CN101590224A - High-efficiency 14-valent pneumococcal conjugate vaccine - Google Patents
High-efficiency 14-valent pneumococcal conjugate vaccine Download PDFInfo
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- CN101590224A CN101590224A CNA2009100406599A CN200910040659A CN101590224A CN 101590224 A CN101590224 A CN 101590224A CN A2009100406599 A CNA2009100406599 A CN A2009100406599A CN 200910040659 A CN200910040659 A CN 200910040659A CN 101590224 A CN101590224 A CN 101590224A
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Abstract
High-efficiency 14-valent pneumococcal conjugate vaccine is capsular polysaccharide and the carrier protein couplet be combined into that extracts with 14 kinds of serotype streptococcus pneumoniae; Described 14 kinds of pneumococcal serotypes of serotype are 1,2,4,5,6A, 6B, 7F, 9N, 9V, 14,18C, 19A, 19F and 23F; Carrier protein is the nontoxic variant protein CRM of diphtherin
197, tetanus toxin albumen and hemophilus influenza D albumen.Compare with existing pneumococcal conjugated vaccine, advantage of the present invention is: (1) serotype is respectively (1,2,4,5,6A, 6B, 7F, 9N, 9V, 14,18C, 19A, 19F and 23F) 14 kinds of streptococcus pneumoniae, and the cross protection rate reaches more than 90%; (2) can produce effective protection to the old people who is subject to the pneumonia puzzlement, and be fit to child below 2 years old.
Description
Technical field:
The present invention relates to field of immunology, be specially the high-efficiency 14-valent pneumococcal conjugate vaccine of prevention pneumococcal infection.
Background technology:
Streptococcus pneumoniae (S.pneumoniae), as shown in Figure 1, the threat that the whole world is caused just increasingly sharpens.Because the diffusion of antibiotics resistance sexually transmitted disease, and pneumococcal pneumonia occurs after the influenza infection of being everlasting, and the probability that the streptococcus pneumoniae disease is shown effect during influenza further increases.The disease that is 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, and concrete statistics as shown in Figure 2.
Show have 1,600,000 people to die from this disease every year on average, according to World Health Organization's statistical data in 2005 comprising the child of 70-100 below 10,000 years old.Global streptococcus pneumoniae disease death distribution situation had been investigated as shown in Figure 3 by World Health Organization (WHO) in 2004.
According to statistics, 15-57 ten thousand routine pneumococcal pneumonias there is approximately every year, 2600-6200 example pneumococcal meningitis is arranged approximately, cause about 40,000 people's death both every year in the U.S..The pneumonia case fatality rate is about 10%, and more than 50 years old old man's case fatality rate up to 28%.The about 25-30% of the case fatality rate of pneumococcal septicemia.The bacillary otitis media of the U.S. has 50-67% to be caused by streptococcus pneumoniae, and the main harm infant generally is difficult to thorough healing, and the relapse rate height.There are 12.5 ten thousand people to cause pneumonia because of streptococcus pneumoniae, nearly 10000 people death every year in France.
World Health Organization (WHO) adds up most deaths and occurs in disadvantaged country, is the principal disease burden of streptococcus pneumoniae disease less than 2 years old child and old people in developed country.In 10 the highest countries of streptococcus pneumoniae disease burden, there are 5 to be positioned at the Asia, be respectively Bangladesh, China, India, Indonesia and Pakistan.The shared ratio in death of child below 2 years old is high especially.In the Europe and the U.S., streptococcus pneumoniae is the topmost cause of disease of the acquired adult's bacterial pneumonia of community.In these zones, the annual morbidity of aggressive streptococcus pneumoniae disease is 10-100 example/100,000 populations.
The case fatality rate of China's pneumonia is 16.4%, wherein more than 50 years old middle-aged and elderly people and infant below 1 years old respectively up to 28.6% and 22.0%.The carrying rate of China streptococcus pneumoniae in healthy children is higher, and statistics show that the carrying rate in the northern area healthy children is 24.2%, and southern area is 31.3%.And this disease is the major reason that causes death of child below 5 years old.Main cause is that baby's immune development is not perfect, baby's immunity a little less than.And the baby that the age is more little, immunity is weak more, the baby below 2 years old particularly, its immunity exists that troops' deficiency, basis are not firm, poor stability three big weakness.
Combined vaccine is present state-of-the-art vaccine technologies, adds protein carrier on specific antigen, can increase its immunogenicity.Protein carrier has T cell dependency characteristic, and the protein binding vaccine 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.The capsular polysaccharide combined vaccine adds protein carrier on polysaccharide, become T cell dependence antigen by T-independent antigen, increases its immunogenicity.The antibody that combined vaccine inoculation back produces in quality and quantitatively be generation vaccine 400-1000 doubly, as Fig. 4 and IgG part shown in Figure 5, the generation immunoprotection is wider stronger, guard time is more permanent, reaches efficient protection.
The nearly 90 kinds of serotypes of streptococcus pneumoniae (bacterial strain), several serotypes were followed successively by before the statistics of China showed the pneumococcal infection bacterial strain: 5,6,1,19,23,14,2,4 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 wherein, 6B 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 the easiest morbific pathogenic bacterias in native country; separate the polysaccharide on the purification streptococcus pneumoniae pod membrane; be mixed in proportion and make vaccine, play antipneumonic effect, protective rate is 50-70%.This means that two the general one-mans of people of the every inoculation of crowd more than 2 years old produce protection, and the peak age of onset of pneumonia is the 6-12 monthly age.
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, even do not produce immunoreation, and immunoreation strengthens with the growth at age; (2) antibody of generation low-affinity is mainly IgM; (3) only produce of short duration immunoreation, do not possess immunological memory and immune-enhancing effect when inoculating repeatedly; (4) be easy to generate immunologic tolerance; (5) common adjuvant is difficult for playing the effect of immunostimulant to this antigen.
Polysaccharide vaccine is inoperative to infant to be because the immune system of child below 2 years old is not physically well developed as yet, may be relevant with the hypoevolutism of IgG 2 class bone-marrow-derived lymphocyte strains, and bacterial polysaccharides can only stimulate the bone-marrow-derived lymphocyte of comparative maturity.Yet children Streptococcus is sent out age bracket well really below 2 one full year of life, and the peak age of onset is the 6-12 monthly age, and therefore infant below 2 years old can only use the vaccine that contains thymus dependent antigen.
Protein carrier can change the polysaccharide antigen of thymus dependent/non-dependent into thymus dependent antigen, and the t helper cell of excitating organism produces a series of immune-enhancing effect.2000, first the 7 valent pneumococcal conjugate vaccine listing of U.S. Wyeth; U.S. Wyeth exploitation infant is (4,6B, 9V, 14,18C, 19F and 23F) valency pneumoprotein vaccine more targetedly 7, and is also effective to child below 5 years old.Capsular polysaccharide protein binding vaccine adds protein carrier on polysaccharide, become T cell dependence antigen by T-independent antigen, can increase its immunogenicity, can be used for above child in 6 ages in week.
Serotype is followed successively by but the data of China shows the pneumococcal infection bacterial strain: 5,6,1,19,23,14,2,4, and Hui Shi 7 valent pneumococcal conjugate vaccines have only 33.9% to China's common pathogen type coverage rate.Hui Shi 7 valent pneumococcal conjugate vaccines need be inoculated 4 injections, and 860 yuan on every pin costs an arm and a leg, and are unfavorable for promoting.These results show that the antibacterial that causes the streptococcus pneumoniae disease constantly changes.The popular streptococcus pneumoniae bacterial strain of China is also followed U.S.'s difference to some extent, the nearly 90 kinds of serotypes of streptococcus pneumoniae (bacterial strain).Main popular pod membrane serotype is different along with age, time and region, and 7 valency combined vaccines of U.S. Wyeth not too are fit to the large-scale children Streptococcus prevention of China.
