CN107405395A

CN107405395A - The method of the composition and treatment urinary tract infections of vaccine and adjuvant

Info

Publication number: CN107405395A
Application number: CN201680016531.7A
Authority: CN
Inventors: 加里·爱尔德里奇; 斯蒂文·M·马丁
Original assignee: Sequoia Sciences Inc
Current assignee: Sequoia Vaccines Inc
Priority date: 2015-03-17
Filing date: 2016-03-16
Publication date: 2017-11-28
Also published as: WO2016149558A3; EP3270963A2; WO2016149558A2; WO2016149417A1; EP3270962A4; CN107530418A; EP3270962A1; EP3270963A4

Abstract

The present invention describes new adjunvant composition and preparation, its refrigerate and at room temperature and reach and about 37 DEG C at there is fabulous stability, can be with significantly low cost production.The present invention describes new vaccine combination and preparation, to treat and prevent as the urinary tract infections caused by gramnegative bacterium includes Escherichia coli and multi-drug resistance Escherichia coli.Invention further describes the method for applying the new vaccine combination and preparation, and the treatment method of prevention and treatment urinary tract infections as caused by gramnegative bacterium includes Escherichia coli and multi-drug resistance Escherichia coli.

Description

The method of the composition and treatment urinary tract infections of vaccine and adjuvant

Background of invention

Invention field

The invention provides new adjunvant composition and preparation, and it is in refrigeration and at room temperature and also in the case where reaching about 37 DEG C With fabulous stability, can be produced with significantly low cost.These new adjunvant compositions and preparation are used for vaccine, exhibition Show the superior property of immune response of the enhancing to antigen, while cause less serious injection position and systemic reaction. Invention further describes new vaccine combination and preparation, come treat and prevent by gramnegative bacterium include Escherichia coli and Urinary tract infections caused by multi-drug resistance Escherichia coli (E.coli).Present invention also offers apply the new bacterin preparation Method, and prevention and treatment urine as caused by gramnegative bacterium includes Escherichia coli and multi-drug resistance Escherichia coli The treatment method of road feel dye.

Description of Related Art

In the U.S. and other countries, protect most of crowds from many communicable diseases by using vaccine.Only lift Several examples, vaccine protects people from communicable disease, for example, diphtheria, lockjaw, pertussis, hepatitis, influenza and gray nucleus It is scorching.The protection that society is provided dependent on vaccine, this make it that these most of communicable diseases are only the one of United States citizen history Part.In fact, in the U.S., Center for Disease Control (CDC) implements pediatric vaccines plan, and it was for 4,000 ten thousand in 2010 Children provide free inoculation.About the 70% of these children adds Medicaid.In order to prevent the outburst of disease With the cost for reducing these communicable diseases for the treatment of, the U.S. has made vaccine inoculation have the country independently of economic scene preferential Property.The vaccine of low cost is to be badly in need of, and in present and foreseeable future, these vaccines have national priority.

In view of the importance of vaccine, it is evident that need continual exploitation new and improved vaccine, to improve our people The health status of group.More crucially need to provide more inexpensive vaccine to help to reduce soaring for U.S. sanitary system Cost.National priority is the cost of U.S. sanitary system to be reduced.

Even in the U.S., it is to meet vaccine to preserve requirement to cause these difficulties.By health and the prison of Department of Public Enterprises (HHS) A research (OEI-04-10-00430) that is that Cha Chang offices are carried out and being reported in 2012 finds, participates in CDC children The supplier of vaccine program：1.) at a temperature of vaccine is exposed to beyond the approved temperature range of these vaccines：2.) by epidemic disease Seedling is stored in refrigerator and freezer at a temperature of being stored in outside their approved temperature ranges；And 3.) will be overdue Vaccine is saved together with not yet due vaccine.

The other problemses of vaccine are that vaccine may have short storage life, easily expire before the use.

In addition, the studies above that Office, The Inspector General is carried out is found, 46 U.S. sanitary suppliers of pediatric vaccines plan In the overdue vaccine of 16 stars be saved together with undue vaccine.On an average, these overdue vaccines are expired About 6 months.For example, 4,000 ten thousand parts of untapped families of about 200,000,000 6 thousand ten thousand dollars of production are consumed on July 1st, 2010 according to reports Swine flu vaccine expires and destroyed.In the U.S., annual vaccine, which expires, generates huge economic loss.

Adjuvant enhances the immune response to the antigen of vaccine.There is provided in the works in the pediatric vaccines that the U.S. is implemented by CDC In 34 kinds of vaccines, 20 kinds contain adjuvant.This 20 kinds have in the vaccine of adjuvant, and 19 kinds of vaccines contain alum adjuvant, a kind of vaccine Adjuvant is used as containing the monophosphoryl lipid A (GSK MPL) for being adsorbed onto alum.

Although the whole industry is attempted to develop new adjuvant, only have at present in the U.S. alum and GSK MPL be used for ratify epidemic disease Seedling.In the U.S., many adjuvant exploitations meet with failure, but still very high to the demand of new and effective adjuvant.

GlaxoSmithKline PLC (GSK) containing the 3'-O- deacylation base -4'- monophosphoryl lipid As for being adsorbed onto alum Cervarix vaccines are approved for preventing the cervix cancer as caused by HPV in the U.S..Due to producing MPL Parent material be isolated from salmonella (Salmonella minnesota), final products are six acyl groups, five acyl groups and four acyls The dynamic complex mixture of base analog；Each of these analogs is all different in terms of biological activity.As a result, 3'- O- deacylation base -4'- monophosphoryl lipid As present manufacture, test and use upper challenge, and it greatly improves the cost of vaccine And supply problem.

In addition to preservation problem, vaccine injection is often pain for subject.Can after the administration of vaccine Can occur injection site it is rubescent, expand, itch and touch a tender spot.The Cervarix vaccine regimens letter of GSK containing MPL and alum adjuvant Breath lists local detrimental event, and it may include pain, rubescent and swelling.Receiving GSK Cervarix vaccines or list About percent 8 report the local pain for hindering activities of daily life in the subject of only adjuvant alum.Contain MPL applying Include headache, fatigue, heating, fash, courbature, joint with the Systemic reaction observed after the vaccine of alum adjuvant Bitterly, nettle rash and gastrointestinal symptom, including nausea,vomiting,diarrhea and/or stomachache.

In addition, " the Clinical Review of Human Papillomavirus Bivalent (Types 16 in And 18) vaccine [GSK's Cervarix Vaccine], Recombinant, Biologies License Application Efficacy Supplement " descriptions, four research reports local detrimental event, it is included in about The local pain of motion is hindered in percent 16 subject.It is reported in about percent 3 subject more than 50mm Swelling.For arthralgia, fatigue, stomach and intestine, headache and courbature, this four researchs report 2.4 to 7.8 percent whole body The serious adverse event of property.

Injection site reaction and the severity of systemic reaction be it is important, it is necessary to be related to anesthetic use, IV aquations or The disposal that other doctors implement, and come from and prevent from being brought by diarrhoea, courbature, fatigue, headache and the daily routines of vomiting Work-loss costs.

It is immune to put into practice consultative committee (Advisory Committee on Immunization Practices) foundation The suggestion of country's strategy of influenza.This strategy is included " to all United States residents in 6 months of declaration of being very popular Pandemic disease vaccine is provided：Pandemic disease vaccine (600,000,000 doses) [country's tactful (in November, 2005) of pandemic influenza and HHS Pandemic influenza plans (in November, 2005) " demand, and need to use adjuvant attempt can not close on this surface The vaccination targets of realization.Due to the U.S. be used for general influenza vaccines approval adjuvant, American National vaccine Deposit does not select, and spends about 500,000,000 dollars to buy MF59 adjuvant from Novartis.Due to " being observed in 9 to 12 years old children Height vaccine reaction originality, the scheme for studying V7P29 are modified to eliminate the children less than 9 years old ", MF59 adjuvant is in Fluad It is interrupted in the clinical research of paediatrics.The evidence is supported during national pandemic disease is declared, due to using MF59 adjuvant will The substantial amounts of severe reaction of generation, and need extra medical nursing.

The synthetic analogues of monophosphoryl lipid A are during about 2005 by Avanti Polar Lipids (Alabaster, Alabama, USA) is introduced.This synthetic analogues are named as six acyl group phosphorus by Avanti Polar Lipids Disaccharides (PHAD) is acidified, also referred to as " GLA ".The PHAD of Avanti Polar Lipids supplies provides as single compound, Show in fig. 1, about 98% purity, the dalton of molecular weight 1763.PHAD purity and GSA MPL form notable contrast, As described above, MPL is isolated from salmonella, exist as dynamic complex compound.Different from GSA MPL, PHAD production Process, supply, use and stability closely can be monitored and controlled as pure compound.

Whether the combination of specific adjuvant or adjuvant is unpredictable by the immune response strengthened for every kind of specific antigen 's.For example, GSK Cervarix vaccines contain monophosphoryl lipid A and alum, because this combination is better than single alum (Giannini et al.Vaccine, 2006,24, p.5937-5949).The vaccine of HbsAg is used The similar effect of observation improves tables 6 of (Vaccine, 1998,16 (7), the p.708-714) in United States Patent (USP) 6,889,885 In show another example, its present antigen-adjuvant combination produce for specific antigen immune response in terms of can Denaturation.These inventors present, compared with single white sail or monophosphoryl lipid A, QS-21 adjuvants, and it is individually white Alum adds the adjuvant combination of monophosphoryl lipid A to generate the bigger antibody response for 74kD protein.In addition in 2009, Novartis Vaccines Derek T.O'Hagan and Ennio De Gregorio are disclosed on the comprehensive of adjuvant exploitation State.June 2009, p.541-551 (Drug Discovery Today, 14 (11/12)) they report, for some eggs White matter or antigen, alum are relatively weak adjuvants, it is still desirable to new adjuvant.

2004, growing number of antibiotic resisted in communicable disease association of the U.S. (IDSA) advanced warning world wide The crisis of property bacterium, its generation can be resisted without new horizon antibiotic.2009, IDSA was identified to current all anti- The raw occurent bacterium infection for being known as resistance, most warning antibiotic-resistant bacteria is gramnegative bacterium, bag Include Escherichia coli.2010, IDSA was claimed, although effort has been made in many private, public's and government laboratories, was studied Do not produce the new selection for the treatment of antibiotic-resistant bacteria, it is now desired to global input.The urging of IDSA has obtained Ge Lan The support of the scientist of plain SmithKline, they predict, any antibiotics for treating gram-negative bacterial infections open With need before more than ten to 15 years (Payne et al.Nature Reviews Drug Discovery.2007,6, p.29-40)。

Failure trial of their prediction based on 34 companies exploitation antibiotics.Known together in scientific circles, It is badly in need of method of the new treatment for bacterium infection in the U.S..Adam L.Hersh are with colleague in 2012 in Clinical (Hersh et al.CID.2012.54 (11), 1677-8) is reported to whole beautiful in Infectious Disease periodical The investigation of 562 infectious disease doctors of state, in last year, 63% medical treatment is excessively resistant to all known antibiotic Bacterial infection patients.These data highlight the demand to the new treatment means of bacterium infection.Identified newly in this area Therapeutic agent selection have recorded well to prevent and treat the failure of gram-negative bacterial infections.

In addition, stopped in recent years to prevent or treat at least five kinds of vaccines of S. aureus infection in exploitation Only.These include STAPHVAX, Veronate, Aurexis, Aurograb and V710.New vaccine is identified to prevent and control The failure for treating bacterium infection have recorded well.

Urinary tract infections (UTI) is one of most common communicable disease in world wide, in the U.S. be that women is met with the The communicable disease of one.It is annual in the U.S. that UTI symptom includes dysuria (urination pain), urgent urination and suprapubic pain It is estimated to be 7 million to 1 1,000 1 million women and acute uncomplicated UTI occurs.All adult females' exceedes half in their one One or many UTI will be met with life, 25-44% women undergoes recurrent UTI.In fact, in the U.S. about 1,000,000 Name women and male's experience UTI acute attacks three times or more every year.In addition, in spite of appropriate antibiosis extract for treating and urine The obvious removing of middle primary infection, recurrence usually occur in 30 to 90 days of infection.

Although having new progress in terms of UTI epidemiology and pathology, actually prevent at us or treat this Also without relatively much progress recently in terms of the ability infected a bit.The women for undergoing the 25-44% with UTI of recurrent infection needs Extra treatment, extra-pay are wanted, and to need extensive urology to assess in some cases more serious to prevent Complication.Thus, there is the safely and effectively vaccine for the potentiality for improving patient convenience and reducing expense for patient, supply Business and healthcare facility are quite attractive.In the UTI colonies of recurrence, because treatment option is constantly reduced, resist micro- The problem of biocide resistance is important.Thus, it is badly in need of developing new method for UTI prevention and treatment, it is less dependent on The use of antimicrobial.

UTI is most commonly caused by urinary tract enteropathogenic E. Coli (UPEC), and it may be obtained to the community for being up to 85% Property UTI is responsible for.The pathogenic cascade of key is disclosed, UPEC escapes host defense and the rapid expansion number in urinary tract by it Amount causes disease.This work supports the medical need to UTI vaccines.

FimH plays an important role in the several stages for causing a disease cascade, and this causes it to turn into main vaccine target spot.Lack The UPEC bacterial strains of weary FimH adhesins can not effectively colonize bladder.Infected for FimH vaccine by host defense is activated All stages identification and remove UPEC, even protected in IBC or intracellular banks.

Contain the FimCH vaccines of squalene using MF59 as adjuvant by Medlmmune Inc. and Scott Hultgren Scientist's joint invention (United States Patent (USP) 6,500,434 in the laboratory of professor；It is completely integrated herein).FimH albumen and FimC albumen exists in the form of non-covalent protein complex FimCH.FimC stabilizes FimH, and antibody is directed to two hatching eggs White matter produces, but shows that the Escherichia coli that bladder is reduced in animal colonize only for FimH antibody.Thus in vaccine It is middle that the limitation for needing effective adjuvant is received using FimCH as antigen.

FimCH vaccines (oil-in-water emulsion) with the MF59 adjuvant containing squalene trigger in 1 clinical trial phase Immune response (U.S. Patent application 20030138449, all merges herein).Reuse the MF59 assistants containing squalene Agent has carried out 2 clinical trial phases in two independent colonies, but women does not produce in any one of these experiments For FimH related IgG titres.Due to these disappointed results, the Medlmmune of MF59 adjuvant FimCH is used The exploitation of vaccine is stopped.When some antigens are used together, the MF59 adjuvant with squalene, which has, causes serious part Injection site and the history of systemic reaction.During these 2 clinical trial phases, women experienced serious injection site reaction With serious systemic reaction.Due to this failure, in the U.S. also without the vaccine of the treatment or prevention for UTI.

For prevent and treat UTI vaccine there is lasting demand.Medlmmune is with other people in exploitation UTI epidemic diseases Failure on seedling demonstrates the difficulty in terms of exploitation is used for the novel vaccine of bacterium infection.Medlmmune demonstrates alum deficiency To strengthen the immune response to FimCH.Medlmmune does not have clear and definite adjuvant selection to match FimCH antigens.

Thus, it is used for strengthening urgent need to the adjuvant of the immune response of antigen be present for vaccine and in vaccine Ask.Vaccine and the adjuvant for vaccine are needed, it has the stability of extension without sacrificing effect.Especially, in room temperature There is urgent and generally acknowledged demand for the vaccine and adjuvant of lower stabilization.Moreover, it may be desirable to possess in the temperature higher than room temperature Vaccine, adjuvant and the composition of lower stabilization.

Moreover, it is necessary to produce less serious injection site and the adjuvant and pharmaceutical composition of systemic reaction.

