CN106459225A - O-acetylated high molecular weight polygalacturonic acids and their use as vi polysaccharide vaccine - Google Patents
O-acetylated high molecular weight polygalacturonic acids and their use as vi polysaccharide vaccine Download PDFInfo
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- CN106459225A CN106459225A CN201580023307.6A CN201580023307A CN106459225A CN 106459225 A CN106459225 A CN 106459225A CN 201580023307 A CN201580023307 A CN 201580023307A CN 106459225 A CN106459225 A CN 106459225A
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0045—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Galacturonans, e.g. methyl ester of (alpha-1,4)-linked D-galacturonic acid units, i.e. pectin, or hydrolysis product of methyl ester of alpha-1,4-linked D-galacturonic acid units, i.e. pectinic acid; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/025—Enterobacteriales, e.g. Enterobacter
- A61K39/0275—Salmonella
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
Abstract
The instant disclosure provides O-acetylated high molecular weight polygalacturonic acids or pharmaceutically acceptable salts thereof, having at least one of: (a) a molecular weight greater than 1 x 106 Da; (b) a degree of methylation less than about 10% per mole; and (c) an intervening rhamnose content ranging from about 2% to about 15% per mole, useful as a synthetic immunogenic Vi antigen. The instant disclosure further provides methods of preparing an O-acetylated high molecular weight polygalacturonic acid or pharmaceutically acceptable salt thereof of, pharmaceutical compositions and/or vaccine compositions comprising the same, and methods of immunization using any of the foregoing.
Description
This application claims the rights and interests of the U.S. Provisional Application No. 61/990,493 submitted on May 8th, 2014, it is all interior
Appearance is incorporated herein by.
Technical field
The disclosure relates generally to medical domain, more particularly to microbiology, immunology and vaccine, relates more specifically to hinder
Cold vaccine.
Background of invention
Typhoid fever is a kind of acute and threat to life febrile disease.It is by salmonella typhi (Salmonella
Typhi) cause.According to estimates, annual generation 1600-3300 million new typhoid fever (typhoid fever) case and 500,000-
600,000 death.Contaminated food and water are the main sources infecting.Sanitary condition difference developing country, for example
Asia, Africa and Latin America, the risk highest of infection.
Antibacterial penetrates the mucosal barrier in small intestinal after ingesting.Then, they reach liver and spleen by blood circulation and draw
Play disease.Treat typhoid fever using several different antibiotherapies.However, occurring in that the drug of salmonella typhi
Resistant strain, and make failing to respond to any medical treatment of these costlinesses.Therefore, effectively vaccine remains minimizing typhoid fever shadow to widely use low cost
The most effective way rung.
At present, there is the two kinds of vaccine that can be used for preventing and treating typhoid fever, with the Vi polysaccharide vaccine of single dose parenteral administration
With oral attenuation Ty21a vaccine alive.Vi polysaccharide vaccine is licensed for >=people in 2 years old age, and provide about 70% protection, continue
3 years.Liquid oral live vaccine is licensed for 2 years old people more than age, and gives the protection of about 53-78% after three or four doses.
The live oral vaccine of another kind of capsule form is approved for 5 years old people more than age, and provides similar level after four doses
Protection.
Persistently the high incidences of typhoid fever in many areas, and the increase of Resistant strain and diffusion, lead to WHO 2000
Year recommend typhoid fever be substantive public health issue area with these vaccine immunity school age populations.In the U.S., vaccine is high
Expensive, and mainly give traveller and army personnel.
Vi polysaccharide vaccine is based on Vi capsular polysaccharide (Fig. 1).By variable O- acetylation (60-90% on C3;Szu and
Bystricky,Enzymol.363:552-567 (2003)) and form the cracking protecting bacteria from complement-mediated and phagocytosis
Capsular polysaccharide exactly (α 1-4), 2- deoxidation -2-N- acetyl galactose aldehydic acid.Develop Vi vaccine after studying for many years, final card
Bright Vi polysaccharide is protective antigen, and can be produced without the Vi polysaccharide of degeneration with improved purification process.At present, in the U.S.
There are two kinds of Vi polysaccharide vaccines with Europe, one kind (Typhim being manufactured by Sanofi-Pasteur) and by GSK manufacture another
A kind ofEach vaccine dose is prepared with 25 μ g Vi polysaccharide with as the phenol of preservative, and by intramuscular or
Subcutaneous injection is applied.Recommend every two years vaccination again in the U.S..Vi polysaccharide vaccine also in other countries, including India and China
Manufacture.
These vaccines pass through the large scale fermentation of wild type Salmonella typhimurium bacteria strain Ty2 and sink from culture supernatants
Shallow lake polysaccharide, then carries out further Downstream purification stages (product inset, TyphimWith) producing.Raw
Product technology is in 20 century 70s and the exploitation eighties in 20th century.Because salmonella typhi is gram negative bacteria,
By endotoxin or lipopolysaccharide (cell-wall component of gram negative bacteria) the pollution always potential security risk to Vi vaccine.
The Vi vaccine similar to the vaccine based on polysaccharide for the great majority is T independent antigen, and in vaccination again
When do not cause reinforcement or memory response (WHO/IVB/12.02).It is invalid to the baby below 2 years old age or child.Therefore, pass through
The covalent attachment exploitation Vi polysaccharide-protein conjugate vaccine with protein carrier for the Vi polysaccharide.Conjugate vaccine is that T dependency resists
Former, and in vaccination again, there is reinfocing effect.
O- acetylation and molecular weight are the immunogenic key determinant of Vi polysaccharide.Research is it has been shown that on C3
The removing of O- acetyl group reduces its immunogenicity.Jarvis et al.,J.Bacteriol.94:1406-1410(1967);
Szewczyk and Taylor,Infect.Immun.29:539–544(1980);Szu et al.,Infect.Immun.59:
4555–4561(1991);Rijpkema et al.,Biologicals.32:11-6(2004).On structural modeling display C3
Large volume nonpolar O- acetyl group by both sides in a row project and constitute the most surfaces of polysaccharide molecule, and carboxyl and
N- acetyl group (in C2) group is multi-embedding greatly or is located near axle.Szu et al.,Infect.Immun.59:4555–4561
(1991).This is that dominant immune immunogenic determinant is consistent with O- acetyl group.The acetylizad amount of O- is expressed as O- degree of acetylation
(ratio [moles/mole] of DOAc, O- acetyl group/Gal UA) or acetyl content (μm ole)/mg polysaccharide.N- second at C2
Acyl group also plays an important role in immunogenicity.
Research also shows, the immunogenicity of Vi polysaccharide reduces when its molecular weight reduces.Martin et al.,
J.Bacteriol.94:1411–1416(1967);Szu et al.,Infect.Immun.59:4555–4561(1991).This
Consistent with the discovery being obtained with model polysaccharide antigen glucosan B512 (dx), the immunogenicity indicating this polysaccharide antigen is with molecular weight
Reduction and reduce.Gonzalez-Fernandez et al.,Vaccine.26:292-300(2008).
O- acetyl content and molecular weight are the efficacy measures of current Vi vaccine (product inset, Typhim Vi).Effectively
The production of Vi polysaccharide vaccine depends on the preservation of Vi polysaccharide structures.Produce effective Vi polysaccharide many early stages attempt unsuccessfully, because
Degrade in purge process for polysaccharide.
Plant pectin shares identical main chain with Vi polysaccharide.They are the polygalacturonics that variable methylated α 1-4 connects
Sour (PGA).Those from Fructus Mali pumilae and citrus fruit are used most widely in food industry.The methylating by methanol of pectin
Make the carboxyl esterification in galacturonic acid residues and natural generation.Have<The pectin of 50% degree of methylation (DM) is defined as
Low-methoxy (LM) pectin, and have>The pectin of 50% DM is high methoxyl (HM) pectin.DM is less than 10% LM pectin quilt
It is considered PGA.As final product, PGA is generally prepared with sodium-salt form or polygalacturonic acid na form.Therefore, term is " poly-
Galacturonic acid " is herein used interchangeably with " Polygalacturonate ".LM pectin and PGA generally pass through in alkaline pH bar
Under part, the demethylation of HM pectin obtains.Although these conditions eliminate methyl, their always scissionable polymers main chains, this
Molecular weight is led to reduce.
