CN103007263A - Vaccine composition - Google Patents
Vaccine composition Download PDFInfo
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- CN103007263A CN103007263A CN2012102805434A CN201210280543A CN103007263A CN 103007263 A CN103007263 A CN 103007263A CN 2012102805434 A CN2012102805434 A CN 2012102805434A CN 201210280543 A CN201210280543 A CN 201210280543A CN 103007263 A CN103007263 A CN 103007263A
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Abstract
The present invention relates to immunogenic compositions and vaccines for the treatment and prevention of Neisserial disease. Immunogenic compositions of the invention contain combinations of antigens selected from at least two different classes of antigens including adhesins, autotransporter proteins, toxins, iron acquisitions proteins and membrane-associated protein (preferably integral outer membrane protein)s. Such combinations of antigens are able to target the immune response against different aspects of the neisserial life cycle, resulting in a more effective immune response.
Description
The application is to be on February 19th, 2004 applying date, and application number is dividing an application of application for a patent for invention 03818648.9, that denomination of invention is identical with the present invention.
Technical field
The present invention relates to Neisseria gonorrhoeae (Neisserial) immunogenic composition and vaccine, their preparation method and the purposes of this compositions in medicine.More specifically, it relates to the vaccine combination that comprises the antigen combination, and as measured in protection algoscopy or serum sterilizing algoscopy, it has can make vaccine of the present invention cause surprising good immunoreactive character.
Background
Neisseria gonorrhoeae bacterial strain in the antibacterial is the pathogen of a lot of human pathological changes, therefore need to develop the effective vaccine that can resist these pathogen.Particularly, Diplococcus gonorrhoeae (Neisseria gonorrhoeae) and the caused pathological changes of Neisseria meningitidis (Neisseriameningitidis) can be treated by vaccination.
Diplococcus gonorrhoeae is the pathogen of gonorrhea, and gonorrhea is one of modal sexually transmitted disease (STD) of reporting of the whole world, and 62,000,000 cases morbidities (people such as Gerbase, 1998 Lancet 351 are arranged according to estimates every year; (Suppl 3) 2-4).The clinical manifestation of gonorrhea comprises urogenital tract, throat or mucous membrane of rectum inflammation and neonate eye infection.Women gonococcus (gonococcal) infect to rise and can cause infertile, extrauterine pregnacy, chronic pelvic inflammatory disease and tubo-ovarian abscess to form.Septicemia, arthritis, endocarditis and meningitis are all relevant with the concurrency gonorrhea.
A large amount of gonococcus strains produce drug resistance and cause sickness rate and the complication relevant with gonorrhea to increase.To utilize vaccination and prevent gonorrhea with the attracting replacement scheme of antibiotic therapy gonorrhea.The vaccine that present also not anti-Diplococcus gonorrhoeae infects.
Neisseria meningitidis is a kind of important pathogen, particularly in child and youngster.Septicemia and meningitis are the forms of the serious threat life of invasive meningococcosis (IMD).This disease is because its higher M ﹠ M has become global health problem.
According to the antigenic difference of capsular polysaccharide, identified 13 kinds of Neisseria meningitidis serum group, the most generally A, B and C, they cause this disease in the whole world 90%.Serum group B is Europe, the common reason of meningococcosis in the several countries of the U.S. and Latin America.
Developed the vaccine based on the capsular polysaccharide of serum group A, C, W and Y, they show (people such as Peltola, 1985 Pediatrics76 of breaking out that can control meningococcosis; 91-96).Yet the immunogenicity of serum group B is relatively poor, and only mainly induce the IgM isotype of short duration antibody response (Ala ' Aldeen D and Cartwright K 1996, J.Infect.33; 153-157).Therefore, at present also there is not to resist the meningococcal extensively effective vaccine of the serum group B that causes these diseases of great majority in the country of most temperate zone.Particularly serious is that the incidence rate of serotypes B disease in Europe, Australia and America increases day by day, mainly is in the child below 5 years old.The development of the meningococcal vaccine of antiserum group B has proposed a distinctive difficulty, namely because the immunology similarity of polysaccharide capsule and human nerve cell adhesion molecule causes its immunogenicity relatively poor.The scheme of production of vaccine is concentrated on the surface exposed structure of meningococcus adventitia, but hindered by the significant variation of these antigens between the bacterial strain.
Further the vaccine that is made of the adventitia vesicle is adopted in research, and it comprises the albumen of a lot of formation bacterial membrane normal contents.These one of them is anti-Neisseria meningitidis serum group B and C
Cuban vaccine (people such as Rodriguez, 1999 Mem Inst.Oswaldo Cruz, Rio de Janeiro 94; 433-440).Design this vaccine and be used for the invasive meningococcosis of anti-Cuba, this disease can not be eliminated by the vaccine program that uses capsular polysaccharide AC vaccine to carry out.Popular serum group is B and C, and
Vaccine can successfully have been controlled and break out, the usefulness of the anti-Neisseria meningitidis serum group of this vaccine B bacterial strain is the 83% (people such as Sierra according to estimates, 1990 In Neisseria, Walter Gruyter, Berlin, the people such as m.Atchman (eds) p 129-134, the people such as Sierra, 1991, NIPH Ann 14; 195-210).This vaccine can break out by effectively anti-specificity, yet the immunoreation that causes can not resist other bacterial strain of Neisseria meningitidis.
Subsequently in Latin America, the efficacy study that carries out in the epidemic diseases process that is caused by homology and allos serum group B meningococcus bacterial strain shows some effects in slightly large child of age and adult, but its effectiveness obvious (people such as Milagres on the low side in the child risk of infection maximum, young, 1994, Infect.Immun.62; 4419-4424).Such as the UK effect how also uncertainly this vaccine has the endemic country of many bacterial strains.The immunogenic research of antagonism allos bacterial strain has only confirmed limited cross reactivity serum bactericidal activity, particularly in the baby (people such as Tappero, 1999, JAMA 281; 1520-1527).
The second adventitia vesicle vaccine is in Norway, uses (people such as Fredriksen, 1991, NIPH Ann, 14 of those the general serotypes B separators developments popular in the Scandinavia; 67-80).This vaccine is tested in clinical trial, and after finding 29 months, and it has 57% protection effect (people such as Bjune, 1991, Lancet, 338; 1093-1096).
Yet the use of adventitia vesicle in vaccine is relevant with some problem.For example, OMV comprises that poisonous lipopolysaccharide and they can comprise immunodominant antigen, and these antigens or bacterial strain are specific, or variable expression.Several methods of having described all can be used for overcoming some problems of adventitia vesicle goods vaccine.WO 01/09350 has described for example by reducing toxicity and modifying the method that the antigen that exists on the adventitia vesicle solves some problem.
WO 01/52885 has described the probability that adventitia vesicle and other antigen group are combined and has comprised and surpass 2,000 kinds of potential Neisseria gonorrhoeae albumen tabulations, can infer from this tabulation and develop the vaccine that wider serotype scope is all had effect.
Present used resisting-meningococcus vaccine exists various problems.Adventitia vaccine based on albumen tends to specificity and only effective to the minority bacterial strain.Polysaccharide vaccine also is suboptimal, and this is because they tend to cause relatively poor and of short duration immunoreation, particularly to the serum group B (people 1986 such as Lepow; Peltola 1998, and Pediatrics 76; 91-96).
Neisseria gonorrhoeae infects and to have proposed an important health care problem, namely for Diplococcus gonorrhoeae, does not also have available vaccine, or for Neisseria meningitidis, only the effect of limited anti-allos bacterial strain and the vaccine of ability.Obviously need the anti-Neisseria gonorrhoeae of development to infect better vaccine, they can improve effect and the anti-wider bacterial strain of present used vaccine.
Accompanying drawing is described
Fig. 1 .-is to the TbpA among the OMV that is expressed the restructuring Neisseria meningitidis bacterial strain preparation of being raised by tbpA and hsf and the detection of Hsf.Analyze (4-20% gradient gel) by the SDS-PAGE with coomassie brilliant blue staining and separate OMV goods (10 μ g).
The detection of restructuring Hsf passerby (passenger) domain that Fig. 2 .-produces in escherichia coli (E.coli), Hsf passerby's albumen of 10 μ g purification (the 2nd with 3 roads) is by separating on the 12%SDS-PAGE gel, and compares with molecular weight marker (the 1st road).
The analysis of Fig. 3 .-Hap passerby purity, this is that (B) silver dyes by (A) coomassie dyeing, (C) anti--His 5 Western blottings and (D) anti--E. coli detection.10 μ g purifying antigens separate by SDS-PAGE on 4-20% acrylamide gradient gel.
Fig. 4 .-is from FrpA and the total sequence similarity district of FrpC albumen of Neisseria meningitidis bacterial strain FAM20 separation.(A) domain of Neisseria meningitidis bacterial strain FAM20RTX albumen FrpA and FrpC forms.(B) the FrpA/C amplified production that obtains from Neisseria meningitidis bacterial strain H44/76.
Fig. 5 .-restructuring Frp23 (the FrpA/C conserved region with 23 recurring units) antigen Expression in Escherichia coli.Non-the inducing (NI) of the e. coli bl21 DE3 that transforms with control vector (pET24d) or recombinant precursor (being respectively Frp3, Frp13 and Frp23) and induce the SDS-PAGE analysis of total cell extract of (I).With Coomassie blue (A) with gel-colored or gel transferred on the nitrocellulose and with resist-the His6 mice serum carries out immune detection.
Fig. 6 .-is at the FHAB 2/3 of expression in escherichia coli
RdThe preferred DNA sequence of fragment.
Fig. 7 .-derives from the restructuring FHAB 2/3 that induces escherichia coli B121DE3 extract
RdPurification.(A) key step in the purge process.(B) SDS-PAGE that carries out in the protein fractions of purge process different step sampling is analyzed.
Fig. 8 .-is for the FHAB 2/3 of Neisseria meningitidis
Rd, Hap, Hap passerby's domain, Hsf and Hsf passerby's domain antigen anti--the adhesion blocking-up of serum is active.
Fig. 9 .-represents the Coomassie blue stain gel of Hsf, TbpA and the expression of NspA in the little brewage of the adventitia that derives from different Neisseria meningitidis bacterial strains.The 1st road-molecular weight marker; The adventitia vesicle of the bacterial strain H44/76 preparation in the 2nd road-reduced from capsular polysaccharide; The adventitia vesicle of the bacterial strain H44/76 preparation in the 3rd road-reduced from capsular polysaccharide and PorA; The 4th road-from the adventitia vesicle of capsular polysaccharide and PorA is reduced and NspA is raised bacterial strain H44/76 preparation; The 5th road-from the adventitia vesicle of capsular polysaccharide and PorA is reduced and Hsf is raised bacterial strain H44/76 preparation; The 6th road-from the adventitia vesicle of capsular polysaccharide and PorA is reduced and TbpA is raised bacterial strain H44/76 preparation; The 7th road-from the adventitia vesicle of capsular polysaccharide and the bacterial strain H44/76 preparation that PorA is reduced and TbpA and Hsf are raised; The 8th road-from the adventitia vesicle of capsular polysaccharide and the bacterial strain H44/76 preparation that PorA is reduced and TbpA and NspA are raised.
Figure 10 .Hap passerby purification flow process.
Describe in detail
The invention discloses the particular combinations of Neisseria gonorrhoeae antigen, when they were combined, the effect that the anti-Neisseria gonorrhoeae of vaccine is infected improved surprisingly.
Neisseria gonorrhoeae infects will experience some different stages.For example, meningococcal biocycle comprise nose because of settle down, mucosa adheres to, enter blood flow, in blood propagation, induce toxic shock, pass blood/brain barrier and in cerebrospinal fluid and/or meninges, breeding.Different molecular on the bacterium surface participates in the different phase of infectious cycle.By using effective dose to participate in the combination results immunoreation of the various process specific antigen that Neisseria gonorrhoeae infects, the Neisseria gonorrhoeae vaccine that can obtain to have astonishing higher effect.
Particularly, the combination that derives from different types of specific antigen can cause for the immunoreation of infecting a plurality of stages, and in these different types of specific antigens, some participation is attached to host cell, some participates in the acquisition of ferrum, and some is from the body transport protein and some is toxin.This combination of antigen can improve the effect that the anti-Neisseria gonorrhoeae of (preferred collaborative improve) vaccine infects surprisingly, wherein the more than function by the immunoreation directed toward bacteria in the best way.
The effect of vaccine can be estimated by the many measure method.The protection algoscopy of carrying out in animal model is well known in the art.In addition, serum sterilizing algoscopy (SBA) is the most universally recognized amynologic label, with the effect of estimating meningococcus vaccine people such as (, J Infect Dis.1998,177:683-691) Perkins.
Some combination of antigen (for example, some is from the combination of body transport protein and some ferrum acquisition albumen) can cause the protection in the animal model algoscopy to improve and/or the collaborative higher SBA of generation tires.Do not wish to be bound by theory, this synergistic combination of antigen can produce immunoreactive numerous characteristics by the antigen combination and realize.Antigen is originally normally surface exposed on the Neisseria gonorrhoeae cell, tends to guard and be not present on the superficial cell with the required sufficient quantity of best bactericidal reaction, and this best bactericidal reaction is produced by the antibody that separately anti-this antigen causes.The preparation that antigen group of the present invention is produced altogether can cause the favourable combination of bactericidin, and the interaction of these bactericidins and Neisseria gonorrhoeae cell has exceeded threshold limit value.Under this critical level, the well-behaved antibody of capacity is combined with bacterium surface, thereby also therefore sees higher levels of bactericidal action by the effective killing bacteria of complement.
Because serum sterilizing algoscopy (SBA) can reflect the effect of vaccine candidate object closely, the good SBA that therefore obtains by the antigen combination tires and can characterize well the protection effect of the vaccine that comprises this antigen combination.That the present invention relates to separate or jointly be rich in the purposes of the two kinds of antigens combination in mixture with another antigen.When combining, this antigen combination advantageously interacts, and the preferred collaborative higher immunoreation (as measured in serum sterilizing algoscopy or SBA) of bactericidal activity that causes, and preferably be higher than the additive reaction that is caused separately by antigen, more preferably be higher than by at least 1.2,1.5,2,3,4,5,6,7,8,9 factors the additive reaction that is most preferably caused by at least 10 factors.
Other advantage of the present invention is that the antigen combination of the present invention that derives from different protein families in the immunogenic composition can resist wider bacterial strain.
The present invention relates to comprise the immunogenic composition of a lot (two or more) albumen, described albumen is selected from least two different types of albumen, and they have different functions in Neisseria gonorrhoeae.The example of this albumen kind is adhesin, obtains albumen from body transport protein, toxin, integration outer membrane protein and Fe.Vaccine group of the present invention is combined in anti-homology Neisseria gonorrhoeae bacterial strain (bacterial strain of the antigen of deriving) and is also preferably demonstrating astonishing raising aspect the efficacy of vaccines of anti-allos Neisseria gonorrhoeae bacterial strain.
The invention provides immunogenic composition, it comprises at least or lucky two kinds, three kinds, four kinds, five kinds, six kinds, seven kinds, eight kinds, nine kinds or ten kinds of different antigens that these antigens are selected from least following or lucky two, three, four or whole five antigens that are selected from following kind:
At least a Neisseria gonorrhoeae adhesin;
At least a Neisseria gonorrhoeae is from the body transport protein;
At least a Neisseria gonorrhoeae toxin;
At least a Neisseria gonorrhoeae Fe obtains albumen;
At least a Neisseria gonorrhoeae embrane-associated protein (preferred outer membrane protein is particularly integrated outer membrane protein).
Preferably, the invention provides and comprise at least or the immunogenic composition of lucky two kinds, three kinds, four kinds, five kinds, six kinds, seven kinds, eight kinds, nine kinds or ten kinds different Neisseria gonorrhoeae antigens.Most preferably these antigens be selected from following at least or lucky two groups, three groups, four groups or five groups be selected from following albumen:
Be selected from least a Neisseria gonorrhoeae adhesin of FhaB, NspA, PilC, Hsf, Hap, MafA, MafB, Omp26, NMB0315, NMB 0995, NMB 1119 and NadA;
Be selected from least a Neisseria gonorrhoeae of Hsf, Hap, IgA protease, AspA and NadA from the body transport protein;
Be selected from one of FrpA, FrpC, FrpA/C, VapD, NM-ADPRT and LPS immunologic pattern L2 and LPS immunologic pattern L3 or both at least a Neisseria gonorrhoeae toxin;
At least a Neisseria gonorrhoeae Fe that is selected from TbpA, TbpB, LbpA, LbpB, HpuA, HpuB, Lipo28 (GNA2132), Sibp, NMB0964, NMB0293, FbpA, Bcp, BfrA, BfrB and P2086 (XthA) obtains albumen; Or
Be selected from least a Neisseria gonorrhoeae embrane-associated protein of PilQ, OMP85, FhaC, NspA, TbpA, LbpA, TspA, TspB, TdfH, PorB, MltA, HpuB, HimD, HisD, GNA1870, OstA, HlpA (GNA1946), NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA and PldA, preferred outer membrane protein is preferably integrated outer membrane protein.
Antigen of the present invention all separates, and namely they are by the manual change.Preferably, they are purified to a certain degree, most preferably surpass 40,50,60,70,80,90,95 or 99% purity (before other combination of components of immunogenic composition of the present invention).
Preferred immunogenic composition of the present invention comprises that at least a Neisseria gonorrhoeae adhesin and at least a Neisseria gonorrhoeae obtain albumen or Neisseria gonorrhoeae embrane-associated protein (preferably integrating outer membrane protein) from body transport protein and optional Neisseria gonorrhoeae toxin, the Neisseria gonorrhoeae Fe of comprising.Preferred described antigen is selected from above-mentioned antigen.
Preferred immunogenic composition of the present invention comprises that at least a Neisseria gonorrhoeae adhesin and at least a Neisseria gonorrhoeae toxin and the optional Neisseria gonorrhoeae that comprises obtain albumen or Neisseria gonorrhoeae embrane-associated protein (preferably integrating outer membrane protein) from body transport protein, Neisseria gonorrhoeae Fe.Preferred described antigen is selected from above-mentioned antigen.
Preferred immunogenic composition of the present invention comprises that at least a Neisseria gonorrhoeae adhesin and at least a Neisseria gonorrhoeae Fe obtain albumen and choose wantonly to comprise that Neisseria gonorrhoeae toxin, Neisseria gonorrhoeae are from body transport protein or Neisseria gonorrhoeae embrane-associated protein (preferably integrating outer membrane protein).Preferred described antigen is selected from above-mentioned antigen.
Preferred immunogenic composition of the present invention comprises that at least a Neisseria gonorrhoeae adhesin and at least a Neisseria gonorrhoeae embrane-associated protein (preferably integrating outer membrane protein) and optional Neisseria gonorrhoeae toxin, Neisseria gonorrhoeae Fe acquisition albumen or the Neisseria gonorrhoeae of comprising are from the body transport protein.Preferred described antigen is selected from above-mentioned antigen.
Preferred immunogenic composition of the present invention comprises that at least a Neisseria gonorrhoeae is from body transport protein and at least a Neisseria gonorrhoeae toxin and optional Neisseria gonorrhoeae adhesin, Neisseria gonorrhoeae Fe acquisition albumen or the Neisseria gonorrhoeae embrane-associated protein (preferably integrating outer membrane protein) of comprising.Preferred described antigen is selected from above-mentioned antigen.
Preferred immunogenic composition of the present invention comprises that at least a Neisseria gonorrhoeae obtains albumen and optional Neisseria gonorrhoeae adhesin, Neisseria gonorrhoeae toxin or the Neisseria gonorrhoeae embrane-associated protein (preferably integrating outer membrane protein) of comprising from body transport protein and at least a Neisseria gonorrhoeae Fe.Preferred described antigen is selected from above-mentioned antigen.
Preferred immunogenic composition of the present invention comprises that at least a Neisseria gonorrhoeae is from body transport protein and at least a Neisseria gonorrhoeae embrane-associated protein (preferably integrating outer membrane protein) and optional Neisseria gonorrhoeae adhesin, Neisseria gonorrhoeae Fe acquisition albumen or the Neisseria gonorrhoeae toxin of comprising.Preferred described antigen is selected from above-mentioned antigen.
Preferred immunogenic composition of the present invention comprises that at least a Neisseria gonorrhoeae toxin and at least a Neisseria gonorrhoeae Fe obtain albumen and choose wantonly to comprise that Neisseria gonorrhoeae adhesin, Neisseria gonorrhoeae are from body transport protein or Neisseria gonorrhoeae embrane-associated protein (preferably integrating outer membrane protein).Preferred described antigen is selected from above-mentioned antigen.
Preferred immunogenic composition of the present invention comprises at least a Neisseria gonorrhoeae toxin and at least a Neisseria gonorrhoeae embrane-associated protein (preferably integrating outer membrane protein) and optionally comprises that Neisseria gonorrhoeae adhesin, Neisseria gonorrhoeae are from body transport protein or Neisseria gonorrhoeae toxin.Preferred described antigen is selected from above-mentioned antigen.
Preferred immunogenic composition of the present invention comprises that at least a Neisseria gonorrhoeae Fe obtains albumen and at least a Neisseria gonorrhoeae embrane-associated protein (preferably integrating outer membrane protein) and choose wantonly to comprise that Neisseria gonorrhoeae adhesin, Neisseria gonorrhoeae are from body transport protein or Neisseria gonorrhoeae toxin.Preferred described antigen is selected from above-mentioned antigen.
Whole 5 groups of antigens preferably are provided in immunogenic composition of the present invention.
When specifically mentioning albumen, the preferably albumen of natural, total length and natural variant thereof (that is, from Neisseria gonorrhoeae, the native protein that preferred meningococcus bacterial strain obtains), it also can comprise its antigenicity fragment (particularly in subunit vaccine).These are to contain or comprise at least 10 aminoacid that obtain continuously on the Argine Monohydrochloride sequence, preferred 20 aminoacid, more preferably 30 aminoacid, more preferably 40 aminoacid or most preferably 50 amino acid whose fragments (usually specifically described herein).In addition, the antigenicity fragment refers to carry out immunoreactive fragment with the antibody of anti-Neisseria gonorrhoeae full-length proteins generation or by the antibody that infects the mammalian hosts generation with Neisseria gonorrhoeae.The antigenicity fragment also comprises when using with effective dose; can infect the fragment that causes protective immunological reaction for Neisseria gonorrhoeae; more preferably it can prevent that Neisseria meningitidis and/or Diplococcus gonorrhoeae from infecting, and most preferably it can prevent that Neisseria meningitidis serum group B from infecting.
The present invention also comprises the recombination fusion protein of Neisseria gonorrhoeae albumen of the present invention, or its fragment.These can be with different Neisseria gonorrhoeae albumen or its fragment combination in same polypeptide.Optionally, the present invention also comprises the individual fusion rotein of Neisseria gonorrhoeae albumen or its fragment, namely has heterologous sequence, such as the supplier of T-cell epitope or purification labelling, for example: the fusion rotein of beta galactosidase, glutathione-S-transferase, green fluorescent protein (GFP), epitope tag such as FLAG, myc labelling, polyhistidyl or virus surface proteins such as influenza virus haemagglutinin, tetanus toxoid, diphtheria toxoid, CRM197.
Antigen of the present invention
The NMB reference marks refers to can be from the sequence reference marks of www.neisseria.org access.
1. adhesin
Adhesin comprises FhaB (WO 98/02547), NadA (J.Exp.Med (2002) 195:1445; NMB 1994), also can be called Hsf (NMB 0992) (WO 99/31132), Hap (NMB1985) (WO 99/55873), NspA (WO96/29412), MafA (NMB 0652) and MafB (NMB 0643) (the Annu Rev Cell Dev Biol.16 of NhhA; 423-457 (2000); Nature Biotech 20; 914-921 (2002)), Omp26 (NMB 0181), NMB 0315, NMB 0995, NMB 1119 and PilC (Mol.Microbiol.1997,23; 879-892).These are to participate in the albumen that Neisseria gonorrhoeae is combined with host cell surface.Hsf is one of them example of adhesin, also is from the body transport protein simultaneously.Therefore immunogenic composition of the present invention can comprise Hsf and other combination from the body transport protein, and wherein Hsf brings into play the effect of its adhesin.These adhesins can derive from Neisseria meningitidis or Diplococcus gonorrhoeae or other Neisseria gonorrhoeae bacterial strain.The present invention also comprises other adhesin that derives from Neisseria gonorrhoeae.
FhaB
The description in WO 98/02547SEQ ID NO:38 (nucleotide 3083-9025) of this antigen-also can be referring to NMB 0497.The inventor have been found that FhaB induce independent anti--adhere to aspect the antibody effective especially, and during particularly with other antigen combination of the present invention.Although can use total length FhaB, the inventor has been found that the terminal truncate of specific C-at least surprisingly effectively and preferred even more effective aspect the effect of intersection-bacterial strain.Advantageously demonstrated the easier clone of this truncate.FhaB truncate of the present invention usually and the N-end 2/3rds of FhaB molecule, be preferably placed at the 1200-1600 position, more preferably the 1300-1500 position, most preferably the new C-end of 1430-1440 position is corresponding.Specific embodiment has the C-end at 1433 or 1436 places.Therefore, this FhaB truncate of the present invention and comprise that the vaccine of this truncate is independent aspects of the present invention and is the component that the present invention makes up immunogenic composition.The N-end also can be reached 10,20,30,40 or 50 aminoacid by truncate.
2. from the body transport protein
From the body transport protein usually by signal sequence, passerby's domain and the anchoring structure territory that is used for adhering to adventitia form.From the example of body transport protein comprise Hsf (WO 99/31132) (NMB0992), HMW, Hia (people such as van Ulsen, Immunol.Med.Microbiol.2001
3253-64), (WO 99/55873 for Hap (NMB1985); The people such as van Ulsen, Immunol.Med.Microbiol.2001
3253-64), UspA, UspA2, NadA (NMB1994) (people such as Comanducci, J.Exp.Med.2002
1951445-1454), AspA (Infection and Immunity 2002,70 (8); 4447-4461; NMB 1029), Aida-1 sample albumen, SSh-2 and Tsh.NadA (J.Exp.Med (2002) 195:1445) is another example from the body transport protein, also is adhesin simultaneously.Therefore immunogenic composition of the present invention comprises the combination of NadA and adhesin, and wherein NadA is as playing a role from the body transport protein.These albumen can derive from Neisseria meningitidis or Diplococcus gonorrhoeae or other Neisseria gonorrhoeae bacterial strain.The present invention also comprise derive from Neisseria gonorrhoeae other from the body transport protein.
