Factor h binding protein variants and methods of use thereof

Abstract

To provide variant factor H binding proteins that can elicit antibodies that are bactericidal for at least one strain of Neisseria meningitidis, compositions comprising such proteins, and methods of use of such proteins.SOLUTION: This invention relates to variants of factor H binding protein (fHbp), wherein: the variant binds human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp for human fH; and the variant of fHbp induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 028,123, filed Jul. 23, 2014, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Introduction Neisseria meningitidis is a Gram-negative bacterium that colonizes the human upper respiratory tract and is the cause of sporadic and periodic epidemics worldwide, most notably epidemics of meningitis and sepsis. Its incidence and prevalence are highest in children under 2 years of age. Like other Gram-negative bacteria, Neisseria meningitidis typically has a cytoplasmic membrane, a peptidoglycan layer, and an outer membrane, which together with capsular polysaccharide constitute the bacterial cell wall, and also has pili that project into the external environment. Capsule-bearing strains of Neisseria meningitidis are the major cause of bacterial meningitis and sepsis in children and young adults. The epidemic and economic importance of invasive Neisseria meningitidis infections have prompted research for an effective vaccine that can confer immunity across different strains, particularly across genetically diverse serogroup B strains that have different serotypes or serosubtypes.

[0003] Factor H-binding protein (fHbp, also known as lipoprotein 2086 in the art (Fletcher et al (2004) Infect Immun 72:2088-2100 (Non-Patent Document 1)), genomic Neisseria antigen (GNA) 1870 (Masignani et al. (2003) J Exp Med 197:789-99 (Non-Patent Document 2)) or "741") is a protein of N. meningitidis (meningococcus) that is expressed in the bacterium as a surface-exposed lipoprotein. An important function of fHbp is to bind to human complement factor H (fH), which downregulates complement activation. The binding of fH to the bacterial surface is an important mechanism by which the pathogen survives in non-immune human serum or blood and escapes innate host defenses. In recent years, genetic mutations in the human factor H gene cluster have been found to affect susceptibility to the development of meningococcal disease (Davila S et al. (2010) Nat Genetics doi:10.1038 / ng.640 (Non-Patent Document 3)). The binding of fH to fHbp is specific for human fH and some non-human primates, which can partially explain why Neisseria meningitidis is a human-specific pathogen. fHbp occurs in a state of many natural sequence variants, which are represented by the registration (ID) numbers given in the fHbp database on the Internet, pubmlst(dot)org / neisseria / fHbp.

[0004] There is a need for fHbp polypeptides that can induce an effective bactericidal antibody response.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

[0006] Summary Provided are a mutant factor H-binding protein capable of inducing an antibody bactericidal against at least one strain of Neisseria meningitidis, a composition containing such a protein, and a method of using such a protein.

[0007] Features The present disclosure provides variants of factor H-binding protein (fHbp) ID1. The present disclosure provides variants of fHbp that: (a) have an amino acid substitution at glutamine (Q38) at amino acid 38; (b) have an amino acid substitution at glutamic acid (E92) at amino acid 92; (c) have a glycine substitution for arginine at amino acid 130 (R130G); (d) have an amino acid substitution at serine (S223) at amino acid 223; and (e) provide variants that include an amino acid substitution selected from at least one of a histidine substitution for leucine at amino acid 248 (H248L), wherein the amino acid substitution is relative to fHbp ID1 (SEQ ID NO: 1), the variant includes an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO: 1, the variant fHbp binds to human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID1 for human fH, and the variant induces a bactericidal antibody response against at least one strain of Neisseria meningitidis in a mammalian host. In some examples, the amino acid substitution at Q38 is Q38R, Q38K, Q38H, Q38F, Q38Y, or Q38W. In some examples, the amino acid substitution at E92 is E92K, E92R, E92H, E92F, E92Y, or E92W. In some examples, the amino acid substitution at S223 is S223R, S223K, S223H, S223F, S223Y, or S223W. In some examples, the variant fHbp may further include a substitution of R41S or R41A relative to fHbp ID1. For example, the variant fHbp may include a substitution of R41S or R41A and a substitution at S223 relative to fHbp ID1, such as R41S / S223R. In other examples, the variant fHbp may further include a substitution of R41S or R41A and a substitution of H248L relative to fHbp ID1. In one example, the variant fHbp may include two, three, or more of the substitutions disclosed herein. In a specific example, the variant fHbp may include the substitutions S223R and H248L relative to fHbp ID1. In some examples, the variant fHbp binds to human fH with an affinity that is 25% or less of the affinity of fHbp ID1 for human fH.In some examples, the mutant fHbp binds to human fH with an affinity that is 10% or less of the affinity of fHbp ID1 for human fH. In some examples, the mutant fHbp binds to human fH with an affinity that is 5% or less of the affinity of fHbp ID1 for human fH.

[0008] The present disclosure provides mutants of fHbp ID22. The present disclosure provides mutants of fHbp comprising at least one amino acid substitution selected from: (a) an isoleucine substitution for asparagine at amino acid 115 (N115I); (b) a glycine substitution for aspartic acid at amino acid 121 (D121G); (c) a threonine substitution for serine at amino acid 128 (S128T); (d) an amino acid substitution at valine (V131) at position 131; (e) an amino acid substitution at lysine (K219) at position 219; (f) an amino acid substitution at glycine (G220) at position 220, wherein the amino acid substitution is relative to fHbp ID22 (SEQ ID NO: 2), the mutant comprises an amino acid sequence having an amino acid sequence identity higher than 85% with SEQ ID NO: 2, the mutant fHbp binds to human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID22 for human fH, and the mutant induces a bactericidal antibody response in a mammalian host. In some examples, the mutant fHbp binds to human fH with an affinity that is 25% or less of the affinity of fHbp ID22 for human fH. In some examples, the mutant fHbp binds to human fH with an affinity that is 10% or less of the affinity of fHbp ID22 for human fH. In some examples, the mutant fHbp binds to human fH with an affinity that is 5% or less of the affinity of fHbp ID22 for human fH. In some examples, the amino acid substitution at V131 is V131D, V131E, V131K, V131R, V131H, V131F, V131Y, or V131W. In some examples, the amino acid substitution at K219 is K219N, K219Q, K219D, K219E, K219F, K219Y, or K219W. In some examples, the amino acid substitution at G220 is G220S, G220N, G220Q, G220D, G220E, G220K, G220R, G220H, G220F, G220Y, or G220W.

[0009] In some examples, the mutant fHbp comprises double mutations that enhance the thermal stability of the mutant fHbp compared to the wild-type (WT) fHbp, e.g., the thermal stability of WT fHbp ID22. In some examples, the mutant fHbp may comprise the substitutions L130R and G133D relative to fHbp ID22 (SEQ ID NO: 2), the mutant fHbp comprises an amino acid sequence having an amino acid sequence identity higher than 85% with SEQ ID NO: 2, the mutant fHbp binds to human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID22 for human fH, the mutant induces a bactericidal antibody response in a mammalian host, and the mutant has a higher thermal stability compared to the thermal stability of fHbp ID22. In some examples, the mutant fHbp may comprise a combination of substitutions, e.g., L130R, G133D, and at least one amino acid substitution selected from: (a) N115I; (b) D121G; (c) S128T; (d) V131; (e) K219 (e.g., K219N); and (f) G220 (e.g., G220S), and the amino acid substitution is relative to fHbp ID22 (SEQ ID NO: 2), the mutant fHbp comprises an amino acid sequence having an amino acid sequence identity higher than 85% with SEQ ID NO: 2, the mutant fHbp binds to human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID22 for human fH, and the mutant induces a bactericidal antibody response in a mammalian host. The thermal stability of the mutant fHbp may be at least 5 °C, 10 °C, 15 °C, 20 °C, or higher, e.g., 5 °C to 30 °C, 5 °C to 25 °C, 5 °C to 20 °C, 10 °C to 20 °C, or 15 °C to 20 °C higher than that of WT fHbp (e.g., fHbp ID22). As used herein, "thermal stability" refers to the stability of a protein when exposed to high temperatures; a thermally stable mutant protein maintains its three-dimensional structure at a higher temperature than the wild-type protein. For example, a mutant fHbp comprising double mutations that enhance thermal stability compared to the thermal stability of wild-type (WT) fHbp, e.g., WT fHbp ID22, may denature at a higher temperature compared to WT fHbp. In one example, the N-terminal domain of the mutant fHbp may denature at a higher temperature than the N-terminal domain of WT fHbp (e.g., fHbp ID22).

[0010] An fHbp variant is also disclosed herein that contains a mutation that increases thermal stability compared to WT fHbp and further contains an additional mutation known to reduce binding of fH, for example, a mutation disclosed in US2011 / 0256180. In certain embodiments, a variant of factor H binding protein (fHbp) is disclosed, the variant comprising at least one of the amino acid substitutions L130R and G133D, and substitutions R80A, D211A, E218A, E248A, G236I, T221A, and H223A relative to fHbp ID22 (SEQ ID NO: 2), the variant comprising an amino acid sequence having an amino acid sequence identity greater than 85% to SEQ ID NO: 2, the variant fHbp binding to human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID22 for human fH, the variant inducing a bactericidal antibody response in a mammalian host.

[0011] The present disclosure provides variants of fHbp ID55. The present disclosure provides variants of fHbp comprising at least one amino acid substitution selected from the group consisting of: (a) an amino acid substitution at position 92 of glutamic acid (E92); (b) an amino acid substitution at position 223 of serine (S223); and (c) an amino acid substitution at position 248 of histidine (H248), wherein the amino acid substitution is relative to fHbp ID55 (SEQ ID NO: 3), the variant comprises an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO: 3, the variant fHbp binds to human factor H (fH) with an affinity less than 50% of the affinity of fHbp ID55 for human fH, and the variant induces a bactericidal antibody response in a mammalian host. In some examples, the variant fHbp binds to human fH with an affinity of 25% or less of the affinity of fHbp ID55 for human fH. In some examples, the variant fHbp binds to human fH with an affinity of 10% or less of the affinity of fHbp ID55 for human fH. In some examples, the variant fHbp binds to human fH with an affinity of 5% or less of the affinity of fHbp ID55 for human fH. In some examples, the amino acid substitution at E92 is E92K, E92R, E92H, E92F, E92Y, or E92W. In some examples, the amino acid substitution at S223 is S223R, S223K, S223H, S223F, S223Y, or S223W. In some examples, the amino acid substitution at H248 is H248L, H248I, H248V, H248D, H248E, H248F, H248Y, or H248W.

[0012] The present disclosure provides an immunogenic composition comprising a variant fHbp of the present disclosure. The present disclosure provides an immunogenic composition comprising: (a) a variant fHbp described in any one of paragraphs 0007 - 0011 above; and (b) a pharmaceutically acceptable excipient. In some examples, the fHbp variant is in a vesicle preparation prepared from a Neisseria meningitidis strain. In some examples, the pharmaceutically acceptable excipient comprises an adjuvant; for example, the adjuvant is aluminum phosphate or aluminum hydroxide. In some examples, the pharmaceutical composition further comprises Neisseria surface protein A.

[0013] The present disclosure provides a nucleic acid encoding a variant fHbp described in any one of paragraphs 0007 - 0011 above. The present disclosure provides a recombinant expression vector comprising a nucleic acid encoding a variant fHbp described in any one of paragraphs 0007 - 0011 above. The present disclosure provides an in vitro host cell comprising a nucleic acid encoding a variant fHbp described in any one of paragraphs 0007 - 0011 above. The present disclosure provides an in vitro host cell comprising a recombinant expression vector comprising a nucleic acid encoding a variant fHbp described in any one of paragraphs 0007 - 0011 above.

[0014] The present disclosure provides a method of inducing an antibody response in a mammal, the method comprising administering the immunogenic composition of paragraph 0012 above to the mammal. In some examples, the mammal is a human. In some examples, the antibody response is a bactericidal antibody response against one or more strains of N. meningitidis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015]

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Mode for Carrying Out the Invention

[0016] Definition The "factor H-binding protein" (fHbp), also known in the literature as GNA1870, GNA1870, ORF2086, LP2086 (lipoprotein 2086), and "741", refers to a class of N. meningitidis polypeptides. It is found in nature as a lipoprotein on the surface of N. meningitidis bacteria. fHbp is subdivided into three groups of fHbp variants (in some reports (Masignani et al. (2003) J Exp Med 197:789-99), called variant group 1 (v.1), variant group 2 (v.2), and variant group 3 (v.3), and in other reports (see, for example, Fletcher et al. (2004) Infect Immun 72:2088-2100), called subfamilies A and B) based on amino acid sequence diversity and immunological cross-reactivity (Masignani et al. (2003) J Exp Med 197:789-99). fHbp can also be classified into one of six most common fHbp modular groups designated modular groups I - VI, as shown in Figure 2 of Vu et al. (2012) Sci. Reports 2:341. Each unique fHbp found in N. meningitidis is also assigned an fHbp peptide ID according to the website pubmlst.org / neisseria / fHbp / . The lengths of the fHbp proteins of variant 2 (v.2) (derived from strain 8047, fHbp ID77) and variant 3 (v.3) (derived from strain M1239, fHbp ID28) differ from those derived from the MC58 strain (fHbp ID1) by -1 and +7 amino acid residues, respectively. Therefore, the numbers used herein to refer to residues in the v.2 and v.3 fHbp proteins differ from the numbers based on the actual amino acid sequences of these proteins. Thus, for example, a reference to the leucine residue (L) at position 166 in the fHbp sequence of v.2 or v.3 refers to the residue at position 165 in the v.2 protein and position 173 in the v.3 protein. Unless otherwise specified, the numbers of amino acid substitutions present in these fHbp variants conform to the numbers of amino acid residues of fHbp ID1.

[0017] As used herein, human factor H (“human fH”) refers to a protein comprising the amino acid sequence shown in FIG. 18 (SEQ ID NO: 4), and its naturally occurring human allelic polymorphisms.

[0018] “Derived from” in relation to an amino acid sequence or a polynucleotide sequence (e.g., an amino acid sequence “derived from” fHbp ID1) is intended to indicate that a polypeptide or nucleic acid has a sequence based on the sequence of a reference polypeptide or nucleic acid (e.g., a naturally occurring fHbp protein or the encoding nucleic acid), and is not intended to be limiting with respect to the origin or method by which the protein or nucleic acid was made. Non-limiting examples of reference polypeptides and reference polynucleotides from which an amino acid sequence or a polynucleotide sequence can be “derived” include naturally occurring fHbp, fHbp ID1, and non-naturally occurring fHbp. “Derived from” in relation to a bacterial strain is intended to indicate that the strain was obtained by in vivo passage or in vitro culture of a parental strain and / or that the strain is a recombinant cell obtained by modification of a parental strain.

[0019] “Conservative amino acid substitution” refers to substituting one amino acid residue in order to further share the chemical and physical properties (e.g., charge, size, hydrophobicity / hydrophilicity) of the amino acid side chain. “Conservative substitutions” are intended to include substitutions within the following groups of amino acid residues: gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Guidance for such substitutions can be obtained from an amino acid sequence alignment of a polypeptide presenting the epitope of interest.

[0020] The term "protective immunity" means that a vaccine or immunization program administered to a mammal induces an immune response that prevents, delays the progression of, or reduces the severity of a disease caused by Neisseria meningitidis, or decreases or completely eliminates the symptoms of such disease. Protective immunity may be accompanied by the production of bactericidal antibodies. It should be noted that the production of bactericidal antibodies against Neisseria meningitidis is accepted in the art as a predictor of the protective effect of vaccines in humans (Goldschneider et al. (1969) J. Exp. Med. 129:1307; Borrow et al. (2001) Infect Immun. 69:1568).

[0021] The expression "disease caused by a Neisseria meningitidis (meningococcus) strain" encompasses any clinical symptom or combination of clinical symptoms that exist in a human infected with Neisseria meningitidis. These symptoms include, but are not limited to, colonization of the upper respiratory tract (e.g., the mucosa of the nasopharynx and tonsils) by a pathogenic strain of Neisseria meningitidis, invasion of the bacteria into the mucosa and submucosal vascular bed, sepsis, septic shock, inflammation, hemorrhagic skin lesions, activation of fibrinolysis and blood coagulation, organ dysfunction, e.g., renal, pulmonary, and heart failure, adrenal hemorrhage and muscle infarction, capillary leakage, edema, peripheral limb ischemia, respiratory distress syndrome, pericarditis, and meningitis.

[0022] The expressions "specifically binds to an antibody" or "specifically immunoreactive" in relation to an antigen (e.g., a polypeptide antigen) refer to a binding reaction based on the presence of the antigen in a sample that may further contain other heterogeneous molecular populations and / or serves as evidence of the presence. Thus, under specified conditions, a particular antibody or a particular plurality of antibodies binds to a particular antigen or a particular plurality of antigens in the sample and does not bind, in a significant amount, to other molecules present in the sample. "Specifically binds to an antibody" or "specifically immunoreactive" in relation to an epitope of an antigen (e.g., an epitope of a polypeptide) refers to a binding reaction based on the presence of the epitope in an antigen (e.g., a polypeptide) that may further contain other heterogeneous epitope populations and heterogeneous antigen populations and / or serves as evidence of the presence. Thus, under specified conditions, a particular antibody or a particular plurality of antibodies binds to a particular epitope of the antigen and does not bind, in a significant amount, to other epitopes present in the antigen and / or in the sample.

[0023] The expression "in an amount sufficient to induce an immune response" means that there is a detectable difference between the immune response indicators measured before and after administration of a particular antigen preparation. Examples of immune response indicators include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), bactericidal assay, flow cytometry, immunoprecipitation, Ouchterlony immunodiffusion; binding detection assays such as spot, Western blot or antigen array; antibody titer or antibody specificity detected by assays such as cytotoxicity assays.

[0024] "Surface antigen" is an antigen present in the surface structure of Neisseria meningitidis (e.g., outer membrane, capsule, pili, etc.).

[0025] "Isolated" refers to an entity of interest that is in an environment different from that in which it may naturally occur. "Isolated" is intended to include a compound in a sample that is substantially enriched with respect to the compound of interest and / or in which the compound of interest is partially or substantially purified. In some examples, an isolated component (e.g., a polypeptide such as an fHbp variant of the present disclosure; a nucleic acid of the present disclosure; a recombinant vector of the present disclosure) is purified. For example, the isolated component has a purity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or more than 99%.

[0026] "Enriched" means that a sample is non-naturally (e.g., by an experimenter or clinician) manipulated such that the compound of interest is present at a higher concentration (e.g., at least 3-fold higher, at least 4-fold higher, at least 8-fold higher, at least 64-fold higher, or higher) than the concentration of the compound in a starting sample such as a biological sample (e.g., a sample in which the compound naturally occurs or a sample in which it is present after administration) or a starting sample in which the compound is produced (e.g., as a bacterial polypeptide, antibody, nucleic acid, etc.).

[0027] Before further describing the present invention, it is to be understood that the present invention is not limited to the specific embodiments described, and thus may naturally vary. It is also to be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting, as the scope of the present invention is defined only by the appended claims.

[0028] When a range of values is given, unless the context clearly indicates otherwise, each intervening value between the upper and lower limits of that range, to one tenth of the unit of the lower limit, as well as any other stated value or intervening values in that stated value, are understood to be included within the scope of the present invention. The upper and lower limits of these smaller ranges may independently be included within these smaller ranges, and are likewise included within the scope of the present invention, depending upon any particular excluded bounds of the stated range. When the stated range includes one or both of the bounds, ranges excluding either or both of those included bounds are also included within the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar to or equivalent to those described herein can also be used in the practice or testing of the present invention, but the preferred methods and materials are described herein. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials cited therein.

[0030] It should be noted that the singular forms "a", "an", and "the" as used in this specification and the appended claims include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a factor H binding protein" includes reference to a plurality of such factor H binding proteins, and reference to "the immunogenic composition" includes reference to one or more immunogenic compositions and equivalents thereof known to those of ordinary skill in the art. It is further noted that the claims may be drafted to exclude any optional element. Accordingly, the specification is intended to serve as a basis for use of the exclusive terms "solely", "only", etc. in connection with the recitation of claim elements, or use of "negative" limitations.

[0031] It is understood that certain features of the invention described in separate embodiments for clarity may also be presented in combination in a single embodiment. Conversely, various features of the invention described in a single embodiment for brevity may also be presented separately or in any suitable sub-combination. All combinations of embodiments related to the invention are clearly embraced by the invention and are disclosed herein as if each and every combination were individually and explicitly disclosed. Further, all sub-combinations of these various embodiments and their elements are also clearly embraced by the invention and are disclosed herein as if each and every such sub-combination were individually and explicitly disclosed herein.

[0032] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such publications by virtue of prior invention. Further, the dates of the publications provided may be different from the actual publication dates, which may need to be independently confirmed.

[0033] Detailed Description The present disclosure provides a mutant factor H binding protein (fHbp) that can induce antibodies that are bactericidal against at least one strain of Neisseria meningitidis. The present disclosure provides compositions including immunogenic compositions comprising the mutant fHbp of the present disclosure. The present disclosure provides methods of using the mutant fHbp of the present disclosure, or a composition comprising the mutant fHbp of the present disclosure.

[0034] Variant fHbp The present disclosure provides variant fHbp having an amino acid sequence that differs from the fHbp of wild-type N. meningitidis by 1 to 10 amino acids (e.g., from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), 10 to 15 amino acids, 15 to 20 amino acids, 20 to 30 amino acids, 30 to 40 amino acids, or 40 to 50 amino acids such that the variant fHbp exhibits a reduced affinity for human factor H (fH) compared to reference fHbp, and the variant fHbp, when administered to a mammalian host, induces a bactericidal immune response against one or more N. meningitidis strains. In some examples, the variant fHbp has an amino acid sequence that differs from the fHbp of reference wild-type N. meningitidis by only 1 to 10 amino acid substitutions. In some examples, the variant fHbp has an amino acid sequence that differs from the fHbp of reference wild-type N. meningitidis by only one amino acid substitution.

[0035] In some examples, the variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the reference fHbp sequence; the variant fHbp comprises one or more amino acid substitutions relative to the reference fHbp sequence such that the variant fHbp exhibits an affinity for human fH that is 85% or less of the binding affinity of the reference fHbp for human fH, e.g., the variant fHbp has a binding affinity for human fH that is about 85% to about 75%, about 75% to about 65%, about 65% to about 55%, about 55% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1% of the binding affinity of the reference fHbp for human fH; and the variant fHbp, when administered to a mammalian host (e.g., a human; or a non-human animal model), induces a bactericidal immune response against at least one strain of N. meningitidis.

