CN106075420A - Multivalent pneumococcal Type B hemophilus influenza combined vaccine - Google Patents
Multivalent pneumococcal Type B hemophilus influenza combined vaccine Download PDFInfo
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- CN106075420A CN106075420A CN201610511433.2A CN201610511433A CN106075420A CN 106075420 A CN106075420 A CN 106075420A CN 201610511433 A CN201610511433 A CN 201610511433A CN 106075420 A CN106075420 A CN 106075420A
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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
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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
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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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
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
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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
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6068—Other bacterial proteins, e.g. OMP
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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 provides a kind of multivalent pneumococcal Type B hemophilus influenza combined vaccine, wherein every dosage μ g/ml Han S. pneumoniae capsular saccharide 50～500, Type B hemophilus influenza capsular saccharides 10～60 μ g/ml；Pneumococcal serotype is one or more in following serotype: 1,3,4,5,6A, 6B, 7F, 8,9N, 9V, 12F, 14,15B, 18C, 19A, 19F, 22F or 23F.Compared with prior art, the combined vaccine that the present invention provides is safe and reliable, and good stability, its application prospect is the most wide.
Description
Technical field
The present invention relates to vaccine, specifically, relate to a kind of combined vaccine, especially a kind of multivalent pneumococcal-Type B stream
Haemophilus influenza combined vaccine.
Background technology
One, bacterial pneumonia
Bacterial pneumonia (BP) is modal pneumonia, is also one of modal infectious disease.Cause the cause of disease of BP
Strain class is more, common are streptococcus pneumoniae, staphylococcus aureus, klebsiella pneumoniae, hemophilus influenza, Aerugo
Pseudomonass etc. are very big to the health threat of child and old people.The diagnosis of this kind of disease depends on medical history and iconography
Checking, Isolation and culture of agent contributes to knowing selection antibiotic but not prompt enough.
In general, BP patient generally occur within cough, expectoration, generate heat, shiver with cold, the symptom such as chest pain.Pneumonia major part exists
Imaging examination (X-ray film, CT etc.) can find that the characteristic such as the infiltration of patch shape changes, bacillus pyocyaneus, Staphylococcus aureus
Bacterium and horse flood coccus (often showing as the unilateral lobe of the lung to get involved) often can cause tuberculoid cavity sample to change.It is noted that stream
When haemophilus influenza infects, fluoroscopic examination often finds the performance (infiltration of double lung diffusivities) of similar PCP, needs to be checked by other
Differentiated.There is no specific serological index at present to diagnose dependency BP, be the most still to rely on clinical medical history, look into
The methods such as body, image and fiber bronchial lavage biopsy.Wherein fiber bronchial lavage can directly obtain deep sputum and carries out
Antibacterial culturing and drug sensitive test, thus instruct clinical application, but due to incubation time long (patient often has started to experiential therapy),
Its application is the most extensive.Other are as higher in molecular Biological Detection technology for detection pathogenic infection sensitivity, and this kind of inspection flower
Take higher, be difficult to promote in economic backward area.Therefore prevent before patient is infected, a kind of good reply of can yet be regarded as is arranged
Execute.
Two, the limitation of prior art
Vaccine is unique weapon that the mankind can utterly destroy a certain disease at present, vaccination be considered as most effective,
Most economical disease prevention means.Vaccine refer to all kinds of pathogenic microorganisms make for premunitive biological product.Its
The middle vaccine made of antibacterial or spirillum is also known as vaccine.Vaccine is divided into live vaccine and killed vaccine two kinds.Conventional live vaccine
There are bacillus calmette-guerin vaccine, poliomyelitis vaccine, Measles Vaccine, plague vaccine etc..Conventional killed vaccine has pertussis vaccine, typhoid fever bacterium
Seedling, Meningococcal Vaccine, cholera vaccine etc..Vaccination is to prevent and control infectious disease public health intervention most economical, effective to arrange
Execute, be also to reduce the effective means that member's disease occurs, reduces medical expense for family.According to estimates, immunity inoculation is every
Year is avoided that the death that 200 ten thousand to 300 ten thousand examples cause because of diphtheria, tetanus, pertussis and measles.Along with the kind of vaccine is continuous
Increasing, for reducing inoculation times and palliating the agonizing sufferings, the mankind have developed again combined vaccine.Combined vaccine refers to by two kinds or two
The single bacterin preparation that antigen independent more than kind is made after being mixed by physical method, including multiple vaccines and polyvalent vaccine two
Kind, this is by multiple infectious disease or of the same race but the immunization strategy of different serotypes infectious disease, the most multi-joint epidemic disease of inoculating against for 1 time
Seedling can prevent different diseases, and polyvalent vaccine can prevent the same disease of different subtype or serotype.The preparation of combined vaccine
Not being being simply mixed of various different antigenic component, occurred between various compositions in combined vaccine is the most mutual
Effect can cause all kinds of antigen immunogenicity to change, and therefore needs to solve problems with in preparation process: whether (1) can
Affect the immunogenic response effect of various antigen；(2) whether there is not incompatibility between synantigen or interfere；(3) different
Whether antagonistic effect can occur between antigenic component；(4) other compositions of vaccine (such as adjuvant etc.) and the mixed proportion of various antigens
The most appropriate.By multiple antigen combined in same vaccinating agent, need in clinical trial, confirm various constituent vaccine
Safety and immunogenicity will not substantially reduce, and in some cases it was necessary to maintain its effect.
