Method for enhancing immunogenicity of recombinant protein antigen and application

Abstract

The invention discloses a method for enhancing recombinant protein antigen immunogenicity and application. The method comprises the following steps: coupling polysaccharide with a recombinant protein antigen to form a nanocrystallization protein antigen; the polysaccharide is selected from sodium hyaluronate, chitosan, glucan, fucoidin or sodium alginate; the recombinant protein antigen is a protein antigen derived from bacteria, and the bacteria belong to Staphylococcus, Neisseria, Klebsiella, Escherichia, Clostridium, Salmonella, Shigella, Pseudomonas, Acinetobacter, Bordetella, Enterococcus, Haemophilus, Mycobacterium or Streptococcus. The immunogenicity of the recombinant protein antigen is improved, and meanwhile, the defects of competitive immunoregulation and poor immunoenhancement effect caused by bacterial capsular polysaccharide modification are overcome. The invention also has the advantages of high clinical application value, simple raw material acquisition, low cost, good process stability, easy amplification, high safety and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for enhancing the immunogenicity of a recombinant protein antigen and its application. Background Art

[0002] As of the beginning of 2023, there are 966 vaccines in the clinical stage worldwide, mainly using recombinant protein vaccines, nucleic acid vaccines, conjugate vaccines, viral vector vaccines, inactivated vaccines and attenuated vaccines, of which recombinant protein vaccines account for the highest proportion (22%). Recombinant protein vaccines have shown excellent application prospects among many vaccine technologies due to their safety, high efficiency, mature technology platform and easy large-scale production.

[0003] Recombinant protein vaccines, also known as genetically engineered recombinant subunit vaccines, are vaccines made using genetic engineering technology to modify important components of pathogenic microorganisms that have immune activity. The parts that have immune activity are usually surface antigens of pathogenic microorganisms, toxin antigens that have host cell killing effects, and antigens that bind to receptor cells. These protein antigens usually have a molecular weight of 10 to 100 kDa. For protein antigens with a small molecular weight or fewer antigenic epitopes, they often have weak immunogenicity and cannot induce a high level of immune response, thus failing to provide effective immune protection.

[0004] Polysaccharide conjugate vaccines use protein antigens with T cell epitopes to couple with polysaccharide antigens, overcoming the defect that polysaccharide antigens alone cannot induce T cell immune responses, stimulating the immune responses of T cells and B cells, converting IgM with low antibody affinity into IgG with high affinity, and producing memory B cells and plasma cells. Inspired by this principle, the applicant found that the use of 9V type pneumococcal capsular polysaccharides coupled to the truncated S protein (RBD protein) of the new coronavirus (SARS-CoV-2) can significantly enhance its immunogenicity (CN113150084A); CN116113644A also found that type 14 and 7F pneumococcal capsular polysaccharides and glucans have an immune-enhancing effect on the immunogenicity of RBD protein.

[0005] The inventors speculate that the main principle of polysaccharide antigens to enhance the immunogenicity of recombinant proteins is to increase the molecular size of protein antigens and improve the efficiency of antigen presentation, thereby improving the response level of the immune system. However, when exogenous polysaccharide antigens such as pneumococcal capsular polysaccharides, meningococcal capsular polysaccharides, and Haemophilus influenzae capsular polysaccharides are used as immune enhancement matrices for recombinant protein vaccines, there are the following shortcomings: (1) Competitive immunoregulation occurs with protein antigens. As an exogenous substance, capsular polysaccharides themselves have certain immunogenicity. After being coupled with proteins and immunized to the body at the same time, competitive immunoregulation occurs, which is particularly evident in multi-component vaccines and cannot exert the ideal immune enhancement effect. (2) It is impossible to share the vaccination population with the marketed vaccines containing the polysaccharide antigens. If the same age group is vaccinated with the marketed polysaccharide vaccine or polysaccharide combination vaccine, there will be repeated vaccination of polysaccharide antigens, excessive immune doses, interference with the immune program, etc., which will have a great impact on the immune effect of the marketed polysaccharide vaccine or polysaccharide combination vaccine, and will also bring great safety risks to the vaccination population.

[0006] Therefore, based on the shortcomings of the above-mentioned technologies, developing an immune enhancement method that can significantly enhance the immunogenicity of recombinant protein antigens without affecting the safety and effectiveness of marketed vaccines is an issue that urgently needs to be addressed. Summary of the invention

[0007] In order to solve the above technical problems, the present invention discloses a method for enhancing the immunogenicity of a recombinant protein antigen and its application.

[0008] Specifically, the first aspect of the present invention provides a method for enhancing the immunogenicity of a recombinant protein antigen, the method comprising coupling a polysaccharide with the recombinant protein antigen to form a nanosized protein antigen.

[0009] In some embodiments, the polysaccharide is selected from the group consisting of sodium hyaluronate (HA), chitosan, dextran, fucose and sodium alginate.

[0010] In some specific embodiments, the polysaccharide is sodium hyaluronate.

[0011] In some embodiments, the recombinant protein antigen is a bacterial protein antigen, and the bacteria belongs to the genus Staphylococcus, Neisseria, Klebsiella, Escherichia, Clostridium, Clostridioides, Salmonella, Shigella, Pseudomonas, Acinetobacter, Bordetella, Enterococcus, Haemophilus, Mycobacterium, Corynebacterium or Streptococcus.

[0012] In some specific embodiments, the bacteria are selected from one or more of the following: Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), Staphylococcus saprophyticus (S. saprophyticus), Neisseria meningitidis (N. meningitidis), Neisseria gonorrhoeae (N. gonorrhoeae), Klebsiella pneumoniae (K. pneumoniae), Escherichia coli (E. coli), Clostridium tetani (C. tetani), Clostridium botulinum (C. botulinum), Clostridium difficile (C. difficile) and Clostridium perfringens (C. perfringens), Salmonella typhi (S.typhi), Salmonella paratyphi (S.paratyphi), Shigella flexneri (S.flexneri), Shigella sonnei (S.sonnei), Pseudomonas aeruginosa (P.aeruginosa), Acinetobacter baumannii (A.baumanii), Bordetella pertussis (B.pertussis), Enterococcus faecium (E.faecium), Enterococcus faecalis (E.faecalis), Haemophilus influenzae (H.influenzae), Mycobacterium tuberculosis (M.tuberculosis), Mycobacterium bovis (M.bovis), Corynebacterium diphtheriae (C.diphtheriae), Group A Streptococcus (Group A Streptococcus), Group B Streptococcus (Group B Streptococcus) and Streptococcus pneumoniae (S.pneumoniae).

[0013] In some specific embodiments, the bacteria are selected from one or more of the following: Staphylococcus aureus (S. aureus), Group B meningococcal, Corynebacterium diphtheriae (C. diphtheriae), Streptococcus pneumoniae (S. pneumoniae) and Haemophilus influenzae (H. influenzae).

