CN101553246A

CN101553246A - Protein matrix vaccines and methods of making and administering such vaccines

Info

Publication number: CN101553246A
Application number: CNA2007800375458A
Authority: CN
Inventors: J·J·梅卡拉诺斯
Original assignee: Harvard College
Current assignee: Harvard College
Priority date: 2006-08-07
Filing date: 2007-08-07
Publication date: 2009-10-07
Anticipated expiration: 2027-08-07
Also published as: CN110064053A; CN101553246B; ZA200900899B

Abstract

The invention relates to vaccine compositions having a carrier protein and an antigen of interest entrapped in a complex, methods of making such vaccines, and methods of vaccine administration.

Description

The preparation of protein matrix vaccines and this vaccine and medication

The cross reference of related application

The claimed benefit from U.S. Provisional Application serial number of submitting on August 7th, 2,006 60/835,944 and the U.S. Provisional Application serial number 60/933,764 submitted on June 8th, 2007 of this application, its description is attached to herein by reference.

Statement about federal funding research

The present invention is finished by government-funded under the Grant No U54AI057159 that National Institute of Health (National Institutes of Health) (NIH) authorizes.Government has some right in the present invention.

Background of invention

The present invention relates to vaccine combination, vaccine production method and vaccine and give method.

A lot of antigens particularly those antigens relevant with pathogen pod membrane layer stimulate very little to immunne response or do not stimulate, and make generation become complicated at the effort of those antigenic effective vaccines.Pod membrane is the surface composition of microorganism, is made up of the polymer (polymer) of organic compound such as saccharide, aminoacid or alcohols usually.Pod membrane is chemically widely different.Constituting the monomer unit (for example saccharide) of pod membrane can various molecular configurations link together and further uses phosphate (phosphate), nitrogen, sulfate (sulfate) and the replacement of other chemical modification group.These chemical changes allow pod membrane that numerous antigen targets is presented to the microorganism surface, thereby allow to avoid these targets of host immune system targeting.Pod membrane can also be the virulence factor that prevents that microorganism from being engulfed and killing by host's macrophage and multinuclear leucocyte.Antibody at pod membrane provides in the microorganism surface at the biological effectively defence of pod membrane by making complement fixation, and complement can cause microorganism dissolving or its opsonic action and quilt to be had phagocytotic host immune cell to absorb and kill.At the most effective antibody of pod membrane is IgG antibody.Can not induce the pod membrane of significant level IgG to be called T-independent antigen.The covalent coupling of protein and pod membrane causes it to become " T is dependent " and this antigen can to cause IgG and reply.

Exist safety, easily synthesize, have cost-benefit, can not cause at pod membrane and other that strong immunization is replied or the demand of the T-independent antigen vaccine of IgG antibody.Need such vaccine prevention various by the infectious disease due to anthrax bacillus, streptococcus pneumoniae, influenza B, meningococcus and the streptococcal infection.

Summary of the invention

The present invention relates to contain the vaccine combination of the target antigen that is carried by the carrier protein bag in the complex, the method for this vaccine of preparation and the method that gives of vaccine.

Therefore, first aspect the invention describes the vaccine combination that contains target antigen and carrier protein, and it is crosslinked that wherein (i) is no more than 50% target antigen and carrier protein, and (ii) wherein said antigen suppressed by vector albumen bag carries the formation complex.

In the embodiment of first aspect present invention phase need, described complex diameter is between 10nm-100 μ m.In the first aspect present invention embodiment that more phase needs, described complex diameter is between about 100nm-100 μ m.In the first aspect present invention embodiment that also more the phase needs, described complex diameter is between about 100nm-10 μ m.

In the embodiment of other phase need of first aspect present invention, when giving mammal, cause the immunne response that the T-cell relies in the mammal with complex.

In the embodiment of the other phase need of first aspect present invention, the mol ratio of antigen and carrier protein is between 1: 10 and 10: 1.Ideally, described carrier protein is polymer (multimer), for example, comprises the polymer of at least 5 subunits.In the embodiment of other phases need, described polymer is same polymer.

In the embodiment of the further phase need of first aspect present invention, described carrier protein and at least one other carrier protein are covalently bound.Ideally, the peptide bond between the carboxyl of the described covalently bound primary amino radical that is included in lysine side-chain and agedoite or glutamine side chain.In the embodiment of other phase need, the described covalently bound formula that comprises

Chemical compound, R wherein _nBe aromatic moieties, a 3-10 atom of a direct-connected or alkine, a 5-10 carbon atom of direct-connected or branched-chain alkene, a 2-12 carbon atom of direct-connected or branched heteroalkyl groups (heteroalkyl), a 2-12 carbon atom of direct-connected or branched alkyl, a 1-12 atom of 1-12 carbon atom the ring-type system ,-(CH ₂CH ₂O) _qCH ₂CH ₂-wherein q is 1-4 or the chemical bond that connects two aldehyde radicals.In the embodiment that the phase needs in addition, described covalently bound glutaraldehyde, dimaleoyl imino benzoyl-N-hydroxysuccinimide eater (m-maleimidobenzoyl-N-hydroxysuccinimide ester), carbodiimide or the BDB of containing.Also has in the embodiment that needs in other phase the described covalently bound bi-functional cross-linking agent (cross linker) that comprises.Ideally, described bi-functional cross-linking agent is glutaraldehyde, two [sulfonic group succinimido] suberate (bis[sulfosuccinimidyl] suberate) or oneself two imidic acid dimethyl esters.

In the embodiment of other phase need of first aspect present invention, described carrier protein right and wrong are covalently bound.In the embodiment of phase need, described non-covalent connection comprises hydrophobic interaction, ionization, model ylid bloom action or hydrogen bond.

In the embodiment of the other phase need of first aspect present invention, described carrier protein is diphtheria toxin, diphtherotoxin 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 (for example vibrio cholera (Vibriocholerae) flagellin); pneumolysin; Neisseria meningitidis (Neisseriamenningitidis) outer membrane protein; Pseudomonas aeruginosa (Pseudomonas aeruginosa) Hcp1 albumen; escherichia coli (Escherichia coli) heat labile enterotoxin; shiga-like toxin (for example shigella (Shigella) SltB2 albumen); people LTB albumen; pneumolysin; Listerella lysin O (or associated protein); the protein extract of intact bacterial cell (for example Pseudomonas aeruginosa (Pseudomonas aeruginosa) or streptococcus (Streptococcal) cell); the negative mutant (DNI) of the antigenic dominance of Bacillus anthracis (Bacillus anthracis) protection; or escherichia coli (Escherichia coli) beta galactosidase.In the embodiment that specifically phase needs, described carrier protein is pneumolysin, Listerella lysin O, diphtheria toxin, diphtherotoxin, diphtheria toxoid, tetanus toxin or tetanus toxoid.

In the embodiment of other phase need of first aspect present invention, described target antigen is polysaccharide, polyhydric alcohol or polyamino acid.Ideally, described polysaccharide comprises at least 18 residues.In the embodiment of other phase need, described polysaccharide is that streptococcus pneumoniae (Streptococcuspneumoniae) polysaccharide, soil draw hot Frances Salmonella (Francisella tularensis) polysaccharide, Bacillus anthracis (Bacillus anthracis) polysaccharide, Haemophilus influenzae (Haemophilusinfluenzae) polysaccharide, salmonella typhi (Salmonella typhi) polysaccharide, Salmonella (Salmonella species) polysaccharide, shigella (Shigella) polysaccharide or Neisseria meningitidis (Neisseria menningitidis) polysaccharide.In the embodiment that specifically phase needs, described streptococcus pneumoniae (Streptococcus pneumoniae) polysaccharide is any in the 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 of other concrete phase need, it is O antigen that described soil draws hot Frances Salmonella (Francisella tularensis) polysaccharide.

In the embodiment of the further phase need of first aspect present invention, described target antigen is a microorganism pod membrane polymer.Ideally, described microorganism pod membrane polymer is the poly--γ-D-glutamic acid from Bacillus anthracis (Bacillus anthracis).

In the embodiment of other phase need of first aspect present invention, the organic polymer that described target antigen is made up of the monomer with at least three atoms, wherein each atom is independently selected from carbon, oxygen, hydrogen, phosphate, nitrogen and sulfate.Ideally, described organic polymer stems from microorganism.In the embodiment of other phase need, described organic polymer is not naturally occurring.

In the embodiment that the phase needs in addition, described vaccine combination further comprises second target antigen.Ideally, described vaccine combination also comprises the 3rd target antigen.

In second aspect, the invention describes the method for preparing vaccine combination.This method comprises that (i) mixes target antigen with carrier protein and forms antigen and carrier protein mixture and (ii) carry target antigen with the carrier protein bag, and it is crosslinked wherein to be no more than 50% target antigen and the carrier protein in the vaccine combination.

In the embodiment of second aspect present invention phase need, described vaccine combination further comprises pharmaceutically acceptable excipient.

In the embodiment of other phase need of second aspect present invention, described bag carries and comprises Precipitation Antigen and carrier protein from mixture.Ideally, described precipitation comprises: the pH of mixture is changed; In mixture, add trichloroacetic acid (TCA) or ammonium sulfate; Change the ionic strength of mixture by the concentration that increases or reduce inorganic salt in the mixture; Heating blends condenses carrier protein and/or antigen; Perhaps the ionizing radiation irradiation mixture with enough flux causes crosslinked.

In the embodiment of second aspect present invention phase need, the antigen of described vaccine combination and the mol ratio of carrier protein are 1: 10-9: between 10.

In the embodiment of the other phase need of second aspect present invention, described carrier protein is a polymer.Ideally, polymer comprises at least 5 subunits.In the embodiment of other phase need, described polymer is same polymer.

In the embodiment of the further phase need of second aspect present invention, the non-covalent connection of described carrier protein.Ideally, described non-covalent connection comprises hydrophobic interaction, ionization, model ylid bloom action or hydrogen bond.

In the embodiment of the other phase need of second aspect present invention, described carrier protein is diphtheria toxin, diphtherotoxin 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 (for example vibrio cholera (Vibriocholerae) flagellin); pneumolysin; Listerella lysin O; Neisseria meningitidis (Neisseria menningitidis) outer membrane protein; Pseudomonas aeruginosa (Pseudomonasaeruginosa) Hcp1 albumen; escherichia coli (Escherichia coli) heat labile enterotoxin; shiga-like toxin (shigella (Shigella) SltB2 albumen); people LTB albumen; protein extract from intact bacterial cell (for example Pseudomonas aeruginosa (Pseudomonas aeruginosa) or streptococcus (Streptococcal) cell); the dominant negative mutant (DNI) of Bacillus anthracis (Bacillus anthracis) protective antigen; or escherichia coli (Escherichia coli) beta galactosidase.In the embodiment that specifically phase needs, described carrier protein is pneumolysin, Listerella lysin O, diphtheria toxin, diphtherotoxin, diphtheria toxoid, tetanus toxin or tetanus toxoid.

In the embodiment of other phases need of second aspect present invention, described target antigen is polysaccharide, polyhydric alcohol or polyamino acid.Ideally, described polysaccharide comprises at least 18 residues.In the embodiment of other phase need, described polysaccharide is that streptococcus pneumoniae (Streptococcuspneumoniae) polysaccharide, soil draw hot Frances Salmonella (Francisella tularensis) polysaccharide, Bacillus anthracis (Bacillus anthracis) polysaccharide, Haemophilus influenzae (Haemophilusinfluenzae) polysaccharide, salmonella typhi (Salmonella typhi) polysaccharide, shigella (Shigella) polysaccharide, Salmonella (Salmonella species) polysaccharide or Neisseria meningitidis (Neisseria meningitidis) polysaccharide.In the embodiment that specifically phase needs, described streptococcus pneumoniae (Streptococcus pneumoniae) polysaccharide is any in the 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 of other concrete phase need, it is O antigen that described soil draws hot Frances Salmonella (Francisella tularensis) polysaccharide.

In the embodiment of the other phase need of second aspect present invention, described target antigen is a microorganism pod membrane polymer.Ideally, microorganism pod membrane polymer is the poly--γ-D-glutamic acid from Bacillus anthracis (Bacillus anthracis).

In the embodiment of other phase need of first aspect present invention, the organic polymer that described target antigen is made up of the monomer that contains three atoms at least, wherein each atom is independently selected from carbon, oxygen, hydrogen, phosphate, nitrogen and sulfate.Ideally, described organic polymer derives from microorganism.In the embodiment of other phase need, described organic polymer is not that nature exists.

In the embodiment of the further phase need of second aspect present invention, the mixing in the step (i) comprises second target antigen and even the 3rd target antigen.

In the third aspect, the invention describes the method that another prepares vaccine combination.This method comprises that (i) mixes target antigen and (ii) add with carrier protein makes the crosslinked joint of carrier protein (linker), and it is crosslinked wherein to be no more than 50% target antigen and the carrier protein in the vaccine combination.

In the embodiment of third aspect present invention phase need, described vaccine combination further comprises pharmaceutically acceptable excipient.In the embodiment of other phase need of third aspect present invention, the mol ratio of antigen and carrier protein is between 1: 10 and 10: 1 in the described vaccine combination.In the embodiment of the other phase need of third aspect present invention, described carrier protein is a polymer.Ideally, described polymer comprises at least 5 subunits.In the embodiment of other phase need, described polymer is same polymer.

In the embodiment of the further phase need of third aspect present invention, described method comprises the Schiff's base in the reduction carrier albumen.In the third aspect present invention embodiment that further phase needs, described carrier protein and at least a other carrier protein are covalently bound.Ideally, the peptide bond between the carboxyl of the described covalently bound primary amino radical that is included in lysine side-chain and agedoite or glutamine side chain.In the embodiment of other phase need, the described covalently bound bi-functional cross-linking agent that comprises.Ideally, described bi-functional cross-linking agent is glutaraldehyde, two [sulfonic group butanimide] suberate or oneself two imidic acid dimethyl esters.

In the embodiment of the other phase need of third aspect present invention, described joint is a formula

Chemical compound, R wherein _nBe aromatic moieties, a 3-10 atom of a direct-connected or alkine, a 5-10 carbon atom of direct-connected or branched-chain alkene, a 2-12 carbon atom of direct-connected or branched heteroalkyl groups, a 2-12 carbon atom of direct-connected or branched alkyl, a 1-12 atom of 1-12 carbon atom the ring-type system ,-(CH ₂CH ₂O) _qCH ₂CH ₂-wherein q is 1-4 or the chemical bond that connects two aldehyde radicals.

In the embodiment of other phase need of third aspect present invention, described joint is glutaraldehyde, a dimaleoyl imino benzoyl-N-N-Hydroxysuccinimide base ester, carbodiimide or BDB.

In the embodiment of the other phase need of third aspect present invention, described carrier protein is diphtheria toxin, diphtherotoxin 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 (vibrio cholera (Vibriocholerae) flagellin); pneumolysin; Listerella lysin O; Neisseria meningitidis (Neisseria menningitidis) outer membrane protein; Pseudomonas aeruginosa (Pseudomonasaeruginosa) Hcp1 albumen; escherichia coli (Escherichia coli) heat labile enterotoxin; shiga-like toxin (shigella (Shigella) SltB2 albumen); people LTB albumen; protein extract from intact bacterial cell (Pseudomonas aeruginosa (Pseudomonas aeruginosa) or streptococcus (Streptococcal) cell); the dominant negative mutant (DNI) of Bacillus anthracis (Bacillus anthracis) protective antigen or escherichia coli (Escherichia coli) beta galactosidase.

