SA519401232B1 - A composition of multivalent pneumococcal capsular polysaccharide-carrier protein conjugates and use thereof - Google Patents

Abstract

A composition of multivalent pneumococcal capsular polysaccharide-carrier protein conjugates and use thereof Abstract The present invention relates to a vaccine composition for preventing pneumococcal diseases and an immunogenic composition against Streptococcus pneumoniae, which contains 13 or 14 types of capsular polysaccharide-carrier protein conjugates.

Description

A composition of multivalent pneumococcal capsular polysaccharide-carrier protein conjugates and use thereof Full description Background of the invention

The present invention relates to a vaccine composition to prevent pneumococcal disease and an immunostimulating composition directed against Streptococcus pneumoniae ¢ containing 13 or 14 les 5 conjugates of the aureus capsule rheumatoid arthritis and rutin is a capsular polysaccharide-carrier

.protein conjugates encephalomeningitis Streptococcus pneumonia is the main cause of severe encephalitis and meningitis and severe invasive diseases pneumonia for infants and children, as it causes other pneumonia. More than 1.6 million people die annually from invasive diseases, especially infectious diseases (according to the World Health Organization in 2008). And on the 0 invasiveness that occurs due to Streptococcus pneumonia is high in young children that are due to low (eld age less than 5 years and elders whose age is from 65 years

immunity in their bodies. There are more than 90 serotypes of Streptococcus pneumoniae, depending on the structural and immunological characteristics of the polysaccharides in the capsule; It is the causative agent of the disease that surrounds the bacteria. add to that; ald is among those serotypes; A score of 20 or slightly more is associated with a 0 to 9690 chance of inducing disease. Humans are the only hosts for Streptococcus pneumoniae, as this bacterium is present in the nasopharyngeal cavity1 in the pathogenicity of healthy individuals (20 to 9640 children and 965 to 9610 adults). In 2005, the Centers for Disease Control and Prevention (CDC) reported that there were about 1 million children under the age of 5 in the United States.

developing die of pneumonia; Within this month (JULY) 1.2 million jib babies have died from bacterial infections. add to that; CDC also stated that in the United States; Encephalitis, meningitis, and septicemia caused by Streptococcus pneumoniae occur in approximately 3,000 dls and 50,000 dlls, respectively (Peters TR et al. (2007) JAMA Invasive pneumococcal disease 5:297(1825-6). According to what is reported in the pneumoACTION databases in the year 2000, the incidence of gl infection in young children in South Korea was 24047 and cle 47 of them were (www.pneumoadip.org) and also » In the 'Studies on Streptococcus pneumoniae Serotype Analysis in Young Children and Adolescents in Korea', a 10 recent study published by the Korea Centers for Disease Control and Prevention, it was reported that Streptococcus pneumoniae is the most common (9643.7) cause of infection. For infants and children from 3 months to 59 months In addition to coll, Streptococcus pneumoniae causes infectious diseases around call) and with the increase in the number of drug-resistant bacteria, which are resistant not only to penicillin but also to three or more drugs, this causes an increase in the severity of Treatment of infectious diseases caused by Streptococcus pneumonia.Edly, there is an ongoing need for a vaccine against Streptococcus pneumonia for young children and those with

Larger; And those who are exposed to infectious diseases that occur due to Streptococcus pneumonia. In order to prevent rheumatic diseases; Vaccines containing polysaccharides and polyvalents against Streptococcus pneumoniae have been developed and approved since 1977; These capsule polysaccharide vaccines have been shown to be useful in preventing pneumonia-0 disease in older or more susceptible patients. anyway; It is because the immune system can be lower cla that when polysaccharide vaccines are given alone; The immune system of infants and young children does not recognize the polysaccharide as a foreign antigen. Therefore; ald It is difficult to anticipate that vaccines from multiple sugars can work as complete vaccines to cover all different age groups. In order to solve the problem of low immune response to polysaccharide vaccines for infants 5 and children [lal], a conjugate vaccine directed against the seven-valent pneumonia bacteria (Prevenar” (© 7700) was developed and used); Conjugation of a polysaccharide encapsulated with a carrier protein has been developed and used to augment the immune response; Its effect has been demonstrated in the prevention of invasive diseases and inflammation

middle ear. Although the use of the seven-valent vaccine has led to a decrease in the level of invasive diseases that occur due to the serotypes used in the vaccine; The serotypes that are not included in the vaccine will increase proportionally. in this context; Synflorix®, which is a polycapsular sugar-protein conjugate vaccine, deca-B, and Prevenar13® 5, which is a polycapsular sugar-protein conjugate vaccine, has been developed and is trivalent, in which 6 serotypes were added. additive to the starting formulation of Prevenar® and is commercially available. However, it has been reported that some serotypes of those included may not be sufficient to elucidate optimal efficacy as a vaccine component (Poolman J et al. (2011) Clean.

Vaccine. Immunol. (18) 327; Impact of the Conjugation Method on the Immunogenicity of Streptococcus pneumoniae Serotype 19F Polysaccharide in Conjugate Vaccines / 0 (EMEA Assessment Report for Prevenar 13 (2009) EMA/798877/2009) In addition, there is ongoing dala for developing vaccine formulations A new ally could have better field efficiency

wider coverage. On the one hand (gal), there is a noticeable phenomenon after the introduction of the conjugate vaccine for hepatitis B bacteria, which is an increase in the incidence of invasive diseases by replacing the serotype due to serotypes of gill bacteria other than those of the vaccine, which are not contained in the vaccine. These serotypes are not Vaccines are believed to be prevalent in the suprapharyngeal region by replacing vaccine serotypes, which mainly accumulate there before the introduction of conjugate vaccines; og that; non-vaccinated serotypes are considered a new threat in causing pneumonia diseases. dag log in particular; the 0 Considering the general distribution of Streptococcus pneumoniae, the occurrence of serotype 2 is more prevalent than some serotypes “GAY (4) TF and 3) contained in [Oss Prevenar] 3® is the 11th most abundant serotype in the world lag (Johnson HL et al. (2010) PLoS Medicine, 7:10, 1000348: Systematic Evaluation of Serotypes Causing Invasive Pneumococcal Disease among Children Under Five: The Pneumococcal (Global Serotype Project 5). In addition, as mentioned in a recent study (Saha SK et al. PLoS One, 7:3, 32134: Streptococcus pneumoniae serotype-2 childhood (2012) (meningitis in Bangladesh: a newly recognized pneumococcal infection threat). OB type

serogroup 2 caused 9620 meningitis (230 cases) in children younger than 5 years of age in Bangladesh from 2001 to 2009. In this context; Since serotype 2 causes invasive pneumococcal diseases (IPD), this serotype is of great interest on the basis of severity as well as geographic abundance. The serotype 2 capsular polysaccharide antigen is not included in any of the pneumococcal vaccines that include [Prevenar]3®, a 13-valent ASHE vaccine from Pfizer; which currently has the highest serotype coverage. There is also no report of transient activity between other serotypes in Prevenar13®. Accordingly; The inventors of this invention have worked hard to develop a vaccine to prevent inflammatory diseases, as this vaccine has greater immunological activity or wider coverage of the serotype compared to the existing vaccine combinations. As a result of (SIM), the inventors of this invention have developed a new 13-valent BSS pneumococcal conjugate vaccine that contains a serotype identical to the existing 13-valent vaccine but with better immunogenic activity; and a 14-valent pneumococcal conjugate vaccine containing two additional antigens 5 of the polycapsular sugar to increase coverage without loss of immunogenic activity, thus completing this invention 5. The 13-valent rheumatoid conjugate vaccine of the present invention contains 13 leg of polycapsulated glucose antigens; and has better immunogenic activity compared to the The conjugate directed against 13-valent rheumatoid aureus bacteria.In addition to (SIN), the 14-valent pneumococcal conjugate vaccine of this invention contains 14 types of capsular polyglucose antigens and enters into a 0-transformation process ideal for hydrolysis during conjugation. A vaccine composition with wider coverage and better immunogenic activity is obtained compared to the existing 13-valent vaccine.As a result, the 14-valent rheumatoid conjugate vaccine of the present invention is expected to have excellent effects on ge rheumatic diseases. GENERAL DESCRIPTION OF THE INVENTION 5 TECHNICAL PROBLEM

