Factor H binding protein variants and methods of use thereof

Abstract

Mutant factor H binding proteins capable of inducing antibodies that are bactericidal against at least one strain of Neisseria meningitidis (meningococcus), compositions containing such proteins, and methods of using such proteins are provided. [Solution] A mutant of factor H binding protein (fHbp) that binds to human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp for human fH, and which induces a bactericidal antibody response against at least one strain of Neisseria meningitidis in a mammalian host.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 028,123, filed July 23, 2014, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Introduction Neisseria meningitidis (meningococcus) is a Gram-negative bacterium that colonizes the human upper respiratory tract and is responsible for sporadic and periodic outbreaks worldwide, most notably those of meningitis and sepsis. Its attack rate and prevalence are highest in children under 2 years of age. Like other Gram-negative bacteria, Neisseria meningitidis typically possesses a cytoplasmic membrane, a peptidoglycan layer, and an outer membrane, which, together with capsular polysaccharides, constitute the bacterial cell wall. It also possesses glandular trichomes that protrude into the external environment. Encapsulated strains of Neisseria meningitidis are the leading cause of bacterial meningitis and sepsis in children and young adults. The prevalence and economic importance of invasive Neisseria meningitidis infections have prompted research into effective vaccines that can confer immunity across different strains, particularly across genetically diverse serogroup B strains with different serotypes or serosubtypes.

[0003] Factor H binding protein (fHbp, also known in the art as lipoprotein 2086 (Fletcher et al. (2004) Infect Immun 72:2088-2100), genome-derived neisserial antigen (GNA) 1870 (Masignani et al. (2003) J Exp Med 197:789-99), or "741") is a protein of Neisseria meningitidis that is 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 the pathogen survives in non-immune human serum or blood and evades innate host defenses. Recently, genetic variation in the human factor H gene cluster has been 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 for some non-human primates, partially explaining why Neisseria meningitidis is exclusively a human pathogen. fHbp occurs in many natural sequence variants, which are represented by accession (ID) numbers provided in the internet fHbp database, pubmlst(dot)org / neisseria / fHbp.

[0004] There is a need for fHbp polypeptides that are capable of eliciting effective bactericidal antibody responses. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Fletcher et al (2004) Infect Immun 72:2088-2100 [Non-patent document 2] Masignani et al. (2003) J Exp Med 197:789-99 [Non-patent document 3] Davila S et al. (2010) Nat Genetics doi:10.1038 / ng.640 Summary of the Invention

[0006] overview Mutant factor H binding proteins capable of inducing antibodies that are bactericidal against at least one strain of Neisseria meningitidis, compositions containing such proteins, and methods of using such proteins are provided.

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

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

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

[0010] Also disclosed herein are fHbp mutants that contain mutations that increase thermostability compared to WT fHbp and further contain additional mutations known to reduce fH binding, such as those disclosed in US 2011 / 0256180. In one embodiment, a mutant factor H binding protein (fHbp) is disclosed, wherein the mutant contains amino acid substitutions L130R and G133D, and at least one of the substitutions R80A, D211A, E218A, E248A, G236I, T221A, and H223A relative to fHbp ID22 (SEQ ID NO: 2), the mutant comprises an amino acid sequence having greater than 85% amino acid sequence identity to SEQ ID NO: 2, the mutant fHbp binds to human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID22 for human fH, and the mutant induces a bactericidal antibody response in a mammalian host.

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

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

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

[0014] 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 0012 above. In some examples, the mammal is a human. In some examples, the antibody response is a bactericidal antibody response against one or more strains of N. meningitidis. [The present invention 1001] 1. A mutant of factor H binding protein (fHbp), wherein the mutant is: (a) Amino acid substitution of glutamine at amino acid 38 (Q38); (b) amino acid substitution of glutamic acid at amino acid 92 (E92); (c) glycine to arginine substitution at amino acid 130 (R130G); (d) an amino acid substitution of serine at amino acid 223 (S223); and (e) histidine to leucine substitution at amino acid 248 (H248L) and comprising an amino acid substitution selected from at least one of: the amino acid substitution is relative to fHbp ID1 (SEQ ID NO: 1); the variant comprises an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 1; the mutant fHbp binds to human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID1 for human fH; A variant, wherein the variant induces a bactericidal antibody response in a mammalian host against at least one strain of Neisseria meningitidis (meningococcus). [The present invention 1002] 1001. The mutant fHbp of the present invention, wherein said amino acid substitution at Q38 is Q38R, Q38K, Q38H, Q38F, Q38Y, or Q38W. [The present invention 1003] 1001. The mutant fHbp of the present invention, wherein said amino acid substitution at E92 is E92K, E92R, E92H, E92F, E92Y, or E92W. [The present invention 1004] 1001. The mutant fHbp of the present invention, wherein said amino acid substitution at S223 is S223R, S223K, S223H, S223F, S223Y, or S223W. [The present invention 1005] 1001. A mutant fHbp of the invention that binds to human fH with an affinity that is 25% or less of the affinity of fHbp ID1 for human fH. [The present invention 1006] 1001. A mutant fHbp of the invention that binds to human fH with an affinity that is 10% or less of the affinity of fHbp ID1 for human fH. [The present invention 1007] 1001. A mutant fHbp of the invention that binds to human fH with an affinity that is 5% or less of the affinity of fHbp ID1 for human fH. [The present invention 1008] The mutant fHbp of any of claims 1001 to 1007, further comprising an amino acid substitution selected from the group consisting of R41S and R41A. [The present invention 1009] A mutant fHbp according to any one of 1001 to 1008 of the present invention, further comprising the following amino acid substitutions: S223R and H248L. [The present invention 1010] 1. A mutant of factor H binding protein (fHbp), wherein the mutant is: (a) isoleucine substitution for asparagine at amino acid 115 (N115I); (b) glycine to aspartic acid substitution at amino acid 121 (D121G); (c) threonine to serine substitution 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) and comprising an amino acid substitution selected from the amino acid substitution is relative to fHbp ID22 (SEQ ID NO: 2); the variant comprises an amino acid sequence having greater than 85% amino acid sequence identity with SEQ ID NO: 2; the mutant fHbp binds to human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID22 for human fH; A mutant, wherein the mutant induces a bactericidal antibody response in a mammalian host. [The present invention 1011] 1010. A mutant fHbp of the invention that binds to human fH with an affinity that is 25% or less of the affinity of fHbp ID22 for human fH. [The present invention 1012] 1010. A mutant fHbp of the invention that binds to human fH with an affinity that is 10% or less of the affinity of fHbp ID22 for human fH. [The present invention 1013] 10. A mutant fHbp of the invention that binds to human fH with an affinity that is 5% or less of the affinity of fHbp ID22 for human fH. [The present invention 1014] 10. The mutant fHbp of the present invention, wherein the amino acid substitution at V131 is V131D, V131E, V131K, V131R, V131H, V131F, V131Y, or V131W. [The present invention 1015] 1010. The mutant fHbp of the invention, wherein the amino acid substitution at K219 is K219N, K219Q, K219D, K219E, K219F, K219Y, or K219W. [The present invention 1016] 10. The mutant fHbp of the invention, wherein the amino acid substitution at G220 is G220S, G220N, G220Q, G220D, G220E, G220K, G220R, G220H, G220F, G220Y, or G220W. [The present invention 1017] 10. The mutant fHbp of any one of claims 1010 to 1016, further comprising the following substitutions: L130R and G133D, said amino acid substitutions being relative to fHbp ID22. [The present invention 1018] 10. A mutant fHbp of the present invention comprising the substitutions L130R, G133D, and N115I. [The present invention 1019] 10. A mutant fHbp of the present invention comprising the substitutions L130R, G133D, and D121G. [The present invention 1020] 10. A mutant fHbp of the invention 1010 comprising the substitutions L130R, G133D, and K219N, or the substitutions L130R, G133D, and D211A. [The present invention 1021] 10. A mutant fHbp of the present invention comprising the substitutions L130R, G133D, and G220S. [The present invention 1022] 1. A mutant of factor H binding protein (fHbp), wherein the mutant is: (a) amino acid substitution of glutamic acid at position 92 (E92); (b) an amino acid substitution of serine at position 223 (S223); and (c) Amino acid substitution of histidine at position 248 (H248) and comprising an amino acid substitution selected from the amino acid substitution is relative to fHbp ID55 (SEQ ID NO: 3); the variant comprises an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO: 3; the mutant fHbp binds to human factor H (fH) with an affinity that is less than 50% of the affinity of the fHbp ID55 for human fH; A mutant, wherein the mutant induces a bactericidal antibody response in a mammalian host. [The present invention 1023] A mutant fHbp of the present invention that binds to human fH with an affinity that is 25% or less of the affinity of the fHbp ID55 for human fH. [The present invention 1024] A mutant fHbp of the present invention that binds to human fH with an affinity that is 10% or less of the affinity of the fHbp ID55 for human fH. [The present invention 1025] A mutant fHbp of the present invention that binds to human fH with an affinity that is 5% or less of the affinity of the fHbp ID55 for human fH. [The present invention 1026] 1022. The mutant fHbp of the present invention, wherein the amino acid substitution at E92 is E92K, E92R, E92H, E92F, E92Y, or E92W. [The present invention 1027] 1022. The mutant fHbp of the present invention, wherein the amino acid substitution at S223 is S223R, S223K, S223H, S223F, S223Y, or S223W. [The present invention 1028] 1022. The mutant fHbp of the invention, wherein the amino acid substitution at H248 is H248L, H248I, H248V, H248D, H248E, H248F, H248Y, or H248W. [The present invention 1029] (a) any one of the mutant fHbps 1001 to 1028 of the present invention; and (b) pharmaceutically acceptable excipients An immunogenic composition comprising: [The present invention 1030] 1029. The immunogenic composition of the present invention, wherein the mutant fHbp is in a vesicle preparation prepared from a Neisseria meningitidis strain. [The present invention 1031] The immunogenic composition of claim 1029 or claim 1030, wherein said pharmaceutically acceptable excipient comprises an adjuvant. [The present invention 1032] The immunogenic composition of any one of claims 1029 to 1031, further comprising surface protein A of Neisseria. [The present invention 1033] The immunogenic composition of the present invention 1031, wherein the adjuvant is aluminum phosphate or aluminum hydroxide. [The present invention 1034] A nucleic acid encoding any one of the mutant fHbps of the present invention 1001 to 1028. [This invention 1035] A recombinant expression vector comprising the nucleic acid of the present invention. [The present invention 1036] An in vitro host cell comprising a nucleic acid of the invention 1034 or a recombinant expression vector of the invention 1035. [This invention 1037] A method for inducing an antibody response in a mammal, comprising administering to the mammal the immunogenic composition of any one of claims 1029 to 1033. [The present invention 1038] 1037. The method of claim 1037, wherein the mammal is a human. [This invention 1039] 1037. The method of claim 1037, wherein said antibody response is a bactericidal antibody response against one or more strains of N. meningitidis (meningococcus). [The present invention 1040] A variant of factor H binding protein (fHbp), the variant comprising the amino acid substitutions L130R and G133D relative to fHbp ID22 (SEQ ID NO: 2); the variant comprises an amino acid sequence having greater than 85% amino acid sequence identity with SEQ ID NO: 2; the mutant fHbp binds to human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID22 for human fH; A mutant, wherein the mutant induces a bactericidal antibody response in a mammalian host. [The present invention 1041] 1040. A mutant fHbp of the invention further comprising one or more of the following substitutions: R80A, D211A, E218A, E248A, G236I, T221A, and H223A. [The present invention 1042] A mutant fHbp of the present invention 1040, further comprising the substitution R80A. [This invention 1043] A mutant fHbp of the invention 1040 or 1042, further comprising the substitution D211A. [This invention 1044] A mutant fHbp of the invention 1040 or 1043, further comprising the substitution E218A. [This invention 1045] A mutant fHbp of the invention 1040 or 1044, further comprising the substitution E248A. [The present invention 1046] A mutant fHbp of the invention 1040 or 1045, further comprising the substitution G236I. [This invention 1047] A mutant fHbp of the invention 1040 or 1046, further comprising the substitutions T221A and H223A. [This invention 1048] A mutant fHbp according to any one of 1040 to 1047 of the present invention, which comprises an amino acid sequence having an amino acid sequence identity of more than 90% with SEQ ID NO:2. [This invention 1049] A mutant fHbp according to any one of 1040 to 1047 of the present invention, which comprises an amino acid sequence having an amino acid sequence identity of more than 95% with SEQ ID NO:2. [The present invention 1050] A mutant fHbp according to any one of 1040 to 1047 of the present invention, which comprises an amino acid sequence having an amino acid sequence identity of more than 99% with SEQ ID NO:2. [Brief explanation of the drawings]

