WO2018048141A1 - Composition comprising multivalent capsular polysaccharide-transport protein and use thereof - Google Patents

Abstract

The present invention relates to a vaccine composition comprising 13 or 14 kinds of capsular polysaccharide-transport protein conjugates for prevention of pneumococcal diseases and an immunogenic composition for Streptococcus pneumoniae.

Description

Compositions comprising the multivalent capsular polysaccharide-carrying protein and uses thereof

The present invention relates to vaccine compositions for the prevention of pneumococcal disease comprising 13 or 14 capsular polysaccharide-carrying protein conjugates and immunogenic compositions against pneumococci.

Streptococcus pneumonia is a leading cause of meningitis, pneumonia and severe invasive diseases in infants and children around the world. More than 1.6 million people die each year from pneumococcal disease (2008 International Health Organization), and the incidence of invasive infectious diseases caused by pneumococcal in children under 5 years old and elderly people 65 years or older with low immunity high.

Pneumococci are classified into more than 90 serotypes according to the structural and immunological characteristics of the capsular polysaccharides, the main pathogenic factors surrounding them, of which 20 to 80% in humans It is known to be associated with pathogenicity. The only host of pneumococci is humans, and they usually exist in colonies in the nasopharynx of healthy normal people (20-40% in infants, 5-10% in adults). In 2005, the US Centers for Disease Control and Prevention (CDC) estimated that approximately 2.1 million children under 5 years of age died from pneumonia, and 1.2 million of them died from developing countries alone annually. In the United States, pneumococcal meningitis and sepsis are reported to be about 3,000 and 50,000 cases per year, respectively (Peters TR, et al . (2007) JAMA (297) 1825-6; Invasive pneumococcal disease). . In addition, pneumoACTION, which is a database of pneumococcal disease, showed that 24,047 cases of pneumococcal infections occurred in Korean children in 2000 and 47 of them died (www.pneumoadip.org). Furthermore, according to a recent study on the analysis of pneumococcal serotypes in children and adolescents published by the Korea Centers for Disease Control and Prevention, pneumococci are the most common cause of invasive infections (43.7%) in infants aged 3 to 59 months. appear. In addition, in pneumococci causing invasive infectious diseases all over the world, multidrug resistant bacteria that are resistant to not only penicillin but also to three or more drugs are increasing, further increasing the difficulty of treating pneumococcal infectious diseases. Therefore, the need for pneumococcal vaccination for children and the elderly, which is a high risk group of pneumococcal infectious diseases, has been continuously raised.

To prevent pneumococcal disease, multivalent pneumococcal polysaccharide vaccines have been developed and approved since 1977, and these capsular polysaccharide vaccines have proven useful in preventing pneumococcal disease in elderly and high-risk patients. However, in the case of infants and children, since the maturity of the immune system is lower than that of adults, when only the polysaccharide vaccine is received, the immune system does not recognize the polysaccharide antigen as an external invading factor, so it is difficult to expect a role as a vaccine. To solve the problem of lowering immunogenicity of polysaccharide vaccines in infants and children, a 7-valent pneumococcal conjugate vaccine, a capsular polysaccharide-protein conjugated vaccine conjugated with a carrier protein that increases immunogenicity to polysaccharide antigens ( 7vPnC, Prevenar ^® (Prevenar ^®)) has been used in the development, has been reported to be effective for the prevention of invasive disease and otitis media in infants and children in many materials. However, the use of the 7-valent vaccine induced a decrease in invasive disease caused by vaccine serotypes used in the vaccine, but also showed a relative increase in pneumococcal disease caused by some non-vaccine serotypes. Thus, 10 the capsular polysaccharide-protein conjugate vaccine in renal flow Rix ^® (Synflorix ^®) and the landscape in Prevenar the vena ^® A 13 add serotypes 6 species in the primary serotype of Streptococcus pneumoniae conjugate vaccine 13 ^® (Prevenar13 ^® Have been developed and are now commercially available, but the possibility that the efficacy as a vaccine may not be sufficient for some of the serotypes included is mentioned [Andrews NJ et al , (2014) Lancet Infec Dis (14) 839; EMEA Assessment Report for Prevenar 13 (2009) EMA / 798877/2009], showing higher and more stable titers due to the risk of developing serotype replacement by non-vaccinating pneumococcal serotypes not included in the vaccine. There is also a continuing need for the development of new vaccine formulations that can cover more diverse serotypes.

On the other hand, one of the notable phenomena since the introduction of pneumococcal conjugate vaccines is an increase in invasive disease due to serotype replacement by non-vaccinating pneumococcal serotypes not included in the vaccine. These non-vaccine serotypes are a new threat to the development of pneumococcal disease by pushing away vaccine serotypes that predominantly formed before the introduction of the conjugate vaccine and gaining colony advantage in the nasopharyngeal cavity. In particular, serotype 2 has a higher incidence than some serotypes (4, 7F, 3) included in Prevena13 ^® in terms of global distribution of pneumococci, and according to the literature, it is the 11th most common in the world. Serotype (Johnson HL et al . (2010) PLoS Medicine, 7:10, e1000348; Systematic Evaluation of Serotypes Causing Invasive Pneumococcal Disease among Children Under Five: The Pneumococcal Global Serotype Project). In addition, as reported in recent reports that 20% of 230 cases of invasive meningitis in infants under 5 years of age in Bangladesh between 2001 and 2009 were caused by serotype 2 (Saha SK et al. (2012) PLoS One. , 7: 3, e32134; Streptococcus pneumoniae serotype-2 childhood meningitis in Bangladesh: a newly recognized pneumococcal infection threat, as well as its frequent regional incidence, as well as invasive pneumococcal (IPD) induced Severity and importance can be said. Pribena 13 ^® , the highest serotype coverage of Pfizer's 13-valent vaccine, does not contain serotype 2 capsular polysaccharides, and cross-reactivity of the other 13 capsular polysaccharide antigens It is known to be absent.

Accordingly, the present inventors have made efforts to develop a vaccine for preventing pneumococcal disease having a higher and more stable titer and higher coverage than a conventionally developed vaccine formulation, and as a result, the same serotype as the conventionally released 13-valent vaccine. However, we have developed a novel trivalent pneumococcal conjugate vaccine that is more effective by applying a conjugation process different from the existing trivalent vaccine, and also contains serotype 2 capsular polysaccharide antigen in 13 species of the trivalent vaccine. The present invention has been completed by developing a 14-valent pneumococcal conjugate vaccine, which has expanded coverage and optimized immunogenicity through optimization of the conjugation process. The 13-valent pneumococcal conjugate vaccine of the present invention contains 13 kinds of capsular polysaccharide antigens, and has a higher titer than the known 13-valent pneumococcal conjugate vaccine, and the 14-valent pneumococcal conjugate vaccine contains 14 kinds of capsular polysaccharide antigens. Including, it can provide a higher coverage and improved coverage compared to the existing vaccine, it is excellent in preventing invasive pneumococcal disease.

One object of the invention comprises 13 capsular polysaccharide-carrying protein conjugates, wherein said 13 conjugates are Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A , 13B capsular polysaccharides derived from 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F are covalently conjugated to a carrier protein, the carrier protein is CRM197 protein, and the conjugate Is to provide a vaccine composition for the prevention of pneumococcal disease, in which the capsular polysaccharide and the transport protein have a structure linked by -OC (NH) -NH- group using a cyanylation method.

Another object of the present invention comprises 14 capsular polysaccharide-carrying protein conjugates, wherein the 14 conjugates are Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, Each of the 14 capsular polysaccharides from 14, 18C, 19A, 19F, and 23F is covalently conjugated to a carrier protein, the carrier protein is CRM197 protein, and the conjugate is capsular polysaccharide and transport using a cyanylation method. It is to provide a vaccine composition for the prevention of pneumococcal disease, in which the protein has a structure linked by -OC (NH) -NH- group.