Summary of the invention
The objective of the invention is deficiency at the above Pnu-Imune 23 existence; a kind of take all factors into consideration domestic generally popular bacterial type, child and factors such as old man's susceptible bacterial type, drug resistance bacterial type and cross protection are provided; can cover China's overwhelming majority common pathogen type or drug resistance bacterial type; can produce effective protection to the old people who is subject to the pneumonia puzzlement; and be fit to child's below 2 years old; the coverage rate height, the high-efficiency 14-valent pneumococcal conjugate vaccine that price is low.
The present invention is achieved in that high-efficiency 14-valent pneumococcal conjugate vaccine, is to combine the back mixed in equal amounts with carrier protein couplet with the capsular polysaccharide on 14 kinds of serotype streptococcus pneumoniae pod membranes of separation purification to form; Described 14 kinds of pneumococcal serotypes of serotype are 1,2,4,5,6A, 6B, 7F, 9N, 9V, 14,18C, 19A, 19F and 23F; Carrier protein is selected the nontoxic variant protein CRM of diphtherin for use
197, tetanus toxin albumen and hemophilus influenza D albumen.
It is with 1-amino-4-dimethyl-pyridine-tetrafluoride boron (CDAP) and triethylamine activation that described capsular polysaccharide combines with carrier protein couplet, connects combination by the isourea key.
The bonded concrete steps of described capsular polysaccharide and carrier protein couplet are: in 10ml concentration is that to add with the dissolved concentration of 1ml acetonitrile respectively in 14 kinds of pneumococcal capsular polysaccharides of serotype of 10mg/ml be 100mg/ml 1-amino-4-dimethyl-pyridine-tetrafluoride boron (CDAP), adding 4ml concentration behind the 1min is the triethylamine activation of 0.2mol/L, 2.5min the back adds the 5g carrier protein, reaction is 1 hour under the room temperature, 4 ℃ were reacted 10～15 hours again, add the ethanolamine cessation reaction, again through Sepharose CL-4B chromatography media purification, with wavelength A
206And A
280Synchronous monitoring, the sodium chloride solution eluting, the eluent of collecting two wavelength fused peakss place is combined vaccine.
Described serotype 6A, 6B, 14,19A, 19F and 23F type separate the capsular polysaccharide of purifying and adopt 1-amino-4-dimethyl-pyridine-tetrafluoride boron and triethylamine activation, by isourea key coupling hemophilus influenza D albumen.
Described serotype 1,2 and 7F type separate the capsular polysaccharide of purifying and adopt 1-amino-4-dimethyl-pyridine-tetrafluoride boron and triethylamine activation, by isourea key coupling tetanus toxoid protein.
Described serotype 4,5,9N, 9V separate the capsular polysaccharide of purifying and adopt 1-amino-4-dimethyl-pyridine-tetrafluoride boron and triethylamine activation with the 18C type, by the nontoxic variant protein of isourea key coupling diphtheria toxin, diphtherotoxin.
The present invention is in view of a kind of 14 valent pneumococcal conjugate vaccines that can cover China overwhelming majority common pathogen type or drug resistance bacterial type of pneumonia to China's pneumonia bacterial strain occurred frequently characteristics development.Compare with existing pneumococcal conjugated vaccine, advantage of the present invention is: (1) serotype is respectively 14 kinds of (1,2,4,5,6A, 6B, 7F, 9N, 9V, 14,18C, 19A, 19F and 23F) streptococcus pneumoniae, and the cross protection rate reaches more than 90%; (2) can produce effective protection to the old people who is subject to the pneumonia puzzlement, and be fit to child below 2 years old.
Description of drawings:
Fig. 1 is pneumococcal sem photograph;
Fig. 2 is the whole world causal investigation of death of child below 5 years old datagram (showing among the figure that pneumonia is global child's No.1 killer) in 2004;
Fig. 3 is the regional scattergram of whole world child below 5 years old of World Health Organization's statistics in 2004 because of pneumonia death, and wherein the shared ratio in Southeast Asia is only second to Africa;
Fig. 4 is in the mouse experiment, and coupling has the PPS4-CRM of protein carrier
197With the antibody comparison diagram that the PPS4 immunity produces, (coupling has the PPS4-CRM of protein carrier among the figure
197Be about 1000 times of the antibody that produces of PPS4 immunity);
Fig. 5 is in mouse experiment, the IgG statistics comparison diagram that the immunity of capsular polysaccharide combined vaccine produced after 42 days.
In Fig. 6, the mouse experiment, the antibody concentration statistics comparison diagram that produces after Chengdu 23 valency vaccines, Hui Shi 7 valency combined vaccines and the immunity of high-efficiency 14-valent combined vaccine (wherein,
Chengdu 23 valency generation vaccines,
The secondary vaccine of Hui Shi 7 valencys and
High-efficiency 14-valent pneumococcal conjugate vaccine);
In Fig. 7, the mouse experiment, the antibody titer statistics comparison diagram that produces after Chengdu 23 valency vaccines and the immunity of high-efficiency 14-valent combined vaccine various dose (
A generation 23 valency vaccine 0.5ug,
Secondary 14 valency combined vaccine 0.5ug,
A generation 23 valency vaccine 1ug and
Secondary 14 valency combined vaccine 1ug);
In Fig. 8, the mouse experiment, the IgG/IgM ratio statistics comparison diagram that produces after Chengdu 23 valency vaccines, Hui Shi 7 valency combined vaccines and the immunity of high-efficiency 14-valent combined vaccine (wherein:
Chengdu 23 valency generation vaccines,
The secondary vaccine of Hui Shi 7 valencys and
High-efficiency 14-valent pneumococcal conjugate vaccine).
The specific embodiment
Below specifically the present invention will be described in detail in conjunction with the embodiments.
High-efficiency 14-valent pneumococcal conjugate vaccine is to combine the back mixed in equal amounts with carrier protein couplet with the capsular polysaccharide on 14 kinds of serotype streptococcus pneumoniae pod membranes of separation purification to form.14 kinds of serotype streptococcus pneumoniae choose that China domestic modal serotype is 1,2,4,5, the pulmonitis strain of 6A, 6B, 7F, 9N, 9V, 14,18C, 19A, 19F and 23F.Carrier protein is chosen the nontoxic variant protein CRM of international endorsement protein carrier diphtherin
197, tetanus toxin albumen and hemophilus influenza D albumen.The nontoxic variant protein CRM of diphtherin
197, tetanus toxin albumen and hemophilus influenza D albumen combines with capsular polysaccharide, can improve immunogenicity of antigens, thereby start the immunologic mechanism of infant, protein carrier has the effect of activating immune cell simultaneously, 6 ages in week, above baby just can inoculate, produce antibody early, the prevention pneumococcal infection.
Capsular polysaccharide and the coupling of carrier protein are combined into respectively the capsular polysaccharide that will extract on the pneumococcal pod membrane of 14 kinds of serotypes (1,2,4,5,6A, 6B, 7F, 9N, 9V, 14,18C, 19A, 19F and 23F) and combine carrier protein (the nontoxic variant protein CRM of diphtherin with 1-amino-4-dimethyl-pyridine-tetrafluoride boron (CDAP) and the coupling of triethylamine activation method
197, tetanus toxin albumen and hemophilus influenza D albumen).This reaction is the coupled reaction of isourea key.When high pH, the hydroxyl reaction on CDAP and the polysaccharide residue radical changes into cyanate, and the reaction rapid with it of the amino on the carrier protein forms the isourea key.This method is simple to operate, repeats easily.Concrete steps are as follows: adding with the dissolved concentration of 1ml acetonitrile respectively in detecting the capsular polysaccharide of above-mentioned 14 kinds of serotype streptococcus pneumoniae that qualified 10ml concentration is 10mg/ml is 100mg/ml CDAP, adding 4ml concentration behind the 1min is the triethylamine activation of 0.2mol/L, 2.5min the back adds the 5g carrier protein, reaction is 1 hour under the room temperature, 4 ℃ were reacted 10～15 hours again, add the ethanolamine cessation reaction, again through SepharoseCL-4B chromatography media purification, with wavelength A
206And A
280Synchronous monitoring, the sodium chloride solution eluting, the eluent of collecting two wavelength fused peakss place is combined vaccine.
