CN104039350A - Antibody recognizing arbitrarily designed epitope of three or more amino acid residues in a peptide and method of generating thereof - Google Patents
Antibody recognizing arbitrarily designed epitope of three or more amino acid residues in a peptide and method of generating thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1036—Retroviridae, e.g. leukemia viruses
- C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
- C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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Abstract
Peptide vaccine that is a mixture of different peptide species, where each species has a number of fixed amino acid residues and a number of randomized residues. The fixed resides are the same amino acid residues at the corresponding positions in each species of the mixture while the randomized residues are randomly any available candidate amino acids chosen by design. The degree of randomization may be also been chosen according to the design under a particular situation. This type of peptide vaccines have shown to be able to induce highly specific antibodies against epitopes that are otherwise difficult to induce antibodies in vitro, for example the GPG triplet in the V3 of HIV-1 gpl20.
Description
The cross reference relevant to application
The application requires the priority of U.S. Provisional Application, and the application number of this U.S. Provisional Application is 61/535,988, within 17th, submits in JIUYUE in 2011.By reference, its full content is all contained in this.
Technical field:
The present invention and immunological technique domain-specific.This invention is devoted to by the epitope antibodies that polypeptide vaccine designs and immunogen design induction forms for any amino acid residue, and the method can induce very difficult with derivative antibody in vitro.
Background technology:
The progress of polypeptide treatment has caused the new interest of people to polypeptide vaccine.Synthetic polypeptide vaccine has following advantage compared with in the past traditional deactivation or attenuated vaccine: easily preparation, high security, is not seldom even similar to the harm that deactivation or attenuated microorganisms vaccine bring immune system.Polypeptide immunogen has been widely used in the antibody induction for known specificity epitope.The amino acid residue of immunodominant epitope and flanking amino acid residue determine the induction of specificity epitope antibody.Virus that these Dominant Epitopes are present in highly sudden change is as HIV (human immunodeficiency virus) (human immunodeficiency virus, HIV), hepatitis C virus (hepatitis C virus, HCV), because they are all height mutational site conventionally, so conventionally seldom there is therapeutic value for the antibody of these Dominant Epitopes.
In the course of infection of HIV-1, main antibody response is removed to induce usually used as a bait in gp120 variable region, the neutralization of antibody but virus can be escaped effectively by suddenling change.Even if but after these region sudden changes, HIV-1 still has replication capacity, infects new cell.The conservative region of Gp120 is not Dominant Epitopes conventionally, is therefore difficult to induce very strong antibody response yet.But there are some researches show for the antibody capable of conserved epitope and well suppress viral infection.Before studies show that the cell adapted (T-cell-line-adapted at laboratory T, TCLA) the 3rd variable region (the third variable in HIV-1gp120, V3) be in main antibody and region (principal neutralizing determinant, PND); But the aminoacid sequence alterable height in this region makes the antibody of induction only have the specificity of identification identical sequence, is difficult to obtain wide spectrum neutralizing antibody, the inhibition of the viral V3 specificity neutralizing antibody of also can escaping easily; These character in PND region have greatly limited its probability as vaccine targets, and therefore, in the research strategy of vaccine, PND can only become limited potential target.In four amino acid residues on PND top, only having three amino acid residues is high conservatives, and the 4th aminoacid can suddenly change.First three amino acid residue is respectively glycine, proline and glycine (glycine, proline and glycine, GPG).By relatively more than 2000 part of HIV-1 memebrane protein sequence discovery, GPG is contained on the PND top in 95% above V3 district, but the 4th amino acid residue is made up of different amino acid residues in different virus.The frequency that wherein arginine (arginine, R) occurs is 34%, and the frequency that glutamine (Glutamine, Q) occurs is 54%, and the frequency that lysine (lysine, K) occurs is 4%.GPG is nonpolar, hydrophobic amino acid residue, and the 4th amino acids is polarity or alkaline hydrophilic amino acid residue.Functional study shows that PND is the major decision district of biting property of HIV-1, and the sudden change of the single amino acids residue in this region can stop the activity of HIV-1 infection Supt1 and cem cell; These conservative amino acid residues that studies show that particular sequence are being brought into play vital effect in viral function.Anti-HIV-1 vaccine research is for many years devoted to the wide spectrum neutralizing antibody of induction identification conserved region always, but only screen the antibody of at least four amino acid residues on top, specific binding V3 district at present, do not screen the antibody of three amino acid residues (GPG) of a specific binding top high conservative.For inducing the immunogen of this region antibody varied, there is virus-like particle (virus like particle, VLP), restructuring gp120, polypeptide or mixed polypeptide storehouse; The strategy of immunity also has a variety of, as cocktail polypeptide method, builds (human rhinovirus, HRV) embedded virus storehouse immunization of ERC group virus, continuous V3 polypeptide immune method etc.; But what these immunogens and immunization strategy all failed induces specificity wide spectrum neutralizing antibody, or for the antibody of GPG.Although, failed up to now good immunogen and immunization strategy can induce for this high conservative of similar GPG but weak immunogenic epitope antibodies suppressing may to bring into play very important effect in virus infection as this important conserved epitope of GPG of HIV.We are badly in need of a kind of brand-new immunogen or immunization strategy and go induction for picture this high conservative of GPG but weak immunogenic functional epitope antibody in sum.