Hsf
Hsf has and the structure that shares from the body transport protein.For example, derive from unique sequence that the Hsf of Neisseria meningitidis bacterial strain H44/76 is comprised of amino acid/11-51, surface exposed and comprise the aminoterminal Head Section of maturation protein (aminoacid 52-479), neck region (aminoacid 480-509) of variable region (aminoacid 52-106,121-124,191-210 and 230-234), hydrophobic alpha-helix district (aminoacid 518-529) and wherein 4 cross-film chains cross over the anchoring structure territory (aminoacid 539-591) of adventitias.
Although total length Hsf can be used in the immunogenic composition of the present invention, various Hsf truncate and disappearance thing also can advantageously use, and this depends on the type of vaccine.
When Hsf is used for subunit vaccine, preferably use the part of solubility passerby domain; For example the complete lattice territory of aminoacid 52-479, most preferably its conservative part, for example particularly advantageous sequence of amino acid/11 34-479.The preferred form of Hsf can be by truncate, with disappearance disclosed albumen variable region in WO 01/55182.Preferred variant can be included in the disappearance of 1,2,3,4 or 5 variable region that limits among the WO 01/55182.In any one or both ends of N or C-terminal, above-mentioned sequence and the following describes those can be stretched or truncate reaches 1,3,5,7,10 or 15 aminoacid.
Therefore the preferred fragment of Hsf comprises the whole Head Section of Hsf, preferably comprises aminoacid 52-473.The preferred fragment of other of Hsf comprises surface exposed Head Section, and it comprises one or more following amino acid sequences: 52-62,76-93,116-134,147-157,157-175,199-211,230-252,252-270,284-306,328-338,362-391,408-418,430-440 and 469-479.
When Hsf was present in the little brewage of adventitia, it can be expressed as full-length proteins or preferably be expressed as the favourable variant (producing the mature outer membrane protein of aminoacid sequence 134-C end) that the fusant by amino acid/11-51 and 134-591 forms.The preferred form of Hsf can be by truncate, thus disclosed albumen variable region among the disappearance WO 01/55182.Preferred variant can be included in the disappearance of 1,2,3,4 or 5 variable region that limits among the WO01/55182.Preferred first and second variable regions disappearance.Preferred variant is the residue disappearance that makes between aminoacid sequence 52-237 or the 54-237, more preferably makes the residue disappearance between aminoacid 52-133 or the 55-133.Described maturation protein can lack signal peptide.
Hap
The computer analysis that the Hap-sample albumen that derives from Neisseria meningitidis is carried out demonstrates at least 3 domains.Take the Hap-sample sequence that derives from bacterial strain H44/76 as example, comprise amino acid/11-42
Domain 1Coding comprises aminoacid 43-950's from the sec dependent signals peptide of body transport protein family characteristic
Domain 2Coding is probably surface exposed and easily enter immune passerby's domain, comprises residue 951-C's terminal (1457)
Domain 3Predicted coding may be assembled into bucket-spline structure and be anchored at beta chain in the adventitia.Because domain 2 may be surface exposed, good conservative (surpassing 80% in all test strains) and can be used as subunit antigen and produce in escherichia coli, so it has represented the target vaccine candidate object.Because that domain 2 and 3 is likely is surface exposed and good conservative people such as (, (2000), Science 287:1816-1820) Pizza, so they have represented the target vaccine candidate object.Domain 2 is known as passerby's domain.
Immunogenic composition of the present invention can comprise total length Hap albumen, preferably is incorporated in the OMV goods.Immunogenic composition of the present invention also can comprise passerby's domain of Hap, and in bacterial strain H44/76, it is comprised of amino acid residue 43-950.This fragment of Hap can be particularly advantageous in the subunits combination thing of the present invention.In any one or both ends of N or C-terminal, the above-mentioned sequence of Hap passerby's domain can be stretched or truncate reaches 1,3,5,7,10,15,20,25 or 30 aminoacid.
3. ferrum obtains albumen
Ferrum acquisition albumen comprises TbpA (NMB 0461), and (WO 92/03467, US5912336, WO93/06861 and EP 586266), TbpB (NMB 0460) (WO 93/06861 and EP 586266), LbpA (NMB 1540) (Med Microbiol (1999) 32:1117), LbpB (NMB1541) (WO/99/09176), HpuA (U73112.2) (Mol Microbiol.1997,23; 737-749), HpuB (NC.003116.1) (Mol Microbiol.1997,23; 737-749), also be known as the P2086 (NMB 0399) (13 of XthA
ThInternational Pathogenic Neisseria Conference 2002), FbpA (NMB 0634), FbpB, BfrA (NMB 1207), BfrB (NMB 1206), also be known as GNA2132 Lipo28 (NMB 2132), Sibp (NMB1882), HmbR, HemH, Bcp (NMB 0750), Iron (III) abc transport albumen-permease albumen (people such as Tettelin, Science 287; 1809-1815 2000), Iron (III) abc transport albumen-pericentral siphon (people such as Tettelin, Science 287; 1809-1815 2000), TonB dependency receptor (NMB 0964 and NMB 0293) (people such as Tettelin, Science 287; 1809-1815 2000) and the transferrin binding protein associated protein (people such as Tettelin, Science 287; 1809-1815 2000).These albumen can derive from Neisseria meningitidis, Diplococcus gonorrhoeae or other Neisseria gonorrhoeae bacterial strain.The present invention comprises that also other ferrum that derives from Neisseria gonorrhoeae obtains albumen.
TbpA
TbpA and TbpB interact and form protein complexes at the Neisseria gonorrhoeae adventitia, and it is in conjunction with transferrins.Structurally, TbpA comprises N-end structure territory and piston structure territory in the born of the same parents with TonB case, this piston structure territory be a plurality of by short born of the same parents' internal ring be connected the cross-film β chain that long born of the same parents' outer shroud connects.
Distinguished two families of TbpB, they have respectively higher molecular weight and lower molecular weight.The height of TbpB and the different families combination of low-molecular-weight form from TbpA can homology be basis this difference family of difference.Although their molecular weight is similar, but they are called high molecular and low-molecular-weight family, this is because they are combined (people such as Rokbi, FEMS Microbiol.Lett.100 with the high or low molecular weight form of TbpB; 51,1993).Term TbpA (height) and TbpA (low) are used for these two kinds of forms of expression TbpA, and similar to TbpB.The ferrum acquisition albumen that immunogenic composition of the present invention can comprise TbpA and the TbpB that derives from Neisseria meningitidis serum group A, B, C, Y and W-135 and derive from other antibacterial that comprises Diplococcus gonorrhoeae.Transferrin binding protein TbpA and TbpB have been respectively referred to as Tbp1 and Tbp2 (people such as Cornelissen, Infection and Immunity 65; 822,1997).
TbpA comprises some different districts.For example, with regard to the TbpA that derives from Neisseria meningitidis bacterial strain H44/76, aminoterminal 186 aminoacid form inner globular domain, and 22 β chains are crossed over film, form the β barrel structure.These are to be connected with larger born of the same parents' outer shroud by short born of the same parents' internal ring.Born of the same parents' outer shroud 2,3 and 5 has the sequence variability of top and encircles 5 is surface exposed.Ring 5 and 4 participates in ligand binding.
The preferred fragment of TbpA comprises born of the same parents' outer shroud of TbpA.Use derives from the TbpA sequence of Neisseria meningitidis bacterial strain H44/76, the aminoacid 707-723 of the aminoacid 609-625 of the aminoacid 438-471 of the aminoacid 226-303 of these rings and ring 1 aminoacid 200-202, ring 2, the aminoacid 348-395 of ring 3, ring 4, the aminoacid 512-576 of ring 5, ring 6, the aminoacid 661-671 of ring 7, ring 8, the aminoacid 769-790 of ring 9, encircle 10 aminoacid 814-844 and encircle 11 aminoacid 872-903 corresponding.After the sequence contrast, the corresponding sequence in other Tbp albumen also can consist of preferred fragment.Most preferred fragment can comprise ring 2, ring 3, the ring 4 that consists of Tbp or encircle 5 aminoacid sequence.
When immunogenic composition of the present invention comprised TbpA, it preferably comprised TbpA (height) and TbpA (low) simultaneously.
Although preferred TbpA is present in the OMV vaccine, it also can be the part of subunit vaccine.It also is (WO00/25811) well known in the art that the ferrum that for example, can be introduced in the separation in the immunogenic composition of the present invention obtains albumen.They can be expressed in bacterial host, use detergent to extract (such as 2%Elugent) and by affinity chromatograph or use standard column chromatographic technique purification well known in the art (people such as Oakhill, Biochem J.2002 364; 613-6).
When TbpA was present in the OMV vaccine, its expression can be raised by gene technology discussed herein, or can preferably grow under the limited condition of following ferrum by parental strain and be raised.The method also can cause variable ferrum-adjusting albumen, the particularly rise of FrpB, it can be changed into immunodominant and it therefore can be by following described expression of advantageously reducing this albumen (particularly FrpB) gene delection of this albumen of coding (and preferably make), causes the anti-immunoreation that is present in the antigen in the Neisseria gonorrhoeae bacterial strain on a large scale to guarantee immunogenic composition of the present invention.It preferably has TbpA (height) and the TbpA (low) that is present in the immunogenic composition, and preferably this is to mix by the OMV that will derive from two kinds of bacterial strains of expressing the TbpA optional form to obtain.
4. toxin
Toxin comprises FrpA, and (NMB 0585; NMB 1405), FrpA/C (seeing following definition), (NMB 1415 for FrpC; NMB 1405) and (WO 92/01460), NM-ADPRT (NMB 1343) (people such as 13th International Pathogenic Neisseria Conference 2002Masignani, p135), VapD (NMB 1753), lipopolysaccharide (LPS; Also be known as lipooligosaccharide or LOS) immunologic pattern L2 and LPS immunologic pattern L3.The contained district of FrpA and FrpC is that guard between these two albumen and the preferred fragment of described albumen is the polypeptide that contains this conservative fragments, preferably contains the aminoacid 227-1004 of FrpA/C sequence.These antigens can derive from Neisseria meningitidis or Diplococcus gonorrhoeae or other Neisseria gonorrhoeae bacterial strain.The present invention also comprises other toxin that derives from Neisseria gonorrhoeae.
In embodiment optionally, toxin can comprise participate in the antigen that toxicity is regulated, the OstA that for example works in lipopolysaccharide is synthetic.
FrpA and FrpC
Two kinds of RTX albumen of Neisseria meningitidis coding, they are known as FrpA and FrpC, secrete (people such as Thompson, (1993) J.Bacteriol.175:811-818 under the limited condition of ferrum; The people such as Thompson, (1993) Infect.Immun.61:2906-2911).RTX (repetition toxin) protein family has a series of 9 aminoacid recurring units of following concensus sequence in the C-end near them: Leu Xaa Gly Gly Xaa Gly (Asn/Asp) Asp Xaa (LXGGXGN
/ DDX).Recurring unit among the escherichia coli HlyA is considered to Ca
2+In conjunction with the site.As depicted in figure 4, meningococcus FrpA and FrpC albumen, as what in bacterial strain FAM20, characterize, at their center and the total widely amino acid similarity of C-end region but very limited at the similarity (if any) of N-end.In addition, because the repetition of 9 amino acid motifs (repeat in FrpA 13 times, repeat 43 times in FrpC), district conservative between FrpA and FrpC demonstrates some polymorphism.
Immunogenic composition of the present invention can comprise total length FrpA and/or FrpC or preferred, contains the fragment of sequence conservative between FrpA and FrpC.Described conserved sequence is made of 9 amino acid whose recurring units.Immunogenic composition of the present invention preferably includes more than 3 recurring units, more than 10 recurring units, more than 13 recurring units, more than 20 recurring units or more than 23 recurring units.
This truncate has than the more favourable character of full-length molecule and comprises this antigen and the vaccine that is incorporated in the immunogenic composition of the present invention forms independent aspects of the present invention.
Sequence conservative between FrpA and FrpC is named as FrpA/C, and when the component of FrpA or FrpC formation immunogenic composition of the present invention, can advantageously use FrpA/C.The aminoacid 277-1004 of FrpA sequence is preferred conserved region.In any one or both ends of N or C-terminal, above-mentioned sequence can be stretched or truncate reaches 1,3,5,7,10,15,20,25 or 30 aminoacid.
LPS
LPS (lipopolysaccharide also is known as the LOS-lipooligosaccharide) is the endotoxin on the Neisseria gonorrhoeae adventitia.The polysaccharide part of known LPS can be induced bactericidin.
Heterogeneity in the LPS oligosaccharide part produces structure and antigen multiformity (people such as Griffiss, Inf.Immun.1987 between different Neisseria gonorrhoeae bacterial strains; 55:1792-1800).This be used to the meningococcus bacterial strain be subdivided into 12 immunologic patterns (people such as Scholtan, J Med Microbiol 1994,41:236-243).Immunologic pattern L3, L7 are being identical aspect the immunology and structurally are being that therefore similar (or or even identical) also be named as L3,7,9 (or the purpose for this description is referred to as " L3 ") with L9.Meningococcus LPS L3,7,9 (L3), L2 and L5 can pass through sialylated, or are modified by adding cytidine 5 '-one phosphoric acid-N-acetylneuraminic acid.Although L2, L4 and L6LPS are diacritic aspect immunology, they are structurally similar, and when mentioning L2 herein, L4 or L6 can be substituted by optional within the scope of the invention.See the people such as M.P.Jennings, Microbiology 1999,145,3013-3021 and Mol Microbiol2002, and 43:931-43 wherein further for example understands structure and the heterogeneity of LPS.
Work as LPS, when preferred meningococcus LPS is included in the vaccine of the present invention, preferably and advantageously have one of immunologic pattern L2 and L3 or both.LPS preferably is present in the adventitia vesicle (preferably with the detergent that hangs down percentage ratio, more preferably 0-0.5%, 0.02-0.4%, 0.04-0.3%, 0.06-0.2%, 0.08-0.15% or 0.1%, when most preferably dexycholate [DOC] extracts vesicle), can also be the part of subunit vaccine.LPS can use the process of knowing, and comprises the hot water-separation of phenol process (Wesphal and Jann Meth.Carbo.Chem.5; 83-91 1965).Be also shown in the people such as Galanos, Eur J Biochem 9:245-249, and the people such as Wu, 1987, Anal Bio Chem 160:281-289.LPS can simply use or with the source of T-cell epitope, put together use such as tetanus toxoid, diphtheria toxoid, CRM-197 or OMV outer membrane protein.The technology that the LOS of separation is puted together also is known (see that for example, EP 941738, it is incorporated herein by reference).
As LOS (particularly LOS of the present invention) when being present in the bleb preparation, preferably by making LOS and also being present in the method that one or more outer membrane protein (for example, the PorA in the meningococcus or PorB) on the bleb preparation put together the LOS original position is puted together.
The method can advantageously improve the stability of LOS antigen in the bleb preparation and/or immunogenicity (the T-cell help is provided) and/or antigenicity-therefore provides T-cell help-such as LOS in its natural surroundings, on the meningococcus outer membrane face take the strongest conformation of its protectiveness as non-T-dependency oligosaccharide immunogen.In addition, can cause LOS detoxifcation (lipid A part is imbedded in the adventitia by stable, therefore seldom causes toxicity) LOS puting together in bleb.Therefore, mention here from htrB
-Or msbB
-Separate bleb in the mutant, or (see WO 93/14115, the people such as WO 95/03327, Velucchi by the detoxification without the phallotoxins functional equivalents that in compositions, adds polymyxin B, (1997) J Endotoxin Res 4:1-12, with EP 976402, the further describing-the particularly use of (sequence KTKCKFLKKC, wherein 2 cysteine form disulfide bond) peptide SAEP 2 without the phallotoxins functional equivalents of polymyxin B) may not be essential (but it can be added in the combination for increasing safety).Therefore, the inventor has been found that the compositions that comprises bleb can be formed for treating or the vaccine that prevents the biological institute by the bleb of deriving to cause disease is basic, the LOS that wherein is present in the bleb puts together with bleb internal schema and the outer membrane protein that is present in the bleb simultaneously, and wherein this vaccine is nontoxic basically and can induces the T-dependency bactericidal reaction that resists the LOS in its natural surroundings.
Therefore the present invention further provides the meningococcus bleb goods that LOS puts together in this bleb.
Whether this bleb goods can be separated from the antibacterial (seeing WO 01/09350) that positive pole is discussed, and then make group (for example, NH on the LOS oligosaccharide part through the known chemical process of puting together
2Or COOH) with the bleb outer membrane protein on group (for example, NH
2Or COOH) puts together also in discussion.Can use the crosslinking technological that utilizes glutaraldehyde, formaldehyde or glutaraldehyde/formaldehyde mixture, but preferably use the stronger chemical process of selectivity such as EDAC or EDAC/NHS (J.V.Staros, R.W.Wright and D.M.Swingle.Enhancement by N-hydroxysuccinimide of water-soluble carbodiimide-mediated coupling reactions.Analytical chemistry 156:220-222 (1986); With Bioconjugates Techniques.Greg T.Hermanson (1996) pp 173-176).Spendable, can put together chemical process or process in EP 941738 and describe at other that generates covalent bond between LOS and the protein molecular.
Preferred bleb goods are puted together under the condition that does not have capsular polysaccharide to exist.Described bleb can be never (according to following natively described or via sudden change) produce in the bacterial strain of capsular polysaccharide and separate, maybe can be from most and preferably all pollute purification the capsular polysaccharides.By this way, the LOS conjugation reaction is more effective in the bleb.
Preferably be present in the bleb surpass 10,20,30,40,50,60,70,80,90 or 95% LOS be crosslinked/put together.
Put together 1,2 or all three steps that should preferably integrate following method in the bleb: puting together pH should be greater than pH 7.0, preferably greater than or equal to pH 7.5 (most preferably pH 9 times); In course of reaction, should keep 1-5%, preferred 2-4%, the condition of 3% sucrose most preferably from about; In conjugation reaction, NaCl should be dropped to minimum, preferred 0.1M, 0.05M, 0.01M, 0.005M, 0.001M and most preferably exist.All these method features can be guaranteed all to keep stable and remain in the solution in the whole bleb of puting together in the process.
The EDAC/NHS conjugation methods is the method for optimizing of puting together in the bleb.EDAC/NHS is better than formaldehyde, and the formaldehyde crosslinkable adversely affects filtration rate thus to very high degree.EDAC and carboxylic acid (such as the KDO among the LOS) reaction produces the active ester intermediate.Under the condition that has amine nucleopilic reagent (such as outer membrane protein, such as the lysine among the PorB) to exist, amido link is to use the release of isourea by-product to form.Yet the usefulness of the reaction of EDAC-mediation can increase by the formation of Sulfo-NHS ester intermediate.The time that the Sulfo-NHS ester exists in aqueous solution is longer than the independent active ester with carboxylate reaction formation of EDAC.Therefore, more the formation of high yield amido link can use this dual stage process to realize.EDAC/NHS is conjugated in J.V.Staros, R.W.Wright and D.M.Swingle.Enhancement by N-hydroxysuccinimide of water-soluble carbodiimide-mediated coupling reactions.Analytical chemistry 156:220-222 (1986); With discuss among Bioconjugates Techniques.Greg T.Hermanson (1996) the pp 173-176.Preferably in reaction, use 0.01-5mg EDAC/mg bleb, more preferably 0.05-1mg EDAC/mg bleb.The amount of used EDAC depends on the LOS amount that is present in the sample, and it depends on successively for dexycholate (DOC) % that extracts bleb.Under low %DOC (for example, 0.1%), use the EDAC (1mg/mg and more than) of larger consumption, yet under high %DOC (for example 0.5%), use the EDAC (0.025-0.1mg/mg) of less consumption to avoid crosslinked between too many bleb.
Therefore method of the present invention is preferably for generation of the method for puting together LOS (preferred meningococcus) in the bleb, the method is included in EDAC/NHS existence, pH 7.0-pH 9.0 (preferably about pH 7.5), 1-5% (preferred about 3%) sucrose and chooses wantonly under the condition that there is no NaCl (as mentioned above) bleb is puted together, and puts together the step of bleb in the separate reacted mixture.
Anti--LOS is used in described reaction subsequently, and (for example, anti--L2 or anti--L3) mAb carries out at the Separation of Proteins gel of reactant mixture, and it shows the carrying out along with the response time, and the ratio of LOS increases in the bleb, and the molecular weight of LOS increases.
Use the callable bleb productive rate 99% of this technology.
It is found that EDAC is a kind of fabulous bleb internal crosslinker, this is because it can make LOS and OMP full cross-linked, be used for to improve LOS T-dependent immunity originality, but can not make it be cross-linked to very high degree and cause that crosslinked problem occurs between, gathering relatively poor such as filtration rate and bleb.The morphology of the bleb that produces similar to unconjugated bleb (utilizing ultramicroscope).In addition, such scheme can avoid occuring excessively crosslinked (it can reduce the natural lip-deep protectiveness OMP of bleb, for example immunogenicity of TbpA or Hsf of being present in).
The meningococcus of the bleb of preferably deriving is the mutant that can not produce capsular polysaccharide (for example, following mutant wherein a kind of, particularly siaD
-).Also preferably effectively the immunogenic composition of meningococcemia disease comprise simultaneously L2 and L3 bleb, wherein L2 and L3 LOS all put together with the bleb outer membrane protein.In addition, put together in the preferred bleb in the bleb the LOS structure with from 1gtB
-Consistent (as described below) that the meningococcus bacterial strain is derived.Most preferably immunogenic composition comprises the bleb of puting together in the bleb: derive from and can not produce capsular polysaccharide and be the sudden change meningococcus bacterial strain of 1gtB; Comprise the L2 and the L3 bleb that derive from the sudden change meningococcus bacterial strain that can not produce capsular polysaccharide; Comprise and derive from 1gtB
-L2 and the L3 bleb of sudden change meningococcus bacterial strain; Or most preferably comprise to derive from and to produce capsular polysaccharide and be 1gtB
-L2 and the L3 bleb of sudden change meningococcus bacterial strain.
Can be used for general L3 meningococcus bacterial strain of the present invention is the H44/76menB bacterial strain.General L2 bacterial strain is B16B6menB bacterial strain or 39E meningococcus C type bacterial strain.
As mentioned above, bleb of the present invention is detoxified extremely to a certain degree by the effect of puting together, and need not further detoxifcation, yet further detoxification can be used for increasing safety, for example, uses to derive from htrB
-Or msbB
-The bleb of meningococcus bacterial strain or add in the bleb compositions polymyxin B without phallotoxins functional equivalents [having molecule than high-affinity with lipid A] (preferred SEAP 2) (as mentioned above).
In said method, provide meningococcus to steep and contain the immunogenic composition of bleb, it has important antigen LOS, this antigen is basically nontoxic, there is not the autoimmunity problem, have the T-dependence-producing property, be present in its natural surroundings, and can induce the anti-bactericidin reaction (in the situation of L2+L3 compositions) that surpasses 90% meningococcus bacterial strain.
LOS puts together 1,2 or all three steps that should integrate following method in the preferred bleb: puting together pH should be greater than pH 7.0, preferably greater than or equal to pH 7.5 (most preferably pH 9 times); In course of reaction, should keep 1-5%, preferred 2-4%, the condition of 3% sucrose most preferably from about; In conjugation reaction, NaCl should be dropped to minimum, preferred 0.1M, 0.05M, 0.01M, 0.005M, 0.001M and most preferably exist.All these method features can be guaranteed to keep stable and remain in the solution in the whole bleb of puting together in the process.
Although can LOS and outer membrane protein be puted together in bleb by various technology and chemical process, the EDAC/NHS conjugation methods be the method for optimizing of puting together in the bleb.EDAC/NHS is better than formaldehyde, and the formaldehyde crosslinkable has adverse influence to filtration rate thus to very high degree.EDAC and carboxylic acid reaction produce the active ester intermediate.Under the condition that has the amine nucleopilic reagent to exist, amido link is to use the release of isourea by-product to form.Yet the usefulness of the reaction of EDAC-mediation can increase by the formation of Sulfo-NHS ester intermediate.The time that the Sulfo-NHS ester exists in aqueous solution is longer than the independent active ester with carboxylate reaction formation of EDAC.Therefore, more the formation of high yield amido link can use this dual stage process to realize.EDAC/NHS is conjugated in J.V.Staros, R.W.Wright and D.M.Swingle.Enhancement by N-hydroxysuccinimide of water-soluble carbodiimide-mediated coupling reactions.Analytical chemistry 156:220-222 (1986); With discuss among Bioconjugates Techniques.Greg T.Hermanson (1996) the pp 173-176.
Therefore method of the present invention is preferably for generation of the method for puting together LOS (preferred meningococcus) in the bleb, the method is included in pH, 1-5% (the preferred about 3%) sucrose of EDAC/NHS existence, pH 7.0-pH 9.0 (preferably about pH 7.5) and chooses wantonly under the condition that there is no NaCl (as mentioned above) bleb is puted together, and separates the step of puting together bleb from reactant mixture.
Anti--LOS is used in described reaction subsequently, and (for example, anti--L2 or anti--L3) mAb carries out at the separating gel of reactant mixture, and it shows the carrying out along with the response time, and the ratio of LOS increases in the bleb, and the molecular weight of LOS increases.
Use the callable bleb productive rate 99% of this technology.
It is found that EDAC is a kind of fabulous bleb internal crosslinker, this is because it can make LOS and OMP full cross-linked, be used for to improve LOS T-dependent immunity originality, but can not make it be cross-linked to very high degree and cause problem crosslinked between and bleb relatively poor such as filtration rate to occur.In addition, should avoid excessively crosslinked, thereby avoid the natural lip-deep protectiveness OMP of bleb that is present in, for example immunogenic any reduction of Tbp.
