ES2363182T3 - VACCINES COMBINED AGAINST MENINGITIS. - Google Patents

Abstract

An aqueous immunogenic composition which, after administration to a subject, can induce an immune response that is (a) bactericidal against at least serogroup W135 of N. meningitidis and (b) protective against H. influenzae type b disease, wherein the composition comprises: (i) a conjugated capsular saccharide antigen of serogroup W135; and (ii) a conjugated capsular saccharide antigen of H. influenzae type b ('Hib'); and in which the saccharide of serogroup W135 is conjugated with a diphtheria toxoid.

Description

Technical field

The present invention relates to immunization against bacterial meningitis, and particularly to combined immunization against bacterial meningitis produced by multiple pathogens.

Prior art

N. meningitidis is a non-mobile Gram-negative human pathogen that colonizes the pharynx and causes meningitis (and, occasionally, septicemia in the absence of meningitis). It causes both endemic and epidemic disease. Following the introduction of the conjugate vaccine against Haemophilus influenzae type b (Hib), N. meningitidis is the leading cause of bacterial meningitis in the US. A third pathogen responsible for bacterial meningitis is Streptococcus pneumoniae, but an effective vaccine is now available (PrevNar ™ [1]). Like the Hib vaccine, the pneumococcal vaccine is based on conjugated capsular saccharide antigens.

Based on the organism's capsular polysaccharide, various serogroups of N. meningitidis have been identified, including (A, B, C, H, I, K, L, 29E, W135, X, Y and Z). Serogroup A is the pathogen almost always implicated in epidemic disease in sub-Saharan Africa. Serogroups B and C are responsible for the vast majority of cases in the United States and in most developed countries. Serogroups W135 and Y are responsible for the rest of the cases in the US. and developed countries. Although the capsular polysaccharide is an effective protective immunogen, each serogroup requires a separate saccharide antigen, and this solution is not suitable for immunizing against serogroup B. Therefore, recent success with conjugated saccharide vaccines against serogroup C (Menjugate ™ [2], Meningitec ™ and NeisVac-C ™) had no impact on the disease caused by serogroups A, B, W135 or Y; on the contrary, they present a selective pressure towards the emergence of these serogroups as the main cause of meningococcal disease.

An injectable tetravalent vaccine of capsular polysaccharides of serogroups A, C, Y and W135 [3,4] has been known for many years and is authorized for human use. The polysaccharides in this vaccine are unconjugated and are present at a 1: 1: 1: 1 [5] weight ratio with 50 µg of each purified polysaccharide. Although effective in adolescents and adults, it induces a poor immune response and short duration of protection and cannot be used in infants [eg, ref. 6]. In addition, vaccines have the disadvantage of requiring reconstitution from lyophilized forms at the time of use.

A vaccine has proven elusive for serogroup B. Vaccines based on vesicles of the outer membrane have been tested [eg, ref. 7], but protection is normally restricted to the strain used to prepare the vaccine.

Therefore, there is still a need for a vaccine that protects against meningococcal serogroups A, C, W135 and Y in children, and also one that does not require reconstitution before administration. In addition, there is still a need for a vaccine that widely protects against serogroup B.

WO 02/00249 describes a multivalent vaccine composition, a conjugate of the H. influenzae type b capsular polysaccharide and two or more additional bacterial polysaccharides.

WO 2004/067030 discloses an injectable immunogenic composition comprising capsular saccharides of at least two of serogroups A, C, W 135 and Y of Neisseria meningitidis. Capsular saccharides are conjugated to carrier protein (s) and / or are oligosaccharides. In the composition, (i) the composition comprises <50 µg of meningococcal saccharide per dose and / or (ii) the composition further comprises an antigen of one or more of the (a) serogroup B of N. meningitidis; (b) Haemophilus influenzae type b; and / or (c) Streptococcus pneumoniae. The saccharide antigens in the compositions are generally conjugated with a vehicle.

Disclosure of the invention

The invention satisfies all these diverse needs and is based on eight separate findings. First, the inventors have found that meningococcal conjugated capsular saccharides of serogroups C, W135 and Y are safe and immunogenic in humans when combined in a single dose. Second, they have found that this effect is retained when a conjugated capsular saccharide of serogroup A. is added. Third, they have found that these conjugated antigens can be stably combined in a single aqueous dose without the need for lyophilization. Fourth, they have found that broad protection against serogroup B infection can be achieved using a small number of defined polypeptide antigens. Fifth, they have found that these polypeptide antigens can be combined with saccharide antigens without loss of protective efficacy for any of the five serogroups. Sixth, they have found that efficacy is retained even if a Hib conjugate is added. Seventh, they have found that the efficacy of a W135 serogroup conjugate is enhanced by the addition of protein antigens derived from a serogroup B strain. Finally, they have found that the addition of a Hib conjugate to meningococcal conjugates enhances the overall activity against serogroup W135 of meningococcus.

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The invention provides the aqueous immunogenic composition of any of claims 1-6. The composition may additionally include conjugated capsular saccharide antigens of serogroups C and Y and, optionally, A. Additionally it includes serogroup B polypeptide antigens of N. meningitidis.

Preferred saccharide antigens are oligosaccharides.

Serogroups C, W135 and Y

Techniques for preparing capsular polysaccharides from meningococci have been known for many years and usually involve a process comprising the steps of precipitation of polysaccharides (for example, using a cationic detergent), fractionation with ethanol, extraction in cold phenol (to remove the protein) and ultracentrifugation (to eliminate LPS) [for example, see ref. 8].

A more preferred procedure [9] involves precipitation of polysaccharides followed by solubilization of the precipitated polysaccharide using a lower alcohol. Precipitation can be achieved using a cationic detergent such as tetrabutylammonium and cetyltrimethylammonium salts (eg, bromide salts), or hexadimethrin bromide and myristyltrimethylammonium salts. Cetyltrimethylammonium bromide ('CTAB') is particularly preferred [10]. The solubilization of the precipitated material can be achieved using a lower alcohol such as methanol, propan-1-ol, propan-2-ol, butan-1-ol, butan-2-ol, 2-methyl-propan-1-ol, 2 -methyl-propan-2-ol, diols, etc., but ethanol is particularly suitable for solubilizing CTAB-polysaccharide complexes. Ethanol can be added to the precipitated polysaccharide giving a final ethanol concentration (based on the total content of ethanol and water) of between 50% and 95%.

After resolubilization, the polysaccharide can be further treated to remove contaminants. This is particularly important in situations where even minor contamination is not acceptable (for example, for the production of human vaccines). This will normally involve one or more filtration stages, for example, deep filtration, active carbon filtration, size filtration and / or ultrafiltration can be used.

Once it has been filtered to remove contaminants, the polysaccharide can be precipitated for further treatment and / or processing. This can be conveniently achieved by exchanging cations (for example, by adding calcium or sodium salts).

After purification, the capsular saccharides are conjugated to carrier proteins as described below.

Other alternative procedures and procedures for the purification and conjugation of meningococcal saccharides are disclosed in references 11 and 12.

As an alternative to purification, the capsular saccharides of the present invention can be obtained by total or partial synthesis, for example, the synthesis of Hib is disclosed in ref. 13 and the synthesis of MenA in ref. 14.

The saccharide may be chemically modified, for example, it may be O-acetylated or de-O-acetylated. Any such de-O-acetylation or hyper-acetylation may be at specific positions in the saccharide. For example, most strains of serogroup C have O-acetyl groups in the C-7 and / or C-8 position of sialic acid residues, but approximately 15% of clinical isolated strains lack these O- groups. acetyl [15,16]. Acetylation does not appear to affect protective efficacy (for example, unlike the Menjugate ™ product, the NeisVac-C ™ product uses a de-O-acetylated saccharide, but both vaccines are effective). The saccharide of serogroup W135 is a polymer of disaccharide units of sialic acid-galactose. The saccharide of serogroup Y is similar to saccharide of serogroup W135, except that the disaccharide repeat unit includes glucose instead of galactose. Like serogroup C saccharides, MenW135 and MenY saccharides have variable Oacetylation, but at positions 7 and 9 of sialic acid [17]. Any such chemical modification takes place preferably before conjugation, but may alternatively or additionally take place during conjugation.

The saccharides of different serogroups are preferably purified separately, and then they can be combined, both before and after conjugation.

Serogroup A

Compositions of the invention may include a conjugated capsular saccharide antigen of serogroup A. The saccharide can be purified and conjugated in the same manner as for serogroups C, W135 and Y (see above), although it is structurally different - while the capsules of serogroups C, W 135 and Y are based on sialic acid (N-acetyl-neuraminic acid, NeuAc), the serogroup A capsule is based on Nacetyl-mannosamine, which is the natural precursor of sialic acid. The saccharide of serogroup A is particularly susceptible to hydrolysis, and its instability in aqueous media means that (a) the immunogenicity of liquid vaccines against serogroup A decreases over time, and (b) quality control is more difficult due to the release of products from the hydrolysis of saccharides in the vaccine.

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The native MenA capsular saccharide is a homopolymer of N-acetyl-D-mannosamine-1-phosphate bound in (α1 → 6) with partial O-acetylation at C3 and C4. The main glycosidic bond is a 1-6 phosphodiester bond that involves the hemiacetal C1 group and the C6 alcohol group of D-mannosamine. The average chain length is 93 monomers. It has the following formula:

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The inventors have prepared a modified saccharide antigen that retains the immunogenic activity of the saccharide of native serogroup A, but which is much more stable in water. The hydroxyl groups bound in the

10 carbons 3 and 4 of the monosaccharide units are replaced by a blocking group [ref. 18].

The number of monosaccharide units that have blocking groups instead of hydroxyls may vary. For example, all or substantially all monosaccharide units may have blocking groups. Alternatively, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the monosaccharide units may have blocking groups. At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,

15 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 units of monosaccharides may have blocking groups.

Also, the number of blocking groups in a monosaccharide unit may vary. For example, the number of blocking groups in any particular monosaccharide unit may be 1 or 2.

The terminal monosaccharide unit may or may not have a blocking group in place of its native hydroxyl. It is preferred to retain a free anomeric hydroxyl group in a terminal monosaccharide unit in order to

20 provide manipulation for other reactions (eg, conjugation). The anomeric hydroxyl groups can be converted into amino groups (-NH2 or -NH-E in which E is a nitrogen protecting group) by reductive amination (using, for example, NaBH3CN / NH4Cl), and then can be regenerated after others hydroxyl groups have become blocking groups.

The blocking groups to replace hydroxyl groups can be directly accessible by a reaction

25 derivatization of the hydroxyl group, that is, replacing the hydrogen atom of the hydroxyl group with another group. Derivatives of suitable hydroxyl groups acting as blocking groups are, for example, carbamates, sulfonates, carbonates, esters, ethers (for example, silyl ethers or alkyl ethers) and acetals. Some specific examples of such blocking groups are allyl, Aloc, benzyl, BOM, t-butyl, trityl, TBS, TBDPS, TES, TMS, TIPS, PMB, MEM, MOM, MTM, THP, etc. Other blocking groups that are not directly accessible and that replace

The entire hydroxyl group includes C1-12 alkyl, C3-12 alkyl, C5-12 aryl, C5-12 aryl-C1-6 alkyl, NR1R2 (R1 and R2 are defined in the following paragraph), H, F, Cl, Br, CO2H, CO2 (C1-6 alkyl), CN, CF3, CCl3, etc.

Preferred blocking groups are of the formula: -O-X-Y or -OR3 in which: X is C (O), S (O) or SO2; Y is C1-12 alkyl, C1-12 alkoxy, C3-12 cycloalkyl, C5-12 aryl or C5-12 aryl-C1-6 alkyl, each of which may be optionally substituted with 1, 2 or 3 groups independently selected from F, Cl, Br, CO2H, CO2 (C1-6 alkyl), CN, CF3 or CCl3; or Y is NR1R2; R1 and R2 are independently selected from H, C1-12 alkyl, C3-12 cycloalkyl, C5-12 aryl, C5-12 aryl-C1-6 alkyl; or R1 and R2 can join to form a saturated C3-12 heterocyclic group; R3 is C1-12 alkyl or C3 cycloalkyl

image4

12, each of which may be optionally substituted with 1, 2 or 3 groups independently selected from F, Cl, Br, CO2 (C1-6 alkyl), CN, CF3 or CCl3; or R3 is C5-12 aryl or C5-12 aryl-C1-6 alkyl, each of which may be optionally substituted with 1, 2, 3, 4 or 5 groups selected from F, Cl, Br, CO2H, CO2 (C1- alkyl 6), CN, CF3 or CCl3. If R3 is C1-12 alkyl or C3-12 cycloalkyl, it is usually substituted with 1, 2 or 3 groups as defined

5 above. If R1 and R2 join to form a saturated C3-12 heterocyclic group it means that R1 and R2 together with the nitrogen atom form a saturated heterocyclic group containing any number of carbon atoms between 3 and 12 (for example, C3, C4 , C5, C6, C7, C8, C9, C10, C11, C12). The heterocyclic group may contain 1 or 2 heteroatoms (such as N, O or S) other than the nitrogen atom. Examples of the saturated C3-12 heterocyclic group are pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, imidazolidinyl, azetidinyl and aziridinyl.

