CN101553246B - The preparation and medication of protein matrix vaccines and this vaccine - Google Patents

The preparation and medication of protein matrix vaccines and this vaccine Download PDF

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Publication number
CN101553246B
CN101553246B CN200780037545.8A CN200780037545A CN101553246B CN 101553246 B CN101553246 B CN 101553246B CN 200780037545 A CN200780037545 A CN 200780037545A CN 101553246 B CN101553246 B CN 101553246B
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antigen
carrier protein
polysaccharide
vaccine
protein
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CN101553246A (en
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J·J·梅卡拉诺斯
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Harvard College
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Harvard College
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Priority to CN201910333309.5A priority Critical patent/CN110064053A/en
Priority claimed from PCT/US2007/017528 external-priority patent/WO2008021076A2/en
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention relates to carrier protein and contain the vaccine composition of target antigen in the composite, prepare the method for this vaccine and give the method for vaccine.

Description

The preparation and medication of protein matrix vaccines and this vaccine
Cross reference to related applications
Claimed 60/835,944 He of provisional application Serial number submitted for 7th from August in 2006 of this application The benefit for the provisional application Serial number 60/933,764 that on June 8th, 2007 submits, specification are incorporated by reference into this Wen Zhong.
Statement about federal funding research
The present invention is authorized in National Institute of Health (National Institutes of Health) (NIH) It is completed under Grant No U54AI057159 by government-funded.Government possesses certain rights in the present invention.
Background of invention
The present invention relates to vaccine composition, vaccine preparation method and vaccine administration ways.
Many antigens are especially those antigens relevant to cause of disease precursor clostridium layer and stimulate very little to immune response or do not stimulate, And the effort for generating the effective vaccine for being directed to those antigens is made to become complicated.Pod membrane is the surface composition of microorganism, usually by having The polymer (polymer) of machine compound such as carbohydrate, amino acid or alcohols forms.Pod membrane is widely different in chemistry.Constitute pod The monomer unit (such as carbohydrate) of film can be linked together with various molecular configurations and further with phosphate (phosphate), Nitrogen, sulfate (sulfate) and other chemical modification groups replace.These chemical changes allow pod membrane by numerous antigenic targets It is presented to antimicrobial surface, to allow that host immune system is avoided to target these targets.Pod membrane can also be to prevent microorganism By the virulence factor of host macrophage and multinuclear leucocyte phagocytosis and kill.For pod membrane antibody by make complement fixation in Antimicrobial surface provides the defence for pod membrane biologically effective, complement can lead to biosolubilization or its opsonic action and by There is the host immune cell of phagocytosis to absorb and kills.It is IgG antibody for the most effective antibody of pod membrane.It not can induce significant The pod membrane of horizontal IgG is called T-independent antigen.The cause of the covalent coupling of protein and pod membrane becomes " T dependence " and this antigen can cause IgG response.
There is to safely, be easily-synthesized, have it is cost-benefit, for pod membrane and other will not cause strong immunization response Or the demand of the T-independent antigen vaccine of IgG antibody.Need such vaccine prevention various by bacillus anthracis, pneumonia Infectious disease caused by coccus, influenza B, meningococcus and streptococcal infection.
Summary of the invention
The present invention relates to containing by the vaccine composition for the target antigen that the carrier protein in compound contains, prepare it is this The method of vaccine and the administration way of vaccine.
Therefore, in a first aspect, the present invention describes the vaccine composition containing target antigen and carrier protein, wherein (i) Target antigen no more than 50% and carrier protein are crosslinked, and (ii) wherein the antigen contained by carrier protein to be formed it is compound Object.
In the embodiment that the first aspect present invention phase needs, the compound diameter is between 10nm-100 μm.At this In the embodiment that the invention first aspect more phase needs, the compound diameter is between about 100nm-100 μm.In the present invention first In the embodiment that the aspect also more phase needs, the compound diameter is between about 100nm-10 μm.
In the embodiment that first aspect present invention other phases need, when giving compound to mammal, causes and feed The immune response that T- cell relies in newborn animal.
In the embodiment that the first aspect present invention other phase needs, the molar ratio of antigen and carrier protein is in 1: 10 and 10 : between 1.It is desirable that the carrier protein is polymer (multimer), for example, the polymer comprising at least five subunit.? In the embodiment that other phases need, the polymer is same polymer.
In the embodiment that the first aspect present invention further phase needs, the carrier protein and at least one other carrier Albumen is covalently attached.It is desirable that the covalent linkage includes the primary amino group and asparagine or glutamine in lysine side-chain Peptide bond between the carboxyl of side chain.In the embodiment that other phases need, described be covalently attached includes formulaChemical combination Object, wherein RnIt is the direct-connected or branched heteroalkyl groups of the direct-connected or branched alkyl of 1-12 carbon atom, 1-12 atom (heteroalkyl), direct-connected or branched alkyne, the 5-10 of the direct-connected or branched-chain alkene of 2-12 carbon atom, 2-12 carbon atom The aromatic moieties of a carbon atom, the cyclic annular system of 3-10 atom ,-(CH2CH2O)qCH2CH2Wherein q is 1-4 or connection two The chemical bond of aldehyde radical.In the embodiment that the other phase needs, described be covalently attached contains glutaraldehyde, dimaleoyl imino benzene first Acyl group-N-hydroxy-succinamide ester (m-maleimidobenzoyl-N-hydroxysuccinimide ester), carbon two are sub- Amine or bisazo benzidine.There are also in the embodiment that other phases need, described be covalently attached includes bi-functional cross-linking agent (cross linker).It is desirable that the bi-functional cross-linking agent is glutaraldehyde, bis- [sulfonic group succinimido] suberates (bis [sulfosuccinimidyl] suberate) or dimethyl diimidate.
In the embodiment that first aspect present invention other phases need, the carrier protein is non-covalent linking.In the phase In the embodiment needed, the non-covalent linking includes hydrophobic effect, ionization, model ylid bloom action or hydrogen bond.
In the embodiment that the first aspect present invention other phase needs, the carrier protein is diphtheria toxin or its mutation Body, diphtheria toxoid, tetanus toxin or its mutant, tetanus toxoid, pseudomonas aeruginosa (Pseudomonas Aeruginosa) exotoxin A or its mutant, b subunit of cholera toxin, Tetanus toxin fragment C, bacterial flagellin (such as Comma bacillus (Vibriocholerae) flagellin), pneumolysin, Neisseria meningitidis (Neisseriamenningitidis) outer membrane protein, pseudomonas aeruginosa (Pseudomonas aeruginosa) Hcp1 egg White, Escherichia coli (Escherichia coli) heat labile enterotoxin, shiga-like toxin (such as Shigella (Shigella) SltB2 albumen), people LTB albumen, pneumolysin, Listeria lysin O (or GAP-associated protein GAP), whole bacterial cells (example Such as pseudomonas aeruginosa (Peudomonas aeruginosa) or streptococcus (Streptococcal) cell) Protein Extraction Object, the dominant negative mutant (DNI) of Bacillus anthracis (Bacillus anthracis) protection antigen or Escherichia coli (Escherichia coli) beta galactosidase.In the embodiment that the specific phase needs, the carrier protein is pneumococcus Hemolysin, Listeria lysin O, diphtheria toxin, diphtheria toxoid, tetanus toxin or tetanus toxoid.
In the embodiment that first aspect present invention other phases need, the target antigen is polysaccharide, polyalcohol or poly- ammonia Base acid.It is desirable that the polysaccharide includes at least 18 residues.In the embodiment that other phases need, the polysaccharide is pneumonia chain Coccus (Streptococcuspneumoniae) polysaccharide, soil draw hot Francisella (Francisella tularensis) Polysaccharide, Bacillus anthracis (Bacillus anthracis) polysaccharide, Hemophilus influenzae (Haemophilusinfluenzae) are more Sugar, salmonella typhi (Salmonella typhi) polysaccharide, salmonella (Salmonella species) polysaccharide, shiga Bacterium (Shigella) polysaccharide or Neisseria meningitidis (Neisseria menningitidis) polysaccharide.It is needed in the specific phase In embodiment, streptococcus pneumonia (Streptococcus pneumoniae) polysaccharide is any one in 1-48 type pod membrane Kind, for example, 3,4,6B, 7A, 7B, 7C, 7F, 9A, 9L, 9N, 9V, 12A, 12B, 12F, 14,15A, 15B, 15C, 15F, 17,18B, 18C, 19F, 23F, 25A, 25F, 33F, 35,37,38,44 or 46.In the embodiment that other specific phases need, the soil draws heat Francisella (Francisella tularensis) polysaccharide is O antigen.
In the embodiment that the first aspect present invention further phase needs, the target antigen is microbial capsular polymerization Object.It is desirable that the microbial capsular polymer is the poly- γ-from Bacillus anthracis (Bacillus anthracis) D-Glu.
In the embodiment that first aspect present invention other phases need, the target antigen is by having at least three atoms Monomer composition organic polymer, wherein each atom is independently selected from carbon, oxygen, hydrogen, phosphate, nitrogen and sulfate.It is ideal Ground, the organic polymer are derived from microorganism.In the embodiment that other phases need, the organic polymer is not naturally to deposit ?.
In the embodiment that the other phase needs, the vaccine composition further includes the second target antigen.It is desirable that institute Stating vaccine composition also includes third target antigen.
In second aspect, the present invention describes the method for preparation vaccine composition.This method include (i) by target antigen with Carrier protein is mixed to form antigen and carrier protein mixture and (ii) contains target antigen with carrier protein, wherein no more than Carrier protein crosslinking in 50% target antigen and vaccine composition.
In the embodiment that the second aspect of the present invention phase needs, the vaccine composition further includes pharmaceutically acceptable Excipient.
It is described to contain including the Precipitation Antigen from mixture in the embodiment that second aspect of the present invention other phases need And carrier protein.It is desirable that the precipitating includes: to change the pH of mixture；Add trichloroacetic acid (TCA) or sulphur into mixture Sour ammonium；Concentration by increasing or decreasing inorganic salts in mixture changes the ionic strength of mixture；Heating mixture makes carrier Albumen and/or antigen condensation；Or cause to be crosslinked with the ionizing radiation mixture of enough flux.
In the embodiment that the second aspect of the present invention phase needs, the antigen of the vaccine composition and mole of carrier protein Than between 1: 10-9: 10.
In the embodiment that the second aspect of the present invention other phase needs, the carrier protein is polymer.It is desirable that poly Body includes at least five subunit.In the embodiment that other phases need, the polymer is same polymer.
In the embodiment that the second aspect of the present invention further phase needs, the carrier protein non-covalent linking.It is desirable that The non-covalent linking includes hydrophobic effect, ionization, model ylid bloom action or hydrogen bond.
In the embodiment that the second aspect of the present invention other phase needs, the carrier protein is diphtheria toxin or its mutation Body, diphtheria toxoid, tetanus toxin or its mutant, tetanus toxoid, pseudomonas aeruginosa (Pseudomonas Aeruginosa) exotoxin A or its mutant, b subunit of cholera toxin, Tetanus toxin fragment C, bacterial flagellin (such as Comma bacillus (Vibriocholerae) flagellin), pneumolysin, Listeria lysin O, Neisseria meningitdis ball Bacterium (Neisseria menningitidis) outer membrane protein, pseudomonas aeruginosa (Pseudomonasaeruginosa) Hcp1 egg White, Escherichia coli (Escherichia coli) heat labile enterotoxin, shiga-like toxin (Shigella (Shigella) SltB2 albumen), people LTB albumen, come from whole bacterial cells (such as pseudomonas aeruginosa Pseudomonas aeruginosa) Or streptococcus (Streptococcal) cell) protein extract, Bacillus anthracis (Bacillus anthracis) protect The dominant negative mutant (DNI) or Escherichia coli (Escherichia coli) beta galactosidase of shield property antigen.Specific In the embodiment that phase needs, the carrier protein is pneumolysin, Listeria lysin O, diphtheria toxin, diphtheria class poison Element, tetanus toxin or tetanus toxoid.
In the embodiment that other phases of second aspect of the present invention need, the target antigen is polysaccharide, polyalcohol or poly- ammonia Base acid.It is desirable that the polysaccharide includes at least 18 residues.In the embodiment that other phases need, the polysaccharide is pneumonia chain Coccus (Streptococcuspneumoniae) polysaccharide, soil draw hot Francisella (Francisella tularensis) Polysaccharide, Bacillus anthracis (Bacillus anthracis) polysaccharide, Hemophilus influenzae (Haemophilusinfluenzae) are more Sugar, salmonella typhi (Salmonella typhi) polysaccharide, Shigella (Shigella) polysaccharide, salmonella (Salmonella species) polysaccharide or Neisseria meningitidis (Neisseria meningitidis) polysaccharide.Specific In the embodiment that phase needs, streptococcus pneumonia (Streptococcus pneumoniae) polysaccharide is in 1-48 type pod membrane Any one, for example, 3,4,6B, 7A, 7B, 7C, 7F, 9A, 9L, 9N, 9V, 12A, 12B, 12F, 14,15A, 15B, 15C, 15F, 17,18B, 18C, 19F, 23F, 25A, 25F, 33F, 35,37,38,44 or 46.In the embodiment that other specific phases need, institute Stating soil and drawing hot Francisella (Francisella tularensis) polysaccharide is O antigen.
In the embodiment that the second aspect of the present invention other phase needs, the target antigen is microbial capsular polymer. It is desirable that microbial capsular polymer is the poly- γ-D- paddy ammonia from Bacillus anthracis (Bacillus anthracis) Acid.
In the embodiment that first aspect present invention other phases need, the target antigen is by containing at least three atoms Monomer composition organic polymer, wherein each atom is independently selected from carbon, oxygen, hydrogen, phosphate, nitrogen and sulfate.It is ideal Ground, the organic polymer derive from microorganism.In the embodiment that other phases need, the organic polymer is not that nature is deposited ?.
In the embodiment that the second aspect of the present invention further phase needs, the mixing in step (i) is anti-including the second target Former or even third target antigen.
In the third aspect, the present invention describes the method for another preparation vaccine composition.This method includes (i) by target Antigen is mixed with carrier protein and the connector (linker) for being crosslinked carrier protein is added in (ii), wherein no more than 50% mesh The carrier protein marked in antigen and vaccine composition is crosslinked.
In the embodiment that the third aspect present invention phase needs, the vaccine composition further includes pharmaceutically acceptable Excipient.In the embodiment that third aspect present invention other phases need, antigen and carrier protein in the vaccine composition Molar ratio between 1: 10 and 10: 1.In the embodiment that the third aspect present invention other phase needs, the carrier protein is Polymer.It is desirable that the polymer includes at least five subunit.In the embodiment that other phases need, the polymer is Same polymer.
In the embodiment that the third aspect present invention further phase needs, the method includes the seats in reduction carrier albumen Husband's alkali.In the embodiment that the third aspect present invention further phase needs, the carrier protein and at least one other carrier Albumen is covalently attached.It is desirable that the covalent linkage includes the primary amino group and asparagine or glutamine in lysine side-chain Peptide bond between the carboxyl of side chain.In the embodiment that other phases need, described be covalently attached includes bi-functional cross-linking agent.It is ideal Ground, the bi-functional cross-linking agent are glutaraldehyde, bis- [sulfonic group succinimide] suberates or dimethyl diimidate.