The concrete manufacture method of high-efficiency 14-valent pneumococcal conjugate vaccine is as follows:
(1) streptococcus pneumoniae of choosing 14 kinds of serotypes (1,2,4,5,6A, 6B, 7F, 9N, 9V, 14,18C, 19A, 19F and 23F) is cultivated.
(2) the strong capsular polysaccharide of antigenicity in above 14 kinds of serotype streptococcus pneumoniae of purifying respectively: centrifugal collection supernatant after the streptococcus pneumoniae deactivation, through ultrafiltration and concentration, add in right amount (volume fraction be 70%) pre-cooled ethanol respectively according to each streptococcus pneumoniae serotype characteristic, centrifugal collection gets rough polysaccharide; Rough polysaccharide is dissolved in the sodium acetate solution, then in 1: 2 ratio with cold phenol mixing, centrifugal removal albumen, phenol is carried 5-6 time repeatedly, collects supernatant, uses distill water dialysis, dialysis back liquid adds the 2mol/L calcium chloride solution, adds ethanol and stirs centrifugal removal nucleic acid, collect supernatant, add ethanol (final concentration 80% stirs), centrifugal collecting precipitation is with ethanol, washing with acetone precipitation, be refining capsular polysaccharide behind the dehydrate, it is standby to put-20 ℃ of preservations.
(3) get the capsular polysaccharide that detects qualified various streptococcus pneumoniae, obtain combined vaccine in conjunction with carrier protein with 1-amino-4-dimethyl-pyridine-tetrafluoride boron (CDAP) and triethylamine activation method.
The pneumococcal cultivation of embodiment 1:14 kind serotype
Take all factors into consideration domestic generally popular bacterial type, child and factors such as old man's susceptible bacterial type, drug resistance bacterial type and cross protection, choose the streptococcus pneumoniae of 14 kinds of serotypes (1,2,4,5,6A, 6B, 7F, 9N, 9V, 14,18C, 19A, 19F and 23F) and cultivate;
1,2,4,5,6A, 6B, 7F, 9N, 9V, 14,18C, 19A, 19F and 23F amount to 14 serum type strains (1) strain:, can be available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute (Chinese medicine antibacterial preservation administrative center), deposit number is respectively 31401,31402,31404,31405,31406,31409,31414,31419,31423,31426,31451,31456,31457 and 31468.Can adopt fluorescently-labeled method to differentiate earlier, adopt earlier from special anti-the hatching of the serotype of rabbit, adopt the goat anti-rabbit igg (two is anti-) of Rhodamine Red-X labelling to hatch then, normal result is the spherical fluorescence of chain of red color visible.
(2) culture medium: solid medium is the plain agar culture medium that contains 10% defiber Sanguis caprae seu ovis; Fluid medium PN96 contains tryptone, aminoacid, glucose, vitamin, catalase and plurality of inorganic salt ion.Above culture medium can be available from Beijing bispin microbiological culture media products factory.
(3) cultivate in the streptococcus pneumoniae fermentation tank: the freeze-drying lactobacillus of breakdown (1,2,4,5,6A, 6B, 7F, 9N, 9V, 14,18C, 19A, 19F and 23F) serotype streptococcus pneumoniae is inoculated in 10% defiber Sanguis caprae seu ovis plain agar culture medium respectively, at CO
2After 37 ℃ of cultivations, with the static cultivation of solid culture (Nat'l Pharmaceutical ﹠ Biological Products Control Institute's 109 culture medium) inoculation two generation fluid mediums (N96 of Nat'l Pharmaceutical ﹠ Biological Products Control Institute culture medium), dyeing microscopic examination is blue " positive " diplococcus of leather in the environment.In same bacterial strain, amount by the expressed capsular polysaccharide of streptococcus pneumoniae is also different, phenotype can take place between at least two kinds of forms most of streptococcus pneumoniae separators changes, these two kinds of forms can be distinguished by the opacity of bacterium colony, transparent (T) variant of opaque type (O) bacterium colony and identical bacterial strain difference aspect the amount of synthetic pod membrane, (O) bacterium colony of type can produce a large amount of CPS usually.After selecting the bacterium colony of smooth transparent (O) type of pod membrane to carry out biochemical reaction, carry out the serum coagulation.After serum coagulation experiment, the seed lot strain carries out solid medium and goes down to posterity and examine and determine.Judge qualified after, be inoculated on the synthetic medium of improvement.
(4) by inoculum concentration about 10
8CFU/ml culture medium meter, bacterium liquid is inoculated in the fermentation tank that contains the 45L fluid medium in 37 ℃ and carries out stir culture, after cultivating 2h, every the 1h sampling once, measure bacterial concentration, regulate sugared concentration with 50% glucose solution, regulate pH to 7.5 with 7.5mol/L NaOH.Intermittently feed compressed air 7～9h and stop cultivating, when antibacterial culturing to OD more than or equal to 1.5 the time, stop to continue to cultivate, centrifugal with the formaldehyde sterilization, remove thalline, collect supernatant.Bacterial concentration is measured: adopt spectrophotometer to survey the OD value under the 600nm wavelength.
Embodiment 2: purification pneumococcal capsular polysaccharide and Quality Identification thereof
1. the purification of pneumococcal capsular polysaccharide:
(1) the bacterium liquid after the sterilization carries out centrifugal removal thalline with centrifuge, obtains stock solution-slightly poly-polysaccharide, and the thalline that centrifugalize is come out sterilizes with formaldehyde.(2) obtain stock solution and assemble the thick liquid of polysaccharide after, add cetyl trimethyl ammonium bromide and precipitate complex polysaccharide work, and precipitate centrifugal gathering polysaccharide.(3) finish the centrifugal collection complex polysaccharide of precipitation after, carry out the calcium chloride purification that dissociates.Subsequently, adding concentration is that 95% ethanol carries out enucleation acid.Afterwards, continuing to add concentration is that 95% ethanol precipitates polysaccharide.(4) finish precipitation and receive polysaccharide after, add phenol and carry out phenol and discard protein, remove unnecessary protein ingredient in the stock solution polysaccharide antigen.(5) finish phenol and discard albumen after, utilize high speed centrifuge to carry out ultracentrifugation and remove endotoxin.Afterwards, carry out reuse ethanol and precipitate polysaccharide and polysaccharide purification, finish refining polysaccharide operation and handle.(6) through after the above operation processing, obtain the polysaccharide stock solution of purification refine.To this moment, polysaccharide stock solution is collected work of treatment and is finished, and only need carry out vacuum drying removal part moisture and aseptic filtration again and handle.
2. capsular polysaccharide Quality Identification:
(1) biochemistry detection: press the refining polysaccharide of 6mg/ml dilution with distilled water respectively, the 0.22um membrane filtration is submitted sample.Protein content is measured with the Lorry method; the nucleic acid content spectrophotometry; total nitrogen is measured with Kjeldahl; phosphorus content is measured with the ammonium molybdate method; the O-acetyl group is measured with alkaline oxammonium hydrochloride .-tri-chlorination iron processes, and glucuronic acid content is measured with carbazole-alcoholic solution method, aminohexose content diaminobenzene formaldehyde determination of color; methylpentose content cysteine determination of color, molecular size is used the SDS-polyacrylamide gel and is filtered.
