MENINGOCOCCAL POLYPEPTIDES fHbp MODIFIED

专利摘要:
The inventors have identified residues in variant 2 and variant 3 of meningococcal fHbp which can be modified to improve their properties.
公开号:BE1022641B1
申请号:E2015/5456
申请日:2015-07-16
公开日:2016-06-23
发明作者:Matthew Bottomley；Vega Masignani
申请人:Glaxosmithkline Biologicals Sa；
IPC主号:

专利说明:

MENINGOCOCCAL POLYPEPTIDES fHbp MODIFIED TECHNICAL FIELD
This invention is in the field of protein engineering, and particularly relates to the factor H-binding meningococcal protein (fHbp), which is known to be a useful vaccine immunogen.
CONTEXT
Neisseria meningitidis is a Gram-negative capsulated bacterium that colonizes the upper respiratory tract of about 10% of the human population. Conjugate vaccines are available against serogroups A, C, W135 and Y, but the only vaccine available to protect against serogroup B in general is BEXSERO ™, which was approved in 2013.
One of the protective immunogens in BEXSERO ™ is fHbp, also known as "741" protein (SEQ ID NO: 2536 in ref 1, SEQ ID 1 herein), "NMB1870", "GNA1870" [2-4], "P2086", "LP2086" or "ORF2086" [5 7]. The 3D structure of this protein is known [8, 9], and has two ß barrels joined by a short linker ("linker"). Many publications have reported the protective efficacy of this protein in meningococcal vaccines, see p. ex. references 10-14. The lipoprotein fHbp is expressed in various strains covering all serogroups. The fHbp sequences have been grouped into three variants [2] (designated v1, v2 and v3 herein), and it has been found generally that a serum directed against a given variant is bactericidal to strains expressing that variant. variant, but is not active against strains that express one of the other two variants, ie, there is cross-protection within the variant, but no cross-protection between variants (except for a few cases of cross-reactivity) v2 and v3).
To increase inter-family cross-reactivity, the fHbp sequence was re-engineered to contain specificities related to the three variants [15]. Protein engineering was also used to eliminate the interaction of fHbp with siderophores [16] and with human factor H [17-25]. The breakdown of fH interaction has been reported for all three variants and is postulated to obtain a higher immunogen immunogen [22, 26]. For v2 polypeptides, however, references 23 and 24 report inherent instability that is also observed in mutants with broken fH binding. Instability appears to be coming from the N-terminal β-barrel domain, and reference 23 warns that any substitution in this barrel may promote instability. Mutations to improve the stability of v2 sequences are described in reference 27.
One of the objects of the invention is to propose mutants v2 and v3 of fHbp having improved properties.
SUMMARY OF THE INVENTION
In one aspect, the present invention relates to a polypeptide comprising an amino acid sequence having a sequence identity of at least 80% with SEQ ID NO: 5, wherein the amino acid sequence differs from SEQ ID NO: 5 at residues 123 and 240 relative to SEQ ID NO: 5, and the polypeptide may, after administration to a human subject, elicit the production of antibodies capable of recognizing a polypeptide consisting of SEQ ID NO: 4.
In another aspect, the invention is a polypeptide comprising an amino acid sequence having a sequence identity of at least 80% with SEQ ID NO: 5, wherein the amino acid sequence differs from SEQ ID NO: At residues 32, 123 and 240 relative to SEQ ID NO: 5, and the polypeptide may, after administration to a human subject, elicit the production of antibodies capable of recognizing a polypeptide consisting of SEQ ID NO: 4 .
In another aspect, the invention is a polypeptide comprising an amino acid sequence having a sequence identity of at least 80% with SEQ ID NO: 17, wherein the amino acid sequence differs from SEQ ID NO: 17 at residues 126 and 243 relative to SEQ ID NO: 17, and the polypeptide may, after administration to a human subject, elicit the production of antibodies capable of recognizing a polypeptide consisting of SEQ ID NO: 40.
According to another aspect, the invention is a polypeptide comprising an amino acid sequence having a sequence identity of at least 80% with SEQ ID NO: 17, wherein the amino acid sequence differs from SEQ ID NO At residues 32, 126 and 243 relative to SEQ ID NO: 17, and the polypeptide may, after administration to a human subject, elicit the production of antibodies capable of recognizing a polypeptide consisting of SEQ ID NO: 40 .
In another aspect, the invention is a polypeptide comprising an amino acid sequence having a sequence identity of at least 90% with SEQ ID NO: 47, wherein with respect to SEQ ID NO: 47, the residue 123 is not leucine and residue 240 is not glutamate; and wherein, when administered to a human subject, the polypeptide may elicit an antibody response that is bactericidal to a meningococcus that expresses fHbp v2.
In another aspect, the invention is a polypeptide comprising an amino acid sequence having a sequence identity of at least 90% with SEQ ID NO: 47, wherein with respect to SEQ ID NO: 47, the residue 32 is not serine, residue 123 is not leucine and residue 240 is not glutamate; and wherein, when administered to a human subject, the polypeptide may elicit an antibody response that is bactericidal to a meningococcus that expresses fHbp v2.
In another aspect, the invention is a polypeptide comprising an amino acid sequence having a sequence identity of at least 90% with SEQ ID NO: 48, wherein with respect to SEQ ID NO: 48, the residue is not leucine and residue 243 is not glutamate; and wherein, when administered to a human subject, the polypeptide may elicit an antibody response that is bactericidal to a meningococcus that expresses fHbp v3.
In another aspect, the invention is a polypeptide comprising an amino acid sequence having a sequence identity of at least 90% with SEQ ID NO: 48, wherein with respect to SEQ ID NO: 48, the residue 32 is not serine, residue 126 is not leucine and residue 243 is not glutamate; and wherein, when administered to a human subject, the polypeptide may elicit an antibody response that is bactericidal to a meningococcus that expresses fHbp v3.
In another aspect, the invention relates to fusion polypeptides which, when administered to a human subject, elicit an antibody response which is bactericidal to both a meningococcus that expresses fHbp v2 and a meningococcus that expresses a fHbp v3.
In another aspect, the invention is an immunogenic composition comprising a pharmaceutically acceptable carrier and a fusion polypeptide or polypeptide of the invention.
In another aspect, the invention is a method for eliciting an antibody response in a human subject, comprising administering to said human subject an immunogenic composition of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the SPR response of wild-type fHbp v 2 (top curve), mutant E266 of v2 (bottom curve) and mutant S58V / L149R of v2 (middle curve) bound to immobilized fH. The y-axis indicates relative units, and the x-axis the time (seconds, 0 corresponding to the injection of the sample).
Figure 2 shows the DSC results of wild type fHbp v2 and S58V / L149R mutant of v2. The C-terminal domain is not affected by the mutation, but the Tm of the N-terminal domain has increased by> 20 ° C (shown by the arrow). The y-axis indicates the Cp (kcal / mol / ° C), and the x-axis, the temperature (° C).
Figure 3 shows the DSC results of wild-type fHbp v2 and E266A mutant of v2. The N-terminal transition disappears in the mutant, but the Tm of the C-terminal domain increased by> 16 ° C.
Figure 4 shows the SPR response of the "wild-type" fHbp v2-v3-v1 fusion polypeptides (top curve, dotted line) and the "SNB mutant" (bottom curve, solid line).
Figure 5 shows the DSC results of the "wild-type" and (B) "SNB mutant" fHbp v2-v3-v1 (A) fusion polypeptides.
Figure 6 shows the SPR response of fHbp v3, either in its wild-type (high) form, or carrying various mutations.
Figure 7 shows the structures of fHbp v2 (Figure 7a) and v3 (Figure 7b) determined by X-ray crystallography in the absence of factor H after stabilization of fHbp using mutations S58 and L149.
Figure 8 compares the stability of the "wild-type" (SEQ ID NO: 18) fusion with that of the non-binding stabilized fusion (SEQ ID NO: 27) in extracts of E. coli analyzed by Western blot transfer. Figure 8a shows that truncated forms of non-binding stabilized fusion are less prevalent. Figure 8b demonstrates that stabilized, non-binding fusion is less prone to cleavage by chymotrypsin than "wild-type" fusion.
Figure 9 is a schematic representation of the 231 SNB merger.
DETAILED DESCRIPTION
The full length fHbp from strain 2996 in v2 has the following amino acid sequence (SEQ ID NO: 2):
MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQS VRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQ IYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKA FSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRY GSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ
Mature lipoprotein lacks the first 19 amino acids of SEQ ID NO: 2 (underlined, gives SEQ ID NO: 4, starting with Cys-20). It is also known to produce an "AG" form of fHbp in which the N-terminus is truncated to residue 26 (i.e., which removes the poly-glycine portion and begins instead with Val-27), thus giving SEQ ID NO: 5.
The full length fHbp from strain M1239 in v3 has the following amino acid sequence (SEQ ID NO: 3):
MNRTAFCCLSLTTALILTACSSGGGGSGGGGVAADIGTGLADALTAPLDHKDKGLKSL TLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTI TLASGEFQIYKQNHSAWALQIEKINNPDKTDSLINQRSFLVSGLGGEHTAFNQLPGG KAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTLEQNVELAAAELKADEKSHA VILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ
Mature lipoprotein lacks the first 19 amino acids of SEQ ID NO: 3 (underlined, gives SEQ ID NO: 40) and the AG form of SEQ ID NO: 3 is devoid of the first 31 amino acids (SEQ ID NO: 17) .
The inventors have studied two different types of mutations in v2 and v3. First, they identified residues in SEQ ID NO: 2 and SEQ ID NO: 3 that can be modified to increase the stability of the polypeptide. Second, they identified residues that reduce binding to human factor H (fH). The invention relates to fHbp polypeptide mutants carrying both types of mutations with improved properties, thereby obtaining fHbp polypeptides having improved properties. More specifically, fHbp mutants that do not bind H-factor but retain immunogenicity are advantageous because the resulting antibody responses are directed against epitopes on or near the fH binding site. After vaccination with wild-type fHbp vaccine antigens, these epitopes can be masked by factor H binding.
The amino acids with the most interest, numbered according to the full-length sequences (SEQ ID NO: 1 & 3) and also according to the AG sequences (SEQ ID NO: 5 & 17) are as follows:
When only one of these residues is mutated, the preference is for leucine
Mutant fHbp v2
Therefore, according to a first aspect, the invention relates to a polypeptide comprising a mutant fHbp v2 amino acid sequence, wherein: (a) the amino acid sequence has at least a sequence identity of at least k% with SEQ ID NO: 5, and / or comprises a fragment of SEQ ID NO: 5; but (b) the amino acid sequence differs from SEQ ID NO: 5 at residues L123 and E240 (and optionally, at residue S32) (amino acids numbered with respect to SEQ ID NO: 5).
When feature (a) relates to a fragment, the fragment will contain the two (or possibly the three) residues indicated in (b), but these residues will differ in terms of positions relative to SEQ ID NO: 5. An acid sequence The mutant fHbp v2 amino may have a sequence identity of at least k% with SEQ ID NO: 5, and include several fragments thereof, each of these fragments being at least 7 amino acids in length. These fragments will typically contain at least one epitope from SEQ ID NO: 5. Epitope identification and mapping is established for fHbp [11; 28-32].
The value of k may be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more. It is preferably 90 (i.e., the mutant fHbp v2 amino acid sequence has an identity of at least 90% with SEQ ID NO: 5) and more preferably 95.
The polypeptide may, after administration to a suitable host animal (such as a mouse or a human subject), elicit the production of antibodies capable of recognizing a wild-type meningococcal polypeptide consisting of SEQ ID NO: 4. Such antibodies will include certain antibodies that do not recognize a v1 or v3 polypeptide (eg that will not recognize a wild-type meningococcal polypeptide consisting of SEQ ID NO: 46 and a wild-type meningococcal polypeptide consisting of SEQ ID NO: 40 ), although they may also include certain antibodies cross-reactive with v1 and / or v3 polypeptides. Ideally, the antibodies are bactericidal to a meningococcal strain that expresses a fHbp v2, p. ex. towards strain M2091 (see below).
The polypeptide has, under the same experimental conditions, a greater stability to the same polypeptide but without the sequence differences indicated in (b), p. ex. a higher stability than a wild-type meningococcal polypeptide consisting of SEQ ID NO: 4. The improvement in stability can be evaluated by differential scanning calorimetry (DSC), e.g. ex. as described in references 33 & 34. DSC has previously been used to evaluate the stability of fHbp v2 [24]. Suitable conditions for assessing DSC stability may include 20 μl of the polypeptide in a buffered solution (eg, 25 mM Tris) at a pH of between 6 and 8 (eg, 7-7.5) with 100-200. mM NaCl (e.g., 150 mM).
Ideally, the increase in stability is at least 5 ° C, e.g. ex. at least 10 ° C, 15 ° C, 20 ° C, 25 ° C, 30 ° C, 35 ° C or higher. These temperatures refer to the increase in the midpoint (Tm) of the thermal transition, as evaluated by DSC. The wild-type fHbp has two DSC peaks during the deployment (one corresponding to the N-terminal domain and one corresponding to the C-terminal domain) and, when a polypeptide according to the invention contains these two domains, the increase refers to the N-terminal domain stability, which may appear even below 40 ° C with wild-type v2 sequences [24] (whereas C-terminal domains may have a Tm of 80 ° C or higher). Therefore, the amino acid sequence of the mutant fHbp v2 according to the invention preferably has an N-terminal domain having a Tm of at least 45 ° C, e.g. ex. > 50 ° C,> 55 ° C,> 60 ° C,> 65 ° C,> 70 ° C,> 75 ° C, or even> 80 ° C.
In addition to this increased stability, the polypeptide has, under the same experimental conditions, a lower affinity for human fH than the same polypeptide but without the sequence differences indicated in (b), p. ex. a lower affinity than a wild-type meningococcal polypeptide consisting of SEQ ID NO: 4. The alteration of the affinity can be quantitatively evaluated by surface plasmon resonance (SPR), e.g. ex. as described in references 18 and 21-24 with immobilized human fH. A reduction in affinity (i.e., an increase in dissociation constant, Kd) of at least 10 fold, and ideally at least 100 fold, is preferred.
In some embodiments, the polypeptide is truncated with respect to SEQ ID NO: 5. Compared to the mature wild-type sequence, SEQ ID NO: 5 is already truncated at its N-terminus to, and including, the polyglycine sequence (compare SEQ ID NO: 4 and 5), but SEQ ID NO: 5 may be truncated at its C-terminus and / or further truncated at its N-terminus. .
Mutant fHbp v3
According to a second aspect, the invention relates to a polypeptide comprising a mutant fHbp v3 amino acid sequence, wherein: (a) the amino acid sequence has a sequence identity of at least 1% with SEQ ID NO: 17, and / or comprises a fragment of SEQ ID NO: 17; but (b) the amino acid sequence differs from SEQ ID NO: 17 at residues L126 and E243 (and optionally, at residue S32) (amino acids numbered with respect to SEQ ID NO: 17).
When feature (a) relates to a fragment, the fragment will contain the two (or possibly the three) residues indicated in (b), but these residues will differ in terms of positions relative to SEQ ID NO: 17. An acid sequence The mutant fHbp v3 amino may have at least one sequence identity with SEQ ID NO: 17, and include several fragments thereof, each of these fragments being at least 7 amino acids in length. These fragments will typically contain at least one epitope from SEQ ID NO: 17. Epitope identification and mapping are established for fHbp [11; 28-32].
The value of j may be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more. It is preferably 90 (i.e., the amino acid sequence, mutant fHbp v3 has an identity of at least 90% with SEQ ID NO: 17) and more preferably 95.
The polypeptide may, after administration to a suitable host animal (such as a mouse or a human subject), elicit the production of antibodies capable of recognizing a wild-type meningococcal polypeptide consisting of SEQ ID NO: 40. These antibodies will include certain antibodies which do not recognize a v1 or v2 polypeptide (e.g., which will not recognize a wild-type meningococcal polypeptide consisting of SEQ ID NO: 46 and a wild-type meningococcal polypeptide consisting of SEQ ID NO: 4), although they may also include certain antibodies cross-reactive with v1 and / or v2 polypeptides. Ideally, the antibodies are bactericidal to a meningococcal strain that expresses a fHbp v3, p. ex. towards strain M01-240355 (see below).
The polypeptide has, under the same experimental conditions, a greater stability to the same polypeptide but without the sequence differences indicated in (b), p. ex. superior stability to a wild-type meningococcal polypeptide consisting of SEQ ID NO: 40. The improvement in stability can be evaluated by differential scanning calorimetry (DSC), e.g. ex. as described in references 33 & 34. DSC has previously been used to evaluate the stability of fHbp v3 [23]. Suitable conditions for assessing DSC stability may include 20 μl of the polypeptide in a buffered solution (eg, 25 mM Tris) at a pH of between 6 and 8 (eg, 7-7.5) with 100-200. mM NaCl (e.g., 150 mM).
Ideally, the increase in stability is at least 5 ° C, e.g. ex. at least 10 ° C, 15 ° C, 20 ° C, 25 ° C, 30 ° C, 35 ° C or higher. These temperatures refer to the increase in the midpoint (Tm) of the thermal transition, as evaluated by DSC. The wild-type fHbp has two DSC peaks during the deployment (one corresponding to the N-terminal domain and one corresponding to the C-terminal domain) and, when a polypeptide according to the invention contains these two domains, the increase refers to the N-terminal domain stability, which may occur in the vicinity of 60 ° C or less with wild-type v3 sequences [24] (whereas C-terminal domains may have a Tm of 80 ° C or higher). Therefore, the amino acid sequence of the mutant fHbp v3 according to the invention preferably has an N-terminal domain having a Tm of at least 65 ° C. ex. > 70 ° C,> 75 ° C, or> 80 ° C.
In addition to this increased stability, the polypeptide has, under the same experimental conditions, a lower affinity for human fH than the same polypeptide but without the sequence differences indicated in (b), p. ex. a lower affinity than a wild-type meningococcal polypeptide consisting of SEQ ID NO: 40. The alteration of affinity can be quantitatively evaluated by surface plasmon resonance (SPR), e.g. ex. as described in references 18 and 21-24 with immobilized human fH. A reduction in affinity (i.e., an increase in dissociation constant, Kd) of at least 10 fold, and ideally at least 100 fold, is preferred.
In some embodiments, the polypeptide is truncated with respect to SEQ ID NO: 17. Compared to the mature wild-type sequence, SEQ ID NO: 17 is already truncated at its N-terminus to, and including, the polyglycine sequence (compare SEQ ID NO: 40 and 17), but SEQ ID NO: 17 may be truncated at its C-terminus and / or further truncated at its N-terminus.
Relative Mutations Φ SEQ ID NO: 5 • The polypeptides according to the first aspect of the invention comprise an amino acid sequence having an identity of at least k% with SEQ ID NO: 5, and / or comprising a fragment of SEQ ID NO: 5. Compared with SEQ ID NO: 5, however, this amino acid sequence has a modification at least at amino acid residues L123 and E240 (and optionally also at residue S32). These residues are numbered according to SEQ ID NO: 5; to coincide with the nascent wild-type sequence (SEQ ID NO: 2), the numbering should include +26 (i.e. Ser-32 in SEQ ID NO: 5 is Ser-58 in SEQ ID NO: 2), and to coincide with the mature wild-type sequence (SEQ ID NO: 4), the numbering should include +7 (which also allows easy comparison with ref 25).
