KR20200033525A - Novel Group B Streptococcus Serotype Strain and Immunogenic Composition Comprising the Same - Google Patents

Abstract

The present invention relates to a novel group B Streptococcus serotype strain, which can simultaneously evaluate the immunogenicity of several serotypes included in type B Streptococcus vaccine in one experiment, and to an immunogenic composition comprising the same. The group B Streptococcus serotype strain of accession numbers KCTC 13365BP and KCTC 13366BP of the present invention has high serotype specific antigenicity and selective antibiotic resistance, thereby being usefully used to evaluate the serotype specific immunity of GBS vaccine by a multiplexed opsonophagocytic assay (MOPA).

Description

Novel Group B Streptococcus Serotype Strain and Immunogenic Composition Comprising the Same}

The present invention relates to a new group B streptococci serotype strain and an immunogenic composition comprising the same, which can simultaneously evaluate the immunogenicity of several serotypes included in the type B streptococcus vaccine in a single experiment. No. KCTC 13365BP or KCTC 13366BP relates to a group B streptococci serotype strain and an immunogenic composition for preventing diseases caused by group B streptococci comprising the strain.

Invasive infectious diseases (Hib, pneumococcal, etc.) in infants and children have been significantly reduced by vaccination, but group B streptococci (GBS) still has a very high rate of mortality and complications in newborns and infants.

According to recent research results in Europe and the United States, it has been reported that there are increasing cases of severe infections caused by streptococci in adults and the elderly.

Currently, vaccine development to prevent GBS infection (protein vaccine, protein-polysaccharide conjugate vaccine) and clinical trials have been actively conducted around large pharmaceutical companies, but tests to evaluate the immunogenicity of GBS still in Korea and abroad Since the law has not been properly established, it is thought that it will be very difficult to respond effectively if a vaccine is spread in the future.

Basic research related to infection mechanism, immunological response, and genomic research according to serotypes for the development of GBS vaccines is actively being conducted, and it is possible to provide important information for basic research on vaccines in the development of cotton relevance evaluation technology.

Therefore, in order to evaluate the immunity to GBS in the high-risk group and to introduce a clear immunogenicity of the vaccine after the introduction of the GBS vaccine, it is essential to establish a standardized test method.

Recently, prenatal GBS culture screening for high-risk pregnant women and neonatal antibiotic prophylaxis (IAP) have been effective in reducing early-onset disease (EOD), but not in removing them (Phares CR et al., JAMA ., 299 (17): 2056-65, 2008; Van Dyke MK et al., N Engl J Med ., 360 (25): 2626-36, 2009). Emerging infections program network. [Accessed 2013 Apr 28] .https: //www.cdc.gov/abcs/reports-findings/survreports/gbs13.html).

Therefore, considering the pitfalls of the current prenatal strategy and the burden of GBS infection in the elderly, it is urgently needed to develop an effective GBS vaccine and to evaluate the immunogenicity of the vaccine.

Thus, the present inventors tried to find a group B streptococcal serotype Ia, III, and V strains having serotype specific antigenicity and selective antibiotic resistance, as a result, group B streptococcus serum of accession numbers ATCC 12401, KCTC 13365BP and KCTC 13366BP Since the type strain has high immunogenicity, it was confirmed that it exhibits a high antibody titer, and the present invention was completed.

An object of the present invention is to provide a group B streptococcus serotype strain that can efficiently evaluate vaccine immunogenicity.

Another object of the present invention is to provide an immunogenic composition for preventing diseases caused by group B streptococci, including the group B streptococci serotype strain.

Another object of the present invention is to provide an immunoassay method comprising the step of determining whether or not immunity to the subject after immunizing the group B streptococcus serotype strain.

Another object of the present invention is to provide a vaccine composition for preventing diseases caused by group B streptococci, including the group B streptococci serotype strain.

In order to achieve the above object, the present invention provides a group B streptococcal serotype strain selected from the group consisting of accession numbers ATCC 12401, KCTC 13365BP and KCTC 13366BP.

The present invention also provides an immunogenic composition for preventing diseases caused by group B streptococcus, comprising at least one group B streptococci serotype strain selected from the group consisting of accession numbers ATCC 12401, KCTC 13365BP and KCTC 13366BP.

The present invention also provides an immunoassay method comprising the step of determining whether immunity after immunizing a subject with at least one group B streptococcal serotype strain selected from the group consisting of accession numbers ATCC 12401, KCTC 13365BP and KCTC 13366BP. .

The present invention also provides a vaccine composition comprising an immunogenic composition for preventing diseases caused by group B streptococci including one or more group B streptococci serotype strains selected from the group consisting of accession numbers ATCC 12401, KCTC 13365BP and KCTC 13366BP to provide.

Group B streptococcal serotype strains of accession numbers ATCC 12401, KCTC 13365BP and KCTC 13366BP of the present invention have high serotype specific antigenicity and selective antibiotic resistance, thereby multiplying the functional characteristics of the serotype specific immunity of the GBS vaccine. It can be usefully used to evaluate by an experimental method (multiplexed opsonophagocytic assay, MOPA). In addition, the vaccine composition for preventing diseases caused by group B streptococcus containing the strain not only suppresses the GBS colony of the mother when inoculating the GBS vaccine to women or mothers of childbearing age, but also delivers antibodies from the mother to the newborn through the placenta. Prevention of even invasive GBS infection in infants can be expected.

1 is a result of observing the complement concentration optimized for the GBS (group B Streptococcus) MOPA (multiplexed opsonophagocytic killing assay) test of the present invention.
2 is a result confirming the specificity (specificity) of the GBS MOPA test of the present invention.
3 is a result confirming the accuracy (accuracy) of the GBS MOPA test method of the present invention.
4 is a result confirming the accuracy (accuracy) of the GBS MOPA test method of the present invention through a multi-center.
Figure 5 is a comparison of the basic OPA titer (opsonic index, OI) for GB S serotype Ia in the high-risk group, such as neonatal / infant, pregnant women, aged 65 years or older of the present invention ((A) average value comparison, (B) OI Of distributions).
Figure 6 is a comparison of the basic OPA titer (opsonic index, OI) for GB S serotype III in the high-risk group, such as neonatal / infant, pregnant women, aged 65 years or older of the present invention ((A) average value comparison, (B) OI Of distributions).
Figure 7 is a comparison of the basic OPA titer (opsonic index, OI) for GB S serotype V in the high-risk group, such as neonatal / infant, pregnant women, aged 65 years or older of the present invention ((A) average value comparison, (B) OI Of distributions).
8 is a comparison of the basic OPA titer (opsonic index, OI) for each GBS serotype in pregnant women: comparison of OI distribution according to whether GBS perineum colonies occurred during prenatal examination at 35-37 weeks of pregnancy (GBS colony group, blue circle; GBS) Unpopulated group, red circle).
9 is an analysis of the correlation between the GBS MOPA and ELISA test results of the present invention.