The object of this invention is to prepare a vaccine composition to prevent diseases caused by pneumococcal bacteria; Where this composition contains 13 types of conjugates consisting of polysaccharides of the capsule Cus cela Gig pg that in the thirteen types of conjugates, each of the ten ADA types of polysaccharides of the capsule is derived from serotypes of Streptococcus pneumoniae 3 4 5 23F 5 » 19F » 19A » 18C » 14 “OV” TF 6 68 “6A where covalently conjugated to a carrier protein; (ss The carrier protein is protein 811197©; each of the conjugates has a structure in which both the polysaccharides of the capsule and the carrier protein are linked by -O-C(NH)-NH-group using the cyanide method. Another object of the invention is It is the preparation of a vaccine formulation to prevent diseases caused by pneumococcus 0 bacteria, which contains 14 types of conjugates consisting of capsule polysaccharides and a carrier protein »whereas in the fourteen types of conjugates »each of the fourteen types of capsule polysaccharides derived from species serogroup Streptococcus pneumoniae 1-2 3 4 6A < 5 « 68 75 Lao 14 « 19F » 198 « 18C »and 23 is covalently coupled to a carrier protein; the carrier protein is protein 0814197; each of the conjugates has a structure in which 5 is Both the polysaccharides of the capsule and the carrier protein are crosslinked by -O-C(NH)-NH- group using the cyanide method.Another objective of this invention is to prepare an immunostimulating composition directed against pneumococcal bacteria;which contains 13 species Of conjugations consisting of polysaccharides of the capsule and Cus als protein that in the thirteen types of conjugates; Each of the thirteen species of 0 polysaccharides of the capsule derived from serotypes of Streptococcus pneumoniae 1 23 4 5; 23F 5 ¢ 19F « 19A » 18© « 14 9177 775 « 68 » 6A is covalently coupled to a carrier protein; The carrier protein is protein 011197; Each of the conjugates has a structure in which both the polysaccharides of the capsule and the carrier protein are bonded via the -O-C(NH)-NH- group using the cyanide method. 5 Still another object of this invention is to prepare an immunostimulating composition directed against pneumococcal bacteria; Which contains 14 kinds of conjugates consisting of polysaccharides of the capsule and protein

cals, since in the fourteen types of conjugations, each of the fourteen types of

Capsule polysaccharides derived from serotypes of Streptococcus pneumoniae 1-2 3 4

23F 5 ¢ 19F » 198 » 18C » 14 < 9V 78 « 6B » 6A < 5 covalently coupled to a carrier protein;

The carrier protein is protein 0814197; Each of the conjugates has a structure in which both the polysaccharides of the capsule and the carrier protein are bonded via -O-C(NH)-NH- group.

using the cyanide method.

There is still a target AT for this invention dae] a way to prevent a disease from the diseases that occur

caused by pneumococcal bacteria by administering the vaccine formulation or the immunostimulating formulation to a person in need

for this installation.

0 Still an object AT of this invention is to prepare a method for using a vaccine formulation preparation to prevent disease caused by aureus sill containing 13 types of conjugates consisting of capsule polysaccharides and a carrier protein; Whereas, in the thirteen species of conjugates, each of the species EN 10 polysaccharides of the capsule derived from serotypes of Streptococcus pneumoniae 1 3 4 5 “9V TF 68 6A 14 180

5F 198 ¢ 5 23F is covalently conjugated to a carrier protein; Oey The carrier protein is protein 7; Each of the conjugates has a structure in which both the polysaccharides of the capsule and the carrier protein are bonded via the -O-C(NH)-NH- group using the cyanide method. Still another object of this invention is to prepare a method for using a vaccine formulation to prevent a disease caused by pneumococcal bacteria; Which contains 14 kinds of

0 conjugates consisting of capsular polysaccharides and protein ala wherein in the fourteen species of conjugates each of the da species has ten capsule polysaccharides derived from serotypes of Streptococcus pneumoniae 0 2 3 4 5 TF 68 “6A HQ 14 0180 19F 198 ¢ and 2357 covalently coupled to a carrier protein; As the carrier protein is protein 7; Each of the conjugates has a structure in which each of the polysaccharides of the capsule

5 and the carrier protein is conjugated to Goh group -1011-(0-0011-) using the cyanide method. [Technical Solution]

In this invention; It is to develop a new vaccine formulation that has the ability to prevent a disease caused by pneumococcal bacteria with a higher efficiency; A composition containing 13 types of couplings consisting of polysaccharides of the capsule and a carrier protein is prepared, as the conjugation method for these couplings differs from the existing thirteen-valent vaccine. In particular dag, the composition contains 13 types of conjugates consisting of the polysaccharides of the capsule and a carrier protein; which is prepared by covalent conjugation of each of the thirteen types of capsule polysaccharides derived from serotypes of Streptococcus pneumoniae 1 03 4 5 ¢TF 68 «6A 23F5 » 198 198 «18C» 14 OV with a carrier protein. Since the 13 types of polysaccharides of the capsule have increased immunogenic activity compared to the existing 13-0 conjugate vaccine HIS, the composition of this invention is expected to have a significant effect on preventing diseases caused by pneumococcal bacteria. Lady, although there are many reports of a decrease in the incidence of invasive diseases caused by vaccine serotypes contained in a gil-protein conjugate vaccine after the vaccine became commercially available, the probability of a disease caused by 5 pneumococcal bacteria according to the serotypes of the vaccine does not reach zero due to the geographical distribution of the serotype (Lee L.H et al. (2014) Vaccines, 2 (112); Towards New Broader Spectrum Pneumococcal Vaccines: The Future of Pneumococcal Disease leg (Prevention) Therefore; It is known that the immune activation of conjugate vaccines can be affected by the addition of more serotypes due to the immunological interference that occurs through interactions between their components such as polysaccharides or carrier proteins (Dagan R et al. (2010) Vaccines, 28(5513); Glycoconjugate vaccines (and immune interference: A review On any (Ja) it has been confirmed that the 14-valent vaccine in this invention; in which a cyanide addition method is used and in an optimized hydrolysis method; 5 has an enhanced immunogenic activity in all serotypes and/or Better coverage compared to the already existing thirteen CHK vaccine

Each description and ideal embodiment in this invention can be applied to any AT “ag” and to any other ideal embodiment. Whereas all combinations of the various elements described in this invention fall within the scope of this invention. In addition; The scope of this invention cannot be considered limited by the following particular description.

as a manifestation of previous goals; This invention works to prepare a vaccine composition to prevent diseases that occur due to aureus (ugh) bacteria, as this vaccine contains 13 types of conjugates consisting of polysaccharides of the capsule and Jala drotin. In particular, the thirteen types of conjugates They can be those in which each of the thirteen types of capsule polysaccharides are derived from Stretococcus

0 pneumoniae serotypes 1« 3 4 5« 19A 180 4 «OV »TF «6B« 6A ¢ 198 and 2385 are covalently conjugated with 7 + which is a carrier protein. Accordingly, this invention is the composition of a vaccine that contains 13 different types of conjugates consisting of a polysaccharide and a protein, as each of these conjugates contains polysaccharides belonging to the capsule derived from Streptococcus pneumoniae of different serotypes conjugated with a protein.

5 1 Carrier 3 Whereas, the polysaccharides of the capsule are prepared from serotypes 1-3 “+ 4 < 9V “TF” 6B < 6A < 5 4 1180 of 198 and 235. In addition, in the form of al, this invention works to prepare a vaccine composition to prevent diseases caused by rheumatoid bacteria; It contains 14 types of conjugates that consist of the polysaccharide of the capsule - a carrier protein.

0 In particular, the fourteen types of conjugates could be those in which each of the fourteen types of capsule polysaccharides derived from Streptococcus pneumoniae serotypes 1« 2 3 4k5« 70168 ¢ 6A kOq 14 180 19A ¢ 191” and 7 are covalently coupled to “CRM197, a carrier protein. Accordingly, this invention consists of a vaccine containing 14 different types of conjugates

5 consists of a polysaccharide and a protein” since each of these conjugations contains a polysaccharide

for a capsule derived from Streptococcus pneumoniae from different serotypes conjugated with a carrier protein; WHEREAS the capsule specific polysaccharides are prepared from serotypes 1.2; 3; 4 <9V “TF 68 6A” 5 4K80 for 198 and 2385 . In order to find a way to improve the immunogenicity of the Vaccine for Staphylococcus bacteria; The inventors of this invention compared two ideal coupling reactions, namely the amine by reduction and the addition of cyanide. da, therefore; It has been confirmed that the cyanide addition method is better than the reductive amine method on the basis of coupling yield and reaction time. On the basis of the aforementioned, the optimal conjugation was carried out with each serotype using the cyanide addition method; lly differs from Prevenar13® to the same thirteen identical Prevenar13® serotypes (ie of which 0 currently has the highest coverage of serotypes. As a result, the IgG concentration of all serotypes in the composition is confirmed to be 13valent or 14valent is higher than that concentration in Prevenar13® and in the case of salvalent in the multivalent immune construct; thermal crosstalk can occur due to increased interactions between components (Dagan R et al. (2010) Vaccines, leg ¢(28(5513); Glycoconjugate vaccines and immune interference: A review) Therefore, a person with normal experience in this field may need greater efforts to optimize and improve overall immune activity in the 13valent or 14valent formulation. In addition, the inventors of this invention have added the serotype 2 strain to increase the coverage area, which is an area that cannot be covered with Prevenar13® and since the immunogenic crosslinking that is activated by the interaction of its components in a multivalent immune complex can also be increased due to Adding one or more type 0 Aad serotypes (Type 2) the improvement in immunogenic activity in all 14 serotypes of this invention compared with Prevenarl3® could be considered a surprising result. In this invention; the conjugates have a structure in which the capsular polysaccharide and carrier protein are bonded by means of an eg -O-C(NH)-NH- group; It is based on differences in composition from pneumococcal conjugate vaccine (PCV) which is prepared 5 by other methods usually to the optimum level of the detailed conjugation method; The composition of the 13-valent or 14-valent vaccine of this invention can give an excellent immunogenic concentration in all subtypes.