Another object of the invention comprises 13 capsular polysaccharide-carrying protein conjugates, wherein said 13 conjugates are Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, Each of 13 capsular polysaccharides derived from 18C, 19A, 19F and 23F is covalently conjugated to a carrier protein, the carrier protein is CRM197 protein, and the conjugate is used to obtain the capsular polysaccharide and carrier protein using a cyanation method. It is to provide an immunogenic composition for pneumococcal, which has a structure linked by -OC (NH) -NH- group.

Another object of the present invention comprises 14 capsular polysaccharide-carrying protein conjugates, wherein the 14 conjugates are Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, Each of the 14 capsular polysaccharides from 14, 18C, 19A, 19F, and 23F is covalently conjugated to a carrier protein, the carrier protein is CRM197 protein, and the conjugate is capsular polysaccharide and transport using a cyanylation method. It is to provide an immunogenic composition for pneumococcal, in which the protein has a structure linked by -OC (NH) -NH- group.

Still another object of the present invention is to provide a method for preventing pneumococcal disease by administering the vaccine composition or immunogenic composition to a subject in need thereof.

Another object of the present invention comprises 13 capsular polysaccharide-carrier protein conjugates, wherein the 13 conjugates are Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F. Each of 13 capsular polysaccharides from 9V, 14, 18C, 19A, 19F and 23F is covalently conjugated to a carrier protein, the carrier protein is CRM197 protein, and the conjugate is cyanylated ( It is to provide a use for the use of the composition for the preparation of a vaccine composition for the prevention of pneumococcal disease, wherein the capsular polysaccharide and the carrier protein has a structure linked by -OC (NH) -NH- group using a cyanylation) method .

Another object of the invention comprises 14 capsular polysaccharide-carrier protein conjugates, wherein the 14 conjugates are Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B 14 capsular polysaccharides derived from 7F, 9V, 14, 18C, 19A, 19F and 23F are covalently conjugated to a carrier protein, the carrier protein is CRM197 protein, and the conjugate is Provided for use of the composition in the preparation of a vaccine composition for the prevention of pneumococcal disease, wherein the capsular polysaccharide and the carrier protein have a structure linked by -OC (NH) -NH- groups using a cyanylation method. It is.

In the present invention, in order to develop a novel vaccine formulation that can prevent pneumococcal disease with a more excellent effect, it differs from the conventionally released 13-valent vaccine and includes 13 capsular polysaccharide-carrying protein conjugates. A composition was prepared. Specifically, the composition is each of 13 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F It includes 13 kinds of capsular polysaccharide-carrying protein conjugates prepared by covalently conjugation to CRM197, which is a carrier protein. Since the 13 kinds of capsular polysaccharides have remarkably superior titers compared to the known 13-valent pneumococcal conjugate vaccine, the composition of the present invention can be expected to have a very good effect for the prevention of pneumococcal disease.

Furthermore, there are many reports on the reduction of invasive disease caused by vaccine serotypes included in the vaccine after commercialization of the pneumococcal protein conjugate vaccine, but the incidence of invasive pneumococcal disease according to the vaccine serotype is still regional serotypes. It is incomparable with specific distribution (Lee LH et al . (2014) Vaccines, 2 (112) Towards New Broader Spectrum Pneumococcal Vaccines: The Future of Pneumococcal Disease Prevention). Therefore, there is also a need to expand the scope of application by adding a new serotype while maintaining the serotype of the existing pneumococcal conjugate vaccine.

Serotype 2 is a more frequent serotype than some serotypes (4, 7F, 3) included in Prevena 13 ^® . In developing a vaccine formulation for preventing pneumococcal disease, the present inventors may indicate titer and / or functional antibody titer when the capsular polysaccharide derived from Streptococcus pneumoniae serotype 2 is further included in the trivalent vaccine formulation. It was confirmed. In the case of polysaccharide-protein conjugate vaccines containing pneumococcal conjugate vaccines, immunogenicity may be lowered due to interference between the serotypes of capsular polysaccharides or carrier proteins. In the case of the 14-valent vaccine prepared by using the cyanylation method according to the method, applying the serotype-specific polysaccharide hydrolysis method, and optimizing the conjugation process, it was confirmed that the improved efficacy and / or coverage of the conventional 13-valent vaccine could be shown. It was.

Each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of the present invention is not to be limited by the specific description described below.

One aspect of the present invention for achieving the above object is a vaccine composition for the prevention of pneumococcal disease comprising 13 capsular polysaccharide-carrying protein conjugates.

Wherein the 13 conjugates are 13 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. Each of the capsular polysaccharides may be covalently conjugated to the carrier protein CRM197.

That is, the present invention relates to a vaccine composition comprising 13 different polysaccharide-protein conjugates, each conjugate comprising a different serotype of the capsular polysaccharide from Streptococcus pneumoniae conjugated to a carrier protein, wherein the capsular polysaccharide is Serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.

Another aspect of the invention is a vaccine composition for the prevention of pneumococcal disease comprising 14 species of capsular polysaccharide-carrying protein conjugates.

Wherein the 14 conjugates are each of 14 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. It may be covalently conjugated to the carrier protein CRM197.

In other words, the present invention provides a vaccine composition comprising 14 different polysaccharide-protein conjugates, each conjugate comprising a different serotype of a capsular polysaccharide derived from Streptococcus pneumoniae conjugated to a carrier protein, wherein the capsular polysaccharide is Serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.

The present inventors compared two representative methods of conjugation, reductive amination and cyanylation, to find a method for enhancing the immunogenicity of pneumococcal vaccines. It was confirmed that the cyanation method is much superior to the reductive amination reaction in terms of the bonding yield and the time required. Based on this, 13 serotypes were optimized for serotype-optimized conjugation using different cyanation reactions from prevena 13 ^® using the same 13 serotypes as prevena 13 ^® with the highest coverage. Has confirmed that the titers of all serotypes are very good compared to the prevenna13 ^® standard. In the case of multivalent immunogenic compositions, there may be interference from the combination of singers (Dagan R et al . (2010) Vaccines, 28 (5513) Glycoconjugate vaccines and immune interference: A review), 13 or 14 used Increasing immunogenicity simultaneously in all serotypes requires a great deal of effort by one skilled in the art.

In particular, in the case of the present 14-valent conjugate vaccine, in order to increase the coverage of the existing pneumococcal vaccine, a new non-vaccinated serotype No. 2 not covered by Prevena 13 ^® was added. The conjugation process was optimized for improvement, resulting in superior titers in all serotypes. Since this may further increase the disturbance due to the hydrolysis combination in the multivalent immunogenic composition by adding a new serotype 2, simultaneously increasing immunogenicity in all 14 serotypes used may be a surprising effect for those skilled in the art. .

In the present invention, the conjugate is characterized by having a structure in which the capsular polysaccharide and the transport protein are connected by -O-C (NH) -NH- group using a cyanylation method. Thus, on the basis of structural differences with the preparation of Pneumococcal conjugate vaccines (PCV) prepared by other methods and optimization of detailed conjugation practices, the 13- or 14-valent vaccine compositions of the present invention can be conjugated by known reductive amination. It is possible to show significantly better titers in all serotypes compared to the 13- or 14-valent vaccine composition.

In the present invention, the term “pneumonia” is a kind of acute inflammatory disease of the lung parenchyma, and the infectious agents are mainly Streptococcus pneumoniae and Klebsiella pneumoniae . In particular, pneumococcal pneumonia accounts for about 50% of all pneumonia, severe chills, fever, cough and chest pain, sputum is often bloody, complications that can cause pleurisy, meningitis, endocarditis, peritonitis Stein GE et al . (2001 Mar) Diagn. Microbiol.Infect, Dis 39: 181-185; Comparative serum bactericidal activity of clarithromycin and azithromycin against macrolide-sensitive and resistant strains of Streptococcus pneumoniae).

