Patent Application
20060147466
All documents cited herein are hereby incorporated by reference in their entirety.
This application relates to mucosal meningitis vaccines, especially intranasal vaccines.
Meningitis is the inflammation of the tissues which cover the brain and spinal cord. It may have a bacterial cause or a viral cause, with bacterial meningitis generally being more serious.
The main pathogen responsible for bacterial meningitis is Neisseria meningitidis (meningococcus), but other relevant pathogens include Streptococcus pneumoniae (pneumococcus), Haemophilus influenzae (Hib), and Streptococcus agalactiae (GBS). N. meningitidis also causes meningococcal septicaemia, which is the main life-threatening aspect of infection.
Vaccines to protect against Hib infection have been available for many years. A vaccine that protects against serogroup C meningococcus (‘MenC’) was introduced in several European countries in 1999-2000. A pneumococcal vaccine entered into routine use in America in 2000.
The vaccines against these three pathogens are based on antigenic capsular polysaccharides, with conjugation to carrier proteins being used to enhance the polysaccharides' immunogenicity. These vaccines are administered by injection, although investigations into mucosal delivery have been described for mice e.g. reference 1 describes the intranasal administration of Hib conjugate vaccines and reference 2 describes intranasal administration of MenC conjugate vaccines. (see also ref. 3). Mucosal delivery of vaccines represents an attractive approach to overcome the problem of the high number of injections administered to young children. In addition, as most pathogens initially infect at mucosal surfaces, inducing mucosal immunity at the site of infection would likely contribute to optimal protective immunity.
Intranasal and oropharyngeal delivery of vesicle-based vaccines against serogroups B meningococcus (‘MenB’) has also been described [e.g. ref 4], as has the intranasal delivery of B. pertussis bacteria which express N. meningitidis transferrin-binding protein B [5]. Reference 6 describes the intranasal delivery of pneumococcal conjugate vaccines [see also refs. 7 & 8].
It is an object of the invention to provide improvements in the mucosal delivery of meningitis vaccines.
The invention provides a composition for mucosal delivery, comprising two or more of the following: (a) an antigen which induces an immune response against Haemophilus influenzae; (b) an antigen which induces an immune response against Neisseria meningitidis; and (c) an antigen which induces an immune response against Streptococcus pneumoniae.
Combining different antigens reduces the number of different doses which need to be administered in order to immunise against multiple pathogens. This is typically seen as an advantage for injectable vaccines, where the number of painful injections is reduced, but it is less important in mucosal vaccines (e.g. intranasal vaccines) because of the lower discomfort levels associated with delivery. However, combined antigen compositions are advantageous even for mucosal delivery because patient compliance is improved and transport/storage of medicines is facilitated.
Although combining antigens into a single dose is attractive [e.g. refs. 9 to 12], it presents difficulties due to interactions between the various components once combined, particularly in liquid formulations [13]. Issues which arise include antigen interference, antigen competition [14,15], antigen degradation, epitope suppression, and adjuvant compatibility. Quality control of mixtures is also more difficult. Furthermore, existing knowledge on combining antigens focuses on injectable, not mucosal, vaccines.
Despite these difficulties, the inventors have surprisingly found that antigens from Haemophilus influenzae, Neisseria meningitidis and/or Streptococcus pneumoniae can be combined for mucosal delivery without the negative consequences which would haven been expected. Combining antigens from these three organisms is also advantageous because it allows a single dose to deal with three separate causes of a common disease, namely bacterial meningitis. Combined meningitis vaccines of this type have previously been reported [16], but mucosal administration was not reported.
Mucosal Delivery
The composition of the invention is for mucosal delivery.
Of the various mucosal delivery options available, the intranasal route is the most practical as it offers easy access with relatively simple devices that have already been mass produced. In addition, intranasal immunisation appears to be more potent that alternative routes. Thus the preferred route for mucosal delivery is the intranasal route, and the composition of the invention is preferably adapted for intranasal administration, such as by nasal spray, nasal drops, gel or powder [e.g. refs 17 & 18].
Alternative routes for mucosal delivery of the vaccine are oral, intragastric, pulmonary, intestinal, rectal, ocular, and vaginal routes.
(a) Haemophilus influenzae Antigen
The H. influenzae antigen in the composition will typically be a capsular saccharide antigen. Saccharide antigens from H. influenzae b are well known.
