WO2009150543A2 - Conjugated vi saccharides - Google Patents
Conjugated vi saccharides Download PDFInfo
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- WO2009150543A2 WO2009150543A2 PCT/IB2009/006285 IB2009006285W WO2009150543A2 WO 2009150543 A2 WO2009150543 A2 WO 2009150543A2 IB 2009006285 W IB2009006285 W IB 2009006285W WO 2009150543 A2 WO2009150543 A2 WO 2009150543A2
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- conjugate
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- 0 *OC(CN(C(CO)C1O)C2*1O)C2O Chemical compound *OC(CN(C(CO)C1O)C2*1O)C2O 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the invention relates to vaccines, more particularly those against typhoid fever.
- Typhoid fever is a common serious disease in many parts of the world.
- Purified capsular polysaccharide from Salmonella typhi (Vi) is used as a vaccine, providing about 70% protection against typhoid fever in individuals 5- to 45-years-old.
- the vaccine is unable to establish immunological memory and is ineffective in infants or toddlers [I ].
- a conjugate vaccine of Vi coupled to recombinant mutant Pseudomonas aeruginosa exoprotein A (Vi-rEPA) gave a booster response in young children and was highly efficacious [2],
- the inventors have devised a new method for manufacturing Vi-conjugates and have also produced a new conjugate comprising Vi coupled to CRMi 97 .
- a first aspect of the invention provides a method for the preparation of a Vi conjugate.
- a linker such as adipic acid dihydrazide (ADH), and a carbodiimide, such as l-ethyl-3(3- dimethylaminopropyl)carbodiimide (EDAC) are simultaneously added to a solution containing a carrier protein, such as CRM 197 or tetanus toxoid (TT), to give a derivatised carrier protein.
- a linker such as adipic acid dihydrazide (ADH)
- EDAC l-ethyl-3(3- dimethylaminopropyl)carbodiimide
- TT tetanus toxoid
- a buffer such as 2-(N-morpholino)ethanesulfonic acid (MES) may be added to the solution containing the carrier protein prior to the addition of ADH and EDAC.
- the weight ratio of the carbodiimide to the protein is typically 0.1 to 0.15, as higher carbodiimide/protein ratios can cause aggregate formation.
- any excess linker ⁇ e.g. ADH is removed by, for example, dialysis or tangential flow filtration (TFF).
- Vi is also activated with a carbodiimide and is subsequently combined with the derivatised carrier protein.
- various ratios of Vi and carbodiimide can be used.
- a 1 : 1 molar ratio (COOH groups of Vi to carbodiimide) can be used, but to reduce the amount of residual unconjugated carbodiimide derivatives ⁇ e.g. ureas such as EDU; N-ethyl-N'-(3- dimethylaminopropyl)urea, a soluble reaction product of EDAC coupling) higher ratios can be used i.e. with a molar excess of Vi e.g.
- ureas such as EDU
- N-ethyl-N'-(3- dimethylaminopropyl)urea a soluble reaction product of EDAC coupling
- the method comprises the steps of: a) Simultaneously combining a linker, a carbodiimide and a carrier protein. b) Reacting Vi with a carbodiimide c) Reacting the product of step a) with the product of step b). Steps a) and b) may be performed in any order.
- step a but before step c), any excess linker may be removed.
- This aspect of the invention also provides a method for preparing a Vi-CRM ⁇ 7 conjugate, wherein Vi is combined with derivatised CRM 197 .
- the method comprises the steps of: a) Reacting Vi with a carbodiimide b) Reacting the product of step a) with derivatised CRM 197 .
- This aspect of the invention also provides a method for preparing a Vi-CRM) 97 conjugate, comprising the step of: a) Reacting activated Vi with derivatised CRM 197 .
- Reference 3 describes a process for preparing conjugates of Vi with rEPA and bovine serum albumin (BSA) by carbodiimide-mediated synthesis with ADH as the linker.
- BSA bovine serum albumin
- this process does not involve the simultaneous combination of rEPA/BSA, ADH and EDAC. Rather, rEPA/BSA and ADH are combined and mixed prior to the addition of EDAC.
- a second aspect of the invention provides a Vi-CRMw conjugate.
- This conjugate may be prepared by the above method, or may be obtained by other means.
- Vi is the capsular saccharide of Salmonella typhi (previously classified as a species itself, but now referred to as the typhi serovar of S.enterica). Vi may also be found in other serovars of Salmonella (such as S.enterica serovar paratyphi C or serovar dublin) and in other bacteria, such as Citrobacter ⁇ e.g. C.freundii and C.youngae).
- the Vi polysaccharide is a linear homopolymer of a hexosaminuronic acid, ⁇ l ⁇ -N-acetylgalactos-aminouronic acid, which is 60 - 90% acetylated at the C-3 position [4-9].
- the O-acetyl substitution on Vi is a factor in its ability to elicit a protective immune response [10].
- the immunogenicity of Vi is closely related to its degree of O-acetylation. Partial de-O-acetylation can slightly increase immunogenicity; complete de-O-acetylation eliminates the immunogenicity of Vi [ H].
- the Vi saccharide used in the present invention may be chemically modified relative to the capsular saccharide as found in nature.
- the Vi saccharide may be partially de-O-acetylated, de- ⁇ -acetylated (partially or fully), ⁇ -propionated (partially or fully), etc. De-acetylation may occur before, during or after conjugation, but preferably occurs before conjugation.
- the effect of de-acetylation etc. can be assessed by routine assays.
- the Vi saccharide may be hydrolysed to form shortened polysaccharides (e.g. with a degree of polymerisation (DP) of at least 10, e.g. 20, 30, 40, 50, 60 or more) or oligosaccharides (e.g. with a degree of polymerisation of from 2 to 10). Oligosaccharides are preferred to polysaccharides for use in vaccines.
- the average degree of polymerisation can conveniently be measured by ion exchange chromatography or by colorimetric assays [ 12].
- pectin when O-acetylated at C-2 and C-3, is antigenically identical to Vi.
- the structure of Vi differs from that of pectin in that it is N- acetylated at C-2 and O-acetylated at C-3.
- O-acetylated pectin conjugated to tetanus toxoid elicited Vi antibodies in mice, and reinjection elicited a booster response [13,14]. Accordingly, O-acetylated pectin may be used in the invention in place of Vi.
- Vi conjugates have been shown to be significantly more immunogenic than their O-acetylated pectin analogs, and so Vi from natural sources is preferred [13]. Nevertheless, it will be understood that references to “Vi” may include "O- acetylated pectin” and any other molecules that may be structurally or antigenically identical to Vi and are capable of eliciting antibodies that recognise native Vi. "Vi” may refer to a Vi polysaccharide (e.g. with a degree of polymerisation of at least 10, e.g. 20, 30, 40, 50, 60 or more) or a Vi oligosaccharide (e.g. with a degree of polymerisation of from 2 to 10) and may have been chemically modified. Oligosaccharides may be the result of depolymerisation and/or hydrolysis of a parent polysaccharide.