5. integration outer membrane protein
Other kind of Neisseria gonorrhoeae albumen also can be the material standed for that is included in the Neisseria gonorrhoeae vaccine of the present invention, and can be with astonishing effective means and the combination of other antigen.Embrane-associated protein, particularly AQP-CHIP and outer membrane protein are the most advantageously particularly integrated outer membrane protein and be can be used in the compositions of the present invention.The example of this albumen be also be known as Omp1A Pld A (NMB 0464) (WO00/15801), it is Neisseria gonorrhoeae phospholipase outer membrane protein.Other example is TspA (NMB 0341) (Infect.Immun.1999,67; 3533-3541) and TspB (T-cytositimulation albumen) (WO 00/03003; NMB 1548, NMB 1628 or NMB1747).Other example comprise OMP85 (NMB0182) that Pi1Q (NMB 1812) (WO99/61620), also is known as D15-(WO00/23593), NspA (U52066) (WO96/29412), FhaC (NMB 0496 or NMB 1780), PorB (NMB 2039) (Mol.Biol.Evol.12:363-370,1995), HpuB (NC.003116.1), (Microbiology 2001,147 for TdfH (NMB1497); 1277-1290), OstA (NMB0280), (NMB 0532 also to be known as MltA (NMB0033), the HtrA of GNA33 and Lipo30; WO99/55872), HimD (NMB 1302), HisD (NMB 1581), GNA 1870 (NMB 1870), HlpA (NMB 1946), NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, TbpA (NMB 0461) (WO 92/03467) (be also shown in top ferrum and obtain albumen) and LbpA (NMB 1541).
OMP85
Immunogenic composition of the present invention can comprise total length OMP85, preferably as the part of OMV goods.The fragment of OMP85 also can be used in the immunogenic composition of the present invention, particularly, preferably will be incorporated in the subunit component of immunogenic composition of the present invention by the surface exposed domain of OMP85 that amino acid residue 1-475 or 50-475 form.In any one or both ends of N or C-terminal, the above-mentioned sequence of the surface exposed domain of OMP85 can be stretched or truncate reaches 1,3,5,7,10,15,20,25 or 30 aminoacid.The signal sequence of preferably deletion OMP85 fragment.
OstA
OstA can play a role in lipopolysaccharide synthetic and can be considered to the toxicity regulator.OstA optionally is included in the endotoxin, and wherein the toxin kind is enlarged to and comprises toxicity regulator and toxin.
Immunogenic composition
Immunogenic composition is the compositions that comprises at least a antigen, and when giving the host, described antigen can produce immunoreation.Preferably, this immunogenicity goods can produce anti-Neisseria gonorrhoeae, the protective immunological reaction that preferred anti-Neisseria meningitidis or Diplococcus gonorrhoeae infect.
The present invention relates to comprise the immunogenic composition of at least two kinds of antigens, it preferably can cause Synergistic biocidal, protectiveness or adhesion blocking reaction one or more.
SBA sterilization algoscopy of the present invention
This concerted reaction can characterize by the SBA that is caused by antigen combination, and the SBA that the antigen combination causes wants height at least 50%, 2 times, 3 times, preferred 4 times, 5 times, 6 times, 7 times, 8 times, 9 times and most preferably 10 times than the SBA that is caused separately by each antigen.Preferred SBA measures with respect to the homology bacterial strain of the antigen of deriving, and preferably measure with respect to one group of heterologous bacterial strain.(see following representative group, for example belong to the BZ10 (B:2b:P1.2) that A-4 clusters; The B16B6 (B:2a:P1.2) that belongs to the ET-37 complex; And H44/76 (B:15:P1.7,16)).SBA is the most universally recognized amynologic label, can estimate the effect (people such as Perkins, J Infect Dis.1998,177:683-691) of meningococcus vaccine.Gratifying SBA can measure by any known method.SBA can utilize from the serum of animal model or human experimenter's acquisition and carry out (seeing embodiment 17-20).
The method for optimizing that carries out SBA with human serum is as follows.Before for the first time inoculation, inoculated rear 2 months for the second time and inoculate for the third time rear 1 month (carrying out 3 inoculations in 1 year is general human primary vaccination program, for example the 0th, 2 and 4 months or the 0th, 1 or give 6 months the time) the collection blood sample.This human primary vaccination program can be carried out to the baby below 1 years old (for example, carrying out simultaneously with Hib inoculation), or inoculates to test the SBA of this primary vaccination program also can for child or the teenager in 2-4 year.If be fit to, can be behind 6-12 behind the primary vaccination month and booster dose again gather blood sample in 1 month.
If behind (the primary vaccination program) the 3rd vaccine administration one month (in 2-4 year child or teenager, but preferred in the baby of life First Year), the SBA (antibody dilution) of the meningococcus bacterial strain of the anti-antigen of the present invention of deriving tire (comparing with tiring before the inoculation) increase by experimenter's percentage ratio of 4 times and surpass 30%, preferably surpass 40%, more preferably surpass 50%, most preferably surpass 60% experimenter, for the antigen with homology bactericidal activity or bleb goods, SBA is gratifying so.
Certainly, if it also can cause gratifying SBA with respect to its meningococcus bacterial strain of deriving, the antigen or the bleb goods that then have the allos bactericidal activity also can consist of the bleb goods with homology bactericidal activity.
If behind (the primary vaccination program) the 3rd vaccine administration one month (in 2-4 year child or teenager, but preferred in the baby of life First Year), the SBA (antibody dilution) of 3 kinds of allos bacterial strains of meningococcemia tire (comparing with tiring before the inoculation) increase by experimenter's percentage ratio of 4 times and surpass 20%, preferably surpass 30%, more preferably surpass 35%, most preferably surpass 40% experimenter, for the antigen with allos bactericidal activity or bleb goods, SBA is gratifying so.This test is to have the well-characterized whether antigen of allos bactericidal activity or bleb goods can induce the intersection bactericidin of anti-various meningococcus bacterial strains.Described three kinds of allos bacterial strains should preferably have and differ from one another and preferred (see the people such as Maiden with the electrophoretype (ET) for preparing or the bacterial strain of derive antigen with allos bactericidal activity or bleb goods is different-complex or polygenes seat sequence typing (MLST) pattern, PNAS USA 1998,95:3140-5).Those skilled in the art should measure three kinds of bacterial strains with different ET-complexs at an easy rate, it can be reflected between the meningococcus, the gene diversity of particularly observing between the meningococcal B type bacterial strain, these bacterial strains are considered to the reason of important diseases burden and/or the MenB severe toxicity pedigree (seeing the people such as Maiden, on seeing) of representative approval.For example, spendable three kinds of bacterial strains are following: belong to the BZ10 (B:2b:P1.2) that A-4 clusters; The B16B6 (B:2a:P1.2) that belongs to the ET-37 complex; With the H44/76 that belongs to the ET-5 complex (B:15:P1.7,16), or belong to any other bacterial strain that same ET/ clusters.This bacterial strain can be used for testing antigen or the bleb goods that prepare or derive, have the allos bactericidal activity from the meningococcus bacterial strain CU385 (B:4:P1.15) that for example belongs to the ET-S complex.Spendable other sample bacterium source is in the popular clone of pedigree 3 (for example, NZ124[B:4:P1.7,4]).Another ET-37 bacterial strain is NGP165 (B:2a:P1.2).
The method of measuring the SBA activity is known in the art.For example, spendable method is described in the embodiment of WO99/09176 10C.In ordinary circumstance, tested strain culture is in the logarithmic (log) phase of growth (preferably in the condition in ferrum disappearance-by adding iron chelating agent such as EDDA in growth medium) growth.It can be suspended in the culture medium (as having the Hanks culture medium of 0.3%BSA) with BSA, in order to obtain to transfer to the test cell suspending liquid of about 20000CFU/ml.The series reaction mixture can be by with the test serum (preferably 56 ℃ of hot deactivations 30 minutes) of a series of 2 times of dilutions [for example, 50 μ l/ pore volumes] and the meningococcus bacterial strain suspension (for example, 25 μ l/ pore volumes) of 20000CFU/ml test mix and prepare.Should cultivate (for example, 37 ℃ 15 minutes) and jolting (for example, 210rpm) reaction bottle.In addition, end reaction mixture [for example, 100 μ l volumes] also can comprise complement source [such as the prerun children rabbit anteserum of, 25% final volume], and according to top described cultivation [for example, 37 ℃ 60 minutes].Microtitration flat board in 96-hole can be used for this algoscopy at the bottom of the aseptic polystyrene U-.Can use multichannel pipettor from each hole, to gather aliquot [for example, 10 μ l], drop in Mueller-Hinton agar plate (preferably containing 1%Isovitalex and 1% heat-inactivated horse serum) and go up and cultivate (for example, at 37 ℃, 5%CO
2Middle cultivation 18 hours).Preferably, independent bacterium colony is up to the 80CFU/ aliquot.With following three kinds of test specimens with comparing: buffer+antibacterial+complement; Buffer+antibacterial+deactivation complement; Serum+antibacterial+deactivation complement.Service routine is directly calculated but SBA tires, thus this program can process data and obtain dilution measured value, this measured value is corresponding with 50% cell death that obtains by regression Calculation.
The animal protection algoscopy
Optionally, concerted reaction can characterize by the effect that antigen group is combined in the animal protection algoscopy.For example, can use the algoscopy of describing in embodiment 12 or 13.Preferably, compare with the suboptimum dosage of independent use antigen, particularly antigen, the size of animal that is protected by the antigen combination obviously increases.
Be used for preventing that the successful vaccine that Diplococcus gonorrhoeae infects from may need a more than following key element: the generation of serum and/or mucoantibody is dead with the gonococcus that promotes complement-mediated, and/or by leukocyte such as polymorphonuclear leukocyte raising phagocytosis and kill microorganisms, and/or prevent that gonococcus is attached to host tissue; The immunoreation of inducing cell mediation, but their also participation protectives.
The raising of bleb gonorrhea vaccine product effect of the present invention can be estimated by serum and/or mucoantibody immunoreation that analysis is induced, they have anti-adhesive and/or conditioning property and/or bactericidal activity, such as (people such as McChesneyD, Infect.Immun.36:1006,1982 as described in other people; The people such as Boslego J: Efficacy trial of a purified gonococcl pilus vaccine, in Program and Abstracts of the 24th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, American Society for Microbiology, 1984; The people such as Siegel M, J.Infect.Dis 145:300,1982; De la Pas, Microbiology, 141 (Pt4): 913-20,1995).
Recently described by the genital infection mouse model due to the Diplococcus gonorrhoeae (Plante M, J.Infect.Dis., 182:848-55,2000).The raising of bleb gonorrhea efficacy of vaccines of the present invention also can be estimated by its ability that prevents or reduce that Diplococcus gonorrhoeae is settled down in this mouse infection model.
Adhere to the blocking-up algoscopy
Optionally, concerted reaction can characterize by the effect that antigen group be combined in the adhesion blocking-up algoscopy.For example, can use the algoscopy of describing among the embodiment 11.Preferably with the antiserum that uses anti-antigen alone to produce, particularly the antibody of suboptimum dosage is compared, and the blocking-up degree of being induced by the antiserum of antigen combination results significantly improves.
The subunits combination thing
Immunogenic composition of the present invention can be a kind of subunits combination thing.The subunits combination thing is before component mix is formed antigenic composition, wherein component separated and be purified at least 50%, the compositions of preferred at least 60%, 70%, 80%, 90% purity.
Immunogenicity subunits combination thing of the present invention preferably includes and is selected from least 2 kinds of following antigens: one of passerby's domain, FrpA, FrpC, TbpA, TbpB, LbpA, LbpB, HpuA, HpuB, TspA, TspB, PldA, PilQ, FhaC, NspA and the LPS immunologic pattern L2 of passerby's domain of FhaB, PilC, Hsf, Hap, NadA, OMP85, IgA protease, AspA, AspA, passerby's domain of Hsf, Hap and LPS immunologic pattern L3 or both.
The subunits combination thing can be the aqueous solution of water-solubility protein.They can comprise detergent, preferably non--ion, amphion or ion detergent, in order to make the hydrophobic part dissolving of antigen.They can comprise lipid, so just can form liposome structure, and antigen is presented with the structure of crossing over adipose membrane.
The little brewage of adventitia
Neisseria meningitidis serum group B (menB) thus the adventitia bleb of secretion capacity makes it possible to make them with commercial scale.The adventitia vesicle also can be via the method preparation (for example seeing that EP 11243) of extracting bacterial cell with detergent.
Immunogenic composition of the present invention also can comprise the little brewage of the adventitia with at least two kinds of antigens, and described antigen is raised, and perhaps reorganized rise or raised by alternate manner is included under ferrum-deletion condition and grows.The example of the antigen that is raised in the little brewage of this adventitia comprises: NspA, Hsf, Hap, OMP85, TbpA (height), TbpA (low), LbpA, TbpB, LbpB, PilQ, AspA, TdfH, PorB, HpuB, P2086, NM-ADPRT, MafA, MafB and PldA.This goods also can be chosen wantonly and comprise one of LPS immunologic pattern L2 and LPS immunologic pattern L3 or both.
The preparation that derives from the bleb goods of Neisseria gonorrhoeae bacterial strain can obtain by any method well known to those skilled in the art.Preferably use at EP 301992, US5,597,572, EP 11243 or US4,271,147, the people such as Frederikson (NIPH Annals[1991], 14:67-80), the people such as Zollinger, (J.Clin.Invest.[1979], 63:836-848), the people such as Saunders, (Infect.Immun.[1999], 67:113-119), the people such as Drabick (Vaccine[2000], disclosed method 18:160-172) or among the WO01/09350 (embodiment 8).Usually, use detergent, preferably dexycholate extraction OMV, and nucleic acid is chosen wantonly enzymatic and is removed.Purification is by behind the ultracentrifugation, optional carries out then that size exclusion chromatography realizes.If comprise 2 kinds of the present invention or multiple different bleb, then they can be combined to form multivalence goods of the present invention (although goods also are considered to multivalence in single container, if different bleb of the present invention is the independent compositions that is in the independent container, but they are that [by same doctor] gives the host's at one time].The OMV goods normally filter by 0.2 μ m filter and sterilizes, and preferably are stored in the sucrose solution (for example 3%), and the known bleb goods that make of this sucrose solution dissolve.
The rise of albumen in adventitia vesicle goods can be inserted into by the additional copies with gene in the Neisseria gonorrhoeae bacterial strain of the OMV goods of deriving and obtain.Optionally, the gene promoter in the Neisseria gonorrhoeae bacterial strain of the OMV goods of deriving can be exchanged into stronger promoter.This technology is described in WO01/09350.Compare with the protein level that exists the OMV that Neisseria meningitidis (for example bacterial strain H44/76) from unmodified derives, the rise of albumen will cause the protein level that exists among the OMV higher.It is high 1.5,2,3,4,5,7,10 or 20 times that preferred described level is wanted.
When LPS was additional antigen among the OMV, the scheme of using low concentration to extract detergent (for example, dexycholate or DOC) can be preferred in the OMV preparation method, so as to keep high-caliber remove especially in conjunction with LPS poisonous in conjunction with relatively poor LPS.The preferred 0-0.5%DOC of the concentration of used DOC, 0.02-0.4%DOC, 0.04-0.3%DOC, more preferably 0.06%-0.2%DOC or 0.08-0.15%DOC, most preferably from about or accurately 0.1%DOC.
" strong promoter sequence " refers to increase the adjusting control element of the genetic transcription of coding target antigen.
" up-regulated expression " refers to for unmodified (namely naturally occurring) bleb, can improve any mode that target antigen is expressed.The amount that should understand ' rise ' depends on specific target antigen, but can not exceed the amount of the film integrality that destroys bleb.The rise of antigen refers to than the expression of the bleb height 10% of unmodified at least.Be preferably up to few 50%.Be more preferably up to few 100% (2 times).Most preferably high 3,4,5,7,10,20 times.Optionally or additionally, up-regulated expression refers to change speech expression right and wrong-opportunistic with regard to metabolism or nutrition, particularly in the situation of TbpA, TbpB, LbpA and LbpB.Preferably when from the antibacterial that the limited condition of ferrum, grows, deriving bleb, evaluation expression level (for example under the condition that has iron chelating agent to exist).
Again for purpose clearly, term ' produces less described antigen with bacterial isolates transformation ' or downward modulation refers to for (being naturally occurring bleb) of unmodified, preferably make target antigen express any mode that (or expression of functioning gene product) reduces by disappearance, express like this and will hang down at least 10% than the bleb of unmodified.Preferred low at least 50% and most preferably lack fully.If down-regulation protein is enzyme or functional protein, then downward modulation can obtain by introducing the one or many sudden change, causes enzyme or functional activity to reduce by 10%, 20%, 50%, 80% or preferred 100%.
Regulating the required adaptation step of Neisseria gonorrhoeae protein expression can carry out with variety of way well known by persons skilled in the art.For example, but insertion sequence (for example, promoter or open reading frame), and by transposon insertion technology destruction promoter/gene.For example, during the expression of up-regulated gene, can via transposon with strong promoter be inserted into gene start codon until 2kb upstream (more preferably 200-600bp upstream, most preferably from about 400bp upstream).Also utilisation point suddenlys change or disappearance (particularly the downward modulation of gene being expressed).
Yet, this method possibility rather unstable or uncertain, and therefore preferred adaptation step is to carry out via the homologous recombination event.Preferably, this event occur in the sequence (luring recombination zone) of at least 30 nucleotide on the bacterial chromosome and the carrier that in bacterial strain, transforms between the sequence (second lures recombination zone) of at least 30 nucleotide.Preferred this district is 40-1000 nucleotide, more preferably 100-800 nucleotide, most preferably 500 nucleotide.These lure the recombination zone should be enough similar, and they just can each other hybridization under very strict condition like this.
Be used for carrying out genetic modification event described herein method (as by recombination event gene is raised or downward modulation and other gene order is incorporated into the Neisseria gonorrhoeae genome) in WO01/09350, describe.Can in Neisseria gonorrhoeae, integrated general strong promoter be porA, porB, lgtF, Opa, p110, lst and hpuAB.PorA and PorB are preferred composing type strong promoters.Determined that the PorB promoter activity is included in the fragment corresponding with the nucleotide 1-250 of PorB upstream from start codon.
Raise the expression of antigen by growth in the limited culture medium of ferrum
The rise of some antigen preferably realizes by the adventitia vesicle that is separated in the Neisseria gonorrhoeae parental strain of growing under the limited condition of ferrum in the little brewage of adventitia of the present invention.The ferrum of low concentration will cause participating in the protein expression increase that ferrum obtains in the culture medium, comprise TbpA, TbpB, LbpA, LbpB, HpuA, HpuB and P2086.These protein expressions are raised thus and be need not the related gene of recombinant modified, for example by inserting the additional copies of stronger promoter or insertion gene.The present invention comprises that also raising ferrum by growth in the limited culture medium of ferrum obtains albumen, and wherein said gene is reorganized modification also.
Ferrum is limited to be realized by add iron chelating agent in culture medium.Suitable iron chelating agent comprises the two pyridines of 2,2-, and EDDHA (ethylenediamine-two (o-hydroxyl phenylacetic acid) and Desferal (deferoxamine mesylate, Sigma).Desferal be preferred iron chelating agent and with 10-100 μ M, preferred 25-75 μ M, more preferably 50-70 μ M, most preferably the concentration of 60 μ M joins in the culture medium.The ferrum inclusions of culture medium is mainly derived from yeast extract and soya peptone component, and the consumption that exists can be according to batch changing.Therefore, in the culture medium that difference is criticized, the Desferal of variable concentrations is best for the rise that obtains ferrum acquisition albumen.Those skilled in the art should measure optium concentration at an easy rate.Under basic condition, should add sufficient iron chelating agent in the culture medium, thereby raise required ferrum-adjusting protein expression, but can not be excessive and adversely affect the growth of antibacterial.
Preferably will obtain by the ferrum of under the limited condition of ferrum, growing the rise of albumen and the restructuring rise combination of other antigen, thereby obtain adventitia vesicle of the present invention.
The downward modulation of variable and non--dominant antigen of protective immunity/remove
In bacterial isolates, a lot of surface antigens all are variable and therefore can only resist one group of limited bacterial strain that is closely related.One aspect of the present invention relates to adventitia vesicle of the present invention, and wherein other protein expression reduces, or the gene delection of optimized encoding variable surface albumen.The bacterial isolates that this disappearance causes producing bleb is when with the vaccine form administration, and the potential of anti-various bacterial strain cross reactivities is stronger, and this is because conservative protein (being deposited on the adventitia) has applied stronger impact to inoculator's immune system.Can in bleb immunogenic composition of the present invention, be comprised PorA, PorB, Opa by the example of this variable antigen in the Neisseria gonorrhoeae of reducing.
Other type gene that can be reduced or cut off is the gene that can be connected at an easy rate by antibacterial in vivo (expression) or cut off.Because the outer membrane protein by this gene code always is not present on the antibacterial, therefore the existence of this albumen in the bleb goods also can be harmful to the vaccine effectiveness for above-mentioned reasons.The preferred example of downward modulation or disappearance is Neisseria gonorrhoeae Opc albumen.Only have limited protective capability by vaccine-induced the resisting of the bleb that contains Opc-Opc immunity, this is because infection biological is easy to become Opc
-
For example, these variable or non--protecting groups because of expression can be reduced, or finally be cut off.Like this then have immune system is concentrated on advantage on the better antigen, described antigen only is present on the bleb outer surface on a small quantity.Downward modulation also refers to surface exposed, and the variable immundominance ring of above-mentioned outer membrane protein can be changed or lack, thereby produces the lower outer membrane protein of immundominance.
The method that downward modulation is expressed is open in WO01/09350.The optimization protein combination of being reduced in bleb immunogenic composition of the present invention comprises PorA and OpA, PorA and OpC, OpA and OpC, PorA and OpA and OpC.
Known have 4 kinds of different Opa genes to be present in the meningococcus genome (people such as Aho, 1991Mol.Microbiol.5:1429-37) in, therefore when the expression that mention Opa by lower timing, it refer to preferably be present in 1 in the meningococcus, 2,3 or (preferably) whole 4 kinds of genes all reduced.This downward modulation usually can be carried out or be undertaken by searching that be very easy to find, natural, stable meningococcus bacterial strain according to the described genetic engineering of WO01/09350, and they are lower or do not have from the expression of Opa locus.This bacterial strain can use the scientific discovery of people (1985J.Med.Micro.19:203-209) descriptions such as Poolman, wherein Opa
-Cell has the phenotype different from the cell of expressing Opa, this can by on flat board or microscopically observation of cell outward appearance see.In case find, fermenting so that after the Opa disappearance, to demonstrate this bacterial strain be stable Opa by carry out Western blotting at cell inclusion
-
When the rise of some antigen in the adventitia vesicle be by growth under the limited condition of ferrum realize the time, (Microbiology 142 for variable protein Fr pB; 3269-3274, (1996); J.Bacteriol.181; 2895-2901 (1999)) also can be raised.The inventor has been found that described according to WO01/09350 is favourable by reducing whole protein expressions or reducing the FrpB expression by making FrpB variable region disappearance under these environment.Can guarantee that so the immunoreation that caused by immunogenic composition is for the antigen that is present in the bacterial strain on a large scale.In bleb immunogenic composition of the present invention, the downward modulation of FrpB is preferably made up with the downward modulation of PorA and OpA, PorA and OpC, OpA and OpC, PorA and OpA and OpC.
Optionally in the embodiment, the FrpB in the adventitia vesicle is reduced in the present invention, and described adventitia vesicle is never to be the Neisseria gonorrhoeae bacterial strain preparation of growing under the limited condition of ferrum.
The LPS detoxifcation
Bleb in the immunogenic composition of the present invention can be via the disclosed LPS detoxification detoxifcation of WO01/09350.The concrete grammar of LPS of the present invention detoxifcation comprises makes among the WO01/09350 disclosed htrB and/or msbB enzyme be reduced/lack.The deletion mutation that the msbB of Neisseria gonorrhoeae and htrB gene also are known as respectively lpxL1 and lpxL2 (WO00/26384) and these genes is to characterize by the msbB-sudden change LOS that loses a secondary acyl chain and the htrB-sudden change LOS that loses two secondary acyl chains and from the phenotype aspect.WO 93/14155 and WO 95/03327 described the polymyxin B that can be used in the present composition without the phallotoxins functional equivalents.
This method preferably with bleb extracting method combination, the bleb extracting method comprises and uses low-level DOC, preferred 0-0.3%DOC, more preferably 0.05%-0.2%DOC, 0.1%DOC most preferably from about or accurately.
Other method of LPS detoxifcation comprise as mentioned above add in the bleb goods polymyxin B without phallotoxins functional equivalents (preferred SAEP 2).
The cross reactivity polysaccharide
With the bacterial outer membrane bleb from encapsulated gram negative bacteria, separate usually cause capsular polysaccharide altogether-purification.In some cases, this " pollution " material can show usefulness, and this is because polysaccharide can improve the immunoreation that is produced by other bleb component.Yet in other cases, contaminative polysaccharide material existing in antibacterial bleb goods is provable harmful to the application of bleb in vaccine.For example, demonstrated at least in the situation of Neisseria meningitidis, serum group B capsular polysaccharide can not bring the protection immunity and be easy to induce disadvantageous autoimmune response in the mankind.Therefore, adventitia vesicle of the present invention can be separated the production for bleb from bacterial isolates, it has been transformed into does not have capsular polysaccharide.So bleb namely is applicable to the mankind.The more preferred example of this bleb goods is to derive from the bleb goods that do not have the Neisseria meningitidis of capsular polysaccharide serum group B.
This can realize by bleb producing bacterial strain of use modifying, wherein pod membrane biosynthesis and/or export required gene and damage.The deactivation of the gene of coding capsular polysaccharide biosynthesis or output can make control zone, coding region or both sudden changes (point mutation, disappearance or insertion) by (preferably using above-mentioned homologous recombination technique), or realizes by any alternate manner that reduces this gene enzyme function.In addition, the deactivation of pod membrane biosynthesis gene also can realize by antisense overexpression or transposon mutagenesis.Method for optimizing is part or all disappearance that makes the polysaccharide biosynthesis and export required Neisseria meningitidis Lps gene.With regard to this purpose, displacement plasmid pMF121 (people such as Frosh, described in 1990, the Mol.Microbiol.4:1215-1218) can be used for transmitting makes cpsCAD (+galE) the gene sudden change of disappearance that clusters.
