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MODIFIED FACTOR H BINDING PROTEIN

20220112249 · 2022-04-14

    Inventors

    Cpc classification

    International classification

    Abstract

    The invention relates to a modified factor H binding protein (fHbp), comprising fHbp, or a variant thereof, modified with the addition of at least one exogenous peptide loop; and associated nucleic acid, compositions, and uses. The invention further relates to treatment or prevention of a pathogenic infection or colonisation of a subject using the modified factor H binding protein (fHbp).

    Claims

    1. A modified factor H binding protein (fHbp), comprising fHbp, or a variant thereof, modified with the addition of at least one exogenous peptide loop.

    2. The modified factor H binding protein according to claim 1, wherein the fHbp is meningococcal or gonococcal fHbp, or a variant thereof.

    3. The modified factor H binding protein according to claim 1 or claim 2, wherein the modified fHbp is modified such that it is not capable of binding factor H, or at least has reduced factor H binding activity.

    4. The modified factor H binding protein according to any preceding claim, wherein the exogenous peptide loop(s) is immunogenic.

    5. The modified factor H binding protein according to any preceding claim, wherein the exogenous peptide loop(s) is derived from an integral outer membrane protein.

    6. The modified factor H binding protein according to any preceding claim, wherein the exogenous peptide loop(s) is prokaryotic in origin.

    7. The modified factor H binding protein according to any preceding claim, wherein the exogenous peptide loop(s) is derived from a membrane protein.

    8. The modified factor H binding protein according to any preceding claim, wherein the exogenous peptide loop(s) is derived from a meningococcal protein.

    9. The modified factor H binding protein according to any preceding claim, wherein the exogenous peptide loop(s) is derived from a gonococcal protein.

    10. The modified factor H binding protein according to any preceding claim, wherein the exogenous peptide loop(s) comprise a fragment of PorA, or a variant thereof.

    11. The modified factor H binding protein according to any preceding claim, wherein the exogenous peptide loop(s) are between 8 and 20 amino acids in length.

    12. The modified factor H binding protein according to any preceding claim, wherein the exogenous peptide loop(s) are about 16 amino acids in length.

    13. The modified factor H binding protein according to any preceding claim, wherein the exogenous peptide loop(s) are selected from any one of the PorA loops 1 to 7, or fragments thereof; and/or variants thereof; and/or combinations thereof; or wherein the exogenous peptide loop(s) are selected from any one of the PorA loops provided in Table 1 herein (e.g. any of SEQ ID NOs: 45 to 79), or variant thereof.

    14. The modified factor H binding protein according to any preceding claim, wherein the modified fHbp comprises two or more exogenous peptide loops.

    15. The modified factor H binding protein according to any preceding claim, wherein the modified fHbp comprises between 1 and 7 exogenous peptide loops.

    16. The modified factor H binding protein according to any preceding claim, wherein the modified fHbp, or variant thereof, is modified with an exogenous peptide loop in at least two positions.

    17. The modified factor H binding protein according to any preceding claim, wherein the peptide loop is inserted into fHbp, or a variant thereof, at one or more of amino acid positions selected from 49-54, 83-88, 114-124, 199-206, 227-233, and 240-246.

    18. The modified factor H binding protein according to any preceding claim, wherein the modified fHbp is immunogenic.

    19. The modified factor H binding protein according to any preceding claim, wherein the modified fHbp is a fusion protein.

    20. The modified factor H binding protein according to any preceding claim, wherein the modified fHbp comprises the sequence of any one of SEQ ID NOs: 1 to 13; or a variant thereof.

    21. A nucleic acid encoding the modified fHbp according to any of claims 1 to 20.

    22. The nucleic acid according to claim 21, wherein the nucleic acid is a vector, optionally a viral vector.

    23. A composition comprising the modified fHbp according to any of claims 1 to 10 or the nucleic acid according to claim 21 or 22.

    24. The composition according to claim 23, wherein the composition comprises two or more different modified fHbp molecules.

    25. The composition according to claim 23 or claim 24, wherein the composition comprises a pharmaceutically acceptable carrier.

    26. The composition according to any of claims 23 to 25, wherein the composition further comprises an adjuvant.

    27. The composition according to any of claims 23 to 26, wherein the composition further comprises at least one other prophylactically or therapeutically active molecule; and optionally wherein the at least one other prophylactically or therapeutically active molecule comprises: a monovalent protein:capsule polysaccharide vaccine; or a conjugate vaccine, wherein antigen(s) comprising the fHbp scaffold bearing exogenous peptide loops is incorporated as the protein carrier molecule in the conjugate vaccine.

