RU2333007C2 - Polypeptide vaccines for wide protection against rows of meningococcus generations with increased virulence - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: essence of the invention lies in a composition which, after introduction to an individual, is capable of inducing a humoral immune response at the given individual the humoral immune response performing bactericidal effect against two or three of supervirulent rows of generations A4, ET5 and a number of generations of the 3rd serogroup in N.meningitidis. of an external membrane. Five albuminous antigens are used, i.e. (1) protein "NadA"; (2) protein "741"; (3) protein "936"; (4) protein of "953" and (5) protein "287".

EFFECT: development of a vaccine against two or more rows of supervirulent meningococcus generations.

29 cl, 1 tbl

Description

All documents described in the present description are fully incorporated into it by reference.

FIELD OF THE INVENTION

The invention relates to the field of immunology and vaccinology. In particular, it relates to antigens from Neisseria meningitidis (meningococcus) and their use in immunization.

State of the art

N. meningitidis is a motionless, 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 Haemophilus influenzae conjugate vaccine, N. meningitidis is the main cause of bacterial meningitis in the United States.

Based on the capsular polysaccharide of the body, various serogroups of N. meningitidis were identified. Serogroup A is a pathogen that most often causes epidemic morbidity in sub-Saharan Africa. Serogroups B and C are responsible for a huge number of cases in the United States and in most developed countries. Serogroups W135 and Y are responsible for the remaining cases in the United States and developed countries. After the serogroup, the classification includes the serotype, seropodip, and then the immunotype and the standard nomenclature lists the serogroup, serotype, seropodip and immunotype, each of which is separated by a colon, for example, B: 4: P1.15: L3,7,9. Within the serogroup B, some generational series cause the disease often (superinvasive), some generational series (sequences) cause more severe forms of the disease than others (supervirulent ones), and others rarely cause the disease. Seven supervirulent series of generations are recognized, namely, subgroups I, III, and IV-1, the ET-5 complex, the ET-37 complex, the A4 cluster, and the generational series 3. They were determined by multi-locus enzyme electrophoresis (MLEE), but multi-locus sequence typing ( MLST) was also used to classify meningococci (Ref. 1).

The polysaccharide vaccine against serogroups A, C, W135 and Y has been known for many years [2,3], but it was not possible to obtain a vaccine against serogroup B. Outer vesicle-based vaccines have been tested (for example, see reference 4), but the protection provided by these vaccines is usually limited to the strain used to make the vaccine. Therefore, there remains a need for a widely effective vaccine against serogroup B.

Genomic sequences were reported for meningococcal serogroups A [5] and B [6,7], and the sequence of serogroup B was examined to identify vaccine antigens (eg, refs. 8-13). Promising antigens were manipulated to improve heterologous expression (refs. 14-16).

The objective of the invention is to obtain additional and improved compositions for providing immunity against meningococcal disease and / or infection, and, in particular, to provide broad immunity against meningococcus serogroup B.

Disclosure of invention

Vaccines against pathogens such as hepatitis B virus (HBV), diphtheria and tetanus pathogens contain one protein antigen (for example, HBV surface antigen, or tetanus toxoid). In contrast, acellular whooping cough vaccines typically contain at least three B.pertussis proteins and the Prevenar ^TM pneumococcal vaccine contains seven separate conjugated saccharide antigens. Other vaccines, such as the pertussis cell vaccine, measles vaccine, inactivated polio vaccine (IPV) and meningococcal OMV vaccines, are complex mixtures of a large number of antigens.

Therefore, whether protection against infection by a pathogen can be caused by a single antigen, a small number of specific antigens or a complex mixture of undefined antigens depends on the pathogen in question. The invention is based on the discovery that a small number of certain antigens can provide a wide range of protection against meningococcal infection, and the invention includes a composition which, after administration to an individual, is capable of eliciting (inducing) an antibody response (humoral immune response) in that individual, wherein the humoral immune response has a bactericidal action against two or more (for example, 2 or 3) supervirulent rows of generations A4, ET-5 and a series of generations 3 of serogroup B N.meningitidis.

Preferably, the composition of the invention does not consist of a single antigen, but includes a mixture of 10 or less (e.g., 9, 8, 7, 6, 5, 4, 3, 2) purified antigens, and it is particularly preferred that the composition does not include complex or undefined mixtures of antigens, for example, it is preferable that the composition does not include vesicles of the outer membrane.

For serogroup B meningococcus, a mixture of five specific protein antigens of proteins has been found to elicit a good protective immune response. Thus, the invention includes a composition comprising the following five meningococcal protein antigens: (1) NadA protein; (2) 741 protein; (3) protein "936"; (4) protein "953" and (5) protein "287". These antigens are referred to herein as “5 major antigens”.

Protein "NadA"

"NadA" (Neisseriae adhesin A) from serogroup B. N.meningitidis is disclosed as protein "961" in reference 10 (SEQ ID NO 2943 and NO 2944) and as "NMB1994" in reference 6 (see also gene access numbers Bank 11352904 and 7227256). A detailed description of the protein can be found in reference 17. Serogroup A does not have a corresponding protein (5, 17).

When used in accordance with the present invention, NadA can take various forms. Preferred forms of NadA are truncated or deletion variants, such as those disclosed in references 14-16. In particular, NadA is preferred without its C-terminal membrane anchor (for example, deletion of residues 351-405 for strain 2996 [SEQ ID NO 1]), which is sometimes defined herein using superscript “C”, for example, NadA ^(C) . Expression of NadA without its membrane anchor domain (eg, SEQ ID NO 1) in E. coli results in secretion of the protein into the supernatant of the culture with the simultaneous removal of its 23-dimensional leader peptide (for example, to leave 327-measure for strain 2996 [SEQ ID NO 2]). Polypeptides without their leader peptides are sometimes defined in the present description using the superscript "NL", for example, NadA ^(NL) or NadA ^{(C) (NL)} .

Preferred NadA sequences have an identity of 50% or more (e.g., 60%, 70%, 80%, 90%, 95%, 99% or more) with SEQ ID NO 2. It includes NadA variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.). Allelic forms of NadA are shown in FIG. 9 of reference 18.

Other preferred NadA sequences include at least n consecutive amino acids from SEQ ID NO 1, where n is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments include an epitope from NadA. 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) from the C-terminus and / or N-terminus of SEQ ID NO 1 (e.g., NadA ^(C) , NadA ^(NL) or NadA ^{(C) (NL)} ). When N-terminal residues are eliminated, it is preferred that the deletion does not eliminate the ability of NadA to adhere to human epithelial cells. A preferred fragment of SEQ ID NO 1 is SEQ ID NO 2.

Secreted NadA can conveniently be obtained in a highly purified form from the culture supernatant in a manner comprising the steps of: concentration and diafiltration against buffer by ultrafiltration; anion column chromatography; hydrophobic column chromatography; hydroxyapatite ceramic column chromatography; diafiltration against buffer and filtration sterilization. Additional details of the method are presented in the examples.

NadA is preferably used in oligomeric form (for example, in trimeric form).

Protein 741

Protein “741” from serogroup B is disclosed in reference 10 (SEQ ID NO 2535 & 2536) and as “NMB1870” in reference 6 (see also GI gene bank access number: 7227128). The corresponding protein in serogroup A (5) has a gene bank access number of 7379322. 741 in structure is a lipoprotein.

When used in accordance with the present invention, protein 741 may take various forms. Preferred forms 741 are truncated or deletion variants, such as the forms disclosed in references 14-16. In particular, the N-terminus 741 may undergo deletion to and including the poly-glycine sequence (ie, deletion of residues 1-72 for strain MC58 [SEQ ID NO 3]), which is sometimes defined in the present description using the prefix "ΔG" . This deletion may enhance expression. Deletion also removes the 741 lipidation site.

Preferred sequences 741 have an identity of 50% or more (e.g., 60%, 70%, 80%, 90%, 95%, 99% or more) with SEQ ID NO 3. It includes variants 741 (e.g. allelic variants, homologues, orthologs, paralogs, mutants, etc.). Allelic forms 741 can be found in SEQ ID NO from 1 to 22 of reference 16 and in SEQ ID NO from 1 to 23 of reference 19. In SEQ ID NO 1-299 of reference 20, additional sequences 741 are provided.

Other preferred sequences 741 include at least n consecutive amino acids from SEQ ID NO 3, where n = 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments include an epitope of 741. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and / or the N-terminus of SEQ ID NO 3.

Protein 741 is an extremely effective antigen for eliciting a humoral immune response against meningococci, and it is expressed in all meningococcal serogroups. Phylogenetic analysis shows that the protein is split into 2 groups and that one of them is split again to obtain only three variants (21), and although the serum produced against this variant is bactericidal within the group of the same variant, it is not active against strains that express one of the other two options, i.e. there is cross-protection within options, but cross-protection between options. Therefore, for maximum efficiency between strains, it is preferable that the composition includes more than one protein variant 741. An illustrative sequence of each variant is presented in SEQ ID NOS 10, 11 and 12, starting from the N-terminal cysteine residue to which the lipid will be covalently attached. in lipoprotein form 741.

Therefore, it is preferable that the composition includes at least 2 of: (1) a first protein comprising an amino acid sequence having at least a % identity with the sequence of SEQ ID NO 10 and / or comprising an amino acid sequence consisting of a fragment, at least x adjacent amino acids from SEQ ID NO 10; (2) a second protein comprising an amino acid sequence having at least b % identity with SEQ ID NO 11 and / or comprising an amino acid sequence consisting of a fragment of at least the adjacent amino acids from SEQ ID NO 11; and (3) a third protein, comprising an amino acid sequence having at least c% sequence identity with SEQ ID NO 12 and / or comprising an amino acid sequence consisting of a fragment of, at least, z contiguous amino acids from 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. Values a, b, and c are not 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 y value 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 x, y and z are not interconnected with each other.

Preferably, each given amino acid sequence 741 does not fall into more than one category (1), (2) and (3). Thus, any given sequence 741 should fall under only one of the categories (1), (2) and (3). Thus, it is preferable that: protein (1) has less than i % sequence identity with protein (2); protein (1) had less than j % sequence identity with protein (3); and protein (2) had less than k % sequence identity with protein (3). The value of i is 60 or more (e.g. 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 is a maximum of a . The value of j is 60 or more (e.g., 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 is a maximum of b . The value of k is 60 or more (e.g., 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 is at most c. Values i, j and k are not related to each other.

Protein 936

Protein "936" from serogroup B is disclosed in reference 10 (SEQ ID NO 2883 and 2884) and in the form of "NMB2091" in reference 6 (see also accession number of gene bank GI: 7227353). The corresponding gene in serogroup A (5) has a gene bank access number of 7379093.

When used in accordance with the present invention, protein 936 may take various forms. Preferred forms 936 are truncated or deletion variants, such as those disclosed in references 14-16. In particular, the N-terminal leader peptide 936 can be deleted (ie, deletion of residues 1-23 for strain MC58 [SEQ ID NO 4]) to obtain 936 ^(NL) .

Preferred sequences 936 have an identity of 50% or more (eg, 60%, 70%, 80%, 90%, 95%, 99% or more) with SEQ ID NO 4. They include variants (eg, allelic variants, homologues, orthologs , paralogs, mutants, etc.).

Other preferred sequences 936 include at least n consecutive amino acids from SEQ ID NO 4, where n = 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments include an epitope of 936. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and / or the N-terminus of SEQ ID NO 4.

Protein 953

Protein "953" from serogroup B is disclosed in reference 10 (SEQ ID NO 2917 and 2918) and in the form of "NMB1030" in reference 6 (see also accession number of gene bank GI: 7226269). The corresponding protein in serogroup A (5) has a gene bank access number of 7380108.

When used in accordance with the present invention, protein 953 may take various forms. Preferred forms 953 are truncated or deletion variants, such as those disclosed in references 14-16. In particular, the N-terminal leader peptide 953 may be deleted (i.e., deleted of residues 1-19 for strain MC58 (SEQ ID NO 5) to obtain 953 ^(NL) .

