OA19655A

OA19655A - Modified factor H binding protein.

Info

Publication number: OA19655A
Application number: OA1201900083
Authority: OA
Inventors: Christoph M. Tang
Original assignee: Oxford University Innovation Limited
Priority date: 2016-08-31
Filing date: 2017-08-31
Publication date: 2020-12-31

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).

Description

MODIFIED FACTOR H BINDING PROTEIN

This invention relates to a modified factor H binding protein (fHbp) and its use to elicit an immune response against pathogenic infection or colonisation, such as against Neisseria meningitidis or Neisseria gonorrhoeae.

Neisseria meningitidis (Nm) remains a leading cause of sepsis and bacterial meningitis in children and young adults. The onset of disease can be extremely rapid, with fatality rates of around 10% for septicaemic disease¹, while those that survive can suffer significant disabilities including loss of limbs and neurological déficits¹. Therefore prophylactic immunisation is the best way to protect individuals from meningococcal infection. Vaccines are available based on the bacterial capsule², but there are only partially effective vaccines available for endemic serogroup B infection, which causes over 80% of cases in the UK currently³; the polysaccharide of serogroup B capsule Nm is poorly immunogenic as it has structural identity with a human glycoprotein in neural tissue and could induce autoimmunity if used as a vaccine⁴. Therefore there is an urgent need to generate novel vaccines, and there are intense efforts in academia and industry to achieve this important goal.

The major target of the immune response elicited against meningococcal outer membrane vesicles (OMVs) is PorA¹⁶’¹⁷’¹⁸, an intégral outer membrane protein (OMP) in the meningococcus¹⁹. However, the sequence of this protein is diverse and the prevalence of particular variants differs by géographie région, and OMV vaccines are largely PorA-specific. Variants of PorA are identified by sequences in the variable-regions (VR) of the protein, which are located in the surface-exposed loops of the protein and are the target of immune responses . PorA has seven extracellular loops; the fourth loop is variable région 2 (VR2) and is the target of most sérum bactericidal activity (SBA) generated by PorA following natural infection and after immunisation with OMVs ’ . SBA is a known correlate of protection against meningococcal disease. Despite sequence diversity, around 70 % of UK isolâtes would be covered by vaccines containing six PorA proteins (http://pubmlst.org/neisseria/PorA/).

A major obstacle for bacterial vaccine development is the difficulty in producing quantities of intégral OMPs, such as PorA, in their native conformation. This is because OMPs contain hydrophobie domains which span the membrane and do not fold correctly when expressed as soluble recombinant proteins. Correct folding is critical for PorA as SBA is elicited by conformational, and not linear, epitopes of the protein . Previous attempts to use PorA peptides as vaccines hâve not been successful because they hâve not been sufficiently immunogenic and do not présent the immunogenic portion of PorA in its correct conformation. Consequently, the only PorA-based vaccines under development are OMVs, which hâve limited efficacy in infants , are reactogenic²¹, and poorly defined providing regulatory issues. OMVs as immunogens are not favoured because consistency and toxicity can be problematic during manufacture. For example, OMVs may contain toxic lipopolysaccharide (LPS).

Meningococcal factor H binding protein (fHbp) is a surface-exposed lipoprotein that consists of two β-barrels⁵ (Fig. IA). The N-terminal β-barrel of fHbp has a relatively open structure, while the C-terminal β-barrel is stabilised by extensive hydrogen bonding between the seven β-strands, which form this barrel⁵.

Importantly, fHbp is a key antigen in vaccines against serogroup B Nm under development by pharmaceutical companies such as Pfizer, GSK, and others, and is included in next génération OMV vaccines⁶’⁷’⁸. The Pfizer vaccine consists of two fHbps, while the GSK vaccine has a single fHbp in a cocktail of other antigens which includes an OMV. fHbp binds human, but not murine, factor H (fH)⁹’¹⁰, an abundant plasma protein that down régulâtes the complément system¹¹, a critical aspect of immunity against Nmⁿ. Immunisation of adolescents and adults with fHbp elicits SBA .

fHbp has been categorised into different schemes based on its predicted amino acid sequence. In the présent application, three variant groups (vl, v2 and v3 ) and peptide numbers (www.mlst.org) are recognised. Importantly, sérum raised against vl fHbp does not médiate SBA against Nm expressing v2 and v3 proteins, and vice versa. The GSK vaccine contains a single vl protein (vl.1), while the Pfizer formulation includes a vl and v3 fHbp^13,14. Therefore no current vaccine includes a v2 fHbp even though between a significant proportion of disease in the UK is currently caused by strains expressing fHbp from this variant group^3,15.

WO2011024072 is a patent application that describes the use of fHbp which is selected or engineered to hâve a sequence which can elicit broad-spectrum bactericidal anti-meningococcal antibodies after administration to a host animal. This document teaches that additional meningococcal antigens may be provided with the engineered fHbp in the form of a N- or C-terminal fusion protein. However, such a proposai is unlikely to produce a protein that would présent the immunogenic portion of many meningococcal antigens, such as PorA, in correct conformation and they would not be sufficiently immunogenic.

An aim of the présent invention is to provide an alternative and improved immunogenic molécules for vaccination against pathogenic organisme, particularly to prevent or reduce meningococcal or gonococcal infection or colonisation.

According to a first aspect of the invention, there is provided a modified factor H binding protein (fHbp), comprising fHbp, or a variant thereof, to act as a molecular scaffold by modification with the addition of at least one exogenous peptide loop from a different antigen.

It has been shown herein that immunogenic peptides, such as those from PorA, can be introduced into factor H binding protein (fHbp), which acts as a molecular scaffold. The peptides that are introduced into fHbp are presented to the immune System and are able to elicit protective responses such as SBA. Advantageously, the fHbp molécule provides an idéal molecular scaffold for stable inclusion of peptide loops for the display of epitopes, particularly for epitopes that are difficult to stabilise and display in their native conformation, for example loops from intégral OMPs such as PorA. This is in contrast to teachings such as in WO2011024072, where simple N- or C- terminal fusions of fHbp and additional antigen would not solve inhérent stability and solubility difficulties with some antigens. In particular, many OMPs, such as PorA, are difficult to express because of the insolubility of their membrane spanning domains. PorA has a 16-beta stranded barrel structure with the surface-exposed loops between strands 1 and 2 (loop 1), strands 7 and 8 (loop 4), strands 9 and 10 (loop 5) and strands 11 and 12 (loop 7) demonstrated to be the most effective antigens. fHbp contains two beta barrels, therefore the peptide loop sequences from OMPs can be inserted into the tips of the loops between betastrands of fHbp to présent the extra-cellular loop fragments from intégral OMPs, in their native conformations for immunisation. Therefore, the modified fHbp scaffold molécule of the invention may be used as a prophylactic or a therapeutic vaccine directed to Nm or the gonococcus in which a single protein présents key epitopes from two different antigens.

In one embodiment, the fHbp is meningococcal fHbp. In another embodiment, the fHbp is gonococcal fHbp. The fHbp may comprise any one of variants vl, v2 and v3. In one embodiment, the fHbp may comprise fHbp vl. In another embodiment, the fHbp may comprise fHbp v2. In another embodiment, the fHbp may comprise fHbp v3.

In one embodiment, the fHbp variant vl may be variant vl.l, vl.13, vl.14, vl.15, vl.4, or vl.55. In one embodiment, the fHbp variant vl may not be vl.l. In one embodiment, the fHbp variant vl may not be vl.55. In one embodiment, the fHbp variant vl may not be vl.l or vl.55. In one embodiment, the fHbp variant v2 may be variant v2.16, v2.19, v2.22, or v2.25. In one embodiment, the fHbp variant v3 may be variant v3.45. In one embodiment, the fHbp comprises any one of fHbp variants vl.4, v2.25 or v3.45.

A variant of fHbp may comprise an orthologue of fHbp. For example, a variant of fHbp may comprise Ghfp, the Gonococcal homologue of fHbp. Ghfp is nonfunctional and closely related to V3 fHbps (>95% aa identity, dissociation constant KD >100 μΜ with factor H).

In one embodiment, the fHbp which is to be further modified with the at least one exogenous peptide loop may comprise or consist of the sequence of CSSGGGGVAA DIGAGLADAL TAPLDHKDKG LQSLTLDQSV RKNEKLKLAA QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QIEVDGQLIT LESGEFQVYK QSHSALTAFQ TEQIQDSEHS GKMVAKRQFR IGDIAGEHTS FDKLPEGGRA TYRGTAFGSD DAGGKLTYTI DFAAKQGNGK IEHLKSPELN VDLAAADIKP DGKRHAVISG SVLYNQAEKG SYSLGIFGGK AQEVAGSAEV KTVNGIRHIG LAAKQ (SEQ ID NO: 1, fHbp VI.1 GI:316985482).

In another embodiment, the fHbp which is to be further modified with the at least one exogenous peptide loop may comprise or consist of the sequence of CSSGGGGVAA DIGAGLADAL TAPLDHKDKS LQSLTLDQSV RKNEKLKLAA

QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QIEVDGQLIT LESGEFQVYK

QSHSALTALQ TEQVQDSEHS GKMVAKRQFR IGDIAGEHTS FDKLPEGGRA

TYRGTAFGSD DASGKLTYTI DFAAKQGHGK IEHLKSPELN VDLAASDIKP

DKKRHAVISG SVLYNQAEKG SYSLGIFGGQ AQEVAGSAEV ETANGIRHIG

LAAKQ (SEQ ID NO: 2, fHbp Vl.4 GE989557230).

In another embodiment, the fHbp which is to be further modified with the at least one exogenous peptide loop may comprise or consist of the sequence of CSSGGGGVAA DIGAGLADAL TAPLDHKDKG LQSLTLDQSV RKNEKLKLAA QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QIEVDGKLIT LESGEFQVYK QSHSALTALQ TEQVQDSEDS GKMVAKRQFR IGDIAGEHTS FDKLPKGGSA

TYRGTAFGSD DAGGKLTYTI DFAAKQGHGK IEHLKSPELN VELATAYIKP

DEKRHAVISG SVLYNQDEKG SYSLGIFGGQ AQEVAGSAEV ETANGIHHIG

LAAKQ (SEQ ID NO: 3, fHbp VI.13 GE752774533).

In another embodiment, the fHbp which is to be further modified with the at least one exogenous peptide loop may comprise or consist of the sequence of CSSGGGGVAA DIGAGLADAL TAPLDHKDKS LQSLTLDQSV RKNEKLKLAA QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QIEVDGQLIT LESGEFQVYK QSHSALTALQ TEQEQDPEHS GKMVAKRRFK IGDIAGEHTS FDKLPKDVMA TYRGTAFGSD DAGGKLTYTI DFAAKQGHGK IEHLKSPELN VELATAYIKP DEKHHAVISG SVLYNQDEKG SYSLGIFGGQ AQEVAGSAEV ETANGIHHIG LAAKQ (SEQ ID NO: 4, fHbp VI.14 GL630057376).