The safety that has resisted L 3 or L2 antibody that LPS produces has produced query, and this is because the existence of the structure similar to the breast-N-neotetraose oligosaccharide group (Gal β l-4GlcNAc β l-3Gal β l-4Glc β l-) in being present in people's glycosyl sphingolipid.Although the vesicle vaccine that contains residual volume L3LPS that has had a lot of people to inoculate safely dexycholate to extract (people such as G.Bjune, Lancet (1991), 338,1093-1096; The people such as GVG.Sierra, NIPH anb (1991), 14,195-210), the disappearance of LOS sugar end portion can advantageously prevent any cross reaction with the structure that is present in people's tissue surface.In preferred embodiments, the deactivation of lgtB gene produces intermediate LPS structure, wherein lacks terminal galactose residues and sialic acid (sudden change stays the 4GlcNAc β l-3Gal β l-4Glc β l-structure among L2 and the L3LOS).This intermediate can obtain in L3 and L2LPS bacterial strain.Alternative and not too preferred (weak point) version of LPS can obtain by closing the lgtE gene.Other selectivity of LPS and not too preferred version can obtain by closing the lgtA gene.If select this lgtA
-Sudden change prevents from forming non-immunogenic L1 immunologic pattern thereby preferably also can close the lgtC expression.
The LgtB-mutant is most preferred, and this is because the inventor has been found that this is the best truncate that still keeps simultaneously the LPS protectiveness oligosaccharide epi-position that can induce the bactericidin reaction for the solution safety issue.
Therefore, the immunogenic composition of the present invention or the meningococcus bleb goods that also comprise L2 or L3 goods (purification or in the bleb of separating) advantageously derive from Neisseria gonorrhoeae bacterial strain (preferred meningococcus) usually, they have been derived from the expression of the functioning gene product of lgtB, lgtA or lgtE gene by the permanent downward modulation of genetic engineering modified one-tenth, preferably by cutting off gene, most preferably by making all or part of disappearance of gene promoter and/or open reading frame.
When the above-mentioned immunogenic composition of the present invention derives from the meningococcal B bacterial strain, also preferably remove capsular polysaccharide (it also comprises people-sample sugar structure).Although a lot of genes can be cut off to realize this purpose, but the inventor advantageously show preferably the bleb producing bacterial strain has been carried out genetic engineering modified functioning gene product with permanent downward modulation siaD gene expression (namely, the activity of downward modulation α-2-8 Polysialic acid transferring enzyme), preferably by cutting off gene, most preferably by making all or part of disappearance of gene promoter and/or open reading frame.This deactivation is described in WO 01/09350.SiaD (also being known as synD) sudden change is best in the various mutations; can remove the people of capsular polysaccharide-similar epi-position; this is because a kind of not effect of biosynthesis to the LOS protective epitope is only arranged in the sudden change; therefore in the method for final use LOS as protective antigen, be favourable, and minimum to the effect of bacterial growth.Therefore preferred aspect of the present invention is above-mentioned bleb immunogenicity goods, and it derives from lgtE
-SiaD
-, lgtA
-SiaD
-Or preferred lgtB
-SiaD
-The meningococcal B mutant.Described bacterial strain itself is another aspect of the present invention.
Although siaD for above-mentioned reasons
-Sudden change is preferred, cuts off other synthetic sudden change of meningococcal B capsular polysaccharide but also can use.Therefore, the bleb producing bacterial strain can be carried out the genetic engineering modified expression that derives from the functioning gene product of following one or more genes with permanent downward modulation: ctrA, ctrB, ctrC, ctrD, synA (being equivalent to synX and siaA), synB (being equivalent to siaB) or synC (being equivalent to siaC) gene, preferably by cutting off gene, most preferably by making all or part of disappearance of gene promoter and/or open reading frame.LgtE
-Sudden change can with one or more combinations of these sudden changes.Preferred lgtB
-One or more combinations of sudden change and these sudden changes.Therefore the present invention is above-mentioned bleb immunogenicity goods on the other hand, and it derives from this combinatorial mutagenesis strain of meningococcal B.Described bacterial strain itself is another aspect of the present invention.
Contain various lgt genes, comprise that the Neisseria gonorrhoeae locus of lgtB and lgtE and sequence thereof are known in the artly (to see the people such as M.P.Jennings, Microbiology 1999,145,3013-3021 and the list of references of wherein quoting as proof, and J.Exp.Med.180:2181-2190[1994]).
When total length (not-truncate) LOS is used for finished product, wish that LOS is not by sialylated (because this LOS can produce anti-most immunoreation dangerous, invasive meningococcal B bacterial strain, described bacterial strain is by sialylated yet).In this case, the pod membrane negative strain that use has disappearance synA (being equivalent to synX and siaA), synB (being equivalent to siaB) or synC (being equivalent to siaC) gene is favourable, and this is because this sudden change also can cause the menB LOS can not be by sialylated.
In the bleb goods, particularly in the goods that extract with low concentration DOC, can use LPS as the antigen in the immunogenic composition of the present invention.Yet, can be advantageously with lgtE, lgtA (particularly with the lgtC combination) or the preferably enzyme function downward modulation/disappearance/deactivation of lgtB gene/gene outcome, in order to remove proper manners breast-N-neotetraose structure.The Neisseria gonorrhoeae locus (and sequence) that comprises for the lgt gene of biosynthesis LPS oligosaccharide structure is the (people such as Jennings known in the art, Microbiology 1999 145:3013-3021 and the list of references of wherein quoting as proof, and J.Exp.Med.180:2181-2190[1994]).Preferred lgtB (or functioning gene product) is reduced/is lacked, and this is because it can stay complete LPS protective epitope.
In Neisseria meningitidis serum group B bleb goods of the present invention, the downward modulation of siaD and lgtB/disappearance is preferred, (although, in meningococcal B bleb producing bacterial strain, also can use lgtB
-With ctrA
-, ctrB
-, ctrC
-, ctrD
-, synA
-(be equivalent to synX
-And siaA
-), synB
-(be equivalent to siaB
-) or synC
-(be equivalent to siaC
-) in any one combination), obtain having the bleb goods that maximum security and LPS protective epitope keep.
Another aspect of the present invention is above-mentioned bleb immunogenicity goods, and it derives from this combinatorial mutagenesis strain of meningococcal B.Described bacterial strain itself is another aspect of the present invention.
Immunogenic composition of the present invention can comprise at least 1,2,3,4 or 5 kind of little brewage of different adventitias.When comprising two or more OMV goods, at least one antigen of the present invention is raised in each OMV.This OMV goods can derive from the Neisseria gonorrhoeae bacterial strain of identical type and serum group or preferably derive from the Neisseria gonorrhoeae bacterial strain of variety classes, serum group, serotype, inferior serotype or immunologic pattern.For example, immunogenic composition can comprise the little brewage of one or more adventitias, the little brewage of one or more adventitias that it contains the LPS of immunologic pattern L2 and contains the LPS of immunologic pattern L3.L2 or L3OMV goods preferably derive from stable bacterial strain, and it has the minimum phase transformation opposite sex at LPS oligosaccharide synthetic gene seat.
Adventitia vesicle with the combination of subunits combination thing
Immunogenic composition of the present invention also can comprise subunits combination thing and adventitia vesicle.Some antigens are arranged owing to their dissolubility is particularly suitable for being included in the subunits combination thing.The example of this albumen comprises: passerby's domain of FhaB, NspA, Hsf, passerby's domain of Hap, passerby's domain of AspA, AspA, OMP85, FrpA, FrpC, TbpB, LbpB, PilQ.The little brewage of adventitia has and is selected from following at least a not synantigen, and it is reorganized rise in the adventitia vesicle: NspA, Hsf, Hap, OMP85, TbpA (height), TbpA (low), LbpA, TbpB, LbpB, NadA, TspA, TspB, PilC, PilQ, TdfH, PorB, HpuB, P2086, NM-ADPRT, MafA, MafB and PldA; And optional comprise one of LPS immunologic pattern L2 and LPS immunologic pattern L3 or both.
The immunogenic composition that the present invention is concrete
In following concrete combination, when the antigen combination was present in the bubble, this antigen combination should be raised as mentioned above.
Particularly preferred embodiment of the present invention comprises from body transport protein and ferrum acquisition albumen, more preferably Hsf and TbpA (height) and/or TbpA (low).This immunogenic composition can preferably further comprise at least a among OMP 85, FrpA, FrpC, LbpA, LbpB, Lipo28, Sibp, NMB0964, NMB0293, TspA, NadA, TspB, PilQ, FhaC, NspA, PldA, HimD, HisD, GNA1870, OspA, HlpA, FhaB, PilC, Omp26, NMB0315, NMB0995, NMB1119, TdfH, PorB, HpuB, P2086, NM-ADPRT, VapD and the Hap.All above-mentioned immunogenic compositions also can comprise one of LPS immunologic pattern L2 and LPS immunologic pattern L3 or both.
Another preferred embodiment of the present invention comprises Hsf and is selected from following at least a other antigen: FrpA, FrpC, NM-ADPRT, VapD, LbpB, LbpA, TbpB, TbpA, P2086, HpuA, HpuB, Lipo28, Sibp, Hap, AspA, IgA protease, OMP85, NspA, PilQ, HimD, HisD, GNA1870, OspA, HlpA, FhaC, NadA, PldA, TspA, TspB, TdfH, PorB and FhaB.All above-mentioned immunogenic compositions also can comprise one of LPS immunologic pattern L2 and LPS immunologic pattern L3 or both.Preferred compositions comprises Hsf and OMP85 (optional with Hap, FrpA or LbpB one or more); Hsf and Hap (optional with FrpA, LbpB or OMP85 one or more); Hsf and FrpA (optional with Hap, LbpB or OMP85 one or more); Hsf and LbpB (optional with Hap, OMP85 or FrpA one or more).Because Hsf is adhesin is again from the body transport protein, therefore particularly preferred combination comprises Hsf, OMP85, TbpA, LPS immunologic pattern L2 and/or L3, and preferably in multivalence bleb goods, member of all 5 groups of antigens that provide is provided for it.Preferred TbpA (low) and TbpA (height) all exist.
Another immunogenic composition of the present invention comprises FhaB and is selected from following at least a other antigen: FrpA, FrpC, NM-ADPRT, VapD, LbpB, LbpA, TbpB, HpuA, HpuB, P2086, Lipo28, Sibp, NMB0964, NMB0293, TdfH, PorB, PldA, Hap, IgA protease, AspA, PilQ, HimD, HisD, GNA1870, OspA, HlpA, OMP85, NspA, PilC, Omp26, NMB0315, NMB0995, NMB 1119, NadA, PldA, TbpA, Hsf, TspA and TspB, and one of LPS immunologic pattern L2 and LPS immunologic pattern L3 or both.Preferred compositions comprises FhaB and Hsf (optional with OMP85, LbpB, Hap or FrpA one or more); FhaB and OMP85 (optional with LbpB, Hap or FrpA one or more); FhaB and LbpB (optional with Hap or FrpA one or more); FhaB and Hap (optional and FrpA).Preferred compositions comprises FhaB, LbpB, Hsf (as OMP) and FrpA, and member of the whole 5 groups of antigens that provide is provided for it.
Another immunogenic composition of the present invention comprises NspA and is selected from following at least a other antigen: one of FrpA, FrpC, NM-ADPRT, VapD, LbpB, LbpA, TbpB, TbpA, HpuA, HpuB, P2086, Lipo28, Sibp, NMB0964, NMB0293, Hap, OMP85, PilQ, AspA, IgA protease, NadA, PldA, Hsf, Hap, TspA, TspB, TdfH, PorB and LPS immunologic pattern L2 and LPS immunologic pattern L3 or both.Preferred compositions comprises NspA and Hsf (optional with OMP85, Hap, LbpA or TbpA one or more); NspA and OMP85 (optional with Hap, LbpA or TbpA one or more); NspA and Hap (optional with LbpA or TbpA one or more); NspA and LbpA (optional and TbpA).Particularly preferred combination comprises NspA, Hsf, TbpA, LPS immunologic pattern L2 and/or L3, and preferably in multivalence bleb goods, member of the whole 5 groups of antigens that provide is provided for it.Preferred TbpA (low) and TbpA (height) all exist.
The present invention does not have the immunogenic composition of indivedual combinations of disclosed antigen among the claimed WO of having 00/25811.Preferably, if their antigen inclusions only is comprised of (or in the bleb vaccine transferrin binding protein and NspA, poly-antigen inclusions that raise or rich only is comprised of transferrin binding protein and NspA), then this para-immunity originality compositions or vaccine are not included in the present invention, yet can comprise specific antigen (or the antigen that the raises) combination that is formed or comprised NspA and TbpA (height) and TbpA (low) by NspA and TbpA (height) and TbpA (low).Randomly, there be not claimed compositions or the vaccine that comprises the combination (subunit) of transferrin binding protein and NspA or raise (bleb).
Another immunogenic composition of the present invention comprises NadA and is selected from following at least a other antigen: FrpA, FrpC, NM-ADPRT, VapD, LbpB, LbpA, TbpB, TbpA, P2086, Lipo28, Sibp, NMB0964, NMB0293, Hap, OMP85, NspA, PilQ, HimD, HisD, GNA1870, OspA, HlpA, HpuA, HpuB, AspA, IgA protease, PldA, Hsf, TspA, TspB, TdfH, PorB, and one of LPS immunologic pattern L2 and LPS immunologic pattern L3 or both.
Another immunogenic composition of the present invention comprises TbpA (low) and is selected from following at least a antigen: FrpA, FrpC, NM-ADPRT, VapD, LbpB, LbpA, TbpB, IgA protease, NspA, HpuA, HpuB, Hap, OMP85, NspA (when making up with TbpA (height)), PilQ, HimD, HisD, GNA1870, OspA, HlpA, PilC, Omp26, NMB0315, NMB0995, NMB1119, MafA, MafB, AspA, NadA, PldA, Hsf, TspA, TspB, TdfH, PorB and haB, and one of LPS immunologic pattern L2 and LPS immunologic pattern L3 or both.Preferred compositions comprises TbpA (low) and Hsf and LbpA; TbpA (low) and OMP85 (optional with one of LbpA and Hap or both); TbpA (low) and LbpA and Hap.
Another immunogenic composition of the present invention comprises TbpA (height) and is selected from following at least a other antigen: FrpA, FrpC, NM-ADPRT, VapD, LbpB, LbpA, TbpB, Hap, OMP85, NspA (when making up with TbpA (low)), PilC, Omp26, NMB0315, NMB0995, NMB 1119, PilQ, HimD, HisD, GNA1870, OspA, HlpA, MafA, MafB, AspA, IgA protease, PldA, FhaB, NadA, PldA, Hsf, TspA, TspB, TdfH, PorB and FhaB, and one of LPS immunologic pattern L2 and LPS immunologic pattern L3 or both.Preferred compositions comprises TbpA (height) and Hsf and LbpA; TbpA (height) and OMP85 (optional with one of LbpA and Hap or both); TbpA (height) and LbpA and Hap.
Another immunogenic composition of the present invention comprises LbpA and is selected from least one following other antigen: FrpA, FrpC, NM-ADPRT, VapD, LbpB, TbpB, Hap, OMP85, NspA, PilC, Omp26, NMB0315, NMB0995, NMB1119, NadA, PldA, TbpA, Hsf, TspA, TspB, MafA, MafB, IgA protease, AspA, FhaB, PilQ, HimD, HisD, GNA1870, OspA, HlpA, TdfH, one of PorB and FhaB and LPS immunologic pattern L2 and LPS immunologic pattern L3 or both.Preferred compositions comprises LbpA and Hsf (optional and Hap).
Another immunogenic composition of the present invention comprises LbpB and is selected from least one following other antigen: FrpA, FrpC, NM-ADPRT, VapD, LbpA, TbpB, Hap, OMP85, NspA, PilC, Omp26, NMB0315, NMB0995, NMB1119, NadA, PldA, TbpA, Hsf, TspA, TspB, MafA, MafB, IgA protease, AspA, FhaB, PilQ, HimD, HisD, GNA1870, OspA, HlpA, TdfH, PorB and FhaB, and one of LPS immunologic pattern L2 and LPS immunologic pattern L3 or both.Preferred compositions comprises LbpB and Hsf (optional with OMP85, Hap or FrpA one or more); LbpB and OMP85 (optional with Hap or FrpA one or more); LbpB and Hap (optional and FrpA).
Another immunogenic composition of the present invention comprises OMP85 and is selected from least one following other antigen: FrpA, FrpC, NM-ADPRT, VapD, LbpB, LbpA, TbpB, TbpA, HpuA, HpuB, P2086, Lipo28, Sibp, NMB0964, NMB0293, Hap, IgA protease, AspA, Hsf, NspA, PilC, Omp26, NMB0315, NMB0995, NMB1119, MafA, MafB, NadA, PldA, Hsf, TspA, TspB, PilQ, TdfH, PorB and FhaB, and one of LPS immunologic pattern L2 and LPS immunologic pattern L3 or both.Preferred compositions comprises OMP85 and Hsf (optional with LbpA or NspA one or more); OMP85 and LbpA (optional with Hap and NspA one or more); OMP85 and Hap (optional and NspA).
Another immunogenic composition of the present invention comprises Hap and is selected from least one following other antigen: FrpA, FrpC, NM-ADPRT, VapD, LbpB, LbpA, TbpB, TbpA, HpuA, HpuB, P2086, Lipo28, Sibp, NMB0964, NMB0293, PilQ, HimD, HisD, GNA1870, OspA, HlpA, NspA, IgA protease, AspA, OMP85, NspA, PilC, Omp26, NMB0315, NMB0995, NMB1119, MafA, MafB, NadA, PldA, Hsf, TspA, TspB, TdfH, PorB and FhaB, and one of LPS immunologic pattern L2 and LPS immunologic pattern L3 or both.
Another immunogenic composition of the present invention comprises FrpA and is selected from least one following other antigen: LbpB, LbpA, TbpA, TbpB, HpuA, HpuB, P2086, Lipo28, Sibp, NMB0964, NMB0293, PilQ, HimD, HisD, GNA1870, OspA, HlpA, TspA, TspB, Hap, IgA protease, AspA, NadA, FhaB, PilQ, HimD, HisD, GNA1870, OspA, HlpA, OMP85, NspA, PilC, Omp26, NMB0315, NMB0995, NMB1119, MafA, MafB, PldA, Hsf, TspA, TspB, TdfH, PorB and FhaB, and one of LPS immunologic pattern L2 and LPS immunologic pattern L3 or both.
Another immunogenic composition of the present invention comprises FrpC and is selected from least one following other antigen: LbpB, LbpA, TbpA, TbpB, HpuA, HpuB, P2086, Lipo28, Sibp, NMB0964, NMB0293, PilQ, HimD, HisD, GNA1870, OspA, HlpA, TspA, TspB, Hap, IgA protease, AspA, NadA, FhaB, OMP85, NspA, PilC, Omp26, NMB0315, NMB0995, NMB1119, MafA, MafB, PldA, Hsf, TspA, TspB, TdfH, PorB and FhaB, and one of LPS immunologic pattern L2 and LPS immunologic pattern L3 or both.
Another immunogenic composition of the present invention comprises one of LPS immunologic pattern L2 and LPS immunologic pattern L3 or both and is selected from least one following other antigen: LbpB, LbpA, TbpA, TbpB, HpuA, HpuB, P2086, Lipo28, Sibp, NMB0964, NMB0293, PilQ, HimD, HisD, GNA1870, OspA, HlpA, TspA, TspB, Hap, IgA protease, AspA, NadA, FhaB, OMP85, NspA, PilC, Omp26, NMB0315, NMB0995, NMB1119, MafA, MafB, PldA, Hsf, TspA, TspB, TdfH, PorB and FhaB.
The combination of preferred antigens in the immunogenic composition of the present invention comprise following combination, this combination comprise ferrum obtain albumen, from body transport protein and FhaB; Ferrum obtains albumen, from body transport protein and PilC; Ferrum obtains albumen, from body transport protein and NadA; Ferrum obtains albumen, from body transport protein and FrpA; Ferrum obtains albumen, from body transport protein and PilQ; Ferrum obtains albumen, from body transport protein and TspA; Ferrum obtains albumen, from body transport protein and TspB; Ferrum obtains albumen, from body transport protein and NspA; Ferrum obtains albumen, from body transport protein and FrpC; More preferably comprise ferrum obtain albumen, from body transport protein and Hap; Ferrum obtains albumen, from body transport protein and FrpA/C; Ferrum obtains albumen, from body transport protein and LbpB; Ferrum obtains albumen, from body transport protein and OMP85 (D15).Most preferably, mix OMP85 (D15) as the part of the little brewage of adventitia.
The immunogenic composition of the present invention that comprises LPS preferably has and T-helper epitopes source, the LPS that optimization protein is puted together, and with regard to the LPS among the OMV, preferred outer membrane protein.Particularly preferred embodiment comprises the LPS that (as mentioned above) original position (in the preferred bleb) is puted together in adventitia vesicle goods with OMP.
Immunogenic composition of the present invention can comprise the antigen (albumen, LPS and polysaccharide) that derives from Neisseria meningitidis serum group A, B, C, Y, W-135 or Diplococcus gonorrhoeae.
Preferred immunogenic composition of the present invention or vaccine be can't help any single combination that embodiment 1-11 describes among the particular combinations of listed SEQ ID in WO00/71725 page 3 the 18th row-the 52nd page the 2nd row table and/or the WO00/71725 and are formed and/or contain these combinations.
Preferably, the present invention does not require disclosed indivedual combinations among the protection WO01/52885.
Other combination
Immunogenic composition of the present invention also can comprise bacterial capsule polysaccharide or oligosaccharide.Capsular polysaccharide or oligosaccharide can derive from following one or more: Neisseria meningitidis serum group A, C, Y and/or W-135, hemophilus influenza b (Haemophilus influenzae b), streptococcus pneumoniae (Streptococcus pneumoniae), A group B streptococcus, B group B streptococcus, staphylococcus aureus (Staphylococcas aureus) and staphylococcus epidermidis (Stapylocoaus epidermidis).
The present invention is the vaccine combination that contains antigenic composition of the present invention and other antigen on the other hand, and it can be advantageously used in anti-some disease condition, comprises those relevant with virus or gram positive bacteria.
Therein in preferred compositions, antigenic composition of the present invention is with 1,2,3 or preferred whole 4 kinds of preparations of following meningococcal capsular polysaccharide or oligosaccharide, its can be simply or and protein carrier: A, C, Y or W-135 put together.Preferred immunogenic composition of the present invention is with A and C; Or C; Or C and Y preparation.This containing is derived from Neisseria meningitidis, and the vaccine of the albumen of preferred serum group B can be advantageously used for general meningococcus vaccine.
In another preferred embodiment, antigenic composition of the present invention, preferably with 1,2,3 or the whole 4 kinds of preparations (as mentioned above) of simple or the meningococcal capsular polysaccharide puted together or oligosaccharide A, C, Y or W-135, be with hemophilus influenza b capsular polysaccharide or the oligosaccharide puted together, and/or one or more are simple or the pneumococcal capsular polysaccharide puted together or oligosaccharide preparation.Randomly, described vaccine also can comprise one or more proteantigens, and they can protect the host to avoid streptococcus pneumoniae infection.This vaccine can be advantageously used for general meningitis vaccines.
In another preferred embodiment, immunogenic composition of the present invention is with the capsular polysaccharide that derives from Neisseria meningitidis, hemophilus influenza b, streptococcus pneumoniae, A group B streptococcus, B group B streptococcus, golden yellow pus staphylococcus or the staphylococcus epidermidis one or more or oligosaccharide preparation.Pneumococcal capsule polysaccharide antigen is preferably selected from serotype 1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F (most preferably be selected from 1,3,4,5,6B, 7F, 9V, 14,18C, 19F and 23F).Further preferred embodiment comprises the PRP capsular polysaccharide of hemophilus influenza.Further preferred embodiment comprises 5 types, 8 types or 336 capsular polysaccharides of staphylococcus aureus.Further preferred embodiment comprises I type, II type or the III type capsular polysaccharide of staphylococcus epidermidis.Further preferred embodiment comprises Ia type, Ic type, II type or the III type capsular polysaccharide of B group B streptococcus.Further preferred embodiment comprises the capsular polysaccharide of A group B streptococcus, preferably also comprises at least a M albumen and more preferably polytype M albumen.
This capsular polysaccharide of the present invention can be non-with carrier protein such as tetanus toxoid, tetanus toxoid fragment C, diphtheria toxoid, CRM197, pneumolysin, protein D (US6342224)-and put together or put together.Described polysaccharide conjugates can be by any known coupling technology preparation.For example, can make the polysaccharide coupling via thioether bond.This conjugation methods relies on 1-cyano group-4-dimethylamino Tetrafluoroboric acid pyridine
(CDAP) activated polysaccharide and form cyanate.Activated polysaccharide therefore can be with carrier protein on amino directly coupling or via the spacer groups coupling.Preferably, use to comprise that the assorted complexation chemical process that forms thioether bond makes cyanate and hexamethylene diamine coupling, make polysaccharide and the carrier protein couplet of amino-derive.This conjugate is openly applied for describing among the WO93/15760Uniformed Services University at PCT.
Conjugate also can be by the direct-reduction amination method preparation as describing among US 4365170 (Jennings) and the US4673574 (Anderson).Other method is described in EP-0-161-188, EP-208375 and EP-0-477508.Other method comprises polysaccharide and the protein carrier coupling (people such as Chu C., Infect.Immunity, 1983245256) that makes the cyanogen bromide-activated that adipic acid hydrazides (ADH) derives by the carbodiimide condensation.When comprising oligosaccharide, they are conjugated in together.
