CN107530418A

CN107530418A - The method of the composition and treatment urinary tract infections of vaccine and adjuvant

Info

Publication number: CN107530418A
Application number: CN201680016475.7A
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: 2016-03-17
Publication date: 2018-01-02
Also published as: WO2016149558A3; EP3270962A1; EP3270962A4; WO2016149417A1; EP3270963A2; EP3270963A4; CN107405395A; WO2016149558A2

Abstract

The present invention describes new adjunvant composition and preparation, refrigeration, 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 has in the case where reaching 37 DEG C There is fabulous stability, can be produced with significantly low cost.These new adjunvant compositions and preparation are used for vaccine, show The superior property of immune response of the enhancing to antigen, while cause less serious injection position and systemic reaction.This Invention also describes new vaccine combination and preparation, includes Escherichia coli and more by gramnegative bacterium to treat and prevent Urinary tract infections caused by drug resistance Escherichia coli (E.coli).Present invention also offers apply the new bacterin preparation Method, and prevention and treatment urinary tract as caused by gramnegative bacterium includes Escherichia coli and multi-drug resistance Escherichia coli The treatment method of infection.

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 about 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 control general of Department of Public Enterprises A research (OEI-04-10-00430) that is that office is carried out and being reported in 2012 finds, participates in CDC pediatric vaccines The supplier of plan：1.) at a temperature of vaccine is exposed to beyond the approved temperature range of these vaccines：2.) vaccine is existed It is stored at a temperature of outside their approved temperature ranges in refrigerator and freezer；And 3.) by overdue vaccine and not The vaccine that expires is saved together.

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 The rubescent of injection site, swelling can occur, 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, in " the Clinical Review of Human Papillomavirus Bivalent (and of Types 16 18)vaccine[GSK's Cervarix Vaccine],Recombinant,Biologies License Application Described in Efficacy Supplement ", four research reports local detrimental event, it is included in about percent 16 The local pain of motion is hindered in subject.The swelling more than 50mm is also reported in about percent 3 subject.For Arthralgia, fatigue, stomach and intestine, headache and courbature, this four researchs, which report 2.4 to 7.8 percent systemic, serious to be had Evil event.

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 (phosphorylated hexaacyl disaccharide) " PHAD " is acidified, also referred to as " GLA ".By Avanti The PHAD of Polar Lipids supplies is as single compound offer, shown in Figure 1, the dalton of molecular weight 1763, about 98% purity.PHAD purity and GSA MPL form notable contrast, as described above, MPL is isolated from salmonella, as dynamic Complex compound exist.Different from GSA MPL, PHAD production process, supply, use and stability can be used as purifying Compound is closely monitored and controlled.

Whether specific adjuvant or adjuvant combination are unpredictalbe by the immune response strengthened for every kind of specific antigen. 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 It was observed that similar effect improves.(Vaccine,1998,16(7),p.708-714).In the table 6 of 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)。

Trial failure 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 Reported in Infectious Disease periodical (Hersh et al.CID.2012.54 (11), 1677-8) to entire United States The investigation of 562 infectious disease doctors, in last year, excessively resistant to all known antibiotic thin of 63% medical treatment Bacterium infected patient.These data highlight the demand to the new treatment means of bacterium infection.New therapeutic agent is identified in this area Selection have recorded well to prevent and treat the failure of gram-negative bacterial infections.

In addition, at least five kinds of vaccines of the prevention or treatment S. aureus infection in exploitation are stopped in recent years .These include Staph VAX, Veronate, Aurexis Aurograb and V710.New vaccine is identified to prevent and treat The failure of 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 indicates 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 guarantor 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 produce minimum injection site and systemic reaction.Desirably gather around There is the preparation of 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, the adjuvant strengthens to the immune of the Escherichia coli of the patient with UTI Response.

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 presented room temperature stability more than about 6 months, And in the case where reaching about 37 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 aobvious in the case where 2 DEG C to 8 DEG C and room temperature reach about 37 DEG C The stability of work.

In one aspect of the invention, composition include a kind of synthetically produced acylphosphate disaccharides of adjuvant six or it Derivative, the acylphosphate disaccharides of 3- deacylations base-six and buffer solution, the buffer solution is selected from by about 25mM to about 50mM, excellent The group that the 28mM of choosing to about 50mM and most preferred 30mM to about 50mM citrate, succinate and phosphate is formed. These new sugar composites of six acylphosphateization two are preferably the suspension of aqueous buffered.The composition can in vaccine and Used in a variety of ways under medicine situation.The composition, preferably without other compositions, significantly improve six acyl group phosphorus Disaccharides or derivatives thereof stability of the acylphosphate disaccharides of 3- deacylations base-six in suspension is acidified, is realized at room temperature With reach and about 37 DEG C at superior stability.The composition also presents fabulous stability at refrigerated temperatures.It is logical To cross and a kind of efficient and economic sugar composite of six acylphosphateization two is provided, it need not be refrigerated for long-time stability, This demonstrate the 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, buffer solution, the buffer solution are selected from about 10mM to about 50mM, and preferably from about 25mM is to about The citrate, succinate and phosphate of 50mM, preferred 28mM to about 50mM and most preferred 30mM to about 50mM The group of composition, and a kind of preferable synthetically produced phosphatidyl choline.When adding phosphatidyl choline, preferable buffer solution is dense Degree can expand to about 10mM to about 50mM, and realize significant stability described herein.These new adjuvant formulations are excellent Selection of land is the suspension of aqueous buffered.The adjuvant formulation is stored refrigerated and have when being stored at room temperature fabulous steady in a long-term Property, and reach and about 37 DEG C at there is fabulous stability.These preparations can be produced with significantly low cost.

For room temperature stability or reach and about 37 DEG C at stability, new adjuvant formulation described herein need not Lyophilized or equivalent processing.The adjuvant formulation includes specific buffer solution, and optionally and preferably one or more of It is synthetically produced selected from the group being made up of DMPC, DPPC, DSPC, DOPC and POPC, preferable DPPC phosphatidyl choline, and A kind of synthetically produced adjuvant, six acylphosphate disaccharides or its acylphosphate disaccharides of derivative 3- deacylations base-six, mole Than about 1:1 to 40:1 (phosphatidyl choline：Six acylphosphate disaccharides), preferably from about 1:1 to 20:1 (phosphatidyl choline：Six acyls Base phosphorylation 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).

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.

In another aspect of the present invention, new adjuvant formulation includes adjuvant, and the adjuvant is by single phosphorus described herein Being acidified five acyl groups or six acyl group didextrose amine, (it can also be generally referred to by those skilled in the art as the analog of six acylphosphate disaccharides Or derivative；It is described herein as five to six acyl group didextrose amine adjuvants), about 10mM is to about 50mM, preferably from about 25mM to about The citrate buffer of 50mM, preferred 28mM to about 50mM and most preferred 30mM to about 50mM, and a kind of tax Shape agent, a kind of preferable synthetically produced phosphatidyl choline composition.These new adjuvant formulations are preferably the outstanding of aqueous buffered Supernatant liquid.The adjuvant formulation has fabulous long-time stability in refrigeration and preservation at room temperature.These adjuvant formulations can be Preserve at room temperature more than 60 days.These preparations can be produced with significantly low cost.It is right in view of those skilled in the art Various preparation works containing MLA and MPL failed to produce the liquid system for allowing stability described herein more than 30 years Agent, this aspect of the invention is significant.The room temperature stability of vaccine adjuvant is that the height of those skilled in the art is pursued.To the greatest extent Pipe has many effort, and the preparation of prior art does not make successfully PtHA adjuvants steady in temperature and time section described herein It is fixed.Invention described herein solves this problem.

Another embodiment of the invention includes nonionic surfactant, preferable polysorbate

80, to reduce the particle aggregation of the present 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 that adjuvant formulation there is no that metabolizable oil includes squalene and there is no cholesterol.In the art, extensively Recognize generally in order that cholesterol is adjuvant formulation or necessary to liposome realizes function.Present invention produces institute 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 less Serious injection site and systemic reaction, while strengthen the immune response to antigen；And lower production or manufacturing cost, Use the material or composition of less material or composition and lower concentration.The adjuvant formulation of the present invention provides these three combinations Principal benefits, this be as the synthetic analogues to the replacement of the adjuvant based on alum, MLA or MPL adjuvant it is previous not It can realize.