Preferred sequences 953 are identical with SEQ ID NO 5 of 50% or more (e.g., 60%, 70%, 80%, 90%, 95%, 99% or more). These include variants 953 (e.g., allelic variants, homologs, orthologs, paralogs, mutants, etc.). Allelic forms 953 are shown in FIG. 19 of reference 12.

Other preferred sequences 953 include at least n consecutive amino acids from SEQ ID NO 5, where n = 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments include an epitope of 953. 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) from the C-terminus and / or the N-terminus of SEQ ID NO 5.

Protein 287

Protein “287” from serogroup B is disclosed in reference 10 (SEQ ID NO 3103 and 3104) and in the form of “NMB2132” in reference 6 and in the form of “GNA2132” in reference 13 (see also GI gene bank access number: 7227388). The corresponding protein in serogroup A (5) has a gene bank access number of 7379057.

When used in accordance with the present invention, protein 287 may take various forms. Preferred forms 287 are truncated or deletion variants, such as those disclosed in references 14-16. In particular, the N-terminus 287 may be deleted to its polyglycine sequence and including it (ie, deletion of residues 1-24 for strain MC58 (SEQ ID NO 6)), which is sometimes identified in the present description using the prefix "ΔG" . Deletion may enhance expression.

Preferred sequences 287 are identical with SEQ ID NO 6 of 50% or more (e.g., 60%, 70%, 80%, 90%, 95%, 99% or more). These include variants 287 (e.g., allelic variants, homologs, orthologs, paralogs, mutants, etc.). Allelic forms 287 are shown in FIGS. 5 and 15 of reference 12 and in Example 13 and FIG. 21 of Reference 10 (SEQ ID NO 3179-3184).

Other preferred sequences 287 include at least n consecutive amino acids from SEQ ID NO 6, where n = 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments include 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) from the C-terminus and / or the N-terminus of SEQ ID NO 6.

Fusion proteins

5 antigens may be present in the composition as 5 separate proteins, but it is preferable that at least 2 of the antigens are expressed as a single polypeptide chain (fusion (“hybrid”) protein (refs. 14-16)), for example, 5 antigens from less than 5 polypeptides. Hybrid proteins provide 2 main advantages: first, a protein that can be unstable or weakly expressed by itself can be helped by adding a suitable hybrid partner that overcomes this problem; secondly, industrial production is simplified, since it is necessary to use only one expression and purification for the production of two separately used proteins.

A fusion protein included in a composition of the invention may include 2 or more (i.e., 2, 3, 4 or 5) of the 5 major antigens. Hybrids consisting of 2 of 5 major antigens are preferred.

Within a combination of 5 major antigens, an antigen may be present in more than one fusion protein and / or as a non-fusion protein. However, it is preferable that the antigen is present either as a hybrid or not a hybrid, but not both, although it may be useful to include protein 741 both as a hybrid and as a non-hybrid (preferably lipoprotein) antigen, in particular when use more than one option 741.

Two antigenic hybrids for use in the invention include: 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 297; 953 and 287. Preferred two antigenic hybrids include: 741 and 936; 953 and 287.

Hybrid proteins can be represented by the formula NH ₂ -A - [- XL-] _n -B-COOH, where: X is the amino acid sequence of one of the five major antigens; L is an optional linker amino acid sequence; A is an optional N-terminal amino acid sequence; B is an optional C-terminal amino acid sequence and n = 2, 3, 4 or 5.

If the -X- part has a leader peptide sequence in its wild-type form, then it can be incorporated into or omitted from the fusion protein. In some embodiments, leader peptides will be deleted, except for those portion -X- located at the N-terminus of the fusion protein, i.e., leader peptide X ₁ will be retained, but leader peptides X ₂ ... X _n will be omitted . This represents the equivalent of a deletion of all leader peptides and the use of leader peptide X ₁ as part of -A-.

For every n cases of [-XL-], the linker amino acid sequence -L- may or may not be present. For example, when n = 2, the hybrid may be NH ₂ -X ₁ -L ₁ -X ₂ -L ₂ -COOH, NH ₂ -X ₁ -X ₂ -COOH, NH ₂ -X ₁ -L ₁ -X ₂ -COOH, NH ₂ -X ₁ -X ₂ -L ₂ -COOH, etc. The linker amino acid sequence (s) -L- will usually be short (for example, of 20 or less amino acids, i.e., 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include short peptide sequences that facilitate cloning, polyglycine linkers (i.e., including Cly _n , where n = 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) and histidine tags (t .e., His _n , where n = 3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable linker amino acid sequences will be apparent to those skilled in the art. A useful linker is GSGGGG (SEQ ID NO 9), wherein the Gly-Ser dipeptide is formed from the BamHI restriction site, thereby facilitating cloning and manipulation, and the tetrapeptide (Gly) ₄ is a typical polyglycine linker. If X _{n + 1} is a ΔG protein and L _n is a glycine linker, then it may be equivalent to X _{n + 1} , which is not a ΔG protein, and is absent L _n .

-A- is an optional N-terminal amino acid sequence. It will usually be short (for example, of 40 or less amino acids, i.e. 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 leader sequences for directing protein transport or short peptide sequences that facilitate cloning or purification (e.g., histidine tags, i.e., His _n , where 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 X _{1 is} devoid of its own N-terminus methionine, -A- is preferably an oligopeptide (for example, with 1, 2, 3, 4, 5, 6, 7, or 8 amino acids) that ensures the presence of methionine at the N-terminus.

-B- is an optional C-terminal amino acid sequence. It will usually be short (for example, of 40 or less amino acids, i.e., 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 for directing protein transport, short peptide sequences that facilitate cloning or purification (e.g., histidine tags, i.e., His _n , where 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.

Most preferably, n = 2. Two preferred proteins of this type are: X ₁ represents 936 and X ₂ represents 741; X ₁ represents 287 and X ₂ represents 953.

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

n A X ₁ L ₁ X ₂ L ₂ B [SEQ ID] 2 MA ΔG287 GSGGGG 953 ^(NL) - - 7 2 M 936 ^(NL) GSGGGG ΔG741 - - 8

These 2 proteins can be used in combination with NadA (in particular, with SEQ ID NO 2).

Hybrid 936-ΔG741 can conveniently be obtained in highly purified form when it is expressed in E. coli in a manner comprising the steps of: homogenizing; centrifugation; cationic column chromatography; anion column chromatography; hydrophobic column chromatography; diafiltration against buffer; and sterilization by filtration. Additional details of the method are presented in the examples.

Sequence and

The invention includes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO 1-8. It also includes polypeptides having an amino acid sequence identical with an amino acid sequence selected from the group consisting of SEQ ID NO 1-8. As described above, the degree of sequence identity is preferably greater than 50% (e.g., 60%, 70%, 80%, 90%, 95%, 99% or more).

The invention also includes a polypeptide containing a fragment of the NadA sequence of N. meningitidis, where the specified fragment retains the ability of adhesion of NadA to human epithelial cells. Thus, fragments that retain the amino acids 24-87 NadA full length are preferred. Preferred fragments lack the N-terminal leader peptide of said NadA and / or C-terminal membrane anchor domain of said NadA. The present invention does not include within its scope any of the NadA fragments disclosed in the prior art, for example, in references 6-18. In full-length NadA (17), SEQ ID NO 1 lacks the membrane anchor domain, and SEQ ID NO 2 lacks the leader peptide.

The invention also includes nucleic acid encoding such polypeptides. In addition, the invention includes a nucleic acid that can hybridize to a given nucleic acid, preferably under “stringent conditions” (for example, 65 ° C in 0.1xSSC, 0.5% solution of SDS, sodium dodecyl sulfate).

The polypeptides of the invention can be obtained by various means (e.g., recombinant expression, purification from cell culture, chemical synthesis (at least partially), etc.) and in various forms (e.g., native, hybrid, non-glycosylated, lipidated, etc. d.). They are preferably prepared in substantially pure form (i.e., substantially devoid of proteins of another N. meningitidis or host cell proteins).

Nucleic acid in accordance with the invention can be obtained in many ways (for example, by chemical synthesis (at least partially) from genomic or cDNA libraries, from the body itself, etc.), and it can take various forms (for example, single-stranded, double-stranded , vectors, probes, etc.). They are preferably prepared in substantially pure form (i.e., substantially lacking the nucleic acids of another N. meningitidis or the nucleic acids of the host cells).

The term "nucleic acid" includes DNA and RNA, as well as their analogues, such as analogues containing modified scaffolds (eg, phosphorothioates, etc.), as well as peptide nucleic acids (PNA), etc. The invention includes a nucleic acid comprising sequences complementary to those described above (for example, for antisense or probing purposes).

The invention also includes a method for producing a polypeptide of the invention, comprising the step of culturing a host cell transformed with a nucleic acid of the invention under conditions that cause expression of the polypeptide.

The invention includes a method for producing a polypeptide of the invention, comprising the step of synthesizing at least a portion of the polypeptide by chemical means.

The invention includes a method for producing a nucleic acid according to the invention, comprising the step of amplifying a nucleic acid using a primer (seed) amplification method (eg, PCR, polymerase chain synthesis reaction).

The invention includes a method for producing a nucleic acid according to the invention, comprising the step of synthesizing at least a portion of the nucleic acid by chemical means.

Strains

Preferred proteins of the invention include the amino acid sequence found in N. meningitidis serogroup B. Within serogroup B, preferred strains are 2996, MC58, 95N477 and 394/98. Strain 394/98 is sometimes referred to herein as “NZ" because it is a New Zealand strain.

Protein 287 preferably comes from strain 2996 or, more preferably, from strain 394/98.

Protein 741 preferably originates from strains MC58, 2996, 394/98 or 95N477 of serogroup B or from strain 90/18311 of serogroup C. Preferably, strain MC58.

Proteins 936, 953 and NadA preferably come from strain 2996.

The strains can be indicated in the form of subscripts, for example, 741 _MC58 is a protein 741 from strain MC58. In the absence of other indications, the proteins indicated in the present description (for example, without superscript) are obtained from strain 2996 N.meningitidis, which can be taken as a “reference” strain. However, it should be understood that the invention as a whole is not limited to this strain. As indicated above, generic protein references (eg, “287”, “919”, etc.) may be used to include said protein from any strain. It will usually have a sequence identity with 2996 90% or more (for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more).

When the composition includes a specific protein antigen (for example, 741 or 287), the composition may include the specified antigen in the form of several options, for example, the same protein, but from several strains. These proteins may be included as tandem or single proteins.

When fusion proteins are used, individual antigens within the hybrid (i.e., individual parts —X—) may be from one or more strains. For example, when n = 2, X ₂ may be from the same strain as X ₁ or from another strain. When n = 3, the strain may be (i) X ₁ = X ₂ = X ₃ (ii) X ₁ = X ₂ ≠ X ₃ (iii) X ₁ ≠ X ₂ = X ₃ (iv) X ₁ ≠ X ₂ ≠ X3 or (v) X ₁ = X ₃ ≠ X ₂ , etc.

Supervirulent generations and bactericidal antibody reactions

In general, the compositions of the invention are capable of eliciting bactericidal reactions of serum antibodies after administration to an individual. These reactions can be measured in mice and they are a standard indicator of vaccine efficacy (for example, see end note 14 of reference 13). Serum bactericidal activity (SBA) measures complement kill mediated bacterial kill and can be analyzed using human complement or rabbit complement. WHO standards require that a vaccine induce at least a 4-fold increase in SBA in more than 90% of recipients.

Instead of providing narrow protection, the compositions of the invention can induce a bactericidal humoral immune response against several supervirulent series of generations of serogroup B. In particular, they can cause bactericidal reactions against 2 or 3 of the following three supervirulent series of generations: (i) cluster A4; (ii) the ET5 complex and (ii) a series of generations 3. They can additionally induce bactericidal humoral immune responses against one or more supervirulent series of generations of subgroup I, subgroup III, subgroup IV-1 or the ET-37 complex and against other generations, for example , ultra-invasive series of generations.