[0015] [Figure 1] Purified recombinant fHbp ID1 mutants stained with Coomassie blue on a polyacrylamide gel: Lane 1, Kaleidoscope molecular weight marker (Bio-Rad Laboratories); 2, fHbp ID1 wild-type; 3, Q38R; 4, E92K; 5, R130G; 6, S223R; 7, H248L. [Figure 2] 2A and 2B show the binding of fHbp ID1 mutants to human fH as measured by ELISA. The mean and range of replicate measurements are shown. [Figure 3] Figures 3A-3E show the binding of fHbp ID1 mutants to human fH, as measured by surface plasmon resonance. For reference, the same data for the ID1 wild-type (WT) protein are shown in each of Figures 3A-3E. [Figure 4] 4A-4E show binding of mouse anti-fHbp monoclonal antibodies (mAbs) to fHbp ID1 mutant proteins as measured by ELISA. Means and ranges of replicate measurements are shown. [Figure 5] Figures 5A and 5B show the bactericidal activity of serum from mice immunized with fHbp ID1 mutants. Each symbol represents the titer for each mouse, and the horizontal bar represents the geometric mean titer. Figure 5A shows the bactericidal activity of serum from wild-type mice immunized with fHbp ID1 mutants. Figure 5B shows the bactericidal activity of serum from human fH transgenic mice immunized with fHbp ID1 mutants. [Figure 6] Figures 6A and 6B show the characterization of single and double mutants of fHbp ID1. Figure 6A shows the binding of human fH to the fHbp ID1 double mutant. Figure 6B shows the binding of the mouse anti-fHbp monoclonal antibody (mAb) JAR4 to the mutants. [Figure 7] Figure 1 shows the bactericidal activity of sera from wild-type mice immunized with single or double mutants of fHbp ID1. Each symbol represents the titer for an individual mouse, and the horizontal bar represents the geometric mean titer. [Figure 8] Figures 8A and 8B show the characterization of fHbp ID55 mutants. Figure 8A shows the binding of human fH to immobilized fHbp ID55 mutants as measured by ELISA, and Figure 8B shows the binding of the mouse anti-fHbp monoclonal antibody (mAb) JAR41 to fHbp mutant ID55 protein as measured by ELISA. The mean and range of two to four replicates are shown. [Figure 9] Figure 1 shows the bactericidal activity of sera from wild-type mice immunized with the fHbp ID55 mutant. Each symbol represents the titer for an individual mouse, and the horizontal bar represents the geometric mean titer. [Figure 10] Figures 10A and 10B show the bactericidal activity of sera from mice immunized with fHbp ID55. Figure 10A shows the bactericidal activity of sera from human fH transgenic mice immunized with the licensed Trumenba vaccine or the investigational fHbp ID55 mutant S223R. Figure 10B shows the relationship between the human fH concentration in each transgenic mouse serum and the bactericidal activity titer of the serum (circle symbols). For comparison, titers from wild-type (WT) mice are shown (squares). [Figure 11] Figures 11A-11D show the characterization of fHbp ID22 mutants. Figures 11A-11C show the binding of fHbp ID22 mutants to human fH, as measured by ELISA. The means and ranges of two to four replicates are shown. Figure 11D shows the binding of the mouse anti-fHbp monoclonal antibody (mAb) JAR4 to fHbp mutant ID22 protein, as measured by ELISA. fHbp ID22 wild-type (WT) and the D211A mutant are shown as controls. The means and ranges of replicates are shown. [Figure 12] Figures 12A and 12B show the bactericidal activity of serum from mice immunized with fHbp ID22 mutants. Each symbol represents the titer for an individual mouse, and the horizontal bar represents the geometric mean titer. Figures 12A and 12B show the bactericidal activity of serum from wild-type mice in two experiments examining different fHbp ID22 mutants. [Figure 13] Figure 1 shows the bactericidal activity of sera from human fH transgenic mice immunized with the fHbp ID22 mutant. [Figure 14] 1 shows the thermal denaturation of fHbp ID22 wild-type (WT) and the L130R / G133D double mutant measured by differential scanning microcalorimetry. [Figure 15] Figure 15 shows characterization of the fHbp ID22 triple mutant. Figure 15A shows binding of human fH to the fHbp ID22 triple mutant. DM refers to the L130R / G133D double mutant. Figure 15B shows binding of the mouse anti-fHbp monoclonal antibody (mAb) JAR4 to the fHbp ID22 triple mutant. [Figure 16] Figure 1 shows the bactericidal activity of sera from human fH transgenic mice immunized with the fHbp ID22 triple mutant. DM refers to the L130R / G133D double mutant. [Figure 17] 1 shows a table of exemplary fHbp mutants with reduced binding of human fH. [Figure 18] The amino acid sequence of wild-type human factor H is shown. [Figure 19] 1 shows the amino acid sequences of fHbp ID1, ID22, and ID55 from N. meningitidis strains. [Figure 20] The amino acid sequence of the fHbp ID1 mutant is shown. [Figure 21] The amino acid sequence of the fHbp ID1 mutant is shown. [Figure 22] The amino acid sequence of the fHbp ID1 mutant is shown. [Figure 23] The amino acid sequence of the fHbp ID1 mutant is shown. [Figure 24] The amino acid sequence of the fHbp ID1 mutant is shown. [Figure 25] The amino acid sequence of the fHbp ID22 mutant is shown. [Figure 26] The amino acid sequence of the fHbp ID22 mutant is shown. [Figure 27] The amino acid sequence of the fHbp ID22 mutant is shown. [Figure 28] The amino acid sequence of the fHbp ID22 mutant is shown. [Figure 29] The amino acid sequence of the fHbp ID22 mutant is shown. [Figure 30] The amino acid sequence of the fHbp ID22 mutant is shown. [Figure 31] The amino acid sequence of the fHbp ID55 mutant is shown. [Figure 32] The amino acid sequence of the fHbp ID55 mutant is shown. [Figure 33] The amino acid sequence of the fHbp ID55 mutant is shown. [Figure 34] The amino acid sequence of the fHbp ID1 double mutant is shown. [Figure 35] The amino acid sequence of the fHbp ID1 double mutant is shown. [Figure 36] The amino acid sequence of the fHbp ID1 double mutant is shown. [Figure 37] The amino acid sequences of the fHbp ID22 double and triple mutants are shown. [Figure 38] The amino acid sequences of the fHbp ID22 double and triple mutants are shown. [Figure 39] The amino acid sequences of the fHbp ID22 double and triple mutants are shown. [Figure 40] The amino acid sequence of NspA is shown. DETAILED DESCRIPTION OF THE INVENTION