[0014] In some specific embodiments, the Staphylococcus aureus (S. aureus) antigens include one or more of recombinant Staphylococcus aureus enterotoxin A (rSEA), recombinant enterotoxin B (rSEB), recombinant enterotoxin C2 (rSEC2), toxic shock syndrome toxin 1 (rTSST1), α-hemolysin (rHlα) and recombinant Panton-Valentine leucocidin S subunit (rLukS).

[0015] In some specific embodiments, the Group B meningococcal antigen comprises recombinant factor H binding protein variant 1 (rfHBPv1).

[0016] In some specific embodiments, the C. diphtheriae antigen comprises a recombinant diphtheria toxin fragment B (rDTB).

[0017] In some specific embodiments, the pneumococcal (S.pneumoniae) antigens include recombinant pneumolysin (rPly) and / or recombinant pneumococcal surface protein A (rPspA).

[0018] In some specific embodiments, the H. influenzae antigen includes recombinant Haemophilus influenza protein D (rPD);

[0019] In some embodiments, the Staphylococcus aureus (S. aureus) antigens include one or more of Staphylococcus aureus enterotoxin A (rSEA), enterotoxin B (rSEB), enterotoxin C2 (rSEC2), and toxic shock syndrome toxin 1 (rTSST1).

[0020] In some specific embodiments, the Staphylococcus aureus (S. aureus) antigens are Staphylococcus aureus enterotoxin A (rSEA), enterotoxin B (rSEB), enterotoxin C2 (rSEC2), and toxic shock syndrome toxin 1 (rTSST1).

[0021] In some embodiments, the coupling method includes one or more of the following:

[0022] (1) Direct linkage of polysaccharides to recombinant proteins;

[0023] (2) The polysaccharide activated by 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate (CDAP) was modified with adipic dihydrazide (ADH) and then bridged with the recombinant protein using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDAC);

[0024] (3) The CDAP-activated polysaccharide was modified with ADH, the recombinant protein was modified with succinic anhydride, and then bridged with EDAC;

[0025] (4) Bridge the CDAP-activated polysaccharide with the ADH-modified recombinant protein.

[0026] In some specific embodiments, the coupling method is direct connection of the polysaccharide to the recombinant protein.

[0027] In some specific embodiments, the method comprises the following:

[0028] (1) The mass ratio of polysaccharide to protein antigen is 1:(1-5);

[0029] (2) The reaction pH of the polysaccharide and protein antigen coupling is 6.5 to 8.5;

[0030] (3) The reaction time is 1 to 8 hours.

[0031] In some embodiments, the polysaccharide in (1) is a CDAP-activated polysaccharide.

[0032] In some specific embodiments, the activation ratio of the polysaccharide to CDAP in (1) is 1:(0.05-0.5).

[0033] In some embodiments, the method further comprises the step of purifying the obtained nanosized protein antigen;

[0034] In some specific embodiments, the step of purifying the nanosized protein antigen comprises ultrafiltration replacement of the nanosized protein antigen using a 300 kDa ultrafiltration membrane, the replacement buffer is 0.85% NaCl solution, collecting the ultrafiltration replacement retentate, and filtering through a 0.2 μm filter to obtain the purified nanosized protein antigen.

[0035] In some specific embodiments, the method includes: hydrolyzed sodium hyaluronate obtained after hydrolysis of injection-grade sodium hyaluronate, with a molecular weight of 200kDa to 400kDa. The activation reaction concentration of the hydrolyzed sodium hyaluronate is 5mg / mL, the activation time is 2min, and the activation pH is 8.3. The feed mass ratio of hydrolyzed sodium hyaluronate to CDAP is 1:0.1, 1:0.2 or 1:0.3. The feed mass ratio of hydrolyzed sodium hyaluronate to recombinant protein after activation is 1:1, 1:2 or 1:3, and the reaction time is 2 to 8h before obtaining the coupling reaction product. The coupling reaction product is ultrafiltered and replaced with a 300kDa ultrafiltration membrane bag, and the replacement buffer is a 0.85% NaCl solution, and the ultrafiltration replacement retentate is collected. The retentate is filtered through a 0.2μm filter to obtain a nano-antigen.

[0036] In some specific embodiments, the polysaccharide is sodium hyaluronate.

[0037] In some specific embodiments, the molecular weight of the sodium hyaluronate is 1000 kDa to 4000 kDa.

[0038] In some specific embodiments, the molecular weight of the sodium hyaluronate is 1800 kDa to 2000 kDa.

[0039] In some specific embodiments, the polysaccharide is hydrolyzed sodium hyaluronate.

[0040] In some specific embodiments, the molecular weight of the hydrolyzed sodium hyaluronate is 200 kDa-400 kDa.

[0041] In some specific embodiments, the hydrolyzed sodium hyaluronate has a molecular weight of 300 kDa.

[0042] The second aspect of the present invention provides a nanosized protein antigen prepared by the method described in any one of the first aspects of the present invention.

[0043] The third aspect of the present invention provides the use of the nanosized protein antigen as described in the second aspect of the present invention in the preparation of bacterial vaccines.

[0044] In some embodiments, the bacteria is of the genus Staphylococcus, Neisseria, Klebsiella, Escherichia, Clostridium, Clostridioides, Salmonella, Shigella, Pseudomonas, Acinetobacter, Bordetella, Enterococcus, Haemophilus, Mycobacterium, Corynebacterium, or Streptococcus.

[0045] The fourth aspect of the present invention provides the use of the nanosized protein antigen as described in the second aspect of the present invention in treating or preventing bacterial infection.

[0046] In some embodiments, the bacteria is of the genus Staphylococcus, Neisseria, Klebsiella, Escherichia, Clostridium, Clostridioides, Salmonella, Shigella, Pseudomonas, Acinetobacter, Bordetella, Enterococcus, Haemophilus, Mycobacterium, Corynebacterium, or Streptococcus.

[0047] The fifth aspect of the present invention provides a method for treating or preventing bacterial infection, the method comprising administering a therapeutically effective amount of the nanosized protein antigen as described in the second aspect of the present invention to a subject in need thereof.

[0048] The therapeutically effective dose for the pharmaceutical composition of the present invention can be initially estimated in cell culture experiments in animal models such as rodents, rabbits, dogs, pigs and / or primates. Animal models can also be used to determine appropriate concentration ranges and routes of administration. They can then be used to determine useful doses and routes for administration in humans. In general, the determination and adjustment of an effective amount or dose for administration and the assessment of when and how to make such adjustments are known to those skilled in the art.

[0049] In some embodiments, the bacteria is of the genus Staphylococcus, Neisseria, Klebsiella, Escherichia, Clostridium, Clostridioides, Salmonella, Shigella, Pseudomonas, Acinetobacter, Bordetella, Enterococcus, Haemophilus, Mycobacterium, Corynebacterium, or Streptococcus.