- Vi polysaccharide e.g. with a degree of polymerisation of at least 10, e.g. 20, 30, 40, 50, 60 or more
- Capsular saccharides can be purified by known techniques, as described in the references herein.
- a typical process involves base extraction, centrifugation, filtration, RNase/DNase treatment, protease treatment, concentration, size exclusion chromatography, ultrafiltration, anion exchange chromatography, and further ultrafiltration.
- a process for purifying Vi may comprise the steps of (a) precipitation of Vi, followed by (b) solubilisation of the precipitated Vi using an alcohol, such as ethanol.
- the detergents preferably have the following general formula: I R 3 - N + - R 2 X " R 4 wherein: R), R 2 and R 3 are the same or different and each signifies alkyl or aryl; or R
- R 4 signifies alkyl or aryl
- X " signifies an anion
- Particularly preferred detergents for use in the method are tetrabutylammonium and cetyltrimethylammonium salts (e.g. the bromide salts).
- Cetyltrimethylammonium bromide ( 1 CTAB') is particularly preferred [16].
- CTAB is also known as hexadecyltrimethylammonium bromide, cetrimonium bromide, Cetavlon and Centimide.
- Other detergents include hexadimethrine bromide and myristyltrimethylammonium salts.
- the starting material for precipitation will typically be the supernatant from a centrifuged bacterial culture or will be a concentrated culture. This material may be filtered to remove turbidity.
- the precipitation step may be selective for Vi, but it will typically also co-precipitate other components (e.g. proteins, nucleic acid etc.).
- other components e.g. proteins, nucleic acid etc.
- Precipitated Vi may be collected by centrifugation prior to solubilisation.
- Vi (typically in the form of a complex with the cationic detergent) is re-solubilised. It is preferred to use a solvent which is relatively selective for Vi in order to minimise contaminants (e.g. proteins, nucleic acid etc.). Ethanol has been found to be advantageous in this respect, and it is highly selective for the CTAB-Vi complex. Other lower alcohols may be used (e.g.
- the alcohol is preferably added to the precipitated Vi to give a final alcohol concentration (based on total content of alcohol and water) of between 50% and 99% (e.g. around 55%, 60%, 65%, 70%, 75%, 80%, 85%, or around 90%), and preferably between 75% and 95%.
- the alcohol may be added to the precipitated Vi in pure form or may be added in a form diluted with a miscible solvent (e.g. water).
- a miscible solvent e.g. water
- Preferred solvent mixtures are alcohohwater mixtures, with a preferred ratio of between around 70:30 and around 95:5 (e.g. 75:25, 80:20, 85:15, 90: 10).
- the process uses cationic detergent rather than anionic detergent.
- precipitation does not require an inert porous support.
- an alcohol is used to re-solubilise Vi rather than to precipitate it.
- Vi is further treated to remove contaminants because, in human vaccine production, even minor contamination is not acceptable.
- This treatment may include centrifugation of the solubilised CTAB-Vi complex, followed by precipitation of Vi from the obtained supernatant by exchanging cations ⁇ e.g. by the addition of calcium or sodium salts) to give a Vi precipitate that is insoluble in alcohol but soluble in water.
- This precipitate may be collected by centrifugation and further washed in alcohol and re-solubilised in an aqueous solution, if desired.
- the treatment process will also typically involve one or more steps of filtration. Depth filtration may be used. This is particularly useful for clarification.
- Filtration through activated carbon may be used. This is useful for removing pigments and trace organic compounds. It can be repeated until, for example, OD 275nm ⁇ 0.2. Size filtration or ultrafiltration may be used.
- the hydrolysate will generally be sized in order to remove short-length oligosaccharides. This can be achieved in various ways, such as ultrafiltration followed by ion- exchange chromatography.
- the invention is not limited to saccharides purified from natural sources, however, and the saccharides may be obtained by other methods, such as total or partial synthesis.
- Vi may be presented to the immune system as a Vi-carrier conjugate.
- the use of conjugation to carrier proteins in order to enhance the immunogenicity of carbohydrate antigens is well known [e.g. reviewed in refs. 19 to 27 etc.] and is used in particular for paediatric vaccines [28].
- a saccharide may be conjugated to a carrier protein or to a mixture of different carrier proteins.
- a carrier protein may carry a saccharide or a mixture of different saccharides, i.e. multiple different saccharides [29].
- the invention provides a conjugate of (i) Vi, and (ii) CRMi 97 as a carrier protein.
- the CRMi 97 may be covalently conjugated to Vi directly or via a linker. Any suitable conjugation reaction can be used, with any suitable linker where necessary. Oligosaccharides will typically be sized prior to conjugation. Where the composition of the invention includes a depolymerised saccharide, it is preferred that depolymerisation precedes conjugation.
- Attachment of Vi to CRM 197 is preferably via a -NH 2 group e.g. in the side chain of a lysine residue in CRM 197 , or of an arginine residue. Attachment to CRM 197 may also be via a -SH group e.g.
- Vi may be attached to CRM 19 7 via a linker molecule as described below. Vi will typically be activated or functionalised prior to conjugation.
- a preferred technique uses carbodiimides (e.g. l-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDAC)).
- EDAC l-ethyl-3(3-dimethylaminopropyl)carbodiimide
- Other suitable techniques use hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDAC, TSTU (see also the introduction to reference 30).
- Linkages via a linker group to carrier proteins in general may be made using any known procedure, for example, the procedures described in references 31 and 32.
- a useful type of linkage is an adipic acid linker, which may be formed by coupling a free -NH 2 group (e.g. introduced to Vi by amination) with adipic acid (using, for example, diimide activation), and then coupling a protein to the resulting saccharide-adipic acid intermediate [23, 33, 34].
- Another useful type of linkage is a carbonyl linker, which may be formed by reaction of a free hydroxyl group of a modified Vi with CDI [35, 36] followed by reaction with a protein to form a carbamate linkage.
- a useful linker is adipic acid dihydrazide ADH [37].
- the carrier protein may be derivatised with ADH (for example, by carbodiimide coupling at a carboxylic acid side group) and subsequently attached to Vi [3] (again, for example, by carbodiimide coupling).
- Other linkers include ⁇ -propionamido [38], nitrophenyl- ethylamine [39], haloacyl halides [40], glycosidic linkages [41 ], 6-aminocaproic acid [42], N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) [43], C 4 to Ci 2 moieties [44], etc.
- Carbodiimide condensation can also be used [45].
- a useful process for linking Vi to CRM 197 involves the thiolation of Vi with cystamine or cysteamine (carbodiimide coupling) and subsequent reaction with CRM 197 derivatised with SPDP [46].
- adipic diester e.g. adipic acid N-hydroxysuccinimido diester
- a bifunctional linker may be used to provide a first group for coupling to an amine group that has been introduced into Vi and a second group for coupling to the carrier (typically for coupling to an amine in the carrier).
- the first group in the bifunctional linker is thus able to react with an amine group (-NH 2 ) on Vi. This reaction will typically involve an electrophilic substitution of the amine's hydrogen.