Business LM pectin or PGA by O- acetylation to make great efforts to analyze the Vi polysaccharide epidemic disease of the antigenicity of Vi polysaccharide and synthesis
The generation of Seedling.Find that gained acetylate and natural Vi polysaccharide have identical antigenicity.Szewczyk and Taylor,
Infect.Immun.29:539–544(1980);Szu et al.,Infect.Immun.62:5545–5549(1994).So
And, it is not immunogenic in animal.Szu et al.,Infect.Immun.62:5545–5549(1994).This attribution
In with 1-2x106The natural Vi polysaccharide of Da is compared, low-molecular-weight (the about 4x10 of the business LM pectin being used5Da).Ibid.Many
The immunogenicity of carbohydrate antigen is proved with other polysaccharide antigens to the dependency of molecular weight.Gonzalez-Fernandez et
al.,Vaccine.26:292-300(2008)..However, once being conjugated with protein carrier, O- acetylation LM pectin is immunity
Originality although its antibody response level be far below Vi polysaccharide-protein conjugate level.Szu et al.,
Infect.Immun.62:5545–5549 1994);Kossaczka et al.,Infect.Immun.67:5806-5810
(1997).Accordingly, it would be desirable to exploitation for typhoid fever new synthesis Vi polysaccharide vaccine, it is immunogenic, in addition not with egg
In the case that white matter carrier is conjugated.
Summary of the invention
Present disclose provides can be used as the O- acetylation high molecular polygalacturonic acid of the immunogenicity Vi antigen of synthesis
(OAc-HPGA) or its pharmaceutically acceptable salt, its have following at least one:A () is more than 1x106The molecular weight of Da;B () is often rubbed
You are less than about 10% methylation;(c) scope is every mole about 2% to about 15% of Fructus rhamni (Rhamnus davurica Pall.) sugared content between two parties
(intervening rhamnose content).The disclosure further provide preparation O- acetylation Poly Gal A Galacturonan or
The method of its pharmaceutically acceptable salt, is comprised its pharmaceutical composition and/or vaccine combination, and is entered using any aforementioned substances
The immunization method of row.
Pectin from different plant origins has different chemically and physically characteristics.From aloe vera (Aloe vera
High molecular polygalacturonic acid (HPGA) L.) (Including its pharmaceutically acceptable salt) as U.S. Patent number
Identified described in 5,929,051,7,705,135 and 7,691,986 (they are incorporated by reference into) and manufactured.Aloe vera is
A kind of plant being extensively planted in world subtropical and tropical zones.HPGA has the feature of uniqueness.It has natural low first
Epoxide content (" DM "<10%), very high molecular weight (>1x106Da) and high Gal UA (sodium salt) content (>90%).
The chemical and physical features of HPGA are summarized in Table 1.HPGA is dissolved in water, but directly in saline or buffer saline (150mM
NaCl dissolve poor in).As LM pectin, it can form gel with calcium ion.When mixing with calcium, occur solidifying immediately
Gel.HPGA generally has>99% purity and the neutral sugar containing minimum and protein (<0.5%).These are superior
HPGA is made a distinction by chemical and physical features with all other existing pectin or PGA.
As defined herein, HPGA is the high molecular polygalacturonic acid with least one following characteristics:(1) it is more than
1x106The molecular weight of Da, (2) less than about 10%/mole methylation, and (3) scope is every mole about 2% to about 15%
Fructus rhamni (Rhamnus davurica Pall.) sugared content between two parties.
As defined herein, OAc-HPGA is that the O- with least one following characteristics is acylated high molecular polygalacturonic
Acid:(1) it is more than 1x106The molecular weight of Da, (2) less than about 10%/mole methylation, and (3) scope be every mole about
2% to about 15% Fructus rhamni (Rhamnus davurica Pall.) sugared content between two parties.
In some embodiments of the disclosure, HPGA isIt is from aloe vera under cGMP
Plant manufactures.This manufacture method includes mulch material, extracts, and clarification is filtered with 0.2 μm, followed by as United States Patent (USP) 7,
The purification step describing in detail in 691,986.Final product is dry matter.Commercially, the HPGA of gained is also referred to asPolymer.On EDMF (DMF) submitted to FDA in 2005, and update once new every year
Product information.
Present disclose provides synthesize and immunogenic Vi antigen, it can serve as new antityphoid vaccine, in safety,
Effectiveness with become present aspect there is the remarkable advantage better than existing Vi vaccine.The Vi antigen of synthesis, the acetylizad high molecular of O-
Polygalacturonic acid (OAc-HPGA, such as GelSite-OAcTM) or its pharmaceutically acceptable salt pass through the poly- gala of novel high polymer amount
Alduronic acid (HPGA, for example,) O acetylation produce.HPGA new property discussed above becomes by O- second
The preferable substrate of the Vi polysaccharide analogs of acylated synthesis.
OAc-HPGA, such as GelSite-OAcTMBy chemical method with very high yield (weight yield>100%) produce
Raw.It is in O- acetyl group/Gal UA [moles/mole] (at C2 or C3 position>100% or>50% (moles/mole)) ratio
In rate or every mg polysaccharide acetyl content (μm ole) (>4.6 μm of ole/mg) on there is height O- acetylation (DOAc) and height
Molecular weight (>1x106Da), therefore exceeded the effect specification of current Vi vaccine.GelSite-OAcTMTo Vi polysaccharide have similar
Or substantially the same antigenicity, and itself has high degree of immunogenicity.Importantly, this synthetic antigen is with lethal
It is protection completely in the animal that the salmonella typhi alive of dosage is attacked.Additionally, OAc-HPGA has reinfocing effect or memory
Immunne response, exhibits more than 2 times of antibody titer rising in second immunity.This is very unique in polysaccharide antigen
, and potentially make it effective in the people below 2 years old age in the case of not being conjugated with protein carrier.
Therefore, the purpose of the disclosure is to provide to have following at least one O- acetylation high molecular polygalacturonic acid
Or its pharmaceutically acceptable salt (OAc-HPGA):
A () is more than 1x106The molecular weight of Da,
B the methylation that every mole less than about 10% of (), and
C () scope is every mole about 2% to about 15% of Fructus rhamni (Rhamnus davurica Pall.) sugared content between two parties.
In some embodiments, OAc-HPGA or its pharmaceutically acceptable salt have more than 1x106The molecular weight of Da.
In some embodiments, the O- degree of acetylation (DOAc) of OAc-HPGA or its pharmaceutically acceptable salt is in C2 or C3
It is more than 100% at position, or be more than 50%.
In some embodiments, OAc-HPGA has more than 1x106The molecular weight of Da, less than about 10%/mole first
Base degree, and it is more than 100% O- degree of acetylation at C2 or C3 position, or the O- degree of acetylation more than 50%.
In some embodiments, OAc-HPGA is the immunogenicity Vi antigen of synthesis.
In some embodiments, OAc-HPGA or its pharmaceutically acceptable salt have substantially the same with Vi polysaccharide vaccine
Antigenicity and be immunogenic.
In some embodiments, OAc-HPGA or its pharmaceutically acceptable salt induction of antibodies titre in second immunity
2 times or more large rising.
In some embodiments, OAc-HPGA or its pharmaceutically acceptable salt be not in the case of being conjugated with protein carrier
In the people below 2 years old age, the immunogenicity Vi antigen as synthesis is effective.
In some embodiments, OAc-HPGA or its pharmaceutically acceptable salt are effective as the vaccine for typhoid fever.
