CN110520154A - Multivalence Streptococcus pneumoniae vaccine composition - Google Patents

Multivalence Streptococcus pneumoniae vaccine composition Download PDF

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CN110520154A
CN110520154A CN201880025013.0A CN201880025013A CN110520154A CN 110520154 A CN110520154 A CN 110520154A CN 201880025013 A CN201880025013 A CN 201880025013A CN 110520154 A CN110520154 A CN 110520154A
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capsular polysaccharide
polysaccharide
vaccine composition
serotype
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CN110520154B (en
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赵成济
崔淑英
朴惠梶
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LG Chem Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine

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  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
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Abstract

The present invention relates to a kind of streptococcus pneumonia (Streptococcus pneumoniae) vaccine compositions, more specifically provide a kind of vaccine composition, it includes: (i) capsular polysaccharide-protein conjugate；(ii) 2- phenoxetol (2-PE)；(iii) formaldehyde (HCHO), and provide the method for preparing the vaccine composition.

Description

Multivalence Streptococcus pneumoniae vaccine composition
Technical field
The present invention relates to the vaccine groups for being directed to pneumococcus (streptococcus pneumonia (Streptococcus pneumoniae)) Object is closed, systems a kind of vaccine composition, it includes (i) capsular polysaccharide-protein conjugate, (ii) 2- phenoxy groups Ethyl alcohol (2-PE) and (iii) formaldehyde (HCHO), and the method for preparing the vaccine composition.
Background technique
Pneumococcus (streptococcus pneumonia (Streptococcus pneumonia)) is a kind of Gram-positive and haemolysis Strains of streptococcus, and the whole world be baby, in children and the elderly meningitis, pneumonia and serious invasive infection master Want reason.1,600,000 people are had more than every year to die of pneumonia coccus disease (2008, the World Health Organization (International Health Organization) data), especially, in the elderly of immunity low 5 years old children below and over-65s In, the disease incidence of the invasive infection disease as caused by pneumococcus is high.
Pneumococcus is according to the structure of the capsular polysaccharide as Major Virulence Factors for surrounding its external (cell membrane) and exempts from Immunological features are classified as known to about 20 kinds about more than 90 kinds of serotypes, and in them in the mankind 80~90% Pathogenic correlation.Unique host of pneumococcus is the mankind, and they are usually lived in concentrated communities in the normal nasopharyngeal (20~40% of health Baby, 5~10% adult).In 2005, disease prevention and control centre (Centers for Disease Control and Prevention) (CDC) report, only just there are about 2,100,000 5 years old children below every year in developing country It dying of pneumonia, 1,200,000 people in them die of pneumonia coccus infection, and in the U.S., the meninx as caused by pneumococcus every year Scorching and septicemia occurs about 3,000 and 50 respectively, 000 (Peters TR, Poehling KA etc., aggressive pneumococcus disease Sick (Invasive pneumococcal disease), JAMA 2007；297:1825-6).In addition, according to as pneumococcus The pneumoACTION database of the database of disease, in 2000, in the morbidity number of South Korea's pneumococcal infection in children It is annual 24,047, and 47 death (www.pneumoadip.org) in them.In addition, according to by disease prevention and Newest " South Korea children and the blueness of control centre (Centers for Disease Control and Prevention) publication The research of the serum type analysis of pneumococcus in teenager " (study on the serotype analysis of Pneumococcus in children and adolescents in the country), pneumococcus is 3 months~59 Most common (43.7%) the invasive infection reason in baby between a month.For causing whole world invasive infection disease For pneumococcus, other than penicillin, the multidrug resistance bacterium for showing drug resistance to three kinds or more drugs increases, and Therefore keep the treatment of pneumococcal infection disease more difficult.
Therefore, to being carried out in the children and the elderly of the high risk with pneumococcal infection disease for pneumonia ball The vaccine inoculation of bacterium, there is demands always.
In order to prevent pneumococcal infection disease, is developed from 1977 and had approved multivalent pneumococcal polysaccharide epidemic disease Seedling, and these capsular polysaccharide vaccines have been found for preventing for pneumococcal disease in the elderly and high-risk patient It is useful.However, the maturity due to immune system in baby and children is lower than adult, polysaccharide vaccine is only being administered In the case of, expectation is played the role of vaccine and is difficult, because polysaccharide antigen cannot be identified as external aggression by immune system The factor.In order to solve the problems, such as that polysaccharide vaccine reduces this of immunogenicity in baby and children etc., develops and used 7 valences Pneumococcal conjugate vaccine (7vPnC,), this is a kind of carrier protein couplet of immunogenicity of improving to more The capsular polysaccharide of sugar antigens-protein conjugate vaccine, and reported it in baby and children in many references It is effective for affecting conditions and inflammation in middle prevention middle ear.However, the use of the 7 valence vaccine causes by for institute The reduction of affecting conditions caused by the vaccine serotype of vaccine is stated, but additionally shows to be caused by part nonvaccine serotype Pneumococcal disease relative increase the phenomenon that.Therefore, it has developed at present and commercially available 10 valence capsular polysaccharide-protein conjugate epidemic disease SeedlingWherein toBasal serum type be added to 6 kinds of serotypes 13 valence pneumococcus coupling Object vaccineBut for by comprising certain serotypes for, have reported they as vaccine drug effect not A possibility that sufficient [Andrews NJ etc., (2014) Lancet Infec Dis (14) 839；The EMEA of Prevenar 13 assesses report Accuse (EMEA Assessment Report for Prevenar 13), (2009) EMA/798877/2009], therefore, to display The exploitation of the new vaccine preparation of higher and more stable threshold value out, there is demands always.
Meanwhile vaccine injection dosage form should prevent the pollution of microorganism using preservative.It is deficient for being exported to by UN etc. For the polyvalent vaccine product of developed country, due to environment, allocator, the cost etc. of country, wherein more comprising preservative Agent product is preferred.There are thimerosal, 2- phenoxetol (2-PE), phenol etc. as be used for vaccine product preservative, And there are the amounts of preservative commonly used in the art.For example, thimerosal is used with the concentration of 10 μ g/mL, 2-PE is with 5mg/mL's Concentration uses, and if including that their multi-agent vaccine product has passed through EP-B (European Pharmacopoeia B class) or USP (United States Pharmacopeia) The bactericidal aptitude tests of standard, then can be commercialized.
Thimerosal (merthiolate) is a kind of ethyl mercury derivative compound, since nineteen thirty is for early stage by with Make the preservative of multi-agent vaccine injecta.Thimerosal has been used for preventing from polluting micro- life during the storage and use of vaccine product The growth of object and the purpose for maintaining aseptic condition, and many 5 valence liquid polyvalent vaccines (packets for obtaining WHO PQ (preliminary hearing is qualified) Include D, T, P, Hib, HBsAg) contain this thimerosal as preservative.
2- phenoxetol (2-PE) is used as the preservative of cosmetics and transdermal drug, and also serves as vaccine injecta Preservative.
However, since the amount of these bactericidal active preservative components for realizing vaccine may be enough to cause toxicity And/or side effect, therefore to reducing their usage amount and providing the exploitation of enough bactericidal active technologies, there is need It asks.
Summary of the invention
[technical problem]
One example provides a kind of Pnu-Imune 23 composition, and it includes (i) streptococcus pneumonias Capsular polysaccharide-the protein conjugate of (Streptococcus pneumoniae), (ii) 2- phenoxetol (2-PE), and (iii) formaldehyde (HCHO).The vaccine composition can be the multi-dose vaccine composition for multiple dosing.
Another example provide it is a kind of for preventing or treating the pharmaceutical composition of pneumococcal infection disease, it includes The Pnu-Imune 23 composition.The pharmaceutical composition for preventing or treating can be the multi-agent for multiple dosing Measure pharmaceutical composition.
Other examples provide a kind of be used to prepare with the stability and/or anti-corrosion ability (or bactericidal activity) improved Pnu-Imune 23 composition method, the method includes prepare streptococcus pneumonia (Streptococcus Pneumoniae the step of capsular polysaccharide-protein conjugate), and by the capsular polysaccharide-protein conjugate and 2- phenoxy group The step of ethyl alcohol (2-PE) and formaldehyde (HCHO) mix；And a kind of stability for improving Pnu-Imune 23 and/or anti- The method of rotten ability (or bactericidal activity).The vaccine composition can be the multi-dose vaccine combination for multiple dosing Object.Described the step of preparing capsular polysaccharide-protein conjugate may include execute cyanalation method with by the capsular polysaccharide and Albumen passes through the step of-O-C (NH)-NH- connection.
[technical solution]
One embodiment provides a kind of Pnu-Imune 23 composition or pneumococcal immunogenic composition, packet Containing following components or substantially it is made of following components:
(i) capsular polysaccharide-protein conjugate, wherein the capsular polysaccharide is derived from streptococcus pneumonia (Streptococcus Pneumoniae),
(ii) 2- phenoxetol (2-PE), and
(iii) formaldehyde (HCHO).
Herein, pneumococcal immunogenic composition means induction for the combination of the immune response of pneumococcus Object, and unless otherwise specified, otherwise with meaning identical with Pnu-Imune 23 composition use.
Pnu-Imune 23 composition provided herein can be (to kill thin with the stability and/or anti-corrosion ability improved Bacterium ability) immunogenic composition, wherein by the inclusion of the pod of streptococcus pneumonia (Streptococcus pneumoniae) Film polysaccharide and 2- phenoxetol and formaldehyde as preservative, induction are (immune for the efficiency of the immune response of pneumococcus Originality) it is able to maintain for a long time.
It is more that the capsular polysaccharide can be the pod membrane from streptococcus pneumonia (Streptococcus pneumoniae) Sugar.Specifically, the capsular polysaccharide can be from streptococcus pneumonia (Streptococcus pneumoniae) serum The pod of such as 5 kinds or more, 7 kinds of two or more of type or more, 9 kinds or more, 11 kinds or more, 13 kinds or more or 14 kinds or more serotype Film polysaccharide.In one embodiment, the capsular polysaccharide may include from streptococcus pneumonia (Streptococcus Pneumoniae) 13 kinds to 24 kinds, 13 kinds to 19 kinds, 13 kinds to 17 kinds, 13 kinds to 15 kinds, 14 kinds to 24 kinds, 14 of serotype It plants to the capsular polysaccharide of 19 kinds, 14 kinds to 17 kinds or 14 kinds to 15 kinds serotype.For example, the capsular polysaccharide may include and be originated from In selected from streptococcus pneumonia (Streptococcus pneumoniae) serotype 1,2,3,4,5,6A, 6B, 7F, 8,9V, 9N, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,13 kinds to 24 kinds of 22F, 23F and 33F, 13 kinds to 19 kinds, 13 It plants to the pod membrane of 17 kinds, 13 kinds to 15 kinds, 14 kinds to 24 kinds, 14 kinds to 19 kinds, 14 kinds to 17 kinds or 14 kinds to 15 kinds serotype Polysaccharide.In one embodiment, the capsular polysaccharide may include from streptococcus pneumonia (Streptococcus Pneumoniae) serotype 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F 13 kinds of capsular polysaccharides, be derived from Streptococcus pneumonia (Streptococcus pneumoniae) serotype 1,2,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 14 kinds of capsular polysaccharides of 19F and 23F, be derived from streptococcus pneumonia (Streptococcus pneumoniae) serotype 1,3,4, 5,15 kinds of capsular polysaccharides of 6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F, 2 and 12F, or it is derived from streptococcus pneumonia (Streptococcus pneumoniae) serotype 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F, 2 and 15B 15 kinds of capsular polysaccharides, or be derived from streptococcus pneumonia (Streptococcus pneumoniae) serotype 1,3,4,5,6A, 17 kinds of capsular polysaccharides of 6B, 7F, 9V, 12F, 14,15B, 18C, 19A, 19F, 22F, 23F and 33F, but not limited to this.
