CN1759121B - Purification process for bacterial cytolysin - Google Patents

Abstract

The present invention relates to a method for purifying bacterial cytolysins such as pneumococcal pneumolysin. A single chromatography step produces excellent purification of the cytolysin by binding soluble aggregated cytolysin to a hydrophobic interaction chromatography material in the presence of detergent and high salt.

Description

The method of purification of bacterial cytolysin

Technical field

The present invention relates to the field of bacterium cytolysin purifying, relate in particular to the purification process of pneumolysin.Pneumolysin is the protein with good resistance originality characteristics from streptococcus pneumoniae, and it is suitable as, and anti-streptococcus pneumoniae is infected or the vaccine component of otitis media.Method of the present invention has been described with the uncommon of the single chromatographic step purifying pneumolysin by pneumolysin being bonded to the hydrophobic interaction post when stain remover and high salt exist and step easily.This method has been utilized the bacterium cytolysin especially under the aggegation situation aromatics of similar cholesterol to be had a high affinity easily and therefore has been applied to the characteristic of this toxin family member purifying usually.

Mercaptan activatory cytolysin forms tangible bacteriotoxin group, wherein streptolysin O be its prototype (Billington etc., FEMS Microbiol.Lett. (2000), 182; 197-205).These toxin dissolve eukaryotic cell by form aperture on cytolemma.Oxygenant influences its cell lysis activity unfriendly and reductive agent can reducing activity.These group memberships' one-level aminoacid sequence presents the 30-60% similarity and comprise the following sequence of almost constant decapeptide near the C-end.Cholesterol is the main receptor in target cell of these toxin.Cytolysin is striden fenestra in conjunction with the cholesterol that comprises film and oligomer to form to the 30nm diameter and by what 40-80 monomer subunit formed.The combination of film cholesterol causes the monomeric conformational change of toxin and promotes oligomerization, the secondary incident that film embeds and aperture forms.

Streptococcus pneumoniae (Streptococcus pneumoniae) is the pathogenic agent that some human diseasess comprise pneumonia, septicemia, meningitis, otitis media and sinusitis paranasal sinusitis.Although microbiotic be effectively sometimes these diseases still can cause death.The appearance of streptococcus pneumoniae strains has aggravated the problem that caused by this pathogenic agent.Based on this, the vaccine of developing effective anti-streptococcus pneumoniae is very important.

The polyvalent pneumococcal vaccine that comprises the capsular polysaccharide of purifying uses for many years.Its application is subjected to especially immunogenicity low in comprising pregnant woman, the elderly's excessive risk colony and brings sickle-cell anemia, multiple myeloma, liver cirrhosis or crapulent restriction.Its specific protection of serotype also is provided and in 90 known serotype only 23 cover by existing preparation.This will protect in the U.S. population serotype of finding 90% and only protect and in the population of Asia, find about 70% of serotype.Recently can use a kind of seven-valency vaccine of puting together, it has the problem of anti-all streptococcus pneumoniae strains equally.

Pneumolysin (Ply) is 53kD mercaptan-activatory cytolysin of finding in all streptococcus pneumoniae strains, and it is released and produces autolysis and be the pathogenesis of streptococcus pneumoniae.Except there being a few amino acids to replace, be very conservative between the Ply protein of different serotypes.Pneumolysin has the conservative property of height and its immunogenicity to make it become the possible material standed for of vaccine component.Yet wild-type Ply is not suitable for incorporating into the vaccine that is used for the people owing to its toxicity.Ply causes the destruction of cell membrane by with film-combine the interaction of cholesterol and oligomer and form aperture on film.At the terminal conservative halfcystine of finding of C--comprise primitive to participate in lytic activity.Pointed out the sudden change Ply and reduce its toxicity (WO90/06951, WO99/03884).

Lock etc. have described the two step method of pneumolysin purifying, and (MicrobialPathogenesis (1996) 21; 71-83).Use the pneumolysin of combination purification of Recombinant from culture of Escherichia coli of ion-exchange and gel permeation chromatography.That this method comprises the preparation extract and the DEAE Sepharose post of flowing through, the step of the Sephacryl S200-HR post of flowing through subsequently.This method can be used for purification of Recombinant or natural pneumolysin.

Kuo etc. have described method (the Infection and Immunity (1995) 63 of the GST-pneumolysin fusion rotein of purification of Recombinant; 2706-2713).This fusion rotein is expressed in culture of Escherichia coli and cell lysate is splined on the glutathione agarose gel.Zymoplasm can be used for this fusion rotein of cracking to fusion rotein with the gsh wash-out.This protein is flowed through gsh-agarose column again to remove GST.The pneumolysin of affinity purification is further used the hydroxyapatite column purification.

(BBA (1989) 1007 for Mitchell etc.; 67-72) method of utilizing hydrophobic interaction chromatography purifying pneumolysin has been described.Under the condition of its use (250mM salt), although slow down its process and pneumolysin with broad peak by wash-out, pneumolysin also fails to be closely adhered on the post.Need to determine which component comprises pure pneumolysin, concentrates positive component, upper prop and do not combine closely to the problem of column material to overcome pneumolysin with the other step of small volume of water wash-out again.

Still keep improving the consistent demand of anti-Streptococcus pneumoniae vaccine.Although this proteinic toxicity remains a problem, this demand has been satisfied in the combination of Ply component.Also need to develop the quick and effective means that is used for pneumolysin purifying in enormous quantities.The method of Miao Shuing comprised that utilization was together with a plurality of purification steps and the enrichment step of getting involved test in the past.The invention provides the purification process more efficiently that has utilized single chromatographic step easily, it can be used for the large batch of pneumolysin of purifying.

Accompanying drawing is described

The SDS-PAGE gel of Fig. 1-demonstration pneumolysin purifying.Following sample is splined on the SDS-PAGE gel :-swimming lane 1-molecular weight standard, the supernatant liquor of swimming lane 2-cell extract, the swimming lane 3-phenyl-sepharose of flowing through, swimming lane 4 phenyl sepharose gels wash first, swimming lane 5-phenyl-sepharose secondary washing, swimming lane 6 phenyl-sepharose washs with 0.5MNaCl, swimming lane 7 phenyl-sepharose low salt buffer wash-out, the pneumolysin of swimming lane 8 behind the folding step of sex change/again, the pneumolysin of swimming lane 9-behind sterilising filtration.

Dull and stereotyped A shows the gel after the Coomassie blue stain.Dull and stereotyped B shows the gel after the Western blotting of-intestinal bacteria antibody anti-through utilizing is surveyed contaminating protein matter.

The SDS-PAGE of pneumolysin-Coomassie blue stain that Fig. 2-GMBS (N-(γ-maleimide butyryl acyloxy) succinimide ester) modifies analyzes.

Following sample is splined on the SDS-PAGE gel :-swimming lane 1-molecular weight standard, the pneumolysin that swimming lane 2-is not modified, the PLY that swimming lane 3-handles with GMBS/ Methionin 4/1 mol ratio with GMBS, swimming lane 4-handles with GMBS/ Methionin 4/1 mol ratio with GMBS and at 37 ℃ of PLY of hatching 7 days, the PLY that swimming lane 5-handles with GMBS/ Methionin 8/1 mol ratio with GMBS, swimming lane 6-is at 37 ℃ of PLY that use GMBS to handle with GMBS/ Methionin 8/1 mol ratio after hatching 7 days, the PLY that swimming lane 7-handles with NHS/ Methionin 10/1 mol ratio with sulfo group-NHS acetate, the PLY that swimming lane 8-handles with NEM, swimming lane 9-is at 37 ℃ of PLY that handle with NEM after hatching 7 days.

Fig. 3-intranasal gives the pneumolysin toxicity of the GMBS processing of mouse.The mouse survival rate of attacking with the natural pneumolysin of 2 μ g with the curve representation of diamond indicia.The mouse survival rate that the pneumolysin of handling with 10 μ g GMBS with the curve representation of square marks is attacked.

The provide protection that the pneumolysin of handling by GMBS in the mouse of Fig. 4-attack with natural pneumolysin intranasal causes.With the curve display of rectangle mark separately with the mouse survival rate of adjuvant inoculation.With the curve representation of diamond indicia mouse survival rate with natural pneumolysin inoculation.Ask the mouse survival rate of bacterium hemolysin inoculation with the pneumonia of GMBS processing with the curve representation of square marks.

Fig. 5-inoculate the provide protection that causes with the pneumolysin of handling by PhtD and GMBS in the mouse of 2 type D39 streptococcus pneumoniae strain intranasals attack.The independent mouse survival rate of curve representative with the rectangle mark with the adjuvant inoculation.Curve with diamond indicia is represented the mouse survival rate of inoculating with PhtD.Use the mouse survival rate of the pneumolysin inoculation of PhtD and GMBS processing with the curve representative of square marks.

Describe in detail

Method

Method of the present invention is to be used for for example method of the bacterium cytolysin of pneumolysin of purifying.Cytolysin, for example pneumolysin uses only single column chromatography step to need not to adorn post again and purifying.Protein when stain remover and salt exist with the aggegation form in conjunction with the hydrophobic interaction post.Almost there is not protein column and make cytolysin under this condition with the single step purifying.

For cytolysin solubility agglomeration of the present invention, preferred pneumolysin is 30, and 000g is retained in the aggegation form of the cytolysin in the supernatant liquor after centrifugal 20 minutes.This solubility agglomeration is at high salt, and preferred 1M is retained on the hydrophobic interaction chromatography material when existing, preferred phenyl-sepharose.Optionally, this solubility agglomeration is gelationus.

Cytolysin, preferred pneumolysin is with solubility agglomeration column.Because various reasons comprise strainer or column blocking and loss of material, it is uncommon that agglomeration is loaded on the post.Yet, dwindle the agglomeration size to form the stain remover of solubility agglomeration by use, find these agglomeratioies post of under the stain remover condition, combining closely, but can with analyze by SDS-PAGE at least 50%, 60%, 70%, 80%, preferred 90%, 95%, more preferably 97%, 98% or 99% purity and do not have the disadvantageous effect of coupled columns strainer and wash-out.This method preferably obtains every liter of fermented liquid at least 100,200,500,700, and more preferably 1000,1500,1700 or the cytolysin of 1900mg, preferred pneumolysin.Preferred at least 1%, 2%, 5%, 7%, 9% or 10% from the protein of the fermenting culture cytolysin as purifying, preferred pneumolysin and reclaiming.

