CN104174019A - Quadrivalent meningococcal polysaccharide carrier protein conjugate vaccine - Google Patents
Quadrivalent meningococcal polysaccharide carrier protein conjugate vaccine Download PDFInfo
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- CN104174019A CN104174019A CN201410324895.4A CN201410324895A CN104174019A CN 104174019 A CN104174019 A CN 104174019A CN 201410324895 A CN201410324895 A CN 201410324895A CN 104174019 A CN104174019 A CN 104174019A
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Abstract
The invention discloses a quadrivalent meningococcal polysaccharide carrier protein conjugate vaccine. The vaccine comprises a chemical conjugate of meningococcal polysaccharide and carrier protein, an immuno-activating agent and adjuvant, wherein the immuno-activating agent is oligodeoxynucleotide of which the sequence includes two copied 5'-CGTTCGAG-3'; and the oligodeoxynucleotide contains 18-38 nucleotides. The quadrivalent meningococcal polysaccharide carrier protein conjugate vaccine is a meningococcal polysaccharide vaccine capable of improving the immune effect of a vaccine, reducing dosage of a vaccine antigen, reducing the number of immune injection times and reducing vaccine cost.
Description
Technical field
The present invention relates to bacterization vaccine, be specifically related to a kind of tetravalence meningococcal polysacharide carrier protein combined vaccine.
Background technology
Epidemic cerebrospinal meningitis, is to infect by meningococcus the respiratory infectious disease that meninges or meninges cause, clinical manifestation mainly contains hyperpyrexia, headache, spurting vomiting, neck and hardens.Also can cause septicemia, there is purple congestion, ecchymosis in skin, and meningitis can cause brain injury and leave over the sequela such as auditory dysesthesia or deafness, mental retardation.Case fatality rate is 5%～10%.Epidemic encephalitis winter-spring season case is occurred frequently, and general 11～December case starts to increase, and be onset peak period 2～May of Second Year.This disease is that sickness rate is high, dangerous large, is the infectious disease of serious harm children's health.According to the chemical constitution of capsular polysaccharide, meningococcus can be divided into tens sero-groups, and wherein A, B, C, Y and W135 are topmost Epidemic bacterial floras.Vaccination is the main path of prevention epidemic encephalitis.
With vaccine prepared by meningococcus capsular polysaccharide, there is certain preventive effect, but it is a kind of T cell dependent/non-dependent antigen, very poor to 2 years old following infant immune effect, and can not induce immunological memory.Polysaccharide antigen can be changed into T cell dependence antigen with carrier protein couplet, not only effective to infant, and can induce immunological memory.But the production technology more complicated of this GL-PP combined vaccine, output is lower, and price is more expensive, can not meet the extensive demand of planned immunization far away.Therefore, be necessary to develop a kind of novel meningococcus vaccine.
In order to solve above-mentioned deficiency of the prior art, the present invention proposes a kind of new solution.
Summary of the invention
The object of this invention is to provide a kind of tetravalence meningococcal polysacharide carrier protein combined vaccine.
For reaching above-mentioned purpose, the technical solution adopted in the present invention is: a kind of tetravalence meningococcal polysacharide carrier protein combined vaccine is provided, comprises: the chemical coupling thing of A group meningitis cocci capsular polysaccharide and carrier protein; The chemical coupling thing of C group meningitis cocci capsular polysaccharide and carrier protein; The chemical coupling thing of Y group meningitis cocci capsular polysaccharide and carrier protein; The chemical coupling thing of W135 group meningitis cocci capsular polysaccharide and carrier protein;
Immune-active agent, this immune-active agent is oligodeoxynucleotide, the 5 '-CGTTCGAG-3 ' that contains two copies in its sequence; The length of described oligodeoxynucleotide is 18 ~ 38 nucleotide; Wherein CpG right and wrong are methylated; Adjuvant, this adjuvant comprises aluminium hydroxide and lactose.
In one embodiment of the invention, a kind of immune effect that can improve vaccine is provided, reduce vaccine antigen consumption, reduce immune pin time and reduce the meningococcus vaccine of vaccine cost.
