Factor H binding protein variants and methods of use thereof

Abstract

Factor H binding protein variants and methods of use thereof are provided. Variant factor H binding proteins that can elicit antibodies that are bactericidal against at least one strain of Neisseria meningitidis, compositions comprising the proteins, and methods of using the proteins are provided.

Description

[0001] This application is a re-divisional application of the divisional application of the invention application with the application date of July 22, 2015, the Chinese application number of 202110192031.1, and the invention name of “Factor H Binding Protein Variants and Methods of Use Thereof” (the original application number is 201580043542.X).

[0002] Cross-references

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 028,123, filed on July 23, 2014, which is incorporated herein by reference in its entirety.

[0004] introduction

[0005] Neisseria meningitidis is a gram-negative bacterium that colonizes the human upper respiratory tract and is most notably responsible for sporadic and periodic epidemic outbreaks worldwide of meningitis and septicemia. The invasion and morbidity rates are highest in children under 2 years of age. Like other gram-negative bacteria, Neisseria meningitidis usually has a cytoplasmic membrane, a peptidoglycan layer, an outer membrane that forms the bacterial wall together with the capsular polysaccharide, and pili that protrude into the external environment. Encapsulated strains of Neisseria meningitidis are the main causes of bacterial meningitis and septicemia in children and young adults. The prevalence and economic importance of invasive Neisseria meningitidis infections have promoted research into effective vaccines that can confer immunity across different strains, particularly across genetically diverse serogroup B with different serotypes or serosubtypes.

[0006] Factor H binding protein (fHbp, also known in the art as lipoprotein 2086 (Fletcher et al. (2004) Infect Immun 72:2088-2100), genomic derived Neisseria antigen (GNA) 1870 (Masignani et al. (2003) J Exp Med 197:789-99) or "741") is a Neisseria meningitidis protein expressed in the bacterium as a surface-exposed lipoprotein. An important function of fHbp is to bind human complement factor H (fH), which downregulates complement activation. Binding of fH to the bacterial surface is an important mechanism by which pathogens survive in non-immune human serum or blood and evade innate host defenses. Recently, genetic variation in the human factor H gene cluster was found to influence susceptibility to developing meningococcal disease (Davila S et al. (2010) Nat Genetics doi:10.1038 / ng.640). Binding of fH to fHbp is specific for human fH and several non-human primates and may explain in part why Neisseria meningitidis is strictly a human pathogen.fHbp occurs in many natural sequence variants designated by identification (ID) numbers, as specified in the fHbp database on the Internet at pubmlst(dot)org / neisseria / fHbp.

[0007] There remains a need for fHbp polypeptides that can elicit effective bactericidal antibody responses.

[0008] Overview

[0009] Variant Factor H binding proteins that can elicit antibodies that are bactericidal against at least one strain of Neisseria meningitidis, compositions comprising the proteins, and methods of using the proteins are provided.

[0010] feature

[0011] The present disclosure provides variants of factor H binding protein (fHbp) ID 1. The present disclosure provides variants of fHbp, wherein the variant comprises at least one amino acid substitution selected from the group consisting of: 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 substitution of arginine with glycine at amino acid 130 (R130G); d) an amino acid substitution of serine (S223) at amino acid 223; and e) a substitution of leucine with histidine at amino acid 248 (H248L), wherein the amino acid substitution is relative to fHbp ID 1 (SEQ ID NO: 1), wherein the variant comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO: 1, wherein the variant fHbp binds to human factor H (fH) with an affinity of 50% or less of the affinity of fHbp ID 1 for human fH, and wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host. In some cases, the amino acid substitution at Q38 is Q38R, Q38K, Q38H, Q38F, Q38Y or Q38W. In some cases, the amino acid substitution at E92 is E92K, E92R, E92H, E92F, E92Y or E92W. In some cases, the amino acid substitution at S223 is S223R, S223K, S223H, S223F, S223Y or S223W. In some cases, variant fHbp may also include R41S or R41A substitutions relative to fHbpID 1. For example, relative to fHbp ID 1, variant fHbp may include R41S or R41A substitutions and substitutions at S223, such as R41S / S223R. In other cases, variant fHbp may also include R41S or R41A substitutions and H248L substitutions relative to fHbp ID 1. In some cases, the variant fHbp may comprise two, three or more substitutions disclosed herein. In a specific example, the variant fHbp may comprise the following substitutions relative to fHbp ID 1: S223R and H248L. In some cases, the variant fHbp binds to human fH with an affinity of 25% or less of the affinity of fHbp ID 1 to human fH. In some cases, the variant fHbp binds to human fH with an affinity of 10% or less of the affinity of fHbp ID 1 to human fH. In some cases, the variant fHbp binds to human fH with an affinity of 5% or less of the affinity of fHbp ID 1 to human fH.

[0012] The present disclosure provides variants of fHbp ID 22. The present disclosure provides variants of fHbp, wherein the variant comprises at least one amino acid substitution selected from the group consisting of: a) substitution of asparagine with isoleucine at amino acid 115 (N115I); b) substitution of aspartic acid with glycine at amino acid 121 (D121G); c) substitution of serine with threonine at amino acid 128 (S128T); d) amino acid substitution of valine at position 131 (V131); e) amino acid substitution of lysine at position 219 (K219); f) amino acid substitution of glycine at position 220 (G220), wherein the amino acid substitutions are relative to fHbp ID 22 (SEQ ID NO:2), wherein the variant comprises an amino acid sequence having greater than 85% amino acid sequence identity to 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 ID 22 for human fH, and wherein the variant induces a bactericidal antibody response in a mammalian host. In some cases, the variant fHbp binds to human fH with an affinity of 25% or less of the affinity of fHbp ID 22 for human fH. In some cases, the variant fHbp binds to human fH with an affinity of 10% or less of the affinity of fHbp ID 22 for human fH. In some cases, the variant fHbp binds to human fH with an affinity of 5% or less of the affinity of fHbp ID 22 for human fH. In some cases, the amino acid substitution at V131 is V131D, V131E, V131K, V131R, V131H, V131F, V131Y, or V131W. In some cases, the amino acid substitution at K219 is K219N, K219Q, K219D, K219E, K219F, K219Y, or K219W. In some cases, the amino acid substitution at G220 is G220S, G220N, G220Q, G220D, G220E, G220K, G220R, G220H, G220F, G220Y, or G220W.

[0013] In some cases, the variant fHbp includes a double mutation that increases the thermostability of the variant fHbp compared to the thermostability of a wild-type (WT) fHbp (e.g., WT fHbp ID 22). In some cases, the variant fHbp may include substitutions L130R and G133D relative to fHbp ID 22 (SEQ ID NO: 2), wherein the variant fHbp includes an amino acid sequence having greater than 85% amino acid sequence identity to 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 ID 22 for human fH, wherein the variant induces a bactericidal antibody response in a mammalian host, and wherein the variant has a higher thermostability compared to the thermostability of fHbp ID 22. In some cases, the variant fHbp may comprise a combination of substitutions, such as L130R, G133D and at least one amino acid substitution selected from the group consisting of: a) N115I; b) D121G; c) S128T; d) V131; e) K219 (e.g., K219N); and f) G220 (e.g., G220S), wherein the amino acid substitutions are relative to fHbp ID 22 (SEQ ID NO:2), wherein the variant fHbp comprises an amino acid sequence having greater than 85% amino acid sequence identity to 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 ID 22 for human fH, and wherein the variant induces a bactericidal antibody response in a mammalian host. The thermal stability of variant fHbp can be at least 5°C, 10°C, 15°C, 20°C or higher than WT fHbp (e.g., fHbp ID 22), for example, 5°C-30°C, 5°C-25°C, 5°C-20°C, 10°C-20°C or 15°C-20°C. As used herein, "thermostability" refers to the stability of a protein when exposed to a higher temperature; a thermostable variant protein maintains its conformation at a temperature higher than that of a wild-type protein. For example, a variant fHbp comprising a double mutation that increases thermal stability compared to the thermal stability of wild-type (WT) fHbp (e.g., WT fHbp ID 22) can be unfolded at a higher temperature than WT fHbp. In some cases, the N-terminal domain of a variant fHbp can be unfolded at a higher temperature than the N-terminal domain of WT fHbp (e.g., fHbp ID 22).

[0014] Also disclosed herein are fHbp variants comprising mutations that enhance thermal stability compared to WT fHbp and further comprising additional mutations known to reduce fH binding, such as those disclosed in US2011 / 0256180. In certain embodiments, variants of factor H binding protein (fHbp) are disclosed, wherein relative to fHbp ID 22 (SEQ ID NO: 2), the variant comprises amino acid substitutions L130R and G133D and at least one of the following substitutions: R80A, D211A, E218A, E248A, G236I, T221A, and H223A, wherein the variant comprises an amino acid sequence having greater than 85% amino acid sequence identity to 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 ID 22 for human fH, and wherein the variant induces a bactericidal antibody response in a mammalian host.

[0015] The present disclosure provides variants of fHbp ID 55. The present disclosure provides variants of fHbp, wherein the variant comprises at least one amino acid substitution selected from the following: 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 amino acid substitution is relative to fHbp ID 55 (SEQ ID NO: 3), wherein the variant comprises an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO: 3, wherein the variant fHbp binds to human factor H (fH) with an affinity of less than 50% of the affinity of fHbp ID 55 for human fH, and wherein the variant induces a bactericidal antibody response in a mammalian host. In some cases, the variant fHbp binds to human fH with an affinity of 25% or less of the affinity of fHbp ID 55 for human fH. In some cases, the variant fHbp binds to human fH with an affinity of 10% or less of the affinity of fHbp ID 55 for human fH. In some cases, the variant fHbp binds to human fH with an affinity of 5% or less of the affinity of fHbp ID 55 for human fH. In some cases, the amino acid substitution at E92 is E92K, E92R, E92H, E92F, E92Y, or E92W. In some cases, the amino acid substitution at S223 is S223R, S223K, S223H, S223F, S223Y, or S223W. In some cases, the amino acid substitution at H248 is H248L, H248I, H248V, H248D, H248E, H248F, H248Y, or H248W.

[0016] The present disclosure provides immunogenic compositions comprising variant fHbp of the present disclosure. The present disclosure provides immunogenic compositions comprising: a) variant fHbp according to any of paragraphs 0006-0010 above; and b) a pharmaceutically acceptable excipient. In some cases, the fHbp variant is in a vesicle formulation prepared from a strain of Neisseria meningitidis. In some cases, the pharmaceutically acceptable excipient includes an adjuvant; for example, wherein the adjuvant is aluminum phosphate or aluminum hydroxide. In some cases, the pharmaceutical composition further comprises Neisseria surface protein A.

[0017] The present disclosure provides nucleic acids encoding variant fHbp according to any of paragraphs 0006-0010 above. The present disclosure provides recombinant expression vectors comprising nucleic acids encoding variant fHbp according to any of paragraphs 0006-0010 above. The present disclosure provides in vitro host cells comprising nucleic acids encoding variant fHbp according to any of paragraphs 0006-0010 above. The present disclosure provides in vitro host cells comprising recombinant expression vectors comprising nucleic acids encoding variant fHbp according to any of paragraphs 0006-0010 above.

[0018] The present disclosure provides a method of eliciting an antibody response in a mammal, the method comprising administering to the mammal the immunogenic composition of paragraph 0011 above. In some cases, the mammal is a human. In some cases, the antibody response is a bactericidal antibody response to one or more strains of Neisseria meningitidis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Purified recombinant fHbp ID 1 mutants stained with Coomassie blue on a polyacrylamide gel are depicted. Lane 1, Kaleidoscope molecular weight marker (Bio-Rad Laboratories); 2, fHbp ID 1 wild type; 3, Q38R; 4, E92K; 5, R130G; 6, S223R; 7, H248L.

[0020] Figure 2 (Panels A and B) depicts binding of fHbp ID 1 mutants to human fH measured by ELISA. Means and ranges of replicate measurements are shown.

[0021] Figure 3 (Panels AE) depicts the binding of fHbp ID 1 mutants to human fH measured by surface plasmon resonance. As a reference, the same data for ID 1 wild-type (WT) protein are shown in each of Figure 3 Panels AE.

[0022] Figure 4 (Panels AE) depicts the binding of murine anti-fHbp monoclonal antibodies (mAbs) to fHbp ID 1 mutant proteins measured by ELISA. Means and ranges of replicate measurements are shown.

[0023] Figure 5 (panels A and B) depicts the bactericidal activity of sera from mice immunized with fHbp ID 1 mutants. Each symbol represents the titer of a single mouse, and the horizontal bar represents the geometric mean titer. Figure 5 panel A depicts the bactericidal activity of sera from wild-type mice immunized with fHbp ID 1 mutants. Figure 5 panel B depicts the bactericidal activity of sera from human fH transgenic mice immunized with fHbp ID 1 mutants.

[0024] Figure 6 (Panels A and B) depict characterization of fHbp ID 1 single or double mutants. Figure 6 Panel A depicts binding of human fH to fHbp ID 1 double mutants. Figure 6 Panel B depicts binding of murine anti-fHbp monoclonal antibody (mAb) JAR4 to the mutants.

[0025] Figure 7 Depicted are the bactericidal activities of sera from wild-type mice immunized with single or double mutants of fHbp ID 1. Each symbol represents the titer of an individual mouse, and the horizontal bars represent the geometric mean titer.

[0026] Figure 8 (panels A and B) depicts the characterization of fHbp ID 55 mutants. Figure 8 panel A shows the binding of human fH to fixed fHbp ID 55 mutants measured by ELISA, and Figure 8 panel B shows the binding of mouse anti-fHbp monoclonal antibody (mAb) JAR 41 to fHbp mutant ID 55 protein measured by ELISA. The average and range of 2 to 4 replicates are shown.

[0027] Fig. 9 Depicted are the bactericidal activities of sera from wild-type mice immunized with the fHbp ID 55 mutant. Each symbol represents the titer of an individual mouse, and the horizontal bar represents the geometric mean titer.

[0028] Figure 10 (panels A and B) depicts the bactericidal activity of sera from mice immunized with fHbp ID 55. Figure 10 panel A depicts the bactericidal activity of sera from human fH transgenic mice immunized with licensed Trumenba vaccine or investigational fHbp ID 55 mutant S223R. Figure 10 panel B depicts the relationship between serum human fH concentration and serum bactericidal antibody titers (circular symbols) in individual transgenic mice. For comparison, titers of wild-type (WT) mice are shown (squares).

[0029] Figure 11 (panels AD) depicts the characterization of fHbp ID 22 mutants. Figure 11 panels AC depict the binding of fHbp ID 22 mutants to human fH measured by ELISA. The average and range of 2 to 4 replicates are shown. Figure 11 panel D depicts the binding of mouse anti-fHbp monoclonal antibody (mAb) JAR 4 to fHbp mutant ID 22 protein measured by ELISA. fHbp ID 22 wild type (WT) and D211A mutants are shown as controls. The average and range of replicate measurements are shown.

[0030] Figure 12 (panels A and B) shows the bactericidal activity of sera from mice immunized with fHbp ID 22 mutants. Each symbol represents the titer of a single mouse, and the horizontal bar represents the geometric mean titer. Figure 12 panels A and B show the bactericidal activity of sera from wild-type mice in two experiments testing different fHbp ID 22 mutants.

[0031] Fig.13 Depicted are the bactericidal activity of sera from human fH transgenic mice immunized with the fHbp ID 22 mutant.

[0032] Fig.14 Depicted are thermal unfolding of fHbp ID 22 wild type (WT) and L130R / G133D double mutant measured by differential scanning microcalorimetry.

[0033] Figure 15 (Panels A and B) depict characterization of the fHbp ID 22 triple mutant. Figure 15 Panel A depicts binding of human fH to the fHbp ID 22 triple mutant. DM refers to the L130R / G133D double mutant. Figure 15 Panel B depicts binding of the murine anti-fHbp monoclonal antibody (mAb) JAR 4 to the fHbp ID 22 triple mutant.

[0034] Fig.16 Depicted are the bactericidal activity of sera from human fH transgenic mice immunized with the triple mutant of fHbp ID 22. DM refers to L130R / G133D double mutant.

[0035] Fig.17 A table of exemplary fHbp mutants with reduced human fH binding is provided.

[0036] Fig.18 The amino acid sequence of wild-type human Factor H is provided.

[0037] Fig.19 The amino acid sequences of fHbp ID 1, ID 22 and ID 55 from Neisseria meningitidis strains are provided.

[0038] Figure 20-24 Amino acid sequences of fHbp ID 1 variants are provided.

[0039] Figure 25-Figure 30 The amino acid sequence of the fHbp ID 22 variant is provided.

[0040] Figure 31-Figure 33 The amino acid sequence of the fHbp ID 55 variant is provided.

[0041] Figure 34-Figure 36 Amino acid sequences of fHbp ID 1 double mutant variants are provided.

[0042] Figure 37-Figure 39 Amino acid sequences of fHbp ID 22 double and triple mutation variants are provided.

[0043] Fig.40 The amino acid sequence of NspA is provided.

