KR20180046893A - A multivalent immunogenic composition with improved IgG titer and use thereof - Google Patents

A multivalent immunogenic composition with improved IgG titer and use thereof Download PDF

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KR20180046893A
KR20180046893A KR1020170140845A KR20170140845A KR20180046893A KR 20180046893 A KR20180046893 A KR 20180046893A KR 1020170140845 A KR1020170140845 A KR 1020170140845A KR 20170140845 A KR20170140845 A KR 20170140845A KR 20180046893 A KR20180046893 A KR 20180046893A
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derived
composition
capsular polysaccharide
serotype
polysaccharide
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KR1020170140845A
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Korean (ko)
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김태현
제훈성
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주식회사 엘지화학
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/09Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505Inorganic adjuvants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/60Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031Proteins

Abstract

More particularly, the present invention relates to a vaccine composition for the prevention of pneumococcal disease comprising a capsular polysaccharide-carrying protein conjugate and an immunogenic composition for pneumococcal composition .

Description

A multivalent immunogenic composition with improved IgG titer and use < RTI ID = 0.0 >

The present invention relates to a multivalent immunogenic composition having improved IgG titers and uses thereof, and more particularly to a vaccine composition for the prevention of pneumococcal disease including a capsular polysaccharide-carrier protein conjugate, ≪ / RTI >

Streptococcus pneumonia is a major cause of meningitis, pneumonia and serious invasive disease in infants and children worldwide. More than 1.6 million people die each year from pneumococcal disease (2008, data from the International Health Organization), and the incidence of invasive infectious diseases due to pneumococcal infection in children aged <5 years and> 65 years, high.

Streptococcus pneumoniae is classified as more than 90 serotypes depending on the structural and immunological characteristics of the capsular polysaccharide, which is a major virulence factor surrounding the outside, of which about 20 to 80% It is known to be associated with pathogenicity. The only host of Streptococcus pneumoniae is human beings and they are usually populated by healthy nasopharynx (20-40% of infants, 5-10% of adults). In 2005, the Centers for Disease Control and Prevention (CDC), in developing countries, estimated that 2.1 million children under age 5 died of pneumonia per year, of which 1.2 million died of pneumococcal infection (JAMA 2007; 297: 1825-6; Invasive pneumococcal disease) in the United States, and in the United States, meningitis and sepsis caused by pneumococci have occurred in about 3,000 and 50,000 cases per year, respectively (Peters TR, Poehling KA et al . . According to the pneumoACTION database, which is a database of pneumococcal disease, 2000 cases of pneumococcal infectious disease occurred in Korea in 24,047 cases per year, of which 47 cases were death (www.pneumoadip.org). Furthermore, according to a recent study by the Center for Disease Control and Prevention, 'A Study on the Serotype Analysis of Pneumococci in Children and Adolescents in Korea', pneumococci were the most common cause of invasive infections (43.7%) in infants between 3 months and 59 months appear. In addition to penicillin, multidrug-resistant bacteria that are resistant to three or more drugs are increasing in pneumococci that cause invasive infectious diseases globally, thus adding to the difficulty of treating pneumococcal infection diseases. Therefore, there is a continuing need for pneumococcal vaccination for children and elderly people who are at high risk for pneumococcal disease.

To prevent pneumococcal disease, a multivalent pneumococcal polysaccharide vaccine has been developed and approved since 1977, and this clamshell polysaccharide vaccine has proved to be useful in preventing pneumococcal disease in elderly and high-risk patients. However, in infants and children, the maturity of the immune system is lower than that of adults. Therefore, if the polysaccharide vaccine alone is met, the vaccine can not be expected to play a role because the immune system does not recognize the polysaccharide antigen as an external invading factor. In order to solve the immunogenicity problem of the polysaccharide vaccine in infants and children, a 7-pneumococcal conjugate vaccine (conjugate polysaccharide-protein conjugate vaccine conjugated with a carrier protein that increases the immunogenicity of the polysaccharide antigen) 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 led to the reduction of invasive disease caused by the vaccine serotypes used in the vaccine, but also caused by the serotype replacement, Showed the disease increase phenomenon. These non-vaccine serotypes have emerged as new threats to the pathogenesis of pneumococcal disease by boosting vaccine serotypes that predominantly clustered prior to introduction of conjugation vaccines and securing the dominance of noninflammatory populations (Hanage WP2007 ; The Journal of Infectious Diseases, 196: 9, 1282; Serotype Replacement in Invasive Pneumococcal Disease: Where Do We Go from Here?). Thus, 10 the capsular polysaccharide in order to provide a wider coverage - a protein conjugate vaccine in renal flow Rix ® (Synflorix ®), and the free the base serotypes of vena ® A 13 add serotypes 6 kinds of pneumococcal conjugate vaccine Prevenar 13 ® (Prevenar13 ®) one of these has been developed, but presently on the market, resulting tried monitoring pneumococcal disease onset into after market, also 10 a and 13 a serotype other than the non-vaccine serotypes included in the vaccine (Weinberger DM et al ., 2011; Lancet, 378: 9807, 1962; Serotype replacement in disease following pneumococcal vaccination: A discussion of the evidence).

Another problem that may be encountered when developing a multivalent pneumococcal conjugation vaccine is that it can be increased by adding serotypes and is generally caused by immune interference which may be caused by interaction between the capsular polysaccharide in the vaccine or carrier proteins (Dagan R et al . (2010) Vaccines, 28 (5513); Glycoconjugate vaccines and immune interference: A review). In fact, the 13 Ga-free clinical data of the vena 13 ® also immunogenicity of serotypes common to the Prevenar ® the control group of 7 Prevenar vaccine ® (EMEA Assessment Report for Prevenar 13, 2009. EMA / 798877/2009), and therefore, this phenomenon is likely to be caused by a serotype change phenomenon, It can be an important issue to be overcome in the development of a mycobacterial vaccine.

Accordingly, the present inventors have made efforts to develop a vaccine for the prevention of pneumococcal disease having a higher and more stable IgG titer and a higher valency than the conventionally developed vaccine preparation, and as a result, A novel pneumococcal conjugation vaccine comprising one pneumococcal conjugation vaccine, specifically 17 serotypes, has been developed and the present invention has been completed. The pneumococcal conjugation vaccine of the present invention contains more than 15 kinds of snack polysaccharide antigens, specifically 17 kinds of snack polysaccharide antigens, and can have a broader serotype coverage than the known pneumococcal conjugate vaccine. Therefore, Can be prevented.

It is an object of the present invention to provide novel polymorphic compositions.

Specifically, one object of the present invention is to provide a capsular polysaccharide-carrying protein conjugate, wherein said conjugate is selected from the group consisting of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B A vaccine composition for the prevention of pneumococcal disease in which each of 15 kinds of barnyard polysaccharides derived from 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F and 23F is covalently bonded to a carrier protein, and To provide an immunogenic composition against S. pneumoniae.

More specifically, one object of the present invention is to provide a conjugate polysaccharide-carrier protein conjugate, wherein said conjugate is selected from the group consisting of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, Wherein 15 kinds of the capsular polysaccharides derived from 14, 15B, 18C, 19A, 19F and 23F are covalently bonded to a carrier protein, Of the serotypes 6A, 14, 19A and 19F were greater than 0.25 and less than 0.95, respectively, and the content of serotyped 6B-derived capsular polysaccharides was greater than 0.5 and less than 1.9 A vaccine composition for the prevention of pneumococcal disease, and an immunogenic composition for pneumococcus.

