Pentavalent neisseria meningitidis conjugate vaccine as well as preparation method and application thereof

Abstract

The invention relates to a pentavalent neisseria meningitidis capsular polysaccharide protein conjugate vaccine. The neisseria meningitidis capsular polysaccharide protein conjugate comprises a group A neisseria meningitidis capsular polysaccharide protein conjugate, a group C neisseria meningitidis capsular polysaccharide protein conjugate, a group Y neisseria meningitidis capsular polysaccharide protein conjugate, a group W135 neisseria meningitidis capsular polysaccharide protein conjugate and a group X neisseria meningitidis capsular polysaccharide protein conjugate. According to the neisseria meningitidis pentavalent vaccine provided by the invention, on the basis of an ACYW135 neisseria meningitidis tetravalent polysaccharide conjugate vaccine, the protection range is expanded, the vaccination times of infants, teenagers and adults are reduced, and the vaccination cost is reduced.

Description

Technical Field

[0001] The present disclosure belongs to the field of biotechnology, and particularly relates to a preparation method and application of a novel pentavalent conjugate vaccine for preventing or treating bacterial meningitis caused by Neisseria meningitidis serogroup ACYW135X. Background Art

[0002] Epidemic cerebrospinal meningitis (referred to as meningitis) is a bacterial vaccine-preventable disease. The pathogen is Neisseria meningitidis, and humans are the only host. The carriage rate in the healthy population is 1%-10%, and it can be as high as 30% in epidemic areas. The pathogen can invade the bloodstream through the nasopharynx, form septicemia, and finally be confined to the meninges and spinal meninges, forming suppurative cerebrospinal meningitis lesions, with a high fatality rate. Due to the popularization of meningitis vaccines, the incidence has decreased significantly. However, even under the conditions of having vaccines for prevention and antibiotics for treatment, meningitis still has a fatality rate of over 10% and a high disability rate of 30%. Its disease burden ranks among the top in the infectious disease spectrum and cannot be underestimated. According to the specificity of the capsular polysaccharide of Neisseria meningitidis, Neisseria meningitidis can be divided into thirteen serogroups: A, B, C, D, 29E, H, I, K, L, W135, X, Y, and Z. Bacteria of all serogroups can cause diseases, but serogroups A, B, C, W135, Y, and X are the most virulent. The above 6 serogroups account for more than 95% of the cases and are the most common strains causing meningitis epidemics. However, infections and outbreaks caused by serogroup X of Neisseria meningitidis, especially in the African meningitis belt with the highest incidence of meningitis diseases globally.

[0003] Neisseria meningitidis spreads directly through contact or by airborne droplets, invades the upper respiratory tract, and grows and multiplies in the nasopharyngeal mucosa. Meningitis can present as latent infection without any clinical symptoms, and only a few people develop clinical symptoms. Humans are generally susceptible to meningitis. Newborns obtain maternal bactericidal antibodies against meningitis through the placenta and maintain effective levels within 6 months of age. During the period from 6 to 18 months of age, the bactericidal antibody level drops to the lowest, while the incidence rate rises to the highest.

[0004] Capsular polysaccharides can shield the functional components on the surface of bacterial cells, protecting them from being recognized by the host immune system, preventing the complement system from being activated by the proteins on the bacterial surface, and avoiding phagocytosis by immune cells. If the bacteria are phagocytosed by immune cells, the capsular polysaccharides can also prevent the bacteria from being killed. The capsular polysaccharide CPS of the meningococcal polysaccharide vaccine is a thymus-dependent antigen TI-Ag. By conjugating the polysaccharide with a protein carrier, the thymus-independent antigen TI-Ag can be transformed into a thymus-dependent antigen TD-Ag, thereby changing the antigenic properties of the capsular polysaccharide CPS, enhancing its immunogenicity, and inducing immune memory. The active ingredient of the conjugate vaccine is the conjugate of bacterial capsular polysaccharide and carrier protein. The antibodies induced by the capsular polysaccharide antigen can bind to the capsule of the bacteria and cooperate with complement to kill the invading bacteria. The conjugate protein can promote the maturation of antibodies, increasing their affinity. At the same time, it can induce immunological memory in the vaccinated individuals against the capsular polysaccharide, extending the effective time of protection.

[0005] A multivalent conjugate vaccine refers to a vaccine containing two or more live, inactivated organisms or purified antigens, such as the pneumococcal polysaccharide conjugate vaccine, which is formulated by the manufacturer for the prevention of multiple diseases. A conjugate vaccine is by no means a simple combination vaccine. Each conjugate vaccine is an independent vaccine that has undergone scientific research. The purpose of developing conjugate vaccines is to prevent more types of diseases while reducing the number of vaccine injections. Its significance lies not only in improving the vaccine coverage rate and vaccination rate, reducing the physical and psychological pain brought to infants and parents by multiple injections, reducing the difficulties in vaccine management, and lowering the vaccination and management costs; but also in reducing the doses of preservatives and adjuvants that must be contained in vaccine production, and reducing the adverse reactions of vaccines. Conjugate vaccines are not assembled from existing vaccines in the factory. Instead, on the premise of considering the solubility, physical compatibility, and antigen stability among the antigen components in the conjugate vaccine, some potential problems need to be solved, such as antigen competition, expression inhibition, and aggravated adverse reactions. China has specifically formulated the "Technical Guidelines for Preclinical and Clinical Research of Conjugate Vaccines", requiring that conjugate vaccines must undergo safety, immunogenicity, and efficacy studies before being marketed. Currently, there are AC bivalent Neisseria meningitidis conjugate vaccines and ACW135Y quadrivalent conjugate vaccines on the market, but there is no pentavalent conjugate vaccine that simultaneously contains the ACYW135X group Neisseria meningitidis conjugate vaccine. There is an urgent need for a conjugate vaccine that can prevent or treat bacterial meningitis and scurvy caused by Neisseria meningitidis in a wider range. Summary of the Invention

[0006] The purpose of the present invention is to provide a pentavalent Neisseria meningitidis capsular polysaccharide conjugate vaccine, its preparation method, and its application.

[0007] The pentavalent Neisseria meningitidis capsular polysaccharide conjugate vaccine provided by the present invention comprises capsular polysaccharides and carrier protein conjugates of Neisseria meningitidis of 5 serotypes, and the 5 serotypes are serogroup A, serogroup C, serogroup Y, serogroup W135 and serogroup X respectively.

[0008] In some embodiments, the conjugate of the Neisseria meningitidis capsular polysaccharide and the carrier protein is the direct conjugation of the corresponding capsular polysaccharide and the carrier protein of each Neisseria meningitidis or the indirect conjugation with a cross-linking agent.

