RU2811942C2 - Options for h-binding protein factor and methods of their use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to variants of factor H-binding proteins, and can be used in medicine to induce a humoral response against N. meningitidis in a mammal. The resulting protein is a variant factor of H-binding protein (fHbp) of A subfamily, which contains the amino acid substitution G220S and optional amino acid substitutions L130R and G133D; wherein the amino acid substitutions are substitutions in relation to fHbp ID 22, where the amino acid positions are numbered relative to the amino acid sequence of fHbp ID1.

EFFECT: invention provides an fHbp variant that binds human factor H (fH) with an affinity that is 50% or less of the fHbp ID 22 affinity for human fH, and the variant produces a bactericidal humoral response in a mammal.

21 cl, 40 dwg, 3 ex

Description

Cross reference

This application claims priority to U.S. Provisional Patent Application No. 62/028123, filed July 23, 2014, which is incorporated herein by reference in its entirety.

Introduction

Neisseria meningitidis is a Gram-negative bacterium that colonizes the human upper respiratory tract and is responsible for worldwide sporadic and cyclical epidemic outbreaks, primarily meningitis and sepsis. The highest rate of damage and morbidity is observed in children under 2 years of age. Like other Gram-negative bacteria, Neisseria meningitidis typically has a cytoplasmic membrane, a layer of peptidoglycan, an outer membrane that, together with the capsular polysaccharide, constitutes the bacterial wall, and fimbriae that project into the external environment. Encapsulated strains of Neisseria meningitidis are a leading cause of bacterial meningitis and sepsis in children and young people. The prevalence and economic importance of invasive Neisseria meningitidis infections has prompted the search for effective vaccines that can provide immunity against different strains and especially against genetically distinct serogroup B strains with different serotypes or serological subtypes.

Factor H-binding protein (fHbp, also known in the art as lipoprotein 2086 (Fletcher et al. (2004) Infect Immun 72:2088-2100), Neisseria genomic antigen (GNA) 1870 (Masignani et al. (2003) J Exp Med 197: 789-99) or "741") is a protein of N. 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 down-regulates complement activation. Binding of fH to the bacterial surface is an important mechanism by which the pathogen survives in nonimmunized human serum or blood and evades innate host defenses. Recently, a genetic change in the human factor H gene cluster has been identified that affects susceptibility to the development of meningococcal disease (Davila S et al. (2010) Nat Genetics doi:10.1038/ng.640). The binding of fH to fHbp is specific to human fH and several non-human primates and may partly explain why Neisseria meningitidis is strictly a human pathogen. fHbp appears to have many naturally occurring sequence variants, which are designated by accession numbers (IDs) assigned in the fHbp database on the Internet at pubmlst(dot)org/neisseria/fHbp.

There remains a need for an fHbp polypeptide that can elicit effective bactericidal antibody responses.

The essence of the invention

Provided are variants of factor H-binding proteins that can induce the synthesis of antibodies that are bactericidal for at least one strain of Neisseria meningitidis, compositions containing such proteins, and methods of using such proteins.

Peculiarities

The present invention provides variants of factor H binding protein (fHbp) ID 1. The present invention provides a variant of fHbp, the variant comprising an amino acid substitution selected from at least one of: a) a glutamine amino acid substitution at amino acid position 38 (Q38); b) replacing the amino acid glutamic acid at amino acid position 92 (E92); c) replacing glycine with arginine at amino acid position 130 (R130G); d) replacing the amino acid serine at amino acid position 223 (S223); and e) a histidine to leucine substitution at amino acid position 248 (H248L), wherein the amino acid substitution is close to fHbp ID 1 (SEQ ID NO: 1), wherein the variant contains an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 1, wherein the fHbp variant binds human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID 1 for human fH, and wherein the variant causes a bactericidal humoral response to at least one strain of Neisseria meningitidis in a mammal. In some cases, the amino acid substitution at Q38 is Q38R, Q38K, Q38H, Q38F, Q38Y, or Q38W. In some cases, the amino acid substitution at E92 is E92K, E92R, E92H, E92F, E92Y, or E92W. In some cases, the amino acid substitution at S223 is S223R, S223K, S223H, S223F, S223Y, or S223W. In some cases, the fHbp variant may additionally contain an R41S or R41A substitution close to fHbp ID 1. For example, the fHbp variant may contain an R41S or R41A substitution and a substitution at position S223, for example, R41S/S223R, close to fHbp ID 1. In other cases, the fHbp variant fHbp may further contain an R41S or R41A substitution and a substitution at position H248L close to fHbp ID 1. In other cases, an fHbp variant may contain two, three or more of the substitutions described herein. In a specific example, the fHbp variant may contain the following substitutions: S223R and H248L close to fHbp ID 1. In some cases, the fHbp variant binds human fH with an affinity that is 25% or less of the affinity of fHbp ID 1 of human fH. In some cases, the fHbp variant binds human fH with an affinity that is 10% or less of the fHbp ID 1 affinity of human fH. In some cases, the fHbp variant binds human fH with an affinity that is 5% or less of the fHbp ID 1 affinity of human fH.

The present invention provides variants of fHbp ID 22. The present invention provides a variant of fHbp, the variant comprising at least one amino acid substitution selected from: a) replacing isoleucine with asparagine at amino acid position 115 (N115I); b) replacing glycine with aspartic acid at amino acid position 121 (D121G); c) replacing threonine with serine at amino acid position 128 (S128T); d) amino acid substitution of valine at position 131 (V131); e) amino acid substitution of lysine at position 219 (K219); f) an amino acid substitution of glycine at position 220 (G220), wherein the amino acid substitutions are close to fHbp ID 22 (SEQ ID NO: 2), provided that the variant contains an amino acid sequence having more than 85% amino acid sequence identity with SEQ ID NO : 2, wherein the fHbp variant binds human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID 22 for human fH, and wherein the variant causes a bactericidal humoral response in a mammal. In some cases, the fHbp variant binds human fH with an affinity that is 25% or less of the fHbp ID 22 affinity of human fH. In some cases, the fHbp variant binds human fH with an affinity that is 10% or less of the fHbp ID 22 affinity of human fH. In some cases, the fHbp variant binds human fH with an affinity that is 5% or less of the fHbp ID 22 affinity of human fH. In some cases, the amino acid substitution at V131 is V131D, V131E, V131K, V131R, V131H, V131F, V131Y, or V131W. In some cases, the amino acid substitution at K219 is K219N, K219Q, K219D, K219E, K219F, K219Y, or K219W. In some cases, the amino acid substitution at G220 is G220S, G220N, G220Q, G220D, G220E, G220K, G220R, G220H, G220F, G220Y, or G220W.

In some cases, the fHbp variant includes a double mutation that increases the thermal stability of the fHbp variant compared to the thermal stability of wild-type (WT) fHbp, for example, WT fHbp ID 22. In some cases, the fHbp variant may include substitutions L130R and G133D close to fHbp ID 22 (SEQ ID NO: 2), wherein the fHbp variant contains an amino acid sequence having greater than 85% amino acid sequence identity with SEQ ID NO: 2, wherein the fHbp variant binds human factor H (fH) with an affinity that is 50% or less than the affinity of fHbp ID 22 for human fH, wherein the variant produces a bactericidal humoral response in a mammal and wherein the variant has greater thermal stability compared to the thermal stability of fHbp ID 22. In some cases, the fHbp variant may include a combination of substitutions such as such as, L130R, G133D and at least one amino acid substitution selected from: a) N115I; b) D121G; c) S128T; d) V131; e) K219 (for example, K219N); and f) G220 (e.g., G220S), wherein amino acid substitutions are similar to fHbp ID 22 (SEQ ID NO: 2), wherein the fHbp variant contains an amino acid sequence having greater than 85% amino acid sequence identity to SEQ ID No: 2, wherein the fHbp variant binds human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID 22 for human fH and the variant induces a bactericidal humoral response in a mammal. The thermal stability of the fHbp variant may be higher than WT fHbp (eg fHbp ID 22) by at least 5°C, 10°C, 15°C, 20°C or more, e.g. 5°C higher - 30°C, 5°C-25°C, 5°C-20°C, 10°C-20°C or 15°C-20°C. As used herein, the term “thermal stability” refers to the stability of a protein when exposed to high temperature; The thermally stable variant protein maintains its conformation at a higher temperature than the wild-type protein. For example, an fHbp variant that includes a double mutation that increases thermal stability relative to the thermal stability of wild-type (WT) fHbp, such as WT fHbp ID 22, may unfold at a higher temperature compared to WT fHbp. In some cases, the N-terminal domain of the fHbp variant may unfold at a higher temperature than the N-terminal domain of WT fHbp (eg, fHbp ID 22).

Also described herein are variants of fHbp that include mutations that increase thermal stability compared to WT fHbp and further include additional mutations that are known to reduce fH binding, such as those disclosed in US 2011/0256180. In some embodiments, a factor H-binding protein (fHbp) variant is described, the variant comprising amino acid substitutions L130R and G133D and at least one of substitutions R80A, D211A, E218A, E248A, G236I, T221A, and H223A close to fHbp ID 22 (SEQ ID NO: 2), wherein the variant contains an amino acid sequence having greater than 85% amino acid sequence identity to SEQ ID NO: 2, wherein the fHbp variant binds human factor H (fH) with an affinity that is 50% or less from the affinity of fHbp ID 22 for human fH and wherein the variant causes a bactericidal humoral response in a mammal.

The present invention provides variants of fHbp ID 55. The present invention provides a variant of fHbp, the variant 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) amino acid substitution of serine at position 223 (S223); and c) a histidine amino acid substitution at position 248 (H248), wherein the amino acid substitution is close to fHbp ID 55 (SEQ ID NO: 3), wherein the variant contains an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO: 3, wherein the fHbp variant binds human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID 55 for human fH, and wherein the variant causes a bactericidal humoral response in a mammal. In some cases, the fHbp variant binds human fH with an affinity that is 25% or less of the fHbp ID 55 affinity of human fH. In some cases, the fHbp variant binds human fH with an affinity that is 10% or less of the fHbp ID 55 affinity of human fH. In some cases, the fHbp variant binds human fH with an affinity that is 5% or less of the fHbp ID 55 affinity of human fH. In some cases, the amino acid substitution at E92 is E92K, E92R, E92H, E92F, E92Y, or E92W. In some cases, the amino acid substitution at S223 is S223R, S223K, S223H, S223F, S223Y, or S223W. In some cases, the amino acid substitution at H248 is H248L, H248I, H248V, H248D, H248E, H248F, H248Y, or H248W.

The present invention provides immunogenic compositions containing the fHbp variant of the present invention. The present invention relates to an immunogenic composition containing: a) an fHbp variant according to one of paragraphs 0006-0010 above; and b) a pharmaceutically acceptable excipient. In some cases, the fHbp variant is found inside drug vesicles derived from a strain of Neisseria meningitidis. In some cases, the pharmaceutically acceptable excipient contains an adjuvant; for example, the adjuvant is aluminum phosphate or aluminum hydroxide. In some cases, the pharmaceutical composition further includes Neisseria surface protein A.

The present invention provides a nucleic acid encoding an fHbp variant according to any one of paragraphs 0006-0010 above. The present invention provides a recombinant expression vector containing a nucleic acid encoding an fHbp variant according to any one of paragraphs 0006-0010 above. The present invention provides an in vitro host cell containing a nucleic acid encoding an fHbp variant according to any one of paragraphs 0006-0010 above. The present invention provides an in vitro host cell containing a recombinant expression vector containing a nucleic acid encoding an fHbp variant according to any one of paragraphs 0006-0010 above.

The present invention provides a method of inducing a humoral response in a mammal, the method comprising administering to the mammal the immunogenic composition of paragraph 0011 above. In some cases, the mammal is a human. In some cases, the humoral response is a bactericidal humoral response to one or more strains of N. meningitidis.

Brief description of graphic materials

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

Fig. 2A and 2B illustrate the binding of fHbp ID 1 mutants to human fH analyzed by ELISA. The mean and range for repeated measurements are given.

Fig. 3A-3E illustrate the binding of fHbp ID 1 mutants to human fH analyzed by surface plasmon resonance. For reference, the same data for wild type (WT) protein ID 1 are shown in each of FIG. 3A-3E.

Fig. 4A-4E illustrate the binding of mouse anti-fHbp monoclonal antibodies (mAbs) to fHbp ID 1 mutant proteins analyzed by ELISA. The mean and range for duplicate measurements are shown.

Fig. 5A and 5B illustrate the bactericidal activity of serum from mice immunized with fHbp ID 1 mutants. Each symbol represents an individual mouse's caption, and the horizontal bars represent geometric mean captions. Fig. 5A illustrates the bactericidal activity of serum from wild-type mice immunized with fHbp ID 1 mutants. Fig. 5B illustrates the bactericidal activity of serum from human fH transgenic mice immunized with fHbp ID 1 mutants.

Fig. 6A and 6B illustrate the characterization of fHbp ID 1 single and double mutants. FIG. 6A illustrates binding of human fH to fHbp ID 1 double mutants. FIG. 6B illustrates the binding of mouse anti-fHbp monoclonal antibody JAR 4 (mAb) to mutants.

Fig. 7 illustrates the bactericidal activity of serum from wild-type mice immunized with fHbp ID 1 single and double mutants. Each symbol represents an individual mouse's caption, and the horizontal bars represent geometric mean captions.

Fig. 8A and 8B illustrate the characterization of fHbp ID 55 mutants. FIG. 8A illustrates the binding of human fH to immobilized fHbp ID 55 mutants analyzed by ELISA and FIG. 8B illustrates the binding of mouse anti-fHbp monoclonal antibody JAR 41 (mAb) to fHbp ID 55 mutant proteins analyzed by ELISA. The mean and range of two to four replicate measurements are given.

Fig. 9 illustrates the bactericidal activity of serum from wild-type mice immunized with fHbp ID 55 mutants. Each symbol represents an individual mouse's caption, and the horizontal bars represent geometric mean captions.

Fig. 10A and 10B illustrate the bactericidal activity of serum from mice immunized with fHbp ID 55. FIG. 10A illustrates the bactericidal activity of sera from human fH transgenic mice immunized with the licensed Trumenba vaccine or the experimental fHbp ID 55 mutant S223R. Fig. 10B illustrates the relationship between human fH serum concentrations of individual transgenic mice and serum bactericidal antibody titers (circular symbols). For comparison, titers from wild-type (WT) mice are shown (squares).

Fig. 11A-11D illustrate the characterization of fHbp ID 22 mutants. FIG. 11A-11C illustrate the binding of fHbp mutant ID 22 to human fH analyzed by ELISA. The mean and range of two to four repeated measurements are given. Fig. 11D depicts the binding of mouse anti-fHbp monoclonal antibody JAR 4 (mAb) to fHbp ID 22 mutant proteins analyzed by ELISA. fHbp ID 22 wild type (WT) and D211A mutant are shown as the control group. The mean and range of duplicate measurements are shown.

Fig. 12A and 12B show the bactericidal activity of serum from mice immunized with fHbp ID 22 mutants. Each symbol represents an individual mouse's caption, and the horizontal bars represent geometric mean captions. Fig. 12A and FIG. 12B depicts the bactericidal activity of serum from wild-type mice in two experiments testing different fHbp ID 22 mutants.

Fig. 13 illustrates the bactericidal activity of serum from human fH transgenic mice immunized with fHbp ID 22 mutants.

Fig. 14 illustrates thermal unfolding of fHbp ID 22 wild type (WT) and L130R/G133D double mutant analyzed by differential scanning microcalorimetry.