Onlooker's interference effect refers to the immunity simultaneously of multiple polysaccharide conjugate vaccine, can affect unconjugated antigen or combine it
The immunization of the combined vaccine of his carrier protein.Possible mechanism includes that (such as antigen obtains to limited resources in lymph node
Approach, chemotactic factor, activation signals, follicular dendritic cell and folliculus t helper cell etc.) competition, Th1/Th2/Th0 balances
Change and/or the induction of T cell regulatory mechanism.Suffered by different vaccines, the impact of onlooker's interference effect is different, second
The immunoreation of hepatovirus and Hib is most sensitive to this effect.Most probable hypothesis is exactly that the requirement to T cell balance is tightened up.
Since Britain introduced Hib combined vaccine from 1992, almost eliminate the disease caused by Hib infection.But from 1999,
Hib combined vaccine more than 1 years old in child the case of immuning failure increase year by year.Between 2000 to 2002 years, Britain with
DTaP-Hib replaces DTwPHib, increases MenC-CRM simultaneously197, this interference effect is more and more significant.But in immune programme for children
Increase IPV, it is possible to strengthen the immunogenicity of Hib and HBV.Clinical study results shows, DTaPIPV-HBV/Hib-TT immunity connects
After Zhong, the serological conversion rate of Hib and HBV is lower than DTaP-IPV/Hib-TT+HBV.DTaP-HBV-IPV/Hib-TT and PCV7-
CRM197Inoculating, the HBV of inoculation more independent than DTaP-HBV-IPV/Hib-TT and the serological conversion rate of Hib-TT and GMC all omit simultaneously
There is reduction, this is because receive the CRM of immunity simultaneously197The impact of conjugate.To 13 valency pneumococcal Polysaccharide Conjugate Vaccines
Clinical study results shows, immunity, PCV together with DTaP-IPV/Hib (2,3 and 4 months)+MenC (2 and 4 months)13-CRM197
The Hib serological conversion rate of group compares PCV7-CRM197Organize low.The clinic of 10 valency pneumococcal Polysaccharide Conjugate Vaccines (PHiD-CV) is ground
Study carefully result to show, DTaP-HBV-IPV/Hib-TT+PCV7-CRM197In the baby of inoculation, serological conversion rate and the GMC of HBV are general
All over higher than DTaP-HBV-IPV/Hib-TT+PHiD-CV.
Traditional vaccine majority uses oil emulsion formulation, generally needed to be added adjuvant to play its immune effect.But these epidemic diseases
The time that Seedling active concentration in vivo maintains is comparatively short, it is impossible to enough realize the purpose of controllable sustained-release, it is generally required to duplicate injection.Existing
It is that one utilizes modern molecular biology technique for vaccine, uses antigen to produce specific immune response with in advance by induction body
Prevent and treat disease or reach certain specific goals of medicine biological preparation.Modern vaccination is more safer than traditional vaccine, but its
Lack immunogenicity.Therefore, it is badly in need of developing immunological adjuvant effectively and safely and vaccine mucosal delivery system, and is applied to
In a new generation's vaccine.Utilize inactivation or attenuated pathogens, cellular component (such as capsular polysaccharide) or nonactive bacteriotoxin composition
Traditional vaccine generally can induce body generation neutralizing antibody to prevent to infect, and immunological adjuvant plays important in the process
Effect.Adjuvant refers to enhancing body antigen immune response or changes the material of type of immune response.It is mainly adjusted by immunity
Joint (modification of cytokine network), antigen presentation (maintenance of antigen conformation), cytotoxic T lymphocyte (CTL) are induced, are resisted
The modes such as former targeting and storage induce body and produce immunoreation long-term, efficient, improve body protective ability, reduce again simultaneously
The consumption of immune material, reduces production of vaccine cost.Traditional vaccine mostly is pathogen or its lysate, and immunogenicity is strong.But with
The development of modern biotechnology, the new generation vaccine antigen such as genetic engineering subunit vaccine, synthetic peptide vaccine, DNA vaccination pure
Spending more and more higher, immunogenicity is the most weak, needs to use adjuvant to improve the protective effect to antigen.But be approved at present
The most few for the vaccine adjuvant of human trial, classical Alum adjuvant is exactly one of them.Alum adjuvant is improving antibody water
Gentle secure context has obtained long-term practice and has confirmed, but its with many polypeptide vaccine antigen co-immunizations recombinantly or synthetically time
Fail to excite effective immunne response, make it difficult to meet the needs of new generation vaccine development.Therefore, to being applicable to clinical practice
The research and development of new adjuvant are imperative.