This method utilized cytolysin for example pneumolysin in conjunction with the ability of cholesterol and other aromatics.This is combined in the cytolysin aggegation, allows cytolysin especially tight bonded the time when stain remover exists.Owing to all members all have the ability that forms aperture in conjunction with aromatics, this method may extend to other members of cytolysin family.In fact this method can be used for purifying those in conjunction with cholesterol or other aromatics and/or form aperture, preferably both other protein familieses.

Therefore, in first embodiment, be provided for the method for bacterium cytolysin purifying, said method comprising the steps of:

A) culture of the cell of culture expression bacterium cytolysin;

B) from the culture that comprises the bacterium cytolysin, prepare extract;

C) the solubility agglutinative bacterium cytolysin that will be contained in when existing in the extract at stain remover (preferred aliphat stain remover) is bonded to the hydrophobic interaction chromatography material under high salt (preferred 0.5-2M salt) condition;

D) wash-out bacterium cytolysin under stain remover (preferred aliphat stain remover) less salt when existing (preferred 0-0.2M salt) condition.

In second embodiment, be provided for the method for bacterium cytolysin purifying, said method comprising the steps of:

A) culture of the cell of culture expression bacterium cytolysin;

B) from the culture that comprises the bacterium cytolysin, prepare extract;

C) the bacterium cytolysin that will be contained in the extract when the solution that comprises 0.5-2M salt and 0.1%-5% stain remover exists is bonded to the hydrophobic interaction chromatography material;

D) utilize less salt (preferred 0-0.2M salt) the eluant solution bacterium cytolysin that comprises the 0.1-5% stain remover.

In above-mentioned two embodiments, method of the present invention is preferably further comprising the steps of:

E) from the bacterium cytolysin, remove stain remover

F) by adding denaturing agent dissolution of bacteria cytolysin;

G) remove denaturing agent from the bacterium cytolysin.

Method of the present invention can be advantageously used in the purifying pneumolysin.Other cytolysins of available method purifying of the present invention comprise the Actinomyces pyogenes hemolysin from Actinomyces pyogenes (A.pyogenes), Bacillus cereus hemolysin from Bacillus cereus (B.cereus), bacillus thuringiensis hemolysin O from bacillus thuringiensis (B.thuringiensis), lateral bud spore bacillus hemolysin from lateral bud spore bacillus (B.latersporus), two enzyme brood cell clostridium hemolysins from two enzyme brood cell clostridiums (C.bifermentans), meat poison brood cell clostridium hemolysin from meat poison brood cell clostridium (C.botulinum), Shore clostridium hemolysin from Shore clostridium (C.chauvoel), clostridium histolyticum's hemolysin from clostridium histolyticum (C.histolyticum), A type Nuo Shi clostridium hemolysin from Nuo Shi clostridium (C.novyi), come the aerogenesis folder film bacillus hemolysin O of automatic gas-producing folder film bacillus (C.perfringens), the clostridium hemolysin O that relieves internal heat from the clostridium that relieves internal heat (C.septicum), Soxhlet brood cell clostridium hemolysin from Soxhlet brood cell clostridium (C.sordellii), tetanus haemolysis hemolysin from clostridium tetani (C.tetani), Ivan slave husband listeria bacteria hemolysin from Ivan slave husband listeria bacteria (L.ivanovi), Listeria monocytogenes hemolysin O from Listeria monocytogenes (L.monocytogenes), Listera seeligeri hemolysin O from Listera seeligeri (L.seeligeri), honeycomb series bacillus hemolysin from honeycomb series bacillus (P.alvei), from streptococcus pyogenes (S.pyogenes), the streptolysin O of dog's leash coccus (S.canis) or streptococcus equisimilis (S.equisimilis), middle streptolysin from intermediate chain coccus (S.intermedius), from the swine streptococcus hemolysin of swine streptococcus (S.suis) or from the pneumolysin of streptococcus pneumoniae (S.Pneumoniae), its be wild-type or as the low-level toxic genetic modification toxin of above-mentioned PdA or PdB.

Pneumolysin or Ply represent: from the natural pneumolysin of streptococcus pneumoniae or the mutant of the pneumolysin of reorganization, wild-type pneumolysin or pneumolysin (for example described in WO90/06951 and the WO99/03884).Randomly, pneumolysin also can be represented any fragment of pneumolysin or any variant of pneumolysin, itself and wild-type pneumolysin sequence have at least 70,80,90 or 95% amino acid sequence identity, and as those of skill in the art were easy to determine, it still keeps can be by the ability of the inventive method purifying.

In an embodiment preferred of the present invention, identical stain remover preferably is present in step b) and c with the concentration of 0.1%-5% (w/v)), b) and d) in, more preferably at step b), c) and d) in.For the present invention, aliphatic stain remover is defined as not having sufficient aromatic series feature to stop the substantial aliphatics stain remover of cytolysin column in the step c).Preferred this aliphatics stain remover has one or aromatic ring still less, does not most preferably have aromatic ring.In step d), the cytolysin agglomeration that stain remover will be bigger is smashed and obtained the solubility agglomeration to less agglomeration is of great use.At step c) and d) in, stain remover has been kept the solubility agglomeration state of cytolysin effectively, make its under high salt condition with the high affinity column.

Cytolysin, preferred pneumolysin be at bacterial cell, preferred streptococcus pneumoniae, intestinal bacteria or express in the culture of yeast cell, insect cell, mammalian cell or any expression system that is fit to its expression.In the expression system that reaches the pneumolysin high yield, pneumolysin often is an automatic agglutinative and method of the present invention is an ideal for its purifying.It occupies above 2,3,4,5,7 or 10% of total protein preferred pneumolysin in expression system to reach with the high yield expression.Preferred pneumolysin is an agglomeration form and therefore farthest lack hemolytic activity.For example, the expression under lambda particles phage promotor or other promotors that produces high expression level are controlled in the Escherichia coli fermentation jar is well-known to those skilled in the art.

Preferred cytolysin extracts from expression system with agglomeration.Perhaps, the expression system than low-yield can provide the solubility cytolysin.In this case, comprise cytolysin, the extract of preferred pneumolysin is adjusted to pH and is lower than 7.5, and it makes cytolysin at least 8 hours periods, preferably aggegation at least 24 hours.

The preparation of extract preferably includes one or more with the mechanical means smudge cellses and/or with the step of detergent-treatment cell in the step b).If make by high-yield process, pneumolysin keep the form of agglomeration but agglomeration should be enough little so that under the required condition of the insoluble cell debris of precipitation the centrifugal back of sample its be retained in the supernatant liquor.Being used for preferred stain release agent of the present invention is the aliphatics stain remover, and it does not comprise aromatic nucleus, preferred ion type stain remover, more preferably positively charged ion or anionic detergent and most preferably, stain remover is a sodium N-lauroyl sarcosinate.Preferred stain remover can dissolve pneumolysin, as long as allow it be in the little agglomeration form that does not cause the filter blocks that is attached to this post in conjunction with the hydrophobic interaction post.The size that preferred stain remover can dwindle pneumolysin makes the pneumolysin agglomeration enough little so that 30, is retained in the supernatant liquor after 20 minutes samples of 000g are centrifugal.This solubility agglomeration is purifiable equally on the hydrophobic interaction post.Stain remover is with 0.1% to 5%, and preferred 0.5% to 3% (w/v) is preferred 0.75% to 2%, and more preferably from about 1% concentration exists.Preferably, stain remover is dialyzable.

Along with cracking mechanical means and/or stain remover of culture in step b), method of the present invention comprise the centrifugal of cellular material and collect supernatant liquor as extract to be splined in the chromatographic material in the step c).Cytolysin preferably is present in the supernatant liquor with the solubility agglomeration.

Method utilization hydrophobic interaction chromatography of the present invention is with single step purifying pneumolysin.The column material that uses in the step c) preferably comprises aromatic group, preferably phenyl and more preferably phenyl-sepharose.

Go up the solution that uses during the wash-out of sample and post in step c) and/or the step d) and comprise ionic detergent, preferred cationic or anionic detergent, preferred soluble stain remover in the above salt concn of 0.5M, most preferably stain remover is a sodium N-lauroyl sarcosinate.Used stain remover can dwindle cytolysin, and the size of preferred pneumolysin agglomeration allows cytolysin to be present in the sample with the solubility agglomeration so that it can rather than irreversibly rest on the post in conjunction with the hydrophobic interaction column material.Stain remover is with 0.1% to 5%, preferred 0.5% to 3% (w/v), and more preferably 0.75% to 2%, most preferably from about 1% concentration exists.

Step c) and/or d) in solutions employed comprise salt, be preferably selected from the salt and the preferred buffer pH 6-8 of sodium-chlor, magnesium chloride, ammonium chloride, sodium sulfate, sal epsom, ammonium sulfate, sodium phosphate, trimagnesium phosphate, ammonium phosphate, preferably about pH7.Can use and anyly can keep the damping fluid of pH between pH5 and 9.

Usedly in the inventive method comprise high salt concn in conjunction with pneumolysin to the solution of post, preferred 0.6-2M, more preferably from about 1M.Select salt concn so that pneumolysin is in solubility agglutinative form and can analyses material by bonded hydrophobic layer.

Randomly, step c) can comprise the additional step that can remove the salt concn washing column of any insufficient bonded impurity with the intermediate salt conditioned disjunction of about 0.5M salt.

The salt gradient that method utilization of the present invention is successively decreased wash-out pneumolysin from the post.Preferred steps d) the low salts solution that is used to make salt gradient in comprises 0-0.1M salt, more preferably 0-40mM salt.Perhaps, can utilize the used 0-0.2M salt that comprises in the step d), more preferably the low salt buffer of 0-40mM salt carries out gradient elution.