In the present invention, since the group B streptococcal serotype strains of accession numbers KCTC 13365BP and KCTC 13366BP have serotype specific antigenicity and selective antibiotic resistance, the serotype specific immunity of the GBS vaccine is multifunctional antibody titer assay (multiplexed opsonophagocytic assay, MOPA).

Therefore, in one aspect, the present invention relates to group B streptococci serotype strain of accession number KCTC 13365BP or KCTC 13366BP.

In the present invention, the strain may be characterized by displaying the 16s RNA nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In the present invention, the accession number KCTC 13365BP strain may be characterized as having spectinomycin antibiotic resistance, and may be characterized as serotype III.

In the present invention, the accession number KCTC 13365BP strain is characterized in that Streptococcus agalactiae KGP0296.

In the present invention, the accession number KCTC 13365BP strain is characterized by being represented by the 16s RNA nucleotide sequence of SEQ ID NO: 1, and was named SPEC-III.

In the present invention, the accession number KCTC 13366BP strain may be characterized as having kanamycin antibiotic resistance, and may be characterized as serotype V.

In the present invention, the accession number KCTC 13366BP strain is characterized in that Streptococcus agalactiae KGP0307.

In the present invention, the accession number KCTC 13366BP strain is characterized by being represented by the 16s RNA nucleotide sequence of SEQ ID NO: 2, and was named KANA-V.

In the present invention, group B streptococci (GBS) is a Gram-positive, beta-hemolytic cocci, and infections have been reported to humans since 1910, and severe meningitis in children and infants mainly in the United States and Europe in the 1970s. And a major pathogen that causes pulmonary disease. Since the late 1980s, Streptococcus b has been transformed into pathogens that cause meningitis and septicemia in various organisms, including freshwater fish, dolphins, and crocodiles, as well as cows and humans.

The cell wall carbohydrate antigen or peptidoglycan of GBS is the same in all strains, but Ia, Ⅰb, Ⅱ, Ⅲ, Ⅳ, Ⅴ, Ⅵ, by the second cell wall polysaccharide (CWPS) 혈청, Ⅷ, Ⅸ are classified into serotypes. Each capsule polysaccharide has different structure and immunogenicity as shown in Figure X.

In order to prevent diseases caused by GBS, capsular polysaccharide is being developed as a protein-polysaccharide vaccine, as in pneumococcus, but the possibility of cross-protection has emerged due to the structural similarity of antigens of several types of streptococcus CWPS, but immunogenicity test method It has not been constructed and has not been proved experimentally.

In another aspect, the present invention relates to an immunogenic composition for preventing diseases caused by group B streptococci including at least one group B streptococcal serotype strain selected from the group consisting of accession numbers ATCC 12401, KCTC 13365BP and KCTC 13366BP.

In the present invention, accession numbers ATCC 12401 and KCTC 13365BP, or ATCC 12401 and KCTC 13366BP, or KCTC 13365BP and KCTC 13366BP, or ATCC 12401, KCTC 13365BP and KCTC 13366BP may be characterized.

In the present invention, the accession number ATCC 12401 strain may be characterized as having streptomycin antibiotic resistance, and the accession number ATCC 12401 strain may be characterized as being serotype Ia.

In the present invention, the accession number ATCC 12401 strain is characterized by being represented by the 16s RNA nucleotide sequence of SEQ ID NO: 3, and was named STREP-Ia.

In the present invention, the strains may be characterized by inactivation and loss of pathogenicity, and the strains may be characterized by attenuation.

The term "immunogenic" of the present invention refers to the ability of a composition to elicit an immune response in a mammal against a particular pathogen. The immune response is a cellular immune response primarily mediated by cytotoxic T-cells and cytokine-producing T-cells, or humoral immunity that produces antibodies by activating B-cells primarily mediated by helper (helper) T-cells It can be a reaction.

The immunogenic composition of the present invention can be administered in an effective amount. "Effective amount" means the amount required to alleviate or eliminate the symptoms to be treated or prevented, and those skilled in the art can appropriately select. For example, the therapeutically or prophylactically effective amount of the group B streptococcal serotype strain expressing the heterologous antigen protein is 1x10 ⁶ to 1x10 ¹⁰ cfu / mL per day, preferably 1x10 ⁷ to 1x10 per day. It can be administered in a range of ⁹ cfu / mL, more preferably 1x10 ⁷ to 1x10 ⁸ cfu / mL per day.

The immunogenic composition of the present invention may further include a pharmaceutically acceptable carrier or adjuvant in addition to the group B streptococcal serotype strain.

In the immunogenic composition of the present invention, the carrier is used as a meaning including a diluent, a buffer, and a preservative. The carrier may include one or more components selected from the group consisting of excipients, disintegrants, binders, and lubricants known in the art, but is not limited to these examples.

