Patent Application
20100266626
The invention relates to vaccines, more particularly those against fungal infections and disease.
The use of β-glucans as anti-fungal vaccines is reviewed in reference 1.
Reference 2 reports the use of various β-glucans in immunisation studies, including laminarin, pustulan and ‘GG-zym’ (soluble C. albicans glucans obtained by digesting glucan ghosts with β-1,3-glucanase). The GG-zym and laminarin glucans were both conjugated to carrier proteins to improve their immunogenicity. Further information on the conjugated laminarin are reported in reference 3.
In reference 2 the GG-zym conjugate was administered with both complete and incomplete Freund's adjuvants. More generally, the β-glucan-containing compositions are disclosed as optionally containing adjuvants. In reference 3 the laminarin conjugate was administered with complete Freund's adjuvant or with cholera toxin adjuvant.
It is an object of the invention to provide further and better glucan-based compositions for inducing protective and/or therapeutic immune responses against infections.
The present invention relates to immunogenic compositions comprising: (a) a glucan containing β-1,3-linkages and/or β-1,6-linkages; and (b) an adjuvant, provided that component (b) is not complete Freund's adjuvant and is not cholera toxin. The glucan may be a single molecular species. In one embodiment, the glucan is conjugated to a carrier protein. In a particular embodiment, the glucan is conjugated to the carrier protein directly. In another particular embodiment, the glucan is conjugated to the carrier protein via a linker.
The present invention also relates to immunogenic compositions comprising a glucan containing β-1,3-linkages and/or β-1,6-linkages, wherein said glucan is a single molecular species and is conjugated to a carrier protein. In one embodiment, the glucan is conjugated to a carrier protein. In a particular embodiment, the glucan is conjugated to the carrier protein directly. In another particular embodiment, the glucan is conjugated to the carrier protein via a linker.
The carrier protein in the immunogenic compositions of the invention can be a bacterial toxin or toxoid, or a mutant thereof. In a particular embodiment, the carrier protein is CRM197.
In certain embodiments, the glucan has a molecular weight of less than 100 kDa (e.g. less than 80, 70, 60, 50, 40, 30, 25, 20, or 15 kDa). In other embodiments, the glucan has 60 or fewer glucose monosaccharide units.
The glucan can be a β-1,3 glucan with some β-1,6 branching. In a particular embodiment, the glucan is a laminarin. In another particular embodiment, the glucan β-1,3-linked glucose residues and β-1,6-linked glucose residues, with a ratio of β-1,3 linked glucose residues to β-1,6-linked residues of at least 8:1 and/or there are one or more sequences of at least five adjacent non-terminal residues linked to other residues only by β-1,3 linkages. For example, the glucan comprises both β-1,3-linked glucose residues and β-1,6-linked glucose residues, with a ratio of β-1,3 linked glucose residues to β-1,6-linked residues of at least 8:1.
The glucan can have exclusively β-1,3 linkages. In a particular embodiment, the glucan is a curdlan. The immunogenic compositions of the invention can include a pharmaceutically acceptable carrier.
The adjuvant can comprise one or more of: an aluminium salt, such as an aluminium hydroxide; an oil-in-water emulsion; an immunostimulatory oligonucleotide; and/or an α-glucosylceramide. The adjuvant can comprise an outer membrane vesicle (OMV). The adjuvant can comprise an immunostimulatory oligonucleotide and a polycationic oligopeptide.
The present invention also relates to methods for raising an immune response in a mammal, comprising administering to the mammal a composition of the invention.
The present invention also relates to processes for purifying glucan comprising a step of separating phlorotannin from the glucan, and to glucans having reduced phlorotannin contamination.
The present invention also relates to methods for making a glucan conjugated to a carrier protein,
wherein the step of conjugation is carried out in a phosphate buffer with >10 mM phosphate; and to conjugates obtained by these methods.
According to the invention, a glucan containing β-1,3-linkages and/or β-1,6-linkages is administered in combination with one or more adjuvant(s). The use of adjuvants has been shown to provide a stronger immune response than when adjuvant is absent. The glucan is preferably used in the form of a conjugate. The adjuvant preferably includes one or more of: an aluminium salt, such as an aluminium hydroxide; an oil-in-water emulsion; an immunostimulatory oligonucleotide; and/or an α-glucosylceramide.
If the glucan is a laminarin then the adjuvant is not complete Freund's adjuvant. Moreover, if the glucan is a laminarin and is for intranasal or intravaginal administration then the adjuvant is not cholera toxin. More generally, the adjuvant is preferably neither complete Freund's adjuvant nor cholera toxin.
Thus the invention provides an immunogenic composition comprising: (a) a glucan containing β-1,3-linkages and/or β-1,6-linkages; and (b) an adjuvant.
The present invention also provides an immunogenic composition comprising a glucan containing β-1,3-linkages and/or β-1,6-linkages, wherein said glucan is a single molecular species and is conjugated to a carrier protein. The inventors have found that glucans that are single molecular species may be more immunogenic than more polydisperse glucans, particularly when the composition also comprises an adjuvant. Preferably, the composition therefore comprises an adjuvant in addition to the glucan.
The present invention also relates to processes for purifying glucan comprising a step of separating phlorotannin from the glucan, and to glucans having reduced phlorotannin contamination. The glucan may be any of the glucans described herein. The inventors have found that glucans purified by known methods may be contaminated by phlorotannin. Phlorotannins are polyphenolic compounds that are found in brown algae and seaweed. Phlorotannins typically comprise a dibenzo-1,4-dioxin skeleton, although many different forms of phlorotannin are known. Phlorotannins have been shown to have a variety of biological effects, including radioprotective and antioxidative effects ([4] and [5]). In particular, phlorotannins have been shown to have effects on the immune system, including anti-inflammatory and anti-allergic effects ([5] and [6]). The anti-immune effects of phlorotannin mean that it may be an undesirable contaminant in glucans, particularly when the glucans are for use as immunogens. The present invention provides processes for purifying glucan comprising a step of separating phlorotannin from the glucan, and to glucans having reduced phlorotannin contamination.
The present invention also provides a method for making a glucan conjugated to a carrier protein, wherein the step of conjugation is carried out in a phosphate buffer with >10 mM phosphate; and to a conjugate obtained by this method. The glucan may be any of the glucans described herein. The inventors have found that glucans conjugated by these methods may provide greater protection when used as vaccines than glucans conjugated by the methods of the prior art (e.g. the method described in references 2 and 3). Without wishing to be bound by theory, it is thought that this effect may be because of reduced aggregation of the resultant conjugate.
Glucans are glucose-containing polysaccharides found inter alia in fungal cell walls. The α-glucans include one or more α-linkages between glucose subunits, whereas β-glucans include one or more β-linkages between glucose subunits. The glucan used in accordance with the invention includes β linkages, and may contain only β linkages (i.e. no a linkages).
The glucan may comprise one or more β-1,3-linkages and/or one or more β-1,6-linkages. It may also comprise one or more β-1,2-linkages and/or β-1,4-linkages, but normally its only 13 linkages will be β-1,3-linkages and/or β-1,6-linkages.
The glucan may be branched or linear.