The term “pneumococcus” in the present invention refers to Streptococcus pneumoniae and is generally a commensal organism that colonizes the mucosal surface of human nasopharynx. If the host's factor allows access to the lower respiratory tract of the organism, then a vigorous inflammatory response follows, which causes dense consolidation when the alveolar space fills the exudate, resulting in pneumonia May cause. The pneumococci can synthesize more than 90 structurally unique capsular polysaccharides, and the serotypes of pneumococci are classified according to the structural and immunological characteristics of these capsular polysaccharides. Thus, when a vaccine preparation is prepared using pneumococcal capsular polysaccharide, the immune response may be different depending on the type of capsular polysaccharide, that is, the serotype of pneumococcal from which capsular polysaccharide is derived. The vaccine composition of the present invention specifically contains 13 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. It can be prepared using. In addition, the vaccine composition of the present invention contains 14 kinds of capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. It can be prepared using.

The capsular polysaccharide is recognized as an antigen when administered in the body, so that it can produce an antibody against it, thereby preparing a vaccine composition for preventing pneumococci. As used herein, the term “antigen” refers to a substance that can specifically induce an immune response when the substance invades the body. In the present invention, 13 kinds of capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F are antigens. Can work. In addition, in the present invention, 14 types of capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F may act as antigens. Can be.

The capsular polysaccharide can be prepared by standard techniques known to those skilled in the art, and are not particularly limited in its method. The capsular polysaccharide can be reduced in size through hydrolysis to reduce viscosity and induce effective immunogenicity.

In one specific embodiment of the present invention, Streptococcus pneumoniae having 14 different serotypes (1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F). Each was lysed using sodium deoxycholate and the polysaccharide bound to the cells was liberated. Then, 12 serotypes 1, 2, 3, 4, 5, 6A, 6B, 9V, 18C, 19A, 19F and 23F were purified by performing CTAB process because they can be ion-bonded with CTAB (cetyltrimethylammonium bromide). Two serotypes 7F and 14, which do not react with CTAB, were purified using an aluminum phosphate gel (Algel) solution.

In the case of using the capsular polysaccharide as a vaccine composition, infants and children whose immune system is lower than adults may not recognize this as an antigen, and thus an immune response may not occur. Thus, in the present invention, the conjugate protein and the capsular polysaccharide are conjugated. It was prepared and used.

In the present invention, the term "carrier protein" refers to a protein that can be covalently conjugated with the capsular polysaccharide to increase the immunogenicity of the polysaccharide antigen. In the present invention, CRM197 was used. The carrier protein may be conjugated with the capsular polysaccharide through a standard conjugation method, and the capsular polysaccharide-carrying protein conjugate formed therefrom may be one or a plurality of capsular polysaccharides conjugated to one carrier protein.

The term “CRM197” in the present invention is a non-toxic variant of diphtheria toxin (ie, toxoid) isolated from the culture of Corynebacterium diphtheriae strain C7 (β197). CRM197 can be purified via ultrafiltration, ammonium sulphate precipitation and ion exchange chromatography. In addition, the CRM197 may be recombinantly prepared according to US Pat. No. 5,614,382.

All known methods for preparing conjugates of capsular polysaccharides and carrier proteins can be included within the scope of the present invention, wherein the conjugates are linked to the -OC (NH) -NH- group by using the cyanylation method. Has a structure. The cyanation method may be appropriately performed by those skilled in the art through a known method, for example, may be performed using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or CNBr, but is not limited thereto.

As an example for preparing conjugates of capsular polysaccharides and carrier proteins, purified capsular polysaccharides can be chemically activated and each chemically activated capsular polysaccharide can be conjugated to the carrier protein one by one to form a glycoconjugate. Cyanation activity by treatment with CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) converts the hydroxyl group of the capsular polysaccharide to a cyanate group, thereby covalently binding to the amino group of the carrier protein CRM197. Can form. The cyanation reaction by the CDAP may be specifically terminated by adding 3 molar equivalents of glycine (glycine) solution to 1 molar equivalent of CDAP and adjusting the pH to 9.0, but is not limited thereto. The reaction solution and reaction conditions can be adjusted accordingly.

The capsular polysaccharide-carrying protein conjugates obtained can be purified by various methods. Examples of these methods include concentration / dialysis filtration processes, column chromatography and multilayer filtration. Purified polysaccharide-protein conjugates can be mixed and formulated into vaccine compositions of the invention and used respectively. Formulations of vaccine compositions of the invention can be carried out using methods known in the art. For example, 13 individual capsular polysaccharide-carrying protein conjugates can be formulated with a physiologically acceptable vehicle to make a composition. Examples of such vehicles may include, but are not limited to, water, buffered saline, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycols) or dextrose solutions.

In a specific embodiment of the present invention, 1) dissolution and hydrolysis of 13 capsular polysaccharides, 2) conjugation reaction process of each capsular polysaccharide and CRM197 using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate), 3) After the conjugation reaction was terminated, 4) ultrafiltration, 5) bactericidal filtration, and 6) adsorption, 13 or 14 capsular polysaccharide-carrying protein conjugates were prepared.

In another specific embodiment of the present invention, 1) dissolution of 14 capsular polysaccharides and 14 hydrolysis, 2) conjugation reaction of each capsular polysaccharide with CRM197 using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) 14 kinds of capsular polysaccharide-carrying protein conjugates were prepared through a process, 3) termination of conjugation reaction, 4) ultrafiltration, 5) bactericidal filtration, and 6) adsorption.

As used herein, the term “vaccine” refers to a biological agent containing an antigen that immunizes a living body, and refers to an immunogen or antigenic substance that immunizes the living body by administering it to a human or an animal to prevent infection.

The vaccine composition may further include one or more selected from the group consisting of an adjuvant, a preservative, a buffer, a cryoprotectant, a salt, a divalent cation, a nonionic detergent, and a free radical oxidation inhibitor.

The term "adjuvant" in the present invention refers to a substance used to increase the immunogenicity of the immunogenic composition of the present invention. The adjuvant is often provided to enhance the immune response, which is well known to those skilled in the art. Adjuvants suitable for increasing the effectiveness of the vaccine composition of the present invention include, but are not limited to:

(1) aluminum salts (alum) (eg, aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.);

(2) Oil-in-water emulsion formulations (with or without muramyl peptide (defined below) or other specific immune stimulants such as bacterial cell wall components), for example (a) MF59 (WO 90/14837): Contains 5% Squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally contains varying amounts of MTP-PE (although not required, see below)), Model 110Y Micro Formulated into submicron particles using a microfluidizer such as a microfluidizer (Microfluidics, Newton, Mass.), (B) SAF: 10% squalene, 0.4% Tween 80, 5% Pluronic -Block polymer L121 and thr-MDP (see below), microfluidized with a submicron emulsion, or vortexed to form a large particle size emulsion, and (c) Ribi ™ adjuvant System (RAS) (Corixa, Hamilton, MT): 2% squalene, 0. 2% Tween 80 and from the group consisting of 3-O-deacylated monophosphoryl lipid A (MPL ™) (Corixa), trehalose dimicholate (TDM) and cell wall backbone (CWS) described in US Pat. No. 4,912,094. Contains at least one bacterial cell wall component, preferably MPL + CWS (Detox ™);

(3) Saponin adjuvant, such as Quill A or STIMULON ™ QS-21 (Antigenics, Framingham, MA, US Pat. No. 5,057,540), may be used or produced from particles such as ISCOM ( Immunostimulatory complexes));

(4) bacterial lipopolysaccharides, synthetic lipid A homologues (eg, aminoalkyl glucoseamine phosphate compounds (AGP)), or derivatives or homologues thereof (commercially available from Corixa and described in US Pat. No. 6,113,918; An example of is 2-[(R) -3-tetradecanoyloxytetradecanoylamino] ethyl 2-deoxy-4-O-phosphono-3-O-[(R) -3-tetradecanoyloxy Tetradecanoyl] -2-[(R) -3-tetradecanoyloxytetradecanoylamino] -bD-glucopyranoside, which is also known as 529 (formerly also known as RC529), which is aqueous or Formulated as a stable emulsion),

(5) synthetic polynucleotides (eg oligonucleotides containing CpG motifs (US Pat. No. 6,207,646));

(6) cytokines, such as interleukins (eg, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL 12, IL-15, IL-18, etc.) Interferon (eg gamma interferon), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (MCSF), tumor necrosis factor (TNF), costimulatory molecules B7-1 and B7-2, and the like;

(7) Wild type cholera toxin (CT) or mutant cholera toxin (Ze WO) wherein, for example, glutamic acid at amino acid position 29 according to WO-2000 / 18434 is replaced with another amino acid, specifically histidine. -2002/098368 and WO-2002 / 098369), pertussis toxin (PT), or E. coli heat-labile toxin (LT), in particular LT-K63, LT-R72, CT-S109, PTK9 / G129 (WO Detoxified mutants of bacterial ADP-ribosylated toxins such as -93/13302 and WO-92 / 19265; And

(8) Complement component C3d, the same complement as the trimer.