Need new vaccine to prevent and treat bacterium infection, particularly for UTI prevention and treatment vaccine.

Desirably possess vaccine, and the adjuvant for the immune response of enhancement antigen in vaccine, there is the storage improved Deposit the life-span and can be produced in a manner of low cost.Such vaccine and adjuvant are greatly reduced the hygienic cost in the U.S., special It is not if they can be preserved without influenceing their stability under room temperature or higher temperature.

Desirably possess adjuvant and vaccine, they need to produce the adjuvant of minimum injection sites and systemic reaction and Vaccine.Desirably possess the preparation that needs have excipient as few as possible.

Desirably possess vaccine and the adjuvant for vaccine, the immune response of the adjuvant enhancing treatment bacterium infection. Desirably possess vaccine and the adjuvant for vaccine, patient of the adjuvant enhancing with UTI should to the immune of Escherichia coli Answer.

The content of the invention

The present invention solves many problems of the adjuvant of prior art described here, vaccine and pharmaceutical composition.This hair Bright to provide new liquid adjuvant composition and preparation, it provides many unexpected and beneficial property, its in adjuvant and It is unknown in the field of pharmaceutical composition.

In one aspect, there is provided liquid adjuvant composition and preparation, it presents room temperature stability about more than 6 months, And reach at about 37b DEG C about 60 days or more long.The new liquid adjuvant composition and preparation can be stored in refrigerated storage temperature Or under room temperature condition, be advantageous to the storage life during accumulating and reduce cost of transportation.

By allowing a kind of low cost and unexpectedly and significantly stable adjuvant formulation, it is with lower serious note Penetrate position and systemic reaction enhances immune response to bacillus coli antigen, the adjuvant formulation solution of invention described herein Determine many current obstacles in vaccine administration.

Data exhibiting described herein, adjuvant formulation of the invention are enhanced to including other of bacterium and viral antigen The immune response of antigen.

Invention described herein by treatment as gramnegative bacterium include Escherichia coli caused by urinary tract infections be This difficulty is reduced to contribute.

In one aspect, new adjunvant composition is disclosed, it has in 2 DEG C to 8 DEG C and room temperature and at reaching about 37 DEG C There is significant stability.

In one aspect of the invention, a kind of composition, a kind of synthetically produced acylphosphate disaccharides of adjuvant six is included Or its acylphosphate disaccharides of derivative 3- deacylations base-six, and selected from by about 25mM to about 50mM, preferable 28mM is to about The buffer solution for the group that 50mM and most preferred 30mM to about 50mM citrate, succinate and phosphate is formed.These are new The sugar composite of six acylphosphateization two be preferably the suspension of aqueous buffered.The composition can be in vaccine and medicine feelings Used in a variety of ways under border.The composition, preferably without other compositions, significantly improve six acylphosphateizations two The stability of sugar or its acylphosphate disaccharides of derivative 3- deacylations base-six in suspension, at room temperature and reaches peace treaty Superior stability is realized at 37 DEG C.The composition also presents fabulous stability at refrigerated temperatures.Offer is provided A kind of efficient and economic sugar composite of six acylphosphateization two, it need not be refrigerated for long-time stability, this representative Marked improvement in terms of adjuvant and pharmaceutical technology.

In another aspect of the present invention, there is provided the new adjuvant formulation as the suspension of aqueous buffered.One In individual embodiment, the adjuvant formulation includes a kind of synthetically produced the acylphosphate disaccharides of adjuvant six or its derivative 3- The acylphosphate disaccharides of deacylation base-six, selected from by about 10mM to about 50mM, preferably from about 25mM to about 50mM, more preferably The buffering for the group that 28mM to about 50mM and most preferred 30mM to about 50mM citrate, succinate and phosphate is formed Liquid, and a kind of preferable synthetically produced phosphatidyl choline.When adding phosphatidyl choline, preferable buffer concentration can be with About 10mM is expanded to about 50mM, and realizes significant stability described herein.These new adjuvant formulations are preferably The suspension of aqueous buffered.The adjuvant formulation be stored in refrigeration and at room temperature when there are fabulous long-time stability, up to Arrive and about 37 DEG C at there is fabulous stability.These preparations can be produced with significantly low cost.

In order to room temperature stability or reach and about 37 DEG C at stability, new adjuvant formulation described herein need not freeze Dry or equivalent processing.The adjuvant formulation includes a kind of specific buffer solution, and optionally and preferably one or more of It is kind synthetically produced selected from the group being made up of DMPC, DPPC, DSPC, DOPC and POPC, preferable DPPC phosphatidyl choline, with And a kind of synthetically produced acylphosphate disaccharides of adjuvant six or its acylphosphate disaccharides of derivative 3- deacylations base-six, rub Er Biyue 1:1 to 40:1 (phosphatidyl choline:Six acylphosphate disaccharides), preferably from about 1:1 to 20:1 (phosphatidyl choline:Six Acylphosphate disaccharides), more preferably from about 2:1 to 5:1 (phosphatidyl choline:Six acylphosphate disaccharides), and most preferably About 2:1 to 5:1(DPPC:Six acylphosphate disaccharides).

One of aspect of most worthy of the present invention is that the adjuvant formulation only includes being in prescribed concentration described herein Citrate, succinate or the acylphosphate disaccharides of single adjuvant six in phosphate buffer or its derivative 3- take off The acylphosphate disaccharides of acyl group-six, and preferable single phosphatidyl choline.Other compositions are not needed to produce in room temperature Under unexpected long-time stability.In addition, the adjuvant formulation can be produced with low cost.In this, these adjuvants The long-time stability of preparation at room temperature are significant, without the use of cholesterol, phosphatidyl glycerol, phosphatidyl-ethanolamine, single acyl Base glycerol, freeze drying protectant and it is metabolizable oil and realize.It is conventional in the case of without using these one or more of compositions Prior art adjuvant can not realize stabilization.

Another aspect of the present invention is the assistant for not needing two or more phosphatidyl cholines or adding phosphatidyl glycerol Agent formulation.As shown in embodiment, two or more phosphatidyl cholines or one or more of phosphatidyl glycerols can add It is added in these preparations, but realize the significant long-time stability showed herein preferably without it.

But without being limited by theory, the preferable buffer concentration of citrate, succinate or phosphate buffer About 10mM to about 50mM is expanded to realize the notable stability of invention described herein, it is considered to be due to being to retouch herein The phosphatidyl choline and six acylphosphate disaccharides or the present invention of its acylphosphate disaccharides of derivative 3- deacylations base-six stated The restriction mol ratio of preferred aspect adds preferable excipient, preferably phosphatidyl choline.It is furthermore preferred that preferable buffering Liquid concentration is about 25mM to about 50mM, even more preferred 28mM to about 50mM, most preferred 30mM to about 50mM.Preferably, pH Value is in the range of about 4.0 to about 7.5, preferably from about 4.5 to about 6.5, more preferably from about 5.0 to about 6.0.

As shown in embodiment, when preparation is in water, the acetate buffer equal to or more than 100mM, PBS or lemon When in hydrochlorate or phosphate buffer, in the absence of this superior room temperature stability.On the contrary, about 10mM is to about 50mM, but preferably About 25mM to about 50mM, citrate, the amber of preferred 28mM to about 50mM, most preferred 30mM to about 50mM concentration Hydrochlorate or phosphate generate stability.

Another embodiment of the invention includes nonionic surfactant, preferable polyoxyethylene sorbitan monoleate, to reduce this The particle aggregation of invention.

The advantages of being notable and unexpected breakthrough need not be freezed, because the step of eliminating many high costs and wind Danger.Another aspect of the present invention is, compared with prior art, these adjuvant formulations are in terms of enhancing is to the immune response of antigen It is superior, while causes significantly less serious injection site and systemic reaction during administration.The present invention another Aspect is to there is no that metabolizable oil includes squalene and there is no the adjuvant formulation of cholesterol.In the art, What is be widely understood that is necessary to cholesterol is adjuvant formulation or liposome realizes function.Present invention produces described herein Benefit without cholesterol.

Thus, include the advantages of adjuvant formulation described herein and pharmaceutical composition：Suspension as aqueous buffered is extremely The room temperature stability of few 6 months, and/or reach and about 37 DEG C at the stability of about 60 days or more long；Apply every time lower Serious injection site and systemic reaction, while strengthen the immune response to antigen；And using less material or composition with And the material or composition of lower concentration, lower production or manufacturing cost.The adjuvant formulation of the present invention provides these three The principal benefits of combination, this is the adjuvant as the synthetic analogues to the option of the adjuvant based on alum, MLA or MPL It is previous to fail what is realized.

In another aspect of the present invention, there is provided the new vaccine combination containing the new adjuvant formulation, be used for Treat or prevent the urinary tract infections as caused by gramnegative bacterium includes Escherichia coli and multi-drug resistance Escherichia coli.Also carry Supplied the method using the new vaccine combination, and prevention and treatment by gramnegative bacterium include Escherichia coli and The method of urinary tract infections caused by multi-drug resistance Escherichia coli.

In another aspect of the present invention, there is provided induce generation to be directed in the mankind with palindromic urinary tract infection The method of FimH antibody.

Another aspect of the present invention is vaccine combination, and it induces generation in the mankind with palindromic urinary tract infection For FimH antibody.

In another aspect of the present invention, there is provided include the sugar composite of six acylphosphateization two or preparation or vaccine group Compound, and apply the vaccine kit for instructing and storing explanation.For room temperature and reach and about 37 DEG C at the six acyl groups phosphorus The exposure for being acidified two sugar composites or preparation provides the explanation.These explanations for describing preservation, transport and Exposure Temperature can To include U.S. FDA and European drugs administration approved by government administration section.Preferably, one or more of Kit components It is the sugar composite of six acylphosphateization two or preparation being in syringe.

Another aspect of the present invention is, PHAD compositions and preparation and vaccine combination are sterile composition and nothing The pharmaceutical composition of bacterium, it is preferred that the sterile sugar composite of six acylphosphateization two and preparation are contained in aseptic injection In device.

Brief description of the drawings

Fig. 1 shows a kind of chemical constitution of salt of six acylphosphate disaccharides.

Fig. 1 a show the preferable acylphosphate disaccharides salt of 3- deacylations base-six.

Fig. 2 shows DPPC chemical constitution.

Fig. 3 is figure of the explanation using the anti-FimH of IgG to FimCH and Q133K indirect ELISAs.

Fig. 4 is the figure for illustrating to analyze FimCH and Q133K Potency Analysis.

Fig. 5 is the figure for illustrating to assess little molecules in inhibiting thing in titration.Two kinds of small molecules, 4-methyl umbelliferone acyl Base-α-D mannopyranes glucosides (UFMP) and methyl-α-D mannopyranes glucosides (MDMP) suppress mannose and FimH combination.

Fig. 6 is the representational chromatogram by CEX-HPLC FimCH drug substance samples.

Fig. 7 is the figure for illustrating to be protected from coli-infection after the FimCH/PHAD of mouse is immune.

Fig. 8 is DPPC and PHAD HPLC example chromatograms.

Embodiment

Definition

On " about " referring to institute's number of columns when six acylphosphate disaccharides quantity or buffer concentration (unless defining) Add and subtract 10%.

" about 25 DEG C " refer to 20 DEG C to 30 DEG C of temperature.

" about 37 DEG C " refer to 34 DEG C to 40 DEG C of temperature.

" about 50mM " refers to buffer concentration described herein in 50mM and less than between 100mM.As shown in embodiment , 100mM or higher specified buffer solution is invalid in terms of long-term room-temperature stability is allowed.The upper limit one of buffer concentration As be assessed as twice of increase.During with reference to about 50mM, the buffer concentration specified of the invention is preferably less than 90mM, preferably Less than 80mM, even more preferably less than 70mM, more preferably less than 60mM.

" about 10mM " refers to buffer concentration described herein between 6mM to 10mM.

" acceptable carrier " refers to a kind of carrier, and it is harmless for the other compositions of composition, and for wanting The material applied therewith is harmless.

" adjuvant " refers to improve a kind of antigen reactive reagent in the presence of effective dose；Strengthen the immune response to antigen Material；Or stimulate the reagent of the antibody producing for antigen.A variety of naming rules or term in this area be present.Without reference to specific UNC, adjunvant composition described herein can be simply referred as adjuvant formulation or adjuvant formulation.

" administration " is directed to subject and provides compound or any mode of composition.

" colloid " refers to the one or moreization microscopically disperseed in the solution or other materials of whole aqueous buffered Learn material, compound or material.Adjuvant formulation described herein can also be described as colloid.One example of colloidal dispersion is Fungizone, it is made up of amphotericin B-deoxysodium cholate for parenteral administration.

" critical micellar group concentration " refers to the concentration of surfactant, forms micelle group higher than the concentration, is added to system All extra surfactants enter micelle group.

" DLPC " refers to 1,2- dilauroyl-sn- glyceryl -3- phosphocholines.

" DMPC " refers to the myristoyl-sn- glyceryl -3- phosphocholines of 1,2- bis-.

" DOPC " refers to DOPC.

" DPPC " refers to palmityl-sn- glyceryl -3- phosphocholines (the molecular formula C of 1,2- bis-₄₀H₈₀NO₈P (MW= 734Da) (chemical constitution is shown in Figure 2).

" DPPG " refers to palmityl-sn- glyceryl -3- phosphoric acid-(the 1'-rac- glycerine) of 1,2- bis-.

" DSPC " refers to 1,2- distearyl acyl group-sn- glyceryl -3- phosphocholines.

" effective dose " refer to be administered in the vaccine combination of the mankind sufficient amount of FimCH or the FimH of truncation or other Antigen, to trigger the immune response for FimH or other antigens, or sufficient amount of adjuvant, preferable six acylphosphateization two Sugar or its acylphosphate disaccharides of derivative 3- deacylations base-six, to trigger the immune response of the raising for antigen.

" there is no " material, additive, chemical substance or excipient refer to the material, additive, chemical substance Or excipient is not added in the composition or preparation of the present invention, but there may be and certain mix horizontal quantity.

" there is no serious injection site and systemic reaction " refers to that 2 percent or less people undergoes these It is attributable to adjunvant composition or the serious injection site of preparation and systemic reaction.

" injection site reaction " refers in the pain of the position of administration or injection site, tenderness, rubescent and/or swelling.

" invention " refers at least some of embodiment of the present invention；Referenced herein " invention " or " present invention " it is various Feature does not mean that the embodiment of all prescriptions or method include mentioned feature.

" mark " or " label " refers on any article or its any container or packing material, or with such article All labels and the material of other hand-written, printings or figure, thus, include the vaccine or adjunvant composition with the present invention Or any package insert or information page of preparation.

" less serious injection site and systemic reaction " refer to herein and in its product information file The commercial vaccine Cervarix of detailed description, and grinding vaccine assistant described in this paper and Treanor et al. (Vaccine 2013) Agent GLA-SE is compared, less serious or 3 grades of injection site reaction and/or systemic reaction.

" liposome " generally refers to vesicle, and it around the bimolecular lamellar lipid membrane of hydrophily core by forming.

" low cost " refers to a composition, its have composition under the least concentration for the new features for being enough to realize the present invention or Material.

" freeze drying protectant " refers to material, chemical substance or excipient, mainly for the protection of material from manufacturing, preserve and Frostbite during use or other damages, or improve reconstruct, including allow the appropriate dissolving before use, in addition to oozed for modification These materials, chemical substance or the excipient of degree of rising are oozed in pressure or regulation thoroughly, including but not limited to sorbierite, mannitol, mannose, Erythrite, xylitol, glycerine, sucrose, glucose, trehalose, maltose, lactose and cellobiose.