Preferred pneumoprotein antigen is at those pneumoproteins (can be identified by host immune system at least part of biocycle of streptococcus pneumoniae) that the streptococcus pneumoniae outer surface exposes, or by streptococcus pneumoniae secretion or the albumen that discharges.Most preferably, described albumen is the lipoprotein of toxin, adhesin, 2-constituent signals transducin or streptococcus pneumoniae, or its fragment.Particularly preferred albumen comprises, but be not limited to: pneumolysin (preferably by chemical treatment or sudden change the detoxifcation) [people such as Mitchell, Nucleic Acids Res.1990 July 11: 18 (13): 4010 " Comparison of pneumolysin genes and proteins from Streptococcus pneumoniae types 1 and 2. ", the people such as Mitchell, Biochim Biophys Acta on January 23rd, 1989; 1007 (1): 67-72, " Expression of the pneumolysin gene in Escherichia coli:rapid purification and biological properties. ", WO 96/05859 (A.Cyanamid), WO 90/06951 (people such as Paton), WO 99/03884 (NAVA)]; PspA and cross-film thereof disappearance variant people such as () US 5804193-Briles; PspC and cross-film thereof disappearance variant people such as () WO 97/09994-Briles; PsaA and cross-film disappearance variant (Berry and Paton, Infect Immun in December, 1996; 64 (12): 5255-62, " Sequence heterogeneity of PsaA, a 37-kilodalton putative adhesin essential for virulence of Streptococcuspneumoniae "); Streptococcus pneumoniae choline binding protein and cross-film disappearance variant thereof; CbpA and cross-film disappearance variant (WO97/41151, WO 99/51266) thereof; GAPDH (Infect.Immun.1996 64:3544); HSP70 (WO 96/40928); PcpA (people such as Sanchez-Beato, FEMS Microbiol Lett 1998,164:207-14); M sample albumen (EP 0837130) and adhesin 18627 (EP 0834568).Further preferred pneumoprotein antigen is those that select among those disclosed among the WO98/18931, particularly WO98/18930 and the PCT/US99/30390.
Immunogenic composition/vaccine of the present invention also can be chosen wantonly and comprise by other gram negative bacteria, for example the little brewage of adventitia of morazella catarrhalis (Moraxella catarrhalis) or hemophilus influenza preparation.
Mucositis is rubbish Salmonella bleb goods not
Immunogenic composition of the present invention also can comprise the OMV goods that derive from morazella catarrhalis.As described in WO01/09350, genetic engineering modified OMV goods can derive from morazella catarrhalis.Preferably with one or more rises of following gene (coding protection antigen): OMP106 (WO97/41731 and WO96/34960); HasR (PCT/EP99/03824); PilQ (PCT/EP99/03823); OMP85 (PCT/EP00/01468); lipo06 (GB9917977.2); lipo10 (GB 9918208.1); lipo11 (GB 9918302.2); lipo18 (GB 9918038.2); P6 (PCT/EP99/03038); ompCD; CopB (people such as Helminen ME, (1993) Infect.Immun.61:2003-2010); D15 (PCT/EP99/03822); OmplAl (PCT/EP99/06781); Hly3 (PCT/EP99/03257); LbpA and LbpB (WO 98/55606); TbpA and TbpB (WO97/13785 and WO 97/32980); OmpE; UspA1 and UspA2 (WO 93/03761); and Omp21.As being incorporated into gene in other gram negative bacterias by allos, they also are preferred.
Preferably with one or more downward modulations of following gene: CopB, OMP106, OmpB1, TbpA, TbpB, LbpA and LbpB.
Preferably with one or more downward modulations of following gene: htrB, msbB and lpxK.
Preferably with one or more rises of following gene: pmrA, pmrB, pmrE and pmrF.
Hemophilus influenza bleb goods
Immunogenic composition of the present invention also can comprise the OMV goods that derive from hemophilus influenza.As described in WO01/09350, genetic engineering modified OMV goods can derive from hemophilus influenza.Preferably with one or more rises of following gene (coding protection antigen): D15 (WO94/12641), P6 (EP 281673), TbpA (WO96/40929, WO95/13370), TbpB (WO96/40929; WO95/13370), P2, P5 (WO94/26304), OMP26 (WO97/01638), HMW1, HMW2, HMW3, HMW4, Hia, Hsf, Hap, Hin47 and Hif (all genes in this operon all should be raised that pilin is raised).As being incorporated into gene in other gram negative bacterias by allos, they also are preferred.
Preferably with one or more downward modulations of following gene: P2, P5, Hif, IgAl-protease, HgpA, HgpB, HMW1, HMW2, Hxu, htrB, msbB and lpxK.
Preferably with one or more rises of following gene: pmrA, pmrB, pmrE and pmrF.
Immunogenic composition/vaccine of the present invention also can choose wantonly comprise diphtheria, tetanus and pertussis Bao Te Salmonella (Bordetella pertussis) infect in one or more antigen.Pertussis Bao Te Salmonella component can be killed, no matter be whole cell pertussis Bao Te Salmonella (Pw) or acellular pertussis Bao Te Salmonella (Pa), it comprises at least a antigen that derives from PT, FHA and 69kDapertactin (preferred 2 or all 3 kinds).Usually, providing the antigen of diphtheria and tetanus protection is diphtheria toxoid and tetanus toxoid.Toxoid is the toxin of chemical ablation or passes through to introduce point mutation and the toxin of deactivation.
Immunogenic composition/vaccine comprises optionally that also one or more protection hosts avoid the antigen of non--typing (non-typeable) hemophilus influenza, rsv infection and/or one or more can protect the host to avoid the antigen of influenza infection.This vaccine can be advantageously used for general otitis media vaccine.
Preferred typeable Haemophilus influenzae proteantigen comprises fimbrin (US 5766608) and contains fusion rotein (for example, LB1 fusion rotein) (US5843464-Ohio State Research Foundation), OMP26, P6, protein D, TbpA, TbpB, Hia, Hmw1, Hmw2, Hap and D15 from the peptide of fimbrin.
That preferred influenza antigen comprises is complete, virus that live or deactivation, influenza virus comes off, it is grown in ovum or mdck cell, or African green monkey kidney cell strain cell or complete influenza virus body are (such as R.Gluck, Vaccine, 1992,10,915-920 is described) or its purification or recombiant protein, such as HA, NP, NA or M albumen or its combination.
Preferred RSV (respiratory syncytial virus) antigen comprises F glycoprotein, G glycoprotein, HN albumen, M albumen or derivatives thereof.
Immunogenic composition of the present invention can comprise morazella catarrhalis albumen, comprises TbpA (WO97/13785; WO99/52947), TbpB (WO97/13785; WO99/52947; The people such as Mathers, FEMS Immunol Med Microbiol 1,997 19; 231-236; The people Infect Immun 1,998 66 such as Myers; 4183-4192), LbpA, LbpB (people such as Du, Infect Immun 1,998 66; 3656-3665), UspA1, UspA2 (people such as Aebi, Infect Immun.1997 65; 4367-4377), OMP106 (US6214981), Ton-B dependency receptor (WO00/78968), CopB (people such as Sethi, Infect.Immun.199765; 3666-3671) and HasR receptor (WO00/78968); The albumen of hemophilus influenza comprises HMW (people such as St Geme, Infect Immun199866; 364-368), Hia (people such as St Geme, J.Bacteriol.2000 182; 6005-6013), Tbp1 (WO96/40929; WO95/13370), Tbp2 (WO96/40929; WO95/13370; The people such as Gray-Owen, Infect Immun 1,995 63; 1201-1210), LbpA, LbpB (people such as Schryvers, 1989,29:121-130), HasR, Ton B-dependency receptor (people such as Fleishmann, Science 1,995 269; 496-512), hemoglobin-in conjunction with albumen, HhuA (people such as Cope, Infect Immun 200068; 4092-4101), HgpA (people such as Maciver, Infect Immun 199664; 3703-3712), HgbA, HgbB and HgbC (people such as Jin, Infect Immun 199664; 3134-3141), HxuA (people such as Cope, Mol Microbiol 1,994 13; 863-873), HxuC (people such as Cope, Infect Immun 200169; 2353-2363); Derive from the albumen of Neisseria meningitidis, comprise Tbp1, Tbp2, FbpA, FbpB, BfrA, BfrB (people such as Tettelin, Science 2,000 287; 1809-1815), LbpA, LbpB and HmbR.
Bacterin preparation
The preferred embodiments of the invention are the bacterin preparations that also can contain the immunogenic composition of the present invention of pharmaceutically acceptable excipient or carrier.
The preparation that derives from the little brewage of adventitia of above-mentioned any modification bacterial strain can realize by any method well known to those skilled in the art.Preferred EP 301992, the US5 of using, 597,572, disclosed method among EP 11243 or the US 4,271,147.More preferably use the method for describing among the WO01/09350.
Vaccine product is described (" The subunit and adjuvant approach " (eds Powell M.F.﹠amp usually in vaccine design; Newman M.J.) (1995) Plenum Press New York).
Can apply adjuvant to antigenic composition of the present invention in the bacterin preparation of the present invention.Suitable adjuvant comprises aluminum salt such as gel aluminum hydroxide (Alumen) or aluminum phosphate; but can also be calcium salt (particularly calcium carbonate), iron salt or zinc salt; maybe can be acidylate tyrosine or acidylate sugar, the insoluble suspension of the polysaccharide of cation or anionic derivative or polyphosphazene.
Spendable suitable Th1 adjuvant system comprise single phosphoryl lipid A, particularly 3-take off-the single phosphoryl lipid A of O-acidylate and single phosphoryl lipid A, preferred 3-take off-combination of the single phosphoryl lipid A of O-acidylate (3D-MPL) and aluminum salt (preferably phosphoric acid aluminum).The system that strengthens comprises the combination of disclosed QS21 and 3D-MPL in the combination, particularly WO 94/00153 of single phosphoryl lipid A and saponin derivative, or such as the lower compositions of disclosed reactionogenicity among the WO 96/33739, wherein uses cholesterol quencher QS21.Described effective especially, as to comprise the O/w emulsion of QS21 3D-MPL and tocopherol adjuvant among the WO 95/17210, and it is preferred preparation.
Described vaccine can comprise saponin, more preferably QS21.It also can comprise O/w emulsion and tocopherol.The unmethylated CpG (WO 96/02555) that contains oligonucleotide is the preferred derivant of TH1 reaction, and is applicable to the present invention.
Vaccine product of the present invention can be used for protection or treats susceptible mammal, wherein gives described vaccine via whole body or mucosal route.These administrations can comprise via intramuscular, intraperitoneal, Intradermal or subcutaneous route injection; Or give oral cavity/digestion, breathing, urogenital tract via mucosa.Therefore one of them aspect of the present invention is to make the human host immunity and the method for the caused disease of anti-gram positive bacterial infection, and the method comprises the OMV goods of the present invention that give described host immune protection dosage.
Select the antigen amount in each vaccine dosage form, but its induction of immunity protective reaction and do not have the obvious side effect of general vaccine.How this amount can and provide according to used specific immunogen changes.Usually each dosage form of expection comprises 1-100 μ g proteantigen or OMV goods, preferred 5-50 μ g, and the most general 5-25 μ g.
The optimum amount of specific vaccine can determine that by research on standard this research on standard comprises the suitable immunoreation of observing among the experimenter.Behind the primary vaccination, the experimenter can accept once or the booster immunization of several times appropriate intervals.
Vaccine of the present invention is immune protective and nontoxic preferably, and is suitable for child and teenager use.
When using to department of pediatrics, it refers to the child less than 4 years old.
Immune protective refers to satisfy satisfactorily above-mentioned SBA and/or animal protection model and/or adheres to the blocking-up algoscopy.
Nontoxic referring to measured by LAL and the pyrogen algoscopy known, and the activity of endotoxin level in the vaccine is not more than gratifying level.
Polynucleotide of the present invention
" polynucleotide " typically refer to any polyribonucleotide or polydeoxyribonucleotide, and it can be the RNA of unmodified or RNA or the DNA of DNA or modification." polynucleotide " include but not limited to strand and double-stranded DNA, the DNA that strand and double stranded region mix, strand and double-stranded RNA, the RNA that strand and double stranded region mix, comprise strand or, the more generally DNA that mixes with double stranded region of two strands or strand and the hybrid molecule of RNA.In addition " polynucleotide " three sequences of referring to comprise RNA or DNA or comprising simultaneously RNA and DNA.The term polynucleotide also comprise DNA or the RNA that contains one or more modified bases, and have because stability or other DNA or RNA former thereby adorned main chain." modification " base comprises, for example, and the base of tritylation and special base such as inosine.DNA and RNA various modifications have been carried out; Therefore, " polynucleotide " comprise chemistry, enzyme or the metabolism modified forms of general naturally occurring polynucleotide, and virus and the DNA of cells characteristic and the chemical species of RNA." polynucleotide " also comprise relatively short polynucleotide, are commonly referred to as oligonucleotide.
The present invention relates to immunity/bacterin preparation on the other hand, and it comprises one or more polynucleotide.This technology is known in the art, sees, and such as people such as Wolff, Science (1990) 247:1465-8.
This vaccine comprises one or more polynucleotide of the coding multiple protein corresponding with the invention described above protein combination.
The protein expression that derives from this polynucleotide will be subject to the control of eukaryotic promoter, and this promoter can drive in mammalian cell and express.Described polynucleotide can comprise the sequence of other antigen of encoding in addition.The example that can drive the eukaryotic promoter of expression comprises the viral promotors that derives from virus, comprises adenovirus promoter, reverse transcription disease virus promoter.Optionally, mammalian promoter can be used for driving expression.
Other side of the present invention
The present invention comprises the method that is used for the treatment of or prevents the Neisseria gonorrhoeae disease on the other hand, when being included in the host and needing, gives the vaccine of the present invention of its protection dosage (or effective dose).Neisseria meningitidis serum group A, B, C, Y or W135 and/or Diplococcus gonorrhoeae infect and can advantageously be prevented or treat.
The present invention also comprises the purposes of vaccine of the present invention in the medicine that infects for the preparation for the treatment of or prevention Neisseria gonorrhoeae.Neisseria gonorrhoeae infects and comprises the infection that is caused by Neisseria meningitidis serum group A, B, C, Y, W-135 and/or Diplococcus gonorrhoeae in addition.
The present invention is the genetic engineering modified Neisseria gonorrhoeae bacterial strain of adventitia vesicle of the present invention (as mentioned above, having at least two kinds of albumen that the present invention recombinates and raises) of can deriving on the other hand.This Neisseria gonorrhoeae bacterial strain can be Neisseria meningitidis or Diplococcus gonorrhoeae.
Described bacterial strain also can be transformed (as mentioned above) to reduce other Neisseria gonorrhoeae protein expression, comprises among LgtB, LgtE, SiaD, OpC, OpA, PorA, FrpB, msbB and the HtrB 1,2,3,4,5,6,7 or 8 expression.The preferred compositions that is used for downward modulation comprises the downward modulation (preferred disappearance) of at least LgtB and SiaD, at least downward modulation, at least downward modulation and at least downward modulation of PorA, OpA and OpC of PorA and OpA of PorA and OpC.
The present invention is the method for preparing immunogenic composition of the present invention or vaccine on the other hand.These methods comprise and derive from the separation antigen of Neisseria gonorrhoeae or the step that albumen mixes with at least two kinds, it can exist with the bleb form that derives from Neisseria gonorrhoeae bacterial strain of the present invention, thereby prepare immunogenic composition of the present invention, another method for preparing vaccine of the present invention comprises the step with immunogenic composition of the present invention and pharmaceutically acceptable carrier combinations.
The method for preparing immunogenic composition of the present invention that is also included among the present invention comprises the step of separating the adventitia vesicle in the Neisseria gonorrhoeae culture.This method can comprise the another step that at least two kinds of adventitia vesicle goods are mixed, and the preferred wherein little brewage of at least a adventitia comprises the LPS of immunologic pattern L2 and the LPS that the little brewage of at least a adventitia comprises immunologic pattern L3.The present invention also comprises this method, and the little brewage of wherein said adventitia is to extract by the DOC with 0-0.5% concentration to separate.The DOC of 0.3%-0.5% concentration is used for LPS content is reduced to minimum.In the OMV goods, wherein LPS guards as antigen, and 0-0.3%, preferred 0.05%-0.2%, most preferably from about the DOC of 0.1% concentration is used for extracting.
Ghost or killed whole-cell vaccines
Inventor's expection can extend to ghost or killed full cell product and vaccine (having identical advantage) at an easy rate to the above-mentioned improvement of bleb goods and vaccine.Can also can be used for preparing ghost and killed full cell product from the Gram-negative bacteria strain that its present invention who prepares the bleb goods modifies.Preparing blood shadow goods (ghost with complete adventitia) from the Gram-negative bacteria strain is (seeing for example WO 92/01791) well known in the art.Kill full cell also to know for the preparation of the method for the as killed cells goods in the vaccine.Term ' bleb [or OMV] goods ' and ' bleb [or OMV] vaccine ' and the described method of the literature therefore, for the purpose of the object of the invention, can be applied to respectively term of the present invention ' blood shadow goods ' and ' blood shadow vaccine ' and ' killed full cell product ' and ' killed whole-cell vaccines '.
Anti-strain and passive immunity
The present invention is for the preparation of the method for the immunoglobulin of prevention or the infection for the treatment of Neisseria gonorrhoeae on the other hand, comprises the step that makes the receptor immunity and separate immunoglobulin in the receptor with vaccine of the present invention.Immunoglobulin by the method preparation be this aspect on the other hand.The pharmaceutical composition that comprises immunoglobulin of the present invention and pharmaceutically acceptable carrier is another aspect of the present invention, and it can be for the preparation of the medicine for the treatment of or prevention Neisseria gonorrhoeae disease.The method that is used for the treatment of or prevents Neisseria gonorrhoeae to infect is another aspect of the present invention, and the method comprises the pharmaceutical preparation of the present invention that gives patient's effective dose.
Be used for the inoculum of polyclonal antibody generation normally by antigenic composition being dispersed in the diluent that is suitable for human physiology's tolerance of using, in normal saline or other adjuvant, prepare thereby form Aquo-composition.Give mammal with the inoculum of immunostimulation amount, then make the mammal of inoculation keep a period of time, be enough to during this period of time make antigenic composition to induce protection antibody.
Can be by the technology of knowing, such as the affinity chromatograph separation antibody to required degree (Harlow and Lane Antibodies; A laboratory manual 1988).
Antibody can comprise and derive from normally used various animal, for example, and goat, primate, donkey, pig, horse, Cavia porcellus, rat or people's antiserum goods.Give described animal blood drawing and reclaim serum.
The immunoglobulin of producing according to the present invention can comprise complete antibody, antibody fragment or subfragrnent.Antibody can be any kind, the complete immunoglobulin of IgG, IgM, IgA, IgD or IgE for example, chimeric antibody or two or more antigens of the present invention are had the hybrid antibody of dual specificity.They can also be fragments, and such as F (ab ') 2, Fab ', Fab, Fv etc. comprises hybridized fragment.Immunoglobulin also comprises natural, synthetic or genetic engineering modified albumen, and they can be by playing a role as antibody in conjunction with forming complex with specific antigen.
Can give receptor with vaccine of the present invention, then receptor plays a role as the source of immunoglobulin, and this immunoglobulin is to produce when the attack of replying specificity vaccine.Then the plasma separation method via routine obtains hyperimmune globulin from the blood plasma that the experimenter who treated like this contributes.Give another experimenter with described hyperimmune globulin, thereby produce resistance anti-or that the treatment Neisseria gonorrhoeae infects.Hyperimmune globulin of the present invention is used in particular for treating or prevent the child, non-responsiveness is individual or the needs treatment to and individual when replying immunoprophylaxis the busy Neisseria gonorrhoeae that produces antibody sick.The present invention is a kind of pharmaceutical composition on the other hand, and it comprises at least two kinds of components of immunogenic composition of the present invention are had reactive two or more monoclonal antibodies (or its fragment; Preferred people or humanized), this pharmaceutical composition can be used for treatment or prevention gram negative bacteria, preferred Neisseria gonorrhoeae, more preferably Neisseria meningitidis or Diplococcus gonorrhoeae, the most preferably caused infection of Neisseria meningitidis serum group B.
This pharmaceutical composition comprises monoclonal antibody, and they can be any kinds, the complete immunoglobulin of IgG, IgM, IgA, IgD or IgE for example, chimeric antibody or two or more antigens of the present invention are had specific hybrid antibody.They can also be fragments, and such as F (ab ') 2, Fab ', Fab, Fv etc. comprises hybridized fragment.
The method for preparing monoclonal antibody is well known in the art, and comprises splenocyte and myeloma cell's fusant (Kohler and Milstein 1975 Nature 256; 495; Antibodies-a laboratory manual Harlow and Lane 1988).Optionally, monoclonal Fv fragment can obtain by screening suitable phage display library (people such as Vaughan TJ, 1998 Nature Biotechnology 16; 535).Utilize known method, monoclonal antibody can be humanized or part is humanized.
All lists of references or the patent application of quoting as proof in the patent specification are all incorporated herein by reference.
The inventor points out that the term in each example herein " comprises (comprising) ", " comprising (comprise) " and " comprising (comprises) " can choose wantonly respectively with " by form (consisting of) ", " by form (consist of) " and " by form (consists of) " alternative.
Industry application method thereof of the present invention
Unless have a detailed description in addition, the following examples are that the use those skilled in the art know and conventional standard technique is carried out.Embodiment only illustrates, rather than limitation of the present invention.
Embodiment 1:
Make up the method for the Neisseria meningitidis serum group B that is used for the little brewage of adventitia
The method detailed that WO01/09350 is provided for preparing the adventitia vesicle and processes the bacterial isolates of the adventitia vesicle of deriving.When the adventitia vesicle remains with lipoprotein such as TbpB or lipopolysaccharide, preferably use low-level or do not use the separation method of dexycholate.
Embodiment 2:Hsf proteantigen is lacking functional cps gene but is expressing rise in the restructuring meninges class Neisseria gonorrhoeae serum group B bacterial strain of PorA
Described in WO01/09350 embodiment, in some country, the existence of PorA in the adventitia vesicle may be favourable, and can strengthen the efficacy of vaccines of restructuring improvement bubble.In the following example, we use pCMK (+) carrier of modification to raise the Hsf proteantigen to lack functional cps gene but express expression in the bacterial strain of PorA.Original pCMK (+) carrier is included in expresses lacl
qEscherichia coli host in suppressed, but be the chimeric porA/lacO promoter of transcriptional activity in Neisseria meningitidis.In the pCMK (+) that modifies, natural porA promoter is used for driving transcribing of hsf gene.The gene of coding Hsf is to use the HSF01-NdeI and the HSF 02-NheI oligonucleotide primers that provide in the following table to carry out pcr amplification.Because the sequence of HSF01-NdeI primer, 5 ' end of expressed Hsf albumen comprises two methionine residues.The used condition of pcr amplification is those (HiFi archaeal dna polymerase, Boehringer Mannheim, GmbH) that described by supplier.Thermal cycle is as follows: 25 times (94 ℃ 1 minute, 48 ℃ 1 minute, 72 ℃ 3 minutes) and 1 time (72 ℃ 10 minutes, 4 ℃ until reclaim).In pCMK (+) delivery vectors respective limits site, clone subsequently corresponding amplicon.In this recombiant plasmid, namely among designed pCMK (+)-Hsf, we make the lacO disappearance that is present in the chimeric porA/lacO promoter by the recombinant PCR strategy.PCMK (+)-Hsf plasmid is used as template so that following 2 independent dna fragmentation pcr amplifications:
-
Fragment 1Comprise that porA 5 ' lures recombination zone, kalamycin resistance gene and porA promoter.Used oligonucleotide primers RP1 (SacII) and RP2 provide in following table.RP1 primer and tight sequence homology in lac operon upstream.
-
Fragment 2Comprise the Shine-Dalgarno sequence, hsf gene and the porA 3 ' recombination zone that derive from the porA gene.Used oligonucleotide primers RP3 and RP4 (ApaI) provide in following table.RP 3 primers and tight sequence homology in lac operon downstream.It is overlapping that 3 ' 5 ' end terminal and fragment 2 of fragment 1 has 48 bases.The whole PCR that each 500ng PCR (1 and 2) is used for using primer RP1 and RP4 to carry out reacts.The whole amplicon of gained is subcloned in the pSL1180 carrier of using SacII and ApaI restriction enzyme action.Use QIAGEN maxiprep test kit large scale purification to modify plasmid pCMK (+)-Hsf, and 2 these materials of μ g are used for transforming the Neisseria meningitidis serum group B bacterial strain that lacks functional cps gene.In order to keep the expression of porA, select the integration of single exchange generation by the combination of PCR and Western blotting screening technique.To be proved to be positive kalamycin resistance by porA-specific PCR and Western blotting is cloned in-70 ℃ and stores with glycerol stock solution and to be used for further research.Antibacterial (is equivalent to about 5.10
8Individual antibacterial) be resuspended in the 50 μ l PAGE-SDS buffer, then freezing (20 ℃)/boiling (100 ℃) 3 times separates at 12.5% gel by the PAGE-SDS electrophoresis.The expression of Hsf is to derive from NmB[Cps-, PorA+] or NmB[Cps-, PorA+, Hsf+] full cell antibacterial lysate (WCBL) in detect.Coomassie dyeing can detect the remarkable increase (being equivalent to endogenous Hsf level) that Hsf expresses.It is functional and the expression that can successfully be used for raising outer membrane protein that this result confirms to modify pCMK (+)-Hsf carrier, can not eliminate the generation of main PorA Outer membrane protein antigen.