[0044] definition

[0045] "Factor H binding protein" (fHbp), also referred to in the literature as GNA1870, GNA 1870, ORF2086, LP2086 (lipoprotein 2086) and "741" refers to a class of Neisseria meningitidis polypeptides. It is found in nature as a lipoprotein on the surface of bacterial Neisseria meningitidis. Based on amino acid sequence variability and immune cross-reactivity (Masignani et al. (2003) J Exp Med 197: 789-99), fHbp has been subdivided into three fHbp variant groups (referred to as variant group 1 (v.1), variant group 2 (v.2) and variant group 3 (v.3)) in some reports (Masignani et al. (2003) J Exp Med 197: 789-99) and in other reports into subfamilies A and B (see, e.g., Fletcher et al. (2004) Infect Immun 72: 2088-2100)). fHbp can also be divided into one of the six most common fHbp module groups, referred to as module group I to module group VI, as shown in Figure 2 of Vu et al. (2012) Sci. Reports 2:341. According to the pubmlst.org / neisseria / fHbp / website, each unique fHbp found in Neisseria meningitidis is also assigned an fHbp peptide ID. Because the length of variant 2 (v.2) fHbp protein (from strain 8047, fHbp ID 77) and variant 3 (v.3) fHbp (from strain M1239, fHbp ID 28) differs from the fHbp protein (fHbp ID 1) of strain MC58 by -1 and +7 amino acid residues, the numbering of the residues used to refer to v.2 and v.3 fHbp proteins herein is different from the numbering based on the actual amino acid sequences of these proteins. Thus, for example, reference to the leucine residue (L) at position 166 of a v.2 or v.3 fHbp sequence refers to residue 165 of the v.2 protein and residue 173 of the v.3 protein. Unless otherwise indicated, the numbering of amino acid substitutions present in fHbp variants is by reference to the numbering of amino acid residues in fHbp ID 1.

[0046] As used herein, human factor H ("human fH") refers to a protein comprising Fig.18 (SEQ ID NO: 4) and its naturally occurring human allelic variants.

[0047] "Derived from" in the context of an amino acid sequence or polynucleotide sequence (e.g., an amino acid sequence "derived from" fHbp ID 1) is intended to mean that the 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 encoding nucleic acid), and does not imply a limitation on the source or method of making the protein or nucleic acid. Non-limiting examples of reference polypeptides and reference polynucleotides from which an amino acid sequence or polynucleotide sequence may be "derived from" include naturally occurring fHbp, fHbp ID 1, and non-naturally occurring fHbp. In the context of a bacterial strain, "derived from" means that the strain is obtained by in vivo passage or in vitro culture of a parent strain, and / or is a recombinant cell obtained by modifying a parent strain.

[0048] "Conservative amino acid substitution" refers to the substitution of one amino acid residue by another amino acid residue that shares the chemical and physical properties (e.g., charge, size, hydrophobicity / hydrophilicity) of the amino acid side chain. "Conservative substitution" is intended to include substitutions within the following amino acid residue groups: gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Guidance for such substitutions can be extracted from alignments of amino acid sequences of polypeptides that present an epitope of interest.

[0049] The term "protective immunity" refers to a vaccine or immunization regimen administered to a mammal that induces an immune response that prevents disease caused by Neisseria meningitidis, delays the development of disease caused by Neisseria meningitidis, or reduces the severity of disease caused by Neisseria meningitidis, or reduces or completely eliminates the symptoms of the 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 recognized in the art as predictive of the protective effect of the vaccine in humans. (Goldschneider et al. (1969) J. Exp. Med. 129: 1307; Borrow et al. (2001) Infect Immun. 69: 1568).

[0050] The phrase "disease caused by a strain of Neisseria meningitidis" encompasses any clinical symptom or combination of clinical symptoms present in human infection with Neisseria meningitidis. These symptoms include, but are not limited to, colonization of the upper respiratory tract (e.g., mucosa of the nasopharynx and tonsils) by pathogenic strains of Neisseria meningitidis, bacterial penetration into the mucosal and submucosal vascular beds, sepsis, septic shock, inflammation, hemorrhagic skin lesions, activation of fibrinolysis and blood coagulation, organ dysfunction, such as renal, pulmonary and heart failure, adrenal hemorrhage and muscle infarction, capillary leakage, edema, peripheral limb ischemia, respiratory distress syndrome, pericarditis and meningitis.

[0051] In the context of an antigen (e.g., a polypeptide antigen), the phrase "specifically binding antibody" or "specifically immunoreacting with" refers to a binding reaction based on and / or demonstrating the presence of an antigen in a sample, which may also include a heterogeneous population of other molecules. Thus, under specified conditions, the specified antibody or antibodies bind to one or more specific antigens in the sample and do not bind to other molecules present in the sample in significant amounts. In the context of an epitope of an antigen (e.g., an epitope of a polypeptide), "specifically binding antibody" or "specifically immunoreacting with" refers to a binding reaction based on and / or demonstrating the presence of an epitope in an antigen (e.g., a polypeptide), which may also include a heterogeneous population of other epitopes, as well as a heterogeneous population of antigens. Thus, under specified conditions, the specified antibody or antibodies bind to a specific epitope of an antigen and do not bind to other epitopes present in the antigen and / or sample in significant amounts.

[0052] The phrase "in an amount sufficient to elicit an immune response" means that there is a detectable difference between immune response indicators measured before and after administration of a particular antigen preparation. Immune response indicators include, but are not limited to: antibody titer or specificity detected by assays such as enzyme-linked immunosorbent assay (ELISA), bactericidal assays, flow cytometry, immunoprecipitation, Ouchterlony immunodiffusion; binding detection assays such as spots, Western blots or antigen arrays; cytotoxicity assays, etc.

[0053] "Surface antigens" are antigens present in surface structures of Neisseria meningitidis (eg, outer membrane, capsule, pili, etc.).

[0054] "Isolated" refers to an entity of interest in an environment different from that in which the compound may naturally occur. "Isolated" is intended to include a compound that is substantially enriched for the compound of interest and / or in which the compound of interest is partially or substantially purified. In some cases, 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, e.g., an isolated component is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or greater than 99% pure.

[0055] "Enriched" refers to the non-natural manipulation (e.g., by an experimenter or clinician) of a sample so that the compound of interest is present in a concentration that is greater (e.g., at least 3-fold greater, at least 4-fold greater, at least 8-fold greater, at least 64-fold greater, or more) than the concentration of the compound in the starting sample, such as a biological sample (e.g., a sample in which the compound occurs naturally or in which it is present following administration), or in which the compound is prepared (e.g., as in bacterial polypeptides, antibodies, nucleic acids, etc.).

[0056] Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described, and therefore can certainly change. It should also be understood that the terminology used herein is only used for the purpose of describing specific embodiments, and is not intended to be limiting, as the scope of the present invention will only be limited by the appended claims.

[0057] Where a range of values ​​is provided, it will be understood that unless the context clearly indicates otherwise, each intermediate value (to one tenth of the lower limit unit) between the upper and lower limits of the range, and any other described range or intermediate values ​​within the described range are included within the present invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range, and are also included within the present invention, subject to any specifically excluded limitations in the described range. When the described range includes one or two limitations, the scope excluding one or both of those included limitations is also included within the present invention.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials associated with the cited publications.

[0059] It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "Factor H binding protein" includes a plurality of such Factor H binding proteins, and reference to an "immunogenic composition" includes reference to one or more immunogenic compositions and equivalents thereof known to those skilled in the art, and so forth. It should also be noted that the claims may be drafted to exclude any optional elements. Therefore, this specification is intended to serve as a reference basis for the use of exclusive terminology such as "solely", "only", etc., in connection with the recitation of claim elements, or for the use of a "negative" limitation.

[0060] It should be understood that certain features of the invention described in the context of separate embodiments for the sake of clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for the sake of brevity may also be provided individually or in any suitable sub-combination. All combinations of embodiments belonging to the invention are specifically encompassed by the invention and disclosed herein, just as each combination is individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically encompassed by the invention and disclosed herein, just as each such sub-combination is individually and explicitly disclosed herein.

[0061] The publications discussed herein are provided solely because they were disclosed prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to preempt such disclosures by virtue of prior invention. In addition, the dates of the publications provided may differ from the actual publication dates which may require independent confirmation. DETAILED DESCRIPTION

[0062] The present disclosure provides variant factor H binding proteins (fHbp) that can elicit antibodies that are bactericidal to at least one strain of Neisseria meningitidis. The present disclosure provides compositions comprising the variant fHbp of the present disclosure, including immunogenic compositions. The present disclosure provides methods of using the variant fHbp of the present disclosure or compositions comprising the variant fHbp of the present disclosure.

[0063] Variant FHBP

[0064] The present disclosure provides variant fHbp, which differs from wild-type meningitidis fHbp in amino acid sequence by 1 to 10 amino acids (e.g., from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids), 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 30 amino acids, 30 amino acids to 40 amino acids or 40 amino acids to 50 amino acids, such that compared with the reference fHbp, the variant fHbp exhibits reduced affinity with human factor H (fH), and wherein the variant fHbp induces a bactericidal immune response to one or more meningitidis strains when administered to a mammalian host. In some cases, the variant fHbp differs from the reference wild-type meningitidis fHbp in amino acid sequence by no more than 1 to 10 acid substitutions. In some cases, the variant fHbp differs from the reference wild-type meningitidis fHbp by only one amino acid substitution in amino acid sequence.

[0065] In some cases, 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 a reference fHbp sequence; wherein 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 exhibits about 10% or less of the binding affinity of the reference fHbp for human fH. 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%, or about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1% affinity for human fH; and when administered to a mammalian host (e.g., a human; or a non-human animal model), the variant fHbp induces a bactericidal immune response to at least one strain of Neisseria meningitidis.

[0066] When the reference fHbp is in its native conformation, the variant fHbp of the present disclosure maintains substantially the same conformation of the reference (e.g., wild-type) fHbp bound to human fH. Whether the variant fHbp of the present disclosure maintains substantially the same conformation of the reference (e.g., wild-type) fHbp bound to human fH can be determined using an antibody that binds to wild-type fHbp when the wild-type fHbp is in its native conformation. Such antibodies include, for example, JAR 41; JAR 4; and JAR 31. See, for example, Vu et al. (2012) Sci. Reports 2:341. The hybridoma producing the JAR 4 monoclonal antibody has an American Type Culture Collection (ATCC) number PTA-8943; see also USPN 8,470,340. For example, in some cases, a variant fHbp of the disclosure retains binding to JAR4; for example, a variant fHbp of the disclosure retains at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the binding of a reference fHbp (e.g., fHbp ID 1, fHbp ID 22 or fHbp ID 55) to JAR 4 in its native conformation.

[0067] Variant 1 of fHbp ID

[0068] Sources A "reference fHbp" for a variant fHbp of the present disclosure is, in some cases, fHbp ID 1. The amino acid sequence of fHbp ID 1 is shown below.

[0069] fHbp ID 1:

[0070] CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQF RIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIGLAAKQ(SEQ ID NO:1).

[0071] In some cases, the variant fHbp of the present disclosure is a variant group 1 fHbp. In some cases, the variant fHbp of the present disclosure is a variant group 1 fHbp and is a module group I fHbp. In some cases, 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; wherein the variant fHbp comprises one or more amino acid substitutions relative to fHbp ID 1, such that the variant fHbp exhibits an affinity for human fH that is 85% or less of the binding affinity of fHbp ID 1 for human fH, e.g., the variant fHbp exhibits an affinity for human fH that is 85% or less of the binding affinity of fHbp ID 1 for human fH. 1 has a binding affinity of 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, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1% of the affinity for human fH; and when administered to a mammalian host (e.g., a human; or a non-human animal model), the variant fHbp induces a bactericidal immune response to at least one strain of Neisseria meningitidis.

[0072] In some cases, 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 of 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 ID 1 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein based on the fHbp ID1, the variant fHbp comprises an amino acid substitution selected from at least one of 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 substitution of arginine with glycine at amino acid 130 (R130G); d) an amino acid substitution of serine (S223) at amino acid 223; and e) a substitution of leucine with histidine at amino acid 248 (H248L).

[0073] In some cases, 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 of 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 ID 1 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp comprises an amino acid substitution of glutamine (Q38) at amino acid 38. In some cases, the variant fHbp comprises a Q38R substitution. Other amino acids with positively charged or aromatic side chains, such as lysine, histidine, phenylalanine, tyrosine or tryptophan, may also be substituted at this position. Thus, in some cases, the variant fHbp comprises a Q38K substitution, a Q38H substitution, a Q38F substitution, a Q38Y substitution or a Q38W substitution. As an example, the variant fHbp of the present disclosure may comprise Fig. 20 and the amino acid sequence shown in SEQ ID NO:5.

[0074] In some cases, 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 of 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 ID 1 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp comprises an amino acid substitution of glutamic acid (E92) at amino acid 92. In some cases, the fHbp variant comprises an E92K substitution. Other amino acids with positively charged or aromatic side chains, such as arginine, histidine, phenylalanine, tyrosine or tryptophan, may also be substituted at this position. Thus, for example, in some cases, the fHbp variant comprises an E92R substitution, an E92H substitution, an E92F substitution, an E92Y substitution or an E92W substitution. As an example, the variant fHbp of the present disclosure may comprise Fig.21 and the amino acid sequence shown in SEQ ID NO:6.

[0075] In some cases, 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 of 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 ID 1 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp comprises a substitution of glycine for arginine at amino acid 130 (R130G). For example, a variant fHbp of the present disclosure may comprise Fig. 22and the amino acid sequence shown in SEQ ID NO: 7. Other amino acids with negatively charged or aromatic side chains, such as aspartic acid, glutamic acid, phenylalanine, tyrosine or tryptophan, may also be substituted at R130. Thus, for example, in some cases, 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 of 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 ID 1 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp comprises a R130D substitution, a R130E substitution, a R130F substitution, a R130Y substitution, or a R130W substitution.

[0076] In some cases, 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 of 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 ID 1 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp comprises an amino acid substitution of serine (S223) at amino acid 223. In some cases, the fHbp variant comprises an S223R substitution. Other amino acids with positively charged or aromatic side chains, such as lysine, histidine, phenylalanine, tyrosine or tryptophan, may also be substituted at this position. Thus, for example, in some cases, the fHbp variant comprises a S223K substitution, a S223H substitution, a S223F substitution, a S223Y substitution or a S223W substitution. As an example, the variant fHbp of the present disclosure may comprise Fig.23 The amino acid sequence shown in SEQ ID NO: 8.

[0077] In some cases, 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 of 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 ID 1 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp comprises a substitution of histidine for leucine at amino acid 248 (H248L). For example, a variant fHbp of the present disclosure may comprise Fig.24 , and the amino acid sequence shown in SEQ ID NO: 9. Other amino acids with non-polar, negatively charged or aromatic side chains, such as isoleucine, valine, aspartic acid, glutamic acid, phenylalanine, tyrosine or tryptophan may also be substituted at H248. Thus, in some cases, 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 of 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 ID 1 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp comprises a H248I substitution, a H248V substitution, a H248D substitution, a H248E substitution, a H248F substitution, a H248Y substitution, or a H248W substitution.

[0078] Combinations of amino acid substitutions

[0079] In some cases, 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 of 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 ID 1 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein based on the fHbp ID1, the variant fHbp comprises two or more amino acid substitutions 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 substitution of arginine with glycine at amino acid 130 (R130G); d) an amino acid substitution of serine (S223) at amino acid 223; and e) a substitution of leucine with histidine at amino acid 248 (H248L).

[0080] A combination of substitutions may be included, wherein the two substitutions are in different domains, and each independently reduces the binding of fH to fHbp (e.g., a substitution in the N-terminal domain, combined with an amino acid substitution in the C-terminal domain). In some cases, the variant fHbp of the present disclosure comprises a first amino acid substitution in the N-terminal domain; and a second amino acid substitution in the C-terminal domain. In some cases, the variant fHbp of the present disclosure comprises a first amino acid substitution in the N-terminal domain; and a second amino acid substitution in the N-terminal domain. In some cases, the variant fHbp of the present disclosure comprises a first amino acid substitution in the C-terminal domain; and a second amino acid substitution in the C-terminal domain.

[0081] For example, in some cases, 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 ID 1 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp is a polypeptide having at least 1% amino acid sequence identity to SEQ ID NO: 1. 1, the variant fHbp comprising: a) an amino acid substitution of glutamine (Q38) at amino acid 38 and b) an amino acid substitution of glutamic acid (E92) at amino acid 92; or wherein the variant fHbp comprises: a) an amino acid substitution of glutamine (Q38) at amino acid 38, and c) a substitution of arginine with glycine at amino acid 130 (R130G); or wherein the variant fHbp comprises a) an amino acid substitution of glutamine (Q38) at amino acid 38 and d) an amino acid substitution of serine (S223) at amino acid 223; or wherein the variant fHbp comprises a) an amino acid substitution of glutamine (Q38) at amino acid 38, and e) a substitution of leucine with histidine at amino acid 248 (H248L).