- the second group in the bifunctional linker is able to react with an amine group on the carrier. This reaction will again typically involve an electrophilic substitution of the amine.
- a bifunctional linker for example a homobi functional linker of the formula X-L-X, where: the two X groups are the same as each other and can react with the amines; and where L is a linking moiety in the linker.
- a useful X group is N-oxysuccinimide.
- L may have formula L'-L 2 -L', where L' is carbonyl.
- Useful L 2 groups are straight chain alkyls with 1 to 10 carbon atoms (e.g. Ci, C 2 , C 3 , C 4 ,
- X groups are those which form esters when combined with HO-L-OH, such as norborane, p-nitrobenzoic acid, and sulfo-N-hydroxysuccinimide.
- Further bifunctional linkers for use with the invention include acryloyl halides (e.g. chloride) and haloacylhalides.
- the linker will generally be added in molar excess to modified Vi.
- Preferred carrier proteins are bacterial toxins, such as diphtheria or tetanus toxins, or toxoids or mutants thereof. These are commonly used in conjugate vaccines.
- the CRMi 97 diphtheria toxin mutant is particularly preferred [47].
- carrier proteins include the N. meningitidis outer membrane protein complex [48], synthetic peptides [49,50], heat shock proteins [51 ,52], pertussis proteins [53,54], cytokines [55], lymphokines [55], hormones [55], growth factors [55], artificial proteins comprising multiple human CD4 + T cell epitopes from various pathogen-derived antigens [56] such as N19 [57], protein D from ⁇ .influenzae [58-60], pneumolysin [61] or its non-toxic derivatives [62], pneumococcal surface protein PspA [63], iron-uptake proteins [64], toxin A or B from C. difficile [65], recombinant Pseudomonas aeruginosa exoprotein A (rEPA) [66], etc. It is possible to use mixtures of carrier proteins.
- a single carrier protein may carry multiple Vi saccharides [67].
- Conjugates may have excess carrier protein (w/w) or excess Vi (w/w) e.g. in the ratio range of 1 :5 to 5: 1. Conjugates with excess carrier protein are typical e.g. in the range 0.2: 1 to 0.9: 1, such as 0.5: 1, or with equal weights (1 : 1). In some embodiments the Vi:protein ratio is between 0.4: 1 and 1.2: 1.
- the composition may also comprise free carrier protein [68].
- the Vi moiety in the conjugate is preferably a low molecular weight Vi polysaccharide or an oligosaccharide, as defined above. Oligosaccharides will typically be sized prior to conjugation.
- the protein- Vi conjugate is preferably soluble in water and/or in a physiological buffer.
- compositions of the invention may be prepared in various forms.
- the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
- the composition may be prepared for topical administration e.g. as an ointment, cream or powder.
- the composition be prepared for oral administration e.g. as a tablet or capsule, or as a syrup (optionally flavoured).
- composition may be prepared for pulmonary administration e.g.
- the composition may be prepared as a suppository or pessary.
- the composition may be prepared for nasal, aural or ocular administration e.g. as drops, as a spray, or as a powder [e.g. 70].
- the composition may be included in a mouthwash.
- the composition may be lyophilised.
- the pharmaceutical composition is preferably sterile. It is preferably pyrogen-free. It is preferably buffered e.g. at between pH 6 and pH 8, generally around pH 7.
- a composition of the invention may comprise a Vi conjugate and saline.
- the invention also provides a delivery device containing a pharmaceutical composition of the invention.
- the device may be, for example, a syringe or an inhaler.
- compositions of the invention are preferably immunogenic compositions, in that they comprise an immunologically effective amount of Vi immunogen.
- immunologically effective amount it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
- a dose of between l ⁇ g and 20 ⁇ g of saccharide is expected e.g. about 5 ⁇ g/dose.
- Dosage treatment may be a single dose schedule or a multiple dose schedule (e.g. including booster doses).
- the composition may be administered in conjunction with other immunoregulatory agents.
- compositions of the invention can be administered directly to the subject.
- the subjects to be treated can be animals; in particular, human subjects can be treated.
- Immunogenic compositions of the invention may be used therapeutically (i.e. to treat an existing infection) or prophylactically (i.e. to prevent future infection).
- an immunogenic composition may be unadjuvanted.
- an immunogenic composition may include an adjuvant, which can function to enhance the immune responses (humoral and/or cellular) elicited in a patient who receives the composition.
- adjuvants that can be used with the invention include, but are not limited to:
- a mineral-containing composition including calcium salts and aluminum salts (or mixtures thereof).
- Calcium salts include calcium phosphate (e.g. the "CAP" particles disclosed in ref. 71).
- Aluminum salts include hydroxides, phosphates, sulfates, etc., with the salts taking any suitable form (e.g. gel, crystalline, amorphous, etc.). Adsorption to these salts is preferred.
- the mineral containing compositions may also be formulated as a particle of metal salt [72].
- the adjuvants known as aluminum hydroxide and aluminum phosphate may be used. These names are conventional, but are used for convenience only, as neither is a precise description of the actual chemical compound which is present (e.g. see chapter 9 of reference 155).
- the invention can use any of the "hydroxide” or "phosphate” adjuvants that are in general use as adjuvants.
- the adjuvants known as "aluminium hydroxide” are typically aluminium oxyhydroxide salts, which are usually at least partially crystalline.
- aluminium phosphate are typically aluminium hydroxyphosphates, often also containing a small amount of sulfate (i.e. aluminium hydroxyphosphate sulfate). They may be obtained by precipitation, and the reaction conditions and concentrations during precipitation influence the degree of substitution of phosphate for hydroxyl in the salt.
- the invention can use a mixture of both an aluminium hydroxide and an aluminium phosphate. In this case there may be more aluminium phosphate than hydroxide e.g. a weight ratio of at least 2: 1 e.g. >5: 1, >6: 1, >7: 1, >8: 1, >9:1 , etc.
- the concentration Of Al 4 ⁇ + in a composition for administration to a patient is preferably less than lOmg/ml e.g. ⁇ 5 mg/ml, ⁇ 4 mg/ml, ⁇ 3 mg/ml, ⁇ 2 mg/ml, ⁇ 1 mg/ml, etc.
- a preferred range is between 0.3 and 1 mg/ml.
- a maximum of 0.85mg/dose is preferred.
- Saponins [chapter 22 of ref. 155], which are a heterologous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponin from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be commercially obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and Saponaria officianalis
- Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOMs.
- QS21 is marketed as StimulonTM.
- Saponin compositions have been purified using HPLC and RP-HPLC. Specific purified fractions using these techniques have been identified, including QS7, QS 17, QS 18, QS21, QH-A, QH- B and QH-C.
- the saponin is QS21.
- a method of production of QS21 is disclosed in ref. 73.
- Saponin formulations may also comprise a sterol, such as cholesterol [74].
- ISCOMs immunostimulating complexs
- phospholipid such as phosphatidylethanolamine or phosphatidylcholine.
- saponin can be used in ISCOMs.