Full-length native β-glucans are insoluble and have a molecular weight in the megadalton range. It is preferred to use soluble glucans in immunogenic compositions of the invention. Solubilisation may be achieved by fragmenting long insoluble glucans. This may be achieved by hydrolysis or, more conveniently, by digestion with a glucanase (e.g. with a β-1,3-glucanase or a β-1,6-glucanase). As an alternative, short glucans can be prepared synthetically by joining monosaccharide building blocks.
Low molecular weight glucans are preferred, particularly those with a molecular weight of less than 100 kDa (e.g. less than 80, 70, 60, 50, 40, 30, 25, 20, or 15 kDa). It is also possible to use oligosaccharides e.g. containing 60 or fewer (e.g. 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4) glucose monosaccharide units. Within this range, oligosaccharides with between 10 and 50 or between 20 and 40 monosaccharide units are preferred.
The glucan may be a fungal glucan. A ‘fungal glucan’ will generally be obtained from a fungus but, where a particular glucan structure is found in both fungi and non-fungi (e.g. in bacteria, lower plants or algae) then the non-fungal organism may be used as an alternative source. Thus the glucan may be derived from the cell wall of a Candida, such as C. albicans, or from Coccidioides immitis, Trichophyton verrucosum, Blastomyces dermatidis, Cryptococcus neoformans, Histoplasma capsulatum, Saccharomyces cerevisiae, Paracoccidioides brasiliensis, or Pythiumn insidiosum.
There are various sources of fungal β-glucans. For instance, pure β-glucans are commercially available e.g. pustulan (Calbiochem) is a β-1,6-glucan purified from Umbilicaria papullosa. β-glucans can be purified from fungal cell walls in various ways. Reference 7, for instance, discloses a two-step procedure for preparing a water-soluble β-glucan extract from Candida, free from cell-wall mannan, involving NaClO oxidation and DMSO extraction. The resulting product (‘Candida soluble β-D-glucan’ or ‘CSBG’) is mainly composed of a linear β-1,3-glucan with a linear β-1,6-glucan moiety. Similarly, reference 2 discloses the production of GG-zym from C. albicans. Such glucans from C. albicans, include (a) β-1,6-glucans with β-1,3-glucan lateral chains and an average degree of polymerisation of about 30, and (b) β-1,3-glucans with β-1,6-glucan lateral chains and an average degree of polymerisation of about 4.
In some embodiments of the invention, the glucan is a β-1,3 glucan with some β-1,6 branching, as seen in e.g. laminarins. Laminarins are found in brown algae and seaweeds. The β(1-3):β(1-6) ratios of laminarins vary between different sources e.g. it is as low as 3:2 in Eisenia bicyclis laminarin, but as high as 7:1 in Laminaria digititata laminarin [8]. Thus the glucan used with the invention may have a β(1-3):β(1-6) ratio of between 1.5:1 and 7.5:1 e.g. about 2:1, 3:1, 4:1, 5:1, 6:1 or 7:1. Optionally, the glucan may have a terminal mannitol subunit, e.g. a 1,1-α-linked mannitol residue [9]. The glucan may also comprise mannose subunits.
In other embodiments, the glucan has exclusively or mainly β-1,3 linkages, as seen in curdlan. The inventors have found that these glucans may be more immunogenic than glucans comprising other linkages, particularly glucans comprising β-1,3 linkages and a greater proportion of β-1,6 linkages. Thus the glucan may be made solely of β-1,3-linked glucose residues (e.g. linear β-D-glucopyranoses with exclusively 1,3 linkages). Optionally, though, the glucan may include monosaccharide residues that are not β-1,3-linked glucose residues e.g. it may include β-1,6-linked glucose residues. The ratio of β-1,3-finked glucose residues to these other residues should be at least 8:1 (e.g. ≧9:1, ≧10:1, ≧11:1, ≧12:1, ≧13:1, ≧14:1, ≧15:1, ≧16:1, ≧17:1, ≧18:1, ≧19:1, ≧20:1, ≧25:1, ≧30:1, ≧35:1, ≧40:1, ≧45:1, ≧50:1, ≧75:1, ≧100:1, etc.) and/or there are one or more (e.g. ≧1, ≧2, ≧3, ≧4, ≧5, ≧6, ≧7, ≧8, ≧9, ≧10, ≧11, ≧12, etc.) sequences of at least five (e.g. ≧5, ≧6, ≧7, ≧8, ≧9, ≧10, ≧11, ≧12, ≧13, ≧14, ≧15, ≧16, ≧17, ≧18, ≧19, ≧20, ≧30, ≧40, ≧50, ≧60, etc.) adjacent non-terminal residues linked to other residues only by β-1,3 linkages. By “non-terminal” it is meant that the residue is not present at a free end of the glucan. In some embodiments, the adjacent non-terminal residues may not include any residues coupled to a carrier molecule, linker or other spacer as described below. The inventors have found that the presence of five adjacent non-terminal residues linked to other residues only by β-1,3 linkages may provide a protective antibody response, e.g. against C. albicans.
In further embodiments, a composition may include two different glucans e.g. a first glucan having a β(1-3):β(1-6) ratio of between 1.5:1 and 7.5:1, and a second glucan having exclusively or mainly β-1,3 linkages. For instance a composition may include both a laminarin glucan and a curdlan glucan.
Where a β-glucan includes both β-1,3 and β-1,6 linkages at a desired ratio and/or sequence then this glucan may be found in nature (e.g. a laminarin), or it may be made artificially. For instance, it may be made by chemical synthesis, in whole or in part. Methods for the chemical synthesis of β-1,3/β-1,6 glucans are well known in the art, for example from references 10-20. β-glucan including both β-1,3 and β-1,6 linkages at a desired ratio may also be made starting from an available glucan and treating it with a β-1,6-glucanase (also known as glucan endo-1,6-β-glucosidase, 1,6-β-D-glucan glucanohydrolase, etc.; EC 3.2.1.75) or a β-1,3-glucanase (such as an exo-1,3-glucanase (EC 3.2.1.58) or an endo-1,3-glucanase (EC 3.2.1.39) until a desired ratio and/or sequence is reached.
When a glucan containing solely of β-1,3-linked glucose is desired then β-1,6-glucanase treatment may be pursued to completion, as β-1,6-glucanase will eventually yield pure β-1,3 glucan. More conveniently, however, a pure β-1,3-glucan may be used. These may be made synthetically, by chemical and/or enzymatic synthesis e.g. using a (1→3)-β-D-glucan synthase, of which several are known from many organisms (including bacteria, yeasts, plants and fungi). Methods for the chemical synthesis of β-1,3 glucans are well known in the art, for example from references 21-24. As a useful alternative to synthesis, a natural β-1,3-glucan may be used, such as a curdlan (linear β-1,3-glucan from an Agrobacterium previously known as Alcaligenes faecalis var. myxogenes; commercially available e.g. from Sigma-Aldrich catalog C7821) or paramylon (β-1,3-glucan from Euglena). Organisms producing high levels of β-1,3-glucans are known in the art e.g. the Agrobacterium of refs. 25 & 26, or the Euglena gracilis of ref. 27.