" metabolizable oil " is primarily referred to as the squalene for being used as adjuvant in bacterin preparation or adjuvant formulation, or is closely related Squalene analog, but also refer in vaccine or adjuvant formulation used as excipient or for produce adjuvant effect or Produce the triglycerides of the middle chain of emulsion, including Miglyol 810 and the oil from plant, animal or fish.Embodiment includes Grape-kernel oil, soybean oil, coconut oil, olive oil, sunflower oil, corn oil and dogfish oil.

" micelle group " refers to be dispersed in the aggregation of the surfactant molecule in the solution of aqueous buffered, hydrophilic head Portion region contacts with the solution of the aqueous buffered of surrounding, by micelle cluster centre it is hydrophobic individually tail region every From.

" MLA " refers to monophosphoryl lipid A.

" MPL " refers to 3'-O- deacylation base -4'- monophosphoryl lipid As.

" pharmaceutically acceptable carrier " refers to a kind of carrier, and it is harmless for the other compositions of composition, and It is harmless for its mankind or other animal subjects.Under the situation of the other compositions of composition, " harmless " refers to The carrier will not be reacted with other compositions or other compositions of degrading, or disturbs their effect.However, the effect of interference component Power does not refer to pure component diluent.

" six acylphosphate disaccharides " is a kind of activator of Toll-like receptor 4, refers to six acylphosphate disaccharides, its acid Or six acylphosphate disaccharides other pharmaceutically acceptable salts.The structure of preferable six acylphosphates disaccharides salt is in accompanying drawing Shown in 1, it can be obtained (PHAD) from Avanti Polar Lipids.As used herein, six acylphosphate disaccharides can be with Be be wholly or partially synthetic or non-synthetic, and completely synthetic is preferable.

" the acylphosphate disaccharides of 3- deacylations base-six " is a kind of activator of Toll-like receptor 4, refers to the acyl of 3- deacylations base-six Other pharmaceutically acceptable salts of base phosphorylation disaccharides, its acid or the acylphosphate disaccharides of 3- deacylations base-six.Preferable 3- The structure of the acylphosphate disaccharides salt of deacylation base-six shows that it can be obtained from Avanti Polar Lipids in accompanying drawing 1A (PHAD).As used herein, the acylphosphate disaccharides of 3- deacylations base-six can be wholly or partially synthetic or non-synthetic , and completely synthetic is preferable.

" pharmaceutical composition " refers to a kind of composition, its be given with mammal with scheme treat or prevent disease, or for For vaccine combination, the immunogenic response for treating or preventing disease is produced, reduces symptom, or provide certain form for the treatment of Benefit, or for adjunvant composition, strengthen the immune response to one or more of antigens.

" phosphate buffer " or " phosphate " refers to the phosphate buffer selected from following group：Disodium hydrogen phosphate, phosphorus Acid dihydride sodium, potassium dihydrogen phosphate and dipotassium hydrogen phosphate, or its certain combination.Preferably, " phosphate " is by disodium hydrogen phosphate, phosphorus Acid dihydride sodium and potassium dihydrogen phosphate composition.Unless otherwise indicated, refer to that phosphate buffer especially excludes ammonium phosphate.

" PBS " refers to phosphate buffer (Na₂HPO₄And/or KH₂PO₄), the phosphorus of the general composition of potassium chloride and sodium chloride Hydrochlorate buffered saline.Typical PBS compositions include about 10mM phosphate buffers (Na₂HPO₄And/or KH₂PO₄), 2.7mM chlorine Change potassium and 0.14M sodium chloride, pH7.4,25 DEG C.

" Phosphate Citrate Buffer " refers to the phosphate buffer containing citric acid and sodium phosphate, and wherein pH value passes through The balance of citrate/citric acid and phosphate/phosphor acid hydrogen salt maintains.Phosphate can include, such as Na₂HPO₄And/or KH₂PO₄, sodium citrate can be used.

" phosphatidyl choline " (also referred to as " PC ") refers to the lipid containing choline.Example includes but is not limited to DMPC (1,2- Two myristoyl-sn- glyceryl -3- phosphocholines), DPPC (1,2- bis- palmityl-sn- glyceryl -3- phosphocholines), DSPC (1,2- distearyl acyl group-sn- glyceryl -3- phosphocholines), DOPC (DAG base -3- phosphoric acid Choline) and POPC (POPA choline).Phosphatidyl choline can be from Avanti Polar Lipids are obtained.

" phosphatidyl-ethanolamine " refers to the lipid containing the phosphate group for being attached to monoethanolamine, for example, 1,2- dioleoyl- Sn- glyceryl -3- phosphoethanolamines.

" phosphatidyl glycerol " refers to the lipid containing glycerine.Embodiment includes but is not limited to the myristoyl-sn- of 1,2- bis- Glyceryl -3- phosphoric acid-(1'-rac- glycerine) (DMPG), 1,2- bis- Palmitoyl-sn-Glycero -3- phosphoric acid (1'-rac- glycerine And 1,2- distearyl acyl group-sn- glyceryl -3- phosphoric acid-(1 '-rac- glycerine) (DSPG) (DPPG).

" POPC " refers to POPA choline.

Palindromic urinary tract infection " refers to that people suffered from 3 to 4 urinary tract infections in about one year.

" refrigeration " refers to 2 DEG C to 8 DEG C of temperature range.

" room temperature " refers to 19 DEG C (66 °F) to the scope of 25 DEG C (77 °F).

" salt solution " refers to the about 125mM to about 155mM NaCl in the aqueous solution of buffering.For example, PBS is typically contained 137mM NaCl, Tris buffered salines can contain 150mM.

" serious injection site reaction " refers to below one or more：Need narcotic-based painkillers or hinder The pain of daily routines；Cause the tenderness of notable discomfort when static；It is rubescent more than 10cm, and more than 10cm or hinder daily work Dynamic swelling.

" serious systemic reaction " refers to following one or more：Nausea/vomiting, it hinders daily routines or needed Want external IV aquations；In 24 hours by 6 times or more it is time watery just and/or>800 grams of diarrhoea, or need external IV aquations；Weight Degree uses narcotic analgesic medicine or the headache of obstruction daily routines；Fatigue that is significant or hindering daily routines；And it is significant or Hinder the courbature of daily routines.

The cholesterol " there is no " referred to refers to, if it exists, cholesterol is 0.3mM or less.

The monoacylglycerol " there is no " referred to refers to, if it exists, monoacylglycerol is 0.5mM or more It is few.The example of monoacylglycerol is MPG.

If the phosphatidyl glycerol or phosphatidyl-ethanolamine " there is no " referred to refers to that fruit is present, these materials are 0.1mM or less.

The freeze drying protectant " there is no " referred to refers to if it exists, these materials are 0.5% or less Composition or formulation concentrations.

" there is no salt solution " refers to be less than 30mM NaCl in the composition or preparation of the present invention.

" systemic reaction " refers to nausea/vomiting, diarrhoea, headache, fatigue and/or courbature.

" Succinate Buffer " or " " succinate " refers to disodium succinate or the alkali of sodium succinate two.Fourth can be used Diacid potassium, but be less preferable.

When the adjuvant, activity, protein, antigen or the medicine that refer to " stability " or " stable " refer to material or product from Protected in a period of time that its build date starts, under the influence of some variables such as temperature and/or humidity for its intended purpose Hold acceptable quality.The stability of material proves often through analyze data (or other suitable evidences).

" trisodium citrate " refers to citrate buffer (also referred to as " citrate "), for example, the water of trisodium citrate two Compound, sodium citrate, the buck compound of sodium citrate three or trisodium citrate monocalcium salt compound, as supplier includes Sigma- Alleged by Aldrich and BDH Chemicals.Potassium citrate, sodium citrate list alkali and the alkali of sodium citrate two can be used, still They are relatively low preferable.For example, the product Imiglucerase (imiglucerase) for injection used trisodium citrate and The combination of DisodiumHydrogen Citrate.The combination of these types is acceptable.Citric acid, it is slow that CAS 77-92-9 can be used for regulation The pH value of fliud flushing, but it can not replace buffer solution listed here.

" FimH " of truncation refers at least about 25 of preceding 175 amino acid being truncated including from FimH to about The FimH albumen of the truncation of 175 amino acid residues.For the FimH of truncation, the FimH albumen of truncation preferably includes FimH eggs White at least 9%, at least the 30% of preferred FimH albumen, at least the 60% of most preferred FimH albumen.

" urinary tract infections " refers to the medical diagnosis characterized by one or more of following S＆Ss：Irritating row Urine, such as frequent micturition, urgent urination and dysuria；Gross hematuria disease；Or touched a tender spot on the pubis triggered when checking；And/or it is a kind of or More kinds of following experimental results：Come self-cleaning collection or the positive Urine test paper inspection of conduit urine specimen；Carry out self-cleaning collection Or the microscope urinalysis (there may be leucocyte, bacterium and tube) of conduit urine specimen；Or net collection or conduit urine Escherichia coli >=10 in the urine culture of sample³CFU/ml。

" vaccine " or " vaccine combination " refers to improve the immunity to the immunity of disease.The vaccine combination is to draw Hair diagnoses the immune response of antigen and the immunogenic composition of antibody producing of the composition.

Invention embodiment

In one embodiment, there is provided pharmaceutical composition or pharmaceutically acceptable carrier.Described pharmaceutical composition Or pharmaceutically acceptable carrier includes six acylphosphate disaccharides or its acylphosphateization two of derivative 3- deacylations base-six Sugar, and buffer solution (be referred to as " sugar composite of six acylphosphateization two " or " composition containing six acylphosphate disaccharides " or " composition and carrier containing six acylphosphate disaccharides ", these are also individually applicable its derivative (3- deacylations base-six Acylphosphate disaccharides).The sugar composite of six acylphosphateization two of the present invention is preferably the suspension of aqueous buffered.It is described slow Fliud flushing is selected from by about 25mM to about 50mM, preferred 28mM to about 50mM, most preferred 30mM to about 50mM citrate, The group that succinate and phosphate are formed.Preferably, pH value is about 4.0 to about 7.5, preferably from about 4.5 to about 6.5, more preferably About 5.0 to about 6.0 in the range of.With this combination (six acylphosphate disaccharides or its acyl of derivative 3- deacylations base-six Base phosphorylation disaccharides and the buffer solution) pharmaceutical composition or carrier present as the basic fabulous of pharmaceutical composition Stability, and improve the general stability of the sugar composite of six acylphosphateization two.Specifically, these contain six acylphosphates The composition and carrier of disaccharides at room temperature and reach and realize stability at about 37 DEG C.These of the present invention contain six acyl group phosphorus The composition and carrier for being acidified disaccharides are also presented in refrigerated storage temperature to fabulous long-time stability at room temperature.

(as previously described it includes six acyl group phosphorus for pharmaceutical composition and pharmaceutical carrier containing six acylphosphate disaccharides It is acidified disaccharides or its acylphosphate disaccharides of derivative 3- deacylations base-six and specific buffer solution) can optionally it include pair In vaccine, adjuvant formulation and the typical other compositions of other drugs composition, for example, excipient, dressing agent, surfactant and Additive.For example, phosphatidyl choline (as described in more detail below) can be optionally added, individually or with other lipids carry Body combines.In one embodiment, the phosphatidyl choline of the natural generation from soybean or egg can be added, or from soybean Or the phosphatidyl choline of the hydrogenation of egg, synthesis or natural mixing phosphatidyl choline.

In a preferred embodiment, one or more of vaccine antigens are added to containing six acylphosphate disaccharides Vaccine is formed in composition.Vaccine antigen can be any antigen used in vaccine, including but not limited to diphtheria, broken wound Wind, pertussis, polio, hepatitis and/or the antigen product of influenza virus.Preferably, vaccine antigen is that following article is detailed The FimCH protein complexes of description.

Composition and carrier containing six acylphosphate disaccharides can be prepared with any concentration, usually with about 0.005 To about 1.0mg/ml six acylphosphate disaccharides, preferably from about 0.05 to 1.0mg/ml six acylphosphate disaccharides are made It is standby, usually no more than about 2.5mg/ml six acylphosphate disaccharides.

Composition containing six acylphosphate disaccharides can be administered to animals or humans as the adjuvant of vaccine, preferably Give every dose of six acylphosphate disaccharides of about 10 microgram and arrive every dose of the acylphosphate disaccharides of about 50 microgram six in ground.Definite dosage can To be changed according to the antigen used.It is furthermore preferred that using six acylphosphate disaccharides of about 20 microgram every dose arrive about 50 micrograms six Every dose of acylphosphate disaccharides.Even more preferred, using six acylphosphate disaccharides of about 40 microgram, every dose is arrived the acyl of about 50 microgram six Every dose of base phosphorylation disaccharides.

As shown herein, when preparing in water, acetate buffer, PBS or citrate equal to or more than 100mM or When in phosphate, the composition containing six acylphosphate disaccharides at room temperature and reaches unexpected stability at 37 DEG C and is It is not present.The sugar composite of six acylphosphateization two contains citrate, succinate or phosphate buffer and exceeded to produce The stability of expectation, preferably from about 25mM are to about 50mM, and preferred 28mM is to about 50mM, and most preferably 30mM is to about 50mM。

More specifically, the preferable buffer solution of the sugar composite of six acylphosphateization two, the concentration of buffer solution is selected from following structure Into group：About 25mM to about 50mM；25mM to about 50mM；About 30mM to about 50mM；28mM to about 50mM；30mM to about 50mM； About 30mM；About 40mM to about 50mM；40mM to about 50mM；About 40mM；And about 50mM.

In addition, shown by the embodiment of following article, in composition and preparation containing six acylphosphate disaccharides The stability features of the new feature of the present invention, the particularly present invention are not showed using PBS.By contrast, use is special herein Citrate, succinate and the phosphate buffer specified allow the new stability features of the present invention.

The sugar composite of six acylphosphateization two of the present invention does not have squalene preferably substantially.

The sugar composite of six acylphosphateization two of the present invention is preferably substantially not used as metabolizable oil of adjuvant.

The sugar composite of six acylphosphateization two of the present invention is preferably substantially without metabolizable oil.

The sugar composite of six acylphosphateization two of the present invention is preferably the suspension of aqueous buffered, and preferably these are water-based The suspension of buffering, which has, is less than 150nm, more preferably less than 130nm granular size, it is preferred that these six acylphosphates Two sugar composites are not oil in water emulsion.

The sugar composite of six acylphosphateization two of the present invention is preferably substantially without the second adjuvant, including alum, spiny dogfish Alkene, QS21, MF59, the activator of Toll-like receptor 9, and other adjuvants, including the adjuvant based on squalene.Second adjuvant has There is the possibility that more serious locally and systemically property reactions are produced in the mankind, change without further improving immune response The benefit of kind treatment results.

The sugar composite of six acylphosphateization two preferably contains less than 5mM cholesterol, and more preferably lower than 1mM courage is consolidated Alcohol, it is even more preferred generally without cholesterol, it most preferably there is no cholesterol.

Preferably, the sugar composite of six acylphosphateization two there is no phosphatidyl glycerol, more preferably there is no Phosphatidyl glycerol.

Preferably, the sugar composite of six acylphosphateization two does not have substantially more preferably generally without phosphatidyl-ethanolamine There is phosphatidyl-ethanolamine.

Preferably, the sugar composite of six acylphosphateization two more preferably there is no generally without monoacylglycerol Monoacylglycerol.

The sugar composite of six acylphosphateization two is preferable to contain the NaCl for being less than 20mM preferably generally without salt solution, The preferred NaCl contained less than 10mM, it is even more preferred to there is no NaCl.

The sugar composite of six acylphosphateization two is even more preferred to there is no preferably generally without freeze drying protectant Freeze drying protectant.

