Components of a multivalent pneumococcal polysaccharide conjugate vaccine and uses thereof

Abstract

The present invention relates to a multivalent pneumococcal polysaccharide protein conjugate and its immunogenicity, specifically, to an immunogenic composition comprising capsular polysaccharides and a carrier from different serotypes of Streptococcus pneumoniae, said serotypes including at least 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F and 33F. The immunogenic composition of the present invention can improve the immunogenicity of the polysaccharides of different serotypes and effectively prevent invasive infections caused by various serotypes of Streptococcus pneumoniae.

Description

[Technical field]

[0001] The present invention relates to the biomedical field, and in particular to the prevention of bacterial pathogen infections through immunization with multivalent vaccines. [Background technology]

[0002] Streptococcus pneumoniae (S. pneumoniae) is a gram-positive, capsule-bearing diplococcus. It is classified into nearly 100 serotypes based on the composition of capsular polysaccharides, of which capsular polysaccharides are an important pathogenic factor. Streptococcus pneumoniae usually parasitizes the nasopharynx of healthy people, but when the parasitic environment changes, for example, when the body's immune system is weakened and the body is infected with respiratory viruses such as measles or influenza, or when the body is malnourished, old, or weak, it can break through the mucosal defense system and cause invasive infections. For example, it can enter the lower respiratory tract and cause pneumonia, pass through the blood-brain barrier and cause bacterial meningitis, break through alveolar epithelial cells and invade vascular endothelial cells to cause bacteremia, migrate from the nasopharynx to the paranasal sinuses and cause sinusitis, enter the middle ear through the pharyngeal tympanic tube and cause otitis media, and spread non-invasively to other respiratory sites.

[0003] Pneumococcal disease is one of the major public health problems worldwide. According to the World Health Organization (WHO) estimates, in 2005, approximately 1.6 million people died from pneumococcal disease each year, including 700,000 to 1 million children under the age of 5, many of whom live in developing countries. This shows that pneumococcus continues to pose a serious threat to children's health. In developed countries, pneumococcal disease occurs mainly in children under the age of 2, the elderly, or people of all age groups with weakened immune systems.

[0004] According to the site of pneumococcus infection, pneumococcal disease is broadly divided into invasive pneumococcal disease (IPD) and noninvasive pneumococcal disease (NIPD). The common treatment method is antibiotic therapy, but globally, pneumococci are becoming resistant to commonly used antibacterial drugs, which is becoming an increasingly serious problem. From many years of clinical practice, pneumococcal vaccination has been proven to be the most economical and effective method to prevent pneumococcal disease.

[0005] There are two types of pneumococcal vaccines currently available on the market: pneumococcal polysaccharide vaccine (PPSV) and pneumococcal polysaccharide protein conjugate vaccine (PCV). The 23-valent pneumococcal polysaccharide vaccine (PPSV23) has low immunogenicity and no immune memory or enhancement effect, making it unsuitable for infants and people with weakened immune systems. Commercially available pneumococcal polysaccharide conjugate vaccines for use in infants include PCV7, PCV10, and PCV13, which have narrow serotype coverage, low immune protection coverage, and may cause immunosuppressive effects. For example, a meta-analysis of literature on the distribution of Streptococcus pneumoniae serotypes in mainland China showed that the serotype coverage rate of PCV10 was 52.3% and the serotype coverage rate of PCV13 was 68.4% during the period 2000-2016. It has also been found that the incidence of non-vaccine serotype diseases has increased after the use of the vaccine. In addition, because the carrier proteins used in polysaccharide-protein conjugate vaccines are also widely used for immunization of infants, the immunosuppressive effects of high doses or duplicated use of carrier proteins are also potential risks in the clinical use of polysaccharide-protein conjugate vaccines.

[0006] Therefore, the serotype coverage rate of the current polysaccharide-protein conjugate vaccine is still not ideal. Developing a multivalent pneumococcal conjugate vaccine to cover a wider range of pathogenic serotypes of pneumococcus, increase the non-vaccine serotypes, improve the coverage protection rate and immunogenicity of the new multivalent vaccine, and reduce the risk of immunosuppressive effects caused by carrier proteins is of great clinical value. Summary of the Invention

[0007] In order to solve the problems of poor immunogenicity of pneumococcal polysaccharide vaccines, low serotype coverage of polysaccharide conjugate vaccines, low immunogenicity, immunosuppression, etc., the present invention provides a multivalent immunogenic composition with broader applicability, stronger immunogenicity, and reduced immunosuppression.

[0008] A first aspect of the invention provides an immunogenic composition comprising capsular polysaccharides and a carrier from different serotypes of Streptococcus pneumoniae, said serotypes including at least 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F and 33F.

[0009] In one or more embodiments, the serotypes include 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 serotypes selected from 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, and the serotypes include at least 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F. Preferably, the serotypes include 20 serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, or 24 serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.

[0010] In one or more embodiments, the carrier is one or more selected from saline, Ringer's solution, or phosphate buffered saline.

[0011] In one or more embodiments, the immunogenic composition further comprises an adjuvant.

[0012] In one or more embodiments, the adjuvant is an aluminum-based adjuvant.

[0013] In one or more embodiments, the adjuvant is one or more selected from aluminum phosphate, aluminum sulfate, aluminum hydroxide, monophosphoryl lipid A, QS21, CpG, MF59, stearyl tyrosine, Fraser's adjuvant and other mucosal adjuvants.

[0014] In one or more embodiments, the weight ratio of capsular polysaccharide from serotype 3, 6B or 12F to any other capsular polysaccharide in the composition is from 10:1 to 1:10, such as from 5:1 to 1:5, preferably 2:1.

[0015] In one or more embodiments, the composition is a formulation, wherein the capsular polysaccharides from serotypes 3, 6B and 12F are each independently at a concentration of 1-8 μg / dose (preferably 4 μg / dose) and the other capsular polysaccharides are each independently at a concentration of 0.5-5 μg / dose (preferably 2 μg / dose), and the aluminum phosphate adjuvant is at a concentration of 0.125 mg / dose to 0.5 mg / dose.

[0016] In one or more embodiments, the composition further comprises a surfactant, such as Tween 20 or Tween 80. Preferably, the concentration of surfactant in the composition is 100-300 μg / dose, preferably 70-120 μg / dose.

[0017] The present invention further provides a multivalent immunogenic composition comprising a plurality of polysaccharide-protein conjugates and a pharma- ceutically acceptable adjuvant, each polysaccharide-protein conjugate comprising capsular polysaccharides from different serotypes of Streptococcus pneumoniae conjugated to a carrier protein.

[0018] In one or more embodiments, the carrier protein comprises at least two carrier proteins.

[0019] In one or more embodiments, the carrier protein comprises (1) CRM197 and (2) TTD or a variant thereof.

[0020] In one or more embodiments, the TTD is the C-terminal domain of TT.

[0021] In one or more embodiments, the TTD has the sequence shown in SEQ ID NO:2, and the TTD variant is a sequence having at least 90% sequence identity to SEQ ID NO:2.

[0022] In one or more embodiments, the serotypes include at least 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F. Preferably, the serotypes include 20 serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, or 24 serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F.

[0023] In one or more embodiments, in the polysaccharide-protein conjugate, one or more or all of the capsular polysaccharides from at least serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F, respectively, are conjugated to the carrier protein TTD or a variant thereof.

[0024] In one or more embodiments, the serotypes include the 20 serotypes, of which capsular polysaccharides from serotypes 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, including serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F, are each conjugated to the carrier protein TTD or a variant thereof.

[0025] In one or more embodiments, the serotypes comprise the 20 serotypes, of which capsular polysaccharides from serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F are each conjugated to the carrier protein TTD or a variant thereof, and of which capsular polysaccharides from serotypes 1, 4, 6A, 7F, 8, 9V, 10A, 11A, 14, 19A, 22F and 33F are each conjugated to the carrier protein CRM197.

[0026] In one or more embodiments, the serotypes include the 24 serotypes, of which capsular polysaccharides from serotypes 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, including serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F, are each conjugated to the carrier protein TTD or a variant thereof.

[0027] In one or more embodiments, the serotypes comprise the 24 serotypes, of which capsular polysaccharides from serotypes 3, 5, 6A, 6B, 9N, 11A, 12F, 15B, 17F, 18C, 19A, 19F, 20, 23F and 33F are each conjugated to the carrier protein TTD or a variant thereof, and capsular polysaccharides from serotypes 1, 2, 4, 7F, 8, 9V, 10A, 14 and 22F are each conjugated to the carrier protein CRM197.

[0028] In one or more embodiments, the serotypes comprise the 24 serotypes, of which capsular polysaccharides from serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F are each conjugated to the carrier protein TTD or a variant thereof, and capsular polysaccharides from serotypes 1, 2, 4, 6A, 7F, 8, 9N, 9V, 10A, 11A, 14, 17F, 19A, 20, 22F and 33F are each conjugated to the carrier protein CRM197.

[0029] In one or more embodiments, the weight ratio of capsular polysaccharide from serotype 3, 6B or 12F to any other capsular polysaccharide in the composition is from 10:1 to 1:10, such as from 5:1 to 1:5, preferably 2:1.

[0030] In one or more embodiments, the composition further comprises an adjuvant, such as an aluminum-based adjuvant.

[0031] In one or more embodiments, the adjuvant is selected from aluminum phosphate, aluminum sulfate and aluminum hydroxide, preferably aluminum phosphate.