[0050] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0051] The reagents and raw materials used in the present invention are commercially available.

[0052] The positive and progressive effects of the present invention are:

[0053] The present invention obtains nano-antigens by coupling recombinant protein antigens from bacterial pathogens with polysaccharides. The immunogenicity of the recombinant protein antigens coupled with polysaccharides is greatly improved, which overcomes the deficiency of poor immunogenicity enhancement effect caused by competitive immunoregulation between capsular polysaccharides and recombinant protein antigens in the body, and also solves the safety and effectiveness problems caused by repeated inoculation, over-inoculation, and interference with immune procedures of the capsular polysaccharide antigens after the products formed with the capsular polysaccharide antigens as nano-matrix are put on the market. In addition, the present invention also has the advantages of simple raw material acquisition and low cost, good process stability and easy amplification, and high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This is a comparative study on the immune effects of antigens modified with different nanostructured matrices. DETAILED DESCRIPTION

[0055] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0056] The following embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the following embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement modes and shall be included in the protection scope of the present invention.

[0057] Example 1 Construction of recombinant protein antigen expression strain

[0058] According to the method shown in Table 1, the amino acid sequences of each recombinant protein antigen are shown in SEQ ID NO: 1 to 11, and the biotechnology company constructs the recombinant expression strain business. In short, the amino acid sequence of the recombinant protein antigen is converted into a nucleotide sequence, optimized according to the codon preference of Escherichia coli, and restriction sites are added to the N-terminus and C-terminus, respectively, and the whole fragment is chemically synthesized; the synthesized nucleotide sequence is connected to the T vector and transformed into DH5α competent cells, and the plasmid is extracted from the correctly sequenced bacterial solution using a kit, and then the plasmid is connected to the pET28a / pET30a vector, transformed into BL21 (DE3) competent cells, and the plasmid is extracted again to confirm that the sequencing is correct.

[0059] Table 1 Construction information of four recombinant superantigen expression strains

[0060] Protein name Amino acid sequence N-terminal tag Expression vector Commissioned construction unit R SEQ ID NO:1 none pET28a Shanghai Medicilon Biopharmaceutical Co., Ltd. R SEQ ID NO:2 none pET28a Shanghai Medicilon Biopharmaceutical Co., Ltd. rSEC2 SEQ ID NO:3 none pET30a Nanjing GenScript Biotechnology Co., Ltd. rTSST1 SEQ ID NO:4 none pET28a Nanjing GenScript Biotechnology Co., Ltd. rHlα SEQ ID NO:5 none pET28a Shanghai Medicilon Biopharmaceutical Co., Ltd. R SEQ ID NO:6 none pET28a Suzhou Nearshore Protein Technology Co., Ltd. rfHBPv1 SEQ ID NO:7 none pET30a Suzhou Nearshore Protein Technology Co., Ltd. dB SEQ ID NO:8 none pET30a Suzhou Nearshore Protein Technology Co., Ltd. rP SEQ ID NO:9 none pET30a Suzhou Nearshore Protein Technology Co., Ltd. rPspA SEQ ID NO:10 none pET27b Suzhou Nearshore Protein Technology Co., Ltd. PD SEQ ID NO:11 none pET30a Suzhou Nearshore Protein Technology Co., Ltd.

[0061] Example 2 Fermentation of recombinant protein antigen expression strain

[0062] The first generation seed liquid culture medium was prepared according to Table 2, and after the preparation was completed, 0.5 mL of 30 mg / mL kanamycin sulfate mother solution was added.

[0063] Table 2 Formula of first generation seed liquid culture medium

[0064]

[0065]

[0066] Note: The kanamycin sulfate in Table 2 is a pre-prepared stock solution with a concentration of 30 mg / mL.

[0067] Take a frozen glycerol bacteria tube, thaw it completely, and then take 1.0 mL of it and inoculate it into a triangular flask containing 0.5 L of seed liquid culture medium for cultivation. Set the shaking temperature to 32°C and the speed to 140 rpm. Shake overnight and the OD 600 Reach between 1 and 3.

[0068] Prepare the basal culture medium according to Table 3. After preparation, set the temperature to 35±0.5°C. Under flame protection and sterile conditions, add defoamer, 2.5 mL of kanamycin sulfate mother solution, K2HPO4 solution, MgSO4·7H2O solution in sequence, and then add the first-generation seed solution. The inoculation ratio of seed solution to culture medium is 1:2.5.

[0069] Table 3 Basic culture medium formula

[0070]

[0071] Note: The defoamer in the above table is added before inoculation.

[0072] The fermentation process parameters were set as follows: pH 7.0, rotation speed controlled at 100-600 rpm, deep aeration set at 50 L / h, tank pressure controlled at 0.05 MPa, and dissolved oxygen ≥ 30%.

[0073] When OD 600 When the value reaches 1 or above, batch feeding begins. Feed once every hour in stages and complete the feeding in the logarithmic phase. OD is measured every hour. 600 Determination and staining microscopy.

[0074] When OD 600 When the value reaches about 10, start to cool down to 20-32°C, add inducer IPTG to a final concentration of 0.1mmol / L, and induce for 4-18 hours before fermentation ends.

[0075] Stop fermentation, discharge and harvest the fermentation liquid.

[0076] Example 3 Crude purification of Escherichia coli fermentation broth

[0077] 2.5 L of fermentation broth was harvested and crushed using a homogenizer at 900 bar for 3 times.

[0078] The disrupted liquid was filtered using a 500 kDa membrane package (Millipore), and the clarified flow-through was collected.

[0079] Use a 3kDa or 10kDa membrane package (Cobite) to concentrate the clarified flow-through liquid. After concentrating to a certain volume, use a replacement fluid to wash and replace the concentrated retentate. After the replacement is completed, collect the sample, add a certain volume of replacement fluid to rinse the membrane package once, and collect the crude sample.

[0080] The above-mentioned replacement solution is not completely consistent due to different antigens. In principle, it can be kept consistent with the initial purification equilibrium buffer in column chromatography purification.

[0081] Example 4 Column chromatography purification of recombinant protein antigen

[0082] 4.1 Preparation of rSEA, rSEB, rSEC2 and rTSST1 protamine

[0083] (1) Preliminary purification: DEAE Bestarose FF from Boglon Corporation was used as the filler material, and the sample loading amount was 5-10 mg / ml gel. Purification was performed in flow-through mode. 50 mmol / L Tris-HCl, pH 8.0, containing 1 mmol / L EDTA equilibration solution was used for 3 CV, and then the crude sample obtained in Example 3 was loaded, and then eluted with the equilibration solution for 3 CV. The load flow-through and the elution flow-through were collected and combined.

