CN1409725B - Peptide minics of conserved gonococcal epitopes and methods and compositions using them - Google Patents
Peptide minics of conserved gonococcal epitopes and methods and compositions using them Download PDFInfo
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- CN1409725B CN1409725B CN008170983A CN00817098A CN1409725B CN 1409725 B CN1409725 B CN 1409725B CN 008170983 A CN008170983 A CN 008170983A CN 00817098 A CN00817098 A CN 00817098A CN 1409725 B CN1409725 B CN 1409725B
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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
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- 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
-
- 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
Abstract
The present invention relates to peptide mimics of a conserved gonoccocal epitode of <i>Neisseria gonorrhoeae</i>, 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 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 human blood type antigen.The present invention also relates to use these and intend the infectious method and composition of peptide prevention gonorrhoea.
Background of invention
The sexually transmitted disease (STD) gonorrhoea causes worldwide danger as one of transmissible disease of the most often reporting.Gonorrhoea is caused by a kind of gram-negative diplococci neisseria gonorrhoeae.Although this pathogenic agent mainly infects 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 tangible disease syndrome.More serious syndrome comprises, for example, and the propagated gonococcal infection among the male sex and the women (" DGI "), and salpingitis among the women or pelvic inflammatory disease (" PID ").Itself can cause secular sequela salpingitis or PID, comprises ectopic pregnancy and sterile.Other important sequela (needing surgical operation sometimes) 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 of treatment PID and relevant ectopic pregnancy and infertility added up 2,600,000,000 dollars (53) in 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 gonococcal infection and the effective applicability of antibiotic therapy in U.S.'s public health, annual CDC (" CDC ") still reports to be had near 315000 routine gonorrhoeas (12).Have significant proportion to occur in the symptomless infection individuality in all gonorrhoea cases, they are sources (6) of most of new cases in the colony.The increasing popular treatment that makes this infection of strains is complicated (10,11,52) more.
Neisseria gonorrhoeae has multiple virulence factor.The surface composition of this pathogenic agent plays an important role adhering to and invade in the host cell, and potential target is provided for host immune response.Gonococcal infection causes showing as 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 bacterium surface exposes antigenic several kinds of compositions.Pili, Opa, Por and LOS all with adhere to and to invade the host relevant, its surperficial exposure zone all shows sizable variation (26,45,46).Variation causes infecting and the hypothesis of the potential of lasting virulence with biological about the tissue specificity of different sites again in the bacterial strain of gonococcus surface composition and between bacterial strain.
In symptom and asymptomatic patient were arranged, gonococcal infection shows can stimulate anti-gonococcus serum immunoglobulin level to improve.The periphery humoral response mainly is IgG (great majority is subclass IgG3), and more a spot of IgM and IgA (13) are arranged.Quantitatively, antibody response is primarily aimed at pili, Opa albumen and LOS.Local antibody is present in the sexual organ secretory product, but content reduces (48), possibly be to serum in different antigens target (27).The main antibody classification that exists in the secretion also is IgG (mainly being IgG3), rather than secretory IgA (" SIgA ") (7).Also have anti-LOS antibody, but its amount is less than the antibody of antibiotic hair, Por and Opa.Possibly show that to the antigenic antibody response of multiple gonococcus isolating neisseria gonorrhoeae has resistance (38) to the germicidal action of normal human serum (" NHS ") and most of reconvalescent's serum from propagated infection (DGI) patient although infect the patient of neisseria gonorrhoeae.This seroresistance phenotype is called stable seroresistance (" SR "), can make bioenergy avoid local defense, passes mucosal barrier, and propagates through blood flow.
After the cultivation of going down to posterity, many gonococcus bacterial strains become phenotype (38) responsive or the serum sensitivity to killing and wounding of NHS.These biologies are called as serum responsive (" SS ") or unsettled seroresistance.These biologies separate from the women of performance of local inflammation severe or clinical tangible PID usually.Acute salpinitis, the relative disease of the pathology of PID (being caused by the SS gonococcus) seldom develops into microbemia or DGI.The violent local inflammation reaction that this prompting is caused by the SS gonococcus possibly be enough to comprise to be infected and the prevention microbemia, although will pay the cost of damage local organization.The SS gonococcus produces the complement deutero-chenotactic peptide C5a (16) of remarkable volume than SR gonococcus.This can cause the inflammatory reaction of polymorphonuclear leukocyte (" the PMN ") mediation that the SS gonococcus produces.
The control more and more difficult that the development of the antibiotics resistance strain of neisseria gonorrhoeae makes this infection.The potential of insufficient treatment gonococcal infection has quickened the needs of antagonism gonococcus vaccine.The prevention of the severe complication of gonococcal infection, particularly PID has been many researchists' a target.But the effort of the effective anti-gonococcus vaccine of ongoing research and development runs into many difficulties.