Capsular polysaccharide-the protein conjugate can be the capsular polysaccharide above-mentioned from two or more serotype By common method for example by covalent bond separately with capsular polysaccharide-protein conjugate of albumen coupling.In a reality It applies in mode, the conjugate, which can have, wherein uses cyanalation method by each capsular polysaccharide (specifically, hydroxyl of polysaccharide Base) with albumen (specifically, the amino of albumen) pass through structure ([polysaccharide]-O-C (NH)-NH- of-O-C (NH)-NH- connection [albumen]).When the capsular polysaccharide and albumen using cyanalation method to be coupled when, it can maintain to exempt from advantageously after coupling Epidemic focus, because being unlikely to occur the structural modification of capsular polysaccharide compared with other methods (such as reduction amination method etc.). Although being combined for example, having reported when by reduction amination method by certain serotypes (19F etc.) and albumen coupling according to reaction Six saccharide ring structures are cut open and are therefore likely to form open architecture, to reduce immunogenicity, but working as will by cyanalation method When it is with albumen coupling, it is impossible to this problem occur.
The albumen can be carrier protein, such as can be and show non-toxic and anergy and can be with enough Amount and purity collect albumen.Capsular polysaccharide from various different serotypes can respectively with identical or different albumen Such as same protein coupling.
In one embodiment, the albumen can be CRM197 albumen.CRM197 albumen is from corynebacterium diphtheriae (Corynebacterium diphtheria) bacterial strain C7 (CRM197, such as it is grown in casamino acid and yeast extract In basal medium) culture medium separation diphtheria toxin a kind of non-toxic variant, i.e., a kind of toxoid.CRM197 albumen can To pass through ultrafiltration, ammonium sulfate precipitation and ion-exchange chromatography from corynebacterium diphtheriae (Corynebacterium diphtheria) bacterium Strain C7 culture medium purifying, or recombinated and obtained by common method, such as (be included in this with reference to U.S. Patent number 5,614,382 In text as reference) disclosed in method.In one embodiment, CRM197 albumen may include GenBank registration number The amino acid sequence of 1007216A, or with the amino acid sequence have 90% or more, 95% or more, 98% or more, 99% with Above, the amino acid sequence of 99.5% or more or 99.9% or more homology.
In addition, being derived from the corynebacterium diphtheriae except corynebacterium diphtheriae (Corynebacterium diphtheria) bacterial strain C7 Toxoid, can be used as carrier protein.
In addition to this, available carrier protein can be selected from least one following:
The bacteriotoxin of inactivation, for example, tetanus toxoid, pertussis toxoid, cholera toxoid (such as it is international specially Disclosed in sharp application publication number WO2004/083251), be derived from bacillus coli (Escherichia coli) (E.coli) LT, bacillus coli (Escherichia coli) ST and pseudomonas aeruginosa (Psendomonas Aeruginosa exotoxin A)；
Bacterial outer membrane proteins, such as outer membrane complex c (OMPC), porin, transferrin binding protein, pneumococcus Hemolysin, Pneumococal surface protein A (PspA), pneumococcal adhesin protein (PsaA), from A group or B group of streptococcus C5a peptase, haemophilus influenzae (Haemophilus influenza) protein D；
Ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) (BSA), the purifying protein of tuberculin are derivative Object (PPD)；
The variant (toxoid) of diphtheria toxin is such as CRM197, CRM173, CRM228, CRM45.
Capsular polysaccharide-the protein conjugate can be multivalence polysaccharide-protein conjugate, including wherein be derived from pneumonia chain Two or more such as 5 kinds or more, 7 kinds or more, 9 kinds or more, 11 kinds of coccus (Streptococcus pneumoniae) serotype Above, each of 13 kinds or more or 14 kinds or more capsular polysaccharides of serotype with carrier protein above-mentioned for example The conjugate of CRM197 albumen coupling.In one embodiment, the capsular polysaccharide-protein conjugate can be 13 valences to 24 Valence, 13 valences to 19 valences, 13 valences to 17 valences, 13 valences to 15 valences, 14 valences to 24 valences, 14 valences to 19 valences, 14 valences to 17 valences or 14 valences are extremely 15 valence polysaccharide-protein conjugates, wherein being derived from the 13 of streptococcus pneumonia (Streptococcus pneumoniae) serotype Kind to 24 kinds, 13 kinds to 19 kinds, 13 kinds to 17 kinds, 13 kinds to 15 kinds, 14 kinds to 24 kinds, 14 kinds to 19 kinds, 14 kinds to 17 kinds or Each of capsular polysaccharide of 14 kinds to 15 kinds serotype and the carrier protein such as CRM197 albumen coupling.For example, institute Stating capsular polysaccharide-protein conjugate can be 13 valences to 24 valences, 13 valences to 19 valences, 13 valences to 17 valences, 13 valences to 15 valences, 14 valences extremely 24 valences, 14 valences to 19 valences, 14 valences to 17 valences or 14 valences are selected from pneumonia streptococcus wherein being derived to 15 valence polysaccharide-protein conjugates Bacterium (Streptococcus pneumoniae) serotype 1,2,3,4,5,6A, 6B, 7F, 8,9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20,13 kinds to 24 kinds of 22F, 23F and 33F, 13 kinds to 19 kinds, 13 kinds to 17 kinds, 13 kinds extremely Each of capsular polysaccharide of 15 kinds, 14 kinds to 24 kinds, 14 kinds to 19 kinds, 14 kinds to 17 kinds or 14 kinds to 15 kinds serotype with The carrier protein such as CRM197 coupling.In one embodiment, the capsular polysaccharide-protein conjugate may is that 13 Valence polysaccharide-protein conjugate, it includes be derived from streptococcus pneumonia (Streptococcus pneumoniae) serotype 1,3, 4, each of 13 kinds of capsular polysaccharides of 5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F and carrier protein be for example CRM197；14 valence polysaccharide-protein conjugates, it includes be derived from streptococcus pneumonia (Streptococcus pneumoniae) blood Each of the 14 kinds of capsular polysaccharides and carrier egg of clear type 1,2,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F White such as CRM197；15 valence polysaccharide-protein conjugates, it includes be derived from streptococcus pneumonia (Streptococcus Pneumoniae) serotype 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F, 2 and 12F 15 kinds of capsular polysaccharides Each of and carrier protein such as CRM197；15 valence polysaccharide-protein conjugates, it includes be derived from streptococcus pneumonia (Streptococcus pneumoniae) serotype 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F, 2 and 15B Each of 15 kinds of capsular polysaccharides and carrier protein such as CRM197；Or 17 valence polysaccharide-protein conjugates, it includes be originated from In streptococcus pneumonia (Streptococcus pneumoniae) serotype 1,3,4,5,6A, 6B, 7F, 9V, 12F, 14,15B, Each of 17 kinds of capsular polysaccharides of 18C, 19A, 19F, 22F, 23F and 33F and carrier protein such as CRM197, but be not limited to This.
Pnu-Imune 23 composition provided herein is the pneumococcus comprising the capsular polysaccharide-protein conjugate Conjugate vaccines (PCV).The Pnu-Imune 23 composition can be polyvalent pneumococcal vaccine composition, the composition Comprising be wherein derived from two or more such as 5 kinds or more of streptococcus pneumonia (Streptococcus pneumoniae) serotype, 7 kinds or more, 9 kinds or more, 11 kinds or more, 13 kinds or more or each of 14 kinds or more the capsular polysaccharides of serotype with mention above The polysaccharide-protein conjugate of the carrier protein arrived such as CRM197 albumen coupling.In one embodiment, the pneumococcus Vaccine composition can be 13 valences to 24 valences, 13 valences to 19 valences, 13 valences to 17 valences, 13 valences to 15 valences, 14 valences to 24 valences, 14 valences extremely 19 valences, 14 valences to 17 valences or 14 valences to 15 valence Pnu-Imune 23 compositions, the composition includes 13 kinds to 24 kinds, 13 kinds extremely 19 kinds, 13 kinds to 17 kinds, 13 kinds to 15 kinds, 14 kinds to 24 kinds, 14 kinds to 19 kinds, 14 kinds to 17 kinds or 14 kinds to 15 kinds polysaccharide- Protein conjugate, wherein 13 kinds to 24 kinds from streptococcus pneumonia (Streptococcus pneumoniae) serotype, 13 kinds to 19 kinds, 13 kinds to 17 kinds, 13 kinds to 15 kinds, 14 kinds to 24 kinds, 14 kinds to 19 kinds, 14 kinds to 17 kinds or 14 kinds to 15 kinds Each of capsular polysaccharide of serotype is coupled with the carrier protein such as CRM197.Such as the Pnu-Imune 23 group Close object can be 13 valences to 24 valences, 13 valences to 19 valences, 13 valences to 17 valences, 13 valences to 15 valences, 14 valences to 24 valences, 14 valences to 19 valences, 14 valences to 17 valences or 14 valences to 15 valence Pnu-Imune 23 compositions, the composition include wherein to be derived to be selected from pneumonia streptococcus Bacterium (Streptococcus pneumoniae) serotype 1,2,3,4,5,6A, 6B, 7F, 8,9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20,13 kinds to 24 kinds of 22F, 23F and 33F, 13 kinds to 19 kinds, 13 kinds to 17 kinds, 13 kinds extremely Each of capsular polysaccharide of 15 kinds, 14 kinds to 24 kinds, 14 kinds to 19 kinds, 14 kinds to 17 kinds or 14 kinds to 15 kinds serotype with The polysaccharide-protein conjugate of the carrier protein such as CRM197 albumen coupling.
In one embodiment, the Pnu-Imune 23 composition may is that
(1) 13 valence Pnu-Imune 23 composition, it includes 13 kinds of polysaccharide-protein conjugates, wherein being derived from pneumonia chain Coccus (Streptococcus pneumoniae) serotype 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F Each of 13 kinds of capsular polysaccharides and the carrier protein such as CRM197 albumen coupling；
(2) 14 valence Pnu-Imune 23 compositions, it includes 14 kinds of polysaccharide-protein conjugates, wherein being derived from pneumonia chain Coccus (Streptococcus pneumoniae) serotype 1,2,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F Each of 14 kinds of capsular polysaccharides and the carrier protein such as CRM197 albumen coupling；
(3) 15 valence Pnu-Imune 23 compositions, it includes 15 kinds of polysaccharide-protein conjugates, wherein being derived from pneumonia chain Coccus (Streptococcus pneumoniae) serotype 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F, 2 With each of 15 kinds of capsular polysaccharides of 12F and the carrier protein such as CRM197 albumen coupling；
(4) 15 valence Pnu-Imune 23 compositions, it includes 15 kinds of polysaccharide-protein conjugates, wherein being derived from pneumonia chain Coccus (Streptococcus pneumoniae) serotype 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F, 2 With each of 15 kinds of capsular polysaccharides of 15B and the carrier protein such as CRM197 albumen coupling；Or
(5) 17 valence Pnu-Imune 23 compositions, it includes 17 kinds of polysaccharide-protein conjugates, wherein being derived from pneumonia chain Coccus (Streptococcus pneumoniae) serotype 1,3,4,5,6A, 6B, 7F, 9V, 12F, 14,15B, 18C, 19A, Each of 17 kinds of capsular polysaccharides of 19F, 22F, 23F and 33F and the carrier protein such as CRM197 albumen coupling.