Compared with the composition of the 13valent or 14valent vaccine prepared by the aforementioned amine reduction method. As used here, the term "pneumonia" refers to an acute inflammatory disease of the spongy tissue of the lung; Which is mainly caused by Streptococcus pneumonia and Klebsiella pneumonia. In particular, pneumonia caused by pneumococcal bacteria accounts for about 9650 of all cases of inflammation (geil) and its symptoms are a feeling of extreme cold; fever; cough; and chest pain. add to that; it is possible that there will be bloody sputum; Complications such as pneumonia can occur; meningitis; endocarditis»; Inflammation (sind to sal Stein GE et al. (2001 Mar) Diagn.

Microbiol.

Infection, Dis 39:181-185; Comparative ) serum bactericidal activity of clarithromycin and azithromycin against macrolide- 10 (sensitive and resistant strains of Streptococcus pneumoniae. It is a symbiotic organism that aggregates and colonizes on the surfaces of the mucous membranes of the human nasopharynx and nasopharynx.When factors specific to host 5 cause the organism to reach the lower gall of the respiratory tract, this causes an intense inflammatory response, which leads to filling the voids in the respiratory tract. Alveoli with secretions, which leads to activation of pneumonia.Rhachymacoccal bacteria produce 90 or more unique polysaccharides on the capsule.”The serotype of pneumococcal bacteria is classified according to the structural and immunological properties of the polysaccharides of the capsule.Therefore, when preparing a vaccine composition using 0 Polysaccharides of the pneumococcal bacteria capsule; the immune response can vary depending on the type of polysaccharide on the capsule, which is the serotype of pneumococcal bacteria from which the capsule polysaccharides are derived. On day in particular the vaccine composition of the present invention can be prepared on dag in particular using 13 least capsular polypeptides derived from Streptococcus pneumoniae serotypes 1 3 4 5” (TF 168 “6A 18C 14, 97 25 « 198 - 23F 5 » 19F In addition, the vaccine composition of this invention can be specially prepared using 14 types of special polypeptides.

capsule and derived from Streptococcus pneumoniae serotypes [2 3« 4 5 (TF 68 « 6A 18C » 14 9V « 19F » and 235). The polysaccharides of the capsule are marked as antigen upon administration to the body so that an antibody can be produced is directed against this antigen; therefore, a vaccine composition can be prepared for the prevention of rheumatoid arthritis. As used herein », the term “antigen” refers to a substance which can specifically activate an immune response when the substance enters the body. Invention », each of the thirteen types of capsule-specific polypeptides derived from Streptococcus pneumoniae serotypes 1 3 4 5 “OV” TF 68 “6A 14 ¢ 180 Air 198 » 23F5 can act as an antigen. In this invention, each of the 14 types of capsule-specific polypeptides derived from Streptococcus pneumoniae serotypes 1 “2 3 ¢ 4” 5 “6A-68” 19A “18C” 14 97 “TF 198” The 2385 capsule polysaccharides can be prepared with standard techniques known to a person with ordinary experience in the field; preparation methods are not limited to specific dag methods in particular. 5 The volume of the polysaccharide of the capsule can be reduced by hydrolysis in order to reduce the viscosity and to make an effective immunostimulator. In an exemplary embodiment of this invention, each of the 14 different serotypes of Streptococcus pneumoniae (01 2 3 4 5 “¢19F” 198 180 14 “OV “TF 68” 6A and 23F) were hydrolysed using sodium deoxycholate After that, polysaccharides associated with 0 were liberated by cells. Then, the following 12 serotypes 0 3 3 4 5 “6B” 6A or 186 198 – 198 ¢ and 2387 were purified by using cetyltrimethylammonium bromide ( CTAB) because the serotypes have the ability to ionic bond with (CTAB) and then the two serotypes do not interact with “CTAB 77 and 14; using a solution of 1p1m. When a vaccine composition is prepared using only capsular polysaccharides,” the device Immuno5 for infants and young children, which is weaker than the adult immune system, cannot recognize it

antigens As a result, an immune response may not arise. Accordingly (SUA) the vaccine was prepared in the form of a conjugation in which a dala protein and a polycapsular sugar were conjugated and used. As used in this invention, the term “carrier protein dels” refers to a protein which can It is covalently conjugated with a capsule-specific polysaccharide to increase the immunoreactivity of a polysaccharide antigen, and 0814197 was used as a carrier protein in this invention. The conjugation of the carrier protein to the capsule polysaccharide can be done by standard conjugation and the conjugation consisting of a polysaccharide-carrier protein can be A constituent is that in which one or more capsular polysaccharides are conjugated to a carrier protein.As used herein; the term “0814197” denotes a non-toxic (Ged) toxoid 0 heterologous to diphtheria toxin separated from Culture of Corynebacterium diphtheria strain C7 (0197). 0814197 can be purified by ultrafiltration, ammonium sulphate precipitation and ion exchange chromatography. In addition, (Say) prepared 0814197 by genetic engineering method as stated in the US patent No. 5,614,382; This is for example but not limited to. Any of the known methods for preparing a conjugation consisting of a polysaccharide-5-capsular carrier protein may be included within the scope of this invention” and the coupling shall have a structure in which the polysaccharide and the carrier protein are bound by -O-C(NH)-NH- group using a method Add cyanide. The cyanide method can be carried out by the person with ordinary experience in this field by the method known in the art; For example, the cyanide method can be implemented using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) or CNBr but 0 to name a few. As an example of preparing a conjugation consisting of a polysaccharide-capsule carrier protein, the glycoconjugate can be formed by chemical activation of the purified polysaccharides; This is followed by the conjugation of each of the chemically activated capsular polysaccharides with a carrier protein. Cyanide activation due to treatment with 1-cyano-4-dimethylaminopyrbidenium tetrafluoroborate (CDAP) 5 can modify the hydroxyl group of the capsular polypeptide to a cyanate; This can be used to form a covalent bond with the amino group of 0811197; As

Ble is a carrier protein. The cyanide reaction can be carried out by means of CDAP by adding to the dag in particular a glycine solution with three molar equivalents with respect to (0/87 in molar equivalents aly) and adjusting the pH to 9, for example but not limited to. In addition, the person Those with regular experience in this field can adequately adjust the reaction solution and reaction conditions according to the desired objective.The obtained conjugate of polysaccharide capsular-carrier protein can be purified by several methods.Examples of these methods include the method of concentration/filtration by Shall column chromatography; field. Olid, the composition can be prepared by making formulas of each of the thirteen types of conjugation of the polysaccharide-capsule carrier protein with a physiologically acceptable carrier. An example of such a carrier could be a pH-controlled cold salt solution ; Polyols (glycerol; propylene glycol; polyethylene glycol liquid); or to a dextrose solution; This is for example, but not limited to 5. In the perfect embodiment of this invention; Thirteen types of conjugations consisting of capsular polysaccharides-carrier protein were prepared by 1) dissolution and hydrolysis of the thirteen types of capsular polysaccharides. 2) coupling reaction method for each of the capsular polysaccharides and carrier protein using 1-cyano-4-di die aminopyridinium tetrafluoroborate 0 (2©0p); 3) termination of the coupling reaction » 4) ultrafiltration; 5) filtration by sterilization and 6) adsorption. In another perfect embodiment of this invention; 14 types of conjugation consisting of a polysaccharide-capsule-carrier protein for Lgl (1 Gob) and hydrolysis of the 14 types of capsular polysaccharides were prepared. Dimethylaminopyridinium Tetrafluoroborate (CDAP) 3) Terminated OLY) Jeli 5 4) Ultrafiltration; 5) sterilization and filtration, and 6) adsorption.

— 5 1 — as used herein; The term 'vaccine' means a biological sale that contains an antigen that is used to immunize the body of an oa, and thus it means an immunostimulant or an antigen material that provides the body with immunity by administering this vaccine to an animal or to a person to prevent diseases infectious.