The disclosure further provides the method producing OAc-HPGA, and it mixes the peculiar property of the HPGA of the disclosure with letter
Change method simultaneously guarantees high-recovery or yield.Can from the parent material based on plant (for example) a large amount of productions
OAc-HPGA (for example, GelSite-OAcTM).
Therefore, another object of the present disclosure is to provide O- acetylizad high molecular polygalacturonic acid (OAc-HPGA)
Or its pharmaceutically acceptable salt, it makes high molecular polygalacturonic acid (HPGA) or its salt and acetic acid and acetic anhydride by inclusion
The method of mixture reaction is formed, and wherein HPGA has following at least one:
A () is more than 1x106The molecular weight of Da,
B the methylation that every mole less than about 10% of (), and
C () scope is every mole about 2% to about 15% of Fructus rhamni (Rhamnus davurica Pall.) sugared content between two parties.
Another object of the present disclosure is to provide the method preparing OAc-HPGA or its pharmaceutically acceptable salt, including making high score
Son amount polygalacturonic acid (HPGA) or the mixture reaction of the acid of its salt and acetic acid and acetic anhydride, wherein HPGA have following extremely
Few one kind:
A () is more than 1x106The molecular weight of Da,
B the methylation that every mole less than about 10% of (), and
C () scope is every mole about 2% to about 15% of Fructus rhamni (Rhamnus davurica Pall.) sugared content between two parties.
In some embodiments, the method be additionally included in the mixture reaction of acetic acid and acetic anhydride before with HPGA or
Its salt formation acid gel.
In some embodiments, the method also includes making HPGA or its salt react with perchloric acid.
In some embodiments, the HPGA using in the method is derived from aloe vera.
Another object of the present disclosure is the OAc-HPGA or its providing and passing through the method formation according to any methods described
Pharmaceutically acceptable salt.
The drug regimen that another object is that OAc-HPGA that offer comprises the disclosure or its pharmaceutically acceptable salt of the disclosure
Thing, it optionally comprises at least one pharmaceutical acceptable excipient.
In some embodiments, pharmaceutical composition also comprises protein carrier.
In some embodiments, protein carrier and OAc-HPGA are conjugated.
Another object of the present disclosure is the method providing for typhoid fever immunized subject, including to experimenter in need
Apply OAc-HPGA or its pharmaceutically acceptable salt of effective dose.
In some embodiments, the disclosure additionally provides by applying the many polygalacturonics of O- acetylation to animal or people
Sour (OAc-HPGA) method to induce the protective immune response for salmonella typhi.For example, present disclose provides pin
Method to salmonella typhi and/or typhoid fever immunized subject, including the basis applying effective dose to experimenter in need
The OAc-HPGA of any aforementioned item of the disclosure or its pharmaceutically acceptable salt or pharmaceutical composition.
In some embodiments, the administration induction of antibodies response of pharmaceutical composition.
In some embodiments, 2 times of induction of antibodies titre or more when being applied in second immunity of pharmaceutical composition
Big rising.
The purpose of the disclosure is the purposes providing OAc-HPGA or its pharmaceutically acceptable salt or pharmaceutical composition, and it is used as tool
There are the antigenicity substantially the same with Vi polysaccharide vaccine and the immunogenicity Vi antigen for immunogenic synthesis.
Another object of the present disclosure is to provide OAc-HPGA or its pharmaceutically acceptable salt or pharmaceutical composition to use in preparation
Purposes in the medicine for the vaccination of typhoid fever.
The disclosure additionally provides with optionally comprising being prepared with OAc-HPGA of at least one pharmaceutical acceptable excipient
Antityphoid vaccine.OAc-HPGA can be optionally conjugated with protein carrier, potentially to further enhance its immunogenicity.
1kg OAc-HPGA can be prepared 2000-4000 ten thousand vaccinating agent effectively with 25-50 μ g/ dosage.It is to be based on
Plant, and therefore do not contain endotoxin (lipopolysaccharide, LPS), it is from gram negative bacteria, such as salmonella typhi purification
Vaccine in the most dangerous pollutant.Compared with existing Vi polysaccharide vaccine, OAc-HPGA can be much more secure, less expensive
With more effective vaccine.Economic advantages also make in the whole world, particularly expand typhoid fever epidemic disease in the Prevalent district of developing country
The production of Seedling and use become easier to and more afford.
Brief description
Fig. 1 shows Vi polysaccharide, and HPGA is (for example) and OAc-HPGA (GelSite-OAcTM) structure.Aobvious
Vi polysaccharide, alkaline galacturonic acid (Gal UA) residue of HPGA and OAc-HPGA are shown.
Fig. 2 shows for producing OAc-HPGA (GelSite-OAcTM) O- acetylation process.Asterisk represents optionally to be made
With a small amount of perchloric acid as catalyst.
Fig. 3 showsAnd GelSite-OAcTMSize exclusion chromatography spectrum together with dextran standard.Make
Dextran standard is 1.597,214.8 and 39.9kDa.
Fig. 4 shows O- acetylation Poly Gal A Galacturonan (GelSite-OAcTM) antigenicity, such as survey in immunodiffusion
Test in fixed.Every hole accepts 20 μ l polysaccharide (200 μ g/ml) or reference serum (centre bore).Agarose plate is kept in moist chamber
Overnight.It is full of hole as follows:1)2) O- acetylation polygalacturonic acid 1x;3) O- acetylation Poly Gal A Galacturonan
2x;4) O- acetylation Poly Gal A Galacturonan 3x;And C) Vi polysaccharide.
Fig. 5 shows DOAc to immunne response GelSite-OAcTMAny effect.With 2.5 μ g/ mices with having difference
The Vi vaccine of DOAc or GelSite-OAcTMImmune Balb/c mice twice, is spaced 4 weeks.Using Vi polysaccharide (A) or GelSite-
OAcTM(B) measure specific IgG as antigen.
Fig. 6 shows GelSite-OAcTMAny dose dependent effect.Use GelSite-OAcTM(DOAc, 1.75)
With different dose indicating immunity Balb/c mices twice, it is spaced 6 weeks.Vi polysaccharide (A) or GelSite-OAc (B) conduct are used
Antigen, measures specific IgG by ELISA.
Fig. 7 shows any cross reactivity.From different seminar combining anteserum sample with(A),
GelSite-OAcTM(B) or Vi polysaccharide (C) reaction.
Fig. 8 shows intersection reinfocing effect.Using Vi polysaccharide (A) or GelSite-OAcTM(B) measurement specific IgG resists
Body.
Fig. 9 shows that IgG subclass is distributed.Measure the specific IgG antibodies of different subclass by ELISA.True by terminal
Fixed (higher than 2 times of background, >=0.2OD) titre.With Vi polysaccharide (A) or GelSite-OAcTM(B) measure antibody as antigen.
Figure 10 shows the protection for the lethal challenge being carried out with the salmonella typhi that lives.With 2.5 μ g/ mices with having
The Vi vaccine of different DOAc or GelSite-OAcTMImmune Balb/c mice twice, is spaced 4 weeks.The 2nd week after second immunity,
Attack animal with the salmonella typhi of 100LD 50.A, % are survived;B, average weight.
Figure 11 shows DOAc and GelSite-OAcTMImmunogenic dependency.With having different DOAc's
GelSite-OAcTMWith 2.5 μ g/ mouse immune Balb/c mice (n=6) twice, it is spaced 4 weeks.Using Vi polysaccharide (A) or
GelSite-OAcTM(B) as the antigen measurement single blood serum sample that the 2nd week (w2) collects after for the first time and second immunity
In specific IgG.
Figure 12 shows DOAc and GelSite-OAcTMProtection dependency.With having the GelSite- of different DOAc
OAc TM with 2.5 μ g/ mouse immune Balb/c mice (n=6) twice, is spaced 4 weeks.Use 100LD after second immunity within 3rd week
50 salmonella typhi attacks animal.A, % are survived;B, average weight.
