CN101638433B - Peptide mimics of conserved gonococcal epitopes and methods and compositions using them - Google Patents
Peptide mimics of conserved gonococcal epitopes and methods and compositions using them Download PDFInfo
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- CN101638433B CN101638433B CN200910136894.6A CN200910136894A CN101638433B CN 101638433 B CN101638433 B CN 101638433B CN 200910136894 A CN200910136894 A CN 200910136894A CN 101638433 B CN101638433 B CN 101638433B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention relates to peptide mimics of a conserved gonoccocal epitode of Neisseria gonorrhoeae, which epitope is not found on human blood group antigens. This invention also relates to methods and compositions using such peptide mimics for the prophylaxis of gonorrheal infections.
Description
The application is dividing an application of No. 00817098.3, the application for a patent for invention submitted on October 27th, 2000 " the plan peptide of conserved gonococcal epitope and apply their method and composition ".
Invention technical field
The present invention relates to the plan peptide (peptide mimics) of the conserved epitope of neisseria gonorrhoeae (Neisseria gonorrhoeae), these epi-positions are not found on Antigen of human blood group.The present invention also relates to apply these and intend the method and composition that peptide prevention gonorrhoea infects.
Background of invention
Sexually transmitted disease (STD) gonorrhoea, as one of transmissible disease of the most often reporting, causes worldwide danger.Gonorrhoea is caused by a kind of gram-negative diplococci neisseria gonorrhoeae.Although this pathogenic agent main infection mucous membrane, it can be invaded tissue and avoid host's defence.Neisseria gonorrhoeae is the pathogenic agent of multiple sequela.From the male sex and women's asymptomatic mucosal infections to obvious disease syndrome.More serious syndrome comprises, for example, and the propagated gonococcal infection in the male sex and women (" DGI "), and the salpingitis in women or pelvic inflammatory disease (" PID ").Itself can cause long-term sequela salpingitis or PID, comprises ectopic pregnancy and sterile.Other important sequela (sometimes needing surgical operation) comprises recurrent infection, chronic pelvic pain, dyspareunia, pelvic adhesion and other inflammatory residue.
According to estimates, in the U.S., the direct and overhead cost for the treatment of PID and relevant ectopic pregnancy and infertility adds up 2,600,000,000 dollars (53) for 1984.Nineteen ninety, total direct expenses is estimated to reach 21.8 hundred million dollars, and overhead cost is 15.4 hundred million dollars.The sickness rate of supposing monetary inflation and PID is constant, and total cost of this disease is estimated will reach 8,000,000,000 dollars (9) in 2000.
Although be devoted to control the applicability of gonococcal infection and effective antibiotics treatment in U.S.'s public health, annual Center for Disease Control (" CDC ") has still been reported and has been approached 315000 routine gonorrhoeas (12).In all gonorrhoea cases, have significant proportion to occur in symptomless infection individuality, they are the sources (6) of most of new cases in colony.Strains is more and more popular makes the treatment of this infection more complicated (10,11,52).
Neisseria gonorrhoeae has multiple virulence factor.The surface composition of this pathogenic agent plays an important role in adhering to and invading host cell, and provides potential target for host immune response.Gonococcal infection causes part and general body fluid and cellullar immunologic response, particularly pili, porin (" Por ") or protein I (" PI "), muddy related protein (" Opa ") or protein I I, Rmp or protein I II and the fat oligosaccharides (" LOS ") (7) that exposes several compositions of antigen to showing as bacterium surface.Pili, Opa, Por and LOS all with adhere to and to invade host relevant, its surperficial exposure zone all shows sizable variation (26,45,46).In the bacterial strain of gonococcus surface composition and bacterium inter-strain variation cause about the tissue specificity of different sites and biological infection again and the hypothesis of the potential of lasting virulence.
In having symptom and asymptomatic patient, gonococcal infection shows can stimulate anti-gonococcus serum immunoglobulin level to improve.Periphery humoral response is mainly IgG (great majority is subclass IgG3), has IgM and the IgA (13) of small amount.Quantitatively, antibody response is mainly for pili, Opa albumen and LOS.Local antibody is present in sexual organ secretory product, but content reduces (48), may be for the different antigen target (27) from serum.The main antibody isotype existing in secretion is also IgG (being mainly IgG3), rather than secretory IgA (" sIgA ") (7).Also have anti-LOS antibody, but its amount is less than the antibody of antibacterial hair, Por and Opa.Although infect the patient of neisseria gonorrhoeae, may show the antibody response to multiple gonococcus antigen, neisseria gonorrhoeae separated from propagated infection (DGI) patient has resistance (38) to the germicidal action of normal human serum (" NHS ") and most of reconvalescent's serum.This seroresistance phenotype, is called stable seroresistance (" SR "), can make bioenergy avoid local defense, through mucosal barrier, and propagates by blood flow.
After the cultivation of going down to posterity, many gonococcus bacterial strains become (38) phenotype sensitivity or serum sensitivity to killing and wounding of NHS.These biologies are called as serum sensitivity (" SS ") or unsettled seroresistance.These are biological conventionally separated from the women of the performance of local inflammation severe or clinical obvious PID.Acute salpinitis, the relative disease of pathology (being caused by SS gonococcus) of PID, seldom develops into microbemia or DGI.The violent local inflammation reaction that this prompting is caused by SS gonococcus may be enough to comprise to be infected and prevents microbemia, although will pay the cost of damage local organization.SS gonococcus produces the derivative chenotactic peptide C5a (16) of complement of remarkable volume than SR gonococcus.This can cause the inflammatory reaction of polymorphonuclear leukocyte (" the PMN ") mediation that SS gonococcus produces.
The development of the antibiotics resistance strain of neisseria gonorrhoeae makes the control of this infection more and more difficult.The potential of insufficient treatment gonococcal infection has accelerated the needs of antagonism gonococcus vaccine.The prevention of the severe complication of gonococcal infection, particularly PID has been many researchists' target.But the effort of the effective anti-gonococcus vaccine of ongoing research and development runs into many difficulties.
The individual surface composition of application pathogenic agent is not succeeded as the trial of the target of conventional vaccine, and this is the antigenic variability due to them.Pili vaccine only infects and has protectiveness homologous strain (being used for producing pili vaccine), and Por inoculation is even also unsuccessful in human experimentation is attacked.In addition, neisseria gonorrhoeae shows obvious phenotype unhomogeneity, generally with 10
3in individual biology, the frequency of > 1 converts (49,50) from a kind of antigen form to another kind, and this makes the surface of this biology become the moving target of most of vaccine schemes.Although vaccine candidate strain causes antibody response, the antibody producing and immunne response do not have protectiveness widely.