- the ISCOM includes one or more of
- Bacterial ADP-ribosylating toxins e.g. the E.coli heat labile enterotoxin "LT”, cholera toxin “CT”, or pertussis toxin “PT”
- Bacterial ADP-ribosylating toxins e.g. the E.coli heat labile enterotoxin "LT”, cholera toxin “CT”, or pertussis toxin “PT”
- the use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in ref. 81 and as parenteral adjuvants in ref. 82.
- Bioadhesives and mucoadhesives such as esterified hyaluronic acid microspheres [83] or chitosan and its derivatives [84]
- Microparticles ⁇ i.e. a particle of ⁇ 100nm to ⁇ 150 ⁇ m in diameter, more preferably ⁇ 200nm to ⁇ 30 ⁇ m in diameter, or ⁇ 500nm to -lO ⁇ m in diameter) formed from materials that are biodegradable and non-toxic ⁇ e.g. a poly( ⁇ -hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.), with poly(lactide-co-glycolide) being preferred, optionally treated to have a negatively-charged surface ⁇ e.g. with SDS) or a positively-charged surface ⁇ e.g. with a cationic detergent, such as CTAB).
- a negatively-charged surface ⁇ e.g. with SDS
- a positively-charged surface ⁇ e.g. with a cationic detergent, such as CTAB
- Liposomes Chopers 13 & 14 of ref. 155. Examples of liposome formulations suitable for use as adjuvants are described in refs. 85-87.
- Muramyl peptides such as N-acetylmuramyl-L-threonyl-D-isoglutamine (“thr-MDP"), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylglucsaminyl-N- acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalrnitoxy propylamide (“DTP-DPP", or "TheramideTM), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1 '-2'dipalmitoyl- sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (“MTP-PE").
- thr-MDP N-acetylmuramyl-L-threonyl-D-isoglutamine
- a polyoxidonium polymer [88,89] or other N-oxidized polyethylene-piperazine derivative • A polyoxidonium polymer [88,89] or other N-oxidized polyethylene-piperazine derivative.
- a CDId ligand such as an ⁇ -glycosylceramide [90-97] ⁇ e.g. ⁇ -galactosylceramide), phytosphingosine-containing ⁇ -glycosylceramides, OCH, KRN7000 [(2S,3S,4R)-l-O-( ⁇ -D- galactopyranosyl)-2-(N-hexacosanoylamino)- 1 ,3,4-octadecanetriol], CRONY- 101, 3"-O- sulfo-galactosylceramide, etc.
- ⁇ -glycosylceramide ⁇ e.g. ⁇ -galactosylceramide
- phytosphingosine-containing ⁇ -glycosylceramides OCH
- KRN7000 [(2S,3S,4R)-l-O-( ⁇ -D- galactopyranosyl)-2-(N-hexacosan
- An oil-in-water emulsion typically include at least one oil and at least one surfactant, with the oil(s) and surfactant(s) being biodegradable (metabolisable) and biocompatible.
- the oil droplets in the emulsion are generally less than 5 ⁇ m in diameter, and may even have a sub-micron diameter, with these small sizes being achieved with a microfluidiser to provide stable emulsions. Droplets with a size less than 220nm are preferred as they can be subjected to filter sterilization.
- An immunostimulatory oligonucleotide such as one containing a CpG motif (a dinucleotide sequence containing an unmethylated cytosine residue linked by a phosphate bond to a guanosine residue), or a CpI motif (a dinucleotide sequence containing cytosine linked to inosine), or a double-stranded RNA, or an oligonucleotide containing a palindromic sequence, or an oligonucleotide containing a poly(dG) sequence.
- Immunostimulatory oligonucleotides can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or (except for RNA) single-stranded.
- References 99, 100 and 101 disclose possible analog substitutions e.g. replacement of guanosine with T- deoxy-7-deazaguanosine.
- the adjuvant effect of CpG oligonucleotides is further discussed in refs. 102-107.
- a CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT [108].
- the CpG sequence may be specific for inducing a ThI immune response, such as a CpG-A ODN (oligodeoxynucleotide), or it may be more specific for inducing a B cell response, such a CpG-B ODN.
- CpG-A and CpG-B ODNs are discussed in refs. 109-11 1.
- the CpG is a CpG-A ODN.
- the CpG oligonucleotide is constructed so that the 5' end is accessible for receptor recognition.
- two CpG oligonucleotide sequences may be attached at their 3' ends to form "immunomers". See, for example, references 108 & 1 12-1 14.
- CpG7909 also known as ProMuneTM (Coley Pharmaceutical Group, Inc.).
- CpGl 826 Another is CpGl 826.
- TpG sequences can be used [1 15], and these oligonucleotides may be free from unmethylated CpG motifs.
- the immunostimulatory oligonucleotide may be pyrimidine-rich. For example, it may comprise more than one consecutive thymidine nucleotide (e.g. TTTT, as disclosed in ref. 1 15), and/or it may have a nucleotide composition with >25% thymidine (e.g.
- cytosine nucleotide e.g. CCCC, as disclosed in ref. 1 15
- nucleotide composition with >25% cytosine (e.g. >35%, >40%,
- oligonucleotides may be free from unmethylated CpG motifs.
- Immunostimulatory oligonucleotides will typically comprise at least 20 nucleotides. They may comprise fewer than 100 nucleotides.
- an adjuvant used with the invention may comprise a mixture of (i) an oligonucleotide (e.g. between 15-40 nucleotides) including at least one (and preferably multiple) CpI motifs, and (ii) a polycationic polymer, such as an oligopeptide (e.g. between 15-40 nucleotides).
- a polycationic polymer such as an oligopeptide
- the oligonucleotide may be a deoxynucleotide comprising 26-mer sequence 5'-(IC) 13 -3'.
- the polycationic polymer may be a peptide comprising 1 1-mer amino acid Lys-
- 3dMPL 3-O-deacylated monophosphoryl lipid A
- '3dMPLTM' 3-O-deacylated monophosphoryl lipid A
- 3dMPL can form micellar aggregates or particles with different sizes e.g. with a diameter ⁇ 150nm or >500nm. Either or both of these can be used with the invention, and the better particles can be selected by routine assay. Smaller particles (e.g. small enough to give a clear aqueous suspension of 3dMPL) are preferred for use according to the invention because of their superior activity [121].
- Preferred particles have a mean diameter less than 220nm, more preferably less than 200nm or less than 150nm or less than 120nm, and can even have a mean diameter less than lOOnm. In most cases, however, the mean diameter will not be lower than 50nm.
- MIMP Methyl inosine 5'-monophosphate
- An imidazoquinoline compound such as Imiquimod (“R-837”) [124,125], Resiquimod (“R-848”) [126], and their analogs; and salts thereof (e.g. the hydrochloride salts). Further details about immunostimulatory imidazoquinolines can be found in references 127 to 131.
- a thiosemicarbazone compound such as those disclosed in reference 132. Methods of formulating, manufacturing, and screening for active compounds are also described in reference 132. The thiosemicarbazones are particularly effective in the stimulation of human peripheral blood mononuclear cells for the production of cytokines, such as TNF- ⁇ .
- a tryptanthrin compound such as those disclosed in reference 133.