[0032] In one or more embodiments, the weight ratio of conjugate to adjuvant in the composition is from 1:10 to 1:2, preferably from 54:500 to 54:125.

[0033] In one or more embodiments, the composition is a formulation, wherein the concentrations of capsular polysaccharides from serotypes 3, 6B and 12F are each independently 1-8 μg / dose (preferably 4 μg / dose), the concentrations of the other capsular polysaccharides are each independently 0.5-5 μg / dose (preferably 2 μg / dose), and the concentration of the aluminum phosphate adjuvant is from 0.125 mg / dose to 0.5 mg / dose.

[0034] In one or more embodiments, the composition further comprises a surfactant, such as Tween 20 or Tween 80. Preferably, the concentration of surfactant in the composition is 100-300 μg / dose, preferably 70-120 μg / dose.

[0035] In one or more embodiments, the pH of the composition is 5.0-7.0, preferably 5.0-6.2.

[0036] The invention further provides the use of an immunogenic composition according to the first aspect in the manufacture of a medicament for inducing an immune response against a pneumococcal capsular polysaccharide conjugate and / or an immune response against a tetanus toxin.

[0037] In one or more embodiments, the medicament is used to prevent or treat pneumococcal and / or tetanus toxoid infections.

[0038] The present invention further provides a method of inducing an immune response against a pneumococcal capsular polysaccharide conjugate and / or an immune response against a tetanus toxin comprising administering to a subject an immunologically effective amount of an immunogenic composition according to the first aspect of the invention.

[0039] The invention further provides an immunological composition for generating passive immunity, comprising bactericidal antibodies targeting pneumococcus, 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, said bactericidal antibodies are present in serum, a gamma globulin fraction, or a purified antibody preparation.

[0040] Advantages of this invention: The immunogenic composition of the present invention can effectively prevent invasive infection caused by 24 different serotypes of pneumococcus, and can induce relatively balanced and high immunogenicity against all 24 serotypes, and the immunogenicity is related to the content ratio of each serotype conjugate.The immunogenic composition of the present invention contains different carrier proteins, and these two carrier proteins are non-toxic, so no detoxification treatment is required.The dual carrier design reduces potential safety risks and immunosuppression risks.The immunogenic composition also has a protective effect against tetanus toxin infection. [Brief description of the drawings]

[0041] [Figure 1] FIG. 1 is a comparison of the immunogenicity of type 5 pneumococcal polysaccharide conjugates of different carriers. [Diagram 2]FIG. 2 shows a comparison of the immunogenicity in mice of the 24-valent pneumococcal polysaccharide conjugate vaccine at different adjuvant doses (the same 13 serotypes as the positive vaccine). [Diagram 3] FIG. 3 shows a comparison of the immunogenicity in mice of the 24-valent pneumococcal polysaccharide conjugate vaccine at different adjuvant doses (11 serotypes other than the 13 serotypes in the positive vaccine). [Figure 4] FIG. 4 shows the comparison of immunogenicity of 24-valent pneumococcal polysaccharide conjugate vaccines with different pH in mice. [Diagram 5] FIG. 5 shows a comparison of the immunogenicity of the 13-valent antibody and the same serotype (D35) in mice. [Figure 6] FIG. 6 shows a comparison of serotype polysaccharide antibody titers (D35) in mice other than the PCV13 vaccine. [Figure 7] FIG. 7 shows a comparison of 13-valent and same serotype polysaccharide antibody titers in rabbits (D35). [Figure 8] FIG. 8 shows a comparison of antibody titers (D35) of serotype polysaccharides other than the 13 types in rabbits. [Figure 9] FIG. 9. Serum antibody titer levels after two immunizations with dual carrier and single carrier 24-valent pneumococcal polysaccharide conjugate vaccine. [Figure 10] Figure 10 shows a comparison of serum antibody titers after two immunizations with PCV20 dual-carrier and single-carrier vaccines. [Figure 11] Figure 11 shows a comparison of serum antibody titers after two immunizations with PCV24 dual-carrier and single-carrier vaccines. [Figure 12] FIG. 12 is a survival curve of the protective effect of the 24-valent pneumococcal polysaccharide conjugate vaccine against type 3 pneumococcal challenge in mice. [Figure 13] FIG. 13 shows survival curves for the protective effect of the 24-valent pneumococcal polysaccharide conjugate vaccine against serotype 22F pneumococcus. [Figure 14] Figure 14 shows a comparison of TT antibody titers after two immunizations and before immunization. Group A is physiological saline, Group B is DPT vaccine, and Group C is 24-valent pneumococcal polysaccharide conjugate vaccine. [Figure 15] FIG. 15 shows the survival curves after tetanus toxoid challenge. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] The present invention provides a novel 20- or 24-valent pneumococcal capsular polysaccharide-protein conjugate and corresponding immunogenic composition and vaccine formulation. The vaccine formulation of the present invention induces higher antibody titers in some serotypes compared with the commercially available 13-valent polysaccharide conjugate vaccine, and also shows good immunogenicity against serotypes other than the 13 serotypes, and further induces better immune protection in the body compared with the 13-valent polysaccharide conjugate vaccine and the 23-valent polysaccharide vaccine. The inventors also found that the use of different carriers for different conjugates can induce higher antibody titers than the single-carrier vaccine group.

[0043] The present invention first provides an immunogenic composition for inducing an immune response in a mammal against a pneumococcal capsular polysaccharide conjugate and protecting the mammal from pneumococcal infection, the immunogenic composition comprising capsular polysaccharides from different serotypes of Streptococcus pneumoniae, the serotypes including at least 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F and 33F. Preferably, the serotypes include 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.

[0044] The pneumococcal capsular polysaccharides present in the immunogenic composition may be in the form of free polysaccharide or may be present as a component of a conjugate in which the polysaccharide is covalently bound to a protein. In addition to the polysaccharide or polysaccharide-protein conjugate, the immunogenic composition may further comprise a pharma- ceutically acceptable carrier, such as saline, Ringer's solution or phosphate buffered saline.

[0045] In a preferred embodiment of the invention, the capsular polysaccharide is covalently linked to a protein to form a conjugate. Thus, any protein or fragment thereof that is tolerated by an individual and capable of inducing an immune cell (e.g., T cell) dependent response is suitable for conjugation with pneumococcal capsular polysaccharide. Essentially, any protein can be used as a conjugated protein. The protein selected in particular must have at least one free amino group in order to be conjugated to the polysaccharide. Preferred proteins are any natural or recombinant bacterial proteins, which are themselves immunogens that induce 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 and CRM197 or variants thereof. Other potential conjugated proteins include toxins or toxoids of enterotoxigenic bacteria, including pseudomonas, staphylococci, streptococci, pertussis and Escherichia coli.

[0046] In a preferred embodiment of the invention, pneumococcal capsular polysaccharides are covalently linked to proteins to form conjugates. Thus, any protein or fragment thereof that is tolerated by an individual and capable of inducing an immune cell (e.g., T cell) dependent response is suitable for conjugation with pneumococcal capsular polysaccharides. Essentially, any protein can be used as a conjugated protein. The protein selected in particular must have at least one free amino group in order to be conjugated to the polysaccharide. Preferred proteins are any natural or recombinant bacterial proteins that are themselves immunogens that induce T cell dependent responses in young and adult mammals. Examples of such proteins include, but are not limited to, tetanus toxoid, cholera toxin and diphtheria toxoid. Other potential conjugated proteins include toxins or toxoids of enterotoxigenic bacteria, including pseudomonas, staphylococcus, streptococcus, pertussis and Escherichia coli.

[0047] The protein to be conjugated to the pneumococcal capsular polysaccharide (i.e., carrier protein) can be any protein or fragment thereof that is tolerated by an individual and is capable of inducing an immune cell (e.g., T cell) dependent response. Essentially, any protein can be used as the conjugated protein. The protein selected in particular must have at least one free amino group in order to be conjugated to the polysaccharide. Preferred proteins are any natural or recombinant bacterial proteins that are themselves immunogens that induce T cell dependent responses in young and adult mammals. Examples of such proteins include, but are not limited to, tetanus toxoid, cholera toxin, and diphtheria toxoid. Other potential conjugated proteins include toxins or toxoids of enterotoxigenic bacteria, including pseudomonas, staphylococcus, streptococcus, pertussis, and E. coli.

[0048] In addition, non-toxic mutants of protein toxins, such as CRM197, can also be used. Preferably, such mutants retain the epitopes of the native toxin. These mutated toxins are called "cross-reactants" or CRMs. CRM197 carrier protein (NCBI: AMV91693.1, SEQ ID NO:3) is a non-toxic mutant of diphtheria toxoid, which is non-toxic and retains the immunogenicity of diphtheria toxoid (DT). Its fermentation and purification methods are available in the literature and patents (US5614382). Non-toxic mutant protein of diphtheria toxoid (CRM197) is a carrier protein that has been proven safe and effective in the clinic and is widely used in commercially available pneumococcal polysaccharide conjugate vaccines. Other diphtheria toxin mutants are also suitable as carrier proteins. Furthermore, fragments of these proteins can be conjugated with pneumococcal polysaccharides, but these fragments must be long enough, i.e., preferably at least 10 amino acids, to identify T-cell epitopes.