[0084] (2) The medium purification filler was Phenyl Bestarose HP from Boglon, and the purification was performed in dissociation mode. The sample was equilibrated with a 50mmol / L Tris-HCl, pH 8.0, 1mmol / L EDTA, and 1.8mol / L (NH4)2SO4 balance solution for 3CV. The combined flow-through in step (1) was adjusted to have the same conductivity as the balance solution with a 3.5mol / L (NH4)2SO4 solution, and then the sample was loaded. The balance solution was used for 3CV for elution. The sample was then removed with a 50mmol / L Tris-HCl, pH 8.0, 1mmol / L EDTA, and 148mS / cm to 165mS / cm (NH4)2SO4 buffer for 2CV. The sample was then eluted with a 50mmol / L Tris-HCl, pH 8.0, 1mmol / L EDTA, and 119mS / cm to 129mS / cm (NH4)2SO4 buffer for 4CV, and the eluate was collected.

[0085] During the intermediate purification process, there is no impurity removal step for rSEB and rSEC2 proteins.

[0086] (3) The fine purification filler was Polar MC30 HIC Butyl from Saifen Technology Co., Ltd., and the purification was carried out in the dissociation mode. The 50mmol / L Tris-HCl, pH8.0, 1mmol / L EDTA, and 1.8mol / L (NH4)2SO4 balance solution was used for 3CV, and the conductivity of the eluate in step (2) was adjusted to be consistent with the balance solution with 3.5mol / L (NH4)2SO4 solution, and then the sample was loaded, and then the balance solution was used for 3CV. The impurities were removed by 50mmol / L Tris-HCl, pH8.0, 1mmol / L EDTA, and 148mS / cm~158mS / cm (NH4)2SO4 buffer for 4CV, and then eluted by 50mmol / L Tris-HCl, pH8.0, 1mmol / L EDTA, and 119mS / cm~185mS / cm (NH4)2SO4 buffer for 4CV, and the eluate was collected.

[0087] During the fine purification process, there was no impurity removal step for rSEB and rSEC2 proteins.

[0088] (4) Ultrafiltration Replacement The finely purified eluate obtained in step (3) was subjected to ultrafiltration replacement using a 3 kDa ultrafiltration membrane package from Cobetter. The replacement solution was 0.2 mol / L borate, pH 8.4, containing 0.85% NaCl. The protein solution after replacement was filtered using a 0.2 μm syringe filter. The filtered protein was protamine, which was frozen at -80°C for later use.

[0089] 4.2 Preparation of rHla protamine

[0090] (1) Preliminary purification: DEAE Sepharose FF from Cytiva was used as filler, and the sample loading amount was 5-10 mg / ml gel. Purification was performed in flow-through mode. 50 mmol / L Tris-HCl, pH 9.0, containing 1 mmol / L EDTA and 2.25 mS / cm NaCl balance solution was used for 3 CV, and then the crude sample obtained in Example 3 was loaded, and then eluted with the balance solution for 3 CV. The load flow-through and the elution flow-through were collected and combined.

[0091] (2) The intermediate purification medium was Q Sepharose FF from Cytiva, and the purification was performed in flow-through mode. The purification method was the same as step (1).

[0092] (3) Fine purification filler was Phenyl Bestarose HP from Boglon, and purification was performed in dissociation mode. 50mmol / L Tris-HCl, pH 8.5, containing 1mmol / L EDTA, and 1.5mol / L (NH4)2SO4 was used for equilibration for 3CV. The flow-through in step (2) was adjusted to have the same conductivity as the equilibration solution with 3.5mol / L (NH4)2SO4 solution, and then the sample was loaded, and then the equilibration solution was used for 3CV. The sample was then eluted with 50mmol / L Tris-HCl, pH 8.5, containing 1mmol / L EDTA, and 104mS / cm (NH4)2SO4 buffer for 4CV, and the eluate was collected.

[0093] (4) Ultrafiltration replacement is the same as step (4) of Example 4.1.

[0094] 4.3rLukS protamine preparation

[0095] (1) Preliminary purification: The filler was SP Sepharose FF from Cytiva, and the loading amount was 5-10 mg / ml gel. The purification was performed in dissociation mode. The crude sample obtained in Example 3 was loaded with a 50 mmol / L Tris-HCl, pH 7.5, containing 1 mmol / L EDTA and 11 m S / cm NaCl equilibration solution for 3 CV, and then the equilibration solution was used for 3 CV elution. The 50 mmol / L Tris-HCl, pH 7.5, containing 1 mmol / L EDTA and 17.5 mS / cm NaCl buffer was used for 3 CV elution, and the eluate was collected.

[0096] (2) The intermediate purification filler was Diamond Butyl Mustang from Boglon, and the purification was performed in dissociation mode. The equilibration solution was 50mmol / L Tris-HCl, pH 7.5, containing 1mmol / L EDTA and 1.0mol / L (NH4)2SO4 for 3CV. The conductivity of the eluate in step (1) was adjusted to be consistent with that of the equilibration solution with 3.5mol / L (NH4)2SO4 solution, and then the sample was loaded, and then the equilibration solution was used for 3CV. Then, the sample was eluted with 50mmol / L Tris-HCl, pH 7.5, containing 1mmol / L EDTA and 61mS / cm (NH4)2SO4 buffer for 4CV, and the eluate was collected.

[0097] (3) Fine purification filler was Q Sepharose FF from Cytiva, and purification was performed in flow-through mode. 50mmol / L Tris-HCl, pH 8.5, 1mmol / L EDTA equilibrated for 3CV, and the eluate in step (2) was replaced by ultrafiltration with the equilibrated solution. The pore size of the replacement membrane was 3kDa, until the conductivity was consistent with that of the equilibrated solution, then the sample was loaded, and the equilibrated solution was used for 3CV to elute, and the flow-through was collected.

[0098] (4) Ultrafiltration replacement is the same as step (4) of Example 4.1.

[0099] 4.4 Preparation of rDTB protamine

[0100] (1) Preliminary purification The filler was SP Sepharose FF from Cytiva, and the purification was performed in dissociation mode. The crude sample obtained in Example 3 was loaded with a 50mmol / L PB, pH 7.0, 1mmol / L EDTA, 1mmol / L DTT equilibration solution for 3CV, and then eluted with the equilibration solution for 3CV. The impurities were then removed with a 50mmol / L PB, pH 7.0, 1mmol / L EDTA, 1mmol / L DTT, 10mS / cm NaCl buffer for 3CV, and then eluted with a 50mmol / L PB, pH 7.0, 1mmol / L EDTA, 1mmol / L DTT, 18mS / cm NaCl buffer for 3CV, and the eluate was collected.