[0082] As additional non-limiting examples, in some cases, 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 ID 1 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, wherein the variant is based on the fHbp ID NO: 1. 1, the variant fHbp comprising: b) an amino acid substitution of glutamic acid (E92) at amino acid 92 and c) a substitution of glycine for arginine at amino acid 130 (R130G); or wherein 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 wherein the variant fHbp comprises b) an amino acid substitution of glutamic acid (E92) at amino acid 92 and e) a substitution of histidine for leucine at amino acid 248 (H248L); or wherein the variant fHbp comprises c) a substitution of glycine for arginine at amino acid 130 (R130G), and d) an amino acid substitution of serine at amino acid 223 (S223); or wherein the variant fHbp comprises c) a substitution of glycine for arginine at amino acid 130 (R130G), and e) a substitution of histidine for leucine at amino acid 248 (H248L); or wherein the variant fHbp comprises d) an amino acid substitution of serine at amino acid 223 (S223) and e) a substitution of histidine for leucine at amino acid 248 (H248L).

[0083] As additional non-limiting examples, in some cases, 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 of 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 ID 1 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein, based on numbering of fHbp ID 1, the variant fHbp comprises: i) a Q38R substitution; and ii) a R130G substitution.

[0084] Also provided herein are variant fHbp proteins comprising one or more substitutions relative to the amino acid sequence of fHbp ID 1 as described above, and further comprising the substitution R41 S. Exemplary variant fHbps comprise an R41S substitution relative to fHbp ID 1 and a substitution at S223, e.g., R41S / S223R, or an R41S substitution relative to fHbp ID 1 and an H248L substitution. In some cases, 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 of 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 ID 22 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp is a polypeptide having at least 1% affinity to human fH. 1, the variant fHbp comprises two or more of the following amino acid substitutions: a) substitution of arginine with serine at amino acid 41 (R41S); b) substitution of serine with arginine at amino acid 223 (S223R); c) substitution of histidine with leucine at amino acid 248 (H248L).

[0085] Also disclosed herein are variant fHbp proteins comprising one or more substitutions relative to the amino acid sequence of fHbp ID 1 as described above, and further comprising the substitutions disclosed in US 2011 / 0256180, which is incorporated herein by reference in its entirety.

[0086] Variants of fHbp ID 22

[0087] The "reference fHbp" from which the variant fHbps of the present disclosure are derived is in some cases fHbp ID 22. The amino acid sequence of fHbp ID 22 is shown below.

[0088] fHbp ID 22:

[0089] CSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSF LVSGLGGEHTAFNQLPSGKAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREKVHEIGIAGKQ(SEQ ID NO:2).

[0090] In some cases, the variant fHbp of the present disclosure is a variant group 2 fHbp. In some cases, the variant fHbp of the present disclosure is a variant group 2 fHbp and is a module group III fHbp. In some cases, 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; wherein the variant fHbp comprises one or more amino acid substitutions relative to fHbp ID 22, such that the variant fHbp exhibits an affinity for human fH that is 85% or less of the binding affinity of fHbp ID 22 for human fH, such as the variant fHbp exhibits an affinity for human fH that is 85% or less of the binding affinity of fHbp ID 22 for human fH. 22 has a binding affinity for human fH of 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%, or about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1% of the affinity for human fH; and when administered to a mammalian host (e.g., a human; or a non-human animal model), the variant fHbp induces a bactericidal immune response to at least one strain of Neisseria meningitidis.

[0091] In some cases, 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, 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 ID 22 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and 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 ID 22 for human fH 22, the variant fHbp comprises an amino acid substitution selected from at least one of the following: a) substitution of asparagine with isoleucine at amino acid 115 (N115I); b) substitution of aspartic acid with glycine at amino acid 121 (D121G); c) substitution of serine with threonine at amino acid 128 (S128T); d) amino acid substitution of valine at position 131 (V131); e) amino acid substitution of lysine at position 219 (K219); f) amino acid substitution of glycine at position 220 (G220). As described herein, the numbering of amino acid residues is based on the numbering of fHbp ID 1.

[0092] In some cases, 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, wherein the variant fHbp binds to human fH with an affinity of 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 ID 22 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp comprises a substitution of asparagine for isoleucine at amino acid 115 (N115I). For example, a variant fHbp of the present disclosure may comprise Fig.25 and the amino acid sequence shown in SEQ ID NO: 10. Other amino acids with non-polar, positively charged or aromatic side chains, such as valine, leucine, lysine, arginine, histidine, phenylalanine, tyrosine or tryptophan may also be substituted at N115. Thus, for example, in some cases, 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, 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 ID 22 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp has a bactericidal antibody response to at least one strain of Neisseria meningitidis relative to fHbp ID 22. The amino acid sequence of 22 comprises N115V substitution, N115L substitution, N115K substitution, N115R substitution, N115H substitution, N115F substitution, N115Y substitution or N115W substitution.

[0093] In some cases, 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, wherein the variant fHbp binds to human fH with an affinity of 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 ID 22 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp comprises a substitution of aspartic acid with glycine at amino acid 121 (D121G). For example, a variant fHbp of the present disclosure may comprise Fig.26 and the amino acid sequence shown in SEQ ID NO: 11. Other amino acids with non-polar, positively charged or aromatic side chains, such as leucine, isoleucine, valine, lysine, arginine, histidine, phenylalanine, tyrosine or tryptophan may also be substituted at D121. Thus, for example, in some cases, 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, 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 ID 22 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp has a bactericidal antibody response to at least one strain of Neisseria meningitidis relative to fHbp ID 22. The amino acid sequence of 22 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.

[0094] In some cases, 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, wherein the variant fHbp binds to human fH with an affinity of 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 ID 22 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp comprises a substitution of threonine for serine at amino acid 128 (S128T). For example, a variant fHbp of the present disclosure may comprise Fig. 27 and the amino acid sequence shown in SEQ ID NO: 12. Other amino acids with polar, charged or aromatic side chains, such as methionine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine or tryptophan, may also be substituted at S128. Thus, for example, in some cases, 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, 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 ID 22 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and 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 ID 22 for human fH 22, 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.

[0095] In some cases, 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, wherein the variant fHbp binds to human fH with an affinity of 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 ID 22 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp comprises an amino acid substitution of valine (V131) at position 131. In some cases, the fHbp variant comprises a V131D substitution. Other amino acids with charged or aromatic side chains, such as glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine or tryptophan may also be substituted at this position. Thus, for example, in some cases, 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 may comprise an amino acid sequence relative to fHbp ID 22. Fig.28 and the amino acid sequence shown in SEQ ID NO:13.

[0096] In some cases, 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, wherein the variant fHbp binds to human fH with an affinity of 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 ID 22 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp comprises an amino acid substitution of lysine at position 219 (K219). In some cases, the fHbp variant comprises a K219N substitution. Other amino acids with polar, 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 cases, 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 may comprise Fig.29 and the amino acid sequence shown in SEQ ID NO:14.

[0097] In some cases, 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, wherein the variant fHbp binds to human fH with an affinity of 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 ID 22 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp comprises an amino acid substitution of glycine at position 220 (G220). In some cases, the fHbp variant comprises a G220S substitution. Other amino acids with polar, charged or aromatic side chains, such as asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine or tryptophan may also be substituted at this position. Thus, for example, in some cases, 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 may comprise Fig.30 and the amino acid sequence shown in SEQ ID NO:15.

[0098] Combinations of amino acid substitutions

[0099] In some cases, 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, 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 ID 22 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and 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 ID 22 for human fH 22, the variant fHbp comprising two or more amino acid substitutions selected from the following: a) substitution of asparagine with isoleucine at amino acid 115 (N115I); b) substitution of aspartic acid with glycine at amino acid 121 (D121G); c) substitution of serine with threonine at amino acid 128 (S128T); d) amino acid substitution of valine at position 131 (V131); e) amino acid substitution of lysine at position 219 (K219); f) amino acid substitution of glycine at position 220 (G220). As described herein, the numbering of residues is based on the numbering of amino acids in fHbp ID 1.

[0100] A combination of substitutions may be included, wherein the two substitutions are in different domains, and each independently reduces the binding of fH to fHbp (e.g., a substitution in the N-terminal domain, combined with an amino acid substitution in the C-terminal domain). In some cases, the variant fHbp of the present disclosure comprises a first amino acid substitution in the N-terminal domain; and a second amino acid substitution in the C-terminal domain. In some cases, the variant fHbp of the present disclosure comprises a first amino acid substitution in the N-terminal domain; and a second amino acid substitution in the N-terminal domain. In some cases, in some cases, the variant fHbp of the present disclosure comprises a first amino acid substitution in the C-terminal domain; and a second amino acid substitution in the C-terminal domain.

[0101] For example, in some cases, 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, 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 ID 22 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, wherein the variant fHbp is based on the amino acid sequence of fHbp ID 22 relative to that of fHbp ID 22. 1, the variant fHbp comprising: a) a substitution of asparagine with isoleucine at amino acid 115 (N115I), and b) a substitution of aspartic acid with glycine at amino acid 121 (D121G); or wherein the variant fHbp comprises: a) a substitution of asparagine with isoleucine at amino acid 115 (N115I), and c) a substitution of serine with threonine at amino acid 128 (S128T); or wherein the variant fHbp comprises: a) a substitution of asparagine with isoleucine at amino acid 115 (N115I), and c) a substitution of serine with threonine at amino acid 128 (S128T); or wherein the variant fHbp comprises: a) a substitution of asparagine at amino acid 115 with isoleucine (N115I) for asparagine, and d) an amino acid substitution of valine at position 131 (V131); or wherein the variant fHbp comprises: a) a substitution of asparagine at amino acid 115 with isoleucine (N115I), and e) an amino acid substitution of lysine at position 219 (K219); or wherein the variant fHbp comprises: a) a substitution of asparagine at amino acid 115 with isoleucine (N115I), and f) an amino acid substitution of glycine at position 220 (G220).

[0102] As another non-limiting example, in some cases, 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, 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 ID 22 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant is 1% to about 0.1% relative to fHbp ID 22. 22, the variant fHbp comprising: b) substitution of aspartic acid with glycine at amino acid 121 (D121G), and c) substitution of serine with threonine at amino acid 128 (S128T); or wherein the variant fHbp comprises: b) substitution of aspartic acid with glycine at amino acid 121 (D121G), and d) amino acid substitution of valine at position 131 (V131); or wherein the variant fHbp comprises: b) substitution of aspartic acid with glycine at amino acid 121 (D121G), and e) amino acid substitution of lysine at position 219 (K219); or wherein the variant fHbp comprises: b) substitution of aspartic acid with glycine at amino acid 121 (D121G), and f) amino acid substitution of glycine at position 220 (G220). The numbering of the substituted residues is based on the numbering of the amino acid sequence of fHbp ID 1.

[0103] As another non-limiting example, in some cases, 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, 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 ID 22 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant is 1% to about 0.1% relative to fHbp ID 22. 22, the variant fHbp comprising: c) a substitution of serine with threonine at amino acid 128 (S128T), and d) an amino acid substitution of valine at position 131 (V131); or wherein the variant fHbp comprises: c) a substitution of serine with threonine at amino acid 128 (S128T), and e) an amino acid substitution of lysine at position 219 (K219); or wherein the variant fHbp comprises: c) a substitution of serine with threonine at amino acid 128 (S128T), and f) an amino acid substitution of glycine at position 220 (G131); 220); or wherein the variant fHbp comprises: d) an amino acid substitution of valine (V131) at position 131 and e) an amino acid substitution of lysine (K219) at position 219; or wherein the variant fHbp comprises: d) an amino acid substitution of valine (V131) at position 131 and f) an amino acid substitution of glycine (G220) at position 220; or wherein the variant fHbp comprises: e) an amino acid substitution of lysine (K219) at position 219 and f) an amino acid substitution of glycine (G220) at position 220. The numbering of the substituted residues is based on the numbering of the amino acid sequence of fHbp ID 1.

[0104] Combinations of substitutions can be included where the two substitutions are in different domains and each independently reduces fH binding 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)).

[0105] For example, in some cases, 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, wherein the variant fHbp binds to human fH with an affinity of 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 ID 22 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp comprises: i) an N115I substitution; and ii) a D211A substitution.

[0106] As another example, in some cases, 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, wherein the variant fHbp binds to human fH with an affinity of 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 ID 22 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp comprises: i) an N115I substitution; and ii) a K219N substitution.

[0107] As another example, in some cases, 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, wherein the variant fHbp binds to human fH with an affinity of 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 ID 22 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp comprises: i) an N115I substitution; and ii) a G220S substitution.

[0108] Also disclosed herein are variant fHbp polypeptides with increased thermal stability compared to wild-type fHbp ID 22. In some cases, the variant fHbp may comprise substitutions L130R and G133D relative to fHbp ID 22 (SEQ ID NO: 2), wherein the variant fHbp comprises an amino acid sequence having greater than 85% amino acid sequence identity to 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 ID 22 for human fH, wherein the variant induces a bactericidal antibody response in a mammalian host, and the variant has a higher thermal stability than WT fHbp ID 22. The thermal stability of the variant fHbp may be at least 5°C, 10°C, 15°C, 20°C, or more, such as 5°C-30°C, 5°C-25°C, 5°C-20°C, 10°C-20°C, or 15°C-20°C higher than WT fHbp (e.g., fHbp ID 22). As used herein, "thermostability" refers to the stability of a protein when exposed to a higher temperature; a thermostable variant protein maintains its conformation at a higher temperature than a wild-type protein. For example, a variant fHbp comprising a double mutation that increases thermostability compared to the thermostability of a wild-type (WT) fHbp (e.g., WT fHbp ID 22) can be unfolded at a higher temperature than WT fHbp. In some cases, the N-terminal domain of a variant fHbp can be unfolded at a higher temperature than the N-terminal domain of WT fHbp (e.g., fHbp ID 22).

[0109] In certain embodiments, a variant of a factor H binding protein (fHbp) is disclosed, wherein the variant comprises amino acid substitutions L130R and G133D and at least one of the following substitutions relative to fHbp ID 22 (SEQ ID NO:2): R80A, N115I, D121G, S128T, V131, D211A, E218A, K219 (e.g., K219N), G220 (e.g., G220S), E248A, G236I, T221A, and H223A, wherein the variant comprises an amino acid sequence having greater than 85% amino acid sequence identity to SEQ ID NO:2, wherein the variant fHbp binds human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID 22 for human fH, and wherein the variant induces a bactericidal antibody response in a mammalian host.

[0110] In some cases, the variant fHbp may include a combination of substitutions, such as L130R, G133D and at least one amino acid substitution selected from the following: a) N115I; b) D121G; c) S128T; d) V131D; e) K219 (e.g., K219N); and f) G220 (e.g., G220S), wherein the amino acid substitutions are relative to fHbp ID 22 (SEQ ID NO:2), wherein the variant fHbp comprises an amino acid sequence having greater than 85% amino acid sequence identity to SEQ ID NO:2, wherein the variant fHbp binds human factor H (fH) with an affinity of 50% or less of the affinity of fHbp ID 22 for human fH, and wherein the variant induces a bactericidal antibody response in a mammalian host.

[0111] In certain embodiments, a variant of a factor H binding protein (fHbp) is disclosed, wherein the variant comprises amino acid substitutions L130R and G133D and at least one of the following substitutions: R80A, D211A, E218A, E248A, G236I, T221A, and H223A relative to fHbp ID 22 (SEQ ID NO:2), wherein the variant comprises an amino acid sequence having greater than 85% amino acid sequence identity to SEQ ID NO:2, wherein the variant fHbp binds human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID 22 for human fH, and wherein the variant induces a bactericidal antibody response in a mammalian host.

[0112] Exemplary variant fHbp include polypeptides having an amino acid sequence having greater than 85% amino acid sequence identity (e.g., at least 90% identity, at least 95%, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity) to SEQ ID NO:2, and comprising 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.

[0113] Also disclosed herein are variant fHbp proteins comprising one or more substitutions relative to the amino acid sequence of fHbp ID 22 as described above, and further comprising the substitutions disclosed in US 2011 / 0256180, which is incorporated herein by reference in its entirety.

[0114] Variants of fHbp ID55

[0115] The "reference fHbp" from which the variant fHbps of the present disclosure are derived is in some cases fHbp ID 55. The amino acid sequence of fHbp ID 55 is shown below.

[0116] fHbp ID 55:

[0117] CSSGGGGSGGGGVTADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQEQDPEHSEKMVAKR RFRIGDIAGEHTSFDKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAVAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGEKAQEVAGSAEVETANGIHHIGLAAKQ(SEQ ID NO:3).

[0118] In some cases, a variant fHbp of the present disclosure is a variant group 1 fHbp. In some cases, a variant fHbp of the present disclosure is a variant group 1 fHbp and is a module group IV fHbp.

[0119] In some cases, 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: 3; wherein the variant fHbp comprises one or more amino acid substitutions relative to fHbp ID 55, such that the variant fHbp exhibits an affinity for human fH that is 85% or less of the binding affinity of fHbp ID 55 for human fH, e.g., the variant fHbp exhibits fHbp ID 55% 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 affinity for human fH; and when administered to a mammalian host (e.g., a human; or a non-human animal model), the variant fHbp induces a bactericidal immune response to at least one strain of Neisseria meningitidis.

