CN106039301A - Preparation method of pneumococcus-type b haemophilus influenzae-diphtheria-pertussis-tetanus combined vaccine - Google Patents
Preparation method of pneumococcus-type b haemophilus influenzae-diphtheria-pertussis-tetanus combined vaccine Download PDFInfo
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Classifications
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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
- 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
- A61K39/02—Bacterial antigens
- A61K39/05—Actinobacteria, e.g. Actinomyces, Streptomyces, Nocardia, Bifidobacterium, Gardnerella, Corynebacterium; Propionibacterium
-
- 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
- A61K39/08—Clostridium, e.g. Clostridium tetani
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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
- A61K39/02—Bacterial antigens
- A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
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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
- A61K2039/70—Multivalent vaccine
Abstract
The invention relates to a pneumococcus-type b haemophilus influenzae-diphtheria-pertussis-tetanus combined vaccine. A preparation method is used for preparing a combined vaccine containing a pneumococcus-type b haemophilus influenza combined vaccine and a diphtheria-pertussis-tetanus vaccine; pneumococcus carrier protein and type b haemophilus influenzae carrier protein, type b haemophilus influenzae capsular polysaccharide and pneumococcus capsular saccharide are added; and the mass ratio of the added capsular polysaccharide to the added type b haemophilus influenzae carrier protein ranges from (5: 1) to (1: 5). With adoption of the combined vaccine, five types of antigens including the pneumococcus are immunized at the same time, and pneumococcus protein is used as a virulence factor for inducing a relatively strong immune protection effect; the capsular saccharide is used for supplementing immunization, so that the immune protection effect of the vaccine is enhanced; and meanwhile, self protein of the pneumococcus can be prevented from immune conflicts with other vaccines, such as a diphtheria vaccine and a tetanus vaccine, in vivo.
Description
Technical field
The present invention relates to production of vaccine preparation field, particularly relate to combined vaccine, specifically refer to a kind of streptococcus pneumoniae-b type
The preparation method of hemophilus influenza-DTP vaccine.
Background technology
Streptococcus pneumoniae (Streptococcus pneumoniae) is called for short streptococcus pneumoniae (Pneumococcus), is lodged in
In the cavum nasopharyngeum of normal person, it it is the main pathogenic fungi of bacillary lobar pneumonia, meningitis, otitis media, pneumonia, bronchitis.
The disease that streptococcus pneumoniae the causes public health problem that always whole world is serious, have in worldwide higher sickness rate and
Case fatality rate, especially child and the old man to less than 2 years old.The S. pneumoniae capsular saccharide vaccine listed at present and capsular glycoprotein
Matter combined vaccine, its design is all based on S. pneumoniae capsular saccharide, covers the most common serotype causing pneumococcal disease.
But S. pneumoniae capsular saccharide is thymus independent antigen (Thymus independent antigen, TI-Ag), antibody response
Depend on the linear epitope of its recurring unit composition, direct and bone-marrow-derived lymphocyte table in the case of assisting without T lymphocyte
The IgM receptor crosslinking in face, the antibody induced is mainly IgM and IgG2, lacks preferable complement activation ability, and antibody horizontal is not
The sufficiently long time can be maintained, and can not inducing immunological memory, it is impossible to generation immunoprotection in child less than 2 years old.Capsular saccharides
Complicated structure causes the immunogenicity difference of each serotype, it is impossible to the different crowd generation for different regions enough has
The immunne response of effect.Pneumococcal conjugated vaccine includes that Serotypes is many, and each type is different for the specificity structure combined, and leads
The associated methods causing each of which type is different.The carrier protein that streptococcus pneumoniae is conventional at present includes diphtheria toxoid；Tetanus
Toxin, DT-Pa, cholera toxoid, E.coli LT, escherichia coli ST, exotoxin A from Pseudomonas aeruginosa；
Outer membrane complex c (OMPC), porin, transferrin binding protein, the C5a peptidase from A group or B group B streptococcus, influenza
Influenzae Protein D；Ovalbumin；Keyhole limpet hemocyanin (KLH)；Bovine serum albumin (BSA) or the purification of tuberculin
Protein derivatives (PPD)；And the high conservative albumen etc. of part streptococcus pneumoniae self.Capsular saccharides is repaiied by streptococcus pneumoniae
Decorations and with the combination of carrier protein will ensure the special group of capsular saccharides not lose, antigenicity and immunogenicity impregnable before
Put and carry out, simultaneously in order to avoid excessively crosslinking and the requirement of conjugate aseptic filtration of sugar chain, capsular saccharides and conjugate are divided
The size of son should have certain control.Whole technical process needs add multiple chemical reagent and participates in reaction, and capsular saccharides
Low and nonvaccine serotype pneumococcal infection disease the increase of serotype coverage rate of protein conjugate vaccines makes more grinding
The person of studying carefully begins to focus on the Pnu-Imune 23 exploitation in other directions.
Pneumoprotein vaccine has become the research and development main flow of pneumovax nearly ten years, has species specificity antigen and is
The vaccine on basis is extensively paid attention to by research staff, has attracted substantial amounts of sight.But the formation and physic-chemical property due to albumen itself
Restriction, a lot of pneumoproteins cannot be used directly for human immunity, needs to spend substantial amounts of man power and material to study
And clinical verification.
The serotype that in hemophilus influenza, b type hemophilus influenza aggressivity is the strongest, is to cause children's seriously to feel
The important pathogenic bacteria of dye, it infects object and is mainly less than 5 years old child, the infant of especially less than 2 years old.Hib is by susceptible
Person's respiratory infectious, is colonizated in nasopharynx part, can behave as asymptomatic carrier, periods of months, and small part crowd then occurs Hib to attack
Property disease.It is meningitis and pneumonia that Hib infects modal disease, additionally includes osteomyelitis, septic arthritis, epiglottitis
And septicemia etc..The most important virulence factor of Hib is its capsular polysaccharide PRP, can infect at Hib and provide existence in cloning procedure
Advantage, the phagocytosis of opposing complement-mediated, suppresses serum bactericidal activity, escapes nasal mucosal immune, promote that antibacterial is in crowd
Propagation.