- Methods of formulating, manufacturing, and screening for active compounds are also described in reference 133.
- the thiosemicarbazones are particularly effective in the stimulation of human peripheral blood mononuclear cells for the production of cytokines, such as TNF- ⁇ .
- a nucleoside analog such as: (a) Isatorabine (ANA-245; 7-thia-8-oxoguanosine):
- An aminoalkyl glucosaminide phosphate derivative such as RC-529 [143,144].
- a phosphazene such as poly[di(carboxylatophenoxy)phosphazene] ("PCPP") as described, for example, in references 145 and 146.
- Antigens and adjuvants in a composition will typically be in admixture.
- Compositions may include two or more of said adjuvants.
- they may advantageously include both an oil-in-water emulsion and 3dMPL, etc.
- oil-in-water emulsion adjuvants useful with the invention include, but are not limited to:
- a submicron emulsion of squalene, Tween 80, and Span 85 A submicron emulsion of squalene, Tween 80, and Span 85.
- the composition of the emulsion by volume can be about 5% squalene, about 0.5% polysorbate 80 and about 0.5% Span 85. In weight terms, these ratios become 4.3% squalene, 0.5% polysorbate 80 and 0.48% Span 85.
- This adjuvant is known as 'MF59' [152-154], as described in more detail in Chapter 10 of ref.
- the MF59 emulsion advantageously includes citrate ions e.g. 1 OmM sodium citrate buffer.
- An emulsion of squalene, a tocopherol, and Tween 80 may include phosphate buffered saline. It may also include Span 85 (e.g. at 1%) and/or lecithin. These emulsions may have from 2 to 10% squalene, from 2 to 10% tocopherol and from 0.3 to 3% Tween 80, and the weight ratio of squalene: tocopherol is preferably ⁇ 1 as this provides a more stable emulsion. Squalene and Tween 80 may be present volume ratio of about 5:2.
- One such emulsion can be made by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90ml of this solution with a mixture of (5g of DL- ⁇ -tocopherol and 5ml squalene), then microfluidising the mixture.
- the resulting emulsion may have submicron oil droplets e.g. with an average diameter of between 100 and 250nm, preferably about 180nm.
- An emulsion of squalene, a tocopherol, and a Triton detergent e.g. Triton X-IOO
- the emulsion may also include a 3d-MPL (see below).
- the emulsion may contain a phosphate buffer.
- An emulsion comprising a polysorbate (e.g. polysorbate 80), a Triton detergent (e.g. Triton X-100) and a tocopherol (e.g. an ⁇ -tocopherol succinate).
- the emulsion may include these three components at a mass ratio of about 75: 1 1: 10 (e.g.
- the emulsion may also include squalene.
- the emulsion may also include a 3d-MPL (see below).
- the aqueous phase may contain a phosphate buffer.
- An emulsion of squalane, polysorbate 80 and poloxamer 401 ("PluronicTM Ll 21").
- the emulsion can be formulated in phosphate buffered saline, pH 7.4. This emulsion is a useful delivery vehicle for muramyl dipeptides, and has been used with threonyl-MDP in the
- SAF-I adjuvant [157] (0.05-1% Thr-MDP, 5% squalane, 2.5% Pluronic Ll 21 and 0.2% polysorbate 80). It can also be used without the Thr-MDP, as in the "AF” adjuvant [158] (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80). Microfluidisation is preferred.
- An emulsion having from 0.5-50% of an oil, 0.1-10% of a phospholipid, and 0.05-5% of a non-ionic surfactant.
- preferred phospholipid components are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin. Submicron droplet sizes are advantageous.
- Additives may be included, such as QuiLA saponin, cholesterol, a saponin-lipophile conjugate (such as GPI-0100, described in reference 160, produced by addition of aliphatic amine to desacylsaponin via the carboxyl group of glucuronic acid), dimethyidioctadecylammonium bromide and/or N,N-dioctadecyl- N,N-bis (2-hydroxyethyl)propanediamine.
- An emulsion in which a saponin (e.g. QuilA or QS21) and a sterol (e.g. a cholesterol) are associated as helical micelles [161 ].
- the invention also provides a Vi conjugate of the invention, for use in medicine e.g. for use in raising an antibody response in a mammal.
- the invention also provides a method for raising an immune response in a mammal, comprising administering a Vi conjugate or pharmaceutical composition of the invention to the mammal.
- the invention also provides the use of a Vi conjugate of the invention in the manufacture of a medicament for preventing or treating typhoid fever in a mammal.
- the immune response raised by these methods and uses will generally include an antibody response, preferably a protective antibody response.
- Methods for assessing antibody responses after saccharide immunisation are well known in the art.
- the antibody response is preferably an IgA or IgG response.
- the immune response may be prophylactic and/or therapeutic.
- the mammal is preferably a human.
- compositions of the invention will generally be administered directly to a patient.
- Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral, vaginal, topical, transdermal, intradermal, ocular, nasal, aural, or pulmonary administration. Injection or intranasal administration is preferred.
- the invention may be used to elicit systemic and/or mucosal immunity.
- Vaccines prepared according to the invention may be used to treat both children (including infants) and adults.
- a subject may be less than 1 year old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old.
- Preferred subjects for receiving the vaccines are the young (e.g. ⁇ 5 years old).
- the vaccines are not suitable solely for these groups, however, and may be used more generally in a population.
- Treatment can be by a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. In a multiple dose schedule the various doses may be given by the same or different routes e.g. a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc. Administration of more than one dose (typically two doses) is particularly useful in immunologically naive patients. Multiple doses will typically be administered at least 1 week apart (e.g. about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.). An example schedule provides a first dose at 6 weeks of age and a second dose at 10 weeks of age, to coincide with existing infant immunisations (co-administration with EPI vaccines). This primary schedule may be followed by a booster dose after a child's first birthday.
- a parenteral prime and mucosal boost e.
- Conjugates of the invention may be combined with non-Vi antigens into a single composition for simultaneous immunisation against multiple pathogens.
- conjugates may be administered to patients at substantially the same time as (e.g. during the same medical consultation or visit to a healthcare professional or vaccination centre) other vaccines.
- Antigens for use in these combination vaccines or for concomitant administration include, for instance, immunogens from Streptococcus ⁇ g ⁇ l ⁇ cti ⁇ e, Staphylococcus aureus and/or Pseudomonas aeuruginosa, hepatitis A virus, hepatitis B virus, Neisseria meningitidis (such as saccharides or conjugated saccharides, for serogroups A, C, Wl 35 and/or Y), Streptococcus pneumoniae (such as saccharides or conjugated saccharides), etc.
- a composition may comprise a Vi conjugate of the invention in combination with a Salmonella paratyphi A antigen, such as an H or O antigen (e.g. an O:2 saccharide antigen), to provide a bivalent typhoid vaccine.
- a composition may comprise a Vi conjugate of the invention in combination with a Salmonella typhimurium antigen, such as an H or O antigen (e.g. an O:9 saccharide).