Summary of the invention
First aim of the present invention is the protection antibody with treatment meaning that the polypeptide immunogen that designs by novel theory is induced, and these antibody can suppress the infection as the height mutated viruses of HIV type.
Second target of the present invention is to induce therapeutic antibodies by novel polypeptide vaccine to be applied to the infected's interior therapeutic.
The 3rd target of the present invention is the antibody that induces three amino acid residues of energy specific binding; The epi-position that these three amino acid residues form can be to be made up of three continuous amino acid residues, also can be by other aminoacid three discontinuous Amino acid profiles spaced apart.In the present invention, a special representative example is the HIV-1 wide spectrum neutralizing antibody of inducing specific in conjunction with gp120 V3 district GPG residue.
In the present invention, all targets will realize by application motif immunogen.Motif immunogen is the polypeptide libraries of a mixing, is made up of the amino acid residue of fixing and random amino acid residue.Fixing amino acid residue is present in the fixed position in any polypeptide in peptide library; What random amino acid residue was random appears at optional position in polypeptide chain.Fixing amino acid residue can be continuous or be interrupted appear in polypeptide chain.Fixing amino acid residue normally designs according to the amino acid residue of target epi-position.Depend on antibody and the application purpose thereof that will induce, selected fixed amino acid residue can be also the artificial sequence building.In motif immunogen, fixing amino acid residue is usually located in the middle of polypeptide chain, and flank is made up of random amino acid.The most suitable length of motif immunogen polypeptide is 15 amino acid residues; The motif immunogen polypeptide of 10-50 amino acid residue formation also can induce specific antibody.In fact this invention is applicable to the immunogenic antibody induction of motif of any length.Motif immunogen can be synthetic by conventional method, also can allow producer's business synthetic; Therefore the immunogenic synthetic method of motif is not the protection target of this invention.
The present invention does not rely on specific theory in the past, but is based upon in the scientific hypothesis of self.The scientific hypothesis of this invention is that the randomization that the randomization of (1) amino acid residue can shield immunodominant epitope (2) amino acid residue can help fixed amino acid residue to become immunodominant epitope; The epi-position that fixing amino residue forms is present in all polypeptide chains in all motif immunogen polypeptide libraries, but in the polypeptide chain that is present in minute quantity that can only be random by random amino acid epi-position that residue forms; From statistically analyzing in the immunogenic polypeptide libraries of motif, by the quantity of epi-position that fixed amino acid forms by the quantity by random amino acid epi-position that residue forms that is greater than of significance.By motif immunogen library strategy, the quantity of the target epi-position that can form with fixed amino acid residue without any the quantity of epi-position in library forms competition, even therefore the immunogenicity of target epi-position very a little less than, but because the quantity of the target epi-position quantity higher than other epi-position far away in the immunogenic library of motif, target epi-position also can become Dominant Epitopes in motif immunogen.In sum because motif immunity proper energy allows immune system concentrate on specific target Dominant Epitopes, so motif immunogen can induce specific antibody in conjunction with the epi-position being made up of arbitrary amino acid residue, can also induce non-existent specific antibody in the blood in human body or in normal animal body.Here the different objects according to user are made corresponding adjustment by the degree of randomization that it must be emphasized that flanking amino acid, thereby affect the composition of randomization aminoacid sequence.Random amino acid residue can be natural, can be also non-natural or through manually modified; In some specific positions are reduced degree of randomization or changed randomized sequence, the composition of some specific amino acids, also can affect the submission of fixed sequence program, thereby induce required antibody.Represent in example of the present invention, by 20 kinds of natural Amino acid profile random amino acids.In fact be less than the random natural amino acid of 20 kinds by use or add some other artificial aminoacid the antigenicity that also can reach the antigenicity that strengthens target epi-position and weaken simultaneously the competition epi-position that random amino acid forms, finally reach the effect that allows target epi-position become Dominant Epitopes.If 1 locational residue in polypeptide chain is made up of 20 kinds of random amino acids, this polypeptide libraries will contain 201=20 peptide species chain so; If 2 locational residues in polypeptide chain are made up of 20 kinds of random amino acids, this polypeptide libraries will contain 202=400 peptide species chain; If 3 locational residues in polypeptide chain are made up of 20 kinds of random amino acids, this polypeptide libraries will contain 203=8000 peptide species chain so; The like, if 10 locational residues in polypeptide chain are made up of 20 kinds of random amino acids, this polypeptide libraries will contain 2010=1.024x1013 peptide species chain so.Under given conditions, thus use conventional technology go to change composition that the ratio of particular amino acid residue in polypeptide synthesis material change random peptide library and immunogenicity and then remove to induce different immunoreation and specific antibody.By routine techniques can coupling glycosyl to one or more fixing amino acid residues, thereby synthetic glycopeptide removes linear antibody or the conformation antibody of inducing specific for glycopeptide again; Also can remove to modify specific amino acid residue by means such as sulfo groups and go to induce corresponding specific antibody.Introduce unconventional aminoacid to the random amino acid region in polypeptide chain, can increase significantly the stability of polypeptide, and then improve induction and the therapeutic effect of antibody.For the same reason, this fixing amino acid residue neither be absolute.Because specific position occurs that a certain proportion of amino acid residue can allow the enough advantages of these amino acid residues remove to reach Dominant Epitopes in polypeptide libraries; So fixing position in whole polypeptide libraries, occurs that 100% amino acid residue just will reach better immune effect.In sum, the application of this invention is not limited to the degree of randomization of random amino acid residue and the immobilization degree of fixing amino acid residue and the type of amino acid residue.Theoretical foundation of the present invention is to go the immunodominance district or the antigenicity that highlight fixing amino acid residue to remove to induce antibody by randomization amino acid residue; The present invention can need go to adjust fixed amino acid residue and the amino acid whose random degree of randomization according to reality.