[0101] (2) The fine purification filler was Capto Q ImpRes from Cytiva, and the purification was performed in dissociation mode. The equilibration solution of 50mmol / L Tris-HCl, pH8.5, containing 1mmol / L EDTA, 1mmol / L DTT, and 6mS / cm NaCl was equilibrated for 3CV, and the eluate in step (1) was replaced by ultrafiltration with the equilibration solution. The pore size of the replacement membrane was 10kDa until the conductivity was consistent with that of the equilibration solution. Then, the sample was loaded, and the equilibration solution was used for 3CV. Then, 50mmol / L Tris-HCl, pH8.5, containing 1mmol / L EDTA, 1mmol / L DTT, and 12mS / cm NaCl were used for 4CV, and the eluate was collected.

[0102] (3) Ultrafiltration replacement is the same as step (4) in Example 4.1.

[0103] 4.5 Preparation of rfHBPv1 Protamine

[0104] (1) Preliminary purification The filler was Q Sepharose FF from Cytiva, and the purification was performed in dissociation mode. The crude sample obtained in Example 3 was loaded with 50mmol / L Tris-HCl, pH8.0, 1mmol / L EDTA equilibration solution for 3CV, and then eluted with the equilibration solution for 3CV. Then, impurities were removed with 50mmol / L Tris-HCl, pH8.0, 1mmol / L EDTA, 12.5mS / cm NaCl buffer for 2CV, and then eluted with 50mmol / L Tris-HCl, pH8.0, 1mmol / L EDTA, 17.5mS / cm NaCl buffer for 3CV, and the eluate was collected.

[0105] (2) Fine purification filler is Phenyl Bestarose HP from Boglon, and purification is performed in dissociation mode. 50mmol / L Tris-HCl, pH8.5, containing 1mmol / L EDTA, and 1.8mol / L (NH4)2SO4 balance solution is used for 3CV, and the eluate in step (1) is adjusted to have the same conductivity as the balance solution with 3.5mol / L (NH4)2SO4 solution, and then the sample is loaded, and then the balance solution is used for 3CV, and then 50mmol / L Tris-HCl, pH8.5, containing 1mmol / L EDTA, and 176mS / cm (NH4)2SO4 buffer is used for 4CV, and the eluate is collected.

[0106] (3) Ultrafiltration replacement is the same as step (4) in Example 4.1.

[0107] 4.6 Preparation of rPly protamine

[0108] (1) Preliminary purification: DEAE Sepharose FF from Cytiva was used as filler, and the loading amount was 5-10 mg / ml gel. Purification was performed in elution mode. 20 mmol / L Tris-HCl, pH 8.0, containing 1 mmol / L EDTA, and containing 0.062 mol / L NaCl balance solution was used for 3 CV, and then the crude sample obtained in Example 3 was loaded, and then eluted with the balance solution for 3 CV, and then eluted with 20 mmol / L Tris-HCl, pH 8.0, containing 1 mmol / L EDTA, and containing 0.12 mol / L NaCl for 3 CV, and the eluate was collected.

[0109] (2) The medium purification filler is Phenyl Bestarose HP from Boglon, and the purification is carried out in dissociation mode. The sample is equilibrated for 3CV with 20mmol / L PB, pH 7.5, 1mmol / L EDTA, and 0.6mol / L (NH4)2SO4. The combined flow-through in step (1) is adjusted to have the same conductivity as the equilibrium solution with 3.5mol / L (NH4)2SO4 solution. Then, the sample is loaded, and the sample is washed with the equilibrium solution for 3CV. Then, the sample is eluted with 20mmol / L PB, pH 7.5, and 1mmol / L EDTA buffer for 3CV, and the eluate is collected.

[0110] (3) Fine purification filler was Q Sepharose FF from Cytiva, and purification was performed in dissociation mode. 100mmol / L Tris-HCl, pH8.5, 1mmol / L EDTA balance solution was used for 3CV, the eluate in step (2) was diluted with the balance solution to adjust the conductivity to be consistent with the balance solution, and then the sample was loaded, and then eluted with the balance solution for 3CV, and impurities were removed with 100mmol / L Tris-HCl, pH8.5, 1mmol / L EDTA, 0.12mol / L NaCl buffer for 4CV, and then eluted with 50mmol / L Tris-HCl, pH8.0, 1mmol / L EDTA, 119mS / cm~185mS / cm (NH4)2SO4 buffer for 3CV, and the eluate was collected.

[0111] (4) Ultrafiltration replacement is the same as step (4) of Example 4.1.

[0112] 4.7 Preparation of rPspA protamine

[0113] (1) Preliminary purification: DEAE Sepharose FF from Cytiva was used as filler, and the loading amount was 5-10 mg / ml gel. Purification was performed in elution mode. The crude sample obtained in Example 3 was loaded with a 20 mmol / L PB, pH 6.5, containing 1 mmol / L EDTA and 0.2 mol / L NaCl equilibration solution for 3 CV, and then the sample was loaded with the equilibration solution for 3 CV. The sample was then eluted with 20 mmol / L PB, pH 6.5, containing 1 mmol / L EDTA and 0.28 mol / L NaCl for 3 CV, and the eluate was collected.

[0114] (2) The medium purification filler was Phenyl Bestarose HP from Boglon, and the purification was performed in dissociation mode. The sample was equilibrated for 3CV with a 20mmol / L PB, pH 7.5, 1mmol / L EDTA, and 1.4mol / L (NH4)2SO4 balance solution. The combined flow-through in step (1) was adjusted to have the same conductivity as the balance solution with a 3.5mol / L (NH4)2SO4 solution. Then, the sample was loaded, and the balance solution was used for 3CV. The sample was then eluted with a 20mmol / L PB, pH 7.5, 1mmol / L EDTA, and 0.5mol / L (NH4)2SO4 buffer for 3CV. The eluate was collected.

[0115] (3) The fine purification filler was Cytiva's SP Sepharose FF, and the purification was performed in dissociation mode. 50mmol / L NaAC, pH 4.8, containing 1mmol / L EDTA balance solution was used for 3CV, and the eluent in step (2) was diluted and replaced with the balance solution to adjust the conductivity to be consistent with the balance solution, and then the sample was loaded, and then the balance solution was used for 3CV, and 50mmol / L NaAC, pH 4.8, containing 1mmol / L EDTA, containing 0.097mol / L NaCl was used for 3CV to elute, and the eluent was collected.

[0116] (4) Ultrafiltration replacement is the same as step (4) of Example 4.1.

[0117] 4.8 Preparation of rPD Protamine

[0118] (1) Preliminary purification: DEAE Sepharose FF from Cytiva was used as the filler, and the sample loading amount was 5-10 mg / ml gel. Purification was performed in flow-through mode. The crude sample obtained in Example 3 was loaded with 50 mmol / L PB, pH 6.5, and 1 mmol / L EDTA equilibration solution for 3 CV, and then the equilibration solution was used for 3 CV elution, and the flow-through solution was collected.