Laminarin and curdlan are typically found in nature as high molecular weight polymers e.g. with a molecular weight of at least 100 kDa. They are often insoluble in aqueous media. In their natural forms, therefore, they are not well suited to immunisation. Thus the invention may use a shorter glucan e.g. those containing 60 or fewer glucose monosaccharide units (e.g. 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4). A glucan having a number of glucose residues in the range of 2-60 may be used e.g. between 10-50 or between 20-40 glucose units. A glucan with 25-30 glucose residues is particularly useful. Suitable glucans may be formed e.g. by acid hydrolysis of a natural glucan, or by enzymatic digestion e.g. with a glucanase, such as a β-1,3-glucanase. A glucan with 11-19, e.g. β-19 and particularly 15 or 17, glucose monosaccharide units is also useful. In particular, glucans with the following structures (A) or (B) are specifically envisaged for use in the present invention:
The glucan (as defined above) is preferably a single molecular species. In this embodiment, all of the glucan molecules are identical in terms of sequence. Accordingly, all of the glucan molecules are identical in terms of their structural properties, including molecular weight etc. Typically, this form of glucan is obtained by chemical synthesis, e.g. using the methods described above. For example, reference 22 describes the synthesis of a single β-1,3 linked species. Alternatively, in other embodiments, the glucan may be obtained from a natural glucan, e.g. a glucan from L. digitata, Agrobacterium or Euglena as described above, with the glucan being purified until the required single molecular species is obtained. Natural glucans that have been purified in this way are commercially available. A glucan that is a single molecular species may be identified by measuring the polydispersity (Mw/Mn) of the glucan sample. This parameter can conveniently be measured by SEC-MALLS, for example as described in reference 28. Suitable glucans for use in this embodiment of the invention have a polydispersity of about 1, e.g. 1.01 or less.
Solubility of natural glucans, such as curdlan, can be increased by introducing ionic groups (e.g. by sulfation, particularly at O-6 in curdlan). Such modifications may be used with the invention, but are ideally avoided as they may alter the glucan's antigenicity.
When glucans are isolated from natural sources, they may be isolated in combination with contaminants. For example, the inventors have found that glucans may be contaminated with phlorotannin, which is identifiable by ultraviolet-visible (UV/VIS) spectroscopy. This problem is particularly common when the glucan is isolated from a brown alga or seaweed. For example, the UV spectrum of a commercially-available laminarin extracted from Laminaria digitata includes an absorption peak resulting from the presence of phlorotannin contamination (
The presence of phlorotannin in a sample of glucan may affect the biological properties of the glucan. Accordingly, it may be desirable to remove phlorotannin from the sample, especially when the glucan is for medical or nutritional use.
In another aspect, the invention provides a process for purifying glucan comprising a step of separating phlorotannin from the glucan. The glucan may be any of the glucans described above. The process of the invention produces glucan having reduced phlorotannin contamination. Accordingly, in another aspect, the invention also provides glucan having reduced phlorotannin contamination. The glucan of the invention may be obtained by, or obtainable by, the process of the invention. The process of the invention may also be used to separate other contaminants from the glucan, for example other organic molecules that may be present in the glucan.
The step of separating phlorotannin from the glucan may be carried out before the glucan is processed for use. For example, this step may be carried out before conjugation of the glucan to a carrier protein, as described below. In particular, this step may be carried our before the glucan is activated or functionalised prior to conjugation. In another example, this step is carried out before processing of the glucan into a nutritional product.
The glucan of these aspects of the invention has reduced phlorotannin contamination. Typically, phlorotannin contamination is measured by UV/VIS spectroscopy. A UV absorbance peak at ˜270 nm is generally indicative of phlorotannin contamination. When this phlorotannin contamination is present, the method of the invention will decrease absorbance at ˜270 nm. Similarly, the glucan will have reduced absorbance at ˜270 nm. Preferably, the glucan demonstrates little UV absorbance at 270 nm. This is a particular advantage over glucans of the prior art, which show an absorbance peak at ˜270 nm. For example, the glucan has a UV absorbance at 270 nm of less than 0.17 at 1 mg/ml in water. An absorbance of <0.15, <0.10 or even <0.05 is preferred. The UV absorbance spectrum of the glucan may be characterised in other ways. For example, between 220 nm and 300 nm the UV spectrum does not exhibit either a shoulder or peak at around 270 nm; between 250 nm and 275 nm the UV spectrum does not increase; and/or between 250 nm and 275 nm the UV spectrum has neither a maximum point nor a point of inflexion. A UV absorbance peak in the 280 to 320 region (e.g. at ˜310 nm) may also be indicative of phlorotannin contamination ([29]). When this phlorotannin contamination is present, the method of the invention will decrease absorbance in the 280 to 320 region. Similarly, the glucan will have reduced absorbance in the 280 to 320 region. Preferably, the glucan demonstrates little UV absorbance in this region. For example, the glucan has a UV absorbance at 310 nm of less than 0.10 at 1 mg/ml in water.
Any suitable method of carrying out UV/VIS spectroscopy may be used to measure the phlorotannin contamination of the glucan. For example, the inventors have found that suitable UV/VIS spectra may be obtained using a Perkin-Elmer LAMBDA™ 25 spectrophotometer at room temperature and pressure, using a quartz cell with a 1 cm path length containing glucan at 1 mg/ml in water. The skilled person would be capable of selecting other suitable methods and conditions for obtaining UV/VIS spectra.
Other methods of measuring phlorotannin are known in the art. For example, high performance liquid chromatography was used for the detection and quantification of phlorotannins in reference 30. The skilled person would be capable of selecting methods that are suitable for measuring phlorotannin contamination in the present invention.
The process of this aspect of the invention may further comprise a subsequent step of measuring the residual phlorotannin contamination of the glucan. In this way, it may be verified that the glucan has reduced phlorotannin contamination. The phlorotannin contamination may be measured by any suitable method, including those methods discussed above.
The phlorotannin may be separated from the glucan by any suitable method. For example, filtration methods may be used. The skilled person would be capable of selecting filters that have suitable properties for separating the phlorotannin from the glucan. Typically, the filter used to separate the phlorotannin from the glucan is a depth filter. Depth filters are well known to the person skilled in the art. Suitable depth filters include Cuno™ SP filters, which have a filter medium composed of an inorganic filter aid, cellulose and a resin that imparts a positive charge on the filter matrix. For example, the inventors have found that the Cuno™ 10 SP filter is particularly effective. However, other depth filters and other methods of filtration may also be used. For example, gel filtration may be used. Carbon-based filters may also be suitable. Carbon-based filters are well known to the person skilled in the art. They typically comprise a loose granular activated carbon bed or a pressed or extruded activated carbon block, which acts as a filter for purification of a sample. Alternatively, chromatographic procedures may be used to separate phlorotannin from the glucan. For example, affinity resin chromatography wherein the phlorotannin or the glucan is retained on the resin may be used.
The separation of phlorotannin from the glucan may be carried out in one (or 2, 3, 4, 5, 6, 7, 8, 9, 10 etc.) or more sub-steps. For example, the inventors have found that three sub-steps of filtration using a depth filter (e.g. a Cuno™ 10 SP filter) are particularly effective for reducing phlorotannin contamination.
The person skilled in the art is capable of identifying other separation techniques and conditions that result in the required reduction in phlorotannin contamination. For example, separation techniques and conditions may be optimised by carrying out a test separation step and then measuring residual phlorotannin contamination by the methods described above.