In the embodiment that the third aspect present invention other phase needs, the connector is formulaCompound, Middle RnIt is the direct-connected or branched alkyl of 1-12 carbon atom, the direct-connected or branched heteroalkyl groups of 1-12 atom, 2-12 carbon atom Direct-connected or branched-chain alkene, the direct-connected or branched alkyne of 2-12 carbon atom, the aromatic moieties of 5-10 carbon atom, 3-10 it is former The cyclic annular system of son ,-(CH2CH2O)qCH2CH2Wherein q is 1-4 or the chemical bond for connecting two aldehyde radicals.
In the embodiment that third aspect present invention other phases need, the connector is glutaraldehyde, dimaleoyl imino Benzoyl-N-HOSu NHS base ester, carbodiimide or bisazo benzidine.
In the embodiment that the third aspect present invention other phase needs, the carrier protein is diphtheria toxin or its mutation Body, diphtheria toxoid, tetanus toxin or its mutant, tetanus toxoid, pseudomonas aeruginosa (Pseudomonas Aeruginosa) exotoxin A or its mutant, b subunit of cholera toxin, Tetanus toxin fragment C, bacterial flagellin (cholera Vibrios (Vibriocholerae) flagellin), pneumolysin, Listeria lysin O, Neisseria meningitidis (Neisseria menningitidis) outer membrane protein, pseudomonas aeruginosa (Pseudomonasaeruginosa) Hcp1 egg White, Escherichia coli (Escherichia coli) heat labile enterotoxin, shiga-like toxin (Shigella (Shigella) SltB2 albumen), people LTB albumen, from whole bacterial cells (pseudomonas aeruginosa (Pseudomonas aeruginosa) or Streptococcus (Streptococcal) cell) protein extract, Bacillus anthracis (Bacillus anthracis) protect The dominant negative mutant (DNI) or Escherichia coli (Escherichia coli) beta galactosidase of shield property antigen.
In the embodiment that the third aspect present invention further phase needs, the target antigen is polysaccharide, polyalcohol or poly- Amino acid.It is desirable that the polysaccharide includes at least 18 residues.In the embodiment that other phases need, the polysaccharide is pneumonia Streptococcus (Streptococcuspneumoniae) polysaccharide, soil draw hot Francisella (Francisella Tularensis) polysaccharide, Bacillus anthracis (Bacillus anthracis) polysaccharide, Hemophilus influenzae (Haemophilusinfluenzae) polysaccharide, salmonella typhi (Salmonella typhi) polysaccharide, Shigella (Shigella species) polysaccharide, salmonella (Salmonella species) polysaccharide or Neisseria meningitidis (Neisseria meningitidis) polysaccharide.In the embodiment that the specific phase needs, the streptococcus pneumonia (Streptococcus pneumoniae) polysaccharide is any one of 1-48 type pod membrane, for example, 3,4,6B, 7A, 7B, 7C, 7F、9A、9L、9N、9V、12A、12B、12F、14、15A、15B、15C、15F、17、18B、18C、19F、23F、25A、25F、33F、 35,37,38,44 or 46.In the embodiment that other specific phases need, the soil draws hot Francisella (Francisella Tularensis) polysaccharide is O antigen.
In the embodiment that third aspect present invention other phases need, the target antigen is microbial capsular polymer. It is desirable that the microbial capsular polymer comes from the poly- γ-D- paddy ammonia of Bacillus anthracis (Bacillus anthracis) Acid.
There are also in the embodiment that third aspect present invention other phases need, the target antigen is by having at least three The organic polymer of the monomer composition of atom, wherein each atom is independently selected from carbon, oxygen, hydrogen, phosphate, nitrogen and sulfate.Reason Think that ground, the organic polymer derive from microorganism.In the embodiment that the other phase needs, the organic polymer is not nature It is existing.
In the embodiment that the third aspect present invention further phase needs, the mixing in step (i) is anti-including the second target Former or even third target antigen.
In the fourth aspect, the present invention, which is described, carries out the side that infectious agent is resisted in vaccine inoculation to study subject Method.This method includes giving the vaccine composition of the first aspect present invention of study subject sufficient amount, to induce study subject to produce Raw antibody.In the embodiment that the fourth aspect present invention phase needs, the method includes second to give step, wherein by sufficient The vaccine composition for the first aspect present invention enough measured gives study subject, generates antibody to reinforce study subject.It is desirable that Fourth aspect present invention, the generation of antibody are T cell dependences.In the embodiment that fourth aspect present invention other phases need In, the generation of the antibody is enough to prevent or reduce infection of the infectious agent to study subject.It is desirable that the infectiousness Pathogen is pneumococcus, meningococcus, influenza B haemophilic bacterium (Haemophilus influenza Type B), verdigris Pseudomonad (Pseudomonas aeruginosa), soil draw hot Francisella (Francisella tularensis), Shigella (Shigella species), salmonella (Salmonella species), acinetobacter calcoaceticus (Acinetobacter species), bulkholderia cepasea (Burkholderia species) or Escherichia coli (Escherichia coli)。
In the embodiment that fourth aspect present invention other phases need, the method includes second to give step, wherein will The second vaccine composition containing target antigen of sufficient amount is supplied to study subject and reinforces study subject generation antibody.It is ideal Ground, the generation of the antibody are enough to prevent or reduce infection of second infectious agent to study subject.
In the embodiment that the fourth aspect present invention phase needs, the antibody is IgG antibody.Fourth aspect present invention into In the embodiment that one step phase needed, the study subject is people.
In the embodiment that the present invention needs any one aspect phase, the streptococcus pneumonia (Streptococcus Pneumoniae) polysaccharide be the pod membrane type described in Kong etc. (J.Med.Microbiol.54:35-356,2005) it One.For example, streptococcus pneumonia (Streptococcus pneumoniae) the polysaccharide capsule type phase needs is 1 (for example, 1-g or 1-q), 2 (for example, 2-g, 2-q or 2-41A), 3 (for example, 3-g, 3-q, 3-c or 3-nz), 4,5 (such as 5-q, 5-c, 5-qap or 5-g), 6A (for example, 6A-g, 6A-c1,6A-c2,6A-n, 6A-qap, 6A-6B-g, 6A-6B-q or 6A-6B-s), 6B (for example, 6B-c, 6A-6B-g, 6A-6B-q or 6A-6B-s), 7F (for example, 7F-7A), 7A (such as 7A-cn or 7F-7A), 7B (for example, 7B-40), 7C (for example, 7C-19C-24B), 8 (for example, 8-g or 8-s), 9A (for example, 9A-9V), 9L, 9N, 9V are (for example, 9A- 9V), 9V and 14,10F (for example, 10F-q, 10F-ca or 10F-10C), 10A (for example, 10A-17A or 10A-23F), 10B (example Such as, 10B-10C), 11F, 11A (for example, 11A-nz or 11A-11D-18F), 11B (for example, 11B-11C), 11C (for example, 11B-11C or 11C-cn), 11D (for example, 11A-11D-18F), 12F (for example, 12F-q or 12F-12A-12B), 12A (for example, 12A-cn, 12A-46 or 12F-12A-12B), 12B (for example, 12F-12A-12B), 13 (for example, 13-20), 14 (for example, 14- G, 14-q, 14-v or 14-c), 15F (for example, 15F-cn1 or 15F-cn2), 15A is (for example, 15A-ca1,15A-ca2 or 15A- Chw), 15B (for example, 15B-c, 15B-15C, 15B-15C-22F-22A), 15C (for example, 15C-ca, 15C-q 1,15C-q2, 15C-q3,15C-s, 15B-15C or 15B-15C-22F-22A), 16F (for example, 16F-q or 16F-nz), 16A, 17F (for example, 17F-n and 17F-35B-35C-42), 17A (for example, 17A-ca or 10A-17A), 18F is (for example, 18F-ca, 18F-w or 11A- 11D-18F), 18A (for example, 18A-nz or 18A-q), 18B (for example, 18B-18C), 18C (for example, 18B-18C), 19F (example Such as, 19F-g1,19F-g2,19F-g3,19F-q, 19F-n or 19F-c), 19A (for example, 19A-g, 19A- or 19A-ca), 19B, 19C (for example, 19C-cn1,19C-cn2 or 7C-19C-24B), 20 (for example, 13-20), 21 (for example, 21-ca or 21-cn), 22F (for example, 15B-15C-22F-22A), 23F (for example, 23F-c, 10A-23F or 23F-23A), 23B (for example, 23B-c or 23B-q), 24F (for example, 24F-cn1,24F-cn2 or 24F-cn3), 24A, 24B (for example, 7C-19C-24B), 25F (for example, 25F-38), 25A, 27,28F (for example, 28F-28A or 28F-cn), 28A (for example, 28F-28A), 29 are (for example, 29-ca Or29-q), 31,32F (for example, 32F-32A), 32A (for example, 32A-cn or 32F-32A), 33F (for example, 33F-g, 33F-q, 33F-chw, 33F-33B or 33F-33A-35A), 33A (for example, 33F-33A-35A), 33B (for example, 33B-q, 33B-s or 33F-33B), 33D, 34 (for example, 34-ca or 34s), 35F (for example, 35F-47F), 35A (for example, 33F-33A-35A), 35B (for example, 17F-35B-35C-42), 36,37 (for example, 37-g or37-ca), 38 (for example, 25F-38), 39 are (for example, 39-cn1 Or 39-cn2), 40 (for example, 7B-40), 41F (for example, 41F-cn or 41F-s), 41A (for example, 2-41A), 42 (for example, 17B- 35B-35C-42), 43,44,45,46 (for example, 46-s or 12A-46), 47F (for example, 35F-47F), 47A, 48 are (for example, 48- Cn1 or 48-cn2) or the GenBank number of logging in AF532714 or AF532715.
Definition
" administration " being used in combination herein with vaccine, it is intended that provide the vaccine-induced tested of sufficient dosage for study subject The intracorporal immune response of object, wherein the immune response cause with vaccine contained by antibody in conjunction with antigen-specific generation.Phase It need to be administered including intramuscular injection, intracutaneous injection or percutaneous injection, and the phase need to include giving immunologic adjuvant appropriate.Administration can wrap Include the single-dose or multiple dose administration of vaccine.It is desirable that study subject is reinforced in second of administration of design generates antibody, prevention is passed The infection of metachromia pathogen.The frequency and amount of vaccine administration depend on the specific activity of vaccine and are easy to true by routine test It is fixed.
" crosslinking " means the formation of the covalent bond between two molecules, macromolecular or molecular combinations such as carrier protein, The formation of the covalent bond is directly realized by by using " zero-length " connector, or using third molecule, that is, chemical linker come Realize, there are two functional groups for chemical linker tool, each can with one of two individual molecules formation covalent bond, or same It is formed between the independent group of two of one molecule and is covalently attached (i.e. they will form " ring " that can surround polymer).It is illustrative Connector include the bifunctional linker that can be crosslinked two carrier proteins.Crosslinking may also occur between antigen and carrier protein.
" antigen " used herein means any molecule specifically bound with antibody or antibody fragment or molecular combinations.
" bifunctional linker " used herein means the compound there are two functional group, and each functional group can be respectively with two A individual molecule, atom or elements collection form covalent bond.For example, G.T.Hermanson (Bioconjugate Techniques, Academic Press, 1996) and Dick and Beurret (Conjugate Vaccines.Contribu.M Icrobiol.Immunol., Karger, Basal 10:48-114,1989) describe illustrative bifunctional linker.It is ideal Ground, bifunctional linker are glutaraldehyde, bis- [sulfonic group succinimide] suberates or dimethyl diimidate.
" connector " used herein means the compound or chemical bond that covalently connect two or more molecules.It is ideal Ground, connector are glutaraldehyde or formulaCompound, wherein RnIt is the direct-connected or branched alkyl, 1-12 of 1-12 carbon atom The direct-connected or branch of the direct-connected or branched heteroalkyl groups of a atom, the direct-connected or branched-chain alkene of 2-12 carbon atom, 2-12 carbon atom Alkine, the aromatic moieties of 5-10 carbon atom, the cyclic annular system of 3-10 atom ,-(CH2CH2O)qCH2CH2Wherein q is 1- 4 or connect two aldehyde radicals chemical bond.It can be directly attached without using connection molecule.For example, can with carbodiimide chemist or With the transglutaminase enzymatic catalysis for being catalyzed this cross-linking reaction, be directly connected to the carboxyl of protein with its amino.
" reinforcing antibody to generate " means that the activation in the memory B cell being exposed to for the second time in Process of Antigen occurs, referred to as " booster response " shows " secondary " anamnestic immune response of Shou Qichang, the antibody of Shou Qichang is caused to generate.
" carrier protein " means the protein for vaccine, causes to its own and/or to compound with carrier protein The immune response of antigen.It is desirable that the antigen by carrier protein by being contained in the composite come non-covalent with carrier protein Association.Even so, antigen and carrier protein can also covalent bonds each other.It is desirable that the carrier protein includes thin by T- The epitope of born of the same parents' identification.Multiple antigenic peptide (MAP) as branched peptide also includes by the definition of " carrier protein ".It is desirable that MAP packet Include lysine.It includes toxin and toxoid (chemical or gene) that the illustrative phase, which needs carrier protein, can be mutant.Reason Think that ground, carrier protein are diphtheria toxin or its mutant, diphtheria toxoid, tetanus toxin or its mutant, tetanus poison Element, pseudomonas aeruginosa (Pseudomonas aeruginosa) exotoxin A or its mutant, b subunit of cholera toxin, tetanus Toxin Fragment C, bacterial flagellin, pneumolysin, Listeria lysin O (and relevant molecule), Neisseria meningitdis Coccus (Neisseria menningitidis) outer membrane protein, pseudomonas aeruginosa (Pseudomonas aeruginosa) Hcp1 albumen, shiga-like toxin, people LTB albumen, has come from Escherichia coli (Escherichia coli) heat labile enterotoxin The dominant negative of the protein extract of whole bacterial cell, Bacillus anthracis (Bacillus anthracis) protective antigens Mutant (DNI) or Escherichia coli (Escherichia coli) beta galactosidase or it is any other can by connector be crosslinked Protein.
" DNI " means dominant negative mutant (DNI) albumen, is that Bacillus anthracis (B.anthracis) protectiveness is anti- The mutant form of former (PA), such as Benson (Biochemistry37:3941-3948,1998) are described.
" containing " used herein in relation to antigen means to be retained in physiological conditions and carrier protein is formed by Antigen in compound.It is formed by compound it is desirable that the antigen is contained in carrier protein, in antigen and carrier It is covalently attached between albumen there is no significant.As used in this article, there is no significant are covalently attached to refer to no more than 50% Antigen and carrier protein covalent bond.It is desirable that covalent no more than 40%, 30%, 10% or 5% antigen and carrier protein Connection.