The sugar composite of six acylphosphateization two need not freeze or equivalent processing comes for storage life or stably Property keep the concentration of six acylphosphate disaccharides or its acylphosphate disaccharides of derivative 3- deacylations base-six.Therefore, described six The sugar composite of acylphosphateization two is not preferably lyophilized or not freezed；The sugar composite of six acylphosphateization two is preferred Do not dried after the preparation on ground；The sugar composite of six acylphosphateization two preferably without after the preparation use liquid or Buffer solution reconstructs from dry material；The sugar composite of six acylphosphateization two is protected before preferably applying after the fabrication Hold the suspension for aqueous buffered.

Preferably, the sugar composite of six acylphosphateization two is generally without cholesterol, phosphatidyl glycerol and phosphatidyl ethanol Amine, and there is no metabolizable oil and the second adjuvant.

It is furthermore preferred that the sugar composite of six acylphosphateization two is generally without cholesterol, phosphatidyl glycerol and phosphatidyl second Hydramine, and there is no metabolizable oil and the second adjuvant.

Preferably, the sugar composite of six acylphosphateization two of the invention realizes the present invention's generally without described herein The unwanted material of new feature institute or excipient, and the even more preferred sugar composite of six acylphosphateization two of the invention is substantially There is no new feature the institute unwanted material or excipient described herein for realizing the present invention

Preferably, the sugar composite of six acylphosphateization two of the invention is generally without every kind of composition described herein A kind of, two kinds, three or more materials or excipient, and it is this limit only a kind of material for being not excluded for every kind of composition or Excipient is per composition.

Preferably, the sugar composite of six acylphosphateization two of the invention there is no every kind of compositions described herein one Kind, two kinds, three or more materials or excipient, it is this to limit the only a kind of material or excipient for being not excluded for every kind of composition Per composition.

Preferably, when the composition containing six acylphosphate disaccharides or preparation of the present invention at room temperature or reach about 37 When being saved, transport, keep or applying at DEG C, most preferably it is sterile filtered or is prepared using asptic technique, it is more excellent Choosing, the sterile sugar composite of six acylphosphateization two and preparation are comprised in asepsis injector.

In another embodiment, there is provided new adjuvant formulation.The adjuvant formulation includes a kind of synthetically produced Six acylphosphate disaccharides or its acylphosphate disaccharides of derivative 3- deacylations base-six, selected from by about 10mM to about 50mM's The buffer solution for the group that citrate, succinate and phosphate are formed, and a kind of preferable synthetically produced phosphatidyl choline (it is referred to as " six acylphosphate disaccharides preparations " or " adjuvant formulation containing six acylphosphate disaccharides " or " contains six acyl group phosphorus It is acidified the preparation of disaccharides ", these also refer to its acylphosphate disaccharides of derivative 3- deacylations base-six respectively).Phosphatidyl choline with Six acylphosphate disaccharides or its acylphosphate disaccharides of derivative 3- deacylations base-six are with about 1 (PC) 1 (six acylphosphates of ratio Change disaccharides) about 40 (PC) 1 (six acylphosphate disaccharides) of ratio are arrived, preferably from about 2.5 (PC) are than 1 (six acylphosphate disaccharides) Mol ratio is present.Unless otherwise indicated, the citation to six acylphosphate disaccharides preparations is applied to adjuvant formulation.These new six Acylphosphate disaccharides preparation is preferably the suspension of aqueous buffered.When being stored in refrigerated storage temperature to room temperature and reach about When at 37 DEG C, the adjuvant formulation has fabulous long-time stability.In addition, the adjuvant formulation can be produced with low cost.

In a preferred embodiment, the adjuvant formulation preferably includes a kind of synthetically produced phosphatidyl choline, excellent The DPPC of choosing, a kind of synthetically produced acylphosphate disaccharides of adjuvant six or its acylphosphate of derivative 3- deacylations base-six Disaccharides, its mol ratio about 1：1 to 40：1(DPPC：Six acylphosphate disaccharides), and about 10mM to 50mM but preferably from about The citrate, succinate or phosphorus of 25mM to 50mM, preferred 28mM to about 50mM and most preferred 30mM to about 50mM Phthalate buffer.Preferably, pH value is about 4.0 to about 7.5, preferably from about 4.5 to about 6.5, more preferably from about 5.0 to about 6.0 In the range of.Importantly, the six acylphosphates disaccharides adjuvant formulation can use only a kind of acylphosphate disaccharides of adjuvant six Or its derivative 3- deacylations base-six acylphosphate disaccharides and preferably a kind of phosphatidyl choline produce, so as to allow it With low cost produce.Preferably, the adjuvant formulation contains six acylphosphate disaccharides or its derivative 3- deacylations base-six Acylphosphate disaccharides can use other adjuvants as unique adjuvant.The long-time stability feature of the present invention enters one Step reduces the cost using these adjuvant formulations for containing six acylphosphate disaccharides.

But without being limited by theory, citrate, succinate or phosphorus in the six acylphosphates disaccharides preparation The preferable buffer concentration of phthalate buffer expands to about 10mM to about 50mM to realize the notable of invention described herein Stability, it is considered to be due to being the preferred aspect of the present invention with phosphatidyl choline described herein and six acylphosphate disaccharides Restriction mol ratio add preferable excipient, preferably phosphatidyl choline.

The adjuvant formulation containing six acylphosphate disaccharides of the present invention does not have squalene preferably substantially.

What the adjuvant formulation containing six acylphosphate disaccharides of the present invention was preferably substantially not used as adjuvant can generation The oil thanked.

The adjuvant formulation containing six acylphosphate disaccharides of the present invention is preferably substantially without metabolizable oil.

The adjuvant formulation containing six acylphosphate disaccharides of the present invention is preferably the suspension of aqueous buffered, preferably The suspension of these aqueous buffereds, which has, is less than 150nm, the even more preferred granular size less than 130nm, it is preferred that these contain The adjuvant formulation for having six acylphosphate disaccharides is not oil in water emulsion.

The adjuvant formulation containing six acylphosphate disaccharides of the present invention preferably substantially no second adjuvant, including white Alum, squalene, QS21, MF59, the activator of Toll-like receptor 9, and other adjuvants, including the adjuvant based on squalene.It is described Six acylphosphate disaccharides preparations produce more preferably substantially without other or extra adjuvant because they have in the mankind The possibility of more serious locally and systemically property reactions, without further improving immune response or generally improving treatment results Benefit.

The adjuvant formulation containing six acylphosphate disaccharides preferably contains less than 5mM cholesterol, more preferably Cholesterol less than 1mM, it is even more preferred generally without cholesterol, it most preferably there is no cholesterol.

Preferably, the adjuvant formulation containing six acylphosphate disaccharides is generally without phosphatidyl glycerol, more preferably There is no phosphatidyl glycerol.

Preferably, the adjuvant formulation containing six acylphosphate disaccharides is more excellent generally without phosphatidyl-ethanolamine Choosing there is no phosphatidyl-ethanolamine.

Preferably, the adjuvant formulation containing six acylphosphate disaccharides is generally without monoacylglycerol, more preferably There is no monoacylglycerol.

The adjuvant formulation containing six acylphosphate disaccharides of the present invention preferably contains preferably generally without salt solution NaCl less than 20mM, it is furthermore preferred that the six acylphosphates disaccharides preparation contains the NaCl less than 10mM, it is even more preferred It there is no NaCl.

Preferably, the adjuvant formulation containing six acylphosphate disaccharides is generally without freeze drying protectant, more preferably There is no freeze drying protectant.

Preferably, the adjuvant formulation containing six acylphosphate disaccharides is generally without cholesterol, phosphatidyl glycerol And phosphatidyl-ethanolamine, and there is no metabolizable oil and the second adjuvant.

It is furthermore preferred that the preparation containing six acylphosphate disaccharides there is no cholesterol, phosphatidyl glycerol and phosphatide Acyl monoethanolamine, and there is no metabolizable oil and the second adjuvant.

Preferably, the preparation of the invention containing six acylphosphate disaccharides realizes this hair generally without described herein The bright unwanted material of new feature institute or excipient, and the even more preferred system of the invention containing six acylphosphate disaccharides Agent there is no new feature the institute unwanted material or excipient described herein for realizing the present invention

Preferably, the preparation of the invention containing six acylphosphate disaccharides generally without it is a kind of, two kinds, three kinds or more Multiple material or every part of the excipient preparation described herein containing six acylphosphate disaccharides, this limitation are not excluded for only a kind of Every part of preparation containing six acylphosphate disaccharides of material or excipient.

Preferably, the preparation of the invention containing six acylphosphate disaccharides there is no it is a kind of, two kinds, three kinds or more Multiple material or every part of the excipient preparation described herein containing six acylphosphate disaccharides, this limitation are not excluded for only a kind of Every part of preparation containing six acylphosphate disaccharides of material or excipient.In another aspect, there is provided vaccine and use vaccine The method for treating and preventing disease.

In order to prepare vaccine, one or more of vaccine antigens are added to contains six acylphosphateizations two as described above In the adjuvant formulation of sugar.The antigen can be FimCH protein complexes described herein, or other antigens, including but not It is limited to the antigen related to diphtheria, lockjaw, pertussis, polio, hepatitis or the antigen product of influenza virus.

The vaccine prepared using six acylphosphate disaccharides preparations need not be freezed come in order to which storage life or stability are protected Hold the concentration of six acylphosphate disaccharides.Lyophilized is a kind of dehydration or freeze-dried process for being mainly used in preserving material. The same target of preservation material is realized now with equivalent process.Preparing vaccine described herein and six acylphosphateizations two During sugar composite, it is not necessary to which the advantages of lyophilized vaccine or adjuvant formulation is unexpected and notable is lyophilized by eliminating Necessity, many expensive steps can be removed.First, step of freeze drying is removed in itself, and it is not only eliminated must be good The expensive manufacturing step carried out under the aseptic condition of monitoring, also eliminate examination ＆ approval and assessment to this manufacturing step.Connect down Come, the removal of step represents a kind of continual expense and saved.For example, when often preparing a batch, step of freeze drying is saved .In addition, when preparing bigger batch, or when production process is transferred to another facility, remove the step and save Extra examination ＆ approval step.Second, it is lyophilized to need to manufacture or purchase, transport and the sterile small of diluent is preserved together with freeze-dried products Bottle, for reconstructing.Remove the cost that reconstruction step eliminates this diluent bottle and its supply chain management.3rd, adjuvant The reconstruct of preparation may influence its aseptic, it is necessary to use at once, if not abandoning production using needs within the predetermined time Product.4th, restructuring procedure is easy to malfunction, thus producer loses the control of the exact concentration of the product to being finally administered to patient System.Adjunvant composition and preparation described herein eliminate this four shortcomings by removing lyophilized demand.Therefore, it is described to contain six The preparation of acylphosphate disaccharides is not preferably lyophilized or not freezed；The preparation containing six acylphosphate disaccharides Do not dried after the preparation preferably；The preparation containing six acylphosphate disaccharides is preferably without after the preparation Reconstructed with liquid or buffer solution from dry material；The preparation containing six acylphosphate disaccharides is preferably in manufacture The suspension of aqueous buffered is remained before applying afterwards.

The further remarkable advantage of invention described herein is realized now.It need not freeze or equivalent process, The sugar composite of six acylphosphateization two of the present invention or the preparation containing six acylphosphate disaccharides can after the fabrication immediately Efficiently and at low cost it is packaged into syringe.Preferably, the composition and preparation are sterile preferably described notes Emitter is sterile.These prefilled syringes can refrigerated storage temperature to be transported at room temperature and in the case where reaching about 37 DEG C, Preserve, deliver or shift.This advantage provides most efficient and low cost mode to make the sugar composite of six acylphosphateization two The place applied is reached with the preparation containing six acylphosphate disaccharides.

In another embodiment, nonionic surfactant is added in the adjuvant formulation.Preferably, it is described Nonionic surfactant is polyoxyethylene sorbitan monoleate, but can also use others.The nonionic surfactant typically with About 0.001% to 1.0%, preferable 0.01% to 0.1% concentration is added in the adjunvant composition.This addition can be with Prevent the little aggregates of six acylphosphate disaccharides preparations described in when being stored in room temperature and reaching at about 37 DEG C and average The slight raising of grain size.Preferably, nonionic surfactant derives from the sorbitan of polyethoxylated, including but not It is limited to polysorbate 20 and polyoxyethylene sorbitan monoleate.

Preferable adjuvant formulation, which includes, to be selected from by about 25mM to about 50mM, preferred 28mM to about 50mM, most preferably The specific buffer solution for the group that 30mM to about 50mM citrate, succinate and phosphate is formed, and preferably according to about 1：1 to 40：1 (phosphatidyl choline：Six acylphosphate disaccharides), preferably from about 1：1 to 20：1 (phosphatidyl choline：Six acyl group phosphorus It is acidified disaccharides), more preferably from about 2：1 to 5：1 (phosphatidyl choline：Six acylphosphate disaccharides) and most preferably from about 2：1 to 5：1 (DPPC:Six acylphosphate disaccharides) be selected from by DMPC, DPPC, DSPC, DOPC and POPC group formed one kind synthesize life The phosphatidyl choline of production, preferable DPPC, and a kind of synthetically produced the acylphosphate disaccharides of adjuvant six or its derivative The acylphosphate disaccharides of 3- deacylations base-six.It is furthermore preferred that citrate or Succinate Buffer according to about 10mM to about 50mM, preferably from about 25mM are used for six acyl groups to about 50mM, preferred 28mM to about 50mM, most preferred 30mM to about 50mM In phosphorylation disaccharides preparation.

As described by this paper for the concentration of the preferable buffer solution of six acylphosphate disaccharides preparations, buffer solution it is dense Group of the degree selected from following composition：About 10mM to about 50mM；15mM to about 50mM；20mM to about 50mM；About 25mM to about 50mM； 25mM to about 50mM；About 30mM to about 50mM；28mM to about 50mM；30mM to about 50mM；About 30mM；About 40mM to about 50mM； 40mM to about 50mM；About 40mM；And about 50mM.Preparation described herein containing six acylphosphate disaccharides is preferably substantially Upper no salt solution, preferably contain less than 20mM NaCl, it is furthermore preferred that the sugar composite of six acylphosphateization two contain it is low It is even more preferred to there is no NaCl in 10mM NaCl.

In addition, shown by the embodiment of following article, in composition and preparation containing six acylphosphate disaccharides The stability features of the new feature of the present invention, the particularly present invention are not showed using PBS.By contrast, specify herein Phosphate buffer allow the present invention new stability features.

The six acylphosphates disaccharides preparation is preferably by selecting with the synthetically prepared single phosphatidyl courage of high-purity It is prepared by alkali.However, the phosphatidyl choline of the natural generation from soybean or egg, or the phosphatidyl of the hydrogenation from soybean or egg Choline, or synthesis or the phosphatidyl choline of natural mixing can be used for preparing the adjuvant formulation.

The six acylphosphates disaccharides preparation can be prepared with any concentration, but usually with about 0.005 to about It is prepared by 1.0mg/ml six acylphosphate disaccharides, preferably from about 0.05 to about 1.0mg/ml six acylphosphate disaccharides, But preferably no more than about 2.5mg/ml six acylphosphate disaccharides.The phosphatidyl courage of the six acylphosphates disaccharides preparation Alkali can be prepared with any concentration, it is preferred that arriving about 16mg/ml (0.007mM to 22mM) according to about 0.005, more preferably About 0.05 arrives about 8mg/ml (0.07mM to 11mM), and even more preferred about 0.05 makes to about 0.8mg/ml (0.07mM to 1mM) It is standby.

