CN116898960A - Bacterial polysaccharide protein conjugates and uses thereof - Google Patents

Abstract

The present invention relates to a polysaccharide protein conjugate, in particular, the present invention provides a polysaccharide protein conjugate having immunogenicity, comprising a polysaccharide derived from bacterial meningitis pathogenic bacteria and a carrier protein conjugated thereto, the carrier protein comprising TTD or a variant thereof. The polysaccharide protein conjugate can effectively improve the immunogenicity of bacterial meningitis pathogenic bacteria polysaccharide and induce specific antibodies.

Description

Bacterial polysaccharide protein conjugates and uses thereof

Technical Field

The invention belongs to the field of vaccines, and particularly relates to a bacterial polysaccharide protein conjugate and application thereof.

Background

Bacterial meningitis is caused by infection of pathogenic bacteria, and seriously endangers the life and health of human beings, wherein common pathogenic bacteria are neisseria meningitidis, streptococcus pneumoniae, haemophilus influenzae, listeria and the like. The bacterial meningitis infection has serious consequences and high mortality, and the recovered patients often have permanent sequelae, such as brain injury, hearing loss, learning disorder and the like.

Neisseria meningitidis is a gram negative bacterium. The capsular polysaccharides are divided into 13 serogroups according to their chemical structure. Of these, A, B, C, Y, W group 135 is the predominant epidemic group.

Epidemic cerebrospinal meningitis (epidemic cerebrospinal meningitis for short) is an acute respiratory infectious disease caused by meningococcal infection, and has become one of the global serious public health problems. It is counted that about 50 thousands of cases of brain flow occur annually worldwide, with mortality rates of up to 5% -10% in developed countries and even 20% in developing countries, even after antibiotics and rigorous treatment.

Haemophilus influenzae can cause a variety of infections such as pneumonia, otitis media, bacteremia and meningitis, with haemophilus influenzae type b (Hib) being the major pathogen causing invasive infections, which can be severe lethal.

Vaccination with vaccines is currently the most cost-effective means of preventing bacterial meningitis. There are two classes of current prophylactic vaccines: polysaccharide vaccines and polysaccharide-protein conjugate vaccines. Capsular polysaccharides are the main protective structural components and virulence factors of bacteria, and are important antigens for the development of prophylactic vaccines. However, the capsular polysaccharide is thymus independent antigen, and according to the clinical test results of the bacterial vaccine which is marketed, the capsular polysaccharide vaccine has low immunogenicity and can not induce immunological memory, and has no protective effect on people with low immunity, such as infants and the elderly below 2 years old; the conjugate vaccine formed by coupling the polysaccharide and the carrier is thymus dependent antigen, has stronger immunogenicity, can form immune memory, and has better and longer lasting immune protection effect.

Bacterial meningitis polysaccharide conjugate vaccines currently on the market contain carrier proteins that include primarily Tetanus Toxoid (TT), diphtheria Toxoid (DT), and diphtheria toxin non-toxic variant protein CRM197.TT and DT proteins are prepared by detoxication of tetanus toxin and diphtheria toxin respectively, and have the defects of incomplete detoxication, unstable and nonuniform protein structure after detoxication, allergic reaction initiation and the like. In addition, TT, DT and CRM197 are common antigenic components in multiple vaccines for infants, so that the induced pre-existing antibodies have potential immunosuppressive effects on the carrier proteins in the polysaccharide protein-bound vaccine, thereby affecting the effectiveness of the vaccine. Therefore, the development of the carrier protein of the novel polysaccharide conjugate vaccine has important clinical significance for improving the safety and effectiveness of the vaccine.

Disclosure of Invention

In order to solve the problems of low immunogenicity and poor protective power of polysaccharide and incomplete detoxification and low immunogenicity of carrier proteins in polysaccharide conjugates, the invention provides an immunogenic composition for protecting mammals from bacterial meningitis infection. The bacterial meningitis polysaccharide conjugate provided by the invention can effectively improve the immunogenicity of meningococcal polysaccharide and haemophilus influenzae type b polysaccharide, and can induce higher immunogenicity compared with polysaccharide conjugates prepared by the existing commonly used carrier proteins.

In a first aspect the invention provides an immunogenic polysaccharide protein conjugate comprising a polysaccharide derived from a bacterial meningococcal pathogen and a carrier protein conjugated thereto.

In one or more embodiments, the bacterial meningococcal pathogenic bacteria are selected from neisseria meningitidis and haemophilus influenzae.

In one or more embodiments, the bacterial meningitis pathogenic bacteria is selected from neisseria meningitidis serogroup A, C, Y, W, haemophilus influenzae type b.

In one or more embodiments, the polysaccharide is a capsular polysaccharide.

In one or more embodiments, the carrier protein comprises TTD or a variant thereof.

In one or more embodiments, the TTD has a sequence set forth in SEQ ID NO. 2 and the TTD variant has a sequence that is at least 70%, at least 80%, or at least 90% sequence identity to SEQ ID NO. 2.

In one or more embodiments, the carrier protein is covalently linked to the polysaccharide.

In one or more embodiments, the weight ratio of polysaccharide to carrier protein is from 0.2 to 3.0, such as from 0.5 to 2.5.

The present invention also provides a method of preparing a polysaccharide protein conjugate as described in any of the embodiments herein, comprising the steps of:

(1) Obtaining a polysaccharide derived from a bacterial meningitis pathogen,

(2) Activating the polysaccharide, optionally derivatizing the activated polysaccharide,

(3) Coupling the polysaccharide with a carrier protein to obtain a polysaccharide protein conjugate.

In one or more embodiments, the bacterial meningococcal pathogenic bacteria are selected from neisseria meningitidis and haemophilus influenzae.

In one or more embodiments, the bacterial meningitis pathogenic bacteria is selected from neisseria meningitidis serogroup A, C, Y, W, haemophilus influenzae type b.

In one or more embodiments, step (1) comprises: and (3) inactivating bacteria, filtering to obtain crude sugar solution, adding a polysaccharide precipitant, and purifying to obtain polysaccharide.

In one or more embodiments, step (2) comprises: the polysaccharide is incubated with periodate solution or CDAP. Preferably, step (2) further comprises: the activated polysaccharide was incubated with ADH.

In one or more embodiments, step (3) comprises: polysaccharide protein conjugates were obtained by incubating polysaccharide with carrier protein in the presence of EDAC. Preferably, in step (3), the weight ratio of polysaccharide to carrier protein is 0.2-3.0. Preferably, in step (3), the incubation is continued for at least 30 minutes, preferably at least 2 hours.

In one or more embodiments, the weight ratio of activated polysaccharide to carrier protein in step (3) is from 0.5 to 2.5. Preferably, step (3) further comprises a process of isolating the polysaccharide protein conjugate, such as ultrafiltration or chromatography. In one or more embodiments, the concentration of sodium cyanoborohydride is 10 to 200mM, preferably 50 to 150mM, more preferably about 100mM. In one or more embodiments, the polysaccharide is incubated with the carrier protein for at least 2 hours, preferably at least 12 hours, more preferably 16-48 hours.

The invention also provides an immunogenic composition comprising a polysaccharide protein conjugate as described in any of the embodiments herein and an adjuvant.