The vaccine composition may also contain at least one adjuvant; sale clipboard; buffer solution cold protection complex; A divalent cationic salt, a non-ionic detergent and an oxidation inhibitor to prevent oxidation by free radicals. As used herein, the term 'adjuvant de lod' refers to a substance which increases the immune activity of the immunostimulating composition of the present invention. The assistant is supplied in many

0 conditions to enhance the immune response; It is well known to a person with regular experience in this field. A suitable adjunct to improve the effect of the vaccine formulation of this invention includes, without limitation: (1) aluminum salts (alum) such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.;

5 (ii) a zap-in-water emulsion formulation (with or without other specific immune activating agents such as muramyl peptides (as defined hereinafter) or components of the bacterial cell wall); These include (WO 90/14837) MF59 (1) containing 965 squalene” 960.5 Tween 80; 0.55% 85 Span (optionally containing several amounts of MTP-PE (see below; although not required); prepared as sub-micron particle sizes using a microfluidization unit ie a unit

0 Fluidization 1107 ¢ (Microfluidics, Newton, Mass.) Model (B) SAF containing 7010 Squalene” 960.4 Tween 80% 5% Polyurethane Sealant Polymer thr-MDP 5 “L121 (see next gall) ; either finely fluidized into an emulsion with sub-micron sizes or cycled to an emulsion with larger particle sizes; and (c) (Corixa, Hamilton, Mont.) (RAS) Ribi™ adjuvant system containing 962 squalene” 960.2 Tween 80; and one or more components of the cell wall to be selected from

5 Group consisting of 0-3©-polyphosphoryl lipid A deacetylated die (MPL™) described

— 6 1 — in US Patent No. 4,912,094 o(Corixa) trehalose dimicolate (TDM) and wall structure

cellular (01775); Preferably ¢ (Detox™) MPL+CWS (3) Quil A Jie saponin aids or 058-21 Antigenics, STIMULON™ (Framingham, Mass. (USP No. 5,057,540) or particulate matter thereof) Jie

ISCOMs 5 (Immune Stimulating Complex Compounds);

(4) Bacterial lipopolysaccharides; A lipid analogues such as aminoalkyl glucosamine phosphate (AGP) compounds or derivatives thereof or analogues of Ally are available from Corixa and are described in US Patent No. 6,113,918; one of the AGP is 2-[(8)-3-tetradecanoyloxytetradecanoylamino] Jal -2- dioxy-2-0-4-deoxy-0-4)-phosphano-0-3)- —( R)].

0 3-tetradecanoyl oxytetradecanol]-2[()-3-tetradecanoyloxytetradecanoylamino]-D-b-glucopyranoside ¢ also known as 529 (previously known as RC529 ¢) which is prepared as aqueous or as a stable emulsion;

(5) Jie Synthesized Polynucleotides Oligonucleotides Containing CpG moieties (US Patent No. 6,207,646)

15 (6) Cytokines “such as interleukins (such as IL 18, 11-15, 12” IL 18, 11-15 “etc.); interferons (such as gamma interferon)”; large granulocyte colony-stimulating factor (MLCF) GM-CSF) large leukocyte colony stimulating factor (577©-84); tumor necrosis factor (TNF) activating molecules B7-1 and 37-2, etc.;

0 (7) detoxified mutants aud add ADP-ribosyl Jie cholera toxin (07) either in wild form or in mutant form; For example, “where glutamic acid is replaced at amino acid position 29 by an amino acid AT, in particular histidine” according to International Patent 4 70 (see Lead International Patent | WO 02/098368 and International Patent “(WO 02/098369). Pertussis toxin (PT) or temperature-sensitive (LT) E. coli toxin, in particular

LT-R72 LT-K63 5 « 5109 NO 93/113302 10 1-19/0129i and {WO 92/19265 and

— 7 1 — (8) Components of the supplement such as the trimer of the ingredient component 3d muramyl peptides which can include but not be limited to » 14-acetyl-muramyl-1-threonia-10-isoglutamine (the-MDP) and 11 - acetyl-turmuryl-1.-alanine 2) 1 = 2 = bi-palmitoyl-sn-glycero-3-hydroxyphosphoryl-oxy)-ethylamine (MTP-PE)

The adjuvant which is the ammonium salt can be an alum precipitated pollen or an alum adsorbed pollen. Alum salts may include hydrated alumina; alumina trihydrate (ATH) aluminum hydrate; aluminum trihydrate; hydrogel; Swervos; amphogel; aluminum hydroxide” aluminum hydroxyphosphate sulfate (auxiliary aluminum phosphate ((APA) alumina amorphous; to al, for example, but not limited to JE.

0 Hydroxy Aluminum Phosphate. If aluminum chloride is mixed with sodium phosphate in a ratio of 1:1; It precipitates aluminum hydroxyphosphate sulfate. Precipitate sizes are adjusted to 2 µm to 8 µm using a high shear mixer and dialysis is completed with physiological salt solution; Followed by sterilization. In one embodiment; of and (21)011 available Was (eg (Jal hydrogel or Swerphos) used for adsorption of proteins. Protein can be adsorbed (from 50 g to

5 200 grams) per 1 milligram of aluminum hydroxide; And that ratio depends on the electronegativity point (pI) of the proteins dads the pH of the solvents. Proteins with low PI deg are more strongly adsorbed than proteins with a high 1m value. The aluminum salts can serve to form an antigen reservoir that slowly releases antigens over two or three weeks' lg. It nonspecifically activates macrophages; supplements; and the innate immune mechanism.

0 As used in 'lia' the term 'sald' preservative' refers to Bale's antiviral and/or antimicrobial; which inhibits the growth of microorganisms in a vaccine formulation. liad the preservative can be thimersal » 2-Phenoxyethanol, formaldehyde, or mixtures of terminators, for example and not limited to. In addition, all conventional preservatives can be used. In addition (lI), the composition of the vaccine may include one or more of the acceptable buffer solutions

5 Physiology. For example; if the vaccine formulation is a formulation for administration or for injection; The buffer solution can have a buffer solution capacity at dad pH ranging from 4 to 10 and on dag

particular at dad's pH range from 5 to 9; and the most specific at a pH value ranging from 6 to 8. The buffer solution can be chosen from the group consisting of (TRIS acetate; glutamate lactate; maleate tartrate; lig (hin (lig) glycinate; histidine; glycine succinate, and triethanolamine.

on the particular day”; if the vaccine composition of this invention is for parenteral administration; The buffer solution can be chosen from those buffer solutions accepted in the United States Pharmacopeia (USP) for example; The buffer solution can be chosen from the group consisting of the monobasic acid Jie acetic acid; benzoic acid; gluconic acid; glyceric acid; lactic acid; A dibasic acid is Jie acid

aconitic acid; adipic acid; ascorbic acid; chloronic acid » glutamic acid; malic acid; succinic acid; tartaric acid; polybasic acid Jie a stearic acid and a phosphoric acid; a base such as diethanolamine; Glycine “triethanolamine” 5 TRIS. In addition lll the composition of the vaccine of this invention may include a non-ionic detergent. For example; The vaccine composition of the present invention may include polyoxyethylene sorbitan ester (which

5 (commonly referred to as Twain); and in particular dag » Polysorbate 20 and Polysorbate 60; DOWFAXTM (i) copolymers of ethylene oxide (BO) propylene oxide (20) and butylene oxide (BO) octosplenes with a different number of repeats of the ethoxy group d = Sl (2-ethanediyl); In particular dng, 9-octooxynol (Triton *-100); Ethylphenoxypolyethoxyethanol ((IGEPAL CA-630/NP-40) Phospholipids Jie Lecithin; Nonylphenol Ethoxylate

Such as chain (dap NP Cetyl Stearyl; Polyoxy oleyl (bd fatty acid Fie of alcohol (Brig surfactant); and in particular dag; Triethylene glycol oleyl lauryl ether Brij} 0) and stimulants [Jie sland] sorbitan ether known as ley (SPAN) in particular; Glug. emulsion; mixtures of