Existing adjuvant mainly has a two types, i.e. vaccine delivery system (immunity irritate complex, liposome, can be biological
Polyesters microsphere/the nanoparticle of degraded) and immunity irritate adjuvant (lipopolysaccharide, list phosphinylidyne plastid, muramyl peptide).At present, antigen
Most study in vehicle delivery system, most successful be biodegradable polyesters nanoparticle.Sustained-release micro-spheres technique for packing is opened
Start from the 1950's, be initially applied to the research of drug delivery system.Until 1979, Morris etc. is first by this
Technology is applied at immunological investigation, finds when preparing microspheres vaccine immune mouse, and this system can stimulate with sustained release antigen
Antibody produces, and without induction immunologic tolerance, microsphere adjuvant the most just starts to cause people to pay close attention to greatly.Nanometer due to preparation
Grain and pathogen sizableness, can pass interstice, can pass through the blood capillary that body is minimum, and nanoparticle has biofacies
Capacitive, biodegradable, hypotoxicity, targeting, medicine location transmission and can make medicine or the long-acting release of vaccine antigen and
The features such as expression, this not only can cause the systemic immunity of whole body to react, it is also possible to produces strong local mucosa-immune reaction, because of
This, Biodegradable nanometer grain has been widely used in medicine or vaccine delivery system and gene therapy research.Microsphere adjuvant
As a stable isolation carrier of antigen, play the effect protecting antigen protein, make antigen in vivo from temperature, acid
The destruction of the factors such as alkali, osmotic strength, Digestive system, and microsphere antigen is also avoided that the neutralization of maternal antibody, it is achieved anti-
Former control discharges, and generation is equivalent to conventional vaccine and repeatedly inoculates the immune effect excited, thus extends the vaccine immunity phase, reduces
Immune time.At present, that commonly uses can mainly have chitosan, hyaluronic acid and alginic acid as the macromolecular material of nano-carrier
Sodium etc..Wherein, chitosan wide material sources, cheap, internal not put aside, nonantigenic immunity, biocompatibility is excellent waits spy
Point so that it is become a kind of preferably vaccine adjuvant carrier.The most existing much with chitosan absorption parcel albumen, enzyme and plasmid
The report of DNA etc., many antitumor drug and anti-inflammatory drug have been made into chitosan microball/nanoparticle the most.Research finds, shell gathers
Sugar/plasmid DNA enwrapped granule can cause the local expression of fluorescein marker gene by mouse subcutaneous injection, and produces up to two
The higher antibody titer of more than ten day；It is reported, chitosan nano can make medication the simplest as the carrier of vaccine
Just, it is easier to be used widely；Chew etc. utilize complex coacervation to prepare chitosan DNA nanoparticles oral vaccine；Research shows,
Chitosan nano can promote the interaction between chitosan and DNA, contributes to nanoparticle and carries out the mucosa of gene vaccine and exempt from
Epidemic disease；Yan Chengyun etc. use emulsion-solvent evaporation method to be prepared for N-succinyl-chitosan nanoparticle.
But owing to immunological adjuvant refers to or apply in advance with antigen simultaneously, can non-specifically change or enhancing body antagonism
Former specific immune response, to strengthen the immunogenicity of corresponding antigens or to change immunoreation type, and no antigen own
Material.Research finds, if the different suitably vaccine fit applications of immunological adjuvant, they can not play a role.Therefore,
Select suitable vaccine, including antigen component, immunoreation type, immunisation route etc., negative effect can be avoided, and can carry
The stability of high vaccine and immune effect.
Summary of the invention
The problems referred to above existed for prior art, it is an object of the invention to provide a kind of immunogenicity than prior art more
Good combined vaccine, makes patient once can inoculate streptococcus pneumoniae and haemophilus influenzae type B vaccine, to reach once to inoculate, many
The effect of secondary immunity.
For achieving the above object, the technical solution used in the present invention is as follows:
Multivalent pneumococcal-Type B hemophilus influenza combined vaccine, it is bloodthirsty that it contains S. pneumoniae capsular saccharide, Type B influenza
Bacillus capsular saccharides and carrier protein, carrier protein is used for and capsular saccharides covalent bond.
Preferably, the mass ratio of S. pneumoniae capsular saccharide, Type B hemophilus influenza capsular saccharides and carrier protein be (5～
25): (1～5): 1.
Preferably, S. pneumoniae capsular saccharide content is 50～500 μ g/ml.
It is highly preferred that S. pneumoniae capsular saccharide content is 100～350 μ g/ml.
It is highly preferred that S. pneumoniae capsular saccharide content is 200 μ g/ml.
Preferably, Type B hemophilus influenza capsular saccharides content is 10～60 μ g/ml.
It is highly preferred that Type B hemophilus influenza capsular saccharides content is 15～40 μ g/ml.
It is highly preferred that Type B hemophilus influenza capsular saccharides content is 25 μ g/ml.
Preferably, one or more during pneumococcal serotype is following serotype: 1,3,4,5,6A, 6B, 7F, 8,
9N, 9V, 12F, 14,15B, 18C, 19A, 19F, 22F or 23F.