The muramyl peptides include N-acetyl-muramil-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanine-2- (1'-2 'dipalmityl) -sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (MTP-PE) and the like, but is not limited thereto.

The aluminum salt adjuvant may be an aluminum-precipitated vaccine or an aluminum-adsorbed vaccine. Aluminum salts include hydrated alumina, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate, alhydrogel, Superfos, amphogel, aluminum hydroxide, aluminum hydroxyphosphate adjuvant (APA), Amorphous alumina, and the like, but is not limited thereto. APA refers to a suspension of aluminum hydroxyphosphate. When aluminum chloride and sodium phosphate are mixed at a ratio of 1: 1, aluminum hydroxyphosphate sulfate is precipitated, and the precipitate is made to have a size of 2 to 8 μm using a high shear mixer, followed by dialysis and sterilization with physiological saline. Can be. In one embodiment, commercially available Al (OH) ₃ (eg alhydrogel or Superfos) is used to adsorb the protein. 50 to 200 g of protein can be adsorbed per mg of aluminum hydroxide, and this ratio is dependent on the pH of the protein and the pH of the solvent. Low pI proteins bind more strongly than proteins with high pi. Aluminum salts can form antigen reservoirs that slowly release antigens for two to three weeks to nonspecifically activate macrophages, complement, and innate immune mechanisms.

As used herein, the term “preservative” means an anti-viral and / or antimicrobial agent that inhibits the growth of microorganisms in the vaccine composition, for example, chimerosal, phenoxyethanol, 2-phenoxyethanol. It may be, but is not limited to, formaldehyde, or mixtures thereof, all conventional preservatives used in the art may be used.

In addition, the vaccine composition may comprise one or more physiologically acceptable buffers. For example, when the vaccine composition is an infusion or injectable, the buffer may have buffering capacity at pH 4.0 to 10.0, specifically, pH 5.0 to 9.0, more specifically pH 6.0 to 8.0. The buffer may be selected from the group consisting of TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate, triethanolamine buffer.

In particular, when the vaccine composition of the present invention is intended for parenteral administration, the buffer may be selected from buffers suitable for USP. For example, buffers include monobasic acids such as acetic acid, benzoic acid, gluconic acid, glyceric acid, lactic acid; Dibasic acids such as aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid, tartaric acid; Polybasic acids such as citric acid and phosphoric acid; It may be selected from the group consisting of ammonia, diethanolamine, glycine, triethanolamine, TRIS and the like.

In addition, the vaccine composition of the present invention may include a nonionic detergent. For example, polysorbate 20 and polysorbate 80 in polyoxyethylene sorbitan esters (commonly called Tweens); Copolymers of ethylene oxide (EO), propylene oxide (PO), butylene oxide (BO) (eg DOWFAX ™); Oxoxynols having a different repeating number of oxy-1,2-ethanediyl groups, especially ostoxynol-9 (Triton-100); Ethylphenoxypolyethoxyethanol (IGEPAL CA-630 / NP-40); Phospholipids such as lecithin; Nonylphenol ethoxylates such as NP series; Polyoxyethylene fatty acid ethers (Brij surfactants) derived from lauryl, cetyl, stearyl, oleyl alcohols, in particular triethyleneglycol monolauryl ether (Brij 30); Sorbitan ethers, also known as SPANs, may include, but are not limited to, surfactants such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Tween 80 may be included in the emulsion and a mixture of nonionic detergents such as Tween 80 / Span 85 may be used. Combinations of polyoxyethylene sorbitan esters such as Tween 80 with octocinols such as Triton X-100 are also suitable, and combinations of Laureth 9 with Tween and or octosinol are also useful. Specifically, polyoxyethylene sorbitan esters such as Tween 80 may be used in an amount of 0.01% to 1% (w / v), in particular 0.1%; Octylphenoxy polyoxyethanol or nonylphenoxy polyoxyethanol (such as Triton X-100) may range from 0.001% to 0.1%, in particular from 0.005% to 0.02%; The polyoxyethylene ether (eg laureth 9) may comprise 0.1% to 20%, preferably 0.1% to 10%, in particular 0.1% to 1% or about 0.5%.

The composition of the present invention may be formulated in a single dose dose vial, multiple dose dose vial or prefilled syringe form, and may further comprise a physiologically acceptable carrier. Physiologically acceptable carriers used in liquid formulations include aqueous or non-aqueous solvents, suspensions, emulsions, oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, ethyl oleate. Aqueous carriers include water, alcohol / aqueous solvents, emulsions or suspensions, physiological saline, buffer solutions. Examples of oils include vegetable or animal oils, peanut oils, soybean oils, olive oils, sunflower oils, synthetic oils such as liver oils, marine oils, and lipids derived from milk or eggs. Vaccine compositions of the present invention may be isotonic, hypertonic or hypotonic, and pharmaceutical compositions administered by infusion or injection are preferably but is not limited to isotonicity. On the other hand, isotonicity or hypertension may be advantageous for storage of the composition. If the vaccine composition is hypertonic, it can be diluted to isotonic prior to administration. Isotonic agents for dilution can be ionic tonicity agents such as salts or nonionic tonicity agents such as carbohydrates. Ionic isotonic agents include, but are not limited to, sodium chloride, calcium chloride, potassium chloride, magnesium chloride, and the like. Nonionic isotonic agents include, but are not limited to, sorbitol, glycerol, and the like.

The amount of the conjugate at each vaccine dose may be chosen to be an amount that induces an immunoprotective response without significant side effects, which amount may vary depending on the serotype of pneumococcal. Specifically, the vaccine composition is a serotype 1-derived capsular polysaccharide, serotypes 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F and 23F derived capsular polysaccharides are 0.9 to 1.1 And, the capsular polysaccharide derived from serotype 6B may be 1.8 to 2.2 in content ratio, but is not limited thereto.

In addition, the capsular polysaccharide derived from serotype 2 in the vaccine composition may have a content ratio of 0.9 to 1.1 compared to the capsular polysaccharide derived from serotype 1, but is not particularly limited thereto.

As an example for preparing the conjugates, each conjugate may comprise 0.1 to 100 μg, specifically 0.1 to 10 μg, more specifically 1 to 5 μg polysaccharide. Most specifically, except for the capsular polysaccharide derived from serotype 6B, the remaining polysaccharides, that is, the capsular polysaccharides derived from serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, and 23F, respectively, 2.2 μg, and the capsular polysaccharide derived from serotype 6B may be 4.4 μg, but is not limited thereto. In this case, CRM197 protein in the composition may be 0.1 to 100 μg, specifically 1 to 50 μg, more specifically 28 to 31 μg, most specifically 29.3 μg, but is not limited thereto. Optimal amounts of ingredients for a particular vaccine can be identified by standard studies involving the observation of an appropriate immune response in a subject. For example, the results of animal experiments can be extrapolated to determine the vaccination dose for humans. Those skilled in the art can also empirically determine the dose as needed.

