CN103893751B - A kind of pneumococal polysaccharide Protein Conjugation vaccine and preparation method thereof - Google Patents
A kind of pneumococal polysaccharide Protein Conjugation vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention provides a kind of pneumococal polysaccharide Protein Conjugation vaccine and preparation method thereof, described pneumococal polysaccharide Protein Conjugation vaccine comprises the immunoconjugates that one or more streptococcus pneumoniae capsular polysaccharides and protein are coupled combination, have at least a kind of single streptococcus pneumoniae capsular polysaccharide of a kind of immunoconjugates and two or more protein to be coupled in described immunoconjugates to be formed, compared with existing pneumococcal conjugated vaccine, its immunogenicity is stronger, can cause immunne response in more wide crowd; Simultaneously because 2 kinds of carrier proteins are coupled by polysaccharide covalent, the Protein Epitopes wherein with protectiveness also can induction ratio two kinds of albumen hybrid injections time higher immunoreation, mutual synergism, enhances the immunogenicity of carrier protein further, adds the immunoreation of body to polysaccharide; Its preparation method is simple, is applicable to the needs that large-scale industrial is produced.
Description
Technical field
The present invention relates to field of immunology, especially pneumococal polysaccharide Protein Conjugation vaccine and preparation method thereof.
Background technology
Infection caused by streptococcus pneumoniae (lung chain) is the main cause of whole world M & M.Pneumonia, heat generation bacteremia and meningitis are the most common manifestation forms of invasive pneumococcal disease, and the antibacterial diffusion in respiratory tract can cause middle ear infection, sinusitis or recurrent bronchitis.Compared with invasive disease, the form of expression of Noninvasive is usually so not serious, but more common.Due to the diffusion of antibiotics resistance sexually transmitted disease, and pneumococcal pneumonia occurs after influenza infection of being everlasting, and the probability that pneumococcal disease shows effect during influenza increases further.The disease caused by streptococcus pneumoniae has become the important public health problem in one, the whole world.Streptococcus pneumoniae has become the number one killer of global child.
The case fatality rate of China's pneumonia is 16.4%, and wherein more than 50 years old middle-aged and elderly people and 1 years old Infants Below are respectively up to 28.6% and 22.0%.The carrying rate of China streptococcus pneumoniae in healthy children is higher, and statistics show, and the carrying rate in northern area healthy children is 24.2%, and southern area is 31.3%.And this disease is the major reason causing less than 5 years old death of child.Main cause is that the growth of infant immunisation system is not perfect, and immunity is more weak.And the baby that the age is less, immunity is more weak.
The nearly 90 kinds of serotypes (bacterial strain) of streptococcus pneumoniae, before the statistics display pneumococcal infection bacterial strain of China, several serotype is followed successively by: 5,6,19,23,14,2,4 types.The research display of 860 strain streptococcus pneumoniae separated strains is have collected according to one, 109 strains (12.7%) are had to show as sero-group 6, reach 100% in the erythromycin resistance of Chinese Pneumococcus serotypes 6,6A, 6B and 6C type is wherein respectively 62 strains (56.9%), 38 strains (34.9%) and 9 strains (8.2%).
The 23 valency Pnu-Imune 23s that Chengdu Inst. of Biological Products of Chinese biological technology group produces have chosen 23 kinds of modal pathogenic bacterium (1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F), respectively fermentation culture various polysaccharide on separating-purifying pneumococcal capsule, is mixed and made into vaccine by equal proportion.
The polysaccharide of antibacterial is a kind of thymus independent antigen, and the main distinction of this antigen and thymus dependent antigen is that the former does not need lymphocytic the assisting of T to produce antibody.Therefore there is following problem in polysaccharide vaccine: (1) can only produce faint immunoreation in brood or infants, does not even produce immunoreation, and immunoreation strengthens with the growth at age; (2) antibody of low-affinity is produced; (3) only produce of short duration immunoreation, do not possess immunological memory when repeatedly inoculating and immune-enhancing effect; (4) easily immunologic tolerance is produced; (5) common adjuvant not easily plays the effect of immunostimulant to this antigen.The protective rate that 23 valency polysaccharide vaccines infect for intrusion type lung chain is 50-70%.And crowd's inoculation of more than 2 years old can only be used for, and the peak age of onset of pneumonia is the 6-12 monthly age.