[0009] In some embodiments, the capsular polysaccharide of serogroup A Neisseria meningitidis is from strain CMCC29201 (A4), the capsular polysaccharide of serogroup C Neisseria meningitidis is from strain CMCC29205, the capsular polysaccharide of serogroup Y Neisseria meningitidis is from strain CMCC29028, the capsular polysaccharide of serogroup W135 Neisseria meningitidis is from strain CMCC29037, and the capsular polysaccharide of serogroup X Neisseria meningitidis is from strain CMCC29040.

[0010] In some embodiments, in the conjugate of the capsular polysaccharide and the carrier protein, the capsular polysaccharide of serogroup X is conjugated with the carrier protein of tetanus toxoid (TT), and the capsular polysaccharides of serogroups ACYW135 are conjugated with the carrier protein of non-toxic diphtheria toxin (CRM197), but it is not limited to this conjugation combination. Moreover, the carrier protein can also be selected from the main components of the outer membrane protein of serogroup B Neisseria meningitidis, including class 1 and class 2 or class 1 and class 3 outer membrane proteins. As described above, as long as the type of the carrier protein and the conjugation method with the polysaccharide do not affect the animal experiment effect that can be achieved by the conjugate vaccine of the present invention, then all these types of carrier proteins and conjugation types should be within the scope of the present invention.

[0011] Among them, the mass ratio of the capsular polysaccharide to the carrier protein is (0.3 - 4):1, preferably (0.3 - 1.2):1.

[0012] In some embodiments, the carrier protein is selected from at least one of tetanus toxoid (TT), diphtheria toxoid (DT), non-toxic variant protein of diphtheria toxin (CRM197) or outer membrane protein (OMP) of serogroup B Neisseria meningitidis.

[0013] The present invention also provides a method for purifying Neisseria meningitidis capsular polysaccharide, including:

[0014] 1) Adding a formaldehyde inactivation solution to the Neisseria meningitidis fermentation broth for sterilization;

[0015] 2) Centrifuging to remove the bacterial cells and collecting the supernatant;

[0016] 3) Ultrafiltrating the supernatant obtained in step 2) to remove impurities such as buffer salts in the fermentation broth and collecting the ultrafiltrate;

[0017] 4) Add CTAB to the ultrafiltrate obtained in step 3) to make its concentration reach 1% - 20%, centrifuge, and collect the precipitate;

[0018] 5) Add 5 - 20 times the volume of ethanol to the precipitate obtained in step 4) for extraction, centrifuge, and remove proteins;

[0019] 6) Filter the supernatant obtained in step 5) through activated carbon to obtain a filtrate;

[0020] 7) Add calcium chloride to the filtrate obtained in step 6) to make its final concentration reach 10 - 100 mM, collect the precipitate, redissolve the precipitate with physiological saline, and then ultrafilter to obtain purified Neisseria meningitidis capsular polysaccharide.

[0021] The present invention also provides a method for preparing the conjugate vaccine described in the first aspect, which is characterized by including the following steps:

[0022] 1) Depolymerize the capsular polysaccharides of Neisseria meningitidis serogroup A and Neisseria meningitidis serogroup X by physical or chemical methods to obtain hydrolyzed polysaccharides with a molecular weight of 30 - 250 kDa;

[0023] 2) Perform adipic dihydrazide derivatization reaction on the hydrolyzed polysaccharides of Neisseria meningitidis serogroup A and Neisseria meningitidis serogroup X obtained in step 1) to obtain derivatized polysaccharides. Preferably, 1 - cyano - 4 - (dimethylamino) pyridinium tetrafluoroborate (CDAP) is added to Neisseria meningitidis serogroup X for cyanoesterification reaction before adipic dihydrazide derivatization;

[0024] 3) Perform sodium periodate oxidation reaction on the capsular polysaccharides of Neisseria meningitidis serogroup C, Neisseria meningitidis serogroup Y, and Neisseria meningitidis serogroup W to obtain activated polysaccharides;

[0025] 4) Perform conjugation reactions on the derivatized polysaccharides of Neisseria meningitidis serogroup A and Neisseria meningitidis serogroup X obtained in step 2) and the activated polysaccharides of Neisseria meningitidis serogroup C, Neisseria meningitidis serogroup Y, and Neisseria meningitidis serogroup W135 obtained in step 3) with carrier proteins respectively to obtain a conjugate stock solution;

[0026] 5) Mix the conjugate stock solution obtained in step 4) to obtain the conjugate vaccine.

[0027] In some embodiments, in step 2), during the addition of CDAP, a 0.1 M - 1.0 M 4 - dimethylpyridine buffer salt is used to maintain the stability of the reaction pH for the required buffer system.

[0028] The present invention improves the CDAP process, enhances the stability of the CDAP process reaction, and makes the cyanoesterification reaction stable and controllable.

[0029] In some specific embodiments, the method includes:

[0030] 1) Using acid hydrolysis or physical homogenization methods to depolymerize and pretreat the capsular polysaccharides of Neisseria meningitidis serogroups A and X, reducing their weight-average molecular weight to 30 - 250 kDa.

[0031] 2) For the hydrolyzed polysaccharide of serogroup A, utilizing the hemiacetal property after hydrolysis, using adipic dihydrazide (ADH) as a bifunctional nucleophilic spacer to form a polysaccharide adipic dihydrazide derivative, and then covalently binding it to the CRM197 carrier protein through the condensation mediated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC). After ammonium sulfate precipitation and filtration, the crude binding solution of the polysaccharide-protein conjugate of serogroup A is obtained.

[0032] 3) For the capsular polysaccharide of serogroup CYW135, using its unique cis - vicinal dihydroxy structure of the structural unit, performing an oxidation reaction with sodium periodate oxidant to generate an activated polysaccharide with aldehyde group reactive sites, then conducting a nucleophilic reaction with the CRM197 carrier protein, and purifying it by hydrophobic chromatography. After filtration, the crude binding solution of the polysaccharide-protein conjugate of serogroup CYW135 is obtained.

[0033] 4) Activating the capsular polysaccharide of serogroup X with 1 - cyano - 4 - (dimethylamino)pyridinium tetrafluoroborate (CDAP), using ADH as a bifunctional nucleophilic spacer to form a polysaccharide adipic dihydrazide derivative, and then covalently binding it to the TT carrier protein through the condensation mediated by EDAC. Purifying it by molecular sieve chromatography and filtering to obtain the crude binding solution of the polysaccharide-protein conjugate of serogroup X.

[0034] 5) Mixing the above-prepared monovalent conjugates of ACYW135X groups to obtain the pentavalent polysaccharide conjugate vaccine of Neisseria meningitidis.

[0035] There are many known methods for the conjugation of capsular polysaccharides and carrier proteins. The conjugation method described in the present invention is just one of the methods and processes, and various equivalent methods can be used to achieve the conjugation process of capsular polysaccharides and carrier proteins. Therefore, any method, process, or procedure for molecular conjugation may be used to prepare the pentavalent Neisseria meningitidis capsular polysaccharide - carrier protein conjugate in the present invention.