Another object of the present invention is to provide a conjugate polysaccharide-carrier protein conjugate, wherein said conjugate is selected from the group consisting of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C , 19A, 19F, 22F, 23F and 33F, each of which is covalently conjugated to a carrier protein, and a vaccine composition for the prevention of pneumococcal disease and an immunogenic composition for pneumococcus .

More specifically, another object of the present invention is to provide a composition comprising a clathrate polysaccharide-carrier protein conjugate, wherein said conjugate is selected from the group consisting of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14 , 15B, 18C, 19A, 19F, 22F, 23F and 33F, each of which is covalently conjugated to a carrier protein, wherein the composition comprises serum The comonomer polysaccharides derived from serotype 6A, 14, 19A and 19F, respectively, have a content ratio of more than 0.25 and not more than 0.95, respectively, and a serogroup polysaccharide derived from serotype 6B of more than 0.5 and less than 1.9 A vaccine composition for preventing pneumococcal disease, and an immunogenic composition for pneumococcus.

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

A further object of the present invention is to provide a method for the preparation of a conjugated polysaccharide-carrier protein conjugate comprising the steps of: (a) 18C, 19A, 19F and 23F, each of which is covalently conjugated to a carrier protein, for use in the preparation of a vaccine composition for the prevention of pneumococcal disease.

More specifically, another object of the present invention is to provide a conjugate polysaccharide-carrier protein conjugate, wherein said conjugate is selected from the group consisting of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, Wherein 15 kinds of the capsular polysaccharides derived from 14, 15B, 18C, 19A, 19F and 23F are covalently bonded to a carrier protein, Of the capsular polysaccharides from serotype 6A, 14A, 19A and 19F, respectively, have a content ratio of more than 0.25 and less than 0.95, and the content of serous polysaccharide from serotype 6B is greater than 0.5 and less than 1.9 To provide a use for use in the preparation of a vaccine composition for the prevention of pneumococcal disease.

A further object of the present invention is to provide a method for the preparation of a conjugated polysaccharide-carrier protein conjugate comprising the steps of: (a) 18C, 19A, 19F, 22F, 23F and 33F, each of which is covalently conjugated to a carrier protein, for use in the preparation of a vaccine composition for the prevention of pneumococcal disease .

More specifically, another object of the present invention is to provide a conjugate polysaccharide-carrier protein conjugate, wherein said conjugate is selected from the group consisting of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, A vaccine composition for the prevention of Streptococcus pneumoniae, wherein each of 17 kinds of snack polysaccharides derived from 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F is covalently bonded to a carrier protein, The concentration of the capsular polysaccharides derived from serotypes 6A, 14, 19A and 19F was greater than 0.25 and less than 0.95, respectively, compared to the serotyped polysaccharides derived from serotype 1, while the serotypes 6B derived from serotypes 6B exceeded 0.5 and 1.9 By weight of the composition is used for the preparation of a vaccine composition for the prevention of pneumococcal disease.

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

One aspect of the present invention for achieving the above object is a novel multivalent immunogenic composition. Specifically, one embodiment of the present invention is a vaccine composition for the prevention of S. pneumoniae disease comprising fifteen species of capsular polysaccharide-carrier protein conjugates.

Another aspect of the present invention for achieving the above object is a vaccine composition for preventing pneumococcal disease comprising 17 kinds of the capsular polysaccharide-carrier protein conjugates.

The fifteen kinds of conjugates are composed of 15 kinds of capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F and 23F Each of which is covalently bonded to the carrier protein.

In addition, in the case of the vaccine composition comprising the 17 kinds of conjugates, in addition to the above-mentioned 15 kinds of conjugates, each of the secretory polysaccharides derived from Streptococcus pneumoniae serotype 22F and 33F was covalently conjugated to each transport protein May further comprise a conjugate of the species.

Specifically, the composition is a vaccine composition comprising fifteen different polysaccharide-protein conjugates wherein each conjugate comprises a narrow-spectrum polysaccharide derived from Streptococcus pneumoniae of different serotypes conjugated to a carrier protein, Are produced from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F and 23F.

In addition, the composition is a vaccine composition comprising 17 different polysaccharide-protein conjugates. In addition to the above-mentioned 15 kinds of conjugates, the capsular polysaccharides derived from Streptococcus pneumoniae serotype 22F and 33F are covalently bonded to respective transport proteins The present invention further relates to a method of manufacturing the same.

In the present invention, as a result of increasing the 13-valent contrast, 15-valent or 17-valent valence in a polyparagonal composition containing various serotypes of the capsular polysaccharide, the overall decrease in IgG titers was confirmed, and 15 or 17 immunogenic compositions It was confirmed that the IgG titers can be actually increased by simultaneously controlling the amount of the serotypes 6A, 6B, 14, 19A and 19F, as compared to the serotyped polysaccharides derived from serotype 1. As the composition of the present invention, the capsular polysaccharides derived from serotype 6A, 14, 19A and 19F have a content ratio exceeding 0.25 and not more than 0.95, respectively, relative to the serous polysaccharide derived from serotype 1, and specifically 0.3 to 0.95, 0.9, 0.4 to 0.95, 0.4 to 0.9, 0.45 to 0.95, 0.45 to 0.9, 0.5 to 0.95, or 0.5 to 0.9. Namely, the concentration of the capsular polysaccharide derived from serum type 1 is 1: 0.3 to 0.95, 1: 0.3 to 0.9, 1: 0.4 to 0.95, 1: 0.4 to 0.9, 1: 0.95, 1: 0.45 to 0.9, 1: 0.5 to 0.95, or 1: 0.5 to 0.9. Also, the serotyped 6B-derived capsular polysaccharide may have a content ratio of greater than 0.5 and less than or equal to 1.9 relative to the serous polysaccharide derived from serotype 1. Specifically, it can show a content ratio of 0.6 to 1,9, 0.6 to 1.8, 0.8 to 1.9, 0.8 to 1.8, 0.9 to 1.9, 0.9 to 1.8, 1.0 to 1.9, or 1.0 to 1.8. Namely, the concentration of the capsular polysaccharide derived from serotype 1: serotype 6B derived capsular polysaccharide was 1: 0.6-1.9, 1: 0.6-1.8, 1: 0.8-1.9, 1: 0.8-1.8, 1: 0.9-1.9, 1: 0.9 To 1.8, 1: 1 to 1.9, or 1: 1 to 1.8. (From serotypes 3, 4, 5, 7F, 9V, 12F, 15B, 18C, 22F, 23F and 33F) other than the above-described types of capsular polysaccharides 1 ratio). The immunogenic compositions provided by the present invention comprising the capsular polysaccharide in the above contents are characterized by a specific content ratio of the above five serotypes and the serotypes 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, and 23F (in addition, 22F and 33F in the case of 17Ag) can exhibit superior immunogenicity compared to immunoconjugate compositions present in equal amounts.

