CN103893751A - Pneumococcal polysaccharide and protein conjugated vaccine and preparation method thereof - Google Patents
Pneumococcal polysaccharide and protein conjugated vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention provides a pneumococcal polysaccharide and protein conjugated vaccine and a preparation method thereof. The pneumococcus polysaccharide protein conjugated vaccine comprises one or more streptococcus pneumoniae capsular polysaccharide and protein coupled immune conjugates, and at least one of the immune conjugates is formed by coupling a single streptococcus pneumoniae capsular polysaccharide with two or more proteins. Compared with the existing pneumococcal conjugated vaccine, the pneumococcal polysaccharide and protein conjugated vaccine has stronger immunogenicity, and can cause immune response in a wider crowd; meanwhile, the two carrier proteins are covalently coupled through the polysaccharide, the protective protein antigen epitope induces higher immune response compared with the mixed injection of the two proteins, and through mutual synergy, the immunogenicity of the carrier protein is further improved and the immune response of the body to the polysaccharide is improved; the preparation method is simple, and meets the requirement of large-scale industrial production.
Description
Technical field
The present invention relates to field of immunology, especially pneumococal polysaccharide albumen conjugate vaccines and preparation method thereof.
Background technology
The main cause of whole world M & M by the caused infection of streptococcus pneumoniae (lung chain).Pneumonia, heat generation bacteremia and meningitis are the common manifestation forms of invasive streptococcus pneumoniae property disease, and 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 conventionally so not serious, but more common.Due to the diffusion of antibiotics resistance sexually transmitted disease, and pneumococcal pneumonia occurs after the influenza infection of being everlasting, and the probability that streptococcus pneumoniae disease is shown effect during influenza further increases.The disease being caused by streptococcus pneumoniae has become the important public health problem in the whole world.Streptococcus pneumoniae has become global child's No.1 killer.
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, statistics demonstration, and the carrying rate in northern area healthy children is 24.2%, southern area is 31.3%.And this disease is the major reason that causes 5 years old following death of child.Main cause is that the immune growth of infant is not perfect, immunity a little less than.And the baby that the age is less, immunity is more weak.
The nearly 90 kinds of serotypes of streptococcus pneumoniae (bacterial strain), before the statistics of China shows pneumococcal infection bacterial strain, several serotype is followed successively by: 5,6,19,23,14,2,4 types.According to studies show that of collecting 860 strain streptococcus pneumoniae separated strains, there are 109 strains (12.7%) to show as sero-group 6, erythromycin resistance in Chinese streptococcus pneumoniae serotype 6 reaches 100%, and 6A, 6B wherein and 6C type are 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 are to 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), the various polysaccharide on fermentation culture separating-purifying streptococcus pneumoniae pod membrane, is mixed and made into vaccine by equal proportion respectively.
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 that T is lymphocytic assists to produce antibody.Therefore there is following problem in polysaccharide vaccine: (1) can only produce faint immunoreation in brood or infants, even do not produce immunoreation, and immunoreation strengthens with the growth at age; (2) antibody of generation low-affinity; (3) only produce of short duration immunoreation, do not possess immunological memory and immune-enhancing effect while repeatedly inoculation; (4) easily produce immunologic tolerance; (5) common adjuvant is difficult for playing 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 can only be used for above crowd's inoculation in 2 years old, 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, adds protein carrier on specific antigen, can increase its immunogenicity.Protein carrier has T cell dependency characteristic, and GL-PP conjugate vaccines can change the polysaccharide antigen of non-T cell pauper character into the antigen of T cell pauper character, and the t helper cell of excitating organism produces a series of immune-enhancing effect.Capsular polysaccharide conjugate vaccine adds protein carrier on polysaccharide, becomes T cell dependence antigen from T-independent antigen, increases its immunogenicity.The antibody producing after combined vaccine inoculation in quality and be quantitatively generation vaccine 400-1000 doubly, generation immunoprotection is more extensively stronger, guard time is more permanent, reaches efficient protection.