LOS is a kind of important virulence determinative of neisseria gonorrhoeae.There is considerable evidence to support LOS as the effect (2,16,18,37,47) of the main target of the bactericidin for neisseria gonorrhoeae surface.LOS antibody has several important functions: kill bacterial activity, by the complement activation (2) of classics or bypass complement pathway, and OA (16).In addition, LOS is considered to the most effective gonococcus antigen (51) that induction is replied homology and the gonococcal functional antibodies of allos.
Monoclonal antibody (" mAb ") 2C7 (30) detects the LOS that seems extensively to guard and express in gonococcus clinical separation strain and derives oligosaccharides (" OS ") epi-position.Carbohydrate is generally the antigen that does not rely on T cell.When using separately as immunogen, they only cause that primary antibody replys conventionally.In addition, oligosaccharides less (< 10Tang unit) (19), may need that other biological chemistry is derivative makes them have immunogenicity.Therefore, these oligosaccharides are restricted aspect several as the purposes of vaccine candidate object.
Once image (internal image) determinant (36) in proposing to use in vaccine.Utilize mAb technology can produce the protection antibody (Ab1) for object epi-position in pathogenic agent.Energy purifying specific antibodies (Ab1), subsequently as immunogen, being used for producing may be the antiidiotypic antibody (Ab2) of the interior image of original epi-position in pathogenic agent.
According to Jerne " network " theoretical (23) prediction, with antiidiotypic antibody (Ab2) immunity of anti-primary antibody (Ab1) antigen binding site, can cause the humoral immunoresponse(HI) to name antigen-specific.The anti-antiidiotypic antibody (or Ab3) producing should with initial elementary antigen-reactive.If elementary antigen is a kind of oligosaccharides (therefore estimating to cause the immunne response that does not rely on T cell), use Ab2 (the suitable thing of protein) immunity can cause replying of T cell dependence.
The anti-idiotope that has proved mAb 2C7 produces anti-LOS antibody in mouse and rabbit, and they are gonococcida together with complement, from the also Opsonin phagolysis (20) of backer PMN of serum of the animal of this antiidiotypic antibody immunity.
Also show, the synthetic peptide of simulating name antigen by being combined with the specific antibody of an antigen of defying orders also can cause the immunne response (29,24,54) for name antigen.
Have can be used for preventing the needs of the medicament of gonorrhoea, object is to prevent gonococcus salpingitis---a kind of may with infection, infertility and ectopic pregnancy (42) old and feeble and that chronic pelvic is bitterly relevant.Another free-revving engine is pre-anti-biological from infecting but asymptomatic host propagates to another immunity companion.This is important, because quite a few of all gonorrhoea cases is asymptomatic in masculinity and femininity, and people symptomless infection, that property is active may be the main source of most of new infection.Therefore, a kind of reduction have the gonococcus vaccine of the seriousness of symptom gonorrhoea can cause higher proportion asymptomatic/have symptom case, therefore, this vaccine may promote the diffusion of gonorrhoea, unless also prevented from propagating (41).
Summary of the invention
The present invention, by the plan peptide of the extensively conservative oligosaccharides epi-position of non-existent neisseria gonorrhoeae in Antigen of human blood group is provided, has generally solved the problems referred to above.Also provide and produced according to the method for plan peptide of the present invention.
Useful in the method and composition that plan peptide according to the present invention infects at prevention neisseria gonorrhoeae.
Accompanying drawing summary
Fig. 1 shows the Western engram analysis that mAb 2C7 is combined with escherichia coli cloning.7 independent escherichia coli clonings (PEP1-PEP7) (SEQ ID NOS:1-7) are grown in the IMC substratum that contains 100 μ g/ml penbritins, then abduction delivering fusion rotein.The bacterial lysate of preparing each clone, is loaded onto in 14%SDS-PAGE gel.After electrophoresis, with Biorad electrophoretic transfer device (Biorad, Hercules CA), protein transduction is moved on on Immobilon PVDF transfer film.MAb 2C7 for this film (A) or anti-Trx antibody (B) are detected.Can not clone [SEQ ID NO:9] in contrast in conjunction with the feminine gender of mAb 2C7 for one.
Fig. 2 shows by the plan peptide sequence that can obtain in conjunction with 7 escherichia coli clonings of mAb 2C7.
Fig. 3 shows the facs analysis that mAb 2C7 is combined with the escherichia coli cloning of expressing the fusion of plan peptide.Escherichia coli cloning is grown in the IMC substratum that contains 100 μ g/ml penbritins, then abduction delivering fusion rotein.Bacterial cell is fixed with 1% paraformaldehyde, then, with mAb2C7 dyeing, dyes subsequently with the anti-mouse IgG of FITC-coupling.Can not clone [SEQ ID NO:9] in contrast in conjunction with the feminine gender of mAb 2C7 for one.Digitized representation below escherichia coli cloning compared with the control, in conjunction with the fluorescence intensity intermediate value of the colony of mAb 2C7; The percentage ratio (total group=100%) of cell in numeral colony in bracket.
The inhibition in conjunction with LOS to mAb 2C7 of the escherichia coli cloning of Fig. 4 Explicit Expression fusogenic peptide.Escherichia coli cloning is grown in the IMC substratum that contains 100 μ g/ml penbritins, then abduction delivering fusion rotein.Bacillus coli cells and mAb 2C7 incubation 30 minutes, be loaded onto on the coated flat board of LOS afterwards.Can not clone [SEQ ID NO:9] in contrast in conjunction with the feminine gender of mAb 2C7 for one.The mean value of data at least 2 experiments of representative (diplopore).PEP1 clone shows that the maximum that mAb 2C7 is combined with LOS suppresses (66%) [SEQ ID NO:1].PEP7, PEP3, PEP4, PEP2, PEP6 and PEP5 show in conjunction with suppressing to reduce respectively [being respectively SEQ IDNO:7,3,4,2,6 and 5].
Fig. 5 shows the peptide inhibition in conjunction with LOS to mAb 2C7 that contains consensus sequence (DE_GLF) [SEQ ID NO:8].Data represent the mean value ± SE of 3 experiments (diplopore).Peptide PEP1 suppresses the combination of mAb 2C7 and LOS in the mode of dose response.
Fig. 6 shows the combination of mAb 2C7 and multiple antigenic peptide (" MAP ") MAP1.
Fig. 7 shows multiple antigenic peptide inhibition in conjunction with LOS to mAb 2C7.
Fig. 8 shows the anti-LOS antibody response of IgG of eight MAP1 (octa-MAP1) induction in mouse.(A) dosage of 8 mouse emulsifications in the 0th day is received in freund's adjuvant is eight MAP1 of 50 μ g, and again accepts at the 21st day.(B) 4 mouse use the LOS immunity of purifying as positive control.Mouse use freund's adjuvant immunity (C) or the eight MAP control peptides (D) that have nothing to do are immune as negative control.