Optional step can join in the method for the present invention, if it makes the pneumolysin sex change and folds it again by the removal of denaturing agent subsequently.These optional steps guarantee to obtain to have the pure cytolysin of natural structure, preferred pneumolysin.First optional step e) comprises by diafiltration, dialysis or dilution removal stain remover.This step preferably includes through pH8-10, the diafiltration/dialysis of preferred about 9 damping fluid, and more preferably this damping fluid can cushion the alkaline pH value, and most preferably this damping fluid is DEA.The preferred low ionic strength of solution, preferred 10-50mM, most preferably from about 25mM.Diafiltration or dialysis are preferably carried out but also can at room temperature be carried out at 4 ℃.

In second optional step, cytolysin, preferred pneumolysin be sex change and dissolve by the interpolation of denaturing agent.The denaturing agent that is used for step f) is preferably Guanidinium hydrochloride, more preferably the Guanidinium hydrochloride of 5-8M, the most preferably from about Guanidinium hydrochloride of 6M.Pneumolysin was hatched 10 minutes with Guanidinium hydrochloride at least, and preferably at least 1 hour, more preferably from about one hour.

In the step f), cytolysin, preferred pneumolysin preferred subsequently with the urea of 5-9M, preferably the urea of about 8M contacts.This is by cytolysin, the diafiltration of the preferred anti-urea of pneumolysin or dialysis and finish.Preferably, between the denaturing agent commutation period, keep identical damping fluid and pH.Preferably between the commutation period of denaturing agent, add reductive agent (DTT, 2 mercapto ethanol or gsh).

Preferred steps f) comprise cytolysin, preferred pneumolysin contacts the exchange of Guanidinium hydrochloride and 5-9M urea subsequently with the 5-8M Guanidinium hydrochloride.

At cytolysin, for avoiding unsuitable disulfide linkage formation, guarantee during preferred pneumolysin sex change at step f) and g) at least a portion in the existence of reductive agent be helpful.Preferred reductive agent is the DTT of 0.1-10mM, preferably the DTT of about 1mM.Perhaps use gsh or 2 mercapto ethanol.The preferred concentration of gsh is 1-50mM, more preferably 10-30mM.

Optional step g) comprises that the removal of denaturing agent is so that folding again cytolysin, preferably pneumolysin.Preferably through pH6-11, the preferably diafiltration of the low salt buffer of about pH9 or dialysis and realize.Preferably, cytolysin, the concentration of preferred pneumolysin is kept at least 100 μ g/ml, preferably between 100 μ g/ml and 1000 μ g/ml, more preferably at about 500 μ g/ml.Optionally, diafiltration or dialysis are through comprising 10% to 30%, the damping fluid of preferred about 15% propylene glycol.Preferably, in step g), keep above-mentioned reductive agent.Diafiltration or dialysis are preferably carried out but also can at room temperature be carried out at 4 ℃.

Further optional step h) comprises cytolysin, the removal of the folding again back of preferred pneumolysin reductive agent.This is preferably through pH6-11, and preferably the diafiltration of the low salt buffer of about pH9 or dialysis realize.Optionally, diafiltration or dialysis are through comprising 10% to 30%, the damping fluid of preferred about 15% propylene glycol.Diafiltration or dialysis are preferably carried out but also can at room temperature be carried out at 4 ℃.

In a preferred method of the invention, folding again cytolysin, preferred pneumolysin most preferably extremely surpasses 90% so that its hemolytic activity returns to above 25%, 50%, 75% of normal unfolded protein.For the present invention, ' folding ' protein is the protein with the proteinic tertiary structure that obtains by non-denaturing process.With regard to the wild-type pneumolysin, the hemolytic activity of folding again pneumolysin expection will be 500,000-1,000,000 hemolytic unit/mg pneumolysin.For the point mutation pneumolysin with low hemolytic activity, Zhe Die pneumolysin hemolytic activity is with relatively low again.

The detoxifcation of toxin

With the cytolysin of method purifying of the present invention, preferred pneumolysin can be through further by chemically treated optional detoxifcation step.This additional step is at cytolysin, and preferred pneumolysin is especially useful in the time of will be bestowed the animal or human.The wild-type pneumolysin is highly toxic.Separated the toxic sudden change pneumolysin protein that some have reduction, yet these still keep residual toxicity, (WO99/03884 may be problematic WO90/06951) when the administration of pneumolysin inside for it.It can detoxify by being bonded to polysaccharide (WO96/05859) alternatively.

Method of the present invention can be by chemical treatment the detoxify cytolysin of wild-type or sudden change, for example pneumolysin.Embodiment preferred is used linking agent, more preferably comprises one or more and is selected from the chemical reagent of formaldehyde, glutaraldehyde and comprises the N-hydroxy-succinamide ester and/or the cross-linking reagent of maleimide (as GMBS).

The detoxification basis is as an aspect of of the present present invention and can be used for the bacteriotoxin that detoxifies, preferably the pneumolysin for preparing by additive method.

In one embodiment, detoxification of the present invention has been described and has been comprised and use compound, and preferably to amido, more preferably primary amine groups reacts, preferred reaction according to qualifications, most preferably the cross-linking reagent of specific reaction is handled the bacteriotoxic detoxifcation of toxin.

With regard to the application, linking agent is defined as the compound that has two active groups at least, wherein at least one can with at least one radical reaction on the bacteriotoxin.Another active group can with bacteriotoxin on or the radical reaction of isolated compound (for example amino acid, peptide, polypeptide, sugar or polysaccharide).

Preferred compound or linking agent are reactive to amine and sulfydryl, more preferably according to qualifications reaction, most preferably specific reaction.The primary amine groups reaction of preferred compound and Methionin, the more preferably sulfydryl of the primary amine groups of linking agent and Methionin and halfcystine reaction.With respect to linking agent only with the residual hemolytic activity of Methionin or halfcystine reaction, pneumolysin is the toxicide aspect owing to the modification of halfcystine and lysine residue causes the collaborative reduction of hemolysis levels, this method is especially favourable.

Therefore an optional embodiment provides and has comprised near cysteine residues (the C-end of optional toxin) the bacteriotoxin method for detoxification of modifying participation toxin toxicity (preferred dissolution activity), comprise sulfydryl and another amino acid used toxin, preferably in primary structure, surpass 2,5,10,15,20,30,40 crosslinked linking agents (preferred isodigeranyl functional cross-link agent) of amino acid whose amino acid and handle toxin away from halfcystine.Preferred another amino acid comprise primary amine group and more preferably this amino acid be Methionin.

In some embodiments, after passing through the SDS-PAGE evaluation process, toxin above 50%60%, 70%, 80%, 90% or 95% keeps molecular weight in 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of its initial molecule amount, more preferably at 1-50%, most preferably within 5-10%.Because some amino-acid residues are handled the preferred toxin in back in detoxifcation and are reached higher a little molecular weight by modifying with the compound covalent attachment.Yet method of the present invention does not preferably comprise extensively puting together of toxin, no matter is by being covalently bond to other lps molecules so that forming the toxin with polymer quaternary structure, still by giant protein white matter, polysaccharide or the lipopolysaccharides of covalent attachment toxin to other.Most preferably disclosed method, protein or product are not comprised among the present invention among the WO96/05859.

Method of the present invention can be used for the bacteriotoxin detoxifcation.Preferred toxin comprises the Actinomyces pyogenes hemolysin of mercaptan-activatory from Actinomyces pyogenes (A.pyogenes), Bacillus cereus hemolysin from Bacillus cereus (B.cereus), bacillus thuringiensis hemolysin O from bacillus thuringiensis (B.thuringiensis), lateral bud spore bacillus hemolysin from lateral bud spore bacillus (B.latersporus), two enzyme brood cell clostridium hemolysins from two enzyme brood cell clostridiums (C.bifermentans), meat poison brood cell clostridium hemolysin from meat poison brood cell clostridium (C.botulinum), Shore clostridium hemolysin from Shore clostridium (C.chauvoel), clostridium histolyticum's hemolysin from clostridium histolyticum (C.histolyticum), A type Nuo Shi clostridium hemolysin from Nuo Shi clostridium (C.novyi), come the aerogenesis folder film bacillus hemolysin O of automatic gas-producing folder film bacillus (C.perfringens), the clostridium hemolysin O that relieves internal heat from the clostridium that relieves internal heat (C.septicum), Soxhlet brood cell clostridium hemolysin from Soxhlet brood cell clostridium (C.sordellii), tetanus haemolysis hemolysin from clostridium tetani (C.tetani), Ivan slave husband listeria bacteria hemolysin from Ivan slave husband listeria bacteria (L.ivanovi), Listeria monocytogenes hemolysin O from Listeria monocytogenes (L.monocytogenes), Listera seeligeri hemolysin O from Listera seeligeri (L.seeligeri), honeycomb series bacillus hemolysin from honeycomb series bacillus (P.alvei), from streptococcus pyogenes (S.pyogenes), the streptolysin O of dog's leash coccus (S.canis) or streptococcus equisimilis (S.equisimilis), middle streptolysin from intermediate chain coccus (S.intermedius), from the swine streptococcus hemolysin of swine streptococcus (S.suis) or from the cytolysin of the pneumolysin of streptococcus pneumoniae (S.Pneumoniae), its for wild-type or have the low toxicity level the genetic modification toxin, as above-mentioned PdA or PdB (WO90/06951, WO99/03884).

This method also can be used for detoxifying Neisserial toxin FrpA, (Microbiology 142 for FrpC (WO92/01460), FrpB; 3269-3274, (1996); J.Bacteriol.181; 2895-2901 (1999)), NM-ADPRT (13 ^ThInternational PathogenicNeisseria Conferenee 2002 Masignani et al p135).The preferred fragment that comprises conserved regions and this toxin between two protein of FrpA and FrpC is the polypeptide that comprises this conservative fragments, preferably includes the amino acid 227-1004 of FrpA/C sequence.