In another aspect of the present invention, there is provided the new vaccine combination containing the new adjuvant formulation is used to control Treat or prevent the urinary tract infections as caused by gramnegative bacterium includes Escherichia coli and multi-drug resistance Escherichia coli.Also provide The method for applying the new vaccine combination, and prevention and treatment include Escherichia coli and more by gramnegative bacterium The method of urinary tract infections caused by 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 combination Thing, and apply the vaccine kit for instructing and storing explanation.It is described explanation provide the sugar composite of six acylphosphateization two or Preparation at room temperature and reach and about 37 DEG C at exposure.These explanations for describing preservation, transport and Exposure Temperature can To include US 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 that the sugar composite of six acylphosphateization two and preparation and vaccine combination are sterile Composition and sterile pharmaceutical composition, it is preferred that the sterile sugar composite of six acylphosphateization two and preparation are contained in nothing In bacterium syringe.

Brief description of the drawings

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

Figure 1A shows a kind of chemical constitution of salt of the acylphosphate disaccharides of 3- deacylations base-six.

Figure 1B shows the example of the selection of the chemical constitution of six acyl group didextrose amine adjuvants of Formulas I.

Fig. 1 C show the example of the selection of the chemical constitution of five acyl group didextrose amine adjuvants of Formula II.

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 titration.

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 to illustrate to be protected from coli-infection after the FimCH/PHAD of mouse is immune

Figure.

Fig. 8 is DPPC and PHAD HPLC example chromatograms.

Embodiment

Definition

On six acylphosphate disaccharides quantity or buffer concentration (unless defining), " about " refer to adding for institute's number of columns With 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 to 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 one kind to improve antigen reactive reagent in the presence of effective dose；Strengthen to the immune response of antigen Material；Or stimulate reagent caused by the antibody for antigen.A variety of naming rules or term in this area be present.Without reference to specific Naming rule, 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 other hand-written, printings or the material drawn, 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 of detailed description wish it is auspicious suitable, and herein and et al. grinding vaccine described in Te Leina et al. (Vaccine 2013) Adjuvant 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 that 3'-O- removes acyl group -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 the activator of Toll-like receptor 4, refers to six acylphosphate disaccharides or six acyl group phosphorus It is acidified the pharmaceutically acceptable salt of disaccharides.The structure of preferable six acylphosphates disaccharides salt shows in fig. 1, and it can be with Obtained from Avanti Polar Lipids (PHAD).As used herein, six acylphosphate disaccharides can be complete or partial Synthesis or non-synthetic, and completely synthetic is preferable.

" the acylphosphate disaccharides of 3- deacylations base-six " is the activator of Toll-like receptor 4, refers to the acyl group phosphorus of 3- deacylations base-six It is acidified the pharmaceutically acceptable salt of disaccharides or the acylphosphate disaccharides of 3- deacylations base-six.The preferable acyl group phosphorus of 3- deacylations base-six The structure of acidifying disaccharides salt shows that it can be obtained from Avanti Polar Lipids in accompanying drawing 1A.As used herein, The acylphosphate disaccharides of 3- deacylations base-six can be wholly or partially synthetic or non-synthetic, and completely synthetic is excellent Choosing.The acylphosphate disaccharides of 3- deacylations base-six is five acyl group disaccharides of phosphorylation.

" 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, 7.4,25 DEG C of pH.

" five to six acyl group didextrose amine adjuvants " or " five to six acyl groups " (" PtHA ") adjuvant refer to connect as shown in Formulas I to VI It is connected to five acyl groups or six acyl group carbochains of mono-phosphorylated didextrose amine support, preferable ten two to ten six carbon, most preferred 12 To the adjuvant of 14 carbon composition, or the pharmaceutically acceptable salt of PtHA adjuvants, excludes six acylphosphate disaccharides or it spreads out The biological acylphosphate disaccharides of 3- deacylations base-six (it is five acyl group disaccharides of phosphorylation).As used herein, PtHA adjuvants can To be wholly or partially synthetic or be isolated from natural origin, and what is synthesized is preferable.

" 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, for example, Na2HPO4 and/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 but is not limited to DMPC (myristoyl-sn- glyceryl -3- phosphocholines of 1,2- bis-), the DPPC (palmityl-sn- glyceryls -3- of 1,2- bis- Phosphocholine), DSPC (1,2- distearyl acyl group-sn- glyceryl -3- phosphocholines), DOPC (1,2- dioleoyl-sn- glycerine Base -3- phosphocholines) 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 temperature range 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 needs External application IV is hydrated；In 24 hours by 6 times or more it is time watery just and/or>800 grams of diarrhoea, or need external application IV to be hydrated；Severe Use narcotic analgesic medicine or the headache of obstruction daily routines；Fatigue that is significant or hindering daily routines；And significant or harm 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 composition or preparation Concentration is 0.5% or less.

" 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 two can be used Sour 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 composition to the immunity of disease.Vaccine combination is to trigger pin The immunogenic composition of immune response and antibody producing to the antigen 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 (are referred to as " sugar composite of six acylphosphateization two " or " composition for containing six acylphosphate disaccharides " or " contained The composition and carrier of six acylphosphate disaccharides ", these are also separately from its acylphosphate of derivative 3- deacylations base-six Disaccharides).The sugar composite of six acylphosphateization two of the present invention is preferably the suspension of aqueous buffered.The buffer solution be selected from by About 25mM to about 50mM, more preferably from about 28mM are to about 50mM, citrate, succinates of the most preferred 30mM to about 50mM The group formed with phosphate.Preferably, pH value arrives about 4.0 to about 7.5, preferably from about 4.5 to about 6.5, more preferably from about 5.0 In the range of about 6.0.With this combination (six acylphosphate disaccharides or its acylphosphate of derivative 3- deacylations base-six Disaccharides and the buffer solution) pharmaceutical composition or carrier present basic fabulous stability as pharmaceutical composition, And improve the general stability of the sugar composite of six acylphosphateization two.Specifically, these groups for containing six acylphosphate disaccharides Compound and carrier realize in room temperature and reach the stability at about 37 DEG C.These of the present invention contain six acylphosphate disaccharides Composition and carrier also present 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, it is individually used for and other lipids Carrier combinations.In one embodiment, the phosphatidyl choline of the natural generation from soybean or egg can be added, or is come arrogant The phosphatidyl choline of the hydrogenation of beans or egg, or 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 prepare in water, acetate buffer, PBS or citrate or phosphate buffer, etc. When 100mM, the composition containing six acylphosphate disaccharides is in room temperature and reaches unexpected steady at 37 DEG C It is qualitative to be not present.The sugar composite of six acylphosphateization two contains citrate, succinate or phosphate buffer To produce unexpected stability, preferably from about 25mM to about 50mM, preferred 28mM to about 50mM, most preferred 30mM 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 has the granular size less than 150nm, more preferably less than 130nm granular size, it is preferred that these six The sugar composite of acylphosphateization two is 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 more preferably there is no generally without phosphatidyl glycerol 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 is come in order to preserve life-span 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；And the sugar composite of six acylphosphateization two preferably applies it after the fabrication Before remain the suspension of 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 there is no cholesterol, phosphatidyl glycerol and phosphatide Acyl monoethanolamine, 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 there is no 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.

Preferably, when the composition containing six acylphosphate disaccharides or preparation of the present invention in room temperature or reach about 37 DEG C Under when being saved, transport, keep or applying, most preferably it is sterile filtered or prepared using asptic technique, more preferably The sterile sugar composite of six acylphosphateization two or preparation be 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 and 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) exist to about 40 (PC) than the mol ratio of 1 (six acylphosphate disaccharides), preferably from about 2.5 (PC) 1 (six acyl groups of ratio Phosphorylation disaccharides).Unless otherwise indicated, the citation to six acylphosphate disaccharides preparations is applied to adjuvant formulation.These are new Six acylphosphate disaccharides preparations are preferably the suspension of aqueous buffered.When being stored in refrigerated storage temperature to room temperature and reach When at about 37 DEG C, the adjuvant formulation has fabulous long-time stability.In addition, the adjuvant formulation can be raw with low cost Production.