    28. A modified fHbp according to any of claims 1 to 20, a nucleic acid according to claim 20 or 21, or a composition according to any of claims 23 to 27, for use as a medicament.

    29. A modified fHbp according to any of claims 1 to 20, a nucleic acid according to claim 21 or 22, or a composition according to any of claims 23 to 27, for use in the treatment or prevention of a pathogenic infection or colonisation of a subject.

    30. A method of treatment or prevention of a pathogenic infection or colonisation of a subject, comprising the administration of a modified fHbp according to any of claims 1 to 20, a nucleic acid according to claim 21 or 22, or a composition according to any of claims 23 to 27, to the subject.

    31. A method of vaccination, comprising the administration of a modified fHbp according to any of claims 1 to 20, a nucleic acid according to claim 21 or 22, or a composition according to any of claims 23 to 27, to a subject.

    32. A combination of the modified fHbp according to any of claims 1 to 20, a nucleic acid according to claim 21 or 22, or a composition according to any of claims 23 to 27 and at least one other prophylactically or therapeutically active molecule.

    33. The combination according to claim 32, wherein at least one other prophylactically or therapeutically active molecule comprises a monovalent protein:capsule polysaccharide vaccine.

    34. The combination according to claim 33 or composition according to claim 26, wherein the monovalent protein:capsule polysaccharide vaccine comprises any of serogroup C or A capsule with bacterial toxoids, bi-valent vaccines (with serogroup C and A capsular polysaccharide conjugated to bacterial toxoids), quadri- (serogroups A, C, Y, W) or penta- (A, C, Y, W, X) valent conjugate vaccines.

    35. The combination according to claim 32 or composition according to claim 26, wherein the at least one other prophylactically or therapeutically active molecule comprises a conjugate vaccine, wherein antigen(s) comprising the fHbp scaffold bearing exogenous peptide loops is incorporated as the protein carrier molecule in the conjugate vaccine, optionally wherein the conjugate vaccine comprises any of serogroup capsular polysaccharides selected from A, C, Y, W, or X strains, or combinations thereof.

    36. Use of factor H binding protein (fHbp) as an epitope display scaffold.

    37. The modified fHbp, nucleic acid, composition, method or use essentially as described herein, optionally with reference to the accompanying figures.

    Description

    [0137] Embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying drawings.

    [0138] FIG. 1 Design of chimeric fHbp:PorAs [0139] (A) Schematic of the surface of N. meningitidis, showing the pre-dominant outer membrane features, lipo-oligosaccharide and type 4 pili, and the important antigens, fHbp and PorA. The immunogenic PorA VR2 loop is highlighted and fHbp is shown interacting with domains 6 and 7 of human CFH. [0140] (B) Structure of V1 fHbp with CFH domains 6-7 showing the six positions (P1-5, and P7) used to generate chimeric fHbp:PorAs into which we have inserted PorA loops. N.B. position 5 is in the fHbp:CFH interface.

    [0141] FIG. 2: Use of fHbp as a molecular scaffold [0142] A) Protein structure of fHbp V1.1 (grey, ribbon representation) with the six positions (P1-5, and P7) used to generate chimeric fHbp:PorAs. B) Secondary structure of fHbp V1.1 (grey, arrows represent β-sheets, rectangles represent α-helices). Locations of the VR2 P1.16 PorA insertion sites are indicated by solid black lines, numbers indicate the residue range that the PorA VR2 loops may be inserted (corresponds with residue numbers in 1D). C) Analysis of purified fHbp:PorAs with the PorA P1.16 VR2 loop in positions 1-5 or 7 of fHbp, the wild-type (WT V1.1) by SDS-PAGE and Western blot. Blots were probed with α-V1 fHbp pAb and an α-P1.16 mAb. D) Primary sequence (SEQ ID NO: 1) of V1.1 fHbp indicating the locations (underlined) of positions 1-5 and 7 into which loops from other proteins can be inserted.