Compared with the polyvaccine (such as 13 valence vaccines) routinely developed, polyvalent pneumococcal vaccine group provided herein Close 13 valences to 24 valences, 13 valences to 19 valences, 13 valences to 17 valences, 13 valences to 15 valences, 14 valences to 24 valences, 14 valences of for example above-mentioned composition of object There is significant outstanding threshold value to 19 valences, 14 valences to 17 valences or 14 valences to 15 valence Pnu-Imune 23s, and be expected very The effect of outstanding prevention and/or treatment pneumococcal infection disease.
The Pnu-Imune 23 composition can be the single dose for being formulated for single-dose or for multiple dosing Multi-agent composition.As used herein, " multi-agent ", which may mean that, is administered comprising (inoculation) can be administered to 1 (inoculation) individual is more than the vaccine dose of primary (such as 2 times or more) or (inoculation) can be administered to 2 or more administrations (inoculation) The preparation unit of vaccine dose that is individual primary or being more than primary (such as 2 times or more).
Other than pneumococcal capsular polysaccharide-protein conjugate of two or more serotype, the Pnu-Imune 23 group Closing object can also include preservative.Can the preservative used in the vaccine composition (such as ejection preparation) can be choosing From 2- phenoxetol, formaldehyde, methaform, metacresol, methyl p-hydroxybenzoate, propylparaben, benzethonium chloride, At least one of benzalkonium chloride, benzoic acid, benzyl alcohol, phenol, thimerosal, phenyl mercuric nitrate etc..
In one embodiment, as polyvalent pneumococcal vaccine composition for example above-mentioned 13 above-mentioned Valence to 24 valences, 13 valences to 19 valences, 13 valences to 17 valences, 13 valences to 15 valences, 14 valences to 24 valences, 14 valences to 19 valences, 14 valences to 17 valences or The optimization form of 14 valences to 15 valence Pnu-Imune 23 compositions, the preservative may include 2- phenoxetol and formaldehyde. Similarly, by the inclusion of 2- phenoxetol and formaldehyde as preservative, the amount of the 2- phenoxetol used is reduced, to drop Low toxicity and/or side effect, and more improved anti-corrosion effect is realized by the synergistic effect of two kinds of components.
It include that the content of the 2- phenoxetol in polyvalent pneumococcal vaccine composition of the invention can be lower than 10mg/ml, lower than 7mg/ml, 6mg/ml or less or 5mg/ml hereinafter, such as 4mg/ml or more and being lower than 10mg/ml, 4mg/ml To 7mg/ml, 4mg/ml or more and lower than 7mg/ml, 4 to 6mg/ml, 4.5mg/ml to 6mg/ml, 5mg/ml to 6mg/ml, 4 to (as used herein, the term used in numberical range " extremely " refers under 5mg/ml or 4.5mg/ml to 5mg/ml It is more than limit value with below upper limit value between range).The content of 2- phenoxetol is preferably described in the vaccine composition More than the lower limit value of range, to show significant anti-corrosion effect, and the upper limit value or described of the preferably less than described range The upper limit value of range is hereinafter, to ensure no or acceptable toxicity and/or side effect.
Include the formaldehyde in polyvalent pneumococcal vaccine composition of the invention content can for 90 to 200 μ g/mL, 90 to 190 μ g/mL, 90 to 180 μ g/mL, 90 to 170 μ g/mL, 100 to 200 μ g/mL, 100 to 190 μ g/mL, 100 to 180 μ The μ of g/mL or 100 to 170 g/mL.It include the content of the formaldehyde in the vaccine composition is preferably the lower limit value of the range More than, to show significant anti-corrosion effect, and the upper limit value of the preferably described range is hereinafter, to ensure without or can connect The toxicity and/or side effect received.
The vaccine composition is characterized in that under common condition of storage, for example, at 2 DEG C to 8 DEG C, 20 DEG C to 25 DEG C or Stabilization about 3 months or more, about 6 months or more, about 1 year or more, about 1.5 years or more, about 2 years or more under the conditions of about 37 DEG C of temperature Or about 2.5 years or more.Herein, the stability of vaccine composition may mean that the vaccine composition by original antigenicity (immunogenicity) maintains equivalent level and/or every kind of component and is maintained without degrading or losing, and/or does not feel Dye is such as bacterium/virus.
Other embodiments provide a kind of for preventing or treating pneumococcal infection or pneumococcal infection disease Pharmaceutical composition, it includes polyvalent pneumococcal vaccine compositions above-mentioned.Other embodiments provide a kind of use In preventing or the method for the treatment of pneumococcal infection or pneumococcal infection disease, the method includes to needing pneumococcus The multivalence lung that the upper surface of infection or the individual administration pharmacy effective dose of the prevention of pneumococcal infection disease or treatment are mentioned Scorching coccus vaccine composition.Include Pnu-Imune 23 composition in described pharmaceutical composition or for the prevention or controls The Pnu-Imune 23 composition for the treatment of method can be for the single dose pharmaceutical composition of single-dose or for multiple dosing Multiple doses composition.
Pneumococcal infection disease means all diseases as caused by pneumococcal infection, and can be pneumonia, Inflammation, nasosinusitis, bacteremia in middle ear etc.." pneumonia " is a kind of acute infectious diseases of pulmonary parenchyma, its source of infection master If streptococcus pneumonia (Streptococcus pneumoniae) and Friedlander's bacillus (Klebsiella pneumoniae).Specifically, pneumococcal pneumonia accounts for about the 50% of all pneumonia, and be wherein occur it is serious shiver with cold, Fever, cough and pectoralgia and the disease of the usual bleeding of sputum, and may cause complication such as pleurisy, meningitis, the heart Intimitis, peritonitis etc. (Diagn.Microbiol.Infect.Dis., 2001,39:181-185).
Herein, term " pneumococcus " refers to streptococcus pneumonia (Streptococcus pneumoniae), and It generally speaking, is a kind of symbiont body colonized in human nasopharynx's mucous membrane surface.When the factor of host allows the organism When close to lower respiratory tract, strong inflammatory response then occurs, and therefore causes fine and close consolidate when alveolar space fills diffusate Knot, to cause pneumonia.Pneumococcus can synthesize the capsular polysaccharide of 90 kinds or more unique structures, and according to capsular polysaccharide These structures and amynologic characteristic classify to the serotype of pneumococcus.Therefore, when the capsular polysaccharide using pneumococcus It, can according to the serotype for the pneumococcus that the type of capsular polysaccharide, i.e. capsular polysaccharide are originated from when preparation is used for the preparation of vaccine It can show different immune responses.
Since the capsular polysaccharide is identified as antigen when being administered into internal, to allow to generate for the anti-of it Body, therefore allow to generate the vaccine composition for preventing pneumococcus (or pneumococcal infection disease) using it.In Herein, term " antigen " means the substance that specific can cause immune response when invading body.In an embodiment party In formula, be derived from streptococcus pneumonia (Streptococcus pneumoniae) serotype 1,2,3,4,5,6A, 6B, 7F, 8,9V, 9N, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,13 kinds to the 24 kinds capsular polysaccharides of 22F, 23F and 33F can be with It is respectively served as antigen.
Other embodiments provide a kind of be used to prepare with the stability and/or anti-corrosion (or bactericidal ability) improved Pnu-Imune 23 composition method or it is a kind of for improving Pnu-Imune 23 stability and/or anti-corrosion ability (or Bactericidal ability) method.The method may include:
(1) capsular polysaccharide-protein conjugate step of streptococcus pneumonia (Streptococcus pneumoniae) is prepared Suddenly, and
(2) step for mixing the capsular polysaccharide-protein conjugate with 2- phenoxetol (2-PE) and formaldehyde (HCHO) Suddenly.
The content of capsular polysaccharide, albumen, conjugate and 2- phenoxetol and formaldehyde is same as described above.
In the method, the capsular polysaccharide can be prepared by the way that well known to a person skilled in the art standard techniques, And the method is not particularly limited in it.The capsular polysaccharide can reduce its size by hydrolyzing, to reduce viscosity and lure Lead effective immunogenicity.
The step of of preparing capsular polysaccharide-protein conjugate (1) may include will be described by executing cyanalation method The step of capsular polysaccharide is connect with albumen by-O-C (NH)-NH-.
In one embodiment, the step of of preparing capsular polysaccharide-protein conjugate (1) may include:
(i) step of simultaneously purified capsular polysaccharide is separated or separated from streptococcus pneumonia (Streptococcus pneumoniae) Suddenly；
(ii) the step of dissolving and/or hydrolyze separated capsular polysaccharide；With
(iii) step being connect the capsular polysaccharide by-O-C (NH)-NH- with albumen by executing cyanalation method Suddenly.
In other embodiments, the step of of preparing capsular polysaccharide-protein conjugate (1) can be by executing cyanogen After the step of capsular polysaccharide is connect with albumen by-O-C (NH)-NH- by base method, further executes and be selected from ultrafiltration The one or more steps of step, aseptic filtration step and adsorption step.CDAP (1- cyanogen can be used in the cyanalation method Base -4-dimethylaminopyridine tetrafluoroborate) or CNBr carry out.
In a particular instance of the invention, being dissolved respectively using NaTDC has 13 kinds or 15 kinds respectively differences Serotype streptococcus pneumonia (Streptococcus pneumoniae), and release the polysaccharide for being connected to cell.Then, Purified by executing CTAB (cetyl trimethylammonium bromide) method from 21 kinds of serotype 1,2,3,4,5,6A, 6B, The polysaccharide of 9V, 8,9N, 10A, 11A, 12F, 15B, 17F, 18C, 19A, 19F, 20,22F and 23F because they and CTAB it Between can form ionic bond, and do not reacted with CTAB using phosphaljel (Algel) Lai Chunhua 3 kinds of serotype 7F, 14 and 33F.The reacting with CTAB can be by adding 0.5 to the 5 weight weight of % or 1 to 3 % (such as 23F to reactant 2 to 3 weight % for the polysaccharide in source, 1 to the 2 weight % for the polysaccharide for the serotype being derived from except 23F) amount Concentration is the CTAB of 1 to 20% (w/v) or 5 to 15% (w/v) to carry out, but not limited to this.After being reacted with CTAB, Ke Yijin Precipitating after one step is centrifuged recycles, using sodium chloride solution (for example, about 100 to precipitating described in 500mM) resuspension and use Sodium iodide removes one or more steps in CTAB ion, but not limited to this.Phosphaljel reaction can pass through to The phosphaljel solution that reactant adds the amount of 1 to the 20 weight weight of % or 5 to 15 % carries out, but not limited to this.
Since when using the vaccine composition merely with the capsular polysaccharide, there is lower siberian crabapple compared with adult The baby and children of system cannot identify it as antigen, it is thus possible to immune response not occur, so preparing in the present invention simultaneously The form for the conjugate for wherein combining carrier protein with the capsular polysaccharide is used.
" carrier protein " refers to can be by improving the immunogenicity of the polysaccharide antigen with capsular polysaccharide covalent bond Albumen, and specific type is same as described above.In a particular implementation, CRM197 can be used.The carrier egg It is white to be coupled by standard coupling procedures and the capsular polysaccharide and even by capsular polysaccharide-carrier protein that it is formed Connection object can be capsular polysaccharide-carrier protein couplet object that one or more capsular polysaccharides are coupled to a carrier protein.
The well known method for being used to prepare capsular polysaccharide and carrier protein couplet object can be included in the scope of the present invention Within, and the capsular polysaccharide and carrier protein are passed through-O-C with the wherein cyanalation method of use by the conjugate (NH) structure of-NH- group connection.The cyanalation method can by those skilled in the art by well known method compatibly It carries out, such as can be carried out by using CDAP (1- cyano -4-dimethylaminopyridine tetrafluoroborate) or CNBr, but It is without being limited thereto.