[0036] The conjugate vaccine provided by the present invention contains, in each 0.5 ml single dose of the combined vaccine composition, 2.5 - 15.0 μg of Neisseria meningitidis serogroup A capsular polysaccharide (such as 2.5 μg, 5.0 μg, 7.5 μg, 10 μg, 12.5 μg, 15 μg or any value therebetween), 2.5 - 15.0 μg of Neisseria meningitidis serogroup C capsular polysaccharide (such as 2.5 μg, 5.0 μg, 7.5 μg, 10 μg, 12.5 μg, 15 μg or any value therebetween), 2.5 - 15.0 μg of Neisseria meningitidis serogroup Y capsular polysaccharide (such as 2.5 μg, 5.0 μg, 7.5 μg, 10 μg, 12.5 μg, 15 μg or any value therebetween), 2.5 - 15.0 μg of Neisseria meningitidis serogroup W135 capsular polysaccharide (such as 2.5 μg, 5.0 μg, 7.5 μg, 10 μg, 12.5 μg, 15 μg or any value therebetween) and 2.5 - 15.0 μg of Neisseria meningitidis serogroup X capsular polysaccharide (such as 2.5 μg, 5.0 μg, 7.5 μg, 10 μg, 12.5 μg, 15 μg or any value therebetween).

[0037] The conjugate vaccine provided by the present invention may further include an adjuvant in each dose. Among them, the adjuvant is an aluminum salt adjuvant, preferably aluminum hydroxide, aluminum phosphate or aluminum sulfate, and the content of the aluminum salt adjuvant (calculated by aluminum content) is 0.2 mg / ml - 0.5 mg / ml of the vaccine, preferably 0.2 mg / ml of the vaccine.

[0038] The pentavalent Neisseria meningitidis capsular polysaccharide conjugate vaccine provided by the present invention has a freeze-dried formulation, or a freeze-dried - liquid combined formulation, or a liquid formulation.

[0039] In some embodiments, in the freeze-dried - liquid combined formulation, the freeze-dried formulation contains the combined stock solution of serogroups A and C, and the liquid formulation is the combined stock solution of serogroups YW135X.

[0040] In some embodiments, in the liquid - freeze-dried combined preparation, the freeze-dried component includes the conjugate of the capsular polysaccharide and the carrier protein of Neisseria meningitidis serogroup A and Neisseria meningitidis serogroup C, and the liquid component includes the conjugate of the capsular polysaccharide and the carrier protein of Neisseria meningitidis serogroup Y, Neisseria meningitidis serogroup W135 and Neisseria meningitidis serogroup X.

[0041] In some embodiments, the freeze-dried component further includes a cryoprotectant, and the cryoprotectant is a mixture including sucrose and mannitol. Preferably, the content of sucrose is 5 - 10 mg per 0.5 ml single dose, and the content of mannitol is 10 mg - 20 mg per 0.5 ml single dose.

[0042] The present invention also provides a method for preparing a freeze-dried dosage form, including: mixing the prepared conjugate stock solution with a cryoprotectant, followed by aliquoting and optionally freeze-vacuum drying. The freeze-vacuum drying includes: pre-freezing at -35 to -40°C for 2 - 3 hours, freezing the product at -35 to -40°C for 3 - 6 hours, preferably 4 - 5 hours, heating and vacuum drying for 8 - 14 hours, preferably 10 - 12 hours, and constant-temperature vacuum drying at below 30°C for 4 - 8 hours, preferably 5 - 6 hours. The total freeze-drying process accumulates to 20 - 24 hours.

[0043] In some embodiments, the osmotic pressure after mixing the liquid component and the freeze-dried component is 280 mOsm / kg ± 65 mOsm / kg, and the pH range is 6 - 7.

[0044] In some embodiments, the freeze-dried preparation further includes a diluent, and the diluent is selected from at least one or more of phosphate buffer salts, Tris buffer salts, boric acid buffer salts, succinate buffer solutions, histidine buffer solutions, sodium chloride solutions, and sodium citrate buffer solutions.

[0045] The present invention also provides the use of the conjugate vaccine in the preparation of a medicament for preventing or treating diseases caused by Neisseria meningitidis, such as bacterial meningitis and / or scurvy.

[0046] The pentavalent Neisseria meningitidis capsular polysaccharide conjugate vaccine provided by the present invention uses five serogroups of A, C, Y, W135, and X, covering all pathogenic serogroups except serogroup B, with a wider coverage range. Description of the Drawings

[0047] Figure 1 It is a comparison diagram of the 1H NMR spectra of the chemically and physically pretreated capsular polysaccharide of serogroup X.

[0048] Figure 2 It is an HPLC-SEC spectrum of the conjugate of the capsular polysaccharide of serogroup A and the carrier protein.

[0049] Figure 3 It is an HPLC-SEC spectrum of the conjugate of the capsular polysaccharide of serogroup C and the carrier protein.

[0050] Figure 4 It is an HPLC-SEC spectrum of the conjugate of the capsular polysaccharide of serogroup Y and the carrier protein.

[0051] Figure 5 It is an HPLC-SEC spectrum of the conjugate of the capsular polysaccharide of serogroup W135 and the carrier protein.

[0052] Figure 6 It is an HPLC-SEC spectrum of the conjugate of the capsular polysaccharide of serogroup X and the carrier protein.

[0053] Figure 7Appearance results of freeze-drying for different concentrations of sucrose and mannitol.

[0054] Figure 8 Appearance results of freeze-drying for different secondary sublimation temperatures. Detailed implementation manners

[0055] The present invention will be further described below through examples. It should be understood that these examples of the present disclosure are only exemplary descriptions of the present disclosure and should not be construed as limitations of the present disclosure.

[0056] Example 1 Preparation of capsular polysaccharide stock solutions of different serogroups

[0057] The bacterial strains used for production are: Neisseria meningitidis serogroup A CMCC29201 (A4) strain, Neisseria meningitidis serogroup C CMCC29205 (C11) strain, Neisseria meningitidis serogroup Y CMCC29028 strain, Neisseria meningitidis serogroup W135 CMCC29037 strain, and Neisseria meningitidis serogroup X CMCC29040 strain. All the strains are derived from the National Institute for the Control of Pharmaceutical and Biological Products, China. The above-mentioned bacterial strains are passaged to establish the original seed bank, the master seed bank, and the working seed bank. The passage numbers are the original seed lot is the 1st generation, the master seed lot is the 4th generation, and the working seed lot is the 8th generation. After the working seed lot is opened, it is inoculated into the semi-synthetic medium for meningococcal meningitis, at a temperature of 35 - 37 °C, and cultured for 16 - 24 hours. Then, it is cultured through three generations of amplification to prepare the production seed, which is inoculated into the fermenter for culture, at a temperature of 35 - 37 °C. After culturing for 6 - 12 hours, the culture is terminated at the late logarithmic growth phase or the early stationary phase. Samples are taken for pure bacteria inspection. After passing the pure bacteria inspection, an appropriate amount of formaldehyde is added to the harvested culture solution to a final concentration of 1% to sterilize for more than two hours to lyse the bacterial cells and release the capsular polysaccharide;

[0058] The sterilized culture solution is centrifuged to collect the supernatant, and the supernatant is ultrafiltered and concentrated 15 - 30 times with a 100 kDa molecular weight cut-off ultrafiltration membrane package to obtain the ultrafiltered supernatant.