5 Non-ionic detergents such as 85 Tween 80/Span It is appropriate to use a union of polyoxyethylene sorbitan ester ie Tween 80 and octoxinol Jie Triton *-100 is also suitable. A combination of Laureth-9, Tween and/or Octoxinol is also suitable. And on the face

particularity; The amount of polyoxyethylene sorbitan ester (Jie Tween 80) that is included can range from 960.01 to 9061 (w/v); dng og specification 960.1; The amount of octylphenoxypolyoxyethanol or nonylphenoxypolyoxyethanol (such as Triton 100-X) that is included can range from 960.001 to 960.1 (w/v); eg in particular from 960.005 to 960.02; and the amount of poly Oxyethylene ether (as laureth 9) which is included may range from 960.1 to 9620 (w/v); and may range from 960.1 to 9610; and in particular from 960.1 to 961 or about 960.5. Image Single-dose vial or multi-dose vial; or prefilled syringe; (Sarg) The composition also includes a physically acceptable carrier. or aqueous or non-aqueous oil. Examples of a non-aqueous solvent include propylene glycol; polyethylene glycol; and ethyl oleate. Aqueous carriers include water; an emulsion; or suspension; a physiological salt solution; and a buffer solution. Examples of an oil include vegetable or animal oil; cu) coconut; soybean oil; olive oil; cu; sunflower; liver oil; Oil 5 Blend Jie Marine Oil; A fat obtained from milk or eggs. The composition of the vaccine of this invention may be similar to osmosis; plus osmosis; or low osmosis. On any Js, it is preferable that the pharmaceutical composition for administration or injection be essentially osmotic, for example, but not limited to. In addition to osmosis, the composition may be diluted to an osmotic level prior to administration Can be an osmotic agent for dilution Can be an ionic osmosis agent Jie Salt or a non-ionic osm like carbohydrate An ionic osmosis agent includes sodium chloride; Magnesium chloride, etc., to name a few. The non-ionic osmotic pressure-adjusting agent includes sorbitol, glycerol, etc., 5, to name a few, but is not limited to. A protective immune response without severe side effects; this amount may vary according to the serotype of bacteria

rheumatoid arthritis. og in particular; in the formulation of the vaccine; For the polyglucose derived from serotype 1; Each of the polysaccharides derived from serotypes 3 » 4 » 5 ¢ 19A » 18C » 14 » 917 » 7 » 6A ¢ 197 » and 2377 can have a content ratio of 0.9 to 1.1; Sug specific capsule-specific multiple derived from serotype 6B 5 can have a content ratio of 1.8 to 2.2; This is for example but not limited to. add to that; In vaccine formulation, the polycapsular sugar derived from serotype 2 can have a content ratio of 0.9 to 1.1 relative to the polycapsulated sugar derived from serotype 1; This is for example but not limited to. As an example of conjugation preparation, each conjugation can contain an amount from 0.1 µg to 0 100 µg; in particular dng from 0.1 µg to 10 µg; og in particular from 1 µg to 5 µg of polysaccharides. Most notably; that in addition to the capsule polysaccharide derived from serotypes 1) 3 4 dm 9V 1 ¢ 6A 14 ©18; 19F ¢ 19A ¢ and 2377 can contain 2.2 µg; the capsule polysaccharide derived from serotype 613 can contain 4.4 µg, by way of example, but not limited to, in which case protein 0181197 in the structure could contain 0.1 µg to 100 µg, in particular from 1 µg to 50 µg, eg in particular from 28 µg to 31 micrograms "and in particular 29.3 micrograms", for example and not limited to. The optimal amounts of components for a particular vaccine can be determined by standard studies, Ally, including observing the appropriate immune response for people. For example, the amount of 0 vaccine for a person can be determined by extrapolating the results of tests on Animals In addition, a person with normal experience in this field can empirically determine the dose, if necessary The composition of the vaccine may also include aluminum chloride and sodium chloride, for example, but not limited to. Contains or does not contain preservative 5 depending on the purpose and use.

The vaccine composition according to this invention can be used to protect persons who are susceptible to infection with pneumococcal bacteria and to prevent gill infections by administering an effective amount of Wass parenterally or on mucous membranes. As used in this invention, the term “ES” refers to all the processes that inhibit or slow down the infection that occurs due to gil-coccal diseases by administering the vaccine composition of this invention. The therapeutically effective quantity defined in this invention refers to the dose required to activate the formation of Antibodies to a level at which Sa reduces the chance of infection with pneumonia or the risk of infection. As used herein, the term “administration” refers to the introduction of a predetermined substance into a person by any appropriate method. The vaccine composition of this invention may be administered by Oral; nasal; rectal; transdermal; or by inhalation using a sprayer; or alternatively, the vaccine formulation may be administered once or may be administered slowly, for example, but not limited to. intramuscularly; into the peritoneal membrane; into the skin or under the skin; or administration into mucous membranes Oral/gastrointestinal tract Respiratory or genitourinary tract In one embodiment: Intranasal administration for gill or otitis media. In that case, it can effectively prevent 5 carrying pneumococcal bacteria; This will reduce the infection in its initial stages. As used in this invention.” The term “person” refers to organisms that can be infected with pathogens; And Bashir on dag in particular for perching vertebrates, for example, but not limited to. In another embodiment of this invention; The composition of this invention can be given once; or 0 can be given as a number 2 3; 4 or more times at appropriately spaced intervals; This is for example but not limited to. For example; the routine regimen for infants and children against invasive diseases caused by Streptococcus pneumoniae can be at 2 ed 6”; And 12 to 15 months of age. add to that; The construct may include one or more Lad proteins that are derived from 5-Metrococcus pneumoniae. Examples of Streptococcus pneumoniae proteins suitable for administration in combination include those defined in International Patent Application No. 053761/2002 (WO) as well as those

mentioned in International Patent Application No. 083855/2002 170; And those proteins can also be made

included within the scope of this invention.

In a perfect embodiment of this invention, the composition of the 13-valent vaccine (so-called

77) to prevent disease gill in a total amount of 0.5 mL; mixing 2.2 µg of each polyconjugate; Except for 4.4 mcg of 63; About 29.3 mcg of carrier protein

7 :)); 0.5 mg elemental aluminum (2 mg ammonium phosphate) as adjuvant;

about 4.25 milligrams sodium chloride (with preservative) or about 3.5 milligrams sodium chloride

sodium (without preservative); about 295 mcg of succinate buffer solution; and about 3

Milligrams of 2-phenoxyethanol and about 60 micrograms of formaldehyde (with preservative).

0 In an exemplary embodiment AT of this pla) a 14-valent vaccine formulation (labeled 8747)) has been prepared to prevent pulmonary disease; in a total amount of 0.5 mL; mixing 2.2 µg of each Su poly; Lad except for 4.4 µg of 6[3; about 31 mcg of carrier protein 7 :));+; 0.5 mg elemental aluminum (2 mg ammonium phosphate) as adjuvant; about 4.25 milligrams sodium chloride (with preservative) or about 3.5 milligrams sodium chloride

5 sodium (without preservative); about 295 mcg of succinate buffer solution; and about 3 milligrams of 2-phenoxyethanol and about 60 micrograms of formaldehyde (with preservative). In another Je embodiment of this invention; It has been confirmed that with regard to the rabbit serum immunized with the vaccine formulations of this invention; The concentration of 150 directed against the serotype was higher than when Prevenarl13® was administered (Table 1a and 1b). add to that; The endocytosis analysis

0 Opsoni (OPA) also confirmed that the vaccine formulation of this invention had higher levels of functional antibodies compared to Prevenarl3® (Table 12 and Table 2b). And accordingly; It can be strongly estimated that the vaccine formulations of this invention have a significant effect on preventing pneumonia diseases. Another manifestation of this invention is an immunostimulant complex directed against coliform bacteria

5 pneumonia; Which contain 13 types or 14 types of conjugates consisting of a polysaccharide of the capsule and a carrier protein.

The thirteen species or fourteen species of associations of pneumococcal bacteria are the same

those aforementioned. The composition of this invention, which contains 13 types of DUE, contains a polysaccharide — carrier protein; It contains the capsule-specific polysaccharide derived from Streptococcus Wes 5 in 13 different serotypes. add to that; when giving the composition into the body; It is recognized as an antigen and due to an immune response so that an antibody directed against that antigen is produced. According to oll, the composition of this invention can be used as an anti-bacterial elie

rheumatoid arthritis. Still another manifestation of this invention is a way to prevent diseases caused by

0 pneumococcal bacteria by giving a vaccine or immunocomplex to the person who needs this treatment. Still another manifestation of this invention is to prepare a method for using a vaccine composition preparation to prevent diseases caused by streptococci; Ally contains 13 types of conjugations consisting of a polysugar capsular-protein dala, as in the ten types of EN conjugates, each

5. ADEN species ten capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1 » 3 » 4 « 5 » 19F » 198 » 180 » 14 » 9177 » TF » 6A » and 2317 are covalently coupled to a carrier protein; The carrier protein is protein 0111197; Each of the conjugates has a structure in which both the capsular polysaccharide and the carrier protein are bonded via the -O-C(NH)-NH- group using the cyanide method.

0 Still another aspect of this invention is to prepare a method for using a vaccine formulation to prevent diseases caused by haemococcal bacteria; Ally contains 14 types of polysaccharide conjugates - a carrier protein; Since it is in the fourteen types of couplings; Each of the fourteen species of capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1” 2 3 2 4 ¢ 5 19A ¢ 18C +14 ¢9V TF ¢ 6A ¢ 19+ and

237 covalently coupled to a carrier protein; The carrier protein is protein 0141197; And everyone who

— 2 4 —

The conjugates have a structure in which both the capsular polysaccharide and the carrier protein are

They are bonded by the -O-C(NH)-NH- group using the cyanide method.

The composition of the vaccine is the composition of the immunostimulant; And prevent pneumonia diseases as already mentioned.

There is still an AT aspect of this invention which is a method of preparing an immunostimulating composition; It contains the conjugation step of each of the thirteen types of separated multi-capsule lull

Derived from Streptococcus pneumoniae serotypes L3 4 5 “<OV”TF “6A 14

19A “18C – 19F” and 23 with “CRM197”, a protein (dela) using the method of adding

cyanide so that the polycapsular sugar and the carrier protein form a structure which binds to

The -O-C(NH)-NH- group path.

