DEVELOPMENTS IN MENINGOCOCCAL OUTER MEMBRANE VESICLES

Abstract

A first aspect of the invention provides meningococcal outer membrane vesicles in which NHBA is over-expressed. A second aspect of the invention provides meningococcal outer membrane vesicles in which NadA is over-expressed. A third aspect of the invention provides a panel of bacterial strains, each member of which is isogenic except for a single gene which in each strain encodes a different variant antigen of interest.

Claims

1. Meningococcal outer membrane vesicles in which NHBA is over-expressed.

2. A meningococcus which over-expresses NHBA.

3. The meningococcus of claim 2, which also over-expresses fHbp.

4. A meningococcus which expresses NHBA, wherein the meningococcus is isogenic with a parental strain, except for a genetic modification which causes the meningococcus to express more NHBA than the parental strain.

5. The meningococcus of claim 4, which includes (i) a gene under the control of a promoter which does not control that gene in the parental strain and/or (ii) a knockout of a gene which is found in the parental strain.

6. The meningococcus of claim 2, wherein expression of NHBA is controlled by an inducible or constitutive promoter, and wherein the promoter optionally includes a CREN.

7. The meningococcus of claim 2, wherein the meningococcus does not express NadR.

8. The meningococcus of claim 2, wherein the bacterium also expresses more fHbp than the parental strain.

9. The meningococcus of claim 2, wherein expression of NHBA is controlled by a strong promoter, NadR is knocked out, and the strain expresses a constitutively active mutant FNR.

10. The meningococcus of claim 2, wherein expression of NHBA is controlled by a strong promoter, expression of fHbp is controlled by a strong promoter, and NadR is knocked out.

11. The meningococcus of claim 2, wherein the bacterium has a knockout of LpxL1.

12. The meningococcus of claim 2, wherein the bacterium does not express an active MltA.

13. The meningococcus of claim 2, wherein the bacterium does not express PorA.

14. The meningococcus of claim 2, wherein the bacterium does not express FrpB.

15. The meningococcus of claim 2, in serogroup B.

16. The meningococcus of claim 2, in immunotype L3.

17. Outer membrane vesicles prepared from the meningococcus of claim 2.

18. A process for preparing a meningococcal strain suitable for OMV preparation, comprising steps of (i) choosing a starting strain which expresses NHBA; and (ii) modifying the starting strain to increase the amount of NHBA which it expresses.

19. A process for preparing a meningococcal strain suitable for OMV preparation, comprising steps of (i) choosing a starting strain which expresses a first amount of NHBA when grown in specific culture conditions, then (ii) modifying the starting strain to provide a modified strain, wherein the modified strain expresses a second amount of NHBA when grown in the same specific culture conditions, wherein the second amount is higher than the first amount.

20. The process of claim 18, including a step (iii) culturing the modified bacteria obtained in step (ii) to provide a bacterial culture.

21. (canceled)

22. A process for preparing a meningococcal vesicle, comprising a step of treating a bacterial culture obtained by the process of claim 20 such that its outer membrane forms vesicles.

23. Outer membrane vesicles prepared by the process of claim 22.

24. An immunogenic pharmaceutical composition comprising the vesicles of claim 1.

25. The composition of claim 24, including one or more capsular saccharides from meningococci.

26. The composition of claim 24, including an antigen from Streptococcus pneumoniae.

27. A method for raising an immune response in a mammal, comprising administering a composition of claim 24 to the mammal.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0138] FIG. 1 illustrates the approach for constructing an isogenic panel by knocking out nadA and nhba (GNA2132) to create a background strain.

[0139] FIG. 2 shows the insertion of fHbp genes into the background strain to make a panel of isogenic strains expressing different fHbp genes under the control of a Ptac promoter.

[0140] FIG. 3A-FIG. 3B shows expression levels of fHbp in the isogenic panel strains described in FIG. 2.

[0141] FIG. 4A-FIG. 4B shows expression of NadA (upper panel) and NadR (lower panel) in eight wild-type strains (FIG. 4A) or their NadR knockout forms (FIG. 4B). The numbers in FIG. 4A show the number of TAAA tetranucleotide repeats in the strain. FIG. 4C-FIG. 4D shows expression of NadA and NadR in 7 strains, in the presence of absence of 4HPA.

[0142] FIG. 5A-FIG. 5C shows (FIG. 5A) starting strain MC58 (FIG. 5B) MC58nhba and (FIG. 5C) MC58nhba transformed with a complementing nhba gene with an upstream CREN and IPTG-inducible promoter.

[0143] FIG. 6 shows NHBA expression by MC58 and derivative strains. The left two lanes show expression in MC58 and MC58nhba. The next 8 lanes show expression in complemented strains at four concentrations of IPTG. The lanes are arranged in pairs, with the right-hand lane being a strain complemented with nhba having an upstream CREN.