GL-PP conjugate vaccines (combined vaccine) is current state-of-the-art vaccine technologies, and specific antigen adds protein carrier, can increase its immunogenicity.Protein carrier has T cell dependency characteristic, and the polysaccharide antigen of non-T cell pauper character can be changed into the antigen of T cell pauper character by GL-PP conjugate vaccines, and the t helper cell of excitating organism produces a series of immune-enhancing effect.Capsular polysaccharide conjugate vaccine, polysaccharide adds protein carrier, becomes T cell dependence antigen from T-independent antigen, increases its immunogenicity.The antibody produced after combined vaccine inoculation quality and be quantitatively generation vaccine 400-1000 doubly, produce immunoprotection wider stronger, guard time more for a long time, reaches efficient protection.
2000, Pfizer Inc.'s first 7 valent pneumococcal conjugate vaccine listing; Pfizer Inc.'s exploitation infant more targetedly 7 (4,6B, 9V, 14,18C, 19F and 23F) valency pneumoprotein vaccine, also effective to less than 5 years old child.Capsular polysaccharide protein conjugate vaccines, polysaccharide adds protein carrier, becomes T cell dependence antigen from T-independent antigen, can increase its immunogenicity, can be used for the child in more than 6 week age.Current Pfizer China's registration 13 valency combined vaccines, add 6 serotypes (1,3,5,7F, 6A, 19A).
But the data of China display pneumococcal infection bacterial strain serotype is followed successively by: 5,6,1,19,23,14,2,4, Pfizer 7 valent pneumococcal conjugate vaccine only has about 50% to China's common causative bacterial type coverage rate, and the coverage rate of 13 valency combined vaccines also only has 70%.Hui Shi 7 valent pneumococcal conjugate vaccine needs inoculation 4 injection, 860 yuan, every pin, expensive, is unfavorable for promoting.13 valency vaccines estimate that its price will be more expensive.Therefore the 7 and 13 valency combined vaccines of Pfizer Inc. are not too applicable to the large-scale children Streptococcus prevention of China.
Although 13 valent pneumococcal conjugate vaccines go on the market, wherein there is the immune effect of several serotype lower relative to other serotypes, as 3 types.And along with the increase of different serotypes and reusing of carrier protein of the same race, make carrier protein consumption increase, its immunogenicity reduces on the contrary.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of pneumococal polysaccharide Protein Conjugation vaccine.
Another technical problem to be solved by this invention is the preparation method providing above-mentioned pneumococal polysaccharide Protein Conjugation vaccine.
For solving the problems of the technologies described above, technical scheme of the present invention is:
A kind of pneumococal polysaccharide Protein Conjugation vaccine, be comprise the immunoconjugates that one or more streptococcus pneumoniae capsular polysaccharides and protein are coupled combination, have at least a kind of single streptococcus pneumoniae capsular polysaccharide of a kind of immunoconjugates and two or more protein to be coupled in described immunoconjugates and formed.
Preferably, above-mentioned pneumococal polysaccharide Protein Conjugation vaccine, comprises the immune composition of multivalent pneumococcal conjugate, is to mix after capsular polysaccharide on separating-purifying various serotype pneumococcal capsule is combined with protein (carrier protein) coupling.
Preferably, above-mentioned pneumococal polysaccharide Protein Conjugation vaccine, the described protein for being coupled combination with capsular polysaccharide is the protein that can be used in directly being coupled with capsular polysaccharide or being undertaken by chemical union joint to be coupled.
Preferably, above-mentioned pneumococal polysaccharide Protein Conjugation vaccine, the described protein for being coupled combination with capsular polysaccharide is diphtheria toxoid, tetanus toxoid, carrier protein CRM197, bloodthirsty hemophilus influenza surface protein HiD, pertussis Prn surface protein, pertussis Fha antigen and/or pneumococcal surface protein PspA.