Hib combined vaccine common are four kinds according to PRP length, carrier protein difference, and its immunogenicity is respectively arranged with feature:
(1) combined vaccine (PRP-diphthena toxoid, PRP-D) with diphtheria toxoid albumen as carrier: with Hib polysaccharide vaccine
Immunogenicity is similar, has certain Age Characteristics, just can produce the antibody of higher level after adult's primary vaccination, and > 15 months
Though child can produce higher level antibody, but act on without long-term holding after booster shot.(2) with B group meningococcus
Membrane-associated protein is the combined vaccine (PRP-OMP) of carrier: PRP-OMP all has preferable immunogenicity to all age group crowds.
After 1st dose of inoculation, great majority can produce high-caliber antibody per capita.The most postvaccinal antibody titer is also more than PRP-D, Hb-
OC, PRP-T, but the time that the antibody of PRP-OMP maintains is short, and antibody peak level relatively Hb-OC, PRP-T is low.(3) with attenuation
The combined vaccine that diphtheria toxoid CRM197 is carrier (Hb-OC, PRP-CRM197): 2 month infants the 1st dose inoculation produce
Antibody horizontal is relatively low, but 4 and 6 monthly ages the most again inject after can induce high-caliber antibody, after 1 year, antibody still can maintain one
Fixed level.(4) combined vaccine (PRP-T) with tetanus toxoid as carrier: similar with Hb-OC, at bigger child and one-tenth
People, the 1st dose of inoculation can show preferable immunogenicity.Young infant is more weak to the 1st dose of immunoprophylaxis reaction, the 2nd dose and the 3rd dose
Again can produce the antibody of higher level after inoculation, and antibody horizontal is held time longer.At present, domestic and main
PRP-CRM197, PRP-T two kinds, PRP-T is used to be applicable to the Hib vaccination of most of age bracket crowd.
The protein carrier used due to pneumococcal polysaccharide-protein vaccine is same or similar with b type hemophilus influenza,
And use identical carrier albumen to cause the immune interference of polysaccharide the most in vivo, i.e. give polysaccharide-protein conjugate or
Polysaccharide-protein conjugate combination is made polyvalent vaccine it is possible that problem.Such as, connect according to standard infant according to reports
Plant timetable, in range of doses, carry out immunity by (dissociating) TT and pneumococal polysaccharide-TT conjugate vaccine simultaneously, and
Tested as H. influenza type b polysaccharide (PRP) vaccine of protein carrier with tetanus toxoid (TT).Work as pneumonia
When the dosage of coccus vaccine increases, reducing the immunne response of the PRP saccharide portion of Hib conjugate vaccine, this shows to be likely to
By using identical carrier albumen to cause the immune interference of polysaccharide.Therefore to avoid the generation of this immune interference, improve
Immunne response, it is simple to epiphytotics prevention and control, researches and develops and a kind of stablizes the primary study side that effective multivalence combined vaccines is this area
To.
DPT vaccine is the pin time vaccine most, most popular of the existing the Immune Programming procedure stipulation of China, bag
Including acellular whooping cough and complete two kinds of combined vaccines of cell whooping cough, tin-free self-polishing anti-fou ling paint is good due to it in recent years
Immunogenicity and safety instead of Whole cells PCR and be widely used in the prophylactic immunization of child, be included into
The routine immunization of many countries is in the works.But DPT vaccine causes it to have multiple secondary work due to the character of its vaccine itself
With, it is difficult to it is used in combination further with other vaccines.The pentavaccine that Sai Nuofei Pasteur S.A. produces once was becoming immunology
Historical new high degree, including DPT vaccine and poliovirus and b type hemophilus influenza capsular saccharides.
Pneumoprotein vaccine has become the research and development main flow of pneumovax nearly ten years, has species specificity antigen and is
The vaccine on basis is extensively paid attention to by research staff, has attracted substantial amounts of sight.But the formation and physic-chemical property due to albumen itself
Restriction, a lot of pneumoproteins cannot be used directly for human immunity, needs to spend substantial amounts of man power and material to study
And clinical verification.Protein vaccine utilizes biotechnology synthetic proteins antigen, and proteantigen is height due to what it was selected
Conservative streptococcus pneumoniae specific proteins, thus eliminate the immunity difference between different serotypes, and owing to need not it
The common protein carrier of his type, eliminating existing Pnu-Imune 23 can not be same with the vaccine of type with universal support albumen
Time the obstacle that is used in combination；Its proteantigen itself can also be puted together with S. pneumoniae capsular saccharide as carrier protein, because of
This immune effect is more preferable, it is achieved that specific immunity combines realization with extensively immunity.
Streptococcus pneumoniae is many due to its serotype, causes the big poor stability of antigenic structure of its vaccine itself, it is difficult to other
The use that coexists combined by vaccine, and existing S. pneumoniae capsular saccharide protein conjugate vaccines is all made with diphtheria or tetanus toxoid
For protein carrier, the capsular saccharides being mainly composed of serotype specificity of this vaccine, to other blood being not included in vaccine
Clear type pneumococcal infection is invalid, i.e. lacks Cross immunogenicity effect；And will be with use in child's routine immunization
Diphtheria and tetanus vaccine produce interference, destroy existing immune effect.Therefore the best energy of a autoantigenic is studied
Enough stable existences can combine the streptococcus pneumoniae-b type hemophilus influenza-hundred of onset with b type influenza vaccines, DPT vaccine white
Broken combined vaccine becomes the task of top priority in vaccine research and development field.
Summary of the invention
It is an object of the invention to the shortcoming overcoming above-mentioned prior art, it is provided that one is capable of five kinds of diseases simultaneously
The preparation method of the streptococcus pneumoniae-b type hemophilus influenza-DTP vaccine of prevention.