[0016] definition "Factor H binding protein" (fHbp), also known in the literature as GNA1870, GNA1870, ORF2086, LP2086 (lipoprotein 2086), and "741," refers to a class of N. meningitidis polypeptides that are found in nature as lipoproteins on the surface of N. meningitidis bacteria. fHbps have 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 as subfamilies A and B in others (see, for example, Fletcher et al. (2004) Infect Immun 72:2088-2100)) based on amino acid sequence diversity and immunological cross-reactivity (Masignani et al. (2003) J Exp Med 197:789-99). fHbps can also be classified into one of the six most common fHbp modular groups, designated modular groups I through VI, as shown in Figure 2 in Vu et al. (2012) Sci. Reports 2:341. Each unique fHbp found in N. meningitidis is also assigned an fHbp peptide ID according to the website pubmlst.org / neisseria / fHbp / . Because the lengths of the variant 2 (v.2) fHbp protein (from strain 8047, fHbp ID 77) and variant 3 (v.3) fHbp (from strain M1239, fHbp ID 28) differ by -1 and +7 amino acid residues, respectively, from that from strain MC58 (fHbp ID 1), the numbers used herein referring to residues in the v.2 and v.3 fHbp proteins differ from those based on the actual amino acid sequences of these proteins. Thus, for example, a reference to a leucine residue (L) at position 166 of the 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 specified, the numbering of amino acid substitutions present in these fHbp mutants refers to the numbering of amino acid residues in fHbp ID1.

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

[0018] "Derived from" in the context of an amino acid sequence or polynucleotide sequence (e.g., an amino acid sequence "derived from" fHbp ID1) is intended to indicate 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 is not intended to be limiting as to the source or manner in which the protein or nucleic acid is made. Non-limiting examples of reference polypeptides and polynucleotides from which an amino acid sequence or polynucleotide sequence may be "derived" include naturally occurring fHbp, fHbp ID1, and non-naturally occurring fHbp. "Derived from" in the context of a bacterial strain is intended to indicate that the strain is obtained by in vivo passage or in vitro cultivation of a parent strain and / or that the strain is a recombinant cell obtained by modification of the parent strain.

[0019] "Conservative amino acid substitution" refers to the substitution of one amino acid residue to further share 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 obtained from amino acid sequence alignments of polypeptides displaying the epitope of interest.

[0020] The term "protective immunity" means that a vaccine or immunization regimen administered to a mammal induces an immune response that prevents, delays the progression of, or reduces the severity of, disease caused by Neisseria meningitidis, or reduces or completely eliminates the symptoms of the disease. Protective immunity can involve the production of bactericidal antibodies. Note that the production of bactericidal antibodies against Neisseria meningitidis is accepted in the field as a predictor of the protective efficacy of a vaccine in humans (Goldschneider et al. (1969) J. Exp. Med. 129:1307; Borrow et al. (2001) Infect Immun. 69:1568).

[0021] The term "disease caused by a Neisseria meningitidis strain" encompasses any clinical symptom or combination of clinical symptoms present in humans infected with Neisseria meningitidis, including, but not limited to, colonization of the upper respiratory tract (e.g., the mucosa of the nasopharynx and tonsils) by pathogenic strains of Neisseria meningitidis, invasion of the mucosa and submucosal vascular beds by the bacteria, sepsis, septic shock, inflammation, hemorrhagic skin lesions, activation of fibrinolysis and blood coagulation, organ dysfunction, e.g., renal, pulmonary, and cardiac failure, adrenal hemorrhage and muscle infarction, capillary leak, edema, peripheral limb ischemia, respiratory distress syndrome, pericarditis, and meningitis.

[0022] The phrase "specifically binds to an antibody" or "specifically immunoreactive" with respect to an antigen (e.g., a polypeptide antigen) refers to a binding reaction that is based on and / or is evidence of the presence of the antigen in a sample that may further contain a heterogeneous population of other molecules. Thus, under specified conditions, a particular antibody or particular antibodies will bind to a particular antigen or particular antigens in a sample and will not bind in significant amounts to other molecules present in the sample. The phrase "specifically binds to an antibody" or "specifically immunoreactive" with respect to an epitope of an antigen (e.g., an epitope of a polypeptide) refers to a binding reaction that is based on and / or is evidence of the presence of an epitope in an antigen (e.g., a polypeptide) that may further contain a heterogeneous population of other epitopes and heterogeneous antigen populations. Thus, under specified conditions, a particular antibody or particular antibodies will bind to a particular epitope of an antigen and will not bind in significant amounts to other epitopes present in the antigen and / or the sample.

[0023] 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, including, but not limited to, antibody titer or antibody specificity as detected in assays such as enzyme-linked immunosorbent assay (ELISA), bactericidal assay, flow cytometry, immunoprecipitation, Ouchterlony immunodiffusion; binding detection assays, e.g., spot, Western blot, or antigen array; cytotoxicity assays, etc.

[0024] A "surface antigen" is an antigen present on the surface structures (eg, outer membrane, capsule, glandular pilus, etc.) of Neisseria meningitidis.

[0025] "Isolated" refers to an entity of interest that is in an environment different from that in which a compound may naturally occur. "Isolated" is intended to include compounds within a sample that are substantially enriched for the compound of interest and / or from which the compound of interest is partially or substantially purified. In some examples, an isolated component (e.g., a polypeptide such as an fHbp variant of the present disclosure; a nucleic acid of the present disclosure; a recombinant vector of the present disclosure) is purified, e.g., the 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.

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

[0027] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0028] Where a range is given, unless the context clearly dictates otherwise, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit, and any other stated value or intervening value therein, is encompassed within the scope of the invention. The upper and lower limits of these smaller ranges may be independently included in those smaller ranges and, subject to any particular excluded boundaries of the stated range, are also encompassed within the scope of the invention. Where a stated range includes one or both of the boundaries, ranges excluding either or both of those included boundaries are also included in the invention.

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

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

[0031] It is understood that certain features of the invention, which are, for clarity, described in separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments relating to the present invention are expressly embraced by the present invention and are disclosed herein exactly as if each and every combination were individually and expressly disclosed. Furthermore, all subcombinations of these various embodiments and elements thereof are also expressly embraced by the present invention and are disclosed herein exactly as if each and every such subcombination were individually and expressly disclosed herein.

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

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

[0034] Mutant fHbp The present disclosure provides mutant fHbps that differ in amino acid sequence from wild-type N. meningitidis fHbp by 1 to 10 amino acids (e.g., from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), 10 to 15 amino acids, 15 to 20 amino acids, 20 to 30 amino acids, 30 to 40 amino acids, or 40 to 50 amino acids, such that the mutant fHbp exhibits reduced affinity for human factor H (fH) compared to the reference fHbp, and the mutant fHbp, when administered to a mammalian host, elicits a bactericidal immune response against one or more N. meningitidis strains. In some examples, the mutant fHbp differs in amino acid sequence from a reference wild-type N. meningitidis fHbp by no more than 1 to 10 amino acid substitutions. In some examples, the mutant fHbp differs in amino acid sequence from a reference wild-type N. meningitidis fHbp by only a single amino acid substitution.

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

[0036] A mutant fHbp of the present disclosure maintains substantially the same conformation as a reference (e.g., wild-type) fHbp that binds to human fH when the reference fHbp is in its native conformation. Whether a mutant fHbp of the present disclosure maintains substantially the same conformation as a reference (e.g., wild-type) fHbp that binds to human fH can be determined using antibodies that bind to the wild-type fHbp when the wild-type fHbp is in its native conformation. Such antibodies include, for example, JAR41, JAR4, and JAR31. See, e.g., Vu et al. (2012) Sci. Reports 2:341. A hybridoma-forming JAR4 monoclonal antibody has American Type Culture Collection (ATCC) No. PTA-8943. See also USPN 8,470,340. For example, in some instances, a variant fHbp of the present disclosure retains binding to JAR4; for example, a variant fHbp of the present disclosure retains at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the binding to JAR4 of a reference fHbp in a native conformation (e.g., fHbp ID1, fHbp ID22, or fHbp ID55).

[0037] fHbp ID1 mutants The "reference fHbp" from which variant fHbps of the present disclosure are derived, in some instances, is fHbp ID1. The amino acid sequence of fHbp ID1 is shown below. TIFF0007719046000001.tif37151

[0038] In some examples, a mutant fHbp of the disclosure is a mutant group 1 fHbp. In some examples, a mutant fHbp of the disclosure is a mutant group 1 fHbp, which is a modular group I fHbp. In some examples, a mutant fHbp of the disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 1; the mutant fHbp is selected from the group consisting of fHbp ID1, fHbp ID2, fHbp ID3, fHbp ID4, fHbp ID5, fHbp ID6, fHbp ID7, fHbp ID8, fHbp ID9, fHbp ID10, fHbp ID11, fHbp ID12, fHbp ID13, fHbp ID14, fHbp ID15, fHbp ID16, fHbp ID17, fHbp ID18, fHbp ID19, fHbp ID20, fHbp ID21, fHbp ID22, fHbp ID23, fHbp ID24, fHbp ID25, fHbp ID26, fHbp ID27, fHbp ID28, fHbp ID29, fHbp ID30, fHbp ID31, fHbp ID32, fHbp ID33, fHbp ID34, fHbp ID35, fHbp ID36, fHbp ID37, fHbp ID38, fHbp ID39, fHbp ID40, fHbp ID41, fHbp ID42, fHbp ID43, fHbp ID44, fHbp ID45, fHbp ID46, fHbp ID47, fHbp ID48, fHbp ID49, fHbp ID50, fHbp ID51, fHbp ID52, fHbp ID53, fHbp ID54, fHbp ID55, f The fHbp comprises one or more amino acid substitutions relative to ID1 such that it exhibits affinity for human fH that is about 85% to about 75%, about 75% to about 65%, about 65% to about 55%, about 55% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1% of the affinity of ID1 for human fH; and the mutant fHbp, when administered to a mammalian host (e.g., a human; or a non-human animal model), induces a bactericidal immune response against at least one strain of N. meningitidis.