Preferably, in above-mentioned combined vaccine carrier protein covalently bound with capsular saccharides selected from tetanus toxoid (TT),
Diphtheria toxoid (DT), diphtheria toxin mutation cross reacting material 197 (CRM197), Pneumococal surface protein A (rPspA),
B group meningitis cocci outer membrane protein (MenB-OMP), hemophilus influenza D albumen (HiD), Pseudomonas aeruginosa Exotoxin A (PEA),
Heat-labile Enterotoxin of E. coli, pneumolysin, pertussis toxin, PT, bacillus pyocyaneus pili, gonococcus P-3-2 pili or
Hepatitis B virus surface antigen.
Interference effect may be caused in body owing to reusing carrier protein or produce competitive inhibitory effect, will be straight
Connecing the immune effect affecting vaccine to inoculation crowd, inventor is carrying out great many of experiments to existing carrier protein, finds to work as
When using specific carrier protein, the generation of above-mentioned Competitive assays situation will be prevented effectively from.
As a kind of preferred embodiment, the carrier of above-mentioned vaccine is Pneumococal surface protein A (rPspA).
As it is another object of the present invention to the preparation method that is to provide above-mentioned combined vaccine, comprise the steps:
Step a1) preparation also separating-purifying carrier protein；
Step b1) in step a1 resulting vehicle albumen, add refined Type B hemophilus influenza capsular saccharides；
Step c1) in step b1 gained mixture, add refined S. pneumoniae capsular saccharide, wherein the adding of 6B type capsular saccharides
Entering quality is 2 times of remaining capsular saccharides addition quality；
Step d1) purification procedures c1 gained capsular saccharides-carrier protein, obtain multivalent pneumococcal-bloodthirsty bar of Type B influenza
Bacterium combined vaccine stock solution.
Another object of the present invention, is to provide a kind of multivalent pneumococcal-Type B hemophilus influenza combined vaccine, and it contains
Having S. pneumoniae capsular saccharide, Type B hemophilus influenza capsular saccharides, carrier protein and adjuvant, carrier protein is for capsular saccharides altogether
Valency combines.
Preferably, the adjuvant of above-mentioned vaccine is selected from chitosan and/or liposome.
It is highly preferred that the deacetylation of chitosan is 50%～60%.
It is highly preferred that liposomal particle size scope is 50nm～1000nm.
It is highly preferred that the mass ratio of chitosan or liposome and capsular saccharides is between 5:1 and 1:5.
Another object of the present invention is to provide the method preparing above-mentioned vaccine, comprise the steps:
Step a2) refine Type B hemophilus influenza capsular saccharides and S. pneumoniae capsular saccharide respectively；
Step b2) preparation also separating-purifying carrier protein；
Step c2) successively polysaccharide-chitosan couplet is combined with step b2 resulting vehicle albumen coupling；
Step d2) purification procedures c2 gained couplet, obtain multivalent pneumococcal-Type B hemophilus influenza associating epidemic disease
Seedling stock solution.
Another object of the present invention is to provide the freeze drying protectant of above-mentioned combined vaccine, described protective agent is single component
Sucrose, and as the lyophilizing skeleton of vaccine, in other words, this protective agent does not contains gelatin and/or alginate.
It is highly preferred that the initial concentration of sucrose is not less than 60%, preferably greater than 70%.
It is highly preferred that the initial mass percentage composition that sucrose is 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%.
It is highly preferred that sucrose selects technical grade analytical pure or pharmaceutical grade.
As another object of the present invention, it is to provide the preparation method of vaccine freeze-drying protective agent, comprises the steps:
Step a3) under gnotobasis, the sucrose mother solution that preparation high temperature sterilize processes, vaccinogen liquid preparation completed adds
In sucrose mother solution, mixing is prepared as containing sucrose protectant vaccinogen liquid semi-finished product；
Step b3) step a3 gained semi-finished product are carried out pre-freeze；
Step c3) semi-finished product after step b3 gained pre-freeze are dried, obtain freeze dried vaccine.
It is highly preferred that during pre-freeze, fast cooling, to-55 DEG C～-50 DEG C, maintains 4h～8h or 15h～20h in step b3.
It is dried it is highly preferred that step c3 includes that the first stage is dried with second stage.
Further, in step c3, the first stage is warming up to-45 DEG C～-33 DEG C and maintains 40h～96h and/or intensification when being dried
12h～22h is maintained to-40 DEG C～-30 DEG C.
Accordingly, as a kind of better embodiment, according to the difference of the vaccine of required preparation, the first rank in step c23
Section is warming up to-45 DEG C～-40 DEG C when being dried, and maintains 50h, then is warming up to-40 DEG C～-33 DEG C, maintains 22h；Or it is warming up to-42 DEG C
～-33 DEG C, maintain 96h；Or it is warming up to-42 DEG C～-38 DEG C, maintain 42h, then be warming up to-35 DEG C～-33 DEG C, maintain 16h；Or
It is warming up to-40 DEG C～-38 DEG C, maintains 44h, then be warming up to-35 DEG C～-30 DEG C, maintain 14h；Also or be warming up to-40 DEG C～-38
DEG C, maintain 40h, then be warming up to-35 DEG C～-30 DEG C, maintain 12h.