Fig. 15 illustrates the characterization of fHbp ID 22 triple mutants. FIG. 15A illustrates the binding of human fH to fHbp ID 22 triple mutants. DM refers to the L130R/G133D double mutant. Fig. 15B illustrates binding of JAR 4 mouse anti-fHbp monoclonal antibody (mAb) to fHbp ID 22 triple mutants.

Fig. 16 illustrates the bactericidal activity of sera from human fH transgenic mice immunized with fHbp ID 22 triple mutants. DM refers to the L130R/G133D double mutant.

Fig. 17 illustrates a table of exemplary fHbp mutants with reduced human fH binding.

Fig. 18 illustrates the amino acid sequence of wild-type human factor H.

Fig. 19 illustrates the amino acid sequences of fHbp ID 1, ID 22 and ID 55 from N. meningitidis strains.

Fig. 20-24 illustrate the amino acid sequences of fHbp ID 1 variants.

Fig. 25-30 illustrate the amino acid sequences of fHbp ID 22 variants.

Fig. 31-33 illustrate the amino acid sequences of fHbp ID 55 variants.

Fig. 34-36 illustrate the amino acid sequences of fHbp ID 1 double mutant variants.

Fig. 37-39 illustrate the amino acid sequences of fHbp ID 22 double mutant and triple mutant variants.

Fig. 40 illustrates the amino acid sequence of NspA.

Definitions

"Factor H-binding protein" (fHbp), which is also known in the literature as GNA1870, GNA 1870, ORF2086, LP2086 (lipoprotein 2086) and "741" belongs to the class of N. meningitidis polypeptides. It occurs naturally as a lipoprotein on the surface of the bacterium N. meningitidis. fHbp are subdivided into three groups of fHbp variants (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 subfamily A and B in other reports (see, for example, Fletcher et al. (2004) Infect Immun 72:2088-2100) based on amino acid sequence variability and immunological cross-reactivity (Masignani et al. ( 2003) J Exp Med 197: 789-99). fHbp can also be classified into one of the six most common modular fHbp groups, designated Modular Group I through Modular Group VI, as shown in FIG. 2 Vu et al. (2012) Sci. Reports 2:341. Each unique fHbp found in N. meningitidis is also designated an fHbp peptide ID according to the website pubmlst.org/neisseria/fHbp/. Since the length of fHbp protein variant 2 (v.2) (from strain 8047, fHbp ID 77) and fHbp variant 3 (v.3) (from strain M1239, fHbp ID 28) differ by -1 to +7 amino acid residues, respectively , from that of strain MC58 (fHbp ID 1), the numbering used herein to designate residues for v.2 and v.3 fHbp proteins differs from the numbering based on the actual amino acid sequences of these proteins. Thus, for example, the designation of a leucine residue (L) at position 166 of the v.2 or v.3 fHbp sequence refers to the residue at position 165 of the v.2 protein and at position 173 of the v.3 protein. Unless otherwise noted, the numbering of amino acid substitutions present in fHbp variants is indicated by the numbering of amino acid residues in fHbp ID 1.

Human factor H ("human fH") as used herein refers to a protein containing the amino acid sequence as illustrated in FIG. 18 (SEQ ID NO: 4), and naturally occurring human allelic variants thereof.

“Derived from” in the context of an amino acid sequence or polynucleotide sequence (e.g., an amino acid sequence “derived from” fHbp ID 1) should be understood to mean that the polypeptide or nucleic acid has a sequence that is based on a reference polypeptide or nucleic acid (e.g. naturally occurring fHbp protein or encoding nucleic acid) and should not be construed as limiting the source or method by which the protein or nucleic acid was obtained. Non-limiting examples of reference polypeptides and reference polynucleotides from which an amino acid sequence or polynucleotide sequence can be "derived from" include naturally occurring fHbp, fHbp ID 1, and non-naturally occurring fHbp. "Derived from" in the context of bacterial strains should be understood to mean that the strain has been obtained by in vivo passage, or in vitro culture, from a parent strain and/or is a recombinant cell obtained by modification of the parent strain.

"Conservative amino acid substitution" refers to the replacement of one amino acid residue with another that shares the same chemical and physical properties of the amino acid side chain (eg, charge, size, hydrophobicity/hydrophilicity). "Conservative substitutions" are intended to include substitutions within the following groups of amino acid residues: gly, ala; val, ile, leu; asp,glu; asn,gln; ser, thr; lys, arg; and phe, tyr. Guidance for such substitutions can be obtained from amino acid sequence alignments of the polypeptides of the epitope of interest.

The term "protective immunity" means that the schedule of vaccination or immunization that is administered to a mammal produces an immune response that prevents, delays the development of, or reduces the severity of a disease that is caused by Neisseria meningitidis, or reduces or eliminates the symptoms of the disease. Protective immunity may be accompanied by the production of bactericidal antibodies. It should be noted that the production of bactericidal antibodies against Neisseria meningitidis as is customary in the art is predictive of the protective effect of the vaccine in humans. (Goldschneider et al. (1969) J. Exp. Med. 129:1307; Borrow et al. (2001) Infect Immun. 69:1568).

The phrase "disease caused by a strain of Neisseria meningitidis" covers any clinical symptom or combination of clinical symptoms that is present when a person is infected with Neisseria meningitidis. These symptoms include, but are not limited to: colonization of the upper respiratory tract (eg, nasopharyngeal and tonsil mucosa) pathogenic strain of Neisseria meningitidis, penetration of bacteria into the mucous membrane and submucosa of the vascular bed, sepsis, septic shock, inflammation, hemorrhagic skin lesions, activation of fibrinolysis and blood coagulation, dysfunction of organs such as kidneys, lungs and heart failure, adrenal bruising and muscle infarction, capillary leak, edema, peripheral ischemia of the lower extremities, respiratory distress syndrome, pericarditis and meningitis.

The phrase “specifically binds to an antibody” or “specifically immunoreactive with” in the context of an antigen (eg, a polypeptide antigen) refers to a binding reaction that is based on and/or provides evidence of the presence of an antigen in a sample, which may also include a heterogeneous population of other molecules. Thus, under these conditions, a particular antibody or antibodies bind to a particular antigen or antigens in the sample and do not bind in significant amounts to other molecules present in the sample. “Specifically binds to an antibody” or “specifically immunoreactive with” in the context of an antigen epitope (e.g., a polypeptide epitope) 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), which may also include heterogeneous a population of other epitopes, as well as a heterogeneous population of antigens. Thus, under the specified conditions, the specified antibody or antibodies bind to a specific epitope of the antigen and do not bind in significant amounts to other epitopes present in the antigen and/or in the sample.

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

"Surface antigen" is an antigen that is present on the surface structure of Neisseria meningitidis (eg, outer membrane, capsule, fimbriae, etc.).

"Isolated" refers to an object of interest that is found in an environment different from that in which the compound may occur naturally. “Isolated” means inclusion of compounds in samples that are substantially enriched in the compound of interest and/or in which the compound of interest is partially or substantially purified. In some cases, the isolated component (e.g., a polypeptide, such as an fHbp variant of the present invention; a nucleic acid of the present invention; a recombinant vector of the present invention) is purified, e.g., an isolated component to at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more than 99% pure.

"Enriched" means that the sample is non-naturally adjusted (eg, by an experimenter or clinician) such that the compound of interest is present at a higher concentration (eg, at least three times more, at least 4 times more, by at least 8 times greater, at least 64 times greater, or more) than the concentration of the compound in a parent sample, such as a biological sample (e.g., a sample in which the compound occurs naturally or in which it is present after administration), or in in which the compound was produced (eg, as a bacterial polypeptide, antibody, nucleic acid, and the like).

Before the present invention is further described, it should be understood that the present invention is not limited to the specific embodiments described, since these may, of course, vary. In addition, it should be understood that the terminology used herein is for the purpose of describing specific embodiments of the invention and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that every intervening value, up to the tenth of the lower limit, unless the context clearly requires otherwise, between the upper and lower limits of that range and any other specified or intervening value within that specified range is covered by limits of the invention. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and are also included in the present invention, subject to any specifically excluded limit within said range. Where a specified range includes one or both limits, ranges excluding either or both of these included limits are also included in the invention.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention, the preferred methods and materials are described at this time. All publications mentioned herein are incorporated herein by reference for the purpose of disclosure and description of the methods and/or materials in connection with which such publications are cited.

It should be noted that in this specification and in the accompanying claims, the singular forms include the plural unless the context clearly requires otherwise. Thus, for example, the designation “factor H-binding protein” includes a plurality of such factor H-binding proteins and the designation “immunogenic composition” includes the designation one or more immunogenic compositions and equivalents known to those skilled in the art, and so on. It is also noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a prior basis for the use of such special terminology as "solely", "only" and the like, in connection with the listing of the elements of the claims or the use of a “negative” limitation.

It should be understood that certain features of the invention, which for clarity are described in the context of individual embodiments, may also be presented in combination in a single embodiment. Conversely, various features of the invention, which for brevity are described in the context of a single embodiment, may also be presented separately or in any suitable subcombination. All combinations of embodiments related to the present invention are specifically embraced by the present invention and are described herein as if each combination were separately disclosed in detail. In addition, all subcombinations of various embodiments and elements thereof are also specifically embraced by the present invention and are described herein as if each such subcombination were separately and fully disclosed herein.

The publications discussed herein are provided solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that the present invention is not entitled to backdate such publication by virtue of a prior invention. In addition, publication dates may differ from actual publication dates, which may require independent confirmation.

Detailed description

The present invention provides variant factor H-binding proteins (fHbp) that can induce the synthesis of antibodies that are bactericidal for at least one strain of Neisseria meningitidis. The present invention provides immunogenic compositions, including compositions containing the fHbp variant of the present invention. The present invention provides methods of using an fHbp variant of the present invention or a composition containing an fHbp variant of the present invention.

fHbp variant

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

In some cases, the fHbp variant of the present invention contains 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 identity sequences with the fHbp reference sequence; wherein the fHbp variant contains one or more amino acid substitutions close to the reference fHbp sequence such that the fHbp variant exhibits an affinity for human fH that is 85% or less of the binding affinity of the reference fHbp for human fH, e.g., the fHbp variant exhibits an affinity for human fH which is from about 85% to about 75%, from about 75% to about 65%, from about 65% to about 55%, from about 55% to about 45%, from about 45% to about 35%, from about 35% to about 25%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1 %, or from about 1% to about 0.1%, or less than 0.1% of the binding affinity to the reference fHbp for human fH; and the fHbp variant induces a bactericidal immune response to at least one strain of N. meningitidis when administered to a mammal (eg, a human, or a non-human animal model).

The fHbp variant of the present invention maintains substantially the same conformation of the reference (eg, wild-type) fHbp in which human fH binds, while the reference fHbp is in the native conformation. By using antibodies that bind wild-type fHbp while the wild-type fHbp is in its native conformation, it can be determined whether the fHbp variant of the present invention maintains substantially the same conformation of the reference (e.g., wild-type) fHbp that binds human fHbp . Such antibodies include, for example, JAR 41; JAR 4; and JAR 31. See, for example, Vu et al. (2012) Sci. Reports 2:341. The hybridome producing JAR 4 monoclonal antibody has the American Type Culture Collection (ATCC) number, PTA-8943; see also USPN 8470340. For example, in some cases, the fHbp variant of the present invention retains binding to JAR 4; for example, a variant fHbp of the present invention retains at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% binding to JAR 4 of the reference fHbp (e.g., fHbp ID 1, fHbp ID 22 or fHbp ID 55) in its native conformation.

Options fHbp ID 1

The “reference fHbp” from which the fHbp variant of the present invention is derived is, in some cases, fHbp ID 1. The amino acid sequence of fHbp ID 1 is set forth below.

In some cases, the fHbp variant of the present invention is group 1 fHbp. In some cases, the fHbp variant of the present invention is a group 1 fHbp and a modular group I fHbp. In some cases, the fHbp variant of the present invention contains 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 identity sequences with SEQ ID NO:1; wherein the fHbp variant contains one or more amino acid substitutions close to fHbp ID 1 such that the fHbp variant exhibits an affinity for human fH that is 85% or less of the binding affinity of fHbp ID 1 for human fH, e.g., the fHbp variant exhibits an affinity for human fH fH, which is from about 85% to about 75%, from about 75% to about 65%, from about 65% to about 55%, from about 55% to about 45%, from about 45% to about 35%, from about 35% to about 25%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%, or less than 0.1% of the binding affinity for fHbp ID 1 for human fH; and the fHbp variant induces a bactericidal immune response to at least one strain of N. meningitidis when administered to a mammal (eg, a human, or a non-human animal model).

In some cases, the fHbp variant of the present invention contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity amino acid sequence of SEQ ID NO: 1, wherein the fHbp variant binds 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 from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 1 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis, in in a mammal, wherein the fHbp variant comprises an amino acid substitution selected from at least one of: a) an amino acid substitution for glutamine at amino acid position 38 (Q38); b) amino acid substitution of glutamic acid at amino acid position 92 (E92); c) replacing glycine with arginine at amino acid position 130 (R130G); d) amino acid substitution of serine at amino acid position 223 (S223); and e) replacing histidine with leucine at amino acid position 248 (H248L), based on fHbp ID 1 numbering.

In some cases, the fHbp variant of the present invention contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity amino acid sequence of SEQ ID NO: 1, wherein the fHbp variant binds 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 from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 1 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis, in in a mammal, and wherein the fHbp variant includes an amino acid substitution for glutamine at amino acid position 38 (Q38). In some cases, the fHbp variant contains 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. In some cases, the fHbp variant contains a Q38K substitution, a Q38H substitution, a Q38F substitution, a Q38Y substitution, or a Q38W substitution. As one example, the fHbp variant of the present invention may contain the amino acid sequence shown in FIG. 20 and set forth in SEQ ID NO:5.

In some cases, the fHbp variant of the present invention contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity amino acid sequence of SEQ ID NO: 1, wherein the fHbp variant binds 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 from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 1 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis, in in a mammal, and wherein the fHbp variant includes an amino acid substitution of glutamic acid at amino acid position 92 (E92). In some cases, the fHbp variant contains an E92K substitution. Other amino acids with positively charged or aromatic side chains, such as arginine, histidine, phenylalanine, tyrosine or tryptophan, may also be substituted at this position. Thus, for example, in some cases, the fHbp variant contains an E92R substitution, an E92H substitution, an E92F substitution, an E92Y substitution, or an E92W substitution. As one example, the fHbp variant of the present invention may contain the amino acid sequence shown in FIG. 21 and set forth in SEQ ID NO:6.

In some cases, the fHbp variant of the present invention contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity amino acid sequence of SEQ ID NO: 1, wherein the fHbp variant binds 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 from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 1 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis, in in a mammal, and the fHbp variant contains a substitution of glycine for arginine at amino acid position 130 (R130G). For example, the fHbp variant of the present invention may contain the amino acid sequence shown in FIG. 22 and set forth in SEQ ID NO:7. Other amino acids with negatively charged or aromatic side chains, such as aspartate, glutamate, phenylalanine, tyrosine or tryptophan, may be substituted at the R130 position. Thus, for example, in some cases, the fHbp variant of the present invention contains 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 fHbp variant binds human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, from about 35% to about 25%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 1 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain N. meningitidis, in a mammal, and wherein the fHbp variant contains an R130D substitution, an R130E substitution, an R130F substitution, an R130Y substitution, or an R130W substitution.