Pure β-glucans are poor immunogens. For protective efficacy, therefore, β-glucans used with the invention are preferably conjugated to a carrier protein. The use of conjugation to carrier proteins in order to enhance the immunogenicity of carbohydrate antigens is well known [e.g. reviewed in refs. 31 to 39 etc.] and is used in particular for paediatric vaccines [40].
The invention provides a composition comprising: (a) a conjugate of (i) a glucan, as defined above, and (ii) a carrier molecule; and (b) an adjuvant, as defined above.
The carrier molecule may be covalently conjugated to the glucan directly or via a linker. Any suitable conjugation reaction can be used, with any suitable linker where necessary.
Attachment of the glucan antigen to the carrier is preferably via a —NH2 group e.g. in the side chain of a lysine residue in a carrier protein, or of an arginine residue. Where a glucan has a free aldehyde group then this can react with an amine in the carrier to form a conjugate by reductive amination. Attachment to the carrier may also be via a —SH group e.g. in the side chain of a cysteine residue. Alternatively the glucan antigen may be attached to the carrier via a linker molecule.
The glucan will typically be activated or functionalised prior to conjugation. Activation may involve, for example, cyanylating reagents such as CDAP (e.g. 1-cyano-4-dimethylamino pyridinium tetrafluoroborate [41, 42, etc.]). Other suitable techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S—NHS, EDC, TSTU (see also the introduction to reference 43).
Direct linkages to the protein may comprise oxidation of the glucan followed by reductive amination with the protein, as described in, for example, references 44 and 45.
Linkages via a linker group may be made using any known procedure, for example, the procedures described in references 46 and 47. Typically, the linker is attached via the anomeric carbon of the glucan. A preferred type of linkage is an adipic acid linker, which may be formed by coupling a free —NH2 group (e.g. introduced to a glucan by amination) with adipic acid (using, for example, diimide activation), and then coupling a protein to the resulting saccharide-adipic acid intermediate [35, 48, 49]. A similar preferred type of linkage is a glutaric acid linker, which may be formed by coupling a free —NH2 group with glutaric acid in the same way. Adipid and glutaric acid linkers may also be formed by direct coupling to the glucan, i.e. without prior introduction of a free group, e.g. a free —NH2 group, to the glucan, followed by coupling a protein to the resulting saccharide-adipic/glutaric acid intermediate. Another preferred type of linkage is a carbonyl linker, which may be formed by reaction of a free hydroxyl group of a modified glucan with CDI [50, 51] followed by reaction with a protein to form a carbamate linkage. Other linkers include β-propionamido [52], nitrophenyl-ethylamine [53], haloacyl halides [54], glycosidic linkages [55], 6-aminocaproic acid [56], N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) [57], adipic acid dihydrazide ADH [58], C4 to C12 moieties [59], etc. Carbodiimide condensation can also be used [60].
A bifunctional linker may be used to provide a first group for coupling to an amine group in the glucan (e.g. introduced to the glucan by amination) and a second group for coupling to the carrier (typically for coupling to an amine in the carrier). Alternatively, the first group is capable of direct coupling to the glucan, i.e. without prior introduction of a group, e.g. an amine group, to the glucan.
In some embodiments, the first group in the bifunctional linker is thus able to react with an amine group (—NH2) on the glucan. This reaction will typically involve an electrophilic substitution of the amine's hydrogen. In other embodiments, the first group in the bifunctional linker is able to react directly with the glucan. In both sets of embodiments, the second group in the bifunctional linker is typically 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 the glucan and the carrier involve amines then it is preferred to use a bifunctional linker. For example, a homobifunctional linker of the formula X-L-X, may be used 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. Similarly, a heterobifunctional linker of the formula X-L-X may be used, where: the two X groups are different and can react with the amines; and where L is a linking moiety in the linker. A preferred X group is N-oxysuccinimide. L preferably has formula L′-L2-L′, where L′ is carbonyl. Preferred L2 groups are straight chain alkyls with 1 to 10 carbon atoms (e.g. C1, C2, C3, C4, C5, C6, C7, C8, C9, C10) e.g. —(CH2)4— or —(CH2)3.
Similarly, where the reaction with the glucan involves direct coupling and the reaction with the carrier involves an amine then it is also preferred to use a bifunctional linker. For example, a homobifunctional linker of the formula X-L-X may be used, where: the two X groups are the same as each other and can react with the glucan/amine; and where L is a linking moiety in the linker. Similarly, a heterobifunctional linker of the formula X-L-X may be used, where: the two X groups are different and one can react with the glucan while the other can react with the amine; and where L is a linking moiety in the linker. A preferred X group is N-oxysuccinimide. L preferably has formula L′-L2-L′, where L′ is carbonyl. Preferred L2 groups are straight chain alkyls with 1 to 10 carbon atoms (e.g. C1, C2, C3, C4, C5, C6, C7, C8, C9, C10) e.g. —(CH2)4— or —(CH2)3—.
Other X groups for use in the bifunctional linkers described in the two preceding paragraphs 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 glucan during coupling to the glucan.
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 [61].
Other suitable carrier proteins include the N. meningitidis outer membrane protein complex [62], synthetic peptides [63,64], heat shock proteins [65,66], pertussis proteins [67,68], cytokines [69], lymphokines [69], hormones [69], growth factors [69], artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen-derived antigens [70] such as N19 [71], protein D from H. influenzae [72-74], pneumolysin [75] or its non-toxic derivatives [76], pneumococcal surface protein PspA [77], iron-uptake proteins [78], toxin A or B from C. difficile [79], recombinant Pseudomonas aeruginosa exoprotein A (rEPA) [80], etc. It is possible to use mixtures of carrier proteins. A single carrier protein may carry multiple different glucans [81].
Conjugates may have excess carrier (w/w) or excess glucan (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, or equal weights. The conjugate may include small amounts of free (i.e. unconjugated) carrier. When a given carrier protein is present in both free and conjugated form in a composition of the invention, the unconjugated form is preferably no more than 5% of the total amount of the carrier protein in the composition as a whole, and more preferably present at less than 2% (by weight).
When the conjugate forms the glucan component in an immunogenic composition of the invention, the composition may also comprise free carrier protein as immunogen [82].
After conjugation, free and conjugated glucans can be separated. There are many suitable methods e.g. hydrophobic chromatography, tangential ultrafiltration, diafiltration, etc. [see also refs. 83, 84 etc.]. Tangential flow ultrafiltration is preferred.
The glucan moiety in the conjugate is preferably a low molecular weight glucan, as defined above. Oligosaccharides will typically be sized prior to conjugation.
The protein-glucan conjugate is preferably soluble in water and/or in a physiological buffer.