This does not necessarily mean that the composition can cause the production of bactericidal antibodies against each and all strains of group B meningococcus within the indicated supervirulent series of generations, for example, rather for each given group of 4 or more serogroup B meningococcus strains within a specific supervirulent series of generations, antibodies, caused by the composition are bactericidal against at least 50% (e.g. 60%, 70%, 80%, 90% or more) of the group. Preferred strain groups 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, 2 ¹⁰ , 2 ¹¹ , 2 ¹² , 2 ¹³ , 2 ¹⁴ , 2 ¹⁵ , 2 ¹⁶ , 2 ¹⁷ , 2 ¹⁸ or higher, preferably at least 2 ¹⁴ ), i.e., serum is capable of killing at least 50% of the tested bacteria of a particular strain when 1/1024 is diluted, as described in reference 13.

Preferred compositions may include bactericidal reactions against the following strains of serogroup B meningococcus: (i) from cluster A4, strain 961-5945 (B: 2b: P1.21,16) and / or strain G2136 (B :-); (ii) from the ET-5 complex, strain MC58 (B: 15: P1.7,16b) and / or strain 44/76 (B: 15: P1.7,16); (iii) from a number of generations 3, strain 394/98 (B: 4: P1.4) and / or strain BZ198 (B: NT :-). More preferred compositions can cause bactericidal reactions against strains 961-5945, 44/76 and 394/98.

Both strains 961-5945 and G2136 are Neisseria MLST reference strains (identification Nos. 638 and 1002 in reference 22). Strain MC58 is widely available (for example, ATCC BAA-335) and was a strain sequenced in reference 6. Strain 44/76 was widely used and characterized (for example, reference 23) and is one of the Neisseria MLST reference strains (identification no. 237 in reference 22; row 32 of Table 2 in reference 1). Strain 394/98 was originally isolated in New Zealand in 1998, and there have been several published studies using this strain (e.g., references 24 and 25). Strain BZ198 is another MLST reference strain (identification no. 409 in ref. 22; row 41 of Table 2 in ref. 1).

The composition may additionally cause a bactericidal reaction against strain LNP17592 (W135: 2a: P1.5,2) of serogroup W135 from the ET-37 complex. It is a Haji strain isolated in France in 2000.

Heterologous host

Although expression of the proteins of the invention may occur in Neisseria, the heterologous host is preferably used. The heterologous host may be prokaryotic (e.g., bacterium) or eukaryotic. It is preferably E. coli, but other suitable hosts include Bacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonella typhimurium, Neisseria lactamica, Neisseria cinerea, Mycobacteria (i.e. M. tuberculosis), yeast, etc.

Thus, the invention includes a composition which, after administration to an individual, is capable of eliciting a humoral immune response in a given individual, the humoral immune response being bactericidal against two or more (for example, 2 or 3) supervirulent rows of generations A4, ET-5 and a series of generations of 3 serogroups In N. meningitidis, and in which the immunogens in the composition that cause the development of a humoral immune response are obtained by recombinant expression in a non-Neisseria host. Thus, the immunogens in the compositions of the invention are preferably recombinant immunogens. Thus, compositions that do not include OMV preparations may be preferred.

Immunogenic compositions and drugs

The compositions of the invention are immunogenic and, more preferably, are vaccine compositions. Vaccines in accordance with the invention can be either prophylactic (i.e., to prevent infection) or therapeutic (i.e., to treat infection), but will usually be prophylactic.

The pH of the composition is preferably from 6 to 8, preferably approximately 7. A stable pH can be maintained using a buffer. When the composition includes aluminum hydroxide, it is preferable to use histidine buffer (26). The composition may be sterile and / or pyrogen free. The compositions of the invention may be isotonic when administered to humans.

The compositions may be presented in vials, or they may be presented as ready-to-use filled syringes. Syringes can be supplied with or without needles. The syringe will include a single dose of the composition, while the bottle may include a single dose or multiple doses. Compositions for injection will usually be a liquid solution or suspension. Alternatively, they may be presented in solid form (e.g., lyophilized) for dissolution or suspension in liquid carriers prior to injection.

The compositions of the invention may be packaged in unit dosage form containing a single dose or multiple doses. For multi-dose forms, vials are preferred over pre-filled syringes. Effective dosage forms can be established by conventional methods, but a typical dose of a composition for injection to humans has a volume of 0.5 ml.

When the composition of the invention is to be prepared immediately before use (for example, when the component is presented in lyophilized form) and it is presented in the form of a kit, the kit may include 2 vials or it may include one ready-to-use syringe and one vial, the contents of the syringe being used to reactivate the contents of the bottle before injection.

The invention also includes a composition of the invention for use as a medicine. The drug is preferably capable of eliciting an immune response in a mammal (i.e., it is an immunogenic composition) and, more preferably, is a vaccine.

The invention also includes the use of a composition of the invention in the preparation of a medicament in order to elicit an immune response in a mammal. It also includes the use of NadA protein, 741 protein, 936 protein, 953 protein and 287 protein (and other optional antigens) in the preparation of a medicament in order to elicit an immune response in a mammal. The drug is preferably a vaccine.

The invention also includes a method for producing an immune response in a mammal, comprising the step of administering an effective amount of a composition of the invention. The immune response is preferably protective and preferably includes antibodies. The method may cause a booster reaction.

The mammal is preferably a human. When the vaccine is intended for prophylactic use, the person is preferably a child (eg, a baby or infant starting to walk); when the vaccine is intended for therapeutic use, the person is preferably an adult. A vaccine intended for children can also be administered to adults, for example, to assess safety, dosage, immunogenicity, etc.

The indicated uses and methods are preferably intended to prevent and / or treat a disease caused by Neisseria (e.g., meningitis, septicemia, bacteremia, gonorrhea, etc.). Prevention and / or treatment of bacterial or meningococcal meningitis is preferred.

One way to verify the effectiveness of therapeutic treatment involves controlling a Neisseria infection after administration of a composition of the invention. One way to test the effectiveness of prophylactic treatment involves monitoring immune responses against 5 major antigens after administration of the composition. The immunogenicity of the compositions of the invention can be determined by administering them to test individuals (eg, children 12–16 months old, or experimental models [27]), and then determining standard parameters, including serum bactericidal antibodies (SBA) and ELISA (GMT) titers, total and antiviral IgG with high avidity. These immune responses in general will be determined approximately 4 weeks after administration of the composition and compared with the values determined before administration of the composition. Preferably, the increase in SBA is at least 4 times or 8 times. When multiple doses of a composition are administered, several determinations can be made after administration.

Preferred compositions of the invention can provide a patient with an antibody titer that is higher than the serological protection criterion for each antigenic component for an acceptable percentage of people. Antigens with an associated antibody titer, above which the host is considered to be serologically converted 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 individuals are serologically converted, more preferably more than 90%, even more preferably more than 93% and most preferably 96-100%.

The compositions of the invention as a whole will be administered directly to the patient. Direct delivery can be accomplished by parenteral injection (e.g., subcutaneously, intraperitoneally, intravenously, intramuscularly or into the interstitial space of the tissue) or rectal, oral, vaginal, local, transdermal, intranasal, ophthalmic, auricular, pulmonary or through another mucous membrane. Intramuscular administration to the thigh or upper shoulder is preferred. Injection can be done with a needle (for example, a hypodermic needle), but alternatively, an injection without a needle can be used. A typical intramuscular dose is 0.5 ml.

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

Dosage treatment may be a regimen comprising administering a single dose or multiple doses. Multiple doses may be used in the primary immunization schedule and / or in the re-immunization schedule. A regimen including a primary dose may be followed by a regimen including a dose for re-immunization. Determining the appropriate time between doses of seeds (for example, from 4 to 16 weeks) and between seeds and re-immunization can be determined in the usual way.

Neisseria infections affect various areas of the body, and therefore the compositions of the invention can be obtained in various forms. For example, the compositions can be obtained in the form of solutions for injection, or in the form of liquid solutions, or in the form of suspensions. Solid forms suitable for dissolution or suspension in liquid carriers prior to injection (e.g., lyophilized composition) can also be prepared. The composition can be obtained for local administration, for example, in the form of an ointment, cream or powder. The composition can be prepared for oral administration, for example, in the form of a tablet or capsule, or in the form of a syrup (optionally flavored). The composition can be obtained for pulmonary administration, for example, in the form of inhalation, using a fine powder or aerosol. The composition can be obtained in the form of a suppository or pessary. The composition can be prepared for intranasal, intraocular or intraocular administration, for example, in the form of an aerosol, drops, gel or powder (for example, refs. 28 and 29). The success of intranasal administration of pneumococcal saccharides (30, 31), pneumococcal polypeptides (32), Hib saccharides (33), MenC saccharides (34) and mixtures of Hib and MenC saccharide conjugates (35) has been reported.

Immunogenic compositions used as vaccines include an immunologically effective amount of antigen (s), as well as, if necessary, any other components. By “immunologically effective amount” is meant that the administration of a given amount of an individual, either in a single dose or 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, the age, taxonomic group of the individual to be treated (for example, not a humanoid ape, monkey, etc.), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, vaccine compositions; evaluations by the attending physician of the medical situation and other relevant factors. The amount is expected to fall within a relatively wide range that can be determined by routine testing, and a typical amount of each meningococcal saccharide antigen per dose is from 1 μg to 20 μg, for example, approximately 1 μg, approximately 2.5 μg, approximately 4 μg, approximately 5 μg, or approximately 10 μg (expressed as saccharide).

Other non-antigenic components of the composition

The composition according to the invention will usually, in addition to the above components, contain one or more "pharmaceutically acceptable carriers", which include any carrier that alone does not cause the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose (36), trehalose (37), lactose and lipid aggregates (such as oil droplets or liposomes ) Such carriers are well known to those of ordinary skill in the art. Vaccines may also contain diluents, such as water, saline, glycerin, etc. In addition, adjuvants such as wetting or emulsifying agents, pH stabilizing buffering agents and the like may be present. A typical carrier is a pyrogen-free physiological saline solution with phosphate buffer. A detailed discussion of pharmaceutically acceptable excipients is given in reference 38.

The compositions of the invention may include an antimicrobial agent, in particular when packaged in multiple dosage formats.

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

The compositions of the invention may include sodium salts (e.g., sodium chloride) to give tonicity. Typical NaCl concentration is 10 ± 2 mg / ml.

The compositions of the invention will generally comprise a buffer. Typical phosphate buffer.

The compositions of the invention may include sugar alcohol (e.g. beckoning) or disaccharide (e.g. sucrose or trehalose), for example, at a concentration of about 15-30 mg / ml (e.g. 25 mg / ml), in particular if they are to be lyophilized, or if they include material that has been dissolved from lyophilized material. The pH of the lyophilization composition can be adjusted to about 6.1 before lyophilization.

The vaccines of the invention can be administered in combination with other immunoregulatory agents. In particular, the compositions will typically include an adjuvant. Adjuvants that can be used in the compositions of the invention include, but are not limited to:

A. Compositions containing minerals

Mineral containing compositions 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 (e.g., oxyhydroxides), phosphates (e.g., hydroxyphosphates, orthophosphates), sulfates, etc. (for example, see chapters 8 and 9 of reference 39), or mixtures of various mineral compounds, the compounds taking any suitable form (for example, gel, crystalline, amorphous, etc.), and adsorption is preferred. Compositions containing minerals can also be formulated as particles of a metal salt (40).

Particularly preferred are aluminum phosphates, especially in compositions that include the H. influenzae saccharide antigen, and a typical adjuvant is amorphous aluminum hydroxyphosphate with a PO ₄ / Al molar ratio of 0.84 to 0.92, included in a concentration of 0.6 mg Al ^{3 +} / ml. Adsorption can be used at a low dose of aluminum phosphate, for example, from 50 to 100 μg Al ³⁺ per conjugate per dose. When there are several conjugates in a composition, not all conjugates must be adsorbed.