2000, first 7 valent pneumococcal conjugate vaccine listing of Pfizer Inc.; Pfizer Inc. exploitation infant is (4,6B, 9V, 14,18C, 19F and 23F) valency pneumoprotein vaccine more targetedly 7, also effective to 5 years old following child.Capsular polysaccharide protein binding vaccine adds protein carrier on polysaccharide, becomes T cell dependence antigen from T-independent antigen, can increase its immunogenicity, can be used for 6 weeks children more than age.The 13 valency combined vaccines of China's registration of Pfizer at present, have increased by 6 serotypes (1,3,5,7F, 6A, 19A).
But the data of China shows pneumococcal infection bacterial strain, serotype is followed successively by: 5,6,1,19,23,14,2,4, Pfizer's 7 valent pneumococcal conjugate vaccines only have 50% left and right to China's common pathogen type coverage rate, and the coverage rate of 13 valency combined vaccines also only has 70%.Hui Shi 7 valent pneumococcal conjugate vaccines need to be inoculated 4 injections, and 860 yuan, every pin is expensive, are unfavorable for promoting.13 valency vaccines estimate that its price will be more expensive.Therefore 7 of Pfizer Inc. and 13 valency combined vaccines 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 are relative other serotypes of immune effect of several serotypes lower, as 3 types.And along with the increase of different serotypes and reusing of carrier protein of the same race, carrier protein consumption is increased, 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 albumen conjugate vaccines.
Another technical problem to be solved by this invention is to provide the preparation method of above-mentioned pneumococal polysaccharide albumen conjugate vaccines.
For solving the problems of the technologies described above, technical scheme of the present invention is:
A kind of pneumococal polysaccharide albumen conjugate vaccines, be to comprise that one or more streptococcus pneumoniae capsular polysaccharides and protein are coupled the immunoconjugates of combination, in described immunoconjugates, have at least a kind of single streptococcus pneumoniae capsular polysaccharide of a kind of immunoconjugates and two or more protein to be coupled and form.
Preferably, above-mentioned pneumococal polysaccharide albumen conjugate vaccines, the immune composition that comprises multivalent pneumococcal conjugate, is to mix after capsular polysaccharide on separating-purifying various serotype streptococcus pneumoniae pod membrane is combined with protein (carrier protein) coupling.
Preferably, above-mentioned pneumococal polysaccharide albumen conjugate vaccines, described is to can be used in the protein that is directly coupled or is coupled by chemical union joint with capsular polysaccharide for the protein that is coupled combination with capsular polysaccharide.
Preferably, above-mentioned pneumococal polysaccharide albumen conjugate vaccines, described is diphtheria toxoid for the protein that is coupled combination with capsular polysaccharide, 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 albumen conjugate vaccines, it is described that for being coupled 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 albumen conjugate vaccines, described for being coupled 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 albumen conjugate vaccines, described streptococcus pneumoniae capsular polysaccharide is the capsular polysaccharide on separating-purifying serotype streptococcus pneumoniae pod membrane, the pneumococcal serotype of described 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 streptococcus pneumoniae pod membrane;
(2) the selected carrier protein of difference separating-purifying;
(3) respectively various capsular polysaccharides are combined with carrier protein couplet, wherein, at least one capsular polysaccharide and two or more protein are coupled combination;
(4) various couplings combinations are mixed to form to 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) CDAP(1-amino-4-dimethyl-pyridine-tetrafluoride boron for capsular polysaccharide) and the coupling of triethylamine activation method in conjunction with carrier protein.This reaction is the coupled reaction of isourea key.In the time of high pH, the hydroxyl reaction on CDAP and polysaccharide residue radical, changes into cyanate, and the amino on carrier protein reacts rapidly with it, forms isourea key.The method is simple to operate, easily repeats; Or
(2) first polysaccharide being carried out to IO4 activation forms free aldehyde radical and is 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 acyls (ADH), and then be connected with albumen under the condition existing at EDAC; Or
(4) be combined with carrier protein couplet can also be by polysaccharide is carried out to fragment for capsular polysaccharide, is derivatively connected with amino or carboxyl on carrier protein after adding chain joint again.