In some cases, the fHbp variant of the present invention contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity amino acid sequence of SEQ ID NO: 1, wherein the fHbp variant binds 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 from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 1 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis, in in a mammal, and wherein the fHbp variant includes an amino acid substitution for serine at amino acid position 223 (S223). In some cases, the fHbp variant contains the S223R substitution. Other amino acids with positively charged or aromatic side chains, such as lysine, histidine, phenylalanine, tyrosine or tryptophan, may also be substituted at this position. Thus, for example, in some cases, the fHbp variant contains an S223K replacement, an S223H replacement, an S223F replacement, an S223Y replacement, or an S223W replacement. As one example, the fHbp variant of the present invention may contain the amino acid sequence shown in FIG. 23 and set forth in SEQ ID NO:8.

In some cases, the fHbp variant of the present invention contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity amino acid sequence of SEQ ID NO: 1, wherein the fHbp variant binds 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 from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 1 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis, in in a mammal, and the fHbp variant contains a substitution of histidine for leucine at amino acid position 248 (H248L). For example, the fHbp variant of the present invention may contain the amino acid sequence shown in FIG. 24 and set forth in SEQ ID NO:9. Other amino acids with non-polar, negatively charged or aromatic side chains, such as isoleucine, valine, aspartate, glutamate, phenylalanine, tyrosine or tryptophan, may also be substituted at position H248. Thus, in some cases, the fHbp variant of the present invention contains 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 fHbp variant binds 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%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1 %, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 1 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis, in a mammal, and wherein the fHbp variant contains an H248I substitution, an H248V substitution, an H248D substitution, an H248E substitution, an H248F substitution, an H248Y substitution, or an H248W substitution.

Combinations of amino acid substitutions

In some cases, the fHbp variant of the present invention contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity amino acid sequence of SEQ ID NO: 1, wherein the fHbp variant binds 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 from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 1 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis, in in a mammal, wherein the fHbp variant comprises an amino acid substitution selected from two or more of: a) an amino acid substitution for glutamine at amino acid position 38 (Q38); b) amino acid substitution of glutamic acid at amino acid position 92 (E92); c) replacing glycine with arginine at amino acid position 130 (R130G); d) amino acid substitution of serine at amino acid position 223 (S223); and e) replacing histidine with leucine at amino acid position 248 (H248L), based on fHbp ID 1 numbering.

Combinations of substitutions may be included, with two substitutions in different structural domains, each independently reducing fH binding to fHbp (for example, a single substitution in the N-terminal domain, combined with an amino acid substitution in the C-terminal domain. In some cases , the fHbp variant of the present invention contains a first amino acid substitution within the N-terminal domain; and a second amino acid substitution within the C-terminal domain. In some cases, the fHbp variant of the present invention contains a first amino acid substitution within the N-terminal domain; and a second amino acid substitution within the N-terminal domain. In some cases, the fHbp variant of the present invention contains a first amino acid substitution within the C-terminal domain; and a second amino acid substitution within the C-terminal domain.

For example, in some cases, the fHbp variant of the present invention contains 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 fHbp variant binds 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%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1% , or from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 1 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis , in a mammal, and wherein the fHbp variant contains: a) an amino acid substitution for glutamine at amino acid position 38 (Q38) and b) an amino acid substitution for glutamic acid at amino acid position 92 (E92); or wherein the fHbp variant contains: a) an amino acid substitution of glutamine at amino acid position 38 (Q38) and c) a substitution of glycine for arginine at amino acid position 130 (R130G); or wherein the fHbp variant contains a) an amino acid substitution for glutamine at amino acid position 38 (Q38) and d) an amino acid substitution for serine at amino acid position 223 (S223); or wherein the fHbp variant contains a) an amino acid substitution of glutamine at amino acid position 38 (Q38) and e) a histidine to leucine substitution at amino acid position 248 (H248L), based on fHbp ID numbering 1.

As additional non-limiting examples, in some cases, the fHbp variant of the present invention contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least at least 99% amino acid sequence identity to SEQ ID NO: 1, wherein the fHbp variant binds 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%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 1 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N .meningitidis, in a mammal, and wherein the fHbp variant contains: b) an amino acid substitution of glutamic acid at amino acid position 92 (E92) and c) a glycine to arginine substitution at amino acid position 130 (R130G); or wherein the fHbp variant contains: b) an amino acid substitution for glutamic acid at amino acid position 92 (E92) and d) an amino acid substitution for serine at amino acid position 223 (S223); or wherein the fHbp variant contains b) an amino acid substitution of glutamic acid at amino acid position 92 (E92) and e) a histidine to leucine substitution at amino acid position 248 (H248L); or wherein the fHbp variant contains c) an amino acid substitution of glycine for arginine at amino acid position 130 (R130G) and d) an amino acid substitution for serine at amino acid position 223 (S223); or wherein the fHbp variant contains c) an amino acid substitution of glycine for arginine at amino acid position 130 (R130G) and e) a histidine for leucine substitution at amino acid position 248 (H248L), or wherein the fHbp variant contains d) an amino acid substitution for serine at amino acid position 223 (S223) and e) substitution of histidine for leucine at amino acid position 248 (H248L), based on fHbp ID 1 numbering.

As further non-limiting examples, in some cases, the fHbp variant of the present invention contains 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 fHbp variant binds human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35% , from about 35% to about 25%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 1 for human fH, wherein the variant causes a bactericidal humoral response to at least one strain of N. meningitidis in a mammal, and wherein the fHbp variant contains: i) a Q38R substitution; and ii) R130G replacement, based on fHbp ID 1 numbering.

Also provided herein are variant fHbp proteins that include fHbp one or more substitutions similar to the amino acid sequence fHbp ID 1 as stated above, and further include the R41S substitution. An exemplary fHbp variant includes an R41S substitution and a substitution at S223, for example, an R41S/S223R similar to fHbp ID 1 or an R41S substitution and an H248L substitution similar to fHbp ID 1. In some cases, the fHbp variant of the present invention contains 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 fHbp variant binds human fH with affinity that is 50% or less (e.g., from about 50% to about 45%, from about 45% to about 35%, from about 35% to about 25%, from about 25% to about 15%, from about 15% up to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%, or less than 0.1 %) of fHbp ID 22 affinity for human fH, wherein the variant causes a bactericidal humoral response to at least one strain of N. meningitidis in a mammal, and wherein the fHbp variant includes two or more of the following amino acid substitutions: a ) replacement of serine with glutamine arginine at amino acid position 41 (R41S); b) replacement of arginine with serine at amino acid position 223 (S223R); c) replacement of leucine with histidine at amino acid position 248 (H248L), based on fHbp ID 1 numbering.

Also disclosed herein are variants of fHbp proteins that include one or more substitutions similar to amino acid sequence fHbp ID 1 as stated above, and further include the substitutions described in US2011/0256180, which are incorporated herein by reference in their entirety.

Options fHbp ID 22

The "reference fHbp" from which the fHbp variant of the present invention is derived is, in some cases, fHbp ID 22. The amino acid sequence of fHbp ID 22 is set forth below.

In some cases, the fHbp variant of the present invention is a group 2 fHbp. In some cases, the fHbp variant of the present invention is a group 2 fHbp and a modular III fHbp. In some cases, the fHbp variant of the present invention contains 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 identity sequences with SEQ ID NO:2; wherein the fHbp variant contains one or more amino acid substitutions close to fHbp ID 22 such that the fHbp variant exhibits an affinity for human fH that is 85% or less of the binding affinity of fHbp ID 22 for human fH, e.g., the fHbp variant exhibits an affinity for human fH fH, which is from about 85% to about 75%, from about 75% to about 65%, from about 65% to about 55%, from about 55% to about 45%, from about 45% to about 35%, from about 35% to about 25%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%, or less than 0.1% of the binding affinity for fHbp ID 22 for human fH; and the fHbp variant induces a bactericidal immune response to at least one strain of N. meningitidis when administered to a mammal (eg, a human, or a non-human animal model).

In some cases, the fHbp variant of the present invention contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity amino acid sequence of SEQ ID NO: 2, wherein the fHbp variant binds 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 from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 22 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis in the body mammal, and wherein the fHbp variant contains an amino acid substitution selected from at least one of: a) an isoleucine to asparagine substitution at amino acid position 115 (N115I); b) replacing glycine with aspartic acid at amino acid position 121 (D121G); c) replacing threonine with serine at amino acid position 128 (S128T); d) replacement of valine at position 131 (V131); e) substitution of the amino acid lysine at position 219 (K219); f) replacing the amino acid glycine at position 220 (G220), close to the amino acid sequence of fHbp ID 22. As indicated herein, the numbering of amino acid residues is based on such fHbp ID 1.

In some cases, the fHbp variant of the present invention contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity amino acid sequence of SEQ ID NO: 2, wherein the fHbp variant binds 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 from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 22 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis in the body mammal, and the fHbp variant contains a substitution of histidine for isoleucine for asparagine at amino acid position 115 (N115I). For example, the fHbp variant of the present invention may contain the amino acid sequence shown in FIG. 25 and set forth in SEQ ID NO:10. Other amino acids with non-polar, positively charged or aromatic side chains, such as valine, leucine, lysine, arginine, histidine, phenylalanine, tyrosine or tryptophan, can also be substituted at the N115 position. Thus, for example, in some cases, the fHbp variant of the present invention contains 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 fHbp variant binds human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, from about 35% to about 25%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 22 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain N. meningitidis, in a mammal, and wherein the fHbp variant contains an N115V substitution, N115L substitution, N115K substitution, N115R substitution, N115H substitution, N115F substitution, N115Y substitution, or N115W substitution close to the amino acid sequence of fHbp ID 22.

In some cases, the fHbp variant of the present invention contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity amino acid sequence of SEQ ID NO: 2, wherein the fHbp variant binds 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 from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 22 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis, in in a mammal, and the fHbp variant contains a substitution of glycine for aspartic acid at amino acid position 121 (D121G). For example, the fHbp variant of the present invention may contain the amino acid sequence shown in FIG. 26 and set forth in SEQ ID NO:11. Other amino acids with non-polar, positively charged or aromatic side chains, such as leucine, isoleucine, valine, lysine, arginine, histidine, phenylalanine, tyrosine or tryptophan, can also be substituted at position D121. Thus, for example, in some cases, the fHbp variant of the present invention contains 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 fHbp variant binds human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, from about 35% to about 25%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 22 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain N. meningitidis, in a mammal, and wherein the fHbp variant contains 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, close to the amino acid sequence of fHbp ID 22.

In some cases, the fHbp variant of the present invention contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity amino acid sequence of SEQ ID NO: 2, wherein the fHbp variant binds 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 from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 22 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis, in in a mammal, and the fHbp variant contains a substitution of threonine for serine at amino acid position 128 (S128T). For example, the fHbp variant of the present invention may contain the amino acid sequence shown in FIG. 27 and set forth in SEQ ID NO: 12. Other amino acids with polar, charged or aromatic side chains, such as methionine, asparagine, glutamine, aspartate, glutamate, lysine, arginine, histidine, phenylalanine, tyrosine or tryptophan, may also be substituted in position S128. Thus, for example, in some cases, the fHbp variant of the present invention contains 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 fHbp variant binds human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, from about 35% to about 25%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 22 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain N. meningitidis, in a mammal, and wherein the fHbp variant contains an S128M substitution, S128N substitution, S128D substitution, S128E substitution, S128K substitution, S128R substitution, S128H substitution, S128F substitution, S128Y substitution, or S128W substitution, close to the amino acid sequence of fHbp ID 22.

In some cases, the fHbp variant of the present invention contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity amino acid sequence of SEQ ID NO: 2, wherein the fHbp variant binds 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 from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 22 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis, in in a mammal, and the fHbp variant includes an amino acid substitution of valine at position 131 (V131). In some cases, the fHbp variant contains a V131D substitution. Other amino acids with charged or aromatic side chains, such as glutamate, lysine, arginine, histidine, phenylalanine, tyrosine or tryptophan, may also be substituted at this position. Thus, for example, in some cases, the fHbp variant contains a V131E replacement, a V131K replacement, a V131R replacement, a V131H replacement, a V131F replacement, a V131Y replacement, or a V131W replacement. As one example, the fHbp variant of the present invention may contain the amino acid sequence shown in FIG. 28 and set forth in SEQ ID NO:13 close to the amino acid sequence of fHbp ID 22.

In some cases, the fHbp variant of the present invention contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity amino acid sequence of SEQ ID NO: 2, wherein the fHbp variant binds 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 from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 22 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis, in in a mammal, and the fHbp variant includes an amino acid substitution of lysine at position 219 (K219). In some cases, the fHbp variant contains a K219N substitution. Other amino acids with polar, negatively charged or aromatic side chains, such as glutamine, aspartate, glutamate, phenylalanine, tyrosine or tryptophan, may also be substituted at this position. Thus, for example, in some cases, the fHbp variant contains a K219Q replacement, a K219D replacement, a K219E replacement, a K219F replacement, a K219Y replacement, or a K219W replacement. As one example, the fHbp variant of the present invention may contain the amino acid sequence shown in FIG. 29 and set forth in SEQ ID NO: 14.

In some cases, the fHbp variant of the present invention contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity amino acid sequence of SEQ ID NO: 2, wherein the fHbp variant binds 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 from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 22 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis, in in a mammal, and wherein the fHbp variant includes an amino acid substitution for glycine at position 220 (G220). In some cases, the fHbp variant contains a G220S replacement. Other amino acids with polar, charged or aromatic side chains, such as asparagine, glutamine, aspartate, glutamate, lysine, arginine, histidine, phenylalanine, tyrosine or tryptophan, may also be substituted at this position. Thus, for example, in some cases, the fHbp variant contains a G220N replacement, a G220Q replacement, a G220D replacement, a G220E replacement, a G220K replacement, a G220R replacement, a G220H replacement, a G220F replacement, a G220Y replacement, or a G220W replacement. For example, the fHbp variant of the present invention may contain the amino acid sequence shown in FIG. 30 and set forth in SEQ ID NO:15.

Combinations of amino acid substitutions

In some cases, the fHbp variant of the present invention contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity amino acid sequence of SEQ ID NO: 2, wherein the fHbp variant binds 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 from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 22 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis in the body mammal, and wherein the fHbp variant contains an amino acid substitution selected from two or more of: a) an isoleucine to asparagine substitution at amino acid position 115 (N115I); b) replacing glycine with aspartic acid at amino acid position 121 (D121G); c) replacing threonine with serine at amino acid position 128 (S128T); d) replacement of valine at position 131 (V131); e) substitution of the amino acid lysine at position 219 (K219); f) replacing the amino acid glycine at position 220 (G220), close to the amino acid sequence of fHbp ID 22. As indicated herein, the numbering of amino acid residues is based on the numbering of amino acids in fHbp ID 1.

Combinations of substitutions may be included, with two substitutions in different structural domains, each independently reducing fH binding to fHbp (eg, one substitution in the N-terminal domain, combined with an amino acid substitution in the C-terminal domain). In some cases, the fHbp variant of the present invention contains a first amino acid substitution within the N-terminal domain; and a second amino acid substitution within the C-terminal domain. In some cases, the fHbp variant of the present invention contains a first amino acid substitution within the N-terminal domain; and a second amino acid substitution within the N-terminal domain. In some cases, the fHbp variant of the present invention contains a first amino acid substitution within the C-terminal domain; and a second amino acid substitution within the C-terminal domain.