B. Oil emulsions

Oil emulsion compositions suitable for use as adjuvants include squalene in water emulsions such as MF59 [Chapter 10 of ref. 39; see also reference 41]) (5% squalene, 0.5% Tween 80 and 0.5% Span 85, compiled into submicron particles using a microfluidizer). You can also use complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA).

C. Compositions of saponin (chapter 22 of reference 39)

Saponin compositions can also be used as adjuvants in the invention. Saponins are a heterologous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, trunks, roots and even flowers of a wide range of plant species. As adjuvants, saponins from the bark of Quillaia saponaria of the Molina tree have been extensively studied. Saponin can also be obtained industrially from Smilax ornate (Aralia), Gypsophilla paniculata ("veil") and Saponaria officianalis (soap root). Saponin adjuvant compositions include purified compositions such as QS21, as well as lipid compositions such as ISCOM compositions. QS21 is commercially available as Stimulon ^TM .

Saponin compositions were purified using reverse phase HPLC and HPLC. Using these techniques, specific purified fractions were identified, including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, the saponin is QS21. A method for producing QS21 is disclosed in reference 42. Saponin compositions may also include sterol, such as cholesterol (43).

Combinations of saponins and cholesterol can be used to form unique particles called immunostimulatory complexes (ISCOMs) (chapter 23 of ref. 39). ISCOMs typically also include a phospholipid, such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOM. Preferably, ISCOM includes one or more of QuilA, QHA, and QHC. ISCOMs are further described in references 43-45. Optionally, ISCOMs may lack an additional detergent (46).

A review of the development of saponin-based adjuvants can be found in references 47 and 48.

D. Virosomes and virus-like particles

Virosomes and virus-like particles (VLPs) can also be used as adjuvants in the invention. These structures are generally one or more proteins from 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, measles virus, Sindbis virus, rotavirus , foot and mouth disease virus, retrovirus, Norfolk virus, human papillomavirus, HIV, RNA phages, Qβ-phage (such as coating proteins), GA-phage, fr-phage, AP205 phage and Ty (such as Ty retrotransposon protein p1). VLPs are further discussed in references 49-54. Virosomes are discussed further, for example, in reference 55.

E. Derivatives of bacteria or germs

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

Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3-O-deacylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. The preferred “fine particles” 3-O-deacylated monophosphoryl lipid A is disclosed in reference 56. Such “small particles” 3dMPL are small enough to be sterilized by filtration through a 0.22 μm membrane (56). Other non-toxic LPS derivatives include monophosphoryl lipid A mimetics, such as aminoalkyl glucosaminide phosphate derivatives, for example, RC-529 (57, 58).

Derivatives of lipid A include derivatives of lipid A from Escherishia coli, such as OM-174. OM-174 are described, for example, in references 59 and 60.

Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a dinucleotide sequence containing unmethylated cytosine bound by a phosphate bond to guanosine). It was also shown that double-stranded RNA and oligonucleotides containing palindromic or poly (dG) sequences are immunostimulatory.

CpGs may include nucleotide modifications / analogs, such as phosphorothioate modifications, and they can be double-stranded or single-stranded. References 61, 62 and 63 disclose possible analog substitutions, for example, substitution of guanosine with 2'-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotides is further discussed in references 64-69.

The CpG sequence can be directed to TLR9, such as GTCGTT or TTCGTT (70). The CpG sequence may be specific for inducing an Th1 immune response, such as a CpG-A ODN, or it may be more specific for inducing a B cell response, such as a CpG-B ODN. CpG-A and CpG-B ODN are discussed in references 71-73. Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotide is designed so that the 5 ′ end is accessible for receptor recognition. Optionally, 2 sequences of CpG oligonucleotide can be attached at their 3 ′ ends to form “immunomers” (see, for example, references 70 and 74-76).

Bacterial ribosylating ADP toxins and their detoxified derivatives can be used as adjuvants in the invention. Preferably, the protein is derived from E. coli (E. coli heat labile enterotoxin "LT"), cholera ("CT"), or pertussis ("PT") bacillus. The use of detoxified ribosylating ADP toxins as mucosal adjuvants is described in reference 77 and as parenteral adjuvants in reference 78. 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 LT detoxified mutant, such as LT-K63, LT-R72 and LT-G192. The use of ADP ribosylating toxins and their detoxified derivatives, in particular LT-K63 and LT-R72, as adjuvants can be found in references 79-86. A digital reference to amino acid sequences is preferably based on combinations of subunits A and B of ribosylating ADP toxins provided in reference 87, which is hereby expressly incorporated by reference in its entirety.

F. Human immunomodulators

Human immunomodulators suitable for use as adjuvants include cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (88) and etc.) (89), interferons (e.g., interferon-γ), macrophage colony stimulating factor, and tumor necrosis factor.

G. Bioadhesive and mucoadhesive substances

Bioadhesive and mucoadhesive substances can also be used as adjuvants in the invention. Suitable bioadhesive substances include esterified hyaluronic acid microspheres (90) or mucoadhesive substances such as crosslinked derivatives of poly (acrylic acid), polyvinyl alcohol, polyvinyl pyrrolidone, polysaccharides and carboxymethyl cellulose. Chitosan and its derivatives can also be used as adjuvants in the invention (91).

H. Microparticles

Microparticles can also be used as adjuvants in the invention. Microparticles are preferred (i.e., particles with a diameter of ≈100 nm to ≈150 nm, more preferably a diameter of ≈200 nm to ≈30 μm, and most preferably a diameter of ≈500 nm to ≈10 μm), formed from materials that are biodegradable and non-toxic (e.g. poly (α-hydroxy acid), polyhydroxybutyric acid, polyorthoester, polyanhydride, polycaprolactone, etc.) with poly (lactide co-glycolide), optionally processed to produce a negatively charged surface (e.g. SDS) or positively charged surfaces (e.g., a cationic detergent such as CTAB (cetyltrimethylammonium bromide)).

I. Liposomes (chapters 13 and 14 of reference 39)

Examples of liposome compositions suitable for use as adjuvants are described in references 92-94.

J. Polyoxyethylene ether and polyoxyethylene ester compositions

Adjuvants suitable for use in the invention include polyoxyethylene ethers and polyoxyethylene esters (95). Such compositions further include polyoxyethylene sorbitan ester surfactants in combination with octoxynol (96), as well as polyoxyethylene alkyl esters or surfactants in combination with at least one additional non-ionic surfactant, such as octoxynol (97). Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-stearyl ether, polyoxyethylene-8-stearyl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35 ether -lauryl and ether polyoxyethylene-23-lauryl.

K. Polyphosphazene (RSRP)

PCPP compositions are described, for example, in references 98 and 99.

L. Muramylpeptides

Examples of muramyl peptides suitable for use as adjuvants in the invention include N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetylnormuramyl-L-alanyl-D-isoglutamine (nor-MDP) and N-acetylmuramyl -L-alanyl-D-isoglutaminyl-L-alanyl-2- (1'2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamine MTP-PE).

M. Imidazoquinolone Compounds

Examples of imidazoquinolone compounds suitable for use as adjuvants in the invention include imichamod and its homologues (eg, “Resimimod 3M”), described further in references 100 and 101.

The invention may also include combinations of aspects of one or more adjuvants identified above. For example, the following adjuvant compositions can be used in the invention: (1) an emulsion of saponin and oil in water (102); (2) saponin (e.g., QS21) + non-toxic derivative of LPS (e.g., 3dMPL) (103); (3) saponin (e.g. QS21) + non-toxic derivative of LPS (e.g. 3dMPL) + cholesterol; (4) saponin (e.g. QS21) + 3dMPL + IL-12 (optional + sterol) (104); (5) combinations of 3dMPL, for example, with QS21 and / or oil-in-water emulsions (105); (6) SAF containing 10% squalane, 0.4% Tween 80 ^TM , 5% pluronic block copolymer L121 and thr-MDP, or microfluidized into a submicron emulsion or mixed using a vortex mixer to create an emulsion with larger particles; (7) an adjuvant system Ribi ^TM (RAS), (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80 and one or more components of the wall of a bacterial cell from the group consisting of monophosphorylipid A (MPL), trehalose dimicolate (TDM ) and cell wall scaffold (CWS), preferably MPL + CWS (Detox ^TM ); and (8) one or more mineral salts (such as an aluminum salt) + a non-toxic derivative of LPS (such as 3dMPL).

Other substances that act as immunostimulating agents are disclosed in chapter 7 of reference 39.

It is particularly preferred to use aluminum hydroxide or aluminum phosphate as adjuvant, and antigens in general are adsorbed to these salts. Preferably, the use of aluminum hydroxide as an adjuvant should be avoided if the composition comprises Hib antigen. When aluminum phosphate is used and it is undesirable that the antigen is not adsorbed to the adjuvant, this is facilitated by the inclusion of free phosphate ions in the solution (for example, using a phosphate buffer). Prevention of adsorption can also be achieved by selecting the correct pH during mixing of the antigen / adjuvant, the adjuvant with the appropriate zero charge point, and the appropriate mixing order for the various antigens in the composition (106).

Another preferred adjuvant is calcium phosphate.

Additional antigens

The compositions of the invention contain 5 major meningococcal protein antigens. They may also include additional antigens, although they may contain meningococcal protein antigens other than the 5 major antigens. Additional antigens for inclusion may be, for example:

- saccharide antigen from Haemophilus influenzae B.

a saccharide antigen from N. meningitidis of serogroup A, C, W135 and / or Y, such as the oligosaccharide disclosed in reference 107, from serogroup C or oligosaccharides of reference 108.

- saccharide antigen from Streptococcus pneumoniae (e.g. 155, 156, 157).

- an antigen from hepatitis A virus, such as an inactivated virus (e.g. 109, 110).

- an antigen from hepatitis B virus, such as surface and / or core antigens (e.g. 110, 111).

a diphtheria antigen, such as a diphtheria toxoid (e.g., chapter 3 of reference 112), for example, a CRM ₁₉₇ mutant (e.g., 113).

- a tetanus antigen, such as a tetanus toxoid (for example, chapter 4 of reference 112).

an antigen from Bordetella pertussis, such as pertussis holotoxin (PT) and filament hemagglutinin (FHA) from B.pertussis, optionally also in combination with pertactin and / or agglutinogens 2 and 3 (for example, refs. 114 and 115). Cellular pertussis antigen may be used.

- preparation of the outer membrane vesicle (OMV) from N.meningitidis serogroup B, such as disclosed in references 4, 116, 117, 118, etc.

- polio antigen (s) (e.g. 119, 120), such as OPV or, preferably, IPV.

The composition may include one or more of these additional antigens. Antigens should usually be present at a concentration of at least 1 μg / ml each. In general, the concentration of any given antigen should be sufficient to elicit an immune response against said antigen. Preferably, the protective efficacy of individual saccharide antigens is not eliminated by combining them, although the actual immunogenicity (for example, ELISA titers) can be reduced.

When a diphtheria antigen is included in the composition, it is also preferable to include tetanus antigen and pertussis antigen. Similarly, when a tetanus antigen is included, it is preferable to also include diphtheria and pertussis antigens. Similarly, when pertussis antigen is included, it is preferable to also include diphtheria and tetanus antigens. Such combinations of DTP can be used to dissolve lyophilized conjugates.

When a saccharide or hydrocarbon antigen is used, it is preferably conjugated to a carrier protein to enhance immunogenicity (see below).

Toxic protein antigens can be detoxified when necessary (e.g., detoxification of pertussis toxin with chemical and / or genetic means (115)).

As an alternative to the use of protein antigens in the composition of the invention, nucleic acid encoding an antigen can be used (e.g., references 121-129). Thus, the protein components of the compositions of the invention can be replaced by a nucleic acid (preferably DNA, for example, in the form of a plasmid) that encodes a protein. Similarly, compositions of the invention may include proteins that mimic saccharide antigens, for example, mimotopes (130) or anti-idiotypic antibodies. They can replace individual saccharide components or can complement them. As an example, a vaccine may include a peptide that mimics the capsular polysaccharide MenC (131) or MenA (132) instead of the saccharide itself.