Fig. 9 shows the anti-LOS antibody response of the IgG in all immune mouses.Show all mouse anti-LOS antibody response of IgG (mean value ± SE) of (comprise and do not show the animal of replying).
Figure 10 only shows the anti-LOS antibody response of the IgG replying in mouse.Antibody response is defined as the anti-LOS of IgG (mean value ± SE) (higher than 4 times of the anti-LOS levels of baseline IgG) higher than 0.4 μ g/ml.Eight MAP1, LOS for mouse, independent freund's adjuvant or eight irrelevant MAP control peptides immunity.The anti-LOS antibody horizontal of IgG inducing is mapped to the time with concentration.
Figure 11 only shows the anti-LOS antibody response of the IgM replying in mouse.Eight MAP1, LOS for mouse, independent freund's adjuvant or eight irrelevant MAP control peptides immunity.The anti-LOS antibody horizontal of IgG inducing is mapped to the time with concentration.
Figure 12 shows and to be exposed to gonococcus 15253 strains of mouse immune serum (100 μ l serum in 67%[150 μ l total reaction volume] add the people's complement from normal people's donor's serum [making people's complement final concentration is 17% volume percent] adding) and the survival rate of lgtG mutant (2C7 epi-position is negative) thereof.Germ experiment carries out (4) with the mAb 2C7 mouse of (A) anti-15253 strains (positive control) and 15253lgtG strain (negative control).15253 strains of 25 μ g/ml mAb 2C7 (100 μ l in 150 μ l total reaction mixture volumes) mediation 100% kill and wound, and do not kill 15253 lgtG strains.(B) normal mouse serum (merging of 20 mice serums, the mean concns of the anti-LOS antibody of IgG is 0.1 μ g/ml) can not kill arbitrary strain.(C) serum (contain the anti-LOS antibody of 5.05 μ g/ml IgG, the blood of being adopted by 7-11 week merges) that picks up from single mouse of eight MAP1 immunity is to the kill rate of 15253 strains demonstrations 92% (8% survival), and 15253 lgtG strains are all survived.(D) serum (contain the anti-LOS antibody of 21.98 μ g/ml IgG, the blood of being adopted by 7-11 week merges) that picks up from single mouse of LOS immunity does not kill 15253 strains (179% survival) and 15253 lgtG strains (133% survival).With the independent freund's adjuvant of negative control antigen (E) or (F) single mouse of eight irrelevant MAP control peptide immunity do not kill arbitrary strain.Figure 12 contrast comprises the complement source (15253 strain 137.9% ± 1.0% survivals (not killing and wounding), 15253 lgtG mutant 132.5% ± 14.3% survivals (not killing and wounding)) that does not conform to antibody.
Figure 13 shows the figure of the anti-LOS antibody concentration of IgG to neisseria gonorrhoeae 15253 strain kill rates.From each anti-LOS antibody horizontal of IgG only in 3 mouse of eight MAP1 immunity, bacterium percentage kill rate is mapped.The mice serum that contains the anti-LOS antibody of 1.38,2.50 and 5.05 μ g/ml shows respectively 31%, 74% and 92% kill rate to 15253 strains.The kill rate of mAb 2C7 shows at 5 minutes other LOS antibody concentration places, as positive control.
Detailed Description Of The Invention
Definition
When this uses, " antibody " is the complete immunoglobulin molecules that contains each two light chain immunoglobulins and heavy chain.Therefore, antibody comprises the complete immunoglobulin (Ig) of IgA, IgG, IgE, IgD, IgM type (and hypotype), and wherein the light chain of immunoglobulin (Ig) can be κ or λ type.
When this uses, " monoclonal antibody " is the monospecific antibody that the initial antibody forming cell by single clone produces.
When this uses, " immunoprophylaxis is effective " refers to that induction is enough to protect this patient at certain hour, to avoid the ability of the immunne response of neisseria gonorrhoeae infection in normal individual.
When this uses, " peptide " refers to a kind of linearity or cyclic amino acid chain, and length is generally at least 4, is less than 50 amino acid.
When this uses, " plan peptide " refers to and shows and to be combined a kind of peptide of figure with the similar immune antibody of known epi-position.
Intend peptide and according to the application in the compositions and methods of the invention
The present invention relates to can with the plan peptide of antibody mediated immunity specific reaction for the conservative oligosaccharides epi-position of neisseria gonorrhoeae, this oligosaccharides epi-position does not exist in Antigen of human blood group.These intend Toplink to be similar to as United States Patent (USP) 5,476, and 784 and 6,099, the mode of the antiidiotypic antibody that 839 (being all incorporated herein by reference) are described is used, and as a kind of Surrogate antigen, causes the immunne response for the T cell dependence of neisseria gonorrhoeae oligosaccharides epi-position.
Can use this plan peptide to the individuality not infecting, to induce biological for gonococcus or to carry the specific immune response of the cell of this oligosaccharides antigen.This immunne response may be immunoprophylaxis in nature, because when acceptor contact gonococcus is biological or carry the cell of this oligosaccharides antigen, it is by preventing infection.
In order to identify that candidate intends peptide, can be according to antibody binding specificity examination random peptide library.This examination technology is known by those skilled in the art.In one approach, can utilize the random peptide library of expressing on coli flagellum to identify the peptide that can be combined with the conservative oligosaccharides epi-position of neisseria gonorrhoeae, this oligosaccharides epi-position does not exist in Antigen of human blood group.For example, can measure with the combination of mAb 2C7 and identify that candidate intends peptide.In conjunction with can or characterizing in conjunction with the competition of LOS mAb 2C7 by solid phase ELISA with Western trace, flow cytometry.
Also can utilize antibody modeling to determine in the complementary determining region of anti-idiotope (CDR) the immunogenicity site corresponding to object epi-position.This analysis can obtain the information about the three-dimensional conformation in immunogenicity site, useful in this design of plan peptide in immunogenicity site.
The special plan peptide once identify and check order, can be by synthetic this plan peptide of producing of the known method in this area.
In order to cause stronger immunne response, also can utilize haptens, utilize adjuvant, by intending peptide, be connected, utilize multiple antigenic peptide with carrier proteins, will intend peptide and complement proteins coupling or modify and intend peptide by other known method of this area.
The preferred medicinal compositions of the present invention is similar to the composition with other peptide immunity people.Plan peptide of the present invention is generally suspended in treatment with in sterile saline solution.Can otherwise prepare medicinal compositions, to control the release of activeconstituents, or extend their existence in patient's system.Many suitable delivery systems are known, comprise, such as: implantable drug delivery system, hydrogel, Walocel MT 20.000PV, microcapsule, liposome, microemulsion, microsphere etc.
Medicinal compositions of the present invention can be used by any suitable method, and for example per os, nose are interior, subcutaneous, intramuscular, intravenously, intra-arterial or parenteral administration.General preferred intravenously (i.v.) or parenteral administration.