(2) identification experiment and serological specificity experiment: adopt the paired antibodies ELISA sandwich assay of various polysaccharide to differentiate various polysaccharide antigen.
(3) toxicity test: with 5 about 350g Cavia porcelluss of body weight, every abdominal cavity injects 3.45mg (2.5ml) unit price polysaccharide, observes must not cause tangible symptom and death in 7 days; Every abdominal cavity of 5 about 20g white mice of body weight injects 0.69mg (0.5ml) unit price polysaccharide, observes 7d and must not cause that tangible symptom and death are qualified products.
Embodiment 3: carrier protein (the bloodthirsty D albumen of influenza, tetanus toxoid protein and the nontoxic variant protein CRM of diphtheria toxin, diphtherotoxin
197) quality control
Adopt the bloodthirsty D albumen of influenza safely and effectively, tetanus toxoid protein and the nontoxic variant protein CRM of diphtheria toxin, diphtherotoxin that have verified by clinical experiment
197As the protein carrier of our combined vaccine,, its characteristic and purity are carried out quality check: adopt the HPLC method to measure the bloodthirsty D albumen of influenza, tetanus toxoid protein and the nontoxic variant protein CRM of diphtheria toxin, diphtherotoxin with reference to existing internationally recognized quality standard
197Purity, purity is being qualified more than 90%; Guarantee that each proteic aminoacid sequence is correct; Guarantee not with diphtheria toxoid in same Workshop Production; Adopt the albumen composition after the SDS-PAGE atlas analysis is measured purification; The content of lipopolysaccharide is up-to-standard less than 8%; The rabbit pyrogen test of carrier protein must be qualified.
Embodiment 4: capsular polysaccharide combines with carrier protein couplet.
Multiple factor affecting the immunogenicity of combined vaccine in zoopery, as the content of free polysaccharide in the selection of protein carrier, the conjugate, in conjunction with factors such as the concentration of the size of preceding polysaccharide molecule, combined vaccine and the glycoprotein in the combined vaccine compare.The present invention selects suitable protein carrier and combined process to mate various pneumococcal polysaccharide according to the characteristic of pneumococcal capsular polysaccharide structure and protein carrier, and is specific as follows:
One, capsular polysaccharide 6A, 6B, 14,19A, 19F and 23F type adopt 1-amino-4-dimethyl-pyridine-tetrafluoride boron and the coupling of triethylamine activation method in conjunction with hemophilus influenza D albumen
(1) gets 6A, 6B, 14,19A, 19F and the 23F type streptococcus pneumoniae capsular polysaccharide 10ml that detects qualified 10mg/ml respectively.
(2) adding with the dissolved concentration of 1ml acetonitrile for 6A, 6B is the CDAP of 100mg/ml, adding 4ml concentration behind the 1min is the triethylamine activation of 0.2mol/L, 2.5min the back adds 5g hemophilus influenza D albumen, reaction is 1 hour under the room temperature, 4 ℃ were reacted 10～15 hours again, add the ethanolamine cessation reaction, again through Sepharose CL-4B chromatography media purification, with wavelength A
206And A
280Synchronous monitoring, the sodium chloride solution eluting, the eluent of collecting two wavelength fused peakss place is standby as combined vaccine.
(3) adding with the dissolved concentration of 0.3ml acetonitrile for 14 type capsular polysaccharides is 100mg/ml CDAP, adding 0.3ml concentration behind the 1min is the triethylamine activation of 0.2mol/L, 2.5min the back adds 5g hemophilus influenza D albumen, reaction is 1 hour under the room temperature, 4 ℃ were reacted 10～15 hours again, add the ethanolamine cessation reaction, again through Sepharose CL-4B chromatography media purification, with wavelength A
206And A
280Synchronous monitoring, the sodium chloride solution eluting, the eluent of collecting two wavelength fused peakss place is standby as combined vaccine.
(4) be the CDAP of 100mg/ml for 19A, 19F and the adding of 23F type capsular polysaccharide with the dissolved concentration of 0.4ml acetonitrile, adding 0.8ml concentration behind the 1min is the triethylamine activation of 0.2mol/L, 2.5min the back adds 5g hemophilus influenza D albumen, reaction is 1 hour under the room temperature, 4 ℃ were reacted 10～15 hours again, add the ethanolamine cessation reaction, again through Sepharose CL-4B chromatography media purification, with wavelength A
206And A
280Synchronous monitoring, the sodium chloride solution eluting, the eluent of collecting two wavelength fused peakss place is standby as combined vaccine.
Two, serotype 1,2 and 7F polysaccharide adopt 1-amino-4-dimethyl-pyridine-tetrafluoride boron and the coupling of triethylamine activation method in conjunction with tetanus toxoid protein:
For 1,2 and 7F type capsular polysaccharide to add with the dissolved concentration of 0.4ml acetonitrile be the CDAP of 100mg/ml, adding 0.8ml concentration behind the 1min is the triethylamine activation of 0.2mol/L, 2.5min the back adds 5g stream tetanus toxoid protein, reaction is 1 hour under the room temperature, 4 ℃ were reacted 10～15 hours again, add the ethanolamine cessation reaction, again through Sepharose CL-4B chromatography media purification, with wavelength A
206And A
280Synchronous monitoring, the sodium chloride solution eluting, the eluent of collecting two wavelength fused peakss place is standby as combined vaccine.
Three, capsular polysaccharide 4,5,9N, 9V and 18C type adopt 1-amino-4-dimethyl-pyridine-tetrafluoride boron and triethylamine activation method, and coupling is in conjunction with the nontoxic variant protein of diphtheria toxin, diphtherotoxin
For 4,5,9N, 9V and 18C type capsular polysaccharide add with the dissolved concentration of 0.4ml acetonitrile is the CDAP of 100mg/ml, adding 0.8ml concentration behind the 1min is the triethylamine activation of 0.2mol/L, 2.5min the back adds the nontoxic variant protein of 5g diphtheria toxin, diphtherotoxin, reaction is 1 hour under the room temperature, 4 ℃ were reacted 10～15 hours again, add the ethanolamine cessation reaction, again through Sepharose CL-4B chromatography media purification, with wavelength A
206And A
280Synchronous monitoring, the sodium chloride solution eluting, the eluent of collecting two wavelength fused peakss place is standby as combined vaccine.
The concentration of above sodium chloride solution can be %0.09, with wavelength A
206And A
280The instrument of synchronous monitoring can be spectrophotometer.
Embodiment 5: immune-affinity chromatography purification conjugate (capsular polysaccharide-hemophilus influenza D albumen, capsular polysaccharide-tetanus toxoid and the nontoxic variant protein of capsular polysaccharide-diphtheria toxin, diphtherotoxin)
MONOCLONAL ANTIBODIES SPECIFIC FOR
At first get capsular polysaccharide-hemophilus influenza D albumen, capsular polysaccharide-tetanus toxoid and the nontoxic variant protein of capsular polysaccharide-diphtheria toxin, diphtherotoxin as antigen, immune mouse, then get mice spleen and the myeloma cell is merged, the screening fused cell, carrying out antibody identifies, assesses, by ascites production, antibody purification is carried out to product and identifies that the back is standby.
The agarose activation
(1) the Sepharose 4B that gets 20ml is placed in the buchner funnel and drains, and adding a spot of concentration is that 0.1mol/L, pH value are 9.0 NaHCO
3The liquid washing changes in the 100ml beaker immediately, and ice bath places on the magnetic stirring apparatus.(2) in fume hood, take by weighing the 2g Bromine cyanide., add water 20ml dissolving, pour into then in the agarose, carefully drip 2mol/L NaOH, pH is remained on about 11, reaction 10min.Adjusting pH value rapidly at 1～2min is 8.0～11.0 to keep 10min.(3) activatory agarose being poured into rapidly in the buchner funnel, taken out with frozen water and wash neutrality, is that 0.1mol/L, pH value are 9.0 NaHCO with the cold concentration of 250ml rapidly again
3Solution is taken out and is washed.
