Hexavalent neisseria meningitidis combination vaccine composition

Abstract

The invention relates to a sexavalent neisseria meningitidis combination vaccine composition. The sexavalent neisseria meningitidis combination vaccine composition comprises a group A neisseria meningitidis capsular polysaccharide, a group C neisseria meningitidis capsular polysaccharide, a group Y neisseria meningitidis capsular polysaccharide, a group W135 neisseria meningitidis capsular polysaccharide, a group X neisseria meningitidis capsular polysaccharide and a group B neisseria meningitidis human factor H binding protein (fHbp). The invention further relates to a preparation method and application of the sexavalent neisseria meningitidis combined vaccine composition in different dosage forms, the immune coverage rate is increased, and the protection range of neisseria meningitidis infection is widened.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a novel combined vaccine composition for simultaneously preventing six serogroups of different Neisseria meningitidis, and a preparation method and application thereof. Background Art

[0002] Epidemic cerebrospinal meningitis (abbreviated as meningitis) is a bacterial vaccine-preventable disease. The pathogen is Neisseria meningitidis, and humans are the only host. The carrier rate in healthy people is 1% to 10%, and it can be as high as 30% in epidemic areas. The pathogen can invade the blood circulation from the nasopharynx, causing sepsis, and finally confined to the meninges and spinal cord membranes, forming purulent meningeal lesions with a high mortality rate. Due to the popularization of meningococcal vaccines, the incidence rate has dropped significantly. However, even with vaccines for prevention and antibiotics for treatment, meningococcal meningitis still has a mortality rate of more than 10% and a high disability rate of 30%. Its disease burden ranks at the forefront of the infectious disease spectrum and should not 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 disease, but A, B, C, W135, Y, and X are the most virulent. The above six serogroups account for more than 95% of the cases and are the most common strains causing meningitis epidemics. From 2005 to 2022, the overall prevalence of meningococcal meningitis in my country was 2.85%. Before 2007, the epidemic of meningococcal meningitis in my country was mainly dominated by group A. From 2007 to 2014, its epidemic flora gradually changed from group A to group C, and group Y and group W135 were sporadically distributed. At present, cases of group B in my country account for 41.62% of the overall prevalence of meningococcal meningitis. Group X accounts for a small proportion of domestic cases, about 0.02%, but a large number of patients have broken out in other countries and regions. This group is an important migratory flora for prevention. In order to survive the attacks of vaccines, antibiotics and other weapons, the bacteria have been constantly migrating and changing. Therefore, the development of multivalent vaccines for meningococcal meningitis must be fast and have a wide coverage.

[0003] Capsular polysaccharide is one of the main antigenic components of Neisseria meningitidis. It is used as the main component of meningococcal polysaccharide vaccine and has a certain degree of protection for older children. However, the antibodies induced by meningococcal polysaccharide vaccine in infants under 2 years old are short-lived and cannot induce immune memory. Therefore, it has no lasting immune effect on infants and young children. Multiple repeated immunizations are required to ensure that infants and young children can safely survive the meningitis epidemic season. The capsular polysaccharide of group B is recognized as a self-antigen in the human body and has poor immunogenicity. It is not suitable as an effective component for the development of group B vaccine. In addition, existing literature has shown that there are certain conversion or escape phenomena between different meningococcal serogroups. Therefore, diseases caused by Neisseria meningitidis are still a major threat to human health, which also prompts iterative research on meningococcal conjugate vaccines.

[0004] Currently, there are already bivalent meningococcal conjugate vaccines against groups A and C, tetravalent conjugate vaccines against ACW135Y, meningococcal group B protein vaccines and their combined vaccines, and pentavalent conjugate vaccines against ACW135YX on the market. Regarding the pentavalent product of group X vaccine, only the Serum Institute of India has successfully launched it. There is no multivalent combined meningococcal vaccine that simultaneously includes meningococcal conjugate vaccines against groups ACYW135X and group B protein vaccine on the market. Summary of the Invention

[0005] Based on the poor applicability of meningococcal group B capsular polysaccharide and other characteristics, through the development of different preparation process methods, the present invention has completed the control of dosage form, dose and process, realized the preparation of the combination of meningococcal group B protein vaccine and other meningococcal polysaccharide-conjugate vaccines, and ensured the product compatibility, stability and immunogenicity. The present disclosure can prevent or treat bacterial meningitis-related diseases caused by Neisseria meningitidis in a larger range, enhance the coverage rate of meningococcal vaccines, and reduce the potential threat of the epidemic of related diseases.

[0006] Therefore, the present invention provides a combined meningococcal vaccine composition that can simultaneously prevent Neisseria meningitidis of six serogroups, namely groups A, B, C, Y, W135 and X. There is a good synergistic protection effect among the antigen components of the combined vaccine composition of the present invention, and the preventive effect on epidemic bacterial meningitis diseases reaches and far exceeds the preventive effect of the previous six injections, reducing the number of vaccinations for infants, adolescents and adults, and reducing the vaccination cost. The vaccine can be used in drugs for preventing or treating diseases caused by Neisseria meningitidis, such as bacterial meningitis and / or sepsis.

[0007] In a first aspect, the present invention discloses and provides a hexavalent meningococcal combined vaccine composition, comprising meningococcal group A capsular polysaccharide, meningococcal group C capsular polysaccharide, meningococcal group Y capsular polysaccharide, meningococcal group W135 capsular polysaccharide, meningococcal group X capsular polysaccharide and meningococcal group B human factor H binding protein (fHbp), wherein: the capsular polysaccharides of Neisseria meningitidis group A, Neisseria meningitidis group C, Neisseria meningitidis group Y, Neisseria meningitidis group W135 and / or Neisseria meningitidis group X exist in the form of a conjugate with a carrier protein, and the H factor binding protein (fHbp) exists in a free form or in a form conjugated with an adjuvant.

[0008] In some embodiments, in the combined vaccine composition, the average molecular weight of the capsular polysaccharide is 100 kDa to 400 kDa.

[0009] In some embodiments, in the combined vaccine conjugate, the average molecular weight of the conjugate formed by the capsular polysaccharide and the carrier protein is 300 kDa to 1200 kDa.

[0010] In some embodiments, the carrier protein is optional but not limited to at least one of tetanus toxoid (TT), diphtheria toxoid (DT), non-toxic mutant protein of diphtheria toxin (CRM197), Neisseria meningitidis outer membrane protein (OMP), human serum albumin, and bovine serum albumin (BSA).

[0011] In some embodiments, the carrier protein is selected from the following combinations: tetanus toxoid (TT) and diphtheria toxoid (DT), tetanus toxoid (TT) and non-toxic mutant protein of diphtheria toxin (CRM197), and more preferably the combination of tetanus toxoid (TT) & non-toxic mutant protein of diphtheria toxin (CRM197).

[0012] In some embodiments, the capsular polysaccharide and the carrier protein are conjugated through a suitable conjugation reaction. In some embodiments, the conjugating agent used in the conjugation reaction is optional but not limited to: carbodiimide compounds, thiol compounds, hydrazide compounds, p-nitrobenzoic acid, N-hydroxysuccinimide, and other compounds with similar functions, or the conjugation process is achieved by existing equivalent methods.

[0013] In some embodiments, the Neisseria meningitidis serogroup B human factor H-binding protein (fHbp) includes factor H-binding protein variant 1 (fHbp-v1 variant protein), factor H-binding protein variant 2 (fHbp-v2 variant protein), and / or factor H-binding protein variant 3 (fHbp-v3 variant protein).

[0014] In some embodiments, the fHbp-v1 variant protein has the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:1, or has one or more amino acid residue substitutions, deletions, or insertions compared with the amino acid sequence shown in SEQ ID NO:1.

[0015] In some embodiments, the fHbp-v2 variant protein has the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:2, or has one or more amino acid residue substitutions, deletions, or insertions compared with the amino acid sequence shown in SEQ ID NO:2.

[0016] In some embodiments, the fHbp-v3 variant protein has the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:3, or has one or more amino acid residue substitutions, deletions, or insertions compared with the amino acid sequence shown in SEQ ID NO:3.

[0017] In some embodiments, the mass ratio of the fHbp-v1 variant protein, the fHbp-v2 variant protein, and the fHbp-v3 variant protein is (80.0 to 120.0):(80.0 to 120.0):(80.0 to 120.0).

[0018] In some embodiments, the mass ratio of the Neisseria meningitidis serogroup A capsular polysaccharide, the Neisseria meningitidis serogroup C capsular polysaccharide, the Neisseria meningitidis serogroup Y capsular polysaccharide, the Neisseria meningitidis serogroup W135 capsular polysaccharide, the Neisseria meningitidis serogroup X capsular polysaccharide, and the Neisseria meningitidis serogroup B human factor H-binding protein is (10 to 30):(10 to 30):(5 to 25):(5 to 25):(5 to 25):(240 to 360).

[0019] In some embodiments, the combined vaccine composition contains Neisseria meningitidis serogroup A capsular polysaccharide at 10 μg / mL to 30 μg / mL (such as 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL or any value therebetween), Neisseria meningitidis serogroup C capsular polysaccharide at 10 μg / mL to 30 μg / mL (such as 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL or any value therebetween), Neisseria meningitidis serogroup Y capsular polysaccharide at 5 μg / mL to 25 μg / mL (such as 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL or any value therebetween), Neisseria meningitidis serogroup W135 capsular polysaccharide at 5 μg / mL to 25 μg / mL (such as 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL or any value therebetween), Neisseria meningitidis serogroup X capsular polysaccharide at 5 μg / mL to 25 μg / mL (such as 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL or any value therebetween), fHbp-v1 variant protein at 80 μg / mL to 120 μg / mL (such as 80 μg / mL, 85 μg / mL, 90 μg / mL, 95 μg / mL, 100 μg / mL, 105 μg / mL, 110 μg / mL, 115 μg / mL, 120 μg / mL or any value therebetween), fHbp-v2 variant protein at 80 μg / mL to 120 μg / mL (such as 80 μg / mL, 85 μg / mL, 90 μg / mL, 95 μg / mL, 100 μg / mL, 105 μg / mL, 110 μg / mL, 115 μg / mL, 120 μg / mL or any value therebetween) and fHbp-v3 variant protein at 80 μg / mL to 120 μg / mL (such as 80 μg / mL, 85 μg / mL, 90 μg / mL, 95 μg / mL, 100 μg / mL, 105 μg / mL, 110 μg / mL, 115 μg / mL, 120 μg / mL or any value therebetween).

[0020] In some embodiments, the adjuvant may be selected from, but not limited to, at least one of aluminum adjuvant, Freund's complete adjuvant, Freund's incomplete adjuvant, MF59 adjuvant, AS01 - AS04 series adjuvants, MPLA adjuvant, CPG adjuvants.

[0021] In some embodiments, the aluminum adjuvant includes at least one of aluminum hydroxide adjuvant, aluminum phosphate adjuvant, or aluminum sulfate adjuvant, and more preferably is aluminum hydroxide adjuvant and / or aluminum phosphate adjuvant. Through practice, it has been found in the present invention that when the aluminum content of the vaccine aluminum adjuvant is 0.2 mg / mL to 1.25 mg / mL, preferably 0.2 mg / mL to 0.8 mg / mL, the titers of various antigens can be at a relatively high quality standard while ensuring the safety of the liquid vaccine composition.

[0022] In some embodiments, the combined vaccine composition further includes formulation excipients.

[0023] In some embodiments, the formulation excipients include at least one of a protectant, a preservative, a stabilizer, a buffer system, or an osmotic pressure maintaining agent.

[0024] In some embodiments, the protectant may optionally include, but is not limited to, at least one of trehalose, sodium citrate, sodium acetate, sucrose, lactose, mannitol, and tromethamine, more preferably at least one of sucrose, lactose, and mannitol, and the preferred addition amount is 5 mg / mL to 20 mg / mL.

[0025] In some embodiments, the preservative may optionally include, but is not limited to, at least one of thimerosal and 2-phenoxyethanol, and the preferred addition amount is 0 mg / mL to 4 mg / mL.