In another embodiment, the fHbp which is to be further modified with the at least one exogenous peptide loop may comprise or consist of the sequence of CSSGGGGSGG GGVAADIGAG LADALTAPLD HKDKGLKSLT LEDSISQNGT LTLSAQGAER TFKAGDKDNS LNTGKLKNDK ISRFDFIRQI EVDGQLITLE

SGEFQVYKQS HSALTALQTE QVQDSEHSGK MVAKRQFRIG DIVGEHTSFG

KLPKDVMATY RGTAFGSDDA GGKLTYTIDF AAKQGHGKIE HLKSPELNVD

LAAADIKPDE KHHAVISGSV LYNQAEKGSY SLGIFGGQAQ EVAGSAEVET

ANGIRHIGLA AKQ (SEQ ID NO: 5, fHbp VI.15 GI:504394462).

In another embodiment, the fHbp which is to be further modified with the at least one exogenous peptide loop may comprise or consist of the sequence of CSSGGGGSGG GGVTADIGTG LADALTAPLD HKDKGLKSLT LEDSISQNGT

LTLSAQGAEK TYGNGDSLNT GKLKNDKVSR FDFIRQIEVD GQLITLESGE

FQVYKQSHSA LTALQTEQEQ DPEHSEKMVA KRRFRIGDIA GEHTSFDKLP

KDVMATYRGT AFGSDDAGGK LTYTIDFAAK QGHGKIEHLK SPELNVDLAV

AYIKPDEKHH AVISGSVLYN QDEKGSYSLG IFGEKAQEVA GSAEVETANG IHHIGLAAKQ (SEQ ID NO: 6, fHbpV 1.55 GE40353481 ).

In another embodiment, the fHbp which is to be further modified with the at least one exogenous peptide loop may comprise or consist of the sequence of CSSGGGGVAA DIGAGLADAL TAPLDHKDKS LQSLTLDQSV RKNEKLKLAA QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QIEVDGQLIT LESGEFQIYK QDHSAVVALQ IEKINNPDKI DSLINQRSFL VSGLGGEHTA FNQLPDGKAE YHGKAFSSDD AGGKLTYTID FAAKQGHGKI EHLKTPEQNV ELAAAELKAD

EKSHAVILGD TRYGSEEKGT YHLALFGDRA QEIAGSATVK IGEKVHEIGI AGKQ (SEQ ID NO: 7, fHbp V2.16 GI:488155511 ).

In another embodiment, the fHbp which is to be further modified with the at least one exogenous peptide loop may comprise or consist of the sequence of CSSGGGGVAA DIGAGLADAL TAPLDHKDKS LQSLTLDQSV RKNEKLKLAA QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QIEVDGQLIT LESGEFQIYK QDHSAVVALQ IEKINNPDKI DSLINQRSFL VSGLGGEHTA FNQLPSGKAE YHGKAFSSDD AGGKLTYTID FAAKQGHGKI EHLKTPEQNV ELASAELKAD EKSHAVILGD TRYGGEEKGT YHLALFGDRA QEIAGSATVK IREKVHEIGI AGKQ (SEQ ID NO: 8, fHbp V2.19 GI:488148626).

In another embodiment, the fHbp which is to be further modified with the at least one exogenous peptide loop may comprise or consist of the sequence of CSSGGGGVAA DIGAGLADAL TAPLDHKDKS LQSLTLDQSV RKNEKLKLAA QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QIEVDGQLIT LESGEFQIYK QDHSAVVALQ IEKINNPDKI DSLINQRSFL VSGLGGEHTA FNQLPSGKAE YHGKAFSSDD PNGRLHYSID FTKKQGYGRI EHLKTPEQNV ELASAELKAD EKSHAVILGD TRYGGEEKGT YHLALFGDRA QEIAGSATVK IREKVHEIGI AGKQ (SEQ ID NO: 9, fHbp V2.22 GI: 120865922).

In another embodiment, the fHbp which is to be further modified with the at least one exogenous peptide loop may comprise or consist of the sequence of CSSGGGGVAA DIGAGLADAL TTPLDHKDKS LQSLTLDQSV RKNEKLKLAA QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QIEVDGQTIT LASGEFQIYK QNHSAVVALQ IEKINNPDKI DSLINQRSFL VSGLGGEHTA FNQLPDGKAE YHGKAFSSDD PNGRLHYSID FTKKQGYGRI EHLKTPEQNV ELASAELKAD EKSHAVILGD TRYGGEEKGT YHLALFGDRA QEIAGSATVK IREKVHEIGI AGKQ (SEQ ID NO: 10, fHbp 2.25 GI:488158712).

In another embodiment, the fHbp which is to be further modified with the at least one exogenous peptide loop may comprise or consist of the sequence of CSSGSGSGGG GVAADIGTGL ADALTAPLDH KDKGLKSLTL EDSISQNGTL TLSAQGAEKT FKVGDKDNSL NTGKLKNDKI SRFDFVQKIE VDGQTITLAS GEFQIYKQDH SAVVALQIEK INNPDKIDSL INQRSFLVSG LGGEHTAFNQ

LPSGKAEYHG KAFSSDDAGG KLTYTIDFAA KQGHGKIEHL KTPEQNVELA SAELKADEKS HAVILGDTRY GSEEKGTYHL ALFGDRAQEI AGSATVKIRE KVHEIGIAGK Q (SEQ ID NO: 11, fHbp V3.45 GE284466869).

In another embodiment, the fHbp which is to be further modified with the at least one exogenous peptide loop may comprise or consist of the sequence of CSSGGGGVAA DIGAGLADAL TAPLDHKDKG LKSLTLEDSI SQNGTLTLSA

QGAEKTFKVG DKDNSLNTGK LKNDKISRFD FVQKIEVDGQ TITLASGEFQ

IYKQNHSAVV ALQIEKINNP DKIDSLINQR SFLVSGLGGE HTAFNQLPGG

KAEYHGKAFS SDDAGGKLTY TIDFAAKQGH GKIEHLKTPE QNVELAAAEL

KADEKSHAVI LGDTRYGSEE KGTYHLALFG DRAQEIAGSA TVKIGEKVHE

ISIAGKQ (SEQ ID NO: 12, fHbp V3.47 GE284466897).

In another embodiment the Ghfp which is to be further modified with the at least one exogenous peptide loop, may comprise or consist of the sequence of MTRSKPVNRT TFCCLSLTAG PDSDRLQQRR GGGGGVAADI GTGLADALTA PLDHKDKGLK SLTLEASIPQ NGTLTLSAQG AEKTFKAGGK DNSLNTGKLK NDKISRFDFV QKIEVDGQTI TLASGEFQIY KQDHSAVVAL RIEKINNPDK IDSLINQRSF LVSDLGGEHT AFNQLPDGKA EYHGKAFSSD DADGKLTYTI DFAAKQGHGK IEHLKTPEQN VELASAELKA DEKSHAVILG DTRYGGEEKG TYRLALFGDR AQEIAGSATV KIGEKVHEIG IADKQ (SEQ ID NO: 13, GHFP).

A variant of fHbp may comprise one or more amino acid residue mutations, including additions, délétions or substitutions, relative to wild type fHbp in addition to the exogenous peptide loop(s) provided on the modified fHbp. For example, the fHbp may act as a scaffold upon which exogenous peptide loops are provided and the variants relative to wild-type may comprise amino acid mutations in the scaffold framework in régions outside of the exogenous peptide loop(s) attachment points. Reference to wild-type fHbp may refer to any one of the variants of fHbp discussed herein, for example any one of SEQ ID NOs: 1 to 13).

A variant of fHbp may comprise at least one amino acid change compared to the amino acid in the wild type protein. A variant of fHbp may comprise no more than one amino acid change compared to the wild type protein. A variant of fHbp may comprise no more than three amino acid changes compared to the wild type protein. A variant of fHbp may comprise no more than four amino acid changes compared to the wild type protein. A variant of fHbp may comprise no more than five amino acid changes compared to the wild type protein. A variant of fHbp may comprise no more than six amino acid changes compared to the wild type protein. In one embodiment, a variant of fHbp is provided which comprises six amino acid mutations compared to the wild type protein.

Amino acid substitutions may be conservative substitutions. For example, a mutated residue may comprise substantially similar properties as the wild-type substituted residue. For example, a substituted residue may comprise substantially similar or equal charge or hydrophobicity as the wild-type substituted residue. For example, a substituted residue may comprise substantially similar molecular weight or steric bulk as the wild-type substituted residue.

In one embodiment a variant fHbp may hâve at least 75% identity with wildtype. In another embodiment a variant fHbp may hâve at least 80% identity with wild-type. In another embodiment a variant fHbp may hâve at least 85% identity with wild-type. In another embodiment a variant fHbp may hâve at least 90% identity with wild-type. In another embodiment a variant fHbp may hâve at least 95% identity with wild-type. In another embodiment a variant fHbp may hâve at least 98% identity with wild-type. In another embodiment a variant fHbp may hâve at least 99% identity with wild-type. In another embodiment a variant fHbp may hâve at least 99.5% identity with wild-type. The above percentage variation is not intended to include percentage identity variation with addition of the exogenous peptide loop(s) (i.e. it is the percentage identity of the fHbp component alone relative to the wild-type), and does not include délétion of fHbp sequence at the site where loops from other proteins are inserted.

The modified fHbp may be modified such that it is not capable of binding factor H, or at least has reduced factor H binding activity. The modified fHbp may be non-functional relative to the function of wild-type fHbp. In one embodiment, the modified fHbp has an impaired capacity to bind CFH with a KD >2 orders of magnitude compared with the wild-type protein. Non-functional fHbps may be provided by mutation of the fHbp sequence. In one embodiment, nonfunctional fHbps may be provided by one or more of the exogenous peptide loops preventing the binding site of factor H.

The amino acid residue mutation(s) may prevent or reduce complément factor H binding of the modified fHbp. In another embodiment, the amino acid residue mutation(s) may not substantially affect the fHbp function. In one embodiment, the amino acid residue mutation(s) in the fHbp, or variants thereof, may be selected from the group consisting of the amino acid at position 85, 133, 134, 135, 136, 204, 206, 211, 212, 213, 222, 225, 227, 231, and 252 on vl.l fHbp or corresponding position in other fHbps.

In one embodiment, the amino acid residue mutation(s) may comprise or consist of a substitution to alanine instead of the wild type residue. In one embodiment, the amino acid residue change(s) may comprise or consist of a substitution to any other amino acid instead of the wild type residue.

Advantageously, providing a non-functional fHbp (i.e. non- or less- binding of factor H) can eliminate or reduce any adverse effects of factor H recruitment on the success of the vaccine.

In one embodiment, the amino acid residue mutation(s) may enhance the stability of the modified fHbp in particular, in an embodiment wherein fHbp V2 is provided, the fHbp V2 may be stabilised by mutations in the fHbp V2 sequence. Details of the mutations for V2 stability may be found in WO2014030003, which is herein incorporated by reference. For example, the amino acid substitution for stabilisation may be at one or more of the amino

H acids at position 35, 36, 42, 43, 46, 107, 112, 114, 137 and 138 in fHbp V2. The substitution for stabilisation may be at one or more of Ser35, Leu36, Asp42, Glu43, Arg46, Aspl07, Vall 12, Leul 14, Serl37, and Glyl38.

In one embodiment, the exogenous peptide loop(s) is immunogenic. The exogenous peptide loop(s) may be derived from an outer membrane/surface exposed protein.