[0059] Then cetyltrimethylammonium bromide (CTAB) is added to a final concentration of 1.0 - 10.0 g / L, stirred well, and left to stand overnight to precipitate the capsular polysaccharide, and the precipitate is collected by centrifugation.

[0060] The precipitated polysaccharide is stirred with calcium chloride solution for 3 hours to dissociate the polysaccharide and CTAB, and the supernatant is collected by centrifugation. Ethanol is added to the supernatant to a final concentration of 25%, and left to stand at 2 - 8 °C overnight. The supernatant is collected by centrifugation. The amount of ethanol added to the supernatant is 5 - 20 times the volume of the precipitate. It is shaken well to precipitate the polysaccharide, left to stand for 2 - 24 hours, and the precipitate is collected by centrifugation;

[0061] Then it is filtered through an activated carbon filter, OD 275nmThe absorbance was controlled below 0.3, and then filtered through a 0.22-μm filter to obtain an activated carbon filtrate;

[0062] Calcium chloride was added to the supernatant filtrate to a final concentration of 10 - 100 mM. The precipitate was collected, washed twice with absolute ethanol, and then redissolved with physiological saline;

[0063] Using a 100-kDa ultrafiltration system, the calcium chloride precipitate re-dissolution solution was subjected to equal-volume tangential flow ultrafiltration to remove additive impurities such as buffer salts remaining in the product, and 5 Neisseria meningitidis capsular polysaccharides of A, C, Y, W135, and X were obtained.

[0064] Example 2 Determination of the molecular weights of Neisseria meningitidis serogroup A and serogroup X capsular polysaccharides

[0065] Pretreatment: Chemically pretreat the Neisseria meningitidis serogroup A capsular polysaccharide stock solution prepared in Example 1. First, control the polysaccharide reaction concentration of the serogroup A and serogroup X polysaccharide stock solutions at 0.8 - 2.0 mg / ml, control the reaction temperature at 55 - 65 °C, adjust the reaction pH to 5.0 - 7.0 with acetate buffer salts, then add hydrogen peroxide to a final concentration of 0.5 - 2%, react for 2 - 6 hours, and ultrafilter to obtain serogroup A hydrolyzed polysaccharide and serogroup X hydrolyzed polysaccharide with molecular weights controlled at 30 - 250 kDa respectively. The 1H NMR spectrum of serogroup X capsular polysaccharide after chemical and physical pretreatment is as Figure 1 shown.

[0066] Example 3 Preparation of serogroup A polysaccharide-carrier protein conjugate

[0067] Derivatization: Add the serogroup A hydrolyzed polysaccharide prepared in Example 2 to a bioreactor, continue to add the ADH stock solution to the reactor, then add the EDAC stock solution, stir for 3 hours, add injection water to make the polysaccharide reaction concentration 6 - 8 mg / ml, then add sodium cyanoborohydride at 100 mg / ml to a final concentration of 2 mg / ml, control the reaction pH at 5 - 7, terminate the activation reaction after reacting for 42 - 48 hours, and ultrafilter and concentrate with a 10-kDa membrane package to obtain activated polysaccharide.

[0068] Coupling: Add the above-activated polysaccharide to a bioreactor, control the amino reaction concentration of the activated polysaccharide at 500 - 900 μmol / ml, then add CRM197 carrier protein to a final concentration of 2 - 4 mg / ml, then add EDAC to a final concentration of 2 mg / ml, and use morpholineethanesulfonic acid (MES) to control the reaction pH at 5 - 6.

[0069] Conjugate purification: Dilute the conjugation reaction solution to <1 mg / ml according to the protein concentration. Slowly add ammonium sulfate powder to it, place it at 2 - 8 °C and let it stand for 1 - 2 hours. Centrifuge to obtain the precipitate, redissolve it with 0.45% normal saline, and filter to obtain the combined stock solution of group A polysaccharide-protein conjugate. Among them, the HPLC-SEC chromatogram of the conjugate of group A capsular polysaccharide and carrier protein is as Figure 2 shown.

[0070] Example 4 Preparation of CYW135 group polysaccharide-carrier protein conjugate (combination of sodium periodate oxidation and reductive amination method)

[0071] Activation: Add the stock solution of the three Neisseria meningitidis capsular polysaccharides of CYW135 group prepared in Example 1 to a bioreactor, then add sodium periodate for oxidation to the polysaccharide. Use acetate buffer salt to control the pH of the reaction system at 5 - 6, and control the reaction duration at 16 - 24 hours. After the reaction, ultrafiltration and concentration are carried out using a 30 - 50 kDa ultrafiltration membrane package to obtain the activated polysaccharide of CYW135 group, and detect the oxidation degree of the activated polysaccharide.

[0072] Conjugation: Put the above-activated polysaccharide of CYW135 group into a bioreactor according to the mass ratio of polysaccharide:CRM197 carrier protein = 1:2 - 4:1. Use phosphate buffer salt to maintain the pH of the reaction system at 7.5 - 9.0, then add sodium cyanoborohydride to the reaction system to a final concentration of 2 mg / ml. After reacting for 16 - 24 hours, add sodium borohydride to terminate the conjugation reaction.

[0073] Purification: Purify the above-terminated conjugation reaction solution using the hydrophobic chromatography packing Toyopearl Phenyl-650M. Adsorb it with 1.0 - 2.0 M ammonium sulfate buffer salt and elute it with 20 mM PB buffer salt. Collect the volume from the starting peak to the falling peak at 280 nm, and then ultrafiltration and concentration are carried out using a 50 kDa ultrafiltration membrane package. After filtration, obtain the combined stock solution of the CYW135 group polysaccharide-protein conjugate. Among them, the HPLC-SEC chromatograms of the conjugates of capsular polysaccharides of groups C, Y, and W135 and carrier protein are respectively as Figures 3 - 5 shown.