Preferably, above-mentioned pneumococal polysaccharide Protein Conjugation vaccine, described for being coupled in two or more protein of combination with capsular polysaccharide, wherein a kind of protein is the antigen with protectiveness, and another albumen is not restriction, can select arbitrarily.
Preferably, above-mentioned pneumococal polysaccharide Protein Conjugation vaccine, described for being coupled in two or more protein of combination with capsular polysaccharide, wherein one is bloodthirsty hemophilus influenza surface protein HiD or PspA albumen.
Preferably, above-mentioned pneumococal polysaccharide Protein Conjugation vaccine, described streptococcus pneumoniae capsular polysaccharide is the capsular polysaccharide on separating-purifying Pneumococcal serotype pod membrane, and the serotype of described Pneumococcal serotype comprises 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and/or 33F.
The preparation method of above-mentioned pneumoprotein vaccine, concrete steps are:
(1) capsular polysaccharide on difference separating-purifying various serotype pneumococcal capsule;
(2) carrier protein respectively selected by separating-purifying;
(3) be combined with carrier protein couplet by various capsular polysaccharide respectively, wherein, at least one capsular polysaccharide and two or more protein are coupled combination;
(4) various coupling combination is mixed to form vaccinogen liquid, wherein, at least one capsular polysaccharide and two or more protein are coupled combination.
The concrete steps of the separation and purification of described capsular polysaccharide are the prior aries that those skilled in the art have grasped; The production of carrier protein is also the prior art that those skilled in the art have grasped.
Preferably, the preparation method of above-mentioned pneumoprotein vaccine, in described step (3), the coupling combination of capsular polysaccharide and carrier protein comprises:
(1) capsular polysaccharide CDAP(1-amino-4-dimethyl-pyridine-tetrafluoride boron) and the coupling of triethylamine activation method in conjunction with carrier protein.This reaction is the coupled reaction of isourea key.When high pH, the hydroxyl reaction on CDAP and polysaccharide residue radical, changes into cyanate, the amino on carrier protein and its react rapidly, form isourea key.The method is simple to operate, easily repeats; Or
(2) first carry out IO4 activation to polysaccharide form free aldehyde radical and be connected combination with the amino on carrier protein; Or
(3) be connected with the carboxyl (or being oxidized obtained carboxyl in polysaccharide) in acidic polysaccharose with adipic acid two hydrazine acyl (ADH), and then be connected with albumen under EDAC existent condition; Or
(4) capsular polysaccharide is combined with carrier protein couplet and by carrying out fragment to polysaccharide, can also derives after adding chain joint and being connected with the amino on carrier protein or carboxyl.
Preferably, the preparation method of above-mentioned pneumococal polysaccharide Protein Conjugation vaccine, on the surface protein that lung chain polysaccharide all can link by 5 kinds of modes that in described step (3), capsular polysaccharide is combined with the coupling of carrier protein, it is preferred that the present invention plants with above-mentioned (2).
Preferably, the preparation method of above-mentioned pneumococal polysaccharide Protein Conjugation vaccine, the connected mode that in described step (3), a kind of capsular polysaccharide and two or more protein are coupled combination comprises:
(1) two kinds of carrier proteins and capsular polysaccharide are joined in reaction vessel simultaneously react by one-step reaction method, and this method is comparatively simple, but in conjugate different albumen ratio and be not easy accurate control; Or
(2) sequential method, first capsular polysaccharide is connected with wherein a kind of carrier protein, then through purification, removes unconjugated floating preteins and free polysaccharide, add the second carrier protein again to connect, the bonded products with stable bond ratio can be produced.