The exogenous peptide loop(s) may be prokaryotic in origin. The exogenous peptide loop(s) may be derived from a protein on the bacterium, such as an outer membrane protein (OMP) of a pathogen. The OMP may be an intégral OMP or a lipoprotein. The exogenous peptide loop(s) may be derived from a meningococcal protein, such as a meningococcal outer membrane protein. The exogenous peptide loop(s) may be derived from an outer membrane protein of another pathogen such as N. gonorrhoeae.

The exogenous peptide loop may comprise a fragment of a transmembrane beta barrel protein. The exogenous peptide loop may comprise a fragment of a beta barrel porin protein. The exogenous peptide loop may comprise a fragment of PorA. In another embodiment, the exogenous peptide loop may comprise a fragment of FetA.

The exogenous peptide loop(s), such as PorA fragments, may be 16 amino acids in length. In one embodiment, the exogenous peptide loop(s), such as PorA fragments, may be between 8 and 20 amino acids in length. In another embodiment, the exogenous peptide loop(s), such as PorA fragments, may be between 8 and 16 amino acids in length. In another embodiment, the exogenous peptide loop(s), such as PorA fragments, may be between 10 and 16 amino acids in length. In another embodiment, the exogenous peptide loop(s), such as PorA fragments, may be between 12 and 16 amino acids in length. In another embodiment, the exogenous peptide loop(s), such as PorA fragments, may be between 14 and 18 amino acids in length. In another embodiment, the exogenous peptide loop(s), such as PorA fragment, may be any length sufficient to provide an immunogenic epitope. In another embodiment, the exogenous peptide loop(s), such as PorA fragments, may be any length sufficient to provide an immunogenic epitope and maintain native conformation relative to the fragment in wild-type.

The exogenous peptide loop(s) may be selected from any one of the PorA loops 1 to 7, or fragments thereof; and/or combinations thereof. The exogenous peptide loop(s) may be selected from any one of the PorA loops of loop 1 (from between beta-strands 1 and 2), loop 4 (from between beta-strands 7 and 8), loop 5 (from between beta-strands 9 and 10) and loop 7 (from between beta-strands 11 and 12; or fragments thereof; and/or combinations thereof.

The exogenous peptide loop may comprise any one peptide selected from PorA loop 1 (between beta-strands 1 and 2); loop 4 (between beta-barrels 7 and 8); and loop 5 (between beta-strands 9 and 10); or fragments thereof; and/or combinations thereof.

The exogenous peptide loop may comprise PorA loop 1 (between beta-barrels 1 and 2), or a fragment thereof. The exogenous peptide loop may comprise PorA loop 4 (between beta-strands 7 and 8), or a fragment thereof. The exogenous peptide loop may comprise PorA loop 5 (between beta-strands 9 and 10), or a fragment thereof.

The skilled person will understand that variant sequences of PorA loops may be provided with minor mutations relative to wild-type and may still function as an epitope. Therefore, the exogenous peptide loop(s) may comprise PorA loop variants. Variants may include one or more amino acid additions, délétions or substitutions relative to the wild-type sequence. In another embodiment, variants may include no more than one amino acid addition, délétion or substitution relative to the wild-type sequence. In another embodiment, variants may include no more than 2, 3, 4 or 5 amino acid additions, délétions or substitutions relative to the wild-type sequence. The substitutions may be conservative substitutions. For example, providing an alternative amino acid residue having substantially similar properties, such as charge, hydrophobicity, steric size or molecular weight. Variants may include sequences having at least 85% sequence identity with wild-type PorA loop sequence. In another embodiment, variants may include sequences having at least 90%, 95%, 98%, 99%, or 99.5% sequence identity with wild-type PorA loop sequence.

In one embodiment, the modified fHbp may comprise two or more exogenous peptide loops. In one embodiment, the modified fHbp may comprise three or more exogenous peptide loops. In one embodiment, the modified fHbp may comprise between 1 and 7 exogenous peptide loops. In one embodiment, the modified fHbp may comprise between 1 and 5 exogenous peptide loops. In one embodiment, the modified fHbp may comprise between 1 and 3 exogenous peptide loops. In one embodiment, the modified fHbp may comprise between 2 and 7 exogenous peptide loops. In one embodiment, the modified fHbp may comprise between 3 and 7 exogenous peptide loops. In one embodiment, the modified fHbp may comprise between 2 and 5 exogenous peptide loops. In one embodiment, the modified fHbp may comprise between 3 and 5 exogenous peptide loops. The modified fHbp, or variants thereof, may be modified with an exogenous peptide loop in at least one position. The modified fHbp, or variant thereof, may be modified with an exogenous peptide loop in at least two positions. The modified fHbp, or variant thereof, may be modified with an exogenous peptide loop in at least three positions. The modified fHbp, or variant thereof, may be modified with an exogenous peptide loop in at least four positions. The modified fHbp, or variant thereof, may be modified with an exogenous peptide loop in at least five positions. The modified fHbp, or variant thereof, may be modified with an exogenous peptide loop in at least six positions. The modified fHbp, or variant thereof, may be modified with an exogenous peptide loop in at least seven positions.

A peptide loop may be provided (for example using fHbp as a scaffold) between two beta sheets of the factor H binding protein. The peptide loop may be inserted into fHbp, or a variant thereof, at one or more of amino acid positions selected from positions where exogenous peptide loops, such as PorA loops, can be inserted (range is inclusive of ail residues in loop) between amino acids 49-54, 83-88, 114-124, 199-206, 227-233, and 240-246 of vl.l fHbp or corresponding positions in other fHbps.

The at least one exogenous peptide loop may not be provided as an N- or Cterminal fusion.

Sequences of fHbp into which the exogenous peptide loop(s) can be inserted may be at any of those positions underlined with regards to fHbp Vl.l primary amino acid sequence below. In an embodiment using an alternative variant fHBP, the insert sites may be at équivalent positions.

Position 1 in fHbp (PI), residues 83-88

P2, residues 199-206

P3, residues 227-233

P4, residues 49-54

P5, residues 114-124

P7, residues 240-246

CSSGGGGVAA DIGAGLADAL TAPLDHKDKG LQSLTLDQSV RKNEKLKLAA

P4

QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QIEVDGQLIT LESGEFQVYK

P4 PI

101 QSHSALTAFQ TEQIQDSEHS GKMVAKRQFR IGDIAGEHTS FDKLPEGGRA

P5

151 TYRGTAFGSD DAGGKLTYTI DFAAKQGNGK 1EHLKSPELN VDLAAADIKP

P2

201 DGKRHAVISG SVLYNQAEKG SYSLGIFGGK AQEVAGSAEV KTVNGIRHIG P2 P3 P7

251 LAAKQ (SEQ ID NO: 14)

In one embodiment, the insert site of an exogenous peptide loop may be at position 1 in fHbp (PI), residues 83-88 (Sequence EVDGQL (SEQ ID NO: 15)). In another embodiment, the insert site of an exogenous peptide loop may be at position P2, residues 199-206 (Sequence KPDGKRHA (SEQ ID NO: 16)). In another embodiment, the insert site of an exogenous peptide loop may be at position P3, residues 227-233 (Sequence FGGKAQE (SEQ ID NO: 17)). In another embodiment, the insert site of an exogenous peptide loop may be at position P4, residues 49-54 (Sequence AAQGAE (SEQ ID NO: 18)). In another embodiment, the insert site of an exogenous peptide loop may be at position P5, residues 114-124 (Sequence IQDSEHSGKM (SEQ ID NO: 19)). In another embodiment, the insert site of an exogenous peptide loop may be at position P7, residues 240-246 (Sequence KTVNGI (SEQ ID NO: 20)).

The exogenous peptide loop insert site at any given position 1-7 may be between any of the residues identified at positions 1 to 7 of fHbp. Alternatively, The exogenous peptide loop insert site at any given position 1-7 may be before the first residue or after the last residue identified at positions 1 to 7 of fHbp. For example if the exogenous peptide loop is inserted at position 4, the insert may be *AAQGAE (SEQ ID NO: 21), A*AQGAE (SEQ ID NO: 22), AA*QGAE (SEQ ID NO: 23), AAQ*GAE (SEQ ID NO: 24), AAQG*AE (SEQ ID NO: 25), AAQGA*E (SEQ ID NO: 26), or AAQGAE* (SEQ ID NO: 27), where * dénotés the insert site.

In another embodiment, the skilled person will understand that the exogenous peptide loop insertion site may be variable such that between 1 and 5 residues in the région of the insertion site may be removed from fHbp (e.g. replaced by the loop) without significantly affecting the fHbp structure. In one embodiment, one or more of the amino acid residues at the identified positions are replaced/substituted by an exogenous peptide loop. In an alternative embodiment, two, three, four, five or more of the amino acid residues at the identified positions are replaced/substituted by an exogenous peptide loop. In another embodiment, the exogenous peptide loop insertion site may be variable such that between 1 and 3 residues in the région of the insertion site may be removed from fHbp (e.g. replaced by the loop) without significantly affecting the fHbp structure. In another embodiment, the exogenous peptide loop insertion site may be variable such that 1 or 2 residues in the région of the insertion site may be removed from fHbp (e.g. replaced by the loop) without significantly affecting the fHbp structure. In another embodiment, the exogenous peptide loop insertion site may be variable such that 1 residue in the région of the insertion site may be removed from fHbp (e.g. replaced by the loop) without significantly affecting the fHbp structure. For example, where an insertion site is amino acid residue 86 at one end of the loop and residue 87 at the other end of the loop, a variant can include a 5, 4, 3, 2, or 1 residue replacement with the loop in the région of the stated insertion site. In an alternative embodiment, six or more of the amino acid residues at the identified positions are replaced/substituted by an exogenous peptide loop. In an alternative embodiment, seven or more (where applicable) of the amino acid residues at the identified positions are replaced/substituted by an exogenous peptide loop. In an alternative embodiment, eight or more (where applicable) of the amino acid residues at the identified positions are replaced/substituted by an exogenous peptide loop. In an alternative embodiment, nine or more (where applicable) of the amino acid residues at the identified positions are replaced/substituted by an exogenous peptide loop. The entire amino acid residues of any of insert positions 1 to 7 may be substituted with an exogenous peptide loop.

For example if the exogenous peptide loop is inserted at position 4 and substitutes one or more residues, the insert may be A*QGAE (SEQ ID NO: 28), AA*GAE (SEQ ID NO: 29), AA*AE (SEQ ID NO: 30), AA*E (SEQ ID NO: 31), AA*, A*GAE (SEQ ID NO: 32), A*AE (SEQ ID NO: 33), AA*E (SEQ ID NO: 34), A*E, A*, *AQGAE (SEQ ID NO: 35), *QGAE (SEQ ID NO: 36), *GAE (SEQ ID NO: 37), *AE, *E, AAQ*AE (SEQ ID NO: 38), AAQ*E (SEQ

ID NO: 39), AAQ* (SEQ ID NO: 40), AAQG*E (SEQ ID NO: 41), AAQG* (SEQ ID NO: 42), AAQGA* (SEQ ID NO: 43), where * dénotés the insert site, and one or more residues are removed from the original sequence.

The skilled person will understand that équivalent combinations of insertion sites and/or substitutions with the exogenous peptide loop may be made with alternative sequences of the other identified insert positions 1 to 7.