In one or more embodiments, the immunogenic composition comprises one or more selected from the group consisting of: (1) a neisseria meningitidis serogroup polysaccharide and carrier protein conjugated thereto, (2) a neisseria meningitidis serogroup polysaccharide and carrier protein conjugated thereto, (3) a neisseria meningitidis serogroup polysaccharide and carrier protein conjugated thereto, (4) a neisseria meningitidis serogroup polysaccharide and carrier protein conjugated thereto, and (5) a haemophilus influenzae type b polysaccharide and carrier protein conjugated thereto. In one or more embodiments, the immunogenic composition comprises (1) - (4) or (1) - (5) above.

In one or more embodiments, the polysaccharide is a capsular polysaccharide.

In one or more embodiments, the carrier protein comprises TTD or a variant thereof.

In one or more embodiments, the TTD has a sequence set forth in SEQ ID NO. 2 and the TTD variant has a sequence that is at least 70%, at least 80%, or at least 90% sequence identity to SEQ ID NO. 2.

In one or more embodiments, the carrier protein is covalently linked to the polysaccharide.

In one or more embodiments, the weight ratio of polysaccharide to carrier protein is from 0.2 to 3.0, such as from 0.5 to 2.5.

In one or more embodiments, the adjuvant includes one or more selected from the group consisting of aluminum hydroxide, aluminum sulfate, aluminum phosphate, monophosphoryl lipid A, QS, stearoyl tyrosine, and freund's adjuvant.

In one or more embodiments, the immunogenic composition further comprises a carrier. Preferably, the carrier is selected from one or more of saline, ringer's solution and phosphate buffered saline.

The invention also provides an immune composition that results in passive immunization comprising bactericidal antibodies that target bacterial meningitis pathogenic bacteria, said antibodies being obtained by immunizing a mammal with an immunogenic composition as described in any of the embodiments herein. In one or more embodiments, the bactericidal antibodies are present in serum, gamma globulin fractions, or purified antibody formulations.

In one or more embodiments, the bacterial meningococcal pathogenic bacteria are selected from neisseria meningitidis and haemophilus influenzae.

In one or more embodiments, the bacterial meningitis pathogenic bacteria is selected from neisseria meningitidis serogroup A, C, Y, W, haemophilus influenzae type b.

The invention also provides a vaccine comprising an immunogenic composition as described in any of the embodiments herein and a pharmaceutically acceptable adjuvant.

The invention also provides the use of a polysaccharide protein conjugate or immunogenic composition as described in any of the embodiments herein in the manufacture of a medicament for providing protection against infection by a bacterial meningococcal pathogen.

In one or more embodiments, the bacterial meningococcal pathogenic bacteria are selected from neisseria meningitidis and haemophilus influenzae.

In one or more embodiments, the bacterial meningitis pathogenic bacteria is selected from neisseria meningitidis serogroup A, C, Y, W, haemophilus influenzae type b.

The invention also provides a method of immunizing a mammal against infection by a bacterial meningococcal pathogen, the method comprising administering to the individual an immunogenic amount of an immunogenic composition or polysaccharide protein conjugate according to any one of the embodiments herein. In one or more embodiments, the mammal is a mouse, a rabbit, or a human.

In one or more embodiments, the bacterial meningococcal pathogenic bacteria are selected from neisseria meningitidis and haemophilus influenzae.

In one or more embodiments, the bacterial meningitis pathogenic bacteria are selected from neisseria meningitidis A, C, Y, W serotype, haemophilus influenzae type b.

The invention has the advantages that:

(1) The invention provides an immune approach to solve the problem of infection by meningococcal pathogenic bacteria (e.g. Neisseria meningococci and haemophilus influenzae type b)

(2) The invention discovers that the recombinant tetanus toxoid fragment (TTD) can be used as carrier protein to effectively improve the immunogenicity of meningitis pathogenic polysaccharide, and more specific polysaccharide functional antibodies can be induced than diphtheria bacteriocin non-toxic variant protein (CRM 197) carrier.

(3) The invention provides an immunogen capable of inducing specific anti-meningitis pathogenic bacteria surface polysaccharide antibody, thereby playing a role in prevention or treatment.

(4) An immunoconjugate is provided wherein a carrier protein is used that is effective to increase the immunogenicity of meningococcal polysaccharide, inducing antibodies specific for meningococcal polysaccharide.

Drawings

Fig. 1: TTD and CRM197 carrier proteins enhance W135 group polysaccharide immunogenicity comparisons.

Fig. 2: immune serum SBA sterilization curves with W135 group polysaccharide conjugates of TTD and CRM197 carrier proteins, respectively.

Fig. 3: TTD and CRM197 carrier proteins enhance Y group polysaccharide immunogenicity comparisons.

Fig. 4: immune serum SBA sterilization curves with group Y polysaccharide conjugates of TTD and CRM197 carrier proteins, respectively.

Fig. 5: TTD and CRM197 carrier proteins enhance group C polysaccharide immunogenicity comparisons.

Fig. 6: immune serum SBA sterilization curves with group C polysaccharide conjugates of TTD and CRM197 carrier proteins, respectively.

Fig. 7: TTD and CRM197 carrier proteins enhance group a polysaccharide immunogenicity comparisons.

Fig. 8: group a polysaccharide conjugates of TTD and CRM197 carrier proteins were used to immunize serum SBA sterilization curves, respectively.

Fig. 9: TTD and CRM197 carrier proteins enhance Hib polysaccharide immunogenicity comparison.

Fig. 10: serum SBA sterilization curves were immunized with Hib polysaccharide conjugates of TTD and CRM197 carrier proteins, respectively.

Fig. 11: immunogenicity comparison of tetravalent meningitis polysaccharide conjugate vaccines of different carrier proteins in mice.

Fig. 12: levels of IgG antibody titers of polysaccharide of each type in the serum of the combination vaccine immune to ACYW135 meningitis polysaccharide conjugate vaccine and HIB in mice.

Detailed Description

The invention provides a novel meningococcal capsular polysaccharide-protein conjugate. The conjugate is prepared by connecting polysaccharide fragments obtained by treating purified and extracted meningococcal capsular polysaccharide to carrier protein in a chemical coupling mode. The conjugate can effectively induce immunogenicity of meningitis pathogenic bacteria polysaccharide, and has good protective effect on bacterial meningitis pathogenic bacteria infection.

The present invention first provides an immunogenic composition for protecting a mammal from infection by a bacterial meningococcal pathogen. The immunogenic composition comprises an immunogenic amount of isolated bacterial meningitis pathogenic capsular polysaccharide.

As used herein, "bacterial meningitis pathogen" or "meningococcal pathogen" refers to a bacterium that causes bacterial meningitis. Including but not limited to neisseria meningitidis and haemophilus influenzae, e.g. neisseria meningitidis A, C, Y, W serogroup 135, 1, 2, 3, 4 or 5 of haemophilus influenzae type b. In some embodiments, the polysaccharide of serogroup A, C, Y, W, haemophilus influenzae type b, has a molecular weight of between 40 and 400 kD. In some embodiments, the bacterial meningococcal pathogen includes neisseria meningitidis A, C, Y and W135 serogroups. In some embodiments, the bacterial meningococcal pathogenic bacteria include neisseria meningitidis A, C, Y, W serogroup and haemophilus influenzae type b.