It will be appreciated by those skilled in the art that, the unitary dose of the plan peptide that the plan peptide of the present invention of immunoprophylaxis significant quantity especially depends on administration schedules, use, intend peptide whether be combined with other therapeutical agent use, immunological status and patient health, the therapeutic activity of the plan peptide used and the judgement for the treatment of doctor.
In order to understand better the present invention, listed the following example.These embodiment, just in order illustrating, should not to regard as by any way and limit the scope of the invention.
Embodiment
The clone's of the peptide that I. coding can specific combination mAb 2C7 evaluation
A. random peptide is shown
Use FliTrx
tMrandom peptide library (Invitrogen, Carlsbad CA) is at the peptide (12-mer) of intestinal bacteria surface expression stochastic sequence.The DNA in this peptide storehouse of coding is inserted in the gene of the active ring of coding Trx, this gene itself inserts in the nonessential region of flagellin gene.The expression of this fusogenic peptide is subject to carrier FliTrx
tMthe main left side of pnagus medius λ promotor (P
l) control.In this system, by adding tryptophane induction P
l.When induction, fusion rotein output, and surface-mounted at bacterial cell be flagellum, prepare this peptide of displaying.
B. can be in conjunction with the screening of the peptide of mAb 2C7
FliTrx
tMpeptide storehouse (1.77 * 10
8elementary clone) at the IMC substratum that contains 100 μ g/ml penbritins (0.2%w/v casamino acids, 0.5%w/v glucose, 42mM Na
2hPO
4, 22mM KH
2pO
4, 8.5mM NaCl, 18.7mM NH
4cl and 1mM MgCl
2) in 25 ℃ of grow overnight.By adding L-Trp to final concentration, be 100 μ g/ml, the expression of induction fusogenic peptide, culture is grown 6 hours at 25 ℃.Then the peptide of induction is merged to coated flat board (the 20 μ g/ml) incubation of library and 2C7mAb-.After incubation 1 hour, the dull and stereotyped IMC substratum washing with containing 100 μ g/ml penbritins and 1% Alpha-Methyl mannoside 5 times.By mechanical shearing or by the purifying LOS with preparing from gonococcus 15253 strains (known mAb 2C7 epi-position is expressed 15253 strains) competition, the intestinal bacteria of elution of bound, then grow overnight at 25 ℃.The 5th, take turns after elutriation, the intestinal bacteria of elution of bound, and at 25 ℃ plating (2%w/v casamino acids, 0.5%w/v glucose, 42mM Na on the RMG agar that contains 100 μ g/ml penbritins
2hPO
4, 22mM KH
2pO
4, 8.5mM NaCl, 18.7mM NH
4cl, 1mM MgCl
2with 1.5% agar).Select the combination (hybridoma cell line of a kind of mAb of secretion 2C7 is by US mode culture collection center [" ATCC "] preservation, and the ATCC preserving number of distribution is HB-11859) with mAb2C7 by Western trace mensuration of indivedual bacterium colonies.
With being coated in mAb 2C7 in 60mm tissue culturing plate, this library is carried out to 5 just taking turns and select, or first use irrelevant IgG3 (Sigma, St.Louis, MO) to bear and select 1 hour, with mAb 2C7, proceed 5 afterwards and just take turns and select.
107 bacterium colonies of random selection, utilize its ability in conjunction with mAb 2C7 of Western trace examination.Identifying 14 can be in conjunction with the clone of mAb 2C7.Then from positive colony, prepare plasmid DNA, utilization can be inserted the primer of the nucleotide sequence 5 ' of peptide and the region of 3 ' side being combined and check order with being positioned at.Identify 7 unique clones, [SEQ ID NOS:1-7] as depicted in figs. 1 and 2.
C. flow cytometry
Positive escherichia coli cloning 25 ℃ of grow overnight in the IMC substratum that contains 100 μ g/ml penbritins, then abduction delivering fusogenic peptide is 6 hours.Bacillus coli cells is fixed 10 minutes on ice with 0.5% paraformaldehyde.200 μ l equal portions of fixed biologically centrifugal 10 minutes with 2000xg.Abandoning supernatant, by pellet resuspended in the sealing damping fluid that contains mAb 2C7 (the IMC substratum that contains 100 μ g/ml penbritins, 1% skim-milk, 150mM NaCl and 1% Alpha-Methyl mannoside).Suspension incubation 30 minutes at 37 ℃, with 2000xg centrifugal 10 minutes afterwards.With 100 μ l lavation buffer solutions (the IMC substratum that contains 100 μ g/ml penbritins and 1% Alpha-Methyl mannoside) washing precipitation, then be resuspended to the anti-mouse IgG (Sigma that contains FITC-coupling, St.Louis, MO) 100 μ l sealing damping fluids in.Mixture incubation 30 minutes at 37 ℃, with 2000xg centrifugal 10 minutes afterwards.Abandoning supernatant, with 100 μ l lavation buffer solution washing precipitations, is resuspended in 1ml PBS afterwards.Application CellQuest software (Becton Dickinson, Franklin Lakes NJ) is analyzed this suspension on FACS.Can not clone in contrast in conjunction with the feminine gender of mAb 2C7 for one.
The combination of observing Bacillus coli cells and mAb 2C7 strengthens (according to fluorescence intensity intermediate value, " MFI ") [SEQ ID NOS:3,4,6,5,2,7,1] from escherichia coli cloning PEP3, PEP4, PEP6, PEP5, PEP2, PEP7 successively to PEP1.Escherichia coli cloning PEP1 shows and the maximum combined of mAb 2C7 (MFI=19.81, with contrast MFI=4.91 compare), [SEQ ID NO:1] as shown in Figure 3.
D. suppress ELISA
Positive escherichia coli cloning 25 ℃ of grow overnight in the IMC substratum that contains 100 μ g/ml penbritins, then abduction delivering fusogenic peptide is 6 hours.Culture is standardized as to identical OD reading (OD
600nm=0.7), add 1% skim-milk, 150mM NaCl and 1% Alpha-Methyl mannoside, to block non-specific binding.Every kind of nutrient solution of 50 μ l equal portions and 50 μ l mAb 2C7 (final concentration 20ng/ml) incubation 30 minutes at 37 ℃, is then loaded onto 100 μ l mixed solutions the hole of using the coated micro plate of the purifying LOS (80 μ g/ml) for preparing from 15253 strains.Hole incubation 1 hour at 37 ℃, then washing.After washing hole, use the mAb 2C7 that detects combination with the anti-mouse IgG of alkaline phosphatase coupling.A kind of can not clone in contrast in conjunction with the feminine gender of mAb 2C7.