The inventors have found that immunogenicity may be improved if there is a spacer between the glucan and the carrier protein. In this context, a “spacer” is a moiety that is longer than a single covalent bond. This spacer may be a linker, as described above. Alternatively, it may be a moiety covalently bonded between the glucan and a linker. Typically, the moiety will be covalently bonded to the glucan prior to coupling to the linker or carrier. For example, the spacer may be moiety Y, wherein Y comprises a straight chain alkyl with 1 to 10 carbon atoms (e.g. C1, C2, C3, C4, C5, C6, C7, C8, C9, C10), typically 1 to 6 carbon atoms (e.g. C1, C2, C3, C4, C5, C6). The inventors have found that a straight chain alkyl with 6 carbon atoms (i.e. —(CH2)6) is particularly suitable, and may provide greater immunogenicity than shorter chains (e.g. —(CH2)2). Typically, Y is attached to the anomeric carbon of the glucan, usually via an —O— linkage. However, Y may be linked to other parts of the glucan and/or via other linkages. The other end of Y is bonded to the linker by any suitable linkage. Typically, Y terminates with an amine group to facilitate linkage to a bifunctional linker as described above. In these embodiments, Y is therefore bonded to the linker by an —NH— linkage. Accordingly, a conjugate with the following structure is specifically envisaged for use in the present invention:
Another a conjugate specifically envisaged for use in the present invention has the following structure:
In one aspect, the invention provides a method for making a glucan conjugated to a carrier protein, wherein the step of conjugation is carried out in a phosphate buffer with >10 mM phosphate; and to a conjugate obtained by this method. The inventors have found that sodium phosphate is a suitable form of phosphate for the buffer. The pH of the buffer may be adjusted to between 7.0-7.5, particularly 7.2. The step of conjugation is typically carried out in a phosphate buffer with between 20-200 mM phosphate, e.g. 50-150 mM. In particular, the inventors have found that a phosphate buffer with 90-110 mM, e.g. about 100 mM, phosphate is suitable. The step of conjugation is usually carried out at room temperature. Similarly, the step of conjugation is usually carried out at room pressure. Typically, the glucan is attached to a linker as described above prior to the step of conjugation. In particular, the glucan may be attached to a bifunctional linker as described above. The free end of the linker may comprise a group to facilitate conjugation to the carrier protein. For example, the inventors have found that the free end of the linker may comprise an ester group, e.g. an N-hydroxysuccinimide ester group.
Even though β-glucans have themselves been reported to be adjuvants, an immunogenic composition may include a separate 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:
These and other adjuvant-active substances are discussed in more detail in references 170 & 171. Specific oil-in-water emulsion adjuvants useful with the invention include, but are not limited to:
Preferred emulsion adjuvants have an average droplets size of ≦1 μm e.g. ≦750 nm, ≦500 nm, ≦400 nm, ≦300 nm, ≦250 nm, ≦220 nm, ≦200 nm, or smaller. These droplet sizes can conveniently be achieved by techniques such as microfluidisation.
In some embodiments, and particularly where the β-glucan is of the laminarin type, a composition is preferably not adjuvanted with complete Freund's adjuvant or with a wild-type cholera toxin.
Antigens and adjuvants in a composition will typically be in admixture.
Where an aluminium salt is used as an adjuvant for a conjugate then it is preferred that at least 50% by mass of the conjugate in a composition is adsorbed to the salt e.g. >60%, >70%, >80%, >90%, >95%, >98%, >99% or 100%. Adsorption of >99% can readily be achieved with a hydroxide salt.
Compositions may include two or more of said adjuvants. As shown in the examples, such combinations can improve the immune response elicited by glucan conjugates. Individual adjuvants may preferentially induce either a Th1 response or a Th2 response, and useful combinations of adjuvants can include both a Th2 adjuvant (e.g. an oil-in-water emulsion or an aluminium salt) and a Th1 adjuvant (e.g. 3dMPL, a saponin, or an immunostimulatory oligonucleotide). For example, compositions may advantageously comprise: both an aluminium salt and an immunostimulatory oligodeoxynucleotide; both an aluminium salt and a compound of formula I, II or III; both an oil-in-water emulsion and a compound of formula I, II or III; both an oil-in-water emulsion and an immunostimulatory oligodeoxynucleotide; both an aluminium salt and an α-glycosylceramide; both an oil-in-water emulsion and an α-glycosylceramide; both an oil-in-water emulsion and 3dMPL; both an oil-in-water emulsion and a saponin; etc.
The invention provides a pharmaceutical composition comprising (a) a glucan or conjugate of the invention, (b) an adjuvant, as described above and (c) a pharmaceutically acceptable carrier. A thorough discussion of such carriers is available in reference 177.
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. 178].
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. The composition may be aqueous, or it may be lyophilised. The inventors have found that liquid formulations of the pharmaceutical compositions of the invention may be unstable (
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 a glucan 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.
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). Therapeutic immunisation is particularly useful for treating Candida infection in immunocompromised subjects.
The invention also provides an adjuvanted glucan or 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 an adjuvanted glucan, conjugate or pharmaceutical composition of the invention to the mammal.
The invention also provides the use of (i) a glucan or conjugate of the invention and (ii) an adjuvant, in the manufacture of a medicament for preventing or treating a microbial infection 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.
Because glucans (and β-glucans in particular) are an essential and principal polysaccharide constituent of almost all pathogenic fungi, particularly those involved in infections in immunocompromised subjects, and also in bacterial pathogens and protozoa, anti-glucan immunity may have efficacy against a broad range of pathogens and diseases. For example, anti-glucan serum raised after immunisation with S. cerevisiae is cross-reactive with C. albicans. Broad spectrum immunity is particularly useful because, for these human infectious fungal agents, chemotherapy is scanty, antifungal drug resistance is emerging and the need for preventative and therapeutic vaccines is increasingly recognized.
The uses and methods of the invention are particularly useful for treating/protecting against infections of: Candida species, such as C. albicans; Cryptococcus species, such as C. neoformans; Enterococcus species, such as E. faecalis; Streptococcus species, such as S. pneumoniae, S. mutans, S. agalactiae and S. pyogenes; Leishmania species, such as L. major; Acanthamoeba species, such as A. castellani; Aspergillus species, such as A. fumigatus and A. flavus; Pneumocystis species, such as P. carinii; Mycobacterium species, such as M. tuberculosis; Pseudomonas species, such as P. aeruginosa; Staphylococcus species, such as S. aureus; Salmonella species, such as S. typhimurium; Coccidioides species such as. C. immitis; Trichophyton species such as T. verrucosum; Blastomyces species such as B. dermatidis; Histoplasma species such as H. capsulatum; Paracoccidioides species such as P. brasiliensis; Pythium species such as P. insidiosum; and Escherichia species, such as E. coli.
The uses and methods are particularly useful for preventing/treating diseases including, but not limited to: candidiasis (including hepatosplenic candidiasis, invasive candidiasis, chronic mucocutaneous candidiasis and disseminated candidiasis); candidemia; aspergillosis, cryptococcosis, dermatomycoses, sporothrychosis and other subcutaneous mycoses, blastomycosis, histoplasmosis, coccidiomycosis, paracoccidiomycosis, pneumocystosis, thrush, tuberculosis, mycobacteriosis, respiratory infections, scarlet fever, pneumonia, impetigo, rheumatic fever, sepsis, septicaemia, cutaneous and visceral leishmaniasis, corneal acanthamoebiasis, cystic fibrosis, typhoid fever, gastroenteritis and hemolytic-uremic syndrome. Anti-C. albicans activity is particularly useful for treating infections in AIDS patients.
Efficacy of immunisation can be tested by monitoring immune responses against β-glucan (e.g. anti-β-glucan antibodies) after administration of the composition. Efficacy of therapeutic treatment can be tested by monitoring microbial infection after administration of the composition of the invention.