" infection " means study subject by microorganism such as bacterium, fungi, helminth or virus attack.Infection may include example Such as, it is typically found in study subject body or the hyper-proliferative of the microorganism of body surface or is generally not present in study subject or table The hyper-proliferative of the microorganism in face.When excessive micropopulation is present in study subject body or body surface or works as micropopulation When the presence of body injures cell or the tissue of study subject is caused pathological symptom occur, infection of the study subject by microorganism.
" infectious agent " means the microorganism for causing infection.
" immunogenicity " means the compound in study subject Immune inducing in vivo immune response.It is desirable that described be immunized is answered Answer be T- cell rely on immune response, the generation including IgG antibody.
" microorganism " is bacterium, fungi, helminth or the virus for referring to that study subject is caused to infect.
" microbial capsular polymer " refers to the polymer for being present in microbial capsular coat inside or surface.It is desirable that Microbial capsular polymer be organic polymer for example polysaccharide, phosphoric acid polysaccharide, with N- acetyl group replace amino sugar polysaccharide, contain There is the sugared, polynary of the polysaccharide of sulfanilyl sugared (sulfanylated sugar), the sugar of other sulfates modification or phosphate modification Alcohol, polyaminoacid, techoic acid and lipopolysaccharides O side chain.
" monomer " is the molecular structure for referring to be formed two or more keys with same monomer, as one as " polymer " When part, the chain or a series of branches, connection chain of repeated monomer structure are often generated.
" organic polymer " refers to that the polymer of the monomer composition by being covalently attached, each monomer contain three or more Following atom: carbon, oxygen, hydrogen, phosphate, nitrogen and sulfate.It is desirable that the organic polymer is polysaccharide, phosphoric acid polysaccharide, has Polysaccharide, the polysaccharide containing sulfanilyl sugar, the sugar of other sulfates modification or the phosphate modification for the amino sugar that N- acetyl group replaces Sugar, sugar, polyalcohol, polyaminoacid, techoic acid and lipopolysaccharides O side chain.
" polyalcohol " refers to that the hydrogenated form of carbohydrate, carbonyl are reduced into primary hydroxyl or secondary hydroxyl.It is illustrative Polyalcohol be polyalkylene oxide (PAO), such as polyalkylene glycols (PAG), including polymethylene ethylene glycol, polyethylene glycol (PEG), methoxy poly (ethylene glycol) (mPEG) and polypropylene glycol；Polyvinyl alcohol (PVA)；Ethylene maleic acid anhydride copolymer；Benzene second Alkene-copolymer-maleic anhydride；Glucan including Sensor Chip CM 5；Cellulose includes methylcellulose, carboxymethyl fibre Tie up element, ethyl cellulose, hydroxyethyl cellulose, carboxyethyl cellulose and hydroxypropyl cellulose；Chitosan hydrolysate；Starch is such as Hydroxyethyl starch and hydroxypropul starch；Glycogen；Agar carbohydrates and their derivative；Guar gum；Amylopectin；Inulin；Xanthan gum；Card Draw glue；Pectin；Seaweed acid hydrolysis products；Sorbierite；Glucitol, mannitol, galactitol, arabite, gulose alcohol, Xylitol, threitol, sorbierite, fructose alcohol, glycerol, maltitol, the alcohol of cellobiose, the alcohol of sucrose, amylopectin alcohol, The alcohol of amylopectin；Or monopropylene glycol (MPG).
" polyaminoacid " refers at least two amino acid being keyed by peptide.It is desirable that the polyaminoacid is containing weight The peptide (i.e. homopolymer) of multiple amino acid sequence or same amino acid chain.
" reduced Schiff base " refers to azomethine or formula R1R2C=N-R3Compound (R1、R2And R3It is chemical structure, usually Contain carbon atom) it is exposed to the reducing agent that schiff bases double bond is saturated with hydrogen atom.The method of reduction is those skilled in the art institute Understand.
" specific binding " used herein about antibody or its segment refer to antibody or antibody fragment to relative to The specific protein such as increased affinity of antigen institute for any other protein of equivalent.It is desirable that according to standard is used Method such as enzyme-linked immunosorbent assay (ELISA) is measured, affinity at least 2 times, 5 of antibody or antibody fragment to its antigen Again, 10 times, 30 times or 100 times other antigens greater than any equivalent, including related antigen.
" study subject " refers to can be by the animal of microorganism infection.It is mammal that phase, which needs study subject, as people, monkey, Dog, cat, mouse, rat, cow, sheep, goat or horse.In the embodiment that the phase needs, the study subject is people, such as small Child.It is desirable that the study subject is baby, toddlers or prepubertal children.
" T- cell independent antigen " refers to the antigen for leading to antibody tormation under the cooperation of no T lymphocyte.It is ideal Ground, the T- cell independent antigen directly stimulate bone-marrow-derived lymphocyte without the cooperation of T lymphocyte.The illustrative phase needs T- cell independent antigen includes the poly- γ-D-Glu (PGA) of capsular antigen, alginic acid sour (alginates), glucan, polysaccharide (PS), polyaminoacid, polyalcohol and nucleic acid.
Advantage
Compared with currently available vaccines, existing vaccines technology, vaccine preparation of the invention is simple, does not tend to have Chemical Problem, does not tend to have immune ask Topic, cheaply, different target antigen and carrier protein is more suitable for than conjugation techniques, more flexiblely for generating polyvaccine (vaccine for resisting a variety of antigens).
Vaccine of the present invention does not need the covalent linkage between carrier protein and the antigen for being intended to induce immune response, thus Preparation method is simplified compared with conjugate vaccines technology and reduces preparation cost.Polysaccharide (PS)-albumen conjugate vaccines due to It is prohibitively expensive and can not be produced and sold in developing country；The chemistry and PS of the profession of the height as needed for every kind of vaccine and The production of carrier protein and purifying cost, traditional conjugate vaccines are difficult to produce cheaply.
Vaccine of the invention also shows the demand to that can induce the previous vaccine of difficult antigen of immune sex resistance safely. These vaccines can be (the inducing immune response with single antigen) of unit price or (exempting from a variety of antigen inductions are a variety of for multivalence Epidemic disease response).Showed that other the immune of difficult antigen was answered in the vaccine induction containing TLR (Toll-like receptor) ligand already It answers, but they have unsafe tendency compared with vaccine of the present invention, because TLR ligand usually inspires inflammation, even if low dose of Also toxic, have reactionogenicity, and may cause ill symptoms.
Other features and advantages of the present invention according to be explained in detail below, drawings and claims will be apparent.
Brief description
Fig. 1 is to induce anti-PS about conjugate vaccines (conjugate prepared between PS and carrier protein tetanus toxoid) The flow chart of the non-limiting proposal sexual approach of IgG immune response.In the model, the antibody receptor of identification PS is only shown B cell is in conjunction with PS protein conjugate.Therefore, carrier protein is incorporated into the B cell surface for showing correct PS binding specificity.
Fig. 2 is the western blot analysis figure of PCMV and control formulation, by sds polyacrylamide gel electrophoresis and with anti- The sero-fast western blot monitoring crosslinking situation of PA.DNI albumen is moved at 84kDa before glutaraldehyde cross-linking.As band Molecular weight is moved to as greater than confirming at 220kDa, PCVM1-PCMV3 (swimming lane 1-3) shows the extensive friendship of DNI albumen Connection.There is no the DNI being individually crosslinked under PGA to also show same high molecular weight species (species) (swimming lane 5).On the contrary, with PGA mixing but the DNI for not having to glutaraldehyde processing are shown and the band of DNI co-migrate or lower molecular weight species (swimming lane 4).
Fig. 3 is shown for measuring by three PCMV preparations (PCMV1-PCMV3, preparation 1-3) and two antigen control systems The ELISA measurement result of the anti-DNI immune response of IgM and IgG specificity in the Mice Body of the immunity inoculation of agent 4 and 5.DNI albumen exists All with the immunogenicity of height in all formulations other than not with the control formulation 4 of glutaraldehyde (glut) crosslinking.However, These special DNI immune responses are uniquely based on IgG.Although even anti-in being also not detected for the 7th day for immunity inoculation DNI IgM, but with 17 days mouse of PCMV preparation immunity inoculation and only with the mouse of DNI (preparation 5) immunity inoculation of crosslinking In can detect that significant anti-DNI IgG response.Noticed that with all formulations include the induction of preparation 4 for DNI at the 30th day Strong booster response.
Fig. 4 is shown for measuring by three PCMV preparations (PCMV1-PCMV3, preparation 1-3) and two antigen control systems The ELISA measurement result of the anti-PGA immune response of IgM specificity in the Mice Body of the immunity inoculation of agent 4 and 5.Anti- PGA IgM response is aobvious Typical capsular polymer mode is shown.Control formulation 4 generated detectable anti-PGA IgM response at the 7th day, but the response exists Do not reinforce within 17th day or the 30th day.All PCMV preparations induced anti-PGA IgM response at the 7th day, then at the 17th day and Stronger anti-PGAIgM response is generated within 30 days only.As expected, control formulation 5 (DNI being only crosslinked), which does not generate, is based on IgM or anti-PGA response based on IgG.
Fig. 5 is shown for measuring by three PCMV preparation (PCMV1-PCMV3；Preparation 1-3) and two antigen control systems The ELISA measurement result of the anti-PGA immune response of IgG specificity in the Mice Body of the immunity inoculation of agent 4 and 5.PCMV1-3 generates strong The anti-PGA response based on IgG, occurred at the 17th day, then strengthened the 30th daybreak is aobvious.
Fig. 6 shows immune preceding and passes through the PCMV (DNI-ALG C, DNI ALG A) containing DNI and alginates and contain " one pot " (" one pot ") trivalent PCMV preparation of compound DNI is immune with alginates (ALG), glucan (DEX) and PGA connects 30 days pooled serum IgM antibody potency after kind.
Fig. 7 display is by the PCMV (DNI-ALG C, DNIALG A) containing DNI and alginates and contains and alginates (ALG), 60 days antigen-specifics after " one pot " trivalent PCMV preparation immunity inoculation of glucan (DEX) and the compound DNI of PGA Titer of serum IgG antibody.
Fig. 8 display is by the PCMV (DNI-ALG C, DNIALG A) containing DNI and alginates and contains and alginates (ALG), 128 days anti-PS IgG are anti-after " one pot " trivalent PCMV preparation immunity inoculation of glucan (DEX) and the compound DNI of PGA Body potency.
Fig. 9 A and 9B are with obtained from American type culture collection and by the streptococcus pneumonia of Merck preparation (S.pneumoniae) the IL-6 measurement that polysaccharide (pss) or the pps obtained from Serum Institute of India (SII) are carried out Figure.
Figure 10 is the (trt 2 of impurity available processes 2 in the pss 6B shown obtained from SII；80 DEG C of heat preservations 1 in 1MNaOH Hour) removal figure.Handling 1 (trt 1) is a succession of 5 step phenol extraction, and protein is removed from polysaccharide.
Figure 11, which shows that the PCMV containing pss 6B is generated in induction IgG, to be comparedMore effectively.BSA=cow's serum Albumin；DT=diphtheria toxin；DTx=diphtheria toxoid；TTx=tetanus toxoid.
Figure 12 show the PCMV containing pss 6B induction IgM generations on andIt is equally effective.
Figure 13, which shows that the PCMV containing pss 6B is generated in induction IgG, to be comparedMore effectively.
Figure 14-16 shows that the PCMV containing pss 14 is generated in induction IgG and is about as much as(DTx=is white Larynx toxoid；TTx=tetanus toxoid).
It is described in detail
The present invention describes vaccine composition, the preparation method and medication of the composition, to provide immunity pair The not dependent antigen of anti-T cell or usually cause the antigen such as polysaccharide (PS) of faint immune response, polyalcohol, polyaminoacid and Other organic polymers.Vaccine of the invention has the effective immunological characteristic of typical case's PS albumen conjugate vaccines, but the phase needs ground Different from conjugate vaccines, difference is connected without significant covalently atom by target antigen such as PS or pod membrane organic polymer Object is connected to carrier protein.On the contrary, containing target antigen such as PS or pod membrane organic polymer with carrier protein.For example, can be can Pass through carrier protein molecule itself covalent cross-linking formation protein base in the presence of dissolubility antigen such as PS or pod membrane organic polymer Matter: these vaccines are referred to as protein matrix vaccines.Highly cross-linked carrier protein, which can be formed, each other can capture antigen and promote Matrix into antigen absorption and to the stimulation for generating antibody in immunocyte.Carrier protein matrix can be to seal antigen " grid " form or in which antigen be " rope " and protein or crosslinking protein compound are the shapes of a series of " beading " of " pearl " Formula.If carrier protein forms the three dimensional network of ring or entanglement antigen around antigen around antigen, carrier protein can be made to contain Antigen.In addition, the carrier and antigen can be crosslinking, such as by making antigen chain and carrier protein be crosslinked in chain. In the embodiment that the phase needs, the antigen and carrier protein are non-covalent linking.This non-covalent linking may include hydrophobic Effect, ionization, model ylid bloom action or hydrogen bond.Non-covalent linking may include antigen and protein complex noncovalent associations Physical geometry (see the metaphor of above " beading ").
Carrier protein is without itself being crosslinked to contain antigen.Such as mixed carrier albumen in aqueous solution can also be passed through With antigen and precipitophore albumen, antigen and protein co-precipitate is thus made to contain antigen.Can also by precipitating from antigen and Carrier protein mixture compound (such as alum, calgon, poly- phosphorus piperazine or by hydrophobic or ionization drive to egg White matter has the other polymers of affinity) with carrier protein contain antigen.The method of precipitating proteins is standard in this field It is changing and including, for example, (1) change the pH of mixture, (2) are by increasing or decreasing the concentration of inorganic salts in mixture Change the ionic strength of solution, (3) or trichloroacetic acid (TCA) or ammonium sulfate is added toward mixture, (4) heating mixture causes Protein coagulating (formed precipitating or gel), the protein of (5) in a certain way in chemical modification mixture prevent it from molten Solution, and (6) cause protein with ionising radiation (ultraviolet light, gamma-rays or β ray) the irradiation protein solution of enough flux Crosslinking and/or precipitating.
When using pathogen protein clostridium, this vaccine is called albumen pod membrane matrix vaccines (PCMV).Such as in embodiment Described in, prepare PCMV comprising be based on mode T cell dependent/non-dependent capsular antigen: poly- γ-D-Glu (PGA) And alginic acid (alginates) and glucan；With the PCMV of illustrative carrier protein DNI.PGA PCMV be easy to largely prepare and It was found that its induction typical case is in the immune response of PGA albumen conjugate vaccines.Can much may be used in the presence of any target antigen with any The connector of energy is crosslinked any many possible carrier proteins to prepare vaccine of the invention.There is discussed herein illustrative and Preferred connector, carrier protein and target antigen.