PEP1 clone shows that the maximum that mAb 2C7 is combined with LOS suppresses (66%) [SEQ IDNO:1].PEP7, PEP3, PEP4, PEP2, PEP6 and PEP5 show in conjunction with suppressing reduction respectively, [SEQ ID NOS:7,3,4,2,6,5] as shown in Figure 4.Suppress ELISA result relevant to flow cytometry result, because PEP1 also shows the maximum combined with mAb 2C7.The combination of Bacillus coli cells and mAb 2C7 and escherichia coli cloning suppress mAb 2C7, and to be combined the reduction of LOS roughly relevant.
II. the synthetic plan peptide that can be combined with mAb 2C7
Synthetic (Boston Biomolecules, MA) sequence corresponding to consensus sequence " DE_GLF " a kind of synthetic peptide (PEP1 of comprising two halfcystine flanking regions (CGP-that lays respectively at N-end and C-end with-GPC residue); IPVLDENGLFAP), be used for by suppressing the specific binding of ELISA assessment and 2C7 mAb, and determine whether the plan peptide that is characterized by Trx-fusion rotein retains the antigenicity [SEQ ID NO:10] that does not rely on fusion content.
Add halfcystine flanking region, estimate whether the cyclisation of intending peptide affects antibodies.At these, intend in peptide, cysteine residues allows to form disulfide linkage between them, produces annular and intends peptide.The peptide that these conformations limit may be similar to the epi-position that they are simulated more, therefore may have more immunogenicity.
By the dilution in sealing damping fluid (0.5M NaCl is in PBS for 1% Protalbinic acid, 0.05% polysorbas20) of these peptides, produce the mixed solution of different concns (0.1,0.5,1mg/ml).(storing concentration is 2 μ g/ml for 50 μ l equal portions of each concentration and 50 μ l mAb 2C7, in sealing, dilute in damping fluid) incubation 1 hour at 37 ℃, then 100 μ l mixed solutions are loaded onto in the hole of using the coated micro plate of the purifying LOS (80 μ g/ml) for preparing from 15253 strains.Hole incubation 1 hour at 37 ℃, then washing.After washing hole, use the mAb 2C7 that detects combination with the anti-mouse IgG of alkaline phosphatase coupling.The purifying LOS preparing from gonococcus 15253 strains is as positive control.The non-reacted 15-mer peptide sequence that above-mentioned random peptide library system produces is as negative control peptide [SEQ ID NO:9].
The dose response mode of take PEP1 suppress mAb 2C7 and LOS combination (for concentration as 0.1,0.5, the PEP1 of 1.0mg/ml, percentage suppresses to equal respectively 17%, 77%, 91%), as shown in Figure 5.Contrast 15-mer peptide synthesizes cyclic peptide (* CKSNPIHIIKNRRNIPC*) [SEQ IDNO:9].This negative control peptide can not suppress the coated dull and stereotyped combination of 2C7 mAb and purifying LOS.
Utilize the known method in this area, ring-type as above is intended peptide can further contain one or more " tail ", for coupling the second reagent, as adjuvant or carrier proteins.
III. improve the immunogenicity of intending peptide
Although little peptide may have immunogenicity, study several times report, some little peptide may lack immunogenicity and cause invalid immunne response (particularly humoral response) (3,43).Applied the immunogenicity that many strategies improve little peptide.Comprise peptide is connected to (54 with carrier proteins, 28,54), by peptide and adjuvant combination (21,22), use multiple antigenic peptide (MAP) to provide and may there is the structure (39) of immunogenic larger configuration more by force, peptide and complement proteins coupling are strengthened to humoral immunoresponse(HI) (15).
A. multiple antigenic peptide is synthetic
Multiple antigenic peptide (MAP) method is a kind of technology (44,8,43) that plan peptide is combined with the lysine residue of dendroid matrix.Peptide is connected with the amino of Methionin framework (scaffold), produces a macromole, and it provides the peptide epitopes of highdensity hope in composite surface.This method can strengthen the immunne response (39,40) to peptide.
The multiple antigenic peptide and the control peptide (Boston Biomolecules, MA) that have synthesized PEP1, and by directly and suppress ELISA and measured the combination with mAb 2C7.
Carry out the combination of solid phase ELISA assessment mAb 2C7 and multiple antigenic peptide.For Salmonella, with coated Immulon 1 flat board of multiple antigenic peptide (1 μ g/ hole), spend the night, and react with the mAb 2C7 of different concns.For suppressing ELISA, flat board is used 37 ℃ of the purifying LOS (80 μ g/ml) that prepare from neisseria gonorrhoeae 15253 strains coated 3 hours.Sealing damping fluid (0.5M NaCl is in PBS for 1% Protalbinic acid, 0.05% polysorbas20) dilution for peptide (linearity or MAP), the mixture of generation different concns.50 μ l equal portions of each concentration and 50 μ l mAb 2C7 (storing concentration is 0.4 μ g/ml, dilutes in sealing damping fluid) incubation 1 hour at 37 ℃, is then loaded onto 100 μ l mixed solutions in the hole of micro plate.Hole incubation 1 hour at 37 ℃, then washing.After the washing of hole, use the mAb 2C7 that detects combination with the anti-mouse IgG of alkaline phosphatase coupling.The purifying LOS preparing from gonococcus 15253 strains in suppressing ELISA as positive control.
The multiple antigenic peptide form of the PEP1 that contains 4 kinds of linear PEP1 molecules (" four MAP1 ") or 8 kinds of linear PEP1 molecules (" eight MAP1 ") shows and the strong combination of mAb 2C7, and contrast MAP does not show combination in Salmonella, as shown in Figure 6.Four MAP1 and eight MAP1 both suppress the combination of mAb 2C7 and LOS more strongly than linear PEP1, as shown in Figure 7.Maximum (the IC that suppresses of half of four MAP1 and eight MAP1
50) at 1.26 μ M and 0.23 μ M place, observe respectively.The IC of linear PEP1
50be 55 μ M.This may be the avidity raising of being combined with mAb2C7 due to MAP1.Contrast MAP does not show obvious inhibition.
In mouse with the anti-LOS antibody response of eight MAP1 immune induction IgG, as shown in Figure 8.Response diagram is shown in Fig. 8 (A), wherein there is no the anti-LOS antibody response of obvious IgG, until strengthen the 3rd week time, shows that eight MAP1 cause the immunne response that T cell relies in replying mouse.These results have proved with intending the immune mankind of peptide (as eight MAP1) resists the prophesy that neisseria gonorrhoeae infects.