Preferably, the preparation method of above-mentioned pneumococal polysaccharide albumen conjugate vaccines, on the surface protein that 5 kinds of modes that in described step (3), capsular polysaccharide is combined with the coupling of carrier protein all can link lung chain polysaccharide, the present invention plants as preferred taking above-mentioned (2).
Preferably, the preparation method of above-mentioned pneumococal polysaccharide albumen conjugate vaccines, the connected mode that in described step (3), a kind of capsular polysaccharide and two or more protein are coupled combination comprises:
(1) one-step reaction method, joins two kinds of carrier proteins and capsular polysaccharide in reaction vessel and reacts simultaneously, and this method is comparatively simple, but the ratio of different albumen be not easy accurate control in conjugate; Or
(2) sequential method, first capsular polysaccharide is connected with a kind of carrier protein wherein, then passes through purification, remove unconjugated floating preteins and free polysaccharide, add again the second carrier protein to connect, can produce the bonded products with stable bond ratio.
Preferably, the preparation method of above-mentioned pneumococal polysaccharide albumen conjugate vaccines, concrete steps are:
(1) streptococcus pneumoniae of 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) the strong capsular polysaccharide of antigenicity in above various serotype streptococcus pneumoniae of purifying respectively: streptococcus pneumoniae, centrifugal collection supernatant after deactivation, through ultrafiltration and concentration, add respectively (volume fraction is 70%) pre-cooled ethanol in right amount according to each streptococcus pneumoniae serotype characteristic, centrifugal collection, obtains rough polysaccharide, rough polysaccharide is dissolved in sodium acetate solution, then mix with cold phenol in 1:2 ratio, centrifugal removal 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 removal nucleic acid, collect supernatant, add ethanol (final concentration 80% stirs), centrifugal collecting precipitation, with ethanol, washing with acetone precipitation, after dehydrate, be refining capsular polysaccharide, putting-20 DEG C saves backup, concrete grammar and parameter can be referring to US Patent No. 2007/0231340[1] (Hui Shi 13 valency patents),
(3) pass through currently available vaccines, existing vaccines explained hereafter CRM197(with reference to 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 (claim again Protein D by HiD, 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 expresses also separation and purification;
(4) get and detect the qualified pneumococcal capsular polysaccharide of various serotypes, with the combinations simultaneously of then with two kinds of albumen of sodium metaperiodate activation method acquisition aldehyde radical, obtain target conjugate; Or will be wherein a kind of albumen and polysaccharide be first coupled, in the situation that not sealing active group, carry out purification, the purified product obtaining and the second albumen are further coupled and obtain target conjugate.
The application of above-mentioned pneumoprotein vaccine aspect the medicine of the immunne response to streptococcus pneumoniae capsular polysaccharide conjugates and carrier protein for the preparation of induction.