Specifically, when the composition comprises 4.4 .mu.g / mL (for example, 2.2 .mu.g / dose) of serotypes 1, 3, 4, 5, 7F, 9V, 12F, 15B, 18C and 23F, , 14, 19A, and 19F were determined to be greater than 1.1 μg / mL, 4.18 μg / mL, 4.18 μg / mL, 1.32 μg / mL to 3.96 μg / mL, 1.76 μg / mL to 4.18 μg / / mL to 3.96 g / mL, 1.98 g / mL to 4.18 g / mL, 1.98 g / mL to 3.96 g / mL, 2.2 g / mL to 4.18 g / mL, or 2.2 g / mL to 3.96 g / mL , Serotype 6B is more than 2.2 μg / mL, 8.36 μg / mL, 2.64 μg / mL to 8.36 μg / mL, 2.64 μg / mL to 7.92 μg / mL, 3.52 μg / mL to 8.36 μg / ML, from 3.96 to 8.36 g / mL, from 3.96 to 7.92 g / mL, from 4.4 g / mL to 8.36 g / mL, or from 4.4 g / mL to 7.92 g / mL &Lt; / RTI &gt; concentration. However, the present invention is not limited thereto, and if it is within the range of the content ratio described above, the concentration can be adjusted according to the object to be produced.

The above composition may further include the same amount of the capsular polysaccharide derived from serotype 22F and the capsular polysaccharide derived from 33F in the same amount as the serous polysaccharide derived from serotype 1.

The term &quot; pneumonia &quot; in the present invention is a type of acute inflammatory disease of the lung parenchyma. The infectious agent is mainly Streptococcus pneumoniae and Klebsiella pneumoniae ). In particular, pneumococcal pneumonia accounts for about 50% of all pneumonia, and severe chills, fever, cough, and chest pain are present, and sputum is often a hemorrhagic complication and can cause pleurisy, meningitis, endocarditis, and peritonitis Stein GE et al .2001; Diagn Microbiol Infect, Dis 39:.. 181; Comparative serum bactericidal activity of clarithromycin and azithromycin against macrolide-sensitive and resistant strains of Streptococcus pneumoniae).

As used herein, the term &quot; pneumococcus &quot; refers to Streptococcus pneumoniae and is a commensal organism that generally colonizes the mucosal surface of human nasopharynx. If the host's factor allows access to the lower respiratory tract, a vigorous inflammatory response will follow, thereby causing dense consolidation when the alveolar space fills the exudate, Lt; / RTI &gt; The pneumococcus can synthesize more than 90 structurally unique capsular polysaccharides, and the serotype of pneumococcus is classified according to the structural and immunological characteristics of the capsular polysaccharide. Therefore, when a vaccine preparation is prepared using the capsular polysaccharide of Streptococcus pneumoniae, the immune response may be different depending on the type of the capsular polysaccharide, that is, the serotype of Streptococcus pneumoniae derived from the capsular polysaccharide. The vaccine composition of the present invention is particularly useful for the production of 15 kinds of capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F and 23F . &Lt; / RTI &gt; In addition, it can be further prepared in a 17 form including serotypes 22F and 33F-derived capsular polysaccharides.

Since the capsular polysaccharide is recognized as an antigen when administered into the body and can produce an antibody against the same, a vaccine composition for the prevention of Streptococcus pneumonia can be prepared. The term &quot; antigen &quot; in the present invention refers to a substance capable of specifically inducing an immune response when a substance enters the body. In the present invention, 15 kinds of capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F and 23F, In addition, 17 capsular polysaccharides can act as antigens, respectively, if 22F and 33F-derived capsular polysaccharides are included.

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

In a specific embodiment of the present invention, 17 different serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F) Were dissolve using sodium deoxycholate, respectively, and the polysaccharide bound to the cells was liberated. In the next 14 serotypes 1, 3, 4, 5, 6A, 6B, 9V, 12F, 15B, 18C, 19A, 19F, 22F and 23F, the CTAB process is performed because CTAB (cetyltrimethylammonium bromide) , And three serotypes 7F, 14 and 33F that did not react with CTAB were purified using Algel phosphate solution.

In case of using as a vaccine composition using only the capsular polysaccharide, infants and children, whose immune system is lower than adults, may not recognize it as an antigen and may not develop an immune reaction. Therefore, in the present invention, a conjugate form of a carrier protein and a capsular polysaccharide And used.

The term &quot; carrier protein &quot; as used herein refers to a protein that can be covalently conjugated to a capsular polysaccharide to increase the immunogenicity of the polysaccharide antigen. The transport protein can be conjugated to the capsular polysaccharide through a standard conjugation method, and the resulting capsular polysaccharide-carrier protein conjugate formed may be one in which one or more capsular polysaccharides are conjugated to one transport protein. The delivery protein may be, but is not limited to, a protein that is specifically non-toxic, non-reactive, and can be obtained in sufficient quantity and purity. In the present invention, the transport protein is not limited thereto, but may be CRM197, for example.

The term &quot; CRM197 &quot; in the present invention is a non-toxic variant (i.e., toxoid) of the diphtheria toxin isolated from the culture of Corynebacterium diphtheriae strain C7 (? 197). CRM197 can be purified through ultrafiltration, ammonium sulfate precipitation and ion exchange chromatography. The CRM197 may also be recombinantly prepared according to U.S. Patent No. 5,614,382, although not limited thereto.

The transport protein may also be selected from the group consisting of tetanus toxoid, pertussis toxoid, cholera toxoid (WO 2004/083251), E. coli LT, E. coli ST or Pseudomonas aeruginosa aeruginosa) light such as exotoxin A derived from an active bacterial toxins, bacterial outer membrane proteins, for example, outer membrane complex c (OMPC), tarpaulins, transferrin binding proteins, New Mori sour pneumococcal surface protein A (PspA), pneumococcal (C5a peptidase from group A or group B streptococci, or Haemophilus influenzae protein D, ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum But are not limited to, variants of diphtheria toxin such as albumin (BSA), purified protein derivative (PPD) of tuberculin, CRM173, CRM228, CRM45.

Any known method for preparing conjugates of the capsular polysaccharide and the carrier protein can be included within the scope of the present invention. As a specific example, in order to find a method for enhancing the immunogenicity of Streptococcus pneumoniae vaccine, the inventors of the present invention, together with a literature review, conducted a reductive amination reaction and cyanylation, two typical conjugation methods, As a result, it was confirmed that the cyanylation method is superior to the reductive amination reaction in terms of the yield of the coupling and the time required. Based on this, we tried to optimize the binding of serotype to 13 serotypes different from those of Prevenar 13 ® using the same serotype as Prevenar 13 ® , which has the highest coverage, We found that all serotype IgG titers were superior to those of Prevenar 13 ® . Thus, in the preparation of the immunogenic composition of the present invention, a binding method using a cyanylation reaction can be used. Increasing immunogenicity at the same time in all serotypes used would require a great deal of effort to those skilled in the art, as there may be a disturbance of immunity in the polymorphic composition.

Specifically, although not limited thereto, the conjugate of the present invention may have a structure in which the capsular polysaccharide and the transport protein are linked by -O-C (NH) -NH- group using a cyanylation method. Based on the structural differences from the PCV (Pneumococcal conjugate vaccine) formulations prepared by other methods and the optimization of the detailed conjugation practice, the vaccine composition of the present invention is superior to the vaccine composition conjugated by the known reductive amination methods IgG titers that are significantly better in serotype can be seen.

The cyanylation can be performed by a person skilled in the art through a known method. For example, the cyanylation can be carried out using, for example, 1-cyano-4-dimethylaminopyridinium tetrafluoroborate or CNBr.

One example for preparing a conjugate of a capsular polysaccharide and a carrier protein is to chemically activate the purified capsular polysaccharide and form glycoconjugates by joining each chemically activated capsular polysaccharide to the carrier protein one by one. The activity of cyanation by the treatment with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) changed the hydroxyl group of the capsular polysaccharide into a cyanate group, and covalently linked the amino group of the carrier protein CRM197 . The cyanation reaction by CDAP may be performed by adding 3 molar equivalents of a glycine solution to 1 equivalent of CDAP and adjusting the pH to 9.0, but the present invention is not limited thereto. Accordingly, the reaction solution and the reaction conditions can be appropriately controlled.