The three specified residues may be deleted, but preferably they are substituted with another amino acid. For example, Leu-123 may be substituted with any of the other 19 naturally occurring amino acids. When a substitution is made, the substitution amino acid may in some embodiments be a single amino acid such as glycine or alanine. In other cases, the substitution amino acid is a conservative substitution, p. ex. it is carried out within the following four groups: (1) acids, namely aspartate, glutamate; (2) basic, namely lysine, arginine, histidine; (3) non-polar, namely alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar, namely glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. In other embodiments, the substitution is non-conservative.
Preferred substitutions at the specified residues are: S32V; L123R; and E240A.
In addition to the mutation (s) indicated above, aimed at increasing the stability and altering the ability of the polypeptide to bind to fH, a polypeptide may contain one or more other mutations, e.g. ex. to alter the interaction of the polypeptide with siderophores. Residues that interact with siderophores can be mutated, following the guidelines in references 16 and 35, p. ex. aligning SEQ ID NO: 5 herein with SEQ ID NO: 4 of reference 16 to identify residues that may interact with siderophores, e.g. ex. with catecholates, hydroxamates or carboxylates.
Reference 24 indicates that certain substitutions in v2 may increase its affinity for fH, and therefore these will generally be avoided, eg, ex. E85 in SEQ ID NO: 5 (residue 157 in ref 24). Other residues may also be mutated provided that, compared to the wild-type sequence (eg, SEQ ID NO: 4), the polypeptide has higher stability, lower affinity for fH, and when it is present. administered to a suitable mammal, it may elicit an antibody response that is bactericidal to meningococcus.
The polypeptide according to the first aspect may comprise SEQ ID NO: 47. In SEQ ID NO: 47, residue 32 is any amino acid, residue 123 is not leucine, and residue 240 is not glutamate. Another option is SEQ ID NO: 39, where residue 32 is not serine, residue 123 is not leucine, and residue 240 is not glutamate. In a preferred embodiment of SEQ ID NO: 47, residue 32 is valine, residue 123 is arginine, and residue 240 is alanine (ie, SEQ ID NO: 50). In another preferred embodiment of SEQ ID NO: 47, residue 32 is serine, residue 123 is arginine, and residue 240 is alanine (ie, SEQ ID NO: 53).
The polypeptide according to the first aspect may comprise SEQ ID NO: 31. In SEQ ID NO: 31, residue 32 is any amino acid, and residue 123 is not leucine. Another option is SEQ ID NO: 37, where residue 32 is not serine, and residue 123 is not leucine. In a preferred embodiment of SEQ ID NO: 31, residue 32 is
valine, and the residue 123 is arginine (ie, SEQ ID NO: 45). In another preferred embodiment of SEQ ID NO: 31, residue 32 is serine, and residue 123 is arginine (ie, SEQ ID NO: 54).
The amino acid residues capable of mutation in a v2 sequence are numbered with respect to SEQ ID NO: 5 which comes from a strain 2996. The corresponding amino acid residues in a fHbp v2 from any other strain can be easily identified by alignment of sequences, being p. ex. the amino acid which, when aligned with SEQ ID NO: 5 using a pair alignment algorithm (eg the Needleman-Wunsch global alignment algorithm, described below ), aligns with the amino acid mentioned herein. Often the amino acid will be identical to that present in SEQ ID NO: 5 (eg residue 32 will be serine), but alignment will readily identify it if it is not.
Relative mutations t SEQ ID NO: 17
The polypeptides according to the second aspect of the invention comprise an amino acid sequence having at least one identity with SEQ ID NO: 17, and / or comprising a fragment of SEQ ID NO: 17. Compared to SEQ ID NO: 17, however, this amino acid sequence has a modification at least at amino acid residues L126 and E243 (and possibly also at residue S32). These residues are numbered according to SEQ ID NO: 17; to coincide with the nascent wild-type sequence (SEQ ID NO: 3), the numbering should include +31 (ie Ser-32 in SEQ ID NO: 17 is Ser-63 in SEQ ID NO: 3), and to coincide with the mature wild type sequence (SEQ ID NO: 40), the numbering should include +12.
The two (or three) specified residues may be deleted, but preferably they are substituted with another amino acid. For example, Leu-126 may be substituted with any of the other 19 naturally occurring amino acids. When a substitution is made, the substitution amino acid may in some embodiments be a single amino acid such as glycine or alanine. In other cases, the substitution amino acid is a conservative substitution, p. ex. it is carried out within the following four groups: (1) acids, namely aspartate, glutamate; (2) basic, namely lysine, arginine, histidine; (3) nonpolar, namely alanine, valine, leucine,. isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar, namely glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. In other embodiments, the substitution is non-conservative.
Preferred substitutions at the specified residues are: S32V; L126R; and E243A.
In addition to the mutation (s) indicated above, aimed at increasing the stability and altering the ability of the polypeptide to bind to fH, a polypeptide may contain one or more other mutations, e.g. ex. to alter the interaction of the polypeptide with siderophores. Residues that interact with siderophores can be mutated, following the guidelines in references 16 and 35, p. ex. aligning SEQ ID NO: 17 herein with SEQ ID NO: 4 of reference 16 to identify residues that may interact with siderophores, e.g. ex. with catecholates, hydroxamates or carboxylates.
Reference 24 indicates that some substitutions in v3 may increase its affinity for fH, and therefore these will generally be avoided, eg, ex. P44 in SEQ ID NO: 17 (residue 106 in ref 24). Other residues may also be mutated provided that, compared to the wild type sequence (eg, SEQ ID NO: 40), the polypeptide has higher stability, lower affinity for fH, and when it is When administered to a suitable mammal, it can elicit an antibody response that is bactericidal to meningococcus.
The polypeptide according to the second aspect may comprise SEQ ID NO: 48. In SEQ ID NO: 48, residue 32 is any amino acid, residue 126 is not leucine, and residue 243 is not glutamate. Another option is SEQ ID NO: 57, where residue 32 is not serine, residue 126 is not leucine, and residue 243 is not glutamate. In a preferred embodiment of SEQ ID NO: 48, residue 32 is valine, residue 126 is arginine, and residue 243 is alanine (ie, SEQ ID NO: 51). In another preferred embodiment of SEQ ID NO: 48, residue 32 is serine, residue 126 is arginine, and residue 243 is alanine (ie, SEQ ID NO: 55).
The polypeptide according to the second aspect may comprise SEQ ID NO: 32. In SEQ ID NO: 32, residue 32 is any amino acid, and residue 126 is not leucine. Another option is SEQ ID NO: 38, where residue 32 is not serine, and residue 126 is not leucine. In a preferred embodiment of SEQ ID NO: 32, residue 32 is valine, and residue 126 is arginine (ie, SEQ ID NO: 44). In another preferred embodiment of SEQ ID NO: 32, residue 32 is serine, and residue 126 is arginine (ie, SEQ ID NO: 56).
The amino acid residues capable of mutation in a sequence v3 are numbered with respect to SEQ ID NO: 17 which comes from an M1239 strain. The corresponding amino acid residues in a fHbp v3 from any other strain can be easily identified by sequence alignment, being p. ex. the amino acid which, when aligned with SEQ ID NO: 17 using a pair alignment algorithm (eg the Needleman-Wunsch global alignment algorithm, described below ), aligns with the amino acid mentioned herein. Often the amino acid will be identical to that present in SEQ ID NO: 17 (eg residue 32 will be serine), but alignment will readily identify it if it is not. Mutant sequences according to the invention
As mentioned above, the polypeptide according to the first aspect of the invention may comprise SEQ ID NO: 47 or SEQ ID NO: 37, and the polypeptide according to the second aspect of the invention may comprise SEQ ID NO: 48 or SEQ. ID NO: 57.
According to a third aspect of the invention, which overlaps with the first aspect, the invention relates to a polypeptide comprising an amino acid sequence having a sequence identity of at least v% with SEQ ID NO: 47, provided that (i) residue 32 is any amino acid, but in some embodiments is not serine, (ii) residue 123 is not leucine, (iii) residue 240 is not glutamate, and provided that ( iv) compared to wild-type sequence, eg SEQ ID NO: 4, the polypeptide has a higher stability and a lower affinity for fH and (v) when administered to a suitable mammal, it can elicit a response to antibody that is bactericidal to a meningococcus that expresses fHbp v2. The numbering of residues from (i) to (iii) is from SEQ ID NO: 47.
The value of v may be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more. It is preferably 90 (i.e., the mutant fHbp v2 amino acid sequence has an identity of at least 90% with SEQ ID NO: 47) and more preferably 95.
According to a fourth aspect of the invention, which overlaps with the second aspect, the invention relates to a polypeptide comprising an amino acid sequence having a sequence identity of at least w% with SEQ ID NO: 48, provided that ( i) residue 32 is any amino acid, but in some embodiments is not serine, (ii) residue 126 is not leucine, (iii) residue 243 is not glutamate, and provided that (iv) ) compared to wild-type sequence, eg SEQ ID NO: 40, the polypeptide has a higher stability and a lower affinity for fH and (v) when administered to a suitable mammal, it can elicit an antibody response which is bactericidal towards a meningococcus that expresses a fHbp v3. The numbering of residues from (i) to (iii) is made from SEQ ID NO: 48.
The value of w may be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more. It is preferably 90 (i.e., the mutant fHbp v3 amino acid sequence has an identity of at least 90% with SEQ ID NO: 48) and more preferably 95.
According to a fifth aspect, the invention relates to a polypeptide comprising the amino acid sequence SEQ ID NO: 47, modified to have 5 individual amino acid changes (i.e. 1, 2, 3, 4 or 5 substitutions, deletions). and / or individual amino acid insertions), provided that (i) residue 32 is any amino acid, but in some embodiments is not serine, (ii) residue 123 is not leucine, (iii) ) residue 240 is not glutamate, and provided that (iv) compared to wild-type sequence, eg SEQ ID NO: 4, the polypeptide has higher stability and lower affinity for fH and that (v) when it is administered to a suitable mammal, it can elicit an antibody response that is bactericidal to a meningococcus that expresses a fHbp v2.
According to a sixth aspect, the invention relates to a polypeptide comprising the amino acid sequence SEQ ID NO: 48, modified up to 5 individual amino acid changes (i.e. 1, 2, 3, 4 or 5 substitutions, deletions and / or individual amino acid insertions), provided that (i) residue 32 is any amino acid, but in some embodiments is not serine, (ii) residue 126 is not leucine, (iii) ) residue 243 is not glutamate, and provided that (iv) compared to wild-type sequence, eg SEQ ID NO: 40, the polypeptide has a higher stability and a lower affinity for fH and that (v) when it is administered to a suitable mammal, it can elicit an antibody response which is bactericidal towards a meningococcus that expresses a fHbp v3. The numbering of residues from (i) to (iii) is made from SEQ ID NO: 48.
These various v2 and v3 polypeptides can be combined to form fusion polypeptides, thereby providing immune responses against both variants with a single polypeptide. Accordingly, a seventh aspect of the invention relates to a polypeptide comprising a fusion consisting of: (i) a polypeptide according to the first, third, or fifth aspects of the invention; and (ii) a polypeptide according to the second, fourth, or sixth aspects of the invention. Advantageously, these polypeptides can, when administered to a suitable mammal, elicit an antibody response that is bactericidal to both a meningococcus that expresses fHbp v2 and a meningococcus that expresses fHbp v3.
Therefore, according to the seventh aspect, the fusion polypeptide comprises: (I) a first amino acid sequence selected from: □ a mutant fHbp v2 amino acid sequence, wherein: (a) the amino acid sequence has a sequence identity of at least k% with SEQ ID NO: 5, and / or comprises a fragment of SEQ ID NO: 5; but (b) the amino acid sequence differs from SEQ ID NO: 5 at residues L123 and E240 (and possibly also at residue S32); An amino acid sequence having at least v% sequence identity with SEQ ID NO: 47, provided that (i) residue 32 is any amino acid, but in some embodiments is not serine (ii) residue 123 is not leucine, (iii) residue 240 is not glutamate; or
The amino acid sequence SEQ ID NO: 47 or SEQ ID NO: 50, optionally modified to the extent of 5 individual amino acid changes (ie 1, 2, 3, 4 or 5 substitutions, deletions and / or insertions) of individual amino acids), provided that (i) residue 32 is any amino acid, but in some embodiments is not serine, (ii) residue 123 is not leucine, (iii) residue 240 not be glutamate; and (II) a second amino acid sequence selected from: □ a mutant fHbp v3 amino acid sequence, wherein: (a) the amino acid sequence has a sequence identity of at least j% with SEQ ID NO: 17, and / or comprises a fragment of SEQ ID NO: 17; but (b) the amino acid sequence differs from SEQ ID NO: 17 at residues L126 and E243 (and, in some embodiments, also at residue S32); An amino acid sequence having at least w% sequence identity with SEQ ID NO: 48, provided that (i) residue 32 is any amino acid, but in some embodiments is not serine (ii) residue 126 is not leucine, (iii) residue 243 is not glutamate; or
The amino acid sequence SEQ ID NO: 48 or SEQ ID NO: 51, optionally modified to the extent of 5 individual amino acid changes (ie 1, 2, 3, 4 or 5 substitutions, deletions and / or insertions) of individual amino acids), provided that (i) residue 32 is any amino acid, but in some embodiments is not serine, (ii) residue 126 is not leucine, (iii) residue 243 is not glutamate, wherein the fusion polypeptides (a) can, when administered to a suitable mammal, elicit an antibody response that is bactericidal to both a meningococcus that expresses fHbp v2 and a meningococcus that expresses a fHbp v3; (b) have superior stability and lower affinity for fH than wild-type meningococcal fHbp consisting of SEQ ID NO: 4; (c) have superior stability and lower affinity for fH than wild-type meningococcal fHbp consisting of SEQ ID NO: 40.
Ideally, the increase in stability is at least 5 ° C, e.g. ex. at least 10 ° C, 15 ° C, 20 ° C, 25 ° C, 30 ° C, 35 ° C or higher, as described above. The lower affinity is at least 10-fold, and ideally at least 100-fold, as described above.
In an embodiment according to the seventh aspect, the invention relates to a polypeptide comprising a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence is SEQ ID NO: 47 or SEQ. ID NO: 39 and the second amino acid sequence is SEQ ID NO: 48 or SEQ ID NO: 57.
The first and second amino acid sequences may be in either order in the N-terminal to C-terminal direction, but it is preferable that the first sequence is upstream of the second.
The first and second amino acid sequences can be joined by a linker sequence. This linker sequence (s) will typically be short (eg 20 amino acids or less, ie 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, poly-glycine linkers (ie Glyn where n = 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (SEQ ID NO: 58)), and histidine tags (ie Hisn where n = 3, 4, 5, 6, 7, 8, 9, 10 or more (SEQ ID NO: 59)). Other suitable linker amino acid sequences will be apparent to those skilled in the art. A useful linker is GSGGGG (SEQ ID NO: 20), the Gly-Ser dipeptide being formed from a BamRI restriction site, thereby facilitating cloning and manipulation. Another useful linker is SEQ ID NO: 21, which may optionally be preceded by a Gly-Ser dipeptide (SEQ ID NO: 22, from BamRI) or a Gly-Lys dipeptide (SEQ ID NO: 23, from HindIII).
The fusion polypeptide may also include a fHbp v1 sequence, thereby obtaining immune responses against the three fHbp variants with a single polypeptide, that is, the polypeptide can, when administered to a suitable mammal, elicit an antibody response. which is bactericidal towards a meningococcus that expresses a fHbp v1, a meningococcus that expresses fHbp v2, and a meningococcus that expresses fHbp v3. Therefore, a polypeptide according to the seventh aspect may also include an amino acid sequence (i) having a sequence identity of at least 1% with SEQ ID NO: 16, and / or (ii) comprising a fragment of SEQ ID NO: 16. The value of i may be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more. It is preferably 90 (i.e., the amino acid sequence has an identity of at least 90% with SEQ ID NO: 16) and more preferably 95. The fragment mentioned in (ii) will generally have at least 7 amino acids, p. ex. 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 24, 26, 28, 40, 45, 50, 55, 60, 65, 70, 75, 80 acids contiguous amines or more from SEQ ID NO: 16. The fragment will typically contain at least one epitope from SEQ ID NO: 16. Sharing at least 30 contiguous amino acids with SEQ ID NO: 16 will be typical, and generally The amino acid sequence of fHbp v1 will contain several (e.g., 2, 3, 4, 5 or more) fragments from SEQ ID NO: 16. Overall, an amino acid sequence of fHbp v1 used according to the seventh aspect may have a sequence identity of at least 1% and contain several fragments from SEQ ID NO: 16.
Advantageously, the fHbp v1 sequence contains a mutation which confers on it a lower affinity (as described above) for human fH than the same polypeptide but without the sequence differences mentioned in (b), p. ex. a lower affinity than a wild-type meninogococcal polypeptide consisting of SEQ ID NO: 46. For example, the amino acid residue Arg-34 in SEQ ID NO: 16 (Arg-60 residue in SEQ ID NO: 1, and Arg -41 in SEQ ID NO: 46) can be mutated to Ser to alter the fHbp / fH interaction [19,21]. Therefore, a preferred fHbp v1 sequence for use in the invention includes SEQ ID NO: 49, wherein residue 34 is not arginine (eg, SEQ ID NO: 52, where residue 34 is serine). ).
When a polypeptide each contains v1, v2 and v3 sequences, these may be present in any order from the N-terminus to the C-terminus, v1-v2-v3, v1-v3- v2, v2-v1-v3, v2-v3-v1, v3-v1-v2, or v3-v2-v1. The most preferred order is v2-v3-vl.
In general, a preferred fHbp fusion polypeptide according to the invention has an amino acid sequence of the formula:
wherein each X is a different fHbp variant sequence as defined herein, L is an optional linker amino acid sequence, A is an optional N-terminal amino acid sequence, and B is an optional C-terminal amino acid sequence.
The three fragments X are a sequence v1, v2, and v3 as described herein, so that the polypeptide can, when administered to a suitable mammal, elicit an antibody response that is bactericidal to a meningococcus that expresses a fHbp v1, a meningococcus that expresses a fHbp v2, and a meningococcus that expresses fHbp v3. As mentioned above, the three variants are preferably in the order v2-v3-v1 in the N-terminal to C-terminal direction. At each occurrence of [-X-L-], a linker amino acid sequence -L- may be present or absent. Suitable linker sequences are described above. . -A- is an optional N-terminal amino acid sequence. It will usually be short (eg 40 amino acids or less, ie 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). As examples, there are leader sequences that direct the transport of proteins. If Xi lacks its own N-terminal methionine, -A- can provide said methionine residue in the translated polypeptide (e.g.