Preferably, the preparation method of above-mentioned pneumococal polysaccharide Protein Conjugation vaccine, concrete steps are:
(1) streptococcus pneumoniae choosing 24 kinds of serotypes is cultivated, and these 24 kinds of serotypes are 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F;
(2) capsular polysaccharide that more than purifying respectively, in various Pneumococcal serotype, antigenicity is strong: streptococcus pneumoniae, collected by centrifugation supernatant after deactivation, through ultrafiltration and concentration, (volume fraction be 70%) pre-cooled ethanol is added in right amount respectively according to each Pneumococcus serotypes characteristic, collected by centrifugation, obtains rough polysaccharide, rough polysaccharide is dissolved in sodium acetate solution, then mix with cold phenol in 1:2 ratio, centrifugal segregation albumen, phenol is carried 5-6 time repeatedly, collect supernatant, with distill water dialysis, after dialysis, liquid adds 2mol/L calcium chloride solution, add ethanol to stir, centrifugal segregation nucleic acid, collect supernatant, add ethanol (final concentration 80% stirs), centrifugal collecting precipitation, with ethanol, washing with acetone precipitates, refining capsular polysaccharide is after dehydrate, put-20 DEG C to save backup, concrete grammar and parameter can see US Patent No. 2007/0231340 [1] (Hui Shi 13 valency patent),
(3) by currently available vaccines, existing vaccines explained hereafter CRM197(reference Chinese patent CN102766647), diphtheria toxoid (with reference to US Patent No. 2005/0002957), tetanus toxoid (with reference to Chinese patent CN102961741), pertussal various antigen (with reference to Chinese patent CN102793915), or by HiD (also known as ProteinD, with reference to European patent EP 0594610 and international monopoly WO2007/071710), PspA albumen (with reference to Chinese patent CN103288936) is cloned into escherichia coli and carries out expression and separation and purification;
(4) get the capsular polysaccharide detecting qualified various Pneumococcal serotype, obtain aldehyde radical then with two kinds of albumen by sodium metaperiodate activation method and combine simultaneously, obtain target conjugate; Or will wherein a kind of albumen and polysaccharide first be coupled, carry out purification when not closing active group, the purified product obtained and the second albumen are carried out being coupled obtaining target conjugate further.
Above-mentioned pneumoprotein vaccine for the preparation of induction to the application in the medicine of the immunne response of streptococcus pneumoniae capsular polysaccharide conjugates and carrier protein.
Beneficial effect of the present invention:
Above-mentioned pneumococal polysaccharide Protein Conjugation vaccine is the immunoconjugates containing two or more different carriers albumen, and compared with existing pneumococcal conjugated vaccine, its immunogenicity is stronger, can cause immunne response in more wide crowd; Simultaneously because 2 kinds of carrier proteins are coupled by polysaccharide covalent, the Protein Epitopes wherein with protectiveness also can induction ratio two kinds of albumen hybrid injections time higher immunoreation, mutual synergism, enhances the immunogenicity of carrier protein further, adds the immunoreation of body to polysaccharide; Its preparation method is simple, is applicable to the needs that large-scale industrial is produced.
Detailed description of the invention
Below in conjunction with specific embodiment, technical scheme of the present invention is further described.
Obtain capsular polysaccharide and carrier protein:
(1) streptococcus pneumoniae choosing 24 kinds of serotypes (1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F) is cultivated;
(2) capsular polysaccharide that more than purifying respectively, in various Pneumococcal serotype, antigenicity is strong: streptococcus pneumoniae, collected by centrifugation supernatant after deactivation, through ultrafiltration and concentration, (volume fraction be 70%) pre-cooled ethanol is added in right amount respectively according to each Pneumococcus serotypes characteristic, collected by centrifugation, obtains rough polysaccharide; Rough polysaccharide is dissolved in sodium acetate solution, then mixes with cold phenol in 1:2 ratio, centrifugal segregation albumen, phenol is carried 5-6 time repeatedly, collects supernatant, with distill water dialysis, after dialysis, liquid adds 2mol/L calcium chloride solution, adds ethanol and stirs, centrifugal segregation nucleic acid, collect supernatant, add ethanol (final concentration 80% stirs), centrifugal collecting precipitation, by ethanol, washing with acetone precipitation, be refining capsular polysaccharide after dehydrate, put-20 DEG C and save backup;
(3) the existing vaccine art by recording in technique scheme produces CRM197, diphtheria toxoid, tetanus toxoid, pertussal various antigen, also HiD, PspA albumen can be cloned into escherichia coli and carry out expression and separation and purification.