- a composition may comprise a Vi conjugate of the invention in combination with a Salmonella enteritidis antigen, such as an H or O antigen (e.g. an 0:4,5 saccharide).
- composition comprising X may consist exclusively of X or may include something additional e.g. X + Y.
- animal (and particularly bovine) materials are used in the culture of cells, they should be obtained from sources that are free from transmissible spongiform encaphalopathies (TSEs), and in particular free from bovine spongiform encephalopathy (BSE). Overall, it is preferred to culture cells in the total absence of animal-derived materials.
- TSEs transmissible spongiform encaphalopathies
- BSE bovine spongiform encephalopathy
- a compound is administered to the body as part of a composition then that compound may alternatively be replaced by a suitable prodrug.
- Figure 1 depicts the structural formula of S. typhi Vi ( ⁇ l,4-N- acetyl galactosaminouronic acid).
- Figure 2 shows a reaction scheme for the preparation of derivatised proteins.
- Figure 3 shows a SEC analysis of CRMi 97 and CRMi 97 derivatised with ADH.
- Figure 4 shows a SEC analysis of tetanus toxoid derivatised with ADH.
- Figure 5 shows SDS page patterns of, from left to right, TT, TT A DH, CRM and CRM A DH-
- Figure 6 shows a reaction scheme for the preparation of a Vi-protein conjugate.
- Figure 7 and Figure 8 show gel filtration profiles of Vi and tetanus toxoid on Sephacryl S-1000.
- Figure 9 shows a gel filtration profile of "pool 1”
- Figure 10 shows a profile of "pool 2”.
- Figure 11 shows a SEC analysis of Vi-TT ADH-
- Figure 12 shows a SDS-PAGE profile (gel 3-8 %) of VI-CRM ADH reaction mixtures after dialysis.
- Lane 2 is CRM ADH (5 ⁇ g)
- Lanes 3 - 5 are 10 ⁇ l of the reactions mixtures after dialysis of Lots 04 - 06 respectively.
- Figure 13 shows the purification of Lot 06 on Sephacryl S-1000.
- Figure 14 shows a SDS-PAGE profile (gel 3-8%) of: "2" - CRM ADH 3 ⁇ g, "3" - reaction mixture 10 ⁇ l and fractions 1 - 11 and 12 - 22 of the purification of Lot 06.
- Figures 15 to 17 show SEC analyses comparing pools 1 obtained from Lots 04 - 06, pools 2 obtained from Lots 04 - 06, and pools 1 and 2 obtained from Lot 06.
- Figure 18 shows the average anti-Vi antibody values.
- Figures 18A and 18B show data from different lots of conjugates, but the control groups are the same in each.
- the precipitate was suspended in ethanol (96%, 1 10 ml) and mixed overnight at RT before subsequent centrifugation at 18000g for 50 minutes, after which the resulting precipitate was discarded.
- 0.1 M NaCl was added to the supernatant to form a gel which was centrifuged at 18000g for 10 minutes.
- the precipitate was collected and washed with ethanol, then solubilised in aqueous NaCl (1 M, 50 ml) and filtered.
- the retentate was brought to 80 % ethanol and centrifuged at 18000g for 10 minutes.
- the precipitate was again washed with ethanol.
- One part of the precipitate remained in suspension (Lot A).
- the precipitate was then washed with water, centrifuged and resuspended in ethanol (85%) and mixed until completely solubilised.
- the solution was passed through SPlO and carbon filters.
- the filtrate was precipitated with NaCl 0.2 M and centrifuged at 18000 g for 5 minutes.
- the precipitate was resuspended in NaCl 1.0 M to give a concentration of 3-5 mg/mL. This solution was diafiltrated against water and 0.22 ⁇ M filtered.
- the purification process has a good yield and provides saccharide with good purity (less than 0.5% protein, less than 0.01% nucleic acid) and is gentle enough to preserve high levels of O-acetylation.
- MES 2-(N- morpholino)ethanesulfonic acid
- the reaction was carried out for 1 hour at room temperature.
- the reaction mixture was dialysed against 0.2 M NaCl, 5 mM MES buffer, pH 7.05, 2-8 0 C and 5 mM MES buffer, pH 7.00, 2-8 0 C. Derivatised tetanus toxoid (86 % yield) was obtained.
- the reaction mixture was dialyzed overnight against 5 mM MES buffer, NaCl 0.2 M, pH 7.0 and then against 5 mM MES buffer, pH 7.0 at 4°C. Protein was measured by microBCA analysis (yield of 75-85%). Sucrose 10 % w/v was added to the product and it was stored at -20 0 C. Rather than store with sucrose, though, it can be stored in 5mM MES, pH 7.0.
- TNBS method derivatisation of the proteins with ADH was verified by a colorimetric method (TNBS method).
- the molar ratio of ADH to TT was measured by MS Maldi-Tof as about 1 1.
- the molar ratio of ADH to CRMi 97 was measured by MS Q-Tof. This showed the formation of several products characterised by the presence of a different number of linkers bound to the protein (from 3 to 10, the principal product containing 6 bound linkers).
- the derivatised proteins were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 3-8% tris-acetate gels (NuPAGE).
- SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- NuPAGE tris-acetate gels
- the samples (5-20 ⁇ l to have a protein content of 5 ⁇ g) were added of DTT 0.5M (1/5 v/v) and of NuPAGE LDS Sample Buffer (1/5 v/v).
- the mixtures were heated at 100 0 C for 1 minute and the samples applied to the wells.
- the gel was subjected to electophoresis at 30 mA in Tris-Acetate SDS Running Buffer (Invitrogen). At the end it was stained with Simply Blue Safe Stain (Invitrogen).
- EDAC (4.4 mg) was added to Vi (4.6 mg) in a buffered solution at pH 6.08 (1.5 ml 200 mM MES buffer) and allowed to react for 2 minutes at room temperature.
- the reaction mixture was dialysed against 0.2 M NaCl, 10 mM phosphate buffer, pH 7.07, 4°C and purified by Sephacryl S-1000 (1.5 x 90 cm) in 10 mM phosphate buffer, 200 mM NaCl, pH 7.00.
- Sephacryl S-1000 1.5 x 90 cm
- two different pools were collected (see Figure 7), which were characterised by a different MW.
- Gel filtration profiles of Vi and of TT on Sephacryl S-1000 show (see Figure 7 and Figure 8):
- Pool 1 should therefore contain less free Vi than pool 2.
- Figure 9 shows the profile on Sephacryl SlOOO (1.6 x 90 cm) of the un-conjugated protein with 10 mM phosphate, NaCl 200 mM, pH 7 at a flow of 0.2 ml/min.
- Figure 10 shows the profile on Sephacryl S 1000 (in the same conditions) of free Vi.
- the conjugate was characterised by SEC analysis (see Figure 1 1, 214 nm, TSK gel 6000; phosphate buffer 10OmM + NaCl 100 mM + 5% CH 3 CN, pH 7.0):
- PSVi-TT ADH pool 1 polysaccharide 26.8 ⁇ g/ml; protein 64.64 ⁇ g/ml
- PSVi-TT ADH pool 2 polysaccharide 82.8 ⁇ g/ml; protein 123.68 ⁇ g/ml
- EDAC (4.4 mg) was added to Vi (4.6 mg, giving a EDAC:Vi molar ratio of about 1.43:1) in a buffered solution at pH 6.0 (1.65 ml 20 mM MES buffer) and mixed for 2 minutes at room temperature.