Advantageously, the Hib saccharide is covalently conjugated to a carrier protein, in order to enhance its immunogenicity, especially in children. The preparation of polysaccharide conjugates in general, and of the Hib capsular polysaccharide in particular, is well documented [e.g. references 19 to 27 etc.]. The invention may use any suitable Hib conjugate.
The saccharide moiety of the conjugate may be a polysaccharide (e.g. full-length polyribosylribitol phosphate (PRP)), but it is preferred to hydrolyse polysaccharides (e.g. by acid hydrolysis) to form oligosaccharides (e.g. MW from ˜1 to ˜5 kDa). If hydrolysis is performed, the hydrolysate may be sorted by size in order to remove oligosaccharides which are too short to be usefully immunogenic. Size-separated oligosaccharides are preferred saccharide antigens.
Preferred carrier proteins are bacterial toxins or toxoids, such as diphtheria or tetanus toxoids. These are commonly used in conjugate vaccines. The CRM197 diphtheria toxoid is particularly preferred [28]. Other suitable carrier proteins include the N. meningitidis outer membrane protein [29], synthetic peptides [30,31], heat shock proteins [32,33], pertussis proteins [34,35], protein D from H. influenzae [36], cytokines [37], lymphokines [37], hormones [37], growth factors [37], toxin A or B from C. difficile [38], iron-uptake proteins [39] etc. It is possible to use mixtures of carrier proteins.
The saccharide moiety may be conjugated to the carrier protein directly or via a linker. Direct linkage may be achieved by oxidation of the polysaccharide followed by reductive amination with the protein, as described in, for example, refs. 40 & 41. Linkage via a linker group may be made using any known procedure, for example, the procedures described in refs. 42 & 43. Suitable linkers include carbonyl, adipic acid, B-propionamido [44], nitrophenyl-ethylamine [45], haloacyl halides [46], glycosidic linkages [47], 6-aminocaproic acid [48], ADH [49], C4 to C12 moieties [50] etc.
The saccharide 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 [51, 52]. Other suitable techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU; see also the introduction to reference 53). Reductive amination is a preferred technique.
A preferred conjugate comprises the Hib saccharide covalently linked to CRM197 via adipic acid succinic diester [54, 55].
Compositions of the invention may comprise more than one Hib antigen.
(b) Neisseria meningitidis Antigen
The N. meningitidis antigen in the composition will typically be a capsular saccharide antigen (e.g. from serogroups A, C, W135 or Y). Saccharide antigens from N. meningitidis are well known. Where the antigen is from serogroup B, however, it is preferred that the antigen is a protein antigen. This is because the native capsular polysaccharide of MenB contains self-antigens. If a saccharide antigen is to be used from serogroup B, it is preferred to use a modified saccharide antigen [e.g. refs. 56, 57, 58) e.g. one modified by N-propionylation. Chemical modification of saccharides from other serogroups is also possible.
The saccharide is preferably an oligosaccharide i.e. a fragment of a capsular polysaccharide. Polysaccharides may be manipulated to give shorter oligosaccharides and these may be obtained by purification and/or sizing of the native polysaccharide (e.g. by hydrolysis in mild acid, by heating, by sizing chromatography etc.). Preferred MenC oligosaccharides are disclosed in references 59 & 60.
The saccharide is preferably conjugated to a carrier protein as described above.
Compositions of the invention may comprise more than one meningococcal antigen. It may be preferred to include capsular saccharide antigens from at least two (i.e. 2, 3 or 4) of serogroups A, C, W135 and Y of N. meningitidis [61].
Where a mixture comprises capsular saccharides from both serogroups A and C, it is preferred that the ratio (w/w) of MenA saccharide:MenC saccharide is greater than 1 (e.g. 2:1, 3:1, 4:1, 5:1, 10:1 or higher). Surprisingly, improved immunogenicity of the MenA component has been observed when it is present in excess (mass/dose) to the MenC component [61].
Where a mixture comprises capsular saccharides from serogroup W135 and at least one of serogroups A, C and Y, it has surprisingly been found that the immunogenicity of the MenW135 saccharide is greater when administered in combination with the saccharide(s) from the other serogroup(s) than when administered alone (at the same dosage etc.) [61]. Thus the capacity of the MenW135 antigen to elicit an immune response is greater than the immune response elicited by an equivalent amount of the same antigen when delivered without association with the antigens from the other serogroups. Such enhanced immunogenicity can be determined by administering the MenW135 antigen to control animals and the mixture to test animals and comparing antibody titres against the two using standard assays such as bactericidal titres, radioimmunoassay and ELISAs etc. Vaccines comprising synergistic combinations of saccharides from serogroup W135 and other serogroups are immunologically advantageous as they allow enhanced anti-W135 responses and/or lower W135 doses.