Detailed Description Of The Invention
The method that O- acetylation process is described based on Schweiger (1964).Due to its high molecular, HPGA is (for example) forming gel under low pH (about pH2.0), this eliminates the needs preparing Ca-deposit, and it is served as reasons
Schweiger,J.Org.Chem.29:The initial step of 2973 2975 (1964) methods describing.This allows with solid or pearl
The HPGA of form is (for example) acetylation, described solid or pearl form allow to be easily processed in acetylation and
Reclaim.At the end of acetylization reaction, dissolve OAc-HPGA pearl by acid pH is increased to neutral pH.Letter due to method
Unisexuality and the addition of acetyl group, acetylation is efficient, yield>100%.
However, acetylation can be realized by other methods, including by Carson and Maclay,
J.Am.Chem.Soc.68:The method that 1015-1017 (1946) describes.
Term " O- acetylation " or " O- is acetylizad " refer to add acetyl group at C2 the and C3 position of saccharide residue.When every
When C2 the and C3 position of individual saccharide residue is all acetylation, reach the O- degree of acetylation (200%) of maximum.
OAc-HPGA (the such as GelSite-OAc of gainedTM) have at least 130% or 100% at C2 or C3 position, or
At least 65% or 50% O- degree of acetylation (DOAc), and high molecular (>1.0x106Da), be therefore very similar to natural
Vi polysaccharide (at C3 60-90%O- acetylation and 1-2x106Da).By extending the response time, can easily DOAc be carried
Up to >=175%.Due to adding acetyl group, OAc-HPGA also has the molecular weight of increase.
The efficacy measures of the Vi vaccine of currently acquired license be O- acetyl content (>=2 μm of ol O- acetyl group [in C3]/
Mg) and Vi polysaccharide molecular weight (50% >=2.5x104The polysaccharide of Da) (the WHO biological standard Committee of Experts, 1993).
Therefore, OAc-HPGA readily satisfy and exceed current Vi vaccine (>2.5 μm of ol O- acetyl group/mg and 70% at C2 or C3
≥1x106The polysaccharide of Da, table 2 and Fig. 3) two kinds of efficacy measures.
With HPGA (for example) different, OAc-HPGA (for example, GelSite OAcTM) no longer form gel with calcium.
Also different from HPGA, OAc-HPGA can be directly dissolved in saline or buffer saline (150mM NaCl).These results indicate
O- acetylation changes chemistry and the functional character substantially of HPGA, and products therefrom OAc-HPGA is new chemical entities.
As with reference to shown in anti-V1 polysaccharide serum, OAc-HPGA is antigenic (Fig. 4).It has and Vi polysaccharide identical
Antigenicity.Importantly, there is no the reaction with HPGA, instruction OAc-HPGA is not intersected with its not acetylizad parent molecules
Reaction.
OAc-HPGA, such as GelSite-OAcTM, it is immunogenic (Fig. 5) in mice.Can be by ELISA Vi
Polysaccharide or OAc-HPGA are as Detection of antigen specific antibody.In the past, found that acetylation LM pectin was not immunogen in mice
Property, this is owing to its low-molecular-weight (4x10 compared with natural Vi polysaccharide5Da).Szu et al.Infect.Immun.62:
5545–5549(1994).OAc-HPGA have much higher molecular weight (>1x106Da), it may partly cause its immunogen
Property.Again, do not observe the cross reactivity with its parent molecules (HPGA), this instruction is resisted by the specificity that OAc-HPGA induces
Body acupuncture is to O acetyl group, the immunogenic determinants of the most critical of Vi polypeptide.
Significantly, OAc-HPGA has reinfocing effect or anamnestic immune response, exhibits more than 2 times in second immunity
Antibody titer increase.Vi vaccine (Typhim for concurrent testing), this reinfocing effect is not observed.Known many
Carbohydrate antigen is T dependent/non-dependent, and lacks immunological memory or reinfocing effect.Polysaccharide antigen is only used in memory or booster immunization response
Realize, described polysaccharide antigen passes through to obtain conjugated to they and protein carrier.Therefore, OAc-HPGA do not have any conjugated
In the case of be highly unique in owned reinfocing effect, this is likely due to its new chemical property.Additionally, not only using
GelSite-OAcTM, and using Vi vaccine as second dose of inducible reinfocing effect.This further demonstrates that OAc-HPGA and Vi
Structural similarity between polysaccharide.This reinfocing effect potentially makes it at 2 years old in the case of not being conjugated with protein carrier
In people below age effectively.
Term " reinfocing effect " is used interchangeably with " anamnestic immune response ", refers in second immunity or vaccination again
Afterwards, about 2 times of immunne response increase.
OAc-HPGA is protection completely in the animal attacked with the salmonella typhi alive of fatal dose, and instruction is by it
The immunne response of induction is protection for salmonella typhi.In a word, these find instruction, and OAc-HPGA can potential be opened
Send out as new Typhoid Vi Vaccine, compared with current Vi vaccine, manufacturing, cost, anamnestic response and not with protein-conjugate
In the case of there is obvious advantage in terms of likely effectiveness in people below 2 years old.OAc-HPGA can be using from plant
The abundant high-quality HPGA in thing source is produced in a large number by simple chemical method.Can be by every kg OAc-HPGA with every dose of 25-
50 μ g polysaccharide prepare 2000-4000 ten thousand vaccinating agent effectively.As plant origin, OAc-HPGA does not contain endotoxin or LPS, its
It is the cell-wall component of gram negative bacteria such as salmonella typhi, and be the most dangerous from the vaccine of these bacteria purifications
Pollutant.Therefore, this synthetic vaccine can be more safer and less expensive than current license Vi vaccine.Economic advantages make
In the whole world, particularly expand the production of antityphoid vaccine in the Prevalent district of developing country and use becomes easier to and more negative
Carry on a shoulder pole and must rise.Additionally, OAc-HPGA can be used for develop conjugate vaccines, its can even more have immunogenicity and to 2 years old age below
Child has protective effect.
Embodiment
Embodiment 1:The O- acetylation of polymer
Assess and be used forThe acetylizad two kinds of different methods of O-:One kind is by Carson and Maclay
(1946) describe, and another kind is described by Schweiger (1964).Both approaches can be byAcetylation.Send out
Existing Schweiger method more effectively and is better suited forPeculiar property and be used for obtaining O- acetylate.
Schweiger method does not use Methanamide or pyridine.Replace, it is used a small amount of perchloric acid as catalyst.Additionally, it is logical
The whole process crossing Schweiger method is carried out at room temperature.Therefore, Schweiger method changing is selected to be used forAcetylation.
Method.Include 6 simple steps (Fig. 2) from the method for Schweiger method modification.With original
Schweiger method is compared, and it passes throughTwo kinds of heterogeneitys (acid cure gel and its sour form in water not
Dissolubility) greatly simplify.The first step be by the HCl of dilution then rinse substrate in glacial acetic acid orFrom
Sodium-salt form changes into its sour form.One of new property be its gelation effectively at a low ph, formed permissible
The strong acid gel beads of tolerance downstream procedures or stock (strand).Original Schweiger method is made first before acid pickling step
Standby calcium deposit.Therefore, forO- acetylation, this is optional, and is eliminated.After acetylation, go from
Detergent gel pearl or stock in sub- water, to remove acetylation reagent in the absence of any losses, because the acetyl of sour form
ChangeWater insoluble.After washing, then by with NaOH and acetylizad to dissolveIn described
It is converted into sodium salt with by it from acid.In addition to last drying steps, whole process can complete in one day.
The all reagent using are all obtained from Sigma Chemical Co. (St.Louis, MO).It is briefly described below based on 1
The basic process of gram quantity level.