As a representative of the present invention, GPG motif polypeptide vaccine is used in mammalian body inducing specific for the antibody of GPG epi-position, in the former research of this antibody-like, is never in the news.Polypeptide chain in this polypeptide libraries is made up of 15 amino acid residues, and sequence is: XXXXX XGPGX XXXXC (Fig. 9).This polypeptide vaccine is named as V3M01 in the present invention.We have induced the antiserum for GPG motif of high titre by conventional technology immune peptide vaccine in Balb/c Mice Body.By monoclonal hybridoma technology, obtain many strains hybridoma monoclonal cell, this cell can be secreted the monoclonal antibody of specificity for GPG epi-position.In HIV pseudovirus neutralization test, the HIV pseudovirus that GPG epi-position is contained in the monoclonal anti physical ability that this GPG epi-position relies on and the top in any HIV V3 district.In addition, can also be by similar method, use motif polypeptide vaccine to remove the particular treatment associated antibodies of induction for poor antigen epi-position.
The present invention also provides some in viral infection person body, the method for this polypeptide vaccine treatment and the effect for the treatment of.
The invention provides the antibody of the epi-position of three amino acid residues formations of specific binding, and the binding specificity of this antibody is not subject to the impact of the flanking amino acid residue of these three amino acid residues.For example specificity, for the antibody of GPG epi-position, plays very important effect in prevention HIV infects.In Seeking for Right, set forth various novelty of the present invention.With the operational advantage of invention and the specific objective of invention, chart and relevant description will continue to list in the back for a better understanding of the present invention, and chart and word will continue to explain the representative instance of this invention.
Brief description of the drawings:
Fig. 1, according to the present invention, qualification motif immunogen V3M01 immune mouse obtains 5 and exempts from rear antiserum.A:Elisa detects sero-fast titre, and what M01-M06 was corresponding is the numbering of immune mouse, and blank-serum refers to not accept the blank serum of immune mice.The numerical value that X-axis shows is multiplied by 1000 times of multiples for mice serum gradient dilution; B: mouse resisting anteserum is in conjunction with the gp120(# of different HIV hypotypes, refers to that serum compared significance with gp120 associated value with the associated value of the bovine serum albumin of serum and negative control, has significant difference).
Fig. 2, sign and Fig. 1 in figure are similar, and this figure shows the sero-fast titre obtaining by the immunity of immunogen V3M01 immunity new zealand white rabbit, and the English that the R in R01-03 is rabbit is write a Chinese character in simplified form, the numbering that 01-03 is rabbit.(using immunogen as antigen).
Fig. 3, rabbit anti-serum binding motif polypeptide after the 5th immunity (A, B and C are respectively 3 sero-fast association reactions of rabbit); After the 5th immunity, rabbit anti-serum is in conjunction with gp120 (D, E and F refer to respectively 3 sero-fast association reactions of rabbit).
Fig. 4, qualification NJU009 binding motif polypeptide (A), HIV polypeptide (B), HIV albumen (C, D).D is NJU009 protein-bonded block diagram in the time that concentration is 100ug/ml.
Fig. 5, in NJU009 monoclonal antibody and the pseudovirus of various different subtypes.
Fig. 6, the rabbit anti-serum (A) that the V2M01 immunogen that appraisal basis immunity of the present invention contains part amino acid residue in V2 sequence is induced and the polyclonal antibody (B) of Protein G affinity purification.
Fig. 7, the polyclonal antibody R182 of V2M01 induction, R183, the association reaction of R184 (being respectively A, B, C) and gp120.
Fig. 8, rabbit anti-serum (A) after intrinsic the 5th immunity of inducing for the designed gp41M01 immunogen of part amino acid residue in gp41 sequence of appraisal basis the present invention immunity, polyclonal antibody (B) and the polyclonal antibody of Protein G affinity purification react with the T20 in gp41.
Fig. 9, the aminoacid sequence of designed polypeptide vaccine in special representative instance in demonstration the present invention.X: represent random arbitrary amino acid residue, the English-word mother of other alphabetical represented amino acid residue writes a Chinese character in simplified form.
Figure 10, from difference clone's memebrane protein relevant information, and in pseudovirus experiment, wide spectrum neutralizing antibody is in pseudovirus and sensitivity.The total antibody concentration of the concentration of antibody for obtaining by Protein G affinity purification antiserum or ascites.Nab concentration or titer refers to antibody concentration in the time that virus infected cell reaches 50% suppression ratio or the dilution factor of serum.Negative control refers to the infection that suppresses MuLv pseudovirus.ND: refer to because serum does not do coherent detection not.
Figure 11, according to the present invention, for influenza virus, hepatitis C virus and chicken ovalbumin design respectively HAM01, and EM01 and OVAM01 show the corresponding sero-fast titre qualification that these immunogens are induced in figure.
Figure 12, V2M01 is according to the design of FYXXD motif, and 2 the random amino acid residues in fixing amino acid residue FY and D interval, represent with X; In this figure, show the association reaction of antibody and V2M01 vaccine polypeptide.