In the embodiment of the further phase need of third aspect present invention, described target antigen is polysaccharide, polyhydric alcohol or polyamino acid.Ideally, described polysaccharide comprises at least 18 residues.In the embodiment of other phase need, described polysaccharide is streptococcus pneumoniae (Streptococcuspneumoniae) polysaccharide, soil draws hot Frances Salmonella (Francisella tularensis) polysaccharide, Bacillus anthracis (Bacillus anthracis) polysaccharide, Haemophilus 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 specifically phase needs, described streptococcus pneumoniae (Streptococcus pneumoniae) polysaccharide is any in the 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 of other concrete phase need, it is O antigen that described soil draws hot Frances Salmonella (Francisella tularensis) polysaccharide.

In the embodiment of other phase need of third aspect present invention, described target antigen is a microorganism pod membrane polymer.Ideally, described microorganism pod membrane polymer is from poly--γ-D-glutamic acid of Bacillus anthracis (Bacillus anthracis).

Also have in the embodiment that needs in other phase of third aspect present invention, the organic polymer that described target antigen is made up of the monomer with at least three atoms, wherein each atom independently is selected from carbon, oxygen, hydrogen, phosphate, nitrogen and sulfate.Ideally, described organic polymer derives from microorganism.In the embodiment that the phase needs in addition, described organic polymer is not that nature exists.

In the embodiment of the further phase need of third aspect present invention, the mixing in the step (i) comprises second target antigen and even the 3rd target antigen.

Aspect the 4th, the invention describes the method for study subject being carried out vaccination opposing infectious agent.This method comprises the vaccine combination of the first aspect present invention that gives the study subject q.s, produces antibody to induce study subject.In the embodiment that needs in the fourth aspect present invention phase, described method comprises that second gives step, and wherein the vaccine combination with the first aspect present invention of q.s gives study subject, produces antibody to strengthen study subject.Ideally, in fourth aspect present invention, production of antibodies is that the T cell is dependent.In the embodiment that needs in other of fourth aspect present invention, described production of antibodies is enough to prevent or reduces the infection of infectious agent to study subject phase.Ideally, described infectious agent is that streptococcus pneumoniae, meningococcus, influenza B haemophilus (Haemophilus influenza Type B), Pseudomonas aeruginosa (Pseudomonas aeruginosa), soil are drawn hot Frances Salmonella (Francisella tularensis), shigella (Shigella species), Salmonella (Salmonella species), acinetobacter calcoaceticus (Acinetobacter species), bulkholderia cepasea (Burkholderia species) or escherichia coli (Escherichia coli).

In the embodiment of other phase need of fourth aspect present invention, described method comprises that second gives step, and wherein second vaccine combination that contains target antigen with q.s offers study subject reinforcement study subject generation antibody.Ideally, described production of antibodies is enough to prevent or reduces the infection of second infectious agent to study subject.

In the embodiment of fourth aspect present invention phase need, described antibody is IgG antibody.In the embodiment of the further phase need of fourth aspect present invention, described study subject is the people.

In the present invention's embodiment that the phase needs aspect any one, described streptococcus pneumoniae (Streptococcus pneumoniae) polysaccharide is one of pod membrane type described in (J.Med.Microbiol.54:35-356,2005) such as Kong.For example, streptococcus pneumoniae (Streptococcus pneumoniae) the polysaccharide pod membrane 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 (5-q for example, 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 (for example 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 (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 (for example, 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 (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-q1,15C-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 (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 (for example, 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 is (for example, 28F-28A or 28F-cn), 28A (for example, 28F-28A), 29 (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 (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 (for example, 48-cn1 or 48-cn2), or the GenBank number of landing AF532714 or AF532715.

Definition

" administration " that is used in combination with vaccine herein means the intravital immunne response of vaccine-induced study subject that sufficient dosage is provided for study subject, and wherein said immunne response causes and the bonded production of antibodies of the contained antigen-specific of vaccine.Phase needs administration comprise intramuscular injection, intradermal injection or percutaneous injection, and the phase need comprise and gives suitable immunological adjuvant.Administration can comprise the single-dose or the multiple dose administration of vaccine.Ideally, study subject generation antibody, the infection of infection prevention sexually transmitted disease (STD) substance are strengthened in design administration for the second time.The frequency of vaccine administration and amount depend on the specific activity of vaccine and be easy to be determined by routine test.

" crosslinked " means in for example formation of the covalent bond between the carrier protein of two molecules, macromole or molecular combinations, the formation of described covalent bond directly realizes by using " distance of zero mark degree " joint, perhaps utilizing the 3rd molecule is that chemical joint is realized, described chemical joint has two functional groups, each can form covalent bond with one of two independent molecules, or forms covalently bound (be them will form can around " ring " of polymer) between with two independent groups of a part.Exemplary joint comprise can crosslinked two carrier proteins bifunctional linker.Crosslinkedly also can occur between antigen and the carrier protein.

This paper employed " antigen " means any and antibody or bonded molecule of antibody fragment specificity or molecular combinations.

This paper employed " bifunctional linker " means the chemical compound of two functional groups, and each functional group can form covalent bond with two independent molecules, atom or elements collection respectively.For example, G.T.Hermanson (Bioconjugate Techniques, Academic Press, 1996) and Dick and Beurret (Conjugate Vaccines.Contribu.Microbiol.Immunol., Karger, Basal 10:48-114,1989) exemplary bifunctional linker has been described.Ideally, bifunctional linker is glutaraldehyde, two [sulfonic group butanimide] suberate or oneself two imidic acid dimethyl esters.

This paper employed " joint " means chemical compound or the chemical bond that covalently connects two or more molecules.Ideally, joint is glutaraldehyde or formula

Chemical compound, R wherein _nBe aromatic moieties, a 3-10 atom of a direct-connected or alkine, a 5-10 carbon atom of direct-connected or branched-chain alkene, a 2-12 carbon atom of direct-connected or branched heteroalkyl groups, a 2-12 carbon atom of direct-connected or branched alkyl, a 1-12 atom of 1-12 carbon atom the ring-type system ,-(CH ₂CH ₂O) _qCH ₂CH ₂-wherein q is 1-4 or the chemical bond that connects two aldehyde radicals.Can not use link molecule directly to connect.For example, available carbodiimide chemistry or with the T-5398 enzymatic catalysis of this cross-linking reaction of catalysis directly is connected proteinic carboxyl and its amino.

" adding powerful antibody produces " means and occurs in the activation that is exposed to the memory B cell in the antigen process for the second time, is called " booster response ", shows " secondary " memory immunne response of Shou Qichang, causes the antibody of Shou Qichang to produce.

" carrier protein " means the protein that is used for vaccine, its cause to himself and/or to the compound antigenic immunne response of carrier protein.Ideally, described antigen is loaded in the complex to come and the non-covalent association of carrier protein by suppressed by vector albumen bag.Even so, also covalent bond each other of antigen and carrier protein.Ideally, described carrier protein comprises by the epi-position of T-cell recognition.Multiple antigenic peptide (MAP) as branched peptide is also comprised by the definition of " carrier protein ".Ideally, MAP comprises lysine.Exemplary phase needs carrier protein to comprise toxin and toxoid (chemistry or gene), can be mutant.Ideally, carrier protein is diphtheria toxin, diphtherotoxin 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; Listerella lysin O (and correlation molecule); Neisseria meningitidis (Neisseria menningitidis) outer membrane albumen; Pseudomonas aeruginosa (Pseudomonas aeruginosa) Hcp1 albumen; escherichia coli (Escherichia coli) heat labile enterotoxin; shiga-like toxin; people LTB albumen; protein extract from the intact bacterial cell; the dominant negative mutant (DNI) of Bacillus anthracis (Bacillus anthracis) protective antigen; or escherichia coli (Escherichia coli) beta galactosidase; or any other can be by the crosslinked protein of joint.

" DNI " means dominant negative mutant (DNI) albumen, is the mutant form of Bacillus anthracis (B.anthracis) protective antigen (PA), and be described as (Biochemistry37:3941-3948,1998) such as Benson.

This paper employed about antigenic " bag carry " mean under physiological condition, be retained in and the formed complex of carrier protein in antigen.Ideally, described antigen bag be stated from the formed complex of carrier protein in, between antigen and carrier protein, do not exist significantly covalently bound.As employed at this paper, not existing significantly, covalently bound being meant is no more than 50% antigen and carrier protein covalent bond.Ideally, it is covalently bound to be no more than 40%, 30%, 10% or 5% antigen and carrier protein.

" infection " means study subject by microorganism such as antibacterial, fungus, parasite or virus attack.Infection can comprise, for example, is present in the study subject body usually or the hyper-proliferative of the microorganism of body surface or be not present in the study subject usually or the hyper-proliferative of the microorganism on surface.In excessive micropopulation is present in the study subject body or body surface or when pathological symptom appearred in the existence of micropopulation injury cell or the tissue that causes study subject, study subject suffered infection by microorganisms.

" infectious agent " means the microorganism that causes infection.

" immunogenic " means the chemical compound of induce immune response in the study subject body.Ideally, described immunne response is the immunne response that the T-cell relies on, and comprises the IgG production of antibodies.

" microorganism " is meant antibacterial, fungus, parasite or the virus that can cause that study subject infects.

" microorganism pod membrane polymer " is meant and is present in the inner or surperficial polymer of microorganism pod membrane coat.Ideally, microorganism pod membrane polymer is organic polymer such as polysaccharide, phosphoric acid polysaccharide, has sugar, polyhydric alcohol, polyamino acid, techoic acid and a lipopolysaccharide O side chain that sugar that polysaccharide, the polysaccharide that contains sulfanilyl sugar (sulfanylated sugar), other sulfate of the amino sugar that the N-acetyl group replaces modify or phosphate are modified.

" monomer " is meant the molecular structure that can form two or more keys with same monomer, when as " polymer " a part of, often produces chain or a series of ramose, chain of connecting of repeated monomer structure.

" organic polymer " is meant the polymer of being made up of covalently bound monomer, and each monomer contains three or more following atoms: carbon, oxygen, hydrogen, phosphate, nitrogen and sulfate.Ideally, described organic polymer is polysaccharide, phosphoric acid polysaccharide, has sugar, sugar, polyhydric alcohol, polyamino acid, techoic acid and a lipopolysaccharide O side chain that sugar that polysaccharide, the polysaccharide that contains sulfanilyl sugar, other sulfate of the amino sugar that the N-acetyl group replaces modify or phosphate are modified.

" polyhydric alcohol " is meant the hydrogenated form of carbohydrate, and its carbonyl is reduced into primary hydroxyl or secondary hydroxyl.Exemplary polyhydric alcohol is polyalkylene oxide (PAO), as polyalkylene glycols (PAG), comprises polymethylene ethylene glycol, Polyethylene Glycol (PEG), methoxy poly (ethylene glycol) (mPEG) and polypropylene glycol; Polyvinyl alcohol (PVA); Ethylene-copolymer-maleic anhydride; Styrene-maleic anhydride copolymer; The glucosan that comprises Sensor Chip CM 5; Cellulose comprises methylcellulose, carboxymethyl cellulose, ethyl cellulose, hydroxyethyl-cellulose, carboxyethyl cellulose and hydroxypropyl cellulose; The chitosan hydrolyzate product; Starch such as hetastarch and hydroxypropyl starch; Glycogen; Agarose and derivant thereof; Guar gum; Amylopectin; Inulin; Xanthan gum; Carrageenan; Pectin; The alginic acid hydrolyzate; Sorbitol; The alcohol of glucitol, mannitol, galactitol, 1,2,3,4,5-pentanepentol, gulose alcohol, xylitol, threitol, sorbitol, fructose alcohol, glycerol, maltose alcohol, cellobiose, the alcohol of sucrose, the alcohol of amylopectin, the alcohol of amylopectin; Or single propylene glycol (MPG).

" polyamino acid " is meant at least two aminoacid that connected by peptide bond.Ideally, described polyamino acid is the peptide (being homopolymer) that contains repetition aminoacid sequence or same amino acid chain.

" reduction Schiff's base " is meant azomethine or formula R ₁R ₂C=N-R ₃Chemical compound (R ₁, R ₂And R ₃Be chemical constitution, contain carbon atom usually) be exposed to Reducing agent with the two keys of hydrogen atom saturated Schiff's base.Reductive method is understood by those skilled in the art.

This paper is employed to be meant that about antibody or its segmental " specificity in conjunction with " antibody or antibody fragment are for the affinity that specified protein such as antigen increased with respect to any other protein of equivalent.Ideally, according to use standard method such as enzyme-linked immunosorbent assay (ELISA) mensuration, antibody or antibody fragment comprise related antigen at least 2 times of its antigenic affinitys, 5 times, 10 times, 30 times or 100 times of other antigens greater than any equivalent.

" study subject " is meant can be by the animal of infected by microbes.It is mammal that phase needs study subject, as people, monkey, Canis familiaris L., cat, mice, rat, cow, sheep, goat or horse.In the embodiment of phase need, described study subject is the people, as child.Ideally, described study subject is the baby, child or preadolescence child learn to walk.

" T-cell dependent/non-dependent antigen " is meant the antigen that causes antibody to generate under the cooperation of T lymphocyte not having.Ideally, described T-cell dependent/non-dependent antigen directly stimulates bone-marrow-derived lymphocyte and need not the lymphocytic cooperation of T.Exemplary phase need T-cell dependent/non-dependent antigen comprise k antigen poly--γ-D-glutamic acid (PGA), alginic acid acid (alginate), glucosan, polysaccharide (PS), polyamino acid, polyhydric alcohol and nucleic acid.

Advantage

Compare with the currently available vaccines, existing vaccines technology, vaccine production of the present invention is simple, is difficult for that Chemical Problem is arranged, be difficult for having immune problem, cheaply, more be adapted to different target antigens and carrier protein, be used to generate polyvalent vaccine (resisting multiple antigenic vaccine) more neatly than conjugation techniques.

Vaccine of the present invention need be at carrier protein and is intended to bring out covalently bound between the antigen of immunne response, thus with conjugate vaccines technology simplified in comparison its preparation method and reduced preparation cost.Polysaccharide (PS)-albumen conjugate vaccines is because too expensive and can't be at developing country's production and selling; Because the chemistry of the height specialty that every kind of vaccine is required and the production and the purification cost of PS and carrier protein, traditional conjugate vaccines are difficult to produce cheaply.

Vaccine of the present invention also shown to can safe induction of immunity sex resistance the demand of previous difficult antigenic vaccine.These vaccines can be monovalent (having the single antigen induce immune response) or polyvalent (having multiple antigen induction panimmunity replys).Shown already that the vaccine that contains TLR (Toll sample receptor) part brought out other difficult antigenic immunne response, but comparing with vaccine of the present invention, they have unsafe tendency, because the TLR part usually inspires inflammation, even low dose is also poisonous, the tool reactionogenicity, and may cause ill symptoms.

Other features and advantages of the present invention will be conspicuous according to following detailed description, accompanying drawing and claim.

The accompanying drawing summary

Fig. 1 is the flow chart of inducing the non-limiting proposal approach of anti-PS IgG immunne response about conjugate vaccines (conjugate for preparing between PS and the carrier protein tetanus toxoid).In this model, have only the B cell of the antibody receptor of showing identification PS to combine with the PS protein conjugate.Therefore, carrier protein is incorporated into the B cell surface of showing correct PS binding specificity.

Fig. 2 is the western blot analysis figure of PCMV and control formulation, monitors crosslinked situation by sds polyacrylamide gel electrophoresis with the sero-fast Western blot of anti-PA.DNI albumen moved to the 84kDa place before glutaraldehyde cross-linking.As band migration to molecular weight greater than the 220kDa place confirm the same, DNI is proteic extensively cross-linked in PCVM1-PCMV3 (swimming lane 1-3) demonstration.Do not exist the down independent crosslinked DNI of PGA to show same high molecular weight species (species) (swimming lane 5) yet.On the contrary, mix with PGA but show and the band of the common migration of DNI or lower molecular weight kind (swimming lane 4) without the DNI that glutaraldehyde is handled.