For example, in some cases, the fHbp variant of the present invention contains 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 fHbp variant binds 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%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 22 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis, in a mammal, and wherein the fHbp variant contains: a) a substitution of isoleucine for asparagine at amino acid position 115 (N115I) and b) a substitution of glycine for aspartic acid at amino acid position 121 (D121G); or wherein the fHbp variant contains: a) a substitution of isoleucine for asparagine at amino acid position 115 (N115I) and c) a substitution of threonine for serine at position 128 (S128T); or wherein the fHbp variant contains: a) an isoleucine to asparagine substitution at amino acid position 115 (N115I) and d) a valine amino acid substitution at amino acid position 131 (V131); or wherein the fHbp variant contains a) an isoleucine to asparagine substitution at amino acid position 115 (N115I) and e) a lysine amino acid substitution at position 219 (K219); or the fHbp variant contains: a) a replacement of isoleucine with asparagine at amino acid position 115 (N115I) and f) an amino acid replacement of glycine at position 220 (G220), close to the amino acid sequence of fHbp ID 22, numbering of substituted residues based on the numbering of the fHbp amino acid sequence ID 1.

As additional non-limiting examples, in some cases, the fHbp variant of the present invention contains 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 fHbp variant binds human fH with an affinity of 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, about 35% to about 25%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 22 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N .meningitidis, in a mammal, and wherein the fHbp variant contains: b) a glycine to aspartic acid substitution at amino acid position 121 (D121G) and c) a threonine to serine substitution at position 128 (S128T); or wherein the fHbp variant contains: b) a glycine to aspartic acid substitution at amino acid position 121 (D121G) and d) a valine amino acid substitution at amino acid position 131 (V131); or wherein the fHbp variant contains b) a glycine to aspartic acid substitution at amino acid position 121 (D121G) and e) a lysine amino acid substitution at amino acid position 219 (K219); or wherein the fHbp variant contains: b) a glycine to aspartic acid substitution at amino acid position 121 (D121G) and f) a glycine amino acid substitution at position 220 (G220), close to the amino acid sequence of fHbp ID 22. Numbering of the substituted residue(s) based on numbering of the amino acid sequence fHbp ID 1.

As additional non-limiting examples, in some cases, the fHbp variant of the present invention contains 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 fHbp variant binds human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, from about 35% to about 25%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 22 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain N. meningitidis, in a mammal, and wherein the fHbp variant contains: c) a threonine to serine substitution at amino acid position 128 (S128T) and d) a valine amino acid substitution at position 131 (V131); or wherein the fHbp variant contains: c) a threonine to serine substitution at amino acid position 128 (S128T) and e) a lysine amino acid substitution at position 219 (K219); or wherein the fHbp variant contains: c) a threonine to serine substitution at position 128 (S128T) and f) a glycine amino acid substitution at position 220 (G220), or wherein the fHbp variant contains: d) a valine amino acid substitution at position 131 (V131) And; e) amino acid substitution of lysine at position 219 (K219); or wherein the fHbp variant contains: d) a valine amino acid substitution at position 131 (V131) and f) a glycine amino acid substitution at position 220 (G220), or wherein the fHbp variant contains: e) a lysine amino acid substitution at position 219 (K219) and f) amino acid substitution of glycine at position 220 (G220), close to the amino acid sequence fHbp ID 22. Numbering of the substituted residue(s) based on the numbering of the amino acid sequence fHbp ID 1.

Combinations of substitutions may be included, with two substitutions in different structural domains, each independently reducing fH binding to fHbp (e.g., one substitution in the N-terminal domain (e.g., N115I, D121G, S128T, or V131D), in combination with one in the C-terminal domain (for example, D211A, K219N, G220S).

For example, in some cases, the fHbp variant of the present invention contains 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 fHbp variant binds 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%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1% , or from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 22 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis in a mammal, and wherein the fHbp variant contains: i) N115I substitution; and ii) D211A replacement.

As another example, in some cases, the fHbp variant of the present invention contains 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 fHbp variant binds human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, from about 35% to about 25%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 22 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain N.meningitidis in a mammal, and the fHbp variant contains: i) N115I substitution; and ii) K219N replacement.

As another example, in some cases, the fHbp variant of the present invention contains 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 fHbp variant binds human fH with an affinity that is 50% or less (e.g., about 50% to about 45%, about 45% to about 35%, from about 35% to about 25%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 22 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain N.meningitidis in a mammal, and the fHbp variant contains: i) N115I substitution; and ii) G220S replacement.

Also described herein are variant fHbp polypeptides with increased thermal stability compared to wild type fHbp ID22. In some cases, the fHbp variant may include substitutions L130R and G133D close to fHbp ID 22 (SEQ ID NO: 2), wherein the fHbp variant contains an amino acid sequence having greater than 85% amino acid sequence identity to SEQ ID NO: 2, wherein the variant fHbp binds human factor H (fH) with an affinity that is 50% or less of that of fHbp ID 22 for human fH, the variant induces a bactericidal humoral response in a mammal, and the variant has greater thermal stability than WT fHbp ID 22. The thermal stability of the fHbp variant may be greater than that of WT fHbp (e.g., fHbp ID 22) by at least 5°C, 10°C, 15°C, 20°C or more, e.g., 5°C higher -30°C, 5°C-25°C, 5°C-20°C, 10°C-20°C or 15°C-20°C. As used herein, the term “thermal stability” refers to the stability of a protein when exposed to high temperature; The thermally stable variant protein maintains its conformation at a higher temperature than the wild-type protein. For example, an fHbp variant that includes a double mutation that increases thermal stability relative to the thermal stability of wild-type (WT) fHbp, such as WT fHbp ID 22, may unfold at a higher temperature compared to WT fHbp. In some cases, the N-terminal domain of the fHbp variant may unfold at a higher temperature than the N-terminal domain of WT fHbp (eg, fHbp ID 22).

In some embodiments, a factor H binding protein (fHbp) variant is described, the variant comprising amino acid substitutions L130R and G133D and at least one of the substitutions R80A, N115I, D121G, S128T, V131, D211A, E218A, K219 (e.g. , K219N), G220 (e.g., G220S), E248A, G236I, T221A, and H223A closely related to fHbp ID 22 (SEQ ID NO: 2), wherein the variant contains an amino acid sequence having greater than 85% amino acid sequence identity with SEQ ID NO: 2, wherein the fHbp variant binds human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID 22 for human fH and wherein the variant causes a bactericidal humoral response in a mammal.

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

In some embodiments, a factor H-binding protein (fHbp) variant is described, the variant comprising amino acid substitutions L130R and G133D and at least one of substitutions R80A, D211A, E218A, E248A, G236I, T221A, and H223A close to fHbp ID 22 (SEQ ID NO: 2), wherein the variant contains an amino acid sequence having greater than 85% amino acid sequence identity to SEQ ID NO: 2, wherein the fHbp variant binds human factor H (fH) with an affinity that is 50% or less from the affinity of fHbp ID 22 for human fH and wherein the variant causes a bactericidal humoral response in a mammal.

An exemplary fHbp variant includes a polypeptide having an amino acid sequence with greater than 85% amino acid sequence identity (e.g., at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least at least 98% identity, at least 99% identity) with SEQ ID NO:2 and including the following substitutions close to the amino acid sequence of SEQ ID NO:2: L130R and G133D; L130R, G133D and K219N; or L130R, G133D and G220S.

Also disclosed herein are variants of fHbp proteins that include one or more substitutions similar to amino acid sequence fHbp ID 22 as stated above, and further include the substitutions described in US2011/0256180, which are incorporated herein by reference in their entirety.

Options fHbp ID 55

The “reference fHbp” from which the fHbp variant of the present invention is derived is, in some cases, fHbp ID 55. The amino acid sequence of fHbp ID 55 is set forth below.

In some cases, the fHbp variant of the present invention is group 1 fHbp. In some cases, the fHbp variant of the present invention is a group 1 fHbp and a modular IV fHbp.

In some cases, the fHbp variant of the present invention contains 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 identity sequences with SEQ ID NO:3; wherein the fHbp variant contains one or more amino acid substitutions close to fHbp ID 55 such that the fHbp variant exhibits an affinity for human fH that is 85% or less of the binding affinity of fHbp ID 55 for human fH, e.g., the fHbp variant exhibits an affinity for human fH fH, which is from about 85% to about 75%, from about 75% to about 65%, from about 65% to about 55%, from about 55% to about 45%, from about 45% to about 35%, from about 35% to about 25%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%, or less than 0.1% of the binding affinity for fHbp ID 55 for human fH; and the fHbp variant induces a bactericidal immune response to at least one strain of N. meningitidis when administered to a mammal (eg, a human, or a non-human animal model).

In some cases, the fHbp variant of the present invention contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity amino acid sequence of SEQ ID NO: 3, wherein the fHbp variant binds 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 from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 55 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis, in in a mammal, wherein the fHbp variant comprises an amino acid substitution selected from at least one of: a) an amino acid substitution of glutamic acid at position 92 (E92); b) amino acid substitution of serine at position 223 (S223); and c) replacing histidine at position 248 (H248); close to the amino acid sequence of fHbp ID 55, while the numbering of amino acid residues is based on the numbering of fHbp ID 1.

In some cases, the fHbp variant of the present invention contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity amino acid sequence of SEQ ID NO: 3, wherein the fHbp variant binds 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 from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 55 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis, in in a mammal, and the fHbp variant includes an amino acid substitution of glutamic acid at position 92 (E92). In some cases, the fHbp variant contains an E92K substitution. Other amino acids with positively charged or aromatic side chains, such as arginine, histidine, phenylalanine, tyrosine or tryptophan, may also be substituted at this position. Thus, for example, in some cases, the fHbp variant contains an E92R substitution, an E92H substitution, an E92F substitution, an E92Y substitution, or an E92W substitution. As one example, the fHbp variant of the present invention may contain the amino acid sequence shown in FIG. 31 and set forth in SEQ ID NO: 16.

In some cases, the fHbp variant of the present invention contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity amino acid sequence of SEQ ID NO: 3, wherein the fHbp variant binds 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 from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 55 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis, in in a mammal, and the fHbp variant includes an amino acid substitution of serine at position 223 (S223). In some cases, the fHbp variant contains the S223R substitution. Other amino acids with positively charged or aromatic side chains, such as lysine, histidine, phenylalanine, tyrosine or tryptophan, may also be substituted at this position. Thus, for example, in some cases, the fHbp variant contains an S223K replacement, an S223H replacement, an S223F replacement, an S223Y replacement, or an S223W replacement. As one example, the fHbp variant of the present invention may contain the amino acid sequence shown in FIG. 32 and set forth in SEQ ID NO: 17.

In some cases, the fHbp variant of the present invention contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity amino acid sequence of SEQ ID NO: 3, wherein the fHbp variant binds 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 from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 55 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis, in in a mammal, and the fHbp variant includes an amino acid substitution of histidine at position 248 (H248). In some cases, the fHbp variant contains an H248L substitution. Other amino acids with non-polar, negatively charged or aromatic side chains, such as isoleucine, valine, aspartate, glutamate, phenylalanine, tyrosine or tryptophan, may also be substituted at this position. Thus, for example, in some cases, the fHbp variant contains an H248I substitution, an H248V substitution, an H248D substitution, an H248E substitution, an H248F substitution, an H248Y substitution, or an H248W substitution. As one example, the fHbp variant of the present invention may contain the amino acid sequence shown in FIG. 33 and set forth in SEQ ID NO: 18.

Combinations of amino acid substitutions

In some cases, the fHbp variant of the present invention contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity amino acid sequence of SEQ ID NO: 3, wherein the fHbp variant binds 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 from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 55 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis, in in a mammal, and wherein the fHbp variant contains two or more amino acid substitutions selected from the group consisting of: a) an amino acid substitution of glutamic acid at position 92 (E92); b) amino acid substitution of serine at position 223 (S223); and c) an amino acid substitution of histidine at position 248 (H248), close to fHbp ID 55; in this case, the numbering of amino acid residues is based on the numbering of fHbp ID 1.

Combinations of substitutions may be included, with two substitutions in different structural domains, each independently reducing fH binding to fHbp (for example, a single substitution in the N-terminal domain, combined with an amino acid substitution in the C-terminal domain. In some cases , the fHbp variant of the present invention contains a first amino acid substitution within the N-terminal domain; and a second amino acid substitution within the C-terminal domain. In some cases, the fHbp variant of the present invention contains a first amino acid substitution within the N-terminal domain; and a second amino acid substitution within the N-terminal domain. In some cases, the fHbp variant of the present invention contains a first amino acid substitution within the C-terminal domain; and a second amino acid substitution within the C-terminal domain.

For example, in some cases, the fHbp variant of the present invention contains 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 fHbp variant binds 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%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1% , or from about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 55 for human fH, wherein the variant produces a bactericidal humoral response to at least one strain of N. meningitidis , in the body of a mammal, and the fHbp variant contains: a) an amino acid substitution of glutamic acid at position 92 (E92); b) amino acid substitution of serine at position 223 (S223); and wherein the fHbp variant contains: a) an amino acid substitution of glutamic acid at position 92 (E92); c) substitution of histidine at position 248 (H248); wherein the fHbp variant contains: b) an amino acid substitution for serine at position 223 (S223) and c) an amino acid substitution for histidine at position 248 (H248); or wherein the fHbp variant contains: a) a glutamic acid amino acid substitution at position 92 (E92) and b) a serine amino acid substitution at position 223 (S223) and c) a histidine amino acid substitution at position 248 (H248); close to fHbp ID 55; in this case, the numbering of amino acid residues is based on the numbering of fHbp ID 1.

Also described herein are variants of fHbp proteins that include one or more substitutions similar to amino acid sequence fHbp ID 55 as stated above, and further include the substitutions described in US 2011/0256180, which are incorporated herein by reference in their entirety.

Fusion polypeptides

The fHbp variant of the present invention may be a fusion polypeptide, for example, a polypeptide comprising the fHbp variant as described above and a heterologous polypeptide (eg, a fusion partner). The fusion partner may be at the N-terminus of an fHbp variant, at the C-terminus of an fHbp variant, or at an internal site within fHbp.

Suitable fusion partners include fusion peptides and polypeptides that confer increased stability in vivo (eg, increased serum half-life); provide ease of cleaning, for example, (His) _n, for example, 6His and the like; ensure secretion of the fusion protein from the cell; provide an epitope tag, e.g., 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; provide a detectable signal, for example, an enzyme that produces a detectable product (eg, β-galactosidase, luciferase), or a protein that can be detected by itself, for example, green fluorescent protein, yellow fluorescent protein, etc.; provided for multimerization, for example, a multimerization domain such as the Fc part of an immunoglobulin; etc.

Methods of obtaining

The fHbp of the present invention can be produced by any suitable method, including recombinant and non-recombinant methods (eg, chemical synthesis). In the case where the fHbp subject is produced using recombinant methods, the methods may include any suitable construct and any suitable host cell, which may be a prokaryotic or eukaryotic cell, typically a bacterial or yeast host cell, more commonly a bacterial cell. Methods for introducing genetic material into host cells include, for example, transformation, electroporation, conjugation, calcium phosphate methods, and the like. The transfer method can be selected to ensure stable expression of the introduced fHbp-encoding nucleic acid. The FHbp-encoding nucleic acid may be provided as a heritable episomal element (eg, a plasmid) or may be integrated into the genome.

The present invention provides nucleic acids (including isolated nucleic acids) that contain a nucleotide sequence encoding an fHbp variant of the present invention. In some embodiments of the present invention, the nucleotide sequence encoding the fHbp variant is operably linked to a transcription control element, such as a promoter. In some cases the promoter is constitutive. In some cases, the promoter is inducible. In some cases, a promoter suitable for use in (eg, active in) a prokaryotic host cell. In some cases, a promoter suitable for use in (eg, active in) a eukaryotic host cell.