Polysaccharide (PS) is the polymer of sugar.PS from pod membrane is to be related to exempting from for the protectiveness of capsular bacterium pathogen The first antigen component (primary antigenic component) of epidemic disease, these bacterial pathogens for example have meningitis Neisser Family name coccus (Neisseria meningitidi), streptococcus pneumonia (Streptococcus pneumoniae), Salmonella typhimurium Bacterium (Salmonella typhi) and influenza B haemophilic bacterium (Haemophilus influenzae Type B).With based on micro- The immunity inoculation teenager and adult of the vaccine of biological PS have succeeded in terms of reducing disease puzzlement, but are proved for mentioning It is lower for effect in terms of child of tender age (i.e. 24 months children below) protective immunity.The adaptive immune system of child of tender age All reach maturity not yet, and the immunogenicity of T cell independent antigen such as pod membrane PS is weak and cannot be young at these Cause long-term protective immune response (i.e. immunological memory response) in vaccine recipient.
It is anti-that T cell independent antigen such as PS can be converted into T cell dependence by the chemical coupling of PS and protein It is former；This chemical coupling process referred to as " is conjugated " and the atom being related in PS structure and the side for being present in " carrier " Argine Monohydrochloride Covalent linkage between chain atom is formed.This " conjugate vaccines " more effectively promote induction B cell maturation and isotype conversion, Cause the antibody for having the level of correct anti-PS protectiveness overview (profile) much higher.Protection antibody is to its PS antigen Have a high-affinity, usually immunoglobulin G (IgG) subclass, with complement fixing activity and opsonin effector activity The antibody of phase in longevity length.
Fig. 1, which is shown, induces anti-PS IgG immune response about the conjugate between PS and carrier protein tetanus toxoid Exemplary, non-limiting approach.In the model, it only shows and can recognize that the B cell of the antibody receptor of PS and PS albumen are conjugated Object combines.Therefore, carrier protein is incorporated in the B cell surface for showing correct PS binding specificity.Albumen-PS compound quilt These B cells are absorbed into intramolecular vacuolar, and carrier is acted on by protein degradation and being processed herein.Derived from protein carrier Peptide be transported and load in the presentation ditch (presentation groove) of MHC II receptoroid (MHC-II).The MHC- II- carrier peptide complexes are showed in the surface of B cell.When MHC-II- peptide complexes are identified by T cell receptor (TCR), T is thin Born of the same parents, which are activated and secreted, provides the cell factor of " auxiliary " for induction B cell differentiation.B cell is quantitatively expanded and is broken up At " thick liquid cell " of ready-made secretory antibody.Initial immunoglobulin M (IgM) is generated by thick liquid cell but the auxiliary of final T cell causes So that thick liquid cell is carried out class switch and generates the antibody such as IgG of other isotypic class.This process is prominent as thick liquid cell is undergone Become and continue, causes to generate the antibody receptor for having more high-affinity to PS protein conjugate.When antigen is removed, only high parent PS protein conjugate activation remaining in the circulating cycle is retained with the thick liquid cell of power.The maturation that the T cell of thick liquid cell relies on Continuation, lead to the expansion of thick liquid cell group, generate the IgG class antibody of high-affinity.It is tested right through immunity inoculation by measuring As the level of PS IgG antibody anti-in such as human serum, dilatation can be easily monitored.
Final mature and conversion process leads to Shou Qichang's and the memory B cell special to PS generation.Memory B cell tool There is unique characteristic, i.e., is activated immediately if being exposed to PS.Activation causes memory B cell to be proliferated and quickly generates anti-PS IgG.Occur to be referred to as " booster response " in the activation for the memory B cell being exposed to for the second time in PS Process of Antigen and shows the phase in longevity Long " secondary " anamnestic immune response.Initial immunity can stimulate the generation of IgM antibody and some IgG antibodies.In secondary immunity, i.e., When " reinforcement " is injected, it is stimulated by the memory cell of initial immunity sequencing and generates a large amount of IgG, is i.e. anamnestic immune Response.
T cell independent antigen does not stimulate lasting immunity, the i.e. generation of IgG antibody usually, but can stimulate more inefficient More provisional IgM antibody generation.Thus, PS antigen does not generate IgG booster response individually usually.However, if with PS protein conjugate carries out initial immunity, and PS will generate booster response, because the memory cell that is induced by conjugate by Sequencing generates IgG.Really, by through vaccine inoculation animal or the intracorporal booster response of people be considered as simulation due to exposure In the aversion response that the microorganism for showing PS generates；This long-term memory is for after the conjugate vaccines immunity inoculation several years The vaccine to work in terms of protecting immunization is very crucial.Therefore, the value of PS protein conjugate is (1) they is lured The ability of the high-level IgG of impedance PS antigen, ability of (2) their inductions for the anamnestic immune response of PS antigen.PS antigen Do not show that these characteristics are thus inferior antigen usually.Difficulty and their production cost in terms of synthesizing conjugated antigen Cause the development progress of the conjugate vaccines for many bacterial diseases (may be wherein protectiveness to the immune response of PS) slow.
Other T cell independent antigens include amino acid homopolymer, such as poly- γ-D-Glu (PGA) and polyalcohol. Really, most of biopolymers are T cell independent antigens.Repeat property of the polymer due to its chemical structure (epitope) It can be crosslinked with their b cell immunoglobulin (Ig) receptor is identified.Therefore polymer can mode same as polysaccharide activate B The IgM of cell generation resistant polymers.For example, amino acid homopolymer, Bacillus anthracis (Bacillusanthracis) it is poly- γ-D-Glu (PGA) is the weak capsular polymer with T cell independent antigen of immunogenicity.By sewing with protein carrier The vaccine of the PGA composition of conjunction is high degree of immunogenicity, can induce anti-PGA IgG and have immunological memory to PGA.Therefore. Most polymers react as PS in terms of their immunogenicity because they can not under MHC-II background quilt Processing and displaying, therefore the auxiliary of T cell cannot be raised.It is referred to as Toll-like receptor in some naturally occurring and another class (TLR) exception is found in the polymer of acceptor interaction.Once being activated, TLR can be by host cell come inducing cell The factor generates and adaptive immune response is made to generate change.Some PS and TLR ligand are covalently attached or are matched body pollution by this (contaminate).For example, lipopolysaccharides (LPS) is high degree of immunogenicity, the PS for inducing IgG and memory response；The rouge A of LPS Part is TLR ligand, can be responsible for immunological characteristic.
In another example, it has been found that a small number of pneumococcus PS show they are not connected with protein carrier to be sewed Certain immunological characteristics of vaccine are closed, i.e., they are induced converts to the isotype of IgG.Recently, business polysaccharide vaccine Pneumovax-23, and individual PS of streptococcus pneumonia (Streptococcus pneumoniae) different strains are come from, hair Body pollution (Sen etc., J.Immunol.175:3084-3091,2005) is now matched by TLR.This discovery can explain why this A little PS preparations can be induced to the isotype of IgG under there is no albumen conjugation and be converted.These pneumococcus PS induce macrophage thin Intracrine IL-6 and TNF-α.Inducing macrophage secretion is then unable to however, extracting by phenol and extracting the PS through being further purified Cell factor.In immune Research, the PS immunogenicity through phenol extraction is weak and no longer induces anti-PS IgG.Therefore, phenol mentions Remove the pollution molecule for being responsible for these abnormal immune characteristics of the PS preparation.Pollution molecule, which seems to be to provide, activates macrophage thin The TLR ligand of born of the same parents' TLR dependent cell factor responsibility.The dirt is eliminated to being further purified for PS by phenol extraction It contaminates TLR ligand and PS is caused to go completely into T cell dependence.
Above example illustrates that PS antigen can play effect as PS- albumen conjugated antigen without protein and carbon The covalent linkage of hydrate.Unfortunately, TLR ligand is usually proinflammatory disease.Even for example, the dosage of LPS very little It is toxic.Therefore, although TLR ligand to be mixed to the immune response that may expand to PS with PS, this method is also possible to produce Raw tool reactionogenicity or cause the vaccine of vaccine recipient's ill symptoms.Conjugate vaccines technology, which remains, to be used to prepare with required The optional method of immunogenicity spectrum and the PS vaccine of safety.
Children's disease as caused by aggressive bacterial pathogens has been greatly reduced with the exploitation of PS- albumen conjugate vaccines Disease burden.It include anti-influenza B haemophilic bacterium (Haemophilus influenzae B Type) for several these less vaccines Vaccine and certain meningococcus and strains of streptococcus are that market can be sold in developed country.These PS- albumen conjugate vaccines exist Developing country can not be produced and sold due to prohibitively expensive in developing country.For example, the septivalency pneumonia ball that business can be sold Bacterium conjugate vaccines are worth about every dose $ 58 (dollar at 2006), and need to take four doses.Only price just makes these vaccines The people for undertaking Disease Spectrum in developing country for those are beyond one's ability.
Production and purifying cost, traditional conjugate vaccines due to related chemistry and PS and carrier protein are difficult to carry out It produces at a low price.Usually require that there is comparable purity in conjugation the former two, chemical reaction is just able to reasonable coupling efficiency Come carry out.In general, conjugation chemistry must various PSs unique to the chemical aspect in selected PS and carrier protein prove effective.This idol Connection chemistry introduces functional group in PS, and the latter can connect by the ε amino side chain of lysine with carrier protein thereafter.Introduce these Epitope that the PS chemical modification of coupling group can destroy on PS simultaneously introduces new epitope (for example, the glycosyl phase with connector or modification The epitope of pass), conspicuousness, which only passes through conscientious careful immune analysis, to be assessed.In addition, for traditional PS- albumen Conjugate vaccines, the property and connection of PS molecule amount, selected carrier that the size of PS, each protein carrier molecules are connected The type of chemistry can influence the immunogenicity of conjugate vaccines.Thus, for example in pneumoccoccosis (wherein known to every kind of 90+ Serotype all has different PS structures (Bentley etc., PLOS Genetics 2 (3): e31262-269,2006)) the case where Under, a kind of single conjugation methods may be not suitable for all serotype.Repeat synthesis (reproduciblysynthesize) It is related to the conjugate vaccines for repeating immunological characteristic (reproducible immunological property): to PS's The property of PS molecule amount, selected carrier that size, each carrier protein molecule are connected and the type of connection chemistry carry out Carefully control, and this increases the cost of manufacture conjugate vaccines significantly.
The appearance of antibiotic resistance makes more prominent to safely and effectively vaccine urgency is developed.Manufacture widely available epidemic disease Seedling, for developing country, it is also necessary to which the vaccine of cost is saved in manufacture.Make for from one or more bacteriums The conjugations of a variety of polysaccharide antigens of type different serotypes joins seedling, in immunization protocol childhood being merged into, this will simplification it is high-risk The vaccine of crowd is administered.However, existing conjugate vaccines technology is not cost-effective, therefore connection seedling (combination is conjugated Conjugate vaccine) it is almost impossible universal in developing country.Really, or even in the hair that powerful market has had been established Up to country, the short supply of nearest Wyeth 7- valence conjugation Pnu-Imune 23 illustrates to produce and store to need complicated conjugation epidemic disease The vaccine of seedling synthetic technology is how difficult.
In the embodiment that the phase needs, vaccine of the invention is multivalence pod membrane matrix vaccines (PCMV), one of or more A bacterial capsule component is contained into multivalent carrier albumen substrate.Preparation PCMV is easy to, because as one of starting material Target antigen, such as pod membrane, it is thus only necessary to purity appropriate.For example, the poly- γ-D-Glu (PGA) of Vedan is also impure (it carries proteinase activity on DNI), as described in this article, behavior are non-dependent exactly as with the T cell suspected Property antigen (embodiment 1).In all three PCMV preparations (protein and PGA ratio are different in its 7 times of range above) PGA to participating in for PCMV be successful.
Because the method for preparing vaccine of the present invention does not need the chemical knowledge of any target antigen such as capsular polysaccharide, the party Method does not depend on the needs for the cross-linking chemistry that exploitation and target antigen and carrier protein chemistry can be held.Although certain antigens still may be with Connector interaction, but this should not detract the efficiency of vaccine, because the crosslinking unintentionally of target antigen and carrier protein is anyway It will can expect with immunogenic attributes.In vaccine of the invention, the crosslinking of target antigen and carrier protein is not to make epidemic disease Seedling is effectively necessary.This forms sharp contrast with traditional conjugate vaccines, therefore is hindered in its preparation and exploitation.This hair The bright vaccine phase need to have at least, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, the carrier protein of 95%, 98% or even 100% crosslinking, and is no more than, for example, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% target antigen and carrier protein are crosslinked.It is desirable that being no more than 10% Antigen and carrier protein crosslinking and at least 50% carrier protein be crosslinked.
The method described herein for preparing vaccine is modified extensively without result in target antigen such as capsular polymer.It is anti- Original generally remains same state, and possible modification is the PS pod membrane that this group is for example carried for the end in polymer chain For reduced sugar reduction (reduction).This small modification can not influence the immunogenicity of most of pod membrane PS, Because polymeric inner residue is 100-1000 times abundanter than end sugar.On the contrary, for traditional conjugate vaccines, it usually needs will connect Head group introduces antigen such as capsular polymer, the covalent linkage point as carrier protein.It needs using connector, because many anti- Former such as capsular polymer is not used as reactive group such as carboxyl or amino of a part of its structure.For example, by linker chemistry Introduce PS can lead to pod membrane epitope destruction and due to its with host's Self Epitopes unknown immune cross-reactivity and in vaccine Undesirable new epitope is generated in product.
Vaccine preparation method described herein is complicated not as good as conjugate vaccines technology, because its chemistry relies only on carrier protein (such as DNI, b subunit of cholera toxin, diphtheria toxin, Tetanus toxin fragment C or Escherichia coli (Escherichia coli) β- Galactosidase) cross-linking chemistry.Although having no effect on friendship for example, capsular polymer influences crosslinking rate when mixing with DNI The mode or degree of connection, the two are more controlled by used protein, concentration and the crosslinking agent (such as penta 2 being added Aldehyde) concentration.These parameters can be easily adjusted, thus reduction prepare vaccine needed for time and effort, save money.
The method described herein for preparing PCMV can be used, together with for example any capsular polymer of any antigen or any Polymer and any carrier protein being crosslinked with a small number of amino or without amino for example can be by boron hydrides also The carrier protein of the critical epitopes of former action breaks down.The carrier protein phase used in method described herein need to have at least 2 A lysine residue or (unblocked) of opening and the other residues that can be crosslinked through chemical modification.Tetanus toxoid It is a kind of possible carrier protein.The toxin is detoxified by using formaldehyde, and formaldehyde is the examination reacted with the amino of protein Agent.The carrier protein that other phases need includes b subunit of cholera toxin (purchased from SBL Vaccin AB), diphtheria toxin, tetanus toxin Segment C (being purchased from SigmaAldrich), DNI or the beta galactosidase from Escherichia coli (Escherichia coli) (are purchased From Sigma Aldrich).