The meningococcal capsular polysaccharide is present in the immunogenic composition as a component of a conjugate in which the polysaccharide is covalently linked to the protein. Natural or recombinant bacterial proteins such as tetanus toxoid, cholera toxin, diphtheria toxoid, or CRM197 are examples of suitable proteins that may be used as conjugates. In addition to the polysaccharide protein conjugate, the immunogenic composition may also comprise a carrier, such as saline, ringer's solution, or phosphate buffered saline.

In one or more embodiments, the immunogenic composition comprises one or more selected from the group consisting of: (1) a neisseria meningitidis serogroup polysaccharide and carrier protein conjugated thereto, (2) a neisseria meningitidis serogroup polysaccharide and carrier protein conjugated thereto, (3) a neisseria meningitidis serogroup polysaccharide and carrier protein conjugated thereto, (4) a neisseria meningitidis serogroup polysaccharide and carrier protein conjugated thereto, and (5) a haemophilus influenzae type b polysaccharide and carrier protein conjugated thereto. In one or more embodiments, the immunogenic composition comprises (1) - (4) or (1) - (5) above.

In a preferred embodiment of the invention, the meningococcal capsular polysaccharide is covalently linked to the protein to form a conjugate. Thus, any protein or fragment thereof that is acceptable to an individual and that is capable of inducing an immune cell (e.g., T-cell) dependent response is suitable for conjugation to the capsular polysaccharide of a meningococcal pathogen. Essentially any protein can be used as the conjugate protein. In particular, the protein of choice must have at least one free amino group for conjugation to the polysaccharide. Preferably the protein is any natural or recombinant bacterial protein and is itself an immunogen that induces T-cell dependent responses in young and adult mammals. Examples of such proteins include, but are not limited to, tetanus toxoid, cholera toxin, diphtheria toxoid. Other candidates for conjugated proteins include toxins or toxoids of pseudomonas, staphylococcus, streptococcus, pertussis, and enterotoxigenic bacteria including escherichia coli.

The protein to which the meningococcal capsular polysaccharide is conjugated (i.e. carrier protein) may be a natural toxin or a detoxified toxin (i.e. toxoid), for example TT, DT. Alternatively, non-toxic mutant forms of the protein toxins may be used. Preferably such mutations retain epitopes of the native toxin. These mutated toxins are referred to as "cross-reactive materials" or CRM. CRM197 carrier protein (NCBI: AMV91693.1, SEQ ID NO: 3) is a nontoxic variant of diphtheria toxin, which is not toxic, but retains the immunogenicity of diphtheria toxin. CRM197 is a component of a conjugate vaccine of haemophilus influenzae, which is a fermentation and purification process available in published articles and patents (US 5614382), and is different from and immunologically indistinguishable from the specific active diphtheria toxin of CRM 197. Fragments of these proteins may also be conjugated to meningococcal capsular polysaccharides, provided that the fragments should be sufficiently long, i.e. preferably at least 10 amino acids, to define a T-cell epitope.

Tetanus toxoid protein (TT) is widely reported in the literature. The inventors have found a variant TTD protein of the TT protein. The protein is not toxic, but retains the immunogenicity of the TT protein. The TTD protein is positioned at the C-terminal end of the heavy chain of the TT protein, has relative molecular weight of 50kDa, is a receptor binding region of toxin, has no toxicity, has good immunogenicity, has lower anaphylaxis than tetanus toxoid, and is a potential tetanus vaccine antigen component and carrier protein. TTD can be purified by recombinant expression (Immunobiology, vol.216, issue 4, 2011, p 485-490).

An exemplary TTD expression purification method includes: cloning the DNA sequence (SEQ ID NO: 1) expressing TTD into a protein expression plasmid pET21 to construct engineering bacteria (such as escherichia coli BL21 (DE 3)) for recombinant expression of TTD protein (SEQ ID NO: 2). Recombinant engineering bacterial monoclonal colonies are picked up and amplified in suitable media and conditions (e.g., BL21 (DE 3) in 10mL LB (Amp) broth, 37 ℃, after 250rpm to OD600 to 0.8), incubated with inducer (e.g., 0.1mM IPTG, at 25 ℃,250rpm for 4 hours) and TTD is isolated from the culture. The process of isolating proteins from cultures is well known in the art, for example the following steps: the culture broth was centrifuged at 8000rpm at 4℃and then, after being resuspended in PBS, the cells were disrupted (sonicated), and the disrupted broth was centrifuged at 8000rpm at 4℃to collect the supernatant containing TTD protein. As a method for purifying TTD protein from the supernatant, a method which is usually used in the art for purifying protein from a liquid, for example, a method of chromatography such as ammonium sulfate precipitation, clarification filtration, combination chromatography such as ion exchange chromatography, composite medium chromatography and/or affinity chromatography, etc., can be used, and the purity is 95% or more. The molecular weight determined by mass spectrometry is consistent with the theoretical molecular weight.

In a preferred embodiment, the conjugate molecule of the invention comprises a polysaccharide and a carrier protein, which are chemically coupled to form a crosslinked polysaccharide-protein conjugate. Each protein is linked to the meningococcal capsular polysaccharide by terminal or non-terminal activation reducing sugar or derivative modification. Such conjugate molecules thus contain on average each carrier protein a plurality of repeat units of the capsular polysaccharide of a meningococcal pathogen. Preferably a plurality of meningococcal capsular polysaccharide repeat units, in particular an average of about 20-400, 30-300, 40-300, 50-250 meningococcal capsular polysaccharide repeat units are attached to each protein. More preferably at least about 50-200 capsular polysaccharide repeat units per protein on average are associated with each protein.

In another embodiment, the meningococcal capsular polysaccharide is linked to the protein by two or more sites on each meningococcal capsular polysaccharide. Since bactericidal epitopes may be present on branches of the meningococcal capsular polysaccharide repeat unit, functionalization of the meningococcal capsular polysaccharide and attachment to the protein should be achieved in such a way that an immunogenic amount of bactericidal epitopes is maintained.

Herein, in the polysaccharide protein conjugate or immunogenic composition, the ratio (w/w) of meningococcal capsular polysaccharide to carrier protein is in the range of 0.2 to 3.0, e.g. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0 or a range between any two of the values mentioned above, preferably in the range of 0.5 to 2.5,0.5-2.0,0.5-1.5, more preferably in the range of 0.6 to 2.5,0.6-2.0,0.6-1.5.