In addition, the adjuvant is a substance used to enhance the immunogenicity of the antigen, and adjuvants suitable for use in the present invention include several adjuvant classes, such as mineral salts such as Alum, aluminum hydroxide, aluminum phosphate. And calcium phosphate; Surfactants and particulates, such as nonionic block polymer surfactants (such as cholesterol), virosomes, saponins (such as Quil A, QS-21 and GPI-0100), proteosomes, immune stimuli Complexes, coclays, quaternary amines (dimethyl dioctadecyl ammonium bromide (DDA)), abridine, vitamin A, vitamin E; Bacterial products, such as the cell wall skeleton (Detox®), muramyl dipeptide (MDP) and tripeptide (MTP), monophos, of RIBI Adjuvant System (Ribi Inc.), Mycobacterum phlei Poryl Lipid A, Bacillus Calmete-Guerin, heat-labile E. coli enterotoxin, cholera toxin, trehalose dimycholate, CpG oligodeoxynucleotide; Cytokines and hormones, such as interleukins (IL-1, IL-2, IL-6, IL-12, IL-15, IL-18), granulocyte colony stimulating factor, dehydroepiandrosterone, 1,25-dihydride Hydroxy vitamin D3; Polyanions such as dextran; Polyacrylics (eg, polymethyl methacrylate, Carbopol 934P); Carriers such as tetanus toxide, dipteria toxide, cholera toxin B subunit, mutant heat of enterotoxicgenic E. coli heat-labile enterotoxin (rmLT), heat shock protein; Oil-in-water emulsions, such as AMPHIGEN (registered trademark) (Hydronics, USA); And water-in-oil emulsions, such as Freund's complete and incomplete adjuvants.

The immunogenic composition of the present invention may be in any form known in the art, for example, liquid and injectable forms, but is not limited to these examples. In the case of liquids or injections, it may contain 10 to 40% of propylene glycol, if necessary, and an amount sufficient to prevent hemolysis (eg, about 1%) of sodium chloride. The solution or injection may include any diluent or buffer known in the art. In addition, the immunogenic composition of the present invention, the group B Streptococcus serotype strain is lyophilized and stored in a container such as a vial, and a carrier or adjuvant, saline, etc., required for injection, is added immediately prior to use. It may be prepared.

In another aspect, the present invention, the identification number of ATCC 12401, KCTC 13365BP and KCTC 13366BP selected from the group consisting of at least one group B Streptococcus serotype strain after immunization to a subject comprising the step of determining whether the immune immunity It is about.

In the present invention, the subject may be a human, cow, pig, goat, dog, sheep, etc., but is not limited to these examples. Preferably, the subject is a human.

The immunogenic composition of the present invention can be administered by any means of administration known in the art. For example, the administration can be administered directly to an individual intravenously, intramuscularly, orally, transdermal, mucosal, intranasal, intratracheal or subcutaneously. The administration may be administered systemically or locally.

The present invention also comprises the step of administering the immunogenic composition according to the present invention to a subject, a method of treating or preventing one or more diseases selected from the group consisting of diseases caused by Streptococcus pneumoniae serotype strains of mammals Gives

In the present invention, in order to confirm the effectiveness of a multiplexed opsonophagocytic killing assay (MOPA), analysis specificity was analyzed for a neutralizing serum sample. By adsorbing serotype antibodies using inactivated serotype GBS, as shown in Figure 2, the OIs of the native serotypes are 1a, III, and V, respectively, 199, 17496, 1275. In contrast, the OI of preadsorbed serum was not detected for Ia and V and 1498 for serotype III, which was 11.7 times lower than natural serum. Pre-adsorption with heterologous GBS serotypes resulted in a 0-30% OI reduction, indicating the possibility of the presence of cross-reactive antibodies in the serum (Figure 2). OI reduction in various serum samples was varied.

In addition, in order to confirm the accuracy, the value of MOPA was investigated by comparing with the value of SOPA previously described. To do this, 35 serum samples (20 former GBS infections and 15 healthy subjects) were tested in both tests and compared MOPA (y-axis) and SOPA (x-axis) OI (FIG. 3). The dashed line and solid line in the graph represent the identity and the 2-fold deviation from the identity, respectively. As a result, 15 of the 105 data points deviated more than 2 times from the identity, but it was confirmed that the OI of the two analyzes correlated well with the R-squared correlation coefficient (r ² ) value exceeding 0.950.

In addition, in order to confirm the precision, the reproducibility of GBS-MOPA under normal operating conditions (variation between assays) was performed in 5 independents by 2 independent experimenters using 5 serum samples for 4 consecutive days. Evaluated by experiment. The average OI, standard deviation, and coefficient of variation of five independent experiments were calculated. As a result, the coefficient of variation (CV) for the three serotypes ranged from 0% to 31.2% (mean 12.5%) (Table 1). For serotype Ia, 4 out of 5 serum samples showed OI <4, consistently undetectable opsonization.

Table 1 below confirms the intermediate analysis precision of GBS MOPA.

Sample STREP-Ia SPEC-III KAN-V Mean ± SD CV (%) Mean ± SD CV (%) Mean ± SD CV (%) Sample 1 2 ± 0 0 599.8 ± 174.3 29.1 753.8 ± 122.4 16.2 Sample 2 87.1 ± 23.4 26.8 6467.4 ± 1328.9 20.5 30.7 ± 27.9 9.1 Sample 3 2 ± 0 0 10.1 ± 1.8 17.9 7.8 ± 3.4 4.3 Sample 4 2 ± 0 0 14.3 ± 3.1 21.8 724.3 ± 230.9 31.9 Sample 5 2 ± 0 0 2.5 ± 0.4 4.3 2.7 ± 1.6 6.1

Since the efficacy of the candidate GBS vaccine in clinical trials was measured only by ELISA, the functional activity of the low antigen antibody and the high antibody antibody may not be accurately distinguished, so OPA is required to evaluate the efficacy of the candidate GBS vaccine.

In order to develop OPA for GBS, (1) the amount of infant serum that can be used for testing should be multiplexed, and (2) the main users of the newly developed GBS-OPA protocol are mainly pneumococcal OPA users. Because it is predicted, it should be compatible with pneumococcal OPA (eg reagents and protocols). Therefore, these users can easily adapt to the proposed OPA for GBS vaccine evaluation.

In the present invention, through systematic meta-analysis, triplex multiplexed GBS-OPA was targeted to serotypes Ia, III, and V with an application range of early-onset disease (EOD) and late-onset disease (LOD) of about 81%. Developed.

Similar to certain pneumococcal serotypes, GBS serotype V showed particularly high levels of NSK. The high level was observed even after cultivation in 5% CO ₂ at the phagocytosis stage, and showed that pneumococcal NSK was significantly reduced.