[0026] In some embodiments, the stabilizer may optionally include, but is not limited to, at least one of surfactant solubilizers, vitamins and their complexes, and amino acids. Among them, the surfactant solubilizers include: polysorbate 80, polysorbate 20, poloxamer 188, polyethylene glycol, span 20, span 80, Triton, etc., preferably polysorbate 80, and the preferred addition amount is 0 μg / mL to 100 μg / mL; the vitamins and their complexes include: vitamin A, pantothenic acid, niacin, biotin, folic acid, vitamin B1 (thiamine), vitamin B2 (riboflavin), pyridoxine (vitamin B6), cyanocobalamin (vitamin B12), vitamin C, vitamin D, vitamin E, etc., preferably vitamin E, and the preferred addition amount is 0 ng / mL to 100 ng / mL; the amino acids include: glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, etc., preferably arginine, histidine, aspartic acid, glutamine, tryptophan, tyrosine, glycine, lysine, and more preferably arginine and histidine, and the preferred addition amount is 50 ppm to 600 ppm.

[0027] In some embodiments, the osmotic pressure maintaining agent is optional but not limited to at least one of sodium chloride, potassium chloride, magnesium chloride, calcium chloride, zinc chloride, manganese chloride, and iron chloride, preferably sodium chloride; preferably, the addition amount is 0.45% (w / w) to 0.9% (w / w).

[0028] In some embodiments, the buffer system is optional but not limited to soluble phosphate buffer solution, acetate buffer solution, citrate buffer solution, Tris-hydrochloric acid buffer solution, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution, etc., preferably phosphate buffer solution; preferably, the addition amount is 5 mM to 25 mM.

[0029] In some embodiments, the pH value range of the combined vaccine composition is 5.5 to 7.5.

[0030] In some embodiments, the osmotic pressure range of the combined vaccine composition is 220 mOsm / kg to 340 mOsm / kg.

[0031] In some embodiments, the combined vaccine composition is a liquid preparation and / or a freeze-dried preparation.

[0032] In some embodiments, the freeze-dried preparation further includes a diluent and / or a protective agent. Preferably, the diluent includes but is not limited to phosphate buffer solution and aqueous sodium chloride solution, etc.; the protective agent includes but is not limited to sucrose, lactose, mannitol, etc.

[0033] According to the properties of the finished products of six serogroups, the combined vaccine composition of the present invention includes different preparation forms, such as a single-dose liquid preparation, a combined freeze-dried preparation and a two-dose diluent, and a two-dose combined liquid preparation and freeze-dried preparation.

[0034] In some embodiments, the liquid preparation comprises a mixture of three variant proteins of Neisseria meningitidis group B fHBP; or comprises a conjugate of Neisseria meningitidis group A capsular polysaccharide - carrier protein, a conjugate of Neisseria meningitidis group C capsular polysaccharide - carrier protein, and a mixture of three variant proteins of Neisseria meningitidis group B fHBP; or comprises a conjugate of Neisseria meningitidis group A capsular polysaccharide - carrier protein, a conjugate of Neisseria meningitidis group C capsular polysaccharide - carrier protein, a conjugate of Neisseria meningitidis group Y capsular polysaccharide - carrier protein, a conjugate of Neisseria meningitidis group W135 capsular polysaccharide - carrier protein, and a mixture of three variant proteins of Neisseria meningitidis group B fHBP; or comprises a conjugate of Neisseria meningitidis group A capsular polysaccharide - carrier protein, a conjugate of Neisseria meningitidis group C capsular polysaccharide - carrier protein, a conjugate of Neisseria meningitidis group Y capsular polysaccharide - carrier protein, a conjugate of Neisseria meningitidis group W135 capsular polysaccharide - carrier protein, a conjugate of Neisseria meningitidis group X capsular polysaccharide - carrier protein, and a mixture of three variant proteins of Neisseria meningitidis group B fHBP, for a total of four liquid preparation combinations.

[0035] In some embodiments, the lyophilized preparation comprises a conjugate of Neisseria meningitidis group A capsular polysaccharide - carrier protein and a conjugate of Neisseria meningitidis group C capsular polysaccharide - carrier protein; or comprises a conjugate of Neisseria meningitidis group A capsular polysaccharide - carrier protein, a conjugate of Neisseria meningitidis group C capsular polysaccharide - carrier protein, a conjugate of Neisseria meningitidis group Y capsular polysaccharide - carrier protein, and a conjugate of Neisseria meningitidis group W135 capsular polysaccharide - carrier protein; or comprises a conjugate of Neisseria meningitidis group A capsular polysaccharide - carrier protein, a conjugate of Neisseria meningitidis group C capsular polysaccharide - carrier protein, a conjugate of Neisseria meningitidis group Y capsular polysaccharide - carrier protein, a conjugate of Neisseria meningitidis group W135 capsular polysaccharide - carrier protein, a conjugate of Neisseria meningitidis group X capsular polysaccharide - carrier protein, and a mixture of three variant proteins of Neisseria meningitidis group B fHBP, for a total of three lyophilized preparation combinations.

[0036] In some embodiments, the five capsular polysaccharide - protein conjugates are formed by conjugating the corresponding Neisseria meningitidis capsular polysaccharides with a physiologically acceptable carrier protein. The three mutants included in Neisseria meningitidis group B fHBP are the fHbp - v1 variant protein (SEQ ID NO:1), the fHbp - v2 variant protein (SEQ ID NO:2), and the fHbp - v3 variant protein (SEQ ID NO:3), and the three purified mutant proteins are mixed.

[0037] In some embodiments, a certain amount of soluble phosphate buffer or analogues may be added to the combined vaccine composition to maintain the pH value within a reasonable range (such as pH 5.8 - 7.2), and a certain amount of sodium chloride or equivalent substances may be added to maintain the osmotic pressure. The present invention also discovers that adding excipients such as histidine or analogues, polysorbate 80 or analogues, 2 - phenoxyethanol or analogues, vitamin E or analogues, etc. to the combined vaccine composition can significantly improve the stability of Neisseria meningitidis group B fHBP protein or / and polysaccharide - protein conjugates of groups A, C, Y, W135, and X in liquid preparations. A certain amount of substances such as sucrose, lactose, or mannitol can also be added to improve the stability of Neisseria meningitidis group B protein or / and polysaccharide - protein conjugates of groups A, C, Y, W135, and X in freeze - dried preparations, thereby improving the overall stability of the combined vaccine composition.

[0038] In a second aspect, the present invention discloses a method for preparing a liquid preparation, which includes proportionally and sequentially formulating the combined stock solutions of Neisseria meningitidis groups A, C, Y, W135, and X and / or the stock solution of Neisseria meningitidis group B human factor H - binding protein, and related preparation excipients, and then performing sub - packaging.

[0039] In some embodiments, the preparation process of the antigen solution of the liquid preparation includes: in each 0.5 mL single - dose liquid preparation, weighing an aluminum adjuvant with a final concentration of 0.2 mg / mL - 0.8 mg / mL into a container, adjusting the rotation speed to 150 rpm - 300 rpm, and sequentially adding an fHbp - v1 variant protein with a final concentration of 40 μg / dose - 60 μg / dose, an fHbp - v2 variant protein with a final concentration of 40 μg / dose - 60 μg / dose (an fHbp - v3 variant protein with a final concentration of 40 μg / dose - 60 μg / dose) to the adjuvant solution, and then sequentially adding a combined stock solution of Neisseria meningitidis group A capsular polysaccharide with a final concentration of 5.0 μg / dose - 15.0 μg / dose, and / or a combined stock solution of Neisseria meningitidis group C capsular polysaccharide with a final concentration of 5.0 μg / dose - 15.0 μg / dose, and / or a combined stock solution of Neisseria meningitidis group Y capsular polysaccharide with a final concentration of 2.5 μg / dose - 12.5 μg / dose, and / or a combined stock solution of Neisseria meningitidis group W135 capsular polysaccharide with a final concentration of 2.5 μg / dose - 12.5 μg / dose, and / or a combined stock solution of Neisseria meningitidis group X capsular polysaccharide with a final concentration of 2.5 μg / dose - 12.5 μg / dose. After each stock solution is added, stir for 3 min - 5 min to ensure that the added components are fully adsorbed or dispersed to obtain the antigen solution.

[0040] In some embodiments, the method for adding formulation excipients to the liquid preparation comprises: after the antigen component is added, adding to the antigen solution the buffer system with a final concentration of 5 mM to 25 mM, the osmotic pressure maintaining agent with a final concentration of 0.45% (w / w) to 0.9% (w / w), the surfactant with a final concentration of 0 μg / mL to 100 μg / mL, the preservative with a final concentration of 0 mg / mL to 4 mg / mL, the vitamin or its complex with a final concentration of 0 ng / mL to 100 ng / mL, and water for injection, controlling the stirring speed at 150 rpm to 300 rpm, and continuing to stir for 20 min after the addition of water for injection is completed to obtain a semi-finished liquid preparation.

[0041] In some embodiments, the method for adding formulation excipients to the liquid preparation comprises: after the antigen component is added, adding to the multi-component antigen solution soluble phosphate buffer or the like (controlling the final concentration at 5 mM to 25 mM), sodium chloride or an equivalent substance (controlling the final concentration at 0.45% (w / w) to 0.9% (w / w)), histidine or the like (controlling the final concentration at 50 ppm to 600 ppm), polysorbate 80 or the like (controlling the final concentration at 0 μg / mL to 100 μg / mL), 2-phenoxyethanol or the like (controlling the final concentration at 0 mg / mL to 4 mg / mL), vitamin E or the like (controlling the final concentration at 0 ng / mL to 100 ng / mL) and other excipients and water for injection, controlling the stirring speed at 150 rpm to 300 rpm, and continuing to stir for 20 min after the addition of water for injection is completed to obtain a semi-finished liquid preparation.

[0042] In a third aspect, the present invention provides a method for preparing a freeze-dried preparation, comprising meningococcal conjugate stock solution of groups A, C, Y, W135 and X and / or human factor H binding protein stock solution of Neisseria meningitidis group B, and related formulation excipients, which are formulated in proportion and sequence (the addition process refers to the liquid preparation), and then subjected to sub-packaging and optionally freeze-vacuum drying.

[0043] In some embodiments, the method for preparing the antigen solution of the freeze-dried preparation is the same as the method for preparing the antigen solvent of the liquid preparation.

[0044] In some embodiments, the diluent of the freeze-dried preparation is a phosphate buffer solution of 5 mM - 15 mM (such as 10 mM) and an aqueous sodium chloride solution of 0.4% - 0.6% (such as 0.45%).

[0045] In some embodiments, the cryoprotectant of the freeze-dried preparation comprises at least one of sucrose, lactose and mannitol, and the preferred addition amount is 5 mg / mL to 20 mg / mL.

[0046] In some embodiments, the freeze-vacuum drying procedure includes: pre-freezing at -35°C to -40°C for 2h to 3h, freezing the product at -35°C to -40°C for 3h to 6h, heating and vacuum drying for 8h to 14h, and constant temperature vacuum drying at below 30°C for 4h to 8h.

[0047] The combined vaccine composition of the present invention can be prepared by conventional methods in the art. For example, the meningococcal polysaccharide-conjugate proteins of groups A, C, Y, W135, and / or X can be prepared by referring to the methods disclosed in CN100556459C, CN102861326A, etc. In addition, the meningococcal polysaccharide conjugate stock solution of group X and the fHBP protein of group B Neisseria meningitidis are both self-made by our company and can also be purchased from commercial enterprises.

[0048] In a fourth aspect, the present invention provides the use of the combined vaccine composition described in the first aspect in a medicament for preventing or treating diseases caused by Neisseria meningitidis.

[0049] In some embodiments, the disease is bacterial meningitis and / or septicemia.