The oligonucleotide that uses in this work
Embodiment 3: utilize promoter replacement to raise the tbpA gene of Neisseria meningitidis serum group B
The purpose of experiment is the internal promoter district with stronger porA promoter replacement tbpA gene, to raise the generation of TbpA antigen.With regard to this purpose, the promoter replacement plasmid makes up with the escherichia coli cloning method.The private Incyte PathoSeq data base who is arranged in DNA district (731bp) that the tbpA coding region lists trip and is at Neisseria meningitidis strains A TCC 13090 finds.This DNA comprises the sequence of coding TbpB antigen.Described gene is organized in operon.The TbpB gene will lack and be replaced by CmR/porA promoter cartridge clip.With regard to this purpose, the dna fragmentation of the 3218bp corresponding with front 483 nucleotide of the intergenic sequence of the tbpB coded sequence of the 509bp 5 ' flanking region of t bpB gene, 2139bp, 87bp and tbpA coded sequence is oligonucleotide BAD16 (the 5 '-GGC CTA that absorbs sequence and NheI and HindIII restriction site (underscore) with containing
GCT AGCCGT CTG AAG CGA TTA GAG TTT CAA AAT TTA TTC-3 ') and BAD17 (5 '-GGC C
AA GCT TCA GAC GGC GTT CGA CCG AGT TTG AGC CTT TGC-3 ' ') increases from Neisseria meningitidis serum group B genomic DNA.With High Pure Kit (Boerhinger Mannheim, Germany) this PCR fragment of purification and with its Direct Cloning in pGemT carrier (Promega, the U.S.).Make this plasmid carry out cycle P CR mutation (Jones and Winistofer (1992)) in order to (i) insert suitable restriction site, make CmR/PorA promoter box clone and the tbpB 5 ' flanking sequence of 209bp and tbpB coded sequence are lacked.Cycle P CR is with BAD 18 (the 5 '-TCC that contain suitable restriction site XmaI, BglII and XhoI (underscore)
CCC GGGA
AG ATC TGG ACG AAA AAT CTC AAG AAA CCG-3 ') and BAD 19 (5 '-GGA
AGA TCTCCG
CTC GAGCAA ATT TAC AAA AGG AAG CCG ATA TGC AAC AGC AAC ATT TGT TCC G-3 ') oligonucleotide carries out.CmR/PorA promoter cartridge clip is to use primer BAD21 (the 5 '-GGA that contains suitable restriction site XmaI, SpeI, BglII and XhoI (underscore)
AGA TCTCCG
CTC GAGACA TCG GGC AAA CAC CCG-3 ') and BAD20 (5 '-TCC
CCC GGG AGA TCTC
AC TAG TAT TAC CCT GTT ATC CC-3 '), according to previous described from pUC D15/Omp85 plasmid amplification.This PCR fragment is cloned in the cycle P CR plasmid.This plasmid is used for transforming Neisseria meningitidis serum group B[cps-] and [cps-porA-] bacterial strain.Integrate the upstream that the porA promoter directly can be inserted into tbpA ATG by in the tbpA upstream, carrying out dual exchange.
The structure of the Neisseria meningitidis serum group B bacterial strain that the expression of 4: two kinds of antigen: TbpA of embodiment and Hsf is raised
Experiment purpose is to raise simultaneously TbpA and the expression of Hsf in same Neisseria meningitidis serum group B bacterial strain.The generation of TbpA is (promoter replacement) that is raised by using stronger its internal promoter district of porA promoter replacement.In this article, be positioned at the tbpB gene delection of tbpA upstream, and TbpB albumen is present in the adventitia no longer.The expression of Hsf is (gene delivery) that is raised by inserting second copy (homologous recombination) of corresponding gene at the porA locus.Two kinds of bacterial strains are described in the WO01/09350 independent patent.Used selected marker (Cm in two kinds of strategies
ROr Kan
R) can make two kinds to be incorporated in the same chromosome.
Extract total genomic dna by the most advanced and sophisticated 500-G scheme of Qiagen Genomic from restructuring Nm.B cps-/TbpA+/PorA+ bacterial strain.With DraIII restriction endonuclease restriction enzyme action 10 μ gDNA, and be used for transforming Neisseria meningitidis serum group B by classical conversion scheme.The cell that is used for transforming is restructuring NmB cps-/Hsf+/PorA+ (exchanging to the homologous recombination that the porA locus carries out by 1 time) or restructuring NmB cps-/Hsf+/PorA-(exchanging to the allele exchange/homologous recombination that carries out in the porA locus by 2 times).Their platings are spent the night at the GC agar that contains 200 μ g/ml kanamycin, at GC fluid medium 10mMMgCl
2In be diluted to DO
650=0.1, and under the condition that firmly stirs with the genomic DNA of 10 μ g DraIII restriction enzyme action in 37 ℃ of incubations 6 hours.The restructuring Neisseria meningitidis of the GC culture medium selection that contains 200 μ g/ml kanamycin and 5 μ g/ml chloromycetin owing to dual exchange event (PCR screening) generation, and analyze TbpA and the expression of Hsf in the OMV goods.As shown in Figure 1, compare with the OMV for preparing from contrast NmB cps-bacterial strain, TbpA and Hsf from the OMV of TbpA/Hsf restructuring NmB bacterial strain preparation produce significantly increase.The overexpression level of each albumen in dual recombinant can be compared with the expression that obtains in corresponding single recombinant.The overexpression level of TbpA and Hsf compares (not providing data) in PorA+ and PorA-bacterial strain.These data have confirmed jointly: (i) TbpA and the Hsf expression in Neisseria meningitidis can be raised jointly and together, and (ii) can obtain to be rich in the restructuring bubble of TbpA and Hsf and to be used for immunity.
The structure of the Neisseria meningitidis serum group B bacterial strain that the expression of 5: two kinds of antigen: TbpA of embodiment and NspA is raised
Experiment purpose is to raise simultaneously TbpA and the expression of NspA in same Neisseria meningitidis serum group B bacterial strain.The generation of TbpA is (promoter replacement) that is raised by using stronger its internal promoter district of porA promoter replacement.The expression of NspA is (gene delivery) that is raised by inserting second copy (homologous recombination) of corresponding gene at the porA locus.Two kinds of bacterial strains are described at independent patent WO01/09350.Used selected marker (Cm in two kinds of strategies
ROr Kan
R) can make two kinds to be incorporated in the same chromosome.
Extract total genomic dna by the most advanced and sophisticated 500-G scheme of Qiagen Genomi c from restructuring NmB cps-/TbpA+/PorA+ bacterial strain.With AatII restriction endonuclease restriction enzyme action 10 μ gDNA, and be used for transforming Neisseria meningitidis serum group B by classical conversion scheme.The cell that is used for transforming is restructuring NmB cps-/NspA+/PorA+.Their platings are spent the night at the GC agar that contains 200 μ g/ml kanamycin, at GC fluid medium 10mM MgCl
2In be diluted to DO
650=0.1, and under the condition that firmly stirs with the genomic DNA of 10 μ g AatII restriction enzyme action in 37 ℃ of incubations 6 hours.The restructuring Neisseria meningitidis of the GC culture medium selection that contains 200 μ g/ml kanamycin and 5 μ g/ml chloromycetin owing to dual exchange event (PCR screening) generation, and analyze TbpA and the expression of NspA in the OMV goods.Compare with the OMV from the preparation of contrast NmB cps-bacterial strain, the TbpA from the OMV of TbpA/NspA restructuring NmB bacterial strain preparation and NspA produce significantly and increase.The overexpression level of each albumen in dual recombinant can be compared with the expression that obtains in corresponding single recombinant.These data have confirmed jointly: (i) TbpA and the expression of Ns pA in Neisseria meningitidis can be raised jointly and together, and (ii) can obtain to be rich in the restructuring bleb of TbpA and NspA also for immunity.
The structure of the meninges class Neisseria gonorrhoeae serum group B bacterial strain that the expression of 6: two kinds of antigen: NspA of embodiment and D15/Omp85 is raised
What test is to raise simultaneously NspA and the expression of D15/Omp85 in same Neisseria meningitidis serum group B bacterial strain.The generation of D15/Omp85 is (promoter replacement) that is raised by using stronger its internal promoter district of porA promoter replacement.The expression of NspA is (gene delivery) that is raised by inserting second copy (homologous recombination) of corresponding gene at the porA locus.Two kinds of bacterial strains are described in independent patent WO01/09350.Used selected marker (Cm in two kinds of strategies
ROr Kan
R) two kinds of integration are combined in the same chromosome.
Extract total genomic dna by the most advanced and sophisticated 500-G scheme of Qiagen Genomic from restructuring NmBcps-/D15-Omp85/PorA+ bacterial strain.With AatII restriction endonuclease restriction enzyme action 10 μ gDNA, and be used for transforming Neisseria meningitidis serum group B by classical conversion scheme.The cell that is used for transforming is restructuring NmB cps-/NspA+/PorA-.Their platings are spent the night at the GC agar that contains 200 μ g/ml kanamycin, at the GC fluid medium, 10mM MgCl
2In be diluted to DO
650=0.1, and under the condition that firmly stirs with the genomic DNA of 10 μ g AatII restriction enzyme action in 37 ℃ of incubations 6 hours.The restructuring Neisseria meningitidis of the GC culture medium selection that contains 200 μ g/ml kanamycin and 5 μ g/ml chloromycetin owing to dual exchange event (PCR screening) generation, and analyze NspA and the expression of D15/Omp85 in the OMV goods.Compare with the OMV from the preparation of contrast NmB cps-bacterial strain, the NspA from the OMV of NspA/D15-Omp85 restructuring NmB bacterial strain preparation and D15/Omp85 produce significantly and increase.The overexpression level of each albumen in dual recombinant can be compared with the expression that obtains in corresponding single recombinant.These data have confirmed jointly: (i) NspA and the Omp85 expression in Neisseria meningitidis can be raised jointly and together, and (ii) can obtain to be rich in the restructuring bubble of NspA and Omp85 and to be used for immunity.
Embodiment 7: generation and the purification of restructuring Hsf form in escherichia coli
The computer analysis that the Hsf-sample albumen that derives from Neisseria meningitidis is carried out has disclosed at least 4 domains.With the Hsf sequence that derives from bacterial strain H44/76 as a reference, comprise amino acid/11-51
Domain 1Coding comprises aminoacid 52-473's from the sec-dependent signals peptide of body transport protein family characteristic
Domain 2Coding is likely surface exposed and easily enters immune passerby's domain, comprises aminoacid 474-534's
Domain 3Encoding proteins oligomeric required coiled coil domain and hinge (cervical region) district of inferring comprises residue 535-C end
Domain 4Predicted coding may be assembled into bucket-spline structure and be anchored at beta chain (people such as Henderson, (1998), Trends Microbiol.6:370-378 in the adventitia; The people such as Hoiczyk, (2000), EMBO 22:5989-5999).Because domain 2 and 3 is likely surface exposed, and finely conservative (in all bacterial strains of testing, surpass 80%; Such as people such as Pizza, (2000), Science 287:1816-1820 is described), they have represented the target vaccine candidate object.With regard to this purpose, domain 2 (being known as Hsf passerby's domain) and domain 2+3 (being known as Hsf neck regions+coiled coil domain) are at expression in escherichia coli and be purified.The dna fragmentation of coded amino acid 52-473 (Hsf passerby) and 52-534 (Hsf n+cc) is to carry out pcr amplification with the oligonucleotide that adds terminal RcaI (forward primer) and XhoI (reverse primer) restriction site.The amplicon of purification is to digest under the condition of supplier's suggestion with RcaI/XhoI; And be cloned into subsequently in the NcoI (compatible with rcaI) of pET24d (Novagen Inc., Madison WI) coli expression carrier/XhoI site.Select recombiant plasmid and for the preparation of the purification of Recombinant plasmid.When carrying out expression study, these carriers (pET-Hsf pas and pET-Hsf ncc) are incorporated among the coli strain B121DE3 (Novagen), wherein, the gene of T7 polymerase is subject to the control of isopropyl-β-D thiogalactoside (IPTG)-adjustable lac promoter.The liquid culture (700ml) of Novablue (DE3) [pET-24b/BASB029] escherichia coli recombinant bacterial strain is grown under 37 ℃ of stirrings, until the optical density of 600nm (OD600) reaches 0.6.At this time point, add IPTG to 1mM final concentration, made the culture regrowth 4 hours.Then 10, centrifugal this culture of 000rpm, will be deposited in-20 ℃ freezing at least 10 hours.After the thawing, before cytolysis, under 22 ℃, utilize the Rannie fracturer will precipitate in the 20mM phosphate buffer that (680ml culture) be resuspended to pH7.0 30 minutes by two kinds of approach.Under 4 ℃, the cell of the centrifugal dissolving of 15,000rpm (Beckman J2-HS centrifuge, JA-20 rotor) made it become precipitation in 30 minutes.In the 20mM Tris-HCl buffer that supernatant is loaded at pH8.0 on the Q-agarose rapid flow post (Pharmacia) of balance.After flowing through, with the 20mM Tris-HCl buffer washing pillar of the pH8.0 of 5 times of column volumes.Utilization is dissolved in the recombiant protein on the 250mM NaCl eluant solution post in the 20mM Tris-HCl buffer of pH8.0.Collect the antigen positive fraction, and with respect to the 20mM phosphate buffer dialysed overnight of pH7.0.Then 0.5M NaCl and 20mM imidazoles are joined in the dialysis sample.Then with sample application on Ni-NTA agarose column (Qiagen), this post is balance in the 20mM phosphate buffer of the 20mM imidazoles that contains 500mMNaCl and pH7.0.After flowing through, contain the 20mM phosphate buffer washing pillar of the pH7.0 of 500mM NaCl and 20mM imidazoles with 5 times of column volumes.With the 100mM imidazoles eluant solution pollutant in the 20mM phosphate buffer that is dissolved in pH7.0.With the 250mM imidazoles eluant solution recombiant protein in the 20mM phosphate buffer that is dissolved in pH7.0.Collect the antigen positive rank and with respect to the 10mM phosphate buffer dialysis of the pH6.8 that contains 150mM NaCl.As shown in Figure 2, rich poly-(purity among the SDS-PAGE of CBB dyeing is higher than 90% according to estimates) Hsf-sample passerby albumen of eluting from post, they are moved to about 47kDa (relative molecular weight of estimation) and locate.This polypeptide is reactive to the mouse monoclonal antibody that 5-histidine motif produces.These data show that jointly Hsf passerby and Hsf ncc gene can be with recombinant forms at expression in escherichia coli and purification.
Embodiment 8: generation and the purification of restructuring Hap passerby in large intestine bar Seedling
The computer analysis that the Hap-sample albumen that derives from Neisseria meningitidis is carried out has disclosed at least 3 domains.With the Hap-sample sequence that derives from bacterial strain H44/76 as a reference, comprise oxygen base acid 1-42's
Domain 1Coding comprises aminoacid 43-950's from the sec-dependent signals peptide of body transport protein family characteristic
Domain 2Coding is likely surface exposed and easily enters immune passerby's domain, comprises aminoacid 951-C's terminal (1457)
Domain 3Predicted coding may be assembled into bucket-spline structure and be anchored at beta chain in the adventitia.Because domain 2 is likely surface exposed, and fine conservative (in all bacterial strains of testing, surpassing 80%) and may in escherichia coli, produce as subunit antigen, so it has represented the target vaccine candidate object.Because domain 2 and 3 is likely surface exposed, and finely conservative (in all bacterial strains of testing, surpass 80%; Such as people such as Pizza, (2000), Science287:1816-1820 is described), so they have represented the target vaccine candidate object.With regard to this purpose, domain 2 (being known as Hap passerby's domain) is at expression in escherichia coli and be purified.The dna fragmentation of coded amino acid 43-950 (Hap passerby) is to carry out pcr amplification with the oligonucleotide that adds terminal NcoI (forward primer) and XhoI (reverse primer) restriction site.The amplicon of purification is to digest under the condition of supplier's suggestion with NcoI/XhoI; And be cloned into subsequently in the NcoI/XhoI site of pET24d (Novagen Inc., Madison WI) coli expression carrier.Select recombiant plasmid and large scale purification.When carrying out expression study, these carriers (pET-Hap pass) are incorporated among the coli strain B121DE3 (Novagen), wherein, the gene of T7 polymerase is subject to the control of isopropyl-β-D thiogalactoside (IPTG)-adjustable lac promoter.
In fermentation tank, cultivate e. coli bl21 [pET-Hap pass]: the aliquot fraction (100 μ l) of main inoculum is spread at FEC013AA dull and stereotyped (soya peptone A3 20g/L, yeast extract 5g/L, NaCl 5g/L, agar 18g/L, distilled water are added to 1L), and 37 ℃ of lower growths 20 hours.The results lawn also is resuspended in the sterilized water that contains 0.9%NaCl.This solution is used for batch mode at the FEC011AC of 20L fermentation tank culture medium (soya peptone 24g/L, yeast extract 48g/L, MgSO
4/ 7H
2O 0.5g/L, K
2HPO
42g/L, NaH
2PO
4/ 2H
2O0.45g/L, glycerol (87%) 40g and distilled water are added to 1L) upper inoculation.Holding temperature (30 ℃), pH (6.8, NaOH 25%/H
3PO
425%), (500mbar) is constant for pressure, divides ventilation with 20L/.In these conditions, by adjusting mixing speed (100-1000rpm) the oxygen pressure of dissolving is maintained 20%.Grow and add inducer (IPTG, 1mM) after 8 hours (OD=27.8).Collect sample (6L) behind 6 hours (OD=49.2) and the 16H 30 (OD=48.6), centrifugal harvested biomass, and at-20 ℃ of corresponding granular substances of storage.
Hap passerby's purification:
HAP passerby with batch mode from the fermentation tank purification.Developed purification flow process (below seeing).
Behind the cell rupture, in centrifugation, be recovered to a large amount of Hap passerbys.Dissolve with 8M carbamide.Although the terminal His-afterbody of N-is arranged, IMAC is invalid as the first step, still carries out at the SP-XL cation exchanger after the first step.On this SP-agarose-XL, at the quantitative eluted protein of mid point of linear NaCl (0-250mM) gradient.IMAC uses Cu
++The chelating agarose FF of-loading carries out, as FHAb.This time, opposite with FHAb, IMAC shows important purification of factor.On SDS-PAGE, HAP2/3 is seemingly pure after the IMAC.Yet under 0-200mM imidazoles gradient, the non-constant width in the peak of HAP2/3, so we attempt by imidazoles step (10mM-100mM) eluting; Yet as if with regard to purity, gradient mode is more effective.As final step, we have tested by the gel infiltration effect and have carried out carbamide-arginine buffer-exchanged, yet in this case, albumen has two eluting peaks.These two peaks demonstrate comparable distribution at SDS-PAGE; Therefore hypothesis is because HAP passerby's part refolding.Then we carry out the classics dialysis of final step for buffer-exchanged.SDS-PAGE analyzes the good purity (seeing Fig. 3) that shows whole material.HAP passerby's purity resists-further confirmation of his by WB.It is by anti--escherichia coli identification.Molecular weight is 96.1KD.
Embodiment 9: generation and the purification of restructuring FrpA/C form in escherichia coli
Two kinds of RTX albumen of Neisseria meningitidis coding, they are known as FrpA and FrpC, secrete (people such as Thompson, (1993) J.Bacteriol.175:811-818 under the limited condition of ferrum; The people such as Thompson, (1993) Infect.Immun..61:2906-2911).RTX (repetition toxin) protein family has a series of 9 the amino acid whose repetitions with following concensus sequence in the C-end near them: Leu Xaa Gly Gly Xaa Gly (Asn/Asp) AspXaa. (LXGGXGN
/ DDX).Recurring unit among the escherichia coli HlyA is considered to Ca
2+Binding site.As shown in Figure 4, the meningococcus FrpA that characterizes in bacterial strain FAM20 and FrpC albumen are at their center and the total widely amino acid similarity of C-end region, but the amino acid similarity of N-end very limited (if any).In addition, district conservative between FrpA and FrpC demonstrates some polymorphism, and this is (in FrpA 13 times, in FrpC 43 times) because the repetition of nine amino acid motif.In order to estimate the vaccine potential of FrpA and FrpC, we in escherichia coli recombinant production between FrpA and FrpC conservative protein region.With regard to this purpose, the dna fragmentation (about Neisseria meningitidis FAM20 peptide sequence) that comprises aminoacid 277-1007 is to carry out pcr amplification with forward primer FrpA-19 (5 '-CTCGAGACCATGGGCAAA TATCATGTCTACGACCCCCTCGC-3 ') and reverse primer FrpA-18 (3 '-GTGCATAGTGTCAGAGTTTTTGTCGACGTCGTAATTATAGACC-3 ') from Neisseria meningitidis serum group B H44/76 genome.Acquisition is respectively 3 amplicons of about 1530bp (3 recurring units), about 2130bp (13 recurring units) and 2732bp (23 recurring units) and uses NcoI and SalI digestion with restriction enzyme.Then these fragments are inserted in NcoI/XhoI (compatible with the Sal I) site of pET24d, and select recombiant plasmid (being respectively pET-Frp3, pET-Frp13 and pET-Frp23) to be used for transforming e. coli bl21 DE3 cell.As shown in Figure 5, all these 3 constructs all can produce restructuring FrpA/C conserved domain after inducing.In addition, the quantity that increases recurring unit can increase the dissolubility of recombiant protein, and this measures (not providing data) by the cell grade compartment analysis.
The purification that comprises the FrpA/C conserved domain (amounting to 911aa) of 23 nonapeptide LXGGXGN/DDX repetitions:Cultivate 3.5 liters of escherichia coli B121DE3[pET-Frp23] and reach at 0.6 o'clock at OD, induced 4 hours by adding 2mM IPTG.Centrifugal cell harvesting and will precipitate accordingly crushing, centrifugal clarification, and with corresponding supernatant loading at Ni
2+On-ionic metal the affinity column (Ni-NTA-agarose, Qiagen GmBh).Imidazoles is used for eluting, and finally extensively dialyses and be removed by 10mM sodium phosphate, 150mM NaCl with respect to pH6.8.
Embodiment 10: generation and the purification of restructuring FHA form in escherichia coli
The FhaB of truncate clones from Neisseria meningitidis
Genomic DNA is from 10 of Neisseria meningitidis serum group B bacterial strain H44/76 with QIAGEN genome DNA extracting reagent kit (Qiagen Gmbh)
10Extract in the individual cell.Then make this material (1 μ g) through the polymerase chain reaction dna amplification, wherein use the Auele Specific Primer of following FhaB gene: JKP:5 ' AAT GGA ATA CAT ATG AAT AAA GGT TTA CAT CGC ATT ATC3 ' and 57JKP 5 ' CCA ACT AGT GTT TTT CGC TAC TTG GAG CTG T3 '.Obtain the dna fragmentation of about 4200bp of front 1433-terminal amino acids of encoding said proteins, with NdeI/SpeI digestion with restriction enzyme and Application standard Protocols in Molecular Biology (Molecular Cloning, a Laboratory Manual, Second Edition, Eds:Sambrook, Fritsch ﹠amp; Maniatis, Cold Spring Harbor press 1989) is inserted in the corresponding site of pMG MCS (pMG derivant, Proc Natl Acad Sci USA1985 January; 82 (1): 88-92).The DNA sequence of clone FhaB fragment is measured (seeing Fig. 1) with Big Dye Cycle sequencing kit (Perkin-Elmer) and ABI 373A/PRISM DNA sequencer.Then make the polymerase chain reaction dna amplification of restructuring pMG-FhaB plasmid (1 μ g) experience, wherein use the FhaB Auele Specific Primer (XJKP035 ' AATGGAATA
CATATGAATAAAGGTTTACATCGCATTATCTTTAG3 ' and XJKP57025 ' GGGGCCA
CTCGAGGTTTTTCGCTACTTGGAGCTGTTTCAG ATAGG 3 ').Obtain the dna fragmentation of 4214bp, utilize NdeI/XhoI digestion with restriction enzyme and Application standard Protocols in Molecular Biology (Molecular Cloning, a Laboratory Manual, Second Edition, Eds:Sambrook, Fritsch ﹠amp; Maniatis, Cold Spring Harbor press 1989) be inserted in the corresponding site of pET-24b clone/expression vector (Novagen).The confirmatory sequencing that contains the restructuring pET-24b of truncate FhaB (pET24b/FhaB2/3) is to use that Big Dyes test kit (Applied biosystems) carries out and under the condition of being described by supplier, analyze in ABI 373/A DNA sequencer.The gained nucleotides sequence is listed among Fig. 6 and provides.
Expression and the purification of FhaB albumen in large intestine lever bacterium of restructuring truncate
PET24b/FhaB2/3 clone/expression vector be structured in top description.This carrier carry from the bacterial strain H44/76 of 6 histidine residues segment compositions the truncate FhaB gene (C-at recombinant product is terminal) separated, it is subject to the control of strong phage t7 gene 10 promoteres.When carrying out expression study, this carrier be introduced in coli strain Novablue (DE3) (Novagen) in, wherein, the gene of T7 polymerase is subject to the control of isopropyl-β-D thiogalactoside (IPTG)-adjustable lac promoter.The liquid culture (100ml) of Novablue (DE3) [pET24b/FhaB2/3] escherichia coli recombinant bacterial strain is grown under 37 ℃ of stirrings, until the optical density of 600nm (OD600) reaches 0.6.At this time point, add IPTG to the 1mM final concentration, and made the culture regrowth 4 hours.Then 10, the centrifugal culture of 000rpm and will be deposited in-20 ℃ freezing at least 10 hours.After the thawing, under 25 ℃ with pellet resuspended in buffer A (6M guanidine hydrochloride, 0.1M NaH
2PO
4, 0.01M Tris, pH 8.0) in 30 minutes, 3 times by pin, and centrifugal clarification (20000rpm, 15 minutes).Then with 1ml/ minute flow velocity sample is loaded to Ni
2+On the Hitrap post of-loading (Pharmacia Biotech).After flowing through, with 40ml buffer B (8M carbamide, 0.1MNaH
2PO
4, 0.01M Tris, pH 8.0), 40ml buffer C (8M carbamide, 0.1M NaH
2PO
4, 0.01M Tris, pH 6.3) the continuous washing pillar.Then the buffer D (8M carbamide, the 0.1M NaH that contain the 500mM imidazoles with 30ml
2PO
4, 0.01M Tris, pH 6.3) the recombiant protein FhaB2/3/His6 on the post is eluted, and collect the fraction of 3ml-size.As shown in Figure 7, move to the rich poly-FhaB-2/3/His6 albumen of height of about 154kDa (relative molecular weight of estimation) from the post under the eluting.This polypeptide is reactive to the mouse monoclonal antibody of 5-histidine motif.These data show that jointly FhaB2/3 can be with recombinant forms at expression in escherichia coli and purification.
With the restructuring FhaB2/3/His to mouse immune
To be expelled to (10 animal/groups) in the Balb/C mice at the 0th, 14 and 29 day three times at the partially purified restructuring FhaB2/3/His6 albumen of expression in escherichia coli.Inject to animal by subcutaneous route with two kinds of different preparations with about 5 μ g antigens: perhaps be adsorbed on 100 μ gAlPO
4Upward or in the SBAS2 emulsion prepare (the SB62 emulsion of each dosage contains 5 μ gMPL and 5 μ gQS21).In addition, also in experiment, add the negative control group that forms with the mice of SBAS2 emulsion immunity by only.The 29th day (behind the II 15 days) and the 35th (behind III 6 days) to the mice blood sampling with detection specificity anti--FhaB antibody.The specificity of measuring pooled serum (10 mice/groups) by purification of Recombinant FhaB2/3/His being carried out ELISA resists-FhaB antibody.