Beneficial effect of the present invention:
Above-mentioned pneumococal polysaccharide albumen conjugate vaccines, is the immunoconjugates containing two or more different carriers albumen, and compared with existing pneumococcal conjugated vaccine, its immunogenicity is stronger, can in more wide crowd, cause immunne response; Simultaneously because 2 kinds of carrier proteins are coupled by polysaccharide covalent, the higher immunoreation also can two kinds of albumen hybrid injections of induction ratio time of the Protein Epitopes wherein with protectiveness, synergism mutually, the immunogenicity of further having promoted carrier protein, has increased 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 of 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) the strong capsular polysaccharide of antigenicity in above various serotype streptococcus pneumoniae of purifying respectively: streptococcus pneumoniae, centrifugal collection supernatant after deactivation, through ultrafiltration and concentration, add respectively (volume fraction is 70%) pre-cooled ethanol in right amount according to each streptococcus pneumoniae serotype characteristic, centrifugal collection, obtains rough polysaccharide; Rough polysaccharide is dissolved in sodium acetate solution, then mixes with cold phenol in 1:2 ratio, centrifugal removal 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 to stir, centrifugal removal nucleic acid, collect supernatant, add ethanol (final concentration 80% stirs), centrifugal collecting precipitation, by ethanol, washing with acetone precipitation, after dehydrate, be refining capsular polysaccharide, put-20 DEG C and save backup;
(3) by the existing vaccine explained hereafter CRM197 recording in technique scheme, diphtheria toxoid, tetanus toxoid, pertussal various antigen, also can be by HiD, and PspA albumen is cloned into escherichia coli and expresses also separation and purification.
The preparation of 1 one kinds of 7F type capsular polysaccharides of embodiment and two kinds of different carriers protein conjugates
5g lung chain 7F type polysaccharide is dissolved in 1L sodium acetate buffer (50mM pH4.5), at room temperature, uses with the magnetic stirrer of magneton 20 minutes, so that polysaccharide dissolves fully, add 0.2g NaIO
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 that generates in course of reaction, the aqueous solution of the 7F activated polysaccharide that to have obtained introducing activation grade be 6.2; And can be by concentrated freeze-dried the polysaccharide having activated.The definite 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 of introducing utilizes Park, J.T. and Johnson, M.J. (1949) journal of biological chemistry (Journal of Biological Chemistry), 18l, the described method of l49-151 page is determined.The structural intergrity of activated polysaccharide is determined by proton 1H and 13C NMR.The purity of activated polysaccharide is by measuring LAL(endotoxin) content determines.
The 7F polysaccharide of each activation is put into respectively to triangular flask, with purified water dilution polysaccharide to 11g/L, add again the aseptic phosphate buffer (500mM) of 1/9 volume, by the pneumonia surface protein PspA powder of the CRM197 protein powder of 5g/L that table 2 adds and 5g/L, and use with the magnetic agitation of magneton and stir, add 2g/L sodium cyanoborohydride, stirring and dissolving, react and add 1.1g/L sodium borohydride after 18 hours, mixing added ammonium sulfate to 1mol/L after 4 hours, with 1M ammonium sulfate+50mM phosphate buffer (pH7.5) balance hydrophobic chromatography post, (filler is Fractogel Propyl (S), Merck), after loading, wash 5 column volumes with above-mentioned buffer again, final with 50mM phosphate buffer eluting and collect polysaccharide-protein conjugate.In the ultrafiltration system of equipment 50KDa filter membrane, with normal saline, polysaccharide-protein conjugate solution is carried out to 15 equal-volume ultrafiltration, collect and reflux and use the filtration sterilization of capsule formula filter, be stored in 4 DEG C.
Be the molecular weight distribution situation that SEC-MALLS method detects GL-PP conjugate by CL-4B; Use different antibody serums to determine albumen and the polysaccharide kind in GL-PP conjugate by the two methods that expand of immunity; Detect the polyoses content of GL-PP conjugate by anthrone method; Lowry method protein content detects the total protein content of GL-PP conjugate, then GL-PP by calculating conjugate is in conjunction with than (Ratio); CRM197, PspA protein concentration detect 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 1 7F type GL-PP conjugate stock solution
The preparation of 2 one kind of 3 type capsular polysaccharide of embodiment and two kinds of different carriers protein conjugates
2g lung chain 3 type polysaccharide are dissolved in 1L sodium phosphate buffer (50mM pH7.2), at room temperature, use with the magnetic stirrer of magneton 20 minutes, so that polysaccharide dissolves fully, add 0.2g NaIO
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 that generates in course of reaction, the aqueous solution of the 3 type activated polysaccharides that to have obtained introducing activation grade be 9.1; And by concentrated freeze-dried the polysaccharide having activated.The definite 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 of introducing utilizes Park, J.T. and Johnson, M.J. (1949) journal of biological chemistry (Journal of Biological Chemistry), 18l, the described method of l49-151 page is determined.The structural intergrity of activated polysaccharide is determined by proton 1H and 13C NMR.The purity of activated polysaccharide is by measuring LAL(endotoxin) content determines.