[0050] The present invention has the following beneficial effects:

[0051] 1. Based on the existing quadrivalent Neisseria meningitidis AYCW135 vaccine, group B Neisseria meningitidis vaccine, and pentavalent Neisseria meningitidis ACYW135X vaccine, the present invention discloses a novel meningococcal combined vaccine composition including six antigens, namely polysaccharide conjugate vaccines of groups A, C, Y, W135, and X of Neisseria meningitidis and group B protein vaccine. The disclosed combined vaccine has a wider coverage range, provides a strategic reserve for coping with outbreaks of group B and group X of Neisseria meningitidis, and has good innovation value.

[0052] 2. In the formulation process of the hexavalent meningococcal combined vaccine composition disclosed in the present invention, considering the current situation of vaccine preparation, transportation, storage, and the stability of single vaccines of different meningococcal serogroups, a variety of dosage form selection spaces for freeze-dried preparations and / or liquid preparations are provided. Among them: the single-dose liquid preparation reduces the number of injections of the multivalent vaccine; the two-dose "freeze-dried preparation and liquid preparation" comprehensively considers the stability attributes of different components and reduces the production process difficulty; the two-dose "freeze-dried preparation and liquid diluent" improves the product shelf life. The disclosed combined vaccine extends the application range of the product and provides a larger application selection space.

[0053] 3. The combined vaccine composition disclosed by the present invention can immunize six meningococcal serogroup antigens at one time. When in use, it can reduce the number of vaccinations for infants, adolescents and adults, especially relieve the pain of infants and the burden of parents, and reduce the vaccination cost. At the same time, the disclosed hexavalent Neisseria meningitidis combined vaccine composition improves the immunization coverage rate and enhances the safety of special working populations. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The purification results of three fHbp lipoproteins of Neisseria meningitidis serogroup B prepared. DETAILED DESCRIPTION OF THE INVENTION

[0055] For the purpose, technical solutions and advantages of the present invention to be more clearly understood, the following examples are used to further elaborate on the present invention in detail. The specific examples described herein are only used to explain the disclosed content of the present invention and do not constitute any limitation to the present invention. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessarily confusing the concept of the present invention, and such structures and technologies are also described in many publications.

[0056] DEFINITIONS

[0057] Unless otherwise defined, all technical terms and scientific and technical terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure belongs. For the purpose of explaining this specification, the following definitions will be applied, and where appropriate, terms used in the singular form will also include the plural form and vice versa.

[0058] Unless the context clearly indicates otherwise, the expressions "a" and "an" used herein include plural references. For example, referring to "a liquid preparation" includes multiple liquid dosage forms not limited to the mentioned one and equivalent dosage forms known to those skilled in the art and so on.

[0059] As used herein, "combined" refers to multiple bacteria, "valence" refers to multiple serotypes of one bacterium, and "combined multiple valences" can effectively reduce the number of injection needles, so as to achieve the effect of preventing multiple disease-causing bacteria and multiple serotype pathogen bacteria of one disease through fewer needle pricks.

[0060] As used herein, a combined vaccine refers to a vaccine that contains two or more live or inactivated organisms or purified antigens, which are formulated by the manufacturer for the prevention of multiple diseases. A combined vaccine is by no means a simple combination of vaccines. Each combined vaccine is an independent vaccine that has undergone scientific research. The purpose of developing combined 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. A combined vaccine is not formed by combining existing vaccines in a factory. Instead, on the premise of considering the solubility, physical compatibility, and antigen stability among the antigen components in the combined vaccine, some potential problems need to be solved, such as antigen competition, expression inhibition, and aggravated adverse reactions. According to the "Technical Guidelines for Preclinical and Clinical Studies of Combined Vaccines", it is required that combined vaccines must undergo safety, immunogenicity, and efficacy studies before being marketed.

[0061] The present invention will be further described below with reference to examples. It should be understood that these examples disclosed in the present invention are only exemplary descriptions of the present invention and should not be construed as limitations on the present invention.

[0062] The preparation methods of the capsular polysaccharide-carrier protein conjugate stock solutions of Neisseria meningitidis of each serogroup involved in the following examples are shown as follows:

[0063] (1) In the present invention, Neisseria meningitidis serogroups A, C, Y, W135, and X were obtained as a gift from the National Institutes for Food and Drug Control of China. In the process of obtaining the polysaccharide stock solution, first, the working seed lot strains of Neisseria meningitidis serogroups A, C, Y, W135, and X were prepared, inoculated into the semi-synthetic medium for meningitis, cultured at a temperature of 35 - 37°C for 16 - 24 hours, and then amplified and cultured through three generations to prepare the seeds for production. These seeds were inoculated into a fermenter for culture at a temperature of 35 - 37°C. After 6 - 12 hours of culture, formaldehyde was added for sterilization; the sterilized culture solution was centrifuged to collect the supernatant, and then cetyltrimethylammonium bromide was added to a final concentration of 1.0 g / L. After sufficient stirring, it was left to stand overnight, and the polysaccharide was collected by centrifugation. The precipitated polysaccharide was stirred with calcium chloride solution for 3 hours to dissociate the polysaccharide and CTAB, and the supernatant was collected by centrifugation. Ethanol was added to the supernatant to a final concentration of 25%, and it was left to stand at 2 - 8°C overnight. The supernatant was collected by centrifugation, and ethanol was added to the supernatant to a final concentration of 75 - 80%. After sufficient shaking to precipitate the polysaccharide, it was left to stand for more than 18 hours, and the precipitate was collected by centrifugation. Then it was washed three times each with absolute ethanol and acetone, and after drying, it was the crude polysaccharide; the crude sugar was dissolved in 10% saturated sodium acetate solution, and cold phenol solution was added in a ratio of 1:1 (v / v). After shaking and mixing evenly, the supernatant was collected by centrifugation. This was repeated 1 - 3 times until the supernatant was clear. The supernatant was collected, and the residual phenol and nucleic acid were removed by ultrafiltration using an ultrafiltration membrane package. 4 mol / L sodium chloride solution was added to the ultrafiltered polysaccharide solution until the final concentration of sodium chloride was 0.3 mol / L, and then 95% ethanol was added until the final concentration of ethanol was 75% (v / v). After sufficient mixing, it was left to stand at 2 - 8°C overnight to precipitate the refined sugar. The precipitate was collected by centrifugation, and then it was washed twice each with absolute ethanol and acetone. After drying, the refined sugars of each serogroup were obtained.

[0064] (2) Dissolve refined sugar in water for injection, add hydrobromic acid solution according to the ratio of polysaccharide: cyanogen bromide = 1:0.5 (w / w), maintain the temperature at 23 ± 3 °C, and react for 30 minutes at pH 10.8 ± 0.2 for activation. After the activation is completed, add 2.8% adipic dihydrazide solution according to the ratio of polysaccharide: adipic dihydrazide = 1:3.5 (w / w), maintain the temperature at 23 ± 3 °C, and react for 15 minutes at pH 8.5 ± 0.2 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, which is the polysaccharide derivative. Calculate the dosage of carrier protein CRM197 according to the ratio of polysaccharide: carrier protein = 1:(0.8 - 1.2) (w / w), add it to the polysaccharide derivative solution, add carbodiimide according to the ratio of polysaccharide: carbodiimide = 1:10 (w / w), maintain the reaction temperature at 5 ± 3 °C and pH 5.7 ± 0.2, react for 60 minutes, 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. Then, purify the polysaccharide-protein conjugate solution with a Sephrose 4FF gel chromatography column, use 0.15 mol / L sodium chloride solution as the mobile phase, detect the absorbance at 280 nm, collect the elution peak near V0, and after combining the eluates harvested from each chromatography, it is the purified conjugate. After sterile filtration, it is the stock solution of the capsular polysaccharide-carrier protein conjugate of each serogroup Neisseria meningitidis.

[0065] The preparation methods of three fHbp mutants of Neisseria meningitidis serogroup B involved in the following examples are as follows:

[0066] (1) Construction of recombinant plasmid

[0067] By genetic engineering means, the coding sequences of three fHbp variant proteins (fHbp-v1 variant protein shown in SEQ ID NO:1, fHbp-v2 variant protein shown in SEQ ID NO:2, and fHbp-v3 variant protein shown in SEQ ID NO:3) were artificially synthesized, and their codon preferences were optimized at the same time. Add restriction enzyme sites XbaI and XhoI at the 5'-end and 3'-end of the sequence. Subsequently, the synthesized sequence was embedded into the pET28a expression vector through molecular construction and transformed into the DH5α cloning strain.

[0068] (2) Extraction of recombinant plasmid

[0069] The DH5α cloning strain containing the recombinant plasmid was inoculated into LB liquid medium with Kan resistance and cultured at 37 °C with 200 rpm for scale-up culture. An appropriate amount of the above bacterial liquid was taken, and the plasmid containing the target gene was extracted using the AxyPrep Plasmid DNA Miniprep Kit (for specific operations, see the instruction manual), and the plasmid concentration was measured by NanoDrop. The extracted plasmid was digested with XbaI and XhoI restriction endonucleases at 37 °C for 30 min, and detected by 1.5% agarose gel electrophoresis. The plasmid identified as positive by enzyme digestion was sent to a sequencing company (BGI) for sequencing. The expression plasmid containing the target fragment was used to transfect competent E. coli BL21(DE3) cells.

[0070] (3) Transformation of the recombinant plasmid

[0071] Take 100 μl of competent E. coli BL21(DE3) cells and place them in an ice bath. If necessary, the freshly thawed cell suspension can be aliquoted into sterile pre-cooled centrifuge tubes and stored while in the ice bath. Add an appropriate amount of the fHBP-pET28a(+) expression plasmid containing the target gene fragment obtained in (2) to the cell suspension, and it is advisable that the amount of plasmid added does not exceed 1 ng. Place the centrifuge tube in a 42 °C water bath for 90 s, then quickly transfer the centrifuge tube to the ice bath to cool the cells for about 2 min. The operation during this process should be gentle and do not shake the centrifuge tube. Then, add 900 μL of sterile SOC or LB medium without antibiotics to each centrifuge tube, mix well, and place it on a shaker at 37 °C and shake culture at 150 rpm for 45 min. The purpose is to resuscitate the bacteria and express the relevant resistance marker genes on the plasmid. Mix the contents of the centrifuge tube evenly, add 100 μL of the transformed competent cells to the LB solid agar medium containing Kan antibiotic, and gently spread the cells evenly with a sterile bent glass rod. Place the plate at room temperature until the liquid is absorbed, and then invert the plate and culture it at 37 °C for 12 - 16 h.

[0072] (4) Expression of the recombinant protein

[0073] Inoculate 1 - 2 μL of the positive monoclonal bacterial solution with a high expression level of the target protein to be activated into 5 mL of LB liquid medium with Kan resistance, and culture at 37°C with 200 rpm. Then, transfer the bacterial solution to 1000 mL of LB liquid medium with Kan resistance, culture at 37°C with 200 rpm until OD = 0.6 - 0.8, and induce with IPTG (1 mM) at 37°C for 3 h. Centrifuge the harvested bacterial solution at 8000 rpm for 5 min and discard the supernatant. Resuspend the bacterial cells with 20 - 30 mL of 10 mM Tris-HCl (pH 8.0) solution and disrupt by sonication (250 W, 5 s / 5 s, 30 min). Take 100 μL of the sonicated bacterial suspension, centrifuge at 12000 rpm for 10 min, take 50 μL of the supernatant after centrifugation into another centrifuge tube, and resuspend the precipitate after removing the supernatant with 50 μL of 10 mM Tris-HCl (pH 8.0) solution. Determine the expression form of each target protein by SDS-PAGE.