As known in the art, the level of bacterial capsule expression and complement concentration are important factors influencing NSK. Therefore, these factors should be optimized to reliably measure opsonin phagocytosis (OPK) of the capsular bacteria by functional antibodies. For example, pneumococci with small stenosis are believed to have a higher sensitivity to complement-mediated NSK, but pneumococci with large stenosis may be relatively resistant to OPK.

In the present invention, very different levels of NSK and OPK were found when OPA was performed with several GBS serotype strains. However, as recommended by the pneumococcal MOPA protocol, the NSK of GBS was significantly and consistently decreased in the analysis by using 9% BRC instead of 12.5% BRC (<30%). Previous studies have found that the optimal complement concentration of pneumococcal OPA varies from 8% to 16% depending on the serotype. Therefore, in the present invention, 9% BRC was used as the optimal concentration of GBS MOPA. NSK at this concentration was consistently lower than the 40% observed for Ia and III. However, at this point it is impossible to determine whether the high level of NSK of serotype V is due to the difference in the level of stenotic expression in other GBS serotypes.

In the present invention, GBS-MOPA was verified in terms of specificity, accuracy and accuracy. Analysis of assay specificity showed that GBS-MOPA was sufficiently specific because adsorption of serum with allogeneic inactive GBS resulted in a significant reduction in OIs. However, also a modest reduction (0-30%) of OI with heterologous serotype adsorption was found (Figure 2). These cross-reactive antibodies are capable of binding conserved antigens expressed on the GBS surface. When Western blotting investigated the reactive surface antigen protein in serum, at least three conserved cell wall-anchoring proteins were found in serum with high OI.

In another aspect, the present invention includes an immunogenic composition for preventing diseases caused by group B streptococci including one or more group B streptococcal serotype strains selected from the group consisting of accession numbers ATCC 12401, KCTC 13365BP and KCTC 13366BP. It relates to a vaccine composition.

In the present invention, the accession number ATCC 12401 strain may be characterized as having streptomycin antibiotic resistance, and may be characterized as being serotype Ia.

In the present invention, accession numbers may include ATCC 12401 and KCTC 13365BP, or ATCC 12401 and KCTC 13366BP, or KCTC 13365BP and KCTC 13366BP, or ATCC 12401, KCTC 13365BP and KCTC 13366BP.

In the present invention, the strains may be characterized by inactivation and loss of pathogenicity, and the strains may be characterized by attenuation.

The term "attenuated" of the present invention means that a toxic strain is modified in such a way that it becomes a less virulent strain or a weaker pathogen than before, which strain may cause clinical disease while still being able to replicate and divide in the host. It means that the ability to do is significantly reduced. The attenuated strain of the present invention is preferably a strain in which toxicity or pathogenicity is reduced to allow administration for vaccine use, more preferably the strain can cause clinical disease while still being able to replicate in the host. It means to attenuate to the extent that there is no.

Individuals to which the vaccine of the present invention can be administered are not limited and include mammals such as rats, mice, livestock, and humans.

The effective dosage range for the vaccine of the present invention is sex, body surface area, type and severity of disease, age, sensitivity to drugs, administration route and discharge rate, administration time, treatment period, target cell, expression level, etc. It can vary depending on various factors well known in the field and can be easily determined by experts in the field.

In addition to the group B streptococcal serotype strain, the vaccine composition of the present invention may further include a pharmaceutically acceptable carrier or adjuvant.

Carriers that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, and crude Vaginal cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the adjuvant that can be used in the present invention can be used without limitation the adjuvant commonly used in the art, preferably the adjuvant is cholera toxin binding unit, aluminum salt, lipid emulsion (MF-59), synthetic Detergents (Tween), microspheres, liposomes, mucoadhesive polymers, and the like, but are not limited to the above examples, and new formulations in this field have been developed, and these may also be used.

The composition according to the present invention may be used in the form of an oral dosage form such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, external preparation, suppository, or sterile injection solution according to a conventional method.

Specifically, when formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient in the compound, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. It can be prepared by mixing. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients. Liquid preparations for oral use include suspensions, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used diluents, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, can be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Non-aqueous solvents, suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

The disease to which the vaccine composition of the present invention is applied refers to any symptoms caused by GBS infection, and may include, for example, sepsis and meningitis in infants, but is not limited thereto.

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

Example 1: Experimental materials and methods

The experiment was conducted in two steps for the development and optimization of GBS MOPA. First, a target bacterial strain was generated, and OPA conditions including agar plate type, complement concentration and reactor-target (E: T) ratio were optimized. Second, as shown in the Pneumococcal MOPA protocol (www.vaccine.uab.edu), analysis specificity, accuracy and precision were investigated. The experiment was approved by Korea University Guro Hospital Ethics Committee (IRB No. KUGH16119).

Serum samples

The experiment of the present invention was approved by the Korea University Guro Hospital Ethics Committee (IRB No. KUGH14106) and was performed according to the Helsinki Declaration and Good Clinical Practice. KUGH's Institutional Review Board requested written consent for the use of unidentified residual serum samples. Serum was mixed from 15 healthy adults 19 years old to prepare a human serum pool (1). The pool was used for quality control of OPA. Twenty serum samples were obtained from subjects recovered from previous GBS infection, and fifteen serum samples were collected from healthy adults who had not previously had GBS infection. All study specimens were taken from subjects who did not receive antibiotics for 3 days prior to blood collection. The collected samples were used to optimize and verify GBS MOPA.

Generation of antibiotic resistant GBS strains and GBS processed strains

The GBS strains used in this study are described in Table 2.

Spectinomycin resistance, streptomycin-resistant and kanamycin resistance variants of the GBS serotypes Ia, III, V were generated by natural selection. Specifically, cells were grown in Todd-Hewitt broth (Becton Dickinson, Sparks, MD) with 0.5% yeast extract (Becton Dickinson) (THY broth) at increasing antibiotic concentration. The antibiotic resistant GBS strain was cultured in THY broth at 37 ° C. until the optical density (OD600) at 600 nm was 0.8 to 1.0. Bacterial strains were frozen at -80 ° C in THY containing 15% glycerol until use. The dilution factor for the MOPA standard target strain was 1,250X for STREPA909 (Ia), 1,250X for SPECNEM316 (III), and 1,000X for KAN10 / 84 (V).