[0074] Example 5 Study on the stability of CDAP cyanation reaction in different buffer systems

[0075] CDAP is extremely easy to degrade in water, and it is very difficult to maintain the stability of the pH of the reaction system, which poses a great challenge to process control. In the present invention, three buffer systems, namely triethylamine, phosphate buffer, and 4-dimethylaminopyridine (DMAP), the degradation product of CDAP, are used to study the pH stability of the CDAP activation reaction system. The polysaccharide and CDAP are added to the reactor in a ratio of 1:0.5, the reaction temperature is maintained at 25 °C, and then a buffer system with a pH of 10.0 is added to the reaction system. After reacting for 30 minutes, the change in pH in the system is monitored. The test results are shown in Table 1:

[0076] Table 1 pH change after CDAP degradation in different buffer systems

[0077] Reaction time (min) Triethylamine Phosphate buffer system DMAP 1 9.8 8.5 9.6 2 9.2 7.6 9.5 3 8.4 6.4 9.5 4 8.1 6.2 9.5 5 8.0 6.2 9.5 30 7.8 6.1 9.5

[0078] As can be seen from Table 1, among the three different buffer systems, DMAP has the best buffering capacity for the CDAP activation reaction system, and phosphate buffer has the worst buffering capacity.

[0079] Example 6 Preparation of group X polysaccharide-conjugate (combination of CDAP oxidation and amide condensation method)

[0080] Derivatization: Add the stock solution of group X Neisseria meningitidis capsular polysaccharide prepared in Example 2 to a bioreactor, add it to water for injection, add a CDAP solution in a ratio of polysaccharide:CDAP = 1:0.5 (w / w), maintain the temperature at 25 ± 3 °C, use DMAP buffer to maintain the pH at 10.8 ± 0.2, and react for 30 minutes for activation. After the activation is completed, add a 2.0 - 3.0% ADH solution in a ratio of polysaccharide:adipic dihydrazide = 1:3.5 (w / w), maintain the temperature at 23 ± 3 °C, and react at pH 8.5 ± 0.2 for 15 minutes for derivatization. After clarification and filtration, use 0.05 mol / L sodium chloride solution as the ultrafiltrate, and ultrafilter with an ultrafiltration membrane package to remove cyanogen bromide to obtain the polysaccharide derivative.

[0081] Conjugation: Calculate the dosage of TT carrier protein according to the ratio of polysaccharide:protein = 1:(0.8 - 1.2) (w / w) for derivatization, add it to the polysaccharide derivative solution, add carbodiimide in a ratio of polysaccharide:carbodiimide = 1:10 (w / w), maintain the reaction temperature at 4 ± 3 °C, pH 5.7 ± 0.2, and react for 2 - 8 hours. Then adjust the pH to 6.9 ± 0.1 to terminate the reaction. After clarification and filtration, ultrafilter with 0.15 mol / L sodium chloride solution to remove carbodiimide to obtain the conjugate washing filtrate.

[0082] Purification: Then, the polysaccharide-protein conjugate solution was purified using a Sephrose 4FF gel chromatography column, with 0.15 mol / L sodium chloride solution as the mobile phase. The absorbance at 280 nm was detected, and the elution peak near V0 was collected. After combining the eluents harvested from each chromatography, the purified conjugate was obtained. After sterile filtration, it was the bulk conjugate of Neisseria meningitidis serogroup X polysaccharide-protein conjugate. Among them, the HPLC-SEC chromatogram of the serogroup X capsular polysaccharide and carrier protein conjugate is as Figure 6 shown.

[0083] Example 7 Quality Control of the Bulk Conjugate of Pentavalent Neisseria meningitidis Capsular Polysaccharide

[0084] According to the structural differences of capsular polysaccharides of different serogroups, the phosphorus content of the bulk conjugates of serogroups A and X was determined, and finally converted into the total polysaccharide content of the whole bulk conjugate. For the bulk conjugates of serogroups C, Y, and W135, the sialic acid content was determined and finally converted into the total polysaccharide content of the bulk conjugate; the protein content was determined by the Lowry method; the content of free polysaccharide in the conjugate was determined by the sodium deoxycholate precipitation method, and the supernatant was measured by colorimetry. Table 2 shows the analysis results of each quality parameter of the conjugate described in the present invention, where the polysaccharide and protein ratio (mass ratio) is in the range of 0.3 - 1.2; the content of free polysaccharide and free carrier protein in different conjugates is also inconsistent, but all free polysaccharides in the conjugate are less than 25%, and the free carrier protein is less than 5%. In this example, the bulk ACYW135X pentavalent conjugate was prepared in three conjugation scales within the range of 20 - 200 mg each.

[0085] Table 2 Characterization of Different Batches of the Bulk Pentavalent Neisseria meningitidis Polysaccharide Conjugate

[0086]

[0087] Example 8 Determination of the Concentration of Lyoprotectant in the Freeze-Dried Preparation Prescription

[0088] Sucrose and mannitol were used as lyoprotectants to study the concentration of lyoprotectant in the preparation prescription. Under the determined sodium chloride concentration condition; according to the experimental design table 3, sucrose and mannitol were formulated into lyophilization systems with different concentrations, dispensed into 2 ml vials, half-capped and placed in a freeze dryer for freeze-drying. After freeze-drying was completed, the vials were crimped, and the appearance, moisture content, and reconstitution effect were detected. The experimental design is shown in Table 3.

[0089] Table 3 Experimental Design of Freeze-Drying of Sucrose and Mannitol with Different Concentrations

[0090]

[0091] Prepare freeze-drying systems with different concentrations of sucrose and mannitol. After dispensing them into freeze-drying vials, stopper them and place them in a freeze-dryer for freeze-drying. After freeze-drying is completed, crimp the caps, and then detect the appearance, moisture content, and reconstitution effect. The test results are shown in Table 4 and the appendix Figure 7 as follows.

[0092] Table 4 Experimental results after freeze-drying of sucrose and mannitol at different concentrations

[0093]

[0094] From the test results in Table 4 and Figure 7 it can be seen that for Samples 1 and 2, the appearance can maintain the volume of the original solution basically unchanged, without collapse or shrinkage, and the surface is smooth and flat; it can be reconstituted within less than 5 seconds after adding the diluent, and the reconstituted solution is clear and free of foreign matters; the moisture contents are 1.54% and 1.52% respectively, and both the appearance and moisture meet the quality requirements. For Sample 3, obvious collapse and shrinkage appear in the appearance and cannot meet the test requirements. In summary, the concentrations of the freeze-drying protectants for Samples 1 and 2 can meet the test requirements, while the concentration of the freeze-drying protectant for Sample 3 cannot meet the test requirements; the concentration of the freeze-drying protectant for Sample 2 is lower than that for Sample 1. Therefore, it is preliminarily determined that the concentration of the freeze-drying protectant is selected as the concentration of the freeze-drying protectant for Sample 2, sucrose 6.50 mg / ml, and mannitol 13 mg / ml.