The vaccine composition may further include aluminum element and sodium chloride, but is not limited thereto. In addition, the vaccine composition may or may not contain a preservative depending on the purpose and use thereof.

The vaccine composition according to the present invention can be used to protect a subject susceptible to pneumococcal and to prevent pneumococcal disease by administering a pharmaceutically effective amount in a systemic or mucosal route. The term "prevention" of the present invention refers to any action that inhibits or delays the infection caused by pneumococcal by administration of the vaccine composition of the present invention. As defined herein, a "pharmaceutically effective amount" refers to a dosage required to elicit an antibody that is capable of significantly reducing the probability of infection or the severity of infection. The term "administration" of the present invention refers to the introduction of certain substances into an individual in any suitable way. The vaccine composition of the present invention may be administered by inhalation route through oral, nasal, rectal, transdermal or aerosol, but may be administered by bolus or infused slowly, but is not limited thereto. The administration may be by injection via an intramuscular, intraperitoneal, intradermal or subcutaneous route; Or mucosal administration to the oral / digestive tract, airway or urogenital tract. In one embodiment, intranasal administration can be used for the treatment of pneumonia or otitis media, in which case more effective prevention of nasopharyngeal carriers of pneumococci can attenuate the infection at an early stage.

The term “individual” of the present invention means a living organism to which a pathogen can be infected, and in particular, may be a higher vertebrate, and more specifically, a mammal, but is not particularly limited thereto.

In another embodiment of the present invention, the composition of the present invention may be administered in a single inoculation, or two, three, four or more times at appropriate intervals, but is not limited thereto. For example, routine inoculation plans for infants and newborns for invasive diseases caused by Streptococcus pneumoniae can be 2, 4, 6 and 12 to 15 months of age.

In addition, the composition may further comprise one or more proteins from Streptococcus pneumoniae. Examples of Streptococcus pneumoniae proteins suitable for inclusion may include all of the proteins identified in WO-2002 / 053855, as well as the proteins described in WO-2002 / 053761, within the scope of the present invention.

In a specific embodiment of the present invention, 2.2 μg of each polysaccharide in a total of 0.5 mL, except that 6B is 4.4 μg; About 29.3 μg CRM197 transport protein; 0.5 mg of elemental aluminum (2 mg aluminum phosphate) adjuvant; About 4.25 mg sodium chloride (without preservatives) or about 3.5 mg (with preservatives); About 295 μg succinate buffer; About 3 mg of 2-phenoxyethanol and about 60 μg of formaldehyde were mixed (including preservatives) to prepare a vaccine composition (named 'LBVE013') for the prevention of 13-valent pneumococcal disease.

In another specific embodiment of the present invention, 2.2 μg of each polysaccharide in a total of 0.5 mL, except that 6B is 4.4 μg; About 31 μg CRM197 transport protein; 0.5 mg of elemental aluminum (2 mg aluminum phosphate) adjuvant; About 4.25 mg sodium chloride (without preservatives) or about 3.5 mg (with preservatives); About 295 μg succinate buffer; About 3 mg of 2-phenoxyethanol and about 60 μg of formaldehyde were mixed (with preservatives) to prepare a vaccine composition for the prevention of 14-valent pneumococcal disease.

In another specific embodiment of the present invention, serum levels of rabbits inoculated with the vaccine composition confirmed higher serotype-specific IgG concentrations than Prevena 13 ^® (Table 1 and Table 2). In addition, it was confirmed that the functional immunogenicity test (Opsonophagocytic assay) shows a superior effect than the prebena 13 ^® (Table 3 and Table 4). Thus, the vaccine composition of the present invention was found to have a very good effect for the prevention of pneumococcal disease.

Another aspect of the invention is an immunogenic composition against pneumococci comprising 13 or 14 species of the capsular polysaccharide-carrying protein conjugate.

The 13 or 14 conjugates and pneumococci are as described above.

The composition comprising 13 capsular polysaccharide-carrying protein conjugates of the present invention comprises Streptococcus pneumoniae- derived capsular polysaccharides having 13 different serotypes, which are recognized as antigens when administered to the body. By causing an immune response to produce an antibody against it, it can be used as an immunogenic composition against pneumococcal.

Another aspect of the invention is a method of preventing pneumococcal disease by administering the vaccine composition or immunogenic composition to a subject in need thereof.

Another aspect of the invention comprises 13 capsular polysaccharide-carrier protein conjugates, wherein the 13 conjugates are Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, Each of 13 capsular polysaccharides derived from 18C, 19A, 19F and 23F is covalently conjugated to a carrier protein, the carrier protein is CRM197 protein, and the conjugate is used to obtain the capsular polysaccharide and carrier protein using a cyanation method. A composition having a structure linked by -OC (NH) -NH- groups is provided for use in the preparation of a vaccine composition for the prevention of pneumococcal disease.

Another embodiment of the present invention comprises 14 capsular polysaccharide-carrier protein conjugates, wherein the 14 conjugates are Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, Each of the 14 capsular polysaccharides from 14, 18C, 19A, 19F, and 23F is covalently conjugated to a carrier protein, the carrier protein is CRM197 protein, and the conjugate is capsular polysaccharide and transport using a cyanylation method. It is to provide a use for the use of the composition in the manufacture of a vaccine composition for the prevention of pneumococcal disease, wherein the protein has a structure linked by -OC (NH) -NH- group.

Vaccine compositions, immunogenic compositions, and prevention of pneumococcal disease are as described above.

Another embodiment of the present invention provides a method for screening each of 13 isolated capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. A method of preparing the immunogenic composition comprising the step of conjugating the capsular polysaccharide and the transport protein to the carrier protein CRM197 to have a structure linked by -OC (NH) -NH- group using a nilation method.

Other embodiments of the invention each comprise 14 isolated capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. It is a method for producing the immunogenic composition comprising the step of conjugating the capsular polysaccharide and the transport protein to the carrier protein CRM197 to have a structure linked by -OC (NH) -NH- group using a cyanylation method.

Immunogenic compositions and methods for their preparation are as described above.

The composition according to the present invention comprises a capsular polysaccharide derived from pneumococcal having 13 different serotypes, or includes a capsular polysaccharide derived from serotype 2 pneumococcal in addition to 13 capsular polysaccharides, which is a CRM197 protein carrier. By conjugation using the cyanation method, excellent serum IgG titers and functional antibody activity can be induced. Therefore, the vaccine composition and the immunogenic composition according to the present invention can be usefully used to prevent diseases caused by pneumococci in infants, infants, children, and adults.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example  One: Streptococcus New Monica  ( Streptococcus Pneumoniae Capsular Polysaccharide Preparation

1-1. Cell Bank Manufacturing

US CDC (Center of Trustees for Streptococcus pneumoniae) with 14 different serotypes (1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F) for Disease Control and Prevention (US) and prepared a cell bank in the following manner.

Streptococcus pneumoniae strains were smeared on blood agar medium to identify pneumococci and the existing media components were removed. Among the 10 or more single colonies, a single good growing colony was selected, inoculated and cultured in a liquid medium containing no animal-derived components, and a research cell bank (RCB) containing synthetic glycerol was prepared.

Master cell bank was prepared by taking out one vial from the cell bank for which the expression of polysaccharides with unique serotypes was confirmed, proliferating the cells in a liquid medium containing no animal-derived components, and adding synthetic glycerol. One vial was taken out of the bank to proliferate cells in a liquid medium containing no animal-derived components, and then synthetic glycerol was added to prepare a cell bank for production. The prepared cell banks were stored under ultra-freezing conditions of -70 deg.

1-2. Fermentation and Polysaccharide Separation

One vial in the cell bank for preparation was thawed and inoculated into a liquid medium containing no animal-derived components. Species were cultured at 37 ± 2 ° C in an unstirred state until reaching a constant cell density (Optical Density, OD ₆₀₀ ). After confirming the contamination of the culture medium is completed inoculation was inoculated into a fermentor containing a liquid medium containing no components derived from the animal.