The preparation of embodiment 1 one kinds of 7F type capsular polysaccharides and two kinds of different carriers protein conjugates
5g lung chain 7F type polysaccharide is dissolved in 1L sodium acetate buffer (50mMpH4.5), at room temperature, by the magnetic stirrer 20 minutes with magneton, to make polysaccharide dissolve fully, adds 0.2gNaIO
4, and under room temperature, reaction is spent the night, and makes two hydroxyls adjacent on polysaccharide be oxidized to aldehyde radical.Carry out 20 equal-volume ultrafiltration by purified water and change liquid, remaining NaIO in reacting
4and the small-molecule substance filtering generated in course of reaction, obtain introducing the aqueous solution that activation grade is the 7F activated polysaccharide of 6.2; And can be concentrated freeze-dried by the polysaccharide activated.The determination of the molecular size of activated polysaccharide utilizes gel permeation chromatography post and utilizes multi-angle laser light scattering to carry out, and utilizes dextran molecule size criteria to calibrate.The content of polysaccharide is determined by anthrone method, the aldehyde group content introduced utilizes Park, J.T. and Johnson, M.J. (1949) journal of biological chemistry (JournalofBiologicalChemistry), the described method of 18l, l49-151 page is determined.The structural intergrity of activated polysaccharide is determined by proton 1H and 13CNMR.The purity of activated polysaccharide is by measuring LAL(endotoxin) content determines.
Each 7F polysaccharide activated is put into triangular flask respectively, with purified water dilution polysaccharide to 11g/L, add the aseptic phosphate buffer (500mM) of 1/9 volume again, by table 2 add the CRM197 protein powder of 5g/L and the pneumonia surface protein PspA powder of 5g/L, and stir by the magnetic agitation with magneton, add 2g/L sodium cyanoborohydride, stirring and dissolving, react and add 1.1g/L sodium borohydride after 18 hours, mix and add ammonium sulfate to 1mol/L after 4 hours, (filler is FractogelPropyl (S) to balance hydrophobic chromatography post with 1M ammonium sulfate+50mM phosphate buffer (pH7.5), Merck), after loading, 5 column volumes are washed again with above-mentioned buffer, final 50mM phosphate buffer eluting also collects polysaccharide-protein conjugate.In the ultrafiltration system of equipment 50KDa filter membrane, with normal saline, 15 equal-volume ultrafiltration are carried out to polysaccharide-protein conjugate solution, collect and reflux and use bellows frit degerming, be stored in 4 DEG C.
The molecular weight distribution situation of GL-PP conjugate is detected by CL-4B and SEC-MALLS method; Albumen in different antibody serum determination GL-PP conjugates and polysaccharide kind is used by the two method expanded of immunity; The polyoses content of GL-PP conjugate is detected by anthrone method; Lowry method protein content detects the total protein content of GL-PP conjugate, then is combined than (Ratio) by the GL-PP calculating conjugate; CRM197, PspA protein concentration is detected by euzymelinked immunosorbent assay (ELISA).The results are shown in Table 1, is the GL-PP concentration of 7F type capsular polysaccharide described in embodiment 1 and two kinds of different carriers protein conjugates.