Figure 13, Gp41M01 is according to the design of LDXW motif, and 1 the random amino acid residue in fixing amino acid residue LD and W interval, represents with X; In this figure, show the association reaction of antibody and gp41M01 vaccine polypeptide.
Detailed description of the invention
immunogen builds
The sequence of immunogen motif polypeptide libraries (novel polypeptide vaccine) is design and illustrate with the special representative instance of figure mono-according to the present invention; X represents any random amino acid residue except cysteine.Motif polypeptide libraries is synthetic by the biochemical company limited of Shanghai gill.At the c-terminus of polypeptide, with underscore indicate cysteine (cysteine, C) be in order to facilitate coupling carrier albumen.Every polypeptide immunogen is coupled to keyhole limpet hemocyanin, and in order to facilitate enzyme-linked immunosorbent assay (enzyme-linked immunesorbent assay, ELISA) to detect, V3M01 is coupled to bovine serum albumin.
immune mouse and new zealand white rabbit
Immunogen is dissolved in phosphate buffer (PBS, pH7.4), and concentration is 1mg/ml.Carried out at the 0th week just exempting from, it is fully emulsified that the immunogen solution that every mice is got 100ul1mg/ml and 100ul Freund's complete adjuvant carry out, and forms water in oil emulsion, carries out the immunity of multiple spot abdominal part hypodermic, immune 6 mices.Respectively the 2nd week, 5 weeks, 8 weeks, 11 weeks booster immunizations 4 times, each polypeptide solution of every mouse immune 50ul1mg/ml and the emulsion of 50ul incomplete Freund's adjuvant; Blood sampling in the 6th week, 9 weeks, 12 weeks.
Immunity new zealand white rabbit, carried out at the 0th week just exempting from, and it is fully emulsified that every polypeptide solution of getting 500ul1mg/ml and 500ul Freund's complete adjuvant carry out, and forms water in oil emulsion, carries out the subcutaneous injection immunity of multiple spot back, immune 3 new zealand white rabbits.Respectively the 2nd week, 5 weeks, 8 weeks, 11 weeks booster immunizations 4 times, each polypeptide solution of every immune 500ul1mg/ml and the emulsion of 500ul incomplete Freund's adjuvant; Blood sampling in the 6th week, 9 weeks, 12 weeks.The antiserum of mice and rabbit obtains antibody by Protein G/A affinity purification.
the preparation of monoclonal antibody
In latter 1 week of last immunity, by the PEG method fusion routinely of the splenocyte of murine myeloma cell (Sp2/0) and immune mouse, limiting dilution assay clone cell, indirect ELISA screening specific antibody, the continuous sub-clone of positive cell 2 times, cell strain after cloning is defined as through going down to posterity after stable cell strain, and liquid nitrogen is preserved.The monoclonal cell strain of acquisition is injected to abdominal cavity, preparation ascites, affinity purification obtains monoclonal antibody, the hypotype of the monoclonal antibody detection kit qualification monoclonal antibody that obtains.
enzyme-linked immunosorbent assay detects antiserum titre
Elisa (enzyme-linked immunoassay, ELISA) check-out console is coated with the concentration of 10ug/ml, every hole 100ul, after 4 DEG C of overnight incubation, seal with 4%BSA, every hole 200ul, hatch 1h for 37 DEG C, wash 4 times, it is normal mouse serum that every hole adds the antiserum 100u(l contrast of gradient dilution), hatch 1h for 37 DEG C, wash 4 times, every hole adds the anti-100ul of sheep anti mouse two of the alkali phosphatase enzyme mark of 1:1000 dilution, hatch 1h for 37 DEG C, wash after 5 times, add 100ul p-NPP substrate, hatch 15min for 37 DEG C, the NaOH stopped reaction of 50ul2M, optical density value (the optical density405nm of working sample under 405nm wavelength, OD405nm).Elisa detects tiring of serum, and to be defined as in the optical density value of 405nm be the above highly diluted multiple of 2 times of blanks (coated detect formerly, be labeled as Neg in figure).
clone's total length membrane protein gene and packaging pseudovirus
The membrane protein gene of pcr amplification total length the proviral DNA of the peripheral blood lymphocytes of not cultivating in the infected's body.PCR set condition be denaturation 94oC2 minute, move 35 loop parameters and arrange as follows: 94 DEG C 15 seconds, 55 DEG C 30 seconds, 68 DEG C 4 minutes; Finally extend 68 DEG C 10 minutes.The primer sequence of different subtype designs according to published conservative hypotype sequence.The fragment of pcr amplification is cloned into the plasmid vector of the pcDNA3.1 expression of handsome company, identifies by direct Sequencing.PNL43R-E-Luciferase plasmid to 293 cell by common transfection expression memebrane protein plasmid and expression skeleton produces the pseudovirus that memebrane protein relies on.The pseudovirus of contrast is for expressing HIV-1HXB2, the pseudovirus of the surface membrane protein of SF162 or JRFL and double biting property Mus source leukemia pseudovirus.After 48 hours, collect the cell conditioned medium after transfection, measure fluorescent value, the consumption of standardization pseudovirus in follow-up function is analyzed.
