Patent Application
20110142876
This application claims the benefit of United Kingdom patent application 0810894.6, filed 13 Jun. 2008, the complete contents of which are incorporated herein by reference.
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. However, the vaccine is unable to establish immunological memory and is ineffective in infants or toddlers [1]. 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].
It is an object of the invention to provide new processes for the production of Vi conjugate vaccines that may be employed on an industrial scale.
The inventors have devised a new method for manufacturing Vi-conjugates and have also produced a new conjugate comprising Vi coupled to CRM197.
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 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDAC), are simultaneously added to a solution containing a carrier protein, such as CRM197 or tetanus toxoid (TT), to give a derivatised carrier protein.
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.
Following the derivatisation of the carrier protein, 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. For Vi activation, 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. >1.5:1 and ideally ≧3:1, such as 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or higher. Ratios up to 200:1 might be used. These ratios are higher than the ones used in reference 3. Vi activation can be performed at room temperature e.g. in about 2 minutes.
Thus, the method comprises the steps of:
Steps a) and b) may be performed in any order.
After 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-CRM197 conjugate, wherein Vi is combined with derivatised CRM197. The method comprises the steps of:
This aspect of the invention also provides a method for preparing a Vi-CRM197 conjugate, comprising the step of:
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. However, 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-CRM197 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, α-1,4-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 [11].
The Vi saccharide used in the present invention may be chemically modified relative to the capsular saccharide as found in nature. For example, the Vi saccharide may be partially de-O-acetylated, de-N-acetylated (partially or fully), N-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].
In addition, it has been found by double immunodiffusion that 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. However, 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.
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 particularly useful method is disclosed in reference 15, which is incorporated herein by reference.
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.
Many techniques for precipitating soluble polysaccharides, such as Vi, are known in the art. Preferred methods use one or more cationic detergents. The detergents preferably have the following general formula:
wherein:
Particularly preferred detergents for use in the method are tetrabutylammonium and cetyltrimethylammonium salts (e.g. the bromide salts). Cetyltrimethylammonium bromide (‘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.
Vi can be released into media during culture. Accordingly, 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.).
Precipitated Vi may be collected by centrifugation prior to solubilisation.
After precipitation, 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. methanol, propan-1-ol, propan-2-ol, butan-1-ol, butan-2-ol, 2-methyl-propan-1-ol, 2-methyl-propan-2-ol, diols etc.)
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). Preferred solvent mixtures are alcohol:water mixtures, with a preferred ratio of between around 70:30 and around 95:5 (e.g. 75:25, 80:20, 85:15, 90:10).
Compared with conventional processes for preparing capsular polysaccharides, the two-step process of precipitation followed by alcohol extraction is quicker and simpler.
In contrast to the process described in ref. 17, the process uses cationic detergent rather than anionic detergent. Unlike the process of ref. 18, precipitation does not require an inert porous support.
Furthermore, unlike prior art processes, an alcohol is used to re-solubilise Vi rather than to precipitate it.
After re-solubilisation, 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, OD275 nm<0.2.
Size filtration or ultrafiltration may be used.
If Vi is hydrolysed, 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.
Pure Vi is a poor immunogen. For protective efficacy, therefore, 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]. As described above, a saccharide may be conjugated to a carrier protein or to a mixture of different carrier proteins. Similarly, 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) CRM197 as a carrier protein.
The CRM197 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 CRM197 is preferably via a —NH2 group e.g. in the side chain of a lysine residue in CRM197, or of an arginine residue. Attachment to CRM197 may also be via a —SH group e.g. in the side chain of a cysteine residue. Alternatively, Vi may be attached to CRM197 via a linker molecule as described below.
Vi will typically be activated or functionalised prior to conjugation. A preferred technique uses carbodiimides (e.g. 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDAC)). 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 —NH2 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 β-propionamido [38], nitrophenyl-ethylamine [39], haloacyl halides [40], glycosidic linkages [41], 6-aminocaproic acid [42], N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) [43], C4 to C12 moieties [44], etc. Carbodiimide condensation can also be used [45].
A useful process for linking Vi to CRM197 involves the thiolation of Vi with cystamine or cysteamine (carbodiimide coupling) and subsequent reaction with CRM197 derivatised with SPDP [46].
Another useful process involves the introduction of amino groups into Vi followed by derivatisation with an adipic diester (e.g. adipic acid N-hydroxysuccinimido diester) and reaction with CRM197.
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 (—NH2) 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.
Where the reactions with both Vi and the carrier protein involve amines then it is preferred to use a bifunctional linker, for example a homobifunctional 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′-L2-L′, where L′ is carbonyl. Useful L2 groups are straight chain alkyls with 1 to 10 carbon atoms (e.g. C1, C2, C3, C4, C5, C6, C7, C8, C9, C1-10) e.g. —(CH2)4—.
Other 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 CRM197 diphtheria toxin mutant is particularly preferred [47].
Other suitable 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 H. 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.
When the conjugate forms the Vi component in an immunogenic composition of the invention, 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.