In some cases, a nucleic acid containing a nucleotide sequence encoding an fHbp variant of the present invention is present in an expression vector. The present invention provides a recombinant expression vector (eg, an isolated recombinant expression vector) that contains a nucleotide sequence encoding an fHbp variant of the present invention. In some embodiments, the nucleotide sequence encoding the fHbp variant is operably linked to a transcription control element, such as a promoter. In some cases the promoter is constitutive. In some cases, the promoter is inducible. In some cases, a promoter suitable for use in (eg, active in) a prokaryotic host cell. In some cases, a promoter suitable for use in (eg, active in) a eukaryotic host cell.

Suitable vectors for transferring fHbp-encoding nucleic acid may vary in composition. Integrative vectors can, under certain conditions, be replicative or suicide plasmids, bacteriophages, and the like. These constructs may include various elements, including, for example, promoters, selectable genetic markers (for example, genes conferring resistance to antibiotics (for example, kanamycin, erythromycin, chloramphenicol or gentamicin)), an origin of replication (to promote replication in the host cell, for example, a bacterial host cell), and the like. The choice of vector will depend on many factors, such as the type of cells in which propagation is desired and the purpose of propagation. Some vectors are used to amplify and produce large quantities of the desired DNA sequence. Other vectors suitable for expression in cells in culture. Still other vectors are suitable for transfer and expression in cells throughout the body. The selection of an appropriate vector is within the competence of one skilled in the art. Many such vectors are commercially available.

In one example, the vector is an expression vector based on episomal plasmids containing selectable drug resistance markers and elements that are designed to replicate autonomously in various host cells (eg, as in E. coli and N. meningitidis). One example of such a shuttle vector is the plasmid pFP10 (Pagotto et al. (2000) Gene 244:13-19).

Constructs (recombinant vectors) can be produced, for example, by inserting a polynucleotide of interest into the backbone of the construct, typically by DNA ligase binding to a restriction enzyme cleavage site in the vector. Alternatively, the desired nucleotide sequence can be inserted by homologous recombination or site-specific recombination. Typically, homologous recombination is accomplished by adding sites of homology to the vector flanking the desired nucleotide sequence, while site-specific recombination can be accomplished by using sequences that facilitate site-specific recombination (e.g., cre-lox, att sites, etc.). d.). Nucleic acid containing such sequences can be added by, for example, ligation of oligonucleotides or by polymerase chain reaction using primers containing both regions of homology and part of the desired nucleotide sequence.

Vectors may be provided for extrachromosomal maintenance in a host cell or may be provided for integration into the genome of a host cell. Vectors are described in detail in numerous publications that are well known in the art, including, for example, Short Protocols in Molecular Biology, (1999) F. Ausubel, et al., eds., Wiley & Sons. Vectors may be provided for expressing nucleic acids encoding an fHbp subject, may be provided for propagating nucleic acids of the subject, or both.

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, pUC 19/18, pUC 118, 119 and M13 series vectors can be used. pET21 is also an expression vector that can be used. Bacteriophage vectors may include the λgt10, λgt11, λgt18-23, λZAP/R and EMBL series of bacteriophage vectors. Additional vectors that may be used include, but are not limited to, pJB8, pCV 103, pCV 107, pCV 108, pTM, pMCS, pNNL, pHSG274, COS202, COS203, pWE15, pWE16 and the charomide 9 vector series.

To express an fHbp entity, an expression cassette can be used. Thus, the present invention provides a recombinant expression vector containing a nucleic acid of a subject. The expression vector provides transcriptional and translational regulatory sequences and may provide for inducible or constitutive expression, wherein the coding region is operably linked under transcriptional control to a transcription initiation site as well as a transcription and translation termination site. These control regions may be native to the fHbp from which the fHbp subject is derived, or may be derived from exogenous sources. In general, transcriptional and translational control sequences may include, but are not limited to, promoter sequences, ribosome binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. Promoters can be either constitutive or inducible, and can be a strong constitutive promoter (eg, T7, and the like).

Expression vectors typically have suitable restriction sites located near the promoter sequence to allow the insertion of nucleic acid sequences encoding proteins of interest. A selectable marker operating in the expression host may be present to facilitate selection of cells containing the vector. In addition, the expression construct may include additional elements. For example, an expression vector may have one or two replication systems, allowing it to be maintained in organisms, such as mammalian or insect cells for expression and prokaryotic hosts for cloning and amplification. In addition, the expression construct may contain a selectable marker gene allowing selection of transformed host cells. Selectable genes are well known in the art and will vary depending on the host cell used.

It should be noted that the fHbp of the present invention may contain additional elements such as a detectable label, for example, a radioactive label, a fluorescent label, a biotin label, an immunologically detectable label (eg, a hemagglutinin label, a polyhistidine label) and the like. Additional fHbp elements may be provided to facilitate isolation (eg, biotin tag, immunologically detectable label) using various methods (eg, affinity capture, etc.). The fHbp entity may optionally be immobilized on the support by covalent or non-covalent binding.

Isolation and purification of fHbp can be carried out in accordance with methods known in the art. For example, fHbp can be isolated from a lysate of cells genetically modified to express fHbp or from a synthetic reaction mixture by immunoaffinity purification, which typically involves contacting the sample with an anti-fHbp antibody (e.g., an anti-fHbp monoclonal antibody (mAb) such as JAR 4 MAb or other appropriate JAR MAb known in the art), washing to remove nonspecifically bound material, and elution of 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 techniques.

Host cells

Any of a variety of suitable host cells can be used to produce fHbp. In general, the fHbp described herein can be expressed in prokaryotes or eukaryotes, for example, bacteria such as Escherichia coli or Neisseria (eg, N. meningitidis) according to conventional methods. Thus, the present invention further provides an in vitro genetically modified host cell that contains a nucleic acid encoding an fHbp entity. The host cells for production (including large-scale production) of an fHbp subject may be selected from any of a variety of available host cells. Examples of host cells for expression include those from prokaryotic or eukaryotic single-celled organisms such as bacteria (eg, Escherichia coli strains), yeast (eg, Saccharomyces cerevisiae, Pichia spp., and the like) and may include host cells originally obtained from a higher organism, such as insects, vertebrates, such as mammals. Suitable mammalian cell lines include, but are not limited to, HeLa cells (eg, American Type Culture Collection (ATCC) number CCL-2), CHO cells (eg, ATCC numbers CRL9618, CCL61, CRL9096), 293 cells (eg, ATCC number CRL-1573), Vero cells, NIH 3T3 cells (eg ATCC number CRL-1658), Huh-7 cells, BHK cells (eg ATCC number CCL10), PC12 cells (ATCC number CRL1721), COS cells, COS- cells 7 (ATCC number CRL1651), RAT1 cells, mouse L cells (ATCC number CCLI.3), human embryonic kidney (HEK) cells (ATCC number CRL1573), HLHepG2 cells and the like). In some cases, bacterial host cells and yeast host cells are of particular interest for producing an fHbp subject.

The fHbp entities may be obtained in substantially pure or substantially isolated form (ie, substantially free from other Neisseria polypeptides or host cell polypeptides) or substantially isolated form. The fHbp entity may be present in a composition that is enriched in the polypeptide relative to other components that may be present (eg, other polypeptides or other host cell components). The purified fHbp entity may be provided such that the polypeptide is present in a composition that contains substantially no other expressed polypeptides, and, for example, less than 90%, typically less than 60%, and more often, typically less than 50% of composition consists of other expressed polypeptides.

Host cells for producing vesicles

In the case where the fHbp entity is to be provided in a membrane vesicle (as described in more detail below), the Neisseria host cell is genetically modified to express the fHbp entity. Any of a variety of strains of Neisseria spp. can be modified to produce an fHbp subject, and, if necessary, that produces or can be modified to produce other antigens of interest, such as PorA, can be used in the methods disclosed herein.

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

Pathogenic strains of Neisseria spp. or strains derived from pathogenic Neisseria spp., in particular strains pathogenic to humans or derived from pathogenic strains or synanthropic to humans, are of particular interest for use in the production of membrane vesicles. Examples of Neisseria spp. include N. meningitidis, N. flavescens, N. gonorrhoeae, N. lactamica, N. polysaccharea, N. cinerea, N. mucosa, N. subflava, N. sicca, N. elongata, and the like.

N. meningitidis strains are of particular interest for genetic modification to express fHbp entities and for use in vesicle production. The strain used to produce the vesicles may be selected according to a variety of different characteristics that may be desired. For example, the strain may be selected according to: the desired PorA type (“serosubtype”), capsular group, serotype, and the like; decreased production of capsular polysaccharide; etc. For example, a production strain may produce any desired PorA polypeptide and may express one or more PorA polypeptides (naturally or through genetic engineering). Examples of strains include those that produce the PorA polypeptide, which confers serosubtype P1.7,16; P1.19.15; P1.7,1; P1.5,2; P1.22a,14; P1.14; P1.5,10; P1.7,4; P1.12,13; as well as variants of such PorA polypeptides that may or may not retain their activity using conventional serological reagents used in serosubtyping. Also of interest are PorA polypeptides characterized according to PorA variable region (VR) typing (see, for example, Russell et al. (2004) Emerging Infect Dis 10:674-678; Sacchi CT et al. (1998) Clin Diagn Lab Immunol 5:845-55; Sacchi et al (2000) J Infect Dis 182:1169-1176). A significant number of different types of VR have been identified, which can be classified into the VR1 and VR2 "prototype" families. Web access to a database describing this nomenclature and its relationship to previous typing schemes is at neisseria.org/nm/typing/pora. Alignments of some PorA VR1 and VR2 types are presented in Russell et al. (2004) Emerging Infect Dis 10:674–678.

Alternatively or in addition, the strain to be produced may be a capsule-deficient strain. Capsule-deficient strains may provide vesicle-based vaccines that provide a reduced risk of inducing a significant autoantibody response in the individual receiving the vaccine (eg, due to the production of antibodies that cross-react with sialic acid on host cell surfaces). "Capsule deficient" or "capsule polysaccharide deficient" as used herein refers to a level of capsule polysaccharide on the bacterial surface that is lower than that of the naturally occurring strain or, if the strain is genetically modified, lower than that of the parent strain. from which the capsule-deficient strain was obtained. A capsule-deficient strain includes strains that have reduced production of surface capsule polysaccharide by at least 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90% or more, and includes strains in which the capsular polysaccharide is not detectable on the bacterial surface (eg, using a whole cell enzyme-linked immunosorbent assay (ELISA) using an antibody against the capsular polysaccharide).

Capsule-deficient strains include those that are capsule-deficient due to natural or recombinantly generated genetic modification. Naturally occurring capsule-deficient strains (see, for example, Dolan-Livengood et al). (2003) J. Infect. Dis. 187: 1616-28), as well as methods for identifying and/or producing capsule-deficient strains (see, for example, 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.

Modification of a Neisseria host cell to cause decreased production of capsule polysaccharide may involve modification of one or more genes involved in capsule synthesis, wherein the modification involves, for example, reduced levels of capsule polysaccharide relative to the parent cell prior to the modification. Such genetic modifications may include changes in the nucleotide and/or amino acid sequences of one or more capsule biosynthesis genes that render the strain capsule deficient (e.g., due to one or more insertions, deletions, substitutions, or the like in one or more capsule biosynthesis genes ). Capsule-deficient strains may lack or be nonfunctional in one or more capsule genes.

Of particular interest are strains deficient in the biosynthesis of sialic acid. Such strains may provide vesicles that have a reduced risk of producing anti-sialic acid antibodies that cross-react with human sialic acid antigens and may further provide increased production safety. Strains that are defective in sialic acid biosynthesis (either due to naturally occurring or genetically engineered modification) may be defective in any of several different genes in the sialic acid biosynthesis pathway. Of particular interest are strains that have defects in the product of the gene encoding N-acetylglucosamine 6-phosphate 2-epimerase (known as synX AAF40537.1 or ORCC AAA20475), with strains having this inactivated gene being of particular interest. For example, in one embodiment, a capsule-deficient strain is produced by defective production of a functional synX gene product (see, eg, Swartley et al. (1994) J Bacteriol. 176:1530-4).

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

While the present invention contemplates the use of two or more strains (eg, to produce antigenic compositions containing fHbp entity-presenting vesicles from different strains), the strains may be selected to differ in one or more strain characteristics, e.g. to provide vesicles, those differ in the fHbp entity that is used, PorA and the like.

Preparation of vesicles

The antigenic compositions contemplated by the present invention typically contain vesicles derived from Neisseria cells that express the subject fHbp. As used herein, the term “vesicles” is intended to include outer membrane vesicles as well as microvesicles (which are also referred to as vesicles).

The antigenic composition may comprise outer membrane vesicles (OMVs) derived from the outer membrane of a cultured strain of Neisseria meningitidis spp. genetically modified to express fHbp. OMVs can be obtained from Neisseria meningitidis grown in broth or solid culture medium, preferably by separating bacterial cells from the culture medium (e.g., by filtration or low-speed centrifugation so as to pellet the cells, or the like), cell lysis (e.g. by adding detergent, osmotic shock, sonication, cavitation, homogenization, or the like) and separating the outer membrane fraction from cytoplasmic molecules (for example, by filtration or by differential precipitation or aggregation of outer membranes and/or outer membrane vesicles, or by affinity methods separations using ligands that specifically recognize outer membrane molecules, or by high-speed centrifugation so as to precipitate outer membranes and/or outer membrane vesicles, or the like); outer membrane fractions can be used to obtain OMVs.

The antigenic composition may comprise microvesicles (MVs) (or “vesicles”) containing fHbp entities, wherein the MVs or vesicles are released during cultivation of a Neisseria meningitidis strain genetically modified to express the fHbp entity. For example, MVs can be obtained by culturing a strain of Neisseria meningitidis in a broth culture medium, by separating whole cells from the broth culture medium (e.g., by filtration or low-speed centrifugation so as to pellet only the cells and not smaller vesicles, or the like) and then collecting those MVs that are present in the cell-free culture medium (eg, by filtration, differential sedimentation or aggregation of MVs, or by high-speed centrifugation so as to sediment vesicles, or the like). Strains for use in MV production can generally be selected based on the number of vesicles that are produced in culture (e.g., bacteria can be cultured in sufficient numbers to produce vesicles suitable for isolation and administration in the methods described in herein), an exemplary strain that produces high levels of vesicles is described in PCT publication WO 01/34642. In addition to vesicle production, strains for use in MV production can also be selected based on NspA production, and strains that produce higher levels of NspA may be of particular interest (for examples of N. meningitidis strains having different levels of NspA production, see ., for example, Moe et al (1999 Infect. Immun. 67: 5664) Other strains of interest for use in vesicle production include strains having an inactivated GNA33 gene, which encodes a lipoprotein essential for cell separation, membrane architecture and virulence (See, for example, ADU-Bobie et al. (2004) Infect Immun.72:1914-1919).

The antigenic compositions of the present invention may contain vesicles from a single strain or from 2, 3, 4, 5 or more strains, and the strains may be homologous or heterologous, usually heterologous to each other. For example, the strains from which the fHbp subject is derived may be homologous or heterologous to PorA and/or fHbp. Vesicles may be derived from strains that express more than one fHbp entity (e.g., 1, 2, 3 or more fHbp entities), which may be comprised of fHbp amino acid sequences from different variants (v.1, v.2, or v.3 ) or sub-variants (for example, sub-variants v.1, v.2 or v.3).