[0049] Tetanus toxoid protein (TT) has been widely reported in the public literature. The inventors have discovered a variant of TT protein, TTD protein, which is non-toxic and retains the immunogenicity of TT protein. TTD protein is located at the C-terminus of the heavy chain of TT protein, has a relative molecular weight of 50 KDa, corresponds to the receptor-binding region of the toxin, is non-toxic, has good immunogenicity, but is less allergenic 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 and purification method includes cloning a DNA sequence expressing TTD (SEQ ID NO:1) into the protein expression plasmid pET21 and constructing an engineered strain (e.g., E. coli BL21(DE3)) that recombinantly expresses TTD protein (SEQ ID NO:2). A single clone colony of the recombinant engineered strain is selected and grown in an appropriate medium and conditions (e.g., BL21(DE3) is cultured in 10 mL of LB(Amp) liquid medium at 37°C and 250 rpm until OD600 reaches 0.8), and an inducer is added and cultured (e.g., 0.1 mM IPTG is added and cultured at 25°C and 250 rpm for 4 hours), and then TTD is isolated from the culture. The process of isolating protein from the culture is well known in the art and includes, for example, the following procedure: the culture is centrifuged at 8000 rpm and 4°C, resuspended in PBS, the cells are disrupted (e.g., ultrasonically disrupted), the disrupted solution is centrifuged at 8000 rpm and 4°C, and the supernatant containing the TTD protein is collected. The method of purifying the TTD protein from the supernatant can be a method commonly used in the art for purifying proteins from liquids, for example, by chromatographic methods such as ammonium sulfate precipitation, clarification filtration, ion exchange chromatography, composite media chromatography and / or affinity chromatography, and the purity reaches 95% or more. The molecular weight determined by mass spectrometry is in agreement with the theoretical molecular weight.

[0050] Other suitable carrier proteins include inactivated bacterial toxins such as tetanus toxoid, pertussis toxoid, cholera toxoid (see, for example, WO2004 / 083251), E. coli LT, E. coli ST, and exotoxin from Pseudomonas aeruginosa. Bacterial outer membrane proteins such as outer membrane complex c (OMPC), porins, transferrin-binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesion protein (PsaA), C5a peptidase from group A or B streptococci, or Haemophilus influenzae protein D can also be used. Other proteins such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or purified protein derivative of tuberculin (PPD) can also be used as carrier proteins.

[0051] A person skilled in the art can obtain a mutant of TTD by modifying one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the TTD of the present invention, provided that the TTD activity (e.g., toxin binding activity) is not substantially affected. These mutants include, but are not limited to, those obtained by deleting, inserting, and / or substituting one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and adding one or more (usually 20 or less, preferably 10 or less, and more preferably 5 or less) amino acids to the C-terminus and / or N-terminus. In the art, conservative substitution with amino acids with close or similar performance usually does not change the function of the protein. Amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. In addition, for example, addition of one or more amino acids to the C-terminus and / or N-terminus usually does not change the function of the protein. These are considered to be within the scope claimed by the present invention. The TTD variants described herein include TTD variants that have at least 90% (e.g., at least 95%, at least 98%, at least 99%) sequence identity with the TTD shown in SEQ ID NO:2 and that retain its toxin-binding activity.

[0052] The inventors have found that by conjugating multiple serotype polysaccharides with different carrier proteins, the immunogenic composition formed after mixing has better immunogenicity and lower immunosuppression risk. Illustratively, the immunogenic composition includes at least two types of carrier proteins (e.g., CRM197 and TTD or variants thereof). In a preferred embodiment, 20 or 24 types of pneumococcal polysaccharides of the present invention are chemically coupled with CRM197 and TTD carrier proteins, respectively, to produce polysaccharide-protein conjugates. The obtained polysaccharide-protein conjugates of different serotypes have excellent physicochemical properties, such as a polysaccharide-protein ratio in the range of 0.5-3.0, a free sugar content of 20% or less, and other residual impurities are controlled in a very low range.

[0053] In some embodiments, in the polysaccharide-protein conjugate, at least one or more or all of the capsular polysaccharides from serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F, respectively, are conjugated to the carrier protein TTD or a variant thereof.Furthermore, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 serotypes, including serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F, respectively, are conjugated to the carrier protein TTD or a variant thereof. Preferably, the capsular polysaccharides from serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F, respectively, are conjugated to the carrier protein TTD or a mutant thereof, and the capsular polysaccharides from serotypes 1, 2, 4, 6A, 7F, 8, 9N, 9V, 10A, 11A, 14, 17F, 19A, 20, 22F and 33F, respectively, are conjugated to the carrier protein CRM197.

[0054] As used herein, in the polysaccharide-protein conjugate or immunogenic composition, the ratio of capsular polysaccharide from different serotypes to carrier protein is in the range of 0.2-3.0, 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 above numbers, preferably 0.5-2.5, 0.5-2.0, 0.5-1.5, more preferably 0.6-2.5, 0.6-2.0, 0.6-1.5.

[0055] In the polysaccharide protein conjugate or immunogenic composition described herein, the weight ratio of capsular polysaccharide from serotype 3, 6B or 12F to any other capsular polysaccharide is in the range of 10:1 to 1:10, such as 5:1 to 1:5, preferably 2:1. For example, the composition is a formulation in which the capsular polysaccharide from serotype 3, 6B and 12F are each independently at a concentration of 1-8 μg / dose (preferably 4 μg / dose), the other capsular polysaccharides are each independently at a concentration of 0.5-5 μg / dose (preferably 2 μg / dose), and the aluminium phosphate adjuvant is at a concentration of 0.125 mg / dose to 0.5 mg / dose.

[0056] The immunogenic composition can be used as a vaccine and can further include an aluminum-based adjuvant (aluminum hydroxide, aluminum phosphate, or aluminum sulfate). The immunogenic compositions described herein also include a surfactant (e.g., Tween 20 or Tween 80). The immunogenic compositions of the invention can induce active and passive protection against pneumococcal infection. For passive protection, immune antibodies are produced by immunizing a mammal with a vaccine prepared from the immunogenic composition of the invention and recovering immune antibodies from the mammal. The invention also provides a method of immunizing a subject against pneumococcal infection by administering an immunologically effective amount of the composition of the invention.

[0057] Capsular polysaccharides are prepared using standard techniques known to those skilled in the art. In the present invention, capsular polysaccharides are prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 20, 22F, 23F and 33F of Streptococcus pneumoniae. These pneumococcal conjugates are prepared by a single method and formulated into single dose formulations.

[0058] Bacterial capsular polysaccharides are an important component of bacterial cells and are also important virulence factors of pathogenic bacteria. Pneumococcal bacterial capsular polysaccharides are released into the culture medium during bacterial cultivation. The pneumococcal bacterial capsular polysaccharides of the present invention are obtained by cultivating, fermenting and purifying different serotypes of pneumococci. This technology is mature and has been used in the industry for many years, and has been reported in many published literature and patents (US4242501). In one embodiment, various pneumococcal polysaccharide serotypes are cultivated in a soy-based medium. Then, each type of polysaccharide is purified using centrifugation, precipitation, ultrafiltration and column chromatography (US4686102).

[0059] A specific method for producing capsular polysaccharides is specifically described in US Patent US5714354. Bacterial culture, fermentation and polysaccharide production of pneumococcus are mature processes (US4686102, US5847112). In brief, different pneumococcus strains are inoculated into shake flasks containing culture medium (e.g., soybean medium) and cultured overnight in a carbon dioxide incubator at 37°C, and the culture liquid is inoculated into a 10-liter fermentation tank and cultured for fermentation for 6-12 hours. After the culture is completed, DOC inactivation agent is added for sterilization and inactivation. A clear culture liquid is obtained by centrifugation, and a CTAB precipitant is added to the clear culture liquid to obtain a crude precipitate. Then, a series of precipitation, washing and chromatography are carried out to obtain purified polysaccharides.

[0060] The purification method of pneumococcal capsular polysaccharide is a conventional technique in the field, for example, it is completed by a method without organic solvent (US Patent US5714354). Usually, the purification process of purified polysaccharide includes, but is not limited to, adding NaCl solution to dissolve complex saccharide, dissolving with NaCl solution and centrifuging, then taking the supernatant, adding alcohol to remove impurities and precipitated saccharide, washing the precipitate several times to remove impurities, or using techniques such as ion exchange column and chromatography using composite packing according to different sugar types to obtain purified polysaccharide. In a specific embodiment, the purification process mainly includes centrifuging the bacteria inactivated liquid to separate the bacterial cells, collecting the centrifuged supernatant, filtering through a 0.22 μm filter membrane, and ultrafiltrating through a 100 KD membrane to concentrate to obtain pneumococcal polysaccharide fermentation concentrate. Add CTAB solution to the pneumococcal polysaccharide fermentation concentrate, stir at room temperature, centrifuging at 8000 rpm, and collecting the precipitate of polysaccharide complex (collecting the supernatant for 7F, 14 and 33F type polysaccharide). The polysaccharide complex is then dissociated with 0.25-0.5M NaCl solution, and then purified by processes such as precipitation, column chromatography, and ultrafiltration to obtain a purified polysaccharide solution, which is then filtered through a 0.22 μm membrane and stored at low temperature.