The immunogenic composition may also be used as a vaccine and may further contain adjuvants such as aluminium sulphate, aluminium hydroxide, aluminium phosphate, monophosphoryl lipid A, QS, cpG, stearoyl cruising acid, incomplete or complete freund's adjuvant. As defined herein, an "adjuvant" is a substance used to enhance the immunogenicity of an immunogenic composition of the invention. Thus, adjuvants are generally administered to boost the immune response and are well known to the skilled artisan. Suitable adjuvants for enhancing the effectiveness of the composition include, but are not limited to: (1) Aluminum salts such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and the like; (2) Oil-in-water emulsion formulations, such as MF59, SAF, ribiTM Adjuvant System (RAS), (cornixa, hamilton, MT); (3) a saponin adjuvant such as Quila or STIMULONTMUS QS-21; (4) Bacterial lipopolysaccharides (e.g., aminoalkyl glucosamine phosphate compounds (AGPs) or derivatives or analogs thereof), synthetic polynucleotides (e.g., oligonucleotides containing CpG motifs); (5) Cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g., gamma interferon), granulocyte macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), tumor Necrosis Factor (TNF), costimulatory molecules B7-1 and B7-2, etc.; (6) Detoxified mutants of bacterial ADP-ribosylating toxins, such as wild-type or mutant Cholera Toxin (CT), pertussis Toxin (PT), or E.coli heat Labile Toxin (LT) (see, e.g., WO93/13302 and WO 92/19265); and (7) other substances that act as immunostimulants to enhance the effectiveness of the composition.

The immunogenic compositions of the invention are capable of inducing active and passive protection against infection by meningococcal pathogenic bacteria. For passive protection, immunogenic antibodies are produced by immunizing a mammal with a vaccine made from the immunogenic composition of the invention, and then recovering the immunogenic antibodies from the mammal. The invention also provides a method of immunizing a mammal against infection by meningococcal pathogenic bacteria by administering an immunogenic amount of a composition of the invention.

The invention also provides a method of preparing a polysaccharide protein conjugate (or polysaccharide-protein conjugate) as described in any of the embodiments herein. The polysaccharide-protein conjugates of the invention can be produced by a number of conjugation methods. During the polysaccharide-protein binding reaction, polysaccharide activation and protein conjugation steps are typically included. The final polysaccharide-protein conjugate is obtained by controlling parameters such as the molecular weight of polysaccharide, the polysaccharide activation reaction, the polysaccharide-protein reaction proportion, the reaction temperature, the reaction time, the pH value of the reaction solution and the like. The polysaccharide-protein conjugate obtained is further purified to remove unreacted substances and impurities, and the polysaccharide-protein crosslinking degree and residual impurities are quantified through mass analysis, so that the consistency and comparability of batches are ensured.

For example, when a single meningococcal capsular polysaccharide links two or more protein molecules, the resulting conjugates are cross-linked to the protein. The degree of cross-linking and the overall size of the conjugate molecule can be adjusted by conventional variations in the conditions used in the coupling or binding reaction, as is known to those of ordinary skill in the art. These variations include, for example, the rate of the conjugation reaction and the ratio of protein present in the reaction mixture to capsular polysaccharide of the meningococcal pathogen. In some embodiments, the ratio (w/w) of meningococcal capsular polysaccharide to carrier protein added to the conjugation reaction system is in the range of 0.2 to 3.0, for example 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0 or a range between any two of the foregoing, preferably in the range of 0.5 to 2.5,0.5-2.0,0.5-1.5, more preferably in the range of 0.6 to 2.5,0.6-2.0,0.6-1.5.

In an exemplary embodiment, the weight ratio of polysaccharide from meningococcal a serogroup to carrier protein is from 0.5 to 1.1, preferably from 0.7 to 0.9; the weight ratio of polysaccharide from meningococcal serogroup C to carrier protein is 1.0 to 1.8, preferably 1.2 to 1.6; the weight ratio of polysaccharide from meningococcal Y serogroup to carrier protein is 1.4-2.3, preferably 1.6-2.1; the weight ratio of polysaccharide from meningococcal W135 serogroup to carrier protein is 0.7-1.4, preferably 0.9-1.2; the weight ratio of polysaccharide derived from haemophilus influenzae type b to carrier protein is 0.8-1.81.0-1.6.

Various chemical methods for conjugating polysaccharides to proteins are known and described in the literature. For example, U.S. patent 4,644,059, incorporated herein by reference, describes conjugates prepared with adipic Acid Dihydrazide (ADH) as the homobifunctional linker. U.S. patent 4,695,624, also incorporated herein by reference, describes a method for preparing polysaccharides and conjugates with a generic spacer. A general study of various methods and factors for designing conjugates is discussed in Contrib.Mrcrobini.Immunol.1989, vol.10, pp.48-114, of Dick, williamE. And MichelBeurret, which is also incorporated herein by reference. A preferred method of conjugating the capsular polysaccharide-protein conjugate of a meningococcal bacterium of the invention is the CDAP method (see, for example, EP0720485, US 5651971).

Herein, a method for preparing a polysaccharide protein conjugate comprises the steps of: obtaining meningitis pathogenic bacteria capsular polysaccharide; activating the polysaccharide using CDAP, and then obtaining a derivatized polysaccharide using adipic Acid Dihydrazide (ADH) solution; combining the derivatized polysaccharide with protein in a buffer, adding a certain amount of EDAC, reacting for a certain time to obtain a polysaccharide protein conjugate, and separating the polysaccharide protein conjugate from the buffer.

Capsular polysaccharides may be obtained from meningococcal pathogenic bacteria using methods well known in the art. Generally comprises: bacteria are inactivated and filtered (e.g. microfiltration or ultrafiltration) to obtain a crude sugar solution, and then polysaccharide precipitants (e.g. ethanol, CTAB) are added and purified (e.g. precipitation, dissociation, centrifugation, ultrafiltration, chromatography) to obtain purified polysaccharide. Methods for mass analysis of the resulting polysaccharide are known in the art, for example by nuclear magnetic resonance.

The meningococcal capsular polysaccharide may then be treated to reduce molecular weight using chemical (e.g. acid or base hydrolysis) or physical means. Illustratively, the polysaccharide may be treated with glacial acetic acid (e.g., at least 0.1M) or hydrochloric acid or high pressure homogenization (e.g., at a pressure of at least 800 Bar) to reduce molecular weight or increase the oxidative binding efficiency of the polysaccharide. In some embodiments, the polysaccharides of different sizes may be further purified separately using chromatography or the like.

Polysaccharides with an immunogen typically require activation and/or derivatization prior to conjugation with a protein. Common polysaccharide activation methods are cyanogen bromide (US 6375846B 1), hydrolysis (US 4761283), 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) (EP 0720485), periodic acid oxidation (US 4711779). The activated polysaccharide may be separated from other impurities by ultrafiltration. Common polysaccharide derivatization methods include the ADH method.

For example, a selective oxidation reaction of a periodate (e.g., sodium periodate) or an equivalent thereof is used to oxidize the hydroxyl groups of the previous sugar moiety to form aldehyde groups. This forms an activated meningococcal capsular polysaccharide, which can now be covalently linked to a protein carrier of choice. For example, about 10mg of polysaccharide is suitably oxidized with about 1ml of about 20mM sodium periodate solution at room temperature for about 10-15 minutes; alternatively, 0.05-2 equivalents of periodate of polysaccharide are added for 12-24 hours. The reaction time may vary with other amounts of periodate to obtain equivalent oxidation. The reduction and ring opening of the sugar activates the ortho-hydroxyl groups of the reducing sugar to aldehyde groups. After the polysaccharide activation is finished, the micromolecular substances in the reaction can be removed by ultrafiltration concentration.

