# Patent

## Title
Promoters for increased protein expression in meningococcus

## Abstract

New promoters are described to drive transcription in meningococcus e.g. for over-expression of protein antigens for retention in membrane vesicles. Modified porA promoters lack the wild-type poly-G sequence which can cause phase variation. Meningococcal rRNA-coding genes (e.g. for 16S rRNA) can be used to drive transcription of a protein-coding gene. These approaches can be used in combination.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a U.S. National Phase of International Patent Application No. PCT/EP2013/052108 filed Feb. 1, 2013 and published in English, which claims the benefit of the U.S. provisional patent application No. 61/594,159, filed Feb. 2, 2012, the complete contents of which are incorporated herein by reference for all purposes.


SEQUENCE LISTING
The application includes a sequence listing that was filed with the PCT application as a part of the specification. The sequence listing is hereby incorporated by reference.
TECHNICAL FIELD
This invention is in the field of promoters for controlling transcription in meningococcal bacteria.
BACKGROUND ART
Neisseria meningitidis is a cause of bacterial meningitis. One approach to meningococcal vaccination relies on outer membrane vesicles (OMVs), as used in the Novartis MENZBTM product and the Norwegian Institute of Public Health MENBVACTM product, both of which are generated by detergent treatment of meningococci.
It is known to change the protein composition of meningococcal outer membranes, and thus to change the composition of OMVs. Knockout of undesirable genes and over-expression of desirable genes have both been described. References 1-3 report pre-clinical studies of an OMV vaccine in which fHbp (also known as GNA1870) is over-expressed (and this over-expression can be combined with knockout of LpxL1 [4]). Reference 5 reported a clinical study of five formulations of an OMV vaccine in which PorA & FrpB are knocked-out and Hsf & TbpA are over-expressed. Reference 6 reports a phase I clinical study of a native outer membrane vesicle vaccine prepared from bacteria having inactivated synX and lpxL1 genes, over-expressed fHbp, two different PorA proteins and stabilised OpcA expression. Reference 7 reports a trivalent native outer membrane vesicle vaccine prepared from bacteria having inactivated synX and lpxL1 genes (and, in two cases, inactivated lgtA), two different PorA proteins, and over-expressed NadA or fHbp. Inactivation of genes such as lpxL1 is important if vesicles will be generated by methods which do not remove LPS.
These changes in protein composition can be effected in various ways. For instance, the bacteria can be growing under iron-limiting conditions in order to stimulate the expression of certain proteins related to iron metabolism. Other techniques can involve engineering the bacteria. For instance, reference 8 suggests that strong promoters (such as the porA, porB, lgtF, or hpuAB promoters) might be used to up-regulate expression of protective outer membrane proteins, or that suppressive transcription control mechanisms might be removed. Reference 9 suggests that genes can be engineered to remove their phase variability. The porA promoter was used in reference 7 to over-express NadA, whereas fHbp was over-expressed using the tac promoter.
It is an object of the invention to provide further and improved ways of modifying protein expression in meningococci, and in particular to provide promoters for driving expression of genes of interest. Up-regulation can be used to increase the levels of useful proteins in OMVs.
DISCLOSURE OF THE INVENTION
A first aspect of the invention uses modified porA promoters to drive transcription. The natural porA promoter, as used in examples 10-16 of reference 8, contains a poly-G sequence between its −35 and −10 regions, but this sequence can cause phase variation and so expression from the natural porA promoter can be unstable. The modified porA promoters of the invention are improved because they lack the wild-type poly-G sequence.
Thus the invention provides a nucleic acid comprising a promoter which includes

    
    
        (i) a −10 region from a meningococcal porA gene promoter and/or
        (ii) a −35 region from a meningococcal porA gene promoter,

wherein the −10 region and the −35 region are separated by an intervening (or spacer) sequence of 12-20 nucleotides, and wherein the intervening sequence either contains no poly-guanidine sequence or includes a poly-guanidine sequence having no more than eight consecutive guanidine nucleotides.

    
    


A second aspect of the invention uses promoters from a meningococcal rRNA-coding gene (such as the 16S rRNA gene) to drive transcription of a protein-coding gene. Thus the invention also provides a nucleic acid comprising a promoter operably linked to a downstream protein-coding gene, wherein the promoter includes (i) a −10 region from a meningococcal rRNA gene promoter and/or (ii) a −35 region from a meningococcal rRNA gene promoter.
The invention also provides a nucleic acid comprising a promoter region from a rRNA gene promoter operably linked to a protein-coding gene having a 5′ UTR which contains translational regulatory elements for expression of the protein. The promoter region can include (i) a −10 region from a meningococcal rRNA gene promoter, (ii) a −35 region from a meningococcal rRNA gene promoter, and (iii) a 5′ UTR from a gene such as the porA gene.
The invention also provides a nucleic acid comprising a promoter which includes SEQ ID NO: 18, or a variant of SEQ ID NO: 18 which differs from SEQ ID NO: 18 by up to 4 (i.e. 1, 2, 3, or 4) single-nucleotide insertions, deletions or substitutions.
The porA and rRNA promoters can be used in hybrid form, for instance to have a −10 region from a porA promoter and a −35 region from a rRNA promoter (which can be a consensus −35 region). Thus the invention also provides a nucleic acid comprising a promoter which includes either

    
    
        (i) a −10 region from a meningococcal rRNA gene and a −35 region from a meningococcal porA gene, or
        (ii) a −10 region from a meningococcal porA gene and a −35 region from a meningococcal rRNA gene.
    
    