Particularly preferred compositions of the invention include 1, 2 or 3 of: (a) saccharide antigens from meningococcal serogroups Y, W135, C and (optionally) A; (b) a saccharide antigen from Haemophilus influenzae type B; and / or (c) an antigen from Streptococcus pneumoniae. Particularly preferred is a composition comprising serogroup B antigens and a Hib conjugate.

Meningococcus serogroups Y, W135, C and (optionally) A

As indicated above, polysaccharide vaccines against serogroups A, C, W135 and Y have been known for many years. These vaccines (MENCEVAX ACWY ^TM and MENOMUNE ^TM ) are based on the capsular polysaccharides of the body, but although they are effective in adolescents and adults, they give a weak immune response and a short duration of protection and cannot be used at an early age.

Unlike unconjugated polysaccharide antigens in these vaccines, recently approved serogroup C vaccines (Menjugate ^TM (133, 107), Meningitec ^TM, and NeisVac-C ^TM ) include conjugated saccharides. Menjugate ^TM and Meningitec ^TM have oligosaccharide antigens conjugated to a CRM ₁₉₇ carrier, while NeisVac-C ^TM uses a complete polysaccharide (de-O-acetylated) conjugated to a tetanus toxoid carrier.

The compositions of the present invention preferably include capsular saccharide antigens from one or more serogroups Y, W135, C and (optionally) A meningococcus, the antigens being conjugated to a carrier protein (s) and / or are oligosaccharides.

A typical amount of each meningococcal saccharide antigen per dose is 1 μg to 20 μg, for example, approximately 1 μg, approximately 2.5 μg, approximately 4 μg, approximately 5 μg, or approximately 10 μg (expressed as saccharide).

When the mixture includes capsular saccharides from both serogroups A and C, the ratio (w / w) of MenA saccharide: MenC saccharide may be more than 1 (e.g. 2: 3, 3: 1, 4: 1, 5: 1, 10: 1 or higher). When the mixture includes capillary saccharides from serogroup Y and one or both of serogroups C and W135, the ratio (w / w) of MenY saccharide: MenW135 saccharide may be more than 1 (e.g. 2: 3, 3: 1, 4: 1 , 5: 1, 10: 1 or higher) and / or the ratio (w / w) of MenY saccharide: MenC saccharide may be less than 1 (e.g. 1: 2, 1: 3, 1: 4, 1: 5 or lower). Preferred ratios (w / w) for saccharides from 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 (w / w) for saccharides from 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 and 2: 1: 1. The use of substantially equal weight of each saccharide is preferred.

Capsular saccharides are generally used in the form of oligosaccharides. They can be formed by fragmentation of the purified capsular polysaccharide (e.g., by hydrolysis), which is usually followed by purification of the fragments of the desired size.

Fragmentation of polysaccharides is preferably performed to obtain a final average degree of polymerization (DP) in the oligosaccharide of less than 30 (for example, from 10 to 20, preferably about 10 for serogroup A; from 15 to 25 for serogroups W135 and Y, preferably about 15- 20; from 12 to 22 for serogroup C, etc.). DP can be suitably measured by ion exchange chromatography or colorimetric analyzes (134).

If hydrolysis is performed, the hydrolyzate should be generally classified by size to remove short-length oligosaccharides (135). This can be achieved in various ways, such as ultrafiltration, followed by ion exchange chromatography. Oligosaccharides with a degree of polymerization of less than about 6 or equal to it are preferably removed for serogroup A, and oligosaccharides of less than about 4 are preferably removed for serogroups W135 and Y.

Preferred MenC saccharide antigens are disclosed in reference 133, as saccharides used in Menjugate ^™ .

Saccharide antigen can be chemically modified. This is especially useful for reducing hydrolysis for serogroup A (136; see below). You can perform de-O-acetylation of meningococcal saccharides. For oligosaccharides, modification may occur before or after depolymerization.

When the composition of the invention comprises a MenA saccharide antigen, the antigen is preferably a modified saccharide in which one or more hydroxyl groups on the native saccharide have been replaced by a blocking group (136). This modification increases the resistance to hydrolysis and means that the antigen of serogroup A can be stored and used in a liquid composition, and it does not require lyophilization.

The number of monosaccharide units having blocking groups 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, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 monosaccharide units may have blocking groups.

Similarly, the number of blocking groups on a monosaccharide unit may vary. For example, the number of blocking groups on the monosaccharide unit may be 1 or 2. The blocking group as a whole is at position 4 and / or position 3 of the monosaccharide units.

The terminal monosaccharide unit may or may not have a blocking group instead of its native hydroxyl. It is preferable to maintain a free anomeric hydroxyl group on the terminal monosaccharide unit to provide a manipulator for further reactions (eg, conjugation). Anomeric hydroxyl groups can be converted to amino groups (-NH ₂ or -NH-E, where E is a nitrogen protecting group) by reductive amination (using, for example, NaBH ₃ CN / NH ₄ Cl), and then can be regenerated afterwards like other hydroxyl groups have been converted into blocking groups.

Blocking groups for the substitution of hydroxyl groups can be directly accessed through the derivatization reaction of the hydroxyl group, i.e. substitution of a hydrogen atom of a hydroxyl group with another group. Suitable derivatives of hydroxyl groups that act as blocking groups are, for example, carbamates, sulfonates, carbonates, esters, ethers (e.g., silyl ethers or alkyl ethers) and acetals. Some specific examples of such blocking groups are allyl, Aloc, benzyl, BOM, tert-butyl, trityl, TBS, TBDPS, TES, TMS, TIPS, PMB, MEM, MOM, MTM, THP, etc. Other blocking groups that are not directly available and which completely replace the hydroxyl group include C _1-12 alkyl, C _3-12 alkyl, C _5-12 aryl, C _5-12 aryl-C _1-6 alkyl, NR ¹ R ² (R ¹ and R ^{2 are} defined in the next paragraph), H, F, Cl, Br, CO ₂ H, CO ₂ (C _1-6 alkyl), CN, CF ₃ , CCl ₃ , etc. Preferred blocking groups are electron removing groups.

Preferred blocking groups are of the formula: —OXY or —OR ³ where X is C (O), S (O) or SO ₂ ; Y represents C _1-12 alkyl, C _1-12 alkoxy, C _3-12 cycloalkyl, C _5-12 aryl or C _5-12 aryl-C _1-6 alkyl, each of which may optionally be substituted with 1, 2 or 3 groups independently selected from F, Cl, Br, CO ₂ H, CO ₂ (C _1-6 alkyl), CN, CF ₃ or CCl ₃ ; or Y represents NR ¹ R ² ; R ¹ and R ^{2 are} independently selected from H, C _1-12 alkyl, C _3-12 cycloalkyl, C _5-12 aryl, C _5-12 aryl-C _1-6 alkyl; or R ¹ and R ² may combine to form a C _3-12 saturated heterocyclic group; R ³ represents C _1-12 alkyl or C _3-12 cycloalkyl, each of which may optionally be substituted with 1, 2 or 3 groups independently selected from F, Cl, Br, CO ₂ (C _1-6 alkyl), CN CF ₃ or CCl ₃ ; or R ³ represents C _5-12 aryl or C _5-12 aryl-C _1-6 alkyl, each of which may optionally be substituted with 1, 2, 3, 4 or 5 groups selected from F, Cl, Br, CO ₂ H, CO ₂ (C _1-6 alkyl), CN, CF ₃ or CCl ₃ . When R ³ is C _1-12 alkyl or C _3-12 cycloalkyl, it is usually substituted with 1, 2 or 3 groups as defined above. When R ¹ and R ² combine to form a C _3-12 saturated heterocyclic group, this means that R ¹ and R ² together with the nitrogen atom form a saturated heterocyclic group containing any number of carbon atoms from 2 to 12 (for example, C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , C ₁₂ ). A heterocyclic group may contain 1 or 2 heteroatoms (such as N, O or S) other than a hydrogen atom. Examples of C _3-12 saturated heterocyclic groups are pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, imidazolidinyl, azetidinyl and aziridinyl.

The blocking groups —OXY and —OR ³ can be obtained from —OH groups by standard derivatizing procedures, such as the reaction of a hydroxyl group with an acyl halide, alkyl halide, sulfonyl halide, etc. Therefore, the oxygen atom in —OXY is preferably an oxygen atom of a hydroxyl group, while the —XY group in —OXY preferably replaces a hydrogen atom of a hydroxyl group.

Alternatively, a blocking group may be available through a substitution reaction, such as Mitsonobu type substitution. These and other methods for producing blocking groups from hydroxyl groups are well known.

Preferably, the blocking group is —OC (O) CF ₃ (137) or the carbamate group —OC (O) NR ¹ R ² , where R ¹ and R ^{2 are} independently selected from C _1-6 alkyl. More preferably, R ¹ and R ^{2 are} both methyl, i.e., the blocking group is —OC (O) NMe ₂ . Carbamate blocking groups have a stabilizing effect on the glycosidic bond, and can be obtained under mild conditions.

Preferred modified MenA saccharides contain n monosaccharide units, where 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 (e.g. 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. Missing —OH groups are preferably blocking groups as defined above.

Other preferred modified MenA saccharides include monosaccharide units in which at least s monosaccharide units do not have —OH at position 3 and do not have —OH at position 4. The value of s is at least 1 (e.g., 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). Missing —OH groups are preferably blocking groups as defined above.

Suitable modified MenA saccharides for use in the invention have the formula:

Where

n is an integer from 1 to 100 (preferably an integer from 15 to 25);

T has the formula (A) or (B);

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

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

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

E represents H or a nitrogen protecting group;

and where more than about 7% (e.g., 8%, 9%, 10% or more) of the Q groups are blocking groups.

Each of the n + 2 Z groups may be the same or different from each other. Similarly, each of the n + 2 Q groups may be the same or different from each other. All Z groups may 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, with 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 about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100% of the Q groups are blocking groups.

Preferred compositions of the invention can be stored for 28 days at 37 ° C, and after this period less than f % of the initial total amount of conjugated MenA saccharide will not be conjugated, where f = 20, 19, 18, 17, 16, 15, 14 , 13, 12, 11, 10, 9, 8, 7, 6, 5 or lower.

Meningococcal capsular polysaccharides are typically prepared by a process comprising the steps of precipitating a polysaccharide (e.g. using a cationic detergent), ethanol fractionation, cold phenolic extraction (to remove protein) and ultracentrifugation (to remove LPS) (e.g. ref. 138). However, a more preferred method (108) involves precipitation of the polysaccharide 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 (e.g. bromides), or hexadimethrin bromide and myristyltrimethylammonium salts. Cetyltrimethylammonium bromide (CTAB) is particularly preferred (139). 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-methylpropan-1-ol, 2-methylpropan- 2-ol, diols, etc., but ethanol is particularly suitable for the solubilization of CTAB polysaccharide complexes. Ethanol is preferably added to the precipitated polysaccharide to obtain a final concentration (based on the total content of ethanol and water) or from 50% to 95%.

After repeated solubilization, the polysaccharide can be further processed to remove impurities. This is especially important in situations where even a little pollution is unacceptable (for example, for the production of human vaccines). This usually should include one or more filtering steps, for example, depth filtering, activated carbon filtering, size filtering and / or ultrafiltration can be used. After filtration to remove impurities, the polysaccharide can be precipitated for further processing and / or processing. This can suitably be achieved by the exchange of cations (for example, by the addition of calcium or sodium salts).

As an alternative to purification, the capsular saccharides of the present invention can be obtained by full or partial synthesis, for example, Hib synthesis is disclosed in reference 140, and MenA synthesis in reference 141.

The compositions of the invention include capsular saccharides of at least two N. meningitidis serogroups. Saccharides are preferably obtained separately (including any fragmentation, conjugation, modification, etc.), and then mixed to obtain a composition according to the invention.