It is highly preferred that second stage is warming up to 0 DEG C～30 DEG C in setting different time when being dried in step c3, different intensifications
Stage continue to 18h～32h (three phasic properties according to water: pressure, boiling point or fusing point, temperature be one interact process,
Under different heating rates, heat radiation speed is different, and last obtained agent product outward appearance and moisture also differ；Tie simultaneously
Conjunction sucrose can produce different glass transition temperatures with different virus；Optimal value is realized by regulation interrogation speed；Therefore
Can be adjusted according to method of the prior art herein, lower same).
Compared with prior art, present invention have the advantage that
1. this combined vaccine is the immunoconjugates containing Type B hemophilus influenza polysaccharide Yu multivalent pneumococcal polysaccharide, its
Immunogenicity is suitable with existing haemophilus influenzae type B vaccine or Pnu-Imune 23, and the polysaccharide antibody level of induction is single
The horizontal sum of only vaccine antibody, can cause immunne response in broader crowd；
2., owing to using a kind of carrier protein, it is effectively prevented from injection process the side reaction occurred；
3. liposome can effectively protect antigen to go directly target cell, and vaccinogen liquid is after by liposomal, modified
-CHO in polysaccharide-chitosan couplet can prevent liposome from aoxidizing in vivo, effectively reduces vaccine slow release rate.
4. the development of the freeze-dried formulation of the combined vaccine that couple present invention provides not only contributes to increase the vaccine holding time, also
It is easy to vaccine transport, is more applicable to the lyophilizing of other combined vaccine；
5. the technical scheme that the present invention provides is avoided in freeze-dried formulation vaccine containing animal derived gelatin macromolecule, food-borne sea
Alginate component protective agent immunity inoculation cause anaphylaxis, it is provided that freeze dried vaccine ease of solubility good, with low cost, preparation hold
Easily, preserving conveniently, after lyophilizing, the stability of vaccine, solubility are significantly better than that prior art like product；
6. with sucrose for the unique protective agent of vaccine freeze-drying preparation and forming agent skeleton, available technical grade analytical pure or medicinal
Grade cane sugar residual impurity, composition is controlled, gets rid of anaphylactogen completely；
7. vaccine preparation method is simple, is suitable for the needs that large-scale industrial produces.
To sum up, multivalent pneumococcal-Type B hemophilus influenza combined vaccine that the present invention provides is safe and reliable, stability
Good, its application prospect is the most wide.
Detailed description of the invention
The present invention is made the most in detail below in conjunction with embodiment and comparative example, intactly illustrates.Embodiment occurs
Reagent or instrument but specified otherwise, are commercially available in a specific embodiment, and operate according to its description, at this not
Repeat.
Embodiment 1-1
One, Type B hemophilus influenza capsular saccharides is prepared
1. use CY culture medium that Hib bacterial strain 37 DEG C is fermented, fermentation liquor formalin-inactivated, after centrifuging and taking supernatant, profit
Precipitate with cetyl trimethylammonium bromide (CTAB).Then with the NaCl of 0.5～1M, complex precipitate is dissociated,
Add ice ethanol and obtain thick to final concentration 25% (v/v), precipitate nucleic acids, ethanol the most on the rocks to final concentration 75% (v/v), precipitation
Sugar.
The most above-mentioned raw sugar sodium acetate solution is dissolved, and cold phenol extracts 5-6 time, is finally concentrated by ultrafiltration and removes phenol residual, warp
75% (v/v) ethanol precipitation obtains refined sugar, puts-20 DEG C and saves backup.
Two, preparation pneumococal polysaccharide
1. choose 7 kinds of serotypes (4,6B, 9V, 14,18C, 19F, 23F) streptococcus pneumoniae cultivate；
Purify the capsular saccharides that in any of the above Pneumococcal serotype, antigenicity is strong the most respectively: centrifugal after streptococcus pneumoniae inactivation
Collect supernatant, through being concentrated by ultrafiltration, be separately added in right amount (volume fraction is 70%) according to each Pneumococcus serotypes characteristic
Pre-cooled ethanol, centrifugal collection, obtain raw sugar；Rough polysaccharide is dissolved in sodium acetate solution, then mixes with cold phenol in 1:2 ratio,
Centrifugal segregation albumen, phenol carries 5-6 time repeatedly, collects supernatant, dialyses with distilled water, and after dialysis, to add 2mol/L calcium chloride molten for liquid
Liquid, adds ethanol stirring, centrifugal segregation nucleic acid, collects supernatant, add ethanol (final concentration 80% stirs), centrifugal collecting precipitation,
By ethanol, washing with acetone precipitation, capsular saccharides must be refined after dehydrate, put-20 DEG C and save backup.