Residue Met only). The Met residue may be at the N-terminus of a linker sequence such as SEQ ID NO: 21 (ie, SEQ ID NO: 24), or at the N-terminus of a short sequence (eg, SEQ ID NO: 25). -B- is an optional C-terminal amino acid sequence. It will usually be short (eg 40 amino acids or less, ie 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). As examples, there are sequences that direct protein transport, short peptide sequences that facilitate cloning or purification (eg, including histidine tags, ie His ^ where n = 3, 4, 5, 6, 7, 8, 9, 10 or more (SEQ ID NO: 59)), or sequences that enhance the stability of the polypeptide. Other suitable C-terminal amino acid sequences will be apparent to those skilled in the art. A suitable -B- fragment is SEQ ID NO: 26, wherein Leu-Glu upstream of the histidine tag is from an XhoI restriction site.
Therefore, in one embodiment, the invention relates to a polypeptide comprising the amino acid sequence of SEQ ID NO: 28. From the N-terminus to the C-terminus, this sequence is constituted by the sequences of following amino acids SEQ ID:
By adding SEQ ID NO: 24 as the -A-N-terminal fragment, the invention also relates to a polypeptide comprising the amino acid sequence of SEQ ID NO: 27.
In another embodiment, the invention relates to a polypeptide comprising the amino acid sequence of SEQ ID NO: 30. From the N-terminus to the C-terminus, this sequence is constituted by the amino acid sequences SEQ IDs: _______
By adding SEQ ID NO: 24 as the -A-N-terminal fragment, the invention also relates to a polypeptide comprising the amino acid sequence of SEQ ID NO: 29.
polypeptides
The polypeptides according to the invention can be prepared by various means, e.g. ex. by chemical synthesis (at least in part), digestion of longer polypeptides with proteases, translation from RNA, purification from a cell culture (eg from recombinant expression or of a N. meningitidis culture), etc. Heterologous expression in an E. coli host is a preferred expression pathway.
Ideally, the polypeptides according to the invention are at least 100 amino acids in length, e.g. ex. 150aa, 175aa, 200aa, 225aa, or more. They contain an amino acid sequence of mutant fHbp v2 and / or v3, and the amino acid sequences of mutant fHbp v2 or v3 will generally have a similar length of at least 100 amino acids, e.g. ex. 150aa, 175aa, 200aa, 225aa, or more. fHbp is naturally a lipoprotein in N. meningitidis. It has also been shown to be lipidated when expressed in E. coli with its native leader sequence or heterologous leader sequences. The polypeptides according to the invention can have an N-terminal cysteine residue, which can be lipidized p. ex. include a palmitoyl group, generally forming a tripalmitoyl-S-glyceryl-cysteine. In other embodiments, the polypeptides are not lipidated.
The polypeptides are preferably prepared in a substantially pure or substantially isolated form (i.e., substantially free of any other neisserial polypeptide or polypeptide derived from the host cell). In general, the polypeptides are provided in a non-natural environment, e.g. ex. they are separated from their natural environment. In some embodiments, the polypeptide is present in a composition that is enriched for the polypeptide compared to the starting material. Therefore, a purified polypeptide is provided, "purified" meaning that the polypeptide is present in a composition that is substantially free of other expressed polypeptides, "substantially free" meaning more than 50% (eg,> 75%, > 80%,> 90%,> 95%, or> 99%) of the total polypeptide in the composition is a polypeptide according to the invention.
The polypeptides can take various forms (eg, native, fusions, glycosylated, non-glycosylated, lipidated, disulfide bridges, etc.).
SEQ ID NO: 4, 5, 17 and 40 do not contain N-terminal methionine. If a polypeptide according to the invention is obtained by translation into a biological host, then a start codon is required, which will provide an N-terminal methionine in most hosts. Therefore, a polypeptide according to the invention will comprise, at least at a nascent stage, a methionine residue upstream of said sequence SEQ ID No.
Cleavage of the nascent sequences means that the mutant fHbp v2 or v3 amino acid sequence can itself be the N-terminus of the polypeptide. In other embodiments, however, a polypeptide according to the invention may comprise an N-terminal sequence upstream of the mutant fHbp v2 or v3 amino acid sequence. In some embodiments, the polypeptide has a single methionine at the N-terminus immediately followed by the mutant fHbp v2 or v3 amino acid sequence, in other embodiments, a longer upstream sequence may be used. This upstream sequence may be short (eg 40 amino acids or less, ie 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). As examples, there are leader sequences that direct the transport of proteins, or short peptide sequences that facilitate cloning or purification (eg, a histidine tag, ie Hisn where n = 4, 5, 6, 7, 8, 9, 10 or more (SEQ ID NO: 60)). Other suitable N-terminal amino acid sequences will be apparent to those skilled in the art, e.g. ex. the native upstream sequences present in SEQ ID NO: 2 or SEQ ID NO: 3.
A polypeptide according to the invention may also comprise amino acids downstream of the final amino acid of the mutant fHbp v2 or v3 amino acid sequence. These C-terminal extensions may be short (eg 40 amino acids or less, ie 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). As examples, there are the sequences that direct the transport of proteins, the short peptide sequences that facilitate cloning or purification (including, for example, a histidine tag, ie Hisn where n = 4, 5, 6 , 7, 8, 9, 10 or more (SEQ ID NO: 60)) or sequences that enhance the stability of the polypeptide. Other suitable C-terminal amino acid sequences will be apparent to those skilled in the art.
The term "polypeptide" refers to amino acid type polymers of any length. The polymer may be linear or branched, may include modified amino acids, and may be interrupted by non-amino acids. The term also encompasses an amino acid-type polymer that has been naturally modified or following an intervention: for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a component marking. Also included in the definition are polypeptides containing, for example, one or more amino acid analogs (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. The polypeptides may be in the form of simple chains or associated chains.
The polypeptides according to the invention can be fixed or immobilized on a support medium.
The polypeptides according to the invention may comprise a detectable marker, e.g. ex. a radioactive label, a fluorescent label, or a biotin label, which are particularly useful in immunoassay techniques.
As described in reference 162, fHbp can be divided into three domains, designated A, B and C. Taking SEQ ID NO: 1, the three domains are (A) 1-119, (B) 120-183 and (C) ) 184-274:
MNRTAFCCLSLTTALILTACSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQS
VRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQ
VYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGT
AFGSDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVL
YNQAEKGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIGLAAKQ
The mature form of the "A" domain, from Cys-20 at its N-terminus to Lys-119, is called "Araature".
Multiple fHbp sequences are known and can be easily aligned using standard methods. With these alignments, one skilled in the art can identify (a) the "A" (and "Amature"), "B" and "C" domains in any given fHbp sequence by comparison with the coordinates in the MC58 sequence, and (b) individual residues in multiple fHbp sequences, p. ex. to identify substitutions. For ease of reference, however, the domains are defined below: - The "A" domain in a given fHbp sequence is the fragment
of that sequence which, when aligned with SEQ ID NO: 1 using a pair alignment algorithm, begins with the amino acid aligned to Met-1 of SEQ ID NO: 1 and ends with amino acid aligned with Lys-119 of SEQ ID NO: 1.
The "Amature" domain in a given fHbp sequence is the fragment of this sequence which, when aligned with SEQ ID NO: 1 using a pair alignment algorithm, begins with the aligned amino acid. on Cys-20 of SEQ ID NO: 1 and ends with the Lys-119 aligned amino acid of SEQ ID NO: 1. - The "B" domain in a given fHbp sequence is the fragment
of this sequence which, when aligned with SEQ ID NO: 1 using a pair alignment algorithm,
starts with the amino acid aligned with Gln-120 of SEQ ID NO: 1 and ends with the amino acid aligned with Gly-183 of SEQ ID NO: 1. - The "C" domain in a given fHbp sequence is the fragment
of this sequence which, when aligned with SEQ ID NO: 1 using a pair alignment algorithm,
begins with the Lys-184 aligned amino acid of SEQ ID NO: 1 and ends with the Gln-274 aligned amino acid of SEQ ID NO: 1. The preferred pair alignment algorithm for defining domains is the global alignment algorithm of
Needleman-Wunsch [156], using the default parameters (eg with a gap opening penalty = 10.0, and a gap extension penalty = 0.5, using the EBLOSUM62 scoring matrix). This algorithm is easy to implement in the "needle" tool of the EMBOSS software [157].
In some embodiments, a mutant fHbp v2 or v3 amino acid sequence according to the invention is truncated to remove its A-domain. In general, however, it is preferred that the mutant fHbp v2 or v3 amino acid sequence contains both an N-terminal β-barrel and a C-terminal β-barrel.
In some embodiments, a polypeptide comprises an amino acid sequence as described above, except that up to 10 amino acids (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) at the N-terminus and / or up to 10 amino acids (ie, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) at the end C-terminal are deleted.
The invention relates to nucleic acids encoding a polypeptide according to the invention as defined above.
The nucleic acids according to the invention can be prepared in many ways, e.g. ex. by chemical synthesis (eg, DNA synthesis by the phosphoramidite method) in whole or in part, by digestion of longer nucleic acids using nucleases (eg restriction enzymes), by assembly of nucleic acids or shorter nucleotides (eg using ligases or polymerases), from genomic DNA or cDNA libraries, etc.
The nucleic acids according to the invention can take various forms, e.g. ex. single-strand, double-stranded, vectors, primers, probes, marked, unmarked, etc.
The nucleic acids according to the invention are preferably in isolated or substantially isolated form.
The term "nucleic acid" includes DNA and RNA, and also analogs thereof, such as those containing modified backbones, and also peptide nucleic acids (PNAs), etc. The nucleic acid according to the invention can be labeled, e.g. ex. with a radioactive or fluorescent label. The invention also relates to vectors (such as plasmids) comprising nucleotide sequences according to the invention (eg cloning or expression vectors, such as those that can be used for nucleic acid immunization) and host cells transformed by these vectors. Bacterial responses
The preferred polypeptides according to the invention can elicit antibody responses which are bactericidal towards meningococci. Bactericidal antibody responses are conveniently measured in mice and are a standard indicator of vaccine efficacy (see eg, footnote 14 of ref 36, also ref 37). Therefore, the antibodies will be bactericidal to a test strain in a suitable serum bactericidal activity (SBA) assay.
The polypeptides according to the first aspect of the invention may preferably elicit an antibody response, e.g. ex. in the mouse, which is bactericidal against a strain of N. meningitidis which expresses a sequence fHbp v2, p. ex. one or more strains 961-5945, 2996, 96217, 312294, 11327, a22, gb013 (= M01-240013), e32, ml90, m4287, 860800, 599, 95N477, 90-18311, cil, m986, m2671, 1000 , ml096, m3279, bz232, dk353, m3697, ngh38, and / or L93 / 4286. The bactericidal responses can for example be evaluated against the strain M2091 var2 (ATCC 13091).
Preferred polypeptides according to the first aspect of the invention may elicit the production of antibodies in mice which are bactericidal to strain M2091 in a serum bactericidal assay.
The polypeptides according to the second aspect of the invention may preferably elicit an antibody response, e.g. ex. in the mouse, which is bactericidal against a strain of N. meningitidis which expresses a sequence fHbp v3, p. ex. one or more strains M1239, 16889, gb355 (= M01-240355), m3369, m3813, ngpl65. The bactericidal responses can for example be evaluated against the strain M01-240355 var3, which is a Neisseria MLST reference strain (id 19265 in ref 38) which has been fully sequenced (see EMBL ID CP002422 [39]).
Preferred polypeptides according to the second aspect of the invention may elicit the production of antibodies in mice that are bactericidal to strain M01-240355 in a serum bactericidal assay.
For example, an immunogenic composition comprising these polypeptides can provide a serum bactericidal titer of 1: 4 by the Goldschneider assay with human serum complement source [40-42], and / or provide a serum bactericidal titer. with as a source of complement the rabbit baby serum.
Immunization
The polypeptides according to the invention can be used as active principle (s) in immunogenic compositions, and therefore the invention relates to an immunogenic composition (eg, a vaccine) comprising a polypeptide according to the invention. . The invention also relates to a method for eliciting an antibody response in a mammal, e.g. ex. mouse or human subject, comprising administering an immunogenic composition according to the invention to said mammal. Preferably, the antibody response is a protective and / or bactericidal antibody response. The invention also relates to polypeptides according to the invention which can be used in these methods. The invention also relates to a method for protecting a mammal, e.g. ex. a mouse or human subject against Neisseria infection (eg, meningococcal) comprising administering to said mammal an immunogenic composition of the invention. The invention relates to polypeptides according to the invention which can be used as medicaments (eg in the form of immunogenic compositions or vaccines) or as diagnostic reagents. It also relates to the use of a nucleic acid or polypeptide according to the invention in the manufacture of a medicament for preventing Neisseria infection (eg meningococcal) in a mammal, e.g. ex. a mouse or a human subject.
The mammal is preferably a human subject. The human subject may be an adult or, preferably, a child. When the vaccine is for prophylactic use, the human subject is preferably a child (eg, a toddler or an infant); when the vaccine is for therapeutic use, the human subject is preferably an adult. A vaccine for children can also be given to adults, eg. ex. to evaluate its safety, dosage, immunogenicity, etc.
Uses and methods are particularly useful for preventing / treating diseases including, but not limited to, meningitis (especially bacterial meningitis, such as meningococcal meningitis) and bacteremia. For example, they lend themselves to active immunization of individuals against invasive meningococcal disease caused by N. meningitidis (eg serogroup B). The effectiveness of the therapeutic treatment can be tested by monitoring the Neisseria infection after administration of the composition according to the invention. The effectiveness of the propylactic treatment can be tested by monitoring the immune responses directed against fHbp following administration of the composition. The immunogenicity of the compositions according to the invention can be determined by administering said compositions to test subjects (eg, children 12-16 months of age, or animal models) and then determining standard parameters including serum bactericidal antibodies ( SBA) and ELISA titers (GMT). In general, these immune responses will be determined about 4 weeks after the administration of the composition, and compared to the values determined before administration of the composition. An SBA increase of at least 4 to 8 times SBA is preferred. When more than one dose of the composition is administered, more than one post-administration determination may be made.
Preferred compositions according to the invention can confer an antibody titer on a human patient that is superior to the seroprotection criterion of each antigenic component in an acceptable percentage of human subjects. Antigens associated with an antibody titer above which a host is considered seroconverted against the antigen are well known, and these are published by organizations such as WHO. Preferably, more than 80% of a statistically significant sample of subjects is seroconverted, more preferably more than 90%, more preferably more than 93% and most preferably 96-100%. The invention can be used to confer systemic and / or mucosal immunity.
In general, the compositions according to the invention will be administered directly to a human patient. Direct administration may be by parenteral injection (eg subcutaneous, intraperitoneal, intravenous, intramuscular, or in the interstitial space of a tissue), or rectally, orally, vaginally, topically, transdermally, intranasal, ocular, auricular, pulmonary or other mucosal administration. Intramuscular administration in the thigh or upper arm is preferred. The injection may be via a needle (eg, a hypodermic needle), but needle-free injection may be used alternatively. A typical intramuscular dose is approximately 0.5 ml (eg as seen in BEXSERO ™).
The dosage may be a single or multiple dose regimen. Multidoses can be used in a primary vaccination schedule and / or a booster dose schedule. A primary vaccination schedule may be followed by a booster dose schedule. The appropriate time interval between primary vaccination doses (eg between 4 and 16 weeks), and between primary vaccination and booster (s) may be determined each time. For example, BEXSERO ™ is given in two or three doses at intervals of at least 1 month or at least 2 months, depending on the subject (eg infants or other).
The immunogenic composition according to the invention will generally contain a pharmaceutically acceptable carrier, which may be any substance which does not itself induce the production of antibodies harmful to the patient receiving the composition, and which can be administered without undue toxicity. Pharmaceutically acceptable carriers may include liquids such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may also be present in these vehicles. An in-depth discussion of suitable vehicles can be found in ref. 43. For example, BEXSERO ™ contains sodium chloride, histidine, sucrose, aluminum hydroxide, and water for injec- tion preparations.
Neisseria infections affect various parts of the body, so that the compositions according to the invention can be prepared in various forms. For example, the compositions can be prepared in the form of injectable preparations, of either liquid solution or suspension type. Solid forms for solution or suspension in pre-injection liquid vehicles may also be prepared. Compositions suitable for parenteral injection (eg in muscle) are most preferred.
The composition is preferably sterile. It is preferably pyrogen-free. It is preferably buffered, e.g. ex. at a pH between pH 6 and pH 8, generally around pH 7. When a composition comprises an aluminum hydroxide salt, it is preferable to use a histidine buffer [44]. The compositions according to the invention can be isotonic with respect to human subjects.
The immunogenic compositions comprise an immunologically effective amount of immunogen, as well as any other specified component, as needed. By "immunologically effective amount" is meant that administration of that amount to an individual, either as a unit dose or as part of a series, is effective for treatment or prevention purposes. This quantity varies according to the health and physical condition of the individual to be treated, his age, the taxonomic group to which the individual to be treated (eg, non-human primate, primate, etc.) belongs. the ability of the individual's immune system to synthesize antibodies, the degree of protection sought, the formulation of the vaccine, the opinion of the attending physician on the medical case, and other relevant factors. This quantity should be in a relatively large range which can be determined by regular tests. The dosage treatment may be a single dose or multi dose regimen (including, for example, booster doses). The composition may be administered together with other immunoregulatory agents.