However, when the composition comprises a capsular saccharide from serogroup A, it is preferred that the serogroup A saccharide is not combined with other saccharide (s) until shortly before use to minimize possible hydrolysis. This can be reasonably achieved by the presence of a component of serogroup A (usually together with appropriate excipients) in lyophilized form, and a component (components) of other serogroups in liquid form (also with corresponding excipients), and the liquid components are used to dissolve the lyophilized component of MenA when ready for use. When an adjuvant in the form of an aluminum salt is used, it is preferable to include the adjuvant in a vial containing a liquid vaccine and lyophilize the MenA component without adjuvant.

Thus, the composition of the invention can be obtained from a kit comprising: (a) a capsular saccharide from N. meningitis serogroup A in lyophilized form; and (b) other antigens from the composition in liquid form. The invention also includes a method for preparing a composition of the invention, comprising mixing a lyophilized capsular saccharide from N. meningitidis serogroup A with other antigens, said other antigens being in liquid form.

The invention also includes a kit comprising: (a) a first container containing capsular saccharides of two or more N.meningitidis serogroups C, W135 and Y, all in lyophilized form; and (b) a second container containing in liquid form (i) a composition which, after administration to an individual, is capable of eliciting a humoral immune response in an individual, wherein the humoral immune response is bactericidal against two or more (e.g. 2 or 3) supervirulent sequences of generations A4, ET-5 and the sequence of generations 3 of N. meningitidis serogroup B, (ii) capsular saccharides from none or from one of the serogroups C, W135 and Y of N. meningitidis, and, optionally (iii) other antigens (see below), which not include meningococcal capsular sugar reads, wherein dissolving the contents of the container (a) with the contents of the container (b) includes a composition of the invention.

Within each dose, the amount of individual saccharide antigen as a whole is from 1 to 50 μg (measured as the mass of saccharide), with each amount preferably about 2.5 μg, 5 μg, or 10 μg. Therefore, with the mass ratios A: C: W135: Y 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, the amount shown in FIG. 1 is preferably about 2.5 μg, 5 μg, or 10 μg. Therefore, for a composition with a ratio of A: C: W135: Y 1: 1: 1: 1 and 10 μg per saccharide, 40 μg of saccharide per dose is administered. Preferably, the compositions have the following μg saccharide per dose:

BUT 10 0 0 0 10 5 2,5 FROM 10 10 5 2,5 5 5 2,5 W135 10 10 5 2,5 5 5 2,5 Y 10 10 5 2,5 5 5 2,5

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

The MenC Menjugate ^™ and NeisVac ^™ conjugates use a hydroxide adjuvant, while Meningitec ^™ uses phosphate. In the compositions of the invention, some antigens can be adsorbed onto aluminum hydroxide, but other antigens may be present in combination with aluminum phosphate. For example, for combinations of a tetravalent serogroup, the following compositions are available:

Serogroup Aluminum salt (H = hydroxide; P = phosphate) BUT P H P H H H P P P H H H P P P H FROM P H H P H H P H H P P H P H P P W135 P H H H P H H P H H P P P P H P Y P H H H H P H H P P H P H P P P

The following compositions are available for combinations of the trivalent serogroup N.meningitidis:

Serogroup Aluminum salt (H = hydroxide; P = phosphate) C P H H H P P P H W135 P H H P H P H P Y P H P H H H P P

Haemophilus influenzae type B

When the composition comprises an H. influenzae type B antigen, it is usually the Hib capsular saccharide antigen. Sugar antigens from H. influenzae b are well known.

Advantageously, Hib saccharide is covalently conjugated to a carrier protein to enhance immunogenicity, especially in children. The preparation of polysaccharide conjugates in general, and Hib capsular polysaccharide in particular, is well documented (e.g., references 142-150, etc.). Any suitable Hib conjugate may be used in the invention. Suitable carrier proteins are described below, and preferred carriers for Hib saccharides are CRM ₁₉₇ ("HbOC"), tetanus toxoid ("PRP-T"), and N.meningitidis outer membrane complex ("PRP-OMP").

The saccharide moiety of the conjugate may be a polysaccharide (e.g., full length polyribosylribitol phosphate (PRP)), but it is preferable to hydrolyze the polysaccharides to form oligosaccharides (e.g., with a molecular weight of from about 1 to about 5 kDa).

A preferred conjugate includes a Hib oligosaccharide covalently linked to CRM ₁₉₇ via an adipic acid linker (151, 152). A tetanus toxoid is also a preferred carrier.

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

The compositions of the invention may include several Hib antigens.

When the composition comprises a Hib saccharide antigen, it is preferable that it also does not include an aluminum hydroxide adjuvant. If the composition includes an aluminum phosphate adjuvant, then the Hib antigen may be adsorbed to the adjuvant (153) or it may not be adsorbed (154).

Hib antigens can be lyophilized, for example, together with meningococcal antigens.

Streptococcus pneumoniae

When the composition comprises an S. pneumoniae antigen, it will usually be a capsular saccharide antigen, which is preferably conjugated to a carrier protein (e.g., references 155-157). It is preferable to include saccharides from several serotypes of S. pneumoniae. For example, mixtures of polysaccharides from 23 different serotypes are widely used, such as conjugate vaccines with polysaccharides from 5-11 different serotypes (158). For example, PrevNar ^™ (159) contains antigens from 7 serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) with each saccharide separately conjugated to CRM ₁₉₇ reductive amination, with 2 μg of each saccharide per dose of 0.5 ml (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. Conjugates can be adsorbed on aluminum phosphate.

As an alternative to using saccharide antigens from pneumococcus, the composition may include one or more polypeptide antigens. There are genomic sequences for several strains of pneumococci (160, 161), and they can be subjected to reverse vaccination (162-165) to identify suitable polypeptide antigens (166, 167). For example, a 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 168. The composition may include several (for example , 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 polypeptide pneumococcal antigens. They can be used in a simple mixture, or the pneumococcal saccharide antigen can be conjugated to the pneumococcal protein. Suitable carrier proteins for such embodiments include the antigens listed in the previous paragraph (168).

Pneumococcal antigens can be lyophilized, for example, together with meningococcal and / or Hib antigens.

Covalent conjugation

Capsular saccharides in the compositions will usually be conjugated to a carrier protein (s). In general, conjugation enhances the immunogenicity of saccharides, since it converts them from T-independent antigens to T-dependent antigens, thus providing priming for immunological memory. Conjugation, in particular, can be used for pediatric vaccines, and it is a well-known technique (for example, reviews in references 169 and 142-150).

Preferred carrier proteins are bacterial toxins or toxoids, such as diphtheria toxoid or tetanus toxoid. Particularly preferred is diphtheria toxoid CRM ₁₉₇ (170-172). Other suitable carrier proteins include the outer membrane protein N.meningitidis (173), synthetic peptides (174, 175), heat shock proteins (176, 177), pertussis proteins (178, 179), cytokines (180), lymphokines (180) , hormones (180), growth factors (180), artificial proteins including multiple human T cell CD4 ⁺ epitopes from antigens derived from various pathogens (181), protein D from H. influenzae (182, 183), pneumococcal surface protein PspA (184), iron capture proteins (185), toxins A or B from C. difficile (186), etc. Preferred carriers are diphtheria toxoid, tetanus toxoid, H. influenzae protein D and CRM ₁₉₇ .

More than one carrier protein may be used within the composition of the invention, for example, to reduce the risk of carrier suppression. Thus, different carrier proteins can be used for different serogroups, for example, serogroup A saccharides can be conjugated to CRM ₁₉₇ , while serogroup C saccharides can be conjugated to a tetanus toxoid. You can also use several carrier proteins for a particular saccharide antigen, for example, serogroup A saccharides can be in two groups, some conjugated to CRM ₁₉₇ , and others conjugated to a tetanus toxoid. However, in general, it is preferable to use the same carrier protein for all saccharides.

A single carrier protein can carry several saccharide antigens (187). For example, a single carrier protein can be conjugated to saccharides from serogroups A and C. To achieve this, saccharides can be mixed before conjugation. However, it is generally preferable to have separate conjugates for each serogroup.

Conjugates with a saccharide: protein ratio (w / w) between 1: 5 (i.e., excess protein) to 5: 1 (i.e., excess saccharide) are preferred. Preferably, the ratio is from 1: 2 to 5: 1, and more preferably the ratio is from 1: 1.25 to 1: 2.5. An excess amount of carrier protein may be preferable for MenA and MenC.

Conjugates can be used in combination with a free carrier protein (188). When a given carrier protein is present in both free and conjugated form in the composition of the invention, the unconjugated form is preferably not more than 5% of the total amount of carrier protein in the composition as a whole, and more preferably is present in an amount of less than 2 wt. .%.

Any suitable conjugation reaction may be used, if necessary with any suitable linker.

The saccharide should usually be activated or functionalized before conjugation. Activation may include, for example, cyanylation reagents such as CDAP (e.g., 1-cyano-4-dimethylaminopyridine tetrafluoroborate (189, 190, etc.)). Other suitable methods use carbodiimides, hydrazides, active esters, norboran, para-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU; see also the introduction to reference 148).

Linkages through a linker group can be obtained using any known procedure, for example, the procedures described in references 191 and 192. One type of linkage involves reductive amination of the polysaccharide, compounding the obtained amino group at one end of the adipic acid linker group, and then linking the protein to the other linker end adipic acid groups (146, 193, 194). Other linkers include B-propionamido (195), nitrophenyl ethylamine (196), haloacyl halides (197), glycosidic bonds (198), 6-aminocaproic acid (199), ADH (200), parts C ₄ -C ₁₂ (201 ) etc. As an alternative to using the linker, you can use a direct link. Direct bonds to the protein may include oxidation of the polysaccharide followed by reductive amination with the protein, as described, for example, in references 202 and 203.

Preferred is a method comprising introducing amino groups into a saccharide (for example, by substituting terminal groups = 0 with —NH ₂ groups) followed by derivatization with an adipic ester (for example, an adipic acid N-hydroxysuccinimido ester) and reaction with a carrier protein. In another preferred reaction, CDAP activation with a carrier protein D is used, for example, MenA or MenC.

After conjugation, free and conjugated saccharides are separated. There are many suitable methods, including hydrophobic chromatography, tangential ultrafiltration, diafiltration, etc. (see also references 204 and 205).

When the composition of the invention comprises a conjugated oligosaccharide, it is preferred that precipitation of the oligosaccharide precedes conjugation.

Complementary and Alternative Serogroup B Polypeptide Antigens

The invention includes a composition which, after administration to an individual, is capable of eliciting a humoral immune response in an individual, the humoral immune response having a bactericidal effect against two or three of the supervirulent rows of generations A4, ET-5 and generation 3 of N. meningitidis serogroup B.

Although NadA, 741, 936, 953, and 287 are preferred antigens for achieving this broad protection, other MenB polypeptide antigens that can be included in compositions of the invention (optionally in combination with one or more of the five major antigens) include antigens comprising one of the following amino acid sequences: SEQ ID NO: 650 of reference 8; SEQ ID NO: 878 from reference 8; SEQ ID NO: 884 from reference 8; SEQ ID NO: 4 from reference 9; SEQ ID NO: 598 from reference 10; SEQ ID NO: 818 from reference 10; SEQ ID NO: 864 from reference 10; SEQ ID NO: 866 from reference 10; SEQ ID NO: 1196 from reference 10; SEQ ID NO: 1272 from reference 10; SEQ ID NO: 1274 from reference 10; SEQ ID NO: 1640 from reference 10; SEQ ID NO: 1788 from reference 10; SEQ ID NO: 2288 from reference 10; SEQ ID NO: 2466 from reference 10; SEQ ID NO: 2554 from reference 10; SEQ ID NO: 2576 from reference 10; SEQ ID NO: 2606 from reference 10; SEQ ID NO: 2608 from reference 10; SEQ ID NO: 2616 from reference 10; SEQ ID NO: 2668 from reference 10; SEQ ID NO: 2780 from reference 10; SEQ ID NO: 2932 from reference 10; SEQ ID NO: 2958 from reference 10; SEQ ID NO: 2970 from reference 10; SEQ ID NO: 2988 from reference 10; or a polypeptide comprising an amino acid sequence that: (a) has 50% or more identity (for example, 60%, 70%, 80%, 90%, 95%, 99% or more) with the indicated sequences; and / or (b) includes a fragment of at least n consecutive amino acids from these sequences, where n = 7 or more (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) include an epitope from a relevant sequence. Several (e.g., 2, 3, 4, 5, 6) of these polypeptides may be included.