Fig. 3 shows and to be used for measuring through three PCMV preparations (PCMV1-PCMV3, preparation 1-3) and two antigen control preparations 4 and the interior IgM of mice body of 5 immunity inoculations and the ELISA measurement result of the anti-DNI immunne response of IgG specificity.DNI albumen except not with the crosslinked control formulation 4 of glutaraldehyde (glut) all preparations in all have the immunogenicity of height.Yet these special DNI immunne response are unique based on IgG.Though even do not detect anti-DNI IgM the 7th day of immunity inoculation yet, with 17 days mouse of PCMV preparation immunity inoculation with only use and can detect significant anti-DNI IgG in the mouse of crosslinked DNI (preparation 5) immunity inoculation and reply.Noticed with all preparations at the 30th day and to comprise preparation 4 inductive intensive booster responses at DNI.

Fig. 4 shows the ELISA measurement result be used for measuring through the interior anti-PGA immunne response of IgM specificity of mice body of three PCMV preparations (PCMV1-PCMV3, preparation 1-3) and two

antigen control preparations

4 and 5 immunity inoculations.Anti-PGA IgM replys and has shown typical pod membrane polymer pattern.Control formulation 4 produced detectable anti-PGA IgM at the 7th day and replys, but this is replied the 17th day or not reinforcement in the 30th day.All PCMV preparations all induced anti-PGA IgM to reply at the 7th day, produced stronger anti-PGAIgM only at the 17th day and 30 days then and replied.As expected, control formulation 5 (only for crosslinked DNI) does not produce based on IgM or based on the anti-PGA of IgG and replys.

Fig. 5 demonstration is used for measuring through three PCMV preparation (PCMV1-PCMV3; Preparation 1-3) the ELISA measurement result of the anti-PGA immunne response of IgG specificity and in the mice body of two

antigen control preparations

4 and 5 immunity inoculations.PCMV1-3 produces intensive anti-PGA based on IgG and replys, and it occurred at the 17th day, seemed to reinforcement at the 30th daybreak then.

Fig. 6 show before the immunity and through containing the PCMV (DNI-ALG C, DNI ALG A) of DNI and alginate and contain and " one pot " (" one pot ") trivalent PCMV preparation immunity inoculation of alginate (ALG), glucosan (DEX) and the compound DNI of PGA after 30 days pooled serum IgM antibody titer.

Fig. 7 show through containing the PCMV (DNI-ALG C, DNIALG A) of DNI and alginate and contain with " one pot " trivalent PCMV preparation immunity inoculation of alginate (ALG), glucosan (DEX) and the compound DNI of PGA afterwards 60 days antigen-specific serum IgG antibody tire.

Fig. 8 show through containing the PCMV (DNI-ALG C, DNIALG A) of DNI and alginate and contain and " one pot " trivalent PCMV preparation immunity inoculation of alginate (ALG), glucosan (DEX) and the compound DNI of PGA after 128 days anti-PS IgG antibody titers.

Fig. 9 A and 9B use available from American type culture collection and by streptococcus pneumoniae (S.pneumoniae) polysaccharide (pss) of Merck preparation or the IL-6 mensuration figure that carries out available from the pps of Serum Institute of India (SII).

Figure 10 shows that (trt 2 available from the impurity available processes 2 among the pss 6B of SII; 80 ℃ of insulations are 1 hour in 1MNaOH) figure that removes.Handling 1 (trt 1) is a succession of 5 step extract with phenol, so that protein is removed from polysaccharide.

Figure 11 shows that the PCMV that contains pss 6B is inducing ratio in the IgG generation

More effective.The BSA=bovine serum albumin; The DT=diphtheria toxin, diphtherotoxin; The DTx=diphtheria toxoid; The TTx=tetanus toxoid.

Figure 12 show the PCMV contain pss 6B induce in IgM generations and Equally effective.

Figure 13 shows that the PCMV that contains pss 6B is inducing ratio in the IgG generation More effective.

Figure 14-16 shows that the PCMV contain pss 14 is about as much as inducing IgG to produce

(DTx=diphtheria toxoid; The TTx=tetanus toxoid).

Describe in detail

The invention describes vaccine combination, described preparation of compositions method and medication, so that not dependence antigen or cause antigen such as polysaccharide (PS), polyhydric alcohol, polyamino acid and other organic polymer of faint immunne response usually of immunity antagonism T cell to be provided.Vaccine of the present invention has the effective immunological characteristic of typical PS albumen conjugate vaccines, but the phase be different from conjugate vaccines with needing, difference is need not significant covalency atom and connects target antigen such as PS or pod membrane organic polymer are connected to carrier protein.On the contrary, carry target antigen such as PS or pod membrane organic polymer with the carrier protein bag.For example, can form protein matrix by carrier protein self covalent cross-linking in the presence of soluble antigen such as PS or pod membrane organic polymer: these vaccines are known as protein matrix vaccines.Highly cross-linked each other carrier protein can form can capture antigen and promote antigen absorption and to producing the substrate of the stimulation of antibody in the immunocyte.Carrier protein substrate can be and seals antigenic " grid " form or antigen wherein is " rope " and protein or crosslinking protein complex are the forms of a series of " beading " of " pearl ".If carrier protein forms around the antigenic ring or the antigenic three dimensional network that tangles around antigen, then can make the carrier protein bag carry antigen.In addition, described carrier and antigen can be crosslinked, and is for example crosslinked by antigen chain and carrier protein are carried out in the chain.In the embodiment of phase need, described antigen and carrier protein right and wrong are covalently bound.This non-covalent connection can comprise hydrophobic interaction, ionization, model ylid bloom action or hydrogen bond.Non-covalent connection can comprise antigen and the non-covalent associating physical geometry of protein complex (seeing the metaphor of above " beading ").

Carrier protein need not self to carry out crosslinked the bag and carries antigen.For example also can antigen and albumen co-precipitation be wrapped carry antigen by mixed carrier albumen and antigen and precipitophore albumen in aqueous solution.Also can come to carry antigen from the chemical compound of antigen and carrier protein mixture (for example Alumen, sodium hexameta phosphate, poly-phosphorus piperazine or by hydrophobic or ionization drives that protein is had other polymer of affinity) by precipitation with the carrier protein bag.The method of precipitating proteins in this area be standardized and comprising, for example, (1) pH of change mixture, (2) change the ionic strength of solution by the concentration that increases or reduce inorganic salt in the mixture, (3) or toward mixture add trichloroacetic acid (TCA) or ammonium sulfate, (4) heating blends causes protein coagulating (promptly forming precipitation or gel), (5) in a certain way the protein in the chemical modification mixture it can not be dissolved and (6) with the ionizing radiation (ultraviolet of enough flux, gamma-rays or β ray) the irradiation protein solution cause proteinic crosslinked and/or the precipitation.

When using the pathogen protein clostridium, this vaccine is called albumen pod membrane matrix vaccines (PCMV).As described in an embodiment, prepare PCMV, it comprises based on pattern T cell dependent/non-dependent k antigen: poly--γ-D-glutamic acid (PGA) and alginic acid (alginate) and glucosan; PCMV with exemplary carrier protein DNI.PGA PCMV be easy to prepare in a large number and find its induce typical case in the immunne response of PGA albumen conjugate vaccines.Can in the presence of any target antigen, carry out the crosslinked vaccine of the present invention for preparing to any a lot of possible carrier proteins with any a lot of possible joints.This paper has discussed exemplary and preferred joint, carrier protein and target antigen.

Polysaccharide (PS) is the polymer of sugar.The PS that derives from pod membrane relates to the first antigen component (primary antigenic component) at the protective immunity of capsular bacterium pathogen, and these bacterial pathogens for example have Neisseria meningitidis (Neisseria meningitidi), streptococcus pneumoniae (Streptococcus pneumoniae), salmonella typhi (Salmonella typhi) and influenza B haemophilus (Haemophilus influenzae Type B).Use based on the immunity inoculation teenager of the vaccine of microorganism PS and adult and reducing success aspect the disease puzzlement, but proved for child of tender age (i.e. child below 24 months) protective immunity aspect effect is provided lower.Child of tender age's adaptive immune system is not all also reached maturity, and a little less than the immunogenicity of T cell dependent/non-dependent antigen such as pod membrane PS and can not in these young vaccine receptors, cause secular protective immune response (being that immunological memory is replied).

T cell dependent/non-dependent antigen such as PS can be converted into T cell dependence antigen by PS and proteinic chemical coupling; This chemical coupling process is called " puting together " and relates to the atom in the PS structure and be present in covalently bound formation between the side chain atom of " carrier " Argine Monohydrochloride.This " conjugate vaccines " more effectively promotes to induce B cell maturation and isotype conversion, causes the much higher antibody of level with correct anti-PS protectiveness overview (profile).Protection antibody has high-affinity to its PS antigen, normally immunoglobulin G (IgG) subclass, have the antibody of the Shou Qichang of complement fixation activity and opsonin effector activity.

Fig. 1 has shown the exemplary non-limiting approach of inducing anti-PS IgG immunne response about the conjugate between PS and the carrier protein tetanus toxoid.In this model, the B cell that has only displaying can discern the antibody receptor of PS combines with the PS protein conjugate.Therefore, carrier protein is incorporated in to the B cell surface that shows correct PS binding specificity.Albumen-PS complex is absorbed intramolecularly vacuole compartment by these B cells, and here carrier is subjected to protein degradation effect processing.Presenting in the ditch (presentation groove) of MHC II receptoroid (MHC-II) transported and loaded to the peptide that derives from protein carrier.This MHC-II-carrier peptides complex is showed in the surface of B cell.When the MHC-II-peptide complexes was discerned by TXi Baoshouti (TCR), the T cell was activated justacrine and is provided the cytokine of " assisting " for inducing the B cell differentiation.The B cell is quantitatively enlarged and is divided into " plasma cell " of ready-made secretory antibody.Initial IgM (IgM) is generated by plasma cell but the auxiliary antibody such as the IgG that causes plasma cell to carry out the classification conversion and generate other isotype classification of final T cell.This process continues along with the sudden change of plasma cell experience, and causing producing has the more antibody receptor of high-affinity to the PS protein conjugate.When antigen is eliminated, have only the plasma cell of high-affinity to be retained in PS protein conjugate activation remaining in the circulation.The continuation of the maturation process that plasmacytic T cell relies on causes plasma cell group's expansion, produces the IgG antibody-like of high-affinity.By measuring the level of anti-PS IgG antibody in the study subject of immunity inoculation such as human serum, can monitor the expansion situation at an easy rate.

Final ripe and transformation process causes Shou Qichang's and to the generation of the special memory B cell of PS.Memory B cell has unique characteristic, if promptly be exposed to PS then be activated immediately.Activation causes memory B cell propagation and produces anti-PS IgG fast.Occur in " secondary " memory immunne response that the activation that is exposed to the memory B cell in the PS antigen process for the second time is called " booster response " and shows Shou Qichang.Initial immunity can stimulate IgM antibody and some IgG production of antibodies.At secondary immunity, i.e. " reinforcement " when injection, be upset and produce a large amount of IgG by the memory cell of initial immunity sequencing, promptly remember immunne response.

T cell dependent/non-dependent antigen does not stimulate lasting immunity usually, i.e. IgG production of antibodies, but can stimulate more provisional IgM production of antibodies than poor efficiency.Thereby PS antigen does not produce the IgG booster response usually separately.Yet if carry out initial immunity with the PS protein conjugate, PS will produce booster response, because produced IgG by sequencing by the inductive memory cell of conjugate.Really, be considered to simulate the protective response that produces owing to the microorganism that is exposed to displaying PS through the intravital booster response of vaccinated animal or human; This secular memory is for being very crucial with the vaccine that works aspect the immune object in protection after the conjugate vaccines immunity inoculation several years.Therefore, the value of PS protein conjugate is (1), and they induce the ability of the antigenic high-level IgG of anti-PS, and they induce ability at the antigenic memory immunne response of PS (2).PS antigen does not show these characteristics usually thereby is semiprecious antigen.Cause at the development progress of the conjugate vaccines of a lot of bacterial diseases (wherein the immunne response to PS may be a protectiveness) slow at the difficulty aspect the synthetic conjugated antigen and their production cost.

Other T cell dependent/non-dependent antigen comprises the aminoacid homopolymer, as poly--γ-D-glutamic acid (PGA) and polyhydric alcohol.Really, most of biopolymers are T cell dependent/non-dependent antigen.Polymer is because the repeat property of its chemical constitution (epi-position) can be crosslinked with their B cell immunoglobulin (Ig) receptor of identification.Therefore polymer can activate the IgM that the B cell produces anti-polymer with the same mode of polysaccharide.For example, the aminoacid homopolymer, poly--γ-D-glutamic acid (PGA) of Bacillus anthracis (Bacillusanthracis) is the weak and antigenic pod membrane polymer of T cell dependent/non-dependent of immunogenicity.The vaccine of being made up of the PGA that puts together with protein carrier is a hyperimmunization originality, can induce anti-PGA IgG and PGA is had immunological memory.Therefore.Most polymers is reacting as PS aspect their immunogenicity, because they are can not be under the MHC-II background processed and show, therefore can not raise the auxiliary of T cell.In being called the polymer of acceptor interaction of Toll sample receptor (TLR), some naturally occurring and another classes find exception.In case be activated, TLR can come the inducing cell factor to produce and adaptive immune response is produced to change by host cell.Some PS and TLR part are covalently bound or be subjected to this part and pollute (contaminate).For example, lipopolysaccharide (LPS) be hyperimmunization originality, induce the PS of IgG and memory response; The fat A of LPS partly is the TLR part, can be responsible for immunological characteristic.

In another example, even have been found that their some immunological characteristics of not linking to each other with protein carrier and also represent conjugate vaccines of minority streptococcus pneumoniae PS, promptly they induce the isotype conversion to IgG.Recently, commercial polysaccharide vaccine Pneumovax-23, and from indivedual PS of streptococcus pneumoniae (Streptococcus pneumoniae) different strains, find to be polluted (Sen etc., J.Immunol.175:3084-3091,2005) by the TLR part.What these PS preparation this discovery can be interpreted as does not exist albumen to put together the following isotype conversion that can induce to IgG.These streptococcus pneumoniae PS induce macrophage secretion IL-6 and TNF-α.Yet the PS that extracts through being further purified by phenol extraction can not induce the macrophage secrete cytokines.In immune Research, a little less than the PS of extract with phenol immunogenicity and no longer induce anti-PS IgG.Therefore, extract with phenol is removed the pollution molecule that these abnormal immunes of being responsible for this PS preparation are learned characteristic.Polluting molecule seemingly provides the TLR part of activating macrophage TLR dependent cell factor responsibility.By extract with phenol being further purified of PS removed described pollution TLR part and caused PS to become T cell dependency fully.

Above example explanation PS antigen can play the effect as PS-albumen conjugated antigen and need not the covalently bound of protein and carbohydrate.Unfortunately, the TLR part is normally urged inflammation.For example, even the very little dosage of LPS also is deleterious.Therefore, though the TLR part is mixed the immunne response that may enlarge PS with PS, this method also may produce the vaccine tool reactionogenicity or that cause vaccine receptor ill symptoms.The conjugate vaccines technology has kept the optional method that is used to prepare the PS vaccine with required immunogenicity spectrum and safety.