[0074] (5) Purification of recombinant lipoprotein

[0075] ① Resuspension: After the induced Escherichia coli precipitate is frozen, resuspend it in buffer A (10 mM HEPES-NaOH, pH 7.4, 1 mM Na2EDTA, 1 μg / mL Pefabloc SC);

[0076] ② Lysis: Lyse with a microfluidizer (110Y) under the condition of 18,000 lb / in2;

[0077] ③ Washing: Centrifuge the lysate at a centrifugal force of 150,000 g - 300,000 g for 1 h, wash the precipitate twice with buffer A, and centrifuge under the same conditions. Finally, freeze the precipitate overnight;

[0078] ④ Extraction: Extract the precipitate with buffer B (10 mM HEPES-NaOH, pH 7.4, 1 - 5 mM MgCl2, 1% Triton X-100) for 30 min, centrifuge at 150,000 g for 30 min, collect the supernatant, and repeat the extraction three times; Triton X-100 can be replaced with Zwittergent 3-12 and 3-14;

[0079] ⑤Chromatography: Select an appropriate chromatography method for chromatographic purification. Anion exchange chromatography: Packed with Poros HQ or Fractogel EMD TMAE, and eluted with a gradient of NaCl buffer containing 1% Zwittergent 3-12. Cation exchange chromatography: Packed with S Fractogel, and eluted with 30 mM sodium phosphate buffer containing 1% Zwittergent 3-12 at different pH gradients. Size exclusion chromatography: Using S-12 molecular sieve, and eluted with HEPES-NaOH or sodium phosphate buffer (pH 7.4) containing 150 mM NaCl and 1% Zwittergent 3-12 (see the results in Figure 1 ).

[0080] Example 1

[0081] This example provides a vaccine preparation of a single-dose "Neisseria meningitidis serogroup A, C, Y, W-135 and X combined vaccine (liquid vaccine) composition" of Neisseria meningitidis serogroup A, C, Y, W-135 and X combined vaccine. The specific preparation process is as follows:

[0082] In the preparation of the combined vaccine of Neisseria meningitidis group ABCYW135 (liquid vaccine), a certain volume of aluminum adjuvant is accurately weighed and placed in the corresponding container, and the aluminum content of the final product is controlled at 0.55 mg / mL. The container containing the aluminum adjuvant is placed on a magnetic stirrer and stirred, and the rotation speed is adjusted to 200 rpm. Three protein solutions of Neisseria meningitidis group B fHBP are added to the adjuvant solution in turn: fHbp-v1 variant protein, fHbp-v2 variant protein, fHbp-v3 variant protein, and the protein concentration of the final product is controlled at 85 μg / mL, 80 μg / mL, and 90 μg / mL respectively. After each component is added, it is stirred for 3 min to 5 min to ensure that the added components are fully adsorbed or dispersed (the same below); then the binding stock solutions of Neisseria meningitidis capsular polysaccharides are added in turn: the binding stock solution of Neisseria meningitidis group A capsular polysaccharide-TT carrier protein (the average molecular weight of the capsular polysaccharide is 235 kDa, and the average molecular weight of the capsular polysaccharide-carrier protein conjugate is 412 kDa), the binding stock solution of Neisseria meningitidis group C capsular polysaccharide-CRM197 carrier protein (the average molecular weight of the capsular polysaccharide is 203 kDa, and the average molecular weight of the capsular polysaccharide-carrier protein conjugate is 355 kDa), the binding stock solution of Neisseria meningitidis group X capsular polysaccharide-TT carrier protein (the average molecular weight of the capsular polysaccharide is 333 kDa, and the average molecular weight of the capsular polysaccharide-carrier protein conjugate is 847 kDa), the binding stock solution of Neisseria meningitidis group Y capsular polysaccharide-CRM197 carrier protein (the average molecular weight of the capsular polysaccharide is 102 kDa, and the average molecular weight of the capsular polysaccharide-carrier protein conjugate is 492 kDa), the binding stock solution of Neisseria meningitidis group W135 capsular polysaccharide-CRM197 carrier protein (the average molecular weight of the capsular polysaccharide is 186 kDa, and the average molecular weight of the capsular polysaccharide-carrier protein conjugate is 696 kDa), and the polysaccharide content concentration of the final product is controlled at 20 μg / mL, 24 μg / mL, 12 μg / mL, 10 μg / mL, and 14 μg / mL respectively; after the addition of the binding stock solution of Neisseria meningitidis group W135 capsular polysaccharide is completed, it is stirred for 4 min to ensure that the added components are fully adsorbed or dispersed, and then the mixed solution of the hexavalent Neisseria meningitidis combined vaccine can be obtained.

[0083] The rotation speed of the magnetic stirrer is adjusted to 260 rpm. Soluble phosphate buffer solution is added to the mixed solution of the hexavalent Neisseria meningitidis combined vaccine to a final concentration of 15 mM and pH = 7.0, sodium chloride solution to a final concentration of 0.85% (w / w), histidine solution to a final concentration of 60 ppm, polysorbate 80 solution to a final concentration of 45 μg / mL, vitamin E solution to a final concentration of 50 ng / mL, and it is made up to volume with water for injection. After the addition of water for injection is completed, it is stirred for another 20 min, and then the semi-finished product of the liquid preparation of the hexavalent Neisseria meningitidis combined vaccine can be obtained.

[0084] The semi-finished product of the hexavalent Neisseria meningitidis combined vaccine liquid preparation is sub-packed by a filling machine to obtain the finished product of the Neisseria meningitidis combined vaccine for groups ABCYW135X (liquid vaccine). The marked dose of the finished product is 0.5 mL / dose per person-time. When in use, it is mixed well and then given by intramuscular injection.

[0085] Example 2

[0086] This example provides a vaccine preparation of a two-dose "freeze-dried formulation + solution diluent" composition of the hexavalent Neisseria meningitidis combined vaccine. The specific preparation process is as follows:

[0087] In the preparation of the Neisseria meningitidis combined vaccine for groups ABCYW135X (freeze-dried vaccine), a certain amount of water for injection is added to a reaction vessel and placed on a magnetic stirrer for stirring, and the rotation speed is adjusted to 150 rpm. Three Neisseria meningitidis group B fHBP protein solutions, namely fHbp-v1 variant protein, fHbp-v2 variant protein, and fHbp-v3 variant protein, are successively added to the aqueous solution, and the final product protein concentrations are controlled to be 90 μg / mL, 80 μg / mL, and 100 μg / mL respectively. After each component is added, it is stirred for 3 min to 5 min to ensure that the added components are fully dispersed (the same below). Then, the Neisseria meningitidis capsular polysaccharide conjugate stock solutions are successively added: Neisseria meningitidis group A capsular polysaccharide-CRM197 conjugate stock solution (the average molecular weight of the capsular polysaccharide is 273 kDa, and the average molecular weight of the capsular polysaccharide-conjugate is 435 kDa), Neisseria meningitidis group C capsular polysaccharide-CRM197 conjugate stock solution (the average molecular weight of the capsular polysaccharide is 225 kDa, and the average molecular weight of the capsular polysaccharide-conjugate is 361 kDa), Neisseria meningitidis group Y capsular polysaccharide-CRM197 conjugate stock solution (the average molecular weight of the capsular polysaccharide is 135 kDa, and the average molecular weight of the capsular polysaccharide-conjugate is 793 kDa), Neisseria meningitidis group W135 capsular polysaccharide-CRM197 conjugate stock solution (the average molecular weight of the capsular polysaccharide is 220 kDa, and the average molecular weight of the capsular polysaccharide-conjugate is 492 kDa), and Neisseria meningitidis group X capsular polysaccharide-TT conjugate stock solution (the average molecular weight of the capsular polysaccharide is 362 kDa, and the average molecular weight of the capsular polysaccharide-conjugate is 896 kDa), and the final product polysaccharide content concentrations are controlled to be 20 μg / mL, 20 μg / mL, 15 μg / mL, 15 μg / mL, and 15 μg / mL respectively. After the Neisseria meningitidis group X capsular polysaccharide conjugate stock solution is added, it is stirred for 4 min to ensure that the added components are fully dispersed, and then the hexavalent Neisseria meningitidis combined vaccine mixture can be obtained.

[0088] Adjust the speed of the magnetic stirrer to 200 rpm, and sequentially add sucrose solution to 15 mg / mL, lactose solution to 5 mg / mL, pH = 6.9, sodium chloride solution to a final concentration of 0.45% (w / w), histidine solution to a final concentration of 30 ppm, and vitamin E solution to a final concentration of 50 ng / mL into the Neisseria meningitidis serogroup A, C, W-135, Y combined vaccine mixture, and make up the volume with water for injection. After the addition of water for injection is completed, continue stirring for 20 min to obtain the semi-finished product of the freeze-dried preparation of the Neisseria meningitidis serogroup A, C, W-135, Y combined vaccine.

[0089] The semi-finished product of the freeze-dried preparation of the Neisseria meningitidis serogroup A, C, W-135, Y combined vaccine is filled into vials by a filling machine, and the filling dose is 0.5 mL / dose per person. The filled vial samples are pre-frozen at -40 °C for 2.5 hours, then frozen at -35 °C for 6 hours, heated and vacuum-dried for 12 hours, and finally vacuum-dried at a constant temperature of 25 °C for 6 hours; after freeze-drying, the finished product of the Neisseria meningitidis serogroup A, C, W-135, Y combined vaccine (freeze-dried vaccine) can be obtained; the diluent of this freeze-dried preparation is 10 mM phosphate buffer and 0.45% sodium chloride aqueous solution. Before use, the freeze-dried preparation is mixed with the diluent preparation and then used for intramuscular injection.

[0090] Example 3

[0091] This example provides a vaccine preparation of a two-dose combination of "freeze-dried AC group + liquid BYW135X group" of the Neisseria meningitidis serogroup A, C, W-135, Y combined vaccine. The specific preparation process is as follows:

[0092] In the preparation of the Neisseria meningitidis serogroup A, C combined vaccine (freeze-dried vaccine), add a certain amount of water for injection to the reaction vessel and place it on a magnetic stirrer for stirring, and adjust the speed to 180 rpm; sequentially add the Neisseria meningitidis capsular polysaccharide conjugate stock solution to the aqueous solution: the A group Neisseria meningitidis capsular polysaccharide-TT carrier protein conjugate stock solution to a medium concentration of 25 μg / mL (the average molecular weight of the capsular polysaccharide is 300 kDa, and the average molecular weight of the capsular polysaccharide-carrier protein conjugate is 489 kDa), and the C group Neisseria meningitidis capsular polysaccharide-CRM197 carrier protein conjugate stock solution to a medium concentration of 25 μg / mL (the average molecular weight of the capsular polysaccharide is 203 kDa, and the average molecular weight of the capsular polysaccharide-carrier protein conjugate is 355 kDa). After adding each component, stir for 3 min to 5 min to ensure that the added components are fully adsorbed or dispersed; adjust the speed of the magnetic stirrer to 200 rpm, and sequentially add sucrose solution to 13 mg / mL, lactose solution to 7 mg / mL, pH = 6.9, sodium chloride solution to a final concentration of 0.3% (w / w), and make up the volume with water for injection. After the addition of water for injection is completed, continue stirring for 20 min to obtain the semi-finished product of the freeze-dried preparation of the Neisseria meningitidis serogroup A, C combined vaccine.

[0093] The semi-finished product of the freeze-dried preparation of the combined meningococcal group A and C vaccine is filled into vials by a filling machine. The filling dose is 0.5 mL / dose per person. The vial samples after filling are pre-frozen at -35 °C for 2.5 hours, then frozen at -40 °C for 5 hours, heated and vacuum-dried for 10 hours, and finally vacuum-dried at a constant temperature of 28 °C for 6 hours. After the freeze-drying is completed, the finished product of the combined meningococcal group A and C vaccine (freeze-dried vaccine) can be obtained. This finished product is used together with the finished product of the combined meningococcal group BYW135X vaccine (liquid vaccine).