[0119] (2) The medium purification filler was Cytiva's SP Sepharose FF, and the purification was performed in dissociation mode. The sample was equilibrated with 50mmol / L PB, pH 6.5, containing 1mmol / L EDTA equilibration solution for 3CV, and the combined flow-through in step (1) was loaded, and then eluted with equilibration solution for 3CV, and then eluted with 20mmol / L PB, pH 7.5, containing 1mmol / L EDTA, and containing 0.14mol / L NaCl buffer for 3CV, and the eluate was collected.

[0120] (3) Fine purification filler was Phenyl Bestarose HP from Boglon, and purification was performed in flow-through mode. 50mmol / L PB, pH 6.5, 1mmol / L EDTA, and 1.0mol / L (NH4)2SO4 were used for 3CV of equilibrium solution. The 3.5mol / L (NH4)2SO4 solution, the eluent in step (2), was adjusted to have the same conductivity as the equilibrium solution, and then the sample was loaded, and then the equilibrium solution was used for 3CV of elution, and the flow-through solution was collected.

[0121] (4) Ultrafiltration replacement is the same as step (4) of Example 4.1.

[0122] 4.9 Quality Characterization of Recombinant Protein Antigens

[0123] The endotoxin content of each protamine antigen was characterized by horseshoe crab reagent, and the purity was characterized by reducing SDS-PAGE and non-reducing SDS-PAGE. The results showed that the purity of each protein antigen was more than 95%, and the endotoxin content was less than 0.1EU / μg.

[0124] Example 5 Preparation of Pneumococcal Capsular Polysaccharide

[0125] The pneumococcal strains producing type 3 (Pn3), type 6B (Pn6B), type 9V (Pn9V), type 14 (Pn14), and type 15B (Pn15B) capsular polysaccharides were cultured in a tryptone medium at 37°C for 10 hours, and the obtained bacterial culture solution was inactivated by 0.5% sodium deoxycholate, and the supernatant was collected after centrifugation at 8000g for 30 minutes. The centrifuged supernatant was concentrated by ultrafiltration using a 100kDa membrane package, and graded precipitation was performed using 25-80% ethanol. The precipitate was collected to obtain a crude polysaccharide, and the crude polysaccharide was freeze-dried. The crude polysaccharide was dissolved in sterile water for injection, and then 100mmol / L Tris-HCl, pH8.0, containing 3.0-4.25mol / LNaCl solution was added, and the sample was obtained after mixing evenly. A Diamond Mix Amustang (Borgron) chromatography column (XK50 / 30) was equilibrated with 100mmol / L Tris-HCl, pH8.0, containing 3.0-4.25mol / LNaCl solution for 2CV at a flow rate of 30cm / h; the sample was loaded onto the Diamond Mix Amustang chromatography column, and after loading, it was eluted with 100mmol / L Tris-HCl, pH8.0, containing 3.0-4.25mol / L NaCl solution for 1CV; the chromatography column was then cleaned with 0.5mol / LNaOH solution for 2CV, and then equilibrated with 100mmol / L Tris-HCl, pH8.0, containing 3.0-4.25mol / L NaCl solution for 4CV. Collect the sample flow-through and the elution flow-through, and use a 10KDa (Millipore) ultrafiltration membrane package for concentration and replacement. The replacement fluid is 0.9% NaCl solution and water for injection. The refined sugar solution is obtained by replacing it with 0.9% NaCl solution 3 times, and then replacing it with water for injection 3 times. Finally, the refined sugar solution after desalination is sterilized and filtered with a 0.2μm capsule filter (Cobetter). The refined sugar solution after filtration is placed in a freeze-drying cup, and the freeze-drying temperature is controlled to be lower than -30°C, the vacuum degree is lower than 1mba, and the freeze-drying time is at least 28h. After the freeze-drying is completed, the freeze-dried refined sugar is weighed into a 250ml reagent bottle and stored below -20°C for standby use.

[0126] Example 6 Preparation of Neisseria meningitidis capsular polysaccharide

[0127] The meningococcal strains producing group A (MenA) and group Y (MenY) capsular polysaccharides were cultured in soytone medium at 35°C for 8-10 hours, the obtained bacterial culture solution was inactivated with 0.5% formaldehyde, precipitated with sodium deoxycholate after dilution, the supernatant was collected by centrifugation, concentrated by ultrafiltration with a 100kDa membrane package, graded precipitation was performed with 25-80% ethanol, the precipitate was collected to obtain crude polysaccharides, and the crude polysaccharides were freeze-dried. The crude polysaccharides were dissolved with sterile water for injection, and then 20mmol / LTris-HCl, pH8.0, containing 1.0-4.25mol / L NaCl solution was added, and the sample was obtained after mixing evenly. The Diamond Mix Amustang (Borgron) chromatography column (XK50 / 30) was equilibrated with 20mmol / L Tris-HCl, pH8.0, containing 1.0-4.25mol / L NaCl solution for 2CV at a flow rate of 30cm / h; the sample was loaded onto the Diamond Mix Amustang chromatography column, and after loading, it was eluted with 20mmol / L Tris-HCl, pH8.0, containing 1.0-4.25mol / L NaCl solution for 1CV; the chromatography column was then washed with 0.5mol / L NaOH solution for 2CV, and then equilibrated with 20mmol / L Tris-HCl, pH8.0, containing 1.0-4.25mol / L NaCl solution for 4CV. The sample flow-through and the elution flow-through were collected and concentrated and replaced with a 10KDa (Millipore) ultrafiltration membrane package. The replacement solution was 20mmol / L Tris-HCl, pH 8.0, containing 0-1.0mol / LNaCl solution to obtain a preliminary purified flow-through. A Diamond Mix Amustang chromatography column (XK50 / 30) was equilibrated with a 20mmol / L Tris-HCl, pH8.0, containing 0-1.0mol / L NaCl solution for 2CV at a flow rate of 30cm / h; the preliminary purified flow-through was loaded onto the Diamond Mix Amustang chromatography column, and after loading, it was eluted with a 20mmol / L Tris-HCl, pH8.0, containing 0-1.0mol / L NaCl solution for 3CV; it was then eluted with a 20mmol / L Tris-HCl, pH8.0, containing 1.0-4.25mol / L NaCl solution for 3CV, and the eluate was collected; the chromatography column was then washed with a 0.5mol / L NaOH solution for 2CV, and then equilibrated with a 20mmol / L Tris-HCl, pH8.0, containing 1.0-4.25mol / L NaCl solution for 4CV.The collected eluate was concentrated and replaced with a 10KDa (Millipore) ultrafiltration membrane package. The replacement solution was 0.9% NaCl solution and water for injection. The refined sugar solution was first replaced with 0.9% NaCl solution 3 times, and then replaced with water for injection 3 times to obtain the refined sugar solution. Finally, the refined sugar solution after desalination was sterilized and filtered with a 0.2μm capsule filter (Cobetter). The refined sugar solution after filtration was placed in a freeze-drying cup. The freeze-drying temperature was controlled to be lower than -30°C, the vacuum degree was lower than 1mba, and the freeze-drying time was at least 28h. After the freeze-drying was completed, the freeze-dried refined sugar was weighed into a 250ml reagent bottle and stored below -20°C for later use.