[0144] FIG. 7 shows NHBA expression by 95N477 and derivative strains. The left two lanes show expression in 95N477 and 95N477nhba. The next 5 lanes show expression in complemented strains at the indicated concentrations of IPTG.

[0145] FIG. 8 shows NHBA expression for five strains in an isogenic panel. From top to bottom the expressed NHBA is from strain NZ98/254, UK013, UK355, 2996 and NM117.

MODES FOR CARRYING OUT THE INVENTION

NHBA

[0146] The endogenous nhba gene is knocked out in various serogroup B strains to create strains MC58nhba, 95N477nhba, NGH38nhba and UK013nhba. These strains are then transformed with pCOMPpind-287 vector containing a gene encoding nhba from strain 394/98, with or without an upstream CREN (contact regulatory element of Neisseria), under the control of an IPTG-inducible promoter. The vectors insert the nhba gene (CREN) between the endogenous nmb1428 and nmb1429 genes by homologous recombination.

[0147] FIG. 5 shows the starting MC58 strain, the MC58nhba strain, and the complemented MC58 strain (+CREN). FIG. 6 shows expression of NHBA by the various MC58 strains with increasing IPTG concentration. The complemented strains show high levels of inducible NHBA expression, with the highest levels seen with the inserted gene has an upstream CREN.

[0148] FIG. 7 shows expression in the 95N477 strains. The endogenous nhba gene in this strain encodes a 427aa protein, whereas the inserted complementing gene has 492aa. Increased expression levels of the larger NHBA protein are clearly visible, and this expression increases with IPTG concentration.

[0149] Although in some strains (e.g. M4407) it was not possible to obtain a nhba knockout using the transformation protocols, for strains which could be transformed these results show that strains which over-express NHBA can readily be obtained.

NadR (NMB1843)

[0150] The nadA gene is present in approximately 50% of meningococcal isolates. NadA exhibits growth-phase dependent expression, with maximal levels in the stationary growth phase of all strains tested. Expression is controlled by a tetranucleotide repeat (TAAA) located upstream of the nadA promoter. The number of repeats can be modified during replication through slipped strand mispairing, and consequently can influence the expression of the nadA gene by creating variants where changes in the repeat number result in promoters with low, medium or high activity.

[0151] An area of the nadA promoterm upstream of the TAAA repeat, is responsible for repression of nadA expression during logarithmic phase of growth. This area is called the GPR region. DNA-affinity fractionation identified a protein present in meningococcus crude extracts which binds to the GPR region. This protein is NadR (NMB1843) and is a member of the MarR family of repressors. NadR binds to three operators (binding sites) in the nadA promoter and results in repression of NadA expression. Knockout of NadR in strains expressing high, medium or low levels of NadA results in almost comparable high level expression in each strain. Thus NadR is the repressor that contributes to the differential expression levels exhibited by meningococcal strains, or phase variants in the same strain, with different numbers of repeats in their promoter. NadR is expressed to similar levels in different strains but can repress more or less efficiently the nadA promoter depending on the number of repeats present in the variant promoter.

[0152] Knockout of NadR in various meningococcus backgrounds results in almost comparable high levels of expression of NadA across the panel. Strains are transformed with the knockout construct for the allelic replacement of nmb1843 with a chloramphenicol cassette. Expression levels in eight different strains are shown in FIG. 4.

[0153] A small molecule ligand 4-hydroxyphenylacetic acid (4HPA) can induce NadA expression in vitro due to derepression of NadR (FIG. 4C). Addition of the molecule to the purified NadR protein in vitro can inhibit the binding activity of the protein for the nadA promoter. 4HPA is a metabolite of the catabolic pathway of the aromatic amino acids and is secreted in human saliva and urine, and so in vivo expression of NadA may be higher than is seen during in vitro growth.

[0154] Thus strains which over-express NadA can readily be obtained by inactivation of NadR and/or by addition of a small molecule inducer to the growth medium.

Isogenic PanelNHBA

[0155] NHBA is an antigen in the 4CMenB product. An isogenic panel was used to study the potential cross protection of NHBA-induced bactericidal antibodies.

[0156] The nhba genes from six different meningococcal strains were amplified to provide the mature form of the polypeptide with a C-terminus histidine tag. These were cloned into the pET-21b+ plasmid vector and expressed in E. coli. The purified NHBA peptides were then used to immunize mice (20 g dose) and obtain mouse antisera. ELISA assays were performed in order to confirm the presence of antibodies in all the mouse sera obtained.