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

[0040] In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 1, wherein the variant fHbp binds to human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID1 for human fH, wherein the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and wherein the variant fHbp comprises an amino acid substitution of glutamine at amino acid 38 (Q38). In some examples, the mutant 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 examples, the mutant fHbp comprises a Q38K substitution, a Q38H substitution, a Q38F substitution, a Q38Y substitution, or a Q38W substitution. As an example, a mutant fHbp of the present disclosure can comprise the amino acid sequence shown in FIG. 20 and set forth in SEQ ID NO:5.

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

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

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

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

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

[0046] Combinations of substitutions may be included, where two substitutions are in different structural domains and each independently reduces binding of fH to an fHbp (e.g., one substitution in the N-terminal domain combined with an amino acid substitution in the C-terminal domain. In some examples, a mutant 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 examples, a mutant 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 examples, a mutant 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.

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

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

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

[0050] Also provided herein are mutant fHbp proteins that contain one or more substitutions relative to the amino acid sequence of fHbp ID1, as described above, and further contain an R41S substitution. Exemplary mutant fHbps contain an R41S substitution and an S223 substitution relative to fHbp ID1, e.g., R41S / S223R, or an R41S substitution and an H248L substitution relative to fHbp ID1. In some examples, a variant fHbp of the 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 ID22 for human fH, wherein the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and wherein the variant fHbp is Based on the ID1 numbering, it contains two or more of the following amino acid substitutions: (a) a serine substitution for arginine at amino acid 41 (R41S); (b) an arginine substitution for serine at amino acid 223 (S223R); (c) a leucine substitution for histidine at amino acid 248 (H248L).

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

[0052] fHbp ID22 mutant The "reference fHbp" from which variant fHbps of the present disclosure are derived, in some instances, is fHbp ID22. The amino acid sequence of fHbp ID22 is shown below. TIFF0007719046000002.tif37152

[0053] In some examples, a mutant fHbp of the disclosure is a mutant group 2 fHbp. In some examples, a mutant fHbp of the disclosure is a mutant group 2 fHbp that is a modular group III fHbp. In some examples, a mutant fHbp of the disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO:2; the mutant fHbp is selected from the group consisting of fHbp ID22, fHbp ID32, fHbp ID42, fHbp ID52, fHbp ID62, fHbp ID72, fHbp ID82, fHbp ID92, fHbp ID102, fHbp ID112, fHbp ID123, fHbp ID132, fHbp ID142, fHbp ID152, fHbp ID162, fHbp ID172, fHbp ID182, fHbp ID19 ... The fHbp comprises one or more amino acid substitutions relative to fHbp ID22 such that it exhibits an affinity for human fH that is about 85% to about 75%, about 75% to about 65%, about 65% to about 55%, about 55% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1% of the affinity of ID22 for human fH; and the mutant fHbp, when administered to a mammalian host (e.g., a human; or a non-human animal model), induces a bactericidal immune response against at least one strain of N. meningitidis.

[0054] In some examples, a variant fHbp of the 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 ID22 for human fH, wherein the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and wherein the variant fHbp is With respect to the amino acid sequence of fHbp ID22, the fHbp ID22 comprises an amino acid substitution selected from at least one of: (a) an isoleucine substitution for asparagine at amino acid 115 (N115I); (b) a glycine substitution for aspartic acid at amino acid 121 (D121G); (c) a threonine substitution for serine at amino acid 128 (S128T); (d) an amino acid substitution for valine at position 131 (V131); (e) an amino acid substitution for lysine at position 219 (K219); and (f) an amino acid substitution for glycine at position 220 (G220). As described herein, the numbering of the amino acid residues is based on that of fHbp ID1.

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

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

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

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

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

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

[0061] Combinations of amino acid substitutions In some examples, a variant fHbp of the 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 ID22 for human fH, wherein the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and wherein the variant fHbp is The fHbp ID22 fragment comprises an amino acid substitution selected from two or more of: (a) an isoleucine substitution for asparagine at amino acid 115 (N115I); (b) a glycine substitution for aspartic acid at amino acid 121 (D121G); (c) a threonine substitution for serine at amino acid 128 (S128T); (d) an amino acid substitution for valine at position 131 (V131); (e) an amino acid substitution for lysine at position 219 (K219); and (f) an amino acid substitution for glycine at position 220 (G220). As described herein, the residue numbering is based on the amino acid numbering of fHbp ID1.

[0062] Combinations of substitutions may be included, where two substitutions are in different structural domains and each independently reduces binding of fH to an fHbp (e.g., one substitution in the N-terminal domain combined with an amino acid substitution in the C-terminal domain. In some examples, a mutant 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 examples, a mutant 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 examples, a mutant 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.

[0063] For example, in some examples, a variant fHbp of the 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 ID22 for human fH, and the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host; Relative to the amino acid sequence of ID22, the mutant fHbp contains: (a) an isoleucine substitution for asparagine at amino acid 115 (N115I) and (b) a glycine substitution for aspartic acid at amino acid 121 (D121G); the mutant fHbp contains: (a) an isoleucine substitution for asparagine at amino acid 115 (N115I) and (c) a threonine substitution for serine at amino acid 128 (S128T); the mutant fHbp contains: (a) an isoleucine substitution for asparagine at amino acid 115 (N115I) and (c) a threonine substitution for serine at amino acid 128 (S128T); or (d) an amino acid substitution of valine at position 131 (V131); the mutant fHbp comprises: (a) an isoleucine substitution for asparagine at amino acid 115 (N115I) and (e) an amino acid substitution of lysine at position 219 (K219); or the mutant fHbp comprises: (a) an isoleucine substitution for asparagine at amino acid 115 (N115I) and (f) an amino acid substitution of glycine at position 220 (G220), the numbering of the substituted residues being based on the numbering of the amino acid sequence of fHbp ID1.

[0064] As a further non-limiting example, in some instances, a variant fHbp of the 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 ID22 for human fH, and the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host; Relative to the amino acid sequence of ID22, the mutant fHbp comprises: (b) a glycine substitution for aspartic acid at amino acid 121 (D121G) and (c) a threonine substitution for serine at amino acid 128 (S128T); the mutant fHbp comprises: (b) a glycine substitution for aspartic acid at amino acid 121 (D121G) and (d) an amino acid substitution of valine at position 131 (V131); the mutant fHbp comprises: (b) a glycine substitution for aspartic acid at amino acid 121 (D121G) and (e) an amino acid substitution of lysine at position 219 (K219); or the mutant fHbp comprises: (b) a glycine substitution for aspartic acid at amino acid 121 (D121G) and (f) an 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 ID1.

[0065] As a further non-limiting example, in some instances, a variant fHbp of the 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 ID22 for human fH, and the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host; Relative to the amino acid sequence of ID22, the mutant fHbp contains: (c) a threonine substitution for serine at amino acid 128 (S128T) and (d) an amino acid substitution of valine at position 131 (V131); the mutant fHbp contains: (c) a threonine substitution for serine at amino acid 128 (S128T) and (e) an amino acid substitution of lysine at position 219 (K219); the mutant fHbp contains: (c) a threonine substitution for serine at amino acid 128 (S128T) and (f) an amino acid substitution of glycine at position 220 (K220); the mutant fHbp comprises: (d) an amino acid substitution at position 131 with valine (V131) and (e) an amino acid substitution at position 219 with lysine (K219); the mutant fHbp comprises: (d) an amino acid substitution at position 131 with valine (V131) and (f) an amino acid substitution at position 220 with glycine (G220); or the mutant fHbp comprises: (e) an amino acid substitution at position 219 with lysine (K219) and (f) an amino acid substitution at position 220 with glycine (G220). The numbering of the substituted residues is based on the numbering of the amino acid sequence of fHbp ID1.

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

[0067] For example, in some examples, 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 ID22 for human fH, and the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, wherein the variant fHbp comprises: (i) an N115I substitution; and (ii) a D211A substitution.

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

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

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

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

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

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

[0074] Exemplary mutant fHbps include polypeptides having an amino acid sequence with 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 containing the following substitutions relative to the amino acid sequence of SEQ ID NO:2: L130R and G133D; L130R, G133D, and K219N; or L130R, G133D, and G220S.

[0075] Also disclosed herein are variant fHbp proteins that contain one or more substitutions relative to the amino acid sequence of fHbp ID22, as described above, and further contain the substitutions disclosed in US2011 / 0256180, which is incorporated by reference in its entirety.

[0076] fHbp ID55 mutants The "reference fHbp" from which variant fHbps of the present disclosure are derived, in some instances, is fHbp ID55. The amino acid sequence of fHbp ID55 is shown below. TIFF0007719046000003.tif37151

[0077] In some examples, a mutant fHbp of the disclosure is a fHbp of Variant Group 1. In some examples, a mutant fHbp of the disclosure is a fHbp of Variant Group 1 and a modular group IV fHbp.

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

[0079] In some examples, a variant fHbp of the 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 the ID55 of fHbp for human fH, wherein the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and wherein the variant fHbp is The amino acid sequence of ID55 contains an amino acid substitution selected from at least one 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), the numbers of the amino acid residues being based on the numbering of fHbp ID1.