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. Subcutaneous or intraperitoneal administration are particularly preferred. Intramuscular administration is also 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 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 elderly (e.g. ≧50 years old, ≧60 years old, and preferably ≧65 years), or 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.). Where a multiple dose schedule is used then at least one dose will include an adjuvanted glucan, but another dose (typically a later dose) may include an unadjuvanted glucan. Similarly, at least one dose may include a conjugated glucan, but another dose (typically later) may include an unconjugated glucan.
Conjugates of the invention may be combined with non-glucan 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. Compositions of the invention may be used in conjunction with anti-fungals, particularly where a patient is already infected. The anti-fungal offers an immediate therapeutic effect whereas the immunogenic composition offers a longer-lasting effect. Suitable anti-fungals include, but are not limited to, azoles (e.g. fluconazole, itraconazole), polyenes (e.g. amphotericin B), flucytosine, and squalene epoxidase inhibitors (e.g. terbinafine) [see also ref. 179]. The anti-fungal and the immunogenic composition may be administered separately or in combination. When administered separately, they will typically be administered within 7 days of each other. After the first administration of an immunogenic composition, the anti-fungal may be administered more than once.
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 means, for example, x±10%.
Unless specifically stated, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.
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.
Curdlan with a starting MW of >100 kDa was treated by acid hydrolysis using HCl (0.5M) in DMSO for 10 minutes at 85° C. The hydrolysate had a DP around 25 units.
Hydrolysed material was neutralised with sodium phosphate buffer (400 mM, pH 6.8) and diluted with water to give a 10:1 dilution of the starting material. The final concentration was 1 mg/ml. After dilution, some precipitation was detectable. The precipitates are probably high MW saccharide.
Ammonium acetate was added and then sodium cyanoborohydride. After adjusting the pH to 7.0 the mixture was incubated at 37° C. for 3-5 days. This treatment introduced a primary amino group at the reducing terminus of the curdlan fragments. The amino-saccharides were then purified by ultrafiltration with a 3 kDa cut-off membrane. Amino groups were estimated by the Habeeb method.
Dried amino-oligosaccharide was solubilised in distilled water at a 40 mM amino group concentration, then 9 volumes of DMSO were added followed by triethyl-amine at a final concentration of 200 mM. To the resulting solution, adipic acid N-hydroxysuccinimido diester was added for a final concentration of 480 mM. Ester groups generated in this way were estimated by analysis of released N-hydroxy-succinimido groups.
Dried activated oligosaccharide was added to CRM197 in 10 mM phosphate buffer pH 7.0. The reaction was maintained under stirring at room temperature overnight. The final material had a ratio of about 50:1 in term of mol of N-hydroxysuccinimido ester per mol of protein.
The conjugate was then purified by ultrafiltration with a 30 kDa cut-off membrane. The conjugate was characterized by SDS-Page, SEC-HPLC and NMR. Also, the saccharide (total and un-conjugated saccharide) and protein content were estimated.
Similar work was carried out using tetanus toxoid as the carrier instead of CRM197.
For five prepared lots of conjugates, the saccharide:protein ratios were as follows (excess carrier):
Laminarin conjugates were prepared as disclosed in references 2 and 3, using CRM197 carrier. One such conjugate had a saccharide:protein mass ratio of 0.4:1. After purification, 0.7% of free glucan (unconjugated) remained present.
Conjugates were prepared in the same way, but with tetanus toxoid as the carrier instead of CRM197.
For five other lots of conjugates, the saccharide:protein ratios were as follows (excess carrier):
A CRM197 conjugate (‘CRM-Lam’) was combined with an aluminium hydroxide salt, at a final adjuvant concentration of 2 mg/ml. Sodium chloride was included at 9 mg/ml, and sodium phosphate at 10 mM. The final composition had pH 7.0 and included 46.6 μg/ml glucan, thus giving 7 μg/dose at a 150 μl dose volume (suitable for mouse studies).
The final composition was analysed by SDS-PAGE. In addition, to determine the degree of adsorption, it was centrifuged and its supernatant was analysed in parallel. TCA precipitation was also used, and the supernatant was again analysed. For comparison, unadsorbed material was also analysed, as was unconjugated CRM197 in both free and adsorbed form. In the presence of the adjuvant, no bands were detected in the supernatants of either CRM-Lam and or unconjugated CRM.
Adsorption was also assessed by analysing the supernatants by HPLC-SEC. Peaks at 214 nm were measured, and the degree of adsorption for CRM-Lam was calculated as 99.7%.
Synthetic curdlan (15-mer) and laminarin (17-mer) conjugates were prepared according to the method described in
The conjugates were then characterized by SDS-Page and SEC-HPLC. The saccharide and protein contents were estimated as follows:
Further lots of laminarin conjugates were prepared as disclosed in references 2 and 3, except that the concentration of the phosphate in the buffer was either a) 10 mM (as per references 2 and 3, hereinafter “lot 9”); b) 25 mM; c) 50 mM or d) 100 mM (“lot 10”). The conjugates were then characterized by SEC-HPLC. Greater aggregation was observed in lot 9 than in the other lots, with no aggregation being detectable in lot 10 (
To test immunogenicity, laminarin conjugates prepared as described in Laminarin conjugation and adsorption (2) were combined with various individual and combined adjuvants and tested in mice.
CD2F1 mice, 4-6 weeks old, were tested in 12 groups of 10. The conjugates were used at a saccharide dose of 7 μg in a dosage volume of 150 μl, administered days 1, 7 and 21. Blood samples were taken on days 0, 21 and 35 for assessing anti-GGZym antibody levels by ELISA [2,3].
Groups 2 & 3 were primed (day 1) using the conjugate in combination with Complete Freund's adjuvant (CFA), solely for comparison purposes. Group 1 received CFA-adjuvanted laminarin and CRM197, but these were not conjugated. Group 4 was primed with unconjugated laminarin plus CFA. Group 5 received CFA alone. These five groups then received a mixture of laminarin and CRM197 with out adjuvant (group 1), unadjuvanted conjugate (group 2), unadjuvanted laminarin (groups 3 & 4) or PBS (group 5) at days 7 and 21.
Groups 7-8 and 9-12 received three identical doses of conjugated laminarin with the following adjuvants: (a) an aluminium hydroxide adjuvant; (b) the MF59 oil-in-water emulsion adjuvant; (c) a CpG oligodeoxynucleotide, CpG 1826; (e) a combination of (a) and (c); (f) a combination of (b) and (c). Groups 6 and 9 received the same as groups 7 and 8, respectively, but the laminarin and CRM197 were not conjugated.
Anti-glucan antibodies (GMT) and the number of responding mice (%) at day 35 are reported in Table 1.
The results show that an optimum immune response requires both conjugation and the presence of at least one adjuvant. Combinations of Th1 and Th2 adjuvants gave good results.
The IgG responses of mice in groups 2 and 11 were studied in more detail. In all three cases IgG subclasses 1 and 3 were present, but subclasses 2a and 2b were absent.
In further work, both curdlan and laminarin conjugates prepared as described in Curdlan conjugation (1) and Laminarin conjugation and adsorption (2) respectively were administered to mice. More than one lot of curdlan conjugates was tested.