[0095] Example 9 Influence of different freeze-drying curves on the preparation process

[0096] During pre-freezing, the temperature of the vaccine preparation liquid is reduced to below its glass transition temperature, and its freezing temperature should be 5 - 10°C lower than the glass transition temperature; the sublimation temperature should neither exceed the disintegration temperature of the dried layer nor exceed the collapse temperature of the frozen layer. If the sublimation temperature is much lower than the collapse temperature, the sublimation rate will be significantly reduced and the sublimation time will be significantly prolonged; if it is higher than the collapse temperature, the material will melt and the freeze-drying will fail. Therefore, the temperature of the material during the sublimation stage should be controlled to be 5 - 10°C below the collapse temperature as much as possible. The glass transition temperature of sucrose is -32 to -35°C, and the collapse temperature is -32 to -34°C. Therefore, the pre-freezing temperature set in the freeze-drying curve is -50°C, and the primary drying temperature is increased in a gradient manner starting from -50°C. In this test, semi-finished products are prepared according to the determined concentration of the freeze-drying protectant. After dispensing them into freeze-drying vials, stopper them and place them in a freeze-dryer for freeze-drying. During the freeze-drying process, the secondary drying temperature is studied under the condition that the pre-freezing and primary drying parameters are the same. After freeze-drying is completed, crimp the caps, and then detect the appearance, moisture content, and reconstitution effect. The test design is shown in Table 5.

[0097] Table 5 Experimental results after freeze-drying of sucrose and mannitol at different concentrations

[0098]

[0099] Prepare the semi-finished product according to the determined concentration of the lyoprotectant, dispense it into lyophilization vials, stopper them, and place them in a lyophilizer. Set the secondary drying temperatures at 28 °C, 32 °C, and 30 °C respectively according to the experimental design for freeze-drying. After freeze-drying is completed, crimp the caps, and detect the appearance, moisture content, and reconstitution effect. The test results are shown in Table 6 and the appendix Figure 8 as follows.

[0100] Table 6 Experimental results after freeze-drying at different secondary sublimation temperatures

[0101]

[0102] From the results in Table 6 and Figure 8 it can be seen that for Samples 1, 2, and 3, the appearance can maintain the volume of the original solution basically unchanged, without collapse or shrinkage, and the surface is smooth and flat; after adding the diluent, they can be reconstituted within less than 5 seconds, and the reconstituted solution is clear and free of foreign matter; the moisture contents are 1.52%, 0.66%, and 1.0% respectively, all meeting the quality standards. Under the condition of the same pre-freezing and primary drying parameters, the higher the secondary sublimation temperature, the lower the moisture content. The secondary sublimation temperatures of 28 °C, 30 °C, and 32 °C can all meet the test requirements, and the secondary sublimation temperature is determined to be 30 ± 2 °C.

[0103] Example 10 Dilution, preparation, dispensing, and freeze-drying of ACYW135X pentavalent Neisseria meningitidis conjugate vaccine (fully freeze-dried formulation)

[0104] Dilution: Mix the ACYW135X group polysaccharide-protein conjugate binding stock solution prepared in Examples 1-4 according to 5.0 μg of Neisseria meningitidis serogroup A capsular polysaccharide-protein conjugate, 5.0 μg of Neisseria meningitidis serogroup C capsular polysaccharide-protein conjugate, 5.0 μg of Neisseria meningitidis serogroup Y capsular polysaccharide-protein conjugate, 5.0 μg of Neisseria meningitidis serogroup W135 capsular polysaccharide-protein conjugate, and 5.0 μg of Neisseria meningitidis serogroup X capsular polysaccharide-protein conjugate, and include sucrose and mannitol as lyoprotectants, with a filling volume of 0.5 ml per dose. Among them, for serogroup A and X, the polysaccharide is calculated according to the phosphorus content of its capsular polysaccharide, and for serogroups C, Y, and W135, the polysaccharide is calculated according to the sialic acid content of its capsular polysaccharide.

[0105] Dispensing of semi-finished product: Quickly perform freeze-vacuum drying on the above-filled vials and store them at 2-8 °C for inspection.

[0106] Preparation of diluent: The diluent can be sterile and pyrogen-free injection water, normal saline, or one or more of phosphate buffer salts, Tris buffer salts, boric acid buffer salts, succinate buffer salts, histidine buffer salts, and sodium citrate buffer salts, and is dispensed in pre-filled syringes.

[0107] Lyophilization: The above-prepared semi-finished products are lyophilized. The steps are as follows: pre-freezing at -35°C for 2 hours, freezing the products at -40°C for 4 - 5 hours, gradually raising the temperature for vacuum freeze-drying for 10 - 12 hours, and then carrying out constant-temperature vacuum freeze-drying at a temperature below 30°C for 5 - 6 hours. The whole process takes a cumulative of 20 - 24 hours. After vacuum plugging and taking out of the freeze-dryer, and crimping the aluminum cap, it is stored at 2 - 8°C to prepare the finished freeze-dried group A, C, Y, W135, and X meningococcal polysaccharide conjugate vaccine, which is stored at 2 - 8°C and comprehensively tested according to the relevant requirements of the current version of the Pharmacopoeia of the People's Republic of China.

[0108] Before using the freeze-dried dosage form product, it is reconstituted with a diluent and then administered by subcutaneous or intramuscular injection.

[0109] Example 11 Dilution, formulation, sub-packaging and lyophilization of group A, C, Y, W135, and X meningococcal polysaccharide conjugate vaccine (lyophilized-liquid combined dosage form)

[0110] Dilution: The ACYW135X group polysaccharide-protein conjugate binding stock solution prepared in Examples 1 - 4 is uniformly mixed according to the mass of 5.0 μg of group A meningococcal polysaccharide-protein conjugate, 2.5 - 15.0 μg of group C meningococcal polysaccharide-protein conjugate, and includes sucrose and mannitol protectants, with a filling volume of 0.5 ml per dose; 5.0 μg of group Y meningococcal polysaccharide-protein conjugate, 5.0 μg of group W135 meningococcal polysaccharide-protein conjugate, and 5.0 μg of group X meningococcal polysaccharide-protein conjugate are uniformly mixed, and include phosphate buffer salts, and are sterilized by filtration through a 0.22 μm filter, with a filling volume of 0.5 ml per dose. Among them, for group A and group X, the polysaccharide is calculated according to the phosphorus content of its capsular polysaccharide, and for group C, group Y, and group W135, the polysaccharide is calculated according to the sialic acid content of its capsular polysaccharide.

[0111] Sub-packaging of semi-finished products: The above-prepared ampoules containing the AC group binding stock solution are quickly subjected to vacuum freeze-drying and stored at 2 - 8°C for inspection.

[0112] Preparation of diluent: The diluent can be one or more of sterile and pyrogen-free water for injection, normal saline, phosphate buffer salts, Tris buffer salts, boric acid buffer salts, succinate buffer salts, histidine buffer salts, and sodium citrate buffer salts, and is sub-packaged in pre-filled syringes.