[0094] In the preparation of the combined meningococcal group BYW135X vaccine (liquid vaccine), a certain volume of aluminum adjuvant is accurately weighed and placed in the corresponding container, and the aluminum content of the final product is controlled at 0.50 mg / mL. The container containing the aluminum adjuvant is placed on a magnetic stirrer and stirred, and the rotation speed is adjusted to 180 rpm. Three types of Neisseria meningitidis group B fHBP protein solutions, namely fHbp-v1 variant protein, fHbp-v2 variant protein, and fHbp-v3 variant protein, are sequentially added to the adjuvant solution, and the protein concentrations of the final product are controlled at 80 μg / mL, 100 μg / mL, and 90 μg / mL respectively. After each component is added, it is stirred for 3 min to 5 min to ensure that the added components are fully adsorbed or dispersed (the same below). Then, the Neisseria meningitidis capsular polysaccharide conjugate stock solutions are sequentially added: the conjugate stock solution of Neisseria meningitidis group X capsular polysaccharide-TT carrier protein (the average molecular weight of the capsular polysaccharide is 351 kDa, and the average molecular weight of the capsular polysaccharide-carrier protein conjugate is 984 kDa), the conjugate stock solution of Neisseria meningitidis group Y capsular polysaccharide-CRM197 carrier protein (the average molecular weight of the capsular polysaccharide is 131 kDa, and the average molecular weight of the capsular polysaccharide-carrier protein conjugate is 526 kDa), and the conjugate stock solution of Neisseria meningitidis group W135 capsular polysaccharide-CRM197 carrier protein (the average molecular weight of the capsular polysaccharide is 202 kDa, and the average molecular weight of the capsular polysaccharide-carrier protein conjugate is 773 kDa), and the polysaccharide content concentrations of the final product are controlled at 10 μg / mL, 10 μg / mL, and 10 μg / mL respectively. After the addition of the conjugate stock solution of Neisseria meningitidis group W135 capsular polysaccharide is completed, it is stirred for 4 min to ensure that the added components are fully adsorbed or dispersed, and the combined meningococcal group BYW135X vaccine mixture can be obtained.

[0095] Adjust the rotation speed of the magnetic stirrer to 240 rpm. Sequentially add soluble citrate buffer to the combined Neisseria meningitidis group BYW135X vaccine mixture to a final concentration of 10 mM and pH = 7.1, sodium chloride solution to a final concentration of 0.6% (w / w), histidine solution to a final concentration of 40 ppm, polysorbate 80 solution to a final concentration of 40 μg / mL, and vitamin E solution to a final concentration of 30 ng / mL, and make up the volume with water for injection. After the addition of water for injection is completed, continue stirring for 20 min to obtain the semi-finished product of the combined Neisseria meningitidis group BYW135X vaccine liquid preparation.

[0096] The semi-finished product of the combined Neisseria meningitidis group BYW135X vaccine liquid preparation is sub-packed by a filling machine to obtain the finished product of the combined Neisseria meningitidis group BYW135X vaccine (liquid vaccine), and the labeled amount of the finished product is 0.5 mL / dose per person per time. Before use, mix the combined Neisseria meningitidis group AC vaccine (lyophilized vaccine) preparation with the combined Neisseria meningitidis group BYW135X vaccine (liquid vaccine) preparation and then use it for intramuscular injection.

[0097] Example 4

[0098] This example provides a vaccine preparation of a two-dose "ACYW135 group freeze-dried dosage form + BX group liquid dosage form" composition of a hexavalent Neisseria meningitidis combined vaccine. The specific preparation process is as follows:

[0099] In the preparation of the combined vaccine of Neisseria meningitidis group A, C, Y and W135 (freeze-dried vaccine), a certain amount of water for injection was added to the reaction vessel, and the mixture was stirred on a magnetic stirrer at a rotation speed of 180 rpm. The capsular polysaccharide conjugate stock solutions of Neisseria meningitidis were successively added to the aqueous solution: the capsular polysaccharide of Neisseria meningitidis group A-TT conjugate stock solution to a medium concentration of 22 μg / mL (the average molecular weight of the capsular polysaccharide was 286 kDa, and the average molecular weight of the capsular polysaccharide-conjugate was 463 kDa), the capsular polysaccharide of Neisseria meningitidis group C-CRM197 conjugate stock solution to a medium concentration of 24 μg / mL (the average molecular weight of the capsular polysaccharide was 179 kDa, and the average molecular weight of the capsular polysaccharide-conjugate was 321 kDa), the capsular polysaccharide of Neisseria meningitidis group Y-TT conjugate stock solution to a medium concentration of 12 μg / mL (the average molecular weight of the capsular polysaccharide was 130 kDa, and the average molecular weight of the capsular polysaccharide-conjugate was 816 kDa), and the capsular polysaccharide of Neisseria meningitidis group W135-CRM197 conjugate stock solution to a medium concentration of 15 μg / mL (the average molecular weight of the capsular polysaccharide was 192 kDa, and the average molecular weight of the capsular polysaccharide-conjugate was 715 kDa). After each component was added, it was stirred for 3 min to 5 min to ensure that the added components were fully adsorbed or dispersed. The rotation speed of the magnetic stirrer was adjusted to 190 rpm, and a sucrose solution was added to the mixture to 12 mg / mL, a lactose solution to 6.5 mg / mL, pH = 7.0, and a sodium chloride solution to a final concentration of 0.4% (w / w), and the volume was made up with water for injection. After the addition of water for injection was completed, stirring was continued for 20 min to obtain the semi-finished freeze-dried preparation of the combined vaccine of Neisseria meningitidis group A, C, Y and W135.

[0100] The semi-finished freeze-dried preparation of the combined vaccine of Neisseria meningitidis group A, C, Y and W135 was sub-packed into vials by a filling machine, and the filling dose was 0.5 mL / dose per person. The filled vial samples were pre-frozen at -35°C for 3 hours, then frozen at -40°C for 5 hours, heated and vacuum-dried for 10 hours, and finally vacuum-dried at a constant temperature of 25°C for 6 hours. After freeze-drying, the finished product of the combined vaccine of Neisseria meningitidis group A, C, Y and W135 (freeze-dried vaccine) was obtained. This finished product was used together with the finished product of the combined vaccine of Neisseria meningitidis group BX (liquid vaccine).

[0101] In the preparation of the combined vaccine of Neisseria meningitidis group B and X (liquid vaccine), a certain volume of aluminum adjuvant is accurately weighed and placed in the corresponding container, and the aluminum content of the final product is controlled at 0.7 mg / mL. The container containing the aluminum adjuvant is placed on a magnetic stirrer and stirred, and the rotation speed is adjusted to 180 rpm. Three Neisseria meningitidis group B fHBP protein solutions, namely fHbp-v1 variant protein, fHbp-v2 variant protein, and fHbp-v3 variant protein, are sequentially added to the adjuvant solution, and the protein concentrations of the final products are controlled at 90 μg / mL, 90 μg / mL, and 90 μg / mL respectively. Then, the combined stock solution of Neisseria meningitidis group X capsular polysaccharide-TT carrier protein is added to the solution to a final concentration of 10 μg / mL (the average molecular weight of the capsular polysaccharide is 367 kDa, and the average molecular weight of the capsular polysaccharide-carrier protein conjugate is 1065 kDa). After each component is added, it is stirred for 3 min to 5 min to ensure that the added components are fully adsorbed or dispersed, and then the combined vaccine mixture of Neisseria meningitidis group B and X can be obtained.

[0102] The rotation speed of the magnetic stirrer is adjusted to 240 rpm. Soluble phosphate buffer solution is sequentially added to the combined vaccine mixture of Neisseria meningitidis group B and X to a final concentration of 8 mM and pH = 7.0, sodium chloride solution to a final concentration of 0.45% (w / w), histidine solution to a final concentration of 30 ppm, polysorbate 80 solution to a final concentration of 48 μg / mL, vitamin E solution to a final concentration of 35 ng / mL, and it is made up to volume with water for injection. After the addition of water for injection is completed, it is continuously stirred for 20 min, and then the semi-finished product of the liquid preparation of the combined vaccine of Neisseria meningitidis group B and X can be obtained.

[0103] The semi-finished product of the liquid preparation of the combined vaccine of Neisseria meningitidis group B and X is sub-packed by a filling machine to obtain the finished product of the combined vaccine of Neisseria meningitidis group B and X (liquid vaccine). The labeled amount of the finished product is 0.5 mL / dose per 1 person-time. Before use, the combined vaccine (lyophilized vaccine) of Neisseria meningitidis group ACYW135 is mixed with the combined vaccine (liquid vaccine) of Neisseria meningitidis group B and X, and then used for intramuscular injection.

[0104] Example 5

[0105] This example provides a vaccine preparation of a two-dose "ACYW135X group freeze-dried dosage form + group B liquid dosage form" composition of the hexavalent Neisseria meningitidis combined vaccine. The specific preparation process is as follows:

[0106] In the preparation of the combined meningococcal ACYW135X vaccine (lyophilized), a certain amount of water for injection is added to the reaction vessel, which is then placed on a magnetic stirrer and stirred at a speed of 210 rpm. The meningococcal capsular polysaccharide conjugate stock solutions are sequentially added to the aqueous solution: the capsular polysaccharide of Neisseria meningitidis serogroup A-CRM197 conjugate stock solution to a medium concentration of 22 μg / mL (the average molecular weight of the capsular polysaccharide is 241 kDa, and the average molecular weight of the capsular polysaccharide-conjugate is 477 kDa), the capsular polysaccharide of Neisseria meningitidis serogroup C-CRM197 conjugate stock solution to a medium concentration of 24 μg / mL (the average molecular weight of the capsular polysaccharide is 255 kDa, and the average molecular weight of the capsular polysaccharide-conjugate is 396 kDa), the capsular polysaccharide of Neisseria meningitidis serogroup Y-CRM197 conjugate stock solution to a medium concentration of 12 μg / mL (the average molecular weight of the capsular polysaccharide is 162 kDa, and the average molecular weight of the capsular polysaccharide-conjugate is 664 kDa), the capsular polysaccharide of Neisseria meningitidis serogroup W135-CRM197 conjugate stock solution to a medium concentration of 10 μg / mL (the average molecular weight of the capsular polysaccharide is 304 kDa, and the average molecular weight of the capsular polysaccharide-conjugate is 615 kDa), and the capsular polysaccharide of Neisseria meningitidis serogroup X-TT conjugate stock solution to a medium concentration of 15 μg / mL (the average molecular weight of the capsular polysaccharide is 289 kDa, and the average molecular weight of the capsular polysaccharide-conjugate is 831 kDa). After each component is added, it is stirred for 3 min to 5 min to ensure that the added components are fully adsorbed or dispersed. The speed of the magnetic stirrer is adjusted to 250 rpm, and a sucrose solution is sequentially added to the mixture to 13 mg / mL, a lactose solution to 5 mg / mL, the pH is adjusted to 6.5, and a sodium chloride solution is added to a final concentration of 0.45% (w / w), and the volume is made up with water for injection. After the addition of water for injection is completed, stirring is continued for 20 min to obtain the semi-finished product of the combined meningococcal ACYW135X vaccine in lyophilized form.

[0107] The semi-finished product of the combined meningococcal ACYW135X vaccine in lyophilized form is dispensed into vials by a filling machine, and the filling dose is 0.5 mL / dose per person. The filled vial samples are pre-frozen at -35°C for 2.5 hours, then frozen at -35°C for 4.5 hours, heated and vacuum-dried for 14 hours, and finally vacuum-dried at a constant temperature of 25°C for 6 hours. After the lyophilization is completed, the finished product of the combined meningococcal ACYW135X vaccine (lyophilized) can be obtained. This finished product is used together with the finished product of the combined meningococcal serogroup B vaccine (liquid).

[0108] In the preparation of the Neisseria meningitidis group B conjugate vaccine (liquid vaccine), a certain volume of aluminum adjuvant is accurately weighed and placed in the corresponding container, and the aluminum content of the final product is controlled at 0.6 mg / mL. The container containing the aluminum adjuvant is placed on a magnetic stirrer for stirring, and the rotation speed is adjusted to 180 rpm. Three Neisseria meningitidis group B fHBP protein solutions, namely fHbp-v1 variant protein, fHbp-v2 variant protein, and fHbp-v3 variant protein, are sequentially added to the adjuvant solution, and the protein concentrations of the final product are controlled at 80 μg / mL, 90 μg / mL, and 100 μg / mL respectively. After each component is added, it is stirred for 3 min to 5 min to ensure that the added components are fully adsorbed or dispersed, and then the Neisseria meningitidis group B conjugate vaccine mixture can be obtained.