Use the individual surface composition of pathogenic agent and do not succeed as the trial of the target of conventional vaccine, this is because their antigenic variability.The pili vaccine only infects homologous strain (being used for producing the pili vaccine) has protectiveness, Por inoculation even also not success in human experimentation is attacked.In addition, neisseria gonorrhoeae performance obvious phenotypes unhomogeneity is generally with 10
3In the individual biology＞to another kind of conversion (49,50), this makes the surface of this biology become the moving target of most of vaccine schemes from a kind of antigen form for 1 frequency.Although the vaccine candidate strain causes antibody response, antibody that produces 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 the effect (2,16,18,37,47) of LOS as the main target of the bactericidin that is directed against the neisseria gonorrhoeae surface.LOS antibody has several important function: kill bacterial activity, the complement activation (2) through classics or bypass complement pathway, and OA (16).In addition, LOS is considered to induce the most effectively gonococcus antigen (51) that homology and the gonococcal functional antibodies of allos are replied.
As if monoclonal antibody (" mAb ") 2C7 (30) detect in the gonococcus clinical separation strain LOS that is extensively conservative and that express oligosaccharides (" the OS ") epi-position of deriving.Carbohydrate generally is the antigen that does not rely on the T cell.When using separately as immunogen, they only cause that usually primary antibody replys.In addition, oligosaccharides less (＜10 sugared unit) (19) possibly need other biological chemistry to derive and make them that immunogenicity arranged.Therefore, these oligosaccharides are restricted aspect several as the purposes of vaccine candidate object.
Once image (internal image) determinant (36) in proposing in vaccine, to use.Utilize the mAb technology can produce protection antibody (Ab1) to purpose epi-position on the pathogenic agent.Ability purifying antibodies specific (Ab1), subsequently as immunogen, being used for producing possibly be the antiidiotypic antibody (Ab2) of the interior image of original epi-position on the pathogenic agent.
According to Jerne " network " theoretical (23) prediction, can cause naming the HI of antigen-specific with antiidiotypic antibody (Ab2) immunity of anti-primary antibody (Ab1) antigen binding site.Anti--the antiidiotypic antibody (or Ab3) that produces 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 the T cell), then use replying of Ab2 (the suitable thing of protein) immunity can initiation T cell dependence.
The anti-idiotope that has proved mAb 2C7 produces anti-LOS antibody in mouse and rabbit, they are gonococcida with complement, from the also Opsonin phagolysis (20) of backer PMN of serum of the immune animal of this antiidiotypic antibody.
Also show, through combining the antigenic synthetic peptide of simulation name also can cause to name antigenic immunne response (29,24,54) with the antigenic specific antibody of name of defying orders.
Have the needs to the medicament that can be used for preventing gonorrhoea, purpose is to prevent the gonococcus salpingitis--a kind of maybe with old and feeble infection, infertility and the ectopic pregnancy (42) relevant bitterly with chronic pelvic.Another free-revving engine is that prevention is 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 the masculinity and femininity, and symptomless infection, the active people of property possibly 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/the symptom case is arranged, therefore, this vaccine possibly promote the diffusion of gonorrhoea, only if also prevent to propagate (41).
Summary of the invention
The present invention has generally solved the problems referred to above through being provided at the plan peptide of the extensively conservative oligosaccharides epi-position of non-existent neisseria gonorrhoeae in the human blood type antigen.The method according to plan peptide of the present invention of producing also is provided.
Plan peptide according to the present invention is useful in the method and composition that the prevention neisseria gonorrhoeae infects.
The accompanying drawing summary
Fig. 1 shows mAb 2C7 and escherichia coli cloning bonded Western engram analysis.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 the abduction delivering fusion rotein.The bacterial lysate for preparing each clone, application of sample is in the 14%SDS-PAGE gel.Behind the electrophoresis, (Biorad, Hercules CA) moves on to protein transduction on the Immobilon PVDF transfer film with Biorad electrophoretic transfer device.This film is detected with mAb 2C7 (A) or anti--Trx antibody (B).The feminine gender clone that can not combine mAb 2C7 is as contrast [SEQ IDNO:9].
Fig. 2 shows the plan peptide sequence that is obtained by 7 escherichia coli clonings that can combine mAb 2C7.
Fig. 3 shows mAb 2C7 and expresses the escherichia coli cloning bonded facs analysis of intending the peptide fusion.Escherichia coli cloning is grown in the IMC substratum that contains 100 μ g/ml penbritins, then the abduction delivering fusion rotein.Bacterial cell is fixed with 1% paraformaldehyde, then with mAb 2C7 dyeing, subsequently with the anti-mouse IgG dyeing of FITC-link coupled.The feminine gender clone that can not combine mAb 2C7 is as contrast [SEQ ID NO:9].Digitized representation below the escherichia coli cloning is compared with contrast, in conjunction with the fluorescence intensity intermediate value of the colony of mAb 2C7; The percentage ratio of cell (total group=100%) in the numeral colony in the bracket.