3 type polysaccharide of each activation are put into respectively to triangular flask, with purified water dilution polysaccharide to 11g/L, add again the aseptic phosphate buffer (500mM of 1/9 volume, pH7.2), add again the CRM197 protein powder of 5g/L, and use with the magnetic agitation of magneton and stir, add 2g/L sodium cyanoborohydride, stirring and dissolving, reacting added ammonium sulfate to 1.5mol/L after 18 hours, with 1.5M ammonium sulfate+50mM phosphate buffer (pH7.5) balance hydrophobic chromatography post, (filler is Fractogel Propyl (S), Merck), after loading, wash 5 column volumes with above-mentioned buffer again, final with 50mM phosphate buffer eluting and collect polysaccharide-protein list carrier conjugates.In the ultrafiltration system of equipment 100KDa filter membrane, with normal saline, polysaccharide-protein conjugate solution is carried out to 5 equal-volume ultrafiltration, then 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, mix after 4 hours ammonium sulfate to 1.5mol/L, with 1.5M ammonium sulfate+50mM phosphate buffer (pH7.5) balance hydrophobic chromatography post, (filler is Fractogel Propyl (S), Merck), after loading, wash 5 column volumes with above-mentioned buffer again, final with 50mM phosphate buffer eluting and collect polysaccharide-protein pair carrier conjugates.In the ultrafiltration system of equipment 100KDa filter membrane, with normal saline, polysaccharide-protein conjugate solution is carried out to 15 equal-volume ultrafiltration, collect and reflux and use the filtration sterilization of capsule formula filter, be stored in 4 DEG C.
Be the molecular weight distribution situation that SEC-MALLS method detects GL-PP conjugate by CL-4B; Use different antibody serums to determine albumen and the polysaccharide kind in GL-PP conjugate by the two methods that expand of immunity; Detect the polyoses content of GL-PP conjugate by anthrone method; Lowry method protein content detects the total protein content of GL-PP conjugate, then GL-PP by calculating conjugate is in conjunction with than (Ratio); CRM197, HiD protein concentration detect 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 solutions
The preparation of 3 one kind of 13 valency pneumococal polysaccharide albumen conjugate vaccines of embodiment
Prepare 4,6B according to known method in background technology, 9V, 14,18C, 19F, single carrier conjugates such as 23F, wherein carrier protein is CRM197.The method of describing according to embodiment 1 or 2, preparation 1,3,5,6A, 7F, two carrier conjugates of 19A type polysaccharide, wherein 1,5, it is Second support that 7F type polysaccharide adopts PspA, 3,6A, 19A employing HiD is Second support.Various combination products are carried out to vaccine preparation according to following table 3.These unit price components are after mixing, and adding ultimate density is the aluminum phosphate adjuvant of 0.5mg/L.
Final content in the carrier protein type of the various antigens of table 3 and preparation
The evaluating drug effect of 4 one kind of 13 valency pneumococal polysaccharide albumen conjugate vaccines of embodiment, and with the comparison of existing single carrier combined vaccine
12-14g BALB/c. in female 3 week age children Mus, is divided into 4 groups at random, and 10 every group, the combined vaccine that lumbar injection is prepared according to embodiment 3 respectively, 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, and HiD albumen 3.1ug, PspA albumen 4.2ug, it is the aluminum phosphate adjuvant of 0.5mg/L that these albumen add ultimate density after mixing.