[0120] In some cases, 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: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 ID 55 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and 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 ID 55 for human fH The variant fHbp comprises an amino acid sequence of 55, wherein the variant fHbp comprises at least one amino acid substitution selected from the group consisting of: a) an amino acid substitution of glutamic acid at position 92 (E92); b) an amino acid substitution of serine at position 223 (S223); and c) an amino acid substitution of histidine at position 248 (H248), wherein the numbering of the amino acid residues is based on the numbering of fHbp ID 1.

[0121] In some cases, 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: 3, wherein the variant fHbp binds to human fH with an affinity of 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 ID 55 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp comprises an amino acid substitution of glutamic acid (E92) at position 92. In some cases, the fHbp variant comprises an E92K substitution. Other amino acids with positively charged or aromatic side chains, such as arginine, histidine, phenylalanine, tyrosine or tryptophan, may also be substituted at this position. Thus, for example, in some cases, the fHbp variant comprises an E92R substitution, an E92H substitution, an E92F substitution, an E92Y substitution or an E92W substitution. As an example, the variant fHbp of the present disclosure may comprise Fig.31 and the amino acid sequence shown in SEQ ID NO:16.

[0122] In some cases, 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: 3, wherein the variant fHbp binds to human fH with an affinity of 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 ID 55 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp comprises an amino acid substitution of serine (S223) at position 223. In some cases, the fHbp variant comprises an S223R substitution. Other amino acids with positively charged or aromatic side chains, such as lysine, histidine, phenylalanine, tyrosine or tryptophan, may also be substituted at this position. Thus, for example, in some cases, the fHbp variant comprises a S223K substitution, a S223H substitution, a S223F substitution, a S223Y substitution or a S223W substitution. As an example, the variant fHbp of the present disclosure may comprise Fig.32 The amino acid sequence shown in SEQ ID NO:17.

[0123] In some cases, 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: 3, wherein the variant fHbp binds to human fH with an affinity of 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 ID 55 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and wherein the variant fHbp comprises an amino acid substitution of histidine at position 248 (H248). In some cases, the fHbp variant comprises an H248L substitution. Other amino acids with non-polar, 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 cases, the fHbp variant comprises a H248I substitution, a H248V substitution, a H248D substitution, a H248E substitution, a H248F substitution, a H248Y substitution or a H248W substitution. As an example, a variant fHbp of the present disclosure may comprise Fig.33 and the amino acid sequence shown in SEQ ID NO:18.

[0124] Combinations of amino acid substitutions

[0125] In some cases, 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: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 ID 55 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and 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 ID 55 for human fH 55, the variant fHbp comprises two or more amino acid substitutions selected from: a) an amino acid substitution of glutamic acid at position 92 (E92); b) an amino acid substitution of serine at position 223 (S223); and c) an amino acid substitution of histidine at position 248 (H248), wherein the numbering of the residues is based on the numbering of the amino acids in the sequence of fHbp ID 1.

[0126] A combination of substitutions may be included, wherein the two substitutions are in different domains, and each independently reduces the binding of fH to fHbp (e.g., a substitution in the N-terminal domain, in combination with an amino acid substitution in the C domain). In some cases, the variant fHbp of the present disclosure comprises a first amino acid substitution in the N-terminal domain; and a second amino acid substitution in the C-terminal domain. In some cases, the variant fHbp of the present disclosure comprises a first amino acid substitution in the N-terminal domain; and a second amino acid substitution in the N-terminal domain. In some cases, the variant fHbp of the present disclosure comprises a first amino acid substitution in the C-terminal domain; and a second amino acid substitution in the C-terminal domain.

[0127] For example, in some cases, 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: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 ID 55 for human fH, wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host, and 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 ID 55 for human fH 55, a variant fHbp comprising: a) an amino acid substitution of glutamic acid (E92) at position 92, and b) an amino acid substitution of serine (S223) at position 223; or wherein the variant fHbp comprises: a) an amino acid substitution of glutamic acid (E92) at position 92, and c) an amino acid substitution of histidine (H248) at position 248; or wherein the variant fHbp comprises: b) an amino acid substitution of serine (S223) at position 223, and c) an amino acid substitution of histidine (H248) at position 248; or wherein the variant fHbp comprises: a) an amino acid substitution of glutamic acid (E92) at position 92, and 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 of fHbp ID 1.

[0128] Also disclosed herein are variant fHbp proteins comprising one or more substitutions relative to the amino acid sequence of fHbp ID 55 as described above, and further comprising the substitutions disclosed in US 2011 / 0256180, which is incorporated herein by reference in its entirety.

[0129] Fusion peptide

[0130] Variant fHbp of the present disclosure may be a fusion polypeptide, such as a polypeptide comprising a variant fHbp as described above and a heterologous polypeptide (eg, a fusion partner). The fusion partner may be at the N-terminus of the variant fHbp, at the C-terminus of the variant fHbp, or at an internal site within the fHbp.

[0131] Suitable fusion partners include peptides and polypeptides that confer enhanced stability in vivo (e.g., increased serum half-life); provide ease of purification, e.g., (His) n , such as 6His, etc.; providing secretion of fusion protein from cells; providing epitope tags, such as GST, hemagglutinin (HA; such as YPYDVPDYA; SEQ ID NO: 26), FLAG (such as DYKDDDDK; SEQ ID NO: 27), c-myc (such as EQKLISEEDL; SEQ ID NO: 28), etc.; providing detectable signals, such as enzymes that produce detectable products (such as β-galactosidase, luciferase) or proteins that can be detected by themselves, such as green fluorescent protein, yellow fluorescent protein, etc.; providing multimerization, such as a multimerization domain, such as the Fc portion of an immunoglobulin; and the like.

[0132] Generation method

[0133] The fHbp of the present disclosure can be produced by any suitable method, including recombinant and non-recombinant methods (e.g., chemical synthesis). When recombinant technology is used to produce the subject fHbp, the method may involve any suitable construct and any suitable host cell, which may be a prokaryotic or eukaryotic cell, typically a bacterial or yeast host cell, more typically a bacterial cell. The method of introducing genetic material into a host cell includes, for example, transformation, electroporation, conjugation, calcium phosphate method, etc. The method of transfer can be selected to provide stable expression of the nucleic acid encoding fHbp introduced. The nucleic acid encoding fHbp can be provided as a heritable free element (e.g., plasmid) or can be genomic integration.

[0134] The present disclosure provides nucleic acids (including isolated nucleic acids) comprising nucleotide sequences encoding fHbp variants of the present disclosure. In some embodiments, the nucleotide sequence encoding the fHbp variant is operably connected to a transcriptional control element, such as a promoter. In some cases, the promoter is constitutive. In some cases, the promoter is inducible. In some cases, the promoter is suitable for prokaryotic host cells (e.g., active in prokaryotic host cells). In some cases, the promoter is suitable for eukaryotic host cells (e.g., active in eukaryotic host cells).

[0135] In some cases, the nucleic acid comprising the nucleotide sequence encoding the fHbp variant of the present invention is present in an expression vector. The present disclosure provides a recombinant expression vector (e.g., an isolated recombinant expression vector) comprising the nucleotide sequence encoding the fHbp variant of the present invention. In some embodiments, the nucleotide sequence encoding the fHbp variant is operably connected to a transcription control element, such as a promoter. In some cases, the promoter is constitutive. In some cases, the promoter is inducible. In some cases, the promoter is suitable for prokaryotic host cells (e.g., active in prokaryotic host cells). In some cases, the promoter is suitable for eukaryotic host cells (e.g., active in eukaryotic host cells).

[0136] Suitable vectors for transferring nucleic acids encoding fHbp may differ in composition. Integration vectors may be conditional replication or suicide plasmids, phages, etc. Constructs may include various elements, including, for example, promoters, selectable genetic markers (genes that confer antibiotic resistance (e.g., kanamycin, erythromycin, chloramphenicol or gentamicin), replication origins (promoting replication in host cells, such as bacterial host cells), etc. The selection of vectors will depend on a variety of factors, such as the cell type and purpose of proliferation in which proliferation is required. Certain vectors can be used to amplify and prepare a large amount of desired DNA sequences. Other vectors are suitable for expression in cells in culture. Other vectors are suitable for transfer and expression in cells throughout animals. The selection of suitable vectors is completely within the technology of the art. Many such vectors are commercially available.

[0137] In one example, the vector is an expression vector based on an episomal plasmid containing selectable drug resistance markers and elements that provide autonomous replication in different host cells (e.g., in both Escherichia coli and Neisseria meningitidis). An example of such a "shuttle vector" is plasmid pFP10 (Pagotto et al. (2000) Gene 244: 13-19).

[0138] Construct (recombinant vector) can be prepared by, for example, inserting the target polynucleotide into the construct backbone, which is usually carried out by attaching to the restriction enzyme site cut in the vector by DNA ligase. Alternatively, the desired nucleotide sequence can be inserted by homologous recombination or site-specific recombination. Usually, homologous recombination is completed by attaching homologous regions with the vector on the flank of the desired nucleotide sequence, and site-specific recombination can be achieved by using sequences (such as cre-lox, att sites, etc.) that promote site-specific recombination. Nucleic acids containing such sequences can be added by, for example, connection of oligonucleotides or by using a polymerase chain reaction of primers containing both homology regions and a part of the desired nucleotide sequence.

[0139] The vector can provide extrachromosomal maintenance in the host cell, or can provide integration into the host cell genome. The vector is fully described in many publications familiar to those skilled in the art, including, for example, Short Protocols in Molecular Biology, (1999) F.Ausubel et al., ed., Wiley & Sons. The vector can provide expression of a nucleic acid encoding a subject fHbp, can provide for the propagation of a subject nucleic acid, or both.

[0140] Examples of vectors that can be used include, but are not limited to, vectors derived from recombinant phage DNA, plasmid DNA or cosmid DNA. For example, plasmid vectors such as pBR322, pUC19 / 18, pUC118, 119 and M13 mp series vectors can be used. pET21 is also an expression vector that can be used. Phage vectors can include λgt10, λgt11, λgt18-23, λZAP / R and EMBL series phage vectors. Other vectors that can be used include, but are not limited to, pJB8, pCV 103, pCV 107, pCV108, pTM, pMCS, pNNL, pHSG274, COS202, COS203, pWE15, pWE16 and charomid 9 series vectors.

[0141] For the expression of theme fHbp, expression cassette can be used. Therefore, the present disclosure provides a recombinant expression vector comprising theme nucleic acid. Expression vector provides transcription and translation regulatory sequences, and can provide inducible or constitutive expression, wherein the coding region is operably connected under the transcription control of transcription initiation region, and transcription and translation termination region. These control regions can be natural fHbp of source theme fHbp, or can be derived from exogenous sources. Generally, transcription and translation regulatory sequences can 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. Promoter can be constitutive or inducible, and can be a strong constitutive promoter (such as T7, etc.).

[0142] The expression vector usually has a convenient restriction site near the promoter sequence to provide the insertion of the nucleic acid sequence encoding the protein of interest. There may be an effective selection marker in the expression host to promote the selection of cells containing the vector. In addition, the expression construct may include other elements. For example, the expression vector may have one or two replication systems, thereby allowing it to be maintained in an organism, such as in mammals or insect cells for expression and in prokaryotic hosts for cloning and amplification. In addition, the expression construct may contain a selection marker gene to allow the selection of transformed host cells. The selection gene is well known in the art and will vary with the host cell used.

[0143] It should be noted that the fHbp of the present disclosure may contain additional elements, such as detectable labels, e.g., radioactive labels, fluorescent labels, biotin labels, immunologically detectable labels (e.g., hemagglutinin labels, polyhistidine tags), etc. Additional elements of fHbp may be provided by various methods (e.g., affinity capture, etc.) to facilitate separation (e.g., biotin labels, immunologically detectable labels). The subject fHbp may optionally be immobilized on a support by covalent or non-covalent attachment.

[0144] The separation and purification of fHbp can be completed according to methods known in the art. For example, fHbp can be separated from the lysate of cells genetically modified to express fHbp or from a synthesis reaction mixture by immunoaffinity purification, which generally involves contacting the sample with an anti-fHbp antibody (e.g., anti-fHbp monoclonal antibody antibody (mAb), such as JAR 4MAb or other suitable JAR MAbs known in the art), washing to remove non-specifically bound substances, and eluting the specifically bound fHbp. The separated fHbp can be further purified by other methods commonly used in dialysis and protein purification methods. In an example, fHbp can be separated using metal chelate chromatography.

[0145] Host cells

[0146] Any of many suitable host cells can be used for the production of fHbp. In general, the fHbp described herein can be expressed in prokaryotes or eukaryotes, such as bacteria, such as Escherichia coli or Neisseria (e.g., Neisseria meningitidis) according to conventional techniques. Therefore, the present disclosure further provides a genetically modified in vitro host cell containing a nucleic acid encoding the subject fHbp. The host cell for producing (including large-scale production) the subject fHbp can be selected from any of a variety of available host cells. Examples of host cells for expression include those of prokaryotic or eukaryotic unicellular organisms, such as bacteria (e.g., Escherichia coli strains), yeast (e.g., Saccharomyces cerevisiae, Pichia species, etc.), and can include host cells originally derived from higher organisms, such as insects, vertebrates, such as mammalian host cells. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC No. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCL1.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, etc.). In some cases, bacterial host cells and yeast host cells are of particular interest for the production of subject fHbp.

[0147] The subject fHbp 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 subject fHbp can be present in a composition that is enriched for the polypeptide relative to other components that may be present (e.g., other polypeptides or other host cell components). The purified subject fHbp can be provided such that the polypeptide is present in a composition that is substantially free of other expressed polypeptides, e.g., less than 90%, typically less than 60%, more typically less than 50% of the composition being composed of other expressed polypeptides.

[0148] Host cells for vesicle production

[0149] When the subject fHbp is to be provided in membrane vesicles (as discussed in more detail below), the Neisserial host cell is genetically modified to express the subject fHbp. Any of a variety of Neisseria species strains that can be modified to produce the subject fHbp, and optionally produce or can be modified to produce other antigens of interest, such as PorA, can be used in the methods disclosed herein.

[0150] Methods and vectors for providing genetic modification of Neisseria strains and expression of desired polypeptides are known in the art. Examples of vectors and methods can be found in WO 02 / 09746 and O'Dwyer et al. (2004) Infect Immun 72:6511-80. Strong promoters, particularly constitutive strong promoters, are of particular interest. Examples of promoters include the promoters of porA, porB, lbpB, tbpB, p110, hpuAB, lgtF, opa, p110, lst, hpuAB and rmp.

[0151] Pathogenic Neisseria or strains derived from pathogenic Neisseria, particularly strains pathogenic to humans or derived from strains pathogenic to humans or commensal, are of particular interest for membrane vesicle production. Examples of Neisseria include N. meningitidis, N. flavescens, N. gonorrhoeae, N. lactamica, N. polysaccharea, N. cinerea, N. mucosa, N. subflava, N. sicca, N. elongata, etc.

[0152] Neisseria meningitidis strains are particularly interesting for genetic modification to express the subject fHbp and for use in vesicle production. Strains for vesicle production can be selected based on many different characteristics that may be desired. For example, strains can be selected based on: desired PorA type ("serosubtype"), capsule group, serotype, etc.; reduced capsular polysaccharide production; and the like. For example, a producing strain can produce any desired PorA polypeptide, and can express one or more PorA polypeptides (naturally or due to genetic engineering). Examples of strains include those that produce PorA polypeptides that confer serosubtypes of 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 variants of such PorA polypeptides, which variants may or may not retain reactivity with conventional serological reagents used in serosubtyping. Also of interest are PorA polypeptides characterized according to PorA variable region (VR) typing (see, e.g., 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 large number of different VR types have been identified, which can be classified as VR1 and VR2 family "prototypes". A web-accessible database describing this nomenclature and its relationship to previous typing schemes can be found at neisseria.org / nm / typing / pora. An alignment of certain PorA VR1 and VR2 types is provided in Russell et al. (2004) Emerging Infect Dis 10:674-678.

[0153] Alternatively or in addition, the producing strain can be a capsule defective strain. Capsule defective strains can provide vesicle-based vaccines, which provide the risk of causing a significant autoantibody response (e.g., due to the production of antibodies cross-reacting with sialic acid on the host cell surface) in the subject administering the vaccine. As used herein, "capsule defective" or "capsule polysaccharide defective" refers to the level of capsular polysaccharide on the bacterial surface lower than the level of naturally occurring strains, or when the strain is genetically modified, its level is lower than the level of the parent strain of the source capsule defective strain. Capsule defective strains include surface capsule polysaccharide production reduction of at least 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90% or more strains, and include strains where capsular polysaccharides are not detected on the bacterial surface (e.g., by whole cell enzyme-linked immunosorbent assay (ELISA) using anti-capsular polysaccharide antibodies).