The six acylphosphate disaccharides preparations of the present invention are prepared to produce mol ratio about 40 as follows:1 to about 1:1 phosphatidyl Choline is than six acylphosphate disaccharides, and preferable DPPC is than six acylphosphate disaccharides, and preferably from about 2.5:1DPPC is than six acyl groups Phosphorylation disaccharides.Six acylphosphate disaccharides are weighed up in appropriate vial, for example, Type1 Plus Schott glass Glass bottle.An appropriate number of phosphatidyl choline that addition is in ethanol, preferable DPPC.By this product ultrasound about 1 minute, Preparation is lightly rotated simultaneously, ethanol is suitably then removed by evaporation.Film citrate, succinate or phosphate Buffer solution, preferable 10mM to about 50mM sodium citrate pH 6.0 are reconstructed, and at about 50 DEG C to 65 DEG C, are surpassed at preferable 55 DEG C Sound about 30 minutes, it can preferably be performed for more than the supersound process of a cycle.The six acylphosphate disaccharides preparations prepared The general granular size with about 60nm to about 500nm.It is sterile filtered to realize, is preferably processed further six acylphosphates Homogeneous granular size that change disaccharides preparation to realize reduction and appropriate, preferably between about 70nm to 130nm.Six acyl group phosphorus It is acidified polycarbonate membrane (Avestin, LFLM-80) Avestin extruder or equivalent of the disaccharides preparation by 80nm apertures Extrusion, passed through for about 7 to about 12 times at about 45 DEG C to 65 DEG C, at preferable 55 DEG C, to realize homogeneous below about 130nm Grain size.In order to ensure the acceptable rate of recovery after aseptic filtration, it is preferred that of the six acylphosphates disaccharides preparation Grain size is 150nm or lower, but even more preferred 130nm or lower.

Then the six acylphosphates disaccharides preparation can optionally use concentration described herein, and preferably from about 25mM is arrived About 50mM, preferred 28mM are to about 50mM, most preferred 30mM to about 50mM, pH6.0 citrate, succinate or phosphorus Acid buffer is diluted, and the buffer solution contains appropriate number of polyoxyethylene sorbitan monoleate to realize preferable 0.02% poly- sorb The final concentration of ester 80, but 0.01% to 0.1% polyoxyethylene sorbitan monoleate is acceptable.Usually, six acylphosphate disaccharides system Agent is diluted to the concentration close to 0.05mg/ml to 0.5mg/ml.Then the six acylphosphates disaccharides preparation passes through 0.2 μm Filter, preferable Sartorius are sterile filtered.The six acylphosphate disaccharides and adjuvant system of invention described herein Agent presents the zeta current potentials that about -20mV arrives -80mV.

Six acylphosphates disaccharides preparation described herein is used in the product of vaccine in one embodiment, as The adjuvant of vaccine is administered to animals and humans.Preferably, the preparation is designed to deliver the acylphosphate of about 10 microgram six Every dose of disaccharides arrives every dose of the acylphosphate disaccharides of about 50 microgram six, and every dose of six acylphosphate disaccharides of preferably from about 20 microgram is to about Every dose of the acylphosphate disaccharides of 50 microgram six, even more preferred, every dose of six acylphosphate disaccharides of about 40 microgram arrives about 50 micrograms Six every dose of acylphosphate disaccharides.

In a preferred embodiment, six acylphosphate disaccharides are provided as single compound, about 98% purity, divided Son 1763 dalton of amount (its structure is shown in fig. 1).One source of preferable six acylphosphates disaccharides is Avanti Polar Lipids (Alabaster, Alabama, USA) (PHAD).However, the sugar composite of six acylphosphateization two of the present invention Cover the pharmaceutically acceptable salt of six acylphosphate disaccharides or six acylphosphate disaccharides.Six acyl groups used in composition Phosphorylation disaccharides can be wholly or partially synthetic or non-synthetic, and completely synthetic is preferable.

In other embodiments, the derivative of six acylphosphate disaccharides, for example, the acylphosphate of 3- deacylations base-six Disaccharides (can obtain from Avanti Polar Lipids；Referred to as 3D-PHAD) it is used alone in the composition and preparation of the present invention Or it is applied in combination with six acylphosphate disaccharides.The acylphosphate disaccharides of 3- deacylations base-six is carried with the purity approached more than 98% For the dalton of molecular weight 1537 (its structure is shown in accompanying drawing 1A).

With PHAD purity sharp contrast be the GSK for being isolated from salmonella monophosphoryl lipid A, it is as six acyls The DYNAMIC COMPLEX mixture of base, five acyl groups and four acyl group analogs is present；Each of these analogs is in biological activity side Face is all different.The technical staff in medicine and vaccine development field knows that PHAD is better than GSK monophosphoryl lipid A, because PHAD manufacturing process, supply, use and stability can closely monitor as pure compound.

The concentration of citrate, succinate or phosphate buffer be used to improve the six acylphosphates disaccharides system Agent at room temperature and reaches the stability at about 37 DEG C.Without being limited by theory, it is believed that be preferable combination PC：Six acyl group phosphorus Disaccharides or the mol ratio of its acylphosphate disaccharides of derivative 3- deacylations base-six are acidified, and with described herein specific The combination of the citrate of concentration range, succinate or phosphate buffer generates synergy.

Importantly, six acylphosphate disaccharides preparations are preferably formulated as generally without various excipient or chemicals Matter.Thus, what addition cholesterol or two or more phosphatidyl cholines or one or more of phosphatidyl glycerols were not required, Preferably it is not included in the preparation of the present invention.It there is no metabolizable oil, including squalene, and generally without The adjuvant formulation of cholesterol is preferable.In addition, although prior art prompting cholesterol is needed for liposome or adjuvant formulation Required chemical substance, adjuvant formulation described herein need not be used for the cholesterol of adjuvant to provide relative to prior art preparation Remarkable advantage, preferably not to the adjuvant formulation add cholesterol.As in the embodiment shown, two or more phosphatidyls Choline or one or more of phosphatidyl glycerols (two or more phosphatidyl cholines or phosphatidyl glycerol altogether) optionally may be used To be added to small concentration in preparation, but for realizing the present invention in room temperature and reaching the stability at about 37 DEG C, or Prevent, lower or reduce serious injection site and systemic reaction while strengthen immune response, it is that these are not required or It is unwanted.

As detailed in this article, those skilled in the art has used the synthesis containing MLA, MPL or these adjuvants similar The liposome of thing and other various preparations more than 20 years, can not produce in water slurry allow it is described herein The preparation of stability.The room temperature of vaccine adjuvant and the stability reached at about 37 DEG C are the mesh that those skilled in the art highly pursue Mark.In spite of many effort, not developing can also be at temperature described herein and duration by MLA, MPL or synthesis class Stable preparation is kept like thing.Aspect described here solves this problem.

The preparation of vaccine

Another embodiment of the invention further describe comprising adjuvant or six acylphosphate disaccharides preparations and The FimCH or FimH of truncation new vaccine combination.This vaccine be used to treat and prevent by gramnegative bacterium bag Include urinary tract infections caused by Escherichia coli and multi-drug resistance Escherichia coli.FimCH is the non-of FimC and FimH recombinant proteins Covalent compound.FimCH is prepared by adding six acylphosphate disaccharides preparations of predetermined in the bottle to FimCH With the vaccine of six acylphosphate disaccharides preparations.

It is the example of vaccine prepared in accordance with the present invention below.Usually, in order to prepare the vaccine for administration, FimCH Or the antigen of the FimH truncated effective dose and the assistant containing the acylphosphate disaccharides of about 0.005mg/ml to about 0.5mg/ml six Agent formulation combines, the six acylphosphate disaccharides to the μ g of human administration per injection about 10 to about 50 μ g.

In fact, it is the example that can be used for process that is produced according to the present invention and applying vaccine by medical worker below. Described condition and process is exemplary, is not limited the scope of the invention.

FimCH bottle takes out from about -20 DEG C of storage, allows to place at room temperature about 20 minutes and reaches close to room Temperature.After FimCH bottles reach approximate room temperature, upset bottle mixes content for several times.Individually, containing six acylphosphates Taken out in the storage container that the bottle of change disaccharides preparation stores from 2 DEG C to 8 DEG C.Overturn the bottle of six acylphosphate disaccharides preparations Content is mixed for several times, then about 0.2mL is extracted out with sterile 1.0mL syringes, through plug is expelled to FimCH bottles In.Bottle is overturn again mixes content for several times.The sterile buffer of aseptic injection water (WFI) or the preferred present invention make 0.2mL is extracted out with sterile 1.0mL syringes, is expelled to through plug in the acylphosphate disaccharides bottles of FimCH/ six.Again Overturn bottle for several times.Finally, the acylphosphateizations two of FimCH/ six of about 0.3mL preparations are extracted out using sterile 1.0mL syringes Saccharide vaccines.The vaccine of preparation contains 50 μ g FimCH and 20 μ g six acylphosphate disaccharides per 0.3mL dosage.Using it The vaccine of preceding this preparation can be in refrigerated storage temperature or room temperature preservation.

Usually, FimCH is preserved for a long time at -70 DEG C, -20 DEG C or 2 DEG C to 8 DEG C, or is even protected in the room temperature short time About 4 days are deposited to two to three weeks.Usually, only sterile product can preserve at room temperature, micro- because if product is not sterile Biological growth is possible, but be cannot be guaranteed.Vaccine is preferably applied by intramuscular injection.Usually, about 5 micrograms FimCH to the FimCH of about 200 micrograms is administered to the mankind, preferably from about 20 micrograms to about 110 micrograms.Usually, about 10 microgram Six every dose of acylphosphate disaccharides are applied to every dose of the acylphosphate disaccharides of about 50 microgram six together with FimCH, more preferably from about Every dose of 20 microgram, six acylphosphate disaccharides arrives every dose of the acylphosphate disaccharides of about 50 microgram six, even more preferred about 40 microgram six Every dose of acylphosphate disaccharides arrives every dose of the acylphosphate disaccharides of about 50 microgram six, but can use more or less.Typically Ground, three to four doses of the FimCH with six acylphosphate disaccharides preparations are administered to the patient of needs.These dosage typically exist Carry out within 0th day, then about 30 to 60 days after applying first, then about 90 to 180 days, then if preferable, about 180 arrived 360 days.As needed, extra injection can be carried out for 12 to 36 months after being inoculated with first.

As described above, the preferred aspect of the present invention is, the sugar composite of six acylphosphateization two and preparation and antigen or FimCH is individually preserved, because the sugar composite of six acylphosphateization two and preparation have fabulous stability, to patient or people Certain time suitably prepares or mixed with antigen or FimCH before class is applied.However, its of mixing, preparation and administration vaccine His method is possible, and will effectively be worked.

Data exhibiting in following trifle, adjuvant formulation of the invention are enhanced to including bacterium and viral antigen The immune response of other antigens.One or more of vaccine antigens can be added to six acylphosphate prepared in accordance with the present invention In disaccharides preparation.These antigens can be FimCH protein complexes described herein, or other antigens, include but is not limited to Diphtheria, lockjaw, pertussis, polio, hepatitis, and/or the antigen product of influenza virus.

In another embodiment, there is provided apply and include adjuvant or six acylphosphate disaccharides preparations and FimCH Or the method for the FimH truncated new vaccine combination.Especially, there is provided prevention and treatment are by gramnegative bacterium bag The method for including urinary tract infections caused by Escherichia coli and multi-drug resistance Escherichia coli.Usually, the FimCH of about 5 micrograms is to about The FimCH of 200 micrograms is administered to the mankind, preferably from about 20 micrograms to about 110 micrograms.Usually, using the acyl of about 10 microgram six Every dose of base phosphorylation disaccharides arrives every dose of the acylphosphate disaccharides of about 50 microgram six, the acylphosphate disaccharides of preferably from about 20 microgram six Every dose is arrived every dose of the acylphosphate disaccharides of about 50 microgram six, and even more preferred every dose of six acylphosphate disaccharides of about 40 microgram is to about Every dose of the acylphosphate disaccharides of 50 microgram six.Depending on the antigen and the situation for the treatment of that use, other dose quantities can be used And dosage regimen.

In another embodiment, there is provided induction produces and is directed to FimH in the mankind with palindromic urinary tract infection Antibody method.

In another embodiment, there is provided induction produces and is directed to FimH in the mankind with palindromic urinary tract infection Antibody vaccine combination.

In another embodiment, there is provided contain six acylphosphate disaccharides or its derivative 3- deacylations base-six The aseptic composite and sterile pharmaceutical composition of acylphosphate disaccharides；Preferably, the combination of the suspension of these aqueous buffereds Thing has the granular size less than 150nm, even more preferred to be less than 130nm.The sterile six acylphosphate disaccharides combination Thing and preparation are stored in medicament reservoir, and the medicament reservoir and the sugar composite of six acylphosphateization two and preparation are direct Contact.The example of these medicament reservoirs is bottle or syringe, it is furthermore preferred that the sterile six acylphosphate disaccharides combination Thing and preparation are comprised in sterile syringe.Accommodate these medicines of the sugar composite of six acylphosphateization two or preparation Container can be stored in the container of the temperature of approval, for example, in incubator, at room temperature.These contain sterile six The medicament reservoir of acylphosphate disaccharides composite preparation can be placed into the shipping container of assembling, at room temperature or reached about Other positions are transferred at 37 DEG C.These shipping containers can be transported by the transportation service of governmental postal services or business. Due to the outstanding stability of invention described herein, the place of the receiving sugar composite of six acylphosphateization two and preparation Can be the place for not refrigerating or being interrupted refrigeration, or without electric power or the only place of uninterruptible power.

In another embodiment, there is provided include six acylphosphate disaccharides or adjunvant composition or preparation or vaccine The vaccine kit of composition.The kit can optionally include the method for preparing and applying the vaccine, and/or in room Temperature is lower and reaches the explanation that the sugar composite of six acylphosphateization two or preparation are preserved and exposed at about 37 DEG C.Describe guarantor Deposit, transport and these explanations of Exposure Temperature can include U.S. FDA and European drugs administration approved by government administration section. Preferably, one or more of Kit components are the sugar composite of six acylphosphateization two or preparation being in syringe.It is excellent Selection of land, the sugar composite of six acylphosphateization two or preparation containing six acylphosphate disaccharides are sterile, and are places In sterile syringe.

The kit can include the six acylphosphate disaccharides or the label of adjunvant composition or preparation of the present invention, its There is provided at room temperature and reach and about 37 DEG C at preserve and the explanation of the exposure sugar composite of six acylphosphateization two or preparation Or limitation.U.S. FDA can be included by government administration section by describing preservation, these labels of transport and Exposure Temperature or explanation With European drugs administration approved.

As it is stated herein that, new feature of the invention allows the sugar composite of six acylphosphateization two and preparation in refrigeration temperature Degree, room temperature, reach the temperature between about 37 DEG C, refrigerated storage temperature and room temperature and manufactured, tested, analyzed, preserve, transported at room temperature Defeated, receiving, mobile, transfer are applied the lasting time described herein.Due to the outstanding stability of invention described herein, The place for receiving the sugar composite of six acylphosphateization two and preparation can be the place for not refrigerating or being interrupted refrigeration, or not have There are electric power or the only place of uninterruptible power.

Embodiment

The some specific aspects and embodiment of present disclosure are explained in greater detail with reference to following examples, its is complete In order to which the purpose of citing provides, and it is not considered as limiting scope of the disclosure in any way.Used quantity is not A kind of limitation is represented, the process can expand to produce bigger batch.

Embodiment 1

It is following to manufacture six acylphosphate disaccharides preparations to produce mol ratio about 2.5：1 DPPC is than six acylphosphateizations two Sugar.