[0036] Variant fHbps of the present disclosure maintain substantially the same three-dimensional structure as a reference (e.g., wild-type) fHbp that binds to human fH when the reference fHbp is in its native three-dimensional structure. Whether a variant fHbp of the present disclosure maintains substantially the same three-dimensional structure as a reference (e.g., wild-type) fHbp that binds to human fH can be determined using an antibody that binds to the wild-type fHbp when the wild-type fHbp is in its native three-dimensional structure. Such antibodies include, for example, JAR41; JAR4; and JAR31. See, for example, Vu et al. (2012) Sci. Reports 2:341. The hybridoma-forming JAR4 monoclonal antibody has American Type Culture Collection (ATCC) number PTA-8943. See also USPN8,470,340. For example, in some examples, variant fHbps of the present disclosure retain binding to JAR4; for example, variant fHbps of the present disclosure retain at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the binding to JAR4 of a reference fHbp (e.g., fHbp ID1, fHbp ID22, or fHbp ID55) in its native three-dimensional structure.

[0037] Variant of fHbp ID1 The "reference fHbp" from which a variant fHbp of the present disclosure is derived is, in some examples, fHbp ID1. The amino acid sequence of fHbp ID1 is shown below. TIFF2025108722000002.tif37151

[0038] In some examples, the variant fHbp of the present disclosure is the fHbp of variant group 1. In some examples, the variant fHbp of the present disclosure is the fHbp of variant group 1 and is the fHbp of modular group I. In some examples, the variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO: 1; the variant fHbp comprises one or more amino acid substitutions relative to fHbp ID1 such that the variant fHbp exhibits an affinity for human fH that is 85% or less of the binding affinity of fHbp ID1 for human fH, for example, the variant fHbp exhibits an affinity for human fH that is about 85% to about 75%, about 75% to about 65%, about 65% to about 55%, about 55% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1% of the binding affinity; the variant fHbp induces a bactericidal immune response against at least one strain of N. meningitidis when administered to a mammalian host (e.g., human; or non-human animal model).

[0039] In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO: 1, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID1 for human fH, the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and based on the numbering of fHbp ID1, the variant fHbp comprises at least one amino acid substitution selected from: (a) an amino acid substitution of glutamine at amino acid 38 (Q38); (b) an amino acid substitution of glutamic acid at amino acid 92 (E92); (c) a glycine substitution for arginine at amino acid 130 (R130G); (d) an amino acid substitution of serine at amino acid 223 (S223); and (e) a histidine substitution for leucine at amino acid 248 (H248L).

[0040] In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO: 1, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., from about 50% to about 45%, from about 45% to about 35%, from about 35% to about 25%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID1 for human fH, the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp comprises an amino acid substitution at glutamine (Q38) at amino acid position 38. In some examples, the variant fHbp comprises a Q38R substitution. Other amino acids having a positive charge or an aromatic side chain, such as lysine, histidine, phenylalanine, tyrosine, or tryptophan, may also be substituted at this position. Thus, in some examples, the variant fHbp comprises a Q38K substitution, a Q38H substitution, a Q38F substitution, a Q38Y substitution, or a Q38W substitution. As an example, a variant fHbp of the present disclosure can be as shown in FIG. 20 and can comprise the amino acid sequence set forth in SEQ ID NO: 5.

[0041] In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO: 1, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID1 for human fH, the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp comprises an amino acid substitution of glutamic acid (E92) at amino acid 92. In some examples, the fHbp variant comprises an E92K substitution. Other amino acids having a positive charge or an aromatic side chain, such as arginine, histidine, phenylalanine, tyrosine, or tryptophan, may also be substituted at this position. Thus, for example, in some examples, the fHbp variant comprises an E92R substitution, an E92H substitution, an E92F substitution, an E92Y substitution, or an E92W substitution. As an example, a variant fHbp of the present disclosure can be as shown in FIG. 21 and comprise the amino acid sequence set forth in SEQ ID NO: 6.

[0042] In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 1, wherein the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID1 for human fH, and the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp comprises a glycine substitution for arginine at amino acid 130 (R130G). For example, a variant fHbp of the present disclosure can be as shown in FIG. 22 and comprise the amino acid sequence set forth in SEQ ID NO: 7. Other amino acids having a negative charge or an aromatic side chain, such as aspartic acid, glutamic acid, phenylalanine, tyrosine, or tryptophan, can also be substituted at R130. Thus, for example, in some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 1, wherein the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID1 for human fH, and the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp comprises an R130D substitution, an R130E substitution, an R130F substitution, an R130Y substitution, or an R130W substitution.

[0043] In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO: 1, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID1 for human fH, the variant induces a bactericidal antibody response against at least 1 strain of N. meningitidis in a mammalian host, and the variant fHbp comprises an amino acid substitution at serine (S223) at amino acid 223. In some examples, the fHbp variant comprises an S223R substitution. Other amino acids having a positive charge or an aromatic side chain, such as lysine, histidine, phenylalanine, tyrosine, or tryptophan, may also be substituted at this position. Thus, for example, in some examples, the fHbp variant comprises an S223K substitution, an S223H substitution, an S223F substitution, an S223Y substitution, or an S223W substitution. As an example, a variant fHbp of the present disclosure can be as shown in FIG. 23 and comprise the amino acid sequence set forth in SEQ ID NO: 8.

[0044] In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 1, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID1 for human fH, the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp comprises a histidine substitution for leucine at amino acid 248 (H248L). For example, a variant fHbp of the present disclosure can be as shown in FIG. 24 and comprise the amino acid sequence set forth in SEQ ID NO: 9. Other amino acids having non-polar, or negatively charged or aromatic side chains, such as isoleucine, valine, aspartic acid, glutamic acid, phenylalanine, tyrosine, or tryptophan, can also be substituted at H248. Thus, in some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 1, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID1 for human fH, the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp comprises an H248I substitution, an H248V substitution, an H248D substitution, an H248E substitution, an H248F substitution, an H248Y substitution, or an H248W substitution.

[0045] Combinations of amino acid substitutions In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO:1, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID1 for human fH, the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and based on the number of fHbp ID1, the variant fHbp comprises: (a) an amino acid substitution of glutamine (Q38) at amino acid 38; (b) an amino acid substitution of glutamic acid (E92) at amino acid 92; (c) a glycine substitution for arginine at amino acid 130 (R130G); (d) an amino acid substitution of serine (S223) at amino acid 223; and (e) a histidine substitution for leucine at amino acid 248 (H248L), and comprises an amino acid substitution selected from two or more of the above.

[0046] Combinations of substitutions may be included, and the two substitutions are in different structural domains, each independently reducing the binding of fH to fHbp (e.g., one substitution in the N-terminal domain in combination with an amino acid substitution in the C-terminal domain. In some examples, a variant fHbp of the present disclosure comprises a first amino acid substitution within the N-terminal domain; and a second amino acid substitution within the C-terminal domain. In some examples, a variant fHbp of the present disclosure comprises a first amino acid substitution within the N-terminal domain; and a second amino acid substitution within the N-terminal domain. In some examples, a variant fHbp of the present disclosure comprises a first amino acid substitution within the C-terminal domain; and a second amino acid substitution within the C-terminal domain.

[0047] For example, in some examples, the variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 1, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID1 for human fH, the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and based on the number of fHbp ID1, the variant fHbp: (a) comprises an amino acid substitution of glutamine at amino acid 38 (Q38) and (b) an amino acid substitution of glutamic acid at amino acid 92 (E92); or the variant fHbp: (a) comprises an amino acid substitution of glutamine at amino acid 38 (Q38) and (c) a glycine substitution for arginine at amino acid 130 (R130G); or the variant fHbp: (a) comprises an amino acid substitution of glutamine at amino acid 38 (Q38) and (d) an amino acid substitution of serine at amino acid 223 (S223); or, the variant fHbp: (a) comprises an amino acid substitution of glutamine at amino acid 38 (Q38) and (e) a histidine substitution for leucine at amino acid 248 (H248L).

[0048] As a further non-limiting example, in some instances, variant fHbps of the present disclosure include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO:1, wherein the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., from about 50% to about 45%, from about 45% to about 35%, from about 35% to about 25%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID1 for human fH, and wherein the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and based on the numbering of fHbp ID1, the variant fHbp comprises: (b) an amino acid substitution of glutamic acid (E92) at amino acid 92 and (c) a glycine substitution for arginine at amino acid 130 (R130G); or the variant fHbp comprises: (b) an amino acid substitution of glutamic acid (E92) at amino acid 92 and (d) an amino acid substitution of serine (S223) at amino acid 223; or the variant fHbp comprises: (b) an amino acid substitution of glutamic acid (E92) at amino acid 92 and (e) a histidine substitution for leucine at amino acid 248 (H248L); or the variant fHbp comprises: (c) a glycine substitution for arginine at amino acid 130 (R130G) and (d) an amino acid substitution of serine (S223) at amino acid 223; or the variant fHbp comprises: (c) a glycine substitution for arginine at amino acid 130 (R130G) and (e) a histidine substitution for leucine at amino acid 248 (H248L); or the variant fHbp comprises: (d) an amino acid substitution of serine (S223) at amino acid 223 and (e) a histidine substitution for leucine at amino acid 248 (H248L).

[0049] As a further non-limiting example, in some instances, variant fHbps of the present disclosure include amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO:1, and the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID1 for human fH, and the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp, based on the numbering of fHbp ID1, includes: (i) a Q38R substitution; and (ii) an R130G substitution.

[0050] Also provided herein are mutant fHbp proteins that contain one or more substitutions relative to the amino acid sequence of fHbp ID1 and further contain the substitution R41S. Exemplary mutant fHbps include, relative to fHbp ID1, the R41S substitution and the substitution of S223, e.g., R41S / S223R, or relative to fHbp ID1, the R41S substitution and the H248L substitution. In some examples, mutant fHbps of the disclosure include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO:1, wherein the mutant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID22 for human fH, and the mutant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the mutant fHbp contains two or more of the following amino acid substitutions based on the numbering of fHbp ID1: (a) a serine substitution for arginine at amino acid 41 (R41S); (b) an arginine substitution for serine at amino acid 223 (S223R); (c) a leucine substitution for histidine at amino acid 248 (H248L).

[0051] Also disclosed herein are mutant fHbp proteins that contain one or more substitutions relative to the amino acid sequence of fHbp ID1 and further contain the substitutions disclosed in US2011 / 0256180, which is incorporated herein by reference in its entirety.

[0052] Variant of fHbp ID22 The “reference fHbp” from which the mutant fHbps of the disclosure are derived is, in some examples, fHbp ID22. The amino acid sequence of fHbp ID22 is shown below. TIFF2025108722000003.tif37152

[0053] In some examples, the variant fHbp of the present disclosure is the fHbp of variant group 2. In some examples, the variant fHbp of the present disclosure is the fHbp of variant group 2 and is the fHbp of modular group III. In some examples, the variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO: 2; the variant fHbp comprises one or more amino acid substitutions relative to fHbp ID22 such that the variant fHbp exhibits an affinity for human fH that is 85% or less of the binding affinity of fHbp ID22 for human fH; for example, the variant fHbp exhibits an affinity for human fH that is about 85% to about 75%, about 75% to about 65%, about 65% to about 55%, about 55% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1% of the binding affinity of fHbp ID22 for human fH; the variant fHbp induces a bactericidal immune response against at least one strain of N. meningitidis when administered to a mammalian host (e.g., a human; or a non-human animal model).

[0054] In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO: 2, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID22 for human fH, the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp comprises an amino acid substitution selected from at least one of: (a) an isoleucine substitution for asparagine at amino acid 115 (N115I); (b) a glycine substitution for aspartic acid at amino acid 121 (D121G); (c) a threonine substitution for serine at amino acid 128 (S128T); (d) an amino acid substitution at valine (V131) at position 131; (e) an amino acid substitution at lysine (K219) at position 219; (f) an amino acid substitution at glycine (G220) at position 220, relative to the amino acid sequence of fHbp ID22. As described herein, the numbering of the amino acid residues is based on that of fHbp ID1.

[0055] In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 2, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID22 for human fH, the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp comprises an isoleucine substitution for asparagine at amino acid 115 (N115I). For example, a variant fHbp of the present disclosure can be as shown in FIG. 25 and comprise the amino acid sequence set forth in SEQ ID NO: 10. Other amino acids having nonpolar, or positively charged or aromatic side chains, such as valine, leucine, lysine, arginine, histidine, phenylalanine, tyrosine, or tryptophan can also be substituted at N115. Thus, for example, in some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 2, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID22 for human fH, the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp comprises an N115V substitution, an N115L substitution, an N115K substitution, an N115R substitution, an N115H substitution, an N115F substitution, an N115Y substitution, or an N115W substitution relative to the amino acid sequence of fHbp ID22.

[0056] In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 2, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID22 for human fH, the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp comprises a glycine substitution (D121G) for the aspartic acid at amino acid 121. For example, a variant fHbp of the present disclosure can be as shown in FIG. 26 and comprise the amino acid sequence set forth in SEQ ID NO: 11. Other amino acids having nonpolar, or positively charged or aromatic side chains, such as leucine, isoleucine, valine, lysine, arginine, histidine, phenylalanine, tyrosine, or tryptophan can also be substituted at D121. Thus, for example, in some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 2, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID22 for human fH, the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp comprises a D121L substitution, a D121I substitution, a D121V substitution, a D121K substitution, a D121R substitution, a D121H substitution, a D121F substitution, a D121Y substitution, or a D121W substitution relative to the amino acid sequence of fHbp ID22.

[0057] In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO:2, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID22 for human fH, the variant induces a bactericidal antibody response against at least 1 strain of N. meningitidis in a mammalian host, and the variant fHbp comprises a threonine substitution for serine at amino acid 128 (S128T). For example, a variant fHbp of the present disclosure can be as shown in FIG. 27 and can comprise the amino acid sequence set forth in SEQ ID NO:12. Other amino acids having polarity, or charged or aromatic side chains, such as methionine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine, or tryptophan can also be substituted at S128. Thus, for example, in some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO:2, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID22 for human fH, the variant induces a bactericidal antibody response against at least 1 strain of N. meningitidis in a mammalian host, and the variant fHbp comprises an S128M substitution, an S128N substitution, an S128D substitution, an S128E substitution, an S128K substitution, an S128R substitution, an S128H substitution, an S128F substitution, an S128Y substitution, or an S128W substitution relative to the amino acid sequence of fHbp ID22.

[0058] In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO: 2, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID22 for human fH, the variant induces a bactericidal antibody response against at least 1 strain of N. meningitidis in a mammalian host, and the variant fHbp comprises an amino acid substitution at valine (V131) at position 131. In some examples, the fHbp variant comprises a V131D substitution. Other amino acids having a charge or aromatic side chain, such as glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine, or tryptophan, may also be substituted at this position. Thus, for example, in some examples, the fHbp variant comprises a V131E substitution, a V131K substitution, a V131R substitution, a V131H substitution, a V131F substitution, a V131Y substitution, or a V131W substitution. As an example, a variant fHbp of the present disclosure can comprise the amino acid sequence shown in FIG. 28 and set forth in SEQ ID NO: 13 relative to the amino acid sequence of fHbp ID22.

[0059] In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO: 2, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID22 for human fH, the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp comprises an amino acid substitution at lysine (K219) at position 219. In some examples, the fHbp variant comprises a K219N substitution. Other amino acids having polar, or negatively charged or aromatic side chains, such as glutamine, aspartic acid, glutamic acid, phenylalanine, tyrosine, or tryptophan, may also be substituted at this position. Thus, for example, in some examples, the fHbp variant comprises a K219Q substitution, a K219D substitution, a K219E substitution, a K219F substitution, a K219Y substitution, or a K219W substitution. As an example, a variant fHbp of the present disclosure can be as shown in FIG. 29 and comprise the amino acid sequence set forth in SEQ ID NO: 14.

[0060] In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO: 2, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID22 for human fH, the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp comprises an amino acid substitution at glycine (G220) at position 220. In some examples, the fHbp variant comprises a G220S substitution. Other amino acids having a polar, or charged or aromatic side chain, such as asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine, or tryptophan can also be substituted at this position. Thus, for example, in some examples, the fHbp variant comprises a G220N substitution, a G220Q substitution, a G220D substitution, a G220E substitution, a G220K substitution, a G220R substitution, a G220H substitution, a G220F substitution, a G220Y substitution, or a G220W substitution. For example, a variant fHbp of the present disclosure can comprise the amino acid sequence shown in FIG. 30 and set forth in SEQ ID NO: 15.

[0061] Combinations of amino acid substitutions In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO: 2, and the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID22 for human fH. The variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host. The variant fHbp comprises amino acid substitutions selected from two or more of the following with respect to the amino acid sequence of fHbp ID22: (a) an isoleucine substitution for asparagine at amino acid 115 (N115I); (b) a glycine substitution for aspartic acid at amino acid 121 (D121G); (c) a threonine substitution for serine at amino acid 128 (S128T); (d) an amino acid substitution at valine (V131) at position 131; (e) an amino acid substitution at lysine (K219) at position 219; (f) an amino acid substitution at glycine (G220) at position 220. As described herein, the numbering of the residues is based on the amino acids of fHbp ID1.

[0062] Combinations of substitutions may be included, and the two substitutions are in different structural domains, each independently reducing the binding of fH to fHbp (e.g., one substitution in the N-terminal domain in combination with an amino acid substitution in the C-terminal domain. In some examples, a variant fHbp of the present disclosure comprises a first amino acid substitution within the N-terminal domain; and a second amino acid substitution within the C-terminal domain. In some examples, a variant fHbp of the present disclosure comprises a first amino acid substitution within the N-terminal domain; and a second amino acid substitution within the N-terminal domain. In some examples, a variant fHbp of the present disclosure comprises a first amino acid substitution within the C-terminal domain; and a second amino acid substitution within the C-terminal domain.

[0063] For example, in some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 2, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID22 for human fH, the variant induces a bactericidal antibody response against at least 1 strain of N. meningitidis in a mammalian host, and with respect to the amino acid sequence of fHbp ID22, the variant fHbp: (a) comprises an isoleucine substitution for asparagine at amino acid 115 (N115I) and (b) a glycine substitution for aspartic acid at amino acid 121 (D121G); or the variant fHbp: (a) comprises an isoleucine substitution for asparagine at amino acid 115 (N115I) and (c) a threonine substitution for serine at amino acid 128 (S128T); or the variant fHbp: (a) comprises an isoleucine substitution for asparagine at amino acid 115 (N115I) and (d) an amino acid substitution of valine (V131) at position 131; or the variant fHbp: (a) comprises an isoleucine substitution for asparagine at amino acid 115 (N115I) and (e) an amino acid substitution of lysine (K219) at position 219; or the variant fHbp: (a) comprises an isoleucine substitution for asparagine at amino acid 115 (N115I) and (f) an amino acid substitution of glycine (G220) at position 220, and the numbers of the substituted residues are based on the numbers of the amino acid sequence of fHbp ID1.

[0064] As a further non-limiting example, in some instances, variant fHbps of the present disclosure include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO:2, wherein the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID22 for human fH, and the variant induces a bactericidal antibody response against at least 1 strain of N. meningitidis in a mammalian host, and relative to the amino acid sequence of fHbp ID22, the variant fHbp: (b) comprises a glycine substitution for aspartic acid at amino acid 121 (D121G) and (c) a threonine substitution for serine at amino acid 128 (S128T); or the variant fHbp: (b) comprises a glycine substitution for aspartic acid at amino acid 121 (D121G) and (d) an amino acid substitution at valine (V131) at position 131; or the variant fHbp: (b) comprises a glycine substitution for aspartic acid at amino acid 121 (D121G) and (e) an amino acid substitution at lysine (K219) at position 219; or the variant fHbp: (b) comprises a glycine substitution for aspartic acid at amino acid 121 (D121G) and (f) an amino acid substitution at glycine (G220) at position 220. The numbering of the substituted residues is based on the numbering of the amino acid sequence of fHbp ID1.

[0065] As a further non-limiting example, in some instances, variant fHbps of the present disclosure include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO: 2, wherein the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID22 for human fH, and the variant induces a bactericidal antibody response against at least 1 strain of N. meningitidis in a mammalian host. With respect to the amino acid sequence of fHbp ID22, the variant fHbp: (c) includes a threonine substitution for serine at amino acid 128 (S128T) and (d) an amino acid substitution at valine (V131) at position 131; or the variant fHbp: (c) includes a threonine substitution for serine at amino acid 128 (S128T) and (e) an amino acid substitution at lysine (K219) at position 219; or the variant fHbp: (c) includes a threonine substitution for serine at amino acid 128 (S128T) and (f) an amino acid substitution at glycine (G220) at position 220; or the variant fHbp: (d) includes an amino acid substitution at valine (V131) at position 131 and (e) an amino acid substitution at lysine (K219) at position 219; or the variant fHbp: (d) includes an amino acid substitution at valine (V131) at position 131 and (f) an amino acid substitution at glycine (G220) at position 220; or the variant fHbp: (e) includes an amino acid substitution at lysine (K219) at position 219 and (f) an amino acid substitution at glycine (G220) at position 220. The numbering of the substituted residues is based on the numbering of the amino acid sequence of fHbp ID1.

[0066] Combinations of substitutions may be included, where the two substitutions are in different structural domains, each independently reducing the binding of fH to fHbp (e.g., one from the N-terminal domain (e.g., N115I, D121G, S128T, or V131D) combined with one from the C-terminal domain (e.g., D211A, K219N, G220S)).

[0067] For example, in some examples, the variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 2, and the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID22 for human fH. The variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp comprises: (i) an N115I substitution; and (ii) a D211A substitution.

[0068] As another example, in some examples, the variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 2, and the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID22 for human fH. The variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp comprises: (i) an N115I substitution; and (ii) a K219N substitution.

[0069] As another example, in some instances, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO: 2, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., from about 50% to about 45%, from about 45% to about 35%, from about 35% to about 25%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID22 for human fH, the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp comprises: (i) an N115I substitution; and (ii) a G220S substitution.