    [0143] FIG. 3 Characterisation of chimeric fHbp:PorAs [0144] (A) Structure of fHbp:PorAs overlaid with the P1.16 loop (black, PDBID: 2 mpa) with the Fab of the α-P1.16 mAb and fHbp:PorAs with the loop in position 1, position 3 and position 7, demonstrating that the epitope is in a conformation recognised by a bactericidal antibody. [0145] (B) Stability of fHbp N-terminal (NT) and C-terminal (CT) beta-barrels of fHbps by Differential Scanning calorimetry analysis performed using a 20-120° C. temperature gradient. Melting temperature is shown for the fHbp N-terminal (NTT) and C-terminal (CTT) barrels. Binding fHbp:PorAs to Complement Factor H (CFH) and mAbs SPR analysis of fHbp and chimeric fHbp:PorAs coupled to a BIAcore CMS chip. CFH (fH67) was flowed over at a dilution range of 0.5-32 nM, and the dissociation constant (K.sub.D) calculated; dissociation constants (KDs) for fHbp:PorAs confirms lack of CFH binding of fHbp with a loop in position 5 which impinges on the fHbp:CFH interface (FIG. 2A). NB=Non-binding.

    [0146] FIG. 4 Repertoire of antigens selected for chimeric fHbp:PorAs [0147] (A, B) Frequency of fHbp variants and PorA VR2 subtypes, respectively, in N. meningitidis disease isolates in the UK between 2010-2015 from the Meningitis Research Foundation Genome Library (http://www.meningitis.org/research/genome), shown as Pie Charts (above) and Tables (below) with the frequency of specific fHbps and PorAs. [0148] N.B. V2 fHbp accounts for 38.9% of isolates.

    TABLE-US-00047 TABLE 2 Exact sequence matches for all N. meningitidis UK isolates between 2009-2015: Antigen Variant % MenB coverage fHbp 1.4 21.5 2.19 9.1 3.45 4.8 PorA P1.4 19.3 P1.9 19.1 P1.14 16.3 P1.16 7.0 P1.15 5.5 P1.1511 5.3 % coverage minus antigen overlap in ALL UK 70.6 strains (isolated between 2009-2015) % coverage minus antigen overlap in UK MenB 79.2 strains (isolated between 2009-2015) [0149] Exact sequence matches for: [0150] Pfizer vaccine, 3.02% [0151] Bexsero fHbp (V1.1) or PorA (P1.16), 15.86% [0152] Chimeric fHbp:PorAs, fHbp or PorA, 72% [0153] (23.5% with fHbp AND PorA)

    [0154] FIG. 5 Recognition of Neisseria meningitidis fHbp (A) and PorA (B) antigens by mouse immune sera. Whole cell lysates from Neisseria meningitidis strains H44/76 (WT), H44/67 ΔfHbp and H44/67 ΔPorA, were separated by SDS-PAGE, transferred to a PVDF membrane and probed with mouse immune sera. Mouse immune sera were obtained by immunising BalbC mice three times with 20 μg of purified fHbp-PorA chimeras with VR2 loops in P1, P2, P4, P5 or P7.

    [0155] FIG. 6 Stabilisation of V2 fHbp: construction and immunogenicity of fHbp:PorAs [0156] (A) Stabilisation of V2 fHbp V2.22 and V2.25 with six (M6) or two (M2) a.a. substitutions. DSC analysis was performed with 20 μM of protein, using a 20-120° C. temperature gradient. Melting temperature is recorded for the fHbp N-terminal (NT.sub.TM) and C-terminal barrels (CT.sub.TM). [0157] (B) Chimeric fHbp:PorAs PorA loops are recognised by corresponding mAbs. PorA VR2 loops from P1.2, P1.4, P1.9, P1.14 and P1.15 were inserted into position 1 of V2.25 fHbp. SDS-PAGE and Western blot analysis of purified wild type V2.25 fHbp and V3.45 fHbp-PorA chimeras. Western blots were probed with fHbp V2.25 pAbs and loop specific PorA mAbs (NIBSC). CFH binding was detected by far Western blot analysis with normal human sera and CFH pAbs.

    [0158] FIG. 7 Chimeric fHbp:PorAs elicit protective immunity. Mice were immunised with chimeric fHbp:PorA i.e. P(1)(1)(13), P(2)(1)(13), and P(3)(1)(13) on three occasions, and SBAs measured against the strains indicated; an SBA >8 is considered protective. The lack of PorA-directed SBA (i.e. SBA 0, against the fHbp mutant) with VR2 loop in position 3 i.e. P(3)(1)(13) is likely because the loop does not protrude from fHbp barrel as far as in pos. 1.