[0154] Capsule-deficient strains include those that are capsule-deficient due to naturally occurring or recombinantly produced genetic modifications. Naturally occurring capsule-deficient strains (see, e.g., Dolan-Livengood et al. (2003) J. Infect. Dis. 187: 1616-28), and methods for identifying and / or producing 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.

[0155] Modification of the Neisseria host cell to provide reduced production of capsular polysaccharides can include modification of one or more genes involved in capsule synthesis, wherein the modification provides, for example, a reduced capsular polysaccharide level relative to the parental cell before modification. Such genetic modifications can include changes in nucleotide and / or amino acid sequences in one or more capsule biosynthetic genes that make the strain capsule defective (e.g., due to one or more insertions, deletions, substitutions, etc. in one or more capsule biosynthetic genes). Capsule-deficient strains can lack one or more capsule genes or be non-functional for one or more capsule genes.

[0156] Of particular interest are strains of sialic acid biosynthesis defects. Such strains can provide the generation of vesicles with reduced risk of causing anti-sialic acid antibodies that cross-react with human sialic acid antigens, and can further provide improved manufacturing safety. The strain with sialic acid biosynthesis defect (due to naturally occurring modification or engineered modification) can be defective in any of the many different genes in the sialic acid biosynthetic pathway. Of particular interest are strains defective in the gene product encoded by N-acetylglucosamine-6-phosphate 2-epimerase gene (referred to as synX AAF40537.1 or siaA AAA20475), wherein the strain of the gene inactivation is of particular interest. For example, in one embodiment, capsule defective strains are produced by destroying the generation of functional synX gene products (see, for example, Swartley et al. (1994) J Bacteriol.176:1530-4).

[0157] Capsule-deficient strains can also be generated from naturally occurring strains using non-recombinant techniques, for example by using bactericidal anti-capsular antibodies to select for strains with reduced levels of capsular polysaccharide.

[0158] When the present disclosure involves the use of two or more strains (e.g., to produce an antigenic composition containing a subject fHbp-presenting vesicle from different strains), the strains can be selected to differ in one or more strain characteristics, e.g., to provide vesicles that differ in the subject fHbp, PorA, etc. used.

[0159] Preparation of vesicles

[0160] Antigenic compositions encompassed by the present disclosure generally include vesicles prepared from Neisserial cells expressing a subject fHbp.As referred to herein, "vesicles" are intended to encompass outer membrane vesicles as well as microvesicles (which are also referred to as blebs).

[0161] The antigenic composition may contain outer membrane vesicles (OMVs) prepared from the outer membrane of a cultured strain of Neisseria meningitidis that is genetically modified to express the subject fHbp. OMVs can be obtained from Neisseria meningitidis cultured in broth or solid medium culture, preferably by separating bacterial cells from the culture medium (e.g., by filtration or by low-speed centrifugation to precipitate the cells, etc.), lysing the cells (e.g., by adding detergents, osmotic shock, sonication, cavitation, homogenization, etc.), and separating the outer membrane fraction from the 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 methods using ligands that specifically recognize outer membrane molecules; or by high-speed centrifugation to precipitate the outer membrane and / or outer membrane vesicles, etc.); the outer membrane fraction can be used to produce OMVs.

[0162] The antigenic composition may contain microvesicles (MVs) (or "vesicles") comprising the subject fHbp, wherein the MVs or vesicles are released during the culture of a strain of Neisseria meningitidis that is genetically modified to express the subject fHbp. For example, the Neisseria meningitidis strain may be cultured in a broth medium, whole cells may be separated from the culture medium (e.g., by filtration, or by low-speed centrifugation, which only precipitates cells, not vesicles, etc.), and then the MVs present in the cell-free culture medium may be collected (e.g., by filtration, differential precipitation or aggregation of the MVs, or by high-speed centrifugation that precipitates the vesicles, etc.). The strain used to produce the MVs may generally be selected based on the amount of vesicles produced in the culture (e.g., the bacteria may be cultured in reasonable quantities to provide vesicles suitable for separation and administration in the methods described herein). Exemplary strains that produce high levels of vesicles are described in PCT Publication No. WO 01 / 34642. In addition to vesicle production, strains for MV production can also be selected based on NspA production, where strains that produce higher levels of NspA may be of particular interest (for examples of N. meningitidis strains with different levels of NspA production, see, e.g., Moe et al. (1999 Infect. Immun. 67:5664). Other strains of interest for use in vesicle production include strains with an inactivated GNA33 gene, which encodes a lipoprotein required for cell separation, membrane structure, and virulence (see, e.g., Adu-Bobie et al. (2004) Infect Immun. 72:1914-1919).

[0163] The antigenic compositions of the present disclosure may contain vesicles from one strain or from 2, 3, 4, 5 or more strains, which may be homologous or heterologous to each other, typically heterologous. For example, the fHbp of the strain may be homologous or heterologous relative to PorA and / or the source subject fHbp. The vesicles may be prepared from strains expressing more than one subject fHbp (e.g., 1, 2, 3 or more subject fHbps), which may be composed of fHbp amino acid sequences from different variants (v.1, v.2 or v.3) or sub-variants (e.g., sub-variants of v.1, v.2 or v.3).

[0164] The antigenic composition may comprise a mixture of OMVs and MVs presenting the same or different subject fHbps, wherein the subject fHbps may optionally present epitopes from different combinations of fHbp variants and / or subvariants, and wherein the OMVs and / or MVs may be from the same or different strains. Vesicles from different strains may be administered as a mixture, or may be administered sequentially.

[0165] When desired (e.g., when the strain used to produce the vesicles is associated with endotoxin or particularly high levels of endotoxin), the vesicles are optionally treated to reduce endotoxin, such as to reduce toxicity after administration. Although less desirable as discussed below, endotoxin reduction can be achieved by using a suitable detergent (e.g., BRIJ-96, sodium deoxycholate, sodium lauroyl sarcosinate, Empigen BB, Triton X-100, non-ionic detergent TWEEN 20 (sorbitan monolaurate polyoxyethylene), non-ionic detergent TWEEN 80 at a concentration of 0.1-10%, such as 0.5-2%, and sodium dodecyl sulfate (SDS)). When detergent extraction is used, it is preferred to use a detergent other than deoxycholate.

[0166] The vesicles of the antigenic composition can be prepared without detergents, for example without deoxycholate. Although detergent treatment can be used to remove endotoxin activity, it can be exhausted by extracting during vesicle production to deplete native fHbp lipoprotein and / or subject fHbp (including lipidated fHbp). Therefore, it may be particularly desirable to use a technique that does not require detergents to reduce endotoxin activity. In one method, a strain of relatively low endotoxin (lipopolysaccharide, LPS) producing bacteria is used to avoid the need to remove endotoxin from the final preparation before being used in humans. For example, vesicles can be prepared from Neisseria mutants, wherein lipooligosaccharides or other antigens that may not be needed in vaccines (e.g., Rmp) are reduced or eliminated.

[0167] Vesicles can be prepared from strains of Neisseria meningitidis containing genetic modifications that result in reduced or undetectable 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). The immunogenic composition can be detoxified by modification of LPS, such as downregulation and / or inactivation of the enzyme encoded by lpxL1 or lpxL2, respectively. The production of pentaacylated lipid A prepared in the 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 found in the 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 GlcNI. Mutations that result in reduced (or absent) expression of these genes (or reduced or absent activity of the products of these genes) result in altered 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 lipid A 4'-kinase encoding gene (lpxK) also reduce the toxic activity of lipid A. Of particular interest for producing vesicles (e.g., MVs or OMVs) are strains of Neisseria meningitidis that are genetically modified to provide reduced or undetectable functional LpxL1-encoded proteins, for example, wherein the Neisseria meningitidis bacteria (e.g., Neisseria meningitidis strains) are genetically modified to provide reduced activity or inactivity of the gene product of the lpxL1 gene. For example, the Neisseria meningitidis bacteria can be genetically modified to have an lpxL1 gene knockout, for example, wherein the lpxL1 gene is disrupted. See, for example, U.S. Patent Publication No. 2009 / 0035328. Neisseria meningitidis bacteria can be genetically modified to provide reduced activity or inactivity of the gene product of the lpxL2 gene. Neisseria meningitidis bacteria can be genetically modified to provide reduced activity or inactivity of the gene products of the lpxL1 gene and the lpxL2 gene. Such vesicles provide reduced toxicity compared to wild-type Neisseria meningitidis strains for LPS production while retaining the immunogenicity of the subject fHbp.

[0168] LPS toxic activity can also be altered by introducing mutations in genes / locuses involved in polymyxin B resistance (this resistance has been associated with the addition of aminoarabinose to the 4' phosphate of lipid A). These genes / locuses may be pmrE encoding UDP-glucose dehydrogenase, or regions of antimicrobial peptide resistance genes common to many enterobacteriaceae that may be involved in the synthesis and transfer of aminoarabinose. The gene pmrF present in this region encodes dolichol phosphate mannosyltransferase (Gunn JS, Kheng, BL, Krueger J., Kim K., Guo L., Hackett M., Miller SI 1998. Mol. Microbiol. 27: 1171-1182).

[0169] Mutations in the PhoP-PhoQ regulatory system, which is a phospho-relay two-component regulatory system (e.g., PhoP constitutive phenotype, PhoPc), or low Mg ++ Environmental or culture conditions (which activate the PhoP-PhoQ regulatory system) result in the addition of aminoarabinose on the 4'-phosphate and 2-hydroxymyristate in place of myristate (hydroxylation of myristate). This modified lipid A exhibits a reduced ability to stimulate E-selectin expression in human endothelial cells and TNF secretion from human monocytes.

[0170] Polymyxin B resistant strains are also suitable for use, as such strains have been shown to have reduced LPS toxicity (see, e.g., van der Ley et al. (1994) In: Proceedings of the ninth international pathogenic Neisseria conference. The Guildhall, Winchester, England). Alternatively, synthetic peptides that mimic the binding activity of polymyxin B can be added to the antigenic composition to reduce LPS toxicity activity (see, e.g., Rustici et al. (1993) Science 259:361-365; Porro et al. (1998) Prog Clin Biol Res. 397:315-25).

[0171] Endotoxins can also be reduced by selecting culture conditions. For example, culturing the strain in a growth medium containing 0.1 mg-100 mg aminoarabinose per liter of culture medium provides reduced lipid toxicity (see, e.g., WO 02 / 097646).

[0172] Compositions and formulations

[0173] For convenience, "composition", "antigenic composition", "antigenic composition" or "immunogenic composition" are used herein to refer generally to a composition comprising a subject fHbp as disclosed herein, which may optionally be conjugated to further enhance immunogenicity. The present disclosure particularly encompasses compositions for eliciting antibodies, e.g., antibodies against Neisseria meningitidis, e.g., bactericidal antibodies against Neisseria meningitidis. The antigenic composition may contain 1, 2 or more different subject fHbps. When there is more than one type of fHbp, each subject fHbp may present epitopes from different combinations of fHbp variants and / or sub-variants.

[0174] The antigenic composition contains an immunologically effective amount of the subject fHbp and may also contain other compatible components as needed. The composition of the present disclosure may contain an fHbp as a low fH binder. The composition contains one or more fHbps, wherein at least one fHbp is a low fH binder. When more than one fHbp is present in the composition, each fHbp may be different (e.g., in amino acid sequence and / or conjugation).

[0175] In some cases, the antigenic composition of the present disclosure comprises only one fHbp variant of the present disclosure. In some cases, the antigenic composition of the present disclosure comprises two or more different fHbp variants of the present disclosure. As a non-limiting example, in some cases, the antigenic composition of the present disclosure comprises:

[0176] 1) a first variant of fHbp ID 1, wherein the first fHbp ID 1 variant comprises an amino acid substitution at Q38 (e.g., Q38R); and a second variant of fHbp ID 1, wherein the second fHbp ID 1 variant comprises an amino acid substitution at E92 (e.g., E92K);

[0177] 2) a first variant of fHbp ID 1, wherein the first fHbp ID 1 variant comprises an amino acid substitution at Q38 (e.g., Q38R); and a second variant of fHbp ID 1, wherein the second fHbp ID 1 variant comprises an amino acid substitution at R130 (e.g., R130G);

[0178] 3) a first variant of fHbp ID 1, wherein the first fHbp ID 1 variant comprises an amino acid substitution at Q38 (e.g., Q38R); and a second variant of fHbp ID 1, wherein the second fHbp ID 1 variant comprises an amino acid substitution at S223 (e.g., S223R);

[0179] 4) a first variant of fHbp ID 1, wherein the first fHbp ID 1 variant comprises an amino acid substitution at Q38 (e.g., Q38R); and a second variant of fHbp ID 1, wherein the second fHbp ID 1 variant comprises an amino acid substitution at H248 (e.g., H248L);

[0180] 5) variants of fHbp ID 22, wherein the fHbp ID 22 variant comprises an amino acid substitution at N115 (e.g., N115I); and variants of fHbp ID 1, wherein the fHbp ID 1 variant comprises an amino acid substitution at Q38 (e.g., Q38R);

[0181] 6) variants of fHbp ID 22, wherein the fHbp ID 22 variant comprises an amino acid substitution at D121 (e.g., D121G); and variants of fHbp ID 1, wherein the fHbp ID 1 variant comprises an amino acid substitution at E92 (e.g., E92K);

[0182] 7) variants of fHbp ID 22, wherein the fHbp ID 22 variant comprises an amino acid substitution at S128 (e.g., S128T); and variants of fHbp ID 1, wherein the fHbp ID 1 variant comprises an amino acid substitution at H248 (e.g., H248L);

[0183] 8) variants of fHbp ID 22, wherein the fHbp ID 22 variant comprises an amino acid substitution at V131 (e.g., V131D); and variants of fHbp ID 1, wherein the fHbp ID 1 variant comprises an amino acid substitution at Q38 (e.g., Q38R);

[0184] 9) variants of fHbp ID 22, wherein the fHbp ID 22 variant comprises an amino acid substitution at K219 (e.g., K219N); and variants of fHbp ID 1, wherein the fHbp ID 1 variant comprises an amino acid substitution at Q38 (e.g., Q38R);

[0185] 10) variants of fHbp ID 22, wherein the fHbp ID 22 variant comprises an amino acid substitution at G220 (e.g., G220S); and variants of fHbp ID 1, wherein the fHbp ID 1 variant comprises an amino acid substitution at Q38 (e.g., Q38R);

[0186] 11) variants of fHbp ID 22, wherein the fHbp ID 22 variant comprises an amino acid substitution at N115 (e.g., N115I); and variants of fHbp ID 55, wherein the fHbp ID 55 variant comprises an amino acid substitution at E92 (e.g., E92K);

[0187] 12) variants of fHbp ID 22, wherein the fHbp ID 22 variant comprises an amino acid substitution at D121 (e.g., D121G); and variants of fHbp ID 55, wherein the fHbp ID 55 variant comprises an amino acid substitution at S223 (e.g., S223R);

[0188] 13) variants of fHbp ID 22, wherein the fHbp ID 22 variant comprises an amino acid substitution at S128 (e.g., S128T); and variants of fHbp ID 55, wherein the fHbp ID 55 variant comprises an amino acid substitution at H248 (e.g., H248L);

[0189] 14) variants of fHbp ID 22, wherein the fHbp ID 22 variant comprises an amino acid substitution at V131 (eg, V131D); and variants of fHbp ID 55, wherein the fHbp ID 55 variant comprises an amino acid substitution at E92 (eg, E92K);

[0190] 15) variants of fHbp ID 22, wherein the fHbp ID 22 variant comprises an amino acid substitution at K219 (e.g., K219N); and variants of fHbp ID 55, wherein the fHbp ID 55 variant comprises an amino acid substitution at E92 (e.g., E92K);

[0191] 16) variants of fHbp ID 22, wherein the fHbp ID 22 variant comprises an amino acid substitution at G220 (e.g., G220S); and variants of fHbp ID 55, wherein the fHbp ID 55 variant comprises an amino acid substitution at E92 (e.g., E92K);

[0192] 17) a first variant of fHbp ID 1, wherein the first fHbp ID 1 variant comprises an amino acid substitution at E92 (e.g., E92K); and a second variant of fHbp ID 1, wherein the second fHbp ID 1 variant comprises an amino acid substitution at H248 (e.g., H248L);

[0193] 18) a first variant of fHbp ID 1, wherein the first fHbp ID 1 variant comprises an amino acid substitution at E92 (e.g., E92K); and a second variant of fHbp ID 1, wherein the second fHbp ID 1 variant comprises an amino acid substitution at S223 (e.g., S223R);

[0194] 19) a first variant of fHbp ID 22, wherein the first fHbp ID 22 variant comprises an amino acid substitution at N115 (e.g., N115I); and a second variant of fHbp ID 22, wherein the second fHbp ID 22 variant comprises an amino acid substitution at D211 (e.g., D211A);

[0195] 20) a first variant of fHbp ID 22, wherein the first fHbp ID 22 variant comprises an amino acid substitution at N115 (e.g., N115I); and a second variant of fHbp ID 22, wherein the second fHbp ID 22 variant comprises an amino acid substitution at K219 (e.g., K219 N);