[0061] Mass spectrometry methods for the obtained polysaccharides are known in the art, for example, the polysaccharides are analyzed by nuclear magnetic resonance. Specifically, the serotypes of the different types of pneumococcal capsular polysaccharides produced are identified with specific serum, the chemical structure of the pneumococcal polysaccharides is analyzed by nuclear magnetic resonance (NMR), and the content of functional groups in the polysaccharides (e.g., components such as rhamnose, uronic acid, and O-acetyl groups) is measured using a chemical color development method. It is confirmed that each physicochemical index of the polysaccharide meets the standards for pneumococcal polysaccharides in the European Pharmacopoeia, and the content of major impurities such as proteins and nucleic acids is less than 1%.

[0062] Various conjugation methods can be used to produce the polysaccharide-protein conjugate of the present invention. The polysaccharide-protein binding reaction process usually includes polysaccharide activation and protein conjugation steps. The polysaccharide is chemically activated by conventional methods and then conjugated to a carrier protein. For example, see U.S. Patent Nos. 4,673,574 and 4,902,506. The final polysaccharide-protein conjugate is obtained by controlling parameters such as the molecular weight of the polysaccharide, the polysaccharide activation reaction, the polysaccharide-protein reaction ratio, the reaction temperature, the reaction time and the pH value of the reaction solution. The obtained polysaccharide-protein conjugate is further purified to remove unreacted materials and impurities, and the crosslinking degree of the polysaccharide-protein and the residual impurities are quantified by mass spectrometry to ensure the consistency and comparability of batches.

[0063] The purified polysaccharides are chemically activated to allow them to react with carrier proteins. Different chemical activation methods can be selected according to the properties of the polysaccharides. Commonly used polysaccharide activation methods include cyanogen bromide method (US6375846B1), hydrolysis (US4761283), 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) (EP0720485), and periodate oxidation method (US4711779). The activated polysaccharides can be separated from other impurities by ultrafiltration. After activation, the polysaccharides have the activity to react with carrier proteins.

[0064] For example, selective oxidation with periodate (e.g., sodium periodate) or equivalents oxidizes the hydroxyl groups of the former sugar moieties to form aldehyde groups. This results in the formation of activated pneumococcal capsular polysaccharides that can be covalently coupled to a selected protein carrier. For example, about 10 mg of polysaccharide is oxidized for about 10-15 minutes with about 1 ml of about 20 mM sodium periodate solution at room temperature, or for 12-24 hours with 0.05-2 equivalents of periodate. The reaction time can be varied with other amounts of periodate to obtain equivalent oxidation. Reduction and ring opening of the sugar activates the ortho-hydroxyl groups of the reduced sugar to aldehyde groups. After the activation of the polysaccharide is complete, ultrafiltration and concentration can be used to remove small molecule substances in the reaction.

[0065] The conjugates are prepared by chemical coupling of polysaccharide fragments obtained by processing purified and extracted pneumococcal outer membrane polysaccharides with carrier proteins. Polysaccharide-protein conjugates can be obtained by direct chemical coupling or by using linkers such as adipic acid dihydrazide. Various chemical methods for conjugating polysaccharides with proteins are known and described in the literature. Common methods include cyanogen bromide, CDAP, and reductive amination (US5952454, EP0720485, US4711779). For example, US Pat. No. 4,644,059, which is incorporated herein by reference, describes conjugates prepared using adipic acid dihydrazide (ADH) as a homobifunctional linker. Also, US Pat. No. 4,695,624, which is incorporated herein by reference, describes methods for preparing polysaccharides and conjugates using intergeneric spacer groups. A comprehensive study of the various manufacturing methods and factors used in conjugate design is discussed in Dick, William E. and Michel Beurret, Contrib. Medic. Immunol. (1989), Vol. 10, pp. 48-114, which is also incorporated herein by reference. Polysaccharide-protein conjugates obtained by coupling can induce relatively strong immunogenicity and can induce specific antibodies against polysaccharides and proteins.

[0066] The preferred method for conjugating each kind of pneumococcal capsular polysaccharide-protein conjugate of the present invention is reductive amination. The activated pneumococcal capsular polysaccharide is conjugated with the selected conjugate protein by coupling the amino group of the carrier protein with the aldehyde group of the pneumococcal capsular polysaccharide in the presence of cyanoborohydride ion or other reducing agent. The pneumococcal capsular polysaccharide-protein conjugate obtained from the reductive amination process is preferably soluble in aqueous solution. This makes the pneumococcal capsular polysaccharide-protein conjugate of the present invention a preferred candidate for use as a vaccine. The conjugation conditions can be adjusted according to the molecular weight of the polysaccharide and the specific protein. In the reductive amination method, the ratio of polysaccharide to carrier protein is controlled in the range of 0.5-3.0, and 1.0-2.0 equivalents of sodium cyanoborohydride solution of polysaccharide is added during the reaction, followed by 2.0 equivalents of sodium borohydride solution. The incubation temperature can be room temperature, and the incubation time is, for example, at least 12 hours. After the reaction is completed, the liquid is exchanged by ultrafiltration or chromatography is used to remove impurities or unreacted substrates during the reaction, and finally, the polysaccharide-protein conjugate solution is sterilized by filtration through a 0.22um filter and stored at 2-8°C.

[0067] After the capsular polysaccharide has been conjugated to the carrier protein, a variety of techniques are used to purify the polysaccharide-protein conjugate, including concentration / diafiltration procedures, precipitation / elution and column chromatography.

[0068] After each complex sugar is purified, they are mixed to prepare the immunogenic composition of the present invention, which can be used as a vaccine. The immunogenic composition of the present invention can be prepared by any method known in the art. For example, each of the 20 or 24 pneumococcal conjugates can be prepared to produce the composition using pharma- ceutically acceptable adjuvants. Examples of such carriers include, but are not limited to, water, buffered saline, polyol (e.g., glycerin, propylene glycol, liquid polyethylene glycol) and glucose solution.

[0069] In some embodiments, the immunogenic composition may include one or more adjuvants. An "adjuvant" as defined herein is a substance for enhancing the immunogenicity of the immunogenic composition of the present invention. Thus, adjuvants are typically administered to enhance immune responses and are known to those skilled in the art. Suitable adjuvants for enhancing the efficacy of the composition include: (1) aluminum salts, such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate; (2) oil-in-water emulsion formulations, such as MF59, SAF, and Ribi™ adjuvant systems (Corixa, Hamilton, MT); (3) saponin adjuvants, such as QuilA or STIMULON™ QS-21; (4) bacterial lipopolysaccharides, such as aminoalkyl glucosamine phosphate compounds (AGPs) or derivatives or analogs thereof, synthetic polynucleotides, such as oligonucleotides containing CpG motifs; (5) Cytokines, such as interleukins (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; (6) mucosal adjuvants, such as protein toxins, recombinant protein subunits and nucleic acids, including detoxified mutants of bacterial ADP-ribosylating toxins (e.g., wild-type or mutant cholera toxin (CT), pertussis toxin (PT), or E. coli heat-labile toxin (LT)), etc. (see, e.g., WO93 / 13302 and WO92 / 19265); (7) other substances that enhance the effect of the composition as an immunostimulant, including, but not limited to, the following. The dosage of the adjuvant can be determined by those skilled in the art based on conventional techniques. For example, the concentration of an adjuvant (eg, aluminum phosphate) in the immunogenic compositions herein ranges from 0.125 mg / dose to 0.5 mg / dose.

[0070] The immunogenic compositions and vaccines of the invention are used to protect or treat mammals (e.g., humans) susceptible to pneumococcal infection, which can be accomplished by administering the vaccine via a systemic or mucosal route. The immunogenic compositions described herein are also used in the manufacture of medicaments that provide protection against various pneumococcal infections.

[0071] The immunogenic compositions of the invention can be used as a means to raise antibodies for prophylactic and diagnostic purposes. Diagnostics are particularly suited for monitoring and detection of various infections and diseases caused by pneumococcus. Another embodiment of the invention is to use the immunogenic compositions as immunogens to provide active and passive immunogenic protection to individuals at risk of pneumococcal infection or disease. The immune antibodies used for passive protection are generated by immunizing a mammal with any of the immunogenic compositions of the invention and then recovering bactericidal antibodies of the gamma-globulin portion from the mammal as serum or specific antibodies. The vaccines of the invention used herein are capable of inducing antibodies that provide protection against pneumococcal infection.

[0072] Additionally, the pneumococcal capsular polysaccharides themselves may be used as an immunizing agent, preferably in combination with an aluminium-based adjuvant to form an immunogenic composition.A further embodiment of the invention is the use of said immunogenic composition for immunogenic protection against pneumococcal infection.

[0073] The immunogenic compositions and vaccines of the present invention are typically formed by dispersing the pneumococcal capsular polysaccharide or conjugate in a suitable pharma- ceutically acceptable excipient. The excipients include, but are not limited to, diluents, carriers, solubilizers, emulsifiers, preservatives and / or adjuvants. The excipients are preferably non-toxic to recipients at the dosages and concentrations employed. For example: saline, buffer, glucose, water, glycerin, ethanol and combinations thereof are contemplated. In some embodiments, the compositions may include substances to improve, maintain or retain, for example, the pH value, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or penetration of the composition. These substances are well known in the art. The optimal pharmaceutical composition can be determined depending on the desired route of administration, delivery mode and required dosage. Additives commonly used in vaccines may also be present, such as stabilizers (e.g., lactose or sorbitol) and adjuvants (e.g., aluminum phosphate, aluminum hydroxide, aluminum sulfate, CpG, monophosphoryl lipid A, QS21, MF59 or stearyl tyrosine).