PHAD Avanti Polar Lipids (Alabaster, Alabama, USA), six acylphosphate disaccharides and DPPC can be from Avanti Polar Lipids (Alabaster, Alabama, USA) as non-GMP or GMP materials (analysis card Bright book provides in table 1) obtain.PHAD is the version of the synthesis of monophosphoryl lipid A.PHAD is prepared together with DPPC to prepare The adjuvant formulation of the present invention.DPPC issue specification provides in table 2.DPPC transition temperature is 41 DEG C.

PHAD is weighed up in appropriate vial, preferable Type 1Plus Schott vials.Addition is in The DPPC solution of an appropriate number of about 2.3mg/ml in ethanol, or equivalent.By this product ultrasound about 1 minute, simultaneously Lightly Rotating article, then suitably carefully remove ethanol by evaporating.Film is reconstructed with pH6.0 10mM sodium citrates, Ultrasound about 30 minutes at about 50 DEG C to 65 DEG C, preferable 55 DEG C.Six acylphosphate disaccharides preparations typically arrive with about 0.5 1.0mg/ml, but it is prepared by no more than about 2.5mg/ml.(as described herein, e.g., from about 13:1DPPC:Six acylphosphates The mol ratio of disaccharides can also be prepared by adjusting DPPC quantity on demand using identical process.

The PHAD preparations of preparation typically show about 60nm to about 500nm granular size.It is sterile filtered to realize, it is necessary to PHAD preparations are usually processed further to realize reduction and suitably homogenous granular size, typically in about 70nm to 130nm Between.Although there is the bibliography of many, including United States Patent (USP) 6,630,161, it was recently reported that high pressure homogenizing is to reduce liposome Granular size preferable method, the high-pressure homogenisation of 25,000psi Avestin homogenizers is reached using pressure to be shown Write ground or relevantly reduce the granular size of these PHAD preparations.This difficulty is unexpected.These PHAD preparations are required The polycarbonate membrane (Avestin, LFLM-80) in 80nm apertures is pressed through about 45 using Avestin extruders or equivalent Passed through for about 7 to about 12 times DEG C to 65 DEG C, at preferable 55 DEG C, to realize the uniform granular size below about 130nm.At 45 DEG C Extruding is important parameter to 65 DEG C.In order to ensure the acceptable rate of recovery after filtration sterilization, six acylphosphate disaccharides The granular size of adjuvant formulation is preferably 150nm or lower, but preferred 130nm or lower.

Then the 10mM sodium citrate pH6.0 containing an appropriate number of polyoxyethylene sorbitan monoleate can be used, dilute six acylphosphates Change disaccharides preparation, to be optimal the 0.02% of choosing polyoxyethylene sorbitan monoleate final concentration, but 0.01% to 0.1% polysorbate 80 be acceptable.Usually, six acylphosphate disaccharides preparations are diluted to about 0.05mg/ml to 0.5mg/ml concentration. These six acylphosphates disaccharides preparations and then by 0.2 μm of filter, preferable Sartorius are sterile filtered.

According to the measure of low-temperature transmission electron microscope, adjuvant formulation described herein is suspension.

Extra or selectable step can increase in said process to prepare six acylphosphate disaccharides preparations.Lift One example, ethanol can evaporate by rotary evaporation or equivalent method, or by nitrogen stream or equivalent method.It is used as other Example, including the ultrasound treatment step of buffer solution of the present invention can be repeated two or more times, in the supersound process repeated Between preparation can be cooled to room temperature or lower temperature, before, during or after the supersound process, preparation can be kept One hour or more long at a temperature of similar to ultrasound treatment step.

Table 1：The analytical proof information of PHAD from Avanti Polar Lipids companies

Table 2：The analytical proof information of DPPC from Avanti Polar Lipids companies

Embodiment 2

The antigen of vaccine can be prepared as follows.

FimCH is the non-covalent compound of FimC and FimH recombinant proteins.The recombinant protein carrys out transgenic Culture of Escherichia coli.FimC and FimH albumen is individually expressed in Escherichia coli, and they spontaneously form non-covalent Compound.The dalton of the molecular weight of FimCH compounds about 51,700.

FimH albumen (the SEQ ID NO of compound：1) there is the molecular weight of 29,065 dalton, it is by following sequence table The 279 amino acid residues composition shown：

Phe Ala Cys Lys Thr Ala Asn Gly Thr Ala Ile Pro Ile Gly Gly Gly Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr Gly Gly Val Leu Ser Asn Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu Pro Asp Tyr Arg Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln Asn Leu Gly Tyr Tyr Leu Ser Gly Thr His Ala Asp Ala Gly Asn Ser Ile Phe Thr Ash Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val Gln Leu Thr Arg Asn Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser Leu Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu Thr Ala Asn Tyr Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile Ile Gly Val Thr Phe Val Tyr Gln

FimC (the SEQ ID NO of compound：2) albumen has the molecular weight of 22,700 dalton, and it is by following sequence table The 205 amino acid residues composition shown：

Gly Val Ala Leu Gly Ala Thr Arg Val Ile Tyr Pro Ala Gly Gln Lys Gln Val Gln Leu Ala Val Thr Asn Asn Asp Glu Asn Ser Thr Tyr Leu Ile Gln Ser Trp Val Glu Asn Ala Asp Gly Val Lys Asp Gly Arg Phe Ile Val Thr Pro Pro Leu Phe Ala Met Lys Gly Lys Lys Glu Asn Thr Leu Arg Ile Leu Asp Ala Thr Asn Asn Gln Leu Pro Gln Asp Arg Glu Ser Leu Phe Trp Met Asn Val Lys Ala Ile Pro Ser Met Asp Lys Ser Lys Leu Thr Glu Asn Thr Leu Gln Leu Ala Ile Ile Ser Arg Ile Lys Leu Tyr Tyr Arg Pro Ala Lys Leu Ala Leu Pro Pro Asp Gln Ala Ala Glu Lys Leu Arg Phe Arg Arg Ser Ala Asn Ser Leu Thr Leu Ile Asn Pro Thr Pro Tyr Tyr Leu Thr Val Thr Glu Leu Asn Ala Gly Thr Arg Val Leu Glu Asn Ala Leu Val Pro Pro Met Gly Glu Ser Ala Val Lys Leu Pro Ser Asp Ala Gly Ser Asn Ile Thr Tyr Arg Thr Ile Asn Asp Tyr Gly Ala Leu Thr Pro Lys Met Thr Gly Val Met Glu

In order to produce transgenic cell line, the FimC genes primer SLC4-28-fimC5 from coli strain J96 Expanded with SLC4-28-FimC3 from the J96 genomic DNAs of purifying and obtain the product of 771 base-pairs.Disappeared with BamHI and EcoRI Change, purifying, be connected in the pTRC99a cut with identical enzyme (BamHI and EcoRI).Connection product is transformed into Escherichia coli In C600 cells, selected on ampicillin, produce plasmid pSJH-32.

Ampicillin antibiotic resistance switches to kanamycins using procedure below：Primer pKD4-pr1 and pKD4-pr2 are used In from pKD4 expand kalamycin resistance gene.This PCR primer T4 polynucleotide kinase phosphorylations, carry out gel-purified. PSJH-32 is cut with ScaI and BglI, is passivated with T4 archaeal dna polymerases, phosphoric acid, Ran Houyu are sloughed with calf intestine alkaline phosphatase The kalamycin resistance gene PCR primer connection of phosphorylation.Then connection product is transformed into Escherichia coli C600 cells, in card Selected on that mycin, produce plasmid pSJH-319.

FimH genes primers F imH5 and FimH3 from bacterial strain J96 expands from the J96 genomic DNAs of purifying to be obtained The product of 978 base-pairs.Digested, purifying, be connected in the pBAD33 with SacI and HindIII digestion with SacI and HindIII. Hereafter, construct is transformed into C600 cells, is selected on chloramphenicol.

Embodiment 3

Biological processing (process for obtaining the antigen of vaccine).

Start biological processing step, master cell bank (MCB) is inoculated into containing band kanamycins (50 μ g/ml) and chloramphenicol In the shaking flask of the APS LB culture mediums of (20 μ g/ml).When OD reaches 2.0-3.0 units (after about growing 15 hours), cell training Foster thing is transferred aseptically to be used to feed-batch fermentation in reactor.APS super broths, about 0.8% sweet will be contained before inoculation The culture medium of oil and antibiotic sterilizes.FimH protein expressions are induced in OD ＞ 10 with IPTG.Add IPTG after five minutes, use Ah Draw uncle's sugar induction FimC protein expressions.Harvesting after about one hour.After harvesting, continuous or in batches centrifugation is passed through Cell is separated from medium component.

Protein Recovery

Restructuring FimCH is expressed in colibacillus periplasm.As gramnegative bacterium, Escherichia coli possess inner and outer Bimolecular lamellar lipid membrane.Space between lipid bilayer is pericentral siphon.After centrifugation immediately using periplasmic preps from thin FimCH is reclaimed in born of the same parents.In the case where sucrose, Tris and EDTA be present 2-8 DEG C by cell with restructuring bacteriolyze enzyme reaction.Then Centrifugal mixture, periplasm protein matter solution caused by collection.Then protein uses ammonium sulfate precipitation, centrifugation, be resuspended in 20mM In MES pH 5.9, then arrived using the dialysis membranes of SpectraPor 2 (Spectrum Labs 132680) by diafiltration In 20mM MES pH 5.9.When electrical conductivity of solution is reduced to about<During 1.5mS/cm, gather solution and be transferred to purification step.

Protein purification

Purifying uses the dialysers of SpectraPor 2 by three column chromatography steps (1.CEX, 2.HIC, 3.CEX) The buffer exchange step that (Spectrum Labs 132680) diafiltration is then filtered, and a final aseptic filtration Step forms.Diafiltration steps are used to exchange to protein in pH 5.9 20mM MES buffer solutions, so as to which it will combine second Individual CEX posts.

Two CEX steps use the Source 15S (GE Healthcare 17-1273-02) in XK26 posts.To two CEX posts, use following condition：Buffer A：20mM MES, pH 5.9；Buffer B：20mM MES/500mM sodium chloride, pH5.9；It is all step 8mL/ minutes, slow with 4CV with 5CV buffer B pre-equilibration in addition to the loading of XK26/10 posts Fliud flushing A balances pillar, using sample pump and does not especially load what is dialysed by chromatographing pump with 5mL/ minutes to XK26 10 FimCH samples, pillar is washed with 4CV buffer A, with the linear 5CV gradient elution pillars of 0-25% buffer Bs, collection Fraction.

For HIC posts, Butyl Sepharose 4FF (GE Healthcare17-0980-01) are used in XK26 posts. Buffer solution C：20mM MES/550mM ammonium sulfate, pH5.9；It it is 8mL/ minutes to XK26/10 in addition to loading, with delaying for 3CV Fliud flushing A (as described above) pre-equilibration pillars, pillar is balanced with 6CV buffer solution C, loads what is concentrated with 5mL/ minutes XK26/10 post FimCH samples, pillar is washed with 6CV buffer solution C, washed with the linear 4CV gradients of 0-100% buffer As and fraction collection De- pillar.FimCH prepares to be prepared in 20mM MES pH 5.9 or 20mM sodium citrates pH 5.4 with 0.3mg/mL concentration. Then it is sterile filtered by 0.2 μm of sterile filters.FimCH is stable, can be preserved about at least 2 years at -20 DEG C.

Embodiment 4

The potency (biological activity for proving FimCH) that external mannose combines

The biological activity of FimCH drug substances (for example, coming from embodiment 3) by external mannose binding analysis come Measure.FimH albumen is that Escherichia coli are plain to combine the bacterial adhesion of the mannose residue on glycosylated protein.In urinary tract During infection, the molten protein of mannosylated urine on FimH adhesin combination Urothelial Cells, it promotes what is combined The internalization of Escherichia coli.The combination of FimCH and the mannosylated molten albumen of urine is that Escherichia coli cause urinary tract infections must not Can be less.In order to monitor FimH mannose-binding activity, observation FimH and enzyme HRPO (HRP) knot in vitro Close.HRP is the glycosylation albumen containing mannose residue, be used to study mammal mannose bind receptor previously.Also Through producing and have studied HRP and agglutinin ConA compound, it combines α-D-MANNOSE base and α-D glucosyl group groups.This A little results show that HRP works as the part of other known mannose-binding protein.Utilize this potency described below Measure, is combined with FimH HRP and shows it is concentration dependent, and is blocked the small molecule suppression that mannose is combined with FimH System.

It is on ELISA flat boards, purifying and effective anti-by being incorporated in FimCH Potency Analysis in vitro FimH antiserums carry out " capture " FimCH.The anti-FimH antiserums used in this analysis have presented (to be made in indirect ELISA For detect antiserum, accompanying drawing 3) and western blot in reference to FimH ability.Then HRP is added, washes excessive HRP, Detect the HRP combined activity.FimCH of the HRP activity measured to adding concentration is proportional (accompanying drawing 4).These result tables Bright, HRP combines FimH in a manner of dose dependent.

In order to prove that HRP and FimH combination need FimH mannose-binding activities, to being also at the compound with FimC In, be referred to as Q133K mannose binding deficient FimH analyzed and compared with FimCH.Q133K and FimH shares identical Amino acid sequence, simply the crucial glutamine at position 133 replaced by lysine.This mutation is in FimH mannose knots Heal up in bag so that Q133K is the defects of functional in terms of mannose and mannosylated protein is combined.Such as institute in accompanying drawing 4 Show, Q133K FimCH do not combine HRP.In indirect ELISA (accompanying drawing 3), the anti-FimH that Q133K mutation compounds are purified resists Serum identifies.This shows that Q133K can not be combined by lacking the antiserum for being not due to purify using Q133K HRP signals；But The mannose residue on HRP can not be combined due to Q133K.These results also indicate that HRP does not combine FimC, because Q133K also locates In the compound with FimC.

As proved in Hung et al. 2002, simple point mutations of the FimH at position 54,133,135 and 140 fully disappears Except mannose combines.According to Hung et al. reported " ... not on the atom directly in conjunction with mannose, in mannose knot Even if most slight change, significantly decreases combination in the bag that heals up ", the mutation for showing in vitro to occur can be limited seriously Or eliminate FimH mannose-binding activities.The combination that HRP lacks to Q133K mutant supports this analysis and is used for assessing FimH The ability of the biological activity combined with mannosylated protein.

Mannose combination FimH several little molecules in inhibiting things have been described.Two kinds in these mortifiers, 4- methyl Umbelliferone acyl-alpha-D mannopyranes glucosides (UFMP) and methyl-α-D- mannopyranes glucosides (MDMP) have been used for further Strengthen this titration.The dissociation constant (Kd) of UFMP combinations FimH report is 20nM, and its 2.2 μM Kd than MDMP is more Powerful about 100 times.According to expectation, added in HRP combination steps these FimH mannose binding inhibitors any one with Dosage-dependent manner has blocked HRP and FimH combination (referring to accompanying drawing 5).For UFMP, observed under 10ng/ml (30nM) Suppress to 50%.For MDMP, observe that 50% suppresses under about 1 μ g/ml (5.1 μM), it is higher than UFMP concentration about 100 times.

These results demonstrate it is this measure be used for assess FimH biological activity and checking manufacturing process batch it Between uniformity ability.In addition, this titration confirms the correct folding of FimH epitopes, for by using FimCH/ The saccharide vaccines of six acylphosphateization two show that the anti-FimH of IgG for reducing Escherichia coli CFU in mouse bladder are predictives to produce 's.