Method of the present invention also can be used for detoxifcation and comprises adenylate cyclase (CyaA) (Glaser (1988) Mol.Microbiol.2; Bordetella 19-30) (Bordetella) toxin, dermatonecrotoxin (Livey (1984J.Med, Microbiol.17; 91-103) and Toxins, pertussis (PT) (Munoz etc. (1981) Infect Immun 33; 820-826).Method of the present invention also can be used for detoxifying tetanus toxin (TT) and diphtheria toxin (DT) and comprise autolysin and staphylolysin (WO0/99499, toxin WO02/59148) from streptococcus aureus (S.aureus) and staphylococcus epidermidis (S.epidermidis).

Method of the present invention causes toxin toxicity and/or hemolytic activity amount at least 90%, preferred 95%, 96%, 98%, 99%, 99.5%, 99.9% or 99.99% recovery.(hemolytic activity uses embodiment 3 methods to measure and toxicity can be measured by the method for embodiment 5.) natural pneumolysin has 500,000-1, the hemolytic activity of 000,000 unit/milligram pneumolysin.Some point-mutation variants of pneumolysin have the toxicity and the hemolytic activity of reduction.The minimizing that may not reach the very big percentage ratio of hemolytic activity than low starting point that the detoxifcation of variant pneumolysin reduces owing to hemolytic activity, however the major part of residual hemolytic activity is removed by method of the present invention predictably.

The detoxifcation step of the inventive method preferably provides a kind of irreversible basically crosslinking reaction.Reversibility preferably surpasses 30 ℃ by after detoxifcation and surpassing 25 ℃, more preferably surpasses 35 ℃, most preferably surpasses 37 ℃ of temperature and hatches the hemolytic activity level of directly monitoring detoxified toxin at least after 5,6,7,8,9 or 10 days and evaluate.Basically irreversible reaction produces irreversible basically detoxifcation and is defined as to improve after wherein the hemolytic activity level is hatched when above-mentioned intensification and is less than 100%, 50%, 40%, 30%, 20%, 10% reaction.Many method for detoxification for example by using formaldehyde treated, cause unsettled and toxicity enhanced detoxifcation in the future.

In the preferred detoxifcation step of the inventive method, surpass 50%, 60%, 70%, 80%, 90%, 95%, or 98% toxin is kept monomeric quaternary structure after crosslinking reaction.Many linking agents form intermolecular cross-linking (for example formaldehyde and glutaraldehyde).Because some antigenic determinants will be hidden in agglomeration inside, this can influence the immunological characteristic of toxin.Method of the present invention preferably includes simple modified amino acid residue, the sulfydryl of preferred amino acid and/or primary amine groups and/or form main intramolecular crosslinking.The monomer quaternary structure permission antigenic determinant of reaction product is kept and is exposed to the toxin surface.

In a preferred embodiment of detoxifcation step, linking agent is the isodigeranyl function.Preferred cross-linking agents comprises with primary amine group reacts according to qualifications, more preferably the N-hydroxy-succinamide ester group of specific reaction.Preferred cross-linking agents comprises with sulfydryl reacts according to qualifications, more preferably the dimaleoyl imino of specific reaction.At pH about 7 o'clock, dimaleoyl imino reacted fast 1000 times with the sulfydryl reaction than itself and amine.Preferred linking agent comprises N-hydroxy-succinamide ester group and dimaleoyl imino.Because this causes relatively poor effective detoxifcation, preferably use the reductive agent can not the cracked linking agent.

Distance between the cross linker active group can influence toxicide and render a service.Preferably in the methods of the invention, and the distance between the cross-linker groups of amine and sulfydryl reaction is between 1.5 and 20 dusts, more preferably 10 dusts between 5 and 15 dusts and most preferably from about.Amino-acid residue on the preferred bacterium toxin is modified by the interpolation that surpasses the long group of 5,7,10,12,15,18,20,50,100,500 dusts.Preferred modification group is between 5 and 100 dusts, more preferably between 10 and 20 dusts.

The detoxifcation step of the inventive method makes enough residues be modified so that steric influence and/or conformational change suppress bacteriotoxic function.At least 5,7,10,12,14,15,20 or 25 amino-acid residues of preferred bacterium toxin are modified.Because unreacted maleimide base group is presented on the linking agent, can use Ellman reaction assessment (indirectly) to be attached to cross-linker molecules number (Ellman 1959 Arch.Biochem.Biophys.82 of each lps molecule; 70).

Preferred cross-linking agents is SMPT, sulfo group-LC-SMPT, sulfo group-KMUS, LC-SMCC, KMUA, sulfo group-LC-SPDP, LC-SPDP, SMPB, sulfo group-SMPB, SMPH, sulfo group-SMCC, SMCC, SIAB, sulfo group-SIAB, GMBS (N-(γ-maleimide butyryl acyloxy) succinimide ester), sulfo group-GMBS, MBS, sulfo group-MBS, sulfo group-EMCS, EMCA, EMCS, BMPS, SPDP, SBAP, BMPA, AMAS, SATP and SIA (Pierce).

In a preferred method of the present invention toxin with compound or linking agent between 5.0 and 9.0, preferred 6.5 to 8.0, most preferably handle under 7.0 to 7.8 the pH condition.In promoting the processing that dimaleoyl imino and sulfydryl react, reacting preferred pH is 6.0 and 8.0, more preferably 6.5 and 7.5.Preferred salt concn is between 100mM and 1M between the reaction period, and more preferably 150mM and 500mM are most preferably between 200mM and the 300mM.Yet the contriver finds to react better sometimes under the low salt concn that does not have sodium-chlor or other salt to add.Carry out because be reflected between pH7.6 and 7.8, this reaction can be chosen wantonly under salt-free interpolation and carry out.Similarly, the GMBS contratoxin more the use of height ratio can under salt-free interpolation between pH value 7.0 and 8.0, carry out.

Preferably use compound or linking agent to each toxin 50-500, more preferably 130-350 or 350-900, most preferably from about 250 times molar excess.Pneumolysin comprises 31 lysine residues.Therefore surpass the compound of 248 times of molar excess of pneumolysin or linking agent and be equivalent to compound or linking agent 8 times of molar excess each lysine residue.Preferred use compound or linking agent are to the 2-20 of lysine residue, more preferably 4-15 or 15-30, most preferably from about 8 times mol ratio in the inventive method.

, between 4 ℃ and 40 ℃, preferably between 15 ℃ and 25 ℃, most preferably at room temperature carried out at least 15 minutes with the processing of linking agent, preferably at least 30 minutes, most preferably from about one hour.Method of the present invention can further comprise the cancellation step of using the compound that comprises sulfydryl, and preferably this quencher has the molecular weight above 50,100 or 120, and more preferably this quencher is a for example halfcystine of amino acid.Perhaps this group can with can and the maleimide peptide or the polysaccharide that react, for example comprise the reactive polypeptide of cysteine residues.This is especially suitable, because unreacted maleimide base group presents before the cancellation step.

The detoxifcation step is suitable for above-mentioned bacteriotoxin.The preferred bacterium toxin is from streptococcus pneumoniae, and most preferably toxin is a pneumolysin.Pneumolysin is natural or recombinant protein or reduce the protein of its toxicity (as mentioned above) through genetic engineering.Toxin, the fusion rotein or the toxin of preferred pneumolysin, the fragment of preferred pneumolysin can be used method detoxifcation of the present invention.

Therefore in a preferred embodiment, toxin (for example pneumolysin) detoxifies with linking agent, it preferably has with the isodigeranyl function of the group of Methionin and cysteine residues reaction and size determines, the active group so that following each or the both that most preferably have 10-20 dust distance are at interval taken place:

A) between 5 and 30, the amino-acid residue of preferred about 12-14 toxin is bonded to Methionin or arginine residues (preferably by the corsslinking molecular preferably covalently, as by Ellman reaction indirect measurement), the other end modified by cancellation (preferably with halfcystine) and/or;

B) toxin relates to another side chain (preferably to Methionin or arginine residues) that the toxic cysteine side chain C-end of toxin (preferably near) is cross-linked to toxin, and it preferably separates with the cysteine residues of the elementary sequence of toxin and surpasses 2,5,10,20,30 or 40 amino acid.

In further preferred embodiment, toxin (preferred pneumolysin) detoxifies with single functional compounds, its preferably with the amino acid that comprises primary amine group, more preferably Methionin reaction, and size is determined, most preferably 10-100 dust so that toxin are by between 5 and 30, and more preferably from about 14 compounds in conjunction with amino-acid residue cover.

Polysaccharide conjugates

A problem relevant with the inoculation of polysaccharide method is that polysaccharide itself lacks immunogen.For overcoming this problem, polysaccharide can be conjugated to protein carrier, and it provides looks on T-cell auxiliary (bystander T-cellhelp).Method of the present invention can advantageously comprise cytolysin, and preferred pneumolysin is conjugated to bacterial polysaccharides, for example the further step of fat-low polysaccharide or preferred capsular polysaccharide.

The preferred conjugate of the present invention comprises the cytolysin that is conjugated to the capsular polysaccharide that derives from streptococcus pneumoniae, preferably the pneumolysin that obtains by the inventive method.Pneumococcal capsular polysaccharide antigen is preferably selected from serotype 1,2,3,4,5,6B, 7F, 8,9N, 9V, 11A, 11A, 12F, 1415B, 17F18C, 19A, 19F, 20,22F, 23F and 33F (most preferably serotype 1,3,4,5,6B, 7F, 9V, 14,18C, 19F and 23F), or the mixture of two or more above-mentioned conjugates (4,7,9,11,13 or 23).

By the cytolysin of method purifying of the present invention, preferred pneumolysin also can preferably be conjugated to the capsular polysaccharide from other bacterial isolateses.Described polysaccharide is separable certainly, for example, hemophilus influenza (H.influenzae), hemophilus influenza Type B (Hib), Neisseria meningitidis (N.meningitidis) A, C, W, Y, the suis except streptococcus pneumoniae are (as B suis B Streptococcus, streptococcus pyogenes S.pyogenes, or the like .), staphylococcus (as streptococcus aureus S.aureus, staphylococcus epidermidis S.epidermidis), intestinal bacteria faecalis (as enterococcus faecalis E.faecalis and faecium E.faecium), or the like.Preferred polysaccharide is from hemophilus influenza Type B (Hib) and/or Neisseria meningitidis A, C, W135 and/or Y.