Antigenic compositions may contain a mixture of OMVs and MVs that present the same or different fHbp entities, wherein the fHbp entities may optionally present epitopes from different combinations of fHbp variants and/or subvariants, and wherein the OMVs and/or MVs may be from the same the same or different strains. Bubbles from different strains may be administered as a mixture or may be administered sequentially.

If necessary (for example, if the strains used to obtain the vesicles are associated with endotoxin or particularly high levels of endotoxin), the vesicles are further processed to reduce endotoxin, for example, to reduce toxicity after administration. Although less desirable, as described below, endotoxin reduction can be achieved by extraction with a suitable detergent (e.g., BRIJ-96, sodium deoxycholate, sodium lauryl sarcosinate, Empigen BB, Triton X-100, TWEEN 20 nonionic detergent (monolaurate sorbitol polyoxyethylene), nonionic detergent TWEEN 80 at a concentration of 0.1-10%, for example 0.5-2%, and sodium dodecyl sulfate (SDS)). Where detergent extraction is used, it is preferable to use a detergent other than deoxycholate.

Vials of antigen compositions can be prepared without detergents, for example, without the use of deoxycholate. Although detergent treatment is used to remove endotoxin activity, it may deplete the native lipoprotein fHbp and/or the fHbp entity (including lipidated fHbp) by extraction during the vesicle production process. Thus, it may be particularly desirable to reduce endotoxin activity using technology that does not require detergent. In one approach, strains that have relatively low endotoxin (lipopolysaccharide, LPS) productivity are used to avoid the need to remove endotoxins from the final preparation before use in humans. For example, vesicles can be obtained from Neisseria mutants in which the amount of lipopolysaccharide or other antigens that may be undesirable in a vaccine (eg, Rmp) is reduced or eliminated.

Vesicles may be derived from strains of N. meningitidis that contain genetic modifications resulting in reduced or inability to detect the toxic activity of Lipid A. For example, such a strain may be genetically modified for lipid 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 undergo detoxification by modification of LPS, such as down-regulation and/or inactivation of enzymes that are encoded by lpxL1 or lpxL2, respectively. The production of penta-acylated lipid A that is formed in lpxL1 mutants indicates that the enzyme encoded by lpxL1 adds a C12 N-linked 3-OH-C14 at the 2' position of GlcN II. The major lipid A species found in lpxL2 mutants is tetra-acylated, indicating that the enzyme encoded by lpxL2 adds another C12, that is, to the N-linked 3-OH-C14 at position 2 of GlcN I. Mutations that result in a decrease (or not) in the expression of these genes (or a decrease or absence of activity of the products of these genes) do not lead to a change in the toxic activity of lipid A (van der Ley et al. (2001) Infect Immun 69:5981-90). Tetra-acylated (lpxL2 mutant) and penta-acylated (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 interest for use in the production of vesicles (eg, MV or OMV) are strains of N. meningitidis that have been genetically modified to reduce or result in an undetectable functional protein encoded by the LpxL1 gene, for example, when a Neisseria bacterium (eg, N. meningitidis strain) is genetically modified to cause reduced or absent activity of the lpxL1 gene product. For example, the bacterium Neisseria can be genetically modified to knock out the lpxL1 gene, for example, the lpxL1 gene is disrupted. See, for example, US Patent Publication No. 2009/0035328. Neisseria bacteria can be genetically modified to reduce or eliminate the activity of the lpxL2 gene product. Neisseria bacteria can be genetically modified to reduce or eliminate the activity of the gene product of the lpxL1 gene and the lpxL2 gene. Such vesicles provide reduced toxicity compared to N. meningitidis strains that are wild type for LPS production, while maintaining the immunogenicity of the fHbp subject.

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

Mutations in the PhoP-PhoQ regulatory system, which is a two-component regulatory system with phosphorylation (eg, PhoP constitutive phenotype, PhoPc), or low Mg ⁺⁺ environmental or culture conditions (which activate the PhoP-PhoQ regulatory system) result in adding aminoarabinose to 4'-phosphate and 2-hydroxymyristate, replacing myristate (hydroxylation of myristate). This modified lipid A exhibits a reduced ability to stimulate E-selectin expression by human endothelial cells and TNF secretion from human monocytes.

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

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

Compositions and composition

“Compositions,” “antigenic composition,” “antigenic composition,” or “immunogenic composition” are used herein for convenience to generically refer to compositions containing an fHbp entity as disclosed herein, the fHbp entity of which may optionally be conjugated to further enhance immunogenicity. Compositions that are used to induce antibodies, for example, antibodies against Neisseria meningitidis, for example, bactericidal antibodies against Neisseria meningitidis, in the human body are specifically provided for by the present invention. Antigenic compositions may contain 1, 2 or more different fHbp entities. Where there is more than one type of fHbp, each fHbp entity may present epitopes from different combinations of fHbp variants and/or subvariants.

The antigenic compositions contain an immunologically effective amount of an fHbp subject and may further include other compatible components as needed. The compositions of the present invention may contain fHbp, which are weak fH binders. The composition contains one or more fHbp, wherein at least one fHbp is a weak fH binder. Where there is more than one fHbp in a composition, each fHbp may be different (eg, in amino acid sequence and/or conjugation).

In some cases, the antigenic composition of the present invention includes only one fHbp embodiment of the present invention. In some cases, the antigenic composition of the present invention includes two or more different fHbp variants of the present invention. By way of non-limiting examples, in some cases the antigenic composition of the present invention contains:

1) the first fHbp ID 1 variant, wherein the first fHbp ID 1 variant contains an amino acid substitution at position Q38 (for example, Q38R); and a second fHbp ID 1 variant, wherein the second fHbp ID 1 variant contains an amino acid substitution at position E92 (eg, E92K);

2) the first fHbp ID 1 variant, wherein the first fHbp ID 1 variant contains an amino acid substitution at position Q38 (for example, Q38R); and a second fHbp ID 1 variant, wherein the second fHbp ID 1 variant contains an amino acid substitution at position R130 (eg, R130G);

3) the first fHbp ID 1 variant, wherein the first fHbp ID 1 variant contains an amino acid substitution at position Q38 (for example, Q38R); and a second fHbp ID 1 variant, wherein the second fHbp ID 1 variant contains an amino acid substitution at position S223 (eg, S223R);

4) the first fHbp ID 1 variant, wherein the first fHbp ID 1 variant contains an amino acid substitution at position Q38 (for example, Q38R); and a second fHbp ID 1 variant, wherein the second fHbp ID 1 variant comprises an amino acid substitution at position H248 (eg, H248L);

5) fHbp ID 22 variant, wherein the fHbp ID 22 variant contains an amino acid substitution at position N115 (for example, N115I); and variant fHbp ID 1, wherein variant fHbp ID 1 contains an amino acid substitution at position Q38 (eg, Q38R);

6) fHbp ID 22 variant, wherein the fHbp ID 22 variant contains an amino acid substitution at position D121 (for example, D121G); and variant fHbp ID 1, wherein variant fHbp ID 1 contains an amino acid substitution at position E92 (eg, E92K);

7) fHbp ID 22 variant, wherein the fHbp ID 22 variant contains an amino acid substitution at position S128 (for example, S128T); and variant fHbp ID 1, wherein variant fHbp ID 1 contains an amino acid substitution at position H248 (eg, H248L);

8) fHbp ID 22 variant, wherein the fHbp ID 22 variant contains an amino acid substitution at position V131 (for example, V131D); and variant fHbp ID 1, wherein variant fHbp ID 1 contains an amino acid substitution at position Q38 (eg, Q38R);

9) fHbp ID 22 variant, wherein the fHbp ID 22 variant contains an amino acid substitution at position K219 (for example, K219N); and variant fHbp ID 1, wherein variant fHbp ID 1 contains an amino acid substitution at position Q38 (eg, Q38R);

10) fHbp ID 22 variant, wherein the fHbp ID 22 variant contains an amino acid substitution at position G220 (for example, G220S); and variant fHbp ID 1, wherein variant fHbp ID 1 contains an amino acid substitution at position Q38 (eg, Q38R);

11) fHbp ID 22 variant, wherein the fHbp ID 22 variant contains an amino acid substitution at position N115 (for example, N115I); and variant fHbp ID 55, wherein variant fHbp ID 55 contains an amino acid substitution at position E92 (eg, E92K);

12) fHbp ID 22 variant, wherein the fHbp ID 22 variant contains an amino acid substitution at position D121 (for example, D121G); and variant fHbp ID 55, wherein variant fHbp ID 55 contains an amino acid substitution at position S223 (eg, S223R);

13) fHbp ID 22 variant, wherein the fHbp ID 22 variant contains an amino acid substitution at position S128 (for example, S128T); and variant fHbp ID 55, wherein variant fHbp ID 55 contains an amino acid substitution at position H248 (eg, H248L);

14) fHbp ID 22 variant, wherein the fHbp ID 22 variant contains an amino acid substitution at position V131 (for example, V131D); and variant fHbp ID 55, wherein variant fHbp ID 55 contains an amino acid substitution at position E92 (eg, E92K);

15) fHbp ID 22 variant, wherein the fHbp ID 22 variant contains an amino acid substitution at position K219 (for example, K219N); and variant fHbp ID 55, wherein variant fHbp ID 55 contains an amino acid substitution at position E92 (eg, E92K);

16) fHbp ID 22 variant, wherein the fHbp ID 22 variant contains an amino acid substitution at position G220 (for example, G220S); and variant fHbp ID 55, wherein variant fHbp ID 55 contains an amino acid substitution at position E92 (eg, E92K);

17) the first fHbp ID 1 variant, wherein the first fHbp ID 1 variant contains an amino acid substitution at position E92 (for example, E92K); and a second fHbp ID 1 variant, wherein the second fHbp ID 1 variant comprises an amino acid substitution at position H248 (eg, H248L);

18) the first fHbp ID 1 variant, wherein the first fHbp ID 1 variant contains an amino acid substitution at position E92 (for example, E92K); and a second fHbp ID 1 variant, wherein the second fHbp ID 1 variant contains an amino acid substitution at position S223 (eg, S223R);

19) the first fHbp ID 22 variant, wherein the first fHbp ID 22 variant contains an amino acid substitution at position N115 (for example, N115I); and a second fHbp ID 22 variant, wherein the second fHbp ID 22 variant comprises an amino acid substitution at position D211 (eg, D211A);

20) the first fHbp ID 22 variant, wherein the first fHbp ID 22 variant contains an amino acid substitution at position N115 (for example, N115I); and a second fHbp ID 22 variant, wherein the second fHbp ID 22 variant comprises an amino acid substitution at position K219 (eg, K219N);

21) the first fHbp ID 22 variant, wherein the first fHbp ID 22 variant contains an amino acid substitution at position N115 (for example, N115I); and a second fHbp ID 22 variant, wherein the second fHbp ID 22 variant contains an amino acid substitution at position G220 (eg, G220S);

22) the first fHbp ID 22 variant, wherein the first fHbp ID 22 variant contains an amino acid substitution at position D121 (for example, D121G); and a second fHbp ID 22 variant, wherein the second fHbp ID 22 variant contains an amino acid substitution at position G220 (eg, G220S);

23) the first fHbp ID 22 variant, wherein the first fHbp ID 22 variant contains an amino acid substitution at position S128 (for example, S128T); and a second fHbp ID 22 variant, wherein the second fHbp ID 22 variant contains an amino acid substitution at position G220 (eg, G220S);

24) the first fHbp ID 22 variant, wherein the first fHbp ID 22 variant contains an amino acid substitution at position V131 (for example, V131D); and a second fHbp ID 22 variant, wherein the second fHbp ID 22 variant contains an amino acid substitution at position G220 (eg, G220S);

25) the first fHbp ID 55 variant, wherein the first fHbp ID 55 variant contains an amino acid substitution at position E92 (for example, E92K); and a second fHbp ID 55 variant, wherein the second fHbp ID 55 variant comprises an amino acid substitution at position S223 (eg, S223R);

26) the first fHbp ID 55 variant, wherein the first fHbp ID 55 variant contains an amino acid substitution at position E92 (for example, E92K); and a second fHbp ID 55 variant, wherein the second fHbp ID 55 variant comprises an amino acid substitution at position H248 (eg, H248L);

27) fHbp ID22 variant containing amino acid substitutions: L130R and G133D and fHbp ID 1 variant, wherein the fHbp ID 1 variant contains an amino acid substitution at position S223 (for example, S223R);

28) fHbp ID22 variant containing amino acid substitutions: L130R and G133D and fHbp ID 1 variant, wherein the fHbp ID 1 variant contains an amino acid substitution at position H248 (for example, H248L);

29) fHbp ID22 variant containing amino acid substitutions: L130R, G133D and K219N and fHbp ID 1 variant, wherein fHbp ID 1 variant contains an amino acid substitution at position S223 (for example, S223R) or H248 (for example, H248L); or

30) fHbp ID22 variant containing amino acid substitutions: L130R, G133D and G220S and fHbp ID 1 variant, wherein fHbp ID 1 variant contains an amino acid substitution at position S223 (for example, S223R) or H248 (for example, H248L).

Immunogenic compositions contemplated by the present invention include, but are not limited to, compositions containing:

1) at least one fHbp variant according to the present invention; And

2) NspA;

wherein fHbp and/or NspA may be provided as recombinant proteins, and/or based on vesicle compositions (eg, OMV or MV). It should be noted that when the composition contains both NspA and fHbp, the bactericidal activity of the antibodies caused by the administration of the composition may result from the interaction of the antibodies with one or both antigens. Examples of immunogenic compositions provided by the present invention include:

a) an immunogenic composition that contains an fHbp variant as described above (for example, where the fHbp variant induces a bactericidal humoral response to at least one strain of Neisseria meningitidis);

b) an immunogenic composition that contains an fHbp variant as described above (eg, wherein the fHbp variant induces a bactericidal humoral response to at least one strain of Neisseria meningitidis); and recombinant NspA protein;

c) an immunogenic composition that contains a native OMV obtained from a genetically modified Neisseria host cell that is genetically modified with a nucleic acid encoding an fHbp variant of the present invention, such that the encoding fHbp variant is produced by the genetically modified host cell, wherein OMV contains a coding variant for fHbp; And

d) an immunogenic composition that contains a native OMV obtained from a genetically modified Neisseria host cell that is genetically modified with a nucleic acid encoding an fHbp variant of the present invention such that the encoding naturally occurring fHbp is produced by the genetically modified cell— host, where OMV contains an fHbp coding variant; and in doing so, the Neisseria host cell produces higher levels of NspA, such that the OMV also contains NspA. For example, the Neisseria host cell may be one that is genetically modified to increase NspA expression.

By "immunologically effective amount" is meant that administration of such amount to an individual, either as a single dose as part of a series of the same or different antigenic compositions, is effective to elicit a humoral response effective for treating or preventing the symptom or disease caused by , for example, infection caused by Neisseria, in particular N.meningitidis, more often group B N.meningitidis. This amount will vary depending on the health and physical condition of the individual being treated, age, the ability of the individual's immune system to produce antibodies, the degree of protection required, vaccine development, the attending physician's assessment of the medical situation, and other relevant factors. The amount is expected to fall within a relatively wide range that can be determined by routine testing.