They are then mixed by synthesizing an other conjugate vaccines first and generate " mixture " by current multivalence conjugate vaccines Conjugate vaccines come prepare (for example, Wyeth septivalency Pnu-Imune 23,).By by the different antigen of chemistry for example Pod membrane organic polymer it is admixed together it is subsequent be crosslinked carrier protein with such as glutaraldehyde, or the sheet by will be respectively synthesized Specific vaccine mixing is invented, the vaccine that the present invention prepares the method preparation multivalence of vaccine can be used.The flexibility is the present invention The method of preparation polyvaccine provides significant advantage.
The illustrative vaccine PCMV vaccine #1-3 of the present invention discussed in embodiment, performance is just like conjugate vaccines, to the greatest extent In fact these vaccines synthesize pipe by the not expected method for generating any covalent bond, and the covalent bond is constituting PGA molecule Atom and constitute DNI albumen atom between formed.Glutaraldehyde exclusively with egg representated by the ε amino as lysine residue White matter amino side chain reacts.PGA polymer does not have free amine group and only has the carboxylic side-chain that do not react with glutaraldehyde.Cause This, immune response caused by the similar conjugate generated as PCMV shows that long PGA molecule by molecule is contained in DNI albumen Matter molecule cross-link content.
According to non-limiting mode, the effect contained is that carrying DNI albumen and PGA enter B cell, and B cell is borrowed Help the Ig receptor of Immune discrimination PGA in conjunction with this matrix.Once being absorbed by these B cells, the matrix with traditional to sew It closes the similar mode of vaccine to be degraded, this will lead to the production of the DNI derived peptide shown on the MHC-II molecule of corresponding B cell It is raw.This raises the attached amplification and maturation helped so as to cause this B cell of T cell immediately, becomes and generates the production special to PGA IgG thick liquid cell and memory cell.Therefore, according to non-limiting mode, PCMV is similar to albumen-conjugated capsular epidemic disease in immune gone up The effect of seedling but it is completely different because PCMV lacks the significant covalent linkage between carrier protein and capsular polymer.
Vaccine of the invention including PCMV can be used for combining, such as pediatric vaccine.In addition, epidemic disease of the invention Seedling can be used for being inoculated with confrontation, for example, pneumococcal infection, (A group and B group) streptococcal infection, influenza B haemophilic bacterium (Haemophilus influenzae Type B) (" HiB ") infection, meningococcus are (for example, Neisseria meningitidis (Neisseriameningitides)) it infects, and can be used as from gramnegative bacterium (for example, pseudomonas aeruginosa (Pseudomonas aeruginosa), soil draw hot Francisella (Francisellatularensis) (Thirumalapura etc., J.Med.Microbiol.54:693-695,2005；Vinogradov and Perry, Carbohydr.Res.339:1643-1648,2004；Vinogradov etc., Carbohydr.Res.214:289-297, 1991), Shigella (Shigella species), salmonella (Salmonella species), acinetobacter calcoaceticus (Acinetobacter species), bulkholderia cepasea (Burkholderia species) and Escherichia coli (Escherichia coli)) O antigen vaccine.
Vaccine of the present invention can be prepared in the following manner: one or more for example in target antigen described herein In the presence of, using it is any for example those connector described herein make it is any for example those in carrier protein described herein Crosslinking.If using a kind of target antigen, albumen substrate vaccine of the present invention if is considered as univalent vaccine.If using being more than one Kind target antigen, albumen substrate vaccine of the invention is then considered as polyvaccine.If microbial capsular polymer is target Antigen, albumen substrate vaccine of the present invention are then considered as albumen pod membrane matrix vaccines (PCMV).
Connector
Carrier protein crosslinking is well known in the art, including glutaraldehyde, maleimidobenzoyl- N-hydroxysuccinimide base ester, carbodiimide and bisazo benzidine.
Using homogeneity is difunctional or heterobifunctional linker is directly crosslinked the conventional method of carrier protein and part has and retouches State, for example, in G.T.Hermanson in Bioconjugate Techniques, Academic Press, 1996 and Dick and Beurret in Conjugate Vaccines.Contribu.Microbiol.Immunol., Karger, Basal 10:48- In 114,1989.For example, theoretically, there is n+1 primary amine for the carrier protein with the lysine moiety that number is n (including terminal amine) can be used for reacting with the carboxyl of illustrative crosslinking agent.Therefore, if using the direct conjugation methods, product is only limited N+1 amido bond is formed by having.
The connector used in the embodiment that the phase of the invention needs is most simply but two carrier proteins of connection Key.The connector can be the molecular skeleton of straight chain, ring-type or branch, has and is covalently attached with two carrier proteins (A) and (B) Side group.Any given carrier protein can all be connect with more than one carrier protein, be resisted to generate and can close (enclose) The carrier protein matrix of former interconnection.
Term linking group refers to generated covalently by reactive moieties and functional group (A) and (B) combination of connector (L) Key.The example of the linking group includes but is not limited to: ester, carbamate, thioesters, imines, disulphide, amide, ether, sulphur Ether, sulfonamide, isourea, isothiourea, imidoate, amidine, phosphoramidate, di-phosphate ester, thioether and hydrazone.
(A) realized with the connection of (B) by covalent mode, including be located at (A) and (B) on one or more functions Group forms key (linking group).The example that can be used for the chemical reaction functional group of this purpose includes but is not limited to: amino, hydroxyl, Sulfydryl, carboxyl, carbonyl, thioether (thioether), guanidine radicals (guanidinyl), imidazole radicals and phenolic group, all these groups are all deposited It is in the natural amino acid in many carrier proteins.
Therefore the available connector (L) containing reactive moieties influences the covalent linkage of (A) and (B), the reactive moieties can with deposit It is the functional group reactions of (A) and (B).The product of the reaction is linking group, contains connection (L) and (A) and connects Connect the key of (L) Yu (B) newly formed.For example, hydroxyl (A) can see below with the derivatives reaction of (L) carboxyl or its activation, Lead to the formation of ester linking group.
The example for the part that can be reacted with sulfydryl includes XCH2The alpha-halogenate acetyl of CO- type (wherein X=Br, Cl or I) Based compound, show it is specifically reactive with sulfydryl, but can also be used in modification imidazole radicals, thioether (thioether), phenol and Amino, this is as described by such as Gurd, MethodsEnzymol.11:532,1967.N- maleimide derivatives are considered There is selectivity to sulfydryl, but coupling amino can be additionally useful under given conditions.If connection by formed disulphide bridges into Row, then such as by amino convert introduce sulfydryl 2- iminothiolane reagent (Traut, Biochemistry 12:3266,1973) it can be considered as mercapto reagent.
The example for the reactive moieties that can be reacted with amino includes, for example, alkanisation and acylating reagent.Representative alkanisation Reagent includes:
(i) alpha-halogenate acetyl compounds show the specificity to amino in the absence of sulfhydryl-group activity and are XCH2CO- type (wherein X=Cl, Br or I), this is described as Wong (Biochemistry 24:5337,1979)；
(ii) N- maleimide derivatives, can by Michael react or by be added cyclocarbonyl acylation with Amino reaction, this is retouched as Smyth etc. (J.Am.Chem.Soc.82:4600,1960 and Biochem.J.91:589,1964) It states；
(iii) aryl halide, such as reactive nitro halogen aromatic compounds；
(iv) alkyl halide, such as such as McKenzie (J.Protein Chem.7:581,1988) are described；
(v) aldehyde and ketone to form schiff bases can be reacted with amino, usually obtain what stable amine made to be formed by reduction Addition product is stablized；
(vi) epoxides can be reacted such as epoxychloropropane and bisoxirane with amino, sulfydryl or phenolic hydroxyl group；
(vii) s- triazine contain chlorine derivative, have very strong reactivity with nucleopilic reagent such as amino, sulfydryl and hydroxyl；
(viii) aziridine of above-mentioned s- triaizine compounds, as Ross (J.Adv.Cancer Res.2:1,1954) is retouched It states, is reacted by open loop with nucleopilic reagent such as amino；
(ix) side's diethyl phthalate as described in Tietze (Chem.Ber.124:1215,1991)；With
(x) alpha-halogenate alkane ether is the Alkylators more more reactive than general alkyl halide, because ether oxygen atom generates work Change effect, this is as described by such as Benneche etc. (Eur.J.Med.Chem.28:463,1993).
Representative amino reacts acylating reagent
(i) it is derivative to be respectively formed stable urea and thiocarbamide for isocyanates and isothiocyanates, especially aromatic derivative Object；
(ii) sulfonic acid chloride is described by Herzig etc. (Biopolymers 2:349,1964)；
(iii) acyl halide；
(iv) active ester, such as nitro phenyl ester or N-hydroxy-succinamide ester；
(v) acid anhydrides, such as mixed acid anhydride, symmetric anhydride or N- carboxylic acid anhydrides；
(Vi) other that useful reagent is formed for amido bond, such as M.Bodansky (Principlesof Peptide Synthesis, Springer-Verlag, 1984) it is described；
(vii) acyl azide, such as the azido generated with sodium nitrite by prefabricated hydrazide derivatives, such as Wetz etc. (Anal.Biochem.58:347,1974) is described；With
(viii) imidoate reacts to form stable amidine with amino, this is such as such as Hunter and Ludwig (J.Am.Chem.Soc.84:3491,1962) is described.
Aldehydes such as glutaraldehyde and ketone can react to form schiff bases with amine, can be acted on and advantageously be carried out surely by reduction amination It is fixed.Alkoxyamino part is easy to generate stable alkoxyamine (alkoxamine) with ketone and aldehyde reaction, this such as such as Webb (Bioconjugate Chem.1:96,1990) is described.
The example for the active part that can be reacted with carboxyl includes diazonium compound such as diazo acid ester and diazo second Amide, they carry out reaction with high degree of specificity and generate ester group, such as Herriot (Adv.Protein Chem.3:169,1947) It is described.It can also be used carboxylic acid to modify reagent such as carbodiimides, amido bond passed through by the formation of O- acylureas again Formation is reacted to obtain.
Functional group in (A) and/or (B), if it is desired, other functional groups can be converted to before the reaction, such as to assign Give other reactivity or selectivity.Method example for this purpose includes: to turn amine using the reagent such as dicarboxylic acid anhydride Change carboxylic acid into；Use reagent such as N- acetyl-homocysteine thiolactone, S- acetyl mercapto succinic anhydride, 2- imino group mercaptan Polymorphs Alkane reaches or succinimidyl derivative containing thiol base makes amine be converted into mercaptan；Use the examination of such as α-halogen acetic acid ester Agent makes mercaptan be converted into carboxylic acid；Reagent using such as aziridine or 2- bromine ethamine makes mercaptan be converted into amine；Use such as carbon two The reagent of imines then makes carboxylic acid be converted into amine using diamines；Thio second is then used using the reagent of such as paratoluensulfonyl chloride Acid esters carries out ester interchange and is hydrolyzed into mercaptan with sodium acetate to make alcohol be converted into mercaptan.
If necessary, so-called distance of zero mark connector can be used according to the present invention, is related to active chemical group (A) and activation Group (B) is learned directly to be covalently attached in the case where not introducing other connecting material.Compound included by example, wherein (L) chemical bond for representing the oxygen atom in connection (A) and the carbonyl or thiocarbonyl moiety that are present on (B), causes linker Group is ester or thioesters.For example, A-L-B can be generated by using carbodiimide chemist, amino (A) is made to be connected to carboxyl (B), wherein L is amido bond or the R-C=O for being connected to N-R, and wherein R is from same or two different proteins molecules amino acid side The carbochain of chain.
However, most commonly, the connector includes two or more active parts connected by above-mentioned spacer element. The presence of spacer allows bifunctional linker to react with the particular functional group in (A) and (B), generates between the two compounds It is covalently attached.Active part in connector (L) can identical (homobifunctional linker) or it is different (heterobifunctional linker, Or wherein there is the heterogeneous multifunction conjunction of several different active parts), this provides and a variety of can generate between (A) and (B) The potential reagent being covalently attached.
Spacer element is usually made of chain, these chains efficiently separate (A) and (B) by following group: 1-10 carbon is former The direct-connected or branched alkyl of son, the direct-connected or branched heteroalkyl groups of 1-10 atom, 2-10 carbon atom direct-connected or branched-chain alkene, The direct-connected or branched alkyne of 2-10 carbon atom, the aromatic moieties of 5-10 carbon atom, the cyclic annular system of 3-10 atom or- (CH2CH2O)nCH2CH2(wherein n is 1-4).
The property of the introduced foreign material of bridging agent may pharmacokinetics to final vaccine product and/or activity have It influences.Therefore, the phase need to introduce the connector of cleavable, it contains between biodegradable or chemical-sensitive or incorporation restriction enzyme site Every arm.
The connector of these cleavables, such as retouched in PCT Publication WO 92/17436 (incorporated herein by reference) It states, is easily biodegradable in vivo.In some cases, linking group is cleaved in the presence of esterase, this enzyme not In the presence of then keep stable.Therefore, it may be advantageous to connect (A) and (B) to allow them to pass through near disease sites Active enzyme and slow release.
Connector can form the linker of biodegradable diester with Formulas I, diamides or diamino acid ester group Group:
--(Z1)o-(Y1)u-(Z2)s-(R11)-(Z3)t-(Y2)v-(Z4)p-
I
Wherein, Z1、Z2、Z3And Z4Each of be independently selected from O, S and NR12(wherein R12It is hydrogen or alkyl)；Y1And Y2In Each be independently selected from carbonyl, thiocarbonyl, sulfonyl, phosphoryl or similar acid and form group；O, in p, s, t, u and v Each stand alone as 0 or 1；R11It is the direct-connected or branched alkyl of 1-10 carbon atom, the direct-connected of 1-10 atom or branch Chain miscellaneous alkyl, direct-connected or branched-chain alkene, direct-connected or branched alkyne, the 5-10 carbon of 2-10 carbon atom of 2-10 carbon atom are former Aromatic moieties, the cyclic annular system of 3-10 atom ,-(CH of son2CH2O)qCH2CH2Wherein q is 1-4 or general-(Z1)o- (Y1)u-(Z2)sIt is connected to-(Z3)t-(Y2)v-(Z4)pChemical bond.
The illustrative phase used in the present invention needs connector (L) that can be described with any one of Formula II-III molecule:
Wherein the connector and oxygen atom (A) and oxygen atom (B) are covalently attached.Therefore, the connector (L) of Formula II-III passes through Two pyrans, ester or carbamate linking group are connect with carrier protein (A) and (B).In these embodiments, R13Represent 1- The direct-connected or branched alkyl of 10 carbon atoms, the direct-connected or branched heteroalkyl groups of 1-10 atom, 2-10 carbon atom direct-connected or Branched-chain alkene, the direct-connected or branched alkyne of 2-10 carbon atom, the aromatic moieties of 5-10 carbon atom, the ring-type of 3-10 atom System, wherein n be 1-4-(CH2CH2O)nCH2CH2Or the chemical bond of two nitrogen of connection or two carbonyls.