In Fig. 8 (A), the dosage of 8 mouse emulsifications in the 0th day is received in freund's adjuvant is eight MAP1 of 50 μ g, and again accepts at the 21st day.Eight MAP1 of simulation 2C7 oligosaccharides epi-position merely hit and induce the anti-LOS antibody of IgG 3 of 8 mouse.The reinforcement for the first time that the anti-LOS of IgG in these 3 mouse replied at the 3rd week significantly raises afterwards, at the 7th week, peaks (next time of measurement), reduces afterwards.Fig. 8 (B) shows positive control experiment, and wherein 4 mouse are immune with the LOS of purifying.In these mouse, the anti-LOS of IgG tires to be increased after immunity for the first time minimum level, after reinforcement, raises.With 4 mouse (being negative control) of freund's adjuvant (C) or irrelevant eight MAP control peptides (D) immunity, cause weak or do not have the anti-LOS of IgG to reply.Fig. 9 has shown the anti-LOS antibody response of average IgG (mean value ± SE comprises and do not show the animal of replying) of all immune mouses (from the experiment shown in Fig. 8).
Figure 10 has only shown the anti-LOS antibody response of IgG of replying mouse (from the experiment shown in Fig. 8).Antibody response is defined as the anti-LOS of IgG (mean value ± SE) (higher than 4 times of the anti-LOS levels of baseline IgG) higher than 0.4 μ g/ml.After initial immunity the 7th week and the 10th week, eight MAP1 immunity reply the anti-LOS antibody horizontal of IgG that mouse produces the antibody horizontal bringing out higher than (p < 0.001) negative control antigen (independent freund's adjuvant or irrelevant eight MAP control peptides).
Figure 11 has only shown the anti-LOS antibody response of IgM of replying mouse (from the experiment shown in Fig. 8).With eight MAP1 immunity, produce the higher anti-LOS level of IgM of mouse that mouse that the anti-LOS of IgG replys can not contrast negative control antigen immune and reply.With LOS (positive control) immunity, produce than the higher anti-LOS antibody horizontal of IgM of animal of eight MAP1 or negative control antigen (independent freund's adjuvant or irrelevant eight MAP control peptides) immunity.From the serum of the mouse of eight MAP1 immunity, to neisseria gonorrhoeae, 15253 strains show the bacterial activity of killing of 2C7 specificity complement-mediated, as shown in figure 12.Figure 12 has shown a figure, shows that being exposed to mouse immune serum (the mouse immune serum final concentration of 67% volume percent) adds neisseria gonorrhoeae 15253 strains of the people's complement (people's complement final concentration of 17% volume percent) obtaining from normal people's donor adding and the survival rate of lgtG mutant (2C7 epi-position is negative) thereof.
15253 strains show 2C7 epi-position.Fat oligosaccharides (LOS) the Transglucosylase G allelotrope that 15253 lgtG strains contain destruction, this enzyme can be transferred to (4) on heptose-2 in LOS core by glucose (by α key).The forfeiture that the destruction of lgtG locus causes 2C7 epi-position to be expressed.
That carries out that standard bacteria-measuring assesses complement-mediated in mice serum kills bacterial activity (11).In this is measured, under people's complement (17% final volume) exists, mice serum (67% final volume) (from different mouse immunity as described below or non-immune) be suspended in about 2.5 * 10 in MorseA substratum (33)
3individual bacterium incubation.Then continuous jolting reaction mixture 30 minutes at 37 ℃.When 0 time and 30 minutes, the equal portions of reaction mixture are inoculated on Chocolate Agar flat board.When survival rate is expressed as 30 minutes, on flat board, the percentage of bacterium colony increases during than 0 minute.Survival rate higher than 100% in mensuration shows the growth between incubation period in 30 minutes.
With mAb 2C7 in contrast, because it can kill neisseria gonorrhoeae 15253 strains together with the complement adding, but can not kill 15253 lgtG mutant strains.As shown in Figure 12 (A), mAb 2C7 has to carrying the gonococcus of 2C7 epi-position the bacterial activity of killing.100% of 25 μ g/ml mAb 2C7 (100 μ l in cumulative volume 150 μ l reaction mixtures) mediation, 15253 strains kill and wound, but do not kill 15253 lgtG strains.
The serum that picks up from single mouse of eight MAP1 immunity (contains the anti-LOS antibody of 5.05 μ g/ml IgG, the blood of being adopted by 7-11 week merges) to the kill rate of 15253 strains demonstrations 92% (8% survival), and 15253 lgtG strains are all survived, as shown in Figure 12 (C).
The normal mouse serum (mean concns of the anti-LOS antibody of IgG is 0.1 μ g/ml) that represents the set of 20 mice serums can not kill any bacterial strain, as shown in Figure 12 (B).The control mice serum containing complement does not show 116.1% ± 4.7% survival rate (not killing and wounding) to 15253 strains, 15253 lgtG mutant is shown to 123.1% ± 3.5% survival rate (not killing and wounding).The complement source containing antibody does not show 137.9% ± 1.0% survival rate (not killing and wounding) to 15253 strains, 15253 lgtG mutant is shown to 132.5% ± 14.3% survival rate (not killing and wounding).
The serum that picks up from single mouse of LOS immunity (contains the anti-LOS antibody of 21.98 μ g/ml IgG, the blood of being adopted by 7-11 week merges) do not kill 15253 strains (179% survival) and 15253lgtG strain (133% survival), as shown in Figure 12 (D).The serum gathering single the mouse as negative control antigen immune from the freund's adjuvant with independent or irrelevant eight MAP control peptides does not kill arbitrary bacterial strain, respectively as shown in Figure 12 (E) and Figure 12 (F).
What the anti-LOS antiserum(antisera) of IgG obtaining from the mouse of eight MAP1 immunity relied on neisseria gonorrhoeae 15253 strain display densities kills and wounds, as shown in figure 13.
Figure 13 shows the figure of the anti-LOS antibody concentration of IgG to neisseria gonorrhoeae 15253 strain kill rates.When each the anti-LOS antiserum(antisera) of IgG level is only mapped to bacterium kill rate in 3 mouse from eight MAP1 immunity, obtain dose-response figure (mice serum that contains the anti-LOS antibody of 1.38,2.50 and 5.05 μ g/ml shows respectively 31%, 74% and 92% kill rate to 15253 strains).Also at 5 minutes other LOS antibody concentration places, show the kill rate of mAb 2C7, as positive control.
B. coupling A intends peptide and complement proteins C3d
Expectation by with complement factor C3d coupling, can further improve the immunogenicity of the plan peptide (eight MAP1 as described here) of gonococcal epitopes.
Studies have shown that in a large number the vital role (1,5,14,17,25,32,34 and 35) of complement proteins C3 in induction humoral immunoresponse(HI).The proteantigen that the mouse that C3 lacks relies on the T cell antibody response (34,35) that demonstration reduces as keyhole relative hemocyanin (" KLH ").The mouse that complement receptor 1-(CR1 or CD35) and complement receptor 2-(CR2 or CD21) lacks has the antibody response (1,14,32) of the T cell dependence of reduction.Further show, with the covalently bound C3d of hen's egg-white lysozyme (" HEL ") cause enhancing to the antibody response of HEL antigen (15).The mouse of the fusion protein immunization being comprised of 3 copy C3d and 1 copy HEL makes anti-HEL antibody response than separately with 10000 times of the antibody response raisings of the mouse of HEL immunity.The anti-HEL antibody response of this fusion rotein induction is approximately higher 100 times than the antibody response of the HEL induction of emulsification in freund's adjuvant.