Adjuvants which can be used in the compositions according to the invention include, inter alia, insoluble metal salts, oil-in-water emulsions (e.g., MF59 or AS03, both containing squalene), saponins, non-toxic LPS derivatives (such as monophosphoryl lipid A or 3-0-deacylated MPL), immunostimulatory oligonucleotides, ADP-ribosylating detoxified bacterial toxins, microparticles, liposomes, imidazoquinolones, or mixtures thereof. Other substances capable of acting as immunostimulatory agents are described in Chapter 7 of ref. 45. The use of an adjuvant of the aluminum hydroxide and / or aluminum phosphate type is particularly preferred, and the polypeptides are generally adsorbed on these salts. These salts include oxyhydroxides and hydroxyphosphates (see eg chapters 8 & 9 of 45). The salts may take any suitable form (eg gel, crystalline form, amorphous, etc.). Al +++ should be present at <1 mg / dose. The most preferred adjuvant is aluminum hydroxide as used in BEXSERO ™. The polypeptides in a composition according to the invention can be adsorbed on this adjuvant, as in BEXSERO ™. It can be incorporated at a rate of about 1 mg / ml Al +++ (ie 0.5 mg per 0.5 ml dose).
Other antigenic components
The compositions according to the invention comprise the mutant fHbp v2 and / or v3 sequence. It is useful that the composition does not contain complex or undefined antigenic mixtures, e.g. ex. it is preferable that it does not contain outer membrane vesicles. The polypeptides according to the invention are preferably expressed by recombinant means in a heterologous host, and then purified.
In addition to a fHbp polypeptide, a composition according to the invention may also contain one or more other neisserial immunogens, since a vaccine that targets more than one immunogen per bacterium reduces the ability to select escape mutants. Therefore, a composition may contain a second polypeptide which, when administered to a suitable mammal, elicits an antibody response which is bactericidal to meningococcus. The second polypeptide may be a meningococcal fHbp, but will often be a polypeptide other than fHbp, e.g. ex. an NHBA sequence, a NadA sequence, etc.
A composition according to the invention may contain an NHBA antigen. The NHBA antigen was included in the published genome sequence of meningococcal serogroup B strain MC58 [46] as NMB2132 gene (GenBank accession number GI: 7227388, SEQ ID NO: 6 herein). NHBA antigen sequences from many strains have since been published. For example, allelic forms of NHBA can be seen in Figures 5 and 15 of Reference 47, and in Example 13 and Figure 21 of Reference 1 (SEQ ID 3179-3184 herein). Various immunogenic fragments of the NHBA antigen have also been reported. Preferred 287 antigens for use in the invention include an amino acid sequence: (a) having an identity of 50% or more (e.g., 60%, 65%, 70%, 75%, 80% 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 6; and / or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 6, 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 in (b) include an epitope derived from SEQ ID NO: 6. The most useful NHBA antigens according to the invention can elicit the production of antibodies which, after administration to a subject, can bind to a polypeptide meningococcal constituted by the amino acid sequence SEQ ID NO: 6. The advantageous NHBA antigens that can be used in the context of the invention can elicit the production of bacterinating antimeningococcal antibodies after administration to the subject.
A composition according to the invention may contain a NadA antigen. The NadA antigen has been included in the published genome sequence of meningococcal serogroup B strain MC58 [46] as NMB1994 gene (GenBank accession number GI: 7227256, SEQ ID NO: 7 herein). NadA antigen sequences from many strains have since been published, and the activity of the protein as Neisseria adhesin is well documented. Various immunogenic fragments of the NadA antigen have also been reported. Preferred NadA antigens for use in the invention include an amino acid sequence: (a) having an identity of 50% or more (e.g., 60%, 65%, 70%, 75%, 80% 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 7; and / or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 7, where "n" is 7 or more (eg 8, 10, 12, 14, 16, 18 , 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). The preferred fragments of (b) comprise an epitope derived from SEQ ID NO: 7. The most useful NadA antigens according to the invention can elicit the production of antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of the amino acid sequence SEQ ID NO: 7. The advantageous NadA antigens that can be used in the context of the invention can elicit the production of bactericidal meningococcal antibodies after administration to a subject. SEQ ID NO: 15 is one of these fragments.
A composition according to the invention may contain a NspA antigen. NspA antigen has been included in the published genome sequence of meningococcal serogroup B strain MC58 [46] as NMB0663 gene (GenBank accession number GI: 7225888, SEQ ID NO: 8 herein). The antigen was already known from references 48 &amp; 49. NspA antigen sequences from many strains have since been published. Various immunogenic fragments of NspA have also been reported. Preferred NspA antigens for use in the invention include an amino acid sequence: (a) having an identity of 50% or more (e.g., 60%, 65%, 70%, 75%, 80% 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 8; and / or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 8, 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). The preferred fragments of (b) comprise an epitope derived from SEQ ID NO: 8. The most useful NspA antigens according to the invention can elicit the production of antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of the amino acid sequence SEQ ID NO: 8. The advantageous NspA antigens that can be used in the context of the invention can elicit the production of bactericidal anti-meningococcal antibodies after administration to a subject.
The compositions according to the invention may contain a meningococcal antigen HmbR. The full length HmbR sequence was included in the published genome sequence of meningococcal serogroup B strain MC58 [46] as NMB1668 gene (SEQ ID NO: 9 herein). The invention may use a polypeptide that includes a full-length HmbR sequence, but will often utilize a polypeptide that includes a partial HmbR sequence. Therefore, in some embodiments, a HmbR sequence used according to the invention may comprise an amino acid sequence having a sequence identity of at least 1% with SEQ ID NO: 9, where the value of i is 50. , 60, 70, 80, 90, 95, 99 or more. In other embodiments, a HmbR sequence used according to the invention may comprise a fragment consisting of at least consecutive amino acids of SEQ ID NO: 9, where the value of j is 7, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more. In other embodiments, a HmbR sequence used according to the invention may comprise an amino acid sequence (i) having a sequence identity of at least 1% with SEQ ID NO: 9, and / or (ii) ) comprising a fragment consisting of at least consecutive amino acids of SEQ ID NO: 9. The preferred amino acid fragments comprise an epitope derived from SEQ ID NO: 9. These epitopes will generally comprise amino acids which are present at the surface of the HmbR. Useful epitopes include those that involve amino acids involved in the binding of HmbR to hemoglobin, since antibodies that bind to these epitopes can block the ability of a bacterium to bind hemoglobin to hemoglobin. 'host. The topology of HmbR, and its critical functional residues, has been studied in reference 50. The most useful HmbR antigens according to the invention can elicit the production of antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of the amino acid sequence SEQ ID NO: 9. The advantageous HmbR antigens that can be used in the context of the invention can elicit the production of bactericidal anti-meningococcal antibodies after administration to a subject.
A composition according to the invention may contain a NhhA antigen. The NhhA antigen was included in the published genome sequence of meningococcal serogroup B strain MC58 [46] as NMB0992 gene (GenBank accession number GI: 7226232, SEQ ID NO: 10 herein). NhhA antigen sequences from many strains have since been published, e.g. ex. refs. 47 &amp; 51, and various immunogenic fragments of NhhA have been reported. The antigen is also known as Hsf. Preferred NhhA antigens for use in the invention include an amino acid sequence: (a) having an identity of 50% or more (e.g., 60%, 65%, 70%, 75%, 80% 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 10; and / or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 10, where "n" is 7 or more (eg 8, 10, 12, 14, 16, 18 , 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). The preferred fragments of (b) comprise an epitope derived from SEQ ID NO: 10. The most useful NhhA antigens according to the invention can elicit the production of antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of the amino acid sequence SEQ ID NO: 10. The advantageous NhhA antigens that can be used in the context of the invention can elicit the production of bacterinizing anti-meningococcal antibodies after administration to a subject.
A composition according to the invention may contain an antigen App. The App antigen has been included in the published genome sequence of meningococcal serogroup B strain MC58 [46] as NMB1985 gene (GenBank accession number GI: 7227246, SEQ ID NO: 11 herein). Sequences of the App antigen from many strains have since been published. Various immunogenic fragments of App have also been reported. Preferred App antigens for use in the invention include an amino acid sequence: (a) having an identity of 50% or more (e.g., 60%, 65%, 70%, 75%, 80% 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 11; and / or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 11, where "n" is 7 or more (eg 8, 10, 12, 14, 16, 18 , 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). The preferred fragments of (b) comprise an epitope derived from SEQ ID NO: 11. The most useful App antigens according to the invention can elicit the production of antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of the amino acid sequence SEQ ID NO: 11. The advantageous App antigens that can be used in the context of the invention can elicit the production of bactericidal anti-meningococcal antibodies after administration to a subject. '
A composition according to the invention may contain an Omp85 antigen. The Omp85 antigen was included in the published genome sequence of meningococcal serogroup B strain MC58 [46] as NMB0182 gene (GenBank accession number GI: 7225401, SEQ ID NO: 12 herein). Omp85 antigen sequences from many strains have since been published. Other information concerning Omp85 is given in references 52 and 53. Various immunogenic fragments of Omp85 have also been reported. Preferred Omp85 antigens for use in the invention include an amino acid sequence: (a) having an identity of 50% or more (e.g., 60%, 65%, 70%, 75%, 80% 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 12; and / or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 12, where "n" is 7 or more (eg 8, 10, 12, 14, 16, 18 , 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). The preferred fragments of (b) comprise an epitope derived from SEQ ID NO: 12. The most useful Omp85 antigens according to the invention can elicit the production of antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of the amino acid sequence SEQ ID NO: 12. The advantageous Omp85 antigens that can be used in the context of the invention can elicit the production of bactericidal anti-meningococcal antibodies after administration to a patient.
A composition according to the invention may contain a 936 antigen. Antigen 936 has been included in the published genome sequence of meningococcal serogroup B strain MC58 [46] as NMB2091 gene (SEQ ID NO: 13 herein). ). The preferred 936 antigens for use in the invention include an amino acid sequence: (a) having an identity of 50% or more (e.g., 60%, 65%, 70%, 75%, 80% 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 13; and / or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 13, 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). Fragments
Preferred of (b) comprise an epitope derived from SEQ ID NO: 13. The most useful 936 antigens according to the invention can elicit the production of antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of the amino acid sequence SEQ ID NO: 13. The 936 antigen is a good fusion partner for fHbp (eg see references 54 & 55).
A composition may comprise: a polypeptide comprising SEQ ID NO: 14; a polypeptide comprising SEQ ID NO: 15; and a polypeptide according to the invention comprising a mutant amino acid sequence fHbp v2 and SEQ ID NO: 13 (see refs 54 & 55).
A composition may comprise: a polypeptide comprising SEQ ID NO: 14; a polypeptide comprising SEQ ID NO: 15; and a polypeptide according to the invention comprising a mutant fHbp v3 amino acid sequence and SEQ ID NO: 13 (see refs 54 & 55).
In some embodiments, a polypeptide according to the invention is combined with another meningococcal fHbp sequence. In particular, a v2 polypeptide may be combined with a v1 and / or v3 polypeptide to increase the coverage spectrum of the strains [160]. Accordingly, a composition may comprise: (i) a polypeptide according to the invention comprising a mutant fHbp v2 amino acid sequence; and (ii) a fHbp v1 polypeptide and / or a -fHbp v3 polypeptide. In other embodiments, a polypeptide according to the invention may comprise (i) a mutant fHbp v2 amino acid sequence and (ii) an fHbp v1 amino acid sequence and / or an fHbp amino acid sequence v3. Therefore, the v1 and / or v3 sequences can be combined with the mutant v2 sequence as separate entities in a composition (or in a fusion polypeptide, as described above).
Similarly, a v3 polypeptide can be combined with a v1 and / or v2 polypeptide to increase the coverage spectrum of the strains [160]. Accordingly, a composition may comprise: (i) a polypeptide according to the invention comprising a mutant fHbp v3 amino acid sequence; and (ii) a fHbp v1 polypeptide and / or a fHbp v2 polypeptide. In other embodiments, a polypeptide according to the invention may comprise (i) a mutant fHbp v3 amino acid sequence and (ii) an fHbp v1 amino acid sequence and / or an fHbp amino acid sequence v2. Therefore, the v1 and / or v2 sequences can be combined with the mutant v3 sequence as separate entities in a composition (or in a fusion polypeptide, as described above). In addition, the mutant v2 and v3 polypeptides can be combined with one another to increase strain coverage. Accordingly, a composition may comprise: (i) a polypeptide according to the invention comprising a mutant fHbp v2 amino acid sequence; (ii) a polypeptide according to the invention comprising a mutant fHbp v3 amino acid sequence; and (iii) a fHbp v1 polypeptide. In other embodiments, a polypeptide according to the invention may comprise (i) a mutant fHbp v2 amino acid sequence, (ii) a mutant fHbp v3 amino acid sequence, and (iii) an acid sequence. amines fHbp vl. Therefore, the mutant v2 and v3 sequences can be combined with a v1 sequence as separate entities in a composition (or in a fusion polypeptide, as described above). The sequence v1 may be a wild-type sequence or a mutant sequence.
An fHbp v1 may comprise (a) an amino acid sequence having an identity of at least k% with SEQ ID NO: 16, and / or (b) a fragment of SEQ ID NO: 16. Information regarding "k "and the fragments are provided above. The fragment will typically contain at least one epitope from SEQ ID NO: 16, and the fHbp v1 polypeptide will contain at least one epitope that is not present in amino acid sequence v2 or v3 according to the invention, so that the antibodies whose production has been elicited by fHbp v1 can recognize strains vl. Ideally, fHbp v1 may elicit the production of antibodies that are bactericidal to strains v1, p. ex. to strain MC58 (available from ATCC under the reference "BAA-335"). FHbp v1 may contain an amino acid mutation that alters its binding capacity to fH.
FHbp v2 may comprise (a) an amino acid sequence having an identity of at least k% with SEQ ID NO: 5, and / or (b) a fragment of SEQ ID NO: 5. Information about "k "and the fragments are provided above. The fragment will typically contain at least one epitope from SEQ ID NO: 5, and the fHbp v2 polypeptide will contain at least one epitope that is not present in the v3 amino acid sequence of the invention, so that the antibodies of which the production was prompted by fHbp v2 can recognize strains v2. Ideally, fHbp v2 may elicit the production of antibodies that are bactericidal to v2 strains, e.g. ex. to strain M2091 (ATCC 13091). FHbp v2 may be a polypeptide according to the first aspect of the invention.
A fHbp v3 may comprise (a) an amino acid sequence having an identity of at least k% with SEQ ID NO: 17, and / or (b) a fragment of SEQ ID NO: 17. Information about "k "and the fragments are provided above. The fragment will typically contain at least one epitope from SEQ ID NO: 17, and the fHbp v3 polypeptide will contain at least one epitope that is not present in the v2 amino acid sequence of the invention, so that the antibodies of which production was stimulated by fHbp v3 to recognize v3 strains. Ideally, fHbp v3 may elicit the production of antibodies that are bactericidal to v3 strains, e.g. ex. to strain M01-240355. FHbp v3 may be a polypeptide according to the second aspect of the invention.
In addition to Neisseria polypeptide antigens, the composition may include antigens to immunize against other diseases or infections. For example, the composition may comprise one or more of the following other antigens: a N. meningitidis serogroup A, C, W135 and / or Y saccharide antigen, such as the saccharide described in ref. 56 serogroup C (see also ref 57) or ref. 58. - Saccharide antigen of Streptococcus pneumoniae [. ex. 59, 60, 61]. an antigen of the hepatitis A virus, such as an inactivated virus [. ex. 62, 63]. an antigen of hepatitis B virus, such as surface and / or heart antigens [. ex. 63, 64]. a diphtheria antigen, such as diphtheria toxoid [. ex. chapter 3 of ref. 65] p. ex. the CRM197 mutant [. ex. 66]. - a tetanus antigen, such as tetanus toxoid (eg Chapter 4 of Ref 65). Bordetella pertussis antigen, such as pertussis holotoxin (PT) and B. pertussis filamentous haemagglutinin (FHA), optionally also in combination with pertactin and / or agglutinogens 2 and 3 (e.g. 67 & 68). a saccharide antigen of Haemophilus influenzae B [. ex. 57]. one or more polio antigens [. ex. 69,70] such as IPV. - antigens from measles, mumps and / or rubella viruses (eg Chapters 9, 10 & 11 of Ref 65). one or more influenza virus antigens (eg, chapter 19 of ref 65), such as haemagglutinin and / or neuraminidase-like surface proteins. - an antigen of Moraxella catarrhalis [. ex. 71]. - a protein antigen of Streptococcus agalactiae (group B streptococcus) [. ex. 72, 73]. a saccharide antigen of Streptococcus agalactiae (group B streptococcus). - an antigen of Streptococcus pyogenes (Group A streptococcus) [. ex. 73, 74, 75]. - Staphylococcus aureus antigen [. ex. 76].
The composition may comprise one or more of these other antigens.
Toxic protein antigens can be detoxified if necessary (eg detoxification of pertussis toxin by chemical and / or genetic means [68]).
When a diphtheria antigen is incorporated into the composition, it is preferable to also include tetanus and pertussis antigens. Similarly, when a tetanus antigen is incorporated, it is preferable to also include diphtheria and pertussis antigens. Similarly, when a pertussis antigen is incorporated, it is preferable to also include diphtheria and tetanus antigens. DTP combinations are therefore preferred.
The saccharide antigens are preferably in the form of conjugates. The carrier proteins for the conjugates are described in more detail below.
The antigens in the composition will typically be present at a concentration of at least 1 μg / ml each. In general, the concentration of any given antigen will be sufficient to elicit an immune response against this antigen.