Where a protein antigen from serogroup B is used, it is preferred to use one of the proteins disclosed in references 62 to 71]. Preferred protein antigens comprise the ‘287’ protein or derivatives (e.g. ΔG287).
It is also possible to use an outer membrane vesicle (OMV) antigen for serogroup B [e.g. 72, 73].
Compositions of the invention may comprise more than one meningococcal antigen.
(c) Streptococcus pneumoniae Antigen
The S. pneumoniae antigen in the composition will typically be a capsular saccharide antigen which is preferably conjugated to a carrier protein as described above [e.g. 74, 75, 76].
It is preferred to include saccharides from more than one serotype of S. pneumoniae. For example, mixtures of polysaccharides from 23 different serotype are widely used, as are conjugate vaccines with polysaccharides from between 5 and II different serotypes [77]. For example, PrevNar™ contains antigens from seven serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) with each saccharide individually conjugated to CRM197 by reductive amination.
Compositions of the invention may thus comprise more than one pneumococcal antigen.
Further Components—Adjuvants
Compositions of the invention will usually comprise a mucosal adjuvant. Mucosal adjuvants include, but are not limited to, (A) E. coli heat-labile enterotoxin (“LT”), or detoxified mutants thereof, such as the K63 or R72 mutants [e.g. Chapter 5 of ref. 78]; (B) cholera toxin (“CT”), or detoxified mutants thereof [e.g. Chapter 5 of ref. 78]; or (C) microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, more preferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to ˜10 μm in diameter) formed from materials that are biodegradable and non-toxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone etc.); (D) a polyoxyethylene ether or a polyoxyethylene ester [79]; (E) a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol [80] or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol [81]; (F) chitosan [e.g. 82]; (G) an immunostimulatory oligonucleotide (e.g. a CpG oligonucleotide), (H) double stranded RNA; (1) a saponin [83]; (J) monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g. RC-529 [84]; or (K) polyphosphazene (PCPP). Other mucosal adjuvants are also available [e.g. see chapter 7 of ref. 85].
Preferred mucosal adjuvants are bacterial ADP-ribosylating toxins or their mutants. For example, cholera toxin (CT) or E. coli heat labile toxin (LT) are potent mucosal adjuvants, as are their detoxified counterparts [86]. CT and LT are homologous and are typically interchangeable.
Detoxification of the CT or LT may be by chemical or, preferably, by genetic means. Suitable examples include LT having a lysine residue at amino acid 63 [‘LT-K63’—ref. 87], and LT having an arginine residue at amino acid 72 [‘LT-R72’—ref. 88]. Other suitable mutants include LT with a tyrosine at residue 63 [‘Y63’—ref. 89] and the various mutants disclosed in reference 90, namely D53, K97, K104 and S106, as well as combinations thereof (e.g. LT with both a D53 and a K63 mutation).
The composition may comprise a bioadhesive [91,92] such as esterified hyaluronic acid microspheres [93] or, in preferred embodiments, a mucoadhesive selected from the group consisting of cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose.
Compositions of the invention may comprise more than one mucosal adjuvant.
Further Components—Antigens
The combination of antigens from H. influenzae, N. meningitidis and S. pneumoniae is advantageous because they all cause bacterial meningitis. Antigens which induce immune responses against further organisms may also be included in compositions of the invention e.g.
Where a conjugate is present, the composition may also comprise free carrier protein [133].
It is preferred that the composition does not include whole bacteria (whether intact or lysed).
Compositions of the invention may comprise proteins which mimic saccharide antigens e.g. mimotopes [134] or anti-idiotype antibodies. These may replace individual saccharine components, or may supplement them. As an example, the vaccine may comprise a peptide mimic of the MenC [135] or the MenA [136] capsular polysaccharide in place of the saccharide itself.
Compositions of the invention may comprise nucleic acid for ‘genetic immunisation’ [e.g. 137]. The nucleic acid will encode a protein component of the composition and may replace individual protein components (including those of the previous paragraph), or may supplement them. As an example, the vaccine may comprise DNA that encodes a tetanus toxin.