To achieve these goals, the system of the streptococcus pneumoniae-b type hemophilus influenza-DTP vaccine of the present invention
Preparation Method has a following composition:
The preparation method of this streptococcus pneumoniae-b type hemophilus influenza-DTP vaccine, it is mainly characterized by, and prepares
Method includes streptococcus pneumoniae-b type hemophilus influenza combined vaccine and the combined vaccine of DPT vaccine, specifically for preparation
For:
A. by streptococcus pneumoniae carrier protein standby for purification and b type hemophilus influenza carrier protein with mass ratio 1:1
Ratio add in preparation system, wherein streptococcus pneumoniae carrier protein is the high conservative expressed of streptococcus pneumoniae and has immunogen
The albumen of property, puts together with having immunogenic S. pneumoniae capsular saccharide, and b type hemophilus influenza carrier protein is that influenza is bloodthirsty
The adventitia D albumen HiD of bacillus；
B. Hib b is added, the capsular polysaccharide of addition and b type hemophilus influenza carrier protein
Mass ratio is between 5: 1 and 1: 5；
C. adding S. pneumoniae capsular saccharide, Pneumococcus serotypes includes 7 kinds～23 kinds of serotypes, wherein 6B type capsular saccharides
Addition quality be that remaining capsular saccharides adds 2 times of quality, S. pneumoniae capsular saccharide adds quality: b type hemophilus influenza pod
It is 1:(1.5～3 that film polysaccharide adds quality)；
D. streptococcus pneumoniae-b type hemophilus influenza combined vaccine is prepared according to the conventional means of this area
E. DPT vaccine is prepared according to the preparation method of the existing vaccine of whooping cough.
Preferably, the serotype of S. pneumoniae capsular saccharide includes: 1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A,
12F, 14,15B, 17F, 18C, 19F, 19A, 20,22F, 23F and/or 33F.
Preferably, carrier protein includes: streptococcus pneumoniae dissolved blood protein and modification derivant thereof, more preferably includes modified lung
Scorching coccus dissolved blood protein Δ A146Ply, streptococcus pneumoniae dissolved blood protein derivant dPly；Pneumococcal surface protein and modification thereof are spread out
Biology, more preferably includes Pneumococal surface protein A, pneumococcal surface protein C；Pneumonia ball surface adhesion albumen and
Modification derivant, more preferably includes Pneumococcal Surface adhesion protein A, Pneumococcal Surface adhesion protein C；Or streptococcus pneumoniae
Three histidine protein family fusion protein, more preferably include streptococcus pneumoniae three histidine protein PhtA, PhtB, PhtD, PhtE,
PhtDE。
As one preferred embodiment, streptococcus pneumoniae-b type hemophilus influenza-DTP vaccine includes lung
Meningococcus-b type haemophilus influenzae combined vaccine and the combined vaccine of DPT vaccine, particularly as follows:
A. streptococcus pneumoniae-b type hemophilus influenza combined vaccine be serotype include 4,6B, 9V, 14,18C, 19F, 23F
7 valency streptococcus pneumoniae-Pneumococal surface protein A (PspA) and b type hemophilus influenza-hemophilus influenza adventitia D albumen
Combined vaccine；
B. acellular pertussis vaccine be pertussis vaccine stock solution, diphtheritic vaccine be diphtheria toxoid and tetanus epidemic disease
Seedling is tetanus toxoid, and DPT vaccine is outstanding mixed liquid.
It is preferably carried out mode, streptococcus pneumoniae-b type hemophilus influenza combined vaccine and DPT vaccine as another kind
Combined vaccine, particularly as follows:
Streptococcus pneumoniae-b type hemophilus influenza combined vaccine be serotype include 4,6B, 9V, 14, the 7 of 18C, 19F, 23F
Valency streptococcus pneumoniae-Pneumococcal Surface adhesion protein A (PsaA) and b type hemophilus influenza-hemophilus influenza adventitia D albumen
Combined vaccine.
It is preferably carried out mode, streptococcus pneumoniae-b type hemophilus influenza combined vaccine and DPT vaccine as another kind
Combined vaccine, particularly as follows:
Streptococcus pneumoniae-b type hemophilus influenza combined vaccine includes 4,6B, 9V, 14, the 7 valency pneumonia balls of 18C, 19F, 23F
Combining of bacterium-modification streptococcus pneumoniae dissolved blood protein Δ A146Ply and b type hemophilus influenza-hemophilus influenza adventitia D albumen
Vaccine.
Being preferably carried out mode as another kind, the streptococcus pneumoniae belonging in the application includes that serotype is 7 valencys～23 valencys,
The combined vaccine of streptococcus pneumoniae-b type hemophilus influenza combined vaccine and DPT vaccine can also be:
Streptococcus pneumoniae-b type hemophilus influenza combined vaccine includes 1,4,5,6B, 7F, 9V, 14, the 10 of 18C, 19F, 23F
Valency streptococcus pneumoniae-PspA/PsaA/ Δ A146Ply or other above-mentioned streptococcus pneumoniae carrier proteins and b type hemophilus influenza-stream
The combined vaccine of haemophilus influenza adventitia D albumen.
Being preferably carried out mode as another kind, the streptococcus pneumoniae belonging in the application includes that serotype is 7 valencys～23 valencys,
The combined vaccine of streptococcus pneumoniae-b type hemophilus influenza combined vaccine and DPT vaccine can also be:
Streptococcus pneumoniae-b type hemophilus influenza combined vaccine includes 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A,
The 13 valency streptococcus pneumoniae-PspA/PsaA/ Δ A146Ply of 19F, 23F or other above-mentioned streptococcus pneumoniae carrier proteins and b type influenza
The combined vaccine of haemophilus-hemophilus influenza adventitia D albumen.
Being preferably carried out mode as another kind, the streptococcus pneumoniae belonging in the application includes that serotype is 7 valencys～23 valencys,
The combined vaccine of streptococcus pneumoniae-b type hemophilus influenza combined vaccine and DPT vaccine can also be:
Streptococcus pneumoniae-b type hemophilus influenza combined vaccine includes 1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A,
12F, 14,15B, 17F, 18C, 19F, 19A, 20, the 23 valency streptococcus pneumoniae-PspA/PsaA/ Δ A146Ply of 22F, 23F and 33F
Or the combined vaccine of other above-mentioned streptococcus pneumoniae carrier proteins and b type hemophilus influenza-hemophilus influenza adventitia D albumen.