For another example, the purified polysaccharide is dissolved in a buffer to prepare a solution, the pH is adjusted to 8.7, CDAP is added with stirring, and the reaction is maintained for 1h. Then ADH is added for maintaining the reaction for 2 hours, and the reactant is ultrafiltered after the reaction is finished, so that the polysaccharide derivative is prepared. The buffers described herein are all commonly used buffers, and can be selected by those skilled in the art based on conventional knowledge.

The conjugate is prepared by connecting polysaccharide fragments obtained by treating purified and extracted meningococcal capsular polysaccharide to carrier protein in a chemical coupling mode. Polysaccharide-protein conjugates can be obtained by direct chemical coupling or by linker implementation, such as coupling using the linker adipic acid dihydrazide. Various chemical methods for conjugating polysaccharides to proteins are known and described in the literature. Typical methods are the cyanogen bromide method, the CDAP method or the reductive amination method (U.S. Pat. No. 3,124, 0720485, U.S. Pat. No. 3, 4711779). For example, U.S. patent 4,644,059, incorporated herein by reference, describes conjugates prepared with adipic Acid Dihydrazide (ADH) as the homobifunctional linker. U.S. patent 4,695,624, also incorporated herein by reference, describes a method for preparing polysaccharides and conjugates with a generic spacer. A general study of various methods and factors for designing conjugates is discussed in Contrib.Mrcrobini.Immunol.1989, vol.10, pp.48-114, of Dick, williamE. And MichelBeurret, which is also incorporated herein by reference. The polysaccharide-protein conjugate obtained by coupling can induce stronger immunogenicity, and can simultaneously induce specific antibodies against polysaccharide and protein.

A preferred method of conjugating the capsular polysaccharide-protein conjugate of the meningococcal bacteria of the invention is the activated ester method (EP 0720485). In the coupling reaction, the ratio of polysaccharide to carrier protein is controlled between 0.5-2.5, the ratio of derivative polysaccharide to carrier protein is regulated during the reaction, and then a certain amount (for example, 10 mM-30 mM) of carbodiimide (EDAC) is added for reaction for at least half an hour. Removing impurities or unreacted substrate by ultrafiltration liquid exchange or chromatography after the reaction, and finally aseptically filtering by a 0.22um filter, and storing the polysaccharide-protein conjugate solution at 2-8deg.C. All polysaccharide-protein conjugates are controlled to be within 20 percent of free polysaccharide content, within 2 percent of free protein content, between 0.5 and 2.5 polysaccharide-protein ratio, and endotoxin and other impurities content are controlled to be within safe and acceptable ranges through mass analysis.

Illustratively, the method of obtaining a capsular polysaccharide-protein conjugate of a meningococcal pathogen of the invention comprises: the polysaccharide is linked to the ADH connector after CDAP activation, and the meningococcal capsular polysaccharide is conjugated to the selected conjugated protein by coupling the carrier protein to a meningococcal capsular polysaccharide derivative. The resulting capsular polysaccharide-protein conjugate of a meningococcal pathogen is preferably soluble in an aqueous solution. This makes the meningococcal capsular polysaccharide-protein conjugates of the invention preferred candidates for use as vaccines. The conditions of conjugation may be adjusted depending on the molecular weight of the polysaccharide and the specific protein. In the polysaccharide protein binding reaction, the ratio of the activated polysaccharide to the carrier protein is controlled between 0.5 and 3.0. Removing impurities or unreacted substrate by ultrafiltration liquid exchange or chromatography after the reaction, and finally aseptically filtering by a 0.22um filter, and storing the polysaccharide-protein conjugate solution at 2-8deg.C.

After conjugation of the capsular polysaccharide to the carrier protein, the polysaccharide-protein conjugate is purified by a variety of techniques. These techniques include concentration/diafiltration operations, precipitation/elution and column chromatography.

After purifying the complex carbohydrates, they are mixed to formulate the immunogenic compositions of the invention, which can be used as vaccines. The formulation of the immunogenic compositions of the invention may be accomplished using methods well known in the art. For example, a bacterial meningococcal pathogen conjugate may be formulated with pharmaceutically acceptable adjuvants to prepare the composition. Examples of such carriers include, but are not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and dextrose solutions.

The present invention provides immunogenic compositions useful for increasing antibodies for prophylactic and diagnostic purposes. It is another object of the invention to provide a method of immunizing a mammal against meningococcal pathogenic bacteria by administering an immunogenic amount of a meningococcal capsular polysaccharide. Use of a polysaccharide protein conjugate or immunogenic composition as described herein for the manufacture of a medicament for providing protection against infection by a meningococcal pathogen.

The present invention provides methods of immunizing mammals, particularly humans, against infection by meningococcal pathogenic bacteria using the polysaccharide protein conjugates or immunogenic compositions described herein. The polysaccharide protein conjugate is formed by covalently linking a capsular polysaccharide of a meningococcal pathogen to a suitable protein.

The immunogenic compositions of the invention are useful as a means of increasing antibodies for prophylactic and diagnostic purposes. Diagnostics are particularly useful for monitoring and detecting various infections and diseases caused by meningococcal pathogens the vaccines of the invention as used herein are capable of inducing antibodies for providing protection against infection by meningococcal pathogens.

The immunogenic compositions and vaccines of the invention can typically be formed by dispersing the meningococcal capsular polysaccharide or conjugate in a suitable pharmaceutically acceptable adjuvant. Such adjuvants include, but are not limited to, diluents, carriers, solubilizers, emulsifiers, preservatives and/or adjuvants. The adjuvant is preferably non-toxic to the recipient at the dosage and concentration employed, for example: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, the composition may contain substances for improving, maintaining or retaining, for example, pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or permeation of the composition. These substances are known from the prior art. The optimal pharmaceutical composition can be determined depending on the intended route of administration, the mode of delivery and the dosage required. Additives commonly used in vaccines may also be present, for example stabilizers such as lactose or sorbitol and adjuvants such as aluminium phosphate, aluminium hydroxide, aluminium sulphate, cpG, monophosphoryl lipid A, QS or stearoyl tyrosine.

The protective or therapeutic effects of the immunogenic compositions and vaccines of the present invention may be accomplished by administering the vaccine via systemic or mucosal routes. Such administration includes parenteral administration, for example by intramuscular, intraperitoneal, intradermal or subcutaneous injection, or by mucosal administration to the mouth/digestive tract, respiratory tract.

The dose of the immunogenic composition should be such that an immunogenic effect is achieved. The amount of conjugate in each vaccine dose was chosen as the amount that induced immune protection without significant adverse effects. Such amounts may vary depending on the bacterial serogroup. Typically, each dose will contain from 0.1 μg to 100 μg of polysaccharide or conjugate, preferably from 1 μg to 50 μg, more preferably from 5 μg to 25 μg. A series of optimal immunization doses may be given. The vaccine in unit dosage form may contain a substantial amount of meningococcal capsular polysaccharide or conjugate, for example, from 1 μg to 30 μg, or from 5 μg to 25 μg, from about 0.1 μg to about 100 μg.