Exploitation with PS-albumen conjugate vaccines has reduced the children disease burden that is caused by the aggressive bacterial pathogens widely.For these less vaccines of number comprise that anti-influenza B haemophilus (Haemophilus influenzae B Type) vaccine and some meningococcus and strains of streptococcus are that the market can be sold in developed country.These PS-albumen conjugate vaccines in developing country because too expensive and can't be at developing country's production and selling.For example, septivalency streptococcus pneumoniae conjugate vaccines that commerce can be sold is worth every approximately every $58 (2006 time dollar), and need take four doses.Only price just makes these vaccines beyond one's ability the people that developing country bears the disease burden for those.

Because being difficult to carry out low price, the related chemistry and the production and the purification cost of PS and carrier protein, traditional conjugate vaccines produce.Usually all need to have suitable purity puting together the former two, chemical reaction just is able to carry out with rational coupling efficiency.Usually, the coupling chemistry must prove effective to the various PS in uniqueness aspect selected PS and carrier protein chemical.This coupling chemistry is introduced functional group in PS, the latter can be connected with carrier protein through the ε amino side chain of lysine thereafter.The PS chemical modification of introducing these coupling group can destroy the epi-position on the PS and introduce new epi-position (for example, the epi-position relevant with the glycosyl of joint or modification), and its significance has only by conscientious careful immune analysis and could assess.In addition, for traditional PS-albumen conjugate vaccines, the type of the character of the size of PS, the PS molecule number that each protein carrier molecule is connected, selected carrier and connection chemistry can influence the immunogenicity of conjugate vaccines.Therefore, for example (wherein every kind of known serotype of 90+ all has different PS structure (Bentley etc. in pneumoccoccosis, PLOS Genetics 2 (3): e31 262-269,2006)) situation under, a kind of single conjugation methods may be not suitable for all serotype.Repeating to synthesize the conjugate vaccines that (reproduciblysynthesize) have repetition immunological characteristic (reproducible immunological property) relates to: the character of the size of PS, the PS molecule number that each carrier protein molecule is connected, selected carrier and the type that connects chemistry are carefully controlled, and this has increased the cost of making conjugate vaccines significantly.

The appearance of antibiotic resistance makes that the vaccine urgency is more outstanding safely and effectively to developing.Make widely available vaccine, especially for developing country, also need to make cost-effective vaccine.Make at from the multiple polysaccharide antigen of one or more bacterial species different serotypes put together the connection Seedling, in the immunization protocol the childhood period of merging to, this will simplify high-risk group's vaccine administration.Yet existing conjugate vaccines technology is not calculate, and therefore puts together connection Seedling (combination conjugate vaccine) and may popularize in developing country hardly.Really, even in the developed country that has set up powerful market, the Wyeth 7-valency short supply of puting together Pnu-Imune 23 has recently illustrated that how difficult the vaccine of producing and store the complicated conjugate vaccines synthetic technology of needs is.

In the embodiment of phase need, vaccine of the present invention is a multivalence pod membrane matrix vaccines (PCMV), and wherein a kind of or a plurality of bacterial capsule components are downloaded in the multivalence carrier protein substrate by bag.Preparation PCMV is easy to, because as the target antigen of one of parent material, for example pod membrane only needs suitable purity.For example, it also is impure (it carries proteinase activity on DNI) that Vedan gathers γ-D-glutamic acid (PGA), and as described herein, its behavior is fully as the T cell dependent/non-dependent antigen of being suspected (embodiment 1).All three kinds of PCMV preparations (protein and PGA ratio its more than 7 times scope different) in PGA be successful to participating in of PCMV.

Because the method for preparing vaccine of the present invention is without any need for the target antigen chemical knowledge of capsular polysaccharide for example, this method does not rely on the needs of the cross-linking chemistry that exploitation and target antigen and carrier protein chemistry can hold.Though some antigen still may interact with joint, the usefulness of this vaccine that should not detract, in any case because target antigen and carrier protein be not intended to crosslinkedly can expect to have the immunogen characteristic.In vaccine of the present invention, the crosslinked of target antigen and carrier protein is not to make vaccine effectively necessary.This and traditional conjugate vaccines form sharp contrast, are therefore hindered in its preparation and exploitation.The vaccine phase of the present invention need have at least, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% and even 100% crosslinked carrier protein for example, and be 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.Ideally, be no more than 10% antigen and carrier protein is crosslinked and at least 50% carrier protein is crosslinked.

The method for preparing vaccine described herein do not cause target antigen for example the pod membrane polymer extensively modified.Antigen keeps same state usually, and possible modification is for example reduction (reduction) of reducing sugar for the PS pod membrane that the end at polymer chain carries this group.This small modification can not have influence on the immunogenicity of most of pod membrane PS, because the inner residue of polymer enriches 100-1000 doubly than terminal sugar.On the contrary, for traditional conjugate vaccines, the joint group need be introduced for example pod membrane polymer of antigen usually, as the covalently bound point of carrier protein.Need to use joint because a lot of antigen for example the pod membrane polymer not as the reactive group of the part of its structure for example carboxyl or amino.For example, joint chemistry is introduced destruction and because its new epi-position that generation is not expected in vaccine product with the immune cross-reactivity of host self epi-position the unknown that PS can cause the pod membrane epi-position.

Vaccine production method described herein is not as the conjugate vaccines technical sophistication, because its chemistry only relies on carrier protein (DNI for example, b subunit of cholera toxin, diphtheria toxin, diphtherotoxin, tetanus toxin fragment C or escherichia coli (Escherichia coli) beta galactosidase) cross-linking chemistry.For example,, do not influence crosslinked pattern or degree, cross-linking agent (for example glutaraldehyde) concentration that the two is controlled by employed protein, concentration more and is added though the pod membrane polymer influences crosslinking rate when mixing with DNI.These parameters be can easily adjust, thereby required time and the effort of preparation vaccine, cost saving reduced.

Can use the method for the PCMV of preparation described herein, together with for example any pod membrane polymer of any antigen or any have minority amino or do not have that amino polymer and any carrier protein that is crosslinked for example do not have can be by the carrier protein of the destructive critical epitopes of borohydride reduction effect.The carrier protein phase of using in method described herein need have (unblocked) of at least 2 lysine residues or opening and can carry out other crosslinked residue through chemical modification.Tetanus toxoid is a kind of possible carrier protein.This toxin detoxifies by using formaldehyde, and formaldehyde is and the proteinic amino reagent that reacts.The carrier protein that other needs phase comprises b subunit of cholera toxin (available from SBL Vaccin AB), diphtheria toxin, diphtherotoxin, tetanus toxin fragment C (available from SigmaAldrich), DNI or from the beta galactosidase (available from Sigma Aldrich) of escherichia coli (Escherichia coli).

Current multivalence conjugate vaccines passes through at first synthetic discrete conjugate vaccines, subsequently their mixing generation " mixture " conjugate vaccines are prepared (for example, Wyeth septivalency Pnu-Imune 23,

).For example the pod membrane organic polymer is admixed together makes carrier protein crosslinked with for example glutaraldehyde subsequently by the antigen that chemistry is different, perhaps mix by distinguishing the specific vaccine of synthetic the present invention, the method that can use the present invention to prepare vaccine prepares polyvalent vaccine.This motility provides significant advantage for the method that the present invention prepares polyvalent vaccine.

The exemplary vaccine PCMV of the present invention who is discussed vaccine #1-3 in an embodiment, its performance is just like conjugate vaccines, although in fact these vaccines are not through expecting that the method that produces any covalent bond synthesizes, described covalent bond is at the atom that constitutes the PGA molecule and constitute between the proteic atom of DNI and form.Glutaraldehyde reacts with protein amino side chain by the amino representative of the ε of lysine residue single-mindedly.The PGA polymer does not have free amine group and only has not carboxylic side-chain with the glutaraldehyde reaction.Therefore, the immunne response that similar conjugate produced that is produced by PCMV shows that long PGA molecule is loaded in DNI cross linking of protein molecule substrate inside by molecule ground bag.

According to non-limiting pattern, the effect that described bag carries is to carry DNI albumen and PGA enters the B cell, and the B cell combines with this substrate by the Ig receptor of immunity identification PGA.In case absorbed by these B cells, described substrate is degraded in the mode similar to traditional conjugate vaccines, this can cause the generation of the DNI derived peptide of showing on the MHC-II of corresponding B cell molecule.Thereby attached amplification and the maturation that causes this B cell that help that this raises the T cell immediately becomes generation product IgG plasma cell and the memory cell special to PGA.Therefore, according to non-limiting pattern, but PCMV works to be similar to albumen-put together the pod membrane vaccine in immunity completely different, because PCMV lacks significantly covalently bound between carrier protein and the pod membrane polymer.

The vaccine of the present invention that comprises PCMV can be used for combination, for example is used for the department of pediatrics vaccine.In addition, vaccine of the present invention can be used for the inoculation antagonism, for example, pneumococcal infection, (A group and B group) streptococcal infection, influenza B haemophilus (Haemophilus influenzae TypeB) (" HiB ") infects, meningococcus (for example, Neisseria meningitidis (Neisseriameningitides)) infects, and (for example can be used as from gram negative bacteria, Pseudomonas aeruginosa (Pseudomonas aeruginosa), soil draws hot Frances Salmonella (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)) the O antigen vaccine.

Vaccine of the present invention can prepare in the following manner: for example in the presence of target antigen described herein, use that any for example those make at joint described herein that any for example those are crosslinked at carrier protein described herein at one or more.If use a kind of target antigen, albumen substrate vaccine of the present invention then is considered to univalent vaccine.If use more than a kind of target antigen, albumen substrate vaccine of the present invention then is considered to polyvalent vaccine.If microorganism pod membrane polymer is a target antigen, albumen substrate vaccine of the present invention then is considered to albumen pod membrane matrix vaccines (PCMV).

Joint

Carrier protein is crosslinked known in the present technique field, comprises glutaraldehyde, a dimaleoyl imino benzoyl-N-N-Hydroxysuccinimide base ester, carbodiimide and BDB.

Use the conventional method and the existing description of part of the direct crosslinked carrier protein of the difunctional or heterogeneous bifunctional linker of homogeneity, for example at G.T.Hermanson in Bioconjugate Techniques, Academic Press, 1996 and Dick and Beurret in Conjugate Vaccines.Contribu.Microbiol.Immunol., Karger, Basal 10:48-114 is in 1989.For example, for having the carrier protein of lysine part that number is n, in theory, there be n+1 primary amine (comprising terminal amine) to can be used for carboxyl reaction with exemplary cross-linking agent.Therefore, if use this direct conjugation methods, product only limits to have a formed n+1 amido link.

Employed joint in the embodiment of phase need of the present invention, but the simplest, be the key that connects two carrier proteins.Described joint can be the molecular skeleton of straight chain, ring-type or side chain, has and two carrier proteins (A) and (B) covalently bound side group.Any given carrier protein all can be connected more than a carrier protein, can seal (enclose) antigenic interconnected carrier protein substrate thereby produce.

The term linking group is meant that reactive moieties by joint (L) with functional group (A) and (B) combines the covalent bond that is produced.The example of described linking group includes but not limited to: ester, carbamate, thioesters, imines, disulphide, amide, ether, thioether, sulfonamide, isourea, isothiourea, imino-ester, amidine, phosphoramidate, di-phosphate ester, thioether and hydrazone.

(A) realize with the mode that is connected by covalency of (B), comprise be positioned at (A) and (B) on one or more functional groups formation key (linking group).The example that can be used for the chemical reaction functional group of this purpose includes but not limited to: amino, hydroxyl, sulfydryl, carboxyl, carbonyl, thioether (thioether), guanidine radicals (guanidinyl), imidazole radicals and phenolic group, all these groups all are present in the natural amino acid in many carrier proteins.

Therefore available joint (L) influence (A) of reactive moieties covalently bound with (B) that contain, this reactive moieties can be present in (A) and described functional group reactions (B).The product of this reaction is a linking group, the key that it contains connection (L) and (A) and is connected (L) and (B) new formation.For example, hydroxyl (A) can with (L) carboxyl or its activatory derivatives reaction, vide infra, cause the formation of ester linking group.

Can comprise XCH with the example of the part of sulfydryl reaction ₂The alpha-halogen acetyl compounds of CO-type (wherein X=Br, Cl, or I); its demonstration and the specific reactivity of sulfydryl, but also can be used for modifying imidazole radicals, thioether (thioether), phenol and amino, this is as for example Gurd; MethodsEnzymol.11:532,1967 descriptions.The N-maleimide derivatives is considered to that sulfydryl is had selectivity, but can be used for coupling amino in addition under given conditions.Carry out if connect by forming disulphide bridges, so for example the reagent (Traut etc., Biochemistry 12:3266,1973) of introducing the 2-imino group tiacyclopentane of sulfydryl by amino conversion can be considered as sulfhydryl reagent.

Can comprise with the example of the reactive moieties of amino reaction, for example, alkanisation and acylating reagent.

Representative alkanisation reagent comprises:

(i) alpha-halogen acetyl compounds shows the specificity of amino when sulfhydryl-group activity does not exist and is XCH ₂CO-type (wherein X=Cl, Br or I), this describes as Wong (Biochemistry 24:5337,1979);

(ii) N-maleimide derivatives can be by Michael reaction or acylation and amino reaction by adding cyclocarbonyl, and this describes as Smyth etc. (J.Am.Chem.Soc.82:4600,1960 and Biochem.J.91:589,1964);

(iii) aryl halide, for example reactive nitro halogen aromatic compounds;

(iv) alkyl halide is described as (J.Protein Chem.7:581,1988) such as for example McKenzie;

(v) can form the aldehyde and the ketone of Schiff's base with the amino reaction, obtain stable amine by reduction usually and make the addition product of formation stable;

(vi) epoxides as epoxychloropropane and bisoxirane, can react with amino, sulfydryl or phenolic hydroxyl group;

(vii) the s-triazine contain chlorine derivative, as amino, sulfydryl and hydroxyl very strong reactivity is arranged with nucleopilic reagent;

(aziridine of viii) above-mentioned s-triaizine compounds is described as Ross (J.Adv.Cancer Res.2:1,1954), reacts by open loop and nucleopilic reagent such as amino;

(ix) as the described side's diethyl phthalate of Tietze (Chem.Ber.124:1215,1991); With

(x) alpha-halogen alkane ether is to have more reactive alkanisation reagent than general alkyl halide, because ether oxygen atom produces activation, this describes as (Eur.J.Med.Chem.28:463,1993) such as for example Benneche.

Representative amino reaction acylating reagent comprises:

(i) isocyanates and isothiocyanate, especially aromatic derivative form stable urea and thiourea derivative respectively;

(ii) sulfonic acid chloride is described by (Biopolymers 2:349,1964) such as Herzig;

(iii) acyl halide;

(iv) active ester, for example nitro phenyl ester or N-hydroxy-succinamide ester;

(v) anhydride is as mixed acid anhydride, symmetric anhydride or N-carboxylic acid anhydrides;

(vi) other forms useful reagent for amido link, and is described as M.Bodansky (Principlesof Peptide Synthesis, Springer-Verlag, 1984);

(vii) acyl azide for example with the azido of sodium nitrite by ready-formed hydrazide derivatives generation, is described as (Anal.Biochem.58:347,1974) such as Wetz; With

(viii) imino-ester, it forms stable amidine with amino reaction, and this describes as for example Hunter and Ludwig (J.Am.Chem.Soc.84:3491,1962).

Aldehydes for example glutaraldehyde and ketone can form Schiff's base with the amine reaction, can advantageously stablize by the reduction amination effect.The amino part of alcoxyl is easy to produce stable alkoxyamine (alkoxamine) with ketone and aldehyde reaction, and this is described as (Bioconjugate Chem.1:96,1990) such as for example Webb.