Polysaccharide can by any known method (for example, by Likhite, United States Patent (USP) 4,372,945 and by Armor etc. United States Patent (USP) 4,474,757) be connected to cytolysin, preferred pneumolysin.Preferably, carry out CDAP and put together (WO95/08348).For improving immunogenicity, polysaccharide can add adjuvant and/or freeze-drying.Polysaccharide of the present invention can be fully size or purifying after adjust size to less polysaccharide or oligose.

Method of the present invention preferably includes the preparation cytolysin, and preferred pneumolysin becomes the further step of vaccine.

Protein and immunogenic composition

The present invention further embodiment is the cytolysin by method purifying of the present invention, preferred pneumolysin.This comprises the pneumolysin-bacterial capsule polysaccharide conjugates by method preparation of the present invention.

The present invention further embodiment is to comprise the cytolysin that obtains by method of the present invention (as mentioned above), preferred pneumolysin or pneumolysin-bacterial capsule polysaccharide ground immunogenic composition.

Immunogenic composition of the present invention preferably further comprises one or more members of streptococcus pneumoniae choline binding protein family, one or more members of preferred choline binding protein A or its immunogenic fragments and/or polyhistidyl three families (comprising its fusion rotein), preferred PhtA, PhtB, PhtD or PhtE or its immunogenic fragments.

For choline binding protein family (CbpX), this family member is accredited as the pneumoprotein matter that can pass through choline-affinity chromatography purifying at first.The fat teichoic acid of the phosphorylcholine part of all choline-conjugated proteins and cell walls teichoic acid and film-relevant is non--covalent attachment.Structurally, all family has some common zones, though its proteinic definite character (aminoacid sequence, length, or the like) can change.Usually, choline binding protein matter comprises N-terminal zone (N), conservative repeat region (R1 and/or R2), proline(Pro) rich region (P) and the choline binding zone (C) of guarding, repeat to constitute by many times, it comprised about proteinic half.As used in this application, term " choline binding protein family (CbpX) " is selected from the choline binding protein matter that WO97/41151 identifies, PbcA, SpsA, PspC, CbpA, CbpD and CbpG.CbpA is open in WO97/41151.CbpD and CbpG are open in WO00/29434.PspC is open in WO97/09994.PbcA is open in WO98/21337.SpsA is a disclosed choline binding protein in WO98/39450.Preferred choline binding protein matter is selected from CbpA, PbcA, SpsA and PspC.

Another embodiment preferred is the CbpX truncate, and wherein " CbpX " as above defines and the CbpX protein in " truncate " hypodactylia 50% or more choline binding zone (C).Preferred this protein lacks whole choline binding zones.More preferably, this protein truncate lacks (i) choline binding zone and (ii) and half part of protein N-end, but still keeps at least one repeat region (R1 or R2).Still more preferably, truncate has 2 repeat regions (R1 and R2), and more preferably this truncate keeps proline(Pro) rich region (P).The example of described embodiment is NR1 * R2 and R1 * R2 and the NR1 * R2P that exemplifies among WO99/51266 or the WO99/51188, yet other choline binding protein matter that lack similar choline binding zone are also included within the scope of invention.

LytX family is the embrane-associated protein relevant with cytolysis.N-end structure territory comprises the choline binding structural domain, and therefore LytX family is not considered to different with CbpX family yet LytX family does not have all characteristics found in the above-mentioned CbpA family.Compare with CbpX family, C-end structure territory comprises the catalyst structure domain of LytX protein families.This family comprises LytA, B and C.For LytX family, LytA is at .Eur J Biochem such as Ronda, and is open among the 164:621-624 (1987).LytB is open in WO98/18930, and is also referred to as Sp46.LytC is also open in WO98/18930, and is also referred to as Sp91.The preferred member of this family is LytC.

Another embodiment preferred is the LytX truncate, and wherein " LytX " as above defines and the LytX protein in " truncate " hypodactylia 50% or more choline binding zone.Preferred this protein lacks whole choline binding zones.An example of this truncate is present in the embodiments of the invention part.

Another preferred embodiment of invention is CbpX truncate-LytX truncate chimeric protein (or fusion).Preferred this comprises NR1 * R2 (or R1 * R2, or NR1 * R2P) and the C-terminal portions (the C-end promptly lacks the choline binding structural domain) (as the C-end of LytC or the C-end of Sp91) of LytX of CbpX.More preferably CbpX is selected from CbpA, PbcA, SpsA and PspC.Remain more preferably, it is CbpA.Preferred LytX is LytC (being also referred to as Sp91).

Another embodiment of the invention is for being expressed as PspA or the PsaA with the fusion rotein of LytX arbitrarily, or lacks the truncate of choline binding structural domain (C).Preferred LytX is LytC.

Pht (polyhistidyl triplet) family comprises protein PhtA, PhtB, PhtD and PhtE.This family has following feature: lipid sequence (lipidation sequence), by separated two districts of proline(Pro)-rich region and several Histidine triplets (may be relevant) with the activity of the combination of metal or nucleosides or enzyme, (3-5) coiled coil district, the terminal and allogenic C-end of conservative N-.It is present in all streptococcus pneumoniae bacterial strains of having checked.Its homologous protein is found in other suis and Neisseria.The preferred member of this family comprises PhtA, PhtB and PhtD.More preferably, it comprises PhtA or PhtD.Be appreciated that term PhtA, B, D and E are meant the albumen with disclosed sequence in following citing document, with and natural (with artificial preparation) variant, described variant and reference protein have at least 90% sequence homology.Preferably at least 95% is identical, and most preferably at least 97% is identical.

Immunogenic composition of the present invention can comprise the proteinic fusion rotein of Histidine triplet.Preferred fusion protein comprises i) PhtD or its fragment or the PhtB that ii) is connected or its fragment that are connected with PhtE or its fragment with PhtE or its fragment.

For PhtX albumen, PhtA is open in WO98/18930, is also referred to as Sp36.As mentioned above, it is the albumen of a kind of polyhistidine triplet family, and has II type signal motif LXXC.

PhtD is open in WO00/37105, and is also referred to as Sp036D.As mentioned above, it also is a kind of albumen of polyhistidine triplet family, also has II type signal motif LXXC.PhtB is open in WO00/37105, and is also referred to as Sp036B.Another member of PhtB family is the polypeptide of C3-degraded, and is open in WO00/17370.This albumen also is polyhistidine triplet family member, has II type signal motif LXXC.Preferred immunologic function equivalent is Protein S p42, and is open in WO98/18930.PhtB truncate (about 79kD) is open in WO99/15675, and it also is considered to the PhtX family member.

PhtE is open in WO00/30299, is called BVH-3.

In order to produce immunogenic composition of the present invention, it can cause comprising the immunne response that surpasses a kind of pathogenic agent of otitis media, and immunogenic composition of the present invention further comprises from streptococcus pneumoniae, do not determine that (non-typable) hemophilus influenza, morazella catarrhalis, RSV, parainfluenza virus and/or the influenza virus of type one or more (2,3,4,5,6) are useful.

The present invention also relates to combined vaccine, described vaccine can provide the provide protection of resisting different pathogens.At present, many paediatrics vaccines provide to reduce the number of times of children's injection with the form of combination-vaccine.Therefore, other antigens that derive from other pathogenic agent also can be mixed with the paediatrics vaccine with vaccine of the present invention.For example, vaccine of the present invention can be prepared (or the administration respectively of same time) with following material: known " trivalent " combination-vaccine, comprise diphtheria toxoid (DT), Toxoid,tetanus (TT) and the Whooping cough composition [pertussis toxin of detoxification (PT) normally, filamentous hemagglutinin (FHA) and alternative PRN (PRN) and/or lectin 1+2], for example, comprise DT, TT, PT, the antigenic commercially available vaccine INFANRIX-DTPa of PHA and PRN ^TM(SmithKlineBeecham Biologicals), or Whooping cough intact cell composition are as the Tritanrix of SmithklineBeecham Biologicals sale ^TMDescribed combination-vaccine also can comprise other antigen, as hepatitis B virus surface antigen (HBsAg), poliovirus antigen (the trivalent poliovirus of deactivation-IPV) for example, morazella catarrhalis (Moraxella catarrhalis) outer membrane protein, do not determine the albumen of (non-typeable) Haemophilus influenzae of type, the outer membrane protein of Neisseria meningitidis B.

The example (especially for preventing otitis media) that can be included in the preferred morazella catarrhalis proteantigen in the combination-vaccine is: OMP106[WO97/417319 (Antex) and WO96/34960 (PMC)]; OMP21; LbpA and/or LbpB[WO98/55606 (pmc)]; TbpA and/or TbpB[WO97/13785 and WO97/32980 (PMC)]; CopB[HelminenME, etc. (1993) Infect.Immun.61:2003-2010]; UspA1 and/or UspA2[WO93/03761 (UniversityofTexas)]; OmpCD; HasR (PCT/EP99/03824); PilQ (PCT/EP99/03823); OMP85 (PCT/EP00/01468); Lipo06 (GB 9917977.2); Lipo10 (GB 9918208.1); Lipo11 (GB 9918302.2); Lipo18 (GB 9918038.2); P6 (PCT/EP99/03038); D15 (PCT/EP99/03822); OmplA1 (PCT/EP99/06781); Hly3 (PCT/EP99/03257) and OmpE.The examples of antigens (especially be used for preventing otitis media) that can be included in the Haemophilus influenzae that can not determine type in the combination-vaccine comprises: fimbrin [(US5766608-Ohio State ResearchFoundation)] and comprise to come syzygy [LB1 (f) the peptide syzygy for example of this proteic peptide; US5843464 (OSU) or WO99/64067]; OMP26[WO97/01638 (Cortecs)]; P6[EP281673 (State University of NeWYork)]; TbpA and/or TbpB; Hia; Hsf; Hin47; Hif; Hmw1; Hmw2; Hmw3; Hmw4; Hap; D15 (WO94/12641); Protein D (EP594610); P2; And P5 (WO94/26304).