Another goal of the invention of the present invention is that protection is a kind of by above-mentioned streptococcus pneumoniae-b type hemophilus influenza-whooping cough
The preparation-obtained combined vaccine of preparation method of combined vaccine.
Preferably, streptococcus pneumoniae-b type hemophilus influenza combined vaccine is freeze-dried formulation, for as streptococcus pneumoniae epidemic disease
The freeze drying protectant of the lyophilized formulations of Seedling for sucrose.
It is highly preferred that the initial concentration that sucrose is in lyophilizing stock solution is not more than 20%；The initial concentration of sucrose is not less than
60%, preferably greater than 70%.
Preferably, sucrose initial mass percentage composition in lyophilizing stock solution is no more than 20%；It is preferred that be 4～
20%；As one preferably embodiment, according to the vaccine of required preparation be not all 7～10%, 8～10%, 10～
15% or 12～15%；Most preferably 7%, 8%, 10% or 12%.
Preferably, sucrose selects technical grade analytical pure or pharmaceutical grade.
It is further preferred that streptococcus pneumoniae-b type hemophilus influenza-DTP vaccine is before use by whooping cough
Outstanding mixed liquid adds in streptococcus pneumoniae-b type hemophilus influenza combined vaccine lyophilized formulations, by the streptococcus pneumoniae-b bloodthirsty bar of type influenza
Bacterium combined vaccine redissolves, intramuscular injection after mix homogeneously.
Preferably, the serum of antibody level of serum >=0.35 μ g/mL, the b type hemophilus influenza of streptococcus pneumoniae multi-vaccine
Antibody horizontal >=0.15 μ g/mL.
It is highly preferred that acellular pertussis vaccine titer >=4.0IU；Diphtheria vaccine titer >=30IU；Tetanus vaccine is imitated
Valency >=40IU.
Compared with prior art, what the present invention initiated achieves the five kinds of antigens that can simultaneously realize including streptococcus pneumoniae
While immunity streptococcus pneumoniae-b type hemophilus influenza-DTP vaccine, the wherein albumen-pod of Pnu-Imune 23
Film sugar coalition can play induction Cross immunogenicity effect between different serotype；And albumen itself as virulence because of
Son can induce higher immanoprotection action, capsular saccharides rises in immunity supplementary function, thus enhances the immunity guarantor of vaccine
Protect effect；The most pneumococcal oneself protein can be avoided producing in vivo with other vaccines such as diphtheria vaccine, tetanus vaccine
Raw immunity conflict so that being implemented as in order to possible of this combined vaccine.This combined vaccine improves the streptococcus pneumoniae sense of inoculator
The immunological memory response of dye；The addition of pneumoprotein makes pneumococcal immunity need to rely on thymus immunity, therefore makes
Obtain pneumococcal immunity and achieve long-acting, the immunity of all standing formula of full age bracket and whole serotype；S. pneumoniae capsular saccharide
With improve the speed of vivo immunization response after pneumoprotein coupling and extend the degree of depth and the range of its immunity, the two
Although synergism between does not has the effective of independent every kind of vaccine, but decreases inoculation times, improves inoculation efficiency, subtracts
The light body burden of vaccinee.Experimental data shows, the streptococcus pneumoniae in the present invention and the pod membrane of b type hemophilus influenza
Whether this combined vaccine is successfully prepared particularly significant, in the case of being wherein initially charged S. pneumoniae capsular saccharide by sugar addition sequence
Owing to it all can occur to put together at random with two kinds of carrier proteins, therefore cannot realize the preparation purpose of b type influenzae vaccine, only
Have be initially charged b type hemophilus influenza capsular saccharides or two kinds of capsular saccharides are simultaneously introduced but b type hemophilus influenza is far in excess in
During S. pneumoniae capsular saccharide, realize while two kinds of vaccines can be realized, DPT vaccine can be coordinated the most in use,
Achieve immunity while pentavaccine.Streptococcus pneumoniae and b type Haemophilus influenzae vaccine, the combining from source of DPT vaccine
On solve a series of Lower respiratory tract infection, along with the development of pneumoprotein capsular saccharides combined vaccine, should
Combined vaccine has to be promoted and practical value widely, and the research to streptococcus pneumoniae and related vaccines has far-reaching influence.
Detailed description of the invention
In order to more clearly describe the technology contents of the present invention, carry out further below in conjunction with specific embodiment
Describe.
Owing to using streptococcus pneumoniae oneself protein as the carrier protein of S. pneumoniae capsular saccharide in the present invention, therefore solve
S. pneumoniae capsular saccharide and Hib b cannot the technical problem that coexists of temperature, therefore the following example
In the part that only relates to present invention improving be described in detail, other not mentioned steps or raw material sources compared with technology,
This does not repeats.
Embodiment 1
1. prepare pneumococcal capsular polysaccharide
A. take existing 7 valency pneumococcal common causative serotype 4,6B, 9V, 14, the pneumonia ball of 18C, 19F and 23F
Bacterium is cultivated；
Purify the most respectively capsular polysaccharide 4 that in any of the above Pneumococcal serotype, antigenicity is strong, 9V, 14,19F and 23F,
Oligosaccharide 18C and polysaccharide 6B；
C. centrifugal collection supernatant after streptococcus pneumoniae inactivation, through being concentrated by ultrafiltration, divides according to each Pneumococcus serotypes characteristic
Not Jia Ru (volume fraction is 70%) pre-cooled ethanol in right amount, centrifugal collect, obtain rough capsular saccharides；Rough capsular saccharides is dissolved in second
In acid sodium solution, then mixing with cold phenol in 1:2 ratio, centrifugal segregation albumen, phenol carries 5-6 time repeatedly, collects supernatant, with distillation
Water is dialysed, and after dialysis, liquid adds 2mol/L calcium chloride solution, adds ethanol stirring, centrifugal segregation nucleic acid, collects supernatant, add second
Alcohol stirring (final concentration 80%), centrifugal collecting precipitation, by ethanol, washing with acetone precipitation, capsular saccharides after dehydrate, must be refined,
Put-20 DEG C to save backup.