The blocking groups -O-X-Y and -OR3 can be prepared from -OH groups by standard derivatization procedures such as reaction of the hydroxyl group with an acyl halide, alkyl halide, sulfonyl halide, etc. Therefore, the oxygen atom in -O-X-Y is preferably the oxygen atom of the hydroxyl group, while the group -X-Y in -O-X-Y preferably substitutes the hydrogen atom of the hydroxyl group. Alternatively, the blocking groups may be accessible by a substitution reaction such as a

15 Mitsonobu type replacement. These and other methods of preparing blocking groups from hydroxyl groups are well known.

More preferably, the blocking group is -OC (O) CF3 [19], or a carbamate -OC (O) NR1R2 group in which R1 and R2 are independently selected from C1-6 alkyl. More preferably, R1 and R2 are both methyl, that is, the blocking group is -OC (O) NMe2. Carbamate blocking groups have a stabilizing effect on the bond

20 glycosidic and can be prepared under mild conditions.

Preferred modified MenA saccharides contain n monosaccharide units in which at least h% of the monosaccharide units do not have -OH groups in both positions 3 and 4. The value of h is 24 or more (for example, 25 , 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99 or 100) and is preferably 50 or more. Absent -OH groups are preferably blocking groups as defined above.

Other preferred modified MenA saccharides comprise monosaccharide units in which at least s of the monosaccharide units do not have -OH in position 3 and do not have -OH in position 4. The value of s is at least 1 (per example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90). Absent -OH groups are preferably blocking groups as defined above.

The modified MenA saccharides suitable for use with the invention have the formula:

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wherein n is an integer from 1 to 100 (preferably an integer from 5 to 25, more preferably 15-25); T is of formula (A) or (B): each group Z is independently selected from OH or a blocking group as defined above; Y

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image3

each group Q is independently selected from OH or a blocking group as defined above;

And it is selected from OH or a blocking group as defined above;

E is H or a nitrogen protecting group;

and in which more than about 7% (for example, 8%, 9%, 10% or more) of the Q groups are blocking groups.

Each of the n + 2 Z groups can be the same or different from each other. Also, each of the n + 2 groups Q may be the same or different from each other. All Z groups can be OH. Alternatively, at least 10%, 20, 30%, 40%, 50% or 60% of the Z groups may be OAc. Preferably, approximately 70% of the Z groups are OAc, the rest of the Z groups being OH or blocking groups as defined above. At least about 7% of the Q groups are blocking groups. Preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100% of the Q groups are blocking groups.

Preferred blocking groups are electron acceptor groups. Without wishing to adhere to any theory, it is believed that glycosidic bonds are unstable to hydrolysis due to the help of an intramolecular nucleophilic attack of a hydroxyl saccharide group on the glycosidic bond (i.e., by the formation of a cyclic intermediate) . The greater the nucleophilia of the hydroxyl group, the greater the tendency to intramolecular nucleophilic attack. An electron acceptor blocking group has the effect of delocalising the pair of oxygen ions, thereby decreasing the nucleophilia of oxygen and increasing the tendency to intramolecular nucleophilic attack.

Therefore, to protect against serogroup A, aqueous compositions may include a modified MenA saccharide as defined above.

Preferred compositions of the invention may be stored for 28 days at 37 ° C and, after that period, less than f% of the initial total amount of conjugated MenA saccharide will be unconjugated, where f 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or less.

Covalent conjugation

Capsular saccharides in compositions of the invention will normally be conjugated to carrier protein (s). In general, conjugation enhances saccharide immunogenicity as it converts them from T-independent antigens to T-dependent antigens, thus allowing priming of immunological memory. Conjugation is particularly useful for pediatric vaccines and is a well-known technique [for example, reviewed in refs. 20 to 29].

Preferred carrier proteins are bacterial toxins or toxoids such as diphtheria toxoid or tetanus toxoid, or the mutant of the diphtheria toxin CRM197 [30-32]. Other suitable carrier proteins include the outer membrane protein of N. meningitidis [33], synthetic peptides [34,35], heat shock proteins [36,37], pertussis proteins [38,39], cytokines [40], lymphokines [40], hormones [40], growth factors [40], artificial proteins comprising epitopes of multiple human CD4 + T lymphocytes of various pathogen-derived antigens [41] such as protein N19 [42], protein D of H. influenzae [43,44], pneumolysin [45], Pneumococcal surface PspA protein [46], iron uptake proteins [47], C. difficile toxin A or B [48], mutant bacterial toxins (by example, cholera toxin 'CT' or heat-labile toxin of E. coli 'LT') such as a CT with a substitution in Glu-29 [49], etc., preferred vehicles are diphtheria toxoid, tetanus toxoid , H. influenzae protein D and particularly CRM197.

Within a composition of the invention it is possible to use more than one carrier protein, for example, to reduce the risk of vehicle suppression. Therefore, different carrier proteins for different serogroups can be used, for example, serogroup A saccharides could be conjugated with CRM197, while serogroup C saccharides could be conjugated with tetanus toxoid. It is also possible to use more than one carrier protein for a particular saccharide antigen, for example, serogroup A saccharides could be in two groups, with some conjugates with CRM197 and others conjugated with tetanus toxoid. However, it is generally preferred to use the same carrier protein for all serogroups, with CRM197 being the preferred choice.

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A single carrier protein could carry more than one saccharide antigen [50]. For example, a single carrier protein could have been conjugated with saccharides from serogroups A and C. To achieve this goal, saccharides can be mixed before the conjugation reaction. However, it is generally preferred to have separate conjugates for each serogroup.

Conjugates with a saccharide: protein ratio (weight / weight) of between 1: 5 (i.e. excess protein) and 5: 1 (i.e. excess saccharide) are preferred. Relationships between 1: 2 and 5: 1 are preferred since the ratios between 1: 1.25 and 1: 2.5 are more preferred. Excess carrier protein may be preferred for MenA and MenC.

Conjugates can be used in conjunction with free carrier protein [51]. When a given carrier protein is present in both free and conjugated form in a composition of the invention, the unconjugated form is preferably not more than 5% of the total amount of the carrier protein in the composition as a whole, and more preferably it is present at less than 2% by weight.

Any suitable conjugation reaction can be used, with any suitable connector if necessary.

The saccharide will be activated or functionalized normally before conjugation. Activation may involve, for example, cyanylation reagents such as CDAP (for example, 1-cyano-4-dimethylaminopyridinium tetrafluoroborate [52, 53, etc.]). Other suitable techniques use carbodiimides, hydrazides, active esters, norbornano, pnitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU; see also the introduction to reference 27).

The linkages by means of a linker group can be made using any known method, for example, the procedures described in references 54 and 55. One type of linkage involves reductive amination of the polysaccharide, coupling of the resulting amino group with one end of a linkage group. adipic acid and then coupling a protein to the other end of the adipic acid binding group [25, 56, 57]. Other connectors include B-propionamido [58], nitrophenyl-ethylamine [59], haloacyl halide [60], glycosidic bonds [61], 6aminocaproic acid [62], ADH [63], C4 to C12 residues [64], etc. . As an alternative to using a connector, the direct link can be used. Direct links to the protein may comprise oxidation of the polysaccharide followed by reductive amination with the protein, as described in, for example, references 65 and 66.

A process involving the introduction of amino groups into the saccharide (for example, replacing = O-terminal groups with -NH2) followed by derivatization with an adipic diester (eg, N-hydroxysuccinimido diester of adipic acid) and reaction with carrier protein is preferred. Another preferred reaction uses the activation of CDAP with a protein D vehicle, for example, for MenA or MenC.

After conjugation, free and conjugated saccharides can be separated. There are many suitable procedures that include hydrophobic chromatography, tangential ultrafiltration, diafiltration, etc. [see also refs. 67 and 68, etc.].

If the composition of the invention includes a conjugated oligosaccharide, it is preferred that the oligosaccharide preparation precede conjugation.

After conjugation, the methods of the invention may include a step of measuring the level of unconjugated carrier protein. One way of making this measurement involves capillary electrophoresis [69] (for example, in free solution), or micellar electrokinetic chromatography [70].

After conjugation, the methods of the invention may include a step of measuring the level of unconjugated saccharide. One way of doing this measurement involves HPAEC-PAD [67].

After conjugation, the methods of the invention may include a step of separating the conjugated saccharide from the unconjugated saccharide. One way to separate these saccharides is to use a process that selectively precipitates a component. Selective precipitation of conjugated saccharide is preferred to leave saccharide unconjugated in solution, for example, by treatment with deoxycholate [67].

After conjugation, the methods of the invention may include a step of measuring the molecular size and / or molar mass of a conjugate. In particular, distributions can be measured. One way of making these measurements involves size exclusion chromatography with detection by multi-angle light scattering photometry and differential refractometry (SEC-MALS / RI) [71].

Oligosaccharides

Capsular saccharides will generally be used in the form of oligosaccharides. These are conveniently formed by fragmentation of purified capsular polysaccharide (for example, by hydrolysis), which will normally be followed by purification of the fragments of the desired size.

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Polysaccharide fragmentation is preferably performed giving a final average degree of polymerization (GP) in the oligosaccharide of less than 30 (for example, between 10 and 20, preferably about 10 for serogroup A; between 15 and 25 for serogroups W 135 and Y, preferably about 15-20; between 12 and 22 for serogroup C; etc.). The GP can be conveniently measured by ion exchange chromatography or by colorimetric assays [72].

If hydrolysis is performed, the hydrolyzate will generally be sized in order to remove short-length oligosaccharides [73]. This can be achieved in various ways such as ultrafiltration followed by ion exchange chromatography. Oligosaccharides with a degree of polymerization of less than or equal to about 6 are preferably removed for serogroup A, and those of less than about 4 are preferably removed for serogroups W 135 and Y.

The chemical hydrolysis of saccharides generally involves treatment with both acid and base under conditions that are standard in the art. Conditions for depolymerization of capsular saccharides in their constituent monosaccharides are known in the art. A depolymerization process involves the use of hydrogen peroxide [11]. The hydrogen peroxide is added to a saccharide (for example, giving a final H2O2 concentration of 1%), and then the mixture is incubated (for example, at about 55 ° C) until a desired chain length reduction has been achieved . The reduction over time can be followed by sampling the mixture and then measuring the molecular size (average) of the saccharide in the sample. Depolymerization can then be stopped by rapid cooling once a desired chain length has been reached.

Serogroup B

Vaccines against pathogens such as hepatitis B virus, diphtheria and tetanus usually contain a single protein antigen (for example, the HBV surface antigen or a tetanus toxoid). In contrast, the acellular pertussis vaccine usually contains at least three B. pertussis proteins and the PrevNar ™ pneumococcal vaccine contains seven separate conjugated saccharide antigens. Other vaccines such as cell pertussis vaccines, measles vaccine, inactivated polio vaccine (IPV) and meningococcal OMV vaccines are by their very nature complex mixtures of a large number of antigens. Whether or not the protection can be caused by a single antigen, a small number of defined antigens, or a complex mixture of undefined antigens, therefore, depends on the pathogen in question.

As mentioned earlier, a serogroup B meningococcal vaccine has proven evasive. OMV-based vaccines show a narrow efficacy. In addition, the large number of undefined antigens present in a VMO, combined with their variable nature, means that VMOs have various quality control problems. The inventors have found that broad protection against serogroup B infection can be achieved, and that this can be achieved using a small number of defined serogroup B polypeptide antigens and, therefore, the compositions of the invention include one or more antigens. of polypeptides so that the composition can induce an immune response that is bactericidal against two or more (i.e., 2 or 3) of the hypervirulent lineages A4, ET-5 and lineage 3 of serogroup B of N. meningitidis.

Genome sequences have been reported for meningococcal serogroups A [74] and B [75,76] and suitable antigens can be selected from the encoded polypeptides [eg, refs. 77-82]. Candidate antigens have been manipulated to improve heterologous expression [refs. 83 to 85].

A preferred composition includes a Tbp protein and an Hsf protein [86]. Hsf is an autotransporter protein [87-89] also known as nhhA [89], GNA0992 [77] or NMB0992 [75]. Tbp is a transferrin binding protein [90-93] and encompasses both TbpA and TbpB and the high molecular weight and low molecular weight forms of TbpA and TbpB. Tbp encompasses the individual proteins described above and protein complexes and any other protein or complex thereof that can bind transferrin. Although Tbp may refer to both the high and low molecular weight forms of TbpA or TbpB, it is preferred that both high molecular weight and low molecular weight forms of TbpA and / or TbpB are present. Most preferably, high molecular weight and low molecular weight TbpA is present.

Another preferred composition includes the lipooligosaccharide of serogroup B (LOS) [94]. The LOS can be used in addition to the serogroup B polypeptide (s) or it can be used instead of him / them.