Experiments were essentially the same as in the first study, but conjugates were used at a saccharide dose of 5 μg. Conjugates were administered either without adjuvant or with the following adjuvants: (a) an aluminium hydroxide adjuvant; (b) the MF59 oil-in-water emulsion adjuvant; (c) a combination of (a) with 10 μg of a CpG oligodeoxynucleotide, CpG1826; (e) a combination of (b) with a CpG oligodeoxynucleotide.
Anti-glucan antibodies (GMT) and the number of responding mice (%) at day 35 are reported in Table 2.
Although individual adjuvants were able to increase both the GMT and proportion of responders, for both laminarin and curdlan, combinations of adjuvants were particularly effective. Combinations of Th1 and Th2 adjuvants gave good results.
IgG responses in all groups were, as seen above, primarily in subclasses 1 and 3.
In further experiments, laminarin and curdlan conjugates prepared as described in Laminarin conjugation and adsorption (2) and Curdlan conjugation (1) respectively were also adjuvanted with α-galactosylceramide (100 ng) or LT-K63 (2 μg), either alone or in combination with other adjuvants. The CpG adjuvant was also tested at three different doses (0.5 μg, 5 μg and 10 μg). Details were as in the previous immunogenicity study, but with 8 mice per group. Results are in Table 3.
Again, adjuvant combinations gave the best results, except that α-GalCer was useful on its own for the curdlan conjugate. The best results from the CpG adjuvant were achieved at the middle dose.
Mice, in groups of 16, were immunized intraperitoneally (IP) three times with laminarin conjugated to CRM197 (Lam-CRM, prepared as described in Laminarin conjugation and adsorption (2)) in combination with different adjuvants. The conjugates were used at a saccharide dose of 5 μg in a dosage volume of 150 μl, administered at days 1, 14 and 28. Blood samples were taken on days 0, 28 and 42 for assessing anti-GGZym antibody levels by ELISA. Anti-laminarin antibody levels were also measured by substituting laminarin for GG-Zym in the ELISA, as described in reference 3.
IgG GMTs against Candida cell wall glucan at day 35 are shown in
The results show the immunogenicity of the glycoconjugate vaccine Lam-CRM in several different adjuvant formulations.
In further work, laminarin or curdlan conjugated to either CRM197 or tetanus toxoid were combined with various individual and combined adjuvants and administered to mice by subcutaneous or intraperitoneal administration. The conjugates were prepared as described in Laminarin conjugation and adsorption (2) and Curdlan conjugation (1) respectively.
CD2F1 mice, 4-6 weeks old, were tested in 12 groups of 10. The conjugates were used at a saccharide dose of 5 μg in a dosage volume of 150 μl, administered days 1, 14 and 28 by subcutaneous or intraperitoneal administration. Blood samples were taken on days 0, 28 and 42 for assessing anti-GGZym antibody levels by ELISA.
Groups 1-3 received three identical doses of laminarin conjugated to CRM197 with the following adjuvants: (a) an aluminium hydroxide adjuvant (300 μg); (b) a combination of (a) and a CpG oligodeoxynucleotide, CpG1826 (10 μg); and (c) the MF59 oil-in-water emulsion adjuvant (75 μl), respectively. Groups 4-6 were treated in the same way as groups 1-3 respectively, except that the glucan was curdlan instead of laminarin. Groups 7-9 were treated in the same way as groups 1-3 respectively, except that the laminarin was conjugated to tetanus toxoid instead of CRM197. Similarly, groups 10-12 were treated in the same way as groups 4-6 respectively, except that the curdlan was conjugated to tetanus toxoid instead of CRM197.
Anti-glucan antibodies (GMT) at day 42 after intraperitoneal administration of laminarin conjugates to the mice are shown in
Similarly, anti-glucan antibodies (GMT) at day 42 after intraperitoneal administration of curdlan conjugates are shown in
In another study, laminarin or curdlan conjugated to CRM197 were administered to mice using different doses of saccharide. The conjugates were prepared as described in Laminarin conjugation and adsorption (2) and Curdlan conjugation (1) respectively. CD2F1 mice, 4-6 weeks old, were tested in 12 groups of 8. The conjugates were used at a saccharide doses of 10 μg, 5 μg, 1 μg or 0.1 μg in a dosage volume of 150 μl, administered days 1, 14 and 28. Blood samples were taken on days 0, 28 and 42 for assessing anti-GGZym and anti-laminarin antibody levels by ELISA.
Group 1 received three identical doses of laminarin conjugated to CRM197 with no adjuvant and a saccharide dose of 5 μg. Group 2 received three identical doses of laminarin conjugated to CRM197 with an aluminium hydroxide adjuvant (300 μg) and a saccharide dose of 5 μg. A phosphate buffer had been used during the purification of the conjugate administered to this group. Groups 3-6 received three identical doses of laminarin conjugated to CRM197 with an aluminium hydroxide adjuvant (300 μg) and a saccharide dose of 10 μg, 5 μg, 1 μg or 0.1 μg, respectively. A histidine buffer had been used during the purification of the conjugates administered to these groups, as described in reference 180.
Groups 7-12 were treated in the same way as groups 1-6, except that the glucan was curdlan instead of laminarin.
Anti-glucan antibodies (GMT) at day 42 after administration of laminarin conjugates at various saccharide doses are shown in
Anti-glucan antibodies (GMT) at day 42 after administration of the curdlan conjugates are shown in
Anti-glucan antibodies (GMT) at day 42 after administration of the laminarin conjugates are shown in
In further work, laminarin conjugated to CRM197 was combined with various individual adjuvants and administered to mice by intraperitoneal, by subcutaneous or intramuscular administration. The conjugate was prepared as described in Laminarin conjugation and adsorption (2).
CD2F1 mice, 4-6 weeks old, were tested in 6 groups of 16. The conjugates were used at a saccharide dose of 5 μg in a dosage volume of 150 μl, administered days 1, 14 and 28 by intraperitoneal, subcutaneous or intramuscular administration. Blood samples were taken on days 0, 28 and 42 for assessing anti-GGZym and anti-laminarin antibody levels by ELISA.
Groups 1-2 received three identical doses of laminarin conjugated to CRM197 by intraperitoneal administration with the following adjuvants: (a) the MF59 oil-in-water emulsion adjuvant (75 μl); and (b) (4) IC31 at a high dose (49.5 μl of a sample having over 1000 nmol/ml oligodeoxynucleotide and 40 nmol/ml peptide), respectively. Groups 3-4 were treated in the same way as groups 1-2 respectively, except that the conjugate was administered by subcutaneous administration. Groups 5-6 were treated in the same way as groups 1-2 respectively, except that the conjugate was administered by intramuscular administration.
Anti-glucan (anti-GGZym) antibodies (GMT) at day 42 after administration of laminarin conjugates are shown in
In another study, the ability of antibodies induced by laminarin conjugated to CRM197 combined with aluminium hydroxide, IC31 or MF59 adjuvants to inhibit the growth of C. albicans in vivo was tested. The conjugate was prepared as described in Laminarin conjugation and adsorption (2).