2. prepare the high conservative of streptococcus pneumoniae expression and there is immunogenic protein Δ A146Ply
The Ply gene (sequence accession number: X52474) announced according to GenBank, modified streptococcus pneumoniae dissolved blood protein Δ
The design primer sequence of A146Ply is as follows:
| Primer | Sequence (5 '-3 ') |
| F-ΔA146Ply-N | GGAATTCCATATGGCAAATAAAGCAGTAAAT |
| R-ΔA146Ply-N | CGAGCTCGTCATTTTCTACCTTATCCTCT |
| F-ΔA146Ply-C | CGGCGGCCGCATGGCAAATAAAGCAGTAAATG |
| R-ΔA146Ply-C | CCCTCGAGTTACTAGTCATTTTCTACCTTATCCTCT |
Wherein dashed part is corresponding restriction enzyme digestion sites.
After Δ A146Ply gene PCR being expanded according to normal experiment method, 1% gel electrophoresis, reclaim target DNA fragments,
Target DNA fragments is connected in pET-28a plasmid, in competent escherichia coli cell BL21, convert extracting recombinant DNA matter
Grain, carries out PAGE gel electroresis appraisal, carries out Ni-NTA tree after determining expression and expression-form after IPTG abduction delivering
Fat purification；Use 0.2% formalin solution to remove endotoxin, i.e. hemolytic activity, obtain the Δ A146Ply after removing endotoxin
Albumen.Δ A146Ply albumen is the modified protein of the 146th deletion of alanine of Ply albumen, and purity of protein reaches more than 80%,
Detection proves that the endotoxin content with immunogenicity and residual is less than 0.1EU/ μ g.
3. prepare Hib b
A. using CY culture medium to ferment Hib bacterial strain 1842 at 37 DEG C, fermentation liquor formalin-inactivated, in centrifuging and taking
After Qing, cetyl trimethylammonium bromide (CTAB) is utilized to precipitate.Then with the NaCl of 0.5～1M, complex precipitate is entered
Row dissociates, and adds ice ethanol and to final concentration 75% (v/v), sinks to final concentration 25% (v/v), precipitate nucleic acids, ethanol the most on the rocks
Shallow lake obtains crude polysaccharides.
The most above-mentioned crude polysaccharides sodium acetate solution is dissolved, and cold phenol extracts 5-6 time, is finally concentrated by ultrafiltration and removes phenol residual,
Obtain refined sugar through 75% (v/v) ethanol precipitation, put-20 DEG C and save backup.
4. preparation b type hemophilus influenza surface protein HiD
HiD albumen is cloned into escherichia coli carry out expressing and isolated and purified, specifically includes:
A. the optimization of genes of interest and the structure of recombinant expression plasmid
In GenBank, obtain HiD gene order and be optimized, after adding His label, carrying out full genome synthesis, will close
The sequence become after Sac I and Nde I double digestion, the expression vector pET-30a of directed cloning to same double digestion (+) in, convert
Competence e. coli bl21 Star (DE3), 37 DEG C of incubated overnight, picking positive monoclonal bacterium colony, extracts matter after amplification cultivation
Grain, identifies with Nde I and Sac I double digestion, and send order-checking, by the named pET-30a-HiD of recombinant expression plasmid correct for order-checking.
B. the abduction delivering of recombiant protein and purification
Recovery engineering bacteria, inoculates 2 × LB culture medium, at 37 DEG C, amplification culture under 237r/min with the ratio of 1:100, when
When thalline value is about 12, adding IPTG to final concentration of 1mmol/L, 37 DEG C of induction 4h, sampling carries out SDS-PAGE analysis centrifugal
Collect induction thalline, add the resuspended washing of normal saline 2 times, add 05mol/LNaCl5mmol/L with the ratio of 1:10 (g/mL)
The resuspended thalline of buffer of imidazoles 20mmol/LPB (pH7.4), ultrasonic disruption thalline, 8000 × g is centrifuged 40min, in collection
Clearly, be purified in nickel ion chromatographic column, by specification operation purified product and analyze by carrier pET-43.1a (+) turn
After SDS-PAGE separates, electrotransfer is extremely with by upper step purification of samples for e. coli bl21 Star (DE3) whole cell (comparison) changed
On nitrocellulose filter, close 2h with 5% defatted milk powder shaking table slight oscillatory；Addition His Mus source monoclonal antibody (1: 800 dilution), 4 DEG C
Overnight；TBST cleans 3 times, adds the sheep anti-mouse igg (1: 2000 dilution) of HRP labelling, incubated at room 1h；Washing 3 times, DAB shows
Color.
5. polysaccharide-protein is puted together, and prepares streptococcus pneumoniae-b type hemophilus influenza combined vaccine
A. Δ A146Ply and Hid standby for purification is added in preparation system with the ratio of mass ratio 1:1；
B. adding Hib b, the capsular polysaccharide of addition and Hid mass ratio are 5: 1；
C. adding 7 valency S. pneumoniae capsular saccharide, wherein the addition quality of 6B type capsular saccharides is that remaining capsular saccharides adds quality
2 times, S. pneumoniae capsular saccharide add quality: Hib b add quality be 1:1.5.
By preparation 10mL vaccinogen liquid as a example by, add capsular polysaccharide 4,9V, 14, each 40 μ g of 19F and 23F, oligosaccharide 18C2 μ g
And polysaccharide 6B 80 μ g, Hib capsular polysaccharide 60 μ g；Hid protein 12 μ g；Δ A146Ply protein 12 μ g.Pure through gel chromatography column
Carrying out this combined vaccine of efficient liquid phase chromatographic analysis after change, result shows that all serotype all has an obvious chromatographic peak, adjacent two
Separating degree R ＞ 1.5 between peak, tailing factor T are in the range of 0.95～1.05, and capsular saccharides content reaches, and " the China people are altogether
With state's pharmacopeia " minimum requirements content in 2010 editions and the present application content.
It is pointed out that owing to pneumoprotein with S. pneumoniae capsular saccharide, b hemophilus influenza with Hid is
Homology, there is the most therebetween synergism, but puting together between albumen and the capsular saccharides in the present invention is not limited only to
State amino reducing process, as long as the method that can realize this coupling result all should be included in present invention scope.