The coupling monoclonal antibody
20ml Sepharose 4B liquid is equivalent to the solid phase carrier of half.(1) will need in advance link coupled antibody (capsular polysaccharide-hemophilus influenza D protein antibodies, capsular polysaccharide-tetanus toxoid protein antibody and the nontoxic variant protein antibody of capsular polysaccharide-diphtheria toxin, diphtherotoxin) respectively 200mg place 0.1mol/L pH 9.0 NaHCO
3Dialyse a few hours in the liquid (general coupling amount is 10～30mg/g carrier).(2) activatory agarose is poured into rapidly in the antibody liquid and (in 1.5min, washed coupling from taking out), 4 ℃ were slowly stirred 12 hours, antibody are combined with activatory agar.
The dress post
(1) select post: should not be excessive, generally can adorn the about 30ml of agarose of coupling antibody with the chromatographic column of 1.5 * 15cm.(2) dress post: will be pack in the chromatographic column, tighten the end opening folder, allow its sinking, unclamp the end opening folder after several minutes, solution is flowed out with the speed of 1ml/min approximately with the agarose of antibody coupling.(3) wash post: with 0.2mol/L pH9.0 NaHCO
3(containing 0.1mol/L NaCl) washs the OD to eluate
280Till＜0.02.Collect whole eluents, the OD that records
280Total milliliter of number with eluent on duty is the not content of coupling protein, can calculate the coupling rate thus.
Adsorption and desorption is attached
(1) add the various polysaccharide to be purified and the conjugate 3ml of carrier protein, concentration is 1～2%, slowly adds antigen to be purified, is 7.4 PBS liquid eluting with concentration 0.01mol/L, pH value, and flow velocity is 1ml/min, to eluate OD
280Till＜0.02.(2) adding concentration is that 0.1mol/L, pH value are glycine-HCl buffer of 2.4, and flow velocity 1ml/min collects the composition that desorption is got off, immediately with 1mol/L NaHCO
3Neutralization is in order to avoid the antibody degeneration.(3) with the 7mol/L carbamide washing of two volumes, reuse concentration is that 0.01mol/L, pH value are 7.4 PB liquid or normal saline washing, after the balance, can continue to use.It is 0.02%NaN that preservation can add concentration
3In 4 ℃ of preservations.Prevent freezing and dry and cracked.
Embodiment 6: the capsular polysaccharide combined vaccine is identified
(1) anthrone method is measured polyoses content.
Sugar can generate furfural or Hydroxymethylfurfural through dehydration under the concentrated sulphuric acid effect, the furfural of generation or Hydroxymethylfurfural can generate the aeruginous furfural derivatives with anthrone reaction, and within the specific limits, the depth of color is directly proportional with the content of sugar.The coloring matter that saccharide and anthrone reaction generate is 630nm at the absworption peak of visible region, carries out colorimetric under this wavelength.Content by production standard curve calculation soluble sugar.
(2) the Lowry method is measured protein content.
Protein is its peptide bond and Cu in alkaline solution
2+Chelating, form protein-copper composition, this complex makes the phosphomolybdic acid reduction of phenol reagent, produces blue chemical compound, under the absworption peak wavelength, carry out colorimetric, utilize the linear relationship of absorbance value and protein concentration to make proteinic concentration in standard curve and the working sample.
(3) the SDS-polyacrylamide gel electrophoresis is measured the polysaccharide conjugate vaccine molecular weight.
SDS is a kind of anion surfactant, and under certain conditions, it can open protein hydrogen bonds and hydrophobic bond, and is attached to the electronegative protein-SDS complex of formation on these protein molecules pari passu, and every gram protein is generally in conjunction with 1.4g SDS.SDS makes protein-SDS complex all with going up identical negative charge with proteinic definite proportion combination, and it is measured considerably beyond the original quantity of electric charge of protein, thereby has covered original charge differences between protein.In aqueous solution, protein-SDS complex has identical conformation, and the approximate ellipse garden of cigar-shaped length rod (minor axis is 1.8nm, and major axis is then with the variation that is directly proportional of proteinic molecular weight) has overcome original shape difference between protein.Protein-the mobility of SDS complex in gel no longer is subjected to the influence of original electric charge and shape like this, and is the function of protein molecular weight.Measuring proteinic molecular weight with this law only needs just can learn molecular weight according to the relative position of testing protein at the standard protein of known molecular amount.The discontinuous vertical electrophoresis method of SDS-is adopted in this experiment: after 1) gel took off most background color, colour band is clear to be showed.Describe or photograph electrophoresis pattern by full pattern; 2) according to the ratio of each albumen migration distance and dye migration distance, obtain each proteic relative mobility (mr), make the lgMr-mr graph of a relation then, from figure, find out agnoprotein relative mobility corresponding molecular weight.
The quality control of embodiment 7:14 kind polysaccharide-protein conjugate stock solution
Polysaccharide and carrier protein combined process reactions steps are more, the conditional request height.In order to ensure the stability of polysaccharide-protein conjugate, safety and batch between concordance, set up following corresponding method of quality control.Because association reaction may influence the structure of polysaccharide, polysaccharide-protein carries out the identification experiment of polysaccharide in conjunction with back (before the unit price conjugate is unmixed).In addition, polysaccharide-protein conjugate behind the purification is carried out following detection: 1. adopt suitable detection method, guarantee polysaccharide-protein conjugate stock solution through behind the purification, employed reagent in the polysaccharide-protein association reaction (chemical reagent such as CDAP or triethylamine) has been eliminated; 2. measure respectively in the conjugate in conjunction with polysaccharide and protein-bonded content, liken to judging an indirect indicator of association reaction by polysaccharide/albumen in the conjugate; 3. after activation stopped, guaranteeing in the conjugate that polysaccharide or carrier protein are no longer residual had a unconjugated functional group in activation back; 4. adopt the method for gel filtration to separate with free polysaccharide, guarantee that not combined dissociation amylase content is no more than 5% in conjunction with polysaccharide; 5. adopt the method for mass spectrum (HPLC) to measure conjugated protein and not protein-bonded content in the polysaccharide-protein conjugate; Guarantee that not combined carrier protein content is no more than 5%; 6. adopt SDS-polyacrylamide gel vertical electrophoresis each batch polysaccharide conjugate vaccine stock solution to be carried out the mensuration of average molecular size; 7. " requirement of Chinese pharmacopoeia is carried out pyrogen test and endotoxin content mensuration to polysaccharide conjugate vaccine stock solution, guarantees that the toxicity test of vaccine is qualified by current edition.
Embodiment 8: the initial stage simulation experiment of single kind polysaccharide conjugated antigen in mouse model
Behind 14 kinds of polysaccharide conjugated antigen difference immunity inoculation children Mus, antibody component, concentration, affinity, the phagocytic activity that produces compared, thereby determine suitable immunizing dose.
(1) polysaccharide conjugate vaccine immunogenicity experiments:
The young Mus of female 3 BALB/c in age in week of 12-14g is divided into two groups at random, and 20 every group, lumbar injection different sugar albumen is injected 2 times in the 0th week and the 4th all programs than combined vaccine respectively, and every young Mus 0.5ml contains (1ug conjugate).Each experimental group 14 days before first pin injection and after the injection of second pin is respectively got 10 young Mus and is taken a blood sample, separation of serum, and it is standby to put-20 ℃ of preservations.