In program injection in the 0th, 2,4 weeks 3 times, every young Mus 0.5ml is containing (0.5ug conjugate).Each experimental group is the blood samplings in 7 days after the 3rd pin injection respectively, and separation of serum, puts-20 DEG C and save backup.
Adopt respectively indirect elisa method for capsular polysaccharide and the detection of carrier protein antibody titer, by various pneumonia polysaccharide, carrier protein is dissolved in the carbonate buffer solution of 0.05mol/L pH9.6, and with 20ug/ml concentration coated elisa plate, 2% BSA fluid-tight is closed.When experiment by test serum 100,200,400,800,1600,3200,6400,12800,25600,51200, after 102400,204800,409600 times of dilutions, add ELISA Plate, put 37 DEG C of reaction 40min, after conventional washing, add the sheep anti mouse two of horseradish peroxidase-labeled anti-.Add the buffer that does not contain serum as negative control simultaneously.With the colour developing of tetra-amino-biphenyl amine, microplate reader 450nm wavelength place reads A value.Calculating A value and the worth ratio of negative hole A in every hole, is the antibody titer in serum but ratio is greater than the extension rate of 2.1 epoch maximums, the titre of 10 mices is carried out to geometric average, and calculate logarithm value taking 10 end of as, the results are shown in Table 4.
Two kinds of dosage form vaccines of table 4 and the comparison of carrier protein mixture mouse immune originality
Find out from data, taking (to add * mark, 1,3,5,6A, 7F, 19A) after two carriers, mice significantly strengthens the immunoreation of lung chain polysaccharide, and wherein 3 types are especially remarkable.In addition, with the protein mixture contrast of same dosage, two carrier conjugates have also shown that its carrier protein 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; can list several embodiment according to institute's limited range; therefore in the variation and the amendment that do not depart under general plotting of the present invention, within should belonging to protection scope of the present invention.
Claims (11)
1. a pneumococal polysaccharide albumen conjugate vaccines, it is characterized in that: be to comprise that one or more streptococcus pneumoniae capsular polysaccharides and protein are coupled the immunoconjugates of combination, in described immunoconjugates, have at least a kind of single streptococcus pneumoniae capsular polysaccharide of a kind of immunoconjugates and two or more protein to be coupled and form.
2. pneumococal polysaccharide albumen conjugate vaccines according to claim 1, it is characterized in that: the immune composition that comprises multivalent pneumococcal conjugate is to mix after capsular polysaccharide on separating-purifying various serotype streptococcus pneumoniae pod membrane is combined with protein coupling.
3. pneumococal polysaccharide albumen conjugate vaccines according to claim 1, it is characterized in that: described is diphtheria toxoid for the protein that is coupled combination with capsular polysaccharide, tetanus toxoid, carrier protein CRM197, bloodthirsty hemophilus influenza surface protein HiD, pertussis Prn surface protein, pertussis Fha antigen and/or pneumococcal surface protein PspA.
4. pneumococal polysaccharide albumen conjugate vaccines according to claim 1, is characterized in that: described for being coupled two or more protein of combination with capsular polysaccharide, wherein a kind of protein is the antigen with protectiveness.
5. according to the pneumococal polysaccharide albumen conjugate vaccines described in claim 1 or 4, it is characterized in that: described for being coupled two or more protein of combination with capsular polysaccharide, wherein one is bloodthirsty hemophilus influenza surface protein HiD or PspA albumen.
6. pneumococal polysaccharide albumen conjugate vaccines according to claim 1, is characterized in that: described streptococcus pneumoniae capsular polysaccharide is the capsular polysaccharide on separating-purifying serotype streptococcus pneumoniae pod membrane, and the pneumococcal serotype of described 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.