In one embodiment, the région of the insertion site may be shifted +/- 5 residues up or downstream of any the positions 1 to 7. Alternatively, the région of the insertion site may be shifted +/- 4 residues up or downstream of any the positions 1 to 7. Alternatively, the région of the insertion site may be shifted +/- 3 residues up or downstream of any the positions 1 to 7. Alternatively, the région of the insertion site may be shifted +/- 2 residues up or downstream of any the positions 1 to 7. Alternatively, the région of the insertion site may be shifted +/- 1 residue up or downstream of any the positions 1 to 7.

Additionally or alternatively, the peptide loop(s) may be provided in a position that sterically prevents fHbp:CFH interaction. These include an exogenous peptide loop inserted into any one or more site between residues 114-124,199206, or 240-246 (e.g. Positions 2, 5 and 7), underlined on VI primary sequence below:

CSSGGGGVAA DIGAGLADAL TAPLDHKDKG LQSLTLDQSV RKNEKLKLAA

QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QIEVDGQLIT LESGEFQVYK

101 QSHSALTAFQ TEQIQDSEHS GKMVAKRQFR IGDIAGEHTS FDKLPEGGRA

151 TYRGTAFGSD DAGGKLTYTI DFAAKQGNGK IEHLKSPELN VDLAAADIKP

201 DGKRHAVISG SVLYNQAEKG SYSLGIFGGK AQEVAGSAEV KTVNGIRHIG

51 LAAKQ (SEQ ID NO: 44)

In one embodiment of the invention, the modified fHbp is immunogenic. The modified fHbp may be a recombinant protein. The modified fHbp may be a fusion protein, such as a recombinant fusion protein. The modified fHbp may be an isolated modified fHbp molécule. The modified fHbp molécule of the invention may be described as a single protein, multi-valent vaccine. The modified fHbp could be included in an OMV vaccine.

In embodiments where more than one exogenous peptide loop is inserted into fHbp, or variants thereof, the exogenous peptide loops may be the same, e.g. the same sequence, or substantially similar. For example, some epitopes such as PorA epitopes, may not elicit sufficient functional responses when displayed singly on fHbp. In this instance, the présent invention may be used to provide the same epitope at multiple sites on the same modified fHbp molécule, thereby enhancing the immunogenic récognition of the epitope.

Alternatively, the exogenous peptide loops may be different relative to each other. For example, where the exogenous peptide loops are derived from a single protein, such as PorA, the different exogenous peptide loops may be from distinct régions of the protein, such as PorA. In one embodiment, the different exogenous peptide loops may be derived from overlapping and distinct régions of the protein, such as PorA.

In embodiments where more than one exogenous peptide loop is inserted into fHbp, or variants thereof, the exogenous peptide loops may be derived from different species or strains. For example, when a multivalent vaccine is desired for multiple different antigens including different organisms.

The PorA peptide loop sequence may be selected from any of the sequences provided in Table 1 (e.g. any of SEQ ID NOs: 45 to 79). Combinations of different PorA sequences of Table 1 may be provided (one loop per site) in any of insertion sites PI to P7 described herein. Additionally or alternatively, two or more of the same PorA sequences of Table 1 may be provided (one loop per site) in any of insertion sites PI to P7 described herein.

Table 1 : sequences of PorA VR2 loops, any of which may be inserted into any variant of fHbp to make a chimeric fHbp-PorA protein.

PorA VR2 Loop	Primary sequence
Pl.l	YVAVENGVAKKVA (SEQ ID NO: 45)
PI.2	HFVQQTPKSQPTLVP (SEQ ID NO: 46)
P1.2_2	HFVQQTPQSQPTLVP (SEQ ID NO: 47)
PI.3	TLANGANNTIIRVP (SEQ ID NO: 48)
P1.3_5	TLAKGANNTIIRVP (SEQ ID NO: 49)
PI.4	HVVVNNKVATHVP (SEQ ID NO: 50)
PI.9	YVDEQSKYHA (SEQ ID NO: 51)
PI.10	HFVQNKQNQRPTLVP (SEQ ID NO: 52)
PI. 101	HFVQNKQNQPPTLVP (SEQ ID NO: 53)
P1.10_2	HFVQDKKGQPPTLVP (SEQ ID NO: 54)
PI.10_8	HFVQNKQNQQNQPPTLVP (SEQ ID NO: 55)
P1.10_4	HFVQNKQNKQNQPPTLVP (SEQ ID NO: 56)
PI. 107	HFVQNKQNKPPTLVP (SEQ ID NO: 57)
PI.13	YWTTVNTGSATTTTTFVP (SEQ ID NO: 58)
PI.13_1	YWTTVNTGSATTTTFVP

	(SEQ ID NO: 59)
PI. 132	YWTTVNTGSATTTFVP (SEQ ID NO: 60)
PI. 13_4	YYTTVTQGSATTTTFVP (SEQ ID NO: 61)
PI.14	YVDEKKMVHA (SEQ ID NO: 62)
P1.14_6	YVDEKQVSHA (SEQ ID NO: 63)
P1.14_26	YVDEKKVVHA (SEQ ID NO: 64)
PI.15	HYTRQNNADVFVP (SEQ ID NO: 65)
PI. 15_1	HYTRQNNTDVFVP (SEQ ID NO: 66)
PI .15_11	HYTRQNNIDVFVP (SEQ ID NO: 67)
PI.16	YYTKDTNNNLTLVP (SEQ ID NO: 68)
PI .16_3	YYTKDKNDNLTLVP (SEQ ID NO: 69)
PI. 164	YYTKDKNDKLTLVP (SEQ ID NO: 70)
P1.1626	YYTNTNNNLTLVP (SEQ ID NO: 71)
PI.23	HWNTVYNTNGTTTTFVP (SEQ ID NO: 72)
P1.23_2	HWNTVYNTNGTTTTTFVP (SEQ ID NO: 73)
PI.25	TYTVDSSGVVTPVP (SEQ ID NO: 74)
P1.26	HFVADSQGKITRVP (SEQ ID NO: 75)

P1.28	YYYTTATNSSTSTTFVP (SEQ ID NO: 76)
PI.30	HYTTVYNATTTTTTFVP (SEQ ID NO: 77)
P1.30_2	HYTTVYNATTTTTTTFVP (SEQ ID NO: 78)
PI.34	YVDDQGKVKGP (SEQ ID NO: 79)

The skilled person will understand that variants involving one or two or more amino acid substitutions, additions or délétions may be provided for the PorA sequences of Table 1 without substantially removing the immunogenic function. Substitutions may be to similar amino acid residues, for example having similar MW, charge, hydrophobicity or moieties, or synthetic analogues. The skilled person will further understand that variants may be truncations of the PorA sequences of Table 1, wherein the truncated variants provide sufficient amino acid residues to form a recognisable epitope. In one embodiment the PorA sequence has at least 80% identity to any one of the sequences in Table 1. In another embodiment the PorA sequence has at least 85% identity to any one of the sequences in Table 1. In another embodiment the PorA sequence has at least 90% identity to any one of the sequences in Table 1. In another embodiment the PorA sequence has at least 95% identity to any one of the sequences in Table 1. In another embodiment the PorA sequence has at least 98% identity to any one of the sequences in Table 1.

In one embodiment, the fHbp which is to be further modified with an exogenous peptide loop may comprise or consist of the sequence:

LAAKQ (SEQ ID NO: 80, fHbp Vl.l GI:316985482), wherein the sequence of any PorA VR2 loop in Table 1 may replace any amino acid or groups of amino acids highlighted by a box.

In one embodiment, the fHbp which is to be fùrther modified with an exogenous peptide loop may comprise or consist of the sequence:

CSSGGGGVAA DIGAGLADAL TAPLDHKDKS LQSLTLDQSV RKNEKLKL^ |QGAEK|TYGNG DSLNTGKLKN DKVSRFDFIR Ql|EVDGQL|lT LESGEFQVYK

QSHSALTALQ TEQjVQDSEHS GKMVjAKRQFR IGDIAGEHTS FDKLPEGGRA

TYRGTAFGSD DASGKLTYTI DFAAKQGHGK IEHLKSPELN VDLAASDI^P]

[DKKRHAjVISG SVLYNQAEKG SYSLGl|FGGQ AQE|VAGSAEV |ETANGl|RHIG

LAAKQ (SEQ ID NO: 81, fHbp VI.4 GE989557230), wherein the sequence of any PorA VR2 loop in Table 1 may replace any amino acid or groups of amino acids highlighted by a box.

CSSGGGGVAA DIGAGLADAL TAPLDHKDKG LQSLTLDQSV RKNEKLKL^Â] |QGAEK|TYGNG DSLNTGKLKN DKVSRFDFIR Ql[EVDGKL|lT LESGEFQVYK

QSHSALTALQ TEQ|VQDSEDS GKMV|AKRQFR IGDIAGEHTS FDKLPKGGSA

LAAKQ (SEQ ID NO: 82, fHbp VI.13 GE752774533), wherein the sequence of any PorA VR2 loop in Table 1 may replace any amino acid or groups of amino acids highlighted by a box.

CSSGGGGSGG GGVAADIGAG LADALTAPLD HKDKGLKSLT LEDSISQNGT ltl|saqgâër| tfkagdkdns lntgklkndk isrfdfirqi ^VDGQL^TLE SGEFQVYKQS HSALTALQTE Q(VQDSEHSGK MV|AKRQFRIG DIVGEHTSFG KLPKDVMATY RGTAFGSDDA GGKLTYTIDF AAKQGHGKIE HLKSPELNVD LAAADI^PDE KHHA|VISGSV LYNQAEKGSY SLGl|FGGQAQ E^AGSAEV^ |ANGl|RHIGLA AKQ (SEQ ID NO: 84, fHbp V1.15 GI:504394462), wherein the sequence of any PorA VR2 loop in Table 1 may replace any amino acid or groups of amino acids highlighted by a box.

KDVMATYRGT AFGSDDAGGK LTYTIDFAAK QGHGKIEHLK SPELNVDLAV ayikp|dekhh a|visgsvlyn qdekgsyslg i|fgekaqe|va gsaev|tâng] 0HHIGLAAKQ (SEQ ID NO: 85, fHbpV 1.55 GE40353481), wherein the sequence of any PorA VR2 loop in Table 1 may replace any amino acid or groups of amino acids highlighted by a box.

PorA VR2 loop in Table 1 may replace any amino acid or groups of amino acids highlighted by a box.

CSSGGGGVAA DIGAGLADAL TAPLDHKDKS LQSLTLDQSV RKNEKLKL|ÂÂ| |qgaek|tygng dslntgklkn dkvsrfdfir qi[evdgql|it LESGEFQIYK QDHSAVVALQ IEK|lNNPDKI DSLl[NQRSFL VSGLGGEHTA FNQLPSGKAE YHGKAFSSDD AGGKLTYTID FAAKQGHGKI EHLKTPEQNV ELASAELKA0 |eksha|vilgd tryggeekgt yhlal^gdra q^iagsatv|k^IREKV^EIGI AGKQ (SEQ ID NO: 87, fHbp V2.19 GI:488148626), wherein the sequence of any PorA VR2 loop in Table 1 may replace any amino acid or groups of amino acids highlighted by a box.