    [0159] FIG. 8. Frequency of PorA VR2 (A) and fHbp variants (B) in N. meningitidis serogroup B strains (n=243) isolated in 2016 in the UK. Data downloaded from the Meningococcal Research Foundation, 27 Jun. 2017. Other: remaining alleles that occur in <4 isolates. (C) Analysis of recombinant Chimeric antigens by SDS-PAGE and Western blot. Immunoblots are probed with α-PorA VR2 mAbs: P1.4, P1.9, P1.14 and P1.15. (D) Detection of PorA in a panel of N. meningitidis serogroup B isolates by mouse polyclonal antisera from Chimeric Antigens fHbp.sup.V1.4: PorA.sup.151/P1.1.10_1, fHbp.sup.V1.4: PorA.sup.151/P1.14 and fHbp.sup.V1.4:PorA.sup.151/P1.15, fHbp.sup.V3.45:PorA.sup.158/P1.4 and fHbp.sup.V3.45:PorA.sup.158/P1.9.

    EXAMPLE 1

    [0160] It has been shown that immunogenic peptides can be introduced into factor H binding protein (fHbp), and the peptides are presented to the immune system and are able to elicit protective responses (FIGS. 5 and 7). Peptides have been used from the integral membrane protein PorA for proof-in principle of this approach. PorA is difficult to express because of the insolubility of its membrane spanning domains. The immunogenic portions of the molecule reside in extracellular loops which are exposed to the immune system. However effective immune responses are only generated against the loops in their right conformation; linear peptide sequences do not elicit functional immune responses. Through knowledge of the structure of fHbp, it is possible to introduce PorA loops into fHbp and generate relevant responses against PorA. This results in a chimeric molecule, based on fHbp and PorA sequences in specific sites to generate a chimeric molecule. This approach can be used for any other immunogenic integral outer membrane protein.

    [0161] It has been shown that the likely reason for the exclusion of v2 fHbp from vaccines is the inherent instability of its N-terminal β-barrel: i) it was not possible to determine the atomic structure of this portion of v2 fHbp.sup.10, ii) v2 fHbp is sensitive to protease digestion (mass spectrometry demonstrates that the cleaved sites reside in the N-terminal β-barrel, not shown), and iii) differential scanning calorimetry confirms that the instability lies in this region of v2 fHbp.sup.10.

    [0162] Stable v2 fHbps have been successfully generated. Mutagenesis affecting the N-terminal barrel has been undertaken, substituting amino acids (a.a.s) singly or in combination. Substitution of six amino acids in M6 fHbp stabilises v2 fHbp (i.e. 6 changes in c 130 a.a.s of this β-barrel, <0.5%) (see WO2014030003 for details of the mutations). This is evident from differential scanning calorimetry (DSC) and protease sensitivity (see WO2014030003 for details). The side chains of the altered residues promote interactions between the β-sheets of the N-terminal barrel, so are orientated towards the centre of the molecule; the changes do not affect the immunogenicity of the protein as expected (no difference in SBA, or α-fHbp IgG levels not shown).

    [0163] Chimeric v1.1 fHbp has been generated incorporating the 13 amino acid VR2 from P1.16 PorA which elicits SBA in recipients of OMV vaccines.sup.16. While integral membrane proteins contain hydrophobic (thence insoluble) β-barrels, fHbp contains two barrels which can be expressed and purified to high levels. The VR2 sequence has been introduced into six different positions of fHbp (FIG. 2B); these sites were selected on the basis of similar spacing of flanking β-sheets in PorA.sup.22 and fHbp to reduce the likelihood of the insertion disrupting the overall structure of fHbp (FIG. 2B for predicted effect of the insertions). The immunogenicity of three fHbps with insertion of VR2 into a different of fHbp (FIG. 2B) has been assessed. All proteins elicit antibody responses that recognise against fHbp and PorA (FIGS. 5 and 7), and importantly both proteins tested so far (with the VR2 in site 1 or 2) elicit SBA against fHbp and PorA independently (SBA for Nm H44/76, 512; and for Nm H44/76αƒHbp, 256 for both fHbps). This provides proof of principle for this approach.

