcus pneumoniae, and Haemophilus influenzae.

3. The method as described in claim 1 or 2, characterized in that, The Staphylococcus aureus antigen includes one or more of Staphylococcus aureus enterotoxin A, enterotoxin B, enterotoxin C2, toxic shock toxin syndrome toxin 1, α-hemolysin, and leukocidin S; the Neisseria meningitidis group B antigen includes v1 type H factor binding protein; the Corynebacterium diphtheriae antigen includes a fragment of diphtheria toxin B; the Pneumococcus antigen includes pneumococcal hemolysin and / or pneumococcal surface protein A; and the Haemophilus influenzae antigen includes outer membrane protein D. Preferably, the Staphylococcus aureus antigen includes one or more of Staphylococcus aureus enterotoxin A, enterotoxin B, enterotoxin C2, and toxic shock toxin syndrome toxin 1.

4. The method according to any one of claims 1-3, characterized in that, The coupling method includes one or more of the following: (1) Polysaccharides are directly linked to recombinant proteins; (2) The CDAP-activated polysaccharide was modified with adipic acid dihydrazide and then bridging was performed with carbodiimide to bind the recombinant protein. (3) The CDAP-activated polysaccharide was modified with adipic acid dihydrazide, the recombinant protein was modified with succinic anhydride, and then bridging was performed using carbodiimide; (4) The CDAP-activated polysaccharide was bridged with the recombinant protein modified with adipic acid dihydrazide; Preferably, the coupling method involves direct linking of the polysaccharide to the recombinant protein.

5. The method as described in claim 4, characterized in that, The method includes the following: (1) The mass ratio of polysaccharide to protein antigen is 1:(1-5); preferably, the polysaccharide is CDAP-activated polysaccharide; more preferably, the activation ratio of the polysaccharide to CDAP is 1:(0.05-0.5); (2) The reaction pH for the coupling of the polysaccharide with the protein antigen is 6.5–8.5; (3) The reaction time is 1 to 8 hours.

6. The method as described in claim 5, characterized in that, The method also includes a step of purifying the obtained nano-sized protein antigen; Preferably, the step of purifying the nano-sized protein antigen includes ultrafiltration replacement of the nano-sized protein antigen using a 300kDa ultrafiltration membrane, wherein the replacement buffer is a 0.85% NaCl solution, the ultrafiltration replacement retentate is collected, and the purified nano-sized protein antigen is obtained by filtration through a 0.2μm filter.

7. The method as described in claim 6, characterized in that, The polysaccharide is sodium hyaluronate, preferably hydrolyzed sodium hyaluronate, and more preferably the hydrolyzed sodium hyaluronate has a molecular weight of 200kDa-400kDa.

8. The nanomaterialized protein antigen prepared by the method according to any one of claims 1-7.

9. The use of the nano-sized protein antigen as described in claim 8 in the preparation of bacterial vaccines.

10. The application as described in claim 9, characterized in that, The bacteria belong to the genera Staphylococcus, Neisseria, Klebsiella, Escherichia, Clostridium, Salmonella, Shigella, Pseudomonas, Acinetobacter, Bordetella, Enterococcus, Haemophilus, Mycobacterium, or Streptococcus.

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