CSSGGGGVAA DIGAGLADAL TAPLDHKDKS LQSLTLDQSV RKNEKLKL|AÂ| |QGAEK|TYGNG DSLNTGKLKN DKVSRFDFIR QI^VDGQL|lT LESGEFQIYK QDHSAVVALQ IEK|lNNPDKI DSLI^QRSFL VSGLGGEHTA FNQLPSGKAE YHGKAFSSDD PNGRLHYSID FTKKQGYGRI EHLKTPEQNV ELASAELKA0 ^ksha|vilgd tryggeekgt yhlal|fgdra QlEIAGSATV^IREKV^IEIGI AGKQ (SEQ ID NO: 88, fHbp V2.22 GI: 120865922), wherein the sequence of any PorA VR2 loop in Table 1 may replace any amino acid or groups of amino acids highlighted by a box.

CSSGSGSGGG GVAADIGTGL ADALTAPLDH KDKGLKSLTL EDSISQNGTL tl|saqgaek]t FKVGDKDNSL NTGKLKNDKI SRFDFVQKI[E VDGQT|lTLAS GEFQIYKQDH SAVVALQIEK| INNPDKIDSL ijNQRSFLVSG LGGEHTAFNQ

LPSGKAEYHG KAFSSDDAGG KLTYTIDFAA KQGHGKIEHL KTPEQNVELA

SAELKA[DEKS HA|VILGDTRY GSEEKGTYHL ALjFGDRAQÎEI AGSATV[KÏRË| |KV]HEIGIAGK q (SEQ ID NO: 90, fHbp V3.45 GE284466869), wherein the sequence of any PorA VR2 loop in Table 1 may replace any amino acid or groups of amino acids highlighted by a box.

CSSGGGGVAA DIGAGLADAL TAPLDHKDKG LKSLTLEDSI SQNGTLTL|SÂ| |QGAEK|TFKVG DKDNSLNTGK LKNDKISRFD FVQKl[EVDGQ TjlTLASGEFQ IYKQNHSAVV ALQIEK^NNP DKIDSLÏjNQR SFLVSGLGGE HTAFNQLPGG

ISIAGKQ (SEQ ID NO: 91, fHbp V3.47 GE284466897), wherein the sequence of any PorA VR2 loop in Table 1 may replace any amino acid or groups of amino acids highlighted by a box.

The modified fHbp may comprise the sequence of any one of SEQ ID NOs: 92 to 109.

The modified fHbp may comprise the sequence of SEQ ID NO: 92 to 109, wherein the sequence YYTKDTNNNLTLVP (SEQ ID NO: 68) is replaced by any PorA VR2 loop sequence, for example any VR2 loop sequence provided in Table 1.

In one embodiment, the modified fHbp may comprise or consist of the sequence:

CSSGGGGVAA DIGAGLADAL TAPLDHKDKG LQSLTLDQSV RKNEKLKLAA

QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QIEVDYYTKDT NNNLTLVPQL ITLESGEFQV YKQSHSALTA FQTEQIQDSE HSGKMVAKRQ FRIGDIAGEH

TSFDKLPEGG RATYRGTAFG SDDAGGKLTY TIDFAAKQGN GKIEHLKSPE

LNVDLAAADI KPDGKRHAVI SGSVLYNQAE KGSYSLGIFG GKAQEVAGSA

EVKTVNGIRH IGLAAKQ (SEQ ID NO: 92, fHbp VI.I GI:316985482, PorA VR2 Pl. 16 in Pl), or the same sequence whereby the sequence YYTKDTNNNLTLVP (SEQ ID NO: 68) is replaced by any PorA VR2 loop sequence, for example any PorA loop sequence as provided in Table 1.

In one embodiment, the modified fHbp may comprise or consist of the sequence:

CSSGSGSGGG

TLSAQGAEKT

NLTLVPQTIT

VSGLGGEHTA

EHLKTPEQNV

QEIAGSATVK

GVAADIGTGL FKVGDKDNSL LASGEFQIYK FNQLPSGKAE ELASAELKAD IREKVHEIGI

ADALTAPLDH NTGKLKNDKI QDHSAVVALQ YHGKAFSSDD EKSHAVILGD AGKQ (SEQ

KDKGLKSLTL

SRFDFVQKIE

IEKINNPDKI

AGGKLTYTID

TRYGSEEKGT

ID NO: 93,

EDSISQNGTL VDYYTKDTNN DSLINQRSFL FAAKQGHGKI YHLALFGDRA fHbp V3.45

GI:284466869, PorA VR2 P 1.16 in Pl), or the same sequence whereby the sequence YYTKDTNNNLTLVP (SEQ ID NO: 68) is replaced by any PorA VR2 loop sequence, for example any PorA loop sequence as provided in Table 1.

In one embodiment, the modified fHbp may comprise or consist of the sequence:

CSSGGGGVAA	DIGAGLADAL	TAPLDHKDKS	LQSLTLDQSV	RKNEKLKLAA
QGAEKTYGNG	DSLNTGKLKN	DKVSRFDFIR	QIEVDYYTKD	TNNNLTLVPQ
LITLESGEFQ	IYKQDHSAVV	ALQIEKINNP	DKIDSLINQR	SFLVSGLGGE
HTAFNQLPSG	KAEYHGKAFS	SDDAGGKLTY	TIDFAAKQGH	GKIEHLKTPE
QNVELASAEL	KADEKSHAVI	LGDTRYGGEE	KGTYHLALFG	DRAQEIAGSA
TVKIREKVHE	IGIAGKQ (SEQ ID NO: 94, fHbp V2.19 GI:488148626, PorA VR2 Pl. 16 in

Pl), or the same sequence whereby the sequence YYTKDTNNNLTLVP (SEQ ID NO: 68) is replaced by any PorA VR2 loop sequence, for example any PorA loop sequence as provided in Table 1.

In one embodiment, the modified fHbp may comprise or consist of the sequence:

CSSGGGGVAA DIGAGLADAL TAPLDHKDKG LQSLTLDQSV RKNEKLKLAA

QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QIEVDGQLIT LESGEFQVYK

QSHSALTAFQ

TYRGTAFGSD

DYYTKDTNNN

TEQIQDSEHS

DAGGKLTYTI

LTLVPKRHAV

GKMVAKRQFR

DFAAKQGNGK

ISGSVLYNQA

IGDIAGEHTS

IEHLKSPELN

EKGSYSLGIF

FDKLPEGGRA

VDLAAADIKP

GGKAQEVAGS

AEVKTVNGIR HIGLAAKQ (SEQ ID NO: 95, fHbp Vl.l GI:316985482, PorA VR2 PI.16 in P2), or the same sequence whereby the sequence YYTKDTNNNLTLVP (SEQ ID NO: 68) is replacer! by any PorA VR2 loop sequence, for example any PorA loop sequence as provided in Table 1.

In one embodiment, the modified fHbp may comprise or consist of the sequence:

CSSGSGSGGG	GVAADIGTGL	ADALTAPLDH	KDKGLKSLTL	EDSISQNGTL
TLSAQGAEKT FKVGDKDNSL NTGKLKNDKI SRFDFVQKIE VDGQTITLAS GEFQIYKQDH SAVVALQIEK INNPDKIDSL INQRSFLVSG LGGEHTAFNQ LPSGKAEYHG KAFSSDDAGG KLTYTIDFAA KQGHGKIEHL KTPEQNVELA SAELKADYYT KDTNNNLTLV PKSHAVILGD TRYGSEEKGT YHLALFGDRA QEIAGSATVK IREKVHEIGI AGKQ (SEQ ID NO: 96, fHbp V3.45 GI:284466869, PorA VR2 P 1.16 in P2), or the same sequence whereby the sequence YYTKDTNNNLTLVP (SEQ ID NO: 68) is replaced by any PorA VR2 loop sequence, for example any PorA loop sequence as provided in Table 1.
In one embodiment, the modified fHbp may comprise or consist of the sequence: CSSGGGGVAA DIGAGLADAL TAPLDHKDKS LQSLTLDQSV RKNEKLKLAA QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QIEVDGQLIT LESGEFQIYK QDHSAVVALQ IEKINNPDKI DSLINQRSFL VSGLGGEHTA FNQLPSGKAE YHGKAFSSDD AGGKLTYTID FAAKQGHGKI EHLKTPEQNV ELASAELKAD YYTKDTNNNL TLVPKSHAVI LGDTRYGGEE KGTYHLALFG DRAQEIAGSA TVKIREKVHE IGIAGKQ (SEQ ID NO: 97, fHbp V2.19 GL488148626, PorA VR2 PI.16 in P2), or the same sequence whereby the sequence YYTKDTNNNLTLVP (SEQ ID NO: 68) is replaced by any PorA VR2 loop sequence, for example any PorA loop sequence as provided in Table 1.
In one embodiment, the modified fHbp may comprise CSSGGGGVAA DIGAGLADAL TAPLDHKDKG QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QSHSALTAFQ TEQIQDSEHS GKMVAKRQFR TYRGTAFGSD DAGGKLTYTI DFAAKQGNGK	or consist of the sequence: LQSLTLDQSV RKNEKLKLAA QIEVDGQLIT LESGEFQVYK IGDIAGEHTS FDKLPEGGRA IEHLKSPELN VDLAAADIKP

DGKRHAVISG

SVLYNQAEKG

SYSLGIFGYY

TKDTNNNLTL

VPKAQEVAGS

AEVKTVNGIR HIGLAAKQ (SEQ ID NO: 98, fHbp Vl.l GI:316985482, PorA VR2

PI. 16 in P3), or the same sequence whereby the sequence YYTKDTNNNLTLVP (SEQ ID NO: 68) is replaced by any PorA VR2 loop sequence, for example any PorA loop sequence as provided in Table 1.

In one embodiment, the modified fHbp may comprise or consist of the sequence:

CSSGSGSGGG	GVAADIGTGL	ADALTAPLDH	KDKGLKSLTL	EDSISQNGTL
TLSAQGAEKT	FKVGDKDNSL	NTGKLKNDKI	SRFDFVQKIE	VDGQTITLAS
GEFQIYKQDH	SAVVALQIEK	INNPDKIDSL	INQRSFLVSG	LGGEHTAFNQ
LPSGKAEYHG	KAFSSDDAGG	KLTYTIDFAA	KQGHGKIEHL	KTPEQNVELA
SAELKADEKS	HAVILGDTRY	GSEEKGTYHL	ALFGYYTKDT	NNNLTLVPRA
QEIAGSATVK	IREKVHEIGI AGKQ	(SEQ ID NO:	99, fHbp V3.45 GE284466869, PorA
VR2 P1.16 in	P3), or the same sequence whereby the sequence YYTKDTNNNLTLVP

(SEQ ID NO: 68) is replaced by any PorA VR2 loop sequence, for example any PorA loop sequence as provided in Table 1.