[0080] In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity 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 the fHbp ID55 for human fH, and the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp comprises an amino acid substitution of glutamic acid at position 92 (E92). In some examples, the fHbp mutant comprises an E92K substitution. Other amino acids with positively charged or aromatic side chains, such as arginine, histidine, phenylalanine, tyrosine, or tryptophan, can also be substituted at this position. Thus, for example, in some examples, the fHbp mutant comprises an E92R substitution, an E92H substitution, an E92F substitution, an E92Y substitution, or an E92W substitution. As an example, a mutant fHbp of the present disclosure can comprise the amino acid sequence shown in Figure 31 and set forth in SEQ ID NO: 16.

[0081] In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity 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 the fHbp ID55 for human fH, and the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp comprises an amino acid substitution of serine at position 223 (S223). In some examples, the fHbp mutant comprises a S223R substitution. Other amino acids with positively charged or aromatic side chains, such as lysine, histidine, phenylalanine, tyrosine, or tryptophan, can also be substituted at this position. Thus, for example, in some examples, the fHbp mutant comprises a S223K substitution, a S223H substitution, a S223F substitution, a S223Y substitution, or a S223W substitution. As an example, a mutant fHbp of the present disclosure can comprise the amino acid sequence shown in FIG. 32 and set forth in SEQ ID NO: 17.

[0082] In some examples, a variant fHbp of the present disclosure comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity 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 the fHbp ID55 for human fH, and the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and the variant fHbp comprises an amino acid substitution of histidine at position 248 (H248). In some examples, the fHbp mutant comprises an H248L substitution. Other amino acids having 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 examples, the fHbp mutant comprises an H248I, H248V, H248D, H248E, H248F, H248Y, or H248W substitution. As an example, a mutant fHbp of the present disclosure can comprise the amino acid sequence shown in FIG. 33 and set forth in SEQ ID NO: 18.

[0083] Combinations of amino acid substitutions In some examples, a variant fHbp of the 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 the ID55 of fHbp for human fH, wherein the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and wherein the variant fHbp is Relative to ID55: (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), the residue numbers being based on the amino acid numbering in the sequence for fHbp ID1.

[0084] Combinations of substitutions may be included, where two substitutions are in different structural domains and each independently reduces binding of fH to an fHbp (e.g., one substitution in the N-terminal domain combined with an amino acid substitution in the C-terminal domain. In some examples, a mutant 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 examples, a mutant 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 examples, a mutant 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.

[0085] For example, in some examples, a variant fHbp of the 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 the fHbp ID55 for human fH, and the variant induces a bactericidal antibody response against at least one strain of N. meningitidis in a mammalian host, and Relative to ID55, the mutant 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; the mutant 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; the mutant 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 the mutant fHbp comprises: (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, the residue numbers being Based on the number of amino acids in the sequence relative to ID1.

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

[0087] Fusion Polypeptides A mutant fHbp of the present disclosure can be a fusion polypeptide, e.g., a polypeptide comprising a mutant fHbp as described above and a heterologous polypeptide (e.g., a fusion partner). The fusion partner can be at the N-terminus of the mutant fHbp, the C-terminus of the mutant fHbp, or an internal site of the fHbp.

[0088] Suitable fusion partners include those that provide improved in vivo stability (e.g., increased serum half-life); those that provide ease of purification, e.g., (His) n , such as 6His; providing secretion of the fusion protein from cells; providing an epitope tag, such as GST, hemagglutinin (HA; e.g., YPYDVPDYA; SEQ ID NO: 26), FLAG (e.g., DYKDDDDK; SEQ ID NO: 27), c-myc (e.g., EQKLISEEDL; SEQ ID NO: 28), etc.; providing a detectable signal, such as an enzyme that generates a detectable product (e.g., β-galactosidase, luciferase), or a protein that is itself detectable, 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.

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

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

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

[0092] Suitable vectors for transferring nucleic acids encoding fHbp can vary in composition. Integrating vectors can be conditionally replicating or suicide plasmids, bacteriophages, etc. These constructs can include various elements, such as promoters, selectable gene markers (e.g., genes that confer resistance to antibiotics (e.g., kanamycin, erythromycin, chloramphenicol, or gentamicin)), and origins of replication (to facilitate replication in host cells, e.g., bacterial host cells). The choice of vector depends on various factors, such as the type of cell in which propagation is desired and the purpose of propagation. Some vectors are useful for amplifying and mass-producing the desired DNA sequence. Other vectors are suitable for expression in cultured cells. Still other vectors are suitable for transfer and expression in whole animal cells. Selection of an appropriate vector is well within the skill of one of ordinary skill in the art. Many such vectors are commercially available.

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

[0094] Constructs (recombinant vectors) can be prepared, for example, by inserting a polynucleotide of interest into the backbone of the construct, typically by attachment to a cleavable restriction enzyme site of the vector using DNA ligase. Alternatively, the desired nucleotide sequence can be inserted by homologous recombination or site-specific recombination. Typically, homologous recombination is achieved by adding homologous regions to the vector on both sides of the desired nucleotide sequence, while site-specific recombination can be achieved by using sequences that promote site-specific recombination (e.g., cre-lox, att sites, etc.). Nucleic acids containing such sequences can be added, for example, by oligonucleotide ligation or by polymerase chain reaction using primers that contain both a homologous region and a portion of the desired nucleotide sequence.

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

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

[0097] An expression cassette may be used for expression of a target fHbp. Thus, the present disclosure provides a recombinant expression vector comprising a target nucleic acid. The expression vector provides transcriptional and translational regulatory sequences that can confer inducible or constitutive expression, and the coding region is operably linked under the transcriptional control of a transcriptional initiation region and a transcriptional and translational termination region. These regulatory regions may be native to the fHbp from which the target fHbp is derived or may be derived from an exogenous source. Generally, transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. The promoter may be constitutive or inducible, or may be a strong constitutive promoter (e.g., T7, etc.).

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

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

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

[0101] host cell Any of a number of suitable host cells can be used to produce fHbp. Generally, the fHbps described herein can be expressed in prokaryotes or eukaryotes, e.g., bacteria such as Escherichia coli or Neisseria (e.g., N. meningitidis), according to conventional techniques. Thus, the present disclosure further provides genetically modified in vitro host cells containing nucleic acids encoding the subject fHbp. Host cells for production (including large-scale production) of the subject fHbp can be selected from any of a variety of available host cells. Examples of host cells for expression include prokaryotic or eukaryotic unicellular organisms, e.g., bacteria (e.g., Escherichia coli strains), yeast (e.g., Saccharomyces cerevisiae, Pichia species, etc.), and may also include host cells originally derived from higher organisms, e.g., insects, or vertebrates, e.g., mammals. 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 Nos. 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. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, etc. In some instances, bacterial and yeast host cells are particularly important for production of the subject fHbp.

[0102] 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 substantially isolated form. The subject fHbp can be present in a composition in which the polypeptide is enriched relative to other components that may be present (e.g., other polypeptides or other host cell components). A 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%, usually less than 60%, and more usually less than 50% of the composition is made up of other expressed polypeptides.

[0103] Host cells for producing vesicles When the fHbp of interest is provided in membrane vesicles (described in more detail below), Neisseria host cells are genetically modified to express the fHbp of interest. Any of a variety of strains of Neisseria species may be modified to produce the fHbp of interest, and optionally, may also produce or be modified to produce other antigens of interest, such as PorA, and used in the methods disclosed herein.

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

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

[0106] N. meningitidis strains are particularly interesting for genetic modification to express a fHbp of interest and for use in vesicle production. Strains used for vesicle production can be selected for many different characteristics that may be desired. For example, strains can be selected for: a desired PorA type ("serosubtype"), capsular group, serotype, etc.; reduced capsular polysaccharide production; etc. For example, a production strain can produce any desired PorA polypeptide and can express one or more PorA polypeptides (naturally or by genetic engineering). Exemplary strains include those that produce PorA polypeptides that confer serosubtypes P1.7,16; P1.19,15; P1.7,1; P1.5,2; P1.22a,14; P1.14; P1.5,10; P1.7,4; and P1.12,13; and variants of such PorA polypeptides that may or may not retain reactivity with conventional serological reagents used for serosubtyping. Equally important 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 number of different VR types have been identified, which can be grouped into "prototype" VR1 and VR2 families. 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 specific PorA VR1 and VR2 types is provided in Russell et al. (2004) Emerging Infect Dis 10:674-678.

[0107] Alternatively or additionally, the producer strain may be a capsule-deficient strain. Capsule-deficient strains can provide vesicle-based vaccines that reduce the risk of eliciting a significant autoantibody response in subjects to which the vaccine is administered (e.g., due to the production of antibodies that cross-react with sialic acid on the host cell surface). As used herein, "capsule-deficient" or "capsular polysaccharide-deficient" refers to a level of capsular polysaccharide on the bacterial surface that is lower than that of a naturally occurring strain, or, if the strain is genetically modified, a level of capsular polysaccharide on the bacterial surface that is lower than that of the parent strain from which the capsule-deficient strain is derived. Capsule-deficient strains include strains that have at least 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90% or more reduced production of surface capsular polysaccharides and strains in which capsular polysaccharides are not detectable on the bacterial surface (e.g., by whole-cell enzyme-linked immunosorbent assay (ELISA) using anti-capsular polysaccharide antibodies).

[0108] Capsule-deficient strains include those that are naturally occurring or recombinantly produced by genetic modification to be capsule-deficient. 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 generating capsule-deficient strains (see, e.g., Fisseha et al. (2005) Infect. Immun. 73:4070-4080; Stephens et al. (1991) Infect Immun 59:4097-102; Frosch et al. (1990) Mol Microbiol.4:1215-1218) are known in the art.