[0196] 21) a first variant of fHbp ID 22, wherein the first fHbp ID 22 variant comprises an amino acid substitution at N115 (e.g., N115I); and a second variant of fHbp ID 22, wherein the second fHbp ID 22 variant comprises an amino acid substitution at G220 (e.g., G220S);

[0197] 22) a first variant of fHbp ID 22, wherein the first fHbp ID 22 variant comprises an amino acid substitution at D121 (e.g., D121G); and a second variant of fHbp ID 22, wherein the second fHbp ID 22 variant comprises an amino acid substitution at G220 (e.g., G220S);

[0198] 23) a first variant of fHbp ID 22, wherein the first fHbp ID 22 variant comprises an amino acid substitution at S128 (e.g., S128T); and a second variant of fHbp ID 22, wherein the second fHbp ID 22 variant comprises an amino acid substitution at G220 (e.g., G220S);

[0199] 24) a first variant of fHbp ID 22, wherein the first fHbp ID 22 variant comprises an amino acid substitution at V131 (eg, V131D); and a second variant of fHbp ID 22, wherein the second fHbp ID 22 variant comprises an amino acid substitution at G220 (eg, G220S);

[0200] 25) a first variant of fHbp ID 55, wherein the first fHbp ID 55 variant comprises an amino acid substitution at E92 (e.g., E92K); and a second variant of fHbp ID 55, wherein the second fHbp ID 55 variant comprises an amino acid substitution at S223 (e.g., S223R);

[0201] 26) a first variant of fHbp ID 55, wherein the first fHbp ID 55 variant comprises an amino acid substitution at E92 (e.g., E92K); and a second variant of fHbp ID 55, wherein the second fHbp ID 55 variant comprises an amino acid substitution at H248 (e.g., H248L);

[0202] 27) variants of fHbp ID 22 comprising the amino acid substitutions: L130R and G133D, and variants of fHbp ID 1, wherein the fHbp ID 1 variant comprises an amino acid substitution at S223 (e.g., S223R);

[0203] 28) variants of fHbp ID22 comprising the amino acid substitutions: L130R and G133D, and variants of fHbp ID 1, wherein the fHbp ID 1 variant comprises an amino acid substitution at H248 (eg, H248L);

[0204] 29) a variant of fHbp ID 22 comprising the amino acid substitutions: L130R, G133D and K219N, and a variant of fHbp ID 1, wherein the fHbp ID 1 variant comprises an amino acid substitution at S223 (eg, S223R) or H248 (eg, H248L); or

[0205] 30) variants of fHbp ID22 comprising the amino acid substitutions: L130R, G133D and G220S, and variants of fHbp ID 1, wherein the fHbp ID 1 variant comprises an amino acid substitution at S223 (eg S223R) or H248 (eg H248L).

[0206] Immunogenic compositions encompassed by the present disclosure include, but are not limited to, compositions comprising:

[0207] 1) at least one variant fHbp of the present disclosure; and

[0208] 2) NspA;

[0209] Wherein fHbp and / or NspA can be provided as recombinant proteins and / or in vesicle-based compositions (e.g., OMV or MV). It should be noted that when the composition comprises both NspA and fHbp, the bactericidal activity of the antibody caused by administering the composition can be derived from the cooperation of antibodies against one or both antigens. Examples of immunogenic compositions provided by the present disclosure include:

[0210] a) an immunogenic composition comprising a fHbp variant as described above (eg, wherein the variant fHbp elicits a bactericidal antibody response to at least one strain of Neisseria meningitidis);

[0211] b) an immunogenic composition comprising a fHbp variant as described above (e.g., wherein the variant fHbp elicits a bactericidal antibody response to at least one strain of Neisseria meningitidis); and a recombinant NspA protein;

[0212] c) an immunogenic composition comprising a naturally occurring OMV obtained from a genetically modified Neisserial host cell, the host cell being 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, wherein the OMV comprises the encoded variant fHbp; and

[0213] d) An immunogenic composition comprising a natural OMV obtained from a genetically modified Neisserial host cell, the host cell being 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, wherein the OMV comprises the encoded variant fHbp; and wherein the Neisserial host cell also produces higher levels of NspA, such that the OMV also comprises NspA. For example, the Neisserial host cell can be a cell that has been genetically modified to achieve increased expression of NspA.

[0214] An "immunologically effective amount" refers to an amount administered to an individual in a single dose, as part of a series of the same or different antigenic compositions, effective to elicit an antibody response that is effective to treat or prevent symptoms or diseases caused by, for example, Neisseria, particularly Neisseria meningitidis infection, more particularly Neisseria meningitidis type B infection. This amount varies with the health and physical condition of the individual to be treated, age, the ability of the individual's immune system to produce antibodies, the degree of protection desired, the formulation of the vaccine, the treating clinician's assessment of the medical condition, and other relevant factors. It is expected that the amount will fall within a relatively wide range that can be determined by routine experiments.

[0215] The amino acid sequences of NspA polypeptides are known in the art. See, for example, WO 96 / 29412; and Martin et al. (1997) J. Exp. Med. 185:1173; GenBank Accession No. U52066; and GenBank Accession No. AAD53286. An "NspA polypeptide" may comprise a polypeptide having the same amino acid sequence as that in Fig.40 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 to a contiguous stretch of about 75 amino acids to about 100 amino acids, about 100 amino acids to about 150 amino acids, or about 150 amino acids to about 174 amino acids depicted in and set forth in SEQ ID NO: 25. An "NspA polypeptide" may comprise a polypeptide 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 to a contiguous stretch of about 75 amino acids to about 100 amino acids, about 100 amino acids to about 150 amino acids, or about 150 amino acids to about 174 amino acids. Fig.40 In some cases, the NspA polypeptide lacks a signal sequence; in other cases (e.g., for expression in a host cell), the NspA polypeptide comprises a signal sequence.

[0216] The dosage regimen can be a single dose schedule or a multi-dose schedule (e.g., including booster doses), wherein the unit dosage form of the antigenic composition is administered at different times. As used herein, the term "unit dosage form" refers to physically dispersed units suitable as unit doses 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 in combination with a pharmaceutically acceptable excipient (e.g., a pharmaceutically acceptable diluent, carrier or excipient). The antigenic composition can be administered in combination with other immunomodulators.

[0217] Antigenic compositions can be provided in a pharmaceutically acceptable excipient, which can be a solution, such as a sterile aqueous solution, typically a saline solution, or they can be provided in powder form. Such excipients can be substantially inert if desired.

[0218] In some embodiments, a subject immunogenic composition comprises a subject fHbp present in a vesicle. In some embodiments, a subject immunogenic composition comprises a subject fHbp present in a MV. In some embodiments, a subject immunogenic composition comprises a subject fHbp present in an OMV. In some embodiments, a subject immunogenic composition comprises a mixture of MVs and OMVs comprising a subject fHbp. Vesicles, such as MVs and OMVs, are described above.

[0219] The antigenic composition may further contain an adjuvant. Examples of known suitable adjuvants that can be used in humans include, but are not necessarily limited to, aluminum adjuvants (such as aluminum phosphate or aluminum hydroxide), MF59 (4.3% w / v squalene, 0.5% w / v Tween 80 TM , 0.5% w / v Span 85), CpG-containing nucleic acids (in which cytosine is unmethylated), QS21, MPL, 3DMPL, extracts from Aquilla, ISCOMS, LT / CT mutants, poly (D,L-lactide-co-glycolide) (PLG) microparticles, Quil A, interleukins, etc. For experimental animals, Freund's adjuvant (Freund's incomplete adjuvant; complete Freund's adjuvant), N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE) and RIBI can be used, which contains three components extracted from bacteria, namely monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene / Tween 80 emulsion. The effectiveness of an adjuvant can be determined by measuring the amount of antibodies directed against the immunogenic antigen or its antigenic epitope.

[0220] Other 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 immunostimulants, such as muramyl peptides (see below) or bacterial cell wall components), such as, for example, (a) MF59 (WO 90 / 14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, Powell and Newman, eds., Plenum Press 1995) containing 5% squalane, 0.5% Tween 80 and 0.5% Span 85 (optionally containing MTP-PE), which is formulated into submicron particles using a microfluidizer, (b) SAF containing 10% squalane, 0.4% Tween 80, 5% pluronic blocking polymer L121 and thr-MDP, which is microfluidized into a submicron emulsion or vortexed to produce an emulsion of larger particle size, and (c) SAF containing 2% squalane, 0.2% Tween 80, 0.5% Span 85 (optionally containing MTP-PE). 80 and one or more bacterial cell walls, such as monophospholipid A (MPL), trehalose dimycolate (TDM) and cell wall skeleton (CWS) RIBI adjuvant system (RAS) (Ribi Immunochem, Hamilton, Mont.), such as MPL + CWS (Detox TM); (2) saponin adjuvants, such as QS21 or Stimulon TM(Cambridge Bioscience, Worcester, Mass.) or particles produced therefrom, such as ISCOMs (immunostimulatory complexes), which may be free of additional detergents, such as WO 00 / 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)), 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), such as GB-2220221, EP-A-0689454, optionally in the substantial absence of alum when used with pneumococcal saccharides, such as WO 00 / 56358; (6) 3dMPL in combination with, for example, QS21 and / or an oil-in-water emulsion, for example, EP-A-0835318, EP-A-0735898, EP-A-0761231; (7) oligonucleotides comprising a CpG motif (see, for example, WO 98 / 52581), for example, oligonucleotides containing at least one CG dinucleotide in which the cytosine is unmethylated; (8) polyoxyethylene ethers or polyoxyethylene esters (see, for example, WO 99 / 52549); (9) combinations of polyoxyethylene sorbitan ester surfactants with octoxynol (WO 01 / 21207) or polyoxyethylene alkyl ether or ester surfactants with at least one further nonionic surfactant, such as octoxynol (WO 01 / 21152); (10) saponins and immunostimulatory oligonucleotides (e.g., CpG oligonucleotides) (WO 00 / 62800); (11) immunostimulants and metal salt particles, for example WO 00 / 23105; (12) saponin and oil-in-water emulsions, for example WO 99 / 11241; (13) saponin (e.g. QS21) + 3dMPL + IM2 (optionally + sterol), for example WO 98 / 57659; (14) other substances that act as immunostimulants to enhance the efficacy of the composition. Muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamyl (thr-MDP), N-25 acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine-L-Alanine-2-(1 '-2 '-dipalmitoyl--sn-glyceryl-3-hydroxyphosphoryloxy)-ethamine MTP-PE) etc.The adjuvant that is suitable for people's administration is particularly interested.In some cases, adjuvant is aluminum salt adjuvant (for example aluminum phosphate or aluminum hydroxide).

[0221] Antigenic compositions may contain other components, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, talcum, cellulose, glucose, sucrose, magnesium, carbonates, etc. As required, compositions may contain pharmaceutically acceptable auxiliary substances to approach physiological conditions, such as pH adjustment and buffering agents, toxicity regulators, etc., for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.

[0222] The concentration of the subject fHbp in the formulation can vary widely (e.g., from less than about 0.1%, e.g., at or at least about 2% to as much as 20% to 50% or more by weight), and will generally be selected primarily based on liquid volume, viscosity, and patient-based factors according to the particular mode of administration selected and the patient's needs.

[0223] The formulation containing fHbp can be provided in the form of a solution, suspension, tablet, pill, capsule, powder, gel, cream, lotion, ointment, aerosol, etc. It is recognized that oral administration may require protection of the composition from digestion. This is generally achieved by combining the composition with an agent that makes it resistant to acid and enzymatic hydrolysis or by packaging the composition in a carrier with appropriate resistance. Methods for preventing digestion are well known in the art.

[0224] A formulation containing fHbp can also be provided to increase the serum half-life of fHbp after administration. For example, when the separated fHbp is formulated for injection, fHbp can be provided in a liposome formulation, prepared as a colloid or other conventional techniques for extending the serum half-life. Various methods can be used to prepare liposomes, such as, for example, Szoka et al., Ann.Rev.Biophys.Bioeng.9:467 (1980), such as U.S. Patent Nos. 4,235,871, 4,501,728 and 4,837,028. The preparation can also be provided in a controlled release or slow release form.

[0225] Methods of inducing an immune response

[0226] The present disclosure provides methods of inducing an immune response to at least one Neisseria strain in a mammalian host. The methods generally involve administering an effective amount of a subject immunogenic composition to an individual in need thereof.

[0227] Antigenic compositions containing fHbp are usually applied to human subjects at risk of acquiring Neisseria disease to prevent or at least partially prevent the development of the disease and its complications. An amount sufficient to achieve this is defined as a "therapeutically effective dose". The effective amount applied to the treatment will depend on, for example, the antigenic composition, the mode of administration, the patient's weight and general health, and the judgment of the attending physician. Single or multiple doses of the antigenic composition can be administered according to the dosage and frequency and administration route required and tolerated by the patient.

[0228] Antigenic compositions containing fHbp are usually administered in an amount that effectively induces an immune response in the host, particularly a humoral immune response, such as a bactericidal antibody response. As described above, the amount of immunity will vary, and can generally be about 1 μg to 100 μg per 70kg patient, generally 5 μg to 50 μg / 70kg. Substantially higher dosages (e.g., 10 mg to 100 mg or more) can be suitable for oral, nasal or topical administration routes. After initial administration, the same antigenic compositions containing different fHbp can be boosted. In some cases, vaccination involves at least one booster, including two boosters in some cases.

[0229] In general, immunization can be administered by any suitable route, including oral, intranasal, nasopharyngeal, parenteral, enteral, gastric, topical, transdermal, subcutaneous, intramuscular, in tablet, solid, powder, liquid, aerosol form, topical or systemic administration of the composition with or without added excipients. Actual methods for preparing parenterally administrable compositions are known or apparent to those skilled in the art and are described in more detail in publications such as Remington's Pharmaceutical Science, 15th edition, Mack Publishing Company, Easton, Pa. (1980).

[0230] The anti-fHbp immune response can be assessed by known methods (e.g., by obtaining serum from the individual before and after the initial immunization and demonstrating changes in the individual's immune status, such as immunoprecipitation assays, ELISA or bactericidal assays, Western blot assays, or flow cytometry assays, etc.).

[0231] Whether the variant fHbp of the present disclosure causes a bactericidal response to one or more strains of Neisseria meningitidis in a mammalian host can be determined using any known assay. For example, human fH transgenic mice can be used, wherein the mice express human fH (e.g., human fH is present in mouse serum at a concentration of about 100 μg / ml or greater than 100 μg / ml). The variant fHbp of the present disclosure is administered to human fH transgenic mice. After a period of time, the bactericidal activity of serum from mice against one or more strains of Neisseria meningitidis is tested. Suitable controls include, for example, fHbp ID 1. Examples of suitable assays are described in Vu et al. (2012) Sci. Reports 2: 341.

[0232] Antigenic compositions can be administered to an immunologically active mammalian host (e.g., a human subject) with respect to Neisseria meningitidis. In a specific embodiment, the subject is a human child about 5 years old or younger, preferably about 2 years old or younger, and the antigenic compositions are administered at any one or more of the following times: 2 weeks after birth, 1, 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 old.

[0233] It may often be desirable to initiate immunization prior to the first sign of disease symptoms or at the first sign of potential or actual exposure to infection or disease (eg, due to exposure or infection with Neisseria).

[0234] Example

[0235] The following examples are presented to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered. 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 may be used, such as bp, base pairs; kb, kilobase; pl, picoliter; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids; kb, kilobase; bp, base pairs; nt, nucleotides; im, intramuscular (n); ip, intraperitoneal (n); sc, subcutaneous (n); and the like.

[0236] Example 1: Identification and characterization of fHbp ID1 mutants

[0237] Materials and methods

[0238] Library screening

[0239] A random mutant fHbp library was generated by error-prone polymerase chain reaction (PCR), and the PCR product was subsequently cloned into a pET28 expression plasmid, which includes a signal sequence that allows surface display on E. coli. Fluorescence-activated cell sorting was used to separate mutant clones with low binding of human fH and high binding of control anti-fHbp monoclonal antibodies, which ensured the full expression and correct folding of fHbp mutants. The collected cells were spread on agar plates (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 amplicon was purified (PCR purification kit; Qiagen), and subjected to DNA sequencing of the fHbp gene using primers annealed to the T7 promoter and T7 terminator. The approach of screening random mutant fHbp libraries has the potential to identify: 1) positions that affect fH binding that could not be predicted from the crystal structure alone; and 2) substitutions other than alanine that affect fH binding in situations where alanine substitution does not result in a sufficient reduction in fH binding.

[0240] Selection of mutants for further study

[0241] The positions of the amino acid substitutions identified from the FACS experiments were examined in the crystal structure of fHbp in complex with a human fH fragment. Site-specific mutagenesis was performed on mutants of the library. Recapitulation of the library mutants by site-specific mutagenesis was necessary to create a soluble recombinant fHbp protein for further characterization. This approach also removed undesired secondary mutations that were present in many of the sorted clones and were far from the fH binding site. Site-specific mutants were constructed using the Phusion Directed Mutagenesis Kit (Thermo Scientific, Inc.).