[0070] Also disclosed herein are mutant fHbp polypeptides that have higher thermal stability compared to wild-type fHbp ID22. In some examples, the mutant fHbp may include the substitutions L130R and G133D relative to fHbp ID22 (SEQ ID NO: 2), the mutant fHbp includes an amino acid sequence having an amino acid sequence identity higher than 85% with SEQ ID NO: 2, the mutant fHbp binds to human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID22 for human fH, the mutant induces a bactericidal antibody response in a mammalian host, and the mutant has higher thermal stability than WT fHbp ID22. The thermal stability of the mutant fHbp may be at least 5 °C, 10 °C, 15 °C, 20 °C, or more higher than that of WT fHbp (e.g., fHbp ID22), for example, 5 °C to 30 °C, 5 °C to 25 °C, 5 °C to 20 °C, 10 °C to 20 °C, or 15 °C to 20 °C higher. As used herein, "thermal stability" refers to the stability of a protein when exposed to high temperatures; a thermally stable mutant protein maintains its three-dimensional structure at a higher temperature than the wild-type protein. For example, a mutant fHbp containing double mutations that enhance thermal stability compared to the thermal stability of wild-type (WT) fHbp, e.g., WT fHbp ID22, may denature at a higher temperature compared to WT fHbp. In one example, the N-terminal domain of the mutant fHbp may denature at a higher temperature than the N-terminal domain of WT fHbp (e.g., fHbp ID22).

[0071] In certain embodiments, a mutant of factor H binding protein (fHbp) is disclosed, the mutant includes the amino acid substitutions L130R and G133D relative to fHbp ID22 (SEQ ID NO: 2), and at least one of the substitutions R80A, N115I, D121G, S128T, V131, D211A, E218A, K219 (e.g., K219N), G220 (e.g., G220S), E248A, G236I, T221A, and H223A, the mutant includes an amino acid sequence having an amino acid sequence identity higher than 85% with SEQ ID NO: 2, the mutant fHbp binds to human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID22 for human fH, and the mutant induces a bactericidal antibody response in a mammalian host.

[0072] In some examples, the variant fHbp may include a combination of substitutions, such as L130R, G133D, and at least one amino acid substitution selected from: (a) N115I; (b) D121G; (c) S128T; (d) V131D; (e) K219 (e.g., K219N); and (f) G220 (e.g., G220S), wherein the amino acid substitution is relative to fHbp ID22 (SEQ ID NO: 2), the variant fHbp includes an amino acid sequence having an amino acid sequence identity higher than 85% with SEQ ID NO: 2, the variant fHbp binds to human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID22 for human fH, and the variant induces a bactericidal antibody response in a mammalian host.

[0073] In certain embodiments, a variant of factor H binding protein (fHbp) is disclosed, wherein the variant includes amino acid substitutions L130R and G133D relative to fHbp ID22 (SEQ ID NO: 2), and at least one of the substitutions R80A, D211A, E218A, E248A, G236I, T221A, and H223A, the variant includes an amino acid sequence having an amino acid sequence identity higher than 85% with SEQ ID NO: 2, the variant fHbp binds to human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID22 for human fH, and the variant induces a bactericidal antibody response in a mammalian host.

[0074] Typical variant fHbps have an amino acid sequence identity with SEQ ID NO: 2 that exceeds 85% (e.g., have an amino acid sequence having at least 90% identity, at least 95%, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity), and include polypeptides that include the following substitutions relative to the amino acid sequence of SEQ ID NO: 2: L130R and G133D; L130R, G133D, and K219N; or L130R, G133D, and G220S.

[0075] A mutant fHbp protein that contains one or more substitutions relative to the amino acid sequence of fHbp ID22 and further contains the substitutions disclosed in US2011 / 0256180, which is incorporated herein by reference in its entirety, is also disclosed herein.

[0076] Variant of fHbp ID55 The "reference fHbp" from which the mutant fHbp of the present disclosure is derived is, in some examples, fHbp ID55. The amino acid sequence of fHbp ID55 is shown below. TIFF2025108722000004.tif37151

[0077] In some examples, the mutant fHbp of the present disclosure is an fHbp of mutant group 1. In some examples, the mutant fHbp of the present disclosure is an fHbp of mutant group 1 and is an fHbp of modular group IV.

[0078] In some examples, the mutant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 3; the mutant fHbp contains one or more amino acid substitutions relative to fHbp ID55 such that the mutant fHbp exhibits an affinity for human fH that is 85% or less of the binding affinity of fHbp ID55 for human fH, e.g., the mutant fHbp has a binding affinity for human fH that is about 85% to about 75%, about 75% to about 65%, about 65% to about 55%, about 55% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1% of the binding affinity of fHbp ID55 for human fH; and the mutant fHbp induces a bactericidal immune response against at least one strain of N. meningitidis when administered to a mammalian host (e.g., a human; or a non-human animal model).

[0079] In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO: 3, wherein the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID55 for human fH, the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp comprises an amino acid substitution selected from at least one of: (a) an amino acid substitution of glutamic acid (E92) at position 92; (b) an amino acid substitution of serine (S223) at position 223; and (c) an amino acid substitution of histidine (H248) at position 248, wherein the numbering of the amino acid residues is based on the numbering of fHbp ID1.

[0080] In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO: 3, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID55 for human fH, the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp comprises an amino acid substitution at glutamic acid (E92) at position 92. In some examples, the fHbp variant comprises an E92K substitution. Other amino acids having a positive charge or an aromatic side chain, such as arginine, histidine, phenylalanine, tyrosine, or tryptophan, may also be substituted at this position. Thus, for example, in some examples, the fHbp variant comprises an E92R substitution, an E92H substitution, an E92F substitution, an E92Y substitution, or an E92W substitution. As an example, a variant fHbp of the present disclosure can be as shown in FIG. 31 and comprise the amino acid sequence set forth in SEQ ID NO: 16.

[0081] In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO: 3, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., from about 50% to about 45%, from about 45% to about 35%, from about 35% to about 25%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID55 for human fH, the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp comprises an amino acid substitution at serine (S223) at position 223. In some examples, the fHbp variant comprises an S223R substitution. Other amino acids having a positive charge or an aromatic side chain, such as lysine, histidine, phenylalanine, tyrosine, or tryptophan, may also be substituted at this position. Thus, for example, in some examples, the fHbp variant comprises an S223K substitution, an S223H substitution, an S223F substitution, an S223Y substitution, or an S223W substitution. As an example, a variant fHbp of the present disclosure can be as shown in FIG. 32 and comprise the amino acid sequence set forth in SEQ ID NO: 17.

[0082] In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO: 3, the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID55 for human fH, the variant induces a bactericidal antibody response against at least 1 strain of N. meningitidis in a mammalian host, and the variant fHbp comprises an amino acid substitution at histidine (H248) at position 248. In some examples, the fHbp variant comprises an H248L substitution. Other amino acids having non-polar, or negatively charged or aromatic side chains, such as isoleucine, valine, aspartic acid, glutamic acid, phenylalanine, tyrosine, or tryptophan, may also be substituted at this position. Thus, for example, in some examples, the fHbp variant comprises an H248I substitution, an H248V substitution, an H248D substitution, an H248E substitution, an H248F substitution, an H248Y substitution, or an H248W substitution. As an example, a variant fHbp of the present disclosure can be as shown in FIG. 33 and comprise the amino acid sequence set forth in SEQ ID NO: 18.

[0083] Combinations of amino acid substitutions In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO: 3, wherein the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID55 for human fH, and the variant induces a bactericidal antibody response against at least 1 strain of N. meningitidis in a mammalian host, and the variant fHbp comprises two or more amino acid substitutions selected from the group consisting of: (a) an amino acid substitution of glutamic acid (E92) at position 92; (b) an amino acid substitution of serine (S223) at position 223; and (c) an amino acid substitution of histidine (H248) at position 248, wherein the numbering of the residues is based on the numbering of the amino acids in the sequence relative to fHbp ID1.

[0084] Combinations of substitutions may be included, and the two substitutions are in different structural domains, each independently reducing the binding of fH to fHbp (e.g., one substitution in the N-terminal domain in combination with an amino acid substitution in the C-terminal domain. In some examples, a variant fHbp of the present disclosure comprises a first amino acid substitution within the N-terminal domain; and a second amino acid substitution within the C-terminal domain. In some examples, a variant fHbp of the present disclosure comprises a first amino acid substitution within the N-terminal domain; and a second amino acid substitution within the N-terminal domain. In some examples, a variant fHbp of the present disclosure comprises a first amino acid substitution within the C-terminal domain; and a second amino acid substitution within the C-terminal domain.

[0085] For example, in some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO: 3, and the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID55 for human fH, and the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and with respect to fHbp ID55, the variant fHbp: (a) comprises an amino acid substitution of glutamic acid (E92) at position 92 and (b) an amino acid substitution of serine (S223) at position 223; or the variant fHbp: (a) comprises an amino acid substitution of glutamic acid (E92) at position 92 and (c) an amino acid substitution of histidine (H248) at position 248; or the variant fHbp: (b) comprises an amino acid substitution of serine (S223) at position 223 and (c) an amino acid substitution of histidine (H248) at position 248; or, the variant fHbp: (a) comprises an amino acid substitution of glutamic acid (E92) at position 92, (b) an amino acid substitution of serine (S223) at position 223, and (c) an amino acid substitution of histidine (H248) at position 248, where the numbering of the residues is based on the numbering of the amino acids in the sequence relative to fHbp ID1.

[0086] Also disclosed herein are variant fHbp proteins that contain one or more substitutions relative to the amino acid sequence of fHbp ID55 as described above and further contain the substitutions disclosed in US2011 / 0256180, which is incorporated herein by reference in its entirety.

[0087] Fusion polypeptide Variant fHbps of the present disclosure can be polypeptides that include a fusion polypeptide, for example, the variant fHbps described above, and a heterologous polypeptide (for example, a fusion partner). The fusion partner can be at the N-terminus of the variant fHbp, at the C-terminus of the variant fHbp, or at an internal site of fHbp.

[0088] Suitable fusion partners include those that result in improved stability in vivo (for example, improved half-life in serum); those that result in ease of purification, for example, (His) n , for example, 6His, etc.; those that result in secretion of the fusion protein from cells; those that result in an epitope tag, for example, GST, hemagglutinin (HA; for example, YPYDVPDYA; SEQ ID NO: 26), FLAG (for example, DYKDDDDK; SEQ ID NO: 27), c-myc (for example, EQKLISEEDL; SEQ ID NO: 28), etc.; those that result in a detectable signal, for example, an enzyme that produces a detectable product (for example, β-galactosidase, luciferase), or a protein that is itself detectable, for example, green fluorescent protein, yellow fluorescent protein, etc.; those that result in multimerization, for example, a multimerization domain such as the Fc portion of an immunoglobulin; and the like peptides and polypeptides.

[0089] Production method The fHbps of the present disclosure can be produced by any suitable method including recombinant and non-recombinant methods (for example, chemical synthesis). When the fHbp of interest is produced using recombinant technology, the method can include any suitable construct and any suitable host cell, and the host cell can be a prokaryotic cell or a eukaryotic cell, and can usually be a bacterial or yeast host cell, and more usually a bacterial cell. Methods for introducing genetic material into host cells include, for example, transformation, electroporation, conjugation, calcium phosphate method, etc. The transfer method can be selected to result in stable expression of the nucleic acid encoding the introduced fHbp. The nucleic acid encoding fHbp can be provided as an episomal element (for example, a plasmid) or can be genomically integrated.

[0090] The present disclosure provides a nucleic acid (including an isolated nucleic acid) comprising a nucleotide sequence encoding an fHbp variant of the present disclosure. In some embodiments, the nucleotide sequence encoding the fHbp variant is operably linked to a transcriptional regulator, such as a promoter. The promoter is, in some examples, constitutive. The promoter is, in some examples, inducible. In some examples, the promoter is suitable for use in a prokaryotic host cell (e.g., is active therein). In some examples, the promoter is suitable for use in a eukaryotic host cell (e.g., is active therein).

[0091] In some examples, the nucleic acid comprising the nucleotide sequence encoding the fHbp variant of the present disclosure is present within an expression vector. The present disclosure provides a recombinant expression vector (e.g., an isolated recombinant expression vector) comprising a nucleotide sequence encoding an fHbp variant of the present disclosure. In some embodiments, the nucleotide sequence encoding the fHbp variant is operably linked to a transcriptional regulator, such as a promoter. The promoter is, in some examples, constitutive. The promoter is, in some examples, inducible. In some examples, the promoter is suitable for use in a prokaryotic host cell (e.g., is active therein). In some examples, the promoter is suitable for use in a eukaryotic host cell (e.g., is active therein).

[0092] The vectors suitable for transferring the nucleic acid encoding fHbp can vary in composition. The integrating vectors can be, for example, conditionally replicating or suicide plasmids, bacteriophages, etc. These constructs can contain various elements such as, for example, a promoter, a selectable gene marker (e.g., a gene conferring resistance to an antibiotic (e.g., kanamycin, erythromycin, chloramphenicol, or gentamicin)), an origin of replication (to facilitate replication in a host cell, e.g., a bacterial host cell), etc. The choice of vector depends on various factors, such as the type of cell in which growth is desired and the purpose of the growth. Some vectors are useful for amplification and large-scale production of the desired DNA sequence. Other vectors are suitable for expression in cultured cells. Still other vectors are suitable for transfer and expression in cells of the whole animal. The selection of the appropriate vector is well within the skill of those in the art. Many such vectors are commercially available.

[0093] In one example, the vector is an expression vector based on an episomal plasmid that contains a selectable drug resistance marker and elements that result in self-replication in different host cells (e.g., both in E. coli and N. meningitidis). An example of such a "shuttle vector" is the pFP10 plasmid (Pagotto et al. (2000) Gene 244:13-19).

[0094] Constructs (recombinant vectors) can be prepared, for example, by inserting the polynucleotide of interest into the backbone of the construct, usually by attachment to the restriction enzyme site of the vector cleaved by DNA ligase. As another method, the desired nucleotide sequence can be inserted by homologous recombination or site-specific recombination. Usually, homologous recombination is achieved by attaching homologous regions on both sides of the desired nucleotide sequence to the vector, and site-specific recombination can be achieved by the use of sequences that facilitate site-specific recombination (e.g., cre-lox, att sites, etc.). Nucleic acids containing such sequences can be added, for example, by ligation reactions of oligonucleotides or by polymerase chain reaction using primers that contain both the homologous regions and a portion of the desired nucleotide sequence.

[0095] The vector can be maintained episomally within the host cell or integrated into the host cell genome. The vector is well described in many publications well known to those skilled in the art, such as, for example, Short Protocols in Molecular Biology, (1999) F. Ausubel, et al., eds., Wiley & Sons. The vector may result in the expression of the nucleic acid encoding the fHbp of interest, may result in the propagation of the nucleic acid of interest, or both.

[0096] Examples of vectors that can be used include, but are not limited to, those derived from recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA. For example, plasmid vectors such as pBR322, pUC19 / 18, pUC118, 119, and vectors of the M13mp series can be used. pET21 is also an expression vector that can be used. Examples of bacteriophage vectors may include λgt10, λgt11, λgt18-23, λZAP / R, and bacteriophage vectors of the EMBL series. Further vectors that can be utilized include, but are not limited to, pJB8, pCV103, pCV107, pCV108, pTM, pMCS, pNNL, pHSG274, COS202, COS203, pWE15, pWE16, and vectors of the charomid 9 series.

[0097] For the expression of the target fHbp, an expression cassette may be used. Thus, the present disclosure provides a recombinant expression vector comprising a nucleic acid of interest. The expression vector provides transcriptional and translational regulatory sequences, which can result in inducible or constitutive expression, and the coding region is operably linked under the transcriptional regulation of a transcription initiation region as well as transcription and translation termination regions. These regulatory regions may originally be present in the fHbp from which the target fHbp is derived, or may be of foreign origin. Generally, the transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or activator sequences. The promoter may be constitutive or inducible, and may be a strong constitutive promoter (e.g., T7, etc.).

[0098] Expression vectors generally have restriction sites located near the promoter sequence that facilitate the insertion of the nucleic acid sequence encoding the protein of interest. A selectable marker that functions in the expression host may be present to facilitate the selection of cells containing the vector. Furthermore, the expression construct may contain additional elements. For example, the expression vector may have one or two replication systems to enable its maintenance in an organism, e.g., for expression in mammalian or insect cells and for cloning and amplification in a prokaryotic host. Furthermore, the expression construct may contain a selectable marker gene to enable the selection of transformed host cells. Selectable genes are well known in the art and vary depending on the host cell used.

[0099] It should be noted that the fHbp of the present disclosure may include additional elements such as a detectable label, e.g., a radioactive label, a fluorescent label, a biotin label, an immunologically detectable label (e.g., a hemagglutinin tag, a polyhistidine tag), etc. The additional elements of fHbp may be provided (e.g., a biotin tag, an immunologically detectable tag) to facilitate isolation by various methods (e.g., affinity capture, etc.). The target fHbp may optionally be immobilized on a support by covalent or non-covalent binding.

[0100] The isolation and purification of fHbp can be achieved according to methods known in the art. For example, fHbp can generally be isolated from cell lysates genetically modified to express fHbp or from synthetic reaction mixtures by immunoaffinity purification, which involves contacting a sample with an anti-fHbp antibody (e.g., an anti-fHbp monoclonal antibody (mAb), e.g., JAR4 MAb or other suitable JAR MAb known in the art), washing to remove non-specifically bound material, and eluting the specifically bound fHbp. The isolated fHbp can be further purified by dialysis and other methods commonly used in protein purification methods. In one example, fHbp can be isolated using a metal chelate chromatography method.

[0101] Host cell Any of a number of suitable host cells can be used for the production of fHbp. Generally, the fHbp described herein can be expressed in prokaryotes or eukaryotes, such as bacteria like Escherichia coli or Neisseria (e.g., N. meningitidis), according to conventional techniques. Thus, the disclosure further provides a genetically modified in vitro host cell comprising a nucleic acid encoding the fHbp of interest. The host cell for the production of the fHbp of interest (including large-scale production) can be selected from any of a variety of available host cells. Examples of host cells for expression include prokaryotic or eukaryotic single-celled organisms, such as bacteria (e.g., Escherichia coli strains), yeasts (e.g., Saccharomyces cerevisiae (Saccharomyces cerevisiae), Pichia species, etc.), and may also include host cells originally from higher organisms, such as insects, vertebrates, e.g., mammals. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) number CCL-2), CHO cells (e.g., ATCC numbers CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC number CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC number CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC number CCL10), PC12 cells (ATCC number CRL1721), COS cells, COS-7 cells (ATCC number CRL1651), RAT1 cells, mouse L cells (ATCC number CCLI.3), human embryonic kidney (HEK) cells (ATCC number CRL1573), HLHepG2 cells, etc.). In some examples, bacterial host cells and yeast host cells are particularly important for the production of the fHbp of interest.

[0102] The fHbp of interest can be prepared in a substantially pure or substantially isolated form (i.e., substantially free of other Neisseria or host cell polypeptides) or in a substantially isolated form. The fHbp of interest can be present in a composition in which the polypeptide is concentrated relative to other components that may be present (e.g., other polypeptides or other host cell components). The purified fHbp of interest can be provided in a composition in which the polypeptide is substantially free of other expressed polypeptides, e.g., a composition in which less than 90%, usually less than 60%, and more usually less than 50% of the composition is composed of other expressed polypeptides.

[0103] Host cell for vesicle production When the fHbp of interest is provided in membrane vesicles (described in more detail below), the Neisseria host cell is genetically modified to express the fHbp of interest. Any of a variety of strains of Neisseria species can be modified to produce the fHbp of interest, and optionally, these can produce or be modified to produce other antigens of interest, e.g., PorA, and can be used in the methods disclosed herein.

[0104] Methods and vectors for genetically modifying Neisseria strains and resulting in the expression of a desired polypeptide are known in the art. Examples of vectors and methods can be found in WO02 / 09746 and O’Dwyer et al. (2004) Infect Immun 72:6511-80. Strong promoters, particularly strong constitutive promoters, are particularly important. Examples of promoters include the promoters of porA, porB, lbpB, tbpB, p110, hpuAB, lgtF, opa, p110, lst, hpuAB, and rmp.

[0105] Pathogenic Neisseria species or strains derived from pathogenic Neisseria species, particularly strains pathogenic to humans, or strains derived from strains that are pathogenic or symbiotic to humans are particularly important for use in membrane vesicle production. Examples of Neisseria species include N. meningitidis, N. flavescens, N. gonorrhoeae, N. lactamica, N. polysaccharea, N. cinerea, N. mucosa, N. subflava, N. sicca, N. elongata, and the like.

[0106] N. meningitidis strains are particularly important for genetic modification and use in vesicle production to express the fHbp of interest. The strain used for vesicle production can be selected according to many different desired characteristics. For example, the strain can be selected according to: the desired PorA type ("serosubtype"), capsular group, serotype, etc.; reduced capsular polysaccharide production; etc. For example, the production strain can produce any desired PorA polypeptide and can express one or more PorA polypeptides (naturally or by genetic manipulation). Examples of strains include PorA polypeptides that confer serosubtypes P1.7,16; P1.19,15; P1.7,1; P1.5,2; P1.22a,14; P1.14; P1.5,10; P1.7,4; P1.12,13; and those that produce variants of such PorA polypeptides that may or may not retain reactivity with conventional serological reagents used for serosubtyping. Also important are PorA polypeptides characterized according to PorA variable region (VR) typing (see, for example, Russell et al. (2004) Emerging Infect Dis 10:674-678; Sacchi CT et al. (1998) Clin Diagn Lab Immunol 5:845-55; Sacchi et al (2000) J. Infect Dis 182:1169-1176). A number of different VR types have been identified and these can be classified into "prototypes" of the VR1 and VR2 families. A web-accessible database that describes this nomenclature and its relationship to previous typing schemes can be found at neisseria.org / nm / typing / pora. Alignments of specific PorA VR1 and VR2 types are provided in Russell et al. (2004) Emerging Infect Dis 10:674-678.

[0107] Alternatively or additionally, the production strain may be a capsule-deficient strain. Capsule-deficient strains can provide vesicle-based vaccines that result in a reduced risk of inducing a significant autoantibody response (e.g., by production of antibodies that cross-react with sialic acid on the surface of host cells) in the subject to whom the vaccine is administered. As used herein, "capsule-deficient" or "deficiency of capsular polysaccharide" refers to a level of capsular polysaccharide on the bacterial surface that is lower than that of a naturally occurring strain, or, if the strain is genetically modified, lower than that of the parental strain from which the capsule-deficient strain is derived. Capsule-deficient strains include those in which polysaccharide production of the surface capsule is reduced by at least 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, or more, as well as strains in which capsular polysaccharide cannot be detected on the bacterial surface (e.g., by whole-cell enzyme-linked immunosorbent assay (ELISA) using anti-capsular polysaccharide antibodies).