[0157] To evaluate the immunogenicity and the contribution of amino acid sequence variability to vaccine coverage, a starting strain was engineered to be susceptible to bactericidal killing only by anti-NHBA antibodies (rather than the other antigens in 4CMenB). N. meningitidis strain 5/99 naturally expresses high levels of NadA, but very low levels of NHBA and fHbp. Its nadA and nhba genes were respectively replaced by ery and kan resistance cassettes (5/99). The nhba gene to be complemented was then inserted in the intergenic region between the open reading frames nmb1428 and nmb1429. Thus the final strain panel was isogenic except for the chosen nhba gene, and this gene should be inducable for expression at equal levels in all members of the panel.

[0158] FACS showed that the panel members showed a comparable amount of the different NHBA polypeptides in each strain (FIG. 8). Several mouse antisera raised against the different NHBA polypeptides were tested in western blot and the detection appeared to be variant-specific, showing a stronger recognition for the homologous variant.

[0159] The panel was also used for testing the bactericidal effect of the mouse antisera. As the strains were isogenic then any difference in bactericidal effect should arise only from the different expressed NHBA polypeptides. In parallel the sera were tested against wild-type strains which express the relevant NHBA polypeptide sequence, to see if the common genetic background of the isogenic panel did enable the detection of differences which would be concealed by natural variation if wild-type strains were used. Results were as follows:

TABLE-US-00001 Antiserum NHBA 5/99 Wild-type NZ98/254 >8192 8192 WC58 8192 512 UK013 256 128 UK355 128 256 2996 128 256 NM117 2048 4096

[0160] Thus the panel does seem to compensate for variability which is unrelated to the NHBA antigen itself. For instance, serum raised against the MC58 sequence is much more effective against the MC58 polypeptide in the isogenic panel than against the wild-type MC58 strain.

Isogenic PanelfHbp

[0161] Sequencing of the fHbp gene in a large collection of meningococcal isolates revealed three variants with low levels of cross-protective bactericidal response. A serum bactericidal assay was used to evaluate the cross-protective capabilities of human antibodies raised against different fHbp variants, but the killing mediated by bactericidal antibodies in this assay is dependent by several factors. Thus the potential coverage of a single antigen may be difficult to estimate.

[0162] A genetic approach was used to overcome variability due to strain-specific serum susceptibility, limitations of compatible complement sources, and variable expression of fHbp and other surface-exposed factors affecting resistance to serum (e.g. the capsule). A well-characterized meningococcal isolate (5/99) was engineered to generate isogenic strains expressing ten different fHbp sub-variants from a constitutive heterologous promoter. The fHbp genes were inserted between endogenous nmb1428 and nmb1429 genes. This panel was then used as the test strain in a serum bactericidal antibody (SBA) assay to assess the ability of a single fHbp variant to elicit a broadly-protective immune response.

[0163] In order to have a genetic background to express different fHbp sub-variants without the interfering action of the other antigens, the nadA and nhba genes in the starting 5/99 strain were inactivated by insertion of erm and kan resistance cassettes, respectively (FIG. 1). The resulting double mutant strain (named 5/99) was manipulated to express different fHbp subvariants under the control of a Ptac promoter to standardize the amount of fHbp expressed (FIGS. 1 & 2). In total, ten different fHbp coding sequences were cloned in the pComp-RBS vector and transferred to the 5/99 genetic background.

[0164] To evaluate the expression of fHbp in the recombinant strains, we performed FACS analysis using a mouse polyclonal serum against a single fHbp variant. The analysis showed a comparable amount of the different fHbp sub-variants on the surface of the recombinant strains generated (FIG. 3).

[0165] The recombinant strains were analyzed for their susceptibility to killing by bactericidal antibodies from mice in a SBA using rabbit complement. Pooled sera from mice immunized with the universal vaccine of reference 19 or with its GNA2091-fHbp component were tested for their ability to kill the 5/99 wild-type, the intermediate 5/99 strain expressing neither NHBA nor NadA antigens, and the ten recombinant strains. The 5/99 strain was killed by sera raised against the universal vaccine, but not by sera raised against the single antigen GNA2091-fHbp. The 5/99 strain was resistant to killing by all sera. All of the complemented strains except one showed significant susceptibility to sera derived from mice immunized with the universal vaccine or with GNA2091-fHbp antigen. The single surviving strain expressed a fHbp in family III, confirming the absence of cross-reactivity between the fHbp families. The nine susceptible strains confirm that the specific fHbp sequence in the universal vaccine can raise antibodies which are broadly protective across the whole of fHbp family 1.

[0166] The panel was also tested using sera obtained from human adults who were immunised with 4CMenB. The results were comparable to those seen using mice.

[0167] 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 end spirit of the invention.

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