Another preferred composition includes at least one antigen selected from each of the at least two different categories of proteins that have different functions within Neisseria. Examples of such protein categories are adhesins, autotransporter proteins, toxins, integral outer membrane proteins and iron acquisition proteins. These antigens can be selected as follows using the nomenclature of reference 95: at least one Neisseria adhesin selected from the group consisting of FhaB, NspA PiIC, Hsf, Hap, MafA, MafB, Omp26, NMB0315, NMB0995, NMB 1119 and NadA ; at least one Neisseria autotransporter selected from the group consisting of Hsf, Hap, IgA protease, AspA and NadA; at least one Neisseria toxin selected from the group consisting of FrpA, FrpC, FrpA / C, VapD, NM-ADPRT (NMB 1343) and one or both of LPS immunotype L2 and LPS immunotype L3; at least one Neisseria Fe acquisition protein selected from the group consisting of TbpA, TbpB, LbpA, LbpB, HpuA, HpuB, Lipo28 (GNA2132), Sibp, NMB0964, NMB0293, FbpA, Bcp, BfrA, BfrA and P2086 (Xth ); at least one Neisseria membrane-associated protein, preferably outer membrane protein, particularly integral outer membrane protein, selected from the group consisting of PilQ, OMP85, FhaC, NspA, TbpA, LbpA, TspA, TspB, TdfH, PorB, MItA, HpuB, HimD, HisD, GNA1870, OstA, HlpA (GNA1946), NMB1124, NMB 1162, NMB1220, NMB1313, NMB 1953, HtrA and PLDA (OMPLA). It is said that these combinations of Neisseria antigens lead to a surprising enhancement of the efficacy of the vaccine against Neisseria infection [95]. Particularly preferred compositions include one or more of the following five antigens [96]: (1) a 'NadA' protein, preferably in oligomeric form (eg, in trimeric form); (2) a '741' protein; (3) a '936' protein; (4) a '953' protein; and (5) a '287' protein.

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'NadA' (Neisseria adhesin A) from MenB is disclosed as '961' protein in reference 80 (SEQ ID 2943 and 2944) and as 'NMB1994' in reference 75 (see also GenBank accession numbers: 11352904 and 7227256). A detailed study of the protein can be found in reference 97. If used according to the present invention, NadA can take various forms. Preferred forms of NadA are truncation or deletion variants such as those disclosed in references 83 to 85. In particular, NadA is preferred without its C-end membrane anchor (eg, residue deletion 351-405 for strain 2996 giving SEQ ID NO: 1 in this document), which is sometimes distinguished in this document by the use of a superscript 'C', for example, NadA (C). The expression of NadA without its membrane anchor domain in E. coli results in the secretion of the protein in the culture supernatant with concomitant removal of its 23mer conducting peptide (for example, to leave a number for strain 2996 [SEQ ID NO. : 2 in this document]). Polypeptides without their conductive peptides are sometimes distinguished herein by the use of a superscript 'NL', for example, NadA (NL) or NadA (C) (NL). Preferred NadA polypeptides have an amino acid sequence that: (a) has 50% or more identity (eg, 60%, 70%, 80%, 90%, 95%, 99% or more) with SEQ ID Nº: 2; and / or (b) comprise a fragment of at least n consecutive amino acids of SEQ ID NO: 1, in which n is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments for (b) lack one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) of the C and / end or from the N-terminus of SEQ ID NO: 1 (for example, NadA (C), NadA (NL), NadA (C) (NL)). Other preferred fragments comprise an epitope of SEQ ID 1, and a particularly preferred fragment of SEQ ID 1 is SEQ ID 2. These various sequences include NadA variants (eg, allelic variants, homologs, orthologs, paralogs, mutants, etc.) . Various sequences of NadA are shown in Figure 9 of reference 98.

MenB's '741' protein is disclosed in reference 80 (SEQ ID 2535 and 2536) and as 'NMB1870' in reference 75 (see also GenBank accession number G1: 7227128). The corresponding protein in serogroup A

[74] has a GenBank accession number 7379322. 741 is naturally a lipoprotein. If used according to the present invention, protein 741 can take various forms. Preferred forms of 741 are truncation or deletion variants such as those disclosed in references 83 to 85. In particular, the N-terminus of 741 can be deleted up to and include its polyglycine sequence (i.e., deletion of residues 1 to 72 for strain MC58 [SEQ ID NO: 3 in this document]), which is sometimes distinguished here by the use of a prefix 'ΔG'. This deletion can enhance expression. The deletion also eliminates the lipidation site of 741. Preferred sequences of 741 have an amino acid sequence that: (a) has 50% or more identity (eg, 60%, 70%, 80%, 90%, 95%, 99% or more) with SEQ ID NO: 3; and / or (b) comprises a fragment of at least n consecutive amino acids of SEQ ID NO: 3 in which n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30 , 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments for (b) comprise an epitope of 741. Other preferred fragments lack one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) of end C and / or end N of SEQ ID NO: 3. These sequences include 741 variants (for example, allelic variants, homologs, orthologs, paralogs, mutants, etc.). Various sequences of 741 can be found in SEQ ID 1 to 22 of reference 85, in SEQ ID 1 to 23 of reference 99 and in SEQ ID 1-299 of reference 100.

The '936' protein of serogroup B is disclosed in reference 80 (SEQ ID 2883 and 2884) and as 'NMB2091' in reference 75 (see also GenBank GI accession number: 7227353). The corresponding gene in serogroup A [74] has the GenBank accession number 7379093. If used according to the present invention, protein 936 can take various forms. Preferred forms of 936 are truncation or deletion variants such as those disclosed in references 83 to 85. In particular, the N-terminus conducting peptide of 936 may be deleted (eg, deletion of residues 1 to 23 for the strain MC58 giving 936 (NL) [SEQ ID NO: 4 in this document]). Preferred 936 sequences have an amino acid sequence that: (a) has 50% or more identity (e.g., 60%, 70%, 80%, 90%, 95%, 99% or more) with SEQ ID Nº: 4; and / or (b) comprises a fragment of at least n consecutive amino acids of SEQ ID NO: 4 in which n is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20, 25, 30 , 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments for (b) comprise an epitope of 936. Other preferred fragments lack one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) of end C and / or end N of SEQ ID NO: 4. These sequences include variants of 936 (for example, allelic variants, homologs, orthologs, paralogs, mutants, etc.).

The '953' protein of serogroup B is disclosed in reference 80 (SEQ ID 2917 and 2918) and as 'NMB1030' in reference 75 (see also GenBank GI accession number: 7226269). The corresponding protein in serogroup A [74] has GenBank accession number 7380108. If used according to the present invention, protein 953 can take various forms. Preferred forms of 953 are truncation or deletion variants such as those disclosed in references 83 to 85. In particular, the N3-conducting conductor peptide of 953 may be deleted (eg, deletion of residues 1 to 19 for the strain MC58 giving 953 (NL) [SEQ ID NO: 5 herein.] Preferred 953 sequences have an amino acid sequence that: (a) has 50% or more identity (eg, 60%, 70% , 80%, 90%, 95%, 99% or more) with SEQ ID NO: 5; and / or (b) comprises a fragment of at least n consecutive amino acids of SEQ ID NO: 5 in which n is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more) Preferred fragments for (b) they comprise an epitope of 953. Other preferred fragments lack one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more ) of the C-terminus and / or the N-terminus of SEQ ID NO: 5. These sequences include variants of 9 36 (for example, allelic variants, homologs, orthologs, paralogs, mutants, etc.). Allelic forms of 953 can be seen in Figure 19 of reference 82.

image9

The '287' protein of serogroup B is disclosed in reference 80 (SEQ ID 3103 and 3104), as 'NMB2132' in reference 75 and as 'GNA2132' in reference 77 (see also GenBank GI accession number: 7227388). The corresponding protein in serogroup A [74] has the GenBank accession number 7379057. If used according to the present invention, protein 287 can take various forms. Preferred forms of 287 are truncation or deletion variants such as those disclosed in references 83 to 85. In particular, the N-terminus of 287 can be deleted up to and include its polyglycine sequence (eg, deletion of residues 1 to 24 for strain MC58 giving ΔG287 [SEQ ID NO: 6 herein] This deletion can enhance expression Sequences of 287 preferred have an amino acid sequence that: (a) has 50% or more identity (per example, 60%, 70%, 80%, 90%, 95%, 99% or more) with SEQ ID NO: 6; and / or (b) comprises a fragment of at least n consecutive amino acids of SEQ ID NO: 6 where n is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more.) Preferred fragments for (b) comprise an epitope of 287. Other preferred fragments lack one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) of the C end and / or the N end of SEQ ID NO: 6. These sequences include 287 variants (eg allelic variants, homologs, orthologs, paralogs, mutants, etc.). Allelic forms of 287 can be seen in Figures 5 and 15 of reference 82, and in Example 13 and Figure 21 of reference 80 (SEQ ID 3179 to 3184).

Preferred MenB antigens comprise an amino acid sequence found in one of strains 2996, MC58, 95N477 and 394/98. Protein 287 is preferably from strain 2996, or more preferably from strain 394/98. Protein 741 is preferably from strains MC58, 2996, 394/98 or 95N477 from serogroup B, or from strain 90/18311 from serogroup C. Strain MC58 is more preferred. Proteins 936, 953 and NadA are preferably of strain 2996. If a composition includes a particular protein antigen (for example, 741 or 287), the composition may include that antigen in more than one variant form, for example, the same protein, but of more than one strain. These proteins can be included as tandem or separated proteins.

However, in some embodiments, the composition of the invention includes the same protein, but of more than one strain. This solution has been found to be effective with the 741 protein. This protein is an extremely effective antigen for eliciting anti-meningococcal antibody responses and is expressed throughout all meningococcal serogroups. The phylogenetic analysis shows that the protein is divided into two groups, and that one of these is divided again giving three variants in total [101] and, while the serum produced against a given variant is bactericidal within the same variant group, it is not active against strains that express one of the other two variants, that is, there is intra-variant cross protection, but no inter-variant cross protection [99, 101]. Therefore, for maximum cross-strain efficacy it is preferred that a composition includes more than one variant of 741 protein. An exemplary sequence of each variant is given in SEQ ID NO: 10, 11 and 12 herein, starting with a cysteine residue of the N-terminus to which the lipid will covalently bind in the form of native lipoprotein. Therefore, it is preferred that the composition may include at least two of: (1) a first protein comprising an amino acid sequence having at least a% sequence identity with SEQ ID NO: 10 and / or comprising a amino acid sequence consisting of a fragment of at least x contiguous amino acids of SEQ ID NO: 10; (2) a second protein comprising an amino acid sequence having at least b% sequence identity with SEQ ID NO: 11 and / or comprising an amino acid sequence consisting of a fragment of at least and contiguous amino acids of SEQ ID NO: 11; and (3) a third protein, which comprises an amino acid sequence that has at least c% sequence identity with SEQ ID NO: 12 and / or that comprises an amino acid sequence consisting of a fragment of at least z amino acids. contiguous of SEQ ID NO: 12. The value of a is at least 85, for example, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or more. The value of b is at least 85, for example, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or more. The value of c is at least 85, for example, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or more. The values of a, b and c are not intrinsically related to each other. The value of x is at least 7, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The value of y is at least 7, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The value of z is at least 7, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The values of x, y and z are not intrinsically related to each other. It is preferred that any given 741 amino acid sequence is not in more than one of the categories (1), (2) and (3). Therefore, any given sequence of 741 will be found in only one of the categories (1), (2) and (3). Therefore, it is preferred that: the protein (1) has less than i% sequence identity with the protein (2); the protein (1) has less than j% sequence identity with the protein (3); and the protein (2) has less than k% sequence identity with the protein (3). The value of i is 60 or more (for example, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, etc.) and at most it is a. The value of j is 60 or more (for example, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, etc.) and at most is b. The value of k is 60 or more (for example, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, etc.) and at most is c. The values of i, j and k are not intrinsically related to each other.

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The compositions of the invention include a small number (eg, less than t antigens, being t 10, 9, 8, 7, 6, 5, 4 or 3) of purified serogroup B antigens. It is particularly preferred that the composition cannot include complex or undefined mixtures of antigens, for example, it is preferred that vesicles of the outer membrane are not included in the composition. The antigens are preferably expressed recombinantly in a heterologous host and then purified. For a composition that includes MenB t antigens they may be separate t polypeptides but, to reduce complexity even further, it is preferred that at least two of the antigens be expressed as a single polypeptide chain (a 'hybrid' protein [refs. 83 to 85]), that is, so that the t antigens form less than t polypeptides. Hybrid proteins offer two main advantages: first, a protein that can be unstable or express itself badly can be helped by adding a suitable hybrid component to overcome the problem; second, commercial manufacturing is simplified since only one expression and purification need to be employed in order to produce two separately useful proteins. A hybrid protein included in a composition of the invention may comprise two or more (ie, 2, 3, 4 or 5) of the five antigens listed above. Hybrids consisting of two of the five antigens are preferred.

Within the combination of five basic antigens (NadA, 741, 953, 936 and 287), an antigen may be present in more than one hybrid protein and / or as a non-hybrid protein. However, it is preferred that an antigen be present as both a hybrid and a non-hybrid, but not as both, although it may be useful to include protein 741 as both a hybrid and a non-hybrid antigen (preferably lipoprotein), particularly when used more than one variant of 741.

Hybrid proteins can be represented by the formula NH2-A - [- X-L-] n-B-COOH in which: X is an amino acid sequence of one of the five basic antigens; L is an optional connective amino acid sequence; A is an optional N-terminus amino acid sequence; B is an amino acid sequence of the optional C-terminus; and n is 2, 3, 4 or 5.

Most preferably, n is 2. Hybrids of two antigens for use in the invention comprise: NadA and 741; NadA and 936; NadA and 953; NadA and 287; 741 and 936; 741 and 953; 741 and 287; 936 and 953; 936 and 287; 953 and 287. Two preferred proteins are: X1 is 936 and X2 is 741; X1 is 287 and X2 is 953.

If an -X- moiety has a naturally occurring conductive peptide sequence, it can be included or omitted in the hybrid protein. In some embodiments, the conductive peptides will be deleted, except that of the -X moiety located at the N-terminus of the hybrid protein, that is, the X1 conductive peptide will be conserved, but the X2 conductive peptides ... Xn will be omitted. This is equivalent to deleting all the conductive peptides and using the X1 conducting peptide as the remainder -A-.