In one embodiment, the modified fHbp may comprise or consist of the sequence:

CSSGGGGVAA DIGAGLADAL TAPLDHKDKS LQSLTLDQSV RKNEKLKLAA

QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QIEVDGQLIT LESGEFQIYK

QDHSAVVALQ IEKINNPDKI DSLINQRSFL VSGLGGEHTA FNQLPSGKAE

YHGKAFSSDD AGGKLTYTID FAAKQGHGKI EHLKTPEQNV ELASAELKAD

EKSHAVILGD TRYGGEEKGT YHLALFGYYT KDTNNNLTLV PRAQEIAGSA

TVKIREKVHE IGIAGKQ (SEQ ID NO: 100, fHbp V2.19 GE488148626, PorA VR2 PI.16 in P3), or the same sequence whereby the sequence YYTKDTNNNLTLVP (SEQ ID NO: 68) is replaced by any PorA VR2 loop sequence, for example any PorA loop sequence as provided in Table 1.

In one embodiment, the modified fHbp may comprise or consist of the sequence:

CSSGGGGVAA

QYYTKDTNNN

LITLESGEFQ

HTSFDKLPEG

ELNVDLAAAD

DIGAGLADAL

LTLVPAEKTY

VYKQSHSALT

GRATYRGTAF

IKPDGKRHAV

TAPLDHKDKG GNGDSLNTGK AFQTEQIQDS GSDDAGGKLT ISGSVLYNQA

LQSLTLDQSV

LKNDKVSRFD

EHSGKMVAKR

YTIDFAAKQG

EKGSYSLGIF

RKNEKLKLAA FIRQIEVDGQ QFRIGDIAGE NGKIEHLKSP GGKAQEVAGS

AEVKTVNGIR HIGLAAKQ (SEQ ID NO: 101, fHbp VI.1 GE316985482, PorA VR2 PI. 16 in

P4), or the same sequence whereby the sequence YYTKDTNNNLTLVP (SEQ ID NO: 68) is replaced by any PorA VR2 loop sequence, for example any PorA loop sequence as provided in Table 1.

In one embodiment, the modified fHbp may comprise or consist of the sequence:

CSSGSGSGGG	GVAADIGTGL	ADALTAPLDH	KDKGLKSLTL	EDSISQNGTL
TLSAQYYTKD TNNNLTLVPA EKTFKVGDKD NSLNTGKLKN DKISRFDFVQ KIEVDGQTIT LASGEFQIYK QDHSAVVALQ IEKINNPDKI DSLINQRSFL VSGLGGEHTA FNQLPSGKAE YHGKAFSSDD AGGKLTYTID FAAKQGHGKI EHLKTPEQNV ELASAELKAD EKSHAVILGD TRYGSEEKGT YHLALFGDRA QEIAGSATVK IREKVHEIGI AGKQ (SEQ ID NO: 102, fHbp V3.45 GI:284466869, PorA VR2 P 1.16 in P4), or the same sequence whereby the sequence YYTKDTNNNLTLVP (SEQ ID NO: 68) is replaced by any PorA VR2 loop sequence, for example any PorA loop sequence as provided in Table 1.
In one embodiment, the modified fHbp may comprise or consist of the sequence: CSSGGGGVAA DIGAGLADAL TAPLDHKDKS LQSLTLDQSV RKNEKLKLAA QYYTKDTNNN LTLVPAEKTY GNGDSLNTGK LKNDKVSRFD FIRQIEVDGQ LITLESGEFQ IYKQDHSAVV ALQIEKINNP DKIDSLINQR SFLVSGLGGE HTAFNQLPSG KAEYHGKAFS SDDAGGKLTY TIDFAAKQGH GKIEHLKTPE QNVELASAEL KADEKSHAVI LGDTRYGGEE KGTYHLALFG DRAQEIAGSA TVKIREKVHE IGIAGKQ (SEQ ID NO: 103, fHbp V2.19 GE488148626, PorA VR2 P1.16 in P4), or the same sequence whereby the sequence YYTKDTNNNLTLVP (SEQ ID NO: 68) is replaced by any PorA VR2 loop sequence, for example any PorA loop sequence as provided in Table 1.
In one embodiment, the modified fHbp may comprise CSSGGGGVAA DIGAGLADAL TAPLDHKDKG QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QSHSALTAFQ TEQIQDSYYT KDTNNNLTLV HTSFDKLPEG GRATYRGTAF GSDDAGGKLT ELNVDLAAAD IKPDGKRHAV ISGSVLYNQA	or consist of the LQSLTLDQSV QIEVDGQLIT PHSGKMVAKR YTIDFAAKQG EKGSYSLGIF	sequence: RKNEKLKLAA LESGEFQVYK QFRIGDIAGE NGKIEHLKSP GGKAQEVAGS

HIGLAAKQ (SEQ ID NO: 104, fHbp VI.1 GE316985482, PorA VR2 Pl. 16

AEVKTVNGIR in P5), or the same sequence whereby the sequence YYTKDTNNNLTLVP (SEQ ID NO: 68) is replaced by any PorA VR2 loop sequence, for example any PorA loop sequence as provided in Table 1.

In one embodiment, the modified fHbp may comprise or consist of the sequence:

CSSGSGSGGG GVAADIGTGL ADALTAPLDH KDKGLKSLTL EDSISQNGTL

TLSAQGAEKT FKVGDKDNSL NTGKLKNDKI SRFDFVQKIE VDGQTITLAS

GEFQIYKQDH

VSGLGGEHTA

EHLKTPEQNV

SAVVALQIEK

FNQLPSGKAE

ELASAELKAD

INNPYYTKDT

YHGKAFSSDD

EKSHAVILGD

NNNLTLVPKI

AGGKLTYTID

TRYGSEEKGT

DSLINQRSFL

FAAKQGHGKI

YHLALFGDRA

QEIAGSATVK IREKVHEIGI AGKQ (SEQ ID NO: 105, fHbp V3.45 GI:284466869, PorA

VR2 PI. 16 in P5), or the same sequence whereby the sequence YYTKDTNNNLTLVP (SEQ ID NO: 68) is replaced by any PorA VR2 loop sequence, for example any PorA loop sequence as provided in Table 1.

In one embodiment, the modified fHbp may comprise or consist of the sequence:

CSSGGGGVAA DIGAGLADAL TAPLDHKDKS LQSLTLDQSV RKNEKLKLAA QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QIEVDGQLIT LESGEFQIYK QDHSAVVALQ IEKINNPYYT KDTNNNLTLV PKIDSLINQR SFLVSGLGGE HTAFNQLPSG KAEYHGKAFS SDDAGGKLTY TIDFAAKQGH GKIEHLKTPE QNVELASAEL KADEKSHAVI LGDTRYGGEE KGTYHLALFG DRAQEIAGSA TVKIREKVHE IGIAGKQ (SEQ ID NO: 106, fHbp V2.19 GI:488148626, PorA VR2 PI.16 in

P5), or the same sequence whereby the sequence YYTKDTNNNLTLVP (SEQ ID NO: 68) is replaced by any PorA VR2 loop sequence, for example any PorA loop sequence as provided in Table 1.

In one embodiment, the modified fHbp may comprise or consist of the sequence:

CSSGGGGVAA DIGAGLADAL TAPLDHKDKG LQSLTLDQSV RKNEKLKLAA

QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QIEVDGQLIT LESGEFQVYK

QSHSALTAFQ TEQIQDSEHS GKMVAKRQFR IGDIAGEHTS FDKLPEGGRA

TYRGTAFGSD DAGGKLTYTI DFAAKQGNGK IEHLKSPELN VDLAAADIKP

DGKRHAVISG SVLYNQAEKG SYSLGIFGGK AQEVAGSAEV KTVYYTKDTN

NNLTLVPGIR HIGLAAKQ (SEQ ID NO: 107, fHbp VI.1 GI:316985482, PorA VR2

PI. 16 in P7), or the same sequence whereby the sequence YYTKDTNNNLTLVP (SEQ ID NO: 68) is replaced by any PorA VR2 loop sequence, for example any PorA loop sequence as provided in Table 1.

In one embodiment, the modified fHbp may comprise or consist of the sequence:

CSSGSGSGGG

TLSAQGAEKT

GEFQIYKQDH

LPSGKAEYHG

SAELKADEKS

GVAADIGTGL

FKVGDKDNSL

SAVVALQIEK

KAFSSDDAGG

HAVILGDTRY

ADALTAPLDH

NTGKLKNDKI

INNPDKIDSL

KLTYTIDFAA

GSEEKGTYHL

KDKGLKSLTL

SRFDFVQKIE

INQRSFLVSG

KQGHGKIEHL

ALFGDRAQEI

EDSISQNGTL

VDGQTITLAS

LGGEHTAFNQ

KTPEQNVELA

AGSATVKIRY

YTKDTNNNLT LVPKVHEIGI AGKQ (SEQ ID NO: 108, fHbp V3.45 GE284466869, PorA VR2 P 1.16 in P7), or the same sequence whereby the sequence YYTKDTNNNLTLVP (SEQ ID NO: 68) is replaced by any PorA VR2 loop sequence, for example any PorA loop sequence as provided in Table 1.

In one embodiment, the modified fHbp may comprise or consist of the sequence:

CSSGGGGVAA	DIGAGLADAL	TAPLDHKDKS	LQSLTLDQSV	RKNEKLKLAA
QGAEKTYGNG	DSLNTGKLKN	DKVSRFDFIR	QIEVDGQLIT	LESGEFQIYK
QDHSAVVALQ	IEKINNPDKI	DSLINQRSFL	VSGLGGEHTA	FNQLPSGKAE
YHGKAFSSDD	AGGKLTYTID	FAAKQGHGKI	EHLKTPEQNV	ELASAELKAD
EKSHAVILGD	TRYGGEEKGT	YHLALFGDRA	QEIAGSATVK	IRYYTKDTNN
NLTLVPKVHE	IGIAGKQ (SEQ ID NO: 109, fHbp V2.19 GI:488148626, PorA VR2 PI. 16 in

P7), or the same sequence whereby the sequence YYTKDTNNNLTLVP (SEQ ID NO: 68) is replaced by any PorA VR2 loop sequence, for example any PorA loop sequence as provided in Table 1.

The skilled person will understand that one, two, three or four or more amino acid substitutions, délétions or additions may be made to the modified fHbp of the invention herein without substantially removing its immunogenic function or affecting stability. Substitutions may be to similar amino acid residues, for example having similar MW, charge, hydrophobicity or moieties, or synthetic analogues. Such modifications are envisaged as part of the invention. In one embodiment the modified fHbp may hâve at least 75% identity with any one of the modified fHbp described herein. In one embodiment the modified fHbp may hâve at least 80% identity with any one of the modified fHbp described herein. In one embodiment the modified fHbp may hâve at least 85% identity with any one of the modified fHbp described herein. In one embodiment the modified fHbp may hâve at least 90% identity with any one of the modified fHbp described herein. In one embodiment the modified fHbp may hâve at least 95% identity with any one of the modified fHbp described herein. In one embodiment the modified fHbp may hâve at least 98% identity with any one of the modified fHbp described herein. In one embodiment the modified fHbp may hâve at least 99% identity with any one of the modified fHbp described herein.

In one embodiment the modified fHbp may hâve at least 99.5% identity with any one of the modified fHbp described herein.

According to another aspect of the invention, there is provided a nucleic acid encoding essentially or at least the modified fHbp according to the invention herein.

The nucleic acid may be in a vector, such as a viral vector.

According to another aspect of the invention, there is provided a composition comprising the modified fHbp according the invention herein.