The nucleic acids of the invention can be present in a bacterium, and in particular in a meningococcus. The promoters can drive expression of downstream protein-coding genes (in particular, genes encoding outer membrane proteins) to which they are operably linked, and the bacteria can be used to prepare vaccines (in particular vesicle-based vaccines). The invention also provides these bacteria, these vesicles, and these vaccines.
Promoters of the Invention
The two essential sequences in bacterial promoters are the −10 region (also known as the Pribnow box) and the −35 region. These are separated by an intervening sequence, and between the −10 region and the transcription start site (+1) is a short non-transcribed upstream sequence.
The PorA promoter has been studied in detail. Reference 10 reports a 6-mer −35 region, followed by a 16-mer of 17-mer intervening sequence, then a 7-mer Pribnow box, then a 7-mer non-transcribed upstream sequence, followed by transcribed nucleotide +1. The start codon in the wild-type porA gene is at nucleotide +59 of the transcript, and this 59-mer spacing was confirmed in reference 8.
Where a promoter of the invention includes a −10 region from a meningococcal porA gene promoter, this is typically a 6-mer TATAAT i.e. a typical −10 region 6-mer.
Where a promoter of the invention includes a −35 region from a meningococcal porA gene promoter, this is typically a 6-mer TGGTTT or ATGGTT.
The intervening sequence between a −35 region and a −10 region can be between 12-20 nucleotides e.g. 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. An intervening sequence of 16, 17 or 18 nucleotides is typical.
The short non-transcribed upstream sequence is typically between 5-10 nucleotides e.g. 6, 7 or 8 nucleotides. It can be G:C-rich e.g. at least 1⁄2 of the nucleotides in the sequence are G or C e.g. at least 3 G/C residues in a 6-mer non-transcribed upstream sequence.
Thus a promoter can include a 6-mer −35 region, a 16-mer or 17-mer intervening sequence, a 6-mer −10 region, and a 6-mer or 7-mer non-transcribed upstream sequence, giving 34, 35 or 36 nucleotides of core promoter in total.
A promoter can, of course, continue upstream of the −35 region. Sequences upstream of the −35 region can be important for high-level expression from the promoter, of for regulation of expression from the promoters. For instance, in rRNA promoters sequences upstream of the core promoter called UP-elements can account for their exceptional strength increasing transcription as much as 300-fold [11,12]. These can be recognised by the α-subunit of the RNA polymerase core. Thus a promoter of the invention can include an upstream UP-element, which will usually be A:T-rich. Similarly, a promoter of the invention can include an upstream CREN (contact regulatory element of Neisseria) [13].
Where a promoter of the invention includes a −35 region and/or a −10 region from a porA gene promoter, the short non-transcribed upstream sequence can be TGAAGAC.
Where a promoter of the invention includes a −35 region and/or a −10 region from a porA gene promoter, the intervening sequence can be varied, as shown in the examples. For instance, it can be TTTGCGGGGGGGGGGG (wild-type 16-mer; SEQ ID NO: 26) or TTTGCGGGGGGGGGGGGG (wild-type 18-mer; SEQ ID NO: 17) but these wild-type sequences are not preferred. Rather, the 11-mer or 13-mer poly-G sequence in this 16-mer or 18-mer can be interrupted by non-G nucleotide(s). Two preferred intervening sequences are TTTGCGAGGGAGGTGG (16-mer; SEQ ID NO: 27) and TTTTGCGAGGGAGGTGG (17-mer; SEQ ID NO: 28).
In some embodiments the intervening sequence contains no poly-guanidine sequence i.e. there is no GG dinucleotide within the intervening sequence. In other embodiments the intervening sequence can contain a poly-guanidine sequence, but it contains no more than eight consecutive guanidine nucleotides i.e. it does not include a sequence GGGGGGGG. Ideally, the intervening sequence does not include a sequence GGGGGGG. Ideally, the intervening sequence does not include a sequence GGGGGG. Ideally, the intervening sequence does not include a sequence GGGGG. Ideally, the intervening sequence does not include a sequence GGGG. The intervening sequence can include a GGG trinucleotide or a GG dinucleotide. Thus a poly-G sequence within the intervening sequence would extend for no more than 8 nucleotides in total, and ideally has no more than three consecutive guanidine residues.
Where a promoter of the invention includes a −10 region from a meningococcal rRNA gene promoter, this is typically a 6-mer TATAAT i.e. a typical −10 region 6-mer.
Where a promoter of the invention includes a −35 region from a meningococcal rRNA gene promoter, this is typically a 6-mer TTGACA (which is a consensus −35 region).
Where a promoter of the invention includes a −35 region and/or a −10 region from a meningococcal rRNA gene promoter, the intervening sequence can be GGCGGAAGGAATACTT (SEQ ID NO: 29).
Where a promoter of the invention includes a −35 region and/or a −10 region from a rRNA gene promoter, the short non-transcribed upstream sequence can be TCGCAAC.
The porA and rRNA promoters can be used in hybrid form, and a useful promoter includes a −35 region from a rRNA gene (e.g. TTGACA), an intervening sequence modified from a porA gene (e.g. TTTGCGAGGGAGGTGG; SEQ ID NO: 27), a typical Pribnow box (TATAAT) and a short non-transcribed upstream sequence from a porA gene (e.g. TGAAGAC). Thus the promoter can include SEQ ID NO: 30 (TTGACATTTGCGAGGGAGGTGGTATAATTGAAGAC).
In some embodiments the promoter includes SEQ ID NO: 18 (a 35-mer fragment immediately before the transcription start site of the wild-type meningococcal 16S rRNA promoter). This sequence can be modified by 1, 2, 3, or 4 single-nucleotide insertions, deletions or substitutions within the 35-mer, as shown herein.
When present in functioning DNA form within a bacterium these promoters are operably linked to a sequence which encodes a protein of interest, whose transcription is controlled by the promoter. The invention can be used to express any protein of interest, but the protein is advantageously an outer membrane protein which can be retained in vesicles. Suitable examples of outer membrane proteins are given below, but the invention is preferably not used to drive expression of a transcript which encodes a PorA outer membrane protein.
In a coding DNA sequence, downstream of a promoter of the invention the DNA includes a transcription start site, followed by a 5′ untranslated region (typically including a Shine-Dalgarno sequence) and then a start codon for the encoded protein of interest.
Where the promoter is from a rRNA gene, the start of the transcribed sequence is ideally derived from a protein-coding gene rather than from a rRNA gene. Thus the rRNA promoter (e.g. SEQ ID NO: 18 or variants) can be fused to the 5′ UTR from a meningococcal protein-coding gene. In some embodiments, the rRNA promoter is fused to the 5′ UTR from a porA gene (a 58-mer), and this 5′ UTR is then fused to the coding sequence of a gene of interest. The 5′ UTR should include translational regulatory elements which permit translation of protein from the protein-coding gene. Thus this embodiment uses the rRNA promoter to produce a protein-coding transcript which can be translated. Sequences downstream of the promoter, inside the 5′ UTR, may be important for regulation of transcription or post-transcriptional effects on expression of downstream genes.
The 5′ UTR of a porA gene can also be used with a modified porA promoter of the invention, such that the modified porA promoter is linked to a 5′ UTR from a porA gene, which is then fused to the coding sequence for a protein of interest.
A useful porA 5′ UTR can include the nucleotide sequence of SEQ ID NO: 39, namely GTATCGGGTGTTTGCCCGATGTTTTTAGGTTTTTATCAAATTTACAAAAGGAAGCC, or a sequence which differs from SEQ ID NO: 39 by up to 5 (i.e. 1, 2, 3, 4, or 5) single-nucleotide insertions, deletions or substitutions.
It will be appreciated that discussions of promoter sequences herein refer to the sense strand. In double-stranded DNA, from which transcription takes place, the promoter has a complementary strand. Where two promoter elements are said to be separated by ‘n’ nucleotides, this is again a reference to the number of nucleotides in the sense strand; in double-stranded DNA the element will be separated by ‘n’ base pairs. If a promoter element is said to lack a guanidine residue, this is again a reference to the sense strand; in double-stranded DNA, for example, a guanidine-free promoter could include a guanidine residue, but only as a complementary base in the antisense strand for a cytosine residue in the sense strand. Although the invention is described by reference to the sense strand, the scope of the invention includes double-stranded nucleic acids including such sense strands, as well as single-stranded nucleic acids including such sense strands or including antisense forms of these sense strands (these antisense strands can, of course, be used to prepare the sense strands, by standard techniques).
Vesicles
The invention is particularly useful when preparing meningococcal vesicles i.e. any proteoliposomic vesicle obtained by disruption of or blebbing from a meningococcal outer membrane to form vesicles therefrom that retain antigens from the outer membrane. Thus the term includes, for instance, OMVs (sometimes referred to as ‘blebs’), microvesicles (MVs [14]) and ‘native OMVs’ (‘NOMVs’ [15]).
MVs and NOMVs are naturally-occurring membrane vesicles that form spontaneously during bacterial growth and are released into culture medium. MVs can be obtained by culturing Neisseria in broth culture medium, separating whole cells from the smaller MVs in the broth culture medium (e.g. by filtration or by low-speed centrifugation to pellet only the cells and not the smaller vesicles), and then collecting the MVs from the cell-depleted medium (e.g. by filtration, by differential precipitation or aggregation of MVs, by high-speed centrifugation to pellet the MVs). Strains for use in production of MVs can generally be selected on the basis of the amount of MVs produced in culture e.g. refs. 16 & 17 describe Neisseria with high MV production.
Another useful technique for spontaneous outer membrane vesicle production is to inactivate the mltA gene in a meningococcus, as disclosed in reference 18. These mutant bacteria release vesicles into their culture medium during growth.
OMVs are prepared artificially from bacteria, and may be prepared using detergent treatment (e.g. with deoxycholate), or by non-detergent means (e.g. see reference 19). Techniques for forming OMVs include treating bacteria with a bile acid salt detergent (e.g. salts of lithocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, cholic acid, ursocholic acid, etc., with sodium deoxycholate [20 & 21] being preferred for treating Neisseria) at a pH sufficiently high not to precipitate the detergent [22]. Other techniques may be performed substantially in the absence of detergent [19] using techniques such as sonication, homogenisation, microfluidisation, cavitation, osmotic shock, grinding, French press, blending, etc. Methods using no or low detergent can retain useful antigens such as NspA [19]. Thus a method may use an OMV extraction buffer with about 0.5% deoxycholate or lower e.g. about 0.2%, about 0.1%, <0.05% or zero. A useful process for OMV preparation is described in reference 23 and involves ultrafiltration on crude OMVs, rather than instead of high speed centrifugation. The process may involve a step of ultracentrifugation after the ultrafiltration takes place.
A convenient way of purifying vesicles is the dual filtration method disclosed in reference 24.
Preferred vesicles are those which form spontaneously during bacterial culture, rather than those which are released only after physical or chemical treatment of the bacteria. Growth of meningococci having an inactivated MltA is a preferred way of producing vesicles in this way. Reference 47 discloses other ways of producing hyperblebbing strains. For spontaneously-released vesicles it is preferable to use bacteria which do not produce intact LOS (detergent-extracted vesicles have reduced levels of potentially reactogenic LOS).
If lipo-oligosaccharide (LOS) is present in a vesicle it is possible to treat the vesicle so as to link its LOS and protein components (“intra-bleb” conjugation [48]).
The vesicles may lack LOS altogether, or they may lack hexa-acylated LOS e.g. LOS in the vesicles may have a reduced number of secondary acyl chains per LOS molecule [25]. For example, the vesicles may from a strain which has a lpxL1 deletion or mutation which results in production of a penta-acylated LOS [2,43]. LOS in a strain may lack a lacto-N-neotetraose epitope e.g. it may be a 1st and/or lgtB knockout strain [5]. LOS may lack at least one wild-type primary O-linked fatty acid [26]. The LOS may have no a chain. The LOS may comprise GlcNAc-Hep2phosphoethanolamine-KDO2-Lipid A [27].
The vesicles may include one, more than one, or (preferably) zero PorA serosubtypes. Modification of meningococcus to provide multi-PorA OMVs is known e.g. from reference 28, which discloses the construction of vesicles from strains modified to express six different PorA subtypes, and from reference 29. Conversely, modification to remove PorA is also known e.g. from reference 5.
The vesicles may be free from one of both of PorA and FrpB. Preferred vesicles are PorA-free.
The vesicles may lack capsular saccharide. For instance they may be derived from a strain that has one or more of the genes for capsule biosynthesis and/or export deactivated (e.g. synX).
The invention may be used with mixtures of vesicles from different strains. For instance, reference 30 discloses vaccine comprising multivalent meningococcal vesicle compositions, comprising a first vesicle derived from a meningococcal strain with a serosubtype prevalent in a country of use, and a second vesicle derived from a strain that need not have a serosubtype prevent in a country of use. Reference 31 also discloses useful combinations of different vesicles. A combination of vesicles from strains in each of the L2 and L3 immunotypes may be used in some embodiments.
Over-Expression