    [0164] fHbp as a Scaffold for Multi-Valent Vaccines

    [0165] Non-functional fHbps as vaccines—The function of fHbp was not known when clinical trials of fHbp-containing vaccines began; fHbp displays high affinity interactions with fH (dissociation constant <5 nM) irrespective of variant group, with fH engaging a large area of fHbp.sup.5. This interaction could impair the use of fHbp as a vaccine by i) blocking immunogenic epitopes and preventing the generation of antibodies that could compete with fH, and ii) reducing complement activation (through fH recruitment) and thence B cell activation at the site of immune induction. The use of non-functional fHbps circumvents these problems. Key amino acids of v1, 2 and 3 fHbps have been identified that are necessary for fH interactions.sup.10, and modification of single a.a.s of v1, v2 and v3 fHbps which prevent fH binding have been shown to retain or even enhance immunogenicity of this important vaccine antigen.sup.10,23.

    [0166] To Generate and Evaluate Single Protein, Multi-Valent Vaccine Candidates:

    [0167] Protective PorA epitopes from prevalent serosubtypes (which are defined by their PorA sequence) of Nm′.sup.5 have been introduced into v1, stable v2 and v3 proteins; their stability and recognition by PorA and fHbp mAbs has been determined. PorA sequences have been selected to cover the diversity of isolates in the UK but data from any collection of meningococcal strains can be used.

    [0168] Methods of Research

    [0169] Generation and characterisation of vaccine candidates—Recombinant fHbps were constructed and expressed in E. coli using standard plasmid vectors; proteins were affinity purified using the polyHis tag in the protein, anion exchange and gel filtration.sup.10 of chimeric v1, V2 and V3 fHbps as these are either in existing vaccines (v1.1 and 3.45) or because of experience with the protein (v2), or because of their prevalence in Nm strains. PorA loops have been introduced into fHbp by standard methods, and we have demonstrated that the fusion proteins bind mAbs against common serosubtypes of PorA. This strategy has been used to compare native and designed sequences, and perform fine mapping of antigenic and fH-binding of the candidates. DSC has been carried out using a VP Capillary DSC (GEHealthcare) and SPR with a Biacore 3000 (GE Healthcare) or ProteOn XPR36 (BioRad) as previously.sup.10.