The invention provides a pharmaceutical composition comprising (a) Vi conjugate, and (b) a pharmaceutically acceptable carrier. A thorough discussion of such carriers is available in ref. 69.
Microbial infections affect various areas of the body and so the compositions of the invention may be prepared in various forms. For example, 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). The composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray. 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.
Pharmaceutical compositions of the invention are preferably immunogenic compositions, in that they comprise an immunologically effective amount of Vi immunogen. By ‘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 1 μg and 20 μg of saccharide is expected e.g. about 5 μg/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.
Once formulated, the 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. In other embodiments, though, 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 particularly useful adjuvant based around immunostimulatory oligonucleotides is known as IC31™ [116]. Thus 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 5-20 amino acids) including at least one (and preferably multiple) Lys-Arg-Lys tripeptide sequence(s). The oligonucleotide may be a deoxynucleotide comprising 26-mer sequence 5′-(IC)13-3′. The polycationic polymer may be a peptide comprising 11-mer amino acid Lys-Leu-Lys-Leu5-Lys-Leu-Lys.
These and other adjuvant-active substances are discussed in more detail in references 155 & 156.
Antigens and adjuvants in a composition will typically be in admixture.
Compositions may include two or more of said adjuvants. For example, they may advantageously include both an oil-in-water emulsion and 3dMPL, etc.
Specific oil-in-water emulsion adjuvants useful with the invention include, but are not limited to:
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, intraperitoneally, 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. Thus 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 naïve 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.
Conjugates of the invention may be combined with non-Vi antigens into a single composition for simultaneous immunisation against multiple pathogens. As an alternative to making a combined vaccine, 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 agalactiae, Staphylococcus aureus and/or Pseudomonas aeuruginosa, hepatitis A virus, hepatitis B virus, Neisseria meningitidis (such as saccharides or conjugated saccharides, for serogroups A, C, W135 and/or Y), Streptococcus pneumoniae (such as saccharides or conjugated saccharides), etc.
In one embodiment, 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 0:2 saccharide antigen), to provide a bivalent typhoid vaccine. In another embodiment, 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 0:9 saccharide). In another embodiment, 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 O:4,5 saccharide).
The term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X+Y.
The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.
The term “about” in relation to a numerical value x is optional and means, for example, x+10%.
Where 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.
Where a compound is administered to the body as part of a composition then that compound may alternatively be replaced by a suitable prodrug.
The supernatant of a 5 L sample of C. freundii WR7001 in mod-LB was concentrated (20 times) to 250 ml with a 100K membrane. The sample was diafiltered against NaCl 1 M (2.5 L) and then with water (1.5 L), again with a 100K membrane, and the permeate discarded. A 0.22 μm filter was used to remove turbidity. Subsequently, 0.9% CTAB was added to form a Vi-CTAB precipitate. This was centrifuged at 18000 g for 15 minutes and the supernatant discarded.
The precipitate was suspended in ethanol (96%, 110 ml) and mixed overnight at RT before subsequent centrifugation at 18000 g 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 18000 g 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 18000 g for 10 minutes. The precipitate was again washed with ethanol. One part of the precipitate remained in suspension (Lot A). The two parts were collected separately as Lot A (97 mg) and Lot B (170 mg).
In a modified process, the supernatant was concentrated to 8.0-10.0 g/L with a 100K membrane. The sample was diafiltered against NaCl 1 M, Tris 0.1M, EDTA 0.02M pH 7.3 (2.5 L) and then with water (2.5 L), again with a 100K membrane, and the permeate was discarded. Subsequently, 2.0% CTAB was added to form a Vi-CTAB precipitate. The precipitate was centrifuged at 18000 g for 15 minutes and the supernatant discarded. The precipitate was then washed with water, centrifuged and resuspended in ethanol (85%) and mixed until completely solubilised. The solution was passed through SP10 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 μM 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 0-acetylation.
ADH (3.25 per mg of protein) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-hydrochloride (EDAC) (EDAC/protein=0.152 w/w) were added to tetanus toxoid (in 2-(N-morpholino)ethanesulfonic acid (MES) buffer 50-100 mM, pH 6.18 to 6.20). 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° C. and 5 mM MES buffer, pH 7.00, 2-8° C. Derivatised tetanus toxoid (86% yield) was obtained.
ADH (3.5 mg per mg of protein) and EDAC (EDAC/protein=0.15 w/w) were added to CRM197 (11.5 mg/ml in MES buffer 50-100 mM, pH 6.0). The reaction was carried out for 1 hour at room temperature, pH 6.0-6.2. Maintenance of the pH in this range prevented protein precipitation.
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° C. Rather than store with sucrose, though, it can be stored in 5 mM MES, pH 7.0.
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-T of as about 11.
The molar ratio of ADH to CRM197 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 (and, subsequently, the conjugates) were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 3-8% tris-acetate gels (NuPAGE). The samples (5-20 μl to have a protein content of 5 μg) were added of DTT 0.5M (⅕ v/v) and of NuPAGE LDS Sample Buffer (⅕ v/v). The mixtures were heated at 100° 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).