In a preferred embodiment, the adjuvant formulation includes a kind of preferable synthetically produced phosphatidyl choline, excellent The DPPC of choosing, and a kind of synthetically produced acylphosphate disaccharides of adjuvant six or its acyl group phosphorus of derivative 3- deacylations base-six It is acidified disaccharides, its mol ratio about 1:1 to 40:1(DPPC:Six acylphosphate disaccharides), and about 10mM to 50mM, but preferably Citrate, the succinate of about 25mM to 50mM, preferred 28mM to about 50mM and most preferred 30mM to about 50mM Or phosphate buffer.Preferably, pH value arrives about about 4.0 to about 7.5, preferably from about 4.5 to about 6.5, more preferably from about 5.0 In the range of 6.0.Importantly, the six acylphosphates disaccharides adjuvant formulation can use only a kind of acylphosphateization two of adjuvant six Sugar or its acylphosphate disaccharides of derivative 3- deacylations base-six, and a kind of preferable phosphatidyl choline produce, so as to permitting Perhaps they are produced with low cost.Preferably, the adjuvant formulation contains six acylphosphate disaccharides or its derivative 3- deacylations The acylphosphate of base-six disaccharides can use other adjuvants as unique adjuvant.The long-time stability feature of the present invention It reduce further 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 highly preferred that the preparation containing six acylphosphate disaccharides there is no cholesterol, phosphatidyl glycerol and Phosphatidyl-ethanolamine, 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 the every kind of preparation described herein containing six acylphosphate disaccharides of excipient, this limitation are not excluded for often containing The only a kind of material of preparation or excipient of six acylphosphate disaccharides.

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 the every kind of preparation described herein containing six acylphosphate disaccharides of excipient, this limitation are not excluded for often containing The only a kind of material of preparation or excipient of six acylphosphate disaccharides.

In another aspect of the present invention, new adjuvant formulation includes one or more of PtHA adjuvants, and about 10mM is to about The lemon of 50mM, preferably from about 25mM to about 50mM, preferred 28mM to about 50mM and most preferred 30mM to about 50mM Phthalate buffer, and excipient, a kind of preferable synthetically produced phosphatidyl choline.These new PtHA adjuvant formulations are preferred Ground is the suspension of aqueous buffered.The PtHA adjuvant formulations are in refrigeration and have when preserving at room temperature fabulous steady in a long-term Property.These PtHA adjuvant formulations can preserve more than 60 days at room temperature.These PtHA preparations can be produced with significantly low cost.

An embodiment of the invention provides the PtHA adjuvants of below formula (I),

Wherein R¹Preferably C₃To C₇；Condition is to be not all C₅；Preferred wherein R¹It is C₃To C₅, condition is to be not all C₅；Most preferred R¹It is C entirely₃、C₄、C₆Or C₇。

Another embodiment of the invention provides the PtHA adjuvants of below formula (II),

Another embodiment of the invention provides the PtHA adjuvants of below formula (III),

Another embodiment of the invention provides the PtHA adjuvants of below formula (IV),

Another embodiment of the invention provides the PtHA adjuvants of below formula (V),

Another embodiment of the invention provides the PtHA adjuvants of below formula (VI),

More specifically, the PtHA preparations citrate buffer concentration is selected from the group of following composition：About 10mM is to about 50mM；10mM to about 50mM；15mM to about 50mM；20mM to about 50mM；About 25mM to about 50mM, 30mM are to about 50mM；About 40mM to about 50mM；40mM to about 50mM；About 40mM；And about 50mM.

Although without being limited by theory, as in the embodiment shown, six acylphosphate disaccharides and its derivative 3- are taken off The forced degradation research of the acylphosphate disaccharides of acyl group-six has shown that preparation of the invention prevents or significantly reduced acyl bond water Solve as didextrose amine support.As a result show, preparation of the invention will also prevent or significantly decrease acyl bond and be hydrolyzed to PtHA assistants The didextrose amine support of agent.Embodiment proves that preparation of the invention improves the stability of PtHA adjuvants.In view of this area skill Art personnel have been investigated the preparation containing MLA and MPL more than 30 years, are failed to produce and are allowed stability described herein Liquid preparation, this aspect of the invention is significant.In spite of many effort, the preparation of prior art does not make successfully PtHA adjuvants are stable in temperature and time section described herein.Invention described herein solves this problem.

Determined by such as in Fendrix European directive applications, MLA may the impurity containing bacterial origin, it is including free Aliphatic acid, nucleic acid, protein, the LPS of partial hydrolysis, 2-keto-3-deoxyoctanoic acid salt and monoethanolamine.In addition, fermentation process helps Additional impurities in MLA, include but is not limited to, culture medium derived peptide, protein and its respective catabolite.Different is pure Change process may or may not can remove some in these impurity.When these compounds are present in complex mixture such as MLA When, any one of these components may have adverse effect to PtHA stability.It is of the invention although as described herein Specific preparation also improves PtHA stability in the presence of these impurity (such as MLA).However, in the excellent of the present invention Select in embodiment, PtHA compositions of the invention, either single compound or mixture, all do not contain these impurity. These PtHAs, either single compound or mixture, are most commonly obtained by building-up process.

Formulas I and II can utilize the acylphosphate disaccharides of commercial production six and the acylphosphate disaccharides of 3- deacylations base-six The building-up process of (both of which can be obtained commercially from Avanti Polar Lipids or AMRI (Albany, NY)) obtains.May be used also To use the process described in Imoto et al. (Bull.Chem.Soc.Jpn.1987,60,2205-2214).

Formula III can obtain from Sigma and Avanti Polar Lipids MLA or MPL as described herein to VI, Or from Hagen et al. (J.Chromatography A, 1997,767,53-61) or Qureshi et al. The process of (J.Biol.Chem.1985,260 (9), 5271-5278) obtains.Formula III and IV can also be according to Johnson et al. The process production of (J.Carbohydrate Chem.1998,17 (9), 1421-1426).

The preparation of the present invention does not have squalene preferably substantially.

The adjuvant formulation containing PtHA of the present invention is preferably substantially not used as metabolizable oil of adjuvant.

The adjuvant formulation containing PtHA of the present invention is preferably substantially without metabolizable oil.

The adjuvant formulation containing PtHA of the present invention is preferably the suspension of aqueous buffered, preferably these aqueous buffereds Suspension have be less than 150nm, the even more preferably granular size less than 130nm, it is preferred that these contain PtHA assistant Agent formulation is not oil in water emulsion.

The adjuvant formulation containing PtHA of the present invention preferably substantially without the second adjuvant, including alum, squalene, QS21, MF59, the activator of Toll-like receptor 9, and other adjuvants, including the adjuvant based on squalene.The PtHA preparations are excellent Selection of land there is no other or extra adjuvant, because they have the more serious locally and systemically property of generation in the mankind anti- The possibility answered, without further improving immune response or significantly improving the benefit for the treatment of results.

The adjuvant formulation containing PtHA 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 adjuvant formulation containing PtHA more preferably there is no generally without phosphatidyl glycerol Phosphatidyl glycerol.

Preferably, the adjuvant formulation containing PtHA does not have substantially more preferably generally without phosphatidyl-ethanolamine There is phosphatidyl-ethanolamine.

Preferably, the adjuvant formulation containing PtHA more preferably there is no generally without monoacylglycerol Monoacylglycerol.

The adjuvant formulation containing PtHA of the present invention is preferable to contain less than 20mM's preferably generally without salt solution NaCl, it is furthermore preferred that the PtHA preparations contain the NaCl less than 10mM, it is even more preferred to there is no NaCl.

Preferably, the adjuvant formulation containing PtHA more preferably there is no generally without freeze drying protectant Freeze drying protectant.

Preferably, the adjuvant formulation containing PtHA 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 preparation containing PtHA there is no cholesterol, phosphatidyl glycerol and phosphatidyl-ethanolamine, And it there is no metabolizable oil and the second adjuvant.

Preferably, the preparation of the invention containing PtHA is generally without the new feature institute described herein for realizing the present invention Unwanted material or excipient, and the even more preferred preparation of the invention containing PtHA there is no reality described herein New feature the institute unwanted material or excipient of the existing present invention

In another aspect, there is provided vaccine and with vaccine therapy and prophylactic method.In order to prepare vaccine, one Kind or more kind vaccine antigen is added to adjuvant formulation or PtHA preparations as described above containing six acylphosphate disaccharides In.The antigen can be FimCH protein complexes described herein, or other antigens, including but not limited to diphtheria, broken The related antigen of cold, pertussis, polio, hepatitis or the antigen product of influenza virus.