The GL-PP concentration of table 17F type GL-PP conjugate stock solution
The preparation of embodiment 2 one kind of 3 type capsular polysaccharide and two kinds of different carriers protein conjugates
2g lung chain 3 type polysaccharide is dissolved in 1L sodium phosphate buffer (50mMpH7.2), at room temperature, by the magnetic stirrer 20 minutes with magneton, to make polysaccharide dissolve fully, adds 0.2gNaIO
4, and under room temperature, reaction is spent the night, and makes two hydroxyls adjacent on polysaccharide be oxidized to aldehyde radical.Carry out 20 equal-volume ultrafiltration by purified water and change liquid, remaining NaIO in reacting
4and the small-molecule substance filtering generated in course of reaction, obtain introducing the aqueous solution that activation grade is the 3 type activated polysaccharides of 9.1; And by concentrated freeze-dried for the polysaccharide activated.The determination of the molecular size of activated polysaccharide utilizes gel permeation chromatography post and utilizes multi-angle laser light scattering to carry out, and utilizes dextran molecule size criteria to calibrate.The content of polysaccharide is determined by anthrone method, the aldehyde group content introduced utilizes Park, J.T. and Johnson, M.J. (1949) journal of biological chemistry (JournalofBiologicalChemistry), the described method of 18l, l49-151 page is determined.The structural intergrity of activated polysaccharide is determined by proton 1H and 13CNMR.The purity of activated polysaccharide is by measuring LAL(endotoxin) content determines.
Each 3 type polysaccharide activated are put into triangular flask respectively, with purified water dilution polysaccharide to 11g/L, add the aseptic phosphate buffer (500mM of 1/9 volume again, pH7.2), add the CRM197 protein powder of 5g/L again, and stir by the magnetic agitation with magneton, add 2g/L sodium cyanoborohydride, stirring and dissolving, react and add ammonium sulfate to 1.5mol/L after 18 hours, (filler is FractogelPropyl (S) to balance hydrophobic chromatography post with 1.5M ammonium sulfate+50mM phosphate buffer (pH7.5), Merck), after loading, 5 column volumes are washed again with above-mentioned buffer, final 50mM phosphate buffer eluting also collects polysaccharide-protein list carrier conjugates.In the ultrafiltration system of equipment 100KDa filter membrane, with normal saline, 5 equal-volume ultrafiltration are carried out to polysaccharide-protein conjugate solution, then carrying out being concentrated into polysaccharide concentration is 5g/L, add the HiD albumen of 5g/L, 1g/L sodium cyanoborohydride, stirring and dissolving, react and add 0.7g/L sodium borohydride after 48 hours, to mix after 4 hours ammonium sulfate to 1.5mol/L, (filler is FractogelPropyl (S) to balance hydrophobic chromatography post with 1.5M ammonium sulfate+50mM phosphate buffer (pH7.5), Merck), after loading, 5 column volumes are washed again with above-mentioned buffer, final 50mM phosphate buffer eluting also collects polysaccharide-protein complex carries conjugate.In the ultrafiltration system of equipment 100KDa filter membrane, with normal saline, 15 equal-volume ultrafiltration are carried out to polysaccharide-protein conjugate solution, collect and reflux and use bellows frit degerming, be stored in 4 DEG C.
The molecular weight distribution situation of GL-PP conjugate is detected by CL-4B and SEC-MALLS method; Albumen in different antibody serum determination GL-PP conjugates and polysaccharide kind is used by the two method expanded of immunity; The polyoses content of GL-PP conjugate is detected by anthrone method; Lowry method protein content detects the total protein content of GL-PP conjugate, then is combined than (Ratio) by the GL-PP calculating conjugate; CRM197, HiD protein concentration is detected by euzymelinked immunosorbent assay (ELISA).The results are shown in Table 2, for implementing
The GL-PP concentration of 3 type capsular polysaccharides and two kinds of different carriers protein conjugates described in example 2.
The GL-PP concentration of table 23 type GL-PP conjugate stock solution
The preparation of embodiment 3 one kind of 13 valency pneumococal polysaccharide Protein Conjugation vaccine
Prepare 4,6B according to known method in background technology, 9V, single carrier conjugates such as 14,18C, 19F, 23F, wherein carrier protein is CRM197.According to the method that embodiment 1 or 2 describes, the complex carries conjugate of preparation 1,3,5,6A, 7F, 19A type polysaccharide, wherein 1,5,7F type polysaccharide adopts PspA to be Second support, and 3,6A, 19A adopt HiD to be Second support.Vaccine formulation is carried out in conjunction with product according to following table 3 by various.These monovalent component upon mixing, add the Aluminium phosphate adjuvant that ultimate density is 0.5mg/L.