As an example for being used to prepare capsular polysaccharide and carrier protein couplet object, can be purified by chemical activation The capsular polysaccharide of each chemical activation is simultaneously coupled to the carrier protein to form sugared coupling by capsular polysaccharide one by one Object.Handling the cyanalation activity carried out using CDAP (1- cyano -4-dimethylaminopyridine tetrafluoroborate) can be by pod The hydroxyl of film polysaccharide is changed to cyanate group, and comes to form covalent bond with the amino of carrier protein CRM197 by using it. The cyanalation reaction carried out by CDAP specifically can be by adding 3 molar equivalents compared with the CDAP of 1 molar equivalent PH is simultaneously adjusted to 9.0 to terminate by glycine solution, but not limited to this, and those skilled in the art can be suitble to according to purpose Ground controls reaction solution and reaction condition.
The capsular polysaccharide of collection-carrier protein couplet object can be purified by a variety of different methods.Those methods Example includes concentration/percolation method, column chromatography and multiple filtration.By the polysaccharide-protein conjugate that will be purified mix respectively come It prepares and uses vaccine composition of the invention.For example, can by by 13 kinds of individual capsular polysaccharide-carrier protein couplet objects with Physiologically acceptable medium is prepared to prepare composition.The example of this medium can be water, buffered saline solution, note It penetrates and uses water, polyalcohol (such as glycerol, propylene glycol, liquid macrogol) or dextrose solution, but not limited to this.
In a particular instance of the invention, 13 kinds of capsular polysaccharide-carrier conjugates are made by carrying out following step It is standby: 1) dissolution and hydrolysis of 13 kind capsular polysaccharide, 2) by using CDAP (1- cyano -4-dimethylaminopyridine tetrafluoro boron Hydrochlorate) every kind of capsular polysaccharide of Lai Jinhang and the coupling reaction process of CRM197,3) termination of coupling reaction, 4) ultrafiltration, 5) it removes Bacterium filtering and 6) adsorption step.
Herein, term " vaccine " is the biological agent containing antigen of immunity to be provided for living body, and mean to lead to Cross the immunogene or antigenicity object for prophylaxis against infection diseases for being administered into mankind or animal and generating immunity in the living body Matter.
The vaccine composition can also comprising selected from adjuvant, preservative, buffer, cryoprotector, salt, divalent sun from One of son, non-ionic detergent and free-radical oxidation inhibitor or more persons.
Herein, term " adjuvant " means the object of the immunogenicity for improving immunogenic composition of the invention Matter.In general, providing the adjuvant is to enhance immune response, and it is well known to the skilled artisan. The adjuvant for being suitable for the effect of improving vaccine composition of the invention may include selected from least one following:
(1) aluminium salt (alum) (such as aluminium hydroxide, aluminum phosphate, aluminum sulfate etc.)；
(2) emulsion formulations of water-in-oil type are ((below with or without other specific immunostimulating agents such as muramyl peptide Middle definition) or bacterial cell wall components), such as (a) MF59 (No.WO90/14837): containing 5% (w/v) squalene, 0.5% (w/v) Tween 80 and 0.5% (w/v) Span 85 (randomly also containing various different amounts of MTP-PE), and use microfluidizer If 110Y type microfluidizer (Microfluidics, Newton, MA) is configured to submicron particle, (b) SAF: contain 10% (w/ V) squalene, 0.4% (w/v) Tween 80,5% (w/v) pluronics block polymer L121 and thr-MDP (see below), And the lotion of big particle size is formed as by sub-micron emulsion or cavity by microjet, and (c) RibiTMAdjuvant system (RAS) (Corixa, Hamilton, MT): containing from following one or more bacterial cell wall components: 2% (w/v) squalene, 3-O- deacylation monophosphoryl lipid A (MPL disclosed in 0.2% (w/v) Tween 80 and U.S. Patent number 4,912,094TM) (Corixa), trehalose dimycolate (TDM) and cell wall skeleton (CWS), preferably MPL+CWS (DetoxTM)；
(3) saponin adjuvant such as Quil A or STIMULONTM(Antigenics, Framingham, MA, the U.S. are special by QS-21 The sharp number particle (such as ISCOM (immunostimulating compound)) 5,057,540) or by it formed；
(4) bacteria lipopolysaccharide, synthesis lipid A homologue (such as: aminoalkyl glucosamine phosphate compounds Or derivatives thereof (AGP)) or homologue (this can buy from Corixa and be disclosed in U.S. Patent number No, in 6,113,918； An example of the AGP is 2- [(R) -3- myristoyl oxygroup myristoyl amino] ethyl 2- deoxidation -4-O- phosphono -3-O- [(R) -3- myristoyl oxygroup myristoyl base] -2- [(R) -3- myristoyl oxygroup myristoyl amino]-b-D- glucopyranoside, and And it is also referred to as 529 (being also referred to as RC529 in the past), and it is formulated into aqueous form or stable lotion；
(5) polynucleotides (such as oligonucleotides (U.S. Patent number 6,207,646) containing CpG motif) synthesized；
(6) cell factor, for example, interleukin (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL 12, IL-15, IL-18 etc.), interferon (such as interferon), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony thorn Swash the factor (MCSF), tumor necrosis factor (TNF), costimulatory molecules B7-1 and B7-2 etc.；
(7) wild-type cholera toxin (CT) or the glutamic acid being for example wherein located in the 29th amino acid position are by other amino The cholera toxin (WO-2002/098368 and WO-2002/098369) of the acid especially mutation of histidine replacement, pertussis toxin (PT) or the thermolabile toxin of bacillus coli (LT), the variant of the detoxification of especially bacterial ADPribosylating toxin Such as LT-K63, LT-R72, CT-S109, PTK9/G129 (WO-93/13302 and WO-92/19265)；With
(8) tripolymer of complement such as complement component C3d,
But not limited to this.
The muramyl peptide may include N- acetyl group-muramyl different gucosamine of-L- Threonyl-D- (thr-MDP), N- Positive muramyl-l-Alanine -2- (1 ' -2 '-two palmityl-sn- glyceryl -3- hydroxyl phosphinylidyne the oxygroup)-ethamine of acetyl group - (MTP-PE) etc., but not limited to this.
The Alum adjuvant can be the vaccine of alum precipitate or the vaccine of alum absorption.The aluminium salt may include hydration Aluminium oxide, hydrated alumina, hibbsite (ATH), hydrated aluminum, aluminum trihydrate, aluminium glue, syperfos, aluminium hydroxide Gel, aluminium hydroxide, sulfuric acid Adju-Phos (Aluminium phosphate adjuvant (APA)), amorphous alumina etc., but not limited to this.APA meaning Taste the suspension of Adju-Phos.It can be prepared as follows: aluminium chloride and sodium phosphate are mixed with the ratio (by volume) of 1:1 So as to sulfate precipitate Adju-Phos, and make 2~8 μm of size of the sediment, then it is dialysed simultaneously with saline solution Degerming.As an embodiment, use commercially available Al (OH)3(such as aluminium glue or superfos) carrys out adhesion protein.Every 1mg Aluminium hydroxide can adsorb 50~200g albumen, and the ratio depends on the pI of albumen and the pH of solvent.With low pI's Albumen is more strongly combined than the albumen with high pI.The aluminium salt can be by forming the storage of 2~3 weeks antigen of slow release antigen It deposits a little, non-specifically activated macrophage, complement, innate immune mechanisms.
Herein, term " antibacterial agent (or preservative) " means to inhibit the proliferation of microorganism in the vaccine composition Antiviral and/or antimicrobial material, and can be such as or mixtures thereof thimerosal, 2- phenoxetol, formaldehyde, but It is without being limited thereto, and all Determination of common preservatives used in the art can be used.
In addition, the vaccine composition may include one or more physiologically acceptable buffers.For example, when described When vaccine composition is transfusion or injection, the buffer can pH 4.0 to 10.0, particularly pH 5.0 to 9.0, More particularly pH 6.0 to 8.0 has buffer capacity.The buffer can be selected from TRIS, acetate, glutamate, lactic acid Salt, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate, At least one of triethanolamine buffer.
Specifically, when vaccine composition of the invention is intended to parenteral administration, the buffer can be suitable for It is selected in the buffer of USP.For example, the buffer can be selected from monoacid such as acetic acid, benzoic acid, gluconic acid, glycerol Acid, lactic acid, binary acid such as aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid, tartaric acid, alkali is such as At least one of ammonia, diethanol amine, glycine, triethanolamine, TRIS.
In addition, vaccine composition of the invention may include non-ionic detergent.For example, it may include surface-active Agent such as polysorbate 20 and polysorbate 80, especially polyoxyethylene sorbitan esters (commonly known as tween)； The copolymer (such as DOWFAXTM) of ethylene oxide (EO), propylene oxide (PO) and butylene oxide (BO)；It is respectively provided with ethyoxyl (oxygen Base -1,2- second diyl) group different repeat numbers oxtoxinol, especially oxtoxinol-9 (Triton-100)；Ethyl Phenoxypolyethoxy ethanols (IGEPAL CA-630/NP-40)；Nonylphenol ethoxylate such as NP series；From laruyl alcohol, whale The polyoxyethylene fatty acid ether (Brij surfactant) that ceryl alcohol, stearyl alcohol, oleyl alcohol generate, especially triethylene glycol list lauryl Ether (Brij 30)；The Sorbitan alcohol ether of referred to as SPAN, especially anhydrosorbitol trioleate (Span 85), but It is without being limited thereto.
Tween 80 can be contained in lotion, and can be used with non-ionic detergent such as Tween 80/Span 85 mixture.The combination of polyoxyethylene sorbitan esters such as Tween 80 and oxtoxinol such as Triton X-100 are suitable It closes, and the combination of Laureth 9 and tween or oxtoxinol are also useful.Specifically, 0.01% (w/v) to 1% (w/v), particularly the polyoxyethylene sorbitan esters (such as Tween 80) of 0.1% (w/v)；0.001% (w/v) to 0.1% (w/v), particularly the Octylphenoxy polyoxyethanols or nonylphenoxy polyoxyethanols of 0.005% (w/v) to 0.02% (w/v) (such as Triton X-100)；With 0.1% (w/v) to 20% (w/v), preferably 0.1% (w/v) to 10% (w/v), particularly The polyoxyethylene ether (such as laureth9) of 0.1% (w/v) to 1% (w/v) or about 0.5% (w/v).
Vaccine composition of the invention can be formulated into the form of single dose bottle, multi-agent bottle or prefilled syringe.Therefore, one A embodiment is provided for example to be filled with more than the bottle of the vaccine composition of dose or in advance comprising single dose or multi-agent Emitter.The vaccine composition can also include physiologically acceptable carrier.
Herein, " multi-agent " may mean that it is more than primary for (inoculation) individual administration (inoculation) being administered to 1 Vaccine dose, or the vaccine dose of (inoculation) individual administration (inoculation) more than once can be administered to 2 or more.
As the physiologically acceptable carrier for liquid preparation, there is aqueous or non-aqueous solvent, suspension, lotions And oil.The example of non-aqueous solvent includes propylene glycol, polyethylene glycol and ethyl oleate.The aqueous carrier includes water, alcohol/water Property solvent, lotion or suspension, saline solution and buffer solution.Oil example include synthetic oil such as plant or animal oil, peanut oil, Soybean oil, sunflower oil, liver oil and marine animal oil, and the lipid obtained from milk or egg.Vaccine composition of the invention can It to be isotonic, hypertonic or hypotonic, and can be preferably isotonic by the pharmaceutical composition of infusion or drug administration by injection, but not It is limited to this.Meanwhile it is isotonic or hypertonic can be advantageous for the storage of the composition.When the vaccine composition is When hypertonic, it can be diluted before administration to become isotonic.The isotonic agent can be ionic isotonic agent such as salt or non- Ionic isotonic agent such as carbohydrate.The ionic isotonic agent includes sodium chloride, calcium chloride, potassium chloride, magnesium chloride etc., but is not limited to This.The non-ionic isotonic agent includes D-sorbite, glycerol etc., but not limited to this.