- the amount of EDAC was reduced, using molar ratios of 5: 1 and 9: 1.
- CRMl 97 conjugates obtained with these derivatised saccharides were better characterisable and reproducible with ⁇ 5% of unconjugated CRM-ADH.
- the ratio 5:1 was better than 1.4: 1
- the ratio 9:1 was better than 5:1).
- CRM ADH is added in 5 mM MES buffer, pH 7. It is necessary that the final mixture is in MES buffer not lower than 50-60 mM at pH 6 to maintain the pH constant during the reaction itself.
- reaction mixture was purified by Sephacryl S lOOO column (1.5 x 90 cm) in 10 mM sodium phosphate buffer, 200 mM NaCl, pH 7 at 4 0 C.
- CRM ADH can be removed from the conjugate by tangential ultrafiltration (10OK or 300K membrane) as follows: reaction mixture diluted from 15 to 50 ml with 10 mM phosphate buffer pH 7.2; membrane: Vivaflow 200 cm 2 IOOK (regenerated cellulose); P 1n : 1.2-1.3 bar; P oul : 0.4 bar; flow: 22.4 ml/min; permeate volume: 1.4 L; (28 cycles with 10 mM phosphate buffer pH 7.2); final retentate volume of 152 ml.
- the conjugate was characterised by microBCA (protein content), acridine orange titration and NMR/HPAEC-PAD (saccharide content), 1 H NMR (O-acetyl level and EDAC derivative quantification), HPLC and SDS-PAGE.
- Figure 13 shows the purification of Lot 06 on Sephacryl S-1000 (Sephacryl SlOOO 1.6cm x 90cm; Flow: 0.2 ml/min, Eluent: 200 mM NaCl, 10 mM NaH 2 PO 4 , pH 7.0).
- Fractions (1-1 1 and 12-22 for Lot 06) were collected for each Lot in two different pools, distinguished by MW. The first pool (earlier fractions) was purified of free saccharide, whilst the second contained an undetermined amount of free saccharide.
- the pools were dialyzed against 2 mM NaH 2 PO ⁇ pH 7.5, at 4°C, overnight and contents were:
- Figures 15 to 17 show comparative SEC analyses of these pools (TSKgel 6000PW (TosoHaas) analytical column (7.5 mm x 30.0 cm), eluent: 100 mM NaCl, 100 mM NaH 2 PO 4 , 5% CH 3 CN, pH 7.2; Flow: 0.5 mL/min; V 0 : -13.5 min - V 1 : -27.8 min).
- the conjugate was sterile filtered (0.22 ⁇ m), aliquoted and stored at -80 0 C (4°C). Determination ⁇ /in vivo activity
- Vi-TT conjugates prepared in the previous example were used.
- VJ-CRM ADH Lot 03 was prepared using substantially the same conditions as VJ-CRM ADH Lots 04 to 06.
- Vi-CRMADH Lot 01 was also prepared using substantially the same conditions as VI-CRM ADH Lots 04 to 06, but with the difference that the ratio CRM ADH /V ⁇ (W) was equal to 1.
- the hyperimmune mouse serum immunized with Vi-rEPA was obtained from the NIH.
- mice Balb/c female mice were divided in fourteen groups of eight mice each and were subcutaneously immunized with 2.5 ⁇ g of Vi, Vi-conjugate, or a physical mixture of Vi and ADH-derivatized carrier protein. Only groups 13 and 14 received 10 ⁇ g of immunization dose.
- Groups 9 to 12 received alum as adjuvant, while the adjuvant for groups 13 & 14 was complete Freund's adjuvant (CFA, 1 st injection) and incomplete Freund's adjuvant (IFA, 2nd & 3rd injection).
- CFA complete Freund's adjuvant
- IFA incomplete Freund's adjuvant
- 96-well ELISA plates (Maxisorp, Nunc) were coated with 100 ⁇ l of l ⁇ g/ml Vi in carbonate buffer (0.05M, pH: 9.6) and left overnight at 4°C. Vi used for coating was purified from Citrobacter freundii WR701 1. The following morning, the plates were blocked with 200 ⁇ l/well of 5% fat-free milk in PBS-Tween 20 (PBST, 0.05%) for 1 hour at room temperature (RT). After washing with PBST, 100 ⁇ l/well of mouse sera (1 :200 diluted in PBST with 0.1% BSA) were incubated for 2 hours at RT.
- PBST PBS-Tween 20
- alkaline phosphatase-conjugated goat anti- mouse IgG secondary antibody (Sigma A3438, 1 : 10000 diluted in PBST, 0.1% BSA) was incubated at 100 ⁇ l/well for 1 hour at RT.
- Alkaline phosphatase substrate (p-NPP, Sigma N2765) was solved in diethanolamine buffer (I M, pH: 9.8) and was incubated after another wash for 1 hour at RT. Plates were read at 405 and 490 nm using an ELISA reader. Absorbance values used for antibody units determination were obtained by subtracting 490 nm to 405 nm values. Antibody units are expressed relative to a hyperimmune mouse anti-Vi standard serum, after Hill Plot analysis.
- a hyperimmune mouse serum immunized with Vi-rEPA was used as internal positive control.
- Vaccine Adjuvants Preparation Methods and Research Protocols (Volume 42 of Methods in Molecular Medicine series). ISBN: 1-59259-083-7. Ed. O'Hagan.