The immunogenic compositions of the invention may be used for therapeutic purposes (i.e., to treat an existing infection) or prophylactically (that is, to prevent future infection).
As an alternative to the use of protein antigens in the immunogenic compositions according to the invention, a nucleic acid (which could be RNA, such as self-replicating RNA, or DNA, such as a plasmid) coding for the antigen can be used.
In some embodiments, a composition according to the invention comprises, in addition to the fHbp sequence, conjugated capsular saccharide antigens from 1, 2, 3 or 4 meningococcal serogroups A, C, W135 and Y. In other embodiments, a composition according to the invention comprises, in addition to the fHbp sequence, at least one conjugated pneumococcal capsular saccharide antigen.
Meningococci of serogroups Y, W135, C and A
Current meningococcal serogroup C vaccines (MENJUGATE ™ [56, 77], MENINGITEC ™ and NEISVAC-C ™) include conjugated saccharides. MENJUGATE ™ and MENINGITEC ™ contain oligosaccharide antigens conjugated to a CRM197 carrier protein, while NEISVAC-C ™ uses the complete polysaccharide (des-O-acetylated) conjugated to a tetanus toxoid carrier protein. The MENACTRA ™ vaccine contains conjugated capsular saccharide antigens from each of the Y, W135, C and A serogroups.
The compositions of the invention may contain capsular saccharide antigens from one or more of the meningococcal Y, W135, C and A serogroups wherein the antigens are conjugated to one or more carrier proteins and / or are oligosaccharides. For example, the composition may contain capsular saccharide antigen from serogroup C; serogroups A and C; serogroups A, C and W135; serogroups A, C and Y; serogroups C, W135 and Y; or from the four serogroups A, C, W135 and Y.
A typical amount of each meningococcal saccharide antigen per dose is between 1 and 20 μg, e.g. ex. about 1 μg, about 2.5 μg, about 4 μg, about 5 μg, or about 10 μg (expressed in terms of saccharide).
When a mixture comprises capsular saccharides from both serogroups A and C, the MenC: MenC saccharide ratio (w / w) may be greater than 1 (eg, 2: 1, 3: 1, 4: 1, 5 : 1, 10: 1 or more). When a mixture comprises capsular saccharides from serogroup Y and one or both serogroups C and W135, the MenW: saccharide MenW: MenW135 saccharide ratio (w / w) may be greater than 1 (eg, 2: 1, 3 : 1, 4: 1, 5: 1, 10: 1 or higher) and / or the MenC: MenC saccharide ratio (w / w) may be less than 1 (eg 1: 2, 1: 3, 1: 4, 1: 5, or less). Preferred (w / w) ratios 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 (w / w) ratios 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 an essentially equal weight of each saccharide is preferred.
Capsular saccharides can be used in the form of oligosaccharides. These are conveniently formed by fragmentation of purified capsular polysaccharide (e.g., by hydrolysis), normally followed by purification of fragments of the desired size.
The fragmentation of the polysaccharides is preferably carried out to obtain a final average degree of polymerization (DP) in the oligosaccharide of less than 30 (eg between 10 and 20, preferably about 10 for serogroup A, between 15 and For serogroups W135 and Y, preferably about 15-20, between 12 and 22 for serogroup C, etc.). DP can be conveniently measured by ion exchange chromatography or colorimetric assays [78].
If hydrolysis is performed, the hydrolyzate will generally be sized to remove short-chain oligosaccharides [57]. This can be done in various ways, such as by ultrafiltration followed by ion exchange chromatography. Oligosaccharides having a degree of polymerization of less than or equal to about 6 are preferably removed from serogroup A, and those having a degree of polymerization of less than about 4 are preferably removed from serogroups W135 and Y.
The preferred MenC saccharide antigens used in MENJUGATE ™ are described in reference 77. The saccharide antigen can be chemically modified. This is particularly useful for reducing the hydrolysis of serogroup A [79]. De-O-acetylation of the menongococcal saccharides can be performed. For oligosaccharides, the modification may be carried out before or after depolymerization.
When a composition according to the invention contains a MenA saccharide antigen, the antigen is preferably a modified saccharide in which one or more of the hydroxyl groups on the native saccharide has been or has been replaced by a blocking group [79]. This modification improves the resistance to hydrolysis.
Covalent conjugation
In general, the capsular saccharides in the compositions according to the invention will be conjugated to carrier proteins. In general, conjugation enhances the immunogenicity of saccharides in that it converts them from T-independent antigens into T-dependent antigens, thereby enabling the priming of an immunological memory. Conjugation is particularly useful for pediatric vaccines and is a well-known technique.
Typical carrier proteins are bacterial toxins, such as diphtheria or tetanus toxins, or toxoids or mutants thereof. The CRM197 [80] diphtheria toxin mutant is useful, and is the carrier protein of PREVNAR ™. Other suitable carrier proteins include N. meningitidis outer membrane protein complex [81], synthetic peptides [82, 83], heat shock proteins [84, 85], pertussis proteins [86, 87], cytokines [88], lymphokines [88], hormones [88], growth factors [88], artificial proteins comprising multiple human CD4 + T epitopes from various pathogen-derived antigens [89] such as N19 [ 90], H. influenzae protein [91-93], pneumolysin [94] or non-toxic derivatives [95], pneumococcal surface protein PspA [96], iron-absorbing proteins [97], toxin A or B of C. difficile [98], recombinant exoprotein A of P. aeruginosa (rEPA) [99], etc.
Any suitable conjugation reaction may be used, with any suitable linker, where necessary.
The saccharide will typically be activated or functionalized prior to conjugation. Activation may involve, for example, cyanylation reagents such as CDAP (eg 1-cyano-4-dimethylaminopyridinium tetrafluoroborate [100, 101, etc.]]. Other suitable techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxy-succinimide, S-NHS, EDC, TSTU, and the like.
Linkages via a linker group can be formed using any known procedure, for example the procedures described in references 102 and 103. One type of binding involves reductive amination of the polysaccharide, the coupling of the amino group obtained at one end of the adipic acid linker group, then the coupling of a protein at the other end of the adipic acid linker group [104, 105] . Other linkers include β-propionamido [106], nitrophenyl-ethylamine [107], haloacyl halides [108], glycosidic linkages [109], 6-aminocaproic acid [110], ADH [111], CU to C12 fragments [112], etc. As an alternative to using a linker, the direct link can be used. Direct binding to the protein may include oxidation of the polysaccharide, followed by reductive amination with the protein, as described, for example, in references 113 and 114.
A process involving the introduction of amino groups into the saccharide (eg by replacing the groups = 0 terminals by -NH2), followed by the formation of derivatives with an adipic diester (eg N-hydroxysuccinimidodiester of acid adipic) and reaction with the carrier protein is preferred. Another preferred reaction utilizes activation of CDAP using a carrier protein D, p. ex. for MenA or MenC. Outer membrane vesicles (OMV)
It is preferred that the compositions according to the invention do not contain complex or undefined antigenic mixtures, which are typical characteristics of OMVs. However, the invention may be used together with OMVs, insofar as the fHbp has been found to improve their efficacy [4], either by simple mixing or overexpression of the polypeptides according to the invention in the strains used for the preparation of OMVs.
In general, this approach can be used to improve microvesicle preparations of N. meningitidis serogroup B [115], "native" OMVs [116], "blisters" or outer membrane vesicles (p '. eg refs 117 to 123, etc.).
Typical outer membrane vesicles are artificially prepared from bacteria, and can be prepared by detergent treatment (eg deoxycholate), or by non-detergent means (see, eg, reference 127). Techniques for forming OMVs include treating bacteria with a bile acid salt detergent (eg, salts of lithocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, acid). cholic, ursocholic acid, etc., sodium deoxycholate [124 & 125] being preferred for treating Neisseria) at a sufficiently high pH not to precipitate the detergent [126]. Other techniques can be used essentially in the absence of detergent [127, 128] such as sonication techniques, homogenization, microfluidization, cavitation, osmotic shock, grinding, French press, mixing, etc. Methods using little or no detergent can retain useful antigens such as NspA and fHbp [127]. Therefore, the OMVs used in the context of the invention can be prepared using an OMV extraction buffer containing about 0.5% deoxycholate or less, e.g. ex. about 0.2%, about 0.1%, <0.05% or not at all.
Vesicles known as MV (membrane vesicles) and NOMV (native outer membrane vesicles) are natural membrane vesicles that form spontaneously during bacterial growth and are released into the culture medium. MVs can be obtained by culturing Neisseria in broth-type culture medium, separating whole cells from smaller MVs in broth culture medium (eg by filtration or low speed centrifugation to form only cell pellets and no smaller vesicles), and then collect MV from the depleted cell medium (eg, by filtration, differential precipitation, or aggregation of MVs, by high speed centrifugation to form MV pellets) . In general, the strains that can be used in the production of MV can be chosen according to the amount of MV produced in culture, the refs 135 &amp; 136 describing p. ex. Neisseria as a high-production bacteria of MV.
The vesicles can be prepared from bacteria that have been genetically engineered [139-132] p. ex. to increase immunogenicity (eg, hyperexpressed immunogens), reduce toxicity, inhibit capsular polysaccharide synthesis, under-regulate PorA expression, etc. They can be prepared from hyperproducer strains of vesicles [133-136]. Vesicles from bacteria with different class I outer membrane protein subtypes can be used, e.g. ex. six different subtypes [137, 138] obtained from two different genetically modified vesicle populations each having three subtypes, or nine different subtypes obtained from three different genetically modified vesicle populations, each with three subtypes; types, etc. Useful subtypes include: PI.7,16; PI.5-1,2-2; PI.19,15-1; PI. 52.10; PI.12-1.13; PI.7-2.4; PI.22.14; PI.7-1.1; PI.18-1,3,6. In general, however, it is preferable for the present invention to prepare OMVs from a wild-type meningococcal strain.
The vesicles that can be used in the context of the invention can therefore be prepared from any wild-type meningococcal strain. The vesicles will generally come from a serogroup B strain, but it is possible to prepare them from serogroups other than B (reference 126 describes eg a method for serogroup A), such as A, C, W135. or Y. The strain can be any serotype (eg, 1, 2a, 2b, 4, 14, 15, 16, etc.), any sero-subtype (eg PI.4), and any immunotypes (eg, L1, L2, L3, L3,7, L3,7,9, L10, etc.). Meningococci can come from any suitable lineage, including hyperinvasive and hypervirulent lineages, e.g. ex. of any of the following seven hypervirulent lines: subgroup I; subgroup III; subgroup IV-1; ET-5 complex; complex ET-37; A4 cluster; 3. Most preferably, OMVs are prepared from strain NZ98 / 254, or other strain of sero-subtype PI. 4 PorA.
Advantageously, the invention uses the same OMVs as those used in the BEXSERO ™ and MENZB ™, prepared from the strain NZ98 / 254.
In general, the vesicles will contain lipooligosaccharides (LOS, also known as LPS, lipopolysaccharides), but the pyrogenic effect of LOS in OMV is much lower, in equal amounts, than that of purified LOS, and The adsorption of OMVs on the aluminum hydroxide further reduces their pyrogenicity. LOS levels are expressed in International Units (IU) of endotoxin and can be determined by the LAL assay (Limulus amoebocyte lysate). Preferably, the LOS are present at less than 2000 IU per μg of OMV protein.
When LOS are present in a vesicle, it is possible to treat the vesicle so as to bind its LOS and protein components ("intra-bleb" conjugation [139]).
A useful method of OMV purification is described in reference 140 and involves ultrafiltration on raw OMV rather than high speed centrifugation. The method may involve an ultracentrifugation step after ultrafiltration. OMVs can also be purified by the two-step size exclusion filtration method described in ref. 152.
Usefully, the OMVs can be suspended in a sucrose solution once they have been prepared.
Hollow cells The invention. relates to a bacterium that expresses a polypeptide according to the invention. The bacterium can be a meningococcus or an E. coli. The bacterium may express the polypeptide constitutively, but in some embodiments the expression may be under the control of an inducible promoter. The bacterium can hyper-express the polypeptide (see ref 141). Ideally, the expression of the polypeptide is not variable in phases. The invention also relates to outer membrane vesicles prepared from a bacterium according to the invention (in particular, from a meningococcus). It also relates to a process for producing vesicles from a bacterium according to the invention. The vesicles prepared from these strains preferably contain the polypeptide according to the invention, which will be in an immunoaccessible form in the vesicles, namely an antibody capable of binding to a purified polypeptide according to the invention must also be able to bind to polypeptide that is present in the vesicles.
The bacteria according to the invention may, in addition to coding for a polypeptide according to the invention, comprise one or more other modifications. For example, they may contain a modified gene [142]. NspA expression can be overregulated with concomitant inactivation of porA and cps. Other mutant strains of N. meningitidis for the production of OMV that have been obtained by gene inactivation techniques are described, e.g. ex. in reference 139. Reference 143 describes the construction of vesicles from strains modified to express six different PorA subtypes. Mutant Neisseria strains with low level of endotoxin, obtained by inactivation of the enzymes involved in LPS biosynthesis, can also be used [144, 145]. Neisseria mutant strains manipulated to reduce or even neutralize the expression of at least one gene involved in the toxification of the lipid A portion of LPS, in particular the lpxl 1 gene, may be used in the context of the invention [146]. Similarly, mutant Neisseria strains engineered to reduce or even neutralize the expression of at least one gene involved in the synthesis or export of capsular polysaccharides, in particular synX and / or ctrA genes, can be used in the scope of the invention. These mutants or others may all be used within the scope of the invention.
In some embodiments, a strain may have been underregulated for expression of PorA, e.g. ex. so that PorA expression is reduced by at least 20% (eg> 30%,> 40%,> 50%,> 60%,> 70%,> 80%,> 90%,> 95%, etc.), or even inactivated, relative to wild-type levels (eg relative to strain H44 / 76).
In some embodiments, a strain may hyper-express certain proteins (relative to the corresponding wild-type strain). For example, strains can hyper-express NspA, the protein 287 [117], fHbp [141] (including the fHbp according to the invention), TbpA and / or TbpB [147], Cu, Zn-superoxide dismutase, HmbR, etc.
A gene encoding a polypeptide according to the invention may be integrated into the chromosome of the bacterium or may be present in an episomal form, e.g. ex. in a plasmid.
Advantageously for the production of vesicles, a meningococcus may be genetically modified to ensure that expression of the polypeptide is not phase-varied. Methods for reducing or even eliminating the phase variability of gene expression in meningococci are described in reference 148. For example, a gene may be placed under the control of a constitutive or inducible promoter, or DNA pattern that is responsible for the variability of the phases can be eliminated or replaced.
In some embodiments, a strain may include one or more of the inactivation and / or hyper-expression mutations described in references 122, 129, 133, and 139. For example, according to guidelines and nomenclature in these four documents, the genes useful for under-regulation and / or inactivation include: (a) Cps, CtrA, CtrB, Ctrc, Ctrl, FrpB, GalE, HtrB / MsbB, LbpA, LbpB, LpxK, Opa, Ope, PilC, PorB, SiaA, SiaB, SiaC, SiaD, TbpA, and / or TbpB; (b) CtrA, CtrB, CtrC, CtrD, FrpB, GalE, HtrB / MsbB, LbpA, LbpB, LpxK, Opa, Ope, PhoP, PilC, PmrE, PmrF, SiaA, SiaB, SiaC, SiaD, TbpA, and / or TbpB; (c) ExbB, ExbD, rmpM, CtrA, CtrB, CtrD, GalE, LbpA, LpbB, Opa, Ope, PilC, PorB, SiaA, SiaB, SiaC, SiaD, TbpA, and / or TbpB; or (d) CtrA, CtrB,
CtrD, FrpB, OpA, OpC, PilC, PorB, SiaD, SynA, SynB, SynX and / or SynC.
When a mutant strain is used, in some embodiments, there may be one or more or all of the following characteristics: (i) LgtB and / or GalE subregulated or inactivated to truncate meningococcal LOS; (ii) over-regulated TbpA; (iii) NhhA overregulated; (iv) Omp85 over-regulated; (v) over-regulated LbpA; (vi) Over-regulated NspA; (vii) inactivated PorA; (viii) Subregulated or inactivated FrpB; (ix) Under-regulated or inactivated opa; (x) Subregulated or inactivated ope; (xii) deleted eps gene complex. A truncated LOS may be of the type that does not contain a sialyl-lacto-N-neotetraose epitope, and may be p. ex. LOS deficient in galactose. The LOS may be devoid of a chain.
Depending on the meningococcal strain used to prepare the vesicles, they may or may not contain the native fHbp antigen of the strain [149].
In a preferred embodiment, a meningococcal does not express functional Mit A protein. As explained in refs 150 &amp; 151, in meningococci, the inactivation of MltA (membrane-bound lytic transglycosylase, also known as GNA33) gives bacteria which spontaneously release large amounts of membrane vesicles into the culture medium, from which they can be easily purified. For example, the vesicles can be purified using the two step size exclusion filtration method of ref. 152, comprising: (i) a first filtration step wherein the vesicles are separated from the bacteria according to their size differences, the vesicles passing through the filtrate; and (ii) a second filtration step in which the vesicles are retained in the retentate. The MltA mutation (under-regulation or inactivation) has been used in "GMMA" vaccines [153], and can conveniently be combined with other subregulations or inactivations, in particular, of at least one gene involved in the toxification of the lipid A portion of LPS, in particular of the lpxll gene and / or of at least one gene involved in the synthesis or export of capsular polysaccharides, in particular synX and / or ctrA genes.
A preferred meningococcal strain for a GMMA (Generalized Modules for Membrane Antigens) vaccine using this approach expresses a mutant fHbp v2 according to the first, third or fifth aspect and / or mutant fHbp v3 according to the second, fourth or the sixth aspect according to the invention, and the expression can be regulated by strong promoters. The vesicles released by this strain contain the mutant fHbp v2 and / or v3 proteins in an immunogenic form, and vesicle administration can elicit a bactericidal antibody response as described in reference 153. The strain can also express a fHbp v1. or, instead, a fHbp v1 may be provided as a separate recombinant protein in a soluble form (and the fHbp v1 may be a wild-type or mutant sequence, eg, mutated to alter its fH binding capacity. as described above). The invention relates to these strains, and also relates to the vesicles released by these strains, e.g. ex. in purified form from the culture medium after growth of the strains. A preferred v2 mutant for expression in these strains carries a mutation at residues L123 and E240 (and optionally S32) as described herein, and a preferred v3 mutant for expression in these strains carries a mutation at the residues L126 and E243 (and optionally S32) as described herein. Therefore, vesicles prepared from meningococci expressing these mutant fHbp v2 and v3 sequences are particularly preferred immunogens for use in vaccines according to the invention. A wild-type v2 sequence useful for this type of mutagenesis comprises SEQ ID NO: 35 or SEQ ID NO: 33 (including SEQ ID NO: 34, AG form), and a wild-type v3 sequence useful for this type of mutagenesis includes SEQ ID NO: 36.