Can comprise diazonium compound such as diazo acid ester and diazo acetamide with the example of the active part of carboxyl reaction, they react the generation ester group with high degree of specificity, describe as Herriot (Adv.Protein Chem.3:169,1947).Also can use carboxylic acid to modify for example carbodiimides of reagent, its formation by the O-acylureas is reacted by the formation of amido link and is obtained.

At (A) and/or the functional group (B), if needed, can before reaction, change into other functional group, for example to give other reactivity or selectivity.The method example that is used for this purpose comprises: use the reagent as dicarboxylic acid anhydride to make amine convert carboxylic acid to; The butanimide radical derivative that uses reagent such as N-acetyl-homocysteine thiolactone, S-acetyl mercapto succinic anhydrides, 2-imino group tiacyclopentane reaches or contain thiol makes amine convert sulfur alcohol to; For example using, the reagent of α-halogen acetic acid ester makes mercaptan convert carboxylic acid to; Use the reagent of aziridine for example or 2-bromine ethamine to make mercaptan convert amine to; For example using, the reagent of carbodiimide uses diamidogen to make carboxylic acid convert amine to subsequently; For example using, the reagent of paratoluensulfonyl chloride carries out ester interchange and is hydrolyzed into mercaptan with sodium acetate making alcohol convert mercaptan to thiacetate subsequently.

If necessary, can use so-called distance of zero mark joint according to the present invention, its relate to activity chemistry group (A) and activity chemistry group (B) do not introduce directly carry out under the other situation that is connected material covalently bound.The chemical compound that example is included, wherein (L) representative connects the oxygen atom and the carbonyl or the thiocarbonyl chemical bond partly that are present on (B) on (A), and causing linking group is ester or thioesters.For example, can produce A-L-B by using the carbodiimide chemistry, make amino (A) be connected to carboxyl (B), wherein L is amido link or the R-C=O that is connected to N-R, and wherein R is the carbochain from the amino acid side chain of same or two different proteins molecules.

Yet modal is that described joint comprises two or more active parts that connected by above-mentioned spacer element.The existence of sept allow bifunctional linker with (A) and the reaction of the particular functional group (B), produce covalently bound between these two chemical compounds.Active part in the joint (L) can identical (homogeneity bifunctional linker) or different (heterogeneous bifunctional linkers or wherein have the heterogeneous multifunction conjunction of several different active parts), this provide multiple can be at (A) and the covalently bound potential reagent of generation (B).

Spacer element is made up of chain usually, and these chains separate (A) and (B) effectively by following group: the ring-type system of a direct-connected or alkine of the direct-connected or branched heteroalkyl groups of the direct-connected or branched alkyl of 1-10 carbon atom, a 1-10 atom, the direct-connected or branched-chain alkene of a 2-10 carbon atom, a 2-10 carbon atom, the aromatic moieties of a 5-10 carbon atom, a 3-10 atom or-(CH ₂CH ₂O) _nCH ₂CH ₂-(wherein n is 1-4).

The character of the foreign material that bridging agent is introduced may be influential to the pharmacokinetics and/or the activity of final vaccine product.Therefore, the phase need be introduced the joint of cleavable, and it contains biodegradable or chemosensitive or mixes the spacerarm of restriction enzyme site.

The joint of these cleavables is for example described in the open WO 92/17436 (being attached to herein by reference) of PCT, in vivo easily by biodegradation.In some cases, linking group is cleaved in the presence of esterase, and is stable in the next maintenance of the non-existent situation of this kind of enzyme.Therefore, can advantageously (A) be connected with (B) to allow them to pass through near the enzyme active disease sites and slowly discharge.

Joint can form the linking group of biodegradable diester, diamides or diamino acid ester group with formula I:

--(Z ¹) _o-(Y ¹) _u-(Z ²) _s-(R ₁₁)-(Z ³) _t-(Y ²) _v-(Z ⁴) _p-

I

Wherein, Z ¹, Z ², Z ³And Z ⁴In each independently be selected from O, S and NR ₁₂(R wherein ₁₂Be hydrogen or alkyl); Y ¹And Y ²In each independently be selected from carbonyl, thiocarbonyl, sulfonyl, phosphoryl or similarly acid form group; Among o, p, s, t, u and the v each all independently is 0 or 1; R ₁₁Be aromatic moieties, a 3-10 atom of a direct-connected or alkine, a 5-10 carbon atom of direct-connected or branched-chain alkene, a 2-10 carbon atom of direct-connected or branched heteroalkyl groups, a 2-10 carbon atom of direct-connected or branched alkyl, a 1-10 atom of 1-10 carbon atom the ring-type system ,-(CH ₂CH ₂O) _qCH ₂CH ₂-wherein q is 1-4 or general-(Z ¹) _o-(Y ¹) _u-(Z ²) _s-be connected to-(Z ³) _t-(Y ²) _v-(Z ⁴) _p-chemical bond.

The exemplary phase used in the present invention needs any one molecule among the available formula II-III of joint (L) to describe:

Wherein said joint and oxygen atom (A) and oxygen atom (B) are covalently bound.Therefore, the joint of formula II-III (L) is connected with (B) with carrier protein (A) by two pyrans, ester or carbamate linking group.In these embodiments, R ₁₃Represent the ring-type system of aromatic moieties, a 3-10 atom of a direct-connected or alkine, a 5-10 carbon atom of direct-connected or branched-chain alkene, a 2-10 carbon atom of direct-connected or branched heteroalkyl groups, a 2-10 carbon atom of direct-connected or branched alkyl, a 1-10 atom of 1-10 carbon atom, wherein n be 1-4-(CH ₂CH ₂O) _nCH ₂CH ₂-or connect the chemical bond of two nitrogen or two carbonyls.

The joint that designs for formation hydrazone key has Formula I V:

Z wherein ⁵Be selected from O, S or NR ₁₆R ₁₆It is hydrogen or alkyl; R ₁₅Be selected from hydrogen, alkyl or assorted alkyl; Y ³Be selected from carbonyl, thiocarbonyl, sulfonyl, phosphoryl or the similar acid formation group covalently bound with oxygen atom (A); W is 0 or 1; R ₁₄Be that the direct-connected or branched heteroalkyl groups of direct-connected or branched alkyl, a 1-10 atom of 1-10 carbon atom, the direct-connected or branched-chain alkene of a 2-10 carbon atom, a direct-connected or alkine of a 2-10 carbon atom, the aromatic moieties of a 5-10 carbon atom, ring-type system, the n of a 3-10 atom are 1-4-(CH ₂CH ₂O) _nCH ₂CH ₂-or general-(Y ³)-(Z ⁵) _w-be connected to

Chemical bond; And X ₄Be hydrazone, by the condensation reaction generation of (B) that contain hydrazide group and joint II precursor, wherein X ₄It is the oxygen atom of ketone or aldehyde radical.

Carrier protein

Generally speaking, any carrier protein that can carry with the antigen bag under physiological condition can use in the present invention.Ideally, described antigen and carrier protein bag are loaded in the complex, and do not exist significant when covalently bound between antigen and carrier protein.Do not exist covalently boundly significantly, mean and be no more than 50% antigen and be connected with carrier.In the embodiment that needs in the phase, it is covalently bound to be no more than 40%, 30%, 10% or 5% antigen and carrier protein.Described antigen/carrier protein complex can contain another kind of chemical compound such as Alumen, and this another kind of chemical compound, in the embodiment of phase need, can wrap and carry an antigen and a carrier protein.

Ideally, the carrier protein that is used for vaccine of the present invention be those independent or with the bonded protein of antigen, it causes the immunne response of study subject.Ideally, described carrier protein contains at least one by the epi-position of T cell recognition.Ideally, described epi-position can be induced the t cell response of study subject, and induces the antibody of B cell generation at whole target antigen.Be used to describe epi-position of the present invention, comprise and be responsible for carrying out any determinant on the antigen of specific reaction with antibody molecule or its fragment.The epi-position determinant is made up of the chemically reactive surface bunch (grouping) such as the molecule of aminoacid or sugared side chain usually, has specific Three Dimensions Structure and specific charge characteristic.In order to have immunogenicity, protein or polypeptide can stimulate the T cell usually.Yet shortage also can be had immunogenicity by the carrier protein of the epi-position of T cell recognition.

By the known carrier protein that causes that strong immunization is replied is selected, can treat the study subject of different groups with PCMV as herein described.The carrier protein phase needs the strong immunization of vaccine to be replied irrelevant (sufficiently foreign) fully with causing.Typically, employed carrier protein is to give target antigen immunogenic molecule.In the embodiment of phase need, carrier protein had hyperimmunization originality originally.Therefore, having immunogenicity highly also can make at the maximized carrier protein of compound antigenic antibody production is ideal with it.

Various carrier protein of the present invention comprises, for example, toxin and toxoid (chemistry or hereditary), it can be or not be mutant, as anthrax toxin, PA and DNI (PharmAthene, Inc.), diphtheria toxoid (Massachusetts State BiologicalLabs; Serum Institute of India, Ltd.) or CRM 197, tetanus toxin, tetanus toxoid (Massachusetts State Biological Labs; Serum Institute of India, Ltd.), tetanus toxin fragment Z, Pseudomonas aeruginosa (Pseudomonas aeruginosa) exotoxin A or exotoxin A mutant, bacterial flagellin, pneumolysin, (bacterial strain provides (U.S. typical case's culture center by ATCC to Neisseria meningitidis (Neisseria meningitidis) outer membrane protein, Manassas, VA)), Pseudomonas aeruginosa (Pseudomonasaeruginosa) Hcp1 albumen, escherichia coli (Escherichia coli) heat labile enterotoxin, shiga-like toxin, people LTB albumen, protein extract from the intact bacterial cell, can carry out crosslinked any other albumen by joint.Ideally, described carrier protein be b subunit of cholera toxin (providing), diphtheria toxin, diphtherotoxin by SBL Vaccin AB (Connaught, Inc.), tetanus toxin fragment C (providing), DNI or from the beta galactosidase (providing) of escherichia coli (Escherichiacoli) by Sigma Aldrich by Sigma Aldrich.The carrier protein that other phase needs comprises bovine serum albumin (BSA), P40 and chicken riboflavin.(unless otherwise noted, exemplary carrier protein is buied from Sigma Aldrich) other exemplary carrier protein is the MAP (multiple antigenic peptide) as branched peptide.By using MAP, crosslink density is owing to a plurality of ramose amino acid residues reach maximization.The exemplary aminoacid that can be used for forming MAP for but be not limited to lysine.

BSA and keyhole limpet hemocyanin (KLH) all are used as carrier usually in the vaccine development of testing with animal.Be used to prepare the carrier protein for the treatment of vaccine and included, but are not limited to many pathogenic bacterium toxin and their toxoid.Example comprises the corresponding toxoid that diphtheria and tetanus toxin and they are medically acceptable.Other candidate's carrier protein is on the antigen and is called the similar protein of bacteriotoxin of cross reaction material (CRM).Carrier protein of the present invention also can comprise any inhuman source with anyone group food in non-existent albumen.

In the embodiment of phase need of the present invention, the protein that forms loop sample structure is used to prepare PCMV.These albumen comprise the Hcp1 albumen of Pseudomonas aeruginosa (Pseudomonas aeruginosa), nontoxic " B subunit ", escherichia coli (Escherichia coli) heat labile enterotoxin and the shiga-like toxin of cholera toxin.This ring sample albumen composition can form " beading ", and the PS chain of its neutral line penetrates the central groove of these annular albumen compositions.After the albumen generation is crosslinked, predict that this complex is stable especially.Proteic structured data shows that these central grooves are enough big so that it is entered easily for the PS chain.For example, the central groove of Hcp1 six aggressiveness rings is 42 dusts (A), and it is enough wide so that can hold the polysaccharide chain that several width are 5.5 dusts (Mougous etc., Science 312 (5779): 1526-1530,2006) easily.Perhaps, the albumen ring can assemble (for example, under specific physical and chemical condition, entering ring from the monomer carrier protein subunit of natural assembling) around PS.This monomeric protein that fits into ring is known and comprise in the present technique field, for example, and pneumolysin (Walker etc., Infect.Immun.55 (5): 1184-1189,1987; Kanclerski and Mollby, J.Clin.Microbiol.25 (2): 222-225,1987), Listerella lysin O (Kayal and Charbit, FEMSMicrobiol.Rev.30:514-529,2006; Mengaud etc., Infect.Immun.55 (12): 3225-3227,1987), DNI, anthrax PA, Hcp1, b subunit of cholera toxin, shiga toxin B subunit, flagellin and countless at the correlation molecule by various microorganisms preparations known in the art.

In the embodiment of another phase need, use Toll sample receptor (TLR) agonist as carrier protein.Toll sample receptor (TLR) activation is important forming acquired immunity aspect replying, and is playing a role aspect affinity maturation, isotype conversion and the immunological memory of antibody response.Vibrio cholera (Vibrio cholerae) flagellin (FLA) is the TLR agonist.The FLA albumen that surpasses 20mg from recombination bacillus coli (Escherichia coli) purification obtain, induce at IL-6 macrophage as herein described to be shown as effective TLR activator in the analysis.In addition, streptococcus pneumoniae (Streptococcus pneumoniae) albumen that is called the high conservative of " pneumolysin " also shows can activate TLR4, but also is protective antigen.Therefore, this albumen also can be used as the PCMV carrier protein.

In addition; outer membrane protein (OMP) mixture (for example; the OMP of Neisseria meningitidis (Neisseriameningitidis)) is used as the carrier protein of the HIB conjugate vaccines of producing by Merck, has to the small part protectiveness and in animal infection modal, be shown as from the protein extract of complete streptococcus pneumoniae streptococcus (Streptococcal pneumoniae) bacterial cell.In the embodiment of phase need of the present invention, these protein mixtures are sources of PCMV carrier protein.

In the embodiment that needs in the phase, described PCMV method is used with carrier protein, and described carrier protein has, for example, and at least 2 lysine residues or open and can carry out other crosslinked residue through chemical modification.In the embodiment of another phase need, described carrier protein is polymer (polymer that for example, contains at least 5 subunits).Ideally, described polymer is same polymer.

In another embodiment, use DNI as carrier protein, because it is nontoxic, needn't be before use with the albumen detoxification.In addition, the use of DNI is ideal, because DNI except that the protective immune response of inducing target antigen, also can induce the immunne response to Bacillus anthracis (B.anthracis).In addition, DNI does not have inner disulfide bond.This key is to borohydrides reduction sensitivity, and the latter can make albuminous degeneration and cause inducing the forfeiture of the epi-position of anthrax toxin neutralizing antibody.

Target antigen

Vaccine combination of the present invention and preparation and the method that gives this vaccine are applicable to any target antigen, for example, and polysaccharide, polyhydric alcohol or polyamino acid.Ideally, described target antigen does not carry can be by the destructive elementary group of the chemical reaction that the vaccine production method is adopted (primary group), and described chemical reaction for example destroys the antigen disulfide bond by the borohydrides reduction and causes antigenic degeneration.Exemplary target antigen includes organic polymer such as polysaccharide (polysaccharide that at least 18 residues are for example arranged), phosphoric acid polysaccharide, has the polysaccharide of the amino sugar of N-acetyl group replacement, contains the polysaccharide of sulfanilyl sugar, the sugar of other sulfate modification or sugar, polyhydric alcohol, polyamino acid, techoic acid and the lipopolysaccharide O side chain that phosphate is modified.Exemplary target antigen comprise those by microorganism such as antibacterial, fungus, parasite, virus is synthetic and the pod membrane organic polymer of purifying from this biogenetic derivation with standard method.Exemplary target antigen comprises microorganism pod membrane organic polymer, those pod membrane organic polymers that comprise purification from following microorganism: bacterium living beings body, bacillus cereus (Bacillus species) (comprising Bacillus anthracis (B.anthracis)) (Wang and Lucas for example, Infect.Immun.72 (9): 5460-5463,2004), streptococcus pneumoniae (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), shigella (Shigella) (Zhao etc., Carbohydr.Res.342 (9): 1275-1279, Epub 2007), Haemophilus influenzae (Haemophilus influenzae), Neisseria meningitidis (Neisseria meningitidis), staphylococcus aureus (Staphylococcus aureu), salmonella typhi (Salmonellatyphi), streptococcus pyogenes (Streptococcus pyogenes), escherichia coli (Escherichia coli) (Zhao etc., Carbohydr.Res.342 (9): 1275-1279, Epub 2007) and Pseudomonas aeruginosa (Pseudomonas aeruginosa); Fungal organism such as Cryptococcus and candida mycoderma; And much other microorganisms (are seen Ovodov for example, 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).Exemplary target antigen also comprise non-natural that exist with polymer abiotic source.