For each case of n of [-X-L-], the amino acid linker sequence -L- may be present or absent. For example, if n = 2 the hybrid can be NH2-X1-L1-X2-L2-COOH, NH2-X1-X2-COOH, NH2-X1-L1-X2-COOH, NH2-X1-X2-L2COOH, etc. . The connecting amino acid sequence (s) -L- will normally be short (for example, 20 or less amino acids, that is, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples comprise short peptide sequences that facilitate cloning, polyglycine connectors (i.e., comprising Glyn in which n = 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) and tags of histidine (that is, Hisn in which n = 3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable connecting amino acid sequences will be apparent to those skilled in the art. A useful connector is GSGGGG (SEQ ID 9), the Gly-Ser dipeptide being formed by a BamHI restriction site, thus aiding in cloning and manipulation, and the tetrapeptide (Gly) 4 being a typical polyglycine connector. If Xn + 1 is a ΔG protein and Ln is a glycine linker, this can be equivalent to Xn + 1 with no ΔG protein and Ln being absent.

-A- is an optional N-terminus amino acid sequence. This will normally be short (for example, 40 or less amino acids, that is, 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, 3, 2, 1). Examples include conductive sequences to direct protein traffic, or short peptide sequences that facilitate cloning or purification (eg, histidine tags, that is, Hisn in which n = 3, 4, 5, 6, 7, 8 , 9, 10 or more). Other suitable N-terminal amino acid sequences will be apparent to those skilled in the art. If X1 lacks its own methionine of the N-terminus, -A- is preferably an oligopeptide (for example, with 1, 2, 3, 4, 5, 6, 7 or 8 amino acids) that provides a methionine of the N-terminus.

-B- is an optional C-terminal amino acid sequence. This will normally be short (for example, 40 or less amino acids, that is, 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, 3, 2, 1). Examples include sequences to direct protein traffic, short peptide sequences that facilitate cloning or purification (eg, comprising histidine tags, that is, Hisn in which n = 3, 4, 5, 6, 7, 8 , 9, 10 or more) or sequences that enhance protein stability. Other suitable C-terminal amino acid sequences will be apparent to those skilled in the art.

image11

Two particularly preferred hybrid proteins of the invention are as follows:

n

TO X1 L1 X2 L2 B SEQ ID NO:

2

MA ΔG287 GSGGGG 953 (NL) - - 7

2

M 936 (NL) GSGGGG ΔG741 - - 8

10 These two proteins can be used in combination with NadA (particularly with SEQ ID NO: 2). Thus, a preferred composition of MenB antigens for use with the invention includes a first polypeptide comprising the amino acid sequence SEQ ID NO: 2, a second polypeptide comprising the amino acid sequence SEQ ID NO: 7 and a third polypeptide comprising the amino acid sequence SEQ ID NO: 8. This is a preferred group of MenB antigens for use with the invention.

As mentioned above, the compositions of the invention can induce a serum bactericidal antibody response that is effective against two or three of the hypervirulent lineages of MenB A4, ET-5 and lineage 3. Additionally, they can induce bactericidal antibody responses. against one or more of the hypervirulent lineages of subgroup I, subgroup III, subgroup IV-1 or ET-37 complex, and against other lineages, for example, hyperinvasive lineages. These antibody responses are conveniently measured in mice and are an indicator

20 vaccine efficacy standard [for example, see final note 14 of reference 77]. Serum bactericidal activity (SBA) measures complement-mediated bactericidal destruction and can be tested using human or rabbit baby complement. WHO standards require a vaccine to induce at least a 4-fold increase in SBA in more than 90% of recipients.

The composition does not need to induce bactericidal antibodies against all strains of MenB within these lineages.

25 hypervirulent; rather, for any given group of four or more serogroup B meningococcal strains within a particular hypervirulent lineage, the antibodies induced by the composition are bactericidal against at least 50% (eg, 60%, 70%, 80% , 90% or more) of the group. Preferred groups of strains will include strains isolated in at least four of the following countries: GB, AU, CA, NO, IT, US, NZ, NL, BR and CU. The serum preferably has a bactericidal titer of at least 1024 (for example, 210, 211, 212, 213, 214, 215, 216, 217, 218 or

30, preferably at least 214), that is, the serum can destroy at least 50% of the test bacteria of a particular strain when diluted 1/1024, as described in reference 77.

Preferred compositions can induce bactericidal responses against the following serogroup B meningococcal strains: (i) of the A4 set, strain 961-5945 (B: 2b: P1.21,16) and / or strain G2136 (B: - ); (ii) of the ET5 complex, strain MC58 (B: 15: PI.7,16b) and / or strain 44/76 (B: 15: P1.7,16); (iii) of lineage 3, strain 394/98 (B: 4: P1.4) and / or strain BZ198 (B: NT :-). More preferred compositions may induce bactericidal responses against strains 9615945, 44/76 and 394/98. Strains 961-5945 and G2136 are both reference strains by Neisseria MLST [identifications 638 and 1002 in ref. 102]. Strain MC58 is widely available (for example, ATCC BAA335) and was the strain sequenced in reference 75. Strain 44/76 has been widely used and characterized (for example, ref. 103) and is one of the strains of reference by MLST of Neisseria [identification 237 in ref. 102; row

40 32 of Table 2 in ref. 104]. Strain 394/98 was originally isolated in New Zealand in 1998 and there have been several studies using this strain (for example, refs. 105 and 106). Strain BZ198 is another reference strain by MLST [identification 409 in ref. 102; row 41 of Table 2 in ref. 104]. The composition can further induce a bactericidal response against strain LNP17592 of serogroup W135 (W135: 2a: P1.5,2) of the ET-37 complex. This is a Haji strain isolated in France in 2000.

Other MenB polypeptide antigens that may be included in the compositions of the invention include those comprising one of the following amino acid sequences: SEQ ID NO: 650 of ref. 78; SEQ ID NO: 878 of ref. 78; SEQ ID NO: 884 of ref. 78; SEQ ID NO: 4 of ref. 79; SEQ ID NO: 598 of ref. 80; SEQ ID NO: 818 of ref. 80; SEQ ID NO: 864 of ref. 80; SEQ ID NO: 866 of ref. 80; SEQ ID NO: 1196 of ref. 80; SEQ ID NO: 1272 of ref. 80; SEQ ID NO: 1274 of ref. 80; SEQ ID NO: 1640 of ref. 80; SEQ ID NO: 1788 of ref. 80; SEC

50 ID Nº: 2288 of ref. 80; SEQ ID NO: 2466 of ref. 80; SEQ ID NO: 2554 of ref. 80; SEQ ID NO: 2576 of ref. 80; SEQ ID NO: 2606 of ref. 80; SEQ ID NO: 2608 of ref. 80; SEQ ID NO: 2616 of ref. 80; SEQ ID NO: 2668 of ref. 80; SEQ ID NO: 2780 of ref. 80; SEQ ID NO: 2932 of ref. 80; SEQ ID NO: 2958 of ref. 80; SEQ ID NO: 2970 of ref. 80; SEQ ID NO: 2988 of ref. 80, or a polypeptide comprising an amino acid sequence that: (a) has 50% or more identity (eg, 60%, 70%, 80%, 90%, 95%, 99% or more) with said sequences; and / or (b) comprises a fragment of at least n consecutive amino acids of said sequences in which n is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments for (b) comprise an epitope of the relevant sequence. More than one (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more) of these polypeptides may be included.

image12

Other antigen components

Non-meningococcal and non-Neisseria antigens, preferably those that do not decrease the immune response against meningococcal components, can also be included in compositions of the invention. The ref. 107, for example, discloses combinations of oligosaccharides from serogroups B and C of N. meningitidis together with saccharide Hib. Particularly preferred non-meningococcal antigens include:

-

a diphtheria antigen such as a diphtheria toxoid [eg, chapter 3 of ref. 108].

-

a tetanus antigen such as a tetanus toxoid [eg, chapter 4 of ref. 108].

- Pertussis holotoxin (PT) and filamentous hemagglutinin (FHA) of B. pertussis, optionally also in combination with pertactin and / or agglutinogens 2 and 3 [eg, refs. 109 and 110].

- pertussis cell antigen.

-

a hepatitis A virus antigen such as inactivated virus [eg, 111, 112].

-

a hepatitis B virus antigen such as surface and / or core antigens [eg 112,113], the surface antigen being preferably adsorbed onto an aluminum phosphate [114].

- polio antigen (s) [eg, 115, 116] such as IPV.

The mixture may comprise one or more of these other antigens that may be detoxified if necessary (for example, detoxification of pertussis toxin by chemical and / or genetic means).

If a diphtheria antigen is included in the mixture it is also preferred to include tetanus antigen and pertussis antigens. Similarly, if a tetanus antigen is included it is also preferred to include diphtheria and pertussis antigens. Similarly, if a pertussis antigen is included it is also preferred to include diphtheria and tetanus antigens.

The antigens in the mixture will normally be present at a concentration of at least 1 µg / ml each. In general, the concentration of any given antigen will be sufficient to elicit an immune response against that antigen. It is preferred that the protective efficacy of individual saccharide antigens is not eliminated by combining them, although actual immunogenicity (eg, ELISA titers) can be reduced.

As an alternative to the use of protein antigens in the mixture, nucleic acid encoding the antigen can be used. Thus, the protein components of the mixture can be replaced by nucleic acid (preferably DNA, for example, in the form of a plasmid) that encodes the protein. Similarly, the compositions of the invention may comprise proteins that mimic saccharide antigens, for example, mimotopes [117] or anti-idiotypic antibodies. These may replace individual saccharide components or may complement them. As an example, the vaccine can comprise a MenC [118] or MenA [119] capsular polysaccharide peptide mimetic instead of the saccharide itself.

A non-meningococcal antigen that is included in compositions of the invention is one that protects against H. influenzae type b (Hib). A preferred non-meningococcal antigen for further inclusion in compositions of the invention is one that protects against Streptococcus pneumoniae.

H. influenzae type b (Hib)

The composition includes an H .influenzae type b antigen, specifically a conjugated Hib capsular saccharide antigen. H. influenzae b saccharide antigens 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 Hib capsular polysaccharide in particular, is very well documented [eg, references 21-29, etc.]. The invention can use any suitable Hib conjugate. Suitable carrier proteins are described above, and the preferred vehicles for Hib saccharides are CRM197 ('HbOC'), tetanus toxoid ('PRP-T') and the outer membrane complex of N. meningitidis ('PRP-OMP').

The saccharide moiety of the conjugate may be a polysaccharide (for example, full length polyiribosylribitol phosphate (PRP)), but it is preferred to hydrolyze polysaccharides to form oligosaccharides (eg, MW from -1 to -5 kDa).

A preferred conjugate comprises a Hib oligosaccharide covalently linked to CRM197 via an adipic acid linker [120, 121]. Tetanus toxoid is also a preferred vehicle.

Administration of the Hib antigen preferably produces an anti-PRP antibody concentration of ≥0.15 µg / ml, and more preferably ≥1 µg / ml.

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With the Hib saccharide antigen in the composition it is preferred not to include an aluminum hydroxide adjuvant. If the composition includes an aluminum phosphate adjuvant, then the Hib antigen can be adsorbed to the adjuvant [122] or it can be non-adsorbed [123]. Adsorption prevention can be achieved by selecting the correct pH during antigen / adjuvant mixing, an adjuvant with an appropriate zero charge point and an appropriate mixing order for the various different antigens in a composition [124].

The compositions of the invention may comprise more than one Hib antigen. Hib antigens can be lyophilized, for example, for reconstitution by meningococcal compositions of the invention.

Streptococcus pneumoniae

If the composition includes an S. pneumoniae antigen, it will normally be a capsular saccharide antigen that is preferably conjugated to a carrier protein [eg, refs. 125 to 127]. It is preferred to include saccharides of more than one S. pneumoniae serotype. For example, mixtures of polysaccharides of 23 different serotypes are widely used since they are conjugate vaccines with polysaccharides of between 5 and 11 different serotypes [128]. For example, PrevNar ™ [I] contains seven serotype antigens (4, 6B, 9V, 14, 18C, 19F and 23F), each saccharide being individually conjugated to CRM197 by reductive amination, with 2 µg of each saccharide per 0.5 ml of dose (4 µg of serotype 6B), and with conjugates adsorbed on an aluminum phosphate adjuvant. The compositions of the invention preferably include at least serotypes 6B, 14, 19F and 23F. The conjugates can be adsorbed on an aluminum phosphate.

As an alternative to the use of pneumococcal saccharide antigens, the composition may include one or more polypeptide antigens. Genome sequences for several strains of pneumococci are available [129, 130] and can undergo reverse vaccinology [131-134] to identify suitable polypeptide antigens [135, 136]. For example, the composition may include one or more of the following antigens: PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp130, as defined in reference 137. Composition may include more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13 or 14) of these antigens.

In some embodiments, the composition may include both saccharide and pneumococcal polypeptide antigens. These can be used in a simple mixture, or the pneumococcal saccharide antigen can be conjugated to a pneumococcal protein. Suitable carrier proteins for such embodiments include the antigens listed in the previous paragraph [137].

Pneumococcal antigens can be lyophilized, for example, together with Hib antigen.