    REFERENCES

    [0170] 1. Rosenstein, N. E., B. A. Perkins, D. S. Stephens, T. Popovic, and J. M. Hughes. 2001. Meningococcal disease. N. Engl. J. Med. 344:1378-1388. [0171] 2. Tan, L. K., Carlone, G. M., and Borrow, R. 2010. Advances in the development of vaccines against Neisseria meningitidis. N Engl J Med 362: 1511-1520 [0172] 3. http://www.meningitis.org/research/genome [0173] 4. Finne, J., M. Leinonen, and P. H. Makela. 1983. Antigenic similarities between brain c omponents and bacteria causing meningitis. Implications for vaccine development and pathogenesis. Lancet 2:355-357. [0174] 5. Schneider, M. C., B. E. Prosser, J. J. Caesar, E. Kugelberg, S. et al. 2009. Neisseria meningitidis recruits factor H using protein mimicry of host carbohydrates. Nature 458:890-893. [0175] 6. Fletcher, L. D., L. Bernfield, V. Barniak, J. E. Farley, et al. 2004. Vaccine potential of the Neisseria meningitidis 2086 lipoprotein. Infect. Immun. 72:2088-2100 [0176] 7. Masignani, V., Comanducci, M., Giuliani, M., Bambini, S., et al. 2003. Vaccination against Neisseria meningitidis using three variants of the lipoprotein GNA1870. J. Exp. Med. 197:789-799. [0177] 8. Beernink, P. T., Shaughnessy, J., Pajon, R., Braga, E. M., et al. 2012. The Effect of Human Factor H on Immunogenicity of Meningococcal Native Outer Membrane Vesicle Vaccines with Over-Expressed Factor H Binding Protein. PLoS Pathogens 8: e1002688 [0178] 9. Granoff, D. M., Welsch, J. A., and Ram, S. 2009. Binding of complement factor H (fH) to Neisseria meningitidis is specific for human fH and inhibits complement activation by rat and rabbit sera. Infect. Immun. 77: 764-769. [0179] 10. Johnson, S., Tan, L., van der Veen, S., Caesar, J., et al. (2012) Design and evaluation of meningococcal vaccines through structure-based modification of host and pathogen molecules. PLoS pathogens 8: e1002981 [0180] 11. Zipfel, P. F., Skerka, C., Hellwage, J., Jokiranta, S. T., et al. 2002. Factor H family proteins: on complement, microbes and human diseases. Biochem. Soc. Trans. 30: 971-978. [0181] 12. Schneider, M. C., Exley, R. M., Ram, S., Sim, R. B., and Tang, C. M. 2007. Interactions between Neisseria meningitidis and the complement system. Trends Microbiol 15: 233-240 [0182] 13. Richmond, P. C., Marshall, H. S., Nissen, M D., Jiang, Q., et al. 2012. Safety, immunogenicity, and tolerability of meningococcal serogroup B bivalent recombinant lipoprotein 2086 vaccine in healthy adolescents: a randomised, single-blind, placebo-controlled, phase 2 trial. Lancet infect Dis. 12: 597-607. [0183] 14. Gorringe A R, Pajón R 2011. Bexsero: a multicomponent vaccine for prevention of meningococcal disease. Expert Opin Biol Ther. 11: 969-85. [0184] 15. Lucidarme, J., Comanducci, M., Findlow, J., Gray, S. J., et al. 2010. Characterization of fHbp, nhba (gna2132), nadA, porA, and sequence type in group B meningococcal case isolates collected in England and Wales during January 2008 and potential coverage of an investigational group B meningococcal vaccine. Clin Vaccine Immunol. 2010 17: 919-29 [0185] 16. Rosenqvist, E., Høiby, E. A., Wedege, E. Caugant, D. A., et al. 1993. A new variant of serosubtype P1.16 in Neisseria meningitidis from Norway, associated with increased resistance to bactericidal antibodies induced by a serogroup B outer membrane protein vaccine. Microb Pathog. 15: 197 [0186] 17. Martin, S. L., Borrow, R., van der Ley, P., Dawson, M., Fox, A. J., and Cartwright, K. A. 2000. Effect of sequence variation in meningococcal PorA outer membrane protein on the effectiveness of a hexavalent PorA outer membrane vesicle vaccine. Vaccine 18: 2476-81. [0187] 18. Martin, D. R., Ruijne, N., McCallum, L., O'Hallahan, J., and Oster, P. 2006. The VR2 epitope on the PorA P1.7-2,4 protein is the major target for the immune response elicited by the strain-specific group B meningococcal vaccine MeNZB. Clinical and Vaccine Immunology 13: 486-491. [0188] 19. McGuinness, B., Barlow, A. K., Clarke, I. N., Farley, J. E. et al. 1990 Deduced amino acid sequences of class 1 protein (PorA) from three strains of Neisseria meninintidis. Synthetic peptides define the epitopes responsible for serosubtype specificity. J Exp Med. June 171: 1871-82. [0189] 20. Christodoulides, M., McGuinness, B. T., Heckels, J. E. 1993. Immunization with synthetic peptides containing epitopes of the class 1 outer-membrane protein of Neisseria meningitidis: production of bactericidal antibodies on immunization with a cyclic peptide. J Gen Micro 139: 1729 [0190] 21. Gossger, N., Snape, M. D., Yu, L. M., Finn, A., et al. 2012. Immunogenicity and tolerability of recombinant serogroup B meningococcal vaccine administered with or without routine infant vaccinations according to different immunization schedules: a randomized controlled trial. JAMA. 307: 573-82. [0191] 22. van den Elsen, J. M. H., Herron, J. N., Hoogerhout, P., Poolman, J. T., et al. 1997. Bactericidal antibody recognition of a PorA epitope of Neisseria meningitidis: Crystal structure of a Fab fragment in complex with a fluorescein-conjugated peptide. Proteins: Structure, Function, and Bioinformatics 29: 113-125. [0192] 23. Beernink, P. T., Shaughnessy, J., Braga, E. M., Liu, Q., et al. 2011. A meningococcal factor H binding protein mutant that eliminates factor H binding enhances protective antibody responses to vaccination. J. Immunol. 186: 3606-3614. [0193] 24. Ufret-Vincenty, R. L., Aredo, B., Liu, X., McMahon, A., et al. 2010. Transgenic mice expressing variants of complement factor H develop AMD-like retinal findings. Invest Ophthalmol. Vis. Sci. 51: 5878-5887.

    [0194] All references are herein incorporated by reference.