Water will be dissolved inSolution (200ml, 5mg/ml;1 gram altogether) instill in 1 liter of 0.1M HCl to produceGel beads.Reclaim gel beads, and washing three times in 100ml acetic acid, 15 minutes every time.Gel beads are suspended in
200ml acetic acid/acetic anhydride (1:1) in mixture.During churning, it is gradually added into 2ml perchloric acid (70%) in 2 hours, its
In at 0,30 minute, 0.5ml was added with 0.1ml part in 60 minutes and 90 minutes.Then stainless steel filter is used to return at 120 minutes
Receive gel beads, and fully washed with water.They are suspended in 300ml water and molten to about 7 by adjusting pH with 0.1M NaOH
Solution, then with ethanol precipitation and be dried under vacuum.
Method is evaluated.The method is evaluated already in connection with the key parameter including yield and DOAc.
1) yield.Weight yield is consistent to be higher than 100%.Such high yield ensure that effective production, and makes raw material and examination
Using of agent maximizes.It is consistent with following facts:Acetyl group is added to increaseMolecular weight, and the method is permissible
Carried out with minimum loss.The diameter of acid gel beadlet is at least 2mm, and can easily reclaim in acetylation.
2) O- degree of acetylation (DOAc).DOAc is GelSite-OAcTMKey parameter.Acetylation is efficient,
Produce after only one wheel reaction>130% DOAc.DOAc can pass through the persistent period of acetylization reaction and the second being used
The concentration of anhydride is controlling.With the increase in response time, keep the 30 minutes intervals of identical adding perchloric acid, DOAc simultaneously
Can increase further.Obtain the DOAc of up to 175% or 7.5 μm of ole/mg.When C2 and C3 site is all completely by acetyl
During change, maximum DOAc is 200%.On the other hand, by the concentration of acetic anhydride being reduced to about the 10% of reactant mixture, also may be used
To obtain relatively low DOAc (such as 80%), therefore allow to produce the GelSite-OAc with even broader scope DOAcTM, with
For evaluating.
3) molecular weight.After acetylation, molecular weight unanimously increases (table 2 and Fig. 3).This with toAdd acetyl group one
Cause, and indicate that the method does not cause parent moleculesDegraded, thereby, it is ensured that retain final product GelSite-
OAcTMHigh molecular.
4) acid cure gel.Forming strong acid gel in the HCl of dilution is important for the efficiency of acetylation.Observe
Business PGA to low-molecular-weight is only capable of forming soft gel, its disintegrate during downstream process.Therefore,Macromolecule
Amount is most important for method efficiency.
5) perchloric acid.Cause acetylation by adding a small amount of perchloric acid to be used as catalyst.Find 20% perchloric acid and 70%
Perchloric acid is equally effective.Additionally, have 1 gram discussed aboveReactant mixture in, only using 0.125ml
20% perchloric acid can be readily available high DOAc (>100%).Moreover, it has been found that when use under same amount of perchloric acid
When, 1% perchloric acid being dissolved in acetic acid is equally effective.70% perchloric acid is strong acid.Therefore, made using the perchloric acid of 1% or 20%
Using of hazardous agents minimizes, and improves method security.
6) it is dried.At the end of the method, GelSite-OAc can be easily dried by lyophilizationTM, therefore eliminate
The use of ethanol precipitation.
Embodiment 2:The sign of acetylation GelSite polymer
1. molecular weight
Measure GelSite-OAc using the HPLC SEC (size exclusion chromatography) with dextran standardTMMolecule
Amount.Now commonly used more advanced HPLC-MALLS (multiple angle laser light scattering) method measures the molecule of polymer such as polysaccharide
Amount.However, using this HPLC SEC with dextran standard, because it is most widely used for studying in disclosed document
Vi polysaccharide, and for the measurement of Vi vaccine potency, therefore allow result directly comparable with those being previously reported by.In short, string
Connection uses Shodex OH pak SB-806HQ (300x8mm), Shodex OHpak SB-805HQ (300x8mm) and Shodex
OHpak SB-804HQ (300x8mm) post, with 0.1M ammonium acetate and 200ppm Hydrazoic acid,sodium salt as mobile phase, flow velocity is 0.5ml/
min.As compared to dextran standard (Phenomenex broad MWD, 8 kinds of standard substance, 10-200kDa) it was recently reported that peak maximum
Molecular weight values (Fig. 3).In the batch of all inspections, find GelSite-OAcTMMolecular weight with initial's
Molecular weight same or higher (>2x106Da) (table 2).This is consistent with by adding acetyl group to increase molecular weight.
2.O- degree of acetylation (DOAc)
Using by Hestrin, J.Biol.Chem.180:The method that 249-261 (1949) describes, is wherein modified.
All reagent are all obtained from Sigma Chemical Co (St.Louis, MO), including acetylcholine, oxammonium hydrochloride. and iron chloride.?
It is measured method in 96 orifice plates.With the duplicate test sample of about 0.2mg/ml (w/v).Acetylcholine is used as standard substance.
DOAc is expressed as mol ratio or the percentage ratio of acetyl group and Gal UA residue, or μm ole/mg- is used for the DOAc of current Vi vaccine
The unit of specification.Gal UA content is based onProduct discharges test result.
Based on above-mentioned initial methods condition, obtain the GelSite-OAc of the DOAc with 134%-153%TM, it corresponds to
67%-76% (table 2) at C2 or C3 position.DOAc can also represent (table 2) based on a μm ole/mg.By acetylation is anti-
Answer time lengthening, DOAc up to 175% or at C2 or C3 position 87.5% can be improved further, close to theoretical maximum
Value 200%,.On the other hand, the concentration by adjusting reaction condition or reduce acetic anhydride in reactant mixture, it is also possible to obtain relatively
Low DOAc such as 80% (embodiment 5).
3. calcium gelation
By inciting somebody to actionAnd GelSite-OAcTMSolution mixes to measure with calcium solutionWith
GelSite-OAcTMThe ability of gelation in the presence of calcium.Therefore, will about 0.2% (w/v) sample solution (2-3ml) and 0.5-
1% calcium chloride solution of 1ml directly mixes.Solution forms gel immediately, and uses GelSite-OAcTMDo not occur
Gel formation.Gel is become by whole sample solution and it is no longer able to free-flowing and indicates gel formation.
GelSite-OAcTMSolution remains the solution of free-flowing with calcium chloride after mixing.
Additionally, withDifference, GelSite-OAcTMSaline or buffer saline (150mM can be directly dissolved in
NaCl in).The instruction O- acetylation of these results changesChemistry and functional character substantially, and products therefrom
GelSite-OAcTMIt is new chemical entities.
4. antigenicity
As Szu et al.Infect.Immun.62:5545 5549 (1994) is described, carries out immunity in 1% agarose
Diffusion measurement.From the reference Vi polysaccharide antigen of citrobacter freundii (Citrobacter freundii) with for typhoid fever sand
The reference donkey serum of door Salmonella is available from National Institute of Child Health and Human
Development(NICHD).GelSite-OAcTMForm positive deposits line (Fig. 4) with reference serum.The intensity of precipitation line with
The increase of DOAc and increase, as with Carson and Maclay method 1,2 and 3 wheel acetylations after obtain shown in sample (Fig. 4).
GelSite-OAcTMShare identical or substantially the same antigenicity with Vi polysaccharide, such as by from GelSite-OAcTMAnd Vi
The merging of the precipitation line of polysaccharide is proved;Be not observed with not acetylizadReaction (Fig. 4).As herein
Used, term " with Vi polysaccharide identical or essentially identical antigenicity " refers to GelSite-OAcTMWith anti-V1 polysaccharide serum or
Antibody response.For example, as see in Fig. 4 with discussed above, in the immunodiffusion assays of Szu et al., GelSite-OAcTM
Form positive deposits line with reference serum, it measures precipitation line with the immunodiffusion of Vi polysaccharide and merges.