Embodiment 11: the mice that anti-FHA, Hap and Hsf antigen produce and the adhesion of rabbit anteserum blocking-up are active
Before be described to important toxicity determiner and mediated pertussis Bao Te Salmonella (FHA) and the bacterial adhesion of hemophilus influenza (Hap and Hsf) with the albumen of meningococcus FHAB-sample, Hsf-sample and Hap-sample homology.Known adhesion with epithelium and endotheliocyte is most important for blood brain barrier is settled down and penetrated to meningococcal nasopharynx.Therefore, hinder meningococcus a kind of of great value method that provides is provided, settle down and infect with the control meningococcus.Here we have tested for meningococcus FHAB2/3
Rd, Hap-sample and Hsf-sample antigen anti--whether serum can hinder the adhesion of Neisseria meningitidis and endotheliocyte.Use following experimentation:
The inhibition that HUVEC adheres to:In this research used meningococcus test strain be bacterial strain NmA8013 without pod membrane, without Opa-and the Opc-derivant of pili.Under 37 ℃, meningococcus cell (the 2.10E5 colony forming unit (CFU) of NmA8013 derivant) is cultivated 30 minutes with test adhesion barrier in the culture medium that is made of 40 μ lRPMI, 50 μ l hyclones and 50 μ l serum.Then this mixture is placed in the hole that contains human umbilical vein's endotheliocyte (HUVEC) confluent monolayer, is removed before the culture medium wherein.At 37 ℃, 5%CO
2Lower culture of bacteria and HUVEC cell 4 hours.Then use fresh RPMI serum washed cell monolayer 3 times, subsequently strike-off stick.Then the CFU relevant with HUVEC measured in serial dilution and cell lysates is seeded on the GC flat board.37 ℃ of culture plates 48 hours are to reclaim and to make the growth of cell related brain meningococcus.
The anti-restructuring FHAB2/3 of mice and rabbit anteserum Rd , Hap and hsf antigen adhesion-blocking-up is active: anti--FHA 2/3, anti--Hsf total length (described in the WO99/58683) and anti--Hap total length (WO99/55873) antibody, and can hinder meningococcus and endothelium HUVEC cell adhesion for the resisting of corresponding Hsf and Hap passerby's domain-serum.Fig. 8 illustrates with independent adjuvant and compares, by at AlPO
4The specific antibody that the FHA 2/3 of middle preparation induces can suppress Neisseria meningitidis B and HUVEC cell adhesion.Compare (not having antigen, 4 groups) with independent SBAS2 adjuvant, anti--FHA 2/3abs (SBAS2 preparation) remains effectively, is lower than AlPO but render a service
4Independent SBAS2 adjuvant (not having antigen) can not be induced the antibody that can hinder adhesion.Compare with 4 groups, anti--Hap antibody (1 group) may have slight inhibitory action.In 5 groups, when testing the mixture of anti--FHA 2/3, anti--Hsf and anti--Hap antibody, the inhibition of adhesion is better than independent resisting-FHA 2/3, shows that anti--Hap and anti--Hsf antibody have produced synergism.Suppress to compare (3 and 4 groups) with negative control in the experiment (Fig. 2) at second group, can partly suppress Neisseria meningitidis B for the specificity rabbit anti-serum of anti-OMV overexpression Hsf (as candidate albumen) and be fixed on the endotheliocyte.This rabbit anti-serum be proved comprise very high specificity anti--the Hsf antibody titer.Antibody for rec Hsf (Hsf passerby and Hsf total length) also can the adhesion of anti-bacteria on the HUVEC cell.All be such with mice serum (5-6 group) with rabbit anteserum (laser expansion (laserextend)) (7-8 group).In this second experiment, confirmed the specificity in first experiment, tested anti--rec FHA 2/3 antibody (1 group) has very high inhibitory action.These results show these specific antigens (Hap, FHA2/3 and Hsf), and the still combination that no matter separates all is the target vaccine antigen.
Embodiment 12: the protective effect of restructuring OMV in the mice attack model
In the Balb/C mice, estimate the protective effect of some restructuring OMV after deadly attack.After this active immne model is included in and carries out a series of immunity by subcutaneous route, will derive from meningococcus (being suspended in the iron deficiency TSB culture medium) peritoneal injection of some bacterial strains in grow up Balb/C or OF1 mice (6-8 week is large).It is necessary and cause infection animal dead seemingly to keep bacteremia as the ferridextran in outside ferrum source.Although but this mice IP model has demonstrated the effect in infection of effective evaluation virulence, immunoprotection and ferrum, they can not enter the pharynx fortune stage that occured before human bacteremia and meningitis.This model has been used for some OMV material standed fors of screening overexpression NspA, TbpA or Hsf.In following experiment, at the 0th, 14 and 28 day, be at Al (OH) with the rec OMV of 3 (PV00N049)-5 μ g (PV00N035 and PV00N043 experiment) overexpression Hsf, NspA or TbpA to Balb/C (inbreeding) or 3 OMV of OF1 (outbreeding) mouse immune inoculation by subcutaneous route
3(100 μ g Al (OH)
3/ animal) (PV00N035 and PV00N043) or at AlPO
4(100 μ g A1PO
4/ animal) upward prepare.Then (behind the III 7 days) gave described animal blood taking the 28th day (behind the II 14 days) and the 35th day, was used for specificity Ab and estimated.The 35th day, in attack front 1 hour, peritoneal injection 10mg ferridextran.Attack about 1.10e7CFU/ animal (seeing accurate challenge dose result's table) with H44/76 (B:15:P1.7,16) or CU-385 (B:4:P1.19,15) bacterial strain.The allos bacterial strain that uses the CU-385 bacterial strain to carry out is more strict than the use homologous strain.Record 1-5 days mortality rate.Table 1 afterwards illustrates with the middle OMV porA (-) of less expansion (lesser extend) and OMV porA (+) and compares, and uses OMV TbpA (+) (1/10 and 3/5 porA (-) and 9/10 and 3/5 porA (+)), OMV NspA (+) (4/10 and 4/5) and OMVHsf (+) (3/10,2/10 and 3/5) to observe better protection.This is the observed result that we obtain in these three experiments jointly.These Data supports be important at TbpA, Hsf and the NspA antigen of bleb surface expression to following menB vaccine.
Table 1: the prolection in the restructuring adventitia vesicle mouse model. this table has been summarized the result (PV00N35, PV00N043 and PV00N049) who obtains in 3 experimentations
OMVs (bleb)
NT: not test
Embodiment 13: the protective effect of recombinant subunit antigen in the mice attack model
Cause death after the attack, in the Balb/C mice, estimate the protective effect of some restructuring purifying proteins.After this active immne model is included in and carries out a series of immunity by subcutaneous route, will derive from meningococcus (being suspended in the TSB culture medium of the iron deficiency) peritoneal injection of some bacterial strains in grow up Balb/C or OF1 mice (6-8 week is large).
It is necessary and cause infection animal dead seemingly to keep bacteremia as the ferridextran in outside ferrum source.Although but this mice IP model has demonstrated the effect in infection of effective evaluation virulence, immunoprotection and ferrum, they can not enter the pharynx fortune stage that occurs before human bacteremia and meningitis.This model has been used for screening some menB subunit vaccine material standed fors, such as restructuring FrpC, TbpA, FHA2/3 and Hap molecule.
In this experiment, at the 0th, 14 and 28 day, by subcutaneous route, there are being 10 μ g MPL (every animal) when existing, to be used in AlPO
4These albumen of 5 μ g (PV00N050 experiment) of (100 μ g) upper preparation are to OF1 (outbreeding) mouse immune inoculation 3 times.
Then (behind the III 7 days) gave described animal blood taking the 28th day (behind the II 14 days) and the 35th day, was used for specificity Ab and estimated, and attacked them at the 35th day simultaneously.Attack that day, in attack front 1 hour, peritoneal injection 10mg ferridextran.Attack with CU-385 (B:4:P1.19,15) bacterial strain, it is allos in this case, in fact, except the TbpA sequence from the B16B6 bacterial strain (B:2a:P1.2), described antigen sequence is all from H44/76 (B:15:P1.7,16).
The listed result of table 2 shows in this model, FrpC, TbpA, FHAB2/3
Rd, Hap induces remarkable protection: after the attack, have 2-4 only to survive in 5 mices, and only use only 1/5 survival of adjuvant.Except one group, in all groups, tire all higher (specificity anti--TbpA tire moderate) of specific antibody.All these Data supports FrpC, the FrpA, FrpA/C conserved domain, TbpA, the FHAB2/3 that provide with subunit antigen, separation or combining form
Rd, Hap can be used for developing the menB vaccine.
Table 2: the prolection in the mouse model of restructuring adventitia vesicle. this table has been summarized therein the result (PV00N050) who obtains in the experimentation.
Subunit antigen
Embodiment 14: the synergistic method of expression vaccine antigen combination
Aspect the synergism that detects this combination of vaccine candidate object, can test alone or in combination resulting different restructuring OMV (OMVs porA (+) rmp-LbpB, OMVs porA (-) TbpA (+) Hsf (+), OMVs porA (-) TbpA (+), OMVs porA (-) NspA (+), OMVs porA (-) Hsf (+), OMVs porA (-) TbpA (+) NspA (+)) with from statistically determining best of breed.Also the combination of available subunit antigen and the combination of subunit antigen+restructuring OMV are carried out in this work.Give serum sterilizing and opsonic activity in 32 groups 5 OF1 mices/group injection and the test mouse model, active and passive protection (if needing to use time good each antigen).If the level of protection that produces after the combination immunity is higher than the summation of each antigen, then represent the synergism of antigen combination.
Embodiment 15: the Hsf of the SDS-PAGE of adventitia vesicle Coomassie blue stain and TbpA content analysis
The albumen that 15 μ g are dissolved in the little brewage of adventitia dilutes in containing the sample buffer of beta-mercaptoethanol, and wherein two quilts of Hsf or TbpA or Hsf and TbpA raise, and 95 ℃ of lower heating 10 minutes.Then make sample pass through SDS-PAGE polyacrylamide gel (Novex4-20%Tris-glycine 1.5mm 2Dwell SDS Page), and in Coomassie blue, dyeed 1 hour, and decolour by several times decolouring washing.The result represents that in Fig. 9 it shows that in the little brewage of the adventitia that derives from Neisseria meningitidis, Hsf and TbpA level are obviously higher, and wherein their expression is improved.
The immunogenicity of the OMV that embodiment 16:Hsf and/or TbpA are raised
At the 0th, 21 and 28 day, give the group immunity 3 times of 20 mices with OMV by the intramuscular approach.Each inoculation comprises that 5 μ g (protein content) are containing the AlPO of MPL
4The OMV of upper preparation.Described OMV derives from Neisseria meningitidis bacterial strain H44/76, and it is transformed into capsular polysaccharide and PorA is reduced.The OMV that Hsf wherein, TbpA, Hsf and two quilts of TbpA raise or all do not raised.At the 41st day, gather blood sample, analyze by ELISA or serum sterilizing algoscopy.
Detect the ELISA of anti-Hsf antibody
Under 4 ℃, be dissolved in specific antigen among the PBS with 100 μ l, 1 μ g/ml and cover 96 holes trace dull and stereotyped (Nunc, Maxisorb) and spend the night.After 150mM NaCl 0.05%Tween 20 washing, under the room temperature jolting, make saturated 30 minutes of flat board with the PBS-BSA of 100 μ l 1%.(jolting was carried out 30 minutes under the room temperature, and used 0.2%PBS-BSA as dilution buffer liquid) between each step, by removing excess reagent with NaCl-Tween 20 washings.In each micro-hole, add the blood serum sample that 100 μ l dilute.Binding antibody is by biotinylated resisting-mice Ig (Prosan) (1/2000) identification.Antigen-antibody complexes is by showing with streptavidin-biotinylated peroxidase conjugated thing (Amersham) (1/4000) incubation.O-phenylenediamine/H
2O
2(4mg/10ml citrate buffer 0.1M pH4.5+5 μ l H
2O
2) be used for showing and measure.Before the cessation reaction, the room temperature incubation is dull and stereotyped 15 minutes in the dark by adding 50 μ l 1N HCl.Read the absorbance at 490nm place.
Result shown in the upper table shows that the higher and equal antibody titer of anti-Hsf is to improve by the OMV immunity of all being raised with Hsf wherein or Hsf and two of TbpA.In fact, with independent adjuvant or wherein Hsf or TbpA are not all raised or only have the antibody that does not detect anti-Hsf in the serum that produces after the OMV inoculation that TbpA raised.
Embodiment 17: the sero-fast serum bactericidal activity that the OMV that anti-Hsf and/or TbpA are raised produces
Relatively with the serum bactericidal activity of the mouse resisting anteserum of OMV inoculation, wherein the Hsf among the OMV, TbpA, Hsf and two quilts of TbpA raise or are not raised in the algoscopy of using homologous strain H44/76 or allos bacterial strain Cu385.The serum sterilizing algoscopy has demonstrated with the good related of protective effect and therefore has been the good indication of candidate set compound significant degree aspect the initiation protective immunological reaction.
At 37 ℃+5%CO
2Lower, incubated overnight Neisseria meningitidis serum group B wild-type strain on MH+1%Polyvitex+1% horse serum culture dish (H44/76 bacterial strain=B:15P1.7,16L3,7,9 and CU385 bacterial strain=B:4P1.19,15L3,7,9).Under 37 ℃ of joltings, in the liquid TSB culture medium of having replenished 50 μ M Desferal (iron chelating agent), they are gone down to posterity and cultivated 3 hours, to reach about 0.5 optical density at the 470nm place.
Under 56 ℃, will mix or independent inactivating blood serum 40 minutes.1/100 dilute serum sample in 0.3%HBSS-BSA, then 2 times of serial dilutions (diluting 8 times) in the round bottom trace flat board of 50 μ l volumes.
The antibacterial of suitable OD is diluted in 0.3%HBSS-BSA, obtain 1.310e4CFU/ml.37.5 these diluents of μ l are joined in the serum dilution, and under 37 ℃ of joltings, cultivated trace dull and stereotyped 15 minutes.Then, 12.5 μ l rabbit complements are joined in each hole.37 ℃ of joltings were cultivated after 1 hour, micro-flat board were placed on stop on ice killing.
Use method of tilting, every hole 20 μ l are coated in the MH+1%Polyvitex+1% horse serum culture dish 37 ℃+CO
2Overnight incubation.The percentage ratio that calculates CFU and kill.Serum bactericidal titer is to reach 〉=the 50% last dilution factor that kills.
In two other similar experiments, to obtain to those similar results shown in the upper table.
Sterilization to homologous strain and allos tire (GMT) significantly increase and be to see after with the OMV inoculation that wherein Hsf and TbpA are raised.By relatively, the sterilization GMT that measures the mice of the OMV inoculation of raising with Hsf or TbpA with use those of contrast OMV Mice Inoculated acquisition similar.
The benefit of dual rise also can very clearly observing (tiring greater than 1/100) aspect the mice percentage ratio that produces the significant level bactericidin, particularly be used the experiment of allos bacterial strain.
Embodiment 18: mix anti--Hsf and anti--TbpA serum to the effect of bactericidal activity
At the 0th, 21 and 28 day, give the group immunity 3 times of 20 mices with OMV by the intramuscular approach.Each inoculation comprises that 5 μ g (protein content) are containing the AlPO of MPL
4The OMV of upper preparation.Described OMV derives from Neisseria meningitidis bacterial strain H44/76, and it is transformed into capsular polysaccharide and PorA is reduced.Give wherein one group of mouse immune with the contrast OMV that does not wherein have albumen to raise.In second group, Hsf expresses and is raised, and in the 3rd group, TbpA expresses and raised, and in the 4th group, the expression of Hsf and TbpA is all raised.
Pooled serum, namely or use the serum of mice on the same group or mix the serum that from the group that wherein Hsf is independent or TbpA is raised separately, separates.Measure the serum bactericidal activity of respectively organizing pooled serum, the result provides in following table.
Result in the upper table shows anti--Hsf and anti--TbpA antiserum mixed and can cause the higher serum bactericidal activity that obtains separately than any antiserum.Synergism exists seemingly owing to anti-Hsf and TbpA antibody the time and to obtain.
Embodiment 19: the Hsf albumen of truncate can with the TbpA synergistic combination
The Hsf construct of a series of truncates prepares with the standard molecular biology method.These comprise that coding comprises the amino acid/11-54 of Hsf signal sequence and the construct (TrlHsf) of Hsf amino acid/11 34-592.The Hsf of second truncate comprises the amino acid/11-53 of Hsf signal sequence, then is the aminoacid 238-592 (Tr2Hsf) of Hsf.The Hsf construct of these two truncates and total length Hsf are incorporated among Neisseria meningitidis B bacterial strain MC58 siaD-, Opc-, the PorA-their expression will be raised like this, and the adventitia vesicle is produced with said method.
The little brewage of adventitia is adsorbed on Al (OH)
3On, and be expelled in the mice at the 0th, 21 and 28 day.At the 42nd day, to mice blood sampling and preparation serum.With described serum with mixes from the serum with the mice of the TbpA OMV immunity of raising, and according to the top described serum sterilizing mensuration of carrying out.
The result
Serum bactericidal titer
Result shown in the upper table shows that for the first time truncate (TrlHsf) can cause immunoreation, its can with the antiserum combination results of anti-TbpA than using the higher serum bactericidal activity of total length Hsf.Yet the degree of truncate is very important, and the truncate that produces in Tr2 compares with total length Hsf, has illeffects.With respect to two kinds of used bacterial strains, the bactericidal activity that all can see TrlHsf improves.
Embodiment 20: the serum bactericidal activity of anti-TbpA, Hsf and the third meninges class coccus protein antibodies
The Neisseria meningitidis bacterial strain H66/76 that above-mentioned wherein PorA and capsular polysaccharide are reduced is as using above-mentioned Fang Han to raise the background bacterial strain of TbpA and Hsf, LbpB, D15, PilQ or NspA.The adventitia vesicle is from above-mentioned each bacterial strain preparation.Restructuring FHAb, FrpC, FrpA/C and Hap prepare (described in PCT/EP99/02766, WO92/01460 and WO98/02547) with previously described technology and technology known in the art.
The little brewage of adventitia and recombiant protein are adsorbed onto Al (OH)
3On, and be expelled in the mice at the 0th, 21 and 28 day.The 42nd day, to mice blood sampling and preparation serum.The serum of the OMV that anti-TbpA and Hsf are raised and the mice serum that derives from the OMV inoculation of LbpB, the D15, PilQ or the NspA that contain rise or restructuring FHAb, FrpC, FrpA/C or Hap mix, and measure according to the top described serum sterilizing that carries out.
The result
The result provides in following table.In the algoscopy of using homology H44/76 bacterial strain, except FrpC, the antibody that adds anti-tritencepehalon meningococcus antigen can not produce than the higher serum bactericidal titer of using independent anti-TbpA and Hsf antibody to produce.
Yet, be favourable in the serum sterilizing algoscopy that is added in use allos bacterial strain of anti-the third antigen-antibody.The antibody of anti-D15 (OMP85), Hap, FrpA/C and LbpB can increase the serum bactericidal titer of anti-CU385 bacterial strain especially effectively.
Serum bactericidal titer
Embodiment 21: the FrpB KO in the adventitia vesicle causes the effect of the immunoreactive ability of sterilization to them in homology and allos bacterial strain
Described according to WO01/09350, use 0.1%DOC to extract, for the preparation of the little brewage of adventitia, LOS content is approximately 20% like this with two kinds of bacterial strains of H44/76 Neisseria meningitidis.Bacterial strain B1733 is siaD (-), PorA (-), and rise and lgtB with Trl Hsf (embodiment 19) are knocked.Bacterial strain B1820 B1733 is siaD (-), PorA (-), has the rise of Trl Hsf, and lgtB is knocked, and FrpB also is knocked.Two kinds of bacterial strains all are to cultivate in the culture medium of having replenished 60 μ M Desferal, and albumen that ferrum regulates such as LbpA/B and TbpA/B are raised.
The bleb goods are adsorbed on Al (OH)
3On, and at the 0th and 21 day, in the group with 5 μ g intramuscular injection to 30 mice.Gathered blood sample at the 28th day.
Described according to embodiment 17, at 3 kinds of L3 bacterial strains (homology wild-type strain H44/76 and two kinds of allos L3 bacterial strains; NZ124 and M97250687) the enterprising promoting the circulation of blood algoscopy that sterilizes clearly.
The result
GMT represents the serum geometric mean titer among the SBA.
SC represents mice serum turnover number (SBA tire＞1/100).
Described result clearly illustrates that FrpB KO (B 1820) bleb can induce better allos intersection-bactericidal reaction than FrpB (+) bleb (B1733).In bacterial strain M97250687 and NZ124, the SBA ratio higher and the seroconversion mice of tiring is higher.When FrpB lacked, the result of homology bacterial strain was not fine.These data show that FrpB drives immunoreation, but because this outer membrane protein is alterable height, the antibody of therefore anti-this albumen only can be induced and be killed homologous strain.
Claims (91)
1. one kind comprises two or more not immunogenic compositions of synantigen, and wherein said antigen is selected from least two of following kind of apoplexy due to endogenous wind:
A) at least a Neisseria gonorrhoeae adhesin;
B) at least a Neisseria gonorrhoeae is from the body transport protein;
C) at least a Neisseria gonorrhoeae toxin;
D) at least a Neisseria gonorrhoeae Fe obtains albumen; Or
E) at least a Neisseria gonorrhoeae embrane-associated protein is preferably integrated outer membrane protein.
2. immunogenic composition according to claim 1, wherein said antigen is selected from least two of following kind of apoplexy due to endogenous wind:
A) be selected from least a Neisseria gonorrhoeae adhesin of FhaB, NspA, PilC, Hsf, Hap, MafA, MafB, Omp26, NMB0315, NMB 0995, NMB 1119 and NadA;
B) be selected from least a Neisseria gonorrhoeae of Hsf, Hap, IgA protease, AspA and NadA from the body transport protein;
C) be selected from one of FrpA, FrpC, FrpA/C, VapD, NM-ADPRT and LPS immunologic pattern L2 and LPS immunologic pattern L3 or both at least a Neisseria gonorrhoeae toxin;
D) be selected from the TbpA height, TbpA is low, TbpB is high, TbpB is low, at least a Neisseria gonorrhoeae Fe of LbpA, LbpB, P2086, HpuA, HpuB, Lipo28, Sibp, FbpA, BfrA, BfrB, Bcp, NMB 0964 and NMB 0293 obtains albumen; Or
E) be selected from least a Neisseria gonorrhoeae embrane-associated protein of PilQ, OMP 85, FhaC, NspA, TbpA (height), TbpA (low), LbpA, HpuB, TspA, TspB, TdfH, PorB, HimD, HisD, GNA 1870, OstA, HlpA, MltA, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA and PldA, preferably integrate outer membrane protein.
3. immunogenic composition according to claim 1 and 2, it is the subunits combination thing.
4. immunogenic composition according to claim 3, it comprises one of surface exposed domain, FrpA, FrpC, TbpB, LbpB, PldA, PilC, Lipo28 and the LPS immunologic pattern L2 of passerby's domain, OMP 85 of passerby's domain of being selected from FhaB, NspA, Hsf, Hap and LPS immunologic pattern L 3 or both at least 2 kinds of antigens.
5. immunogenic composition according to claim 1 and 2, it comprises the little brewage of adventitia, wherein said antigen is raised by (preferred restructuring) in the adventitia vesicle.
6. immunogenic composition according to claim 5, it is included at least two kinds of following antigens that are selected from that raised in the adventitia vesicle: NspA, Hsf, Hap, OMP 85, AspA, HpuA, HpuB, TspA, TspB, FhaC, TbpA (height), TbpA (low), LbpA, TbpB, LbpB, PilQ, NM-ADPRT, P2086, TdfH, PorB, MafA, MafB, HimD, HisD, GNA 1870, OstA, HlpA, MltA and PldA; And optional comprise one of LPS immunologic pattern L2 and LPS immunologic pattern L3 or both.
7. immunogenic composition according to claim 1 and 2, it comprises the subunits combination thing with one or more antigens, and has the little brewage of adventitia of at least a antigen that has been raised in the adventitia vesicle.
8. immunogenic composition according to claim 7, it comprises subunits combination thing and the little brewage of adventitia, wherein said subunits combination thing comprises and is selected from following at least a antigen: FhaB, NspA, passerby's domain of Hsf, passerby's domain of Hap, the surface exposed domain of OMP 85, FrpA, FrpC, TbpB, LbpB, PilC, Lipo28, and the little brewage of described adventitia have in a adventitia vesicle reorganized rise be selected from following at least a not synantigen: NspA, Hsf, Hap, OMP 85, AspA, HpuA, HpuB, TspA, TspB, FhaC, TbpA (height), TbpA (low), LbpA, TbpB, LbpB, PilQ, NM-ADPRT, P2086, TdfH, PorB, MafA, MafB, HimD, HisD, GNA 1870, OstA, HlpA, MltA and PLdA; And optional comprise one of LPS immunologic pattern L2 and LPS immunologic pattern L3 or both, preferably in adventitia vesicle goods.
9. described immunogenic composition according to claim 5-8, it comprises at least two kinds of little brewages of different adventitias of claim 5 or 6.
10. immunogenic composition according to claim 9, wherein the little brewage of a kind of adventitia is that immunologic pattern L2 and the little brewage of another kind of adventitia are immunologic pattern L3.
11. according to claim 1,2,5,6,7,8,9 or 10 described immunogenic compositions, wherein select Hsf and TbpA (height).
12. according to claim 1-2 or the described immunogenic composition of 5-11, wherein select Hsf and TbpA (low).
13. wherein also be selected from according to claim 11 or 12 described immunogenic compositions, one or more the additional antigen of Hap, LbpB, OMP 85 and FrpA.
14. described immunogenic composition is wherein also selected LPS immunologic pattern L2 according to claim 11-13.
15. described immunogenic composition is wherein also selected LPS immunologic pattern L3 according to claim 11-14.