[0109] The rotation speed of the magnetic stirrer is adjusted to 230 rpm. Sequentially add soluble phosphate buffer solution to the Neisseria meningitidis group B conjugate vaccine mixture to a final concentration of 10 mM, pH = 7.5, sodium chloride solution to a final concentration of 0.45% (w / w), 2-phenoxyethanol to a final concentration of 3 mg / mL, histidine solution to a final concentration of 40 ppm, polysorbate 80 solution to a final concentration of 70 μg / mL, vitamin E solution to a final concentration of 35 ng / mL, and make up the volume with water for injection. After the addition of water for injection is completed, continue to stir for 20 min, and then the semi-finished product of the Neisseria meningitidis group B conjugate vaccine liquid preparation can be obtained.

[0110] The semi-finished product of the Neisseria meningitidis group B conjugate vaccine liquid preparation is sub-packed by a filling machine to obtain the finished product of the Neisseria meningitidis group B conjugate vaccine (liquid vaccine), and the labeled amount of the finished product is 0.5 mL / dose per person. Before use, mix the ACYW135 group Neisseria meningitidis conjugate vaccine (lyophilized vaccine) preparation with the Neisseria meningitidis group B conjugate vaccine (liquid vaccine) preparation, and then use it for intramuscular injection.

[0111] Example 6

[0112] This example provides a vaccine preparation of a six-valent Neisseria meningitidis conjugate vaccine in a two-dose package of "ACX group lyophilized dosage form + BYW135 group liquid dosage form", and the specific preparation process is as follows:

[0113] In the preparation of the combined meningococcal vaccine (freeze-dried) of groups A, C, and X, a certain amount of water for injection is added to the reaction vessel, and the vessel is placed on a magnetic stirrer for stirring. The rotation speed is adjusted to 200 rpm. Then, the meningococcal capsular polysaccharide conjugate stock solutions are successively added to the aqueous solution: the capsular polysaccharide of Neisseria meningitidis group A-CRM197 conjugate stock solution to a medium concentration of 20 μg / mL (the average molecular weight of the capsular polysaccharide is 215 kDa, and the average molecular weight of the capsular polysaccharide-carrier protein conjugate is 376 kDa), the capsular polysaccharide of Neisseria meningitidis group C-TT conjugate stock solution to a medium concentration of 24 μg / mL (the average molecular weight of the capsular polysaccharide is 211 kDa, and the average molecular weight of the capsular polysaccharide-carrier protein conjugate is 338 kDa), and the capsular polysaccharide of Neisseria meningitidis group X-TT conjugate stock solution to a medium concentration of 10 μg / mL (the average molecular weight of the capsular polysaccharide is 395 kDa, and the average molecular weight of the capsular polysaccharide-carrier protein conjugate is 1165 kDa). After each component is added, stirring is carried out for 3 min to 5 min to ensure that the added components are fully adsorbed or dispersed. Then, the rotation speed of the magnetic stirrer is adjusted to 250 rpm, and a sucrose solution is added to the mixture to a concentration of 10 mg / mL, a lactose solution is added to a concentration of 5 mg / mL, the pH is adjusted to 7.5, and a sodium chloride solution is added to a final concentration of 0.6% (w / w), and the volume is made up with water for injection. After the addition of water for injection is completed, stirring is continued for 20 min to obtain the semi-finished freeze-dried preparation of the combined meningococcal vaccine of groups A, C, and X.

[0114] The semi-finished freeze-dried preparation of the combined meningococcal vaccine of groups A, C, and X is sub-packed into vials by a filling machine, and the filling dose is 0.5 mL / dose per person. The filled vial samples are pre-frozen at -35°C for 2 hours, then frozen at -35°C for 4 hours, heated and vacuum-dried for 12 hours, and finally vacuum-dried at a constant temperature of 25°C for 6 hours. After freeze-drying is completed, the finished product of the combined meningococcal vaccine (freeze-dried) of groups A, C, and X can be obtained. This finished product is used together with the finished product of the combined meningococcal vaccine (liquid) of groups B, Y, W135.

[0115] In the preparation of the combined vaccine of Neisseria meningitidis group BYW135 (liquid vaccine), a certain volume of aluminum adjuvant is accurately weighed and placed in the corresponding container, and the aluminum content of the final product is controlled to be 0.6 mg / mL. The container containing the aluminum adjuvant is placed on a magnetic stirrer for stirring, and the rotation speed is adjusted to 190 rpm. Three Neisseria meningitidis group B fHBP protein solutions, namely fHbp-v1 variant protein, fHbp-v2 variant protein, and fHbp-v3 variant protein, are sequentially added to the adjuvant solution, and the protein concentrations of the final products are controlled to be 110 μg / mL, 100 μg / mL, and 90 μg / mL respectively. After mixing evenly, the combined stock solution of Neisseria meningitidis group Y capsular polysaccharide-CRM197 carrier protein is added to a medium concentration of 10 μg / mL (the average molecular weight of the capsular polysaccharide is 234 kDa, and the average molecular weight of the capsular polysaccharide-carrier protein conjugate is 673 kDa), and the combined stock solution of Neisseria meningitidis group W135 capsular polysaccharide-CRM197 carrier protein is added to a medium concentration of 14 μg / mL (the average molecular weight of the capsular polysaccharide is 277 kDa, and the average molecular weight of the capsular polysaccharide-carrier protein conjugate is 772 kDa). After each component is added, it is stirred for 3 min to 5 min to ensure that the added components are fully adsorbed or dispersed, and then the combined vaccine mixture of Neisseria meningitidis group BYW135 can be obtained.

[0116] The rotation speed of the magnetic stirrer is adjusted to 230 rpm. Sequentially, soluble phosphate buffer solution is added to the combined vaccine mixture of Neisseria meningitidis group BYW135 to a final concentration of 11 mM, pH = 6.8, sodium chloride solution to a final concentration of 0.40% (w / w), histidine solution to a final concentration of 30 ppm, polysorbate 80 solution to a final concentration of 70 μg / mL, vitamin E solution to a final concentration of 40 ng / mL, and it is made up to volume with water for injection. After the addition of water for injection is completed, stirring is continued for 20 min, and then the semi-finished product of the liquid preparation of the combined vaccine of Neisseria meningitidis group BYW135 can be obtained.

[0117] The semi-finished product of the liquid preparation of the combined vaccine of Neisseria meningitidis group BYW135 is sub-packed by a filling machine, and then the finished product of the combined vaccine of Neisseria meningitidis group BYW135 (liquid vaccine) is obtained. The labeled amount of the finished product is 0.5 mL / dose per person per time. Before use, the combined vaccine (lyophilized vaccine) of Neisseria meningitidis group ACX is mixed with the combined vaccine (liquid vaccine) of Neisseria meningitidis group BYW135, and then used for intramuscular injection.

[0118] Example 7

[0119] This example provides a vaccine preparation of a two-dose combination of hexavalent Neisseria meningitidis vaccine in the form of "freeze-dried ABC group + liquid YW135X group". The specific preparation process is as follows:

[0120] In the preparation of the combined meningococcal vaccine against groups A, B and C (lyophilized vaccine), a certain amount of water for injection is added to the reaction vessel, and the vessel is placed on a magnetic stirrer for stirring. The rotation speed is adjusted to 160 rpm. Then, the meningococcal capsular polysaccharide conjugate stock solutions are successively added to the aqueous solution: the capsular polysaccharide of Neisseria meningitidis serogroup A - TT conjugate stock solution to a medium concentration of 20 μg / mL (the average molecular weight of the capsular polysaccharide is 262 kDa, and the average molecular weight of the capsular polysaccharide - carrier protein conjugate is 431 kDa), and the capsular polysaccharide of Neisseria meningitidis serogroup C - CRM197 conjugate stock solution to a medium concentration of 20 μg / mL (the average molecular weight of the capsular polysaccharide is 242 kDa, and the average molecular weight of the capsular polysaccharide - carrier protein conjugate is 366 kDa). After mixing evenly, three fHBP protein solutions of Neisseria meningitidis serogroup B are successively added: fHbp - v1 variant protein, fHbp - v2 variant protein, and fHbp - v3 variant protein, and the final protein concentrations of the product are controlled to be 120 μg / mL, 120 μg / mL, and 80 μg / mL respectively. After each component is added, stirring is carried out for 3 min to 5 min to ensure that the added components are fully adsorbed or dispersed. The rotation speed of the magnetic stirrer is adjusted to 230 rpm, and a sucrose solution is added to the mixed solution to 13 mg / mL, a lactose solution to 6.5 mg / mL, pH = 7.0, and a sodium chloride solution to a final concentration of 0.45% (w / w), and the volume is made up with water for injection. After the addition of water for injection is completed, stirring is continued for 20 min to obtain the semi - finished product of the combined meningococcal vaccine against groups A, B and C in lyophilized form.

[0121] The semi - finished product of the combined meningococcal vaccine against groups A, B and C in lyophilized form is sub - packed into vials by a filling machine. The filling dose is 0.5 mL / dose per person - time. The filled vial samples are pre - frozen at - 40°C for 3 hours, then frozen at - 35°C for 4 hours, heated and vacuum - dried for 14 hours, and finally vacuum - dried at a constant temperature of 25°C for 5 hours. After the lyophilization is completed, the finished product of the combined meningococcal vaccine against groups A, B and C (lyophilized vaccine) can be obtained. This finished product is used together with the finished product of the combined meningococcal vaccine against groups YW135X (liquid vaccine).

[0122] In the preparation of the combined meningococcal vaccine of Neisseria meningitidis group YW135X (liquid vaccine), a certain volume of water for injection is accurately weighed and placed in a corresponding container, and the container is placed on a magnetic stirrer for stirring, and the rotation speed is adjusted to 200 rpm. Then, the combined stock solution of Neisseria meningitidis group Y capsular polysaccharide - CRM197 carrier protein is added to the aqueous solution to a medium concentration of 10 μg / mL (the average molecular weight of the capsular polysaccharide is 125 kDa, and the average molecular weight of the capsular polysaccharide - carrier protein conjugate is 682 kDa), the combined stock solution of Neisseria meningitidis group W135 capsular polysaccharide - CRM197 carrier protein is added to the aqueous solution to a medium concentration of 10 μg / mL (the average molecular weight of the capsular polysaccharide is 215 kDa, and the average molecular weight of the capsular polysaccharide - carrier protein conjugate is 724 kDa), and the combined stock solution of Neisseria meningitidis group X capsular polysaccharide - CRM197 carrier protein is added to the aqueous solution to a medium concentration of 10 μg / mL (the average molecular weight of the capsular polysaccharide is 313 kDa, and the average molecular weight of the capsular polysaccharide - carrier protein conjugate is 946 kDa). After each component is added, it is stirred for 3 min to 5 min to ensure that the added components are fully adsorbed or dispersed, and then the combined vaccine mixture of Neisseria meningitidis group YW135X can be obtained.

[0123] The rotation speed of the magnetic stirrer is adjusted to 240 rpm. Then, soluble phosphate buffer solution is added to the combined vaccine mixture of Neisseria meningitidis group YW135X to a final concentration of 10 mM and pH = 6.8, sodium chloride solution is added to a final concentration of 0.45% (w / w), and it is made up to volume with water for injection. After the addition of water for injection is completed, it is continuously stirred for 20 min, and then the semi - finished liquid preparation of the combined meningococcal vaccine of Neisseria meningitidis group YW135X can be obtained.

[0124] The semi - finished liquid preparation of the combined meningococcal vaccine of Neisseria meningitidis group YW135X is sub - packed by a filling machine, and then the finished product of the combined meningococcal vaccine of Neisseria meningitidis group YW135X (liquid vaccine) is obtained. The labeled amount of the finished product is 0.5 mL / dose per 1 person - time. Before use, the combined vaccine (lyophilized vaccine) of Neisseria meningitidis group ABC is mixed with the combined vaccine (liquid vaccine) of Neisseria meningitidis group YW135X, and then used for intramuscular injection.