General Provisions

The term “comprising” means “comprising” as well as “consisting”, for example, a composition “comprising” X may consist solely of X, or may include something additional, for example, X + Y.

The term “approximately” with respect to a numerical value x means, for example, x ± 10%.

The word “essentially” does not exclude “completely”, for example, a composition that is “substantially lacking” Y can be completely lacking Y. If necessary, the word “essentially” can be omitted from the definition of the invention.

References to percent sequence identity between two amino acid sequences mean that when combined, this percentage of amino acids is the same when comparing two sequences. This alignment and percent homology or sequence identity can be determined using software known in the art, for example, the software described in section 7.7.18 of reference 206. The preferred alignment is determined by the Smith-Waterman homology search algorithm using affinity gap search with a fine for open gap 12 and a fine for widening gap 2, the BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is referred to in reference 207.

The term “alkyl” refers to alkyl groups in both straight and branched forms. The alkyl group can be interrupted by 1, 2 or 3 heteroatoms selected from —O—, —NH— or —S—. The alkyl group can also be interrupted by 1, 2 or 3 double and / or triple bonds. However, the term "alkyl" usually refers to alkyl groups that do not have heteroatomic interruptions or interruptions by double or triple bond. When reference is made to C _1-12 alkyl, this means that the alkyl group can contain any number of carbon atoms from 1 to 12 (for example, C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , C ₁₂ ). Similarly, when reference is made to C _1-6 alkyl, this means that the alkyl group can contain any number of carbon atoms from 1 to 6 (for example, C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ ) .

The term “cycloalkyl” includes cycloalkyl, polycycloalkyl and cycloalkenyl groups, as well as combinations of these groups with alkyl groups such as cycloalkylalkyl groups. The cycloalkyl group may be interrupted by 1, 2 or 3 heteroatoms selected from —O—, —NH— or —S—. However, the term “cycloalkyl” usually refers to a cycloalkyl group that does not have heteroatom “interruptions”. Examples of cycloalkyl groups include cyclopentyl, cyclohexyl, cyclohexenyl, cyclohexylmethyl and adamantyl groups. When reference is made to C _3-12 cycloalkyl, this means that the cycloalkyl group can contain any number of carbon atoms from 3 to 12 (for example, C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , C ₁₂ ).

The term “aryl” refers to an aromatic group such as phenyl or naphthyl. When reference is made to C _5-12 aryl, this means that the aryl group can contain any number of carbon atoms from 5 to 12 (for example, C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , C ₁₂ ).

The term “C _5-12 aryl-C _1-6 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, tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Z or Cbz), 9-fluorenylmethoxycarbonyl (Fm ), 2- (trimethylsilyl) ethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl (Troc)), sulfonyl derivatives (such as β-trimethylsilylethanesulfonyl (SES)), sulfenyl derivatives, C _1-12 alkyl, benzyl, benzhydryl, trityl, 9 phenyl fluorenyl, etc. A preferred nitrogen protecting group is Fmoc.

Sequences included to facilitate cloning or purification, etc., do not necessarily contribute to the invention and may be omitted or deleted.

It should be understood that sugar rings can exist in open and closed form and that although closed forms are shown in structural formulas in the present description, open forms are also encompassed by the invention.

The implementation of the invention

Hybrid Protein ΔG287-953

The DNA coding protein 287 from strain 394/98 of meningococcal serogroup B and the protein 953 from strain 2996 of meningococcal serogroup B were digested and ligated together with a short linker sequence to obtain a plasmid encoding the amino acid sequence of SEQ ID NO 7. The plasmid was transfected into E. coli, and bacteria were grown for protein expression.

After adequate growth, the bacteria were collected and purified protein. Bacteria were centrifuged from the culture, and the precipitate obtained after centrifugation was homogenized in the presence of 50 mM acetate buffer (pH 5) with a ratio between the volumes of sediment and buffer 1: 8. Lysis was performed using a high pressure homogenizer (AVESTIN, 4 cycles at 14,000 psi). After lysis, urea was added at a final concentration of 5M, followed by stirring for 1 h at room temperature. The pH was lowered from 6 to 5 using 200 mM acetate buffer (pH 4) + 5 M urea. The mixture was centrifuged at 16800 g for 60 min at 2-8 ° C. The supernatant was collected and filtered with SARTOBRAN P (0.45-0.22 μm SARTORIUS).

The protein in the filtered supernatant was stable for ≥30 days at -20 ° C and for ≥15 days at 2-8 ° C.

The protein was further purified on a cation exchange column (SPFF, Amersham Biosciences) eluting with 350 mM NaCl + 50 mM acetate + 5 M urea at pH 5.00. Most of the impurities were present in the flowing fluid. Washing before elution using a lower concentration of NaCl (180 mM) predominantly eliminated 2 contaminating E. coli proteins.

The pH of the eluted material was adjusted to 8 (using 200 mM TRIS / HCl + 5 M urea at pH 9) and then purified on a Q Sepharose HP column (Amersham) by elution using 150 mM NaCl + 20 mM TRIS / HCl at pH 8.00 in 5 M urea. Again, washing before elution was used using a lower salt concentration (90 mM) to remove impurities.

Filtered eluted material from an HP Q column was diluted 1: 2 using PBS (saline with phosphate buffer) 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 diafiltration, the material was concentrated 1.6 times to approximately 1.2 mg / ml total proteins. By using a 30,000 Da cut-off membrane (50 cm ² regenerated cellulose membranes, Millipore PLCTK 30), the material could be dialyzed in approximately 90% yield.

Hybrid Protein 936-ΔG741

The DNA coding protein 936 from strain 2996 of meningococcal serogroup B and protein 741 from strain MC58 of meningococcal serogroup B were digested and ligated together with a short linker sequence to obtain a plasmid encoding the amino acid sequence of SEQ ID NO 8. The plasmid was transfected into E. coli and bacteria were grown for protein expression. The recombinant protein was not secreted, but remained soluble inside the bacteria.

After adequate growth, the bacteria were centrifuged to obtain a wet paste and processed as follows:

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

- Centrifugation and clarification by orthogonal filtration.

- Chromatography on a cationic column (SP Sepharose Fast Flow) with elution with 150 mm NaCl in 20 mm sodium phosphate at pH 7.00.

- Chromatography on an anion column (Q Sepharose XL) with flow collection.

- Chromatography on a hydrophobic column (Phenyl Sepharose 6 Fast Flow High Sub) with elution with 20 mm sodium phosphate at pH 7.00.

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

- Final sterilizing filtration and storage at -20 ° C.

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

NadA Protein ^{(NL) (C)}

The NadA DNA encoding protein from strain 2996 of meningococcal serogroup B was digested to remove the C-terminus sequence to obtain a plasmid encoding the amino acid sequence of SEQ ID NO 1. The plasmid was transfected into E. coli and bacteria were grown to express the protein. The recombinant protein was secreted into the culture medium, and the leader peptide was absent in the secreted protein (SEQ ID NO 2). The supernatant was treated as follows:

- Concentration of 7 times and diafiltration against a buffer of 20 mm TRIS / HCl at pH 7.6 by ultrafiltration in a cross flow (cut-off 30 kDa).

- Chromatography on an anion column (Q Sepharose XL) with elution with 400 mm NaCl in 20 mm TRIS / HCl at pH 7.6.

- Chromatography on a hydrophobic column (Phenyl Sepharose 6 Fast Flow High Sub) with elution with 50 mm NaCl in TRIS / HCl at pH 7.6.

- Chromatography on a hydroxyapatite ceramic column (HA Marco. Prep) with elution with 200 mM sodium phosphate at pH 7.4.

- Diafiltration (cut-off 30 kDa) against PBS at pH 7.4.

- Final sterilizing filtration and storage at -20 ° C.

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 mass spectrometry (e.g., MALDI-TOF), indicating a molecular weight loss of up to 10 kDa. Decomposition products were separated from native NadA by gel filtration (for example, using a TSK 300SWXL column, TSKSWXL pre-column, TOSOHAAS). This filtration gives 3 peaks: (i) the first peak with a retention time of 12.637 min and a apparent molecular weight of 885036 Da; (ii) with a retention time of 13.871 min and an apparent molecular weight of 530,388 Da; (iii) with a retention time of 13.871 min and a apparent molecular weight of 530,388 Da. The light scattering analysis of the three peaks reveals the actual molecular weight values: (i) 208,500 Da, (ii) 98,460 Da, (iii) 78,760 Da. Thus, the first peak contains NadA aggregates, and the third peak contains decomposition products.

Since the predicted molecular weight of NadA ^{(NL) (C)} is 34113 Da, peak (ii) contains a trimeric protein, which is the desired antigen.

Antigenic combinations

Mice were immunized with a composition comprising 3 proteins and an aluminum hydroxide adjuvant. For comparison purposes, 3 proteins were also tested separately. Used 10 mice per group. The mixture could cause high bacterial titers against various strains:

Meningococcal strain ^(Serogroup) 2996 ^(B) MS58 ^(B) NGH38 394/98 ^(B) H44 / 76 ^(B) F6124 ^(A) BZ133 ^(C) C11 ^(C) (one) 32000 16000 130,000 16000 32000 8000 16000 8000 (2) 256 131000 128 16000 32000 8000 16000 <4 (3) 32000 8000 - - - 8000 - 32000 Mixture 32000 32000 65,000 16000 260,000 65,000 > 65000 8000 “-” indicates that this strain does not contain the NadA gene

Examining individual mice, the triple mixture caused high and consistent bacterial titers against the three strains of serogroup B, from which individual antigens were obtained:

# one 2 3 four 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

Combination and comparison with OMV

In further experiments, adjuvanted antigens (20 μg of each antigen per dose) were administered in combination with 10 μg of OMV obtained from either strain H44 / 76 (Norway) or strain 394/98 (New Zealand). Positive controls were the anticapsular antibody SEAM-3 mAb for serogroup B or the capsular saccharides conjugated with CRM197 for other strains. The results (bacterial titers) are shown in table 1. The mixture almost always gives better titers than simple OMV, and in addition, adding the mixture to OMV almost always significantly increases the effectiveness of OMV. Moreover, in many cases, a mixture of antigens corresponds or exceeds the reaction observed in a positive control.

Tests of supervirulent generations

The following antigens were tested against various strains of serogroup B from various supervirulent generations:

(a) NadA ^{(NL) (C)}

(b) ΔG287-953

(c) 936-ΔG741

(d) a mixture of (a), (b) and (c)

(e) OMV obtained from strain H44 / 76 (Norway)

(f) OMV obtained from strain 394/98 (New Zealand)

(g) A mixture of ΔG287 and (e)

(h) A mixture of (d) and (e)

(i) A mixture of (d) and (f)

SEAM-3 was used as a positive control.

The following results were obtained, expressed as the percentage of strains in the indicated supervirulent sequence of generations, where the serum bactericidal titer exceeded 1024:

strains (a) (b) (c) (d) (e) (f) (g) (h) (i) S-3 A4 four fifty fifty 0 one hundred 25 25 25 one hundred one hundred + ET-5 8 25 75 88 one hundred 71 fourteen 71 one hundred one hundred + Generation Sequence 3 13 0 75 fifteen 93 8 85 8 92 93 + ET-37 four eleven 22 0 33 0 0 0 22 25 +

The following bactericidal titers were obtained against specific reference strains:

strains (a) (b) (c) (d) (e) (f) (g) (h) (i) S-3 A4 961-5945 128 2048 <8 2048 262144 8192 262144 262144 4096 8192 ET-5 44/76 <4 2048 32768 131072 524288 8192 524288 524288 524288 16384 Generation Sequence 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

Therefore, compositions (d), (h) and (i) elicit bactericidal humoral immune responses against a wide variety of serogroup B meningococcus strains within the supervirulent sequences of generations A4, ET-5 and generation sequence 3. Captions using compositions (h) and (i ) were generally higher than when using (d), but the coverage of the strains within the supervirulent sequences of generations A4, ET-5 and sequences of generations 3 was not better.