Further Components—Formulation
The composition of the invention preferably includes a pharmaceutically acceptable carrier.
‘Pharmaceutically acceptable carriers’ include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, trehalose [138] lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. The vaccines may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. The carrier will be compatible with mucosal administration. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences.
The composition of the invention is preferably sterile.
The composition of the invention is preferably buffered.
The composition of the invention is preferably pyrogen-free.
The composition of the invention may be packaged with its components (a), (b) and/or (c) in admixture, or these components may remain separate until they are to be administered to a patient, at which stage they will be combined. Where separate, the individual components may each be in lyophilised form or in solution/suspension. Where mixed, the components will all be in lyophilised form or all in solution/suspension. Lyophilised components will be re-suspended (e.g. in buffer) prior to administration to a patient. Components such as adjuvants may be present in the buffer or in the lyophilised material.
Immunogenic Compositions
The composition of the invention is preferably an immunogenic composition (e.g. a vaccine). Formulation of vaccines based on saccharides or saccharide-protein conjugates is well known in the art.
Immunogenic compositions comprise immunologically effective amounts of antigens, as well as any other of other specified components, as needed. 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.
Methods of Treatment
Once formulated, the compositions of the invention can be administered directly to a patient, which will generally be a human. The human is preferably a child or a teenager. A further preferred class of patient is an adult woman, and particularly a woman of child-bearing age or a pregnant woman. Compositions of the invention are particularly suited for passively immunising children via the maternal route.
Antigens in the composition induce immune responses against certain bacteria. These immune responses are preferably protective i.e. they protect the patient from later infection by the bacteria. Thus the compositions of the invention are preferably used for prophylaxis (i.e. to prevent infection), although they may also be used for therapeutic purposes (i.e. to treat disease after infection). The immune responses preferably involve the production of bactericidal antibodies in the patient.
The invention provides a method of raising an immune response in a patient, comprising administering to a patient a vaccine according to the invention via a mucosal route (e.g. intranasally). The immune response is preferably protective against bacterial meningitis and/or bacteremia caused by Haemophilus influenzae, Neisseria meningitidis and/or Streptococcus pneumoniae. The individual antigenic components of the compositions are preferably administered simultaneously and in combination. In other embodiments, however, they may be administered separately, either simultaneously or sequentially. When they are administered separately, the components are preferably delivered to the same mucosal surface.
The invention also provides a composition of the invention for use as a medicament.
The invention also provides the use of: (a) an antigen which induces an immune response against Haemophilus influenzae; (b) an antigen which induces an immune response against Neisseria meningitidis; and (c) an antigen which induces an immune response against Streptococcus pneumoniae, in the manufacture of a medicament for immunising a patient.
These methods and uses of the invention may involve a prime/boost regime. The methods and uses of the invention may be a priming dose which will be followed by a booster dose, where the booster dose may be by a mucosal or parenteral route. Similarly, the methods and uses of the invention may raise a booster response in a patient that has already been immunologically primed, where the primer dose may have been by a mucosal or parenteral route. Booster doses may comprise fewer antigens than priming doses e.g. they may use a single antigen.
Dosage treatment at priming and/or boosting may be a single dose or a multiple dose schedule. Compositions of the invention may be presented in unit dose form.
Manufacturing Methods
The invention provides a method for producing a composition of the invention, comprising the steps of mixing two or more of the following: (a) an antigen which induces an immune response against Haemophilus influenzae; (b) an antigen which induces an immune response against Neisseria meningitidis; and (c) an antigen which induces an immune response against Streptococcus pneumoniae, and formulating the mixture for mucosal delivery.
Combined Hib/MenC Composition
Neisseria meningitidis serogroup C capsular oligosaccharide was produced by selective end-reducing group activation of sized oligosaccharide. The same method was used for Haemophilus influenzae type B. The saccharides were conjugated to protein carrier CRM197 through a hydrocarbon spacer [139] (Chiron Siena, Italy). The conjugates were diluted in phosphate-buffered saline (PBS) and combined with (i) mutant E. coli heat-labile enterotoxin LTK63 or LTR72), (ii) aluminium hydroxide (Superfos Biosector a/s) or (iii) Cholera toxin (CT) from Sigma. For combined administration, these formulations were mixed prior to use.