Fig. 4 shows that the escherichia coli cloning of expressing fusogenic peptide combines the inhibition of LOS to mAb 2C7.Escherichia coli cloning is grown in the IMC substratum that contains 100 μ g/ml penbritins, then the abduction delivering fusion rotein.Bacillus coli cells and mAb 2C7 incubation 30 minutes are on the flat board that application of sample encapsulates to LOS afterwards.The feminine gender clone that can not combine mAb 2C7 is as contrast [SEQ ID NO:9].The MV of data represented at least 2 experiments (diplopore).The PEP1 clone shows mAb 2C7 and LOS bonded largest inhibition (66%) [SEQ IDNO:1].PEP7, PEP3, PEP4, PEP2, PEP6 and PEP5 show that combination suppresses to reduce respectively [being respectively SEQ ID NO:7,3,4,2,6 and 5].
Fig. 5 shows that the peptide that contains consensus sequence (DE_GLF) [SEQ ID NO:8] combines the inhibition of LOS to mAb2C7.MV ± the SE of data represented 3 experiments (diplopore).Peptide PEP1 suppresses combining of mAb 2C7 and LOS with the mode of dose response.
Fig. 6 shows combining of mAb 2C7 and multiple antigenic peptide (" MAP ") MAP1.
Fig. 7 shows that multiple antigenic peptide combines the inhibition of LOS to mAb 2C7.
Fig. 8 shows that eight MAP1 (octa-MAP1) inductive IgG resists-the LOS antibody response in the mouse.(A) 8 mouse are received at the 0th day that emulsive dosage is eight MAP1 of 50 μ g in the freund's adjuvant, and accept once more at the 21st day.(B) 4 mouse with the LOS immunity of purifying as positive control.Mouse with freund's adjuvant immunity (C) or irrelevant eight MAP control peptides (D) immunity as negative control.
Fig. 9 shows that the IgG in all immune mouses resists-the LOS antibody response.The IgG that shows all mouse (comprise and do not show the animal of replying) is anti--and the LOS antibody response (MV ± SE).
Figure 10 only shows that the IgG that replys in the mouse resists-the LOS antibody response.Antibody response is defined as the IgG that is higher than 0.4 μ g/ml anti--LOS (MV ± SE) (be higher than baseline IgG anti--4 times of LOS levels).Mouse is with eight MAP1, LOS, independent freund's adjuvant or irrelevant eight MAP control peptides immunity.The IgG that induces is anti--and the LOS antibody horizontal maps to the time with concentration.
Figure 11 only shows that the IgM that replys in the mouse resists-the LOS antibody response.Mouse is with eight MAP1, LOS, independent freund's adjuvant or irrelevant eight MAP control peptides immunity.The IgG that induces is anti--and the LOS antibody horizontal maps to the time with concentration.
Figure 12 demonstration is exposed to gonococcus 15253 strains of mouse immune serum (67% [100 μ l serum in the 150 μ l total reaction volume] add the people's complement from normal people's donor's serum [making that people's complement final concentration is 17% volume percent] of adding) and the survival rate of IgtG two mutants (the 2C7 epi-position is negative) thereof.Germ experiment carries out (4) with the mAb 2C7 mouse of (A) anti-15253 strains (positive control) and 15253IgtG strain (negative control).15253 strains of 25 μ g/ml mAb 2C7 (100 μ l in the 150 μ l total reaction mixture volumes) mediation 100% kill and wound, and do not kill the 15253IgtG strain.(B) normal mouse serum (merging of 20 mice serums, IgG is anti--mean concns of LOS antibody is 0.1 μ g/ml) can not kill arbitrary strain.(C) pick up from the kill rate (8% survival) of the serum (contain 5.05 μ g/ml IgG and resist-LOS antibody, merge) of single mouse of eight MAP1 immunity, and the 15253IgtG strain is survived all to 15253 strains demonstration 92% by the blood of adopting in 7-11 week.(D) serum (contain 21.98 μ g/ml IgG and resist-LOS antibody, merged by the blood of adopting in 7-11 week) that picks up from single mouse of LOS immunity does not kill 15253 strains (179% survival) and 15253IgtG strain (133% survival).With the independent freund's adjuvant of negative control antigen (E) or (F) single mouse of irrelevant eight MAP control peptides immunity do not kill arbitrary strain.Figure 12 contrast comprises the complement source (15253 strains, 137.9% ± 1.0% survival (not killing and wounding), 132.5% ± 14.3% survival (not killing and wounding) of 15253IgtG two mutants) that does not contain antibody.
Figure 13 show IgG anti--the LOS AC is to the figure of neisseria gonorrhoeae 15253 strain kill rates.From each IgG only in 3 mouse of eight MAP1 immunity anti--the LOS antibody horizontal maps to bacterium percentage kill rate.Contain 1.38,2.50 and 5.05 μ g/ml anti--mice serum of LOS antibody shows 31%, 74% and 92% kill rate respectively to 15253 strains.The kill rate of mAb 2C7 shows at other LOS AC place of 5 branches, as positive control.
Detailed Description Of The Invention
Definition
When this used, " antibody " was the complete immunoglobulin molecules that contains each two light chain immunoglobulin and heavy chain.Therefore, antibody comprises the complete Tegeline of IgA, IgG, IgE, IgD, IgM type (and hypotype), and wherein the light chain of Tegeline can be κ or λ type.
When this used, " monoclonal antibody " was the initial monospecific antibody that is produced by single clone's antibody forming cell.