[0074] The protective or therapeutic effect of the immunogenic compositions and vaccines of the present invention can be achieved by administering the vaccines via systemic or mucosal routes. Such administration includes parenteral administration, such as intramuscular, intraperitoneal, intradermal or subcutaneous injection, or mucosal administration to the oral / alimentary tract, respiratory tract or urogenital tract. In one embodiment, intranasal administration is used to treat pneumonia or otitis media. The amount of immunogenic composition used should be an effective amount to induce an immune effect. The amount of conjugate in each vaccine dose is selected as an amount that induces immune protection without significant side effects. Such amount may vary depending on the serotype of pneumococcus. Generally, each dose contains 0.01 μg to 100 μg, for example, 0.1 μg to 10 μg, or 1 μg to 5 μg of polysaccharide or conjugate. The dosage is usually in the range of about 0.01 μg to about 10 μg per kilogram of body weight. An optimal immune dosage range can be suggested. A vaccine in unit dosage form can contain about 0.01 μg to about 100 μg equivalent, for example, 0.1 μg to 10 μg, or 1 μg to 5 μg of meningococcal capsular polysaccharide or conjugate.

[0075] The optimal amount of the specific vaccine components is determined by standard studies involving observation of appropriate immune responses in subjects. After the initial vaccination, subjects can receive one or more booster immunizations at sufficient intervals. The immunogenic composition of the present invention is suitable for use in infants, children, adolescents and adults. The immunization schedule can be determined by an experienced clinician.

[0076] In a specific embodiment of the invention, the 20 or 24 valent vaccine is a sterile liquid formulation of pneumococcal capsular polysaccharides of serotypes 1, 2, 4, 6A, 7F, 8, 9N, 9V, 10A, 11A, 14, 17F, 19A, 20, 22F and 33F, respectively, conjugated to CRM197, and pneumococcal capsular polysaccharides of serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F, respectively, conjugated to TTD. Each dose is formulated to contain 2 μg of each saccharide, except for 3, 6B or 12F, which have 4 μg polysaccharide, and also contains about 20-80 μg of CRM197 carrier protein, about 10-40 μg of TTD carrier protein and 0.125 mg-0.5 mg of aluminum phosphate adjuvant. The liquid is filled into a preservative-free single-use syringe. After shaking, the vaccine becomes a homogenous white suspension and is ready for intramuscular administration.

[0077] Exemplary specific embodiments of the present invention: 1. An immunogenic composition comprising capsular polysaccharides and a carrier from different serotypes of Streptococcus pneumoniae, said serotypes including at least 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F and 33F; Preferably, the serotypes include 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 serotypes selected from 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, and the serotypes include at least 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F and 33F.

[0078] 2. The serotypes include 20 serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F; or 24 serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F; Preferably, the carrier is one or more selected from physiological saline, Ringer's solution, or phosphate buffered saline.

[0079] 3. The immunogenic composition further comprises an adjuvant; Preferably, the immunogenic composition according to item 1 or 2, characterized in that the adjuvant is one or more selected from aluminum-based adjuvants, monophosphoryl lipid A, QS21, CpG, MF59, stearyl tyrosine, Fraser's adjuvant and other mucosal adjuvants.

[0080] 4. the weight ratio of capsular polysaccharide from serotype 3, 6B or 12F to any other capsular polysaccharide is within the range of 10:1 to 1:10, for example 5:1 to 1:5; 3. Preferably, the immunogenic composition according to item 1 or 2, characterized in that the composition is a formulation, the concentrations of the capsular polysaccharides from serotypes 3, 6B and 12F are each independently 1-8 μg / dose and the concentrations of the other capsular polysaccharides are each independently 0.5-5 μg / dose.

[0081] 5. A multivalent immunogenic composition comprising a plurality of polysaccharide-protein conjugates and a pharma- ceutically acceptable adjuvant, each polysaccharide-protein conjugate comprising a capsular polysaccharide from a different serotype of Streptococcus pneumoniae conjugated to a carrier protein; Preferably, the carrier protein comprises at least two carrier proteins, Preferably, the serotypes include at least 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F and 33F, more preferably, the serotypes include 20 serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, or the 24 serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.

[0082] 6. The carrier protein comprises (1) CRM197 and (2) TTD or a variant thereof; Preferably, TTD is the C-terminal domain of TT, More preferably, the multivalent immunogenic composition according to item 5, characterized in that the TTD has the sequence shown in SEQ ID NO. 2 and the TTD variant has a sequence having at least 90% sequence identity with SEQ ID NO: 2.

[0083] 7. In said polysaccharide-protein conjugate, one or more or all of the capsular polysaccharides from at least serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F are conjugated to a carrier protein TTD or a variant thereof, respectively; Preferably, The serotypes include the 20 serotypes, of which the capsular polysaccharides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 serotypes, including serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F, are conjugated to the carrier protein TTD or a variant thereof, respectively; or the serotypes include the 24 serotypes, among which the capsular polysaccharides from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24 serotypes, including serotypes 3, 5, 6B, 12F, 15B, 18C, 19F, and 23F, are conjugated to a carrier protein TTD or a mutant thereof; More preferably, the serotypes include the 20 serotypes, in which the capsular polysaccharides from serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F are each conjugated to the carrier protein TTD or a variant thereof, and the capsular polysaccharides from serotypes 1, 4, 6A, 7F, 8, 9V, 10A, 11A, 14, 19A, 22F and 33F are each conjugated to the carrier protein CRM197; or the serotypes include the 24 serotypes, of which the capsular polysaccharides from serotypes 3, 5, 6A, 6B, 9N, 11A, 12F, 15B, 17F, 18C, 19A, 19F, 20, 23F and 33F are each conjugated to the carrier protein TTD or a variant thereof, and the capsular polysaccharides from serotypes 1, 2, 4, 7F, 8, 9V, 10A, 14 and 22F are each conjugated to the carrier protein CRM197; or 7. The multivalent immunogenic composition according to item 5 or 6, characterized in that the serotypes comprise the 24 serotypes, among which the capsular polysaccharides from serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F are conjugated to the carrier protein TTD or a mutant thereof, and the capsular polysaccharides from serotypes 1, 2, 4, 6A, 7F, 8, 9N, 9V, 10A, 11A, 14, 17F, 19A, 20, 22F and 33F are conjugated to the carrier protein CRM197,

[0084] 8. The multivalent immunogenic composition further comprises the following characteristics: in said composition the weight ratio of capsular polysaccharide from serotype 3, 6B or 12F to any other capsular polysaccharide is from 10:1 to 1:10, for example from 5:1 to 1:5; the composition is a formulation, and the concentrations of capsular polysaccharides from serotypes 3, 6B and 12F are each independently 1-8 μg / dose, and the concentrations of the other capsular polysaccharides are each independently 0.5-5 μg / dose; the composition comprises an adjuvant, preferably the adjuvant being one or more selected from aluminum-based adjuvants, monophosphoryl lipid A, QS21, CpG, MF59, stearyl tyrosine, Fraser's adjuvant and other mucosal adjuvants; In the composition, the weight ratio of the conjugate to the adjuvant is 1:10 to 1:2; the composition comprises a surfactant, preferably at a concentration of 100-300 μg / dose; 7. A multivalent immunogenic composition according to item 5 or 6, characterized in that it has one or more characteristics selected from the pH of the composition being 5.0-7.0.

[0085] 9. Use of the immunogenic composition according to any one of items 1 to 8 in the manufacture of a medicament for inducing an immune response against a pneumococcal capsular polysaccharide conjugate and / or an immune response against a tetanus toxin, comprising: Preferably, the medicament is used for the prevention or treatment of pneumococcal and / or tetanus toxoid infection.

[0086] 10. An immunological composition for generating passive immunity, comprising bactericidal antibodies targeting Streptococcus pneumoniae, said antibodies being obtained by immunizing a mammal with an immunogenic composition according to any one of items 1 to 8, Preferably, said bactericidal antibody is present in serum, a gamma globulin fraction or a purified antibody preparation. The present invention will now be described with reference to specific examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Unless otherwise specified, the methods and materials used in the examples are conventional materials and methods in the art. EXAMPLES

[0087] Experimental Method 1. Production of pneumococcal capsular polysaccharides Pneumococcal bacterial culture, fermentation and polysaccharide production are mature processes (US4686102, US5847112). Pneumococcal strains of different serotypes (United States Type Culture Collection) are cultured to create a bacterial strain library, and one tube of pneumococcal strain from the bacterial strain library is inoculated into a shaking flask containing soybean medium and cultured overnight in an incubator at 37℃ and 5-10% CO2. After the culture is completed, a microscopic examination is performed, and if the results are normal, the culture is inoculated into a 10L fermentation tank and cultured for fermentation at pH 7.0 and 37℃. After the culture is completed, 10% DOC solution is added to ensure that the pneumococci are completely inactivated.

[0088] The purification of pneumococcal capsular polysaccharides is completed by a method without organic solvents (US Patent US5714354). Its production and purification process mainly involves centrifuging the bacterial inactivated liquid to separate the bacterial cells, collecting the centrifugal supernatant, filtering through a 0.22μm filter membrane, and then ultrafiltrating and concentrating through a 100KD membrane to obtain pneumococcal polysaccharide fermentation concentrate. The pneumococcal polysaccharide fermentation concentrate is added with CTAB solution, stirred at room temperature, centrifuged at 8000rpm, and the precipitate of polysaccharide complex is collected (for 7F, 14 and 33F type polysaccharides, the supernatant is collected). The polysaccharide complex is then dissociated with 0.25-0.5M NaCl solution, and then purified by processes such as precipitation, column chromatography, and ultrafiltration to obtain a purified polysaccharide solution. The polysaccharide solution is filtered through a 0.22μm membrane and then stored at low temperature.