6. preparation DPT vaccine
Owing to the combined vaccine in the present invention is with Pnu-Imune 23 and b type Haemophilus influenzae vaccine, DPT vaccine
Minimizing influences each other, and can produce immunity for main improvement direction simultaneously, therefore can use prior art in the present embodiment
Preparation DPT vaccine, therefore not to repeat here for concrete grammar.But it should be noted that nothing in the DPT vaccine in the present embodiment
Cellular pertussis vaccine titer >=4.0IU, diphtheria vaccine titer >=30IU and tetanus vaccine titer >=40IU, and the least
Liquid drugs injection dosage form, wherein the titer Han acellular pertussis vaccine is not less than 4.0IU, and diphtheria vaccine titer is not less than 30IU, tetanus
Vaccine valence is not less than 40IU.
Above-mentioned streptococcus pneumoniae-b type hemophilus influenza the combined vaccine obtained respectively and DPT vaccine are continued to employ, with
The Pharmacopoeia of the People's Republic of China 2010 editions requires carry out the assessment experiment of the vaccine valence such as immunogenicity, Conversion rate, tool respectively
Body experimental result sees below.
Embodiment 2
The present embodiment differs only in embodiment 1: streptococcus pneumoniae-b type hemophilus influenza combined vaccine is lyophilizing
Dosage form, wherein sucrose is as lyophilizing skeleton, and wherein sucrose initial mass percentage composition in lyophilizing stock solution is not more than 20%, freezes
Streptococcus pneumoniae-b type hemophilus influenza combined vaccine lyophilized formulations is obtained after Gan.Therefore the present embodiment and embodiment 1 is further
Difference is, DPT vaccine can i.e. face use as the redissolution liquid of streptococcus pneumoniae-b type hemophilus influenza associating freeze dried vaccine
Front by outstanding for whooping cough mixed liquid addition streptococcus pneumoniae-b type hemophilus influenza lyophilized formulations, redissolution, after mix homogeneously, muscle is noted
Penetrate.
Step of freeze drying particularly as follows:
1. compound concentration be 70%, through the sucrose mother solution of 121 DEG C of sterilization treatment 15min；
2. streptococcus pneumoniae-b type hemophilus influenza combined vaccine isolated and purified for embodiment 1 gained is placed in sterile chamber
In, add the sucrose mother solution that step 1 is configured to, make sucrose concentration to 10%, obtain mixing and be prepared as containing the protectant disease of sucrose
Toxogen liquid semi-finished product, more respectively with the specification fill of 1.0ml/ bottle in cillin bottle to carry out follow-up lyophilizing technique；
3. step 2 gained semi-finished product being carried out pre-freeze, during pre-freeze, fast cooling is to-55 DEG C, maintains 15h-20h；
4. semi-finished product after step 4 gained pre-freeze carry out the first stage to be dried: is warming up to-45 DEG C～-40 DEG C and maintains 50h,
It is warming up to-40 DEG C～-33 DEG C and maintains 22h；
5. step 4 gained first stage dried semi-finished product are carried out second stage to be dried: heat up in setting different time
To 0 DEG C～30 DEG C, the different temperature rise periods continue to 28h；Prepare again streptococcus pneumoniae that sucrose concentration in lyophilizing stock solution is 10%-
B type hemophilus influenza combined vaccine, the preparation method of freeze dried vaccine refers to prior art and prepares, do not repeats at this.
Above-mentioned streptococcus pneumoniae-b type hemophilus influenza the combined vaccine obtained respectively and DPT vaccine are continued to employ, with
The Pharmacopoeia of the People's Republic of China 2010 editions requires carry out the assessment experiment of the vaccine valence such as immunogenicity, Conversion rate, tool respectively
Body experimental result sees below.
Embodiment 3
The present embodiment is with the difference of embodiment 1, and the polysaccharide order of addition in the present embodiment is for first adding streptococcus pneumoniae
Capsular saccharides, then add Hib b.After order exchange, Hib b is without chromatograph
Peak, it was demonstrated that S. pneumoniae capsular saccharide can be puted together with Hid for carrier protein, streptococcus pneumoniae be initially charged or two kinds of polysaccharide by
Former ratio is simultaneously introduced and can form competitive relation, affects the normal conjugation procedure of Hib b, therefore without
Method realizes the technical program.
Embodiment 4
The present embodiment is with the difference of embodiment 3, the Hib b in the present embodiment and pneumonia
S. pneumoniae capsular saccharide is simultaneously introduced, and Hib:ps mass ratio is (4～10): 1, i.e. Hib b is far in excess in
S. pneumoniae capsular saccharide, when addition is puted together in system at the same time, competitive relation is weakened by great disparity in mass.Pure through gel chromatography column
Carrying out this combined vaccine of efficient liquid phase chromatographic analysis after change, result shows that all serotype all has an obvious chromatographic peak, adjacent two
Separating degree R ＞ 1.5 between peak, tailing factor T in the range of 0.95～1.05, and capsular saccharides content reaches, and " the China people are altogether
With state's pharmacopeia " minimum requirements content in 2010 editions and the present application content.
Above-mentioned streptococcus pneumoniae-b type hemophilus influenza the combined vaccine obtained respectively and DPT vaccine are continued to employ, with
The Pharmacopoeia of the People's Republic of China 2010 editions requires carry out the assessment experiment of the vaccine valence such as stability, immunogenicity, tool respectively
Body experimental result sees below.
Embodiment 5
The present embodiment is with the difference of embodiment 1, and streptococcus pneumoniae carrier protein is different.The albumen used in the present embodiment
Carrier is Pneumococcal Surface adhesion protein A (PsaA).Remaining step is identical, is not repeated herein.
Prepare the high conservative of streptococcus pneumoniae expression and there is immunogenic protein Pneumococcal Surface adhesion protein A
(PsaA)
As follows according to PsaA gene (sequence accession number: U53509) the design primer sequence that GenBank announces:
| Primer | Sequence (5 '-3 ') |
| F-PasA | CATGCCATGGCTGCTAGCGGAAAAAAAGAT |
| R-PasA | CGCAAGCTTTTATTTTGCCAATCCTTCAG |
Wherein dashed part is corresponding restriction enzyme digestion sites.