Embodiment 5

According to CEX-HPLC FimCH drug substance impurityes

CEX-HPLC be used to determine FimCH compounds in final FimCH drug substances, uncombined FimC and mixed Debris.Use 0.3M in 20mM MES pH of buffer 6.2 (buffer B) (buffer A be 20mM MES buffer solutions, pH6.2) NaCl gradient elutes protein from GE Healthcare Mono S 5/50GL posts.In T=0, mobile phase is 100% Buffer A, in T=22 minutes, mobile phase is 100% buffer B.Uncombined FimC and impurity are determined according to peak area Relative amount.Representational chromatogram is provided in figure 6.

Embodiment 6

FimCH and PHAD preparations：In rabbit 85 days muscle it is endotoxic/immunogenicity research, the convalescence (GLP) of 21 days

The GLP toxicity research of key is carried out in doe, to assess the PHAD preparations (DPPC containing the present invention:PHAD- is about 2.4:1 mol ratio) FimCH vaccines toxicity and immunogenicity.The research checks ophthalmology discovery, antibody assessment and pathology solution Cut open.Doe continued 13 weeks saline control (N=for applying 5 doses altogether by the IM injections (the 1st, 22,43,64 and 85 day) of every 3 weeks 6), only 40 to 50 μ g PHAD (N=12), 100 μ g FimCH add 20 μ gPHAD (N=6；Low dosage), or 125 μ g FimCH add 40 to 50 μ g PHAD (N=12；High dose).Three days (the 88th days) after the 5th dose, every group of 6 rabbits progress are peaceful and comfortable Extremely, remaining with 6 rabbits in PHAD groups and FimCH high dose groups after 3 week convalescence (the 106th day) carry out it is peaceful and comfortable Extremely.

According to clinical observation, ophthalmology, body temperature, body weight, feed intake, clinicopathologia, gross necropsy, organ weight and disease Anatomical data is managed to assess toxicity.2 before and after per injection, obtain body temperature within 4,6,24,48 and 72 hours.Except mark Outside accurate Clinicopathological Parameters (before administration, the 2nd and 88 day), C- proteins C reactives (CRP) are assessed within 2 days and 7 days after administration And fibrinogen.Per 24,48 and 72 hours after one, possible injection site reaction was using Draize scales to oedema and red Spot is scored, and assesses other performances (that is, eschar, bubble, ulcer and hemotoncus) of local toxicity.To the 1st, 22,43,64 and 85 Before its administration, the blood serum sample that is gathered before the postmortem of the 88th and 106 day, before being administered for the 64th day, the 88th and 106 day corpse The urine sample gathered before is examined, the vagina washings of collection is resisted before administration in the 1st day, before postmortem in the 88th and 106 day FimH antibody assessments.The antibody assessment of urine and vagina washing sample is analyzed qualitatively using titular MSD-ECL.Use Verify the antibody level in effective MSD-ECL analyses measure serum.In 18 rabbits being immunized with FimCH and PHAD, About the 88th day, 17 presented about 1:3,200,000 anti-FimH IgG titres.

When the postmortem of plan is arrived in all rabbit survivals.Preliminary data shows that FimCH adds PHAD and single PHAD It is resistant to well.These discoveries demonstrate, for clinical observation, body weight, feed intake, body temperature, clinicopathologia or Organs Weight Amount, without obvious PHAD individually influence or the influences of vaccine correlation.According to somatoscopy, local reaction is limited.

Using according to DPPC:PHAD about 1:FimCH prepared by 3.9 mol ratios has carried out similar rabbit with PHAD and ground Study carefully.During this investigation it turned out, at the about the 43rd day, only 5 of 16 rabbit present 1:400,000 to 1:800,000 it is anti- FimH IgG titres.By contrast, with DPPC:PHAD about 2.4:FimCH and PHAD prepared by 1 mol ratio is (outlined above ) 16 in 18 rabbit of immunity inoculation presented 1 at the about the 43rd day:400,000 to 3,200,000 anti-FimH IgG titres.These immunogenicity differences are consistent with the research described herein carried out in mouse, show about 2.4:1 DPPC: PHAD mol ratios are better than about 1:3.9 DPPC:PHAD mol ratios.

Embodiment 7

It is used for systemic vaccines inoculation as adjuvant to test six acylphosphate disaccharides in mouse UTI infection models Effect, C3H/HeN mouse insert art infection about 1 × 10 after IM is immune by transurethral catheter⁸CFU clinical cystitis is big Enterobacteria isolate UTI89.Female C3H/HeN mouse (about 9 week old) are purchased from Charles River laboratories (Wilmington, MA).Mouse passes through flesh in the case where light isoflurane anaesthetizes (Henry Schein, Melville, NY) in right leg Approach (IM) is injected using 30 specification syringe needles with 50 μ l volumes in meat.In these effect research, 12.5 μ g of mouse PHAD/15 μ g FimCH are immunized.With individually receiving with the mouse of adjuvant immunity and first the mouse of experiment (using PHAD's Experiment is shown in the figure 7) compare, show after infection one as the mouse of antigen immune by the use of PHAD as adjuvant and FimCH It or the Escherichia coli CFU in two days bladders reduction statistically significantly.These as shown by data, with six acylphosphateizations two Sugar generates antibody as the FimCH vaccines of adjuvant in mouse, and the Escherichia coli for which reducing bladder colonize.These data carry Evidence is supplied, i.e. use six acyl groups that are being prepared according to compositions described herein and being used according to method described herein Phosphorylation disaccharides gives human patientses in need to will also decrease the large intestine bar in human bladder as the FimCH vaccine administrations of adjuvant Bacterium colonizes.

Embodiment 8

The FimH (FimHt) of the truncation of adjuvant is used as by the use of six acylphosphate disaccharides preparations or Freund's adjuvant：In rabbit Immunogenicity research

Female rabbits are injected at by IM to be applied 3 doses of 100 μ g FimHt altogether on the the 0th, 21 and 42 day and adds about 50 μ g this hair Bright PHAD preparations (N=2), or 5 doses of 100 μ g FimHt add Freund's adjuvant (inoculation first are complete, about the altogether 14th, each reinforcing of 21,49 and 70 days is incomplete) (N=2).In this experiment, the FimH of truncation has a series of groups Propylhomoserin is histidine-tagged, it is understood to one skilled in the art that the FimH of other truncation versions can also be used, most preferably Need mannose binding structural domain.The FimH of truncation in this embodiment is by with histidine-tagged (the SEQ ID of C- ends 6- NO：3) FimH residues 1 to 175 form.FimH sequence describes in example 2.To the about the 30th day or the about the 35th and 56 day The blood serum sample of collection carries out anti-FimH antibody assessments.Antibody in serum is determined using ELISA described herein.This reality The capture antigen tested is no histidine-tagged FimH considerably truncated.The rabbit being inoculated with two kinds of preparations, which presents, to be more than 1:1,600,000 (preimmune serums<1:10,000) anti-FimH IgG.The FimH versions of previous truncation disclose, and one Individual example is in United States Patent (USP) 6,737,063, and especially it is completely integrated herein.

Embodiment 9

It is administered to the FimCH vaccines with six acylphosphate disaccharides preparations of rabbit

Using 50 μ g FimCH with and without 0.1% polyoxyethylene sorbitan monoleate 54 μ g PHAD (PHAD preparations of the invention, 10mM sodium citrates, pH 6.0) by IM be injected at the 0th day it is immune, strengthened two groups of rabbit (N=3) at the 21st, 42 day.Use Antibody level in ELISA measure serum described herein.It is included in the about the 30th day and gathers serum from two groups.In two groups In, the anti-FimH titres of IgG are about as much as or more than 1:1,600,000 (the serum before immune<1:10,000).It is described here As shown by data, generated with and without the FimCH vaccines of the six acylphosphate disaccharides preparations of the invention of polyoxyethylene sorbitan monoleate Equal immunogenic response.

Embodiment 10

PHAD and DPPC HPLC analyses in composition

PHAD and DPPC concentration in PHAD preparations is using Agilent Eclipse XBD C18,1.8 μm, and 4.6mm × 50mm posts are analyzed by HPLC-ELSD.Mobile phase is as follows：MP A：The acetic acid of 20mM ammonium acetates in water/1%；MP B：First The acetic acid of 20mM ammonium acetates in alcohol/1%；And MP C：The acetic acid of 20mM ammonium acetates in methanol/chloroform (50/50)/1%.Method 1：Gradient was 100%MP B at 2 minutes since 5%MP A and 95%MP B, was 100%MP C at 8 minutes.Method 2：Gradient was 100%MP B at 2 minutes since 5%MP A and 95%MP B, was 100%MP C at 15 minutes.Sample Diluent 1：85:15 (have the 75 of the acetic acid of 20mM ammonium acetates/1%:15:10 methanol：Chloroform：Water)：(there is 20mM ammonium acetate/1% The 1 of acetic acid：1 methanol：Chloroform).Sample and reference material press 1 with diluents 1：4 dilutions.Diluents 2：There is 20mM second (the 70 of the acetic acid of sour ammonium/1%:25:5) methanol：Chloroform：Water.If using diluents 2, sample and reference material sample are dilute Agent 2 is released by 1：10 dilutions.ELSD gains are 8, temperature 60 C, and nitrogen stream is arranged on about 3.7bars.Application method 2 and sample The example chromatogram of diluent 2 is shown in Figure 8.

Embodiment 11

The stability study of PHAD compositions and preparation

Using the HPLC methods described in embodiment 10, include phosphorus as the different PHAD products of suspension, or conduct The stability of the PHAD preparations of the suspension of phosphatidylcholine is by analyzing the PHAD concentration of these products and by itself and initial results Compare to monitor.Mean that PHAD concentration is within the plus/minus 20% of the initial testing result of issue sample by result, this Within boundary in the HPLC methods using EISD (ELSD).At 2 DEG C to 8 DEG C or 25 DEG C or at 37 DEG C During preservation, compare the PHAD concentration of these products to map (estimation) these samples in the several months to the stability between the several years.For For those skilled in the art, 25 DEG C of data are the conditions of moderate/acceleration, and 37 DEG C of data are the conditions accelerated, are used for Storage life under the long-term storage conditions of 2 DEG C to 8 DEG C of mapping.The buffering for the superior stability for allowing PHAD is determined first Liquid；Then these preferable buffer solutions are assessed with the PHAD preparations including phosphatidyl choline.

The PHAD stability of 7 days at 25 DEG C in selected buffer solution of table 3.

		Condition/time point	As a result
				1	0.5mg/ml PHAD in water	25 DEG C/7 days	Failure
2	0.5mg/ml PHAD in 10mM sodium citrates pH6.0	25 DEG C/7 days	Pass through
				3	0.5mg/ml PHAD in 10mM sodium citrates pH5.0	25 DEG C/7 days	Pass through
4	0.5mg/ml PHAD in 50mM sodium citrates pH6.0	25 DEG C/7 days	Pass through
				5	0.5mg/ml PHAD in 100mM sodium citrates pH6.0	25 DEG C/7 days	Failure
6	0.5mg/ml PHAD in 10mM sodium acetates pH6.0	25 DEG C/7 days	Failure
				7	0.5mg/ml PHAD in 10mM disodium succinates pH6.0	25 DEG C/7 days	Pass through
8	0.5mg/ml PHAD in phosphate buffered saline (PBS) (PBS)	25 DEG C/7 days	Failure
				9	200mM Na₂HPO₄With the 0.5mg/ml PHAD in 100mM citric acids pH6.0	It is unavailable	Precipitation
10	20mM Na₂HPO₄With the 0.5mg/ml PHAD in 10mM citric acids pH6.0	25 DEG C/7 days	Pass through
				11	10mM Na₂HPO₄0.5mg/ml PHAD in pH6.0	25 DEG C/7 days	Pass through

Citrate, succinate and the phosphate buffer of as shown by data in form, about 10mM to 50mM are better than institute Other some buffer solutions checked, in the temperature listed and provide stability under phase time listed.

Table 4. continues 30 or 60 days in selected buffer solution at 25 DEG C, and continues 7 days, 60 days or 4 at 37 DEG C The PHAD of individual month stability

As shown by data in table 4, about 30mM to about 50mM citrate and phosphate buffer be better than checked its His buffer solution.Citrate and phosphate buffer in the temperature listed and provide stability under the time cycle listed.Number According to showing citrate and phosphate concn bringing up to about 25mM to about 50mM, preferred 28mM to about 50mM, most preferably 30mM to about 50mM significant stability benefit.According to all data described here, it is shown that succinate is as slow The preferable utilization of fliud flushing.Six acylphosphate disaccharides are at 37 DEG C in 30mM to 50mM citrates and phosphate buffer The stability for continuing 60 days or more long is unknown in prior art, is the important aspect of the present invention.

Table 5. 30 days at 60 days or 37 DEG C, extrudes without polyoxyethylene sorbitan monoleate and not at 25 DEG C, in selected buffer solution With the stability of 0.5mg/ml (or if being labelled with 1.5mg/ml) PHAD preparations of the invention in selected phosphatidyl choline.Press 2.5 to 1 mol ratio than PHAD prepares phosphatidyl choline.

Tables of data in table 5 understands that present invention combination about 10mM to 50mM citrate, succinate and phosphate delays The significant and unexpected benefit of fliud flushing and phosphatidyl choline.The group of the preferable buffer solution and phosphatidyl choline of the present invention Close the superior stability for generating the six acylphosphate disaccharides in preparation compared with single buffer solution.

Table 6.

As shown in table 6, about 10mM citrate and combinations of the DPPC than the specific molar ratio of six acylphosphate disaccharides Provide the long-time stability at about 25 DEG C.

As shown in upper table, the preparation prepared in water is short-term stability when preserving for 2 DEG C to 8 DEG C, however, for a long time Since the target pursued be to reduce cold chain to preserve and management.The adjuvant formulation of invention disclosed herein has in room by providing The adjuvant formulation of the stability extended at warm to about 37 DEG C realizes this target.Data in table 6 clearly demonstrate, in lemon The PHAD concentration of these preparations prepared in lemon phthalate buffer will keep stable, continue at least about 6 months at about 25 DEG C Or more long, 2 to 3 years may be continued at 2 DEG C to 8 DEG C.

To phosphatidyl choline：Six acylphosphate disaccharides preparations addition citrate, succinate or phosphate buffer Significantly and unexpectedly allow preservation at room temperature and reach the exposure at about 37 DEG C.The phosphatidyl courage prepared in water Alkali:Six acylphosphate disaccharides preparations can produce suitable immunogenic response in mouse and rabbit, but at about 25 DEG C Under be unstable.

Embodiment 12

Use MalvernZS90 or Brookhaven Instruments Corp., use ZetaPlus Particle Sizing softwares, granular size and zeta potential are determined to PHAD preparations using dynamics light scattering.Follow producer Instruct and suggest.Table 7 provides representational data.Zeta potential value is used as estimating the qualitative data of the electric charge of bilayer One piece, and use as described herein.The stability of PHAD preparations according to the granular size of preparation and PHAD concentration come Test Shangdi measure.

Table 7.

As it appears from the above, compared with the preparation containing PHAD of the present invention, single DPPC has significantly lower zeta electricity Position, much larger mean particle size.DPPC critical micellar group concentration is about 0.46 nanomole.These data provide card According to, i.e. single DPPC is a kind of and the dramatically different composition of DPPC and PHAD compositions.