Three, Pneumococal surface protein A (rPspA) is prepared
PspA albumen is cloned into escherichia coli carry out expressing and isolated and purified, specifically includes:
1. the optimization of genes of interest and the structure of recombinant expression plasmid
In GenBank, obtain PspA gene order and be optimized, after adding His label, carrying out full genome synthesis, will
The sequence of synthesis after Sac I and Nde I double digestion, the expression vector pET-30a of directed cloning to same double digestion (+) in, turn
Change competence e. coli bl21 Star (DE3), 37 DEG C of incubated overnight, picking positive monoclonal bacterium colony, extract after amplification cultivation
Plasmid, identifies with Nde I and Sac I double digestion, and send order-checking, by the named pET-30a-of recombinant expression plasmid correct for order-checking
rPspA。
2. 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, works as bacterium
When body 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 receipts
Collection induction thalline, adds the resuspended washing of normal saline 2 times, adds 05mol/LNaCl5mmol/L miaow with the ratio of 1:10 (g/mL)
The resuspended thalline of buffer of azoles 20mmol/LPB (pH7.4), ultrasonic disruption thalline, 8000 × g is centrifuged 40min, collects supernatant,
Be purified in nickel ion chromatographic column, by specification operation purified product and analyze by carrier pET-30a (+) convert big
Enterobacteria BL21Star (DE3) whole cell (comparison) with by fine to nitric acid for upper step purification of samples electrotransfer after SDS-PAGE separates
On dimension element film, close 2h with 5% defatted milk powder shaking table slight oscillatory；Adding 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 develops the color.
Four, multivalent pneumococcal-Type B hemophilus influenza combined vaccine is prepared
1. prepare Type B hemophilus influenza capsular saccharides-carrier protein conjugate
Step one gained is refined Type B hemophilus influenza capsular saccharides and adds step 3 gained with the ratio of mass ratio 5:1
In Pneumococal surface protein A.
2. prepare multivalent pneumococcal-Type B hemophilus influenza combined vaccine stock solution
Refined S. pneumoniae capsular saccharide is added above-mentioned Type B hemophilus influenza capsular saccharides-load with the ratio of mass ratio 5:1
In body protein conjugate, S. pneumoniae capsular saccharide adds quality: it is 1.5:1 that Type B hemophilus influenza capsular saccharides adds quality.
3. multivalent pneumococcal-Type B hemophilus influenza combined vaccine is isolated and purified
Carrying out isolated and purified with Superdex200 solvent resistant column (2.6cm × 60cm), eluent is that the phosphoric acid of 20mM delays
Rushing liquid (pH7.4), flow velocity is 3mL/min.
By preparation 5mL vaccinogen liquid as a example by, add capsular polysaccharide 4,9V, 14, each 40 μ g of 19F and 23F, oligosaccharide 18C 20 μ g
And polysaccharide 6B 80 μ g, Hib capsular saccharides 200 μ g；PspA albumen 40 μ g.High-efficient liquid phase color is carried out after purification through gel chromatography column
This combined vaccine of analysis of spectrum, result shows that all serotype all has an obvious chromatographic peak, separating degree R ＞ 1.5 between adjacent two peaks,
Tailing factor T is in the range of 0.95～1.05, and capsular saccharides content reaches the Pharmacopoeia of the People's Republic of China 2010 editions and basis
Minimum requirements content in application summary of the invention.
It is pointed out that owing to pneumoprotein and S. pneumoniae capsular saccharide are homologies, have the most therebetween
There is synergism, but puting together between albumen and the capsular saccharides in the present invention is not limited only to above-mentioned amino reducing process, as long as energy
The method realizing this coupling result all should be included in present invention scope.
Embodiment 1-2
The present embodiment is only that with the difference of embodiment 1-1, and the polysaccharide order of addition in the present embodiment is for adding lung simultaneously
Scorching S. pneumoniae capsular saccharide and Type B hemophilus influenza capsular saccharides.After order exchange, Type B hemophilus influenza capsular saccharides is without chromatographic peak,
Prove to be simultaneously introduced S. pneumoniae capsular saccharide in former ratio and Type B hemophilus influenza capsular saccharides can form competitive relation, affect B
The normal conjugation procedure of type hemophilus influenza capsular saccharides, therefore cannot realize the technical program.
Embodiment 1-3
The present embodiment is only that with the difference of embodiment 1-1, and the polysaccharide order of addition in the present embodiment is for first adding pneumonia
S. pneumoniae capsular saccharide, rear interpolation Type B hemophilus influenza capsular saccharides.After order exchange, Type B hemophilus influenza capsular saccharides is without chromatograph
Peak, it was demonstrated that be initially charged S. pneumoniae capsular saccharide in former ratio and rear addition Type B hemophilus influenza capsular saccharides can form competition and close
System, affects the normal conjugation procedure of Type B hemophilus influenza capsular saccharides, therefore cannot realize the technical program.
Embodiment 1-4
The present embodiment is only that with the difference of embodiment 1-1, also include by preparation multivalent pneumococcal-Type B influenza addicted to
Blood bacillus combined vaccine carries out the step of lyophilizing:
Five, freeze dried vaccine protective agent is prepared
1. compound concentration be 70%, through the sucrose mother solution of 121 DEG C of sterilization treatment 15min；
2. multivalent pneumococcal isolated and purified for embodiment 1-1 gained-Type B hemophilus influenza combined vaccine is placed in nothing
In bacterium container, add the sucrose mother solution that step 1 is configured to, make sucrose concentration to 10%, obtain mixing and be prepared as protecting containing sucrose
The vaccinogen liquid semi-finished product of agent, 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 the multivalence pneumonia that sucrose concentration in lyophilizing stock solution is 10% again
Coccus-Type B hemophilus influenza combined vaccine, the preparation method of freeze dried vaccine refers to prior art and prepares, do not does at this superfluous
State.