Promoters of the invention can be used to over-express a gene of interest in meningococcus. Where the gene encodes an outer membrane protein antigen, this over-expression can be used to provide a vesicle which advantageously retains that antigen. Such vesicles are discussed in more detail below. As a result of the over-expression, vesicles prepared from the modified meningococcus contain higher levels of the over-expressed antigen(s) than seen in a corresponding wild-type meningococcus. The increase in expression in the vesicles is usefully at least 10%, measured in mass of the relevant antigen per unit mass of vesicles, and is more usefully at least 20%, 30%, 40%, 50%, 75%, 100% or more.
Suitable recombinant modifications which can be used to over-express an antigen by using promoters of the invention include, but are not limited to: (i) promoter replacement; (ii) gene addition; and/or (iii) gene replacement. These three techniques can, if desired, be used in conjunction with (iv) repressor knockout.
In promoter replacement, the promoter which controls expression of the antigen's gene in a bacterium is replaced with a promoter of the invention in order to provide higher levels of expression.
In gene addition, a bacterium which already expresses the antigen receives a second copy of the relevant gene. This second copy can be integrated into the bacterial chromosome or can be on an episomal element such as a plasmid. The second copy can be expressed using a promoter of the invention. The effect of the gene addition is to increase the amount of expressed antigen. Where a plasmid is used, it is ideally a plasmid with a high copy number e.g. above 10, or even above 100.
In gene replacement, gene addition occurs but is accompanied by deletion of the existing copy of the gene. For instance, this approach was used in reference 3, where a bacterium's endogenous chromosomal fHbp gene was deleted and replaced by a plasmid-encoded copy (see also reference 32). Expression from the replacement copy, using a promoter of the invention, is higher than from the previous copy, thus leading to over-expression.
In some embodiments gene replacement occurs where gene addition is accompanied by the deletion of another gene. For instance when the knocking out of an endogenous gene is required but is not the gene of interest that is being overexpressed by the promoter of the invention.
In some embodiments, more than one event of gene addition or gene replacement may occur such that expression from multiple copies of the gene of interest by promoters of the invention or combinations of overexpression of different genes of interest by promoters of the invention may take place.
Over-expression of at least one antigen will use a promoter of the invention, but these promoters can be used in conjunction with other techniques. For instance, the promoters can be used in conjunction with repressor knockout, in which a protein which represses expression of an antigen of interest is knocked out. This knockout means that the repression does not occur and the antigen of interest can be expressed at a higher level.
For instance, where NadA is over-expressed, the nadA gene can use a promoter of the invention, but in addition the gene encoding NadR (NMB1843) can be deleted. NadR a transcriptional repressor protein [33] which down-regulates or represses the NadA-encoding gene in all strains tested. Knockout of NadR results in constitutive expression of NadA. An alternative way to over-express NadA is to add 4-hydroxyphenylacetic to the culture medium. Thus promoters of the invention can be used as an over-expression strategy alone, or in combination with other approaches.
In some embodiments a bacterium over-expresses NadA.
In some embodiments a bacterium over-expresses NHBA.
In some embodiments a bacterium over-expresses fHbp.
In some embodiments, a bacterium over-expresses both NHBA and NadA.
In some embodiments, a bacterium over-expresses both fHbp and NadA.
In some embodiments, a bacterium over-expresses both fHbp and NHBA.
In some embodiments, a bacterium over-expresses fHbp, NHBA and NadA.
In addition to over-expressing NHBA and/or NadA, a bacterium may over-express one or more further antigens. For instance, a bacterium may over-express one or more of: (a) NhhA; (b) TbpA; (c) HmbR; (d) TbpB; (e) NspA; (f) Cu,Zn-superoxide dismutase; (g) Omp85; (h) App; and/or (i) fHbp. Over-expression of NhhA is already reported in references 5 and 34. Over-expression of TbpA is already reported in references 5, 34 and 35. Over-expression of HmbR is already reported in reference 36. Over-expression of TbpB is already reported in reference 35. Over-expression of NspA is already reported in reference 37, in combination with porA and cps knockout. Over-expression of Cu,Zn-superoxide dismutase is already reported in reference 35. Over-expression of fHbp is already reported in references 1-3 & 32, and by a different approach (expressing a constitutively-active mutant FNR) in references 38 & 39. Where more than one antigen is over-expressed, at least one antigen will be over-expressed using a promoter of the invention, and in some embodiments more than one antigen will be over-expressed using a promoter of the invention.
In some embodiments a bacterium over-expresses NHBA, NadA and fHbp. These three antigens are components of the “universal vaccine” disclosed in reference 40 or “4CMenB” [41,42]. In one embodiment, expression of NHBA is controlled by a promoter of the invention, NadR is knocked out, and the strain expresses a constitutively active mutant FNR. In another embodiment, expression of NHBA is controlled by a promoter of the invention, expression of fHbp is controlled by a promoter of the invention, and NadR is knocked out.
An over-expressing modified strain will generally be isogenic with its parent strain, except for a genetic modification. As a result of the modification, expression of the antigen of interest in the modified strain is higher (under the same conditions) than in the parent strain.
Bacteria
As mentioned above, vesicles of the invention are prepared from meningococci which over-express the relevant antigen(s) due to genetic modification, involving at least the use of a promoter of the invention. The invention also provides these bacteria. They can be used for preparing vesicles of the invention.
In addition to including a promoter of the invention, the meningococci may include one or more further modifications. For instance, it can have a knockout of one or more of lpxL1, lgtA, lgtB, porA, frpB, synX, mltA and/or 1st. For instance, reference 43 reports a NOMV vaccine prepared from bacteria having inactivated synX, lpxL1, and lgtA genes. Knockout of at least lpxL1 and synX is particularly useful.
The bacterium may have low endotoxin levels, achieved by knockout of enzymes involved in LPS biosynthesis [44,45].
The bacterium may be from any serogroup e.g. A, B, C, W135, or Y. It is preferably serogroup B or serogroup W135.
The bacterium may be of any serotype (e.g. 1, 2a, 2b, 4, 14, 15, 16, etc.), any serosubtype, and any immunotype (e.g. L1; L2; L3; L3,3,7; L10; etc.). Vesicles can usefully be prepared from strains having one of the following subtypes: P1.2; P1.2,5; P1.4; P1.5; P1.5,2; P1.5,c; P1.5c,10; P1.7,16; P1.7,16b; P1.7h,4; P1.9; P1.15; P1.9,15; P1.12,13; P1.13; P1.14; P1.21,16; P1.22,14.
The bacterium may be from any suitable lineage, including hyperinvasive and hypervirulent lineages e.g. any of the following seven hypervirulent lineages: subgroup I; subgroup III; subgroup IV-1; ET-5 complex; ET-37 complex; A4 cluster; lineage 3. These lineages have been defined by multilocus enzyme electrophoresis (MLEE), but multilocus sequence typing (MLST) has also been used to classify meningococci [ref. 46] e.g. the ET-37 complex is the ST-11 complex by MLST, the ET-5 complex is ST-32 (ET-5), lineage 3 is ST-41/44, etc.
In some embodiments a bacterium may include one or more of the knockout and/or over-expression mutations disclosed in references 8, 37, 47 and 48. Suitable genes for modification include: (a) Cps, CtrA, CtrB, CtrC, CtrD, FrpB, GalE, HtrB/MsbB, LbpA, LbpB, LpxK, Opa, Opc, PilC, PorB, SiaA, SiaB, SiaC, SiaD, TbpA, and/or TbpB [8]; (b) CtrA, CtrB, CtrC, CtrD, FrpB, GalE, HtrB/MsbB, LbpA, LbpB, LpxK, Opa, Opc, PhoP, PilC, PmrE, PmrF, SiaA, SiaB, SiaC, SiaD, TbpA, and/or TbpB; (c) ExbB, ExbD, rmpM, CtrA, CtrB, CtrD, GalE, LbpA, LpbB, Opa, Opc, PilC, PorB, SiaA, SiaB, SiaC, SiaD, TbpA, and/or TbpB; and (d) CtrA, CtrB, CtrD, FrpB, OpA, OpC, PilC, PorB, SiaD, SynA, SynB, and/or SynC.
As mentioned above, the meningococcus advantageously does not express an active MltA (GNA33), such that it spontaneously releases vesicles e.g. which contain antigens which are expressed using a promoter of the invention.
Ideally, the bacterium does not express a native LPS e.g. it has a mutant or knockout of LpxL1 and/or of LgtB.
Where the bacterium has a knockout, it is convenient to achieve this knockout by insertion of a sequence including a promoter of the invention controlling transcription of a protein of interest. For example, the genomic synX or lpxL1 gene can be knocked out by inserting within it a sequence which includes a promoter of the invention which controls expression of an outer membrane protein such as fHbp. A single transformation event can thus be used to achieve two goals.
A bacterium of the invention may include a selection marker e.g. an antibiotic resistance marker.
A bacterium may have one or more, or all, of the following characteristics: (i) down-regulated or knocked-out LgtB and/or GalE to truncate the meningococcal LOS; (ii) up-regulated TbpA; (iii) up-regulated NhhA; (iv) up-regulated Omp85; (v) up-regulated LbpA; (vi) up-regulated NspA; (vii) knocked-out PorA; (viii) down-regulated or knocked-out FrpB; (ix) down-regulated or knocked-out Opa; (x) down-regulated or knocked-out Opc; (xii) deleted cps gene complex. A truncated LOS can be one that does not include a sialyl-lacto-N-neotetraose epitope e.g. it might be a galactose-deficient LOS. The LOS may have no a chain.
A bacterium may have one or more, or all, of the following characteristics: (i) down-regulated or knocked-out LgtA; (ii) down-regulated or knocked-out LpxL1; (iii) down-regulated or knocked-out SynX; (iv) more than one PorA subtype, such as two different PorA subtypes; and/or (v) an up-regulated outer membrane antigen, such as NadA or fHbp.
Strain Production
The invention provides a process for preparing a meningococcal strain suitable for vesicle preparation, comprising steps of (i) choosing a starting strain which expresses a first amount of an antigen when grown in specific culture conditions, then (ii) modifying the starting strain to provide a modified strain including a promoter of the invention, wherein the modified strain expresses a second amount of the antigen when grown in the same specific culture conditions, the second amount being higher than the first amount. The second amount is usefully at least 10%, higher than the first amount, measured in mass of the relevant antigen per unit mass of bacteria, and is more usefully at least 20%, 30%, 40%, 50%, 75%, 100% or more.
This process can be followed by a step of (iii) culturing the modified bacteria obtained in step (ii) to provide a bacterial culture.
The process can also involve further steps of genetic manipulation, either before or after the introduction of a promoter of the invention. A promoter of the invention can be introduced into the bacterium more than once, and at more than one site e.g. with multiple controlled genes.
The invention also provides a process for preparing a meningococcal vesicle, comprising a step of treating a bacterial culture obtained by a process of the invention (as described above) such that its outer membrane forms vesicles. This treatment step can use any of the techniques discussed above.
The invention also provides a process for preparing a meningococcal vesicle, comprising a step of treating a meningococcus of the invention such that its outer membrane forms vesicles. This treatment step can use any of the techniques discussed above.
The invention also provides a process for preparing a meningococcal vesicle, comprising a step of culturing a meningococcus of the invention under conditions in which its outer membrane spontaneously sheds vesicles. For instance, the meningococcus might not express active MltA.
Useful starting strains are in meningococcus serogroup B. Three useful starting meningococcal strains for preparing bacteria which over-express an antigen of interest are MC58, NZ98/254, and H44/76. MC58 has PorA serosubtype 1.7,16; NZ98/254 has serosubtype P1.7-2,4; and H44/76 has serosubtype 1.7,16. Other strains with these serosubtypes can also be used.
Vectors Etc.
The invention provides promoters as described above. These can be present in a bacterial chromosome, or in an extra-chromosomal or episomal nucleic acid e.g. within a plasmid. A bacterium can include 1 or more copies of the promoter of the invention. Where a bacterium includes 2 copies, these can be in the chromosome and/or episome(s).
Thus the invention provides a bacterium comprising (i) a chromosome including at least one promoter of the invention and/or (ii) an episome, such as a plasmid, including at least one promoter of the invention.
The invention also provides a nucleic acid vector comprising a promoter of the invention, such as a bacterial expression vector.
These promoters within these vectors or bacteria can be ‘orphan’ promoters without any downstream transcribable sequence, but they will typically be operably linked to a sequence which is transcribed, and which is then translated to express a protein of interest.
Antigens
NHBA (Neisserial Heparin Binding Antigen)
NHBA [51] was included in the published genome sequence for meningococcal serogroup B strain MC58 [78] as gene NMB2132 (GenBank accession number GI:7227388; SEQ ID NO: 9 herein). Sequences of NHBA from many strains have been published since then. For example, allelic forms of NHBA (referred to as protein ‘287’) can be seen in FIGS. 5 and 15 of reference 49, and in example 13 and FIG. 21 of reference 50 (SEQ IDs 3179 to 3184 therein). Various immunogenic fragments of NHBA have also been reported.