[0108] Examples of capsule-deficient strains include those with naturally occurring or recombinantly generated genetic modifications that result in capsule deficiency. Naturally occurring capsule-deficient strains (see, e.g., Dolan-Livengood et al. (2003) J. Infect. Dis. 187:1616-28), as well as methods for identifying and / or generating capsule-deficient strains (see, e.g., Fisseha et al. (2005) Infect. Immun. 73:4070-4080; Stephens et al. (1991) Infect Immun 59:4097-102; Frosch et al. (1990) Mol Microbiol.4:1215-1218) are known in the art.

[0109] Modifications of Neisseria host cells that result in a decrease in the production of capsular polysaccharide may include modification of one or more genes involved in capsule synthesis, where the modification results in a decrease in the level of capsular polysaccharide, for example, relative to the parental cell prior to modification. Such genetic modifications can include changes in the nucleotide and / or amino acid sequence of one or more capsular biosynthesis genes, rendering the strain capsule-deficient (e.g., by one or more insertions, deletions, substitutions, etc. in one or more capsular biosynthesis genes). Capsule-deficient strains may lack one or more capsular genes or may be non-functional with respect to one or more capsular genes.

[0110] Particularly important are strains that are deficient in sialic acid biosynthesis. Such strains can result in the production of vesicles with a reduced risk of inducing anti-sialic acid antibodies that cross-react with human sialic acid antigens and, further, can result in an improvement in manufacturing safety. Strains deficient in sialic acid biosynthesis (either by naturally occurring modification or artificial modification) can have a deficiency in any of a number of different genes in the sialic acid biosynthetic pathway. Particularly important are strains that are deficient in the gene product encoded by the N-acetylglucosamine-6-phosphate 2-epimerase gene (known as synX AAF40537.1 or siaA AAA20475), and strains in which this gene is inactivated are of particular interest. For example, in one embodiment, capsule-deficient strains are generated by interfering with the production of a functional synX gene product (see, e.g., Swartley et al. (1994) J Bacteriol. 176:1530-4).

[0111] Capsule-deficient strains can also be generated from naturally occurring strains using non-recombinant techniques, for example, by selecting strains in which the level of capsular polysaccharide has been reduced using bactericidal anti-capsular antibodies.

[0112] When the present disclosure involves the use of two or more strains (for example, to produce antigenic compositions containing vesicles presenting the target fHbp from different strains), the strains can be selected such that one or more strain characteristics are different, for example, to result in vesicles with differences in the target fHbp, PorA, etc. used.

[0113] Preparation of vesicles The antigenic compositions contemplated by the present disclosure generally contain vesicles prepared from Neisseria cells expressing the target fHbp. As used herein, the term "vesicles" is intended to encompass outer membrane vesicles and microvesicles (also referred to as blebs).

[0114] The antigenic composition can include outer membrane vesicles (OMVs) prepared from the outer membrane of a cultured strain of the Neisseria meningitidis species genetically modified to express the target fHbp. The OMVs can be obtained from Neisseria meningitidis grown in broth or on solid medium, preferably by separating the bacterial cells from the medium (e.g., by filtration or low-speed centrifugation to pellet the cells), lysing the cells (e.g., by addition of a surfactant, osmotic shock, sonication, cavitation, homogenization, etc.), and separating the outer membrane fraction from cytoplasmic molecules (e.g., by filtration; or by differential precipitation or aggregation of the outer membrane and / or outer membrane vesicles, or by affinity separation using a ligand that specifically recognizes outer membrane molecules; or by high-speed centrifugation to pellet the outer membrane and / or outer membrane vesicles, etc.); the outer membrane fraction can be used to produce OMVs.

[0115] The antigenic composition can comprise microvesicles (MVs) (or "blebs") comprising the subject's fHbp, and the MVs or blebs are released during the culture of a Neisseria meningitidis strain that has been genetically modified to express the subject's fHbp. For example, the MVs can be obtained by culturing a strain of Neisseria meningitidis in a broth medium, separating whole cells from the broth medium (e.g., by filtration or by low-speed centrifugation that pellets only the cells and not the smaller blebs, etc.), and then recovering the MVs present in the cell-free medium (e.g., by filtration, differential precipitation or aggregation of the MVs, or by high-speed centrifugation that pellets the blebs, etc.). The strain for MV production can generally be selected based on the amount of blebs produced under culture (e.g., the bacteria can be cultured in a reasonable number that results in the production of blebs suitable for isolation and administration in the methods described herein). Exemplary strains that produce high concentrations of blebs are described in PCT Publication No. WO01 / 34642. In addition to bleb production, the strain used for MV production can likewise be selected based on NspA production, and strains that produce higher concentrations of NspA can be particularly important (see, e.g., Moe et al. (1999 Infect. Immun. 67: 5664) for examples of N. meningitidis strains having different NspA production concentrations). Other strains of interest for use in bleb production include strains having an inactivated GNA33 gene, which encodes a lipoprotein required for cell separation, membrane structure, and pathogenicity (see, e.g., Adu-Bobie et al. (2004) Infect Immun.72:1914-1919).

[0116] The antigenic composition of the present disclosure can include vesicles from one strain or from 2, 3, 4, 5, or more strains, which may be of the same or different species, and are usually different from each other. For example, the strains may be of the same or different species as PorA and / or fHbp from which the fHbp of the subject is derived. The vesicles may consist of amino acid sequences of fHbp from different variants (v.1, v.2, or v.3) or subvariants (e.g., subvariants of v.1, v.2, or v.3) of a plurality of fHbp of the subject (e.g., 1, 2, 3, or more fHbp of the subject) and can be prepared from strains that express them.

[0117] The antigenic composition can include a mixture of OMVs and MVs presenting the same or different fHbp of the subject, and the fHbp of the subject may optionally present epitopes from different combinations of fHbp variants and / or subvariants. The OMVs and / or MVs may be from the same or different strains. Vesicles from different strains can be administered as a mixture or sequentially.

[0118] If necessary (e.g., when the strain used for vesicle production is associated with endotoxin or a specific high concentration of endotoxin), the vesicles can be optionally treated to reduce endotoxin, e.g., to reduce toxicity after administration. Although not desirable as described below, endotoxin reduction can be achieved by extraction with a suitable surfactant (e.g., BRIJ-96, sodium deoxycholate, sodium lauroyl sarcosinate, Empigen BB, Triton X-100, nonionic surfactant TWEEN 20 (polyoxyethylene sorbitan monolaurate), nonionic surfactant TWEEN 80, at a concentration of 0.1 - 10%, e.g., 0.5 - 2%, and sodium dodecyl sulfate (SDS)). When surfactant extraction is used, it is preferred to use a surfactant other than deoxycholate.

[0119] Vesicles of the antigenic composition can be prepared without a surfactant, for example, without using deoxycholate. Surfactant treatment is useful for removing endotoxin activity, but can deplete the native fHbp lipoprotein and / or the fHbp of interest (including lipidated fHbp) by extraction during vesicle production. Thus, it may be particularly desirable to reduce endotoxin activity using techniques that do not require a surfactant. In one approach, the need to remove endotoxin from the final preparation prior to human use is avoided by using a strain that produces relatively low levels of endotoxin (lipopolysaccharide, LPS). For example, the vesicles can be prepared from Neisseria mutants in which lipooligosaccharide and other vaccines (e.g., Rmp), which may be undesirable in the vaccine, are reduced or removed.

[0120] Vesicles can be prepared from N. meningitidis strains that contain genetic modifications that reduce or abolish the toxic activity of lipid A. For example, such strains can be genetically modified in lipid A biosynthesis (Steeghs et al. (1999) Infect Immun 67:4988-93; van der Ley et al. (2001) Infect Immun 69:5981-90; Steeghs et al. (2004) J Endotoxin Res 10:113-9; Fissha et al, (2005) Infect Immun 73:4070). Immunogenic compositions can be detoxified by modification of LPS, for example, by downregulation and / or inactivation of the enzymes encoded by lpxL1 or lpxL2, respectively. The production of pentaacylated lipid A made by an lpxL1 mutant indicates that the enzyme encoded by lpxL1 adds C12 to the N-linked 3-OH C14 at the 2' position of GlcN II. The major lipid A species seen in an lpxL2 mutant is tetraacylated, indicating that the enzyme encoded by lpxL2 adds another C12, i.e., to the N-linked 3-OH C14 at the 2 position of GlcN I. Mutations that reduce (or abolish) the expression of these genes (or reduce or abolish the activity of these gene products) alter the toxic activity of lipid A (van der Ley et al. (2001) Infect Immun 69:5981-90). Tetraacylated (lpxL2 mutant) and pentaacylated (lpxL1 mutant) lipid A are less toxic than wild-type lipid A. Mutations in the gene (lpxK) encoding lipid A 4'-kinase also reduce the toxic activity of lipid A. Of particular importance for use in the production of vesicles (e.g., MV or OMV) are N. meningitidis strains that have been genetically modified to result in reduced or undetectable functional LpxL1-encoding protein, e.g., Neisseria bacteria (e.g., N. meningitidis strains) are genetically modified to reduce or abolish the activity of the gene product of the lpxL1 gene.For example, Neisseria bacteria can be genetically modified to have an lpxL1 gene knockout, for example, the lpxL1 gene is disrupted. See, for example, U.S. Patent Application Publication No. 2009 / 0035328. Neisseria bacteria can be genetically modified to reduce or eliminate the activity of the gene product of the lpxL2 gene. Neisseria bacteria can be genetically modified to reduce or eliminate the activity of the gene products of the lpxL1 gene and the lpxL2 gene. Such vesicles are less toxic compared to N. meningitidis strains that are wild-type for LPS production while retaining the immunogenicity of the fHbp of interest.

[0121] The toxic activity of LPS can also be altered by introducing mutations into genes / loci involved in polymyxin B resistance (such resistance has been associated with the addition of aminoarabinose to the 4'-phosphate of lipid A). These genes / loci can be pmrE, which encodes UDP-glucose dehydrogenase, or can be an antimicrobial peptide resistance gene region common to many Enterobacteriaceae that can be involved in aminoarabinose synthesis and transfer. The pmrF gene present in this region encodes a dolicol-phosphate manosyl transferase (Gunn J. S., Kheng, B. L., Krueger J., Kim K., Guo L., Hackett M., Miller S. I. 1998. Mol. Microbiol. 27: 1171-1182).

[0122] Mutations in the PhoP-PhoQ regulatory system, which is a two-component phosphorelay regulatory system (e.g., PhoP constitutive phenotype, PhoPc), or low Mg ++The environment or culture conditions (which activate the PhoP-PhoQ regulatory system) lead to the addition of 4'-phosphate to aminoarabinose and the substitution of myristate with 2-hydroxymyristate (hydroxylation of myristate). This modified lipid A exhibits a reduced ability to stimulate E-selectin expression by human endothelial cells and TNF secretion from human monocytes.

[0123] Polymyxin B-resistant strains are also suitable for use because such strains have been shown to have reduced LPS toxicity (see, for example, van der Ley et al. (1994): Proceedings of the ninth international pathogenic Neisseria conference. The Guildhall, Winchester, England). Alternatively, a synthetic peptide that mimics the binding activity of polymyxin B may be added to the antigenic composition to reduce the toxic activity of LPS (see, for example, Rustici et al. (1993) Science 259:361-365; Porro et al. (1998) Prog Clin Biol Res.397:315-25).

[0124] Endotoxin can also be reduced by the choice of culture conditions. For example, culturing the strain in a growth medium containing 0.1 mg to 100 mg of aminoarabinose per liter of medium reduces the toxicity of the lipid (see, for example, WO02 / 097646).

[0125] Compositions and formulations "Composition", "antigen composition", "antigenic composition", or "immunogenic composition" are used herein, for convenience, generically to refer to a composition containing an fHbp of the subject matter disclosed herein, where the fHbp of the subject matter may optionally be conjugated to further enhance immunogenicity. In humans, compositions useful for inducing antibodies, e.g., anti-Neisseria meningitidis antibodies, e.g., bactericidal antibodies against Neisseria meningitidis are particularly contemplated by the present disclosure. An antigenic composition can contain one, two, or more different subject fHbps. Where there are two or more fHbps, each subject fHbp can present epitopes from different combinations of fHbp variants and / or subvariants.

[0126] An antigenic composition contains an immunologically effective amount of a subject fHbp and may further contain other compatible components as needed. The compositions of the present disclosure can contain an fHbp that is a low fH binder. The composition contains one or more fHbps where at least one fHbp is a low fH binder. Where there are two or more fHbps in the composition, each fHbp can be different (e.g., in amino acid sequence and / or binding).

[0127] In some examples, the antigenic compositions of the present disclosure contain only one fHbp variant of the present disclosure. In some examples, the antigenic compositions of the present disclosure contain two or more different fHbp variants of the present disclosure. By way of non-limiting example, in some examples, the antigenic compositions of the present disclosure contain: (1) a first variant of fHbp ID1, the first fHbp ID1 variant containing an amino acid substitution at Q38 (e.g., Q38R); and a second variant of fHbp ID1, the second fHbp ID1 variant containing an amino acid substitution at E92 (e.g., E92K); (2) a first variant of fHbp ID1, the first fHbp ID1 variant containing an amino acid substitution at Q38 (e.g., Q38R); and a second variant of fHbp ID1, the second fHbp ID1 variant containing an amino acid substitution at R130 (e.g., R130G); (3) A first variant of fHbp ID1, the first fHbp ID1 variant comprising an amino acid substitution at Q38 (e.g., Q38R); and a second variant of fHbp ID1, the second fHbp ID1 variant comprising an amino acid substitution at S223 (e.g., S223R); (4) A first variant of fHbp ID1, the first fHbp ID1 variant comprising an amino acid substitution at Q38 (e.g., Q38R); and a second variant of fHbp ID1, the second fHbp ID1 variant comprising an amino acid substitution at H248 (e.g., H248L); (5) A variant of fHbp ID22, the fHbp ID22 variant comprising an amino acid substitution at N115 (e.g., N115I); and a variant of fHbp ID1, the fHbp ID1 variant comprising an amino acid substitution at Q38 (e.g., Q38R); (6) A variant of fHbp ID22, the fHbp ID22 variant comprising an amino acid substitution at D121 (e.g., D121G); and a variant of fHbp ID1, the fHbp ID1 variant comprising an amino acid substitution at E92 (e.g., E92K); (7) A variant of fHbp ID22, the fHbp ID22 variant comprising an amino acid substitution at S128 (e.g., S128T); and a variant of fHbp ID1, the fHbp ID1 variant comprising an amino acid substitution at H248 (e.g., H248L); (8) A variant of fHbp ID22, the fHbp ID22 variant comprising an amino acid substitution at V131 (e.g., V131D); and a variant of fHbp ID1, the fHbp ID1 variant comprising an amino acid substitution at Q38 (e.g., Q38R); (9) A variant of fHbp ID22, the fHbp ID22 variant comprising an amino acid substitution at K219 (e.g., K219N); and a variant of fHbp ID1, the fHbp ID1 variant comprising an amino acid substitution at Q38 (e.g., Q38R); (10) Variants of fHbp ID22 comprising an amino acid substitution at G220 (e.g., G220S); and variants of fHbp ID1 comprising an amino acid substitution at Q38 (e.g., Q38R); (11) Variants of fHbp ID22 comprising an amino acid substitution at N115 (e.g., N115I); and variants of fHbp ID55 comprising an amino acid substitution at E92 (e.g., E92K); (12) Variants of fHbp ID22 comprising an amino acid substitution at D121 (e.g., D121G); and variants of fHbp ID55 comprising an amino acid substitution at S223 (e.g., S223R); (13) Variants of fHbp ID22 comprising an amino acid substitution at S128 (e.g., S128T); and variants of fHbp ID55 comprising an amino acid substitution at H248 (e.g., H248L); (14) Variants of fHbp ID22 comprising an amino acid substitution at V131 (e.g., V131D); and variants of fHbp ID55 comprising an amino acid substitution at E92 (e.g., E92K); (15) Variants of fHbp ID22 comprising an amino acid substitution at K219 (e.g., K219N); and variants of fHbp ID55 comprising an amino acid substitution at E92 (e.g., E92K); (16) Variants of fHbp ID22 comprising an amino acid substitution at G220 (e.g., G220S); and variants of fHbp ID55 comprising an amino acid substitution at E92 (e.g., E92K); (17)A first variant of fHbp ID1, the first fHbp ID1 variant comprising an amino acid substitution at E92 (e.g., E92K); and a second variant of fHbp ID1, the second fHbp ID1 variant comprising an amino acid substitution at H248 (e.g., H248L); (18)A first variant of fHbp ID1, the first fHbp ID1 variant comprising an amino acid substitution at E92 (e.g., E92K); and a second variant of fHbp ID1, the second fHbp ID1 variant comprising an amino acid substitution at S223 (e.g., S223R); (19)A first variant of fHbp ID22, the first fHbp ID22 variant comprising an amino acid substitution at N115 (e.g., N115I); and a second variant of fHbp ID22, the second fHbp ID22 variant comprising an amino acid substitution at D211 (e.g., D211A); (20)A first variant of fHbp ID22, the first fHbp ID22 variant comprising an amino acid substitution at N115 (e.g., N115I); and a second variant of fHbp ID22, the second fHbp ID22 variant comprising an amino acid substitution at K219 (e.g., K219N); (21)A first variant of fHbp ID22, the first fHbp ID22 variant comprising an amino acid substitution at N115 (e.g., N115I); and a second variant of fHbp ID22, the second fHbp ID22 variant comprising an amino acid substitution at G220 (e.g., G220S); (22)A first variant of fHbp ID22, the first fHbp ID22 variant comprising an amino acid substitution at D121 (e.g., D121G); and a second variant of fHbp ID22, the second fHbp ID22 variant comprising an amino acid substitution at G220 (e.g., G220S); (23)A first variant of fHbp ID22, the first fHbp ID22 variant comprising an amino acid substitution at S128 (e.g., S128T); and a second variant of fHbp ID22, the second fHbp ID22 variant comprising an amino acid substitution at G220 (e.g., G220S); (24) A first variant of fHbp ID22, which is a first fHbp ID22 variant comprising an amino acid substitution at V131 (e.g., V131D); and a second variant of fHbp ID22, which is a second fHbp ID22 variant comprising an amino acid substitution at G220 (e.g., G220S); (25) A first variant of fHbp ID55, which is a first fHbp ID55 variant comprising an amino acid substitution at E92 (e.g., E92K); and a second variant of fHbp ID55, which is a second fHbp ID55 variant comprising an amino acid substitution at S223 (e.g., S223R); (26) A first variant of fHbp ID55, which is a first fHbp ID55 variant comprising an amino acid substitution at E92 (e.g., E92K); and a second variant of fHbp ID55, which is a second fHbp ID55 variant comprising an amino acid substitution at H248 (e.g., H248L); (27) A variant of fHbp ID22 comprising amino acid substitutions L130R and G133D and a variant of fHbp ID1, which is a fHbp ID1 variant comprising an amino acid substitution at S223 (e.g., S223R); (28) A variant of fHbp ID22 comprising amino acid substitutions L130R and G133D and a variant of fHbp ID1, which is a fHbp ID1 variant comprising an amino acid substitution at H248 (e.g., H248L); (29) A variant of fHbp ID22 comprising amino acid substitutions L130R, G133D, and K219N and a variant of fHbp ID1, which is a fHbp ID1 variant comprising an amino acid substitution at S223 (e.g., S223R) or an amino acid substitution at H248 (e.g., H248L); or (30) A variant of fHbp ID22 comprising amino acid substitutions L130R, G133D, and G220S and a variant of fHbp ID1, which is a fHbp ID1 variant comprising an amino acid substitution at S223 (e.g., S223R) or an amino acid substitution at H248 (e.g., H248L).

[0128] Immunogenic compositions contemplated by the present disclosure include, but are not limited to, (1) At least one variant fHbp of the present disclosure; and (2) A composition comprising NspA are provided, where fHbp and / or NspA can be provided as a recombinant protein and / or in a vesicle-based composition (e.g., OMV or MV). It should be noted that when the composition contains both NspA and fHbp, the bactericidal activity of the antibodies induced by administration of the composition may be due to the cooperation of the antibodies against one or both antigens. Examples of the immunogenic compositions provided by the present disclosure include (a) An immunogenic composition comprising the above fHbp variant (e.g., when the variant fHbp induces a bactericidal antibody response against at least one strain of Neisseria meningitidis); (b) An immunogenic composition comprising the above fHbp variant (e.g., when the variant fHbp induces a bactericidal antibody response against at least one strain of Neisseria meningitidis) and a recombinant NspA protein; (c) A native OMV obtained from a genetically modified Neisseria host cell genetically modified with a nucleic acid encoding a variant fHbp of the present disclosure such that the encoded variant fHbp is produced by the genetically modified host cell, the OMV comprising the encoded variant fHbp comprising, an immunogenic composition; and (d) A native OMV obtained from a genetically modified Neisseria host cell genetically modified with a nucleic acid encoding a variant fHbp of the present disclosure such that the encoded non-naturally occurring fHbp is produced by the genetically modified host cell, the OMV comprising the encoded variant fHbp comprising, an immunogenic composition wherein the Neisseria host cell also produces a high concentration of NspA such that the OMV also contains NspA, a composition is provided. For example, the Neisseria host cell can be one that is genetically modified to increase the expression of NspA.

[0129] "An immunologically effective amount" means that in a single administration as part of a series of identical or different antigenic compositions, administration of that amount to an individual is effective to induce an antibody response effective for the treatment or protection against the symptoms of infection or the disease caused by infection, for example, by Neisseria, particularly N. meningitidis, more particularly group B of N. meningitidis. This amount will vary depending on the health and physical condition of the individual to be treated, age, the antibody-producing ability of the individual's immune system, the degree of protection desired, the formulation of the vaccine, the judgment of the treating clinician's medical situation, and other relevant factors. It is expected that the amount will fall within a relatively wide range that can be determined by routine testing.

[0130] The amino acid sequence of the NspA polypeptide is known in the art. See, for example, WO96 / 29412; and Martin et al. (1997) J. Exp. Med. 185:1173; GenBank accession number U52066; and GenBank accession number AAD53286. "NspA polypeptide" can include a continuous stretch of from about 75 amino acids to about 100 amino acids, from about 100 amino acids to about 150 amino acids, or from about 150 amino acids to about 174 amino acids of the amino acid sequence shown in FIG. 40 and set forth in SEQ ID NO:25, and an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity. "NspA polypeptide" can include a continuous stretch of from about 75 amino acids to about 100 amino acids or from about 100 amino acids to about 155 amino acids of amino acids 20-174 of the amino acid sequence shown in FIG. 40 and set forth in SEQ ID NO:25, and an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity. In some examples, the NspA polypeptide lacks a signal sequence; in other examples (e.g., for expression in a host cell), the NspA polypeptide includes a signal sequence.