[0089] The different types of pneumococcal capsular polysaccharides produced are identified by serotype identification using specific serum, the chemical structure of the pneumococcal polysaccharide is analyzed by nuclear magnetic resonance (NMR), and the content of functional groups in the polysaccharide (e.g., components such as rhamnose, uronic acid, and O-acetyl group) is measured using a chemical color development method. It is confirmed that each physicochemical index of the polysaccharide meets the standards for pneumococcal polysaccharides in the European Pharmacopoeia, and the content of major impurities such as proteins and nucleic acids is less than 1%.

[0090] 2. Preparation of carrier protein TTD The DNA sequence expressing TTD was optimized (SEQ ID NO:1) and cloned into the protein expression plasmid pET21 to construct E. coli BL21(DE3) recombinantly expressing TTD protein (SEQ ID NO:2). A single clone colony of the recombinant BL21(DE3) was selected and placed in 10 mL of LB(Amp) liquid medium and incubated at 37°C, 250 rpm, and OD 600 The culture is then cultured until the affinity of the culture reaches 0.8, after which 0.1 mM IPTG is added and cultured at 25°C and 250 rpm for an additional 4 hours. The culture is centrifuged at 8000 rpm and 4°C to collect the cells, which are then resuspended in PBS and disrupted. The disrupted solution is centrifuged at 8000 rpm and 4°C to collect the supernatant. The TTD protein is purified by a combination of ammonium sulfate precipitation, clarification filtration and chromatography, and the purity reaches 95% or more. The molecular weight determined by mass spectrometry is consistent with the theoretical molecular weight, and the homogeneity of the TTD protein is confirmed by molecular sieving. The amino acid sequence of TTD is as follows:

[0091] KNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSII SSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRI FCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNN EHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND(SEQ ID NO:2).

[0092] 3. Production of diphtheria toxin avirulent mutant CRM197 protein CRM197 carrier protein is a non-toxic mutant of diphtheria toxin (SEQ ID NO: 3), which is non-toxic but retains the immunogenicity of diphtheria toxin. Its fermentation and purification methods have been reported in published literature and patents (US5614382). Usually, Corynebacterium diphtheriae strain is inoculated into a medium, stirred at 37°C for 20-30 hours, centrifuged to remove the bacteria, and the supernatant is retained. The fermentation supernatant is subjected to ultrafiltration to exchange the liquid, and then precipitated by adding ammonium sulfate, and the precipitate is collected. The precipitate is then dissolved, the liquid is exchanged, and further purified by column chromatography to obtain CRM197 protein, which has a purity of 95% or more and is used in the conjugation reaction.

[0093] 4. Preparation of Polysaccharide-Protein Conjugates The preparation of polysaccharide-protein conjugates by coupling bacterial polysaccharides with carrier proteins is an effective method to enhance the immunogenicity of bacterial polysaccharides. Polysaccharide-protein conjugates are widely used in the preparation of bacterial vaccines, such as Haemophilus influenzae type B polysaccharide PRP-TT conjugate vaccine and Neisseria meningitidis A, C, Y, W polysaccharide-carrier protein conjugate vaccine. The preparation of polysaccharide-protein conjugates mainly involves two steps: polysaccharide hydrolysis or activation and polysaccharide-protein coupling. There are different methods for polysaccharide activation, such as the CNBr method (US 4619828), hydrolysis (US 4761283), and sodium periodate oxidation method (US 5306492), and the activated polysaccharide can be chemically coupled to carrier proteins directly (US 4356170) or coupled to carrier proteins via small molecule linkers.

[0094] The preparation of pneumococcal polysaccharide-protein conjugates mainly involves the steps of polysaccharide processing, polysaccharide activation and polysaccharide-protein coupling.

[0095] Pretreatment of pneumococcal polysaccharides: Some serotypes, such as serotypes 1, 3, 4, 6A, 6B, 8, 10A, 11A, 12F, 15B, 18C, and 22F polysaccharides, can be reduced in molecular weight or improved in oxidative coupling efficiency by acid or base hydrolysis (US5847112A). Some polysaccharides, such as serotypes 2, 6B, 7F, 8, 10A, 11A, 12F, 14, 15B, 19A, 19F, and 33F, can be reduced in molecular size by high-pressure homogenization.

[0096] Activation of pneumococcal polysaccharide: Activation of polysaccharide is carried out by adding sodium periodate solution (US4711779). The amount of periodate added is usually 0.05-2 equivalents of polysaccharide, and the oxidation time is 12-24 hours. After the activation of polysaccharide is completed, the small molecular substances in the reaction are removed by ultrafiltration and concentration.

[0097] Preparation of pneumococcal polysaccharide-protein conjugates: Chemical coupling of polysaccharide-protein is carried out by reductive amination method (US5952454, EP0720485, US4711779). Serotype 1, 2, 4, 6A, 7F, 8, 9N, 9V, 10A, 11A, 14, 17F, 19A, 20, 22F and 33F polysaccharides are coupled with CRM197 to prepare polysaccharide-protein conjugates, and serotype 3, 5, 6B, 12F, 15B, 18C, 19F and 23F polysaccharides are coupled with TTD to prepare polysaccharide-protein conjugates. During the coupling reaction, the ratio of polysaccharide to carrier protein is controlled in the range of 0.5-2.5, and 1.0-2.0 equivalents of sodium cyanium borohydride solution is added during the reaction, followed by 2.0 equivalents of sodium borohydride solution. After the reaction is completed, the impurities and unreacted substrates in the reaction are removed by ultrafiltration or chromatography, and finally, the polysaccharide-protein conjugate solution is sterile filtered through a 0.22 μm filter and stored at 2-8° C. All polysaccharide-protein conjugates were analyzed by mass spectrometry and found to have free polysaccharide content within 20%, free protein content within 2%, polysaccharide-protein ratio within 0.5-2.0, and endotoxin and other impurity contents all controlled within safe and acceptable ranges.

[0098] 5. Formulation of Multivalent Pneumococcal Polysaccharide-Protein Conjugate Vaccine There are 24 major pathogenic serotypes of Streptococcus pneumoniae, which are 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F. The stock solution of each type of polysaccharide-protein monovalent conjugate is diluted with a pH 6.0 buffer solution, then mixed, and then aluminum phosphate adjuvant is added and stirred to produce the conjugate. The polysaccharide content of each conjugate is 2-4 micrograms per milliliter, and the aluminum phosphate adjuvant content is 0.125-0.5 milligrams per milliliter.

[0099] 6. Immunogenicity evaluation of polysaccharide-protein conjugates in animals Mouse immunization experiment plan: The polyvalent vaccine stock solution was added to aluminum phosphate adjuvant to prepare the immunization antigen, and 6-8 week old Balb / c mice were selected for intraperitoneal immunization. There were 8 mice in each group, and the immunization volume was 0.5ml each time. The immunization was carried out on days 0, 14, and 21, respectively. Commercially available related vaccines (Prevnar 13 by Pfizer, Weuphoria by Walvax Biotechnology) were used as positive controls, and aluminum phosphate adjuvant was used as negative controls. Blood was collected on day 35 to evaluate the immunogenicity of the polysaccharides.

[0100] Rabbit immunization experiment plan: The polyvalent vaccine stock solution was added to aluminum phosphate adjuvant to prepare the immunization antigen, and 2.0-2.5 kg New Zealand white rabbits were selected for thigh muscle immunization. There were eight rabbits in each group, and the immunization volume was 0.5 ml each time. PCV13 (Prevnar 13) was administered twice as the positive vaccine, and blood was collected on the 35th day to evaluate the immunogenicity of the polysaccharide.

[0101] 7. Evaluation of polysaccharide immunogenicity by ELISA Pneumonia polysaccharide is diluted in coating buffer and coated on a 96-well ELISA plate with 100 μl / well, incubated at 37°C, and then the plate is washed. Mouse and rabbit antisera are treated with adsorbent, and then the serum is first diluted 1:100, then diluted 8 times with a 2.5-fold gradient, and added to the ELISA plate with 50 μl per well, and incubated overnight. The plate is washed, and then the secondary antibody is diluted 1:10,000, and added to the ELISA plate with 100 μl per well, incubated for 2 hours, and washed again. Then, 100 μl of 1 mg / ml PNPP-Na color-developing substrate is added to each well, incubated for 2 hours, and then 50 μl / well of 3M NaOH is added to stop the reaction and measure the OD. 405If the ratio of the OD value of the measurement well to the OD value of the negative well is 2.1 or more, it is judged as positive, and the maximum dilution ratio that results in a positive judgment is taken as the antibody titer of each serum. In addition, the geometric mean of the antibody titers of the immunized animals in each group is calculated, and the statistical significance between different groups is analyzed by T-test.

[0102] Example 1: Comparison of immunogenicity in mice of serotype 5 polysaccharide-CRM197 conjugate and pneumococcal polysaccharide-TTD conjugate The 5PS-CRM197 conjugate and the 5PS-TTD conjugate were added to aluminum phosphate adjuvant, and BALB / c mice (5 mice / group) were immunized with 2 μg / dose on days 0, 14, and 28, and blood was collected on days 21 and 35 to test the antibody titer. The results (Figure 1) show that the titer level of conjugate D35 on CRM197 carrier is 866.43, while the titer level of conjugate D35 on TTD carrier is 16260.67, indicating that the immunogenicity of polysaccharide conjugates with TTD carrier is higher than that of polysaccharide conjugates with CRM197 carrier, with a significant difference (P=0.0079) by Mann-Whitney test.