Preferred NHBA antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 9; and/or (b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 9, wherein ‘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 of (b) comprise an epitope from SEQ ID NO: 9.
The most useful NHBA antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 9. Advantageous NHBA antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.
Over-expression of NHBA has previously been achieved in various ways e.g. introduction of a NHBA gene under the control of an IPTG-inducible promoter [51].
NadA (Neisserial Adhesin A)
The NadA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [78] as gene NMB1994 (GenBank accession number GI:7227256; SEQ ID NO: 10 herein). The sequences of NadA antigen from many strains have been published since then, and the protein's activity as a Neisserial adhesin has been well documented. Various immunogenic fragments of NadA have also been reported.
Preferred NadA antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 10; and/or (b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 10, wherein ‘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 of (b) comprise an epitope from SEQ ID NO: 10.
The most useful NadA antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 10. Advantageous NadA antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject. SEQ ID NO: 6 is one such fragment.
HmbR
The full-length HmbR sequence was included in the published genome sequence for meningococcal serogroup B strain MC58 [78] as gene NMB1668 (SEQ ID NO: 7 herein). Reference 52 reports a HmbR sequence from a different strain (SEQ ID NO: 8 herein), and reference 36 reports a further sequence (SEQ ID NO: 19 herein). SEQ ID NOs: 7 and 8 differ in length by 1 amino acid and have 94.2% identity. SEQ ID NO: 19 is one amino acid shorter than SEQ ID NO: 7 and they have 99% identity (one insertion, seven differences) by CLUSTALW. The invention can use any such HmbR polypeptide.
The invention can use a polypeptide that comprises a full-length HmbR sequence, but it will often use a polypeptide that comprises a partial HmbR sequence. Thus in some embodiments a HmbR sequence used according to the invention may comprise an amino acid sequence having at least i % sequence identity to SEQ ID NO: 7, 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 of at least j consecutive amino acids from SEQ ID NO: 7, 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 at least i % sequence identity to SEQ ID NO: 7 and/or (ii) comprising a fragment of at least j consecutive amino acids from SEQ ID NO: 7.
Preferred fragments of j amino acids comprise an epitope from SEQ ID NO: 7. Such epitopes will usually comprise amino acids that are located on the surface of HmbR. Useful epitopes include those with amino acids involved in HmbR's binding to haemoglobin, as antibodies that bind to these epitopes can block the ability of a bacterium to bind to host haemoglobin. The topology of HmbR, and its critical functional residues, were investigated in reference 53. Fragments that retain a transmembrane sequence are useful, because they can be displayed on the bacterial surface e.g. in vesicles. Examples of long fragments of HmbR correspond to SEQ ID NOs: 21 and 22. If soluble HmbR is used, however, sequences omitting the transmembrane sequence, but typically retaining epitope(s) from the extracellular portion, can be used.
The most useful HmbR antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 7. Advantageous HmbR antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.
fHbp (Factor H Binding Protein)
The fHbp antigen has been characterised in detail. It has also been known as protein ‘741’ [SEQ IDs 2535 & 2536 in ref. 50], ‘NMB1870’, ‘GNA1870’ [54-56], ‘P2086’, ‘LP2086’ or ‘ORF2086’ [57-59]. It is naturally a lipoprotein and is expressed across all meningococcal serogroups. The structure of fHbp's C-terminal immunodominant domain (‘fHbpC’) has been determined by NMR [60]. This part of the protein forms an eight-stranded β-barrel, whose strands are connected by loops of variable lengths. The barrel is preceded by a short α-helix and by a flexible N-terminal tail.
The fHbp antigen falls into three distinct variants [61] and it has been found that serum raised against a given family is bactericidal within the same family, but is not active against strains which express one of the other two families i.e. there is intra-family cross-protection, but not inter-family cross-protection. The invention can use a single fHbp variant, but is will usefully include a fHbp from two or three of the variants. Thus it may use a combination of two or three different fHbps, selected from: (a) a first protein, comprising an amino acid sequence having at least a % sequence identity to SEQ ID NO: 1 and/or comprising an amino acid sequence consisting of a fragment of at least x contiguous amino acids from SEQ ID NO: 1; (b) a second protein, comprising an amino acid sequence having at least b % sequence identity to SEQ ID NO: 2 and/or comprising an amino acid sequence consisting of a fragment of at least y contiguous amino acids from SEQ ID NO: 2; and/or (c) a third protein, comprising an amino acid sequence having at least c % sequence identity to SEQ ID NO: 3 and/or comprising an amino acid sequence consisting of a fragment of at least z contiguous amino acids from SEQ ID NO: 3.
The value of a is at least 85 e.g. 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or more.
The value of b is at least 85 e.g. 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or more.
The value of c is at least 85 e.g. 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or more.
The values of a, b and c are not intrinsically related to each other.
The value of x is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The value of y is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The value of z is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The values of x, y and z are not intrinsically related to each other.
Where the invention uses a single fHbp variant, a composition may include a polypeptide comprising (a) an amino acid sequence having at least a % sequence identity to SEQ ID NO: 1 and/or comprising an amino acid sequence consisting of a fragment of at least x contiguous amino acids from SEQ ID NO: 1; or (b) an amino acid sequence having at least b % sequence identity to SEQ ID NO: 2 and/or comprising an amino acid sequence consisting of a fragment of at least y contiguous amino acids from SEQ ID NO: 2; or (c) an amino acid sequence having at least c % sequence identity to SEQ ID NO: 3 and/or comprising an amino acid sequence consisting of a fragment of at least z contiguous amino acids from SEQ ID NO: 3.
Where the invention uses a fHbp from two or three of the variants, a composition may include a combination of two or three different fHbps selected from: (a) a first polypeptide, comprising an amino acid sequence having at least a % sequence identity to SEQ ID NO: 1 and/or comprising an amino acid sequence consisting of a fragment of at least x contiguous amino acids from SEQ ID NO: 1; (b) a second polypeptide, comprising an amino acid sequence having at least b % sequence identity to SEQ ID NO: 2 and/or comprising an amino acid sequence consisting of a fragment of at least y contiguous amino acids from SEQ ID NO: 2; and/or (c) a third polypeptide, comprising an amino acid sequence having at least c % sequence identity to SEQ ID NO: 3 and/or comprising an amino acid sequence consisting of a fragment of at least z contiguous amino acids from SEQ ID NO: 3. The first, second and third polypeptides have different amino acid sequences.
Where the invention uses a fHbp from two of the variants, a composition can include both: (a) a first polypeptide, comprising an amino acid sequence having at least a % sequence identity to SEQ ID NO: 1 and/or comprising an amino acid sequence consisting of a fragment of at least x contiguous amino acids from SEQ ID NO: 1; and (b) a second polypeptide, comprising an amino acid sequence having at least b % sequence identity to SEQ ID NO: 2 and/or comprising an amino acid sequence consisting of a fragment of at least y contiguous amino acids from SEQ ID NO: 2. The first and second polypeptides have different amino acid sequences.
Where the invention uses a fHbp from two of the variants, a composition can include both: (a) a first polypeptide, comprising an amino acid sequence having at least a % sequence identity to SEQ ID NO: 1 and/or comprising an amino acid sequence consisting of a fragment of at least x contiguous amino acids from SEQ ID NO: 1; (b) a second polypeptide, comprising an amino acid sequence having at least c % sequence identity to SEQ ID NO: 3 and/or comprising an amino acid sequence consisting of a fragment of at least z contiguous amino acids from SEQ ID NO: 3. The first and second polypeptides have different amino acid sequences.
Another useful fHbp which can be used according to the invention is one of the modified forms disclosed, for example, in reference 62 e.g. comprising SEQ ID NO: 20 or 23 therefrom. These modified forms can elicit antibody responses which are broadly bactericidal against meningococci. SEQ ID NO: 77 in reference 62 is another useful fHbp sequence which can be used.
A useful modification of fHbp, including of the sequences mentioned above, is a mutation which reduces or removes the protein's affinity for factor H. For instance, references 63 & 64 disclose such mutations at residues Glu-283 and/or Glu-304 by their numbering (subtract 72 from this numbering scheme to match SEQ ID NO: 1 herein). Similarly, references 65 & 66 discloses mutation (e.g. to Ser) at position Arg-41 by their numbering (subtract 7 to match SEQ ID NO: 1) for variant 1 and at positions 80, 211, 218, 220, 222, and/or 236 by their numbering (subtract 7 to match SEQ ID NO: 2) for variant 2. Alignments will quickly reveal the corresponding residues for any fHbp sequence of interest. The Arg-41-Ser mutation in variant 1 sequences is particularly preferred.
fHbp protein(s) in a OMV will usually be lipidated e.g. at a N-terminus cysteine. In other embodiments they will not be lipidated.
NspA (Neisserial Surface Protein A)
The NspA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [78] as gene NMB0663 (GenBank accession number GI:7225888; SEQ ID NO: 11 herein). The antigen was previously known from references 67 & 68. The sequences of NspA antigen from many strains have been published since then. Various immunogenic fragments of NspA have also been reported.
Preferred NspA antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 11; and/or (b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 11, wherein ‘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 of (b) comprise an epitope from SEQ ID NO: 11.
The most useful NspA antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 11. Advantageous NspA antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.
NhhA (Neisseria Hia Homologue)
The NhhA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [78] as gene NMB0992 (GenBank accession number GI:7226232; SEQ ID NO: 12 herein). The sequences of NhhA antigen from many strains have been published since e.g. refs 49 & 69, and various immunogenic fragments of NhhA have been reported. It is also known as Hsf.
Preferred NhhA antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 12; and/or (b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 12, wherein ‘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 of (b) comprise an epitope from SEQ ID NO: 12.
The most useful NhhA antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 12. Advantageous NhhA antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.
App (Adhesion and Penetration Protein)
The App antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [78] as gene NMB1985 (GenBank accession number GI:7227246; SEQ ID NO: 13 herein). The sequences of App antigen from many strains have been published since then. It has also been known as ‘ORF1’ and ‘Hap’. Various immunogenic fragments of App have also been reported.
Preferred App antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 13; and/or (b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 13, wherein ‘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 of (b) comprise an epitope from SEQ ID NO: 13.
The most useful App antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 13. Advantageous App antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.
Omp85 (85 kDa Outer Membrane Protein)
The Omp85 antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [78] as gene NMB0182 (GenBank accession number GI:7225401; SEQ ID NO: 14 herein). The sequences of Omp85 antigen from many strains have been published since then. Further information on Omp85 can be found in references 70 and 71. Various immunogenic fragments of Omp85 have also been reported.
Preferred Omp85 antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 14; and/or (b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 14, wherein ‘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 of (b) comprise an epitope from SEQ ID NO: 14.
The most useful Omp85 antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 14. Advantageous Omp85 antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.
TbpA
The TbpA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [78] as gene NMB0461 (GenBank accession number GI:7225687; SEQ ID NO: 23 herein). The sequences of TbpA from many strains have been published since then. Various immunogenic fragments of TbpA have also been reported.
Preferred TbpA antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 23; and/or (b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 23, wherein ‘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 of (b) comprise an epitope from SEQ ID NO: 23.
The most useful TbpA antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 23. Advantageous TbpA antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.
TbpB
The TbpB antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [78] as gene NMB1398 (GenBank accession number GI:7225686; SEQ ID NO: 24 herein). The sequences of TbpB from many strains have been published since then. Various immunogenic fragments of TbpB have also been reported.