[0109] Modifications of Neisseria host cells that result in reduced production of capsular polysaccharides can include modification of one or more genes involved in capsule synthesis, e.g., to reduce capsular polysaccharide levels relative to the parent cell prior to modification. Such genetic modifications can include changes in the nucleotide and / or amino acid sequences of one or more capsule biosynthesis genes, rendering the strain capsule-deficient (e.g., by one or more insertions, deletions, substitutions, etc., in one or more capsule biosynthesis genes). Capsule-deficient strains can lack one or more capsule genes or can be non-functional for one or more capsule genes.

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

[0111] Capsule-deficient strains can also be generated from naturally occurring strains using non-recombinant techniques, for example, by selecting for strains with reduced levels of capsular polysaccharide using bactericidal anti-capsule antibodies.

[0112] Where the present disclosure involves the use of two or more strains (e.g., to produce antigenic compositions comprising vesicles displaying a fHbp of interest from different strains), the strains may be selected to differ in one or more strain characteristics, e.g., to result in vesicles that differ in the fHbp of interest, PorA, etc. that are used.

[0113] Preparation of vesicles Antigenic compositions contemplated by the present disclosure generally comprise vesicles prepared from Neisseria cells expressing the fHbp of interest. As referred to herein, "vesicles" are intended to encompass outer membrane vesicles and microvesicles (also called blebs).

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

[0115] The antigenic composition can include microvesicles (MVs) (or "blebs") containing the fHbp of interest, which are released during cultivation of a Neisseria meningitidis strain genetically modified to express the fHbp of interest. For example, MVs may be obtained by culturing a strain of Neisseria meningitidis in a broth medium, separating whole cells from the broth medium (e.g., by filtration, or by low-speed centrifugation to pellet only the cells and not the smaller blebs), and then recovering the MVs present in the cell-free medium (e.g., by filtration, differential sedimentation or aggregation of MVs, or high-speed centrifugation to pellet the blebs). Strains for the production of MVs can generally be selected based on the amount of blebs produced in culture (e.g., bacteria can be cultured at reasonable numbers to result in the production of blebs suitable for isolation and administration in the methods described herein). Exemplary strains that produce high concentrations of blebs are described in PCT Publication No. WO 01 / 34642. In addition to producing blebs, strains for use in MV production may also be selected based on NspA production, with strains producing higher levels of NspA being particularly important (see, e.g., Moe et al. (1999 Infect. Immun. 67:5664) for examples of N. meningitidis strains with different NspA production levels). Other strains of interest for use in producing blebs include strains with an inactivated GNA33 gene, which encodes a lipoprotein required for cell detachment, membrane structure, and virulence (see, e.g., Adu-Bobie et al. (2004) Infect. Immun. 72:1914-1919).

[0116] The antigenic compositions of the present disclosure can include vesicles from one strain or from two, three, four, five, or more strains, which may be homologous or heterologous, and are typically heterologous to each other. For example, the strains may be homologous or heterologous to the PorA and / or fHbp from which the fHbp of interest is derived. Vesicles can be prepared from strains expressing multiple fHbps of interest (e.g., one, two, three, or more fHbps of interest), which may consist of amino acid sequences of fHbps from different variants (v.1, v.2, or v.3) or subvariants (e.g., subvariants of v.1, v.2, or v.3).

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

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

[0119] Vesicles of antigenic compositions can be prepared without detergents, e.g., without deoxycholate. While detergent treatment is useful for removing endotoxin activity, native fHbp lipoproteins and / or the fHbp of interest (including lipidated fHbp) may be depleted by extraction during vesicle production. Therefore, it may be particularly desirable to reduce endotoxin activity using techniques that do not require detergents. One approach involves using a strain with relatively low endotoxin (lipopolysaccharide, LPS) production, avoiding the need to remove endotoxins from the final preparation before human use. For example, vesicles can be prepared from Neisseria mutants with reduced or eliminated lipooligosaccharides or other vaccines (e.g., Rmp) that may be undesirable in vaccines.

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

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

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

[0123] Polymyxin B-resistant bacterial 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): Proceedings of the ninth international pathogenic Neisseria conference. The Guildhall, Winchester, England). Alternatively, synthetic peptides that mimic the binding activity of polymyxin B may be added to the antigenic composition to reduce the toxic activity of LPS (see, e.g., Rustici et al. (1993) Science 259:361-365; Porro et al. (1998) Prog Clin Biol Res. 397:315-25).

[0124] Endotoxins can also be reduced by selection of culture conditions, for example, culturing strains in growth medium containing 0.1 mg to 100 mg of aminoarabinose per liter of medium reduces lipid toxicity (see, e.g., WO 02 / 097646).

[0125] Compositions and Formulations For convenience, the terms "composition," "antigenic composition," "antigenic composition," or "immunogenic composition" are used herein collectively to refer to compositions comprising a subject fHbp disclosed herein, optionally conjugated to further enhance immunogenicity. Compositions useful for eliciting antibodies, e.g., anti-Neisseria meningitidis antibodies, e.g., bactericidal antibodies against Neisseria meningitidis, in humans are specifically contemplated by the present disclosure. An antigenic composition can include one, two, or more different subject fHbps. When there are two or more fHbps, each subject fHbp can present epitopes from a different combination of fHbp mutants and / or subvariants.

[0126] The antigenic composition comprises an immunologically effective amount of a target fHbp and may further comprise other compatible components, as needed. The compositions of the present disclosure can include an fHbp that is a poor fH binder. The compositions include one or more fHbps, at least one of which is a poor fH binder. When there is more than one fHbp in the composition, each fHbp can be different (e.g., in amino acid sequence and / or linkage).

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

[0128] Immunogenic compositions contemplated by the present disclosure include, but are not limited to: (1) at least one mutant fHbp of the present disclosure; and (2) Composition containing NspA and fHbp and / or NspA may be provided as recombinant proteins and / or in vesicle-based compositions (e.g., OMV or MV). Note that when a composition contains both NspA and fHbp, the bactericidal activity of antibodies elicited by administration of the composition may result from cooperation of antibodies against one or both antigens. Examples of immunogenic compositions provided by the present disclosure include: (a) an immunogenic composition comprising the fHbp mutant described above (e.g., where the mutant fHbp elicits a bactericidal antibody response against at least one strain of Neisseria meningitidis); (b) an immunogenic composition comprising the above-described fHbp mutant (e.g., where the mutant fHbp elicits a bactericidal antibody response against at least one strain of Neisseria meningitidis) and a recombinant NspA protein; (c) a naturally occurring OMV obtained from a genetically modified Neisseria host cell that has been genetically modified with a nucleic acid encoding a mutant fHbp of the present disclosure such that the encoded mutant fHbp is produced by the genetically modified host cell, the OMV comprising the encoded mutant fHbp. an immunogenic composition comprising: (d) a naturally occurring OMV obtained from a genetically modified Neisseria host cell that has been genetically modified with a nucleic acid encoding a mutant fHbp of the present disclosure such that the encoded non-naturally occurring fHbp is produced by the genetically modified host cell, the OMV comprising the encoded mutant fHbp. An immunogenic composition comprising wherein the Neisseria host cells also produce high levels of NspA such that the OMVs also contain NspA. For example, the Neisseria host cell can be genetically modified to increase expression of NspA.

[0129] By "immunologically effective amount" is meant that the administration of that amount to an individual, in a single dose as part of a series of the same or different antigenic compositions, is effective to elicit an antibody response effective in treating or protecting against, for example, the symptoms of infection with Neisseria, particularly N. meningitidis, and more particularly group B of N. meningitidis, or the disease caused by such infection. This amount will vary depending on the health and physical condition of the individual being treated, their age, the capacity of their immune system to produce antibodies, the degree of protection desired, the vaccine formulation, the treating clinician's judgment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined by routine testing.

[0130] The amino acid sequence of an NspA polypeptide is known in the art. See, e.g., 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" can comprise 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 with a contiguous stretch of about 75 to about 100 amino acids, about 100 to about 150 amino acids, or about 150 to about 174 amino acids of the amino acid sequence shown in Figure 40 and set forth in SEQ ID NO:25. An "NspA polypeptide" can comprise 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, or about 100 amino acids to about 155 amino acids, from amino acids 20 to 174 of the amino acid sequence set forth in Figure 40 and set forth in SEQ ID NO: 25. In some instances, the NspA polypeptide lacks a signal sequence; in other instances (e.g., for expression in a host cell), the NspA polypeptide includes a signal sequence.

[0131] The dosing regimen may be a single-dose regimen or a multiple-dose regimen (e.g., including booster doses) in which the antigenic composition in unit dosage form is administered at different times. As used herein, the term "unit dosage form" refers to a physically discrete unit suitable as a single dose 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, the composition being provided together with a pharmaceutically acceptable excipient (e.g., a pharmaceutically acceptable diluent, carrier, or vehicle). The antigenic composition may be administered in combination with other immunomodulatory agents.

[0132] The antigenic composition may be provided in a pharmaceutically acceptable vehicle, which may be a solution such as a sterile aqueous solution, often saline, or may be provided in powder form. Such vehicles may optionally be substantially inert.

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

[0134] The antigenic composition may further comprise an adjuvant. Examples of known suitable adjuvants that can be used in humans include, but are not limited to, aluminum adjuvants (e.g., aluminum phosphate or aluminum hydroxide), MF59 (4.3% w / v squalene, 0.5% w / v Tween 80™, 0.5% w / v Span 85), CpG-containing nucleic acids (cytosine unmethylated), QS21, MPL, 3DMPL, Aquilla extract, ISCOMS, LT / CT mutants, poly(D,L-lactide-co-glycolide) (PLG) microparticles, Quil A, interleukins, etc. For experimental animals, Freund's adjuvant (incomplete Freund's adjuvant; complete Freund's adjuvant), N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP11637, also known as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)ethylamine (CGP19835A, also known as MTP-PE), and RIBI, which contains three bacterial extracts: 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.