MOPA strain Serotype No. Resistant antibiotic
and concentration STREP-Ia (A909) Ia ATCC 12401 Streptomycin (300 mg / L) SPEC-III (NEM316) III KCTC 13365BP Spectinomycin (300 mg / L) KAN-V (NCTC10 / 84) V KCTC 13366BP Kanamycin (300 mg / L)

GBS functional pneumococcal antibody analysis method

GBS multiplexed opsonocytes (GBS-MOPA) consisted of a modified version of the pneumococcal MOPA. In summary, HL-60 cells are cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT) and 0.8% dimethylformamide (Fisher Scientific, Pittsburgh, PA) for 5 days. Differentiated with. Differentiated HL-60 cells were diluted with Opsonization Assay Buffer B at 107 cells / ml (OBB; Hanks' buffer supplemented with 0.1% gelatin (Sigma-Aldrich) and 10% FBS). 30 μl of each test serum sample was diluted 3-fold with OBB in a 96-well plate. The assay used 10 μl of stock and diluted serum. 10 μl of heat-inactivated complement was used as a negative control. Frozen processed strains of each of the three target GBS strains were thawed just prior to use, washed twice with OBB, resuspended cells, and adjusted to 1 x 10 ⁵ CFU / mL in OBB. The three GBS strains were mixed equally, then 10 μl of the GBS mixture was added to each well. After incubation for 30 minutes at room temperature, 42.8 μl (4.28 x 10 ⁵ cell / well) of differentiated HL-60 cell suspension and 7.2 μl of young rabbit complement (BRC; Pel-Freez Biological; Rogers, AR) were added to each well. . The mixture was then incubated for 45 minutes at 37 ° C. Then, 10 μl of the final reaction mixture was inoculated into trypsin soybean agar containing 1.5% bacto-agar (TSA; Becton Dickinson) in each well.

Next, after overlaid with 25 mL of THY containing 0.75% bacto agar containing one of three antibiotics and 2,3,4-triphenyltetrazolium chloride (TTC; Sigma-Aldrich), the plate was plated. Was incubated overnight at 37 ° C. The bacterial colonies surviving on the plate were enumerated and the opsonic index (OIs) of the serum sample was determined. OI was defined as the final dilution of the serum sample resulting in a 50% CFU reduction compared to the negative control with 10 μl of heat-inactivated complement instead of diluted serum. Undiluted serum samples killed 50% of GBS, and the OI was 4. Analytical assay reproducibility of control serum samples (pool 1) was included in each assay.

For the GBS single opsonin phagocytosis assay (GBS-SOPA), the same protocol was performed as for GBS-MOPA, except that the GBS strain was prepared as a single GBS serotype rather than a mixture of the three serotypes. Following the same protocol used in GBS-MOPA, 5 μl of the final reaction mixture from each well was inoculated onto trypsin soybean agar containing 1.5% bacto-agar (TSA; Becton Dickinson). Then, 25 mL of THY containing 0.75% bacto agar containing only 2,3,4-triphenyltetrazolium chloride (TTC; Sigma-Aldrich) was overlaid on a spotted plate, and the plate was incubated overnight at 37 ° C. . After overnight incubation, bacterial colonies on agar plates were counted using the National Institute of Standards and Technology, US Integrated Colony Enumerator.

Determination of specificity by adsorption of serum samples

Analysis specificity was analyzed using pre-adsorption of serum samples with formalin-inactivated hetero-serum type or homo-serum type GBS. The indicated GBS serotypes (Ia, III, and V) were cultured in THY medium with an OD600 of 0.5. 50 ml of each GBS culture medium was mixed with 0.2% formalin (100 μl, v / v) and incubated at 37 ° C. for 2 hours. The inactivated GBS cells were then harvested by centrifugation and resuspended in phosphate buffered saline (PBS) with 15% glycerol. An aliquot of 1 ml was stored at -80 ° C. For adsorption, a GBS aliquot was centrifuged at 13,000 x g for 2 minutes, washed twice with OBB, and then resuspended in 1 ml of OBB. 50 µl of inactivated GBS was mixed with 450 µl of a serum sample, followed by shaking culture at 4 ° C for 2 hours. GBS cells were removed by centrifugation (13,000 x g, 5 min) and 400 μl of absorbed serum was collected and used immediately in GBS-MOPA.

Statistical analysis

All statistical analyzes were performed using SPSS 18.0 (SPSS Korea, Seoul, Korea). Accuracy is considered equivalent to the test conditions and is reproducible with CV ≤ 20%. Regarding accuracy, it was considered acceptable in r2≥0.95 in the linear correlation analysis.

Example 2: Experimental results

Reactor-target (E: T) ratio

When the ratio of HL60 cell and complement E: C ratio of 4: 1 (complement concentration 12.5%) was applied in the process of GBS MOPA experiment, non-specific killing for serotype V was repeatedly measured to be higher than 80%. Experiments were conducted to find the optimal ratio (in other serotype V GBS strains that induced resistance to tetracycline, non-specific killing was higher than 80%, and the experiment was performed by changing the complement lot 3 times). . When single serotype OPA is applied at a ratio of 6: 1 (complement concentration 9%) and 8: 1 (complement concentration 7%), non-specific killing is maintained at a level of 40% or less, 20% or less, respectively. Therefore, it was decided to apply the E: C ratio in a 6: 1 ratio (9%) (FIG. 1).

  There have been reports of non-specific killing of bacteria at concentrations above 5% of baby rabbit serum complement. It has been reported that the smaller the capsule size of OPA target bacteria, the higher the non-specific killing may be, and non-speific killing may occur due to various components of bacteria. When non-specific killing was significantly reduced. In the case of pneumococci, there is a difference in non-specific killing depending on the serotype, so the concentration of complement has been applied at 8-16%. In the case of GBS, since the size of the stenosis is smaller than that of pneumococcus, the appropriate complement concentration may be low, and considering the results of this experiment, it was decided to apply a 6: 1 ratio (9%).