[0113] Lyophilization: The above-mentioned semi-finished products packed in portions are subjected to lyophilization. The steps are as follows: pre-freezing at -35°C for 2 hours, freezing the product at -40°C for 4 - 5 hours, gradually raising the temperature for vacuum freeze-drying for 10 - 12 hours, and carrying out constant-temperature vacuum freeze-drying at a temperature below 30°C for 5 - 6 hours. The whole process accumulates for 20 - 24 hours. After vacuum plugging and outputting from the freeze-dryer, and pressing the aluminum cap, it is stored at 2 - 8°C. The finished freeze-dried Neisseria meningitidis polysaccharide conjugate vaccine for groups A, C, Y, W135, and X is prepared and stored at 2 - 8°C, and is comprehensively tested according to the relevant requirements of the current version of the Pharmacopoeia of the People's Republic of China.

[0114] When the finished product is used, the AC freeze-dried product is reconstituted with the YW135X liquid. After complete dissolution, subcutaneous and intramuscular injection is completed.

[0115] Example 12 Dilution, preparation, sub-packaging and lyophilization of the pentavalent Neisseria meningitidis polysaccharide conjugate vaccine for groups A, C, Y, W135, and X (lyophilized-liquid combined dosage form)

[0116] Dilution: The ACYW135X group polysaccharide-protein conjugate binding stock solution prepared in Examples 1 - 4 is mixed evenly according to 5.0 μg of Neisseria meningitidis serogroup A polysaccharide-protein conjugate, 5.0 μg of Neisseria meningitidis serogroup C polysaccharide-protein conjugate, 5.0 μg of Neisseria meningitidis serogroup Y polysaccharide-protein conjugate, 5.0 μg of Neisseria meningitidis serogroup W135 polysaccharide-protein conjugate, and 5.0 μg of Neisseria meningitidis serogroup X polysaccharide-protein conjugate, and phosphate buffer is added. It is sterilized by filtration with a 0.22 μm filter, and the filling volume per dose is 0.5 ml. Among them, for serogroups A and X, the polysaccharide is calculated according to the phosphorus content of its capsular polysaccharide, and for serogroups C, Y, and W135, the polysaccharide is calculated according to the sialic acid content of its capsular polysaccharide.

[0117] The sub-packaging and lyophilization of the semi-finished product are the same as those in Example 1.

[0118] Example 13 Comparative study on the immunogenicity of the pentavalent meningococcal polysaccharide conjugate vaccine

[0119] The antigen component of the vaccine is an exogenous substance from the body of a pathogenic bacterium. When it is inoculated into the body, the body's immune system produces a response, including specific antibodies produced by humoral immunity, cell-mediated immunity, and the synergistic effect of the immune accessory system, achieving the function of preventing diseases. To produce an effective immune response, in addition to the normal state of the body's own immune system and the coordinated immune function state of the system, the nature and function of the exogenous substance - antigen that activates the body's immune function are crucial. The immunogenicity test is a quantifiable detection method for detecting the activation of humoral immunity and the intensity of specific antibody production after this exogenous substance - antigen enters the body.

[0120] The immune response capabilities induced by three different dosage forms of the pentavalent capsular polysaccharide conjugate vaccine in mice were studied. A comparative study on the immunogenicity of the vaccine was carried out using mice.

[0121] In the mouse immunogenicity study, a total of 4 groups of experiments were conducted, with 10 mice in each group (BALB / c female mice (SPF) in good health condition and weighing 12 - 14 g). In these 4 groups, the negative control group was normal saline (0.85% sodium chloride), which was administered at 1 / 4 of the human dosage, that is, 125 μl was subcutaneously injected at multiple points into each mouse. Immunization was carried out on the 1st day, 14th day, and 28th day of immunization respectively, with a total of 3 immunization times. On the 14th day after the last vaccination, blood was taken from the eyes of each mouse. After centrifugation of the blood sample, the supernatant was taken, that is, the serum sample. The serum sample of each mouse was stored separately, and the serum IgG antibody titer of each mouse was measured by ELISA.

[0122] Antibodies against Neisseria meningitidis serogroup A capsular polysaccharide - protein conjugate, Neisseria meningitidis serogroup C capsular polysaccharide - protein conjugate, Neisseria meningitidis serogroup Y capsular polysaccharide - protein conjugate, Neisseria meningitidis serogroup W135 capsular polysaccharide - protein conjugate, and Neisseria meningitidis serogroup X capsular polysaccharide were measured by ELISA method. The above - mentioned antigens were respectively coated on the titration wells of the ELISA microtiter plate, so that each group of polysaccharide and protein antigens were firmly bound to the titration wells of the microtiter plate. The serum sample was incubated with an excess of each antigen firmly bound to the titration wells of the ELISA microtiter plate to form antigen - antibody complexes. After incubation, the titration wells were washed with PBST buffer (0.5 ml of Tween 20 dissolved in 1000 ml of potassium phosphate solution with pH 7.4) to discard the unbound part, and the secondary antibody - enzyme conjugate that could bind to the antigen - antibody complex to be detected was added to the antigen - antibody complex titration wells for incubation. After incubation, the titration wells were washed with PBST solution, and the chemical substrate was added to the antigen - antibody - secondary antibody - enzyme conjugate. After a part of the chemical substrate was hydrolyzed by the enzyme, color formation occurred. The amount of color formation (optical absorbance OD) was proportional to the antibody bound in the titration wells. The antibody titer levels of each serotype after immunization with different experimental conjugate vaccines were statistically analyzed (OD > 2 times the maximum dilution multiple of the OD value of the normal saline control group).

[0123] The Cutoff value was obtained from the A value of the mouse serum in the dilution control group, and the Cutoff value of the mouse serum in the vaccine group was judged as seroconversion. The seroconversion percentage (%) of each group of mice serum was calculated. In addition, according to the measured ELISA OD value of each mouse serum, the geometric mean titer (GMT) of each group of OD values was calculated. The implementation results are shown in Table 7.

[0124] Table 7. Comparison of the immunogenicity of different dosage forms of pentavalent Neisseria meningitidis capsular polysaccharide conjugate vaccine in mice

[0125]

[0126]

[0127] It can be seen from the results in Table 7 that for the serum ELISA titers of the combined vaccines with different doses of immunization responses using the polysaccharide vaccine as a control, the antibody titers of each serogroup of the combined vaccines are significantly better than those of the polysaccharide vaccine. The carrier protein in the polysaccharide-protein conjugate vaccine promotes the immunogenicity of the polysaccharide antigen. At the same time, it can be seen from the titers of the three-dose responses of different dosage forms that the antibody titers produced by the freeze-dried, freeze-dried-liquid, and liquid dosage forms are not very different, indicating that the dosage form is not a factor affecting immunogenicity.

[0128] The preferred embodiments of the present disclosure have been described in detail above. However, the present disclosure is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure. In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without conflict. To avoid unnecessary repetition, the present disclosure will not separately describe various possible combination methods. In addition, any combination can be made between the various different embodiments of the present disclosure, as long as it does not violate the idea of the present disclosure, it should also be regarded as the content disclosed by the present disclosure.