[0103] Example 2: Comparison of immunogenicity in mice of 24-valent pneumococcal polysaccharide conjugate vaccines with different adjuvant doses The immunogenicity of 24-valent pneumonia polysaccharide conjugate vaccines with aluminum phosphate adjuvant at 0.5 mg / dose, 0.25 mg / dose, and 0.125 mg / dose was evaluated in BALB / c mice. BALB / c female mice aged 6-8 weeks were randomly divided into groups of 8 mice / group and immunized once with 0.5 ml / dose on days 0, 14, and 28, respectively, and blood was collected on day 35 to test antibody titer levels. The immunogenicity data are shown in Figures 2 and 3. The results showed that the immunogenicity of the 24-valent pneumococcal polysaccharide conjugate vaccine with different adjuvant doses against the 13 serotypes (serotypes included in the positive vaccine) was comparable to that of the commercially available 13-valent conjugate vaccine, and some serotypes, such as 6B, 18C and 23F, showed higher antibody titer levels than the 13-valent conjugate vaccine. In addition, 11 serotypes other than the 13 serotypes also achieved good immunogenicity.

[0104] Example 3: Comparison of immunogenicity in mice of 24-valent pneumococcal polysaccharide conjugate vaccines under different pH conditions The immunogenicity of 24-valent pneumococcal polysaccharide conjugate vaccines with different pH values ​​was evaluated in BALB / c mice. The 24-valent pneumococcal polysaccharide conjugate vaccine was evaluated in 6-8 week-old female BALB / c mice, which were randomly divided into groups of 8 mice per group. The study consisted of two immunizations on D0 and D14, one dose per time, and serum was collected on D0 and D21 to measure serum polysaccharide antibody levels by ELISA. The serum antibody titers of the 24-valent pneumococcal polysaccharide conjugate vaccine with a pH range of 5.0-6.2 were compared one week after the second immunization (D21) (see Figure 4). After immunization with the 24-valent pneumococcal polysaccharide conjugate vaccine with a pH range of 5.0-6.2, there was no significant difference in the antibody titers of each serotype between the groups (P>0.05), indicating that all of them could induce good immunogenicity.

[0105] Example 4: Comparison of immunogenicity in mice between 24-valent pneumococcal polysaccharide conjugate vaccine and positive vaccine There are two types of 13-valent pneumonia conjugate vaccines currently on the market: Prevnar 13, manufactured by Pfizer in the United States and using CRM197 as the carrier protein, and Weuphoria, manufactured by Walvax Biotechnology and using tetanus toxoid (TT) as the carrier protein. BALB / c mice were immunized with the 24-valent pneumonia conjugate vaccine along with Prevnar 13, Weuphoria, and an adjuvant control group on days 0, 14, and 28, respectively, and immunogenicity was evaluated on day 35 (see Figures 5 and 6). According to the results: (1) The 24-valent pneumococcal conjugate vaccine was significantly more immunogenic against serotype 18C than Prevnar 13, which is based on the CRM197 carrier protein, and also had higher antibody titers against serotypes 5, 6A, 7F and 23F than the control group. (2) The 24-valent pneumococcal conjugate vaccine was compared with Weuphoria based on the TT carrier protein, and the D35 serum antibody titers were examined by the non-parametric Kruskal-Wallis test. Significant differences were observed for serotypes 4, 6A, and 23F, and the immunogenicity was higher than that of Weuphoria. Furthermore, the antibody titers for 5, 7F, and 9V were also higher than those of the control group. (3) The 24-valent pneumococcal conjugate vaccine was also able to induce good immunogenicity against non-PCV13 vaccine serotypes (see Figure 6). When the antibody titers on day 35 of the 24-valent pneumococcal conjugate vaccine (PCV24) and adjuvant control groups were analyzed using the Mann-Whitney test, significant differences were observed for serotypes 2, 8, 9N, 11A, 15B, 17F, 20, and 33F (P<0.05), indicating that the PCV24 vaccine has good immunogenicity against serotypes other than PCV13. When the 10A vaccine group was compared with the adjuvant control group, no significant difference was observed (P=0.3068). This is thought to be due to the high basal antibody titer of 10A in experimental mice.

[0106] Example 5: Comparison of immunogenicity in rabbits between 24-valent pneumococcal polysaccharide conjugate vaccine and positive vaccine The PCV24 pneumococcal polysaccharide conjugate vaccine was prepared and used to immunize rabbits (8 rabbits / group). PCV13 (Prevnar 13) was used as a positive control and saline was used as a negative control. The rabbits were immunized at 3-week intervals after the first immunization, and blood was collected 2 weeks after the second immunization (D35) to test the antibody titers for each type of polysaccharide in the serum (see Figures 7 and 8). Compared with PCV13 (Prevnar 13) (Figure 7), after two immunizations with PCV24, the immunogenicity of the 13 serotypes was tested and analyzed by T-test, which showed that the immunogenicity reached the efficacy of the commercially available vaccine. In addition, 11 serotypes other than the 13 serotypes (Figure 8) also showed good immunogenicity.

[0107] Example 6: Immunogenicity comparison of dual-carrier and single-carrier 24-valent pneumococcal polysaccharide conjugate vaccines To compare the immunogenicity of 24-valent pneumococcal polysaccharide conjugate vaccines using two carrier proteins versus one carrier protein, two different 24-valent pneumococcal polysaccharide conjugate vaccines were prepared. In group A vaccines, 8 serotypes (3, 5, 6B, 12F, 15B, 18C, 19F, and 23F) used TTD as the carrier protein, and the remaining 16 serotypes used CRM197 as the carrier protein, while in group B vaccines, all 24 serotypes used CRM197 as the carrier protein. The above two groups of vaccines were immunized into New Zealand rabbits, eight in each group, for a total of two immunizations. Serum was collected at three-week intervals before immunization and two weeks after the second immunization to test the antibody titers for each type of polysaccharide. The vaccine titers of the two groups two weeks after the second immunization were converted to logarithms and then subjected to statistical analysis using a multiple t-test. The results are shown in Figure 9. 9 shows that the dual-carrier vaccine group can induce higher antibody titers than the single-carrier vaccine group in all 24 serotypes, and the induced antibody titers were significantly different in 7 serotypes (serotypes 4, 5, 9V, 12F, 19A, 19F and 23F) (P<0.05), indicating that the dual-carrier vaccine can avoid the immunosuppressive effect of the single carrier.

[0108] Example 7: Immunogenicity comparison of dual-carrier and single-carrier 20-valent pneumococcal polysaccharide conjugate vaccines Two different 20-valent (1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F) pneumococcal polysaccharide conjugate vaccines were prepared. In group A vaccine, 8 serotypes (3, 5, 6B, 12F, 15B, 18C, 19F and 23F) used TTD as the carrier protein, and the remaining 12 serotypes used CRM197 as the carrier protein, while in group B vaccine, all 20 serotypes used CRM197 as the carrier protein. The two vaccine groups were used to immunize New Zealand rabbits, eight in each group, twice in total, and serum samples were taken before immunization and two weeks after the second immunization at three-week intervals to test the antibody titers of each type of polysaccharide. The serum titers two weeks after the second immunization were converted to logarithmic values ​​and then statistically analyzed using multiple t-test. The results are shown in Figure 10. The results showed that in the dual-carrier vaccine group, eight serotypes used the TTD carrier (3, 5, 6B, 12F, 15B, 18C, 19F and 23F), and the antibody titer levels of these eight serotypes were all higher than those in the single-carrier vaccine group, and there was a significant difference in type 5 and type 23F, P<0.05. Of the 20 serotypes in the dual-carrier vaccine, titers for 16 serotypes (except 6A, 7F, 22F, and 33F) were all higher than those in the single-carrier vaccine group.

[0109] Example 8: Immunogenicity comparison of dual-carrier and single-carrier 24-valent pneumococcal polysaccharide conjugate vaccines Two different 24-valent (1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F) pneumococcal polysaccharide conjugate vaccines were prepared. In group A, 15 serotypes (3, 5, 6A, 6B, 9N, 11A, 12F, 15B, 17F, 18C, 19A, 19F, 20, 23F, and 33F) were used as the carrier protein with TTD, and the remaining 9 serotypes were used as the carrier protein with CRM197, while in group B, all 24 serotypes were used as the carrier protein with CRM197. The two vaccine groups were used to immunize New Zealand rabbits, eight in each group, twice in total, and serum samples were taken before immunization and two weeks after the second immunization at three-week intervals to test the antibody titers of each polysaccharide. The serum titers two weeks after the second immunization were converted to logarithmic values ​​and then statistically analyzed using multiple t-test. The results are shown in Figure 11. The results showed that in the dual-carrier vaccine group, 15 serotypes used the TTD carrier (3, 5, 6A, 6B, 9N, 11A, 12F, 15B, 17F, 18C, 19A, 19F, 20, 23F and 33F), of which the antibody titer levels of 12 serotypes were higher than those of the single-carrier vaccine group, and a significant difference was observed for 23F, P<0.05. Of the 24 serotypes in the dual-carrier vaccine, antibody titer levels for 16 serotypes (excluding 1, 2, 3, 4, 8, 10A, 14, and 19F) were higher than those in the single-carrier vaccine group.