Pharmaceutical compositions

The composition of the invention will normally comprise, in addition to the components mentioned above, one or more "pharmaceutically acceptable carriers" that include any vehicle that by itself does not induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are normally slowly metabolized large macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose [138], trehalose [139], lactose and lipid aggregates (such as oil droplets or liposomes ). Such vehicles are well known to those skilled in the art. Vaccines may also contain diluents such as water, saline, glycerin, etc. Additionally, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may be present. Physiological saline buffered with pyrogen-free sterile phosphate is a typical vehicle. A thorough discussion of pharmaceutically acceptable excipients is available in reference 140.

The compositions of the invention are in aqueous form, that is, solutions or suspensions. The liquid formulation of this type allows the compositions to be administered directly from their packaged form without the need for reconstitution in an aqueous medium and are therefore ideal for injection. The compositions may be presented in vials, or they may be presented in syringes already filled. Syringes can be supplied with or without needles. A syringe will include a single dose of the composition, while a vial may include a single dose or multiple doses.

The liquid compositions of the invention are also suitable for reconstituting other vaccines in a lyophilized form, to reconstitute lyophilized Hib or DTP antigens. If a composition of the invention is to be used for such extemporaneous reconstitution, the invention provides a kit that may comprise two vials, or may comprise an already filled syringe and a vial, the contents of the syringe being used to reactivate the contents of the vial before the injection.

The compositions of the invention can be packaged as a unit dose or as a multiple dose. For multiple dose forms, the vials are preferred to the previously filled syringes. Effective dosage volumes can be routinely established, but a typical human dose of the composition for injection has a volume of 0.5 ml.

The pH of the composition is preferably between 6 and 8, preferably about 7. The stable pH can be maintained by using a buffer. If a composition comprises an aluminum hydroxide salt, it is preferred to use a histidine buffer [141]. The composition may be sterile and / or pyrogen free. The compositions of the invention may be isotonic with respect to humans.

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The compositions of the invention are immunogenic and are more preferably vaccine compositions. Vaccines according to the invention can be both prophylactic (i.e. to prevent infection) and therapeutic (i.e., to treat the infection), but they will usually be prophylactic. The immunogenic compositions used as vaccines comprise an immunologically effective amount of antigen (s), in addition to any other component, as needed. By 'immunologically effective amount' it is indicated that the administration of that amount to an individual, both in a single dose and part of a series, is effective for treatment or prevention. This amount varies depending on the health and physical condition of the individual to be treated, age, the taxonomic group of the individual to be treated (for example, non-human primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the evaluation of the practical physician of the medical situation and other relevant factors. The quantity is expected to be in a relatively wide range that can be determined by routine tests.

Within each dose, the amount of an individual saccharide antigen will generally be between 1-50 µg (measured as a saccharide mass), for example, about 1 µg, about 2.5 µg, about 4 µg, about 5 µg or approximately 10 µg.

Each saccharide may be present at substantially the same amount per dose. However, the ratio (weight / weight) of MenY saccharide: MenW135 saccharide may be greater than 1 (eg, 2: 1, 3: 1, 4: 1, 5: 1, 10: 1

or higher) and / or the ratio (weight / weight) of MenY saccharide: MenC saccharide may be less than 1 (for example, 1: 2, 1: 3, 1: 4, 1: 5, or less).

Preferred ratios (weight / weight) for saccharides of serogroups A: C: W135: Y are: 1: 1: 1: 1; 1: 1: 1: 2; 2: 1: 1: 1; 4: 2: 1: 1; 8: 4: 2: 1; 4: 2: 1: 2; 8: 4: 1: 2; 4: 2: 2: 1; 2: 2: 1: 1; 4: 4: 2: 1; 2: 2: 1: 2; 4: 4: 1: 2; and 2: 2: 2: 1. Preferred ratios (weight / weight) for saccharides of serogroups C: W135: Y are: 1: 1: 1; 1: 1: 2; 1: 1: 1; 2: 1: 1; 4: 2: 1; 2: 1: 2; 4: 1: 2; 2: 2: 1; Y

2: 1: 1. It is preferred to use a substantially equal mass of each saccharide.

Preferred compositions of the invention comprise less than 50 µg of meningococcal saccharide per dose. Other preferred compositions comprise ≤40 µg of meningococcal saccharide per dose. Other preferred compositions comprise ≤30 µg of meningococcal saccharide per dose. Other preferred compositions comprise ≤25 µg of meningococcal saccharide per dose. Other preferred compositions comprise ≤20 µg of meningococcal saccharide per dose. Other preferred compositions comprise ≤10 µg of meningococcal saccharide per dose but, ideally, the compositions of the invention comprise at least 10 µg of total meningococcal saccharide per dose.

The compositions of the invention may include an antimicrobial agent, particularly when packaged in the form of multiple doses.

The compositions of the invention may comprise detergent, for example, a Tween (polysorbate) such as Tween 80. Detergents are generally present at low levels, for example, <0.01%.

Compositions of the invention may include sodium salts (eg, sodium chloride) to give tonicity. A concentration of 10 + 2 mg / ml NaCl is typical.

The compositions of the invention will generally include a buffer. It is typical phosphate buffer.

The compositions of the invention will generally be co-administered with other immunoregulatory agents. In particular, the compositions will normally include one or more adjuvants. Such adjuvants include, but are not limited to:

A. Compositions containing minerals

Compositions containing minerals suitable for use as adjuvants in the invention include mineral salts such as aluminum salts and calcium salts. The invention includes mineral salts such as hydroxides (for example, oxyhydroxides), phosphates (for example, hydroxyphosphates, orthophosphates), sulfates, etc. [for example, see chapters 8 and 9 of ref. 142], or mixtures of different mineral compounds, the compounds taking any suitable form (for example, gel, crystalline, amorphous, etc.), and being preferred with adsorption. Mineral-containing compositions can also be formulated as a metal salt particle [143].

B. Oil emulsions

Oil emulsion compositions suitable for use as adjuvants in the invention include squalene-water emulsions such as MF59 [Chapter 10 of ref. 142; see also ref. 144] (5% squalene, 0.5% Tween 80 and 0.5% Span 85, formulated in submicron particles using a microfluidizer). Freund's complete adjuvant (CFA) and incomplete Freund's adjuvant (IFA) can also be used.

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C. Saponin formulations [chapter 22 of ref. 142]

Saponin formulations can also be used as adjuvants in the invention. Saponins are a heterologous group of sterol glycosides and triterpenoids found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponin from the bark of the Quillaja saponaria Molina tree has been extensively studied as adjuvants. Saponin can also be obtained commercially from Smilax ornata (sarsaparilla), Gypsophila paniculata (bridal veil) and Saponaria officinalis (soap root). Saponin adjuvant formulations include purified formulations such as QS21, in addition to lipid formulations such as ISCOM. QS21 is marketed as Stimulon ™.

Saponin compositions have been purified using HPLC and RP-HPLC. Specific purified fractions have been identified using these techniques that include QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, the saponin is QS21. A production process of QS21 is disclosed in ref. 145. Saponin formulations may also comprise a sterol, such as cholesterol [146].

Combinations of saponins and cholesterols can be used to form unique particles called immunostimulatory complexes (ISCOM) [Chapter 23 of ref. 142]. ISCOMs also normally include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin in ISCOMs can be used. Preferably, the ISCOM includes one or more of QuilA, QHA and QHC. ISCOMs are further described in refs. 146-148. Optionally, ISCOMs may lack additional detergent [149].

A review of the development of saponin-based adjuvants can be found in refs. 150 and 151.

D. Virosomes and virus-like particles

Virosomes and virus-like particles (VLP) can also be used as adjuvants in the invention. These structures generally contain one or more proteins of a virus optionally combined or formulated with a phospholipid. They are generally non-pathogenic, non-replicating and generally do not contain any of the native viral genomes. Viral proteins can be recombinantly produced or isolated from whole viruses. These viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), hepatitis B virus (such as core or capsid proteins), hepatitis E virus, virus from measles, Sindbis virus, rotavirus, glossopeda virus, retrovirus, Norwalk virus, human papillomavirus, HIV, RNA phages, phage Qß (such as cover proteins), GA phage, fr phage, AP205 and Ty phage (such as the P1 protein of the Ty retrotransposon). VLPs are further treated in refs. 152-157. Virosomes are further treated in, for example, ref. 158.

E. Bacterial or microbial derivatives

Adjuvants suitable for use in the invention include bacterial or microbial derivatives such as non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), lipid A derivatives, immunostimulatory oligonucleotides and ADP ribosilating toxins and detoxified derivatives thereof.

Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and MPL 3-O-deacylated (3dMPL). 3dMPL is a mixture of monophosphoryl lipid A 3-des-O-acylated with 4, 5 or 6 acylated chains. A preferred "small particle" form of monophosphoryl lipid A 3-de-O-acylated is disclosed in ref. 159. Such "small particles" of 3dMPL are small enough to be sterilized by filtration through a 0.22 µm membrane [159]. Other non-toxic LPS derivatives include monophosphoryl lipid A mimetics such as aminoalkylglucosaminide phosphate derivatives, for example, RC-529 [160, 161].

Derivatives of lipid A include derivatives of lipid A of Escherichia coli such as OM-174. OM-174 is described, for example, in refs. 162 and 163.

Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a dinucleotide sequence containing an unmethylated cytosine linked by a phosphate bond to a guanosine). It has also been shown that double stranded RNAs and oligonucleotides containing palindromic or poly (dG) sequences are immunostimulators.

CpGs may include nucleotide modifications / analogs such as phosphorothioate modifications and may be double stranded or single stranded. References 164, 165 and 166 disclose possible analogous substitutions, for example, substitution of guanosine with 2'-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotides is further discussed in refs. 167-172.

The CpG sequence can be directed to TLR9, such as the GTCGTT or TTCGTT motif [173]. The CpG sequence may be specific to induce a Th1 immune response such as a CpG-A ODN, or it may be more specific to induce a B lymphocyte response such as CpG-B ODN. The CpG-A and CpG-B ODNs are discussed in refs. 174-176. Preferably, the CpG is the CpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5 'end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences can be joined at their 3 'ends to form "immunomers." See, for example, refs. 173 and 177-179.

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Bacterial ADP ribosilant toxins and detoxified derivatives thereof can be used as adjuvants in the invention. Preferably, the protein is derived from E. coli (heat labile enterotoxin from E. coli "LT"), cholera ("CT") or Pertussis ("PT"). The use of detoxified ADP ribosilant toxins as mucosal adjuvants is described in ref. 180 and as parenteral adjuvants in ref. 181. The toxin or toxoid is preferably in the form of a holotoxin comprising both A and B subunits. Preferably, subunit A contains a detoxifying mutation; preferably subunit B is not mutated. Preferably, the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72 and LT-G192. The use of ADP ribosilant toxins and detoxified derivatives thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in refs. 182-189. The numerical reference for amino acid substitutions is preferably based on the alignments of subunits A and B of ADP ribosilant toxins set forth in ref. 190.

F. Human immunomodulators

Human immunomodulators suitable for use as adjuvants in the invention include cytokines such as interleukins (eg, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 [191] , etc.) [192], interferons (for example, interferon ), macrophage colony stimulating factor, tumor necrosis factor.

G. Bioadhesives and mucoadhesives

Bioadhesives and mucoadhesives can also be used as adjuvants in the invention. Suitable bioadhesives include esterified hyaluronic acid microspheres [193] or mucoadhesives such as crosslinked derivatives of poly (acrylic acid), polyvinyl alcohol, polyvinylpyrrolidone, polysaccharides and carboxymethyl cellulose. Chitosan and derivatives thereof can also be used as adjuvants in the invention [194].

H. Microparticles

The microparticles can also be used as adjuvants in the invention. Microparticles (ie, 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 (for example, a poly (-hydroxy acid), a polyhydroxybutyric acid, a polyortoester, a polyanhydride, a polycaprolactone, etc.), with poly (lactide-co-glycolide), optionally treated to have a negatively charged surface (for example, with SDS) or a positively charged surface (for example, with a cationic detergent such as CTAB).

I. Liposomes (chapters 13 and 14 of ref. 142)

Examples of liposome formulations suitable for use as adjuvants are described in refs. 195-197.

J. Formulations of polyoxyethylene ethers and polyoxyethylene esters

Adjuvants suitable for use in the invention include polyoxyethylene ethers and polyoxyethylene esters [198]. Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol [199], in addition to polyoxyethylene alkyl ether ether or surfactants in combination with at least one additional nonionic surfactant such as an octoxynol [200]. Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether-23-polyoxyethylene

K. Polyphosphazene (PCPP)

PCPP formulations are described, for example, in refs. 201 and 202.

L. Muramilpeptides

Examples of suitable muramylpeptides for use as adjuvants in the invention include N-acetyl-muramyl-Ltreonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP) and N- acetylmuramyl-L-alanylD-isoglutaminyl-L-alanine-2- (1'-2'-dipalmitoyl-sn-glycerol-3-hydroxyphosphoryloxy) -ethylamine (MTP-PE).

M. Imidazoquinolone Compounds

Examples of imidazoquinolone compounds suitable for use as adjuvants in the invention include imiquimod and its counterparts (eg, "Resiquimod 3M"), further described in refs. 203 and 204.