The vaccine prepared using the six acylphosphates disaccharides preparation or PtHA preparations need not be freezed come in order to preserve Life-span or stability keep the concentration of six acylphosphate disaccharides.It is lyophilized be a kind of dehydration for being mainly used in preserving material or Freeze-dried process.The same target of preservation material is realized now with equivalent process.Prepare vaccine described herein and When the sugar composite of six acylphosphateization two or PtHA preparations, it is not necessary to which lyophilized vaccine or adjuvant formulation is unexpected and aobvious The advantages of work.By eliminating lyophilized necessity, many expensive steps can be cancelled.First, step of freeze drying is cancelled in itself, It not only eliminates the expensive manufacturing step that must be carried out under the aseptic condition well monitored, also eliminates to this manufacture The examination ＆ approval and assessment of step.Saved next, the removal of step represents a kind of continual expense.For example, often prepare one During batch, step of freeze drying is all saved.In addition, when preparing bigger batch, or production process is transferred to another and set Shi Shi, remove the step and save extra examination ＆ approval step.Second, freeze need manufacture or purchase, transport and with lyophilized system Product preserve the sterile vials of diluent together, for reconstructing.Remove reconstruction step and eliminate this diluent bottle and its confession Answer the cost of chain management.3rd, the reconstruct of adjuvant formulation may influence its aseptic, it is necessary to use at once, if not pre- Use needs disposable product in the fixed time.4th, restructuring procedure is easy to malfunction, thus producer is lost to being finally administered to The control of the exact concentration of the product of patient.Adjunvant composition and preparation described herein eliminate this by removing lyophilized demand Four shortcomings.Therefore, the preparation containing six acylphosphate disaccharides or PtHA preparations be not lyophilized preferably or without It is lyophilized；The preparation containing six acylphosphate disaccharides or PtHA preparations are not dried preferably after the preparation；It is described to contain Have six acylphosphate disaccharides preparation or PtHA preparations preferably without using liquid or buffer solution after the preparation from drying Material in reconstruct；And the preparation containing six acylphosphate disaccharides or PtHA preparations are preferably applied after the fabrication The suspension of aqueous buffered is remained before.

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 or PtHA preparations can made Efficiently and at low cost it is packaged into immediately in syringe after making.Preferably, the composition and preparation be sterile, excellent Syringe described in selection of land is sterile.These prefilled syringes can be transported in refrigerated storage temperature to room temperature and at reaching about 37 DEG C Defeated, preservation, deliver or shift.This advantage provides most efficient and low cost mode combine six acylphosphate disaccharides Thing and the preparation containing six acylphosphate disaccharides or the place of PtHA preparations arrival administration.

In another embodiment, nonionic surfactant is added in adjuvant formulation described herein.It is preferred that Ground, the nonionic surfactant are polyoxyethylene sorbitan monoleates, but can also use others.The nonionic surfactant Typically it is added to about 0.001% to 1.0%, preferable 0.01% to 0.1% concentration in the adjunvant composition.It is this to add The six acylphosphates disaccharides preparation or PtHA preparations can be prevented when preserving at room temperature and reaching at about 37 DEG C by adding The slight increase of little aggregates or mean particle size.Preferably, nonionic surfactant derives from the mountain of polyethoxylated Pears sugar alcohol acid anhydride, including but not 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 a kind of preferable conjunction Into the phosphatidyl choline of production, the phosphatidyl choline is selected from the group being made up of DMPC, DPPC, DSPC, DOPC and POPC, preferably DPPC, and a kind of synthetically produced acylphosphate disaccharides of adjuvant six or its acylphosphate of derivative 3- deacylations base-six Change disaccharides, its mol ratio about 1：1 to 40：1 (phosphatidyl choline：Six acylphosphate disaccharides), preferably from about 1：1 to 20：1 (phosphorus Phosphatidylcholine：Six acylphosphate 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).It is furthermore preferred that citrate or Succinate Buffer are pressed According to about 10mM to about 50mM, preferably from about 25mM to about 50mM, preferred 28mM to about 50mM, most preferred 30mM is to about 50mM is used in six acylphosphate disaccharides preparations.

As described by this paper for the concentration of six acylphosphate disaccharides or the preferred buffer of PtHA preparations, buffer solution Concentration be selected from following composition group：About 10mM to about 50mM；15mM to about 50mM；20mM to about 50mM；About 25mM is to about 50mM；25mM to about 50mM；About 30mM to about 50mM；28mM to about 50mM；30mM to about 50mM；About 30mM；About 40mM is to about 50mM；40mM to about 50mM；About 40mM；And about 50mM.Preparation described herein containing six acylphosphate disaccharides or PtHA preparations preferably contain less than 20mM NaCl, it is furthermore preferred that six acylphosphate preferably generally without salt solution Change two sugar composites and preparation or PtHA preparations contain NaCl less than 10mM, it is even more preferred to there is no NaCl.

In addition, shown in the embodiment of following article, in the composition containing six acylphosphate disaccharides and preparation or The stability features of the new feature, the particularly present invention of the present invention are not showed in PtHA preparations using PBS.By contrast, exist This specially appointed phosphate buffer allows the new stability features of the present invention.

Six acylphosphate disaccharides preparations preferably by select with the synthetically prepared single phosphatidyl choline of high-purity come Prepare.However, the phosphatidyl choline of the natural generation from soybean or egg, or the phosphatidyl courage of the hydrogenation from soybean or egg Alkali, or synthesis or the phosphatidyl choline of natural mixing can be used for preparing the adjuvant formulation.

Six acylphosphate disaccharides preparations or PtHA preparations can be prepared with any concentration, usually with about 0.005 to about 1.0mg/ml six acylphosphate disaccharides or PtHA, preferably from about 0.05 to about 1.0mg/ml six acylphosphate disaccharides or Prepared by PtHA, but preferably no more than about 2.5mg/ml six acylphosphate disaccharides or PtHA.Six acylphosphate The phosphatidyl choline of disaccharides preparation or PtHA preparations can be prepared with any concentration, it is preferred that according to about 0.005 to about 16mg/ml (0.007mM to 22mM), more preferably from about 0.05 arrives about 8mg/ml (0.07mM to 11mM), it is even more preferred about 0.05 prepares to about 0.8mg/ml (0.07mM to 1mM).

The six acylphosphate disaccharides preparations and PtHA preparations of the present invention are prepared to produce about 40 as follows:1 to about 1:1 phosphorus Phosphatidylcholine is than six acylphosphate disaccharides or PtHA, and preferable DPPC is than six acylphosphate disaccharides or PtHA, preferably from about 2.5:Mol ratios of 1 DPPC than six acylphosphate disaccharides or PtHA.Six acylphosphate disaccharides or PtHA are weighed up suitable When vial in, for example, Type 1Plus Schott vials.An appropriate number of phosphatide that addition is in ethanol Phatidylcholine, preferable DPPC.By this product ultrasound about 1 minute, while preparation is lightly rotated, then by evaporating suitably Remove ethanol.Film citrate, succinate or phosphate buffer, preferable 10mM to about 50mM sodium citrate pH 6.0 reconstruct, at about 50 DEG C to 65 DEG C, ultrasound about 30 minutes, can preferably be performed for more than a week at preferable 55 DEG C The supersound process of phase.PtHA preparations are prepared using citrate buffer described herein.The six acylphosphate disaccharides prepared Preparation typically has about 60nm to about 500nm granular size.It is sterile filtered to realize, is preferably processed further six acyl groups Phosphorylation disaccharides preparation or PtHA preparations are preferably arrived in about 70nm come homogeneous granular size that realize reduction and appropriate Between 130nm.Six acylphosphate disaccharides preparations and PtHA preparations by the polycarbonate membrane in 80nm apertures (Avestin, LFLM-80) extruded with Avestin extruders or equivalent, at about 45 DEG C to 65 DEG C, about 7 to about 12 times at preferable 55 DEG C Pass through, to realize the homogeneous granular size below about 130nm.It is excellent in order to ensure the acceptable rate of recovery after aseptic filtration Choosing, the granular size of the six acylphosphates disaccharides preparation and/or PtHA preparations is 150nm or lower, but even more preferably 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 or PtHA preparations described herein are used for vaccine in one embodiment In product, the adjuvant as vaccine is administered to animals and humans.Preferably, the preparation is designed to deliver about 10 micrograms six Every dose of acylphosphate disaccharides arrives every dose of the acylphosphate disaccharides of about 50 microgram six, the acylphosphateization two of preferably from about 20 microgram six Every dose of sugar or PtHA arrive every dose of the acylphosphate disaccharides of about 50 microgram six or PtHA, even more preferred, the acyl group phosphorus of about 40 microgram six Every dose of acidifying disaccharides or PtHA arrive every dose of the acylphosphate disaccharides of about 50 microgram six or PtHA.