Other combination is the combination of streptococcus pneumoniae PS and albumen of the present invention and virus antigen, and described virus antigen is for example from influenza virus (the attenuation type is separated build (split) or its subunit) [for example, surface glycoprotein neuraminidase (NA) and hemagglutinin (HA).Referring to, ChaloupkaI. etc. for example., Eur.Jounal.lin.Microbiol.Infect.Dis.1996,15:121-127], RSV (for example F and G antigen or F/G syzygy, referring to for example, SchmidtA.C. etc., J.Virol, May 2001, p4594-4603), PIV3 (for example, HN and F albumen are referring to Schmidt etc. the same), varicella virus (Varicella) (attenuation type for example, glycoprotein I-V, etc.), and MMR (Measles virus, mumps virus, rubella virus) any (or all) compositions.

Vaccine

A further embodiment of the present invention is to comprise the cytolysin that obtains by the inventive method, and preferred pneumolysin or pneumolysin-bacterial capsule polysaccharide conjugates and medicine can be accepted vehicle and optional adjuvant.

Vaccine of the present invention can comprise that immunogenic composition and medicine that the present invention is above-mentioned can accept vehicle.

Vaccine of the present invention can produce the protective immune response to streptococcus pneumoniae infection and/or otitis media.

The further embodiment of the present invention comprises the cytolysin that is made by the inventive method by getting, and preferred pneumolysin and medicine can accept that vehicle and further any above-mentioned one or more psma ligands are made vaccine and the method for preparing vaccine.

The present invention further embodiment comprises infectation of bacteria, and the treatment or the prevention method of preferred streptococcus pneumoniae infection or otitis media comprise the administration of vaccine of the present invention or immunogenic composition.

The present invention is the cytolysin of embodiment for obtaining by method of the present invention further, preferred pneumolysin and/or pneumolysin-bacterial capsule polysaccharide conjugates are used for the treatment of or prevents infectation of bacteria, the preferably purposes of the vaccine of streptococcus pneumoniae infection or otitis media in preparation.

Vaccine of the present invention preferably contains adjuvant.Suitable adjuvant comprises aluminium salt such as aluminium hydroxide gel (alum) or aluminum phosphate; but also can be calcium salt, magnesium salts, molysite or zinc salt; maybe can be insoluble acidylate tyrosine suspended substance, or positively charged ion or the anionic derivative or the polyphosphonitrile of the sugar of acidylate, polysaccharide.

Preferred described adjuvant is the preferential inductor that the TH1 type is replied.This high-caliber Th1 cytokines is more prone to the immunne response that inducing cell mediates at given antigen, and high-caliber Th2 cytokines tends to induce at this antigenic humoral immunoresponse(HI).

Remember that importantly the difference between Th1 type and the Th2 type immunne response is not absolute.In fact, individual immunne response can be based on Th1, or based on Th2.But judge cytokine family (Mosmann with Mosmann and Coffman at the description of mouse CD4+T cell clone for simplicity usually, T.R.and Coffman, R.L (1989) Th1 and TH2cells:different patterns of iymphocine secretion lead todifferent functional properties.Annual Review of Immunology, 7, p145-173).Usually, the Th1 type is replied relevant with the IL-2 cytokine with T lymphocyte generation INF-γ.Directly do not produce generally speaking, as IL-12 by the T cell with other relevant cytokine of inducing of Th1 type immunne response.On the contrary, the Th2 type is replied relevant with the secretion of IL-4, IL-5, IL-6, IL-10.The proper adjuvant system that promotes significant Th1 to reply comprises: the single phosphatidyl lipid A (3D-MPL) (it prepares referring to GB 2220211A) of single phosphatidyl lipid A or derivatives thereof, particularly 3-deoxidation acidylate; And single phosphatidyl lipid A, the single phosphatidyl lipid A of preferred 3-deoxidation acidylate with the combination of aluminium salt (for example aluminum phosphate or aluminium hydroxide), perhaps makes up with oil-in-water emulsion.In described combination, antigen and 3D-MPL are included in the same microgranular texture jointly, make the transmission of antigen signals and immunostimulating signal more effective.Studies show that 3D-MPL can further improve immunogenicity of antigens [.Vaccine (1998) 16:708-14 such as Thoelen that has adsorbed alum; EP689454-B1].

A raising system relates to combination, the especially QS21 of single phosphatidyl lipid A and saponin derivative and the combination of 3D-MPL, and it is disclosed in WO94/00153, perhaps relates to a kind of composition of low reactivity, and WO96/33739 is seen in wherein QS21 cholesterol cancellation.

A kind of potent especially adjuvant goods relate to and contain QS21 in oil-in-water emulsion, and 3D-MPL and tocopherol are described in WO95/17210, and this kind preparation is preferred preparation.

Preferred vaccine also comprises saponin(e, more preferably QS21.Described preparation also can comprise oil-in-water emulsion and tocopherol (WO95/17210).

The present invention also provides a kind of method for preparing vaccine preparation, and this method comprises cytolysin of the present invention and pharmaceutically acceptable vehicle, mixes as 3D-MPL.

The oligonucleotide (WO96/02555) that comprises non-methylated CpG also is the preferential inductor that TH1 replys, and is suitable for the present invention.

The present invention provides immunogen that is used for medical science or vaccine as described herein on the other hand.In one embodiment, the invention provides the method for a kind of prevention or alleviation the elderly (+55 years old) pneumonia, this method comprises the vaccine of the present invention to described gerontal patient's administration safety and significant quantity, and optional Th1 adjuvant.

In another embodiment, the invention provides the method for a kind of prevention or alleviation baby (maximum) or child's (normally 24 months to 5 years old) otitis media to 24 months, it comprises the vaccine to described baby or child's administration safety and significant quantity, described vaccine comprises cytolysin, preferred pneumolysin of the present invention, and further above-mentioned any one or multiple antigen and optional Th1 adjuvant.

Easily infected Mammals (preferred people experimenter) be protected or be treated to vaccine product of the present invention can by whole body or mucosal route administration.These administering modes comprise intramuscular injection, peritoneal injection, intracutaneous or subcutaneous route; Or by mucosa delivery to oral cavity/digestive tube, respiratory tract, reproductive tract.The intranasal administration that is used for the treatment of the vaccine of pneumonia or otitis media is preferred (because can carry streptococcus pneumoniae by more effective prevention pharynx nasalis, therefore just can alleviate infection in very early time).Although vaccine of the present invention can single agent administration, but its composition is Combined Preparation simultaneously also, or (for example at the different time Combined Preparation, can coordinate better in order to make between the immunne response, pneumococcal polysaccharide antigen can be in the bacterioprotein composition administration of vaccine or 1-2 administration respectively after week).Except single route of administration, can also use 2 different route of administration.For example, any virus antigen can pass through intradermal administration (ID), and bacterioprotein can pass through intramuscular administration (IM) or intranasal administration (IN).Polysaccharide can intramuscular administration (or intradermal administration), and bacterioprotein can intranasal administration (or intradermal administration).In addition, vaccine of the present invention can the intramuscular administration carry out initial immunity, and intranasal administration carries out booster immunization.

The antigenic amount of puting together in every dose of typical vaccine should be able to induce protective immune response, and does not have significant side effects.This amount will change according to employed specific immunogens and the different of route of administration.The content of proteantigen is usually in the scope of 1-100 μ g in the vaccine, preferred 5-50 μ g, 5-25 μ g most preferably usually.If comprise polysaccharide, usually, every dose should comprise polysaccharide 0.1-100 μ g, preferred 0.1-50g, preferred 0.1-10 μ g, most preferably 1-5 μ g.

Concerning concrete vaccine, the optimum quantity of each composition can determine that described research relates to the corresponding immunne response of observing the experimenter by research on standard.After the inoculation, the experimenter is carried out the booster immunization of one or many with the competent timed interval for the first time.Usually, vaccine comprises antigen (protein), adjuvant and vehicle or pharmaceutical acceptable carrier.

Vaccine product is described in Vaccine Design (" The subunit and adjuvantapproach " (eds PowellM.F.﹠amp usually; Newman M.J.) (1995) Pleun Press NewYork).Fullerton, United States Patent (USP) 4235877 have described and have carried out encapsulated with liposome.

Although vaccine of the present invention can pass through any administration, described vaccine carries out intracutaneous (ID) administration and has constituted one embodiment of the invention.Human skin comprises outside " cutin " epidermal area, is called stratum corneum, and it has covered epidermis.Be skin corium under this epidermis, skin corium has covered subcutis.The investigator finds the intradermal injection vaccine, but particularly intradermal vaccinate immune stimulatory is replied, and also follows other multiple advantages.Intradermal vaccination vaccine described herein has constituted a preferred feature of the present invention.

The routine techniques of intradermal injection " mantoux test (mantouxprocedure) " may further comprise the steps: cleaning skin, launch a hand, and the inclined-plane that makes small size syringe needle (26-31 number) then inserts a needle into the angle of 10-15 degree up.In case after the inclined-plane of pin inserted, syringe descended, further propulsive slightly exerting pressure simultaneously makes it lift subcutaneous.Then liquid is injected very lentamente, form bag or protuberance, slowly pin is withdrawed from then at skin surface.

Recently, intracutaneous or wear skin and be disclosed for the specific device with liquid preparation, for example: this device is disclosed in WO99/34850 and EP1092444, and jet injection device is disclosed in WO01/13977, US5,480,381, US5,599,302, US5,334,144, US5,993,412, US5,649,912, US5,569,189, US5,704,911, US5,383,851, US5,893,397, US5,466,220, US5,339,163, US5,312,335, US5,503,627, US5,064,413, US5,520,639, US4,596,556, US4,790,824, US4,941,880, US4,940,460, WO97/37705 and WO97/13537.The intradermal administration alternative method of this vaccine preparation comprises conventional syringe and pin, or the ballistic doser (WO99/27961) of solid vaccine, or through skin patch (WO97/48440; WO98/28037); Or skin surface administration (percutaneous dosing WO98/20734; WO98/28037).

When vaccine of the present invention is when being given to skin, when more specifically saying so corium, give and the gobbet vaccine, specifically be about between 0.05ml and the 0.2ml.