The amount of conjugate can be selected to induction immune protection response without significant side effect in every kind of vaccine dose Amount, and this amount can change according to the serotype of pneumococcus.Specifically, with the pod membrane from serotype 1 The poidometer of polysaccharide, the vaccine composition may include 0.8 to 1.2 or 0.9 to 1.1 weight ratio (that is 0.8 to 1.2 Parts by weight or 0.9 to 1.1 parts by weight) be derived from serotype 2,3,4,5,6A, 7F, 8,9V, 9N, 10A, 11A, 12F, 14, The capsular polysaccharide of 15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F, and may include 1.6 to 2.4,1.8 to 2.2 or 1.9 to 2.1 weight ratios (that is 1.6 to 2.4 parts by weight, 1.8 to 2.2 parts by weight or 1.9 to 2.1 parts by weight) are derived from The capsular polysaccharide of serotype 6B, but not limited to this.
As an example for being used to prepare the conjugate, every kind of conjugate may include 0.1 to 100 μ g, particularly The polysaccharide of 0.1 to 10 μ g, more particularly 1 to 5 μ g.
In addition, most particularly, other polysaccharide other than the capsular polysaccharide from serotype 6B, that is to say, that be originated from In serotype 1,2,3,4,5,6A, 7F, 8,9V, 9N, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F It, can be with 2 to the 2.4 μ μ of g or 2.1 to the 2.3 g for example, about amount of 2.2 μ g by comprising and described being originated from the capsular polysaccharide of 33F It can be wrapped in the capsular polysaccharide of serotype 6B with the amount of 4 to 4.8 μ g, 4.2 to the 4.6 μ μ of g or 4.3 to 4.5 g for example, about 4.4 μ g Contain, but not limited to this.
The optimized amount of component for certain vaccines can confirm by research on standard, and the research on standard is included in pair As the immune response that middle observation is suitable.For example, the vaccination doses for the mankind can pass through the result for zoopery of extrapolating To determine.In addition, dosage can be empirically determined according to site requirement in those skilled in the art.
The vaccine composition can also include aluminium element and sodium chloride, but not limited to this.
It is effectively measured by the way that pharmacy is administered through whole body or mucosal route, vaccine composition of the invention can be used for protecting possibility By the individual of pneumococcal infection and prevent pneumococcal disease.Term " prevention " of the invention means epidemic disease through the invention Seedling composition is administered to inhibit or postpone all behaviors of the infection as caused by pneumococcus." pharmacy defined in the present invention Effective quantity " means to significantly reduce by a possibility that pneumococcal infection or needed for the horizontal generation antibody of the seriousness of infection Dosage.Term " administration " of the invention means that formulatory agents are introduced into individual by certain suitable methods.The present invention Vaccine composition can by mouth, nose, rectum, percutaneous or be administered via the approach of aerosol sucking, but not limited to this.It is described Administration can be selected from and be injected by intramuscular, intraperitoneal, intradermal or subcutaneous route, or pass through oral cavity/alimentary canal, air flue or life Grow the mucosa delivery of the urinary tract.As an embodiment, intranasal administration can be used for treating the inflammation in pneumonia or middle ear, and In this case, by more effectively preventing nasopharynx pneumococcus carrier, the inflammation can be weakened in early stage.
The administration object " individual " of vaccine composition or pharmaceutical composition of the invention may mean that pathogenic bacteria can be with Organism in the work wherein infected or the cell from its separation, tissue or its culture medium, and the organism can be height Equal vertebrates, more specifically can be mammal such as mankind etc., but be not particularly limited to this.
In other embodiments of the invention, composition of the invention can be administered by single inoculation, or with suitable The doses at intervals of conjunction two, three, four or more times, but not limited to this.For example, for baby and neonatal for by lung The regular inoculation schedule of affecting conditions caused by scorching streptococcus (Streptococcus pneumoniae) can be 2,4,6 With 12 to 15 monthly ages.
In addition, the composition can also be comprising being derived from streptococcus pneumonia (Streptococcus pneumoniae) One or more albumen.As be suitable for by comprising streptococcus pneumonia (Streptococcus pneumoniae) albumen reality Example, not only albumen disclosed in WO-2002/053761, but also the albumen identified in WO-2002/083855 can be wrapped It includes within the scope of the present invention.
In a particular instance of the invention, illustrate a kind of multivalence such as 13 valences to 24 valences, 13 valences to 19 valences, 13 valences To 17 valences, 13 valences to 15 valences, 14 valences to 24 valences, 14 valences to 19 valences, 14 valences to 17 valences or 14 valences to 15 valence Pnu-Imune 23 groups Object is closed, includes 2.2 every kind of polysaccharide of μ g in 0.5mL vaccine composition in total, but the polysaccharide from 6B is 4.4 μ g, about 29.3 μ g CRM197 carrier proteins, 0.5mg aluminium element (2mg aluminum phosphate) adjuvant, about 4.25mg is (in the feelings for not including preservative Under condition) or about 3.5mg (comprising preservative) sodium chloride, about 295 μ g succinate buffer solution, and about 3mg 2- phenoxetol and about 60 μ g formaldehyde.
In another particular instance of the invention, in the serum of rabbit for being wherein inoculated with the vaccine composition, blood Clear type specificity IgG concentration ratioHigher level (table 1).Furthermore, it was confirmed that it shows to compareOutstanding effect, even (conditioning cell gulps down in the iunctional immunogenic validation test carried out to it Bite measuring method) in (table 2).Therefore, it can be seen that vaccine composition of the invention is for the purposes of prevention pneumococcal disease Have the effect of very outstanding.
Other aspects of the present invention are a kind of immunogenic compositions for pneumococcus, it includes 13 kinds to 24 kinds, 13 kinds to 19 kinds, 13 kinds to 17 kinds, 13 kinds to 15 kinds, 14 kinds to 24 kinds, 14 kinds to 19 kinds, 14 kinds to 17 kinds or 14 kinds to 15 kinds Capsular polysaccharide-carrier protein couplet object.The conjugate and pneumococcus are same as described above.
Of the invention includes 13 kinds to 24 kinds, 13 kinds to 19 kinds, 13 kinds to 17 kinds, 13 kinds to 15 kinds, 14 kinds to 24 kinds, 14 Kind includes to be derived to have to 19 kinds, 14 kinds to 17 kinds or 14 kinds to 15 kinds capsular polysaccharide-carrier protein couplet object compositions 13 kinds to 24 kinds, 13 kinds to 19 kinds, 13 kinds to 17 kinds, 13 kinds to 15 kinds, 14 kinds to 24 kinds, 14 kinds to 19 kinds, 14 kinds to 17 kinds Or the capsular polysaccharide of the streptococcus pneumonia (Streptococcus pneumoniae) of 14 kinds to 15 kinds respective different serotype, And antigen is identified it as when being administered in vivo, is directed to its antibody so as to cause immune response to generate, and because This it may be used as immunogenic composition for pneumococcus.
Other aspects of the present invention are a kind of by the way that the vaccine composition or immunogenicity group is administered to the individual of needs Object is closed to prevent the method for pneumococcal disease.
Other aspects of the present invention, which are provided, to be used comprising 13 kinds of capsular polysaccharides-carrier protein couplet object composition in preparation Purposes in the vaccine composition of prevention pneumococcal disease, wherein 13 kinds of conjugates are will to be derived from streptococcus pneumonia (Streptococcus pneumoniae) serotype 1,3,4,5,6A, 6B, 7F, 9V, 14,13 kinds of 18C, 19A, 19F and 23F Each of capsular polysaccharide covalent coupling is to the conjugate of the carrier protein, and the carrier protein is CRM197 egg It is white, and the capsular polysaccharide and carrier protein are passed through-O-C (NH)-with the wherein cyanalation method of use by the conjugate The structure of NH- group connection.
Vaccine composition, the prevention etc. of immunogenic composition and pneumococcal disease are same as described above.
Other aspects of the present invention are a kind of methods for being used to prepare the immunogenic composition, and the method includes will From streptococcus pneumonia (Streptococcus pneumoniae) serotype 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, The step of each of the capsular polysaccharide of 13 kinds of separation of 19A, 19F and 23F is coupled to carrier protein CRM197, so that described Capsular polysaccharide has the structure connected by-O-C (NH)-NH- group with carrier protein.
Immunogenic composition and preparation method thereof is same as described above.
[beneficial effect]
Polyvalent pneumococcal vaccine composition of the invention includes that capsular polysaccharide-albumen with unique coupled structures is more Valence conjugate, and in the combination and content of the optimization wherein comprising 2- phenoxetol and formaldehyde as preservative, to tie up It holds outstanding immunogenicity and there is outstanding stability and/or anti-corrosion ability.Therefore, vaccine composition of the invention and immune Immunogenic Compositions can be safer and be usefully used for preventing the disease as caused by pneumococcus in baby, children and adult Disease.
[mode of the invention]
It hereinafter, will the present invention will be described in more detail by following embodiments.However, provide these embodiments be for The purpose of explanation, and it is not intended to limit the scope of the present invention.
Preparation example 1: the preparation of capsular polysaccharide
The preparation of 1-1. cell bank
Obtaining from commission tissue US CDC (U.S.'s Disease Control and Prevention Center) has 16 kinds of respective different serotypes Streptococcus pneumonia (the Streptococcus of (1,2,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F, 12F and 15B) Pneumoniae), and by following methods cell bank is prepared.
Streptococcus pneumonia (Streptococcus pneumoniae) bacterial strain is coated on blood agar culture-medium to confirm Pneumococcus simultaneously removes existing nutrient media components.After well-grown single colonie of selection 10 or more single colonies, by it It is inoculated with fluid nutrient medium (soy peptone (Kerry Bio-Science) or the ferment in the component for not including animal origin wherein The culture medium in female extract (Bio-springer) source) in, it cultivates and is proliferated, then add the glycerol of synthesis, to prepare Research cell bank (RCB) containing the synthetic glycerine.
It is taking out the research cell bank that has been found of expression of 1 bottle of polysaccharide with intrinsic serotype and is being free of cell After being proliferated in the fluid nutrient medium of the component of animal origin, synthetic glycerine is added, thus prepares master cell bank, and taking out 1 Cell simultaneously after proliferation, is added synthetic glycerine in the fluid nutrient medium of the component without animal origin by bottle master cell bank, by This prepares the cell bank being used to prepare.
Prepared cell bank is stored under -70 DEG C or lower temperature with super freezing state, it is following to be used for Test.
1-2. fermentation and separation of polysaccharides
The cell bank that 1 bottle is used to prepare is melted and is seeded in the fluid nutrient medium of the component without animal origin.Institute It states seed culture to carry out under the conditions of not stirring at 37 ± 2 DEG C, until reaching constant cell concentration (optical density, OD 600). After whether the culture solution that the seed culture is completed in confirmation is contaminated, it is seeded in containing the component for not including animal origin The fluid nutrient medium (training in soy peptone (Kerry Bio-Science) or the source yeast extract (Bio-springer) Support base) fermentor in (OD600=2.5 ± 0.2).