SDS-PAGE patterns and SEC profiles (214 nm, TSK gel 4000; phosphate buffer 100 mM+NaCl 100 mM+5% CH3CN, pH 7.0) of the derivatised proteins were found to be similar to those of the native proteins (see
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.
Derivatised tetanus toxoid (9.2 mg) in a buffered solution at pH 7.0 (1.5 ml 5 mM MES buffer) was allowed to react with the activated Vi for 3 hours at room temperature ([TTADH]=3.15 mg ml−1, [Vi]=1.53 mg ml−1, Ratio TTADH/Vi (w)=2, [EDAC]=1.5 mg ml−1).
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 (1.5×90 cm) in 10 mM phosphate buffer, 200 mM NaCl, pH 7.00. During conjugate purification, two different pools were collected (see
Pool 1 should therefore contain less free Vi than pool 2.
Pools 1 and 2 were dialysed against 2 mM phosphate buffer pH 7.0 and their contents were as follows:
The conjugate was characterised by SEC analysis (see
Three different Vi-CRMADH Lots (“04”, “05”, and “06”) were prepared as follows:
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. (In later experiments, for comparison, the amount of EDAC was reduced, using molar ratios of 5:1 and 9:1. CRM197 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, and the ratio 9:1 was better than 5:1).
Derivatised CRM197 (9.2 mg) in a buffered solution at pH 7.0 (1.085 ml 5 mM MES buffer) was allowed to react with the activated Vi for 3 hours at room temperature ([CRMADH]=3.07 mg ml−1, [Vi]=1.53 mg ml−1, Ratio CRMADH/Vi(w)=2, [EDAC]=1.47 mg ml−1), during which the pH was maintained at 6.0-6.20 by using MES buffer in order to avoid precipitation.
As noted above, CRMADH 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.
The reaction mixture was purified by Sephacryl S1000 column (1.5×90 cm) in 10 mM sodium phosphate buffer, 200 mM NaCl, pH 7 at 4° C.
As an alternative means of purification, CRMADH can be removed from the conjugate by tangential ultrafiltration (100K or 300K membrane) as follows: reaction mixture diluted from 15 to 50 ml with 10 mM phosphate buffer pH 7.2; membrane: Vivaflow 200 cm2 100K (regenerated cellulose); Pin: 1.2-1.3 bar; Pout: 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), 1H NMR (O-acetyl level and EDAC derivative quantification), HPLC and SDS-PAGE.
The pools were dialyzed against 2 mM NaH2PO4, pH 7.5, at 4° C., overnight and contents were:
The conjugate was sterile filtered (0.22 μm), aliquoted and stored at −80° C. (4° C.).
The Vi-TT conjugates prepared in the previous example were used. Vi-CRMADH Lot 03 was prepared using substantially the same conditions as Vi-CRMADH Lots 04 to 06. Vi-CRMADH Lot 01 was also prepared using substantially the same conditions as Vi-CRMADH Lots 04 to 06, but with the difference that the ratio CRMADH/Vi (w) was equal to 1. The hyperimmune mouse serum immunized with Vi-rEPA was obtained from the NIH.
aconjugates were adjuvanted with alum
b1st immunization was adjuvanted with CFA, 2nd and 3rd immunizations with IFA
Balb/c female mice were divided in fourteen groups of eight mice each and were subcutaneously immunized with 2.5 μg of Vi, Vi-conjugate, or a physical mixture of Vi and ADH-derivatized carrier protein. Only groups 13 and 14 received 10 μg of immunization dose.
Three injections of 200 μl each were given every two weeks, with bleedings two weeks after each immunization.
Groups 9 to 12 received alum as adjuvant, while the adjuvant for groups 13 & 14 was complete Freund's adjuvant (CFA, 1st injection) and incomplete Freund's adjuvant (IFA, 2nd & 3rd injection).
The wells of 96-well ELISA plates (Maxisorp, Nunc) were coated with 100 μl of 1 μg/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 WR7011. The following morning, the plates were blocked with 200 μl/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 μl/well of mouse sera (1:200 diluted in PBST with 0.1% BSA) were incubated for 2 hours at RT. After three more washes, alkaline phosphatase-conjugated goat anti-mouse IgG secondary antibody (Sigma A3438, 1: 10000 diluted in PBST, 0.1% BSA) was incubated at 100 μl/well for 1 hour at RT. Alkaline phosphatase substrate (p-NPP, Sigma N2765) was solved in diethanolamine buffer (1M, 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.
Each mouse serum was run in triplicate on three different ELISA plates and data are presented as arithmetic means and standard errors. One antibody unit is defined as the reciprocal of the dilution of the standard sera that gives an optical density equal to 1 in a standard ELISA.
The results are plotted in
Various modifications and variations of the present disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific preferred embodiments, it should be understood that the claims should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure, which are understood by those skilled in the art are intended to be within the scope of the claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0810894.6 | Jun 2008 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2009/006285 | 6/12/2009 | WO | 00 | 2/25/2011 |