The optimal amounts of components of a particular vaccine are determined by standard studies involving observation of the appropriate immune response in the subject. Following initial vaccination, the subject may receive one or several booster immunizations at sufficient intervals. The immunogenic compositions of the invention are suitable for infant, pediatric, adolescent and adult use. The immunization schedule may be determined by an experienced clinician.

The invention will be illustrated by way of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the invention. The methods and materials used in the examples are those conventional in the art, unless otherwise indicated.

Examples

Experimental method

Neisseria meningococcal capsular polysaccharide culture, fermentation and purification

Inoculating meningococcal strain (American type biological resource collection) on common agar medium (chocolate medium) containing 5-10% of sheep blood, culturing at 35-37 ℃ in carbon dioxide environment for 16-20 hours until the lawn is visible to naked eyes, scraping the lawn, transferring the lawn into a epidemic solid medium, culturing at 35-37 ℃ for 14-16 hours until the lawn is visible to naked eyes, then scraping the lawn again, transferring the lawn into a shake flask containing improved semi-integrated liquid medium, culturing overnight at 35-37 ℃, microscopic examination, and inoculating the culture solution into a 10L fermentation tank for 8-12 hours after the culture is finished and the result is normal. After the cultivation is completed, 1% formaldehyde is added for inactivation, then a clarified culture solution is obtained through centrifugation, and then a crude sugar culture solution is obtained through microfiltration and ultrafiltration (Huang Zhen, et al A, C, W, development of Y-group meningococcal polysaccharide vaccine [ J ]. Chinese biol. No. 20No. 4:273-275).

Adding polysaccharide precipitant into the crude sugar culture solution to obtain compound polysaccharide, and subjecting the compound polysaccharide to a series of purification steps such as ethanol precipitation, dissociation, ultrafiltration, sodium deoxycholate treatment, chromatography, etc. to obtain refined polysaccharide. The purification process of the purified polysaccharide generally includes, but is not limited to, ethanol precipitation, dissociation, centrifugation, ultrafiltration, sodium deoxycholate treatment, chromatography, etc., wherein the chromatography may be performed by using different chromatography methods depending on the polysaccharide to obtain the purified polysaccharide (Biologicals, volume 43,Issue 5,September 2015,Pages383-389). The obtained polysaccharide confirms the effective components of meningococcal capsular polysaccharide by analyzing sialic acid, O-acetyl and phosphorus content, and further confirms the size and structure of the meningococcal capsular polysaccharide by SEC-HPLC and nuclear magnetic resonance detection, and in addition, the indexes of main impurities such as nucleic acid, protein and the like are controlled within 1%, so that the meningococcal capsular polysaccharide meets pharmacopoeia standards.

Culturing, fermenting and purifying of haemophilus influenzae type B capsular polysaccharide

Inoculating the frozen strain into Hib comprehensive solid culture medium, activating and culturing in carbon dioxide environment at 35-37deg.C for 15-20 hr until the lawn is visible, scraping the lawn, transferring into shake flask containing Hib comprehensive liquid culture medium, and culturing in shake flask at 35-37deg.C and 250 rpm. After culturing until OD 600.5-2.5, inoculating the culture solution into a 10L fermentation tank for fermentation culture, wherein the culture temperature is 35-37 ℃, the air flow rate is 2-3L/min, stirring is carried out at 200-300rpm, and culturing is carried out for about 8-9 hours. After fermentation, 1% formaldehyde is added for inactivation, and then a clarified culture solution is obtained by centrifugation, and then a crude sugar culture solution (Wu Quanzhong and the like: fermentation culture of haemophilus influenzae type b and purification of PRP polysaccharide) is obtained by microfiltration and ultrafiltration.

Adding a cationic surfactant CTAB into the crude sugar culture solution to obtain a polysaccharide complex, and obtaining refined polysaccharide through a series of purification steps such as ethanol precipitation, ultrafiltration, chromatography and the like after the polysaccharide complex is dissociated (Yin Shanshan and the like. Optimization of a purification method of the b type haemophilus influenzae capsular polysaccharide, animal medicine development, 2009,30 (1): 50-52.). The refined polysaccharide confirms the effective components of the haemophilus influenzae type b capsular polysaccharide by detecting the ribose and the phosphorus content, the nuclear magnetic resonance detection confirms the structure of the polysaccharide, and the content of main impurity proteins and nucleic acids is controlled within 1 percent, so that the refined polysaccharide accords with the Chinese pharmacopoeia standard.

Carrier protein preparation

1. Preparation of diphtheria toxin non-toxic variant protein (CRM 197)

CRM197 carrier protein is a non-toxic variant of diphtheria toxin, which is non-toxic but retains the immunogenicity of diphtheria toxin. Methods for fermentation and purification are reported in both the published articles and patents (US 5614382). Typically, the culture medium is inoculated with a Corynebacterium diphtheriae strain, stirred at 37℃for 20-30 hours, and then the cells are removed by centrifugation to leave the supernatant. And (3) after ultrafiltration and liquid exchange of the fermentation supernatant, adding ammonium sulfate for precipitation, and collecting a precipitate. And then dissolving the precipitate, changing the liquid, purifying by column chromatography to obtain CRM197 protein, wherein the purity of the CRM197 protein is more than 95 percent, and the CRM197 protein is used for a combination reaction.

2. Preparation of the Carrier protein TTD

TTD carrier protein is obtained by genetic engineering recombinant protein technology (Immunobiology, vol.216, issue 4, 2011, P485-490). The method comprises the following specific steps: cloning the DNA sequence for expressing TTD (SEQ ID NO: 1) into a protein expression plasmid pET21 to construct escherichia coli BL21 (DE 3) for recombinant expression of TTD protein (SEQ ID NO: 2). Recombinant BL21 (DE 3) monoclonal colonies were picked up in 10mL of LB (Amp) liquid medium, cultured at 37℃and 250rpm to OD ₆₀₀ After reaching 0.8, 0.1mM IPTG was added and the incubation continued at 25℃and 250rpm for 4h. The culture broth was centrifuged at 8000rpm at 4℃to collect the cells, which were then resuspended in PBS and then disrupted. The disruption solution was centrifuged at 8000rpm at 4℃to collect the supernatant containing TTD protein. The TTD protein is purified by ammonium sulfate precipitation, clarifying filtration, and combined chromatography (ion exchange chromatography, composite medium chromatography, affinity chromatography, etc.), and the purity is above 95%. The molecular weight determined by mass spectrometry is consistent with the theoretical molecular weight.

3. Preparation of polysaccharide-protein conjugates

The bacterial polysaccharide herein was prepared as a polysaccharide protein conjugate by chemical coupling with CRM197 and TTD carrier proteins, respectively. The obtained polysaccharide-protein conjugate of different serogroups has good physicochemical properties, such as polysaccharide-protein ratio of 0.5-2.5, free sugar content below 20%, and other residual impurities controlled in very low range.

The preparation of the bacterial meningitis pathogenic bacteria polysaccharide-protein conjugate mainly comprises the following steps: polysaccharide activation derivatization and coupling of derivatized polysaccharide-proteins.