Then, the main culture was performed while maintaining the pH of the medium by using sterilized potassium hydroxide solution with minimal stirring at 37 ± 2 ° C. Sampling was carried out after 2 hours after incubation to determine the cell concentration in the culture medium and the glucose concentration in the medium. The culture was terminated when glucose in the medium was depleted.

After incubation, an appropriate amount of sterile 12% sodium deoxycholate was added to the culture to a final 0.12% to lyse the cells and release the polysaccharides bound to the cells.

1-3. Purification of Capsular Polysaccharides

Phosphoric acid was added to the sample treated with sodium deoxycholate for a long time, and the supernatant was recovered by centrifugation. The recovered supernatant was passed through a depth filter and then buffer exchanged with concentrated and phosphate buffer. After the buffer exchange, the sample was passed through an active carbon filter, and then impurities were removed by the following two methods.

1) In the case of 12 serotypes 1, 2, 3, 4, 5, 6A, 6B, 9V, 18C, 19A, 19F and 23F, CTAB (cetyltrimethylammonium bromide) can be ion-bonded, so the CTAB process was performed. CTAB treatment, centrifugation, sodium chloride (NaCl) and sodium iodide (NaI) treatment, and centrifugation were performed.

2) Two serotypes 7F and 14 that do not react with CTAB were reacted with the addition of an aluminum phosphate gel (Algel) solution, and then the supernatant obtained through centrifugation was used.

After completing the two types of impurities removal process, the sample was subjected to a depth filter and ultrafiltration (UF / DF), and then stored in a raw form while controlling the amount of ethanol and sodium chloride.

Example  2: Streptococcus New Monica  Capsular Polysaccharides Protein Conjugate Preparation

2-1: Reductive Amination Process Cyanylated  Joining method comparison

Using the serotype 9V purified polysaccharide and CRM197 carrier protein, a conjugation method of reductive amination and cyanylation was attempted to compare the conjugation yields of the two conjugation methods. Specific bonding process is as follows.

(1) reductive amination

11.7 mg of sodium periodate was added to a 9V polysaccharide stock solution to activate the polysaccharides while stirring at 21 to 25 ° C. Oxidized polysaccharides were concentrated and diafiltered using a 100 KDa ultrafiltration filter and WFI, and the remaining polysaccharides were lyophilized by mixing CRM197 with saccharides / CRM197 = 0.5. Lyophilized complexes were thawed and stabilized (equilibrated) at 21-25 ° C. The equilibrated complex was dissolved by incubation (37 ± 2 ° C.) in sodium phosphate (Na ₃ PO ₄ ) buffer solution at a rate of 0.1 M per 20 g of saccharides, followed by addition of cyanoborohydride (100 mg / mL). The conjugation reaction was started. After incubation at 37 ± 2 ° C. for about 44-52 hours, the temperature was lowered to 23 ± 2 ° C. and 1 mL of 0.9% NaCl solution was added to the reactor. Sodium borohydride solution (100 mg / mL) is added to 1.8 to 2.2 molar equivalents of sodium borohydride per mole of saccharide and the mixture is stirred at 23 ± 2 ° C. and incubated to remove any unreacted aldehyde present in the saccharide. Was reduced. The mixture was diluted with 5 mL of 0.9% aqueous sodium chloride solution and the diluted conjugate mixture was diafiltered using a 100 kDa MWCO membrane.

(2) cyanylation

A 2M NaCl polysaccharide solution was prepared by adding sodium chloride powder to a 9V polysaccharide stock solution prepared without hydrolysis. In order to activate polysaccharide, CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) was dissolved at a ratio of 0.5 w / w% to polysaccharide, added to a 9V polysaccharide solution, and stirred for 15 minutes to induce a polysaccharide activation reaction. The sodium hydroxide solution was then raised until the pH was 9.5 ± 0.1 ° C. and then stirred for 3 minutes so that the hydroxyl groups of the polysaccharides could be sufficiently activated by CDAP. CRM197 was added at a ratio of CRM197 1.0 w / w% to polysaccharide in the polysaccharide solution that had undergone polysaccharide activation, and the conjugation reaction was performed at room temperature for 1 hour. The conjugation reaction was terminated by adding 2 M glycine solution in 3 molar equivalents to 1 molar equivalent of CDAP and incubating overnight at room temperature with pH adjusted to 9.0. The terminated conjugate was concentrated and diafiltered into an ultrafiltration filter through a buffer containing 0.9% sodium chloride.

As a result, it was confirmed that the conjugate produced by the cyanylation method is 4 times or more in yield than the conjugate produced by the reductive amination method. Thus, we prepared conjugates using the capsular polysaccharide and the cyanylation method with CRM197.

2-2. Conjugation and Purification of Conjugates

Preparation of the conjugate of the capsular polysaccharide and CRM197 of Streptococcus pneumoniae was carried out through the following six steps.

Step 1-1. Dissolution and Hydrolysis of 13 Capsular Polysaccharides

The original capsular polysaccharide originated from each serotype was dissolved in water for injection so that the final concentration range was within the range described below and filtered through a 0.45 μm filter.

Specifically, it was dissolved in the range of 0.8-2.0 mg / ml for serotypes 1, 3 and 4, 4-8 mg / ml for serotypes 5, 6B, 9V, 18C and 19F, and 8 for serotypes 6A and 19A. Filtration proceeded with 12 mg / mL, 2-4 mg / mL for serotypes 7F, 14 and 23F.

Hydrolysis was performed by incubating the solution at the pH and temperature ranges described below for each serotype. Specifically, phosphoric acid at 70-80 ° C. overnight for serotypes 1, 3, 5, 6B, 7F, 14 and 23F, 70-80 ° C. for 1-4 hours for serotypes 6A and 19F, for serotypes 9V and 18C. The solution was incubated at pH 2.0, 65-80 ° C. for 1-3 hours. The hydrolysis was then stopped by cooling to 21-24 ° C. and adding sodium hydroxide to a target pH of 6.0 ± 1.0. Serotypes 4 and 19A did not undergo hydrolysis.

Step 1-2. Dissolution and Hydrolysis of 14 Capsular Polysaccharides

The original capsular polysaccharide originated from each serotype was dissolved in water for injection so that the final concentration range was within the range described below and filtered through a 0.45 μm filter.

Specifically dissolved in the range of 0.8-2.0 mg / ml for serotypes 1, 2, 3 and 4, 4-8 mg / ml for serotypes 5, 6B, 9V, 18C and 19F, serotypes 6A and 19A Was dissolved in the range of 8-12 mg / ml, 2-4 mg / ml for 14 and 23F, and in the range of 6-10 mg / ml for serotype 7F, followed by filtration.

In general, the immunogenicity of conjugate vaccines is known to be subject to immune interference induced by interactions between polysaccharide or carrier proteins involved (Dagan R et al . (2010) Vaccines, 28). (5513) Glycoconjugate vaccines and immune interference: A review. In order to prevent such degradation of the immunogenicity in the 14-valent vaccine, the present inventors applied the 13-valent conjugate vaccine and other polysaccharide hydrolysis methods in serotypes 3, 4, 6A, and 7F as follows. The joining process was optimized.

Hydrolysis of the polysaccharide was carried out by incubation of the solution in the pH and temperature ranges described below for each serotype. Specifically, overnight at 70-80 ° C for serotypes 1, 2, 4, 5, 6A, 6B, 14 and 23F, 70-80 ° C for 1-4 hours for serotype 19F, serotypes 3, 7F, 9V and 18C In the case of using a phosphoric acid solution, the incubation process was performed at pH 2.0, 65-80 ℃ for 1-3 hours. The hydrolysis was then stopped by cooling to 21-24 ° C. and adding sodium hydroxide to a target pH of 6.0 ± 1.0. 19A did not proceed with hydrolysis.