(2) the various capsular polysaccharide specificity of mice serum:
Indirect elisa method is adopted in the IgG antibody test, and various pneumonia polysaccharide is dissolved in the carbonate buffer solution of 0.05mol/L pH 9.6, and with 20ug/ml concentration coated elisa plate, 2% BSA fluid-tight is closed.To add ELISA Plate after 20 times of dilutions of serum to be checked during experiment, put 37 ℃ of reaction 40min, the sheep anti mouse two of conventional washing back adding horseradish peroxidase-labeled is anti-.With the colour developing of tetramino benzidine, microplate reader 450nm wavelength place reads the A value.
(3) evaluation of humoral immunoresponse(HI):
Carry out the evaluation of humoral immunoresponse(HI) after the immunity inoculation, thus the concentration of the quantitative anti-PPS immunoglobulin of serum.For example: in the PPS4 evaluation experimental, detect IgG concentration with indirect elisa method.Collect the concentration of anti-PPS antibody in each experimental group serum, arithmetic average and standard deviation in the confidence interval of calculating 95%.To each immunity inoculation group, antibody horizontal (after the data transaction logarithmic form) is compared after antibody horizontal and the immunity inoculation before immunity, to eliminate the difference between the mice individuality.With the difference between the more paired different immunity inoculation groups of standard t check, the Excel of Microsoft form software is used for editing data and statistical analysis.
In order to detect and estimate the antibody secreting cell of inducing generation after the immunity inoculation in the spleen, before the 1st dose of immunity inoculation and two time points results spleens behind the 2nd dose of immunity inoculation first quarter moon.Isolated lymphocytes is with ELISpot assay vaccine.
Embodiment 9: the initial stage simulation experiment of mixing polysaccharide conjugated antigen in mouse model
14 kinds of polysaccharide conjugate vaccine mixed in equal amounts, each injection contain mixed polysaccharide conjugate vaccine 2ug.Behind the immunized mice, the antibody component, concentration, affinity and the opsonophagocytosis ability that produce are analyzed.Thereby determine the pneumococcal conjugated vaccine component of best results.High-efficiency 14-valent pneumococcal Polysaccharide Conjugate Vaccine immune effect in mouse experiment is better than the 7 valency polysaccharide conjugate vaccines of Hui Shi and the 23 valency polysaccharide vaccines in Chengdu, as sees Fig. 6, Fig. 7 and shown in Figure 8.
The formulating of vaccine: the prescription to vaccine is studied, and the stabilizer element of guaranteeing to add in the vaccine, buffer, adjuvant etc. are far longer than negative effect to vaccine in the positive influences aspect stability, storage life, immunogenicity and the safety.
Pharmacology, toxicity and bio distribution: carry out the pharmacological experiment of pneumococcal conjugated vaccine, comprise relation, immune programme for children and the route of inoculation of action principle, biological value and dosage that vaccine takes place and the experiments such as relation of effect; Set up suitable experiment and the detection method of a cover and estimate vaccine, optimize immune programme for children and route of inoculation by estimating.
Embodiment 10: serotype is the capsular polysaccharide combined vaccine of 4 types
Be the streptococcus pneumoniae of 4 types with serotype earlier, the single culture amplification; Purify polysaccharide on its pod membrane, centrifugal collection supernatant after the 4 type streptococcus pneumoniae deactivations through ultrafiltration and concentration, adds an amount of pre-cooled ethanol (70%), centrifugal collection, rough polysaccharide; Raw sugar is dissolved in the sodium acetate solution with suitable concentration, then in 1: 2 ratio with cold phenol mixing, centrifugal removal albumen, phenol is carried 5-6 time repeatedly, collects supernatant, uses distill water dialysis, dialysis back liquid aliquot adds the 2mol/L calcium chloride solution, adds an amount of ethanol and stirs centrifugal removal nucleic acid, collect supernatant, add ethanol (final concentration 80% stirs), centrifugal collecting precipitation is with ethanol, washing with acetone precipitation, be refining polysaccharide behind the dehydrate, it is standby to put-20 ℃ of preservations.
Adopt the external nontoxic variant protein of diphtheria toxin, diphtherotoxin safely and effectively (CRM197) by the clinical experiment checking,, its characteristic and purity are carried out quality check with reference to external corresponding quality standard; (10mg/ml 10ml), adopts CDAP (1-amino-4-dimethyl-pyridine-tetrafluoride boron) and triethylamine activation method in conjunction with carrier protein (the nontoxic variant protein CRM of diphtherin to get 4 qualified type streptococcus pneumoniae capsular polysaccharides of detection
197).At first add with the dissolved 100mg/ml CDAP of 1ml acetonitrile, the triethylamine activation that adds 4ml 0.2mol/L behind the 1min, 2.5min the back adds the 5g carrier protein, reaction is 1 hour under the room temperature, 4 ℃ were reacted 12 hours again, added 1.5ml 0.5mol/L, with the dissolved ethanolamine cessation reaction of pH7.5 0.75mol/L HEPES, again through Sepharose CL-4B chromatography media purification, with wavelength A
206And A
280Synchronous monitoring, the sodium chloride solution eluting, the eluent of collecting two wavelength fused peakss place is combined vaccine.
1. mice and immunity inoculation: the female BALB/c mouse of 10-11 weeks (Guangdong Province's Experimental Animal Center), subcutaneous vaccination 1ug PPS4, PPS4-CRM
197(being dissolved among the aseptic no endotoxic PBS of 200ul).Antigen same after 28 days carries out the immunity inoculation second time, and secondary inoculation is heart puncturing extracting blood after 14 days.
2. indirect ELISA is measured the anti-PPS4 antibody concentration: PPS4-IgM and the special indirect elisa method of PPS4-IgG are used for measuring immune serum anti-PPS4 antibody concentration.The anti-PPS4 monoclonal antibody IgM 9.2 of purification and IgG44.2 are the standard control things in the analyzing and testing.In the experimental port of ELISA Plate, the purification PPS4 that every hole adds 100ul 10ug/ml (is dissolved in 0.1mol/L pH 9.6 NaHCO
3In the solution) the bag quilt, with the 200ul PBS incubated at room 1h sealing that contains 1%BSA and 0.1% sodium azide.Wash plate with the PBS that contains 0.1%Tween-20.Experimental port adds the 100ul sample of suitable dilution (100-1000 doubly), utilizes standard substance to do standard curve.It is anti-to wash the sheep anti mouse two that adds alkali phosphatase enzyme mark behind the plate, hatches 1h for 37 ℃.Wash plate at last, with the colour developing of Sigma-104 phosphatase substrate, the 405nm wavelength reads light absorption value on the microplate reader.By standard curve function calculation antibody concentration.The result shows that coupling has the PPS4-CRM of protein carrier
197The antibody that immunity produces is about 1000 times of PPS4 immunity, see Fig. 4 and Fig. 5 shown in the IgG part.
3. IgG purification: the blood serum sample of secondary immunity after 14 days, use PPS4-CRM
197The PPS4-IgG antibody concentration of immunity inoculation is about 77.3 ± 26.9ug/ml.With 1ml G albumen HiTrap affinity chromatographic column purified blood serum IgG.G albumen post eluting is collected the part that contains IgG, the enrichment of YM10 membrane filtration subsequently.The IgG of enrichment dissolves with PBS.