the pseudovirus neutralization test of antiserum and monoclonal antibody
The serum of collecting, the wide spectrum neutralizing antibody 4E10 of the antibody of purification and purchase, 447-52D, b12 is used to do in pseudovirus and experimental analysis simultaneously.All blood serum samples before detection all through 56 DEG C of hot deactivations of 1 hour.The pseudovirus of 200TCID50 and serum or antibody, in 96 orifice plates, repeat 3 holes, hatch one hour for 37 DEG C; Add 1 × 10
4individual GHOST (3) X4/R5 cell suspension in each hole, 37 DEG C of 5%CO
2cultivate 48hr.Judge that by detecting the activity of luciferase neutralization is active.In calculating and suppression ratio with in and titre, suppression ratio=[1-(the RLU average of RLU average-cell matched group CC of sample sets)/(the RLU average of RLU average-cell matched group CC of virus control group VC)] × 100%.In and titre (ID
50) dilution inverse while being expressed as 50% suppression ratio.
v3M01 antiserum titer determination
2 exempt from after, 6 mices (M01-06) are all successfully induced specificity for immunogenic antiserum (M01-06
srm); After five immunity, the antiserum ELISA titre of 6 mices is all greater than 10,000, and blank serum does not react (blank with immunogen
srm), and the antiserum titre of each mice is without significant difference (figure .1.A).The demonstration of ELISA serological analysis, this antiserum can be identified HIV-1 envelope protein (gp120
aDAand gp120
iIIB), but do not contain GPG epi-position with negative control protein B SA() in conjunction with (figure .1.B).In sum 6 mices can be induced out high titre for immunogenic antiserum, and can have very strong association reaction from the memebrane protein that derives from different strains that contains this epi-position.
in NJU009 monoclonal antibody and active qualification
NJU009 is the wide spectrum neutralizing antibody that the neutralization activity in the monoclonal antibody filtering out according to monoclonal hybridoma technology is stronger.
In pseudovirus, check the neutralization activity of NJU009 with system; Pseudovirus is made up of 11 primary separation strains and 2 laboratory adapted strains.Its virus subtype has the pseudovirus of the different subtypes such as B, B`, C, B`C, CRF07_BC, CRF08_BC, CRF01_AE.11 strain pseudoviruss in 13 strains can well be neutralized by NJU009, its 50% in and concentration (50%Neutralization Dose, ND
50) scope be to suppress the 3.7ug/ml of B ' C subtype C NE16 to the 20.5ug/ml that suppresses C subtype C NE58; Meanwhile NJU009 can not in and pseudovirus CNE6 and CNE11.JR-FL pseudovirus is the strain that CCR5 relies on, and always this strain is not had to very strong neutralization activity in conjunction with the antibody of V3.In NJU009 and the ND of JR-FL pseudovirus strain
50for 27.8ug/ml, the ND50 of the HXB2 pseudovirus strain relying on for CXCR4 is 34ug/ml.
NJU009 can neutralize the HIV-1 strain of nearly all different subtype, and neutralization curve gathers together closely and illustrates that the neutralization activity of NJU009 is not subject to the impact of GPG flanking amino acid residue variation.Wherein CNE6 and CNE11(are B ' hypotype) completely insensitive to the neutralization of NJU009, sequence alignment finds in V3PND district, and 2 viral V3PND districts are extremely rare sequence, and CNE6 is GLG, and CNE11 is GQG, the two is not all common GPG sequence.This presentation of results, the neutralization activity of NJU009 is mediated by GPG sequence-specific.
In pseudovirus and in experiment, we are not to rely on the negative contrast of MuLv pseudovirus of CD4 receptor and accessory receptor, we are using the wide spectrum neutralizing antibody that identifies immunological properties as positive control antibody, and the wide spectrum neutralizing antibody of choosing has 447-52D, b12 and 4E10; The neutralization activity of these positive antibodies neutralization and other document detection effect is in this experiment basically identical, proves in pseudovirus of the present invention fine with experimental repeatability.447-52D is up to now in the neutralizing epitope for V3 (GPGR), in and the widest monoclonal antibody of broad spectrum activity; For the ND of HXB2 and two strains of JR-FL
50be respectively 0.737ug/ml and 19ug/ml.447-52D can be well in and the CNE40 pseudovirus strain of CFR07_BC hypotype, this strain is in our this pseudovirus neutralized system to be the strain being the most easily neutralized.447-52D suppresses the ND of CNE40
50for 0.14ug/ml.But 447-52D can not neutralize other strain.B12 is the specific antibody in conjunction with epi-position for CD4,7 strains in energy and in 13 strain pseudovirus strains, ND
50scope be to suppress the 0.119ug/ml of HXB2 to suppressing the 23.07ug/ml of CNE5.4E10 is the wide spectrum neutralizing antibody for gp41 membrane-proximal region epi-position, can neutralize 13 all strain pseudoviruss.