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

NadA N-End Domain Analysis

It is known that the purified N. meningitidis NadA protein binds to human epithelial cells (17) (e.g., Chang cells, HeLa cells, Hep-2 cells), and recombinant E. coli that express NadA exhibit an attachable phenotype (18). These E. coli are also able to invade epithelial cells, and intracellular NadA ^{+ ve} E. coli can be detected in Chang cells by immunofluorescence (after induced membrane permeability) and electron microscopy. Therefore, it is believed that NadA functions as adhesin and invasin for epithelial cells.

Based on the analysis of the secondary structure, mature NadA was subdivided into 3 putative domains: the N-terminal globular domain (aa 24-87), the α-helical inner region (aa 88-350) with a high tendency to twisted spirals and the C-terminal membrane anchor ( aa 351-405). The role of the N-terminal globular domain in the interaction of the host cell was investigated.

The truncated nadA gene encoding a protein lacking amino acids 30-87 was cloned into the pET-21 vector (pET-NadAΔ30-87) and expressed in E. coli strain BL21 (DE3). Amino acids 24-29 were retained to allow processing of the leader peptide and correction of protein maturation. Analysis of Western blot and FACS (laser cell analyzer by fluorescence intensity) confirmed that NadAΔ30-87 was expressed and formed oligomers on the cell surface of E. coli, i.e. a deletion of the N-terminal domain does not interfere with the expression, export, and membrane localization of NadA. However, the recombinant E. coli strain completely lost its ability to adhere to Chang epithelial cells. Thus, the N-terminal domain is involved in adhesin activity.

For further investigation of which part of the N-terminal domain is involved in the interaction, the region was further divided into 3 putative subdomains: amino acids 24-42 containing the predicted α-helical region with hydrophobic residues; amino acids 43-70, the inner part without a predicted specific secondary structure; and amino acids 71-87 containing another predicted α-helical structure. 3 constructs were created, each coding protein with a deletion of one subdomain, and then introduced into E. coli BL21 (DE3), obtaining the following strains: BL21 (DE3) / pET-NadAΔ24-42, BL21 (DE3) / pET-NadAΔ43-70 and BL21 (DE3) / pET-NadAΔ71-87. Superficial localization of oligomers was confirmed by Western blotting and FACS analysis, but adherence to Chang epithelial cells was not better than that of the control E. coli BL21 (DE3) / pET strain. These results, also confirmed using immunofluorescence microscopy analysis, indicate that the entire globular N-terminal NadA domain is important when interacting with human cells.

Combination with Meningococcus and / or Hib conjugates

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

Component Quantity per dose 0.5 ml Serogroup C conjugate 10 mcg saccharide + 12.5-25 mcg CRM ₁₉₇ Serogroup conjugate W135 10 mcg saccharide + 6.6-20 mcg CRM ₁₉₇ Serogroup Y conjugate 10 mcg saccharide + 6.6-20 mcg CRM ₁₉₇ ΔG287-953 20 mcg polypeptide 936-ΔG741 20 mcg polypeptide Nada 20 mcg polypeptide

A similar vaccine is prepared comprising the MenB conjugate (10 μg saccharide + 12.5-33 μg CRM ₁₉₇ ) and / or the HbOC Hib conjugate (10 μg saccharide + 2-5 μg CRM ₁₉₇ ).

Using Modified MenA Saccharide

The capsular polysaccharide was purified from MenA and hydrolyzed to obtain 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 (135). After hydrolysis, the solution was dried by rotary evaporation.

The oligosaccharide was activated using the following reaction scheme:

The oligosaccharide was dissolved in DMSO (dimethyl sulfoxide) to give a saccharide concentration of 10 mg / ml. According to a molar ratio between the oligosaccharide and CDI of 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. According to calculations by determining the ratio between free imidazole and bound imidazole, the reaction activation efficiency was approximately 67.9%.

In a second reaction step, the MenA-CDI oligosaccharide was solubilized in DMSO at a saccharide concentration of about 10 mg / ml. In accordance with a molar ratio of 1: 100 between MenA-CDI and DMA (dimethylamine), 36.288 g of 99% dimethylamine hydrochloride (i.e., R ¹ and R ² = Me) was added and the reaction mixture was stirred for 16 hours at room temperature. The reaction product was lyophilized and redissolved in an aqueous solution at a concentration of 10 mg / ml.

To remove the interaction of the reagent with a low molecular weight (in particular, dimethylamine (DMA)) from the oligosaccharide preparation step was performed through a dialysis membrane MWCO 3,5 kDa (Spectra / Por ^TM). There were 4 stages of dialysis: (i) 16 hours against 2 L of 1 M sodium chloride (dialysis factor 1:20), (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 (dialysis factor 1:20). To improve purification, a diafiltration step through a 1 kDa MWCO membrane (Centricon ^™ ) was also performed.

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

To obtain conjugates of the modified MenA saccharide (MenA-CDI-DMA), in general, the method was as follows:

- hydrolysis of the polysaccharide to obtain oligosaccharide fragments

- size classification of oligosaccharide fragments

- reductive amination of terminal aldehyde groups on sized oligosaccharides

- protection of the -NH ₂ end groups by the Fmoc group before the CDI reaction

- endogenous deprotection of -NH ₂ groups during the DMA reaction

-activation of the terminal groups -NH ₂ SIDEA (N-hydroxysuccinimidadipic acid)

- covalent attachment to protein CRM ₁₉₇

The modified MenA oligosaccharide conjugate is 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, compared with 23.5% for the natural antigen. Moreover, titers caused by modified oligosaccharides are not significantly lower than titers obtained using native sugar structures.

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

Pneumococcal combinations

3 combined MenB proteins are mixed with pneumococcal saccharide conjugates to obtain a final concentration of 2 μg / dose of each of the pneumococcal serotypes (double for serotype 6B). Thus, a dissolved vaccine contains the following antigens:

Component Quantity per dose 0.5 ml Serogroup A conjugate 5 μg saccharide + 6.25-16.5 μg CRM ₁₉₇ Serogroup C conjugate 5 μg saccharide + 6.25-12.5 μg CRM ₁₉₇ Serogroup conjugate W135 5 μg saccharide + 3.3-10 μg CRM ₁₉₇ Serogroup Y conjugate 5 μg saccharide + 3.3-10 μg CRM ₁₉₇ Serotype 4 pneumococcus conjugate 2 μg saccharide + 2.5 μg CRM ₁₉₇ Conjugate pneumococcus serotype 9V 2 μg saccharide + 2.5 μg CRM ₁₉₇ Conjugate pneumococcus serotype 14 2 μg saccharide + 2.5 μg CRM ₁₉₇ Pneumococcus conjugate serotype 18C 2 μg saccharide + 2.5 μg CRM ₁₉₇ Pneumococcus conjugate serotype 19F 2 μg saccharide + 2.5 μg CRM ₁₉₇ Pneumococcus conjugate serotype 23F 2 μg saccharide + 2.5 μg CRM ₁₉₇ Conjugate pneumococcus serotype 6B 4 μg saccharide + 5 μg CRM ₁₉₇

It should be understood that the invention has been described only as an example and modifications can be made without going beyond the scope and essence of the invention.

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Claims (30)

1. A composition which, after administration to an individual, is capable of inducing a humoral immune response in a given individual, wherein the humoral immune response has a bactericidal effect against two or more supervirulent rows of generations A4, ET-5 and a series of generations 3 of N.meningitidis serogroup B, where the composition contains five meningococcal antigens: (1) a "NadA" protein represented by the sequence of SEQ ID NO 1 or a sequence that is 85% or more identical to SEQ ID NO 2; (2) the protein "741" represented by a sequence that is 85% or more identical to SEQ ID NO 3; (3) the protein "936" represented by a sequence that is 85% or more identical to SEQ ID NO 4; (4) the protein "953" represented by a sequence that is 85% or more identical to SEQ ID NO 5; and (5) the protein "287" represented by a sequence that is 85% or more identical to SEQ ID NO 6. 2. The composition according to claim 1, in which components that induce a bactericidal humoral immune response obtained by recombinant expression. 3. Composition for inducing a humoral immune response in a mammal, containing five meningococcal antigens: (1) protein "NadA"; (2) the protein "741"; (3) protein "936"; (4) protein "953" and (5) protein "287". 4. The composition according to claim 3, in which the NadA protein has an identity of 85% or more with SEQ ID NO 2. 5. The composition according to claim 4, in which the NadA protein comprises SEQ ID NO 2. 6. The composition according to claim 3, in which the protein 741 has an identity of 85% or more with SEQ ID NO 3. 7. The composition according to claim 6, in which the protein 741 includes SEQ ID NO 3. 8. The composition according to claim 3, in which the protein 936 has an identity of 85% or more with SEQ ID NO 4. 9. The composition of claim 8, in which protein 936 includes SEQ ID NO 4. 10. The composition according to claim 3, in which the protein 953 has an identity of 85% or more with SEQ ID NO 5. 11. The composition of claim 10, in which protein 953 comprises SEQ ID NO 5. 12. The composition according to claim 3, in which the protein 287 has an identity of 85% or more with SEQ ID NO 6. 13. The composition according to item 12, in which protein 287 includes SEQ ID NO 6. 14. The composition according to claim 3, in which at least two of the antigens (1) through (5) are expressed as a single polypeptide chain. 15. The composition according to claim 3, containing a polypeptide that comprises a pair of antigens within the same polypeptide chain, selected from the group consisting of: 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. 16. The composition according to claim 3, containing a polypeptide of the formula NH ₂ -A - [- XL-] _n -B-COOH, where X is the amino acid sequence of one of the five antigens from (1) to (5); L is an optional linker amino acid sequence; A is an optional N-terminal amino acid sequence; B is an optional C-terminal amino acid sequence and n = 2, 3, 4 or 5. 17. The composition according to clause 16, in which n = 2, X ₁ represents a protein 936, and X ₂ represents a protein 741. 18. The composition according to clause 16, in which n = 2, X ₁ represents a protein 287, and X ₂ represents a protein 953. 19. The composition according to claim 3, containing a protein comprising SEQ ID NO 7. 20. The composition according to claim 3, containing a protein comprising SEQ ID NO 8. 21. The composition according to claim 3, additionally containing saccharide antigens from serogroups of meningococcus Y, W135, C and, optionally, A. 22. The composition according to claim 3, additionally containing saccharide antigens from Haemophilus influenzae type B. 23. The composition according to item 21 or 22, in which the saccharide antigen is conjugated to a carrier selected from: diphtheria toxoid, tetanus toxoid, CRM ₁₉₇ or protein D. H. influenzae. 24. The composition of claim 3, further comprising an antigen from Streptococcus pneumoniae. 25. The composition according to claim 3 for use as a medicine. 26. The use of a composition according to any one of the preceding paragraphs in the manufacture of a medicament for preventing and / or treating a disease caused by Neisseria. 27. A method of inducing a humoral immune response in a mammal, comprising the step of administering an effective amount of a composition according to any one of claims 1 to 26. 28. A polypeptide for inducing a humoral immune response in a mammal having an amino acid sequence selected from the group consisting of SEQ ID NOs from 1 to 8. 29. A method for purifying soluble NadA from a culture medium, comprising the steps of concentration and diafiltration against an ultrafiltration buffer; anion column chromatography; hydrophobic column chromatography; hydroxyapatite ceramic column chromatography; diafiltration against buffer and sterilization through a filter. Priority on points: 10/11/2002 to 03/13/2003 and 04/22/2003 according to claims 1-29.