Eight MAP1 can with C3d coupling, method is that eight MAP1 DNA sequence dnas are cloned in C3d fusion rotein box, and transforms expression system with this member.So eight MAP1-C3d fusion roteins can be expressed, purifying is also used as immunogen.In addition,, according to the known method in this area, eight MAP1-C3d gene fusion also can be used as DNA vaccination with the form of DNA.
The example that produces the hybridoma of antiidiotypic antibody (it shows and the similar immunoreactivity of plan peptide of the present invention) has on March 26th, 1993 at ATCC (10801 UniversityBoulevard, Manassas, Va.20110-2209 U.S.A.) preservation, distribute the cell culture of ATCC preserving number HB 11311.
The example of the hybridoma 2C7 of secretion mAb 2C7 (it shows and the similar immunoreactivity of plan peptide of the present invention) has March 9 nineteen ninety-five at the cell culture of the called after 2C7 of ATCC preservation.This culture distributes ATCC preserving number HB-11859.
Although we have described many embodiments of the present invention hereinbefore, obviously our basic structure can change, so that other embodiment of application method and composition of the present invention to be provided.Therefore, should be appreciated that scope of the present invention is limited the claims by adding, rather than limited by the specific embodiments of above describing in the mode of embodiment.
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Sequence table
<110>Rice，Peter
The plan peptide of <120> conserved gonococcal epitope and apply their method and composition
<130>BOS-3
<140> is to be allocated
<141>2000-10-27
<150>60/162491
<151>1999-10-29
<160>10
<170>PatentIn version 3.0
<210>1
<211>12
<212>PRT
<213> is artificial/the unknown
<220>
<221> peptide
<222>(1)..(12)
<223> synthesizes construct
<400>1
Ile Pro Val Leu Asp Glu Asn Gly Leu Phe Ala Pro
1 5 10
<210>2
<211>12
<212>PRT
<213> is artificial/the unknown
<220>
<221> peptide
<222>(1)..(12)
<223> synthesizes construct
<400>2
Trp Gly Leu Asp Tyr Glu Arg Gly Asn Tyr Glu Glu
1 5 10
<210>3
<211>12
<212>PRT
<213> is artificial/the unknown
<220>
<221> peptide
<222>(1)..(12)
<223> synthesizes construct
<400>3
Asp Ala Leu Ala Val Asp Gln Met Gly Arg Phe Gly
1 5 10
<210>4
<211>12
<212>PRT
<213> is artificial/the unknown
<220>
<221> peptide
<222>(1)..(12)
<223> synthesizes construct
<400>4
Val Leu Val Gly Glu Lys Gly Leu Phe Glu Gly Gly
1 5 10
<210>5
<211>12
<212>PRT
<213> is artificial/the unknown
<220>
<221> peptide
<222>(1)..(12)
<223> synthesizes construct
<400>5
Glu Ala Leu Val Leu Asp Thr Asn Gly Leu Met Ser
1 5 10
<210>6
<211>12
<212>PRT
<213> is artificial/the unknown
<220>
<221> peptide
<222>(1)..(12)
<223> synthesizes construct
<400>6
Ala Asp Arg Thr Gln Gly Leu Gly Trp Gly Ala Ser
1 5 10
<210>7
<211>12
<212>PRT
<213> is artificial/the unknown
<220>
<221> peptide
<222>(1)..(12)
<223> synthesizes construct
<400>7
Glu Glu Val Gly Ser Ile Leu Tyr Gly Leu Gly Gly
1 5 10
<210>8
<211>6
<212>PRT
<213> is artificial/the unknown
<220>
<221> peptide
<222>(3)..(3)
The arbitrary amino acid of <223>X=
<220>
<221> peptide
<222>(1)..(6)
<223> synthesizes construct
<400>8
Asp Glu Xaa Gly Leu Phe
1 5
<210>9
<211>17
<212>PRT
<213> is artificial/the unknown
<220>
<221> peptide
<222>(1)..(17)
<223> synthesizes construct
<400>9
Cys Lys Ser Asn Pro Ile His Ile Ile Lys Asn Arg Arg Asn Ile
Pro
1 5 10 15
Cys
<210>10
<211>15
<212>PRT
<213> is artificial/the unknown
<220>
<221> peptide
<222>(1)..(15)
<223> synthesizes construct
<400>10
Cys Gly Pro Ile Pro Val Leu Glu Asn Gly Leu Phe Gly Pro Cys
1 5 10 15
Claims (18)
1. a kind of plan peptide that there is no the conserved gonococcal epitope found on Antigen of human blood group, wherein this is intended Toplink and in Mammals, induces the immunne response to this conserved gonococcal epitope, the aminoacid sequence of wherein said plan peptide as shown in SEQ ID NO:7,
Optionally wherein, described plan peptide and adjuvant combination,
Described plan peptide is connected with carrier proteins,
Described plan peptide and complement proteins coupling;
Described plan peptide provides with multiple antigenic peptide form, or
Described plan peptide further connects at least one for the tail with the second reagent coupling.
2. according to the plan peptide of claim 1, wherein immunne response is that T cell relies on.
3. according to the plan peptide of claim 1, wherein this plan peptide further connects at least one for the tail with the coupling of the second reagent.
4. according to the plan peptide of claim 3, wherein the second reagent is a kind of adjuvant.
5. according to the plan peptide of claim 1 or 2, wherein this plan peptide is connected with adjuvant combination or with carrier proteins.
6. according to the plan peptide of claim 1 or 2, the part that wherein this plan peptide is a kind of multiple antigenic peptide.
7. according to the plan peptide of claim 1 or 2, wherein this plan peptide and gonococcus LOS competition are in conjunction with the monoclonal antibody 2C7 of the hybridoma generation that is HB-11859 by preserving number.
8. according to the plan peptide of claim 1, wherein this plan Toplink is combined with the monoclonal antibody 2C7 that the hybridoma that is HB-11859 by preserving number produces.
9. according to the plan peptide of claim 1, wherein this intends Toplink in conjunction with the monoclonal antibody of passing through to produce with a kind of anti-idiotype monoclonal antibodies immune animal, or its fragment, and the hybridoma cell line that wherein said monoclonal antibody is HB11311 by ATCC preserving number produces.
10. according to the plan peptide of claim 1, the part that wherein this plan peptide is a kind of multiple antigenic peptide.