[0125] Example 8

[0126] This example provides a vaccine preparation of a combined hexavalent meningococcal vaccine in a two - dose package of "freeze - dried form of group ABCX + liquid form of group YW135". The specific preparation process is as follows:

[0127] In the preparation of the combined meningococcal vaccine (lyophilized) for groups ABCX, a certain amount of water for injection was added to the reaction vessel and stirred on a magnetic stirrer, and the rotation speed was adjusted to 190 rpm. The meningococcal capsular polysaccharide conjugate stock solutions were successively added to the aqueous solution: the capsular polysaccharide of Neisseria meningitidis serogroup A-CRM197 conjugate stock solution to a medium concentration of 18 μg / mL (the average molecular weight of the capsular polysaccharide was 277 kDa, and the average molecular weight of the capsular polysaccharide-carrier protein conjugate was 484 kDa), the capsular polysaccharide of Neisseria meningitidis serogroup C-CRM197 conjugate stock solution to a medium concentration of 22 μg / mL (the average molecular weight of the capsular polysaccharide was 323 kDa, and the average molecular weight of the capsular polysaccharide-carrier protein conjugate was 406 kDa), the capsular polysaccharide of Neisseria meningitidis serogroup X-TT conjugate stock solution to a medium concentration of 12 μg / mL (the average molecular weight of the capsular polysaccharide was 383 kDa, and the average molecular weight of the capsular polysaccharide-carrier protein conjugate was 946 kDa). After mixing evenly, three kinds of Neisseria meningitidis serogroup B fHBP protein solutions were successively added: fHbp-v1 variant protein, fHbp-v2 variant protein, fHbp-v3 variant protein, and the final product protein concentrations were controlled to be 100 μg / mL, 100 μg / mL, and 120 μg / mL respectively. After each component was added, it was stirred for 3 min to 5 min to ensure that the added components were fully adsorbed or dispersed. The rotation speed of the magnetic stirrer was adjusted to 220 rpm, and a sucrose solution was added to the mixed solution to 13 mg / mL, a lactose solution to 7.0 mg / mL, pH = 7.0, and a sodium chloride solution to a final concentration of 0.4% (w / w), and it was made up to volume with water for injection. After the addition of water for injection was completed, stirring was continued for 20 min, and the semi-finished product of the combined meningococcal vaccine (lyophilized) for groups ABCX could be obtained.

[0128] The semi-finished product of the combined meningococcal vaccine (lyophilized) for groups ABCX was sub-packed into vials by a filling machine, and the filling dose was 0.5 mL / dose per person. The filled vial samples were pre-frozen at -40°C for 2.5 hours, then frozen at -35°C for 4 hours, heated and vacuum-dried for 13 hours, and finally vacuum-dried at a constant temperature of 25°C for 6 hours. After the lyophilization was completed, the finished product of the combined meningococcal vaccine (lyophilized) for groups ABCX could be obtained; this finished product was used together with the finished product of the combined meningococcal vaccine (liquid) for groups YW135.

[0129] In the preparation of the combined vaccine of Neisseria meningitidis serogroups Y and W135 (liquid vaccine), a certain volume of water for injection was accurately weighed and placed in the corresponding container, and the container was placed on a magnetic stirrer for stirring, and the rotation speed was adjusted to 200 rpm. The combined stock solution of Neisseria meningitidis serogroup Y capsular polysaccharide-CRM197 carrier protein was added to the aqueous solution in sequence to a medium concentration of 10 μg / mL (the average molecular weight of the capsular polysaccharide was 115 kDa, and the average molecular weight of the capsular polysaccharide-carrier protein conjugate was 593 kDa), and the combined stock solution of Neisseria meningitidis serogroup W135 capsular polysaccharide-CRM197 carrier protein was added to the aqueous solution in sequence to a medium concentration of 10 μg / mL (the average molecular weight of the capsular polysaccharide was 224 kDa, and the average molecular weight of the capsular polysaccharide-carrier protein conjugate was 692 kDa). After the addition of each component, stirring was carried out for 3 min to 5 min to ensure that the added components were fully adsorbed or dispersed, and the combined vaccine mixture of Neisseria meningitidis serogroups Y and W135 could be obtained.

[0130] The rotation speed of the magnetic stirrer was adjusted to 240 rpm, and soluble phosphate buffer solution was added to the combined vaccine mixture of Neisseria meningitidis serogroups Y and W135 to a final concentration of 10 mM and pH = 6.8, and sodium chloride solution was added to a final concentration of 0.45% (w / w), and the volume was made up with water for injection. After the addition of water for injection was completed, stirring was continued for 20 min, and the semi-finished product of the liquid preparation of the combined vaccine of Neisseria meningitidis serogroups Y and W135 could be obtained.

[0131] The semi-finished product of the liquid preparation of the combined vaccine of Neisseria meningitidis serogroups Y and W135 was sub-packed by a filling machine, and then the finished product of the combined vaccine of Neisseria meningitidis serogroups Y and W135 (liquid vaccine) was obtained. The labeled amount of the finished product was 0.5 mL / dose per 1 person-time. Before use, the combined vaccine preparation of Neisseria meningitidis serogroups ABCX (lyophilized vaccine) was mixed with the combined vaccine preparation of Neisseria meningitidis serogroups Y and W135 (liquid vaccine), and then used for intramuscular injection.

[0132] The statistical results of the active ingredients of the combined vaccine compositions in Examples 1 to 8 are shown in Table 1 below.

[0133] Table 1

[0134]

[0135]

[0136] Note: The content of each component above is in μg / mL.

[0137] Example 9

[0138] Immune evaluation of the combined vaccine compositions in Examples 1 to 8

[0139] This example studied the immune response ability induced by the 1-8 combined vaccine composition in mice. By comparing the antibody titers after immunizing mice with the vaccines (Examples 1-8), the immunogenicity evaluation of the vaccine was achieved. In addition, Example 5: ACYW135 group meningococcal combined vaccine (lyophilized vaccine) was selected as the pentavalent meningococcal positive control, group B meningococcal combined vaccine (liquid vaccine) was selected as the group B meningococcal monovalent control, and physiological saline was selected as the negative control. The quality of the product of the disclosed example of the present invention was evaluated with the antibody titer as the index.

[0140] In the mouse immunogenicity study, a total of 11 groups of experiments were carried out (Examples 1-8 experimental groups, pentavalent meningococcal positive control group, group B meningococcal monovalent control group, and physiological saline control group), with 10 mice in each group (BALB / c female mice (SPF) in good health condition and weighing 12-14 g). They were administered at 1 / 4 of the human dose, that is, each mouse was subcutaneously injected at multiple points with 0.125 mL. Immunizations were carried out on the 1st day, 14th day, and 28th day of immunization respectively, with a total of 3 immunization times. Blood was taken from the eyes of each mouse on the 14th day after the last vaccination. After centrifuging the blood sample, the supernatant was taken, that is, the serum sample, and the serum sample of each mouse was stored separately.

[0141] The antibodies against group A meningococcal capsular polysaccharide, group C meningococcal capsular polysaccharide, group Y meningococcal capsular polysaccharide, group W135 meningococcal capsular polysaccharide, group X meningococcal capsular polysaccharide, and group B meningococcal lipoprotein fHbp were determined by ELISA method. The above antigens were respectively coated on the titration wells of the ELISA microtiter plate, so that each group of polysaccharide and protein antigens were fixedly combined on the titration wells of the microtiter plate. The serum sample was incubated with an excess of each antigen fixedly combined on the titration wells of the ELISA microtiter plate to form an antigen-antibody complex. 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 the antigen-antibody complex to be detected was added to the titration wells of the antigen-antibody complex 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 the conjugated vaccine in different experiments were statistically analyzed (OD > the maximum dilution multiple of the OD value of the physiological saline control group by 2 times). The experimental results are shown in Table 2 below.

[0142] Table 2. Antibody titers in the immune sera of mice of the hexavalent combined vaccine composition (calculated as Log10)

[0143]

[0144] As can be seen from the results in Table 2, compared with the negative control group, the hexavalent Neisseria meningitidis conjugate vaccines of Examples 1-8 can induce corresponding levels of antibody content in mice, all showing good immunogenicity. There are certain differences among different sample groups. Among them, the comprehensive antibody titer value of the liquid hexavalent meningococcal conjugate vaccine composition in Example 1 is the highest; this dosage form vaccine has better immune effects against strains of groups A, C, Y, W135, and X compared with the positive control group of the pentavalent meningococcal conjugate vaccine composition; this dosage form vaccine has higher immune effects against strains of fHbp-v1, fHbp-v2, and fHbp-v3 sequences compared with the positive control group of the group B meningococcal conjugate vaccine composition, indicating that the liquid hexavalent meningococcal conjugate vaccine composition in Example 1 has good synergistic effects compared with the conjugate vaccine composition of the positive control group under the stimulation of adjuvants and the functional effects of related excipients, enhancing the immunogenicity of the single-dose liquid vaccine. It shows that the hexavalent Neisseria meningitidis conjugate vaccine disclosed in the present invention has good feasibility, practicability, and applicability.

[0145] Example 10

[0146] Verification of the bactericidal ability of epidemic strains of the conjugate vaccine compositions of Examples 1-8:

[0147] In this example, the bactericidal effect of the antibody serum after immunizing mice with the conjugate vaccine compositions of 1-8 was studied. By comparing the in vitro bactericidal ability results of the immune mouse sera, the immunogenicity evaluation of the vaccine was achieved. In addition, the ACYW135X group Neisseria meningitidis conjugate vaccine (lyophilized vaccine) of Example 5 was selected as the pentavalent meningococcal positive control, the group B Neisseria meningitidis conjugate vaccine (liquid vaccine) was selected as the group B meningococcal positive control, and normal saline was selected as the negative control. The immunogenicity of the products of the disclosed examples of the present invention was evaluated with the antibody titer as the evaluation index.

[0148] Preparation of target bacteria: Six epidemic Neisseria meningitidis strains were cultured on a 8-12% sheep blood agar plate at 37°C in 6-10% carbon dioxide for 16-18 hours. The bacterial lawn was scraped into physiological saline, and the bacteria were counted by turbidimetry. According to the count, the target bacteria were diluted to 1x10 6 CFU / mL.

[0149] Complement of the experimental groups of Examples 1-8, the pentavalent meningococcal positive control group, the group B meningococcal monovalent control group, and the normal saline control group were added to the test sera, and they were serially diluted on a 96-well culture plate and 10 μL of target bacteria was added. After mixing, it was cultured at 37°C for 2-4 hours.

[0150] Spotting: The cultured mixed bacterial solution was spotted onto a solid agar medium in an amount of 10 μL and cultured overnight at 37°C.

[0151] Color development: Cover the solid agar medium with soft agar containing 150 μg / mL - 300 μg / mL of TTC and develop color at an appropriate temperature.

[0152] Counting: Use image scanning technology to analyze and calculate the number of bacterial colonies, and calculate the bactericidal titer with the software for bactericidal efficacy results. The experimental results are shown in Table 3 below.

[0153] Table 3. Results of SBA bactericidal efficacy test for epidemic strains of groups A, B, C, Y, W135, and X of Neisseria meningitidis (calculated in Log10)

[0154]

[0155]

[0156] As can be seen from the results in Table 3, compared with the control experimental group, the hexavalent meningococcal conjugate vaccines of Examples 1 - 8 showed good in vitro bactericidal effects, and each group of samples had good in vitro bactericidal effects. In addition, there were certain differences among the sample groups. Among them, the liquid - dosage - form hexavalent meningococcal conjugate vaccine composition in Example 1 had better comprehensive in vitro bactericidal effects; this dosage - form vaccine had better in vitro bactericidal effects against strains of groups A, C, Y, W135, and X compared with the positive control group of the pentavalent meningococcal conjugate vaccine composition; this dosage - form vaccine had good in vitro bactericidal effects against strains with fHbp - v1, fHbp - v2, and fHbp - v3 sequences compared with the positive control group of the group B meningococcal conjugate vaccine composition. This indicates that under the stimulation of the adjuvant and the functional effects of related excipients, the liquid - dosage - form hexavalent meningococcal conjugate vaccine composition in Example 1 had good synergistic effects compared with the conjugate vaccine composition of the positive control group, enhancing the immunogenicity of the single - dose liquid vaccine. From the data of the negative experimental group, it can be seen that the vaccines of different examples all had in vitro bactericidal effects. This data result was similar to the antibody immunity result, had good reproducibility, and the overall performance difference was small. Further indicating that the hexavalent meningococcal conjugate vaccine disclosed in the present invention had good immune effects.