Antigenic content can be similar to the dosage of intramuscular vaccine routine in skin of the present invention or the intradermal vaccine.Therefore, proteantigen is at 1-100 μ g, in the preferred 5-50 μ g scope in the intradermal vaccine.Similarly, the common expection of the antigenic amount of polysaccharide conjugates comprises polysaccharide 0.1-100 μ g in every vaccinating agent, preferred 0.1-50 μ g, and preferred 0.1-10 μ g, and may be between 1 and 5 μ g.But described skin or intradermal vaccine are " low dose " preparation.Therefore every dose of proteantigen amount that contains is preferably 0.1-10 μ g in this " low dose " vaccine, and preferred every dose contains proteantigen 0.1-5 μ g; And every dose of antigenic amount of the polysaccharide conjugates that contains is 0.01-1 μ g, preferred 0.01-0.5 μ g.

Term used herein " intradermal administration " is meant the dermal zone that vaccine is given to skin.But vaccine not necessarily exists only in intradermal.Skin corium is apart from the skin layer between the human body skin about 1.0mm in surface and the about 2.0mm, but there are differences between the individuality and between the different sites of same individuality.Usually, the degree of depth of projected distance skin surface 1.5mm reaches skin corium.Being stratum corneum and epidermis above the corium, is subcutaneous layer below.According to the difference of mode of administration, this vaccine is finally existed only in or mainly be present in intradermal, or finally be distributed in epidermis and corium.

Immunogenic composition of the present invention and vaccine can be assessed in various animal models or with human serum.Illustrate, following animal model can be used for assessing pneumococcal infection.15 μ g s.c. protein immunizations of the available interpolation 50 μ l CFA adjuvants of C3H/HeJ mouse (6 to 8 week size), 2-4 injects the 15 μ g albumen that IFA is an adjuvant after week.In order to verify the passive of systemic infection and initiatively protection, 8-10 is during week, mouse by available immune serum before with the attack of 15 to 90LD50 streptococcus pneumoniae peritoneal injections or protein through peritoneal administration.In addition, protein can be at mouse nasopharynx clustered model (Microbial Pathogenesis 1997 such as Wu; Check 23:127-137).

Except mouse, young mouse be susceptible and clustered and pass through streptococcus pneumoniae infection.In the research of passive protection thing, the administration of mouse immune serum in the young mouse son of 2-5 days sizes (100 μ l i.p. or 10 μ l i.n.) can be finished before streptococcus pneumoniae (10 μ l) intranasal administration is attacked.Clustered can be measured by the washing (20-40 μ l instils, and 10 μ l cancel) of inoculation nasal cavity.

The interaction that (or protein and polysaccharide) is useful between the protein component of this combined vaccine can by administration with in the univalent vaccine serve as time-this vaccine of protectiveness in the dosage of every kind of albumen (or protein and polysaccharide) confirm.This combined vaccine is renderd a service owing to interaction useful between component with respect to the protection of univalent vaccine enhanced.

The present invention is in the following example illustrated.Embodiment uses standard technique to carry out, unless describe in detail in addition, it is well known to those skilled in the art and is conventional.Embodiment is intended to exemplify explanation, and unrestricted the present invention.

Embodiment

Embodiment 1

The purifying culture of Escherichia coli of pneumolysin is by after improving 18 hours of temperature to 39.5 ℃ and inducing, and intestinal bacteria are 17, centrifugal 1 hour of 000g and precipitating.Throw out is resuspended in the diethanolamine of 25mM pH9.0 and once finishes with the broken intestinal bacteria of mechanical means with 500PSI in the Rannie device.1% sodium N-lauroyl sarcosinate (SLS) adds in the broken intestinal bacteria and mixture was at room temperature hatched 1 hour, and subsequently 30, centrifugal 20 minutes of 000g is so that the cell debris precipitation.Supernatant liquor dilutes 2.5 times to the 20mM phosphoric acid salt of the pH7.0 that comprises 1M NaCl and 1%SLS, is splined on subsequently with same damping fluid (the 20mM phosphoric acid salt=level pad that comprises the pH7.0 of 1MNaCl and 1%SLS) equilibrated phenyl-sepharose HP post.Post is with the washing of the level pad of 4 times of column volumes, and the 20mM phosphate buffered saline buffer with the pH7.0 that comprises 0.5MNaCl and 1%SLS of 2 times of column volumes washs subsequently.Pneumolysin by using low salt buffer from the post wash-out, damping fluid comprises the 20mM phosphate buffered saline buffer that contains 1%SLSpH7.0.The component that comprises pneumolysin is utilized the SDS-PAGE Analysis and Identification and is collected, and damping fluid utilizes diafiltration to be exchanged for the 25mM diethanolamine of pH9.0.

Pneumolysin dissolves by interpolation solid Guanidinium hydrochloride to 6M final concentration sex change and hatched one hour.It is with after comprise the 25mM diethanolamine that 1mM DTTpH9.0 contains 8M urea and filter thoroughly.Pneumolysin is folding again by the saturating filter of the pH9.0 ground 20mM borate that comprises 1mM DTT.After the renaturation, DTT filters thoroughly by the 20mM borate buffer solution of pH9.0.

The pneumolysin purity that is reached is by last sample SDS-PAGE and coomassie brilliant blue staining analysis.Separation gel is analyzed to detect Escherichia coli protein level residual in the pneumolysin prepared product of purifying by utilizing anticolibacillary antibody protein blotting.Utilize the in vitro biological activity of the pneumolysin of hemolytic test evaluation purifying.

The result

As shown in Figure 1, aforesaid method can reach the efficiently purifying of pneumolysin after single chromatographic step.Show can be with the 53kD band of very pure form wash-out corresponding to pneumolysin from the post with the low salt buffer wash-out post that does not add sodium-chlor for the gel of Coomassie blue stain among the plate A.The very weak band of about 45kD is also thought pneumolysin, because this second band is in conjunction with the antibody (result does not show) of anti--pneumolysin and can not be in conjunction with anticolibacillary antibody (shown in plate B).The western blotting of plate B is for detecting the high-sensitivity method of any contaminating protein matter in the pneumolysin that remains in purifying.This method can detect to be lower than few pollutent that coomassie dyeing detection level exists.Therefore pneumolysin is purified to the purity of 98-100%.

The output of this purification process of classic test determination is every liter of fermented liquid 1900mg pneumolysin approximately, also is good.Reclaim from the protein of fermenting culture about 10% pneumolysin as purifying.

The removal folding again back evaluation that the activity of pneumolysin is handled with Guanidinium hydrochloride/urea and passed through denaturing agent at pneumolysin in the hemolytic test.In reducing to the diluent of the concentration 1.3ng/ml that shows that hemolytic activity has been rebuild, the pneumolysin of purifying detects hemolytic activity.This is equivalent to every milligram 500,000 and 1,000 of wild-type pneumolysin, between 000 hemolytic unit.

Embodiment 2-pneumolysin utilizes the detoxifcation of GMBS

The pneumolysin of purifying detoxifies to the modification of sulfydryl and primary amine groups by utilizing NHS ester-maleimide amine crosslinker GMBS (N-(γ-maleimide butyryl acyloxy) succinimide ester).0.5mg/ml the pneumolysin of concentration is through the phosphate buffered saline buffer dialysis of 50mM pH7.0.GMBS is dissolved at first among the DMSO and with GMBS248-molar excess doubly and adds pneumolysin.Handle and at room temperature continue one hour.Excessive GMBS and byproduct are removed by the 100mM sodium phosphate dialysis of pH6.8.Further, maleimide base group is by at room temperature reacting two hours and cancellation with the 0.6mg/ml halfcystine.In order to remove excessive halfcystine, sample is dialysed through the 2mM of pH7.15 sodium phosphate.

The evaluation of embodiment 3-detoxifcation pneumolysin

Hemolytic activity

Hemolytic test is used to evaluate the residual toxicity of toxicide pneumolysin.Pneumolysin and sheep red blood cell (SRBC) that continuous 2-doubly dilutes hatch.After centrifugal, supernatant liquor is transferred to the immunity flat board and is utilized the optical density readings at 405nm place to measure the oxyphorase that discharges.The result is expressed as the ng/ml pneumolysin, is equivalent to the mid point of OD curve.37 ℃ hatch the toxicide pneumolysin after 7 days revision test with monitoring toxicide reversibility.

As shown in table 1, can reduce the hemolytic activity of PLY basically to the reduction that realizes 3,000 times of hemolytic activities with the processing of GMBS.In this experiment, the more high molar ratio of GMBS/ Methionin can better be removed with 4/1 and 5/1 best ratio realization hemolytic activity.This processing estimates to have caused the modification of about 14 lysine residues.Less lysine residue is modified, and then the reduction of hemolytic activity is just relatively poor.

ELISA

Antigenicity by ELISA evaluation toxicide pneumolysin.The ELISA flat board with cavy anti--antibody sandwich of pneumolysin.The sample that comprises the pneumolysin dilution was hatched under room temperature 1 hour in flat board.After the washing, utilize the rabbit polyclonal antibody of the anti-pneumolysin that is conjugated to horseradish peroxidase to detect the bonded pneumolysin.Behind the washing flat board, utilize enzyme substrates to react and evaluate the pneumolysin amount that is bonded to each hole.

As shown in table 1, just, cause some antigenic losses with the GMBS processing as by the ELISA evaluation.Yet about 66% the ELISA reading that can reach given unprocessed PLY shows many antibody and still can discern the pneumolysin of modified.

SDS-PAGE-analyzes

Toxicide pneumolysin protein is splined on SDS-PAGE (Novex4-20% polyacrylamide gel Invitrogen) and shows this protein with Xylene Brilliant Cyanine G.As shown in Figure 2, the processing with GMBS causes the slight raising of PLY molecular weight from 53kD to about 56kDa.This improves owing to the modification of a plurality of amino-acid residues with GMBS.It seems that from the appearance of the faint band of about 110kD and 170kD molecular weight the PLY of small proportion becomes the polymer form, yet most of PLY keep monomeric basically form.Hatch the influence that any material alterations that did not cause PLY appearance on the SDS-PAGE in PLY7 days shows that the PLY of modification is not formed by the polymer of Degradation or covalency-connection subsequently at 37 ℃.