16. described immunogenic composition according to claim 1-15, wherein select FhaB, and be selected from PilC, MafA, MafB, Omp26, NMB 0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, Hsf, LbpB, FrpA, FrpC, FrpA/C, NadA, OMP85, PldA, LbpA, TbpA (low), TbpA (height), TbpB (low), TbpB (height), HpuA, HpuB, Hap, IgA protease, AspA, PilQ, MltA, HimD, HisD, GNA1870, OstA, HlpA, NspA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 0315, NMB 1119, TdfH, PorB, NM-ADPRT, one of VapD and LPS immunologic pattern L2 and LPS immunologic pattern L3 or both at least a other antigen.
17. described immunogenic composition according to claim 1-16, wherein select NspA, and be selected from PilC, MafA, MafB, Omp26, NMB0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, Hsf, LbpB, FrpA, FrpC, FrpA/C, NadA, OMP85, PldA, LbpA, TbpA (low), TbpA (height), TbpB (low), TbpB (height), HpuA, HpuB, Hap, IgA protease, AspA, PilQ, MltA, HimD, HisD, GNA1870, OstA, HlpA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 0315, NMB 1119, TdfH, PorB, NM-ADPRT, one of VapD and LPS immunologic pattern L2 and LPS immunologic pattern L 3 or both at least a other antigen.
18. described immunogenic composition according to claim 1-17, wherein select NadA, and be selected from PilC, MafA, MafB, Omp26, NMB 0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, Hsf, LbpB, FrpA, FrpC, FrpA/C, OMP85, PldA, LbpA, TbpA (low), TbpA (height), TbpB (low), TbpB (height), HpuA, HpuB, Hap, IgA protease, AspA, PilQ, MltA, HimD, HisD, GNA1870, OstA, HlpA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 0315, NMB 1119, TdfH, PorB, NM-ADPRT, one of VapD and LPS immunologic pattern L2 and LPS immunologic pattern L3 or both at least a other antigen.
19. described immunogenic composition according to claim 1-18, wherein select TbpA (low), and be selected from PilC, MafA, MafB, Omp26, NMB 0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, Hsf, LbpB, FrpA, FrpC, FrpA/C, OMP85, PldA, LbpA, TbpA (height), TbpB (low), TbpB (height), HpuA, HpuB, Hap, IgA protease, AspA, PilQ, MltA, HimD, HisD, GNA1870, OstA, HlpA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 0315, NMB 1119, TdfH, PorB, NM-ADPRT, one of VapD and LPS immunologic pattern L2 and LPS immunologic pattern L3 or both at least a other antigen.
20. described immunogenic composition according to claim 1-19, wherein select TbpA (height), and be selected from PilC, MafA, MafB, Omp26, NMB 0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, Hsf, LbpB, FrpA, FrpC, FrpA/C, OMP85, PldA, LbpA, TbpB (low), TbpB (height), HpuA, HpuB, Hap, IgA protease, AspA, PilQ, MltA, HimD, HisD, GNA1870, OstA, HlpA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 0315, NMB 1119, TdfH, PorB, NM-ADPRT, one of VapD and LPS immunologic pattern L2 and LPS immunologic pattern L3 or both at least a other antigen.
21. described immunogenic composition according to claim 1-20, wherein select LbpB, and be selected from PilC, MafA, MafB, Omp26, NMB 0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, Hsf, FrpA, FrpC, FrpA/C, OMP85, PldA, LbpA, TbpB (low), TbpB (height), HpuA, HpuB, Hap, IgA protease, AspA, PilQ, MltA, HimD, HisD, GNA1870, OstA, HlpA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 0315, NMB 1119, TdfH, PorB, NM-ADPRT, one of VapD and LPS immunologic pattern L2 and LPS immunologic pattern L3 or both at least a other antigen.
22. described immunogenic composition according to claim 1-21, wherein select OMP85, and be selected from PilC, MafA, MafB, Omp26, NMB 0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, Hsf, FrpA, FrpC, FrpA/C, PldA, LbpA, TbpB (low), TbpB (height), HpuA, HpuB, Hap, IgA protease, AspA, PilQ, MltA, HimD, HisD, GNA1870, OstA, HlpA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 0315, NMB 1119, TdfH, PorB, NM-ADPRT, one of VapD and LPS immunologic pattern L2 and LPS immunologic pattern L3 or both at least a other antigen.
23. described immunogenic composition according to claim 1-22, wherein select Hap, and be selected from PilC, MafA, MafB, Omp26, NMB 0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, Hsf, FrpA, FrpC, FrpA/C, PldA, LbpA, TbpB (low), TbpB (height), HpuA, HpuB, IgA protease, AspA, PilQ, MltA, HimD, HisD, GNA1870, OstA, HlpA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 0315, NMB 1119, TdfH, PorB, NM-ADPRT, one of VapD and LPS immunologic pattern L2 and LPS immunologic pattern L3 or both at least a other antigen.
24. described immunogenic composition according to claim 1-23, wherein select Hsf, and be selected from PilC, MafA, MafB, Omp26, NMB 0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, FrpA, FrpC, FrpA/C, PldA, LbpA, TbpB (low), TbpB (height), HpuA, HpuB, IgA protease, AspA, PilQ, MltA, HimD, HisD, GNA1870, OstA, HlpA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 0315, NMB 1119, TdfH, PorB, NM-ADPRT, one of VapD and LPS immunologic pattern L2 and LPS immunologic pattern L3 or both at least a other antigen.
25. described immunogenic composition according to claim 1-24, wherein select FrpA, and be selected from PilC, MafA, MafB, Omp26, NMB0 995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, FrpC, FrpA/C, PldA, LbpA, TbpB (low), TbpB (height), HpuA, HpuB, IgA protease, AspA, PilQ, MltA, HimD, HisD, GNA1870, OstA, HlpA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 0315, NMB 1119, TdfH, PorB, NM-ADPRT, one of VapD and LPS immunologic pattern L2 and LPS immunologic pattern L3 or both at least a other antigen.
26. described immunogenic composition according to claim 1-25, wherein select FrpC, and be selected from PilC, MafA, MafB, Omp26, NMB 0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, PldA, LbpA, TbpB (low), TbpB (height), HpuA, HpuB, IgA protease, AspA, PilQ, MltA, HimD, HisD, GNA1870, OstA, HlpA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 0315, NMB 1119, TdfH, PorB, NM-ADPRT, one of VapD and LPS immunologic pattern L2 and LPS immunologic pattern L3 or both at least a other antigen.
27. described immunogenic composition according to claim 1-26, wherein select LPS immunologic pattern L2, and be selected from PilC, MafA, MafB, Omp26, NMB 0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, PldA, LbpA, TbpB (low), TbpB (height), HpuA, HpuB, IgA protease, AspA, PilQ, MltA, HimD, HisD, GNA1870, OstA, HlpA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 0315, NMB 1119, TdfH, PorB, NM-ADPRT, at least a other antigen of VapD and LPS immunologic pattern L3.
28. described immunogenic composition according to claim 1-27, wherein select LPS immunologic pattern L3, and be selected from PilC, MafA, MafB, Omp26, NMB0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, PldA, LbpA, TbpB (low), TbpB (height), HpuA, HpuB, IgA protease, AspA, PilQ, MltA, HimD, HisD, GNA1870, OstA, HlpA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 0315, NMB 1119, TdfH, PorB, at least a other antigen of NM-ADPRT and VapD.
29. described immunogenic composition according to claim 1-28, wherein select PilQ, and be selected from PilC, MafA, MafB, Omp26, NMB 0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, PldA, LbpA, TbpB (low), TbpB (height), HpuA, HpuB, IgA protease, AspA, MltA, HimD, HisD, GNA1870, OstA, HlpA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 0315, NMB 1119, TdfH, PorB, at least a other antigen of NM-ADPRT and VapD.
30. described immunogenic composition according to claim 1-29, wherein select HlpA, and be selected from PilC, MafA, MafB, Omp26, NMB 0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, PldA, LbpA, TbpB (low), TbpB (height), HpuA, HpuB, IgA protease, AspA, MltA, HimD, HisD, GNA1870, OstA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 0315, NMB 1119, TdfH, PorB, at least a other antigen of NM-ADPRT and VapD.
31. described immunogenic composition according to claim 1-30, wherein select MltA, and be selected from PilC, MafA, MafB, Omp26, NMB 0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, PldA, LbpA, TbpB (low), TbpB (height), HpuA, HpuB, IgA protease, AspA, HimD, HisD, GNA1870, OstA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 0315, NMB 1119, TdfH, PorB, at least a other antigen of NM-ADPRT and VapD.
32. described immunogenic composition according to claim 1-31, wherein select GNA1870, and be selected from PilC, MafA, MafB, Omp26, NMB 0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, PldA, LbpA, TbpB (low), TbpB (height), HpuA, HpuB, IgA protease, AspA, HimD, HisD, OstA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 0315, NMB 1119, TdfH, PorB, at least a other antigen of NM-ADPRT and VapD.
33. described immunogenic composition according to claim 1-32, wherein select NM-ADPRT, and be selected from PilC, MafA, MafB, Omp26, NMB 0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, PldA, LbpA, TbpB (low), TbpB (height), HpuA, HpuB, IgA protease, AspA, HimD, HisD, OstA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 0315, NMB 1119, TdfH, at least a other antigen of PorB and VapD.
34. described immunogenic composition according to claim 1-33, wherein select MafA, and be selected from PilC, MafB, Omp26, NMB 0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, PldA, LbpA, TbpB (low), TbpB (height), HpuA, HpuB, IgA protease, AspA, HimD, HisD, OstA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 0315, NMB 1119, TdfH, at least a other antigen of PorB and VapD.
35. described immunogenic composition according to claim 1-34, wherein select MafB, and be selected from PilC, MafB, Omp26, NMB 0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, PldA, LbpA, TbpB (low), TbpB (height), HpuA, HpuB, IgA protease, AspA, HimD, HisD, OstA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 0315, NMB 1119, TdfH, at least a other antigen of PorB and VapD.
36. described immunogenic composition according to claim 1-35, wherein select NMB 0315, and be selected from PilC, MafB, Omp26, NMB 0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, PldA, LbpA, TbpB (low), TbpB (height), HpuA, HpuB, IgA protease, AspA, HimD, HisD, OstA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 1119, TdfH, at least a other antigen of PorB and VapD.
37. described immunogenic composition according to claim 1-36, wherein select NMB 1119, and be selected from PilC, MafB, Omp26, NMB 0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, PldA, LbpA, TbpB (low), TbpB (height), HpuA, HpuB, IgA protease, AspA, HimD, HisD, OstA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 1119, TdfH, at least a other antigen of PorB and VapD.
38. described immunogenic composition according to claim 1-37, wherein select HisD, and at least a other antigen that is selected from PilC, MafB, Omp26, NMB 0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, PldA, LbpA, TbpB (low), TbpB (height), HpuA, HpuB, IgA protease, AspA, HimD, OstA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 1119, TdfH, PorB and VapD.
39. described immunogenic composition according to claim 1-38, wherein select LbpA, and at least a other antigen that is selected from PilC, MafB, Omp26, NMB 0995, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, PldA, TbpB (low), TbpB (height), HpuA, HpuB, IgA protease, AspA, HimD, OstA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 1119, TdfH, PorB and VapD.
40. described immunogenic composition according to claim 1-39, wherein select NMB0995, and at least a other antigen that is selected from PilC, MafB, Omp26, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, PldA, TbpB (low), TbpB (height), HpuA, HpuB, IgA protease, AspA, HimD, OstA, TspA, TspB, P2086, Lipo28, Sibp, NMB 0964, NMB 0293, NMB 1119, TdfH, PorB and VapD.
41. described immunogenic composition according to claim 1-40, wherein select Lipo28, and at least a other antigen that is selected from PilC, MafB, Omp26, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, PldA, TbpB (low), TbpB (height), HpuA, HpuB, IgA protease, AspA, HimD, OstA, TspA, TspB, P2086, Sibp, NMB 0964, NMB 0293, NMB 1119, TdfH, PorB and VapD.
42. described immunogenic composition according to claim 1-41, wherein select HimD, and at least a other antigen that is selected from PilC, MafB, Omp26, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, PldA, TbpB (low), TbpB (height), HpuA, HpuB, IgA protease, AspA, OstA, TspA, TspB, P2086, Sibp, NMB 0964, NMB 0293, NMB 1119, TdfH, PorB and VapD.
43. described immunogenic composition according to claim 1-42, wherein select NMB 1313, and at least a other antigen that is selected from PilC, MafB, Omp26, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, NMB 1953, HtrA, PldA, TbpB (low), TbpB (height), HpuA, HpuB, IgA protease, AspA, OstA, TspA, TspB, P2086, Sibp, NMB 0964, NMB 0293, NMB 1119, TdfH, PorB and VapD.
44. described immunogenic composition according to claim 1-43, wherein select NMB1953, and at least a other antigen that is selected from PilC, MafB, Omp26, FhaC, FbpA, Bcp, NMB 1124, NMB 1162, NMB 1220, HtrA, PldA, TbpB (low), TbpB (height), HpuA, HpuB, IgA protease, AspA, OstA, TspA, TspB, P2086, Sibp, NMB0964, NMB 0293, NMB 1119, TdfH, PorB and VapD.
45. described immunogenic composition according to claim 5-44, the host cell of the little brewage of adventitia of wherein deriving by engineered to reduce one or more lgtB or lgtE, preferably the former expression.
46. described immunogenic composition according to claim 5-45, the host cell of the little brewage of adventitia of wherein deriving can not synthesize capsular polysaccharide and preferably by engineered to reduce one or more siaD, ctrA, ctrB, ctrC, ctrD, synA (being equivalent to synX and siaA) or synB (being equivalent to siaB) and synC (being equivalent to siaC), the expression of preferred siaD.
47. described immunogenic composition according to claim 5-46, the host cell of the little brewage of adventitia of wherein deriving by engineered to reduce one or more OpC, OpA or PorA, the expression of preferred PorA.
48. described immunogenic composition according to claim 5-47, the host cell of the little brewage of adventitia of wherein deriving are by engineered expression with downward modulation FrpB.
49. described immunogenic composition according to claim 5-48, the host cell of the little brewage of adventitia of wherein deriving by engineered with downward modulation msbB and/or htrB, the expression of preferred msbB.
50. described immunogenic composition according to claim 5-49, the little brewage of wherein said adventitia comprises the LPS that puts together with outer membrane protein (OMP).
51. described immunogenic composition according to claim 50, wherein LPS and OMP put together at adventitia vesicle goods situ (in the preferred bubble).
52. described immunogenic composition according to claim 1-51, it comprises the antigen of deriving from Neisseria meningitidis (Neisseria meningitidis), preferred serum group B.
53. described immunogenic composition according to claim 1-52, it comprises the antigen of deriving from Diplococcus gonorrhoeae.
54. described immunogenic composition according to claim 1-52, wherein all Neisseria gonorrhoeae antigens all derive from Neisseria meningitidis, preferred serum group B.
55. described immunogenic composition according to claim 1-54, it also comprises one or more bacterial capsule polysaccharide or oligosaccharide.
56. 5 described immunogenic compositions according to claim 5, wherein said capsular polysaccharide or oligosaccharide derive from and are selected from Neisseria meningitidis serum group A, C, Y and W-135, hemophilus influenza b (Haemophilus influenzae b), streptococcus pneumoniae (Streptococcus pneumoniae), the A group B streptococcus, the B group B streptococcus, the antibacterial of staphylococcus aureus (Staphylococcas aureus) and staphylococcus epidermidis (Stapylocoaus epidermidis).
57. the described immunogenic composition of 5-56 according to claim 5, wherein said capsular polysaccharide or oligosaccharide and albumen are puted together.
58. described immunogenic composition according to claim 1-57, it comprises adjuvant.
59. 8 described immunogenic compositions according to claim 5, it comprises aluminum salt, preferably phosphoric acid aluminum.
60. 8 or 59 described immunogenic compositions according to claim 5, it comprises 3D-MPL.
61. one kind comprises the immunogenic composition of claim 1-60 and the vaccine of pharmaceutically acceptable carrier.
62. a vaccine that comprises one or more polynucleotide, two or more different albumen of described polynucleotide encoding, its expression is driven by eukaryotic promoter, and wherein said albumen is selected from least two of following kind of apoplexy due to endogenous wind:
A) be selected from least a Neisseria gonorrhoeae adhesin of FhaB, NspA, PilC, Hsf, Hap, MafA, MafB, Omp26, NMB 0315, NMB 0995, NMB 1119 and NadA;
B) be selected from least a Neisseria gonorrhoeae of Hsf, Hap, IgA protease, AspA and NadA from the body transport protein;
C) be selected from least a Neisseria gonorrhoeae toxin of FrpA, FrpC, FrpA/C, VapD and NM-ADPRT;
D) be selected from the TbpA height, TbpA is low, TbpB is high, TbpB is low, at least a Neisseria gonorrhoeae Fe of LbpA, LbpB, P2086, HpuA, HpuB, Lipo28, Sibp, FbpA, BfrA, BfrB, Bcp, NMB0964 and NMB0293 obtains albumen; Or
E) be selected from least a Neisseria gonorrhoeae embrane-associated protein of PilQ, OMP85, FhaC, NspA, TbpA (height), TbpA (low), LbpA, HpuB, TspA, TspB, TdfH, PorB, HimD, HisD, GNA1870, OstA, HlpA, MltA, NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA and PldA, preferably integrate outer membrane protein.
63. a method that is used for the treatment of or prevents the Neisseria gonorrhoeae disease is included in and gives the vaccine that it protects the claim 61-62 of dosage when the host needs.
64. 3 described methods according to claim 6, wherein Neisseria meningitidis infects and is prevented or treat.
65. 3 described methods according to claim 6, wherein Diplococcus gonorrhoeae infects and is prevented or treat.
66. the vaccine of claim 61-62 is for the preparation of the purposes of the medicine for the treatment of or the infection of prevention Neisseria gonorrhoeae.
67. 6 described purposes according to claim 6, wherein Neisseria meningitidis infects and is prevented or treat.
68. 6 described purposes according to claim 6, wherein Diplococcus gonorrhoeae infects and is prevented or treat.
69. the genetic engineering modified Neisseria gonorrhoeae bacterial strain of the little brewage of adventitia of the claim 5-60 that derives.
70. a method for preparing the immunogenic composition of claim 1-60 comprises the step that at least two kinds of antigens that derive from Neisseria gonorrhoeae are mixed.
71. a method for preparing the immunogenic composition of claim 5-60 comprises the step of separating the adventitia vesicle in the Neisseria gonorrhoeae culture.
72. 1 described method also comprises the step that at least two kinds of adventitia vesicle goods are mixed according to claim 7.
73. 2 described methods according to claim 7, wherein the little brewage of at least a adventitia comprises the LPS of immunologic pattern L2, and the little brewage of at least a adventitia comprises the LPS of immunologic pattern L3.
74. the described method of 1-73 according to claim 7, wherein said adventitia vesicle are to extract by the DOC with 0-0.5% concentration to separate.
75. 4 described methods according to claim 7, wherein said adventitia vesicle be by with 0.02%-0.4%, 0.04%-0.3%, 0.06%-0.2%, 0.08%-0.15% or preferably approximately or accurately the DOC of 0.1% concentration extract and separate.
76. a method for preparing the vaccine of claim 61 comprises the step that the immunogenic composition with claim 1-60 mixes with pharmaceutically acceptable carrier.
77. the method for an immunoglobulin that infects for the preparation of prevention or treatment Neisseria gonorrhoeae comprises that the vaccine with claim 61 makes receptor immune, and from receptor the step of separating immune globulin.
78. immunoglobulin by the method preparation of claim 77.
79. a pharmaceutical composition, it comprises immunoglobulin and the pharmaceutically acceptable carrier of claim 78.
80. one kind is used for the treatment of or prevents the method that Neisseria gonorrhoeae infects, and comprises the step of the pharmaceutical preparation of the claim 79 that gives patient's effective dose.
81. the pharmaceutical preparation of claim 79 is in the purposes for the preparation of the medicine for the treatment of or prevention Neisseria gonorrhoeae disease.
82. described immunogenic composition according to claim 5-60, it comprises that the meningococcus bubble of immunologic pattern L2 and the meningococcus of immunologic pattern L3 steep.
83. 2 described immunogenic compositions according to claim 8, wherein TbpA (height) is raised in a bubble therein.
84. 2 or 83 described immunogenic compositions according to claim 8, wherein TbpA (low) is raised in a bubble therein.
85. the described immunogenic composition of 2-84 according to claim 8, wherein Hsf is raised in a bubble therein.
86. the described immunogenic composition of 2-85 according to claim 8, wherein OMP85 is raised in a bubble therein.
87. the described immunogenic composition of 2-86 according to claim 8, wherein said bubble are from not producing capsular polysaccharide, preferred siaD
-The meningococcus bacterial strain in separate.
88. the described immunogenic composition of 2-87 according to claim 8, wherein L2 and/or L3LPS oligosaccharide structure be truncated to from lgtB
-The bubble of separating in the meningococcus bacterial strain is consistent.
89. the described immunogenic composition of 2-88 according to claim 8, wherein said bubble are to separate from the meningococcus bacterial strain that downward modulation msbB expresses.
90. the described immunogenic composition of 2-89 according to claim 8, wherein L2 and/or L3LPS oligosaccharide part is integrated in the outer membrane protein bubble with bubble and is puted together.
91. the described immunogenic composition of 2-89 according to claim 8, wherein said bubble derive from the meningococcus bacterial strain that downward modulation one or more FrpB, PorA, Opa or Opc express.
Applications Claiming Priority (22)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB0218037.0 | 2002-08-02 | ||
| GB0218036A GB0218036D0 (en) | 2002-08-02 | 2002-08-02 | Vaccine |
| GB0218035.4 | 2002-08-02 | ||
| GB0218035A GB0218035D0 (en) | 2002-08-02 | 2002-08-02 | Vaccine composition |
| GB0218051A GB0218051D0 (en) | 2002-08-02 | 2002-08-02 | Vaccine composition |
| GB0218037A GB0218037D0 (en) | 2002-08-02 | 2002-08-02 | Vaccine composition |
| GB0218036.2 | 2002-08-02 | ||
| GB0218051.1 | 2002-08-02 | ||
| GBGB0220197.8A GB0220197D0 (en) | 2002-08-30 | 2002-08-30 | Refolding method |
| GB0220197.8 | 2002-08-30 | ||
| GBGB0220199.4A GB0220199D0 (en) | 2002-08-30 | 2002-08-30 | Mutant protein and refolding method |
| GB0220199.4 | 2002-08-30 | ||
| GB0225524A GB0225524D0 (en) | 2002-11-01 | 2002-11-01 | Vaccine composition |
| GB0225524.8 | 2002-11-01 | ||
| GB0225531.3 | 2002-11-01 | ||
| GB0225531A GB0225531D0 (en) | 2002-11-01 | 2002-11-01 | Vaccine |
| GB0230168.7 | 2002-12-24 | ||
| GB0230168A GB0230168D0 (en) | 2002-12-24 | 2002-12-24 | Vaccine composition |
| GB0230170.3 | 2002-12-24 | ||
| GB0230164A GB0230164D0 (en) | 2002-12-24 | 2002-12-24 | Vaccine composition |
| GB0230164.6 | 2002-12-24 | ||
| GB0305028.3 | 2003-03-05 |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN038186489A Division CN1674933B (en) | 2002-08-02 | 2003-07-31 | Vaccine composition |
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| Publication Number | Publication Date |
|---|---|
| CN103007263A true CN103007263A (en) | 2013-04-03 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012102805434A Pending CN103007263A (en) | 2002-08-02 | 2003-07-31 | Vaccine composition |
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| Country | Link |
|---|---|
| CN (1) | CN103007263A (en) |
| BR (2) | BR0313100A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107602697A (en) * | 2017-08-29 | 2018-01-19 | 杭州医学院 | A kind of application treated haemophilus influenzae and cause the serum and the serum of nasosinusitis |
| CN107722120A (en) * | 2017-08-29 | 2018-02-23 | 杭州医学院 | A kind of application treated haemophilus influenzae and cause the serum and the serum of tympanitis |
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|---|---|---|---|---|
| WO1999031132A1 (en) * | 1997-12-12 | 1999-06-24 | The University Of Queensland | NOVEL SURFACE PROTEIN OF $i(NEISSERIA MENINGITIDIS) |
| WO2000071725A2 (en) * | 1999-05-19 | 2000-11-30 | Chiron S.P.A. | Combination neisserial compositions |
| WO2001009350A2 (en) * | 1999-08-03 | 2001-02-08 | Smithkline Beecham Biologicals S.A. | Genetically engineered bleb vaccine |
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2003
- 2003-07-31 BR BR0313100-9A patent/BR0313100A/en active Search and Examination
- 2003-07-31 BR BRPI0313120-3A patent/BR0313120A/en not_active IP Right Cessation
- 2003-07-31 CN CN2012102805434A patent/CN103007263A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999031132A1 (en) * | 1997-12-12 | 1999-06-24 | The University Of Queensland | NOVEL SURFACE PROTEIN OF $i(NEISSERIA MENINGITIDIS) |
| WO2000071725A2 (en) * | 1999-05-19 | 2000-11-30 | Chiron S.P.A. | Combination neisserial compositions |
| WO2001009350A2 (en) * | 1999-08-03 | 2001-02-08 | Smithkline Beecham Biologicals S.A. | Genetically engineered bleb vaccine |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107602697A (en) * | 2017-08-29 | 2018-01-19 | 杭州医学院 | A kind of application treated haemophilus influenzae and cause the serum and the serum of nasosinusitis |
| CN107722120A (en) * | 2017-08-29 | 2018-02-23 | 杭州医学院 | A kind of application treated haemophilus influenzae and cause the serum and the serum of tympanitis |
| CN107602697B (en) * | 2017-08-29 | 2020-05-05 | 杭州医学院 | Serum for treating nasosinusitis caused by haemophilus influenzae and application of serum |
| CN107722120B (en) * | 2017-08-29 | 2020-07-14 | 杭州医学院 | A kind of serum for treating otitis media caused by Haemophilus influenzae and application of the serum |
Also Published As
| Publication number | Publication date |
|---|---|
| BR0313100A (en) | 2005-06-21 |
| BR0313120A (en) | 2007-07-17 |
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