Abstract
Description
Claims
Priority Applications (14)
Application Number | Priority Date | Filing Date | Title |
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AU2009259017A AU2009259017B2 (en) | 2008-06-13 | 2009-06-12 | Conjugated Vi saccharides |
NZ590089A NZ590089A (en) | 2008-06-13 | 2009-06-12 | Conjugated vi saccharides |
EP09762071.0A EP2303333B1 (en) | 2008-06-13 | 2009-06-12 | Conjugated vi saccharides |
SI200931193T SI2303333T1 (en) | 2008-06-13 | 2009-06-12 | Conjugated vi saccharides |
US12/997,411 US20110142876A1 (en) | 2008-06-13 | 2009-06-12 | Conjugated vi saccharides |
DK09762071.0T DK2303333T3 (en) | 2008-06-13 | 2009-06-12 | Conjugated Vi saccharides |
CA2727565A CA2727565C (en) | 2008-06-13 | 2009-06-12 | Conjugated vi saccharides |
BRPI0915517A BRPI0915517B8 (en) | 2008-06-13 | 2009-06-12 | method for producing a vi conjugate, conjugate, e, pharmaceutical composition |
CN2009801278952A CN102089009B (en) | 2008-06-13 | 2009-06-12 | Conjugated Vi saccharides |
PL09762071T PL2303333T3 (en) | 2008-06-13 | 2009-06-12 | Conjugated vi saccharides |
ES09762071.0T ES2537019T3 (en) | 2008-06-13 | 2009-06-12 | Saccharides Vi conjugates |
ZA2010/09304A ZA201009304B (en) | 2008-06-13 | 2010-12-23 | Conjugated vi saccharides |
US13/661,409 US9475846B2 (en) | 2008-06-13 | 2012-10-26 | Conjugated Vi saccharides |
HRP20150527TT HRP20150527T1 (en) | 2008-06-13 | 2015-05-18 | Conjugated vi saccharides |
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GBGB0810894.6A GB0810894D0 (en) | 2008-06-13 | 2008-06-13 | Conjugated saccharide |
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US12/997,411 A-371-Of-International US20110142876A1 (en) | 2008-06-13 | 2009-06-12 | Conjugated vi saccharides |
US13/661,409 Continuation US9475846B2 (en) | 2008-06-13 | 2012-10-26 | Conjugated Vi saccharides |
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US (2) | US20110142876A1 (en) |
EP (1) | EP2303333B1 (en) |
CN (1) | CN102089009B (en) |
AU (1) | AU2009259017B2 (en) |
BR (1) | BRPI0915517B8 (en) |
CA (1) | CA2727565C (en) |
CY (1) | CY1116379T1 (en) |
DK (1) | DK2303333T3 (en) |
ES (1) | ES2537019T3 (en) |
GB (1) | GB0810894D0 (en) |
HR (1) | HRP20150527T1 (en) |
HU (1) | HUE025512T2 (en) |
NZ (1) | NZ590089A (en) |
PL (1) | PL2303333T3 (en) |
PT (1) | PT2303333E (en) |
SI (1) | SI2303333T1 (en) |
WO (1) | WO2009150543A2 (en) |
ZA (1) | ZA201009304B (en) |
Cited By (7)
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CN102120761A (en) * | 2010-12-24 | 2011-07-13 | 新疆维吾尔自治区畜牧科学院兽医研究所 | Acapsular type staphylococcus aureus extracellular polysaccharide-protein conjugate and preparation method thereof |
WO2013038375A2 (en) | 2011-09-14 | 2013-03-21 | Novartis Ag | Methods for making saccharide-protein glycoconjugates |
WO2015029056A1 (en) | 2013-08-24 | 2015-03-05 | Bharat Biotech International Limited | A bacterial vaccine and methods for manufacture thereof |
EP2870974A1 (en) * | 2013-11-08 | 2015-05-13 | Novartis AG | Salmonella conjugate vaccines |
WO2017187448A1 (en) * | 2016-04-25 | 2017-11-02 | National Institute Of Immunology | A novel conjugate for vaccination against typhoid comprising chemical conjugate of vi polysaccharide and flagellin, a process for producing the same and a composition comprising the conjugate |
WO2021099982A1 (en) | 2019-11-22 | 2021-05-27 | Glaxosmithkline Biologicals Sa | Dosage and administration of a bacterial saccharide glycoconjugate vaccine |
US11147866B2 (en) | 2016-09-02 | 2021-10-19 | Sanofi Pasteur Inc. | Neisseria meningitidis vaccine |
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CN102120761A (en) * | 2010-12-24 | 2011-07-13 | 新疆维吾尔自治区畜牧科学院兽医研究所 | Acapsular type staphylococcus aureus extracellular polysaccharide-protein conjugate and preparation method thereof |
CN102120761B (en) * | 2010-12-24 | 2013-02-06 | 新疆维吾尔自治区畜牧科学院兽医研究所 | Preparation method of acapsular type staphylococcus aureus extracellular polysaccharide-protein conjugate |
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KR20200133398A (en) * | 2013-08-24 | 2020-11-27 | 브하라트 바이오테크 인터내셔날 리미티드 | A bacterial vaccine and methods for manufacture thereof |
EP3035957A4 (en) * | 2013-08-24 | 2017-05-03 | Bharat Biotech International Limited | A bacterial vaccine and methods for manufacture thereof |
KR102183569B1 (en) * | 2013-08-24 | 2020-11-26 | 브하라트 바이오테크 인터내셔날 리미티드 | A bacterial vaccine and methods for manufacture thereof |
EP3329935A1 (en) | 2013-08-24 | 2018-06-06 | Bharat Biotech International Limited | A bacterial vaccine and methods for manufacture thereof |
EP3335727A1 (en) | 2013-08-24 | 2018-06-20 | Bharat Biotech International Limited | A bacterial vaccine and methods for manufacture thereof |
WO2015068129A1 (en) * | 2013-11-08 | 2015-05-14 | Novartis Ag | Salmonella conjugate vaccines |
EA033491B1 (en) * | 2013-11-08 | 2019-10-31 | Glaxosmithkline Biologicals Sa | Salmonella conjugate vaccines |
US10098947B2 (en) | 2013-11-08 | 2018-10-16 | Glaxosmithkline Biologicals Sa | Salmonella conjugate vaccines |
JP2016536317A (en) * | 2013-11-08 | 2016-11-24 | グラクソスミスクライン バイオロジカルズ ソシエテ アノニム | Salmonella conjugate vaccine |
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WO2017187448A1 (en) * | 2016-04-25 | 2017-11-02 | National Institute Of Immunology | A novel conjugate for vaccination against typhoid comprising chemical conjugate of vi polysaccharide and flagellin, a process for producing the same and a composition comprising the conjugate |
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WO2021099982A1 (en) | 2019-11-22 | 2021-05-27 | Glaxosmithkline Biologicals Sa | Dosage and administration of a bacterial saccharide glycoconjugate vaccine |
Also Published As
Publication number | Publication date |
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EP2303333A2 (en) | 2011-04-06 |
PL2303333T3 (en) | 2015-11-30 |
BRPI0915517A2 (en) | 2017-06-20 |
CN102089009A (en) | 2011-06-08 |
BRPI0915517B1 (en) | 2020-10-06 |
US20110142876A1 (en) | 2011-06-16 |
HRP20150527T1 (en) | 2015-06-19 |
AU2009259017A1 (en) | 2009-12-17 |
CA2727565C (en) | 2017-01-10 |
WO2009150543A3 (en) | 2010-05-06 |
SI2303333T1 (en) | 2015-06-30 |
US20130142822A1 (en) | 2013-06-06 |
ES2537019T3 (en) | 2015-06-01 |
GB0810894D0 (en) | 2008-07-23 |
CY1116379T1 (en) | 2017-02-08 |
NZ590089A (en) | 2012-12-21 |
CN102089009B (en) | 2013-12-04 |
DK2303333T3 (en) | 2015-05-26 |
HUE025512T2 (en) | 2016-05-30 |
US9475846B2 (en) | 2016-10-25 |
ZA201009304B (en) | 2012-08-29 |
EP2303333B1 (en) | 2015-02-18 |
BRPI0915517B8 (en) | 2021-05-25 |
PT2303333E (en) | 2015-06-08 |
AU2009259017B2 (en) | 2014-12-11 |
CA2727565A1 (en) | 2009-12-17 |
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