Preferred TbpB antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 24; and/or (b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 24, wherein ‘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 of (b) comprise an epitope from SEQ ID NO: 24.
The most useful TbpB antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 24. Advantageous TbpB antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.
Cu,Zn-Superoxide Dismutase
The Cu,Zn-superoxide dismutase antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [78] as gene NMB1398 (GenBank accession number GL7226637; SEQ ID NO: 25 herein). The sequences of Cu,Zn-superoxide dismutase from many strains have been published since then. Various immunogenic fragments of Cu,Zn-superoxide dismutase have also been reported.
Preferred Cu,Zn-superoxide dismutase antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 25; and/or (b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 25, wherein ‘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 of (b) comprise an epitope from SEQ ID NO: 25.
The most useful Cu,Zn-superoxide dismutase antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 25. Advantageous Cu,Zn-superoxide dismutase antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.
Pharmaceutical Compositions
Vesicles of the invention are useful as active ingredients in immunogenic pharmaceutical compositions for administration to a patient. These will typically include a pharmaceutically acceptable carrier, and a thorough discussion of such carriers is available in reference 72.
Effective dosage volumes can be routinely established, but a typical human dose of the composition has a volume of about 0.5 ml e.g. for intramuscular injection. The RIVM OMV-based vaccine was administered in a 0.5 ml volume [73] by intramuscular injection to the thigh or upper arm. MeNZBTM is administered in a 0.5 ml by intramuscular injection to the anterolateral thigh or the deltoid region of the arm. Similar doses may be used for other delivery routes e.g. an intranasal OMV-based vaccine for atomisation may have a volume of about 100 μl or about 130 μl per spray, with four sprays administered to give a total dose of about 0.5 ml.
The pH of a composition of the invention is usually between 6 and 8, and more preferably between 6.5 and 7.5 (e.g. about 7). The pH of the RIVM OMV-based vaccine is 7.4 [74], and a pH <7.5 is preferred for compositions of the invention. The RIVM OMV-based vaccine maintains pH by using a 10 mM Tris/HCl buffer, and stable pH in compositions of the invention may be maintained by the use of a buffer e.g. a Tris buffer, a citrate buffer, phosphate buffer, or a histidine buffer. Thus compositions of the invention will generally include a buffer.
The composition may be sterile and/or pyrogen-free. Compositions of the invention may be isotonic with respect to humans.
Compositions of the invention for administration to patients are immunogenic, and are more preferably vaccine compositions. Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection), but will typically be prophylactic. Immunogenic compositions used as vaccines comprise an immunologically effective amount of antigen(s), as well as any other components, as needed. By ‘immunologically effective amount’, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. The antigen content of compositions of the invention will generally be expressed in terms of the amount of protein per dose. A dose of about 0.9 mg protein per ml is typical for OMV-based intranasal vaccines.
Compositions of the invention may include an immunological adjuvant. Thus, for example, they may include an aluminium salt adjuvant or an oil-in-water emulsion (e.g. a squalene-in-water emulsion). Suitable aluminium salts include hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), (e.g. see chapters 8 & 9 of ref. 75), or mixtures thereof. The salts can take any suitable form (e.g. gel, crystalline, amorphous, etc.), with adsorption of antigen to the salt being preferred. The concentration of Al+++ in a composition for administration to a patient is preferably less than 5 mg/ml e.g. ≦4 mg/ml, ≦3 mg/ml, ≦2 mg/ml, ≦1 mg/ml, etc. A preferred range is between 0.3 and 1 mg/ml. A maximum of 0.85 mg/dose is preferred. Aluminium hydroxide adjuvants are particularly suitable for use with meningococcal vaccines.
Meningococci affect various areas of the body and so the compositions of the invention may be prepared in various liquid forms. For example, the compositions may be prepared as injectables, either as solutions or suspensions. The composition may be prepared for pulmonary administration e.g. by an inhaler, using a fine spray. The composition may be prepared for nasal, aural or ocular administration e.g. as spray or drops. Injectables for intramuscular administration are typical.
Compositions of the invention may include an antimicrobial, particularly when packaged in multiple dose format. Antimicrobials such as thiomersal and 2-phenoxyethanol are commonly found in vaccines, but it is preferred to use either a mercury-free preservative or no preservative at all.
Compositions of the invention may comprise detergent e.g. a Tween (polysorbate), such as Tween 80. Detergents are generally present at low levels e.g. <0.01%.
Compositions of the invention may include residual detergent (e.g. deoxycholate) from OMV preparation. The amount of residual detergent is preferably less than 0.4 μg (more preferably less than 0.2 μg) for every jug of MenB protein.
If a composition of the invention includes LOS, the amount of LOS is preferably less than 0.12 μg (more preferably less than 0.05 μg) for every jug of protein.
Compositions of the invention may include sodium salts (e.g. sodium chloride) to give tonicity. A concentration of 10±2 mg/ml NaCl is typical e.g. about 9 mg/ml.
In addition to vesicles of the invention, immunogenic compositions can include soluble protein antigens. For instance, a useful composition can include vesicles of the invention in combination with one or more of (i) a NHBA antigen (ii) a NadA antigen and (iii) a fHbp antigen. For example, the vesicles can be mixed with the “4CMenB” vaccine (see above). In one useful embodiment, the composition includes fHbp both in soluble form and also in over-expressed vesicular form, with the two forms being different fHbp variants e.g. variant 1 in soluble form (as in 4CMenB) and variant 2 and/or 3 present in the surface of a vesicle, prepared from bacteria which express the variant 2 or 3 sequence from a promoter of the invention.
Thus one composition of the invention includes: (i) vesicles of the invention, such as spontaneously-released vesicles which display a fHbp sequence; (ii) a soluble NHBA antigen, such as SEQ ID NO: 4; (iii) a soluble fHbp antigen, such as SEQ ID NO: 5; and (iv) a soluble NadA antigen, such as SEQ ID NO: 6.
Methods of Treatment
The invention also provides a method for raising an immune response in a mammal, comprising administering a composition of the invention to the mammal. The immune response is preferably protective and preferably involves antibodies. The method may raise a booster response in a patient that has already been primed against N. meningitidis. 
The mammal is preferably a human. Where the vaccine is for prophylactic use, the human is preferably a child (e.g. a toddler or infant) or a teenager; where the vaccine is for therapeutic use, the human is preferably an adult. A vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc.
The invention also provides vesicles of the invention for use as a medicament. The medicament is preferably used to raise an immune response in a mammal (i.e. it is an immunogenic composition) and is more preferably a vaccine.
The invention also provides the use of vesicles of the invention in the manufacture of a medicament for raising an immune response in a mammal.
These uses and methods are preferably for the prevention and/or treatment of a disease caused by N.meningitidis e.g. bacterial (or, more specifically, meningococcal) meningitis, or septicemia.
One way of checking efficacy of therapeutic treatment involves monitoring Neisserial infection after administration of the composition of the invention. One way of checking efficacy of prophylactic treatment involves monitoring immune responses against antigens after administration of the composition. Immunogenicity of compositions of the invention can be determined by administering them to test subjects (e.g. children 12-16 months age, or animal models [76]) and then determining standard parameters including serum bactericidal antibodies (SBA) and ELISA titres (GMT). These immune responses will generally be determined around 4 weeks after administration of the composition, and compared to values determined before administration of the composition. A SBA increase of at least 4-fold or 8-fold is preferred. Where more than one dose of the composition is administered, more than one post-administration determination may be made.
In general, compositions of the invention are able to induce serum bactericidal antibody responses after being administered to a subject. These responses are conveniently measured in mice and are a standard indicator of vaccine efficacy. Serum bactericidal activity (SBA) measures bacterial killing mediated by complement, and can be assayed using human or baby rabbit complement. WHO standards require a vaccine to induce at least a 4-fold rise in SBA in more than 90% of recipients. MeNZBTM elicits a 4-fold rise in SBA 4-6 weeks after administration of the third dose.
Preferred compositions can confer an antibody titre in a human subject patient that is superior to the criterion for seroprotection for an acceptable percentage of subjects. Antigens with an associated antibody titre above which a host is considered to be seroconverted against the antigen are well known, and such titres are published by organisations such as WHO. Preferably more than 80% of a statistically significant sample of subjects is seroconverted, more preferably more than 90%, still more preferably more than 93% and most preferably 96-100%.
Compositions of the invention will generally be administered directly to a patient. Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by any other suitable route. The invention may be used to elicit systemic and/or mucosal immunity. Intramuscular administration to the thigh or the upper arm is preferred. Injection may be via a needle (e.g. a hypodermic needle), but needle-free injection may alternatively be used. A typical intramuscular dose is 0.5 ml.
Dosage treatment can be a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. A primary dose schedule may be followed by a booster dose schedule. Suitable timing between priming doses (e.g. between 4-16 weeks), and between priming and boosting, can be routinely determined. The OMV-based RIVM vaccine was tested using a 3- or 4-dose primary schedule, with vaccination at 0. 2 & 8 or 0, 1, 2 & 8 months. MeNZBTM is administered as three doses at six week intervals.
Compositions of the invention may be used to induce bactericidal antibody responses against more than one hypervirulent lineage of meningococcus. In particular, they can preferably induce bactericidal responses against two or three of the following three hypervirulent lineages: (i) cluster A4; (ii) ET5 complex; and (iii) lineage 3. They may additionally induce bactericidal antibody responses against one or more of hypervirulent lineages subgroup I, subgroup III, subgroup IV-1 or ET-37 complex, and against other lineages e.g. hyperinvasive lineages. This does not necessarily mean that the composition can induce bactericidal antibodies against each and every strain of meningococcus within these hypervirulent lineages e.g. rather, for any given group of four of more strains of meningococcus within a particular hypervirulent lineage, the antibodies induced by the composition are bactericidal against at least 50% (e.g. 60%, 70%, 80%, 90% or more) of the group. Preferred groups of strains will include strains isolated in at least four of the following countries: GB, AU, CA, NO, IT, US, NZ, NL, BR, and CU. The serum preferably has a bactericidal titre of at least 1024 (e.g. 210, 211, 212, 213, 214, 215, 216, 217, 218 or higher, preferably at least 214) e.g. the serum is able to kill at least 50% of test bacteria of a particular strain when diluted 1/1024.
Useful compositions can induce bactericidal responses against the following strains of serogroup B meningococcus: (i) from cluster A4, strain 961-5945 (B:2b:P1.21,16) and/or strain G2136 (B:−); (ii) from ET-5 complex, strain MC58 (B:15:P1.7,16b) and/or strain 44/76 (B:15:P1.7,16); (iii) from lineage 3, strain 394/98 (B:4:P1.4) and/or strain BZ198 (B:NT:−). More preferred compositions can induce bactericidal responses against strains 961-5945, 44/76 and 394/98.
Strains 961-5945 and G2136 are both Neisseria MLST reference strains (ids 638 & 1002 in ref. 77). Strain MC58 is widely available (e.g. ATCC BAA-335) and was the strain sequenced in reference 78. Strain 44/76 has been widely used and characterised (e.g. ref. 79) and is one of the Neisseria MLST reference strains [id 237 in ref. 77; row 32 of Table 2 in ref. 46]. Strain 394/98 was originally isolated in New Zealand in 1998, and there have been several published studies using this strain (e.g. refs. 80 & 81). Strain BZ198 is another MLST reference strain (id 409 in ref. 77; row 41 of Table 2 in ref. 46).
Further Antigenic Components
In addition to vesicles of the invention, an immunogenic composition can include further non-vesicle antigens.
In some embodiments, a composition includes one or more capsular saccharides from meningococci e.g. from serogroups A, C, W135 and/or Y. These saccharides will usually be conjugated to a protein carrier. A composition of the invention may include one or more conjugates of capsular saccharides from 1, 2, 3, or 4 of meningococcal serogroups A, C, W135 and Y e.g. A+C, A+W135, A+Y, C+W135, C+Y, W135+Y, A+C+W135, A+C+Y, A+W135+Y, A+C+W135+Y, etc. Components including saccharides from all four of serogroups A, C, W135 and Y are ideal.
As well as containing antigens from N.meningitidis, compositions may include antigens from further pathogens. For example, the composition may comprise one or more of the following further antigens:

    
    
        an antigen from Streptococcus pneumoniae, such as a saccharide (typically conjugated)
        an antigen from hepatitis B virus, such as the surface antigen HBsAg.
        an antigen from Bordetella pertussis, such as pertussis holotoxin (PT) and filamentous haemagglutinin (FHA) from B.pertussis, optionally also in combination with pertactin and/or agglutinogens 2 and 3.
        a diphtheria antigen, such as a diphtheria toxoid.
        a tetanus antigen, such as a tetanus toxoid.
        a saccharide antigen from Haemophilus influenzae B (Hib), typically conjugated.
        inactivated poliovirus antigens.
    
    


Where a diphtheria antigen is included in the composition it is preferred also to include tetanus antigen and pertussis antigens. Similarly, where a tetanus antigen is included it is preferred also to include diphtheria and pertussis antigens. Similarly, where a pertussis antigen is included it is preferred also to include diphtheria and tetanus antigens. DTP combinations are thus preferred.
If a Hib saccharide is included (typically as a conjugate), the saccharide moiety may be a polysaccharide (e.g. full-length polyribosylribitol phosphate (PRP) as purified from bacteria), but it is also possible to fragment the purified saccharide to make oligosaccharides (e.g. MW from  ̃1 to  ̃5 kDa) e.g. by hydrolysis. The concentration of Hib conjugate in a composition will usually be in the range of 0.5 μg to 50 μg e.g. from 1-20 μg, from 10-15 μg, from 12-16 μg, etc. The amount may be about 15 g, or about 12.5 μg in some embodiments. A mass of less than 5 μg may be suitable [82] e.g. in the range 1-5 μg, 2-4 μg, or about 2.5 μg. As described above, in combinations that include Hib saccharide and meningococcal saccharides, the dose of the former may be selected based on the dose of the latter (in particular, with multiple meningococcal serogroups, their mean mass). Further characteristics of Hib conjugates are as disclosed above for meningococcal conjugates, including choice of carrier protein (e.g. CRM197 or tetanus toxoid), linkages, ratios, etc.
If a S.pneumoniae antigen is included, this may be a polypeptide or a saccharide. Conjugates capsular saccharides are particularly useful for immunising against pneumococcus. The saccharide may be a polysaccharide having the size that arises during purification of the saccharide from bacteria, or it may be an oligosaccharide achieved by fragmentation of such a polysaccharide. In the 7-valent PREVNARTM product, for instance, 6 of the saccharides are presented as intact polysaccharides while one (the 18C serotype) is presented as an oligosaccharide. A composition may include a capsular saccharide from one or more of the following pneumococcal serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and/or 33F. A composition may include multiple serotypes e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or more serotypes. 7-valent, 9-valent, 10-valent, 11-valent and 13-valent conjugate combinations are already known in the art, as is a 23-valent unconjugated combination. For example, an 10-valent combination may include saccharide from serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. An 11-valent combination may further include saccharide from serotype 3. A 12-valent combination may add to the 10-valent mixture: serotypes 6A and 19A; 6A and 22F; 19A and 22F; 6A and 15B; 19A and 15B; r 22F and 15B; A 13-valent combination may add to the 11-valent mixture: serotypes 19A and 22F; 8 and 12F; 8 and 15B; 8 and 19A; 8 and 22F; 12F and 15B; 12F and 19A; 12F and 22F; 15B and 19A; 15B and 22F. etc. Further characteristics of pneumococcal conjugates are as disclosed above for meningococcal conjugates, including choice of carrier protein (e.g. CRM197 or tetanus toxoid), linkages, ratios, etc. Where a composition includes more than one conjugate, each conjugate may use the same carrier protein or a different carrier protein. Reference 83 describes potential advantages when using different carrier proteins in multivalent pneumococcal conjugate vaccines.
General
The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., references 84-90, etc.
The term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X+Y.
The term “about” in relation to a numerical value x is optional and means, for example, x±10%.
Where the invention concerns an “epitope”, this epitope may be a B-cell epitope and/or a T-cell epitope, but will usually be a B-cell epitope. Such epitopes can be identified empirically (e.g. using PEPSCAN [91,92] or similar methods), or they can be predicted (e.g. using the Jameson-Wolf antigenic index [93], matrix-based approaches [94], MAPITOPE [95], TEPITOPE [96,97], neural networks [98], OptiMer & EpiMer [99, 100], ADEPT [101], Tsites [102], hydrophilicity [103], antigenic index [104] or the methods disclosed in references 105-109, etc.). Epitopes are the parts of an antigen that are recognised by and bind to the antigen binding sites of antibodies or T-cell receptors, and they may also be referred to as “antigenic determinants”.
References to a percentage sequence identity between two amino acid sequences means that, when aligned, that percentage of amino acids are the same in comparing the two sequences. This alignment and % homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of ref. 110. A preferred alignment is determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is disclosed in ref. 111.
The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 illustrates fHbp expression levels in the same background strain (NZ98/254) using five different promoters and the benchmark strain overexpressing the same fHbp allele in the same background [112,113]. A variant 2 allele of fHbp was overexpressed in the benchmark strain or from either 2 wildtype phase variants of the porA promoter with either 11 Gs or 13 Gs in the spacer tract or with the modified ST1, ST2 and ST3 porA promoters as indicated. Western blotting of equivalent quantities of total protein were loaded from 0.5× to 2× as indicated for each strain, and the arrow shows fHbp.
FIG. 2 illustrates comparative analysis of fHbp expression from porA and 16S modified promoters. The NZ98/254 strain expressing fHBP variant 2 under the control of the indicated promoters was grown on plates (A) and in liquid to logarithmic (B) and stationary phase (C) and levels of fHBP expression were measured by western blot from duplicate cultures (#1, #2) or from the benchmark overexpression strain. The unbroken arrow shows fHbp, whereas the lower broken arrow shows a loading control.
FIG. 3 shows expression of fHbp from three strains: (a) a ΔlpxL1 knockout; (b) a ΔsynX knockout; and (c) a ΔlpxL1ΔsynX double knockout. In all cases the knocked out gene(s) was replaced by fHbp under the control of a modified PorA promoter. Expression of fHbp was assessed by western blot on crude cell extracts using anti-fHbp serum, and was compared to Hfq as a control. FIG. 3A shows the western blot, and FIG. 3B quantifies expression in arbitrary units.
FIG. 4 shows fHbp levels in vesicles isolated from (i) ΔlpxL1ΔsynX and (ii) ΔlpxL1ΔsynXΔmltA knockout strains. FIG. 4A shows yield in mg/OD, and FIG. 4B is a western blot of 0.5 μg vesicles.
FIG. 5 shows expression of fHbp in two strains that overexpress either variant 1 or variant 2 fHbp in distinct loci (lanes c & d) or a strain that overexpresses both variant 1 and variant 2 in respective loci (lane e). Lane a is the wild-type strain, and lane b is a ΔfHbp knockout of that strain.
FIG. 6 shows silver-stained SDS-PAGE of vesicles purified from the triple knockout.