Embodiment 1-5
1,5 the present embodiment is only that with the difference of embodiment 1-1, and the serotype of the S. pneumoniae capsular saccharide of preparation is:,
6B, 7F, 9V, 14,18C, 19F and 23F.
Embodiment 2-1
The present embodiment is only that with the difference of embodiment 1-1, and the concrete operations of step 4 are:
1. prepared by chitosan
Preparing the chitosan that deacetylation is 55%, step refers to prior art, does not repeats at this.
2. capsular saccharides and chitosan coupling altogether
Under 0.1MTBS solution buffer, 4 DEG C, under pH8.55-8.60, will the S. pneumoniae capsular saccharide of preparation and Type B influenza
Haemophilus capsular saccharides respectively with the chitosan coreaction 20 hours of deacetylation 55%, for ensureing vaccine efficacy, capsular saccharides
With chitosan mass ratio for 1:1), reaction is fully dialysed with bag filter after terminating, and removes unreacted chitosan, collects multivalence pneumonia
Coccus-Type B hemophilus influenza combined vaccine stock solution.
3. couplet is modified
Take step 2 capsular saccharides-chitosan couplet 30mL, under stirring condition, be slowly added dropwise 2mL25% 1,3-propanedicarboxylic acid, 200r/
Min stirring reaction 6 hours；After completion of the reaction, after washing three times with 0.01MPBS, it is settled to 30mL, makes capsular saccharides-chitosan coupling
-the OH that surface is not reacted with capsular saccharides is modified as-CHO；N2,4 DEG C save backup.
4. with the coupling altogether of PspA carrier protein
Under 0.1MTBS solution buffer, 4 DEG C, under pH4.0-9.0, by step 3 gained multivalent pneumococcal capsular saccharides-shell
Glyean body and Type B hemophilus influenza capsular saccharides-chitosan couplet and PspA albumen coreaction 24 hours, wherein multivalence
S. pneumoniae capsular saccharide-chitosan couplet: Type B hemophilus influenza capsular saccharides-chitosan couplet: PspA albumen quality ratio
For 5:5:2.
5. multivalent pneumococcal-Type B hemophilus influenza combined vaccine is isolated and purified
Carrying out isolated and purified with Superdex200 solvent resistant column (2.6cm × 60cm), eluent is that the phosphoric acid of 20mM delays
Rushing liquid (pH7.4), flow velocity is 3mL/min.
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.
Embodiment 2-2
The present embodiment is only that with the difference of embodiment 2-1, also include by preparation multivalent pneumococcal-Type B influenza addicted to
Blood bacillus combined vaccine carries out lyophilizing.
Embodiment 2-3
1,5 the present embodiment is only that with the difference of embodiment 2-1, and the serotype of the S. pneumoniae capsular saccharide of preparation is:,
6B, 7F, 9V, 14,18C, 19F and 23F.
Embodiment 3-1
The present embodiment is only that with the difference of embodiment 2-1, and step 4 also prepares liposome, and concrete operations refer to existing
Technology, does not repeats at this.100 μ g liposomees are taken former with multivalent pneumococcal-Type B hemophilus influenza combined vaccine before using
Subcutaneous injection after liquid mix homogeneously.
Embodiment 3-2
1,5 the present embodiment is only that with the difference of embodiment 3-1, and the serotype of the S. pneumoniae capsular saccharide of preparation is:,
6B, 7F, 9V, 14,18C, 19F and 23F.100 μ g liposomees and multivalent pneumococcal-Type B hemophilus influenza connection is taken before using
Close subcutaneous injection after vaccinogen liquid mix homogeneously.
Protection and Study On Immunogenicity
Combined vaccine (freeze dried vaccine stored refrigerated is after 12 months) prepared by above-described embodiment with people by dose dilution 100
The NIH mice of immunity 14-18g, often group 10 again, each experimental vaccine uses two groups, subcutaneous injection knot combined vaccine, negative control
Group injection 0.5ml physiological saline solution, immunity in every 2 weeks 1 time, each 0.5ml, totally 3 times.After initial immunity, according to Chinese Pharmacopoeia
2005 editions, using agar diffusion method to measure average cumulative release (%), result is as shown in table 1；Blood sampling in 4th week, uses ELISA method inspection
Survey serum and resist the immunogenicity of above-mentioned all capsular saccharides；Within 8th week, attack with streptococcus pneumoniae and Type B hemophilus influenza gavage, meter
Calculate vaccine protective rate, vaccine protective rate (%)=(matched group sickness rate-immune group sickness rate) × 100%.Gained Conversion rate is tied
Fruit is shown in Table 2, and titre is shown in Table 3, and vaccine protective rate is shown in Table 4.
Table 1
Table 2
Table 3
Note: embodiment 1-4 and embodiment 2-2 there was no significant difference (P ＞ with embodiment 1-1 and embodiment 2-1 data respectively
0.05), do not repeat at this.