Then at 37 ± 2 DEG C under minimum stirring condition, the pH of culture medium is maintained using the potassium hydroxide solution of degerming To 7.2 ± 0.2, main culture is carried out.By sampling 2 hours afterwards since culture, the concentration of cell in the culture solution is measured With the concentration for the glucose for including in the culture medium.When the glucose in the culture medium is depleted, culture is completed.
After the completion of the culture, 12% (w/v) NaTDC for preparing the degerming of suitable amount keeps its final concentration of 0.12% (w/v), then adds it to culture, thus lytic cell and releases polysaccharide with the cell combination.
The purifying of 1-3. capsular polysaccharide
Phosphoric acid is added for a long time in the sample handled with NaTDC obtained into preparation example 1-3 and is titrated to pH 3.5 ± 0.3, then by after standing and reacting it, by be collected by centrifugation supernatant (17,000xG, room Temperature, 1 hour).The supernatant of collection is passed through into depth filter (0.55-9.0um) and is concentrated, enforcement phosphate buffer of going forward side by side Buffer replacement.
After buffer replacement, by sample by active carbon filter, impurity then is carried out by following two methods Removal:
1) due to for serotype 1,2,3,4,5,6A, 6B, 9V, 12F, 15B, 18C, 19A, 19F and 23F totally 14 kinds of serum Ionic bonding for type with CTAB (cetyl trimethylammonium bromide) is available, therefore has carried out CTAB method, specifically For, with 10 weight %CTAB solution with the 1.5 weight % (other than 23F) of poidometer of Process liquor or The amount of 2.5% (in the case where 23F) is handled, so that it is reacted ± 10 minutes 1 hour.Then by with 17,000xG's Rate is centrifuged 1 hour to collect sediment.Then, in order to use sodium chloride (NaCl) and sodium iodide (NaI) processing, using 200~ 300mM sodium chloride (NaCl) solution will handle the sediment resuspension collected by CTAB, and used using corresponding to The sodium iodide (NaI) of 50 weight % of the amount of CTAB removes CTAB ion.However, using 350mM for serotype 3 NaCl solution.After with sodium chloride and sodium iodide processing, by the way that supernatant is collected by centrifugation and for subsequent process.2) for blood For both serotypes for react with CTAB of clear type 7F and 14, addition phosphaljel (Algel) solution simultaneously reacts, then The phosphaljel solution that 10 weight % are calculated as to total Process liquor is handled and is reacted 1 hour.Collect from centrifugation (17, Be centrifuged 1 hour under the conditions of 000xG) obtain supernatant and be used for subsequent process.
By the sample after the completion of two kinds of impurity minimizing technology by depth filter and ultrafiltration (UF/DF) process, so They are manufactured into powder form and are stored by the amount of control ethyl alcohol and sodium chloride afterwards.
Preparation example 2: streptococcus pneumonia (Streptococcus pneumoniae) capsular polysaccharide-protein conjugate preparation
2-1: the comparison of reductive amination method and the coupling method of cyanalation method
Use purified polysaccharide and CRM197 carrier protein (the GenBank registration number 1007216A of serotype 9V；SEQ ID NO:1；mgaddvvdssk sfvmenfssy hgtkpgyvds iqkgiqkpks gtqgnydddw kefystdnky daagysvdne nplsgkaggv vkvtypgltk vlalkvdnae tikkelglsl teplmeqvgt eefikrfgdg asrvvlslpf aegsssveyi nnweqakals veleinfetr gkrgqdamye ymaqacagnr vrrsvgssls cinldwdvir dktktkiesl kehgpiknkm sespnktvse ekakqyleef hqtalehpel selktvtgtn pvfaganyaa wavnvaqvid setadnlekt taalsilpgi gsvmgiadga vhhnteeiva qsialsslmv aqaiplvgel vdigfaaynf vesiinlfqv vhnsynrpay spghktqpfl hdgyavswnt vedsiirtgf qgesghdiki taentplpia gvllptipgk ldvnkskthi svngrkirmr craidgdvtf crpkspvyvg Ngvhanlhva fhrsssekih sneissdsig vlgyqktvdh tkvnsklslf feiks), attempt reduction amination and Cyanalation coupling method, to compare the coupling yield of two kinds of coupling methods.Specific coupling method is as described below.
2-1-1. reduction amination
11.7mg sodium metaperiodate is added to the undiluted solution of the polysaccharide of serotype 9V, in 21 to 25 DEG C of stirrings to activate The polysaccharide.After the polysaccharide of oxidation is concentrated and is percolated using 100KDa ultrafiltration filter and WFI (water for injection), by reservation Remaining polysaccharide and CRM197 albumen are mixed and are freeze-dried with sugar/CRM197=0.5 ratio (weight ratio).The freezing is dry Dry compound melts and in 21 to 25 DEG C of stabilisation (balance).By by the compound of the balance in sodium phosphate (Na3PO4) in buffer with the ratio isothermal processes (37 ± 2 DEG C) of 0.1M/20g sugar it to be cracked after, cyano hydroboration is added Object (100mg/mL), thus causes the coupling reaction between the albumen and sugar.Isothermal processes about 44 to 52 are small at 37 ± 2 DEG C Temperature is reduced to 23 ± 2 DEG C, and adds 1mL 0.9% (w/v) NaCl solution to reactor by Shi Hou.It is molten to add sodium borohydride Liquid (100mg/mL) is so that every 1 mole of sugared sodium borohydride is 1.8 to 2.2 molar equivalents, and obtained reaction mixture is existed Lower isothermal processes are stirred, any unreacted aldehyde present in the sugar is thus restored.To obtained sugar-albumen coupling mixture 5mL 0.9% (w/v) sodium chloride solution is added to dilute it, and the diluted coupling is mixed using 100kDa MWCO film Object is dialysed and is filtered.
2-1-2. cyanalation
Sodium chloride powder is added to the undiluted solution of the polysaccharide of the serotype 9V prepared without hydrolysis process process, with system Standby 2M NaCl polysaccharide solution.In order to activate the polysaccharide, CDAP (1- cyano -4- diformazan is being added to serotype 9V polysaccharide solution Base aminopyridine tetrafluoroborate) solution, so that passing through stirring 15 minutes after concentration is calculated as 0.5% (w/w) with the polysaccharide To induce polysaccharide priming reaction.The mixture addition sodium hydroxide solution to reacting of obtaining and pH is increased to 9.5 ± After 0.1, it is stirred 3 minutes, so that the hydroxyl of polysaccharide is sufficiently activated by CDAP.To the polysaccharide for passing through polysaccharide activation process CRM197 is added, so that the ratio of CRM197 and polysaccharide is 1.0% (w/w) (CRM197 weight/polysaccharide weight), thus in room temperature It is lower to carry out coupling reaction 1 hour.
By adding the 2M glycine solution of 3 molar equivalents in terms of the CDAP of 1 molar equivalent, pH is adjusted to 9.0 and is incited somebody to action It is incubated overnight, to complete the coupling reaction.The conjugate of completion is concentrated in ultrafiltration filter and by containing 0.9% (w/w) buffer solution of sodium chloride is percolated.
As a result, it was confirmed that the yield of the conjugate prepared by cyanalation method is prepared by reduction amination method 4 times or more of yield of conjugate.Therefore, the present inventor by using cyanalation method using capsular polysaccharide and CRM197 prepares conjugate.
The purifying of 2-2. coupling and conjugate
The preparation of the conjugate of the capsular polysaccharide and CRM197 of streptococcus pneumonia (Streptococcus pneumoniae) It is carried out by following process steps.
The dissolution and hydrolysis of step 1. capsular polysaccharide
The capsular polysaccharide powder for being derived from every kind of serotype is dissolved in water for injection respectively, so that ultimate density range Within the scope of being mentioned below, and pass through 0.45 μm of filter filtering:
1) in the case where serotype 1,2 and 4,0.8 to 2.0mg/ml range,
2) in the case where serotype 5,6B, 9V, 18C and 19F, 4 to 8mg/ml range,
3) range that serotype 6A, 12F and 19A is 8 to 12mg/ml, and
4) in the case where serotype 3,7F, 14,15B and 23F, 2 to 4mg/ml range.
It is carried out in the pH and temperature range that the process of the isothermal processes of the solution is mentioned according to serotype below:
1) in the case where serotype 1,2,4,5,6B, 7F, 14 and 23F, 70 to 80 DEG C are overnight,
2) in the case where serotype 6A and 19F, 70 to 80 DEG C 0.5 to 4 hour,
3) in the case where serotype 3,9V, 12F and 18C, Isothermal Treatment Process is using phosphoric acid solution in pH 2.0 and 65 It is carried out 0.5 to 4 hour to 80 DEG C,
4) in the case where serotype 15B, 19A, without hydrolysis.
Then, it is cooled to 21 DEG C to 24 DEG C and adds sodium hydroxide to target pH, thus stop hydrolysis.
The coupling reaction process of step 2. capsular polysaccharide and CRM197
Sodium chloride powder is added to all serotypes, thus prepares 2M NaCl polysaccharide solution.It will be suitable for every kind of serotype CDAP (1- cyano -4-dimethylaminopyridine tetrafluoroborate) with every 1/1 acetonitrile of 1ml/water for injection (v/v) solution The ratio of 100mg CDAP dissolves, the amount addition that the solution is hereafter mentioned according to serotype.Specifically,
It is dissolved with following ratios
1) in the case where serotype 6A, 9V and 14, CDAP is 1 to 1.5 (w/w) compared with polysaccharide,
2) in the case where serotype 2 and 4, CDAP is 2 (w/w) compared with polysaccharide,
3) in the case where serotype 1,3,7F, 15B, 19F and 19A, CDAP is 3 (w/w) compared with polysaccharide,
4) in the case where serotype 5,6B, 18C, 23F, CDAP is 4 (w/w) compared with polysaccharide, and
5) in the case where serotype 12F, CDAP is 5 (w/w) compared with polysaccharide,
And it is added to every kind of polysaccharide solution.
Then, it adds sodium hydroxide solution and pH is increased to 9.5, then stir it 3 to 7 minutes, so that polysaccharide Hydroxyl is sufficiently activated by CDAP.CRM197 is added to every kind of serotype with the amount that CRM197 is 0.75 (w/w) compared with polysaccharide Thus polysaccharide solution carries out 2 hours coupling reactions.Then, using the conversion ratio of SE-HPLC measurement reaction, and if any must Further to add CDAP.
Step 3. coupling reaction terminates
For all serotypes, addition and the CDAP of added 1 molar equivalent are comparably 3 to 6 molar equivalents Glycine solution, and pH is adjusted to 9.0, thus terminate reaction.The conjugate solution is being stirred 1 hour at 21 DEG C to 24 DEG C Afterwards, it is stored in a low temperature of 2 to 8 DEG C overnight.
Step 4. ultrafiltration
The diluted conjugate mixtures are concentrated in ultrafiltration filter, and use the buffer solution of minimum 20 times of volumes (the 5mM succinate buffer solution (pH 5.8) comprising 150mM NaCl) diafiltration.Herein, using maintenance pH 5.5 to 6.5 Range and include the buffer solution of 0.9% (w/w) sodium chloride as buffer solution.Segment is used for all serotypes Molecular weight is the ultrafiltration filter of 300kDa, and dialysis solution is destroyed.
Step 5. aseptic filtration
Remaining solution buffer solution (the 5mM succinate buffer solution (pH comprising 150mM NaCl after being percolated 5.8) it) is diluted to and 0.4g/L is calculated as with the concentration of polysaccharide content hereinafter, it is then passed through 0.22 μm of filter filtering.It is preparing (sugared content, residual DMAP) is controlled to filtered product in the process.During the preparation process to filtered residual solution It is controlled, thereby determines whether to need other concentration, diafiltration and/or dilution.