7. the preparation method of the pneumoprotein vaccine described in one of claim 1-6, is characterized in that: concrete steps are:
(1) capsular polysaccharide on difference separating-purifying various serotype streptococcus pneumoniae pod membrane;
(2) the selected carrier protein of difference separating-purifying;
(3) respectively various capsular polysaccharides are combined with carrier protein couplet, wherein, at least one capsular polysaccharide and two or more protein are coupled combination;
(4) various couplings combinations are mixed to form to vaccinogen liquid, wherein, at least one capsular polysaccharide and two or more protein are coupled combination.
8. the preparation method of pneumoprotein vaccine according to claim 7, is characterized in that: in described step (3), the coupling combination of capsular polysaccharide and carrier protein comprises:
(1) capsular polysaccharide uses CDAP and the coupling of triethylamine activation method in conjunction with carrier protein.This reaction is the coupled reaction of isourea key.In the time of high pH, the hydroxyl reaction on CDAP and polysaccharide residue radical, changes into cyanate, and the amino on carrier protein reacts rapidly with it, forms isourea key.The method is simple to operate, easily repeats; Or
(2) first polysaccharide being carried out to IO4 activation forms free aldehyde radical and is connected combination with the amino on carrier protein; Or
(3) with adipic acid two hydrazine acyls with carboxyl in acidic polysaccharose or be oxidized obtained carboxyl be connected in polysaccharide, and then be connected with albumen under the condition existing at EDAC; Or
(4) be combined with carrier protein couplet can also be by polysaccharide is carried out to fragment for capsular polysaccharide, is derivatively connected with amino or carboxyl on carrier protein after adding chain joint again.
9. the preparation method of pneumococal polysaccharide albumen conjugate vaccines according to claim 7, is characterized in that: the connected mode that in described step (3), a kind of capsular polysaccharide and two or more protein are coupled combination comprises:
(1) one-step reaction method, joins two kinds of carrier proteins and capsular polysaccharide in reaction vessel and reacts simultaneously, and this method is comparatively simple, but the ratio of different albumen be not easy accurate control in conjugate; Or
(2) sequential method, first capsular polysaccharide is connected with a kind of carrier protein wherein, then passes through purification, remove unconjugated floating preteins and free polysaccharide, add again the second carrier protein to connect, can produce the bonded products with stable bond ratio.
10. the preparation method of pneumoprotein vaccine according to claim 7, is characterized in that: concrete steps are:
(1) streptococcus pneumoniae of 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) the strong capsular polysaccharide of antigenicity in above various serotype streptococcus pneumoniae of purifying respectively: streptococcus pneumoniae, centrifugal collection supernatant after deactivation, through ultrafiltration and concentration, adding respectively volume fraction according to each streptococcus pneumoniae serotype characteristic is 70% pre-cooled ethanol, centrifugal collection, obtains rough polysaccharide; Rough polysaccharide is dissolved in sodium acetate solution, then mixes with cold phenol in 1:2 ratio, centrifugal removal 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 to stir, centrifugal removal nucleic acid, collect supernatant, add ethanol, final concentration 80% stirs, centrifugal collecting precipitation, by ethanol, washing with acetone precipitation, after dehydrate, be refining capsular polysaccharide, put-20 DEG C and save backup;
(3) produce CRM197, diphtheria toxoid, tetanus toxoid, pertussal various antigen, or by HiD, PspA albumen is cloned into escherichia coli and expresses also separation and purification;
(4) get and detect the qualified pneumococcal capsular polysaccharide of various serotypes, with the combinations simultaneously of then with two kinds of albumen of sodium metaperiodate activation method acquisition aldehyde radical, obtain target conjugate; Or will be wherein a kind of albumen and polysaccharide be first coupled, in the situation that not sealing active group, carry out purification, the purified product obtaining and the second albumen are further coupled and obtain target conjugate.
The application of pneumococal polysaccharide albumen conjugate vaccines described in one of 11. claim 1-5 aspect the medicine of the immunne response to streptococcus pneumoniae capsular polysaccharide conjugates and carrier protein for the preparation of induction.
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