When this used, " immunoprophylaxis is effective " is meant in normal individual, to induce was enough to protect this patient to avoid the ability of the immunne response of neisseria gonorrhoeae infection at certain hour.
When this used, " peptide " was meant a kind of linearity or cyclic amino acid chain, and length is generally at least 4, is less than 50 amino acid.
When this used, " plan peptide " was meant and shows a kind of peptide that combines 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 and can guard the plan peptide of the antibody mediated immunity specific reaction of oligosaccharides epi-position with being directed against neisseria gonorrhoeae, this oligosaccharides epi-position does not exist in human blood type antigen.These intend Toplink to be similar to like USP 5,476 784 and 6; 099; The mode of the described antiidiotypic antibody of 839 (all being incorporated herein by reference) is used, and as a kind of alternative antigen, causes the immunne response to the T cell dependence of neisseria gonorrhoeae oligosaccharides epi-position.
Can use this plan peptide to the individuality that does not infect, biological or carry the specific immune response of the antigenic cell of this oligosaccharides to induce to gonococcus.This immunne response possibly be immunoprophylaxis in nature, because when acceptor contacted the gonococcus biology or carries the antigenic cell of this oligosaccharides, it was with preventing infection.
In order to identify that the candidate intends peptide, can be according to antibody binding specificity examination random peptide library.This examination technology is that those skilled in the art institute is known.In one approach, can be utilized in the random peptide library of expressing on the coli flagellum and identify and to guard oligosaccharides epi-position bonded peptide with neisseria gonorrhoeae that this oligosaccharides epi-position does not exist in human blood type antigen.For example, can measure with mAb2C7 combine identify that the candidate intends peptide.In conjunction with using Western trace, flow cytometry or combining the competition of LOS to characterize to mAb 2C7 through solid phase ELISA.
Also can utilize the antibody modeling to confirm in the complementary determining region of anti-idiotope (CDR) immunogenicity site corresponding to the purpose epi-position.This analysis can obtain the information about the three-dimensional conformation in immunogenicity site, and this is useful in the plan peptide design in immunogenicity site.
In case identify and the special plan peptide that checks order, can should intend peptide through synthetic production of the known method in this area.
In order to cause stronger immunne response, also can utilize haptin, utilize adjuvant, will intend peptide and be connected, utilize multiple antigenic peptide with carrier proteins, will intend peptide and complement proteins coupling or modify and intend peptide through other known method of this area.
The preferred medicinal compsns of the present invention is similar to the compsn with other peptide immunity people.Plan peptide of the present invention generally is suspended in treatment with in the sterile saline solution.Can otherwise prepare medicinal compsns,, or prolong their existence in the patient system with the release of control activeconstituents.Many suitable delivery systems are known, comprise, for example: implantable drug delivery system, hydrogel, Walocel MT 20.000PV, microcapsule, liposome, microemulsion, microsphere etc.
Medicinal compsns of the present invention can be used through 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, used, intend whether peptide combines with other therapeutical agent to use, immunological status and patient health, the therapeutic activity of the plan peptide used and the judgement of treating the doctor.
In order to understand the present invention better, listed the following example.These embodiment should not regard the scope of the present invention that limits by any way as just in order to explain.
Embodiment
But the I. clone's of the peptide of coding specific combination mAb 2C7 evaluation
A. peptide is showed at random
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, and this gene itself inserts in the nonessential region of flagellin gene.The expression of this fusogenic peptide receives carrier FliTrx
TMThe main left side of pnagus medius λ promotor (P
L) control.In this system, induce P through adding tryptophane
LWhen inducing, fusion rotein output, and surface-mounted at bacterial cell be flagellum, prepare this peptide of displaying.
B. can combine 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, 42mMNa
2HPO
4, 22mM KH
2PO
4, 8.5mM NaCl, 18.7mM NH
4Cl and 1mMMgCl
2) in 25 ℃ of grow overnight.Through adding L-tryptophane to final concentration is 100 μ g/ml, induces the expression of fusogenic peptide, and culture was grown 6 hours down at 25 ℃.Then the inductive peptide is merged flat board (the 20 μ g/ml) incubation that library and 2C7mAb-encapsulate.Behind the incubation 1 hour, dull and stereotyped with the IMC substratum washing that contains 100 μ g/ml penbritins and 1% Alpha-Methyl mannoside 5 times.Perhaps through competing with the purifying LOS (known mAb 2C7 epi-position is expressed 15253 strains) for preparing from gonococcus 15253 strains, the intestinal bacteria of elution of bound are then 25 ℃ of following grow overnight through mechanical shearing.The 5th take turns elutriation after, the intestinal bacteria of elution of bound, and at 25 ℃ of following platings (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, 22mMKH
2PO
4, 8.5mM NaCl, 18.7mM NH
4Cl, 1mM MgCl
2With 1.5% agar).Select indivedual bacterium colonies through of combine (hybridoma cell line of a kind of secrete mAb 2C7 by US mode culture collection center [" the ATCC "] preservation, the ATCC preserving number of distribution be HB-11859) of Western trace mensuration with mAb 2C7.