Activation derivative of bacterial meningitis pathogenic polysaccharide: polysaccharide activation was performed by adding CDAP solution (US 5651971). Dissolving purified polysaccharide in buffer solution to prepare solution, regulating pH to 8.7, adding CDAP under stirring for maintaining reaction for 1h, adding ADH for maintaining reaction for 2h, and ultrafiltering the reactant after the reaction is finished to obtain polysaccharide derivative.

Preparation of bacterial meningitis pathogenic bacteria polysaccharide-protein conjugate: chemical coupling of polysaccharide-proteins is carried out by the activated ester method (EP 0720485). In the coupling reaction, the ratio of polysaccharide to carrier protein is controlled between 0.5-2.5, and during the reaction, the derivative polysaccharide and carrier protein are mixed and regulated in proportion, and then a certain amount (for example, 10 mM-30 mM) of EDAC is added for 2h. Removing impurities or unreacted substrate by ultrafiltration liquid exchange or chromatography after the reaction, and finally aseptically filtering by a 0.22um filter, and storing the polysaccharide-protein conjugate solution at 2-8deg.C. All polysaccharide-protein conjugates are controlled to be within 20 percent of free polysaccharide content, within 2 percent of free protein content, between 0.5 and 2.5 polysaccharide-protein ratio, and endotoxin and other impurities content are controlled to be within safe and acceptable ranges through mass analysis.

4. Preparation of polysaccharide-protein conjugate vaccine

Monovalent polysaccharide-protein conjugate vaccines are obtained by mixing a monovalent polysaccharide-protein conjugate stock solution with an adjuvant. The multivalent polysaccharide-protein conjugate vaccine is prepared by diluting and mixing a polysaccharide-protein monovalent conjugate stock solution with a buffer solution with pH of 6.0, and then adding a certain amount of aluminum adjuvant and stirring.

5. Immunogenicity evaluation of polysaccharide-protein conjugates

ELISA method for evaluating polysaccharide immunogenicity: animals were immunized with the polysaccharide-protein conjugate, and after antisera were collected, ELISA was performed. Bacterial polysaccharide is coated on 100 mu l/hole of an ELISA plate, the plate is washed after 2-8 ℃ overnight (A, C, Y, W group polysaccharide needs to be coated after being adsorbed with mHSA, hib polysaccharide takes HbO-HA from NIBSC as coating antigen), after the ELISA plate is closed by new born calf serum, the mouse antiserum is treated by the method that 1:100 dilution, 2.5-fold gradient dilution, 8 dilutions, and 100 μl/well in the elisa plate were added and incubated overnight. After washing the plates, the secondary antibodies were used as 1: after 2-thousand dilutions, 100. Mu.l of the plate was added to the ELISA plate, incubated for 2 hours, then 1mg/ml PNPP-Na chromogenic substrate was added, 100. Mu.l of the plate was added to the plate, incubated for 2 hours, and after 50. Mu.l of 3M NaOH was added to the plate to stop the reaction, and the OD405 value was read on the machine. The ratio of the OD value of the measurement hole to the OD value of the negative hole which is greater than or equal to 2.1 is judged as positive, the reciprocal of the dilution with the maximum multiple of dilution being positive is determined as the antibody titer of each serum, and the geometric average value of the antibody titer of each group of immunized animals is calculated.

6. Meningococcal capsular polysaccharide and haemophilus influenzae type b capsular polysaccharide specific antibody bactericidal assay (SBA)

The reference SBA method evaluates the bactericidal efficacy of polysaccharide-specific antibodies. (Maslaska SE, bioesting L L, liButti D E, et al Standard and a multilaboratory comparison of Neisseria meningitidis serogroup A and C serum bactericidal assays.the Multilaboratory Study Group [ J ]. Clinical & Diagnostic Laboratory Immunology,1997,4 (2): 156.)

(1) Plate a: preparation of seed in flat-bottomed 96-well plates

Meningitis bacterial strain is recovered by flat plate resuscitating and flat plate streaking culturing, then washed by D-PBS buffer solution, and is regulated to bacterial concentration of 4000-4800 CFU/ml, and 25 μl/well is added into 96-well tissue culture plate (flat bottom). 222286Z1 1CNCN

(2) Plate B: dilution of serum samples in U-bottom 96-well plates

In plate B (96 well tissue culture plate, U-shaped bottom) 100 μl of test serum inactivated at 56℃for 30min was used as the sample at 1: 4-fold gradient dilutions (total of 8 gradients) were performed, 50 μl per well.

(3) Plate C: the diluted serum in plate B was pipetted into a flat-bottomed 96-well culture plate by 25 μl/well.

(4) Co-incubation of complement with thallus

An appropriate amount of diluted complement was added to 25. Mu.l/well of plate A containing 25. Mu.l/well of diluted cells, and mixed well in a shaker at 37℃for 10min.

(5) Taking 25 μl/well from the plate A in the step (4) to the plate C, and incubating for 60-90 min at 37 ℃.

(6) 12.5 μl/well was plated with an eight-channel row gun, incubated overnight at 37deg.C with 5% CO2, and colonies were counted.

(in vitro sterilization rate of serum specific antibody= (1 serogroup colony count of test sample/negative serum control colony count) ×100%).

Example 1 evaluation of the immune Effect of meningococcal polysaccharide conjugate W135 group polysaccharide-TTD in mice

The W135 group polysaccharide-CRM 197 conjugate and the W135 group polysaccharide-TTD conjugate are respectively added into aluminum phosphate adjuvant to prepare immune antigens, and physiological saline group is used as a control. Balb/c mice of 6-8 weeks of age were selected for intraperitoneal immunization, 5 mice per group, each at 5 micrograms of immunization dose, on days 0, 14, 21, respectively, and their polysaccharide immunogenicity and SBA sterilization assays were evaluated by blood withdrawal on day 35 (FIGS. 1 and 2).

Table 1: immunogenicity comparison of W135 polysaccharide-conjugates of different carrier proteins

As can be seen from table 1, the W135 group polysaccharide conjugate with TTD as carrier protein had a higher functional antibody bactericidal titer than the CRM197 carrier, although the immune serum antibody titer level was lower than that of the CRM197 carrier.

Example 2 evaluation of the immune Effect of meningococcal polysaccharide conjugate group Y polysaccharide-TTD in mice

Adding aluminium phosphate adjuvant into Y group polysaccharide-TTD conjugate and Y group polysaccharide-CRM 197 conjugate to prepare immune antigen, and taking physiological saline as negative control. Balb/c mice of 6-8 weeks of age were selected for intraperitoneal immunization, 5 mice per group, each at 5 micrograms, on days 0, 14, 21, and blood drawn on day 35 to evaluate their polysaccharide immunogenicity (FIG. 3) and serum SBA bactericidal titres (FIG. 4).

Table 2: comparison of immunogenicity of Y polysaccharide-conjugates of different carrier proteins

As can be seen from table 2, the group Y polysaccharide conjugates have higher antibody titer levels and bactericidal titers with TTD as carrier protein than CRM197 carrier protein.