5. the comparison with Vi polysaccharide and vaccine
GelSite-OAcTMHave at C2 or C3 position>130% (5.58 μm/mg) or>The O- of 65% (2.79 μm/mg)
Degree of acetylation (under equally distributed hypothesis between this two O- acetylation sites), and>1x106The molecular weight (table 2) of Da.
Natural Vi polysaccharide has 1-2x106The molecular weight of Da and C3 position 60-90% O- degree of acetylation (Szu and Bystricky,
2003).Therefore, based on DOAc and molecular weight (immunogenic two key determinant), GelSite-OAcTMVery similar
In Vi polysaccharide.
The efficacy measures of current license Vi vaccine be Vi polysaccharide O- acetylation content (>=2 μm of ole/mg [in C3]) and
Molecular weight (50% >=2.5x10 of polysaccharide4Da) (the WHO biological standard Committee of Experts, 1993;Keitel etc., 1994).With
Borrow and easily have at C2 or C3 position>2.5μmol/mg(>65%) DOAc and >=1x106Da molecular weight (for >=
70% polysaccharide) (table 2 and Fig. 3), GelSite-OAcTMMore than two kinds of effect specifications.
Embodiment 3:O- acetylation(GelSite-OAcTM) immunogenicity
Vi vaccine (Typhim with licenseSanofi-Pasteur) compare, check in Balb/c mice
GelSite-OAcTMImmunogenicity.By in right hind intramuscular injection, with specified dosage with 50 μ l PBS
GelSite-OAcTMOr Typhim Vi vaccine immune mouse group (n=10).By animal immune 2 times, it is spaced 4 weeks, and every two weeks
Collect blood serum sample.
As it was previously stated, specific antibody is measured by ELISA in 96 orifice plates.Szu et al.,Enzymol.363:
552-567(2003).It is used as to be coated the antigen of elisa plate from the Vi glycocalix of NICHD and International Vaccine Institute (IVI),
Both known mechanisms in typhoid fever research.Reference serum is the hyperimmune of the 40 ELISA unit/ml obtaining from NICDH
Mice serum.Using CDC ELISA program (http://www.cdc.gov/ncidod/dbmd/bimb/elisa.htm) determine
Antibody titer, wherein with reference to serum as standard substance.
1.DOAc and the impact of antigen dose
The impact of DOAc:Test have different DOAc (136%, 155%, or 175% moles/mole, or 5.6,6.5 or
7.5 μm of ole/mg) three GelSite-OAcTMSample.Result shows, finds level and the DOAc phase of specific IgG response
Close:DOAc is higher, immunne response higher (Fig. 5).This is especially true after immunity for the first time, either using Vi antigen (Fig. 5 A)
Or synthetic antigen GelSite-OAcTM(Fig. 5 B) measures specific IgG.There is the GelSite- of minimum DOAc (136%)
OAcTMUnanimously produce minimum titre in most of time point.There is 155% or 175%DOAc GelSite-OAcTMSample
Show closely similar reaction level, point out the GelSite-OAc with 155% or 175%DOAcTMCan be equally effective.Logical
Cross and evaluate GelSite-OAc with as little as 80% DOAcTM, prove this DOAc dependency impact in immunne response further
(embodiment 5).
The impact of antigen dose:Also using the GelSite-OAc with 175%DOAcTM, in four kinds of various dose levels
GelSite-OAc is tested under (1,2.5,5 and 10 μ g)TMThe impact of antigen dose.With Vi or GelSite-OAcTMThe antibody of measurement
Response is that antigen dose is dependent, particularly after second immunity (Fig. 6).
Comparison with Vi vaccine:Compared with Vi vaccine, there is 155% or 175%DOAc GelSite-OAcTMAs one man
Induce equal or higher antibody titer, except after immunity for the first time and in addition to the time point when measure with Vi antigen (Fig. 5 with
Fig. 6).When with GelSite-OAcTMDuring antigen measurement, by GelSite-OAcTMThe Specific antibody titre of induction is than by Vi vaccine
Up to 10 times of the Specific antibody titre height of induction.This is likely due in GelSite-OAcTMIn the case of exist higher
DOAc or more acetyl group.In a word, these results indicate when compared with Vi vaccine, GelSite-OAcTMIt is that hyperimmunization is former
Property.
2. cross reactivity
By GelSite-OAcTMInduction antibody specificity by with its parent moleculesReaction is many with Vi
Sugar and GelSite-OAcTMCompare assessment.The antigen-agent that the combining anteserum sample that 8th week collects after second immunity is carried out
The result of amount dependency experiment (Fig. 6) shows in the figure 7.With 1:40 low initial dilution test sera sample, and test
In also include the reference serum obtaining from NICDH (with 1:2000 initial dilution).In GelSite-OAcTMAny dosage water
All be not observed under flat withReaction (Fig. 7 A), and be directed to GelSite-OAcTMObtain the anti-of high saturated level
Answer (Fig. 7 B).These results are consistent with the result being obtained by above-mentioned immunodiffusion.They indicate, for GelSite-
OAcTMThe reaction producing is directed to GelSite-OAcTMOn O- Acetyl Groups.This is by using having different DOAc
(134%-175%) GelSite-OAc TM as antigen ELISA in be further characterized by, its show antibody response level
Related to DOAc.
3. reinfocing effect
In all zooperies carrying out, in second immunity, use GelSite-OAcTMIt is consistently observed uniqueness
Reinfocing effect or anamnestic immune response.As it can be seen in figures 5 and 6, compared with the highest titre after for the first time immunity, for Vi or
GelSite-OAcTMAll three GelSite-OAc of antigen measurementTMThe titre of group increases at least 2 times after second immunity
And up to>4 times, and those becoming than Vi vaccine are much higher.Such reinfocing effect is not observed with Vi vaccine.With having
The GelSite-OAc of the DOAc of wide scopeTMThe evaluation further carrying out shows, the DOAc as little as 100% can obtain enhancing
Effect and completely protection (embodiment 5).
In order to be further characterized by this new reinfocing effect, carry out intersecting and strengthen experiment, wherein exempted from a kind of antigen first
Epidemic disease animal, then carries out booster immunization (Fig. 8) twice with identical or different antigen.Result shows, uses GelSite-OAcTMDraw
The all animals sending out are with GelSite-OAcTMOr Vi vaccine carries out showing reinfocing effect (Fig. 8) during second immunity.When with
During the measurement of Vi polysaccharide, it is higher than to use GelSite-OAc with the reinforcement level that Vi vaccine obtainsTMThe reinforcement level (Fig. 8 A) obtaining.This
The additive effect that the reinforcement based on O- acetyl group can be reflected and induce the antibody of other parts for Vi polysaccharide.
The animal only being caused with Vi vaccine and strengthening does not show reinfocing effect.Caused with Vi vaccine and use GelSite-
OAcTMThose strengthened show reinfocing effect really although degree is less, and only ought measure for GelSite-
OAcTMAntibody when just can detect that (Fig. 8 B).Further reinfocing effect is not observed in third time immunity.These knots
Fruit show together, can occur with Vi polysaccharide intersect reinforcement, particularly work as GelSite-OAcTMDuring for causing immunity, therefore
It is further characterized by GelSite-OAcTMNew reinfocing effect and its structural similarity with Vi polysaccharide.