0 No Jin There is an AT aspect of this invention which is a method of preparing an immunostimulating composition; It contains the conjugation step of each of the fourteen types of capsular, uncapsulated polysaccharides derived from Streptococcus pneumoniae serotypes 01 2 3 4 5 Z10 A 14 19A “18C-19F” and 23 with “CRM197,” a protein (dela) by using the cyanide addition method so that the polycapsular sugar and the carrier protein form a structure that binds to

5 group road -0-00111-1111- . The composition of the immunostimulant and the method of preparation are described as previously mentioned. [Favourable Effects] The formulations according to this invention contain a capsule polysaccharide derived from Streptococcus pneumoniae of 13 different serotypes; Or Lad contains a poly-capsular sugar derived from

0 Metretococcus pneumoniae serotype 2, as each of these capsular polysaccharides conjugates with “CCRMI197”, which is a protein (dela) using the cyanide method; this leads to an increase in the level of 150 directed against it and the activity of the functional antibody. And on ld The vaccine and immunogen formulation according to this invention can be used preferentially to prevent diseases caused by pneumococcal bacteria in infants, young children and adults.

5 Detailed Description:

Lad Chay follows this invention in detail using the accompanying exemplary embodiments shown herein for illustrative purposes only and should not be considered to be specific to the scope of this invention. EXAMPLE 1: Polysaccharide preparation of the Streptococcus pneumonia capsule 1-1 Cell bank preparation Streptococcus pneumonia strains were isolated on LAL blood medium to separate a single colony. After selecting a single colony with a good growth rate out of 10 or more terminators, the colony was then inoculated and cultured in Bile medium which did not contain any animal-derived components; a Research Cell Bank (RCB) was set up to create a stock Of glycerol One vial was taken from the research cell bank, which was confirmed by gene expression to produce polysaccharides 0 of the desired serotype, after which the cells were cultured in a liquid medium that did not contain any animal-derived components, and after that; glycerol was added Reductase to prepare the main cell bank In addition, one vial was taken from the main cell bank, and the cells were multiplied in a liquid medium that did not contain any animal-derived ingredients. Storage of the cell bank prepared under ultralow conditions 5 Freezing at -70 °C or less used 2-1 fermentation and polysaccharide separation One vial of the working cell bank was melted and this was placed in a liquid medium containing no ingredients derived from animals.The culture was seeded at 2437°C without stirring until obtaining the desired optical density (01600). And after making sure that the center of the farm is; at which 0 the seeding of the farm is completed; was contaminated, the product was fortified in a fermentation unit containing JL medium that did not contain animal-derived ingredients. cell aay, the main culture was carried out while maintaining the pH of the medium using a sterile potassium hydroxide solution at 2237 °C with minimal stirring conditions. The concentration of cells in the culture medium and the concentration of glucose contained in the medium were measured every 2 hours and culture was completed at set sé 5 when the glucose concentration in the medium was low.

— 2 6 —

And after the completion of agriculture; An appropriate amount of sterile sodium deoxycholate 9612 was added to the culture to a final concentration of 960.12 to activate immunolysis; dag, the cell-bound polysaccharides are released. 3-1 Purification of the polysaccharides of the capsule

Phosphorbic acid was added to samples treated with sodium deoxycholate for one hour; The supernatant was obtained by centrifugation. The supernatant solution that was obtained was passed ade through a filter (Gee with pla) that was concentrated and the buffer was replaced with a buffer solution of phosphoric acid 0 and the buffer was replaced by ¢ 28 samples were passed through an activated SS filter and removed bugs in the following two ways.

1) Since 2 serotypes, 21 3 4 5 “6A Qo 9 0180 19A 19F and 2317, had the ability to bind ionically with cetyltrimethylammonium bromide (CTAB), the method was implemented CTAB Treatment was carried out by centrifugation (CTAB; sodium chloride (NaCl) and sodium iodide (Nal) and centrifugation methods).

2) Two serotypes interacted »147717; And who did not react with “CTAB” by adding a gel solution

5 ammonium phosphate (Algel) and then the supernatant obtained from centrifugation was used. As for the two aforementioned impurities removal steps, the samples were entered into a process of deep filtration and ultrafiltration/diafiltration (UF/DF). The samples were delle treated with ethanol and sodium chloride to form a powder of polycapsular sugar.

0 - Example 2: Conjugation preparation of Streptococcus pneumoniae and protein 1-2 Comparison of the reductive and cyanide method The conjugation productivity (ie, amine reduction and cyanide) was compared using purified polysaccharide 7 and carrier protein 018/1197. The specific associations are as follows.

(1) Amina reduction method

11.7 mg periodate was added to the concentrated solution of polysaccharide (OV) and the polysaccharides were activated with the mixture stirred at a temperature ranging from 21 °C to 25 °C. The oxidized polysaccharides were concentrated and filtered using an ultrafiltration filter (100 dang that; the remaining polysaccharide was mixed with CRM197 at a ratio of polysaccharide (181/197) = 0.5 and lyophilized. The lyophilized complex was melted and fixed (ah) at a temperature ranging from 21 °C to 25 °C. The equilibrium complex was dissolved and incubated (2437 degrees (Lge) in a buffer containing sodium phosphate (B0fold21) at a ratio of 0.1 M per 20 ala of sugars; after that, cyanoborohydride (100 mg/mL) was added to start the conjugation reaction. the incubation at 2237 °C for about 44 0 hours to 52 hours; The temperature was reduced to +23°C and a 960.9 solution of sodium chloride (del) was added to the reactor. Sodium borohydride solution (100 mg/mL) was added to obtain 1.8 molar to 2.2 molar equivalents of sodium borohydride per 1 mole of sugars. The mixture was then incubated with stirring at 23+2°C to reduce any amount of unreacted aldehydes present in the sugars. The mixture was diluted with a 9060.9 solution of 5 NaCl (5 mL); dag the dilute conjugate mixture was filtered using a MWCO membrane (100 kDa). (2) Method of adding cyanide A 2 M solution of polysaccharide sodium chloride was prepared by adding sodium chloride powders to a concentrated solution of polysaccharide 917, which was prepared without hydrolysis. methylaminopyridinoem tetrafluorourate (CDAP) 960.5 w/w for polysaccharides was added to a concentrated solution of OV polysaccharide and then stirred for 15 minutes to activate the polysaccharide After that, sodium hydroxide solution was added until The pH was reached at 0.1 + 9.5; after that, it was stirred for 3 minutes to fully activate the hydroxyl group of the polysaccharides by CDAP. of polysaccharides” and the OEY reaction was carried out at room temperature for one hour. In the (GUE) reaction a 2 M solution of glycine was added in the amount of 3 equivalents

— 8 2 — molarity with respect to 1 molar equivalent of “CDAP” and the pH was adjusted to 9.0 followed by the incubation of the results throughout (Jalil) and thus the reaction was completed. After the conjugation reaction was completed, it was concentrated and filtered through the FW filtrate with a buffer solution containing 0.9% sodium chloride.

As a result; It was confirmed that the productivity of the conjugates prepared by the cyanide method was at least 4 times the ef of the conjugates prepared by the reductive amine method. Accordingly, the inventors of this invention prepared the conjugations of encapsulated polysaccharides and 81/197© using the cyanide method.

2-2 Conjugation and conjugation purification 0 The method for preparing the conjugation of the encapsulated polysaccharides of Streptococcus pneumoniae 5 CRM197 is carried out in the following six steps. Step 1-1 Dissolution and hydrolysis of the thirteen capsule polysaccharides al. The polysaccharides of the capsules obtained from each type of serum were dissolved in water for injection so that the final concentration was as described by can Lad, and then filtered through 5 filter (0.45 μm). In particular, dag, the filtration process was carried out by dissolving capsule polysaccharide powders in the range of 0.8 mg/ml to 2.0 mg/ml for serotypes (1 033) and 4; in the range of 4 mg/mL to 8 mg/mL for serotypes 5; 9V ¢ 6B; 180 and 1917; within ranges from 8 mg/mL to 12 mg/mL for serotypes 6A and /19; and in the range of 0 from 2 mg/mL to 4 mg/mL; for serotypes (TF 14 and 2315). The solution was incubated within the limits of pH and temperature described later for each serotype; after that hydrolysis was carried out. dag deg in particular; for serotypes 23F ¢ 14 « 6B ¢ 5 ¢ 3 1 The incubation was carried out at a temperature ranging from 70 °C to 50 °C for a period of 1 hour to 4 hours; for serotypes 17 and 18 the incubation was carried out at a pH value of 2 at a temperature of 65 °C