In a preferred embodiment, six acylphosphate disaccharides are as about 98% purity, the dalton of molecular weight 1763 Single compound provide (its structure is shown in Figure 1).One source of preferable six acylphosphates disaccharides is Avanti Polar Lipids (Alabaster, Alabama, USA) (PHAD).However, six acylphosphateizations two of the present invention Sugar composite covers the pharmaceutically acceptable salt of six acylphosphate disaccharides or six acylphosphate disaccharides.Make in composition Six acylphosphate disaccharides can be wholly or partially synthetic or non-synthetic, and completely synthetic is preferable.

In other embodiments, six acylphosphate disaccharides are to derivative, for example, the acylphosphate of 3- deacylations base-six Disaccharides (can be obtained from Avanti Polar Lipids) the present invention composition and preparation in be used alone or with six acyl group phosphorus Acidifying disaccharides is applied in combination.3- deacylations base-six acylphosphate disaccharides is to approach the purity and 1537 dalton more than 98% Molecular weight provides (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 is used for six acylphosphate disaccharides preparations of improvement and existed Room temperature and reach the stability at about 37 DEG C.Without being limited by theory, it is believed that be preferable combination PC：Six acylphosphateizations two The mol ratio of sugar or its acylphosphate disaccharides of derivative 3- deacylations base-six, and there is certain concentration model described herein The combination of the citrate, succinate or the phosphate buffer that enclose generates synergy.

Importantly, six acylphosphate disaccharides preparations or PtHA preparations are preferably formulated as generally without various taxes Shape agent or chemical substance.Thus, add cholesterol or two or more phosphatidyl cholines or one or more of phosphatidyls are sweet What oil was not required, preferably it is not included in the preparation of the present invention.Metabolizable oil, including squalene are there is no, And generally without the adjuvant formulation of cholesterol it is preferable.Although in addition, prior art prompting cholesterol be liposome or Required chemical substance needed for adjuvant formulation, the cholesterol that adjuvant formulation described herein need not be used for adjuvant are relative to provide In the remarkable advantage of prior art preparation, preferably cholesterol is not added to the adjuvant formulation.As in the embodiment shown, two kinds Or more kind phosphatidyl choline or one or more of phosphatidyl glycerols (two or more phosphatidyl cholines or phosphatidyl altogether Glycerine) it can be optionally added to small concentration in preparation, but for realizing the present invention in room temperature and reaching about 37 DEG C Under stability, or prevent, lower or reduce serious injection site and systemic reaction while strengthen immune response, these are not It is required or 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 or PtHA preparations and FimCH or the FimH of truncation new vaccine combination.This vaccine be used to treat and prevent by leather orchid Family name's negative bacteria includes urinary tract infections caused by Escherichia coli and multi-drug resistance Escherichia coli.FimCH is FimC and FimH weights The non-covalent compound of histone matter.By the six acylphosphate disaccharides systems that predetermined is added in the bottle to FimCH Agent prepares FimCH and six acylphosphate disaccharides preparations vaccine.

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 administrations typically exist Carry out within 0 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 or PtHA systems Agent individually preserves with antigen or FimCH, because the sugar composite of six acylphosphateization two and preparation and PtHA preparations have pole Good stability, suitably prepared or is mixing with antigen or FimCH to certain time before patient or human administration.However, Mixing, the other method for preparing and applying vaccine are possible, and will effectively worked.

Data exhibiting in following chapters and sections, 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 or PtHA preparations.These antigens can be FimCH protein complexes described herein, or other antigens, bag Include but be 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 or PtHA preparations And the method for the FimCH or FimH of truncation new vaccine combination.Especially, there is provided prevention and treatment are by gram-negative Property bacterium include the method for urinary tract infections caused by Escherichia coli and multi-drug resistance Escherichia coli.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, using about 10 Every dose of six acylphosphate disaccharides of microgram arrives every dose of the acylphosphate disaccharides of about 50 microgram six, the acyl group phosphorus of preferably from about 20 microgram six Every dose of acidifying disaccharides or PtHA preparations arrive every dose of the acylphosphate disaccharides of about 50 microgram six or PtHA, even more preferably preferably Every dose of the acylphosphate disaccharides of about 40 microgram six or PtHA arrive every dose of the acylphosphate disaccharides of about 50 microgram six or PtHA.Depend on The antigen and the situation for the treatment of used, can use other dose quantities 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 sterile composition of acylphosphate disaccharides and sterile pharmaceutical composition；Preferably, the suspension of these aqueous buffereds Composition has the granular size less than 150nm, even more preferred to be less than 130nm.The six sterile acylphosphate disaccharides Composition and preparation are stored in medicament reservoir, the medicament reservoir and the sugar composite of six acylphosphateization two and preparation Directly contact.The example of these medicament reservoirs is bottle or syringe, it is furthermore preferred that the six sterile acylphosphate disaccharides Composition and preparation are comprised in sterile syringe.Accommodate these of the sugar composite of six acylphosphateization two or preparation Medicament reservoir can be stored in the container of the temperature of approval, for example, in incubator, at room temperature.These contain sterile The medicament reservoirs of six acylphosphate disaccharides composite preparations can be placed into the shipping container of assembling, at room temperature or reach Other positions are transferred to about 37 DEG C.These shipping containers can be transported by the transportation service of governmental postal services or business It is defeated.Due to the outstanding stability of invention described herein, the sugar composite of six acylphosphateization two and preparation are received Place 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 13:1 DPPC:Six acylphosphates Changing 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 processed further to realize reduction and suitably homogenous granular size, typically between about 70nm to 130nm.Though So there are the bibliography of many, including United States Patent (USP) 6,630,161, it was recently reported that high pressure homogenizing is that the particle of reduction liposome is big Small preferable method, the high-pressure homogenisation that 25,000psi Avestin homogenizers are reached using pressure can not significantly or phase Ground is answered to reduce the granular size of these PHAD preparations.This difficulty is unexpected.These PHAD preparations must use The polycarbonate membrane (Avestin, LFLM-80) that Avestin extruders or equivalent are pressed through 80nm apertures arrives at about 45 DEG C Passed through for about 7 to about 12 times at 65 DEG C, preferable 55 DEG C, to realize the uniform granular size below about 130nm.At 45 DEG C to 65 Extruding is important parameter at DEG C.In order to ensure the acceptable rate of recovery after aseptic filtration, six acylphosphate disaccharides adjuvants The granular size of preparation is preferably 150nm or lower, but preferred 130nm or lower.

Then the 10mM sodium citrates (pH6.0) containing an appropriate number of polyoxyethylene sorbitan monoleate can be used, dilute six acyl group phosphorus Disaccharides preparation is acidified, to be optimal the 0.02% of choosing polyoxyethylene sorbitan monoleate final concentration, but 0.01% to 0.1% poly- sorb Ester 80 is acceptable.Usually, six acylphosphate disaccharides preparations are diluted to the dense of about 0.05mg/ml to 0.5mg/ml Degree.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 present invention buffer solution ultrasound treatment step can repeat to come two or more times, repeat ultrasound at Preparation can be cooled to room temperature or lower temperature between reason, and before, during or after the supersound process, preparation can be protected Hold at a temperature of similar to ultrasound treatment step one hour or more long.