epi-position qualification
For being mediated instead of participated in by the amino acid residue of periphery by GPG with activity in the wide spectrum of definite NJU009, we analyze NJU009 and antigen reactive specificity.In the time that GPG3 amino acid residue sported to 3 alanine (Alanine, A), NJU009 completely be combined with the polypeptide of sudden change (Fig. 2) illustrate that NJU009 is GPG specificity highly to the identification of V3, this is consistent with active observation with pseudovirus.This result also illustrates that tri-residues of GPG may be all direct ingredients of NJU009 target position simultaneously.NJU009 can be in conjunction with the V3 polypeptide from different subtype and the different subtype gp120 that contains GPG, as gp120
aDA(B hypotype), gp120
bC(BC hypotype) and gp120
iIIB(B hypotype).These results all illustrate that NJU009 is the monoclonal antibody that not affected by flanking amino acid residue that GPG epi-position relies on.By RT-PCR, we have recorded the aminoacid sequence in 6 monoclonal antibody Fv districts.Wherein NJU001, NJU003, the light chain variable region amino acid sequence of tri-monoclonal antibodies of NJU005 is in full accord, NJU007, the light chain variable region sequence of NJU008 and NJU009 is also consistent.These results show NJU001, NJU003, and NJU005 probably derives from same precursor hybridoma; NJU007, NJU008 and NJU009 derive from another precursor hybridoma.Main Differences sequence between these clones is to be positioned at complementary determining region and variable region of light chain c-terminus sequence.
NJU009 and 447-52D all can identify the aminoacid sequence on the top in V3 district.447-52D competition NJU009 shows that in conjunction with the competitive ELISA result of V3 polypeptide competition binding curve is two-phase curve (bi-phasic), in the time that 447-52D concentration is less than 6.25ug/ml, along with the raising of 447-52D concentration, NJU009 weakens successively in conjunction with the amount of gp120, the combination of the highest minimizing 85%.Need the 447-52D that is reached 15 times of concentration to go competition to fall (Fig. 2) but be combined in upper remaining 15% the NJU009 of gp120.These presentation of results NJU009 has and intersects with the antibody epitope of 447-52D.There is the combination activity of about 10%NJU009 not to be subject to high concentration 447-52D impact.
the immunogenic inducibility of motif and animal specificity
For whether the immune effect of identifying V3M01 has species specificity, 3 new zealand white rabbits (R01-03) are by the immunization strategy inoculation V3M01 immunogen of similar mice.Result has induced has similar specific serum antibody, and 5 exempt to tire is 42,000(Fig. 2).All serum antibodys all can be identified gp120
iIIBand gp120
aDA, and gp120
b ' C.These serum have similar neutralization activity and GPG epi-position dependency equally.This immunogen induction antibody of these presentation of results is not subject to the restriction of animal species, can in the animal body of which kind of genus in office, induce similar wide spectrum neutralizing antibody.
the extensive use of motif immunization method
In order to illustrate that the motif immunity targeted induction antibody in the present invention is not only applicable to the polypeptide of induction for GPG epi-position, can also be applied to the targeting antibodies induction of other epi-position, we have selected the epi-position of upper other sequences of gp120 as targeted induction; These epi-positions are the known immunogenic epi-positions that have, but are difficult to induce the antibody for this epi-position by conventional immunization method.First variable region (Variable Region1, V1) on HIV memebrane protein surface and second variable region (Variable Region2, V2) mediation wide spectrum cross-neutralization activity, be induced out but seldom have for the antibody of these domains.We have selected FYXXD as targeting epi-position on the top of V2, and reason is as follows: (1) recently from having separated the neutralizing antibody of two wide spectrums in the infected's body, PG9 and PG16, and these two antibody epitopes are in V2He V3 district; Aminoacid point mutation discovery, when the 176th phenylalanine (Phenylalanine, F) residue sported to A, the IC of the inhibition viral infection of two antibody
505000 times and 7000 times are increased respectively; The aspartic acid (Aspartic acid, D) of (2) the 180th is the amino acid residue in integrin alpha 4 β 7 binding sequence LDL, and integrin alpha 4 β 7 are the special receptors of intestinal mucosa in conjunction with periphery T cell; The leucine (Leucine, L) that the lysine (Lysine, K) and the 179th of (3) the 178th is is not high conservative in HIV strain, only has F176 on V2 top, Y177 and D180 are high conservatives, and its conservative rate is respectively 93%, 91% and 96%.Therefore these three amino acid residues are structured in the motif immunogen for V2.By the polypeptide libraries of 15 amino acid residues of chemosynthesis, in polypeptide libraries, Y177 and D180 are spaced apart two amino acid residues, and polypeptide libraries is named as V2M01.At new zealand white rabbit subcutaneous inoculation V2M01, after 5 immunity, sero-fast ELISA titre is greater than 80000(Fig. 9 and Figure 12).The gp120 albumen of this antiserum and different subtype (is respectively gp120
aDA, gp120
bCand gp120
iIIB) there is very strong association reaction, can also be in conjunction with the V2 glycoprotein of restructuring; But these antibody do not show very strong neutralization activity.Gp41 membrane-proximal region (Membrane Proximal External Region, MPER) is a wide spectrum neutralizing epitope, has 4E10 and 2F5 for the wide spectrum neutralizing antibody of this epi-position.These two antibody are all to screen and obtain in the infected's body, have a large amount of tests go to attempt induction for this epi-position be similar to 4E10 and 2F5 antibody, but all fail.The antibody epitope of 2F5 is ELDKWA, and the conservative amino acid residue of this epi-position camber is L663, D664, W666; Its conservative rate is respectively 98%, 97% and 99%.Therefore we have designed and have contained these amino acid residues, LDXW, 15 amino acid whose polypeptide libraries remove to induce antibody, the antibody capable of inducing is well in conjunction with T20(Fig. 9 and Figure 13).