Step 6. absorption
Make ultimate density be calculated as 1mg/mL with aluminium ion to the solution of aseptic filtration addition aluminium salt (aluminum phosphate), and inhales It is attached, additional salt is added to maintain 5.5 to 6.5 pH range.Quality examination is carried out to the undiluted solution for completing absorption, by This confirmation quality suitability, and it is refrigerated at 2 to 8 DEG C until using.
The preparation of 1. multivalent pneumococcal conjugate vaccines of embodiment
Prepared from preparation example 2 be derived from streptococcus pneumonia (Streptococcus pneumoniae) serotype 1,2, 3, each of capsular polysaccharide of 4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F, 12F and 15B and CRM197 albumen Capsular polysaccharide-protein conjugate of coupling is prepared for following combinations of multivalent pneumococcal polysaccharide-protein conjugate:
(1) 13 valence conjugate: serotype 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F；
(2) 15 valence A conjugates: serotype 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F, 2 and 12F；
(3) 15 valence B conjugates: serotype 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F, 2 and 15B.
The demand of conjugate bulk solution is on the basis of the polysaccharide concentration of batch volume and the undiluted solution of conjugate It calculates.It is identical as the step 6 of preparation example 2, after the conjugate bulk solution addition aluminum phosphate to every kind of serotype, addition 0.85% (w/w) saline solution and 5mM succinate buffer solution (pH 5.8), thus prepare containing 0.85% sodium chloride, 5mM The sheet of every kind of conjugate of succinate buffer solution (pH 5.8) and 1mg/mL aluminium element (concentration of aluminium element in aluminum phosphate) Liquid solution.Finally, as preservative, add thimerosal (85 or 100ug/ml) or 2- phenoxetol (2-PE) (0,5.0 or It 10.0mg/ml) and formaldehyde (0,10,25,40,50,80,100,120 or 170ug/ml) and is slowly mixed together, as above-mentioned Serotype combination, thus prepares the multivalent pneumococcal conjugate vaccines composition of preparation.At this point, the multivalent vaccine composition The concentration of every kind of serotype capsular polysaccharide be 4.4 μ g/mL (only serotype 6B is 8.8 μ g/mL).Check pH, if it is desired, It is adjusted to pH 5.8.The undiluted solution of the final vaccine composition of prepared preparation is packed into 1 type borosilicate glass bottle.It will The vaccine composition of the bottling is stored in 2 to 8 DEG C.
Embodiment 2: the estimation of the immunogenicity of multivalent pneumococcal conjugate vaccines
Select in 0.5mL vaccine preparation in total containing 2.2 every kind of capsular polysaccharides of μ g (only serotype 6B capsular polysaccharide for 4.4 μ g), about 29.3 μ g CRM197 carrier proteins, 0.5mg aluminium element (2mg aluminum phosphate) adjuvant, about 4.25mg sodium chloride, about The polyvalent pneumococcal vaccine composition of 295 μ g succinate buffer solution, about 3mg 2- phenoxetol and about 60 μ g formaldehyde (being named as LBVE013) as in the vaccine composition prepared in embodiment 1 13 valences (serotype 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F) vaccine, thereby confirm that whether it has the ability that immune response is induced in rabbit.This Kind immunogenicity is confirmed and in the feelings of antibody function in the case where serotype IgG concentration by antigentic specificity ELISA It is confirmed under condition by conditioning cell phagocytosis measuring method (OPA).
By vaccine composition LBVE013 or as positive controlsThe 0th week, 2 weeks and 4 weeks, with Muscle of Human clinical's dosage (every kind of 2.2 μ g of polysaccharide, except that 4.4 μ g of the 6B) immunity inoculation of plan in New Zealand White Rabbit In, and serum was collected with 2 weeks time intervals after inoculation.It the use of ELISA is that the IgG measurement result that the serum collected obtains is shown Out in table 1.It is described in detail as follows.
2-1. serotype specificity IgG measurement of concetration
The capsular polysaccharide of 13 kinds in total of every kind of serotype is handled on 96 orifice plates with the amount in 5 holes μ g/, thus by it It is coated with 16 hours at room temperature.In order to minimize heterogenetic antigen-antibody response of serum, every is collected in the same amount The serum of body, to merge identical group of serum.By the serum consolidated material and 333.3 μ g/mL C-PS (cell wall polysaccharides； SSI (staten Serum Institute)) and the capsular polysaccharide (PnPs22F) of 333.3 μ g/mL serotype 22F it is anti-in room temperature Answer 30 minutes to adsorb it after, it is used for the diluted buffer solution of antibody with comprising polysorbas20 with suitable dilution rate (about 1:100~1:40000) dilution.Coated plate cleaning is cleaned into 4 times, and the blood that will be adsorbed and diluted with buffer solution Clear 50 μ l is placed in coated orifice plate, then reacts it at room temperature 1 hour.The plate hole reacted is passed through into identical method Cleaning 4 times, and goat anti-rabbit IgG antibody-HRP conjugate (1:20000) is placed in each hole, then it is being reacted at room temperature 30 minutes.Plate is cleaned 4 times by identical method, and by 100 μ l stabilized tmb substrate solution (3,3', 5,5'- tetramethyls Base benzidine；Sigma, St.Louis, MO, USA) it is placed in each hole at room temperature, then by it in room temperature reaction 15 minutes. After by placing the stopping reaction of 100 μ l 1N sulfuric acid solutions, uses 650nm as reference wavelength, measure the suction at 450nm Luminosity.In order to carry out objective estimation to immunogenicity, analyzed by identical method used as control groupThe blood sample of collection.
As a result it is disclosed in following table 1.
[table 1]
As shown in table 1, it was confirmed that multivalence (13 valence) Pnu-Imune 23 composition (LBVE013) according to serotype Immunogenic response mode is definitely different fromMode.WithIt compares, is being inoculated with this reality It applies in the rabbit of 13 valence vaccine compositions (LBVE013) of example and illustrates higher threshold level in all serotypes, especially Serotype 1,6B, 7F, 9V, 14 and 19F show ratioHigh 2~6 times of effect.
2-2. iunctional immunogenic validation test (conditioning cell swallows measuring method, OPA)
By carrying out OPA analysis to the serum obtained from rabbit, the function of the antibody by serotype induction is had evaluated.
Specifically, same amount of serum is collected from each individual, thus merges identical group of serum.By every kind of serotype Streptococcus pneumonia (Streptococcus pneumoniae) in THY culture medium, (Todd-Hewitt meat soup contains 2w% ferment Female extract) in culture, and using conditioning buffer be diluted to 200 to 300CFU/10 μ l.By the diluted serum of 20 μ l and 10 μ l Diluted streptococcus pneumonia (Streptococcus pneumoniae) mixing, and in room temperature reaction 30 minutes.Then, 50 are added The mixture (cell: complement=4:1) of the HL-60 cell and complement that have broken up of μ l, and in CO2(37 DEG C) reactions in culture medium 45 minutes.
Stop cell phagocytosis by reducing temperature, and 10 μ l reaction solutions are coated on and have dried 30 to 60 minutes agar In culture medium.Then, by it in CO2It is cultivated 12 to 18 hours for (37 DEG C) in culture medium, and colony count is counted.OPA Threshold value is represented as observing dilution rate when 50% death.
Obtained result is shown in following table 2:
[table 2]
In table 2, for example, OPA titre be represented as 2187 be even if in most dilute part compared with negative control group Also it is not up to 50% horizontal situation, and it means that the significant ground of threshold value is high.Pass through above-mentioned test result, it was confirmed that this implementation Example polyvalent pneumococcal vaccine composition withCompared to significant outstanding serum IgG threshold value.Therefore, originally The polyvalent pneumococcal vaccine composition of embodiment can be by highly usefully for preventing the disease as caused by pneumococcus.
Reference example: the process and standard of the bactericidal ability test of vaccine
The bactericidal ability test of vaccine is carried out according to EP-B standard, and the standard is United States Pharmacopeia in the present embodiment (USO) and the World Health Organization (WHO) of European Pharmacopoeia (EP) is to standard required by vaccine product.
If specifically summarizing the content of the test, by 2 kinds of Pseudomonas aerugifzosa (Pseudomonas Aeruginosa) (ATCC No.9027, PA), staphylococcus aureus (Staphylococcus aureus) (ATCC No.6538, SA), saccharomycete candida albicans (Candida albicans) (ATCC No.10231, CA) and fungus Aspergillus niger (Aspergillus niger) (ATCC No.16404, AN) totally four kinds of germs 105To 106CFU/mL(CFU；Bacterium colony forms list Position) vaccine composition is inoculated at 0 respectively.By sampling and by it at 24 hours, 7 days, 14 days and 28 days in solid It cultivates in culture medium, colony count is counted at 3 to 5 days.
For the result according to the above method, the standard of the bactericidal ability of the pharmacopeia of EP-B and each country is shown In following table 3.
[table 3]
* NR: unrecovered
* NI: do not increase
As that can see in table 3, EP is required than United States Pharmacopeia (USP) or Japanese Pharmacopoeia (JP) more strictly, and Be divided into A and B class according to preparation, and WHO level that vaccine product is required be EP-B (EP 5.1.3. antimicrobial it is anti- Rotten effect (Efficacy of antimicrobial perserbation), USP 37-51, antimicrobial efficiency assay (Antimicrobial effectiveness testing))。
In the present embodiment, it in order to develop multivalent pneumococcal polysaccharide-protein conjugate vaccines, adds commonly used in the art Vaccine preservative thimerosal and 2-PE, thus execute bactericidal ability test, and confirm and thimerosal or low is being used alone The standard of the bactericidal ability is unsatisfactory in the case where concentration 2-PE.
Further, since other companies have submitted patent application to the case where using high concentration 2-PE (7mg/mL or more), Therefore it cannot be used in our companies.
Therefore, as ongoing experiment as a result, the present inventor obtains following result, so that exploitation can be with It minimizes the content of 2-PE and improves bactericidal effect to meet the bactericidal capability standard without violating other companies Patent new composition, the preservative as multivalent pneumococcal protein conjugate vaccine.
Embodiment 3: the screening of the bactericidal ability of multivalent pneumococcal polysaccharide-protein conjugate vaccines
In 2 kinds of Pseudomonas aerugifzosas (Pseudomonas aeruginosa) (ATCC No.9027, PA), golden yellow Staphylococcus (Staphylococcus aureus) (ATCC No.6538, SA), saccharomycete candida albicans (Candida Albicans) (ATCC No.10231, CA) and fungus Aspergillus niger (Aspergillus niger) (ATCC No.16404, AN) In totally four kinds of germs, it is not easy staphylococcus aureus (the Staphylococcus killed by 2-PE well known to selection Aureus) (ATCC No.6538, SA) is as the bacterium for screening bactericidal ability.The purpose tested be by using The sample sampled at 24 hours estimates bactericidal ability, to select the combination of the 2-PE for meeting EP-B and formaldehyde.Therefore, described Test is carried out according to the test method of EP-B.
Specifically, by reference to the preparation method of the multivalent pneumococcal polysaccharide-protein conjugate vaccines of embodiment 1, It is even that the multivalent pneumococcal albumen of vaccine composition 1 to 12 is prepared by adding thimerosal or 2-PE and/or formaldehyde according to table 2 Join object vaccine composition, and by 105To 106The staphylococcus aureus of CFU/mL (CFU=Colony Forming Unit) (Staphylococcus aureus) (ATCC No.6538, SA) germ is inoculated into these preparations at 0.By at 0 and 24 Hour samples and cultivates in solid medium, counts at 3 to 5 days to colony count, thus calculates pair that bacterium colony is reduced Number (Log).As a result it shows in following table 4.