[0131] The dosing regimen may be a single-dose schedule or a multiple-dose schedule (e.g., including a booster dose) in which the antigenic composition in a unit dosage form is administered at different times. As used herein, the term "unit dosage form" refers to physically discrete units suitable as a single dosage for human and animal subjects, each unit containing a predetermined amount of the antigenic composition of the present disclosure in an amount sufficient to produce the desired effect, and the composition is provided with a pharmaceutically acceptable excipient (e.g., a pharmaceutically acceptable diluent, carrier, or vehicle). The antigenic composition may be administered in combination with other immunomodulatory agents.

[0132] The antigenic composition may be provided in a pharmaceutically acceptable excipient, which may often be a solution such as a sterile aqueous solution that is physiological saline, or it may be provided in powder form. Such excipients may be substantially inert, if necessary.

[0133] In some embodiments, the immunogenic composition of the subject comprises the subject's fHbp present in vesicles. In some embodiments, the immunogenic composition of the subject comprises the subject's fHbp present in MVs. In some embodiments, the immunogenic composition of the subject comprises the subject's fHbp present in OMVs. In some embodiments, the immunogenic composition of the subject comprises a mixture of MVs and OMVs containing the subject's fHbp. Vesicles such as MVs and OMVs are described above.

[0134] The antigenic composition can further contain an adjuvant. Examples of known suitable adjuvants that can be used in humans include, but are not necessarily limited to, aluminum adjuvants (e.g., aluminum phosphate, or aluminum hydroxide), MF59 (4.3% w / v squalene, 0.5% w / v Tween80™, 0.5% w / v Span85), CpG-containing nucleic acids (wherein the cytosine is not methylated), QS21, MPL, 3DMPL, Aquilla extract, ISCOMs, LT / CT mutants, poly(D,L-lactide-co-glycolide) (PLG) microparticles, Quil A, interleukin, etc. For experimental animals, Freund's adjuvant (incomplete Freund's adjuvant; complete Freund's adjuvant), N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP11637, called nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)ethylamine (CGP19835A, called MTP-PE), and RIBI containing monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS), which are three components extracted from bacteria in a 2% squalene / Tween80 emulsion, can be used. The effectiveness of the adjuvant can be determined by measuring the amount of antibody against the immunogenic antigen or its antigenic epitope.

[0135] Additional exemplary adjuvants for enhancing the effectiveness of the composition include, but are not limited to, (1) oil-in-water emulsion formulations (with or without other specific immunopotentiating substances such as muramyl peptides (see below) or bacterial cell wall components), for example, (a) MF59 which contains 5% squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing MTP-PE) and is formed into submicron particles using a microfluidizer (WO90 / 14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995), (b) SAF which contains 10% squalene, 0.4% Tween 80, 5% pluronic block polymer L121, and thr-MDP and is microfluidized into a submicron emulsion or vortexed to produce an emulsion with a larger particle size, and (c) the RIBI adjuvant system (RAS) (Ribi Immunochem; Hamilton, Montana) which contains 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components, such as monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), for example, MPL + CWS (Detox (trademark)); (2) saponin adjuvants, such as QS21 or Stimulon (trademark) (Cambridge Bioscience, Worcester, Massachusetts) may be used or particles formed therefrom, such as ISCOM (immunostimulating complex) which may be without additional surfactant, for example, WO00 / 07621; (3) complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA); (4) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (WO99 / 44636), etc.), interferons (e.g., gamma interferon), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.;(5) Monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL), for example, GB-2220221, EP-A-0689454, and when used optionally with pneumococcal saccharides, in the substantial absence of alum, for example, WO00 / 56358; (6) Combinations of 3dMPL with, for example, QS21 and / or oil-in-water emulsions, for example, EP-A-0835318, EP-A-0735898, EP-A-0761231; (7) Oligonucleotides containing CpG motifs (see, for example, WO98 / 52581), for example, oligonucleotides containing at least one CG dinucleotide and in which cytosine is not methylated; (8) Polyoxyethylene ethers or polyoxyethylene esters (see, for example, WO99 / 52549); (9) Combinations of polyoxyethylene sorbitan ester surfactants and octoxynol (WO01 / 21207) or combinations of polyoxyethylene alkyl ether or ester surfactants and at least one additional nonionic surfactant such as octoxynol (WO01 / 21152); (10) Saponin and immunostimulatory oligonucleotides (for example, CpG oligonucleotides) (WO00 / 62800); (11) Immunostimulants and metal salt particles, for example, WO00 / 23105; (12) Saponin and oil-in-water emulsions, for example, WO99 / 11241; (13) Saponin (for example, QS21) + 3dMPL + IM2 (optionally + sterol), for example, WO98 / 57659; (14) Other substances that act as immunostimulants to enhance the effectiveness of the composition. Examples of muramyl peptides include N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)ethylamine MTP-PE), etc. Adjuvants suitable for administration to humans are particularly important. In some examples, the adjuvant is an aluminum salt adjuvant (for example, aluminum phosphate or aluminum hydroxide).;

[0136] The antigen composition may contain other components, for example, pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium, carbonate, etc. The composition may contain pharmaceutically acceptable auxiliary substances such as pH adjusters and buffers, toxicity adjusters, etc., for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc., as necessary to approach physiological conditions.

[0137] The concentration of fHbp in the formulation of the subject can vary widely (for example, from less than about 0.1% by weight, for example, from about 2% by weight or at least about 2% by weight to 20% - 50% by weight or more), and is usually selected mainly based on the liquid volume, viscosity, and patient-based factors depending on the specific administration method selected and the needs of the patient.

[0138] The fHbp-containing formulation can be provided in the form of solutions, suspensions, tablets, pills, capsules, powders, gels, creams, lotions, ointments, aerosols, etc. It is recognized that oral administration may require protection of the composition from digestion. This is usually achieved either by associating the composition with an agent that renders it resistant to acid and enzymatic hydrolysis or by appropriately packaging the composition in a durable carrier. Means for protecting from digestion are well known in the art.

[0139] The fHbp-containing formulation can also be provided to extend the serum half-life of fHbp after administration. For example, when isolated fHbp is formulated for injection, the fHbp may be provided as a colloid in a liposome formulation or by other conventional techniques for extending the serum half-life. As described in Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Patent Nos. 4,235,871, 4,501,728, and 4,837,028, various methods are available for the preparation of liposomes. These preparations can also be provided in controlled-release or sustained-release forms.

[0140] Method for inducing an immune response The present disclosure provides a method for inducing an immune response against at least one strain of Neisseria in a mammalian host. The method generally comprises administering an effective amount of an immunogenic composition of interest to an individual in need thereof.

[0141] fHbp-containing antigenic compositions are generally administered to human subjects at risk of developing Neisseria disease to prevent or at least partially arrest the onset of the disease and its complications. The amount appropriate to achieve this is defined as a "therapeutically effective dose". The amount effective for therapeutic use will depend, for example, on the antigenic composition, the method of administration, the weight and general health of the patient, and the judgment of the prescribing physician. Single or multiple administrations of the antigenic composition can be administered depending on the dose and frequency required and tolerated by the patient and the method of administration.

[0142] fHbp-containing antigenic compositions are generally administered in an amount effective to induce an immune response, particularly a humoral immune response, such as a bactericidal antibody response, in the host. As noted above, the amount for immunization varies and can generally range from about 1 μg to 100 μg per 70 kg patient, usually 5 μg to 50 μg / 70 kg. Considerably higher doses (e.g., 10 mg to 100 mg or more) may be suitable for oral, nasal, or topical routes of administration. After the initial administration, booster immunizations with the same antigenic composition containing different fHbps can be performed. In some examples, vaccination includes at least one booster immunization, and in some examples, two booster immunizations.

[0143] Generally, immunization can be achieved by administering the composition orally, nasally, nasopharyngeally, parenterally, enterally, gastrally, locally, transdermally, subcutaneously, intramuscularly, in any suitable route including, in the form of tablets, solids, powders, liquids, aerosols, locally or systemically, with or without added excipients. Practical methods for preparing compositions administrable parenterally are known or apparent to those skilled in the art and are described in more detail by publications such as Remington’s Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980).

[0144] The immune response against fHbp can be evaluated by known methods (e.g., by collecting sera from an individual before and after primary immunization and showing the change in the immune state of the individual, e.g., by immunoprecipitation assay, ELISA, or bactericidal assay, Western blot assay, or flow cytometry assay, etc.).

[0145] Whether the variant fHbp of the present disclosure induces a bactericidal response against one or more strains of N. meningitidis in a mammalian host can be determined using any well-known assay. For example, a human fH transgenic mouse expressing human fH (e.g., where human fH is present in the serum of the mouse at a concentration of about 100 μg / ml or more than 100 μg / ml) can be used. The variant fHbp of the present disclosure is administered to the human fH transgenic mouse. After a certain period of time, the serum derived from the mouse is tested for bactericidal activity against one or more strains of N. meningitidis. Suitable controls include, for example, fHbp ID1. Examples of suitable assays are described in Vu et al. (2012) Sci. Reports 2:341.

[0146] The antigenic composition can be administered to a mammalian host (e.g., a human subject) that is immunologically naive with respect to Neisseria meningitidis. In certain embodiments, the subject is a human infant less than about 5 years old, preferably less than about 2 years old, and the antigenic composition is administered one or more times at any of the following time points: 2 weeks, 1 month, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 months, or 1 year or 15, 18, or 21 months, or 2, 3, 4, or 5 years of age.

[0147] Generally, it may be desirable to initiate immunization prior to the initial signs of disease symptoms or at the time of initial signs of potential or actual exposure to infection or disease (e.g., due to exposure or infection by Neisseria).

Examples

[0148] The following examples are described to provide a complete disclosure and description of the methods of making and using the invention to those skilled in the art, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Attempts have been made to ensure accuracy with respect to the numerical values used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations, e.g., bp, base pair; kb, kilobase; pl, picoliter; s or sec, second; min, minute; h or hr, hour; aa, amino acid; kb, kilobase; bp, base pair; nt, nucleotide; i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); etc. may be used.

[0149] Example 1: Identification and characterization of fHbp ID1 variant Materials and Methods Library Screening A random mutant fHbp library was generated by error-prone polymerase chain reaction (PCR), followed by cloning the PCR products into a pET28 expression plasmid containing a signal sequence that enables surface display in E. coli. Using fluorescence-activated cell sorting, mutant clones with low binding of human fH and high binding of a control anti-fHbp monoclonal antibody were isolated to ensure sufficient expression and proper folding of the fHbp mutants. The recovered cells were seeded on an agar plate (LB agar containing 50 μg / ml kanamycin sulfate) and incubated overnight at 37°C. A single E. coli colony was used as a template for PCR amplification, and the DNA amplification product was purified (PCR purification kit; Qiagen) and subjected to DNA sequencing of the fHbp gene using primers that anneal to the T7 promoter and T7 terminator. This screening method for the random mutant fHbp library has the potential to identify (1) positions that act on fH binding that cannot be predicted from its crystal structure alone; and (2) substitutions other than alanine that act on fH binding when alanine substitution does not result in a sufficient decrease in fH binding.

[0150] Selection of mutants for detailed study The positions of the amino acid substitutions identified in the FACS experiment were examined in the crystal structure in the complex of fHbp and the human fH fragment. Mutants that were proximal (<5 Å) to the fH binding interface were selected for site-directed mutagenesis. Iterative generation of mutants of the library by site-directed mutagenesis was necessary for the production of soluble recombinant fHbp protein for further characterization. This approach also eliminated unwanted secondary mutations that were present in many of these selected clones and were distant from the fH binding site. This site-directed mutant was constructed using the Phusion site-directed mutagenesis kit (Thermo Scientific, Inc.).

[0151] Expression and purification of soluble mutant fHbp Soluble recombinant fHbp was expressed in E. coli, and the lysate was prepared as described above. fHbp was purified by nickel affinity chromatography using a HiTrap Chelating HP column (5 ml; GE Life Sciences, Inc.) and an Akta Purifier chromatography system (GE Life Sciences). The binding and elution buffers using an imidazole gradient were prepared according to the protocol of the column manufacturer. The fractions containing purified fHbp were combined, dialyzed against PBS containing 3% sucrose, and stored at -80 °C until use.

[0152] For the examination of immunogenicity in mice, a second purification step using ion exchange chromatography on a HiTrap SP HP column (5 ml; GE Life Sciences) was performed. These binding and elution buffers were 25 mM MES at pH 5.5 containing 150 mM and 750 mM NaCl, respectively. The bound fHbp was eluted from this SP column with a linear gradient formed by these binding and elution buffers. The fractions containing purified fHbp were combined, dialyzed against PBS containing 3% sucrose, and stored at -80 °C until use.

[0153] Purification of human factor H (fH) Human fH was purified using an fHbp affinity column. This column was prepared by binding 5 mg of fHbp ID1 to an NHS-activated HP column (5 ml; GE Life Sciences) using the manufacturer's protocol. Human serum from healthy donors was diluted 1:1 with phosphate-buffered saline (PBS). This serum was loaded onto the column, and the column was washed with 10 column volumes (i.e., 50 ml) of PBS. The bound fH was eluted with 5 column volumes of 0.1 M glycine-HCl at pH 2.7. The eluted fractions were collected into tubes containing 50 μl of 1 M Tris-HCl at pH 9.0. Fractions containing fH were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (4–12% NuPAGE; Invitrogen). Electrophoresis was performed at 200 V for 45 minutes using 1×MES running buffer (Invitrogen). These proteins were visualized by staining with Coomassie G-250 (SimplyBlue SafeStain; Invitrogen). Fractions containing fH were pooled, dialyzed against PBS, and the aliquots of fH were stored at -30 °C until use.

[0154] Characterization of fHbp mutants SDS-PAGE. The size and purity of the purified fHbp mutant proteins were evaluated by SDS-PAGE using a 4–12% polyacrylamide gradient gel (NuPAGE; Invitrogen, Inc.). 2 μg of each protein was loaded onto this gel. SDS-PAGE for fH was performed as described above.

[0155] Binding of fH to fHbp by enzyme-linked immunosorbent assay (ELISA). Wells of a 96-well microtiter plate (Immulon 2HB; Thermo Scientific) were coated with 2 μg / ml of purified recombinant wild-type fHbp (positive control) or mutant fHbp (experiment). Nonspecific binding to these wells was blocked with PBS containing 1% BSA (Lifeblood Medical, Inc.) or 5% non-fat dry milk (Carnation; Nestle, Inc.). Purified human fH serially diluted 5-fold in the range of 25 to 0.0016 μg / ml in dilution buffer (PBS containing 0.1% Tween-20, 0.01% sodium azide, and 1% BSA) was added to these wells, and the plate was incubated at room temperature for 2 hours. After washing 3 times with PBS containing 0.1% Tween-20 (Sigma) and 0.01% sodium azide (Sigma), bound fH was detected with sheep anti-human fH (1:7,000; Abcam, Inc.) in dilution buffer. The plate was incubated at room temperature for 1 hour. After washing these wells again, the bound primary antibody was detected with alkaline phosphatase-conjugated donkey anti-sheep IgG (1:5,000; Sigma-Aldrich, Inc.) in dilution buffer. The plate was incubated at room temperature for 1 hour and these wells were washed again. This ELISA was developed using a substrate for phosphatase (1 mg / ml p-nitrophenyl phosphate; Sigma) in substrate buffer (50 mM sodium carbonate, 1 mM MgCl2, pH 9.8). After incubation at room temperature for 30 minutes, the absorbance at 405 nm was measured with a UV-VIS plate reader (Spectromax 190; Molecular Devices, Inc.).

[0156] Binding of anti-fHbp monoclonal antibody to fHbp by ELISA. Wells of a microtiter plate were coated with fHbp and blocked and washed as described above for the fH ELISA. Mouse anti-fHbp monoclonal antibody (mAb) serially diluted 5-fold from 25 to 0.0016 μg / ml in dilution buffer was added, and the plate was incubated at room temperature for 1 hour. After washing these wells, the primary antibody was detected with alkaline phosphatase-conjugated goat anti-mouse IgG (1:5,000; Sigma-Aldrich). The ELISA was developed and read as described above.

[0157] Binding of fH to fHbp by surface plasmon resonance (SPR). SPR experiments were performed on a Biacore X100 Plus instrument (GE Life Sciences). 3000 response units of purified human fH were coupled to a CM5 chip (GE Life Sciences) using an amine coupling kit (GE Life Sciences). fH was immobilized in flow cell 2, and a blank immobilization (without fH) was performed in flow cell 1 for reference. The chip surface was conditioned with three startup cycles consisting of HEPES buffered saline containing 3 mM EDTA and 0.05% Surfactant P-20 (GE Life Sciences) and regeneration with 100 mM glycine, 3 M NaCl, pH 2.0. Dilutions of purified recombinant fHbp ranging from 100 to 1 nM (wild type) or 316 to 3.16 nM were injected for 150 seconds. Dissociation was monitored for 300 seconds, and these data were analyzed with Biacore X100 evaluation software.

[0158] Immunogenicity in mice. Wild-type CD-1 mouse groups (N = 14 - 21) were immunized with fHbp vaccine adsorbed to aluminum hydroxide. Each dose of the vaccine contained 10 μg of fHbp and 600 μg of Alhydrogel (Brenntag Biosector) in 10 mM histidine, 150 mM NaCl, pH 6.5. Two doses were given 3 weeks apart, and blood was collected by cardiac puncture 3 weeks after the second administration. The blood was processed to obtain serum, which was maintained at -80 °C for long-term storage (> 2 weeks) or 4 °C for short-term storage (< 2 weeks).

[0159] Human fH transgenic BALB / c mice were first screened, and animals with a human fH concentration in serum > 240 μg / ml were identified using an fHbp ELISA and a standard curve of purified human fH. This ELISA was performed using purified fHbp ID1 immobilized on the plate, and the primary and secondary antibodies for detecting fH were the same as above (see "Binding of fH to fHbp by ELISA").

[0160] Transgenic mouse groups (N = 11 - 21) were immunized with fHbp vaccine adsorbed to aluminum hydroxide (the same amount of antigen and adjuvant as for the wild-type CD-1 mice above). Three doses were administered at 3-week intervals, and blood was collected 3 weeks after the third vaccine administration. The serum was processed and stored as described above.

[0161] Serum bactericidal antibody (SBA) response. The human complement-mediated SBA response was measured against meningococcal strains with fHbp sequences identical or nearly identical to each vaccine antigen. These bacteria were grown to mid-log phase (OD620nm = 0.6) in standard Frasch medium (Frasch et al. “Outer membrane protein vesicle vaccines for meningococcal disease.” In Methods in Molecular Medicine, v. 66. Meningococcal Vaccines: Methods and Protocols. Edited by Pollard, A.J. and Maiden, M.C. Humana Press Inc. Totowa, NJ) containing 4 mM lactate and 0.02 mM CMP-NANA. These bacteria were diluted 1:25,000 in Dulbecco's PBS (Equitech Bio.) containing 1% BSA. Human complement was from donors without endogenous bactericidal antibodies and IgG antibodies were depleted using a HiTrap Protein G column (5 ml; GE Life Sciences). Each reaction contained 25% human complement, approximately 400 cfu of bacteria, and a dilution of the test antiserum or control antibody. The SBA titer was calculated as the serum dilution at which cfu decreased by 50% compared to negative control wells after incubation at 37°C for 60 minutes. Protein purification: Recombinant fHbp was expressed in E. coli with a C-terminal hexahistidine tag and purified by metal chelate chromatography (HiTrap Chelating HP; GE Life Sciences) followed by ion exchange chromatography (HiTrap SP; GE Life Sciences). These proteins (2 μg each) were separated on a 4–12% NuPAGE gel (Invitrogen) using MES running buffer (Invitrogen) and visualized by Coomassie blue staining (Simply Blue Safe Stain; Invitrogen).

[0162] Results We developed a method based on a random mutant library and identified fHbp mutants with reduced binding to human fH. This method was able to identify mutations that led to reduced binding of fH, which may not be predictable based on structural information alone, and was also able to cause multiple amino acid substitutions at any given position. This method contrasts with the alanine substitution method at common selection positions that sometimes results in a small decrease in fH binding.

[0163] Using this random mutant library method, we identified five promising new fHbp ID1 mutants. These purified recombinant mutant fHbp ID1 antigens Q38R, E92K, R130G, S223R, and H248L are shown in Figure 1.

[0164] Figure 1. Purity of fHbp ID1 mutants. Recombinant fHbp was expressed in E. coli with an N-terminal hexahistidine tag and purified by metal chelate chromatography (HiTrap Chelating HP; GE Life Sciences) followed by ion exchange chromatography (HiTrap SP; GE Life Sciences). These proteins (2 μg each) were separated on a 4–12% NuPAGE gel (Invitrogen) using MES running buffer (Invitrogen) and visualized by Coomassie blue staining (Simply Blue Safe Stain; Invitrogen). Lane 1, Kaleidoscope molecular weight marker (Bio-Rad Laboratories); 2, fHbp ID1 wild type; 3, Q38R; 4, E92K; 5, R130G; 6, S223R; 7, H248L.

[0165] These mutants showed a decrease in fH binding ranging from approximately 10-fold (R130G) to approximately 20-fold (Q38R) to approximately 100-fold (E92K, S223R, and H248L) (Figure 2).

[0166] Figures 2A and 2B. fH binding of fHbp ID1 variants by ELISA. Wells of microtiter plates were coated with purified recombinant ID 1 wild-type (WT) or one of six different variant proteins. Different concentrations of purified human fH were added to these wells. Bound fH was detected with sheep anti-human fH (Abcam) and alkaline phosphatase-conjugated donkey anti-sheep IgG (Sigma). A, fHbp ID1 wild-type (WT) protein, a positive control with high binding of human fH. fHbp ID1 R41S variant, a negative control with low binding of fH. B, New fHbp ID1 variants with reduced binding of fH. The R130G variant showed moderate binding of fH, Q38R showed low binding, and E92K, S223R, and H248L showed significantly lower binding than that of R41S. Means and standard deviations of repeated measurements are shown.

[0167] A similar pattern of reduced binding of fH to these variant proteins, in which the R130G and Q38R variants showed some binding and the other three variants showed no detectable binding, was obtained in surface plasmon experiments. (Figures 3A and 3B).