Table 1: by the PLY detoxifcation test of GMBS

Handle (PLY is 0.68mg/ml) except last test with 3mg, test is finished with 1mg PLY (1mg/ml).

The reactionogenicity assessment of embodiment 4 toxicide lung class coccus hemolysins in rat

Three OFA rat groups are by using salt, adjuvant QS21 (US5,057,540), the pneumolysin of pneumolysin, interpolation adjuvant, formaldehyde toxicide pneumolysin, the formaldehyde toxicide pneumolysin that adds adjuvant, GMBS toxicide pneumolysin, the GMBS toxicide pneumolysin that adds adjuvant, NHS-acetate toxicide pneumolysin or add NHS-acetate toxicide pneumolysin intramuscular injection (tibialis) inoculation of adjuvant and immunity once.All rats are put to death in back three days of immunity and the preparation tibialis is used for histological examination.Tibialis fixes and is cut into the thin section of 2mm in formalin, it is through dehydration and paraffin embedding.Before test under microscope, be cut into the section of 7 μ m and dye with Trichrome Masson method.

With four criterion evaluation reactionogenicities; Degeneration/necrosis, endomysium inflammation, hemorrhage and aponeurosis (aponeuroses) inflammation.Based on each histology standard, each muscle divided rank of each group is calculated the average pathology grade of each group subsequently.Grade 0=is normal, and 1=is atomic, and 2=is slight, the 3=moderate, 4=is significant and 5=is serious.

The result

Check the weave construction of section.Table 2 shows degenerations/necrosis, endomysium inflammation, the average rank of hemorrhage and aponeurosis (aponeuroses) inflammation.

Table 2

Inoculation	Degeneration/necrosis	The endomysium inflammation	Hemorrhage	The aponeurosis (aponeuroses) inflammation
NaCl	0	0.5	0	0
					Ply	3.6	3.8	3.0	1.4
GMBS-Ply	0.6	1.3	1.3	0.4
					Adjuvant	2.9	3.9	2.8	2.8
The Ply+ adjuvant	4.2	3.9	4.6	1.8
					The GMBS-Ply+ adjuvant	2.9	3.9	3.8	1.6

Natural and the histology grade toxicide pneumolysin that does not add adjuvant comparison shows that GMBS is used for especially effectively linking agent of pneumolysin toxicide, and it makes degeneration/necrosis, endomysium inflammation, hemorrhage and aponeurosis (aponeuroses) inflammation reduce in a large number.

Adjuvant to the interpolation (50 μ g aluminum phosphates and 5 μ g MPL) of inoculum as the bystander cell of stimulating immune system and improved the reactionogenicity value.Pneumolysin makes degenerations/necrosis be reduced to the level that reaches by adjuvant separately with the detoxifcation of GMBS, its than by inoculate with natural pneumolysin be up to the standard low.GMBS toxicide pneumolysin reaches, and to go out blood level lower than what reach by natural pneumolysin.The level of endomysium inflammation improves by adjuvant and this level still exists when the natural or GMBS toxicide pneumolysin that adds adjuvant exists.Along with the slight reduction of aponeurosis (aponeuroses) level of inflammation after pneumolysin is handled with GMBS, the aponeurosis (aponeuroses) inflammation begins to reduce from the level that is produced by adjuvant separately by natural or GMBS toxicide pneumolysin.

The toxicity assessment of the pneumolysin that GMBS handles in the embodiment 5-mouse

The pneumolysin intranasal that 20 OF1 mouse group is handled with natural pneumolysin or GMBS-is attacked and mouse is monitored subsequently 9 days.

As shown in Figure 3, all mouse cause rapid death with the natural pneumolysin attack of 2 μ g in that group.The pathology that pneumolysin causes spreads all over respiratory system, and it causes expiratory dyspnea and dead.On the contrary, the pneumolysin of GMBS processing has the toxicity that reduces in fact to inculcating all mouse of attacking and surviving with the pneumolysin of 2 μ g, 5 μ g or 10 μ g GMBS processing.

Embodiment 6-utilizes the protection research of toxicide lung class coccus hemolysin

20 OF1 mouse group 0 day, 14 days and 28 days with 5 μ g pneumolysins and 50 μ g aluminum phosphates and 5 μ gMPL as adjuvant intramuscularly immunity 3 times.Control mice is immune separately with adjuvant.Pneumolysin is undressed or utilizes above-mentioned GMBS to handle toxicide.

At the 42nd day, mouse gave that 2 μ g are natural pneumolysin intranasal, lethal attack.The mouse of monitoring survival in 9 days subsequently.

The result

This lethal attack mode causes 90% death (Fig. 4) in control mice.Produce good provide protection with the immunity of GMBS toxicide pneumolysin, in subsequently 9 days, have only 5% dead mouse.This is with suitable with the provide protection of natural pneumolysin inoculation back 10% dead mouse.

Embodiment 7 toxicide pneumolysins associatings PhtD is in mouse causes death attack model

Assessment

20 OF1 mouse group a) independent adjuvant or b) 1 μ g PhtD and adjuvant or c) 1 μ g PhtD and 5 μ g GMBS toxicide pneumolysins and adjuvant intramuscularly and immunity.Used adjuvant is made up of 50 μ g aluminum phosphates and 5 μ gMPL, and carries out immunity at the 0th day and the 14th day.Mouse is attacked with the 5.105CFU lethal dose of the serotype 2 streptococcus pneumoniae strain D39 of intranasal and in subsequently 10 days monitoring survival rates.

The result

As shown in Figure 5, the attack with bacterial classification D39 causes 75% to cause death in control mice after 10 days.Fail to provide significant provide protection with the immunity of PhtD separately, 70% dead mouse (p=0.29) after 10 days in this group.With PhtD and the obvious better protection effect of immunity generation together of GMBS toxicide pneumolysin, lethality rate is reduced to 50% (p=0.04).

The detoxifcation of formaldehyde of embodiment 8 pneumolysins

The pneumolysin mother liquor of the purifying of the about 0.4mg/ml of concentration was handled 21 days at 40 ℃ with 50m ML-Methionin and 0.1% formaldehyde (w/v) in the pH7.025mM potassium phosphate buffer.

Claims (27)

1. method that is used for bacterium cytolysin purifying said method comprising the steps of:

A) culture of the cell of culture expression bacterium cytolysin;

B) from the culture that comprises the bacterium cytolysin, prepare extract;

The solubility agglutinative bacterium cytolysin that will be contained in the extract when c) having 0.1-5% (w/v) aliphatics stain remover under the high salt condition of 0.6-2M under pH6-8 is bonded to phenyl-sepharose hydrophobic interaction chromatography material;

D) wash-out bacterium cytolysin when under pH6-8, having 0.1-5% (w/v) aliphatics stain remover under the 0-0.2M low-salt conditions;

E) from the bacterium cytolysin, remove stain remover;

F) contain the denaturing agent dissolution of bacteria cytolysin of Guanidinium hydrochloride by interpolation;

G) from the bacterium cytolysin, remove denaturing agent.

2. the process of claim 1 wherein that the bacterium cytolysin is a pneumolysin.

3. claim 1 or 2 method, wherein step b) comprises and uses the mechanical means smudge cells.

4. each method among the claim 1-3, wherein step b) comprises and uses detergent-treatment.

5. each method among the claim 1-4, wherein same stain remover is present in step c) and d) in.

6. the method for claim 4, wherein same stain remover is present in step b), c) and d) in.

7. each method of aforementioned claim, wherein step b) comprises the disruptive cellular material is centrifugal and collect the extract of supernatant liquor as step c).

8. each method of aforementioned claim, wherein stain remover is a sodium N-lauroyl sarcosinate.

9. each method of aforementioned claim wherein is used for step c) and/or d) solution comprise the salt that is selected from sodium-chlor, magnesium chloride, ammonium chloride, sodium sulfate, sal epsom, ammonium sulfate, sodium phosphate, trimagnesium phosphate, ammonium phosphate.

10. each method of aforementioned claim, the condition that wherein is used for step c) and/or step d) is about pH7.

11. each method of aforementioned claim, wherein the used condition of step d) comprises the salt of 0-0.1M.

12. the method for claim 11, wherein the used condition of step d) comprises the salt of 0-40mM.

13. each method of aforementioned claim, wherein step e) comprises by diafiltration or dialysis and removes stain remover.

14. the method for claim 13, wherein diafiltration/dialysis pH8-10, preferably the low salt buffer of about pH9 carries out.

15. each method among the claim 1-14, wherein step f) comprises that the interpolation by denaturing agent comprises by progressively removing this denaturing agent again folding bacterium cytolysin with sex change of bacterium cytolysin and step g).

16. the process of claim 1 wherein that the Guanidinium hydrochloride that uses 5-8M is as the denaturing agent in the step f).

17. the method for claim 16, wherein the bacterium cytolysin contacts with 5-9M urea in step f).

18. the method for claim 17, wherein step f) comprises contacting of bacterium cytolysin and 5-8M Guanidinium hydrochloride, subsequently Guanidinium hydrochloride is replaced with the urea of 5-9M.

19. each method among the claim 16-18 is wherein at step f) and g) during small part, have reductive agent.

20. the method for claim 19, wherein reductive agent is the DTT of 0.1-10mM, preferably the DTT of about 1mM.

21. each method among the claim 1-20, wherein step g) comprises by diafiltration or dialysis removal denaturing agent.

22. the method for claim 21, wherein diafiltration or dialysis are carried out with the solution of pH7-9.

23. each method of aforementioned claim, described method also comprise by carry out the detoxify step of bacterium cytolysin of chemical treatment with linking agent.

24. the method for claim 23, wherein chemical treatment comprise use can with the linking agent of amine and mercapto groups reaction.

25. the method for claim 23, wherein linking agent comprises that one or more are selected from the chemical reagent of formaldehyde, glutaraldehyde, N-hydroxy-succinamide ester and GMBS.

26. each method of aforementioned claim, described method also comprises the step that the bacterium cytolysin is conjugated to the bacterial capsule polysaccharide.

27. each method of aforementioned claim, described method also comprise the step that bacterium cytolysin and pharmaceutical acceptable excipient is made into vaccine composition.