Accompanying drawing explanation
Fig. 1 is the antibody titer that in experimental example 1, A1 vaccine and B1 vaccine produce specific antibody;
Fig. 2 is the antibody titer that in experimental example 2, A2 vaccine and B2 vaccine produce specific antibody;
Fig. 3 is the specificity of the specific antibody that in experimental example 1 and experimental example 2, vaccine produces;
Wherein, with leg-of-mutton curve, be the specificity curve of the specific antibody that in experimental example 2, B2 group vaccine produces, with foursquare curve, be the specificity curve of the specific antibody that in experimental example 1, B1 group vaccine produces.
The specific embodiment
In one embodiment of the invention, A group, C group, Y group and W135 group meningitis cocci, the combination of these four kinds of meningococcal capsular polysaccharides and carrier protein realizes by the following method:
Step 1: respectively four kinds of meningococcal polysacharides are carried out to priming reaction and react with derivation, when reaction, first use cyanogen bromide-activated meningococcal polysacharide, then use the meningococcal polysacharide of ethylenediamine derivation cyanogen bromide-activated, finally use reagent derivation polysaccharide.
Step 2: the sulfhydrylation of carrier protein, sulfhydrylization reagent used includes but not limited to 2-imino group sulfane.
Step 3: the meningococcal polysacharide of derivation is combined with the carrier protein of sulfhydrylation.
The coupling method of meningococcus blooming polysaccharide and carrier protein is open by a plurality of documents, and therefore, this is coupled method and is not limited only to method disclosed by the invention, adopts other coupling methods can reach equally enforcement object of the present invention.
In another embodiment of the invention, the chemical coupling thing of meningococcal capsular polysaccharide and carrier protein is one or more capsular polysaccharides and tetanus toxoid or diphtheria toxoid or other any chemical coupling thing that is applicable to the carrier protein of vaccine for man in meningococcus serogroups A group, C group, Y group and W135 group preferably.Tetanus toxoid herein or diphtheria toxoid are all schematically to illustrate of the present inventionly, do not form any limitation of the invention, and any carrier protein that is applicable to vaccine for man is all applicable to the present invention
In one embodiment of the invention, immune-active agent is oligodeoxynucleotide, the 5 '-CGTTCGAG-3 ' that contains two copies in its sequence; The length of described oligodeoxynucleotide is 18 ~ 38 nucleotide; Wherein CpG right and wrong are methylated.Optimize, the nucleotides sequence of immune-active agent is classified 5 '-TCGC CGTTCGAG CCTA CGTTCGAG CTTG-3 ' as.Further optimize, the nucleotides sequence of immune-active agent is classified 5 '-TTCTGAC CGTTCGAG TGCACTA CGTTCGAG CAGTTG-3 ' as.When implementing this programme, can prepare tetravalence meningococcus blooming polysaccharide according to existing either method, be then combined with the carrier protein that can receive, in conjunction with and conjugate simply can be mixed with immune-active agent and adjuvant, make vaccine.
In one embodiment of the invention, in every vaccine, the chemical coupling thing total content of meningococcus blooming polysaccharide and carrier protein is 10ug ~ 100ug, and in every vaccine, in adjuvant, content is 1mg ~ 100mg.
In one embodiment of the invention, in every vaccine, also comprise pharmaceutically acceptable excipient, such as freeze-dried excipient, forming agent etc.
Experimental example 1
The reinforced effects research of combined vaccine to mice A group meningitis cocci immunne response
By prior art, preparing tetravalence meningococcal polysacharide and the endotoxic chemical coupling thing of tetanus, is vaccine A1 hereinafter to be referred as this conjugate, adds adjuvant aluminium hydroxide and immune-active agent in vaccine A1, obtains vaccine B1.In vaccine A1, A group, C group, Y group and W135 group's polyoses content is identical, and polysaccharide total content is 80ug/ml; In vaccine B1, polysaccharide total content is 80ug/ml, and the content of adjuvant aluminium hydroxide is 5mg/ml.Immune-active agent sequence in vaccine B1 is 5 '-TCGC CGTTCGAG CCTA CGTTCGAG CTTG-3 '.Through synthetic, and carry out full chain thio-modification, polyacrylamide gel electrophoresis purification, is dissolved in normal saline, under the condition of subzero 20 ℃, saves backup.
Choose test mice, be divided into two groups, be i.e. vaccine A1 group and vaccine B1 group; Inject respectively 80ul and carry out immunne response experiment, after injection 3 weeks, with the same manner and Isodose, carry out again immunity, now gather mice and gather blood separation serum, and again gathering blood separation serum after immune 2 weeks again.Adopt respectively PBS solution to carry out 3 times of dilutions to serum separated serum, adopt ELISA method to detect antibody titer.