[0135] Further exemplary adjuvants for enhancing the effectiveness of the compositions include, but are not limited to, (1) oil-in-water emulsion formulations (with or without muramyl peptides (see below) or other specific immunostimulants such as bacterial cell wall components), such as (a) MF59, containing 5% squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing MTP-PE) formed into submicron particles using a microfluidizer (WO 90 / 14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press, 2009). (1995), (b) SAF, which contains 10% squalene, 0.4% Tween 80, 5% Pluronic block polymer L121, and thr-MDP and is microfluidized to a submicron emulsion or vortexed to produce a larger particle size emulsion, and (c) RIBI adjuvant system (RAS) (Ribi Immunochem; Hamilton, Montana), which contains 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components, such as monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), e.g., MPL+CWS (Detox™); (2) saponin adjuvants, such as QS21 or Stimulon™ (Cambridge (2) immunoglobulins, such as erythrocyte-derived immunoglobulins (IGN), ...Cat. No. 100200229, WO 00 / 07621), or particles formed therefrom, such as ISCOMs (immunostimulating complexes) without added surfactant, e.g., 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 (WO 99 / 44636), etc.), interferons (e.g., gamma interferon), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.;(5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL), e.g., GB-2220221, EP-A-0689454, optionally in the substantial absence of alum when used with pneumococcal saccharides, e.g., WO 00 / 56358; (6) 3dMPL in combination with, e.g., QS21 and / or oil-in-water emulsions, e.g., EP-A-0835318, EP-A-0735898, EP-A-0761231; (7) oligonucleotides containing CpG motifs (see, e.g., WO 98 / 52581), e.g., oligonucleotides containing at least one CG dinucleotide in which the cytosines are not methylated; (8) polyoxyethylene ethers or polyoxyethylene esters (see, e.g., WO 99 / 52549); (9) a combination of a polyoxyethylene sorbitan ester surfactant and an octoxynol (WO 01 / 21207) or a combination of a polyoxyethylene alkyl ether or ester surfactant and at least one additional nonionic surfactant such as an octoxynol (WO 01 / 21152); (10) a saponin and an immunostimulatory oligonucleotide (e.g., a CpG oligonucleotide) (WO 00 / 62800); (11) an immunostimulant and a metal salt particle, e.g., WO 00 / 23105; (12) a saponin and an oil-in-water emulsion, e.g., WO 99 / 11241; (13) a saponin (e.g., QS21) + 3dMPL + IM2 (optionally + a sterol), e.g., WO 98 / 57659; (14) other substances that act as immunostimulants to enhance the effectiveness of the composition. Muramyl peptides include N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), and N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)ethylamine (MTP-PE). Adjuvants suitable for human administration are particularly important. In some cases, the adjuvant is an aluminum salt adjuvant (e.g., aluminum phosphate or aluminum hydroxide).

[0136] The antigen composition may contain other ingredients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium, carbonates, etc. The composition may contain pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, toxicity adjusting agents, etc., to approximate physiological conditions as needed, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.

[0137] The concentration of the target fHbp in the formulation can vary widely (e.g., from less than about 0.1% by weight, e.g., from about or at least about 2% by weight to 20%-50% by weight or more) and is typically selected based primarily on fluid volume, viscosity, and patient-based factors depending on the particular method of administration selected and the patient's needs.

[0138] Formulations containing fHbp may be provided in the form of solutions, suspensions, tablets, pills, capsules, powders, gels, creams, lotions, ointments, aerosols, and the like. It is recognized that oral administration may require the composition to be protected from digestion. This is typically accomplished either by associating the composition with an agent that renders it resistant to acid and enzymatic hydrolysis, or by packaging the composition in a suitable durable carrier. Means of protection from digestion are well known in the art.

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

[0140] Methods for inducing immune responses The present disclosure provides methods for inducing an immune response to at least one strain of Neisseria in a mammalian host, the methods generally comprising administering an effective amount of a subject immunogenic composition to an individual in need thereof.

[0141] Antigenic compositions containing fHbp are generally administered to human subjects at risk for Neisseria disease to prevent or at least partially halt the development of the disease and its complications. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Amounts effective for therapeutic use will depend, for example, on the antigenic composition, the method of administration, the patient's weight and general health, and the judgment of the prescribing physician. Single or multiple administrations of the antigenic composition can be administered, depending on the dosage and frequency, and method of administration, as required and tolerated by the patient.

[0142] The fHbp-containing antigenic composition is generally administered in an amount effective to elicit an immune response, particularly a humoral immune response, e.g., a bactericidal antibody response, in the host. As noted above, immunization amounts vary and generally range from about 1 μg to 100 μg per 70 kg patient, usually 5 μg to 50 μg / 70 kg. Much higher doses (e.g., 10 mg to 100 mg or more) may be suitable for oral, nasal, or topical administration. A primary administration can be followed by booster immunizations with the same antigenic composition containing a different fHbp. In some cases, vaccination includes at least one booster immunization, and in some cases, two booster immunizations.

[0143] Generally, immunization may be achieved by administering the composition by any suitable route, including oral, nasal, nasopharyngeal, parenteral, enteral, gastric, topical, transdermal, subcutaneous, or intramuscular administration, in tablet, solid, powder, liquid, or aerosol form, locally or systemically, with or without added excipients. Practical methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in publications such as Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980).

[0144] The anti-fHbp immune response can be assessed by known methods (e.g., by collecting serum from an individual before and after an initial immunization and demonstrating a change in the individual's immune status, e.g., by immunoprecipitation assay, ELISA, or bactericidal assay, Western blot assay, or flow cytometry assay, etc.).

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

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

[0147] Generally, it may be desirable to begin immunization prior to the earliest signs of disease symptoms or at the earliest signs of potential or actual exposure to infection or disease (e.g., by exposure or infection with Neisseria). [Example]

[0148] The following examples are provided to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present 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 following experiments 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 error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations, such as bp, base pair; kb, kilobase; pl, picoliter; s or sec, second; min, minute; h or hr, hour; aa, amino acid; kb, kilobase; bp, base pair; nt, nucleotide; im, intramuscular; ip, intraperitoneal; sc, subcutaneous; etc., may be used.

[0149] Example 1: Identification and characterization of fHbp ID1 mutants Materials and Methods Library screening A random fHbp mutant library was generated by error-prone polymerase chain reaction (PCR), followed by cloning of the PCR products into the pET28 expression plasmid, which contains a signal sequence enabling surface display in E. coli. Fluorescence-activated cell sorting was used to ensure sufficient expression and proper folding of the fHbp mutants. Mutant clones exhibiting low binding of human fH and high binding of a control anti-fHbp monoclonal antibody were isolated. Recovered cells were plated onto 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. The DNA amplification product was purified (PCR purification kit; Qiagen) and subjected to DNA sequencing of the fHbp gene using primers annealing to the T7 promoter and T7 terminator. This approach to screening random mutant fHbp libraries has the potential to identify: (1) positions affecting fH binding that are not predicted from the crystal structure alone; and (2) substitutions other than alanine that affect fH binding in cases where alanine substitutions do not result in a sufficient reduction in fH binding.

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

[0151] Expression and purification of soluble mutant fHbp Soluble recombinant fHbp was expressed in E. coli, and lysates were prepared as previously described. fHbp was purified by nickel affinity chromatography using a HiTrap Chelating HP column (5 ml; GE Life Sciences, Inc.) and an Äkta Purifier chromatography system (GE Life Sciences). Binding and elution buffers using an imidazole gradient 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 until use.

[0152] For mouse immunogenicity studies, a second purification step was performed using ion-exchange chromatography on 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 with a linear gradient formed using the binding and elution buffers. Fractions containing purified fHbp were combined, dialyzed against PBS containing 3% sucrose, and stored at -80°C until use.

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

[0154] Characterization of fHbp mutants SDS-PAGE. The size and purity of the purified fHbp mutant proteins were 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 for fH was performed as described above.

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

[0156] Binding of anti-fHbp monoclonal antibodies to fHbp by ELISA. Microtiter plate wells were coated with fHbp and blocked and washed as described above for the fH ELISA. Five-fold serial dilutions of mouse anti-fHbp monoclonal antibodies (mAbs) in dilution buffer from 25 to 0.0016 μg / ml were added, and the plates were incubated for 1 h at room temperature. After washing the wells, the primary antibody was detected with alkaline phosphatase-conjugated goat anti-mouse IgG (1:5,000; Sigma-Aldrich). The ELISA was developed and read as described above.

[0157] Surface plasmon resonance (SPR) analysis of fH binding to fHbp. SPR experiments were performed on a Biacore X100 Plus instrument (GE Life Sciences). 3,000 response units of purified human fH were coupled to a CM5 chip (GE Life Sciences) using an amine coupling kit (GE Life Sciences). fH was immobilized on flow cell 2, and a blank (no fH) was immobilized on flow cell 1 for reference. The chip surface was conditioned by three start-up cycles consisting of HEPES-buffered saline containing 3 mM EDTA and 0.05% Surfactant P-20 (GE Life Sciences) followed by regeneration with 100 mM glycine, 3 M NaCl, pH 2.0. Dilutions of purified recombinant fHbp ranging from 100 to 1 nM (wild-type) or 316 to 3.16 nM were injected for 150 s. Dissociation was monitored for 300 s, and these data were analyzed using the Biacore X100 evaluation software.