Final content in the carrier protein type of the various antigen of table 3 and preparation
The evaluating drug effect of embodiment 4 one kind of 13 valency pneumococal polysaccharide Protein Conjugation vaccine, and with the comparing of existing single carrier combined vaccine
12-14g BALB/c. in female 3 week age children Mus, is divided into 4 groups at random, often organizes 10, the combined vaccine that difference lumbar injection is prepared according to embodiment 3, commercially available 13 valency lung chain combination vaccines, blank (aluminum phosphate) and carrier protein mixture.Wherein carrier protein the ingredients of a mixture is: CRM19730ug, HiD albumen 3.1ug, PspA albumen 4.2ug, and these albumen add the Aluminium phosphate adjuvant that ultimate density is 0.5mg/L upon mixing.
In the 0th, 2,4 week programmed injection 3 times, often only young Mus 0.5ml containing (0.5ug conjugate).Each experimental group is respectively at the blood samplings in 7 days after the 3rd pin injection, and separation of serum, puts-20 DEG C and save backup.
Detect for capsular polysaccharide and carrier protein antibodies titre and adopt indirect elisa method respectively, by various pneumonia polysaccharide, carrier protein is dissolved in the carbonate buffer solution of 0.05mol/LpH9.6, and with 20ug/ml concentration coated elisa plate, the BSA fluid-tight of 2% is closed.By test serum 100 during experiment, 200,400,800,1600,3200,6400,12800,25600,51200,102400,204800, add ELISA Plate after 409600 times of dilutions, put 37 DEG C of reaction 40min, the sheep anti mouse two adding horseradish peroxidase-labeled after conventional washing resists.Add simultaneously not containing the buffer of serum as negative control.With the colour developing of tetra-amino-biphenyl amine, microplate reader 450nm wavelength place reads A value.Calculate A value and negative hole A in every hole and be worth ratio, but ratio is greater than 2.1 epoch maximum extension rate is antibody titer in serum, geometric average is carried out to the titre of 10 mices, and with 10 for the end calculates logarithm value, the results are shown in Table 4.
Table 4 two kinds of dose vaccine and carrier protein mixture Mouse immunogenicity compare
Find out from data, after taking complex carries, (add * mark, 1,3,5,6A, 7F, 19A), the immunoreation of mice to lung chain polysaccharide significantly strengthens, and wherein 3 types are especially remarkable.In addition, contrast with the protein mixture of same dosage, complex carries conjugate also show its carrier protein and has more excellent immunogenicity (adding * mark).
Above-mentioned detailed description of this pneumoprotein vaccine and preparation method thereof being carried out with reference to embodiment; illustrative instead of determinate; several embodiments can be listed according to institute's limited range; therefore in the change do not departed under general plotting of the present invention and amendment, should belong within protection scope of the present invention.
Claims (8)
1. a pneumococal polysaccharide Protein Conjugation vaccine, it is characterized in that: be comprise the immunoconjugates that one or more streptococcus pneumoniae capsular polysaccharides and protein are coupled combination, have at least a kind of immunoconjugates to be that a kind of single streptococcus pneumoniae capsular polysaccharide and two kinds of protein are coupled and the complex carries conjugate formed in described immunoconjugates, these two kinds of protein are selected from: bloodthirsty hemophilus influenza surface protein HiD, PspA albumen, carrier protein CRM197.
2. pneumococal polysaccharide Protein Conjugation vaccine according to claim 1, it is characterized in that: the immune composition comprising multivalent pneumococcal conjugate, is mix after capsular polysaccharide on separating-purifying various serotype pneumococcal capsule and protein are coupled combination.
3. pneumococal polysaccharide Protein Conjugation vaccine according to claim 1, is characterized in that: described streptococcus pneumoniae capsular polysaccharide is the capsular polysaccharide on separating-purifying Pneumococcal serotype pod membrane, and the serotype of described Pneumococcal serotype comprises 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and/or 33F.