As shown in Figure 1, in vaccinate 3 weeks and inoculation after 2 weeks again, the anti-polysaccharide IgG titre that the vaccine A1 of vaccinate A1 group produces injection after 3 weeks with 2 weeks after all a little less than.Contrary, injection adds the anti-polysaccharide IgG titre of the vaccine B1 group of vaccine activity agent and adjuvant obviously to increase, and compares with vaccine A1 group, and the IgG titre of the vaccine B1 group in injection after 3 weeks is compared and has been increased by 15.2 times with the vaccine A1 of the same period; Inoculation is after two weeks again, and the IgG titre that the IgG titre of vaccine B1 group is compared the vaccine A1 of the same period has increased by 10.5 times.This shows, after vaccinate, vaccine B1 can produce immunne response fast, and can induce the higher antibody titer of generation, and immune effect strengthens.
Experimental example 2
The reinforced effects research of combined vaccine to mice C group meningitis cocci immunne response
By prior art, preparing tetravalence meningococcal polysacharide and the endotoxic chemical coupling thing of diphtheria, is vaccine A2 hereinafter to be referred as this conjugate, adds adjuvant aluminium hydroxide and immune-active agent in vaccine A2, obtains vaccine B2.In vaccine A2, A group, C group, Y group and W135 group's polyoses content is identical, and polysaccharide total content is 100ug/ml; In vaccine B2, polysaccharide total content is 100ug/ml, and the content of adjuvant aluminium hydroxide is 5mg/ml.Immune-active agent sequence in vaccine B2 is 5 '-TTCTGAC CGTTCGAG TGCACTA CGTTCGAG CAGTTG-3 '.Through synthetic, and carry out full chain thio-modification, polyacrylamide gel electrophoresis purification, is dissolved in normal saline, under the condition of subzero 20 ℃, saves backup.
Choose test mice, be divided into two groups, be i.e. vaccine A2 group and vaccine B2 group; Inject respectively 80ul and carry out immunne response experiment, after injection 3 weeks, with the same manner and Isodose, carry out again immunity, now gather mice and gather blood separation serum, and again gathering blood separation serum after immune 2 weeks again.Adopt respectively PBS solution to carry out 3 times of dilutions to serum separated serum, adopt ELISA method to detect antibody titer.
As shown in Figure 2, in vaccinate 3 weeks and inoculation after 2 weeks again, the anti-polysaccharide IgG titre that the vaccine A2 of vaccinate A2 group produces injection after 3 weeks with 2 weeks after all a little less than.Contrary, injection adds the anti-polysaccharide IgG titre of the vaccine B2 group of vaccine activity agent and adjuvant obviously to increase, and compares with vaccine A2 group, and the IgG titre of the vaccine B2 group in injection after 3 weeks is compared and has been increased by 15.2 times with the vaccine A2 of the same period; Inoculation is after two weeks again, and the IgG titre that the IgG titre of vaccine B2 group is compared the vaccine A2 of the same period has increased by 10.5 times.This shows, after vaccinate, vaccine B2 can produce immunne response fast, and can induce the higher antibody titer of generation, and immune effect strengthens.
Experimental example 3
The specificity of polysaccharide specific antibody
By 200 times of the mice plasma dilutions of the vaccine group of the vaccine group of inoculation B1 vaccine and B2 vaccine.Mice plasma to 2 kinds of dilutions adds respectively A group meningitis polysaccharide and C group meningitis polysaccharide, detects the antibody horizontal of anti-polysaccharide in mice plasma by ELISA method.As shown in Figure 3, along with the increase of polysaccharide addition, polysaccharide specific antibody reduces gradually in conjunction with the ability of coated polysaccharide in 96 orifice plates.When the polysaccharide adding reaches 15 microgram, the Disability of the antibodies polysaccharide of two groups of blood plasma.This antipolysaccharide antibody that shows that mice produces can be specifically in conjunction with polysaccharide.