Table 4
In summary, the combined vaccine that the present invention provides is provided simultaneously with multiple antigen, and Conversion rate has reached more than 85%, because of
The combined vaccine that this technical scheme using the present invention to provide obtains can significantly induce Type B haemophilus capsular saccharides, streptococcus pneumoniae
The generation of capsular saccharides antibody, protective rate is high, and immunne response is effective.And the antigen release of the combined vaccine of embodiment 3 offer
Considerably slower than other embodiments, there is preferable slow release effect.
Claims (10)
1. multivalent pneumococcal-Type B hemophilus influenza combined vaccine, it is characterised in that: containing S. pneumoniae capsular saccharide, Type B
Hemophilus influenza capsular saccharides and carrier protein, carrier protein is used for and capsular saccharides covalent bond.
2. the combined vaccine described in claim 1, it is characterised in that: S. pneumoniae capsular saccharide, Type B hemophilus influenza capsular saccharides
It is (5～25) with the mass ratio of carrier protein: (1～5): 1.
3. the combined vaccine described in claim 1, it is characterised in that: pneumococcal serotype is the one in following serotype
Or multiple: 1,3,4,5,6A, 6B, 7F, 8,9N, 9V, 12F, 14,15B, 18C, 19A, 19F, 22F or 23F.
4. the combined vaccine described in claim 1, it is characterised in that: carrier protein covalently bound with capsular saccharides in described vaccine
Selected from tetanus toxoid, diphtheria toxoid, diphtheria toxin mutation cross reacting material 197, Pneumococal surface protein A, B
Group meningitis cocci outer membrane protein, hemophilus influenza D albumen, Pseudomonas aeruginosa Exotoxin A, gram negative bacteria adventitia egg
In vain, Heat-labile Enterotoxin of E. coli, pneumolysin, pertussis toxin, PT, bacillus pyocyaneus pili, gonococcus P-3-2 bacterium
Hair or hepatitis B virus surface antigen.
5. the combined vaccine described in claim 4, it is characterised in that: the carrier of vaccine is Pneumococal surface protein A.
6. the method preparing the arbitrary described combined vaccine of Claims 1 to 5, it is characterised in that comprise the steps:
Step a1) preparation also separating-purifying carrier protein；
Step b1) in step a1 resulting vehicle albumen, add refined Type B hemophilus influenza capsular saccharides；
Step c1) in step b1 gained mixture, add refined S. pneumoniae capsular saccharide, the wherein addition matter of 6B type capsular saccharides
Amount is 2 times that remaining capsular saccharides adds quality；
Step d1) purification procedures c1 gained capsular saccharides-carrier protein, obtain multivalent pneumococcal-Type B hemophilus influenza connection
Close vaccinogen liquid.
7. multivalent pneumococcal-Type B hemophilus influenza combined vaccine, it is characterised in that: containing S. pneumoniae capsular saccharide, Type B
Hemophilus influenza capsular saccharides, carrier protein and adjuvant, carrier protein is used for and capsular saccharides covalent bond.
8. the combined vaccine described in claim 7, it is characterised in that: adjuvant is selected from chitosan and/or liposome.
9. the combined vaccine described in claim 7, it is characterised in that: the mass ratio of chitosan or liposome and capsular saccharides is between 5:
Between 1 and 1:5.
10. the method preparing the arbitrary described combined vaccine of claim 7～9, it is characterised in that comprise the steps:
Step a2) refine Type B hemophilus influenza capsular saccharides and S. pneumoniae capsular saccharide respectively；
Step b2) preparation also separating-purifying carrier protein；
Step c2) successively capsular saccharides-chitosan couplet is combined with step b2 resulting vehicle albumen coupling；
Step d2) purification procedures c2 gained couplet, obtain multivalent pneumococcal-Type B hemophilus influenza combined vaccine former
Liquid.
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| CN110251667A (en) * | 2018-05-11 | 2019-09-20 | 武汉博沃生物科技有限公司 | A kind of immune combination preparation and its preparation method and application |
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| CN101785857A (en) * | 2010-03-05 | 2010-07-28 | 郑玉清 | Novel pneumococcal conjugate vaccine and preparation method thereof |
| CN104837505A (en) * | 2012-12-11 | 2015-08-12 | Sk化学公司 | Multivalent pneumococcal polysaccharide-protein conjugate composition |
| CN105342982A (en) * | 2015-11-19 | 2016-02-24 | 上海现代药物制剂工程研究中心有限公司 | Influenza vaccine immune preparation for nasal administration and preparation method thereof |
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|---|---|---|---|---|
| CN101785857A (en) * | 2010-03-05 | 2010-07-28 | 郑玉清 | Novel pneumococcal conjugate vaccine and preparation method thereof |
| CN104837505A (en) * | 2012-12-11 | 2015-08-12 | Sk化学公司 | Multivalent pneumococcal polysaccharide-protein conjugate composition |
| CN105342982A (en) * | 2015-11-19 | 2016-02-24 | 上海现代药物制剂工程研究中心有限公司 | Influenza vaccine immune preparation for nasal administration and preparation method thereof |
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| CN110251667A (en) * | 2018-05-11 | 2019-09-20 | 武汉博沃生物科技有限公司 | A kind of immune combination preparation and its preparation method and application |
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