This library is carried out 5 just take turns and select with encapsulating mAb 2C7 in 60mm tissue culturing plate, perhaps at first (Sigma, St.Louis MO) bear selection 1 hour, proceed 5 with mAb 2C7 afterwards and just take turns and select with irrelevant IgG3.
Select 107 bacterium colonies at random, it combines the ability of mAb 2C7 to utilize the examination of Western trace.Identify 14 clones that can combine mAb 2C7.From positive colony, prepare DNA then, utilization can be checked order with the regional bonded primer of nucleotide sequence 5 ' that is positioned at the insertion peptide and 3 ' side.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, the abduction delivering fusogenic peptide is 6 hours then.Bacillus coli cells is being fixed 10 minutes with 0.5% paraformaldehyde on ice.200 μ l equal portions of fixed biologically centrifugal 10 minutes with 2000 * g.Abandoning supernatant, with 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 is 37 ℃ of following incubations 30 minutes, with 2000 * g 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; Be resuspended to then and contain the anti-mouse IgG of FITC-link coupled (Sigma; St.Louis is in 100 μ l sealing damping fluid MO).Mixture is 37 ℃ of following incubations 30 minutes, with 2000 * g centrifugal 10 minutes afterwards.Abandoning supernatant with 100 μ l lavation buffer solution washing precipitations, is resuspended among the 1ml PBS afterwards.Use CellQuest software (Becton Dickinson, Franklin Lakes NJ) and on FACS, analyze this suspension.The feminine gender clone that can not combine mAb 2C7 is as contrast.
Observe Bacillus coli cells and mAb 2C7 combine strengthen (according to the 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 the maximum combined (MFI=19.81, MFI=4.91 compares with contrast) with mAb 2C7, [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, the abduction delivering fusogenic peptide is 6 hours then.Culture is standardized as identical OD reading (OD
600nm=0.7), adds 1% skim-milk, 150mM NaCl and 1% Alpha-Methyl mannoside, with the blocking-up non-specific binding.Every kind of nutrient solution of 50 μ l equal portions and 50 μ l mAb 2C7 (final concentration 20ng/ml) are 37 ℃ of following incubations 30 minutes, then with 100 μ l mixed solution application of samples to the hole of using the micro plate that encapsulates from the purifying LOS (80 μ g/ml) of 15253 strains preparation.The hole is 37 ℃ of following incubations 1 hour, washing then.Behind the washing hole, use with the anti-mouse IgG of SEAP link coupled and detect bonded mAb 2C7.A kind of feminine gender clone that can not combine mAb 2C7 is as contrast.
The PEP1 clone shows mAb 2C7 and LOS bonded largest inhibition (66%) [SEQ IDNO:1].PEP7, PEP3, PEP4, PEP2, PEP6 and PEP5 show and combine to suppress to reduce [SEQ ID NOS:7,3,4,2,6,5] as shown in Figure 4 respectively.It is relevant with the flow cytometry result to suppress ELISA result, because PEP1 also shows the maximum combined with mAb 2C7.Bacillus coli cells suppresses mAb 2C7 with the combination of mAb 2C7 with escherichia coli cloning and combines the reduction of LOS roughly relevant.
II. can synthesize with mAb 2C7 bonded and intend peptide
Synthetic (Boston Biomolecules, MA) sequence corresponding to consensus sequence " DE_GLF " and comprise two halfcystine flanking regions (lay respectively at N-end and C-end CGP-and-the GPC residue) a kind of synthetic peptide (PEP1; IPVLDENGLFAP), be used for combining with the specificity of 2C7mAb, and confirm whether the plan peptide that is characterized by Trx-fusion rotein keeps the antigenicity [SEQID NO:10] that does not rely on the fusion content through suppressing the ELISA assessment.
Add the halfcystine flanking region, estimate whether the cyclisation of intending peptide influences antibodies.Intend in the peptide at these, cysteine residues allows between them, to form disulfide linkage, produces annular and intends peptide.The peptide that these conformations limit possibly be similar to their institute's mimic epi-positions more, therefore possibly have more immunogenicity.
With 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; Dilute in the damping fluid in sealing) 37 ℃ of following incubations 1 hour, then with 100 μ l mixed solution application of samples in the hole of the micro plate of using the purifying LOS (80 μ g/ml) that from 15253 strains, prepares to encapsulate.The hole is 37 ℃ of following incubations 1 hour, washing then.Behind the washing hole, use with the anti-mouse IgG of SEAP link coupled and detect bonded mAb 2C7.The purifying LOS that from gonococcus 15253 strains, prepares 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].
PEP1 with the dose response mode suppress the combining of mAb 2C7 and LOS (for concentration be 0.1,0.5, the PEP1 of 1.0mg/ml, percentage suppresses to equal 17%, 77%, 91% respectively), as shown in Figure 5.Contrast 15-mer peptide synthesize cyclic peptide (
*CKSNPIHIIKNRRNIPC
*) [SEQ ID NO:9].This negative control peptide can not suppress combining of flat board that 2C7 mAb and purifying LOS encapsulate.