4.IgG subclass
GelSite-OAcTMUnique reinfocing effect point out its be probably T dependence antigen.T- dependent immune response
One of feature be in the case of polysaccharide conjugates vaccine subclass after booster immunization or compared with holosaccharide antigen change or
Conversion.Therefore, the combining anteserum sample collected for the 2nd week after for the first time and second immunity is used for checking IgG by ELISA
Subclass (IgG1, IgG2a, IgG2b, IgG3) and the distribution of IgM.Injection GelSite-OAcTMAnimal once or twice immunity
After represent high-caliber IgG1 (Fig. 9).After second immunity, the titre of all IgG increases >=2 times.After second immunity,
Observe the bigger increase of the titre of IgG1, IgG2a and IgG2b, wherein IgG2a shows highest amplitude titre increases (figure
9).The spectrum prompting of this IgG subclass, the T cell activation of aTh2- preference can participate in for GelSite-OAcTMResponse.Reaction class
It is similar to be conjugated with Vi- conjugate vaccine (An et al., 2012) and streptococcus pneumoniae (Streptococcus pneumonia) polysaccharide
The reaction that thing vaccine (Safari et al., 2011) obtains.With GelSite-OAcTMDifference, Vi vaccine represents after second immunity
Titre that is identical for all IgG subclass or reducing, in addition to IgG2a (increase of its 2 times of display).With any one vaccine all not
Observe the significant change of IgM level.
Embodiment 4:O- acetylated polymerProtected effect
As discussed previously (Park et al., 2002), with live salmonella typhi carry out attack experiment in evaluate by
GelSite-OAcTMThe protective effect of the specific immune response of induction.By intramuscular injection twice, with 2.5 μ g/ mice apparatus
There is the GelSite-OAc of different DOAc (136% or 155%)TMOr 15 6 to 8 week old female Balb/c mices of Vi vaccine immunity
Group, be separated by surrounding.Matched group accepts buffer solution.The 2nd week after second immunity, with 0.5ml 5% hog gastric mucin
100LD50 (1,000CPU/ mice) antibacterial in every group of 10 mouse peritoneums attack.Antibacterial used is that enteritis is husky
Door Salmonella subspecies (Salmonella enterica subsp).Enterica serovar typhi (salmonella typhi) obtains
From ATCC (production code member 19430).
Result shows that the animal of all immunity is protected from, including two GelSite-OAcTMGroup and Vi vaccine group (figure
10).All non-immune control mice are dead in three days.There is no difference (Figure 10) between protected group, indicate GelSite-
OAcTMIt is protectiveness as Vi vaccine.Additionally, having two GelSite-OAc of 136% or 155% DOAcTMGroup
Do not show difference in terms of protection, instruction has the GelSite-OAc of 136%DOAcTMCan be similarly protectiveness.
All shielded mices the 1-3 days slight body weight of experience after attack of short duration mitigation (<10%), but recovered later to just
Ordinary water puts down (Figure 10 B).As also described above, with Vi or GelSite-OAcTMAntigen measures specific antibody, and obtains and Fig. 5 institute
The similar result of the result shown.
Embodiment 5:DOAc and the further dependency of immunogenicity and protection
DOAc is GelSite-OAcTMCritical specification.It is therefore important that proving further between DOAc and protection
Dependency.Therefore, produce a series of other GelSite-OAc of DOAc (80%-153%) with relative broad rangeTMSample,
And screening immunogenicity and protection in mice.Result shows, antibody titer and level of protection are in directly related increase with DOAc
(Figure 11 and 12).There is the not acetylizad of 0%DOAcDo not induce for Vi polysaccharide or GelSite-OAcTMAppoint
What detectable antibody, does not provide any protection yet, indicates by GelSite-OAc furtherTMThe antibody of induction is directed to O- acetyl
Base.Importantly, having the GelSite-OAc of 80%DOAcTMShow minimum antibody titer after second immunity and do not have
There is reinfocing effect, and only provide part to protect, and there are other GelSite-OAc of higher DOAc (>=100%)TMProduce high
Antibody titer much (>10 times), there is stiffening effect, and comprehensive protection is provided.On the other hand, that is, using low antibody titer
(it is directed to Vi 0.239U/ml after second immunity;Figure 11 A), there is the GelSite-OAc of 80%DOAcTMStill provide 83%
Protection (Figure 12 A), has pointed out by GelSite-OAcTMThe antibody of induction is highly protective property.In a word, these results prompting
There is the GelSite-OAc of 100% or higher DOAcTMCan be complete protectiveness, and there is reinfocing effect, therefore be formed
For setting up the solid foundation of the DOAc specification of vaccine.
Consider description disclosed herein and practice, other embodiments of the disclosure will for those skilled in the art
It is obvious.Be intended that description and embodiments be to be considered only as exemplary, the true scope of the disclosure and spirit by
Appended claims indicate.
Claims (37)
1.O- acetylation high molecular polygalacturonic acid (OAc-HPGA) or its pharmaceutically acceptable salt, it has following at least one
Kind:
A () is more than 1x 106The molecular weight of Da,
B the methylation that every mole less than about 10% of (), and
C () scope is every mole about 2% to about 15% of Fructus rhamni (Rhamnus davurica Pall.) sugared content between two parties.
2. OAc-HPGA according to claim 1 or its pharmaceutically acceptable salt, it has more than 1x 106The molecular weight of Da.
3. the OAc-HPGA according to any one of claim 1 or 2 or its pharmaceutically acceptable salt, wherein O- degree of acetylation is in C2
Or be more than 100% at C3 position or be more than 50%.
4. the OAc-HPGA according to any one of claims 1 to 3 or its pharmaceutically acceptable salt, wherein said OAc-HPGA is to close
The immunogenicity Vi antigen becoming.
5. the OAc-HPGA according to any one of Claims 1-4 or its pharmaceutically acceptable salt, wherein said OAc-HPGA or its
Pharmaceutically acceptable salt has the antigenicity substantially the same with Vi polysaccharide vaccine and is immunogenic.
6. the OAc-HPGA according to any one of claim 1 to 5 or its pharmaceutically acceptable salt, wherein said OAc-HPGA or its
The 2 times or more large rising of pharmaceutically acceptable salt induction of antibodies titre in second immunity.
7. the OAc-HPGA according to any one of claim 1 to 6 or its pharmaceutically acceptable salt, wherein said OAc-HPGA or its
Pharmaceutically acceptable salt immunogenicity as synthesis in the people below 2 years old age in the case of not being conjugated with protein carrier
Vi antigen is effective.
8. the OAc-HPGA according to any one of claim 1 to 7 or its pharmaceutically acceptable salt, wherein said OAc-HPGA or its
Pharmaceutically acceptable salt is effective as the vaccine for typhoid fever.
9. the OAc-HPGA according to any one of claim 1 to 8 or its pharmaceutically acceptable salt, it has more than 1x 106Da's
Molecular weight, every mole less than about 10% of degree of methylation, and it is more than 100% O- degree of acetylation at C2 or C3 position, or
O- degree of acetylation more than 50%.
10.O- acetylation high molecular polygalacturonic acid (OAc-HPGA) or its pharmaceutically acceptable salt, it makes height by inclusion
Molecular weight polyisoprene galacturonic acid (HPGA) or its salt are formed with the method for acetic acid and the mixture reaction of acetic anhydride, wherein said
HPGA has at least one of:
A () is more than 1x 106The molecular weight of Da,
B the methylation that every mole less than about 10% of (), and
C () scope is every mole about 2% to about 15% of Fructus rhamni (Rhamnus davurica Pall.) sugared content between two parties.
11. OAc-HPGA according to claim 10 or its pharmaceutically acceptable salt, wherein said method be additionally included in acetic acid and
With described HPGA or its salt formation acid gel before the mixture reaction of acetic anhydride.
12. also wrap according to the OAc-HPGA of any one of claim 10 or 11 or its pharmaceutically acceptable salt, wherein said method
Include and so that described HPGA or its salt is reacted with perchloric acid.
13. are derived from according to the OAc-HPGA of any one of claim 10 to 12 or its pharmaceutically acceptable salt, wherein said HPGA
Aloe vera (Aloe vera).
14. according to the OAc-HPGA of any one of claim 10 to 13 or its pharmaceutically acceptable salt, wherein said OAc-HPGA
O- degree of acetylation at C2 or C3 position be more than 100% or be more than 50%.