The composition may comprise two or more different modified fHbp molécules (e.g. different forms/species thereof) according to the invention herein. For example, the composition may comprise two or more different variants of the modified fHbp according to the invention. For example, the composition may comprise fHbp variants vl and v2. The composition may comprise fHbp variants v2 and v3. In another embodiment, the composition comprises at least fHbp variant v2.

According to another aspect of the invention, there is provided a composition comprising the nucleic acid according the invention herein.

The composition may comprise a pharmaceutically acceptable carrier. The composition may further comprise an adjuvant.

According to a further aspect of the invention, there is provided a modified fHbp, nucleic acid, or composition according to the invention, for use as a médicament.

According to a further aspect of the invention, there is provided a modified fHbp, nucleic acid, or composition according to the invention, for use in the treatment or prévention of a pathogenic infection or colonisation of a subject.

According to a further aspect of the invention, there is provided a method of treatment or prévention of a pathogenic infection or colonisation of a subject, comprising the administration of a modified fHbp, nucleic acid, or composition according to the invention to the subject.

According to a further aspect of the invention, there is provided a method of vaccination, comprising the administration of a modified fHbp, nucleic acid, or composition according to the invention to a subject.

The administration may be provided in a therapeutically effective amount. A skilled person will be capable of determining an appropriate dosage and répétitions for administration.

The subject may be mammalian, such as human.

The infection may be a bacterial infection. For example, the infection may be meningitis, such as Neisseria meningitidis, or Neisseria gonorrhoeae.

According to a further aspect of the invention, there is provided a single protein, multi-valent vaccine comprising the modified factor H binding protein (fHbp) according to the invention.

The vaccine may be used as a prophylactic or a therapeutic vaccine directed to Nm.

The use may be with a pharmaceutically acceptable carrier. Additionally or alternatively, the use may be with an adjuvant. Suitable pharmaceutically acceptable carriers and adjuvants are well known to the skilled person.

According to a further aspect of the invention, there is provided a combination of the modified fHbp according to the invention and at least one other prophylactically or therapeutically active molécule.

The at least one other prophylactically or therapeutically active molécule may comprise a vaccine or antigen different to the modified fHbp according to the invention herein. The combination may be used in a combination vaccine or therapy. For example, the combination may be used in a combination vaccine or therapy where another meningococcal antigen is provided.

In one embodiment, the at least one other prophylactically or therapeutically active molécule comprises a monovalent protein:capsule polysaccharide vaccine. The monovalent protein:capsule polysaccharide vaccine may comprise 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.

Alternatively, the at least one other prophylactically or therapeutically active molécule may comprise a conjugate vaccine, wherein antigen(s) comprising the fHbp scaffold bearing exogenous peptide loops (such as PorA loops) may be incorporated as the protein carrier molécule in the conjugate vaccine. The conjugate vaccine may comprise any of serogroup capsular polysaccharides from A, C, Y, W, or X strains individually or in combination.

According to another aspect of the invention, there is provided the use of factor H binding protein (fHbp) as an epitope display scaffold.

The use as an epitope display scaffold may comprise the use of a factor H binding protein (fHbp) comprising any of the modifications described herein.

In addition to their potential use as vaccines, compositions or modified fHbps according to the invention may be useful as diagnostic reagents and as a measure of the immune compétence of a vaccine.

The term “immunogenic” means that the molécule is capable of eliciting an immune response in a human or animal body. The immune response may be protective.

The immune response elicited by the modified fHbp of the invention may affect the ability of Neisseria meningitidis (Nm) to infect a subject immunised with the modified fHbp of the invention. Preferably, the ability of Nm to infect a subject immunised with the modified fHbp of the invention is impeded or prevented. The immune response elicited may recognise and destroy Nm. Alternatively or additionally, the immune response elicited may impede or prevent réplication of Nm. Alternatively or additionally, the immune response elicited may impede or prevent Nm causing disease in the human or non-human animal.

The term “peptide loop” used herein is intended to refer to a single chain polypeptide sequence anchored at both ends (e.g. anchored to a scaffold such as fHbp). The term “loop” does not infer or require any particular secondary structure adopted by the polypeptide.

The term “exogenous” used in the context of “exogenous peptide loop” is understood to mean that the peptide loop is derived from a different source relative to the fHbp protein (i.e. it is not fHbp or a fragment thereof). However, it may be from the same organism as the fHbp. For example a modified fHbp may include an N. meningitidis fHbp modified with (exogenous) peptide loop(s) derived from N. meningitidis PorA.

The term “fusion protein” used herein is understood to mean a polypeptide comprising a combination of sequences from different gene products or sources.

Term “fusion protein” may be used interchangeably with the term “chimeric molécule”.

Reference to sequence “identity” used herein may refer to the percentage identity between two aligned sequences using standard NCBI BLASTp parameters (http://blast.ncbi.nlm.nih.gov).

The term isolated, when applied to the modified fHbp of the présent invention means a protein: (i) encoded by nucleic acids using recombinant DNA methods or a viral vector; or (ii) synthesized by, for example, Chemical synthetic methods; or (iii) separated from biological materials, and then purified. An isolated polypeptide of the invention includes a protein expressed from a nucléotide sequence encoding the protein, or from a recombinant vector containing a nucléotide sequence encoding the protein.

The term protective means prévention of a disease, a reduced risk of disease infection, transmission and/or progression, reduced severity of disease, a cure of a condition or disease, an alleviation of symptoms, or a réduction in severity of a disease or disease symptoms.

The term “prophylaxis” means prévention of or protective treatment for a disease. The prophylaxis may include a reduced risk of infection, transmission and/or progression, or reduced severity of disease.

The term “treatment”, means a cure of a condition or disease, an alleviation of symptoms, or a réduction in severity of a disease or disease symptoms.

The skilled person will understand that optional features of one embodiment or aspect of the invention may be applicable, where appropriate, to other embodiments or aspects of the invention.

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

Figure 1 Design of chimeric fHbp:PorAs (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.

(B) Structure of VI fHbp with CFH domains 6-7 showing the six positions (PI-5, and P7) used to generate chimeric fHbp:PorAs into which we hâve inserted PorA loops. N.B. position 5 is in the fHbp:CFH interface.

Figure 2: Use of fHbp as a molecular scaffold

A) Protein structure of fHbp Vl.l (grey, ribbon représentation) with the six positions (PI-5, and P7) used to generate chimeric fHbp:PorAs. B) Secondary structure of fHbp Vl.l (grey, arrows represent β-sheets, rectangles represent ahelices). Locations of the VR2 PI. 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 ID). C) Analysis of purified fHbp:PorAs with the PorA P 1.16 VR2 loop in positions 1-5 or 7 of fHbp, the wild-type (WT Vl.l) by SDS-PAGE and Western blot. Blots were probed with α-Vl fHbp pAb and an a-P1.16 mAb. D) Primary sequence (SEQ ID NO: 1) of Vl.l fHbp indicating the locations (underlined) of positions 1-5 and 7 into which loops from other proteins can be inserted.

Figure 3 Characterisation of chimeric fHbp:PorAs (A) Structure of fHbp:PorAs overlaid with the P 1.16 loop (black, PDBID: 2mpa) with the Fab of the a-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.

(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 température gradient. Melting température is shown for the fHbp N-terminal (NT_TM) and C-terminal (CT_TM) barrels. Binding fHbp:PorAs to

Complément Factor H (CFH) and mAbs SPR analysis of fHbp and chimeric fHbp:PorAs coupled to a BIAcore CM5 chip. CFH (fH67) was flowed over at a dilution range of 0.5-32 nM, and the dissociation constant (K_D) calculated; dissociation constants (K_Ds) 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.

Figure 4 Répertoire of antigens selected for chimeric fHbp:PorAs (A, B) Frequency of fHbp variants and PorA VR2 subtypes, respectively, in N.

meningitidis disease isolâtes 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 spécifie fHbps and PorAs.

N.B. V2 fHbp accounts for 38.9% of isolâtes.

Table 2. Exact sequence matches for ail N. meningitidis UK isolâtes 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
PI.9	19.1
PI.14	16.3
PI.16	7.0
PI.15	5.5
PI.1511	5.3
% coverage minus antigen overlap in ALL UK strains (isolated between 2009-2015)	70.6
% coverage minus antigen overlap in UK MenB strains (isolated between 2009-2015)	79.2

Exact sequence matches for:

- Pfizer vaccine, 3.02%

- Bexsero fHbp (V1.1 ) or PorA (PI.16), 15.86%

- Chimeric fHbp:PorAs, fHbp or PorA, 72% ( 23.5% with fHbp AND PorA)

Figure 5 Récognition of Neisseria meningitidis fHbp (A) and PorA (B) antigens by mouse immune sera. Whole cell lysâtes from Neisseria meningitidis strains H44/76 (WT), H44/67 AfHbp and H44/67 ΔΡογΑ, 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 pg of purified fHbp-PorA chimeras with VR2 loops in PI, P2, P4, P5 or P7.

Figure 6 Stabilisation of V2 fHbp: construction and immunogenicity of fHbp:PorAs (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 μΜ of protein, using a 20120°C température gradient. Melting température is recorded for the fHbp Nterminal (NT_Tm) and C-terminal barrels (CT_TM)· (B) Chimeric fHbp:PorAs PorA loops are recognised by corresponding mAbs. PorA VR2 loops from PI.2, PI.4, PI.9, PI.14 and PI.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 spécifie PorA mAbs (NIBSC). CFH binding was detected by far Western blot analysis with normal human sera and CFH pAbs.

Figure 7 Chimeric fHbp:PorAs elicit protective immunity. Mice were immunised with chimeric fHbp:PorA i.e. P(l)(l)(13), P(2)(l)(13), and P(3)(l)(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)(l)(13) is likely because the loop does not protrude from fHbp barrel as far as in pos. 1.

Figure 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 June 2017. Other: remaining alleles that occur in <4 isolâtes. (C) Analysis of recombinant Chimeric antigens by SDS-PAGE and Western blot. Immunoblots are probed with aPorA VR2 mAbs: PI.4, PI.9, PI.14 and PI.15. (D) Détection of PorA in a panel of N. meningitidis serogroup B isolâtes by mouse polyclonal antisera from Chimeric Antigens fHbp^{vl 4}:PorA^{151pl 1 10J}, fHbp^{vl 4}:PorA^{l51/PI 14} and fHbp^vl'⁴:PorA^I51/pl '⁵, ffibp^{v3 45}:PorA^{158/pl 4} and fHbp^V3'⁴⁵:PorA^l58/P1·⁹.

Example 1

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 (Fig. 5 and 7). Peptides hâve been used from the intégral 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 molécule résidé 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 molécule, based on fHbp and PorA sequences in spécifie sites to generate a chimeric molécule. This approach can be used for any other immunogenic intégral outer membrane protein.

It has been shown that the likely reason for the exclusion of v2 fHbp from vaccines is the inhérent instability of its N-terminal β-barrel: i) it was not possible to détermine the atomic structure of this portion of v2 fHbp¹⁰, ii) v2 fHbp is sensitive to protease digestion (mass spectrometry demonstrates that the cleaved sites résidé in the N-terminal β-barrel, not shown), and iii) differential scanning calorimetry confirms that the instability lies in this région of v2 fHbp¹⁰.