Example 3 evaluation of immune Effect in meningococcal polysaccharide conjugate group C polysaccharide-TTD mice

Group C polysaccharide-TTD conjugate and group C polysaccharide-CRM 197 conjugate were used as negative control with physiological saline. Balb/c mice of 6-8 weeks of age were selected for intraperitoneal immunization, 5 mice per group, each at 5 micrograms, on days 0, 14, 21, and blood drawn on day 35 to evaluate their polysaccharide immunogenicity (FIG. 5) and serum SBA bactericidal titres (FIG. 6).

Table 3: comparison of immunogenicity of C polysaccharide-conjugates of different Carrier proteins

As can be seen from Table 3, the group C polysaccharide conjugates have higher antibody titer levels (P < 0.05) and bactericidal titers with TTD as carrier protein than CRM197 carrier protein.

Example 4 evaluation of the immune Effect of meningococcal polysaccharide conjugate group A polysaccharide-TTD in mice

Group a polysaccharide-TTD conjugate, group a polysaccharide-CRM 197 conjugate, were selected for intraperitoneal immunization in 6-8 week old Balb/c mice, each group of 5 mice at 1 μg, immunized on days 0, 14, 28, respectively, and blood drawn on day 42 to evaluate their polysaccharide immunogenicity (fig. 7) and serum SBA bactericidal titer (fig. 8).

Table 4: comparison of immunogenicity of polysaccharide A-conjugates of different Carrier proteins

As can be seen from table 4, the group a polysaccharide conjugate induced higher levels of antibody titres with CRM197 carrier than TTD carrier, but the immune serum bactericidal effect of the two carrier protein vaccines was comparable.

EXAMPLE 5 evaluation of the immune Effect of influenza b polysaccharide conjugate Hib polysaccharide-TTD in mice

The Hib polysaccharide-TTD conjugate and the Hib polysaccharide-CRM 197 conjugate were subjected to intraperitoneal immunization by taking physiological saline as a negative control, selecting Balb/c mice of 6-8 weeks of age, immunizing at a dose of 2 micrograms each for 5 mice of each group, respectively performing immunization on 0 day, 14 days and 28 days, and drawing blood for 42 days to evaluate the immunogenicity of the polysaccharide (as shown in figure 9) and the sterilization titer of serum SBA (as shown in figure 10).

Table 5: comparison of immunogenicity of Hib polysaccharide-conjugates of different Carrier proteins

As can be seen from table 5, hib group polysaccharide conjugates induced higher levels of antibody titers and serum bactericidal titers with TTD vector than CRM197 vector.

Example 6 evaluation of the immune Effect of tetravalent bacterial polysaccharide conjugate vaccine in mice

A, C, Y, W135 tetravalent meningitis polysaccharide conjugate vaccine with TTD and CRM197 as carrier proteins respectively was used as negative control with physiological saline. Balb/c mice of 6-8 weeks of age were selected for intraperitoneal immunization, 5 mice per group, each at 1 microgram dose, were immunized on days 0, 14, 28, and their polysaccharide immunogenicity was assessed by bleeding before immunization, on days 14, 28, 42.

As shown in fig. 11, on day 42 serum titers were significantly higher for MCV4 vaccine with TTD vector, group Y and group W135 immune serum IgG antibody titers than for MCV4 vaccine with CRM197 vector (P < 0.05) by t-test assay analysis; immune serum IgG antibody titer levels of both group a and group C vectors were comparable (P > 0.05).

Example 7 evaluation of the immune Effect of combination vaccine against ACYW135 meningitis polysaccharide conjugate vaccine with Hib in mice

A, C, Y, W135, hib polysaccharide conjugate using TTD as carrier protein is formulated into combined vaccine, and physiological saline is used as negative control. Balb/c mice of 6-8 weeks of age were selected for intraperitoneal immunization, 8 mice per group, each at 1 microgram dose, on days 0, 14, 28, and on day 42 were bled to evaluate their polysaccharide immunogenicity.

As shown in FIG. 12, after 42 days, TTD is A, C, Y, W and Hib combined polysaccharide conjugate vaccine of carrier protein, and has better immunogenicity after three doses of immunization, and the titer level of each polysaccharide antibody is obviously higher than that of a normal saline group (P < 0.05) through T-test statistical analysis.

Claims (10)

1. An immunogenic polysaccharide protein conjugate comprising a polysaccharide derived from a bacterial meningococcal pathogen and a carrier protein conjugated thereto, the carrier protein comprising TTD or a variant thereof,

preferably, the TTD is the C-terminal domain of TT,

more preferably, the TTD has the sequence shown in SEQ ID NO. 2 and the TTD variant has a sequence having at least 70% sequence identity to SEQ ID NO. 2.

2. The polysaccharide protein conjugate of claim 1, wherein the bacterium is selected from the group consisting of Neisseria meningococci and Haemophilus influenzae,

preferably, the carrier protein is covalently linked to the polysaccharide.

3. The polysaccharide protein conjugate of claim 1 or 2, wherein the bacterium is selected from the group consisting of Neisseria meningococci A, C, Y, W serogroup, haemophilus influenzae type b,

preferably, the weight ratio of polysaccharide to carrier protein is 0.2-3.0.

4. A method of preparing the polysaccharide protein conjugate of any one of claims 1-3, comprising the steps of:

(1) Obtaining the polysaccharide derived from the bacterial meningococcal pathogenic bacteria,

(2) Activating the polysaccharide, optionally derivatizing the activated polysaccharide,

(3) Coupling polysaccharide with carrier protein to obtain polysaccharide protein conjugate,

preferably, the method comprises the steps of,

the step (2) comprises: incubating the polysaccharide with periodate solution or CDAP to obtain an activated polysaccharide, and/or

The step (3) comprises: polysaccharide protein conjugates were obtained by incubating polysaccharide with carrier protein in the presence of EDAC.

5. The method of claim 4, wherein,

the step (1) comprises: inactivating bacteria, filtering to obtain crude sugar solution, adding polysaccharide precipitant, and purifying to obtain polysaccharide, and/or

Step (2) further comprises: incubating the activated polysaccharide with ADH, and/or

In the step (3), the weight ratio of the polysaccharide to the carrier protein is 0.2-3.0.

6. An immunogenic composition comprising the polysaccharide protein conjugate of any one of claims 1-4 and an adjuvant,

preferably, the adjuvant comprises one or more selected from aluminium hydroxide, aluminium phosphate, monophosphoryl lipid A, QS, cpG, stearoyl tyrosine and freund's adjuvant.

7. An immunogenic composition for use in a method of producing a passive immunity comprising bactericidal antibodies targeting bacterial meningitis pathogens, said antibodies being obtained by immunizing a mammal with an immunogenic composition according to claim 6,

preferably, the bactericidal antibodies are present in serum, gamma globulin fractions or purified antibody preparations.

8. A vaccine comprising the immunogenic composition of claim 6 and a pharmaceutically acceptable adjuvant.

9. Use of the polysaccharide protein conjugate of any one of claims 1-4 or the immunogenic composition of claim 6 in the manufacture of a medicament for providing protection against infection by a bacterial meningococcal pathogen.

10. The use according to claim 9, wherein the bacteria are selected from the group consisting of Neisseria meningococci and Haemophilus influenzae,

preferably, the bacteria are selected from the group consisting of neisseria meningitidis serogroup A, C, Y, W, haemophilus influenzae type b.