MODES FOR CARRYING OUT THE INVENTION
Modified PorA Promoters
The sequences upstream of the transcription start site of two phase variants of the wild-type meningococcal porA promoter, containing 11 and 13 consecutive Gs in the spacer region, are as follows, with the −35 and −10 regions underlined:









(SEQ ID NO: 31)



AAAAATGGTTTTTTGCGGGGGGGGGGGTATAATTGAAGAC



 



(SEQ ID NO: 40)



AAAAATGGTTTTTTGCGGGGGGGGGGGGGTATAATTGAAGAC






The intervening sequence between the −35 and −10 regions includes a G11 poly-G sequence which is connected to phase variation of PorA expression. To stabilise expression from this promoter, three modifications were tested (modifications in lower case):









(′′ST1′′; SEQ ID NO: 32)



AAAAATGGTTTTTTGCGaGGGaGGtGGTATAATTGAAGAC



 



(′′ST2′′; SEQ ID NO: 33)



AAAAATtGacaTTTGCGaGGGaGGtGGTATAATTGAAGAC



 



(′′ST3′′; SEQ ID NO: 34)



AAAAtTGacaTTTTGCGaGGGaGGtGGTATAATTGAAGAC






Thus in “ST1” the G11 sequence was interrupted in three places, leaving no more than 3 consecutive G residues. In “ST2” the same intervening sequence was used, but the −35 region was substituted by a consensus −35 region TTGACA. In “ST3” an extra T residue was inserted immediately downstream of the −35 region.
These three promoters were used to drive expression of a fHbp sequence (fHbp variant 2) in the NZ98/258 strain. All three modified promoters resulted in high level overexpression of fHBP in the strain when compared to an equivalent overexpressing strain which has been reported in the literature [112,113]. The best expression was seen with ST2 (FIGS. 1 & 2).
Modified rRNA Promoters
In further experiments the 16S rRNA promoter was fused to the 5′ UTR of the porA gene:









(SEQ ID NO: 35)



ATATCTTGACAGGCGGAAGGAATACTTTATAATTCGCAAC . . .