[0157] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above - mentioned embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions disclosed in the present invention, and these simple modifications all fall within the protection scope of the present invention. Additionally, it should be noted that in the above - described specific embodiments, the various specific technical features described can be combined in any appropriate way without contradiction. To avoid unnecessary repetition, the present invention does not separately describe various possible combination methods. Furthermore, any combination can be made among the various different embodiments disclosed in the present invention as long as it does not violate the disclosed idea of the present invention, and it should also be regarded as the content disclosed in the present invention.

Claims

1. A hexavalent Neisseria meningitidis conjugate vaccine composition, the active ingredients of which comprise: capsular polysaccharide of Neisseria meningitidis group A, capsular polysaccharide of Neisseria meningitidis group C, capsular polysaccharide of Neisseria meningitidis group Y, capsular polysaccharide of Neisseria meningitidis group W135, capsular polysaccharide of Neisseria meningitidis group X, and human factor H binding protein (fHbp) of Neisseria meningitidis group B, wherein: The capsular polysaccharides of Neisseria meningitidis group A, Neisseria meningitidis group C, Neisseria meningitidis group Y, Neisseria meningitidis group W135, and / or Neisseria meningitidis group X exist in the form of being conjugated with a carrier protein, and the factor H-binding protein (fHbp) exists in a free form or in a form conjugated with an adjuvant.

2. The conjugate vaccine composition according to claim 1, characterized in that In the combined vaccine composition, the average molecular weight of the capsular polysaccharide is 100 kDa to 400 kDa; Preferably, in the combined vaccine conjugate, the average molecular weight of the conjugate formed by the capsular polysaccharide and the carrier protein is 300 kDa to 1200 kDa.

3. The conjugate vaccine composition according to claim 1 or 2, characterized in that The carrier protein includes at least one of tetanus toxoid (TT), diphtheria toxoid (DT), non-toxic mutant protein of diphtheria toxin (CRM197), outer membrane protein of Neisseria meningitidis (OMP), human serum albumin, and bovine serum albumin (BSA); Preferably, the carrier protein is selected from the following combinations: tetanus toxoid (TT) and diphtheria toxoid (DT), tetanus toxoid (TT) and non-toxic mutant protein of diphtheria toxin (CRM197), and more preferably selected from tetanus toxoid (TT) and non-toxic mutant protein of diphtheria toxin (CRM197); Preferably, the capsular polysaccharide and the carrier protein are conjugated through a coupling reaction. More preferably, the coupling agent used in the coupling reaction includes at least one of carbodiimide compounds, thiol compounds, hydrazide compounds, p-nitrobenzoic acid, and N-hydroxysuccinimide.

4. The conjugate vaccine composition according to any one of claims 1 to 3, characterized in that The human factor H-binding protein (fHbp) of Neisseria meningitidis group B includes factor H-binding protein variant 1 (fHbp-v1 variant protein), factor H-binding protein variant 2 (fHbp-v2 variant protein), and / or factor H-binding protein variant 3 (fHbp-v3 variant protein); Preferably, the fHbp-v1 variant protein, fHbp-v2 variant protein, and fHbp-v3 variant protein have the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 in sequence, or amino acid sequences having at least 85% sequence identity with the corresponding amino acid sequences, or amino acid sequences having substitution, deletion, or insertion of one or more amino acid residues with the corresponding amino acid sequences.

5. The conjugate vaccine composition according to claim 4, characterized in that The mass ratios of the fHbp-v1 variant protein, fHbp-v2 variant protein, and fHbp-v3 variant protein are (80.0 to 120.0):(80.0 to 120.0):(80.0 to 120.0) in sequence; Preferably, the mass ratios of the capsular polysaccharides of Neisseria meningitidis group A, Neisseria meningitidis group C, Neisseria meningitidis group Y, Neisseria meningitidis group W135, Neisseria meningitidis group X, and the human factor H-binding protein of Neisseria meningitidis group B are (10 to 30):(10 to 30):(5 to 25):(5 to 25):(5 to 25):(240 to 360) in sequence.

6. The conjugate vaccine composition according to claim 5, characterized in that The combined vaccine composition contains Neisseria meningitidis serogroup A capsular polysaccharide at a concentration of 10 μg / mL to 30 μg / mL, Neisseria meningitidis serogroup C capsular polysaccharide at a concentration of 10 μg / mL to 30 μg / mL, Neisseria meningitidis serogroup Y capsular polysaccharide at a concentration of 5 μg / mL to 25 μg / mL, Neisseria meningitidis serogroup W135 capsular polysaccharide at a concentration of 5 μg / mL to 25 μg / mL, Neisseria meningitidis serogroup X capsular polysaccharide at a concentration of 5 μg / mL to 25 μg / mL, fHbp-v1 variant protein at a concentration of 80 μg / mL to 120 μg / mL, fHbp-v2 variant protein at a concentration of 80 μg / mL to 120 μg / mL, and fHbp-v3 variant protein at a concentration of 80 μg / mL to 120 μg / mL.

7. The conjugate vaccine composition according to any one of claims 1 to 6, characterized in that The adjuvant includes at least one of aluminum adjuvant, Freund's complete adjuvant, Freund's incomplete adjuvant, MF59 adjuvant, AS01 - AS04 series adjuvants, MPLA adjuvant, CPG adjuvants; Preferably, the aluminum adjuvant includes at least one of aluminum hydroxide adjuvant, aluminum phosphate adjuvant, or aluminum sulfate adjuvant, more preferably aluminum hydroxide adjuvant and / or aluminum phosphate adjuvant; Preferably, the aluminum content in the aluminum adjuvant is 0.2 mg / ml to 1.25 mg / ml, preferably 0.2 mg / ml to 0.8 mg / ml.

8. The conjugate vaccine composition according to any one of claims 1 to 7, characterized in that The combined vaccine composition further includes formulation excipients. Preferably, the formulation excipients include at least one of a cryoprotectant, a preservative, a stabilizer, a buffer system, and an osmotic pressure maintainer; Preferably, the cryoprotectant includes at least one of trehalose, sodium citrate, sodium acetate, sucrose, lactose, mannitol, and tromethamine, more preferably at least one of sucrose, lactose, and mannitol; Preferably, the preservative includes thimerosal and / or 2 - phenoxyethanol; Preferably, the stabilizer includes at least one of surfactant solubilizers, vitamins or their complexes, and amino acids. More preferably, the surfactant solubilizers include at least one of polysorbate 80, polysorbate 20, poloxamer 188, polyethylene glycol, span 20, span 80, Triton, preferably polysorbate 80; the vitamins or their complexes include at least one of vitamin A, pantothenic acid, niacin, biotin, folic acid, vitamin B1 (thiamine), vitamin B2 (riboflavin), pyridoxine (vitamin B6), cyanocobalamin (vitamin B12), vitamin C, vitamin D, vitamin E, preferably vitamin E; the amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, preferably at least one of arginine, histidine, aspartic acid, glutamine, tryptophan, tyrosine, glycine, lysine, more preferably arginine and histidine; Preferably, the osmotic pressure maintainer includes at least one of sodium chloride, potassium chloride, magnesium chloride, calcium chloride, zinc chloride, manganese chloride, iron chloride, preferably sodium chloride; Preferably, the buffer system includes at least one of phosphate buffer solution, acetate buffer solution, succinate buffer solution, citrate buffer solution, and Tris-HCl buffer solution; Preferably, the pH value range of the combined vaccine composition is 5.5 to 7.5; Preferably, the osmotic pressure range of the combined vaccine composition is 220 mOsm / kg to 340 mOsm / kg.

9. The conjugate vaccine composition according to any one of claims 1 to 8, characterized in that The combined vaccine composition is a liquid preparation and / or a freeze-dried preparation; Preferably, the liquid preparation is prepared by a method comprising the following steps: after formulating each Neisseria meningitidis conjugate stock solution and / or Neisseria meningitidis serogroup B human factor H binding protein stock solution and formulation excipients, performing sub-packaging; Preferably, the preparation process of the antigen solution of the liquid preparation includes: in each 0.5 mL single-dose liquid preparation, weighing an aluminum adjuvant with a final concentration of aluminum of 0.2 mg / mL to 0.8 mg / mL in a container, adjusting the rotation speed to 150 rpm to 300 rpm, and sequentially adding to the adjuvant solution a fHbp-v1 variant protein with a final concentration of 40 μg / dose to 60 μg / dose, a fHbp-v2 variant protein with a final concentration of 40 μg / dose to 60 μg / dose (, a fHbp-v3 variant protein with a final concentration of 40 μg / dose to 60 μg / dose, and then sequentially adding a Neisseria meningitidis serogroup A capsular polysaccharide conjugate stock solution with a final concentration of 5.0 μg / dose to 15.0 μg / dose, and / or a Neisseria meningitidis serogroup C capsular polysaccharide conjugate stock solution with a final concentration of 5.0 μg / dose to 15.0 μg / dose, and / or a Neisseria meningitidis serogroup Y capsular polysaccharide conjugate stock solution with a final concentration of 2.5 μg / dose to 12.5 μg / dose, and / or a Neisseria meningitidis serogroup W135 capsular polysaccharide conjugate stock solution with a final concentration of 2.5 μg / dose to 12.5 μg / dose, and / or a Neisseria meningitidis serogroup X capsular polysaccharide conjugate stock solution with a final concentration of 2.5 μg / dose to 12.5 μg / dose, wherein each stock solution is stirred for 3 min to 5 min after addition to obtain an antigen solution; Preferably, the method for adding formulation excipients to the liquid preparation comprises: after adding the antigen component, adding the buffer system with a final concentration of 5 mM to 25 mM, the osmotic pressure maintainer with a final concentration of 0.45% (w / w) to 0.9% (w / w), the surfactant with a final concentration of 0 μg / mL to 100 μg / mL, the preservative with a final concentration of 0 mg / mL to 4 mg / mL, the vitamin or its complex with a final concentration of 0 ng / mL to 100 ng / mL, and water for injection to the antigen solution, controlling the stirring speed at 150 rpm to 300 rpm, and continuing to stir for 20 min after adding the water for injection to obtain a semi-finished liquid preparation; more preferably, adding a soluble phosphate buffer or analog with a final concentration of 5 mM to 25 mM, sodium chloride or an equivalent substance with a final concentration of 0.45% (w / w) to 0.9% (w / w), histidine or an analog with a final concentration of 50 ppm to 600 ppm, polysorbate 80 or an analog with a final concentration of 0 μg / mL to 100 μg / mL, 2-phenoxyethanol or an analog with a final concentration of 0 mg / mL to 4 mg / mL, vitamin E or an analog with a final concentration of 0 ng / mL to 100 ng / mL, and water for injection to the antigen solution; Preferably, the freeze-dried preparation is prepared by a method comprising the following steps: formulating each Neisseria meningitidis conjugate stock solution and / or Neisseria meningitidis serogroup B human factor H binding protein stock solution, and formulation excipients to obtain a liquid preparation solution, and sub-packaging the liquid preparation solution and optionally performing freeze-vacuum drying; Preferably, the method for formulating the antigen solution of the freeze-dried preparation is the same as the method for formulating the antigen solvent of the liquid preparation; Preferably, the freeze-vacuum drying program comprises: pre-freezing at -35°C to -40°C for 2 h to 3 h, freezing at -35°C to -40°C for 3 h to 6 h, heating and vacuum drying for 8 h to 14 h, and maintaining a constant temperature and vacuum drying at below 30°C for 4 h to 8 h; Preferably, the diluent of the freeze-dried preparation comprises 0.4% - 0.6%, preferably 0.45% aqueous sodium chloride solution and 5 mM - 15 mM, preferably 10 mM phosphate buffer.

10. Use of the combined vaccine composition according to any one of claims 1 to 9 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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