800: 1 (4 * 10 ⁷ CFU / mL), 400: 1 (2 * 10) using serum (FGBS 5) of patients recovering from GBS infection to find the optimal ratio of HL60 cell and target strain concentration during the GBS MOPA experiment ⁷ CFU / mL), 200: 1 (1 * 10 ⁷ CFU / mL), 150: 1 (1.5 * 10 ⁷ CFU / mL) and 100: 1 (0.5 * 10 ⁷ CFU / mL) were compared. The E: T ratio between 150: 1 and 800: 1 was stably maintained without change, so it was decided to apply the E: T ratio of 200: 1 to the standard test method (Table 3).

Table 3 shows the results of comparative evaluation of opsonic titer according to Effector: target ratio.

Opsonic titers E: T ratio Serotype Ia Serotype Ⅲ Serotype Ⅴ 800: 1 (4 * 10 ⁷ ) 207 17.496 2,105 400: 1 (2 * 10 ⁷ ) 212 7,470 2,054 200: 1 (1 * 10 ⁷ ) 213 7,290 1,911 150: 1 (1 * 10 ⁷ ) 230 7,370 1,805 100: 1 (0.5 * 10 ⁷ ) 143 5,133 41

Specificity evaluation

After pretreatment to neutralize serotype-specific antibodies using three strains of Ia, III and V, MOPA was performed to evaluate the specificity of the assay. OPA titers against specific serotypes were completely inhibited when adsorbed using serotype Ia or V dead strains (FIG. 2). On the other hand, when adsorbed using the serotype III GBS strain, the OPA titer for serotype III GBS tended to decrease slightly but was not completely suppressed.

[Neutralization inhibition assay using Serotype Ia GBS bacteria strain]

OPA titers against serotype Ia GBS were completely inhibited when adsorbed using serotype Ia GBS dead strain. For other serotypes, OPA titers tended to decrease somewhat.

[Neutralization inhibition assay using Serotype III GBS dead strain]

OPA titers against serotype III GBS tended to decrease when adsorbed using serotype III GBS dead strain, but were not completely inhibited. It seems that the opsonic antibody titer against serotype III GBS is too high to be completely inhibited. OPA titers for other serotypes also showed a slight decrease, but because they are within a very low range, they do not appear to be meaningful changes, but it is possible that non-specific opsonophagocytic activity against teichoic acid and surface protein of the GBS strain is inhibited. In the preparation phase of MOPA implementation, it may be necessary to perform adsorption with a GBS strain without stenosis.

[Neutralization inhibition assay using Serotype V GBS dead strain]

OPA titers against serotype V GBS were completely inhibited when adsorbed using serotype V GBS dead strain. For other serotypes, there was no apparent change in OPA titers.

Precision evaluation

 To verify the precision of the MOPA test method, five validation samples were used to verify the precision of the MOPA test method. The coefficient of variation (CV) of the test values measured 8 times was calculated by measuring 2 opsonic titers 4 times each at 1 week intervals. The CV value was 0-31.2%, and the average CV was maintained at less than 20% (12.5%), and a specific serotype did not show a high CV (Table 4).

Table 4 below summarizes the precision validation of the GBS MOPA test method.

Sample Serotype Ia Serotype III Serotype V mean ± SD CV (%) mean ± SD CV (%) mean ± SD CV (%) One 2 0 599.8 ± 174.3 29.1 753.8 ± 122.4 16.2 2 87.1 ± 23.4 26.8 6467.4 ± 1328.9 20.5 30.7 ± 27.9 9.1 3 2 0 10.1 ± 1.8 17.9 7.8 ± 3.4 4.3 4 2 0 14.3 ± 3.1 21.8 724.3 ± 230.9 31.9 5 2 0 2.5 ± 0.4 4.3 2.7 ± 1.6 6.1

Accuracy evaluation

To verify the accuracy of the MOPA assay, the correlation of the opsonic titer obtained through the MOPA test and the SOPA (single serotype opsonophagocytic killing assay) test was evaluated using 35 validation samples. The results of experiments with MOPA and SOPA showed good agreement, and all three serotypes showed high correlation with r value of 0.9 or higher. Even when the linear regression analysis was performed, r ² values showed good correlation with 0.996, 0.986, and 0.956, respectively (FIG. 3).

Multi-center validation of MOPA test methods

In order to verify the accuracy of the results of the MOPA test conducted by the multi-center, 30 elderly samples over 65 years old were used to evaluate the correlation of the opsonic titer obtained through the MOPA test at the Guro Hospital and the Nuclear Research Institute of Korea University (FIG. 4). .

When a linear regression analysis was performed for serotypes Ia, III, and V, the r ² values were 0.88, 0.60, and 0.35, respectively, which did not show a sufficiently high level of agreement. However, when the opsonic titer measured by Korea University Guro Hospital and Atomic Energy Research Institute was corrected based on the median value of the repeated QC serum, the r ² values for serotypes Ia, III, and V improved to 0.89, 0.73, and 0.77, respectively. Became (Fig. 4).

Evaluation of the basic immunity by serotype in the high-risk group of GBS infection

Serum-type specific basic immunity to GBS was evaluated in three high-risk groups, including neonatal / infant, pregnant women, and elderly people over 65 years (Table 4, Table 5, and FIGS. 5 to 7). The average age of the neonatal / infant, pregnant and elderly groups was 1.3 months (1-3 months), 31.9 years (23-41 years) and 68.8 years (65-76 years), respectively.

Table 5 below shows the results of comparing the basic OPA titer (opsonic index) by serotype in high-risk groups such as newborns / infants, pregnant women, and elderly people over 65 years old.

Serotypes Groups Opsonic index (mean) 95% CI p-value Ia neonates / infants 137 33-241 0.596 pregnant women 285 17-552 old adults 231 3-459 III neonates / infants ^a 338 105-570 0.002 pregnant women ^b 1377 941-1813 old adults ^b 1350 700-2000 V neonates / infants ^a 161 -5-327 <0.001 pregnant women ^b 9414 6825-12003 old adults ^c 3669 1579-5759

 For Serotype Ia, there was no statistically significant difference in opsonic index (OI) between the three trial groups. The average OI of each high-risk group showed statistically significant differences between newborns / infants within 3 months of age (mean ± standard deviation, 137 ± 278), pregnant women (285 ± 717), and seniors over 65 (231 ± 610). (P = 0.596), but showed relatively low OI in neonates / infants (Table 10 and FIG. 5). When the OI level was divided into 4 sections of less than 100, 100-499, 500-999, 1,000 or more, and comparing the fractions of each high-risk group, 70% of newborns / infants, 80% of pregnant women, and 65 years old or older were OI 100 It showed a low titer of less than, and overall, low immunity was a factor that did not show a significant difference (Table 6, p = 0.156).