Useful promoters for use in these strains include those described in references 154 and 155. For example, the promoters may be: (a) promoters of porin genes, preferably porA or porB, particularly N. meningitidis ; or (b) an rRNA gene promoter (such as a 16S rRNA gene), particularly N. meningitidis. When a meningococcal porin promoter is used, it is preferably derived from porA, and more particularly from a -10 region of a porA meningococcal gene promoter, and / or a -35 region of a promoter. of porA meningococcal gene (preferably wherein the region -10 and the -35 region are separated by an intervening sequence of 12-20 nucleotides, and wherein the intervening sequence is either not containing a poly-G sequence, or contains a sequence poly-G having no more than eight nucleotides G). When an rRNA gene promoter is used, it may more particularly include (i) a region -10 from a meningococcal rRNA gene promoter and / or (i i) a -35 region from a meningococcal rRNA gene promoter. It is also possible to use a hybrid of (a) and (b), for example containing a region -10 from a porA promoter and a region -35 from an rRNA promoter (which may be a region -35 consensus). A useful promoter can therefore be a promoter that contains either (i) a region -10 from an rRNA gene (in particular, meningococcal) and a -35 region from a porA gene (in particular, meningococcal), or (ii) a region -10 from a porA gene (in particular, meningococcal) and a region -35 from an rRNA gene (in particular, meningococcal). Overview
The term "comprising" includes "containing" as well as "constituted by", eg ex. a composition "comprising" X may consist exclusively of X or may contain something else, e.g. ex. X + Y. The references to "comprising" (or "includes", etc.) may possibly be replaced by references to "consisting of" (or "consisting of", etc.).
The term "about" in relation to a numerical value x is optional and refers, for example, x ± 10%.
The term "substantially" does not exclude "completely", p. ex. a composition that is "substantially free" of Y may be completely Y-free. Where necessary, the term "substantially" may be omitted from the definition of the invention. "Sequence identity" is preferably determined by the Needleman-Wunsch global alignment algorithm [156], using the default parameters (eg with gap opening penalty = 10.0 , and a gap extension penalty = 0.5, using the notation matrix EBLOSUM62). This algorithm is easy to implement in the needle tool of the EMBOSS software [157]. When the application refers to a sequence identity with a particular SEQ ID, the identity must be calculated over the entire length of that SEQ ID.
After serogroup, meningococcal classification includes serotype, serotype subtype, then immunotype, and standard nomenclature lists serogroup, serotype, serotype subtype, and immunotype, each separated by a colon, p. ex. B: 4: PI. 15: L3, 7, 9. In serogroup B, some lines often cause the disease (hyperinvasive), some lines cause more severe forms of the disease than others (hypervirulent), and others rarely cause the disease. Seven hypervirulent lines are recognized, namely subgroups I, III and IV-1, complex ET-5, complex ET-37, cluster A4 and line 3. They were defined by multilocus electrophoresis of enzymes ( MLEE), but multilocus sequence typing (MLST) was also used to classify meningococci. The four main hypervirulent clusters are the ST32, ST44, ST8 and ST11 complexes.
In general, the invention does not encompass the various fHbp sequences specifically described in references 2, 3, 5, 6, 7, 158, 159, 160, 161, 162, 163, 164 and 165.
EXAMPLES
Example 1: Mutagenesis for the link Φ fH
The wild-type v2 protein (SEQ ID NO: 2) shows strong fH binding when assessed by surface plasmon resonance (SPR) with immobilized human fH (Figure 1, top curve). To alter the ability of fHbp to bind to fH, Glu-266 in v2 (E240 in SEQ ID NO: 5, corresponds to E248 in references 19 & 25) and Glu-274 in v3 (E243 in SEQ ID NO: 17) were transferred to Ala. The E266A mutation in v2 strongly reduced the binding to fH (Figure 1, bottom curve).
Similarly, the known "R41S" mutation of fHbp v1 was introduced (SEQ ID NO: 52).
Example 2: Mutagenesis for Increasing Stability fHbp v2 and v3 are both significantly less stable than v1, particularly in their N-terminal domains, and v2 is the least stable of the three. To improve the stability in v2, two residues were mutated: Ser-58 of
SEQ ID NO: 2 (S32 in SEQ ID NO: 5) and Leu-149 of SEQ ID NO: 2 (L123 in SEQ ID NO: 5) were mutated to Val and Arg, respectively. The mutant v2 protein (SEQ ID NO: 19) was analyzed by DSC and, compared to the wild-type SEQ ID NO: 2 sequence, the Tm of the C-terminal domain was unaffected by the mutation. The increase in the Tm of the N-terminal domain is> 20 ° C (Figure 2, increase shown by the arrow). Equivalent mutations have also been introduced in v3 (SEQ ID NO: 44).
Surprisingly, although S58V and L149R mutations have been introduced to improve stability, and have indeed fulfilled this function, Figure 1 (middle curve) shows that the mutant polypeptide (SEQ ID NO: 19) (even without the E266A mutation) also showed a very reduced binding to fH. In addition, in a serum bactericidal assay, this mutant was able to compete for binding to human antibodies that were directed against SEQ ID NO: 18:
The S58V / L149R stabilizing mutation in v2 had a surprising effect on fH binding, so the effect of E266A on stability was also studied. Unexpectedly, this mutation reduced the stability of the N-terminal domain, but increased that of the C-terminal domain by> 15 ° C (from 83 to 99 ° C, as shown in Figure 3), compared to the wild-type, suggesting a potential stabilization of the beta-barrel.
The effects of individual S58V and L149R mutations on the fH binding were studied in v3. Therefore, numbered according to SEQ ID NO: 17, mutation S32V or L126R was introduced in sequence v3. These two mutants were compared to two different wild-type v3 sequences, and also to the "E313A" mutant that is known to alter fH binding in v3 [23].
As shown in Figure 6, the two wild type v3 bind fH (the two top curves). The S58V mutation, which was designed to improve stability, reduced the SPR peak by about 2-fold. More surprisingly, the L149R mutation (again, designed to improve stability) reduced the affinity for fH to a level similar to that of the known E313A mutant (the two bottom curves).
The S58V and L149R mutations in v3 were also studied by DSC, and were found to increase the N-terminal Tm of 5.5 ° C (S58V) or 6.7 ° C (L149R). The Tm of each mutant was greater than that observed in the S58V / L149R v2 double mutant. The L149R v3 mutant also showed a higher Tm value for its C-terminal domain, whereas there was hardly any shift for the S58V v3 mutant.
Example 3: Fusion Polypeptides
The stability and binding mutations of fHbp were combined into mutant forms of v2 (SEQ ID NO: 50) and v3 (SEQ ID NO: 51). They were fused to the mutant v1 sequence (SEQ ID NO: 52) in the order v2-v3-v1 and joined using linkers to obtain SEQ ID NO: 27 ("SNB"). ). Therefore, compared to the three wild-type sequences, this fusion polypeptide contains a total of 7 point mutations (Figure 9). The SNB fusion was compared to a "wild-type" fusion polypeptide devoid of these point mutations (SEQ ID NO: 18, SEQ ID NO: 36 in reference 163). Extracts of E. coli expressing these two forms of the protein were analyzed by Western blotting, and the forms of degradation of the protein were much less visible with the stabilized non-binding forms of the fusion (SEQ ID NO: 27) ( Figure 8).
The binding of SNB fusion to fH was studied by SPR, and compared to "wild-type" fusion. Figure 4 shows that the "wild-type" fusion shows strong fH binding (top curve), whereas the SNB mutant does not interact significantly with fH (bottom curve).
The stability of the two fusion polypeptides was studied by DSC (Figure 5). The "wild type" merge thermogram (Figure 5A) contained no N-terminal transitions attributable to v3, suggesting that this domain was not correctly folded. In contrast, with the mutant "SNB", the thermogram showed transitions for the 6 domains (3 of each for the N- and C-termini), indicating that they were all correctly folded (Figure 5B). Separately, the stability mutations in v2 (SEQ ID NO: 45) and v3 (SEQ ID NO: 44) were fused to the mutant v1 sequence "R41S" (SEQ ID NO: 52) in order v2-v3- v1 and joined with linkers to obtain SEQ ID NO: 29. Therefore, compared to the three wild type sequences, this fusion polypeptide contains a total of 5 point mutations.
The ability of non-binding forms of fHbp to elicit SBA titres was tested in
transgenic mice (Tg): _
These data indicate that non-binding forms of fHbp may be more immunogenic.
Example 4: 3D Structures
So far, the structure of fHbp var.3 has only been split in complex with fH. For the fHbp v2-fH complex, only the C-terminal domain of fHbp was detectable in previous studies.
Crystals of the fHbp V2 and V3 mutants were prepared as follows: Crystallization experiments were conducted using a Gryphon crystallization robot (Art Robbins Instruments). X-ray diffraction data were collected by the Pilatus 2M X06DA X-ray beams of the Swiss Light Source (Paul Scherrer Institute, Villigen, Switzerland) or by the BM30A X-ray beams of the European synchrotron radiation (ESRF) from Grenoble, France. All diffraction data were processed with iMosflm, scaled with Aimless, and crystallographic manipulations were implemented using CCP4 software
.__
Stabilization mutations potentiate the determination of the N-terminal domain structure of var.2 and the X-ray structure of fHbp var.3 S58V was resolved in the absence of fH. fHbp var.2 and var.3 are characterized by less stable folding compared to var.l. Given this observation, the complete structure of fHbp var2 and var3 was difficult to determine. The stabilization of the protein results in the conservation of both the structure and the functionality, coupled with the establishment of a better thermodynamic equilibrium with the (micro) environment. Consequently, the stabilization of the protein often makes it possible to obtain crystals suitable for determining the structure. The stabilized S58V and L149R substitutions allowed the determination of the complete crystal structure of fHbp var.3 and the splitting of the 81-254 segment of fhbp var.2. By introducing stabilizing mutations, the almost complete N-terminal domain structure was obtained in the absence of fH (Figure 7).
Example 5 Surface Plasmon Resonance Analysis (SPR)
SPR was used to analyze the binding of chimeric 231 proteins to fH proteins. All SPR experiments were performed on a Biacore T200 at 25 ° C (GE Healthcare). Briefly, a carboxymethyl dextran functionalized biosensor (CM-5, GE Healthcare) was prepared in which similar densities (~ 400-500 response units (RUs)) of 231 proteins were immobilized by amine coupling. The immobilized proteins were: -231 wt (SEQ ID NO: 18) MenB 547 purification (0.26 mg / ml) immobilized on 2-231 S flow cell, comprising R41S, S58V and L149 for both v2 and v3 (SEQ ID NO: 29) MenB 532 purification (0.68 mg / ml) immobilized on SNB flow cell, including R41S, S58V and L149 for both v2 and v3, E252A and E255A (SEQ ID NO: 29). NO: 27) MenB 512 purification (0.78 mg / ml) immobilized on flow cell 4
These proteins were diluted to 5 μg / ml in pH 5.5 acetate and an amine coupling protocol was followed to achieve the target density. Flow cell 1 was prepared like the others but no protein was used. It was then used as a reference cell and the signal obtained was subtracted from the signal from the other flow cells. The migration buffer contained 10 mM Hepes, 150 mM NaCl, 0.05% (vol / vol) P20 surfactant, pH 7.4 (HBS-P-GE-Healthcare). Then the fH proteins were injected in a range of five injections with increasing concentration of analyte in 2-fold dilutions (62.5 nM to 1 DM) for the binding experiments. The following fH constructs were tested: full-length H factor (Calbiochem) and factor H comprising only the 6-7 domains (Schneider et al., Nature 458, 890-893) provided by C. Tang.
After each injection, the surfaces were regenerated by a 20 second injection of 10 mM glycine pH 1.7. A blank injection consisting solely of the buffer was removed from each curve, and the reference sensorgrams were removed from the experimental sensorgrams to obtain the curves representing the specific binding. The illustrated data represent two independent experiments. The SPR data was analyzed using the Biacore T200 (GE Healthcare) evaluation software. The obtained sensorgrams were fitted using the Langmuir 1: 1 binding model, with a term to account for potential mass transfer, and to obtain the individual kon and koff kinetic constants; the individual values were then combined to derive the individual averaged KD values (Kd = K0ff / K0n) reported. Equilibrium analysis was also used to obtain thermodynamic dissociation constants (KD) at pH 7.4. The results of the titration with the injection of the 6-7 domains of the fH are indicated below:
Based on binding assays, a strong reduction of at least 90% in fH binding was observed for 231 S protein compared to 231 wt and at least 98% for 231 SNB protein compared to 231 wt .
The full-length fH titration results are shown below: _ ____ _
Based on binding assays, a strong reduction of at least 90% in fH binding was observed for 231 S protein compared to 231 wt and 98% for 231 SNB protein compared to 231 wt.
It will be understood that the invention is described above only by way of examples and that modifications can be made while remaining within the scope and spirit of the invention.
REFERENCES
[1] WO99 / 57280.
[2] Masignani et al. (2003) J Exp Med 197: 789n799.
[3] Welsch et al. (2004) J Inminol 172: 5605 (015.
[4] Hou et al. (2005) J Infect Dis 192 (4): 580n90.
[5] WO03 / 063766.
[6] Fletcher et al. (2004) Infect Immun 72: 2088n2100.
[7] Zhu et al. (2005) Infect Immun 73 (10): 6838n45.
[8] Cendron et al. (2011) Acta Crystallogr Sect Struct Biol Cryst Common. 67: 531 n5.
[9] Mascioni et al. (2009) J. Biol Chem284: 8738046.
[10] Pizza et al. (2008) Vaccine26 Suppl 8: I46n8.
[11] Malitoet al. (2013) PNASUSM10: 3304n9.
[12] Marshall et al. (2012) Pediatr Infect Dis J 31: 1061n8.
[13] McNeil et al. (2013) Microbiol Mol Biol Rev 77: 234052.
[14] Serruto et al. (2012) Vaccine 30 Suppl 2: B87O97.
[15] Scarselli et al. (2011) Sei Transi Med3: 91ra62.
[16] Ruan et al. (1990) J Immunol 145: 3379 (03384.
[17] European patent 0301992.
[18] Bjune et al. (1991) Lancet 338 (8775): 1093ril096.
[19] Frasch et al. (2001) chapter 7 of Methods in Molecular Medicine, volume 66 (Meningococcal Vaccines: Methods and Protocolsi, Pollard & Maiden).
[20] Fukasawa et al. (1999) Vaccine 17: 2951 (02958.
[21] W002 / 09746.
[22] European Patent 0449958.
[23] EPfiAn0996712.
[24] WO99 / 59625.
[25] US Patent 6,180,111.
[30] Giuntini et al. (2012) PLoSOne7: e34272.
[31] Vu et al. (2012) Sei Rep2: 341.
[32] Faleri et al. (2013) FASEB J, 13, 239012.
[33] Johnson (2013) Arch Biochem Biophys531: 100fi9.
[34] Bruylants et al. (2005) Current Medicinal Chemistry 12: 2011 (020.
[35] Veggi et al. (2012) Biochemistry 51: 9384n93.
[36] Pizza et al. (2000) Science287: 1816fil820.
[37] WO2007 / 028408.
[38] http://pubmlst.org/ neisseria / [39] Budroni et al. (2011) PNASUS 108: 4494099.
[40] Goldschneider et al. (1969) J. Exp. Med. 129: 1307026.
[41] Santos et al. (2001) Clinical and Diagnostic Laboratory Immunology 8: 616023.
[42] Frasch et al. (2009) Vaccine27S: B1 12n6.
[43] Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472.
[44] W003 / 009869.
[45] Vaccine DesignÔ (1995) eds. Powell &amp; Newman. ISBN: 030644867X. Plenum.
[46] Tettelinet al. (2000) Science 287: 1809n1815.
[47] WO00 / 66741.
[48] Martin et al. (1997) J Exp Med 185 (7): 1173fi83.
[49] WO96 / 29412.
[50] PerkinsfiBalding et al. (2003) Microbiology 149: 3423n35.
[51] WO01 / 55182.
[52] W001 / 38350.
[53] WO00 / 23595.
[54] Giuliani et al. (2006) Proc Natl Acad Sci U SA. 103: 1083409.
[55] WO2004 / 032958.
[56] Costantino et al. (1992) Vaccine 10: 691n698.
[57] Costantino et al. (1999) Vaccine 17: 1251nl263.
[58] W003 / 007985.
[59] Watson (2000) Pediatr Infect Dis J 19: 331n332.
[60] Rubin (2000) Pediatr Clin North Am 47: 269-285, c.
[61] Jedrzejas (2001) Microbiol Mol Biol Rev 65: 1870207.
[62] Bell (2000) Pediatr Infect Dis J 19: 1187nl 188.
[63] Iwarson (1995) APMIS 103: 321 n326.
[64] Gerlich et al. (1990) Vaccine8 Suppl: S63n68 &amp; 79n80.
[65] Vaccines (1988) eds. Plotkin &amp; Mortimer. ISBN 0ή7216η1946ή0.
[66] Del Guidice et al. (1998) Molecular Aspects of Medicine, 19: 1970.
[67] Gustafsson et al. (1996) N. Engl. J. Med. 334: 3490355.
[68] Rappuoli et al. (1991) Tl BTECH 9: 232n238.
[69] Sutter et al. (2000) Pediatr Clin North Am 47: 287fi308.
[70] Zimmerman &amp; Spann (1999) AmFamPhysician59: 1130118, 1250126.
[71] McMichael (2000) Vaccine 19 Suppl 1: S101n107.
[72] Schuchat (1999) Lancet 353 (9146): 5106.
[73] WO02 / 34771.
[74] Dale (1999) Disfect DisClin North Am 13: 227043, viii.
[75] Ferrettiet al. (2001) PNASUS 98: 4658O4663.
[76] Kurodaet al. (2001) Lancet 357 (9264): 1225n1240; see also pages 1218Ö1219.
[77] Jones (2001) Curr Opin Investig Investigations2: 47049.
[78] Ravenscroft et al. (1999) Vaccine 17: 280202816.
[79] W003 / 080678.
[80] Research Disclosure ^ 453077 (Jan 2002).
[81] ΕΡήΑή0372501.
[82] EPRAn0378881.
[83] ΕΡηΑή0427347.
[84] W093 / 17712.
[85] WO94 / 03208.
[86] WO98 / 58668.
[87] ΕΡηΑη0471177.
[88] W091 / 01146.
[89] Falugi et al. (2001) Eur J Immunol 31: 381603824.
[90] Baraldo et al. (2004) Infect Immun 72 (8): 4884n7.
[91] EPnAR0594610.
[92] Ruan et al. (1990) J Immunol 145: 337903384.
[93] WO00 / 56360.
[94] Kuo et al. (1995) Infect Immun 63: 2706n13.
[95] Michon et al. (1998) Vaccine. 16: 1732n41.
[96] W002 / 091998.
[97] WO01 / 72337.
[98] WO00 / 61761.
[99] WO00 / 33882 [100] Leesetal. (1996) Vaccine 14: 190-198.
[101] WO95 / 08348.
[102] US patent 4,882,317 [103] US Patent 4,695,624 [104] Porro et al. (1985) Mol Immunol 22: 907n919.s [105] EPn20208375 [106] WO00 / 10599 [107] Gever et al. Med. Microbiol. Immunol. 165: 171, 288 (1979).
[108] US patent 4,057,685.
[109] US patents 4,673,574; 4,761,283; 4808700.
[110] US patent 4,459,286.
[111] US patent 4,965,338 [112] US patent 4,663,160.
[113] US Patent 4,761,283 [114] US Patent 4,356,170 [115] W002 / 09643.
[116] Katial et al. (2002) Infect Immun 70: 702n707.
[117] WO01 / 52885.
[118] European patent 0301992.
[119] Bjune et al. (1991) Lancet 338 (8775): 1093fil096.
[120] Frasch et al. (2001) chapter 7 of Methods in Molecular Medicine, volume 66 (Evolvingococcal Vaccines: Methods and Protocolsi, eds Pollard & Maiden).
[121] Fukasawaet al. (1999) Vaccine 17: 295ln2958.
[122] W002 / 09746.
[123] Rosenqvist et al. (1998) Dev. Biol. Stand. 92: 323n333.
[124] European patent 0011243.
[125] Fredriksen et al. (1991) NI PH Ann. 14 (2): 67fi80.
[126] WO01 / 91788.
[127] WO2004 / 019977.
[128] US patent 6,558,677.
[125] W001 / 09350.
[130] European patent 0449958.
[131] ΕΡήΑή0996712.
[132] EPnAfi0680512.
[133] WO02 / 062378.
[134] WO99 / 59625.
[135] US patent 6,180,111.
[136] WO01 / 34642.
[137] Peeters et al. (1996) Vaccine 14: 1008 (11015.
[138] Vermont et al. (2003) Infect Immun 71: 1650hl655.
[139] WO2004 / 014417.
[140] WO2005 / 004908.
[141] WO2006 / 081259.
[142] WO98 / 56901.
[143] Claassen et al. (1996) 14 (10): 1001 n8.
[144] WO99 / 10497.
[145] Steeghs et al. (2001) The EM BO Journal 20: 6937-16945.
[146] Fisseha ei al. (2005) Infect Immun 73: 4070ή80.
[147] WO00 / 25811.
[148] WO2004 / 015099.
[149] WO2004 / 046177.
[150] WO2006 / 046143.
[151] AdunBobie et al. (2004) Infect Immun 72: 1914nl9.
[152] WO2011 / 036562.
[153] Koeberling et al. (2014) Vaccine32: 2688n95.
[154] WO2013 / 033398.
[155] WO2013 / 113917.
[156] Needleman &amp; Wunsch (1970) J. Mol. Biol. 48, 4430453.
[157] Rice et al. (2000) Trends Genet 16: 276-27.
[158] WO01 / 64920.
[159] W003 / 020756.
[160] WO2004 / 048404.
[161] WO2004 / 094596 [162] WO2006 / 024954.
[163] WO2007 / 060548.
[164] WO2009 / 104097.
[165] WO2013 / 132452. '