Stable v2 fHbps hâve been successfully generated. Mutagenesis affecting the Nterminal 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 évident 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 molécule; the changes do not affect the immunogenicity of the protein as expected (no différence in SBA, or α-fHbp IgG levels not shown).

Chimeric vl.l fHbp has been generated incorporating the 13 amino acid VR2 from P 1.16 PorA which elicits SBA in récipients of OMV vaccines¹⁶. While intégral membrane proteins contain hydrophobie (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 Banking β-sheets in PorA 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. Ail proteins elicit antibody responses that recognise against ffibp and PorA (Fig. 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 H44/76o/W6/2, 256 for both fHbps). This provides proof of principle for this approach.

fHbp as a scaffold for multi-valent vaccines

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 fHbp⁵. This interaction could impair the use of fHbp as a vaccine by i) blocking immunogenic epitopes and preventing the génération of antibodies that could compete with fH, and ii) reducing complément 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 vl, 2 and 3 fHbps hâve been identified that are necessary for fH interactions¹⁰, and modification of single a.a.s of vl, v2 and v3 fHbps which prevent fH binding hâve been shown to retain or even enhance immunogenicity of this important vaccine antigen^10,23.

To generate and evaluate single protein, multi-valent vaccine candidates'.

Protective PorA epitopes from prévalent serosubtypes (which are defined by their PorA sequence) of Nm¹⁵ hâve been introduced into vl, stable v2 and v3 proteins; their stability and récognition by PorA and fHbp mAbs has been determined. PorA sequences hâve been selected to cover the diversity of isolâtes in the UK but data from any collection of meningococcal strains can be used.

Methods of research

Génération 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 filtration¹⁰ of chimeric vl, V2 and V3 fHbps as these are either in existing vaccines (vl.l and 3.45) or because of expérience with the protein (v2), or because of their prevalence in Nm strains. PorA loops hâve been introduced into fHbp by standard methods, and we hâve 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 previously¹⁰.

References

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.

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

3. http://www.meningitis.org/research/genome

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.

5. Schneider, M. C., B. E. Presser, J. J. Caesar, E. Kugelberg, S. et al. 2009. Neisseria meningitidis recruits factor H using protein mimicry of host carbohydrates. Nature 458:890-893.

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

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.

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: el002688

9. Granoff, D. M., Welsch, J. A., and Ram, S. 2009. Binding of complément factor H (fH) to Neisseria meningitidis is spécifie for human fH and inhibits complément activation by rat and rabbit sera. Infect. Immun. 77: 764-769.

ΙΟ. Johnson, S., Tan, L., van der Veen, S., Caesar, J., et al. (2012) Design and évaluation of meningococcal vaccines through structure-based modification of host and pathogen molécules. PLoS pathogens 8: el002981

11. Zipfel, P. F., Skerka, C., Hellwage, J., Jokiranta, S. T., et al. 2002. Factor H family proteins: on complément, microbes and human diseases. Biochem. Soc. Trans. 30: 971-978.

12. Schneider, M. C., Exley, R. M., Ram, S., Sim, R. B., and Tang, C. M. 2007. Interactions between Neisseria meningitidis and the complément System. Trends Microbiol 15: 233-240

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.

14. Gorringe AR, Pajôn R 2011. Bexsero: a multicomponent vaccine for prévention of meningococcal disease. Expert Opin Biol Ther. 11: 969-85.

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 isolâtes 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

16. Rosenqvist, E., Hoiby, E. A., Wedege, E., Causant, D. A., et al. 1993. A new variant of serosubtype P 1.16 in Neisseria meningitidis from Norway, associated with increased résistance to bactericidal antibodies induced by a serogroup B outer membrane protein vaccine. Microb Pathog. 15: 197

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.

18. Martin, D. R., Ruijne, N., McCallum, L., O'Hallahan, J., and Oster, P. 2006. The VR2 epitope on the PorA P 1.7-2,4 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.

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 meningitidis. Synthetic peptides define the epitopes responsible for serosubtype specificity. J Exp Med. Jun 171: 1871-82.

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

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.

22. van den Elsen, J. M. H., Herron, J. N., Hoogerhout, P., Poolman, J. T., et al. 1997. Bactericidal antibody récognition of a PorA epitope of Neisseria meningitidis: Crystal structure of a Fab fragment in complex with a fluoresceinconjugated peptide. Proteins: Structure, Function, and Bioinformatics 29: 113125.

23. Beernink, P. T., Shaughnessy, J., Braga, E. M., Liu, Q., et al. 2011. A meningococcal factor H binding protein mutant that éliminâtes factor H binding enhances protective antibody responses to vaccination. J. Immunol. 186: 36063614.

24. Ufret-Vincenty, R. L., Aredo, B., Liu, X., McMahon, A., et al. 2010. Transgenic mice expressing variants of complément factor H develop AMD-like retinal findings. Invest Ophthalmol. Vis. Sci. 51: 5878-5887.

Ail references are herein incorporated by reference.

Example 2 - Chimeric Antigens containing an expanded range of PorA VR2 loops generate immune responses

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 hâve exact sequence matches to the most common antigens in a given région. In 2016, the most prévalent PorA VR2s in serogroup B N. meningitidis isolâtes were PI.4 (15.2%), PI.14 (15.2%), PI.9 (12.8%), P 1.16 (11.1%) and P 1.15 (5.8%, Figure 8B). VR2 P1.101 was présent in 1.6% serogroup B isolâtes. The most prévalent variant 1, variant 2 and variant 3 fHbps were VI.4, V2.19 and V3.45, présent in 21.8%, 5.3% and 4.9% of serogroup B N. meningitidis isolâtes respectively (Figure 8C). Five different Chimeric antigens were constructed, in which a PorA VR2 was inserted position 151 (VI.4) or position 158 (V3.45, Figure 8A). Following Chimeric antigen expression and purification, Western blot analyses confirmed these Chimeric antigens ail retained epitopes recognised by their cognate a-VR2 mAb and afHbp pAbs (Figure 8D). The thermal stability of wild type fHbps Vl.l, VI.4 and V3.45 and the Chimeric antigens was determined by differential scanning calorimetry (DSC, Table 3).

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 isolâtes. Figure 8E demonstrates that ail Chimeric antigens elicited α-PorA antibodies that recognised their cognate PorA VR2. To evaluate α-PorA SBA responses, Sérum 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, 780786 (2003)) (Table 4).

Table 3: Stability of wild type fHbp and Chimeric Antigens

Protein	Cp (kcal mole'^{1 O}C⁴)
N-terminal T_m	C-Terminal T_m
fHbp VI.1	69.8	87.9
fHbp VI.4	64.0	89.0
fHbp V3.45	41.0	83.0
fHbp^vl'⁴:PorA^151/P1110-¹	54.0	89.0
fHbp^v,4:PorA^{151/pl 14}	55.0	88.0
fHbp^v,4:PorA^{151/P1 15}	55.0	89.0
fHbp^V3'⁴⁵:PorA^158/P1·⁴	40.0	81.0
fHbp^{V3 45}:PorA^{158/pl 9}	39.0	80.0

Melting température, T_m

Table 4: Sérum bactericidal assay titres

Pooled antisera	Serogroup isolate	B fHbp variant	PorA VR2	SBA titre
fHbp^^PorA¹^¹·⁴	Ml 0240123	VI.92*	PI.4	1/160
fHbp^{V3 45}:PorA^158/P19	Ml 1240431	V2.19	PI.9	1/1280
fHbp^vl'⁴:PorA^151/P1110-¹	M11240189	V3.84	PI.10_l	1/20
ÎHbp^vl'⁴:PorA^151/P1·¹⁴	M15240853	V3.45	PI.14	1/640

α-PorA SBA titres generated using pooled Chimeric Antigen/alum antisera and serogroup B N. meningitidis isolâtes with mismatched fHbp variants. * fHbp truncated at residue 242.

fHbp^{vl 4}:PorA^{151/pl 15} not tested, as it was needed to generate kfHbp strains, as the fHbp in strains with PorA VR2 P 1.15 is not mismatched.

Claims

1. A modified factor H binding protein (fHbp), comprising wild-type fHbp or the wild-type gonococcal orthologue of fHbp (Ghfp), or a variant thereof 5 having at least 75% identity with any of the sequences of SEQ ID NOs: 1-13, which is further modified with the addition of at least one exogenous peptide loop, wherein the at least one exogenous peptide loop is provided between two beta sheets of the factor H binding protein.

10

2. The modified factor H binding protein according to claim 1, wherein the exogenous peptide loop is between 8 and 20 amino acids in length and wherein the exogenous peptide loop is inserted at an amino acid position that is corresponding to any one of the amino acid residue numbers 49-54, 83-88, 114124, 199-206, 227-233, and 240-246 of SEQ ID NO: 1.

3. The modified factor H binding protein according to claim 1, 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.

20

4. The modified factor H binding protein according to any preceding claim, wherein the exogenous peptide loop(s) has a sequence of an intégral outer membrane protein.

5. The modified factor H binding protein according to any preceding claim, 25 wherein the exogenous peptide loop(s) has a sequence of a prokaryotic polypeptide; optionally wherein the prokaryotic polypeptide is a meningococcal protein or a gonococcal protein.

6. The modified factor H binding protein according to any preceding claim, 30 wherein the exogenous peptide loop(s) comprises a fragment of PorA;

or wherein the exogenous peptide loop(s) are selected from any one of the PorA loops 1 to 7, or variants thereof having no more than 1, 2, 3, 4 or 5 amino acid additions, délétions or substitutions; and combinations thereof; or wherein the exogenous peptide loop(s) are selected from any one of the PorA loops comprising a sequence according to any of SEQ ID NOs: 45 to 79, or variant thereof having no more than 1, 2, 3, 4 or 5 amino acid additions, délétions or substitutions.

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

8. 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.

9. 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, wherein at least one exogenous peptide loop is an additional insert within the sequence of any of SEQ ID No. 1-13.

10. A nucleic acid encoding the modified fHbp according to any of claims 1 to 9; optionally wherein the nucleic acid is a vector, further optionally a viral vector.

11. A composition comprising the modified fHbp according to any of claims 1 to 9 or the nucleic acid according to claim 9;

optionally wherein the composition comprises two or more different modified fHbp molécules; and/or wherein the composition comprises a pharmaceutically acceptable carrier and/or an adjuvant.

12. The composition according to claim 11, wherein the composition further comprises at least one other prophylactically or therapeutically active molécule; and optionally wherein the at least one other prophylactically or therapeutically active molécule 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 molécule in the conjugate vaccine.

13. A modified fHbp according to any of claims 1 to 9, a nucleic acid according to claim 10, or a composition according to any of claims 11 to 12, for use as a médicament.

14. A modified fHbp according to any of claims 1 to 9, a nucleic acid according to claim 10, or a composition according to any of claims 11 to 12, for use in the treatment or prévention of a pathogenic infection or colonisation of a subject.

15. A combination of the modified fHbp according to any of claims 1 to 9, a nucleic acid according to claim 10, or a composition according to any of claims 11 to 12 and at least one other prophylactically or therapeutically active molécule;

optionally wherein at least one other prophylactically or therapeutically active molécule comprises a monovalent protein:capsule polysaccharide vaccine.

16. Use of the factor H binding protein (fHbp) according to any of claims 1-9 as an epitope display scaffold.