 In the case of Serotype III, the neonatal / infant OI (mean ± standard deviation, 338 ± 623) was statistically significantly lower than that of pregnant women (1377 ± 1167) and elderly people over 65 years of age (1350 ± 1741) (p = 0.002). Even when the OI level was less than 100, 100-499, 500-999, and 1,000 or more were stratified to compare the fractions of each high-risk group, 60% of newborns / infants had an OI titer of less than 100, and pregnant women and elderly people over 65 years of age. There was a significant difference (Table 6, p <0.001).

In the case of Serotype V, OI was significantly lower in the order of neonatal / infant (mean ± standard deviation, 161 ± 445), seniors over 65 (3669 ± 5597), and pregnant women (9414 ± 6394). There was a significant difference (Table 5 and Table 6, p <0.001) .The level of OI was less than 100, 100-499, 500-999, 1,000 or more, stratified into 4 sections, and compared with the fraction of each high-risk group. While the OI titer of 76.7% of infants was less than 100, 93.3% of pregnant women and 70% of the elderly aged 65 years or older showed a high OI of 1,000 or more (Table 6, p <0.001).

Table 6 below shows the comparison of the basic OPA titer (opsonic index) by serotype in the high-risk group, such as neonatal / infant, pregnant women, and the elderly aged 65 years or older: a comparison result of the fractions of stratified ranges of opsonic index (OI).

Serotype Opsonic index Case No. (%) p-value neonates / infants pregnant women old adults Ia <100 21 (70) 24 (80) 25 (83.3) 0.156 100-499 6 (20) 2 (6.7) 2 (6.7) 500-999 2 (6.7) 0 (0) 0 (0) > 1,000 1 (3.3) 4 (13.3) 3 (10) III <100 18 (60) 4 (13.3) 4 (13.3) <0.001 100-499 4 (13.3) 4 (13.3) 6 (20) 500-999 6 (20) 6 (20) 7 (23.3) > 1,000 2 (6.7) 16 (53.3) 13 (43.3) V <100 23 (76.7) 0 (0) 2 (6.7) <0.001 100-499 5 (16.7) 0 (0) 2 (6.7) 500-999 1 (3.3) 2 (6.7) 5 (16.7) > 1,000 1 (3.3) 28 (93.3) 21 (70)

 Prenatal GBS screening was performed in 26 of 30 pregnant women who participated in the study at 35-37 weeks of pregnancy, and 4 out of 26 (15.4%) showed GBS positive.

Serotype Ia (1230 ± 1467 vs. 139 ± 416, p = 0.234), Ⅲ (1705 ± 1569 vs. 1327 ± 1124, p = 0.556), Ⅴ ( The difference of OI for 6318 ± 7465 vs. 9890 ± 6879, p = 0.346) was not able to confirm statistical significance due to numerical limitation (FIG. 8).

When the level of OI was less than 100, 100 ~ 499, 500 ~ 999, 1,000 or more, and stratified into 4 sections, when comparing the OI distribution fraction between the GBS colonized pregnant group and the non-collapsed pregnant group, there was no statistically significant difference, and colonization was not found. Regardless of this, serotype Ⅲ showed a higher titer of OI 1,000 or higher at 50% or higher and serotype Ⅴ at 90% or higher (Table 7). Considering the low OI of newborns / infants while showing high OI titers in pregnant women, in order to propagate humoral immunity through the placenta to a real newborn, a subclass antibody with a high placental passage efficiency is required, and a higher level of immunity It may be necessary. In addition, it is not possible to exclude the possibility that the titer was measured higher than the actual with nonspecific opsonophagocytic activity of teichoic acid and surface protein of the GBS strain in adults who were repeatedly exposed to GBS.

Table 7 below shows the comparison of the basal OPA titer (opsonic index) by serotype in pregnant women: 35-37 weeks prenatal examination, results of comparison of fractions by stratified range of opsonic index (OI) according to colony of GBS perineum.

Serotype Opsonic index Case No. (%) p-value GBS-positive (N = 4) GBS-negative (N = 26) Ia <100 2 (50) 22 (84.6) 0.065 100-499 0 (0) 2 (7.7) 500-999 0 (0) 0 (0) > 1,000 2 (50) 2 (7.7) III <100 0 (0) 4 (15.4) 0.764 100-499 1 (25) 3 (11.5) 500-999 1 (25) 5 (19.2) > 1,000 2 (50) 14 (53.9) V <100 0 (0) 0 (0) 0.566 100-499 0 (0) 0 (0) 500-999 0 (0) 2 (7.7) > 1,000 4 (100) 24 (92.3)

 Serum type analysis of GBS strains isolated from 4 pregnant women with confirmed GBS colonies was performed, OI of colonized serotypes was high, and OI for serotype V showed a non-specific high tendency (Table 8 and FIG. 8). ).

Table 8 below shows the results of evaluating the association between colony serotypes and OPA titers (opsonic index) in pregnant women with GBS colonies.

No. Age Serotypes Opsonic index (OI) Serotype Ia Serotype Ⅲ Serotype Ⅴ One 30 NT 2 11 2,773 2 34 Ⅰa 2,001 3,218 3,020 3 29 Ⅲ, Ⅷ 2,916 2,876 17,496 4 27 Ⅰb 2 614 1,984

Evaluation of the correlation of GBS immunity measured by MOPA and ELISA

For 90 serum samples from 3 high-risk groups, including neonatal / infant, pregnant women, and elderly people over 65 years of age, serotype-specific basic immunity to GBS was measured by MOPA and ELISA, respectively, and correlation was evaluated. The antibody titers for serotypes Ia, III, and V measured by ELISA were very high in all three high-risk groups, and when correlation analysis was performed, the correlation coefficient r was very low, less than 0.5 for all three serotypes. Level correlation was shown (Fig. 9).