— 9 2 — to 80 °C s.d. for 1 to 3 hours using a phosphoric acid solution. The hydrolysis process was then interrupted by cooling the solution to a temperature of 21°C to 24°C and adding NaOH until a target pH value of 1<6 was reached. Sl lysis was not performed for serotypes 4 19A. Step 2.1 Dissolution and Hydrolysis of the 14 polysaccharides of the capsule al The polysaccharides of the capsules obtained from each type of serum were dissolved in water for injection so that the final concentration was as described below; Then it was filtered through a filter (0.45 μm). In particular, dag » 208 The filtration process was carried out by dissolving capsule polysaccharide powders in the range from 0.8 mg/ml to 2.0 mg/ml for serotypes 1, 2, 6, 3 + and 4; in the range of 4 mg/mL to 8 mg/mL for serotypes 5; 9V ¢ 6B; 180 and 1917; within ranges from 8 mg/mL to 12 mg/mL for serotypes 6A and 19/8; and within limits ranging from 2 mg/mL to 4 mg/mL; for serotypes (TF 14; and 2317; within ranges from 6 mg/mL to 10 mg/mL for serotype TF 5) It has been known that conjugate vaccine-induced immune activation is generally negatively affected by an increase in parity due to crosstalk. Vaccines, 28(5513); Glycoconjugate vaccines and immune (2010) (interference: A review) In order to reduce the loss of immune activity and improve the immunogenic activity of a (LBVEO14) The inventors of this invention used different Sle hydrolysis steps of serotype 0 specific to polysaccharides in dime serotypes (serotypes 3 4 “TF” 6A) to optimize the overall immune activity. The hydrolysis was performed for the solution within the limits of a pH value and a certain temperature, and for the conditions of the total sample of the serotype for hydrolysis are as described later. temperature ranging from 70 ° C to 80 ° C overnight; and for serotype “191; incubation was carried out at a temperature ranging from 70 ° C to

0 °C for 1 to 4 hours; for serotypes 3 «OV » TF and 180; The incubation was carried out at pH 2 at a temperature of 65 °C to 80 °C for 1 to 3 hours using a phosphoric acid solution. Hydrolysis was stopped by cooling the solution to 21°C to 24°C and adding NaOH until the target pH value of 1+6 was reached. Hydrolysis of serotype/19 was not performed. Step 2 Conjugation Reaction of the Capsular Polysaccharide 5 CRM197 A 2 M solution of polysaccharide in NaCl was prepared by adding NaCl powder to each serotype. Dissolve 1-cyano-4-dimethylaminopyridinium tetrafluorosorbate 0 appropriate (CDAP) per serum in a ratio of 1 aba of CDAP per 100 mL of 50/50 acetonitrile/Water for injection (vol/vol). og in particular; CDAP dissolved at a ratio of 1 w/96 for the polysaccharide of serotypes 6/8 and 14; CDAP was dissolved in a ratio of 962 w/w for serotypes 2 and 4; CDAP was solubilized at a ratio of 963 w/w for serotypes 19A5 » 7F » 68 » 3 1 ; CDAP had a solubilization ratio of 964 w/w for 5 serotypes; 18C 97 5 ; 197; and 237. Each of them was added after dissolution to each polysaccharide solution. dag a period of 1 minute to 3 minutes; Sodium hydroxide was added to the solutions to raise the pH to 9.4 to 9.7; The mixture was stirred for 3 minutes to 7 minutes so that the hydroxyl group of the polysaccharide was sufficiently activated by 17/00. 0.55 w/w was added to 901 w/w from 01821197 for polysaccharides; For each serotype of the 0 polysaccharide solutions; The conjugation reaction was carried out for 1 to 4 hours. add to that; The conversion rate of the reaction was measured using HPLC-SEC and additional CDAP was added as necessary. Step 3 Completion of the coupling reaction Add 3 molars to 6 molars of glycine solution with respect to 1 molarity of 5 molars of CDAP which was added to all serotypes, and then the reaction was completed by adjusting the pH to 9. The coupling solution was stirred at a temperature of 21°C.

— 1 3 — Refrigerate at 24°C for 1 hour and then store overnight at a low temperature of 2°C to 8°C. Step 4 Ultrafiltration The dilute conjugate solution was concentrated and double-filtrated through an ultrafiltration filter with a minimum volume of 20 volumes of buffer. and especially dag; The buffer solution was kept at a pH value ranging from 5.5 to 6.5 and the buffer containing 760.9 NaCl was used. The molecular weight cutoff of the ultrafiltration filter was 300 kDa for all serotypes; The permeable solution has been discarded. Step 5: Sterile filtrate 0" galls retained after double filtration were diluted to a concentration of less than 0.4 g/L on a polysugar concentration basis using buffer; filtered through a filter (0.22 microns). Quality control has been implemented at Process oli (sugar content, DMAP, residual) on the filtered product Quality control of the method is performed on the filtered residue to determine if additional concentration, double filtration and/or dilution is needed 5 Step 6: Adsorption Aluminum salt (which mainly consists of aluminum phosphate) was added to the sterile filtrate so that the final concentration of aluminum ion became 1 mg/mL, and additional amounts of salt were added to maintain the pH value from 5.5 to 6.5._ The concentrated solution was evaluated, in which the Adsorption completed, to determine if it is of adequate quality and cooled at a temperature of 2 0 °C to 8 °C until use Example 3: Vaccine formulation with conjugation of P. aureus formulations 1-3: Streptococcus conjugate formulation pneumoniae thirteen valence (LBVEO13) The required amount of final mass concentration was calculated based on the batch size and the mass polysaccharide concentration. After adding the required amount of 960.85 NaCl; Succinate solution

regulator » 2-phenoxyethanol; formaldehyde to the previously labeled mounting vessel; Mass focus has been added. This is considered; The products were mixed well and filtered through a filter (0.22 μm). The prepared solution has been mixed slowly; Ammonium phosphate bulk (cad) was added, followed by good mixing of the mixture. The pH has been checked and adjusted where necessary. The processed mass product was stored at a temperature ranging from 2°C to 8°C. The composition of the vaccine obtained (hereinafter referred to as 1.317120137) contained; as a total volume in 0.5 mL; Contains 2.2 µg of each sugar Lad canis minus 4.4 µg of 6B about 29.3 µg_ of carrier protein 7:+; 0.5 mg elemental aluminum (2 mg aluminum phosphate) adjuvant; 0 and about 4.25 mg NaCl; about 295 mcg of succinate buffer solution; and about 3 milligrams of *2-phenoxyethanol and about 60 micrograms of *formaldehyde (*used when a preservative is added). 2.3 Composition of Pneumococcal Conjugate Vaccine XIV <$ (LBVEO14) The required amount of final mass concentration was calculated based on the batch size and the poly-mass 5 glucose concentration. After adding the required amount of 960.85 NaCl; succinate buffer solution » 2-phenoxyethanol; formaldehyde to the previously labeled mounting vessel; Mass focus has been added. This is considered; The products were mixed well and filtered through a filter (0.22 μm). The prepared solution has been mixed slowly; Ammonium phosphate bulk (cad) was added, followed by good mixing of the mixture. The pH has been checked and adjusted where necessary. The prepared 0 "mass product was stored at a temperature ranging from 2 °C to 8 °C. The obtained inoculum composition (hereinafter referred to as 1.317120147) contained; as a whole at 0.5 « de contains 2.2 µg of each connie sugar except for 4.4 µg of 6B; about 31 µg of carrier protein 811197; 0.5 mg of cofactor aluminum (2 mg of aluminum phosphate); about 4.25 5 mg of sodium chloride; and about 295 mcg of succinate buffer; approximately 3 mg of *2-phenoxyethanol and approximately 60 mcg of *formaldehyde (*used when preservative is added).