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

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

[0160] Groups of transgenic mice (N = 11-21) were immunized with aluminum hydroxide-adsorbed fHbp vaccine (same amounts of antigen and adjuvant as for wild-type CD-1 mice). Three doses were administered at 3-week intervals, and blood was collected 3 weeks after the third vaccination. Serum was processed and stored as described above.

[0161] Serum bactericidal antibody (SBA) responses. Human complement-mediated SBA responses were measured against meningococcal strains with identical or nearly identical fHbp sequences compared to the respective vaccine antigens. Bacteria were grown to mid-logarithmic phase (OD = 0.6) in standard Franz 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, AJ and Maiden, MC Humana Press Inc., Totowa, NJ) containing 4 mM lactate and 0.02 mM CMP-NANA. Bacteria were diluted 1:25,000 in Dulbecco's PBS containing 1% BSA (Equitech Bio.). Human complement was derived from a donor with no endogenous bactericidal antibodies and depleted of IgG antibodies using a HiTrap Protein G column (5 ml; GE Life Sciences). Each reaction contained 25% human complement, approximately 400 cfu of bacteria, and a dilution of the test antiserum or control antibody. The SBA titer was calculated as the serum dilution that resulted in a 50% reduction in cfu compared to negative control wells after 60 min of incubation at 37°C. Protein purification: Recombinant fHbp was expressed in E. coli with a C-terminal hexahistidine tag and purified by metal chelation chromatography (HiTrap Chelating HP; GE Life Sciences) followed by ion exchange chromatography (HiTrap SP; GE Life Sciences). These proteins (2 μg each) were separated on a 4–12% NuPAGE gel (Invitrogen) using MES running buffer (Invitrogen) and visualized with Coomassie blue stain (Simply Blue Safe Stain; Invitrogen).

[0162] result We developed a random mutant library-based approach to identify fHbp mutants with reduced binding of human fH. This approach was capable of identifying mutations that reduced fH binding, which may not be predictable based on structural information alone, and was also capable of generating multiple amino acid substitutions at any given position. This approach contrasts with alanine substitutions at commonly selected positions, which sometimes result in small reductions in fH binding.

[0163] Using this random mutant library approach, we identified five promising new fHbp ID1 mutants, Q38R, E92K, R130G, S223R, and H248L, as shown in Figure 1.

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

[0165] These mutants 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).

[0166] Figures 2A and 2B. fH binding of fHbp ID1 mutants by ELISA. Microtiter plate wells were coated with purified recombinant ID1 wild-type (WT) or one of six different mutant proteins. Different concentrations of purified human fH were added to the wells. Bound fH was detected with sheep anti-human fH (Abcam) and alkaline phosphatase-conjugated donkey anti-sheep IgG (Sigma). A, Positive control fHbp ID1 wild-type (WT) protein exhibits high human fH binding. Negative control fHbp ID1 R41S mutant exhibits low fH binding. B, New fHbp ID1 mutants exhibiting reduced fH binding. The R130G mutant exhibited moderate fH binding, Q38R exhibited low binding, and E92K, S223R, and H248L exhibited significantly lower binding than R41S. Averages and standard deviations of replicate measurements are shown.

[0167] A similar pattern of reduced binding of fH to these mutant proteins was observed in surface plasmon experiments, with the R130G and Q38R mutants showing some binding and the other three mutants showing no detectable binding (Figures 3A and 3B).

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

[0169] All five of these mutant fHbp ID1 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 Figure 4.

[0170] Figures 4A-4E. Binding of mouse anti-fHbp monoclonal antibodies to fHbp mutant proteins measured by ELISA. The similar concentration-dependent binding of the anti-fHbp monoclonal antibodies indicated that the wild-type and mutant fHbp proteins were present in similar amounts in the microtiter plate wells, suggesting that the mutant fHbp proteins retained the conformational epitopes recognized by the five different monoclonal antibodies. The secondary antibody was alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma). Means and standard deviations of replicate measurements are shown.

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

[0172] Figures 5A and 5B. Bactericidal antibody responses to fHbp ID1 mutants in mice. Figure 5A. Groups of 12–14 wild-type mice were immunized intraperitoneally with two doses of purified recombinant fHbp (10 μg per dose) three weeks apart. Serum was collected 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 the test strain. H44 / 76 expresses fHbp ID1, which matches the control fHbp ID1 WT vaccine. Each symbol represents the titer of an individual mouse, and the horizontal bar represents the geometric mean titer. The differences between the WT and each of these mutant groups were not statistically significant (p > 0.4 by t-test). Figure 5B. Groups of 14–15 human fH transgenic mice were immunized intraperitoneally with three doses of purified recombinant fHbp (10 μg per dose) at 3-week intervals. Serum was collected 3 weeks after the third dose. Serum bactericidal activity was measured as described above for wild-type mice.

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

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

[0175] result Three promising mutants identified in fHbp ID1 were similarly constructed in fHbp ID55; these contained E92K, S223R, and H248L. All three fHbp ID55 mutants significantly reduced fH binding (Fig. 8A). These mutants preserved the conformational integrity as judged by binding of the mouse anti-fHbp monoclonal antibody JAR41 (Fig. 8B).

[0176] Figures 8A and 8B. fH binding of fHbp ID55 mutants. A. Binding of fH to immobilized fHbp ID55 mutants by ELISA. This experiment was performed as described in the legend to Figure 2. Means and ranges for two to four replicates are shown. B. Concentration-dependent binding of the anti-fHbp monoclonal antibody (mAb) JAR41 suggested that these recombinant fHbps were present in similar amounts in the microtiter plate wells and had a conserved conformation in the region of the epitope recognized by JAR41 (the N-terminal domain; Vu et al. (2012) Sci. Reports, supra). The secondary antibody was alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma).

[0177] Figure 9. Bactericidal antibody responses to fHbp ID55 mutants in wild-type mice. Groups of 12 mice were immunized and their serum bactericidal antibody responses were measured as described above for Figure 5A. Bactericidal activity was measured against mutants of strain H44 / 76 expressing fHbp ID55.

[0178] Figure 10A. Bactericidal antibody responses to the fHbp ID55 S223R mutant in human fH transgenic mice. Groups of 11–12 transgenic mice were immunized with three doses of purified recombinant fHbp (12 μg per dose) or one-tenth the human dose of the licensed Trumenba (Pfizer) vaccine, which contains a total of 12 μg of fHbp. These transgenic mice were immunized, and their serum bactericidal antibody responses were measured as described above for Figure 5B. Figure 10B. Bactericidal antibody responses to Trumenba in wild-type and human fH transgenic mice in relation to serum human fH concentrations. Serum human fH concentrations were measured by ELISA as previously described (Beernink et al. (2011) Journal of Immunology 186(6):3606-14).

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

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

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

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

[0183] Figures 12A-12B. Bactericidal antibody responses to fHbp ID22 library variants in wild-type mice. Library variants with low binding of human fH were selected for immunization. Groups of 10-21 mice were immunized with two doses of purified recombinant fHbp (10 μg per dose) given three weeks apart. Serum was collected 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 the test strain. This strain expresses fHbp ID23, which closely matches the control fHbp ID22 WT vaccine. Figure 12A. Experiments investigating the new variants V131D and K219N. The D211A variant, which did not reduce immunogenicity, was used as a control mutant fHbp vaccine. Each symbol represents the titer of an individual mouse, and the horizontal bar represents the geometric mean titer. Figure 12B, Second experiment examining new mutants D121G, S128T, F129S, and G220S. No significant reduction in immunogenicity was observed for these new mutant fHbp, except for a slight reduction for V131D.

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

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

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

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

[0188] A summary of the exemplary fHbp ID1 and ID22 mutants described above is provided in the table of FIG.

[0189] While the present invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various modifications may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. Furthermore, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

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

Claims

1. A subfamily A factor H binding protein (fHbp), comprising amino acid substitutions at positions corresponding to L130 and G133 of fHbp ID22 (SEQ ID NO: 2), the substitutions being L130R and G133D, the numbering of L130 and G133 being based on the numbering of amino acid residues in SEQ ID NO: 1; the fHbp comprises an amino acid sequence having greater than 95% amino acid sequence identity to SEQ ID NO:2; and the fHbp binds to human factor H (fH) with an affinity that is 50% or less of the affinity of fHbpID22 for human fH, and the fHbp induces a bactericidal antibody response in a mammalian host; Subfamily A factor H binding protein (fHbp).

2. The fHbp of claim 1, wherein the fHbp comprises an amino acid sequence having greater than 99% amino acid sequence identity with SEQ ID NO:

2.

3. (a) a fHbp according to claim 1 or 2; and (b) a pharmaceutically acceptable excipient An immunogenic composition comprising:

4. The immunogenic composition of claim 3 , wherein the fHbp is in a vesicle preparation prepared from a Neisseria meningitidis strain.

5. The immunogenic composition of claim 3 or 4, further comprising Neisseria surface protein A.

6. The immunogenic composition of any one of claims 3 to 5, wherein the pharmaceutically acceptable excipient comprises an adjuvant.

7. The immunogenic composition of claim 6, wherein the adjuvant is aluminum phosphate or aluminum hydroxide.

8. A nucleic acid encoding the fHbp of claim 1 or 2.

9. A recombinant expression vector comprising the nucleic acid of claim 8.

10. 10. An in vitro host cell comprising the nucleic acid of claim 8 or the recombinant expression vector of claim 9.

11. 8. The immunogenic composition of any one of claims 3 to 7 for use in a method for eliciting an antibody response against N. meningitidis in a mammal.

12. The immunogenic composition of claim 11, wherein the mammal is a human.

13. 13. The immunogenic composition of claim 11 or 12, wherein the antibody response is a bactericidal antibody response against one or more strains of N. meningitidis.

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