4. opsonophagocytosis experiment: flow cytometry is used to analyze the opsonophagocytosis situation of IgG, owing to be marked with the streptococcus pneumoniae of Fluorescein isothiocyanate fluorescent quenching can take place, bacterium surface fluorescence Alexa488 labelling in the experiment.Prepare serotype 4 streptococcus pneumoniae of height pod membrane bag quilt, making it be in the bacterial growth third phase is balance period.Bacterium surface is with EZ-Link SulfoNHS-LC-Biotin biotinylation, and gives a baby a bath on the third day after its birth time with PBS.Biotinylated streptococcus pneumoniae hatches the 1h deactivation for 60 ℃, washes once, and Alexa488 is hatched with fluorescein, wash once after, the HBSS of calcium ion, magnesium ion, 1%BSA is resuspended with containing ,-20 ℃ of stored frozen.Fluorescein Alexa488 is easy to just stop under the situation that adds the 4mg/ml trypan blue.The tool pod membrane integrity of labelling and antigenic PPS4 and PPS4-IgG 44.2 or PPS4-IgM 9.2 are hatched, and suitable two anti-in conjunction with what have phycoerythrin, the up flow type cell instrument detects, and observes on the perhaps rose-red mark fluorescent microscope.Be marked with the antibacterial of fluorescein Alexa488, developed with the Nikon-E600 fluorescence microscope and confirm the combination of anti-PPS4 antibody, perhaps confirm, and confirm that antibacterial keeps k antigen after hot deactivation by the Two Colour Fluorescence flow cytometry.
Mouse macrophage RAW264.7 cultivates the use of going down to posterity after three days with the DMEM culture medium culturing (from ATCC) that contains 10% hyclone.Cell labelling 7-aminoactinomycin D before engulfing the experiment use carries out the vigor evaluation, and vigor is being qualified more than 90%.Antibacterial with 5ul fluorescein Alexa488 conditioning labelling, combine from the mice PPS4-IgG44.2 monoclonal antibody of the IgG purification of immune mouse or 20ul purification with unlabelled antibacterial and 20ul and to do internal contrast, the HBSS that adding contains 1% BSA is to final volume 50ul, and the PPS4-IgG final concentration is 0.25～2.0ug/ml in two tests.Mixture in shaking bath 37 ℃ hatch 55min.Add RAW264.7 cell (50ul, cell density 5 * 10 in the mixture
6/ ml) 37 ℃ continue to hatch 30min.Wash twice at 4 ℃ of cells with the cold HBSS that contains 1% BSA then, 0.5ml PBS is resuspended.Each test adds the trypan blue of isopyknic 8mg/ml, mixes to hatch 15s, and the up flow type cell instrument detects immediately.The result shows the antibody activity titre that polysaccharide conjugate vaccine inoculation back produces, and is 10-100 times of polysaccharide vaccine.
Pneumococcal capsular polysaccharide combined vaccine for other 13 kinds of serotypes (1,2,5,6A, 6B, 7F, 9N, 9V, 14,18C, 19A, 19F and 23F) is made and effect detection such as embodiment 10 described embodiment basically identicals.
Claims (6)
1, high-efficiency 14-valent pneumococcal conjugate vaccine is characterized in that: be to combine the back mixed in equal amounts with carrier protein couplet with the capsular polysaccharide on 14 kinds of serotype streptococcus pneumoniae pod membranes of separation purification to form; Described 14 kinds of pneumococcal serotypes of serotype are 1,2,4,5,6A, 6B, 7F, 9N, 9V, 14,18C, 19A, 19F and 23F; Carrier protein is selected the nontoxic variant protein CRM of diphtherin for use
197, tetanus toxin albumen and hemophilus influenza D albumen.
2, shown in claim 1, high-efficiency 14-valent pneumococcal conjugate vaccine is characterized in that: it is with 1-amino-4-dimethyl-pyridine-tetrafluoride boron and triethylamine activation that described capsular polysaccharide combines with carrier protein couplet, connects combination by the isourea key.
3, shown in claim 2, high-efficiency 14-valent pneumococcal conjugate vaccine, it is characterized in that: the bonded concrete steps of described capsular polysaccharide and carrier protein couplet are: in 10ml concentration is that to add with the dissolved concentration of 1ml acetonitrile respectively in 14 kinds of pneumococcal capsular polysaccharides of serotype of 10mg/ml be 100mg/ml1-amino-4-dimethyl-pyridine-tetrafluoride boron, adding 4ml concentration behind the 1min is the triethylamine activation of 0.2mol/L, 2.5min the back adds the 5g carrier protein, reaction is 1 hour under the room temperature, 4 ℃ were reacted 10～15 hours again, add the ethanolamine cessation reaction, again through Sepharose CL-4B chromatography media purification, with wavelength A
206And A
280Synchronous monitoring, the sodium chloride solution eluting, the eluent of collecting two wavelength fused peakss place is combined vaccine.
4, shown in the claim 2, high-efficiency 14-valent pneumococcal conjugate vaccine, it is characterized in that: described serotype 6A, 6B, 14,19A, 19F and 23F type separate the capsular polysaccharide of purifying and adopt 1-amino-4-dimethyl-pyridine-tetrafluoride boron and triethylamine activation, by isourea key coupling hemophilus influenza D albumen.
5, shown in the claim 2, high-efficiency 14-valent pneumococcal conjugate vaccine, it is characterized in that: described serotype 1,2 and 7F type separate the capsular polysaccharide of purifying and adopt 1-amino-4-dimethyl-pyridine-tetrafluoride boron and triethylamine activation, by isourea key coupling tetanus toxoid protein.
6, shown in the claim 2, high-efficiency 14-valent pneumococcal conjugate vaccine, it is characterized in that: described serotype 4,5,9N, 9V separate the capsular polysaccharide of purifying and adopt 1-amino-4-dimethyl-pyridine-tetrafluoride boron and triethylamine activation with the 18C type, by the nontoxic variant protein of isourea key coupling diphtheria toxin, diphtherotoxin.
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| US10034949B2 (en) | 2012-06-20 | 2018-07-31 | Sk Chemicals Co., Ltd. | Polyvalent pneumococcal polysaccharide-protein conjugate composition |
| US10058607B2 (en) | 2012-06-20 | 2018-08-28 | Sk Chemicals Co., Ltd. | Polyvalent pneumococcal polysaccharide-protein conjugate composition |
| CN104755102A (en) * | 2012-06-20 | 2015-07-01 | Sk化学公司 | Polyvalent pneumococcal polysaccharide-protein conjugate composition |
| JP2015520227A (en) * | 2012-06-20 | 2015-07-16 | エスケー ケミカルズ カンパニー, リミテッドSk Chemicals Co., Ltd. | Polyvalent pneumococcal polysaccharide-protein conjugate composition |
| WO2013191459A1 (en) * | 2012-06-20 | 2013-12-27 | 에스케이케미칼주식회사 | Polyvalent pneumococcal polysaccharide-protein conjugate composition |
| EP2865392B1 (en) | 2012-06-20 | 2016-11-16 | SK Chemicals Co., Ltd. | Polyvalent pneumococcal polysaccharide-protein conjugate composition |
| EP2865392A4 (en) * | 2012-06-20 | 2016-01-20 | Sk Chemicals Co Ltd | COMPOSITION OF POLYSACCHARIDE CONJUGATES OF MULTIPURPOSE PNEUMOCOCCUS-PROTEIN |
| EP2865392B2 (en) † | 2012-06-20 | 2020-02-12 | SK Bioscience Co., Ltd. | Polyvalent pneumococcal polysaccharide-protein conjugate composition |