The invention may also comprise combinations of aspects of one or more of the adjuvants identified above. For example, the following adjuvant compositions may be used in the invention: (1) a saponin and an oil-in-water emulsion [205]; (2) a saponin (for example, QS21) + a non-toxic LPS derivative (for example, 3dMPL) [206]; (3) a saponin (for example, QS21) + a non-toxic LPS derivative (for example, 3dMPL) + a cholesterol; (4) a saponin (for example, QS21) + 3dMPL + IL-12 (optionally + a sterol) [207]; (5) combinations of 3dMPL with, for example, QS21 and / or oil-in-water emulsions [208]; (6) SAF, containing 10% squalene, 0.4% Tween 80 ™, 5% L121 Pluronic block polymer and thr-MDP, both microfluidized in a submicron emulsion and vortexed to generate an emulsion of larger particle size; (7) Ribi ™ adjuvant system (RAS), (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80 and one or more components of the bacterial cell wall of the group consisting of monophosphoryl lipid A ( MPL), trehalose dimicolate (TDM) and cell wall skeleton (CWS), preferably MPL + CWS (Detox ™); (8) one or more mineral salts (such as an aluminum salt) + a non-toxic LPS derivative (such as 3dMPL).

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Other substances acting as immunostimulatory agents are disclosed in chapter 7 of ref. 142.

The use of aluminum salt adjuvants is particularly preferred, and the antigens are generally adsorbed to such salts. MenC Menjugate ™ and NeisVac ™ conjugates use a hydroxide adjuvant, while Meningitec ™ uses a phosphate. In compositions of the invention it is possible to adsorb some antigens to an aluminum hydroxide but to have other antigens in association with an aluminum phosphate. However, in general, it is only preferred to use a single salt, for example, a hydroxide or a phosphate, but not both. Aluminum hydroxide is preferably avoided as an adjuvant, particularly if the composition includes a Hib antigen. Therefore, compositions that do not contain aluminum hydroxide are preferred. Rather, aluminum phosphates can be used, and a typical adjuvant is amorphous aluminum hydroxyphosphate with the molar ratio of PO4 / Al between 0.84 and 0.92, including 0.6 mg of Al3 + / ml. Adsorption can be used with a low dose of aluminum phosphate, for example, between 50 and 100 µg of Al3 + per dose conjugate. If an aluminum phosphate is used and it is desired that an antigen is not adsorbed to the adjuvant, this is favored by including free phosphate ions in solution (for example, using a phosphate buffer).

Not all conjugates need to be adsorbed, that is, some or all may be free in solution.

Calcium phosphate is another preferred adjuvant.

Treatment procedures

A composition of the invention may be to produce an antibody response in a mammal by administering a pharmaceutical composition of the invention to the mammal.

The composition may be for use in a process for producing an immune response in a mammal comprising the step of administering an effective amount of the composition of the invention. The immune response is preferably protective and preferably involves antibodies. The procedure can produce a booster response.

The mammal is preferably a human being. If the vaccine is for prophylactic use, the human being is preferably a child (for example, a small or nursing child) or a teenager; If the vaccine is for therapeutic use, the human being is preferably an adult. A vaccine intended for children can also be administered to adults, for example, to assess safety, dosage, immunogenicity, etc.

The invention also provides a composition of the invention for use as a medicament. The medicament may preferably produce an immune response in a mammal (ie, it is an immunogenic composition) and is more preferably a vaccine.

The invention also provides the use of a composition of the invention in the preparation of a medicament for producing an immune response in a mammal.

These uses and procedures are preferably for the prevention and / or treatment of a disease caused by a Neisseria (for example, meningitis, septicemia, bacteremia, gonorrhea, etc.). Prevention and / or treatment of bacterial and / or meningococcal meningitis is preferred.

One way to verify the efficacy of therapeutic treatment involves monitoring Neisseria infection after administration of the composition of the invention. One way to verify the efficacy of prophylactic treatment involves monitoring immune responses against the five basic antigens after administration of the composition. The immunogenicity of compositions of the invention can be determined by administering them to test subjects (eg, children 12-16 months of age, or animal models [209]) and then determining standard parameters including serum bactericidal antibodies (SBA) and titres of ELISA (GMT) of total and high affinity anti-capsule IgG. These immune responses will generally be determined approximately 4 weeks after administration of the composition, and in comparison with values determined before administration of the composition. An increase in SBA of at least 4 times or 8 times is preferred. If more than one dose of the composition is administered, more than one post-treatment determination can be made.

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Preferred compositions of the invention may confer an antibody titer on a patient that is greater than the criteria for seroprotection for each antigen component for an acceptable percentage of human subjects. Antigens with an associated antibody titer above which a host is considered to be seroconverted against the antigen are well known, and such titers are published by organizations such as WHO. Preferably more than 80% of a statistically significant sample of subjects is seroconverted, more preferably more than 90%, still more preferably more than 93%, and most preferably 96-100%. The compositions of the invention will generally be administered directly to a patient. Direct administration can be carried out by parenteral injection (for example, subcutaneously, intraperitoneally, intravenously, intramuscularly or to the interstitial space of a tissue), or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, auditory, pulmonary administration or other mucosa. Intramuscular administration to the thigh or upper arm is preferred. The injection can be by means of a needle (for example, a hypodermic needle), but alternatively needleless injection can be used. A typical intramuscular dose is 0.5 ml.

The invention can be used to cause systemic and / or mucosal immunity.

The dosage treatment may be a single dose schedule or a multiple dose schedule. The multiple dose can be used in a primary immunization program and / or in an immunization booster program. A primary dose schedule may be followed by a booster dose schedule. The appropriate time between two sensitization doses (for example, between 4-16 weeks), and between sensitization and reinforcement, can be determined routinely.

Neisseria infections affect various areas of the body and, therefore, the compositions of the invention can be prepared in various ways. For example, the compositions can be prepared as injectables, both as liquid solutions and suspensions. The composition can be prepared for pulmonary administration, for example, as an inhaler, using a fine powder or a spray. The composition can be prepared as a suppository or pessary. The composition may be prepared for nasal, auditory or ocular administration, for example, as spray, drops, gel or powder [eg, refs. 210 and 211]. Success has been reported with nasal administration of pneumococcal saccharides [212, 213], pneumococcal polypeptides [214], Hib saccharides [215], MenC saccharides [216] and mixtures of Hib and MenC saccharide conjugates [217] .

Storage stability

The compositions of the invention offer improved stability, particularly for the saccharide component of serogroup A. The present application discloses a process for preparing a vaccine composition comprising the steps of: (1) mixing (i) a capsular saccharide antigen serogroup C conjugate, (ii) a serogroup W135 conjugated capsular saccharide antigen, (iii) a serogroup Y conjugated capsular saccharide antigen and (iv) one or more serogroup B polypeptide antigens; (2) save the composition resulting from step (1) for at least 1 week; (3) prepare a syringe containing the stored composition of step (2) ready for injection to a patient; and, optionally (4) inject the composition into the patient.

Step (I) may also involve mixing (v) a serogroup A conjugated capsular saccharide antigen. In one aspect of the invention it also involves mixing (vi) a Hib conjugated capsular saccharide antigen. It may also involve mixing (vii) a pneumococcal antigen. Step (2) preferably involves at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks or more of storage. The storage stage (2) may or may not be below room temperature (for example, at 10 ± 10 ° C).

The application also discloses a process for preparing a vaccine composition comprising the steps of: (1) mixing (i) a conjugated capsular saccharide antigen of serogroup C, (ii) a conjugated capsular saccharide antigen of serogroup W135, (iii ) a conjugated capsular saccharide antigen of serogroup Y and (iv) one or more polypeptide antigens of serogroup B; and (2) extract a unit dose volume of the antigens; and (c) pack the extracted unit dose in a tightly closed container.

Step (I) may also involve mixing (v) a serogroup A conjugated capsular saccharide antigen. In one aspect of the invention it also involves mixing (vi) a Hib conjugated capsular saccharide antigen. It may also involve mixing (vii) a pneumococcal antigen. The tightly closed container can be a vial or syringe.

The invention provides a hermetically sealed container containing a composition of the invention.

General information

The term "comprising" means "including", in addition to "consisting of", for example, a composition "comprising" X may consist exclusively of X or may include something additional, for example, X + Y.

The term "approximately" in relation to a numerical value x means, for example, x ± 10%.

The word "substantially" does not exclude "completely", for example, a composition that is "substantially free" of Y may be completely free of Y. If necessary, the word "substantially" can be omitted from the definition of the invention.

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References to a percentage sequence identity between two amino acid sequences means that, when aligned, that percentage of amino acids is the same by comparing the two sequences. This alignment and the percentage homology or sequence identity can be determined using software programs known in the art, for example, those described in section 7.7.18 of reference 218. A preferred alignment is determined by the homology search algorithm of Smith-Waterman using a related hollow search with an open hole penalty of 12 and a gap extension penalty of 2 of the BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is taught in reference 219.

The term "alkyl" refers to alkyl groups in both linear and branched forms. The alkyl group may be interrupted with 1, 2 or 3 heteroatoms selected from -O-, -NH-or -S-. The alkyl group may also be interrupted with 1, 2 or 3 double and / or triple bonds. However, the term "alkyl" usually refers to alkyl groups that do not have heteroatom interruptions or double or triple bond interruptions. When referring to C1-12 alkyl it is indicated that the alkyl group may contain any number of carbon atoms between 1 and 12 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10 , C11, C12). Similarly, when referring to C1-6 alkyl it is indicated that the alkyl group may contain any number of carbon atoms between 1 and 6 (for example, C1, C2, C3, C4, C5, C6).

The term "cycloalkyl" includes cycloalkyl, polycycloalkyl and cycloalkenyl groups, in addition to combinations thereof with alkyl groups such as cycloalkylalkyl groups. The cycloalkyl group may be interrupted with 1, 2 or 3 heteroatoms selected from -O-, -NH- or -S-. However, the term "cycloalkyl" usually refers to cycloalkyl groups that do not have heteroatom interruptions. Examples of cycloalkyl groups include cyclopentyl, cyclohexyl, cyclohexenyl, cyclohexylmethyl and adamantyl groups. When referring to C3-12 cycloalkyl it is indicated that the cycloalkyl group may contain any number of carbon atoms between 3 and 12 (for example, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12 ).

The term "aryl" refers to an aromatic group such as phenyl or naphthyl. When referring to C5-12 aryl it is indicated that the aryl group may contain any number of carbon atoms between 5 and 12 (for example, C5, C6, C7, C8, C9, C10, C11, C12).

The term "C5-12 aryl-C1-6 alkyl" refers to groups such as benzyl, phenylethyl and naphthylmethyl.

Nitrogen protecting groups include silyl groups (such as TMS, TES, TBS, TIPS), acyl derivatives (such as phthalimides, trifluoroacetamides, methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl (Boc), benzyloxycarbonyl (Z or Cbz), 9-fluorenylmethoxycarbonyl (Fmoc), 2- (trimethylsilyl) ethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl (Troc), sulfonyl derivatives (such as β-trimethylsilylethanesulfonyl (SES)), sulfenyl derivatives, C1-12 alkyl, benzyl, benzhydryl, trityl, 9-phenylfluorenyl, etc. A preferred nitrogen protecting group is Fmoc.

The sequences included to facilitate cloning or purification, etc., do not necessarily contribute to the invention and can be omitted or deleted.

It will be appreciated that there may be open and closed sugar rings and that, while the closed forms are shown in structural formulas herein, the open forms are also encompassed by the invention.

The polypeptides of the invention can be prepared by various means (e.g., recombinant expression, cell culture purification, chemical synthesis (at least in part), etc.) and in various forms (e.g., native, fusions, non-glycosylated, lipidated , etc.). They are preferably prepared in substantially pure form (ie, substantially free of other N. meningitidis proteins or host cells). While expression of the polypeptide can take place in Neisseria, a heterologous host is preferred. The heterologous host can be prokaryotic (for example, a bacterium) or eukaryotic. Preferably it is E. coli, but other suitable hosts include Bacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonella typhimurium, Neisseria lactamica, Neisseria cinerea, Mycobacteria (eg, M. turberculosis), yeast, etc.

The nucleic acid according to the invention can be prepared in many ways (for example, by chemical synthesis (at least in part) from genomic libraries or cDNA, from the organism itself, etc.) and can take various forms (for example , single chain, double stranded, vectors, probes, etc.). They are preferably prepared in substantially pure form (ie, substantially free of other N. meningitidis nucleic acids or host cells). The term "nucleic acid" includes DNA and RNA, and also their analogs, such as those containing modified skeletons (eg, phosphorothioates, etc.), and also peptide nucleic acids (PNA), etc. The invention includes nucleic acid comprising sequences complementary to those described above (for example, for antisense or probing purposes).