Utilize the known method in this area, aforesaid ring-type is intended peptide can further contain one or more " tail ", is used for second kind of reagent of coupling, like adjuvant or carrier proteins.
III. improve the immunogenicity of intending peptide
Although little peptide has immunogenicity, research several times reports that some little peptide possibly lack immunogenicity and cause invalid immunne response (particularly humoral response) (3,43).Used the immunogenicity that many strategies improve little peptide.Comprise peptide is connected (54,28,54) with carrier proteins,, use multiple antigenic peptide (MAP) to provide and to have the structure (39) of stronger immunogenic big configuration, peptide and complement proteins coupling are strengthened HI (15) peptide and adjuvant combination (21,22).
A. multiple antigenic peptide is synthetic
Multiple antigenic peptide (MAP) method is a kind of lysine residue bonded technology (44,8,43) that will intend peptide and 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.
Synthesized PEP1 multiple antigenic peptide and control peptide (Boston Biomolecules, MA), and through directly with suppress ELISA and measured and the combining of mAb 2C7.
Carry out combining of solid phase ELISA assessment mAb 2C7 and multiple antigenic peptide.For direct ELISA, encapsulate Immulon 1 flat board with multiple antigenic peptide (1 μ g/ hole) and spend the night, and with the mAb 2C7 of different concns reaction.For suppressing ELISA, flat board is used from the purifying LOS (80 μ g/ml) of neisseria gonorrhoeae 15253 strains preparation and was encapsulated 3 hours for 37 ℃.Peptide (linearity or MAP) produces the mixture of different concns with sealing damping fluid (0.5M NaCl is in PBS for 1% Protalbinic acid, 0.05% polysorbas20) dilution.50 μ l equal portions of each concentration and 50 μ l mAb 2C7 (storing concentration is 0.4 μ g/ml, in the sealing damping fluid, dilutes) are 37 ℃ of following incubations 1 hour, then with 100 μ l mixed solution application of samples in the hole of micro plate.The hole is 37 ℃ of following incubations 1 hour, washing then.After the washing of hole, use with the anti-mouse IgG of SEAP link coupled and detect bonded mAb 2C7.The purifying LOS that from gonococcus 15253 strains, prepares in suppressing ELISA as positive control.
The multiple antigenic peptide form that contains the PEP1 of 4 kinds of linear PEP1 molecules (" four MAP1 ") or 8 kinds of linear PEP1 molecules (" eight MAP1 ") shows and combines strongly with mAb 2C7, and contrast MAP is not directly showing combination among the ELISA, and is as shown in Figure 6.Four MAP1 and eight MAP1 boths suppress combining of mAb 2C7 and LOS more doughtily than linear PEP1, and are as shown in Figure 7.Half largest inhibition (the IC of four MAP1 and eight MAP1
50) observe at 1.26 μ M and 0.23 μ M place respectively.The IC of linear PEP1
50Be 55 μ M.This possibly be because MAP1 and mAb 2C7 bonded avidity improve.Contrast MAP does not show obvious suppression.
In mouse, resist-the LOS antibody response with eight MAP1 immune induction IgG, as shown in Figure 8.Response diagram is seen Fig. 8 (A), does not wherein have tangible IgG to resist-the LOS antibody response, when the 3rd week, strengthens, and shows that eight MAP1 cause the immunne response that the T cell relies in replying mouse.These results have proved with intending the prophesy that the human antagonism of peptide (like eight MAP1) immunity neisseria gonorrhoeae infects.
In Fig. 8 (A), 8 mouse are received at the 0th day that emulsive dosage is eight MAP1 of 50 μ g in the freund's adjuvant, and accept once more at the 21st day.Eight MAP1 of simulation 2C7 oligosaccharides epi-position merely hit 3 of 8 mouse and induce IgG to resist-LOS antibody.IgG in these 3 mouse is anti--and LOS replys and strengthens the back in the first time in the 3rd week and significantly raise, and peaks in the 7th week (next time of measurement), reduce afterwards.Fig. 8 (B) shows the positive control experiment, and wherein 4 mouse are immune with the LOS of purifying.In these mouse, IgG is anti--and LOS tires in minimally ground, immunity back increase for the first time, after reinforcement, raises.With 4 mouse (being negative control) of freund's adjuvant (C) or irrelevant eight MAP control peptides (D) immunity cause more weak or do not have IgG anti--LOS replys.Fig. 9 has shown that the average IgG of all immune mouses (from the experiments shown in Fig. 8) resists-LOS antibody response (MV ± SE comprises not showing the animal of replying).
Figure 10 has only shown that the IgG that replys mouse (from experiment shown in Figure 8) resists-the LOS antibody response.Antibody response is defined as the IgG that is higher than 0.4 μ g/ml anti--LOS (MV ± SE) (be higher than baseline IgG anti--4 times of LOS levels).In the 7th week and the 10th week behind initial immunity, the mouse of replying of eight MAP1 immunity produces the IgG that is higher than the antibody horizontal that (p＜0.001) negative control antigen (independent freund's adjuvant or irrelevant eight MAP control peptides) brings out and resists-the LOS antibody horizontal.