[0242] Expression and purification of soluble mutant fHbp

[0243] Soluble recombinant fHbp was expressed in E. coli, and lysates were prepared as described previously. fHbp was purified by nickel affinity chromatography using HiTrap chelating HP columns (5 ml; GE Life Sciences, Inc.) and Akta Purifier chromatography systems (GE Life Sciences). Buffers for binding and elution using imidazole gradients were prepared according to the column manufacturer's protocol. Fractions containing purified fHbp were combined, dialyzed against PBS containing 3% sucrose, and stored at -80°C before use.

[0244] For mouse immunogenicity studies, a second purification step was performed using ion exchange chromatography with a HiTrap SP HP column (5 ml; GE Life Sciences). The binding and elution buffers were 25 mM MES, pH 5.5, containing 150 mM and 750 mM NaCl, respectively. The bound fHbp was eluted from the SP column using a linear gradient formed by the binding and elution buffers. The fractions containing the purified fHbp were combined, dialyzed against PBS containing 3% sucrose, and stored at -80 °C before use.

[0245] Purification of human factor H (fH)

[0246] Human fH was purified on an fHbp affinity column. 5mg fHbp ID 1 was coupled to an NHS-activated HP column (5ml; GE Life Sciences) using the manufacturer's protocol to prepare the column. Human serum from healthy individual donors was diluted 1:1 in phosphate buffered saline (PBS). Serum was applied to the column and the column was washed with 10 volumes of PBS (i.e., 50ml). The bound fH was eluted with 5 column volumes of 0.1M glycine-HCl, pH 2.7. The eluted fractions were collected in a tube containing 50 μl 1M TRIS-HCl, pH 9.0. The fractions containing fH were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (4-12% NuPAGE; Invitrogen). Electrophoresis was performed at 200V for 45 minutes using 1x MES running buffer (Invitrogen). Proteins were visualized by staining with Coomassie G-250 (SimplyBlue SafeStain; Invitrogen). Fractions containing fH were pooled and dialyzed against PBS, and aliquots of fH were stored at -30 °C before use.

[0247] Characterization of fHbp mutants

[0248] SDS-PAGE. The size and purity of the purified fHbp mutant proteins were assessed by SDS-PAGE using 4-12% polyacrylamide gradient gels (NuPAGE; Invitrogen, Inc.). 2 μg of each protein was loaded onto the gel. SDS-PAGE was performed as described above for fH.

[0249] fH was bound to fHb by enzyme-linked immunosorbent assay (ELISA). Wells of a 96-well microtiter plate (Immulon 2HB; Thermo Scientific) were coated with 2 μg / ml purified recombinant wild-type fHbp (positive control) or mutant fHbp (experimental). Nonspecific binding to the wells was blocked with PBS containing 1% BSA (Lifeblood Medical, Inc.) or 5% skim milk powder (Carnation; Nestle, Inc.). Five-fold serial dilutions of purified human fH in dilution buffer (PBS containing 0.1% Tween-20, 0.01% sodium azide, and 1% BSA) (ranging from 25 to 0.0016 μg / ml) were added to the wells and incubated for 2 hours at room temperature. After washing three 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 plates were incubated for 1 hour at room temperature. After washing the wells again, bound primary antibodies were detected with donkey anti-sheep IgG conjugated with alkaline phosphatase (1:5,000; Sigma-Aldrich, Inc.) in dilution buffer. The plates were incubated for 1 hour at room temperature and the wells were washed again. The plates were incubated for 1 hour at room temperature with PBS containing 0.1% Tween-20 (Sigma) and 0.01% sodium azide (Sigma). 2 The ELISA was developed with a phosphatase substrate (1 mg / ml p-nitrophenyl phosphate; Sigma) in PBS (pH 9.8). After incubation at room temperature for 30 minutes, the absorbance at 405 nm was measured in a UV-VIS plate reader (Spectromax 190; Molecular Devices, Inc.).

[0250] Anti-fHbp monoclonal antibodies were bound to fHbp by ELISA. The wells of the microtiter plate were coated with fHbp and blocked and washed as described above for fH ELISA. A 5-fold serial dilution of mouse anti-fHbp monoclonal antibody (mAb) 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 the wells, the primary antibody was detected with goat anti-mouse IgG (1; 5,000; Sigma-Aldrich) conjugated with alkaline phosphatase. ELISA was developed and read as described above.

[0251] fH is bound to fHbp by surface plasmon resonance (SPR). SPR experiments were performed on a Biacore X100Plus 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 fixed in flow cell 2, and blank fixation (without fH) was performed in flow cell 1 as a reference. Three startup cycles consisting of HEPES buffered saline containing 3mM EDTA and 0.05% surfactant P-20 (GE Life Sciences) and regeneration with 100mM glycine, 3M NaCl, pH 2.0 were performed to condition the chip surface. Dilutions of purified recombinant fHbp ranging from 100 to 1nM (wild type) or 316 to 3.16nM were injected for 150 seconds. Dissociation was monitored for 300 seconds, and data were analyzed using Biacore X100 evaluation software.

[0252] Mouse immunogenicity. Groups of wild-type CD-1 mice (N=14 to 21) were immunized with fHbp vaccines adsorbed with aluminum hydroxide. Each dose of vaccine contained 10 μg fHbp and 600 μg Alhydrogel (Brenntag Biosector) in 10 mM histidine, 150 mM NaCl, pH 6.5. Two doses were given three weeks apart, and blood was collected by cardiac puncture three weeks after the second dose. Blood was processed to obtain serum, which was kept at -80°C for long-term storage (>2 weeks) or 4°C for short-term storage (<2 weeks).

[0253] Human fH transgenic BALB / c mice were first screened to identify animals with serum human fH concentrations >240 μg / ml using fHbp ELISA and a standard curve of purified human fH. ELISA was performed using purified fHbp ID 1 immobilized on a plate, and the primary and secondary antibodies detecting fH were the same as above (see "Binding of fH to fHbp by ELISA").

[0254] Groups of transgenic mice (N=11 to 21) were immunized with fHbp vaccine adsorbed with aluminum hydroxide (same amount of antigen and adjuvant as described above for wild-type CD-1 mice). Three doses were administered at three-week intervals, and blood was collected three weeks after the third dose of vaccine. Serum was processed and stored as described above.

[0255] Serum bactericidal antibody (SBA) response. Human complement-mediated SBA responses were measured for meningococcal strains with identical or closely matched fHbp sequences compared to the corresponding vaccine antigens. Bacteria were grown in conventional Frantz 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, MC. Humana Press Inc. Totowa, NJ) containing 4 mM lactate and 0.02 mM CMP-NANA to mid-exponential phase (OD620 nm = 0.6). Bacteria were diluted 1:25,000 in Dulbecco's PBS containing 1% BSA (Equitech Bio.). Human complement was from a donor without intrinsic bactericidal antibodies and IgG antibodies were depleted using HiTrap Protein G columns (5 ml; GE Life Sciences). Each reaction contained 25% human complement, approximately 400 cfu of bacteria, and dilutions of test antiserum or control antibodies. SBA titers were calculated as the serum dilution that resulted in a 50% reduction in cfu relative to the negative control wells after incubation for 60 minutes at 37°C. Protein purification: Recombinant fHbp with a C-terminal hexahistidine tag was expressed in Escherichia coli and purified by metal chelate chromatography (HiTrap Chelate HP; GE Life Sciences) followed by ion exchange chromatography (HiTrap SP; GE Life Sciences). Proteins (2 μg each) were separated on 4-12% NuPAGE gels (Invitrogen) using MES running buffer (Invitrogen) and visualized with Coomassie blue stain (Simply Blue Safe Stain; Invitrogen).

[0256] result

[0257] A random mutant library-based approach was developed to identify fHbp mutants with reduced human fH binding. This approach is able to identify mutations that lead to reduced fH binding that might not be predicted based on structural information alone, and is able to generate multiple amino acid substitutions at any given position. This approach contrasts with the common approach of alanine substitutions at selected positions, which sometimes results in small reductions in fH binding.

[0258] Using a random mutant library approach, we identified five promising novel fHbp ID 1 mutants. Figure 1 Purified recombinant mutant fHbp ID 1 antigens Q38R, E92K, R130G, S223R and H248L are shown in FIG.

[0259] Figure 1 : Purity of fHbp ID 1 mutants. Recombinant fHbp with a C-terminal hexa-histidine tag was expressed in E. coli and purified by metal chelate chromatography (HiTrap Chelating HP; GE Life Sciences) followed by ion exchange chromatography (HiTrap SP; GE Life Sciences). Proteins (2 μg each) were separated on 4-12% NuPAGE gels (Invitrogen) using MES running buffer (Invitrogen) and visualized with Coomassie blue stain (Simply Blue Safe Stain; Invitrogen). Lane 1, Kaleidoscope molecular weight marker (Bio-Rad Laboratories); 2, fHbp ID 1 wild type; 3, Q38R; 4, E92K; 5, R130G; 6, S223R; 7, H248L.

[0260] These mutants exhibited reductions in fH binding ranging from about 10-fold (R130G) to about 20-fold (Q38R) to about 100-fold (E92K, S223R, and H248L) ( FIG. 2 ).

[0261] Fig. 2, panels A and B. fHbp ID 1 mutants were bound to fH by ELISA. The wells of a microtiter plate were coated with purified recombinant ID 1 wild type (WT) or one of six different mutant proteins. Different concentrations of purified human fH were added to the wells. The bound fH was detected with sheep anti-human fH (Abcam) and donkey anti-sheep IgG (Sigma) conjugated to alkaline phosphatase. A, positive control fHbp ID 1 wild type (WT) protein, with high binding of human fH. Negative control fHbp ID 1 R41S mutant, with low binding of fH. B, novel fHbp ID 1 mutant with reduced fH binding. R130G mutant showed moderate fH binding, Q38R showed low binding, and E92K, S223R and H248L showed significantly lower binding than R41S binding. The mean and standard deviation of repeated measurements are shown.

[0262] A similar pattern of reduced binding of fH to the mutant proteins was obtained from surface plasmon experiments, with the R130G and Q38R mutants showing some binding, while the other three mutants showed no detectable binding (Fig. 3 Panels A and B).

[0263] Fig. 3, panels AE. fH binding of fHbp ID 1 mutants obtained by surface plasmon resonance. 3000 response units of purified human fH were coupled to a CM5 chip (GE Life Sciences), and 316nM purified recombinant fHbp150 seconds were injected. For reference, the same data for ID 1 wild-type (WT) protein are shown in each figure. The same binding pattern as in ELISA was observed (Fig. 2, above); moderate binding of the R130G mutant to fH, low binding of Q38R, and very low binding of E92K, S223R, and H248L. All experiments used HBS-EP running buffer and Biacore X100 Plus surface plasmon resonance instrument. Data were analyzed using Biacore X100 evaluation software.

[0264] All five mutant fHbp ID 1 proteins retained the conformational epitope recognized by the anti-fHbp monoclonal antibodies. The concentration-dependent binding of the five anti-fHbp monoclonal antibodies to wild-type or mutant fHbp is shown in FIG4 .

[0265] Fig. 4 panels AE. As measured by ELISA, the binding of mouse anti-fHbp monoclonal antibodies to fHbp mutant proteins. Similar concentration-dependent binding of anti-fHbp monoclonal antibodies shows that wild-type and mutant fHbp are present in the wells of microtiter plates in similar amounts, and mutant fHbp retains conformational epitopes recognized by five different monoclonal antibodies. The second antibody is a goat anti-mouse IgG conjugated to alkaline phosphatase (Sigma). The mean and standard deviation of repeated measurements are shown.

[0266] The mutant proteins also retained thermal stability similar to wild-type fHbp ID 1, except for the E92K mutant, which had slightly reduced stability. Finally, when tested against serogroup B strain H44 / 76, the mutants elicited similar bactericidal antibody responses in wild-type CD-1 mice (Figure 5 Panels AB).

[0267] Fig. 5 panels A and B. Bactericidal antibody response to fHbp ID 1 mutants in mice. Fig. 5 panel A, a group of 12 to 14 wild-type mice were immunized intraperitoneally with two doses of purified recombinant fHbp (10 μg / dose) given at three-week intervals. Serum was obtained three weeks after the second dose. Serum bactericidal activity was measured using IgG-depleted human serum as a complement source and serogroup B strain H44 / 76 as a test strain. H44 / 76 expresses fHbp ID 1, which matches the control fHbp ID 1WT vaccine. Each symbol represents the titer of a single mouse, and the horizontal bar represents the geometric mean titer. There was no statistically significant difference between the WT group and each mutant group (by t test, p>0.4). Fig. 5 panel B, a group of 14 to 15 human fH transgenic mice were immunized intraperitoneally with three doses of purified recombinant fHbp (10 μg per dose) given at three-week intervals. Serum was obtained three weeks after the third dose. Serum bactericidal activity was measured as described above for wild-type mice.

[0268] Figure 6 Panels A and B. Binding of human fH to fHbp ID 1 single and double mutants by ELISA. The experiments were performed as described above for Figure 2 Panels AB. Figure 7 Bactericidal antibody responses to fHbp ID 1 single and double mutants in mice. Groups of 20 wild-type mice were immunized and serum bactericidal antibody responses were determined as described above for Figure 5, Panel A.

[0269] Example 2: Characterization of fHbp ID55 mutants

[0270] Materials and methods

[0271] The experiment was performed as described in Example 1.

[0272] result

[0273] Three promising mutants identified in fHbp ID 1 were also constructed in fHbp ID 55; these included E92K, S223R and H248L. All three fHbp ID 55 mutants significantly reduced the binding of fH (Fig. 8 panel A). The mutants retained conformational integrity, as judged by the binding of mouse anti-fHbp monoclonal antibody JAR 41 (Fig. 8 panel B).

[0274] Figure 8, panels A and B. fH binding of fHbp ID 55 mutants. A. Binding of fH obtained by ELISA to fixed fHbp ID 55 mutants. The experiment was performed as described in the legend of Figure 2. The average and range of two to four replicates are shown. B. Concentration-dependent binding of anti-fHbp monoclonal antibody (mAb) JAR41 shows that recombinant fHbp is present in similar amounts in the wells of the microtiter plate and retains the conformation in the epitope region recognized by JAR41 (N-terminal domain; Vu et al. (2012) Sci. Reports, supra). The second antibody is goat anti-mouse IgG conjugated to alkaline phosphatase (Sigma).

[0275] Fig. 9 Bactericidal antibody responses to fHbp ID 55 mutants in wild-type mice. Groups of 12 mice were immunized and serum bactericidal antibody responses were determined as described above for Figure 5, panel A. Bactericidal activity was measured against mutants of strain H44 / 76 expressing fHbp ID 55.

[0276] Figure 10A, bactericidal antibody response of human fH transgenic mice to fHbp ID 55S223R mutant. Groups of 11 to 12 transgenic mice were immunized with three doses of purified recombinant fHbp (12 μg / dose) or one-tenth of the human dose of the licensed Trumenba (Pfizer) vaccine containing a total of 12 μg fHbp. Transgenic mice were immunized and serum bactericidal antibody responses were determined as described above for Figure 5, panel B. Figure 10, panel B, bactericidal antibody response to Trumenba associated with serum human fH concentrations in wild-type and human fH transgenic mice. Serum human fH concentrations were measured by ELISA as previously described (Beernink et al. (2011) Journal of Immunology 186 (6): 3606-14).

[0277] Example 3: Identification and characterization of fHbp ID22 mutants

[0278] Materials and methods

[0279] The experiment was performed as described in Example 1.

[0280] result

[0281] An independent search for random fHbp mutants with reduced fH binding was performed using fHbp ID 22, which is in variant group 2 (subfamily A). This screen yielded six promising novel mutants ( Fig.17). Binding of fH to ID 22 wild type and the aforementioned D211A mutant by ELISA is shown in Figure 11 Panel A. Binding of fH to six novel mutant ID 22 proteins is shown in Figure 11 Panel B.

[0282] Figure 11 panels AD. fH binding of fHbp ID 22 library mutants. Figure 11 panel A, positive control fHbp ID 22 wild-type (WT) protein with high binding of human fH. Negative control fHbp ID 22D211A mutant with low binding of fH. Figure 11 panel B, novel fHbp ID 22 mutants with reduced fH binding. All mutants showed low binding, and V131D showed very low binding similar to D211A. The experiment was performed as described in the legend of Figure 2. The average and range of 2 to 4 replicates are shown. Figure 11 panel C, fH binding of a subset of fHbp ID 22 mutants with fH concentrations up to 100 μg / ml. Figure 11 panel D, binding of anti-fHbp monoclonal antibody JAR4 to a subset of mutants (same symbols used in Figure 11 panel C). The novel mutant K219N retained JAR4 binding, while the G220S mutant had reduced JAR4 binding. All mutants had normal binding to another anti-fHbp monoclonal antibody JAR31 (data not shown). The mean and standard deviation of replicate measurements are shown.