After the serogroup, the meningococcal classification includes serotype, serosubtype and then immunotype, and the standard nomenclature lists serogroup, serotype, serosubtype and immunotype, each separated by a colon, for example, B: 4: P1.15: L3.7, 9. Within serogroup B, some lineages frequently produce disease (hyperinvasive), some lineages produce more severe forms of disease than others (hypervirulent), and others rarely produce disease at all. Seven hypervirulent lineages are recognized, namely subgroups I, III and IV-1, complex ET-5, complex ET-37, set A4 and lineage 3. These have been defined by multilocus enzyme electrophoresis (MLEE), but they have also been used multilocus sequence typing (MLST) to classify meningococci [ref. 104].

image20

Modes for carrying out the invention

Δ G287-953 hybrid protein

Protein 287 encoding DNA from strain 394/98 meningococcal serogroup B and protein 953 from meningococcal strain 2996 serogroup B were digested and ligated, together with a short linker sequence, giving a plasmid encoding the amino acid sequence SEQ ID 7. The plasmid was transfected into E. coli and the bacteria were cultured to express the protein. After proper growth, the bacteria were collected and the protein was purified. From the culture, the bacteria were centrifuged and the sediment was homogenized in the presence of 50 mM acetate buffer (pH 5) with a volume ratio of pellet: buffer of 1: 8. Lysis was performed using a high pressure homogenizer (AVESTIN, 4 cycles at 14000 psi). After lysis, urea was added at a final concentration of 5 M, followed by stirring for 1 hour at room temperature. The pH was reduced from 6 to 5 using 200 mM acetate buffer (pH 4) + 5 M urea. The mixture was centrifuged at 16800 g for 60 minutes at 2-8 ° C. The supernatant was collected and filtered by SARTOBRAN P (0.45-0.22 µm SARTORIUS). The protein in the filtered supernatant was stable for at least 30 days at -20 ° C and for at least 15 days at 2-8 ° C.

The protein was further purified on a cation exchange column (SPFF, Amersham Biosciences) with elution using 350 mM NaCl + 50 mM acetate + 5 M urea at pH 5.00. Most of the impurities were present in the continuous flow. A pre-elution wash using a lower concentration of NaCl (180 mM) advantageously removed two contaminating E. coli proteins.

The eluted material was adjusted to pH 8 (using 200 mM TRIS / HCl + 5 M urea at pH 9) and further purified on a Q Sepharose HP (Amersham) column with elution using 150 mM NaCI + 20 mM TRIS / HCl a pH 8.00 in 5 M urea. Again, a pre-elution wash with reduced salt (90 mM) was useful for removing impurities.

The filtered eluted material from column Q HP was diluted 1: 2 using PBS at pH 7.00 (150 mM NaCl + 10 mM potassium phosphate, pH 7.00) and then diafiltered against 10 volumes of PBS at pH 7, 00 by tangential ultrafiltration. At the end of the diafiltration the material was concentrated 1.6 times to approximately 1.2 mg / ml of total proteins. Using a 30,000 Da membrane of cut (50 cm2 regenerated cellulose membrane, Millipore PLCTK 30) it was possible to dialyze the material with a yield of approximately 90%.

936-ΔG741 hybrid protein

Protein 936 encoding DNA of meningococcal strain 2996 of serogroup B and protein 741 of meningococcal strain MC58 of serogroup B were digested and ligated, together with a short linker sequence, giving a plasmid encoding the amino acid sequence SEQ ID 8. The plasmid was transfected into E. coli and the bacteria were cultured to express the protein. The recombinant protein was not secreted, but remained soluble within the bacteria.

After proper growth, the bacteria were centrifuged giving a wet paste and treated as follows:

- Homogenization by means of a high pressure system in the presence of 20 mM sodium phosphate at pH 7.00.

- Centrifugation and clarification by orthogonal filtration.

-

Cationic column chromatography (SP Sepharose Fast Flow), with elution with 150 mM NaCl in 20 mM sodium phosphate at pH 7.00.

-

Anionic column chromatography (Q Sepharose XL) with continuous flow collection.

-

Hydrophobic column chromatography (Phenyl Sepharose 6 Fast Flow High Sub) with elution with 20 mM sodium phosphate, pH 7.00.

- Diafiltration against PBS at pH 7.4 with a cut of 10 Kd.

- Sterilization by final filtration and storage at -20ºC

The protein in the final material was stable for at least 3 months at both -20 ° C and 2-8 ° C.

NcidA protein (NL) (C)

The NadA protein encoding DNA of serogroup B meningococcal strain 2996 was digested to remove the sequence encoding its C-terminus giving a plasmid encoding the amino acid sequence SEQ ID 1. The plasmid was transfected into E. coli and the bacteria were They cultivated to express the protein. The recombinant protein was secreted in the culture medium and the conductive peptide was absent from the secreted protein (SEQ ID 2). The supernatant was treated as follows:

image21

5 - 7X concentration and diafiltration against 20 mM TRIS buffer / HCI at pH 7.6 per cross flow UF (30 Kd cut).

- Anionic column chromatography (Q Sepharose XL) with elution with 400 mM NaCl in 20 mM TRIS / HCl at pH 7.6.

- Hydrophobic column chromatography stage (Phenyl Sepharose 6 Fast Flow High Sub) with elution with 50 mM NaCl in TRIS / HCl at pH 7.6.

- Ceramic column chromatography of hydroxylapatite (HA Macro. Prep) with elution with 200 mM sodium phosphate at pH 7.4.

- Diafiltration (30 Kd cut) against PBS at pH 7.4

- Sterilization by final filtration and storage at -20ºC

The protein in the final material was stable for at least 6 months at both -20 ° C and 2-8 ° C.

The NadA protein is susceptible to degradation, and truncated forms of NadA can be detected by Western blotting or by mass spectrometry (eg, by MALDI-TOF) indicating up to a loss of 10 kDa of MW. The degradation products can be separated from the native NadA by gel filtration (for example, using the TSK 300SWXL column, TSKSWXL pre-column, TOSOHAAS). Such filtration gives three peaks: (i) a first peak with

20 retention time of 12,637 min and apparent MW of 885,036 Da; (ii) retention time of 13,871 min and apparent MW of 530,388 Da; (iii) retention time 13,871 min and apparent MW of 530,388 Da. The light scattering analysis of the three peaks reveals real MW values of (i) 208500 Da, (ii) 98460 Da, (iii) 78760 Da. Therefore, the first peak contains NadA aggregates and the third peak contains degradation products.

Since the predicted molecular weight of NadA (NL) (C) is 34,113 Da, peak (ii) contains a trimeric protein that is the desired antigen.

Antigenic combinations

Mice were immunized with a composition comprising the three proteins and, for comparison, all three proteins were also tested individually. Ten mice were used per group. The mixture was able to induce high bactericidal titers against various strains:

Meningococcal strain (serogroup)

2996 (B)

MC58 (B) NGH38 394/98 (B) H44 / 76 (B) F6124 (A) BZ133 (C) C11 (C)

(one)

32000 16000 130000 16000 32000 8000 16000 8000

(2)

256 131000 128 16000 32000 8000 16000 <4

(3)

32000 8000 - - - 8000 - 32000

Mixture

32000 32000 65000 16000 260000 65000 > 65000 8000

'-' indicates that this strain does not contain the NadA gene

Observing individual mice, the triple mixture induced high and consistent bactericidal titers against the three strains of serogroup B from which the individual antigens were derived:

#

one 2 3 4 5 6 7 8 9 10

2996

32768 16384 65536 32768 32768 65536 65536 32768 65536 8192

MC58

65536 32768 65536 65536 65536 8192 65536 32768 32768 65536

394/98

65536 4096 16384 4096 8192 4096 32768 16384 8192 16384

image22

Combination and comparison with OMV

In other experiments, the antigens (20 µg of each antigen per dose) were administered in combination with 10 µg of OMV prepared both from strain H44 / 76 (Norway) and strain 394/98 (New Zealand). The controls

5 positive were the capsular anti-SEAM-3 mAb for serogroup B or the capsular CRM 197-saccharide conjugates for other strains. The mixture almost always gave better titres than simple OMVs, and the addition of the mixture to OMV almost always significantly enhanced the effectiveness of OMVs. In many cases, the mixture of antigens matched or exceeded the response observed with the positive control.

Hypervirulent lineage tests

10 The following antigens against a variety of serogroup B strains were tested from a variety of hypervirulent lineages:

(to)

 NadA (NL) (C)

(b)

ΔG287-953

(c) 936-ΔG741 15 (d) a mixture of (a), (b) and (c)

(and)

 OMV prepared from strain H44 / 76 (Norway)

(F)

 OMV prepared from strain 394/98 (New Zealand)

(g)

 a mixture of ΔG287 and (e)

(h)

 a mixture of (d) and (e)

20 (i) a mixture of (d) and (f) SEAM-3 was used as a positive control. The results were as follows, expressed as the percentage of strains in the hypervirulent lineage

indicated when the serum bactericidal titer exceeded 1024:

No. of strains

(to) (b) (C) (d) (and) (F) (g) (h) (i) S-3

A4

4 fifty fifty 0 100 25 25 25 100 100 +

ET-5

8 25 75 88 100 71 14 71 100 100 +

Lineage 3

13 0 75 fifteen 93 8 85 8 92 93 +

ET-37

4 eleven 22 0 33 0 0 0 22 25 +

Against particular reference strains, bactericidal titers were as follows:

image23

Strain

(to) (b) (C) (d) (and) (F) (g) (h) (i) S-3

A4

961-5945 128 2048 <8 2048 262144 8192 262144 162144 4096 8192

ET-5

44/76 <4 2048 32768 131072 524288 8192 524288 524288 52428 8 16384

Lineage 3

394/98 <4 1024 32 4096 <4 16384 256 16384 16384 16384

ET-37

LPN17592 2048 1024 256 4096 <8 <8 512 16384 65536 1024

image24

Thus, compositions (d), (h) and (i) induce bactericidal antibody responses against a wide variety of serogroup B meningococcal strains within the hypervirulent A4, ET-5 and lineage 3 lineages. Titles using the Compositions (h) and (i) were generally superior to with (d), but the coverage of the strains within the hypervirulent A4, ET-5 and lineage 3 lineages was not better.

The coverage of untyped strains was also high with compositions (d), (h) and (i).

Combination with meningococcal and / or Hib conjugates

The triple MenB composition is combined with a mixture of oligosaccharide conjugates for serogroups C, W135 and Y giving a vaccine containing the following antigens:

Component

Amount per 0.5 ml dose

Serogroup C conjugate

10 µg saccharide + 12.5-25 µg CRM197

W135 serogroup conjugate

10 µg saccharide + 6.6-20 µg CRM197

Serogroup Y conjugate

10 µg saccharide + 6.6-20 µg CRM197

ΔG287-953

20 µg polypeptide

936-ΔG741

20 µg polypeptide

Any

20 µg polypeptide

10

A similar vaccine is prepared that includes MenA conjugate (10 µg saccharide + 12.5-33 µg CRM197) and / or a HbOC Hib conjugate (10 µg saccharide + 2-5 µg CRM197).

In a series of tests the conjugates of serogroups C, W135 and Y were combined, each conjugate being present at 40 µg / ml (measured as saccharide). For storage before use with MenB antigens, the combined 15 conjugates were lyophilized [-45 ° C for 3 hours, -35 ° C for 20 hours at 6.65 MPa under vacuum, 30 ° C for 10 hours at 6.65 MPa, 30 ° C for 9 hours at 125 mTorr] in the presence of 15 mg sucrose, 10 mM phosphate buffer (pH 7.2). The final volume before lyophilization was 0.3 ml. Therefore, after resuspension in 0.6 ml of aqueous solution, saccharides are present at 12 µg per serogroup. Lyophilization was used for convenience only, and neither the efficacy nor the stability during normal storage of the final product require

20 lyophilization.

A second batch of material was prepared in the same way, but also including the conjugate of serogroup A at the same saccharide dosage as for serogroups C, W135 and Y.

A third batch of material was prepared in the same way (serogroups A, C, W135 and Y), but also including a Hib-CRM197 conjugate at the same saccharide dosage as for meningococci.

For comparison, lyophilized preparations of serogroup A and C conjugates were prepared. The MenA material was lyophilized with 15 mg sucrose giving a dose of 12 µg saccharide after reconstitution as described above. The MenC material was lyophilized with 9 mg of mannitol giving a dose of 12 µg saccharide after reconstitution.

These materials were combined with 600 µl of the serogroup mixture (d) (or, as a control, that is, groups 2 and 30 3, in an identical composition but lacking antigens) giving eight compositions:

Components

one 2 3 4 5 6 7 8

NadA (NL) (C) µg / dose

twenty twenty twenty twenty twenty twenty

936-741 µg / dose

twenty twenty twenty twenty twenty twenty

287-953 µg / dose

twenty twenty twenty twenty twenty twenty

MenA-CRM µg / dose *

2.4 2.4 2.4 2.4 2.4

image25

MenC-CRM µg / dose *

2.4 2.4 2.4 2.4 2.4 2.4

MenW-CRM µg / dose *

2.4 2.4 2.4 2.4 2.4

MenY-CRM µg / dose *

2.4 2.4 2.4 2.4 2.4

image26

(cont)

Components

one 2 3 4 5 6 7 8

Hib-CRM µg / dose *

2.4 2.4

Aluminum hydroxide mg / dose

0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15

MM histidine

10 10 10 10 10 10 10 10

Sucrose mg / dose

3 3 3 3 3 3

Mannitol mg / dose

1.8

Potassium phosphate at pH 7.2 mM

3 3 3 3 3 3

Sodium phosphate at pH 7.2 mM

3

Sodium Chloride mg / dose

1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8

* The amount shown is saccharide

These compositions were administered intraperitoneally in a volume of 200 µl to CD / l mice (8 per group) on days 0, 21 and 35, with a final bleeding on day 49. The sera on day 49 were tested in SBA assays against a variety of meningococcal strains in serogroups A, B, C, W135 and Y. The results were:

image27

Group

B 2996 MC58 394/98 44/76 A F6124 C C11 312294 C4678 M1569 W135 LPN17592 And 860800

1 2 3 4 5 6 7 8

1024 4096 1024 8192 <4 <4 128 <16 <4 <4 <4 <16 64 4096 512 8192 256 4096 1024 8192 128 1024 256 8192 256 512 512 16384 256 2048 512 8192 2048 4096 4096 8192 256 128 1024 1024 2048 <16 * 64 * 128 * 8192 --- 16384 --- 128 --- 8192> 8192> 8192> 8192 8192 8192> 8192> 8192 8192 4096> 8192> 8192 8192 2048> 8192> 8192 512 32 512 256 512 512 1024 512 65536 32768 32768 32768 32768 16384 16384 32768

image28

Therefore, the meningococcal protein antigen remains effective even after the addition of the meningococcal and Hib conjugated saccharide antigens. Similarly, meningococcal conjugates retain efficacy even after the addition of protein antigens. In fact, the data suggests that the addition of protein antigens to the conjugates enhances the anti-MenW135 efficacy (see groups 2 and 7).