Figure 11 has only shown that the IgM that replys mouse (from experiment shown in Figure 8) resists-the LOS antibody response.With eight MAP1 immunity, produce IgG anti--mouse that LOS replys can not contrast the higher IgM of the mouse of negative control antigen immune, and anti--LOS level is replied.Resist-the LOS antibody horizontal with the immune higher IgM of animal that produces than eight MAP1 or negative control antigen (independent freund's adjuvant or irrelevant eight MAP control peptides) immunity of LOS (positive control).From the serum of eight MAP1 mice immunized neisseria gonorrhoeae 15253 strains are shown the bacterial activity of killing of 2C7 specificity complement-mediated, shown in figure 12.Figure 12 has shown a figure, shows to be exposed to neisseria gonorrhoeae 15253 strains and the survival rate of IgtG two mutants (the 2C7 epi-position is negative) thereof that mouse immune serum (the mouse immune serum final concentration of 67% volume percent) adds the people's complement (people's complement final concentration of 17% volume percent) that obtains from normal people's donor of adding.
15253 strains show the 2C7 epi-position.The 15253IgtG strain contains destructive fat oligosaccharides (LOS) Transglucosylase G allelotrope, and this enzyme can be transferred to glucose (through the α key) on the heptose-2 in the LOS core (4).The forfeiture that the destruction of IgtG locus causes the 2C7 epi-position to be expressed.
Carry out the standard bacteria-measuring and assess the bacterial activity (11) extremely of complement-mediated in the mice serum.In this is measured, under people's complement (17% final volume), mice serum (67% final volume) (from being described below immune or non-immune different mouse) and be suspended in about 2.5 * 10 in the MorseA substratum (33)
3Individual bacterium incubation.Descended continuous jolting reaction mixtures 30 minutes at 37 ℃ then.Equal portions with reaction mixture when 0 time and 30 minutes are inoculated on the Chocolate Agar flat board.When survival rate is expressed as 30 minutes during than 0 minute on the flat board percentage of bacterium colony increase.Be higher than 100% survival rate in the mensuration and show the growth between incubation period in 30 minutes.
As contrast,, but can not kill the 15253IgtG mutant strain with mAb 2C7 because it can kill neisseria gonorrhoeae 15253 strains with the complement that adds.Shown in Figure 12 (A), mAb 2C7 has bacterial activity extremely to the gonococcus of carrying the 2C7 epi-position.100% of 25 μ g/ml mAb2C7 (100 μ l in the TV 150 μ l reaction mixtures) mediation, 15253 strains kill and wound, but do not kill the 15253IgtG strain.
The serum (contain 5.05 μ g/ml IgG and resist-LOS antibody, merged by the blood of adopting in 7-11 week) that picks up from single mouse of eight MAP1 immunity shows 92% kill rate (8% survival) to 15253 strains, and the 15253IgtG strain is all survived, shown in Figure 12 (C).
Represent the normal mouse serum (IgG is anti--mean concns of LOS antibody is 0.1 μ g/ml) of the set of 20 mice serums can not kill any bacterial strain, shown in Figure 12 (B).The control mice serum that does not contain complement shows 116.1% ± 4.7% survival rate (not killing and wounding) to 15253 strains, the 15253IgtG two mutants is shown 123.1% ± 3.5% survival rate (not killing and wounding).The complement source that does not contain antibody shows 137.9% ± 1.0% survival rate (not killing and wounding) to 15253 strains, the 15253IgtG two mutants is shown 132.5% ± 14.3% survival rate (not killing and wounding).
The serum (contain 21.98 μ g/ml IgG and resist-LOS antibody, merged by the blood of adopting in 7-11 week) that picks up from single mouse of LOS immunity does not kill 15253 strains (179% survival) and 15253IgtG strain (133% survival), shown in Figure 12 (D).From not killing arbitrary bacterial strain, respectively shown in Figure 12 (E) and Figure 12 (F) with the serum of gathering independent freund's adjuvant or irrelevant single the mouse of eight MAP control peptides as the negative control antigen immune.
The IgG that from eight MAP1 mice immunized, obtains is anti--and the LOS antiserum(antisera) kills and wounds what neisseria gonorrhoeae 15253 strain display densities relied on, and is shown in figure 13.
Figure 13 show IgG anti--the LOS AC is to the figure of neisseria gonorrhoeae 15253 strain kill rates.In from 3 mouse of eight MAP1 immunity each IgG only anti--when LOS antiserum(antisera) level map to the bacterium kill rate, obtain dose-response figure (contain 1.38,2.50 and 5.05 μ g/ml to resist-mice serum of LOS antibody 15253 strains are shown 31%, 74% and 92% kill rate respectively).The kill rate that also shows mAb 2C7 at other LOS AC place of 5 branches is as positive control.
B. coupling A intends peptide and complement proteins C3d
Expectation through with complement factor C3d coupling, can further improve the immunogenicity of the plan peptide (so locating described eight MAP1) of gonococcal epitopes.