The resulting capsular polysaccharide-carrier protein conjugate can be purified by various methods. Examples of these methods include concentration / dialysis filtration processes, column chromatography and multi-layer filtration. The purified polysaccharide-protein conjugates may be formulated into a vaccine composition of the present invention by mixing them, and they may be used. The formulation of the vaccine composition of the present invention may be carried out using methods known in the art. For example, a composition may be formulated by formulating individual capsular polysaccharide-transfer protein conjugates together with a physiologically acceptable vehicle. Examples of such vehicles include, but are not limited to, water, buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol), or dextrose solution.

In a specific embodiment of the present invention, 1) dissolution and hydrolysis of each of the capsular polysaccharides, 2) reaction process of CRM197 with each of the narrow-spectrum polysaccharides using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) Termination of the reaction, 4) ultrafiltration, 5) bactericidal filtration, and 6) adsorption step to prepare the respective capsular polysaccharide-carrier protein conjugates.

The term &quot; vaccine &quot; in the present invention refers to a biological agent containing an antigen that immunizes a living body. The term &quot; vaccine &quot; refers to an immunogenic agent or an antigenic substance that causes immunity to a living body by administration to a human or animal for prevention of infection.

The vaccine composition may further comprise at least one member 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" as used herein refers to a substance used to increase the immunogenicity of an immunogenic composition of the 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 efficacy of the vaccine compositions of the present invention include, but are not limited to,

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

(2) a water-in-oil emulsion formulation (with or without other specific immunostimulants such as muramyl peptides (defined below) or bacterial cell wall components), such as (a) MF59 (WO90 / 14837) 5% Squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally containing various amounts of MTP-PE (but not necessarily, see below)), Model 110Y Micro (B) SAF: 10% squalene, 0.4% Tween 80, 5% pluronic. The microfluidizer was formulated as submicron particles using a microfluidizer such as a microfluidizer (Microfluidics, Newton, MA) - block polymer L121 and thr-MDP (see below), microfluidized with a submicron emulsion, or vortexed to form a large particle size emulsion, and (c) System (RAS) (Corixa, Hamilton, MT): 2% Squalene, 0. 2% tween 80 and 3-O-deacylated monophosphoryl lipid A (MPL ™) (Corixa), trehalose dimeric cholate (TDM) and cell wall skeleton (CWS) described in US Pat. No. 4,912,094 Of one or more bacterial cell wall components, preferably MPL + CWS (Detox ™);

(3) Saponin adjuvants such as Quil A or STIMULON ™ QS-21 (Antigenics, Framingham, MA, US Pat. No. 5,057,540) are used or produced from particles such as ISCOM Immunostimulatory complex));

(4) Bacteriostatic lipopolysaccharides, synthetic lipid A analogs, such as aminoalkylglucosamine amine phosphate compounds (AGP), or derivatives or homologues thereof, which are available from Corixa and are described in U.S. Patent No. 6,113,918; (R) -3-tetradecanoyloxy] ethyl 2-deoxy-4-O-phosphono-3-O - [(R) Which is also known as 529 (formerly known as RC529), which can be either male-shaped or &lt; RTI ID = 0.0 &gt; Formulated as a stable emulsion),

(5) synthetic polynucleotides such as oligonucleotides containing CpG motifs (US Patent No. 6,207,646);

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 (MCSF), tumor necrosis factor (TNF), co-stimulating molecules B7-1 and B7-2 and the like;

(7) a wild-type cholera toxin (CT) or a mutant cholera toxin in which glutamic acid at position 29 of the amino acid according to WO 2000/18434 is substituted with another amino acid, specifically histidine (WO2002 / 098368 LT-R72, CT-S109, PTK9 / G129 (see WO93 / 13302 and WO92 / 09236), pertussis toxin (PT), or E. coli heat-labile toxin (LT) / 19265); &lt; RTI ID = 0.0 &gt; a &lt; / RTI &gt; non-toxic mutant of the ADP-ribosylated toxin; And

(8) Complement component Complement such as trimer of C3d.

The muramyl peptides include N-acetyl-muramyl-L-tryonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L- -sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (MTP-PE), and the like.

The aluminum salt adjuvant may be an aluminum-precipitated vaccine or an alum-adsorbed vaccine. Aluminum salts include hydrated alumina, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate, Alhydrogel, Superfos, Ampogel, Aluminum Hydrophosphate Adjuvant (APA) Amorphous alumina, and the like, but are 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. The precipitate is sized to 2 ~ 8 ㎛ using a high shear mixer, then dialyzed with physiological saline and sterilized . In one embodiment, the protein is adsorbed using commercially available Al (OH) 3 (e.g., Alhydrogel or Superfos). 50 to 200 g of protein can be adsorbed per 1 mg of aluminum hydroxide, which is dependent on the pH of the protein and the pH of the solvent. Low pI proteins bind strongly to high pI proteins. Aluminum salts can activate the macrophage, complement, and innate immune mechanisms nonspecifically by forming an antigen reservoir that slowly releases the antigen for 2 to 3 weeks.

The term &quot; preservative &quot; in the present invention means an anti-viral and / or antimicrobial substance which inhibits the growth of microorganisms in the vaccine composition. For example, thimerosal, 2-phenoxyethanol, , Formaldehyde, or mixtures thereof, but any conventional preservative used in the art may be used without limitation.

In addition, the vaccine composition may comprise one or more, physiologically acceptable buffering agents. For example, when the vaccine composition is an infusion or injecting agent, the buffer may have a buffering capacity at a pH of 4.0 to 10.0, specifically, a pH of 5.0 to 9.0, more specifically a pH of 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, the buffer may be a monobasic acid 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; Ammonia, diethanolamine, glycine, triethanolamine, TRIS, and the like.

In addition, the vaccine composition of the present invention may comprise a nonionic detergent. For example, polyoxyethylene sorbitan esters (commonly called Tweens), especially polysorbate 20 and polysorbate 80; Copolymers of ethylene oxide (EO), propylene oxide (PO), butylene oxide (BO) (e.g. DOWFAX TM); Oxytoxynols, in particular the octyloxynol-9 (Triton-100), in which the number of repeats of the oxy-1,2-ethanediyl group is different; Ethylphenoxypolyethoxyethanol (IGEPAL CA-630 / NP-40); Phospholipids such as lecithin; Nonylphenol ethoxylate such as NP series; Polyoxyethylene fatty acid ethers (Brij surfactants) derived from lauryl, cetyl, stearyl, oleyl alcohol, especially triethylene glycol monolauryl ether (Brij 30); But are not limited to, sorbitan ethers known as SPANs, especially surfactants such as sorbitan triolate (Span 85) and sorbitan monolaurate. Tween 80 may be included in the emulsion, and mixtures of nonionic detergents such as Tween 80 / Span 85 may also be used. A combination of a polyoxyethylene sorbitan ester such as Tween 80 and an octocinol such as Triton X-100 is also suitable, and a combination of Laureth 9, Tween, and or Octocinol is also useful. Specifically 0.01% to 1% (w / v), in particular 0.1%, of polyoxyethylene sorbitan esters (e.g. Tween 80); Octylphenoxy polyoxyethanol or nonylphenoxy polyoxyethanol (e.g. Triton X-100) in an amount of 0.001% to 0.1%, in particular 0.005% to 0.02%; Polyoxyethylene ethers (such as laureth 9) may comprise from 0.1% to 20%, preferably from 0.1% to 10%, especially from 0.1% to 1% or about 0.5%.