Mucosal Administration of the Composition
Two identical administration studies were performed simultaneously. Groups of 10 female BALB/C mice 6-10 weeks old were immunised intranasally with 10 μg of MenC or Hib alone, combined with CT (1 μg), or with the LT mutants (1 μg and 10 μg). For comparison, an additional group of mice was immunised IM with 10 μg of MenC or Hib adsorbed to Alum. The compositions were prepared on the same day as immunisation and mice were immunised on Days 0, 21, and 35. 50 μl of the compositions were injected into the thigh or instilled into alternate nostrils in unaneesthetised mice. Blood samples were taken on Day 49 along with terminal nasal wash samples (NW).
To evaluate if the immunogenicity of the conjugates was impaired when the two were mixed, a third study was performed concurrently in which the two vaccines were administered simultaneously to the same groups of mice, at the same doses and regimen described above.
Immunological Responses to the Compositions
Antibody responses against the MenC conjugate were measured by ELISA using a modified procedure as previously described [140]. Briefly, ELISA plates were coated with adipidic dihydrazide-derivatised MenC saccharide over night at 4° C. Specific antibodies were developed with goat anti Mouse IgG-horseradish peroxidase conjugate. MenC IgG antibody titres for the test samples and the internal control were expressed as the reciprocal of the serum dilution giving OD=1.0. Each serum sample was assayed in duplicate, and the average value was used to calculate the geometrical mean and the standard deviation within one standard error. The antibody responses against Hib PRP were determined similarly to the MenC ELISA, except that the plates were coated with BSA conjugated PRP (PRP-BSA). Titres were expressed as OD450nm for serum diluted 1:50.
Nasal washes were assayed for IgA anti-MenC using a bioluminescent assay (BIA) [141]. Briefly, identical reagents and coating procedure to measure serum IgG against MenC was used. Then, a biotinylated Goat anti-Mouse IgA specific was added as a first antibody. Titres represent the logarithmic dilution values extrapolated from the log RLU data at the cutoff value calculated at least two standard deviations above mean background.
Complement-mediated bactericidal activity against MenC bacteria was measured in pooled serum samples as previously described [140]. Titres were determined by calculating the serum dilution showing a 50% reduction in the number of CFU after 1 hour incubation.
The levels of bactericidal antibodies induced by intranasal immunisation with LT mutants closely correlate with the ELISA serum IgG responses, and were again comparable to the responses induced by CT, or intramuscular immunisation with alum adsorbed vaccine (
Samples obtained from nasal wash following intranasal immunisation with MenC with either LT mutant showed higher IgA titers than those obtained by intranasal immunisation in the absence of adjuvants (
Potent serum antibody responses against N. meningitidis and H. influenzae can be induced by intranasal immunisation with conjugate vaccines in combination with mucosal adjuvants. Moreover, for the MenC antigen, the antibodies induced by intranasal immunisation had potent bactericidal activity, which is known to correlate with protective immunity [142]. In addition, IgA responses in the nasal cavity were induced only in animals immunised through the intranasal route. Inducing secretory immunity is important because the upper respiratory tract is the portal of entry for several pathogens, including N. meningitidis and H. influenzae.
Based on antibody titres obtained with conjugate vaccines given alone and in combination, and on the bactericidal activity measured against MenC, the combination of two vaccines co-administered with mucosal adjuvant did not negatively influence the antibody responses against MenC or Hib. The results thus suggest that intranasal immunisation is an effective route of immunisation for polysaccharide-protein conjugate vaccines in combination with mucosal adjuvants such as LT mutants.
The same dose of LT mutants was sufficient to significantly enhance the immunogenicity of both conjugate vaccines administered simultaneously. This is particularly important as it would reduce the amount of adjuvant needed and the risks associated with potential toxicity. Importantly, pre-existing immunity against the LTK63 mutant does not affect the ability of the mutant to act as an adjuvant for a second antigen [2]. Furthermore, the potency of mucosally-delivered vaccines may be further improved by formulating the vaccines in bioadhesive delivery systems [91].
In conclusion, combining polysaccharide-protein conjugate vaccines with LT mutants for intranasal immunisation is an effective approach to mucosal immunisation for pediatric use.
It will be understood that the invention is described above by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB03/02648 | 5/14/2003 | WO | 8/24/2005 |
| Number | Date | Country | |
|---|---|---|---|
| 60380675 | May 2002 | US |