This promoter was subjected to mutation and three variants downstream of the −35 region were sequenced as follows:









(′′wt′′; SEQ ID NO: 35)



ATATCTTGACAGGCGGAAGGAATACTTTATAATTCGCAAC



 



(′′#5′′; SEQ ID NO: 36)



ATATCTTGACAGGCGGAAGGAATACTTTATATTCGCAAC . . .



 



(′′#6′′; SEQ ID NO: 37)



ATATCTTGACAGGCGGAAGGAATACTTTTAATTCGCAC . . . 



 



(′′#8′′; SEQ ID NO: 38)



ATATCTTGACAGGCGGAAGGAAACTTTATAATTCGCAAC . . .






Thus in variants #5 and #6 the −10 sequence is a 5-mer, and variant #6 also lost a nucleotide between the −10 region and the transcription start site. In variant #8 the −10 region and its downstream sequence are the same as wt, but the intervening sequence between the −35 and −10 regions is one nucleotide shorter than wt.
Expression of fHbp from Modified Promoters
These promoters were fused to the 5′UTR region of the porA gene and used to drive expression of the fHbp coding sequence. As seen in FIG. 2, variants #5 and #6, variants #5 and #6 showed the strongest expression, approaching levels seen with ST2.
The complete region spanning the wild-type promoter (SEQ ID NO: 35), extending downstream into the transcribed region and also upstream, was as follows (SEQ ID NO: 41; 358mer), with the −35 and −10 regions underlined, and nucleotide +1 (following reference 10) double-underlined:








(SEQ ID NO: 41)



. . . CGTCTGAGTCCCCGAGTTTCAGACAGCATATTCACAAAGGCGCACCAGCCGGAGGAGGGAGAGGAAAG



 


GATTGTTGGAGGCGGCGCAGTATTTAGCAGAAATAAAAAACCTTATCCGACAGCGACATGACGAATTTCCC


 


CAAAAAAATCCCGCTGAAAGCATTGACCGTTTTTCCCTGTGGGCGTATAGTTCGGTTCTTCGCTGCTGCAG


 


AAGTGGCGGACGAACTGAAAAGTATAGCACAGAATGTTGGGGATATCGAGAGATATCTTGACAGGCGGAAG


 


GAATACTTTATAATTCGCAACGTATCGGGTGTTTGCCCGATGTTTTTAGGTTTTTATCAAATTTACAAAAG


 


GAAGCC . . .






Similarly, the surrounding sequences for variants #5 and #6 were as follows:








(SEQ ID NO: 42)



#5: . . . CGTCTGAGTCCCCGAGTTTCAGACAGCATATTCACAAAGGCGCACCAGCCGGAGGAGGGAGAGG



 


AAAGGATTGTTGGAGGCGGCGCAGTATTTAGCAGAAATAAAAAACCTTATCCGACAGCGACATGACGAATT


 


TCCCCAAAAAAATCCCGCTGAAAGCATTGACCGTTTTTCCCTGTGGGCGTATAGTTCGGTTCTTCGCTGCT


 


GCAGAAGTGGCGGACGAACTGAAAAGTATAGCACAGAATGTTGGGGATATCGAGAGATATCTTGACAGGCG


 


GAAGGAATACTTTATATTCGCAACGTATCGGGTGTTTGCCNNANGTTTTTAGGTTTTTATCAAATTTCAAA


 


AGGAAGCC . . .


 


(SEQ ID NO: 43)



#6: . . . CGTCTGAGTCCCCGAGTTTCAGACAGCATATTCACAAAGGCGCACCAGCCGGAGGAGGGAGAGG



 


AAAGGATTGTTGGAGGCGGCGCAGTATTTAGCAGAAATAAAAAACCTTATCCGACAGCGACATGACGAATT


 


TCCCCAAAAAAATCCCGCTGAAAGCATTGACCGTTTTTCCCTGTGGGCGTATAGTTCGGTTCTTCGCTGCT


 


GCAGAAGTGGCGGACGAACTGAAAAGTATAGCACAGAATGTTGGGGATATCGAGAGATATCTTGACAGGCG


 


GAAGGAATACTTTTAATTCGCACGTATCGGGTGTTTGCCCGATGTTTTTAGGTTTTTATTAAATTTACAAA


 


AGGAAGCCCATANGAATCGAACTGC . . .






Any of these longer sequences can be used with the invention, although modifications as discussed herein may, of course, be made.
A fHbp gene under the control of a variant promoter was stably inserted into the chromosome of a meningococcus (strain NZ98/254) in place of the endogenous lpxL1 and/or synX gene(s). Slightly higher expression levels were seen at the synX locus, and expression for the strain with both insertions (ΔlpxL1ΔsynX double knockout) was the sum of the expression from the individual loci (FIG. 3).
Strains were also made in which two different fHbp variants (1 & 2) were expressed under the control of a modified PorA promoter. The co-expression of both variants has no significant negative effect on the expression from each distinct locus, and instead resulted in an additive effect (FIG. 5).
Knockout of the endogenous mltA (GNA33) gene in the ΔlpxL1ΔsynX double knockout further increased expression levels, and had no negative impact on the localisation of fHbp protein to the strain's vesicles. FIG. 4 shows fHbp levels in vesicles isolated from these strains, showing (A) the yield of protein (mg) relative to the culture's optical density (OD), and (B) an anti-fHbp western blot against of 0.5 μg of the vesicles. SDS-PAGE shows that the fHbp band in the triple knockout strain was one of the most abundant proteins in the vesicles, along with PorA and PorB (FIG. 6).
It will be understood that the invention is described above by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.
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## Claims

1. A nucleic acid comprising a promoter operably linked to a heterologous coding sequence, wherein the promoter is selected from the group consisting of:
(a) a promoter comprising
(i) a −10 region from a meningococcal porA gene promoter comprising TATAAT and
(ii) a −35 region comprising the nucleic acid sequence TGGTTT or the nucleic acid sequence TTGACA,


wherein the −10 region and the −35 region are separated by an intervening sequence comprising 12-20 nucleotides, wherein the intervening sequence either comprises no poly-G sequence or includes a poly-G sequence comprising no more than five consecutive G nucleotides; and
(b) a promoter comprising a sequence selected from the group consisting of
(i) the sequence of SEQ ID NO: 18 and
(ii) a variant of SEQ ID NO: 18 which differs from SEQ ID NO: 18 by up to 4 single-nucleotide insertions, deletions, or substitutions.

2. The nucleic acid of claim 1, wherein
the promoter of (a) has a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34.

3. The nucleic acid of claim 1, wherein the intervening sequence does not comprise a sequence GGGGG.

4. The nucleic acid of claim 1, wherein non-transcribed sequence downstream of the −10 sequence and upstream of the transcription start site comprises 5-10 nucleotides.

5. The nucleic acid of claim 1, wherein the promoter of (a) comprises the sequence of SEQ ID NO: 30.

6. The nucleic acid of claim 1, wherein the promoter of (b) is selected from the group consisting of: nucleotides 6-39 of SEQ ID NO: 36, nucleotides 6-38 of SEQ ID NO: 37, and nucleotides 6-39 of SEQ ID NO: 38.

7. A bacterial expression vector comprising a DNA sequence comprising the nucleic acid of claim 1.

8. A meningococcus comprising a DNA sequence comprising the nucleic acid of claim 1.

9. The meningococcus of claim 8, wherein the heterologous coding sequence encodes an outer membrane protein.

10. The meningococcus of claim 9, wherein the outer membrane protein is a fHbp.

11. The meningococcus of claim 8, wherein the meningococcus does not express an active MltA.

12. The meningococcus of claim 8, wherein the meningococcus comprises a knockout of at least one of SynX and LpxL1.

13. The meningococcus of claim 8 comprising a meningococcus selected from the group consisting of serogroup B and serogroup W135.

14. The meningococcus of claim 8 comprising a lipopolysaccharide of immunotype L3.

15. The nucleic acid of claim 4, wherein the non-transcribed sequence comprises a sequence selected from the group consisting of TGAAGAC and TCGCAAC.