EXAMPLE 4: EVALUATION OF THE ABILITY OF LBVEO14 5 LBVEOI3 TO ACTIVATE THE IMMUNE SYSTEM Research was carried out to evaluate whether the polyvalent pneumococcal (LBVEO14 SLBVEO13) vaccine formulations prepared in Example 3 have the potential to activate the immune response in rabbits These immunogenic effects were confirmed by measuring the concentration of 180 in serum by antigen-targeted ELISA and the level of functional antibodies by Oyson endocytosis analysis (OPA) New Zealand white rabbits vaccinated intramuscularly at week 0, week 2 and week 4 at a planned dose of one of the two combinations 1317285013 or 13172014 administered to humans or the comparator 0” Prevenarl3® (2.2 µg of all Ld amie Su except 6B at 4.4 µg). Sera samples were taken every two weeks after immunization All 186 serum concentrations were measured using ELISA analysis dug Results are shown in Tables 1 and 1 [Ap] These results are described in detail below 5 1-4: Determination of directed IgG concentration Against the administered vaccine, each of the capsule polysaccharides of each of the two serotypes of haemophilus bacteria was placed in a 96-well plate at 5 μg/eye at room temperature for 16 hours. In order for the nonspecific response between antigen and antibody to be reduced to a minimum level; Serum from each rabbit was adsorbed with 333.3 µg/mL C-PS and 333.3 0 µg/mL serotype 221 capsule-specific polysaccharide (PnPs 22F) at room temperature s.a.d. 30 min. dag that; The products were appropriately diluted with buffer buffer containing antibody dilution containing Tween 20. The coated plate was washed 4 times with buffer wash; Then 50 µL of pre-adsorbed and diluted serum was added to the plate; Follow this up by letting the reaction proceed at room temperature for 1 hour. The reaction plate 5 was washed 4 times in the same way; aang that added conjunctions 150-117 of goat

— 4 3 — and directed against rabbit (1: 2000) for each eye. “day”; the reaction was carried out at room Sha degree for 30 minutes. The plate was washed 4 times in the same way as previously mentioned. 100 were added μl of TMB solution fixed at room temperature per eye and allowed to react at room temperature for 15 min 100 μl of 1 N solution of sulfuric acid was added to stop the reaction Absorbances were measured at 450 nm and 650 nm As a comparator The serum obtained by immunizing a rabbit with Prevenar13® was also analyzed.As a result, it was unexpectedly found that the pattern of immune response in each of the 3 serotypes was significantly different from that of Prevenarl®3 (Table 2). i and 1[b); which shows that LBVEOI3 is an immunologically different composition than Prevenarl3® despite 0 sharing the same serotype; and that cohorts vaccinated with 1.317013 have a higher level of IgG concentration in each J of serotypes compared to Prevenarl® 3. In particular, dag » 1 TF » 6B » 957 » 14 » and 1917 serotypes were found to have produced 2 and 6 times higher concentrations of IgG compared to Prevenar13® (Table 1a and Table 1b). In ob 131711014 dl serotypes 1” 4 ¢ 5 ¢ 71 » 319A ¢ 18C ™ 14 ¢ 9V 19F 5 produced 2- and 6-fold higher concentrations of 1850 compared to serotypes 1" 2660213 (Table 1a and Table 1) B). [Table 1a] gl concentration 180 | gall | Serum IgG concentration | Kia) and g/ml) | worshiper | (µg/ml) AH |

— 5 3 — 19F 59.5 6B 17.6 [Table]=[ LBVEO!4 Prayer | (μg/mL) | worshiper | (µg/ml) 1203 © _ © 9 -

sms | a | aq es [5[ 27 [2] - *Serotype 2 is not included in Prevenar13®: 2-4OPA) Determination of functional antibody level (Oisonic endocytosis analysis) Levels of functional antibody activated by antigens were assessed that belong to a particular serotype by performing OPA analysis with serum obtained from rabbits immunized with B

Prevenar13®4 LBVEO14 JLBVEO13 5 . In particular, equal amounts of samples were taken from each rabbit and grouped. Streptococcus pneumoniae was cultured in THY medium (Todd-Hewitt's medium with 2% yeast extract) of each serotype and diluted with Osonic endocytosis buffer to 200 cfu/10 μl to 300 cfu 0 colony/10 µL._ 20 µL_ of diluted serum and 10 µL of diluted Streptococcus pneumoniae were mixed and incubated at room temperature for 30 min. This is considered; 50 ily Sie was added from a mixture of previously differentiated WIS 111-60 and complement (cell:complement ratio = 4:1) and the reaction was incubated in crion dioxide (37 °C) for 45 minutes. The temperature was reduced to terminate endocytosis and 10 μL of the reaction solution was smeared onto a pre-dried agar plate for 30 min to 60 min. The plate was incubated in a carbon dioxide incubation (37 °C) for a period of 12 hours to 18 hours ay so that the colonies were counted. The OPA concentration was expressed as the dilution rate at which a kill ratio of 9650 was observed (Table 2: OPA concentration equal to 2187 shows that the concentration was not able to reach the level of 9650 even in the most dilute region compared to the negative comparator; Wherever it is

0 concentration is too high). [Table 2]

Serotype

evening

(nw || cells eg) It was confirmed from previous experimental results that the OPA concentration of the 13-valent immunoactivating complex of this invention had a higher level of working antibodies compared to what was found with 2:67602139. Accordingly. The immunostimulating combination of this invention can be very effective for preventing the disease caused by Streptococcus pneumoniae.

5 ([Table 2b]

Urgent revenarl 3 LBVEOI14

Cr[we[ee| Ce [en [we |] Based on the results from Table 2, it is clear that the level of each functional antibody in the rabbit serum immunized with B1.31712014 is higher than the level of the functional antibody in the rabbit serum immunized with B©2:67602:13. According to UN, the combination of immunostimulation based on this invention can be highly effective in preventing diseases caused by Strettococcus pneumoniae.

Whilst this invention is described with reference to particular illustrated embodiments; It will be understood by those with experience in the field of this invention that this invention can be implemented in a certain other form Og deviating from the spirit and scope or basic characteristics of this invention. And on that; The aforementioned embodiments are illustrative and not specific to the scope of this invention. And also; The scope of this invention is defined by the appended claims rather than a full description; It shall be understood that all modifications and alterations derived from the meanings and scope of this invention shall be made

included in the scope of the appended claims.

Claims (1)

Hamama elements 1. Formulating a vaccine to prevent Cus pneumococcal diseases that includes 14 types of capsular polysaccharide-carrier protein conjugates. .protein conjugates where; In the 14 species of conjugates, each of the 14 species of capsular polysaccharides is derived from the serotypes of Streptococcus (TF “6B” 6A “5” 4 3 2 “1 Streptococcus pneumoniae ge gah s14 ¢ 18C ¢ drug 23F 5 » 19F is covalently conjugated with a carrier protein ¢carrier protein The carrier protein is protein 814197©; each of the conjugates has a structure in which each of the polysaccharides is 0 The capsular polysaccharides and the carrier protein are linked via the -O-C(NH)-NH- group using the a cyanylation method. 2. Synthesis according to claim [Gus] that the cyanylation method is carried out using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) or :03. 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) or CNBr 5 3 Composition according to claim 1; which includes at least one chosen from the set consisting of the sale adjuvant preservative ; buffer solution ¢ cryoprotectant; salt; cdivalent cation non-ionic detergent non- jonic detergent 0 » and free radical oxidation inhibitor 4 Composition Gy of protection element 3 where the adjuvant is chosen from the group consisting of Caluminum phosphate, caluminum sulfate, aluminum hydroxide, and mixtures thereof. 5. Composition according to claim 3) wherein the preservative is 2-phenoxyethanol formaldehyde or mixtures thereof. 6 Composition Wy of claim 1 which includes Load dela Pharmaceutically acceptable .physiologically acceptable carrier 5 7. Composition Gy of claim 1 wherein; As for the capsular polysaccharides derived from “serotype 1 from serotype capsular polysaccharide, each of the capsular polysaccharides is derived from serotypes 0 32 4” Mf TF “Tag 14-19F” 19A “18C” and 2385 It has a content ratio of 0.9 to 1. The capsular polysaccharide derived from serotype 68 has a ratio of 2.2 from 1.8 to sina legs 14 Streptococcus pneumoniae includes an immunocomplex against Streptococcus pneumoniae 8. 5 capsular polysaccharide-carrier protein ¢conjugates whereby; In the fourteen species of conjugates, each of the fourteen species of capsular polysaccharides is derived from serotypes of Streptococcus pneumoniae 0 1 2 3 qv 5 (TF 6B 6A s14 ¢ 180 drug 23F 5 » 19F covalently conjugate with a carrier protein ¢ The carrier protein is protein 814197©; each of the conjugates has a structure in which each of the capsular polysaccharides The polysaccharides and the carrier protein are linked via the -O-C(NH)-NH- group 5 using the cyanylation method 8. 9. A method for preparing an immunoogenic composition in accordance with claim 8; Cus The conjugating step includes each of the 14 species of isolated capsular polysaccharides — 2 4 — isolated capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1” 4 3” 4K 5” 6A “qw 19A 180 ¢ 14 ¢ 9V “TF ¢ 23F 3 ¢ 19F with “CRMI197 is a carrier protein ¢ using the cyanylation method so that the capsular polysaccharide and the carrier protein form a structure that binds through the group -1111-(0-0011- . The Saudi Authority for Intellectual Property Sweden Authority for Intellectual Property pW RE .¥ + \ A 0 § Um 5 + < Ne ge “Benaj > Aye Ki Jada Li Days TEE Bbha Nase eg + Ed - 2 - 3 .++ .* provided that the annual financial fee is paid for the patent and that it is not null and void due to its violation of any of the provisions of the patent system, layout designs of integrated circuits, plant varieties and industrial designs or its implementing regulations. »> Issued by + BB 0.B Saudi Authority for Intellectual Property > > > “+ PO Box 1011 .| for ria 1*1 uo ; Kingdom | Arabic | For Saudi Arabia, SAIP@SAIP.GOV.SA