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 answer 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 Asn 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 albumen (the SEQ ID NO of compound：2) there is the molecular weight of 22,700 dalton, 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 Broth, about will be contained before inoculation The culture medium of 0.8% glycerine and antibiotic sterilizes.FimH protein expressions are induced in OD ＞ 10 with IPTG.Five points after addition IPTG Clock, FimC protein expressions are induced with arabinose.Harvesting after about one hour.After harvesting, by it is continuous or point The centrifugation criticized separates cell 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 0-100% buffer As and the linear 4CV gradients for collecting cut 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, Fig. 3) and western blot in reference to FimH ability.Then HRP is added, washes excessive HRP, is examined Survey the HRP combined activity.FimCH of the HRP activity measured to adding concentration is proportional (accompanying drawing 4).These results indicate that 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 (Fig. 3), Q133K is mutated the anti-blood of anti-FimH that compound is purified Clear identification.This shows that Q133K HRP signals, which lack the antiserum for being not due to purify, can not combine Q133K；And it is due to Q133K can not combine the mannose residue on HRP.These results also indicate that HRP does not combine FimC because Q133K be also at In FimC compound.

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, combined in mannose Even if most slight change, significantly decreases combination in pocket ", the mutation for showing in vitro to occur can seriously limit or Eliminate FimH mannose-binding activities.HRP lack combination to Q133K mutant support this analysis be used for assessing FimH with The ability for the biological activity that mannosylated protein combines.

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), PHAD individually adds 20 μ gPHAD (N=6 in only 40 to 50 μ g PHAD (N=12), 100 μ g FimCH；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 Son is euthanized, and remaining 6 rabbit in single PHAD groups and FimCH high dose groups are the (the 106th after 3 week convalescence My god) be euthanized.

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 aqueous solution of 20mM ammonium acetates/1%；MP B： The methanol solution of the acetic acid of 20mM ammonium acetates/1%；And MP C：Methanol/chloroform (50/50) of the acetic acid of 20mM ammonium acetates/1% is molten Liquid.Method 1：Gradient was 100%MP B at 2 minutes, was 100%MP at 8 minutes since 5%MP A and 95%MP B C.Method 2：Gradient was 100%MP B at 2 minutes, was 100%MP at 15 minutes since 5%MP A and 95%MP B C.Diluents 1：85:15 (have the 75 of the acetic acid of 20mM ammonium acetates/1%:15:10 methanol：Chloroform：Water)：(there is 20mM acetic acid The 1 of the acetic acid of ammonium/1%：1 methanol：Chloroform).Sample and reference material press 1 with diluents 1：4 dilutions.Diluents 2：Have (the 70 of the acetic acid of 20mM ammonium acetates/1%:25:5) methanol：Chloroform：Water.If using diluents 2, sample and reference material are used Diluents 2 presses 1：10 dilutions.ELSD gains are 8, temperature 60 C, and nitrogen stream is arranged on about 3.7bars.Application method 2 It is shown in Figure 8 with the example chromatogram of diluents 2.

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 release 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 The preservation life-span 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.

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 Citrate and phosphate concn are brought up into about 25mM to about 50mM according to indicating, preferred 28mM is optimal to about 50mM The 30mM of choosing to about 50mM significant stability benefit.According to all data described here, it is shown that succinate conduct The preferable utilization of buffer solution.Six acylphosphate disaccharides are at 37 DEG C in 30mM to 50mM citrates and phosphate buffer Under continue 60 days or more long stability be unknown in prior art, be 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 combination of the preferable buffer solution and phosphatidyl choline of the present invention Generate the superior stability of 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 potentials are determined to PHAD preparations using dynamics light scattering.Follow producer Guidance and suggestion.Table 7 provides representational data.Zeta potential values are used as estimating the qualitative number of the electric charge of bilayer According to one piece, and use as described herein.The stability of PHAD preparations is dense according to the granular size of preparation and PHAD's Degree determines to test Shangdi.

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 PHAD preparations of example 1, to map stability of (estimation) the PHAD preparations during 12 to 36 months.Prepare multiple batches PHAD preparations, instant 70 are typically presented after extruding between 100nm.The target of these PHAD preparations is to ensure that them Mean effective diameter be preferably held in during its preservation life-span less than 150nm, it is even more preferred to be less than 130nm, base Estimated in data storage life described here is 2 to 3 years or more long.It is important that under centre/acceleration environment, such as 25 DEG C The lower mean effective diameter for concluding cycle certain time, carrys out particle of the aid forecasting at 2 DEG C to 8 DEG C at about 2 years or more long Size.

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.

Sample	Time point/condition	Mean effective diameter (nm)
			The adjuvant formulation of embodiment 1, without polyoxyethylene sorbitan monoleate	1 month/25 DEG C	99
The adjuvant formulation of embodiment 1, there is 0.1% polyoxyethylene sorbitan monoleate	1 month/25 DEG C	104
			The adjuvant formulation of embodiment 1	1 month/2-8 DEG C	79
The adjuvant formulation of embodiment 1	1 month/25 DEG C	82
			The adjuvant formulation of embodiment 1, there is 0.01% polyoxyethylene sorbitan monoleate	1 month/25 DEG C	105
The adjuvant formulation of embodiment 1, without polyoxyethylene sorbitan monoleate	4 months/2-8 DEG C	83
			The adjuvant formulation of embodiment 1, without polyoxyethylene sorbitan monoleate	4 months/25 DEG C	114
The adjuvant formulation of embodiment 1, there is 0.1% polyoxyethylene sorbitan monoleate	4 months/2-8 DEG C	94
			The adjuvant formulation of embodiment 1, there is 0.1% polyoxyethylene sorbitan monoleate	4 months/25 DEG C	106
The adjuvant formulation of embodiment 1, there is 0.01% polyoxyethylene sorbitan monoleate	4 months/2-8 DEG C	90
			The adjuvant formulation of embodiment 1, there is 0.01% polyoxyethylene sorbitan monoleate	4 months/25 DEG C	114
The adjuvant formulation of embodiment 1	4 months/25 DEG C	86
			The adjuvant formulation of embodiment 1	5 months/2-8 DEG C	82
The adjuvant formulation of embodiment 1	6 months/25 DEG C	101
			The adjuvant formulation of embodiment 1	6 months/2-8 DEG C	94
The adjuvant formulation of embodiment 1, without polyoxyethylene sorbitan monoleate	8 months/2-8 DEG C	117

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 preservation life-span 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.

Adjuvant/preparation	Antibody dilution titer
		Without adjuvant；N=6 mouse	1:20,000
Experiment 1：With reference to adjuvant：Freund's adjuvant；N=6 mouse	1:200,000
		Experiment 2：With reference to adjuvant：Freund's adjuvant；N=7 mouse	1:200,000
Experiment 1：DPPC:PHAD(1:3.9) aqueous formulation；N=8 mouse	1:200,000
		Experiment 2：DPPC:PHAD(1:3.9) aqueous formulation；N=10 mouse	1:200,000
DPPC:PHAD(2.6:1) adjuvant formulation；N=10 mouse	1:400,000
		DPPC:PHAD(13:1) adjuvant formulation；N=10 mouse	1:400,000
DPPC:DPPG:PHAD(2.6:0.3:1) adjuvant formulation；N=10 mouse	1:400,000
		DPPC:DPPG:PHAD(2.6:2.6:1) adjuvant formulation；N=10 mouse	1:400,000
DPPC:The acylphosphate disaccharides (2.6 of 3- deacylations base-six:1) adjuvant formulation；N=3 mouse	1:200,000

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.

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, cause about 8% to the subject more than 10% using the Cervarix vaccines containing adjuvant MPL and alum Show serious injection site reaction and systemic reaction.Such as Treanor et al. (Vaccine, 2013,31 (48), 5760-5) Publication in report, PHAD is prepared in the different preparations with squalene and is administered to the mankind.According to this report Accuse, the PHAD of 5 μ g in this preparation cause serious local reaction, tremble with it is stiff, thus limit the research in future It is middle using this preparation only containing 2 micrograms or less PHAD.In this preparation under the PHAD of 1 microgram, still observe tight The injection site reaction of weight and systemic reaction.In Treanor etc. this preparation under 2 micrograms or less PHAD, assistant The benefit that agent PHAD produces immune response is limited, thus because poor preparation has deprived PHAD potentiality.However, this hair Bright adjuvant formulation overcomes this limitation using superior preparation, and it allows to apply up to 50 μ g six acylphosphateizations two Sugar, or may be more, less serious injection site and systemic reaction.These human datas provide evidence, i.e. six acyls Base phosphorylation disaccharides can be administered to the mankind with preparation described herein with 100 μ g or more.The adjuvant formulation of the present invention is better than Those preparations being earlier attempted to.