15. according to the OAc-HPGA of any one of claim 10 to 14 or its pharmaceutically acceptable salt, wherein said OAc-HPGA
Or its pharmaceutically acceptable salt is the immunogenicity Vi antigen of synthesis.
16. according to the OAc-HPGA of any one of claim 10 to 15 or its pharmaceutically acceptable salt, wherein said OAc-HPGA
There is the antigenicity substantially the same with Vi polysaccharide vaccine and it is immunogenic.
17. according to the OAc-HPGA of any one of claim 10 to 16 or its pharmaceutically acceptable salt, wherein said OAc-HPGA
Or the immunity as synthesis in the people below 2 years old age in the case of not being conjugated with protein carrier of its pharmaceutically acceptable salt
Originality Vi antigen is effective.
18. according to the OAc-HPGA of any one of claim 10 to 17 or its pharmaceutically acceptable salt, wherein said OAc-HPGA
Or its pharmaceutically acceptable salt is effective as the vaccine for typhoid fever.
19. according to the OAc-HPGA of any one of claim 10 to 18 or its pharmaceutically acceptable salt, wherein said OAc-HPGA
The 2 times or more large rising of induction of antibodies titre in second immunity.
20. methods preparing O- acetylation high molecular polygalacturonic acid (OAc-HPGA) or its pharmaceutically acceptable salt, including
Make the mixture reaction of high molecular polygalacturonic acid (HPGA) or its salt and acetic acid and acetic anhydride, wherein said HPGA has
At least one of:
A () is more than 1x 106The molecular weight of Da,
B the methylation that every mole less than about 10% of (), and
C () scope is every mole about 2% to about 15% of Fructus rhamni (Rhamnus davurica Pall.) sugared content between two parties.
21. methods according to claim 20, be additionally included in the mixture reaction of acetic acid and acetic anhydride before with HPGA or its
Salt formation acid gel.
22., according to the method for any one of claim 20 or 21, also include making described HPGA or its salt react with perchloric acid.
23. are derived from aloe vera according to the method for any one of claim 20 to 22, wherein said HPGA.
24. pass through the O- acetylation high-molecular-weight poly galactose that the method according to any one of claim 20 to 23 is formed
Aldehydic acid (OAc-HPGA) or its pharmaceutically acceptable salt.
25. OAc-HPGA according to claim 24 or its pharmaceutically acceptable salt, the O- acetylation journey of wherein said OAc-HPGA
Degree is more than 100% at C2 or C3 position, or is more than 50%.
26. according to the OAc-HPGA of any one of claim 24 or 25 or its pharmaceutically acceptable salt, wherein said OAc-HPGA
Or its pharmaceutically acceptable salt is the immunogenicity Vi antigen with the antigenic synthesis substantially the same with Vi polysaccharide vaccine, and
And be immunogenic.
27. according to the OAc-HPGA of any one of claim 24 to 26 or its pharmaceutically acceptable salt, wherein said OAc-HPGA
Or the immunity as synthesis in the people below 2 years old age in the case of not being conjugated with protein carrier of its pharmaceutically acceptable salt
Originality Vi antigen is effective.
28. according to the OAc-HPGA of any one of claim 24 to 27 or its pharmaceutically acceptable salt, wherein said OAc-HPGA
Or its pharmaceutically acceptable salt is effective as the vaccine for typhoid fever.
29. according to the OAc-HPGA of any one of claim 24 to 28 or its pharmaceutically acceptable salt, wherein said OAc-HPGA
The 2 times or more large rising of induction of antibodies titre in second immunity.
30. pharmaceutical compositions, it comprises OAc-HPGA according to any one of claim 1 to 19 or 24 to 29 or its pharmacy can
Accept salt and at least one pharmaceutical acceptable excipient.
31. pharmaceutical compositions according to claim 30, it also comprises protein carrier.
32. are conjugated with described OAc-HPGA according to the pharmaceutical composition of claim 31, wherein said protein carrier.
33. methods being directed to typhoid fever immunized subject, apply wanting according to right of effective dose including to subject in need
Ask any one of 1 to 19 or 24 to 29 OAc-HPGA or its pharmaceutically acceptable salt, or according to arbitrary in claim 30 to 32
The pharmaceutical composition of item.
34. according to the method for claim 33, the administration induction of antibodies response of wherein said pharmaceutical composition.
35. according to the method for any one of claim 33 or 34, wherein said pharmaceutical composition be applied in second immunity
When induction of antibodies titre 2 times or more large rising.
36. according to the O- acetylation high molecular polygalacturonic acid (OAc- of any one of claim 1 to 19 or 24 to 29
HPGA) or its pharmaceutically acceptable salt or according to the pharmaceutical composition of any one of claim 30 to 32 as synthesis immunogen
The purposes of property Vi antigen, wherein said Vi antigen has the antigenicity substantially the same with Vi polysaccharide vaccine and is immunogenicity
's.
37. according to the O- acetylation high molecular polygalacturonic acid (OAc- of any one of claim 1 to 9 or 24 to 29
) or its pharmaceutically acceptable salt or use in medicine is being prepared according to the pharmaceutical composition of any one of claim 30 to 32 HPGA
On the way, described medicine is used for the vaccination for typhoid fever.
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US201461990493P | 2014-05-08 | 2014-05-08 | |
US61/990,493 | 2014-05-08 | ||
PCT/US2015/029979 WO2015172077A1 (en) | 2014-05-08 | 2015-05-08 | O-acetylated high molecular weight polygalacturonic acids and their use as vi polysaccharide vaccine |
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EP (1) | EP3140324A1 (en) |
JP (1) | JP2017514961A (en) |
CN (1) | CN106459225A (en) |
AU (1) | AU2015255693A1 (en) |
CA (1) | CA2946900A1 (en) |
WO (1) | WO2015172077A1 (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US7691986B2 (en) * | 1998-05-13 | 2010-04-06 | Nanotherapeutics, Inc. | High molecular weight, low methoxyl pectins, and their production and uses |
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Publication number | Priority date | Publication date | Assignee | Title |
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US5929051A (en) | 1998-05-13 | 1999-07-27 | Carrington Laboratories, Inc. | Aloe pectins |
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2015
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- 2015-05-08 JP JP2016566668A patent/JP2017514961A/en active Pending
- 2015-05-08 EP EP15723634.0A patent/EP3140324A1/en not_active Withdrawn
- 2015-05-08 CA CA2946900A patent/CA2946900A1/en not_active Abandoned
- 2015-05-08 WO PCT/US2015/029979 patent/WO2015172077A1/en active Application Filing
- 2015-05-08 CN CN201580023307.6A patent/CN106459225A/en active Pending
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US7691986B2 (en) * | 1998-05-13 | 2010-04-06 | Nanotherapeutics, Inc. | High molecular weight, low methoxyl pectins, and their production and uses |
Non-Patent Citations (3)
Title |
---|
BOGUSLAW SZEWCZYK等: ""Immunochemical Properties of Vi Antigen from Salmonella typhi Ty2: Presence of Two Antigenic Determinants"", 《INFECTION AND IMMUNITY》 * |
RICHARD G. SCHWEIGER: ""Acetyl Pectates and Their Reactivity with Polyvalent Metal Ions"", 《J. ORG. CHEM.》 * |
SHOUSUN C. SZU等: ""Synthesis and Some Immunologic Properties of an O-Acetyl Pectin [Poly(l1->4)-C-D-GalpA]-Protein Conjugate as a Vaccine for Typhoid Fever"", 《INFECTION AND IMMUNITY》 * |
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EP3140324A1 (en) | 2017-03-15 |
CA2946900A1 (en) | 2015-11-12 |
WO2015172077A1 (en) | 2015-11-12 |
AU2015255693A1 (en) | 2016-11-03 |
JP2017514961A (en) | 2017-06-08 |
WO2015172077A8 (en) | 2016-01-28 |
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