[0110] Example 9: Protection test of 24-valent pneumococcal polysaccharide conjugate vaccine against type 3 pneumococcus in BALB / c mice In BALB / c mice, PCV13 (Weuphoria from Walvax Biotechnology) polysaccharide conjugate vaccine was used as a positive control and saline as a negative control, and 2 × 10 5 Mice were infected with CFU of type 3 Streptococcus pneumoniae via nasal dropwise infection. The survival rate was observed for 2 weeks after challenge, and the protective effect of the 24-valent pneumococcal polysaccharide conjugate vaccine against type 3 Streptococcus pneumoniae challenge was analyzed. The results (Figure 12) show that the antibodies induced by the 24-valent pneumococcal polysaccharide conjugate vaccine have 100% immune protection, and its efficacy is superior to that of the commercially available 13-valent pneumococcal polysaccharide conjugate vaccine, which has a protection rate of 89.9%.

[0111] Example 9: Protection test of 24-valent pneumococcal polysaccharide conjugate vaccine against Streptococcus pneumoniae type 22F in BALB / c mice The test was carried out in BALB / c mice using 23-valent pneumococcal polysaccharide vaccine (PPV23) as a positive control and saline as a negative control, with 1 × 10 8 Mice were intraperitoneally infected with CFU of serotype 22F Streptococcus pneumoniae, and the protective effect of the 24-valent polysaccharide conjugate vaccine against serotype 22F Streptococcus pneumoniae was analyzed based on survival after challenge. The results (Figure 13) showed that the survival rate of the 24-valent vaccine was 100%, the survival rate of the PPV23 polysaccharide vaccine as a control was 30%, and the survival rate of saline was 20%. The Log-rank (Mantel-Cox) test showed that there was a significant difference between the 24-valent vaccine and the control, P<0.05 (P value 0.0027). These results suggest that the 24-valent pneumococcal polysaccharide conjugate vaccine has a more pronounced protective effect against serotype 22F pneumococcus than the PPV23 polysaccharide vaccine.

[0112] Example 10: Testing the protective effect of 24-valent pneumococcal polysaccharide conjugate vaccine against tetanus toxin The 24-valent pneumococcal polysaccharide conjugate vaccine contains the TTD carrier protein, and vaccine efficacy was evaluated by a tetanus toxin challenge protection test in mice. BALB / c mice were immunized with the 24-valent pneumococcal polysaccharide conjugate vaccine by intraperitoneal injection on days 0 and 14, with DPT vaccine as the positive control and saline as the negative control. Blood was collected on day 28 to examine the antibody titer level against tetanus toxoid (TT) in the serum (in FIG. 14, group A is saline, group B is DPT vaccine, and group C is 24-valent pneumococcal polysaccharide conjugate vaccine). Tetanus toxoid (20 LD 50 ) was intraperitoneally injected into the mice, and the survival status of the mice was observed for two weeks (FIG. 15). As can be seen from Figure 14, the TT antibody titers of groups B, C, and D increased significantly after two immunizations compared to before immunization, with P<0.05. The TT antibody titer of group B was higher than that of group C, possibly because the DPT vaccine consists of TT, whereas the 24-valent pneumococcal polysaccharide conjugate vaccine uses TTD. As can be seen from Figure 15, all mice in the saline group died on the first day after intraperitoneal infection with tetanus toxin, while the protective rate of the 24-valent pneumococcal polysaccharide conjugate vaccine was 80%, and the protective rate of the control group's DPT vaccine was 100%, indicating that the 24-valent pneumococcal polysaccharide conjugate vaccine has a protective effect against infection with tetanus toxin, and its effect is obviously superior to that of the negative control.

Claims

1. An immunogenic composition comprising capsular polysaccharides from different serotypes of Streptococcus pneumoniae and a carrier, said serotypes including at least 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F and 33F; Preferably, the serotypes include 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 serotypes selected from 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, and the serotypes include at least 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F and 33F.

2. The serotypes include 20 serotypes, 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, or 24 serotypes, 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F; The immunogenic composition according to claim 1, characterized in that the carrier is preferably one or more selected from physiological saline, Ringer's solution, or phosphate buffered saline.

3. The immunogenic composition further comprises an adjuvant; The immunogenic composition according to claim 1 or 2, characterized in that the adjuvant is preferably one or more selected from the group consisting of aluminum-based adjuvants, monophosphoryl lipid A, QS21, CpG, MF59, stearyl tyrosine, Fraser's adjuvant and other mucosal adjuvants.

4. the weight ratio of capsular polysaccharide from serotype 3, 6B or 12F to any other capsular polysaccharide is in the range of 10:1 to 1:10, for example 5:1 to 1:5; 3. An immunogenic composition according to claim 1 or 2, characterized in that the composition is a formulation, the concentrations of capsular polysaccharides from serotypes 3, 6B and 12F being each independently 1-8 μg / dose and the concentrations of the other capsular polysaccharides being each independently 0.5-5 μg / dose.

5. A multivalent immunogenic composition comprising a plurality of polysaccharide-protein conjugates and a pharma- ceutically acceptable adjuvant, each polysaccharide-protein conjugate comprising a capsular polysaccharide from a different serotype of Streptococcus pneumoniae conjugated to a carrier protein; Preferably, the carrier protein comprises at least two carrier proteins, Preferably, the serotypes include at least 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F and 33F, more preferably, the serotypes include 20 serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, or the 24 serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.

6. the carrier protein comprises (1) CRM197 and (2) TTD or a variant thereof; Preferably, TTD is the C-terminal domain of TT, More preferably, the multivalent immunogenic composition according to claim 5, characterized in that the TTD has the sequence shown in SEQ ID NO. 2 and the TTD variant has a sequence having at least 90% sequence identity with SEQ ID NO:

2.

7. in said polysaccharide-protein conjugate, one or more or all of the capsular polysaccharides from at least serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F, respectively, are conjugated to a carrier protein TTD or a variant thereof; Preferably, The serotypes include the 20 serotypes, of which the capsular polysaccharides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 serotypes, including serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F, are conjugated to the carrier protein TTD or a variant thereof, respectively; or the serotypes include the 24 serotypes, among which the capsular polysaccharides from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24 serotypes, including serotypes 3, 5, 6B, 12F, 15B, 18C, 19F, and 23F, are conjugated to a carrier protein TTD or a mutant thereof; More preferably, the serotypes include the 20 serotypes, in which the capsular polysaccharides from serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F are each conjugated to the carrier protein TTD or a variant thereof, and the capsular polysaccharides from serotypes 1, 4, 6A, 7F, 8, 9V, 10A, 11A, 14, 19A, 22F and 33F are each conjugated to the carrier protein CRM197; or the serotypes include the 24 serotypes, of which the capsular polysaccharides from serotypes 3, 5, 6A, 6B, 9N, 11A, 12F, 15B, 17F, 18C, 19A, 19F, 20, 23F and 33F are each conjugated to the carrier protein TTD or a variant thereof, and the capsular polysaccharides from serotypes 1, 2, 4, 7F, 8, 9V, 10A, 14 and 22F are each conjugated to the carrier protein CRM197; or 7. The multivalent immunogenic composition according to claim 5 or 6, characterized in that the serotypes comprise the 24 serotypes, among which the capsular polysaccharides from serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F are conjugated to the carrier protein TTD or a mutant thereof, respectively, and the capsular polysaccharides from serotypes 1, 2, 4, 6A, 7F, 8, 9N, 9V, 10A, 11A, 14, 17F, 19A, 20, 22F and 33F are conjugated to the carrier protein CRM197, respectively.

8. The multivalent immunogenic composition further comprises the following characteristics: in said composition the weight ratio of capsular polysaccharide from serotype 3, 6B or 12F to any other capsular polysaccharide is from 10:1 to 1:10, for example from 5:1 to 1:5; the composition is a formulation, and the concentrations of capsular polysaccharides from serotypes 3, 6B and 12F are each independently 1-8 μg / dose, and the concentrations of the other capsular polysaccharides are each independently 0.5-5 μg / dose; the composition comprises an adjuvant, preferably the adjuvant being one or more selected from aluminum-based adjuvants, monophosphoryl lipid A, QS21, CpG, MF59, stearyl tyrosine, Fraser's adjuvant and other mucosal adjuvants; In the composition, the weight ratio of the conjugate to the adjuvant is 1:10 to 1:2; the composition comprises a surfactant, preferably at a concentration of 100-300 μg / dose; 7. A multivalent immunogenic composition according to claim 5 or 6, characterized in that it has one or more characteristics selected from the fact that the pH of the composition is 5.0-7.

0.

9. Use of an immunogenic composition according to any one of claims 1 to 8 in the manufacture of a medicament for inducing an immune response against a pneumococcal capsular polysaccharide conjugate and / or an immune response against a tetanus toxin, comprising: Preferably, the medicament is used for the prevention or treatment of pneumococcal and / or tetanus toxoid infection.

10. An immunological composition for eliciting passive immunity, comprising bactericidal antibodies targeting Streptococcus pneumoniae, said antibodies being obtained by immunizing a mammal with an immunogenic composition according to any one of claims 1 to 8, Preferably, said bactericidal antibody is present in serum, a gamma globulin fraction or a purified antibody preparation.

Images