Vaccine combination

The vaccine of the present invention that comprises PCMV can be used for combination, for example is used for the department of pediatrics vaccine.In addition, vaccine of the present invention can be used for inoculation and resists for example pneumococcal infection, influenza B haemophilus (Haemophilus influenzae Type B) (" HiB ") infects, (group A and group B) streptococcal infection, meningococcus (for example Neisseria meningitidis (Neisseria meningitides)) infects, and can be used as from gram negative bacteria (Pseudomonas aeruginosa (Pseudomonasaeruginosa) for example, soil draws hot Frances Salmonella (Francisella tularensis), shigella (Shigella species), Salmonella (Salmonella species), acinetobacter calcoaceticus (Acinetobacter species), bulkholderia cepasea (Burkholderia species) and escherichia coli (Escherichia coli)) the O antigen vaccine.

The bacterin preparation phase need comprise at least a carrier protein, one or more target antigen and pharmaceutically acceptable carrier or excipient (for example aluminum phosphate, sodium chloride and sterilized water).Vaccine combination also can comprise and be used to strengthen the immunogenic adjuvant system of preparation, for example oil-in-water system and at other system known in the art or pharmaceutically acceptable other excipient.Insoluble carrier under physiological condition/antigenic compound phase needs give behind the study subject released antigen lentamente.This complex phase need be discharged in the suspension that contains pharmaceutically acceptable excipient.Yet carrier/antigenic compound also may be solubility under physiological condition.

The volume that is generally used for hypodermic vaccine is about 0.5mL, and intradermal injection is 0.1mL, and percutaneous dosing is 0.002-0.02mL.0.5mL the vaccine of dosage can contain the antigen of about 2-500 μ g that useful about 2-500 μ g carrier protein bag carries.In the embodiment of phase need, in the dosage of 0.5mL, with the antigen of about 10 μ g carrier protein Bao Zaiyue, 10 μ g.Mol ratio phase of antigen and carrier protein needs between 1: 10 (for example, 1 part of antigen to 2 parts of carriers or 1 part of antigen to 3 parts of carriers) to 10: 1 (for example, 3 parts of antigen to 1 part of carrier or 2 parts of antigens to 1 part of carrier).In the embodiment of phase need, the mol ratio of antigen and carrier is 1: 1.Perhaps, the dry weight of antigen and carrier protein is between 1: 10 to 10: 1 (for example 1: 1 dry weight) than reason.

Because peptide or conjugate can be degraded under one's belt, the therefore described vaccine phase needs through parenteral (for example, by subcutaneous injection, intramuscular injection, intravenous injection, intradermal injection).When current need were passed medicine (for example, pin, aerosol apparatus or galling) by the method for physics infiltration skin corium, vaccine of the present invention also can carry out administration by Transdermal absorption.

Especially, vaccine of the present invention can be inoculated by for example intramuscular injection, intradermal injection or transcutaneous immune together with suitable immunological adjuvant and give study subject.Vaccine of the present invention can often comprise the secondary administration of design to promote that study subject antibody produces through in single or divided doses, infects to prevent infectious agent.The frequency of vaccine administration and amount depend on the given activity of vaccine, and can easily determine by normal experiment.

For example, for the baby, inoculation program can be: three each dosage of 0.5mL dosage are interval (since two monthly ages) with about 4-8 week, then the dosage with the 4th 0.5mL at about 12-15 monthly age.For some vaccines, the phase needs between four years old to six years old with the 5th dosage.

Though at normally two months age of administration for the first time, can when 6 ages in week, give the baby with vaccine.For the child who exceeds conventional baby's inoculation plan age, can give vaccine of the present invention according to following exemplary planning chart.

The first administration age	The inoculation plan
The first administration age	The inoculation plan	7-11 month	Three 0.5mL dosage altogether; Two at least at interval around, the 3rd after second at least two months
12-23 month	Two 0.5mL dosage altogether, each was separated by two months at least	7-11 month
12-23 month		24 months to 9 years old	A 0.5mL dosage

For the adult, two or more 0.5mL dosage in 2-8 week is enough to provide secular protection usually at interval.Through the adult of immunity inoculation and the child more than 11 years old, the phase needs per ten years to a booster dose for previous.

Described preparation can be deposited in ampoule and the bottle that for example seals in the container of single dose or multiple dose, can preserve under lyophilizing (lyophilization) condition, only need add aseptic liquid-carrier immediately before using.Vaccine of the present invention can be formulated in the pharmaceutically acceptable carrier, described carrier is gel aluminum hydroxide, adjuvant formulation or saline for example, then by for example intramuscular injection, intradermal injection or utilize the transdermal immune of suitable immunological adjuvant to inoculate and carry out administration.

The present invention also comprises and contains vaccine described herein (for example, medicine box PCMV).Medicine box of the present invention also comprises the medicine box operation instructions of inoculation method described herein.

Can determine the effect of immunity inoculation plan by the antibody titer of measuring study subject with standard method.Generally speaking, think the average antibody of about 1 μ g/mL tire (phase is required to be IgG and tires) show that long-term protective effect is arranged.

The diameter phase that is used for the antigen/carrier protein complex of vaccine combination described herein needs between 10nm-100 μ m.The virus diameter can be 100nm and has immunogenicity.The diameter of intact bacterial is 1-10 μ m and also has immunogenicity.The diameter of one little antibacterial is about 100 μ m.In specific embodiment, the diameter phase of the antigen of described vaccine combination/carrier protein complex needs between 100nm-10 μ m.This complex can be solubility or insolubility.

By reference certain embodiments, embodiment and accompanying drawing, hereinafter the present invention is made description.Following examples are not interpreted as restricted.

Embodiment

Embodiment 1. vaccines and control formulation

Pod membrane gathers γ-D-glutamic acid (PGA) available from Vedan (Taiwan) or carry out purification by the method for (Proc.Natl.Acad.Sci.USA 100:10925-10930,2003) such as Rhie and obtain.Dominant negative mutant (DNI) is the mutant form of Bacillus anthracis (B.anthracis) protection antigen (PA), makes from escherichia coli (Escherichia coli) with the method (Biochemistry 37:3941-3948,1998) of Benson etc.PGA and DNI albumen use 0.05M, pH 7.4 sodium phosphate buffers (SP7.4) thoroughly to dialyse before use.DNI storing solution concentration is 30mg/mL.PGA storing solution concentration is 134mg/mL.The joint glutaraldehyde is 25% storing solution available from Pierce.Albumen pod membrane matrix vaccines (PCMV) and contrast compile (assemble) in the reaction of table 1.

Table 1. is used to prepare the compiling of reaction (assembly) of PCMV preparation 1-3 and

contrast

4 and 5

Reaction #DNI PGA dH ₂O 25% glutaraldehyde

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 contrast

5 16 08 0.8 P only contrast

Under room temperature, five reactions are not packed under the situation of glutaraldehyde having.When T=0, add the glutaraldehyde (G25) of 0.1mL 25% in the shown reaction.Thereafter per G25 and repetition that added other 0.1mL in 30 seconds reaches the G25 of 0.8mL altogether up to reaction shown in each.The molecule crosslinked situation of DNI of being undertaken by difunctional glutaraldehyde molecule can be by being observed visually, this is according to producing different turbidity and judging according to the insoluble particle sample " gel " (" gel " like particle) that following order is arranged: the highest and gel formation situation of turbidity, reaction 1＞2＞3＞4, reaction 5 keeps fully transparent and solvable.After 1 hour, 0.5M sodium borate buffer liquid pH 9.3 (SBH) that 2mL contained the 1M sodium borohydride join in all six reactions, to reduce the Schiff's base that forms between DNI molecules of ammonia base side chain and difunctional glutaraldehyde molecule.Silicone defoaming agent (0.01mL) is joined each reaction to be formed with the foam in the control course of reaction.Reactant was preserved 72 hours in 4 ℃.Then all reactants were fully dialysed 48 hours with SP7.4.With the centrifugal insoluble substance and stand-by of removing of end product in 4 ℃ of preservations.

By using water miscible carbodiimide, EDAC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide) amino coupled with carboxyl and the BSA of PGA, to synthesize the traditional conjugate between bovine serum albumin (BSA) and PGA, step is as follows: the water that makes 5mL contain 30mg/mL BSA mixes with the NP7.5 that 1mL contains 134mg/mL PGA, add 50mg EDAC, allow reaction at room temperature to carry out 3 hours.Make to react on 4 ℃, only in SP7.4, dialysed 24 hours then in 4 ℃ with SP7.4 dialysis blocking-up in the 18 hours active group that contains the 1mM glycine.End-product is called as the PGA-BSA conjugate.

After synthetic and dialysis PCMV and control formulation, check the proteic molecularity of DNI confirming no matter whether to exist different content PGA, glutaraldehyde really and albumen taken place molecule crosslinked.By SDS (dodecyl sodium sulfate) polyacrylamide gel electrophoresis with the described crosslinked situation of the sero-fast Western blot of anti-PA monitoring PCMV and control formulation.As shown in Figure 2, before glutaraldehyde cross-linking, DNI albumen moves to the 84kDa place.Confirm that as the migration band of molecular weight PCVM1-PCMV3 (swimming lane 1-3) shows that DNI albumen is crosslinked widely greater than 220kDa.Do not exist DNI albumen crosslinked alone under the PGA to show same high molecular weight species (swimming lane 5) yet.On the contrary, mix with PGA but show the band that moves altogether with DNI or lower molecular weight kind (swimming lane 4) without the DNI that glutaraldehyde is handled.Therefore, as if be subjected to protease activity contact scar from the PGA preparation of Vedan (Taiwan) to DNI.Vedan PGA sample carries out electrophoresis in swimming lane 6, yet does not show the contaminating protein of high-caliber and anti-PA antiserum reaction, shows that observed band is the DNI derivative products of each reaction.

In addition, with PGA and one or more as antigenic streptococcus pneumoniae PS whether be used for probing into FLA (vibrio cholera (Vibrio cholerae) flagellin) be a kind of under the PCMV background than the better carrier protein of DNI.By measuring, estimate the effect of these carrier proteins with the resulting carrier protein usefulness of calculating based on albumen weight at IgG level and the carrier protein of PS and PGA of various PCMV immunity inoculations.

Can also prepare PCMV by not using bi-functional cross-linking agent directly to make amino and carboxyl carry out crosslinked method.Particularly, prepare PCMV with the amino and the carboxyl of carbodiimide chemistry by crosslinked carrier protein.This chemistry makes between the carboxyl of the primary amino radical of lysine side-chain and agedoite and glutamine side chain and forms peptide bond.Though amino major part is subjected to through the sealing of the toxoid of formaldehyde treated, formaldehyde fully not with carboxyl reaction.Therefore, the carbodiimide chemistry can be used for preparing PCMV with fluid toxoid, and described fluid toxoid can have resistance to glutaraldehyde cross-linking.Be easy to crosslinked situation is detected by SDS-PAGE.The existence that depends on the high-molecular-weight protein " stain " that cross-linking agent (as glutaraldehyde) adds show taken place crosslinked.

The carrier protein that table 2. is determined by the SDS-PAGE analytic process is crosslinked

Glutaraldehyde is not

Pod membrane polymer-PGA-PGA+PGA+PS 6B+PS 23F

BSA	-	+	+	+	+
BSA	-	+	+	+	+	Diphtheria toxin, diphtherotoxin	-	+	+	n.d.	n.d.
Diphtheria toxoid	-	-	-	n.d.	n.d.	Diphtheria toxin, diphtherotoxin	-	+	+	n.d.	n.d.
Diphtheria toxoid	-	-	-	n.d.	n.d.	Tetanus toxoid	-	+	+	n.d.	n.d.

+ number expression detects crosslinked by SDS-PAGE ,-number expression albumen migration no glutaraldehyde contrast seen in to situation be as good as.N.d.-(not determined) does not measure (not analyzing).

For the experiment shown in the table 2, in 50mM HEPES pH 7.5, carry out the reaction of 200 microlitres and make it at room temperature be incubated 2 hours.With the reaction of going out of 120mM hydroboration Na temper.Add glutaraldehyde to 64mM, use bovine serum albumin (BSA), use diphtheria toxin, diphtherotoxin, diphtheria toxoid and tetanus toxoid extremely, add PGA, add 6B and 23F type streptococcus pneumoniae PS with 4mg/mL with 13.4mg/mL with about 5mg/mL with 15mg/mL.

As shown in table 2, some albumen through formaldehyde treated (for example, diphtheria toxoid) can not be crosslinked well with glutaraldehyde, therefore, needs to use other cross-linking chemistry to prepare PCMV.Other protein carrier as tetanus toxoid can with glutaraldehyde cross-linking, but different with the crosslinking degree of unmodified protein such as diphtheria toxin, diphtherotoxin and bovine serum albumin.

The analysis of embodiment 2. immunity inoculations and anti-DNI and anti-PGA immunne response

The soluble product of adjustment form 1 described 5 reactions is to same protein concentration (based on the absorptance at its 280nm place).To be used for described all the immunity inoculation researchs of Fig. 2 from the BALB/c mouse in about 5-7 age in week of Charles River.Contrasted 4 and 5 with 20 μ g DNIs proteic dosage immunized mice by peritoneal injection with PCMV vaccine 1-3 and antigen preparation at the 0th day.At the 7th day all mices are carried out blood-letting, at the 10th day antigen preparation it is carried out booster immunization then with onesize dosage.The 17th day once more to the mice blood-letting, then at the 20th day once more to its booster immunization.Again mice was carried out blood-letting at the 30th day and this time with its execution.Treat behind the blood clotting from blood sample collection serum and in-20 ℃ of storages.Use enzyme-linked immunosorbent assay (ELISA) to analyze the level of anti-PGA serum antibody and anti-DNI serum antibody.In brief, by volume the 0.1M sodium carbonate buffer (pH9.6) that contains 0.5 μ g/ hole BSA-PGA or DNI in 100 μ L/ holes to Immulon 2HB ELISA (VWR) microtitration plate bag.After 4 ℃ of incubated overnight, by with 3%BSA (w/v) (being dissolved in TBS-0.1% tween (TBST)) in room temperature 1 hour or 4 ℃ of night incubation, seal and be coated with antigenic plate.The blood serum sample that will merge from four mice groups when the time point behind each booster immunization carries out serial dilution with TBST, it is joined be coated with in the antigenic plate, hatches at least 1 hour.Use at Mus IgG that puts together with alkali phosphatase (Zymed) or the rabbit anti-serum of IgM, measure anti-DNI antibody response and anti-PGA antibody response.Be added to substrate p-nitrophenyl phosphate (PNPP) in each hole and measure the absorptance of each reaction in the 405nm place with spectrophotography.The form report that data are tired reciprocal with terminal point, the latter is defined as the OD of acquisition ₄₀₅Reading surpass two standard deviations of negative control reading greatest dilution.