Separate pools of sera were obtained from mice used in Immunogenicity study (1), Immunogenicity study (4) and Immunogenicity study (7) above. A further pool of sera from pre-immune mice was obtained. Prior to use, the sera were inactivated by treatment at 56° C. for 30 minutes.
CD2F1 mice, 4-6 weeks old, were tested in groups of 4-5. 0.5 ml of pooled sera was administered to each mouse by intraperitoneal administration. After two hours, each mouse was infected with 0.2 ml of a culture of C. albicans by intravenous administration via the caudal vein such that each mouse received 5×105 CFU. After two days, each mouse was sacrificed and the left kidney removed. Each kidney was homogenised in the presence of 0.5 ml PBS with 0.1% Triton X. Serial dilutions of the homogenates were plated onto Sabourad's agar and incubated for 48 h at 28° C.
The accumulation of C. albicans in the kidneys of the mice treated with the pre- and post-immunization sera is shown in
A 1 mg/ml aqueous solution of a commercially-available laminarin extracted from Laminaria digitata (L-9634, Sigma) was analysed by UV/VIS spectroscopy. The UV/VIS spectrum was obtained using a Perkin-Elmer LAMBDA™ 25 spectrophotometer at room temperature and pressure, using a quartz cell with a 1.00 cm path length. The same material was analysed after one, two or three steps of filtration using a depth filter (a Cuno™ 10 SP filter). The results are shown in
Phlorotannin contamination (as indicated by a UV absorbance peak at ˜270 nm) was reduced after each step of filtration.
The stability of glucan conjugates formulated as liquids with various adjuvants was compared. The conjugate was prepared as described in Laminarin conjugation and adsorption (2). Samples were stored at 37° C. for 4 weeks (
Lam-CRM 20 μg/mL in 10 mM histidine buffer at pH7, 0.9% NaCl, 2 mg/mL Al(OH)3, 0.05% Tween 20;
Lam-CRM 20 μg/mL in 10 mM phosphate buffer at pH7, 0.9% NaCl, 2 mg/mL Al(OH)3,0.05% Tween 20;
Lam-CRM 20 μg/mL in MF59; and
Lam-CRM 20 μg/mL in 10 mM phosphate buffer at pH7, 0.9% NaCl.
The results show that glucan conjugates combined with MF59 are more stable than glucan conjugates combined with aluminium hydroxide (in phosphate or histidine buffer).
The stability of a lyophilised formulation of glucan conjugates was also measured. Samples were stored at 4, 25 or 37° C. for up to 3 months (
Lam-CRM 10 μg/mL, sodium chloride 3.5 mg, sodium phosphate monobasic monohydrate 0.092 mg, sodium phosphate dibasic dihydrate 0.48 mg, mannitol 7.3 mg.
In another study, conjugates prepared as described in Conjugation (3) and laminarin conjugated to CRM197 were combined with various individual and combined adjuvants and administered to mice by intraperitoneal administration. The laminarin conjugated to CRM197 was prepared as described in Laminarin conjugation and adsorption (2), except for an alternative lot of laminarin to CRM197 (lot 11AD) which was prepared without an amination step prior to conjugation.
CD2F1 mice, 4-6 weeks old, were tested in 11 groups of 16. The conjugates were used at a saccharide dose of 5 μg in a dosage volume of 150 μl, administered by intraperitoneal administration at days 1, 14 and 28. Blood samples were taken on days 0, 28 and 42 for assessing anti-laminarin antibody levels by ELISA.
Groups 1-3 received three identical doses of a) 17-mer-C2 β(1-3)-CRM conjugate; b) 15-mer-C6 β(1-3)-CRM conjugate; or c) 15-mer-C2 β(1-3)-CRM conjugate respectively, all with no adjuvant. Groups 4-6 received three identical doses of a) 17-mer-C2 β(1-3)-CRM conjugate; b) 15-mer-C6 β(1-3)-CRM conjugate; or c) 15-mer-C2 β(1-3)-CRM conjugate respectively, all with the MF59 oil-in-water emulsion adjuvant (75 μl). Groups 7-8 received three identical doses of laminarin conjugated to CRM197 with a) no adjuvant; or b) the MF59 oil-in-water emulsion adjuvant (75 μl) respectively. Groups 9-10 received three identical doses of laminarin conjugated to CRM197 with a) the MF59 oil-in-water emulsion adjuvant (75 μl) combined with IC31 at a high dose (49.5 μl of a sample having over 1000 nmol/ml oligodeoxynucleotide and 40 nmol/ml peptide); or b) an aluminium hydroxide adjuvant (300 μg), respectively. Group 11 received three identical doses of a different preparation of laminarin conjugated to CRM197 with the MF59 oil-in-water emulsion adjuvant (75 μl).
Anti-laminarin antibodies (GMT) at day 42 after administration of the conjugates are shown in
In another study, the ability of mice receiving glucans conjugated to CRM197 combined with MF59 adjuvant to survive challenge with C. albicans was tested. The conjugates were prepared as described in Laminarin conjugation (4) (lots 9 and 10) and Curdlan conjugation (1).
Female, four-week old CD2F1 mice (Harlan) were immunized with three doses of laminarin or curdlan conjugated to CRM197, each dose consisting of 10 μg polysaccharide in 0.2 ml of PBS:MF59 (1:1 v/v) per mouse.
The immunization schedule was:
Protection endpoints were measured in terms of mortality (median survival time (MST) and ratio of dead/total challenged mice).
Survival was greater in mice receiving lot 10 than in mice receiving lot 9.
Survival was greater in mice receiving curdlan conjugated to CRM197 than in mice receiving laminarin conjugated to CRM197.
In a similar study, the ability of mice receiving synthetic glucans conjugated to CRM197 combined with MF59 adjuvant to survive challenge with C. albicans was tested. The conjugates were prepared as described in Conjugation (3). In this study, fungal challenge was by intravenous administration of 5.0×105 cells.
Treatment with 15-mer-C2 β(1-3)-CRM resulted in increased survival, while treatment with 17-mer-C2 β(1-3)-CRM did not seem to have any effect. This result suggests that the epitope responsible for inducing a protective antibody response in glucan comprises at least five adjacent non-terminal residues linked to other residues only by β-1,3 linkages. Without wishing to be bound by theory, it is though that this effect may contribute to the greater protective antibody response seen in mice receiving curdlan conjugated to CRM197 than in mice receiving laminarin conjugated to CRM197 in Active protection study (1). The curdlan conjugated to CRM197 (wherein the glucan comprises β-1,3-linked residues only) may contain a greater proportion of protective epitopes than the laminarin conjugated to CRM197 (wherein the glucan comprises β-1,3-linked residues and β-1,6-linked residues).
It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.
This application is a §371 filing from PCT/IB2008/003582, filed Nov. 26, 2008, and claims the benefit under 35 U.S.C. §119(e)(1) of U.S. Provisional Application Ser. No. 61/004,396, filed on 26 Nov. 2007; and U.S. Provisional Application Ser. No. 61/133,738, filed on 1 Jul. 2008, which applications are incorporated herein by reference in their entireties and from which applications priority is claimed pursuant to the provisions of 35 U.S.C. §§119/120.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2008/003582 | 11/26/2008 | WO | 00 | 5/25/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 61004396 | Nov 2007 | US | |
| 61133738 | Jul 2008 | US |