[0168] Figures 3A - 3E. fH binding of fHbp ID1 variants by surface plasmon resonance. 3000 response units of purified human fH were immobilized on a CM5 chip (GE Life Sciences), and 316 nM of purified recombinant fHbp was injected for 150 seconds. For reference, the same data for the ID 1 wild-type (WT) protein are shown in each panel. The same pattern of binding as in ELISA (Figure 2, above); moderate binding to fH for the R130G variant, low binding for Q38R, and very low binding for E92K, S223R, and H248L were observed. All experiments were performed using HBS-EP running buffer and a Biacore X100 Plus surface plasmon resonance instrument. Data were analyzed with Biacore X100 evaluation software.

[0169] All five of these mutant fHbp ID1 proteins retained the conformational epitopes recognized by anti-fHbp monoclonal antibodies. The concentration-dependent binding of five anti-fHbp monoclonal antibodies to wild-type or mutant fHbp is shown in Fig. 4.

[0170] Figs. 4A-4E. Binding of mouse anti-fHbp monoclonal antibodies to fHbp mutant proteins measured by ELISA. Similar concentration-dependent binding of the anti-fHbp monoclonal antibodies suggested that these wild-type and mutant fHbps were present in similar amounts in the wells of the microtiter plate and that these mutant fHbps retained the conformational epitopes recognized by five different monoclonal antibodies. The secondary antibody was alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma). The mean and standard deviation of repeated measurements are shown.

[0171] These mutant proteins also retained similar thermal stability to wild-type fHbp ID1, except for the E92K mutant, which had slightly reduced stability. Finally, these mutants induced similar bactericidal antibody responses in wild-type CD-1 mice when tested against serogroup B strain H44 / 76 (Figs. 5A-5B).

[0172] Figures 5A and 5B. Bactericidal antibody responses to fHbp ID1 variants in mice. Figure 5A, Groups of 12 - 14 wild - type mice were immunized by intraperitoneal administration of 2 doses of purified recombinant fHbp (10 μg per dose) at 3 - week intervals. Three weeks after the second dose, sera were collected. The bactericidal activity of the sera was measured using human serum depleted of IgG as a complement source and serum group B strain H44 / 76 as a test strain. H44 / 76 expresses fHbp ID1, which is identical to the control fHbp ID1 WT vaccine. Each symbol represents the titer of an individual mouse, and the horizontal bar represents the geometric mean titer. The difference between each of the WT group and these variant groups was not statistically significant (p > 0.4 by t - test). Figure 5B, Groups of 14 - 15 human fH transgenic mice were immunized by intraperitoneal administration of 3 doses of purified recombinant fHbp (10 μg per dose) at 3 - week intervals. Three weeks after the third dose, sera were collected. The bactericidal activity of the sera was measured as described above for wild - type mice.

[0173] Figures 6A and 6B. Binding of human fH to fHbp ID1 single and double mutants by ELISA. These experiments were performed as described above for Figures 2A - 2B. Figure 7. Bactericidal antibody responses to fHbp ID1 single and double mutants in mice. Groups of 20 wild - type mice were immunized, and the bactericidal antibody responses of these sera were measured as described above for Figure 5A.

[0174] Example 2: Characterization of fHbp ID55 variant Materials and Methods These experiments were performed as described in Example 1.

[0175] Results Three promising variants identified with fHbp ID1 were similarly constructed with fHbp ID55; these included E92K, S223R, and H248L. All three of these fHbp ID55 variants had significantly reduced binding to fH (Figure 8A). These variants preserved the structural integrity as judged by the binding of the mouse anti - fHbp monoclonal antibody JAR41 (Figure 8B).

[0176] fH binding of the fHbp ID55 variant in FIGS. 8A and 8B. A. Binding of fH to immobilized fHbp ID55 variant by ELISA. This experiment was conducted as described in the description for FIG. 2. The mean and range for 2 - 4 replicates are shown. B. The concentration - dependent binding of the anti - fHbp monoclonal antibody (mAb) JAR41 suggested that these recombinant fHbps were present in similar amounts within the wells of the microtiter plate and had a conserved three - dimensional structure in the region of the epitope recognized by JAR41 (N - terminal domain; Vu et al. (2012) Sci. Reports, supra). The secondary antibody was alkaline phosphatase - conjugated goat anti - mouse IgG (Sigma).

[0177] FIG. 9. Bactericidal antibody response to fHbp ID55 variant in wild - type mice. A group of 12 mice was immunized and the bactericidal antibody response of their sera was measured as described above for FIG. 5A. Bactericidal activity was measured against the variant of strain H44 / 76 expressing fHbp ID55.

[0178] FIG. 10A. Bactericidal antibody response to the fHbp ID55 S223R variant in human fH transgenic mice. A group of 11 - 12 transgenic mice was immunized with 3 doses of purified recombinant fHbp (12 μg per dose) or one - tenth of the human dose of the approved Trumenba (Pfizer) vaccine containing a total of 12 μg of fHbp. These transgenic mice were immunized and the bactericidal antibody response of their sera was measured as described above for FIG. 5B. FIG. 10B. Bactericidal antibody response to Trumenba in wild - type and human fH transgenic mice in relation to serum human fH concentration. Serum human fH concentration was measured by ELISA as described previously (Beernink et al. (2011) Journal of Immunology 186(6):3606 - 14).

[0179] Example 3: Identification and characterization of fHbp ID22 variant Materials and Methods These experiments were conducted as described in Example 1.

[0180] Result An independent search for random fHbp mutants with reduced fH binding was performed using fHbp ID22 in mutant group 2 (subfamily A). This screening yielded six promising new mutants (Figure 17). The fH binding to the wild-type of ID22 and the previously described D211A mutant by ELISA is shown in Figure 11A. The fH binding to these six new mutant ID 22 proteins is shown in Figure 11B.

[0181] Figures 11A - 11D. fH binding of fHbp ID22 library mutants. Figure 11A, fHbp ID22 wild-type (WT) protein, a positive control with high binding of human fH. fHbp ID22 D211A mutant, a negative control with low fH binding. Figure 11B, new fHbp ID22 mutants with reduced fH binding. All of these mutants showed low binding, and V131D showed extremely low binding similar to D211A. This experiment was performed as described in the caption for Figure 2. The mean and range of 2 - 4 replicates are shown. Figure 11C, fH binding of a subset of fHbp ID22 mutants at fH concentrations of 100 μg / ml or less. Figure 11D, binding of anti-fHbp monoclonal antibody JAR4 to this subset of mutants (using the same symbols as in Figure 11C). The new mutant K219N retains JAR4 binding, while the G220S mutant shows reduced JAR4 binding. All of these mutants had normal binding to another anti-fHbp monoclonal antibody JAR31 (data not shown). The mean and standard deviation of repeated measurements are shown.

[0182] The bactericidal antibody responses of wild-type CD-1 mice to these new mutants V131D and K219N, along with the control wild-type ID22 protein and the previously characterized mutant D211A, are shown in Figure 12.

[0183] Figures 12A - 12B. Bactericidal antibody responses to fHbp ID22 library variants in wild - type mice. Library variants with low human fH binding were selected for immunization. Groups of 10 - 21 mice were immunized with two doses of purified recombinant fHbp (10 μg per dose) at 3 - week intervals. Three weeks after the second dose, sera were collected. Serum bactericidal activity was measured using human serum depleted of IgG as a complement source and serum group B strain CH597 as a test strain. This strain expresses fHbp ID23, which is nearly identical to the control fHbp ID22 WT vaccine. Figure 12A, experiment examining new variants V131D and K219N. This D211A variant, which did not reduce immunogenicity, was used as a control variant fHbp vaccine. Each symbol represents the titer of an individual mouse, and the horizontal bar represents the geometric mean titer. Figure 12B, second experiment examining new variants D121G, S128T, F129S, and G220S. Except for a slight decrease for V131D, no significant decrease in immunogenicity was observed for these new variant fHbps.

[0184] Figure 13. Bactericidal antibody response to fHbp ID22 library variant K219N in human fH transgenic mice. The control variant D211A showed a higher response, and the K219N variant showed a response similar to the fHbp ID22 wild - type (WT) antigen.

[0185] Figure 14. Thermal stability of fHbp ID22 measured by differential scanning microcalorimetry. fHbp ID22 wild - type (WT, solid line) undergoes denaturation transitions at 38 °C (N - terminus) and 81 °C (C - terminal domain). The fHbp ID22 L130R / G133D double mutant exhibits a 19 °C higher thermal stability for the N - terminal domain compared to ID22 WT.

[0186] Figures 15A - 15B. fHbp ID22 triple mutants combining the stabilizing substitutions L130R and G133D (double mutant, DM) with library - derived mutants to reduce fH binding. A, fH binding to the fHbp ID22 triple mutant. B, binding of the control mouse anti - fHbp monoclonal antibody (mAb) JAR4 (same symbols as in panel A. All of these stabilizing mutants bind better to JAR4 than fHbp ID22WT).

[0187] Figure 16. Bactericidal antibody responses to the fHbp ID22 triple mutant in human fH transgenic mice. These two test mutants combine the stability double mutant (DM) with K219N and G220S, respectively. These triple mutants induced responses 8 - fold and 18 - fold higher than the control ID22 WT antigen.

[0188] An overview of the exemplary fHbp ID1 and ID22 mutants described above is shown in the table of Figure 17.

[0189] Although the present invention has been described with reference to its specific embodiments, it should be understood by those skilled in the art that various changes can be made and equivalents can be substituted without departing from the true spirit and scope of the present invention. Furthermore, many changes can be made to adapt a particular situation, material, composition, process, process step or steps to the objective, that is, to the spirit and scope of the present invention. All such changes are intended to be within the scope of the appended claims of this specification.