The connector designed to form hydrazone bond has Formula I V:
Wherein Z5Selected from O, S or NR16；R16It is hydrogen or alkyl；R15Selected from hydrogen, alkyl or miscellaneous alkyl；Y3Selected from carbonyl, thio Carbonyl, sulfonyl, phosphoryl form group with the similar acid that oxygen atom (A) is covalently attached；W is 0 or 1；R14It is 1-10 The direct-connected or branched alkyl of carbon atom, the direct-connected or branched heteroalkyl groups of 1-10 atom, 2-10 carbon atom direct-connected or branch Alkene, the direct-connected or branched alkyne of 2-10 carbon atom, the aromatic moieties of 5-10 carbon atom, 3-10 atom ring bodies System, n are 1-4-(CH2CH2O)nCH2CH2Or general-(Y3)-(Z5)wIt is connected toChemical bond；And X4It is Hydrazone is generated, wherein X by the condensation reaction of (B) containing hydrazide group and connector II precursor4It is the oxygen atom of ketone or aldehyde radical.
Carrier protein
In general, any carrier protein that can be contained in physiological conditions with antigen can be used in the present invention. It is desirable that the antigen and carrier protein contain in the composite, and there is no significant total between antigen and carrier protein When valence connects.It is covalently attached there is no significant, it is intended that the antigen no more than 50% is connect with carrier.In the embodiment that the phase needs In, antigen and carrier protein no more than 40%, 30%, 10% or 5% are covalently attached.Antigen/the carrier protein complex Can be containing another compound such as alum, and this another compound, in the embodiment that the phase needs, can contain antigen and Carrier protein.
It is desirable that the carrier protein for vaccine of the present invention is those individual or protein with antigen binding, draw Play the immune response of study subject.It is desirable that the carrier protein contains at least one epitope identified by T cell.It is desirable that The epitope can induce the t cell response of study subject, and B cell is induced to generate the antibody for being directed to entire target antigen.With In describing epitope of the invention, any decision on the antigen of idiosyncrasy is carried out with antibody molecule or its segment including being responsible for Cluster.Epitopic determinants are usually made of the chemical active surface cluster (grouping) of the molecule of such as amino acid or carbohydrate side chain, tool There are specific three-dimensional structural feature and specific charge characteristic.In order to which with immunogenicity, protein or polypeptide usually can Stimulate T cell.However, immunogenicity can also be had by the carrier protein for the epitope that T cell identifies by lacking.
By selecting the known carrier protein for causing strong immunization response, can be treated with PCMV as described herein The study subject of different groups.The carrier protein phase need to cause the strong immunization response to vaccine completely irrelevant (sufficiently foreign).Typically, used carrier protein is point that can assign target antigen immunogenicity Son.In the embodiment that a phase needs, carrier protein inherently high degree of immunogenicity.Therefore, with the immunogene of height Property and the maximized carrier protein of antibody production for the antigen compound with it can be made to be ideal.
Various carrier proteins of the invention include, for example, toxin and toxoid (chemical or hereditary), can be or Person is not mutant, such as Anthrax toxin, PA and DNI (PharmAthene, Inc.), diphtheria toxoid (Massachusetts State BiologicalLabs；Serum Institute of India, Ltd.) or it is CRM 197, tetanus toxin, broken Wind toxoid (Massachusetts State Biological Labs；Serum Institute of India, Ltd.), It is tetanus toxin fragment Z, pseudomonas aeruginosa (Pseudomonas aeruginosa) exotoxin A or exotoxin A mutant, thin Bacterium flagellin, pneumolysin, Neisseria meningitidis (Neisseria meningitidis) outer membrane protein (bacterium Strain by ATCC provide (American Type culture center, Manassas, VA)), pseudomonas aeruginosa (Pseudomonasaeruginosa) Hcp1 albumen, Escherichia coli (Escherichia coli) heat labile enterotoxin, will are congratulated Sample toxin, people LTB albumen, the protein extract from whole bacterial cells, any other egg that can be crosslinked by connector It is white.It is desirable that the carrier protein is b subunit of cholera toxin (being provided by SBL Vaccin AB), diphtheria toxin (Connaught, Inc.), Tetanus toxin fragment C (being provided by Sigma Aldrich), DNI come from Escherichia coli (Escherichiacoli) beta galactosidase (being provided by Sigma Aldrich).The carrier protein that other phases need includes ox Seralbumin (BSA), P40 and chicken riboflavin.(unless otherwise noted, illustrative carrier protein is purchased from Sigma Aldrich ) other illustrative carrier proteins are MAP (multiple antigenic peptide) as branched peptide.By using MAP, crosslink density is due to multiple The amino acid residue of branch and reach maximization.The Exemplary amino acid that can be used to form MAP is but is not limited to lysine.
BSA and keyhole limpet hemocyanin (KLH) are usually all used as carrier in the vaccine development tested with animal. The carrier protein for having been used for preparation treatment vaccine includes, but are not limited to the toxoid of many pathogenic verticillium toxins and they.Example packet Include diphtheria and tetanus toxin and their medically acceptable corresponding toxoids.Other candidate vectors albumen are on antigen With protein as the Bacterial toxin of referred to as cross reaction material (CRM).Carrier protein of the invention may also include any non- The albumen being not present in source of people and any human foods.
In the embodiment that the phase of the invention needs, the protein for forming loop sample structure is used to prepare PCMV.These albumen packets Include the Hcp1 albumen of pseudomonas aeruginosa (Pseudomonas aeruginosa), nontoxic " B subunit ", the large intestine bar of cholera toxin Bacterium (Escherichia coli) heat labile enterotoxin and shiga-like toxin.This ring sample albumen composition can form " string Pearl ", wherein linear PS chain penetrates the central groove of these annular albumen compositions.After albumen crosslinks, predict this multiple It is particularly stable to close object.The structured data of albumen show these central grooves be big enough to for PS chain can make its easily into Enter.For example, the central groove of six aggressiveness ring of Hcp1 be 42 angstroms (A), it is sufficiently wide so that can accommodate easily several width be 5.5 Angstrom polysaccharide chain (Mougous etc., Science 312 (5779): 1526-1530,2006).Alternatively, albumen ring can around PS into Luggage matches (for example, under specific physical and chemical condition, entering ring from the monomeric carrier protein protomer naturally assembled).It is this can Fit into ring monomeric protein it is known in the art and including, for example, pneumolysin (Walker etc., Infect.Immun.55 (5): 1184-1189,1987；Kanclerski and Mollby, J.Clin.Microbiol.25 (2): 222-225,1987), Listeria lysin O (Kayal and Charbit, FEMSMicrobiol.Rev.30:514-529,2006； Mengaud etc., Infect.Immun.55 (12): 3225-3227,1987), DNI, anthrax PA, Hcp1, cholera toxin B are sub- Base, Shiga toxin B subunit, flagellin and countless in the relevant molecule known in the art prepared by various microorganisms.
In the embodiment that another phase needs, use Toll-like receptor (TLR) agonist as carrier protein.Toll-like by Body (TLR) activation is important in terms of forming Acquired immune response, is converted in affinity maturation, the isotype of antibody response It plays a role in terms of immunological memory.Comma bacillus (Vibrio cholerae) flagellin (FLA) is TLR agonist.It is more than Purifying obtains the FLA albumen of 20mg from recombination bacillus coli (Escherichia coli), huge in IL-6 as described herein Effective TLR activator is shown as in phagocyte induction assays.In addition, being referred to as the highly conserved lung of " pneumolysin " Albumen is also shown scorching streptococcus (Streptococcus pneumoniae) can activate TLR4, but also is protective antigens.Cause This, which also is used as PCMV carrier protein.
In addition, outer membrane protein (OMP) mixture is (for example, Neisseria meningitidis (Neisseriameningitidis) OMP) be used as by Merck produce HIB conjugate vaccines carrier protein, and come from whole S. pneumoniae streptococcus The protein extract of (Streptococcal pneumoniae) bacterial cell is shown as having at least in animal infection modal Partial protection.In the embodiment that the phase of the invention needs, these protein mixtures are the sources of PCMV carrier protein.
In the embodiment that the phase needs, the PCMV method is used together with carrier protein, and the carrier protein has, example Such as, at least two lysine residue or opening and the other residues that can be crosslinked through chemical modification.In the reality that another phase needs It applies in scheme, the carrier protein is polymer (for example, polymer containing at least five subunit).It is desirable that the polymer It is same polymer.
In another embodiment, use DNI as carrier protein, because it is nontoxic, it is not necessary to preceding by egg using White detoxification.In addition, the use of DNI is ideal, because DNI in addition to induction is to the protective immune response of target antigen, is gone back It can induce the immune response to Bacillus anthracis (B.anthracis).In addition, DNI does not have internal disulfide bonds.This key is to hydrogen Boride reduction is sensitive, and the latter can make albuminous degeneration and lead to the forfeiture for inducing the epitope of anthrax toxin neutralizing antibody.
Target antigen
Vaccine composition and preparation of the invention and the method for giving this vaccine are suitable for any target antigen, for example, Polysaccharide, polyalcohol or polyaminoacid.It is desirable that the target antigen does not carry the chemistry used by vaccine preparation method The primary group (primary group) destroyed is reacted, the chemical reaction is for example, anti-by borohydride reduction action breaks down Former disulfide bond causes the denaturation of antigen.Illustrative target antigen includes that organic polymer such as polysaccharide (such as has at least 18 residues Polysaccharide), phosphoric acid polysaccharide, with N- acetyl group replace amino sugar polysaccharide, the polysaccharide containing sulfanilyl sugar, other sulfuric acid The sugar of base modification or sugar, polyalcohol, polyaminoacid, techoic acid and the lipopolysaccharides O side chain of phosphate modification.Illustrative target is anti- Original includes that those are synthesized by microorganism such as bacterium, fungi, helminth, virus and is purified with standard method from this biological source Pod membrane organic polymer.Illustrative target antigen includes microbial capsular organic polymer, including from following microorganism it is pure Those of change pod membrane organic polymer: bacterial organisms, such as bacillus (Bacillus species) (including anthrax spore Bacillus (B.anthracis)) (Wang and Lucas, Infect.Immun.72 (9): 5460-5463,2004), streptococcus pneumonia (Streptococcus pneumoniae) (Bentley etc., PLoS Genet.2 (3): e31, Epub 2006；Kolkman etc., J.Biochemistry 123:937-945,1998；With Kong etc., J.Med.Micorbiol.54:351-356,2005), will Hayes bacterium (Shigella) (Zhao etc., Carbohydr.Res.342 (9): 1275-1279, Epub 2007), Hemophilus influenzae (Haemophilus influenzae), Neisseria meningitidis (Neisseria meningitidis), golden yellow grape Coccus (Staphylococcus aureu), salmonella typhi (Salmonellatyphi), streptococcus pyogenes (Streptococcus pyogenes), Escherichia coli (Escherichia coli) (Zhao etc., Carbohydr.Res.342 ) and pseudomonas aeruginosa (Pseudomonas aeruginosa) (9): 1275-1279, Epub 2007；Fungal organism is for example hidden Ball yeast and Candida；And many other microorganisms (see, such as Ovodov, Biochemistry (Mosc.) 71 (9): 937-954,2006；Lee etc., Adv.Exp.Med.Biol.491:453-471,2001；And Lee, Mol.Immunol.24 (10): 1005-1019,1987).Illustrative target antigen further includes non-naturally occurring and non-biological origin polymer.
Vaccine composition
Vaccine of the present invention including PCMV can be used for combining, such as pediatric vaccine.In addition, vaccine of the invention It can be used for being inoculated with confrontation such as pneumococcal infection, influenza B haemophilic bacterium (Haemophilus influenzae Type B) (" HiB ") infection, (group A and group B) streptococcal infection, meningococcus (such as Neisseria meningitidis (Neisseria Meningitides it)) infects, and can be used as from gramnegative bacterium (such as pseudomonas aeruginosa (Pseudomonasaeruginosa), soil draws hot Francisella (Francisella tularensis), Shigella (Shigella species), salmonella (Salmonella species), acinetobacter calcoaceticus (Acinetobacter Species), bulkholderia cepasea (Burkholderia species) and Escherichia coli (Escherichia coli)) O Antigen vaccine.
The vaccine preparation phase need to include at least one carrier protein, one or more target antigens and pharmaceutically acceptable load Body or excipient (such as aluminum phosphate, sodium chloride and sterile water).Vaccine composition may also include for enhancing preparation immunogenicity Adjuvant system, such as oil-in-water system and the other systems being known in the art or pharmaceutically acceptable other excipient. In physiological conditions insoluble carrier/antigenic compound phase need to after giving study subject slowly released antigen.It is this compound The object phase need to be discharged into the suspension containing pharmaceutically acceptable excipient.However, carrier/antigenic compound is in physiological condition Under be also possible to be soluble.
Volume commonly used in hypodermic vaccine is about 0.5mL, intracutaneous injection 0.1mL, and percutaneous dosing is 0.002-0.02mL.The vaccine of 0.5mL dosage can contain the antigen for the about 2-500 μ g that useful about 2-500 μ g carrier protein contains.? In the embodiment that phase needs, in the dosage of 0.5mL, the antigen of about 10 μ g is contained with about 10 μ g carrier proteins.Antigen and carrier The molar ratio phase of albumen need in 1: 10 (for example, 1 part of antigen is to 2 parts of carriers or 1 part of antigen to 3 parts of carriers) to 10: 1 (for example, 3 Part antigen is to 1 part of carrier or 2 parts of antigens to 1 part of carrier) between.In the embodiment that the phase needs, the molar ratio of antigen and carrier It is 1: 1.Alternatively, the dry weight of antigen and carrier protein is between 1: 10 to 10: 1 (such as 1: 1 dry weight) than reason.
Because peptide or conjugate can be degraded under one's belt, the vaccine phase need to be parenterally administered (for example, passing through Subcutaneous injection, intramuscular injection, intravenous injection, intracutaneous injection).It is current to carry out passing medicine by the method for Physical penetration skin corium When (for example, needle, sprayer or galling), vaccine of the invention can be also administered by Transdermal absorption.
Particularly, vaccine of the present invention can be together with suitable immunologic adjuvant for example, by intramuscular injection, intracutaneous injection or warp Skin immunity inoculation gives study subject.It is tested to promote that vaccine of the present invention can in single or divided doses, be frequently included design The secondary administration that object antibody generates, to prevent infectious agent from infecting.The frequency and amount of vaccine administration depend on vaccine Given activity, and can be readily determined by routine experiment.
For example, inoculation program can for baby are as follows: three each dosage of 0.5mL dosage were with about 4-8 weeks It is spaced (since two monthly ages), then in the about 12-15 monthly age dosage of the 4th 0.5mL.For some vaccines, the phase is needed With the 5th dosage between four years old to six years old.
Although the age of administration is usually two months for the first time, vaccine can be given to baby in 6 week old.For super The children at conventional baby inoculation plan age out, can give vaccine of the invention according to the planning chart of property illustrated below.
The first administration age Inoculation plan
7-11 months A total of three 0.5mL dosage；First two are at least spaced surrounding, and third is at least two months after second
12-23 months Two 0.5mL dosage in total, is each at least separated by two months
24 months to 9 years old One 0.5mL dosage
For adult, the 2-8 weeks two or more 0.5mL dosage in interval are typically enough to provide long-term protection.It is right In adult and 11 years old or more children previously through immunity inoculation, the phase need every ten years to a booster.