权利要求:
Claims (18)
[1]
A polypeptide comprising an amino acid sequence having a sequence identity of at least 80% with SEQ ID NO: 5, wherein (a) the amino acid sequence differs from SEQ ID NO: 5 at residues 123 and 240 with respect to SEQ ID NO: 5; (b) the polypeptide may, after administration to a human subject, elicit the production of antibodies capable of recognizing a polypeptide consisting of SEQ ID NO: 4; (c) the polypeptide has a higher stability than a polypeptide consisting of SEQ ID NO: 4; and (d) the polypeptide has a lower affinity for human fH than a polypeptide consisting of SEQ ID NO: 4.
[2]
The polypeptide of claim 1, wherein the amino acid sequence differs from SEQ ID NO: 5 also at residue 32 with respect to SEQ ID NO: 5; for example, comprising the amino acid sequence SEQ ID NO: 47.
[3]
A polypeptide according to claim 2 having substitutions S32V, L123R, and E240A with respect to SEQ ID NO: 5, p. ex. SEQ ID NO: 50.
[4]
A polypeptide comprising an amino acid sequence having a sequence identity of at least 80% with SEQ ID NO: 17, wherein (a) the amino acid sequence differs from SEQ ID NO: 17 at the residues 126 and 243 with respect to SEQ ID NO: 17; (b) the polypeptide may, after administration to a human subject, elicit the production of antibodies capable of recognizing a polypeptide consisting of SEQ ID NO: 40; (c) the polypeptide has a higher stability than a polypeptide consisting of SEQ ID NO: 40; and (e) the polypeptide has a lower affinity for human fH than a polypeptide consisting of SEQ ID NO: 40.
[5]
The polypeptide of claim 4, wherein the amino acid sequence differs from SEQ ID NO: 17 also at residue 32 with respect to SEQ ID NO: 17; for example, comprising the amino acid sequence SEQ ID NO: 48.
[6]
6. The polypeptide of claim 4 or claim 5 having substitutions S32V, L126R, and E243A with respect to SEQ ID NO: 17, p. ex. SEQ ID NO: 51.
[7]
A polypeptide comprising: an amino acid sequence having a sequence identity of at least 90% with SEQ ID NO: 47, wherein with respect to SEQ ID NO: 47, the residue 123 is not leucine and the residue 240 is not glutamate; and wherein (i) compared to SEQ ID NO: 4, the polypeptide has higher stability and lower affinity for fH; and (ii) when administered to a human subject, the polypeptide may elicit an antibody response that is bactericidal to a meningococcus that expresses fHbp v2, or: the amino acid sequence SEQ ID NO: 47, modified at height of 5 individual amino acid substitutions, provided that (i) residue 123 is not leucine, (ii) residue 240 is glutamate, (iii) compared to wild-type sequence, e.g. ex. SEQ ID NO: 4, the polypeptide has a higher stability and a lower affinity for fH, and (iv) when administered to a human subject, the polypeptide can elicit an antibody response that is bactericidal to a meningococcus that expresses a fHbp v2.
[8]
The polypeptide of claim 7 wherein residue 32 to SEQ ID NO: 47 is non-serine.
[9]
A polypeptide comprising: an amino acid sequence having a sequence identity of at least 90% with SEQ ID NO: 48, wherein with respect to SEQ ID NO: 48, residue 126 is not leucine and the residue 243 is not glutamate; and wherein (i) compared to SEQ ID NO: 40, the polypeptide has higher stability and lower affinity for fH; and (ii) when administered to a human subject, the polypeptide may elicit an antibody response that is bactericidal to a meningococcus that expresses fHbp v3, or: the amino acid sequence SEQ ID NO: 48, modified at height of 5 individual amino acid substitutions, provided that (i) the residue 126 is not leucine, (ii) the residue 243 is glutamate, (iii) compared to the wild-type sequence, e.g. ex. SEQ ID NO: 40, the polypeptide has a higher stability and lower affinity for fH, and (iv) when administered to a human subject, the polypeptide can elicit an antibody response that is bactericidal to a meningococcus that expresses a fHbp v3.
[10]
The polypeptide of claim 9, wherein the residue 32 with respect to SEQ ID NO: 48 is not serine.
[11]
A fusion polypeptide comprising: (i) a polypeptide as defined in any one of claims 1, 2, 3, 7, or 8; and (ii) a polypeptide as defined in any one of claims 4, 5, 6, 9, or 10.
[12]
12. A fusion polypeptide that (a) can, when administered to a human subject, elicit an antibody response that is bactericidal to both a meningococcus that expresses fHbp v2 and a meningococcus that expresses fHbp v3; (b) has a higher stability and a lower affinity for human fH than a polypeptide consisting of SEQ ID NO: 4; (c) has a higher stability and a lower affinity for human fH than a polypeptide consisting of SEQ ID NO: 40, wherein the polypeptide comprises: (I) a first amino acid sequence selected from: ) an amino acid sequence having a sequence identity of at least k% with SEQ ID NO: 5, and / or comprising a fragment of SEQ ID NO: 5; wherein the amino acid sequence differs from SEQ ID NO: 5 at residues L123 and E240 (and optionally at S32) with respect to SEQ ID NO: 5; (b) an amino acid sequence having a sequence identity of at least v% with SEQ ID NO: 47, wherein, with respect to SEQ ID NO: 47, (i) residue 123 is not leucine (ii) residue 240 is not glutamate, (iii) optionally residue 32 is not serine; and the amino acid sequence SEQ ID NO: 47 or SEQ ID NO: 50, optionally modified to 5 individual amino acid changes (i.e. 1, 2, 3, 4 or 5 substitutions, deletions and / or insertions individual), wherein (i) optionally residue 32 is not serine, (ii) residue 123 is not leucine, and (iii) residue 240 is not glutamate; and (II) a second amino acid sequence selected from: an amino acid sequence wherein: (a) the amino acid sequence has a sequence identity of at least 1% with SEQ ID NO: 17, and / or comprises a fragment of SEQ ID NO: 17; wherein (b) the amino acid sequence differs from SEQ ID NO: 17 at residues L126 and E243 (and optionally at S32) with respect to SEQ ID NO: 17; an amino acid sequence having at least w% sequence identity with SEQ ID NO: 48, wherein (i) residue 126 is not leucine, (ii) residue 243 is not glutamate, and (iii) optionally the residue 32 is not serine; or the amino acid sequence SEQ ID NO: 48 or SEQ ID NO: 51, optionally modified to 5 individual amino acid changes (i.e. 1, 2, 3, 4 or 5 substitutions, deletions and / or insertions individual), wherein (i) residue 32 is any amino acid, possibly different from serine, (ii) residue 126 is not leucine, and (iii) residue 243 is not glutamate.
[13]
The fusion polypeptide of claim 12, wherein either (A) the first amino acid sequence is SEQ ID NO: 47 (e.g., SEQ ID NO: 50) and the second amino acid sequence is SEQ ID NO: 48 (e.g., SEQ ID NO: 51), or (B) the first amino acid sequence is SEQ ID NO: 31 (e.g., SEQ ID NO: 45) and the second amino acid sequence is amino acid is SEQ ID NO: 32 (e.g., SEQ ID NO: 44).
[14]
The fusion polypeptide according to any of claims 11 to 13, further comprising a fHbp v1 sequence, wherein the polypeptide can, when administered to a human subject, elicit an antibody response that is bactericidal to a meningococcus that expresses a fHbp v1, a meningococcus that expresses fHbp v2, and a meningococcus that expresses fHbp v3; and wherein, optionally, the fHbp v1 sequence contains a mutation which confers on it a lower affinity for human fH than a meningococcal polypeptide consisting of SEQ ID NO: 46, p. ex. wherein the sequence fHbp v1 has the amino acid sequence SEQ ID NO: 49 or 52.
[15]
The fusion polypeptide of claim 14, comprising the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30.
[16]
An immunogenic composition comprising a pharmaceutically acceptable carrier and a polypeptide selected from the polypeptides of claims 1 to 10 and the fusion polypeptides of claims 11 to 15.
[17]
17. An immunogenic composition according to claim 16, further comprising an adjuvant.
[18]
A method for eliciting an antibody response in a human subject, comprising administering to the human subject an immunogenic composition of claim 16.