Measure in intravital IgM of mice and special anti-DNI and the anti-PGA immunne response of IgG with the ELISA algoscopy through three kinds of PCMV preparation 1-3 and two antigen control preparations 4 and 5 (Fig. 3-5) immunity inoculation.As shown in Figure 3, except the control formulation 4 without glutaraldehyde cross-linking (no glutaraldehyde), the DNI albumen in all preparations has hyperimmunization originality.Yet these DNI specific immune responses are only based on IgG's.Though even also detect less than anti-DNI IgM the 7th day of immunity inoculation, at the 17th day, in the mice body of crossing through the mice and crosslinked DNI (preparation 5) immunity inoculation of those usefulness of PCMV preparation immunity inoculation, can detect significant anti-DNI IgG and reply.Observed after inoculation comprises all preparations of preparation 4 strong booster response at DNI at the 30th day.

Anti-PGA IgM replys and has shown typical pod membrane polymer pattern (Fig. 4).Control formulation 4 produced detectable anti-PGA IgM at the 7th day and replys, but the 17th or 30 day this do not reply and be reinforced.All PCMV preparations induced anti-PGA IgM to reply at the 7th day, produced exclusive then the 17th day and the 30th day even stronger anti-PGA IgM replys.As expected, control formulation 5 (having only crosslinked DNI) does not produce based on IgM or based on the anti-PGA of IgG and replys.What form sharp contrast is, PCMV1-3 (preparation 1-3) produces intensive anti-PGA based on IgG and replys, and this is replied performance in the 7th day obviously subsequently at the 30th day significantly strengthened (Fig. 5).Finding is similar at replying obviously based on the anti-PGA of IgG of PCMV1-3: the observed conventional P GA-DNI conjugate vaccines of Aulinger etc. report inductive immunne response (Infect.Immun.73:3408-3414 to PGA, 2005) and Rhie etc.) inductive the replying of PA-PGA conjugate vaccines (Proc.Natl.Acad.Sci.USA100:10925-10930,2003 described.Therefore inducing (Bacillus anthracis (B.anthracis) protection antigen (Wang etc. of known T cell dependent/non-dependent at pod membrane PGA; Infect.Immun.72:5460-5463; 2004) IgG) replys the aspect, and PCMV vaccine #1, #2 have and traditional the same good performance of PGA vaccine of puting together with #3.Contain with the control formulation 5 of the blended DNI of PGA (noncrosslinking) and do not induce detectable IgG, show DNI does not serve as the TLR part in the anti-PGA of IgG that stimulates PCMV preparation 1-3 replys effect at PGA.This result has also confirmed the observation in the document, and promptly PGA is the T cell dependent/non-dependent immunogen of reduced immunogenicity, unless it is by covalently bound and protein coupling (Rhie etc., Proc.Natl.Acad.Sci.USA 100:10925-10930,2003).The PCMV method obviously changes into PGA the dependent immunogen of T cell, although in fact this method do not cause DNI albumen directly and the PGA molecule carry out crosslinked.

These data support the PCMV method can prepare characteristic and the similar immunogen of traditional conjugate vaccines.Prepare PGA PCMV easily with method as herein described, and find the typical immunne response that it induces PGA-albumen conjugate vaccines.The small-scale prepared in reaction that describes in detail in the table 1 goes out the dosage of describing based on Fig. 3 enough carries out immunity inoculation to 1000 mices PCMV.These data are supported can be used as the anthrax that vaccine prevention is caused by Bacillus anthracis (Bacillus anthracis) by the PCMV of PGA and DNI preparation.

Preparation and the sign of embodiment 3. additional PCMV

The PCMV technology can be applicable to the k antigen of various structures and ionic charge.23 types of streptococcus pneumoniae (Streptococcus pneumonia) PS is available from American type culture collection (ATCC) and by Merck, and Inc makes.These PS are very different on its molecular structure, comprise the reinforcing YIN-essence ion-type, the PS partial cation type, neutral charge, phosphorylation, linear, that branched structure is arranged and that modify through various alternate manners.In preliminary experiment, get these PS subclass corresponding to 7 pod membrane types among the Wyeth product P revnar (4,6B, 9V, 14,18C, 19F and 23F), measure the ability that its inducing mouse macrophage produces IL-6.The PS of 4 types enlivens in mensuration; Lipopolysaccharide (LPS) is the contrast of TLR agonist.Also measured other PS (for example, 3 types), PGA and draw O antigen PS of hot Frances Salmonella (F.tularensis) and the PCMV vaccine that is prepared from by PGA-DNI and do not have crosslinked contrast from soil.This experiment has shown that 3 type streptococcus pneumoniae PS (degree than PGA is less) also are subjected to the TLR agonist and pollute.Draw the PS of hot Frances Salmonella (F.tularensis) and PCMV equal clean in mensuration from soil.Phenol extraction and ethanol precipitation can be after twice be handled continuously " cleaning " (removing the unknown TLR agonist of remnants) streptococcus pneumoniae (S.pneumoniae) PS 3 types.Therefore, find that 6 kinds of streptococcus pneumoniae (S.pneumoniae) PS and soil draw hot Frances Salmonella (F.tularensis) O antigen PS to clean totally for the generation of IL6, these are probed in experiment as herein described.

Utilize DNI as carrier protein by the method that is similar to described in the embodiment 1, cleaned 7 kinds of clean PS and carried out PCMV and synthesize producing for IL6.Preliminary immunogenicity determining shows that all seven kinds of PCMV have immunogenicity in various degree." unit price " PCMV (14-PCMV) based on DNI that finds streptococcus pneumoniae (S.pneumoniae) PS14 induces the remarkable enhanced height anti-PPS14 IgG that tires after immunity for the third time.Obviously, when six kinds of PCMV of 14-PCMV and other mix generation " mixing " antigen, can see same immunne response.Because

Be " adjuvantization " vaccine of absorption Alumen, so whether also can be determined by adsorbed onto alum adjuvant absorption to sexavalence PCMV mixture.Come observation measurements being exposed to the amount that PCMV or control mixture (identical PS mixes with DNI albumen but do not carry out glutaraldehyde cross-linking) are adsorbed in the streptococcus pneumoniae PS of Alumen afterwards by immunoassay, the result shows, the PS comparison that is adsorbed in Alumen under the PCMV background is according to (the noncrosslinking DNI albumen of PS+) much more (this as further the immunoreactivity higher level of the PCMV of dilution is indicated in immunoassay).

Mice is carried out immunity, the immunogenicity of the septivalency PCMV of the adsorbed onto alum adjuvant of being adsorbed in is arranged or do not have with evaluation.Adsorbed onto alum adjuvant is improved the immunne response kinetics to PS14, takes Zao 7 days with induce the IgG at this PS than no Adjuvanted vaccines.Yet, have more immunogenicity than the non-crosslinked PS+DNI combination contrast that has Alumen at the septivalency PCMV that does not have Alumen.This result has confirmed that the PCMV method makes PS have more immunogenicity for mice, and has supported the PCMV method can be used for preparing the antigen mixture that immune performance is similar to the conjugate vaccines mixture.

In other experiment, use phenol extraction and ethanol precipitation so that contaminative TLR agonist is removed from 23 kinds of pneumococal polysaccharide commercial formulation.Method with standard is come to testing through treated PS, to confirm the removal situation of pollutant according to inducing peritoneal macrophages to produce IL-6.The PS that lacks the IL-6 induced activity is used to prepare PCMV.Other polysaccharide that is used for PCMV comprises from soil and draws the O antigen PS of hot Frances Salmonella (F.tularensis) purification and from the PGA pod membrane of Bacillus anthracis (B.anthracis).25 pod membrane types (23 streptococcus pneumoniae types, each one of tularemia type and anthrax pattern of fever) have altogether been checked.Each of 25 kinds of pod membrane types is passed through embodiment 1 described method substantially, prepares PCMV with DNI albumen.PS with 1: 1 ratio: albumen is used for (about 1: 1 dry weight) these initial p CMV preparation.By SDS-PAGE every kind of preparation is characterized, confirming proteic crosslinked situation, the immunogenicity of various PCMV preparations is relevant fully in protein-crosslinking situation and the preliminary experiment.For some pod membrane type (for example 6B and 23F), use other carrier protein to prepare PCMV.For these same pod membrane types (for example 6B and 23F), can use alternative cross-linking chemistry.Immunogenicity to all PCMV preparations of having confirmed protein-crosslinking (for example in SDS-PAGE) is tested.

For example, below with 10 kinds of different PCMV of 5 kinds of different stromatins and 2 kinds of different antigen preparation.The selection of 5 kinds of stromatins based on: their are current to be used for vaccine of FDA approval, perhaps allows it to be used to measure other characteristic of PCMV preparation stability as tracer.Used following stromatin: (1) b subunit of cholera toxin (SBL Vaccin AB provides), (2) diphtheria toxin, diphtherotoxin, (3) tetanus toxin fragment C, " Frag C " (Sigma Aldrich provides), (4) DNI and (5) are from the beta galactosidase (SigmaAldrich provides) of escherichia coli (Escherichia coli).As k antigen, used k antigen to gather-D-glutamic acid and 14 type streptococcus pneumoniae (Streptococcuspneumoniae) pod membrane (Suarez etc. from Bacillus anthracis (Bacillusanthracis), Appl.Environ.Micobiol.67:969-971,2001).When being used for corresponding PCMV with DNI as stromatin, these two kinds of k antigens all have hyperimmunization originality.10 kinds of different PCMV are made in every kind of k antigen and 5 kinds of each combinations in the stromatin of selection.

As viewed in traditional conjugate vaccines, in the mice body, can test PCMV and induce isotype antibody to convert the ability of IgG to.Can make all antigens be adsorbed in the group that Alumen typically uses corresponding 5 mices of every kind of PCMV preparation then.Obtain at being tried antigenic immunne response baseline in advance the mice blood-letting.Peritoneal injection scheme by standard is to three immunity of mice several (in the 0th, 7,14 days) then, and the 10th, 20,30 and 60 day collection blood in initial immunity after.By the ELISA assay method of standard, mice serum is analyzed according to situation at the IgG of used PS and carrier protein.In these experiments, the control group mice of only accepting the PS immunity inoculation is included, be used for estimating various PCMV preparations with should non-immunogenicity or the low non-PS of puting together of immunogenicity compare the ability of inducing anti-PS IgG.Make promising PCMV (promptly inducing high-level anti-PS IgG) through careful immune analysis, manage to set up kinetics and the dose response situation of mice the PCMV immunne response.

Perhaps, promising PCMV and its corresponding contrast can be delivered to businessman to produce rabbit anti-serum.Carry out similar immunoassay estimate immunogenicity, the classification, the immunne response kinetics of rabbit of inductive antibody.In these experiments, contrast is the commercially available prod

It is the mixture that is adsorbed in Alumen of puting together the PS vaccine with the link coupled 7 kinds of different tradition of CRM197, and CRM197 is the nontoxic protein mutant relevant with diphtheria toxin, diphtherotoxin.

Can be to functional evaluation of the inductive antibody response of PCMV.For example, can come to estimate by measuring anti-PS antibody conditioning pod membrane streptococcus pneumoniae (S.pneumococcus) and causing macrophage to pass through the ability of phagocytosis killing bacteria functional.The deadly attack of avoiding pod membrane streptococcus pneumoniae (S.pneumococcus) that watches for animals is the another kind of method of proof vaccine in the effect in the animal of PCMV immunity.

Embodiment 4.PCMV with

Comparison

Mensuration by Merck available from ATCC or directly from streptococcus pneumoniae (S.pneumoniae) polysaccharide (pps) 6B of Serum Institute of India (SII), 14 with the relative cleannes of 23F.With the indicator of IL-6 expression, with the positive control of LPS as " unclean " as the pps cleannes.Shown in Fig. 9 A,

Merck pps

6B, 14 and 23F be clean, and shown in Fig. 9 B, be " unclean " from the pps 6B of SII.As shown in figure 10, handle 2 (80 ℃ are incubated 1 hour in 1M NaOH) and make SII pps 6B cleaning.Clean pps 6B is used for the immunological characteristic of comparison conjugate and PCMV.As shown in table 3, pollutant are not LPS.

Table 3. polysaccharide level of endotoxin is measured

Polysaccharide	Endotoxin unit/mg polysaccharide
Polysaccharide	Endotoxin unit/mg polysaccharide	SII pps
6B-is untreated	0.75	SII pps
6B-is untreated	0.75	SII pps 23F-is untreated	0.85
SII pps 23F-handles 2	0.24	SII pps 23F-is untreated	0.85
SII pps 23F-handles 2	0.24	Various Merck pps-are untreated	0.1-0.4

Figure 11 and 13 shows

(is adjuvant with Alumen) induces anti-pps 6B IgG antibody, and is PCMV (BSA and the pps 6B of adjuvant with Alumen; Diphtheria toxin, diphtherotoxin and pps 6B; Diphtheria toxoid and pps 6B; Tetanus toxoid and pps 6B) inductive IgG replys than usefulness

Viewed IgG replys better.Equally, as shown in figure 12, reply by the inductive IgM of the PCMV of adjuvant with Alumen and use

Being seen close.

In addition, for pps 14 (

The immunogenic pps of tool), shown in Figure 14-16, the PCMV that is adsorbed in Alumen that contains diphtheria toxoid and pps 14 or tetanus toxoid and pps 14 is inducing IgG almost to be equivalent to aspect replying

Embodiment 5. multivalence PCMV

Multivalent immunogen is former to be prepared by following PCMV method: chemically different pod membrane organic polymers are mixed, make DNI carrier protein crosslinked (" one pot of synthetic reaction ") with glutaraldehyde afterwards.As carrier, prepare this class trivalent immunogen with DNI with three kinds of organic polymer: PGA, alginate and glucosans.These trivalent vaccines have immunogenicity and produce immunne response at three kinds of pod membrane organic polymers, this result is according to shown in the index in the following drawings: before the immunity and the pooled serum IgM (Fig. 6) that analyzed after 30 days, the antigenic specificity serum IgG antibody that occurred in 60 days after the immunity inoculation is tired (Fig. 7), the anti-PS serum antibody titer (Fig. 8) that occurred in 128 days after the immunity inoculation.Shown in Fig. 6-8, unit price alginate PCMV preparation also produces immunne response in mice in addition.Also can prepare multivalence PCMV immunogen by mixing specific PCMV, PCMV makes " mixture " vaccine through separately synthetic last remix earlier together.

Open and other publication of all patents of quoting or relate in this description, patent application, patent application merges to herein by reference, its similar degree just like each patent, patent application, patent application is open or publication indicates by reference one by one or individually and merges to herein.

Claims

1. vaccine combination, it comprises target antigen and carrier protein, and wherein (i) is no more than 50% described target antigen and described carrier protein is crosslinked and (ii) wherein carry described antigen with described carrier protein bag forms complex.

2. the vaccine combination of claim 1, the diameter of wherein said complex is between 10nm-100 μ m.

3. the vaccine combination of claim 2, the diameter of wherein said complex is about 100nm-100 μ m.

4. the vaccine combination of claim 2, the diameter of wherein said complex is about 100nm-10 μ m.

5. the vaccine combination of claim 1, wherein said complex cause the dependent immunne response of T cell in described mammal when giving mammal.

6. the vaccine combination of claim 1, wherein said antigen to the mol ratio of described carrier protein 1: 10-10: between 1.

7. the vaccine combination of claim 1, wherein said carrier protein is a polymer.

8. the vaccine combination of claim 7, wherein polymer comprises at least 5 subunits.