Claims

1. A pentavalent Neisseria meningitidis capsular polysaccharide conjugate vaccine, characterized in that, A conjugate comprising capsular polysaccharides of five Neisseria meningitidis serogroups and a carrier protein, wherein the five serogroups of Neisseria meningitidis are serogroup A, serogroup C, serogroup Y, serogroup W135, and serogroup X.

2. The conjugate vaccine according to claim 1, characterized in that, The conjugate of Neisseria meningitidis capsular polysaccharide and carrier protein is the direct conjugation of the corresponding capsular polysaccharide and carrier protein of each Neisseria meningitidis or the indirect conjugation with a cross-linking agent. Preferably, the capsular polysaccharide of Neisseria meningitidis serogroup A is from strain CMCC29201 (A4), the capsular polysaccharide of Neisseria meningitidis serogroup C is from strain CMCC29205, the capsular polysaccharide of Neisseria meningitidis serogroup Y is from strain CMCC29028, the capsular polysaccharide of Neisseria meningitidis serogroup W135 is from strain CMCC29037, and the capsular polysaccharide of Neisseria meningitidis serogroup X is from strain CMCC29040.

3. The conjugate vaccine according to claim 1, characterized in that, The capsular polysaccharide is purified as follows: 1) Add formaldehyde inactivation solution to the Neisseria meningitidis fermentation broth for sterilization. 2) Centrifuge to remove the bacterial cells and collect the supernatant. 3) Ultrafilter the supernatant obtained in step 2) and collect the ultrafiltrate. 4) Add CTAB to the ultrafiltrate obtained in step 3) to make its concentration reach 1% - 20%, centrifuge, and collect the precipitate. 5) Add 5 - 20 volumes of ethanol to the precipitate obtained in step 4) for extraction, centrifuge, and remove proteins. 6) Filter the supernatant obtained in step 5) through activated carbon. 7) Add calcium chloride to the filtrate obtained in step 6) to make its final concentration reach 10 - 100 mM, collect the precipitate, and obtain the purified Neisseria meningitidis capsular polysaccharide.

4. The conjugate vaccine according to claim 1, characterized in that, The mass ratio of the capsular polysaccharide to the carrier protein is (0.3 - 4):

1. Preferably, the carrier protein is selected from at least one of tetanus toxoid (TT), diphtheria toxoid (DT), non-toxic mutant protein of diphtheria toxin (CRM197), or outer membrane protein of Neisseria meningitidis serogroup B (OMP).

5. A method for preparing the conjugate vaccine according to any one of claims 1 to 4, characterized in that, It includes the following steps: 1) Depolymerize the capsular polysaccharides of Neisseria meningitidis serogroup A and serogroup X by physical or chemical methods to obtain hydrolyzed polysaccharides with a molecular weight of 30 - 250 kDa. 2) Perform adipic dihydrazide derivatization reaction on the hydrolyzed polysaccharides of Neisseria meningitidis serogroup A and serogroup X obtained in step 1) to obtain derivatized polysaccharides. Preferably, 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate (CDAP) is added to Neisseria meningitidis serogroup X for cyanoesterification reaction before adipic dihydrazide derivatization. 3) Perform sodium periodate oxidation reaction on the capsular polysaccharides of Neisseria meningitidis serogroup C, serogroup Y, and serogroup W to obtain activated polysaccharides. 4) Perform binding reactions on the derivatized polysaccharides of Neisseria meningitidis serogroup A and serogroup X obtained in step 2) and the activated polysaccharides of Neisseria meningitidis serogroup C, serogroup Y, and serogroup W135 obtained in step 3) with the carrier protein respectively to obtain the binding stock solution. 5) Mix the binding stock solution obtained in step 4) to obtain the combined vaccine of Neisseria meningitidis. Preferably, in step 2), during the addition of CDAP, a 0.1M to 1.0M 4-dimethylpyridine buffer salt is used to maintain the stability of the reaction pH for the required buffer system of the reaction.

6. The conjugate vaccine according to any one of claims 1 to 4, characterized in that, The vaccine is any one of a liquid preparation, a freeze-dried preparation, or a liquid-freeze-dried combined preparation.

7. The conjugate vaccine according to claim 6, characterized in that, In the liquid-freeze-dried combined preparation, the freeze-dried component includes the conjugate of the capsular polysaccharide and the carrier protein of Neisseria meningitidis group A and Neisseria meningitidis group C, and the liquid component includes the conjugate of the capsular polysaccharide and the carrier protein of Neisseria meningitidis group Y, Neisseria meningitidis group W135, and Neisseria meningitidis group X; Preferably, the freeze-dried component further includes a cryoprotectant, and the cryoprotectant is a mixture including sucrose and mannitol. Preferably, the content of sucrose is 5 - 10 mg per 0.5 ml single dose, and the content of mannitol is 10 mg - 20 mg per 0.5 ml single dose; Preferably, the freeze-drying procedure of the freeze-dried component is as follows: Take the conjugate stock solution of each group of Neisseria meningitidis and the cryoprotectant, pre-freeze at -35°C to -40°C for 2 - 3 hours, freeze at -35°C to -40°C for 3 - 6 hours, preferably 4 - 5 hours, heat up and vacuum freeze-dry for 8 - 14 hours, preferably 10 - 12 hours, and vacuum freeze-dry at a constant temperature below 30°C for 4 - 8 hours, preferably 5 - 6 hours. The total freeze-drying process accumulates for 20 - 24 hours; Preferably, the osmotic pressure after mixing the liquid component and the freeze-dried component is 280 mOsm / kg ± 65 mOsm / kg, and the pH range is 6 - 7.

8. The conjugate vaccine according to claim 6, characterized in that, The freeze-dried preparation further includes a diluent, and the diluent is selected from at least one or more of phosphate buffer salts, Tris buffer salts, boric acid buffer salts, succinate buffer solutions, histidine buffer solutions, sodium chloride solutions, and sodium citrate buffer solutions.

9. The conjugate vaccine according to any one of claims 1 to 4, 6 to 8, characterized in that, Each 0.5 ml single dose of the conjugate vaccine contains 2.5 - 15.0 μg of capsular polysaccharide of Neisseria meningitidis group A, 2.5 - 15.0 μg of capsular polysaccharide of Neisseria meningitidis group C, 2.5 - 15.0 μg of capsular polysaccharide of Neisseria meningitidis group Y, 2.5 - 15.0 μg of capsular polysaccharide of Neisseria meningitidis group W135, and 2.5 - 15.0 μg of capsular polysaccharide of Neisseria meningitidis group X.

10. Use of the conjugate vaccine according to any one of claims 1 to 4, 6 to 8 in the preparation of a medicament for preventing or treating diseases caused by Neisseria meningitidis, such as bacterial meningitis and / or scurvy.

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