The amino acid sequences of NspA polypeptides are known in the art. See, for example, WO 96/29412; and Martin et al. (1997) J. Exp. Med. 185:1173; GenBank accession number U52066; and GenBank accession number AAD53286. An “NspA polypeptide” may comprise an amino acid sequence of 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 region of about 75 amino acids to about 100 amino acids, about 100 amino acids to about 150 amino acids, or from about 150 amino acids to about 174 amino acids of the amino acid sequence shown in FIG. 40 and set forth in SEQ ID NO: 25. “NspA polypeptide” may comprise an amino acid sequence with 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 region of about 75 amino acids to about 100 amino acids, or about 100 amino acids to about 155 amino acids, 20 to 174 amino acids of the amino acid sequence shown in FIG. 40 and specified in SEQ ID NO:25. In some cases, the NspA polypeptide does not have a signal sequence; in other cases (eg, for expression in a host cell), the NspA polypeptide contains a signal sequence.

The dosage regimen may be a single dose or multiple doses (eg, including booster doses) with a single dosage form of the antigenic composition administered at different times. The term "unit dosage form" as used herein refers to physically discrete units suitable as unit dosages for humans or animals, each unit containing a predetermined amount of the antigenic compositions of the present invention in an amount sufficient to produce the desired effect, the compositions of which are presented in combination with a pharmaceutically acceptable excipient (eg, a pharmaceutically acceptable solvent, carrier or excipient). The antigenic composition can be administered in combination with other immunoregulatory agents.

The antigenic compositions may be presented in a pharmaceutically acceptable vehicle, which may be a solution, for example, a sterile aqueous solution, often a saline solution, or they may be presented as a powder. Such fillers may be substantially inert if desired.

In some embodiments, the subject of the immunogenic composition comprises an fHbp subject that is present in the vesicle. In some embodiments, the subject of the immunogenic composition comprises an fHbp subject that is present in the MV. In some embodiments, the subject of the immunogenic composition comprises an fHbp subject that is present in the OMV. In some embodiments, the immunogenic composition subject comprises a mixture of MVs and OMVs containing an fHbp subject. Vesicles such as MV and OMV are described above.

Antigenic compositions may additionally contain an adjuvant. Examples of known suitable excipients that may be used in humans include, but are not necessarily limited to, aluminum as an adjuvant (eg 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 acid (where cytosine is unmethylated), QS 21, MPL, 3dMPL, Aquilla extracts, ISCOMS, LT/CT mutants, poly(D ,L-lactide-co-glycolide) (PLG) microparticles, Quil A, interleukins, and the like. For experimental animals, Freund's adjuvant (incomplete Freund's adjuvant; complete Freund's adjuvant), N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D can be used -isoglutamine (CGP 11637, called nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-DIP-almitoyl-SN-glycero-3-hydroxyphosphoryloxy)ethylamine (CGP 19835A, called MTP-PE) and RIBI, which contains three components extracted from bacteria, 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 an immunogenic antigen or its antigenic epitope.

Further examples of adjuvants to enhance the effectiveness of the composition include, but are not limited to: (1) oil-in-water emulsion compositions (with or without other specific immunostimulants, such as muramyl peptides (see below) or bacterial cell wall components), such as such as (a) MF59 (WO 90/14837; chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press, 1995), containing 5% squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally containing MTP-PE) made into submicron particles using a microfluidizer, (b) SAF containing 10% squalene, 0.4% Tween 80, 5% pluronic-blocked polymer L121 and thr- MDP either microfluidized in a submicron emulsion or vortexed to create a large particle size emulsion, and (c) RIBI Adjuvant System (RAS), (Ribi Immunochem, Hamilton, Montana) containing 2% squalene, 0.2% Tween 80 and one or more bacterial cell wall components such as monophosphoryl lipid A (MPL), trehalose dimycolate (TDM) and cell wall scaffold (CWS), eg MPL+CWS (Detox TM ); (2) saponin adjuvants such as QS21 or Stimulon™ (Cambridge Bioscience, Worcester, MA) can be used or particles formed from them such as ISCOMs (immune stimulating complexes) which can be devoid of additional detergent, for example in WO 00/07621; (3) complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA); (4) cytokines such as interleukins (for example, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (WO 99/44636), etc. ), interferons (eg interferon gamma), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL), for example GB-2220221, EP-A-0689454, optionally, mainly in the absence of aluminum when used with pneumococcal saccharides, for example in WO 00/ 56358; (6) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions, for example, EP-A-0835318, EP-A-0735898, EP-A-0761231; (7) oligonucleotides containing CpG motifs (see, for example, WO 98/52581), for example, an oligonucleotide containing at least one CG dinucleotide, where the cytosine is unmethylated; (8) polyoxyethylene ether or polyoxyethylene ester (see, for example, WO 99/52549); (9) a polyoxyethylene sorbitol ether surfactant in combination with octoxynol (WO 01/21207) or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional nonionic surfactant such as octoxynol (WO 01 /21152); (10) saponin and immunostimulatory oligonucleotide (eg CpG oligonucleotide) (WO 00/62800); (11) an immunostimulant and a metal salt particle, for example in WO 00/23105; (12) saponin and oil-in-water emulsion, for example WO 99/11241; (13) saponin (eg QS21) + 3dMPL + IM2 (optional + sterol), for example in WO 98/57659; (14) other substances that act as immunostimulants to enhance the effectiveness of the composition. Muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-25 normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D -isoglutarninyl-L-alanine-2- (1'-2'-dipalmitoyl-SN-glycero-3-hydroxyphosphoryloxy)ethylamine MTP-PE), etc. Adjuvants suitable for administration to the human body are of particular interest. In some cases, the adjuvant is an adjuvant aluminum salt (eg, aluminum phosphate or aluminum hydroxide).

The antigen compositions may contain other components such as pharmaceutical granules of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate and the like. The compositions may contain pharmaceutically acceptable excipients that are necessary to meet physiological conditions, such as pH adjusting and buffering agents, toxicity controlling agents, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and things like that.

The concentration of the fHbp entity in the composition can vary widely (e.g., from less than about 0.1%, such as at least about 2% to at least 20% to 50% or more by weight ) and is typically selected primarily based on fluid volumes, viscosities, and patient condition factors, consistent with the specific route of administration chosen and the patient's needs.

fHbp-containing compositions can be presented in the form of a solution, suspension, tablet, pill, capsule, powder, gel, cream, lotion, ointment, aerosol and the like. It is generally recognized that when administered orally, it may be necessary to protect the compositions from the effects of digestion. This is typically accomplished either by combining the composition with an agent that makes it resistant to acid and enzymatic hydrolysis, or by packaging the composition in a suitable stable carrier. Digestive system protectants are well known in the art.

fHbp-containing formulations can also be provided in such a way as to increase the serum half-life of fHbp after administration. For example, while isolated fHbp is formulated for injection, fHbp may be formulated into liposomes, formulated as a colloid, or other conventional methods to prolong serum half-life. Various methods are available for the preparation of liposomes, as described, for example, in Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), US Patents No. 4235871, No. 4501728 and No. 4837028. The formulations may also be provided in controlled release or slow release forms.

Methods for inducing an immune response

The present invention provides a method of inducing an immune response to at least one strain of Neisseria in a mammal. The methods generally include administering to an individual in need thereof an effective amount of the subject's immunogenic composition.

fHbp-containing antigen compositions are typically administered to a human subject who is at risk of Neisseria-associated disease in order to prevent or at least partially arrest the progression of the disease and its complications. An amount sufficient to achieve this purpose is defined as a "therapeutically effective dose." Amounts effective for therapeutic use will depend on, for example, the antigenic composition, route of administration, weight and general health of the patient and the judgment of the attending physician. Single or multiple doses of the antigenic compositions can be administered depending on the dose and frequency required and the patient's tolerance and route of administration.

fHbp-containing antigenic compositions are typically administered in an amount effective to induce an immune response, in particular a humoral immune response, for example a bactericidal humoral response, in the host. As noted above, the amounts for immunization may vary, and in general can be from about 1 μg to 100 μg per 70 kg of patient, typically from 5 μg to 50 μg/70 kg. Significantly higher doses (eg, 10 mg to 100 mg or more) may be suitable for oral, nasal or topical routes of administration. The initial administration may be followed by a booster immunization with the same different fHbp-containing antigen composition. Vaccination in some cases includes at least one booster, and in some cases two boosters.

In general, immunization can be achieved by administration by any suitable route, including oral, intranasal, nasopharyngeal, parenteral, enteral, gastric, topical, transdermal, subcutaneous, intramuscular, tablet, solid, powder, liquid, aerosol, topical, or systemically, with or without the addition of excipients. Current methods for preparing parenteral compositions are well 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).

The immune response against fHbp can be assessed by known methods (for example, by obtaining serum from the individual before and after initial immunization, and demonstrating a change in the immune status of the individual, for example, by the results of an immunoprecipitation ELISA or bactericidal assay, Western blot analysis or flow cytometry analysis, or the like).

Whether the fHbp variant of the present invention produces a bactericidal response to one or more strains of N. meningitidis in a mammal can be determined using any well known test. For example, transgenic mice with human fH can be used, wherein the mouse expresses human fH (eg, human fH is present in mouse serum at a concentration of about 100 μg/ml or greater than 100 μg/ml). The fHbp variant of the present invention is administered to human fH transgenic mice. After a certain period of time, serum from mice is tested for bactericidal activity against one or more strains of N. meningitidis. Suitable controls include, for example, fHbp ID 1. An example of a suitable assay is described in Vu et al. (2012) Sci. Reports 2:341.

The antigenic compositions can be administered to a mammal (eg, a human subject) that is immunologically native to Neisseria meningitidis. In a specific embodiment, the subject is a human child about five years of age or younger, and preferably about two years of age or younger, and the antigenic compositions are administered at any one or more of the following: two weeks, one month, 2, 3, 4, 5 6, 7, 8, 9, 10, or 11 months, or one year, or 15, 18, or 21 months after birth, or at 2, 3, 4, or 5 years.

It may be generally desirable to begin immunization before the first sign of disease symptoms, or at the first sign of possible or actual exposure to infection or disease (eg, due to Neisseria exposure or infection).

Examples

The following examples are provided to provide those of ordinary skill in the art with full disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors believe to be their invention, nor are they intended to represent that that the experiments below are all the experiments that were conducted. Care has been taken to ensure accuracy in terms of numbers used (eg quantities, temperatures, etc.), but some experimental errors and deviations must be taken into account. Unless otherwise noted, parts are parts by weight, molecular weight is the average of molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations may be used, for example, bp, base pair (base pairs); kb, kilobase(s); pl, picolitre(s); s or sec, second(s); min, minute(s); h or hour, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair (base pairs); nt, nucleotide(s); i.m., intramuscular(but); i.p., intraperitoneal(but); s.c., subcutaneously, subcutaneous(but); etc.

Example 1: Identification and Characterization of fHbp ID 1 Mutants

Materials and methods

Library screening

A library of random fHbp mutants was generated by error-prone polymerase chain reaction (PCR), followed by cloning of the PCR products into a pET28 expression plasmid that included a signal sequence to allow surface expression of E. coli. Fluorescence-active cell sorting was used to isolate mutant clones with low levels of human fH binding and high levels of control anti-fHbp monoclonal antibody binding, ensuring sufficient expression and proper folding of the fHbp mutants. The collected cells were plated on agar plates (LB agar containing 50 μg/ml kanamycin sulfate), which were incubated overnight at 37°C. Single E. coli colonies were used as templates for PCR amplification, and DNA amplicons were purified (PCR Purification Kit; Qiagen) and subjected to fHbp gene DNA sequencing using primers that hybridized to the T7 promoter and T7 terminator. The approach of screening a library of random fHbp mutants has the potential to identify: 1) positions that affect fH binding that are not predicted by crystal structure alone; and 2) substitutions other than alanine that affect fH binding in cases where the alanine substitution does not result in a significant reduction in the level of fH binding.

Selection of mutants for additional study

The positions of the amino acid substitutions, which were determined from the FACS experiment, were examined in the crystal structure of fHbp in complex with a fragment of human fH. Mutants who were in close proximity with the fH binding surface were selected for site-directed mutagenesis. Replication of the mutant library by site-directed mutagenesis was necessary to generate soluble recombinant fHbp proteins for further characterization. This approach also removed unwanted secondary mutations that were present in many of the sorted clones and that were distant from the fH binding site. Site-directed mutants at specific sites were constructed using the Phusion site-directed mutagenesis kit (Thermo Scientific, Inc.).

Expression and purification of soluble fHbp mutants

Soluble, recombinant fHbp was expressed in E. coli and lysates were prepared as previously described. fHbp was purified by nickel affinity chromatography using HiTrap Chelating HP columns (5 mL; GE Life Sciences, Inc.) and an Akta Purifier chromatography system (GE Life Sciences). Binding and imidazole gradient elution buffers were prepared according to the column manufacturer's protocols. Fractions containing purified fHbp were pooled, dialyzed against PBS containing 3% sucrose, and stored at −80°C until use.

For immunogenicity studies in mice, a second purification step was performed using ion exchange chromatography with HiTrap SP HP columns (5 mL; GE Life Sciences). Binding and elution buffers were 25 mM MES, pH 5.5, containing 150 mM and 750 mM NaCl, respectively. Bound fHbp was eluted from the SP column with a linear gradient formed by binding and elution buffers. Fractions containing purified fHbp were pooled, dialyzed against PBS containing 3% sucrose, and stored at −80°C until use.

Purification of human factor H (fH)

Human fH was purified on an fHbp affinity column. The column was prepared by combining 5 mg fHbp ID 1 with an NHS-activated HP column (5 mL; GE Life Sciences) using the manufacturer's protocols. Human serum from a healthy human donor was diluted 1:1 in phosphate-buffered saline (PBS). The serum was applied to the column and the column was washed with 10 volumes of PBS (ie 50 ml). Bound fH was eluted with 5 column volumes of 0.1 M glycine-HCl, pH 2.7. The eluted fractions were collected in tubes 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 min using 1x MES running buffer (Invitrogen). Proteins were visualized by Coomassie G-250 staining (SimplyBlue SafeStain; Invitrogen). Fractions containing fH were pooled and dialyzed against PBS, and aliquots of Fh were stored at -30°C until use.

Characteristics of fHbp mutants

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

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 purified recombinant wild-type fHbp (positive control) or mutant fHbp (experimental). Nonspecific binding to the wells was blocked with PBS containing 1% bovine serum albumin (Lifeblood Medical, Inc.) or 5% nonfat dry milk (Carnation, Nestle, 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% bovine serum albumin) were added to wells and plate were 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 antibodies (1:7000; Abcam, Inc.) in dilution buffer. The plates were incubated at room temperature for 1 h. After the wells were washed again, primary antibody binding was detected using alkaline phosphatase-conjugated donkey anti-sheep IgG (1:5000, Sigma-Aldrich, Inc.) in dilution buffer . The plates were incubated at room temperature for 1 hour, and the wells were washed again. ELISA was performed with phosphatase substrate (1 mg/ml para-nitrophenyl phosphate; Sigma) in substrate buffer (50 mM sodium carbonate, 1 mM MgCl _2, pH 9.8). After incubation at room temperature for 30 min, absorbance was measured at 405 nm in a UV-VIS reader (Spectromax 190; Molecular Devices, Inc.).

Binding of anti-fHbp monoclonal antibodies to fHbp analyzed by ELISA. Microtiter plate wells were coated with fHbp, blocked, and washed as described above for the fH ELISA. Five-fold serial dilutions of mouse anti-fHbp monoclonal antibody (mAb) were added from 25 to 0.0016 μg/ml in dilution buffer, and the plate was incubated at room temperature for 1 h. After washing the wells, the primary antibody was detected using goat anti-IgG antibodies alkaline phosphatase-conjugated mice (1; 5000, Sigma-Aldrich). ELISA was performed and analyzed as described above.