Based on above result, can induce by the targeting antibodies inductive technology in the present invention the epitope antibodies that unknown epi-position and arbitrary amino acid form.We have designed three polypeptide libraries, are respectively HAM01, OVAM01, EM01; They derive from respectively influenza virus, hepatitis C virus and chicken ovalbumin.In HAM01, three fixing continuous amino acids are HHP, and these three aminoacid are positioned at the 199th to 201 of influenza virus HA1 albumen, are the aminoacid of three high conservatives, are present in the different subtype strain of influenza virus; At present only there is the epi-position of the wide spectrum neutralizing antibody S139/1 of an influenza virus to contain HHP.Three continuous fixed amino acid residues that EM01 contains are HRM, and these three amino acid residues are positioned at the 316th to 318 of hepatitis C virus E1 albumen, and they are also the aminoacid of high conservative.The fixed amino acid that OVAM01 contains is the ERK that is positioned at the 276th to 278 of chicken ovalbumins.These three polypeptide libraries have all induced the antibody (Figure 11) of high titre.
Not as a part of the present invention, just as hypothesis of the present invention; Our hypothesis thinks, strategy of the present invention is to allow selected epi-position highlight by minimizing flanking amino acid sequence on the impact of selected epi-position, is easy to allow immune system recognition.The antibody for GPG epi-position that we obtain is at present unique; Up to now, in the infected's body, do not screen any antibody for GPG epi-position, only have the antibody of part for GPGR and GPGQ epi-position.These presentation of results GPG epi-position, in natural virus infection, be difficult to be only had in other words a small amount of B cell clone can identify GPG epi-position by immune system recognition, but in the time of clonal expansion, such B cell clone may be lost.Mechanism Study shows compared with GPG epi-position, GPGR, and GPGQ or GPGK are Dominant Epitopes in the time of clonal expansion, therefore, in natural infection process, finally can be lost for the clone of GPG epi-position.Nuclear magnetic resonance, NMR is usually used to study the interaction of V3 polypeptide and a Serial relation antibody.0.5 β is the antibody of a typical specificity for V3, can be attached to 16 amino acid residues of V3 polypeptide.447-52D is for the V3 epi-position neutralizing antibody of wide spectrum the most, can interact by the amino acid residue in conjunction with GPGR site and V3 polypeptide; And more with the amino acid residue of the direct combination of 0.5 β, finally at the higher affinity also having shown in the time that V3 polypeptide is combined with in conjunction with rigidity.Because NJU009 is only in conjunction with three amino acid residues on V3 top, and not affected by the amino acid whose of these three amino acid residue peripheries in conjunction with active, thus NJU009 with the cohesive process of V3 polypeptide in, there is better flexibility.This is also perhaps in NJU009 and one of the reason of active wide spectrum the most.
In sum, the present invention is significant by being applied at polypeptide vaccine in the process of clinical treatment.In addition, according to the present invention we can also using chimeric sequence arbitrarily in polypeptide or albumen as detecting or the label of purification.
the antiserum of V2M01 and gp41M01
Fig. 6 A and 8A show that the antiserum after V2M01 and gp41M01 immunity new zealand white rabbit can specific binding motif immunogen.And the antiserum of V2M01 can react with the gp120 of different subtype.The peptide T 20 of gp41 contains the fixed amino acid LDXW in motif; The antiserum of Gp41M01 induction also can be well in conjunction with T20.
Basic innovative point of the present invention is described out according to the representative instance of application.Various omission in representative instance, replaces and changes all and carry out according to thought of the present invention.The present invention is not only limited to the application of above mentioned representative instance; Above-mentioned representative instance is only an example for convenience of description.In fact this invention can also be mentioned in patent right demand in attached sheet, the modification that can use various different modes; Although do not mention concrete example, the present invention has very strong practical value.Because what use in the present invention is all conventional experimental technique, but invention in thought, thinking and viewpoint are innovated.Once thought of the present invention, thinking and viewpoint are set forth out, this invention just can very simply be applied in conventional technical field gone innovation achievement.
Claims (19)
1. polypeptide vaccine comprises its polypeptide of peptide library and has an identical fixing amino acid residue in some positions, is random amino acid residue in other positions.
Polypeptide vaccine in claim 1 wherein the number of fixed amino acid residue be 3.
Polypeptide vaccine in claim 2 wherein fixed amino acid residue be on continuous position.
4. wherein fixed amino acid residue can be on continuous position for the polypeptide vaccine in claim 2, but is separated by one or more random amino acid residue.
Polypeptide vaccine in claim 1 wherein the flank of fixed amino acid residue formed by random amino acid residue.
6. the polypeptide vaccine in claim 1, wherein the number of random amino acid can be between 5 to 50.
7. the polypeptide vaccine in claim 6, wherein the number of random amino acid residue is 11.
8. treat a method for viral infection, wherein using many vaccines is mixtures of polypeptides, and this mixtures of polypeptides is a polypeptide libraries; This polypeptide libraries contains multiple fixing amino acid residues and multiple random amino acid residue.
9. the viral infection mentioned in claim 8 method refers to and is caused by HIV.
10. the viral infection of mentioning in claim 9 method refers in human body.
The manufacture method that contains aforementioned polypeptides vaccine in 11. claim 10 methods and aforementioned polypeptides vaccine are to human body therapy effect.
In 12. claim 8 methods, in said mixture polypeptide, at least contain 400 kinds.
In 13. claim 12 methods, in said mixture polypeptide, at least contain 8000 kinds.