The composition of the present invention may be formulated as a single dose vial, a multi-dose vial or a pre-field syringe, and may further comprise a physiologically acceptable carrier. Physiologically acceptable carriers for use in liquid preparations include aqueous or nonaqueous solvents, suspensions, emulsions, and oils. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, and ethyl oleate. Aqueous carriers include water, alcohol / aqueous solvents, emulsions or suspensions, physiological saline, buffer solutions. Examples of oils are synthetic oils such as vegetable or animal oils, peanut oil, soybean oil, olive oil, sunflower oil, liver oil, marine oil, and lipids obtained from milk or eggs. The vaccine composition of the present invention may be isotonic, hypertensive, or shelf stable, and pharmaceutical compositions administered by infusion or injection are preferably but not limited to isotonicity. On the other hand, isotacticity and toughness may be advantageous for the storage of the composition. If the vaccine composition is malfunctioning, it may be diluted to become isotonic before administration. An isotonic agent for dilution may be an ionic isotonic agent such as a salt or a non-ionic isotonic agent such as a carbohydrate. Ionic isotonic agents include, but are not limited to, sodium chloride, calcium chloride, potassium chloride, magnesium chloride, and the like. Non-ionic isotonic agents include, but are not limited to, sorbitol, glycerol, and the like.

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

The vaccine composition according to the present invention can be used to protect individuals susceptible to pneumococci and prevent pneumococcal disease by administering a pharmacologically effective amount in a systemic or mucosal route. The term " prophylactic " of the present invention refers to any act that inhibits or delays infection by the pneumococcus by administration of the vaccine composition of the present invention. As used herein, the term "pharmaceutically effective amount" as used herein refers to the dosage required to induce antibodies that are capable of significantly reducing the probability of infection or the severity of the infection with S. pneumoniae. The term &quot; administering &quot; of the present invention refers to the introduction of a predetermined substance into an individual by any suitable method. The vaccine composition of the present invention may be administered via the oral, nasal, rectal, transdermal or aerosol inhalation route, and may be administered by bolus or by slow infusion, but is not limited thereto. Such administration may be by intramuscular, intraperitoneal, intradermal or subcutaneous injection; Or mucosal administration to the oral / alimentary tract, airway or genitourinary tract. In one embodiment, intranasal administration can be used for the treatment of pneumonia or otitis media, which can more effectively prevent non-invasive infections of S. pneumoniae and attenuate infection at an early stage.

The term &quot; individual &quot; of the present invention means a living organism in which pathogens can be infected, specifically, a high vertebrate animal, 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 dose, or may be administered two times, three times, four times or more at appropriate intervals, but is not limited thereto. For example, for an invasive disease caused by Streptococcus pneumoniae, the routine vaccination schedule for infants and newborns may be 2, 4, 6 and 12 to 15 months of age.

In addition, the composition may further comprise one or more proteins derived from Streptococcus pneumoniae. Examples of Streptococcus pneumoniae proteins suitable for inclusion include not only the proteins described in WO2002 / 053761 but also the proteins identified in WO2002 / 083855, all within the scope of the present invention.

In a specific embodiment of the present invention 4.4 [mu] g / mL of each polysaccharide in 0.5 mL total, 6.6 [mu] g / mL in 6B; About 29.3 [mu] g CRM197 transport protein; 0.5 mg aluminum element (2 mg aluminum phosphate) adjuvant; Approximately 4.25 mg of sodium chloride (without preservatives) or about 3.5 mg (with preservatives); Succinate buffer solution about 295 쨉 g; About 3 mg of 2-phenoxyethanol and about 60 μg of formaldehyde were mixed (with preservative) to prevent pneumococcal disease of 13 (designated 'LBVE013') and 17 (named 'LBVE017') A vaccine composition was prepared. A vaccine composition was prepared in which the content of the composition was adjusted to 100% and the contents of 6A, 6B, 14, 19A and 19F, which are some serotypes, were regulated.

In a specific alternative embodiment of the present invention as in the serum of the rabbit inoculated with the LBVE013 vaccine composition, Prevenar confirm the high degree of serotype-specific IgG concentrations of more than 13 ® (Table 1), the other serotypes derived from capsular polysaccharides In addition, when 17 transverse compositions were prepared, IgG titers for each serotype were significantly reduced (FIG. 1). In contrast, the amount of serotypes 6A, 6B, 14, 19A and 19F conjugate was decreased simultaneously, and the IgG titers against all serotypes were re-measured as a result, , Especially when the above-mentioned five kinds of conjugates were reduced to 40% to 95% (FIGS. 2 and 3). Furthermore, this result shows that the 17-valent composition is only present in the 13-valent form, and the IgG titer of the 13-valent form is decreased overall as the 5 kinds of conjugates are reduced (FIG. 4).

Another aspect of the invention is an immunogenic composition for S. pneumoniae comprising a capsular polysaccharide-carrier protein conjugate.

The conjugate and pneumococci are as described above.

The composition comprising the capsular polysaccharide-carrier protein conjugate of the present invention comprises 15 or 17 different kinds of secretory polysaccharides derived from Streptococcus pneumoniae having different serotypes. When administered to the body, it is recognized as an antigen, As an immunological reaction to produce antibodies, it can be used as an immunogenic composition against S. pneumoniae.

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

Another aspect of the present invention is a method for the preparation of a vaccine composition for the prevention of pneumococcal disease comprising administering to a patient in need thereof a therapeutically effective amount of Streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B , 15C, 15C, 19A, 19F and 23F of 15 kinds of capsular polysaccharides are covalently conjugated to transport proteins.

In another aspect of the present invention, in addition to the above-mentioned 15 kinds of conjugates for use in the preparation of a vaccine composition for the prevention of pneumococcal disease, each of the secretory polysaccharides derived from Streptococcus pneumoniae serotype 22F and 33F, The present invention also provides the use of 17 kinds of conjugated polysaccharide-carrier protein conjugates further comprising two kinds of conjugates covalently bonded.

Concretely, the composition of the capsular polysaccharides derived from serotypes 6A, 14, 19A and 19F, respectively, has a content ratio of not less than 0.25 and not more than 0.95 with respect to the serous polysaccharide derived from serotype 1, , And may have a content ratio of 1.9 or less.

More specifically, in the composition, the somatic polysaccharide derived from serotype 22F and the capsular polysaccharide derived from 33F may be present in the composition in the same amount as the serous polysaccharide derived from serotype 1.

Vaccine composition, immunogenic composition, and prevention of pneumococcal disease have been described above.

The present invention provides a composition comprising a narrow-spectrum polysaccharide derived from Streptococcus pneumoniae having 15 or more different serotypes, specifically, a narrow-spectrum polysaccharide derived from Streptococcus pneumoniae having 17 different serotypes, Polysaccharides are included in optimal amounts, leading to excellent serum IgG titers and functional antibody activity. Accordingly, the vaccine composition and the immunogenic composition according to the present invention can be effectively used to prevent diseases caused by S. pneumoniae in infants, children, and adults.