After PsaA gene PCR being expanded according to normal experiment method, 1% gel electrophoresis, reclaims target DNA fragments, by mesh
Mark DNA fragmentation connects in pET-28a plasmid, converts extracting recombinant dna plasmid in competent escherichia coli cell BL21,
Carry out PAGE gel electroresis appraisal after IPTG abduction delivering, carry out Ni-NTA resin after determining expression and expression-form pure
Change；Obtaining PsaA albumen, test concentrations, purity of protein reaches more than 80%.
Carrying out this combined vaccine of efficient liquid phase chromatographic analysis after purification through gel chromatography column, result shows all serotype
All having obvious chromatographic peak, between adjacent two peaks, separating degree R ＞ 1.5, tailing factor T are in the range of 0.95～1.05, and pod
Film sugar content reaches the minimum requirements content in the Pharmacopoeia of the People's Republic of China 2010 editions and the present application content.
Above-mentioned streptococcus pneumoniae-b type hemophilus influenza the combined vaccine obtained respectively and DPT vaccine are continued to employ, with
The Pharmacopoeia of the People's Republic of China 2010 editions requires carry out the assessment experiment of the vaccine valence such as stability, immunogenicity, tool respectively
Body experimental result sees below.
Embodiment 6
The present embodiment differs only in embodiment 5, and streptococcus pneumoniae-b type hemophilus influenza combined vaccine is lyophilizing
Dosage form, wherein sucrose is as lyophilizing skeleton, and wherein sucrose initial mass percentage composition in lyophilizing stock solution is not more than 20%, freezes
Streptococcus pneumoniae-b type hemophilus influenza combined vaccine lyophilized formulations is obtained after Gan.Therefore the present embodiment and embodiment 1 the most no
With being, DPT vaccine can be as the redissolution liquid of streptococcus pneumoniae-b type hemophilus influenza associating freeze dried vaccine, the most before use
Outstanding for whooping cough mixed liquid is added in streptococcus pneumoniae-b type hemophilus influenza lyophilized formulations, redissolves, intramuscular injection after mix homogeneously.
Carrying out this combined vaccine of efficient liquid phase chromatographic analysis after purification through gel chromatography column, result shows all serotype
All having obvious chromatographic peak, between adjacent two peaks, separating degree R ＞ 1.5, tailing factor T are in the range of 0.95～1.05, and pod
Film sugar content reaches the minimum requirements content in the Pharmacopoeia of the People's Republic of China 2010 editions and the present application content.
Above-mentioned streptococcus pneumoniae-b type hemophilus influenza the combined vaccine obtained respectively and DPT vaccine are continued to employ, with
The Pharmacopoeia of the People's Republic of China 2010 editions requires carry out the assessment experiment of the vaccine valence such as stability, immunogenicity, tool respectively
Body experimental result sees below.
Comparative example 1
Use commercially available septivalency S. pneumoniae capsular saccharide-Protein Conjugation vaccine (trade name: abundant youngster) as a comparison case, its consumption
And the same description of using method, vaccine valence assessment experiment is carried out in the lump with the various embodiments described above obtained vaccine, specific experiment is tied
Fruit sees below.
Comparative example 2
Use commercially available b type hemophilus influenza combined vaccine (trade name: Act-Hib) as a comparison case, its consumption and use
The same description of method, carries out vaccine valence assessment experiment in the lump with the various embodiments described above obtained vaccine, and specific experiment result sees below.
Comparative example 3
Use commercially available whooping cough (trade name: Nitrogen in absorbed), its consumption and use as a comparison case
The same description of method, carries out vaccine valence assessment experiment in the lump with the various embodiments described above obtained vaccine, and specific experiment result sees below.
Vaccine valence assessment experiment
One, Western-blot analyzes pneumococcal protein antigen
Anti-△ A146Ply, anti-PsaA mice serum are carried out Western-blot analysis, recombinant expressed △ respectively
A146Ply albumen, PsaA albumen can be combined with mice serum generation immunogenicity, at △ A146Ply albumen near 53-kDa
See that destination protein band, PsaA albumen are shown in destination protein band near 37-kDa, show that the albumen expressed has the most anti-
Originality, all remains the epitope of two kinds of albumen.
Two, stability
Liquid streptococcus pneumoniae-b type hemophilus influenza combined vaccine: 4 DEG C keep in Dark Place 24 months, albumen is without degeneration, anti-
Originality is good.
Lyophilized formulations streptococcus pneumoniae-b type hemophilus influenza combined vaccine :-20 DEG C keep in Dark Place 24 months, albumen is without becoming
Property, antigenicity is good.
Three, Study On Immunogenicity
By the combined vaccine of above-mentioned preparation with people with the NIH mice of 10 times of immunity of dose dilution 14～18g, often organize 10,
Each experimental vaccine uses two groups, subcutaneous injection combined vaccine, negative control group injection 0.5ml physiological saline solution.0,14 days 2 pins
Peritoneal immunity, each 0.5ml, blood sampling in the 28th day, use ELISA method detection serum to resist the antibody of above-mentioned all capsular polysaccharides to drip
Degree, testing result is shown in Table 1, and Conversion rate the results are shown in Table 2.
Table 1
Table 2
In this description, the present invention is described with reference to its specific embodiment.But it is clear that still may be made that
Various modifications and alterations are without departing from the spirit and scope of the present invention.Therefore, description is considered as illustrative rather than limits
Property processed.