[table 4]
As that can see in table 4, from killing for the 2-PE 5.0mg/mL and combined SA of 100 μ g/mL of formaldehyde Bacterium ability passes through EP-B standard.On the basis of the result, it was confirmed that about 5.0mg/mL 2-PE and the 100 above first of μ g/mL The combination of aldehyde is suitable for the preservative of multivalent pneumococcal polysaccharide-protein conjugate vaccines.
Embodiment 4: the bactericidal ability test of multivalent pneumococcal polysaccharide-protein conjugate vaccines 1
The bactericidal test is carried out according to the test method of EP-B.By 2 kinds of Pseudomonas aerugifzosas (Pseudomonas aeruginosa) (ATCC No.9027, PA), staphylococcus aureus (Staphylococcus Aureus) (ATCC No.6538, SA), saccharomycete candida albicans (Candida albicans) (ATCC No.10231, CA) With fungus Aspergillus niger (Aspergillus niger) (ATCC No.16404, AN) totally four kinds of germs 105To 106CFU/mL (CFU；Colony Forming Unit) the multivalent pneumococcal protein conjugate vaccine of vaccine composition 13 to 15 is inoculated at 0 respectively Preparation.By at 0, sample and cultivate it in solid medium within 24 hours, 7 days and 28 days, at 3 to 5 days to clump count Mesh is counted, and the logarithm of bacterium colony reduction is thus calculated.Fungi and yeast are at 0,14 days and sampling in 28 days and in solid culture It cultivates in base, then colony count is counted at 3 to 5 days, thus calculate the logarithm of bacterium colony reduction.As a result it shows in table 5 In.
[table 5]
* NR: unrecovered
* NI: do not increase
As shown in table 5, as the multivalent pneumococcal in every kind of serotype wherein comprising 13 valences, 15 valence A and 15 valence B 2 kinds of Pseudomonas aerugifzosa (Pseudomonas are confirmed in polysaccharide-protein conjugate vaccines composition 13 to 15 Aeruginosa) (ATCC No.9027, PA), staphylococcus aureus (Staphylococcus aureus) (ATCC No.6538, SA), saccharomycete candida albicans (Candida albicans) (ATCC No.10231, CA) and fungus Aspergillus niger (Aspergillus niger) (ATCC No.16404, AN) though the bactericidal ability of totally four kinds of germs as a result, serotype How, EP-B standard is met to the bactericidal ability of 4 kinds of germs in all 13 valences, 15 valence A and 15 valence B.It is demonstrate,proved from the result It is real, it include about 5mg/mL 2-PE and about 100 μ g/mL formaldehyde in the preservative of multivalent pneumococcal polysaccharide-protein conjugate vaccines In the case where, meet the bactericidal ability of EP-B standard.
In the art, pneumoprotein conjugate vaccines usually using the 2-PE of 7mg/mL or more as preservative, But the result of the result and embodiment 3 is together illustrated in the case where adding the formaldehyde of amount of 100 μ g/mL or more, 2-PE Content down to 5mg/mL, the bactericidal ability of EP-B also may be implemented.Therefore, multi-agent combination can be provided in the present invention Object, which use the combinations of 2- phenoxetol (2-PE) and the optimization of formaldehyde (HCHO) as multivalent pneumococcal polysaccharide- The preservative of protein conjugate vaccine, and maintain strong and effective bactericidal ability.
Embodiment 5: the stability of multivalent pneumococcal polysaccharide-protein conjugate vaccines composition
It is based on embodiment 4 as a result, being prepared for the new of multivalent pneumococcal polysaccharide-protein conjugate vaccines composition 16 Composition.Specifically, the vaccine composition 16 is prepared by following methods: placing 14 kinds of polysaccharide-protein conjugate (serum Type 1,2,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F) and 1mg/mL aluminum phosphate (in terms of aluminum concentration) and stir with It is sufficiently mixed.5mM succinate and 0.85% (w/v) sodium chloride are dissolved in distilled water for injection, degerming is thus mixed The buffer of filtration.After being homogenized finally by the formaldehyde of the 2-PE of addition 6.0mg/mL concentration and 120 μ g/mL concentration, Final pH is adjusted to 5.8.The concentration of every kind of serotype is similarly 4.4 μ g/mL (only 6B is in the vaccine composition of preparation 8.8μg/mL)。
Knot as the content for testing preservative after storing the vaccine composition 16 of the preparation 6 months at 2 to 8 DEG C Fruit, it was confirmed that the content rate of recovery of 2-PE and formaldehyde is stably maintained in the range of 90 to 110%, not the damage of preservative It loses.
In short, it was confirmed that the combination of the 2-PE and formaldehyde that illustrate in an embodiment of the present invention are effectively to tie up for a long time The bactericidal active outstanding composition of multivalent pneumococcal polysaccharide-protein conjugate vaccines is held, and it can be usefully Bactericidal ability for providing wherein multivalence polysaccharide-protein conjugate vaccines is improved or maintains the bactericidal ability Time span is able to extended multi-agent composition.
From foregoing description, it should be appreciated by those skilled in the art that the present invention can be embodied in other specific forms without carrying on the back From its technical spirit or essential attribute.In this regard, embodiment described above should be treated as illustrative in all respects And not restrictive.The scope of the present invention should be by appended claims rather than foregoing description indicates, and falls into power Therefore the meaning of sharp claim and all changes within equivalency range are intended within the scope of the present invention.
<110>LG Chemical Ltd (LG CHEM, LTD.)
<120>multivalence Streptococcus pneumoniae vaccine composition
<130> OPP20180151KR
<150> KR 10-2017-0032643
<151> 2017-03-15
<160> 1
<170> KoPatentIn 3.0
<210> 1
<211> 536
<212> PRT
<213>artificial sequence
<220>
<223>CRM197 albumen
<400> 1
Met Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu
1 5 10 15
Asn Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile
20 25 30
Gln Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp
35 40 45
Asp Asp Trp Lys Glu Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala
50 55 60
Gly Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly
65 70 75 80
Val Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys
85 90 95
Val Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr
100 105 110
Glu Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe
115 120 125
Gly Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly
130 135 140
Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu
145 150 155 160
Ser Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln
165 170 175
Asp Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val
180 185 190
Arg Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp
195 200 205
Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His
210 215 220
Gly Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser
225 230 235 240
Glu Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu
245 250 255
Glu His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro
260 265 270
Val Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln
275 280 285
Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala
290 295 300
Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly
305 310 315 320
Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu
325 330 335
Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val
340 345 350
Asp Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu
355 360 365
Phe Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly
370 375 380
His Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn
385 390 395 400
Thr Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly
405 410 415
His Asp Ile Lys Ile Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly
420 425 430
Val Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val Asn Lys Ser Lys
435 440 445
Thr His Ile Ser Val Asn Gly Arg Lys Ile Arg Met Arg Cys Arg Ala
450 455 460
Ile Asp Gly Asp Val Thr Phe Cys Arg Pro Lys Ser Pro Val Tyr Val
465 470 475 480
Gly Asn Gly Val His Ala Asn Leu His Val Ala Phe His Arg Ser Ser
485 490 495
Ser Glu Lys Ile His Ser Asn Glu Ile Ser Ser Asp Ser Ile Gly Val
500 505 510
Leu Gly Tyr Gln Lys Thr Val Asp His Thr Lys Val Asn Ser Lys Leu
515 520 525
Ser Leu Phe Phe Glu Ile Lys Ser
530 535

Claims (12)

1. a kind of multivalent vaccine composition for pneumococcus, it includes:
(i) capsular polysaccharide-carrier protein couplet object,
(ii) 4mg/mL or more and the 2- phenoxetol (2-PE) less than 7mg/mL, and
(iii) formaldehyde (HCHO) of 90 μ g/mL to 200 μ g/mL,
Wherein the capsular polysaccharide includes 13 to 24 kinds from streptococcus pneumonia (Streptococcus pneumoniae) blood The capsular polysaccharide of clear type.
2. the multivalent vaccine composition for pneumococcus of claim 1, wherein the capsular polysaccharide includes selected from the following 13 to 24 kinds of capsular polysaccharides: be derived from S. pneumoniae serotypes 1,2,3,4,5,6A, 6B, 7F, 8,9V, 9N, 10A, 11A, The capsular polysaccharide of 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F.
3. the multivalent vaccine composition for pneumococcus of claim 2, wherein the capsular polysaccharide includes
From S. pneumoniae serotypes 1,3,4,5,6A, 6B, 7F, 9V, 14,13 kinds of pod membranes of 18C, 19A, 19F and 23F it is more Sugar；
14 kinds of pod membranes from S. pneumoniae serotypes 1,2,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F Polysaccharide；
From S. pneumoniae serotypes 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 15 kinds of 19F, 23F, 2 and 12F Capsular polysaccharide；
From S. pneumoniae serotypes 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 15 kinds of 19F, 23F, 2 and 15B Capsular polysaccharide；Or
From S. pneumoniae serotypes 1,3,4,5,6A, 6B, 7F, 9V, 12F, 14,15B, 18C, 19A, 19F, 22F, 23F With 17 kinds of capsular polysaccharides of 33F.
4. the multivalent vaccine composition for pneumococcus of claim 1, wherein the carrier protein is CRM197 albumen.
5. the multivalent vaccine composition for pneumococcus of claim 1, wherein the capsular polysaccharide-carrier protein couplet object With the structure for wherein being connected the capsular polysaccharide by-O-C (NH)-NH- group with carrier protein using cyanalation method.
6. the multivalent vaccine composition for pneumococcus of claim 5, wherein the cyanalation method uses CDAP (1- cyanogen Base -4-dimethylaminopyridine tetrafluoroborate) or CNBr carry out.
7. the multivalent vaccine composition for pneumococcus of claim 1, it includes:
In terms of the capsular polysaccharide from serotype 1 of 1 parts by weight,
Every kind of 0.9 to 1.1 parts by weight be derived from serotype 2,3,4,5,6A, 7F, 9V, 14,18C, 19A, 19F, 23F, 12F and The capsular polysaccharide of 15B, and
The capsular polysaccharide from serotype 6B of 1.8 to 2.2 parts by weight.
8. the multivalent vaccine composition for pneumococcus of any one of claim 1 to 7, is used for multiple dosing.
9. a kind of for preventing or treating the pharmaceutical composition of pneumococcal infection or pneumococcal infection disease, it includes rights It is required that any one of 1 to 7 multivalent vaccine composition for pneumococcus.
10. the pharmaceutical composition of claim 9, wherein the pneumococcal infection disease is pneumonia.
11. a kind of method for being used to prepare the multivalent vaccine composition for pneumococcus, which comprises
(1) capsular polysaccharide-protein conjugate of streptococcus pneumonia is prepared, and
(2) capsular polysaccharide-protein conjugate is mixed with 2- phenoxetol (2-PE) and formaldehyde (HCHO),
Wherein the capsular polysaccharide includes 13 to 24 kinds of capsular polysaccharides for being derived from S. pneumoniae serotypes, and
The amount of the 2- phenoxetol used is 4mg/mL or more and is less than 7mg/mL, and the amount of formaldehyde is 90 μ g/mL to 200 μg/mL。
12. the method for being used to prepare the multivalent vaccine composition for pneumococcus of claim 11, wherein it is more to prepare pod membrane The step of sugar-protein conjugate (1) includes executing cyanalation method so that the capsular polysaccharide and albumen are passed through-O-C (NH)- The step of NH- connection.
CN201880025013.0A 2017-03-15 2018-03-14 Multivalent streptococcus pneumoniae vaccine compositions Active CN110520154B (en)

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