Figure 1 shows IgG titers declined in most serotypes with increasing singer number from 13 (LBVE013) to 17 (LBVE017). At this time, 6A, 6B, 14, 19A, 19F (50%) at the same time, the overall IgG titer rise (recovery) phenomenon occurs. 22F, and 33F, the standard used is different from the other serotypes.
Figure 2 shows that when the amounts of serotype 6A, 6B, 14, 19A and 19F conjugates were simultaneously changed to 100%, 90%, 75%, 50%, 25%, 10% and 1% in the 17-valent composition (LBVE017) 17 shows the overall translocation pattern of each serotype. 22F, and 33F, the standard is different from the other serotypes, so they are separated from other serotypes and separately described
Figure 3 is a plot of the dose threshold setting for each serotype 6A, 6B, 14, 19A and 19F conjugate in the 17-valent composition (LBVE017), where all 17 serotypes (LBVE017) When the GMT values of the 6A, 6B, 14, 19A and 19F serotypes of the 6 types of sera were taken as the respective (lower) bases, To 95%.
Figure 4 shows that in the case of 13-valent (LBVE013), immunogenicity is reduced in most serotypes when the amount of conjugate of serotypes 6A, 6B, 14, 19A, Lt; / RTI &gt;

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these examples.

Example  One: Streptococcus Pneumoniae  Manufacture of capsular polysaccharides

1-1. Cell bank manufacturing

Streptococcus pneumoniae with 17 different serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F) The cells were obtained from the Trusted Center, Center for Disease Control and Prevention (US) (CDC), and the cell bank was prepared as follows.

Streptococcus pneumoniae strains were plated on blood agar medium to separate single colonies. A single colony with a good growth rate among 10 or more single colonies was selected and cultured in a liquid medium containing no animal-derived components, and then a research cell bank (RCB) containing synthetic glycerol was prepared.

One of the vials of the study cell bank having confirmed the expression of the polysaccharide having the inherent serotype was taken out, and the cell was grown in a liquid medium not containing an animal-derived component, and synthetic glycerol was added to prepare a master cell bank. One vial of the bank was taken out to proliferate the cells in a liquid medium containing no animal-derived components, and synthetic glycerol was added to prepare a cell bank for production. The prepared cell bank was stored at -70 ° C or lower for use in the next step.

1-2. Fermentation and polysaccharide separation

One vial of the cell bank for production was thawed and inoculated into a liquid medium containing no animal-derived components, and ending fermentation was started. The seed culture was carried out at 37 ± 2 ° C in an untreated half-body until the end of the medium-exponential growth phase reached the optical density (OD 600 ). The main fermentation was started by inoculating the culture obtained from the seed culture into a fermenter containing a liquid medium containing no animal-derived components.

The culture was then carried out at 37 +/- 2 DEG C while adjusting the pH of the medium with potassium hydroxide solution. Optimal cell density and glucose concentration in the medium were measured every 2 hours. Fermentation was terminated when the glucose in the medium was depleted.

After the fermentation was over, 12% sodium deoxycholate was added to the culture to a final concentration of 0.12% for 1 hour to dissolve the cells and free the polysaccharide bound to the cells.

1-3. Purification of capsular polysaccharides

Sodium deoxycholate was treated with phosphoric acid, and the supernatant was recovered by centrifugation. The recovered supernatant was passed through a depth filter, followed by concentration and buffer exchange with phosphoric acid buffer solution. After the buffer exchange, the sample was passed through an activated carbon filter, and impurities were removed by the following two methods.

In the case of 14 serotypes 1, 3, 4, 5, 6A, 6B, 9V, 12F, 15B, 18C, 19A, 19F, 22F and 23F, the CTAB process is possible because of ionic bonding with CTAB (cetyltrimethylammonium bromide) . CTAB treatment, centrifugation, sodium chloride (NaCl) and sodium iodide (NaI) treatment, and centrifugation were performed.

2) Three serotypes 7F, 14, and 33F, which did not react with CTAB, were reacted by adding aluminum phosphate gel (Algel) solution, and then the supernatant obtained by centrifugation was used.

After the two types of impurity removal processes were completed, the samples were subjected to a depth filter and an ultrafiltration (UF / DF) process, and then adjusted to the amount of ethanol and sodium chloride.

Example  2: Streptococcus Pneumoniae  Barrier Polysaccharide - Preparation of protein conjugates

2-1: Reducing Aminolysis Cyanylation  Comparison of bonding methods

The binding yields of the two conjugation systems were compared by using the reductive amination method and the cyanylation conjugation method using serum type 9V purified polysaccharide and CRM197 carrier protein. The concrete bonding process is as follows.

(1) Reductive amination method

11.7 mg of sodium periodate was added to the 9V polysaccharide stock solution and the polysaccharide was activated with stirring at 21 to 25 ° C. The oxidized polysaccharide was concentrated and dialyzed by ultrafiltration filter of 100 kDa and WFI, and the remaining polysaccharide was freeze-dried by mixing CRM197 with saccharide / CRM197 = 0.5. The lyophilized complex was thawed and stabilized (equilibrated) at 21-25 ° C. The equilibrated complex was incubated (37 ± 2 ° C) in a sodium phosphate (Na 3 PO 4 ) buffer solution at a rate of 0.1 M per 20 g of saccharide and dissolved, then cyanoborohydride (100 mg / To initiate the bonding reaction. After incubation at 37 ± 2 ° C for about 44 to 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 give 1.8 to 2.2 molar equivalents of sodium borohydride per 1 mol of saccharide, and the mixture is incubated at 23 +/- 2 DEG C with stirring to yield an unreacted Of aldehyde was reduced. The mixture was diluted with 5 mL of 0.9% aqueous sodium chloride solution and the diluted bound mixture was dialyzed against a 100 kDa MWCO membrane.

(2) Cyanylation

A 2M NaCl polysaccharide solution was prepared by adding sodium chloride powder to the 9V polysaccharide stock prepared without hydrolysis. To activate the polysaccharide, 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) was dissolved at a ratio of 0.5 w / w% relative to the polysaccharide, added to the 9 polysaccharide solution and stirred for 15 minutes to induce the polysaccharide activation reaction. The sodium hydroxide solution was then raised to a pH of 9.5 + 0.1 [deg.] C and then stirred for 3 minutes so that the hydroxyl groups of the polysaccharide were fully activated by CDAP. CRM197 was added to the polysaccharide-activated polysaccharide solution at a ratio of CRM197 1.0 w / w% to the polysaccharide, and the reaction was carried out at room temperature for 1 hour. The conjugation reaction was terminated by adding 2 M glycine solution to 3 molar equivalents to 1 equivalent of CDAP, adjusting the pH to 9.0, and incubating overnight at room temperature. The reaction terminated conjugate was concentrated and dialyzed against an ultrafiltration filter through a buffer containing 0.9% sodium chloride.

As a result, it was confirmed that the yield of the conjugate prepared by the cyanylation method was four times or more as compared with the conjugate prepared by the reductive amination method. Thus, the present inventors prepared conjugates using the cyanylation method with the capsular polysaccharide and CRM197.

2-2. Junction and purification of the conjugate

The preparation of the conjugate of Streptococcus pneumoniae of the collapsible polysaccharide and CRM197 was carried out through the following six steps.

Step 1. Dissolving and hydrolyzing the capsular polysaccharide

The filamentous polysaccharide originated from each serotype was dissolved in the injection water to a final concentration range as described below and filtered through a 0.45 μm filter.