| EP2932979A4 (en) * | 2012-12-11 | 2016-06-01 | Sk Chemicals Co Ltd | COMPOSITION OF A POLYSACCHARIDE-MULTIVALENT PNEUMOCOCCAL PROTEIN CONJUGATE |
| AU2013360596B2 (en) * | 2012-12-11 | 2016-03-31 | Sk Bioscience Co., Ltd. | Multivalent pneumococcal polysaccharide-protein conjugate composition |
| JP2016503015A (en) * | 2012-12-11 | 2016-02-01 | エスケー ケミカルズ カンパニー, リミテッドSk Chemicals Co., Ltd. | Polyvalent pneumococcal polysaccharide-protein conjugate composition |
| US9981029B2 (en) | 2012-12-11 | 2018-05-29 | Sk Chemical Co., Ltd. | Multivalent pneumococcal polysaccharide-protein conjugate composition |
| CN104837505A (en) * | 2012-12-11 | 2015-08-12 | Sk化学公司 | Multivalent pneumococcal polysaccharide-protein conjugate composition |
| WO2014092377A1 (en) * | 2012-12-11 | 2014-06-19 | 에스케이케미칼주식회사 | Multivalent pneumococcal polysaccharide-protein conjugate composition |
| CN106177933B (en) * | 2012-12-20 | 2020-09-29 | 辉瑞公司 | immunogenic composition |
| CN106177933A (en) * | 2012-12-20 | 2016-12-07 | 辉瑞公司 | Immunogenic composition |
| CN103495161B (en) * | 2013-10-08 | 2019-06-18 | 江苏康泰生物医学技术有限公司 | A kind of mixture and preparation method thereof of polynary pneumococcal capsular polysaccharide-protein conjugate |
| CN103495161A (en) * | 2013-10-08 | 2014-01-08 | 江苏康泰生物医学技术有限公司 | Mixture of poly-pneumococcal capsular polysaccharide-protein conjugates and preparation method of mixture |
| TWI586363B (en) * | 2013-10-30 | 2017-06-11 | Sk化學公司 | Multivalent pneumococcal polysaccharide-protein conjugate composition |
| US9969793B2 (en) | 2014-10-28 | 2018-05-15 | Adma Biologics, Inc. | Compositions and methods for the treatment of immunodeficiency |
| US9815886B2 (en) | 2014-10-28 | 2017-11-14 | Adma Biologics, Inc. | Compositions and methods for the treatment of immunodeficiency |
| US11339206B2 (en) | 2014-10-28 | 2022-05-24 | Adma Biomanufacturing, Llc | Compositions and methods for the treatment of immunodeficiency |
| US9107906B1 (en) | 2014-10-28 | 2015-08-18 | Adma Biologics, Inc. | Compositions and methods for the treatment of immunodeficiency |
| US10683343B2 (en) | 2014-10-28 | 2020-06-16 | Adma Biologics, Inc. | Compositions and methods for the treatment of immunodeficiency |
| US11780906B2 (en) | 2014-10-28 | 2023-10-10 | Adma Biomanufacturing, Llc | Compositions and methods for the treatment of immunodeficiency |
| US9714283B2 (en) | 2014-10-28 | 2017-07-25 | Adma Biologics, Inc. | Compositions and methods for the treatment of immunodeficiency |
| IL252915B2 (en) * | 2015-01-15 | 2024-04-01 | Pfizer | Immunogenic preparations for use in pneumococcal vaccines |
| JP2020203950A (en) * | 2015-01-15 | 2020-12-24 | ファイザー・インク | Immunogenic compositions for use in pneumococcal vaccines |
| US11135279B2 (en) | 2015-01-15 | 2021-10-05 | Pfizer Inc. | Immunogenic compositions for use in pneumococcal vaccines |
| IL252915B1 (en) * | 2015-01-15 | 2023-12-01 | Pfizer | Immunogenic compositions for use in pneumococcal vaccines |
| JP2016132667A (en) * | 2015-01-15 | 2016-07-25 | ファイザー・インク | Immunogenic composition for use in a pneumococcal vaccine |
| WO2016113644A1 (en) | 2015-01-15 | 2016-07-21 | Pfizer Inc. | Immunogenic compositions for use in pneumococcal vaccines |
| CN107427568A (en) * | 2015-01-15 | 2017-12-01 | 辉瑞公司 | For the immunogenic composition in Pnu-Imune 23 |
| US10653764B2 (en) | 2015-01-15 | 2020-05-19 | Pfizer Inc. | Immunogenic compositions for use in pneumococcal vaccines |
| JP7033175B2 (en) | 2015-01-15 | 2022-03-09 | ファイザー・インク | Immunogenic composition for use in pneumococcal vaccine |
| JP2021105016A (en) * | 2015-01-15 | 2021-07-26 | ファイザー・インク | Immunogenic compositions for use in pneumococcal vaccines |
| CN107427568B (en) * | 2015-01-15 | 2021-12-14 | 辉瑞公司 | Immunogenic compositions for use in pneumococcal vaccines |
| JP7121155B2 (en) | 2015-01-15 | 2022-08-17 | ファイザー・インク | Immunogenic composition for use in pneumococcal vaccine |
| AU2016207820B2 (en) * | 2015-01-15 | 2020-12-17 | Pfizer Inc. | Immunogenic compositions for use in pneumococcal vaccines |
| CN107810010A (en) * | 2015-06-23 | 2018-03-16 | 生物E有限公司 | Multivalent pneumococcal combined vaccine |
| US11147863B2 (en) | 2015-06-23 | 2021-10-19 | Biological E Limited | Multivalent pneumococcal conjugate vaccine |
| CN108289938A (en) * | 2015-09-10 | 2018-07-17 | 创赏有限公司 | Multivalence VLP conjugates |
| US11547752B2 (en) | 2016-09-30 | 2023-01-10 | Biological E Limited | Multivalent pneumococcal vaccine compositions comprising polysaccharide-protein conjugates |
| WO2018064444A1 (en) | 2016-09-30 | 2018-04-05 | Biological E Limited | Multivalent pneumococcal vaccine compositions comprising polysaccharide-protein conjugates |
| WO2018156468A1 (en) | 2017-02-24 | 2018-08-30 | Merck Sharp & Dohme Corp. | Pneumococcal conjugate vaccine formulations |
| US10259865B2 (en) | 2017-03-15 | 2019-04-16 | Adma Biologics, Inc. | Anti-pneumococcal hyperimmune globulin for the treatment and prevention of pneumococcal infection |
| US11084870B2 (en) | 2017-03-15 | 2021-08-10 | Adma Biologics, Inc. | Anti-pneumococcal hyperimmune globulin for the treatment and prevention of pneumococcal infection |
| US11897943B2 (en) | 2017-03-15 | 2024-02-13 | Adma Biomanufacturing, Llc | Anti-pneumococcal hyperimmune globulin for the treatment and prevention of pneumococcal infection |
| WO2019139692A2 (en) | 2017-12-06 | 2019-07-18 | Merck Sharp & Dohme Corp. | Compositions comprising streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof |
| US12097250B2 (en) | 2017-12-06 | 2024-09-24 | Merck Sharp & Dohme Llc | Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof |
| US11116828B2 (en) | 2017-12-06 | 2021-09-14 | Merck Sharp & Dohme Corp. | Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof |
| US11850278B2 (en) | 2017-12-06 | 2023-12-26 | Merck Sharp & Dohme Llc | Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof |
| CN108524926A (en) * | 2018-06-29 | 2018-09-14 | 康希诺生物股份公司 | A kind of formulation compositions of multivalent pneumococcal combined vaccine and its application |
| WO2020075201A1 (en) | 2018-10-12 | 2020-04-16 | Biological E Limited | Multivalent pneumococcal polysaccharide-protein conjugate vaccine |
| JP7558944B2 (en) | 2018-10-12 | 2024-10-01 | バイオロジカル イー リミテッド | Multivalent pneumococcal polysaccharide-protein conjugate vaccine |
| US11642406B2 (en) | 2018-12-19 | 2023-05-09 | Merck Sharp & Dohme Llc | Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof |
| WO2020131763A2 (en) | 2018-12-19 | 2020-06-25 | Merck Sharp & Dohme Corp. | Compositions comprising streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof |
| US12016914B2 (en) | 2018-12-19 | 2024-06-25 | Merck Sharp & Dohme Llc | Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof |
| CN111735967A (en) * | 2020-08-03 | 2020-10-02 | 天津中逸安健生物科技有限公司 | A method for detection of adsorption completeness of recombinant novel coronavirus vaccine |
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