[0128] Example 7 Preparation of Nanosized Protein Antigen Using Capsular Polysaccharide as Nanosized Matrix

[0129] (1) Hydrolysis of purified capsular polysaccharide

[0130] Weigh 500 mg of refined sugar obtained in Example 5 or Example 6, dissolve it to 5 mg / mL with 0.85% NaCl solution, add 200 μl of degradation solution containing 100 mmol / L ascorbic acid, 10 mmol / L CuSO4.5H2O, and 10 mmol / LFeSO4.7H2O at the beginning of the reaction, react for 1 hour, then add 200 μl of degradation solution, react for 2 hours and the reaction is over. The reaction solution is ultrafiltered with a 300 kDa ultrafiltration membrane bag (Millipore), and the permeate is collected. The permeate is concentrated and replaced with a 10 kDa ultrafiltration membrane bag (Millipore), the replacement liquid is 0.9% NaCl solution, 10 times the volume is replaced 3 times, the retentate after replacement is collected, and filtered into a freeze-drying cup with a 0.45 μm filter, and the freeze-drying machine (CHRIST, Alpha 1-2LDplus) parameters are set to a vacuum degree of less than 0.5 mbar, a freeze-drying temperature of less than -40 ° C, and a freeze-drying time of more than 28 hours. The molecular size of hydrolyzed sugars was determined by SEC-MALLs method.

[0131] (2) Nano-modification of recombinant protein antigens

[0132] Weigh 50 mg of hydrolyzed sugar, dissolve it to 5 mg / mL with 0.85% NaCl solution, add 90 μl 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate (CDAP) solution (100 mg / mL), then use 3% triethylamine (Sigma) solution to maintain pH 7.0-9.0 for 2 min; add 10 mL of 5-20 mg / mL protamine solution and react for 1-8 h; after the reaction, add 0.85% NaCl solution to 1 L, concentrate and replace 3 times with a 300 kDa ultrafiltration membrane package (Millipore), and finally collect the retentate, and filter it with a 0.2 μm filter to obtain the nano-protein antigen.

[0133] (3) Quality attributes of nanosized protein antigens

[0134] The obtained nano-superantigen was used to determine the protein concentration in the conjugate by the Lowry method, the polysaccharide concentration in the conjugate was determined by the orcinol method, the free sugar concentration in the conjugate was determined by the DOC precipitation-orcinol method, and the molecular size of the conjugate was determined by the SEC-MALLs method or the CL-4B method.

[0135] Table 4 Quality attributes of nano-antigens with capsular polysaccharide as nano-matrix

[0136]

[0137] Example 8 Preparation of Nanosized Protein Antigen Using Sodium Hyaluronate as Nanosized Matrix

[0138] Injection grade sodium hyaluronate (HA) was purchased from Shandong Zhongshan Biological. The preparation method of the nanosized protein antigen was the same as that in Example 7. The quality attributes of the obtained nanosized protein antigen are shown in Table 5.

[0139] Table 5 Quality attributes of nano-antigens with sodium hyaluronate as the nano-matrix

[0140]

[0141] Example 9 Comparative study on the effects of different nanostructured matrices on immunogenicity enhancement

[0142] In this study, rTSST1 antigen was used as protein antigen to compare the immune enhancement effects of TSST1 antigen after modification with different capsular polysaccharides and sodium hyaluronate.

[0143] According to the quality attribute information of the nano-protein antigen obtained in Example 7 and Example 8, the nano-protein antigen was diluted and prepared into 25 μg / mL with a sterile 0.85% NaCl solution, and mixed with HA201 liposome adjuvant (Beijing Huanuotai Biopharmaceutical Technology Co., Ltd., the main components are QS-21 (100 μg / mL), 3D-MLA (100 μg / mL), DOPC (2 mg / ml) and cholesterol (0.5 mg / ml)) in equal volumes, and mixed by turning upside down more than ten times to obtain an immune drug. BALB / c mice were used as animal models, 8 mice / group, two injections of immunization procedures (two weeks apart), leg muscle injection of 0.1 mL, that is, the immunization dose of each group was 1.25 μg / dose, and orbital blood was collected 2 weeks after the second injection of immunization.

[0144] The indirect ELISA method was used to detect the titer of rTSST1 antigen-specific IgG. The rTSST1 antigen was diluted to 10 μg / mL, 100 μL / well was coated on the ELISA plate, and the coating was carried out overnight at 4°C; the antiserum of each experimental group was diluted in multiples, 100 μL was added to each well, and the plate was incubated at 37°C for 120 min; then 200 μL of cleaning solution was added to each well to wash the plate, and the plate was washed 5 times in total. After the completion, a 4000-fold diluted sheep anti-mouse alkaline phosphatase-labeled enzyme-labeled secondary antibody (Southern Biotech) was added, and the plate was incubated at 37°C for 120 min; after the incubation was completed, 200 μL of cleaning solution was added to each well to wash the plate, and the plate was washed 5 times in total. After the completion, 100 μL of color development solution (magnesium chloride hexahydrate (Chinese medicine) and diethanolamine (Sigma) were prepared in a ratio of 1 (g): 1 (ml)) was added to each well, and the display was carried out at 20-25°C for 60 min. After the completion, 50 μL of stop solution (3 mol / L NaOH solution); the ELISA plate was read using an ELISA reader, and the Cutoff value was determined to be 0.2 based on the negative serum.

[0145] Table 6 Comparative study on the immunogenicity enhancement effect of different nanomatrices

[0146] Immunization group 1 2 3 4 5 6 7 8 GMT rTSST1-HA 320000 80000 640000 320000 640000 640000 80000 640000 320000 rTSST1-Pn3 80000 80000 80000 40000 40000 40000 160000 40000 61688 rTSST1-Pn15 160000 80000 320000 160000 20000 160000 80000 160000 113137 rTSST1-Pn9V 80000 160000 80000 160000 80000 80000 80000 160000 103747 rTSST1-Pn14 40000 320000 80000 160000 320000 320000 160000 160000 160000 rTSST1-Pn6B 320000 320000 160000 320000 160000 320000 320000 160000 246754 rTSST1-MenA 80000 160000 80000 160000 80000 640000 160000 320000 160000 rTSST1-MenY 80000 160000 160000 160000 160000 320000 160000 640000 190273

[0147] The results are shown in Table 6 and Figure 1 As shown, the immune group with sodium hyaluronate as the nano-matrix was significantly higher than the group coupled with Streptococcus pneumoniae capsular polysaccharide and Neisseria meningitidis capsular polysaccharide (p<0.05, One way ANOVA), indicating that sodium hyaluronate with good biocompatibility will not undergo competitive immune regulation with protein antigens after coupling with protein antigens, thereby having a better immune enhancement effect.