Step 2. Conjugation Reaction Process of Capsular Polysaccharide and CRM197

Sodium chloride powder was added to all serotypes to prepare a 2 M NaCl polysaccharide solution. For each serum, the appropriate CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) was dissolved at a rate of 1 g of CDAP per 100 ml of 50/50 acetonitrile / water for injection (v / v) solution. Specifically, 1 w / w% of CDAP relative to polysaccharide for serotypes 6A and 14, 2 w / w% for serotypes 2 and 4, and 3 w / w for serotypes 1, 3, 6B, 7F, and 19A. % Was dissolved at 4 w / w% ratio for serotypes 5, 9V, 18C, 19F, 23F and added to each polysaccharide solution. After 1 to 3 minutes, sodium hydroxide solution was added to raise the pH to 9.4 to 9.7, followed by stirring for 3 to 7 minutes so that the hydroxyl groups of the polysaccharides could be sufficiently activated by CDAP. Conjugation reaction was performed for 1 hour to 4 hours by adding CRM197 0.5-1.0 w / w% to polysaccharide to each serotype polysaccharide solution. The reaction conversion was measured using HPLC-SEC, and CDAP was added as needed.

Step 3. Termination of conjugation reaction

The reaction was terminated by adding 3-6 molar equivalents of glycine solution relative to 1 molar equivalent of CDAP added for all serotypes and adjusting the pH to 9.0. The conjugation solution was stirred at 21-24 ° C. for one hour and then stored overnight at 2-8 ° C. low temperature.

Step 4. Ultrafiltration

The diluted conjugate mixture was concentrated and diafiltered into an ultrafiltration filter using at least 20 volumes of buffer. Here the buffer was maintained in the range of pH 5.5 to 6.5, and a buffer containing 0.9% sodium chloride was used. Fractional molecular weight of the ultrafiltration filter was carried out using 300 kDa in all serotypes, and the permeate was discarded.

Step 5. Sterile Filtration

The residue after diafiltration was diluted to less than 0.4 g / L based on polysaccharide concentration using a buffer and filtered through a 0.22 μm filter. The filtered product was subjected to in-process control (sugar content, residual DMAP). In-process controls were performed on the filtered residue to determine if further concentration, diafiltration and / or dilution were needed.

Step 6. Adsorption

Aluminum salts (mainly aluminum phosphate) were added to the sterile filtrate so that the final concentration was 1 mg / mL based on aluminum ions, and extra salts were added to maintain a pH range of 5.5 to 6.5. After the adsorption, the stock solution was subjected to quality inspection to confirm quality suitability, and refrigerated at 2 to 8 ° C. until use.

Example  3. Formulation of Multivalent Pneumococcal Conjugate Vaccine

3-1. 13-valent pneumococcal conjugate vaccine ( LBVE013 Formulation of

The required amount of final bulk concentrate was calculated based on batch volume and bulk polysaccharide concentration. The required amount of 0.85% sodium chloride, succinate buffer, 2-phenoxyethanol and formaldehyde were added to a previously labeled formulation vessel, followed by the bulk concentrate. Then it was thoroughly mixed and filtered through a 0.22 μm filter. The formulated bulk liquid was slowly mixed, then bulk aluminum phosphate was added and mixed well. pH was checked and adjusted if necessary. The formulated bulk product was stored at 2-8 ° C. The resulting vaccine composition (hereinafter referred to as 'LBVE013') contains 2.2 μg of each polysaccharide in 0.5 mL total, except that 6B is 4.4 μg; About 29.3 μg CRM197 transport protein; 0.5 mg of elemental aluminum (2 mg aluminum phosphate) adjuvant; About 4.25 mg sodium chloride; About 295 μg succinate buffer; It contains about 3 mg of 2-phenoxyethanol and about 60 μg of formaldehyde (applicable when preservatives are added).

3-2. 14-valent pneumococcal conjugate vaccine ( LBVE014 Formulation of

As in the preparation of LBVE013, the required amount of the final bulk concentrate was calculated based on the batch volume and bulk polysaccharide concentration. The required amount of 0.85% sodium chloride, succinate buffer, 2-phenoxyethanol and formaldehyde were added to a previously labeled formulation vessel, followed by the bulk concentrate. Then it was thoroughly mixed and filtered through a 0.22 μm filter. The formulated bulk liquid was slowly mixed, then bulk aluminum phosphate was added and mixed well. pH was checked and adjusted if necessary. The formulated bulk product was stored at 2-8 ° C. The resulting vaccine composition (hereinafter referred to as 'LBVE014') contains 2.2 μg of each polysaccharide in 0.5 mL total, except that 6B is 4.4 μg; About 31 μg CRM197 transport protein; 0.5 mg of elemental aluminum (2 mg aluminum phosphate) adjuvant; About 4.25 mg sodium chloride; About 295 μg succinate buffer; It contains about 3 mg of 2-phenoxyethanol and about 60 μg of formaldehyde (applicable when preservatives are added).

Example  4. LBVE013  And Of LBVE014  Immunogenicity Assessment

Next, a study was conducted to evaluate whether the multivalent pneumococcal vaccine composition (LBVE013, LBVE014) prepared in Example 3 has an ability to induce an immune response in a laboratory rabbit. This immunogenicity was confirmed by antigen-specific ELISA for serum IgG concentration and by Opsonophagocytic assay (OPA) for the production of functional antibodies.

Formulated LBVE013, LBVE014 or Controlbena13 ^® as a control human dose (2.2 μg of polysaccharide, exception: 6B 4.4 μg) into the muscles of New Zealand White rabbits at Parking 0, Parking 2 and Parking 4 Immunizations were made and serum was taken at two week intervals after inoculation. The results of IgG measurement using ELISA for the collected serum are shown in Table 1 and Table 2. This will be described in detail as follows.

4-1. Serotype specific IgG  Concentration measurement

Capsular polysaccharides for each of the 13 or 14 serotypes were treated in 96-well plates at 5 μg / well and coated for 16 hours at room temperature. In order to minimize non-specific antigen-antibody reactions, serum of each individual was reacted with C-PS 333.3 ㎍ / mL and serotype 22F capsular polysaccharide (PnPs 22F) at 333.3 ㎍ / mL for 30 minutes at room temperature, followed by Tween 20. Diluted with appropriate dilution buffer. The coated plate was washed four times with washing buffer, 50 μl of previously adsorbed and diluted serum was added to the coated well-plate and allowed to react at room temperature for 1 hour. The reacted well-plates were washed four times in the same manner, and each well was added with goat anti-Rabbit IgG-HRP conjugates (1: 20000), followed by reaction at room temperature for 30 minutes. I was. The plate was washed four times in the same manner as above, and 100 μl of the TMB solution stabilized at room temperature was added to each well, followed by reaction at room temperature for 15 minutes. 100 μl of 1 N sulfuric acid solution (Sulfuric acid solution) was added to stop the reaction, and the absorbance was measured at 450 nm and 650 nm. For objective immunogenicity evaluation, blood samples obtained by immunizing prevena 13 ^® as a control were analyzed together.

As a result, it was unexpectedly confirmed that the serotype immunogenic response pattern was clearly different from that of Prevena 13 ^® (Tables 1 and 2), and all rabbits inoculated with LBVE13 and LBVE14 of the present invention. Higher titers were found in serotypes than Prevena13 ^® (Table 1 and Table 2). In particular, in LBVE013, serotypes 1, 6B, 7F, 9V 14, and 19F showed two to six times better effects than prevena13 ^® (Table 1), and in the case of LBVE014, serum levels increased Forms 1, 4, 5, 7F, 9V, 14, 18C, 19A and 19F showed 2-6 times better effect than Prevena 13 ^® (Table 2).