9. the vaccine combination of claim 7, wherein said polymer is same polymer.

10. the vaccine combination of claim 1, other carrier protein of wherein said carrier protein and at least one is covalently bound.

11. the vaccine combination of claim 10, the peptide bond between the wherein said covalently bound carboxyl that is included in lysine side-chain primary amino radical and agedoite or glutamine side chain.

12. the vaccine combination of claim 10, the wherein said covalently bound formula that comprises

13. the vaccine combination of claim 10, wherein said covalently bound glutaraldehyde, dimaleoyl imino benzoyl-N-hydroxysuccinimide eater, carbodiimide or the BDB of comprising.

14. the vaccine combination of claim 10, the wherein said covalently bound bi-functional cross-linking agent that comprises.

15. the vaccine combination of claim 14, wherein said bi-functional cross-linking agent are glutaraldehyde, two [sulfonic group succinimido] suberate or oneself two imidic acid dimethyl esters.

16. the vaccine combination of claim 1, wherein said carrier protein is through non-covalent connection.

17. the vaccine combination of claim 16, wherein said non-covalent connection comprises hydrophobic interaction, ionization, model ylid bloom action or hydrogen bond.

18. the vaccine combination of claim 1, wherein said carrier protein is: diphtheria toxin, diphtherotoxin 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; 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; pneumolysin; Listerella lysin O (or associated protein); protein extract from the intact bacterial cell; the dominant negative mutant (DNI) of Bacillus anthracis (Bacillus anthracis) protective antigen or escherichia coli (Escherichia coli) beta galactosidase.

19. the vaccine combination of claim 18, wherein said intact bacterial cell are Pseudomonas aeruginosa (Pseudomonas aeruginosa) or streptococcus (Streptococcal) cell.

20. the vaccine combination of claim 18, wherein said bacterial flagellin are vibrio cholera (Vibrio cholerae) flagellins.

21. the vaccine combination of claim 18, wherein said shiga-like toxin are shigella (Shigella) SltB2 albumen.

22. the vaccine combination of claim 18, wherein said carrier protein is a pneumolysin.

23. the vaccine combination of claim 18, wherein said carrier protein are Listerella lysin O.

24. the vaccine combination of claim 18, wherein said carrier protein is a diphtheria toxin, diphtherotoxin.

25. the vaccine combination of claim 18, wherein said carrier protein is a diphtheria toxoid.

26. the vaccine combination of claim 18, wherein said carrier protein is a tetanus toxin.

27. the vaccine combination of claim 18, wherein said carrier protein is a tetanus toxoid.

28. the vaccine combination of claim 1, wherein said target antigen are polysaccharide, polyhydric alcohol or polyamino acid.

29. the vaccine combination of claim 28, wherein said polysaccharide comprises at least 18 residues.

30. the vaccine combination of claim 28, wherein said polysaccharide are streptococcus pneumoniae (Streptococcus pneumoniae) polysaccharide, soil draws hot Frances Salmonella (Francisellatularensis) polysaccharide, Bacillus anthracis (Bacillus anthracis) polysaccharide, Haemophilus influenzae (Haemophilus influenzae) polysaccharide, salmonella typhi (Salmonella typhi) polysaccharide, Salmonella (Salmonella species) polysaccharide, shigella (Shigella) polysaccharide or Neisseria meningitidis (Neisseria meningitidis) polysaccharide.

31. the vaccine combination of claim 30, wherein said streptococcus pneumoniae (Streptococcuspneumoniae) polysaccharide are pod membrane 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.

32. it is O antigen that the vaccine combination of claim 30, wherein said soil draw hot Frances Salmonella (Francisella tularensis) polysaccharide.

33. the vaccine combination of claim 1, wherein said target antigen are microorganism pod membrane polymer.

34. the vaccine combination of claim 33, wherein said microorganism pod membrane polymer are the poly--γ-D-glutamic acid from Bacillus anthracis (Bacillus anthracis).

35. the vaccine combination of claim 1, the organic polymer that wherein said target antigen is made up of the monomer with at least 3 atoms, wherein each described atom independently is selected from carbon, oxygen, hydrogen, phosphate, nitrogen and sulfate.

36. the vaccine combination of claim 35, wherein said organic polymer derives from microorganism.

37. the vaccine combination of claim 35, wherein said organic polymer are not naturally occurring organic polymers.

38. the vaccine combination of claim 1, wherein said vaccine combination further comprises second target antigen.

39. the vaccine combination of claim 38, wherein said vaccine combination further comprises the 3rd target antigen.

40. prepare the method for vaccine combination, it comprises that (i) mixes target antigen in the mixture that forms described antigen and described carrier protein and (ii) carry described target antigen with described carrier protein bag with carrier protein, and it is crosslinked wherein to be no more than 50% described target antigen and described carrier protein in described vaccine combination.

41. the method for claim 40, wherein said vaccine combination further comprises pharmaceutically acceptable excipient.

42. carrying, the method for claim 40, wherein said bag comprise described antigen of precipitation and described carrier protein from described mixture.

43. the method for claim 42, wherein said precipitation comprise that the pH that makes described mixture changes.

44. the method for claim 42, wherein said precipitation comprise trichloroacetic acid (TCA) or ammonium sulfate are joined in the described mixture.

45. the method for claim 42, wherein said precipitation comprise by increasing or reduce the ionic strength of inorganic salt concentration change mixture in the mixture.

46. the method for claim 42, wherein said precipitation comprise that heating blends causes carrier protein and/or antigen to condense.

47. the method for claim 42, wherein said precipitation comprise that the ionizing radiation irradiation mixture with enough flux causes crosslinked.

48. the method for claim 40, wherein in described vaccine combination, the mol ratio of described antigen and described carrier protein is 1: 10-9: between 10.

49. the method for claim 40, wherein said carrier protein is a polymer.

50. the method for claim 49, wherein said polymer comprises at least 5 subunits.

51. the method for claim 49, wherein said polymer are same polymers.

52. the method for claim 40, wherein said carrier protein is through non-covalent connection.

53. the method for claim 52, wherein said non-covalent connection comprises hydrophobic interaction, ionization, model ylid bloom action or hydrogen bond.

54. the method for claim 40, wherein said carrier protein is: diphtheria toxin, diphtherotoxin 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; Listerella lysin O; Neisseria meningitidis (Neisseria menningitidis) outer membrane protein; Pseudomonas aeruginosa (Pseudomonas aeruginosa) Hcp1 albumen; escherichia coli (Escherichiacoli) heat labile enterotoxin; shiga-like toxin; people LTB albumen; protein extract from the intact bacterial cell; the dominant negative mutant (DNI) of Bacillus anthracis (Bacillus anthracis) protective antigen or escherichia coli (Escherichia coli) beta galactosidase.

55. the method for claim 54, wherein said intact bacterial cell are Pseudomonas aeruginosa (Pseudomonas aeruginosa) or streptococcus (Streptococcal) cell.

56. the method for claim 54, wherein said bacterial flagellin are vibrio cholera (Vibrio cholerae) flagellins.

57. the method for claim 54, wherein said shiga-like toxin are shigella (Shigella) SltB2 albumen.

58. the method for claim 54, wherein said carrier protein is a pneumolysin.

59. the method for claim 54, wherein said carrier protein are Listerella lysin O.

60. the method for claim 54, wherein said carrier protein is a diphtheria toxin, diphtherotoxin.

61. the method for claim 54, wherein said carrier protein is a diphtheria toxoid.

62. the method for claim 54, wherein said carrier protein is a tetanus toxin.

63. the method for claim 54, wherein said carrier protein is a tetanus toxoid.

64. the method for claim 40, wherein said target antigen are polysaccharide, polyhydric alcohol or polyamino acid.

65. the method for claim 64, wherein said polysaccharide comprises at least 18 residues.

66. the method for claim 64, wherein said polysaccharide are streptococcus pneumoniae (Streptococcus pneumoniae) polysaccharide, soil draws hot Frances Salmonella (Francisellatularensis) polysaccharide, Bacillus anthracis (Bacillus anthracis) polysaccharide, Haemophilus influenzae (Haemophilus influenzae) polysaccharide, salmonella typhi (Salmonella typhi) polysaccharide, shigella (Shigella species) polysaccharide, Salmonella (Salmonella species) polysaccharide or Neisseria meningitidis (Neisseria meningitidis) polysaccharide.

67. the method for claim 66, wherein said streptococcus pneumoniae (Streptococcuspneumoniae) polysaccharide are pod membrane 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.

68. it is O antigen that the method for claim 67, wherein said soil are drawn hot Frances Salmonella (Francisella tularensis) polysaccharide.

69. the method for claim 40, wherein said target antigen are microorganism pod membrane polymer.

70. the method for claim 69, wherein said microorganism pod membrane polymer are the poly--γ-D-glutamic acid from Bacillus anthracis (Bacillus anthracis).

71. the method for claim 40, the organic polymer that wherein said target antigen is made up of the monomer with at least 3 atoms, wherein each described atom independently is selected from carbon, oxygen, hydrogen, phosphate, nitrogen and sulfate.

72. the method for claim 71, wherein said organic polymer derives from microorganism.

73. the method for claim 71, wherein said organic polymer are not naturally occurring organic polymers.

74. the method for claim 40, it further comprises second target antigen.

75. the method for claim 74, it further comprises the 3rd target antigen.

76. prepare the method for vaccine combination, it comprises that (i) mixes target antigen and (ii) add with carrier protein makes the crosslinked joint of described carrier protein, and it is crosslinked wherein to be no more than 50% described target antigen and described carrier protein in described vaccine combination.

77. the method for claim 76, wherein said vaccine combination further comprises pharmaceutically acceptable excipient.

78. the method for claim 76, wherein in described vaccine combination, described antigen to the mol ratio of described carrier protein 1: 10-10: between 1.

79. the method for claim 76, wherein said carrier protein is a polymer.

80. the method for claim 79, wherein said polymer comprises at least 5 subunits.

81. the method for claim 79, wherein said polymer are same polymers.

82. the method for claim 76, wherein said method comprise the Schiff's base in the described carrier protein of reduction.

83. the method for claim 76, wherein said carrier protein and at least one other carrier protein are covalently bound.

84. the method for claim 83, the peptide bond between the carboxyl of the wherein said covalently bound primary amino radical that is included in lysine side-chain and agedoite or glutamine side chain.

85. the method for claim 83, the wherein said covalently bound bi-functional cross-linking agent that comprises.

86. the method for claim 85, wherein said bi-functional cross-linking agent are glutaraldehyde, two [sulfonic group succinimido] suberate or oneself two imidic acid dimethyl esters.

87. the method for claim 76, wherein said joint is a formula

Chemical compound, wherein R _nBe aromatic moieties, a 3-10 atom of a direct-connected or alkine, a 5-10 carbon atom of direct-connected or branched-chain alkene, a 2-12 carbon atom of direct-connected or branched heteroalkyl groups, a 2-12 carbon atom of direct-connected or branched alkyl, a 1-12 atom of 1-12 carbon atom the ring-type system ,-(CH ₂CH ₂O) _qCH ₂CH ₂-wherein q is 1-4, or connect the chemical bond of two aldehyde radicals.

88. the method for claim 76, wherein said joint are glutaraldehyde, dimaleoyl imino benzoyl-N-hydroxysuccinimide eater, carbodiimide or BDB.

89. the method for claim 76, wherein said carrier protein are diphtheria toxin, diphtherotoxin or its mutant; diphtheria toxoid; tetanus toxin or its mutant; the tetanolysin toxoid; Pseudomonas aeruginosa Pseudomonas aeruginosa) exotoxin A or its mutant; b subunit of cholera toxin; tetanus toxin fragment C; bacterial flagellin; pneumolysin; Listerella lysin O; Neisseria meningitidis (Neisseria menningitidis) outer membrane protein; Pseudomonas aeruginosa (Pseudomonas aeruginosa) Hcp1 albumen; escherichia coli (Escherichiacoli) heat labile enterotoxin; shiga-like toxin; people LTB albumen; protein extract from the intact bacterial cell; the dominant negative mutant (DNI) of Bacillus anthracis (Bacillus anthracis) protective antigen or escherichia coli (Escherichia coli) beta galactosidase.

90. the method for claim 89, wherein said intact bacterial cell are Pseudomonas aeruginosa (Pseudomonas aeruginosa) or streptococcus (Streptococcal) cell.

91. the method for claim 89, wherein said bacterial flagellin are vibrio cholera (Vibrio cholerae) flagellins.

92. the method for claim 89, wherein said shiga-like toxin are shigella (Shigella) SltB2 albumen.

93. the method for claim 76, wherein said target antigen are polysaccharide, polyhydric alcohol or polyamino acid.

94. the method for claim 93, wherein said polysaccharide comprises at least 18 residues.

95. the method for claim 93, wherein said polysaccharide are streptococcus pneumoniae (Streptococcus pneumoniae) polysaccharide, soil draws hot Frances Salmonella (Francisellatularensis) polysaccharide, Bacillus anthracis (Bacillus anthracis) polysaccharide, Haemophilus influenzae (Haemophilus influenzae) polysaccharide, salmonella typhi (Salmonella typhi) polysaccharide, shigella (Shigella species) polysaccharide, Salmonella (Salmonella species) polysaccharide or Neisseria meningitidis (Neisseria meningitidis) polysaccharide.

96. the method for claim 95, wherein said streptococcus pneumoniae (Streptococcuspneumoniae) polysaccharide are pod membrane 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.

97. it is O antigen that the method for claim 95, wherein said soil are drawn hot Frances Salmonella (Francisella tularensis) polysaccharide.

98. the method for claim 76, wherein said target antigen are microorganism pod membrane polymer.

99. the method for claim 98, wherein said microorganism pod membrane polymer are the poly--γ-D-glutamic acid from Bacillus anthracis (Bacillus anthracis).

100. the method for claim 76, the organic polymer that wherein said target antigen is made up of the monomer with at least 3 atoms, wherein each described atom independently is selected from carbon, oxygen, hydrogen, phosphate, nitrogen and sulfate.

101. the method for claim 100, wherein said organic polymer derives from microorganism.

102. the method for claim 100, wherein said organic polymer are not naturally occurring organic polymers.

103. the method for claim 76, it further comprises second target antigen.

104. the method for claim 103, it further comprises the 3rd target antigen.

105. be the method for study subject vaccination opposing infectious agent, described method comprises the vaccine combination of the claim 1 that gives the study subject q.s, produces in described study subject to induce antibody.

106. the method for claim 105, wherein said method comprises second dosing step, and wherein the vaccine combination with described claim 1 gives described study subject with enough amounts, produces in described study subject to add powerful antibody.

107. the method for claim 106, wherein said production of antibodies are that the T cell is dependent.

108. the method for claim 105, wherein said production of antibodies are enough to prevent or lower the infection of infectious agent to described study subject.

109. the method for claim 105, wherein said infectious agent is: streptococcus pneumoniae, meningococcus, influenza B haemophilus (Haemophilus influenzae Type B), Pseudomonas aeruginosa Pseudomonas aeruginosa), soil draws hot Frances Salmonella (Francisella tularensis), shigella (Shigella species), Salmonella (Salmonella species), acinetobacter calcoaceticus (Acinetobacter species), bulkholderia cepasea (Burkholderia species) and escherichia coli (Escherichia coli).

110. the method for claim 105, wherein said method comprises second dosing step, and second vaccine combination that wherein will comprise target antigen offers described study subject with enough amounts, produces in described study subject to add powerful antibody.

111. the method for claim 110, wherein said production of antibodies are enough to prevent or lower the infection of second infectious agent to described study subject.

112. the method for claim 105 or 110, wherein said antibody are IgG antibody.

113. the method for claim 105, wherein said study subject is the people.