Big quantity research has proved the vital role (1,5,14,17,25,32,34 and 35) of complement proteins C3 in inducing HI.The mouse that C3 lacks shows the antibody response (34,35) that reduces to proteantigen such as the keyhole relative hemocyanin (" KLH ") that the T cell relies on.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, cause that with the covalently bound C3d of HEL (" HEL ") enhanced is to the antigenic antibody response of HEL (15).The mouse of the fusion protein immunization of being made up of 3 copy C3d and 1 copy HEL makes anti--HEL antibody response than separately with 10000 times of the antibody response raisings of HEL mice immunized.This fusion rotein inductive is anti--and the HEL antibody response is higher approximately 100 times than emulsive HEL inductive antibody response in freund's adjuvant.
Eight MAP1 can with the C3d coupling, method is that eight MAP1 dna sequence dnas are cloned in the C3d fusion rotein box, and transforms expression system with this member.So eight MAP1-C3d fusion roteins can obtain expressing, purifying also is 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 instance 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, the cell culture of Va.20110-2209U.S.A.) preservation, distribution ATCC preserving number HB 11311.
The instance of the hybridoma 2C7 of secretion mAb 2C7 (it shows and the similar immunoreactivity of plan peptide of the present invention) has the cell culture of March 9 nineteen ninety-five at 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 substruction can change, so that other embodiment of using method and composition of the present invention to be provided.Therefore, should be appreciated that scope of the present invention will be limited additional claims, rather than limited the specific embodiments of describing with the mode of embodiment in the preceding text.
The document of quoting
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Claims (20)
1. a kind of plan peptide that on human blood type antigen, does not have the conserved gonococcal epitope of discovery; Wherein this plan Toplink is induced the immunne response to this conserved gonococcal epitope in Mammals; Wherein said plan peptide is made up of the aminoacid sequence of SEQ ID NO:1, and randomly coupling sequence C of described aminoacid sequence GP is terminal in C-in N-end and sequence GPC; Make up with adjuvant; Be connected with carrier proteins; With the complement proteins coupling, perhaps provide with the multiple antigenic peptide form.
2. according to the plan peptide of claim 1, wherein immunne response is that the T cell relies on.
3. according to the plan peptide of claim 1 or 2, wherein the aminoacid sequence of this plan peptide all contains cysteine residues at each end.
4. according to the plan peptide of claim 3, wherein form cyclic peptide through the disulfide linkage between the cysteine residues of this each end of sequence.
5. according to the plan peptide of claim 4, wherein this plan peptide also contains at least one and is used for and second kind of reagent link coupled tail.
6. according to the plan peptide of claim 5, wherein second kind of reagent is a kind of adjuvant.
7. according to the plan peptide of claim 1 or 2, wherein this plan peptide also contains a kind of adjuvant or carrier proteins.
8. according to the plan peptide of claim 1 or 2, this plan peptide part that is a kind of multiple antigenic peptide wherein.
9. according to the plan peptide of claim 1 or 2, wherein combines preserving number with gonococcus LOS competition be the monoclonal antibody 2C7 of the hybridoma generation of HB-11859 to this plan peptide.
10. according to the plan peptide of claim 1, wherein this plan Toplink and preserving number are that the monoclonal antibody 2C7 that the hybridoma of HB-11859 produces combines.
11. plan peptide according to claim 1; Wherein should intend the monoclonal antibody of Toplink combination through producing with a kind of anti-idiotype monoclonal antibodies immunity Mammals; Or its fragment, described anti-idiotype monoclonal antibodies is produced by the hybridoma cell line that is registered as ATCC preserving number HB 11311.
12. one kind is used to resist neisseria gonorrhoeae and infects and the compsn of immunity, its contain the immunoprophylaxis significant quantity according to claim 1-2,4-6,8,10 or 11 each plan peptides.
13. one kind is used to resist neisseria gonorrhoeae and infects and the compsn of immunity, it contains the plan peptide of immunoprophylaxis significant quantity, and this plan peptide is made up of the peptide sequence of SEQ ID NO:1.
14. be used for the purposes of the medicine of immune Mammals antagonism neisseria gonorrhoeae infection in preparation according to the plan peptide of claim 1.
15., wherein should intend peptide and a kind of complement proteins coupling according to the plan peptide of claim 1.
16., wherein should intend peptide and complement proteins C3d coupling according to the plan peptide of claim 15.
17. the plan peptide of claim 16 is used for the purposes of the medicine of immune Mammals antagonism neisseria gonorrhoeae infection in preparation.
18. one kind is used to resist neisseria gonorrhoeae and infects and the compsn of immunity, it contains the plan peptide according to claim 16 of immunoprophylaxis significant quantity.
19. a raising comprises this plan peptide and complement proteins link coupled step according to the method for enhancing antigenicity of the plan peptide of claim 1.
20. according to the method for claim 19, wherein this complement proteins is C3d.
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| US6528061B1 (en) * | 1997-09-04 | 2003-03-04 | Pasteur Institut | Immunogenic polypeptides that mimic a surface polysaccharide antigen of a pathogenic microorganism, method for obtaining the same, and their use in vaccine compositions |
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