The preparation can deposit in the ampoule and bottle of such as sealing in the container of single dose or multi-dose, can be lyophilized It is saved under the conditions of (freeze-drying), sterile liquid-carrier need to be added immediately using preceding.Vaccine of the invention can be prepared In pharmaceutically acceptable carrier, the carrier such as gel aluminum hydroxide, adjuvant formulation or salt water, then for example, by flesh Meat injection, intracutaneous injection are administered using the transdermal immune inoculation of sufficient immune adjuvant.
The invention also includes the medicine boxs for containing vaccine described herein (for example, PCMV).Medicine box of the present invention further includes this paper institute State the medicine box operation instructions of inoculation method.
With standard method by measuring the effect of antibody titer of study subject can determine immunization programs.It is general and Speech, it is believed that the Mean antibody titer (phase need to be IgG potency) of about 1 μ g/mL shows there is Long-Term Protection.
For vaccine composition described herein antigen/carrier protein complex diameter phase need to 10nm-100 μm it Between.Viral diameters can be for 100nm and with immunogenicity.The diameter of intact bacterial is 1-10 μm and also has immunogenicity. The diameter of one small bacterium is about 100 μm.In specific embodiments, antigen/carrier protein of the vaccine composition The diameter phase of compound need to be between 100nm-10 μm.The compound can be soluble or insolubility.
By referring to specific embodiment, embodiment and attached drawing, description hereafter is made to the present invention.Following embodiment is not It is construed to restricted.
Embodiment
1. vaccine of embodiment and control formulation
Poly- γ-the D-Glu (PGA) of pod membrane is purchased from Vedan (Taiwan) or passes through Rhie etc. The method of (Proc.Natl.Acad.Sci.USA 100:10925-10930,2003) is purified to obtain.Dominant negative mutation Body (DNI) is the mutant form of Bacillus anthracis (B.anthracis) protection antigen (PA), with the method for Benson etc. (Biochemistry 37:3941-3948,1998) is made from Escherichia coli (Escherichia coli).PGA and DNI albumen It is thoroughly dialysed using preceding with 7.4 sodium phosphate buffer of 0.05M, pH (SP7.4).DNI Stock concentrations are 30mg/mL. PGA Stock concentrations are 134mg/mL.Connector glutaraldehyde is purchased from Pierce, is 25% stock solution.Albumen pod membrane matrix vaccines (PCMV) and control collects (assemble) in the reaction of table 1.
Table 1. is used to prepare PCMV preparation 1-3 and the reaction of control 4 and 5 collects (assembly)
React # DNI PGA dH225% glutaraldehyde of O
ML mL mL mL title
1 20 1 3 0.8 PCMV1
2 12 4 8 0.8 PCMV2
3 16 2 6 0.8 PCMV3
4 16 260 P+C control
5 16 08 0.8 P are only compareed
Five reactions are packed at room temperature in the case where no glutaraldehyde.In T=0, shown in reaction in The glutaraldehyde (G25) of 0.1mL 25% is added.Thereafter every G25 that other 0.1mL is added for 30 seconds and repetition, until each shown anti- The G25 of 0.8mL in total should be reached.The DNI molecule cross-link situation carried out by difunctional glutaraldehyde molecules can observe by the naked eye It arrives, this is according to insoluble particle sample " gel " (" gel " like for generating different turbidity and arranging according to following order Particle) judge: turbidity highest and gel-forming situation react 1 >, 2 > 3 > 4, and reaction 5 keeps fully transparent and can It is molten.After 1 hour, the 0.5M sodium borate buffer liquid pH 9.3 (SBH) of 2mL sodium borohydride containing 1M is added to all six reactions In, to restore the schiff bases formed between DNI molecule amino side chain and difunctional glutaraldehyde molecules.By organic silicon defoamer (0.01mL) is added to each reaction to control the formation of foam in reaction process.Reactant saves 72 hours in 4 DEG C.Then will All reactants are sufficiently dialysed 48 hours with SP7.4.Final product is centrifuged off insoluble substance and is saved in 4 DEG C stand-by.
By using water-soluble carbodiimide, EDAC (1- ethyl -3- (3- dimethylamino-propyl) carbodiimide) by PGA Carboxyl and BSA amino coupled, to synthesize traditional conjugate between bovine serum albumin(BSA) (BSA) and PGA, step is such as Under: it mixes the water of 5mL BSA containing 30mg/mL with the NP7.5 of 1mL PGA containing 134mg/mL, 50mg EDAC is added, allow anti- It should carry out at room temperature 3 hours.Make to react on 4 DEG C with SP7.4 18 hours blocking activity bases of dialysis containing 1mM glycine Then group only dialyses 24 hours in SP7.4 in 4 DEG C.Final product is referred to as PGA-BSA conjugate.
After synthesizing and dialysing PCMV and control formulation, the molecular state of inspection DNI albumen is to confirm regardless of whether depositing In different content PGA, molecule cross-link is had occurred with albumen really in glutaraldehyde.Pass through SDS (dodecyl sodium sulfate) polyacrylamide Amine gel electrophoresis and the crosslinking situation that PCMV and control formulation are monitored with the sero-fast western blot of anti-PA.As shown in Fig. 2, Before glutaraldehyde cross-linking, DNI albumen is moved at 84kDa.If molecular weight is confirmed greater than the migration band of 220kDa, PCVM1- PCMV3 (swimming lane 1-3) shows that DNI albumen is widely crosslinked.There is no the DNI albumen being crosslinked alone under PGA to also show similarly High molecular weight species (swimming lane 5).The band migrated altogether with DNI is shown on the contrary, mixing with PGA but not having to the DNI that glutaraldehyde is handled Or lower molecular weight species (swimming lane 4).Therefore, the PGA preparation from Vedan (Taiwan) seems by the protease to DNI Activity pollution.Vedan PGA sample carries out electrophoresis in swimming lane 6, however reacts that display is not high-caliber with anti-PA antiserum Contaminating protein, show observed by band be each reaction DNI derivative products.
In addition, using PGA and one or more as the pneumococcus PS of antigen for probe into whether FLA (comma bacillus (Vibrio cholerae) flagellin) it is a kind of carrier protein more better than DNI under PCMV background.By measurement with respectively The carrier that the obtained IgG level and carrier protein for PS and PGA of kind PCMV immunity inoculation is calculated based on protein by weight Albumen efficiency, to evaluate the effect of these carrier proteins.
PCMV can also be prepared by the method for being directly crosslinked amino and carboxyl without using bi-functional cross-linking agent.Tool For body, PCMV is prepared by the amino and carboxyl for being crosslinked carrier protein with carbodiimide chemist.The chemistry makes lysine side-chain Primary amino group and asparagine and the carboxyl of glutamine side chain between form peptide bond.Although amino is most of by through formaldehyde The toxoid of reason is closed, but formaldehyde does not react completely with carboxyl.Therefore, carbodiimide chemist can be used for being made with fluid toxoid Standby PCMV, the fluid toxoid can be resistant to glutaraldehyde cross-linking.By SDS-PAGE be easy to be crosslinked situation carry out Detection.The presence of high-molecular-weight protein " stain " dependent on crosslinking agent (such as glutaraldehyde) addition shows to be crosslinked.
Table 2. is crosslinked by the carrier protein that SDS-PAGE analytic approach determines
Glutaraldehyde is no to be
Capsular polymer-PGA-PGA+PGA+PS 6B+PS 23F
BSA - + + + +
Diphtheria toxin - + + n.d. n.d.
Diphtheria toxoid - - - n.d. n.d.
Tetanus toxoid - + + n.d. n.d.
+ number indicate to detect and be crosslinked by SDS-PAGE, seen in-number expression albumen is migrated and is compareed in no glutaraldehyde The case where be no different.N.d.- (not determined) is not measured and (is not analyzed).
For being tested shown in table 2,200 microlitres are carried out in 50mM HEPES pH 7.5 and reacts and makes it at room temperature Heat preservation 2 hours.It is gone out reaction with 120mM Peng Qingization Na temper.Glutaraldehyde is added to 64mM, bovine serum albumin(BSA) is used with 15mg/mL (BSA), diphtheria toxin, diphtheria toxoid and tetanus toxoid are used extremely with about 5mg/mL, PGA is added with 13.4mg/mL, with 6B and 23F type pneumococcus PS is added in 4mg/mL.
As shown in table 2, some albumen (for example, diphtheria toxoid) through formaldehyde treated cannot be handed over well with glutaraldehyde Connection, therefore, it is necessary to use other cross-linking chemistries to prepare PCMV.Other protein carriers can be with penta 2 as tetanus toxoid Aldehyde crosslinking, but it is different with the crosslinking degree of unmodified protein such as diphtheria toxin and bovine serum albumin(BSA).
The analysis of 2. immunity inoculation of embodiment and anti-DNI and anti-PGA immune response
5 soluble products reacted described in table 1 are adjusted to same protein concentration (based on the extinction at its 280nm Rate).The BALB/c mouse of about 5-7 week old from Charles River is used to all immunity inoculations described in Fig. 2 to grind Study carefully.Exempted from the 0th day by intraperitoneal injection with PCMV vaccine 1-3 and antigen preparation control 4 and 5 with the dosage of 20 μ g DNI albumen Epidemic disease Mice Inoculated.The 7th day to all mouse carry out bloodletting, then at the 10th day with the antigen preparation of onesize dosage to it Carry out booster immunization.At the 17th day again to mouse bloodletting, then at the 20th day again to its booster immunization.It is right again at the 30th day Mouse carries out bloodletting and is put to death this time.It is stored from blood sample collection serum after blood clotting and in -20 DEG C.Use enzyme Linked immunosorbent assay (ELISA) analyzes the level of anti-PGA serum antibody and anti-DNI serum antibody.In brief, right It is 100 holes μ L/ containing 0.5 hole μ g/ BSA-PGA or DNI that Immulon 2HB ELISA (VWR) microtiter plate, which is coated with volume, 0.1M sodium carbonate buffer (pH9.6).After 4 DEG C of incubated overnights, by (being dissolved in TBS-0.1% to spit with 3%BSA (w/v) Warm (TBST)) it was incubated overnight in room temperature 1 hour or 4 DEG C, to close the plate for being coated with antigen.By after each booster immunization when Between put when merge be serially diluted from the four other blood serum samples of mouse group with TBST, add it to the plate for being coated with antigen In, it is incubated at least 1 hour.With for the rabbit anti-serum with alkaline phosphatase (Zymed) mouse IgG or IgM being conjugated, measurement is anti- DNI antibody response and anti-PGA antibody response.Substrate p-nitrophenyl phosphate (PNPP) is added in each hole and at 405nm The absorptance of each reaction is measured with spectrophotometry.Data report that the latter is defined as obtaining in the form of endpoint titers inverse The OD obtained405Reading is more than the greatest dilution that negative control reads two standard deviations.
With the measurement of ELISA measuring method immune through three kinds of PCMV preparation 1-3 and two antigen control preparations 4 and 5 (Fig. 3-5) The special anti-DNI of the mouse of inoculation intracorporal IgM and IgG and anti-PGA immune response.As shown in figure 3, in addition to being handed over without glutaraldehyde Except the control formulation 4 for joining (no glutaraldehyde), the DNI albumen in all formulations has high degree of immunogenicity.However, these DNI specific immune response is only based on IgG.Although even also can't detect anti-DNI at the 7th day of immunity inoculation IgM, but at the 17th day, in the mouse through PCMV preparation immunity inoculation with those only with DNI (preparation 5) immunity inoculation mistake of crosslinking Mice Body in significant anti-DNI IgG response can be detected.It was observed at the 30th day through being inoculated with owning including preparation 4 The strong booster response of DNI is directed to after preparation.
Anti- PGA IgM response shows typical capsular polymer mode (Fig. 4).Control formulation 4 generated detectable at the 7th day Anti- PGA IgM response, but the response was not reinforced at the 17th or 30 day.All PCMV preparations induced anti-PGA at the 7th day Then IgM response generated exclusive even stronger anti-PGA IgM response at the 17th day and the 30th day.As expected, control system Agent 5 (only crosslinking DNI) does not generate the anti-PGA response based on IgM or based on IgG.Form sharp contrast, PCMV1-3 (preparation 1-3) generates the strong anti-PGA response based on IgG, and the response is significant at the 30th day significantly depending on after in performance in the 7th day to be obtained To reinforcement (Fig. 5).Finding is clearly resembled for PCMV1-3's based on the anti-PGA response of IgG: the reports such as Aulinger are observed The immune response (Infect.Immun.73:3408-3414,2005) to PGA that is induced of traditional PGA-DNI conjugate vaccines With Rhie etc.) response (Proc.Natl.Acad.Sci.USA100:10925- that is induced of the PA-PGA conjugate vaccines of description 10930,2003.Therefore the pod membrane PGA (Bacillus anthracis of known T cell dependent/non-dependent is directed in induction (B.anthracis) protect antigen (Wang etc., Infect.Immun. 72:5460-5463,2004)) IgG response in terms of, PCMV vaccine #1, #2 and #3 have good performance as tradition conjugation PGA vaccine.Contain DNI (the non-friendship mixed with PGA Connection) control formulation 5 do not induce the detectable IgG for PGA, show DNI stimulate PCMV preparation 1-3 the anti-PGA of IgG The effect of TLR ligand is not served as in response.The result also demonstrates the observation in document, i.e. PGA is the T cell of low immunogenicity Independent immune is former, unless it is coupled (Rhie etc., Proc.Natl.Acad.Sci.USA with protein by being covalently attached 100:10925-10930,2003).PGA is obviously converted to the immunogene of T cell dependence by PCMV method, despite the fact that should Method is directly crosslinked with PGA molecule without result in DNI albumen.
These data support PCMV method that can prepare the characteristic immunogene similar with traditional conjugate vaccines.With this paper institute The method stated is easy to prepare PGA PCMV, and finds that it induces the typical Western response of PGA- albumen conjugate vaccines.In table 1 in detail The PCMV that the dosage based on Fig. 3 description carries out immunity inoculation to 1000 mouse enough is prepared in the small-scale reaction stated. These data support the PCMV prepared by PGA and DNI to may be used as vaccine prevention by Bacillus anthracis (Bacillus Anthracis anthrax caused by).