In detail, the serotypes 1, 3 and 4 were dissolved in the range of 0.8 - 2.0 mg / ㎖, and 4 - 8 mg / ㎖ serotypes 5, 6B, 9V, 18C and 19F, serotypes 6A, 12F and 19A 8 - 12 mg / ㎖ for serotype 7F, 2 - 4 mg / ㎖ for serotype 7F, 14 and 23F, respectively. In the case of serotypes 15B, 22F and 33F, the solution was dissolved in the range of 2 - 5 mg / ml, and filtration was carried out

The solution was incubated at the pH and temperature ranges described below for each serotype. In the case of serotypes 1, 3, 5, 6B, 7F, 14 and 23F, the temperature was 70-80 ℃ overnight, for serotypes 6A and 19F for 1 - 4 hours 70-80 ℃, for serotypes 9V and 18C, The solution was incubated at pH 2.0 and 65 - 80 ℃ for 1-3 hours. Serum type 22F and 33F were incubated overnight at 75 - 85 ℃ and serum type 12F was incubated at pH 2.0 and 75 - 85 ℃ for 1 to 3 hours with phosphate solution

Serotypes 4, 15B, and 19A did not undergo hydrolysis. The hydrolysis was then stopped by cooling to 21 ° C to 24 ° C and adding sodium hydroxide to a target pH of 6.0 ± 1.0.

Step 2. Capsular polysaccharide and Of CRM197  Bonding reaction process

A 2 M NaCl polysaccharide solution was prepared by adding sodium chloride powder to all serotypes. Appropriate CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) for each serum was dissolved at a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile / water solution (v / v) solution. In detail, in the case of serotypes 6A, 14 and 33F, 1 w / w% of CDAP and 3 w / w% of serotypes 1 and 3, 6B, 7F 15B and 19A, respectively, w% at 4 w / w% for serotypes 5, 9V, 18C, 19F, 22F and 23F and 5 w / w% for serotype 12F 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, and the mixture was stirred for 3 to 7 minutes so that the hydroxyl group of the polysaccharide was fully activated by CDAP. Addition of 0.5-1.0 w / w% of CRM197 to polysaccharide was added to each serogroup polysaccharide solution and the reaction was conducted for 1 hour to 4 hours. The reaction conversion rate was measured using HPLC-SEC, and CDAP was added as needed.

Step 3 . Termination of junction reaction

For each serotype, 3 to 6 molar equivalents of glycine solution was added per 1 molar equivalent of CDAP added and the pH was adjusted to 9.0 to terminate the reaction. The bonding solution was stirred at 21 to 24 ° C for 1 hour and then stored at 2 to 8 ° C at low temperature overnight.

Step 4: Ultrafiltration

The diluted binding mixture was concentrated and dialyzed against an ultrafiltration filter using a minimum of 20 volumes of buffer. Here, the buffer solution was maintained at a pH of 5.5 to 6.5, and a buffer solution containing 0.9% sodium chloride was used. The cutoff molecular weight of the ultrafiltration filter was 300 kDa in all serotypes and the permeate was discarded.

Step 5: Sterilization  percolation

The residual solution after dialysis filtration was diluted to a concentration of less than 0.4 g / L based on the polysaccharide content concentration using a buffer and filtered through a 0.22 탆 filter. The filtered product was subjected to control (saccharide content, residual DMAP) during the manufacturing process. The filtered residue was subjected to control during the manufacturing process to determine whether further concentration, dialysis filtration and / or dilution was necessary.

Step 6: Adsorption

Aluminum salt (mainly aluminum phosphate) was added to the bactericidal filtrate so that the final concentration was 1 mg / mL based on the aluminum ion, adsorbed, and an extra salt was added so as to maintain the pH range of 5.5 to 6.5. After the adsorption was completed, the quality of the stock solution was checked for quality, and the solution was stored at 2 to 8 ° C until used.

Example  3. Formulation of vaccine against polyvalent pneumococcal conjugate

The required amount of final bulk concentrate was calculated based on batch volume and bulk polysaccharide concentration. After adding the required amount of 0.85% sodium chloride, succinate buffer, 2-phenoxyethanol and formaldehyde to the pre-labeled formulation vessel, the bulk concentrate was added. It was then thoroughly mixed and filtered through a 0.22 탆 filter. The formulated bulk liquid was slowly mixed and then bulk aluminum phosphate was added and mixed thoroughly. The pH was checked and adjusted if necessary. The formulated bulk product was stored at 2-8 [deg.] C. The resulting vaccine composition contained 4.4 μg / mL of each polysaccharide (13, (LBVE013 ') at 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F and 23F 15B, 18C, 19A, 19F, 22F, 23F, and 33F), and 6B was 8.8 mu g / ml in the case of LBVE017 / mL; About 29.3 [mu] g CRM197 transport protein; 0.5 mg aluminum element (2 mg aluminum phosphate) adjuvant; About 4.25 mg of sodium chloride; Succinate buffer solution about 295 쨉 g; * About 3 mg of 2-phenoxyethanol and about 60 μg of formaldehyde (* applicable with the addition of a preservative). In the following examples, the contents of some serotypes are controlled (for example, 90%, 75%, 50%, 25%, 10%, and 1% Respectively.

Example  4. Evaluation of the immunogenicity of the multidrug-resistant streptococcal conjugate vaccine

(1) Evaluation method

Studies were conducted to evaluate whether each of the polyvalent pneumococcal vaccine compositions prepared in Example 3 had the ability to induce an immune response in rabbits. This immunogenicity was confirmed by measuring serum IgG concentration through an antigen-specific ELISA.

Each of the formulated polyvalent pneumococcal vaccine compositions or the control group, Prevenar 13 ®, was administered at a planned human clinical dose of 4.4 μg / mL for each polysaccharide, with the exception of 6B 8.8 μg / mL for 100% (Control), and immunized in the muscle of New Zealand White rabbits at 0, 2, and 4 weeks of age. Serum samples were collected every two weeks after inoculation. Serum - specific IgG concentrations were measured using Multiplex bead assay for collected sera. This will be described in detail as follows.

A solution in which 13 or 17 kinds of polysaccharide antigens were conjugated to magnetic beads having magnetic properties was placed in a 96-well plate and adhered thereto. To minimize the nonspecific antigen-antibody reaction, each individual serum was reacted with 1 mg / mL of CWPS multi- solution (CWPS multi ® , Statens Serum Institute) for 30 minutes at room temperature to adsorb the antibody. Was diluted with an appropriate dilution factor. Plates with 13 or 17 polysaccharide conjugate beads attached thereto were washed twice with washing buffer, and 50 μl of the previously adsorbed and diluted serum was added to the plate, followed by reaction at room temperature for 30 minutes.

The reacted plate was washed three times in the same manner, and R-PE gauge anti-rabbit IgG (1: 500) was added to each well, followed by reaction at room temperature for 30 minutes. Plates were washed 3 times by the same method as above, 80 ㎕ of buffer was added to each well, and fluorescence was measured using a Multiplex Reader.

For objective immunogenicity assessment, blood samples taken from immunized Prevenar 13 ® as a control were analyzed.

(2) Evaluation results

First, in the 13-valent vaccine preparation, the immunogenicity of the composition (LBVE013) prepared by the method of Example 3 and Prevnar 13 ® was compared and evaluated. As a result, Example 13 is an immunogenic composition (LBVE013) prepared in the present invention are all relative to Prevenar 13 ® Levels of IgG were found to be higher in Serotype than in Prevnar 13 ® . In particular, serotype 1, 6B, 7F, 9V 14, and 19F showed 2 to 6 times better results than Prevenar 13 ® (Table 1).