    EXAMPLE 2

    Chimeric Antigens Containing an Expanded Range of PorA VR2 Loops Generate Immune Responses

    [0195] To test the adaptability of the fHbp:PorA Chimeric antigens, several Chimeric antigens composed from different combinations of fHbp and PorA VR2 were generated. The comprehensive meningococcal genome data available for strains isolated in the UK (Meningitis Research Foundation Meningococcus Genome Library developed by Public health England, the Wellcome Trust Sanger Institute and the University of Oxford as a collaboration.) enables construction of Chimeric antigens that have exact sequence matches to the most common antigens in a given region. In 2016, the most prevalent PorA VR2s in serogroup B N. meningitidis isolates were P1.4 (15.2%), P1.14 (15.2%), P1.9 (12.8%), P1.16 (11.1%) and P1.15 (5.8%, FIG. 8B). VR2 P1.10_1 was present in 1.6% serogroup B isolates. The most prevalent variant 1, variant 2 and variant 3 fHbps were V1.4, V2.19 and V3.45, present in 21.8%, 5.3% and 4.9% of serogroup B N. meningitidis isolates respectively (FIG. 8C). Five different Chimeric antigens were constructed, in which a PorA VR2 was inserted position 151 (V1.4) or position 158 (V3.45, FIG. 8A). Following Chimeric antigen expression and purification, Western blot analyses confirmed these Chimeric antigens all retained epitopes recognised by their cognate α-VR2 mAb and α-fHbp pAbs (FIG. 8D). The thermal stability of wild type fHbps V1.1, V1.4 and V3.45 and the Chimeric antigens was determined by differential scanning calorimetry (DSC, Table 3).

    [0196] To examine the ability of these fHbp:PorA Chimeric antigens to elicit immune responses, groups of CD1 mice were immunized with each Chimeric antigen/alum; antisera obtained post immunisation were pooled. To assess the resulting PorA immune responses, Western blot were conducted with pooled antisera and a panel of serogroup B N. meningitidis disease isolates. FIG. 8E demonstrates that all Chimeric antigens elicited α-PorA antibodies that recognised their cognate PorA VR2. To evaluate α-PorA SBA responses, Serum Bactericidal Assays were performed with pooled Chimeric antigen/alum antisera and serogroup B N. meningitidis strains with mismatched fHbp variants, to negate fHbp cross-protection. Titres range between ≥20 to ≥1280 and are above the ≥8 threshold for an accepted correlate of protective immunity against N. meningitidis (Andrews, N. et al. Clin Diagn Lab Immunol 10, 780-786 (2003)) (Table 4).

    TABLE-US-00048 TABLE 3 Stability of wild type fHbp and Chimeric Antigens Cp (kcal mole.sup.−1 ° C..sup.−1) N-terminal C-Terminal Protein T.sub.m T.sub.m fHbp V1.1 69.8 87.9 fHbp V1.4 64.0 89.0 fHbp V3.45 41.0 83.0 fHbp.sup.V1.4:PorA.sup.151/P1.1.10.sup.—.sup.1 54.0 89.0 fHbp.sup.V1.4:PorA.sup.151/P1.14 55.0 88.0 fHbp.sup.V1.4:PorA.sup.151/P1.15 55.0 89.0 fHbp.sup.V3.45:PorA.sup.158/P1.4 40.0 81.0 fHbp.sup.V3.45:PorA.sup.158/P1.9 39.0 80.0 Melting temperature, T.sub.m

    TABLE-US-00049 TABLE 4 Serum bactericidal assay titres Serogroup B fHbp PorA SBA Pooled antisera isolate variant VR2 titre fHbp.sup.V3.45:PorA.sup.158/P1.4 M10240123 V1.92* P1.4 1/160 fHbp.sup.V3.45:PorA.sup.158/P1.9 M11240431 V2.19 P1.9  1/1280 fHbp.sup.V1.4:PorA.sup.151/P1.1.10.sup.—.sup.1 M11240189 V3.84 P1.10_1 1/20  fHbp.sup.V1.4:PorA.sup.151/P1.14 M15240853 V3.45 P1.14 1/640 α-PorA SBA titres generated using pooled Chimeric Antigen/alum antisera and serogroup B N. meningitidis isolates with mismatched fHbp variants. *fHbp truncated at residue 242. fHbp.sup.V1.4:PorA.sup.151/P1.15 not tested, as it was needed to generate ΔfHbp strains, as the fHbp in strains with PorA VR2 P1.15 is not mismatched.