11. according to the plan peptide of claim 1, wherein this intends peptide and a kind of complement proteins coupling.
12. according to the plan peptide of claim 11, wherein this intends peptide and complement proteins C3d coupling.
13. 1 kinds for resisting that neisseria gonorrhoeae infects and the composition of immunity, its contain immunoprophylaxis significant quantity according to the plan peptide of claim 1-12 any one.
14. 1 kinds are infected and the composition of immunity for resisting neisseria gonorrhoeae, the plan peptide that it contains immunoprophylaxis significant quantity, and this plan peptide consists of the peptide sequence shown in SEQ ID NO:7.
15. the purposes of the medicine that the plan peptide of claim 1 infects for the preparation of immune Mammals antagonism neisseria gonorrhoeae.
16. the purposes of the medicine that the plan peptide of claim 12 infects for the preparation of immune Mammals antagonism neisseria gonorrhoeae.
17. 1 kinds of raisings, according to the method for enhancing antigenicity of the plan peptide of claim 1, comprise the step of this plan peptide and a kind of complement proteins coupling.
18. according to the method for claim 17, and wherein this complement proteins is C3d.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US16249199P | 1999-10-29 | 1999-10-29 | |
| US60/162,491 | 1999-10-29 |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN008170983A Division CN1409725B (en) | 1999-10-29 | 2000-10-27 | Peptide minics of conserved gonococcal epitopes and methods and compositions using them |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101638433A CN101638433A (en) | 2010-02-03 |
| CN101638433B true CN101638433B (en) | 2014-10-29 |
Family
ID=22585848
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910136894.6A Expired - Fee Related CN101638433B (en) | 1999-10-29 | 2000-10-27 | Peptide mimics of conserved gonococcal epitopes and methods and compositions using them |
| CN008170983A Expired - Fee Related CN1409725B (en) | 1999-10-29 | 2000-10-27 | Peptide minics of conserved gonococcal epitopes and methods and compositions using them |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN008170983A Expired - Fee Related CN1409725B (en) | 1999-10-29 | 2000-10-27 | Peptide minics of conserved gonococcal epitopes and methods and compositions using them |
Country Status (7)
| Country | Link |
|---|---|
| CN (2) | CN101638433B (en) |
| AP (1) | AP1638A (en) |
| AU (1) | AU785022B2 (en) |
| HK (1) | HK1141034A1 (en) |
| NZ (2) | NZ532271A (en) |
| OA (1) | OA12315A (en) |
| WO (1) | WO2001032692A2 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899096B (en) * | 2010-07-07 | 2012-07-04 | 南方医科大学 | Blood group A epitope mimic peptide and application thereof |
| CN101899097B (en) * | 2010-07-07 | 2012-02-01 | 南方医科大学 | Blood group B epitope mimic peptide and application thereof |
| US20220395567A1 (en) * | 2019-09-23 | 2022-12-15 | University Of Massachusetts | Multi-antigenic peptide mimics of gonococcal lipo-oligosaccharide (los) epitopes |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994022479A1 (en) * | 1993-04-06 | 1994-10-13 | Trustees Of Health & Hospitals Of The City Of Boston | Gonococcal anti-idiotypic antibodies and methods and compositions using them |
| WO1997046582A1 (en) * | 1996-06-05 | 1997-12-11 | Peptide Therapeutics Limited | Meningococcal vaccine |
| WO1999011660A2 (en) * | 1997-09-04 | 1999-03-11 | Institut Pasteur | Immunogenic polypeptides that mimic a surface polysaccharide antigen of a pathogenic microorganism, method for obtaining the same, and their use in vaccine compositions |
| WO1999040189A2 (en) * | 1998-02-09 | 1999-08-12 | Genset | Cdnas encoding secreted proteins |
-
2000
- 2000-10-27 CN CN200910136894.6A patent/CN101638433B/en not_active Expired - Fee Related
- 2000-10-27 AP APAP/P/2002/002511A patent/AP1638A/en active
- 2000-10-27 OA OA1200200130A patent/OA12315A/en unknown
- 2000-10-27 CN CN008170983A patent/CN1409725B/en not_active Expired - Fee Related
- 2000-10-27 NZ NZ532271A patent/NZ532271A/en not_active IP Right Cessation
- 2000-10-27 WO PCT/US2000/029749 patent/WO2001032692A2/en active IP Right Grant
- 2000-10-27 NZ NZ518915A patent/NZ518915A/en not_active IP Right Cessation
- 2000-10-27 AU AU12420/01A patent/AU785022B2/en not_active Ceased
-
2010
- 2010-08-03 HK HK10107364.1A patent/HK1141034A1/en not_active IP Right Cessation
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994022479A1 (en) * | 1993-04-06 | 1994-10-13 | Trustees Of Health & Hospitals Of The City Of Boston | Gonococcal anti-idiotypic antibodies and methods and compositions using them |
| WO1997046582A1 (en) * | 1996-06-05 | 1997-12-11 | Peptide Therapeutics Limited | Meningococcal vaccine |
| WO1999011660A2 (en) * | 1997-09-04 | 1999-03-11 | Institut Pasteur | Immunogenic polypeptides that mimic a surface polysaccharide antigen of a pathogenic microorganism, method for obtaining the same, and their use in vaccine compositions |
| WO1999040189A2 (en) * | 1998-02-09 | 1999-08-12 | Genset | Cdnas encoding secreted proteins |
Non-Patent Citations (3)
| Title |
|---|
| DEMPSEY PW, ET AL.C3d OF COMPLEMENT AS A MOLECULAR ADJUVANT: BRIDGING INATE AND ACQUIRED IMMUNITY.《SCIENCE》.1996,第271卷348-350. |
| DEMPSEY PW, ET AL.C3d OF COMPLEMENT AS A MOLECULAR ADJUVANT: BRIDGING INATE AND ACQUIRED IMMUNITY.《SCIENCE》.1996,第271卷348-350. * |
| KIEBER-EMMONS T.PEPTIDE MIMOTOPES OF CARBOHYDRATE ANTIGENS.《IMMUNOLOGIC RESEARCH》.1998,第17卷(第1-2期),95-108. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1409725B (en) | 2012-06-06 |
| AP2002002511A0 (en) | 2002-06-30 |
| OA12315A (en) | 2006-05-15 |
| NZ518915A (en) | 2004-05-28 |
| AU1242001A (en) | 2001-05-14 |
| CN1409725A (en) | 2003-04-09 |
| AP1638A (en) | 2006-07-20 |
| NZ532271A (en) | 2006-03-31 |
| CN101638433A (en) | 2010-02-03 |
| HK1141034A1 (en) | 2010-10-29 |
| WO2001032692A2 (en) | 2001-05-10 |
| AU785022B2 (en) | 2006-08-24 |
| WO2001032692A3 (en) | 2002-03-07 |
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