In a recent European study (DEVANI study), the correlation between OPA titers and ELISA titers for serotypes Ia, Ib, and III was compared, and serum with an ELISA titer of 1 ug / mL was OPA titers 64-128, 70-80 %.

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

<110> Korea University Research and Business Foundation <120> Novel Group B Streptococcus Serotype Strain and Immunogenic          Composition Comprising the Same <130> DHP17-612 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 660 <212> RNA <213> Artificial Sequence <220> <223> KCTC 13365BP 16s RNA sequence <400> 1 cgatgagtgc taggtgttag gccctttccg gggcttagtg ccgcagctaa cgcattaagc 60 actccgcctg gggagtacga ccgcaaggtt gaaactcaaa ggaattgacg ggggcccgca 120 caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aggtcttgac 180 atccttctga ccggcctaga gataggcttt ctcttcggag cagaagtgac aggtggtgca 240 tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaacccc 300 tattgttagt tgccatcatt aagttgggca ctctagcgag actgccggta ataaaccgga 360 ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtgcta 420 caatggttgg tacaacgagt cgcaagccgg tgacggcaag ctaatctctt aaagccaatc 480 tcagttcgga ttgtaggctg caactcgcct acatgaagtc ggaatcgcta gtaatcgcgg 540 atcagcacgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccacga 600 gagtttgtaa cacccgaagt cggtgaggta accttttagg agccagccgc ctaaggtggg 660                                                                          660 <210> 2 <211> 660 <212> RNA <213> Artificial Sequence <220> <223> KCTC 13366BP 16s RNA sequence <400> 2 cgatgagtgc taggtgttag gccctttccg gggcttagtg ccgcagctaa cgcattaagc 60 actccgcctg gggagtacga ccgcaaggtt gaaactcaaa ggaattgacg ggggcccgca 120 caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aggtcttgac 180 atccttctga ccggcctaga gataggcttt ctcttcggag cagaagtgac aggtggtgca 240 tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaacccc 300 tattgttagt tgccatcatt aagttgggca ctctagcgag actgccggta ataaaccgga 360 ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtgcta 420 caatggttgg tacaacgagt cgcaagccgg tgacggcaag ctaatctctt aaagccaatc 480 tcagttcgga ttgtaggctg caactcgcct acatgaagtc ggaatcgcta gtaatcgcgg 540 atcagcacgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccacga 600 gagtttgtaa cacccgaagt cggtgaggta accttttagg agccagccgc ctaaggtggg 660                                                                          660 <210> 3 <211> 660 <212> RNA <213> Artificial Sequence <220> <223> ATCC 12401 16s RNA sequence <400> 3 cgatgagtgc taggtgttag gccctttccg gggcttagtg ccgcagctaa cgcattaagc 60 actccgcctg gggagtacga ccgcaaggtt gaaactcaaa ggaattgacg ggggcccgca 120 caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aggtcttgac 180 atccttctga ccggcctaga gataggcttt ctcttcggag cagaagtgac aggtggtgca 240 tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaacccc 300 tattgttagt tgccatcatt aagttgggca ctctagcgag actgccggta ataaaccgga 360 ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtgcta 420 caatggttgg tacaacgagt cgcaagccgg tgacggcaag ctaatctctt aaagccaatc 480 tcagttcgga ttgtaggctg caactcgcct acatgaagtc ggaatcgcta gtaatcgcgg 540 atcagcacgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccacga 600 atcagcacgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccacga 660                                                                          660

Claims (19)

Group B streptococci serotype strain of accession number KCTC 13365BP or KCTC 13366BP.
The group B streptococcus serotype strain according to claim 1, wherein the strain displays the 16s RNA nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 2.
The group B streptococcus serotype strain according to claim 1, wherein the accession number KCTC 13365BP strain has spectinomycin antibiotic resistance.
The group B streptococcus serotype strain according to claim 1, wherein the accession number KCTC 13365BP strain is serotype III.
The group B streptococcus serotype strain according to claim 1, wherein the accession number KCTC 13366BP strain has kanamycin antibiotic resistance.
The group B streptococcus serotype strain according to claim 1, wherein the accession number KCTC 13366BP strain is serotype V.
An immunogenic composition for preventing diseases caused by group B streptococcus, comprising at least one group B streptococci serotype strain selected from the group consisting of accession numbers ATCC 12401, KCTC 13365BP and KCTC 13366BP.
The immunogenic composition according to claim 7, comprising accession numbers ATCC 12401 and KCTC 13365BP, or ATCC 12401 and KCTC 13366BP, or KCTC 13365BP and KCTC 13366BP, or ATCC 12401, KCTC 13365BP and KCTC 13366BP.
[8] The immunogenic composition according to claim 7, wherein the accession number ATCC 12401 strain has streptomycin antibiotic resistance.
8. The immunogenic composition according to claim 7, wherein the accession number ATCC 12401 strain is serotype Ia.
8. The immunogenic composition of claim 7, wherein the strains are inactivated and lose pathogenicity.
8. The immunogenic composition according to claim 7, wherein the strains are attenuated.
Immunization method comprising the step of determining the immunity after immunizing a subject with at least one group B streptococcal serotype strain selected from the group consisting of accession numbers ATCC 12401, KCTC 13365BP and KCTC 13366BP.
A vaccine composition comprising an immunogenic composition for preventing diseases caused by group B streptococcus, comprising at least one group B streptococcal serotype strain selected from the group consisting of accession numbers ATCC 12401, KCTC 13365BP and KCTC 13366BP.
The vaccine composition according to claim 14, comprising accession numbers ATCC 12401 and KCTC 13365BP, or ATCC 12401 and KCTC 13366BP, or KCTC 13365BP and KCTC 13366BP, or ATCC 12401, KCTC 13365BP and KCTC 13366BP.
The vaccine composition according to claim 14, wherein the accession number ATCC 12401 strain is resistant to streptomycin antibiotics.
The vaccine composition according to claim 14, wherein the accession number ATCC 12401 strain is serotype Ia.
15. The vaccine composition of claim 14, wherein the strains are inactivated and lose pathogenicity.
15. The vaccine composition of claim 14, wherein the strains are attenuated.

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