[0190] Sequence Information SEQUENCE LISTING <110> CHILDREN'S HOSPITAL & RESEARCH CENTER AT OAKLAND <120> Factor H Binding Protein Variants and Methods of Use Thereof <150> US 62 / 028,123 <151> 2014 - 07 - 23 <160> 28 <170> PatentIn version 3.5 <210> 1 <211> 255 <212> PRT <213> Neisseria meningitidis <400> 1 Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly Ala Gly Leu 1 5 10 15 Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys Gly Leu Gln 20 25 30 Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys Leu Lys Leu 35 40 45 Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn 50 55 60 Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe Ile Arg 65 70 75 80 Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly Glu Phe 85 90 95 Gln Val Tyr Lys Gln Ser His Ser Ala Leu Thr Ala Phe Gln Thr Glu 100 105 110 Gln Ile Gln Asp Ser Glu His Ser Gly Lys Met Val Ala Lys Arg Gln 115 120 125 Phe Arg Ile Gly Asp Ile Ala Gly Glu His Thr Ser Phe Asp Lys Leu 130 135 140 Pro Glu Gly Gly Arg Ala Thr Tyr Arg Gly Thr Ala Phe Gly Ser Asp 145 150 155 160 Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp Phe Ala Ala Lys Gln 165 170 175 Gly Asn Gly Lys Ile Glu His Leu Lys Ser Pro Glu Leu Asn Val Asp 180 185 190 Leu Ala Ala Ala Asp Ile Lys Pro Asp Gly Lys Arg His Ala Val Ile 195 200 205 Ser Gly Ser Val Leu Tyr Asn Gln Ala Glu Lys Gly Ser Tyr Ser Leu 210 215 220 Gly Ile Phe Gly Gly Lys Ala Gln Glu Val Ala Gly Ser Ala Glu Val 225 230 235 240 Lys Thr Val Asn Gly Ile Arg His Ile Gly Leu Ala Ala Lys Gln 245 250 255 <210> 2 <211> 254 <212> PRT <213> Neisseria meningitidis <400> 2 Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly Ala Gly Leu 1 5 10 15 Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys Ser Leu Gln 20 25 30 Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys Leu Lys Leu 35 40 45 Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn 50 55 60 Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe Ile Arg 65 70 75 80 Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly Glu Phe 85 90 95 Gln Ile Tyr Lys Gln Asp His Ser Ala Val Val Ala Leu Gln Ile Glu 100 105 110 Lys Ile Asn Asn Pro Asp Lys Ile Asp Ser Leu Ile Asn Gln Arg Ser 115 120 125 Phe Leu Val Ser Gly Leu Gly Gly Glu His Thr Ala Phe Asn Gln Leu 130 135 140 Pro Ser Gly Lys Ala Glu Tyr His Gly Lys Ala Phe Ser Ser Asp Asp 145 150 155 160 Pro Asn Gly Arg Leu His Tyr Ser Ile Asp Phe Thr Lys Lys Gln Gly 165 170 175 Tyr Gly Arg Ile Glu His Leu Lys Thr Pro Glu Gln Asn Val Glu Leu 180 185 190 Ala Ser Ala Glu Leu Lys Ala Asp Glu Lys Ser His Ala Val Ile Leu 195 200 205 Gly Asp Thr Arg Tyr Gly Gly Glu Glu Lys Gly Thr Tyr His Leu Ala 210 215 220 Leu Phe Gly Asp Arg Ala Gln Glu Ile Ala Gly Ser Ala Thr Val Lys 225 230 235 240 Ile Arg Glu Lys Val His Glu Ile Gly Ile Ala Gly Lys Gln 245 250 <210> 3 <211> 260 <212> PRT <213> Neisseria meningitidis <400> 3 Cys Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Val Thr Ala Asp 1 5 10 15 Ile Gly Thr Gly Leu Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys 20 25 30 Asp Lys Gly Leu Lys Ser Leu Thr Leu Glu Asp Ser Ile Ser Gln Asn 35 40 45 Gly Thr Leu Thr Leu Ser Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn 50 55 60 Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg 65 70 75 80 Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu 85 90 95 Glu Ser Gly Glu Phe Gln Val Tyr Lys Gln Ser His Ser Ala Leu Thr 100 105 110 Ala Leu Gln Thr Glu Gln Glu Gln Asp Pro Glu His Ser Glu Lys Met 115 120 125 Val Ala Lys Arg Arg Phe Arg Ile Gly Asp Ile Ala Gly Glu His Thr 130 135 140 Ser Phe Asp Lys Leu Pro Lys Asp Val Met Ala Thr Tyr Arg Gly Thr 145 150 155 160 Ala Phe Gly Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp 165 170 175 Phe Ala Ala Lys Gln Gly His Gly Lys Ile Glu His Leu Lys Ser Pro 180 185 190 Glu Leu Asn Val Asp Leu Ala Val Ala Tyr Ile Lys Pro Asp Glu Lys 195 200 205 His His Ala Val Ile Ser Gly Ser Val Leu Tyr Asn Gln Asp Glu Lys 210 215 220 Gly Ser Tyr Ser Leu Gly Ile Phe Gly Glu Lys Ala Gln Glu Val Ala 225 230 235 240 Gly Ser Ala Glu Val Glu Thr Ala Asn Gly Ile His His Ile Gly Leu 245 250 255 Ala Ala Lys Gln 260 <210> 4 <211> 1231 <212> PRT <213> Homo sapiens <400> 4 Met Arg Leu Leu Ala Lys Ile Ile Cys Leu Met Leu Trp Ala Ile Cys 1 5 10 15 Val Ala Glu Asp Cys Asn Glu Leu Pro Pro Arg Arg Asn Thr Glu Ile 20 25 30 Leu Thr Gly Ser Trp Ser Asp Gln Thr Tyr Pro Glu Gly Thr Gln Ala 35 40 45 Ile Tyr Lys Cys Arg Pro Gly Tyr Arg Ser Leu Gly Asn Val Ile Met 50 55 60 Val Cys Arg Lys Gly Glu Trp Val Ala Leu Asn Pro Leu Arg Lys Cys 65 70 75 80 Gln Lys Arg Pro Cys Gly His Pro Gly Asp Thr Pro Phe Gly Thr Phe 85 90 95 Thr Leu Thr Gly Gly Asn Val Phe Glu Tyr Gly Val Lys Ala Val Tyr 100 105 110 Thr Cys Asn Glu Gly Tyr Gln Leu Leu Gly Glu Ile Asn Tyr Arg Glu 115 120 125 Cys Asp Thr Asp Gly Trp Thr Asn Asp Ile Pro Ile Cys Glu Val Val 130 135 140 Lys Cys Leu Pro Val Thr Ala Pro Glu Asn Gly Lys Ile Val Ser Ser 145 150 155 160 Ala Met Glu Pro Asp Arg Glu Tyr His Phe Gly Gln Ala Val Arg Phe 165 170 175 Val Cys Asn Ser Gly Tyr Lys Ile Glu Gly Asp Glu Glu Met His Cys 180 185 190 Ser Asp Asp Gly Phe Trp Ser Lys Glu Lys Pro Lys Cys Val Glu Ile 195 200 205 Ser Cys Lys Ser Pro Asp Val Ile Asn Gly Ser Pro Ile Ser Gln Lys 210 215 220 Ile Ile Tyr Lys Glu Asn Glu Arg Phe Gln Tyr Lys Cys Asn Met Gly 225 230 235 240 Tyr Glu Tyr Ser Glu Arg Gly Asp Ala Val Cys Thr Glu Ser Gly Trp 245 250 255 Arg Pro Leu Pro Ser Cys Glu Glu Lys Ser Cys Asp Asn Pro Tyr Ile 260 265 270 Pro Asn Gly Asp Tyr Ser Pro Leu Arg Ile Lys His Arg Thr Gly Asp 275 280 285 Glu Ile Thr Tyr Gln Cys Arg Asn Gly Phe Tyr Pro Ala Thr Arg Gly 290 295 300 Asn Thr Ala Lys Cys Thr Ser Thr Gly Trp Ile Pro Ala Pro Arg Cys 305 310 315 320 Thr Leu Lys Pro Cys Asp Tyr Pro Asp Ile Lys His Gly Gly Leu Tyr 325 330 335 His Glu Asn Met Arg Arg Pro Tyr Phe Pro Val Ala Val Gly Lys Tyr 340 345 350 Tyr Ser Tyr Tyr Cys Asp Glu His Phe Glu Thr Pro Ser Gly Ser Tyr 355 360 365 Trp Asp His Ile His Cys Thr Gln Asp Gly Trp Ser Pro Ala Val Pro 370 375 380 Cys Leu Arg Lys Cys Tyr Phe Pro Tyr Leu Glu Asn Gly Tyr Asn Gln 385 390 395 400 Asn Tyr Gly Arg Lys Phe Val Gln Gly Lys Ser Ile Asp Val Ala Cys 405 410 415 His Pro Gly Tyr Ala Leu Pro Lys Ala Gln Thr Thr Val Thr Cys Met 420 425 430 Glu Asn Gly Trp Ser Pro Thr Pro Arg Cys Ile Arg Val Lys Thr Cys 435 440 445 Ser Lys Ser Ser Ile Asp Ile Glu Asn Gly Phe Ile Ser Glu Ser Gln 450 455 460 Tyr Thr Tyr Ala Leu Lys Glu Lys Ala Lys Tyr Gln Cys Lys Leu Gly 465 470 475 480 Tyr Val Thr Ala Asp Gly Glu Thr Ser Gly Ser Ile Thr Cys Gly Lys 485 490 495 Asp Gly Trp Ser Ala Gln Pro Thr Cys Ile Lys Ser Cys Asp Ile Pro 500 505 510 Val Phe Met Asn Ala Arg Thr Lys Asn Asp Phe Thr Trp Phe Lys Leu 515 520 525 Asn Asp Thr Leu Asp Tyr Glu Cys His Asp Gly Tyr Glu Ser Asn Thr 530 535 540 Gly Ser Thr Thr Gly Ser Ile Val Cys Gly Tyr Asn Gly Trp Ser Asp 545 550 555 560 Leu Pro Ile Cys Tyr Glu Arg Glu Cys Glu Leu Pro Lys Ile Asp Val 565 570 575 His Leu Val Pro Asp Arg Lys Lys Asp Gln Tyr Lys Val Gly Glu Val 580 585 590 Leu Lys Phe Ser Cys Lys Pro Gly Phe Thr Ile Val Gly Pro Asn Ser 595 600 605 Val Gln Cys Tyr His Phe Gly Leu Ser Pro Asp Leu Pro Ile Cys Lys 610 615 620 Glu Gln Val Gln Ser Cys Gly Pro Pro Pro Glu Leu Leu Asn Gly Asn 625 630 635 640 Val Lys Glu Lys Thr Lys Glu Glu Tyr Gly His Ser Glu Val Val Glu 645 650 655 Tyr Tyr Cys Asn Pro Arg Phe Leu Met Lys Gly Pro Asn Lys Ile Gln 660 665 670 Cys Val Asp Gly Glu Trp Thr Thr Leu Pro Val Cys Ile Val Glu Glu 675 680 685 Ser Thr Cys Gly Asp Ile Pro Glu Leu Glu His Gly Trp Ala Gln Leu 690 695 700 Ser Ser Pro Pro Tyr Tyr Tyr Gly Asp Ser Val Glu Phe Asn Cys Ser 705 710 715 720 Glu Ser Phe Thr Met Ile Gly His Arg Ser Ile Thr Cys Ile His Gly 725 730 735 Val Trp Thr Gln Leu Pro Gln Cys Val Ala Ile Asp Lys Leu Lys Lys 740 745 750 Cys Lys Ser Ser Asn Leu Ile Ile Leu Glu Glu His Leu Lys Asn Lys 755 760 765 Lys Glu Phe Asp His Asn Ser Asn Ile Arg Tyr Arg Cys Arg Gly Lys 770 775 780 Glu Gly Trp Ile His Thr Val Cys Ile Asn Gly Arg Trp Asp Pro Glu 785 790 795 800 Val Asn Cys Ser Met Ala Gln Ile Gln Leu Cys Pro Pro Pro Pro Gln 805 810 815 Ile Pro Asn Ser His Asn Met Thr Thr Thr Leu Asn Tyr Arg Asp Gly 820 825 830 Glu Lys Val Ser Val Leu Cys Gln Glu Asn Tyr Leu Ile Gln Glu Gly 835 840 845 Glu Glu Ile Thr Cys Lys Asp Gly Arg Trp Gln Ser Ile Pro Leu Cys 850 855 860 Val Glu Lys Ile Pro Cys Ser Gln Pro Pro Gln Ile Glu His Gly Thr 865 870 875 880 Ile Asn Ser Ser Arg Ser Ser Gln Glu Ser Tyr Ala His Gly Thr Lys 885 890 895 Leu Ser Tyr Thr Cys Glu Gly Gly Phe Arg Ile Ser Glu Glu Asn Glu 900 905 910 Thr Thr Cys Tyr Met Gly Lys Trp Ser Ser Pro Pro Gln Cys Glu Gly 915 920 925 Leu Pro Cys Lys Ser Pro Pro Glu Ile Ser His Gly Val Val Ala His 930 935 940 Met Ser Asp Ser Tyr Gln Tyr Gly Glu Glu Val Thr Tyr Lys Cys Phe 945 950 955 960 Glu Gly Phe Gly Ile Asp Gly Pro Ala Ile Ala Lys Cys Leu Gly Glu 965 970 975 Lys Trp Ser His Pro Pro Ser Cys Ile Lys Thr Asp Cys Leu Ser Leu 980 985 990 Pro Ser Phe Glu Asn Ala Ile Pro Met Gly Glu Lys Lys Asp Val Tyr 995 1000 1005 Lys Ala Gly Glu Gln Val Thr Tyr Thr Cys Ala Thr Tyr Tyr Lys 1010 1015 1020 Methionine, Aspartic acid, Glycine, Alanine, Serine, Asparagine, Valine, Threonine, Cysteine, Isoleucine, Asparagine, Serine, Arginine, Tryptophan, Threonine 1025 1030 1035 Glycine, Arginine, Proline, Threonine, Cysteine, Arginine, Aspartic acid, Threonine, Serine, Cysteine, Valine, Asparagine, Proline, Proline, Threonine 1040 1045 1050 Valine, Glutamine, Asparagine, Alanine, Tyrosine, Isoleucine, Valine, Serine, Arginine, Glutamine, Methionine, Serine, Lysine, Tyrosine, Proline 1055 1060 1065 Serine, Glycine, Glutamic acid, Arginine, Valine, Arginine, Tyrosine, Glutamine, Cysteine, Arginine, Serine, Proline, Tyrosine, Glutamic acid, Methionine 1070 1075 1080 Phenylalanine, Glycine, Aspartic acid, Glutamic acid, Glutamic acid, Valine, Methionine, Cysteine, Leucine, Asparagine, Glycine, Asparagine, Tryptophan, Threonine, Glutamic acid 1085 1090 1095 Proline, Proline, Glutamine, Cysteine, Lysine, Aspartic acid, Serine, Threonine, Glycine, Lysine, Cysteine, Glycine, Proline, Proline, Proline 1100 1105 1110 Proline, Isoleucine, Aspartic acid, Asparagine, Glycine, Aspartic acid, Isoleucine, Threonine, Serine, Phenylalanine, Proline, Leucine, Serine, Valine, Tyrosine 1115 1120 1125 Alanine, Proline, Alanine, Serine, Serine, Valine, Glutamic acid, Tyrosine, Glutamine, Cysteine, Glutamine, Asparagine, Leucine, Tyrosine, Glutamine 1130 1135 1140 Leucine, Glutamic acid, Glycine, Asparagine, Lysine, Arginine, Isoleucine, Threonine, Cysteine, Arginine, Asparagine, Glycine, Glutamine, Tryptophan, Serine 1145 1150 1155 Glutamic acid, Proline, Proline, Lysine, Cysteine, Leucine, Histidine, Proline, Cysteine, Valine, Isoleucine, Serine, Arginine, Glutamic acid, Isoleucine 1160 1165 1170 Met Glu Asn Tyr Asn Ile Ala Leu Arg Trp Thr Ala Lys Gln Lys 1175 1180 1185 Leu Tyr Ser Arg Thr Gly Glu Ser Val Glu Phe Val Cys Lys Arg 1190 1195 1200 Gly Tyr Arg Leu Ser Ser Arg Ser His Thr Leu Arg Thr Thr Cys 1205 1210 1215 Trp Asp Gly Lys Leu Glu Tyr Pro Thr Cys Ala Lys Arg 1220 1225 1230 <210> 5 <211> 255 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 5 Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly Ala Gly Leu 1 5 10 15 Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys Gly Leu Gln 20 25 30 Ser Leu Thr Leu Asp Arg Ser Val Arg Lys Asn Glu Lys Leu Lys Leu 35 40 45 Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn 50 55 60 Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe Ile Arg 65 70 75 80 Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly Glu Phe 85 90 95 Gln Val Tyr Lys Gln Ser His Ser Ala Leu Thr Ala Phe Gln Thr Glu 100 105 110 Gln Ile Gln Asp Ser Glu His Ser Gly Lys Met Val Ala Lys Arg Gln 115 120 125 Phe Arg Ile Gly Asp Ile Ala Gly Glu His Thr Ser Phe Asp Lys Leu 130 135 140 Pro Glu Gly Gly Arg Ala Thr Tyr Arg Gly Thr Ala Phe Gly Ser Asp 145 150 155 160 Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp Phe Ala Ala Lys Gln 165 170 175 Gly Asn Gly Lys Ile Glu His Leu Lys Ser Pro Glu Leu Asn Val Asp 180 185 190 Leu Ala Ala Ala Asp Ile Lys Pro Asp Gly Lys Arg His Ala Val Ile 195 200 205 Ser Gly Ser Val Leu Tyr Asn Gln Ala Glu Lys Gly Ser Tyr Ser Leu 210 215 220 Gly Ile Phe Gly Gly Lys Ala Gln Glu Val Ala Gly Ser Ala Glu Val 225 230 235 240 Lys Thr Val Asn Gly Ile Arg His Ile Gly Leu Ala Ala Lys Gln 245 250 255 <210> 6 <211> 255 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 6 Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly Ala Gly Leu 1 5 10 15 Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys Gly Leu Gln 20 25 30 Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys Leu Lys Leu 35 40 45 Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn 50 55 60 Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe Ile Arg 65 70 75 80 Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Lys Ser Gly Glu Phe 85 90 95 Gln Val Tyr Lys Gln Ser His Ser Ala Leu Thr Ala Phe Gln Thr Glu 100 105 110 Gln Ile Gln Asp Ser Glu His Ser Gly Lys Met Val Ala Lys Arg Gln 115 120 125 Phe Arg Ile Gly Asp Ile Ala Gly Glu His Thr Ser Phe Asp Lys Leu 130 135 140 Pro Glu Gly Gly Arg Ala Thr Tyr Arg Gly Thr Ala Phe Gly Ser Asp 145 150 155 160 Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp Phe Ala Ala Lys Gln 165 170 175 Gly Asn Gly Lys Ile Glu His Leu Lys Ser Pro Glu Leu Asn Val Asp 180 185 190 Leu Ala Ala Ala Asp Ile Lys Pro Asp Gly Lys Arg His Ala Val Ile 195 200 205 Ser Gly Ser Val Leu Tyr Asn Gln Ala Glu Lys Gly Ser Tyr Ser Leu 210 215 220 Gly Ile Phe Gly Gly Lys Ala Gln Glu Val Ala Gly Ser Ala Glu Val 225 230 235 240 Lys Thr Val Asn Gly Ile Arg His Ile Gly Leu Ala Ala Lys Gln 245 250 255 <210> 7 <211> 255 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 7 Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly Ala Gly Leu 1 5 10 15 Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys Gly Leu Gln 20 25 30 Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys Leu Lys Leu 35 40 45 Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn 50 55 60 Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe Ile Arg 65 70 75 80 Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly Glu Phe 85 90 95 Gln Val Tyr Lys Gln Ser His Ser Ala Leu Thr Ala Phe Gln Thr Glu 100 105 110 Gln Ile Gln Asp Ser Glu His Ser Gly Lys Met Val Ala Lys Arg Gln 115 120 125 Phe Gly Ile Gly Asp Ile Ala Gly Glu His Thr Ser Phe Asp Lys Leu 130 135 140 Pro Glu Gly Gly Arg Ala Thr Tyr Arg Gly Thr Ala Phe Gly Ser Asp 145 150 155 160 Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp Phe Ala Ala Lys Gln 165 170 175 Gly Asn Gly Lys Ile Glu His Leu Lys Ser Pro Glu Leu Asn Val Asp 180 185 190 Leu Ala Ala Ala Asp Ile Lys Pro Asp Gly Lys Arg His Ala Val Ile 195 200 205 Ser Gly Ser Val Leu Tyr Asn Gln Ala Glu Lys Gly Ser Tyr Ser Leu 210 215 220 Gly Ile Phe Gly Gly Lys Ala Gln Glu Val Ala Gly Ser Ala Glu Val 225 230 235 240 Lys Thr Val Asn Gly Ile Arg His Ile Gly Leu Ala Ala Lys Gln 245 250 255 <210> 8 <211> 255 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 8 Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly Ala Gly Leu 1 5 10 15 Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys Gly Leu Gln 20 25 30 Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys Leu Lys Leu 35 40 45 Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn 50 55 60 Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe Ile Arg 65 70 75 80 Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly Glu Phe 85 90 95 Gln Val Tyr Lys Gln Ser His Ser Ala Leu Thr Ala Phe Gln Thr Glu 100 105 110 Gln Ile Gln Asp Ser Glu His Ser Gly Lys Met Val Ala Lys Arg Gln 115 120 125 Phe Arg Ile Gly Asp Ile Ala Gly Glu His Thr Ser Phe Asp Lys Leu 130 135 140 Pro Glu Gly Gly Arg Ala Thr Tyr Arg Gly Thr Ala Phe Gly Ser Asp 145 150 155 160 Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp Phe Ala Ala Lys Gln 165 170 175 Gly Asn Gly Lys Ile Glu His Leu Lys Ser Pro Glu Leu Asn Val Asp 180 185 190 Leu Ala Ala Ala Asp Ile Lys Pro Asp Gly Lys Arg His Ala Val Ile 195 200 205 Ser Gly Ser Val Leu Tyr Asn Gln Ala Glu Lys Gly Ser Tyr Arg Leu 210 215 220 Gly Ile Phe Gly Gly Lys Ala Gln Glu Val Ala Gly Ser Ala Glu Val 225 230 235 240 Lys Thr Val Asn Gly Ile Arg His Ile Gly Leu Ala Ala Lys Gln 245 250 255 <210> 9 <211> 255 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 9 Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly Ala Gly Leu 1 5 10 15 Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys Gly Leu Gln 20 25 30 Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys Leu Lys Leu 35 40 45 Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn 50 55 60 Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe Ile Arg 65 70 75 80 Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly Glu Phe 85 90 95 Gln Val Tyr Lys Gln Ser His Ser Ala Leu Thr Ala Phe Gln Thr Glu 100 105 110 Gln Ile Gln Asp Ser Glu His Ser Gly Lys Met Val Ala Lys Arg Gln 115 120 125 Phe Arg Ile Gly Asp Ile Ala Gly Glu His Thr Ser Phe Asp Lys Leu 130 135 140 Pro Glu Gly Gly Arg Ala Thr Tyr Arg Gly Thr Ala Phe Gly Ser Asp 145 150 155 160 Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp Phe Ala Ala Lys Gln 165 170 175 Gly Asn Gly Lys Ile Glu His Leu Lys Ser Pro Glu Leu Asn Val Asp 180 185 190 Leu Ala Ala Ala Asp Ile Lys Pro Asp Gly Lys Arg His Ala Val Ile 195 200 205 Ser Gly Ser Val Leu Tyr Asn Gln Ala Glu Lys Gly Ser Tyr Ser Leu 210 215 220 Gly Ile Phe Gly Gly Lys Ala Gln Glu Val Ala Gly Ser Ala Glu Val 225 230 235 240 Lys Thr Val Asn Gly Ile Arg Leu Ile Gly Leu Ala Ala Lys Gln 245 250 255 <210> 10 <211> 254 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 10 Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly Ala Gly Leu 1 5 10 15 Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys Ser Leu Gln 20 25 30 Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys Leu Lys Leu 35 40 45 Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn 50 55 60 Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe Ile Arg 65 70 75 80 Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly Glu Phe 85 90 95 Gln Ile Tyr Lys Gln Asp His Ser Ala Val Val Ala Leu Gln Ile Glu 100 105 110 Lys Ile Ile Asn Pro Asp Lys Ile Asp Ser Leu Ile Asn Gln Arg Ser 115 120 125 Phe Leu Val Ser Gly Leu Gly Gly Glu His Thr Ala Phe Asn Gln Leu 130 135 140 Pro Ser Gly Lys Ala Glu Tyr His Gly Lys Ala Phe Ser Ser Asp Asp 145 150 155 160 Pro Asn Gly Arg Leu His Tyr Ser Ile Asp Phe Thr Lys Lys Gln Gly 165 170 175 Tyr Gly Arg Ile Glu His Leu Lys Thr Pro Glu Gln Asn Val Glu Leu 180 185 190 Ala Ser Ala Glu Leu Lys Ala Asp Glu Lys Ser His Ala Val Ile Leu 195 200 205 Gly Asp Thr Arg Tyr Gly Gly Glu Glu Lys Gly Thr Tyr His Leu Ala 210 215 220 Leu Phe Gly Asp Arg Ala Gln Glu Ile Ala Gly Ser Ala Thr Val Lys 225 230 235 240 Ile Arg Glu Lys Val His Glu Ile Gly Ile Ala Gly Lys Gln 245 250 <210> 11 <211> 254 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 11 Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly Ala Gly Leu 1 5 10 15 Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys Ser Leu Gln 20 25 30 Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys Leu Lys Leu 35 40 45 Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn 50 55 60 Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe Ile Arg 65 70 75 80 Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly Glu Phe 85 90 95 Gln Ile Tyr Lys Gln Asp His Ser Ala Val Val Ala Leu Gln Ile Glu 100 105 110 Lys Ile Asn Asn Pro Asp Lys Ile Gly Ser Leu Ile Asn Gln Arg Ser 115 120 125 Phe Leu Val Ser Gly Leu Gly Gly Glu His Thr Ala Phe Asn Gln Leu 130 135 140 Pro Ser Gly Lys Ala Glu Tyr His Gly Lys Ala Phe Ser Ser Asp Asp 145 150 155 160 Pro Asn Gly Arg Leu His Tyr Ser Ile Asp Phe Thr Lys Lys Gln Gly 165 170 175 Tyr Gly Arg Ile Glu His Leu Lys Thr Pro Glu Gln Asn Val Glu Leu 180 185 190 Ala Ser Ala Glu Leu Lys Ala Asp Glu Lys Ser His Ala Val Ile Leu 195 200 205 Gly Asp Thr Arg Tyr Gly Gly Glu Glu Lys Gly Thr Tyr His Leu Ala 210 215 220 Leu Phe Gly Asp Arg Ala Gln Glu Ile Ala Gly Ser Ala Thr Val Lys 225 230 235 240 Ile Arg Glu Lys Val His Glu Ile Gly Ile Ala Gly Lys Gln 245 250 <210> 12 <211> 254 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 12 Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly Ala Gly Leu 1 5 10 15 Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys Ser Leu Gln 20 25 30 Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys Leu Lys Leu 35 40 45 Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn 50 55 60 Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe Ile Arg 65 70 75 80 Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly Glu Phe 85 90 95 Gln Ile Tyr Lys Gln Asp His Ser Ala Val Val Ala Leu Gln Ile Glu 100 105 110 Lys Ile Asn Asn Pro Asp Lys Ile Asp Ser Leu Ile Asn Gln Arg Thr 115 120 125 Phe Leu Val Ser Gly Leu Gly Gly Glu His Thr Ala Phe Asn Gln Leu 130 135 140 Pro Ser Gly Lys Ala Glu Tyr His Gly Lys Ala Phe Ser Ser Asp Asp 145 150 155 160 Pro Asn Gly Arg Leu His Tyr Ser Ile Asp Phe Thr Lys Lys Gln Gly 165 170 175 Tyr Gly Arg Ile Glu His Leu Lys Thr Pro Glu Gln Asn Val Glu Leu 180 185 190 Ala Ser Ala Glu Leu Lys Ala Asp Glu Lys Ser His Ala Val Ile Leu 195 200 205 Gly Asp Thr Arg Tyr Gly Gly Glu Glu Lys Gly Thr Tyr His Leu Ala 210 215 220 Leu Phe Gly Asp Arg Ala Gln Glu Ile Ala Gly Ser Ala Thr Val Lys 225 230 235 240 Ile Arg Glu Lys Val His Glu Ile Gly Ile Ala Gly Lys Gln 245 250 <210> 13 <211> 254 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 13 Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly Ala Gly Leu 1 5 10 15 Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys Ser Leu Gln 20 25 30 Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys Leu Lys Leu 35 40 45 Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn 50 55 60 Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe Ile Arg 65 70 75 80 Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly Glu Phe 85 90 95 Gln Ile Tyr Lys Gln Asp His Ser Ala Val Val Ala Leu Gln Ile Glu 100 105 110 Lys Ile Asn Asn Pro Asp Lys Ile Asp Ser Leu Ile Asn Gln Arg Ser 115 120 125 Phe Leu Asp Ser Gly Leu Gly Gly Glu His Thr Ala Phe Asn Gln Leu 130 135 140 Pro Ser Gly Lys Ala Glu Tyr His Gly Lys Ala Phe Ser Ser Asp Asp 145 150 155 160 Pro Asn Gly Arg Leu His Tyr Ser Ile Asp Phe Thr Lys Lys Gln Gly 165 170 175 Tyr Gly Arg Ile Glu His Leu Lys Thr Pro Glu Gln Asn Val Glu Leu 180 185 190 Ala Ser Ala Glu Leu Lys Ala Asp Glu Lys Ser His Ala Val Ile Leu 195 200 205 Gly Asp Thr Arg Tyr Gly Gly Glu Glu Lys Gly Thr Tyr His Leu Ala 210 215 220 Leu Phe Gly Asp Arg Ala Gln Glu Ile Ala Gly Ser Ala Thr Val Lys 225 230 235 240 Ile Arg Glu Lys Val His Glu Ile Gly Ile Ala Gly Lys Gln 245 250 <210> 14 <211> 254 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 14 Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly Ala Gly Leu 1 5 10 15 Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys Ser Leu Gln 20 25 30 Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys Leu Lys Leu 35 40 45 Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn 50 55 60 Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe Ile Arg 65 70 75 80 Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly Glu Phe 85 90 95 Gln Ile Tyr Lys Gln Asp His Ser Ala Val Val Ala Leu Gln Ile Glu 100 105 110 Lys Ile Asn Asn Pro Asp Lys Ile Asp Ser Leu Ile Asn Gln Arg Ser 115 120 125 Phe Leu Val Ser Gly Leu Gly Gly Glu His Thr Ala Phe Asn Gln Leu 130 135 140 Pro Ser Gly Lys Ala Glu Tyr His Gly Lys Ala Phe Ser Ser Asp Asp 145 150 155 160 Pro Asn Gly Arg Leu His Tyr Ser Ile Asp Phe Thr Lys Lys Gln Gly 165 170 175 Tyr Gly Arg Ile Glu His Leu Lys Thr Pro Glu Gln Asn Val Glu Leu 180 185 190 Ala Ser Ala Glu Leu Lys Ala Asp Glu Lys Ser His Ala Val Ile Leu 195 200 205 Gly Asp Thr Arg Tyr Gly Gly Glu Glu Asn Gly Thr Tyr His Leu Ala 210 215 220 Leu Phe Gly Asp Arg Ala Gln Glu Ile Ala Gly Ser Ala Thr Val Lys 225 230 235 240 Ile Arg Glu Lys Val His Glu Ile Gly Ile Ala Gly Lys Gln 245 250 <210> 15 <211> 254 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 15 Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly Ala Gly Leu 1 5 10 15 Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys Ser Leu Gln 20 25 30 Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys Leu Lys Leu 35 40 45 Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn 50 55 60 Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe Ile Arg 65 70 75 80 Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly Glu Phe 85 90 95 Gln Ile Tyr Lys Gln Asp His Ser Ala Val Val Ala Leu Gln Ile Glu 100 105 110 Lys Ile Asn Asn Pro Asp Lys Ile Asp Ser Leu Ile Asn Gln Arg Ser 115 120 125 Phe Leu Val Ser Gly Leu Gly Gly Glu His Thr Ala Phe Asn Gln Leu 130 135 140 Pro Ser Gly Lys Ala Glu Tyr His Gly Lys Ala Phe Ser Ser Asp Asp 145 150 155 160 Pro Asn Gly Arg Leu His Tyr Ser Ile Asp Phe Thr Lys Lys Gln Gly 165 170 175 Tyr Gly Arg Ile Glu His Leu Lys Thr Pro Glu Gln Asn Val Glu Leu 180 185 190 Ala Ser Ala Glu Leu Lys Ala Asp Glu Lys Ser His Ala Val Ile Leu 195 200 205 Gly Asp Thr Arg Tyr Gly Gly Glu Glu Lys Ser Thr Tyr His Leu Ala 210 215 220 Leu Phe Gly Asp Arg Ala Gln Glu Ile Ala Gly Ser Ala Thr Val Lys 225 230 235 240 Ile Arg Glu Lys Val His Glu Ile Gly Ile Ala Gly Lys Gln 245 250 <210> 16 <211> 260 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 16 Cys Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Val Thr Ala Asp 1 5 10 15 Ile Gly Thr Gly Leu Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys 20 25 30 Asp Lys Gly Leu Lys Ser Leu Thr Leu Glu Asp Ser Ile Ser Gln Asn 35 40 45 Gly Thr Leu Thr Leu Ser Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn 50 55 60 Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg 65 70 75 80 Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu 85 90 95 Lys Ser Gly Glu Phe Gln Val Tyr Lys Gln Ser His Ser Ala Leu Thr 100 105 110 Ala Leu Gln Thr Glu Gln Glu Gln Asp Pro Glu His Ser Glu Lys Met 115 120 125 Val Ala Lys Arg Arg Phe Arg Ile Gly Asp Ile Ala Gly Glu His Thr 130 135 140 Ser Phe Asp Lys Leu Pro Lys Asp Val Met Ala Thr Tyr Arg Gly Thr 145 150 155 160 Ala Phe Gly Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp 165 170 175 Phe Ala Ala Lys Gln Gly His Gly Lys Ile Glu His Leu Lys Ser Pro 180 185 190 Glu Leu Asn Val Asp Leu Ala Val Ala Tyr Ile Lys Pro Asp Glu Lys 195 200 205 His His Ala Val Ile Ser Gly Ser Val Leu Tyr Asn Gln Asp Glu Lys 210 215 220 Gly Ser Tyr Ser Leu Gly Ile Phe Gly Glu Lys Ala Gln Glu Val Ala 225 230 235 240 Gly Ser Ala Glu Val Glu Thr Ala Asn Gly Ile His His Ile Gly Leu 245 250 255 Ala Ala Lys Gln 260 <210> 17 <211> 260 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 17 Cys Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Val Thr Ala Asp 1 5 10 15 Ile Gly Thr Gly Leu Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys 20 25 30 Asp Lys Gly Leu Lys Ser Leu Thr Leu Glu Asp Ser Ile Ser Gln Asn 35 40 45 Gly Thr Leu Thr Leu Ser Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn 50 55 60 Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg 65 70 75 80 Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu 85 90 95 Glu Ser Gly Glu Phe Gln Val Tyr Lys Gln Ser His Ser Ala Leu Thr 100 105 110 Ala Leu Gln Thr Glu Gln Glu Gln Asp Pro Glu His Ser Glu Lys Met 115 120 125 Val Ala Lys Arg Arg Phe Arg Ile Gly Asp Ile Ala Gly Glu His Thr 130 135 140 Ser Phe Asp Lys Leu Pro Lys Asp Val Met Ala Thr Tyr Arg Gly Thr 145 150 155 160 Ala Phe Gly Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp 165 170 175 Phe Ala Ala Lys Gln Gly His Gly Lys Ile Glu His Leu Lys Ser Pro 180 185 190 Glu Leu Asn Val Asp Leu Ala Val Ala Tyr Ile Lys Pro Asp Glu Lys 195 200 205 His His Ala Val Ile Ser Gly Ser Val Leu Tyr Asn Gln Asp Glu Lys 210 215 220 Gly Ser Tyr Arg Leu Gly Ile Phe Gly Glu Lys Ala Gln Glu Val Ala 225 230 235 240 Gly Ser Ala Glu Val Glu Thr Ala Asn Gly Ile His His Ile Gly Leu 245 250 255 Ala Ala Lys Gln 260 <210> 18 <211> 260 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 18 Cys Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Val Thr Ala Asp 1 5 10 15 Ile Gly Thr Gly Leu Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys 20 25 30 Asp Lys Gly Leu Lys Ser Leu Thr Leu Glu Asp Ser Ile Ser Gln Asn 35 40 45 Gly Thr Leu Thr Leu Ser Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn 50 55 60 Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg 65 70 75 80 Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu 85 90 95 Glu Ser Gly Glu Phe Gln Val Tyr Lys Gln Ser His Ser Ala Leu Thr 100 105 110 Ala Leu Gln Thr Glu Gln Glu Gln Asp Pro Glu His Ser Glu Lys Met 115 120 125 Val Ala Lys Arg Arg Phe Arg Ile Gly Asp Ile Ala Gly Glu His Thr 130 135 140 Ser Phe Asp Lys Leu Pro Lys Asp Val Met Ala Thr Tyr Arg Gly Thr 145 150 155 160 Ala Phe Gly Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp 165 170 175 Phe Ala Ala Lys Gln Gly His Gly Lys Ile Glu His Leu Lys Ser Pro 180 185 190 Glu Leu Asn Val Asp Leu Ala Val Ala Tyr Ile Lys Pro Asp Glu Lys 195 200 205 His His Ala Val Ile Ser Gly Ser Val Leu Tyr Asn Gln Asp Glu Lys 210 215 220 Gly Ser Tyr Ser Leu Gly Ile Phe Gly Glu Lys Ala Gln Glu Val Ala 225 230 235 240 Gly Ser Ala Glu Val Glu Thr Ala Asn Gly Ile His Leu Ile Gly Leu 245 250 255 Ala Ala Lys Gln 260 <210> 19 <211> 255 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 19 Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly Ala Gly Leu 1 5 10 15 Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys Gly Leu Gln 20 25 30 Ser Leu Thr Leu Asp Gln Ser Val Ser Lys Asn Glu Lys Leu Lys Leu 35 40 45 Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn 50 55 60 Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe Ile Arg 65 70 75 80 Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly Glu Phe 85 90 95 Gln Val Tyr Lys Gln Ser His Ser Ala Leu Thr Ala Phe Gln Thr Glu 100 105 110 Gln Ile Gln Asp Ser Glu His Ser Gly Lys Met Val Ala Lys Arg Gln 115 120 125 Phe Arg Ile Gly Asp Ile Ala Gly Glu His Thr Ser Phe Asp Lys Leu 130 135 140 Pro Glu Gly Gly Arg Ala Thr Tyr Arg Gly Thr Ala Phe Gly Ser Asp 145 150 155 160 Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp Phe Ala Ala Lys Gln 165 170 175 Gly Asn Gly Lys Ile Glu His Leu Lys Ser Pro Glu Leu Asn Val Asp 180 185 190 Leu Ala Ala Ala Asp Ile Lys Pro Asp Gly Lys Arg His Ala Val Ile 195 200 205 Ser Gly Ser Val Leu Tyr Asn Gln Ala Glu Lys Gly Ser Tyr Arg Leu 210 215 220 Gly Ile Phe Gly Gly Lys Ala Gln Glu Val Ala Gly Ser Ala Glu Val 225 230 235 240 Lys Thr Val Asn Gly Ile Arg His Ile Gly Leu Ala Ala Lys Gln 245 250 255 <210> 20 <211> 255 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 20 Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly Ala Gly Leu 1 5 10 15 Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys Gly Leu Gln 20 25 30 Ser Leu Thr Leu Asp Gln Ser Val Ser Lys Asn Glu Lys Leu Lys Leu 35 40 45 Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn 50 55 60 Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe Ile Arg 65 70 75 80 Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly Glu Phe 85 90 95 Gln Val Tyr Lys Gln Ser His Ser Ala Leu Thr Ala Phe Gln Thr Glu 100 105 110 Gln Ile Gln Asp Ser Glu His Ser Gly Lys Met Val Ala Lys Arg Gln 115 120 125 Phe Arg Ile Gly Asp Ile Ala Gly Glu His Thr Ser Phe Asp Lys Leu 130 135 140 Pro Glu Gly Gly Arg Ala Thr Tyr Arg Gly Thr Ala Phe Gly Ser Asp 145 150 155 160 Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp Phe Ala Ala Lys Gln 165 170 175 Gly Asn Gly Lys Ile Glu His Leu Lys Ser Pro Glu Leu Asn Val Asp 180 185 190 Leu Ala Ala Ala Asp Ile Lys Pro Asp Gly Lys Arg His Ala Val Ile 195 200 205 Ser Gly Ser Val Leu Tyr Asn Gln Ala Glu Lys Gly Ser Tyr Ser Leu 210 215 220 Gly Ile Phe Gly Gly Lys Ala Gln Glu Val Ala Gly Ser Ala Glu Val 225 230 235 240 Lys Thr Val Asn Gly Ile Arg Leu Ile Gly Leu Ala Ala Lys Gln 245 250 255 <210> 21 <211> 255 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 21 Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly Ala Gly Leu 1 5 10 15 Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys Gly Leu Gln 20 25 30 Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys Leu Lys Leu 35 40 45 Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn 50 55 60 Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe Ile Arg 65 70 75 80 Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly Glu Phe 85 90 95 Gln Val Tyr Lys Gln Ser His Ser Ala Leu Thr Ala Phe Gln Thr Glu 100 105 110 Gln Ile Gln Asp Ser Glu His Ser Gly Lys Met Val Ala Lys Arg Gln 115 120 125 Phe Arg Ile Gly Asp Ile Ala Gly Glu His Thr Ser Phe Asp Lys Leu 130 135 140 Pro Glu Gly Gly Arg Ala Thr Tyr Arg Gly Thr Ala Phe Gly Ser Asp 145 150 155 160 Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp Phe Ala Ala Lys Gln 165 170 175 Gly Asn Gly Lys Ile Glu His Leu Lys Ser Pro Glu Leu Asn Val Asp 180 185 190 Leu Ala Ala Ala Asp Ile Lys Pro Asp Gly Lys Arg His Ala Val Ile 195 200 205 Ser Gly Ser Val Leu Tyr Asn Gln Ala Glu Lys Gly Ser Tyr Arg Leu 210 215 220 Gly Ile Phe Gly Gly Lys Ala Gln Glu Val Ala Gly Ser Ala Glu Val 225 230 235 240 Lys Thr Val Asn Gly Ile Arg Leu Ile Gly Leu Ala Ala Lys Gln 245 250 255 <210> 22 <211> 254 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 22 Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly Ala Gly Leu 1 5 10 15 Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys Ser Leu Gln 20 25 30 Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys Leu Lys Leu 35 40 45 Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn 50 55 60 Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe Ile Arg 65 70 75 80 Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly Glu Phe 85 90 95 Gln Ile Tyr Lys Gln Asp His Ser Ala Val Val Ala Leu Gln Ile Glu 100 105 110 Lys Ile Asn Asn Pro Asp Lys Ile Asp Ser Leu Ile Asn Gln Arg Ser 115 120 125 Phe Arg Val Ser Asp Leu Gly Gly Glu His Thr Ala Phe Asn Gln Leu 130 135 140 Pro Ser Gly Lys Ala Glu Tyr His Gly Lys Ala Phe Ser Ser Asp Asp 145 150 155 160 Pro Asn Gly Arg Leu His Tyr Ser Ile Asp Phe Thr Lys Lys Gln Gly 165 170 175 Tyr Gly Arg Ile Glu His Leu Lys Thr Pro Glu Gln Asn Val Glu Leu 180 185 190 Ala Ser Ala Glu Leu Lys Ala Asp Glu Lys Ser His Ala Val Ile Leu 195 200 205 Gly Asp Thr Arg Tyr Gly Gly Glu Glu Lys Gly Thr Tyr His Leu Ala 210 215 220 Leu Phe Gly Asp Arg Ala Gln Glu Ile Ala Gly Ser Ala Thr Val Lys 225 230 235 240 Ile Arg Glu Lys Val His Glu Ile Gly Ile Ala Gly Lys Gln 245 250 <210> 23 <211> 254 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 23 Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly Ala Gly Leu 1 5 10 15 Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys Ser Leu Gln 20 25 30 Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys Leu Lys Leu 35 40 45 Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn 50 55 60 Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe Ile Arg 65 70 75 80 Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly Glu Phe 85 90 95 Gln Ile Tyr Lys Gln Asp His Ser Ala Val Val Ala Leu Gln Ile Glu 100 105 110 Lys Ile Asn Asn Pro Asp Lys Ile Asp Ser Leu Ile Asn Gln Arg Ser 115 120 125 Phe Arg Val Ser Asp Leu Gly Gly Glu His Thr Ala Phe Asn Gln Leu 130 135 140 Pro Ser Gly Lys Ala Glu Tyr His Gly Lys Ala Phe Ser Ser Asp Asp 145 150 155 160 Pro Asn Gly Arg Leu His Tyr Ser Ile Asp Phe Thr Lys Lys Gln Gly 165 170 175 Tyr Gly Arg Ile Glu His Leu Lys Thr Pro Glu Gln Asn Val Glu Leu 180 185 190 Ala Ser Ala Glu Leu Lys Ala Asp Glu Lys Ser His Ala Val Ile Leu 195 200 205 Gly Asp Thr Arg Tyr Gly Gly Glu Glu Asn Gly Thr Tyr His Leu Ala 210 215 220 Leu Phe Gly Asp Arg Ala Gln Glu Ile Ala Gly Ser Ala Thr Val Lys 225 230 235 240 Ile Arg Glu Lys Val His Glu Ile Gly Ile Ala Gly Lys Gln 245 250 <210> 24 <211> 254 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 24 Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly Ala Gly Leu 1 5 10 15 Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys Ser Leu Gln 20 25 30 Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys Leu Lys Leu 35 40 45 Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn 50 55 60 Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe Ile Arg 65 70 75 80 Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly Glu Phe 85 90 95 Gln Ile Tyr Lys Gln Asp His Ser Ala Val Val Ala Leu Gln Ile Glu 100 105 110 Lys Ile Asn Asn Pro Asp Lys Ile Asp Ser Leu Ile Asn Gln Arg Ser 115 120 125 Phe Arg Val Ser Asp Leu Gly Gly Glu His Thr Ala Phe Asn Gln Leu 130 135 140 Pro Ser Gly Lys Ala Glu Tyr His Gly Lys Ala Phe Ser Ser Asp Asp 145 150 155 160 Pro Asn Gly Arg Leu His Tyr Ser Ile Asp Phe Thr Lys Lys Gln Gly 165 170 175 Tyr Gly Arg Ile Glu His Leu Lys Thr Pro Glu Gln Asn Val Glu Leu 180 185 190 Ala Ser Ala Glu Leu Lys Ala Asp Glu Lys Ser His Ala Val Ile Leu 195 200 205 Gly Asp Thr Arg Tyr Gly Gly Glu Glu Lys Ser Thr Tyr His Leu Ala 210 215 220 Leu Phe Gly Asp Arg Ala Gln Glu Ile Ala Gly Ser Ala Thr Val Lys 225 230 235 240 Ile Arg Glu Lys Val His Glu Ile Gly Ile Ala Gly Lys Gln 245 250 <210> 25 <211> 174 <212> PRT <213> Neisseria meningitidis <400> 25 Met Lys Lys Ala Leu Ala Thr Leu Ile Ala Leu Ala Leu Pro Ala Ala 1 5 10 15 Ala Leu Ala Glu Gly Ala Ser Gly Phe Tyr Val Gln Ala Asp Ala Ala 20 25 30 His Ala Lys Ala Ser Ser Ser Leu Gly Ser Ala Lys Gly Phe Ser Pro 35 40 45 Arg Ile Ser Ala Gly Tyr Arg Ile Asn Asp Leu Arg Phe Ala Val Asp 50 55 60 Tyr Thr Arg Tyr Lys Asn Tyr Lys Ala Pro Ser Thr Asp Phe Lys Leu 65 70 75 80 Tyr Ser Ile Gly Ala Ser Ala Ile Tyr Asp Phe Asp Thr Gln Ser Pro 85 90 95 Val Lys Pro Tyr Leu Gly Ala Arg Leu Ser Leu Asn Arg Ala Ser Val 100 105 110 Asp Leu Gly Gly Ser Asp Ser Phe Ser Gln Thr Ser Ile Gly Leu Gly 115 120 125 Val Leu Thr Gly Val Ser Tyr Ala Val Thr Pro Asn Val Asp Leu Asp 130 135 140 Ala Gly Tyr Arg Tyr Asn Tyr Ile Gly Lys Val Asn Thr Val Lys Asn 145 150 155 160 Val Arg Ser Gly Glu Leu Ser Val Gly Val Arg Val Lys Phe 165 170 <210> 26 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 26 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 <210> 27 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 27 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 28 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 28 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10