LBVE013 Prevena 13 ^® Serotype IgG (μg / mL) Serotype IgG (μg / mL) 19F 82.4 6A 46.5 6A 64.7 6B 22.4 14 61.1 23F 18.3 6B 59.5 19F 17.6 19A 26.7 19A 17.2 23F 21.8 14 10.1 7F 13.4 5 8.4 5 11.4 4 6.7 4 9.1 7F 6.3 One 5.3 18C 4.4 18C 4.7 3 2.7 3 3.0 One 1.6 9 V 2.7 9 V 1.2

LBVE014 Prevena 13 ^® Serotype IgG (μg / mL) Serotype IgG (μg / mL) 18C 157.3 23F 35.6 19A 120.3 6A 34.3 23F 75.7 18C 26.5 7F 65.6 6B 21.2 19F 56.2 19A 18.9 9 V 54.8 7F 12.3 6B 50.0 19F 10.2 6A 48.0 4 8.9 2 42.6 14 7.9 14 38.6 5 7.8 4 38.5 9 V 6.8 5 29.5 One 3.6 One 10.8 3 0.8 3 2.7 2* 0.4

* Prevena 13 ^® Does not contain serotype 2

4-2. Functional immunogenicity test Opsonophagocytic  assay, OPA )

Serum obtained from rabbits was subjected to OPA analysis to evaluate the functionality of antibodies induced by the serotypes of LBVE013 and LBVE014.

Specifically, the same amount of serum was taken for each individual and the same groups were pooled. Streptococcus Pneumoniae was incubated in THY medium (Todd-Hewitt Broth w / 2% Yeast Extract) for each serotype and diluted with Opsonization buffer to 200 to 300 CFU / 10 μl. 20 μl of diluted serum and 10 μl of diluted Streptococcus pneumoniae were mixed and reacted at room temperature for 30 minutes. Then, 50 μl of a mixture of pre-differentiated HL-60 cells and complement (cells: complement = 4: 1) was added and reacted for 45 minutes in a CO ₂ incubator (37 ° C.). Phagocytosis was stopped by lowering the temperature, and 10 μl of the reaction solution was plated in dried agar medium for 30 to 60 minutes in advance. Then, the cells were incubated for 12 to 18 hours in a CO ₂ incubator (37 ° C.) and the number of colonies was counted. OPA titers were expressed as dilution folds where 50% killing was observed (Table 3; OPA titers labeled 2187 indicate that the titers were very high, even in the most diluted sections, where the 50% level was not reached compared to the negative control). .

Serotype OPA titier LBVE013 / Prevena 13 ^® LBVE013 Prevena 13 ^® One 208 44 4.7 3 218 190 1.1 4 1775 836 2.1 5 493 228 2.2 6A 1606 1375 1.2 6B 1751 784 2.2 7F 2187 2187 1.0 9 V 1032 512 2.0 14 2187 1761 1.2 18C 1222 1035 1.2 19A 2187 1468 1.5 19F 2187 847 2.6 23F 752 320 2.4

Therefore, through the above test results, it was confirmed that LBVE013, the thirteen immunogenic composition of the present invention, has a significantly superior functional antibody compared to Prevena 13 ^® . Therefore, the immunogenic composition according to the present invention can be very useful for preventing diseases caused by pneumococci.

Serotype OPA titier LBVE014 / Prevena 13 ^® LBVE014 Prevena 13 ^® One 1626 392 4.1 2 3729 20 186.5 3 1853 801 2.3 4 6798 3395 2.0 5 7357 904 8.1 6A 11207 4623 2.4 6B 16694 7001 2.4 7F 7811 2215 3.5 9 V 4543 1178 3.9 14 5991 2001 3.0 18C 14327 1587 9.0 19A 16131 2067 7.8 19F 5941 1205 4.9 23F 6636 5351 1.2

In addition, when looking at the results of the above (Table 4), it was confirmed that the functional antibody value is significantly superior to the prebena 13 ^{® in the} case of LBVE014 as in LBVE013. Therefore, the immunogenic composition according to the present invention can also be very useful for preventing diseases caused by pneumococcal.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (13)

13 capsular polysaccharide-carrying protein conjugates, Wherein the 13 conjugates are 13 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. each of the capsular polysaccharide is covalently conjugated to the carrier protein, The carrier protein is CRM197 protein, The conjugate has a structure in which the capsular polysaccharide and the carrier protein are connected by -O-C (NH) -NH- group using a cyanation method, Vaccine composition for the prevention of pneumococcal disease. The method of claim 1, wherein the composition further comprises a conjugate in which the capsular polysaccharide derived from Streptococcus pneumoniae serotype 2 is covalently conjugated to CRM197 protein, which is a carrier protein, The conjugate is characterized by having a structure in which the capsular polysaccharide and the carrier protein are connected by -O-C (NH) -NH- group using a cyanylation method, The composition further comprises a conjugate wherein the capsular polysaccharide derived from Streptococcus pneumoniae serotype 2 is covalently conjugated to CRM197 protein, which is a carrier protein. Characterized in that it comprises 14 kinds of capsular polysaccharide-carrying protein conjugates, Vaccine composition for the prevention of pneumococcal disease. The method according to claim 1 or 2, wherein the cyanylation method is performed using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or CNBr. Vaccine composition for the prevention of pneumococcal disease. 3. The composition of claim 1, wherein the composition further comprises at least one selected from the group consisting of adjuvant, preservative, buffer, cryoprotectant, salt, divalent cation, nonionic detergent and free radical oxidation inhibitor. Vaccine composition for the prevention of pneumococcal disease. The method of claim 4, wherein the adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate, aluminum hydroxide, and mixtures thereof. Vaccine composition for the prevention of pneumococcal disease. The vaccine composition of claim 4, wherein the preservative is phenoxyethanol, formaldehyde, or a mixture thereof. The method according to claim 1 or 2, The vaccine composition further comprises a physiologically acceptable carrier, Vaccine composition for the prevention of pneumococcal disease. 3. The capsular polysaccharide of claim 1, wherein the composition is serotype 1-derived. Capsular polysaccharides derived from serotypes 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F and 23F are 0.9 to 1.1, respectively, Capsular polysaccharide derived from serotype 6B has a content ratio of 1.8 to 2.2, Vaccine composition for the prevention of pneumococcal disease. The vaccine composition of claim 2, wherein the composition comprises a capsular polysaccharide derived from serotype 2 relative to a capsular polysaccharide derived from serotype 1 in a content ratio of 0.9 to 1.1. 13 capsular polysaccharide-carrying protein conjugates, Wherein the 13 conjugates are 13 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F ( each capsular polysaccharide is covalently conjugated to a carrier protein, The carrier protein is CRM197 protein, The conjugate has a structure in which the capsular polysaccharide and the carrier protein are connected by -O-C (NH) -NH- group using a cyanation method, Immunogenic Compositions against Pneumococci. 11. The composition of claim 10, wherein the composition further comprises a conjugate wherein the capsular polysaccharide derived from Streptococcus pneumoniae serotype 2 is covalently conjugated to CRM197 protein, which is a carrier protein. The conjugate is characterized by having a structure in which the capsular polysaccharide and the carrier protein are connected by -O-C (NH) -NH- group using a cyanylation method. The composition further comprises a conjugate wherein the capsular polysaccharide derived from Streptococcus pneumoniae serotype 2 is covalently conjugated to CRM197 protein, which is a carrier protein, Characterized in that it comprises 14 kinds of capsular polysaccharide-carrying protein conjugates, Immunogenic Compositions against Pneumococci. 13 isolated capsular polysaccharides from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F, respectively. Using a cyanylation method to conjugate the capsular polysaccharide and the transport protein to the transport protein CRM197 to have a structure linked by -OC (NH) -NH- group, A method for preparing the immunogenic composition of claim 10. 14 isolated capsular polysaccharides from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F ) Using a cyanylation method to conjugate each of the capsular polysaccharides and the transport protein with a -OC (NH) -NH- group to a transport protein CRM197. A method for producing the immunogenic composition of claim 11.