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

WO2011126863A1|2010-03-30|2011-10-13|Children's Hospital & Research Center Oakland|Factor h binding proteins with altered properties and methods of use thereof|

---

WO2014030003A1|2012-08-23|2014-02-27|Isis Innovation Limited|Neisseria meningitidis fhbp variant and its use for vaccination|

---

GB0227346D0|2002-11-22|2002-12-31|Chiron Spa|741|

---

GB0419408D0|2004-09-01|2004-10-06|Chiron Srl|741 chimeric polypeptides|

---

GB0524066D0|2005-11-25|2006-01-04|Chiron Srl|741 ii|

---

US20130022633A1|2009-10-27|2013-01-24|University Of Florence|MENINGOCOCCAL fHBP POLYPEPTIDES|

---

US20130011429A1|2010-03-10|2013-01-10|Jan Poolman|Immunogenic composition|

---

BR112014031386A2|2012-06-14|2017-08-01|Pasteur Institut|serogroup x meningococcal vaccines|

---

PL3110442T3|2014-02-28|2021-04-19|Glaxosmithkline Biologicals Sa|Modified meningococcal fhbp polypeptides|

---

US20180214531A1|2014-07-17|2018-08-02|Glaxosmithkline Biologicals, Sa|Meningococcus vaccines|TR201802933T4|2010-03-30|2018-03-21|Childrens Hospital & Res Center At Oakland|The modified factor h binding proteinsand their method of use.|

---

PL3110442T3|2014-02-28|2021-04-19|Glaxosmithkline Biologicals Sa|Modified meningococcal fhbp polypeptides|

---

KR20170028442A|2014-07-23|2017-03-13|칠드런즈 하스피틀 앤드 리써치 센터 앳 오클랜드|Factor h binding protein variants and methods of use thereof|

---

JP2020500859A|2016-11-25|2020-01-16|グラクソスミスクライン バイオロジカルズ ソシエテ アノニム|nOMV-antigen conjugates and uses thereof|

---

EP3607967A1|2018-08-09|2020-02-12|GlaxoSmithKline Biologicals S.A.|Modified meningococcal fhbp polypeptides|

---

WO2021126421A1|2019-12-19|2021-06-24|Dow Technology Investments Llc|Processes for preparing isoprene and mono-olefins comprising at least six carbon atoms|

法律状态:
2019-04-01| FG| Patent granted|Effective date: 20160623 |

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2019-04-01| MM| Lapsed because of non-payment of the annual fee|Effective date: 20180731 |

优先权:

---

申请号 | 申请日 | 专利标题

---

EP14177564|2014-07-17|

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EP14177564.3|2014-07-17|

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