Claims

**Claim 1** A variant of the H factor-binding protein (fHbp), wherein the variant comprises at least one amino acid substitution selected from the following: (a) an amino acid substitution of glutamine (Q38) at amino acid 38; (b) an amino acid substitution of glutamic acid (E92) at amino acid 92; (c) a glycine substitution for arginine at amino acid 130 (R130G); (d) an amino acid substitution of serine (S223) at amino acid 223; and (e) a histidine substitution for leucine at amino acid 248 (H248L) wherein the amino acid substitution is relative to fHbp ID1 (SEQ ID NO: 1), the variant comprises an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 1, the variant fHbp binds to human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID1 for human fH, and the variant induces a bactericidal antibody response against at least one strain of Neisseria meningitidis in a mammalian host. **Claim 2** The variant fHbp according to claim 1, wherein the amino acid substitution at Q38 is Q38R, Q38K, Q38H, Q38F, Q38Y, or Q38W. **Claim 3** The variant fHbp according to claim 1, wherein the amino acid substitution at E92 is E92K, E92R, E92H, E92F, E92Y, or E92W. **Claim 4** The variant fHbp according to claim 1, wherein the amino acid substitution at S223 is S223R, S223K, S223H, S223F, S223Y, or S223W. **Claim 5** The variant fHbp according to claim 1, which binds to human fH with an affinity that is 25% or less of the affinity of fHbp ID1 for human fH. **Claim 6** The variant fHbp according to claim 1, which binds to human fH with an affinity that is 10% or less of the affinity of fHbp ID1 for human fH. **Claim 7** The variant fHbp according to claim 1, which binds to human fH with an affinity that is 5% or less of the affinity of fHbp ID1 for human fH. **Claim 8** The variant fHbp according to any one of claims 1 to 7, further comprising an amino acid substitution selected from the group consisting of R41S and R41A. **Claim 9** The variant fHbp according to any one of claims 1 to 8, further comprising the following amino acid substitutions S223R and H248L. **Claim 10** ​ A variant of factor H-binding protein (fHbp), wherein the variant is as follows: (a) an isoleucine substitution for asparagine at amino acid 115 (N115I); (b) a glycine substitution for aspartic acid at amino acid 121 (D121G); (c) a threonine substitution for serine at amino acid 128 (S128T); (d) an amino acid substitution at valine (V131) at position 131; (e) an amino acid substitution at lysine (K219) at position 219; (f) an amino acid substitution at glycine (G220) at position 220 comprising an amino acid substitution selected from: wherein the amino acid substitution is with respect to fHbp ID22 (SEQ ID NO: 2); wherein the variant comprises an amino acid sequence having an amino acid sequence identity higher than 85% with SEQ ID NO: 2; wherein the variant fHbp binds to human factor H (fH) with an affinity of 50% or less of the affinity of fHbp ID22 for human fH; wherein the variant induces a bactericidal antibody response in a mammalian host.

11. The variant fHbp according to claim 10, which binds to human fH with an affinity of 25% or less of the affinity of fHbp ID22 for human fH.

12. The variant fHbp according to claim 10, which binds to human fH with an affinity of 10% or less of the affinity of fHbp ID22 for human fH.

13. The variant fHbp according to claim 10, which binds to human fH with an affinity of 5% or less of the affinity of fHbp ID22 for human fH.

14. The variant fHbp according to claim 10, wherein the amino acid substitution at V131 is V131D, V131E, V131K, V131R, V131H, V131F, V131Y, or V131W.

15. The variant fHbp according to claim 10, wherein the amino acid substitution at K219 is K219N, K219Q, K219D, K219E, K219F, K219Y, or K219W.

16. The variant fHbp according to claim 10, wherein the amino acid substitution at G220 is G220S, G220N, G220Q, G220D, G220E, G220K, G220R, G220H, G220F, G220Y, or G220W.

17. The variant fHbp according to any one of claims 10 to 16, further comprising the following substitutions L130R and G133D, wherein the amino acid substitution is with respect to fHbp ID22.

18. The mutant fHbp according to claim 10, comprising the substitutions L130R, G133D, and N115I.

19. The mutant fHbp according to claim 10, comprising the substitutions L130R, G133D, and D121G.

20. The mutant fHbp according to claim 10, comprising the substitutions L130R, G133D, and K219N, or the substitutions L130R, G133D, and D211A.

21. The mutant fHbp according to claim 10, comprising the substitution L130R, G133D, and G220S.

22. A mutant of factor H-binding protein (fHbp), wherein the mutant comprises the following: (a) an amino acid substitution at position 92 of glutamic acid (E92); (b) an amino acid substitution at position 223 of serine (S223); and (c) an amino acid substitution at position 248 of histidine (H248) selected from the amino acid substitutions, the amino acid substitution being relative to fHbp ID55 (SEQ ID NO: 3), the mutant comprising an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO: 3, the mutant fHbp binding to human factor H (fH) with an affinity less than 50% of the affinity of fHbp ID55 for human fH, the mutant inducing a bactericidal antibody response in a mammalian host.

23. The mutant fHbp according to claim 22, which binds to human fH with an affinity of 25% or less of the affinity of fHbp ID55 for human fH.

24. The mutant fHbp according to claim 22, which binds to human fH with an affinity of 10% or less of the affinity of fHbp ID55 for human fH.

25. The mutant fHbp according to claim 22, which binds to human fH with an affinity of 5% or less of the affinity of fHbp ID55 for human fH.

26. The mutant fHbp according to claim 22, wherein the amino acid substitution at E92 is E92K, E92R, E92H, E92F, E92Y, or E92W.

27. The mutant fHbp according to claim 22, wherein the amino acid substitution at S223 is S223R, S223K, S223H, S223F, S223Y, or S223W.

28. The mutant fHbp according to claim 22, wherein the amino acid substitution at H248 is H248L, H248I, H248V, H248D, H248E, H248F, H248Y, or H248W.

29. (a) A variant fHbp according to any one of claims 1 to 28; and (b) A pharmaceutically acceptable excipient An immunogenic composition comprising.

30. The immunogenic composition according to claim 29, wherein the variant fHbp is in a vesicle preparation prepared from a Neisseria meningitidis strain.

31. The immunogenic composition according to claim 29 or claim 30, wherein the pharmaceutically acceptable excipient comprises an adjuvant.

32. The immunogenic composition according to any one of claims 29 to 31, further comprising Neisseria surface protein A.

33. The immunogenic composition according to claim 31, wherein the adjuvant is aluminum phosphate or aluminum hydroxide.

34. A nucleic acid encoding a variant fHbp according to any one of claims 1 to 28.

35. A recombinant expression vector comprising the nucleic acid according to claim 34.

36. An in vitro host cell comprising the nucleic acid according to claim 34 or the recombinant expression vector according to claim 35.

37. A method for inducing an antibody response in a mammal, comprising administering the immunogenic composition according to any one of claims 29 to 33 to the mammal.

38. The method according to claim 37, wherein the mammal is a human.

39. The method according to claim 37, wherein the antibody response is a bactericidal antibody response against one or more strains of N. meningitidis (meningococcus).

40. A variant of factor H binding protein (fHbp), the variant comprising amino acid substitutions L130R and G133D relative to fHbp ID22 (SEQ ID NO: 2), the variant comprises an amino acid sequence having an amino acid sequence identity higher than 85% with SEQ ID NO: 2, the variant fHbp binds to human factor H (fH) with an affinity of 50% or less of the affinity of fHbp ID22 for human fH, the variant induces a bactericidal antibody response in a mammalian host.

41. The variant fHbp according to claim 40, further comprising one or more of the following substitutions: R80A, D211A, E218A, E248A, G236I, T221A, and H223A.

42. The variant fHbp according to claim 40, further comprising the substitution R80A.

43. The variant fHbp according to claim 40 or 42, further comprising the substitution D211A.

44. The mutant fHbp according to claim 40 or 43, further comprising the substitution E218A. **Claim 45** The mutant fHbp according to claim 40 or 44, further comprising the substitution E248A. **Claim 46** The mutant fHbp according to claim 40 or 45, further comprising the substitution G236I. **Claim 47** The mutant fHbp according to claim 40 or 46, further comprising the substitutions T221A and H223A. **Claim 48** The mutant fHbp according to any one of claims 40 to 47, comprising an amino acid sequence having an amino acid sequence identity higher than 90% with SEQ ID NO:

2. **Claim 49** The mutant fHbp according to any one of claims 40 to 47, comprising an amino acid sequence having an amino acid sequence identity higher than 95% with SEQ ID NO:

2. **Claim 50** The mutant fHbp according to any one of claims 40 to 47, comprising an amino acid sequence having an amino acid sequence identity higher than 99% with SEQ ID NO: 2.

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