[0283] The bactericidal antibody responses of wild-type CD-1 mice to the novel mutants V131D and K219N, as well as the control wild-type ID 22 protein and the previously characterized mutant D211A are shown in FIG. 12 .

[0284] Figure 12, panels AB. Bactericidal antibody responses to fHbp ID 22 library mutants in wild-type mice. Library mutants with low binding to human fH were selected for immunization. Groups of 10 to 21 mice were immunized using two doses of purified recombinant fHbp (10 μg / dose) given at intervals of three weeks. Serum was obtained three weeks after the second dose. Serum bactericidal activity was measured using IgG-depleted human serum as a complement source and serogroup B strain CH597 as a test strain. The strain expresses fHbpID 23, which is closely matched with the control fHbp ID 22WT vaccine. Figure 12, panels A, experiments testing novel mutants V131D and K219N. The D211A mutant was used as a control mutant fHbp vaccine that does not reduce immunogenicity. Each symbol represents the titer of a single mouse, and the horizontal bar represents the geometric mean titer. Figure 12, panels B, the second experiment testing novel mutants D121G, S128T, F129S and G220S. Aside from a modest loss of V131D, no significant loss of immunogenicity was observed for the novel mutant fHbp.

[0285] Fig.13 Bactericidal antibody responses to fHbp ID 22 library mutant K219N in human fH transgenic mice. The control mutant D211A gave a higher response, and the K219N mutant produced a response similar to the fHbp ID 22 wild-type (WT) antigen.

[0286] Fig.14 . Thermostability of fHbp ID 22 measured by differential scanning microcalorimetry. fHbp ID 22 wild type (WT, solid line) undergoes an unfolding transition at 38 (N-terminus) and 81 °C (C-terminal domain). fHbp ID22 L130R / G133D double mutant exhibits 19 °C higher N-terminal domain thermostability compared to ID 22 WT.

[0287] Figure 15 Panels AB. fHbp ID 22 triple mutants that combine stabilizing substitutions L130R and G133D (double mutants, DM) with library-derived mutants to reduce fH binding. A, fH binding to fHbp ID 22 triple mutants. B, control mouse anti-fHbp monoclonal antibody (mAb) JAR4 binding (same symbols as in panel A. All stable mutants bind JAR 4 better than fHbp ID22WT.

[0288] Fig.16 Bactericidal antibody responses to fHbp ID 22 triple mutants in human fH transgenic mice. The two mutants tested combined a stabilizing double mutant (DM) with K219N and G220S, respectively. The triple mutant elicited 8-fold and 18-fold higher responses than the control ID22 WT antigen.

[0289] exist Fig.17 An overview of the exemplary fHbp ID 1 and ID 22 mutants described above is presented in the Table in .

[0290] The present invention relates to the following embodiments.

[0291] 1. A factor H binding protein (fHbp) variant, wherein the variant comprises an amino acid substitution selected from at least one of the following:

[0292] a) amino acid substitution of glutamine at amino acid 38 (Q38);

[0293] b) amino acid substitution of glutamic acid at amino acid 92 (E92);

[0294] c) substitution of glycine for arginine at amino acid 130 (R130G);

[0295] d) an amino acid substitution of serine at amino acid 223 (S223); and

[0296] e) substitution of histidine for leucine at amino acid 248 (H248L),

[0297] wherein the amino acid substitutions are relative to fHbp ID 1 (SEQ ID NO: 1), wherein the variant comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO: 1, wherein the variant fHbp binds human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID 1 for human fH, and wherein the variant induces a bactericidal antibody response to at least one strain of Neisseria meningitidis in a mammalian host.

[0298] 2. The variant fHbp of embodiment 1, wherein the amino acid substitution at Q38 is Q38R, Q38K, Q38H, Q38F, Q38Y or Q38W.

[0299] 3. The variant fHbp of embodiment 1, wherein the amino acid substitution at E92 is E92K, E92R, E92H, E92F, E92Y or E92W.

[0300] 4. The variant fHbp of embodiment 1, wherein the amino acid substitution at S223 is S223R, S223K, S223H, S223F, S223Y or S223W.

[0301] 5. The variant fHbp of embodiment 1, wherein the variant fHbp binds human fH with an affinity that is 25% or less of the affinity of fHbp ID 1 for human fH.

[0302] 6. The variant fHbp of embodiment 1, wherein the variant fHbp binds human fH with an affinity that is 10% or less of the affinity of fHbp ID 1 for human fH.

[0303] 7. The variant fHbp of embodiment 1, wherein the variant fHbp binds human fH with an affinity that is 5% or less of the affinity of fHbp ID 1 for human fH.

[0304] 8. The variant fHbp of any one of embodiments 1-7, further comprising an amino acid substitution selected from the group consisting of R41S and R41A.

[0305] 9. The variant fHbp of any one of embodiments 1-8, further comprising the following amino acid substitutions: S223R and H248L.

[0306] 10. A factor H binding protein (fHbp) variant, wherein the variant comprises an amino acid substitution selected from the group consisting of:

[0307] a) substitution of isoleucine for asparagine at amino acid 115 (N115I);

[0308] b) substitution of aspartic acid with glycine at amino acid 121 (D121G);

[0309] c) substitution of threonine for serine at amino acid 128 (S128T);

[0310] d) amino acid substitution of valine at position 131 (V131);

[0311] e) amino acid substitution at lysine at position 219 (K219);

[0312] f) an amino acid substitution of glycine at position 220 (G220),

[0313] wherein the amino acid substitutions are relative to fHbp ID 22 (SEQ ID NO:2), wherein the variant comprises an amino acid sequence having greater than 85% amino acid sequence identity to SEQ ID NO:2, wherein the variant fHbp binds human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID 22 for human fH, and wherein the variant induces a bactericidal antibody response in a mammalian host.

[0314] 11. The variant fHbp of embodiment 10, wherein the variant fHbp binds human fH with an affinity that is 25% or less of the affinity of fHbp ID 22 for human fH.

[0315] 12. The variant fHbp of embodiment 10, wherein the variant fHbp binds human fH with an affinity that is 10% or less of the affinity of fHbp ID 22 for human fH.

[0316] 13. The variant fHbp of embodiment 10, wherein the variant fHbp binds human fH with an affinity that is 5% or less of the affinity of fHbp ID 22 for human fH.

[0317] 14. The variant fHbp of embodiment 10, wherein the amino acid substitution at V131 is V131D, V131E, V131K, V131R, V131H, V131F, V131Y or V131W.

[0318] 15. The variant fHbp of embodiment 10, wherein the amino acid substitution at K219 is K219N, K219Q, K219D, K219E, K219F, K219Y or K219W.

[0319] 16. The variant fHbp of embodiment 10, wherein the amino acid substitution at G220 is G220S, G220N, G220Q, G220D, G220E, G220K, G220R, G220H, G220F, G220Y or G220W.

[0320] 17. The variant fHbp of any one of embodiments 10-16, further comprising the following substitutions: L130R and G133D, wherein the amino acid substitutions are relative to fHbp ID 22.

[0321] 18. The variant fHbp of embodiment 10, comprising the substitutions: L130R, G133D and N115I.

[0322] 19. The variant fHbp of embodiment 10, comprising the substitutions: L130R, G133D and D121G.

[0323] 20. The variant fHbp according to embodiment 10, comprising the substitutions: L130R, G133D and K219N or the substitutions L130R, G133D and D211A.

[0324] 21. The variant fHbp of embodiment 10, comprising the substitutions: L130R, G133D and G220S.

[0325] 22. A factor H binding protein (fHbp) variant, wherein the variant comprises an amino acid substitution selected from the group consisting of:

[0326] a) amino acid substitution of glutamic acid at position 92 (E92);

[0327] b) amino acid substitution of serine at position 223 (S223); and

[0328] c) an amino acid substitution of histidine at position 248 (H248),

[0329] wherein the amino acid substitutions are relative to fHbp ID 55 (SEQ ID NO:3), wherein the variant comprises an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO:3, wherein the variant fHbp binds human factor H (fH) with an affinity that is less than 50% of the affinity of fHbp ID 55 for human fH, and wherein the variant induces a bactericidal antibody response in a mammalian host.

[0330] 23. The variant fHbp of embodiment 22, wherein the variant fHbp binds human fH with an affinity that is 25% or less of the affinity of fHbp ID 55 for human fH.

[0331] 24. The variant fHbp of embodiment 22, wherein the variant fHbp binds human fH with an affinity that is 10% or less of the affinity of fHbp ID 55 for human fH.

[0332] 25. The variant fHbp of embodiment 22, wherein the variant fHbp binds human fH with an affinity that is 5% or less of the affinity of fHbp ID 55 for human fH.

[0333] 26. The variant fHbp of embodiment 22, wherein the amino acid substitution at E92 is E92K, E92R, E92H, E92F, E92Y or E92W.

[0334] 27. The variant fHbp of embodiment 22, wherein the amino acid substitution at S223 is S223R, S223K, S223H, S223F, S223Y or S223W.

[0335] 28. The variant fHbp of embodiment 22, wherein the amino acid substitution at H248 is H248L, H248I, H248V, H248D, H248E, H248F, H248Y or H248W.

[0336] 29. An immunogenic composition comprising:

[0337] a) the variant fHbp according to any one of embodiments 1-28; and

[0338] b) pharmaceutically acceptable excipients.

[0339] 30. The immunogenic composition of embodiment 29, wherein the variant fHbp is in a vesicle formulation prepared from a strain of Neisseria meningitidis.

[0340] 31. The immunogenic composition of embodiment 29 or embodiment 30, wherein the pharmaceutically acceptable excipient comprises an adjuvant.

[0341] 32. The immunogenic composition of any one of embodiments 29-31, further comprising Neisseria surface protein A.

[0342] 33. An immunogenic composition according to embodiment 31, wherein the adjuvant is aluminum phosphate or aluminum hydroxide.

[0343] 34. A nucleic acid encoding the variant fHbp according to any one of embodiments 1-28.

[0344] 35. A recombinant expression vector comprising the nucleic acid according to embodiment 34.

[0345] 36. An in vitro host cell comprising the nucleic acid according to embodiment 34 or the recombinant expression vector according to embodiment 35.

[0346] 37. A method of eliciting an antibody response in a mammal, the method comprising administering to the mammal an immunogenic composition according to any one of embodiments 29-33.

[0347] 38. The method of embodiment 37, wherein the mammal is a human.

[0348] 39. The method of embodiment 37, wherein the antibody response is a bactericidal antibody response to one or more strains of Neisseria meningitidis.

[0349] 40. A factor H binding protein (fHbp) variant, wherein the variant comprises amino acid substitutions L130R and G133D relative to fHbp ID 22 (SEQ ID NO: 2), wherein the variant comprises an amino acid sequence having greater than 85% amino acid sequence identity to SEQ ID NO: 2, wherein the variant fHbp binds human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID 22 for human fH, and wherein the variant induces a bactericidal antibody response in a mammalian host.

[0350] 41. The variant fHbp of embodiment 40, wherein the variant further comprises one or more of the following substitutions: R80A, D211A, E218A, E248A, G236I, T221A and H223A.

[0351] 42. The variant fHbp of embodiment 40, wherein the variant further comprises the substitution R80A.

[0352] 43. The variant fHbp of any one of embodiments 40 or 42, wherein the variant further comprises the substitution D211A.

[0353] 44. The variant fHbp of any one of embodiments 40 or 43, wherein the variant further comprises the substitution E218A.

[0354] 45. The variant fHbp of any one of embodiments 40 or 44, wherein the variant further comprises the substitution E248A.

[0355] 46. ​​The variant fHbp of any one of embodiments 40 or 45, wherein the variant further comprises the substitution G236I.

[0356] 47. The variant fHbp of any one of embodiments 40 or 46, wherein the variant further comprises substitutions T221A and H223A.

[0357] 48. The variant fHbp of any one of embodiments 40-47, wherein the variant comprises an amino acid sequence having greater than 90% amino acid sequence identity to SEQ ID NO:2.

[0358] 49. The variant fHbp of any one of embodiments 40-47, wherein the variant comprises an amino acid sequence having greater than 95% amino acid sequence identity to SEQ ID NO:2.

[0359] 50. The variant fHbp of any one of embodiments 40-47, wherein the variant comprises an amino acid sequence having greater than 99% amino acid sequence identity to SEQ ID NO:2.

[0360] Although the present invention has been described with reference to specific embodiments of the present invention, it will be appreciated by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present invention. In addition, many modifications may be made to adapt specific circumstances, materials, material compositions, processes, one or more process steps to the purpose, spirit and scope of the present invention. All of these modifications are within the scope of the appended claims.

Claims

1. A factor H binding protein (fHbp) variant, wherein the variant comprises an amino acid substitution selected from the group consisting of: a) amino acid substitution of histidine at amino acid 248 (H248); b) an amino acid substitution of serine at amino acid 223 (S223); and c) amino acid substitution of glutamic acid at amino acid 92 (E92); wherein the amino acid substitutions are relative to fHbp ID 55 (SEQ ID NO:3), wherein the variant comprises an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO:3, wherein the variant fHbp binds human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID 55 for human fH, and wherein the variant induces a bactericidal antibody response in a mammalian host.

2. The variant fHbp of claim 1, comprising an amino acid substitution at H248, wherein the amino acid substitution at H248 is H248L, H248I, H248V, H248D, H248E, H248F, H248Y or H248W.

3. The variant fHbp of claim 2, wherein the amino acid substitution at H248 is H248L.

4. The variant fHbp of claim 1, comprising an amino acid substitution at S223, wherein the amino acid substitution at S223 is S223R, S223K, S223H, S223F, S223Y or S223W.

5. The variant fHbp of claim 4, wherein the amino acid substitution at S223 is S223R.

6. The variant fHbp of claim 1, comprising an amino acid substitution at E92, wherein the amino acid substitution at E92 is E92K, E92R, E92H, E92F, E92Y or E92W.

7. The variant fHbp of claim 6, wherein the amino acid substitution at E92 is E92K.

8. The variant fHbp of any one of claims 1-7, wherein the variant comprises an amino acid sequence having at least 95% amino acid sequence identity to SEQ ID NO:

3.

9. The variant fHbp of any one of claims 1-7, wherein the variant comprises an amino acid sequence having at least 99% amino acid sequence identity to SEQ ID NO:

3.

10. A factor H binding protein (fHbp) variant, wherein the variant comprises an amino acid substitution selected from the group consisting of: a) substitution of isoleucine for asparagine at amino acid 115 (N115I); b) substitution of aspartic acid with glycine at amino acid 121 (D121G); c) substitution of threonine for serine at amino acid 128 (S128T); d) amino acid substitution of valine at position 131 (V131); e) amino acid substitution at lysine at position 219 (K219); f) an amino acid substitution of glycine at position 220 (G220), wherein the amino acid substitutions are relative to fHbp ID 22 (SEQ ID NO:2), wherein the variant comprises an amino acid sequence having greater than 85% amino acid sequence identity to SEQ ID NO:2, wherein the variant fHbp binds human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID 22 for human fH, and wherein the variant induces a bactericidal antibody response in a mammalian host.

11. The variant fHbp of claim 10, wherein the amino acid substitution at V131 is V131D, V131E, V131K, V131R, V131H, V131F, V131Y or V131W; the amino acid substitution at K219 is K219N, K219Q, K219D, K219E, K219F, K219Y or K219W; or the amino acid substitution at G220 is G220S, G220N, G220Q, G220D, G220E, G220K, G220R, G220H, G220F, G220Y or G220W.

12. The variant fHbp according to claim 10 or 11, further comprising the following substitutions: L130R and G133D, wherein the amino acid substitutions are relative to fHbp ID 22.

13. The variant fHbp of claim 10, comprising the substitutions L130R, G133D and G220S; L130R, G133D and K219N; L130R, G133D and N115I; L130R, G133D and D121G; or L130R, G133D and D211A.

14. An immunogenic composition comprising: a) a variant fHbp according to any one of claims 1-13; and b) pharmaceutically acceptable excipients.

15. The immunogenic composition of claim 14, wherein the variant fHbp is in a vesicle formulation prepared from a strain of Neisseria meningitidis.

16. The immunogenic composition of claim 14 or 15, wherein the pharmaceutically acceptable excipient comprises an adjuvant.

17. The immunogenic composition according to any one of claims 14 or 15, further comprising Neisseria surface protein A.

18. The immunogenic composition of claim 16, further comprising Neisseria surface protein A.

19. The immunogenic composition of claim 16, wherein the adjuvant is aluminum phosphate or aluminum hydroxide.

20. A nucleic acid encoding the variant fHbp according to any one of claims 1-13. A recombinant expression vector comprising the nucleic acid according to claim 20.

22. An in vitro host cell comprising the nucleic acid according to claim 20 or the recombinant expression vector according to claim 21.

23. Use of a variant fHbp according to any one of claims 1 to 13 in the manufacture of a medicament for use in a method of eliciting an antibody response in a mammal.

24. The use according to claim 23, wherein the mammal is a human.

25. The use of claim 23, wherein the antibody response is a bactericidal antibody response to one or more strains of Neisseria meningitidis.

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