Embodiment 13

Compare and be stored at 2 DEG C to 8 DEG C or about 25 DEG C, with and without polyoxyethylene sorbitan monoleate or glycerine come the implementation for preparing The granular size of the PHAD preparations of example 1, to map the stabilization of granular size of (estimation) the PHAD preparations during 12 to 36 months Property.The PHAD preparations of multiple batches are prepared, typically present after extruding instant 70 to the granular size between 100nm.This The target of a little PHAD preparations is to ensure that their mean effective diameter is preferably held in during its storage life and is less than 150nm, even more preferred to be less than 130nm, estimated based on data storage life described here is 2 to 3 years or more long.Important It is the mean effective diameter that cycle certain time is concluded under centre/acceleration environment, such as at 25 DEG C, carrys out aid forecasting at 2 DEG C Granular size to 8 DEG C at about 2 years or more long.

Use MalvernZS90 or Brookhaven Instruments Corp., use ZetaPlus Particle Sizing softwares, use dynamics determination of light scattering granular size and zeta potentials.Follow the guidance of instrument producer And suggestion.

Table 8.

The data presented in the table 8 of this embodiment have mapped, without polyoxyethylene sorbitan monoleate, in 2 DEG C to the 8 DEG C PHAD preserved The granular size of preparation will remain in about minimum 2 years less than 150nm, imply that as described below possible 3 years.At this It is stable to refer to that what mean effective diameter was maintained at instrument limits it only for the purpose of this test in specific embodiment Interior is less than 150nm.To those skilled in the art, 25 DEG C of data are the conditions of middle/acceleration, for mapping 2 DEG C to the storage life under 8 DEG C of long-term storage conditions.These data clearly have mapped, the granular size of these PHAD preparations Stable at least six moon or more long will be kept at about 25 DEG C, may be 3 years at 2 DEG C to 8 DEG C.As described herein, these PHAD This stability of the preparation at 25 DEG C is that prior art is unknown and unexpected.

Embodiment 14

The preparation of the present invention and comparison of other adjuvant formulations in terms of the anti-FimH antibody of inducing mouse

The PHAD preparations or aqueous formulation (the water-based system in this embodiment of the present invention is prepared as described in Example 1 Agent refers to the mol ratio of the lipid and adjuvant described in United States Patent (USP) 6,491,919 and U.S. Patent application 20080131466), There is following exception.Using the lipid and/or mol ratio (in bracket show) specified, according to the needs for realizing homogeneous suspension Ultrasound is carried out at about 45 DEG C about 30 minutes to 2 hours.As previously described, all lipids are purchased from Avanti Polar Lipids。

Female C3H/HeN mouse (about 9 week old) are purchased from Charles River laboratories (Wilmington, MA).Mouse 30 rule are used by intramuscular route (IM) in right leg in the case where light isoflurane anaesthetizes (Henry Schein, Melville, NY) Lattice syringe needle is injected with 50 μ l volumes.The 1st day and the 29th day, mouse 12.5 μ g PHAD or its derivative 3- deacylations The acylphosphate of base-six disaccharides or Freund's adjuvant (subcutaneous administration) and 15 μ g FimCH immunity inoculations.Such as those skilled in the art It is known, when in use, complete Freund's adjuvant was given at the 1st day, was incomplete Freund's adjuvant at the 29th day.

ELISA for Serum Antibody Detection：Serum is gathered from mouse when putting to death, is resisted by the anti-FimH of elisa assay Body.The T3 that FimH is truncated is attached to Immulon 4HBX flat boards (ThermoFisher) with 2 μ g/ml in PBS, at 4 DEG C overnight. After being washed with PBS+0.05%Tween 20, open bound site is blocked with the 1.5%BSA (Sigma Aldrich) in PBS Point 1 hour.After wash, Sample serum dilution (with 0.05%Tween 20,0.1%BSA, 0.5% methyl- In the PBS of α-D- mannopyrane glucosides) it is incubated 2 hours.After wash, 1 in sample dilution buffer:The biology of 500 dilutions Elementization goat anti-mouse IgG detection antiserum (Sigma Aldrich) 4 DEG C of overnight incubations in reacting hole.After wash, sample 1 in product dilution buffer:The avidin-horseradish peroxidase (HRP, Sigma Aldrich) of 25,000 dilutions exists It is incubated 20 minutes in reacting hole.After wash, using the tmb substrate (Sigma Aldrich) in phosphate citrate buffer Detect HRP activity.Optical density is read at 630nm using VERSAMAX PLUS ELIASAs, is analyzed using SoftMax Pro soft Part (Molecular Devices, Sunnyvale, California) is analyzed.Antibody titer is defined as higher than the back of the body The highest dilution of the signal of scape.

Table 9.

As clearly showed that in table 9, with no adjuvant, Freund's adjuvant or 1:The DPPC of 3.9 mol ratios is more water-based than PHAD Preparation is compared, and about 2:1 to about 13:1 DPPC than PHAD mol ratio in strengthening mouse to FimH immune response in terms of be excellent More.Freund's adjuvant is considered as the standard adjuvant used in animal experiment.Data show, described herein about 2:1 to 13: 1 mol ratio is better than this conventional preceding clinical adjuvant.This is that the present invention presents one of the superiority to prior art preparation Individual aspect.Data in table 9 are also shown that the desired use for not weakening PHAD preparations to these preparations addition DPPC, i.e. enhancing pair FimH immune response.

Embodiment 15

The preparation of the present invention kept the measure of homogeneous mixture in 24 hours.The process of embodiment 1 be used to prepare about 2.5 to 1 DPPC:The PHAD preparations of PHAD mol ratios, do not add polyoxyethylene sorbitan monoleate simply.Bottle containing PHAD preparations is gently Ground is overturn about three to five times.Then, PHAD preparations are allowed to be maintained at room temperature about 24 hours.At 24 hours later, do not overturn, shake Or agitation PHAD preparations, small aliquot is carefully taken out from the top of PHAD suspension, centre and bottom.By previous herein The HPLC of description analyzes the PHAD concentration of these aliquots.As a result show, the aliquot from top, centre and bottom contains phase Deng PHAD concentration.These results prove that PHAD preparations of the invention did not deposited in 24 hours.

Embodiment 16

The preparation of the invention used in Human clinical's research

The adjuvant formulation and FimCH of embodiment 1 are prepared according to cGMP, to contain the Human clinical of about 21 to 64 years old women Research.The PHAD preparations by adding predetermined to FimCH bottle as described in herein previously, it is excellent to obtain each bottle FimCH the and PHAD concentration of choosing, to prepare the vaccine of FimCH and PHAD preparations.IM (intramuscular) injects the preparation of proper volume Vaccine, apply 50 μ g or about 107 μ g FimCH to each female subjects, and 10 μ g, 20 μ g or about 40 μ g PHAD。

These IM injections in the mankind, vaccine presents, with some other known adjuvants used in the mankind Preparation is compared, and adjuvant formulation of the invention and FimCH generate less serious injection site and systemic anti-in the mankind Should.This is the significant aspect of the present invention.Injection site and the intermediate data of systemic reaction show it is as follows, frequency injection be to Untill during the intermediate analysis of every women.Human research is carried out, and the women plan in research receives 4 injections.

Table 10.

	Women number	By the FimCH/PHAD dosage of microgram	The number of injection	Serious injection site and systemic reaction
					Group 1	5	107μg/0μg	3 to 4	Nothing
Group 2	8	50μg/10μg	2 to 3	Nothing
					Group 3	16	50μg/20μg	1 to 2	Nothing
Group 4	8	50μg/40μg	1	Nothing

In interim analysis, 37 women are carried out with more than 60 times IM injections, has not observed serious injection part Position and systemic reaction.When this interim analysis, the women in group 2 presents after the injections of IM twice to be resisted to FimH Precursor reactant, it is bigger 10 times than the initial value before vaccine inoculation.These as shown by data, vaccine are generated for expected from FimH Antibody response.Women in this research will receive 4 vaccine IM injections.Group 5 and 6 is by there is the women of recurrent UTI medical histories It is open.

Compare, wishing auspicious suitable vaccine using the Cervarix containing adjuvant MPL and alum causes about 8% to arrive more than 10% Subject shows serious injection site reaction and systemic reaction.As Treanor et al. (Vaccine, 2013,31 (48), Reported in publication 5760-5), PHAD is prepared in the different preparations with squalene and is administered to the mankind.According to This report, the PHAD of 5 μ g in this preparation cause serious local reaction, tremble with it is stiff, thus limit in the future Research in using this preparation only containing 2 micrograms or less PHAD.In this preparation under the PHAD of 1 microgram, still see Observe serious injection site reaction and systemic reaction.In 2 micrograms or less PHAD in Treanor etc. this preparation Under, the benefit that adjuvant PHAD produces immune response is limited, thus because poor preparation has deprived PHAD potentiality.So And adjuvant formulation of the invention overcomes this limitation using superior preparation, it allows to apply up to 50 μ g six acyl group phosphorus Disaccharides is acidified, or may be more, less serious injection site and systemic reaction.These human datas provide evidence, That is, six acylphosphate disaccharides can be administered to the mankind with preparation described herein with 100 μ g or more.The adjuvant system of the present invention Agent is better than those preparations being earlier attempted to.

All bibliography quoted in this specification, including but not limited to herein cited all newspapers, publication, drill Say, textbook, report, manuscript, handbook, internet are posted, journal article, weekly etc..The discussion of this paper bibliography is only It is intended to the opinion for summarizing their author, does not recognize that any bibliography forms prior art.Inventor rights reserved is challenged The accuracy and appropriateness of the bibliography of citation.

Desirably the theme of all patentabilities disclosed herein is required right, and the theme of these patentabilities is not contributed To the public.Accordingly it is desirable to be that claim is broadly understood according to the intention.In addition, unless based on context it is obvious Contradiction, it is desirable to " one ", all references of "one", and then later refer to as " one " indicated by "one" Corresponding reference when first basic to " described ", the meaning according to " at least one " are broadly understood.Similarly, except not according to Context be it is clearly contradicted, word "or" to selectable specified element in use, being intended to broadly be read as, can In option, specify any one of element, specify any subset of element or all specified elements.

According to above, it will be seen that realize several advantages of the present invention, obtain other beneficial results.Will Understand, foregoing embodiment is only many possibility for representing the principle application of the present invention only for exemplary purpose Embodiment citing.Therefore, it is possible to various change is carried out in the above method and composition without departing from the present invention Scope, it is desirable to all the elements contained in the description above shown with accompanying drawing should be interpreted it is illustrative, and It is not restricted.

In addition, one of ordinary skill in the art can carry out variations and modifications to make its adaptation various to the present invention Purposes and condition, including those do not deployed specifically herein, without departing from the spirit and scope of the present invention.Thus, those changes Change and modification properly, equitably and be intended in invention disclosed and described herein equivalent full breadth it It is interior.

Claims

1. a kind of composition, the composition includes

Six acylphosphate disaccharides, the acylphosphate disaccharides of 3- deacylations base-six, or its pharmaceutically acceptable salt；

Phosphatidyl choline；With

About 10mM to about 50mM citrate, succinate or phosphate buffer, wherein the phosphate buffer is selected from Disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, the group of dipotassium hydrogen phosphate and its mixture, and wherein described composition It is stable containing the NaCl less than 30mM, and when exposed to 19 DEG C to 25 DEG C of temperature range 60 days or more long.

2. composition as claimed in claim 1, wherein, the composition includes six acylphosphate disaccharides or six acyl group phosphorus It is acidified the pharmaceutically acceptable salt of disaccharides.

3. composition as claimed in claim 1, wherein, the buffer concentration is 20mM to about 50mM.

4. composition as claimed in claim 1, wherein, the buffer concentration is 30mM to about 50mM.

5. composition as claimed in claim 1, wherein, the buffer solution is citrate.

6. composition as claimed in claim 1, wherein, the buffer solution is citrates of the 15mM to about 50mM.

7. composition as claimed in claim 1, wherein, the buffer solution is citrates of the 25mM to about 50mM.

8. composition as claimed in claim 1, wherein, the composition has about 4.5 to about 6.5 pH value.

9. composition as claimed in claim 8, wherein, the composition has 150 nanometers or smaller of mean particle size.

10. composition as claimed in claim 9, wherein, the composition is the suspension of aqueous buffered and containing being less than 10mM NaCl.

11. composition as claimed in claim 1, wherein, the buffer solution is citrates of the 25mM to about 50mM, Yi Jisuo State the suspension that composition is the aqueous buffered of 150 nanometers or smaller of mean particle size.

12. composition as claimed in claim 11, wherein, the composition includes six acylphosphate disaccharides or six acyl groups The pharmaceutically acceptable salt of phosphorylation disaccharides, there are about 4.5 to about 6.5 pH value, and the phosphatidyl choline and six acyls The mol ratio of base phosphorylation disaccharides is about 1:1 to about 20:1.

13. a kind of vaccine combination, the vaccine combination includes：

Adjuvant formulation；With,

The antigen of effective dose,

Wherein described adjuvant formulation includes：

Six acylphosphate disaccharides, the acylphosphate disaccharides of 3- deacylations base-six or its pharmaceutically acceptable salt of effective dose；

Phosphatidyl choline；With

About 10mM to about 50mM citrate, succinate or phosphate buffer,

Wherein described phosphate buffer is selected from disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and it is mixed The group of compound, and wherein described adjuvant formulation contain the NaCl less than 30mM.

14. vaccine combination as claimed in claim 13, wherein, the antigen is FimCH.

15. vaccine combination as claimed in claim 13, wherein, the composition includes six acylphosphate disaccharides or six The pharmaceutically acceptable salt of acylphosphate disaccharides.

16. a kind of composition, comprising six acylphosphate disaccharides, the acylphosphate disaccharides of 3- deacylations base-six or its pharmaceutically may be used The salt of receiving, and 15mM is to about 50mM citrate buffer.

17. composition as claimed in claim 16, wherein, the buffer concentration is 20mM to about 50mM.

18. composition as claimed in claim 16, wherein, the buffer concentration is 25mM to about 50mM.

19. composition as claimed in claim 16, wherein, the buffer concentration is 30mM to about 50mM.

20. composition as claimed in claim 16, wherein, the composition has about 4.5 to about 6.5 pH value.

21. composition as claimed in claim 16, wherein, the composition is big with 150 nanometers or smaller of average grain It is small.

22. composition as claimed in claim 21, wherein, the composition is the suspension of aqueous buffered and containing being less than 10mM NaCl.

23. composition as claimed in claim 16, wherein, the composition is 150 nanometers or smaller of mean particle size The suspension of aqueous buffered.

24. composition as claimed in claim 16, wherein, the composition includes phosphatidyl choline, has about 4.5 to about 6.5 pH value, and the phosphatidyl choline and six acylphosphate disaccharides, the acylphosphate disaccharides of 3- deacylations base-six or its The mol ratio of pharmaceutically acceptable salt is about 1:1 to about 20:1.

25. composition as claimed in claim 16, wherein, the composition includes six acylphosphate disaccharides or six acyl groups The pharmaceutically acceptable salt of phosphorylation disaccharides.

26. composition as claimed in claim 25, wherein, the buffer concentration is 20mM to about 50mM.

27. composition as claimed in claim 25, wherein, the buffer concentration is 25mM to about 50mM.

28. composition as claimed in claim 25, wherein, the buffer concentration is 30mM to about 50mM.

29. composition as claimed in claim 25, wherein, the composition has about 4.5 to about 6.5 pH value.

30. composition as claimed in claim 25, wherein, the composition is big with 150 nanometers or smaller of average grain It is small.

31. composition as claimed in claim 30, wherein, the composition is the suspension of aqueous buffered and containing being less than 10mM NaCl.

32. composition as claimed in claim 25, wherein the composition is the water of 150 nanometers or smaller of mean particle size Property buffering suspension.

33. composition as claimed in claim 25, wherein, the composition includes phosphatidyl choline, has about 4.5 to about 6.5 pH value, and the mol ratio of the phosphatidyl choline and six acylphosphate disaccharides are about 1:1 to about 20:1.