Embodiment 17

Six acylphosphate disaccharides and its acylphosphate disaccharides of derivative 3- deacylations base-six and natural derivative MLA forced degradation research

To six acylphosphate disaccharides and its acylphosphate disaccharides of derivative 3- deacylations base-six, and it is natural derivative MLA compound carry out forced degradation experiment, to illustrate the preparation of the present invention is prevented or significantly reduced mechanism of degradation.

Experiment 1.

0.5mg/ml PHAD is prepared in potassium citrate, pH 8.1, in 37 DEG C of preservations.PH 8.1 is preferred the present invention's Outside scope, induced degradation.By the way that 200 μ L CHCl3,200 μ L methanol and 100 μ L ammonium acetates are added into 80:20 methanol/waters The middle sample prepared for analysis.After blending, bottom is shifted, with 100 μ L methanol dilution.By direct infusion with negative norm Electron spray flight time (TOF) mass spectrum (MS) the analysis sample of formula.By the TOF-MS all analyses carried out it is possible that every The mass of ion trueness error of its about 1 to 2 mass unit.In addition, it will be appreciated by those skilled in the art that during each analysis, Various adduct ions may occur daily.Therefore, molecular formula is based on the ion considered observed by daily systematic error. Show within 0 day the single leading ion of six acylphosphate disaccharides at 1745.By 1,7,13 and 20 day, go out at 1309 and 1519 Now obvious ion, continued to improve from 1 day to 20 days.Two carboxyl groups on MS ions representative didextrose amine supports at 1309 Loss (molecular formula C₂₈H₅₃O₃；437 dalton), single carboxyl groups on the MS ions representative didextrose amine supports at 1519 Loss (molecular formula C₁₄H₂₇O₂；227 dalton).By 7 days, the mass intensity of 1519 ions brought up to 1745 mass ions The 46% of mass intensity.At 0 day, the mass intensity of 1519 ions was the 4% of 1745 mass ions.These as shown by data, six acyls One of principal degradation mechanism of base phosphorylation disaccharides is the loss of carboxyl groups or two carboxyl groups.As shown by data herein, The preparation of the present invention prevents these carboxyl groups from being lost from six acylphosphate disaccharides.

Experiment 2.

The 0.5mg/ml acylphosphate disaccharides of 3- deacylations base-six is prepared in potassium citrate, pH 8.1, in 37 DEG C of guarantors Deposit.The progress of all preparations and analysis as listed by experiment 1.The acylphosphate of 3- deacylations base six at the as shown by data 1521 of the 0th day The single leading ion of disaccharides.By the 1st, 13 and 22 day, occur obvious ion at 1084, continue to carry from the 1st day by 22 days It is high.(molecular formula is C to two carboxyl groups of MS ions representatives at 1084₂₈H₅₃O₃；437 dalton) lost from didextrose amine support Lose.By the 13rd day, the intensity of 1084 mass ions brought up to the 24% of the intensity of 1521 mass ions.At the 0th day, 1084 mass The intensity of ion is the 3% of the intensity of 1521 mass ions.These as shown by data, the master of the acylphosphate disaccharides of 3- deacylations base six Want the loss that one of degradation mechanism is two carboxyl groups.Six acylphosphate disaccharides and the acylphosphate disaccharides of 3- deacylations base six Mainly degraded by losing carboxyl groups.The preparation of the present invention prevents the loss of these carboxyl groups.

Experiment 3.

Data according to caused by above, and the fact that MLA is a kind of complex mixture of PtHA adjuvants, from MLA point Separate out four acyl group MLA, five acyl group MLA and six acyl group MLA (Sigma, from Salmonella enterica serotypes Ming Nisu Up to the monophosphoryl lipid A of Re 595 (Re mutant), identification symbol L6895).Five acyl group MLA and six are identified from natural MLA Acyl components are with coming from Hagen et al. (J.Chromatog.A, 1997,767,53-61) and GSK in its Fendrix vaccines Data in European regulatory application are all consistent.NMR and MS as shown by data, the compound of both separation is by the Portugal of five acyl group two Osamine and six acyl group didextrose amine composition.Compound is separated using following condition by HPLC：Mobile phase A：Methanol/water (95:5) In 20mM ammonium acetates and 1% (v/v) acetic acid；Mobile phase B：Ethanol/methylene (50:50) the 20mM ammonium acetates and 1% in (v/v) acetic acid.MLA is dissolved in A:B(3:1) in.Compound separates on Betamax Neutral C18HPLC posts (8x250mm), Use A:B(95:5) eluted with 3mL/ minutes, be followed by A:B(20:80) gradient was more than 60 minutes.Peak is detected using ELSD. More than 12 kinds compounds are observed during this separation, it is the complex mixture of compound to show MLA.

Four acyl group MLA compositions of separation have 1280 mass ion.Five acyl group MLA compositions of separation have 1506 matter Ion is measured, six acyl group MLA compositions of separation have 1717 mass ion.Because they are all pure and abundant, five are selected Its mechanism of degradation of acyl group MLA composition detections.When testing beginning, in five acyl group MLA compounds of purifying, MS is not detected (C is lost to corresponding to single acyl group₁₄H₂₇O₂；227 dalton) significant impurity.Degradation experiment enters in PBS, pH 7.4 OK, in 37 DEG C of preservations.It is because the use of salt solution not being preferred embodiment of the invention to select this buffered saline system.It is logical Cross MS and analyze five acyl group MLA PBS solutions (pH7.4), the early increase that the mass ion at 1280 was detected to 11 days, show list Individual acyl chain (C₁₄H₂₇O₂；227 dalton) loss.These as shown by data, the degradation pathway that this buffered saline system allows With in the embodiments herein to pH 8.1 prepare six acylphosphate disaccharides and the acylphosphate disaccharides of 3- deacylations base-six in show That shows is identical.These data further support the assessment of MLA mechanism of degradations described below.These data provide evidence card Bright PtHA adjuvants have similar Major degradation pathways, and it includes losing one or two acyl chain from didextrose amine support. Because the preparation of the present invention prevents or significantly decreased acyl chain from six acylphosphate disaccharides and the acylphosphate of 3- deacylations base-six Change and lost on disaccharides, preparation of the invention will prevent acyl chain from being lost from PtHA adjuvants, because they have the Portugal of identical two Osamine support and suitable acyl chain covalent bonding.

Embodiment 18

Based on the experiment using the MLA obtained from Sigma, obtain from Avanti Polar Lipids (Lipid A Detoxified, 699200P, Salmonella minnesota R595) MLA.Avanti MLA preliminary MS analyses are aobvious Show significant mass ion at 1280,1490,1506 and 1717, represent four acyl group MLA, two kinds of five independent acyl groups respectively MLA and six acyl group MLA, is integrally incorporated in this complex mixture.Due to the principal degradation mechanism of six acyl groups and five acyl group MLA It is to lose one or two acyl chain, produces more four acyl groups MLA of mass ion 1280, and due to obtained from Avanti's MLA contains the significant mass ion at 1280,1490,1506 and 1717, the ratio of these mass ions from MS analyses The stability for determining five acyl groups and six acyl group MLA compounds can be used for.Therefore, Avanti MLA is selected to be used to check respectively The stability experiment of kind preparation.

Avanti MLA sample is prepared in PBS, pH 7.4 with 0.5mg/ml, or in 25mM citrates, pH6.0 Middle preparation.Sample storage is at 37 DEG C.Sample preparation and analysis are carried out as described herein.

Table 11.

Data in table 11 list the mass ion relative intensity compared with 1,717 six acyl group MLA mass ions, real Border mass intensity is listed in bracket.Mass ion and intensity are analyzed by TOF-MS to be produced.For two kinds shown in table 11 Buffer solution, compared with reference mass ion, four acyl group MLA mass ions significantly increase (0.9 to 2.5 times), show from five acyls One and two acyl chains are lost on base and six acyl group MLA.In the sample prepared in PBS 1,490 five acyl group MLA mass from Son also significantly increases (1.3 times).As shown in table 11,25mM citrates, pH 6.0 buffer solution are better than PBS, but it It is not enough to reach 20 days in 37 DEG C of independent stable Avanti MLA compound.Therefore, these buffer solutions can not individually improve five The stability of acyl group and six acyl group MLA.Without being limited by theory, MLA seems a kind of complex mixture, therefore it needs embodiment Process described in 1, it includes excipient, preferable phosphatidyl choline.It is most important, these as shown by data, use TOF-MS can be determined in various preparations to detect the increased this process of mass ion 1280 (four acyl group didextrose amine) The stability of PtHA adjuvants.