4. the preparation method of the pneumoprotein vaccine that one of claim 1-3 is described, is characterized in that: concrete steps are:
(1) capsular polysaccharide on difference separating-purifying various serotype pneumococcal capsule;
(2) carrier protein respectively selected by separating-purifying;
(3) respectively various capsular polysaccharide and carrier protein are coupled combination, wherein, at least one capsular polysaccharide and two kinds of protein are coupled and are combined into complex carries conjugate;
(4) be coupled combination be mixed to form vaccinogen liquid by various.
5. the preparation method of pneumoprotein vaccine according to claim 4, is characterized in that: in described step (3), the combination that is coupled of capsular polysaccharide and carrier protein comprises:
(1) capsular polysaccharide CDAP and triethylamine activation method are coupled in conjunction with carrier protein, and this reaction is the coupled reaction of isourea key, when high pH, hydroxyl reaction on CDAP and polysaccharide residue radical, changes into cyanate, the amino on carrier protein and its react rapidly, form isourea key, or
(2) first carry out IO4 activation to polysaccharide form free aldehyde radical and be connected combination with the amino on carrier protein; Or
(3) with adipic acid two hydrazine acyl and the carboxyl in acidic polysaccharose or be oxidized obtained carboxyl be connected in polysaccharide, and then be connected with albumen under EDAC existent condition.
6. the preparation method of pneumococal polysaccharide Protein Conjugation vaccine according to claim 4, is characterized in that: in described step (3), the connected mode being coupled combination of capsular polysaccharide and carrier protein comprises:
(1) two kinds of carrier proteins and capsular polysaccharide are joined in reaction vessel simultaneously react by one-step reaction method, or
(2) sequential method, is first connected capsular polysaccharide with wherein a kind of carrier protein, then through purification, removes unconjugated floating preteins and free polysaccharide, then adds the second carrier protein and connect.
7. the preparation method of pneumoprotein vaccine according to claim 4, is characterized in that: concrete steps are:
(1) streptococcus pneumoniae choosing 24 kinds of serotypes is cultivated, and these 24 kinds of serotypes are 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F;
(2) capsular polysaccharide that more than purifying respectively, in various Pneumococcal serotype, antigenicity is strong: streptococcus pneumoniae, collected by centrifugation supernatant after deactivation, through ultrafiltration and concentration, the pre-cooled ethanol that volume fraction is 70% is added respectively according to each Pneumococcus serotypes characteristic, collected by centrifugation, obtains rough polysaccharide; Rough polysaccharide is dissolved in sodium acetate solution, then mixes with cold phenol in 1:2 ratio, centrifugal segregation albumen, phenol is carried 5-6 time repeatedly, collects supernatant, with distill water dialysis, after dialysis, liquid adds 2mol/L calcium chloride solution, adds ethanol and stirs, centrifugal segregation nucleic acid, collect supernatant, add ethanol, final concentration 80% stirs, centrifugal collecting precipitation, by ethanol, washing with acetone precipitation, be refining capsular polysaccharide after dehydrate, put-20 DEG C and save backup;
(3) produce CRM197, or HiD, PspA albumen is cloned into escherichia coli and carries out expressions also separation and purification;
(4) get the capsular polysaccharide detecting qualified various Pneumococcal serotype, obtain aldehyde radical then with two kinds of albumen by sodium metaperiodate activation method and combine simultaneously, obtain target conjugate; Or will wherein a kind of albumen and polysaccharide first be coupled, carry out purification when not closing active group, the purified product obtained and the second albumen are carried out being coupled obtaining target conjugate further.
8. the pneumococal polysaccharide Protein Conjugation vaccine that one of claim 1-3 is described for the preparation of induction to the application in the medicine of the immunne response of streptococcus pneumoniae capsular polysaccharide conjugates and carrier protein.
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| PCT/CN2015/074925 WO2015144031A1 (en) | 2014-03-26 | 2015-03-24 | Pneumococcus polysaccharide protein conjugated vaccine and preparation method therefor |
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| WO2018048141A1 (en) * | 2016-09-06 | 2018-03-15 | 주식회사 엘지화학 | Composition comprising multivalent capsular polysaccharide-transport protein and use thereof |
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