5 In addition, there is a level of cross-reactivity, in particular for serogroup Y, since protein antigens alone give a good anti-MenY titer [see reference 220] as groups 4 and 5.

The data also indicates that the addition of a Hib conjugate to meningococcal conjugates (see groups 2 and 3) enhances anti-W135 activity.

Use of modified MenA saccharide

The capsular polysaccharide was purified from MenA and hydrolyzed to give the MenA oligosaccharide. The polysaccharide (2 g) was hydrolyzed at 50 ° C in 50 mM sodium acetate buffer, pH 4.75, at a polysaccharide concentration of 10 mg / ml for approximately 4 hours [73]. After hydrolysis, the solution was dried by rotary evaporation.

The oligosaccharide was activated using the following reaction scheme:

image29

The oligosaccharide was dissolved in DMSO giving a saccharide concentration of 10 mg / ml. According to an oligosaccharide: CDI molar ratio that is 1:20, then 21,262 g of CDI was added and the reaction mixture was stirred for 16 hours at room temperature. The resulting MenA-CDI compound was purified by selective precipitation in a mixture of 80:20 (v / v) acetone: DMSO followed by centrifugation. The reaction efficiency of

The activation was approximately 67.9% determining the ratio of free imidazole to bound imidazole.

In the second reaction stage, the MenA-CDI oligosaccharide was solubilized in DMSO at a saccharide concentration of approximately 10 mg / ml. According to a mole ratio of MenA-CDI: DMA unit that is 1: 100, 36,288 g of 99% dimethylamine hydrochloride (i.e., R1 and R2 = Me) were added and the reaction mixture was stirred for 16 hours at temperature ambient. The reaction product was lyophilized and resolubilized in 10 mg / ml of

25 water solution.

To remove the low molecular weight reaction reagent (in particular dimethylamine (DMA)) from the oligosaccharide preparation, a dialysis step was performed through a 3.5 kDa MWCO membrane (Spectra / Por ™). Four dialysis stages were carried out: (i) 16 hours against 2 l of 1 M sodium chloride (1:20 dialysis factor), (ii) 16 hours against 2 l of 0.5 M sodium chloride (dialysis factor 1:20), (iii) and (iv) 16 hours against 2 l of WFI (factor of

30 dialysis 1:20). To improve purification, a diafiltration stage was also performed through a 1 kDa MWCO membrane (Centricon ™).

The purified MenA-CDI-DMA product was buffered to pH 6.5 in 25 mM L-histidine (Fluka ™).

To prepare conjugates of the modified MenA saccharide (MenA-CDI-DMA), the overall procedure was as follows:

35 -hydrolysis of the polysaccharide giving oligosaccharide fragments

- sizing of oligosaccharide fragments

- reductive amination of terminal aldehyde groups on the sized oligosaccharides

- protection of terminal -NH2 groups by the Fmoc group before the CDI reaction

- intrinsic deprotection of -NH2 groups during the DMA reaction

40 -activation of terminal -NH2 groups by SIDEA (N-hydroxysuccinimide-adipic acid)

image30

- covalent binding to CRM197 protein

The modified MenA oligosaccharide conjugate was much more resistant to hydrolysis than its natural counterpart at elevated temperatures. After 28 days at 37 ° C, for example, the percentage of saccharide released is 6.4% for the modified oligosaccharide versus 23.5% for the natural antigen. In addition, the titles induced by the

5 modified oligosaccharides are not significantly smaller than those obtained using native sugar structures.

The modified MenA conjugate is combined with MenC, MenW135 and MenY conjugates as a substitute for the unmodified oligosaccharide conjugate. This tetravalent mixture is mixed with the three MenB polypeptides giving an effective vaccine against serogroups A, B, C, W135 and Y of N. meningitidis in a single dose.

10 Pneumococcal combinations

The three MenB proteins combined are mixed with pneumococcal saccharide conjugates giving a final concentration of 2 µg / dose of each of the pneumococcal serotypes (double for serotype 6B). Therefore, the reconstituted vaccine contains the following antigens:

Component

Amount per 0.5 ml dose

Serogroup A conjugate

5 µg saccharide + 6.25-16.5 µg CRM197

Serogroup C conjugate

5 µg saccharide + 6.25-12.5 µg CRM197

Conjugate of serogroup W 135

5 µg saccharide + 3.3-10 µm CRM197

Serogroup Y conjugate

5 µg saccharide + 3.3-10 µg CRM197

Pneumococcal serotype 4 conjugate

2 µg saccharide + 2.5 µg CRM197

Pneumococcal 9V serotype conjugate

2 µg saccharide + 2.5 µg CRM197

Pneumococcal serotype 14 conjugate

2 µg saccharide + 2.5 µg CRM197

Pneumococcal serotype 18C conjugate

2 µg saccharide + 2.5 µg CRM197

Pneumococcal serotype 19F conjugate

2 µg saccharide + 2.5 µg CRM197

Pneumococcal serotype 23F conjugate

2 µg saccharide + 2.5 µg CRM197

Conjugate of pneumococcal serotype 6B

4 µg saccharide + 5 µg CRM197

fifteen

It will be understood that the invention has only been described by way of example and that modifications may be made as long as they remain within the scope of the invention, which is defined in the appended claims.

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SEQUENCE LIST

SEC ID

one

NadA of strain 2996, with deletion of end C

2

NadA of strain 2996, with deletion of the C-terminus and processed conductive peptide

3

ΔG741 of strain MC58

4

936 of strain MC58 with processed conductor peptide

5

953 of strain MC58 with processed conductor peptide

6

ΔG287 of strain MC58

image37

SEC ID

7

Hybrid of 287-953

8

936-741 hybrid

9

Connector

10

Sequence of 741

eleven

Sequence of 741

12

Sequence of 741

<110> NOVARTIS VACCINES AND DIAGNOSTICS SRL

<120> LIQUID VACCINES FOR MULTIPLE MENINGOCOTIC SEROGROUPS

<130> P050770EP 5 <140> EP-08

<141> 04-10-2004

<150> PCT / IB2004 / 003373

<151> 04-10-2004

<150> GB0323102.4 10 <151> 02-10-2003

<150> GB0412052.3

<151> 05-28-2004

<160> 12

<170> SeqWin99, version 1.02 15 <210> 1

<211> 350

<212> PRT

<213> Neisseria meningitidis

<400> 1

image38

image39

image3

image3

<210> 2

<211> 327

<212> PRT

<213> Neisseria meningitidis

<400> 2

image40

image3

image3

<210> 3

<211> 248

<212> PRT

<213> Neisseria meningitidis

<400> 3

image41

image3

<210> 4

<211> 179

<212> PRT

image42

<213> Neisseria meningitidis

<400> 4

image3

<210> 5

<211> 168

<212> PRT

<213> Neisseria meningitidis

<400> 5

image43

image3

<210> 6

<211> 464

<212> PRT

<213> Neisseria meningitidis

<400> 6

image44

image3

image3

<210> 7

<211> 644

<212> PRT

<213> Neisseria meningitidis

<400> 7

image45

image3

image46

image3

image3

<210> 8

<211> 434

<212> PRT

<213> Neisseria meningitidis

<400> 8

image3

image47

image3

<210> 9

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Connector sequence

<400> 9

image3

10 <210> 10 <211> 255

image48

<212> PRT

<213> Neisseria meningitidis

<400> 10

image49

<210> 11 <211> 254

image50

<212> PRT

<213> Neisseria meningitidis

<400> 11

image49

<210> 12

image51

<211> 262

<212> PRT

<213> Neisseria meningitidis

<400> 12

image52

image3

Claims (24)

1. An aqueous immunogenic composition which, after administration to a subject, can induce an immune response that is (a) bactericidal against at least serogroup W135 of N. meningitidis and (b) protective against H. influenzae type disease b, in which the composition comprises: (i) a conjugated capsular saccharide antigen of serogroup W135; Y (ii) a conjugated capsular saccharide antigen of H. influenzae type b ('Hib'); and in which the saccharide of serogroup W135 is conjugated with a diphtheria toxoid. 2. An aqueous immunogenic composition which, after administration to a subject, can induce an immune response that is (a) bactericidal against at least serogroup W135 of N. meningitidis and (b) protective against H. influenzae type disease b, in which the composition comprises: (i) a conjugated capsular saccharide antigen of serogroup W135; Y (ii) a conjugated Hib capsular saccharide antigen; and in which the saccharide of serogroup W135 is conjugated with a tetanus toxoid. 3. An aqueous immunogenic composition which, after administration to a subject, can induce an immune response that is (a) bactericidal against at least serogroup W135 of N. meningitidis and (b) protective against H. influenzae type disease b, in which the composition comprises: (i) a conjugated capsular saccharide antigen of serogroup W135; Y (ii) a conjugated Hib capsular saccharide antigen; and in which the saccharide of serogroup W135 is conjugated with a protein H. of H. influenzae. 4. An aqueous immunogenic composition which, after administration to a subject, can induce an immune response that is (a) bactericidal against at least serogroup W135 of N. meningitidis and (b) protective against H. influenzae type disease b, in which the composition comprises: (i) a conjugated capsular saccharide antigen of serogroup W135; Y (ii) a conjugated Hib capsular saccharide antigen; and in which the saccharide Hib is conjugated with a tetanus toxoid. 5. An aqueous immunogenic composition which, after administration to a subject, can induce an immune response that is (a) bactericidal against at least serogroup W135 of N. meningitidis and (b) protective against H. influenzae type disease b, in which the composition comprises: (i) a conjugated capsular saccharide antigen of serogroup W135; Y (ii) a conjugated Hib capsular saccharide antigen; and wherein the composition includes an aluminum phosphate adjuvant. 6. An aqueous immunogenic composition which, after administration to a subject, can induce an immune response that is (a) bactericidal against at least serogroup W135 of N. meningitidis and (b) protective against H. influenzae type disease b, in which the composition comprises:

(i)

 a conjugated capsular saccharide antigen of serogroup W135; Y

(ii)

 a conjugated Hib capsular saccharide antigen;

and wherein the saccharide of serogroup W135 is conjugated to a mutant of the diphtheria toxin CRM197 and the composition comprises ≤30 µg of meningococcal saccharide per dose.

7.

 The composition of any preceding claim further comprising conjugated capsular saccharide antigens of serogroups C and Y and, optionally, A.

8.

 The composition of any preceding claim further comprising one or more polypeptide antigens of serogroup B of N. meningitidis.

9.

 The composition of any one of claims 1-7, wherein the composition consists essentially of (i), (ii) and conjugated capsular saccharide antigens of serogroups C and Y and, optionally, A.

10.

 The composition of any preceding claim, wherein the saccharide of serogroup W135 has a polymerization degree of less than 30.

eleven.

 The composition of any preceding claim when it depends on claim 4 or claim 5, wherein the saccharide of serogroup W135 is conjugated to a diphtheria toxoid, a tetanus toxoid or a mutant of the diphtheria toxin CRM197.

12.

 The composition of any preceding claim, wherein the same carrier protein is used for all serogroups.

13.

 The composition of any preceding claim, wherein the saccharide of serogroup W135 has a saccharide: protein (weight / weight) ratio between 1: 5 and 5: 1.

14.

 The composition of any preceding claim, wherein the saccharide of serogroup W135 is conjugated by a connector.

fifteen.

 The composition of any one of claims 1 to 13, wherein the saccharide of serogroup W135 is directly conjugated.

16.

 The composition of any preceding claim when it depends on one of claims 1-3 or 5-6, wherein the Hib saccharide is conjugated to a diphtheria toxin CRM197 mutant, a tetanus toxoid or a complex of the outer membrane of N. meningitidis

17.

 The composition of any preceding claim, wherein the composition comprises an adjuvant.

18.

 The composition of any preceding claim, wherein the composition does not include an aluminum hydroxide adjuvant.

19.

 The composition of any preceding claim when it depends on one of claims 1-4 or 6, wherein the composition includes an aluminum phosphate adjuvant.

twenty.

 The composition of claim 5 or 19, wherein the Hib antigen is adsorbed to aluminum phosphate.

twenty-one.

 The composition of claim 5 or 19, wherein the Hib antigen is not adsorbed to aluminum phosphate.

22

 The composition of any preceding claim, wherein the Hib antigen is initially lyophilized.

2. 3.

 The composition of any preceding claim, wherein the administration of the Hib antigen produces a concentration of polypyribosylribitol anti-phosphate antibody of ≥0.15 µg / ml.

24.

 The composition of any preceding claim, wherein the conjugates have a saccharide: protein (weight / weight) ratio of between 1: 5 and 5: 1.