14. 1 antibody capable specific recognition are only by the epi-position of three Amino acid profiles, and not affected by the amino acid whose flanking amino acid residue of epi-position; 3 amino acid residues above mentioning also can not be subject to the impact of flanking amino acid in the time of derivative antibody recognition.
Antibody in 15. claim 14,3 amino acid residues mentioning refer to GPG.
Antibody in 16. claim 14, the antibody of mentioning is antiserum.
Antibody in 17. claim 14, the antibody of mentioning is polyclonal antibody and monoclonal antibody.
Polypeptide vaccine in 18. claim 1, polypeptide libraries contains at least 400 kinds of different classes of polypeptide.
Polypeptide vaccine in 19. claim 18, polypeptide libraries contains at least 800 kinds of different classes of polypeptide.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201161535988P | 2011-09-17 | 2011-09-17 | |
| US61/535,988 | 2011-09-17 | ||
| PCT/US2012/055771 WO2013040564A2 (en) | 2011-09-17 | 2012-09-17 | Antibody recognizing arbitrarily designed epitope of three or more amino acid residues in a peptide and method of generating thereof |
Publications (2)
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| EP (1) | EP2755687A4 (en) |
| JP (2) | JP2014527085A (en) |
| CN (1) | CN104039350B (en) |
| AU (2) | AU2012308212A1 (en) |
| CA (1) | CA2855781A1 (en) |
| HK (1) | HK1200359A1 (en) |
| WO (1) | WO2013040564A2 (en) |
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| CN111153996A (en) * | 2020-01-10 | 2020-05-15 | 苏州睿瀛生物技术有限公司 | Antibody of G protein coupled receptor, preparation method thereof and G protein coupled receptor kit |
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| US10245313B2 (en) | 2014-10-24 | 2019-04-02 | Versitech Limited | DNA motif compounds and methods for inducing specific antibodies and cellular immunity |
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| WO2009046984A1 (en) * | 2007-10-09 | 2009-04-16 | Technologie Integrale Ltd. | Hiv preventive vaccine based on hiv specific antibodies |
| US20100291145A1 (en) * | 1999-09-14 | 2010-11-18 | Antigen Express, Inc. | Ii-KEY/ANTIGENIC EPITOPE HYBRID PEPTIDE VACCINES |
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| JPH09504296A (en) * | 1993-10-26 | 1997-04-28 | シンテロ インコーポレイテッド | Prevention of HIV mucosal infection |
| EP0725838A4 (en) * | 1993-10-26 | 1997-02-26 | United Biomedical Inc | Structured synthetic antigen libraries as diagnostics, vaccines and therapeutics |
| US6441140B1 (en) * | 1998-09-04 | 2002-08-27 | Cell Signaling Technology, Inc. | Production of motif-specific and context-independent antibodies using peptide libraries as antigens |
| FR2809402A1 (en) * | 2000-05-26 | 2001-11-30 | Dev Des Antigenes Combinatoire | CONVERGENT COMBINATORY PEPTIDE LIBRARIES AND THEIR APPLICATION TO VACCINATION AGAINST HEPATITIS C VIRUS |
| AU2005211424A1 (en) * | 2004-02-02 | 2005-08-18 | Mixture Sciences, Inc. | Peptide mixtures with immunomodulatory activity |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100291145A1 (en) * | 1999-09-14 | 2010-11-18 | Antigen Express, Inc. | Ii-KEY/ANTIGENIC EPITOPE HYBRID PEPTIDE VACCINES |
| WO2009046984A1 (en) * | 2007-10-09 | 2009-04-16 | Technologie Integrale Ltd. | Hiv preventive vaccine based on hiv specific antibodies |
Non-Patent Citations (3)
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| CLAUDIA CHARLES-NIÑO等: "Variable epitope libraries:New vaccine immunogens capable of inducing broad human immunodeficiency virus type 1 neutralizing antibody response", 《VACCINE》 * |
| RESNICK D.A.等: "Libraries of human rhinovirus-based HIV vaccines generated using random systematic mutagenesis", 《ADIS RESEARCH&HUMAN RETROIRUSES》 * |
| SIROIS S.等: "HIV-1 gp120 V3 loop for structure-based drug disign", 《CUURENT PROTEIN AND PEPTIDE SCIENCE》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111153996A (en) * | 2020-01-10 | 2020-05-15 | 苏州睿瀛生物技术有限公司 | Antibody of G protein coupled receptor, preparation method thereof and G protein coupled receptor kit |
| CN111153996B (en) * | 2020-01-10 | 2021-12-14 | 苏州睿瀛生物技术有限公司 | Antibody of G protein coupled receptor, preparation method thereof and G protein coupled receptor kit |
Also Published As
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| AU2012308212A1 (en) | 2014-03-27 |
| AU2017251715A1 (en) | 2017-11-09 |
| CA2855781A1 (en) | 2013-03-21 |
| WO2013040564A2 (en) | 2013-03-21 |
| HK1200359A1 (en) | 2015-08-07 |
| EP2755687A4 (en) | 2015-04-08 |
| JP2017141291A (en) | 2017-08-17 |
| EP2755687A2 (en) | 2014-07-23 |
| CN104039350B (en) | 2019-07-05 |
| JP2014527085A (en) | 2014-10-09 |
| WO2013040564A3 (en) | 2013-06-27 |
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