LBVE013 Prevenar13
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
9V 2.7 9V 1.2

Next, the immunogenicity of LBVE017 prepared by adding four or more serotypes to LBVE013 was compared with LBVE013. As a result, it was confirmed that overall immunoglobulin composition decline in IgG titers as immunoglobulin compositions were increased from 13 to 17 (Fig. 1). In order to overcome the problem of immunogenicity loss of the multivalent immunogenic composition, the present inventors measured the capacity of each of the 6A, 6B, 14, 19A and 19F conjugates, which had relatively high IgG titers at the 13th position on the 17 immunoconjugate composition 50%, respectively. As a result, it was confirmed that the overall increase in immunogenicity occurred (Fig. 1). In particular, in the case of serotypes 5, 6A, 6B, 14, 18C, and 19A, higher IgG titers than 13 were confirmed.

Based on the above results, we have found that by simultaneously reducing the input dose for the particular serotypes 6A, 6B, 14, 19A and 19F, the overall serotype specific IgG titers of the polyclonal pneumococcal conjugate vaccine can be increased , And the effective dosing range of the five conjugates was confirmed by adjusting the dosage of the five conjugates at a level of 1 to 90%. As a result, when the input capacities of the five junctions were simultaneously decreased from 95% or less to 25% or more, specifically 50 to 90% (i.e. 17% 90%, 17% 75%, 17% 50% , It was confirmed that the immunogenicity of 13 was restored to a level generally similar to that of the immunogenic composition (Figs. 2 and 3). Therefore, in the present invention, when the conjugate dose of the serotypes 6A, 6B, 14, 19A, and 19F is simultaneously reduced to a certain level in the preparation of the immunogenic composition of the present invention, overall immunogenicity is restored Respectively. Accordingly, the immunogenic composition according to the present invention can be very useful for developing a pneumococcal conjugation vaccine by providing a practical method for overcoming the possible immune interference phenomenon that may adversely affect the overall immunogenicity of the vaccine.

Comparative Example  One. In the 13th  Evaluation of immunogenicity when dose is adjusted

We wanted to see if the recovery of the immune response as identified in the 17-mer composition could similarly be observed in the 13-mer composition by simultaneously reducing the input dose of each of the conjugates. Therefore, in the 13th group, the amount of the above-mentioned five serotypes of administration conjugate was reduced to 50% or 25%, respectively, to prepare a 13-immunogen composition, and its immunogenicity was compared with 100% of the 13-ary immunogenic composition of the present invention (LBVE013 ) And Prevenar 13 ® . As a result, unlike the results of the above 17 trials, the 13 immunoglobulins did not recover (rather decreased) the overall immunogenicity (Fig. 4), even though they decreased the capacity of 5 serotypes. This result implies that the results obtained by reducing the dose of some serotypes in the immunogenic compositions of the present invention are beyond the predictable level by those skilled in the art.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (16)

17 capsular polysaccharide-carrying protein conjugates,
Wherein said conjugate is selected from the group consisting of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F A vaccine composition for the prevention of pneumococcal disease, wherein each of 17 capsular polysaccharides is covalently conjugated to a respective carrier protein.
The method according to claim 1, wherein the carrier protein is selected from the group consisting of tetanus toxoid, pertussis toxoid, cholera toxoid, E. coli LT, E. coli ST, exotoxin A derived from Pseudomonas aeruginosa , outer membrane complex c (OMPC) (PspA), pneumococcal adhesin protein (PsaA), C5a peptidase from Group A or Group B streptococci, Hemophilus influenzae (H. pneumoniae) Haemophilus selected from the group consisting of influenzae protein D, ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), purified protein derivatives of tuberculin (PPD), CRM173, CRM228, CRM45 and CRM197.
Vaccine composition for prevention of pneumococcal disease.
2. The vaccine composition according to claim 1, wherein the carrier protein is CRM197 protein.
4. The vaccine composition according to claim 3, wherein the conjugate has a structure in which the capsular polysaccharide and the transport protein are linked by -OC (NH) -NH- group using a cyanylation method.
5. The method of claim 4, wherein the cyanylation process is performed using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or CNBr.
Vaccine composition for prevention of pneumococcal disease.
2. The composition according to claim 1, wherein the composition comprises, in comparison with the serous polysaccharide derived from serotype 1,
The capsular polysaccharides derived from serotypes 6A, 14, 19A and 19F have a content ratio exceeding 0.25 and not more than 0.95, respectively,
The serogroup polysaccharide derived from serotype 6B has a content ratio of more than 0.5 and not more than 1.9,
The capsular polysaccharide derived from serotype 22F and the capsular polysaccharide derived from 33F are the same as those of serogroup 1-derived capsular polysaccharide,
Vaccine composition for prevention of pneumococcal disease.
The composition according to claim 1, wherein the composition comprises the serotype 1 -derived polysaccharide derived from serotype 1,
The content of the capsular polysaccharides derived from serotypes 6A, 14, 19A and 19F is 0.4 to 0.95,
The content of the capsular polysaccharide derived from serotype 6B is 0.8 to 1.9,
The capsular polysaccharide derived from serotype 22F and the capsular polysaccharide derived from 33F are the same as those of serogroup 1-derived capsular polysaccharide,
Vaccine composition for prevention of pneumococcal disease.
The composition of claim 1, wherein the composition further comprises at least one 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.
Vaccine composition for prevention of pneumococcal disease.
9. The method of claim 8, wherein the adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide.
Vaccine composition for prevention of pneumococcal disease.
The vaccine composition according to claim 8, wherein the preservative is phenoxyethanol, formaldehyde, or a mixture thereof.
The method according to claim 1,
Wherein said vaccine composition further comprises a physiologically acceptable carrier.
Vaccine composition for prevention of pneumococcal disease.
17 capsular polysaccharide-carrying protein conjugates,
Herein, the above-mentioned conjugate is composed of 17 kinds of clusters derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F Wherein each of the polysaccharides is covalently conjugated to a carrier protein.
13. The immunogenic composition of claim 12, wherein the delivery protein is a CRM197 protein.
14. The composition of claim 13, wherein the conjugate has a structure in which the capsular polysaccharide and the carrier protein are linked by-OC (NH) -NH- groups using a cyanylation method.
13. The composition of claim 12, wherein the composition comprises a mixture of serotyped polysaccharides derived from serotype 1,
Serotypes 6A, 14, 19A and 19F each have a content ratio of greater than 0.25 and not greater than 0.95,
Serotype 6B has a content ratio of greater than 0.5 and less than or equal to 1.9,
The capsular polysaccharide derived from serotype 22F and the capsular polysaccharide derived from 33F are the same as those of serogroup 1-derived capsular polysaccharide,
Immunogenic compositions for pneumococci.
13. The composition of claim 12, wherein the composition comprises, in comparison with the serotype 1 derived capsular polysaccharide,
The content of the capsular polysaccharides derived from serotypes 6A, 14, 19A and 19F is 0.4 to 0.95,
The content of the capsular polysaccharide derived from serotype 6B is 0.8 to 1.9,
The capsular polysaccharide derived from serotype 22F and the capsular polysaccharide derived from 33F are the same as those of serogroup 1-derived capsular polysaccharide,
Immunogenic compositions for pneumococci.
KR1020170140845A 2016-10-28 2017-10-27 A multivalent immunogenic composition with improved IgG titer and use thereof KR20180046893A (en)

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