Based on data above, Avanti MLA sample, with 0.5mg/ml, do not extrude as described in Example 1 Or polyoxyethylene sorbitan monoleate, prepared together with DPPC in 4mM or 25mM citrates pH 6.0, and do not have as described in Example 1 There are extruding or polyoxyethylene sorbitan monoleate, prepared in 25mM mM citrates pH 6.0 together with DPPG.Sample storage is straight at room temperature To analysis.

Table 12.

To the as shown by data of 4mM citrate samples in table 12, the ratio of mass ion 1490 to 1717 has in 30 days 11% improves.If for these Data Extrapolations by 60 days, this sample will not meet the stability criterion of embodiment 11.Therefore, have The 4mM citrates pH 6.0 for having DPPC is not suitable for improving the stability of the complex mixture of MLA adjuvants.It is not bound by the limit of opinion System, MLA as described above is complex mixture, thus the formula for improving MLA stability includes excipient, preferable phosphatidyl courage Alkali, for example, obtain during described in embodiment 1.

The table 12 of data in to(for) the 25mM citrate samples with DPPC meet the stability criterion of embodiment 11. 61 days, according to the stability criterion of embodiment 11, the ratio of mass ion did not significantly improve.Therefore, there is DPPC 25mM Citrate pH 6.0 improves the stability of the PtHA adjuvants of the complex mixture including MLA adjuvants.Especially, these data Show, preparation stabilization of the invention five acyl groups to six acyl group didextrose amine adjuvants.

Mass ion 1280, which compares, in 30 days is shown to the data of the 25mM citrate samples with DPPG in table 12 1717 ratio has 13% raising.If for these Data Extrapolations by 60 days, this sample will not meet the stability of embodiment 11 Standard.Therefore, the 25mM citrates pH 6.0 with DPPG is not suitable for improving the stabilization of the complex mixture of MLA adjuvants Property.Without being limited by theory, MLA as described above is complex mixture, thus the formula for improving MLA stability includes excipient, Preferable phosphatidyl choline, for example, obtain during described in embodiment 1.

Data in table 12 support evidence previous caused by this paper, i.e. have phosphatidyl choline, 10mM citric acids Salt buffer, preferably from about 25mM can improve the stability of PtHA in the solution to about 50mM.These data support previously Evidence, i.e. preparation of the invention prevent or significantly reduce PtHA adjuvants carboxyl groups lose.

All bibliography quoted in this specification, including but not limited to herein cited all newspapers, publication, drill Say, textbook, report, manuscript, handbook, internet dispatch, 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 The citing of embodiment.Therefore, it is possible to various change is carried out in the above method and composition without departing from the present invention's Scope, it is desirable to all the elements contained in the description above shown with accompanying drawing should be interpreted it is illustrative, without It is 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：

Corresponding at least one compound of following structure：

Or its pharmaceutically acceptable salt；

Phosphatidyl choline；And

About 10mM to about 50mM citrate buffer, and wherein described composition contain the NaCl less than 30mM, when sudden and violent It is stable when being 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 compound includes

3. composition as claimed in claim 1, wherein, the compound includes

4. composition as claimed in claim 1, wherein, the compound includes

5. composition as claimed in claim 1, wherein, the compound includes

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

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

8. composition as claimed in claim 1, wherein, the buffer concentration is about 25mM to about 50mM.

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

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

11. composition as claimed in claim 10, wherein, the composition is big with 150 nanometers or smaller of average grain It is small.

12. composition as claimed in claim 11, wherein, the composition is the suspension of aqueous buffered, and contains and be less than 10mM NaCl.

13. 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.

14. composition as claimed in claim 13, wherein, the composition has about 4.5 to about 6.5 pH value, Yi Jisuo It is about 1 to state phosphatidyl choline and the mol ratio of the compound of claim 1:1 to about 20:1.

15. a kind of vaccine combination, comprising：Adjuvant formulation；With the antigen of effective dose, wherein, the adjuvant formulation includes effective The compound of the claim 1 of amount；Phosphatidyl choline, and about 10mM is to about 50mM citrate buffer.

16. vaccine combination as claimed in claim 15, wherein, the antigen is FimCH.

17. a kind of composition, comprising：

Corresponding at least one compound of following structure：

Or its pharmaceutically acceptable salt；

Phosphatidyl choline, and,

About 10mM to about 50mM citrate buffer, and

Wherein described composition contains the NaCl less than 30mM, and when the temperature range 60 days or more long exposed to 19 DEG C to 25 When be stable.

18. composition as claimed in claim 17, wherein, the compound includes

19. composition as claimed in claim 17, wherein, the compound includes

20. composition as claimed in claim 17, wherein, the compound includes

21. composition as claimed in claim 17, wherein, the compound includes

22. composition as claimed in claim 17, wherein, the buffer concentration is 15mM to about 50mM.

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

24. composition as claimed in claim 17, wherein, the buffer concentration is about 25mM to about 50mM.

25. composition as claimed in claim 17, wherein, the buffer concentration is 30mM to about 50mM.

26. composition as claimed in claim 17, wherein, the composition has about 4.5 to about 6.5 pH value.

27. composition as claimed in claim 26, wherein, the composition is big with 150 nanometers or smaller of average grain It is small.

28. composition as claimed in claim 27, wherein, the composition is the suspension of aqueous buffered, and

Contain the NaCl less than 10mM.

29. composition as claimed in claim 17, wherein, the buffer solution is citrates of the 25mM to about 50mM, and The composition is the suspension of the aqueous buffered of 150 nanometers or smaller of mean particle size.

30. composition as claimed in claim 29, wherein, the composition has about 4.5 to about 6.5 pH value, Yi Jisuo It is about 1 to state phosphatidyl choline and the mol ratio of the compound of claim 20:1 to about 20:1.

31. a kind of vaccine combination, the seedling composition includes：Adjuvant formulation；With, the antigen of effective dose, wherein, the adjuvant Preparation includes the compound of the claim 17 of effective dose；Phosphatidyl choline, and about 10mM is to about 50mM Citrate buffer Liquid.

32. vaccine combination as claimed in claim 31, wherein, the antigen is FimCH.

33. a kind of composition, the composition includes

Corresponding at least one compound of following structure：

Or its pharmaceutically acceptable salt；

Phosphatidyl choline, and

About 10mM to about 50mM citrate buffer, and wherein described composition contain the NaCl less than 30mM, and It is stable when exposed to 19 DEG C to 25 DEG C of temperature range 60 days or more long.

34. composition as claimed in claim 33, wherein, the compound includes

35. composition as claimed in claim 33, wherein, the compound includes

36. composition as claimed in claim 33, wherein, the compound includes

37. composition as claimed in claim 33, wherein, the compound includes

38. composition as claimed in claim 33, wherein, the buffer concentration is 15mM to about 50mM.

39. composition as claimed in claim 33, wherein, the buffer concentration is 20mM to about 50mM.

40. composition as claimed in claim 33, wherein, the buffer concentration is about 25mM to about 50mM.

41. composition as claimed in claim 33, wherein, the buffer concentration is 30mM to about 50mM.

42. composition as claimed in claim 33, wherein, the composition has about 4.5 to about 6.5 pH value.

43. composition as claimed in claim 42, wherein, the composition is big with 150 nanometers or smaller of average grain It is small.

44. composition as claimed in claim 43, wherein, the composition is the suspension of aqueous buffered, and contains and be less than 10mM NaCl.

45. composition as claimed in claim 33, wherein, the buffer solution is citrates of the 25mM to about 50mM, and The composition is the suspension of the aqueous buffered of 150 nanometers or smaller of mean particle size.

46. composition as claimed in claim 45, wherein, the composition has about 4.5 to about 6.5 pH value, Yi Jisuo It is about 1 to state phosphatidyl choline and the mol ratio of the compound of claim 36:1 to about 20:1.

47. a kind of vaccine combination, comprising：Adjuvant formulation；With, the antigen of effective dose, wherein, the adjuvant formulation includes effective The compound of the claim 33 of amount；Phosphatidyl choline, and about 10mM is to about 50mM citrate buffer.

48. vaccine combination as claimed in claim 47, wherein, the antigen is FimCH.