Claims (10)
1. the preparation method of streptococcus pneumoniae-b type hemophilus influenza-DTP vaccine, it is characterised in that preparation side
Method includes streptococcus pneumoniae-b type hemophilus influenza combined vaccine and the combined vaccine of DPT vaccine, pneumonia ball for preparation
The preparation method of bacterium-b type hemophilus influenza combined vaccine includes:
A. by streptococcus pneumoniae carrier protein standby for purification and b type hemophilus influenza carrier protein with the ratio of mass ratio 1:1
Example adds in preparation system；
B. Hib b is added, the capsular polysaccharide of addition and b type hemophilus influenza carrier protein quality
Ratio is between 5: 1 and 1: 5；
C. S. pneumoniae capsular saccharide is added, wherein the addition quality of 6B type capsular saccharides be that remaining capsular saccharides adds quality 2 times, lung
Scorching S. pneumoniae capsular saccharide adds quality: it is 1:(1.5～3 that Hib b adds quality)；
Wherein,
Pneumococcus serotypes includes 7 kinds～23 kinds of serotypes, and b type hemophilus influenza carrier protein is hemophilus influenza
Adventitia D albumen, streptococcus pneumoniae carrier protein is the high conservative expressed of streptococcus pneumoniae and has immunogenic albumen, and has
Immunogenic S. pneumoniae capsular saccharide is puted together.
The preparation method of streptococcus pneumoniae-b type hemophilus influenza-DTP vaccine the most according to claim 1, its
Being characterised by, the serotype of S. pneumoniae capsular saccharide includes: 1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,
15B, 17F, 18C, 19F, 19A, 20,22F, 23F and/or 33F.
The preparation method of streptococcus pneumoniae-b type hemophilus influenza-DTP vaccine the most according to claim 1, its
Being characterised by, carrier protein includes: streptococcus pneumoniae dissolved blood protein and modification derivant, pneumococcal surface protein and modification thereof
Derivant, pneumonia ball surface adhesion albumen and modification derivant thereof or streptococcus pneumoniae three histidine protein family fusion protein.
The preparation method of streptococcus pneumoniae-b type hemophilus influenza-DTP vaccine the most according to claim 3, its
Being characterised by, pneumoprotein is particularly as follows: modified streptococcus pneumoniae dissolved blood protein Δ A146Ply, streptococcus pneumoniae dissolved blood protein spread out
Biological dPly, Pneumococal surface protein A, Pneumococcal Surface adhesion protein A or streptococcus pneumoniae three histidine protein PhtA,
PhtB、PhtD、PhtE、PhtDE。
5. according to the system of the streptococcus pneumoniae-b type hemophilus influenza-DTP vaccine described in any one of claims 1 to 3
Preparation Method, it is characterised in that streptococcus pneumoniae-b type hemophilus influenza-DTP vaccine includes streptococcus pneumoniae-b type influenza
Haemophilus combined vaccine and the combined vaccine of DPT vaccine, particularly as follows:
A. streptococcus pneumoniae-b type hemophilus influenza combined vaccine is 7 valency streptococcus pneumoniae-Pneumococal surface protein A and b type stream
The combined vaccine of haemophilus influenza-hemophilus influenza adventitia D albumen；
B. acellular pertussis vaccine is that pertussis vaccine stock solution, diphtheritic vaccine are diphtheria toxoid and tetanus vaccine is
Tetanus toxoid, and DPT vaccine is for hanging mixed liquid.
The preparation method of streptococcus pneumoniae-b type hemophilus influenza-DTP vaccine the most according to claim 5, its
It is characterised by, streptococcus pneumoniae-b type hemophilus influenza combined vaccine and the combined vaccine of DPT vaccine, particularly as follows:
Streptococcus pneumoniae-b type hemophilus influenza combined vaccine is 7 valency streptococcus pneumoniae-Pneumococcal Surface adhesion protein A and b type
The combined vaccine of hemophilus influenza-hemophilus influenza adventitia D albumen.
The preparation method of streptococcus pneumoniae-b type hemophilus influenza-DTP vaccine the most according to claim 5, its
It is characterised by, streptococcus pneumoniae-b type hemophilus influenza combined vaccine and the combined vaccine of DPT vaccine, particularly as follows:
Streptococcus pneumoniae-b type hemophilus influenza combined vaccine is 7 valency streptococcus pneumoniae-modification streptococcus pneumoniae dissolved blood protein Δ
The combined vaccine of A146Ply Yu b type hemophilus influenza-hemophilus influenza adventitia D albumen.
8. the preparation side of streptococcus pneumoniae-b type hemophilus influenza-DTP vaccine according to claim 1
The preparation-obtained combined vaccine of method, it is characterised in that: combined vaccine is prepared by claim 1～7 any one preparation method.
Streptococcus pneumoniae-b type hemophilus influenza-DTP vaccine the most according to claim 8, it is characterised in that:
Streptococcus pneumoniae-b type hemophilus influenza combined vaccine is freeze-dried formulation, for freezing as the lyophilized formulations of Pnu-Imune 23
Dry protectant for sucrose.
Streptococcus pneumoniae-b type hemophilus influenza-DTP vaccine the most according to claim 9, it is characterised in that:
Sucrose initial concentration in lyophilizing stock solution is not more than 20%.
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| CN101112619A (en) * | 2001-04-03 | 2008-01-30 | 葛兰素史密丝克莱恩生物有限公司 | Vaccine composition |
| CN102813917A (en) * | 2012-08-17 | 2012-12-12 | 武汉生物制品研究所有限责任公司 | Diphtheria, tetanus and acellular pertussis/Haemophilus influenzae type b - group A and C meningococcus combined vaccine |
| CN103533954A (en) * | 2011-03-02 | 2014-01-22 | 诺华股份有限公司 | Combination vaccines with lower doses of antigen and/or adjuvant |
| CN104383531A (en) * | 2014-11-18 | 2015-03-04 | 北京智飞绿竹生物制药有限公司 | Combined vaccine for preventing meningitis in children and preparation method of combined vaccine |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101112619A (en) * | 2001-04-03 | 2008-01-30 | 葛兰素史密丝克莱恩生物有限公司 | Vaccine composition |
| CN103533954A (en) * | 2011-03-02 | 2014-01-22 | 诺华股份有限公司 | Combination vaccines with lower doses of antigen and/or adjuvant |
| CN102813917A (en) * | 2012-08-17 | 2012-12-12 | 武汉生物制品研究所有限责任公司 | Diphtheria, tetanus and acellular pertussis/Haemophilus influenzae type b - group A and C meningococcus combined vaccine |
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