Claims (6)
1. a tetravalence meningococcal polysacharide carrier protein combined vaccine, comprising:
The chemical coupling thing of A group meningitis cocci capsular polysaccharide and carrier protein;
The chemical coupling thing of C group meningitis cocci capsular polysaccharide and carrier protein;
The chemical coupling thing of Y group meningitis cocci capsular polysaccharide and carrier protein;
The chemical coupling thing of W135 group meningitis cocci capsular polysaccharide and carrier protein;
Immune-active agent, this immune-active agent is oligodeoxynucleotide, the 5 '-CGTTCGAG-3 ' that contains two copies in its sequence; The length of described oligodeoxynucleotide is 18 ~ 38 nucleotide; Wherein CpG right and wrong are methylated;
Adjuvant, this adjuvant comprises aluminium hydroxide and lactose.
2. vaccine as claimed in claim 1, is characterized in that: described chemical coupling thing is one or more capsular polysaccharides and tetanus toxoid, diphtheria toxoid or other any chemical coupling thing that is applicable to the carrier protein of vaccine for man in meningococcus serogroups A group, C group, Y group and W135 group.
3. vaccine as claimed in claim 1, is characterized in that: the nucleotides sequence of described immune-active agent is classified 5 '-TCGC CGTTCGAG CCTA CGTTCGAG CTTG-3 ' as.
4. vaccine as claimed in claim 1, is characterized in that: the nucleotides sequence of described immune-active agent is classified 5 '-TTCTGAC CGTTCGAG TGCACTA CGTTCGAG CAGTTG-3 ' as.
5. vaccine as claimed in claim 1, is characterized in that: in every vaccine, the chemical coupling thing total content of meningococcus blooming polysaccharide and carrier protein is 10ug ~ 100ug, and in every vaccine, in adjuvant, content is 1mg ~ 100mg.
6. vaccine as claimed in claim 1, is characterized in that: further comprise pharmaceutically acceptable excipient.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104998255A (en) * | 2015-06-30 | 2015-10-28 | 北京祥瑞生物制品有限公司 | Novel ACYW135 meningococcal conjugate vaccine and preparation method thereof |
| CN113274488A (en) * | 2021-04-30 | 2021-08-20 | 山东省药学科学院 | Oligosaccharide vaccine for specifically preventing fungal infection and preparation method thereof |
| CN117679500A (en) * | 2023-11-22 | 2024-03-12 | 罗益（无锡）生物制药有限公司 | Preparation method of tetravalent epidemic cerebrospinal meningitis vaccine |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| EP1090642A2 (en) * | 1995-06-07 | 2001-04-11 | SMITHKLINE BEECHAM BIOLOGICALS s.a. | Vaccines comprising a polysaccharide antigen-carrier protein conjugate and free carrier protein |
| WO2004011027A1 (en) * | 2002-07-30 | 2004-02-05 | Baxter International Inc. | Chimeric multivalent polysaccharide conjugate vaccines |
| CN101559225A (en) * | 2008-04-18 | 2009-10-21 | 北京生物制品研究所 | Meningococcus vaccine |
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2014
- 2014-09-23 CN CN201410324895.4A patent/CN104174019A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1090642A2 (en) * | 1995-06-07 | 2001-04-11 | SMITHKLINE BEECHAM BIOLOGICALS s.a. | Vaccines comprising a polysaccharide antigen-carrier protein conjugate and free carrier protein |
| WO2004011027A1 (en) * | 2002-07-30 | 2004-02-05 | Baxter International Inc. | Chimeric multivalent polysaccharide conjugate vaccines |
| CN101559225A (en) * | 2008-04-18 | 2009-10-21 | 北京生物制品研究所 | Meningococcus vaccine |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104998255A (en) * | 2015-06-30 | 2015-10-28 | 北京祥瑞生物制品有限公司 | Novel ACYW135 meningococcal conjugate vaccine and preparation method thereof |
| CN113274488A (en) * | 2021-04-30 | 2021-08-20 | 山东省药学科学院 | Oligosaccharide vaccine for specifically preventing fungal infection and preparation method thereof |
| CN113274488B (en) * | 2021-04-30 | 2023-08-29 | 山东省药学科学院 | Oligosaccharide vaccine for specifically preventing fungal infection and preparation method thereof |
| CN117679500A (en) * | 2023-11-22 | 2024-03-12 | 罗益（无锡）生物制药有限公司 | Preparation method of tetravalent epidemic cerebrospinal meningitis vaccine |
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Application publication date: 20141203 |