[0148] Example 10 Study on the Immunogenicity Enhancement Effect of Sodium Hyaluronate on Different Protein Antigens

[0149] In this study, sodium hyaluronate (HA) was used as the nanostructured matrix to investigate the immune enhancement effect of sodium hyaluronate on different protein antigens.

[0150] The nanosized protein antigen prepared in Example 8 was diluted with a sterile 0.85% NaCl solution to a certain concentration, and an adjuvant was added or not to prepare an immune drug. BALB / c mice were used as animal models, 8 mice / group, two injections of immunization program (two weeks apart), leg muscle injection of 0.1 mL, and orbital blood was collected 2 weeks after the second injection of immunization. The indirect ELISA method was used to detect the specific IgG titer of each protein, and the method was the same as in Example 9.

[0151] The results are shown in Table 7. Under the condition of no adjuvant, the IgG GMT of 1 / 10 dose of nano-sized protein antigen is 5 to 34896 times higher than that of recombinant protein antigen. Under the condition of AlPO4 adjuvant, the IgG GMT of 1 / 2 dose of nano-sized protein antigen is 6 to 371 times higher than that of recombinant protein antigen. For large molecular proteins, under the condition of Al(OH)3 adjuvant, the IgG GMT of 1 / 5 dose of nano-sized protein antigen is equivalent to that of full dose of recombinant protein antigen. This study fully demonstrates the effect of sodium hyaluronate nano-modification on enhancing the immunogenicity of recombinant protein antigen.

[0152] Table 7 Comparative study on the immunogenicity enhancement effect of sodium hyaluronate on different protein antigens

[0153]

[0154] Note: The aluminum content in the aluminum adjuvants in each group in the above table is 50 μg / dose.

[0155] Example 11 Comparative study on the immunogenicity of four-component nanoantigens

[0156] In this study, four recombinant proteins and four nano-proteins were mixed to prepare four-component protein immune drugs and four-component nano-protein immune drugs, and the immune enhancement effect of sodium hyaluronate modification in multi-component vaccines was compared.

[0157] The rSEA, rSEB, rSEC2 and rTSST1 recombinant protein antigens prepared in Example 4 were diluted with sterile 0.85% NaCl solution to make the concentration of each antigen in the four-component antigen mixture 400 μg / mL, and mixed with the HA201 liposome adjuvant by inverting more than ten times to obtain a four-component protein vaccine.

[0158] The rSEA-HA, rSEB-HA, rSEC2-HA and rTSST1-HA nano-protein antigens prepared in Example 8 were diluted with a sterile 0.85% NaCl solution so that the concentration of each antigen in the four-component antigen mixture was 25 μg / mL, and mixed with the HA201 liposome adjuvant by inverting it upside down more than ten times to obtain a four-component nano-protein vaccine.

[0159] BALB / c mice were used as animal models, 8 mice / group, two injections (two weeks apart), 0.1 mL of the above two vaccines were injected into the leg muscles, and blood was collected from the orbits 4 weeks after the second injection. The indirect ELISA method was used to detect the specific IgG titer of each protein, and the method was the same as in Example 9.

[0160] 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. The same adjuvant was used. The results showed that the IgGGMT of each antigen in the nano-protein vaccine was significantly 6 to 30 times higher than that in the protein vaccine group (p<0.01, One way ANOVA), indicating that after the recombinant protein was nano-modified with sodium hyaluronate, it could induce extremely high immune response levels at a lower immunization dose, which further improved the safety of the vaccine.

[0161] Table 8 Comparative study on immunogenicity of four-component protein vaccine and four-component nano-protein vaccine

[0162]

[0163] Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that these are only examples, and various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the protection scope of the present invention is limited by the appended claims.

Claims

1. A method for enhancing the immunogenicity of a recombinant protein antigen, characterized in that: The method comprises forming a nanosized 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-derived protein antigen, and the bacteria belong to the genus Staphylococcus, Neisseria, Klebsiella, Escherichia, Clostridium, Salmonella, Shigella, Pseudomonas, Acinetobacter, Bordetella, Enterococcus, Haemophilus, Mycobacterium or Streptococcus.

2. The method according to 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 sonnetii, Pseudomonas aeruginosa, Acinetobacter baumannii, Bordetella pertussis, Enterococcus faecium, Enterococcus faecalis, Haemophilus influenzae, Mycobacterium tuberculosis and Mycobacterium bovis, Group A Streptococcus, Group B Streptococcus 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 according to 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, alpha hemolysin and leukocidin S; the group B Neisseria meningitidis antigen includes v1 type H factor binding protein; the Corynebacterium diphtheriae antigen includes diphtheria toxin B fragment; the pneumococcal antigen includes pneumolysin and / or pneumococcal surface protein A; the Haemophilus influenzae antigen includes outer membrane protein D; Preferably, the Staphylococcus aureus antigens include one or more of Staphylococcus aureus enterotoxin A, enterotoxin B, enterotoxin C2 and toxic shock syndrome toxin 1.

4. The method according to any one of claims 1 to 3, characterized in that: The coupling method includes one or more of the following: (1) Direct linkage of polysaccharides to recombinant proteins; (2) The CDAP-activated polysaccharide was modified with adipic acid dihydrazide and then bridged with the recombinant protein using carbodiimide; (3) The CDAP-activated polysaccharide was modified with adipic acid dihydrazide, and the recombinant protein was modified with succinic anhydride, and then bridged with carbodiimide; (4) bridging the CDAP-activated polysaccharide with the adipic acid dihydrazide-modified recombinant protein; Preferably, the coupling method is direct connection of the polysaccharide and the recombinant protein.

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

6. The method according to claim 5, characterized in that The method further comprises the step of purifying the obtained nanosized protein antigen; Preferably, the step of purifying the obtained nanosized protein antigen comprises ultrafiltration replacement of the nanosized protein antigen using a 300kDa ultrafiltration membrane, the replacement buffer is a 0.85% NaCl solution, collecting the ultrafiltration replacement retentate, and filtering through a 0.2μm filter to obtain the purified nanosized protein antigen.

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

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

9. Use of the nanosized protein antigen as claimed in claim 8 in the preparation of bacterial vaccines.

10. The use according to claim 9, characterized in that The bacteria belongs to the genus Staphylococcus, Neisseria, Klebsiella, Escherichia, Clostridium, Salmonella, Shigella, Pseudomonas, Acinetobacter, Bordetella, Enterococcus, Haemophilus, Mycobacterium or Streptococcus.

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