Embodiment 3. adds the preparation and characterization of PCMV
PCMV technology can be applied to the capsular antigen of various structures and ionic charge.23 seed type streptococcus pneumonias (Streptococcus pneumonia) PS is manufactured purchased from American type culture collection (ATCC) and by Merck, Inc. These PS are very different on its molecular structure, including strong anion type, partial cation type, neutral charge, The PS that phosphorylation, linear, to have branched structure sum is modified through various other modes.In preliminary experiment, take corresponding to Wyeth These PS subsets (4,6B, 9V, 14,18C, 19F and 23F) of 7 Buccal mucosa flaps, it is huge to measure its inducing mouse in product Prevnar The ability of phagocyte generation IL-6.The PS of 4 types is active in the assay；Lipopolysaccharides (LPS) is the control of TLR agonist.Also Determine other PS (for example, 3 types), PGA and from soil draw hot Francisella (F.tularensis) O antigen PS, with And by the PGA-DNI PCMV vaccine being prepared and without the control of crosslinking.The experiment shows 3 type pneumococcus PS (than PGA's Degree is smaller) also polluted by TLR agonist.The PS and PCMV of hot Francisella (F.tularensis) are drawn from soil It is same clean in the assay.Phenol extraction and ethanol precipitation " cleaning " (can remove the unknown of remnants after continuous processing twice TLR agonist) 3 type of streptococcus pneumonia (S.pneumoniae) PS.Thus, it is found that 6 kinds of streptococcus pneumonias (S.pneumoniae) PS and soil draw hot Francisella (F.tularensis) O antigen PS clean for the generation of IL6, these are Through being probed into experiment as described herein.
It is produced to for IL6 by being similar to described method in embodiment 1 using DNI as carrier protein 7 kinds of clean PS have carried out PCMV synthesis for life.Preliminary immunogenicity measurement shows that all seven kinds of PCMV have not With the immunogenicity of degree.It was found that " unit price " PCMV (14- based on DNI of streptococcus pneumonia (S.pneumoniae) PS14 PCMV) the anti-PPS14IgG of high-titer significantly increased is induced after third time is immune.Obviously, as 14-PCMV and other six kinds When PCMV mixing generates " mixing " antigen, it is seen that same immune response.BecauseAdsorb " the adjuvant of alum Change " vaccine, so being determined to whether sexavalence PCMV mixture can also be adsorbed by alum adjuvant.Determined by immunoassays Property measurement be exposed to the suction later of PCMV or control mixture (identical PS mix with DNI albumen but without glutaraldehyde cross-linking) The amount of the streptococcus pneumonia PS of alum is invested, the results show that being adsorbed in the PS of alum under PCMV background than control (the non-friendship of PS+ The DNI albumen of connection) want it is much more (this as the immunoreactivity higher level of further diluted PCMV in immunoassay It is indicated).
Mouse is immunized, to evaluate the immunogenicity with or without the septivalency PCMV for being adsorbed in alum adjuvant.Alum assistant Agent improves the immune response dynamics to PS14, induces the IgG for this PS 7 days early with than no Adjuvanted vaccines.However, There is no the septivalency PCMV ratio of alum, there are the non-crosslinked PS+DNI of alum combination controls to have more immunogenicity.The result confirms PCMV method makes PS have more immunogenicity for mouse, and support PCMV method can be used to prepare immune performance be similar to sew Close the antigen mixture of vaccine mixture.
In other experiment, using phenol extraction and ethanol precipitation with by pollution TLR agonist from 23 kinds of pneumonia balls It is removed in granulose commercial formulation.IL-6 is generated with the method for standard according to induction peritoneal macrophages come to through processed PS It is tested, to confirm the removal situation of pollutant.The PS for lacking IL-6 induced activity is used to prepare PCMV.For PCMV's Other polysaccharide include the O antigen PS of hot Francisella (F.tularensis) purifying being drawn from soil and from Bacillus anthracis (B.anthracis) PGA pod membrane.Examine 25 pod membrane types (23 pneumococcus types, tularemia type and anthraxs in total Each one of heat type).Each of 25 kinds of Buccal mucosa flaps is prepared essentially by method described in embodiment 1 with DNI albumen PCMV.The PS: albumen of 1: 1 ratio is used for (about 1: 1 dry weight) these initial ps CMV preparation.By SDS-PAGE to every kind of preparation It is characterized, to confirm that the crosslinking situation of albumen, protein-crosslinking situation and the immunogenicity of PCMV preparations various in preliminary experiment are complete Total correlation.For certain Buccal mucosa flaps (such as 6B and 23F), PCMV is prepared using other carrier proteins.For it is same these Buccal mucosa flap (such as 6B and 23F), can be used alternative cross-linking chemistry.To confirming protein-crosslinking (such as in SDS-PAGE) The immunogenicity of all PCMV preparations is tested.
For example, the following antigen different with 5 kinds of different stromatins and 2 kinds prepares 10 kinds of different PCMV.5 kinds of matrix The selection of albumen is based on: they are currently used in the vaccine of FDA approval, or allow it as tracer for measuring PCMV preparation Other characteristics of stability.Used following stromatin: (1) b subunit of cholera toxin (SBL Vaccin AB offer), (2) are white Larynx toxin, (3) Tetanus toxin fragment C, " Frag C " (Sigma Aldrich offer), (4) DNI, and (5) come from large intestine bar The beta galactosidase (SigmaAldrich offer) of bacterium (Escherichia coli).As capsular antigen, uses and come from The poly- D-Glu of capsular antigen of Bacillus anthracis (Bacillusanthracis) and 14 type streptococcus pneumonias (Streptococcuspneumoniae) pod membrane (Suarez etc., Appl.Environ.Micobiol.67:969-971, 2001).When with the DNI as stromatin in corresponding PCMV, both capsular antigens all have hyperimmunization original Property.Every kind of capsular antigen is combined with each in 5 kinds of seleced stromatins, and 10 kinds of different PCMV are made.
Turn as PCMV induction isotype antibody what is observed in traditional conjugate vaccines, can be tested in Mice Body Change the ability of IgG into.All Antigen adsorptions can be made in alum then typically using corresponding 5 mouse of every kind of PCMV preparation Group.The immune response baseline for being directed to test antigen is obtained to mouse bloodletting in advance.Then pass through the intraperitoneal injection side of standard Case mouse is immunized three times several (in the 0th, 7,14 days), and the 10th, 20, the 30 and 60 day collection blood after initial immunity Liquid.By the ELISA measuring method of standard, mice serum is carried out according to the case where the IgG for being directed to PS used and carrier protein Analysis.In these experiments, the control group mice for only receiving PS immunity inoculation is included, for evaluating various PCMV preparations With should compared with non-immunogenicity or the low unconjugated PS of immunogenicity the anti-PS IgG of induction ability.Make promising PCMV (i.e. the anti-PS IgG of induced high levels) passes through careful immune analysis, tries to establish mouse to the dynamics of PCMV immune response With dose response situation.
Alternatively, can send promising PCMV and its corresponding control to businessman to produce rabbit anti-serum.It carries out similar Immunoassay is come the classification for the antibody evaluating immunogenicity, being induced, the immune response dynamics of rabbit.In these experiments, right According to being commercial productIt is from CRM197 coupling 7 kinds it is different tradition conjugation PS vaccines be adsorbed in alum mix Object is closed, CRM197 is nontoxic protein mutant relevant to diphtheria toxin.
The antibody response functionality induced with PCMV can be evaluated.For example, can be by measuring anti-PS antibody tune Reason pod membrane streptococcus pneumonia (S.pneumococcus) and the ability for causing macrophage to kill bacterium by phagocytosis, next pair Functionality is evaluated.Protect animal from the lethal challenge of pod membrane streptococcus pneumonia (S.pneumococcus) be prove vaccine Another method of effect in the animal being immunized through PCMV.
Embodiment 4.PCMV withComparison
Measurement is obtained from ATCC or the pneumonia chain directly from Serum Institute of India (SII) by Merck The relative clean degree of coccus (S.pneumoniae) polysaccharide (pps) 6B, 14 and 23F.Use IL-6 expression as the finger of pps cleannes Show agent, uses LPS as the positive control of " unclean ".As shown in Figure 9 A, Merck pps 6B, 14 and 23F are clean, and are such as schemed Shown in 9B, the pps 6B from SII is " unclean ".As shown in Figure 10, processing 2 (keeping the temperature 1 hour for 80 DEG C in 1M NaOH) makes SII pps 6B cleaning.Clean pps 6B is for comparing conjugate and the immunological characteristic of PCMV.As shown in table 3, pollutant is not It is LPS.
The measurement of 3. polysaccharide level of endotoxin of table
Polysaccharide Endotoxin unit/mg polysaccharide
SII pps 6B- is untreated 0.75
SII pps 23F- is untreated 0.85
SII pps 23F- processing 2 0.24
Various Merck pps- are untreated 0.1-0.4
The display of Figure 11 and 13The anti-pps 6B IgG antibody of (using alum as adjuvant) induction, and be with alum PCMV (BSA the and pps 6B of adjuvant；Diphtheria toxin and pps 6B；Diphtheria toxoid and pps 6B；Tetanus toxoid and pps IgG response ratio 6B) induced is usedObserved IgG response is more preferable.Equally, as shown in figure 12, it is with alum The IgM response and use that the PCMV of adjuvant is inducedIt is seen close.
In addition, for pps 14 (The most pps of immunogenicity), as illustrated in figures 14-16, contain diphtheria class The PCMV for being adsorbed in alum of toxin and pps 14 or tetanus toxoid and pps 14 are almost equivalent in terms of inducing IgG response In
5. multivalence PCMV of embodiment
Multivalent immunogen original is prepared by following PCMV method: chemically being mixed different pod membrane organic polymers, later DNI carrier protein is set to be crosslinked (" one-pot synthesis reaction ") with glutaraldehyde.Use DNI as carrier, with three kinds of organic polymers: PGA, Alginates and glucan prepare this kind of trivalent immunogene.These trivalent vaccines, which have immunogenicity and generate, is directed to three kinds of pod membranes The immune response of organic polymer, the result is according to the index in the following drawings: that is analyzed before immune and after 30 days is mixed It closes serum IgM (Fig. 6), 60 days antigen-specific serum IgG antibody potency (Fig. 7) occurred after immunity inoculation, after immunity inoculation 128 days anti-PS serum antibody titers (Fig. 8) occurred.Furthermore as shown in figs 6-8, monovalent alginates PCMV preparation is also in mouse Generate immune response.Also multivalence PCMV immunogene can be prepared by mixing specific PCMV, PCMV, which is first passed through, to be separately synthesized most After remix " mixture " vaccine be made together.
All patents, patent application, patent application publication and other publications cited or be related to are logical in this specification Reference is crossed to be merged into herein, one by one just like each patent, patent application, patent application publication or publication similar to degree Or it individually indicates and is incorporated by reference into herein.

Claims (16)

1. the method for preparing vaccine composition, the composition include the antigen contained by carrier protein matrix, the antigen with The albumen substrate noncovalent associations, which comprises
(I) mixes the target antigen selected from polysaccharide, polyalcohol and polyaminoacid homopolymer with carrier protein；It is wherein described polynary Alcohol is the hydrogenated form of carbohydrate, and carbonyl has been reduced into primary hydroxyl or secondary hydroxyl；With
The connector for being crosslinked the carrier protein by the sulfydryl, amino or carboxyl of carrier protein molecule is added in (II), and
(iii) it is crosslinked the carrier protein and forms carrier protein matrix.
2. the method for claim 1 wherein the vaccine compositions to further include pharmaceutically acceptable excipient.
3. the antigen exists to the molar ratio of the carrier protein the method for claim 1 wherein in the vaccine composition Between 1:10-10:1.
4. the method for claim 1 wherein the carrier protein is polymer, wherein the polymer includes at least five subunit, Or in which the polymer is same polymer.
5. the method for claim 1 wherein the method includes restoring the schiff bases in the carrier protein.
6. the method for claim 1 wherein the carrier proteins to be covalently bonded at least one other carrier proteins, wherein institute Stating covalent cross-linking includes the peptide bond between the primary amino group and asparagine of lysine side-chain or the carboxyl of glutamine side chain, or Wherein described be covalently attached includes bi-functional cross-linking agent, wherein the bi-functional cross-linking agent is glutaraldehyde, bis- [sulfonic group succinyls Imido grpup] suberate or dimethyl diimidate.
7. the method for claim 1 wherein the connector is formulaCompound, wherein RnIt is the straight of 1-12 carbon atom Even or branched alkyl, the direct-connected or branched heteroalkyl groups of 1-12 atom, direct-connected or branched-chain alkene, the 2-12 of 2-12 carbon atom are a The direct-connected or branched alkyne of carbon atom, the aromatic moieties of 5-10 carbon atom, the cyclic annular system of 3-10 atom ,-(CH2CH2O)qCH2CH2Wherein q is 1-4 or in which the connector is glutaraldehyde, maleimidobenzoyl-N- hydroxysuccinimidyl acyl Imines ester, carbodiimide or bisazo benzidine.
8. the method for claim 1 wherein the carrier proteins to be: diphtheria toxin or its mutant, diphtheria toxoid, tetanus Toxin or its mutant, tetanus toxoid, pseudomonas aeruginosa (Pseudomonas aeruginosa) exotoxin A or its Mutant, b subunit of cholera toxin, Tetanus toxin fragment C, bacterial flagellin, pneumolysin, Listeria lysin O, Neisseria meningitidis (Neisseria menningitidis) outer membrane protein, pseudomonas aeruginosa (Pseudomonas Aeruginosa) Hcp1 albumen, Escherichia coli (Escherichia coli) heat labile enterotoxin, shiga-like toxin, people LTB albumen, the protein extract from whole bacterial cells, Bacillus anthracis (Bacillus anthracis) protectiveness are anti- Former dominant negative mutant (DNI) or Escherichia coli (Escherichia coli) beta galactosidase.
9. method for claim 8, wherein the whole bacterial cells are pseudomonas aeruginosa (Pseudomonas Aeruginosa) or streptococcus (Streptococcal) cell or in which the bacterial flagellin are comma bacillus (Vibrio cholerae) flagellin.
10. the method for claim 1 wherein the shiga-like toxin is Shigella (Shigella) SltB2 albumen.
11. being pneumonia streptococcus the method for claim 1 wherein the polysaccharide includes at least 18 residues or in which the polysaccharide Bacterium (Streptococcus pneumoniae) polysaccharide, soil draw hot Francisella (Francisella tularensis) more Sugar, Bacillus anthracis (Bacillus anthracis) polysaccharide, Hemophilus influenzae (Haemophilus influenzae) are more Sugar, salmonella typhi (Salmonella typhi) polysaccharide, Shigella (Shigellaspecies) polysaccharide, salmonella (Salmonella species) polysaccharide or Neisseria meningitidis (Neisseria meningitidis) polysaccharide.
12. the method for claim 11, wherein the streptococcus pneumonia (Streptococcus pneumoniae) polysaccharide is pod Membranous type 3,4,6B, 7A, 7B, 7C, 7F, 9A, 9L, 9N, 9V, 12A, 12B, 12F, 14,15A, 15B, 15C, 15F, 17,18B, 18C, 19F, 23F, 25A, 25F, 33F, 35,37,38,44 or 46 or in which the soil draw hot Francisella (Francisella Tularensis) polysaccharide is O antigen.
13. the method for claim 1 wherein the polyaminoacid homopolymer is microbial capsular polymer.
14. the method for claim 13, wherein the microbial capsular polymer is from Bacillus anthracis (Bacillus Anthracis poly- γ-D-Glu).
15. method of claim 1 further includes the second target antigen.
16. method of claim 1 further includes third target antigen.
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