Binding of fH to fHbp by surface plasmon resonance (SPR). SPR experiments were performed on a Biacore X100 Plus instrument (GE Life Sciences). Three thousand response units of purified human fH were coupled to the CM5 chip (GE Life Sciences) using the Amine Coupling Kit (GE Life Sciences). fH was immobilized in flow cell 2, and an immobilization blank (without fH) was performed in flow cell 1 as a reference. Three run cycles consisting of HEPES-buffered saline containing 3 mM EDTA and 0.05% Surfactant P-20 (GE Life Sciences) and regeneration with 100 mM glycine, 3 M NaCl, pH 2.0, were performed to create chip surface conditions. Dilutions of purified recombinant fHbp ranging from 100 to 1 nM (wild type) or 316 to 3.16 nM were administered over 150 seconds. Dissociation was observed for 300 s and data were analyzed using Biacore X100 evaluation software.

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

BALB/c human fH transgenic mice were first screened to identify animals with serum human Fh concentrations >240 μg/ml using ELISA and an fHbp standard curve of purified human fH. ELISA was performed using purified fHbp ID 1 immobilized on the plate, and the primary and secondary antibodies to detect fH were the same as described above (see “Binding of Fh to fHbp by ELISA”).

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

Serum bactericidal humoral (SBA) responses. Complement-mediated human SBA responses were measured against meningococcal strains with identical or closely matching fHbp sequences compared to the corresponding vaccine antigen. Bacteria were grown in regular Frantz medium (Frasch et al. “Outer membrane protein vesicle vaccines for meningococcal disease.” In Methods in Molecular Medicine, v. 66. Meningococcal Vaccines: Methods and Protocols. Edited by Pollard, A.J. and Maiden, M.C. Humana Press Inc. Totowa, NJ) containing 4 mM lactate and 0.02 mM CMP-NANA until mid-exponential phase (OD620 nm = 0.6). Bacteria were diluted 1:25,000 in Dulbecco's PBS containing 1% bovine serum albumin (Equitech Bio.). Human complement was from a donor without any intrinsic bactericidal antibodies and was depleted of IgG antibodies using a HiTrap Protein G column (5 ml; GE Life Sciences). Each reaction contained 25% human complement, ∼400 CFU of bacteria, and dilutions of test antiserum or control antibodies. The SBA titer was calculated as the serum dilution that resulted in a 50% reduction in CFU relative to negative control wells after 60 min of incubation at 37°C. Protein purification: Recombinant fHbp were expressed in E. coli with a C-terminal hexa-histidine tag and purified by metal chelate chromatography (HiTrap Chelating HP, GE Life Sciences) followed by ion exchange chromatography (HiTrap SP; GE Life Sciences ). Proteins (2 μg each) were separated on a 4–12% NuPAGE gel (Invitrogen) using MES running buffer (Invitrogen), and staining was visualized with Coomassie blue (Simply Blue Safe Stain; Invitrogen).

results

A random mutant library approach was developed to identify fHbp mutants with reduced human fH binding. This approach was able to identify mutations resulting in decreased fH binding that may not be predictable based on structural information alone, and was able to make multiple amino acid substitutions at any given position. This approach is contrary to the general approach of replacing alanine at selected positions, which sometimes results in a slight decrease in fH binding.

Using a random mutant library approach, we identified five promising new fHbp ID 1 mutants. The purified antigens of the recombinant fHbp ID 1 mutant Q38R, E92K, R130G, S223R and H248L are shown in FIG. 1.

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

These mutants exhibit decreased fH binding ranging from ∼10-fold (R130G) to ∼20-fold (Q38R) to ∼100-fold (E92K, S223R, and H248L) (Fig. 2).

Fig. 2A and 2B show binding of fHbp ID 1 mutants analyzed by ELISA. Microtiter plate wells were coated with purified recombinant wild-type (WT) ID 1 or one of six different mutant proteins. Various concentrations of purified human fH were added to the wells. Bound fH was detected with sheep anti-human fH antibody (Abcam) and alkaline phosphatase-conjugated donkey anti-sheep IgG antibody (Sigma). A, Positive control, wild-type (WT) fHbp ID 1 protein with high levels of human fH binding. Negative control fHbp ID 1 mutant R41S with low fH binding. B, New fHbp ID 1 mutants with reduced levels of fH binding. The R130G mutant showed moderate fH binding, Q38R showed low binding, and E92K, S223R, and H248L showed significantly lower binding than R41S. The mean and standard deviation for repeated measurements are shown.

A similar pattern of reduced levels of fH binding to mutant proteins was obtained from surface plasmon experiments, in which the R130G and Q38R mutants showed some level of binding, while the other three mutants showed no detectable binding (Figures 3A and 3B).

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

All five fHbp ID 1 mutant proteins retained conformational epitopes recognized by anti-fHbp monoclonal antibodies. Concentration-dependent binding of five anti-fHbp monoclonal antibodies to wild-type or fHbp mutants is demonstrated in FIG. 4.

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

The mutant proteins also retain thermal stability similar to that of wild type fHbp ID 1, with the exception of the E92K mutant, which had slightly reduced stability. Finally, the mutants induced similar bactericidal humoral responses in wild-type CD-1 mice when tested against serogroup B strain H44/76 (Fig. 5A-5B).

Fig. 5A and 5B. Bactericidal humoral responses to fHbp ID 1 mutants in mice. Fig. 5A, groups of 12 to 14 wild-type mice were immunized intraperitoneally with two doses of purified recombinant fHbp (10 μg per dose) given at three-week intervals. Serum was obtained three weeks after the second dose. Serum bactericidal activity was measured using IgG-depleted human serum as the complement source and serogroup B strain H44/76 as the test strain. H44/76 expressed fHbp ID 1, which corresponds to the fHbp ID 1 WT control vaccine. Each symbol represents an individual mouse's caption, and the horizontal bars represent geometric mean captions. The differences between the WT group and each of the mutant groups were not statistically significant (p>0.4 by T test). Fig. 5B, groups of 14 to 15 human fH transgenic mice were immunized intraperitoneally with three doses of purified recombinant fHbp (10 μg per dose) administered at three-week intervals. Serum was obtained three weeks after the third dose. Serum bactericidal activity was measured as described above for wild-type mice.

Fig. 6A and 6B. Binding of human fH to fHbp ID 1 single and double mutants analyzed by ELISA. Experiments were performed as described above for FIG. 2A-2B. Fig. 7. Bactericidal humoral responses to fHbp ID 1 single and double mutants in mice. Groups of 20 wild-type mice were immunized and serum bactericidal humoral responses were determined as described above for FIG. 5A.

Example 2. Characterization of fHbp ID 55 mutants

Materials and methods

The experiments were carried out as described in example 1.

results

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

Fig. 8A and 8B show binding of fHbp ID 55 mutants. A. Binding of fH to immobilized fHbp ID 55 mutants analyzed by ELISA. The experiment was carried out as described above in the legend to FIG. 2. The mean and range are given for two to four repeated measurements. B. Concentration-dependent binding of monoclonal antibody (mAb) JAR 41 against fHbp showed that recombinant fHbp was present in quantities similar to those in microtiter plate wells and maintained a conformation in the region of the epitope recognized by JAR 41 (N-terminal domain; Vu et al (2012) Sci. Reports, supra). The secondary antibody was alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma).

Fig. 9. Bactericidal humoral responses to fHbp ID 55 mutants in wild-type mice. Groups of 12 mice were immunized and serum bactericidal humoral responses were determined as described above for FIG. 5A. Bactericidal activity was measured against the mutant strain H44/76, which expresses fHbp ID 55.

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

Example 3: Identification and Characterization of fHbp ID 22 Mutants

Materials and methods

The experiments were carried out as described in example 1.

results

An independent search for random fHbp mutants with decreased levels of fH binding was performed using fHbp ID 22, which is a group 2 variant (subfamily A). This search resulted in six new promising mutants (Fig. 17). fH binding to the wild type ID 22 and the previously described D211A mutant ELISA is illustrated in FIG. 11A. fH binding to six new mutant ID 22 proteins is illustrated in FIG. 11B.

Fig. 11A-11D. fH binding fHbp ID 22 mutant library. Fig. 11A. Positive control, wild type (WT) fHbp protein ID 22 with high levels of human fH binding. Negative control fHbp ID 22 mutant D211A with low fH binding. Fig. 11B, New fHbp ID 22 mutants with reduced levels of fH binding. All mutants showed low levels of binding and V131D showed very low levels of binding similar to D211A. The experiment was carried out as described above in the legend to FIG. 2. The mean and range of two to four repeated measurements are given. Fig. 11C, fH binding of a subset of fHbp ID 22 mutants at fH concentrations up to 100 μg/ml. Fig. 11D, Binding of anti-fHbp monoclonal antibody JAR 4 to a subset of mutants (same symbols used in Fig. 11C). The new mutant K219N retains the level of binding to JAR 4, whereas the G220S mutant decreases the level of binding to JAR 4. All mutants had normal levels of binding of the other anti-fHbp monoclonal antibody JAR 31 (data not shown). The mean and standard deviation for repeated measurements are shown.

The bactericidal humoral responses of wild-type CD-1 mice to the novel mutants V131D and K219N, along with the wild-type control protein ID 22 and the previously characterized mutant D211A, are illustrated in FIG. 12.

Fig. 12A-12B. Bactericidal humoral responses to fHbp ID 22 libraries of wild-type mutant mice. A library of mutants with low levels of human fH binding were selected for immunization. Groups of 10 to 21 mice were immunized with two doses of purified recombinant fHbp (10 μg per dose) given at three-week intervals. Serum was obtained three weeks after the second dose. Serum bactericidal activity was measured using IgG-depleted human serum as the complement source and serogroup B strain CH597 as the test strain. This strain expressed fHbp ID 23, which corresponds to the control vaccine fHbp ID 22 WT. Fig. 12A, experiment testing new mutants V131D and K219N. The D211A mutant was used as a control for a mutant fHbp vaccine that did not reduce immunogenicity. Each symbol represents an individual mouse's caption, and the horizontal bars represent geometric mean captions. Fig. 12B, Second experiment testing new mutants D121G, S128T, F129S and G220S. With the exception of the modest loss of immunogenicity of V131D, no significant loss of immunogenicity was observed for the new fHbp mutants.

Fig. 13. Bactericidal humoral responses to the fHbp ID 22 library of the K219N mutant in transgenic mice with human fH. The control mutant D211A exhibited higher levels of responses, and the K219N mutant exhibited responses similar to the wild-type (WT) fHbp ID 22 antigen.

Fig. 14. Thermal stability of fHbp ID 22 measured by differential scanning microcalorimetry. Wild-type FHbp ID 22 (WT, solid line) undergoes unfolding transitions at 38 (N-terminal) and 81°C (C-terminal domain). The FHbp ID 22 L130R/G133D double mutant exhibits 19°C higher thermal stability for the N-terminal domain compared to that of ID 22 WT.

Fig. 15A-15B. fHbp ID 22 triple mutants combining the stabilizing substitutions L130R and G133D (double mutant, DM) with a library of resulting mutants to reduce fH binding. A, fH binding to fHbp ID 22 triple mutants. B, control binding of anti-fHbp mouse monoclonal antibody (mAb) 4 JAR (same symbols as in panel A). All stabilized mutants bind JAR 4 better than fHbp ID 22 WT.

Fig. 16. Bactericidal humoral responses to fHbp ID 22 triple mutants in human fH transgenic mice. Both mutants were tested by combining double mutant (DM) stability with K219N and G220S, respectively. The triple mutants elicited eight- and 18-fold higher levels of responses than the WT control antigen ID 22.

A brief description of the exemplary fHbp ID 1 and ID 22 mutants described above is presented in the table in FIG. 17.

While the present invention has been described with reference to specific embodiments, those skilled in the art will understand that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process steps or stage to the object, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

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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

15

<210> 27

<211> 8

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 27

Asp Tyr Lys Asp Asp Asp Asp Lys

15

<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

sequence list

<110> Beernink, Peter T

<120> Factor H Binding Protein Variants and Methods of Use Thereof

<130>CHOR-076WO

<150> US 62/028,123

<151> 2014-07-23

<160> 28

<170> Patent In 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

15

<210> 27

<211> 8

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 27

Asp Tyr Lys Asp Asp Asp Asp Lys

15

<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 (27)

1. A protein that is a variant factor H-binding protein (fHbp) subfamily A, which contains: amino acid replacement of glycine at amino acid position 220 with serine (G220S); and optional amino acid substitutions L130R and G133D; wherein the amino acid substitutions are substitutions with respect to fHbp ID NO: 22 (SEQ ID NO: 2), wherein the amino acid positions are numbered relative to the amino acid sequence of fHbp ID1 presented in SEQ ID NO: 1, and the variant contains an amino acid sequence having more than 85% amino acid sequence identity with SEQ ID NO: 2, wherein the fHbp variant binds human factor H (fH) with an affinity that is 50% or less of the affinity of fHbp ID 22 for human fH, and the variant causes a bactericidal humoral response in a mammal. 2. The fHbp variant of claim 1, which binds human fH with an affinity that is 25% or less of the fHbp ID 22 affinity for human fH. 3. The fHbp variant of claim 1, which binds human fH with an affinity that is 10% or less of the fHbp ID 22 affinity for human fH. 4. The fHbp variant of claim 1, which binds human fH with an affinity that is 5% or less of the fHbp ID 22 affinity for human fH. 5. The fHbp variant according to claim 1, which additionally includes substitutions L130R and G133D, where amino acid substitutions are relative to fHbp ID 22. 6. The fHbp variant according to claim 1, wherein the amino acid sequence has at least 90% amino acid sequence identity with SEQ ID NO: 2. 7. The fHbp variant of claim 1, wherein the amino acid sequence has at least 95% amino acid sequence identity with SEQ ID NO: 2. 8. The fHbp variant of claim 1, wherein the amino acid sequence has at least 99% amino acid sequence identity with SEQ ID NO: 2. 9. An immunogenic composition for inducing a humoral response against N. meningitidis in a mammal, containing: a) a therapeutically effective amount of the fHbp variant according to any one of claims. 1-8, wherein the fHbp variant is optionally present in a vesicle preparation derived from a Neisseria meningitidis strain; And b) a pharmaceutically acceptable excipient, where the excipient optionally constitutes an adjuvant, and if necessary c) Neisseria surface protein A. 10. The immunogenic composition of claim 9, wherein the fHbp variant is a vesicular preparation derived from a strain of Neisseria meningitidis. 11. The immunogenic composition according to claim 9, wherein said pharmaceutically acceptable excipient is an adjuvant. 12. The immunogenic composition according to claim 10, wherein said pharmaceutically acceptable excipient is an adjuvant. 13. The immunogenic composition according to claim 9, additionally containing Neisseria surface protein A. 14. The immunogenic composition according to claim 11, in which the adjuvant is aluminum phosphate or aluminum hydroxide. 15. Immunogenic composition according to claim 12, in which the adjuvant is aluminum phosphate or aluminum hydroxide. 16. Nucleic acid encoding the fHbp variant according to any one of paragraphs. 1-8. 17. Recombinant expression vector comprising the nucleic acid according to claim 16. 18. An in vitro host cell for expressing the fHbp variant according to any one of claims. 1-8 and containing the recombinant expression vector according to claim 17, where the host cell is not a human embryonic stem cell. 19. A method for inducing a humoral immune response against N. meningitidis in mammals, comprising administering to the mammal a therapeutically effective amount of an immunogenic composition according to any one of claims. 9-15. 20. The method of claim 19, wherein the mammal is a human. 21. The method of claim 19, wherein the antibody response is a bactericidal antibody response to one or more strains of N. meningitidis.